[
  {
    "caption": "(a) Intracellular levels of spermidine (Spd) in chronologically ageing wild-type yeast. Data represent means ± s.e.m. (n = 3; *P 0.05 and *P 0.001).",
    "molecules": "spermidine"
  },
  {
    "caption": "(b) Survival determined by clonogenicity during chronological ageing of wild-type yeast (BY4741) cells with (o) and without (▪) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 5).",
    "molecules": "spermidine"
  },
  {
    "caption": "(c) Intracellular levels of spermidine in chronologically ageing wild-type yeast cells cultured with (open bar) or without (closed bar) spermidine (4 mM) for 5 days. Data represent means ± s.e.m. (n = 3; **P 0.001).",
    "molecules": "spermidine"
  },
  {
    "caption": "(d) Replicative lifespan analysis of BY4741 wild-type yeast cells after separation into old (fraction V) and young (fraction II) cells by elutriation centrifugation. The remaining lifespan with or without (control) spermidine (1 mM, applied after elutriation) on 2% glucose synthetic complete medium is shown.",
    "molecules": "glucose, spermidine"
  },
  {
    "caption": "(e) Survival determined by the age-specific number of dead individuals of female Drosophila with and without (control) supplementation of food with various concentrations of spermidine (as indicated). A representative ageing experiment of at least 50 flies per sample is shown.",
    "molecules": "food, spermidine"
  },
  {
    "caption": "(f) Endogenous spermidine of female Drosophila fed for 48 h with food supplemented by 1 mM spermidine, compared with normal food (control). Data represent means ± s.e.m. (n = 3; *P 0.01).",
    "molecules": "food, spermidine"
  },
  {
    "caption": "(g) Survival determined by annexin V/7-AAD co-staining (unstained cells were considered as viable) of human immune cells (PBMC) cultured for 6 and 12 days in the absence (black bar) or presence (white bars) of various spermidine concentrations (as indicated). Data represent means ± s.e.m. of 3 independent experiments (each performed on PBMC from different donors). *P 0.05 and **P 0.01.",
    "molecules": "spermidine"
  },
  {
    "caption": "(h, i) Free thiol group (RSH) concentration in serum (h) and intracellular spermidine concentration in hepatocytes (i) of ageing mice with (open bars) or without (closed bar) supplementation of drinking water with spermidine (0.3 and 3 mM) for 200 days. Data represent mean ± s.e.m. (n = 3; *P 0.05 and **P 0.01).",
    "molecules": "spermidine, Free thiol group (RSH), water"
  },
  {
    "caption": "(a) Intracellular spermidine of five-day-oldΔspe1 cells, compared with wild-type cells. Data represent means ± s.e.m. (n = 3; *P 0.001).",
    "molecules": "spermidine"
  },
  {
    "caption": "(b) Chronological ageing of wild-type (▪, o) and polyamine-depleted Δspe1 (▴, ▿) yeast cells with (open symbols) and without (closed symbols) supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4). Cells were tested for cell death markers at day 3.",
    "molecules": "polyamine, spermidine"
  },
  {
    "caption": "(c) Fluorescence microscopy of DHE→Eth conversion in wild-type and Δspe1 cells indicating ROS production. Scale bars, 10 μm.",
    "molecules": "Eth, ROS"
  },
  {
    "caption": "(d) Quantification (FACS analysis) of ROS accumulation using DHE→Eth conversion of wild-type (WT) and Δspe1 cells with and without supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4; *P 0.001).",
    "molecules": "Eth, ROS, spermidine"
  },
  {
    "caption": "(e) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using annexin V/PI co-staining (at day 5) and of DNA fragmentation using TUNEL staining (at day 3) of chronologically ageing wild-type (WT) and Δspe1 cells with or without supplementation of 0.1 mM spermidine as indicated. Data represent means ± s.e.m. (n = 3; *P 0.001).",
    "molecules": "spermidine"
  },
  {
    "caption": "(a) Fluorescence microscopy of DHE→Eth conversion and annexin V (green)/PI (red) co-staining of wild-type cells at day 18 of the chronological ageing experiment shown in Fig. 1b. Scale bars, 10 μm. (b, c) Quantification of DHE→Eth conversion (b) and annexin V/PI co-staining (c) using FACS analysis performed at indicated time-points of the chronological ageing experiment shown in Fig. 1b. Data represent means ± s.e.m. (n = 3; *P 0.001).",
    "molecules": "Eth"
  },
  {
    "caption": "(d) Electron microscopy of young log-phase cells (day 0) and of 20-day-old wild-type cells aged without (control) or with spermidine (4 mM). Representative cells are shown (for an overview see Supplementary Information, Fig. S3c).",
    "molecules": "spermidine"
  },
  {
    "caption": "(e) Fluorescence microscopy of chronologically aged wild-type cells (day 3 and 14) expressing an EGFP-tagged version of the yeast HMGB1 homologue (Nhp6A-EGFP) with or without (control) addition of 4 mM spermidine. Scale bars, 5 μm.",
    "molecules": "spermidine"
  },
  {
    "caption": "(a) Relative acetylation (normalized to controls at each day) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis. A representative blot using site specific antibodies is shown in b. Data represent means ± s.e.m. (n = 3). Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification (examples are shown in Supplementary Information, Fig. S4). For calculation details see section on Methods. P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (b) Immunoblot of whole-cell extracts of wild-type cells chronologically aged to designated time-points with (+) or without (−) spermidine application. Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues. Full scans of blots are available in Supplementary Information, Fig. S5.",
    "molecules": "spermidine"
  },
  {
    "caption": "(e) Relative acetylation (normalized to controls) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine. Data represent quantification of two independent experiments performed with cells obtained from two different donors.",
    "molecules": "spermidine"
  },
  {
    "caption": "(f) Immunoblot analysis of liver cell extracts (applied in serial dilutions) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age. Blots were probed with antibodies recognizing total histone H3 (C-terminal epitope) or specific for acetylated lysine 18 residue (N-terminal epitope). Full scans of blots are available in Supplementary Information, Fig. S5.",
    "molecules": "spermidine"
  },
  {
    "caption": "(a) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b. Data represent means ± s.e.m. (n = 3; *P 0.05). (b) Chronological ageing of wild-type (▪, o) and Δiki3Δsas3 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4). (c) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity (annexinV/PI costaining) as well as ROS production (DHE->Eth conversion) performed at day 20 of the experiment shown in b. Data represent means ± s.e.m. (n = 4; *P 0.001).",
    "molecules": "Eth, ROS, spermidine"
  },
  {
    "caption": "(d) Relative inhibition of histone acetyltransferase activity (HAT-activity) by spermidine determined by an in vitro HAT-activity assay of yeast nuclear extracts of wild-type cells. Data represent means ± s.e.m. of three independent experiments (*P = 0.024).",
    "molecules": "spermidine"
  },
  {
    "caption": "(g) Relative change of ATG7, ATG11 and ATG15 mRNA levels by spermidine supplementation (normalised to controls) after ten days of chronological ageing as determined by reverse transcriptase real-time PCR. Data represent means ± s.e.m. (n = 3; *P 0.05 and **P 0.01).",
    "molecules": "spermidine"
  },
  {
    "caption": "(a) Fluorescence microscopy of wild-type yeast cells expressing an EGFP-Atg8p fusion protein with or without (control) treatment of 4 mM spermidine for 48 h. White arrows indicate vacuolar localization of EGFP-Atg8p indicative of autophagy. Scale bars, 5 μm.",
    "molecules": "spermidine"
  },
  {
    "caption": "(b) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with (open bars) or without (closed bars) application of spermidine (4 mM). Data represent means ± s.e.m. (n = 3 *; P 0.01 and **P 0.001).",
    "molecules": "spermidine"
  },
  {
    "caption": "(d) Chronological ageing of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4).",
    "molecules": "spermidine"
  },
  {
    "caption": "(e) Chronological ageing on water of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (8 mM) at day 0. Data represent means ± s.e.m. (n = 4).",
    "molecules": "spermidine, water"
  },
  {
    "caption": "(f) Quantification (fluorescence reader) of ROS production (DHE→Eth conversion) by wild-type and Δatg7 cells with and without supplementation of spermidine (8 mM), obtained from the ageing experiment shown in panel e. Data represent means ± s.e.m. (n = 4; *P 0.07 and **P 0.01).",
    "molecules": "Eth, ROS, spermidine"
  },
  {
    "caption": "(a) Fluorescence microscopy of Hoechst-counterstained HeLa cells transiently transfected with LC3-GFP subjected to 100 μM spermidine for 6 h. Representative pictures are shown. (b) Percentage of adherent cells exhibiting a clear LC3-GFP relocalization into cytoplasmic vacuoles. Numbers were determined using micrographs of Hoechst-counterstained HeLa cells as representatively shown in a. Data represent means ± s.d. (n = 3).",
    "molecules": "spermidine"
  },
  {
    "caption": "(c) LysoTracker Red staining of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days, compared with controls (without spermidine). Nuclei were visualized by Hoechst staining. Scale bars, 10 μm.",
    "molecules": "spermidine"
  },
  {
    "caption": "(d) Quantification of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of female flies fed with supplementation of 1 mM spermidine or with 10% glucose (starved) for 48 h, compared with normal food (control). Data represent means ± s.e.m. of at least 20 flies for each group (*P 0.01).",
    "molecules": "food, glucose, spermidine"
  },
  {
    "caption": "(e, f) Survival of Drosophila during ageing without (control) and with supplementation of food at various concentrations of spermidine (as indicated). Autophagy-deficient flies (f) homozygous mutant for Atg7 (Δatg7) were compared with flies capable of autophagy (e) and heterozygous for Atg7 (control). For details of strains and additional wild-type controls, see Supplementary Information Methods and Fig. S6h, i.",
    "molecules": "food, spermidine"
  },
  {
    "caption": "(g) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRED::LGG-1 fusion protein indicative of autophagic activity. Shown are two representative pictures of embryos untreated (control) or treated with spermidine (0.2 mM) supplementation of food. (h) Quantification of autophagic activity through measurement of DsRED::LGG-1 pixel intensity from images of wild-type animals shown in g, and bec-1 RNAi knockdown animals (bec-1 RNAi). Data represent means ± s.e.m. (n = 3) with at least 25 images processed for each trial.",
    "molecules": "food, spermidine"
  },
  {
    "caption": "(i) Survival of C. elegans during ageing with and without (control) supplementation of food (UV-killed E. coli) with spermidine (0.2 mM). Wild-type (N2) animals were compared with bec-1 RNAi animals deficient in autophagy induction. For mean life spans see Supplementary Information, Table S3.",
    "molecules": "food, spermidine"
  },
  {
    "caption": "(F) Quantification of the average number of ATTO-550α-synucleinfibrils per acceptor cell after 24 hours co-culture with donor cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α-synuclein puncta in acceptor cells; n = 3 independent experiments (**, p < 0.01 by two-tailed Mann Whitney test).",
    "molecules": "DiI"
  },
  {
    "caption": "(C) ALYREF RIP-qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P< 0.001, n.s., not significant.",
    "molecules": "tRNA"
  },
  {
    "caption": "(D) The distribution of histone reads in 5' UTR, CDS and 3' UTR in rRNA-depleted nuclear RNA-seq and ALYREF-iCLIP-seq.",
    "molecules": "rRNA"
  },
  {
    "caption": "(B) List of histone genes with defective 3'-end processing detected by polyA+ and polyA- RNA-seq. PolyA+ ALYREF/Cntl shows polyA+ RNA-seq RPM ratio of each histone gene in ALYREF KD versus Cntl.",
    "molecules": "polyA, PolyA"
  },
  {
    "caption": "(E, F) RT-qPCRs to detect histone mRNA levels in the polyA+ (E) or the polyA- fraction (F) in cells treated with Cntl, ALYREF or ARS2 siRNA. The arrowhead and arrows indicate the cleavage site and primer location, respectively. The bars show the relative abundance of polyA+ forms to the actin mRNA (E) and that of polyA- forms relative to the 18S rRNA (F). Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.",
    "molecules": "polyA"
  },
  {
    "caption": "RT-qPCRs to detect polyA+ histone mRNA levels (H) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The bars show the relative abundance of histone mRNAs to the GAPDH mRNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.",
    "molecules": "polyA"
  },
  {
    "caption": "(A) (Top) Illustration of the HIST2H2AA3 reporter construct. (Bottom) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. DAPI staining served as a nuclear marker. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B. Quantification of oryzalin root growth assays in single (A) and double (B) mutants. DAG = days after germination. Graphs show mean ± SD of three biological replicates with at least 10 plants per genotype per replicate. Asterisks indicate a significant difference in root length in a two-way ANOVA followed by Tukey's multiple comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001).",
    "molecules": "oryzalin"
  },
  {
    "caption": "A, B. DAPI staining of pollen in cycb1 mutants, including pollen configurations found in cycb1;1-/- cycb1;2+/- cycb1;3+/- cycb1;4-/- mutants (B). Scale bars: 5 μm. C. Quantification of DAPI-stained pollen configurations in different cycb1 mutant combinations, n = 420-616 pollen grains per genotype. Different letters indicate significant differences in the proportion of abnormal pollen (uni- and bicellular) in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Alexander staining of mature pollen indicating pollen viability. Scale bars: 5 μm. E. Quantification of Alexander-stained pollen viability, n = 403-498 pollen grains per genotype. Different letters indicate significant differences in the proportion of dead pollen in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.",
    "molecules": "Alexander"
  },
  {
    "caption": "A, B, C. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). White arrowheads indicate laggards in the metaphase stage. Scale bars: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). Scale bars: 5 μm. B. Quantification of wildtype (WT) and abnormal phragmoplasts. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. C. Quantification of the different mitotic stages in roots of the different genotypes. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Kinase assays against Histone H1. Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. B. Kinase assays against GIP1. Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of GIP1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. C. Western blot against StrepIII-tagged proteins to show loaded amounts of the CDKs. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. Double CDKB2;2 bands are likely due to a truncation of the expressed protein.",
    "molecules": "CBB, StrepIII"
  },
  {
    "caption": "(A) BMDMs  were treated with E. coli  (MOI=100) for 30 minutes and further cultured for various length of time  as indicated. The dynamic of ENT3  expression was determined by measuring Slc29a3  transcripts via RT-qPCR. (B) Phagocytosis inhibitor, Cytochalasin D (CytoD), pretreated BMDMs  were incubated with E. coli (MOI=100) and subjected for Slc29a3  expression analysis via RT-qPCR  6 hours post-infection. Data information: The expression level was calculated relative to Rpl19  and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "Cytochalasin D, CytoD"
  },
  {
    "caption": "(D) Different bacterial components were applied as stimulants to BMDMs. Slc29a3  expression was analyzed after 30 minutes of stimulation and a total of 6 hours incubation. (E) BMDMs  were treated with different doses of LPS  for 30 minutes, harvested 6 hours post-stimulation, and subjected for RT-qPCR  analysis. Data information: The expression level was calculated relative to Rpl19  and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) WT or (D) MyD88-/- BMDMs  were stimulated with 10 ng/ml LPS  or live  E. coli  (MOI=100) in the presence of 10 µM TBK  inhibitor, MRT 67307, and analyzed for Slc29a3 expression. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "LPS, MRT 67307"
  },
  {
    "caption": "(E) The 10 ng/ml LPS-treated BMDMs  were examined for Ifnb1 expression  after 30 minutes of stimulation and a total of 6 hours incubation. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "(B-E) BMDMs  from WT, IFNAR1-/- (B), or STAT1-/- (C) mice  were treated with 10 ng/ml LPS, live E. coli (MOI=100), or 100 U/ml IFN-β, harvested and examined for Slc29a3  induction. BMDMs  from WT, IFNAR1-/- (D) or STAT1-/- (E) mice  were treated with 100 U/ml  IFN-β, 50 µg/ml poly(I:C), or EMCV  (MOI=10), harvested and examined for Slc29a3  induction. Data information: The expression  level was calculated relative to Rpl19  and normalized to untreated group as 1. The results shown in B to E are from three biological replicates (n=3) Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "LPS, poly(I:C)"
  },
  {
    "caption": "(A-C) (A) BMDMs  were infected with EMCV  (MOI=10) and harvested at indicated time points. (B) THP-1 derived macrophages  were challenged with EV71  (MOI=5) and collected at different time points. (C) BMDMs  were co-cultured with 50 µg/ml  poly (I:C)  with various length of time  and harvested. The mRNA expression  of Slc29a3  or SLC29A3  was measured by RT-qPCR. The expression level was calculated relative to Rpl19  or RPLP0  and normalized to untreated group at time  0 as 1. Results are combined from three biological replicates (n=3) Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "poly (I:C)"
  },
  {
    "caption": "(G, H) The replication of EMCV  in WT (G) and ENT3-/- BMDMs (H) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides  (A, U, C, G) 9 hours post-infection  (MOI=10). The expression of EMCV-2A2B   was determined by RT-qPCR. Shown are results from three to four biological replicates (n=3-4). Data information: In G, H) the EMCV-2A2B  expression level was calculated relative to Rpl19  and normalized to untreated group at time  0 as 1. Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.",
    "molecules": "ribonucleosides"
  },
  {
    "caption": "(A) Illustration of strategy to track the viral uncoating upon infection. (B-D) (B) WT and ENT3-/- BMDMs  were infected with Syto82-labeled EMCV  (MOI=1) and subjected to  live imagining 30, 60 and 90 minutes post-infection. The arrowheads indicated the Syto82+ virus puncta in the cell. Scale bars represent 10 µm. (C) Quantification of Syto82  signal in BMDMs  after 60 minutes was performed by enumerating Syto82+ cells (%) and (D) the mean fluorescent intensity (MFI) per Syto82+ cell (D). Results were generated from images of three biological replicates (n=3), each with 4 fields analyzed. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "Syto82"
  },
  {
    "caption": "(E) WT and ENT3-/- BMDMs  were infected with Syto82-labeled EMCV  (MOI=1) and co-stained  with 100 nM Lysotracker Green, subjected to live imagining 60 minutes post-infection. Data were the representative images of two biological replicates (n=2). Scale bars represent 5 µm. (F) WT and ENT3-/- BMDMs   were infected with EMCV  (MOI=10) for 2 hours and then enriched the endosomes  and lysosomes. EMCV  particles in endosome- or lysosome-enriched fractions were quantified by  plaque assay. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "Syto82, Lysotracker Green"
  },
  {
    "caption": "(G-K) (G) Calu-3  cells were infected with  SARS-CoV-2  original strain or delta  variant at MOI=0.1 for 48 hours, harvested, and examined for SLC29A3  induction. (H) ENT3  knockdown in Calu-3  cells was performed by lentiviral  shRNA system. The knockdown efficiency was confirmed by RT-qPCR. ENT3  knockdown Calu-3 cells were infected with SARS-CoV-2 delta  variant and subjected for the viral replication analysis  24 hours post-infection. The virus released out of cells was measured by plaque assay (I), and the viral titer in the cells was measured by RT-qPCR (J). (K) The replication of SARS-CoV-2 delta  variant in scramble and ENT3  knockdown  Calu-3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides  (A, U, C, G) 24 hours post-infection  (MOI=0.1). The SLC29A3  expression level was calculated relative to RPLP0  and normalized to control as 1. The SARS-CoV-2 E gene expression level was calculated relative to RPLP0  to normalize the cell number. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.",
    "molecules": "ribonucleosides"
  },
  {
    "caption": "(a) Confocal analysis of the subcellular localization of HA-PLD1 (green) and endogenous LC3 (red) in HeLa cells in normal medium and under nutrient deprivation conditions. Left panels: normal medium condition (Nm); middle panels: 1 h with nutrient-deprived medium (1 h St); right panels: 2 h with nutrient-deprived medium (2 h St). Insets denote high magnification of the selected area. Scale bar, 5 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and subjected to 1 h of nutrient deprivation in the presence (St+B) or absence (St) of bafilomycin (50 nM). Insets denote high magnification of the selected area. Scale bar, 500 nm. Right panel: Histogram showing morphometric analysis of the relative distribution of GFP-PLD1-positive gold particles on the outer (arrows) and inner (arrowheads) membranes of the autophagosomal-like structures. Values denote means±s.e.m. (n=14). **P0.01.",
    "molecules": "gold particles, bafilomycin, nutrient"
  },
  {
    "caption": "Confocal analysis of the subcellular localization of HA-PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h. Organelle markers used were as follows (from top to bottom panels): Lamp1 (late endosomes/lysosomes), giantin (Golgi-TGN), Vps35 (retromer-positive endosomes), GFP-Rab5 (early endosomes), GFP-UVRAG (early and late endosomes). Insets show high magnification of the selected areas. The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.",
    "molecules": "nutrients"
  },
  {
    "caption": "(a) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h. Cells were transfected with HA-PLD1 and GFP-ATG14L (upper panels) or with HA-PLD1 alone (lower panels). Upper panels: HA-PLD1 (red), GFP-ATG14L (green), endogenous LC3 (blue). Lower panels: HA-PLD1 (green), endogenous ATG16L (red) and LC3 (blue). Insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) Confocal analysis showing lack of co-localization of HA-PLD1 (green) with the isolation membrane marker ATG16L (blue) in HeLa cells expressing the strawberry-ATG4-C74A mutant (red) and subjected to 1 h of nutrient deprivation. Insets denote high magnification of the selected area. Scale bar, 5 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(c) EM analysis showing that GFP-PLD1 does not localize to the isolation membrane (arrows) in cells expressing the strawberry-ATG4-C74A mutant. Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and pStrawberry-ATG4-C74A and subjected to 1 h of nutrient deprivation. Inset denotes high magnification of the selected area. Scale bar, 250 nm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05.",
    "molecules": "ethanol, nutrient, phosphatidylserine, phosphatidylcholine, PEtOH, phosphatidylethanol, phosphatidylethanolamine, phospholipid, wortmannin"
  },
  {
    "caption": "(b) Western blot analysis of HA-PLD1 and endogenous LC3 levels in post-nuclear supernatant (PNS, upper panel), as well as in cytosolic/soluble (S) and particulate (P) fractions prepared from PNS after high-speed centrifugation (middle panel). GAPDH was used as loading control in PNS fractions. HeLa cells were transfected with expression vectors encoding HA-PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin (100 nM). Lower panel: quantitative analysis of HA-PLD1 immunoreactivity expressed as P/S ratio. Conditions were as follows: normal medium (Nm); normal medium with wortmannin (Nm+W); starvation condition (St); starvation condition+wortmannin (St+W). Values denote means±s.e.m. (n=3).",
    "molecules": "nutrient, wortmannin"
  },
  {
    "caption": "(c) Confocal analysis of the co-localization between HA-PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence (ST+W) or absence (St) of wortmannin 100 nM. Insets show high magnification of the selected areas. Scale bar, 5 μm.",
    "molecules": "nutrient, wortmannin"
  },
  {
    "caption": "(a) Western blot analysis showing the effects of siRNA-mediated Vps34 (siVps34) downregulation on the expression levels of HA-PLD1 and Vps34 compared with control cells (siCtrl). Cells were incubated in normal medium (Nm) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin (St+B). HeLa cells were transfected for 48 h with siRNA directed to Vps34 (siVps34) or a control sequence (siCtrl) and then transfected for 24 h with a plasmid encoding HA-PLD1.",
    "molecules": "bafilomycin, nutrient"
  },
  {
    "caption": "(b) Confocal imaging showing the effects of Vps34 (siVps34) downregulation on HA-PLD1 (green), endogenous LC3 (blue) and Lamp1 (red) on 2 h of nutrient deprivation compared with control treatment (siCtrl). Scale bar, 5 μm. (c) Confocal analysis showing the subcellular localization of HA-PLD1 mutated in its PX domain (F120A/R179Q), so as to abolish phosphoinositide binding (HA-PXmut). HeLa cells were transiently transfected with a plasmid encoding HA-PXmut and fixed after 2 h of nutrient deprivation. HA-PXmut (green), endogenous LC3 (blue) and Lamp1 (red). Scale bar, 10 μm. In b and c, insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3. CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum (normal medium, Nm) or in the absence of nutrients (starvation, St). Insets denote high magnification of the selected areas. Scale bar, 10 μm. (b) Quantification of the average number, size and surface area of LC3-positive compartments per cell after FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=180-285 cells for Nm; n=449-519 cells for St). **P0.01.",
    "molecules": "DMSO, FIPI, nutrients"
  },
  {
    "caption": "(c) Western blot analysis showing levels of LC3-I and LC3-II in control- and FIPI-treated CHO cells grown in normal medium (Nm), after nutrient starvation (St) or after nutrient starvation in the presence of 50 nM bafilomycin (St+B). Treatments were for 3 h. Right panel: quantification of protein signal intensities showing LC3-II protein levels in extracts from nutrient-starved and bafilomycin-treated CHO cells following FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=4).",
    "molecules": "bafilomycin, DMSO, FIPI, nutrient"
  },
  {
    "caption": "(d) Western blot analysis of the effects of PLD inhibition on the levels of total Tau, phosphorylated Tau (p-Tau) and detergent-insoluble Tau. Organotypic brain slices from hTau mice were treated with either DMSO (D) or 750nM FIPI (F) and harvested after 5 days of treatment. Left panel: western blot analysis showing immunoreactivities for total human Tau (CP27), hyperphosphorylated tau (p-Tau, ser202/ser205, CP13), p62 and tubulin from the supernatant fraction and aggregated tau (CP27) and p62 from the sarkosyl-insoluble fraction. Two representative pairs are displayed. Right panel, quantification of total proteins (Tau, p-Tau, p62 and tubulin) and sarkosyl-insoluble aggregates of tau (insoluble Tau) and p62 (insoluble p62). Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=7). **P0.01; *, P0.05. For the statistical analysis of FIPI-treated samples, tau and p62 protein levels were compared with tubulin.",
    "molecules": "DMSO, FIPI, sarkosyl"
  },
  {
    "caption": "a) Western blot analysis showing reduced levels of LC3-II in KO MEFs. (b) Quantification of protein signal intensities from western blots showing LC3-II levels in MEFs extracts after normalization to the control protein GAPDH. In a and b, MEFs were grown in normal medium (Nm), treated for 2 h with 50 nM bafilomycin in normal medium (B), or grown in nutrient-deprived medium in the absence (St) or presence of 50 nM bafilomycin (St+B). Values denote means±s.e.m. (n=4).",
    "molecules": "bafilomycin, nutrient"
  },
  {
    "caption": "(c) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3-positive compartment in KO MEFs. The DAPI staining is shown in blue. Cells were deprived of nutrients for 2 h in the absence (St, upper panels) or presence of bafilomycin (St+B, lower panels). Scale bar, 5 μm. (d) Quantification of the average size (top panel) and number of LC3-positive compartments per cell (bottom panel) in Pld1 WT and KO MEFs starved for 2 h. Values denote means±s.e.m. (n=6, number of cells=26-45 for all the conditions). **P0.01; *P0.05.",
    "molecules": "bafilomycin, nutrients"
  },
  {
    "caption": "(c) Electron microscopic analysis showing a reduction in the size and number of autophagosome/amphisome-like structures (AP) in PLD1-deficient mice after food restriction. Representative electron micrographs from animals analysed in a showing APs (arrows) and autophagolysosomes/lysosomes (AL, arrowheads) in liver sections from starved WT and KO mice. Insets show high magnification of the selected areas. Scale bar, 1 μm. (d) Morphometric analysis of the number (per 100 μm2 cytoplasmic area) and relative surface area (% cytoplasmic area) of APs and ALs. Values denote means±s.e.m. (n=36 electron micrograms, from a total of 3,600 μm2 cytoplasmic area per genotype). **P0.01. (c) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1-deficient mice after food restriction. Values denote means±s.e.m. (n=60). **P0.01.",
    "molecules": "food"
  },
  {
    "caption": "(E) Representative coronal section images of Golgi-Cox staining  of Fibcd1  WT and KO hippocampi  (left), Neurolucida tracing of  hippocampal CA1 pyramidal neurons (middle) and apical dendrites with spines  (right). Scale bars as indicated.",
    "molecules": "Golgi-Cox"
  },
  {
    "caption": "(B) Immunoblot  analysis (left) and quantification of signal intensity (right) of Fibcd1  WT (blue) versus Fibcd1  KO littermates (red) adult hippocampi  with antibodies against CS-0S, CS-4S, CS-6S and actin  as a loading control. Each lane represents an independent animal (n = 3). Protein marker sizes are indicated. Data information: For panel B, each data point represent hippocampal  protein isolates from an individual mouse, Data are shown as mean values +/- SEM. p values were calculated using paired Student's t-test (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.",
    "molecules": "CS-4S, CS-6S, CS-0S"
  },
  {
    "caption": "(F) Confocal  images depicting internalisation  of FITC-tagged CS-4S  by FIBCD1-overexpressing HEK293T  lines compared to untransduced cells and unstained cells. Left, representative images; right, quantification. Data is plotted as total puncta per condition (n = 5). Cells are further stained  with CellMask Orange (cellular membrane) and Hoechst  (nuclei). Scale bar = 50µm. Data information: for panels F, each data point represents an individual cell preparation. Data are shown as mean values +/- SEM. p values were calculated using 1-way ANOVA (panels F, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.",
    "molecules": "CellMask Orange, Hoechst, CS-4S, FITC"
  },
  {
    "caption": "(A) Left, representative image of immunofluorescent staining  (MAP2, red; DAPI, blue) of primary hippocampal  cultures at 2 days in vitro (DIV), plated on +/- CSPG  coating with and without prior digestion with ChABC, as indicated. Right, quantification of DIV2 images, showing the number of protruding cells per field normalised to untreated condition. n(Fibcd1  WT) = 4; n(Fibcd1  KO) = 3. Scale bar = 250µm. (B) Left, representative images of DIV14 neurons, same conditions as in (A). Right, quantification of DIV14 images, representing the percentage of cells per field that are clumped. n(Fibcd1  WT) = 3; n(Fibcd1  KO) = 2. Scale bar = 250µm. Data information: For panels A and B each data point represents an individual preparations of primary cell culture. Data are represented as mean and error bars represent SEM. p values were calculated by 1-way ANOVA. * = p≤0.05; ** =p≤0.01; *** =p≤0.001.",
    "molecules": "DAPI"
  },
  {
    "caption": "A αSMA mRNA levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. *P = 0,001.",
    "molecules": "4μ8C"
  },
  {
    "caption": "B Collagen1α2 mRNA levels in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C. *P = 0,01.",
    "molecules": "4μ8C"
  },
  {
    "caption": "D Western blot of CHOP protein levels in TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C.",
    "molecules": "4μ8C"
  },
  {
    "caption": "E Agarose gel showing the presence of spliced and unspliced XBP-1 mRNA in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C.",
    "molecules": "4μ8C"
  },
  {
    "caption": "F Immunohistochemistry staining of αSMA in TGFβ-treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C. Scale bars = 20μm.",
    "molecules": "4μ8C"
  },
  {
    "caption": "G Quantification of secreted soluble collagen proteins produced by TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C. *P = 0,049.",
    "molecules": "4μ8C"
  },
  {
    "caption": "A miR-150 levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. * P = 0.046 compared to control.",
    "molecules": "4μ8C"
  },
  {
    "caption": "M miR-150 expression in HFL-1 cells that were pre-treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D (ActD) for 4 and 24 hours. * P = 0.027 compared to TGFβ.",
    "molecules": "ActD, Actinomycin D, 4μ8C"
  },
  {
    "caption": "A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence (Ctrl) or presence of the IRE1α inhibitor 4μ8C, or in HFL1 cells transfected with a siRNA control siRNA (siCtrl) or with siRNA targeting XBP-1 (siXBP-1). The ER was visualized with ER tracker Red and nuclei stained with Hoechst (left, scale bars = 20 μm) or by electron microscopy (right, scale bars = 200 nm) where red lines mark the ER.B Quantification of ER size relative to the nucleus from the experimental setup in A. * P = 0.0001 in Ctrl, and * P = 0.0223 in siCtrl.",
    "molecules": "4μ8C"
  },
  {
    "caption": "A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL/6mice as measured by the histological metavir score. Ctrl (n = 8) ; Ctrl + 4μ8C (n = 6) ; CCl4 (n = 6); CCl4 + 4μ8C (n = 8). * P = 0,0077.B Representative liver tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars= 50 μm.C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A. * P = 0,036.",
    "molecules": "CCl4, 4μ8C"
  },
  {
    "caption": "F Representative gel showing levels of spliced and unspliced XBP-1 in liver tissue from control and CCl4-induced cirrhotic mice and the effect of 4μ8C treatment.",
    "molecules": "CCl4, 4μ8C"
  },
  {
    "caption": "H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C. * P = 0,031, n = 3.",
    "molecules": "4μ8C"
  },
  {
    "caption": "I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C. * P = 0,008, n = 3.",
    "molecules": "4μ8C"
  },
  {
    "caption": "L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ±4μ8C.",
    "molecules": "4μ8C"
  },
  {
    "caption": "A The effect of the IRE1α inhibitor 4μ8C on TGFβ-induced skin fibrosis in C57BL/6 mice as measured by the percentage of sirius red areas in the hypodermis. Ctrl (n = 6); Ctrl + 4μ8C (n = 5); TGFβ (n = 8); TGFβ + 4μ8C (n = 8). * P = 0,046.B Representative skin tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars = 50 μm.C Percentage αSMA-positive cells in the hypodermis of mice from the experimental setup in A. * P = 0,024.",
    "molecules": "4μ8C"
  },
  {
    "caption": "D Representative image of the area selected for laser capture microdissection (scale bars = 100 μm).E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection. Ctrl (n = 5), Ctrl + 4μ8C (n = 5), TGFβ (n = 5), TGFβ + 4μ8C (n = 5). * P = 0,044.F miR-150 expression levels in the same samples as shown in E.",
    "molecules": "4μ8C"
  },
  {
    "caption": "G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C. Scale bars = 20 μm.",
    "molecules": "4μ8C"
  },
  {
    "caption": "H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C. * P = 0,049, n = 4.",
    "molecules": "4μ8C"
  },
  {
    "caption": "I Agarose gel showing levels of spliced and unspliced XBP-1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.",
    "molecules": "4μ8C"
  },
  {
    "caption": "J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C. * P = 0,00007 for TGFβ vs. Ctrl and P = 0,00002 for TGFβ vs. TGFβ + 4μ8C. n=4.",
    "molecules": "4μ8C"
  },
  {
    "caption": "K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.",
    "molecules": "4μ8C"
  },
  {
    "caption": "L miR-150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. * P = 0,00031 compared to untreated control, n = 3.",
    "molecules": "4μ8C"
  },
  {
    "caption": "A CTGF mRNA expression levels in skin (dSSc) and lung (LSSc) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C. * P = 0,018 for dSSc (n = 6) and P = 0,003 for LSSc (n = 3).",
    "molecules": "4μ8C"
  },
  {
    "caption": "D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C.",
    "molecules": "4μ8C"
  },
  {
    "caption": "E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or asthma. * P = 0,00001 for asthma vs. healthy controls, and P = 0,00003 for ctrl vs. 4μ8C in asthma patients.",
    "molecules": "4μ8C"
  },
  {
    "caption": "F Quantification of mRNA levels for BiP in same samples as in E. * P = 0,0023 between asthma and healthy controls and P = 0,0031 between 4μ8C-treated and control treatment in fibroblasts from asthma patients.",
    "molecules": "4μ8C"
  },
  {
    "caption": "(B) Flow cytometry  analysis of WT to Il21r-/- splenic  SWHEL B cells over time. (C) WT to Il21r-/- SWHEL B cell ratio within CTV  division peaks. Data information: Data in (B) is representative of 8-9 biological replicates (n = 8-9) pooled from two independent experiments with statistical analysis by one-way ANOVA with Tukey's post-test. ** p ≤ 0.01; **** p ≤ 0.0001. Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.",
    "molecules": "CTV"
  },
  {
    "caption": "(E) Rate of apoptosis   measured by detecting active caspase 3 by flow cytometry across CTV  division peaks. Data information: Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.",
    "molecules": "CTV"
  },
  {
    "caption": "(B) Flow cytometry  analysis showing WT to Il21r-/- SWHEL B cell  ratio of cells that have been in S phase and thus incorporated BrdU. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.",
    "molecules": "BrdU"
  },
  {
    "caption": "(D) Ratio of the proportion of BrdU  positive WT and Il21r-/- SWHEL B cells with 2N DNA  content 4, 10 and 12 hours post BrdU  pulse on day 3.5 post immunization. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.",
    "molecules": "BrdU, DNA"
  },
  {
    "caption": "(C) Rate of de novo  cell cycle entry 10 hours post in vivo IL-21 (2 µg) or saline  pulse. The ratio of WT to Il21r-/- cells not in S/G2 at the time of pulse (BrdU- cells) and containing >2N DNA  content 10 hours after pulse is shown. Data information: Data are representative of 10-12 biological replicates (n = 10-12) from two independent experiments. Statistical analysis by one-sample t test. * p ≤ 0.05.",
    "molecules": "BrdU, DNA, saline"
  },
  {
    "caption": "Phosphoflow analysis  of naïve WT or Il21r-/- B cells following in vitro culture for 3 hours with or without IL-21 (20 ng/mL) and/or in the presence of BCR  cross-linking (biotinylated anti-Igκ  + anti-Igλ  and avidin-mediated cross-linking) or agonistic anti-CD40. (A) Exemplary p-AKT  (S473) staining  and (B) quantification of p-AKT  median fluorescence intensity (MedFI). (C) Exemplary p-S6  (Ser235/236) staining  and (D) quantification of frequency of p-S6  positive cells Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by one-way ANOVA with Tukey's post-test (D), t test (B, E) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.",
    "molecules": "avidin"
  },
  {
    "caption": "(F, In vitro cell culture of CTV  labeled SWHEL B cells  with or without  IL-21 (20 ng/mL) and anti-CD40  for three days and analysis by  flow cytometry. (F) Exemplary CTV  division peaks.",
    "molecules": "CTV"
  },
  {
    "caption": "G) In vitro cell culture of CTV  labeled SWHEL B cells  with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by  flow cytometry. (G) Quantification of the proportion SWHEL B that had divided  and thus diluted CTV. Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by two-way ANOVA with correction for multiple comparison using Šidák method (G). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.",
    "molecules": "CTV"
  },
  {
    "caption": "Analysis of splenic  WT and Il21r-/- SWHEL B cells  by  flow cytometry. (D) CTV  profile of and (E) absolute  cell count in pooled CTV  division peaks (0-4, 5-8, 9-11+). Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. D, show concatenated data representative of 3-5 biological replicates. Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm-Šídák method. * p ≤ 0.05; ** p ≤ 0.01.",
    "molecules": "CTV"
  },
  {
    "caption": "(F) Cell surface expression  (median fluorescence intensity, MedFI) of CD38, IgD  and FAS  across individual CTV  division peaks. (G) Representative  flow cytometry plot of  plasma cell differentiation  indicated by CD138 expression. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. F and G show concatenated data representative of 3-5 biological replicates.",
    "molecules": "CTV"
  },
  {
    "caption": "Substrate was synthesized in reticulocyte lysate in the presence of 35S-methionine and mixed with SecA and SecYEG proteoliposomes. After incubation with ATP, the samples were treated with proteinase K to degrade any non-translocated material. Where indicated, the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X-100 (TX-100). Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT, generating protease-protected full-length (FL) substrate or a translocation intermediate (IM), respectively. All samples were analyzed by SDS PAGE followed by autoradiography. Lane 1 shows 10% of the input sample. The gel is representative of three replicates. Molecular weight markers are estimated from Coomassie-stained gels run in parallel.",
    "molecules": "DTT, ATP, 35S-methionine, NaTT, Triton X-100, TX-100"
  },
  {
    "caption": "Translocation intermediates (IM) were incubated in the presence of different nucleotides (Nuc.) for different time periods and then treated with proteinase K (Prot. K). One sample received hexokinase and glucose (HKG) to generate ADP, other samples ADP•BeFx or ATPγS. The gel is representative of two replicates.   Quantification of (C). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show the average of replicates. Colors correspond to different nucleotides as shown in the inset box.   As in (C), but DTT was added concurrently with HKG or nucleotides. The gel is representative of two replicates.   Quantification of (E) as in (D), except intensities were calculated for the FL bands.   ",
    "molecules": "DTT, ADP, BeFx, ATPγS, glucose"
  },
  {
    "caption": "Imidazole (imi) and either WT or mutant SecA were added to translocation intermediates (IM) pre-formed with SecA dN20::H6. The samples were incubated for the indicated times before proteolysis with proteinase K (Prot. K). The gel is representative of two replicates, except for the experiment with the R792G/K797G mutant and the 30-min time points. Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to different SecA mutants as shown in the inset box. As in (B), but ATPγS was added together with imidazole. The gel is representative of two replicates. Quantification of (E) as in (C). As in (D), but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA. The gel is representative of two replicates.   Quantification of (G) as in (F), except that both the intensities of the IM (darker colors) and FL (lighter colors) bands were measured.   ",
    "molecules": "H6, DTT, ATPγS, imi, Imidazole, imidazole"
  },
  {
    "caption": "A. Arginine methylase inhibitor decreases SIRT7 methylation. HEK293T cells expressing Flag-tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs. After immunoprecipitated with Flag-beads, SIRT7 was analyzed by western blot and detected by an antibody against mono-methylarginine (α-me1). Relative methylation ratio (Ratio) was calculated by normalizing arginine methylation level against the Flag-SIRT7 protein. WCL, whole cell lysate",
    "molecules": "AdOx, arginine, me1, mono-methylarginine"
  },
  {
    "caption": "C. PRMT inhibitor reduces arginine methylation of wildtype SIRT7, but not its RK/RF mutants. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. Arginine methylation of immunopurified SIRT7 was determined by western blot",
    "molecules": "AdOx, arginine, Arginine"
  },
  {
    "caption": "D. SIRT7 is mono- and asymmetrically di-methylated at R388. Flag-tagged SIRT7 and its RK/RF mutants were expressed in HEK293T cells. Immunopurified SIRT7 was blotted with antibodies against mono-methylarginine (α-me1), asymmetric di-methylarginine (α-me2a), and symmetric di-methylarginine (α-me2s), respectively",
    "molecules": "me2s, symmetric di-methylarginine, asymmetric di-methylarginine, me2a, me1, mono-methylarginine"
  },
  {
    "caption": "H. SIRT7 is methylated at R388. HEK293T cells ectopically expressing Flag-tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated. SIRT7 protein was pulled down by Flag-beads. Arginine methylation of SIRT7 was detected with a site-specific antibody against R388 methylation [α-meSIRT7(R388)]",
    "molecules": "AdOx, Arginine"
  },
  {
    "caption": "I-J. AdOx treatment decreases R388 methylation of SIRT7. MEF and L02 cells were treated with AdOx for 24 hrs. Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot (I). The band intensity of western blots was measured to quantify relative R388 methylation ratio (J) (mean ± SD, n=3 experimental replicates, ***p < 0.001, unpaired two-tailed t-test)",
    "molecules": "AdOx"
  },
  {
    "caption": "G. PRMT6 inhibitor reduces arginine methylation of wildtype SIRT7, but not RK/RF mutants. Wildtype SIRT7 or its RK/RF mutants were stably expressed in MEF cells. After treating cells with or without PRMT6-specific chemical inhibitor (PRMT6i) for 24 hrs, SIRT7 was immunoprecipitated and blotted with R388-specific methylation antibody",
    "molecules": "PRMT6i, arginine"
  },
  {
    "caption": "I. Treatment with PRMT6 inhibitor reduces R388 methylation of endogenous SIRT7 in a dose-dependent manner. MEF and HEK293T cells were treated with increasing doses of PRMT6i for 24 hrs as indicated. R388 methylation of immunoprecipitated endogenous SIRT7 was determined by western blot",
    "molecules": "PRMT6i"
  },
  {
    "caption": "L. Recombinant PRMT6 directly methylates SIRT7 at R388 in vitro. Bacterially expressed PRMT6 and SIRT7 proteins were incubated with SAM or PRMT6i as indicated. Heat-inactivated PRMT6 was included as negative control R388 methylation of recombinant SIRT7 protein was determined by western blot",
    "molecules": "PRMT6i, SAM"
  },
  {
    "caption": "A. RF, but not RK mutant, suppresses H3K18 deacetylase activity of SIRT7. Flag-SIRT7 and its RK/RF mutants were expressed in HEK293T and MEF cells, respectively. Cell lysates were subjected to western blot to detect histone H3K18 acetylation. H3K27ac and H3K56ac were included as negative control. The level of each histone acetylation marker was normalized against histone H3 (Ratio)",
    "molecules": "H3K56ac, H3K27ac"
  },
  {
    "caption": "B. R388 methylation of SIRT7 decreases its H3K18 deacetylase activity. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. The SIRT7 enzyme was immunopurified with Flag-beads and further incubated with extracted chromatin in vitro. R388 methylation of immunopurified SIRT7 and H3K18ac of in vitro deacetylated chromatin were determined by western blot",
    "molecules": "AdOx"
  },
  {
    "caption": "Wildtype SIRT7, but not its RF mutant, restores mitochondria mas in Sirt7-deficient MEF cells Mitochondria mass was determined by Mito Tracker Red staining (B) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)",
    "molecules": "Mito Tracker Red"
  },
  {
    "caption": "Wildtype SIRT7, but not its RF mutant, restore ATP leve in Sirt7-deficient MEF cells Cellular ATP was quantified and normalized to cell number (C) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)",
    "molecules": "ATP"
  },
  {
    "caption": "Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs. The H3K18ac level at the promoter of SIRT7 target genes (Gfm2, Mrps31, Mrps33, and Mrpl40) was determined by ChIP-qPCR, Rabbit IgG was used as a negative control (G) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test",
    "molecules": "PRMT6i"
  },
  {
    "caption": "Inhibition of Prmt6 decreases mitochondria mas of control cells, but not Sirt7-knockdown MEF cells Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red staining (H) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test",
    "molecules": "PRMT6i, Mito Tracker Red"
  },
  {
    "caption": "Inhibition of Prmt6 decrease ATP leve of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Cellular ATP was quantified and normalized to cell number (I) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test",
    "molecules": "ATP, PRMT6i"
  },
  {
    "caption": "Inhibition of Prmt6 decrease the respiration flux of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated (J). ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test",
    "molecules": "PRMT6i"
  },
  {
    "caption": "A. Glucose depletion reduces SIRT7 methylation. MEF cells stably expressing Flag-SIRT7 were cultured in medium depleted with glucose (12 hrs), FBS (12 hrs) or glutamine (24 hrs). R388 methylation of immunopurified SIRT7 and Ampk phosphorylation were determined by western blot. R388 methylation level was normalized to Flag-SIRT7 (Ratio)",
    "molecules": "glucose, Glucose, glutamine"
  },
  {
    "caption": "B. Glucose starvation decreases R388 methylation of SIRT7 in a time-dependent manner. MEF cells stably expressing Flag-SIRT7 were cultured in glucose-free media as indicated. R388 methylation level of immunopurified SIRT7 was determined by western blot",
    "molecules": "Glucose, glucose"
  },
  {
    "caption": "C. Glucose dose-dependently upregulates R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were cultured with increasing concentrations of glucose as indicated for 12 hrs. R388 methylation of SIRT7 and Ampk phosphorylation were determined by western blot",
    "molecules": "Glucose, glucose"
  },
  {
    "caption": "D. AICAR treatment decreases R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were treated with or without AICAR for 12 hrs. R388 methylation of immunoprecipitated SIRT7 was determined by western blot",
    "molecules": "AICAR"
  },
  {
    "caption": "E-F. AICAR reduces the percentage of R388-methylated endogenous SIRT7 in MEF cells. R388-methylated endogenous SIRT7 was captured from control and AICAR-treated MEF cells by using R388 site-specific methylation antibody (E). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (F) (mean±SD, n=3 experimental replicates)",
    "molecules": "AICAR"
  },
  {
    "caption": "H. AMPK is required for glucose starvation-induced R388 hypomethylation of SIRT7. Ampk wildtype (WT) and Ampk α1/α2 double-knockout (DKO) MEF cells stably expressing Flag-SIRT7 were cultured with or without glucose for 12 hrs. SIRT7 was purified with Flag-beads and R388 methylation was determined by western blot",
    "molecules": "glucose"
  },
  {
    "caption": "I. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Flag-tagged SIRT7 was stably expressed in scramble control MEF cells or cells expressing two independent shRNAs targeting Prmt6 (#1 and #2). Cells were cultured with or without glucose for 12 hrs. R388 methylation of immunopurified SIRT7 was determined by western blot",
    "molecules": "glucose"
  },
  {
    "caption": "J. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Wildtype MEFs and Ampk DKO cells were treated with PRMT6i for 24 hrs. SIRT7 was purified by Flag-beads, and R388 methylation of SIRT7 was determined by western blot",
    "molecules": "PRMT6i"
  },
  {
    "caption": "K. Both AICAR treatment and glucose depletion impair the association between Prmt6 and Sirt7. MEF cells were cultured with or without glucose and treated with AICAR as indicated. Co-immunoprecipitation was performed using SIRT7 antibody. Input and immunoprecipitates were analyzed by western blot",
    "molecules": "AICAR, glucose"
  },
  {
    "caption": "L. R388 methylation of SIRT7 does not modulate its phosphorylation. MEF cells stably expressing Flag-tagged wildtype SIRT7 or RK mutant were treated with PRMT6i (24 hrs) and cultured with or without glucose (12 hrs) as indicated. SIRT7 protein was immunopurified with Flag-beads, R388 methylation and phosphorylation of SIRT7 were determined by western blot",
    "molecules": "PRMT6i, glucose"
  },
  {
    "caption": "Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined by western blot (A Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µm",
    "molecules": "Glucose, glucose"
  },
  {
    "caption": "Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined b immunohistochemical staining (B). Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µm",
    "molecules": "Glucose, glucose"
  },
  {
    "caption": "AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs. R388 methylation of SIRT7 was determined by western blot (C) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test",
    "molecules": "AICAR, PRMT6i"
  },
  {
    "caption": "AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs H3K18ac level at SIRT7-target gene promoters were determined by ChIP-qPCR. Normal rabbit IgG was used as a negative control (D) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test",
    "molecules": "AICAR, PRMT6i"
  },
  {
    "caption": "AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs mRNA expression was determined by qPCR analysis (E) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test",
    "molecules": "AICAR, PRMT6i"
  },
  {
    "caption": "AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs Mitochondria mass was determined by Mito Tracker Red staining (F). (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test",
    "molecules": "AICAR, PRMT6i, Mito Tracker Red"
  },
  {
    "caption": "2-NBD-glucose uptake in murine pancreatic endothelial cells (MS1) and primary human umbilical vein endothelial cells (HUVEC) treated for 2 h with VEGF-B167 (B167) or VEGF-B186 (B186) compared to control treated cells. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). Scale bar, 20 μm.",
    "molecules": "2-NBD-glucose"
  },
  {
    "caption": "Trans-cellular 2-NBD-glucose transport kinetics in MS1 cells treated for 2 h with VEGF-B186. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using t-test, p-value: ** <0.01 (compared to untreated control).",
    "molecules": "2-NBD-glucose"
  },
  {
    "caption": "Cardiac glycogen content in 17 weeks old male Vegfb+/+ (n=6), Vegfb+/- (n=6) and Vegfb-/- mice (n=5) presented as mean ± SEM. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05 (compared to Vegfb+/+).",
    "molecules": "glycogen"
  },
  {
    "caption": "Glucose uptake and accumulation (18F-DG PET) in hearts of HFD fed wild-type (WT) mice pre-treated for 1 week with 2H10 antibody (n=4) compared to control antibody treated mice (n=4). Data presented as mean ± SEM. Statistical evaluation using two-way ANOVA, p-value: **** <0.0001.",
    "molecules": "18F-DG, Glucose"
  },
  {
    "caption": "Linear regression analysis of the 18F-DG PET data in D assessing differences in initial (T=0.9-6.5 min) glucose uptake rate. Data presented as mean curve slope (m) ± SEM. Statistical evaluation comparing slope differences using analysis of covariance (ANCOVA), p-value: * <0.05.",
    "molecules": "18F-DG, glucose"
  },
  {
    "caption": "2-NBD-glucose uptake in response to 2 h treatment with VEGF-B186 (B186) in HUVECs exhibiting siRNA-mediated knock-down of FLT1, NRP1, SCL2A1, KDR (VEGFR2) or control siRNA. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using one-way ANOVA and Sidak´s multiple comparisons test, p-value: * <0.05 (comparisons made to respective untreated control).",
    "molecules": "2-NBD-glucose"
  },
  {
    "caption": "Sucrose gradient (15-50% sucrose) fractions of HUVEC cellular lysates after 2 h VEGF-B186 stimulation were analyzed with Western blots shown in Appendix Fig S2. Different cellular sub-compartments were identified by accumulation of specific protein markers: EEA1 for early endosomes, LIMPII for late endosomes/lysosomes, flotillin for plasma membrane/lipid rafts and calnexin for endoplasmic reticulum (ER) (left panels). Amount and sub-cellular distribution of VEGFR1, NRP1 and GLUT1 protein in control or VEGF-B186 stimulated HUVECs shown in panels to the right. Data represent % protein relative to total protein of interest measured from a representative experiment.",
    "molecules": "Sucrose, sucrose"
  },
  {
    "caption": "Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF-B186 stimulation in different sucrose fractions (upper panel). Data from two independent experiments are shown. Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF-B186 stimulation. Changes in GLUT1 content in response to VEGF-B treatment are shown in the table (bottom panel).",
    "molecules": "sucrose"
  },
  {
    "caption": "Representative confocal (grey) and epifluorescence (blue) images of filipin staining in HUVECs in response to 2 h VEGF-B186 stimulation (left panels). Cellular cholesterol content measured by quantification of fluorescent filipin staining. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05. Scale bars, 10 µm.",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "Measurement of cholesterol content (B) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).",
    "molecules": "cholesterol"
  },
  {
    "caption": "Measurement of glucose uptake (C) and FA uptake (D) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).",
    "molecules": "FA, glucose"
  },
  {
    "caption": "Colorimetric quantification of non-esterified cholesterol extracted from HUVECs stimulated with VEGF-B186 for 20 min. Data are presented as mean ± SEM of three individual experiments. Statistical evaluation using t-test, p-value: * <0.05.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Uptake of a fluorescent cholesterol tracer (TopFluor) in HUVECs after VEGF-B186 stimulation for 20 min or 2 h. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01, *** <0.001 (compared to respective untreated control).",
    "molecules": "TopFluor, cholesterol"
  },
  {
    "caption": "Quantification of the major cholesteryl ester (CE) species in HUVECs after VEGF-B186 stimulation for 20 min, analyzed by lipid mass spectrometry. Data presented as mean ± StDev of an experiment performed in triplicates.",
    "molecules": "CE, cholesteryl ester"
  },
  {
    "caption": "Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 1 h or after 15 min treatment with cholesterol-extracting methyl-beta-cyclodextrin (MbCD). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: *** <0.001 (compared to untreated control).",
    "molecules": "cholesterol, glucose, MbCD, methyl-beta-cyclodextrin"
  },
  {
    "caption": "Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 15 min alone or supplemented with soluble methyl-beta-cyclodextrin complexed with cholesterol (5, 10 or 20 μg/ml cholesterol). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to control w/o cholesterol).",
    "molecules": "cholesterol, glucose, methyl-beta-cyclodextrin"
  },
  {
    "caption": "Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).",
    "molecules": "cholesterol"
  },
  {
    "caption": "glucose uptake (C) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).",
    "molecules": "glucose"
  },
  {
    "caption": "Cellular cholesterol content of ECs freshly isolated from Ldlr+/+ and Ldlr-/- mice stimulated for 15 min with VEGF-B186. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: *** <0.001 (compared to Ldlr+/+ control).",
    "molecules": "cholesterol"
  },
  {
    "caption": "Cellular cholesterol content (left panel) and glucose uptake (right panel) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). p-value: # <0.05, ### <0.001 (compared to simvastatin control).",
    "molecules": "cholesterol, glucose, simvastatin"
  },
  {
    "caption": "Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).",
    "molecules": "biotinylated"
  },
  {
    "caption": "Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non-permeabilized cells (white, left panels), Dil-LDL cell surface binding (evaluated at 4 °C, middle panels) and Dil-LDL uptake (evaluated at 37 °C, right panels) in HUVECs in response to VEGF-B186 stimulation for 10 min. Scale bar, 10 µm.",
    "molecules": "Dil, LDL"
  },
  {
    "caption": "Quantification of non-esterified cholesterol content by means of filipin staining in the blood vessel compartment (podocalyxin+) in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- mice (n=7), normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01 (compared to Vegfb+/+ mice).",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- (n=7) mice, normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01 (compared to Vegfb+/+ mice).",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 25-week old male CD1 Flt1+/lacz (n=20) mice, normalized to wildtype CD1 (Flt1+/+) mice (n=16). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using t-test, p-value: ** <0.01.",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 12 to 16-week old male C57BL/6 wildtype (Vegfb+/+) (n=4), BKS.C57BL/6 db/+ Vegfb+/+ (n=8), db/db Vegfb+/- (n=10) and db/db Vegfb-/- (n=4) mice, normalized to db/db Vegfb+/+ (n=6) mice. Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to db/db Vegfb+/+ mice).",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "Left panel: Quantification of non-esterified cholesterol content by means of filipin staining in hearts of C57BL/6 male mice after 25 weeks on HFD, long-term treated with isotype control (n=17) or VEGF-B blocking antibody (2H10, n=17) initiated after 5 weeks on HFD, normalized to age-matched mice on chow diet (n=10). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Right panel: Body weights (mean ± SEM) of HFD (n=17+17) and chow fed littermate mice (n=10). Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to HFD control mice).",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "Quantification of non-esterified cholesterol content by means of filipin staining in hearts of mice on HFD for 17 weeks followed by treatment for 1 week with 2H10 antibody (n=5) compared to control antibody treated mice (n=5). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values.Statistical evaluation using t-test, p-value: ** <0.01.",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "Phenotypic analysis. Seedlings of the indicated genotypes were grown in 1/2 MS with the addition of a series of concentrations of NAA under continuous white light or white plus UV-B (1 W/m2) light for 2 weeks. The lateral root density (number of lateral roots/length of primary root) (A) and average length of lateral roots (B) of the indicated genotypes were measured.",
    "molecules": "NAA"
  },
  {
    "caption": "UV-B inhibits the response of Arabidopsis seedlings to exogenously added NAA in a UVR8-dependent manner, since the response to NAA was not repressed in uvr8. Seedlings of the indicated genotypes were grown in LD (16-h light/ 8-h dark) conditions for 5 days, then transplanted to new plates containing 0.4 μM NAA and kept in continuous white light or white light plus UV-B (1 W/m2) for 7 days. Images are shown in (C); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (D) and average length of lateral roots (E) of the indicated genotypes were measured.",
    "molecules": "NAA"
  },
  {
    "caption": "Phenotypic analysis. WT, GR-UVR8R338A/uvr8 and GR-UVR8W285F/uvr8 seedlings were grown in LD (16-h light/ 8-h dark) for 5 days, then transplanted to new medium with 0.4 μM NAA and with or without 20 μM DEX and kept in white light plus UV-B (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. The lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of indicated genotypes were measured. SDs (n > 8) are indicated.",
    "molecules": "NAA, DEX"
  },
  {
    "caption": "UVR8 inhibited auxin responses under UV-B in a tissue-autonomous way. WT and uvr8 seedlings grown in LD for 5 days were used for reciprocal grafting. Seedlings were kept in LD for 7 days after grafting, then transplanted to new medium containing 0.4 μM NAA and kept in UV-B light (1 W/m2) for 10 days. Images are shown in (D); scale bar = 2 mm. The lateral root density (number of lateral roots/length of primary root) (E) and average length of lateral roots (F) of indicated genotypes were measured. SDs (n > 8) are indicated.",
    "molecules": "NAA, auxin"
  },
  {
    "caption": "Transcriptome analysis of gene expression regulated by auxin, UV-B, and UVR8. (A) Heat map of UV-B-, UVR8-, and auxin-regulated genes. The parameter measured by color key shows the Log-fold change. (B) Auxin signaling genes are up-regulated by auxin treatment but down-regulated by UV-B in a UVR8-dependent manner. Three biological replicates were analyzed and final Log fold-change is shown.",
    "molecules": "auxin, Auxin"
  },
  {
    "caption": "Quantitative RT-PCR results showing that UV-B represses the expression of auxin signaling genes. Six-day-old WT seedlings grown in continuous white light were transferred to UV-B light (2 W/m2) for the indicated period. Error bars are SD of three biological replicates.",
    "molecules": "auxin"
  },
  {
    "caption": "Quantitative RT-PCR analysis of auxin signaling gene expression in wild-type (Col-0), uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 5 days were kept in white light (D) or transferred to white light plus UV-B (E) for 1 day with the addition of 1 μM IAA. Error bars are SD of three biological replicates.",
    "molecules": "auxin, IAA"
  },
  {
    "caption": "qPCR analysis of auxin signaling gene expression in roots of WT, uvr8, and rup1 rup2 seedlings. Seedlings grown in continuous white light for 12 days were kept in white light (F) or transferred to white light plus UV-B (G) for 1 day with the addition of 0.4 μM NAA. Error bars are SD of three biological replicates.",
    "molecules": "NAA, auxin"
  },
  {
    "caption": "Phenotypic analysis. Seedlings of the indicated genotypes were grown in LD conditions (16-h light/ 8-h dark) for 5 days, and then transplanted to new medium containing 0.4 μM NAA and kept in continuous white light or white plus UV-B light (1 W/m2) for 7 days. Images are shown in (A); scale bars = 2 mm. Lateral root density (number of lateral roots/length of primary root) (B) and average length of lateral roots (C) of the indicated genotypes were measured. SDs (n > 8) are indicated.",
    "molecules": "NAA"
  },
  {
    "caption": "Quantitative RT-PCR analysis of auxin signaling gene expression in roots of wild type (Col-0), uvr8, myb73 myb77, and myb73 myb77 uvr8. (D) Results under white light and (E) under white plus UV-B light. Seedlings grown in continuous white light for 12 days were kept in white light or transferred to white light plus UV-B for 1 day with the addition of 0.4 μM NAA, and roots were collected to analyze auxin signaling gene expression. Error bars represent SD of three biological replicates.",
    "molecules": "NAA, auxin"
  },
  {
    "caption": "EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB73(C)",
    "molecules": "DNA"
  },
  {
    "caption": "EMSA results showing that monomeric UVR8 inhibits the DNA-binding activity of MYB77(D).",
    "molecules": "DNA"
  },
  {
    "caption": "A, SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 (SB) or LGK974 (LGK) for 16 h. Total cell extracts were analyzed by WB. Protein quantification was analyzed by densitometry of three different experiments and represented B, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs from three biological replicates are presented in the right panel. Values correspond to fold variation in shRor2 versus shCtl cells (mean ± SD).",
    "molecules": "LGK, LGK974, SB, SB505124"
  },
  {
    "caption": "D, MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D) for the indicated times. TGFβ receptor inhibitor SB505124 (SB, 5 μM) was added when indicated in D. Cells were lysed and proteins analyzed by WB. Quantification of the protein levels from D is represented at the lower panel (mean ± SD of three biological replicates)",
    "molecules": "SB, SB505124"
  },
  {
    "caption": "E, GFP-labelled MSC were seeded in Matrigel-coated transwells with SW620 and HCT116 cells, and supplemented with SB when indicated. After 48 h non-invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed. GFP-labelled cells were counted in five different fields per filter by ImageJ software and represented (bottom). A representative picture of SW620 cells in each condition is shown (top). The bar corresponds to 500 μm. The figure shows the mean ± SD of three experiments.",
    "molecules": "SB"
  },
  {
    "caption": "Cells were seeded for tumor-sphere formation in tumor organoid complete medium. In E, SW620 cells were supplemented with LGK (10 μM), starting the day of seeding. After four days, the wells were stained (top) and the number of colonies counted and represented (bottom) (mean ± SD of three biological replicates). A more detailed micrograph of the colonies is shown in Appendix Fig S7D.",
    "molecules": "LGK"
  },
  {
    "caption": "F, RNA was isolated from SW620 control, stably-depleted of Ror2, treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5, ASCL2, SMOC2 and the differentiation marker KRT20 was assessed by qRT-PCR. The figure shows the mean ± SD of at least three biological replicates (performed in triplicate) represented with respect to the corresponding control (dashed line).",
    "molecules": "LGK974"
  },
  {
    "caption": "F, G, In vivo tumor growth of MTO cells subcutaneously injected in NOD/SCID mice (F) (5x105 cells/flank; eight tumors per condition) and treated with WntC59 (10 mg/kg/day in the drinking water) or vehicle. Tumor weight of NOD/SCID mice at day 26 is presented in G. In F and G, the mean ± SD of the eight tumors is shown.",
    "molecules": "WntC59, water"
  },
  {
    "caption": "N, O, C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle. In N, kinetics of in vivo luminescence (mean ± SD of the values in five mice); in O, the percentage of mice showing in vivo luminescence.",
    "molecules": "WntC59"
  },
  {
    "caption": "A-C, Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h. In B, cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid. The figure shows the mean ± SD of the results of three experiments.",
    "molecules": "cisplatin, LGK"
  },
  {
    "caption": "D, Cells were treated with cisplatin for the indicated times. Total extracts were lysed and analyzed by WB.",
    "molecules": "cisplatin"
  },
  {
    "caption": "C. Immunostaining  for CD31  (green), CD13  (white; pericyte  marker) and αSMA  (cyan) using paraffin  section from WT (top panel) and Glut1iECKO (bottom panel) mice.",
    "molecules": "paraffin"
  },
  {
    "caption": "J. Representative confocal  images of fibrin  (green), CD31  (red) and DAPI  (blue) immunostaining  in the brain vasculature of WT (top panel) and Glut1iECKO (bottom panel) mice  K. Quantification of the number of cadaverine 555 positive brain parenchyma  cells  in WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) L. Quantification of extravascular fibrin  deposits in WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) M. Quantification of lactate  in the cerebrospinal fluid of WT (blue) and Glut1iECKO (orange) mice. (n=3 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)",
    "molecules": "cadaverine 555, DAPI, lactate"
  },
  {
    "caption": "A. Quantification of the mTFP/Venus  fluorescence ratio of laconic (lactate  sensor) showing concentration-dependent lactate  uptake in  HBVPs. (n=8 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM)) B. Representative images showing  mTFP (blue) and Venus  (yellow) fluorescence in control and lactate  (20mM) treated HBVPs.",
    "molecules": "lactate"
  },
  {
    "caption": "A. Permeability  assay  using vibratome  section showing the cadaverine 555 dye (red), CD31  (green) and CD13  (purple) in the  brain vasculature of  Glut1iECKO mice  with (bottom) or without (top) lactate  administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate  supplemented Glut1iECKO mice.",
    "molecules": "cadaverine 555 dye, cadaverine dye, lactate"
  },
  {
    "caption": "B. Quantification of the CD13-positive pericytes  coverage in the brain vasculature  of  Glut1iECKO mice  with (orange) or without (blue) lactate  administration. (n=16 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) C. Quantification of the number of cadaverine 555 positive  brain parenchyma  cells  in the brain vasculature  of  Glut1iECKO mice  with (orange) or without (blue) lactate  administration. (n=24 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) D. Quantification of CSF lactate  level in WT and Glut1iECKO mice  with/without lactate  supplementation. (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)",
    "molecules": "cadaverine 555, lactate"
  },
  {
    "caption": "F. Western blot detection of p62 and LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle and cultured in normal medium (starvation \"-\") or EBSS (starvation \"+\") for 3 h. Similar results were observed in three independent experiments.",
    "molecules": "Baf A1, DMSO"
  },
  {
    "caption": "G. Quantification of GFP-LC3 puncta in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or HBSS (starvation \"+\") for 3 h. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). *p<0.05; two-tailed unpaired t-test. NC, nonsilencing control.",
    "molecules": "Baf A1, DMSO"
  },
  {
    "caption": "A. Representative images of Parkin-mediated clearance of mitochondrial outer membrane protein TOMM20 in HeLa/Parkin cells treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13 oligos. Scale bars, 20 μm.B. Quantification of TOMM20 clearance in the experiment shown in (A). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "C. Representative images of Parkin-mediated clearance of mitochondrial double-stranded DNA (mtDNA) in HeLa/HA-Parkin cells treated with indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA) or DMSO vehicle control. siPEX13 oligo #1 is shown; similar results were observed with three other siPEX13 oligos. Nuclear DNA staining was masked using DAPI. Scale bars, 20 μm.D. Quantification of mtDNA clearance in the experiment shown in (C). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001; Kruskal-Wallis H-test.",
    "molecules": "antimycin A, DMSO, oligomycin"
  },
  {
    "caption": "E. Representative images of TOMM20 fragmentation or compaction around perinuclear region in MEFs of the indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control. White lines indicate cell borders. Scale bars, 20 μm.F. Quantification of percentage of cells in experiment shown in (E) with accumulation of fragmented mitochondria after CCCP treatment. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01; two-tailed unpaired t-test.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "B. Representative images of Parkin-mediated clearance of TOMM20 in HeLa/Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA-resistant plasmid and then treated with CCCP (10 μM, 16 h). Scale bars, 20 μm. See Fig EV3B for representative images of mitochondrial morphology (TOMM20 staining) in control cells treated with DMSO.",
    "molecules": "CCCP"
  },
  {
    "caption": "E. Representative images of Parkin-mediated clearance of TOMM20 in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts treated with Oligomycin (2.5 μM) + Antimycin A (250 nM) for 24 h. Scale bars, 20 μm. W313G cells varied in morphology; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology (see Fig EV3C for higher resolution imaging) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in (F).F. Quantification of experiment shown in (E). Results represent mean ± SEM of 5 groups of 10 images of random fields of cells (>350 cells analyzed per sample). Similar results were observed in two independent experiments. ***p<0.0001; two-tailed unpaired t-test.",
    "molecules": "Antimycin A, Oligomycin"
  },
  {
    "caption": "A-C Representative images of PEX13 and PMP70 (A), PEX13 and TOMM20 (B), or PEX13-Flag and WIPI2 (C) colocalization in HeLa/Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment. Scale bars, 20 μm (A-B) and 5 μm (C).",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "D Western blot detection of endogenous PEX13 in HeLa/Parkin cells treated with 10 μM CCCP for the indicated time.",
    "molecules": "CCCP"
  },
  {
    "caption": "B. Quantification of Parkin-mediated TOMM20 clearance in HeLa/Parkin cells transfected with the indicated siRNA 16 h after treatment with 10 µM CCCP. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.",
    "molecules": "CCCP"
  },
  {
    "caption": "C. Quantification of mtDNA clearance in HeLa/HA-Parkin cells transfected with the indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001, NS= not significant; Kruskal-Wallis H-test.",
    "molecules": "antimycin A, oligomycin"
  },
  {
    "caption": "D. Quantification of GFP-LC3 puncta in HeLa/GFP-LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media. Results represent mean ± SEM in triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01, ***p<0.001, NS= not significant; one-way ANOVA with adjustment for multiple comparisons. Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs. NC siRNA within each treatment group.",
    "molecules": "Baf A1, DMSO"
  },
  {
    "caption": "B. Single-cell lengths were measured from time-lapse microscopy images upon beta-lactam antibiotic treatment. Initial cell length was measured in the first time frame. Elongation ends with cell length shrinkage due to bulging. The longest length of each cell across the time frames was used as the final length. Tracked intermediate cell lengths were shown over time, while initial and final lengths were shown in larger circles. Linear regression of log-scaled lengths over time showed a constant rate of exponential elongation (solid gray line).",
    "molecules": "beta-lactam"
  },
  {
    "caption": "A. Probability density distributions of final lengths of MG1655 cells exposed to carbenicillin. Individual cell lengths were measured from populations exposed to three doses of carbenicillin (20, 50, and 100 µg/ml) at two temperatures (27°C in blue and 37°C in red). Line plots show the measured initial and final lengths over time in log scale, and the first 15 cells were picked in each dataset for presentation among n ≥ 100 of the tracked cells in each condition Probability density distributions (ρL) of final lengths were collected with the same number of bins within the same final cell length range (bottom panels). The peak of ρL shifted to the left with increasing carbenicillin dose.",
    "molecules": "carbenicillin"
  },
  {
    "caption": "C. The probability of lysis increased with the filamentation length of bacteria treated with other beta-lactams. MG1655 cells were treated with cefotaxime (20 and 100 µg/ml) and amoxicillin (6.25 and 25 µg/ml, shown in gray text) at two temperatures (27 °C in blue and 37 °C in red).",
    "molecules": "amoxicillin, beta-lactams, cefotaxime"
  },
  {
    "caption": "D. The probability of lysis increased with the filamentation length of E. coli pathogens treated with a beta-lactam. E. coli pathogens expressing extended beta-lactam resistance were treated with amoxicillin (6.25 or 25 µg/ml, shown in gray text) and clavulanic acid (50 µg/ml) simultaneously at 37 °C. Cells were sensitized by clavulanate acid, which inhibits beta-lactamases.",
    "molecules": "amoxicillin, beta-lactam, clavulanate acid, clavulanic acid"
  },
  {
    "caption": "C. Experimental measurements confirm that τE decreases with the increase of antibiotic dose and growth rates. For growth rate modulation, casamino acid at 0.02% (left panel) and 0.2% (right panel) were added to minimal media. Cells were first cultured without carbenicillin for 3h (dashed line) and then exposed to carbenicillin (antibiotic doses are marked near each plot, µg/ml). The average of four technical repeats (gray lines) was plotted in a colored line. τE points were marked by finding the first time point at which the averaged OD became equal to or less than the OD at the start of carbenicillin exposure.",
    "molecules": "carbenicillin, casamino acid"
  },
  {
    "caption": "B-C. Stochastic simulations and measurements of survivor length. Simulated survivor lengths were plotted using the predicted LC of lower doses of carbenicillin (left panels) at 2h (B) and 6h (C). The corresponding single-cell length measurements in vitro were plotted (right panels). In the simulation, only non-antibiotic treated cells were set to divide, when the cell length reaches the division length in the linear model (Ld= aLi + b, a = 0.871, b = 2.7. In both simulations and measurements, cells in carbenicillin-treated conditions were further elongated than control cells at all time points. At 2h exposure, antibiotic-exposed cells showed similar lengths across antibiotic concentrations since lysis is not highly probable for short filaments. At 6h exposure, survivor lengths decreased with increasing carbenicillin dose. Independent t-tests between non-treated to treated group showed extreme significance, **** P < 0.0001. The box represents the middle 50%, the central line represents the median, and the whiskers represent the lower and upper quartile of the data points (n>300).",
    "molecules": "carbenicillin"
  },
  {
    "caption": "(a) (top) Experimental design. Cultured cortical neurons are stimulated with BDNF, KCl or no treatment (Control) and activated neurons are prepared in three specific time points for RNA-seq and ATAC-seq at early (1h) and late time points (6 and 10h); (bottom) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF (Con = no stimulation). 44 and 42 KDa bands indicated with lines. Nucleolin shown as internal control.",
    "molecules": "KCl"
  },
  {
    "caption": "(d) (top) Association between distal regulatory elements (DREs) and gene expression at the BDNF 1h time-point. Each point indicates the log2-fold change of an ATAC-seq peak (x-axis) and the gene expression of a closest gene (y-axis) with distance between 2-20 Kbp. Colors indicate whether none (gray), only the peak (orange), or both peak and gene (red) show significant changes versus control neurons. (bottom) Enrichment for paired DA-peak and DE-gene in the four quadrants are summarized for BDNF and KCl. Asterisks indicate P values as corrected by a Benjamini Hochberg procedure (* = P < 0.05; ** = P < 0.01).",
    "molecules": "KCl"
  },
  {
    "caption": "(c) Chromosome 15 genome tracks neighboring Arc, displaying ATAC-seq read counts per million (CPM); RNA-seq CPM; H3K27ac normalized signal upon KCl stimulation, H3K4me1(Malik et al, 2014); CTCF(Sams et al, 2016); and Cortical neurons Hi-C data(Bonev et al, 2017). Red bars in ATAC-seq tracks indicate gained DA-peaks in BDNF, and red bars in RNA-seq tracks indicate Arc differential expression in BDNF and KCl 1h. Green blocks in Hi-C tracks indicate anchor points for the calculation of contact scores, using shaman(Cohen et al, 2017). Line and Spearman's rho value indicate counts rank-based correlation between highlighted peaks across all samples (Appendix Figure S4c). TF module names indicate the presence of 8-mers in those peaks.",
    "molecules": "KCl"
  },
  {
    "caption": "C-E) Kinase activity for ERK  (C), JNK   (D), or p38   (E) in three representative single cells (upper panels) in response to 400 ng/ml NCS  (blue) or 300 µM H2O2  (red) as measured by C/N ratio. Lower panel shows mean C/N ratio in response to 400 ng/ml NCS  (blue) or 300 µM H2O2  (red). Thick line represents mean with shaded area=SD. N for each condition and kinase is shown in population average plots.",
    "molecules": "H2O2, NCS"
  },
  {
    "caption": "C-E) Mean kinase activation for ERK  €, JNK   (D), or p38   (E) in both live (grey) and dead (red) cells measured over 24-hours in response to H2O2. Shaded areas=SD. N for each condition is shown.",
    "molecules": "H2O2"
  },
  {
    "caption": "D) Mean JNK   activity as measured by C/N ratio (bold line)±SD (shaded area) in response to 300 µM H2O2  with or without p38   inhibition (grey). Bar graph shows integrated JNK   activity (C/N ratio) over 24-hours. Data represents mean±SD. The number of analyzed cells, n, is shown for each condition. Statistics performed using 2-tailed t-test. Data information: ** p<0.01, *** p<0.001, ns=not significant.",
    "molecules": "H2O2"
  },
  {
    "caption": "A. Measurement of oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and oxidative burst of monocytes from COVID-19 patients (COVID) and healthy controls (CTR). OCR was measured in real time, under basal condition and in response to mitochondria inhibitors: oligomycin (Oligo, 2 μM), cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.5 μM), and antimycin A plus rotenone (Rot/AA, 0.5 μM). Oxidative burst was measured in response to PMA/ionomycin (PMA/Iono, 1 μg/mL). Scatter plots show the quantification of basal respiration (indicated as Basal OCR), ATP-linked respiration (ATP-linked), maximal respiration (Max Resp), proton-leak, spare respiratory capacity (Spare), oxidative burst immediate response and basal ECAR. The maximal respiration kinetic range and the oxidative burst kinetic range are also reported and were obtained by analyzing the area under the curve (AUC) from the sixth to the tenth measurement and from the tenth to the thirteenth measurement, respectively. Data represent individual values, mean and standard error of the mean (CTR, n=12; COVID, n=13). Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure. Representative traces of OCR of monocytes from COVID-19 patients (COVID) and healthy controls (CTR) are also reported.",
    "molecules": "AA, antimycin A, ATP, cyanide-4-(trifluoromethoxy)phenylhydrazone, FCCP, oxygen, Iono, ionomycin, Oligo, oligomycin, PMA, Rot, rotenone"
  },
  {
    "caption": "(a) Different VPS34 complexes were immunoprecipitated (IP) from MEFs in the presence (N) or absence (−A) of amino acids using the indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using the antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel; data shown are mean+s.d.). AR, autoradiography.",
    "molecules": "amino acids"
  },
  {
    "caption": "(b) Immunoprecipitation of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient-rich (N) or starvation (-A) conditions. VPS34-binding partners were analysed by western blotting.",
    "molecules": "nutrient"
  },
  {
    "caption": "(f) LC3B puncta and PtdIns(3)P levels were analysed with anti-LC3B and the biotin-2XFYVE domain probe. Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown (scale bars, 10 μm). (g) Quantification of LC3B puncta from f. Details of quantification are provided in the Methods; Data shown in g-i are mean+s.d. from a minimum of 6 unique fields of view from a representative experiment (see statistical source data in Supplementary Table S1). (h) Total PtdIns(3)P was quantified from the experiment in f. Error bars were calculated as in g. (i) Quantification of PtdIns(3)P that co-localizes with LC3B on amino-acid withdrawal from the experiment described in f.",
    "molecules": "amino-acid, PtdIns(3)P"
  },
  {
    "caption": "(a) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ-32P[ATP]. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blotting was performed (right panels). Results are representative of two unique experiments.",
    "molecules": "32P, ATP"
  },
  {
    "caption": "(e) HEK293 cells were transfected with the indicated plasmids under nutrient-rich conditions. Beclin-1 was immunoprecipitated and immunoblotted with pBeclin-1(Ser 14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below immunoprecipitated samples.",
    "molecules": "nutrient"
  },
  {
    "caption": "(g) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1 and grown under nutrient-rich conditions. ATG14L-containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig. 1j. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) WT MEFs were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blotting (immunoblotting, IB) was performed with the indicated antibodies. Beclin-1 Ser 14 phosphorylation was quantified (shown under top panel) and normalized to total Beclin-1.",
    "molecules": "amino acids"
  },
  {
    "caption": "(c) WT or FIP200−/− MEFs were incubated under nutrient-rich, amino-acid-deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig. 3b.",
    "molecules": "amino-acid, nutrient, Torin-1"
  },
  {
    "caption": "(d) WT or ULK-deficient MEFs were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in a. Two unique experiments were performed. Uncropped images of blots are shown in Supplementary Fig. S4.",
    "molecules": "amino acids"
  },
  {
    "caption": "(c) An ATG14L-FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed.",
    "molecules": "amino acids"
  },
  {
    "caption": "(e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown.",
    "molecules": "amino acids"
  },
  {
    "caption": "(a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies.",
    "molecules": "amino acids, NH4Cl"
  },
  {
    "caption": "(b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments.",
    "molecules": "amino acids"
  },
  {
    "caption": "(c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel).",
    "molecules": "amino acids"
  },
  {
    "caption": "(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm.",
    "molecules": "amino-acid"
  },
  {
    "caption": "(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant.",
    "molecules": "amino-acid, nutrient"
  },
  {
    "caption": "(f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.",
    "molecules": "nutrient"
  },
  {
    "caption": "(F-G) iMEFgt/gt stably expressing Cherry-STING and either ev or V5-tagged m152 were stimulated with 5 µg/ml ISD (F), 10 µg/ml poly(I:C) (G) or mock stimulated with Lipofectamine. 4 hours post stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT-PCR. Data information: (A-G) Data is combined from three independent experiments.",
    "molecules": "Lipofectamine, poly(I:C)"
  },
  {
    "caption": "(H-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 10 µg/ml cGAMP (H), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD. ",
    "molecules": "cGAMP"
  },
  {
    "caption": "c, Immuno-TEM (gold marks LAMP1) of starved NRK cells. Continuity (arrow) illustrated between autolysosome (red star) and tubule.",
    "molecules": "gold"
  },
  {
    "caption": "e-g, LAMP1-YFP NRK-LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h (total, 6 h) or 6 h (total, 8 h) analysed by blotting (e) as in b or microscopy (f; scale bar, 5 µm) with quantification (g). Error bars, s.e.m.; n = 3.",
    "molecules": "rapamycin"
  },
  {
    "caption": "a, LAMP1/LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation.",
    "molecules": "GTPγS"
  },
  {
    "caption": "c, Rab7-CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation.",
    "molecules": "rapamycin"
  },
  {
    "caption": "d, LAMP1-YFP NRK-LC3 cells starved for times shown with 1 µg ml-1 leupeptin and blotted with indicated antibodies. e, Cells in d imaged..",
    "molecules": "leupeptin"
  },
  {
    "caption": "(B) HCT116 cells in which eIF3k was homozygously modified with the mAID domain to trigger auxin-dependent degradation were exposed to 500 μM indole-3-acetic acid (IAA) for the periods shown, and cell lysate was subjected to immunoblotting with the indicated antibodies",
    "molecules": "IAA, indole-3-acetic acid"
  },
  {
    "caption": "(A) Parental HCT116 and the five different conditional eIF3-mAID cell lines were treated with the vehicle DMSO or with IAA for the periods indicated. Metabolic activity (i.e. NAD(P)H) as a proxy of cell proliferation was determined by MTT assay. Graphs represent means ± SD, n = 5 - 6. Asterisks denote: * p < 0.005 , ** p < 0.00005, *** p < 0.000005",
    "molecules": "DMSO, IAA, NAD(P)H"
  },
  {
    "caption": "(D) 1 x 106 eIF3-mAID cells were injected into nude mice. When tumors reached a diameter of ~ 4 mm, mice were treated with vehicle or 500 mg/kg IAA, and tumor growth was measured for 2 weeks. Graphs represent means ± SD, n = 5 to 7. Numbers indicate p values",
    "molecules": "IAA"
  },
  {
    "caption": "(A) The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunits. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Signals were normalized to the reference signal of actin and plotted relative to the vehicle DMSO. Bars represent means ± SD, n = 3. Numbers indicate p values (unpaired Student's t-test).",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(B) The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (M) and polysomal (P) peaks were quantified, and P/M ratios relative to the vehicle DMSO were plotted. Bars represent means ± SD, n = 3. Numbers indicate p values (unpaired Student's t-test).",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(A) eIF3k-mAID cells were maintained in media containing different concentrations of FBS and DMSO or IAA as indicated, and cell numbers were determined at various time points. Data represent means ± SD, n = 3 biological replicates. Numbers indicate p values",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(B) eIF3k-mAID cells were serum starved by maintaining in media containing 0% FBS for 24 h. During the last 12 h of starvation, DMSO or IAA was added as indicated. Cells were re-stimulated with media containing 10% FBS, and the expression of the indicated proteins was followed of a period of 120 minutes. The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(C) eIF3k-mAID cells were exposed to IAA or DMSO for 12 hours, followed by the addition of increasing concentrations of rapamycin for 1 h. The expression of the indicated proteins was determined by immunoblotting. The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).",
    "molecules": "DMSO, IAA, rapamycin"
  },
  {
    "caption": "(C) Total ribosome occupancy of the indicated mRNAs in eIF3k-mAID cells exposed to DMSO or IAA for 12 hours was determined by RT-qPCR of RNA across a sucrose density gradient Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test). Triplicate RT-qPCR data across the sucrose gradient are shown below the bar graphs.",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(A) Parental eIF3k-mAID cells or S15A-eIF3KO cells (clones #45 and # 361) were maintained in media with DMSO or IAA, and cell numbers were determined at various time points. Data represent means ± SD (too small to be visible), n = 3. All p values were > 0.45 except at the single time point where indicated otherwise.",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(D) Total ribosome occupancy of the indicated mRNAs in S15A-eIF3KO (clone #361) cells exposed to DMSO or IAA for 12 hours was determined by RT-qPCR of RNA across a sucrose density gradient Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test). Triplicate RT-qPCR data across the sucrose gradient are shown below the bar graphs.",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(E) RNA immunoprecipitation. eIF3k-mAID and S15A-eIF3KO (clone #361) cells were exposed to DMSO or IAA for 12 hours. Cell lysates were employed in immunoprecipitation with eIF3c antibodies and co-precipitated mRNAs were quantified by qPCR. Bars represent means ± SD, n = 4; numbers indicate p values (unpaired Student's t-test).",
    "molecules": "DMSO, IAA"
  },
  {
    "caption": "(A) The indicated eIF3-mAID cell lines were treated with DMSO or IAA for 12 hours, followed by exposure to 2 μg/ml tunicamycin for up to 72 hours. Metabolic activity (i.e. NAD(P)H) as a proxy of cell viability was determined by CCK8 assay. Graphs represent the percentage of change in cell viability upon downregulating eIF3 subunits with IAA. Date are means ± SD, n = 3. (B) Same experiment as in (A) but cells were exposed to increasing concentrations of the oxidative stress inducer tert-butyl hydroperoxide (TBHP) for 2 hours.",
    "molecules": "DMSO, IAA, NAD(P)H, TBHP, tert-butyl hydroperoxide, tunicamycin"
  },
  {
    "caption": "(C) The indicated eIF3-mAID cell lines were treated with DMSO or IAA for 12 hours, followed by exposure to 2 μg/ml tunicamycin for 24 hours. The expression of individual eIF3 subunits, ribosomal proteins, and the ER stress marker BIP were assessed by immunoblotting. Tubulin is show for reference. The data from triplicate experiments were quantified and to avoid overcrowding of the graph data obtained from IAA-treated cells were not plotted. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).",
    "molecules": "DMSO, IAA, tunicamycin"
  },
  {
    "caption": "survival curves (G) of Cre+ or TKO mice injected subcutaneously with B16F10 OVA cells (n = 7 per genotype). Survival time was defined as the time required for a tumor to reach a volume of 500 mm3.",
    "molecules": "OVA"
  },
  {
    "caption": "(J) Representative plots (left panels) and quantification (right panels) of IFNγ- and TNFα-producing CD8 TILs of B16F10 OVA tumors from Cre+ (n = 10) and TKO (n = 12) animals at day 14.",
    "molecules": "OVA"
  },
  {
    "caption": "(F) Western blot (left panel) and quantification (right panel) of CPEB4 protein expression in OT-I T cells resting or activated ex vivo with OVA/IL-2 at the indicated time-points; β-actin is used as loading control (n = 4).",
    "molecules": "OVA"
  },
  {
    "caption": "(G) Basal oxygen consumption rate (left panel) and maximal oxygen consumption rate (right panel) after mitochondrial stress test analysis. Cre+ and TKO CD8 T cells were activated as in A, but were treated with either Tauroursodeoxycholic Acid (TUDCA, T) 250 μM or vehicle for the last 24 h. Values are normalized to basal Cre+ measurements. n = 7 for all conditions in basal respiration; n = 6 for all conditions in maximal respiration.",
    "molecules": "Tauroursodeoxycholic Acid, TUDCA"
  },
  {
    "caption": "(I) Quantification of IFNγ+ CD8 cells activated and treated as in G (n = 9 for Cre+ and n = 12 for TKO in both conditions). *P = 0.0168, ** P = 0.0014 for Cre+ vs. TKO, P = 0.0029 for Cre+ vs. Cre+ TUDCA, **** P <0.0001.",
    "molecules": "TUDCA"
  },
  {
    "caption": "F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.",
    "molecules": "DAPI"
  },
  {
    "caption": "L-O Number of myonuclei / fiber and of Pax7pos (red) MuSCs (red arrows) on myofibers isolated from flexor digitorum brevis muscle. Nuclei were stained using DAPI (blue). Scale bar in L indicates 50 µm for L and M. (N) The number of myonuclei / isolated fiber is increased in linc-MYH KO compared to WT myofibers (n = 4 KO/4 WT, > 26 fibers/animal; Mann-Whitney test two-tailed, * p < 0.05). (O) Number of MuSCs on isolated fibers from linc-MYH KO and WT mice (n = 4 KO/4 WT animals, Mann-Whitney test two-tailed; ****p < 0.0001). Data are mean ± S.E.M.",
    "molecules": "DAPI"
  },
  {
    "caption": "P-S Number of Pax7 (red) positive MuSCs in cross sections of tibialis anterior muscle (P-R); the relative amount of quiescent MuSCs identified by double staining (yellow arrows) for Pax7 and CalcR (green) does not significantly differ between WT and linc-MYH KO animals (S). Nuclei were stained with DAPI (blue). Scale bar in P indicates 25 µm for P and Q. (n = 3 KO/3 WT animals, Mann-Whitney test one-tailed; *p = 0.05, ns: not significant). All data are mean ± S.E.M.",
    "molecules": "DAPI"
  },
  {
    "caption": "E-I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 (green) and EdU incorporation (red). MuSCs without (green arrows) and with EdU-labeling (red arrows) are indicated. (E, F) Deletion of linc-MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU-positive MuSCs. (G-I) The increase in both the number of MuSCs and EdU positive MuSCs in linc-MYH mutant muscle is abolished by constitutive (G: Pax7-Crepos / INO80-/-, Ino80 KO; H: linc-MYH-/- / Pax7-Crepos / INO80-/-, dKO) and induced (I: linc-MYH-/- / Pax7-CreERT2pos / INO80-/-, dKO CreERT) deletion of INO80 in Pax7 expressing cells.",
    "molecules": "EdU"
  },
  {
    "caption": "C-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.",
    "molecules": "DAPI"
  },
  {
    "caption": "Flow cytometry analysis of PD-L1 expression in (A) KPC and (B) Pan02 cells following treatment with gemcitabine and RT. Gemcitabine was diluted in DMSO (vehicle) and DMSO was used for the 'untreated' control group (mean ± SD, n=3, One-way ANOVA, Bonferroni test).",
    "molecules": "DMSO, gemcitabine, Gemcitabine"
  },
  {
    "caption": "Western blot analysis of indicated proteins in both KPC (C) and Pan02 (D) cells following radiation and chemotherapy ± AG490, a JAK/Stat kinase inhibitor. Actin represents loading control.",
    "molecules": "AG490"
  },
  {
    "caption": "Flow cytometry analysis of PD-L1 after gemcitabine and RT (as described above) in KPC (E) and Pan02 (F) following Stat1 downregulation by siRNA (mean ± SD, n=3, Student's t-test). MFI, mean fluorescence index;",
    "molecules": "gemcitabine"
  },
  {
    "caption": "(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "D Protein localization upon heat shock and during recovery as analyzed by immunofluorescence microscopy. HeLa cells were fixed and immunostained with antibodies against indicated target proteins (green) at different conditions [control, after heat shock (10 minutes at 44°C) and after five hours of recovery from the heat shock]. DNA staining (Hoechst) is shown in blue.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Relative transcript levels of Parkin in iBAT, sWAT and eWAT of mice injected with the β3-AR agonist CL316,243, every day for 1 week. (n=6). Data are presented as means ±s.e.m. *p<0.05, ***p<0.001, CL316,243 treatment vs. saline. Two-tailed unpaired Student's t-test.",
    "molecules": "CL316,243"
  },
  {
    "caption": "Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture treated with norepinephrine (NE) or cAMP for indicated times. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to non-treated cells. One-way ANOVA with Dunnett's post hoc test.",
    "molecules": "cAMP, NE, norepinephrine"
  },
  {
    "caption": "Parkin protein levels in brown adipocytes treated with NE or cAMP for the indicated times in the presence or absence of 3-methyladenine (3-MA). Top: Representative immunoblots. p62 and LC3BII proteins are shown as positive controls of 3-MA treatment. β-actin was use as loading control. Bottom: Parkin protein quantification (performed using 3 immunoblots processed in parallel due to sample size). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to NE/cAMP non-treated cells. One-way ANOVA with Dunnett's post hoc test.",
    "molecules": "cAMP, 3-MA, 3-methyladenine, NE"
  },
  {
    "caption": "Relative transcript levels of Park2 in brown adipocytes treated with NE in the presence or absence of cycloheximide (CHX). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.",
    "molecules": "CHX, cycloheximide, NE"
  },
  {
    "caption": "Relative mRNA expression levels of Parkin in brown adipocytes treated with NE in the presence or absence of actinomycin D (n=3). Data are presented as means ±s.e.m. One-way ANOVA with Tukey's post hoc test comparing treatments.",
    "molecules": "actinomycin D, NE"
  },
  {
    "caption": "Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct treated with or without NE or cAMP. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that in untreated cells. (n=6). Data are presented as means ±s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test",
    "molecules": "cAMP, NE"
  },
  {
    "caption": "Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated with cAMP (12 h) plus H89 (a PKA inhibitor) or SB202190 (a p38 MAPK inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.",
    "molecules": "cAMP, H89, SB202190"
  },
  {
    "caption": "Relative transcript levels of Park2, and concentration of glycerol in the culture medium of brown adipocytes treated with cAMP (24h) plus Atglistatin (an adipose triglyceride lipase inhibitor) or CAY10499 (a hormone sensitive lipase inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.",
    "molecules": "cAMP, Atglistatin, CAY10499, glycerol"
  },
  {
    "caption": "Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated for 24h with a PPARα activator (GW7647), a PPARα inhibitor (GW6471) with or without norepinephrine (NE), or all-trans-retinoic acid (RA). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001. Two-tailed unpaired Student's t-test.",
    "molecules": "all-trans-retinoic acid, RA, GW6471, GW7647, NE, norepinephrine"
  },
  {
    "caption": "Relative transcript levels of Park2 in wild-type and PPARα-KO brown adipocytes treated for 24h with NE (n=3). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.",
    "molecules": "NE"
  },
  {
    "caption": "Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct and, when indicated, with a pSG5-Ppara expression vector. Cells were treated with the PPARα activator GW7647 or NE. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that only transfected with the pGL4-Park2-Luc construct (n=6). Data are presented as means ± s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test.",
    "molecules": "GW7647, NE"
  },
  {
    "caption": ", blood glucose levels and plasma insulin levels from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Immunoblot for p62 and OPTN (left) and its quantification (right) in iBAT. PS was used as the loading control. Arrowhead indicates p62 band and asterisk indicates a non-specific band. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. **p<0.01. ANOVA with Tukey's post hoc test.",
    "molecules": "PS"
  },
  {
    "caption": "Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Mitochondrial DNA (mtDNA) content in iBAT relative to nuclear DNA (nDNA). (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.",
    "molecules": "DNA"
  },
  {
    "caption": "E. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 400-420 days of age, when the study was terminated. Mock transplanted and untreated Ppt1 -/- mice at about 250 days, i.e. humane end point, are shown as reference; 450 days-old untreated wild type Ppt1+/+ are shown as control. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Arrows highlight the surviving Purkinje cells in the cerebellum of IV and ICV+IV transplanted mice; arrowheads highlight the high number of AF+ cells in the Purkinje cell layer of ICV transplanted or Ppt1-/- untreated (UT) mice. Scale bar = 100μm.",
    "molecules": "Neurotrace"
  },
  {
    "caption": "E. Representative fluorescence microscope photomicrographs showing autofluorescent storage material (AF, green) in different brain regions of Ppt1-/- mice transplanted with hPPT1-LV transduced HSPCs at 18-20 weeks of age, analyzed at study termination. Ppt1 -/- mice mock transplanted at 18-20 weeks of age and analyzed at about 250 days, i.e. humane end point, are shown as reference. Neurotrace fluorescent Nissl staining (NT, red) is used to highlight neurons in different brain regions. Scale bar = 100 μm. F. Top panel: quantification of autofluorescent (AF) storage material in different brain regions of untreated or transplanted mice analyzed at study termination. ** = p<0.01; *** = p< 0.001; **** = p< 0.0001; 2-way ANOVA followed by Tukey's post-hoc test. Bottom panel: histograms showing the quantification of cortical thickness. SM = somatomotor; SS = somatosensory; V = visual cortex. * = p <0.05; ** = p<0.01; **** = p<0.0001; Kruskal Wallis followed by Dunn's post-hoc test (for SM and V area) or ANOVA followed by Tukey's post-hoc test (for SS area). S",
    "molecules": "Neurotrace"
  },
  {
    "caption": "A. RT-PCR products with divergent and convergent primers showing circularization of has_circ_0079480 and has_circ_0087391. cDNA, complementary DNA; gDNA, genomic DNA.",
    "molecules": "cDNA, complementary DNA, gDNA, genomic DNA"
  },
  {
    "caption": "E-F. Representative hematoxylin and eosin (H&E) staining and statistical results of the micro-metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days. N=8 per group. **P < 0.01, Student's t-test, mean ± SD.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Pull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead bound His-KIF13A in each pull-down was shown as coomassie gel. *, non-specific bands.",
    "molecules": "His"
  },
  {
    "caption": "IFM images of KIF13A-YFP transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa fluor 594 arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm",
    "molecules": "Alexa fluor 594"
  },
  {
    "caption": "BF and IFM images of control and Rab22A-depleted melanocytes. The colocalization coefficient (r, in mean±SEM) between the two markers indicated separately. Nuclei are stained with Hoechst33258 ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm",
    "molecules": "Hoechst33258"
  },
  {
    "caption": "Graph represents the quantified melanin content in control sh, Rab22A sh and BLOC-1- melanocytes. n=3. The fold-change in melanin content (mean±SEM) indicated separately",
    "molecules": "melanin"
  },
  {
    "caption": "Pull-down of His-Rab22AWT using HeL lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel",
    "molecules": "His"
  },
  {
    "caption": "Pull-down of His-Rab22AWT usin melanocyte (E) lysate. In E, the beads were preloaded with GTPγS or GDP the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel",
    "molecules": "His, GTPγS, GDP"
  },
  {
    "caption": "Pull-down of different His-KIF13A domains using HeLa cell lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel",
    "molecules": "His"
  },
  {
    "caption": "Kinetochore-MT attachments at the prometaphase II stage were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maximum intensity projection through all z-planes containing MTs. Representative chromosomes displaying syntelic, amphitelic or merotelic/lateral attachments are enclosed in white frames and their enlarged single z-plane images are shown to the right. PB: polar body. Scale bar, 10μm. Chromosomes with syntelic (red), amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). Data shown for 156 chromosomes taken from 8 prometaphase II oocytes.",
    "molecules": "DAPI"
  },
  {
    "caption": "Kinetochore-MT attachments in oocytes released from the CSF-dependent metaphase arrest were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maxiumum intensity projection through all z-planes containing MTs. Single z-planes of representative chromosomes displaying amphitelic or merotelic/lateral attachments are shown to the right. Scale bar, 10μm. Chromosomes with amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). The inter-centromere distance in fixed oocytes is 1.2±0.2 μm for amphitelic and 1.0±0.2 μm for merotelic attachments (Mean±SD). Data shown for 95 chromosomes taken from 5 oocytes released from the CSF-dependent metaphase II arrest.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Effects of HDAC6 overexpression or CAMDI co-expression on the centrosomal localization of γ-tubulin. HeLa cells co-transfected with Centrin2-EGFP with/without HDAC6-HA and FLAG-CAMDI were immunostained with anti-EGFP (green) and anti-γ-tubulin antibodies (red). DNA was stained with Hoechst 33258. Scale bar, 1 μm.(D) Quantification of intensity of γ-tubulin at centrosome. n = 39, 16, 17 cells. *, p<0.05, **, p<0.01, One-way ANOVA with Bonferroni's post hoc test. Data are presented as mean ± SEM.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Rescue of a delayed cortical migration in CAMDI-KO mice by Tubastatin A. Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI-KO mice from E12.5-17.5. Scale bar, 100 μm.(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT (vehicle) = 3,628 cells, KO (vehicle) = 2,816 cells, WT (Tubastatin A) = 1,847 cells, KO (Tubastatin A) = 2,364 cells). *, p<0.05, **, p<0.01, Two-way ANOVA followed by Tukey HSD test. Data are presented as mean ± SEM.",
    "molecules": "Tubastatin A"
  },
  {
    "caption": "(C) Enhanced α-tubulin acetylation at P0 by Tubastatin A. Ac-tubulin level was assessed by immunoblot analysis.(D) Quantification of the α-tubulin acetylation. n=3 independent experiments. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test. Data are presented as mean ± SEM.",
    "molecules": "Tubastatin A"
  },
  {
    "caption": "(A) Representative traces from control (WT) and CAMDI-KO (KO) mice in the open field test at P21. Sample traces of locomotor activity were shown.(B) Distance traveled for 10 min. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=6.25, main effect of drug F(1, 43)=2.14, interaction F(1, 43)=12.68). Data are presented as mean ± SEM.",
    "molecules": "Tubastatin A"
  },
  {
    "caption": "(C) Repetitive jumping behavior. n = 13 for WT (vehicle) mice, n = 27 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 18 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 59)=5.04, main effect of drug F(1, 59)=10.13, interaction F(1, 59)=4.98). Data are presented as mean ± SEM.",
    "molecules": "Tubastatin A"
  },
  {
    "caption": "(D) Grooming time in small open field test. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 43)=11.18, main effect of drug F(1, 43)=7.23, interaction F(1, 43)=9.34). Data are presented as mean ± SEM.",
    "molecules": "Tubastatin A"
  },
  {
    "caption": "(E) Distance traveled for 15 min. n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. **, p<0.01, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=1.73, main effect of drug F(1, 28)=3.96, interaction F(1, 28)=11.80). Data are presented as mean ± SEM.",
    "molecules": "Tubastatin A"
  },
  {
    "caption": "(F) Light-dark test. Time in latency to the light chamber (latency). n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. *, p<0.05, Two-way ANOVA followed by Scheffe's post-hoc test (main effect of genotype F(1, 28)=2.34, main effect of drug F(1, 28)=3.82, interaction F(1, 28)=10.64). Data are presented as mean ± SEM.",
    "molecules": "Tubastatin A"
  },
  {
    "caption": "Activity-induced formation of nuclear  infoldings is abolished in SATB2-defficient hippocampal neurons. Bic-induced AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei  in DIV10 hippocampal  cultures derived from Satb2flx/flx mice  but not from  Satb2flx/flx::Nes-Cre mice  (Satb2NesCre) (n = 6 independent primary cultures, two-way ANOVA, F1,20 = 9.51, significant interaction p = 0.0058, simple main effects analysis, Satb2flx/flx cultures, Bic-treated vs untreated p = 0.0000043, Satb2NesCre cultures, Bic-treated vs untreated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, untreated - 680, Satb2flx/flx cultures, Bic-treated - 711, Satb2NesCre, untreated - 755, Satb2NesCre, Bic-treated - 720). Data are presented as mean ± SEM, ***p < 0.001.",
    "molecules": "Bic"
  },
  {
    "caption": "Activity-induced formation of nuclear  infoldings is impaired in  SATB2-deficient  cortical neurons. DIV14 cortical  cultures form Satb2flx/flx and Satb2NesCre mice  were silenced with NBQX  for 1 h followed by stimulation with Bic  for 1 h. The percentage of infolded nuclei  following AP bursting was increased in control Satb2flx/flx cultures but not in SATB2-deficient cultures (n = 3-4 independent primary cultures, two-way ANOVA, F1,10 = 39.124, significant interaction p = 0.000094, simple main effects analysis, Satb2flx/flx cultures: Bic-treated vs NBQX-treated p = 7.66E-07, Satb2NesCre cultures: Bic-treated vs NBQX-treated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, NBQX  - 606, Satb2flx/flx cultures, Bic  - 910; Satb2NesCre cultures, NBQX -604, Satb2NesCre cultures, Bic  - 835). Data are presented as mean ± SEM, ***p < 0.001.",
    "molecules": "NBQX, Bic"
  },
  {
    "caption": "Activity-induced formation of nuclear  infoldings is impaired in LEMD2-depleted cortical neurons. Bic-triggered AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei  in  cortical neurons transduced with AAV-scrambled but not with AAV-shLemd2  virus (n = 5 independent primary cultures, two-way ANOVA, F1,16 = 8.35, significant interaction p = 0.0107, simple main effects analysis: AAV-scrambled-transduced cultures, Bic-treated vs untreated p = 0.0049, AAV-shLemd2-transduced cultures, Bic-treated vs untreated p > 0.99, adjustment for multiple comparisons: Bonferroni). Data are presented as mean ± SEM, **p < 0.01 compared to NBQX-treated cultures.",
    "molecules": "NBQX, Bic"
  },
  {
    "caption": "LEMD2  is required for the nuclear envelope infoldings of CA1 pyramidal neurons in vivo. Top panel: representative confocal  images (z-axis-projected stack) of Lamin B2/DAPI-stained coronal brain   sections from wild-type mice after stereotaxic injection of AAV-shRNA viruses (AAV-shLemd2-mCherry or  AAV-scrambled-mCherry) into the dorsal hippocampus. Merged,  colocalization of the three signals. Scale bars: 50 μm. Middle panel: higher magnification images of AAV-shLemd2-transduced CA1 pyramidal neurons. Arrows show examples of mCherry-positive nuclei that are devoid of nuclear infoldings, whereas stars denote non-transduced infolded nuclei. Lower panel: higher magnification images of AAV-scrambled siRNA-transduced  CA1 pyramidal neurons. Arrows show examples of infolded, mCherry-positive nuclei; stars denote infolded, non-transduced neurons. Scale bars (middle and lower panels): 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Activity-induced formation of nuclear  infoldings is impaired in VPS4-depleted cortical neurons. Upon AP bursting, the percentage of infolded nuclei was increased in non-transfected cultures and in cultures transfected with scramble siRNA (Scr) but not with siVps4 (n = 3-4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,8 = 90.525, p = 1.63E-06,  Bic-treated vs NBQX-treated p = 0.000012, Scr Bic-treated vs NBQX-treated p = 0.000052, siVps4 Bic-treated vs NBQX-treated p = 0.396, siVps4 Bic-treated vs Scr Bic-treated p = 0.000015). Number of analyzed  nuclei: 474 (NBQX), 456 (Bic), 409 (Bic + Scr), 383 (Bic + siVps4). Data are presented as mean ± SEM, ***p < 0.001 compared to NBQX-treated, ###p < 0.001 compared to Scr Bic-treated.",
    "molecules": "NBQX, Bic"
  },
  {
    "caption": "(d,e) Growth of BSF (d) and PCF (e) trypanosomes in the absence (black lines, −Tet) or presence (red lines, +Tet) of 1 μg ml−1 tetracycline for the indicated number of days. Values are mean±s.d. (n=3).",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "(f,g) Northern blot analyses of TbMCU RNAi of BSF (f) and PCF (g) trypanosomes grown in the absence (0) and presence (2-8; 2-10) of tetracycline (top). Tubulin is shown as a loading control (bottom). Markers are shown on the right. The transcript of TbMCU, including the 5′- and 3′-UTR was ~1.5 kb in length.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(h,i) Western blot analyses of TbMCU RNAi of BSF (h) and PCF (i) trypanosomes grown in the absence (0) or presence (2-8; 2-10) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU (top). One band of ~30 kDa was detected in T. brucei homogenates. Membranes were stripped and re-incubated with antibody against the voltage-dependent anion channel (TbVDAC) as a loading control (bottom).",
    "molecules": "tetracycline"
  },
  {
    "caption": "(a) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5-N. After several pulses of Ca2+ (8 μM final concentration) cells (5 × 107 trypanosomes grown in the absence (black tracing) or presence (green tracing) of tetracycline) were added to the reaction medium (2.45 ml) and the reaction was started adding 50 μM digitonin. Ruthenium red (RR, 40 μM, yellow and red tracing), and FCCP (5 μM) were added where indicated. A decrease in fluorescence indicates decreasing medium Ca2+ or increasing vesicular Ca2+. (b) Relative Ca2+ uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1.0 (−Tet) (means±s.d., n=3, **P0.001, Student's t-test).",
    "molecules": "Ca2+, FCCP, digitonin, Ruthenium red, succinate, Tet, tetracycline"
  },
  {
    "caption": "(c) Similar conditions as in a except that further CaCl2 additions (8 μM each time) were added to −Tet trypanosomes to show the high mitochondrial capacity to take up Ca2+. EGTA (8 μM) and CaCl2 (Ca2+) were added where indicated to show the presence of medium Ca2+. (d) Relative initial rate of Ca2+ uptake by +Tet as compared with −Tet trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).",
    "molecules": "CaCl2, Ca2+, EGTA, Tet"
  },
  {
    "caption": "(e) Representative traces of Ca2+ uptake kinetics in digitonin-permeabilized TbMCU BSF trypanosomes cultured in the absence (−Tet) or presence (+Tet) of tetracycline. The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate. Ca2+ uptake was monitored over time using 1 μM Calcium Green-5N. Ruthenium red (RR, 40 μM, yellow and red tracings), oligomycin (Oligo, 2.5 μg ml−1), EGTA (0.4 μM) and CaCl2 (pulses of 0.4 μM) were added where indicated. (f) Relative Ca2+ uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 (−Tet) (means±s.d., n=3, *(+Tet±RR), **(−Tet±RR), ***(−Tet/+Tet) , P0.001, Student's t-test).",
    "molecules": "ATP, CaCl2, Ca2+, digitonin, EGTA, oligomycin, Ruthenium red, Tet, tetracycline, sodium orthovanadate"
  },
  {
    "caption": "(a) PCFtrypanosomes (5 × 107 cells) were added to the reaction buffer (2.45 ml) containing 2 mM succinate, and 5 μM safranine, and the reaction was started with digitonin (50 μM). ADP (10 μM), oligomycin (Oligo, 2 μg ml−1), CaCl2 (40 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (b) Changes in safranine fluorescence after addition of ADP or Ca2+ to −Tet and +Tet PCFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).",
    "molecules": "safranine, ADP, CaCl2, Ca2+, FCCP, digitonin, EGTA, oligomycin, succinate, Tet"
  },
  {
    "caption": "(c) BSFtrypanosomes (2 × 108 cells) were added to the reaction buffer (2.45 ml) containing EGTA (20 μM), ATP (1 mM), sodium orthovanadate (500 μM) and 12.5 μM safranine. The reaction was started with digitonin (40 μM). CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (d,e) Changes in safranine fluorescence after addition of Ca2+ (d) or FCCP (e) to −Tet and +Tet BSFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).",
    "molecules": "safranine, ATP, CaCl2, Ca2+, FCCP, digitonin, EGTA, Tet, sodium orthovanadate"
  },
  {
    "caption": "(a) Comparison of AMP/ATP ratios between PCF trypanosomes grown in the absence (−Tet) or presence (+Tet) of tetracycline to induce RNAi, and with or without glucose (Gl), expressed as fold increase (means±s.d., n=3; *P0.05; **P0.01, Student's t-test).",
    "molecules": "AMP, ATP, glucose, Tet, tetracycline"
  },
  {
    "caption": "(b) Comparison of AMP/ATP ratios between (−Tet) and (+Tet) PCF trypanosomes grown in a glucose-depleted medium (SDM-80) containing 5.2 mM L-proline for 2 days after 2-day growth in SDM-79±tetracycline, expressed as fold increase (means±s.d., n=3, **P0.01, Student's t-test).",
    "molecules": "AMP, ATP, glucose, L-proline, Tet, tetracycline"
  },
  {
    "caption": "(c) Fluorescence microscopic images of TbATG8.2 in PCF trypanosomes cultured in SDM-79 in the absence (WT) or presence (RNAi) of tetracycline to induce RNAi; scale bars, 10 μm. (d) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM-79 or SDM-80 medium in the presence (+Tet) or absence (−Tet) of tetracycline. Over 500 cells from three experiments with 20 random fields/experiment were analysed (means±s.d., n=3, **P0.001, Student's t-test).",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "(e) Western blot analysis of TbATG8.2 or tubulin in PCFtrypanosomes cultured in SDM-79 or SDM-80 in the absence (−Tet) or presence (+ Tet) of tetracycline to induce RNAi. Cell lysates (30 μg) were fractionated in 12% urea-SDS-PAGE gel. Immunoblots were labelled with anti-TcATG8.2 or anti-tubulin antibody. (f) Quantification of ATG8.2-II/(ATG8.2-II+ATG8.2-I) presented as mean ratios±s.d. (n=3, **P0.01, Student's t-test).",
    "molecules": "Tet, tetracycline, urea"
  },
  {
    "caption": "(g) Groups of five mice were infected with WT T. brucei (black line) or trypanosomes transfected with the construct for RNAi of TbMCU (red line). Doxycycline (green line; 200 μg ml−1) was given in the drinking water throughout a 30-day period.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "(a) Growth of TbMCU-KO BSF in the absence (black line) or presence (red line) of 1 μg ml−1 tetracycline for the indicated number of days. Addition of 10 mM threonine to the medium (green line) partially rescued the mutant BSF trypanosomes but had no effect on +Tet cells (yellow line; means±s.d., n=3, **P0.001, Student's t-test).",
    "molecules": "Tet, tetracycline, threonine"
  },
  {
    "caption": "(b) Southern blot analysis of the BSF conditional KOs. Genomic DNA from the T. brucei SM parental strain (+/+), single- (+/−) or double-allele (−/−) KO cells.",
    "molecules": "DNA"
  },
  {
    "caption": "(c) Northern blot analysis of wild-type (WT) and TbMCU-KO BSF cultured in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. Tubulin is shown as a loading control (bottom panel).",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "(d) Western blot analysis of total cell lysates. Proteins from BSF trypanosomes cultured in the presence (+Tet) or absence (−Tet) of tetracycline were detected using anti-HA antibody. TbVDAC was used as a loading control.",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "(e) Immunofluorescence analysis of control (upper panels) and TbMCU-KO (lower panels) trypanosomes. TbMCU co-localized with MitoTracker (MT) in the control parasites (+Tet; PCC=0.808). Scale bar, 10 μm.",
    "molecules": "Tet"
  },
  {
    "caption": "(f) Conditional TbMCU-KO BSF trypanosomes were grown in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. BSF trypanosomes (2 × 108 cells) were incubated as in Fig. 2e. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 20 μM. A representative trace (red or black) of Ca2+ uptake from one of three independent experiments is shown.",
    "molecules": "CaCl2, Ca2+, Tet, tetracycline"
  },
  {
    "caption": "(g) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSF trypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated.",
    "molecules": "CaCl2, FCCP, digitonin, EGTA, Tet, tetracycline"
  },
  {
    "caption": "(h,i) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSFtrypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 25, 50 and 100 μM CaCl2) or FCCP (i) to +Tet and −Tet BSFtrypanosomes (means±s.d., n=3, **P0.001, Student's t-test).",
    "molecules": "CaCl2, FCCP, digitonin, Tet, tetracycline"
  },
  {
    "caption": "(a) Rhod-2 fluorescence in control or overexpressing TbMCU (TbMCU-OE) PCF. Scale bar, 10 μm. (b,c) Rhod-2 (mitochondrial) (b) and Fluo-4 (cytosolic) (c) fluorescence in control (−Tet) and overexpressing TbMCU (+Tet) PCF (means±s.d., n=3, **P0.05, Student's t-test); NS, not significant.",
    "molecules": "Tet"
  },
  {
    "caption": "(d) Ca2+ uptake by digitonin-permeabilized PCF overexpressing TbMCU. Trypanosomes were incubated as in Fig. 2c. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 8 μM. (e) Ca2+ uptake in TbMCU-OE PCF (+Tet) 200 s after addition of Ca2+, as compared with uninduced trypanosomes considered as 1 (−Tet) (means±s.d., n=3, **P0.01, Student's t-test).",
    "molecules": "CaCl2, Ca2+, digitonin, Tet"
  },
  {
    "caption": "(f) Mitochondrial membrane potential of digitonin-permeabilized control (−Tet) and TbMCU-OE (+Tet) PCF. The reactions were incubated as in Fig. 3a.",
    "molecules": "digitonin, Tet"
  },
  {
    "caption": "(g) Changes in safranine fluorescence after addition of ADP, Ca2+ or FCCP to -Tet and +Tet PCF trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).",
    "molecules": "safranine, ADP, Ca2+, FCCP, Tet"
  },
  {
    "caption": "(h) Mitochondrial oxidative stress as measured with MitoSOX Red in control (C) and TbMCU-overexpressing (OE) PCF permeabilized with 50 μM digitonin in the presence of 0.4 mM Ca2+. Controls were treated with 2 μM antimycin A (C+AA), or were trypanosomes exposed to 0.4 mM Ca2+ in the absence of digitonin (−DIG+Ca2+), and trypanosomes permeabilized with digitonin in the absence of Ca2+ (C, yellow line). (i) Quantification of change at 900 s. Results are expressed as mean fluorescence±s.d. (n=3, **P0.001, Student's t-test).",
    "molecules": "antimycin A, Ca2+, digitonin"
  },
  {
    "caption": "(j) Growth of trypanosomes in the absence (control, black lines) or presence (TbMCU-OE, red lines) of 1 μg ml−1 tetracycline (means±s.d., n=3). (k) Dead cells in representative fields of control (−Tet) and TbMCU-OE (+Tet) PCF are indicated (white arrows). Scale bar, 10 μm.",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "(l) Cell viability on apoptotic challenge of control (−Tet) or TbMCU-OE (+Tet) PCF trypanosomes with H2O2 and C2-ceramide. The percentage of cell death was obtained from means±s.d. by analysing >60 fields (including >400 cells) (n=3). **P0.01, Student's t-test.",
    "molecules": "H2O2, C2-ceramide, Tet"
  },
  {
    "caption": "A-D LSM images of proximity ligation assay (PLA) dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jugular lymph sac / primordial thoracic duct (jls/pTD) of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. E-H LSM images of PLA dots composed of VEGFR2 and phosphorylated tyrosine (p-Tyr) on cross-sections through the jls/pTD of E13.5 control and ILK K.O. embryos. Scale bars: 10 µm. I Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 9 embryos per genotype), *P = 0.022. J Quantification of the PLA dots indicating VEGFR2 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype). K Quantification of the total number of PLA dots indicating both VEGFR2 (left) and VEGFR3 (right) with phosphorylated tyrosine (p-Tyr) per jls/pTD section of E13.5 control or ILK K.O. embryos (n = 3 embryos per genotype), *P = 0.005 (VEGFR2/p-Tyr in ILK K.O. versus VEGFR3/p-Tyr in ILK K.O.), *P = 0.013 (VEGFR3/p-Tyr in control versus VEGFR3/p-Tyr in ILK K.O.)",
    "molecules": "Tyr, tyrosine"
  },
  {
    "caption": "LSM images of PLA dots composed of VEGFR3 and phosphorylated tyrosine (p-Tyr) on stained cross-sections through the jls/pTD of control and ILK & β1 integrin K.O. embryos. Arrows point to PLA dots within the Lyve1 stained area. Scale bars: 10 µm",
    "molecules": "Tyr, tyrosine"
  },
  {
    "caption": "Quantification of the PLA dots indicating VEGFR3 with phosphorylated tyrosine (p-Tyr) per LEC of E13.5 control or ILK & β1 integrin K.O. embryos",
    "molecules": "Tyr, tyrosine"
  },
  {
    "caption": "VEGFR3 tyrosine phosphorylation as determined by ELISA of skin lysates of adult control or ILK K.O. mice (n = 3 mice per genotype)",
    "molecules": "tyrosine"
  },
  {
    "caption": "LEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3)",
    "molecules": "BrdU, Cys, Ser"
  },
  {
    "caption": "VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA)",
    "molecules": "Cys, Ser, tyrosine"
  },
  {
    "caption": " SA‐β‐gal staining on spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM at day 7. Scale bar, 100 μm. Quantification of the numbers of senescent cells shown in (K). Error bars represent SEM (n = 3). **P 0.01. Quantification of the numbers of spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM. Error bars represent SEM (n = 3). **P 0.01. Statistical significance was assessed by Student's unpaired t‐test. ",
    "molecules": "GS493"
  },
  {
    "caption": " Western blot analysis of p27, p53, and α‐tubulin in PyMT cells treated with control (DMSO) or inhibitors against Skp2 (MLN4924), Aurora A (VX‐680), or Notch (DAPT) at the indicated concentrations. The numbers below indicate the relative ratios of band intensities between p27 and α‐tubulin or between p53 and α‐tubulin. ",
    "molecules": "DAPT, DMSO, MLN4924, VX‐680"
  },
  {
    "caption": " Quantification of the numbers of senescent cells in PyMT cells treated with control, MLN4924, MLN4924 plus p27 knockdown or MLN4924 plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant. ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Quantification of the numbers of senescent cells in PyMT cells treated with control, VX‐680, VX‐680 plus p27 knockdown, or VX‐680 plus p53 knockdown. Error bars represent SEM (n = 3). *P 0.05; NS, not significant. ",
    "molecules": "VX‐680"
  },
  {
    "caption": " Quantification of the numbers of senescent cells in PyMT cells treated with control, DAPT, DAPT plus p27 knockdown, or DAPT plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant. Statistical significance was assessed by Student's unpaired t‐test. ",
    "molecules": "DAPT"
  },
  {
    "caption": " SA‐β‐gal staining on MLN4924 tumorcells treated with control, Shp2 inhibitor GS493 (15 μM), Src inhibitor PP2 (2.5 μM), Fak inhibitor TAE226 (0.5 μM), or Mek1 inhibitor U0126 (20 μM). Scale bar, 100 μm. ",
    "molecules": "GS493, U0126, TAE226, PP2, MLN4924"
  },
  {
    "caption": " Western blot analysis of pY527, pY416 and total Src, pY925 and total Fak, pT202/pT204 and total Erk1/2, and α‐tubulin in MLN4924 tumorcells with or without treatment with 5 μM GS493. ",
    "molecules": "GS493"
  },
  {
    "caption": "  Kaplan-Meier analyses of tumor formation for control (MLN4924;Shp2fl/fl, blue, n = 33), heterozygous Shp2 mutant (MLN4924;MMTV‐Cre;Shp2+/fl, green, n = 15), and homozygous Shp2 mutant (MLN4924;MMTV‐Cre;Shp2fl/fl, also called MLN4924;coShp2, orange, n = 34) mice. ***P 0.001 (log‐rank test). ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Mammary gland whole‐mounts from control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice at indicated ages. The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads; premalignant lesions are marked by red arrowheads. Scale bar, 4 mm. Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice, as shown in (B). Error bars represent SEM (n = 6). *P 0.05 (Student's unpaired t‐test). ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice at hyperplasia, adenoma, and carcinoma stages. Scale bar, 100 μm. n = 8. ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors (top) and metastases (bottom) from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice. Scale bar, 100 μm. n = 8. ",
    "molecules": "MLN4924"
  },
  {
    "caption": " qRT-PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924;Shp2fl/fl (n = 8), MLN4924;MMTV‐Cre;Shp2+/fl (n = 6), and MLN4924;MMTV‐Cre;Shp2fl/fl (n = 6) mice. Error bars represent upper and lower quartiles. ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Western blot analysis of Shp2protein levels in tumor tissues from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice. ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Immunohistochemistry analysis of EYFP (top) and SA‐β‐gal staining (bottom) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Note that SA‐β‐gal activity was present in the EYFP‐positive (Shp2‐negative) areas (right, arrows) and was absent in the EYFP‐negative (Shp2‐positive) areas (right, arrowhead) of mammary glandtumors of MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Consecutive sections of EYFP and SA‐β‐gal staining for each group are shown. Scale bar: 100 μm. n = 5. ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Immunofluorescence analysis of Ki67, EYFP and CK8 (pan‐epithelial marker) (top panel), p27 and EYFP (middle panel), and p53 and EYFP (bottom panel) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. DAPI was used for nuclear counterstain. Ki67 was absent in EYFP‐positive cells (top right, arrows) and was present in EYFP‐negative cells (top right, arrowheads); p27 was present in EYFP‐positive cells (middle right, arrows) and was absent in EYFP‐negative cells (middle right, arrowhead); and p53 was present in EYFP‐positive cells (bottom right, arrows) and was absent in EYFP‐negative cells (bottom right, arrowhead). Scale bar: top panel, 100 μm; middle and bottom panel, 50 μm. n = 5. ",
    "molecules": "MLN4924"
  },
  {
    "caption": " qRT-PCR analysis of mRNA levels of Shp2, Skp2, Aurka, Dll1, and Hey1 in EYFP+ and EYFP−cells isolated by FACS from MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/fltumors. Error bars represent SEM (n = 5). ",
    "molecules": "MLN4924"
  },
  {
    "caption": " Kaplan-Meier analysis of tumor formation in control (blue, n = 8) and GS493‐pretreated (red, n = 8) MLN4924;MMTV‐Cre;Shp2fl/flmice. The bar indicates the duration of treatment. ",
    "molecules": "GS493, MLN4924"
  },
  {
    "caption": " Tumorgrowth curves for control (blue, n = 5) and GS493‐treated (red, n = 5) MLN4924 mice. Note that after day 24, all mice in the control group were sacrificed due to the tumor size. Error bars represent SEM (n = 5). ",
    "molecules": "GS493, MLN4924"
  },
  {
    "caption": "(A) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12.",
    "molecules": "antimycin A, glucose"
  },
  {
    "caption": "(B) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70).",
    "molecules": "antimycin A"
  },
  {
    "caption": "(C) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually.",
    "molecules": "antimycin, antimycin A"
  },
  {
    "caption": "(C) Concentrations of fructose-1,6-bisphosphate, which correlate with glycolytic flux, are significantly higher in A-strain (ratio = 1.35, nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)",
    "molecules": "fructose-1,6-bisphosphate"
  },
  {
    "caption": "(E) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T- and A-strains (FDR <10%) and are measured in all four conditions (columns). First column: genes ordered by increasing fold-changes for RNAs (computed as log2[T]-log2[A]). Second column: fold-changes for proteins (computed as log2[T]-log2[A]). Third column: fold-changes for RNAs in cells treated with rapamycin & caffeine (TORC1 inhibition; computed as log2[treatment]-log2[control]) (data from ref. (Rallis et al, 2013)). Fourth column: fold-changes for RNAs in cells treated with H2O2 (oxidative stress; computed as log2[treatment]-log2[control]) (data from Chen et al, 2003). Log2 fold-changes are capped at absolute values of 1 for all columns.",
    "molecules": "caffeine, H2O2, rapamycin"
  },
  {
    "caption": "(C) Media samples were taken from the same cultures (n=3 for both strains) at the 8 hr timepoint and the remaining glucose was quantified. The consumed glucose was calculated based on the amount of glucose measured in the same, fresh media and normalised to the OD of each culture at the time of sampling.",
    "molecules": "glucose"
  },
  {
    "caption": "(E) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain. Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "(A) Spot assays on solid media from a three-fold dilution series of exponential cultures at the same cell density in 96-well plates (3 biological replicates of each strain) and spotted in 16 technical replicates (each dilution in 4x4 square). The A-strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide. A control without toxin (left) was included in each batch of spot assays performed and a representative image is shown here.",
    "molecules": "diamide, antimycin A, H2O2"
  },
  {
    "caption": "(B) Fitness (approximated by maximum slope of smoothed growth curves) of A- and T-strains on 12 carbon sources, with either yeast extract (YE) or ammonium (NH4), with or without 0.1% priming glucose to support initial growth. For all 48 conditions, two biological replicates of A- and T-strains were grown in technical quadruplicates each. Dotted lines in panels mark a fitness ratio of 1 (i.e., same fitness).",
    "molecules": "ammonium, NH4, glucose"
  },
  {
    "caption": "(C) Fitness of A- and T-strains on 95 nitrogen sources on Biolog Phenotype MicroArrays. Conditions with no substantial growth were excluded (black circles, maximum slope <0.015). Red lines show arbitrary significance cut-off, put at |log2(A-strain/T-strain)|>0.75.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(D) Fitness of A- and T-strains on 72 different drugs and toxins, at 4 concentrations each, on Biolog Phenotype MicroArrays. (Results for benzamidine were inconclusive, with both strains comparatively resistant in one concentration each.)",
    "molecules": "benzamidine"
  },
  {
    "caption": "(F) Chronological lifespan of A- and T-strains, i.e. the proportion of non-dividing cells in stationary phase that maintain proliferative potential after refeeding. The data show colony forming units (CFUs) per ml of culture over 7 days of stationary phase in glucose-depleted rich media. Three biological repeats were carried out for both strains, with each repeat measured as technical triplicates. Error bars represent standard error of the biological replicates.",
    "molecules": "glucose"
  },
  {
    "caption": "(G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A.",
    "molecules": "antimycin A, H2O2"
  },
  {
    "caption": "A, B After grown in nutrient rich medium to log phase, wild-type (WT) cells were treated with rapamycin (10 μM) or subject to nitrogen starvation (SD-N) for 3 h and 16 h and degradation of autophagic marker GFP-Atg8 was detected. The cycloheximide (CHX, 100 μg/mL,) or thiolutin (10 μg/mL) was simultaneously used to block protein synthesis or gene transcription.",
    "molecules": "CHX, cycloheximide, nitrogen, rapamycin, thiolutin"
  },
  {
    "caption": "Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.",
    "molecules": "nitrogen, rapamycin, thiolutin"
  },
  {
    "caption": "Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.",
    "molecules": "nitrogen, rapamycin, thiolutin"
  },
  {
    "caption": "B N-terminally GFP-tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4 and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls.",
    "molecules": "50Q"
  },
  {
    "caption": "E-I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1, ATG9, ATG13, ATG17 and ATG5 were overexpressed in rpb9∆ yeast cells, GFP-50Q was checked for its autophagic degradation by GFP processing assays after 1, 4 and 16 h starvation.",
    "molecules": "50Q"
  },
  {
    "caption": "B Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP-50Q was checked by GFP-processing assays.",
    "molecules": "50Q"
  },
  {
    "caption": "F-G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP-50Q was detected.",
    "molecules": "50Q"
  },
  {
    "caption": "C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.",
    "molecules": "50Q"
  },
  {
    "caption": "D N-terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.",
    "molecules": "50Q"
  },
  {
    "caption": "G-I. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of Tfh cells (CD4+CD44+PD-1+CXCR5+) in the spleen after primary immunization (day 7) with NIP-OVA (n = 7).",
    "molecules": "NIP"
  },
  {
    "caption": "J, K. ELISA analysis of high- (J) and low-affinity (K) isotype-specific anti-NIP antibodies in sera from OVA/OVA- and OVA/KLH-immunized mice (day 15 post-challenge; n = 5 mice/group). Data representative of one experiment of two.",
    "molecules": "NIP, KLH"
  },
  {
    "caption": "A-C. Analysis of TCR nanoclustering by EM in OT-II WT and CCR5-/- naïve cells (A; n = 6 cells/genotype; WT: 3427, CCR5-/-: 3528 particles), and IL-2- (B; WT, n = 8 cells, 15419 particles; CCR5-/-, n = 6 cells, 5410 particles) or IL-15-expanded lymphoblasts (C; WT, n = 8 cells, 27518 particles; CCR5-/-, n = 7 cells, 22696 particles). A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti-CD3ε-labeled cells; at bottom, quantification (mean ± SEM) of gold particles in clusters of indicated size in WT (gray bars) and CCR5-/- cells (red). Insets show the distribution of clusters of one, two, three, four, or more than four particles, and statistical analysis.",
    "molecules": "gold"
  },
  {
    "caption": "F. Comparison of TCR oligomer size using BN-PAGE and anti-CD3ζ immunoblotting in day 10, IL-2-expanded WT and CCR5-/- OT-II lymphoblasts lysed in buffer containing digitonin or Brij-96. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry (right; n = 5).",
    "molecules": "Brij-96, digitonin"
  },
  {
    "caption": "G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5-/- mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14680 particles; CCR5-/-, red; n = 7 cells, 15374 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.",
    "molecules": "gold"
  },
  {
    "caption": "A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles). B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.",
    "molecules": "gold, TAK-779"
  },
  {
    "caption": "C. OT-II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100. Left, representative EM images showing gold particle distribution in the cell surface replicas. Right, analysis of gold particles in clusters as above, vehicle- (gray bars; n = 6 cells, 12339 particles) and AMD3100-treated cells (black; n = 7 cells, 17059 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.",
    "molecules": "gold, AMD3100"
  },
  {
    "caption": "A. Total Chol levels in WT and CCR5-/- OT-II lymphoblasts (day 10, IL-2-expanded) as determined by a fluorometric assay (n = 6).",
    "molecules": "Chol"
  },
  {
    "caption": "B-D. SM (B), Cer (C) and dhCer (D) levels in WT and CCR5-/- OT-II 10-day lymphoblasts, as determined by UPLC-TOF MS. Values, after normalization with C17 standards and cell number in each sample, are the mean of two independent experiments (n = 6).",
    "molecules": "Cer, dhCer, SM"
  },
  {
    "caption": "H. Relative CerS2 mRNA level in CD4 T cells treated with PTx (n = 3).",
    "molecules": "PTx"
  },
  {
    "caption": "L. Representative immunofluorescence images showing pSer142-GATA-1 staining (green) of OT-II WT and CCR5-/- lymphoblasts. The green channel (top) and the merge with nuclear DAPI staining (blue; bottom) are shown. Scale bar, 10 μm. M. Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI-stained area; n ≥50 cells/condition).",
    "molecules": "DAPI, Ser"
  },
  {
    "caption": "A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition (n = 3). SBP, streptavidin-binding-peptide-tagged TCR.",
    "molecules": "SBP, streptavidin-binding-peptide, lipid"
  },
  {
    "caption": "B. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms).",
    "molecules": "Brij-96, digitonin"
  },
  {
    "caption": "D. Cer levels in OT‑II 10-day lymphoblasts, untreated or treated with SMase (n = 4).",
    "molecules": "Cer"
  },
  {
    "caption": "E. Representative small field images showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black cyan; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles.",
    "molecules": "gold"
  },
  {
    "caption": "F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS.",
    "molecules": "puromycin"
  },
  {
    "caption": "G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F. Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells (n = 3).",
    "molecules": "TAK-779"
  },
  {
    "caption": "I. TCR nanoclustering of shCtrl- and shCerS2 transduced 2B4 cells in the presence of TAK-779 as determined by EM. Representative small field images and quantification (mean ± SEM) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl (gray black bars; n = 6 cells, 12337 particles) and shCerS2 2B4 lymphoblasts (orange; n = 7 cells, 13456 particles). Inset, distribution between clusters of indicated size and statistical analysis.",
    "molecules": "gold, TAK-779"
  },
  {
    "caption": "J. Percentage of CD69+ shCtrl (black) and shCerS2 (orange) 2B4 cells restimulated with plate-bound anti-CD3ε antibody in the presence of TAK-779 (n = 3).",
    "molecules": "TAK-779"
  },
  {
    "caption": "A. Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM. Top, representative small field image showing gold particle distribution in cell surface replicas of anti-CD3ε-labeled cells; bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size in the WT (gray bars; n = 5 cells, 17689 particles) and Δ32/Δ32 cells (light red; n = 4 cells, 16938 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.",
    "molecules": "gold"
  },
  {
    "caption": "B. Normalized SM, Cer and dhCer levels in lymphoblasts obtained as in A. A representative experiment is shown (n = 3 donors/genotype; n = 2 independent experiments)",
    "molecules": "Cer, dhCer, SM"
  },
  {
    "caption": "A, B. RT-qPCR analysis of Zfp207 and Oct4 in mouse ESCs during (A) retinoic acid (RA)-induced and (B) embryoid body (EB)-mediated differentiation. Nestin was used as a neuronal differentiation marker to monitor RA-mediated differentiation. mRNA levels are relative to the expression at day 0.",
    "molecules": "RA, retinoic acid"
  },
  {
    "caption": "(O) Western blot of OCT4 during a 9-h cycloheximide (CHX) time course treatment in shScr, sh1 and sh2 ESCs.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(Q) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "B. Immunostaining of NESTIN (green) and TUJ1 (red) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation. Nuclei were counterstained with DAPI. Scale bar, 20 μM.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) FISH of poly(A)+ RNA distribution in shScr, sh1 and sh2 stained with 5´Cys3-oligo dT(50). Nuclei were counterstained with DAPI. Scale bar, 20 µM.",
    "molecules": "5´Cys3-oligo, DAPI, poly(A)"
  },
  {
    "caption": "Quantification of (F) nuclear, (G) cytoplasmic, and (H) of nuclear/cytoplasmic poly(A)+ FISH signals. (I) Relative nuclear and cytosolic total RNA levels in sh1 and sh2 compared to shScr. Data are presented as mean ± SEM of n ≥ 3 independent biological experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 (shScr vs. sh1 or sh2); unpaired Student's t-test.",
    "molecules": "poly(A)"
  },
  {
    "caption": "(E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.",
    "molecules": "Hoechst, BafA1"
  },
  {
    "caption": "(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.",
    "molecules": "BafA1, nanogold particles"
  },
  {
    "caption": "(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.",
    "molecules": "nanogold particles"
  },
  {
    "caption": "(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).",
    "molecules": "collagen, gold particles"
  },
  {
    "caption": "(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.",
    "molecules": "BafA1"
  },
  {
    "caption": "(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.",
    "molecules": "BafA1"
  },
  {
    "caption": "(B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001.",
    "molecules": "BafA1"
  },
  {
    "caption": "(B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments.",
    "molecules": "BafA1"
  },
  {
    "caption": "(C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.",
    "molecules": "BafA1, CST"
  },
  {
    "caption": "U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.",
    "molecules": "BafA1, CST"
  },
  {
    "caption": "B) Cells infected with UIS4‐mCherry‐expressing parasites were treated with nocodazole for 1.5 h (top) or cytochalasin D for 30 min (bottom) and imaged by confocal microscopy. Imaging occurred 29 and 24 h post‐infection, respectively. UIS4‐positive tubules, static (top) and dynamic (bottom), are indicated with white arrowheads. Displayed cells are representative of five imaged parasites from at least two different experiments.",
    "molecules": "cytochalasin D, nocodazole"
  },
  {
    "caption": "B) Cells infected with UIS4‐mCherry‐expressing parasites were treated with nocodazole for 1.5 h (top) or cytochalasin D for 30 min (bottom) and imaged by confocal microscopy. Imaging occurred 29 and 24 h post‐infection, respectively. UIS4‐positive tubules, static (top) and dynamic (bottom), are indicated with white arrowheads. Displayed cells are representative of five imaged parasites from at least two different experiments.",
    "molecules": "cytochalasin D, nocodazole"
  },
  {
    "caption": "A and B) The bleach was applied to elongated membrane clusters of the LS‐TVN approximately 27 h post‐infection. The UIS4‐mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B).",
    "molecules": "bleach"
  },
  {
    "caption": "C) When the bleach was applied to the PVM at 25 h post‐infection, the signal recovered rapidly in the bleached region. Upon the application of additional bleach pulses in the same area, the bleached area (region #1 measured graphically) repeatedly recovered, and the remaining PVM‐associated UIS4‐mCherry signal depleted (region #2) although the LS‐TVN‐associated signal remained (region #3). When a region of the LS‐TVN was subsequently bleached (yellow arrow, region #4), no recovery was observed. The times are shown in minutes and seconds. Scale bars, 10 µm.",
    "molecules": "bleach"
  },
  {
    "caption": "esgts midguts expressing GFP alone (control), or expressing Notch Intra Cellular Domain (NICD), and NICD + OvoB. In closeups (right), DAPI is shown in purple for improved contrast; the arrow highlights a cell with intermediate phenotype.",
    "molecules": "DAPI"
  },
  {
    "caption": "esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Prospero (red). Closeups correspond to boxed regions, with DAPI shown in purple and GFP-positive cells outlined in yellow.",
    "molecules": "esg, DAPI"
  },
  {
    "caption": "MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green), Scribble (yellow), ß-catenin (purple) and DAPI (Blue). A' pictures display cross sections of the regions shown in A.",
    "molecules": "DAPI"
  },
  {
    "caption": "MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green) and PH3 (red). The graph plots number of mitotic PH3-positive cells per midgut of MyoIAts guts expressing GFP alone (ctrl), or expressing SvbACT , and SvbREP + pri.",
    "molecules": "PH3"
  },
  {
    "caption": "Top: Immunoblot analysis of TFAP4 after transfection of miRNA mimics (miR-291a-5p, miR-291a-3p and miR-291a-5p+miR-291a-3p combined) in miR-290-295_KO mESCs. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of TFAP4 intensity, normalized to Coomassie and relative to the WT sample in three biological replicates. Data information: In I, bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie. Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.",
    "molecules": "Coomassie, Coomassie blue"
  },
  {
    "caption": "Immunoblot validation of siPOOL-mediated knock down of Tfap4. TFAP4 levels were compared between untreated WT versus miR-290-295_KO cells treated with a negative control and a Tfap4-targeted siPOOL. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of two biological replicates.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "Human spheroids established from BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) DLD1 cells, were incubated with 1.25 µM olaparib or 0.5 µM chlorambucil over the indicated period of time.",
    "molecules": "chlorambucil, olaparib"
  },
  {
    "caption": "BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells were infected with lentiviruses expressing control or CHD4 shRNAs, followed by selection with puromycin for 72 hours. Dose-dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days.",
    "molecules": "puromycin"
  },
  {
    "caption": "BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells were incubated with 1 µM chlorambucil (Chl). Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. GAPDH was used as a loading control.",
    "molecules": "Chl, chlorambucil"
  },
  {
    "caption": "Quantification of chromosome aberrations and chromatid/chromosome break frequencies in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h. Data were obtained from three independent experiments and normalised to untreated controls. A minimum of 60 Giemsa-stained metaphases were analysed for each sample. Cis, cisplatin; Chl, chlorambucil.",
    "molecules": "Giemsa, Chl, chlorambucil, Cis, cisplatin"
  },
  {
    "caption": "Quantification of G2/M cell frequency relative to solvent control, using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours. Cis, cisplatin; Chl, chlorambucil.",
    "molecules": "Chl, chlorambucil, Cis, cisplatin"
  },
  {
    "caption": "Nude mice (nu/nu) were injected subcutaneously with 5 x 106 human DLD1 cells, BRCA2-proficient (A) or BRCA2-deficient (B). Tumour-bearing mice were treated with 3 mg/kg daily chlorambucil administered intraperitoneally (i.p.) for a total of ten days. Tumour weight was determined on the indicated days after initiation of the treatment.",
    "molecules": "chlorambucil"
  },
  {
    "caption": "PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with chlorambucil at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER-negative tumour, no BRCA1 alteration; STG201, tumour with BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant)",
    "molecules": "chlorambucil, DMSO, olaparib"
  },
  {
    "caption": "CB17/SCID mice were injected intramuscularly with 5 x 106 human BRCA2-deficient HCT116 cells. Tumour-bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally (i.p.), or talazoparib administered orally (o.s.) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment (day 1). Scale bar, 40 µm. Each experimental group included n = 5 mice. Error bars represent SEM. Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining.",
    "molecules": "chlorambucil, cisplatin, talazoparib"
  },
  {
    "caption": "Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown.",
    "molecules": "chlorambucil, cisplatin, Duramycin, Tc"
  },
  {
    "caption": "HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-Ubiquitin (Ub) and anti-Myc antibodies. Scale bar=10 µm.",
    "molecules": "Ubiquitin"
  },
  {
    "caption": "HeLa cells were co-transfected with Ub-R-GFP and F1-Myc. Mitochondria were isolated and then treated with or without proteinase K (Pro.K), or proteinase K in combination with digitonin or Triton X-100 (TX-100). Samples were then subjected to Immunoblot analysis. HSP60, TIM23 and TOM20 are representatives of mitochondrial matrix-, mitochondrial inner membrane- and mitochondrial outer membrane-localized proteins, respectively.",
    "molecules": "digitonin, Triton X-100, TX-100"
  },
  {
    "caption": "Fractionation analysis of the HeLa cells transfected with Ub-R-GFP and treated with DMSO (control vehicle) or 10 μM MG132 for 4 hours.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Proteinase K protection analysis of the mitochondria isolated from the HeLa cells transfected with Ub-R-GFP and treated with MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "Proteinase K protection analysis of the mitochondria isolated from HeLa cells co-transfected with Ub-R-GFP and Ctrl, TOM20, TOM22 or TOM70 shRNA and treated with MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "Fractionation analysis of HeLa cells stably transfected with Ctrl or LONP1 shRNA and transiently co-transfected with Ub-R-GFP and Myc or LONP1 (shRNA-resistant) and then treated with DMSO or MG132.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Fractionation analysis of HeLa cells stably transfected with Ctrl shRNA, F1 shRNA, or F1 shRNA together with shRNA-resistant F1-Myc or ΔLIR-Myc, and transiently transfected with Ub-R-GFP and then treated with DMSO or MG132.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Fractionation analysis of HeLa cells stably transfected with Ctrl or HSC70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSC70 and then treated with DMSO or MG132.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Fractionation analysis of HeLa cells stably transfected with Ctrl or HSP70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSP70 and then treated with DMSO or MG132.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Mitochondria were isolated from the untransfected cells, cytosols were isolated from the cells transfected with GFP or Ub-R-GFP and treated with DMSO or MG132 for 8 hours, the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Co-IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated.",
    "molecules": "MG132"
  },
  {
    "caption": "Immunoblot analysis of the HeLa cells transfected with or without HA-Bcl-xL and treated with DMSO or MG132 for 6 hours.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "D, E. Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132. Quantification is shown in (E).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "HeLa cells were transfected with GFP-p62, treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti-Ub and anti-γ-Tubulin antibodies. Note that the aggresome (blue arrowhead), rather than the MAPAs (white arrowheads), colocalizes with γ-Tubulin. Scale bar=10 µm.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm.",
    "molecules": "MG132"
  },
  {
    "caption": "B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (C).",
    "molecules": "MG132"
  },
  {
    "caption": "D, E. HeLa cells were transfected with Ctrl, TOM20, TOM22 or TOM70 shRNA vectors including nuclear localized GFP (N-GFP) expressing elements, then treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (E).",
    "molecules": "MG132"
  },
  {
    "caption": "F, G. HeLa cells were transfected with GFP or GFP-Bcl-xL, then treated with MG132 and immunostained with anti-p62 antibodies. Scale bar=10 µm. Quantification is show in (G).",
    "molecules": "MG132"
  },
  {
    "caption": "H, I. HeLa cells were stably transfected with Ctrl or LONP1 shRNA, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (I).",
    "molecules": "MG132"
  },
  {
    "caption": "csCLEM analysis of cells transfected with GFP-p62 and treated with MG132. Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy (LM) and transmission electron microscopy (TEM). The blue arrowhead shows a MAPA observed under LM and TEM.",
    "molecules": "MG132"
  },
  {
    "caption": "TEM analysis of HeLa cells treated with DMSO or MG132. Note the layered structures (red arrowheads) in the mitochondrial matrix (ii, iii), budding from mitochondria (iv), attached to mitochondria (v-vi), away from mitochondria (vii) or inside the MAPAs (viii, ix) in MG132-treated cells. Scale bar=500 nm. M, mitochondrion. N, nucleus.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells treated with MG132 as indicated.",
    "molecules": "MG132, TX-100"
  },
  {
    "caption": "Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells transfected with Ub-R-GFP and treated with MG132 as indicated.",
    "molecules": "MG132, TX-100"
  },
  {
    "caption": "HeLa cells were co-transfected with Mito-OM-cherry and Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 antibody. Note the colocalization of a p62 punctum with a structure that is positive for Mito-OM-cherry but not for Mito-MTX-GFP (white arrowheads) in MG132-treated cells. Scale bar=10 µm.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "HeLa cells were transfected with Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 and anti-TIM23 antibodies. Note the colocalization of a p62 punctum with a structure that is positive for TIM3 but not for Mito-MTX-GFP (white arrowhead) in MG132-treated cells. Scale bar=10 µm.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "HeLa cells were co-transfected with Mito-MTX-GFP and OTC-Myc or OTC-Δ-Myc, treated with DMSO or MG132 and immunostained with anti-Myc and anti-p62 antibodies. Note that the p62-labled MAPAs were colocalized with OTC-∆ (red arrowheads), but not with OTC (white arrowheads) in the MG132 treated cells. Scale bar=10 µm.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "TEM analysis of HeLa cells transfected with indicated vectors, treated with MG132 and stained with DAB. Blue arrowheads show MAPAs, red arrowheads show electron-dense Apex signals. Scale bar=500 nm. M, mitochondrion.",
    "molecules": "DAB, MG132"
  },
  {
    "caption": "Time-lapse analysis of live HeLa cells transfected with OTC-Δ-GFP and Mito-MTX-DsRed and treated with MG132. White arrowheads show a OTC-Δ-GFP-positive Mito-MTX-DsRed-negative structure that eventually segregated from the mitochondrial network.",
    "molecules": "MG132"
  },
  {
    "caption": "Immunoblot analysis of the MG132-induced deposition of FUNDC1, TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA.",
    "molecules": "MG132"
  },
  {
    "caption": "C, D. Immunoblot analysis of the MG132-induced deposition of FUNDC1 in cells transfected with ctrl shRNA, FIS1 shRNA or FIS1 shRNA together with shRNA-resistant Flag-FIS1. Quantification is shown in (D).",
    "molecules": "MG132"
  },
  {
    "caption": "E, F. HeLa cells were stably transfected with Ctrl or FIS1 shRNA, treated with MG132 and immunostained with indicated antibodies. Scale bar=10 µm. Quantification is shown in (F).",
    "molecules": "MG132"
  },
  {
    "caption": "G, H. Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA, then transiently co-transfected with Ub-R-GFP and Flag or Flag-FIS1 and treated with DMSO or MG132. Quantification is shown in (H).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "HeLa cells were transfected with GFP-LC3, treated with DMSO or 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Immunoblot analysis of the TCL of HeLa cells treated with MG132 as indicated.",
    "molecules": "MG132"
  },
  {
    "caption": "Immunoblot analysis of the TCL of HeLa cells stably transfected with Ctrl or ATG5 shRNA and treated with MG132 as indicated.",
    "molecules": "MG132"
  },
  {
    "caption": "Immunoblot analysis of the TCL of HeLa cells stably transfected with the indicated vectors and treated with MG132 as indicated.   Quantification of the results from panels (D) and (E). 3 replicates were analyzed using Student's 2-tailed t-test and are presented as the means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.   ",
    "molecules": "MG132"
  },
  {
    "caption": "HeLa cells were stably transfected with the indicated vectors, transiently transfected with GFP-LC3, then treated with 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.",
    "molecules": "MG132"
  },
  {
    "caption": "TEM analysis of F1 shRNA/F1-Myc or F1 shRNA/ΔLIR-Myc cells treated with MG132 for 8 hours. Yellow arrowheads show the MAPAs. Blue arrows show the places where membrane is absent. White arrowheads show the layered aggregates budding from mitochondria. Scale bar=500 nm; M, mitochondrion.",
    "molecules": "MG132"
  },
  {
    "caption": "A, B. HeLa cells were treated with DMSO or 10 μM MG132 for 8 hours, then washed with fresh medium three times and cultured for another 3 days. Cells were then fixed and subjected to SA-β-gal staining. The senescent cells show SA-β-gal-positive staining (Blue). Quantification is shown in (B).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "C, D. Immunoblot analysis of HeLa cells treated with DMSO or MG132 for 8 hours, then washed and cultured for the indicated times. Quantification is shown in (D).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "E, Quantification of SA-β-gal staining in HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days.",
    "molecules": "DMSO, C.C, Compound C, MG132"
  },
  {
    "caption": "F. HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days. Immunoblot analysis of the cells is shown in (F).",
    "molecules": "DMSO, C.C, Compound C, MG132"
  },
  {
    "caption": "Quantification of SA-β-gal staining of HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "H. HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (H). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Quantification of MG132-induced SA-β-gal staining in HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "J. HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (J). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Cells were then subjected to SA-β-gal staining. The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.",
    "molecules": "DMSO, MG132, Tet, tetracycline"
  },
  {
    "caption": "L. HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Immunoblot analysis of the cells is shown in (L). The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.",
    "molecules": "DMSO, MG132, Tet, tetracycline"
  },
  {
    "caption": "Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": ", N. HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (N).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "P. HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (P).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(A to D) 10- to 11-week-old mice were treated with vehicle control or AT845 at the vector doses indicated and followed for 12 weeks (N = 7 or 8 per cohort). (A) Vector copy number, (B) GAA levels, (C) GAA activity, and (D) glycogen content in cardiac and skeletal muscles. (C to D) Statistical analysis: two-way ANOVA, Dunnett's test. Data are presented as box-and-whisker plots with Tukey whiskers that show minimum, median, and maximum. Asterisks (*) indicate significant differences compared with untreated Gaa-/- mice (black) or wild-type mice (red). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P <0.0001. See also Appendix Table S1.",
    "molecules": "glycogen"
  },
  {
    "caption": "Representative images of H&E (top) and PAS (bottom) staining of the quadriceps in wild-type and Gaa-/- mice untreated or treated with escalating doses of AT845 (3×1013, 1×1014, and 3×1014 vg/kg). Scale bar, 80 µm. (See also Appendix Table S1)",
    "molecules": "PAS"
  },
  {
    "caption": "(A) Substrate-dependent nitrogenase activity, measured through reduction of acetylene into ethylene, in isolated B. diazoefficiens bacteroids upon supplementation of malate, succinate, arginine, and nodule extract. Data represents the mean and standard error of the mean of at least eight independent replciates.",
    "molecules": "acetylene, arginine, ethylene, malate, succinate"
  },
  {
    "caption": "(B) Fold change in intracellular ATP level in isolated B. diazoefficiens bacteroids upon supplementation of succinate, arginine, and nodule extract. The values obtained for each of the 4 individual measurements (points) together with the regression trajectory of each experiment is shown.",
    "molecules": "arginine, ATP, succinate"
  },
  {
    "caption": "(C) Recombinant enzymes involved in arginine catabolism were combined into a single reaction mixture using arginine and pyruvate as substrates. Substrate (S) consumption into the products (P) succinate and alanine, including their intermediates (I1,I2), was determined along the time series using LC-MS/MS.",
    "molecules": "alanine, arginine, pyruvate, succinate"
  },
  {
    "caption": "ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) for 30 minutes (B), followed by stimulation with IL-17 (500 ng/ml) for 2 hours. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < -1, - log10 Benjamini-Hochberg adjusted p-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with - log10 adjusted p-value > 100). Names of several significantly upregulated transcripts are indicated.",
    "molecules": "MRT67307"
  },
  {
    "caption": "F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 hours and induction of mRNA for selected genes was analyzed by real time PCR. Mean + SEM from five independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.",
    "molecules": "MRT67307"
  },
  {
    "caption": "G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting.",
    "molecules": "MRT67307"
  },
  {
    "caption": "D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.",
    "molecules": "7-oxozeanol, MRT67307"
  },
  {
    "caption": "E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band.",
    "molecules": "MRT67307"
  },
  {
    "caption": "F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.",
    "molecules": "MRT67307"
  },
  {
    "caption": "TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting.",
    "molecules": "MRT67307"
  },
  {
    "caption": "TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.",
    "molecules": "MRT67307"
  },
  {
    "caption": "E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.",
    "molecules": "MRT67307, TPCA1"
  },
  {
    "caption": "ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.",
    "molecules": "alanines"
  },
  {
    "caption": "ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.",
    "molecules": "alanines"
  },
  {
    "caption": "A, Representative immunofluorescence images showing the subcellular distribution of AGO1x (red) relative to nuclear and cytoplasmic markers. DAPI was used to mark the nucleus (blue). The co-stained subcellular marker is indicated in each panel in green. SC 35, Lsm4, α-Tubulin, ERP72, p54 (NRB), and Nucleolin serve as markers for nuclear speckles, cytosol and nucleus, cytosol, endoplasmic reticulum, paraspeckle and nucleolus, respectively.",
    "molecules": "DAPI"
  },
  {
    "caption": "F, Representative result of the apoptosis assay using AnnexinV and propidium iodide (PI) staining in the control and the two mutant cell lines (left). The percentage of cells in each quadrant is depicted for each cell line. Quantification of the mean (+/- s.d.) percentage of apoptotic cells (Q1+Q2, AnnexinV+) across the different cell lines (n=4) (right). Shown is the P-value determined by unpaired two-tailed t-test.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "G, Impedance-based mean (+/- s.d.) cell index values at the indicated time points of growth after seeding equal numbers of control, W1A and W6A cells, upon treatment with DMSO or Ruxolitinib. For the DMSO treatment: Control (n=4), W1A (n=3) and W6A (n=3). For the Ruxolitinib treatment: Control (n=6), W1A (n=3) and W6A (n=4). From 6 hours after DMSO treatment, there is a statistically significant difference between control and the two mutants (P < 0.05, two tailed t-test), whereas no statistically significant difference is found after Ruxolitinib treatment.",
    "molecules": "DMSO, Ruxolitinib"
  },
  {
    "caption": "F, Quantification of the relative abundance (mean+/- s.e.m.) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane, non-overlapping foci were counted from 100 cells of each type, and the numbers in three abundance bins (<1, 1-5, >5) are shown.",
    "molecules": "dsRNA"
  },
  {
    "caption": "G, Representative immunofluorescence image of the control and mutant MDA-MB-231 cells stained with AGO1x (green) and J2 antibody (red) and DAPI (blue) to mark the nucleus. Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR-induced mutations.",
    "molecules": "DAPI"
  },
  {
    "caption": "a representative immunofluorescence image of siPNPT1/siControl-treated control and W6A mutant MDA-MB-231 cells stained with J2 antibody (red). DAPI was used to mark the nucleus (blue) (G). The right-most panels show a magnification of the cells enclosed by the white boxes in the middle panels.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) -(D) DCA treatment induces a cholestatic response in primary hepatocytes. Gene expression analysis of genes involved in bile acid metabolism and transport in primary hepatocytes treated with DMSO or 200 µM DCA for 16h. Represented are mean Cq values normalized to Gapdh expression (N=4 biological replicates). One sample t-test *P < 0.05, ***P < 0.001. (C) Abcb11/Bsep, (D) Cyp7a1/Cyp7. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.",
    "molecules": "bile acid, DCA, DMSO"
  },
  {
    "caption": "(I-J) DCA treatment in 3D hepatocyte organoids also induce canalicular dilation and rosette-like canaliculi and Sox9 expression. Hepatocyte organoids were treated with DMSO or 200 µM DCA for 24h (N=3 biological replicates). (I) Maximum projection of full organoids stained for F-actin (green). Scale bar 50 µm. Inset A and B. Maximum projection of zoomed-in parts of the bile canaliculi network in DMSO-treated (A) and DCA-treated (B) hepatocyte organoids stained with F-actin (green), nuclei (white) and the tight junction protein ZO-1 (yellow). Scale bar 10 µm. (J) High-resolution microscopy of individual bile canaliculi in DMSO-treated and DCA-treated hepatocyte organoids stained for F-actin (green). Red arrows indicate apical bulkheads. Scale bar 2 µm.",
    "molecules": "DCA, DMSO"
  },
  {
    "caption": "(K) Maximum projection of F-actin (green) and nuclei (white), and the bile duct cell marker Sox9 (magenta) in DMSO-treated and DCA-treated hepatocyte organoids (N=3 biological replicates). Untreated bile duct cell organoids serve as positive control.Scale bar 50 µm.",
    "molecules": "DCA, DMSO"
  },
  {
    "caption": "(A)Rosette-like canaliculi with reduced number of apical bulkheads can emerge from usual canaliculi with accumulated apical bulkheads. Timelapse imaging with brightfield phase-contrast of primary hepatocytes either untreated (top row), DMSO-treated (middle row) or DCA-treated (bottom row). An image was acquired every 15 minutes. Representative images are shown (N=3 biological replicates). Inserts show magnification of bile canaliculi with red arrows pointing towards apical bulkheads. Red stars mark areas in between apical bulkheads that markedly bulge out. Scale bar 10 µm.",
    "molecules": "DCA, DMSO"
  },
  {
    "caption": "(B) Pharmacological inhibition of canalicular water influx can prevent canalicular dilation upon DCA treatment. Confocal microscopy of CD13 (magenta), F-actin (green) and nuclei (white) in primary hepatocytes treated with DMSO and 200 µM DCA with 200 µM phloretin and 1 µM ouabain for 16h. Scale bar 10 µm. Representative images are shown.",
    "molecules": "DCA, DMSO, ouabain, phloretin"
  },
  {
    "caption": "(C) Quantification of the mean bile canaliculi volume in µm3of primary hepatocyte treated with DMSO and 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h (N=4 biological replicates). Unpaired t-test ***P < 0.001. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.",
    "molecules": "DCA, DMSO, Ouabain, Phloretin"
  },
  {
    "caption": "(D)- (E) Pharmacological inhibitors do not affect key bile acid signalling pathways. Gene expression analysis of different genes involved in bile acid signalling in primary hepatocytes treated with DMSO or 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h. Represented are mean Cq values normalized to Gapdh expression and whiskers show min and max values (N=5 biological replicates). Unpaired t-test. (D) Cyp7a1/Cyp7, (E) Nr0b2/Shp. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.",
    "molecules": "bile acid, DCA, DMSO, Ouabain, Phloretin"
  },
  {
    "caption": "C) Numbers of CTLs (gated on CD8+ T cells) from Ptpn6/11fl/fl or GzmBcre Ptpn6/11fl/fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells.",
    "molecules": "DAPI"
  },
  {
    "caption": "G) Graph illustrates the percentage of DAPI+ dead cells of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (gated on CD8+ T cells) at the indicated days as measured by flow cytometry.",
    "molecules": "DAPI"
  },
  {
    "caption": "Proteoliposomes  containing human ADP/ATP  carrier  were loaded with either 0.10 mM (red traces), 0.25 mM (orange traces), 0.50 mM (green traces), 1.00 mM (light blue traces) or 2.50 mM (dark blue traces) ATP  (ATPint), and transport  was initiated by the externally added radiolabeled  [33P]-ATP  (ATPext) at either 0.50 μM (filled circles), 0.67 μM (filled squares), 1.00 μM (filled upward triangles), 1.50 μM (filled downward triangles), 2.50 μM (open circles), 5.00 μM (open squares) or 15.0 μM (open upward triangles). A schematic representation of the  transport assays is shown. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for the assays with 0.10, 0.25, 0.5 and 1.0 mM of internal ATP, and one biological repeat (based on the average of three technical repeats) for the 2.50 mM ATP  internal assay.",
    "molecules": "ATP, 33P"
  },
  {
    "caption": "Initial rates were estimated by fitting the data with the model in Equation 1. A. Michaelis-Menten plots of 0.10 mM (red traces), 0.25 mM (orange traces), 0.50 mM (green traces), 1.00 mM (light blue traces) and 2.50 mM (dark blue traces) internally-loaded ATP. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.",
    "molecules": "ATP"
  },
  {
    "caption": "Initial rates were estimated by fitting the data with the model in Equation 1. B. Lineweaver-Burk analysis of [33P]-ATP/ATP  homo-exchange (same color scheme as A). Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.",
    "molecules": "ATP, 33P"
  },
  {
    "caption": "Initial rates were estimated by fitting the data with the model in Equation 1. C. Km  plotted against Vmax  for the various substrate gradients. The kinetic  parameters were determined by fitting of the Michaelis-Menten curves through iteration. Data information: The data are represented by the average and standard deviation of three biological repeats (each the average of three technical repeats) for all internal concentrations but 2.50 mM ATP, which is the average of three technical repeats.",
    "molecules": "ATP"
  },
  {
    "caption": "(E) AH109 cells were transformed with two yeast two‐hybrid assay plasmids, AD and BD, which encode the indicated domains fused with Atg30, Atg32 and Atg8 or an empty vector, as negative controls and grown on +His and −His+40 mM 3‐AT plates.",
    "molecules": "3‐AT, His"
  },
  {
    "caption": "(G) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate‐induced peroxisomes after shifting cells to SD‐N. aa, amino acid; AD, activation domain; AIM, Atg8‐family‐interacting motif; AOX, alcohol oxidase; BD, binding domain; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; λPP, λ protein phosphatase.",
    "molecules": "oleate"
  },
  {
    "caption": "(B) Localization of GFP-Atg11 during pexophagy of methanol‐induced peroxisomes in cells expressing WT or mutant Atg30 proteins. White arrows indicate correct localization and yellow arrows indicate mislocalization, or in case of Atg8 localization, indicate absence of phagophore membrane elongation. Peroxisomes were labelled with BFP-SKL and vacuoles with FM4‐64. Scale bar, 5 μm. GFP, green fluorescent protein; WT, wild type.",
    "molecules": "methanol"
  },
  {
    "caption": "(E) S. cerevisiae Δatg36 cells (ΔScatg36) expressing thiolase-GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid‐log growth and then shifted to SD‐N. Pexophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies.",
    "molecules": "oleate"
  },
  {
    "caption": "Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses, immunolabled for BDNF (green), SCG2 (red) and counterstained with DAPI (blue) in orthogonal views (dashed lines) showing colocalization of BDNF and SCG2 in the soma (white arrows).",
    "molecules": "DAPI"
  },
  {
    "caption": "BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.",
    "molecules": "KCl"
  },
  {
    "caption": "Representative photomicrographs of a CamKIIaCRE;Tardbpfx/fx hippocampus after stereotaxic injection of EGFP-expressing lentivirus immunostained for EGFP (green) and NeuN (red), and counterstained with DAPI (blue). The injected area within CA1 is indicated in the merged panel.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative photomicrographs of cultured hippocampal neurons immunostained for TDP-43 (green), FLAG (red) and counterstained with DAPI (blue) in control conditions (untransduced) or infected with lentivirus expressing FLAG-tagged human TDP-43 12xQN construct.",
    "molecules": "DAPI"
  },
  {
    "caption": "BDNF secretion induced by KCl treatment as assessed by in-situ BDNF ELISA in cultured hippocampal neurons untransduced (control) or infected with lentiviruses expressing Tardbp shRNA or human TDP-43 12xQN constructs. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis",
    "molecules": "KCl"
  },
  {
    "caption": "BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs as indicated. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; ***, p<0.001 Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.",
    "molecules": "KCl"
  },
  {
    "caption": "BDNF secretion induced by KCl treatment in cultured hippocampal neurons infected with Tardbp shRNA and mutant human TARDBP lentivirus constructs together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=5 independent experiments; n=5 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.",
    "molecules": "KCl"
  },
  {
    "caption": "Expression of TARDBP (left), total SORT1 (center) and exon 17b SORT1 (right) mRNAs quantified by Q-PCR in human neurons derived from the indicated stem cells carrying either wild type Met337 (grey bars) or disease-associated Val337 (black bars) TARDBP alleles. Results are presented as average ± SEM (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.",
    "molecules": "Met, Val"
  },
  {
    "caption": "BDNF secretion in stem-cell derived human neurons carrying wild type Met337or Val337 TARDBP alleles, infected with proBDNF lentivirus 5 days prior to depolarization by KCl treatment. Results are presented as average ± SEM (N=3 independent experiments; n=5 wells per condition in each experiment). **, p<0.01; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.",
    "molecules": "Met, KCl, Val"
  },
  {
    "caption": "Left panel, growth of HCT116 VPS4B-/- shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n=9 for each group, each mouse bearing one tumor, ±SEM. Two-tailed unpaired t-test; ns - non-significant (p≥0.05), **p<0.01. Right panel, scatter plot representing end-point volumes of single xenografts. Bars represent means ±SEM.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline-treated mice (6 separate xenograft samples for each group). Lysates of HCT116 VPS4B-/- cells transfected with control or VPS4A-targeting siRNA marked VPS4A protein detection. GAPDH served as a loading control.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Growth of HCT116 VPS4B-/- shCTRL#1 cells as xenografts in mice in the presence or absence of doxycycline. The arrow indicates the first day of doxycycline (Dox) administration. n=2 mice for each group, each mouse bearing two tumors, ±SEM.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "Analysis of the impact of RIPK1 inhibitor (necrostatin-1) or pan-caspase inhibitor (Q-VD-Oph) on cell viability of HCT116 VPS4B-/- cells transfected with siRNA (non-targeting siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Cell viability was assessed 72 h after siRNA transfection. Necrostatin-1 (50 µM), Q-VP-Oph (20 µM) or vehicle were added to the medium 48 h before viability assessment. Data are means of 5 independent experiments ±SEM. All values were normalized, viability of siCTRL-transfected and vehicle-treated cells was set as 100%. Two-tailed unpaired t-test; **p<0.01.",
    "molecules": "necrostatin-1, Necrostatin-1, Q-VP-Oph, Q-VD-Oph"
  },
  {
    "caption": "Measurement of ATP released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.",
    "molecules": "ATP"
  },
  {
    "caption": "Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm.",
    "molecules": "DAPI, mitoxantrone"
  },
  {
    "caption": "Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A-depleted HCT116 VPS4B-/- cells 66 h after siRNA transfection (siVPS4A duplexes: #2, #4 or #5 were used). Non-transfected (NT) or siCTRL#1-transfected cells served as negative controls. Left panel, percentage of cells positive for calreticulin in the population of live (DAPI-negative) cells, data are means of 4 independent experiments ±SEM. The Mann-Whitney U test; **p<0.01. Right panel, representative dot plot diagrams of flow cytometric analysis of cell surface-exposed calreticulin. Primary rabbit anti-calreticulin and control isotype IgG antibodies were used for staining, followed by secondary AlexaFluor 647-conjugated antibody.",
    "molecules": "AlexaFluor 647, DAPI"
  },
  {
    "caption": "a, b, THP1 cells were stimulated with rotenone (10 µM), TTFA (10 µM) or antimycin (40 µg ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry.",
    "molecules": "TTFA, antimycin, rotenone"
  },
  {
    "caption": "c, THP1 cells were stimulated for 6 h with the indicated amounts of rotenone, TTFA or antimycin. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated.",
    "molecules": "TTFA, antimycin, rotenone"
  },
  {
    "caption": "d, LPS-primed bone-marrow-derived macrophages (BMDMs) from wild-type (WT), Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU (150 µg ml−1), rotenone (10 µM for THP1 cells and 40 µM for BMDMs) or antimycin (40 µg ml−1 for THP1 cells and 10 µg ml−1 for BMDMs). The release of caspase-1 (western blot) was then determined. Data shown are representative of three independent experiments.",
    "molecules": "antimycin, LPS, rotenone, MSU"
  },
  {
    "caption": "a, BMDMs expressing GFP-LC3 were stimulated with rotenone (40 µM) for 3 h and the co-localization of mitochondria and GFP-LC3 dots were analysed using confocal microscopy.",
    "molecules": "rotenone"
  },
  {
    "caption": "b, THP1 cells stimulated with 3-methyladenine (3-MA, 10 mM) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min, and analysed by flow cytometry.",
    "molecules": "3-MA, 3-methyladenine"
  },
  {
    "caption": "c, LPS-primed BMDMs from wild-type, Nlrp3−/−, Asc−/− or Ipaf −/− knockout mice were stimulated with MSU (150 µg ml−1) or 3-MA (10 mM) for 6 h and the release of active caspase-1 and IL-1b was determined.",
    "molecules": "3-MA, LPS, MSU"
  },
  {
    "caption": "e, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h. Data shown are representative of three independent experiments.",
    "molecules": "nigericin, MSU"
  },
  {
    "caption": "e, THP1 cells were stimulated with MSU or nigericin and then fractionated. The cytosolic, ER, MAM and mitochondrial fractions were analysed by western blotting as indicated in the text.",
    "molecules": "nigericin, MSU"
  },
  {
    "caption": "f, THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e.",
    "molecules": "nigericin"
  },
  {
    "caption": "g, TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS-dependent manner. THP1 cells were stimulated with MSU and nigericin, and TXNIP localization was investigated in the presence or absence of the antioxidant APDC. Data shown are representative of two or three independent experiments. Scale bars: a-g, 20 µm.",
    "molecules": "APDC, nigericin, ROS, MSU"
  },
  {
    "caption": "b, THP1 cells expressing shRNA against VDAC2 were stimulated with MSU, R837, silica, alum or nigericin and analysed for IL-1b secretion.",
    "molecules": "alum, R837, nigericin, silica, MSU"
  },
  {
    "caption": "c, THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin, and NLRP3 localization was analysed by confocal microscopy. Scale bars: 20 µm.",
    "molecules": "nigericin, MSU"
  },
  {
    "caption": "(g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "(h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student's t-test.",
    "molecules": "insulin"
  },
  {
    "caption": "(i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student's t-test",
    "molecules": "triglycerol"
  },
  {
    "caption": "(j) Plasma leptin levels in WT and Atg5 Tg mice (n=8-12). ***P0.0001 versus control; Student's t-test. (",
    "molecules": "leptin"
  },
  {
    "caption": "(k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student's t-test.",
    "molecules": "GSH, GSSG"
  },
  {
    "caption": "(b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin.",
    "molecules": "Baf.A1, rapamycin"
  },
  {
    "caption": "(c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student's t-test.",
    "molecules": "3-MA, Baf.A1, H2O2"
  },
  {
    "caption": "(e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm.",
    "molecules": "H2O2"
  },
  {
    "caption": "(f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.",
    "molecules": "Baf.A1, H2O2"
  },
  {
    "caption": "A) H1299 cell line was transfected with increasing concentrations of MDM2, and the RB+5´UTR exogenous (left panel) and endogenous (right panel) levels were evaluated. B) Evaluation of the RB expression as in (A) but inducing DNA damage with doxorubicin 1 µM during 16 h. ",
    "molecules": "doxorubicin"
  },
  {
    "caption": "C) Evaluation of the RB levels in an MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line. D) Evaluation of the RB levels in a MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line. ",
    "molecules": "doxorubicin"
  },
  {
    "caption": "A) Evaluation of RB mRNA levels, normalised by GAPDH, in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Endogenous levels (upper panel); exogenous levels (middle panel); endogenous levels of RB but using the MDM2(S395D) under normal conditions (lower panel).",
    "molecules": "doxorubicin"
  },
  {
    "caption": "B) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h. The cells were treated with cichlohexamide (CHX) was added to stop translation, under normal conditions (upper panel) and treated with doxorubicin (lower panel). The cells were transfected with MDM2.",
    "molecules": "CHX, cichlohexamide, doxorubicin"
  },
  {
    "caption": "C) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence (upper panel) or in absence (lower panel) of CHX.",
    "molecules": "CHX"
  },
  {
    "caption": "D) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX.",
    "molecules": "CHX, doxorubicin"
  },
  {
    "caption": "Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. A) Evaluation of the small pocket expression in an MDM2 dose-dependent manner. B) Cells were transfected with the RB large pocket construct. ",
    "molecules": "doxorubicin"
  },
  {
    "caption": "Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. C) Cells were transfected with RB full length without the 5´URT (named RB-HA). D) Cells were transfected with the RB full-length plus 260 nt of its own 5´ UTR (named RB+5´UTR). ",
    "molecules": "doxorubicin"
  },
  {
    "caption": "E) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP. Cells were treated with doxorubicin (1 µM for 16 h) or DMSO.",
    "molecules": "DMSO, doxorubicin"
  },
  {
    "caption": "G) PLA assay using anti-RB and anti-MDM2 antibodies, H1299 cell were treated or not with etoposide to induce stress, the endogenous proteins were visualised. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the two proteins. The scale bar corresponds to 20 μm.",
    "molecules": "DAPI, etoposide"
  },
  {
    "caption": "H) RNA-PLA assay using rabbit anti-MDM2 and a mouse biotin-antibody. H1299- RBKO was stable transfected with RB-HA or RB+5´UTR. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the protein and the mRNA. The scale bar corresponds to 20 μm.",
    "molecules": "biotin, DAPI"
  },
  {
    "caption": "A) Cell cycle analysis (FACS) of C33 cell line after treatment with etoposide. The DNA contents of the cells were evaluated by propidium iodide staining, (PE-A; Phycoerythrin). The inset shows the % of the cells in each cell cycle phase.",
    "molecules": "DNA, etoposide"
  },
  {
    "caption": "C) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide, (EV; empty vector). D) The same as in (C) but under normal cellular conditions.",
    "molecules": "etoposide"
  },
  {
    "caption": "E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress.",
    "molecules": "Etoposide, nocodazole"
  },
  {
    "caption": "F) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment. Data information: Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).",
    "molecules": "nocodazole"
  },
  {
    "caption": "D) A representative clonogenic assay. Increasing concentrations of MDM2 were transfected in H1299-RBKO stable transfected with RB-HA or RB+5´UTR. The cells were treated with doxorubicin to induce genotoxic stress conditions. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05).",
    "molecules": "doxorubicin"
  },
  {
    "caption": "E) Evaluation of cyclin E mRNA levels in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, ***p< 0.001).",
    "molecules": "doxorubicin"
  },
  {
    "caption": "(b) Wild type or STG-p62 Hap1 cells were mock or puromycin treated for 2 hours and stained for ubiquitin. The endogenous p62 was stained with an anti-p62 antibody in the parental Hap1 cells, while the fluorescence of the GFP tag was recorded in STG-p62 cells. Arrowheads indicate co-localizing puncta",
    "molecules": "ubiquitin, puromycin"
  },
  {
    "caption": "(c) Quantification of cluster formation assays conducted with the indicated proteins. mCherry-p62 was pre-incubated with the indicated 4xUb variants, followed by the addition of streptavidin immediately before imaging",
    "molecules": "streptavidin"
  },
  {
    "caption": "(d) Representative micrographs of the indicated samples taken 60 minutes after addition of streptavidin",
    "molecules": "streptavidin"
  },
  {
    "caption": "(g) Quantification of cluster formation assays conducted with GFP-p62 and the indicated GST-tagged ubiquitin chains",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(h-i) Quantification of cluster formation assays performed with mCherry-p62 and GST-4xUb in presence of the indicated free ubiquitin chains. Ubiquitin chains were either pre-mixed with p62 before the addition of GST-4xUb (h), or added 3 minutes after the addition of GST-4xUb to p62 (i)",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(j) Quantification of cluster formation assays conducted with mCherry-2xFKBP-p62 in presence of AP20187, GST-4xUB and the indicated Ub chains",
    "molecules": "AP20187"
  },
  {
    "caption": "(c) Top left: Western blots of lysates of CRISPR/Cas9 genome-edited STG-p62 wild type and LIR mutant cells. Clone Lir1B was used for the experiments shown in Figure 6c. Bottom left: Representative pictures of STG-p62 WT or LIR mutant Hap1 cells treated with Puromycin for 90 minutes and stained with anti-Ub conjugates antibody (FK2). Right: quantification of cytoplasmic p62 puncta per cell (top), and of their co-localization upon puromycin treatment (bottom). Averages and SDs from three independent experiments are shown. In total, at least 1440 number of cells were counted per condition",
    "molecules": "Puromycin, puromycin"
  },
  {
    "caption": "(c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control.",
    "molecules": "DMSO, purmorphamine"
  },
  {
    "caption": "(d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph.",
    "molecules": "bafA1, purmorphamine, trehalose"
  },
  {
    "caption": "(e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected.",
    "molecules": "bafA1, DMSO, purmorphamine"
  },
  {
    "caption": "(f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio.",
    "molecules": "purmorphamine"
  },
  {
    "caption": "(g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS-polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.",
    "molecules": "purmorphamine"
  },
  {
    "caption": "(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph.",
    "molecules": "bafA1"
  },
  {
    "caption": "(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) Quantification and statistical analysis is shown in graphs.",
    "molecules": "trehalose"
  },
  {
    "caption": "(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs",
    "molecules": "bafA1"
  },
  {
    "caption": "(f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test: **P0.01; *P0.05; NS, not significant.",
    "molecules": "bafA1"
  },
  {
    "caption": "(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.",
    "molecules": "Pmph, trehalose"
  },
  {
    "caption": "(b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.",
    "molecules": "Pmph, trehalose"
  },
  {
    "caption": "(c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control.",
    "molecules": "purmorphamine"
  },
  {
    "caption": "(d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager.",
    "molecules": "purmorphamine"
  },
  {
    "caption": "(e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. tubulin-II and tubulin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.",
    "molecules": "bafA1, DMSO, purmorphamine"
  },
  {
    "caption": "In vitro pull down of FlagSLC25A51 variants using cardiolipin-bound or control beads and detected with anti-Flag Western Blots.",
    "molecules": "cardiolipin"
  },
  {
    "caption": "Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 25 biological replicates), wildtype FlagSLC25A51 (n = 25 biological replicates), and indicated mutants (n = 4 - 6 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p <0.0001 with post-hoc Dunnett's test compared to empty vector control, *p < 0.05, **p < 0.01. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants using anti-Flag western blot; HSP60, loading control.",
    "molecules": "NAD+"
  },
  {
    "caption": "Quantitation of uptake of 32P-NAD+ after 1 hour by E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) and the indicated mutants; wildtype activity and equivalent, red. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 5 - 8 biological replicates, ANOVA p <0.0001 with post-hoc Dunnett's test compared to wildtype, ***p < 0.001.",
    "molecules": "NAD+, 32P"
  },
  {
    "caption": "Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells co-expressing empty vector control (n = 8 biological replicates), wildtype FlagSLC25A51 (n = 8 biological replicates), and indicated mutants (n = 4 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p < 0.0001 with post-hoc Dunnett's test compared to empty vector control ***p < 0.001. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants using anti-Flag western blot; HSP60, loading control.",
    "molecules": "NAD+"
  },
  {
    "caption": "Relative uptake of 32P-NAD+ after 1 hour by E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) or the E132A mutant when competed by 100 μM (n = 3 biological replicates) and 250 μM (n = 4 biological replicates) unlabeled NAD+ (red) or NADH (black). Data is shown as floating bars with central line at mean and the box represents the min and max value, unpaired two-sided t-test *p < 0.05.",
    "molecules": "NAD, NAD+, NADH, 32P"
  },
  {
    "caption": "Quantitation of 32P-NAD+ uptake after 1 hour in E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) and indicated mutants; wildtype activity (n = 19 biological replicates) and equivalent, red. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 5 - 8 biological replicates, ANOVA p <0.0001 with post-hoc Dunnett's test compared to wildtype ***p < 0.001.",
    "molecules": "NAD+, 32P"
  },
  {
    "caption": "Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 27 biological replicates), wildtype FlagSLC25A51 (n = 27 biological replicates), and indicated mutants (n = 4 - 7 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p < 0.0001, post-hoc Dunnett's test compared to empty vector control ***p < 0.001. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants detected by anti-Flag western blot; HSP60, loading control.",
    "molecules": "NAD+"
  },
  {
    "caption": "Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HEK293 SLC25A51 KO cells expressing empty vector control, wildtype FlagSLC25A51, and indicated mutants. Measurements were taken at 48 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 4 - 6 biological replicates, ANOVA p < 0.0001, post-hoc Dunnett's test compared to empty vector control **p < 0.01, ***p < 0.001. (bottom) Protein expression from HEK293 SLC25A51 KO cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants detected with anti-Flag western blot; HSP60, loading control.",
    "molecules": "NAD+"
  },
  {
    "caption": "EVs were purified from either schistosomal-growing medium (supernatant) or unused schistosomal-medium as control. Both pellets were stained using Thiazole Orange. Purified labeled ~5 × 106 EVs were incubated with 5 × 105 freshly purified Th cells that were stimulated with anti-CD3 and anti-CD28 antibodies for 10 minutes. Images from the same slide were taken by inverted confocal microscopy at the indicated time points. The arrows in the enlarged images point toward cells labeled with EVs. The scale bars in all images represent 20μm. The percentage of labeled cells was calculated by dividing the number of labeled cells by the total numbers of cells in the same image. The mean +/- SEM was calculated from 3 independent images",
    "molecules": "Thiazole Orange"
  },
  {
    "caption": "(H) Whole blood Hb1Ac levels in 16 week old Abca12tm1d mice (mean±SEM, n=4-9 mice per group, **p<0.01 Students t-test).",
    "molecules": "Hb1Ac"
  },
  {
    "caption": "(I) Acute phase insulin secretion after intraperitoneal injection of glucose (mean±SEM, n=4-9 animals per group, white/grey/black = Abca12+/+, cre/+ and Abca12tm1d respectively, mice at 8 weeks of age, **p<0.01 Students t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "(J) Silencing of ABCA12 in MIN6 cells as assessed by Western blot of ABCA12 immuno-precipitate (top panel) or confocal microscopy in cells transfected with Dy547-siRNAABCA12 (lower panel, scale bar = 10μm).",
    "molecules": "Dy547"
  },
  {
    "caption": "(K) Glucose stimulated insulin secretion (GSIS) from MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate, ***p<0.0001, Students t-test).",
    "molecules": "Glucose"
  },
  {
    "caption": "(E) Total cholesterol (COH) or cholesteryl esters (CE) levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=9, 5 and 5 mice per genotype respectively).",
    "molecules": "cholesterol, COH, CE, cholesteryl esters"
  },
  {
    "caption": "(F) Ceramides levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=10, 4 and 5 mice per genotype respectively).",
    "molecules": "Ceramides"
  },
  {
    "caption": "(G) Lipid levels in MIN6 cells in which ABCA12 had been depleted by siRNA treatment (mean±SEM, n=5 (biological replicates assayed in triplicate), FC = free cholesterol, CE = cholesterol ester, Cer = ceramide, SM = sphingomyelin, TG = triacylglycerol, PC = phosphatidylcholine, *p<0.05, **p=<0.01, Students t-test).",
    "molecules": "Cer, ceramide, FC, free cholesterol, CE, cholesterol ester, Lipid, PC, phosphatidylcholine, SM, sphingomyelin, TG, triacylglycerol"
  },
  {
    "caption": "(H) Cholesterol efflux to lipid-free apoA-I (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM)(mean±SEM, n=4, (biological replicates assayed in triplicate), *p=<0.05, Students t-test).",
    "molecules": "Cholesterol, lipid, TO901317"
  },
  {
    "caption": "(I) Cholesterol efflux to HDL (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM); (mean±SEM, n=4 (biological replicates assayed in triplicate)).",
    "molecules": "Cholesterol, HDL, TO901317"
  },
  {
    "caption": "(M) The effect of activation of cells with TO901317 (4 μM) on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test).",
    "molecules": "TO901317"
  },
  {
    "caption": "(C) Circulating glucose levels after administration of intraperitoneal L-arginine in mice at 8 weeks of age, mean±SEM, n =3 mice per genotype, *p=<0.05 of cre relative to tm1d, Students t-test).",
    "molecules": "glucose, L-arginine"
  },
  {
    "caption": "(D) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 15mM glucose (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the lower panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01, ***p=<0.001 of cre relative to tm1d, Students t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "(E) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 20mM KCl (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the right panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01 of cre relative to tm1d, Students t-test).",
    "molecules": "KCl"
  },
  {
    "caption": "(F) Assessment of calcium flux measured by Fluo4 in response to either Glucose or Potassium in isolated islets from 11 week old animals. Fluorescence responses were corrected for basal fill (F/F0). (mean±SEM, each data point represents the average of 10 cells/coverslip for 13-16 coverslips for each of 4 wild type and 5 Abca12tm1d mice)",
    "molecules": "calcium, Fluo4, Glucose, Potassium"
  },
  {
    "caption": "(F) Assessment of total cholesterol (COH) by mass spectroscopy in mice fed normal chow or a high cholesterol diet (HCD)(mean±SEM, n=5 and 4 animals respectively, **p=<0.01, Students t-test).",
    "molecules": "cholesterol, COH"
  },
  {
    "caption": "(J) Glucose tolerance test results following intraperitoneal glucose challenge in Abca12tm1d and cre control mice fed normal chow or HCD (mean±SEM, n=3 mice per genotype, *p=<0.05, for tm1d versus tm1d+HCD, Students t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "(K) Serum insulin in Abca12tm1d +/- HCD mice after i.p glucose challenge (mean±SEM, n=3 mice/genotype, **p=<0.01, Students t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "(A) Detection of lipid rafts in 8 week old islets with recombinant cholera toxin subunit B (CT-B) conjugated to Alexa Fluor 488 (green). Islets are co-stained with DAPI (blue) (scale bar = 50μm.).",
    "molecules": "Alexa Fluor 488, DAPI"
  },
  {
    "caption": "(C) Confocal images of rafts (using CTB-Alexa Fluor 647) in MIN6 cells transfected with Fluorescein-488-labelled siRNAScr or Dy547-labelled siRNAABCA12 (scale bar=10μm). (D) Quantitation by confocal microscopy of the effect of ABCA12-deficincy on CTB binding to MIN6 cells (mean±SEM, *p=<0.05, Students t-test, n=4 biological replicates, 50-100 cells for each). (E) Quantitation by flow cytometry of the effect of ABCA12-deficiency on CTB binding to MIN6 cells (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test). ",
    "molecules": "Dy547, Fluorescein-488, Alexa Fluor 647"
  },
  {
    "caption": "(F) The effect 10 mM MβCD on GSIS in MIN6 cells depleted or not for ABCA12 (incubation for 5 or 10 minutes as indicated, mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test).",
    "molecules": "MβCD"
  },
  {
    "caption": "(H) The effect of ABCA12 deficiency on activation of CDC42 by bradykinin in MIN6 cells (BDK, 100 ng/ml, 4 min; *p=<0.05, Students t-test, significance versus ABCA12-deficient BDK activated cells is indicated, n=4 biological replicates, mean±SEM).",
    "molecules": "BDK, bradykinin"
  },
  {
    "caption": "(Q) Effects of Jasplakinolide on F-actin levels (green) in siRNA treated cells (scale bar=10μm).",
    "molecules": "Jasplakinolide"
  },
  {
    "caption": "(R) Effects of Abca12 knockdown and Jasplakinolide treatment on MIN6 cell in low and high glucose (mean±SEM, n=4 biological replicates, *p=<0.05, **p=<0.01, significance relative to untreated cells, Students t-test)",
    "molecules": "glucose, Jasplakinolide"
  },
  {
    "caption": "(A) Mass spectroscopy analysis of levels of cholesterol and cholesteryl esters in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively; COH=cholesterol, CE=cholesteryl ester).",
    "molecules": "cholesterol, COH, CE, cholesteryl ester, cholesteryl esters"
  },
  {
    "caption": "(B) Mass spectroscopy analysis of lipids families in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, **p=<0.01 Students t-test); CE=cholesteryl ester, Cer=ceramide, DHC (Hex2Cer)=dihexosylceramide, MHC (HexCer)=monohexosylceramide, SM=sphingomyelin, GM3=GM3 ganglioside, dhCer=dihydroceramide, DG=diacylglyerol, TG=triacylglycerol).",
    "molecules": "DHC, dihexosylceramide, Hex2Cer, HexCer, MHC, monohexosylceramide, Cer, ceramide, CE, cholesteryl ester, DG, diacylglyerol, dhCer, dihydroceramide, GM3 ganglioside, GM3, lipids, SM, sphingomyelin, TG, triacylglycerol"
  },
  {
    "caption": "(C) Mass spectroscopy analysis of triglyceride species in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, *p=<0.05, **p=<0.01, Students t-test)",
    "molecules": "triglyceride"
  },
  {
    "caption": "Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing glucagon (H) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).",
    "molecules": "glucagon"
  },
  {
    "caption": "(J) Fasting blood glucose levels coincident with the emergence of inflammatory phenotypes (mean±SEM, n=3, 4 and 5 mice per genotype respectively, *p=<0.05, Students t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "  Bovine heart mitochondria were incubated at the indicated pH in absence or presence of 200 μM DPC, treated with 1% (w/v) digitonin and F-ATP synthase was immunoprecipitated with an anti-F-ATP synthase Ab followed by 15% SDS-PAGE. β subunit and CyPD content were detected by Western blotting. The ratio between CyPD and the corresponding β subunit is reported in the lower part of the panel, referring to pH 7.4 values as 100%. Data are an average ± s.e. of 3 independent experiments.  ",
    "molecules": "DPC, digitonin"
  },
  {
    "caption": "B,C EDTA-SMP were solubilized at the indicated pH values, extracted with 1% (w/v) digitonin and subjected to BN-PAGE in order to separate dimers (Vd) and monomers (Vm), which were identified by Coomassie blue (panel B) or in-gel activity staining (panel C) and analyzed by densitometry, which is reported in the bottom part of each panel. Values report the dimer/monomer ratio, where the ratio at pH 7.4 was taken as 100%. Data are an average ± s.e. of 3 independent experiments. The p-values calculated with the Student t test are shown * p≤0.05 *** p≤0.001.",
    "molecules": "Coomassie blue, digitonin, EDTA"
  },
  {
    "caption": "D Sequence of peptides 95-113 of OSCP before (left panel) and after reaction with DPC (right panel) obtained from tryptic digests of the SDS-PAGE band corresponding to the expected molecular mass of OSCP in immunoprecipitated F-ATP synthase. Fragments of the series b and y identified in the LC-MS/MS analysis are indicated on the sequence of the peptides. H112 and H112CeT are indicated in red boldface. Ions y4-y17 show a mass shift of +72 Da in the modified peptide.",
    "molecules": "DPC"
  },
  {
    "caption": "Permeabilized HEK293T cells expressing wild type (WT) or edited (H112Q or H112Y) OSCP subunit were suspended in KSCN-based media at pH 7.4 (left traces) or pH 6.5 (right traces) and PTP opening was measured as the decrease in absorbance at 540 nm. Where indicated (arrows) 0.1 mM Ca2+ in the absence of further additions (black traces) or in the presence of 0.5 mM DPC (red traces) or 2 µM CsA (blue traces) was added. The bottom panel reports the difference between the absorbance readings before the addition of Ca2+ and at the end of the swelling phase. Data are mean ± s.e. of 5 experiments (triplicate samples) for Ca2+ alone (black bars) and Ca2+ + DPC (red bars) and of 3 experiments (duplicate samples) for Ca2+ + CsA (blue bars). Solid bars, pH 7.4, hatched bars, pH 6.5",
    "molecules": "CsA, Ca2+, DPC"
  },
  {
    "caption": "Mitochondria were prepared from cells, suspended in sucrose buffer at pH 7.4 (solid bars) or 6.5 (hatched bars) in the absence (black bars) or presence (blue bars) of 1.6 µM CsA, extracted with 1% (w/v) digitonin and immunoprecipitated with an F-ATP synthase antibody. The immunoprecipitates were subjected to 12% SDS-PAGE followed by Western blotting for CyPD and α subunit of F-ATP synthase. Each immunodetected band was analyzed by densitometry, the ratio between CyPD and α subunit was measured and expressed relative to the ratio obtained in absence of CsA at pH 7.4, which was taken as 100%. Data are average ± s.e. of 6 independent experiments.",
    "molecules": "digitonin, CsA"
  },
  {
    "caption": "HEK293T cells were incubated in a balanced salt solution containing 2-deoxyglucose at pH 7.4 (solid bars) or 6.5 (hatched bars) and anoxia induced by incubation in a BD GasPak EZ Anaerobe Gas-generating Pouch System (BD Biosciences). Cell death was measured based on LDH release (average ± s.e. from 8 independent experiments). In all panels the p values calculated with the Student t test are shown (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).",
    "molecules": "2-deoxyglucose"
  },
  {
    "caption": "Recordings as in panel B from mitoplasts of cells harboring the OSCP H112Q replacement. Left panel, pH 7.3; middle panel, 440 seconds after acidification to pH 6,5; right panel, 30 seconds after the addition of 5 mM Ba2+ at pH 6.5. Amplitude histograms were obtained from 100 second-long segments and the panels are from the same experiment. Note that both in panels B and C well-visible subconductance states can be detected in the amplitude histograms (peaks between open levels o2 and o3 on left panels). Gaussian fitting has been obtained using the Origin Program set. In all panels c, closed; o1, o2, o3 open states with discrete subconductance.",
    "molecules": "Ba2+"
  },
  {
    "caption": "A - DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and Rx-Dicer mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Time-dependent killing of a panel of bacterial isolates in the presence of meropenem (MEM10, 10 µg/ml). Cells were grown overnight and sub-cultured for 30 minutes at 37°C before being exposed to meropenem at 10 µg/mL and moved to a 37°C stationary incubator. Samples were taken at each indicated time point and spot-titered onto LB medium to quantify colony forming units (CFU/mL) throughout meropenem treatment. (B) images of cells corresponding to the time points in (A) were taken after labeling with membrane stain FM4-64. E. cloacae, Enterobacter cloacae; K. aerogenes, Klebsiella aerogenes; K. pneumoniae, Klebsiella pneumoniae; E. coli, Escherichia coli ",
    "molecules": "FM4-64, MEM, meropenem"
  },
  {
    "caption": "(D) Regulatory modes of representative VxrB-controlled genes in response to PenG. Upper plot shows VxrB binding patterns (assessed via ChIP-Seq), lower panel shows transcriptional response upon overexpression of VxrB D78E (assessed via RNA-Seq). RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG (100 µg/ml for 3 hours). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.",
    "molecules": "PenG"
  },
  {
    "caption": "Reduction of cell wall synthesis gene expression causes a tolerance defect. (C) Survival of a ∆pbp1b ∆pbp1a Piptg:pbp1a strain after 6 hours of PenG exposure. Pup indicates conditions of high IPTG concentrations (100 µM), Pdown low IPTG concentration (2 µM). Survival fraction is cfu/ml of PenG-treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration.",
    "molecules": "IPTG, iptg, PenG"
  },
  {
    "caption": "(D) Time-dependent killing experiment in the presence of PenG (100 µg/ml, 10 x MIC). All data are means (+/- standard error) of 3 independent biological replicates.",
    "molecules": "PenG"
  },
  {
    "caption": "(C) Time-course of iron accumulation in PenG-treated WT and ∆vxrAB cells as measured by ICP-MS. Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗P=0.001; ∗∗∗P<0.0003 (paired t-test).",
    "molecules": "iron, PenG"
  },
  {
    "caption": "(B). To test PenG-mediated induction of Fur-regulated genes, S1 nuclease mapping assays were performed on the small RNA ryhB, the RyhB target sodB, hutA/B, coding for heme transport systems and vc2212, which encodes a component of a ferric citrate uptake system). Cells were grown to mid-exponential phase followed by exposure to H2O2 or PenG for the indicated duration. Numbers represent expression levels normalized to untreated control (averages from three independent replicates). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.",
    "molecules": "H2O2, PenG"
  },
  {
    "caption": "(B) and (C) Iron uptake mutants restore ∆vxrAB growth on 10% sucrose. The indicated strains were grown to exponential phase (OD600~0.5) in LB medium and spot-plated on 10% Sucrose plates with or without additional iron sulfate. An example is shown in (B), quantification of cfu is shown in (C). Data information: Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗∗ P<0.0003; ∗∗∗∗ P<0.0001 (paired t-test).",
    "molecules": "Iron, iron sulfate, sucrose, Sucrose"
  },
  {
    "caption": "(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.",
    "molecules": "glucose, PenG"
  },
  {
    "caption": "(F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines). Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.",
    "molecules": "PenG, ROS"
  },
  {
    "caption": "A) Maximum intensity projections of pediatric (0-12 months) small intestine showing the distribution of CD56, CD90 and CD24 within the ENS. The first image of each row shows a low magnification overview and an inset depicting the DAPI channel in high magnification. The remaining panels correspond to the high magnification inset. Scale bar in overview images = 200 µm. Scale bar in high magnification panels = 40 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B) Representative plot showing full co-expression of TUBB3 (neuronal marker) and SOX10 (glial marker), on ENS cells (bottom) and only minimal and low-intensity background on non-ENS cells (Top) (N=1 representative sample, for stats see Figure 2C). Intracellular staining was performed on formalin fixed cells from ileum, after pre-staining for the extracellular markers. Selection of the ENS cluster in fixed cells is shown in Appendix Figure S2F.",
    "molecules": "formalin"
  },
  {
    "caption": "Confocal images of ENS events sorted as nucleated and non-nucleated, as shown in Figure. 3. (A) Most of the staining belongs to what appears to be non-nucleated (DAPI-) cellular debris (putative neuronal terminations) attached to a nucleated cell (putative glial cell). This is particularly true for TUBB3 and CD24, which show (as expected for neuronal selective markers) a starker distinction than CD56 (also expressed by glial cells). At a very low frequency among sorted ENS cells, we could observe putative neurons as represented in (B), with a more homogenous distribution of the three markers. (C) Examples of sorted Hoechst-negative ENS debris, which are confirmed to be non-nucleated (thus validating our gates on Hoechst 33342-A), but positive for all neuronal markers.",
    "molecules": "Hoechst, Hoechst 33342, DAPI"
  },
  {
    "caption": "(I-L) Control and IRGM knockdown THP-1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN-β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent",
    "molecules": "cGAMP, Poly dA:dT, Poly I:C"
  },
  {
    "caption": "(M-P) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN-β. (Q-T) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN-β.",
    "molecules": "Poly dA:dT, Poly I:C"
  },
  {
    "caption": "(L, M) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP-1 cells treated with DQ-OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP-1 cells with DQ-OVA puncta's",
    "molecules": "DQ-OVA"
  },
  {
    "caption": " D. Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2/3 complex and 3 μM 15% pyrene labeled actin. E. Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in (D) ",
    "molecules": "pyrene"
  },
  {
    "caption": " F. Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2.5 μM SPIN90, 500 nM Arp2/3 complex, 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct ",
    "molecules": "pyrene"
  },
  {
    "caption": " G. Time courses of pyrene actin polymerization as described in panel (D), except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351-722 construct ",
    "molecules": "pyrene"
  },
  {
    "caption": " B. Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90(269-722) to activate Arp2/3 complex. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex, and 20 μM wild type or mutant SPIN90(269-722). C. Plot of maximum polymerization rate versus concentration of SPIN90(269-722) for reactions as shown in panel (B) ",
    "molecules": "pyrene"
  },
  {
    "caption": " A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666 ",
    "molecules": "CK-666, pyrene"
  },
  {
    "caption": "Representative images of EGFP-Nix-FKBP (green) Rapalog-induced recruitment to mtKeima labeled mitochondria (Keima excitation 488, cyan) and subsequent mitophagy (Keima excitation 561, magenta). Scale bar: 10 µm. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition.",
    "molecules": "Rapalog"
  },
  {
    "caption": "Confirmation of decreased mitochondrial proteins in WT but not in FIP200 ko cells expressing CID Nix either untreated or treated for 24 hours with Rapalog by immunoblotting. Quantification of MTCO2 relative to Actin over three independent experiments after treatment with Rapalog over 24 hours by immunoblotting. Cells in this figure express mtKeima-P2A-FRB-Fis1tTand EGFP-Nix1-188-FKBP. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "Mitophagy by mtKeima of Nix1-70 when treated with Torin, Rapalog, or both compared to control. Mitophagy by mtKeima of Nix1-87 when treated with Torin, Rapalog, or both compared to control. Data Information: Area shading indicates standard deviation between wells. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.",
    "molecules": "Rapalog, Torin"
  },
  {
    "caption": "IP western blot of FKBP-EGFP-Nix1-188, Nix1-87, or Nix1-70, comparing 1% input to GFP-pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog. Data Information: Statistics: one-tailed unpaired Student's t-test. Fold change calculated from spectral counts (sc) by (sc1-87+1)/(sc1-70+1) so that zero values could be included. IP Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "HeLa cells expressing FKBP-EGFP- p62327-348 or FKBP-EGFP-WIPI2b were treated for 16 hours with phenanthroline to induce endogenous Nix and BNIP3 expression, then underwent a GFP-pulldown and compared to 1% input by immunoblot. Data Information IP Experiments repeated at least 3 times.",
    "molecules": "phenanthroline"
  },
  {
    "caption": "IP western blot of FKBP-EGFP-Nix1-188 compared to Nix60-188, comparing 1% input to GFP-pulldown and blotted against GFP or WIPI2 after 4 hours of Rapalog. Data Information IP Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "Representative images of WT or 6ko HeLa cells expressing EGFP-Nix1-188-FKBP (green), mtKeima, and FRB-Fis1T with or without 4 hours of Rapalog, immunostained for endogenous Tom20 (red) and WIPI2 (white). Scale bar: 50µm. Automated puncta counting of WIPI2 in WT or 6ko cells normalized to cell number in control conditions or after 4 hours with Rapalog. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "Representative images of WT or WIPI2 ko HeLa cells expressing EGFP-Nix1-188-FKBP (green), mtKeima, and FRB-Fis1T with or without 4 hours of Rapalog immunostained for endogenous Tom20 (red) and GABARAP (white). Scale bar: 50µm. Automated puncta counting of GABARAP in WT or WIPI2 ko cells normalized to cell number in control conditions or after 4 hours with Rapalog. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "GFP pulldown of EGFP-Nix1-188-FKBP in WT, WIPI2 ko, or FIP200 ko cells after 4 hours of Rapalog treatment. FIP200 cropped due to the appearance of non-specific bands after IP. Data Information: Error bars indicate standard deviation. Statistics performed by two-way ANOVA; ** indicates a p-value<0.01, *** indicates a p-value<0.001. ns indicates not significant. Over one thousand cells quantified per condition. Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "Representative images of endogenous Halo -WIPI2 in U2OS cells and stained with JF646 Halo dye (magenta) expressing mtKeima (cyan), FRB-Fis1T, and FKBP-EGFP-Nix1-87­, FKBP-EGFP-Nix60-188, or FKBP-EGFP-Nix1-70 (green) after 16 hours of Rapalog treatment. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA;* indicates a p-value<0.05, ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.",
    "molecules": "JF646, Rapalog"
  },
  {
    "caption": "Automated puncta counting of endogenous Halo -WIPI2 in U2OS cells expressing either FKBP-EGFP-Nix1-87 (LIR and MER), Nix60-188 (MER), or Nix1-70 (LIR) after 16 hours of Rapalog treatment then fixed. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics performed by two-way ANOVA;* indicates a p-value<0.05, ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "Automated puncta counting of endogenous Halo -WIPI2 in U2OS cells expressing FKBP-EGFP-Nix1-188 as WT or with W36A/L39 (LIR) or L75A (MER) mutations after 16 hours of Rapalog treatment then fixed. Data Information: Error bars indicate standard deviation between wells. Over one thousand cells quantified per condition. Statistics: panels C two-way ANOVA; ** indicates a p-value<0.01, **** indicates a p-value<0.0001. ns indicates not significant. Experiments repeated at least 3 times.",
    "molecules": "Rapalog"
  },
  {
    "caption": "GFP pulldown of EGFP alone, EGFP-Nix1-188-FKBP as WT, W36A/L39A, or L75A mutants after 4 hours of Rapalog treatment.",
    "molecules": "Rapalog"
  },
  {
    "caption": "GFP pulldown of FKBP-EGFP-WIPI2 as WT or with R108E/R125E mutations after 24 hours of Phenanthroline treatment.",
    "molecules": "Phenanthroline"
  },
  {
    "caption": "C Heatmap of key CyTOF data. Average activities of selected analytes are given as log fold-change after normalization to DMSO control condition. Range of color scale was adjusted for each analyte. For relative changes between all analytes, see Appendix Fig. S7).",
    "molecules": "DMSO"
  },
  {
    "caption": "A Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external IAA concentration. Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP (in the case of the control) both under the control of a constitutive CaMV 35S promoter. Where indicated, 10 µM NPA was added. B Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external NPA concentration in the presence of 100nM IAA in control GFP- (blue or red) or PIN1- (green or purple) transformed protoplasts. ",
    "molecules": "IAA, NPA"
  },
  {
    "caption": "A, B Native PAGE separation reveals distinct detergent-sensitive PIN1-complexes. Plasma membrane preparations from dark grown Arabidopsis cell suspension cultures were solubilized with (a) Complexiolyte 27 (a mixture of ionic and non-ionic detergents), and (b) Complexiolyte 47 (lower stringency, non-ionic detergent), (both Logopharm); first dimension BN-PAGE, second dimension SDS-PAGE, blots stained with anti-PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions. Values are given in KDa.",
    "molecules": "detergent, detergents"
  },
  {
    "caption": "a) BN-PAGE was performed with PIN1-containing microsomes prepared from either PIN1-expressing HEK293T cells or a dark-grown Arabidopsis cell suspension culture. Prior to solubilization with 1% dodecyl maltoside, microsomes were incubated with either 10 µM NPA or 10 µM quercetin.",
    "molecules": "dodecyl maltoside, NPA, quercetin"
  },
  {
    "caption": "b) PIN1 dimer stability induced by NPA after expression in HEK cells. Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50% (v/v) CL27. Each measurement given (three for each concentration) represents the mean of three gel lanes for wild type (circles) or triple S2523, S253E, S261E phosphomimetic sequences (crosses) (example images are given in the figure inset).",
    "molecules": "NPA"
  },
  {
    "caption": "a) Summed 2D projections of PLA interactions (left) and co-localisation (right) (n=3) between PIN1 and PIN4 laterally bisected 2D-projected heat map. Treatments as indicated. b) Quantification of positive PLA interactions. n.t (no treatment) and 10µM NPA, n=4; 10µM IAA and 10µM IAA+ 10µM NPA; n=3. Error bars indicate standard deviation. ",
    "molecules": "IAA, NPA"
  },
  {
    "caption": "a) Microsomes from HEK293 cells expressing PIN1-RFP were separated under native conditions in the presence of NPA or an anti-PIN1 Fab fragment. 1) PIN1-RFP, 2) PIN1-RFP+Fab, 3) PIN1-RFP+10 µM NPA, 4) PIN1-RFP+Fab+10 µM NPA, 5) PIN1-RFP + 10 µM Quercetin (Q), 6) PIN1-RFP+Fab+10 µM Quercetin (Q). Protein complexes were solubilized and separated under native conditions as described before. Western blots were performed with an anti-RFP monoclonal antibody.",
    "molecules": "NPA, Quercetin"
  },
  {
    "caption": "b) Fourteen-day-old Arabidopsis seedlings (wild type, brown; pPIN1::Mab9B2; blue) grown on AM containing 0.2 µM NPA. Scale bar = 1 cm.",
    "molecules": "NPA"
  },
  {
    "caption": "c) NPA-affected lateral root density of nine-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments (wild type sample sizes were between 16 and 21 plants; Mab9B2 sample sizes were between 12 and 31 plants). Bars indicate standard error.",
    "molecules": "NPA"
  },
  {
    "caption": "d) NPA-affected apical hook angle in dark-grown three-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 38 and 50 plants; Mab9B2 sample sizes were between 33 and 45 plants). e) Gravitropic curvature in four-day old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 15 and 21 plants; Mab9B2 sample sizes were between 14 and 20 plants).",
    "molecules": "NPA"
  },
  {
    "caption": "D) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.",
    "molecules": "991, MRT68921"
  },
  {
    "caption": "D) VPS34 CI components expressed with Ulk1 1-427 WT or KI as indicated. After starvation for 1 hr in the presence or absence of MRT68921 (ULK inhibitor), cells were lysed and VPS34 CI coimmunoprecipitated via ATG14-ZZ. VPS15 S861 phosphorylation was increased in the presence of active Ulk1. VPS15 pS861/total VPS15 was quantified, mean +/- SEM, n=3.",
    "molecules": "MRT68921"
  },
  {
    "caption": "A) HEK293A, VPS15 KO control and stably rescued VPS15 KOs (sgA-B3) were starved for 1 hr before WIPI2 (green), LC3 (red) and DNA (Hoechst, blue) were visualised. Quantification of WIPI2 puncta number per cell, mean +/-SEM, n=5.",
    "molecules": "Hoechst"
  },
  {
    "caption": "B) Stably rescued VPS15 KO (sgA-B3), HEK293A and VPS15 KO control cells were starved for 1 hr with (SB) or without (St) 100nM Bafilomycin A1, or cultured in full media (F). Quantification shows LC3II/Actin, mean +/-SEM, n=5.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "A) HEK293A were treated with VPS34-IN1 (1μM) alone or in the presence of MRT68921 (1μM) for the indicated time. Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot. Identical samples were loaded in parallel for VPS34, ATG13, PRKAB2 analysis, indicated by dashed line.",
    "molecules": "MRT68921, IN1"
  },
  {
    "caption": "B) HEK293A were transfected with siRNAs targeting ATG14, UVRAG or p62, or with non-targeting siRNA (RISC-free, RF) and cells were treated with VPS34-IN1 (IN1; 1μM) for 0, 4 or 18 hrs as indicated. Quantification of ATG13 pS318/Total ATG13, mean +/- SEM, n=3. Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis, indicated by dashed line.",
    "molecules": "IN1"
  },
  {
    "caption": "C) Cells transfected as in B were treated with IN1 for 18 hours as indicated, fixed and labelled for ubiquitin (green), FIP200 (red) and p62 (blue). FIP200-positive bodies were quantified from 4 independent experiments with mean +/- SEM plotted. Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested.",
    "molecules": "ubiquitin, IN1"
  },
  {
    "caption": "6mA/dA levels (mol/mol) of seven stages of Hydractinia development . purple area indicates the background level of 6mA contamination from the digestive enzymes used in the respective experiment. hpf: hours post fertilization. The number of biological replicates used for each sample are indicated by the amount of data points in the graph.",
    "molecules": "dA, 6mA"
  },
  {
    "caption": "Whole-mount immunofluorescence of 6mA from 16- and 64-cell stages of Hydractinia. Samples were RNase-treated. . In D, the scale bare is equal to 20 µm.",
    "molecules": "6mA"
  },
  {
    "caption": "Whole-mount immunofluorescence of anti-6mA in 64-128-cell embryos upon injection of shGFP (as control), shAlkbh1",
    "molecules": "6mA"
  },
  {
    "caption": "Relative quantification of anti-6mA signals from immunofluorescence images Fluorescence intensity were normalized to the highest and lowest measured area in shGFP (n=135), shAlkbh1 (n=168), and rescue (n=140), where n = nuclei numbers. Central band shows the mean, the boxes show lower and upper quartiles and whiskers show minimum and maximum data values.",
    "molecules": "6mA"
  },
  {
    "caption": "Quantification of shAlkbh1-electroporated embryos showing significantly higher level of 6mA/dA (P<0.05) compared to shGFP electroporated embryos and to wild type embryos at 64-128 cell stage.",
    "molecules": "dA, 6mA"
  },
  {
    "caption": "RNase treatment abolishes the EU signal.",
    "molecules": "EU"
  },
  {
    "caption": "Whole-mount image of EU incorporation signals at 64 cells upon injection with shGFP, shAlkbh1, rescue, and mutant-rescue solution Relative quantification of EU signals Fluorescence intensity were normalized to the highest and lowest measured area in shGFP (n=86), shAlkbh1 (n=63), rescue (n=64), and mutant-rescue (n=66) where n = nuclei numbers. Central band shows the mean, the boxes show lower and upper quartiles and whiskers show minimum and maximum data values.",
    "molecules": "EU"
  },
  {
    "caption": "Rapid decline of m6A-marked maternal RNA occurs between the 2- to 16-cell stages, analyzed by UHPLC-QQQ of m6A/A (% in arbitrary unit) from four Hydractinia developmental stages.",
    "molecules": "m6A"
  },
  {
    "caption": "Replication stall at 8-16 nuclei following hydroxyurea treatment. The control shows a normal number of nuclei at the same developmental stage as treated one.",
    "molecules": "hydroxyurea"
  },
  {
    "caption": "Whole-mount immunofluorescence of anti-6mA in 32-cell embryos upon injection of ATP and m6ATP at 20 mM The uninjected control shows a normal level of anti-6mA at 32-cell embryos.",
    "molecules": "ATP, m6ATP, 6mA"
  },
  {
    "caption": " Kinetics of SARS-CoV-2 and SARS-CoV RNA synthesis in infected Vero E6 cells.(a) Hybridization analysis of viral mRNAs isolated from SARS-CoV-2- and SARS-CoV -infected Vero E6 cells, separated in an agarose gel and probed with a radiolabelled oligonucleotide recognizing the genome and subgenomic mRNAs of both viruses. Subsequently, the gel was re-hybridized to a probe specific for 18S ribosomal RNA, which was used as a loading control. (b) Analysis of the relative abundance of each of the SARS-CoV-2 and SARS-CoV transcripts. Phosphorimager quantification was performed for the bands of the samples isolated at 12, 14 and 24 h p.i., which yielded essentially identical relative abundances. The table shows the average of these three measurements. SARS-CoV-2 mRNA sizes were calculated on the basis of the position of the leader and body transcription-regulatory sequences (ACGAAC) in the viral genome (Sawicki and Sawicki 1995, Xu, Hu et al. 2003).",
    "molecules": "18S ribosomal RNA, viral mRNAs"
  },
  {
    "caption": " Cross-reactivity of antisera raised against SARS-CoV structural and non-structural proteins.Selected antisera previously raised against SARS-CoV nsps and structural proteins cross-react with corresponding SARS-CoV-2 proteins. SARS-CoV-2-infected Vero E6 cells (m.o.i. of 0.3) were fixed at 12 or 24 h p.i. For immunofluorescence microscopy, cells were (double)labelled with (a) a rabbit antiserum recognising nsp4 and a mouse mAb recognising dsRNA; (b) anti-nsp4 rabbit serum and a mouse mAb directed against the N protein; (c-e) rabbit antisera recognising against nsp3, nsp13 and the M protein, respectively. Nuclear DNA was stained with Hoechst 33258. Bar, 20 µm. ",
    "molecules": "dsRNA, Hoechst 33258"
  },
  {
    "caption": " Assay to screen for compounds that inhibit SARS-CoV-2 replication.Inhibition of SARS-CoV-2 replication (coloured bars) was tested in Vero E6 cells by developing a CPE-reduction assay and evaluating several previously identified inhibitors of SARS-CoV, which was included for comparison (grey bars). For each compound a two-fold serial dilution series in the low-micromolar range was tested; (a) Remdesivir, (b) chloroquine, (c) Alisporivir and (d) pegylated interferon alpha-2. Cell viability was assayed using the CellTiter 96® Aqueous One Solution cell proliferation assay (MTS assay). Compound toxicity (solid line) was evaluated in parallel using mock-infected, compound-treated cells. The graphs show the results of 3 independent experiments, each performed using quadruplicate samples (mean ± SD are shown). ",
    "molecules": "pegylated interferon alpha-2, Alisporivir, chloroquine, Remdesivir"
  },
  {
    "caption": "(B) Expression levels of total H2B and H2Bub1 were analyzed in wild-type (WT), mog1Δ, ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti-total H2B and anti-H2Bub1 antibodies. Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test. Error bars represent SD. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001)",
    "molecules": "ub1"
  },
  {
    "caption": "(A) Ten-fold serial dilutions of the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea (HU) and were incubated for 2-3 days.",
    "molecules": "HU, hydroxyurea"
  },
  {
    "caption": "(A) Co-purification of Rtf1-PK in cells expressing Mog1-TAP (+,+). A non-tagged strain (-,-) and cells expressing only Rtf1-PK (+,-) were included as negative controls for co-purification. Inputs and IPs are depicted for each strain.",
    "molecules": "PK"
  },
  {
    "caption": "(B) ChIP analysis of the presence of HA-tagged Mog1 compared to a non-tagged strain (WT) at PMA1 and YEF3 promoter (P) and coding regions (5'ORF). The occupancy level was calculated as the signal ratio of the IP samples in relation to the Input signal normalized to an Intergenic region. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test and is presented as a P-value (*, P<0.05).",
    "molecules": "HA"
  },
  {
    "caption": "(D) Mog1-HA immunoprecipitation in cells expressing Bre1-PK (upper panel) and Mog1-TAP co-precipitation in cells expressing Ada2-HA (lower panel) are indicate as in (A) Inputs and IPs are depicted for each strain.",
    "molecules": "HA, PK"
  },
  {
    "caption": "Representative images of the analysis of nuclear mRNA export are shown. The cellular localization of poly(A)+RNA was assessed by in situ hybridization using Cy3-labeled oligo(dT) probes (red) in the indicated strains. In situ hybridization was monitored at 30 ºC and after shifting of the cells to 39 ºC for 3 hours. DNA was stained by DAPI (blue). In all images the scale bar corresponds to 2 μM. Images are representative pictures from 2 independent experiments of approximately 600 cells. The percentages indicate the number of cells showing mRNA export defect from these experiments.",
    "molecules": "Cy3, DAPI, DNA, mRNA, poly(A)+RNA"
  },
  {
    "caption": "(B) The Mog1E65K Ran-binding mutant complements the low H3K4me3 and H2Bub1 levels of mog1Δ. The expression levels of H3K4me3, total H3, H2Bub1, total H2B and PGK1 in whole cell lysates from wild type (WT) or from mog1Δ cells transformed with an empty plasmid (+pEmpty), wild type MOG1 (pMOG1) or a Ran-binding mutant (pMOG1E65K) were analyzed by western blotting using the corresponding antibodies.",
    "molecules": "H3K4me3, ub1"
  },
  {
    "caption": "(C) Gsp2 is not involved in H2Bub1 regulation. The expression levels of total H2B and of H2Bub1 were analyzed in whole cell lysates from wild type (WT) and gsp2Δ strains by western blotting using anti-total H2B and anti-H2Bub1 antibodies.",
    "molecules": "ub1"
  },
  {
    "caption": "Flow cytometric analysis of BrdU incorporation rates in GFPlow and GFPhigh cells from the DPR and VPR. Statistical analysis of BrdU+ in different cell populations. The Data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. *p-value < 0.05; ***p-value < 0.001.",
    "molecules": "BrdU"
  },
  {
    "caption": "Sagittal section of E12.5 Pdx1-Cre; Rosa26-LacZ mouse embryo counterstained for ALB (brown). X-gal staining (blue) showing cells derived from Pdx1-expressing cells. The arrowheads indicate cells co-stained with X-gal and ALB (See Fig EV5D for more images of different sections). Scale bar: 50 µm (left) or 20 µm (right).",
    "molecules": "X-gal"
  },
  {
    "caption": "B: Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two-hour pulse activation (ON/OFF scenario). Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes. Genes were ranked by their model-derived response time.",
    "molecules": "CYHX"
  },
  {
    "caption": "B: pERK2 log2 fold change upon sustained activation (left) and deduced input function (right) used for model fitting. Average pERK2 log2 fold change upon 4OHT treatment equals 100% signalling amplitude.",
    "molecules": "4OHT"
  },
  {
    "caption": "B: Signalling input conditions (left side shows deduced input function, right side shows pERK2 measurements): Sustained ERK signalling (4OHT), two-hour pulse ERK signalling (4OHT+U0126), growth factor signalling (EGF: Epidermal growth factor, FGF: Fibroblast growth factor). Deduced input functions: 100% signalling amplitude corresponds to mean induction in training condition (4OHT). Growth factor-induced input functions are linear interpolations of pERK2 log2 fold changes relative to mean induction in test condition.",
    "molecules": "4OHT, U0126"
  },
  {
    "caption": " Figure 5 - Immediate late genes (ILGs) have long mRNA half-lives, are transcribed immediately and have GC-rich promoters. A: Boxplot comparison of mRNA half-life estimates based on modelling of gene induction (model-derived), transcriptional shutdown (ActD-derived) and metabolic labelling (4SU-derived). Estimates from 4OHT-pretreated HEK293∆RAF1:ER cells (ON panel) are more appropriate to characterise induced genes than estimates from unstimulated cells (OFF panel). Genes not assigned to any cluster are shown in grey. ",
    "molecules": "4SU, ActD, 4OHT"
  },
  {
    "caption": "C: Log2 fold changes of transcription rate in 4OHT-treated HEK293∆RAF1:ER cells derived from metabolic labelling (4SU) RNA-sequencing data document immediate transcription of IEGs and ILGs but delayed transcription of DEGs.",
    "molecules": "4SU, 4OHT"
  },
  {
    "caption": " Figure 6 - Immediate late genes (ILGs) translate signal duration into response amplitude. A: Upper panel: pERK2 log2 fold changes upon different input scenarios (sustained: 4OHT; two-hour pulse: 4OHT+U0126). Lower panel: Response amplitude across temporal clusters and signal durations. Bold lines show median cluster amplitude at each time point. ",
    "molecules": "4OHT, U0126"
  },
  {
    "caption": "C: qPCR validation to test different ERK signal durations. HEK293∆RAF1:ER cells were treated with 4OHT and U0126 for different periods of time to generate signal duration scenarios of 0.5 to 8 hours (cf. Fig EV1 C). mRNAs of IEGs EGR1 and FOS relay signal duration to response duration, whereas ILGs CLU and FOSL1 decode signal duration to response amplitude (qPCR data for all 17 validated mRNAs is shown in Fig EV6 A).",
    "molecules": "4OHT, U0126"
  },
  {
    "caption": "E: Quantification of western blots to present protein log2 fold changes of sample genes upon sustained ERK signalling (4OHT-induced) and transient ERK signalling (EGF-induced) in HEK293∆RAF1:ER cells.",
    "molecules": "4OHT"
  },
  {
    "caption": "F Mutant Rescue: WT and two representative pap8-1 plants were grown in vitro using sucrose and low white light intensity (of 10 µmol.m-2.s-1); scale bar equals 20 mm.",
    "molecules": "sucrose"
  },
  {
    "caption": "I,J Confocal imaging on Arabidopsis cotyledons stably expressing pP8::PAP8ΔNLS-GFP; ΔNLS, deletion of the NLS (I) during skotomorphogenesis and (J) after 24h light; Yellow arrowheads show the GFP signal; the picture is a merge of different channels: GFP in green, Pchl,: protochlorophyllide, or Chl: chlorophyll in magenta marked with arrowheads, and propidium iodide, showing the waxy cuticle in red, the empty space correspond to the layer of highly vacuolated epidermal cells.",
    "molecules": "Chl, chlorophyll, propidium iodide, Pchl, protochlorophyllide"
  },
  {
    "caption": "B Overlay of 1H-15N correlation 2D NMR spectra of free 15N-labelled PAP8 alone (blue) or in complex with PAP5 (red). Grey areas depict changes of signals in the PAP8 spectrum.",
    "molecules": "1H, 15N"
  },
  {
    "caption": "C 15N-Filtered Diffusion Ordered Spectroscopy-NMR measurements to PAP8. Exponential decay curves of PAP8 in absence or in presence of MBP-PAP5 are shown in red and black respectively. The units on the y-axis are normalized values of the integrals of the signal measured in the amide proton region.",
    "molecules": "15N"
  },
  {
    "caption": "(A Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (A) The percentage of cell death at DIV14, after co-transfection of indicated kinases and HTTex1 plasmid in rat primary striatal neurons at DIV9 (n=3),",
    "molecules": "Hoechst dye"
  },
  {
    "caption": "B) Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (B) The percentage of cell death at DIV7, after co-transfection of indicated kinases and HTT N586 plasmid in mouse primary striatal neurons at DIV5 (n=8).",
    "molecules": "Hoechst dye"
  },
  {
    "caption": "(D) The percentage of cell death., quantification by a nuclear condensation assay in mouse primary cortical neurons co-transfected with the indicated HTT N586 and kinases at DIV5, cells were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7. Cells were fixed, and the nuclei were stained with Hoechst dye (n=3).",
    "molecules": "Hoechst dye, MRT 68601"
  },
  {
    "caption": "(A) Representative immunoblot of a filter retardation of HTT (ab-MAB5492) from the insoluble cellular fraction; HEK293T cells co-expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour, and for the last 16 hour, they were treated with the indicated proteasome inhibitor (MG132, 5 µM) or autophagy inhibitor (Baf A1, 200 nM, NH4Cl, 10 mM, 3-MA,5 mM). (B) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A (n=3).",
    "molecules": "3-MA, NH4Cl, Baf A1, DMSO, MG132"
  },
  {
    "caption": "(C) Immunoblot of LC3 (ab48394) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour; for the last hour, Baf A1 (500 nM) was added as indicated. (D) Fold change in LC3-II levels (lower band) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C (n=3). ",
    "molecules": "Baf A1"
  },
  {
    "caption": "(E) Immunoblot of LC3 (ab48394) from soluble rat primary neuronal cells overexpressing lentivirus-mediated TBK1 and TBK1 KD for 96 hour. For the last 1 or 4 hour, Baf A1 (500 nM) was added as indicated. (F) Quantification of the fold change in LC3-II levels (lower band) compared to the kinase dead mutant, which was untreated, normalized to GAPDH from the blots like in E (n=3).",
    "molecules": "Baf A1"
  },
  {
    "caption": "(E-G) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro and treated with Tf-555 for 30 minutes before fixation. Cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). Blue alone channels are shown in black and white images. Lower panels in (E) and (F) are high magnification images indicated by white or blue rectangles in upper panels. The graph in (G) shows the Pearson's correlation coefficient of Tf-555 and Rab11 or APPL1. Each score represents the mean with the individual points. Control: n = 23 cells, Rab21-sh115: n = 18 cells, Rab5-sh232 (Tf - Rab11): n = 23 cells, Rab5-sh232 (Tf - APPL1): n = 22 cells.",
    "molecules": "555"
  },
  {
    "caption": "Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-EGFP , incubated for two days in vitro and treated with BODIPY-LacCer (LacCer) (green) (A-B) for 30 minutes before fixation. White arrows in (A) indicate the perinuclear accumulation of LacCer. Blue alone channels are shown in black and white images. The graphs in (B) show the ratio of cells with perinuclear accumulation of LacCer (B) which was quantified in a blinded counting. Each score represents the mean with the individual points. B: n = 5 biological replicates (Control: 115 cells, Cav1-sh490: 127 cells, Rab21-sh115: 136 cells, Rab5-sh232: 216 cells, Rab21-sh115 + wt-Rab21: 132 cells)",
    "molecules": "BODIPY, LacCer"
  },
  {
    "caption": "(H-I) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro, treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Lamp1, a lysosomal marker, in control and Rab21-sh115-transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21-sh115: n = 22 cells.",
    "molecules": "Baf A1, Bafilomycin A1"
  },
  {
    "caption": "Western blot for p53, phosphorylated p53 at Ser15 (p53 pS15), and vinculin (vinc) in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1, PHD2, or PHD3.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot of p53 pS15, p53, PHD1, and vinculin in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1 or a scrambled (Scr) control.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot for p53, p53 pS15, and vinc in HCT116 silenced for PHD1 with two different constructs (constructs 1 and 2) upon exposure to 300 μM 5-FU for 8 h.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot for p53 pS15, p53, and vinculin in HCT116 silenced for PHD1 and treated with either 200 nM SN-38 or 20 μM oxaliplatin for 8 h.",
    "molecules": "oxaliplatin, SN-38"
  },
  {
    "caption": "Western blot for p53 pS15, p53, and vinc in LIM1215 upon silencing of PHD1 and treatment for 8 h with 200 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "A Western blot for p53, cleaved caspase-3 (cleaved casp3), cleaved parp, and vinculin (vinc) in p53wt/wt and p53−/− HCT116 silenced for PHD1 upon exposure to 300 μM 5-FU for 24 h, showing an increased apoptotic response to 5-FU treatment in PHD1-silenced cells compared to the scrambled control.",
    "molecules": "5-FU"
  },
  {
    "caption": "B Confirmation of these results by FACS analysis of propidium iodide-stained PHD1-silenced p53wt/wt and p53−/− HCT116 cells exposed for 26 h to 300-μM 5-FU treatment. *P = 0.002 toward p53wt/wt HCT116 shScr 5-FU-treated (two-tailed unpaired t-test) with n = 6 for non-treated p53wt/wt HCT116 and n = 3 in all other groups.",
    "molecules": "5-FU"
  },
  {
    "caption": "D, E Apoptosis as detected by Western blot for cleaved casp3 and parp in PHD1-silenced HCT116 treated for 20 h with 200 nM SN-38 (D) or for 24 h with 20 μM oxaliplatin (E).",
    "molecules": "oxaliplatin, SN-38"
  },
  {
    "caption": "F Western blot for cleaved casp3, parp, and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "G Similar apoptosis levels were detected by Western blot for cleaved casp3 and parp with vinc as a loading control in p53S15A→p53−/− HCT116 silenced for PHD1 or scrambled (Scr) control and treated for 20 h with 300 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "Representative images and quantification of the colony formation capacity of p53wt/wt HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct, treated for 24 h with doxycycline with or without additional treatment for 8 h with 300 μM 5-FU. *P = 0.008 toward shScr, two-tailed unpaired t-test and n = 3/group.",
    "molecules": "5-FU, doxycycline"
  },
  {
    "caption": "Tumor volume of p53wt/wtHCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. *P = 0.045 toward shScr 5-FU-treated mice by two-way ANOVA with n = 6 for p53wt/wtHCT116 shScr, n = 7 for p53wt/wtHCT116 shPHD1 and p53wt/wtHCT116 shScr 5-FU-treated, and n = 8 for p53wt/wtHCT116 shPHD15-FU-treated.Tumor volume of p53−/−HCT116 cells transduced with a doxycycline-inducible shScr or shPHD1 silencing construct injected subcutaneously in nude mice and treated with 5-FU. n = 6 for p53−/−HCT116 shScr, p53−/−HCT116 shPHD1, and p53−/−HCT116 shScr 5-FU-treated and n = 8 for p53−/−HCT116 shPHD15-FU-treated.",
    "molecules": "5-FU, doxycycline"
  },
  {
    "caption": "Western blot for p53 pS15, p53, and vinculin (vinc) in PHD1-silenced HCT116 upon treatment with 300 μM 5-FU for 8 h with or without 0.5 mM DMOG for 26 h. Vinculin (vinc) is used as a loading control.",
    "molecules": "5-FU, DMOG"
  },
  {
    "caption": "Western blot for p53 pS15, p53, and vinculin (vinc) in HCT116 treated with 300 μM 5-FU upon silencing of PHD1 and p38α, alone or in combination. Vinculin (vinc) is used as a loading control.",
    "molecules": "5-FU"
  },
  {
    "caption": "Detection by Western blot of p53 and p38 from whole cell extracts (WCE) or after immunoprecipitation of p53 from cell silenced for a Scr control or siPHD1 and treated for 1 h with 300 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "Transcription analysis of CDKN1A, GADD45, MDM2, BAX, PUMA in PHD1-silenced p53wt/wt HCT116 either untreated or treated for 8 h with 300 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot for cleaved parp and vinculin (vinc) in PHD1-silenced HCT116 treated for 20 h with 300 μM 5-FU alone or in combination with 3 μg/ml α-amanitin.",
    "molecules": "α-amanitin, 5-FU"
  },
  {
    "caption": "Western blot for p53 pS15, p53, and vinculin in PHD1-silenced p53R248/− HCT116 upon 8-h treatment with 300 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot for cleaved casp3 and vinculin in p53R248/− HCT116 silenced for PHD1 and treated with 300 μM 5-FU for 24 h.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot for pH2AX and vinculin (vinc) in p53wt/wt and p53−/− HCT116 cells after 24-h treatment with 300 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "Detection of pH2AX and vinc in p53S15A→p53−/− HCT116 cells silenced for PHD1 and treated with 300 μM 5-FU for 24 h.",
    "molecules": "5-FU"
  },
  {
    "caption": "Detection of pH2AX and vinc in HCT116 silenced with a second silencing construct of PHD1 and treated for 24 h with 300 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot for pH2AX and vinc in HCT116 treated for 20 h with 200 nM SN-38 or 24 h with 20 μM oxaliplatin.",
    "molecules": "oxaliplatin, SN-38"
  },
  {
    "caption": "Western blot for pH2AX and vinc in p53R248/− HCT116 cells silenced for PHD1 upon treatment with 300 μM 5-FU for 24 h.",
    "molecules": "5-FU"
  },
  {
    "caption": "Detection by Western blot of p53 and XPB from whole cell extracts (WCE) or after immunoprecipitation of p53 from cells silenced for Scr control or siPHD1 and treated for 4 or 8 h with 300 μM 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "Western blot for pH2AX and vinc in HCT116 silenced for PHD1 alone or in combination with silencing for XPB upon treatment with 300 μM 5-FU for 20 h.",
    "molecules": "5-FU"
  },
  {
    "caption": "A Protein O-glucosyltransferase activity of wild-type (WT) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat (hFIX-EGF). Wild-type shows higher O-glucosyltransferase activity than D233E, and this activity is dependent on the concentration of the acceptor substrate hFIX-EGF repeat (left) and on the concentration of donor substrate UDP-glucose (UDP-Glc) (right). Values indicate mean ± SEM from three independent assays.",
    "molecules": "Glc, glucose, UDP"
  },
  {
    "caption": "C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC. hFIX-EGF repeat was incubated with wild-type or D233E mutant POGLUT1 and donor substrate, UDP-Glc or UDP-xylose (UDP-Xyl), at 37ºC overnight. Values on top of the peaks indicate the measured masses. Addition of Glc (162 Da) or xylose (132 Da) to hFIX-EGF (5696.2 Da) by wild-type POGLUT1 caused a shift to an earlier retention time (left). Similarly, the products exhibited a similar shift after incubation with POGLUT1D233E (right). These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX-EGF repeats and thus has residual enzymatic activity.",
    "molecules": "Glc, glucose, UDP, Xyl, xylose"
  },
  {
    "caption": "C-D C2C12 myogenic cells showed a reduced level of glycosylated α-dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium. This indicates that reducing Notch signaling can affect α-dystroglycan glycosylation in wild-type C2C12 myoblasts cells, which do not harbor any known mutations in α-dystroglycan glycosyltransferases or Poglut1.",
    "molecules": "DAPT, LY3039478"
  },
  {
    "caption": "E-F qRT-PCR experiments show that upon DAPT or LY3039478 treatment, a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation.",
    "molecules": "DAPT, LY3039478"
  },
  {
    "caption": "(A) Remdesivir inhibition of SARS-CoV-2 infections of Vero E6 cells. drugs, , were used at the indicated concentrations. Viral RNA level was determined by qRT-PCR 15 hours after inoculation of SARS-CoV-2 (Swedish isolate, 106 PFU).",
    "molecules": "Remdesivir"
  },
  {
    "caption": "(B) Cell viability of isolated cell clones and WT AN3-12 cells following 72h remdesivir treatment with the indicated doses. Mean ±SEM of 2-4 biological replicates is displayed.",
    "molecules": "remdesivir"
  },
  {
    "caption": " (A) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with Human recombinant soluble ACE 2 (hrsACE2) (200µg/ml) and remdesivir (Remd. 4µM). Viral RNA level was determined at 15 hours after virus inoculation. ",
    "molecules": "Remd, remdesivir"
  },
  {
    "caption": " (B) Treatment of SARS-CoV-2 (106 PFU) infected human kidney organoids with hrsACE2 (200µg/ml) and/or remdesivir (Remd.4µM)). Viral RNA was determined by qRT-PCR 72 hours after the inoculation of 106 PFU of SARS-CoV-2. ",
    "molecules": "Remd, remdesivir"
  },
  {
    "caption": " (C) Treatment of SARS-CoV-2 (106 PFU) infected Vero-E6 cells with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM)). ",
    "molecules": "Remd, remdesivir"
  },
  {
    "caption": " (D) Treatment of SARS-CoV-2 (106 PFU) infected kidney organoids with hrsACE2 (10µg/ml) and remdesivir (4µM). ",
    "molecules": "remdesivir"
  },
  {
    "caption": " (E,F) Progeny virus released from untreated Vero-E6 cells or Vero-E6 cells treated with clinical doses of hrsACE2 (5 and 10µg/ml) and remdesivir (Remd. 4µM). Progeny was determined (E) 15 hours and (F) 48 hours post-infection (hpi). ",
    "molecules": "Remd, remdesivir"
  },
  {
    "caption": "Representative images of single plane phase-contrast and Z-stacked immunofluorescence of empty vector (EV) and CEP55-overexpressing MCF10A cells grown on Matrigel for 14 days. Red: Cytokeratin-19; Green: F-actin stained by Phallodin and Blue: DAPI. Z-stack images were acquired through Zeiss LSM 780 confocal microscope-ZMBH.",
    "molecules": "DAPI, Phallodin, Matrigel"
  },
  {
    "caption": "(A) Polyploidy analysis (>4N DNA contents) determined using FACS in control and CEP55 knockdown MDA-MB-231 cells (left panel). Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4.0 software (right panel). Yellow peaks represent subpopulation of polyploidy/aneuploidy. Graph represents the mean±SEM of three independent experiments.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Representative cytogram of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles following treatment with and without the B12536 (5 nM).",
    "molecules": "B12536"
  },
  {
    "caption": "Percentage of polyploidy (>4N) following B12536 (5 nM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.",
    "molecules": "B12536"
  },
  {
    "caption": "Percentage of polyploidy (>4N) following nocodazole (0.5 µM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with Nocodazole Graph represents the mean±SEM of three independent experiments.",
    "molecules": "Nocodazole"
  },
  {
    "caption": "(F) Percentage of sub-G1 fraction following B12536 (10 nM) or nocodazole (0.5 µM) in control and CEP55 knockdown Hs578T cells. Graph represents the mean±SEM of two independent experiments.",
    "molecules": "B12536, nocodazole"
  },
  {
    "caption": "(G) Representative cytogram of empty vector (EV) and CEP55-overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 (10 nM).",
    "molecules": "B12536"
  },
  {
    "caption": "(H) Percentage of polyploidy following treatment with and without B12536 (10 nM) in empty vector (EV) and CEP55-overepressing MCF10A cells. Graph represents the mean±SEM of three independent experiments.",
    "molecules": "B12536"
  },
  {
    "caption": "(I) Corresponding sub-G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H. Graph represents the mean±SEM of three independent experiments.",
    "molecules": "B12536"
  },
  {
    "caption": "(A) Box and whiskers plot showing average time spent in mitosis and (B) mitotic outcomes in control and CEP55 knockdown MDA-MB-231 cells following treatment with the BI2536 (5 nM). Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis, characterized by membrane blebbing. Graph represents the mean±SEM of two independent experiments. For each experiments n=50 mitotic cells were counted per condition using Olympus Xcellence IX81 time-lapse microscopy.",
    "molecules": "BI2536"
  },
  {
    "caption": "(C, D) Cells were pre-treated for two hours with 50 µM of the pan Caspase inhibitor Z-VAD-FMK, followed by treatment with B12536 (5 nM), before assessment of cell fate (C) and an average time spent in mitosis (D) Graph represents the mean±SEM of two independent experiments.",
    "molecules": "B12536, Z-VAD-FMK"
  },
  {
    "caption": "(E) Cells were synchronized using double thymidine then released into nocodazole (0.5 µM), and protein lysates were collected at the indicated time points. Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of Cdk1 activation/mitotic entry. COX-IV served as a loading control.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "(G) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole (0.5 µM) treatment at indicated time points. Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "(H) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G. Blue: 1 ns; red: 3 ns. Graph represents the mean±SEM of three independent experiments.",
    "molecules": "mCerulean, nocodazole"
  },
  {
    "caption": "(I) Control and CEP55 knockdown MDA-MB-231 cells were synchronized as above, released into culture medium for six hours and then blocked with nocodazole (0.5 µM) for an additional four hours prior to treatment with the Cdk1 inhibitor RO-3306 for an additional 16 hours. Sub-G1 cells were then identified by propidium iodide staining and quantified by FACS. Graph represents the mean±SEM of two independent experiments.",
    "molecules": "nocodazole, RO-3306"
  },
  {
    "caption": "(J) Control and CEP55 knockdown MDA-MB-231 cells were transfected with MAD2 siRNA (5 nM) for 48 hours followed by nocodazole (0.5 µM) treatment for an additional 12 hours. Cells were collected, fixed and sub-G1 analysis was performed as described in methods. Graph represents the mean±SEM of two independent experiments.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(A) Immunoblots analysis was performed to determine CEP55 levels in MDA-MB-231 cells treated with multiple inhibitors targeting the EGFR/HER2 pathway for 24 hour. The following inhibitors were used: MEK1/2i (AZD6244 (1 µM)), the AKT, PI3K/mTORi (BEZ235 (0.5 µM), AKTi VIII (1 µM)), EGFRi and HER2i (Erlotinib (1 µM), Afatinib (0.25 µM), Lapatinib (1 µM), Trastuzumab (10 µg/ml) or the pan VEGFR, PDGFR and RAF kinases (Sorafinib, (1 µM)). COX-IV as a loading control.",
    "molecules": "Afatinib, AKTi VIII, BEZ235, Erlotinib, Lapatinib, AZD6244, Sorafinib, Trastuzumab"
  },
  {
    "caption": "(B) Immunoblots analysis of MDA-MB-231 cells treated with three different MEK1/2 inhibitors at various concentrations (Selumetinib AZD6244, Trametinib GSK1120212 and Binimetinib MEK162) after 24h. COX-IV as a loading control.",
    "molecules": "Binimetinib, MEK162, AZD6244, Selumetinib, GSK1120212, Trametinib"
  },
  {
    "caption": "(C) Relative CEP55 promoter luciferase activity in MDA-MB-231 cells either treated with AZD6244 (1 µM) for 6 hours or ERK1/2 knockdown for 24 hours. PGL basic reporter plasmid was used to normalize basal CEP55 promoter activity. Graph represents the mean±SEM of two independent experiments.",
    "molecules": "AZD6244"
  },
  {
    "caption": "(G) Immunoblots analysis showing CEP55 and MYC levels following 4-hydroxytamoxifen (4OHT) (0.5 µM) induction in MCF10A MYCER cells cultured in 0.1% fetal bovine serum contained media at indicated time points. COX-IV served as a loading control.",
    "molecules": "4-hydroxytamoxifen, 4OHT"
  },
  {
    "caption": "(J) Both control and CEP55 knockdown MDA-MB-231 cells were exposed with different concentrations of PLK1 (BI2536) alone (i) or in combination with AZD6244 (1 µM) (ii-iv), and cell viability was determined after 6 days. The dose-response curve was generated by calculating cell viability relative to untreated control and plotted against drug concentration. Graph represents the mean±SEM of three independent experiments.",
    "molecules": "BI2536, AZD6244"
  },
  {
    "caption": "(K) Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors after 96 h. Graph represents the mean±SEM of two independent experiments.",
    "molecules": "BI2536, AZD6244"
  },
  {
    "caption": "(A) Heat map showing relative cell viability of a panel of human breast cancer lines treated single or in combination with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors and cell viability was determined after 6 days. DMSO treated control was used to calculate percentage of cells affected by individual or combination treatment. NN: near normal. Heat map represents data derived from three independent experiments.",
    "molecules": "BI2536, DMSO, AZD6244"
  },
  {
    "caption": "(B) Immunoblot analysis was performed on MDA-MB-157 and SUM159PT cells treated with single or in combination with AZD6244 and BI2536 inhibitors after 96 hours and cleaved PARP and Caspase-3 were determined along with CEP55, MYC and both phosphorylated and total ERK1/2. COX-IV as a loading control (left panels). Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 and BI2536 inhibitors after 96h. Graph represents the mean±SEM of two independent experiments (middle panels). Representative images of colony forming capacity at 14 days determined using crystal violet staining in cells treated with single and combination inhibitors (middle panels). Combination index (CI) values calculated through CompuSyn software (right panels) where 1 indicative of additive (no interaction), >1 indicative of antagonistic, and <1 indicative of synergistic.",
    "molecules": "BI2536, AZD6244"
  },
  {
    "caption": "(C) Percentage of mitotic outcomes determined using time-lapse microscopy of MDA-MB-231 cells treated with single or in combination drugs of BI2536 (2.5 nM) and AZD6244 (1 µM). Graph represents the mean±SEM of two independent experiments. For each experiments n=50 cells were counted per condition.",
    "molecules": "BI2536, AZD6244"
  },
  {
    "caption": "(D) Immunoblot analysis was performed to determine the expression and activity of mitotic and apoptotic regulators upon single and combination treatment with AZD6244 and BI2536 inhibitors as indicated time points in both MDA-MB-231 and SUM159PT cells. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of CDK1 activation/mitotic entry. COX-IV as a loading control.",
    "molecules": "BI2536, AZD6244"
  },
  {
    "caption": "(A) Left, Six week-old female BALB/c cohorts of mice were injected in the 4th inguinal mammary fat pad with the Cep55-ovexpressing mammary carcinoma cell line 4T1.2. Tumor size (area, mm2) was measured using a digital calliper and mean tumor size of each cohort is presented. Mice were treated with vehicle, AZD6244 (12.5mg/kg BID), BI6727 (12.5mg/kg thrice weekly), or combined AZD6244 and BI2536 treatment. Right, representative excised tumor images are shown. Graph represents the mean tumor area±SEM from six mice/group.",
    "molecules": "BI2536, AZD6244, BI6727"
  },
  {
    "caption": "(C) Six week-old female BALB/c cohorts of mice were pre-treated with single and combination BI6727 and AZD6244 inhibitors as indicated in panel A for 4 days, followed by 4T1.2 cells were injected in the 4th inguinal mammary fat pad. Survival of the mice was then monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P=0.0005); n=6 mice/group.",
    "molecules": "AZD6244, BI6727"
  },
  {
    "caption": "(D) Growth rate (mean tumor size, area, mm2) of MDA-MB-231 xenografts in BALB/c nude mice treated with vehicle, AZD6244, BI6727, or combined AZD6244 and BI6727 treatment",
    "molecules": "AZD6244, BI6727"
  },
  {
    "caption": "F: FISH  images of Kms27 MM  cells depleted or not for  Che-1  expression (siChe-1A) labeled with Hoechst  (cell nuclei) and Quasar 670 (NEAT1  RNA). Scale bar 10 μm.",
    "molecules": "Quasar 670, Hoechst"
  },
  {
    "caption": "F: (Top) S9.6 antibody was used for dot blot analysis to evaluate RNA:DNA hybrids  formation in Kms27 MM  cells by serial dilutions of genomic DNA starting at 1.5 micrograms. (Bottom) Quantification of S9.6 levels by densitometric analysis. Values were normalized to a Methylene blue loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ***P<0.005, ****P<0.001).",
    "molecules": "Methylene blue"
  },
  {
    "caption": "D: (Top) Representative confocal   images of Kms27 MM   cells transfected and treated as in C and immunostained   with S9.6  antibody. Scale bar 10 μm. (Bottom) Box plot showing S9.6  signal nuclear   intensity per cell (Arbitrary Unit-AU). siChe-1A cells are described in dark grey, siChe1A plus CX5461   in green color, while siControl in grey color. Median scores per sample are written at the top of the median red line. P value: <1.30-54. ANOVA test was performed to evaluate statistical significance. The red center line denotes the median value (50th percentile) while the black box contains the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.",
    "molecules": "CX5461"
  },
  {
    "caption": "E: (Top) Dot blot  analysis of decreasing amounts of genomic DNA   extracts from Kms27 MM  cells depleted or not for Che-1 expression (siChe-1A) and treated with CX5461, were probed using abs directed against RNA:DNA hybrids. (Bottom) Quantification of S9.6 levels by  densitometric analysis. Values were normalized to a Methylene blue  loading control. Error bars represent the standard error of three different biological experiments (Two Tailed T test ****P<0.001).",
    "molecules": "CX5461, Methylene blue"
  },
  {
    "caption": "A: Representative S9.6 immunofluorescence  levels in healthy and tumor CD138+ plasma cells purified from patient bone marrow. Nuclei  were visualized by staining  with Hoechst  dye. Scale bar 10 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "B: Dot blot  performed to evaluate the enrichment of  RNA:DNA hybrids  formation using the S9.6 antibody by serial dilutions of genomic DNA   starting at 1.0 micrograms. Methylene blue  normalization of S9.6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd.",
    "molecules": "Methylene blue"
  },
  {
    "caption": "D: WB  analysis with the indicated abs of total extracts from U266 MM  cells treated or not with Thapsigargin  (THAP) 0.5 μg/ml for 16 hours. G: WB  with indicated abs of total extracts from Kms27 MM  cells treated or not with ISRIB  1μM for 6 hours.",
    "molecules": "ISRIB, THAP, Thapsigargin"
  },
  {
    "caption": "E: (Left) U266 MM  cells treated or not with THAP  as in D and immunostained  with  S9.6  antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear  mean intensity per cell. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 1.1-20. T-test was performed to evaluate statistical significance. H: (Left) Immunostaining  with S9.6  antibody of Kms27 MM  cells treated or not with ISRIB  as in G Scale bar 10 μm. (Right)  Dot plot  showing relative S9.6 nuclear  mean intensity per cell. 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line. P value: 4.7-23. T-test was performed to evaluate statistical significance.",
    "molecules": "ISRIB, THAP"
  },
  {
    "caption": "F: RT-qPCR analysis of IFNG  and IFNB  in U266 MM  cells treated or not with THAP. Values were normalized to Actin  expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001) I:  RT-qPCR  analysis of IFNG  and IFNB  levels in Kms27 MM  cells treated as in G. Values were normalized to Actin  expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ****P<0.001).",
    "molecules": "THAP"
  },
  {
    "caption": "(B) Dose-dependent inhibition of nirmatrelvir (left panel) and GC376 (right panel) on SARS-CoV-2 Mpro. Prior to adding the substrate for the biochemical reaction, the protease was preincubated for 30 minutes at 37°C with the indicated concentrations of the compound.",
    "molecules": "GC376, nirmatrelvir"
  },
  {
    "caption": "(C) Dose-dependent antiviral activity of nirmatrelvir in HEK293T-hACE2 cells infected with SARS-CoV-2 D614G (purple symbols), B.1.617.2 (green symbols) or B.1.1.529 (blue symbols). Antiviral activity was determined as percent inhibition of the virus-induced cytopathic effect.",
    "molecules": "nirmatrelvir"
  },
  {
    "caption": "B Inflammatory signaling in wild-type (WT/WT), RIPK2 CRISPR KO (KO/KO) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs. BMDMs were primed with IFNy, stimulated with MDP for indicated times and analysed by immunoblotting.",
    "molecules": "MDP"
  },
  {
    "caption": "C Cytokine production of RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs in response to MDP. BMDMs were left untreated or treated with IFNy or IFNy and MDP over-night and cytokines were measured by ELISA. N = 5-8 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001; two-way ANOVA.",
    "molecules": "MDP"
  },
  {
    "caption": "D Serum cytokines in RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) or homozygous (KI/KI) mice after i.p. MDP administration. Mice were injected i.p. with PBS or MDP, sacrificed after 4h and serum cytokines were measured by ELISA. N = 3-6 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; two-way ANOVA.",
    "molecules": "MDP"
  },
  {
    "caption": "A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs. Protein lysates from FLAG-RIPK2 BMDMs (A) were sequentially subjected to ubiquitin enrichment (UBA, B) and FLAG pulldown (C) prior to protein elution and subsequent mass spectrometry analysis.",
    "molecules": "UBA, ubiquitin"
  },
  {
    "caption": "A Activation of the NF-κB pathway by RIPK2 Lysine- and phosphosite mutants. RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants were stimulated with L18-MDP, harvested at indicated time points and activation of the NF-κB pathway was analyzed by immunoblotting.",
    "molecules": "L18-MDP, Lysine"
  },
  {
    "caption": "B IL-8 production of wild-type THP-1 and RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 4-8 experiments. Shown is average ± SEM. * = P ≤ 0.05; two-way ANOVA.",
    "molecules": "L18-MDP"
  },
  {
    "caption": "C) RIPK2 ubiquitination determined by UBA pulldown in RIPK2-deficient cells reconstituted with wild-type or mutant RIPK2 after stimulation with L18-MDP.",
    "molecules": "L18-MDP, UBA"
  },
  {
    "caption": "D) Detection of K63- and M1-linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type-specific antibodies in RIPK2-deficient cells reconstituted with RIPK2 K209R after stimulation with L18-MDP.",
    "molecules": "L18-MDP, UBA, ubiquitin"
  },
  {
    "caption": "C Activation of the NF-κB pathway by RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type RIPK2 or RIPK2 mutants, stimulated with L18-MDP, harvested at indicated time points and analyzed by immunoblotting. ­",
    "molecules": "L18-MDP"
  },
  {
    "caption": "D IL-8 production of RIPK2-deficient THP-1cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 3-8 experiments. Shown is average ± SEM. ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001; two-way ANOVA.",
    "molecules": "L18-MDP"
  },
  {
    "caption": "E Ubiquitination of RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type or mutant RIPK2, left unstimulated or stimulated with L18-MDP and subjected to UBA pulldown and immunoblotting.",
    "molecules": "L18-MDP, UBA"
  },
  {
    "caption": "(D,E) Representative immunofluorescence microscopy images of the distribution of septin 7 (red) or borg3 or borg4 (green) in young, aged and aged LT-HSCs treated with CASIN (5µM). Nuclei were stained with DAPI (blue), scale bar= 5µm.",
    "molecules": "CASIN, DAPI"
  },
  {
    "caption": "(A) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (B) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the Cdc42-borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ****P<0.0001. (C) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of borg4 and septin 7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (D) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the borg4-septin 7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ***P<0.001, ****P<0.0001. ",
    "molecules": "CASIN, DAPI"
  },
  {
    "caption": "(A,B) Representative immunofluorescence microscopy images of the distribution of septin 7 (red), Cdc42 (red) and tubulin (green) in LT-HSCs from borg4fl/fl or borg4∆/∆ mice. Nuclei were stained with DAPI (blue), scale bar= 5 µm. (C) Percentage of cells with a polar distribution of septin 7, Cdc42 or tubulin in borg4fl/fl or borg4∆/∆ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, ***P<0.001, **P<0.01, *P<0.05. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(D,E) Representative immunofluorescence microscopy images of the distribution of borg4 (red), Cdc42 (red) or tubulin (green) in LT-HSCs from septin 7fl/fl or septin 7∆/∆ mice. Nuclei were stained with DAPI (blue). scale bar= 5 µm. (F) Percentage of cells with a polar distribution of borg4, Cdc42 or tubulin in septin 7fl/fl or septin 7∆/∆ LT-HSCs. At least 3 biological repeats, at least 30 to 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, **P<0.01, *P<0.05. ",
    "molecules": "DAPI"
  },
  {
    "caption": "B Analysis of cell differentiation  (left) and proliferation  (right) in the intestine  after CDK8  deletion as in (A). Olfm4, Lysozyme, PAS  and Dclk1 staining  was used to reveal, respectively, stem, Paneth, goblet  and tuft cells. β-Catenin staining  allows detection of cancer cells (cytoplasmic  vs nuclear localisation). Cell proliferation was assessed by PCNA, Ki-67 and BrdU  (2h pulse) staining. Scatter plots represent the percentage of the area stained  by each antibody (relative to the area occupied by hematoxylin). For Paneth  cells, BrdU, PCNA   and Ki67, only crypts  were analysed. For  goblet  cells, crypts  and villi  were analysed. For Tuft cell quantification, Dclk1-positive cells were counted in 50 villi. Colour code depicts small intestine (green), proximal colon  (blue), and distant colon  (red). Mean ± SEM is shown. P-value of unpaired two-tailed t-test is indicated (ns, not significant; p > 0.05). Scale bars, 25μm (Olfm4, Lysozyme, Dclk1  and  β-Catenin) and 50μm (PAS, BrdU,  Ki-67 and PCNA). (n=9 biological replicates)",
    "molecules": "PAS, BrdU, hematoxylin"
  },
  {
    "caption": "C Analysis of mouse colon  after AOM/DSS  treatment. Quantification of the number of neoplastic lesions  (n = 10 for Cdk8+/+, and n = 7 for Cdk8 -/- mice). P-value of unpaired t-test is indicated: ns, not significant (p > 0.05). Mean ± SD is shown.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "A Genotyping  confirms the loss of Cdk8  exon 2 in Cdk8 -/- and Cdk8 -/-/Cdk19 -/- organoids after 7 days of OH-tamoxifen  treatment.",
    "molecules": "OH-tamoxifen"
  },
  {
    "caption": "B WB  of organoid samples after 7 days of OH-tamoxifen treatment; β-actin  was used as loading control.",
    "molecules": "OH-tamoxifen"
  },
  {
    "caption": "C  Phase contrast images of organoids  before and after 6 days of OH-tamoxifen  treatment. Scale bars, 150μm. D Quantification of organoid  size at day 6, shown in (C) (mean ± SEM are shown). P-value, one-way ANOVA (Bonferroni test): (*) p ≤ 0.05, (***) p ≤ 0.001; ns, not significant (p > 0.05). (n=47 biological replicates)",
    "molecules": "OH-tamoxifen"
  },
  {
    "caption": "E IHC staining  of organoids  (day 7 of OH-tamoxifen treatment) with  Ki-67  antibody. Scale bars, 100μm. F Quantification of Ki-67  positive area (% of the total area of the organoids; mean ± SEM) in the four different genotypes presented in (E). Areas with positive  Ki-67 signal were detected and quantified using QuPath and ImageJ programs. Four technical replicates from 2 biological replicates of each genotype were analysed. Adjusted p-values of ordinary one-way ANOVA (Bonferroni test) are indicated: (**) p-values ≤ 0.01; (*) p-values ≤ 0.05; ns, not significant (p > 0.05).",
    "molecules": "OH-tamoxifen"
  },
  {
    "caption": "C Left, IHC staining  of WT and Cdk8 -/-/Cdk19 -/- organoids  with Olfm4  (stem cells), Lysozyme  (Paneth cells), Muc2  (goblet cells), and Dclk1  (tuft cells) antibodies. Scale bars, 50μm (Olfm4, Muc2, Dclk1) and 25μm (Lysozyme). Right, quantification of Olfm4, Lysozyme  and Muc2  positive area (% of the total organoid   area as indicated by hematoxylin staining; mean ± SEM), or the number of Dclk1  positive cells per organoid. P-value, unpaired two-tailed t-test: (***) p-value ≤ 0.001; ns, not significant (p > 0.05). 20 different organoids  from 2 biological replicates of each genotype were used for the analysis.",
    "molecules": "hematoxylin"
  },
  {
    "caption": "D WB  of indicated proteins extracted from Wt and Cdk8 -/-/Cdk19 -/- organoids. Included are CFTR  positive and negative control cell line samples obtained from the Cystic Fibrosis  Foundation (CFF). Amidoblack  was used as loading control.",
    "molecules": "Amidoblack"
  },
  {
    "caption": "A Histological PAS staining  of organoids  treated for 7 days with OH-tamoxifen. Scale bar, 50μm. B Quantification of PAS  signal (% of total organoid  area; mean ± SD are shown) in the four different genotypes presented in (A). Areas containing positive PAS staining  were detected and quantified using QuPath and ImageJ programs. Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05). Four technical replicates from 2 biological replicates of each genotype were analysed.",
    "molecules": "PAS, OH-tamoxifen"
  },
  {
    "caption": "C Representative  phase contrast  images of organoids  at the indicated  time points  after 7 days of  OH-tamoxifen treatment are shown. Scale bar, 100 μm. D Quantification of the time  needed for mucus  release (observed as a dark staining  in the center of the organoid; mean ± SD are shown). Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05); (n= 17 for Wt, 11 for Cdk8 -/-, 18 for Cdk19 -/-, and 12 for Cdk8 -/-/ Cdk19 -/- , all biological replicates).",
    "molecules": "OH-tamoxifen"
  },
  {
    "caption": "E Fluorescence confocal microscopy images of Calcein  green-labeled WT and Cdk8-/-/Cdk19-/- organoids  treated with forskolin. Scale bars, 100 μm. F Quantification of forskolin-induced swelling  in WT organoids  treated for 1hour or 24 hours with 0.1μM, 1μM or 10μM Senexin B  (SenB), as indicated, or double KO organoids; DMSO  vehicle was used as control. The surface area of individual organoids  at different time points relative to the area at t = 0 (100%) was measured (mean ± SD, n=8 biological replicates). Linear regression lines are shown.",
    "molecules": "Calcein  green, DMSO, forskolin, SenB, Senexin B"
  },
  {
    "caption": "H WB  of indicated proteins extracted from Senexin B-treated (10μM, 2h and 24h) organoids  (left), and WT and Cdk8 -/-/ Cdk19 -/- organoids  (right). Amidoblack  was used as loading control.",
    "molecules": "Amidoblack, Senexin B"
  },
  {
    "caption": "A WB  of indicated proteins extracted from intestinal epithelium of control mice  or mice  treated with Senexin 631  (Snx 631) for 4 or 11 days.",
    "molecules": "Senexin 631, Snx 631"
  },
  {
    "caption": "B Left, immunohistochemical PAS  and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 (Snx 631) treatment; scale bars 50μm. Right, quantification of PAS- and hematoxilin  (Hema)-positive area (mean ± SEM), using QuPath and ImageJ software. Adjusted p-values of unpaired t-test are shown: (*) p < 0.05; ns, not significant (p > 0.05). 8 mice  (4 treated with Senexin 631 and and 4 control) were used in this quantification.",
    "molecules": "PAS, Senexin 631, Snx 631, Hema, hematoxilin, hematoxylin"
  },
  {
    "caption": "A. Viability analysis of GICs treated with the α-AR antagonists prazosin, ARC 239, doxazosin, BMY 7378, and terazosin. GICs were treated with the antagonists or corresponding vehicles for 72 h, and viability was assayed using WST-1. *P<0.05, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "ARC 239, BMY 7378, doxazosin, prazosin, terazosin"
  },
  {
    "caption": "B. Quantification of GIC survival using trypan blue exclusion after 24 h and 72 h of treatment with prazosin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin"
  },
  {
    "caption": "C. Viability analysis of patient-derived GICs (TG1, TG16, GBM5, GBM44) and NSCs (NSC24, NSC25, NSC5031, NSC8853) treated with prazosin for 72 h. *P=0.0286, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin"
  },
  {
    "caption": "D. Analysis of the sphere-forming capabilities of GICs using the extreme limiting dilution assay. Cells were seeded in presence of vehicle or 30 µM prazosin (PRZ). Sphere formation was scored 10 days post-seeding. Frequency of sphere-forming cells: Control=1/3.88 (lower 8.61, upper 1.95); prazosin 1/248 (lower 1003, upper 62), n=12, p= 1.13 10-10. Overall test for difference in stem cell number between groups.",
    "molecules": "prazosin, PRZ"
  },
  {
    "caption": "E. Viability analysis of GICs treated with prazosin after sorting cells according to their expression of EGFR, and the neural stem cell markers CD15 and CD133. Prazosin inhibits cell viability regardless of CD133 or CD15 expression. *P=0.0286, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "CD15, Prazosin, prazosin"
  },
  {
    "caption": "G. Viability analysis of differentiated NSCs and GICs treated with prazosin for 72 h. Diff: differentiation. *P=0.0286, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin"
  },
  {
    "caption": "B, C. In vivo effect of prazosin treatment (1.5mg/Kg) on glioblastoma growth. Tumors were initiated with GBM44 GICs (B) or GMB5 GICs (C). Left panels: Bioluminescent in vivo images of tumors in mice treated with prazosin (PRZ) or vehicle for 45 days. Middle panels: Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P=0.0002 and *P=0.003 for GBM44 and GBM5 respectively, n=8, two-sided Mann-Whitney U-test. Right panels: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle, log-rank Mantel-Cox test. The treatment period is shaded in gray.",
    "molecules": "prazosin, PRZ"
  },
  {
    "caption": "D. Example of hematoxilin/eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin (PRZ) or vehicle. i: tumor infiltration. n: tumor necrosis. Scale bar = 2 mm. GBM44 GICs.",
    "molecules": "prazosin, PRZ"
  },
  {
    "caption": "E. Left panel: Representative flow cytometry plots depicting the percentage of CD133+ glioblastoma cells (GFP+) isolated from mice treated with prazosin (PRZ) or vehicle. Tumors were initiated with GBM44 GICs. Right panel: Quantification of flow cytometry analyses of CD133 expression by GFP+-tumor cells isolated from xenografts of three prazosin-treated (PRZ) mice and three vehicle-treated mice. *P=0.0003, n=6, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin, PRZ"
  },
  {
    "caption": "F. Secondary grafting of tumor cells (GBM44-GFP+) isolated from vehicle or prazosin-treated mice bearing primary tumors. Mice injected with glioblastoma cells isolated from prazosin-treated mice developed tumors at a lower frequency (50% versus 100%). Moreover, mice injected with glioblastoma cells isolated from prazosin-treated mice presented a statistically significant survival benefit. Left Panel: Bioluminescent in vivo images of secondary grafts. Middle panel: Quantification of the bioluminescent signals. *P=0.0004, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle- and prazosin-treated mice.",
    "molecules": "prazosin"
  },
  {
    "caption": "G. Inhibitory effect of low doses of prazosin (0.15 mg/Kg) on glioblastoma growth in vivo. Left panel: Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin (PRZ) for 45 days. Tumors were initiated with GBM44 GICs (compare with panel B). Middle panel: Quantification of the bioluminescent signals. *P=0.0007, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin, log-rank Mantel-Cox test. The treatment period is shaded in gray.",
    "molecules": "prazosin, PRZ"
  },
  {
    "caption": "A. Viability analysis of GL261 treated with prazosin in vitro. *P=0.0286, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin"
  },
  {
    "caption": "C. Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma. Left panel: Bioluminescent in vivo images of tumors in C57/Bl6mice treated with vehicle or prazosin (PRZ) for 14 days.Middle panel: Quantification of the bioluminescent signals. *P=0.0002, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle. log-rank Mantel-Cox test. The treatment period is shaded in gray.",
    "molecules": "Prazosin, prazosin, PRZ"
  },
  {
    "caption": "D. Hematoxilin/eosin staining of C57/Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin. Scale bar = 2 mm.",
    "molecules": "prazosin"
  },
  {
    "caption": "E. Fluorescence imaging of GL261 intra-striatal grafts at 1 and 2 h after an intra-peritoneal injection of BODIPY FL prazosin, the green-fluorescent derivative of prazosin. Scale bar = 2 mm.",
    "molecules": "prazosin"
  },
  {
    "caption": "A. Immunoblotting for pro-caspase-3 (pro-CASP3) and active caspase-3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase-3. ZVAD, a caspase inhibitor, prevents prazosin-induced caspase-3 activation. kDa: kilodaltons.",
    "molecules": "prazosin, PRZ, ZVAD"
  },
  {
    "caption": "B. Immunoblotting for pro-caspase-9 (pro-CASP9) and active caspase-9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase-9.",
    "molecules": "prazosin, PRZ"
  },
  {
    "caption": "C. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin-induced GIC death. *P=0.0286, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin, ZVAD"
  },
  {
    "caption": "D. Prazosin induces glioblastoma cell apoptosis in vivo. GFP+ and GFP- cells were analyzed from tumors in vehicle or prazosin-treated mice. Annexin V / DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation.",
    "molecules": "Prazosin, prazosin"
  },
  {
    "caption": "E. Dose-response curve of prazosin on GIC survival (24h treatment). EC50 was determined with curve fit using nonlinear regression.",
    "molecules": "prazosin"
  },
  {
    "caption": "F. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline, a subtype agonist of the α-ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin-induced GIC death. *P<0.05, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "cirazoline, Cirazoline, prazosin"
  },
  {
    "caption": "G. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing 1-AR. PRZ: prazosin. CRZ: cirazoline.",
    "molecules": "cirazoline, CRZ, prazosin, PRZ"
  },
  {
    "caption": "H. Immunoblotting for phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs.",
    "molecules": "prazosin, Prazosin"
  },
  {
    "caption": "I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs.",
    "molecules": "U0126, prazosin"
  },
  {
    "caption": "J. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 did not counteract prazosin-induced GIC death. Blockade of prazosin-induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin.",
    "molecules": "U0126, prazosin, PRZ"
  },
  {
    "caption": "B. PKCδ by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin-treated (PRZ, 5µM) GICs, as compared to vehicle-treated GICs. PRZ: prazosin. Scale bar: 10 µm.",
    "molecules": "prazosin, PRZ"
  },
  {
    "caption": "C. Total PKCδ (PKCδ 78kDa) and cleaved PKCδ (PKCδ 41kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41kDa active fragment. 2 µM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin-induced PKCδ cleavage. V: vehicle. kDa: kilodaltons.",
    "molecules": "Prazosin, prazosin, PRZ, Rott, rottlerin"
  },
  {
    "caption": "D. Pro-caspase-3 (Pro-CASP3) and activated caspase-3 (CASP3) expression analysis in GICs by immunoblotting. 2 µM rottlerin (Rott) inhibits prazosin (PRZ)-induced caspase-3 activation. V: vehicle. kDa: kilodaltons.",
    "molecules": "prazosin, PRZ, Rott, rottlerin"
  },
  {
    "caption": "E. Inhibition of PKCδ by rottlerin prevents, in a concentration-dependent manner, prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin, rottlerin"
  },
  {
    "caption": "F. Inhibition of PKCδ by the specific anti-PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for Prazosin 1 and 5 µM and P<0.001 for Prazosin 10 µM by two-tailed unpaired Student's t-test, n=4.",
    "molecules": "δV1.1, prazosin, Prazosin"
  },
  {
    "caption": "G. Decreased PKCδ expression using shRNA counteracts prazosin-induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two-tailed unpaired Student's t-test, n=4.",
    "molecules": "prazosin"
  },
  {
    "caption": "H. Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 µM), an inhibitor of the PI3K/AKT pathway, was used as positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P-AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons.",
    "molecules": "LY294002, Prazosin, Terazosin"
  },
  {
    "caption": "I. Prazosin (10 µM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.",
    "molecules": "Prazosin"
  },
  {
    "caption": "J. β-catenin expression, a downstream target of AKT, is decreased in prazosin-treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.",
    "molecules": "prazosin"
  },
  {
    "caption": "K. L. Inhibition of PKCδ using rottlerin (Rott, 2 µM) or δV1.1 (10 µM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.",
    "molecules": "δV1.1, prazosin, Rott, rottlerin"
  },
  {
    "caption": "B. Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA. All mice were treated with prazosin (PRZ) for 45 days.C. Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth. Fold change in total flux represents the ratio: total flux after treatment/total flux prior treatment. *P=0.0007, n=8, two-sided Mann-Whitney U-test.",
    "molecules": "prazosin"
  },
  {
    "caption": "Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1-CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group.",
    "molecules": "oleate"
  },
  {
    "caption": "Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor associated macrophages (TAMs) phenotype, MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay. Data are expressed as mean±SD from two to four independent experiments. Unpaired Student's two-tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups. *p<0.05; **p≤0.01",
    "molecules": "lipid, oleate"
  },
  {
    "caption": "Gr1-CD11b+ cells, polarized by the indicated treatment, were purified for mitochondrial respiration detection. The oxygen consumption rate (OCR) of these cells was monitored after the addition of oligomycin (OA) (1 µM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA) (0.5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.",
    "molecules": "AA, antimycin A, ATP, Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, FCCP, oxygen, OA, oligomycin, rotenone"
  },
  {
    "caption": "Forty µM etomoxir were added starting from day 0 of bone marrow polarization, followed by mitochondrial respiration assay by using the same amount of polarized Gr1-CD11b+ myeloid cells on day 7. The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer.",
    "molecules": "etomoxir"
  },
  {
    "caption": "NO production from the co-culture supernatant was quantified by Griess reaction.",
    "molecules": "NO"
  },
  {
    "caption": "The oxygen consumption rate (OCR) of 1×105 purified Gr1-CD11b+ cells was monitored as described in Figure 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.",
    "molecules": "ATP, oxygen"
  },
  {
    "caption": "The lipid droplets accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index were calculated.",
    "molecules": "BODIPY"
  },
  {
    "caption": "Nitric oxide (NO) concentration in the co-culture supernatant was quantified by Griess reaction.",
    "molecules": "Nitric oxide, NO"
  },
  {
    "caption": "Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression.",
    "molecules": "Rapa, rapamycin, oleate"
  },
  {
    "caption": "Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection.",
    "molecules": "nitric oxide, NO"
  },
  {
    "caption": "The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified.",
    "molecules": "AA, antimycin A, ATP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, FCCP, oxygen, OA, oligomycin, rotenone"
  },
  {
    "caption": "Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206+ (black) and CD206- (grey) cells.",
    "molecules": "BODIPY"
  },
  {
    "caption": "5×105 MCA205 were injected subcutaneously into C57BL/6 mice. PBS, liposome control or iDGAT-liposome was injected into the peritoneum every other day starting at day 7. Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis. Immune cells from spleen, tumor-draining lymph nodes (TDLN) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells.",
    "molecules": "BODIPY"
  },
  {
    "caption": "Tumor infiltrating CD8+ T cells (red) were stained together with nuclei (DAPI/blue) after sacrificing the tumor bearing mice.",
    "molecules": "DAPI"
  },
  {
    "caption": "1×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry.",
    "molecules": "stearate, oleate"
  },
  {
    "caption": "The level of lipid droplets was determined via BODIPY staining.",
    "molecules": "BODIPY"
  },
  {
    "caption": "The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods.",
    "molecules": "oxygen"
  },
  {
    "caption": "(D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay.",
    "molecules": "CFSE"
  },
  {
    "caption": "Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Genome views of RNA read density  profiles on representative loci enriched (left) or low (right) in FLAG-HP1γ RNAchIP (IP) relative to input RNA (inp.) in overlaid replicate samples from HP1γ cells stimulated or not with PMA. Orange arrow, position of the locus analyzed in D.",
    "molecules": "PMA"
  },
  {
    "caption": "F, Gel mobility shift assays of bacterially expressed, purified HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled  RNA  oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.",
    "molecules": "Cy3"
  },
  {
    "caption": "G, Gel mobility shift assays of bacterially expressed, purified  HP1γ-Hinge domain, fused to GST proteins, and tested for its direct interaction with the Cy3-labeled dsDNA oligonucleotide probes  The probes in the gels, either free (white arrowheads) or as shifted  protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.",
    "molecules": "Cy3"
  },
  {
    "caption": "H. Gel mobility shift assays of bacterially expressed, purified  HP1γ-Hinge domain, fused to GST proteins, and tested for its direct  interaction  with the Cy3-labeled RNA  oligonucleotide probes The probes in the gels, either free (white arrowheads) or as shifted protein/RNA complexes (red arrowheads) were detected by their Cy3 fluorescence. Representative of 3 independent experiments.",
    "molecules": "Cy3"
  },
  {
    "caption": "H. North-western blot assay showing direct association between the indicated bacterially expressed, purified GST-fusion proteins and in vitro transcribed, biotinylated RNA probes based on the sequence of the B4 SINE depicted in (G). Top panels, binding of a 256nt probe corresponding to the CACACA-containing B4 SINE sequence, was compared to an identical B4 SINE deleted of its CACACA by truncation of its 3' portion (B4 SINE-Δ3'). RNA probes hybridized on the membranes were detected by their Cy3 fluorescence. Total GST-fusion protein loading was visualized by Ponceau S staining. Representative of two independent experiments.",
    "molecules": "Cy3"
  },
  {
    "caption": "A. Box plot showing the distribution of fold enrichment values per genes sorted by exon number, n= 66, 247, 389, and 574 genes with 1, 2, 3, and 4 exons, respectively. Dashed line represents an  IP/Input ratio of 1, taken as a reference. B. Reads from input and  IP counted in unstimulated and PMA  stimulated triplicates give significant enrichment values of 1.4 and 0.7 on introns and exons, respectively, shown as mean and s.d., n=3 biological replicates. Dots represent individual data points. C. Box plot of reads belonging to spliced transcripts versus all reads in input or RNAchIP samples pooled between + and -PMA, calculated as a ratio between split reads and total reads, n=3 biological replicates. Dots represent individual data points. Data information: All box boundaries represent 25th and 75th percentiles; center line represents median; whiskers indicate ±1.5×IQR; points are values of outliers. All P-values are * P < 0.01, ** P < 0.005, ***P < 0.001; two-tailed Student's t-test.",
    "molecules": "PMA"
  },
  {
    "caption": "D. RNAchIP  read density per kb on the indicated introns for input and  IP in samples from both untreated and  PMA stimulated cells, n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Dots represent individual data points. HP1γ/KO introns are all 272 intronic intervals between exon junctions  differentially detected in HP1γ  and KO. Random introns are a matching library of random intronic intervals. E.  RNAchIP read density per kb on the genes corresponding to the introns tested in (D), and analyzed as in (D), n=6 biological replicates, or 3-times randomized intervals, n=18 samples. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; two-tailed Student's t-test).",
    "molecules": "PMA"
  },
  {
    "caption": "F. Reads from input and  IP  counted on the indicated features in combined unstimulated and PMA stimulated triplicate samples, n=6 biological replicates. Dots represent individual data points. Data information: All graphes represent mean and s.d. (** P < 0.005, ***P < 0.001; one-tailed Student's t-test).",
    "molecules": "PMA"
  },
  {
    "caption": "(C-F) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from brain (C and E) and spinal cord (D and F) of 16-month-old WT and Tmem106b-/- mice. n=5. Data presented as mean ± SEM. Unpaired Student's t test. *, p<0.05, ***, p<0.001, ****, p<0.0001.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "(A, B) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from spinal cord (C5-C8) and brain of 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Spinal cord: n=3; brain: n=4. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05; **, p<0.01***, p<0.001, ****, p<0.0001.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "(C, D) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. The number of p62 or Ub positive puncta was quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "d, Representative images show cilia segmentation based on acetylated tubulin  (Ac tub), polyglutamylated tubulin  (Glu tub), ARL13B and PCNT. The cartoon on the left indicates proximal segments (PS, Glu tub rich-region) and distal segments (DS). DS were determined by calculating the difference between  PS  and total  cilia  length based on ARL13B staining as depicted by the dashed lines on the images. Scale bar: 1.5µm. e, Quantification of d. Comparative analysis of average  cilia length based on Ac tubulin and ARL13B (cilia) staining  shown for specified cilia length groups (2<, 2-3, 3-4 and >4µm). Three biological replicates, n=400 cilia per sample and repetition. The numbers above the cartoons show the ratio of the calculated Ac tub/whole  cilia length. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test.",
    "molecules": "Ac, acetylated, Glu, polyglutamylated"
  },
  {
    "caption": "a, Representative images of 48h serum starved RPE1 cells treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before  immunostaining with the indicated antibodies. Scale bar: 3µm. b, Quantification of (a) showing average length of proximal segment (PS,  Glu tub) and whole cilia (ARL13B). The numbers above the cartoons show the ratio of the calculated distal segment  (DS)/whole cilia length. Three biological replicates, n=100 cilia  per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.",
    "molecules": "cytochalasin D, CytoD, DMSO, Glu, Noco, nocodazole"
  },
  {
    "caption": "e-f, Cells were treated with the indicated siRNAs before  serum starvation for 48h and treatment with DMSO or Noco for 3h before  immunostaining with the indicated antibodies. Representative images (e) and quantification of cilia  length based on ARL13B (f) are shown. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.",
    "molecules": "DMSO, Noco"
  },
  {
    "caption": "f, Effect of nocodazole (Noco) treatment (3h) on the  localisation of endogenous SEPT2 and  SEPT7 at cilia in RPE1 cells. The insets show magnifications of the  cilium. Scale bar: 5µm (large) and 2.5µm (small). g, Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from (f). SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length. Three biological replicates, n>90  cilia per sample and repetition. Data show in (g) includes mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.",
    "molecules": "Noco, nocodazole"
  },
  {
    "caption": "i-j, RPE1 WT and SEPT2-KO cells after 48h SS were treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole  (Noco) for 3h before  immunostaining  with the indicated antibodies. Representative images (i) and quantifications of  PS  (Glu tub) and whole cilia (ARL13B) length (j) are shown. The numbers above the cartoons in (j) show the ratio of the calculated DS/whole cilia length. Scale bar: 3µm. Three biological replicates, n=100  cilia per sample and repetition. Data show include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test",
    "molecules": "cytochalasin D, CytoD, DMSO, Glu, Noco, nocodazole"
  },
  {
    "caption": "c-d, KIF7 localisation  at the  tip and base of cilia (indicated by arrows) in  RPE1 cells after 48h SS treated for 3h with solvent control (DMSO) or nocodazole  (Noco) and quantification of the relative signal intensities for the  ciliary base  and tip of KIF7  (d). Scale bar: 2µm. KIF7 signal intensity was quantified at the ciliary tip or base as indicated in (c) (white arrows). Three biological replicates, at least 80  cilia  per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.",
    "molecules": "DMSO, Noco, nocodazole"
  },
  {
    "caption": "e-f, RPE1 WT and SEPT2-KO cells were treated with control or KIF7  siRNA after 48h SS  and  immunostained with the indicated antibodies. Representative images (e) and quantifications of proximal segment  (Glu tub) and whole  cilia (ARL13B) length (f) are shown. The numbers above the cartoons in (f) show the ratio of the calculated  distal segments /whole  cilia length. Three biological replicates, n=100  cilia per sample and repetition. Scale bar: 2µm. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.",
    "molecules": "Glu"
  },
  {
    "caption": "f-g, Influence of MKS3  depletion on  cilia length of  RPE WT and SEPT2-KO cells. Cells are treated with the indicated siRNA and  serum starved (SS) for 48h before  immunostaining  with the indicated antibodies. Representative images (f) and quantifications of proximal segment  (Glu tub) and whole cilia (ARL13B) length (g) are shown. The numbers above the cartoons in (g) show the ratio of the calculated distal segments/whole cilia  length. Scale bar: 2µm. Three 3 biological replicates, n=100 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.",
    "molecules": "Glu"
  },
  {
    "caption": "g-h, RPE1 SEPT2-KO cells expressing  ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained  with Glu-tub antibody. Representative images (g) and quantifications of  proximal segment (Glu tub), EFHC1  and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100  cilia per sample and repetition. Scale bar: 2µm.",
    "molecules": "Glu"
  },
  {
    "caption": "Gel shift assay of biotin-labeled mascRNA and 5S rRNA with the cytosol (Cyto) and the postnuclear membrane lysate (Mem).",
    "molecules": "biotin"
  },
  {
    "caption": "SDS-PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples. Biotin-labeled mascRNA (S) and the antisense RNA (AS) were used to pull down the RNA-protein complexes.   Representive data of mass spectrometry analysis of the RNA pulldown samples. Proteins most enriched in mascRNA (S) RNA pulldown sample are listed.   ",
    "molecules": "Biotin, SDS"
  },
  {
    "caption": "Gel shift assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.",
    "molecules": "biotin"
  },
  {
    "caption": "Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the acceptor stem mutants (Mut1-8) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.   Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the anticodon stem-loop mutants (Anti-Mut1-9) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.   ",
    "molecules": "Biotin, biotin"
  },
  {
    "caption": "Gel shift assay of biotin-labeled mascRNA or the mutants (Anti-Mut1 and Mut1) with the cytosol.",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel C .   ",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA. Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel F.   ",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.   Quantification of the levels of the labeled nascent proteins in Panel I.   ",
    "molecules": "biotin"
  },
  {
    "caption": "Decay of the pulse Biotin-labeled proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambed RNA (Scramble)",
    "molecules": "Biotin"
  },
  {
    "caption": "Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells, before and after emetine and cycloheximide treatment. β-Tubulin was used as a loading control.   Quantification of the relative QARS protein levels in Panel J.   Quantification of the relative GARS protein levels in Panel J.   ",
    "molecules": "cycloheximide, emetine"
  },
  {
    "caption": "Detection of the newly synthesized proteins in HEK cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel B.   ",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel E.   ",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in mascRNA Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in mascRNA Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins. Quantification of the labeled nascent protein levels in Panel H.",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in wild-type HEK cells (WT) and MALAT1 knockdown cells (sh-MALAT1), or MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in wild-type HEK cells (WT) and MALAT1 knockdown cells (sh-MALAT1), or MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins. Quantification of the labeled nascent protein levels in Panel K.",
    "molecules": "biotin"
  },
  {
    "caption": "Detection of the newly synthesized proteins in QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the scrambled RNA (Scramble). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.   Quantification of the labeled nascent protein levels in Panel M.   ",
    "molecules": "biotin"
  },
  {
    "caption": "Soft agar colony formation assay of HEK, HeLa or MCF-7 cells overexpressing mascRNA or expressing the the scrambled RNA.   Quantification of stained colones in Panel C.   ",
    "molecules": "agar"
  },
  {
    "caption": "Soft agar colony formation assay of HEK cells overexpressing mascRNA, or expressing the mutants (Anti-Mut1 and Mut1) or the the scrambled RNA (Scramble).",
    "molecules": "agar"
  },
  {
    "caption": "Soft agar colony formation assay of QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the the scrambled RNA (Scramble).",
    "molecules": "agar"
  },
  {
    "caption": "Soft agar colony formation assay of MALAT1 knockdown cells (shMALAT1) and the control cells (Control) with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ).",
    "molecules": "agar"
  },
  {
    "caption": "(A) The graphs depict the  Aβ  profiles generated by WT or mutant PSEN1 MEF  cell lines transduced with adenovirus  encoding APPC99. ELISA  quantified secreted Aβ37, Aβ38, Aβ40  and Aβ42  peptide levels are shown as % of total Aβ  (sum of the four peptides). The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40  and Aβ42  peptides in the WT Aβ  profile, respectively. Data are presented as mean ± SD, N ≥ 3 independent experiments.",
    "molecules": "Aβ37, Aβ38, Aβ, Aβ40, Aβ42"
  },
  {
    "caption": "(B) GSEC processivity levels for the cleavage of APPC99 determined in WT and mutant PSEN1/GSEC  cell lines. The mutant Aβ(37+38)/(40+42) ratios are shown as % of the WT cell line. Data presented as mean ± SD, N ≥ 3 independent experiments. As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05); **P < 0.01, ***P < 0.001, ****P < 0.0001.",
    "molecules": "Aβ"
  },
  {
    "caption": "(A)  Aβ profiles  generated by WT or mutant PSEN1 MEFs  lines treated with 1 µM GSM III. The chemical structure of the bicyclic heterocycle  GSM III  is depicted. Data are presented as mean ± SD, N ≥ 3 independent experiments. The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40  and Aβ42  peptides in the WT  Aβ profile, respectively.",
    "molecules": "Aβ37, Aβ38, Aβ40, Aβ42, GSM III"
  },
  {
    "caption": "(B) Fold change in GSEC  processivity of  APPC99 estimated by the Aβ(37+38)/(40+42) ratio and presented as % of the WT response. The data are presented as mean ± SD, N ≥ 3. The symbol # marks mutations that fully abolished the GSEC  response towards GSM III, As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). **P < 0.0001, ****P < 0.0001 (F(DFn, DFd): F (32, 101) = 112.5.",
    "molecules": "Aβ, GSM III"
  },
  {
    "caption": "(D) Aβ profiles generated by WT or mutant PSEN1  cell lines overexpressing APPC99 and treated with increasing concentrations of GSM III. Data are presented as mean ± SD, N = 2 independent experiments.",
    "molecules": "GSM III"
  },
  {
    "caption": "(E) GSEC  processivity estimated by the Aβ(37+38)/(40+42) ratio was determined for the  dose-response  analysis (presented in panel D) and shown as % of DMSO. Data are presented as mean ± SD.",
    "molecules": "Aβ, DMSO"
  },
  {
    "caption": "(A) Aβ profiles generated by WT and indicated mutant MEF  lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.",
    "molecules": "DMSO, GSM III"
  },
  {
    "caption": "(B) WT and mutant GSEC  processivity estimated by the Aβ  (37+38)/(40+42) ratio and presented as % of WT. Data presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.",
    "molecules": "Aβ"
  },
  {
    "caption": "Aβ profiles generated by WT and indicated mutant MEF  lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III  or (C) 1 µM  GSM II. Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.",
    "molecules": "DMSO, GSM II, GSM III"
  },
  {
    "caption": "Analysis of mutant APP  substrates with regards to their effects on the response of GSEC  to the imidazole-based GSM III. Aβ profiles secreted by HEK293T  cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by ELISA  (D) ELISA-based Aβ  profiles are presented as % of total Aβ  (Aβ37+ Aβ38+ Aβ40+ Aβ42) (mean ± SD, N ≥ 3); One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.",
    "molecules": "Aβ37, Aβ38, Aβ, Aβ40, Aβ42, DMSO, GSM III, imidazole"
  },
  {
    "caption": "Analysis of mutant APP  substrates with regards to their effects on the response of GSEC  to the imidazole-based GSM III. Aβ profiles secreted by HEK293T  cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by mass spectrometry (MS). (E) APP  mutation-driven effects on GSEC  processivity according to the Aβ(37+38)/(40+42) ratio are presented as % of the WT condition (mean ± SD). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.",
    "molecules": "Aβ, DMSO, GSM III, imidazole"
  },
  {
    "caption": "(F) MS-based profiles show the proportion of individual Aβ  peptides on the total immunoprecipitated pool. Data shown as mean of 3 independent experiments.",
    "molecules": "Aβ"
  },
  {
    "caption": "(B) Aβ profiles generated by WT or mutant PSEN1  cell lines transduced with APPC99. Dotted lines indicate the levels of Aβ  peptides (%) in WT profiles. Data presented as mean ± SD, N ≥ 3 independent experiments.",
    "molecules": "Aβ"
  },
  {
    "caption": "(C) Pocket filling PSEN1  V236W and Y106W-V236W substitutions lead to increased Aβ(37+38)/(40+42) ratio, indicating the activation of the sequential GSEC-mediated cleavage of  APPC99. Data presented as mean ± SD, N ≥ 3 independent experiments.",
    "molecules": "Aβ"
  },
  {
    "caption": "(D)  Aβ profile  analysis in  dose-response experiments for  GSM III revealed no modulation of the PSEN1  V236W mutant cell line. Data are presented as mean ± SD, N = 2 independent experiments. For comparison purposes, WT and V236W mutant Aβ profiles are presented in panel 6B.",
    "molecules": "GSM III"
  },
  {
    "caption": "(E) GSEC  activity  assay using purified GSEC  and an APP-based fluorogenic peptide as substrate Upper panel: In the non-cleaved state the Dpn  (2,4-dinitrophenyl) quencher group suppresses fluorescent emission from the Nma  (N-methyl-o-aminobenzoic acid) fluorophore through  Förster resonance energy transfer (FRET). Upon GSEC  cleavage, the Nma  group emits at λ = 430 nm a fluorescent signal upon excitation at λ = 355 nm. Lower panel: Specific activities for the purified WT or mutant GSECs   (bearing the indicated substitutions in PSEN1) treated with DMSO  (vehicle), 10 µM GSMIII or GSM II. Data are normalized to the WT GSEC  activity in DMSO  (vehicle). Data are shown as mean ± SD, N ≥ 3 independent experiments. Significance is shown for meaningful comparisons; two-way ANOVA with comparison to WT was used to determine statistical significance; **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.",
    "molecules": "2,4-dinitrophenyl, Dpn, DMSO, GSM II, GSMIII, N-methyl-o-aminobenzoic acid, Nma"
  },
  {
    "caption": "(F) Transition state analogue inhibitor  L-685,458 (Inhibitor X) treatment of WT, V236W or Y240W mutant PSEN1  cell lines. Inhibitory profiles  and derived IC50 values (table) indicate that the PSEN1-V236W significantly increases the affinity for the inhibitor. Dotted line indicates 50% inhibition. Data presented as mean ± SD, N = 3 independent experiments.",
    "molecules": "L-685,458"
  },
  {
    "caption": "A, A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold.",
    "molecules": "leptin"
  },
  {
    "caption": "B, MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n=3.",
    "molecules": "leptin"
  },
  {
    "caption": "C, NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 hours post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies.",
    "molecules": "agarose"
  },
  {
    "caption": "D, OTUB1 interacts with wt NRAS and active NRAS mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V).",
    "molecules": "agarose"
  },
  {
    "caption": "E, GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies.",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "F, OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). C, D, E, F, Immunoprecipitates, IP. Whole cell lysate, WCL.",
    "molecules": "agarose"
  },
  {
    "caption": "A, Suppression of OTUB1 expression increases NRAS mono- and di-ubiquitination. 6xHis-tagged ubiquitin and Flag-NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1. Ubiquitinated NRAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.",
    "molecules": "6xHis, Co2+"
  },
  {
    "caption": "B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.",
    "molecules": "6xHis, Co2+"
  },
  {
    "caption": "A, B, OTUB1 overexpression promotes serum-induced activation of endogenous wt RAS (A) or wt NRAS (B). GTP-bound RAS was pulled down from HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) using recombinant RAF1 RBD conjugated to agarose beads. Input was controlled by immunoblotting using anti-panRAS or anti-Flag antibodies",
    "molecules": "agarose, GTP"
  },
  {
    "caption": "A subplantar injection of 5% of formalin  (20 µl) was administered in the right  hind paw of Balb/c mice  and an equal volume of PBS  was injected in left hind paw. After 1 hour of formalin  injection, Balb/c mice  were treated with a single dose of either PBS  (vehicle control) or Diclofenac  (10 mg/kg) or different concentrations of rPPE2 (2 mg/kg and 3 mg/kg) via intraperitoneal route and after three hours of treatment, mice  were sacrificed and the paw  sections were prepared and stained  with Toluidine blue to quantify mast cell population. (A) Photographs of representative sections were visualized at 20X magnification (scale bar = 50 µm). (B) Counting  of mast cells was performed in  Toluidine blue stained paw  sections using ImageJ software and were normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. For (B, unpaired t-test was applied to calculate p values.",
    "molecules": "Diclofenac, formalin, PBS, Toluidine blue"
  },
  {
    "caption": "(B-E) In another experiment, NIH-3T3 cells (B, C) or MEFs  (D, E) were incubated with either PBS  or rPPE2 (3 µg/ml). After 45 minutes of rPPE2 treatment, cells were fixed, permeabilized, and stained  with anti-PPE2  Ab. DAPI  was used to stain  the nucleus. Nuclear localization  of rPPE2  in NIH-3T3 (C) and MEFs  (E) was measured by calculating the colocalization  coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure C, E unpaired t-test was applied to calculate p values.",
    "molecules": "DAPI, PBS"
  },
  {
    "caption": "(F-G) (F) NIH-3T3  cells were transiently transfected with either pEGFP or pEGFP-PPE2. After 24 hours of transfection, cells were fixed and observed under a confocal microscope. DAPI  was used to stain  the nucleus. (G) Nuclear localization  of rPPE2  was measured by calculating colocalization  coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure G, unpaired t-test was applied to calculate p values.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Varying concentrations of rPPE2 protein were incubated with [γ-32P]-ATP  oligonucleotides of 60 bp spanning SCF  promoter.  DNA-protein  complexes were resolved on 7% native polyacrylamide  gel. In cold  competition reactions, equimolar cold/unlabeled 60 bp oligonucleotides were used (arrow represents PPE2-oligo complex). The data shown are representative of 3 independent experiments.",
    "molecules": "ATP, γ-32P"
  },
  {
    "caption": "A subplantar injection of 5% of formalin  (20 µl) was administered in the right  hind paw of Balb/c mice, and an equal volume of PBS  was injected in left hind paw. After induction of inflammation, Balb/c mice  were treated with PBS  or peptide  (8 mg/kg) via the intraperitoneal route. After 3 hours, paw  tissues were harvested and used for cDNA synthesis. B) (B) Next, Toluidine blue staining  was done to check the mast cell population. Photographs of representative sections were visualized at 20X magnification (scale bar = 100 µm).",
    "molecules": "peptide, formalin, PBS, Toluidine blue"
  },
  {
    "caption": "A subplantar injection of 5% of formalin  (20 µl) was administered in the right hind paw  of Balb/c mice, and an equal volume of PBS  was injected in left  hind paw. After induction of inflammation, Balb/c mice  were treated with PBS  or peptide  (8 mg/kg) via the intraperitoneal route. After 3 hours, paw  tissues were harvested and used for cDNA synthesis. (C) Counting of  mast cells  was performed in Toluidine blue-stained  paw sections using ImageJ software and was normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. unpaired t-test was applied to calculate p values.",
    "molecules": "peptide, formalin, PBS, Toluidine blue"
  },
  {
    "caption": "A, B Representative  time-lapse images (A) and kymograph  (B) of mouse zygotes  expressing H2B-RFP  (magenta) and labelled with SiR-Tubulin   (green). Shown is an example of marked chromosomal  displacement to the cortex. C Changes in chromosome-to-cell centre  distances (d) over time  relative to anaphase-onset (n = 50 blastomeres). Data information: In (A, time  is shown as h post-metaphase  and scale bar = 20 µm. In (C, data are shown as mean ± SEM.",
    "molecules": "SiR"
  },
  {
    "caption": "B, C Representative  time-lapse images of embryos  in which DMSO  (B) or nocodazole  (C) was added after anaphase  completion. D Changes in nucleus-to-cell centre distances (d) over time  relative to time  of drug addition following DMSO- (n = 22 blastomeres) or nocodazole-treatment (n = 26 blastomeres) at anaphase  completion. Data information: In (B,C, time  is shown as h post-drug addition and scale bar = 20 µm. In (D, data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).",
    "molecules": "DMSO, nocodazole"
  },
  {
    "caption": "E-G Representative  time-lapse images of embryos  in which DMSO  (E), Nocodazole  (F) or Cytochalasin D (G) was added when nuclei  had travelled to the cortex. H Changes in nucleus-to-cell centre  distances (d) over time  relative to time  of drug addition DMSO- (n = 20 blastomeres), nocodazole- (n = 12 blastomeres) or cytochalasin D-treatment (n = 14 blastomeres) after nuclei  reached the cortex. Data information: In E - G), time  is shown as h post-drug addition and scale bar = 20 µm. In H), data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).",
    "molecules": "Cytochalasin D, cytochalasin D, DMSO, Nocodazole, nocodazole"
  },
  {
    "caption": "A, B Representative time-lapse images of embryos  that have been washed out of either nocodazole  after nuclei  were prevented from travelling to the cortex  after anaphase  (A, n = 15 embryos) or  cytochalasin D  after nuclei   had been trapped at the cortex  by disrupting actin  (B, n = 22 embryos). C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst   stage or not. Each data point represents the mean maximal d value of the two blastomeres  from the same embryo  (n = 45 embryos). D Graph of time  taken to develop to the  blastocyst  stage in relation to maximal d values (n = 27 embryos). E Graph comparing the distribution of times  taken for nuclei  to re-centre in embryos  that developed to the blastocyst  stage or not (n = 45 embryos). F Graph of time  taken to develop to the blastocyst  stage in relation to times  taken for nuclei  to re-centre (n = 27 embryos). Data information: In (A, B), time  is shown as h post-drug washout and scale bar = 20 µm. In (C - F), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ns: not significant. In (D, F), the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test, ns: not significant.",
    "molecules": "cytochalasin D, nocodazole"
  },
  {
    "caption": "A Representative  time-lapse image of DNA  and SiR-tubulin  in DMSO- and BFA-treated embryos during the first mitotic division and subsequent interphase. Time  is shown as h relative to the first mitotic anaphase. B Changes in nucleus-to-cell centre  distances (d) over time  in BFA-treated embryos, relative to maximal nuclear  displacement to the cortex  (n = 14 blastomeres). Data information: In (A, scale bar = 20 µm.",
    "molecules": "SiR, BFA, DNA, DMSO"
  },
  {
    "caption": "D Representative  time-lapse images of the second mitotic division in DMSO- and BFA-treated embryos. Dashed white lines highlight position of nuclei  shortly before NEBD. Dashed yellow lines outline daughter-cells. Time  is shown as h relative to the second mitotic NEBD. E Dot plot of the duration between first mitotic division anaphase-onset and second mitotic division NEBD  for DMSO- (n = 47 blastomeres) and BFA-treated (n = 47 blastomeres) embryos. F Plot of the duration between NEBD  and anaphase  of the second mitotic division for DMSO- (n = 44 blastomeres) and BFA-treated (n = 43 blastomeres) embryos. G Plot of the spindle-to-cell centre distances against pre-NEBD nucleus-to-cell centre distance (n = 34 blastomeres). Line indicates mathematically regressed fit. Data information: In D), scale bar = 20 µm. In E - G, data are shown as mean ± SEM. In (E, F, statistical significance was assessed by Mann-Whitney test, ****P < 0.0001, ns: not significant. In (G, the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test. Two-tailed p values are reported (**P < 0.01).",
    "molecules": "BFA, DMSO"
  },
  {
    "caption": "A Representative time-lapse images of 2-cell embryos cultured with SiR-DNA   to label nuclei (white circles) and microinjected with oil  droplets (yellow circles). Time  is shown as h:min relative to maximal nuclear  displacement. B Plot of the distances between oil  droplets and nuclei  to the  cell-centre  over time  (n = 14 oil-nucleus  pairs). Data information: Scale bar = 20 µm in A, In (B), data are shown as mean ± SEM.",
    "molecules": "SiR-DNA"
  },
  {
    "caption": "D Representative  time-lapse  images of embryos  expressing H2B-RFP  and labelled with SiR-tubulin  following the addition of dynarrestin  at anaphase  completion (n = 14 embryos). Data information: In (D, time  is shown as h post-drug addition. In D, scale bar = 20 µm.",
    "molecules": "SiR, dynarrestin"
  },
  {
    "caption": "E Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos  (n = 28 blastomeres  per group). Data information: In E, data are shown as mean ± SEM. In E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.",
    "molecules": "DMSO, dynarrestin"
  },
  {
    "caption": "G Representative time-lapse images of embryos  expressing H2B-RFP  and labelled with SiR-tubulin  following the addition of taxol  at maximal nuclear  displacement. Data information: In G, scale bar = 20 µm.",
    "molecules": "SiR, taxol"
  },
  {
    "caption": "H Changes in nucleus-to-cell centre distances (d) over time  relative to the time  of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres  (mean ± SEM). Data information: In H), data are shown as mean ± SEM. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).",
    "molecules": "DMSO, taxol"
  },
  {
    "caption": "I Representative images of embryos immunostained  for β-tubulin and Hoechst  following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos  (n = 9 embryos  per group). Time is shown as h post-drug addition. Scale bar = 20 µm. Data information: In I), scale bar = 20 µm.",
    "molecules": "Hoechst, DMSO, taxol"
  },
  {
    "caption": "(B) Immunofluorescence (IF) validation of mintbody specificity. Mouse MC12 cells, which stably express H3K27me3-mintbody (sfGFP), are labelled with antibodies specific for H3K27me3 (Cy5) and H3K9me3 (Cy3). DNA is stained with Hoechst33342. Single confocal sections are shown. Arrows mark Xi. Scale bar = 10 μm.",
    "molecules": "Cy3, Cy5, DNA, Hoechst33342"
  },
  {
    "caption": "(D) ESCs as in (B) were engineered to express H3K27me3-mintbody (SNAP/JF646) and H4K20me1-mintbody (mCherry). Cells were cultured in the absence of LIF for 2 days. Time-lapse images for 12 z-plane confocal stack were acquired every 1 h. Maximum intensity projection images are shown with elapse time (hh:mm). Arrowheads mark Xi. Scale bar = 10 μm",
    "molecules": "JF646"
  },
  {
    "caption": "(E) H4K20me1 (blue) and H3K27me3 (red) accumulation across the Xi after 12 h of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Below each plot shown is the Xist locus (green bar) and Xist entry sites (black bars).",
    "molecules": "DOX"
  },
  {
    "caption": "C, D Clinical chemistry plasma concentrations of parameters related to acid-base balance (Lactate and Ammonia) and metabolic state (Glucose) n = 15 Uqcrh-/- and 16 WT pups 1-2-week-old (C); n = 15 f, 15 m  Uqcrh-/- and 15 f,15 m WT mice aged 8-9 weeks (D); sex not differentiated. Data shown as scatter dot plots with line at median. * p < 0.05, ** p < 0.01, **** p < 0.0001, Wilcoxon rank sum test.",
    "molecules": "Ammonia, Glucose, Lactate"
  },
  {
    "caption": "C  Western blot analysis of  OXPHOS  components in the  patient (P) and control (n = 2, C1-2) fibroblasts. D BN-PAGE analysis of  OXPHOS complex assembly in enriched mitochondria from  patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]) ",
    "molecules": "DDM"
  },
  {
    "caption": "A  Western blot  analysis of control (n = 2, C1-2) and patient fibroblasts  transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Expression of wild-type UQCRH was induced using various concentrations of doxycycline (dox) up to 20 ng/ml for 72 hours. B BN-PAGE analysis of control (n = 2, C1-2) and  patient fibroblasts  transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Transduced fibroblasts were either uninduced or induced with 20 ng/ml doxycycline (dox) for 72 hours. ",
    "molecules": "dox, doxycycline"
  },
  {
    "caption": "C Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei  (DAPI, blue), in control (NHDF) fibroblasts. D Immunofluorescence for  UQCRC2 (green), VDAC1/porin (red) and  nuclei  (DAPI, blue), in patient fibroblasts. E Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and  nuclei  (DAPI, blue), in control (NHDF) fibroblasts  transduced with wild-type UQCRH. F Immunofluorescence  for UQCRC2 (green), VDAC1/porin (red) and  nuclei  (DAPI, blue), in  patient fibroblasts  transduced with wild-type (WT) UQCRH. G Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei  (DAPI, blue), in control (NHDF) fibroblasts transduced with the pseudogene UQCRHL. H Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei  (DAPI, blue), in patient fibroblasts transduced with the pseudogene UQCRHL. I Graph indicates the staining intensity of  UQCRC2 in control (NHDF) fibroblasts (green) and patient fibroblasts  (red), those respective cell lines transduced (T) with wild-type UQCRH (diagonal lines) and those cell lines transduced with the pseudogene UQCRHL (vertical lines) measured using Image J, Data are given as mean ± SEM. One-way ANOVA (Kruskal-Wallis test), * p < 0.05; n = 3 macroscopic field (10-14 cells for macroscopic field were measured). ",
    "molecules": "DAPI"
  },
  {
    "caption": "Mitochondrial membranes from  fibroblasts of control (WT) and patient fibroblasts (i, immortalised) were solubilised with digitonin and separated on native gradient gels. A BN-PAGE gel  stained  with Coomassie showing molecular weight marker (BHM) and samples from control and  patient (primary fibroblasts, (I)=immortalised  fibroblasts) B BN-PAGE gel stained with NADH:NTB reductase  activity  stain showing molecular weight marker (BHM) and samples from control and patient  (primary fibroblasts, (I)=immortalised  fibroblasts) C Further complexome analysis of the  BN-PAGE gels shown in A and B. Each native lane was cut into even fractions from high to low molecular mass. Proteins in each slice were digested with trypsin  and analysed by quantitative mass spectrometry. Shown here are average IBAQ values of all identified subunits from complexes III, I and IV ",
    "molecules": "Coomassie, digitonin, NADH, NTB, trypsin"
  },
  {
    "caption": "heart tissue from wild-type (WT) and Uqcrh-/- mice  were solubilised with  digitonin and separated on native gradient gels. F BN-PAGE gel stained  with Coomassie (left) and NADH:NTB reductase activity stain  (right) showing molecular weight marker (BHM) and heart  tissue samples from wild-type (WT) and Uqcrh-/- mice. G BN-PAGE and immunoblot using the antibody to CIII  subunit UQCRC2 in heart  tissue samples from wild-type (WT) and Uqcrh-/- mice. H Further  complexome analysis of the BN-PAGE gels shown in F, shown here are average IBAQ values of subunits from complexes III, I and IV ",
    "molecules": "Coomassie, digitonin, NADH, NTB"
  },
  {
    "caption": "Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results.",
    "molecules": "PRN1126, Suc-LLVY-AMC"
  },
  {
    "caption": "Presentation of UTY246-254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results.",
    "molecules": "PRN1126"
  },
  {
    "caption": "Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM.",
    "molecules": "PRN1126"
  },
  {
    "caption": "Splenocytes from C57BL/6 mice (C) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results.",
    "molecules": "PRN1126, DMSO, LPS, ONX 0914"
  },
  {
    "caption": "Splenocytes from human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired student´s t-test.",
    "molecules": "PRN1126, DMSO, LPS, ONX 0914"
  },
  {
    "caption": "Colitis was induced by oral administration of 3 % DSS for 5 d. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± SEM of 15 mice pooled from three independent experiments.",
    "molecules": "PRN1126, DSS, ONX 0914"
  },
  {
    "caption": "Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± SEM (n = 6 per group).",
    "molecules": "PRN1126, ONX 0914"
  },
  {
    "caption": "Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results.",
    "molecules": "Ac-PAL-AMC, PRN1126, ONX 0914"
  },
  {
    "caption": "Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS-PAGE and immunoblotting against indicated proteins was performed. Shown are representative western blots out of three independent experiments with similar outcome.",
    "molecules": "DMSO, Ficoll, ONX 0914"
  },
  {
    "caption": "Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild type (left side) or LMP7-/- (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. **P < 0.01, ***P < 0.001. One-way ANOVA.",
    "molecules": "LU-001i, ML604440, PRN1126, DMSO, ONX 0914"
  },
  {
    "caption": "Splenocytes from C57BL/6 mice (A) or human PBMCs (B) were incubated (continuous treatment) with DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) and stimulated with LPS overnight. The IL-6 concentration in the supernatant was analyzed by ELISA. The highest IL-6 concentration from each experiment was set to 100%. (A) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates. (B) Data are presented as the mean ± SEM of 6 different healthy donors. All data were statistically compared to the DMSO treated group.",
    "molecules": "LU-001i, ML604440, PRN1126, DMSO, LPS, ONX 0914"
  },
  {
    "caption": "The differentiation of CD4+ T cells isolated from the spleens of C57BL/6 mice that were exposed (continuous treatment) to DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+ LU-001i (300 nM each) and that were stimulated with plate bound antibodies to CD3/CD28 in the presence of TGF-β and IL-6 and scavenging antibodies neutralising IL-4 and IFN-γ was measured in 3 days cultures. IL-17 expression was detected by intracellular cytokine staining after a short restimulation with PMA/Ionomycin. IL-17 expression in activated T cells cultured in the absence of Th17-polarizing conditions is shown as a comparison (no cocktail). Values reflect the percentage of CD4+ cells that are IL-17A+. Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates. All data were statistically compared to the DMSO treated group.",
    "molecules": "LU-001i, ML604440, PRN1126, DMSO, Ionomycin, ONX 0914, PMA"
  },
  {
    "caption": "Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. (A) The body weight of individual mice was monitored daily and the percent weight loss (y-axis) was plotted versus time (x-axis). All data were statistically compared to the vehicle treated group. *",
    "molecules": "LU-001i, PRN1126, DSS"
  },
  {
    "caption": "Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. On day 9 after initiation of DSS treatment, colon lengths were measured (n=15). Naïve mice (n=5) were used as healthy controls. All data were statistically compared to the vehicle treated group.",
    "molecules": "LU-001i, PRN1126, DSS"
  },
  {
    "caption": "Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated intermittently with three times a week (s.c.) schedule with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the start of the experiment. Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments. All data were statistically compared to the vehicle treated group.",
    "molecules": "LU-001i, PRN1126"
  },
  {
    "caption": "I Cytoplasmic and  nuclear proteins were extracted from WT and  SNX10 KO  Caco-2 cells treated with or without Tolimidone (10 μM), and the indicated proteins were analyzed by  immunoblots.",
    "molecules": "Tolimidone"
  },
  {
    "caption": "E, WT and SNX10 KO  Caco-2 cells were treated with  OMVs (100 μg/mL) for the indicated  time and  stained with antibodies against LPS  and Rab5 (E). Scale bar: 20 μm.",
    "molecules": "LPS"
  },
  {
    "caption": "G  LPS levels in the cell culture supernatants of WT and SNX10 KO Caco-2 cells stimulated by OMVs (100 μg/mL) for the indicated time were detected by  LAL assay (n = 6 independent experiments). H LPS levels in the  cytosolic and residual fractions of  Caco-2 cells stimulated by OMVs (100 μg/mL) with or without chloroquine (CQ) (200 nM) or bafilomycin A1 (Baf A1) (50 nM) for the indicated time were detected by LAL assay  (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. ",
    "molecules": "Baf A1, bafilomycin A1, chloroquine, LPS"
  },
  {
    "caption": "F  Caco-2 cells were stimulated by OMVs (100 μg/mL) with or without apilimod  (30 nM) for 24h. Proteins of lysates extracted from  Caco-2  cells were subjected to  immunoblots with the indicated antibodies.",
    "molecules": "apilimod"
  },
  {
    "caption": "I LPS levels in the  cytosolic and residual fractions of Caco-2  cells stimulated by OMVs  (100 μg/mL) with or without apilimod (30 nM) for the indicated time were detected by  LAL assay (n = 6 independent experiments). J  Caco-2 cells transfected with control siRNA or CASP5 siRNA were treated with OMVs (100 μg/mL) for the indicated time.  LPS levels in the cytosolic and residual fractions of Caco-2 cells were detected by  LAL assay (n = 6 independent experiments). K LPS levels in the cytosolic  and residual fractions of Caco-2 cells stimulated by  OMVs  (100μg/mL) with or without bafetinib (1μM) for the indicated time were detected by LAL assay (n = 6 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyzes. ***p < 0.001. ",
    "molecules": "apilimod, bafetinib, LPS"
  },
  {
    "caption": "(C) Representative flow cytometry results showing CD71, a cell surface marker, can be efficiently knocked-down in the majority of organoid cells after 5 days of Dox  and TMP  treatment. N = 3 different organoid lines and 3 different gRNAs for CD71  were used. Mean ± SEM is labelled.",
    "molecules": "Dox, TMP"
  },
  {
    "caption": "(B) qRT-PCR  showing that SOX9  gRNAs effectively knocked down SOX9  transcript levels using inducible CRISPRi after 5 days of Dox and TMP  treatment. 3 different organoid lines and 2 different SOX9  gRNAs were used. Error bars: mean ± SEM. Two-sided Student's t-test with equal variance ***p < 0.001.",
    "molecules": "Dox, TMP"
  },
  {
    "caption": "(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX-5461 vehicle: 25 mM NaH2PO­­­4) (V/V; mouse #1-6), 5 mg/kg everolimus (EV; mouse #7-12), 35 mg/kg CX-5461 (mouse #13-18) or both drugs (CX-5461+EV; mouse #19-24) for 2 hours (n=6 per treatment group). Actin was used as a loading control.",
    "molecules": "CX-5461, EV, Everolimus, everolimus, methylcellulose, NaH2PO­­­4"
  },
  {
    "caption": "(C) Enrichment analysis by MetaCore® GeneGO of genes in "translation up" (red) and "translation down" (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX-5461+EV treatment group with the V/V group (n=6). Ratio" values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore®'s database. (D) Genes implicated in "Translation: initiation" and "Translation: Elongation-Termination" processes based on Fig 1C",
    "molecules": "CX-5461, EV"
  },
  {
    "caption": "(E) Intracellular ATP levels determined using the liquid chromatography-mass spectrometry in CTRL, EV, CX and CMB early-passage cell lines.",
    "molecules": "ATP"
  },
  {
    "caption": "(G) Propidium iodide (PI) exclusion analysis of early-passage CX-5461-everolimus combination therapy-resistant (CMB), CX-5461-resistant (CX) and drug-naïve (CTRL) lymphoma cells treated with indicated concentrations of metformin for 48 hours.",
    "molecules": "CX-5461, everolimus, metformin, PI, Propidium iodide"
  },
  {
    "caption": "(H) CellTiterGLO®-based assay measuring cellular ATP levels of the CX-5461-resistant (CX) and CX-5461-everolimus combination therapy-resistant (CMB) cells treated with metformin as indicated for 48 hours.",
    "molecules": "ATP, CX-5461, everolimus, metformin"
  },
  {
    "caption": "(I) PI analysis of CTRL and CMB cells treated with CX-5461 and everolimus in the presence and absence of metformin for 48 hours as indicated.",
    "molecules": "CX-5461, everolimus, metformin, PI"
  },
  {
    "caption": "(J) PI analysis of CTRL and CX cells treated with CX-5461 in the presence and absence of metformin for 48 hours as indicated.",
    "molecules": "CX-5461, metformin, PI"
  },
  {
    "caption": "(B) Abundance of intracellular 3'5'-cyclic AMP as measured by a liquid chromatography (LC)-mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n=3 (5-6 technical replicates each).",
    "molecules": "3'5'-cyclic AMP"
  },
  {
    "caption": "(C) Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP-bound RAP1 levels in the indicated early-passage cells during their log-phase growth period (n=3). Actin and total RAP1 were used as loading controls.",
    "molecules": "GTP"
  },
  {
    "caption": "(D) Propidium iodide (PI) exclusion assays of CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours.",
    "molecules": "CX-5461, everolimus, H89, PI, Propidium iodide"
  },
  {
    "caption": "(E) PI exclusion analysis of early-passage drug-naïve (CTRL) lymphoma cells treated with CX-5461 and everolimus in the presence or absence of selective PKA activator 6-Bnz-cAMP for 48 hours.",
    "molecules": "6-Bnz-cAMP, CX-5461, everolimus, PI"
  },
  {
    "caption": "(F) PI exclusion analysis of the CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI-05 for 48 hours.",
    "molecules": "CE3F4, CX-5461, ESI-05, everolimus, PI"
  },
  {
    "caption": "(G) PI exclusion analysis of early-passage drug-naïve (CTRL) cells treated with CX-5461 and everolimus in the presence or absence of the selective EPAC activator 8-pCPT-2-O-Me-cAMP for 48 hours.",
    "molecules": "8-pCPT-2-O-Me-cAMP, CX-5461, everolimus, PI"
  },
  {
    "caption": "(A) A heatmap illustrating the abundances of polysome-associated mRNAs that are associated with the mRNA translation/protein synthesis based on the Gene Ontology Resource Database in drug-naïve (CTRL) or CX-5461-everolimus combination therapy-resistant (CMB) cell lines (n=3). The colours illustrate the expression values of indicated mRNAs that were normalized using voom (red: high expression; blue: low expression).",
    "molecules": "CX-5461, everolimus"
  },
  {
    "caption": "(B) Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX-5461-resistant (CX) cells (representatives of n=3). (C) Quantitation of polysome:monosome ratio in the indicated early-passage Eµ-Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software. Graph represents mean ± SEM of n=3. ",
    "molecules": "CX-5461"
  },
  {
    "caption": "(A) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP-bound RAP1 in CX and CMB cells (n=3) and its quantitation.",
    "molecules": "GTP, metformin"
  },
  {
    "caption": "(D) Kaplan-Meier curve of C57BL/6 mice transplanted with CX-5461-everolimus-resistant (CMB #8) early-passage Eµ-Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX-5461/metformin vehicle: 25 mM NaH2PO­­­4) (n=6); CX-5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily) (n=8), metformin (400 mg/kg twice daily) (n=6), or CX-5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively) (n=8). Light grey: 5-day metformin pre-treatment period, dark grey: treatment period. Data were analyzed by a log-rank (Mantel-Cox) test. Vehicle vs. CX-5461-everolimus: P = 0.0006, Vehicle vs. CX-5461-everolimus-metformin: P = 0.0001. CX-5461-everolimus vs. CX-5461-everolimus-metformin: P = 0.0003.",
    "molecules": "CX-5461, everolimus, metformin, methylcellulose, NaH2PO­­­4"
  },
  {
    "caption": "(F) Immunoblot  (IB) analysis of whole-cell lysates (WCLs) and immunoprecipitates (IPs) derived from 293T cells transfected with Flag-SIRT5  together with HA-TRIM21 or indicated HA-tagged mutant TRIM21  constructs. Cells were treated with 10 μM MG132  for 4 hours before harvesting. HA, hemagglutinin.",
    "molecules": "MG132"
  },
  {
    "caption": "(G) IB  analysis of WCLs derived from 293T  cells transfected with increasing amounts of Flag-TRIM21  construct. Where indicated, 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(H) 293T  cells transfected with control or TRIM21  siRNA for 48 hours were then treated with 100 ng/mL CHX  for the indicated time  period before harvesting. Protein expression was analyzed by  western blotting (left panel). The SIRT5  protein abundance was quantified by ImageJ and plotted as indicated (right panel). Data information: All WB  data in this work otherwise indicated are representative of at least three independent experiments.",
    "molecules": "CHX"
  },
  {
    "caption": "(I) IB  analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs  treated with 100 ng/ml CHX  for the indicated time  period  before harvesting (left panel). The SIRT5  protein abundance was quantified (right panel).",
    "molecules": "CHX"
  },
  {
    "caption": "(J) IB  analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS  for the indicated time  period before harvesting (top panel). The SIRT5  protein abundance was quantified (bottom panel).",
    "molecules": "LPS"
  },
  {
    "caption": "(A) Immunoblot analysis of WCLs and anti-GST immunoprecipitates derived from 293T  cells transfected with the indicated plasmids for 48 hours, cells were then treated with the proteasome  inhibitor MG132  for another 4 hours prior to harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) WCLs from TRIM21+/+ and TRIM21-/- BMDMs  treated with 100 ng/mL LPS  for 4 hours were immunoprecipitated  with anti-SIRT5  antibody and analyzed by western blotting. 10 μM MG132  was added for 4 hours before harvesting. Data information: All IP  and WB  data in this work otherwise indicated are representative of at least three independent experiments.",
    "molecules": "LPS, MG132"
  },
  {
    "caption": "(A) In vivo ubiquitination assay analysis of WCLs and GST-tagged immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) IB analysis of WCLs and anti-HA immunoprecipitates  of 293T  cells transfected with the indicated plasmids. 48 hours posttransfection, cells were treated with 10 μM MG132  for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(E) WCLs from TRIM21+/+ and TRIM21-/- BMDMs  treated with 100 ng/mL LPS  for 4 hours were immunoprecipitated  with anti-SIRT5  antibody and analyzed by  western blotting using antibodies specifically against Ub-K48 and Ub-K63. 10 μM MG132  was added for 4 hours before harvesting. Data information: All IP  and WB  data in this work otherwise indicated are representative of at least three independent experiments.",
    "molecules": "LPS, MG132"
  },
  {
    "caption": "(D) IB  analysis of WCLs and anti-SIRT5 immunoprecipitates  of BMDMs  treated with 100 ng/ml LPS  for 4 hours. Cells were treated with 10 μM MG132 for 4 hours before harvesting.",
    "molecules": "LPS, MG132"
  },
  {
    "caption": "(F) IB  analysis of WCLs derived from 293T  cells transfected with HA-SIRT5  together with Flag-HAUSP  or vector control constructs (left panel). Cells were treated with 100 ng/ml CHX  for the indicated time  period  prior to harvesting. The SIRT5  protein abundance was quantified (right panel).",
    "molecules": "CHX"
  },
  {
    "caption": "(G, H) WCLs from BMDMs  treated with increasing amounts of P5091(G) or P22077  (H) in the presence or absence of 100 ng/mL LPS  were subjected to IB  analysis using indicated antibodies (left panel). The SIRT5  protein abundance was quantified (right panel).",
    "molecules": "P22077, P5091, LPS"
  },
  {
    "caption": "(A) In vivo ubiquitination assay analysis of WCLs and GST-tagged immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) IB  analysis of WCLs and anti-GST immunoprecipitates  of 293T  cells transfected with the indicated plasmids in the presence or absence of 100 µM P22077  for 24 hours. Cells were treated with 10 μM MG132  for 4 hours before harvesting.",
    "molecules": "P22077, MG132"
  },
  {
    "caption": "(C, IB  analysis of WCLs and GST-tagged immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "D) IB  analysis of WCLs and GST-tagged immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(F) IB  analysis of WCLs and anti-SIRT5 immunoprecipitated  products derived from BMDMs  transfected with control or HAUSP  siRNA for 48 hours. 10 μM MG132  was added for 6 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(G) BMDMs  transfected with control (-) or HAUSP  siRNA for 48 hours were then treated with 100 ng/mL mM LPS  for 4 hours, and 10 μM MG132  was added for 4 hours before harvesting. WCLs and anti-SIRT5 immunoprecipitated  products were prepared and subjected to IB  analysis using indicated antibodies.",
    "molecules": "LPS, MG132"
  },
  {
    "caption": "(A) IB  analysis of WCLs and anti-Flag immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids using anti-Flag, anti-Acetyllysine, anti-Succinyllysine, anti-Glutaryllysine  and anti-Malonyllysine  antibodies",
    "molecules": "Glutaryllysine, Malonyllysine, Acetyllysine, Succinyllysine"
  },
  {
    "caption": "(B) IB  analysis of WCLs and anti-TRIM21 immunoprecipitated  products derived from 293T  cells transfected with control or SIRT5  siRNA using anti-Flag, anti-acetyl Lysine (AcK), anti-succinyl Lysine (SulK), anti-glutaryl Lysine (GluK) and anti-malonyl Lysine (MalK) antibodies.",
    "molecules": "GluK, glutaryl Lysine, MalK, malonyl Lysine, acetyl Lysine, AcK, succinyl Lysine, SulK"
  },
  {
    "caption": "(C) IB  analysis of WCLs and anti-Flag immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(D) IB  analysis of WCLs and anti-TRIM21 immunoprecipitated  products derived from BMDMs  treated 100 ng/mL LPS  or left untreated for 4 hours. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "LPS, MG132"
  },
  {
    "caption": "(F, IB  analysis of WCLs and anti-Flag immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "G) IB  analysis of WCLs and anti-Flag immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "(H) IB  analysis of WCLs and anti-GST immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids in the presence or absence of 3 mM NAM  for 24 hours. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132, NAM"
  },
  {
    "caption": "(J) IB  analysis of WCLs and anti-GST immunoprecipitated  products derived from 293T  cells transfected with the indicated plasmids. 10 μM MG132  was added for 4 hours before harvesting.",
    "molecules": "MG132"
  },
  {
    "caption": "wild-type (WT, n=5), TRIM21-/- (n=5), SIRT5-/-(n=5), and TRIM21-/-SIRT5-/- (n=5) mice  were orally administrated with 2.5% (w/v) DSS  in drinking water for 7 days and regular drinking water for another 2 days. Colon-length (G) changes were measured. Data information: Data are means ± SD. n=5 biological replicates. P values were determined by unpaired two-tailed Student's t-tests. ns, not significant; ***p < 0.001, ****p < 0.0001.",
    "molecules": "DSS"
  },
  {
    "caption": "(A-D) Immunostaining  with dopamine  antibody at late 3rd instar  (96h AEL) in control background (domeMESO-GFP/+). The inset shows DomeGFP+ cells (B') and the corresponding dopamine  levels (C') in them. (E-L) Similar expression  analysis of dopamine  at early 3rd (72h AEL, E-H) and early 2nd instar (48h AEL, I-L). (D, H and L) The spectral mode of iDopamine  representation with blue pixels indicating low and red pixels indicating high intensity. (M) Quantifications of dopamine  levels in DomeGFP+ progenitors  (domeMESO-Gal4, UAS-GFP/+, n= 14 at 48h, n=13 at 72h and n=16 at 96h AEL). Data information: DNA  is stained  with Hoechst  in blue, intracellular Dopamine  (iDopamine) in red, domeMESO expression  in green (representative of progenitors  in the  medullary zone, MZ). The iDopamine channel has been converted to spectral mode in panels D, H and L. All image panels show a 20µm scale bar. The inset represents a 2X zoom of region of interest (ROIs) marked in white dashed square. AEL indicates After Egg Laying. For better representation, the primary lobe  of the lymph gland  has been represented and outlined in white dashed lines while the MZ  is outlined in yellow dashed line for segregation between Dome+ and Dome- cells. The quantification in M shows median with whiskers indicating the maximum and minimum values,. box indicates the lower and upper quartiles. The statistical analysis applied is One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of  lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe  of the  lymph gland has been represented and outlined in white dashed lines while the MZ  is outlined in yellow dashed line.",
    "molecules": "Hoechst, dopamine, Dopamine"
  },
  {
    "caption": "(B-D') A temporal expression  profile of TH  at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+) (E-G') A temporal expression  profile of Ddc  at 48, 72 and 96h AEL lymph glands in control background (domeMESO-Gal4, UAS-GFP/+). (H) Quantification of TH  levels (domeMESO-Gal4, UAS-GFP/+, n=18 at 48h, n= 15, at 72h and n= 23 at 96h AEL). (I) Quantification of Ddc  levels (domeMESO-Gal4, UAS-GFP/+, n= 17 at 48h, n= 33 at 72h and n= 38 at 96h AEL). Data information: DNA  is stained  with Hoechst  in blue, iDopamine in red, Tyrosine hydroxylase (TH) and Dopa decarboxylase  (Ddc) in red, domeMESO (representative of progenitors) expression  in green, AEL indicates After Egg Laying. The quantifications in H, I represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in H, I, is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe  of the lymph gland has been represented and outlined in white dashed lines while the MZ  is outlined in yellow dashed line.",
    "molecules": "Hoechst, Dopamine"
  },
  {
    "caption": "(J-L) Assessment of dopamine  levels upon the knockdown of TH  and Ddc  in the progenitors  using domeMESO-Gal4, UAS-GFP. (M) The mean intensity quantification of dopamine  levels upon TH  and Ddc  loss of function in the progenitors  (domeMESO-Gal4, UAS-GFP/+, n=40, domeMESO-Gal4, UAS-GFP; THRNAi, n= 17, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 34). Data information: DNA  is stained   with Hoechst  in blue, iDopamine in red All image panels show a 20µm scale bar. The quantifications in M represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in M, is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of  lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe  of the lymph gland  has been represented and outlined in white dashed lines",
    "molecules": "Hoechst, dopamine, Dopamine"
  },
  {
    "caption": "(N-P) The loss of progenitor TH  and Ddc  function using domeMESO-Gal4, UAS-GFP  analysed at wandering 3rd instar, 120h AEL. Data information: DNA  is stained  with Hoechst  in blue, domeMESO  (representative of progenitors) expression  in green, P1 (representative of mature plasmatocytes) in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. For better representation, the primary lobe  of the lymph gland has been represented and outlined in white dashed lines",
    "molecules": "Hoechst"
  },
  {
    "caption": "(S) Quantification of the number of PPO  positive crystal cells upon dopamine  synthesis perturbation (domeMESO-Gal4, UAS-GFP/+, n=29, domeMESO-Gal4, UAS-GFP; THRNAi, n= 21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 23). Data information: The quantifications in S represent mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in S is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.",
    "molecules": "dopamine"
  },
  {
    "caption": "(A-C') Expression  profile of dopamine activation  based receptor sensor (DA1m) in the progenitors  using domeMESO-Gal4  driver in a temporal  manner- 48h, (A-A'), 72h, (B-B') and 96h AEL, (C-C'). (D) Quantification of the number  DA1m+ cells, %DA1m positive cells (domeMESO-Gal4; DA1m, n=10 at 96h, n=16 at 72h and n=10 at 48h AEL). Data information: DNA is stained  with Hoechst  in blue, lifeAct-RFP to mark progenitors in red, Dopamine  receptor-based reporter (DA1m) in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in D represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe  of the lymph gland  has been represented and outlined in white dashed lines while the MZ  is outlined in yellow dashed line.",
    "molecules": "Hoechst, dopamine"
  },
  {
    "caption": "(E-G') Temporal expression  analysis of  dopamine transporter, DAT. (H) A line plot representing the trend of DAT expression  at 96h AEL. The distance (x) axis indicates 0µm as a line beginning from the core region to the periphery. Data information: DNA  is stained with Hoechst  in blue,  Dopamine transporter (DAT) in red, domeMESO  (representative of progenitors) expression  in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. For better representation, the primary lobe  of the  lymph gland  has been represented and outlined in white dashed lines while the MZ  is outlined in yellow dashed line.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(I-K) The loss of progenitor Dop2R and DAT  function using domeMESO-Gal4, UAS-GFP  analysed at wandering 3rd instar, 120h AEL. Data information: DNA  is stained  with Hoechst in blue domeMESO (representative of progenitors) expression  in green, P1  (representative of mature plasmatocytes) in red. All image panels show a 20µm scale bar. For better representation, the primary lobe  of the lymph gland has been represented and outlined in white dashed lines",
    "molecules": "Hoechst"
  },
  {
    "caption": "(N) Quantification of the number of PPO  positive  crystal cells upon dopamine  sensing perturbation (domeMESO-Gal4, UAS-GFP/+, n= 29, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 26, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25). Data information: The quantification in N represents mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in N is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of  lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.",
    "molecules": "dopamine"
  },
  {
    "caption": "(A) A temporal  analysis of total number of nuclei  across development in control background (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 31 at 72h, n= 19 at 96h and n= 23 at 120h AEL). The loss of dopamine  modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=19,  domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30 and  domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=33 and domeMESO-Gal4, UAS-GFP;  DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=18, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20 and  domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=25, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=31). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis is Two-way Anova with Tukey's multiple comparisons test while the control comparison, domeMESO-Gal4, UAS-GFP/+, for different  time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying",
    "molecules": "dopamine"
  },
  {
    "caption": "(B) Quantification of total Dome+ nuclei  counts of control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 19 at 96h and n=23 at 120h AEL). The loss of dopamine  modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP;  Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP;  DATRNAi, n=30). (C) A temporal analysis of Dome- nuclei  counts in the control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 18 at 96h, n= 23, at 120h AEL). The perturbation of the dopamine  modules in the progenitors  at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22,  domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32,  domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP;  DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP; DATRNAi, n=30). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test while the control comparison,  domeMESO-Gal4, UAS-GFP/+, for different  time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying",
    "molecules": "dopamine"
  },
  {
    "caption": "(A) Quantification of the Dome+ BrdU  index normalised to Dome+ nuclei  in a time course manner with dopamine modules perturbed in the progenitors  using domeMESO-Gal4, UAS-GFP  driver. At 48h AEL (domeMESO-Gal4, UAS-GFP/+, n=11, domeMESO-Gal4, UAS-GFP; THRNAi, n=13, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=11, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=15, domeMESO-Gal4, UAS-GFP;  DATRNAi, n=5), at 72h AEL (domeMESO-Gal4, UAS-GFP/+, n=34,  domeMESO-Gal4, UAS-GFP; THRNAi, n=31, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=15, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27) and at 96h AEL (domeMESO-Gal4, UAS-GFP/+, n=17, domeMESO-Gal4, UAS-GFP; THRNAi, n=28, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=20,  domeMESO-Gal4, UAS-GFP;  Dop2RRNAi, n=17, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22). (B) Quantification of the Dome+ mitotic index  normalised to Dome+ nuclei  in a  time course manner with loss of dopamine  modules in the progenitors using domeMESO-Gal4, UAS-GFP  driver. At 48h AEL (domeMESO-Gal4, UAS-GFP/+, n=35, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=20), at 72h AEL (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=23, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=31, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27) and at 96h AEL (domeMESO-Gal4, UAS-GFP/+, n=19, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22). Data information: AEL indicates After Egg Laying. The quantifications in A and B represent median with whiskers indicating maximum and minimum value, box indicates the lower and upper quartiles The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of  lymph gland lobes   analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.",
    "molecules": "BrdU, dopamine"
  },
  {
    "caption": "(C) Quantifications of %G1, %S and %G2/M at different time points, in control and dopamine  perturbed backgrounds, color coding scheme to represent the  cell cycle phase as shown through FUCCI  reporter is below the graph. Across development, at 48h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=34, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 13, domeMESO-Gal4; UAS-FUCCI;  DdcRNAi, n= 15,  domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, n= 19, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 17), at 72h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=38, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 22, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 29, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, n= 47, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 29) and at 96h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=55, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 14, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 19, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 19) Data information: FUCCI (fluorescent ubiquitination-based cell cycle indicator) reporter shows G1 phase cells in green, S phase  cells in red and G2/M phase cells in yellow. AEL indicates After Egg Laying. The quantifications in in C represents mean. The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test. The domeMESO-Gal4; UAS-FUCCI  control comparison temporally in C is analysed using One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of  lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.",
    "molecules": "dopamine"
  },
  {
    "caption": "(A-I'') Representative  lymph gland images at 48, 72 and 96h AEL depicting the  lymph gland growth trajectory along with dopamine  level status in the PSC  and remaining  lymph gland. (A-C'') The control profile of lymph gland and PSC  iDopamine levels at 48h (A-A''), 72h (B-B'') and 96h (C-C''). (D-I'') Downregulation of the dopamine  synthesising enzymes TH  and Ddc  in the PSC  at 48h (D-D'' and G-G''), 72h AEL (E-E'' and H-H'') and at 96h AEL (F-F'' and I-I''). (J) Quantification of mean dopamine  intensity in the PSC  at upon PSC  perturbation of dopamine  synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=33, Antp-Gal4; UAS-GFP; THRNAi, n=8, Antp-Gal4; UAS-GFP; DdcRNAi, n=14), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=26, Antp-Gal4; UAS-GFP; THRNAi, n=25, Antp-Gal4; UAS-GFP;  DdcRNAi, n=20) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17). (K) Quantification of mean dopamine  levels in the  lymph gland upon PSC  perturbation of dopamine  synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=28, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=18), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=25, Antp-Gal4; UAS-GFP;  THRNAi, n=24, Antp-Gal4; UAS-GFP;  DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=16, Antp-Gal4; UAS-GFP; DdcRNAi, n=18). Data information: DNA is stained  with Hoechst  in blue, Antp-Gal4; UAS-GFP  (representative of Posterior Signaling Center, PSC) in green, iDopamine in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in J, K represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in J, K is Two-way Anova with Tukey's multiple comparisons test. The control animals Antp-Gal4; UAS-GFP/+ were analysed by One-way Anova with Tukey's multiple comparisons test across time points. 'n' represents the number of  lymph gland lobes  analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe  of the  lymph gland  has been represented and outlined in white dashed lines and the PSC in yellow dashed line.",
    "molecules": "Hoechst, dopamine, Dopamine"
  },
  {
    "caption": "F. Phosphorylation of ErbB2 in Mtmr2-/- Schwann cell/DRG neuron co-cultures after 4 days of ascorbic acid treatment. Each lane is a pool from at least 10 coverslips/DRG per genotype. Representative of two independent experiments using n=4 different pools of coverslips/DRG per genotype, p=0.20, two-tailed Mann Whitney U test.",
    "molecules": "ascorbic acid"
  },
  {
    "caption": "B. ShRNA#1 and 2 downregulate Nrg1 type III expression also in myelin-forming Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, as shown by western blot analysis, representative of two independent experiments. Clx is calnexin.",
    "molecules": "ascorbic acid"
  },
  {
    "caption": "C, D. Titration of shRNA #1 (C) and of shRNA #2 (D) LVs (lentiviral vectors) on WT Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, with quantification of the mean number of Mbp positive segments and Schwann cell number. In (C) n= 20 (SCR, Scramble), n= 11 (LV #sh1, 12.5%), n= 16 (LV #sh1, 25%), n= 9 (LV #sh1, 50%) DRG/coverslips from two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. In (D) n= 14 (SCR), n= 10 (LV #sh2, 12.5%), n= 12 (LV #sh2, 25%), n= 79 (LV #sh2, 50%) DRG/ coverslips, representative of two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.",
    "molecules": "ascorbic acid"
  },
  {
    "caption": "C. Titration of WT co-cultures using different amounts of Niacin, nicotinic acid, with quantification of Mbp positive fibers and Schwann cellnuclei, n=9 (NT), n=8 (1mM), n=9 (5mM), n=8 (10mM), DRGs/ coverslips. Representative of three independent experiments, p=0.0055, nonparametric one-way ANOVA, followed by Dunn's post hoc test.",
    "molecules": "Niacin, nicotinic acid"
  },
  {
    "caption": "D. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 1 mM and 5mM Niacin, with quantification of the percentage of myelin outfoldings; n=31 (NT), n=14 (1mM), and n=29 (5mM) DRGs/coverslips from two independent experiments. A total of 1256, 489, and 1305 fibers, respectively, were quantified, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.",
    "molecules": "Niacin"
  },
  {
    "caption": "D. Tace activity was measured from lysates of Niacin-treated cultures, n=7 (KO NT) and n=8 (KO 5mM Niacin) number of independent plates, each containing from 10-15 DRGs plated. Note that Tace activity was similar between WT and Mtmr2-/- cultures, one-tailed Mann Whitney U test, p=0.037.",
    "molecules": "Niacin"
  },
  {
    "caption": "E. Niacin treatment of Tace-/- explants does not rescue hypermyelination, with quantification, n=8 DRGs/coverslips per condition, p=0.0056, nonparametric one-way ANOVA, followed by Dunn's post hoc test. Representative of three independent experiments.",
    "molecules": "Niacin"
  },
  {
    "caption": "A. Niacin treatment of Vim-/- explants rescues hypermyelination, n=10 DRGs/ coverslips per condition, with quantification, representative of three independent experiments, p=0.0048, nonparametric one-way ANOVA, followed by Dunn's post hoc test.",
    "molecules": "Niacin"
  },
  {
    "caption": "A. Western blot analysis on lysates from treated and not treated explants (at least 10 DRGs/ coverslips per lane) shows that increased Akt activation (S473 phosphorylation) in Vim-/- explants is restored to normal levels following 5 mM Niacin treatment.",
    "molecules": "Niacin"
  },
  {
    "caption": "B. Niaspan treatment of Vim-/- mice does not affect mouse growth, as the growth rate in WT (n=8, saline; n=6 Niaspan) and Vim-/- (n=10 saline and n=10 Niaspan) mice either saline or Niaspan-treated is not significantly different, Linear Mixed Effect (LME) models.",
    "molecules": "Niaspan"
  },
  {
    "caption": "B. Niaspan administration (daily i.p. injection of 160mg/Kg Niaspan starting at P15 for 15 days) enhances Tace activity in Vim-/- nerves at P30, n=6 animals per genotype, p=0.0325, one-tailed Mann Whitney U test.",
    "molecules": "Niaspan"
  },
  {
    "caption": "C. Semithin section and g-ratio analyses of sciatic nerves at P30 shows that Niaspan does not alter myelin thickness in WT nerves, whereas restores myelin thickness to normal values in Vim-/- nerves. G-ratio values: WT saline, 0.71 ± 0.003, 1872 fibers, n= 5 animals; WT Niaspan, 0.71 ± 0.004, 1882 fibers, n= 5 animals; Vim-/- saline 0.69 ± 0.004, 2313 fibers, n= 6 animals; Vim-/- Niaspan 0.72 ± 0.01, 1616 fibers, n=5 animals. WT saline as compared to Vim-/- saline, p=0.0588; Vim-/- saline as compared to Vim-/- Niaspan, p=0.0431, repeated measures ANOVA, representative of two independent experiments. The number of myelinated fibers is similar between the 4 groups as shown, p=0.3042, nonparametric one-way ANOVA, followed by Dunn's post hoc test.",
    "molecules": "Niaspan"
  },
  {
    "caption": "A. Niaspan administration (daily i.p. injection of 160mg/Kg starting at P15 for 60 days) does not affect the growth of Mtmr2-/- mice. The growth rates of Mtmr2-/- and WT both saline-treated are significantly different, p<0.0001, Linear Mixed Effect (LME) models, as already reported (Bolino et al, 2004), n=8 animals per condition.",
    "molecules": "Niaspan"
  },
  {
    "caption": "B. Niaspan administration reduces the percentage of myelin outfoldings (red asterisks) in Mtmr2-/- sciatic nerves without affecting the number of myelinated fibers as assessed by semithin section analysis at P75, n= 8 mice per genotype, results are mean ± SEM, p=0.0093, two-tailed Mann Whitney U test, representative of three independent experiments. Niaspan treatment does not alter myelin thickness in WT or Mtmr2-/- mice as assessed by g-ratio analysis (WT saline-treated, 0.67 ± 0.003, 2014 fibers; Mtmr2-/- Niaspan-treated, 0.67 ± 0.007, 1924 fibers; Mtmr2-/- saline-treated, 0.68 ± 0.003, 2244 fibers; n=4 animals per condition).",
    "molecules": "Niaspan"
  },
  {
    "caption": "C. Representative images of semithin section analysis of sciatic nerves at P45 from Pmp22+/- saline and Pmp22+/- Niaspan-treated mice. Niaspan was administered daily by i.p. injection at 160mg/Kg starting at P15 for 30 days.C'. The percentage of tomacula in sciatic nerves was assessed by ultrastructural analysis, results are mean ± SEM, n= 6 animals per condition, p=0.0022, two-tailed Mann Whitney U test, representative of two independent experiments.D. G-ratio analysis on semithin sections of sciatic nerves shows that increased myelin thickness in Pmp22+/- sciatic nerves at P45 is restored to normal values following Niaspan treatment. Mean g-ratio values, WT saline-treated: 0.69 ± 0.002, 2052 fibers, n=5 animals; Pmp22+/- saline-treated: 0.66 ± 0.003, 2643 fibers, n=6 animals; Pmp22+/- Niaspan-treated, 0.68 ± 0.002, 2311 fibers, n=6 animals. WT saline as compared to Pmp22+/- saline-treated, p<0.0001; Pmp22+/- saline-treated as compared to Pmp22+/- Niaspan-treated, p=0.0002; Pmp22+/- Niaspan-treated as compared to WT saline, p=0.0899; repeated measures ANOVA.E. The number of myelinated fibers is similar between the 3 groups analyzed, p=0.4362, nonparametric one-way ANOVA, followed by Dunn's post hoc test.",
    "molecules": "Niaspan"
  },
  {
    "caption": "C. Representative images of semithin section analysis of WT and Pmp22-/- sciatic nerves at P30. Tomacula are abundant in Pmp22-/- nerves at this age, where both fibers with thicker myelin (in the range of <3-4 μm in diameter) and thinner myelin (with diameters greater than 4 μm, particularly in motor fasicles) can be observed.C'. Niaspan does not ameliorate the phenotype in Pmp22-/- mice, as indicated by the number of tomacula (quantification of the percentage of tomacula in the entire sciatic nerve section, results are mean ± SEM, p=0.7400, Mann Whitney U test, n= 15 animals per condition).",
    "molecules": "Niaspan"
  },
  {
    "caption": "D. Growth curve of Pmp22-/- mice treated with Niaspan administered by i.p. injection daily at 160mg/Kg and starting at P15 for 15 days. A significant time by group effect is noted between WT saline and Pmp22-/- saline (group effect p=0.9907 and time effect p=0.0057), indicating that the two groups start with similar weight values but then the WT saline group (n=6 animals) grows more in time. When Pmp22-/- saline (n=5) and Pmp22-/- Niaspan-treated (n=6) are compared, the group effect is statistically significant (p=0.0076) while the time effect is not (p=0.6677), suggesting that these two groups have a similar growth trend but Pmp22-/- Niaspan-treated group starts with a higher baseline weight.",
    "molecules": "Niaspan"
  },
  {
    "caption": "E. Niaspan does not ameliorate myelin thickness as indicated by g-ratio analysis, with axonal diameter distribution: WT 0.68 ± 0.003, 2945 fibers, n=5 animals; Pmp22-/- 0.64 ± 0.003, 1557 fibers, n= 6 animals; Pmp22-/- Niaspan 0.63 ± 0.003, 1445 fibers, n=6 animals, results are mean ± SEM. WT saline as compared to Pmp22-/- saline-treated p<0.0001; WT saline as compared to Pmp22-/- Niaspan-treated p<0.0001, repeated measures ANOVA. Note the loss of myelinated fibers in Pmp22-/- nerves for all caliber axons.",
    "molecules": "Niaspan"
  },
  {
    "caption": "(D) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.",
    "molecules": "cholesterol"
  },
  {
    "caption": "(C) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm.",
    "molecules": "HPβCD, filipin"
  },
  {
    "caption": "(F) Cells were transfected with siRNAs as indicated, incubated with cholesterol-MβCD complex in serum-starvation media, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of ciliated cells shown in (F). Data represent mean ± SD (n=3experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test. ",
    "molecules": "cholesterol, MβCD"
  },
  {
    "caption": "(A) RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild-type pGFP-Rab8a (WT-Rab8), constitutively active pGFP-Rab8a (Q67L) (CA-Rab8), or DN Rab8 dominant-negative pGFP Rab8a (T22N), incubated in serum-starved media for 12 h, and immunostained for ARL13B (Red), GFP-Rab8 (green), and DAPI (blue). Scale bar, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST-JCF1 (RBD). The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. Bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.",
    "molecules": "GTP"
  },
  {
    "caption": "(D) Cells were transfected and cell lysates were incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected and cell lysates were incubated with purified GST- protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody.",
    "molecules": "GTP"
  },
  {
    "caption": "(E) Cells were transfected with siRNAs as indicated, followed by transfection with Flag-IFT20, incubation in serum-starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag-IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. (F) Quantification of the percentage of ciliated cells with both the Flag-IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n=3 experiments), 150 Flag positive cells were scored per condition per experiment, *P < 0.05, Student's t-test. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Cells were transfected with siRNAs as indicated, followed by further transfection with CA-Rab8, incubation in serum-starvation media for 12 h, and immunostained for acetylated-tubulin. Representative fluorescent images of GFP-Rab8 Q67L (green), acetylated-tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. (H) Quantification of the percentage of GFP -positive ciliated cells (only those cilia having both GFP-Rab8 and acetylated-tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), 200 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) RPE1 cells were transfected as indicated, followed by incubation in serum-starvation media in the presence or absence of cholesterol/MCD for 24 h, and then immunostained for Rab8 (red) and γ-tubulin (green). Scale bar, 10 μm. Representative magnified images are shown from cells labeled with white asterisks.",
    "molecules": "cholesterol, MCD"
  },
  {
    "caption": "(C) (Upper panel) Cells were transfected and cell lysate was incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) The intensity of bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. The bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.",
    "molecules": "GTP"
  },
  {
    "caption": "Insulin (50 µg/mL) induced the translocation of NFAT1-GFP to the nucleus of WM3734 cells. (A) Ratio images (F/F0), (B) corresponding endpoint quantification (n=5).",
    "molecules": "Insulin"
  },
  {
    "caption": "Reduced nuclear translocation of NFAT1-GFP in TMX1 or TMX3 silenced (siRNA) melanoma cells. (C) Images show NFAT1-GFP fluorescence intensity before and after stimulation with thapsigargin (Tg; 1 µM) in WM3734 cells. (D) Corresponding time-dependent nuclear import of NFAT1 as a change of F/F0. (E) Normalized endpoint quantification. Same analysis was performed with Mel Juso cells with (F) images, (G) time-dependent nuclear import and (H) normalized endpoint quantification. data are presented as mean ± SEM (n values: WM3734, control=142, TMX1 kd=116, TMX3 kd=148; Mel Juso, control=75, TMX1 kd=47, TMX3 kd=67).",
    "molecules": "Tg, thapsigargin"
  },
  {
    "caption": "Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones). : In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508).",
    "molecules": "Ringer's buffer, Ca2+, calcium, Fura-2, Tg, Thapsigargin"
  },
  {
    "caption": "Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3. In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52).",
    "molecules": "Ca2+, Fura-2"
  },
  {
    "caption": "Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells. In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.",
    "molecules": "H2O2"
  },
  {
    "caption": "WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0.",
    "molecules": "H2O2, Tg"
  },
  {
    "caption": "The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark).",
    "molecules": "H2O2"
  },
  {
    "caption": "Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h.",
    "molecules": "N-acetylcysteine, NAC, Tg"
  },
  {
    "caption": "Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM). data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22).",
    "molecules": "DTT, catalase, NAC, PEG"
  },
  {
    "caption": "Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation.",
    "molecules": "Tg"
  },
  {
    "caption": "Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM). Quantification of the maximum cytosolic calcineurin activity (plateau-basal).",
    "molecules": "H2O2, Tg"
  },
  {
    "caption": "Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1. data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19).",
    "molecules": "NAC"
  },
  {
    "caption": "Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso. , data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442).",
    "molecules": "H2O2"
  },
  {
    "caption": "Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau). , data are presented as mean ± SEM (n values: control=62, TMX1 kds=45).",
    "molecules": "Ringer's buffer, Ca2+, calcium, Tg"
  },
  {
    "caption": "Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1. The mean mitochondrial volume was modeled based on microscopy, the quantification of mean volume (µm3) and mean surface (µm2) is depicted in (G) and (H) respectively. data are presented as boxplots (n values: control=101, TMX1 kds=90; center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots).",
    "molecules": "MitoTracker DeepRed"
  },
  {
    "caption": "Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1. A peripheral mask was applied based on the membrane staining (CellMask Green) and mitochondria covered area (MitoTracker DeepRed) was evaluated (see Fig. 9). Representative images (I) and quantification (J). In (J), data are presented as mean ± SEM (n values: control=163, TMX1 kds=116).",
    "molecules": "Green, MitoTracker DeepRed"
  },
  {
    "caption": "Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification. In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844).",
    "molecules": "GKT, GKT137831, H2O2"
  },
  {
    "caption": "Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (100 nM).",
    "molecules": "NAC, mTEMPO"
  },
  {
    "caption": "Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (1 µM).",
    "molecules": "NAC, mTEMPO"
  },
  {
    "caption": "Transwell migration (48 h) of melanoma cell lines after preventing NFAT-calcineurin interaction with dipyridamole (40 µM) or inhibition of calcineurin with cyclosporine A (CsA, 2 µM).",
    "molecules": "CsA, cyclosporine A, dipyridamole"
  },
  {
    "caption": "(B-C)  Coimmunoprecipitation of  Ams1 (B) and Ape4 (C) with  Nbr1. Endogenously TAP-tagged Nbr1 was  immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and  immunoprecipitates were examined by immunoblotting (IB).",
    "molecules": "Sepharose"
  },
  {
    "caption": "A Effects of myeloid-specific Lamtor1 deficiency on peritoneal IL-1β production, CD11b+ Ly6C+ monocyte recruitment, and CD11b+ Ly6G+ neutrophil recruitment after an intraperitoneal injection of 600 μL alum solution (20 mg/mL, 6 h), Data are shown as means ± SEM. * indicates P < 0.05 and *** indicates P < 0.001 by Student's t-test, n = 6 mice (peritoneal IL-1β production) and 4 mice (FACS analysis).",
    "molecules": "alum"
  },
  {
    "caption": "B Effects of myeloid-specific Lamtor1 deficiency in an acute gouty arthritis model. MSU crystals (0.5 mg) or the PBS control were injected intra-articularly into the tibia-tarsal joint of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre mice. Mice were assessed for joint swelling using electronic calipers. Images show photomicrographs (upper panel) and hematoxylin and eosin-stained sections (middle and lower panels) of ankle joints obtained at 24 h. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 7 mice each.",
    "molecules": "eosin, hematoxylin, MSU, PBS"
  },
  {
    "caption": "C Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation. ELISA assay shows IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h), followed by nigericin (15 μM, 1 h), ATP (10 mM, 1 h), or GPN (100 nM, 6 h).",
    "molecules": "ATP, GPN, LPS, nigericin"
  },
  {
    "caption": "D Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO different single clones, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).",
    "molecules": "nigericin, PMA"
  },
  {
    "caption": "E, F Effects of Lamtor1 deficiency on IL-1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous ROS (hydrogen peroxide). ELISA assay shows IL-1β secretion in supernatants after treatment with LPS (200 ng/mL), Rotenone (10 μM), or Antimycin (10 μg/mL) for 6 h or followed by hydrogen peroxide (3.86 mM).",
    "molecules": "Antimycin, hydrogen peroxide, LPS, ROS, Rotenone"
  },
  {
    "caption": "G Effects of Lamtor1 reconstitution in Lamtor1-deficient macrophages on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in BMDM supernatants from Lamtor1flox/flox cells, Lamtor1flox/flox LysM-Cre cells, and Lamtor1flox/flox LysM-Cre cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with LPS (200 ng/mL, 4 h).",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "H Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).",
    "molecules": "LPS, nigericin, PMA"
  },
  {
    "caption": "I Effects of Lamtor1 deficiency on IL-18 secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-18 secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).",
    "molecules": "LPS, nigericin, PMA"
  },
  {
    "caption": "J Effects of Lamtor1 deficiency on IL-1β secretion after Pyrin inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with TcdB (0.5 μg/mL, 3 h) after LPS (200 ng/mL, 4 h) stimulation.",
    "molecules": "LPS"
  },
  {
    "caption": "K Effects of Lamtor1 deficiency on IL-1β secretion after AIM2 inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with Poly(dA:dT) (5 μg/mL, overnight) and Lipofectamine 3000 after LPS (200 ng/mL, 4 h) stimulation.",
    "molecules": "Lipofectamine 3000, LPS, Poly(dA:dT)"
  },
  {
    "caption": "Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs (A) (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL, for 4 h (BMDM) stimulation.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. WT and Lamtor1 KO THP-1 cells (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL 2 h (THP-1)] stimulation.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation. Western blot shows GSDMD, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation and the percentage of propidium iodide positive cells were counted by flow-cytometry.",
    "molecules": "LPS, nigericin, propidium iodide"
  },
  {
    "caption": "E Effects of Lamtor1 deficiency on IL-1β dynamics. Western blot shows IL-1β and β-actin. WT cells and Lamtor1 KO cells were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) stimulation.",
    "molecules": "nigericin, PMA"
  },
  {
    "caption": "F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with active form of GSDMD, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).",
    "molecules": "LPS, nigericin, PMA"
  },
  {
    "caption": "H Effects of Lamtor1 deficiency on the priming phase. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with treated with LPS (200 ng/mL, 4 h).",
    "molecules": "LPS"
  },
  {
    "caption": "I Effects of IL-1β and active form of Caspase-1 reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with IL-1β, active form of Caspase-1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).",
    "molecules": "LPS, nigericin, PMA"
  },
  {
    "caption": "A Representative images of mutant Lamtor1. PMA-primed Lamtor1 KO THP-1 macrophages reconstituted with full-length or truncated Lamtor1.",
    "molecules": "PMA"
  },
  {
    "caption": "B Effects of the Lamtor1 point mutation on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion in THP-1 cell supernatants from WT cells, Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full-length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine (G2A); all cell types were then treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).",
    "molecules": "nigericin, PMA"
  },
  {
    "caption": "C Effects of Lamtor1 deficiency on mTORC1 activity. Immunoblot analysis of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h).",
    "molecules": "LPS"
  },
  {
    "caption": "D Effect of the rapamycin treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from PMA (50 nM, overnight) primed WT THP-1 cells treated with rapamycin at indicated concentration for 2 h, then treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h).",
    "molecules": "LPS, nigericin, PMA, rapamycin"
  },
  {
    "caption": "E Effect of rapamycin treatment on Caspase-1 processing. Western blot shows caspase-1/p20. LPS (200 ng/mL, 4 h) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin (15 μM, 1 h).",
    "molecules": "LPS, nigericin, rapamycin"
  },
  {
    "caption": "F Effect of Torin-1 treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from WT BMDMs pretreated with Torin-1 (0, 1, 10, 100, or 250 nM) for 2 h before LPS (200 ng/ml, 4 h) and nigericin (15 μM, 1 h) treatment (left panel). Western blot shows phospho-p70-S6K from BMDMs pretreated with Torin-1 at the indicated concentrations for 2 h before LPS and nigericin treatment (right panel).",
    "molecules": "LPS, nigericin, Torin-1"
  },
  {
    "caption": "A Effects of the HDAC6 inhibitor treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion and caspase-1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM).",
    "molecules": "rocilinostat, tubacin"
  },
  {
    "caption": "H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc-fused Lamtor1 and Halo-tag-fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano-Glo Luciferase Assay Substrate (Promega).",
    "molecules": "Lipofectamine 2000"
  },
  {
    "caption": "Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP-1 cells. ELISA assay showing IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full-length Lamtor1, and Met1-Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).",
    "molecules": "nigericin, PMA"
  },
  {
    "caption": "A, B Representative images of Lamtor1 and ASC. PMA-treated THP-1 cells stably expressing GFP-tagged ASC and td-Tomato-tagged Lamtor1 were primed with LPS (200 ng/mL, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s).",
    "molecules": "LPS, nigericin, PMA"
  },
  {
    "caption": "C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA-primed THP-1 macrophages stably expressing GFP-tagged ASC treated with or without LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm.",
    "molecules": "LPS, nigericin, PMA"
  },
  {
    "caption": "Effects of DL-all-rac-α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL-all-rac-α-tocopherol (B)",
    "molecules": "DL-all-rac-α-tocopherol"
  },
  {
    "caption": "IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL-all-rac-α-tocopherol (C) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).",
    "molecules": "DL-all-rac-α-tocopherol, LPS, nigericin, PMA"
  },
  {
    "caption": "D) IL-6 secretion after overnight pretreatment with DL-all-rac-α-tocopherol. ELISA assay showing IL-6 secretion in supernatants from WT BMDMs treated with LPS (200 ng/mL, overnight) and DL-all-rac-α-tocopherol at the indicated concentrations (overnight).",
    "molecules": "DL-all-rac-α-tocopherol, LPS"
  },
  {
    "caption": "Effects of α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D -α-tocopherol (E)",
    "molecules": "D -α-tocopherol, α-tocopherol"
  },
  {
    "caption": "IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D -α-tocopherol (F) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).",
    "molecules": "D -α-tocopherol, LPS, nigericin, PMA"
  },
  {
    "caption": "G Effects of DL-all-rac-α-tocopherol in an acute gouty arthritis model. MSU crystals (0.5 mg) suspended in 20 μL endotoxin-free PBS or PBS control were injected intra-articularly into the tibia-tarsal joint (ankle) of C57BL/6 WT mice with or without DL-all-rac-α-tocopherol. Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin-stained sections of ankle joints.",
    "molecules": "DL-all-rac-α-tocopherol, eosin, hematoxylin, endotoxin, MSU, PBS"
  },
  {
    "caption": "H Effects of DL-all-rac-α-tocopherol in an MSU-induced peritonitis model. Twelve hours after DL-all-rac-α-tocopherol pretreatment, 100 μL MSU solution (10 mg/mL) was intraperitoneally injected for 4 h. Peritoneal IL-1β production was then measured by ELISA, and CD11b+ Ly6G+ neutrophil recruitment was measured by FACS.",
    "molecules": "DL-all-rac-α-tocopherol, MSU"
  },
  {
    "caption": "A Growth of indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3). B Growth of indicated Y. pestis strains in zinc-replete medium (cPMH2 + 10μM ZnCl2) (n=3). Data information: One-way ANOVA with Dunnett test, *p≤0.05, **p≤0.001, ****p≤0.0001. For A, B growth of mutants was compared to znuBC. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.",
    "molecules": "zinc, ZnCl2"
  },
  {
    "caption": "C Growth of indicated Y. pestis strains in iron-limited (cPMH2 + 0μM FeCl2) or iron-replete (cPMH2 + 10μM FeCl2) media (n=3).",
    "molecules": "iron, FeCl2"
  },
  {
    "caption": "D Growth of znuBC y0702-y0703, znuBC y0704, or y0702-y0703 complemented mutant (pBCSK+y0702-70703) in zinc-limited medium (cPMH2) (n=3). Data information: One-way ANOVA with Dunnett test, *p≤0.05, **p≤0.001, ****p≤0.0001. D, growth of mutants was compared to znuBC. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.",
    "molecules": "zinc"
  },
  {
    "caption": "B Growth of znuBC irp2 pGENlux in co-culture with indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3). Data information: For B, Data represents the mean ±SD relative luminescent units (RLU) from three biological replicates. For some points, error bars are too small to visualize on the graph. One-way ANOVA with Dunnett test compared to znuBC irp2 + Y. pestis; ****p≤0.0001.",
    "molecules": "zinc"
  },
  {
    "caption": "C Absorbance of PAR at 500nm incubated with indicated Y. pestis supernatants (n=3). D OD600 of bacterial cultures at 8 h for LC-MS (n=3). Data information: For One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.",
    "molecules": "PAR"
  },
  {
    "caption": "E LC-MS analysis of Ybt in culture supernatants (note: both peaks are Ybt). F LC-MS quantification (n=3). Data information For One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.",
    "molecules": "Ybt"
  },
  {
    "caption": "Growth of indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3), One-way ANOVA with Dunnett test compared to znuBC mutant, **p≤0.01, ****p≤0.0001. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.",
    "molecules": "zinc"
  },
  {
    "caption": " Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments). A) Levels of TIMP1, MMP7 and TSP2 in plasma of mice bearing orthotopic HuPa8 at 30, 90 and 150 days after tumor transplantation (mean ± SEM; n≥4 for each group). *p<0.05, (Mann-Whitney). The exact n and p-values are indicated in Table S5A ",
    "molecules": "GEM, gemcitabine, NAB-P, Nab-paclitaxel"
  },
  {
    "caption": " Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) B) Magnetic resonance imaging of HuPa8. Representative images are shown, the white dotted lines indicate tumor masses ",
    "molecules": "GEM, gemcitabine, NAB-P, Nab-paclitaxel"
  },
  {
    "caption": ". Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) C) Tumor growth over time measured by MRI; each black arrow indicates one treatment (mean ± SEM; n=4 for each group; 2-way ANOVA with Tukey\"s multiple comparison test) ",
    "molecules": "GEM, gemcitabine, NAB-P, Nab-paclitaxel"
  },
  {
    "caption": " Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) D) Levels of the 3 selected biomarkers in plasma of HuPa8 bearing mice, prior to (day 80; at randomization), during (day 120) and after (day 165) the treatments (mean ± SEM; n≥4 for each group; 2-way ANOVA with Tukey\"s multiple comparison test). The exact n is indicated in Appendix Table S5B ",
    "molecules": "GEM, gemcitabine, NAB-P, Nab-paclitaxel"
  },
  {
    "caption": " Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant, visualized by anti-HA staining (green). Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus, respectively. A novel MS-microscopy approach was used to further define the molecular context of the proteins. This analysis identified possible chromosomal, endosomal and membrane localization of the proteins. The possible endosomal localization is in agreement with the anti-HA immunofluorescence microscopy results. (key: the scale bar for immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-microscopy tool. ",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "B. Quantification of time-course nucleosome H2A-Ub assays using the indicated BRCA1/BARD1 truncations. Data are presented as the normalized fraction of H2A consumed during a time-course nucleosome ubiquitylation assay. Data points represent mean values and error bars are ± 1-s.d. of n=3 independent technical replicate experiments. The residue bounds of BRCA1RING are 1-112, and BARD1RING are 26-140. Additional details about all reagents used in these studies can be found in the methods section.",
    "molecules": "Ub"
  },
  {
    "caption": "C. Nucleosome binding curves from fluorescence-quenching-based measurements using the indicated BRCA1/BARD1 constructs. Data show mean values and error bars are ± 1-s.d. of n=4 independent technical replicate experiments. The reported difference in affinities to BRCA1RING/BARD1RING is likely underestimated, as the minimal RING/RING heterodimer binding data was collected at a lower ionic strength than the other constructs to obtain well-behaved binding data (50 mM vs. 100 mM NaCl).",
    "molecules": "NaCl"
  },
  {
    "caption": "B. 1H15N-HSQC NMR amide chemical shift perturbations (CSPs) observed for 15N-BARD1 141-216 signals when bound to a 36-bp dsDNA fragment (1:1 molar equivalent complex).",
    "molecules": "dsDNA, 1H, 15N"
  },
  {
    "caption": "C. Representative SDS-PAGE gel monitoring in-gel fluorescence (labelled DNA) of UV-induced Bpa crosslinking of the E3 ligase fusion BRCA1-ƒ-BARD1221 (BCƒBD) to nucleosomes (left). In this construct, the C-terminus of BRCA11-104 is genetically fused to the N-terminus of BARD126-221 via a 12-residue GlySer-linker. Quantification of Bpa crosslinking experiments with nucleosomes (right). The intensity of each crosslinked band was normalized to the intensity of the L120Bpa crosslinked band for each replicate experiment. Graph bars show the mean; error bars are ± 1-s.d. and the open circles are the values of individual replicates for of n=3 independent technical replicate experiments.",
    "molecules": "Bpa, DNA"
  },
  {
    "caption": "Quantification of H2A-Ub time-course assays using the indicated BRCA1/BARD1 constructs. Data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.",
    "molecules": "Ub"
  },
  {
    "caption": "C. Inhibition of UV-induced Bpa crosslinking between the indicated BRCA1-f-BARD1221 Bpa-incorporated constructs and NCP185 substrates in the presence of increasing amounts of dsDNA or bubble-DNA competitor. Data show the mean; error bars are ± 1-s.d. of n=3 (W146Bpa) or n=4 (A195Bpa) independent technical replicate experiments.",
    "molecules": "Bpa, dsDNA"
  },
  {
    "caption": "D. 1H15N-HSQC NMR CSPs observed to 15N-BARD1 141-216 signals when bound to a 36-mer dsDNA (blue bars) or bubble-DNA (gray bars with black outlines) fragment (1:1 molar equivalent complex). Signals for residues 143-157 are broadened beyond detection in the bubble-DNA-bound spectrum; for visualization purposes, gray bars are set equal to the CSP value for the W146Ne CSP that was observed; the real CSP values are likely larger that this value",
    "molecules": "dsDNA, 1H, 15N"
  },
  {
    "caption": "F. Single time-point H2A-Ub inhibition assays using heterodimers containing BRCA1RING/BARD1FL (left, 12 min endpoint) or the indicated BARD1 internal deletion mutant (middle and right, 20 min endpoint) and increasing amounts of dsDNA or bubble-DNA competitor. The same E3 concentration (50 nM) was used for each BRCA1/BARD1 truncation. A longer time point was used for the double-deletion mutant as the intrinsic H2A-Ub activity of this mutant was lower, likely due to the deletion of DNA binding regions.",
    "molecules": "dsDNA, Ub"
  },
  {
    "caption": "A. Western blot analysis to detect HA-BARD1 in nuclei isolated from HeLa-shBARD1 cells stably expressing wild-type or Δ194-216 mutant of HA-BARD1, where endogenous BARD1 was depleted by doxycycline-induced shBARD1 expression. The nuclei were salt-fractionated into NS100, NS300, NS420, and NP420 to assess the amount of HA-BARD1 associated with chromatin. Data are representative of n=2 biological replicate experiments.",
    "molecules": "doxycycline, salt"
  },
  {
    "caption": "B. Quantification of cells with >5 HA-BARD1 foci (left panel) and number of HA-BARD1 foci per nucleus (right panel) with and without olaparib treatment. *p<0.05, **p<0.01, ***p<0.001, by Student's t-test. Data show the individual points from n=3 biological replicate experiments.",
    "molecules": "olaparib"
  },
  {
    "caption": "C. Clonogenic survival of HeLa-shBARD1 cells stably expressing wild type or Δ194-216 mutant of HA-BARD1 upon treatment with indicated amount of olaparib and cisplatin. Data points show the mean; error bars represent SEM of n=3 biological replicate experiments.",
    "molecules": "cisplatin, olaparib"
  },
  {
    "caption": "B, C. Quantification of time-course H2A-Ub assays using the indicated BRCA1/BARD1 constructs and chromatin substrates. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.",
    "molecules": "Ub"
  },
  {
    "caption": "D. Quantification of time-course H2A-Ub assays using homogenously modified H2A K15-Ub mono-nucleosome substrates and indicated BRCA1/BARD1 constructs. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments. One time-point from Panel D was determined to be an outlier due to a reaction quenching error and excluded from analysis. The excluded value is reported as source data.",
    "molecules": "Ub"
  },
  {
    "caption": "G, H. Quantification of time-course H2A-Ub assays using the indicated combinations of di-NCP substrate and BRCA1/BARD1 heterodimer. In each case, the y-axis reports on the fraction of H2Aobserve (as shown in Panel F). Lower E3 concentrations were used in these reactions than in those presented in Figure 1 using unmodified mono-nucleosome substrates (15 nM vs. 100 nM), accounting for the slower observed H2A-Ub kinetics for the unmodified di-nucleosome substrate. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.",
    "molecules": "Ub"
  },
  {
    "caption": "A Coolness stimulates intracellular cyclic guanosine monophosphate (cGMP) accumulation in HEK‐293T cells expressing GC‐G. Two days after transfection of empty vector or GC‐G‐encoding plasmid, cells were exposed to ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured.",
    "molecules": "cGMP, cyclic guanosine monophosphate"
  },
  {
    "caption": "D The H+CYC domain is critical for stimulation by coolness. The domain structure of full‐length (FL) GC‐G and its mutant variants is shown in the upper panel. A FLAG tag was added at the N‐terminus of each protein. The ΔECD+KLD mutant protein lacks amino acids 73-455 and 556-833; the ΔCYC variant lacks amino acids 834-1,003. The H+CYC mutant protein contains amino acids 806-1,100. ECD, extracellular domain; KLD, kinase‐like domain; SP, signal peptide; TM, transmembrane region. HEK‐293T cells expressing the indicated GC‐G constructs were incubated at 37 or 15°C for 20 min, then intracellular cGMP concentration was measured (lower panel).",
    "molecules": "cGMP"
  },
  {
    "caption": "In the chimeric GC‐A/G protein, the H+CYC domain of GC‐A was replaced by that of GC‐G (upper panel). HEK‐293T cells expressing the indicated proteins were exposed to ambient temperature of 37 or 15°C for 20 min; then, intracellular cGMP concentration was measured. Data are mean ± SD from three experiments in triplicate. **P 0.01.",
    "molecules": "cGMP"
  },
  {
    "caption": "B, C Protein expression and coolness‐stimulated cGMP synthesis of the above shown divergent C‐terminal fragments of GC‐G with intact or ablated hinge (H) region. The expression plasmids encoding the H+CYC domain or a deletion construct (Δ1-Δ2) tagged with a FLAG epitope were transfected into HEK‐293T cells. Two days after transfection, cells were exposed to the ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured (C). Protein expression of each construct was confirmed by Western blot (WB) analysis with anti‐FLAG antibody (B).",
    "molecules": "cGMP"
  },
  {
    "caption": "HEK‐293T cells expressing CNGA3 alone (upper panel) or together with GC‐G (full‐length in the middle panel and H+CYC domain in the lower panel) were loaded with the Ca2+ indicator Fura‐2 to monitor intracellular [Ca2+]i concentration. Cells co‐expressing GC‐G and CNGA3 showed a rapid increase in [Ca2+]i when the temperature was lowered from 37 to 15°C (middle and lower panel); cells only expressing CNGA3 are not responsive to cool temperatures (top panel). Removal of extracellular Ca2+ (−Ca2+; middle panel) completely suppressed the response to cooling. ΔF represents changes in the ratio of the fluorescence intensity of Fura‐2 at 340/380 nm excitation. The ratiometric Ca2+ responses are representative of 30 cells recorded from three experiments.",
    "molecules": "Ca2+"
  },
  {
    "caption": "J-N Calcium imaging of coronal sections through the GG of olfactory marker protein‐green fluorescent protein (OMP‐GFP)/GC‐G+/+ or OMP‐GFP/GC‐G‐KO pups (P1-P4). High magnification image (J) of a tissue slice through the GG of an OMP‐GFP pup with GG neurons labeled by intrinsic GFP fluorescence (GFP fluorescence was merged with the transmitted‐light channel). Cells analyzed are circled in blue (GFP‐negative) or red (GFP‐positive). NC, nasal cavity. Scale bar: 30 μm. (K-M) Representative ratiometric Ca2+ transients induced by cooling from 37 to 15°C in GFP‐negative non‐neuronal cells (K) and in GFP‐positive GG neurons from OMP‐GFP/GC‐G+/+ (L) and OMP‐GFP/GC‐G‐KO pups (M). The numbers in the bottom right hand corners are the number of cells with Ca2+ transient similar to what is shown in the respective graph (left) and total number of measured cells (right). (N) Quantification of coolness‐induced ΔF in GG neurons from OMP‐GFP/GC‐G+/+ and OMP‐GFP/GC‐G‐KO mice. Coolness‐induced ΔF was calculated by subtracting the baseline fluorescence ratio (340/380 nm) at 37°C from the peak fluorescence ratio (340/380 nm) measured at 15°C. For OMP‐GFP/GC‐G+/+, the 29 coolness‐responsive neurons (black bar) of all 54 analyzed neurons (gray bar) from five slices (obtained from different animals) were analyzed. For OMP‐GFP/GC‐G‐KO, the 50 analyzed neurons (shaded bar) from five slices (obtained from different animals) were analyzed. Data are mean ± SD, **P 0.01.",
    "molecules": "Ca2+, Calcium"
  },
  {
    "caption": "(D) Cells were treated with  sodium arsenite (50 µM) for 45 min, followed by recovery in drug-free medium. Kinetics of SG dissolution are reported. Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 159 (siRNA control); 166 (siRNA Hsp90 α+β).",
    "molecules": "sodium arsenite"
  },
  {
    "caption": "(E) HeLa-Kyoto cells were treated with HS at 43.5˚C for 1 hr. Cells were then allowed to recover at 37 °C for 1 hr in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed, stained for the SG marker TIA-1 and the percentage of cells with  SGs was counted. Number of cells counted: 605 (recovery control); 510 (recovery  VER); 1334 (recovery GA); 468 (recovery 17AAG). n = 3-4 independent experiments, ± sem. p < 0.01 (One-way ANOVA). (F) G3BP2-GFP HeLa-Kyoto cells were treated with MG132 (20 µM) for 3 hrs, followed by recovery for 2 hrs in drug-free medium (control) or in presence of  VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed and the percentage of cells with SGs was counted.  Number of cells counted: 439 (recovery control); 649 (recovery VER); 637 (recovery GA); 649 (recovery 17AAG). n = 3 independent experiments, ± sem. p < 0.0001 (One-way ANOVA). ",
    "molecules": "VER, GA, MG132, 17AAG"
  },
  {
    "caption": "(C) HeLa cells were subjected to proximity ligation assay (PLA) using antibodies specific for endogenous Hsp90 and DYRK3. PLA foci and nuclei were segmented, and  PLA foci/cell were automatically quantified. The mean of PLA/foci in cells incubated with no antibodies (-), only Hsp90 antibody, only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown. Cells incubated with  Hsp90  and DYRK3 antibodies and left untreated were used as control. n = 3 independent experiments, ± sem; 368-504 cells  counted/sample, p < 0.002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies; p = 10-6 between cells incubated with  Hsp90 and DYRK3 antibodies and left untreated (control) or treated for 2 hrs with GA (5 µM) (One-way ANOVA). Scale bar is 10 µm.",
    "molecules": "GA"
  },
  {
    "caption": "(F)  HeLa cells were lipofected with a cDNA encoding for GFP or GFP-DYRK3. 6 hrs post-transfection, cells were incubated in drug-free medium (-) or in presence of GA, 17AAG or VER (concentrations are shown). Total proteins were extracted 16 hrs later. GFP and TUBA4A protein levels were analyzed by immunoblotting. Representative immunoblotting  of 3 independent experiments.",
    "molecules": "VER, GA, 17AAG"
  },
  {
    "caption": "(I) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3. 24 hrs post-transfection, cells were treated for 45 min with arsenite (50 µM) alone (control) or with GA (5 µM) or 17AAG (5 µM). Cell were then fixed and the percentage of cells expressing GFP-DYRK3 with SGs was counted. Total cells counted: 462 (sodium arsenite); 444 (sodium arsenite and GA); 561 (sodium arsenite and 17AAG). n = 3 independent experiments, ± sem; p < 0.001 (One-way ANOVA).",
    "molecules": "GA, arsenite, sodium arsenite, 17AAG"
  },
  {
    "caption": "(A, HeLa cells were left untreated (control) or treated with  GA (5 µM), 17AAG (5 µM) or VER (10 µM) for 8 hrs. Cells were then fixed and immunostained for the nuclear speckle marker  SC35 and  DYRK3. Automatic segmentation of nuclear speckles  is based on SC35. An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 (using a fluorescent ratio  1.5 as threshold). Scale bar is 10 µm.",
    "molecules": "VER, GA, 17AAG"
  },
  {
    "caption": "(F) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3-dN. 24 hrs post-transfection GFP-DYRK3-dN cells were either left untreated or exposed to GA (5 µM) for 4 hrs. In untreated cells (control), GFP-DYRK3-dN is diffusely distributed in the cytosol and in the nucleus. Upon GA treatment, GFP-DYRK3-dN forms perinuclear (PN) aggregates. Representative confocal images are shown. Scale bar is 10 µm.",
    "molecules": "GA"
  },
  {
    "caption": "(I) mCherry-G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP-DYRK3-dN or Sup35-NM-GFP-DYRK3-dN. 24 hrs post-transfection cells were treated for 45 min with sodium arsenite to induce  SGs, followed by recovery for 4 hrs in presence of  GA  (5 µM; rec. GA). Representative confocal microscopy images are shown. Nucleic acid was stained with DAPI. Scale bar is 10 µm.",
    "molecules": "DAPI, GA, sodium arsenite"
  },
  {
    "caption": "(C) Quantitation of the fluorescence intensity recovery after  bleach of GFP-DYRK3 in G3BP1-mCherry HeLa-Kyoto cells 24 h after transfection. Cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, which were visualized with G3BP1-mCherry; then, sodium arsenite was removed and the cells were incubated in drug-free medium or in presence of  GA (5 µM) or of GSK  (5 µM). FRAP  was performed during the stress recovery period. A representative  image of cells treated with arsenite followed by recovery in drug-free medium is shown. Arrowheads and dotted circle indicate the ROI. Scale bar is 5 µm. The mean of 13 FRAP curves (recovery control), 12 FRAP curves (recovery GA) and 14 FRAP  curves (recovery GSK) is shown in red; the sem is shown in gray.",
    "molecules": "GA, GSK, arsenite, sodium arsenite"
  },
  {
    "caption": "(B) Confocal microscopy images of  P525L FUS eGFP MNs treated with sodium arsenite  for 2 hrs and showing colocalization of FUS with TIAR-positive stress granules. Scale bar is 10 µm. (C) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs, followed by recovery in drug-free medium (+ rec. control) or in presence of 17AAG (30 μM; + rec. 17AAG) or of GSK  (5 μM; +rec. GSK) for 6 hrs. Quantitation of the number of SGs per cell in P525L FUS eGFP MNs. Number  of P525L cells counted and n/condition: 2488, n = 13 (arsenite); 1045, n = 12 (arsenite + rec. control); 646, n = 6 (arsenite + rec.  17AAG); 924, n = 3 (arsenite + rec. GSK), ± sem (One-way ANOVA). ",
    "molecules": "GSK, arsenite, sodium arsenite, 17AAG"
  },
  {
    "caption": "(I) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in  lumbar spinal cord α-MNs of healthy subjects (control) and fALS patients  carrying the p.521C mutation in the FUS  gene. Control (upper panel): α-MNs showing uniform cytoplasmic (white arrows) and speckled pattern of strong nuclear immunoreactivity (white arrowhead) of DYRK3, as well as diffuse  nuclear FUS immunoreactivity. fALS (p.R521C-FUS; lower panel): surviving α-MNs harboring large FUS aggregates (yellow arrows) showed markedly reduced  DYRK3 immunoreactivity both in the cytoplasm as well as in the  nucleus (red arrowheads). Instead, α-MNs devoid of  FUS  aggregates (white arrows) showed normal DYRK3 immunoreactivity similar to the one of α-MNs from normal controls. Asterix (*) represents non-specific lipofuscin granules in one of the α-MN in FUS-ALS. Paraffin sections; scale bars is 50 µm. (J) Quantification of α-MNs showing low or absent DYRK3 staining (upper graphic) and FUS aggregates (lower graphic) in lumbar spinal cord from five healthy subjects (control) and five  fALS patients carrying the p.521C mutation in the FUS gene. Total number  of α-MNs analyzed: 403 (control); 132 (fALS). n = 5, ± sem (Student's t-test). ",
    "molecules": "lipofuscin"
  },
  {
    "caption": "(B-E) Immunostaining of germaria from 7 day-old females with anti-Vasa (green, B-D) or anti-GFP (green, E), and anti-Hts (red). DAPI (blue) was used to visualize DNA. (B) aubHN2/+ was used as a control. (C, D) examples of aubHN2/QC42 germ cell loss and tumor, respectively. (E) Phenotypic rescue of aubHN2/QC42 with UASp-GFP-Aub expressed using nos-Gal4. White arrowheads indicate GSCs; the white arrow indicates GSC loss.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(G-H') Germaria containing control (G, G') or aubHN2 mutant (H, H') clonal GSCs stained with anti-GFP (green) and anti-Hts (red), 14 days after clone induction. DAPI (blue) was used to stain DNA. Clonal cells are marked by the lack of GFP. Clonal GSCs and cysts are outlined with dashed line. White arrowheads show clonal GSCs in the control. aub mutant clonal GSCs have been lost (H, H'). Scale bar: 10 μm in B-E and G-H'.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(A, B) Immunostaining of germaria with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of mnkP6 and mnkP6 aubHN2/QC42 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(D-F) Immunostaining of germaria from 7 day-old females with anti-Vasa or anti-GFP (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. aubHN2/QC42; nos-Gal4/+ was used as a negative control. Examples of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-Aub germarium (E), and of lack of rescue in aubHN2/QC42; nos-Gal4/UASp-GFP-AubAA germarium (F). White arrowheads indicate GSCs; white arrows indicate GSC loss in (D) and germ cell loss in (F). Scale bar: 10 μm in A, B and D-F.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(E-G) Genetic interaction between aub and twin in Vasa self-renewal. Immunostaining of germaria with anti-DNA (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. Examples of aubHN2/+, twinDG24102 and aubHN2/+; twinDG24102 germaria are shown. White arrowheads indicate GSCs; the white arrow indicates GSC loss. Scale bar: 10 μm in A-B'' and E-G.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(B-C'') Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B'') or 10F1 (C-C'') monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(B-C'') Immunostaining of mosaic germaria with anti-GFP (green), to identify clonal cells by the lack of GFP, and either 8C4 (B-B'') or 10F1 (C-C'') monoclonal anti-Cbl (red). DAPI was used to visualize DNA. White arrowheads indicate aubHN2/+ control GSCs; yellow arrowheads indicate clonal mutant aubHN2 GSCs. Scale bar: 10 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(A-E) Immunostaining expression in germaria. (C-C') Immunostaining of germaria expressing GFP-Par1 to label spectrosomes with anti-GFP (green) and anti-Cbl 8C4 (red). DAPI (blue) was used to visualize DNA. White arrowheads indicate GSCs; yellow arrowheads indicate cystoblasts.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(K-M) Cbl induces GSC differentiation. Immunostaining of germaria overexpressing Cbl with Hsp83-CblS (L) with anti-Vasa (green) and anti-Hts (red). DNA (blue) was revealed with DAPI. White arrows indicatethe loss of GSCs and germ cells (L).",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(A-B) Immunostaining of germaria from aubHN2/QC42 (A) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(A-B) Immunostaining of germaria from aubHN2/QC42; CblMB/+ (B) females with anti-Vasa (green) and anti-Hts (red). DAPI (blue) was used to visualize DNA. The white arrow indicates the lack of GSCs; white arrowheads indicate GSCs. Scale bar: 10 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "Mitochondrial translational products were labelled with [35S]methionine and anti-FLAG immunoisolation was performed as described in Fig. 1D. Eluates were analyzed by SDS-PAGE and digital autoradiography. Total 1.5 %, Eluate 100 %.",
    "molecules": "[35S]methionine"
  },
  {
    "caption": "Mitochondria from wild type and MITRAC15-/- cells were isolated and analyzed by BN-PAGE. Triton X-100 (left site) or digitonin (right site) were used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates).",
    "molecules": "digitonin, Triton X-100"
  },
  {
    "caption": "Pulse-chase [35S]methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).",
    "molecules": "[35S]methionine"
  },
  {
    "caption": "Mitochondrial translation products were radiolabelled with [35S]methionine after siRNA-mediated depletion of ACAD9 in wild type and MITRAC15-/- cells. Cell lysates were analyzed by SDS-PAGE and digital autoradiography. For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal. (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01 ***p < 0.001; unpaired t-test).",
    "molecules": "[35S]methionine"
  },
  {
    "caption": "After C12orf62 down regulation, [35S]methionine labelling was performed. During labeling mitochondrial translation was inhibited with puromycin (pur) (2 µg/ml). MITRAC15FLAG interacting nascent chains were isolated by FLAG-immunoprecipitation from cell lysates. Eluates were subjected to SDS-PAGE, analyzed by digital autoradiography. Total 2 %, MITRAC15FLAG eluates 100 % (in case of C12ORF62FLAG, 25% of eluate was loaded as a standard). Red asterisks, accumulating nascent chains of ND2. The accumulating nascent chains of ND2 of lane 12 is presented in a magnification. F1-F3 mark nascent chains of COX1 [18].",
    "molecules": "[35S]methionine, pur, puromycin"
  },
  {
    "caption": "HEK293T cells were pulse labelled with [35S]methionine in the presence of puromycin (2 µg/ml). During puromycin treatment, cells were incubated for 5 min and 10 min in medium lacking [35S]methionine. Anti-FLAG immunoisolations were performed as described and eluates analyzed as described in Fig. 4C. Total 1 %, Eluates 100 %. The signal of ND2 was graphed after normalization against the COX2 signal and the signal of mature ND2 in the 5 min chase sample was set as 100 % (error bars indicate SEM from three biological replicates; *p < 0,05; paired t-test). Red asterisks, accumulating nascent chains of ND2.",
    "molecules": "[35S]methionine, puromycin"
  },
  {
    "caption": "Radiolabeling with [35S]methionine, anti-FLAG immunoisolation and sample analyses were performed Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.",
    "molecules": "[35S]methionine"
  },
  {
    "caption": "E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by  PknG or  His-UBA1 at 37°C for 30 min. The reaction products were analyzed by immunoblotting with antibodies against Ub  (top panel), the His-tag on UBA1 (middle panel), and PknG (bottom panel), or by staining with Coomassie Brilliant Blue (bottom). For reducing conditions, samples were treated with 500 mM β-mercaptoethanol  (βME) prior to SDS-PAGE gel analysis.",
    "molecules": "Coomassie Brilliant Blue, β-mercaptoethanol, βME, Ub"
  },
  {
    "caption": "A  MS/MS analysis of UbcH7 in the absence (top) or presence (bottom) of PknG to reveal ubiquitination sites. Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high-confidence peptides possessing  di-Gly modifications. The ubiquitinated residue (Lys82) in the peptide sequence is highlighted in red. The peak heights are the relative abundances of the corresponding fragmentation ions. Matched amino terminus-containing ions (b ions) are indicated in red, and matched carboxyl terminus-containing ions (y ions) are indicated in blue. Only the major peaks that were identified are labeled.",
    "molecules": "di-Gly"
  },
  {
    "caption": "E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.",
    "molecules": "CHX, MG132"
  },
  {
    "caption": "All liposome permeabilization assays  were performed with C-terminally  His-tagged  BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (a-d) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes. (a) Pore formation of 50 nM BakΔTM (red) is activated by 20 nM   cBid   (blue). 20 nM cBid  alone (orange) shows no liposome permeabilization. Addition of 25 nM BclxLΔTM (green) inhibits  pore-formation. (b) Increasing amounts of cBid  are titrated to 50 nM BakΔTM in the absence (blue) and presence (green) of 25 nM BclxLΔTM. (c,d) BakΔTM becomes autoactive at increasing concentrations. The concentrations are color coded as indicated in (d). (c) The averaged kinetics for increasing BakΔTM concentrations. Bar diagrams in (b) and (d) represent data from the kinetic experiments averaged between 9000 -10000 s. The standard deviation was calculated from three measurements. ( ",
    "molecules": "lipid, Ni2+"
  },
  {
    "caption": "All CD measurements were performed with C-terminally  His-tagged BakΔTM and  BclxLΔTM attached to Ni2+-lipid doped MOM-like  liposomes. (e) Far-UV-CD spectra of  BakΔTM +/- liposomes  and the activators  cBid  or Puma-BH3 peptide as indicated.",
    "molecules": "lipid, Ni2+"
  },
  {
    "caption": "All CD  measurements were performed with C-terminally  His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid  doped MOM-like liposomes. (f) CD-detected  thermal melting experiments of  BakΔTM (black) with  liposomes (red) and Puma-BH3 (blue).",
    "molecules": "lipid, Ni2+"
  },
  {
    "caption": "(E) Analysis of different retinoids' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug (Adapalene 1 µM). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against DMSO control: **",
    "molecules": "Adapalene, DMSO, retinoids, tazarotene"
  },
  {
    "caption": "(D) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) simultaneously treated with 10 and 20 µM tazarotene and bexarotene, respectively, up to 21 days. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent treatment times. #### p<0.0001. Difference against 0 days control: * p<0.05, **** p<0.0001.",
    "molecules": "bexarotene, tazarotene"
  },
  {
    "caption": "(E) Analysis and quantification of increased sulfatase activities different to ARSA, namely ARSB, GALNS, and STS in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001, **** p<0.0001.",
    "molecules": "bexarotene, tazarotene"
  },
  {
    "caption": "(F) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations (FGE Gly247Arg, FGE Gly263Val, FGE Ala279Val, FGE Arg349Trp) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ****p<0.0001.",
    "molecules": "bexarotene, tazarotene"
  },
  {
    "caption": "(G) Quantification of ARSA activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 6 independent experiments (biological replicates). Unpaired t-test. **** p<0.0001. (H) Quantification of SGSH activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 3 independent experiments (biological replicates). Unpaired t-test. * p<0.05, *** p<0.001.",
    "molecules": "bexarotene, tazarotene"
  },
  {
    "caption": "(A) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (B) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (C) Quantification of total intensity of LAMP1- green fluorescence. N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), # p<0.05 (MSD DMSO vs MSD treated). RFU: relative fluorescence units. (D) Quantification of LAMP1- green fluorescence vesicle size (µm). N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), ## p<0.01 (MSD DMSO vs MSD treated).",
    "molecules": "bexarotene, DAPI, DMSO, tazarotene"
  },
  {
    "caption": "(A) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RAR receptor antagonist AGN 193109 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 3-11 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01, ### p<0.001. Difference against 0 µM DMSO control (B) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RXR receptor antagonist HX 531 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 4-14 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01. Difference against 0 µM DMSO control: **** p<0.0001.",
    "molecules": "AGN 193109, bexarotene, DMSO, HX 531, tazarotene"
  },
  {
    "caption": "(A) ARSA protein amount quantification after treatment of MSD primary fibroblasts (variant FGE Gly247Arg homozygous) with tazarotene, bexarotene, and tazarotene/bexarotene in combination for 6 days referred to β- actin amounts and normalization of ARSA activity based on ARSA protein amount (specific ARSA activity). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. # p<0.05. Difference against 0/0 µM control: * p<0.05, *** p<0.001.",
    "molecules": "bexarotene, tazarotene"
  },
  {
    "caption": "(C) Quantification of ARSA activities in CRISPR/Cas9 generated ARPE19 SUMF1 -/- cells and appropriate controls (ARPE19 wild type, MSD primary fibroblasts (variant FGE Gly247Arg homozygous)) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM for up to 21 days. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. ## p<0.01. Difference against 0 days control: **** p<0.0001.",
    "molecules": "bexarotene, tazarotene"
  },
  {
    "caption": "(A) Pulse-chase-experiment in HT1080 FGE Ser155Pro cells after pre-treatment with tazarotene/bexarotene and DMSO (control) for 3 days. Upper panel: representative autoradiogram of intracellular (C) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours. Lower panel: quantification of 35S isotope labelled intracellular FGE protein amounts. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ** p<0.01, *** p<0.001.",
    "molecules": "bexarotene, DMSO, 35S, tazarotene"
  },
  {
    "caption": "(A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4h), NeedleTox (4h), Val-boroPro (VbP 22h), ABT737/S63845 (A/S 22h). When indicated, cells were primed with LPS for 2 hours prior to stimulation. When indicated, MCC950 was added to the media 30 minutes prior to the addition of Nigericin. LDH-activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).",
    "molecules": "NeedleTox, ABT737, LPS, MCC950, Nig, Nigericin, S63845, Val-boroPro, VbP"
  },
  {
    "caption": "A) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI-signal. One donor out of two is shown. (B) Representative images were acquired with a 63x objective at the end of the experiment shown in (A) at 16 hours. One donor out of two is shown. Scale bars: 25 µm. ",
    "molecules": "PI"
  },
  {
    "caption": "(C) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.",
    "molecules": "ABT737, S63845, VbP"
  },
  {
    "caption": "(B) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours. * indicates an unspecific band. One representative experiment out of three is shown.",
    "molecules": "Pam3CSK4"
  },
  {
    "caption": "(A) Quantification of EMSAs  (error bars represent the mean ± SD of n=3 biological replicates performed with either  32P-labeled SpoY  or SpoX  (short isoform) with increasing concentrations of the spo0A  target region, respectively. Purified Hfq  was added to facilitate SpoY-spo0A  complex formation. Mutating the respective sRNA seed region (Appendix Figure S4A, SpoY*/SpoX*) abolished the interaction, while introducing compensatory mutations into the spo0A  target region (spo0A*C) slightly rescued the complex formation.",
    "molecules": "32P"
  },
  {
    "caption": "(B) In-line probing of 0.2 pmol of 32P-labeled SpoY  and SpoX  (short isoform) in the absence (lane 4) or presence of increasing concentrations (lane 5-7) of the spo0A  long 5′UTR (starting from pTSS) and first 69 nt of the CDS. RNase T1  and alkali-digested (OH) SpoY  and SpoX  serve as ladders respectively. Secondary structure and predicted seed region are highlighted. A representative image of three independent experiments is shown.",
    "molecules": "32P"
  },
  {
    "caption": "Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids. UBE2D3 is used as a positive control for lysine and cysteine discharge.",
    "molecules": "cysteine, lysine"
  },
  {
    "caption": "Immunoblot from immunoprecipitation (IP) assays to validate the predicted model of UFL1/UFBP1 interaction in cells. UFL1WT-3xFLAG and UFL1L45R-3xFLAG were transiently overexpressed along with UFBP1-SBP in HEK293T-UFL1 KO cells and subjected to separate pulldowns using anti-FLAG antibody or streptavidin. Immunoprecipitated material was run on a 4-12% SDS PAGE gel and analysed by immunoblotting using indicated antibodies.",
    "molecules": "streptavidin"
  },
  {
    "caption": "Single turnover lysine discharge assays to check for activation of UFC1 by UFL1/UFBP1 in the presence and absence of CDK5RAP3. The reaction products were run on a 4-12% SDS PAGE and visualized by Oriole staining.   Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D, n=3 biological replicates,  ",
    "molecules": "Oriole"
  },
  {
    "caption": "C. Dendrite labeling and quantification. Dendritic spines were stained with Dil. Scale bar: 10 μm. Spine density and total spine length are substantially reduced in 3-miR mix treated primary neurons compared to those treated with scramble RNA (n = 49-97 images)",
    "molecules": "Dil"
  },
  {
    "caption": "A Satellite cells were isolated by enzymatic digestion from R3hdml KO (n = 6) and wild type control (n = 6) mice and placed in culture medium. Then, cell proliferation was evaluated by Ki67 staining. Cell nuclei were stained with DAPI. B The ratio of Ki67 positive cells to total cells (DAPI-positive) was evaluated. The number of cells was counted under approximately 60 fields of view for each well. Closed columns, wild type controls; open columns, R3hdml KO mice. * p < 0.05; Student's t-test. ",
    "molecules": "DAPI"
  },
  {
    "caption": "Thirty freshly isolated myofibers from 8-12-week-old R3hdml KO (n = 7) and wild type mice (n = 7) were cultured for 72 h. Then, the isolated myofibers were fixed with 4% PFA followed by immunostaining using anti Pax-7, anti MyoD, and anti Myog antibodies. (C, D) Co-immunostaining parallel cultures for Pax7 and MyoD showed that there were fewer cells with the self-renewing Pax7+/MyoD− phenotype in muscles from R3hdml KO mice compared with control; Pax7+/MyoD− cells (arrowheads), Pax7−/MyoD+ cells (yellow arrows) and Pax7+/MyoD+ cells (white arrows). * p < 0.05; Student's t-test.",
    "molecules": "PFA"
  },
  {
    "caption": "A Seven days after CTX injection, skeletal muscles were dissected, fixed, and embedded in paraffin. Two consecutive sections were utilized, and the first section was subjected to in situ hybridization using an R3hdml sequence-specific RNA probe to detect Rh3dml, while the second section was used for IHC using the anti-MyoD antibody. MyoD protein was visualized with DAB stain (brown). R3hdml expression is highly detected in MyoD-positive cells. Scale bar in low magnification images = 100 µm; in high magnification images = 25µm.",
    "molecules": "CTX, DAB"
  },
  {
    "caption": "B One hundred microliters of 10 µM CTX was intramuscularly injected into the forearm muscle of anesthetized R3hdml KO ([b], n = 10) and wild type control ([a], n = 10) mice. Subsequently, hand grip was evaluated 0, 3, 6, and 14 days after CTX injection. R3hdml genes were overexpressed in the forearm muscle of anesthetized R3hdml KO mice ([c], n = 10) and hand grip was also evaluated 0, 3, 6, and 14 days after CTX injection. Data are expressed as the means ± standard error of the mean (SEM). ** p < 0.01; **** p < 0.0001 vs. wild type control; # p < 0.05; ## p < 0.01 vs. R3hdml KO mice; two-way ANOVA followed by a Bonferroni post hoc comparison of the individual time points.",
    "molecules": "CTX"
  },
  {
    "caption": "C Myofibers were freshly dissected from 8-12-week-old R3hdml KO (n = 4) and control mice (n = 4) 35 days after CTX injection, and the number of satellite cells was evaluated by Pax-7 staining. Arrowheads indicate Pax-7-positive cells. The number of Pax-7-positive cells per myofiber was counted. At least 50 myofibers in each animal were evaluated. Closed columns, wild type controls; open columns, R3hdml KO mice. Data are expressed as the means ± standard error of the mean (SEM). * p < 0.05; Student's t-test.",
    "molecules": "CTX"
  },
  {
    "caption": "B Expression levels of GlcNAc in wild-type (WT) GAS, ΔgacG, H, I, J, K, and L were measured as the sWGA fluorescence intensity (normalized to WT GAS).",
    "molecules": "GlcNAc"
  },
  {
    "caption": "D, E Recruitment of ubiquitin in GAS-infected cells. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (D) Representative confocal images and (E) percentage of cells with ubiquitin-positive GAS. Scale bar, 10 μm.",
    "molecules": "DAPI, ubiquitin"
  },
  {
    "caption": "G, H Localization of ubiquitin in sWGA-treated GAS. HeLa WT were infected with the indicated GAS strains masked with sWGA for 4 h, fixed, and immunostained for ubiquitin (magenta). (G) Representative confocal images and (H) percentages of cell with ubiquitin-positive GAS. Scale bar, 10 μm.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "I, J Recruitment of p62 to gacI mutants. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for p62 (magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (I) Representative confocal images and (J) percentage of cells with p62-positive GAS. Scale bar, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E - J Localization of ubiquitin, LC3 and p62 in FBXO2 KO cells. HeLa WT or FBXO2-KO cells were infected with WT GAS for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta) or LC3 (magenta) or p62 (magenta). Representative confocal images (E, G, and I) and percentages of cells with ubiquitin, LC3, or p62 -positive GAS (F, H, and J).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "M, N GlcNAc recognition of FBXO2 required for GAS xenophagy. Non-transfected HeLa WT cells and HeLa FBXO2-KO cells transfected GFP, GFP-FBXO2, GFP-FBXO2 YW/AA were infected with GAS WT for 4 h, fixed, and immunostained for ubiquitin. Percentages of cells with LC3 (M) or ubiquitin (N) -positive GAS were quantified.",
    "molecules": "GlcNAc, ubiquitin"
  },
  {
    "caption": "A-C Recruitment of LC3 and ubiquitin to intracellular GAS in SKP1/CUL1/ROC1 knockdown cells. HeLa cells transfected with mCherry-LC3 (magenta) and the indicated siRNAs were infected with GAS for 4 h. Cells were immunostained for ubiquitin (FK2); scale bar, 10 μm. Representative confocal images (A) and percentages of cells with ubiquitin (B) and LC3 (C) -positive GAS.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "D, E Neddylated cullin inhibitor: MLN4924-treated HeLa cells were infected with WT GAS, and fixed at 4 h. Cells were immunostained for ubiquitin (FK2: magenta) or LC3 (magenta); scale bar, 10 μm. Representative images and quantification of cells with ubiquitin-positive GAS (D) and LC3-positive GAS (E).",
    "molecules": "MLN4924, ubiquitin"
  },
  {
    "caption": "F Lysates from HeLa cells infected with GAS were immunoprecipitated with an anti-lipoteichoic acid antibody and Protein G-Magnetic Beads. Immunoprecipitates were analyzed by immunoblotting.",
    "molecules": "lipoteichoic acid"
  },
  {
    "caption": "B) Levels of creatine kinase (CK) and creatinine.",
    "molecules": "creatinine"
  },
  {
    "caption": "C) Levels of procalcitonin (PCT) and C-reactive protein (CRP).",
    "molecules": "procalcitonin, PCT"
  },
  {
    "caption": "E) Levels of D-dimer and lactate dehydrogenase (LDH).",
    "molecules": "D-dimer"
  },
  {
    "caption": "Correlation between IL-6 and D-dimer.",
    "molecules": "D-dimer"
  },
  {
    "caption": "Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.",
    "molecules": "oxygen, glucocorticoids"
  },
  {
    "caption": "The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.",
    "molecules": "procalcitonin, PCT"
  },
  {
    "caption": "B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease.",
    "molecules": "PCT, Procalcitonin"
  },
  {
    "caption": "K. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm. L-N. Quantification of area of FITC-dextran-labeled CNV (L), isolectin B4 (IB4 )-stained laser impact area (M) and the ratio of FITC/IB4 per laser-burn (N) relative to LysM-Cre/Nrp1+/+ at D14; n = 23 burns (LysM-Cre/Nrp1+/+), n = 27 burns ( LysM-Cre/Nrp1fl/fl).",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "E. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and isolectin B4 (IB4)-stained laser impact area from Vehicle and NRP1-derived trap treated wildtype mice. Scale bar: 20μm. F-H. Quantification of area of FITC -dextran-labeled CNV (F), IB4-stained laser impact area (G) and the ratio of FITC/IB4 per laser-burn (H) relative to Vehicle at D14; n = 24 burns (Vehicle), n = 26 burns (NRP1-derived trap).",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "J-L. Quantification of area of FITC-dextran-labeled CNV (J), isolectin B4 (IB4)-stained laser impact area (K) and the ratio of FITC/IB4 per laser-burn (L) relative to LysM-Cre/Nrp1+/+ + Vehicle in Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14; n = 16 burns (LysM-Cre/Nrp1+/+ + Vehicle), n = 16 burns (LysM-Cre/Nrp1+/+ + Trap), n = 13 burns (LysM-null/Nrp1fl/fl + Vehicle) , n = 19 burns (LysM-Cre/Nrp1fl/fl + Trap).",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "(D) Representative images of CTX signal in WT or  SOD1G93A primary  MNs before and 2 days after Sema3A  application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A or untreated. Green: denotes  CTX-positive cells. Yellow circles are numbered CTX  positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 30 μm. (E) Quantification of CTX signal in a SOD1G93A explant before and 2 days after  Sema3A  application to the distal compartment in the presence of either Dynein  inhibitor+Sema3A, Dynasore+Sema3A. 3 independent chambers in each condition were analyzed. ~200 neurons were monitored per each condition. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p<0.05, **p<0.01.  Dynein inhibitor and Dynasore treatments were used as a negative control. ",
    "molecules": "Dynasore"
  },
  {
    "caption": "(A) Representative images and insets of P90 WT and SOD1G93A SC cross sections at P90. Blue: denotes  DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Representative images of P90 SOD1G93A mice SC  cross sections that were injected with AAV9-GFP/AAV9-50aa-GFP. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes  activated caspase 3. Scale bar: 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Distribution of the fold-change (FC) in degradation rate of mRNAs (n=13296, red), micropeptide encoding transcripts (n=43, pink), bona fide cytosolic (n=759, blue) and nuclear (n=4299, grey) lncRNAs in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days treatment, horizontal dashed line represents a KO/WT FC in degradation rate of 1. Statistics: NS-p>0.05, *-p-value<0.05. Two-tailed Mann-Whitney U test: p-value=0.044. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.",
    "molecules": "4-OHT, ethanol"
  },
  {
    "caption": "(F) Distribution of the fold-change (FC) in degradation rates of mRNAs (n=29900, red), cytosolic (n=474, blue) and nuclear (n=2348, grey) lncRNAs, in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days of treatment (estimated based on the 10 minutes 4sU pulse), horizontal dashed line represents a KO/WT FC in degradation rate of 1 Statistics: NS-p-value>0.05 anth and 95th quantiles of the distribution. (two-tailed Mann-Whitney U test).",
    "molecules": "4sU, 4-OHT, ethanol"
  },
  {
    "caption": "(D) Distribution of the fold-change (FC) in degradation rate of mRNAs (n=13296, red), micropeptide encoding transcripts (n=43, pink), bona fide cytosolic (n=759, blue) and nuclear (n=4299, grey) lncRNAs in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days treatment, horizontal dashed line represents a KO/WT FC in degradation rate of 1. Statistics: NS-p>0.05, *-p-value<0.05. Two-tailed Mann-Whitney U test: p-value=0.044. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.",
    "molecules": "4-OHT, ethanol"
  },
  {
    "caption": "(B) Expression of GFP, lncRNA-c1, GFP-lncRNA-c1, lncRNA-c2 and GFP-lncRNA-c2 (x-axis) in 8 day 4-OHT treated, miRNA depleted cells (KO) relative to ethanol treated (WT) mESC (y-axis) 24h hours post-transfection. Four independent biological replicates were treated, transfected and analyzed by RT-qPCR. Statistical significance represented on the figure based on comparison of KO/WT fold change in expression of GFP, with GFP-lncRNA-c1 and with GFP-lncRNA-c2, (paired two-tailed t-test p-value= 0.034 and 0.032 respectively) and based on comparison of KO/WT fold change in expression of GFP-lncRNAc1 with lncRNA-c1 and GFP-lncRNA-c2 with lncRNA-c2 (paired two-tailed t-test p-value= 0.012 and 0.018 respectively). Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.",
    "molecules": "4-OHT, ethanol, Neomycin, miRNA"
  },
  {
    "caption": "(C) Expression of GFP-lncRNA-c1 ΔMRE relative to GFP-lncRNA-c1 (y-axis) in ethanol treated (WT, circles) or 4-OHT treated, miRNA-depleted cells (KO, triangles, x-axis). Four independent biological replicates were treated, transfected and analyzed by RTqPCR. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0071 Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.",
    "molecules": "4-OHT, ethanol, Neomycin, miRNA"
  },
  {
    "caption": "(F) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(-30)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted (G) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(-30)-GFP-lncRNA-c1, BoxB (-30)-GFP-lncRNA-c2 (BoxB(-30), triangles) (x-axis) 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates were analyzed. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0494 for GFP-lncRNA-c1 and p-value=0.0355 for GPF-lncRNA-c2Data information: Uncropped blots used for assembly of panels F are provided in Figure 4 Source Data.",
    "molecules": "4-OHT, ethanol, miRNA"
  },
  {
    "caption": "(H) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(+339)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted. (I) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(+339)-GFP-lncRNA-c1, BoxB (+339)-GFP-lncRNA-c2 (BoxB(+339), triangles) (x-axis) in 8 day 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates analyzed.Data information: Uncropped blots used for assembly of panels H are provided in Figure 4 Source Data.",
    "molecules": "4-OHT, ethanol, miRNA"
  },
  {
    "caption": "E-F Schematic diagram of the cell survival experimental design using thymidine analogs (F) and quantification of the percentage of surviving cells in Tau-/- mice as compared to WT mice at each cell age (1-, 2-, 4-, 6- and 8-week-old cells) (F) (mean ± SEM (normalized data); Student´s t-test).",
    "molecules": "thymidine"
  },
  {
    "caption": "C Representative images of 1-week-old newborn granule neurons labeled with CldU (green) belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice. Cell nuclei were labeled with DAPI (blue). Scale bar 50 µm.D-E Quantification of the number of 1-week-old CldU+ cells (D) and apoptotic fractin+ cells (E) in each experimental group.",
    "molecules": "CldU"
  },
  {
    "caption": "E Representative images of 8-week-old newborn granule neurons labeled with IdU (red) belonging to WT CH, WT EE, Tau-/- CH and Tau-/- EE mice.F-G Quantification of the number of 8-week-old IdU+ cells (F) and apoptotic fractin+ cells (G).",
    "molecules": "IdU"
  },
  {
    "caption": "GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (A) Representative maximum intensity projection images of 10 Z-stacks per image. Scale bar: 10 µm.",
    "molecules": "ABA, ACC, NAA, ATP, BL, 6-BA"
  },
  {
    "caption": "GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (B) Quantification of GFP foci per 0.0025 mm2. Values are presented as mean ± standard deviation of the mean and were calculated from at least three independent experiments with 3 individuals per replicate. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.",
    "molecules": "ABA, ACC, NAA, ATP, BL, 6-BA"
  },
  {
    "caption": "GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (C) GFP-ATG8a cleavage immunoblot for plants exposed to the same treatments as in (A). Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.",
    "molecules": "ABA, ACC, NAA, ATP, BL, 6-BA"
  },
  {
    "caption": "GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (D) NBR1 immunoblot for atg2-2 samples for given treatments. Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.",
    "molecules": "ABA, ACC, NAA, ATP, BL, 6-BA"
  },
  {
    "caption": "(A) Seedlings were acclimated for 16 h in MS containing ABA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing flg22. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (B) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in A. Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. ",
    "molecules": "ABA, NAA"
  },
  {
    "caption": "(D) Seedlings were acclimated for 16 h in MS containing NAA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing 6-BA. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (E) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in (D). Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. ",
    "molecules": "ABA, NAA, 6-BA"
  },
  {
    "caption": "(B) Pattern correlation used to find proteins that accumulate upon ABA/NAA treatment and are removed in WT but not in atg2 after swapping to flg22/6-BA.",
    "molecules": "ABA, NAA, 6-BA"
  },
  {
    "caption": "Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described (E) Protein cluster of proteins which accumulate upon NAA treatment and are removed in WT but not in atg2 after swapping to 6-BA (F) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with 6-BA.",
    "molecules": "NAA, 6-BA"
  },
  {
    "caption": "(D-E) Catalytically active sACE22.v2.4-IgG1 and catalytically dead sACE22.v2.4(NN)-IgG1 were aerosolized (7.5 ml protein at 8.3 mg/ml in 25 minutes) and delivered by inhalation to K18-hACE2 transgenic mice at 12 h, 48 h, and 84 h post-inoculation with SARS-CoV-2 gamma variant. 10 mice in each group were observed for survival (D) and weight loss (E). The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1.",
    "molecules": "PBS"
  },
  {
    "caption": "(D-E) Authentic BA.1 omicron virus (isolate USA/MD-HP20874/2021) was incubated with sACE22-IgG1 (D) or sACE22.v2.4-IgG1 (E) for 1 h and added to Calu-3 cells. Infection 48 h later was measured by RT-qPCR for the viral N gene. 3 µM remdesivir (black columns) is a positive neutralization control.",
    "molecules": "remdesivir"
  },
  {
    "caption": "Ca2+‑current-voltage relationship of control CD1B6F1 wild‑type (n=6 IHCs), dual-AAV-TS transduced (n=8 IHCs), and non‑transduced CD1B6F1 Otof-/- (n=10 IHCs) IHCs (P14‑18)",
    "molecules": "Ca2+"
  },
  {
    "caption": "Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV)",
    "molecules": "Ca2+"
  },
  {
    "caption": "Average exocytosis level measured as ΔCm (G) and corresponding Ca2+‑current integrals (QCa2+) (H) in wild‑type (CD1B6F1: n=6 IHCs; B6: n=11 IHCs (B6 data replotted from Strenzke et al, 2016)), dual-AAV-TS transduced Otof-/- (n=8 IHCs), and non‑transduced (n=11 IHCs) Otof-/- IHCs. Transduced IHCs that showed eGFP expression, but had almost no exocytosis are depicted as dashed lines (not included into the average)",
    "molecules": "Ca2+"
  },
  {
    "caption": "C. Heat-annealed ODN/RNA 1-294 complex was incubated with HIV-1 RT and increasing concentrations of recombinant DDX42. Reverse transcription was initiated by addition of the four dNTPs. Extension was for 1, 5, 20 or 60 min and samples were analysed by PAGE 8% (P/T: primer/template; SSDNA: Strong-Stop DNA). A representative autoradiograph is shown.",
    "molecules": "dNTPs, ODN, RNA"
  },
  {
    "caption": "E. PLAs were performed in MDMs infected with HIV-1 or not (N.I. CTRL), using anti-Capsid and anti-DDX42 antibodies (nuclei stained with Hoechst). Images were acquired using a LSM880 Airyscan microscope. Left: representative images, scale-bar: 10 μm. Right: Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD (n>65 cells per condition).",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA using mouse anti-dsRNA (J2) and rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition).",
    "molecules": "Alexa Fluor 546, dsRNA"
  },
  {
    "caption": "Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition). using an anti-N antibody",
    "molecules": "Alexa Fluor 546"
  },
  {
    "caption": "F. The following biotinylated RNAs were used to pull-down recombinant DDX42 poly(I:C), CHIKV G4, TRF2 G4 and a mutated TRF2 G4 sequence (Mut. TRF2 G4), and DDX42 was revealed by an immunoblot.",
    "molecules": "biotinylated, poly(I:C)"
  },
  {
    "caption": " A, B. Coronal CT image of the chest at baseline with pleural fluid and intralobar septal thickening (arrow) (A), and 3 months after starting trametinib (B). ",
    "molecules": "trametinib"
  },
  {
    "caption": " C, D. MR T2 weighted coronal slice of the chest at baseline demonstrating bilateral pleural effusions (arrow heads) and pulmonary interstitial edema (arrows) (C), and 3 months on trametinib therapy (D). ",
    "molecules": "trametinib"
  },
  {
    "caption": " IN-DCMRL coronal MIP of the chest at baseline demonstrating dilated and tortuous TD (arrow) with dilated central lymphatic networks and extensive bilateral pulmonary perfusion (arrowheads) (E), and 6 months after trametinib therapy demonstrating dilated and tortuous TD (arrow) with reduction in the extent of the dilated central lymphatic networks and resolution of bilateral pulmonary perfusion ",
    "molecules": "trametinib"
  },
  {
    "caption": " IN-DCMRL coronal slice of the chest at the level of the carina demonstrating extensive mediastinal and pulmonary interstitial perfusion (arrows) at baseline (G), and after 6 months of treatment with trametinib (H). ",
    "molecules": "trametinib"
  },
  {
    "caption": "(A-C) AFM and TEM analysis of unloaded (u-NPs) and cholesterol loaded NPs (NPs-Chol). AFM \"height\" images (left column), 3D reconstruction (middle column) and TEM micrograph (right column) of u-NPs (A), NPs-Chol1 (B) and NPs-Chol3 (C).",
    "molecules": "Chol, cholesterol, NPs"
  },
  {
    "caption": "(D) Release profile in water of cholesterol (continuous line, -) and NBD-Chol (dotted line, - - -) from NPs-Chol1 and NPs-NBD-Chol1, respectively.",
    "molecules": "Chol, cholesterol, NPs, water"
  },
  {
    "caption": "(E) In vitro release of NBD-Chol from NPs at different time-intervals in NS cells. Data in the graph represent mean (µg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1",
    "molecules": "Chol, NPs"
  },
  {
    "caption": "(A-B) Representative confocal images of liver (A) and brain (B) slices from R6/2 mice ip injected with C-NPs (left) or with g7-NPs (right) and sacrificed after 4 hrs. (C) Quantification of g7-NPs localized in the liver, striatum, and cortex of WT (n=3) and R6/2 mice (n=3). Data represent mean (µg) ± SEM.",
    "molecules": "g7, NPs"
  },
  {
    "caption": "(D-E) g7-NPs in brain slices from R6/2 mice administered with a single ip injection and sacrificed after 24 hrs (D, left) or 2 weeks (D, right) and after multiple ip injections within 1 week (E).",
    "molecules": "g7, NPs"
  },
  {
    "caption": "(F-I) Representative confocal images of immunostaining against IBA1 (F), GFAP (G), calbindin (H), and DARPP-32 (I) on coronal sections of brains isolated from R6/2 mice ip injected with g7-NPs and sacrificed at the indicated time points. White arrowheads indicate intracellular g7-NPs.",
    "molecules": "g7, NPs"
  },
  {
    "caption": "(A) Representative confocal image (crop) of brain slices from R6/2 mice ip injected with rhodamine labeled g7-NPs-NBD-Chol and sacrificed after 12 hrs, showing co-localization of NBD-Chol (green) and Rhodamine (NPs, red). Scale bar: 5m.",
    "molecules": "g7, rhodamine, Rhodamine, Chol, NPs"
  },
  {
    "caption": "(B-C) Representative confocal image (low magnification) of brain slices from R6/2 mice ip injected with g7-NPs-NBD-Chol and sacrificed after 24 hrs (B) or 2 weeks (C) and relative co-localization of NBD-Chol and g7-NPs. Scale bar: 10 m (D) g7-NPs quantification in brain slices at the same time points in (B-C). DAPI was used to counterstain nuclei. Data in (D) are expressed as number of g7-NPs (evaluated based their size) for 100 m2 sem. Statistics: **P<0,01 (48 hrs vs 7 days; 7 days vs 14 days), ***P<0,001 (24 hrs vs 7 days; 7 days vs 14 days) determined by one-way ANOVA followed by Newmann-Keuls multiple comparison test.",
    "molecules": "g7, Chol, NPs"
  },
  {
    "caption": "(A,D) Spontaneous IPSCs (A) and spontaneous EPSCs (D) were recorded from striatal MSNs (WTs=52; R6/2-untreated=27; R6/2-Chol=29) at a holding potential of +10 mV and -70mV respectively. As no differences were found between R6/2 mice treated with saline (R6/2) or with empty g7-NPs (R6/2-emp), data were pooled. (B, E) Amplitude-frequency histogram and average frequency (inset) of IPSCs (B) and EPSCs (E) from R6/2-Chol, R6/2-untreated and WT MSNs. (C,F) Cumulative inter-event histogram showing the release probability of IPSCs (C) and EPSCs (F) in all groups.",
    "molecules": "g7, Chol, NPs"
  },
  {
    "caption": "(A,D) Spontaneous IPSCs (A) and spontaneous EPSCs (D) were recorded from striatal MSNs (WTs=52; R6/2-untreated=27; R6/2-Chol=29) at a holding potential of +10 mV and -70mV respectively. As no differences were found between R6/2 mice treated with saline (R6/2) or with empty g7-NPs (R6/2-emp), data were pooled. (B, E) Amplitude-frequency histogram and average frequency (inset) of IPSCs (B) and EPSCs (E) from R6/2-Chol, R6/2-untreated and WT MSNs. (C,F) Cumulative inter-event histogram showing the release probability of IPSCs (C) and EPSCs (F) in all groups.",
    "molecules": "g7, Chol, NPs"
  },
  {
    "caption": "(B) Fall latency from an accelerating rotarod for 5-11-week-old WT and R6/2 mice during cholesterol supplementation (WT=17; R6/2-untreated=21; R6/2-Chol=13).",
    "molecules": "Chol, cholesterol"
  },
  {
    "caption": "(C) Rearing activity in open field at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).",
    "molecules": "Chol"
  },
  {
    "caption": "(D) global activity in the open field test at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).",
    "molecules": "Chol"
  },
  {
    "caption": "(E) Index of discrimination (%) in WT, R6/2-untreated and R6/2-Chol mice during disease progression, at 8 weeks of age (WT=24; R6/2-untreated=36; R6/2-Chol=21), at 10 weeks of age (WT=25; R6/2-untreated=35; R6/2-Chol=20) and at 12 weeks of age (WT=24; R6/2-untreated=30; R6/2-Chol=19); the index above zero indicates a preference for the novel object; the index below zero indicates a preference for the familiar object. As no differences were found between R6/2 mice treated with saline (R6/2) or treated with empty g7-NPs (R6/2-emp), data were pooled.",
    "molecules": "g7, Chol, NPs"
  },
  {
    "caption": "(A-B) protein levels (A) and relative densitometry quantification (B) of several synaptic proteins in triton-insoluble (synaptic enriched) fractions purified from total brains. Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6/2 mice by cholesterol supplementation.",
    "molecules": "cholesterol"
  },
  {
    "caption": "(C-D-E) mRNA levels for Bdnf (C), Snap25 (D) in cortex and hippocampus; Complexin II (E) in cortex, hippocampus and striatum from a subset of WT (n=4), R6/2-untreated (n=7) and R6/2-chol animals (n=3). As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.",
    "molecules": "g7, chol, NPs"
  },
  {
    "caption": "(F-G) Representative images of Nissl Staining (F) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT (n= 7), R6/2 (n=7), R6/2-emp (n=6), R6/2-Chol (n=8)",
    "molecules": "Chol"
  },
  {
    "caption": "(A-B) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT (n=6), R6/2-untreated (n=7) and R6/2-Chol (n=5) mice. Data represent mean ± SEM.",
    "molecules": "Chol, Lathosterol, 24OHC"
  },
  {
    "caption": "(C-D) mRNA levels of hmgcr and fdft1 in liver and lung of WT (n=7), R6/2-untreated (n=8) and R6/2-Chol (n=4) mice. As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.",
    "molecules": "g7, Chol, NPs"
  },
  {
    "caption": "(E-F) mRNA levels of inflammatory genes in liver and lung of the of WT (n=7), R6/2 (n=4), R6/2-emp (n=4) and R6/2-Chol (n=5) mice.",
    "molecules": "Chol"
  },
  {
    "caption": "E) Larval brains at 0 h ALH from Jupiter-GFP (G147) were labeled with GFP, Msps, and Dpn. F) Larval brains at 24 h ALH from Jupiter-GFP were raised on normal food (fed) and food depleted of amino acids (nutritional restriction/NR) and labelled with GFP and Dpn. ",
    "molecules": "amino acids"
  },
  {
    "caption": " A) Larval brains at 24 h ALH from control (insc-Gal4; UAS-dicer2/UAS-β-Gal RNAi) and msps RNAi (VDRC#21982) controlled under insc-Gal4 were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for both control and msps RNAi. ****p< 0.0001 ",
    "molecules": "EdU"
  },
  {
    "caption": " C) Larval brains at 24 h ALH from wild-type and various msps loss-of-function alleles and msps810 with a genomic rescue construct (g-msps) were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. D) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (C). n=13 BL for control; n=13 BL for msps810; n=17 BL for msps924; n=14 BL for mspsP18; n=6 BL for mspsP; n=12 BL for g-msps; msps924. ****p< 0.0001; p=0.7502 (ns). E) Quantification graph of the diameter of the cell body in NSCs at 24 h ALH from various genotypes. n=238 NSCs for control; n=219 NSC for msps924; n=170 NSCs for mspsP. ****p< 0.0001. F) Quantification graph of the percentage of qNSCs with a primary protrusion in wild-type, msps924, and mspsP18 larval brains. The protrusion was labelled by grh>CD8-GFP. n=5 BL for control; n=6 BL for msps924; n=4 BL for mspsP. ****p< 0.0001 ",
    "molecules": "EdU"
  },
  {
    "caption": " G) Larval brains at 24 h ALH from wild-type and tacc loss-of-function tacc74 were examined for EdU incorporation. NSCs were marked by Dpn and Mira. H) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (G). n=17 BL for control; n=15 BL for tacc74. ****p< 0.0001. I) Quantification graph of qNSCs retaining primary protrusion per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001. J) Quantification graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001 ",
    "molecules": "EdU, PH3"
  },
  {
    "caption": " A) Larval brains at 16 h ALH from wild-type (yw) and E-cadR69 were analyzed for EdU incorporation, and larval brains were labeled with EdU, Dpn and Mira ",
    "molecules": "EdU"
  },
  {
    "caption": " E) Larval brains at 24 h ALH from the wild-type control (yw) and klp64Dk5h/Df, kap3V6 and kap3 V6; g-kap3#11 were stained with EdU, Dpn, and Mira",
    "molecules": "EdU"
  },
  {
    "caption": "A) Larval brains at 24 h ALH from wild-type control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), msps RNAi control (UAS-GFP + UAS-msps RNAi (21982)), and rescued animals (UAS-klp64D+ msps RNAi (21982)) under the control of insc-Gal4 were analyzed for EdU incorporation, and larval brains were labeled with EdU and Dpn. B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for contorl; n=13 BL for msps RNAi; n=20 BL for UAS-klp64D + msps RNAi. ****p<0.0001.",
    "molecules": "EdU"
  },
  {
    "caption": "D) Larval brains at 24 h ALH from wild-type control (UAS-β-Gal RNAi), RNAi control (UAS-msps RNAi (21982)), and rescued animals (g-kap3 + msps RNAi (21982)) driven by insc-Gal4 were examined for EdU incorporation, and larval brains were stained with EdU and Dpn. E) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (D). n=13 BL for control; n=15 BL for msps RNAi; n=19 BL for g-kap3 + msps RNAi. ****p<0.0001. ",
    "molecules": "EdU"
  },
  {
    "caption": "G) 24 h ALH Larval brains from wild-type control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), RNAi control (UAS-GFP + UAS-msps RNAi (21982)), and rescued animals (UAS-E-cad7 + msps RNAi (21982)) were assessed for EdU incorporation, and larval brains were stained with EdU and Dpn. H) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (G). n=21 BL for control; n=21 BL for msps RNAi; n=22 BL for UAS-E-cad7 + msps RNAi. ****p<0.0001. ",
    "molecules": "EdU"
  },
  {
    "caption": "J) 24 h ALH Larval brains from wild-type control (UAS-β-Gal RNAi), mutant control (UAS-β-Gal RNAi + klp64Dk5h), and rescued animals (UAS-E-cad7 + klp64Dk5h) were processed for EdU incorporation, and larval brains were probed by with EdU, Dpn, and Mira. K) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (J). n=10 BL for control; n=12 BL for klp64Dk5h; n=10 BL for UAS-E-cad7 + klp64Dk5h. ****p<0.0001; p=0.3006 (ns). ",
    "molecules": "EdU"
  },
  {
    "caption": "A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured.",
    "molecules": "colchicine"
  },
  {
    "caption": "G-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas.",
    "molecules": "colchicine, DMSO"
  },
  {
    "caption": "-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. (B) RNAi knockdown of klp10A in msps810/mspsP18 mutants restored the retrograde movement pattern of EB1 comets. MT depolymerization or severing induced by Colchicine treatment (D) or overexpression of Kat-60 (F) led to mixed MT orientation in ddaC neurons.",
    "molecules": "Colchicine"
  },
  {
    "caption": "Yeast three-hybrid assays showing that CBF1 represses the interaction of the Pfr-phyB with PIF4 in yeast cells. AD-PIF4, phyB-BD and CBF1 were expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent SD of three independent yeast clones.",
    "molecules": "ONPG, Pfr"
  },
  {
    "caption": "Yeast three-hybrid assays showing that CBF1 represses the interaction of the Pfr-phyB with PIF5 (B) in yeast cells. AD-PIF4, phyB-BD and CBF1 were expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent SD of three independent yeast clones.",
    "molecules": "ONPG, Pfr"
  },
  {
    "caption": "(A) Diauxic growth of WT E. coli (NCM3722) in minimal media containing glucose and acetate, bacterial density measured as optical density (OD600). Growth on glucose is captured by the exponential fit (blue dashed line), and proceeds until glucose runs out (black dashed line) at time = 0 h. This is followed by a period of growth-lag lasting ~3.5h before exponential growth resumes on acetate (red dashed line).",
    "molecules": "acetate, glucose"
  },
  {
    "caption": "(D) Lag-times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 (inset). Addition of chlorotetracycline (cTc) removes the tetR repression and induces aceBA expression. As the expression of aceB/aceA during the response (inducer added at time = 0h) is increased, lag times decrease. Bar plot shows mean lag-times (N=3 biological repeats) for different inducer concentration with error bars denoting the Standard Deviations (SD).",
    "molecules": "chlorotetracycline, cTc"
  },
  {
    "caption": "(A) A plasmid system (inset) is used to control the expression of the non-required gene lacZ using the strain NQ1389. lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion (time = 0 h) using the indicated range of cTc concentrations. Diauxic growth conditions with glucose and acetate, same as in Fig. 1.",
    "molecules": "acetate, cTc, glucose"
  },
  {
    "caption": "(B) For the various gene categories in panel A, the temporal kinetics of expression during the diauxic shift is plotted. Time 0 indicates time when glucose runs out. The series of RNA-seq through the growth transition was performed once.",
    "molecules": "glucose"
  },
  {
    "caption": "(C) Steady-state growth in glucose and acetate for the WT and the motility deletion strains ΔfliC and Δflh. Growth rate differences between WT and the mutants are not significant in glucose (p-value >0.05), but are significant for growth in acetate (p-values<0.02). Additional growth conditions are shown in Fig. EV6.",
    "molecules": "acetate, glucose"
  },
  {
    "caption": "Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT (black) and the motility deletion strains ΔfliC and ΔflhD strain (magenta and green).",
    "molecules": "glucose"
  },
  {
    "caption": "Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT (black) and the motility deletion strains ΔfliC and ΔflhD strain (magenta and green).",
    "molecules": "glucose"
  },
  {
    "caption": "(C, D) Primary cultured neurons were transduced by Semliki Forest virus (SFV) expressing wild type human APP695 and treated with 3 µM of the indicated VHHs. PBS and anti-GFP VHH were used as controls. (C) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (D) ELISA measurements of Aβ1-40 and Aβ1-42 in conditioned media. Values are mean ± S.E.M., n=3 cultures for each analysis. One-way ANOVA, *p<0.5, **p<0.01, *** p<0.0001.",
    "molecules": "CTF⍺, CTFβ, Aβ1-40, Aβ1-42, PBS"
  },
  {
    "caption": "(E, F) Dose-dependent inhibition of BACE1 in primary cultured neurons by VHH-B9. Cultured neurons were transduced by SFV expressing wild type human APP695 and treated with PBS (control: CT) or decreasing concentrations of VHH-B9, ranging from 10 µM to 0.7 nM. (E) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (F) Conditioned media was analyzed by ELISA to assess levels of Aβ1-40. Values are mean ± S.D., n=3 cultures for each analysis. The EC50 value (95 % confidence interval) was estimated as 85.4 nM (52.5-125.6 nM).",
    "molecules": "CTF⍺, CTFβ, Aβ1-40, PBS"
  },
  {
    "caption": "(A) Co-staining with anti-N-Terminal Aβ 82E1 (green) and Thioflavin-S (magenta) reveals Aβ plaques (arrow heads) in regions of interest. Representative images are shown. Scale bar: 50 μm. (B, C) The number of Aβ plaques in regions of interest was significantly reduced by treatment AAV-VHH-B9 (B), as was total plaque area (C). (",
    "molecules": "Aβ, Thioflavin-S"
  },
  {
    "caption": "(A-C) MITOL-HA formed small dot-like structures following CCCP treatment. HeLa cells transiently expressing Flag-Parkin and MITOL-HA were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry. MITOL and Tom20 (mitochondrial marker; A) signals co-localized well without CCCP treatment, whereas the MITOL-positive small dots were not coincident with Tom20, Sec61β (ER marker; B), or LAMP1 (lysosomal marker; C) after CCCP treatment. Scale bars, 10 µm. (D) MITOL-HA co-localized with catalase (peroxisome marker) following CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-HA co-localization with catalase. (E) Correlation statistics for the localization of MITOL-HA and Tom20, Sec61β, LAMP1, or catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. (F) The line-graph shows a line scan of fluorescence through three MITOL-positive peroxisomes (red bar in Fig 1D) that clearly indicates co-localization of MITOL (green line) and catalase (magenta line). ",
    "molecules": "CCCP"
  },
  {
    "caption": "(G) Peroxisomal membrane protein (PMP) 34-FusionRed also co-localized with MITOL-GFP in Parkin-expressing HeLa cells after 3 hours of CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. (H) Correlation statistics for the localization of MITOL-GFP and PMP34-FusionRed. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. ",
    "molecules": "CCCP"
  },
  {
    "caption": "(A) MITOL-HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild-type HeLa cells or HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag-MITOL were transfected with a GFP-Parkin plasmid or the pEGFP-C1 vector, treated with 15 µM CCCP +/- 10 µM MG132 for 3 hours, and then immunoblotted with anti-Flag, anti-ubiquitin, and anti-tubulin antibodies. Black arrowheads indicate 3Flag-MITOL in the upper panel and mono-ubiquitin in the middle panel, respectively.",
    "molecules": "mono-ubiquitin, CCCP, MG132, ubiquitin"
  },
  {
    "caption": "(D) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that Mitofusin2 (MFN2) underwent rapid degradation within 3 hours of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria-rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 hours 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor ZVAD-FMK (10 μM) was added to cells along with valinomycin to prevent cell death.",
    "molecules": "valinomycin, ZVAD-FMK"
  },
  {
    "caption": "(E) Quantification of 3Flag-MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 µM valinomycin + ZVAD-FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m relative to untreated samples in three biological replicates.",
    "molecules": "valinomycin, ZVAD-FMK"
  },
  {
    "caption": "(F) Pre-existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 hours to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 hours, cells were immunostained using anti-Flag, anti-Pex14 (peroxisomal membrane protein), and anti-Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.",
    "molecules": "CCCP, doxycycline"
  },
  {
    "caption": "(A) Wild-type or PINK1 knock out (KO) HeLa cells were transfected with GFP-Parkin wild-type or the C431S mutant, treated with 15 µM CCCP, and then immuno-stained with anti-Flag and anti-catalase antibodies. Wild-type Parkin, but not the catalytically inactive Parkin (C431S) mutant, mediated 3Flag-MITOL translocation to peroxisomes following mitophagy stimulation. In PINK1 KO HeLa cells, the redistribution of MITOL to peroxisomes was not observed even in the presence of wild-type Parkin when mitochondria were damaged. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization observed only in the presence of wild-type Parkin.",
    "molecules": "CCCP"
  },
  {
    "caption": "(B) The redistribution of MITOL from mitochondria to peroxisomes does not require its own E3 activity. The E3-inactive MITOL Cys65Ser/Cys68Ser (CS) and H43W mutants were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 µM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-catalase antibodies. After 3 hours of CCCP treatment, both the CS and H43W mutants co-localized with catalase. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase.",
    "molecules": "CCCP, Cys, Ser"
  },
  {
    "caption": "(B) Peroxisomal translocation of other mitochondrial proteins such as MitoNEET/CISD1, Fis1, Miro1/2, and Tom70 (mitochondrial outer membrane proteins), and Hsp60 (mitochondrial matrix protein) was not observed in CCCP-treated cells. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 hours, and then immuno-stained with anti-MitoNEET/CISD1, anti-Fis1, anti-Miro1/2, anti-Tom70, anti-Hsp60, and anti-catalase antibodies. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) A multi-spanning outer membrane protein PBR (Peripheral Benzodiazepine Receptor) did not co-localize with catalase, but did with Tom20 during mitophagy. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 hours, and then immuno-stained with anti-Flag, anti-Tom20, and anti-catalase antibodies. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) Statistical analysis of the MITOL subcellular localization following 15 µM CCCP treatment for 3 hours in cells treated with control, Tom70, Tom20, Tom40, or Sam50 siRNAs. Su9-GFP was not imported into the mitochondria, but rather localized to the cytosol and nucleus following Tom20 and Tom40 knockdown. The number of HeLa cells with cytosol-localized Su9-GFP or 3Flag-MITOL in each siRNA experiment were determined. Data represent the mean ± s.e.m from > 100 cells in three biological replicates. (In case of Tom40, and Sam50 siRNAs, data represent the mean ± s.e.m from > 60 cells in three biological replicates.) Statistical significance was calculated using a one-tailed Welch's t-test.",
    "molecules": "CCCP"
  },
  {
    "caption": "Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 (B) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase rather than Hsp60 in wild-type HCT116 cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.",
    "molecules": "valinomycin"
  },
  {
    "caption": "Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag anti-Tom20 (C) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. ",
    "molecules": "valinomycin"
  },
  {
    "caption": "(C) MITOL translocation from the mitochondria to peroxisomes is highly dependent on Pex3. HeLa cells stably expressing 3Flag-MITOL and HA-Parkin were transfected with control, PEX3, PEX16, or PEX19 siRNA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A) MITOL did not merge with Hsp60 in control siRNA-treated cells, whereas most MITOL co-localized with Hsp60 in p97/VCP knockdown cells in response to mitophagy stimuli. HeLa cells stably expressing 3Flag-MITOL and HA-Parkin were transfected with control or p97/VCP siRNA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) Overexpression of an p97/VCP ATP hydrolysis-defective mutant, E305Q/E578Q (p97QQ), blocked MITOL redistribution from mitochondria to peroxisomes, while overexpression of wild-type p97/VCP had no effect on MITOL redistribution. HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA and Flag-p97/VCP wild-type or p97QQ, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-catalase antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization that was only observed in the presence of a functional VCP.",
    "molecules": "ATP, CCCP"
  },
  {
    "caption": "(D) NMS-873, a specific inhibitor of p97/VCP, prevented MITOL translocation following CCCP-treatment. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL, treated with 15 µM CCCP in the presence or absence of 10 µM NMS-873 for 3 hours, and then subjected to immuno-staining with anti-Flag, anti-catalase, and anti-HA antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization that was only observed in the presence of a functional VCP.",
    "molecules": "CCCP, NMS-873"
  },
  {
    "caption": "(E) Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.",
    "molecules": "NMS-873"
  },
  {
    "caption": "(A) MITOL was ubiquitylated only in the presence of Parkin when the mitochondrial membrane was decreased. After 15 µM CCCP treatment for 3 hours, HeLa cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then immunoblotted with the indicated antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates 3Flag-MITOL.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) The MITOL K268A and K268R mutants were targeted to mitochondria under steady-state conditions, whereas peroxisomal localization following CCCP treatment was considerably disrupted. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL wild-type, K268A, or K268R mutants, treated with 15 µM CCCP for 3 hours, and then subjected to immuno-staining with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(F) The fold change in ubiquitylation of MITOL K268, K40, and K54 in valinomycin-treated samples versus untreated samples. After 3 hours of valinomycin treatment, PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then subjected to LC-MS/MS analysis for label-free quantification of ubiquitylated peptides. Error bars represent the mean ± s.e.m in three biological replicates.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(B) The MITOL subcellular localization was observed in MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Endogenous MITOL (detectable with an anti-Flag antibody) overlapped with Hsp60 under steady-state conditions, whereas 3 hours of valinomycin (10 µM) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(D) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97/VCP inhibitor NMS-873. Higher magnification images of the boxed regions are shown in the lower panel. Scale bars, 10 µm.",
    "molecules": "NMS-873"
  },
  {
    "caption": "(E) Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.",
    "molecules": "NMS-873"
  },
  {
    "caption": "(F) Endogenous MITOL is ubiquitylated following valinomycin treatment. After MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin were treated with valinomycin for 3 hours, the collected cell lysates were immunoprecipitated with anti-Flag magnetic beads. The immunoprecipitates were blotted using anti-Flag and anti-ubiquitin antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates MITOL-3Flag.",
    "molecules": "ubiquitin, valinomycin"
  },
  {
    "caption": "(B) The recovery ratio of 3Flag-MITOL between the mitochondria-enriched (3,000 g pellet) and peroxisome-enriched fraction (100,000 g pellet) following valinomycin treatment for the indicated times. The ratio of peroxisome-localized 3Flag-MITOL to mitochondria-localized 3Flag-MITOL increased with valinomycin treatment for 3 hours. Graphic data represent results of two biological replicates. In scatter plot, dots indicate individual data points. Black dots indicate the ratio of 3Flag-MITOL collected in the mitochondria-enriched fractions, and red dots are the ratio of 3Flag-MITOL collected in the peroxisome-enriched fractions. Mean values are also shown.",
    "molecules": "valinomycin"
  },
  {
    "caption": "Distribution of endogenous MITOL in the mitochondria‐rich or peroxisome-rich fraction following cellular fractionation. The distribution of endogenous MITOL was examined using MITOL-3Flag knock-in HCT116 cells. Treatment of cells with valinomycin for 3 hours reduced the amount of endogenous MITOL in the mitochondria-enriched fraction, but concomitantly increased endogenous MITOL in the peroxisomes-enriched fraction.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(A) HeLa cells expressing HA-Parkin and wild type 3Flag-MITOL or 3Flag-MITOL lacking C-terminal 8 amino acids (∆C8) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. Expanded peroxisomes were observed in MITOL∆C8-expressing cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(B) 3Flag-MITOL∆C8 with the E3-inactive Cys65Ser/Cys68Ser (CS) and H43W mutations were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 µM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-PMP70 antibodies. After 3 hours of CCCP treatment, both the CS and H43W mutants localized on peroxisomes but expansion was not observed. Scale bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) HeLa cells expressing HA-Parkin and MITOL wild-type or ∆C8 were treated with 15 µM CCCP for 3 hours, and the number of peroxisomes was counted as PMP70-positive dots per 100 μm2. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. The abundance of peroxisomes in cells expressing MITOL∆C8 was significantly decreased as compared with cells expressing wild-type MITOL.",
    "molecules": "CCCP"
  },
  {
    "caption": "(D) MITOL∆C8 causes a drastic expansion of peroxisomes following CCCP treatment. HeLa cells expressing HA-Parkin and wild-type or ∆C8 MITOL were treated with 15 µM CCCP for 3 hours, and the approximate size of peroxisomes was determined as the number of pixels occupied by one peroxisome. The PMP70-positive pixels per 100 µm2 were divided by the number of peroxisomes in the same area. In the box-plots, dots indicate individual data points, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A and B) HeLa cells expressing HA-Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.",
    "molecules": "CCCP, ubiquitin"
  },
  {
    "caption": "C) Western Blot showing the levels of the PgP and control (α-TUBB) in RPE-1 parental and Taxol resistant clones.",
    "molecules": "Taxol"
  },
  {
    "caption": "E) Representative smRNA-FISH images of RPE-1 Parental and clones for the ABCB1 gene and DAPI. The images are projections of 0,5μm sections and a total 5 μm in thickness. Scale bar, 15μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome. Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6. B) TLA analysis for RPE-1 Parental and sg6C9. Zoom in in the region of chr6 with de novo interaction for sg6C9. Image modified from IGV viewer.",
    "molecules": "Taxol"
  },
  {
    "caption": "D) PCR products using the primers in C(1) over the ABCB1 and EEF1A1 regions in RPE-1 Parental and the different Taxol resistant clones. E) PCR products using the primers in C(2) over the specific break site in the ABCB1 promoter in RPE-1 Parental and the different Taxol resistant clones.",
    "molecules": "Taxol"
  },
  {
    "caption": "A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone.",
    "molecules": "Taxol"
  },
  {
    "caption": "B) H3K9me3 ChIP-qPCR in the ABCB1 promoter region. Bar graph show primer pairs amplifying the sgRNA#6 break and spanning this region (+/- base pairs) for each Taxol-resistant clone compared to the parental. Bar plots show the mean of H3K9me3 relative enrichment. Each dot represents a technical replicate (n=3).",
    "molecules": "Taxol"
  },
  {
    "caption": "E Plasma membrane stained with an αSAG1 antibody is separated from the IMC stained with anti-Ty after aerolysin treatment, indicating that PKAr-3Ty localises at the IMC.",
    "molecules": "aerolysin"
  },
  {
    "caption": "G A second copy of DDmyc-PKAc1 is stabilised after 1 hour in presence of Shld-1.",
    "molecules": "Shld-1"
  },
  {
    "caption": " H A second copy of DDmyc-PKAc1 stabilised with Shld-1 is targeted to the IMC in presence of a second copy of PKArWT-Ty. When of a second copy of PKArG2A-Ty is provided, stabilised DDmyc-PKAc1 does not localise at the cell periphery. Data information: Scale bars = 2 μm. ",
    "molecules": "Shld-1"
  },
  {
    "caption": "B A drop in PKAr-TyDD expression is detected 5 hours after Shld-1 removal and a significant parasite growth defect is observed as early as 12 hours later. Profilin (αPRF) served as loading control.",
    "molecules": "Shld-1"
  },
  {
    "caption": "C IFA analysis showed that the down regulation of PKAr-TyDD leads to the abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) 24 hours after Shld-1withdrawal.",
    "molecules": "Shld-1"
  },
  {
    "caption": "D Replication assay of PKAr-TyDD parasites grown for 24 hours +/- Shld-1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation. (100 parasites were counted in three independent replicates).",
    "molecules": "Shld-1"
  },
  {
    "caption": "G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).Data information: In C and G, data are presented as mean ± SEM. Scale bars = 2 μm (C and G), 3 mm (A and E).",
    "molecules": "Shld-1"
  },
  {
    "caption": "G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).",
    "molecules": "Shld-1"
  },
  {
    "caption": "A A drop in PKAc1-Ty-iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours.",
    "molecules": "ATc"
  },
  {
    "caption": "C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Scale bars = 20 μm (C)",
    "molecules": "ATc"
  },
  {
    "caption": "C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Data information: In D, data are presented as mean ± SD.",
    "molecules": "ATc"
  },
  {
    "caption": "E Prematurely egressed PKAc1-Ty-iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle. Scale bars = 2.5 mm (E)",
    "molecules": "ATc"
  },
  {
    "caption": "B Addition of Shld-1 leads to the rapid stabilisation of DDmyc-PKArWT-Ty and DDmyc-PKArG321E-Ty.",
    "molecules": "Shld-1"
  },
  {
    "caption": "C PKAc1 is co-immunoprecipitated with DDmyc-PKArWT-Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [35S]-labeled methionine/cysteine metabolic labelling. Conversely, PKAc1 is co-immunoprecipitated with DDmyc-PKArG321E-Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP. The autoradiogram shown is representative of two independent biological replicates.Data information: In C and E-H, data are presented as mean ± SEM.",
    "molecules": "cAMP, cysteine, methionine, 35S"
  },
  {
    "caption": "E Overexpression of the dominant negative DDmyc-PKArG321E-Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA. Images are representative of ~80% of the parasite population. Blue = DAPI; red = GAP45; green = GRA3.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 10 μm (E)",
    "molecules": "DAPI"
  },
  {
    "caption": "F In heavily infected cells, addition of Shld-1 at 32 hours post-inoculation for 4 hours leads to premature egress of DDmyc-PKArG321E-Ty parasites but not of the DDmyc-PKAr-Ty control line as assessed by IFA.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 5 µm (F)",
    "molecules": "Shld-1"
  },
  {
    "caption": "G Quantification of extracellular DDmyc-PKArG321E-Ty parasites released from heavily infected cells after addition of Shld-1 at 32 hours post-inoculation for 4 hours (100 parasites were counted in three independent replicates).Data information: In C and E-H, data are presented as mean ± SEM.",
    "molecules": "Shld-1"
  },
  {
    "caption": "H A non-selective PKA inhibitor, KT5270, significantly enhances the premature egress of DDmyc-PKArWT-Ty parasites in presence of Shld-1 (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).Data information: In C and E-H, data are presented as mean ± SEM.",
    "molecules": "Shld-1, KT5270"
  },
  {
    "caption": "I Prematurely egressed DDmyc-PKArG321E-Ty parasites after 4 hours of Shld-1 treatment invade and exit host cells, fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle.Scale bars = 2.5 mm (I).",
    "molecules": "Shld-1"
  },
  {
    "caption": "B Upon Shld-1 addition, a putative cGMP-specific PDE, PDE2 was more phosphorylated at Serine 1317 in DDmyc-PKArG321E-Ty parasites compared with DDmyc-PKArWT-Ty parasites suggesting a crosstalk between cAMP and cGMP signaling.",
    "molecules": "Shld-1"
  },
  {
    "caption": "C Chemical inhibition of the cGMP-dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 µM BIPPO, a PDE inhibitor, or by stabilisation of DDmyc-PKArG321E-Ty in presence of Shld-1. A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0.2 µM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only (data is from three independent biological replicates).Data information: In C and D, data are presented as mean ± SD.",
    "molecules": "Compound 1, Compound 2, BIPPO, Shld-1"
  },
  {
    "caption": "B Transferring supernatants of Shld-1 treated cultures from cells infected with DDmyc-PKArG321E-Typarasites at a MOI of 8 induces egress of parasites from cells infected at a MOI of 2 within three hours (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).",
    "molecules": "Shld-1"
  },
  {
    "caption": "C Acidification of the culture medium induces parasite egress in less than 10 minutes. The effect is inhibited in presence of compound 1 (data is from two independent biological replicates).",
    "molecules": "compound 1"
  },
  {
    "caption": " D Neutralisation of cultures with the weak base NH4Cl blocks premature egress induced by DDmyc-PKArG321E-Ty stabilisation. This block is circumvented by addition of the Ca2+ ionophore A23187 or the PDE inhibitor BIPPO (data is from three independent biological replicates). Data information: Data are presented as mean ± SD. ",
    "molecules": "NH4Cl, A23187, BIPPO"
  },
  {
    "caption": "F PKG-3Ty parasites were solubilised in either PBS or 1% Triton X-100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown.",
    "molecules": "Triton X-100, TX100"
  },
  {
    "caption": "G, H The signal of Myc-ACβ-iKD followed by IFA (G) after 48 hours ± ATc.Data information: Scale bars = 2 μm.",
    "molecules": "ATc"
  },
  {
    "caption": "G, H The signal of Myc-ACβ-iKD followed by western blot (H) after 48 hours ± ATc.",
    "molecules": "ATc"
  },
  {
    "caption": "I Invasion of ACα-KO tachyzoites or Myc-Acβ-iKD after 48 hours ± ATc is not affected. However, addition of ATc for 48 hours reduces the invasion efficiency of ACα-KO/ Myc-Acβ-iKD (data is from three independent biological replicates).",
    "molecules": "ATc"
  },
  {
    "caption": " J As for destabilisation of PKAc1-iKD or stabilisation of DDmyc-PKArG321E-Ty, ATc -KO/ Myc-Acβ-iKD tachyzoites treated with lytic cycle for 33 or 40 hours, exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non-treated parasites invade and initiate a new lytic cycle. Data information: Scale bars = 2 μm. ",
    "molecules": "ATc"
  },
  {
    "caption": "A Double immunofluorescence  for GFP  (green) and the BP  marker TBR2  (yellow), combined with DAPI staining  (white), of a 57 days-old chimpanzee cerebral organoid  2 days after electroporation with GFP  expression plasmid plus either control plasmid (top) or ARHGAP11B  expression plasmid (bottom). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells that are TBR2+ in 57 days-old chimpanzee cerebral organoids   2 days after electroporation with GFP  expression plasmid plus either control plasmid (dark red) or ARHGAP11B  expression plasmid (light red). Data are the mean of 9 control and 9 ARHGAP11B-transfected  cerebral organoids   of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "C Triple immunofluorescence  for GFP  (green), the cycling cell marker Ki67  (magenta) and TBR2  (yellow), combined with DAPI staining  (white), of a 59 days-old chimpanzee cerebral organoid  4 days after electroporation  with GFP  expression plasmid plus either control plasmid (top) or ARHGAP11B  expression plasmid (bottom). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+, Ki67+ and TBR2+ triple-positive cells. Scale bar, 50 µ D Quantification of the proportion of GFP+ cells in the SVZ/NL  that are Ki67+ and TBR2+ double-positive in 59 days-old chimpanzee cerebral organoids  4 days after electroporation  with GFP  expression plasmid plus either control plasmid (dark red) or ARHGAP11B  expression plasmid (light red). Data are the mean of 8 control and 8 ARHGAP11B-transfected cerebral organoids  of four independent batches each; error bars indicate SD; *, P <0.05 (one-sided Wilcoxon rank sum test).",
    "molecules": "DAPI"
  },
  {
    "caption": "A Double immunofluorescence  for GFP  (green) and the radial glia marker HOPX  (yellow), combined with DAPI staining  (white), of a 59 days-old chimpanzee cerebral organoid   4 days after electroporation  with GFP  expression plasmid plus either control plasmid (top) or ARHGAP11B  expression plasmid (bottom). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells in the SVZ/NL  that are HOPX+ in 59 days-old chimpanzee cerebral organoids   4 days after electroporation  with GFP  expression plasmid plus either control plasmid (dark red) or ARHGAP11B  expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids  of three independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test). C Double immunofluorescence for GFP  (green) and HOPX  (yellow), combined with DAPI staining  (white), of a 61 days-old chimpanzee cerebral organoid   10 days after electroporation  with GFP  expression plasmid plus either control plasmid (top) or ARHGAP11B  expression plasmid (bottom). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL  that are HOPX+ in 61 days-old chimpanzee cerebral organoids  10 days after electroporation  with GFP  expression plasmid plus either control plasmid (dark red) or ARHGAP11B  expression plasmid (light red). Data are the mean of 5 control and 7 ARHGAP11B-transfected cerebral organoids  of three independent batches each; error bars indicate SD; *, P<0.05 (two-sided Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "A Double immunofluorescence  for GFP (green) and the neuron  marker Hu  (magenta), combined with DAPI staining  (white), of a 61 days-old chimpanzee cerebral organoid  10 days after electroporation  with GFP  expression plasmid plus either control plasmid (top) or ARHGAP11B  expression plasmid (bottom). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and Hu+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells that are Hu+ in 61 days-old chimpanzee cerebral organoids  10 days after electroporation  with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B  expression plasmid (light red). Data are the mean of 7 control and 11 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; **, P <0.01 (two-sided Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "C Double immunofluorescence  for GFP  (green) and the  deep-layer neuron  marker CTIP2  (magenta), combined with DAPI staining  (white), of a 59 days-old chimpanzee cerebral organoid  4 days after electroporation  with GFP  expression plasmid plus either control plasmid (top) or ARHGAP11B  expression plasmid (bottom). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and CTIP2+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL  that are CTIP2+ in 59-days old chimpanzee cerebral organoids  4 days after electroporation  with GFP  expression plasmid plus either control plasmid (dark red) or ARHGAP11B  expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids   of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "E Double immunofluorescence  for GFP  (green) and the upper-layer neuron  marker SATB2  (magenta), combined with DAPI staining  (white), of a 66 days-old chimpanzee cerebral organoid   15 days after electroporation  with GFP  expression plasmid plus either control plasmid (top) or ARHGAP11B  expression plasmid (bottom). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and SATB2+ double-positive cells. Scale bar, 50 µm. F Quantification of the proportion of GFP+ cells in the SVZ/NL  that are SATB2+ in 66 days-old chimpanzee cerebral organoids  15 days after electroporation  with GFP  expression plasmid plus either control plasmid (dark red) or ARHGAP11B  expression plasmid (light red). Data are the mean of 6 control and 4 ARHGAP11B-transfected cerebral organoids   of two independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test).",
    "molecules": "DAPI"
  },
  {
    "caption": "A Double immunofluorescence  for GFP  (green) and the thymidine  analog BrdU  (magenta, combined with DAPI staining  (white), of 57 days-old human (top panels) and chimpanzee  (bottom panels) cerebral organoids   2 days after electroporation  with GFP  expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220  expression plasmid (lower panels). Tick marks indicate the borders of the VZ   and SVZ/NL; arrowheads indicate examples of GFP+ and BrdU+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells in the SVZ/NL  that are BrdU+ in 57 days-old human  (blue) and chimpanzee  (red) cerebral organoids   2 days after electroporation  with GFP  expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 12 human  control, 12 human ARHGAP11A220-transfected, 9 chimpanzee  control and 8 chimpanzee ARHGAP11A220-transfected  cerebral organoids  (grown from one human  and one chimpanzee iPSC  line, respectively) of six (human) and four (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; **, P <0.01 (Kruskal Wallis test).",
    "molecules": "BrdU, DAPI, thymidine"
  },
  {
    "caption": "C Double immunofluorescence  for GFP  (green) and TBR2  (yellow), combined with DAPI staining  (white), of 57 days-old human  (top panels) and chimpanzee  (bottom panels) cerebral organoids   2 days after electroporation  with GFP   expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL  that are TBR2+ in 57 days-old human  (blue) and chimpanzee  (red)  cerebral organoids   2 days after electroporation  with GFP  expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 9 human  control, 9 human ARHGAP11A220-transfected, 10 chimpanzee  control and 10 chimpanzee ARHGAP11A220-transfected cerebral organoids   (grown from one human and one chimpanzee iPSC  line, respectively) of four (human) and three (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05 (Kruskal Wallis test).",
    "molecules": "DAPI"
  },
  {
    "caption": "A Double immunofluorescence  for GFP  (green) and the bRG  marker PTPRZ1  (magenta), combined with DAPI staining  (white), of 60 days-old human forebrain organoids, generated from ARHGAP11A  plus ARHGAP11B  double-knockout clone 16. Images were obtained 10 days after electroporation  with GFP  expression plasmid plus either ARHGAP11A  plus ARHGAP11B  expression plasmids (ARHGAP11A+B-rescue, referred to as Control), ARHGAP11B  expression plasmid only (ARHGAP11B-rescue, referred to as ARHGAP11A-KO), or ARHGAP11A  expression plasmid only (ARHGAP11A-rescue, referred to as ARHGAP11B-KO). Tick marks indicate the borders of the VZ  and SVZ/NL; arrowheads indicate examples of GFP+ and PTPRZ1+ double-positive cells. Scale bar, 50 µm B Quantification of the proportion of GFP+ cells in the SVZ/NL  that are PTPRZ1+ in 60 days-old ARHGAP11A  plus ARHGAP11B  double-knockout (11A+11B KO) human forebrain organoids. Organoids  were generated from clone 16 (left) and clone j (right), and data were obtained 10 days after the indicated electroporation  with GFP  expression plasmid plus either ARHGAP11A  (11A) plus ARHGAP11B  (11B) expression plasmids (referred to as Control, as this constitutes an ARHGAP11A+B double-rescue in the 11A+11B KO organoids; dark blue), ARHGAP11B  (11B) expression plasmid only (referred to as 11A-KO, as this constitutes a selective ARHGAP11B  rescue in the 11A+11B KO organoids; light blue), or ARHGAP11A  (11A) expression plasmid only (referred to as 11B-KO, as this constitues a selective ARHGAP11A  rescue in the 11A+11B KO organoids; dark red). For clone 16, data are the mean of 8 control, 8 11A-KO and 8 11B-KO human forebrain organoids  of two independent batches each; for clone j, data are the mean of 9 control, 10 11A-KO and 8 11B-KO human forebrain organoids   of three independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05; **, P <0.01; ***, P <0.001 (Kruskal Wallis test)",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative image and measurements of lengths for CD31+/LYVE-1+ lacteal, CD31+/LYVE-1- capillary plexus and DAPI+ villus.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm.",
    "molecules": "BODIPY, FA, lipid"
  },
  {
    "caption": "Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm.",
    "molecules": "lipid, lipoproteins"
  },
  {
    "caption": "Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).",
    "molecules": "FFA, free fatty acids, TG, triglyceride"
  },
  {
    "caption": "Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).",
    "molecules": "TG, triglyceride"
  },
  {
    "caption": "Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B, C: JurkatT cells transfected with siRNA control, FIP3.1 or FIP3.2 were allowed to spread on poly-lysine-coated coverslips for the indicated times. Intracellular actin was detected with FITC-coupled Phalloidin. A: A single optical section at the level of the contact surface is shown. Bar, 5 µm. B: Population analyses of the spreading area of cells at the level of the contact surface. Each dot represents one cell. Horizontal bars represent the mean ± SEM. C:Time course representation of B, showing mean ± SEM.",
    "molecules": "poly-lysine"
  },
  {
    "caption": "D-F: Primary humanCD4T cells were transfected with control or FIP3.1 siRNA. D, E: Cells were allowed to spread on poly-lysine-coated coverslips for 5 min. Intracellular Lck was detected by immunofluoresecence. D: A single optical section at the level of the contact surface is shown. Bar, 10 µm. E: Population analyses of the spreading area of cells at the level of the contact surface.",
    "molecules": "poly-lysine"
  },
  {
    "caption": "F: Cells were stained with CMFDA dye, suspended in a paraformaldehyde solution and submitted to centrifugation at 3,724 xg onto poly-lysine-coated coverslips. Confocal optical sections separated by 0.2 µm were acquired. X-Z images were built with ImageJ. The maximal sections in 'x' (width) and 'z' (thickness) were determined for each cell (upper panel). Plots represent the deformability index (x/z). Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments.",
    "molecules": "paraformaldehyde, poly-lysine"
  },
  {
    "caption": "Jurkat T cells were transfected with control or FIP3.1 siRNA, then transfected again with empty vector or with expression vectors encoding Myc-tagged-Rac1WT, -Rac1G12V (constitutively active mutant), or -Rac1Rac1T17N (dominant negative mutant). Cells were allowed to spread on poly-lysine-coated coverslips for the indicated time and the spreading area of cells measured. A-C, H: Population analyses of the spreading area of cells at the level of the contact surface. A-C: Time course plots representing the mean ± SEM of the data shown as in I. D-G: Staining for nucleus (blue = Dapi), F-actin (green = phalloidin) and Rac1-Myc (red = anti-Myc) is shown. A single optical section at the level of the contact surface is shown. Bar, 5 µm.",
    "molecules": "poly-lysine"
  },
  {
    "caption": "A: Jurkat T cells transfected with control or FIP3.1 were treated 1 h with the Rac1 inhibitor NSC23766 at 100 µM and allowed to spread on poly-lysine-coated coverslips for 15 min. F-actin was detected with FITC-Phalloidin and the spreading area of cells at the contact surface was measured.",
    "molecules": "NSC23766, poly-lysine"
  },
  {
    "caption": "Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days.",
    "molecules": "folate"
  },
  {
    "caption": "Example of a single combed DNA molecule labeled with IdU (green) and CldU (red), showing replication from three adjacent origins. Horizontal white arrows indicate fork orientation.",
    "molecules": "DNA"
  },
  {
    "caption": "Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate-free medium.",
    "molecules": "DNA, folate"
  },
  {
    "caption": "Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138).Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361).",
    "molecules": "A, C, folate, G, nucleosides, T"
  },
  {
    "caption": "Fork distance (Kb) distribution. The color code is as in (D). Control (n = 72); −folate (n = 71); −folate + AGCT (n = 75).Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 212); −folate (n = 215); −folate + AGCT (n = 209).",
    "molecules": "A, C, folate, G, T"
  },
  {
    "caption": "A Population doublings (PDs) determined in BJ cells cultured at the indicated folate concentrations for 48 days.",
    "molecules": "folate"
  },
  {
    "caption": "B, C The average replication rate ± SEM (B) and the average fork distance ± SEM (C) in the indicated folate concentrations at 14 and 21 days. At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate. At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance.",
    "molecules": "DNA, folate"
  },
  {
    "caption": "D-G BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation. (D) Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 115); gray bars: BJ cells that were cultured for 14 days in 100 nM folate (n = 117); blue bars: BJ cells cultured for 14 days in 100 nM folate and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 117). (E) Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 352); 100 nM folate (n = 364); 100 nM folate + AGCT (n = 355). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. (F) Fork distance (Kb) distribution. The color code is as in (D). Control (n = 69); 100 nM folate (n = 74); 100 nM folate + AGCT (n = 72). (G) Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 201); 100 nM folate (n = 220); 100 nM folate + AGCT (n = 228). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. **P < 0.001.",
    "molecules": "A, C, folate, G, nucleosides, T"
  },
  {
    "caption": "Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Fork rate (Kb/min) distribution. White bars: BJ cells expressing an empty vector (n = 145); dark gray bars: BJ cells expressing the cyclin E oncogene (n = 147); light gray bars: BJ cells cultured for 7 days in a folate-free medium (n = 135); black bars: BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate-free medium (n = 138).",
    "molecules": "folate"
  },
  {
    "caption": "Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Fork distance distribution (Kb). The color code is as in (A). Empty vector (n = 78); CycE (n = 79); empty vector −folate (n = 71); CycE −folate (n = 80).",
    "molecules": "folate"
  },
  {
    "caption": "Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Percent of origins with the indicated progression ratio between sister forks. Empty vector (n = 158); CycE (n = 155); empty vector −folate (n = 160); CycE −folate (n = 154). *P < 0.05.",
    "molecules": "folate"
  },
  {
    "caption": "Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression (CycE) (n = 65), empty vector (n = 65), folate-free medium for 7 days (empty vector −folate) (n = 67) or oncogene expression under folate-free conditions (CycE −folate) (n = 70). Red: γH2AX; green: 53BP1; blue: DAPI staining.Percent of nuclei with the indicated number of γH2AX-53BP1 co-localized foci. **P < 0.01.",
    "molecules": "folate"
  },
  {
    "caption": "Cyclin E-expressing BJ cells grown for 7 days with and without folate.Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.Protein level quantification.",
    "molecules": "folate"
  },
  {
    "caption": "Cyclin E-expressing BJ cells grown for 7 days with and without folate.Examples of nuclei with RAD51 foci following cyclin E expression (CycE) (n = 67), empty vector (n = 65), folate-free medium for 7 days (−folate) (n = 71) or oncogene expression under folate-free conditions (CycE −folate) (n = 75). Green: RAD51, blue: DAPI staining.Percent of nuclei with the indicated number of RAD51 foci (n = 66). **P < 0.01.",
    "molecules": "folate"
  },
  {
    "caption": "A, B Cyclin E- and Ras (H-RasV12)-expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (A) Examples of anchorage-independent growth in soft agar of 3T3 cells. (B) Average number of colonies per soft agar plate of 3T3 cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments.",
    "molecules": "folate"
  },
  {
    "caption": "C-E Ras (H-RasV12)-expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (C) Examples of anchorage-independent growth in soft agar of MCF10A cells. (D) Average number of colonies per soft agar plate of MCF10A cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. (E) Percentage of tumor-free flanks at the indicated time points after cell injection. Ten mice were injected in both sides in each group.",
    "molecules": "folate"
  },
  {
    "caption": "Optimization of transcriptional output using different concentrations of Mg-glutamate and K-glutamate in 10 bacterial cell-free expression systems with Broccoli as a reporter (solid lines, optimal buffer composition shown in red). No DNA template controls are shown as grey dashed lines. 12.5 nM of DNA template was used in all systems, except L. lactis (50 nM used).",
    "molecules": "Broccoli, DNA, Mg-glutamate, K-glutamate"
  },
  {
    "caption": "(B) Immunoblotting and quantification of brown preadipocytes (WT-1) stimulated with insulin at different doses for 5 min (n=3). The molecular weight corresponded to the I-Afadin isoform.",
    "molecules": "insulin"
  },
  {
    "caption": "(C) Immunoblotting of mature brown adipocytes (WT-1) stimulated with insulin (100nM) for 5 min (n=4).",
    "molecules": "insulin"
  },
  {
    "caption": "(D) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min (n=3).",
    "molecules": "insulin"
  },
  {
    "caption": "(E) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min (n=3).",
    "molecules": "insulin"
  },
  {
    "caption": "(F) Immunoblotting and quantification of isolated brown adipose tissue (BAT) from male C57BL/6J mice injected with 5U insulin for 5 min (n=4-6).",
    "molecules": "insulin"
  },
  {
    "caption": "(H) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12-week old C57BL/6 male mice fed with either chow (CD) or high-fat diet (HFD) for 6-weeks injected with vehicle or insulin (n=6).",
    "molecules": "insulin"
  },
  {
    "caption": "(I) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions (SVF) from lean, obese or Type 2 Diabetic (T2DM) subjects stimulated or not with insulin (n=6). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (G) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (H) Quantifications of Total IRβ/tubulin in BAT, sWAT and Liver (n=5-6). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin"
  },
  {
    "caption": "Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (I) Insulin-induced lipogenesis in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin, Insulin"
  },
  {
    "caption": "(B) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "Oil red O"
  },
  {
    "caption": "(C) Wild-type, Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT I-Afadin or a non-phosphorylable form (S1795A) stimulated with vehicle or insulin (n=4) for Immunoblotting.",
    "molecules": "insulin"
  },
  {
    "caption": "(D) Quantification of insulin-induced Akt phosphorylation from immunoblots. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "insulin"
  },
  {
    "caption": "(E) Real-time measurement and (F) quantification of insulin-induced fatty-acid uptake (n=4). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "fatty-acid, insulin"
  },
  {
    "caption": "(E) IR abundance measured by immunoblotting following 4 hours pre-incubation with the HDAC6 inhibitor (Tubastatin A) in differentiated adipocytes exposed to insulin (100nM) stimulation for 15 min (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.",
    "molecules": "insulin, Tubastatin A"
  },
  {
    "caption": "(F) Immunolocalization of IR (green) and HDAC6 (red) in Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin. Nuclei were stained with DAPI (blue). Co-localization was detected by the appearance of white pixels where the red and green overlap. Scale bars: 25 µm. Representative images are shown. The ratio of IR co-localized with HDAC6 (Mandler's co-localization coefficient) was quantified (98-112 individual cells from two independent experiments). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.",
    "molecules": "DAPI, insulin"
  },
  {
    "caption": "(G) Immunoprecipitation of IR followed by immunoblotting using an anti-acetyl-Lys antibody in adipocytes lacking Afadin or re-expressing Afadin, with or without insulin stimulation (n=1). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.",
    "molecules": "insulin, acetyl-Lys"
  },
  {
    "caption": "Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (C) Sirius Red staining of representative embedded BAT, sWAT and vWAT from mice on HFD and quantification of adipocyte areas (~1000 adipocytes/genotype). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.",
    "molecules": "Sirius Red"
  },
  {
    "caption": "C. HEK293  cells were incubated with 5 µg of purified EVCx43- or EVCx43+ for 24 h, in the presence or absence of Bafilomycin A1 (Baf) in the last 6 h. The levels of LC3 were analyzed by WB  and are depicted on graph (LC3-II; n=4 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.",
    "molecules": "Baf, Bafilomycin A1"
  },
  {
    "caption": "A. Cell free EV biogenesis  was assessed by relative protection of Cx43-luciferase. Reactions were incubated at 4ºC or 30ºC in the presence of membranes  derived from  HEK293Cx43- transiently transfected with Cx43-luciferase  and cytosol  from  HEK293Cx43- cells, except in the negative control. 1% TX100 was used to disrupt membranes, where indicated (n=3 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.",
    "molecules": "TX100"
  },
  {
    "caption": "B. Crude membranes  and cytosol  from broken  HEK293Cx43+ or HEK293Cx43- cells were mixed in an ATP regenerating system, supplemented with synthetic miR-133b  or miR-509-3p, as indicated. Graph depicts the indicated miRNAs levels, assessed by  RT-qPCR and represented as percent protected after RNAse I  digestion of unincorporated miRNAs (n=7-8 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.",
    "molecules": "ATP"
  },
  {
    "caption": "D. WB  analysis for Cx43  in samples derived by miRNAs pulldowns  performed with whole lysates of HEK293Cx43+ cells and biotinylated miR-133b and miR-509-3p (n=5 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.",
    "molecules": "biotinylated"
  },
  {
    "caption": "EMSA  was performed using either Cy3-labeled miR-133b (E , and Cx43  produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled  miR-133b  was used (competitor). Empty nanodiscs  were used to control unspecific binding.",
    "molecules": "lipid nanodiscs, nanodiscs, Cy3"
  },
  {
    "caption": "EMSA  was performed using either Cy3-labeled  miR-133b F) and Cx43  produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1  or GST-hnRNPQ, as indicated.",
    "molecules": "lipid nanodiscs, Cy3"
  },
  {
    "caption": "EMSA  was performed using either Cy3-labeled miR-509-3p (G and Cx43  produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled miR-133b or miR-509-3p  was used (competitor). Empty nanodiscs  were used to control unspecific binding.",
    "molecules": "lipid nanodiscs, nanodiscs, Cy3"
  },
  {
    "caption": "EMSA was performed using either Cy3-labeled miR-509-3p H) and Cx43  produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1  or GST-hnRNPQ, as indicated.",
    "molecules": "lipid nanodiscs, Cy3"
  },
  {
    "caption": "I. EMSA  was performed using either Cy3-labeled wild type or mutant miR-199a-3p, and Cx43-enriched membranes  prepared from C33a  parental cells (Cx43+). Cx43- membranes  were used to control unspecific binding.",
    "molecules": "Cy3"
  },
  {
    "caption": "Representative maximum projection images of microscopy-based miRNA-protein interaction using beads coated with GST-tagged NT/CT-Cx43,  hnRNPA2B1 or hnRNPQ, as indicated, following the addition of Cy3-labelled  miR-133b or miR-509-3p. Graph depicts quantification of total fluorescence/bead, as a measure of  protein-miRNAs binding. Scale bars 50 μm (n=2 biological replicates; average of 23 beads analyzed/condition). Data information: p-values were derived by Kruskal-Wallis (Dunn's post-hoc) test. Bars and error bars indicate mean ± SD in all graphs.",
    "molecules": "Cy3"
  },
  {
    "caption": "A. Equal amounts of EVCx43+ and EVCx43- labelled with SYTO® RNASelect™ green fluorescent stain  were incubated with HEK293Cx43+ cells for 30 min, at 37ºC. Scale bars 10 μm (n=5 biological replicates). p-values were derived by Mann-Whitney test.",
    "molecules": "SYTO® RNASelect™"
  },
  {
    "caption": "(b) GFP signals were observed in these cells before or after starvation. (c) The number of GFP-positive puncta per cell was counted; mean ± SD values are presented. N, nutrient rich; S, starvation.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) HEK293 cells were simultaneously transfected with four plasmids harboring One-Strep-Flag (OSF)-tagged Atg14L, Beclin-1, hVps34, and hVps15, and Atg14L was pulled down using Strep-Tactin Sepharose beads. Empty plasmid was used as control instead of Atg14L. The precipitates were subjected to SDS-PAGE and Coomassie brilliant blue staining.",
    "molecules": "Sepharose"
  },
  {
    "caption": "(e) NRK cells were transiently transfected with adenovirus expressing GFP-Atg14L or GFP-Atg14L4C4A and fixed under nutrient-rich conditions. The cells were stained with anticalnexin antibody as an ER membrane marker. Higher magnification of the boxed areas is shown below. Bars, 10 µm.",
    "molecules": "nutrient"
  },
  {
    "caption": "C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.",
    "molecules": "Thaps"
  },
  {
    "caption": "A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm. B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *. C ",
    "molecules": "Thapsigargin"
  },
  {
    "caption": "A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.",
    "molecules": "Thapsigargin"
  },
  {
    "caption": "A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).",
    "molecules": "Ub"
  },
  {
    "caption": "B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005. ",
    "molecules": "Thaps, Ub"
  },
  {
    "caption": "B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.",
    "molecules": "Thapsigargin"
  },
  {
    "caption": "C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.",
    "molecules": "MG132, o-phenanthroline, PR619, ubiquitin"
  },
  {
    "caption": "(B) Metabolomic  analysis focused on strains with over-expression or deletion of the AYT1  gene shows a significant reduction or accumulation, respectively, in  acetyl-glutamate  compared to the matched control strain of each mutant. Only metabolites changing significantly in at least one of the two conditions are presented. D-EIP  is D-erythro-1-(Imidazole-4-yl) glycerol 3-phosphate.",
    "molecules": "D-EIP, D-erythro-1-(Imidazole-4-yl) glycerol 3-phosphate, acetyl-glutamate"
  },
  {
    "caption": "(D) A  growth assay in a condition that does not induce high levels of  acetyl-CoA in peroxisomes  (glucose) shows that all strains grow similarly to the control until  stationary phase  when peroxisomes  become vital. In stationary phase, the over-expression (OE) of AYT1  grows to a lower density than the control strain, plausibly due to burdening of peroxisomal  functions. (E) A growth assay in a condition expected to elevate acetyl-glutamate  levels in peroxisomes  (oleate  as a sole carbon  source and glutamate  as a nitrogen  source) demonstrates that the AYT1  overexpressing strain grows faster and to a higher density than the control. Data information: The  growth assays in D and E were done in three biologically independent replicates and error bars were plotted. Note that some error bars are shorter than the symbol, hence are not visible in the graph.",
    "molecules": "acetyl-CoA, carbon, glucose, glutamate, acetyl-glutamate, nitrogen, oleate"
  },
  {
    "caption": "(G) A  β-oxidation activity assay of Δlpx1, Δfsh3, and Δlpx1Δfsh3 strains supplemented with labeled  8 carbon- or 18 carbon-free fatty acids in media supplemented with 0.5% glucose  shows a significant reduction in β-oxidation activity compared to the wild type and the two single mutants, suggesting an overlapping role for Fsh3  with Lpx1.This assay was done in three biologically independent replicates. Bars represent the mean and error bars represent the standard deviation.",
    "molecules": "8 carbon, 18 carbon, glucose"
  },
  {
    "caption": "(E)  Western blot  analysis of Fbp1-HA  levels in mutants Δpex3 (no peroxisomes), Δpex5 (abolished targeting of GID subunits to peroxisomes), and Δpex7 (no β-oxidation due to loss of Pot1  targeting) demonstrates that two hours after the transition from glucose  to ethanol  (gluconeogenic conditions), control and Δpex7 cells upregulated Fbp1-HA  levels, while for Δpex3 and Δpex5 cells Fbp1-HA  levels remain low. Antibodies were used against the HA  tag (for Fbp1) and Actin  as a loading control.",
    "molecules": "ethanol, glucose"
  },
  {
    "caption": "(b) Western analysis of whole-cell extracts, membrane, cytosolic and peroxisome fractions from MCF-7 cells stimulated with insulin (200 nM) for 30 min after 1 h of serum starvation and immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively.",
    "molecules": "insulin"
  },
  {
    "caption": "(d) Proteinase K protection assays performed in the presence or absence of Triton X-100 to disrupt peroxisomes on equal masses of peroxisome fractions (HEK293 cells) collected at the indicated time points. The lysates were immunoblotted for TSC2, TSC1, Rheb, catalase and PMP70. Uncropped images of western blots are shown in Supplementary Fig. S6.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(c) Representative immunocytochemistry images using MCF-7 cells transfected with an mRFP-GFP-LC3 construct. Cells treated with 0.4 mM H2O2 were analysed at 0 h (Control), 1 h, 3 h and 6 h. Scale bars, 10 μm. (d) Quantification of autophagosomes (AP, GFP-LC3) and autolysosomes (AL, RFP-LC3) per cell in different conditions as shown in Fig. 4c. The results are represented as the average puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment. **P0.01;***P0.001.",
    "molecules": "H2O2"
  },
  {
    "caption": "(e) Western analysis of FAO cells treated with 50 μM Wy-14643 (WY) or vehicle (DMSO) for PPAR-alpha-inducible proteins (EHHADH and ACAA1), mTORC1 signalling proteins ((pS6 (S235/236), S6, pS6K (T389) and S6K)) and autophagy markers (LC3 and p62). (f) Representative images of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 1 h, with superoxide production detected using dihydroethidium (DHE). Scale bars, 30 μm.",
    "molecules": "DMSO, Wy-14643"
  },
  {
    "caption": "(g) Representative images of LC3 puncta (green) in FAO cells following treatment with 50 μM Wy-14643 (WY) or vehicle (DMSO) for the indicated time periods. Scale bars, 10 μm. (h) Quantification of LC3 puncta per cell in response to Wy-14643 (WY) or vehicle (DMSO). The results are represented as the average LC3 puncta fluorescence per cell (±s.e.m., n = 3 independent experiments) from 100 cells per experiment as shown in Fig. 4a. *P0.05; **P0.01.",
    "molecules": "DMSO, Wy-14643"
  },
  {
    "caption": "(i) Transmission electron microscopy of FAO cells treated with vehicle (DMSO) or 50 μM Wy-14643 (WY) for 24, 48 and 72 h. Peroxisomes and autophagosomes are indicated with red and yellow arrows, respectively. Scale bars, 500 nm. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.",
    "molecules": "DMSO, Wy-14643"
  },
  {
    "caption": "(b) Western analysis of human fibroblasts obtained from a Zellweger (GM13267) or a corresponding control patient (with Ehlers-Danlos syndrome (GM15871)) treated with the indicated doses of H2O2 for 1 h. mTORC1 signalling was assessed by western analysis for pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pATM (S1981), ATM, pAMPK (T172), AMPK, p62 and LC3.",
    "molecules": "H2O2"
  },
  {
    "caption": "(c) Western analysis of human fibroblast (GM13427) cells pre-incubated with 3 mM N-acetyl cysteine (NAC; ROS scavenger) for 1 h before being treated with 0.4 mM H2O2 for 1 h using anti-pS6K (T389), S6K, p62 and LC3 antibodies.",
    "molecules": "H2O2, N-acetyl cysteine, NAC, ROS"
  },
  {
    "caption": "(d) Representative western analysis using cell extracts from human fibroblasts obtained from a Zellweger patient (GM13267) or control fibroblasts (GM15871) treated with amino-acid-free media for 60 min, and stimulated with a mixture of amino acids for 10 min. mTOR signalling was monitored using anti-pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1(T37/46) and 4E-BP1 antibodies. Uncropped images of western blots are shown in Supplementary Fig. S6.",
    "molecules": "amino acids"
  },
  {
    "caption": "(b) TSC2 functional assay was performed in HEK293 cells co-expressing myc-Rheb, Flag-TSC1 and Flag-TSC2 wild type (WT), or Flag-TSC2 mutants (RG, RQ and RW) and HA-S6K (left panel) or HA-4E-BP1 (right panel). Cells transfected with empty vector (EV) with or without myc-Rheb were used as controls. Lysates were further analysed for phospho-S6K (T389), S6K, phospho-4EB-P1 (S65), 4E-BP1 and 32P incorporation into S6.",
    "molecules": "32P"
  },
  {
    "caption": "(f) In vivo guanine nucleotide loading of Rheb was measured using HEK293 cells overexpressing Flag-TSC1 and Flag-TSC2 wild type, or Flag-TSC2 mutants (RQ and RQ-9NT) with or without Rheb as indicated. (g) Graph shows quantification of the percentage of Rheb bound to GTP (indicative of Rheb GTPase activity, ±s.e.m., n = 4 independent experiments). *P0.05;***P0.001.",
    "molecules": "GTP"
  },
  {
    "caption": "(A) Cellulase  functional screening by    Congo Red   assay. Halos on the plate indicate cellulase  activity. Pc: Pseudomonas cellulosa, Bs: Bacillus subtilis. Numbered strains are from the  C. elegans  native gut  microbiome",
    "molecules": "Congo Red"
  },
  {
    "caption": "(B) Production of reducing sugars by cellulose-degrading bacterial strains. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.",
    "molecules": "reducing sugars, cellulose"
  },
  {
    "caption": "(E) C. elegans colonized with cellulolytic  P. cellulosa and incubated with different carbon   sources. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.",
    "molecules": "carbon"
  },
  {
    "caption": "(A) Incorporation of 14C-labelled cellulose. Worms  were first colonized with cellulolytic bacteria and then incubated with 14C-cellulose  to allow carbon  utilization. Antibiotic treatment was applied before 14C  measurement of worms  to eliminate bacterial interference in isotopic reading. 14C-cellulose  incorporation measurement reflected the isotopic carbon    incorporation in C. elegans. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In A, experiments were performed in biological triplicates using 200 worms  per data point. In A error bars represent 95% confidence intervals for the mean.",
    "molecules": "carbon, 14C, cellulose"
  },
  {
    "caption": "(B) Induction of the worm fat-7p::GFP metabolic reporter. Adult worms  pre-colonized with P. cellulosa were incubated in liquid media for 24 h with the indicated carbon  sources, and GFP  fluorescence was read in individual worms  on a BioSorter large object sorter. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In B, error bars represent 95% confidence intervals for the mean. In B, each point represents a different worm  in biological replicates, n=96, n=53, and n=80 from left to right.",
    "molecules": "carbon"
  },
  {
    "caption": "Synchronized adult worms  were colonized for two days on plates containing E. coli OP50 +  P. cellulosa (Pc), E. coli OP50  + Lp, or E. coli OP50  + Pc  + Lp. (B) After 36 h outgrowth of the pathogen, worms  pre-colonized with P. cellulosa in the presence of L. plantarum (E. coli OP50 + Pc  + Lp) showed lower pathogen burden than P. cellulosa  monocolonized worms  when CMC  was used as the carbon  source. Data shown are fold change Salmonella infection  (CFU/worm) relative to the  P. cellulosa-only condition. Error bars represent 95% confidence interval of the mean. CMC  = 0.01% carboxymethylcellulose  (w/v). All experiments were performed in biological triplicates. Student's t test was used for statistical analysis.",
    "molecules": "carbon, carboxymethylcellulose, CMC"
  },
  {
    "caption": "(B) Oxygen consumption rates (OCR) in primary myoblasts and differentiated myotubes. Basal OCR was first measured, followed by administration of 10 mM sodium pyruvate, and 2 μM oligomycin (to inhibit ATP synthase), uncoupler FCCP (2 μM), or rotenone/antimycin (Rot/A) (1 μM) as indicted. n = 3 separate experiments done with 10 biological replicates. *p < 0.0001 (FCCP).",
    "molecules": "antimycin, FCCP, oligomycin, rotenone, sodium pyruvate"
  },
  {
    "caption": "(D) Mitochondrial respiration rates were determined from mitochondria isolated from indicated muscle using pyruvate as substrate. ADP-dependent respiration, oligomycin-induced (oligo), uncoupler FCCP, and antimycin A (AA) are shown. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.05 (ADP).",
    "molecules": "ADP, FCCP, oligomycin, pyruvate"
  },
  {
    "caption": "(F) Bars represent mean blood lactate levels for 3-month and 15-month old male MCK-miR-499 and NTG mice following a 25 min run on a motorized treadmill. For 3-month blood lactate, NTG, n = 9; MCK-miR-499, n = 10. For 15-month blood lactate, NTG, n = 7; MCK-miR-499, n = 5. *p = 0.039 (3-month), *p = 0.018 (15-month).",
    "molecules": "lactate"
  },
  {
    "caption": "(G) Mitochondrial respiration rates were determined from the plantaris muscle of the indicated genotypes using pyruvate/malate as substrate. Pyruvate/malate (Py/M)-stimulated, ADP-dependent respiration, oligomycin-induced (oligo) and the respiratory control ratio (RC) are shown. NTG, n = 6; MCK-miR-499, n = 4. *p = 0.015 (Py/M), *p = 0.017 (ADP), *p = 0.001 (Oligo).",
    "molecules": "ADP, malate, oligomycin, Pyruvate"
  },
  {
    "caption": "(F) The bars represent the mean blood lactate levels from the indicated mice following a 25 min run on a motorized treadmill. NTG, n = 9; 499Tg, n = 7; PGC-1α mKO, n = 6; 499Tg/PGC-1α mKO, n = 9. *p = 0.0009 (NTG vs. 499Tg), p = 0.316 (NS, not significant).",
    "molecules": "lactate"
  },
  {
    "caption": "(B, C) Results of RT-qPCR analysis for WT primary mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNAs as indicated. For C, 48 h post-siRNA transfection, myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest (n = 3 independent experiments). *p < 0.0001 (Fnip1 in B), *p = 0.0001 (PGC-1α in B); *p < 0.0001 (vs. Con siRNA in C), ‡p < 0.0001 (vs. Fnip1 siRNA in C ).",
    "molecules": "DMSO, compound C"
  },
  {
    "caption": "(E) Oxygen consumption rates (OCR) in primary mouse myotubes transfected with Fnip1 siRNA or Con siRNA. n = 7 separate experiments done with 5 biological replicates. *p < 0.05 (Pyruvate), *p < 0.01 (FCCP).",
    "molecules": "FCCP, Pyruvate"
  },
  {
    "caption": "(B) Mitochondrial respiration rates were determined from mitochondria isolated from the hindlimb muscle of the indicated mice using pyruvate as substrate. n = 3 separate experiments done with 7-8 biological replicates. *p < 0.01 (ADP).",
    "molecules": "ADP, pyruvate"
  },
  {
    "caption": "(D) Representative immunoblots for pPERK, cleaved-ATF6, and GAPDH, and relative protein levels of pPERK and cleaved-ATF6 in control (open bars) and Brucella-infected (solid bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. As a positive control for activation of PERK or ATF6, HeLa cells were treated with 5 μg/ml of tunicamycin for 8hr ('Tm'). The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.",
    "molecules": "Tm, tunicamycin"
  },
  {
    "caption": "(A) Representative immunoblots showing co-immunoprecipitation with pIRE1. After 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-pIRE1 antibody (lanes labeled 'pIRE1') or control anti-rabbit IgG (lanes labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with a panel of antibodies against molecules involved in the ER-Golgi vesicular transport pathways or against ER- (Sec61α, HSP47, and calnexin), ERGIC- (ERGIC53) and cis-Golgi- (GM130) resident proteins.",
    "molecules": "Tm"
  },
  {
    "caption": "(B) Representative immunoblot showing the co-immunoprecipitation of pIRE1 with Yip1A. Following 5 hr of Tm treatment, immunoprecipitation was performed on HeLa cell lysates with an anti-Yip1A antibody (lane labeled 'Yip1A') or control anti-rabbit IgG (lane labeled 'IgG'), and the immunoprecipitates were analyzed by Western blotting with an anti-pIRE1 antibody.",
    "molecules": "Tm"
  },
  {
    "caption": "(C) Representative confocal micrographs of HeLa cells double-stained for pIRE1 (green) and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were detected as large foci. Scale bars are 10 μm.",
    "molecules": "Tm"
  },
  {
    "caption": "(D) Representative confocal micrographs of HeLa cells triple-stained for pIRE1 (green), Sec31 (red), and Yip1A (blue) after 5 hr of Tm treatment. Insets are magnifications of the boxed areas on the main image. Co-localized proteins were identified as large, bright foci (arrows). Scale bars are 10 μm.",
    "molecules": "Tm"
  },
  {
    "caption": "HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm to induce the UPR. Cell lysates were prepared at the indicated time points and analyzed by Western blotting. (A) Representative immunoblots for pIRE1, spliced-XBP1, and β-tubulin. β-tubulin was used for normalization. The intensity of the bands was quantified using the MultiGauge software.",
    "molecules": "Tm"
  },
  {
    "caption": "(B-D) Relative protein levels of pIRE1 (B), relative mRNA levels of spliced-XBP1 (C), and relative protein levels of spliced-XBP1 (C) in control (solid circles) and Yip1A-knockdown (open circles) cells. The protein or mRNA levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments.",
    "molecules": "Tm"
  },
  {
    "caption": "(E) Representative immunoblots for Sar1, Sec23, Sec24D, and GAPDH, and relative protein levels of Sar1, Sec23, and Sec24D in control (solid circles), Yip1A-knockdown (open circles), and IRE1-knockdown (solid gray circles) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the line graphs. The protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments.",
    "molecules": "Tm"
  },
  {
    "caption": "(F) Representative confocal micrographs of control (upper panels) or Yip1A-knockdown (lower panels) cells during the Tm treatment. Fixed cells at the indicated time points were stained for pIRE1. Cells are outlined with white dashed lines. Scale bars are 10 μm. The numbers of pIRE1 foci per cell were counted, and shown in the line graph. Data are means ± SD (N = 30).",
    "molecules": "Tm"
  },
  {
    "caption": "(G) Representative immunoblot for pIRE1 after native PAGE, showing two high-order complexes of pIRE1 (pIRE1-I and pIRE1-II). Lane 'S' represents lysate from HeLa cells transfected with control scramble siRNA, and lane 'Y' represents lysate from HeLa cells transfected with Yip1A siRNA. Numbers on the left-hand side correspond to the standard molecular weight. The intensity of the bands was quantified by using the MultiGauge software, and the results are shown in the bar graphs. Protein levels in control cells at the beginning of the Tm treatment were assigned the value 1. Data are means ± SD from three independent experiments. **: p<0.01.",
    "molecules": "Tm"
  },
  {
    "caption": "HeLa cells were transfected with each siRNA for 24 hr, and then treated with Tm for 5 hr to induce the UPR. (A, B) Representative confocal micrographs of control (left-hand panels), Yip1A-knockdown (middle panels), and IRE1-knockdown (right-had panels) cells after 0 hr or 5 hr of Tm treatment (A). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control cells (arrowheads), but not in Yip1A- or IRE1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (B). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.",
    "molecules": "Tm"
  },
  {
    "caption": "(C, D) Representative confocal micrographs of control, Atg9-knockdown, WIPI1-knockdown, and DFCP1-knockdown cells after 0 hr or 5 hr of Tm treatment (C). The ER structure was visualized with an anti-calnexin antibody. Large vacuoles were observed in control and DFCP1-knockdown cells (arrowheads), but rarely seen in Atg9- or WIPI1-knockdown cells. Scale bars are 10 μm. The percentage of cells with vacuoles was counted, and is shown in the bar graph (D). Data are means ± SD from three independent experiments (N = 100). **: p<0.01.",
    "molecules": "Tm"
  },
  {
    "caption": "(E) Representative immunoblots for pPERK, cleaved-ATF6, and GAPDH, and relative protein levels of pPERK and cleaved-ATF6 in control (solid bars) and Yip1A-knockdown (open bars) cells. GAPDH was used for normalization. The intensity of the bands was quantified using the MultiGauge software, and the results are shown in the bar graphs. As a positive control for activation of PERK or ATF6, HeLa cells were treated with 5 μg/ml tunicamycin for 8 hr ('Tm'). The protein levels at time 0 hr were assigned the value 1. Data are means ± SD from three independent experiments.",
    "molecules": "Tm, tunicamycin"
  },
  {
    "caption": "(A) Representative images of necrotic cell death (SYTOX orange - red fluorescence) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in control Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f/f-EsrCRE enteroids without tamoxifen treatment. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (D) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids. ",
    "molecules": "SYTOX orange, tamoxifen"
  },
  {
    "caption": "(A) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.1μg/mL TcdB in HIOs. Scale bar = 250μm. (B) Quantification of necrotic cell death in HIOs. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in kcHIEs. Scale bar = 250μm. (D) Quantification of necrotic cell death in kcHIEs. (E) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in bHIEs. Scale bar = 250μm. (F) Quantification of necrotic cell death in bHIEs. ",
    "molecules": "SYTOX orange"
  },
  {
    "caption": "(A) Validation of anti-phasic Rac1 expression in mouse (mRac1, red) and human enteroids (hRac1, black) by qRT-PCR. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1-KD bHIEs. ",
    "molecules": "SYTOX orange"
  },
  {
    "caption": "(C-D) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f/f;CAGGCre-ERTM without tamoxifen (C) or Rac1 f/f;CAGGCre-ERTM with tamoxifen-treated (D) enteroids.",
    "molecules": "SYTOX orange, tamoxifen"
  },
  {
    "caption": "D: Complex nutrient shift of non-dividing cells (S382) growing on agarose pad (MM+0.02% glucose) in the presence of vancomycin (100 μg/mL). Same quantities as in B, as a function of time since MreB-motion stop. Cells were transiently starved for glucose (through addition of 0.5% (w/v) αMG and 0.25% (w/v) 2DG), followed by addition of excess glucose (1%). Different from A-C, all compounds were added to the agarose pad as droplets and reached cells by diffusion.",
    "molecules": "2DG, agarose, glucose, αMG, vancomycin"
  },
  {
    "caption": "A: Single-cell growth of non-dividing b42 cells (ΔmepS/pBAD30-mepS) grown on LB agarose pads as a function of time after addition of arabinose (0.2%) in form of a droplet. Left: Relative increase of volume, mass, surface, and length. Middle: Elongation rate and surface-growth rate. Right: Relative changes of surface-to-mass ratio and width.",
    "molecules": "agarose, arabinose"
  },
  {
    "caption": "B-C: Cell bending: B: Time-lapses of bending filamentous cells (S290) in donut microchambers (3% agarose; RDM+glu) and grown without (top) or with (bottom) D-cycloserine (1 mM) as a function of time since D-cycloserine treatment, starting >10 min after D-cycloserine treatment (the time of MreB-motion stop D-cycloserine-treated cells were osmotically shocked (NaCl) after 33 min of treatment, which releases a fraction of cells C: Change of cell centerline curvature as a function of cell length between 10 min after start of D-cycloserine treatment and release of cells through osmotic shock",
    "molecules": "agarose, D-cycloserine, glu, NaCl"
  },
  {
    "caption": "D-E: Cell straightening. D: Time-lapses of straightening filamentous cells (S290) previously bent in donuts: control (top) and D-cycloserine treated cells (bottom) as a function of time since simultaneous release from donuts and beginning of drug treatment. E: Change of centerline curvature as a function of relative length from 10 min after D-cycloserine treatment (time of MreB-motion stop).",
    "molecules": "D-cycloserine"
  },
  {
    "caption": "C: Relative change of cell width of non-dividing cells (S257) grown in LB during combined D-cycloserine treatment (5 mM) and a hypo-osmotic ramp as in A (DCS + Hypo ramp), as compared to cells exposed to the hypo-osmotic ramp only (Hypo ramp) or treated with D-cycloserine only (DCS). D-cycloserine was contained in both agarose pads, and treatment started 15 min prior to the shock.",
    "molecules": "agarose, D-cycloserine, DCS"
  },
  {
    "caption": "(B-D) Flow cytometric quantification of DHE>Eth. positive wild type yeast cells (allowing the detection of both dead cells and cells exhibiting sub-lethal oxidative stress) after 42 h (B) or 66 h (C, D) expressing Abeta constructs. Dot plots show all data points along with the mean (bar) ± s.d.. n = 5-12 biologically independent cultures. ***, p < 0.001, **, p < 0.01, *, p < 0.05. ANOVA with Tukey's post-hoc test.",
    "molecules": "DHE, Eth"
  },
  {
    "caption": "(E) Annexin V (AV)/PI co-staining to assess cell death in yeast expressing A42 after 48 h. Data represent means ± s.d. n = 6 biologically independent cultures. ***, p < 0.001 (comparing PI positive populations). Unpaired, two-tailed t-test.",
    "molecules": "A42, PI"
  },
  {
    "caption": "(A) Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial, microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP-Abeta42 (A42), EGFP-Abeta40 (A40) and EGFP empty vector (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Purity of fractions was tested with antibodies against Cyc1 (mitochondria), Sec61 (microsomes) and Pgk1 (cytosol).",
    "molecules": "A40, Abeta40, A42, Abeta42"
  },
  {
    "caption": "(B) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP-A42 (A42) or EGFP empty vector (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 16 biologically independent cultures. **, p < 0.01. Unpaired, two-tailed t-test.",
    "molecules": "A42, ATP"
  },
  {
    "caption": "(C) Relative protein abundance of EGFP-Abeta42 (A42) versus empty vector (ev) expressing wild type yeast cells. The significant subset of cytosolic heat shock proteins (HSP) detected in a proteomics analysis of isolated mitochondria is depicted. Log2 SILAC ratios of A42/ev of two independent proteome measurements are shown. Dot plots show all data points along with the mean (line). Significance was determined using an outlier test (Significance A, p <0.003).",
    "molecules": "A42, Abeta42"
  },
  {
    "caption": "(D) Screen of 123 deletion strains assessing Abeta42 (A42)-induced toxicity (assessed by DHE>Eth. positive cells indicative of the sum of sub-lethal oxidative stress and cell death) in yeast. Relative A42-toxicity normalized to wild type (WT) cells is depicted. Potential hits (mutants that reduce A42 toxicity) are shown in green. Data represent means ± s.d.. n ≥ 3 biologically independent cultures.",
    "molecules": "A42, Abeta42, DHE, Eth"
  },
  {
    "caption": "Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP-A42 (A42) or EGFP empty vector (ev) of wild type (WT) (A) and YDJ1 deleted (Δydj1) cells (B). Mean ± s.d.. n = 5 biologically independent cultures. Comparisons by two-way ANOVA (mixed-design) followed by simple main effects (***, p < 0.001, vs. control).",
    "molecules": "A42, PI"
  },
  {
    "caption": "Immunoblot of whole cell extract (Input) and eluate of FLAG-tagged Ydj1. Immunoprecipitation (IP: FLAG) of YDJ1 deletion strain (Δydj1) cells expressing EGFP-A42 (A42) or EGFP empty vector (ev) and co-overexpressing Ydj1-FLAG using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).",
    "molecules": "A42"
  },
  {
    "caption": "Confocal microscopy of wild type (WT) yeast cells expressing EGFP-A42 (A42) only and co-overexpressing Ydj1-FLAG or with the corresponding vector control after 18 h of expression. Colors indicate fluorescence intensity.",
    "molecules": "A42"
  },
  {
    "caption": "Confocal microscopy of wild type (WT) yeast cells expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG after 18 h of expression. Mitochondria were visualized with MitoTracker Red (magenta). See also Fig EV2J, EV1C.",
    "molecules": "A42, MitoTracker Red"
  },
  {
    "caption": "Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.) and cytosolic (Cyt.) fractions of wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Tom22-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.",
    "molecules": "A42"
  },
  {
    "caption": "Ratio of full length EGFP-A42 in the mitochondrial fraction / cytosolic fraction, using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three/4 independent experiments. Dot plots show all data points along with the mean (line/bar) ± s.d.. n = 3-4 biologically independent cultures. *, p < 0.05. Unpaired, two-tailed t-test.",
    "molecules": "A42"
  },
  {
    "caption": "Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity was measured at two time points (t0 and t2, 2 h after CH administration) and normalized to t0 in wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 8 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc. See also Appendix Fig S2D.",
    "molecules": "A42, CH, cycloheximide"
  },
  {
    "caption": "Quantification of the ratio between EGFP-A42 tetramer and monomer in wild type (WT) and YDJ1 deletion strain (Δydj1) expressing EGFP-A42 (J) as well as between WT expressing EGFP-A42 only and co-overexpressing Ydj1-FLAG (K) at indicated time points. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8(J) n=12(K) biologically independent cultures. ***, p < 0.001; **, p < 0.01; *, p < 0.05. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2A-B.",
    "molecules": "A42"
  },
  {
    "caption": "Quantification of propidium iodide (PI)-staining positive cells indicating cell death at indicated time points of expression of EGFP-A42 (A42) Δydj1 cells with co-expression of DnaJA1-FLAG (DnaJA1) (B) or corresponding empty vector controls (ev) (A). Mean ± SD n = 4-6 biologically independent cultures. Comparisons by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus control).",
    "molecules": "A42, PI"
  },
  {
    "caption": "(C)Immunoblot (WB) of whole-cell extract (Input) and eluate of FLAG-tagged DnaJA1 immunoprecipitation (IP: FLAG) of wild-type strain (WT) expressing EGFP-A42 (A42) and co-expressing DnaJA1-FLAG (A1) using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).",
    "molecules": "A42"
  },
  {
    "caption": "Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity of YDJ1 deletion strain (Δydj1) and wild type (WT) cells expressing EGFP-A42 (A42) only or co-expressing DnaJA1 was measured at two time points (t0 and t2) and normalized to t0. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2C.",
    "molecules": "A42, CH, cycloheximide"
  },
  {
    "caption": "Immunoblot (WB) of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.), and cytosolic (Cyt.) fractions of YDJ1-deleted cells (Δydj1) after 18 h expression of EGFP-A42 (A42) only or co-expressing DnaJA1-FLAG (DnaJA1) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time (Short exp.) exposure (two sections from one immunoblot). Cox4-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.",
    "molecules": "A42"
  },
  {
    "caption": "Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 (A1) and quantified by densitometry in (G).",
    "molecules": "Abeta42"
  },
  {
    "caption": "Quantification of the oligomer-to-monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in (F). Dot plots show all data points along with the mean (line) ± SD n = 3 biologically independent cultures. P-values by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus A42 control). See also Appendix Fig S2D.",
    "molecules": "A42, Abeta42"
  },
  {
    "caption": "A42 beta-sheet-rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ. Data represent means of at least eight measurements. See also Appendix Fig S1E.",
    "molecules": "A42, ThT"
  },
  {
    "caption": "(A) Immunoblot of cytosol-enriched cerebral tissue homogenate (Input) and eluate of Abeta42 or DnaJA1 using Abeta-specific antibody (6E10) and DnaJA1-specific antibody (DnaJA1), respectively, showing optimal and long exposure (Long exp.). Immunoprecipitation using magnetic beads without antibody, DnaJA1 or 6E10 antibody of female (15 months old) wild type (WT) or 3xTg (PS1M146V/APPSwe/tauP301L) mice. Sections showing monomer and low-n oligomers, dodecamer and full-length amyloid precursor protein (APP) are from 1 immunoblot.",
    "molecules": "Abeta42"
  },
  {
    "caption": "(B) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). PonceauS serves as a loading control. (C) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). Representative immunoblot is shown in (B). Dot plots show all data points along with the mean (bar) ± s.d.. n = 4-5 independent patients. *, p < 0.05. Unpaired, two-tailed t-test.",
    "molecules": "PonceauS"
  },
  {
    "caption": "(A) Representative confocal microscopy of 10-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).",
    "molecules": "A42, Abeta42, DAPI"
  },
  {
    "caption": "(B) Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).",
    "molecules": "A42, Abeta42, DAPI"
  },
  {
    "caption": "(E-F) qPCR analysis of Droj2-mRNA levels of 3-6 days old female (E) and male (F) flies expressing human Abeta42 (UAS-A42) of w1118 wild type flies (Droj2+/+) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression (Droj2+/+ ctrl). Reference gene is Rpl32. Dot plots show all data points along with the mean (line) ± s.d.. n = 3. **, p < 0.01. One sample t-test against 1.",
    "molecules": "A42, Abeta42"
  },
  {
    "caption": "(G) Immunoprecipitation (IP: 6E10) of w1118 wild type flies (Droj2+/+) using synthetic Abeta42 added to the fly head extract (Input). Abeta-specific antibody 6E10 was used for Abeta42 and DnaJA1-specific antibody for Droj2 immunoblot detection. Showing input, supernatant, supernatant after washing (WS) and eluate.",
    "molecules": "Abeta42"
  },
  {
    "caption": "(A-B) Survival of female (A) and male (B) w1118 wild type flies (Droj2+/+) and Droj2 knockdown flies (Droj2+/-) with expression of human Abeta42 (UAS-A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl2. Survival was determined at indicated time points. n = 6 with 100-120 flies per experiment. The indicated p-value refers to the interaction (int.) term of a Cox Proportional Hazards model comparing Abeta42 toxicity (UAS-A42 vs. ctrl) and Droj2 expression (Droj2+/+ vs. Droj2+/-) as main factors. The following pairwise comparisons of the indicated groups survival were done by Log Rank test (****, p < 0.0001; ns, p > 0.05).",
    "molecules": "A42, Abeta42, MnCl2, sucrose"
  },
  {
    "caption": "(C) Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies (Droj2+/-) and corresponding isogenic w1118 wild type flies (Droj2+/+) both expressing human Abeta42 (UAS-A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6. *, p < 0.05. Unpaired, two-tailed t-test.",
    "molecules": "A42, Abeta42"
  },
  {
    "caption": "(D) Representative confocal and gSTED deconvolved images of Kenyon cells in 15-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A-specific antibody (ATP5A, green) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).",
    "molecules": "A42, Abeta42"
  },
  {
    "caption": "A Turbidity and bright-field microscope images of indicated groups (left) and the quantitative analysis of turbidity  of samples were measured by absorbance at 600 nm (right). 10 μM cGAS, 5 μM G3BP1, 5 μM  BSA and 100 nM dsDNA  (60 bp) were used in these assays. n = 3 biological replicates. Data information: Representative images are shown Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. Scale bars, 10 μm",
    "molecules": "dsDNA"
  },
  {
    "caption": "F FRAP analysis of DNA-induced droplets of cGAS (1 μM  dsDNA, 10 μM cGAS-mCherry), the yellow dotted circle indicated the region of photobleaching. G FRAP curve of (F). n = 3 biological replicates. H  FRAP analysis of cGAS-G3BP1 droplets. 10 μM  cGAS-mCherry and 5 μM G3BP1 were used. The yellow dotted circle indicated the region of photobleaching. I FRAP curve of (H). n = 3 biological replicates. Data information: Representative images are shown (A, Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. FRAP, Fluorescence recovery after photobleaching. Scale bars, 2 μm (F and H).",
    "molecules": "DNA, dsDNA"
  },
  {
    "caption": "B  Immunofluorescent staining of cGAS in both WT and G3BP1-/- U937 cells. 3D images were reconstituted with Leica LAS X software. C Quantitative analysis of total cGAS puncta number (left) and volume (right) per cell of (B). n = 51 cells. D Data information: Representative images are shown Scale bars, 5 μm.  Hoechst (blue), nuclear staining. Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type.",
    "molecules": "Hoechst"
  },
  {
    "caption": "F ELISA analysis of the secreted IFN-β in HT-DNA-treated WT or G3BP1-/- U937  cells. n = 3 biological replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. HT-DNA, Herring testes DNA.",
    "molecules": "Herring testes DNA, HT-DNA"
  },
  {
    "caption": "G  cGAMP production in ISD-treated WT or G3BP1-/- U937 cells were analyzed by  LC-MS/MRM. ND, not detected. n = 3 technical replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type.  ISD,  interferon stimulatory DNA.",
    "molecules": "interferon stimulatory DNA, ISD, cGAMP"
  },
  {
    "caption": "D cGAS, G3BP1 and Cy5-dsDNA  were incubated. A quantitative analysis of a representative  cGAS-DNA droplet  is shown. Along the white line on the merged image, the fluorescence intensity of  G3BP1, cGAS and dsDNA  channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsDNA were used in the assays.",
    "molecules": "Cy5, DNA, dsDNA"
  },
  {
    "caption": "F cGAS, G3BP1 and Cy5-dsRNA were incubated. Similar quantification analysis as in D was performed. Data information: Representative images are shown Scale bars, 5 μm 45 μM cGAS-mCherry, 10 μM  G3BP1-mEGFP, 2 μM dsRNA  were used in the assays.",
    "molecules": "Cy5, dsRNA"
  },
  {
    "caption": "A, B Recombinant cGAS (10 μM) and the indicated amounts of Cy5-labeled dsDNA were incubated, with or without G3BP1  (2 μM). The  DNA-induced cGAS-DNA liquid droplets  were analyzed with time-lapse imaging. Data information: Representative images are shown Scale bars, 10 μm",
    "molecules": "Cy5, DNA, dsDNA"
  },
  {
    "caption": "D cGAMP production assays. Recombinant cGAS (10 μM) was incubated with the indicated amounts of dsDNA, with or without G3BP1 (10 μM). The produced cGAMP was quantitatively analyzed by  LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.",
    "molecules": "cGAMP, dsDNA"
  },
  {
    "caption": "G qPCR analysis of  IFNB mRNA levels in WT and G3BP1-/- U937 cells transfected with dsDNA as indicated. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.",
    "molecules": "dsDNA"
  },
  {
    "caption": "D, Time-lapse imaging of DNA-induced LLPS of cGAS, in the presence of full length or truncated G3BP1 (2 μM) n = 3 biological replicates.10 μM cGAS, 100 nM dsDNA were used in the assay Data information: Representative images are shown Scale bars, 10 μm",
    "molecules": "DNA, dsDNA"
  },
  {
    "caption": "A Bright-field microscope images of indicated groups (left) and a quantitative analysis of total area of droplets  (right). n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 50 μM KCl, 50 μM MgCl2, 50 μM  ZnCl2  were used. Data information: Representative images are shown Scale bars, 10 μm. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.",
    "molecules": "MgCl2, KCl, ZnCl2"
  },
  {
    "caption": "C Fluorescent images and analysis of  DNA-induced cGAS LLPS in the presence of the indicated reagent. n = 3 biological replicates. 10 μM cGAS, 50 μM of each ion and 100 nM  dsDNA  were used in these assays. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst  (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.",
    "molecules": "Hoechst, DNA, dsDNA"
  },
  {
    "caption": "D Fluorescent images and analysis of DNA-induced LLPS of cGAS in the presence of different amounts of ZnCl2. n = 3 biological replicates. 10 μM cGAS and 100 nM  dsDNA were used in the assay. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst  (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.",
    "molecules": "Hoechst, DNA, dsDNA, ZnCl2"
  },
  {
    "caption": "E Fluorescent images and analysis of DNA-induced  LLPS of  cGAS in the presence of G3BP1 (2 μM) and/or  ZnCl2  (20 μM) as indicated. n = 3 biological replicates. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.",
    "molecules": "Hoechst, DNA, ZnCl2"
  },
  {
    "caption": "A Fluorescent images of cGAS-G3BP1 droplets in the presence or absence of 10 μM  EGCG (left). The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background (right), n = 3 biological replicates. 45 μM cGAS-mCherry and 10 μM G3BP1-mEGFP were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.",
    "molecules": "EGCG"
  },
  {
    "caption": "D Fluorescent images of DNA-induced LLPS  of cGAS, with or without G3BP1, in the absence or presence of 10 μM EGCG (left). The partition coefficient was calculated as in (A), n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.",
    "molecules": "EGCG, DNA, dsDNA"
  },
  {
    "caption": "E cGAMP production assays. Recombinant  cGAS (10 μM) and G3BP1 (10 μM) were incubated, in the presence or absence of dsDNA  (200 nM), with or without EGCG (20 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.",
    "molecules": "EGCG, cGAMP, dsDNA"
  },
  {
    "caption": "F  cGAS-G3BP1 interaction analysis. cGAS was precipitated using anti-cGAS  antibodies in U937  cells treated with EGCG as indicated. IgG served as control. The cGAS-G3BP1 interaction was further analyzed by immunoblotting. Data information: Representative images are shown WCL, whole cell lysate. IP, immunoprecipitation.",
    "molecules": "EGCG"
  },
  {
    "caption": "(E) Cells, either untreated or treated with chloroquine for 24 h, were fixed and stained with anti‐Ret (green) and DAPI (blue). Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma; siRNA, short interfering RNA.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(A) FAK+/+ cells were treated with dasatinib, then lysates were immunoblotted with anti‐FAK, anti‐FAK Y397, anti‐FAK Y925, anti‐PY416 Src, anti‐Src and anti‐actin. FAK was immunoprecipitated and then immunoblotting performed using anti‐Src, anti‐FAK or anti‐Ret.",
    "molecules": "dasatinib"
  },
  {
    "caption": "(B) Cells were treated with dasatinib, then fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (C) FAK−/− cells treated with dasatinib were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions, while broken arrows show lack of co‐localization in puncta. Scale bars, 20 μM. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3).",
    "molecules": "dasatinib"
  },
  {
    "caption": "(D) Lysates from untreated and dasatinib‐treated FAK−/− cells were immunoblotted with anti‐Ret, anti‐PY416 Src, anti‐Src and anti‐actin. Ret was also immunoprecipitated from cells and then immunoblotted using anti‐Src and anti‐Ret. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection.",
    "molecules": "dasatinib"
  },
  {
    "caption": "Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm.",
    "molecules": "CF640R WGA"
  },
  {
    "caption": "Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm.",
    "molecules": "CF640R WGA"
  },
  {
    "caption": "(A) Toluidine blue stainings of semi-thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss. Scale bar=100µm, OC: Organ of Corti.",
    "molecules": "Toluidine blue"
  },
  {
    "caption": "(A-B) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown. (A) HEK293T cells transfected with siRNAs for Elp3, Alkbh8 or control and immunolabelled with LGN, acetylated alpha-tubulin and DAPI. Scale bar=5µm. (B) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified (siRNA Control/Elp3/Alkbh8: n=27/30/27; pooled from 3 independent experiments; mean±SEM).",
    "molecules": "DAPI"
  },
  {
    "caption": "(C-D) The length of the primary cilium is reduced in non-dividing HEK293T cells upon Elp3 or Albh8 knockdown. (C) Control, Elp3 or Alkbh8 siRNA-treated HEK293T cells were immunolabelled for basal body marker ALMS1, axoneme marker Arl13b and DAPI. Scale bar=5µm. (D) Primary cilium length of non-dividing HEK293T was measured and pooled data were plotted (siRNA Control/Elp3/Alkbh8: n=94/81/90; data collected from 3 independent experiments; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, F=21.46, DF=4; minimum, first quartile, median, third quartile and maximum presented).",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Increased formation of aggresome-like structures in Elp3- or Alkbh8-depleted HEK293T. Aggresomes (red); DAPI (blue). Enlarged images depict examples of non-dividing (left) and dividing (right) cells. Scale bar=10µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (H) HEK293T cells treated with DMSO vehicle or MG-132 and stained for aggresomes (left panels) or LGN, F-actin and DAPI (green, red and blue, respectively in right panels). Scale bar=10µm.",
    "molecules": "DAPI, DMSO, MG-132"
  },
  {
    "caption": "Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (I) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells. Mean fluorescence values per percentage of cell length with SEM are plotted. Fluorescence intensity profiles collected from 3 independent experiments (DMSO/MG-132: n= 24/24).",
    "molecules": "DMSO, MG-132"
  },
  {
    "caption": "(J) Proteostat staining of MG132-treated whole-mounted P0 organs of Corti reveals the presence of aggresome-like structures in HCs. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of MG132-treated HCs. Scale bars=5µm. #IHC; *OHC1; **OHC2; ***OHC3",
    "molecules": "MG132"
  },
  {
    "caption": "(K) Localization of LGN at the lateral apical surface of E16.5 WT HCs is disrupted by MG-132. LGN (white/green) and F-actin (red). Scale bars=5µm; magnified views=2µm.",
    "molecules": "MG-132"
  },
  {
    "caption": "(C) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion. HEK293T transfected with Elp3 siRNA were co-stained for aggresomes, Dapi and LGN or Gαi3. Scale bar=5µm.",
    "molecules": "Dapi"
  },
  {
    "caption": "Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (D): DMSO/MG-132/MG-132+4PBA n=555/337/453, pooled from 3 experiments, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.",
    "molecules": "4-PBA, 4PBA, DMSO, LysoTracker, MG-132"
  },
  {
    "caption": "Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (E): siCtl/+4PBA n=622/677; siElp3/+4PBA n=674/577; siAlkbh8/+4PBA n=460/484, pooled from 4 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.",
    "molecules": "4-PBA, 4PBA, LysoTracker"
  },
  {
    "caption": "Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (F): DMSO/MG-132/MG-132+4PBA n=210/225/266, pooled from 3 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.",
    "molecules": "4-PBA, 4PBA, DMSO, LysoTracker, MG-132"
  },
  {
    "caption": "Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (G): WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA n=732/763/730/738, pooled from 4 OCs per condition, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.",
    "molecules": "4-PBA, 4PBA, LysoTracker"
  },
  {
    "caption": "Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (A) LGN and F-actin immunostainings of E16.5+1DIV OCs reveal that MG-132 perturbs LGN distribution at the apical surface of HCs, while co-treatment with 4-PBA ameliorates its enrichment at the lateral edge. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3",
    "molecules": "4-PBA, MG-132"
  },
  {
    "caption": "Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (B) Acetylated alpha-tubulin immunostaining reveals that the kinocilium is centrally shifted by MG-132 and partially restored by 4-PBA in E16.5+1DIV HCs (yellow arrowheads = kinocilium base; scale bar=2µm).",
    "molecules": "4-PBA, MG-132"
  },
  {
    "caption": "Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (C) Kinocilium position plotted as a fraction of HC diameter for each HC row (DMSO/MG-132/MG-132+4PBA IHC: n=57/67/53, ***p<0.001, F=34.52, DF=2; OHC1: n=61/66/53, ***p<0.001, F=57.88, DF=2; OHC2: n=55/73/58, ***p<0.001, F=124.7, DF=2; OHC3: n=48/66/43, ***p<0.001, F=67.16, DF=2; pooled from 4 explants per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).",
    "molecules": "4-PBA, 4PBA, DMSO, MG-132"
  },
  {
    "caption": "In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (D) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3",
    "molecules": "4-PBA, 4PBA"
  },
  {
    "caption": "In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (E) Acetylated alpha-tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4-PBA (yellow arrowheads = base of kinocilium; scale bar=2µm).",
    "molecules": "4-PBA"
  },
  {
    "caption": "In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (F) Kinocilium position plotted as a fraction of HC diameter for each HC row (WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA IHC: n=35/31/35/36, p=0.6812, F=4.510, DF=3; OHC1: n=36/32/37/40, ***p<0.001, **p<0.01, F=27.60, DF=3; OHC2: n=35/33/38/42, ***p<0.001, F=76.62, DF=3; OHC3: n=36/32/38/39, ***p<0.001, *p<0.05, F=27.86, DF=3; pooled from 3 animals per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).",
    "molecules": "4-PBA, 4PBA"
  },
  {
    "caption": "(C) Quantification of Iba1+ macrophages in IHC sections of 99LN-BrM at trial endpoint (Ctrl n=27, WBRT n=18). (D) Representative IF images of 99LN-BrM stained for the macrophage marker Iba1 (red) and the MG marker Tmem119 (white). Higher magnification images present areas in the peri-tumor region and in the tumor core. DAPI was used as nuclear counterstain. Scale bar; 100 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Representative images (and details from boxed regions) of WT MEF  cells transfected with   YFPParkin (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one stage in the process of  mitophagy that has been used to score the progression of mitophagy at different  time-points for the different conditions tested (the specific time-point of each example is indicated in the  image). From top to bottom: i) \"Diffuse Parkin\" defines cells in where  Parkin expression is homogenously distributed throughout the cytoplasm (quantified in B). ii) Soon after  mitochondrial  damage  Parkin appears enriched in small \"puncta\" throughout the cytoplasm localizing within isolated mitochondria (quantified in C). iii) Later in the process these puncta structures aggregate in bigger structures accumulating in perinuclear  regions (quantified in D). iv) The process of Parkin translocation eventually affects all the mitochondrial population and usually collapses in few large structures possibly forming part of big autolysosomes in the perinuclear region (quantified in D). Scoring was performed from 3 independent experiments (n=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01",
    "molecules": "FCCP"
  },
  {
    "caption": "(F) Representative images (and details from boxed regions) of WT MEFs cells transfected with GFPPINK1 (green) and treated with FCCP (10 μM) to induce  mitophagy. Cells were immunostained  with anti-Tom20 (magenta) to reveal the mitochondrial  network. Each row represents one of the three categories of  GFP signal used to describe  PINK1 translocation to  mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and  MiroDKO cells at different time-points shortly after mitochondrial insult (G). Data collected from 3 independent experiments (n=3). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01 ",
    "molecules": "FCCP"
  },
  {
    "caption": "B) Representative  images showing full length control and selected Miro1 lysine mutants (Miro15R, Miro1allR and Miro1R572K) expressing in MiroDKO MEF  cells. All constructs (myc-tag, green) localise to mitochondria (Tom20, red).",
    "molecules": "lysine"
  },
  {
    "caption": "C) Immunoblot showing efficient expression of  Miro1 lysine mutants in   FlagParkin expressing SH-SY5Y cells.",
    "molecules": "lysine"
  },
  {
    "caption": "D) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon  FCCP treatment (1h, 10 μM) in FlagParkin overexpressing SH-SY5Y. The effect is quantified in (E) (n=4 independent experiments for Miro1WT and Miro15R and n=3 for  Miro1allR and Miro1R572K, ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.",
    "molecules": "FCCP, lysine"
  },
  {
    "caption": "F) Representative western blot showing a degradation assay in FlagParkin overexpressing SH-SY5Y  cells transfected with  Miro1WT, Miro15R or Miro1allR constructs and treated with FCCP  (10 μM) for 3 or 6 hours. Quantification of Miro1 levels (G) the matrix protein PDH E1α in (H) and  Parkin levels (I) (n=4 independent experiments; ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001. ",
    "molecules": "FCCP"
  },
  {
    "caption": "(A) Representative images at 3 hours of FCCP treatment (10 μM) of YFPParkin  (green) expressing  MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. A detail of YFPParkin translocation onto the mitochondrial  network is also shown for each condition.",
    "molecules": "FCCP"
  },
  {
    "caption": "(B and C) Quantification of the  fraction of cells showing advanced stages of the mitophagic process  (cells showing mitochondrial aggregation due to advanced  Parkin translocation or the complete translocation of  Parkin onto all the mitochondrial  network) at 1 hour (B) or 3 hours (C) after  FCCP treatment (data collected from at least 3 independent experiments: WT n=7,  MiroDKO n=8, +mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; mycMiro2=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001",
    "molecules": "FCCP"
  },
  {
    "caption": "D) Western blots and quantification of  Miro1 degradation in MiroDKO cells co-expressing YFPParkin and the indicated  myc-tagged Miro1 constructs and treated with  FCCP (10 μM) for the indicated times  (n=3 different experiments; 2-way-ANOVA with Dunnett post test; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs, comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black).",
    "molecules": "FCCP"
  },
  {
    "caption": "E). Representative  images at 24 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the  mitochondrial  network. Expression of both ubiquitin mutants (Miro15R and Miro1allR - arrowheads) protects from the mitophagic clearance of  mitochondria  (arrows) after damage. (F) Quantification of the fraction  of cells showing the complete loss of  Tom20 signal of mitochondria  after 24 hours of FCCP  treatment (data collected from at least 3 independent experiments: MiroDKO n=8, mycMiro1WT=7,   mycMiro15R=3, mycMiro1allR=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001 ",
    "molecules": "FCCP, ubiquitin"
  },
  {
    "caption": "(A) Representative  confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed  after valinomycin treatment at the indicated  time points (scale bars = 5 µm).",
    "molecules": "valinomycin"
  },
  {
    "caption": "(B) Airyscan confocal images  of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal  processes (scale bars = 2.5 µm).",
    "molecules": "valinomycin"
  },
  {
    "caption": "(C)  Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment (lines shown in A).",
    "molecules": "valinomycin"
  },
  {
    "caption": "(D) Quantification of Parkin recruitment to mitochondria (YFPParkin signal overlapping MtDsRed  signal - intensity-adjusted and normalised to t=0) in WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001",
    "molecules": "valinomycin"
  },
  {
    "caption": "(E) Quantification of mitochondrial   occupancy (area of MtDsRed signal in soma / entire area of soma) in the somas of WT and Miro1KO neurons following  valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA).",
    "molecules": "valinomycin"
  },
  {
    "caption": "(F) Quantification of mitochondrial   clearance in  somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment (n=15 cells all conditions per genotype over 3  neuronal preparations; Unpaired t-test).",
    "molecules": "valinomycin"
  },
  {
    "caption": "(G) Representative confocal images of WT,  Miro1KO and PINK1KO cortical neurons immunostained  with MAP2  (cyan) and s65-phospho-ubiquitin (green) after valinomycin treatment (Scale bars = 20µm). (H) Quantification of s65-phospho-ubiquitin signal  intensity within  MAP2 signal (normalised to MAP2 area and t=0) in Miro1WT, Miro1KO and PINK1KO neurons following valinomycin treatment n= 3, 4 and 3  embryos from WT, Miro1KO and PINK1KO embryos, respectively (6 ROIs per condition; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001",
    "molecules": "ubiquitin, valinomycin"
  },
  {
    "caption": "(I) Representative  confocal images of the  soma  of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1,   YFPParkin and MtDsRed without valinomycin treatment (Scale bars = 10 µm). Quantification of parkin colocalization with MtDsRed (YFPParkin signal overlapping MtDsRed  signal - intensity-adjusted) in the soma of Miro1KO and PINK1KO cortical neurons (n=12 cells all conditions per genotype over 3 neuronal preparations; one-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001",
    "molecules": "valinomycin"
  },
  {
    "caption": "(J) Representative confocal images of the soma of  Miro1KO cortical neurons expressing WT and mutant forms of  Miro1, YFPParkin and  MtDsRed after 5 hours of valinomycin treatment (Scale bars = 10 µm). Quantification of  Parkin recruitment to mitochondria (Normalised to t=0) in  Miro1KO and PINK1KO cortical neurons following  valinomycin treatment (n=12 cells all conditions per genotype over 3 neuronal preparations, two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001 ",
    "molecules": "valinomycin"
  },
  {
    "caption": "C) Representative images of  hippocampal regions from control and Miro1CKO crossed with MitoDendra animals.",
    "molecules": "MitoDendra"
  },
  {
    "caption": "(C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month  mitoDendra-crossed WT and Miro1CKO mice  (n=4 mice per genotype; unpaired t-test).",
    "molecules": "mitoDendra"
  },
  {
    "caption": "(E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without  megamitochondria.",
    "molecules": "mitoDendra"
  },
  {
    "caption": "(F) Example zoom images of  neuronal somas with and without megamitochondria from cortical regions of 12-month  mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from  neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).",
    "molecules": "mitoDendra"
  },
  {
    "caption": "(C-F) Two months after a single tail vein injection of rAAV, wild-type mice showed similar body weight (C), blood glucose levels (D), serum alanine aminotransferase (ALT) (E) and aspartate aminotransferase (AST) activities (F) with the mice injected with PBS. n = 8 - 10 for each group.",
    "molecules": "glucose, PBS"
  },
  {
    "caption": "(H-I) Two months after a single injection of rAAV, db/db mice showed similar body weight (H) and blood glucose levels (I) with the mice injected with PBS. n = 10 for each group. (J-K) Serum ALT (J) and AST (K) of mice in (H).",
    "molecules": "glucose, PBS"
  },
  {
    "caption": "(M) The hepatic p-MLKL protein level in normal and necrotic area from mice injected with PBS or rAAV in (H). Ncp, Necroptosis. n = 7 - 8 biological replicates.",
    "molecules": "PBS"
  },
  {
    "caption": "(N) The hepatic mRNA levels of Il-6, Il-1β, Tnf-α and Mcp-1 involved in necroptosis at normal or necroptotic area from mice injected with PBS or rAAV in (H). n = 7 - 8 biological replicates.",
    "molecules": "PBS"
  },
  {
    "caption": "(Q-R) Six months after a single injection of rAAV, db/db mice showed decreased body weight (Q) and similar blood glucose levels (R) compared to the mice injected with PBS. n = 8 - 12. (S-T) Serum ALT (S) and AST (T) activities of mice in (Q). n = 8 - 10.",
    "molecules": "glucose, PBS"
  },
  {
    "caption": "Body weight (B) were measured 6 weeks later, after injection once a day for 5 consecutive days with vehicle (Veh) or streptozotocin (STZ). n = 6 - 19 for each group.",
    "molecules": "streptozotocin, STZ"
  },
  {
    "caption": "blood glucose levels (C) were measured 6 weeks later, after injection once a day for 5 consecutive days with vehicle (Veh) or streptozotocin (STZ). n = 6 - 19 for each group.",
    "molecules": "glucose, streptozotocin, STZ"
  },
  {
    "caption": "Two months after a single injection of rAAV, hyperglycemic mice induced by streptozotocin showed , blood glucose levels (F) compared with those mice injected with PBS. n = 9 (G-H) Serum ALT (G) and AST (H) activities of mice in (D).",
    "molecules": "glucose, PBS, streptozotocin"
  },
  {
    "caption": "blood glucose levels (Q) of obese and euglycemic mice in (L) after a single injection of PBS or rAAV for 2 months. n = 6. Serum ALT (R) of mice in (O).",
    "molecules": "glucose, PBS"
  },
  {
    "caption": "Two months after a single injection of PBS or rAAV blood glucose levels (G) of hyperglycemic and obese mice in (B) were not significantly changed. n = 10 - 11 for each group. (H-I) Serum ALT (H) and AST (I) activities of mice in (E).",
    "molecules": "glucose, PBS"
  },
  {
    "caption": "(K) The hepatic p-MLKL protein level in normal or necrotic area from mice injected with PBS or rAAV in (E).",
    "molecules": "PBS"
  },
  {
    "caption": "blood glucose levels (O) of hyperglycemic and obese mice after a single injection of PBS or rAAV for 6 months. n = 8. (P-Q) Serum ALT (P) and AST (Q) activities of mice in (M).",
    "molecules": "glucose, PBS"
  },
  {
    "caption": "(A) Prednisone treatment markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly(I:C) (pIC). n = 3 in duplicate.",
    "molecules": "pIC, poly(I:C), Prednisone"
  },
  {
    "caption": "Prednisone treatment markedly blocked the increase of p-MLKL level (C, n = 3 in duplicate.) of macrophages induced by poly(I:C) and z-VAD-fmk (zVAD). Scar bar, 100 μm.",
    "molecules": "poly(I:C), Prednisone, z-VAD-fmk, zVAD"
  },
  {
    "caption": "Two months after a single injection of rAAV-si-Pebp1, HFD-induced hyperglycemic and obese mice showed blood glucose levels (C) with the mice injected with rAAV-si-NC. n = 10 - 11. (D-E) Serum ALT (D) and AST (E) activities from mice in (B). The colored dots indicate the mice with high ALT or AST activity, and the same color dot in (D) and (E) indicates the same mouse.",
    "molecules": "glucose"
  },
  {
    "caption": "(A) The effect of si-Pebp1 in primary mouse macrophages. n = 3. (B) Knockdown of Pebp1 markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly(I:C). n = 6.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "Knockdown of Pebp1 markedly blocked the loss of cell membrane integrity (C, n = 6 in duplicate or triplicate.) of mouse macrophages induced by poly(I:C) and z-VAD-fmk.",
    "molecules": "poly(I:C), z-VAD-fmk"
  },
  {
    "caption": "(E) Knockdown of Pebp1 attenuated the increase of p-Tbk1 level induced by poly(I:C). n = 3 biological replicates.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "Knockdown of Tbk1 markedly blocked the loss of cell membrane integrity (H of mouse macrophages induced by poly(I:C) and z-VAD-fmk.",
    "molecules": "poly(I:C), z-VAD-fmk"
  },
  {
    "caption": "After a single injection of rAAV-si-Tbk1 for 11 weeks, the db/db mice showed blood glucose levels (M) with the mice injected with rAAV-si-NC. n = 11. (N-O) Serum ALT (N) and AST (O) activities of mice in (K).",
    "molecules": "glucose"
  },
  {
    "caption": "E Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function in GA of mice receiving PBS or indicated EVs. Data information: Western blot signals are quantified and normalized to Gapdh.",
    "molecules": "PBS"
  },
  {
    "caption": "F RT-qPCR showing relative levels of miR-122 and indicated mRNAs in GA of indicated groups of mice. Data were normalized to U6 (for miR-122) or Ppib (for all mRNAs) and compared to control group receiving PBS (one-way ANOVA, n=4 or 5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.",
    "molecules": "PBS"
  },
  {
    "caption": "H ROS levels in GA from indicated groups of mice detected by DHE staining (red). DAPI (blue) shows the nuclei. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.",
    "molecules": "DAPI, DHE, ROS"
  },
  {
    "caption": "I ROS levels in GA from mice that had received indicated EV treatment for 5 weeks. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.",
    "molecules": "DAPI, DHE, ROS"
  },
  {
    "caption": "D CFSE signals in C2C12 myotubes treated with indicated CFSE-labeled Evs for 24 h, indicating EV uptake. Scale bar: 100 µm. Data information: In bar graph, values are shown as mean ± SD. **P<0.01, ns: not significant.",
    "molecules": "CFSE"
  },
  {
    "caption": "C Relative ATP levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.",
    "molecules": "ATP"
  },
  {
    "caption": "D Relative ROS levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3-5 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.",
    "molecules": "ROS"
  },
  {
    "caption": "A GA were collected from female NSG mice that had received indicated EVs or PBS for 5 weeks. Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function. Data information: Western blot signals are quantified and normalized to Gapdh.",
    "molecules": "PBS"
  },
  {
    "caption": "C RT-qPCR showing indicated mRNAs in GA of indicated groups of mice. Data were normalized to Ppib and compared to control group receiving PBS (one-way ANOVA, n=4 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ns: not significant.",
    "molecules": "PBS"
  },
  {
    "caption": "AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. E ROS levels in GA from indicated groups of mice. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (two-tailed Student's t-test, n=4 mice per group). Scale bar: 50 µm. Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.",
    "molecules": "DAPI, DHE, ROS"
  },
  {
    "caption": "c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3).",
    "molecules": "qVD-oph, DMSO"
  },
  {
    "caption": "e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol.",
    "molecules": "qVD-oph, tempol, BHT, butylated hydroxytoluene, CAT, catalase, ROS"
  },
  {
    "caption": "f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophagecytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm.",
    "molecules": "iron, Iron"
  },
  {
    "caption": "a, Ter119+CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining.",
    "molecules": "PHZ"
  },
  {
    "caption": "b, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 µm.",
    "molecules": "PHZ"
  },
  {
    "caption": "c, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 µm. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; Nix-/-, 0.44 ± 0.036 (n = 35, P = 0.0006).",
    "molecules": "PHZ"
  },
  {
    "caption": "d, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy. Scale bar, 0.5 µm.",
    "molecules": "PHZ"
  },
  {
    "caption": "a, Ter119+CD71+ reticulocytes were sorted from the peripheral blood of wild-type and Nix-/- mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119.",
    "molecules": "ABT-737, FCCP, PHZ"
  },
  {
    "caption": "b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 µm.",
    "molecules": "ABT-737, FCCP"
  },
  {
    "caption": "B Representative pictures of P28 animals that received BrdU   at P3, P7, P14 or P21. For cell type identification of generated progeny, immunostainings  against NESTIN  (white), GFAP  (green), and BrdU (magenta) were performed. rNSCs were identified as NESTIN+/GFAP+/BrdU+ with a radial process (as indicated with arrows), neurons  as NESTIN-/GFAP-/BrdU+ cells, and astrocytes  as NESTIN-/GFAP+/BrdU+ cells in the hilus, GZ, and ML  (arrowheads). Data information: dotted lines label GZ  borders; scale bars = 50 µm.",
    "molecules": "BrdU"
  },
  {
    "caption": "B Confocal  image of an adult DG  populated by distinct cells identified by cell type-specific  protein expression and morphology. All cells newly-generated within the 12 days incorporated BrdU  (magenta);  rNSCs were identified by expression  of SOX2  (cyan), localization  of their  cell bodies to the SGZ, and a GFAP+ radial process (green); IPCs  were located  within the SGZ  and express SOX2  only (cyan); neuroblasts  in the SGZ  and GZ  were labelled by DCX  (white; arrows); astrocytes  in the hilus, GZ, and ML  (arrowheads) expressed SOX2  (cyan). C Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DG. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C n = 5 ; dotted lines border SGZ  and GZ; scale bars = 50 µm (B",
    "molecules": "BrdU"
  },
  {
    "caption": "D Representative picture of astrocytes located  to distinct DG  layers (hilus GZ, ML). Co-expression of BrdU/SOX2/GFAP  revealed that all DG  compartments harboured astrocytes  which were newly generated during adulthood (arrowheads). E Quantification of the proportions of generated astrocytes  per DG  layer out of all generated DG astrocytes. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): E) n = 5, dotted lines border GZ; scale bars = 50 µm D,",
    "molecules": "BrdU"
  },
  {
    "caption": "B Representative images of  BrdU-incorporating  newly-generated cells in control and running hGFAPeGFP animals. BrdU+ cells (magenta) were assigned to distinct cell types: rNSCs  [identified by their SOX2+ (cyan)  cell bodies residing in the SGZ  with a GFAP+ radial process (green)]; IPCs/neuroblasts  [SOX2+ (cyan) nuclei located  to the SGZ, DCX+ cells (white) in the SGZ  and GZ, respectively], and astrocytes  [SOX2+ cells (cyan) in the hilus, GZ, and ML]. Arrows mark newly generated neuronally committed cells (BrdU+/DCX+), while arrowheads indicate BrdU+ astrocytes (SOX2+). Data information: dotted lines border SGZ  and GZ; scale bars = 50 µm.",
    "molecules": "BrdU"
  },
  {
    "caption": "C Quantification of total numbers of generated cell types (BrdU+) per area in the DGs of control and runner animals. rNSCs: **** p < 0.0001; IPCs/neuroblasts: *** p = 0.0007; astrocytes: ** p = 0.0032; unpaired t-test. D Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs  + neuroblasts, astrocytes) in the DGs  of controls and runners; not significant, unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C,D) n = 5 for controls, n = 6 for runners",
    "molecules": "BrdU"
  },
  {
    "caption": "C Quantification of total numbers of generated cell types (BrdU+; rNSCs, IPCs  + neuroblasts, astrocytes) per area in the DGs  of 8 week-, 14 month-, and 21 month-old hGFAPeGFP animals. rNSCs: *** p = 0.00076, ** p = 0.0014; IPCs/neuroblasts  **** p < 0.0001; astrocytes: *** p = 0.0008; * p = 0.0403; one-way ANOVA with Tukey's post hoc test. D Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs  + neuroblasts, astrocytes) in the DG  of 8 week-, 14 month-, and 21 month-old animals. rNSCs: not significant; IPCs/neuroblasts  **** p < 0.0001, *** p = 0.0001; astrocytes: ** p < 0.0019, **** p < 0.0001; one-way ANOVA with Tukey's post hoc test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C,D) n = 5 in 8 week-old mice, n = 6 in 14 month-old mice, n = 5 in 21 month-old mice",
    "molecules": "BrdU"
  },
  {
    "caption": "G Confocal  images of 8 week- and 14 month-old NestinCreERT2; GFP  animals, which received tamoxifen  for five days (10 times, every 12 h) and were killed 12 days after the last tamoxifen  shot. The majority of recombined cells were rNSCs  [SOX2+ cell nuclei  (cyan) residing in the SGZ  with a NESTIN+ radial process (white)] and IPCs/neuroblasts [SOX2+ (cyan) nuclei located  to the SGZ, DCX+ cells (magenta) in the SGZ  and GZ, respectively], and only very few astrocytes  [SOX2+ cells (cyan) in the hilus, GZ, and ML] were generated by initially recombined rNSCs. H Quantification of total numbers of recombined (GFP+) rNSCs, IPCs/neuroblasts, astrocytes  per area in the DGs of 8 week- and 14 month-old animals. rNSCs: * p = 0.0460; IPCs/neuroblasts: **** < 0.0001; astrocytes: not significant; unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): ; (H) n = 3 in 8 week-old mice, n = 5 in 14 month-old mice  ; dotted lines border SGZ   and GZ; scale bars = 50 µm.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1/atf6 RNAi and controls in starved conditions after double immunostaining with anti-tyrosine-tubulin and anti-H3P (****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. indicates not significant using two-sided Chi-square test); n ≥ 5 planarians. Representative images are shown. Arrows indicate abnormal organization or number of spindle poles. j8 displays asymmetrical distribution of chromosome content.",
    "molecules": "tyrosine"
  },
  {
    "caption": "The Kaplan-Meier curve demonstrates increased survival of cct3A RNAi animals when treated with DTT under starved conditions. Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate p < 0.001 with log-rank (Mantel-Cox) test. Images are representative surviving animals for the different conditions at the displayed time point of regeneration. At the bottom are the number of planarians with the phenotype shown. The rest of planarians are dead at the displayed time point.",
    "molecules": "DTT"
  },
  {
    "caption": "Volcano plots displaying shRNA-Cct3- and shRNA-luciferase-infected LSK cells cultured either in normal glucose (upper graph) or low glucose conditions (lower graph). Dots represent all the NanoString analyzed transcripts. Red dots are UPR and ER stress related genes. The UPR and ER stress genes significantly down-regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph. For better visualization two dots with q ≤ 0.001 have been removed from the volcano plot (see them in Dataset EV4). Y-axis indicates the false discovery rate (FDR) (q- value), and the X-axis indicates the log2 fold changes. The FDR-based method of p-value adjustment was conducted to calculate the q-values by nSolver software using Benjamini-Hochberg methods. Significance is established by q-value < 0.05 and indicated by the dotted horizontal line. n= 3 mice.",
    "molecules": "glucose"
  },
  {
    "caption": "The Kaplan-Meier curve demonstrates increased survival of starved cct3A(RNAi) animals when treated with palmitic acid/palmitoylcarnitine (PA/PC). Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate P < 0.001 with log-rank (Mantel-Cox) test.",
    "molecules": "PA, palmitic acid, palmitoylcarnitine, PC"
  },
  {
    "caption": "A. Gentamicin survival  assay showing bacteria captured by DC  or B cells. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Significant differences, analyzed by T test (two samples) or one way ANOVA (more than two samples), are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).",
    "molecules": "Gentamicin"
  },
  {
    "caption": "G. Bacteria-infected and HEL-loaded DCs  were co-incubated with B cells from MD4 mouse. After conjugate formation, BacB cells were re-isolated, and intracellular bacteria were detected by gentamicin survival  assays at different times. Data were normalized compared to time 0. As control of the bacterial fitness, in parallel, HeLa  cells were directly infected.",
    "molecules": "gentamicin"
  },
  {
    "caption": "H. WT B cells were allowed to form conjugates with Listeria-infected DC, after 30 min samples were treated with the indicated inhibitors; Leupeptin  + NH4Cl, pepstatin-A  + E64D, lactacystin, or PBS  for controls. One hour later gentamicin  was added to kill extracellular bacteria and two hours later  B cells were reisolated by negative selection, lysed and plated in agar-containing plates. CFUs  were counted; each colony represents one live intracellular bacterium inside B cells. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Significant differences, analyzed by T test (two samples) or one way ANOVA (more than two samples), are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).",
    "molecules": "E64D, NH4Cl, gentamicin, lactacystin, Leupeptin, pepstatin-A, PBS"
  },
  {
    "caption": "I. Confocal  images of Listeria-WT (left panels) or Listeria-OVA (right panels) BacB cells incubated with naïve OTI CD8+ T cells  previously stained  with CellTraceViolet  (CV, red). Actin  and CD3  fluorescence are shown on a \"fire\" scale and in cyan and white respectively in the merged images. Scale bars = 10 μm. * indicate  CD8+ T cells. Arrow points to the IS. J. Quantification of actin  accumulation at the IS  on CD8+ T cells  conjugated with Listeria-WT or Listeria-OVA  BacB cells, analyzed using Synapse measures software from confocal  images. Each dot corresponds to one IS. Three independent experiments were performed. Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).",
    "molecules": "CellTraceViolet, CV"
  },
  {
    "caption": "H. BacB cells reduce melanoma  growth.  B16 F10 melanoma  cells (4 × 105) were injected s.c. in the mid-right flank of C57BL/6J (WT) host mice  (4 mice/group; treated with BacB or untreated). Once tumors were formed and visible (6 days after imlantation) mice  were treated with Listeria TRP2   BacB cells or with vehicle (PBS) (three consecutive injections in three days). Tumor growth were monitored every 1-3 days. It is shown the mean and the SD of the tumor volume  in each group at the indicated time points. Mice  with tumors ≥ 300 mm3 were sacrified. Data information: Bars in the figure represent the mean. Error bars indicate the SD. Statistical significant differences, analyzed by ANOVA are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns). …",
    "molecules": "PBS"
  },
  {
    "caption": "A. Experimental design and representative traces for analysis of synaptic transmission in retinas from wild-type (black) and RIBEYE KO mice (red). Presynaptic bipolar neurons were depolarized in voltage-clamp mode from -70 mV to -10 mV for 0.5 s as indicated on top to open Ca2+-channels and trigger release. Presynaptic Ca2+-currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces; dotted box displays an expansion of the initial Ca2+-currents and EPSCs.B. RIBEYE KO does not alter presynaptic Ca2+-currents. Summary graphs show average peak Ca2+-currents and Ca2+-current.C. RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization. Plot shows integrated EPSC charge as a function of time; both initial and sustained neurotransmitter release are impaired.D. RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude.E. RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization. Summary graphs display integrated EPSC charges for the initial phase (0-50 ms) and the sustained phase (50-500 ms).F. RIBEYE KO does not alter the kinetics of EPSCs. Summary graphs depict the mean synaptic delays (left), rise times (center) and decay time constants (right) of EPSCs.",
    "molecules": "Ca2+"
  },
  {
    "caption": "C and D. RIBEYE KO renders mEPSCs sensitive to the slow Ca2+-buffer EGTA (C, representative mEPSCs traces recorded from AII cells after 30 minutes incubation in DMSO or EGTA-AM [0.2 mM]; D, summary graphs of the mean mEPSC frequency (left) and amplitude (right) after incubation in DMSO or EGTA-AM).",
    "molecules": "Ca2+, DMSO, EGTA, EGTA-AM"
  },
  {
    "caption": "E and F. mEPSCs in AII amacrine cells are largely triggered by Ca2+-influx via presynaptic L-type Ca2+-channels (E, representative mEPSCs traces recorded in regular [2 mM] or reduced extracellular Ca2+-concentration [0.3 mM], or in the presence of nimodipine [50 µM] in regular Ca2+; F, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for E).",
    "molecules": "Ca2+, nimodipine"
  },
  {
    "caption": "G and H. mEPSCs in AII amacrine cells are suppressed by the fast Ca2+-buffer BAPTA (G, representative mEPSCs traces recorded after 30 min incubations with DMSO or BAPTA-AM [30 µM]; H, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for G).",
    "molecules": "BAPTA, BAPTA-AM, Ca2+, DMSO"
  },
  {
    "caption": "Representative immunofluorescence  images of SCOTIN-KO cells expressing Str-Ii-SCOTIN-SBP-EGFP  with 40 μM D-biotin treatment for the indicated times. B Upper, localization  of RUSH reporters retained on the ER membrane with Sec61β  (red). Bottom, localization  of released RUSH reporters after 240 min of biotin  treatment. The arrow indicates the EGFP  signal on the ER. The arrowhead indicates the EGFP  signal on vesicles. The empty arrowhead indicates the EGFP  signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.",
    "molecules": "biotin, D-biotin, Str"
  },
  {
    "caption": "Representative immunofluorescence  images of SCOTIN-KO cells expressing Str-Ii-VSVG-SBP-EGFP  (green) with 40 μM D-biotin treatment for the indicated times. , Upper, localization  of RUSH reporters retained on the ER membrane with Sec61β  (red). Bottom, localization  of released RUSH reporters after 240 min of biotin  treatment. The arrow indicates the EGFP  signal on the ER. The arrowhead indicates the EGFP  signal on vesicles. The empty arrowhead indicates the EGFP  signal on the plasma membrane. Scale bar: 20 μm. Scale bars in the magnified images: 5 μm.",
    "molecules": "biotin, D-biotin, Str"
  },
  {
    "caption": "(A) Immunoblot  analysis of proteinase K protection assay. HeLa  cells were transfected with SCOTIN-Myc  (Total) and digitonin-permeabilized. The supernatants  (Cytosol) and pellet  (PNSs) were separated by centrifugation. The PNSs  were incubated with PBS  or with proteinase K (+ PK) in the presence or absence of  Triton X-100 (+PK  +Triton X).",
    "molecules": "digitonin, PBS, Triton X, Triton X-100"
  },
  {
    "caption": "(B) Representative images of VAPA-Rab7 PLA  signals taken from Str-Ii-SCOTIN-SBP-EGFP  transfected SCOTIN-KO cells after untreated (-Biotin) or biotin  treatment (+ Biotin  4 h). For the PLA  signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (C) The PLA  signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, **<0.01, ***<0.001, and ****P<0.0001.",
    "molecules": "Biotin, biotin, Str"
  },
  {
    "caption": "(C) HEK293  cells were transfected with SCOTIN-GST  along with empty (pcDNA3.1-Myc), SCOTIN-Myc, or SCOTIN(Δ150-177)-Myc  plasmids for 48 h. SCOTIN-GST  was  pulled down from total cell lysates using glutathione-Sepharose  beads, and the interacting  proteins were analyzed by immunoblotting.",
    "molecules": "Sepharose, glutathione"
  },
  {
    "caption": "(G) Representative images of VAPA-VAPA PLA  signals taken from Str-Ii-SCOTIN(Δ150-177)-SBP-EGFP  transfected SCOTIN-KO cells after untreated (-Biotin) or biotin-treated (+Bition  4 h). For the PLA  signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. (H) The PLA  signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n: number of cells analyzed. Data are shown as bars with dots and represent the means±SEMs. P values were determined using a two-tailed unpaired t test. Significant differences are labeled, ****P<0.0001.",
    "molecules": "Biotin, biotin, Bition, Str"
  },
  {
    "caption": "Representative images of ASY1  (green) and RAD51  (red) immunostaining  in Col and hsbp-3. Nuclei  spreads were stained  with DAPI  (blue). Scale bars: 10 μm. Quantification of RAD51  foci numbers per cell in Col (blue) and hsbp-3 (red). n = 20 cells of biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative images of MLH1  (red) immunostaining  in Col, hsbp-3, hsbp-2, Col/Ler, and meiMIGS-HSBP  Col/Ler. Nuclear  DNA  was stained  with DAPI. Scale bar: 5 μm. Quantification of the number of MLH1  foci per cell shown in (G). n ≥ 32 cells of biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "Hsp12-GFP accumulation (Y axis) vs. time (X axis) in response to different levels of KCl stress. Points reflect the measured values, and lines correspond to best model fit.",
    "molecules": "KCl"
  },
  {
    "caption": "Hsp12-GFP accumulation (Y axis) vs. time (X axis) at 0.4M KCl.",
    "molecules": "KCl"
  },
  {
    "caption": "Representation of the experiments shown in (B,C) in parameter space. Connected line shows the progression in the response to increasing KCl levels The inferred tonparameter (Y axis) vs. KCl concentration (X axis) of the KCl gradient experiment The inferred total open time - toff− ton(Y axis) vs. the inferred production rate (X axis) of the KCl gradient experiment",
    "molecules": "KCl"
  },
  {
    "caption": "Time-lapse microscopy of Hsp12-GFP in response to stress (0.4M KCl). Examples of raw images (top) and traces of individual cells (bottom). Traces are colored according to their basal GFP level.",
    "molecules": "KCl"
  },
  {
    "caption": "Upper: HSP12 mRNA in response to osmotic stress (0.4M KCl). Lower: Estimate of Msn2/4 dependent production of HSP12 mRNA over time (difference between WT and Δmsn2Δmsn4 production profiles, Figure EV4A, Methods).",
    "molecules": "KCl"
  },
  {
    "caption": "Msn2-GFP localization following stress (0.4M KCl) in selected strains. Top: The percentage of nuclear cells (Y axis) over time (X axis). Bottom: count of the number of nuclear import events in live-cell time-lapse microscopy at the two relevant periods.",
    "molecules": "KCl"
  },
  {
    "caption": "D. Effect of LITATS1 knockdown on TGF-β-induced SMAD2 phosphorylation in MDA-MB-231 cells. Cells were serum starved for 16 h and stimulated with TGF-β for the indicated durations. The p-SMAD2 and total SMAD2 (t-SMAD2) levels were analyzed by western blotting. GAPDH, loading control. Representative results from a minimum of three independent experiments are shown. E. Effect of LITATS1 knockdown on E-cadherin, N-cadherin and SNAIL expression in A549 cells. Cells were stimulated with vehicle control (-), SB431542 (SB; 10 μM) or TGF-β (Τβ) for 24 h, and protein expression was analyzed by western blotting. α/β-Tubulin, loading control. Representative results from a minimum of three independent experiments are shown. F",
    "molecules": "SB, SB431542"
  },
  {
    "caption": "C, D. Analysis of TβRI protein stability (as measured by western blotting) in MDA-MB-231 cells with ectopic LITATS1 expression (C) or LITATS knockdown (D). Cells were treated with CHX (50 µg/mL) for the indicated durations. Quantification of the relative TβRI protein level is shown in the lower panel. GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.",
    "molecules": "CHX"
  },
  {
    "caption": "E. TβRI expression in MDA-MB-231 cells with ectopic LITATS1 expression in the absence or presence of lysosome or proteasome inhibitors. Cells were incubated with vehicle control DMSO (-), the lysosome inhibitor BafA1 (20 nM) or HCQ (20 μM), or the proteasome inhibitor MG132 (5 μM) for 8 h. Vinculin, loading control. Representative results from a minimum of three independent experiments are shown. F. Effect of LITATS1 on TβRI ubiquitination. HEK293T cells were transfected with ectopic expression constructs for HA-Ubiquitin (HA-Ub), caTβRI-FLAG and/or LITATS1. TβRI polyubiquitination was analyzed by western blotting. Representative blots from a minimum of three independent experiments are shown. D",
    "molecules": "BafA1, DMSO, HCQ, MG132, Ub, Ubiquitin"
  },
  {
    "caption": "B. Interactions between LITATS1 and TβRI or SMURF2 in MDA-MB-231 cells were detected by the CARPID approach. Cells with stable expression of TurboID-dCasRx were transduced without (Co.) or with (#1 and #2) LITATS1 targeting gRNAs. Cells were stimulated with or without TGF-β (2.5 ng/mL) for 2 h and were then stimulated with biotin (500 μM) for 30 min. Western blotting was performed to detect SMURF2 and TβRI expression in whole-cell lysates (Input) and immunoprecipitates (IP). GAPDH and HA-dCasRx expression levels were measured for equal loading of Input samples and as the negative control or positive control, respectively, for proximity biotinylation in immunoprecipitate (IP) samples. Representative results from a minimum of three independent experiments are shown.",
    "molecules": "biotin"
  },
  {
    "caption": "D. Interactions between LITATS1 and caTβRI or SMURF2 were analyzed by RNA pull-down. Biotinylated 25x poly(A), antisense LITATS1 (LITATS1-AS) or LITATS1 was incubated with lysates from HEK293T cells transfected with the caTβRI-HA or MYC-SMURF2 expression construct. Western blot analysis was performed to detect HA or MYC expression in whole-cell lysates (Input) and immunoprecipitates (IP). Representative blots from a minimum of three independent experiments are shown.",
    "molecules": "poly(A)"
  },
  {
    "caption": "E. In vitro RNA pull-down assays were performed to evaluate the interactions between LITATS1 and SMURF1/2. In vitro-transcribed Antisense LITATS1 (LITATS1-AS) or LITATS1 (LITATS1-S) was incubated with recombinant FLAG-tagged SMURF1 or SMURF2 protein. Western blotting analysis was performed to evaluate FLAG expression in input and IP samples. The amounts of RNA probes used for RNA pull-down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.",
    "molecules": "agarose"
  },
  {
    "caption": "G. Effect of LITATS1 overexpression on SMURF2-mediated TβRI polyubiquitination. HEK293T cells were transfected with expression constructs for HA-Ubiquitin (HA-Ub) and caTβRI-FLAG and ectopic expression constructs for SMURF2 and/or LITATS1. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.",
    "molecules": "Ub, Ubiquitin"
  },
  {
    "caption": "H. Effect of SMURF2 knockdown on LITATS1-mediated TβRI polyubiquitination. MDA-MB-231 cells with stable HA-Ub expression were transduced with expression constructs for LITATS1 and/or two different SMURF2 shRNAs, as indicated. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.",
    "molecules": "Ub"
  },
  {
    "caption": "A. An in vitro RNA pull-down assay was performed to evaluate the interaction between LITATS1 truncation mutants and SMURF2. Recombinant FLAG-SMURF2 protein was incubated with antisense LITATS1 (LITATS1-AS), LITATS1 (LITATS1-S), or LITATS1 truncation mutants (T1-T4). Western blot analysis was performed to evaluate FLAG expression in immunoprecipitates (IP). The amounts of RNA probes used for RNA pull-down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.",
    "molecules": "agarose"
  },
  {
    "caption": "E. SMURF2 expression and localization (as measured by immunofluorescence) upon LITATS1 depletion in A549 cells. DAPI staining was performed to visualize nuclei. Scale bar=23.2 μm. Representative results from two independent experiments are shown.",
    "molecules": "DAPI"
  },
  {
    "caption": "A Cytokine release by PBMCs challenged with heat inactivated S. aureus (hiSa), and/or RNase A (RA) treated solution, or Sa19 (TLR13 activating ORN) upon TLR2 blockade (T2.5; n.p., not performed; **, p ≤ 0.01; n = 3).",
    "molecules": "ORN, Sa19"
  },
  {
    "caption": "C Cytokine release of PBMCs transfected with ORNs including TLR7/8 ligand RNA40 (n = 3).",
    "molecules": "ORN, RNA40"
  },
  {
    "caption": "D, E Activity of PBMCs upon bacterial RNA challenge, pretreatment with chloroquine in (D) only (S, Svedberg; r, ribosomal; tot, total; n = 3).",
    "molecules": "chloroquine, RNA"
  },
  {
    "caption": "F Activity of PBMCs upon mitochondrial (mt) RNA challenge (Hs, human; triangle, increasing doses; LFA, Lipofectamine 2000; n = 3).",
    "molecules": "Lipofectamine, RNA"
  },
  {
    "caption": "G Activity of PBMCs challenged with ORNs and total S. aureus (Sa) RNA (Myd88d/d, mutant MyD88 expression not impairing LPS driven IL-8 production; n = 2).",
    "molecules": "LPS, ORNs, RNA"
  },
  {
    "caption": "H Responsiveness of undifferentiated (undiff) and 3- or 8 days PMA differentiated (ddi) THP1 cells to Sa19 challenge (n = 3).",
    "molecules": "PMA, Sa19"
  },
  {
    "caption": "I Activity of parental and Unc93b1-/--3ddiTHP-1 cells challenged with ORNs (n = 3).",
    "molecules": "ORNs"
  },
  {
    "caption": "A, B Cytokine release of 3 days differentiated (ddi) THP1 cells challenged with (A) ORNs or (B) bacterial RNA fractions (tot, total; S, Svedberg; r, ribosomal).",
    "molecules": "ORNs, RNA"
  },
  {
    "caption": "D Responsiveness of 3ddiTHP1 cells upon challenge with human (Hs) mitochondrial (mt) RNA (triangle, increasing doses).",
    "molecules": "RNA"
  },
  {
    "caption": "E-H NF-B driven relative luciferase activity (Rel. luc. act.) of hTLR8+ HEK293 cells or cytokine release by PBMCs all transfected with RNAs indicated and additional uridine (U) if indicated (n.p., not performed; vector, empty plasmid).",
    "molecules": "uridine"
  },
  {
    "caption": "B PBMC activity upon endosome function inhibition (chloroquine) and TLR2 blockade (T2.5) followed by TLR ligand challenge or bacterial infection.",
    "molecules": "chloroquine"
  },
  {
    "caption": "C Activity of whole blood culture upon endosome function inhibition (chloroquine) and challenge with TLR ligands, heat inactivated (hi), or viable bacteria.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(B) In vitro crosslinking assays were performed for SPOP28-359 and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. (C) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP1, SPOP2 and SPOPn identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.",
    "molecules": "BS3"
  },
  {
    "caption": "(A-H) 3D views of 232 x 232 μm2 fields of view of the AT  (A, G) and endothelial  layer (B, H) from infected LoCs reconstituted without macrophages at 1 (A, B) and 3 dpi (G, H). Orf1ab  antisense RNA, S RNA, ACE2  mRNA, and nuclear staining with DAPI  are false-colored pink, amber, spring green, and electric indigo, respectively. (C, D) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (C) An example of infection and intracellular replication in a cell with no detectable ACE2 expression (white arrow) as well as nuclear localization of viral RNA (white arrowhead). (D) An example of an uninfected cell with ACE2 mRNA expression (yellow arrow). (E, F) Zooms corresponding to the regions in (B) representing increased nucleic acid staining (hyperplasic) and normal levels of nucleic acid staining highlighted with white and yellow boxes, respectively. (E) Examples of infection of hyperplasic endothelial cells both with and without ACE2 expression (F) Examples of endothelial cell  infection with no ACE2 expression, nuclear localization of viral RNA in an infected endothelial cell is indicated (white arrow).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-F) Maximum  intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with (A) and without Tocilizumab treatment (D). Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively, the actin staining is saturated for the  endothelial cell clusters to highlight cells with lower levels of actin. (B, C) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (B) A region with normal vascular profile, (C) A region of endothelial cell cluster formation with increased actin staining. (E, F) Zooms corresponding to the regions in (D) representing regions highlighted with white and yellow boxes, respectively. (E) An example of reduced tight junctions between endothelial cells; bare patches are indicated with arrows. (F) A region of endothelial cell clusters. Scale bar = 20 μm.",
    "molecules": "Tocilizumab"
  },
  {
    "caption": "(G, H) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi,  immunostaining  for the tight junction protein  CD31  is shown in the Spring Green LUT. Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows.",
    "molecules": "Tocilizumab"
  },
  {
    "caption": "Immunoblot with anti-HA Abs of lysates from parental (Ctrl) and tagged lines (TgCRMPa-HA3 and TgCRMPb-HA3) together with inducible-knockdown lines (TgCRMPa-HA3_iKD and TgCRMPb-HA3_iKD) treated with ATc for 0, 24 or 48h. TgROP5 was used as loading control. Two close bands around 300kDa were detected for TgCRMPa. A ~820kDa protein, corresponding to the predicted size for TgCRMPb, was observed together with a ~130kDa band.",
    "molecules": "ATc"
  },
  {
    "caption": "Quantification of microneme secretion in TgCRMPa- and TgCRMPb-depleted tachyzoites was measured by detecting the processed form (arrowhead) of TgMIC2 (arrow) in the media. Control, TgCRMPa_iKD and TgCRMPb_iKD parasites, ATc-treated (+) and untreated (-), were stimulated with propranolol to release microneme contents. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.",
    "molecules": "ATc, Prop, propranolol"
  },
  {
    "caption": "Quantification of microneme secretion in control and Tg277910-depleted tachyzoites was measured as in Fig 2H. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.",
    "molecules": "Prop, propranolol"
  },
  {
    "caption": "Immunofluorescence images of extracellular tachyzoites of untagged, TgCRMPa-HA3 and TgCRMPb-HA3 parasites, incubated either with host cell monolayers for 2min, or with ionophore A23187, to induce natural or artificial conoid extrusion, respectively. Parasites were immunostained with anti-HA Abs; DNA was labeled by Hoechst. CRMPa and CRMPb consistently accumulate at the tip of extruded conoids (arrows). The apexes of A23187-treated parasites were magnified on the right, and increased in brightness and contrast to highlight the apical dots. DIC: differential interference contrast. Quantification of the dot pattern upon A23187 treatment shown in (A). Values are expressed as percentage of parasites showing (dot) or lacking (no dot) the apical accumulation of TgCRMPa and TgCRMPb; n= number of parasites analyzed per line.",
    "molecules": "Hoechst, A23187, ionophore A23187"
  },
  {
    "caption": "Immunofluorescence images of extracellular TgCRMPb-HA3 tachyzoites in presence (-ATc) or absence (+48h ATc) of TgCRMPa-FLAG3 (iKD line). Parasites were stained with anti-HA Abs to visualize CRMPb. TgCRMPb localization at the tip of the extruded conoid (arrow) disappears upon TgCRMPa depletion, but it is still detected in the cytoplasm (lower panel). DNA is labeled by Hoechst. Single focal planes are shown. DIC: differential interference contrast.   Quantification of the dot pattern shown in (C). The values are reported as in (B).  ",
    "molecules": "Hoechst, ATc"
  },
  {
    "caption": "Whole cell lysates from TIR1-expressing parental line (Ctrl), HA3-miniAID-TgCRMPa_iKD (N-term) and TgCRMPa-miniAID-HA3_iKD (C-term) lines were immunoblotted with anti-HA Abs to visualize tagged CRMPa in IAA-treated and untreated samples. CRMPa was undetectable upon 24h incubation with IAA when C-terminally, but not N-terminally, tagged with the miniAID-HA3, suggesting that the C-terminus is the one exposed towards the cytosol. TgMIC2 was used as loading control and detected with anti-MIC2 Abs. MW: molecular weight standards.",
    "molecules": "IAA"
  },
  {
    "caption": "Immunofluorescence images of extracellular A23187-treated parasites expressing either N-or C-terminally HA3-tagged TgCRMPa, and immunostained as in (A). TgCRMPa accumulates at the tip of extruded conoids (arrows) in triton-permeabilized (+) or non-permeabilized (-) parasites. DIC: differential interference contrast. Single focal planes are shown.   Quantification of the dot pattern upon natural (+H F Fibroblasts) or artificial (+A23187) conoid extrusion in parasites expressing either N- or C-terminally HA3-tagged TgCRMPa. Values are reported as in (B).  ",
    "molecules": "A23187, triton"
  },
  {
    "caption": "Ultrastructure Expansion Microscopy of extracellular tachyzoites, either untagged or co-expressing TgCRMPa-HA3/TgCRMPa-TY2 and TgCRMPb-HA3 together or in pairwise combination with TgNd6-TY2. Parasites were treated with A23187 prior to fixation and preparation for U-ExM, and stained with anti-HA, anti-TY and anti-α/β tubulin Abs to label CRMPs, Nd6/CRMPa and microtubules, respectively. Shown are maximum intensity projections of z-stack confocal images. CRMPs overlap with Nd6 at the tip of the extruded conoid (yellow selection). A magnified image of the apical tip of each parasite is shown on the right.",
    "molecules": "A23187"
  },
  {
    "caption": "Extent of colocalization between TgCRMPa-TY2 and TgCRMPb-HA3, and TgCRMPs-HA3 with TgNd6-TY2 in the apical dot shown in B. Pearson's correlation coefficient was measured using the Fiji-JACoP plugin. Values are expressed as mean ± SD. Three-five parasites per line were analyzed. Quantification of the dot pattern for TgCRMPa-HA3 and TgCRMPb-HA3 in TgNd9_iKD lines. CRMPs accumulation at the apical tip of extracellular parasites was measured as in Fig EV4A. TgCRMPs were still found in the apical dot in absence of TgNd9 (+ATc). Numbers are expressed as percentage of parasites showing (dot) or lacking (no dot) the tip accumulation of TgCRMPa and TgCRMPb. The number of parasites (n) analyzed for each line is reported at the top of the column.",
    "molecules": "ATc"
  },
  {
    "caption": "(E) Relative mRNA levels of AR  and the indicated AR  target genes (left) and western blot analysis of AR  (right) in  C4-2 cells treated with increasing concentrations of XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "XMU-MP-1"
  },
  {
    "caption": "(G) Relative mRNA levels of the indicated AR  or YAP  target genes in  C4-2  cells treated with control (siNC) or YAP  siRNA (siYAP_1 or siYAP_2) for 24 hours and with or without 2 μM XMU-MP-1  for 12 hours. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "XMU-MP-1"
  },
  {
    "caption": "(A) Western blot  analysis of AR  (left) and quantification of AR  (right) in C4-2  cells treated CHX  for the indicated time.",
    "molecules": "CHX"
  },
  {
    "caption": "Western blot analysis of AR  and YAP  (I, in C4-2  cells stably expressing Tet-O-YAP-5SA and treated with 0.2 μg/ml doxycycline  (Dox) for the indicated periods of time. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "Dox, doxycycline, Tet"
  },
  {
    "caption": "Western blot analysis of AR  and YAP  L) in C4-2  cells stably expressing Tet-O-YAP-5SAS94A and treated with 0.2 μg/ml doxycycline  (Dox) for the indicated periods of time.",
    "molecules": "Dox, doxycycline, Tet"
  },
  {
    "caption": "(D) Relative mRNA levels of the indicated AR  target genes (left) and western blot  analysis of TEAD  (right) in C4-2  cells expression the control or TEAD4  constructs and treated with vehicle or XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "XMU-MP-1"
  },
  {
    "caption": "(D) YAP-5SA but not YAP-5SAS94A inhibited AR/TEAD interaction. C4-2  cells stably expressing Tet-O-GFP, Tet-O-YAP-5SA or Tet-O-YAP-5SAS94A were treated with 0.2 μg/ml doxycycline  (Dox) for 10 hours, followed by CoIP  and western blot  analyses with the indicated antibodies.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(G-H) ChIP-qPCR analysis of AR  (G) and TEAD  (H) binding  to the promoter/enhancer regions of KLK2, KLK3  and CTGF  in Tet-O-YAP-5SA expressing  C4-2 cells treated with 0.2 μg/ml doxycycline  (Dox) for the indicated time. Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "Dox, doxycycline, Tet"
  },
  {
    "caption": "(A-F) YAP  activation inhibited AR  positive  prostate cancer  growth in xenografts  bearing C4-2  or 22RV1 tumors  that stably express Tet-O-YAP-5SA. Male NOD scid gamma (NSG) mice  bearing C4-2 tumor  cells stably expressing a Tet-O-EGFP  construct were used as control. Male NOD scid gamma (NSG) mice  bearing the indicated tumors  were injected i.p. with PBS  containing Dox  (20mg/kg) or PBS  daily for the indicated time. Tumor growth  curve (A, D), photograph of tumor  samples (B, E), and quantification of  tumor weight  (C, F) at the end of treatment were shown. XMU-MP-1 inhibited AR  positive  prostate cancer  growth in xenografts  bearing C4-2  (G-I), tumors. Male NOD scid gamma (NSG) mice  bearing the indicated tumors  were treated daily with XUM-MP-1 at the indicated concentrations or with vehicle for the indicated periods of time. Tumor growth  curve (G, J, images of tumor samples (H, K, and quantification of tumor weight (I at the end of treatment were shown. Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice  for each group.",
    "molecules": "Dox, PBS, Tet, XMU-MP-1, XUM-MP-1"
  },
  {
    "caption": "XMU-MP-1 inhibited AR  positive prostate cancer growth in xenografts  bearing 22RV1 tumors. Male NOD scid gamma (NSG) mice  bearing the indicated tumors  were treated daily with XUM-MP-1  or with vehicle for the indicated periods of time. Tumor growth curve J, Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice  for each group.",
    "molecules": "XMU-MP-1, XUM-MP-1"
  },
  {
    "caption": "Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.",
    "molecules": "oligonucleotide, ssDNA"
  },
  {
    "caption": "Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.",
    "molecules": "oligonucleotide, ssDNA"
  },
  {
    "caption": "Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.",
    "molecules": "oligonucleotide, ssDNA"
  },
  {
    "caption": "(B-J) ssDNA decatenation assays using TOP3A variants, using substrates constructed as in (A).",
    "molecules": "ssDNA"
  },
  {
    "caption": "(K) Quantification of ssDNA decatenation activity as in (B-J). Data represents the percentage of decatenated substrate expressed as mean values, ±SEM, from independent experiments, n = 3 (WT, p.Ala95Val, p.Met100Val, p.Arg558Trp), n = 4 (p.Ala176Val), n = 5 (p.Met575Val), n = 2 (p.Leu37Val). Symbols represent significance values (one-way ANOVA), colour-coded to the data points. *p<0.05, **p<0.01.",
    "molecules": "ssDNA"
  },
  {
    "caption": "(M) Quantifications of ssDNA decatenation assays as in (L). Data represents the mean percentage of decatenated substrate from 3 independent experiments. Significance values are shown for one-way ANOVA compared to the WT protein. *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "ssDNA"
  },
  {
    "caption": "The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.",
    "molecules": "glucose, glycolytic, lactate, pentose–phosphate"
  },
  {
    "caption": "Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).",
    "molecules": "14C, glucose, lactate"
  },
  {
    "caption": "The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α.",
    "molecules": "glycolytic, lactate"
  },
  {
    "caption": "siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information.",
    "molecules": "glycolytic"
  },
  {
    "caption": "Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF",
    "molecules": "O2, H2O2"
  },
  {
    "caption": "(g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal.",
    "molecules": "O2, GSH-EE, ROS"
  },
  {
    "caption": "(h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).",
    "molecules": "GSH-EE, ROS"
  },
  {
    "caption": "(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).",
    "molecules": "2-deoxyglucose, 2DG, DNA, glucose"
  },
  {
    "caption": "(c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α).",
    "molecules": "DNA"
  },
  {
    "caption": "a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons.",
    "molecules": "O2"
  },
  {
    "caption": "(b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons.",
    "molecules": "DNA"
  },
  {
    "caption": "(H and I) Representative images and quantitative analysis of calcein  double labeling. Scale bars, 20 μm. (n = 6 biological replicates) Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test , I was used for statistical analysis.",
    "molecules": "calcein"
  },
  {
    "caption": "(I and J) Representative images and quantification of the relative pit  resorption    area of hydroxyapatite-coated plates. WT or Sigamr1  gKO BMMs  were seeded on hydroxyapatite-coated plates and treated with 50 ng/mL RANKL  (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with unpaired two-tailed Student's t-test J) were used for statistical analysis.",
    "molecules": "hydroxyapatite"
  },
  {
    "caption": "(A and B) Western blots showing SERCA2  expression  in HEK-293T cells with or without Sigmar1  cotransfection treated with eeyarestatin I  or MG132 at the indicated concentration. All inhibitors were applied to cells 8 hours prior to protein collection. Data information: All results are representative data generated from at least three independent experiments.",
    "molecules": "eeyarestatin I, MG132"
  },
  {
    "caption": "(E) Truncated myc-tagged SERCA2 ubiquitination  levels in  HEK-293T  cells transfected with empty vector or Sigmar1  were analyzed by immunoprecipitation. Cells were treated with MG132  (10 μM) 8 hours before harvest. Data information: All results are representative data generated from at least three independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) Micro-CT images of calvaria  from mice  that received sham or LPS  injection with PBS  or dimemorfan  treatment. Scale bars, 1 mm. (C) Quantification of  bone  volume/tissue volume (BV/TV) of calvaria  from different groups (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C, was used for statistical analysis.",
    "molecules": "dimemorfan, LPS, PBS"
  },
  {
    "caption": "(A) TRAP staining  of hPBMCs  treated with human RANKL  (50 ng/mL) or dimemorfan  for 12 days. Scale bars, 500 μm.",
    "molecules": "dimemorfan"
  },
  {
    "caption": "C. The transfected Hela PTEN-/- cells were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy.",
    "molecules": "DAPI"
  },
  {
    "caption": "To determine the sequences that are critical for the membrane localization of PTENε. Hela were transfected with constructs Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F).",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Images of immunofluorescence staining for PTENε and its interacting proteins (VASP and FSCN1). Hela PTEN-/- cells were co-transfected by a plasmid expressing C-terminal GFP-tagged PTENε with FLAG-tagged targets (FSCN1, VASP) separately, followed by staining with an anti-FLAG antibody and DAPI, and were imaged by confocal microscopy.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Immunofluorescence staining of F-actin in PTENε or PTENε Y210L overexpressed Hela PTEN-/- cells and control cells. PTENε-GFP, PTENε Y210L-GFP, or mock construct was introduced into HeLa PTEN-/- cells respectively. The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm (left panel). White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane. B. Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t-test. ****, p<0.001.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "G. Left panel: immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells. Middle panel: representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model (n=4). H & E, hematoxylin-eosin staining. The scale bars represent 600 mm (upper panel) and 500μm (lower panel) separately. Right panel: the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules (data are presented as mean ± SD based on three independent replicates). The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group. ****, p<0.0001 in the unpaired t-test.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows).",
    "molecules": "Alexa-Fluor647-succinimidyl ester"
  },
  {
    "caption": "G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m.",
    "molecules": "POM1"
  },
  {
    "caption": "C: Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment. Each dot represents an individual experiment. Statistics: Unpaired t-test. **: p<0.01.",
    "molecules": "POM1"
  },
  {
    "caption": "G: Left: partially restored PIKfyve levels in prion-infected tga20 mice treated with LIN5044 (LCP) or vehicle. Right: quantification of the western blot. Each dot represents an individual biological replicate. *: p0.05; unpaired t-test Error bars represent s.e.m.",
    "molecules": "LCP, LIN5044"
  },
  {
    "caption": "H: Left, tga20 COCS were inoculated with RML or NBH, optionally treated with GSK2606414 starting at 21 dpi, and lysed at 35 dpi. Depletion of PIKfyve in prion-infected samples was largely rescued by GSK2606414 (GSK). Right, quantification of western blot. Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01; unpaired t-test; Error bars represent s.e.m.",
    "molecules": "GSK, GSK2606414"
  },
  {
    "caption": "A: Upper row: prion-infected Gt1 cells (75 dpi) immunostained for LAMP1 showing prominent vacuoles. Control: NBH-inoculated cells. Middle row: Gt1 cells transfected with shRNA targeting PIKfyve or luciferase (control) for 5 days and immunostained for LAMP1. Depletion of PIKfyve resulted in LAMP1+ vacuoles (yellow). DAPI: nuclear stain. Lower row: prion-infected and NBH treated cells (75 dpi) contained LAMP2- vacuoles. n=3 individual biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "D: tga20 COCS treated with POM1 or control IgG (10 days) and immunostained with NeuN and LAMP1. Nuclei: DAPI. POM1-treated COCS showed increased LAMP1 expression (quantification: Appendix Fig. S4B). NeuN+ cells exhibited vacuoles (arrowheads).",
    "molecules": "POM1, DAPI"
  },
  {
    "caption": "E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.",
    "molecules": "DAPI"
  },
  {
    "caption": "E: tga20 COCS were infected with RML and optionally treated with bPIP (5µg/ml). At 45 dpi, NeuN morphometry revealed ablation of cerebellar granule layer (CGN) in prion-infected slices and its rescue by bPIP. Control: NBH treated COCS. Each dot represents an individual slice (Statistics: ANOVA). *: p<0.05. **** p<0.0001.",
    "molecules": "bPIP"
  },
  {
    "caption": "F: tga20 COCS were treated with POM1 (optionally pre-blocked with recPrP) and treated with bPIP (5µg/ml). At 14 dpi NeuN morphometry revealed POM1-induced ablation of cerebellar granule layer (CGL) and rescue by bPIP.",
    "molecules": "POM1, bPIP"
  },
  {
    "caption": "A Peroxisomes were induced by growing the WT strain expressing Pot1‐GFP in oleate medium to mid‐log‐phase, then transferred to SD‐N starvation medium with or without GABA to trigger pexophagy for 6 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.",
    "molecules": "GABA"
  },
  {
    "caption": "B Mitochondria were induced by growing the WT strain expressing OM45‐GFP in YPL medium to mid‐log‐phase and subsequently transferring cells to either SD‐N with or without GABA to trigger mitophagy for 12 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.",
    "molecules": "GABA"
  },
  {
    "caption": "C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45‐GFP grown in YPL medium for 12 h to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA for 24 h. Bar, 5 μm.",
    "molecules": "GABA"
  },
  {
    "caption": "D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA, grown to mid‐log‐phase, after which samples were analyzed for Ape1 maturation.",
    "molecules": "GABA"
  },
  {
    "caption": "E Ribophagy was monitored by growing the WT strain expressing Rpl25‐GFP in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 24 h.",
    "molecules": "GABA"
  },
  {
    "caption": "F Autophagy was monitored by growing the WT strain expressing GFP‐Atg8 in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 6 h.",
    "molecules": "GABA"
  },
  {
    "caption": "B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm.",
    "molecules": "GABA, rapamycin"
  },
  {
    "caption": "A WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin).",
    "molecules": "rapamycin"
  },
  {
    "caption": "B WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD).",
    "molecules": "rapamycin"
  },
  {
    "caption": "A,B WT cells were cultured under pexophagy (A) or mitophagy (B) conditions with or without GABA and rapamycin. S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control.",
    "molecules": "GABA, rapamycin"
  },
  {
    "caption": "A,B WT, WT with GABA, WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under (A) pexophagy and (B) mitophagy conditions. After 24 h incubation, cells were stained with DHR−123 and propidium iodide for 1 h. Living cells were analyzed for DHR−123 fluorescence by flow cytometry. Data represent mean + s.d. (n = 3). *P < 0.005, **P < 0.01",
    "molecules": "GABA, GSH, intracellular ROS, rapamycin"
  },
  {
    "caption": "C Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1−/− mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.",
    "molecules": "rapamycin"
  },
  {
    "caption": "D Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1−/− mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.",
    "molecules": "rapamycin"
  },
  {
    "caption": "E Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay.",
    "molecules": "rapamycin"
  },
  {
    "caption": "G Immunofluorescence images showing typical nuclear staining (DAPI, blue) and SOD2 staining (red) from WT, Aldh5a1−/− mice treated with vehicle and Aldh5a1−/− mice treated with rapamycin. Bar, 10 μm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "A Quantification of S6 phosphorylation of liver lysates from WT (n = 5) and Aldh5a1−/− mice treated with vehicle (n = 4) or rapamycin (n = 5) after normalization (WT set to 1).",
    "molecules": "rapamycin"
  },
  {
    "caption": "C Quantification of S6 phosphorylation of brain lysates from WT (n = 2) and Aldh5a1−/− mice treated with vehicle (n = 3) or rapamycin (n = 3) after normalization (WT set to 1).",
    "molecules": "rapamycin"
  },
  {
    "caption": "B. IHLs were isolated from the liver of Tie2-GFP mice intrasplenically injected with NaCl (n=3) or with 5x103 CT26 at the specified time points (day 25 post-injection n=6, day 35 post-injection n=5). The number of GFP+ TEMs was estimated by flow cytometry as 7AAD-/CD45+/CD11b+/CD11c-/GFP+ cells per liver; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.",
    "molecules": "liver, NaCl"
  },
  {
    "caption": "C. Representative images (top panels) and corresponding H&E micrographs (bottom panels) of the liver from control (NaCl-injected, left panels) or Tie2-GFP mice 25 and 35 days (center and right panels, respectively) post-intrasplenic injection of 5x103 CT26. Large metastatic foci in the liver of mice that received CT26 cells are indicated (arrowheads). The dashed line in the H&E panels identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm.",
    "molecules": "NaCl"
  },
  {
    "caption": "D. Liver RNA was extracted from mice transplanted and injected as described in B (NaCl n=4, day 25 n=7, day 35 n=4). The average GFP expression value of NaCl injected mice estimated by quantitative real-time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.",
    "molecules": "NaCl"
  },
  {
    "caption": "E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice that were injected intrasplenically with either NaCl (upper panels) or 5x103 CT26 (bottom panels, 25 days post-injection). Note that TEMs (identified as GFP+, macrophage mannose receptor [MMR]+ and F4/80+ cells) gather in the proximity of CRC metastatic foci (identified by the dashed lines); ✱=CRC metastatic area; scale bars=100μm.",
    "molecules": "NaCl"
  },
  {
    "caption": "F. Eighty-four or 54 days post-NaCl or 5x103 CT26 intrasplenic injection, Sham/CTRL (n=5) or Tie2-IFNα mice (n=7) were infected with LCMV (Armstrong strain, 200 pfu intraperitoneally injected). Eight days post-infection white blood cells were isolated and analyzed for LCMV specific CD8+ T cell response. Left panel: percentage of total CD8+ T cells; middle panel: percentage of CD8+/NP118+ T cells (NP118=recombinant dimeric H-2d/Ig fusion protein complexed with the immune-dominant H-2d-restricted LCMV NP118-126 peptide); right panel: percentage of CD8+/IFNγ+ T cells after in vitro stimulation with virus-specific H-2d-restricted peptide (NP118-126); data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; differences were not statistically significant (by unpaired Student's t-Test).",
    "molecules": "NaCl"
  },
  {
    "caption": "A. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2-GFP or Tie2-IFNαmice that were intrasplenically injected with either NaCl (n=4 and n=4, respectively) or 5x105CT26-RFP and euthanized 5 minutes (n=9, n=5), 3 days (n=6, n=3) and 7 days (n=5, n=3) thereafter. The basal expression of Irf7 estimated in control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed at the following time points post-injection. Data pooled from four independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increase in Irf7 expression levels from the liver of Tie2-GFPmice was statistically significant (p=0.004 by one-way Anova test, not reported on graph).",
    "molecules": "NaCl"
  },
  {
    "caption": "(B, C) Western Blot showing the protein enrichments upon pulldown of the same probes as in (A) incubated with increasing concentrations of purified recombinant GST-Ythdf (B) and His-NT-Fmr1 proteins (C). Quantification of the m6A/A signal intensity is shown below the blot as median +/- SEM of the triplicates. Both proteins bind more efficiently upon methylation.",
    "molecules": "A, m6A"
  },
  {
    "caption": "(D) Results of fluorescence polarization (FP) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites, with or without the methylation, incubated with GST-Ythdf and His-NT-Fmr1. The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8. Binding constants (kd values) were determined by fitting a Michaelis-Menten non-linear regression onto the relative FP values in GraphPad Prism 8, if applicable. The respective binding constants are given in the table next to the graphs.",
    "molecules": "m6A"
  },
  {
    "caption": "(B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA.",
    "molecules": "Benzonase, RNA"
  },
  {
    "caption": "(a,b) Multiple MBds associate with a PC3 cell (a) and a B-lymphoblast (b). Insets: MBd labelling (a) and merged differential interference contrast microscopy image with MBd labelling to show cell boundaries (b). MKLP1, MBd marker (a,b; red); CD44, membrane (a; green); DAPI, DNA (a; blue). Scale bar, 5 μm (a) and 2 μm (b).",
    "molecules": "DNA"
  },
  {
    "caption": "(c,d) Three-dimensional reconstructions of polarized cells in a monolayer (c) and a HeLa cell (d) show intracellular MBds. (c) ZO-1, tight junction; MKLP1, MBds. Scale bar, 2 μm. Enlargement (bottom panel) of the box in the top panel shows five MBds (arrows). (d) Wheat-germ agglutinin, plasma membrane (red); MKLP1-GFP, MBds (green); DAPI, DNA (blue). Scale bar, 5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Electron microscopy of an MBd in a permeabilized MCF-7 cell showing immungold labelling with MKLP1 antibodies. Inset, lower magnification of the MBd (boxed) in the cell; nucleus, right. Scale bar, 200 nm.",
    "molecules": "immungold"
  },
  {
    "caption": "(c,d) Time-lapse images show that the mitotic midbody is preferentially inherited by the daughter cell with the older centrosome in HeLa cells (c) and hESCs (d). Cells were imaged at the indicated times (h:min) from telophase by phase-contrast microscopy (c) and from metaphase by differential interference contrast microscopy (d). Middle panel of (c) and upper left panel of (d): CETN1-GFP at centrosomes; enlargements and integrated intensity profiles show that the daughter cell with the older centrosome (c, upper; d, lower) inherits the MBd (lower right images in c,d). Mitotic midbody and MBds (c,d; arrows). MKLP1, MBd marker (red); α-tubulin, mitotic midbody and cell-boundary marker (green); DAPI, DNA (blue). Scale bars, 10 μm (c,d).",
    "molecules": "DNA"
  },
  {
    "caption": "(d) Representative planes of a neural progenitor cell in the ventricular zone (Sox2+, bottom left panel) of an E13.5 mouse brain show that an intracellular MBd (asterisk) is associated with the ventricle-facing daughter in the asymmetrically dividing cell (top row). The bottom row emphasizes the position of paired chromosomes in a dividing anaphase cell. CD133, midbody/MBd marker (green); Na/K-ATPase, cell-border marker (red); DRAQ5, DNA (blue); DAPI, DNA. v, ventricle. Scale bar, 5 μm. Note that abscission occurs apically in these cells.",
    "molecules": "DNA"
  },
  {
    "caption": "(e) A histological section through a hair follicle (left, phase-contrast microscopy) stained for the stem-cell marker keratin 15 (K15) to identify the bulge region (dotted box), the stem-cell niche. DNA stain (DAPI) and the phase-contrast microscopy image show full follicle architecture. (f) Upper panels show MBd-accumulating cells in the bulge region (boxed) co-labelled with K15 and MKLP1. Enlargements (lower panels) of the boxed region highlight a cell with four MBds (asterisks). N, nucleus. Scale bar, 5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(a) Depiction of FPP assay. Digitonin selectively permeabilizes the plasma membrane but not internal membranes. Proteinase K degrades cytoplasmic components but membranous compartments remain intact. Under these conditions, MKLP1-GFP-labelled MBds (blue ellipse) in the cytoplasm will be degraded whereas those inside membrane-bound compartments will not. (b) MBds in MBd-poor hRPE-1 cells are largely protected (∼90% in membranous compartments, 10 cells analysed), whereas most MBds in HeLa cells are not (∼27%, 11 cells analysed), and are thus degraded in cytoplasm. Scale bar, 5 μm.",
    "molecules": "Digitonin, Proteinase K"
  },
  {
    "caption": "(c) Presence of MBds in lysosomes on chloroquine or E64d-pepstatin A (E64d-PepA) inhibition in hRPE-1 and HeLa cells, but not in MCF-7 and H9 hESCs. Chloroquine treatment of H9 hESCs is not included as it caused differentiation and cell death. A representative image of hRPE-1 cells inhibited by chloroquine is shown, depicting two MBds inside lysosomes. MKLP1 and LAMP2 are used as MBd (red) and lysosome (green) markers, respectively. DAPI, DNA (blue). n=100 MBds per treatment in each of the biological triplicates. Scale bar, 5 μm.",
    "molecules": "chloroquine, Chloroquine, DNA, E64d, pepstatin A"
  },
  {
    "caption": "(e) Both chloroquine and E64d-PepA treatments increase the percentage of MBd+ cells in hRPE-1 cells and HeLa cells (chloroquine, P=0.0021 and P=0.0187, respectively; E64d-PepA, P=0.0022 and P=0.0043, respectively; n=3 for all experiments). In contrast, lysosomal inhibition has no detectable effect on hESCs (H1, H9) or MCF-7 cancer cells. DMSO, dimethylsulphoxide. Data are presented as mean±s.d. (c-e), except mean±s.e.m. in hESCs (e).",
    "molecules": "chloroquine, dimethylsulphoxide, DMSO, E64d, PepA"
  },
  {
    "caption": "(c) Rapamycin (Rapa) and LiCl co-treatment induces autophagy and decreases the percentage of MBd+ cells (left, HeLa; P=0.0056, n=3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by overexpression of Flag-tagged BECN1 reduces the percentage of MBd+ cells (right, MCF-7; P=0.0008, n=4). Ctrl, control.",
    "molecules": "LiCl, Rapamycin"
  },
  {
    "caption": "(d) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d-PepA. U, uninhibited. I, inhibited. Below, the average of the percentage change in LC3-II levels after lysosomal inhibition from three experiments. α-tubulin, loading control.",
    "molecules": "E64d, PepA"
  },
  {
    "caption": "(e-g) Overexpression of CEP55-eGFP (enhanced green fluorescent protein) increases the percentage of MBd+ cells (e; P=0.0007, n=3) and the percentage of NBR1-negative MBds (f; P=0.0568, n=3), presumably by sequestering NBR1 (red) away from MBds in cells expressing CEP55-eGFP (green) as shown in g, and consequently preventing MBd degradation. The dotted box in g is enlarged (top right panel), and the labelling of NBR1 and CEP55-eGFP (middle and bottom right panels) are also presented. DAPI, DNA (blue). Scale bar, 5 μm. The data are presented as mean±s.d. (b,c,e,f). Uncropped images of blots are shown in Supplementary Fig. S6.",
    "molecules": "DNA"
  },
  {
    "caption": "(K) Western blot analysis for phospho-TBK1 (Ser172) in WT and MAVS-knockdown BMMs treated for 4 hr with EVs from uninfected or M.tb-infected macrophages. β-actin served as a loading control. Densitometry of the western blots for (K) are shown.",
    "molecules": "Ser"
  },
  {
    "caption": "(A and B) Quantitative analysis of immunofluorescence microscopy images for colocalization of M.tb with Lamp-1 in WT (A) and Mavs -/- (B) BMMs infected with M.tb for 24 hr and then treated for an additional 24 hr with moxifloxacin (MFX) and/or EVs from M.tb-infected BMMs (EVs+MFX) in the presence of recombinant mouse IFN-ɣ. Mock, no EVs or moxifloxacin treatment; EVs_Control, EVs from uninfected BMMs. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).",
    "molecules": "MFX, moxifloxacin"
  },
  {
    "caption": "(I and J) M.tb CFU analysis in WT (I) and Mavs -/- (J) BMMs infected with M.tb for 24 hr followed by treatment with EVs, moxifloxacin or combination in the presence of IFN-ɣ for 24 and 72 hr. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).",
    "molecules": "moxifloxacin"
  },
  {
    "caption": "Representative histopathological analysis for lung sections of WT (B) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM",
    "molecules": "moxifloxacin, MXF"
  },
  {
    "caption": "M.tb CFU in the lung and spleen of WT (C) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).",
    "molecules": "MXF"
  },
  {
    "caption": "Representative histopathological analysis for lung sections of Mavs -/- (D) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μM",
    "molecules": "moxifloxacin, MXF"
  },
  {
    "caption": "M.tb CFU in the lung and spleen of Mavs -/- (E) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).",
    "molecules": "MXF"
  },
  {
    "caption": "ELISA analysis for, IFN-β, TNF-α, IL-1β and IFN-γ protein level in serum isolate from M.tb-infected WT (F) or Mavs -/- (G) mice treated with EVs, MXF or a combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).",
    "molecules": "MXF"
  },
  {
    "caption": "D MCF-7 cells were transfected with the indicated siRNAs. Cell sensitivity to camptothecin (CPT), hydroxyurea (HU), etoposide (ETO), or ionizing radiation was determined by MTS assays. Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.",
    "molecules": "camptothecin, CPT, ETO, etoposide, HU, hydroxyurea"
  },
  {
    "caption": "B BGL3 promotes RAP80 binding to K63 ubiquitin chain in cells. 293T cells expressing the indicated siRNAs and plasmids were irradiated (10 Gy). Cell lysates were immunoprecipitated with HA beads and subjected to immunoblot with the indicated antibodies.",
    "molecules": "K63, ubiquitin"
  },
  {
    "caption": "A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs. U2OS cells were treated with DMSO, PARP inhibitor (olaparib), ATM inhibitor (KU-55933), ATR inhibitor (VE-821) or caffeine. BGL3 recruitment to DNA damage sites was assessed by RNA-FISH assays. B Quantification of the positive cells. For each group, 100 randomly selected cells were counted. Data are presented as mean ±SD of three biological replicates. Two-tailed Student's t test, **P < 0.01, N: no significant difference. ",
    "molecules": "caffeine, DNA, DMSO, KU-55933, olaparib, VE-821"
  },
  {
    "caption": "C PARP inhibitor mainly affected BGL3 recruitment at an early time point. U2OS cells were treated with DMSO or olaparib, and BGL3 recruitment was examined following laser micro-irradiation at indicated time point. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.",
    "molecules": "DMSO, olaparib"
  },
  {
    "caption": "(B) Representative metaphase spreads from wild-type and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid or EV control. Telomeric signals were detected with Cy3-OO-(CCCTAA)3 and are false colored in red, DNA is stained with DAPI and is false colored in cyan. Scale bar: 5µm.",
    "molecules": "Cy3, DAPI, DNA"
  },
  {
    "caption": "(D) Representative cell cycle profiles from two biological replicates of WT and SMCHD1 KO HeLa cells transfected with shTRF2 plasmid or EV control. Cells were stained with propidium iodide and analyzed by Flow cytometry.",
    "molecules": "propidium iodide"
  },
  {
    "caption": "(A) Western Blot detection of SMCHD1, TRF2 and hnRNPA1 in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) Quantification of telomere fusions in SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated with or without doxycycline for the indicated number of days (d7, d8, d11). Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>1,900 telomeres counted/condition/experiment). (***) P < 0.001, (**) P< 0.01; (*) P< 0.05; unpaired two-tailed Student's t-test.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C) Terminal Restriction Fragment (TRF) analysis of telomeric DNA to detect 3'overhang processing of genomic DNA isolated from SMCHD1 wild-type or SMCHD1 knockout HeLa inducible shTRF2 cells treated as in the experiment in (B). (Left) Radiolabeled (CCCTAA)n probe was hybridized with a short run (upper panel) and long run (lower panel) native DNA gel to detect the signal of the telomeric 3' overhang. Samples used for the short and the long run were from the same digestion split into two. Exo I treatment (+ Exo) was used as a control that single stranded telomeric signal was terminal. (Right) The total TTAGGG signal in the same lane was deteced upon denaturation and hybridization with the same probe.",
    "molecules": "DNA, genomic DNA"
  },
  {
    "caption": "(D) Quantification of the telomeric overhang signal at d11 after doxycyclin addition to SMCHD1 wild-type and SMCHD1 knockout HeLa shTRF2 inducible cells. The bar graph represents the average overhang signal intensity from two biological replicates as percentage of the signal in the cells untreated with doxycycline with error bars representing the standard deviation (SD) of the sample.",
    "molecules": "doxycyclin, doxycycline"
  },
  {
    "caption": "B Representative images for CO-FISH on metaphase spreads in wild type (WT) Hela and U2OS cells. The leading strand telomeres were labeled by PNA-FISH with a (TTAGGG)3-Alexa488 probe (green), the lagging strand with a (CCCTAA)3-Cy5 probe (pink), and the DNA was stained with DAPI. Examples of telomere sister chromatid exchange (t-SCEs) events are marked with white arrows. Scale bar: 5µm",
    "molecules": "Alexa488, Cy5, DAPI, DNA"
  },
  {
    "caption": "(A-C) Representative images for detection of ATM pS9181, γH2AX, and 53BP1 at telomeres in wild-type (WT) and SMCHD1 knockout HeLa cells transfected with shTRF2 plasmid and empty vector (EV) control. Immunofluoresence (IF) for ATM pS1981 (gray), γH2AX (green) and 53BP1 (yellow) was combined with telomeric (CCCTAA)3-FISH (red) and DAPI staining total DNA. Scale bars: 5µm",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(C) Representative metaphase spreads (Scale bar: 5µm) from HeLa cells transfected with indicated shRNAs or EV control treated for 4 days with ATRi (VE-821) and quantification of telomere fusions. Bars represent average number of fused chromosome ends. SDs were obtained from 3 independent experiments (>2,000 telomeres counted/condition/experiment). (***) P < 0.001; (**) P < 0.01; (*) P < 0.05 unpaired two-tailed Student's t-test.",
    "molecules": "VE-821"
  },
  {
    "caption": "Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are given for (A) 48 antihypertensive drugs The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (B) , for the analysis on the 48 antihypertensive drugs CCBs, calcium channel blockers; ARBs, angiotensin II type-I receptor blockers; ACEIs, angiotensin-converting enzyme inhibitors",
    "molecules": "angiotensin II type-I receptor blockers, ARBs, calcium channel blockers, CCBs, ACEIs, angiotensin-converting enzyme inhibitors"
  },
  {
    "caption": "Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The ACE2 expression levels in control and drug-treated groups for the top significant drugs (vorinostat, panobinostat, and isotretinoin) among the 672 clinically approved drugs are shown with boxplots in (D), the ACE2 expression log fold-change values and adjusted P values from limma, as well as the number of samples per group are labeled for each drug. In the boxplot of panel (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots.",
    "molecules": "isotretinoin, panobinostat, vorinostat"
  },
  {
    "caption": "A) Seeding efficiency was compared between control non-neurodegenerative and FTLD-TDP patient samples. Cells were transfected using Lipofectamine after mitotic arrest with AraC and were fixed 3 to 6 days post-transfection with SarkoSpin fractions (Laferrière et al, 2019). Surface masks generated with Imaris show DAPI (blue), nuclear TDP-43 (NuclTDP-43, cyan) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 20 μm as indicated in AraC panel Day 6. B) Higher magnification imaging of cells seeded for 6 days revealed presence of large cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-A and multiple cytoplasmic TDP-43-HA aggregates in cells seeded with FTLD-TDP-C. Surface representation generated with Imaris showing DAPI , TDP-43-HA (green), phosphorylated TDP-43 (pTDP-43, magenta) and neoaggregates (NeoAgg, orange). All images are presented at the same scale, 5 μm as indicated in FTLD-TDP-C top panel.",
    "molecules": "AraC, DAPI, Lipofectamine"
  },
  {
    "caption": "C) TOP: Aggregation kinetics of full length (fl) recombinant human TDP-43 (rec-hTDP-43) measured as an increase in absorbance. The addition of TEV protease induces the cleavage of the MBP tag and subsequent aggregation of rec-hTDP-43. Rec-hTDP-43 aggregates used for seeding were collected 18 hours after the addition of TEV protease. BOTTOM LEFT: rec-hTDP-43 aggregates stained by Thioflavin T (ThT). BOTTOM RIGHT: Transmission Electron Microscopy (TEM) image of the rec-hTDP-43 aggregates. All images are presented at the same scale of 1 μm.",
    "molecules": "Thioflavin T, ThT"
  },
  {
    "caption": "E) Quantification of neoaggregates (left y-axis) and pTDP-43 (right y-axis) at day 5 and day 6 post-transfection for increasing concentrations of rec-hTDP-43. 2-way ANOVA repeated measure, with post-hoc Fisher's LSD, F(7,80)=5.886, p<0.0001, Day 5: AraC vs 50 ng p=0.0497, AraC vs 500 ng p=0.0002, AraC vs 5 µg p=0.0161. 50 ng vs 100 ng p=0.0208, 50 ng vs 500 ng p=0.0169, 50 ng vs 2.5 µg p=0.0272, 100 ng vs 1 µg p<0.0001, 100 ng vs 5 µg p=0.006, 250 ng vs 1 μg p=0.0005, 250 ng vs 5 µg p=0.034, 500 ng vs 1 µg p<0.0001, 500 ng vs 5 µg p=0.0047, 1 µg vs 2.5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0081, Day 6: AraC vs 250 ng p=0.0385, AraC vs 1 µg p=0.0006, 50 ng vs 1 µg p=0.0016, 100 ng vs 1 µg p=0.0101, 100 ng vs 5 µg p=0.0173, 250 ng vs 5 µg p=0.0006, 500 ng vs 1 µg p=0.0099, 500 ng vs 5 µg p=0.0176, 1 µg vs 2.5 µg p=0.0144, 1 µg vs 5 µg p<0.0001, 2.5 µg vs 5 µg p=0.0121. To reduce complexity, only comparisons with AraC are marked in the graph. N=6 biological replicates for each concentration; bar graph showing mean ± SEM.",
    "molecules": "AraC"
  },
  {
    "caption": "qRT-PCR of whole tissue from small intestine and colon. Expression of mRNA (2(−dCt)) calculated relative to GAPDH (N = 3 mice). Statistics: one-sided Mann-Whitney U-test.",
    "molecules": "mRNA"
  },
  {
    "caption": "C, Immunoblot analysis of protein lysates from MEF cells treated with EBSS, 250 nM Torin-1 or 100 μM NaAsO2 for 1h and run under non-reducing conditions. For refeed condition, starved cells were incubated in complete medium for the indicated times.",
    "molecules": "NaAsO2, Torin-1"
  },
  {
    "caption": "C, Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-FLAG-WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h and run under non-reducing condition.",
    "molecules": "NaAsO2"
  },
  {
    "caption": "D, Immunoblot analysis of protein lysates from ARPE-19 cells overexpressing TFEB-FLAG or TFE3-MYC, treated with 250 nM Torin-1 for 1 h and run under non-reducing condition. E, Quantification of immunoblot data shown in (D). Data are presented as mean ± SD using two-tailed t-test (*)p<0.05 from three independent experiments. ",
    "molecules": "Torin-1"
  },
  {
    "caption": "G, Relative quantitative RT-PCR analysis of the mRNA expression of lysosome-related genes (MCOLN1 and ATPV61C1) in ARPE-19 and HeLa cells treated with 100 μM and 50 μM NaAsO2 respectively for the indicated times. Data are presented as mean ± SD using one-way ANOVA (*)p<0.05, (**)p< 0.01, (***)p<0.001 from two independent experiments.",
    "molecules": "NaAsO2"
  },
  {
    "caption": "D, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), lysosome- (ATP6V1C1), oxidative stress response- (TXNRD1 and HMOX1), cell cycle control- (CDKN1a) and cytokine production (IL6) -related genes in TFEB/TFE3-double knockout MEFs cells stably expressing TFEB-FLAG-WT or TFEB-FLAG-C212A and treated with 5 μM arsenic trioxide (ATO) or 12.5 μM NaAsO2 for 12 h. Data are presented as mean ± SD using two-way ANOVA (*)p<0.05, (**)p<0.01, (***)p<0.001, and (****)p<0.0001 from three independent experiments.",
    "molecules": "arsenic trioxide, ATO, NaAsO2"
  },
  {
    "caption": "A, Lifespan assay of hlh-30 mutants at 25°C. Graph represents Kaplan-Meier survival plots of two independent experiments initiated with 100 animals per strain. (**)p<0.01 and (****)p<0.0001 by Log-rank (Mantel-Cox) test compared to wild type control. B, Survival of hlh-30 mutants on 7.5 mM NaAsO2. Data are the mean ± SD of three independent experiments initiated with 120 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control. C, Survival of hlh-30 mutants to infection by Staphylococcus aureus. Data are the mean ± SD of three independent experiments initiated with 100 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control. ",
    "molecules": "NaAsO2"
  },
  {
    "caption": "Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) (in the top well) subjected to the indicated compounds at the indicated concentration; AGN and BMS are antagonists of RAR, DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA, and HX is an antagonist of RXR;   Fraction of differentiated CD142- ASPCs indicated in G, as quantified by the \"adiposcore\"; marker shapes correspond to different biological replicates, n=4-15, 2-5 biological replicates, 2-3 independent wells for each;  ",
    "molecules": "DEAB, AGN, retinaldehyde, BMS, HX, RA"
  },
  {
    "caption": "Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) subjected to control (scr) or siRNA-mediated knockdowns of selected RA signalling pathway genes: Rara, Rarb, Rarg, Rbp1, and Rxrg after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods);   Fraction of differentiated CD142- ASPCs indicated in I, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=24-29, 4 biological replicates, 5-8 independent wells for each.  ",
    "molecules": "RA"
  },
  {
    "caption": "A. Representative FACS profiles of HSPC cell cycle analysis using combinatorial staining for Ki67 and Hoechst 33342. Bar charts depict the average percentage of cells in each phase of the cell cycle for each LSK subset from WT (n=6) and PKCδ KO (n=7) mice. Data compiled from two independent experiments. Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "B. FACS profiles of indicated BM subsets from WT and PKCδ KO mice 20hr after BrdU injection. Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.",
    "molecules": "BrdU"
  },
  {
    "caption": "D. Representative FACS plots and summary of FACS data analyzing the frequency of apoptotic HSPC cells using co-staining for AnnexinV and 7-AAD (WT and PKCδ−/− (n=7 per genotype). Data information: All data are presented as mean±SEM, *p<0.05 and **p<0.001, with significance determined by two-tailed Student's unpaired t-test analysis.",
    "molecules": "7-AAD"
  },
  {
    "caption": "B,C Representative FACS plots of PKCδ fl/fl and cKO BM 4-8 weeks (B), and 20-24-weeks (C) after pIpC treatment. Numbers in plot indicate the absolute numbers of the indicated subset within the gated population (n=8-9 mice per genotype and time point). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.",
    "molecules": "pIpC"
  },
  {
    "caption": "G. Average percentage BrdU+ HPSCs (3 days after BrdU injection into mice reconstituted 24 weeks previously with control or cKO marrow and injected 16 weeks previously with pIpC among donor-derived LSK cells in the control and cKO chimeras BM. Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.",
    "molecules": "BrdU, pIpC"
  },
  {
    "caption": "H. Percentages of apoptotic cells (AnnexinV+/7-AAD-) in donor-derived LSK cells in control and cKO chimera mouse BM at 24 weeks after transplant and 16 weeks after pIpC injection. Data are compiled from two independent experiments (total of n=7-8 mice per genotype). Data information: Data presented as mean ± SEM. *p<0.05, **p<0.005 and ***p<0.001 by two-tailed Student's unpaired t-test analysis.",
    "molecules": "7-AAD, pIpC"
  },
  {
    "caption": "B. Kinetics of LSK cell recovery. Representative FACS profiles of BM LSK cells from WT and cKO mice at the indicated time points after 5-FU treatment. Numbers in the contour plots indicate the percentages of LSK subsets among Lin- live cells. C. Average absolute numbers of BM LSK cells at day13 and day28 after 5-FU treatment. Data are pooled from two independent experiments. (total of n=6-8 mice per time point and genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with Sidak's multiple comparisons tests (C) *p<0.05, **p<0.01 and ***p<0.001.",
    "molecules": "5-FU"
  },
  {
    "caption": "D. Quantification of apoptotic HSPCs at the indicted time points after 5-FU treatment of control and cKO mice (n=6 per genotype). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs by two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001.",
    "molecules": "5-FU"
  },
  {
    "caption": "E. Representative FACS plots showing analysis of in vivo BrdU incorporation assays. Mice were received 2mg of BrdU (i.p.) at d13 (left) or d28 (right) after 5-FU treatment. 20hrs later, BrdU incorporation in BM LSK cells was assessed using anti-BrdU antibodies. Bar chart at right indicates percent of BrdU+ LSK cells at d13 and d28 after 5-FU treatment (n=6 per genotype and time point). Data information: Data presented as mean ± SEM. The statistical significance was assessed using repeated-measures two-way ANOVAs with two-tailed Student's unpaired t-test analysis (D and E). *p<0.05, **p<0.01 and ***p<0.001.",
    "molecules": "BrdU, 5-FU"
  },
  {
    "caption": "F. Kaplan-Meier survival cures of control (n=12) and cKO (n=11) mice after sequential 5-FU treatment. Mice were treated with 5-FU (150mg/kg, i.p.) once each week for three weeks. Arrows indicate time points of 5-FU injection. Data information: Data presented as mean ± SEM. Survival data (F) were analyzed using a log-rank nonparametric test (Mantel-Cox test) (p=0.0141).",
    "molecules": "5-FU"
  },
  {
    "caption": "A. MitoStress test revealed increased oxygen consumption rates (OCR) in PKCδ-deleted LSKs. BM LSK cells were sorted from WT and PKCδ cKO mice 12-weeks after pIpC-treatment. OCR was measured at basal level and after sequential loading of ATP synthase inhibitor Oligomycin (350nm), mitochondrial uncoupler, FCCP (10μM), and electron transport chain inhibitor, Rotenone (1μM) using Seahorse XF24 extracellular flux analyzer. Data are from 3 independent experiments (n=9 mice). In each experiment, bone marrow cells from identical genotypes (n=3 mice per genotype) were pooled and used for LSK cell sorting. Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.",
    "molecules": "FCCP, Oligomycin, pIpC, Rotenone"
  },
  {
    "caption": "(B) Basal OCR was measured before inhibitors treatment (left); and the Maximal OCR capacity after FCCP treatment (right). (n=9 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.",
    "molecules": "FCCP"
  },
  {
    "caption": "D, E. (D) Glycolysis Stress test. Extracellular acidification rates (ECAR) of LSKs were measured at basal conditions (unbuffered assay medium without glucose) and after sequential loading of glucose (7.5mM), Oligomycin (350nm), and 2-deoxyglucose (50mM; 2DG, a glucose analog). (E). AUC of baseline ECAR levels (left), and glycolytic capacity (maximal ECAR) was calculated from the data shown in panel D (n=7-8 per genotype, from 3 independent experiments). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.",
    "molecules": "2-deoxyglucose, 2DG, glucose, Oligomycin"
  },
  {
    "caption": "F. Relative ATP content in indicated HSPC subsets determined using an ATP assay kit (n=6 mice per genotype). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.",
    "molecules": "ATP"
  },
  {
    "caption": "G. Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice. Relative MitoTracker Green mean fluorescence intensity (MFI) is quantified below for n=6 mice of each genotype. Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.",
    "molecules": "MitoTracker Green"
  },
  {
    "caption": "I. ROS levels in control and PKCδ cKO HSPCs as measured by FACS based CM-H2DCFDA staining. Bar graph at right shows the relative CM-H2DCFDA MFI. Data compiled from three independent experiments (n=6-8 per genotype). Data information: The statistical significance of difference was assessed using two-tailed Student's unpaired t-test analysis. All data are presented as mean± SEM., *p<0.05, **p<0.001.",
    "molecules": "CM-H2DCFDA, ROS"
  },
  {
    "caption": "Confocal microscopy reveals typical \"class B\" pattern of βarr1 recruitment for V2R and B2R as reflected by first the localization at the plasma membrane and subsequently, internalization in endosomal vesicles upon agonist-stimulation. HEK-293 cells expressing V2R/B2R and βarr1-mCherry were stimulated with agonist (AVP; 100nM and Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring of images from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 77% of the cells for V2R (221 cells) and 75% of the cells for B2R (662 cells).",
    "molecules": "Bradykinin, AVP"
  },
  {
    "caption": "Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist-stimulation. HEK-293 cells expressing either B2RWT or B2R mutants along with βarr1-mCherry were stimulated with agonist (Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 75% of the cells for B2RWT (662 cells), 74% of the cells for B2RΔG368 (169 cells), 75% of the cells for B2RL370T (130 cells), 85% of the cells for B2RΔG368+L370T (132 cells) and 77% of the cells for B2RΔI374 (116 cells).",
    "molecules": "Bradykinin"
  },
  {
    "caption": "HEK-293 cells expressing B2R constructs with C-terminal SmBiT and βarr1 with N-terminal LgBiT were treated with indicated concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism, and pEC50 (top) and Emax (bottom) were calculated. Data represent mean ± SEM of five independent experiments (three for B2RΔI374), each performed in duplicate.",
    "molecules": "bradykinin"
  },
  {
    "caption": "Knock-down of βarr1 leads to an increase in agonist-induced ERK1/2 phosphorylation for B2RΔG368 and B2RΔI374 but a decrease for B2RL370T and B2RΔG368+L370T mutants. HEK-293 cells expressing the indicated B2R mutants in presence and absence of βarr1 knock-down were stimulated with indicated doses of bradykinin (Brady) for 10 min followed by detection of phosphorylated ERK1/2 using Western blotting. Densitometry-based quantification of data (mean±SEM) from three independent experiments (five for B2RΔG368 and B2RΔI374) is presented as bar graphs in the lower panels. Data are normalized with respect to maximal signal under control condition (treated as 100%), and analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (*p <0.05; **p <0.01; ***p<0.001).",
    "molecules": "Brady, bradykinin"
  },
  {
    "caption": "HEK-293 cells expressing V2R, B2R, B2RΔG/L370Tor B2RL370T along with βarr1-mCherry and YFP-tagged intrabody30 (Ib30-YFP) were stimulated with their respective agonists (AVP, 100nM for V2R) and (bradykinin, 1μM for B2R) and the localization of βarr1 and Ib30 were visualized. Although βarr1 gets recruited for all four constructs, Ib30-YFP does not colocalize with βarr1 either at the surface or upon internalization in case of B2RWT. Representative data from 3-5 independent experiments are shown and the scale bar is 10μm. Visual scoring from 3-5 independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 82% of the cells for V2R (691 cells), 10% for B2RWT (254 cells), 63% for B2RL370T (273 cells) and 83% for B2RΔG368+L370T (158 cells). Pearson's correlation coefficients (PCC) were measured to assess the colocalization of βarr1 and Ib30 using JACoP plugin in ImageJ, and the mean±sem for the basal, surface and internalized panels, respectively, are presented here. B2RWT - 0.32±0.02 from 16 cells, 0.18±0.01 from 32 cells, 0.25±0.02 from 31 cells; B2RΔG/L370T - 0.32±0.03 from 13 cells, 0.92±0.01 from 18 cells, 0.90±0.01 from 16 cells; B2RL370T - 0.26±0.01 from 10 cells, 0.85±0.01 from 11 cells, 0.87±0.01 from 25 cells.",
    "molecules": "bradykinin, AVP"
  },
  {
    "caption": "HEK-293 cells expressing the NanoBiT-Gq protein and indicated B2R constructs were treated different concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism to calculate pEC50 (top) and Emax(bottom) values included in the graph. Data represent mean±SEM of three independent experiments with each performed in duplicate, and normalized with respect to the luminescence signal under basal (no-agonist treatment) condition (treated as 1).",
    "molecules": "bradykinin"
  },
  {
    "caption": "HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).",
    "molecules": "AngII, angiotensin II"
  },
  {
    "caption": "HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five (six for AT1aRΔL337) independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).",
    "molecules": "AngII, angiotensin II"
  },
  {
    "caption": "B. Volcano plot showing DE 1hr after NB1 (top) and IMR32 (bottom) cell treatment with ALKAL2. Dashed lines show DE thresholds. Up-/downregulated genes indicated in blue. Six genes that are DE in both cell lines and sensitive to the ALK inhibitor lorlatinib are indicated and labelled in red.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "D. Temporal dynamics of ALKAL2-induced transcription of ARC, EGR1-3, FOS and FOSB in NB1 and IMR32 cells in the presence and absence of the lorlatinib, as indicated.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "E. Immunoblot validation of ALKAL2 induction of EGR1 and FOS at the protein level in NB1 cells. Cells were treated for 0, 1 and 6h in the presence and absence of lorlatinib as indicated.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "G. Immunoblot analysis of ALKAL2 induction of pSRF in NB1 cells. Cells were treated for 0 and 1h in the presence and absence of lorlatinib as indicated. RNA-Seq analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "B. Volcano plots showing differential protein expression 1hr and 24hr after ALKAL2 +/- lorlatinib stimulation as indicated. Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "C, D. Immunoblot analysis of VGF protein in NB cells. (C) NB1 cells after 24h stimulation with ALKAL2 in the presence or absence of lorlatinib. (D) CLB-BAR and CLB-GE cells after 24h inhibition with lorlatinib.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "G. Graphical representation of FOXO3 dynamics, indicating S253 phosphorylation and total FOXO3 protein levels in response to ALKAL2 stimulation, in the presence or absence of lorlatinib.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "H-I. Immunoblot validation of FOXO3a and STAT3 in response to ALKAL2 stimulation in the presence or absence of lorlatinib as indicated. The slower migrating FOXO3a band in SDS-PAGE in (H) likely reflects FOXO3a phosphorylation that is not seen in the presence of lorlatinib. Proteomic analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.",
    "molecules": "lorlatinib, SDS"
  },
  {
    "caption": "B. The effect of increasing concentrations of brigatintib on cell confluence was analyzed by IncuCyte Live Cell Analysis of both Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) cell lines. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.",
    "molecules": "brigatintib"
  },
  {
    "caption": "C-D. Brigatinib suppressed tumour spheroid formation and spheroid viability. Cells (#3456 or #3540) were treated with brigatinib (0, 150nM) for 4d in ultra-low attachment plates. The spheroid number was analyzed by IncuCyte® Live Cell Analysis Systems. Data are presented as means ± SEM from three independent experiments. *p<0.05, two-tailed unpaired student t-test. Scale bar (C) is 200μm.",
    "molecules": "Brigatinib, brigatinib"
  },
  {
    "caption": "E. Tumour spheroids formed from either Rosa26_Alkal2;Th-MYCN (#3540) or Alk-F1178S;Th-MYCN (#3456) were formed at indicated cell number in ultra-low attachment plates for 3d and followed by brigatinib (0, 150nM) for 10d. Inhibitor was re-fed every other day. Cell viability was determined by CellTiter-Glo 3D cell viability kit and data is presented as mean ± SEM from five independent experiments. *p<0.05, ****p<0.0001, two-tailed unpaired student t-test.",
    "molecules": "brigatinib"
  },
  {
    "caption": "F. Cell viability. Mouse tumour derived cell lines #3540 (Rosa26_Alkal2;Th-MYCN) and #3456 (Alk-F1178S;Th-MYCN) were treated with brigatinib (125nM and 500nM) and viability was evaluated by using a resazurin-based assay. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.",
    "molecules": "brigatinib"
  },
  {
    "caption": "G. Brigatinib treatment (0 or 150 nM) for 6h resulted in inhibition of ALK phosphorylation, and of activation of downstream signaling (ERK1/2), as well as MYCN expression. Cell lysates (Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456)) were immunoblotted with the indicated antibodies.",
    "molecules": "Brigatinib"
  },
  {
    "caption": "A. Cells derived from tumours arising in Rosa26_Alkal2;Th-MYCN (#4953) and Alk-F1178S;Th-MYCN (#4938) mice are sensitive to both lorlatinib and brigatinib. The effect of increasing concentrations of each ALK TKI (as indicated) on cell confluence was analyzed by IncuCyte Live Cell Analysis. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.",
    "molecules": "brigatinib, lorlatinib"
  },
  {
    "caption": "B. Tumour volume changes over time for Rosa26_Alkal2;Th-MYCN mice treated with lorlatinib (10 mg/kg; twice daily) or vehicle control. Tumour volume was measured by ultrasound on Day 0 and 7, and by direct measurement at Day 14. Day 0 (lorlatinib n=7, Ctrl n=6), Day 7 (lorlatinib n=2, Ctrl n=5) and Day 14 (lorlatinib n=7, Ctrl n=6). Data shown represents mean ± SD. **p<0.005; two-tailed unpaired student t-test.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "C. Rosa26_Alkal2;Th-MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls. Data shown represents mean ± SD.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "E. Representative MRI imaging of Rosa26_Alkal2;Th-MYCN tumours in response to lorlatinib at 4d and 14d.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "F. Rosa26_Alkal2;Th-MYCN tumours from lorlatinib or vehicle controls were analysed for pHis. A representative field of view for each tumour at 40x (175740μm2) were manually counted. Data shown represents mean ± 95% CI. p=0.0286; Mann-Whitney test.",
    "molecules": "lorlatinib"
  },
  {
    "caption": "B, C. Distribution of the m6A peaks across host mRNAs (B) and EBV mRNAs (C) was analyzed based on the MeRIP-seq data.",
    "molecules": "m6A"
  },
  {
    "caption": "B. The BZLF1 m6A levels were measured using MeRIP-qPCR in CNE2EBV, Raji and B95-8 cells with latent EBV infection or lytic reactivation. TPA (30 ng/ml) and NaB (2 mM) were used to treat the cells for 24 h to reactivate EBV. The cellular RNA was harvested for MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The mean value of the results in mock-treated cells was normalized to 1.",
    "molecules": "m6A, TPA, NaB"
  },
  {
    "caption": "B. The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated (24 h post NaB treatment) CNE2EBV cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) or mock for 24 h, followed by qRT-PCR analysis. All the gene expression levels were normalized to the housekeeping gene ACTB.",
    "molecules": "NaB"
  },
  {
    "caption": "F. The protein level of YTHDF1, BZLF1 and ACTB in CNE2EBV cells following EBV reactivation (24 h post-NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. The whole-cell protein was extracted at same time as the RNA.",
    "molecules": "NaB"
  },
  {
    "caption": "D. The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2, siYTHDF3 or siNC siRNA following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using YTHDF1-specific antibody or IgG control in these cells. Fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.",
    "molecules": "TPA, NaB"
  },
  {
    "caption": "E, F. The relative m6A and YTHDF1 enrichment of the BZLF1 (E) or BRLF1 (F) mRNAs measured in CNE2EBV cells transfected with the METTL14-specific siRNAs (siM14) or siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h, followed by MeRIP and RIP assays. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.",
    "molecules": "m6A, TPA, NaB"
  },
  {
    "caption": "G. The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP, DDX17, or DCP2 in CNE2EBV cells following EBV reactivation. CNE2EBV cells were transfected with plasmids encoding myc-ZAP, myc-DDX17, myc-DCP2, or vector control. At 24 h post transfection, the cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using Myc-beads. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.",
    "molecules": "TPA, NaB"
  },
  {
    "caption": "A. Maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA (red) combined with immunostaining for L1 ORF1p (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. White arrow heads point to cytoplasmic foci where L1 RNA and ORF1p co-localize. Scatter plot shows the number of co-localized L1 Tf-ORF1 foci in the cytoplasm per cell (n=275 WT, 304 Dicer_KO1, 311 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 RNP foci. Bar graphs are mean values of percentage of cells with large L1 Tf-ORF1 foci co-localizing in the cytoplasm (n=3 technical replicates).",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (red), MOV10 (green) and nuclei stained with DAPI (blue). The grey square marks position of inset in the zoomed image. White arrow heads point to cytoplasmic foci where L1 ORF1p and MOV10 co-localize. Scatter plot shows the number of co-localized ORF1-MOV10 foci in the cytoplasm per cell (n= 293 WT, 295 Dicer_KO1, 295 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 ORF1-MOV10 foci. Bar graphs are mean values of percentage of cells with large ORF1-MOV10 foci co-localizing in the cytoplasm (n=3 technical replicates).",
    "molecules": "DAPI"
  },
  {
    "caption": "A. WT and Dicer_KO mESCs were treated with 0.5mM Sodium Arsenite (NaAsO2) for 20 minutes or left untreated prior to fixation with formaldehyde. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for G3BP1 (red) and MOV10 (green) with nuclei stained with DAPI (blue).",
    "molecules": "DAPI, NaAsO2, Sodium Arsenite"
  },
  {
    "caption": "B. Representative western blots showing low AGO2 protein levels in Dicer_KO as compared to WT mESCs (right side). LAMINB1 served as loading control. On the left, maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA FISH (red) combined with immunostaining for a resident protein of P-bodies, DDX6 (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. Yellow arrow heads point to cytoplasmic foci where L1 RNA and DDX6 protein co-localize.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Representative western blots showing L1 ORF1p and MOV10 levels in the indicated cell lines. Immunoblot with antibody recognizing TUBULIN and coomassie stained membranes depict the loading, asterisk marks position of non-specific band in the ORF1 immunoblot (n=3 technical replicates).",
    "molecules": "coomassie"
  },
  {
    "caption": "B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (green) combined with RNA FISH for L1 Tf RNA (red) and nuclei stained with DAPI (blue) (n=3 technical replicates).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Representative images of BlastR colonies stained with crystal violet blue of indicated cell lines is shown on the left. Cells were transfected with either mutant reporter plasmid (L1N21A) or retrotransposition competent reporter (L1WT) as shown in the scheme with timeline for the experiment on the top. Bar graphs on the right depict the average number of BlastR colonies (n=3 biological replicates).",
    "molecules": "Blast, crystal violet"
  },
  {
    "caption": "A. Scheme of experiment and representative images along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression. Images are maximum intensity projections across Z stacks from indicated mESCs stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10 tagged with HA, and nuclei stained with DAPI (blue). White arrow heads point to cytoplasmic foci where L1 ORF1p and HA-MOV10 co-localize. Data collected from 289 Ctrl, 275 L1UP Cl1 and 296 L1UP Cl2 mESCs (n=3 biological replicates).",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of full length L1 and HA-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10, and nuclei stained with DAPI (blue). The bottom panel shows cells where accumulation of ORF1-MOV10 was observed in relatively large foci compared to cells where co-localization of the two proteins was mostly in relatively small foci (top panel), 68-119 transfected cells were analyzed per experiment (n=3 biological replicates).",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of HA-ORF1 or HA-ORF1 in conjunction with T7-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for HA (green) and T7 (red) to detect ectopically expressed ORF1p and MOV10 respectively, and nuclei stained with DAPI (blue). 100-147 transfected cells were analyzed per experiment (n=3 biological replicates).",
    "molecules": "DAPI"
  },
  {
    "caption": "A and B. hTERT-RPE1  cells were co-transfected with Flag  (V) or FBXL4-Flag  (FL4) and either GFP  (Vect), GFP-BNIP3  or GFP-NIX. Lysates were subjected to immunoprecipitation  (IP) with either GFP-nanobody coupled beads (A) or Flag-antibody coupled agarose  beads (B). IPs  were probed alongside the input samples for Flag  and GFP.",
    "molecules": "agarose"
  },
  {
    "caption": "D. hTERT-RPE1 cells were transfected with empty vector (V), FBXL4-Flag  (WT) or FBXL4- R482W-Flag  (RW). Lysates were subjected to IP  with Flag-antibody coupled agarose  beads and probed for SKP1  and Flag. Note that FBXL4-Flag  presents with an as yet uncharacterized higher molecular weight band (indicated by a blue arrowhead) E. Quantitation of SKP1  protein co-immunoprecipitated with FBXL4-R482W-Flag   relative to FBXL4-Flag   (WT). Data from two independent experiments are shown, normalised to the amount of SKP1  in the input and the FBXL4-Flag  in the IP.",
    "molecules": "agarose"
  },
  {
    "caption": "C. Representative western blot  of lysates prepared from  hTERT-RPE1  cells transfected with siRNA targeting BNIP3  (B3), NIX  or both for 72 hours. Cells were treated with either DMSO  or MLN4924  (1 µM) for the last 24 hours of transfection. Untransfected cells were treated alongside transfected ones with DMSO, antimycin  and oligomycin  (AO; 1 and 10 µM respectively), or MLN4924. D. Quantification of fold change in protein levels calculated from data represented in C for three colour-coded independent experiments; One way ANOVA and Dunnett's multiple comparison test *P<0.05, ****P<0.0001.",
    "molecules": "antimycin, DMSO, oligomycin, MLN4924"
  },
  {
    "caption": "E. Representative western blot  of  hTERT-RPE1 cells treated with either DMSO  or MLN4924  (1 µM) for the indicated  time points.",
    "molecules": "DMSO, MLN4924"
  },
  {
    "caption": "A. Representative western blot  of lysates from control or FBXL4  KO RPE1-Cas9i-mt-mKeima  cells treated with MLN4924  (1 µM) for 6 hours. B. Representative western blot of lysates from control or FBXL4  KO RPE1-Cas9i-mt-mKeima  cells pre-treated without or with MLN4924  (1 µM) for 6 hours followed by a cycloheximide  (CHX) chase (100 µg/ml).",
    "molecules": "CHX, cycloheximide, MLN4924"
  },
  {
    "caption": "BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.F Effect of AAV-BR1 vector on BBB permeability. No vector (left) or empty AAV-BR1 vector (right) was injected i.v. to wild type mice and BBB permeability was assessed by extravasation of the fluorescent tracer sodium fluorescein (n=7 animals per group). No significant difference was detected (n.s.).",
    "molecules": "sodium fluorescein"
  },
  {
    "caption": "D In vivo ookinete differentiation in midgut of the infected mosquitos. Right panel indicates the parasite smear stained with Giemsa solution. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01. Scale bar = 5 μm.",
    "molecules": "Giemsa solution"
  },
  {
    "caption": "E Number of oocysts in mosquito midgut 7 days post blood feeding. n is the number of mosquitoes in each group. The horizontal line shows the mean value of each group, Mann-Whitney test, ****, P<0.0001. Right panel shows mosquito midguts stained with 0.5% mercurochrome. Scale bar = 50 μm. Three biological replicates performed.",
    "molecules": "mercurochrome"
  },
  {
    "caption": "H IFA analysis of tagged ISP1 and ISP3 in zygotes of the complemented parasites. Nuclei are stained with Hoechst 33342. Scale bar = 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "E RT-qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT, Δisp1, Δisp3, and Δisp1/3 parasites. The expression is normalized to the 18s rRNA. Values are means ± SEM (n=4 biological replicates).",
    "molecules": "18s rRNA"
  },
  {
    "caption": "E Substitutions of N-terminal cysteine or glycine residues to alanine (G2A or C7A/C8A) ablates palmitoylation of ISP1. ISP1 was fused with BFP::3V5 and episomally expressed in zygotes. Right panel shows quantification of palmitoylated protein band from two independent repeats, values are means ± SD.",
    "molecules": "alanine, cysteine, glycine"
  },
  {
    "caption": "B Western blot of DHHC2, ISP1, and P28 (plasma membrane protein) of the TTS zygotes treated with PBS, trypsin (Try), or heat-inactivated (HI) trypsin. Upper panel shows the PPM and IMC structures.",
    "molecules": "Try, trypsin"
  },
  {
    "caption": "F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl-RAC method. NH: NH2OH. Right panel indicates quantification of protein band intensity from three replicates, values are means ± SD.",
    "molecules": "NH, NH2OH"
  },
  {
    "caption": "E Palmitoylation of DHHC2::6HA and C4A mutant episomally expressed in zygotes. C4A: substitutions of C-terminal four cysteine to alanine. Right panel indicates the quantification of protein band intensity from two replicates, values are means ± SD.",
    "molecules": "alanine, cysteine"
  },
  {
    "caption": "G IFA of DHHC2 at the dhhc2::6HA zygotes treated with 100 μΜ 2-BP. Scale bar = 5 μm.",
    "molecules": "2-BP"
  },
  {
    "caption": "I Palmitoylation of the episomally expressed DHHC2::6HA with mutation in respective individual cysteine (C255A, C258A, C260A, and C262A). Representative of three independent repeats.",
    "molecules": "cysteine"
  },
  {
    "caption": "O Palmitoylation of DHHC2 ectopically expressed in bacteria treated with 2-BP.",
    "molecules": "2-BP"
  },
  {
    "caption": "D IFA of DHHC2 and P28 in female gametocytes, female gametes, and zygotes of the dhhc2::6HA parasite. Gametocytes were stimulated with XA and 22°C in vitro. P28 is expressed specifically in female gametes and zygotes. Scale bar = 5 μm.",
    "molecules": "XA"
  },
  {
    "caption": "F No polarization of DHHC2 in the dhhc2::6HA gametocytes with only either XA or 22°C stimulation. Three replicates performed, values are means ± SD.",
    "molecules": "XA"
  },
  {
    "caption": "D Co-localization of ISP1::HA and β-Tubulin at ookinete periphery in IFA. Scale bar = 5 μm.",
    "molecules": "HA"
  },
  {
    "caption": "I Membrane association of α- and β-Tubulins in WT ookinete culture after DMSO, 2-BP, or colchicine treatment.",
    "molecules": "2-BP, colchicine, DMSO"
  },
  {
    "caption": "a, AMBRA1-BECN1 interaction by yeast two-hybrid assay. Yeast cells were co-transfected with the indicated plasmids and plated in medium with (+) or without (-) histidine (H) and adenine (A). AD, activation domain; DBD, DNA-binding domain; G4, Gal4; LAM, lamin.",
    "molecules": "adenine, histidine"
  },
  {
    "caption": "b, Rapamycin-induced autophagy requires AMBRA1. After AMBRA1 downregulation, 2FTGH cells were treated with rapamycin; after 48 h, the occurrence of autophagy was analysed by appearance of GFP-LC3 punctate staining or by LC3-I to LC3-II conversion (western blot in inset). Representative results are accompanied by a graph reporting data from three experiments. Scale bar, 20 µm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "d, AMBRA1 overexpression increases basal and rapamycin-induced autophagy. 2FTGH cells were transduced with full-length AMBRA1 (FL), AMBRA1 fragments (F1-3), BECN1 and β-gal (negative control) encoding retroviruses. They were then stimulated with rapamycin or left untreated, and analysed by appearance of GFP-LC3 punctate staining.",
    "molecules": "rapamycin"
  },
  {
    "caption": "c, Rapamycin-induced autophagy is impaired in Ambra1gt/gt murine embryonic fibroblasts (MEFs). Cells dissected from embryos with different genotypes (A-D) were treated with rapamycin, and the occurrence of autophagy was analysed 48 h later by the appearance of GFP-LC3 punctate staining. Representative results are shown in the upper panels, whereas the lower panel shows the data from three experiments. Scale bar, 20 µm.",
    "molecules": "Rapamycin, rapamycin"
  },
  {
    "caption": "A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows.",
    "molecules": "1NIP, NIP15, Calcium"
  },
  {
    "caption": "A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.",
    "molecules": "1NIP, NIP15, Calcium"
  },
  {
    "caption": "E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.",
    "molecules": "1NIP, NIP15, Calcium"
  },
  {
    "caption": "E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.",
    "molecules": "1NIP, NIP15, Calcium"
  },
  {
    "caption": "Calcium flux measured by FACScan for 3046M (A, D), 3046SD (B, E) and 3046D (C, F) cells stimulated with anti-HC (2 μl/ml) (A, B, C), or anti-λ (2 μl/ml) (D, E, F) antibodies with or without 1 min prestimulation with 1NIP-pep (80 nM). The addition of antibodies is indicated by black arrows. The 1NIP-pep was added immediately before recording. Data are representative of three independent experiments.",
    "molecules": "1NIP, Calcium"
  },
  {
    "caption": "A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.",
    "molecules": "1NIP, calcium"
  },
  {
    "caption": "F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.",
    "molecules": "1NIP"
  },
  {
    "caption": " (B) Hematoxylin and (Periodic acid-Shiff) PAS staining of WT (left) and Hnf4g KO (right) intestine. Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS ",
    "molecules": "Hematoxylin, Periodic acid"
  },
  {
    "caption": " (C) Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right)  small intestinal  organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue ",
    "molecules": "Alcian Blue, Fast Red"
  },
  {
    "caption": "A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs",
    "molecules": "Alizarin Red S, ARS"
  },
  {
    "caption": " B, C Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test ",
    "molecules": "calcium"
  },
  {
    "caption": " F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm ",
    "molecules": "H&E"
  },
  {
    "caption": "D Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. ***: p<0.001 by t test",
    "molecules": "serum"
  },
  {
    "caption": "F Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Cells were depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by t test",
    "molecules": "serum"
  },
  {
    "caption": "G Representative images of ALP and ARS staining. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 10 and 21 days, respectively",
    "molecules": "ARS"
  },
  {
    "caption": "H, I Quantitative analyses of the ALP activity (n=4) and calcium mineralization (n=5). Results are shown as mean ± SEM; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test",
    "molecules": "calcium"
  },
  {
    "caption": "L H&E staining of BMP2-induced newly formed bone. Scale bar, 50 μm",
    "molecules": "H&E"
  },
  {
    "caption": " N H&E staining of calvarial organ cultures stimulated with or without 100 ng/ml BMP2. Red dotted lines indicate the interface between original and newly formed bone. Scale bar, 50 μm",
    "molecules": "H&E"
  },
  {
    "caption": " C Immunoblot of Smad1-linked polyUb. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 10 µM MG132 for 4 hours before collection ",
    "molecules": "MG132, Ub"
  },
  {
    "caption": " D Immunoblot analysis of the degradation of FLAG-Smad1 protein. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 50 µg/ml cycloheximide (CHX) before collection at designated time points. Right: relative quantification of FLAG-Smad1 protein levels at different time points",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "G Immunoblot of RUNX2-linked polyUb. Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection. Prx1-Cre(+) indicates MSCs isolated from Prx1-Cre;Usp34f/f mice, and Prx1-Cre(-) indicates those from Usp34f/f control mice",
    "molecules": "MG132, Ub"
  },
  {
    "caption": "H Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were stimulated with BMP2, treated with 50 µg/ml CHX, and collected at designated time points. Right: relative quantification of RUNX2 protein levels at different time points",
    "molecules": "CHX"
  },
  {
    "caption": "I Representative images of ALP and ARS staining. Usp34f/f and Prx1-Cre;Usp34f/f MSCs were transduced with GFP, Smad1, Runx2 or Smad1+Runx2 adenoviruses, and then cultured in osteogenic medium for 5 and 10 days, respectively",
    "molecules": "ARS"
  },
  {
    "caption": "J, K Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test",
    "molecules": "calcium"
  },
  {
    "caption": "L, M H&E staining and quantitative analysis of MSCs-mediated ectopic bone formation. Scale bar, 50 μm. Results are shown as mean ± SEM; n=8; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test",
    "molecules": "H&E"
  },
  {
    "caption": "A Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. *: p<0.05 by ANOVA with Tukey's post hoc test",
    "molecules": "serum"
  },
  {
    "caption": "C Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test",
    "molecules": "serum"
  },
  {
    "caption": " E Representative images of ARS staining and quantification. Cells were cultured with normal medium supplemented with 100 ng/ml BMP2 for 14 days. Results are shown as mean ± SEM; n=8; *: p<0.05, and **: p<0.01 by ANOVA with Tukey's post hoc test ",
    "molecules": "ARS"
  },
  {
    "caption": " F Representative images of ALP and ARS staining. Mouse MSCs were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 and 10 days, respectively",
    "molecules": "ARS"
  },
  {
    "caption": "G, H Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test ",
    "molecules": "calcium"
  },
  {
    "caption": "(D) RNF145 and ADIPOR2 respond to a range of FAs. HEK-293T cells were treated for 6h with saturated FAs (SFAs; 400 µM (C12:0 - C18:0); 200 µM (C20:0)) or unsaturated FAs (UFAs; 400 µM (C18:1-n9); 200 µM (C20:4-n6, C20:5-n3)). * non-specific bands.",
    "molecules": "C20:5-n3, C12:0, C20:0, C20:4-n6, C18:0, C18:1-n9, saturated FAs, SFAs, UFAs, unsaturated FAs"
  },
  {
    "caption": "(E) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels. HEK-293T cells were treated with the SCD1 inhibitor MF-438 (SCDi, 100 nM, 18h) and RNF145/ADIPOR2 expression analysed by immunoblotting. * non-specific bands.",
    "molecules": "MF-438"
  },
  {
    "caption": "(A) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells. Representative 35S pulse-chase analysis in HeLa WT, RNF145 knockout (∆RNF145) or ADIPOR2 knockout (∆AR2) cells. Quantification of 35S-labelled ADIPOR2 normalised to t = 0 h is shown below.",
    "molecules": "35S"
  },
  {
    "caption": "(C) RNF145 binds ADIPOR2. RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). Cells were treated with VCP inhibitor (NMS-873, 10 µM, 90 min) prior to immunoprecipitation with an anti-V5 antibody.",
    "molecules": "NMS-873"
  },
  {
    "caption": "(A) FA-mediated regulation of RNF145 is proteasome-dependent. HEK-293T cells were treated with BSA, PA (400 µM, 6h) ± MG132 (10 µg/ml, 6h) and analysed by immunoblotting. *non-specific bands.",
    "molecules": "PA, MG132"
  },
  {
    "caption": "(B) UBXD8 participates in PA-modulated RNF145 degradation. CRISPR/Cas9-mediated depletion of UBXD8 (gUBXD8) or B2M (gB2M, control) in HEK-293T cells, followed by exposure to PA (400 µM, 6h) or BSA (6h, vehicle control).",
    "molecules": "PA"
  },
  {
    "caption": "(C) PA and OA treatment alter RNF145 ubiquitination. Immunoprecipitation of RNF145 from cells treated with BSA (control), PA or OA (each 200 µM, 3h). During FA treatments, VCP inhibitor (NMS-873, 10 µM) was present to stabilise ubiquitinated proteins. RNF145 ubiquitination (ubi) was detected using an anti-ubiquitin antibody (VU-1).",
    "molecules": "PA, NMS-873, OA, ubi, ubiquitin"
  },
  {
    "caption": "(D) HEK-293T cells were treated with cycloheximide (CHX, 1 µg/ml) and OA (400 µM, 6h) ± MG132 (10 µg/ml) for the indicated times. LE, long exposure; * non-specific bands.",
    "molecules": "CHX, cycloheximide, MG132, OA"
  },
  {
    "caption": "(E) Lysine-less RNF145 is stabilised under PA treatment. RNF145 knockout HEK-293T cells (∆RNF145) were complemented with RNF145-V5 or a lysine-less variant of RNF145 (RNF145-V5 K > R) and treated BSA or PA (400 µM, 6h). Dotted lines demarcate the different cell lines.",
    "molecules": "PA"
  },
  {
    "caption": "(F) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity. Immunoblot of whole-cell lysates from RNF145 knockout HEK-293T (∆RNF145) cells stably complemented with RNF145-V5 or RNF145C552A, H554A-V5 (mRNF145-V5) and treated with BSA or PA (400 µM, 6h). LE, long exposure.",
    "molecules": "PA"
  },
  {
    "caption": "(A) ADIPOR2 regulation by FAs is completely dependent on RNF145. WT or RNF145 knockout HEK-293T cells (∆RNF145) were treated with BSA, PA or OA (400 µM, 6h) and analysed by immunoblotting. Non-specific bands are indicated (*).",
    "molecules": "PA, OA"
  },
  {
    "caption": "(B) RNF145 - ADIPOR2 interaction is FA-modulated. Immunoprecipitation of endogenous RNF145 HEK-293T cells depleted of UBE2G2 (gUBE2G2) and/or RNF145 (gRNF145) and treated with 400 µM of the indicated PA, OA or BSA (control) for 3h. LE, long exposure. * non-specific bands.",
    "molecules": "PA, OA"
  },
  {
    "caption": "(C) Immunoprecipitation (IP) of endogenous ADIPOR2 from WT or ∆RNF145 cells (see (C)) treated with MG132 (5 µg/ml, 6h) ± OA (200 µM, 6h). Ubiquitinated ADIPOR2 was detected using a specific anti-ubiquitin antibody (clone VU-1). Note that ADIPOR2 levels in RNF145 knockout HEK-293T cells are increased.",
    "molecules": "MG132, OA, ubiquitin"
  },
  {
    "caption": "(D) ADIPOR2 knockout HEK-293T cells were stably complemented with ADIPOR2-HA, an ADIPOR2 mutant in which all lysines were mutated to arginines (K-less ADIPOR2-HA), or an empty vector control and exposed to OA (200 µM, 6h) or BSA (control). ADIPOR2 variants were visualised by immunoblotting using a specific anti-HA antibody. LE, long exposure.",
    "molecules": "OA"
  },
  {
    "caption": "(A) Loss of ADIPOR2 sensitises cells to PA-induced cytotoxicity. HEK-293T cells were depleted of RNF145 (purple), ADIPOR2 (blue), B2M (CTR; grey), or both ADIPOR2 and RNF145 (blue + purple), exposed to PA (200 µM, 96h) and stained with crystal violet. Quantification of crystal-violet stained cells is shown alongside a representative micrograph (below). The response of each cell line to PA was normalised to its BSA-treated condition (dotted line). Statistically significant differences to cells containing gCTR cells are indicated (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction). Mean ± SD are shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. N = 3 biological replicates, ** q ≤ 0.01. Double knockdowns were generated by sequential constitutive CRISPR/Cas9-mediated depletion of RNF145 or ADIPOR2. Knockdown efficiencies are shown in Appendix Fig S1A.",
    "molecules": "crystal violet, crystal-violet, PA"
  },
  {
    "caption": "(C) HEK-293T cells depleted of B2M (gCTR) or ADIPOR2 (gADIPOR2) were complemented with empty vector (blue), full-length ADIPOR2-HA (green), or a catalytically inactive variant of ADIPOR2-HA (dead ADIPOR2; red) and treated with PA (200 µM, 96h) or BSA (vehicle control, 96h). Measurements from PA-treated cells were normalised to their BSA-treated condition (dotted line). Cell growth was quantified, and representative crystal violet staining is shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. ** q ≤ 0.01 (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction).",
    "molecules": "crystal violet, PA"
  },
  {
    "caption": "(A) 3D time‐lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron. Neurons were transfected with Mito‐DsRed2, pretreated with the pan‐caspase inhibitor zVAD‐fmk methyl ester (100 μM), and exposed to SNOC (200 μM). Images were 3D iso‐surface rendered. Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure (upper panels; scale bar, 15 μm) and closeup views (lower panels; scale bar, 3 μm). See Supplementary Video 1.",
    "molecules": "SNOC, zVAD‐fmk methyl ester"
  },
  {
    "caption": "(C) NO induces dose‐dependent mitochondrial fission. Neurons expressing Mito‐DsRed2 were exposed to SNOC and fixed at 60 min. The fraction of neurons displaying fragmented mitochondria is shown as the mean±s.e.m.",
    "molecules": "SNOC"
  },
  {
    "caption": "(D) Mito‐DsRed2‐expressing corticalneurons were pretreated with 1 mM nitro‐L‐arginine (20 min) and exposed to 25 μM NMDA. The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean±s.e.m. of quintuplicate samples from a representative experiment (†††significance at P0.001 or n.s.†, not significant compared to control; ***significance at P0.001 as compared to NMDA treatment).",
    "molecules": "NMDA, nitro‐L‐arginine"
  },
  {
    "caption": "Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2.",
    "molecules": "SNOC"
  },
  {
    "caption": "(D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series.",
    "molecules": "SNOC"
  },
  {
    "caption": "(F-G) Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm.",
    "molecules": "SNOC"
  },
  {
    "caption": "(H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test).",
    "molecules": "SNOC"
  },
  {
    "caption": "(I) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).",
    "molecules": "zDEVD‐AMC, SNOC, staurosporine"
  },
  {
    "caption": "Fission is associated with ultrastructural changes in mitochondria. EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission. Cortical cultures were exposed to aged SNOC control (A-D) or 200 μM SNOC (E-L) for 6 h in the presence of 80 μM zVAD‐fmk methyl ester. A slice from each tilt series is shown (A, E and I), followed by orthogonal views of 3D reconstructions after segmentation of the OMM (blue) and IMM (gray), with cristae (various arbitrary colors) (B-D, F-H and J-L). For clarity, 10 representative cristae are shown in (B, D, F, G, J and K), while all cristae are displayed in (C, H and L). The control mitochondrion (A-D) is elongated with a dark matrix, indicating no swelling, and 34 cristae. The NO‐exposed mitochondrion in (E-H) is dividing, and the lower mitochondrion of the couplet shows severe damage, recognized by an outer membrane rupture, a regionally confined inner membrane blowout, slight matrix swelling and cristae fragmentation. Each of the daughter mitochondria is shorter and rounder than controls. Unlike the upper daughter, few of the cristae extend throughout the lower mitochondrion. Additionally, cristae are considerably smaller and regionally confined. The lower mitochondrion of the couplet contains 82 cristae, while the upper contains only 39, indicating cristae fragmentation in the former. The NO‐exposed mitochondrial triplet in (I-L) displays further damage, including membrane degradation, accumulation of an electron dense mass, greater cristae fragmentation, and apparent mitochondrial fragmentation. All cristae are smaller than controls, with 73 in the top mitochondrion, 112 in the middle, and 47 in the bottom. Scale bars, 400 nm. See Supplementary Video 3, 4 and 5 of tomographic reconstruction.",
    "molecules": "SNOC, zVAD‐fmk methyl ester"
  },
  {
    "caption": "(E) Autophagy in neurites at 6 h after SNOC exposure. Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image. The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes. White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome. Scale bars, 500 nm.",
    "molecules": "SNOC"
  },
  {
    "caption": "(A) Mitochondrial fission is linked to a drop in ATP. Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors (2 μM rotenone plus 2 μg/ml oligomycin; Rot/Olig). ATP concentrations are shown as the mean±s.e.m. normalized to the plating density of neurons (n=6)",
    "molecules": "ATP, SNOC, oligomycin, rotenone"
  },
  {
    "caption": "(B) [ATP]/[ADP] ratios in purified corticalneurons exposed to increasing SNOC concentrations (n=4).",
    "molecules": "ADP, ATP, SNOC"
  },
  {
    "caption": "(C) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2.5 h.",
    "molecules": "ethidine, SNOC"
  },
  {
    "caption": "(D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm.",
    "molecules": "hydroethidine, SNOC"
  },
  {
    "caption": "(E) Reduced glutathione (GSH) partially blocks mitochondrial fission. Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester, washed once and then exposed to 35 μM SNOC. Mitochondrial fission was scored after 1 h. Data are means±s.e.m. of quintuplicate samples from one representative experiment of a total of three (†††significance at P0.001 or n.s., †not significant compared to control; *significance at P0.05).",
    "molecules": "glutathione, GSH monoethyl ester, SNOC"
  },
  {
    "caption": "(A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25-35 versus Aβ35-25(10 μM, 6 h), as indicated. Scale bar, 20 μm.",
    "molecules": "SNOC, rotenone"
  },
  {
    "caption": "(B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining.",
    "molecules": "SNOC"
  },
  {
    "caption": "(D) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4).",
    "molecules": "Rotenone"
  },
  {
    "caption": "(E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3).",
    "molecules": "rotenone"
  },
  {
    "caption": "(A) Immunoblot  analysis of NRF1, NRF2-α  and PGC1-α in mouse PMs  infected with VSV  or HSV-1 or treated with poly(I:C) or ISD  for the indicated time points. ACTIN  was used as a loading control. Ratios of target proteins versus loading control normalized to the 0-h time point  of each condition.",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "(B) qPCR  analysis of Nrf1, Gabpa  and Ppargc1a  mRNA in mouse PMs  infected with VSV  or HSV-1 or treated with poly(I:C) or ISD  for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "(F and G) The representative curves of oxygen consumption rate  (OCR) in wild-type and NRF1-KO mouse PMs  infected with VSV  (F) or HSV-1 (G) for 8 h (n=5 biological replicates per group). Oligomycin: 1 µM, FCCP: 4 µM, Rotenone: 1 µM. Quantification of basal and maximal OCR. Data information: Data are from at least three biological replicates and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "FCCP, Oligomycin, Rotenone"
  },
  {
    "caption": "(B and C) Immunoblot  analysis of phosphorylated  and total TBK1, p65  and STAT1  in wild-type and NRF1-KO mouse PMs  infected with VSV  or HSV-1  (B) or stimulated with poly(I:C) or ISD  (C) for the indicated time points. GAPDH  was used as a loading control. Ratios of phosphorylated  proteins versus total proteins normalized to the 0-h or 4-h  time point of each wild-type sample.",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "(D) qPCR  analysis of Ifnb1, Il6, Tnfa  and Cxcl10  mRNA in wild-type and NRF1-KO mouse PMs  infected with VSV  or HSV-1  or stimulated with  poly(I:C)  or ISD  for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ND, not detectable, *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "(A) 8-week-old Nrf1F/F and Nrf1MyeKO mice  (n=3 for PBS  treatment and n=6 for virus treatment) were intravenously infected with VSV  (4×107 PFU/mouse) or HSV-1 (1×107 PFU/mouse). After 24 h, ELISA  quantification of IFN-β, IL6  and TNF-α in sera. Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "PBS"
  },
  {
    "caption": "(A) mtROS levels measured by MitoSOX staining  and  flow cytometry in wild-type and NRF1-KO mouse PMs  infected with VSV  (left) or HSV-1  (right) for the indicated time points. MFI, mean fluorescence intensity (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "MitoSOX, ROS"
  },
  {
    "caption": "(B) DNA was isolated from digitonin  extracts of wild-type and NRF1-KO mouse PMs  infected with VSV  (left) or HSV-1 (right) for the indicated  time points. Cytosolic  translocation of mtDNA  was quantified via qPCR  using the indicated primer sets. 18S rRNA served as a control (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "digitonin"
  },
  {
    "caption": "(C and D) Wild-type and NRF1-KO mouse PMs  infected with VSV  (8 h) or HSV-1 (4 h) were imaged by confocal microscope after staining  with anti-DNA  (DNA) and anti-Prohibitin  (Mito) antibodies. Scale bars, 5 µm (C). (D) The number of cytosolic mtDNA  puncta per cell was quantitated by ImageJ (n=30 cells per group). Data information: Data are from three independent experiments or representative data (C) and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "DNA"
  },
  {
    "caption": "(E) ELISA  quantification of IFN-β (left) and IL6  (right) in wild-type and NRF1-KO mouse PMs  pretreated with or without EtBr  (450 ng/ml, 72 h), followed by infection with VSV  or HSV-1 for 8 h (n=3 biological replicates per group). (F) ELISA  quantification of IFN-β (left) and IL6  (right) in wild-type and NRF1-KO mouse PMs  pretreated with or without MitoQ  (100 nM, 2 h), followed by infection with VSV  or HSV-1 for 8 h (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "EtBr, MitoQ"
  },
  {
    "caption": "(H) qPCR  analysis of Ifnb1  and Il6  mRNA in the lungs  of Nrf1F/F and Nrf1MyeKO mice  pretreated with MitoQ  as in (G) and 1 h later infected i.p. with VSV  (5×108 PFU/mouse) for 24 h (n=6 per group). (I) qPCR  analysis of Ifnb1  and Il6  mRNA in the brains  of Nrf1F/F and Nrf1MyeKO mice  pretreated with MitoQ  as in (G) and 1 h later infected i.p. with HSV-1 (1×107 PFU/mouse) for 24 h (n=6 per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.",
    "molecules": "MitoQ"
  },
  {
    "caption": "C. WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline (DOX) treatment for 5 days.",
    "molecules": "DOX, doxycycline"
  },
  {
    "caption": "D. The 7SK RNA levels in these two clones were detected by qRT-PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels. The error bars represent mean ± SD from three independent experiments.",
    "molecules": "DOX"
  },
  {
    "caption": "E. 1D-TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment.",
    "molecules": "DOX"
  },
  {
    "caption": "F. NE and anti-CDK9IP from NE of inducible DKC1 KD clone 1, which was untreated or treated with DOX, were analyzed by WB for the indicated proteins and PEx for 7SK RNA.",
    "molecules": "DOX"
  },
  {
    "caption": "G. Clone 1 was treated with DOX for 3 days and then transfected with the Tat-Flag cDNA (20 ug/150-mm dish). Two days later, NE and anti-FlagIP from NE were analyzed as in F.",
    "molecules": "DOX"
  },
  {
    "caption": "F. Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10-derived clone inducibly expressing shDKC1 upon exposure to DOX.",
    "molecules": "DOX"
  },
  {
    "caption": "G and H. The inducible DKC1 KD clone, treated or untreated with DOX for 4 days, was incubated with the indicated concentrations of JQ1 (G) or prostratin (H) for 24 h. The GFP expression was measured by flow cytometry and expressed as percentages of GFP(+) cells of the entire population. The error bars in all panels represent mean ± SD from three independent experiments, and the p values determined by the Student's t-test.",
    "molecules": "DOX, JQ1, prostratin"
  },
  {
    "caption": "B - Congo red plates with colonies of E. coli strain MC4100 relA+ and its target mutation csgD:M4-ompR:mut2 derivative, harboring sRNA-producing plasmids pOmrA, pOmrB, or an empty vector (pControl). Incubation at 28°C for 48 h.",
    "molecules": "Congo red"
  },
  {
    "caption": "A - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA (210 nt from +1) by OmrA and OmrB. Inclusion of 30S ribosomes, tRNAfmet, OmrA, and OmrB is indicated. The unrelated IstR-1 sRNA is a negative control. The 30S/ tRNAfmet-dependent reverse transcription block (toeprint), is indicated by an arrow. Sequencing reactions were loaded next to the primer extension products (UCGA). The AUG start codon is indicated by asterisks.",
    "molecules": "fmet, tRNA"
  },
  {
    "caption": "A - 5' end-labeled dgcM mRNA (10 nM) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram. When added, Hfq hexamers were at 50 nM, the sRNAs OmrA and OmrB at 250 nM. The accessibility of each nucleotide was probed with water (lanes 1-4, negative control for RNA integrity), RNase T1, or lead(II) acetate. Lane T1: RNase T1 under denaturing conditions. Lane OH: alkaline ladders. Binding sites of Hfq (green arrows and boxes), and site 1 (red arrows and boxes) and 2 (blue arrows and boxes) of the sRNAs, are highlighted. Note that site 2 is bound differently by OmrA and OmrB (blue boxes on gel). Black boxes indicate Hfq-dependent T1 cleavage enhancements.",
    "molecules": "lead(II) acetate"
  },
  {
    "caption": "B - Northern blot analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry, probed for OmrA (strains containing pOmrA) or OmrB (strains containing pOmrB). 5S rRNA was used as loading control.",
    "molecules": "5S rRNA"
  },
  {
    "caption": "(c) The effect of Atg14L knockout on p62 turnover. WT, mAtg14L−/−, mAtg14L−/− + Atg14L, and mAtg5−/− ES cells were cultured in nutrient-rich (N) or starvation medium (S) for 3 h. Cell lysates were subjected to western blotting with the antibodies indicated.",
    "molecules": "nutrient"
  },
  {
    "caption": "(d) Degradation of long-lived proteins is reduced in Atg14L knockout cells. WT, mAtg14L−/−, and mAtg5−/− ES cells were cultured in N or S medium or S medium with wortmannin (W, 100 nM) for 3 h and degradation of long-lived proteins was measured. The data are the mean ± s.d. of three independent experiments.",
    "molecules": "proteins, wortmannin"
  },
  {
    "caption": "(e) The effect of Atg14L knockout on LC3 turnover. WT, mAtg14L−/− and mAtg5−/− ES cells were cultured in N or S medium for 3 h in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1). Cell lysates were subjected to western blotting with the antibodies indicated. Full scans of the blots in b, c and e are available in Supplementary Information, Fig. S6.",
    "molecules": "E64d, pepstatin A"
  },
  {
    "caption": "(a) The effect of Atg14L knockout (mAtg14−/−) on Atg16L dot formation. Wild-type (WT) and mAtg14−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with an anti-Atg16L antibody. Images of Atg16L staining (grey-scale) and Atg16L merged with Hoechst staining (colour) are shown.(c) Quantification of the number of Atg16L dots in a. The data are the mean ± s.d. of three independent experiments.",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) The effect of Atg14L knockout on LC3 dot formation. WT and mAtg14L−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with anti-LC3 antibody. Images of LC3 staining (grey-scale) and LC3 merged with Hoechst staining (colour) are shown. Scale bars, 20 μm. (d) Quantification of the number of LC3 dots in b. The data are the mean ± s.d. of three independent experiments. For each of the experiments in c and d at least 13 cells were counted.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) Immunofluorescence using an anti-Atg16L antibody. A549 cells were transfected with adenovirus expressing GFP-Atg14L and cultured in nutrient-rich or starvation medium for 4 h. Cells were fixed and stained with an anti-Atg16L antibody.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) Double knockdown of Rubicon and Atg14L. A549 cells stably expressing GFP-LC3 were infected with adenovirus expressing either shRNA against Rubicon and Atg14L or control shRNA (luciferase). The image is available in Supplementary Information, Fig. S5a. The GFP-LC3 puncta were counted in nutrient-rich (N) or starvation (S) conditions. The data are mean ± s.d. of three independent experiments; at least 23 cells were counted for each experiment.",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) A549 cells were infected with adenovirus harbouring shRNA for Rubicon or control (luciferase). The cells were cultured in N or S medium in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) for 1-4 h. Cell lysates were subjected to western blotting with the antibodies indicated.",
    "molecules": "E64d, pepstatin A"
  },
  {
    "caption": "d) Bulk degradation activity of Rubicon knockdown cells. The bulk degradation activity in control and Rubicon knockdown cells in nutrient-rich medium was measured. Wortmannin (W, 100 nM) was added as a negative control. The data are mean ± s.d. of three independent experiments.",
    "molecules": "nutrient, Wortmannin"
  },
  {
    "caption": "(a) The maturation of autophagosomes is enhanced in Rubicon knockdown cells. A549 cells stably expressing GFP-LC3, with Rubicon or control shRNA, were incubated in nutrient-rich medium with or without the protease inhibitors (PI) E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) treatment for 4 h. The cells were stained with an anti-Lamp1 antibody (upper panels) and colocalization efficiency between GFP-LC3 and Lamp1 was counted (lower panel). The data are mean ± s.d. of 30 cells.",
    "molecules": "E64d, nutrient, pepstatin A"
  },
  {
    "caption": "(b). Electron microscopy analysis of Rubicon knockdown cells. Control (luciferase) or Rubicon shRNA A549 cells grown in nutrient-rich medium was subjected to electron microscopy analysis (left panels). The arrows indicate autophagosomes and the arrowheads indicate autolysosomes. Scale bar, 1 μm. Autophagosomes and autolysosomes were counted in 21 cells, and the mean ± s.d. are shown (right panel).",
    "molecules": "nutrient"
  },
  {
    "caption": " K. NESTIN and GFP immunostaining of the cells in (H) at day 5 of neural differentiation. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm. ",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": " C. NDIME interacts with EZH2 (P = 0.0043) and SUZ12 (P = 0.0003), but not EED (P = 0.6720) and EZH1 (P = 0.0951) compared with IgG, as determined by RIP using EZH2, SUZ12, EED, and EZH1 antibodies at day 5 of neural differentiation. Nuclear RNA U1 as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT). ",
    "molecules": "Nuclear RNA U1, U1"
  },
  {
    "caption": " NDIME levels following EZH2-RIP (P = 0.0028) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT). ",
    "molecules": "Nuclear RNA U1, U1"
  },
  {
    "caption": " E. NDIME levels following SUZ12-RIP (P = 0.0015) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT). ",
    "molecules": "Nuclear RNA U1, U1"
  },
  {
    "caption": " H. Expression of neural-lineage genes at day 5 of neural differentiation in the NDIME-knockout cells after adding the EZH2 inhibitor GSK126 (7.5 µM) partially rescued neural differentiation defects, as shown by qRT-PCR; the wild-type and NDIME-knockout cells treated with DMSO. ",
    "molecules": "DMSO, GSK126"
  },
  {
    "caption": " A. qRT-PCR analysis of NDIME (P = 0.0051) and MEF2C (P = 0.0062) in VPA-treated mice and saline-treated mice; n = 9 per group. ",
    "molecules": "VPA"
  },
  {
    "caption": " B. Enrichment of EZH2 at the Mef2c promoter in VPA-treated mice and saline-treated mice (compared with saline-treated mice); n = 9 per group. ",
    "molecules": "VPA"
  },
  {
    "caption": "C. Western blot analysis of the protein levels of MEF2C and GAPDH in the hippocampus of VPA-treated mice (n = 9) and saline-treated mice (n = 10).",
    "molecules": "VPA"
  },
  {
    "caption": " D. Immunofluorescence images for MEF2C in the hippocampus of VPA-treated mice and saline-treated mice. Nuclei were stained with Hoechst 33342 (blue); n = 9 per group. Scale bars, 50 µm. E. Statistical analysis of the grayscale levels of MEF2C in (C) by ImageJ (P = 0.0074); saline-treated mice (n = 10), VPA-treated mice (n = 9). F. Statistical analysis of the Mean MEF2C staining intensity/unit area in (D) by ImageJ (P = 0.0096); n = 9 per group. ",
    "molecules": "Hoechst 33342, VPA"
  },
  {
    "caption": " G. Immunofluorescence images for GFP in the hippocampus of saline-treated mice, VPA-treated mice, and VPA-treated mice with viral-mediated expression of NDIME ",
    "molecules": "VPA"
  },
  {
    "caption": " I-K. Adeno-associated virus overexpression of NDIME in the dentate gyrus (DG) of the dorsal hippocampus of VPA-treated mice rescued autism-like social deficits. Open field test (I), three-chamber test (J), and elevated-plus maze (EPM) test (K). Data in (I-K) are represented as mean ± SEM; n = 9 per group. Asterisks indicate a difference between VPA+AAV-GFP and Saline+AAV-GFP, whereas hash tags indicate a difference between VPA+AAV-NDIME-GFP and VPA+AAV-GFP. #P < 0.05, **/##P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant. ",
    "molecules": "VPA"
  },
  {
    "caption": "b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs.",
    "molecules": "oxygen, CoCl2"
  },
  {
    "caption": "d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µm",
    "molecules": "oxygen, CoCl2"
  },
  {
    "caption": "e) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%.",
    "molecules": "oxygen"
  },
  {
    "caption": "f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.",
    "molecules": "oxygen, bilirubin"
  },
  {
    "caption": "b) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM.",
    "molecules": "oxygen"
  },
  {
    "caption": "c) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors.",
    "molecules": "oxygen"
  },
  {
    "caption": "d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.",
    "molecules": "oxygen"
  },
  {
    "caption": "b) Microscopic assessment of the averaged fluorescence intensity (AFI) in CHO cells stably transfected with the dUnOHR sensor. Hypoxia was induced by incubation in 1% oxygen for 18 hrs, then the culture was switched to normoxia for 24 hrs, followed by another 14 hrs hypoxia and finally normoxia again. As expected UnaG fluorescence is efficiently induced under hypoxia, while mOrange fluorescence only appears after the switch to normoxia only. The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE-mCMV promoter activity. This behavior is repeated in subsequent hypoxia / normoxia cycles. The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period. Plotted is the average of the mean ± SEM.c) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b.) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange. Scale bars = 100 µm.",
    "molecules": "oxygen"
  },
  {
    "caption": "A) z-stacks were acquired from FM4-64 labeled vacuoles in wildtype cells (BJ3505), using a spinning disc microscope (step size 200 nm). 3D reconstructions from these stacks reveal a peripherally located fusion pore. Scale bar 2 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "B) Wildtype (BJ3505) and isogenic pmc1∆/vcx1Δ mutant cells were labeled with FM4-64. The number of vacuoles per cell was determined in 3 independent experiments evaluating 200 cells each. Scale bar: 2 µm. C) Frequency of visible fusion pores. Cells were grown as in (B). The percentage of cells showing visible fusion pore was determined from z-stacks. Means s.d. are shown for 100 stained vacuoles from 3 independent experiments. ",
    "molecules": "FM4-64"
  },
  {
    "caption": "E) Distribution of Vph1-GFP and FM4-64 in vacuole-vacuole contact sites. Vph1-GFP expressing cells, labeled with FM4-64, were analyzed under a spinning disk confocal microscope equipped with two cameras for the simultaneous acquisition of GFP and FM4-64 fluorescence signals. Separate channels are shown on the right. The entire contact surface, but not the pore, is accessible to both probes. Scale bar: 2 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "F) Expansion of a vacuolar fusion pore in real time. pmc1∆/vcx1Δ cells were labeled with FM4-64 and immobilized in a 50 µL flow chamber (Ibidi). The osmotic value of the medium was changed by perfusion with water, using a pump with a flow of 30 µL/s. A frame was acquired every 2 s for a total period of the 30 s, using the laser at minimal intensity.",
    "molecules": "FM4-64, water"
  },
  {
    "caption": "A) Vacuolar membranes of wildtype (BY4741), nyv1∆ vam3∆, ypt7∆ or vma16F190Y cells were labeled with the vital dye FM4-64. A vacuole that was in a sufficiently peripheral location to be selectively photobleached was exposed to a laser pulse (white arrows indicate the bleaching area). Recovery of fluorescence in this area was assayed 10 seconds later by spinning disc confocal microscopy. Scale bar: 2 µm. B) Kinetics of FM4-64 recovery in (A). Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments. ",
    "molecules": "FM4-64"
  },
  {
    "caption": "C) In vivo lipid and content mixing. Vacuoles in living yeast were labeled with the indicated lumenal (CDCFDA) and membrane (FM4-64 or PX-GFP) probes. Note that CRY1 cells lack the ADE2 gene and hence naturally accumulate 5-amino-1-(5-phospho-D-ribosyl)imidazole as fluorescent fluid phase marker in the vacuolar lumen. FRAP-experiments were performed (arrows indicate the bleaching area). 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Scale bar: 2 µm. D and E) Kinetics of fluorescence recovery after photobleaching of the probes in (C). Numbers denote the areas shown in (C): 1. Non-bleached lumenal area. 2. Bleached lumenal area. 3. Bleached membrane area. Means and s.d. are shown for 20 vacuole clusters from 3 independent experiments. ",
    "molecules": "5-amino-1-(5-phospho-D-ribosyl)imidazole, FM4-64, CDCFDA"
  },
  {
    "caption": "A) FM4-64 and GFP excitation/emission spectra.",
    "molecules": "FM4-64"
  },
  {
    "caption": "C) Assay by microscopy. Half of two cultures of cells expressing Vph1-EGFP or Nyv1-EGFP were labeled with FM4-64, washed and mixed with the other, non-labeled half of the same culture. Cells were analyzed by spinning disc confocal microscopy. The intensities of GFP fluorescence were tracked along the dashed lines and compared between neighboring FM4-64-labeled and unlabeled cells.",
    "molecules": "FM4-64"
  },
  {
    "caption": "D) FACS analysis. The strains were labeled washed and analyzes by FACS. Shown are side (SSC-A) and forward scatter (FSC-A), as well as the fluorescence intensity distributions for FM4-64 and for GFP in the absence or presence of FM4-64. E) Comparison of the GFP mean fluorescence from FM4-64-stained cells and unstained cells (20'000 each). ",
    "molecules": "FM4-64"
  },
  {
    "caption": "C,D) FRAP assays for fusion pores. The indicated cells expressing (C) yeGFP-LT or (D) LT-yeGFP were labeled with FM4-64 and subjected to FRAP experiments Cells were imaged before and 0 (bleached) or 30 seconds after photobleaching (recovery). The bleached areas are indicated by an arrow. E,F) FRAP assays for fusion pores were performed as in (C, D), but with nyv1∆ cells. Scale bar: 2 µm. G) The histogram reports the fraction of cells showing FRAP. Means and s.d. are shown from 3 independent experiments, with 100 cells analyzed in each. ",
    "molecules": "FM4-64"
  },
  {
    "caption": "A-F) The indicated yeast strains were transformed with expression plasmids for FYVE2-GFP and their vacuolar membranes were labeled with FM4-64. FRAP was performed using a spinning disk microscope equipped with an emission beam splitter and two synchronized cameras, allowing to simultaneously analyze FM4-64 and GFP fluorescence. The histograms show the kinetics of signal recovery for (A) wildtype (WT), (B) vam3tsf, (C) ypt7T22N, (D) vma16F190Y, (E) vps33P184L, and (F) nyv1∆ cells. Scale bar: 2 µm. Arrows indicate the bleaching area. Means and s.d. are shown for 20 stained vacuoles from 3 independent experiments.",
    "molecules": "FM4-64"
  },
  {
    "caption": "A-D) Wild-type (A, B) or nyv1∆ cells (C,D), which lack the vacuolar R-SNARE Nyv1, were labeled with FM4-64. Cells were immobilized on a chambered slide and subjected to FRAP experiments (C) Vacuole fission induced by hypertonic shift. FRAP experiments were performed before (0') or 15 min after addition of NaCl to 0.5 mol/l. The fluorescence images show the vacuoles inside a single cell. (B, D) Cells in S-phase were selected, which grow a daughter cell and therefore fission their vacuoles into a chain of tubulo-vesicular structures, part of which are transported into the daughter cell. FRAP experiment on vesicles in the inheritance structure were performed. Arrows indicate the bleaching area, dashed orange lines the cell cortex. Scale bar: 2 µm.",
    "molecules": "FM4-64, NaCl"
  },
  {
    "caption": "A) Wildtype (BY4741) cells labeled with FM4-64 show extensive contact zones between vacuoles. Scale bar: 2 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "C) FRAP analysis of FM4-64-labeled wildtype cells was performed by acquiring pictures in continuous acquisition mode after bleaching, for a total period of 5 s. Three representative frames after photobleaching are shown. The bars in the magnified parts of frames 2 and 3 illustrate the positions at which the recovery of FM4-64 fluorescence was quantified. Scale bar: 2 µm. D) Quantification of recovery experiments in (C). A delay of >50 ms between the appearance of the signal on both sides of the photobleached vacuole was considered as asymmetric recovery. The histogram shows means and s.d. from 3 independent experiments evaluating 50 stained vacuoles each. A Schematic view of the various possible positions of a fusion pore and its consequences for our ability to detect asymmetric FM4-64 recovery. The contact zone between the two vacuoles is tilted by 90° in the middle panel, which gives four examples for the positioning of the pore (shown as a black dot). Asymmetric recovery could only be detected if the pore is sufficiently far from the center of the contact zone and close to the optical plane that is imaged (the cases marked in red). ",
    "molecules": "FM4-64"
  },
  {
    "caption": "E Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.034 for basal (using one sample t-test) and *P = 0.018 for maximum respiration (using unpaired Student's t-test).",
    "molecules": "Hoechst, O2"
  },
  {
    "caption": "F Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.01 for basal (using one sample t-test), ***P = 0.0004 for maximum respiration and *P = 0.012 for non-mitochondrial respiration (using unpaired Student's t-test).",
    "molecules": "Hoechst, O2"
  },
  {
    "caption": "A-D Representative single particle tracks of cells expressing MIC60- (A) and MIC10-SNAP (B) in WT Hela cells and MIC60- (C) and MIC10-SNAP (D) in MIC13 KO Hela cells, stained with silicone-rhodamine dye, and imaged at a rate of 33 ms/frame. Single tracks were colour-coded according to temporal appearance. Scale Bar 5 µm. Insets in each image show zoomed-in images of few representative tracks.",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP stained with silicone-rhodamine. Number of tracks analysed (obtained from 2 independent experiments): n = 2541, 2540, 3560 and 1441 in WT Hela cells expressing MIC60-SNAP and MIC10-SNAP, MIC13 KO Hela cells expressing MIC60-SNAP and MIC10-SNAP, respectively. (****P ≤ 0.0001 for all possible comparisons, unpaired Student's t-test).",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "A, C Representative live-cell STED super-resolution images (t=0 s) showing WT (A) and MIC13 KO (C) Hela cells expressing MIC10-SNAP stained with silicone-rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D) respectively. Scale bar 500 nm. B, D Time-lapse image series of a mitochondrion expressing MIC10-SNAP in WT (B) and MIC13 KO (D) Hela cells (2.6 s/frame). Green and magenta asterisks show merging and splitting events of MIC10-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "A Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 2.5 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 2.5 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Cristae mergence events that form structure resembling letter `X´ or `Y´ are marked at 5 s or 14.9 s respectively. Scale bar 500 nm. ",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "C Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 1.3 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 1.3 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. ",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in WT Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green `Y´ represent Y- type imminent mergence event . Scale bar 500 nm. ",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "C Representative live-cell STED super-resolution images (t=0 s) showing MIC13 KO Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in MIC13 KO Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. ",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (B) (red hot LUT). Scale bar 500 nm. B Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green asterisks show merging events and redistribution of TMRM intensity from a high-intensity cristae to a low-intensity cristae (two such instances are shown at 3 and 7.4 s). Green arrows pointing inward connected by solid line at 1.5 and 5.9 s indicate imminent merging events. Scale bar 500 nm. ",
    "molecules": "TMRM"
  },
  {
    "caption": "C Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (D) (red hot LUT). Scale bar 500 nm. D Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green and magenta asterisks show cristae merging and splitting cycles (shown at 5.9 s and 7.4 s). Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. ",
    "molecules": "TMRM"
  },
  {
    "caption": "E Representative time-lapse images of WT Hela cells stained with NAO, and imaged using Airyscan microscope, indicate cristae merging and splitting dynamics. Yellow and magenta asterisks show merging and splitting events of cristae respectively. Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.",
    "molecules": "NAO"
  },
  {
    "caption": "B Dual-colour STED super-resolution images of fused mitochondrion in WT Hela cells (Left Panel) showing colocalization of cristae marked with ATP5I-GFP (using anti-GFP antibody) and ATP5I-SNAP (stained with silicone-rhodamine). (Right Panel) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I-GFP (using anti-GFP antibody) or ATP5I-SNAP (stained with silicone-rhodamine). Arrowheads show merged cristae while arrows show cristae that maintain their individual identity. Scale bar 500 nm. C Boxplot showing quantification of colocalization events per µm of mitochondria in WT and MIC13 KO Hela cells. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. (15 mitochondria were taken from WT and MIC13 KO cells from 4 and 2 independent experiments, respectively, *P = 0.03, unpaired Student's t-test) ",
    "molecules": "silicone-rhodamine"
  },
  {
    "caption": "D, E Images of FRET efficiency in mock-treated (- ant.A, D) and Antimycin A-treated (+ ant. A., E) ubi-AT1.03NL wing discs at time point 2 h. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars= 50 µm. F Line graphs showing the FRET efficiency in individual mock-treated (- ant. A, n=26) and ant. A-treated (+ ant. A., n=19) ubi-AT1.03NL wing discs before (0 h) and after 2 h of treatment. Paired t-test, ns= not significant, ***p≤0.001. ",
    "molecules": "ant. A, ant.A, Antimycin A"
  },
  {
    "caption": "G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001. ",
    "molecules": "ATP"
  },
  {
    "caption": "L Time-controlled knock-down of ecdysoneless (ecd) in the dorsal compartment of ubi-AT1.03NL wing discs using apGalts>ecdRNAi. Image of FRET efficiency in apGalts>ecdRNAi wing disc 48 h after RNAi induction. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm. M Line graph showing the FRET in the dorsal and ventral compartment of apGalts>ecdRNAi (n=28) wing discs. Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ***p≤0.001. ",
    "molecules": "ATP"
  },
  {
    "caption": "A-D''' IF of NIH3T3 cells stably expressing Smo-mEos2 treated with mock (control, A-A'''), the Smo agonist (+ SAG, B-B''') or the glycolytic inhibitors 3-bromopyruvate (+3-BP, C-C''') and 2-deoxyglucose (+ 2-DG, D-D'''). DAPI staining, shown in gray, was used to visualize cell nuclei. Acetylated tubulin (AcTub), used as a ciliary marker, is shown in magenta, Smo-mEos2 is shown in green. Zoomed-in images of cilia marked by white arrows in A'', B'', C'', and D'' are shown in A''', B''', C''', and D'''. Scale bars= 5 μm, but 2 μm in A''', B''', C''', and D'''. E, F Box plot showing quantification of Smo-mEos2 ciliary localization in NIH3T3 cells upon 3-BP (E) and 2-DG (F) treatment, respectively. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to the minimum and maximum values. Sample size is indicated for each perturbation. Treatment with glycolytic inhibitors induces Smo-mEos2 localization to the primary cilium . t-test, ***p≤0.001.",
    "molecules": "SAG, 2-deoxyglucose, 2-DG, 3-BP, 3-bromopyruvate, DAPI"
  },
  {
    "caption": " A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs 48 h or 120 h after RNAi induction, respectively. V/D indicates the boundary between the ventral and dorsal compartments. Next to the maximal projections are shown the sum projections of cross-sections along the A/P axis. Loss of Ecd or Gapdh alters the plasma membrane potential. Scale bars= 50 μm. B Structure of the N-acylethanolamide analogue PAC-NAE. PAC-NAE contains a C15 fatty acid featuring diazirine and alkyne (red) modifications. C-C''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). PAC-NAE uptake was quantified as the ratio of PAC-NAE signal intensity in the dorsal to ventral compartments. Shown is the fold change from control in apGalts>ecdRNAi and apGalts>GapdhRNAi wing discs (C). For each condition, dorsal/ventral PAC-NAE signal intensity was quantified for 5 wing discs. Error bars indicate ± SD. test-test, **p≤0.01, ***p≤0.001. (C'-C''') Representative images of PAC-NAE uptake in control (C'), apGalts>ecdRNAi (C'') and apGalts>GapdhRNAi (C''') wing discs at 48 h or 120 h after RNAi induction, respectively. Loss of Ecd or Gapdh blocks the uptake of PAC-NAE. Dashed green lines mark the D/V boundary. Scale bars= 50 μm.",
    "molecules": "alkyne, DiBAC4, diazirine, fatty acid, N-acylethanolamide, NAE"
  },
  {
    "caption": "D-E'' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A (gA). (D) Quantification of DiBAC4(3) signal intensity in mock-treated (- gA, n=7) and gA-treated (+ gA, n=5) wing discs. (D', D'') Representative images of DiBAC4(3) assay in mock- (D') and gA-treated (D'') wing discs. Next to the maximal projections are shown sum projections of cross-sections along the A/P axis. (E) Quantification of PAC-NAE signal intensity in mock- (n=7) and gA-treated (n=9) wing discs, shown as fold change from control (mock-treated). (E'-E'') Representative images of PAC-NAE uptake in mock- (E') and gA-treated (E'') wing discs. GA treatment strongly reduces the uptake of PAC-NAE. Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. Scale bars= 50 μm.",
    "molecules": "DiBAC4, gA, GA, gramicidin A, NAE"
  },
  {
    "caption": "MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).",
    "molecules": "LPS"
  },
  {
    "caption": "Cells transfected as above were pre-treated or not with nevirapine and transduced with HIV-GFP vectors for 48h. The percentage of GFP+ MoLC was analysed by flow cytometry. Data represent means values ± SD from two different donors.",
    "molecules": "nevirapine"
  },
  {
    "caption": "Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.",
    "molecules": "Nevirapine"
  },
  {
    "caption": "The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV-1-R5 and HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).",
    "molecules": "Nev, Nevirapine"
  },
  {
    "caption": "E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown.",
    "molecules": "CCF2"
  },
  {
    "caption": "Colorimetric HIV-1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations. Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C-containing conditions and shown on graphs.",
    "molecules": "MgCl2"
  },
  {
    "caption": "(A) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor before and after shockwave stimulation in HBSS Ca2+ medium with (yellow line, n = 6, N = 3) or without the Piezo1 expression (black line, n = 6, N = 3). (B) The representative FRET/ECFP ratio images of calcium biosensor in Piezo1-expressed HEK cells before (top left) and after (top right) LIS stimulation. Phase image with laser initiating point (left) and fluorescent image indicating Piezo1 expression (right) were also shown on the bottom panels (Scale bar, 40 μm). Color scale bars in the figures are to show the FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (C) The time course of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2+ medium (n = 6, N = 3).",
    "molecules": "calcium, Ca2+"
  },
  {
    "caption": "(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before (left) and after (right) shockwave stimulation (Scale bar, 40 μm). Color scale bars in the figures are to show the ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized ECFP/FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2+ media with (green line, n = 16, N = 3) or without Piezo1 (black line, n = 10, N = 3). (C) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2+ medium. *P < 0.05 from student two-tailed t-test.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after Yoda1 stimulation with (pink line, n = 10, N = 3) or without Piezo1 (black line, n = 11, N = 3). The grey line represents the DMSO control group (n = 8, N = 3).",
    "molecules": "DMSO, Yoda1"
  },
  {
    "caption": "(C) The representative FRET/ECFP ratio images of the calcium biosensor in the Piezo1-expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (D) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells (purple line, n = 9, N = 3) before and after 25 μM Yoda1 stimulation in culture medium.",
    "molecules": "calcium, Yoda1"
  },
  {
    "caption": "(A)&(B), The time courses (Mean ± SEM) (A) and representative images (Scale bar, 10 μm) of FRET ratio of SHP2 biosensor (Y542F) before and after 25 μM or 0.5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression (red bar, n = 5, N=3; blue bar, n = 5, N=3; purple bar, n = 11, N=3; green bar, n = 5, N=3).",
    "molecules": "Yoda1"
  },
  {
    "caption": "(A-C) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP-LC3. (A) Increased LC3 puncta per cell following β‐catenin knockdown with siRNA compared with a non‐targeting (NT) control siRNA. Columns show autophagosome numbers (mean±s.e.m., n>200 cells in >20 fields of view per experiment of three independent treatments) assessed under normal and starved (2 h) conditions with β‐catenin or non‐targeting (NT) siRNA. Representative images (B) of YFP-LC3 puncta (green) and DAPI nuclei staining (blue) are shown. (C) Upper panel: western blotting showed increased LC3‐II expression in HT29 cells with β‐catenin knockdown compared to control cells after 2 h starvation. Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.011 in nutrient conditions, *P=0.035 under starvation).",
    "molecules": "nutrient"
  },
  {
    "caption": "(D and E) HCT116 β‐cateninWT/− cells overexpressing a control or β‐cateninS33Y plasmid nutrient starved for 24 h. (D) Immunostaining of LC3 (green) with DAPI nuclei staining (blue) showed decreased LC3 puncta with β‐cateninS33Y overexpression. (E) By western blotting, LC3‐II decreased with β‐cateninS33Y overexpression (upper panel). Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.015).",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) LC3 puncta number per cell during starvation and nutrient addback post starvation. Left panel: LC3 puncta number reduces in non‐targeting (NT) siRNA control with nutrient addback. Right panel: LC3 puncta number reduces in β‐catenin knockdown cells with nutrient addback (mean±s.e.m. of three independent treatments; n>200 cells in >20 fields of view per experiment). Western blotting for β‐catenin confirmed knockdown.",
    "molecules": "nutrient"
  },
  {
    "caption": "(D) Western blotting of LC3 and p62 in HT29 cells following β‐catenin knockdown and starvation with lysosomal inhibitors. LC3‐II and p62 protein expression showed an increase with β‐catenin siRNA in the presence of autophagy flux inhibitors chloroquine (10 μM) or bafilomycin A1 (100 nM; applied for the final 30 min of the 8 h) compared to β‐catenin knockdown alone. Quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of four independent experiments). See also Supplementary Figure S1A.Source data for this figure is available on the online supplementary information page.",
    "molecules": "bafilomycin A1, chloroquine"
  },
  {
    "caption": "(C) The increase in p62 protein expression induced by β‐catenin siRNA was attenuated in HT29 cells treated with 10 μg/μl cycloheximide and starvation for 8 and 24 h compared to vehicle control. A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(E) Doxycycline induction (1 μg/ml) of DNTCF4 increased p62 and LC3‐II protein expression in doxycycline‐inducible DNTCF4 LS174T‐L8 cells. LGR5 downregulation by DNTCF4 confirmed inhibition of β‐catenin/TCF4 signalling.",
    "molecules": "Doxycycline, doxycycline"
  },
  {
    "caption": "(F) Relative p62 mRNA levels (48 h) by qRT-PCR increased following doxycycline induction of DNTCF4 in LS174T‐L8 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A) Chromatin immunoprecipitation (ChIP) demonstrates β‐catenin and TCF4 binding to the p62 promoter. HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies (IgG was used as a negative control). Binding of RNA Pol II, β‐catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin. During nutrient deprivation, binding of RNA Pol II to the p62 promoter increased; binding of β‐catenin to the p62 promoter decreased; TCF4 binding did not change (data are from one representative experiment of at least three independent experiments performed in triplicate). PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A.",
    "molecules": "nutrient"
  },
  {
    "caption": "(C, D) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using (C) starvation or (D) 100 nM mTOR inhibitor PP242 (C and D, mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D).",
    "molecules": "PP242"
  },
  {
    "caption": "(G-I) Inhibition of Wnt‐induced TopFlash activity by autophagy induction in (G) HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, **P0.01) and (H) RKO cells (mean±s.e.m., three independent experiments performed in triplicate, *P0.05). (I) Reduction of Wnt3a‐induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 (mean±s.e.m., two independent experiments).",
    "molecules": "PP242"
  },
  {
    "caption": "(N) Western blot showing prevented β‐catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine (10 μM) and bafilomycin A1 (100 nM) compared to starvation alone.",
    "molecules": "bafilomycin A1, chloroquine"
  },
  {
    "caption": "(O) Inhibition of autophagy using wortmannin (50 nM) prevented β‐catenin protein degradation during starvation.",
    "molecules": "wortmannin"
  },
  {
    "caption": "(P) Western blot analysis of RKO cells expressing a myc-tagged β‐catenin mutant (S33A, S37A, T41A, S45A) resistant to proteasomal degradation (myc-β‐cateninAAAA). RKO cells were subject to nutrient starvation for 2, 8 and 24 h, and autophagy induction was confirmed by increased LC3‐II and decreased p62 protein expression. Both endogenous β‐catenin and myc-β‐cateninAAAA protein expression decreased during starvation.",
    "molecules": "nutrient"
  },
  {
    "caption": "(Q) Proteasome inhibition with MG132 (10 μM) did not prevent starvation‐induced decrease in β‐catenin protein expression.Source data for this figure is available on the online supplementary information page.",
    "molecules": "MG132"
  },
  {
    "caption": "LC3 and β‐catenin co‐localise in the mouseintestinal epithelium. (A and B) Immunofluorescence of LC3 (red) and β‐catenin (green) expression in the intestinal epithelium following 2 daystamoxifen treatment in control (A: β‐catenin+/lox‐villin‐creERT2) and β‐catenin deleted (B: β‐catenin−/lox‐villin‐creERT2) mice. (C and D) Magnified areas from B revealing co‐localisation of β‐catenin (red) and β‐catenin (green). Arrowheads indicate co‐localisation of LC3 and β‐catenin. (E) Linescan analyses from C and D showing staining intensity of indicated co‐localised puncta. Red and green channel levels were adjusted post acquisition (equal changes applied across the entire figure) and the blue channel was removed for clarity.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(A) Co‐immunoprecipitation of YFP-LC3 or negative control YFP in HT29 cells. Binding of endogenous β‐catenin to YFP-LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine (10 μM). Input and immunodepleted lysates are shown.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(F) Co‐immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP-LC3 with β‐cateninWT or β‐cateninW504A/I507A starved for 8 h with chloroquine (10 μM). YFP-LC3 immunoprecipitated β‐cateninWT but not β‐cateninW504A/I507A. Input lysates are shown and immunoprecipitated p62 and YFP-LC3 served as positive controls.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(H) Relative cell death in HT29 cells subject to nutrient starvation for 24 h. Atg7 knockdown increased cell survival. The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β‐catenin (double knockdown of Atg7 and β‐catenin). Data are the mean±s.e.m. of three independent experiments performed in triplicate, ***P0.001; **P0.01.Source data for this figure is available on the online supplementary information page.",
    "molecules": "nutrient"
  },
  {
    "caption": "Diploid strain RBY1 and tetraploid strain RBY18 were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 7 days and plated onto 2-DOG medium to monitor for GAL1 loss.",
    "molecules": "2-DOG"
  },
  {
    "caption": "2-DOGR progeny arising from RBY18 on PRE-SPO medium were DNA stained and their ploidy determined via flow cytometry.",
    "molecules": "2-DOG, DNA"
  },
  {
    "caption": "TTC assay results in diploid and tetraploid cells grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively. Cells were grown for 24 h at which point a TTC overlay solution was applied and color allowed to develop for 15 min.",
    "molecules": "TTC"
  },
  {
    "caption": "Diploid and tetraploid strains were spot inoculated onto YPD or PRE-SPO medium supplemented with the pH indicator bromocresol purple and cultured for 24 h at 30°C or 37°C, respectively.",
    "molecules": "bromocresol purple"
  },
  {
    "caption": "Cell size and mean MitoTracker Green signal intensity in diploid and tetraploid cells grown on YPD medium for 24 h at 30°C (n=100 and n=5 for cell volume and MitoTracker Green measurements, respectively, error bars represent S.E.M.).",
    "molecules": "MitoTracker Green"
  },
  {
    "caption": "Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h, stained with CellROX Green, and signal analyzed via flow cytometry.",
    "molecules": "CellROX Green, calcofluor white, DAPI"
  },
  {
    "caption": "Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 (Cap1-mNeonGreen) were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE-SPO medium. "n.d." indicates that no cells with activated Cap1 were detected.",
    "molecules": "calcofluor white, DAPI"
  },
  {
    "caption": "Analysis of cell viability in tetraploid cells after culture on PRE-SPO medium (37°C for 24 h) when supplemented with ascorbic acid, DTT, or glutathione at the indicated concentrations.",
    "molecules": "DTT, ascorbic acid, glutathione"
  },
  {
    "caption": "Analysis of GAL1 loss (2-DOG resistant colonies) in tetraploid strain RBY18 after growth on PRE-SPO medium with or without antioxidants for 7 days at 37°C.",
    "molecules": "2-DOG"
  },
  {
    "caption": "Tetraploid cells expressing Cap1-mNeonGreen were grown on PRE-SPO medium supplemented with 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione for 24 h at 37°C. Cell images were analyzed to determine the percentage of viable cells with activated Cap1.",
    "molecules": "DTT, ascorbic acid, glutathione"
  },
  {
    "caption": "Assay to monitor for loss of GAL1 from tetraploid RBY18 overexpressing the indicated genes. Cells were grown for 7 d at 37°C on PRE-SPO medium in the presence (gene OFF) or absence (gene ON) of doxycycline (DOX).",
    "molecules": "DOX, doxycycline"
  },
  {
    "caption": "Diploid (2n) and tetraploid (4n) strains expressing a Gam-GFP reporter were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence (Gam-GFP ON) or absence (Gam-GFP OFF) of doxycycline (DOX) for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.",
    "molecules": "calcofluor white, DAPI, DOX, doxycycline"
  },
  {
    "caption": "Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence or absence of doxycycline for 24 h.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on PRE-SPO medium with or without 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione at 37°C for 24 h.",
    "molecules": "DTT, ascorbic acid, glutathione"
  },
  {
    "caption": "10-fold dilutions of diploid and tetraploid cells were spot inoculated onto SCD medium with or without the DNA-damaging agents hydroxyurea (HU, 20 mM) or methyl methanesulfonate (MMS, 0.01%). Cells were grown for 48 h at 30°C or 37°C and the plates imaged.",
    "molecules": "HU, hydroxyurea, methyl methanesulfonate, MMS"
  },
  {
    "caption": "Assay to monitor for loss of GAL1 (2-DOG resistant cells) arising from tetraploid strain RBY18 after culture at 37°C on SCD medium alone, SCD supplemented with HU (20 mM) or MMS (0.01%), or SCD medium with exposure to UV light for five seconds a day for 7 days.",
    "molecules": "2-DOG, HU, MMS"
  },
  {
    "caption": "Tetraploid cells were grown on SCD medium with or without the oxidative-stress inducing agents paraquat (PQT; 600 μg/mL), hydrogen peroxide (H2O2; 2 mM), or piperlongumine (PL; 100 μg/mL) for 24 h at 37°C and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.",
    "molecules": "CellROX Green, calcofluor white, DAPI, H2O2, hydrogen peroxide, paraquat, PQT, piperlongumine, PL"
  },
  {
    "caption": "Tetraploid cells expressing Cap1-mNeonGreen were grown on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C. Cell images as in (A). Scale bar = 10 μm.",
    "molecules": "H2O2, PQT, PL"
  },
  {
    "caption": "Tetraploid cells expressing a Gam-GFP reporter were cultured on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C and analyzed via fluorescence microscopy. Cell images as in (A). Scale bar = 10 μm.",
    "molecules": "H2O2, PQT, PL"
  },
  {
    "caption": "Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on SCD with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C.",
    "molecules": "H2O2, PQT, PL"
  },
  {
    "caption": "2-DOG assay to monitor for loss of GAL1 function from tetraploid strain RBY18 after culture on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) at 37°C for 7 d.",
    "molecules": "2-DOG, H2O2, PQT, PL"
  },
  {
    "caption": "C Representative immunoblot  comparing the ability of wild-type and Arg371Ser FICD (at a wide range of enzyme concentrations) to catalyse the accumulation of BiP-AMP  in the presence of physiological ATP  concentrations (5 mM). At low enzyme concentrations (and early time points) the AMPylation defect imposed by Arg371Ser is manifest. At higher enzyme concentrations where the  deAMPylation activity of the wild-type enzyme begins to dominate, the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP-AMP  (relative to the same concentration of wild-type enzyme). D Quantification of the   AMPylated BiP  signals relative to total BiP  of experiments as in C (mean values ± SEM, n = 4, biological replicates).",
    "molecules": "AMP, ATP"
  },
  {
    "caption": "F Representative immunoblot  comparing the accumulation of BiP-AMP  in reactions constituted with the indicated concentrations of wild-type and Arg371Ser  FICD, performed as in A (upper panel). The lower panel is a  Coomassie-stained gel of the reaction constituents. An N-terminally extended version of the wild-type enzyme (residues 45-458was used in lanes 2 and 5-7, to distinguish its migration on the gel from the mutant (residues 104-445). The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F (from n = 3, biological replicates, mean values ± SEM).",
    "molecules": "AMP"
  },
  {
    "caption": "A Immunoblots  of AMPylated  proteins detected with a pan-AMP  antibody, BiP, FICD  and eIF2α  (a loading control) in lysates of  FICD∆ CHO  cells transiently transfected with plasmids encoding the indicated derivatives of FICD. Signals corresponding to AMPylated BiP, auto-AMPylated FICDE234G, total BiP, FICD  and eIF2α  are indicated. The novel species reactive with the anti-FICD  antiserum in lysates of cells transfected with plasmids expressing FICDR371S (*) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369.",
    "molecules": "AMP"
  },
  {
    "caption": "B On the left are immunoblots  of AMPylated  proteins detected with pan-AMP, BiP  and eIF2α  (a loading control) reactive antibodies in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide  (Cx, 100 µg/mL) for the indicated time. In the panel on the right are immunoblots  of samples immunoprecipitated  with a BiP-specific antiserum from the lysates of the samples shown on the left.",
    "molecules": "AMP, Cx, cycloheximide"
  },
  {
    "caption": "C Immunoblots  as in B in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide  (Cx, 100 µg/mL) and/or thapsigargin  (Tg) for the indicated periods prior to harvest. D Quantification of ratio of the BiP-AMP  to total BiP  signal (expressed in arbitrary units) of repeats of the experiment shown in C. Mean ± SD of the BiP-AMP  signal normalized to total BiP  signal from the same samples are depicted, (P values by two tailed t-test, n = 6, biological replicates).",
    "molecules": "AMP, Cx, cycloheximide, Tg, thapsigargin"
  },
  {
    "caption": "Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 5 μM CTV-labelled (CTVbright) 24JK-HER-2 and 0.5 μM CTV-labelled (CTVdim) 24JK sarcoma cells. Antigen-specific target cell lysis was monitored for the depletion of CTVbright 24JK-HER-2 cells by flow cytometry.",
    "molecules": "CTV"
  },
  {
    "caption": "HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. CTV-labelled CD8+ HER-2 CAR-T cells were incubated with 24JK-HER-2 or 24JK sarcoma cells and CTV dilution assessed by flow cytometry to monitor proliferation.",
    "molecules": "CTV"
  },
  {
    "caption": "HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. Lymphocytes were isolated from the tumours on day 16 post adoptive transfer and mCherry+CD45+ CD8+ CAR-T cell numbers were determined by flow cytometry. In (c) TILs were stained for intracellular IFNγ and TNF after PMA/Ionomycin treatment.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "HER-2-E0771 cells generated to inducibly overexpress PTPN2 in response to doxycycline (E0771-HER-2-PTPN2hi) were pre-incubated (24 h) with vehicle or doxycycline (DOX) subsequently stimulated with IFNγ for the indicated times. STAT-1 Y701 phosphorylation (p-STAT-1) and PTPN2 levels were assessed by immunoblotting.",
    "molecules": "DOX, doxycycline"
  },
  {
    "caption": "Cxcl9 and Cxcl10 mRNA levels in vehicle versus DOX-treated and IFNγ-stimulated HER-2-E0771 cells were assessed by quantitative real time PCR.",
    "molecules": "DOX"
  },
  {
    "caption": "E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. Mice were then injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer In (g) CD45+CD8+mCherry+ TILs were quantified by flow cytometry at day 4 post adoptive transfer.",
    "molecules": "DOX"
  },
  {
    "caption": "E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. Mice were then injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and (h) tumour growth was monitored.",
    "molecules": "DOX"
  },
  {
    "caption": "CD8+ LY CAR-T cells generated from human PBMCs were treated with PTPN2-inhibitor (+) or vehicle (-) followed by incubation with plate-bound α-LY, LY-negative MDA-MB-435 cells and LY-expressing OVCAR-3 cells. The proportion of IFNγ+ versus IFNγ+TNF+ CD8+CAR-T cells was determined by flow cytometry.",
    "molecules": "LY"
  },
  {
    "caption": "HER-2 CAR-T cells generated from C57BL/6 and Lck-Cre;Ptpn2fl/fl splenocytes were transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs and intracellular PTPN2 levels were determined by flow cytometry.",
    "molecules": "FITC"
  },
  {
    "caption": "c-d) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 HER-2 CAR-T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs two days before adoptive CAR-T cell transfer. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and tumour growth was monitored.",
    "molecules": "FITC"
  },
  {
    "caption": "HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 HER-2 CAR-T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs two days before adoptive CAR-T cell transfer. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and tumour growth was monitored. CD45+CD3+CD8+ mCherry+ CAR-T cells numbers were determined in HER-2-E0771 positive tumours and spleens by flow cytometry 21 days post adoptive transfer.",
    "molecules": "FITC"
  },
  {
    "caption": "Hydrogen peroxide (H2O2) levels were measured in WT and MCK-KO soleus (E) at rest and after a bout of exercise (n = 6 per group, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).",
    "molecules": "H2O2, Hydrogen peroxide"
  },
  {
    "caption": "(M, N) Mitochondrial respiration was measured in WT and MCK-KO soleus tissue after a bout of low-intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM each of rotenone and antimycin A (complex I inhibitor) (n = 8, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (O, P) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM rotenone and antimycin A (complex I inhibitor) (n = 5, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).",
    "molecules": "antimycin A, FCCP, oligomycin, rotenone"
  },
  {
    "caption": "Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction. NADPH levels (H) and NOX activity (I) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).",
    "molecules": "NADPH"
  },
  {
    "caption": "(J, K) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (J: n = 3 and K: n = 6 for Control, Aldh1l1 KD, Aldh1l1 KD + Dnmt3a KD, n = 5 for Dnmt3a KD, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).",
    "molecules": "H2O2"
  },
  {
    "caption": "(D, E) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC (3mM) or vehicle treatment for 24hrs (n = 5 Control groups n = 3 Dnmt3a KD, n = 4 Dnmt3a KD with NAC treatment groups. means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).",
    "molecules": "NAC"
  },
  {
    "caption": "TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress. (A) ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) left untreated, after 6 h of nutrient starvation or 24 h of metabolic stress. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation), from three independent experiments.",
    "molecules": "nutrient, ROS"
  },
  {
    "caption": "TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.(B) Basal, nutrient starvation-induced (5 h) or metabolic stress-induced (18 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05).",
    "molecules": "nutrient, ROS"
  },
  {
    "caption": "TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) confocal microscopy images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.",
    "molecules": "nutrient"
  },
  {
    "caption": "TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(B) (Left panel) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP-LC3 and transfected with scrambled or TIGAR siRNAs. After 48 h transfection, cells were then left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.",
    "molecules": "nutrient"
  },
  {
    "caption": "TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (C) (Left panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs, and 48 h later exposed to nutrient starvation for 0, 2.5 and 6 h. (Middle panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) and left untreated. (Right panel) Western blot showing the expression levels of p62, COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated. Actin expression was examined as a loading control.",
    "molecules": "nutrient"
  },
  {
    "caption": "TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(D) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0, 1, 3, 5 and 8 h; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).",
    "molecules": "nutrient"
  },
  {
    "caption": "TIGAR expression modulates autophagy independently of p53. (A) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were transfected with scrambled or TIGAR siRNAs, and 48 h after transfection, cells were left untreated (t0) or treated with Bafilomycin A1 (100 nM) for 1 or 2 h (t1 and t2). The percentage of cells with GFP-LC3 puncta was calculated at the indicated time points. Data are shown as the mean and standard deviation from three independent experiments.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(B) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA, and 48 h later exposed to nutrient starvation for 5 h. Actin expression was examined as a loading control.",
    "molecules": "nutrient"
  },
  {
    "caption": "(D) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or DRAM siRNA1/2. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments; * represents significant difference from starved control conditions (P<0.05); @ represents significant difference from untreated control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).",
    "molecules": "nutrient"
  },
  {
    "caption": "(A) U2OS cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.",
    "molecules": "L-ascorbic acid, NAC, AO1, nutrient"
  },
  {
    "caption": "(B) (Left panel) ROS levels in U2OS cells left untreated or treated with 0.5 or 1 mM of H2O2 for 24 h. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation) from three independent experiments. (Right panel) Quantitation of the percentage of GFP-LC3 puncta positive cells for cells treated as described above. Data are shown as the mean and standard deviation from three independent experiments.",
    "molecules": "H2O2, ROS"
  },
  {
    "caption": "(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2. After 48 h transfection, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.",
    "molecules": "L-ascorbic acid, NAC, AO1, nutrient"
  },
  {
    "caption": "(D) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 6 h with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)), AO2 (glutathione ethyl ester (4 mM)) or AO3 (ethyl pyruvate (4 mM)) for 24 h. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmid for Flag-tagged-TIGAR. After 48 h transfection, cells were treated. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.",
    "molecules": "AO3, ethyl pyruvate, glutathione ethyl ester, AO2, L-ascorbic acid, NAC, AO1, nutrient"
  },
  {
    "caption": "(E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).",
    "molecules": "L-ascorbic acid, NAC, AO1, nutrient, ROS"
  },
  {
    "caption": "(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and either scrambled, ATG5 siRNA or ATG10 siRNA. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h.",
    "molecules": "nutrient"
  },
  {
    "caption": "(D) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and scrambled, ATG5 siRNA or ATG10 siRNA, measured by flow cytometry after DCF treatment. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).",
    "molecules": "nutrient, ROS"
  },
  {
    "caption": "(A) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) were left untreated or exposed to nutrient starvation for 6 h, with or without treatment with Z-VAD-FMK for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation of the mean from three independent experiments.",
    "molecules": "nutrient, Z-VAD-FMK"
  },
  {
    "caption": "(B) Apoptosis in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3), as measured by cells with a sub-G1 DNA content. Cells were transfected with either scrambled, TIGAR siRNA1 or TIGAR siRNA2. Cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h.",
    "molecules": "DNA, nutrient"
  },
  {
    "caption": "(C) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled, ATG5 siRNA or ATG10 siRNA, as measured by cells with a sub-G1 DNA content. After 48 h, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. Data are shown as the mean and standard deviation from three independent experiments. In each case, the increase in apoptosis after knockdown of ATG5 or ATG10, compared with the matched control, was statistically significant; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).",
    "molecules": "DNA, nutrient"
  },
  {
    "caption": "MSCs were exposed to increasing LPS concentrations, thereafter washed and co-cultured with PMA activated neutrophils. Incubation of neutrophils with PMA alone served as positive control and DMSO treated neutrophils as negative control. Scale bars: 50μm.",
    "molecules": "DMSO, LPS, PMA"
  },
  {
    "caption": "Representative microphotographs of murine wound sections immunostained for Ly6G (neutrophils, green) and DNA-histone (displayed as expulsed streaks in red indicative of NETs). Wounds injected with LPS primed MSCs (upper row, outer right panel) depict enhanced NET formation (magnified inset) and increased expression of activation markers like neutrophil elastase (NE, lower panel, outer left panel) compared to wounds injected with non-primed MSC (middle panels) or PBS injected wounds (outer left panels). Murine skin wounds treated with PBS injections served as control. Scale bar: 10μm (upper row) and 50μm (lower row). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets.",
    "molecules": "DNA, histone, LPS"
  },
  {
    "caption": "Hierarchical clustering analysis of RNAseq expression profile from MSCs stimulated with LPS for 6 and 24 h. The heat-map shows the gene expression profile of non-primed MSCs and MSCs which were LPS primed for 6 and 24h. Red color represents upregulation, while blue color depicts down regulation in gene expression, each data point represents FPKM in log2 value.",
    "molecules": "LPS"
  },
  {
    "caption": "Validation of the selected genes uncovered from global RNAseq analyses by qRT-PCR analyses displays up-regulation of CXCL-6 after LPS treatment.",
    "molecules": "LPS"
  },
  {
    "caption": "Validation of the selected genes uncovered from global RNAseq analyses by qRT-PCR analyses displays up-regulation of , IL-8 and IL-1β after LPS treatment.",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA results depict an up-regulated expression of CXCL-6 after 24h of LPS treatment",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA results depict a significantly increased IL-8 expression after 6 and 24h of LPS treatment.",
    "molecules": "LPS"
  },
  {
    "caption": "Western blot analysis showed up-regulation of IL-1β expression upon LPS priming of LPS primed MSCs. Actin served as control. Densitometry graph shows significant increase after 6h LPS stimulation compared to non-primed MSCs. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates.",
    "molecules": "LPS"
  },
  {
    "caption": "The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS primed MSCs into wounds increased the expression of CXCL6, IL-8 and IL-1β. Graphs display numbers of double positive cells for h-β2M + CXCL6, h-β2M + IL-8 and h-β2M + IL-1β, respectively. MSCs injected wounds served as controls. Statistical analysis was performed using unpaired t-test, values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50µm.",
    "molecules": "LPS"
  },
  {
    "caption": "Results show that LPS primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL-8 in mice and are known as neutrophil chemoattractant. Similar results were found for IL-1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP-2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double positive cells for Ly6G MIP/KC, IL-1β and LIX, respectively. PBS and non-primed MSCs injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Scale bars: 50µm.",
    "molecules": "LPS"
  },
  {
    "caption": "Representative microphotographs of TLR4 silenced and non-silenced MSCs primed with LPS and co-cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA-histone, red). Of note, TLR4 silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non-silenced, LPS primed MSCs co-cultured with PMA activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co-culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50μm.",
    "molecules": "DNA, DMSO, histone, LPS, PMA"
  },
  {
    "caption": "Quantitative assessment of NET bound elastase employing a specific ELISA in identical experimental setting as described in Figure 1. Similarly, to immunostaining, a significant reduction of NET bound elastase was observed in TLR4 silenced LPS primed MSCs when co-cultured with activated neutrophils as opposed to high NET bound elastase in non-silenced LPS primed MSCs co-cultured with activated neutrophils. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.",
    "molecules": "LPS"
  },
  {
    "caption": "High resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS primed MSC co-cultured with PMA activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non-primed MSCs co-cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4 silenced LPS primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4 silenced and non-primed MSCs display reduced NETs in co-cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA activated neutrophils alone served as positive control with highly enhanced NETs (upper row, outer right panel) and DMSO treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil derived granules. Scale bars: 0.1µm",
    "molecules": "DMSO, LPS, PMA"
  },
  {
    "caption": "Representative clinical pictures of murine wounds at 0, 3, 5, 7 and 10 days after wounding. Enhanced wound healing in LPS primed MSCs group as opposed to all other groups. Statistical analysis of 20 wound areas per group at the indicated time points, expressed as percentage of the initial wound size (day 0), for PBS control, non-primed MSCs, LPS primed MSCs, non-primed scrambled siRNA-treated (Scr) MSCs, LPS primed Scr MSCs and LPS primed TLR4 silenced MSCs. Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments. Statistical analysis was performed using one way ANOVA.",
    "molecules": "LPS"
  },
  {
    "caption": "Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G+ neutrophils (green) and F4/80+ macrophages (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 1 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs Double stained cells indicate phagocytic engulfment of neutrophils by macrophages. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bar: 100µm.",
    "molecules": "DAPI, LPS"
  },
  {
    "caption": "Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double-stained for TGFβ-1 (red) and for F4/80+ macrophages (green). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 5 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs. Scale bar: 50µm. Representative photomicrographs of sections of day 5 wounds immunostained for CD31 (indicative of endothelial cells and newly formed vessels) and for α-SMA (indicative of myofibroblasts differentiation) after injection of LPS primed MSCs, non-primed MSCs, LPS primed TLR4-silenced MSCs or PBS (middle and lower panel). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bars: 50µm.",
    "molecules": "DAPI, LPS"
  },
  {
    "caption": "Quantitative analysis of CD31 positive endothelial cells in sections of wounds injected with PBS, non-primed MSCs, LPS primed MSCs and LPS primed TLR4 silenced LPS Cell counting was performed on immunostained wound sections and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.",
    "molecules": "LPS"
  },
  {
    "caption": "Western blot analysis of lysates from day 5 wounds (left panel) and the corresponding densitometric analysis (right panel) depicts enhanced α-SMA protein expression in wounds injected with LPS primed MSCs as opposed to the respective control groups. Actin served as loading control. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.",
    "molecules": "LPS"
  },
  {
    "caption": "  (C) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled RORA consensus, rs2978980T or rs2978980G probes and HGC-27 or MGC80-3 nuclear extracts. HGC-27 or MGC80-3 left panel: EMSA with RORA consensus or rs2978980T probes. Lane 1-8: from the first lane at the left side to the right side. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5 and 7, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 1 and 5) or RORA consensus probes (lanes 3 and 7). HGC-27 or MGC80-3 right panel: EMSA with rs2978980T or rs2978980G probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3-5 and 8-10, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 5 and 8), rs2978980G (lanes 3 and 10) or RORA consensus probes (lanes 4 and 9).  ",
    "molecules": "biotin"
  },
  {
    "caption": " (B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm. ",
    "molecules": "crystal violet"
  },
  {
    "caption": " (F) Gastric cancer cells after lncPSCA-knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide (CHX) or vehicle for the indicated periods of time. DDX5 levels were analyzed by Western blot. ",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(A) Total to oxidized glutathione ratio (GSH/GSSG) in mock infected or HIV-1 infected CD4+ T-cells (7 dpi, n=4 biological replicates; mean±SEM).",
    "molecules": "glutathione, GSH, GSSG"
  },
  {
    "caption": "representative picture (B) of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH",
    "molecules": "DNA, ROS"
  },
  {
    "caption": "quantification of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH (D). The black dash in (C) indicates the median. Box plots in (D) depict median and 25-75 percentile, while whiskers extend from min to max. n= number of cells. Scale bar = 2μm.",
    "molecules": "ROS"
  },
  {
    "caption": "(F) Nuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA+ and HIV-1 RNA- cells (7dpi) as measured by combining immunofluorescence and HIV-1 RNA FISH. Black dash indicates median, n= number of cells. Scale bar = 2μm",
    "molecules": "RNA"
  },
  {
    "caption": "(I) Relative mRNA expression of Nrf2 downstream antioxidant targets in J-Lat 9.2 cells left untreated (latent) or treated for 24h with 10µM TPA (reactivated). Data are depicted as mean±SEM of 3 biological replicates. raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in TPA-reactivated vs latent cells Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC= glutamate-cysteine ligase  ; TrxR1= thioredoxin reductase 1.",
    "molecules": "TPA"
  },
  {
    "caption": "(F) Percentage of intracellular p24+ gag CD4+ T-cells infected with HIV-1 and left untreated or treated for 48hr with the iron donor Fe-NTA at 150μM concentration (n=3; mean ± range of three biological replicates).",
    "molecules": "Fe-NTA, iron"
  },
  {
    "caption": "(G) Percentage of GFP+ J-Lat cells (clones 9.2 and 15.4) left untreated or treated for 48hr with TPA (10μM), the iron donor FeNT (150μM) or a combination of the two. Data are expressed as mean±SEM of four technical replicates for each cell line and were analyzed by one-way ANOVA followed by Tukey´s post-test. *P<0.05; ***P<0.001; ****P<0.0001.",
    "molecules": "TPA, FeNT, iron"
  },
  {
    "caption": "(E) Western blot and quantification of PML protein in two different clones of J-Lat cells left untreated or treated with TPA for 24h. Data were analyzed by unpaired t-test (mean±SEM; n=3 technical replicates for each cell line).",
    "molecules": "TPA"
  },
  {
    "caption": "(F PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the antioxidant compound NAC (10mM, F)",
    "molecules": "NAC"
  },
  {
    "caption": "G) PML protein expression in CD4+ T-cells infected with HIV-1 or mock infected at 7dpi as measured by western blot. Cells were left untreated or treated with the iron chelator deferiprone (L1, 50μM, G).",
    "molecules": "deferiprone, iron"
  },
  {
    "caption": "B) Representative image and quantification of the number of PML NBs in HIV-1 RNA+ vs HIV-1 RNA- CD4+ T-cells. Scale bars = 2μm. Infected cells were identified with RNA FISH and PML NBs were stained by IF.",
    "molecules": "RNA"
  },
  {
    "caption": "Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Proteasome function was blocked for 4 hrs in both latent and TPA reactivated cells using MG132 (10μM) or Bortezomib (100nM)",
    "molecules": "TPA, Bortezomib, MG132"
  },
  {
    "caption": "Representative immunofluorescence images and quantifications of PML NBs (green, maximal projection) and mab414/nuclear pore (red, one Z stack) in J-Lat 9.2 cells. Cells were left latent or reactivated for 24 hrs with 10μM TPA. Alternatively, ubiquitination was blocked for 4 hrs using TAK243 (10μM)",
    "molecules": "TPA, TAK243"
  },
  {
    "caption": "(E,F) Representative immunofluorescence images (E) and quantification (F) of PML NBs (green) and SUMO 2/3 (red) in J-Lat 9.2 cells left latent or reactivated for 24 hours with 10μM TPA. Cells were imaged by STED microscopy, scale bar 2 μm.",
    "molecules": "TPA"
  },
  {
    "caption": "Left: Representative images of cells were transfected with super-folder GFP (sfGFP) treated with 250ng/mL Tunicamycin (Tm), 50nM Thapsigargin (Tg) and 250ng/mL Brefeldin-A (BFA) for 24 hours. Right: Quantification of the microscopy images of cells expressing ER-targeted sfGFP and the cytosolically localized mCherry. Data values are the mean ± SD of technical replicates (n=10) from 3 independent experiments (****p<0.0001). One-way ANOVA was applied for the statistical analysis through the GraphPad Prism 9 software. Scale bar 15μm.",
    "molecules": "BFA, Brefeldin-A, Tg, Thapsigargin, Tm, Tunicamycin"
  },
  {
    "caption": "Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) protocols, representative western blots are showed.",
    "molecules": "Digitonin, NP40, Tg, Tm"
  },
  {
    "caption": "Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) or differential centrifugation protocols, representative western blots are showed.",
    "molecules": "Digitonin, NP40, Tg, Tm"
  },
  {
    "caption": "Subcellular protein fractionation of several ER resident proteins in (A) GL261, (B) U87 and (C) A549 cells treated with the indicated concentrations of Tm or Tg using Digitonin. (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) Quantification of the subcellular protein fractionation of several ER endogenous proteins in GL261, U87 and A549 cells as in (A-C) respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad).",
    "molecules": "Digitonin, NP40, Tg, Tm"
  },
  {
    "caption": "Representative transmission electron microscopy images of the gold particles distribution (after immunogold labelling with PDIA3 antibodies) in A549 cells treated with DMSO or Tm. In the inserts, gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles. (Scale bar 500 nm). n represents the nucleus and m represents the mitochondria. (C) violin plots of the gold particles distribution from the electron microscopy experiment (immunogold labelling of PDIA3) as shown in Figure 4B and 4C. *P-value=0.0486. n=8 for DMSO and n=8 for Tm. Thick horizontal lines represent mean ± SD -lighter dashed lines. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad),",
    "molecules": "DMSO, gold particles, Tm"
  },
  {
    "caption": "Immunoprecipitation of p53 and AGR2 in the digitonin fraction of A549 cells. A549 cells were treated with Tunicamycin (Tm 100ng/ml), Thapsigargin (Tg 25nM) and Brefeldin A (BFA 0,1 and 0,25nM). Endogenous p53 was immunoprecipitated from the cytoplasmic fraction (digitonin fraction) of A549 cells. Coprecipitated endogenous AGR2 was detected by western blot.",
    "molecules": "BFA, Brefeldin A, digitonin, Tg, Thapsigargin, Tm, Tunicamycin"
  },
  {
    "caption": "A549 were treated with Etoposide (Eto) for 2 hours to induce p53 pathway. Then cells have been challenged with Tm, Tg and BFA at the indicated concentrations for 16 hours. Luciferase experiments were performed after 24 hours of transfection. Graph shows the fold induction of p53 luciferase construct. Western blot experiments for phospho-p53, pan-p53, p21 and HSP90 were performed as control. n=3, biological replicates (mean ± SD; Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. *p<0.05, ***p < 0.001.",
    "molecules": "BFA, Eto, Etoposide, Tg, Tm"
  },
  {
    "caption": "Left: Western blot experiments for phospho-p53, pan-p53, and p21 in AGR2-silenced A549 cells exposed to ER stressors: 100ng/ml Tunicamycin ( Tm), 25nM Thapsigargin (Tg) and 0.25 nM Brefeldin A (BFA), in the presence and absence of etoposide. Right: Quantification of the western blot. n=3, biological replicates, mean ±SD.",
    "molecules": "BFA, Brefeldin A, etoposide, Tg, Thapsigargin, Tm, Tunicamycin"
  },
  {
    "caption": "A549 were transfected with the indicated constructs of differently targeted nanobodies, cells were then treated with Tunicamycin (Tm 100 ng/ml and 250 ng/ml), Thapsigargin (Tg 25 nM) and Brefeldin A (BFA 0,25 nM) for 16 hours at the indicated concentration. Western blot experiments were performed and HSP90 were used as loading control. n=3, biological replicates (mean ± SD of n=3 independent experiments (***p<0,001 and **p<0,01). Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated.",
    "molecules": "BFA, Brefeldin A, Tg, Thapsigargin, Tm, Tunicamycin"
  },
  {
    "caption": "(E) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates+ADP); State 4 (+oligomycin); Uncoupled (+FCCP). (n = 3). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001).",
    "molecules": "ADP, FCCP, glutamate, malate, oligomycin, pyruvate, succinate"
  },
  {
    "caption": "(A) Representative gel of the In organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S-met (1 hour Pulse) or after cold chase (3h Chase). Positions of individual proteins are indicated on the left. Position of full-length proteins (Proficient) and low molecular weight polypeptides (Abortive) are indicated on the right. Note that in WT and ClpP KO low molecular weight polypeptides include ATP8/ND4L proteins.",
    "molecules": "met"
  },
  {
    "caption": "(B) Relative overall protein synthesis and turnover rate.",
    "molecules": "protein"
  },
  {
    "caption": "(E) Relative levels of protein synthesis of Proficient and Abortive polypeptides. (F) Quantification of the turnover rate of full-length (Proficient) de novo synthesized proteins. Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), (n=3-4).",
    "molecules": "protein, proteins"
  },
  {
    "caption": "(C) E-cad-GFP remains downregulated in cells arrested in mitosis with Colchicine. Scale bar ~2µm. n>10 independent biological replicates.",
    "molecules": "Colchicine"
  },
  {
    "caption": "(B) High-magnification view of single mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Scale bar ~1µm. n>10 independent biological replicates. (C) High-magnification view of single UAS.Rok-RNAi expressing mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Note loss of cortical p-MLC staining (purple). Scale bar ~1µm. n>10 independent biological replicates. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) MyoII RLC-GFP (MyoII-GFP) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis (basal section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic accumulation of MyoII-GFP. Quantification shown right (n = 8 independent samples per genotype). Scale bar ~1µm. Mean +/- 1 S.D. shown.",
    "molecules": "Y-27632"
  },
  {
    "caption": "(F) E-cad-GFP is downregulated as cells round up for mitosis (apical section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic downregulation of E-cad-GFP. Extended treatment with Y-27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E-cad-GFP is generally lost. Arrow indicates mitotic cell cortex at maximal rounding. Quantification shown right (n = 6 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.",
    "molecules": "Y-27632"
  },
  {
    "caption": "(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells (pH3, red) in wild-type wing discs, and aurA homozygous mutant wing discs, but not upon inhibition of both AurA and AurB by treatment with VX-680. Note increased numbers of mitotic cells in aurA homozygous mutant wing discs, due to delayed mitotic progression. Scale bars ~20µm (low mag.) and ~1µm (high mag.). n > 15 independent biological replicates.",
    "molecules": "VX-680"
  },
  {
    "caption": "(B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype).",
    "molecules": "VX-680"
  },
  {
    "caption": "(C) Live-imaging of E-cad-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, cytokinesis failure in UAS.aurB-RNAi expressing wing discs, and complete failure of mitotic rounding and E-cad-GFP downregulation upon inhibition of both AurA and AurB by treatment with VX-680. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 9 independent samples per genotype).",
    "molecules": "VX-680"
  },
  {
    "caption": "(A) Striped ptc.Gal4-driven expression of UAS.GFP in a control wing imaginal disc. Scale bars ~20µm. n > 6 independent biological replicates. (B) Striped ptc.Gal4-driven expression of UAS.GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells. n > 7 independent biological replicates. (C) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB-RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei (marked by DAPI; white arrowhead). n > 6 independent biological replicates. (D) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB -RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (E) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.pbl-RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (F) ptc.Gal4-driven expression of UAS.aurB-RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence. n > 6 independent biological replicates. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Live-imaging of MyoII-GFP in a control wing disc epithelium. Asterisk indicates the location of a single mitotic cell about to initiate rounding. Scale bar ~1µm. n > 5 independent biological replicates. (B) Live-imaging of MyoII-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 9 independent biological replicates. ",
    "molecules": "VX-680"
  },
  {
    "caption": "(C) Live-imaging of E-cad-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 6 independent biological replicates.",
    "molecules": "VX-680"
  },
  {
    "caption": "Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of microglia homeostatic (Olfml3, Fcrls, Tmem119, Siglech, Gpr34, P2ry12, Mef2c), phagocytic (Tyrobp, Trem2), and inflammatory (Il1β, Tnf, Ccl2, Mrc1, Arg1) markers were analyzed by qPCR. Bars represent mean of relative expression (% of saline; Gapdh as housekeeping gene) ± SEM (*p < 0.05; **p < 0.01 by two-tailed Student's test; n=4).",
    "molecules": "LPS, saline"
  },
  {
    "caption": "Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis of 5 independent experiments showing CD11b and CD45 positive populations in single viable cells in saline or LPS-injected mouse brains.",
    "molecules": "LPS, saline"
  },
  {
    "caption": "Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Representative multicolour flow cytometry analysis showing the percentage of the mean ± SEM of 5 independent experiments of Ly6C and CD206 expressing cells in CD11b+CD45int cells from saline or LPS-injected mice.",
    "molecules": "LPS, saline"
  },
  {
    "caption": "Three-four months old BL/6 mice were treated with an acute dose of LPS (4 µg/g body) or vehicle (saline). Microglia (pool of 2 mice per group per replicate; 1 female and 1 male) were FACS-sorted 24 hours later. Gene expression levels of the monocytic markers Ly6c1 and Ccr2 in purified microglia (n=4) and isolated bone marrow monocytes (n=2) by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test).",
    "molecules": "LPS, saline"
  },
  {
    "caption": "Primary adult microglia were cultivated in the presence of TGF-β (50 μg/ml) and M-CSF (10 ng/ml), while neonatal cells were stimulated for 24 hours with TGF-β (50 μg/ml) followed by 6 hours stimulation with LPS (1 ng/mL) or left untreated. Expression levels of microglia homeostatic (Olfml3, Tmem119, Gpr34) and inflammatory (Il1β, Tnf, Ccl2) genes were analysed by qPCR. Bars represent mean of relative expression (Gapdh as housekeeping gene) ± SEM (**p < 0.01 by two-tailed Student's test; n=3).",
    "molecules": "LPS"
  },
  {
    "caption": "Representative multicolour flow cytometry analysis of 2 independent experiments showing TMEM119 and P2RY12 expression levels in CD11b+CD45int microglia of saline or LPS-injected mouse brains. For the unconjugated TMEM119 antibody, negative stands for primary antibody without secondary antibody. For P2RY12 antibody, negative represents isotype PE control.",
    "molecules": "LPS, saline"
  },
  {
    "caption": "Heatmap showing examples of specific genes mainly up-regulated in \"main LPS\" (Manf) or \"subset LPS\" (Ash1l) and down-regulated in \"main LPS\" (Mef2c) or \"subset LPS\" (Lamp1) overlaid on the 2D-tSNE space. Bars represent log2 (Count+1).",
    "molecules": "LPS"
  },
  {
    "caption": "F Representative May-Gruenwald-Giemsa-stained pleural fluid cytocentrifugal specimen from a KRASG12D;Trp53f/f mouse showing macrophages (MΦ, black arrow), lymphocytes (LΦ, purple arrow), and neutrophils (NΦ, green arrow) and summary of cellular and biochemical features of effusions of KRASG12D;Trp53f/f mice (n = 10).",
    "molecules": "May-Gruenwald-Giemsa"
  },
  {
    "caption": "G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [n is given in table in (E)].",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Wild-type (Wt), KRASG12D, and Trp53f/f mice were intercrossed, and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad-Cre and were sacrificed when moribund. In parallel, C57BL/6 mice received ten consecutive weekly intraperitoneal injections of 1 g/Kg urethane and were sacrificed after six months. Data summary (heatmap) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma (MPM) and lung adenocarcinoma (LUAD). n = 10 mice/group were analyzed for each marker. Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining. Note the ubiquitous strong expression of Wilms' tumor 1 (WT1), patchy moderate expression of vimentin (VIM), ubiquitous moderate expression of mesothelin (MSLN), ubiquitous strong expression of calretinin (CALB2), podoplanin (PDPN), and osteopontin (SPP1), patchy moderate expression of cytokeratin 5/6 (CK5/6), and the absence of expression of surfactant protein C (SFTPC) in murine KRAS-driven mesotheliomas. Note also the ubiquitous strong expression of WT1, the patchy moderate expression of VIM, the ubiquitous low-level expression of MSLN, the ubiquitous strong expression of CALB2 and SPP1, the ubiquitous low-level expression of PDPN, the variable moderate expression of CK5/6, and the ubiquitous moderate expression of SFTPC in murine KRASG12D-driven and urethane-induced LUAD.",
    "molecules": "hematoxylin, urethane"
  },
  {
    "caption": "F, G C57BL/6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1% DMSO or 15 mg/Kg deltarasin in 1% DMSO at day 9 post-tumor cells. Shown are data summaries of MPE volume (n = 8 and 7 DMSO and deltarasin-treated mice/group, respectively) and pleural fluid nucleated cells at day 19 post-KPM1 cells (F), as well as representative images of pleural effusions (dashed lines) and tumors (t in (G)).",
    "molecules": "deltarasin, DMSO, liposomes"
  },
  {
    "caption": "B-E) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.",
    "molecules": "his, leu"
  },
  {
    "caption": "withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.",
    "molecules": "his, leu"
  },
  {
    "caption": "wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his), quantified in (F) (n=28,22,7), scale bar = 50 μm",
    "molecules": "his, histidine, leu, leucine"
  },
  {
    "caption": "F-H) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his),",
    "molecules": "his, histidine, leu, leucine"
  },
  {
    "caption": "I-K) Representaitve pictures showing that adult nerfin-1 clonal growth was significantly reduced on -his diet compared to CDD (measured at day 0, and day 9) quantified in (K) (n= 27, 110, 136). Scale bar = 75μm. ***p<0.001 L-N) Representaitve pictures showing that larval nerfin-1 clonal growth was significantly reduced on 25% his diet compared to CDD (after 6 days), quantified in (N) (n=53,36). Scale bar = 50 μm. ",
    "molecules": "his"
  },
  {
    "caption": "O-Q) Representaitve pictures showing that adult pros clonal growth was not significantly altered on -his diet compared to CDD (after 7 days), quantified in (Q) (n = 12, 23,16). Scale bar = 75μm. R-T) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction (25% his) compared to CDD (after 6 days), quantified in (T) (n=19,13). Scale bar = 50 μm. ",
    "molecules": "his, histidine"
  },
  {
    "caption": "U-W) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction (25% his) compared to CDD, quantified in (W) (n=27,19). Scale bar = 100 μm.",
    "molecules": "his, histidine"
  },
  {
    "caption": "B) Concentration of histidine and 3-methyl-L-histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or -his diet (n=3,3). Both compounds are reduced to below detection levels (<0.3mM) upon dietary his depletion.",
    "molecules": "his, histidine, 3-methyl-L-histidine"
  },
  {
    "caption": "C) Dietary supplementation of 3-methyl-L-his for 4 days significantly increased adult nerfin-1 clonal growth, and rescued nerfin-1 growth inhibition due to his dietary withdrawal (n =18,14, 7,9).",
    "molecules": "his, 3-methyl-L-his"
  },
  {
    "caption": "D) Dietary supplementation of histamine does not significantly increase adult nerfin-1 clone size, but can rescue nerfin-1 clonal growth inhibition due to his dietary withdrawal (n =42,14,23,25).",
    "molecules": "histamine, his"
  },
  {
    "caption": "A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm. Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7).",
    "molecules": "his, Histidine"
  },
  {
    "caption": "Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav+ neurons (red) quantified in (D) (n = 16,17) and significantly fewer Mira+ NBs (blue), quantified in (E) (n = 12,22),",
    "molecules": "Histidine"
  },
  {
    "caption": "F-G) Larval nerfin-1 clones consisted of significantly greater proportion of differentiated neurons (n=10,7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n=9,11).",
    "molecules": "his"
  },
  {
    "caption": "Representative images of adult nerfin-1 (I-J', green ) and pros clones (K-L', green), scale bar = 5μm. In nerfin-1 clones, the size of the NBs (Ase+, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n=33,20). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n=9,8)",
    "molecules": "histidine"
  },
  {
    "caption": "Cellular growth measured as the ratio of nucleolus /nuclear volume was significantly reduced in larval nerfin-1 NBs after 6 days his dietary reduction (25% his compared to CDD, n=14,6) quantified in (N).",
    "molecules": "his"
  },
  {
    "caption": "In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (O) (n=9,7).",
    "molecules": "histidine"
  },
  {
    "caption": "O) Reducing Myc dosage with the hypomorphic Myc allele, mycP0, abolished the increase in nerfin-1 clone size mediated by 2x histidine dietary intake, 2x his (n=41,18,10,57).",
    "molecules": "his, histidine"
  },
  {
    "caption": "P) Overexpression of myc restored the growth of nerfin-1 clones inhibited by histidine dietary reduction, 25% his (n=51,36,39,29).",
    "molecules": "his, histidine"
  },
  {
    "caption": "A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles.",
    "molecules": "PEG"
  },
  {
    "caption": "B. PEG pellets were subjected to OptiPrep flotation analysis.",
    "molecules": "PEG"
  },
  {
    "caption": "D. sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown. E. Quantification of the NTA data obtained in five independent experiments.",
    "molecules": "glycine"
  },
  {
    "caption": "G. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. H. Quantification of the NTA data obtained in five independent experiments. ",
    "molecules": "glycine"
  },
  {
    "caption": "C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. GPRC5C was detected only in the apical sEV samples",
    "molecules": "PEG"
  },
  {
    "caption": "D. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samples",
    "molecules": "PEG"
  },
  {
    "caption": "C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.",
    "molecules": "glycine"
  },
  {
    "caption": "A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.",
    "molecules": "GW4869"
  },
  {
    "caption": "C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.",
    "molecules": "glycine"
  },
  {
    "caption": "A. Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) treatment as indicated.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "B. Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP. **, P<0.01 by unpaired two-tailed t test (n=3 biological repeats in one experiments).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "F. Flow cytometry analysis for CD63+ exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants. Quantification was done by mean fluorescence intensity. **, P<0.01 by unpaired two-tailed t test (n=4).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "A. Imaging analysis for co-localization of CD63 with IQGAP1 in IQGAP1 (FL) and IQ domain truncation (ΔIQ) restored YAMC cell. Cells were treated with LPS plus ATP. The IQGAP1-CD63 co-localization dots (shown by yellow) were counted, and Pearson's correlation coefficient was calculated for the quantification of co-localization.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "B. Imaging analysis for co-localization of CD63 with GSDMD in IQGAP1 full length (FL) and IQ domain truncation restored YAMC cell (ΔIQ) after LPS plus ATP treatment. The GSDMD-CD63 co-localized dots were counted and Pearson's correlation coefficient was calculated for quantification.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "F. ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "D. Co-localization of CD63 with IQGAP1-GSDMD complex, as measured by PLA, shown by the yellow dots after LPS and ATP treatment.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "A. Total particle count and size distribution of exosomes secreted in the supernatant from IQGAP1 KO (IQ1 KO), IQGAP1 full length restored (FL) and GRD domain truncation restored (ΔGRD) YAMC cells upon LPS or LPS plus ATP treatment.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "C. Colocalization of CD63 with IQGAP1-CDC42 complex (shown by PLA signal) in WT and GSDMD KO cells under LPS treatment. Quantification was performed by counting PLA green dots.",
    "molecules": "LPS"
  },
  {
    "caption": "D. Co-immunoprecipitation analysis of endogenous IQGAP1 with CDC42 in wild-type and GSDMD KO cells upon LPS or LPS plus ATP treatment.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "E. Co-immunoprecipitation analysis of GTP-bound CDC42 in indicated cells following LPS treatment.",
    "molecules": "GTP, LPS"
  },
  {
    "caption": "F. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in wild-type and IQGAP1 KO cells upon LPS or LPS plus ATP treatment.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "G. Western blots for exosome secretion upon CDC42 GTPase inhibition. Cells were pretreated with and without 10µM ML141 for 24 hours to fully block its GTPase activity, followed by LPS and ATP treatment as indicated. H. Particle analysis and size distribution for exosomes collected from cells with the same treatment as panel G.",
    "molecules": "ATP, LPS, ML141"
  },
  {
    "caption": "I. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in vehicle or ML141 (10µM for 24 hours) treated cells following LPS or LPS plus ATP treatment.",
    "molecules": "ATP, LPS, ML141"
  },
  {
    "caption": "A. Histology staining for IQGAP1 and GSDMD in naive and inflamed (3 days of DSS-challenge) colon tissues.",
    "molecules": "DSS"
  },
  {
    "caption": "B. Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls.",
    "molecules": "DSS"
  },
  {
    "caption": "C. Western blot analysis for supernatant collected from colon explants of DSS-treated wild-type (WT), Gsdmd-/-(GSDMD KO) and Iqgap1-/- (IQGAP1 KO) mice. Supernatant was first pulled down by IL-1β antibody and samples were subjected to Western blot analysis. The indicated proteins were probed.",
    "molecules": "DSS"
  },
  {
    "caption": "D. Immunofluorescent staining with anti-GTP-CDC42 in DSS treated colons from Gsdmd+/+ (WT) and Gsdmd-/-(GSDMD KO), IQGAP1+/+ and IQGAP1-/- mice.",
    "molecules": "DSS, GTP"
  },
  {
    "caption": "Analysis of DNA content by flow cytometry.",
    "molecules": "DNA"
  },
  {
    "caption": "Analysis of BrdU incorporation by BrdU-IP-chip 45 minutes after release into S phase in cells treated with 0.1% MMS as described in (A). A representative region on chromosome VII is shown. The position of replication origins is indicated above the map. Asterisks: late origins firing in mrc1∆ cells.",
    "molecules": "BrdU, MMS"
  },
  {
    "caption": "Quantification of the BrdU incorporation (signal log ratio) at 384 early and late replication origins in wild type, rad9∆ and mrc1∆ strains exposed to 0.1 or 0.033% MMS. Red lines indicate median values. Median replication times (Trep) are indicated for bins of 96 origins.",
    "molecules": "MMS"
  },
  {
    "caption": "Wild type, mrc1∆ and rad9∆ cells were released into S phase in the presence of BrdU and 0.033% MMS, as described in Fig. 1A. DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase. Representative DNA fibers are shown. Red: DNA, green: BrdU. Bar is 20 kb.",
    "molecules": "BrdU, DNA, MMS"
  },
  {
    "caption": "Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase, respectively.",
    "molecules": "BrdU"
  },
  {
    "caption": "Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.",
    "molecules": "α-factor, MMS, pronase"
  },
  {
    "caption": "Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored with an in situ phosphorylation assay - ISA (F).",
    "molecules": "α-factor, MMS, pronase"
  },
  {
    "caption": "Analysis of DNA content by flow cytometry.",
    "molecules": "DNA"
  },
  {
    "caption": "Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg/ml Zeocin. IdU tracks (red) correspond to initiation events occurring during the HU treatment. Elongation events after HU removal appear as CldU tracks (yellow) flanking IdU tracks. New initiation events as detected as isolated CldU tracks (asterisks). Bar is 20 kb.",
    "molecules": "IdU, CldU, DNA, HU, Zeocin"
  },
  {
    "caption": "Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in (D). For each sample, at least 65 individual DNA fiber larger than 200 kb were analyzed, for a total of >17 Mb.",
    "molecules": "DNA"
  },
  {
    "caption": "2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212. The experiment was performed as in (A) for the indicated times. Arrows indicate late origin firing.",
    "molecules": "DNA"
  },
  {
    "caption": "Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates.",
    "molecules": "CldU"
  },
  {
    "caption": "2D gel analysis of initiation and elongation in wild type and rad9∆ cells at the early origin ARS719 (probe 1) and at a locus 7 kb away of the origin (probe 2). Time in Zeocin after the HU wash is indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.",
    "molecules": "HU, Zeocin"
  },
  {
    "caption": "2D gel analysis of fork progression in wild type and rad9∆ cells through a 70-kb region of chromosome VI for which all active origins downstream of ARS607 have been deleted. The position of probes and the time after release from HU are indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.",
    "molecules": "HU"
  },
  {
    "caption": "Analysis of DNA content by flow cytometry.",
    "molecules": "DNA"
  },
  {
    "caption": "Analysis of Rad53 phosphorylation in G1 prior to Zeocin addition (G1) and at indicated times. The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.",
    "molecules": "Zeocin"
  },
  {
    "caption": "DNA combing analysis of replication profiles 60 minutes after release into S phase in the presence of Zeocin. Representative DNA fibers are shown. Red: DNA, green: BrdU. The green channel is also shown separately. Bar: 20 kb.",
    "molecules": "BrdU, DNA, Zeocin"
  },
  {
    "caption": "Distribution of BrdU tracks length in wild type, mrc1∆ and mrc1AQ cells. DNA fibers are fully labeled in the case of rad9∆ cells. Median values are indicated in red and correspond to the analysis of at least 150 tracks from two biological replicates. ns: non-significant Mann-Whitney rank sum test.",
    "molecules": "BrdU, DNA"
  },
  {
    "caption": "Cells from the indicated strains were released synchronously into S phase in the presence of Zeocin as described in Fig. 5A and DNA content was analyzed by flow cytometry.",
    "molecules": "DNA, Zeocin"
  },
  {
    "caption": "Analysis of chromosome mobility by Pulse Field Gel Electrophoresis (PFGE) after staining of DNA with ethidium bromide. In G1, the chromosomes are linear and are sorted according to their length. Upon entry into S phase (30 min), they cannot enter the gel due to the presence of replication intermediates. Quantitation of chromosomes re-entering the gel shows completion of DNA replication in 40% of rad9∆, rad53-11 and rad9∆ rad53-11 mutants, but not in wild type cells and mrc1 mutants as determined for a representative experiment. Error bars indicate SD between the signal intensity determined for the three larger chromosomes (arrowheads).",
    "molecules": "ethidium bromide, DNA"
  },
  {
    "caption": "DNA combing analysis of replication profiles 30 minutes after release into S phase in the presence of Zeocin. Representative images are shown. Red: DNA, green: BrdU. Bar is 20 kb.",
    "molecules": "BrdU, DNA, Zeocin"
  },
  {
    "caption": "Length distribution of BrdU tracks measured at that time for wild type, rad9∆, rad53-11 and rad53-11 rad9∆ cells. Median lengths are indicated in red. For each sample, at least 200 tracks were measured from two biological replicates.",
    "molecules": "BrdU"
  },
  {
    "caption": "(E) Mafosphamide dose-response curves of SLF2KO and control SU-DHL-5 cells. Cells were treated with mafosphamide (MAF; 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 ng/ml) for 48 hours and viability was determined by DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments.",
    "molecules": "DAPI, MAF, Mafosphamide, mafosphamide"
  },
  {
    "caption": "(A) Immunoblot analysis of U-2-OS cells following CRISPR/Cas9 mediated knockout of SLF2 upon DRB treatment (0.5 µM) for indicated times with the indicated antibodies.",
    "molecules": "DRB"
  },
  {
    "caption": "(E) Immunoblot analysis of human SU-DHL-5 control and SLF2KO cell lines upon DRB treatment (0.5 µM) for indicated time points with the indicated antibodies.",
    "molecules": "DRB"
  },
  {
    "caption": "(A) TMT-based MS results of chromatin fractions from SU-DHL-5 control and SU-DHL-5 SLF2KO cells. Volcano plot depicting depleted chromatin-associated proteins. Significant hits are shown by red dots (depleted in SLF2KO cells). Proteins belonging to the DDR and cell cycle regulation are labeled. Experiments were performed in triplicates.",
    "molecules": "TMT"
  },
  {
    "caption": "(E) SUMO inhibitor (SUMOi (ML-093); 0, 6.25, 12.5, 25, 50, 100, 200, 400, 800 nM) dose response curves of SLF2KO and control SU-DHL-5 cells. Cells were treated with increasing concentrations of SUMOi for 72 hours and viability was determined by negative DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments.",
    "molecules": "ML-093, DAPI"
  },
  {
    "caption": "(F) Quantification of flow cytometry results for DAPI and Annexin V staining in human SLF2KO and control SU-DHL-5 cells after 12.5 nM of SUMOi treatment for 72 hours. P-value determined by unpaired t-test. Error bars represent SD from three independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Immunoblot analysis of human SU-DHL-5 control and CLSPNKO cell lines upon DRB treatment (0.5µM) for indicated time points with the indicated antibodies.",
    "molecules": "DRB"
  },
  {
    "caption": "(C) Quantification of the p-CHK1 western blots from Figure 7B. Protein expression of p-CHK1 was normalized to β-Actin protein expression. p-CHK1 expression in control cells at 3 hours of doxorubicin treatment was arbitrarily set to 1. Data are presented as mean +/- SD of n=3 independent experiments. P-value determined by two-way ANOVA; Šídák's multiple comparisons test.",
    "molecules": "doxorubicin"
  },
  {
    "caption": "(F) SUMO inhibitor (SUMOi, TAK981; 0, 6.25, 12.5, 25, 50, 100, 200, 400, 800 nM) dose-response curves of CLSPNKO and control SU-DHL-5 cells. Cells were treated with increasing concentrations of SUMOi for 72 hours and viability was determined by negative of DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments. (G) Mafosphamide dose-response curves of CLSPNKO and control SU-DHL-5 cells. Cells were treated with mafosphamide (MAF; 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 ng/ml) for 48 hours and viability was determined by negative of DAPI staining and flow cytometry measurement. Error bars represent SD from three independent experiments.",
    "molecules": "DAPI, MAF, mafosphamide, Mafosphamide, TAK981"
  },
  {
    "caption": "(G) Quantification of cell viability measured by celltiterGlo from primary murine Eµ-Myc lymphoma derived cells co-treated with indicated concentrations of SUMOi and Rabusertib for 72 hours. Bar plots represent cell viability relative to the DMSO treated control cells. Error bars represent SD from three independent experiments. P-value was determined by ANOVA test; Tukey's post hoc test.",
    "molecules": "celltiterGlo, DMSO, Rabusertib"
  },
  {
    "caption": "E-G, Ex vivo treatment of HRDhigh (NMFH-1, OH931, PM197, USZ-21_MFS2 and USZ-21_UPS1) sarcoma models compared with HRDlow (USZ-20_REA1, USZ-21_LG1, USZ-22_EMC2, USZ-20_SFT1 and USZ-21_CIC1) sarcoma models for 4 days with six doses of the chemotherapy agents oxaliplatin, doxorubicin and trabectedin.",
    "molecules": "doxorubicin, oxaliplatin, trabectedin"
  },
  {
    "caption": "M-O, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses olaparib alone and in combination with 1 nM trabectedin. Note that no sensitivity to olaparib in monotherapy was observed at 3 days",
    "molecules": "olaparib, trabectedin"
  },
  {
    "caption": "Q-S, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses adavosertib alone and in combination with 100 nM doxorubicin.",
    "molecules": "adavosertib, doxorubicin"
  },
  {
    "caption": "A, Immunofluorescence showing nuclear expression of the DNA damage marker γH2A.X (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination in HRDhigh and HRDlow sarcoma cell models as well as ovarian carcinoma UWB1.289 cells. B, Immunofluorescence showing RAD51 nuclear foci (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination only in the HRDlow sarcoma cell model (USZ-21_LG1). C, D, Quantification of γH2A.X (C) or RAD51 (D) nuclear intensity per cell compared to untreated control cells. E,",
    "molecules": "olaparib, trabectedin"
  },
  {
    "caption": "(D) Tukey box plot of ZEB1, ZEB2, TWIST1, and MITF mRNA expression according to the IC50 of the drug (µM) administered (BRAFi/MEKi), in melanoma cell lines from the CCLE (n=28) (Student's t-Test). High ZEB1, low ZEB2, and low MITF expression levels were correlated with BRAFi (PLX4720) and MEKi (AZD6244) resistance. PLX4720 is an analog of PLX4032.",
    "molecules": "AZD6244, PLX4032, PLX4720"
  },
  {
    "caption": "(E) IC50 values of PLX4032 (µM) in the panel of BRAFV600 melanoma cells as determined by ATP assay (n=3, mean ± SD). For SKMEL24 and WM793, IC50 was >8µM.",
    "molecules": "ATP, PLX4032"
  },
  {
    "caption": "(C) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from patients, classified in ZEB1 high, int and low subgroups, based on the intensity of ZEB1 staining and on the percentage of cells positive for ZEB1. Scale bar = 80 µm. (D) Pie charts representing the distribution of ZEB1 alone (upper part), or ZEB1 and TWIST1 (lower part) immunohistochemistry staining in tumors according to their initial response to vemurafenib +/- cobimetinib treatment. ZEB1 +/- TWIST1 levels are higher in MAPKi primary resistant melanomas (initial non-responders) compared to tumors that initially respond to treatment (n=70, Fisher's exact test).",
    "molecules": "cobimetinib, vemurafenib"
  },
  {
    "caption": "(E) Representative pictures of ZEB1 and MITF immunostainings, before and after vemurafenib treatment, in the tumor from patient 1, exhibiting primary resistance to BRAFi. Scale bar = 80 µm. Right: Magnification of MITFhigh and MITFlow clones in the resistant tumor under treatment. Arrows indicate endothelial and stromal cells that also show positive staining for ZEB1, besides tumor cells.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "(A) PLX4032 IC50 (µM) of sensitive A375 and SKMEL5 and resistant (A375-R, SKMEL5-R) cell lines, as well as of GOKA and ESPcells, two BRAFi-resistant patient-derived short-term cultures, as determined by ATP assay (n=3, mean ± SD). For ESP, IC50 was >8µM.",
    "molecules": "ATP, PLX4032"
  },
  {
    "caption": "(D) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2, 3 and 4, before and after vemurafenib treatment. Scale bar = 40 µm. For ZEB1 staining in patient 4, the inset shows a magnification. Arrows point at stromal cells (s). All other cells positive for ZEB1 are tumor cells.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "(E) Western blot analyses of ZEB1, MITF, P-ERK, and TWIST1 levels in control or ZEB1-expressing cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.",
    "molecules": "PLX4032"
  },
  {
    "caption": "(F) FACS analyses of p75 cell surface expression upon ZEB1 overexpression, after 10 days treatment with or without 150 nM PLX4032. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's Exact Test).",
    "molecules": "PLX4032"
  },
  {
    "caption": "(H) Clonogenic assay +/- PLX4032 (150 nM), +/- GDC-0973 (5 nM) treatment for 10 days. The graphs represent the mean number of colonies (± SD) in 3 independent experiments (Student's t-test). (I) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1-expressing cells (n=3, Student's t-test).",
    "molecules": "PLX4032, GDC-0973"
  },
  {
    "caption": "(E) Clonogenic assay +/- PLX4032 (100 nM) treatment for 10 days. The graph represents the mean number of colonies in 3 independent experiments (Student's t-test).",
    "molecules": "PLX4032"
  },
  {
    "caption": "(F) A375 cells were infected with an IPTG-inducible ZEB1-shRNA. Left panel: Western blot analyses of ZEB1 expression ± IPTG (100µM) treatment for 6 days. Actin was used as a loading control. Right panel: 2 x 106 IPTG-inducible shRNA-Control or shRNA-ZEB1A375 cells were injected subcutaneously into nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by adding IPTG (10 mM) into the drinking water for 20 days. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).",
    "molecules": "IPTG"
  },
  {
    "caption": "(G) SKMEL5 cells expressing an IPTG-inducible ZEB1-shRNA were treated with IPTG (100µM) for 10 days (+IPTG), then IPTG was removed (-IPTG) and ZEB1, MITF and p75 expression levels were analyzed by Western blot and/or quantitative PCR analyses. GAPDH was used as a protein loading control, mRNA expression levels are represented relatively to untreated cells, in which the levels were fixed at 1 (mean ± SD, n=2).",
    "molecules": "IPTG"
  },
  {
    "caption": "(A) Western blot analyses of ZEB1 and P-ERK in shRNA-control or shRNA-ZEB1-expressing A375 cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.",
    "molecules": "PLX4032"
  },
  {
    "caption": "(B) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 (150 nM). Scale bar = 100 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (C) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA-ZEB1-expressing cells (n=3, Student's t-test).",
    "molecules": "PLX4032"
  },
  {
    "caption": "(D) RPMI7951, A375-R and SKMEL5-R were infected with a retrovirus encoding a constitutive shRNA-ZEB1. Short-term cultures of GOKA and ESP cells, derived from vemurafenib-resistant patients, were infected with a lentivirus encoding an IPTG-inducible shRNA-ZEB1. Western blot analyses showing efficient ZEB1 knock-down in the different models, ±IPTG (200µM), ±PLX4032 (3µM) as indicated. Induction of cell death was assessed by PARP cleavage. GAPDH or actin were used as loading control.",
    "molecules": "PLX4032, IPTG, vemurafenib"
  },
  {
    "caption": "(E) Clonogenic assays in the presence of 3 µM PLX4032, and with or without IPTG (200 µM) as indicated. The graphs represent the mean number of colonies in 3 independent experiments (Student's t-test).",
    "molecules": "PLX4032, IPTG"
  },
  {
    "caption": "(F) Western blot analyses of ZEB1, p75 and quantitative PCR analyses of MITF in shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant patient-derived short-term culture cells. Actin was used as a loading control. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).",
    "molecules": "vemurafenib"
  },
  {
    "caption": "(G) 2,5 x 106 shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant cells were injected subcutaneously in nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by providing mice with IPTG (10 mM) in their drinking water and orally administering vemurafenib (50 mg/kg) daily for 4 weeks. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).",
    "molecules": "IPTG, vemurafenib"
  },
  {
    "caption": "(H) Upper part: Western blot analyses of ZEB1 in shRNA-Control (1 to 4) or shRNA-ZEB1 (5 to 8) ESPxenografttumors, showing efficient ZEB1 knock-down directly in the tumors, after IPTG ±PLX4032 treatment in vivo. Lower part: Representative pictures of ZEB1 immunostaining in shRNA-Control or shRNA-ZEB1tumors, after IPTG treatment in vivo. Scale bar = 40µm.",
    "molecules": "PLX4032, IPTG"
  },
  {
    "caption": "DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL-2nil (32694) ordered by increasing fold change of tag count for TB IL-2 compared to TB IL-2nil. The sequence tag density is also shown in parallel at the same sites for the DNase-Seq analyses of CD4 naïve T cells (TN), TB IL-2 DMSO and TB IL-2 Ruxolitinib and ATAC samples from in vivo derived Th2 (Shih et al., 2016), Th1 and Th17 cells. The location of the 1000 IL-2 pDHSs is shown to the right.",
    "molecules": "DMSO, Ruxolitinib"
  },
  {
    "caption": "UCSC genome browser tracks with examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci. IL-2 pDHSs are highlighted with blue boxes.",
    "molecules": "cytokines"
  },
  {
    "caption": "UCSC genome browser tracks at the Il13/Il4/Il5 Th2 cytokine locus LCR (indicated by the grey box within the Rad50 gene), Gzmb and Il24 showing DNase-Seq and ChIP-Seq for STAT5 and JUND in TB IL-2 and TB IL-2nil. Replicate JUND and STAT5 ChIP-Seq tracks are shown for TB IL-2.",
    "molecules": "cytokine"
  },
  {
    "caption": "Average DHS signal at the 1015 IL-2-dependent inducible DHSs in TB IL-2, TB+ IL-2, TB+ IL-2nil and naïve T cells stimulated with PMA/I (TN+).",
    "molecules": "PMA"
  },
  {
    "caption": "Relative mRNA expression levels in TB+ IL-2 and TB+ IL-2nil (A) and TB+ DMSO and TB+ Ruxolitinib (B). The expression level in the IL-2/DMSO TB+ sample is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)",
    "molecules": "DMSO, Ruxolitinib"
  },
  {
    "caption": "UCSC genome browser tracks showing DNase-Seq and RNA-Seq at the IL-2 regulated genes Csf2/Il3 and at the LCR within the Rad50 gene (grey box) which controls the Th2 cytokines Il13, Il4 and Il5. IL-2 pDHSs are highlighted in blue and IL-2 iDHSs in red.",
    "molecules": "cytokines"
  },
  {
    "caption": "UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci (C) IL-2 pDHSs are highlighted with blue boxes.",
    "molecules": "cytokines"
  },
  {
    "caption": "UCSC genome browser tracks showing ChIP-Seq and DNase-Seq at the LCR within the Rad50 gene which controls the Il4/Il13/Il5 Th2 cytokine locus, and also Il17a, Il12rb2 and Tbx21. The grey boxes indicate IL-2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages.",
    "molecules": "cytokine"
  },
  {
    "caption": "Representative IF staining of Sox2 (green) and Ki67 (red) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI (blue) on a sagittal section. Dotted lines, basement membrane; yellow arrowheads mark approximate stem cell boundaries. SCs, stem cells; TACs, transit amplifying cells; Ant, Anterior; Post, Posterior.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF double staining of E-Cadherin (ECad, green, for cell border) and AcTub (red, for the cores of primary cilia) on a sagittal section. Sample is counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "Tamoxifen induced IFT88 deletion strategy (top panel) and representative images of Sox2 (green) and Ki67 (red) immunostaining on CLE regions of the indicated mouse strains (bottom panels). Dotted lines, basement membrane. Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88.",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop (green) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Sample is counterstained with DAPI (blue). Dotted lines, basement membrane. SCs, stem cells; TACs, transit amplifying cells; Ams, ameloblasts.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF staining of Prom1 (red) and Sox2 (green) in CLESCs in vitro. Sample is counterstained with DAPI (blue). Arrows indicate colocalisation of Prom1 with low-medium level of Sox2 in the cell nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF double staining of Sox 2 (green) and Ki67 (red) on the and transit amplifying cell regions of WT vs. Prom1 KO mice on frontal sections. Samples are counterstained with DAPI (blue). Quantification of Ki67+ cell number in Sox2-positive region based on IF data from WT and Prom1 KO mice (C).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative image of CLESCs harbouring the Ki67-Fucci system showing colored cells at different cell cycle phases.",
    "molecules": "Fucci"
  },
  {
    "caption": "Flow cytometry analysis of Ki67-Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1. As a result, >95% of the events have been included in the analysis. The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1.0 software.",
    "molecules": "Fucci"
  },
  {
    "caption": "Representative IF staining of AcTub (blue) on Ki67-Fucci CLESCs without and with the presence of SHH. Note that the cilium could be visualised (arrows) solely when SHH is present.",
    "molecules": "Fucci"
  },
  {
    "caption": "Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor (Tubacin) and appropriate controls (Nitubacin and DMSO, C) Colonies were stained with crystal violet",
    "molecules": "Nitubacin, DMSO, Tubacin"
  },
  {
    "caption": "siRNA knockdown efficiency of Arl13b (E) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH",
    "molecules": "FUCCI"
  },
  {
    "caption": "siRNA knockdown efficiency of Hdac6 (F) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH",
    "molecules": "FUCCI"
  },
  {
    "caption": "Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Prom1 (AC133) or with anti-FITC antibody as control (L). Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin.",
    "molecules": "FITC"
  },
  {
    "caption": "Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by SEM (B, top panels) or confocal laser scanning microscopy after DAPI staining (B, bottom panels, Individual cells within a given dome are indicated (B, top panels) and were quantified using three different planes (bottom, centre and top) of DAPI-labeled nuclei (B, bottom panels).",
    "molecules": "DAPI"
  },
  {
    "caption": "Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc (A-C) or as indicated (D). confocal laser scanning microscopy after DAPI staining Domes of similar size were evaluated (C). Means and standard deviations are displayed. 15 domes per independent experiment were examined (n = 3). Cell density at a given time of culture (7, 10 and 14 days) was determined by counting DAPI-labeled nuclei per 0.1 mm2 (D, n = 4).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF staining of Gli1 (left panel, red) and Glis2 (right panel, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Dotted lines, basement membrane.",
    "molecules": "DAPI"
  },
  {
    "caption": "3D reconstruction of Glis2 staining (see B, red) and its association with DAPI-labeled nuclei (blue). Nuclear transparency was set at 0 or 70% as indicated.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF double staining of Glis2 (red, bottom panel) and AcTub (green, top and bottom panels) along stem cell-transit amplifying cell axis in lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop (top panels, green) or cytoplasmic C-terminal end (bottom panels, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice. Samples are counterstained with DAPI (blue). Dotted lines, basement membrane. Arrowheads marks approximate boundary of stem cell regions.",
    "molecules": "DAPI"
  },
  {
    "caption": "Time dependent Prom1 and Glis2 co-expression (proximity ligation assay) in the cultured CLESCs starved for 4 days, and then treated with SHH (100 ng/ml) and importazole (40 µM), an inhibitor of importin β1function. DMSO was used as vehicle control. Data are represented triplicated samples as mean and standard deviation.",
    "molecules": "DMSO, importazole"
  },
  {
    "caption": "B. Structured Illumination Microscopy (SIM) images of S. aureus cells labelled with the membrane  dye  Nile Red. White arrowheads point to tetrads  of cells that result from deficient cell splitting. Scale bars, 1μm.",
    "molecules": "Nile Red"
  },
  {
    "caption": "B. Total protein extracts of strains 8325-4 TF-3XFLAG,  8325-4 GFP-FtsK TF-3XFLAG, 8325-4sfgfp-ftsK  and 8325-4 TF-3XFLAG GFP-HU  (Input extracts, bottom panel) were immunoprecipitated  with DYKDDDDK Fab-Trap Agarose  that binds FLAG-tagged proteins. Western blot  analysis of the pulled-downed fractions (top panels) using antibodies against FLAG  and GFP  shows that GFP-FtsK, but not the negative control GFP-HU, is pulled down with TF-3XFLAG, suggesting an interaction between TF  and FtsK. For each assay, a representative image of three independent biological replicates is shown.",
    "molecules": "DYKDDDDK Fab-Trap Agarose"
  },
  {
    "caption": "A. SIM  images of cells expressing TF-GFP  labeled with membrane  dye FM5-95. TF-GFP  forms a gradient towards the septum  in the background of parental strain NCTC8325-4 (8325-4 TF-GFP, top panels). This localization  pattern is lost in cells lacking FtsK  (8325-4∆ftsK TF-GFP, middle panels), but not in 8325-4∆ftsKc TF-GFP  mutant, expressing FtsK  without its C-terminal domain (bottom panels). Scale bars, 1 µm.",
    "molecules": "FM5-95"
  },
  {
    "caption": "B. Deconvolved epifluorescence images of  live cells  of parental strain  8325-4 Sle1-3XFLAG ∆spa and TF  deletion mutant 8325-4 Sle1-3XFLAG ∆spa ∆tig, producing Sle1-3XFLAG as the sole Sle1   copy in the cell. Normal localization  pattern of Sle1   (enriched at midcell, top panel) is altered in the absence of TF  (bottom panel) with the protein becoming more dispersed over the cell surface. See image S1 for more images of Sle1-3XFLAG surface localization  in the parental strain and the negative control of the  immunofluorescence assay. Cells were incubated with fluorescent D-amino acid HADA, which is incorporated into peptidoglycan, for cell wall visualization (colored blue). Cell surface Sle1-3XFLAG  was detected with Anti-FLAG  Monoclonal Antibody-Alexa Fluor 488 (colored green, projection of a 33 slices Z-stack shown). Scale bars, 1 µm.",
    "molecules": "Alexa Fluor 488, HADA, peptidoglycan"
  },
  {
    "caption": "A. Quantification of Sle1  total cellular levels, detected by  western blot (representative gel on the bottom), in S. aureus cultures of wild-type strain NCTC8325-4 grown in the presence (1/2 X MIC) or absence of DNA targeting antibiotics mitomycin C  and nalidixic acid or protein synthesis inhibitor erythromycin. The results show a decrease in Sle1  cellular levels upon DNA replication defects or DNA damage. Graphs show ratio between Sle1  amount in different samples versus the amount in parental strain NCTC8325-4  indicated by dashed line. Data represented in scatter dot plot column graphs, where column height represents mean, and error bars are the standard deviation. Data from four biological replicates. Statistical significance based on a Mann-Whitney U test, where * p< 0.05.",
    "molecules": "erythromycin, MIC, mitomycin C, nalidixic acid"
  },
  {
    "caption": "C. SIM  image and heat map representation of TF-GFP localization  in  8325-4 TF-GFP  cells grown in the presence or absence of  nalidixic acid, showing the loss of TF-GFP  gradient towards the septum  in most cells grown in the presence of the antibiotic. Scale bars, 1 µm. Heatmaps represent the average of TF-GFP localization  in 50 cells in phase 2 of the cell cycle. The color scale depicts the average normalized TF-GFP  fluorescence signal in each pixel, ranging between 0 and 1. The fraction of the area of each cell model where the normalized signal intensity is above 70% of the maximum signal is 0.47 and 0.61 in the absence or presence of  nalidixic acid, respectively, confirming that TF-GFP  becomes more dispersed over the cytoplasm  in the presence of the antibiotic.",
    "molecules": "nalidixic acid"
  },
  {
    "caption": "Validation of upregulated lncRNAs (ΔCT) in PBMC-derived M2-like macrophages  (stimulated with  IL-4  stimulated for 24 hours, M2). n = 6 biological replicates, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with M1-like macrophages (LPS/IFNɣ-stimulated for 24 hours, M1).",
    "molecules": "LPS"
  },
  {
    "caption": "(C, Representative images and quantification of mitochondria  by MitoTracker  (red) in THP1  control and ADPGK-AS1  OE cells. Nuclei  were stained  with DAPI  (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).",
    "molecules": "DAPI, MitoTracker"
  },
  {
    "caption": "D) Representative images and quantification of mitochondria  by TOM20  (green) in THP1  control and ADPGK-AS1 OE cells. Nuclei  were stained  with DAPI  (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).",
    "molecules": "DAPI"
  },
  {
    "caption": "I) full oxygen  consumption plot (n = 8) by the seahorse assay in THP1  control and ADPGK-AS1  OE cells. **p ≤ 0.01, ***p ≤ 0.001 compared with control.",
    "molecules": "oxygen"
  },
  {
    "caption": "(K) Representative confocal  fluorescence images and quantification of mitochondrial  shape with the mitochondrial  marker TOM20  (yellow) in THP1  control and ADPGK-AS1  OE cells. Nuclei  were stained  with DAPI  (blue). n = 35 cells, Scale bar=10µm (left), 5µm (magnification, right).",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Representative images of JC-1    monomers (green) and aggregates (red) in THP1  control and ADPGK-AS1 overexpressing macrophages  transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. Scale bar = 100 µm.",
    "molecules": "JC-1"
  },
  {
    "caption": "(E) Representative images (left panel) and quantification (right panel) of ROS  accumulation in THP1  control and ADPGK-AS1 OE cells transfected with a siRNA against  ADPGK-AS1, MRPL35, or negative control. n = 7. ***p ≤ 0.001 compared to control.",
    "molecules": "ROS"
  },
  {
    "caption": "(D)  Mass spectrometry analysis of TCA cycle metabolites (fumarate, malate, citrate, succinate) in macrophages co-cultured with tumor cells (A549) and macrophage  control (M0). n = 4 biological replicates. *p ≤ 0.05, **p ≤ 0.01 compared with M0.",
    "molecules": "citrate, fumarate, malate, succinate"
  },
  {
    "caption": "(C, D) Representative fluorescence images of PCLS with proliferative  (EdU+) and apoptotic  (TUNEL+) cells (red) and nuclear  dye (DAPI+, blue) in the tumor area (cytokeratin+, green). Scale bar = 250 µm (c, upper panel), 150 µm (d, upper panel), and 50 µm (c and d lower panel, magnification).",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "(B) Detection of soluble, cleaved NrCAM (sNrCAM) and full-length, mature NrCAM (mNrCAM) and soluble APPα (sAPPα) in neuronal supernatants and lysates (prepared from E16 neurons), treated with GI254023x (5 µM), or solvent for 48h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).",
    "molecules": "GI254023x"
  },
  {
    "caption": "HEK 293 cells were transfected with a C-terminally VSV-tagged NrCAM construct or empty vector. Cells were then treated with DAPT (1 µM), GI254023x (5 µM), either substances or solvent for 24 h. The C-terminal NrCAM-fragment was detected with an antibody against the VSV-tag. Representative Western Blots are shown.",
    "molecules": "DAPT, GI254023x"
  },
  {
    "caption": "(A) ADAM10 fl/fl neurons were treated with an iCre (to induce an ADAM10 KO), or a control lentivirus at DIV2. At DIV7, cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAM's surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(B) Primary murine neurons were treated with GI254023x (5 µM) or solvent, for 48h. The cell surface proteins were enriched like in A. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 11). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.",
    "molecules": "GI254023x"
  },
  {
    "caption": "(C) Workflow of the neurite outgrowth assay. (D) Representative pictures of single neurites at time-point 0h (DIV3) and 24h (DIV4). Neurons were infected 4h after plating with lentiviruses encoding GFP (to visualize the neurons) together with shRNA expression cassettes (sh1 and sh2) targeting either NrCAM or carrying a scrambled (scr) control construct. Images of neurites were taken at three days in vitro (DIV3) and 24h later at DIV4. In order to study the effect of ADAM10 on neurite outgrowth, neurons were treated with the ADAM10 inhibitor GI254023x, or vehicle (control), at DIV3, after taking the first pictures with an epifluorescent microscope. The differences in neurite length were calculated as absolute values (neurite length at 24h minus neurite length of 0h) for individual neurites passing through the middle channels of the chambers. Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered. The red arrows indicate the start and the end of the respective length measurements. The scale bar indicates 40 µm. (E) One-way ANOVA with post hoc Dunnett's test. Given are mean +/- the standard error of them mean (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n-numbers for each condition are shown in the graph in 3E).",
    "molecules": "GI254023x"
  },
  {
    "caption": "(A) Wt neurons were kept in culture until DIV5, then the cells were treated with acitretin (4 µM) or vehicle control for 48h, as described earlier (Tippmann et al, 2009). (B) Densitometric quantifications of the Western Blots are shown. Given are mean +/- the standard error of the mean (*** p < 0.001; **** p < 0. 0001, two-sided students t-test n = 9-20). The mean levels of solvent treated cells were set to 1.",
    "molecules": "acitretin"
  },
  {
    "caption": "(C) Detection of sNrCAM and human serum albumin (hSA) in AD patients CSF that had been treated with acitretin (30 mg/d) or placebo for 30 days (n = 9) (Endres et al, 2014). (D) Densitometric quantification of the Western Blots in (C). sNrCAM/hSA ratios were calculated, and then the treatment to baseline ratios (t/b) for every patient were calculated. Two-sided student's t-test (n = 9). Given are mean +/- the standard error of them mean. Representative Western Blots are shown.",
    "molecules": "acitretin"
  },
  {
    "caption": "(A) To ensure proper synapse formation, primary neurons were kept in culture for ten days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 µM), NMDA (50 µM) and GI254023x (5 µM), GI254023x (5 µM) alone or vehicle for 30 minutes. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western Blots are shown. One-way ANOVA with post hoc Dunnett's test (*** p < 0.001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. The membrane was reprobed with the different indicated antibodies.",
    "molecules": "GI254023x, NMDA"
  },
  {
    "caption": "(B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.",
    "molecules": "biotin, NMDA, streptavidin"
  },
  {
    "caption": "Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days. Every circle represents one protein. The log10 transformed t-test p-values (again treatment/baseline for every patient) are plotted against the log2 transformed label-free quantification intensity ratios of acitretin and placebo CSF. ADAM10 substrates are indicated in bold writing. In total, more than 50 ADAM10 substrates were identified, but only the significantly altered substrates as well as NrCAM and APP are indicated.",
    "molecules": "acitretin"
  },
  {
    "caption": "(A) Representative images of the occurrence of GVBD in control, Mcrs1-KD, and Mcrs1-rescue (Mcrs1 siRNA + Mcrs1 mRNA) oocytes at 3 h following release from IBMX. Scale bar, 100 μm. (B) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 155), Mcrs1-KD (n = 157), and Mcrs1-rescue (n = 150) oocytes.",
    "molecules": "IBMX"
  },
  {
    "caption": "(D) Expression levels of CDK1 in Mcrs1-KD and control oocytes. The bars represent the mean (± SEM) of 5 biological replicates. The P-values were calculated using Student's t-test. *P = 0.01. (E) Expression levels of p-CDK1(Y15) in Mcrs1-KD and control oocytes at 1 h following release from IBMX (200 oocytes per sample). **P = 0.006. (F) Expression levels of cyclin B1 in control and Mcrs1-KD oocytes at 1 h following release from IBMX (200 oocytes per sample). The bars represent the mean (± SEM) of 3 biological replicates. The P-values were calculated using Student's t-test. **P = 0.0042.",
    "molecules": "IBMX"
  },
  {
    "caption": "(I) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control oocytes (n = 112), Mcrs1-KD oocytes (n = 98), and Mcrs1-KD oocytes treated with 5 mM WEE1-1N-4 (n = 102). (J) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 123), Mcrs1-KD (n = 134), and Mcrs1 siRNA + cyclin B1-GFP (n = 95) oocytes. (K) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 92), Mcrs1-KD (n = 97), and Mcrs1 siRNA + cyclin B1-GFP oocytes treated with 5 mM WEE1-1N-4 (n = 106).",
    "molecules": "IBMX, WEE1-1N-4"
  },
  {
    "caption": "(B) Detection of newly synthesized RNAs by EU incorporation in control, Mcrs1-KD, and Mcrs1-rescue GV oocytes. The scattergram shows the relative fluorescence intensity of EU in control (n = 43), Mcrs1-KD (n = 52), and Mcrs1-rescue (n = 30) GV oocytes. Scale bar, 10 μm. ***P = 0.0001.",
    "molecules": "EU"
  },
  {
    "caption": "(A) Representative images of spindle assembly at 3 h post-release from IBMX in control and Mcrs1-KD oocytes. Scale bar, 20 μm.",
    "molecules": "IBMX"
  },
  {
    "caption": "Cell cycle analysis of wild type and Sl/Sl E11.5 YS EMPs. The percentage of EMPs (Ter119- Kit+ CD41+) in G0/G1 or S/G2/M was determined by flow cytometry on the basis of BrdU and 7-AAD incorporation. a: apoptotic (percentage not shown). Embryos were analyzed individually. N=6 (+/+), N=5 (Sl/Sl) over 3 independent experiments. E11.5 embryos were staged according to tail somite counts as described in [76] and were: 12-17 (+/+); 12-17 (Sl/Sl). Bar graphs show mean±SD, and FACS plots representative results.",
    "molecules": "7-AAD"
  },
  {
    "caption": "Total cell numbers (nr) of E12.5 wild type (n=7) and Sl/Sl (n=8) FL. Right: CFU-E numbers per wild type and Sl/Sl E12.5 FL. Data are the mean (±SD) of 3 biological replicates analyzed over 2 independent experiments. Each biological replicate consisted of 1-2 FL samples pooled according to genotype and was plated in duplicate into a methylcellulose based medium containing EPO; colonies were scored after 2 days. Total number of FLs analyzed: 4 wild type and 6 Sl/Sl.",
    "molecules": "EPO, methylcellulose"
  },
  {
    "caption": "Percentage of cycling Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid (Early E) cells in the E11.5 FL, determined by flow cytometric analysis of BrdU and 7-AAD incorporation. Data are the mean (±SD) of 6 wild type and 5 Sl/Sl FLs analyzed individually over 3 independent experiments. Tail somite range: 12-17 (+/+); 12-17 (Sl/Sl).",
    "molecules": "7-AAD"
  },
  {
    "caption": "Percentage of early apoptotic EMPs and Early E in wild type and Sl/Sl E11.5 FL, determined by flow cytometric analysis of AnnexinV staining among 7-AAD- Ter119- CD41+ Kit+ CD16/32+ EMPs and 7-AAD- Ter119- Kit+ CD41- CD71+ CD44+ Early E. Data are the mean (±SD) of 9 wild type and 7 Sl/Sl biological replicates analyzed over 3 independent experiments, with each replicate consisting of 1-3 FLs of identical genotypes. A total number of 13 wild type and 9 Sl/Sl embryos were analyzed. Tail somite range: 11-17 for both genotypes.",
    "molecules": "AnnexinV, 7-AAD"
  },
  {
    "caption": "Analysis of apoptosis among pre-HSC type II/HSCs freshly isolated from wild type and Sl/Sl E11.5 AGM. Ter119- VE-Cadherin+ Kit+ CD41+ CD45+ cells were analyzed by flow cytometry for Annexin V binding to detect early apoptotic cells. Percentages are the mean (±SD) of 9 wild type and 7 Sl/Sl biological replicates analyzed over 3 independent experiments. Individual replicates consisted of 1-3 AGMs. A total of 13 wild type and 9 Sl/Sl embryos (11-17 tail sp) were analyzed. The percentage of apoptotic pre-HSC type II/HSCs in the mutant AGM was significantly increased.",
    "molecules": "Annexin V"
  },
  {
    "caption": "Number of phenotypic LSK, HPC and HSC per FL in E12.5 Tie2Cre::Kitl∆/∆and control (Kitlf/f or f/+) FL, determined by flow cytometry and total FL cell counts. Cells were gated as 7-AAD- Lin- (F4/80- CD3e- Nk1.1- Ter119- Gr-1- B220- CD19-). LSK: Lin- Sca1+ Kit+; HPC: Lin- Sca1+ Kit+ CD48+ CD150- and HSC: Lin- Sca1+ Kit+ CD48- CD150+. Gating strategy is shown in Figure EV4E. Data are the mean (±SD) of 12 control and 6 Tie2Cre::Kitl∆/∆ FLs analyzed individually over 3 independent experiments. Total cellularity of E12.5 Kitlf/f or f/+ (n=14) and Tie2Cre::Kitl∆/∆ (n=11) FL. Error bars represent SD.",
    "molecules": "7-AAD"
  },
  {
    "caption": "C) Left. Representative image of coronal striatal slices of fresh frozen brains of wild-type and Tor1a+/- mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. (Right) Graph showing DRD2 binding density in Tor1a+/- striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals",
    "molecules": "3H-spiperone"
  },
  {
    "caption": "B1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190",
    "molecules": "leu, leupeptin"
  },
  {
    "caption": "F) Treatment with bafilomycin A1 (BafA1) induces a significantly more pronounced increase of LC3-II in Tor1a+/- than in wild-type dorsal striatum slices. BafA1-induced increase in LC3-II level measured in Tor1a+/- slices was normalized to the increase observed in the Tor1a+/+ samples of the same experiment (N=4, one sample t test **P=0.0012)",
    "molecules": "BafA1, bafilomycin A1"
  },
  {
    "caption": "A) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT (cyano), DRD2 (red), and the nuclear stain DAPI (white). Bar = 200 μm. B1,B2) Top. Representative ChAT-positive (cyano) cholinergic interneurons, labelled for DRD2 (red), from a Tor1a+/+ (B1) or Tor1a+/- (B2) striatal section. The colocalization mask of the two signals is shown in white (right). Bar = 10 μm. Bottom. Higher magnification images (bar = 5 μm) of representative Tor1a+/+ (B1) and Tor1a+/- (B2) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI (green). A detail at higher magnification is shown in the insets",
    "molecules": "DAPI"
  },
  {
    "caption": "A) As depicted in the cartoon and shown by representative confocal images, patch-clamp recordings were performed in the dorsal striatum, in the area of HSV-LacZ (blue, left) and HSV-RGS9-2 (red, right) viral infection, the latter showing an increased RGS9-2 immunolabelling with respect to the sham contralateral striatum of the same mouse. Representative confocal images at higher magnification show colocalization of RGS9-2 immunolabeling with Lucifer Yellow (LY) fluorescence in two ChIs loaded intracellularly with LY during whole-cell recordings in the infected area",
    "molecules": "Lucifer Yellow, LY"
  },
  {
    "caption": "B) Representative patch-clamp recordings of ChIs from hMT (top, perforated configuration) and Tor1aΔgag/+ (bottom, cell-attached configuration) mice, showing that viral-mediated delivery of RGS9-2 rescues the physiological response to the DRD2 agonist quinpirole (10 µM, 2 min). The dot plot reports the firing frequency changes induced by quinpirole in hMT striata (N=6, HSV-RGS9-2 n=9, HSV-LacZ n=14, t test P<0.0001)",
    "molecules": "quinpirole"
  },
  {
    "caption": "C) Representative traces show a typical response of Tor1a+/+ ChIs to quinpirole, characterized by a reduction in the frequency of the ongoing firing activity of the cell, as opposed to an aberrant increase in firing frequency in Tor1a+/- ChIs, quantified in the plot (right, Tor1a+/+ N=6, n=14; Tor1a+/- N=10, n=27; Tor1a+/+ vs. Tor1a+/- in quinpirole t test ***P=0.0005; Tor1a+/- basal vs. quinpirole paired t test ***P=0.0009)",
    "molecules": "quinpirole"
  },
  {
    "caption": "D) LV-mediated delivery of RGS9-2 rescues the physiological response to quinpirole in Tor1a+/- slices. Dot plot reporting firing frequency changes induced by quinpirole in Tor1a+/+ (N=3) and Tor1a+/- (N=6) striatal ChIs infected with LV/HSV-RGS9-2 or control LV-GFP and HSV-LacZ ChIs (Tor1a+/+ RGS9-2 n=7; Tor1a+/- RGS9-2 n=9; Tor1a+/- LacZ/GFP n=8; Tor1a+/-: LacZ/GFP vs. RGS9-2 t test *P=0.0136)",
    "molecules": "quinpirole"
  },
  {
    "caption": "dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Cells were treated with tetracycline (Tet (A3A)) or/and 300 U/ml IFNα from day 9 to 18 p.i. cccDNA amplicons were analyzed by 3D‑PCR",
    "molecules": "cccDNA, Tet, tetracycline"
  },
  {
    "caption": "A subsequence of the 3D-PCR amplicon was used as (-) ssDNA oligomer, complementary (+) ssDNA and a dU-containing (‑) ssDNA, where all cytosines were replaced by uracils. These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated.",
    "molecules": "cytosines, EDTA, ssDNA, uracils"
  },
  {
    "caption": "dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and at day 7 p.i. transduced with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. Cell lysates were analyzed for cccDNA after T5 digest (B) and total intracellular HBV DNA (C) by qPCR relative to Prnp (n=5-6 biological replicates from two independent experiments).",
    "molecules": "cccDNA, Tet, tetracycline"
  },
  {
    "caption": "dHepaRG-TR-A3A cells were infected with wildtype HBV (HBVwt) or HBV∆x at an MOI of 300 virions/cell and transduced on day 7 p.i. with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. HBeAg (E) was measured by ELISA and cccDNA after T5 digest (F) by qPCR relative to Prnp (n=6 biological replicates from two independent experiments).",
    "molecules": "cccDNA, Tet, tetracycline"
  },
  {
    "caption": "A,B  NAD  and nicotinamide (NAM) levels in heart  of WT (n=5, n=6, respectively), mdx  (n=5, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). C,D NAD  and NAM levels in  diaphragm of WT (n=5, n=6, respectively), mdx (n=4, n=5, respectively) and mdx/CD38-/- mice (n=6, n=5, respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; B: Kruskal-Wallis followed by Dunn's test; D: Kruskal-Wallis followed by Mann-Whitney tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "NAD, NAM, nicotinamide"
  },
  {
    "caption": "E,F levels of ADP-ribose  (ADPR) (E),  cyclic ADP-ribose  (cADPR) (F) expressed as nmol/mg protein in heart of WT (n=6), mdx (n=5) and  mdx/CD38-/- mice (n=5, n=5, n=6 respectively). Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "ADP-ribose, ADPR, cADPR, cyclic ADP-ribose"
  },
  {
    "caption": "H,I Levels of ADPR (H), cADPR (I) expressed as nmol/mg protein in  diaphragm  of WT (n=6), mdx (n=5) and  mdx/CD38-/- (n=5) mice. Data information: Each dot of the graphs represents a mouse and measured in duplicate After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; I: Welch's ANOVA followed by Welch's t-tests Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "ADPR, cADPR"
  },
  {
    "caption": "C Cropped images revealed the    collagen (blue) stained by Masson's trichrome staining  in the  heart  of WT,  mdx, and  mdx/CD38-/- mice. Dot plot showing the quantification of heart collagen staining area (% total area) in 7-month-old WT, mdx, and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: : Each dot of the graphs represents a mouse. in triplicate After normality and variance comparison tests, significance was assessed using ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "trichrome"
  },
  {
    "caption": "D Isoproterenol-induced  heart failure in 3-month-old mice. The Kaplan-Meier  curve shows the survival rate of mdx mice  (NaCl, n=5); mdx  (n=19) and mdx/CD38-/- (n=9) mice following isoproterenol (subcutaneous injection) at 2.5 mg/kg/d for 10 days. Data information: one value/mouse. After normality and variance comparison tests, significance was assessed using D: Log-rank Mantel-Cox test and Log-rank test for trend and Gehan-Breslow-Wilcoxon test ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "Isoproterenol, isoproterenol, NaCl"
  },
  {
    "caption": "E Histogram showing isoproterenol-induced heart hypertrophy in mdx NaCl (n=5) mice, surviving mdx  (n=9), mdx/CD38-/- NaCl  (n=9) and mdx/CD38-/- (n=9) mice, expressed as heart weight/body weight ratio (HW/BW). Data information: one value/mouse. After normality and variance comparison tests, significance was assessed using Kruskal-Wallis followed by Mann-Whitney tests Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "isoproterenol, NaCl"
  },
  {
    "caption": "F Plasma levels of cardiac  stress biomarkers: brain natriuretic peptide (BNP) (F1) and  cardiac troponin I (cTnI) (F2) following isoproterenol treatment in mdx NaCl  (n=5), surviving mdx  (n=8),  mdx/CD38-/- NaCl  (n=5) and mdx/CD38-/- (n=8) mice. Data information: in duplicate After normality and variance comparison tests, significance was assessed using F2 Kruskal-Wallis followed by Mann-Whitney tests; F1: ANOVA followed by Student's/ Welch's t-tests; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "isoproterenol, NaCl"
  },
  {
    "caption": "G,H Time-lapse images recorded by confocal microscopy in \"line scanning mode\", showing Ca2+ sparks and waves in  cardiomyocytes at rest, extracted from  mdx and mdx/CD38-/- mice; scale bars: 10 µM (horizontal), 500 ms (vertical). Bar graphs showing the averaged Ca2+ sparks (G) and waves (H) frequencies in cardiomyocytes  isolated from WT (n=21 cells), mdx (n=28 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3  mice of each group. After normality and variance comparison tests, significance was assessed using Kruskal-Wallis followed by Dunn's test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "Ca2+"
  },
  {
    "caption": "I Bar graph showing the fractional release following caffeine application in  cardiomyocytes from WT (n=24 cells), mdx (n=33 cells) and mdx/CD38-/- (n=32 cells) mice. Data information: experiments performed in 3 mice of each group. After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; ; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "caffeine"
  },
  {
    "caption": "D Images revealing the collagen (blue) by Masson's trichrome staining in  diaphragm of WT, mdx, and mdx/CD38-/- mice. Quantification of collagen staining area (% total area) in the diaphragm of WT, mdx  and mdx/CD38-/- mice (n=4 per group). Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. D in duplicate; After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "trichrome"
  },
  {
    "caption": "A NAD+ levels in  limb muscles of 20-month-old WT (n=7), mdx  (n=5) and mdx/CD38-/- (n=6) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "NAD+"
  },
  {
    "caption": "F Images displaying the collagen (blue) revealed by Masson's  trichrome staining in limb  of WT, mdx, and mdx/CD38-/- mice. The dot plot shows the quantification of collagen staining area (% total area) in limb of WT (n=4), mdx  (n=5) and mdx/CD38-/- (n=5)  mice. Scale bars: 200 µm. Data information: Each dot of the graphs represents a mouse. in triplicate. After normality and variance comparison tests, significance was assessed using: : ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "trichrome"
  },
  {
    "caption": "A Young  mdx mice and treatment with an  CD38 inhibitor. 6-week-old mdx mice were treated for 5 weeks with K-rhein at 2.5 mg/kg/d by intraperitoneal injection. Dot plots showing measurement of the grip duration  (A1) and the force (A2) of WT (n=9), mdx (n=7) and  K-rhein-treated mdx (n=10) mice. Data information: Each dot of the graphs represents a mouse. A1 one value/mouse; A2, in triplicate. After normality and variance comparison tests, significance was assessed using: A1: Welch's ANOVA followed by Welch's t-tests; A2 ANOVA followed by Fisher's LSD test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "K-rhein"
  },
  {
    "caption": "B, New-born double knockout utrophin-dystrophin (mdx/utr-/-) mice and treatment with an  CD38  inhibitor. Mdx/utr-/- mice were subcutaneously injected with  K-rhein (0.6 and 2.5 mg/kg/d) for 4 weeks. B: Dot plots showing the treadmill performances of K-rhein-treated  mdx/utr-/- mice: distance traveled (B1), maximum speed reached (B2) and maximum running time (B3) (WT (NaCl) and mdx/utr-/-+ K-rhein 2.5 mg/kg/d n=10 mice per group; mdx/utr-/-(NaCl) and mdx/utr-/-+ K-rhein 0.6 mg/kg/d, n=12 mice per group). Data information: Each dot of the graphs represents a mouse. one value/mouse; After normality and variance comparison tests, significance was assessed using: ANOVA followed by Fisher's LSD test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001, #: p<0.001 vs mdx/utr-/- .",
    "molecules": "K-rhein, NaCl"
  },
  {
    "caption": "C New-born double knockout utrophin-dystrophin  (mdx/utr-/-) mice and treatment with an CD38 inhibitor. Mdx/utr-/- mice were subcutaneously injected with K-rhein  (0.6 and 2.5 mg/kg/d) for 4 weeks. C: Measurement of the grip duration (grid test) in WT (n=10), mdx/utr-/- (n=8) and K-rhein-treated mdx/utr-/- mice (n=10, 6 mice, respectively for the 0.6 mg/kg/d and 2.5 mg/kg/d doses). Data information: Each dot of the graphs represents a mouse. in triplicate. After normality and variance comparison tests, significance was assessed using: : Kruskal-Wallis followed by Dunn's test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001, #: p<0.001 vs mdx/utr-/- .",
    "molecules": "K-rhein"
  },
  {
    "caption": "D, Adult mdx mice and long-term treatment with an CD38 inhibitor. Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c (10 mg/kg/d). D: Histogram showing  NAD+ levels in  limb of mdx (n=4) and 78c-treated mdx (n=5) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: D: unpaired Student's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "78c, NAD+"
  },
  {
    "caption": "E, Adult mdx mice and long-term treatment with an CD38 inhibitor. Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c  (10 mg/kg/d). E: Histogram showing grip duration (E1) (n=8 mdx and n=11) in the inverted grid test and performances in chronic treadmill test  (E2) at days 1, 6 and 7 after treatment of mdx (n= 5 excepted for D7 n=4) and 78c-treated mdx  (n= 7) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: E1: unpaired Mann-Whitney test; E2: two-way ANOVA; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "78c"
  },
  {
    "caption": "F Adult mdx mice and long-term treatment with an CD38 inhibitor. Mdx mice were evaluated after 6 months of intraperitoneal injection with the CD38 inhibitor 78c (10 mg/kg/d). F: Dot plots showing barometric plethysmography measures of the tidal (F1) and minute (F2) volumes of mdx (n=4) and 78c-treated mdx (n=6) mice. Data information: Each dot of the graphs represents a mouse. in duplicate; After normality and variance comparison tests, significance was assessed using: F2, unpaired Mann-Whitney test ; F1 unpaired Welch's t-test; Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "78c"
  },
  {
    "caption": "G Time-lapse confocal  imaging of calcium dynamics in  human healthy and DMD myotubes loaded with the  Ca2+-sensitive dye Fluo-4 (white arrows show the active cells) (G1). Traces illustrating recordings from region of interest (ROI) in an inactive healthy myotube (black line) and in a DMD myotube displaying Ca2+ spiking activity (red line) (G2). Histogram showing the percentage of myotubes displaying spontaneous  Ca2+  waves (G3): healthy myotubes (n=91 cells),  DMD myotubes  (n=186 cells). Scale bars: 200 µm. Data information: After normality and variance comparison tests, significance was assessed using: G3 Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "Ca2+, calcium, Fluo-4"
  },
  {
    "caption": "H Human DMD myotubes treated by SAR650984  (isatuximab), a humanized anti-CD38  antibody. Fluorescence trace illustrating a recording of DMD myotubes treated with 10 µg/ml of  SAR650984 (blue). Histogram showing the percentage of spontaneous  Ca2+  waves in DMD myotubes untreated (n=740 cells) or treated with 10 µg/ml of SAR650984 (n=279 cells) (H1). Histogram showing the Ca2+ wave inter-spike duration (interval between spikes) in myotubes  treated with 10 µg/ml of SAR650984 (n=43 cells vs 91 for the untreated DMD myotubes) (H2). Data information: After normality and variance comparison tests, significance was assessed using: H2: unpaired Welch's t-test; H1: Chi-square test Values are expressed as means ± SEM. Significance: *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "Ca2+, isatuximab, SAR650984"
  },
  {
    "caption": "B WT HeLa cells were incubated with HPG for 30 minutes in the presence or absence of Harr and/or Chloramphenicol (Chl). After fixation, AF647-azide (magenta) was used for click reaction (see A), and cells were immunostained for TOM20 as mitochondrial marker (green). Representative epifluorescent microscopy images are shown. Magenta images shown in the presence of only Chl and in the absence of both inhibitors (none) are presented at lower brightness level than the other panels, duo to their very strong intensities. An inset of Harr panel is zoomed in the panel below for better visualization. Met, methionine. Scale bars, 50 µm and 10 µm for the zoom panel.",
    "molecules": "AF647-azide, Chl, Chloramphenicol, Harr, HPG, Met, methionine"
  },
  {
    "caption": "A Mitochondrial translation products were labeled in WT HeLa cells for indicated time lengths (heat map) and cells immunostained for TOM20 (gray). Representative confocal images are shown for each time point. The same 5-minute image was used in both rows with different brightness as a reference for intensity comparison of the other time points in the row. Scale bars, 10 µm. Mitochondrial protein synthesis was quantified over time. 30 cells from N = 3 independent experiments were analyzed for each time point (Average of the HPG intensity in the mitochondrial area was calculated and the baseline was set to zero by subtracting the value of time point 0 from all the values. Error bars, +/- SEM). A hyperbolic graph was fitted to the data. R-squared, 0.97.",
    "molecules": "HPG"
  },
  {
    "caption": "C Mitochondrial translation products were labeled for 30 minutes and clicked with AF647 (magenta) and cells immunostained for TOM20 (green) in fibroblasts, iPSCs-derived cardiomyocytes, and hippocampal neurons. Representative epifluorescent microscopy images are shown. The correlation of HPG and TOM20 channels is represented in fire lookup table, after calculating the Pearson Correlation values over each pixel of the images. Scale bars, 25 µm.",
    "molecules": "AF647, HPG"
  },
  {
    "caption": "A Mitochondrial translation products were labeled using a FUNCAT approach and [35S]Met labeling in control and TSFM patient fibroblasts. Representative epifluorescent microscopy and the autoradiography images are shown. Scale bars, 50 µm. Changes in mitochondrial protein synthesis were quantified for both approaches. For microscopy image analysis, >300 cells from N = 3 independent experiments were analyzed for each cell type. Mann-Whitney U test, ****p < 0.0001. For radiolabeling analyses, N =3 independent samples were quantified for each cell type. Student t test, **p < 0.01. Error bars, +/- SEM. B As in A using control and COA6 KO HEK cells. Scale bars, 25 µm. For microscopy image analysis, ****p < 0.0001, and for radiolabeling experiments, *p < 0.05 were calculated. Error bars, +/- SEM.",
    "molecules": "Met, 35S"
  },
  {
    "caption": "A Mitochondrial translation products were labeled with HPG in hippocampal neurons, immunostained for ANK-G (initial axon segment), and plasma membrane was stained with DiO. Representative epifluorescent images and zoomed insets of neural branches are shown. In both axon (ANK-G positive, arrow) and dendrite (star), HPG incorporation revealing mitochondrial protein synthesis was detected. Scale bars, 20 µm.",
    "molecules": "DiO, HPG"
  },
  {
    "caption": "B Representative confocal images of a dendrite (top) and an axon (bottom) in which mitochondrial translation products were labeled with HPG (magenta). HOMER1 and VGLUT1 were used as post- and presynapse markers, respectively (green) and DiO stained randomly neurites (gray). In each neurite, an exemplary region was selected and the DiO was outlined to mark the outer edge of the region. As a control, cells were treated with Chloramphenicol (Chl) to inhibit mitochondrial translation. Scale bars, 5 µm.",
    "molecules": "Chl, Chloramphenicol, DiO, HPG"
  },
  {
    "caption": "(c) Blockage of pseudouridylation by deletion of specific pseudouridylase genes. RNAs isolated from wild-type and pseudouridylase-deletion strains (indicated on the top) were used for U2 pseudouridylation assay (CMC modification followed by primer-extension). Primer-extension pauses/stops correspond to ψ sites (indicated on the left and right). Note: The above-background signal of ψ42 in the snr81Δ lane (and to some extent, in the snr81Δ pus7Δ lane) is likely caused by the presence of strong ψ44 band. This is a primer-extension artifact, which often occurs when there is a strong ψ signal in the neighboring position. Alternatively, it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 (albeit inefficiently) (Perhaps Pus1, which is responsible for ψ44 formation, can be a bit \"processive\"). Lanes 1-8 and lanes 9-16 are from two separate gels.",
    "molecules": "CMC"
  },
  {
    "caption": "(a) Growth assay using the ACT1-CUP1 reporter system. Yeast cells carrying the ACT1-CUP1 reporter were deleted of pseudouridylase genes (indicated on the left), and were then assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).",
    "molecules": "Cu2+"
  },
  {
    "caption": "(b) Growth rescue by restoration of pseudouridylation. The wild-type strain [from (a)] or the snr81Δ pusΔ strain [also from (a)], which exhibited the most severe growth-deficiency phenotype, was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42, position 44, or both (indicated). The resulting cells were assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).",
    "molecules": "Cu2+"
  },
  {
    "caption": "(d) Splicing assay using the wild-type and mutant ACT1-CUP1 reporters. RNAs isolated from the wild-type and the snr81Δ pusΔ strains (indicated on the top), which carry the wild-type ACT1-CUP1 reporter pre-mRNA or any of the mutant ACT1-CUP1 reporter pre-mRNAs [indicated, also see (c)], were used for splicing assay (primer-extension analysis). The un-spliced pre-mRNA, lariat intermediate, and spliced mRNA are indicated. A primer complementary to U6 was also used (as an internal control) in the assay, and the U6 band is indicated as well. In addition, the growth phenotype of each strain (in various concentrations of [Cu2+]) is also shown. Lanes 1-12, lanes 13 and 14, and lanes 15 and 16 are from separate gels.",
    "molecules": "Cu2+, mRNA"
  },
  {
    "caption": "(e) Quantification of ACT1-CUP1 mRNA levels. Spliced ACT1-CUP1 mRNA levels were calculated relative to U6 in each lane. The quantification was based on three independent experiments.",
    "molecules": "mRNA"
  },
  {
    "caption": "(f) Relative improvement/reduction for the mutant reporters. Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency (mRNA/U6) of each mutant reporter to that of WT reporter (set at 1).",
    "molecules": "mRNA"
  },
  {
    "caption": "Figure 3. Synthetic lethality assay. The Prp5 domain structure, and the mutations in some of the domains (indicated) are schematized at the top. Each of these PRP5 genes (wild-type or mutants, plasmid-borne) was used to replace the chromosomal PRP5. The numbers in parentheses are the relative ATPase activities of the wild-type (SAT) Prp5 and some of the Prp5 mutants tested before (Xu & Query, 2007). The U2 status, either containing Ψ42 and Ψ44 (WT-U2 and snR81Δ pus1Δ with pΨ42 or Ψ44) or lacking Ψ42 and Ψ44 (snR81Δ pus1Δ), is indicated (on the right). Upon transformation with the plasmids carrying PRP5 genes (wild-type or any of the mutants), cells were grown on the regular medium (-FOA) or on medium containing 5-FOA (+FOA).",
    "molecules": "5-FOA, FOA"
  },
  {
    "caption": "(a) Prp5-U2 co-immunoprecipitation (IP). Wild-type (lanes 1 and 2) and snR81Δ pus1Δ (lane 3) strains, each containing a FLAG-tagged PRP5 gene (indicated on the top), were used for anti-FLAG IP in the absence (lane 1) or presence (lanes 2 and 3) of ATP. The precipitated Prp5 (WT Prp5-FLAG) as well as co-precipitated U1 and U2, detected by primer-extension, are indicated.",
    "molecules": "ATP"
  },
  {
    "caption": "d) Depletion of Prp5. Wild-type (lanes 1, 3 and 4) and snR81Δ pus1Δ (lanes 2 and 5) cells containing a FLAG-tagged PRP5 were lysed, and anti-FLAG IP was performed at a high concentration of salt. Cell extracts, before (lanes 1 and 2), and after [lanes 3 (mock), 4 and 5] anti-FLAG IP, were used for western analysis using anti-FLAG. The Prp5 band is indicated.",
    "molecules": "salt"
  },
  {
    "caption": "(e) IP of reconstituted Prp5 (GAR)-U2. An equal amount of FLAG-tagged Prp5 (GAR) was added to the wild-type (lane 1) and snR81Δ pus1Δ (lane 2) cell extracts depleted of endogenous Prp5 [see (c)]. After the salt concentration was brought back to its original level, anti-FLAG IP was carried out. The Prp5 (GAR) band, detected by western, is indicated. The co-precipitated U1 and U2 bands, detected by primer-extension, are also indicated.",
    "molecules": "salt"
  },
  {
    "caption": "(g) Prp5-U2-pre-mRNA co-IP. Reconstitution was carried out as described in (d). Prior to IP, an equal amount of radiolabeled pre-mRNA was also added to each reaction. Anti-FLAG IP was then performed in the presence of ATP. The precipitated Prp5 (WT and GAR) band, detected by western, is indicated. The co-precipitated pre-mRNA, directly visualized after electrophoresis, is also indicated. The pre-mRNA input is also shown. In lanes 1 and 2, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with the wild-type FLAG-Prp5. In lanes 3 and 4, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with mutant FLAG-Prp5 (GAR).",
    "molecules": "ATP"
  },
  {
    "caption": "(a) Prp5's ATPase activity assay using synthetic U2. The 5' sequence of S. cerevisiae U2 is shown (top). The three ψs (35, 42 and 44) as well as the branch site recognition sequence (underlined) are indicated. In the ATPase assay (bottom), no RNA (lane 3), or an equal amount of LiCl-precipitated RNA (lane 1) or tRNA (lane 2), a short U2 fragment (nts 1-76) (lane 4), a fully pseudouridylated short U2 fragment (nts 1-76) (lane 5), a long U2 fragment (nts 1-120) (lane 6), or a fully pseudouridylated long U2 fragment (nts 1-120) (lane 7), was used. The un-hydrolyzed ATP and hydrolyzed product ADP are indicated. Lanes 1 and 2, lanes 3-5, and lanes 6 and 7 are from separate gels.",
    "molecules": "tRNA, ADP, ATP, LiCl, RNA"
  },
  {
    "caption": "(b) Quantification of Prp5's ATPase activity activated by in vitro transcribed U2. Prp5's ATPase activity shown in (a) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (b) correspond to the lane numbers of (a). Quantification was based on three independent experiments.",
    "molecules": "ADP, ADT, ATP"
  },
  {
    "caption": "(e) Prp5's ATPase activity assay using cellular U2. U2 RNA isolated from the wild-type strain (lanes 2 and 4) or from the snR81Δ pus1Δ strain (lanes 6 and 8) was used for the ATPase activity assay. In odd-numbered lanes, no RNA was added. Both the wild-type Prp5 (lanes 1-4) and the mutant Prp5 (GAR) (lanes 5-8) were tested.",
    "molecules": "RNA"
  },
  {
    "caption": "(f) Quantification of Prp5's ATPase activity activated by U2 from cells. Prp5's ATPase activity shown in (e) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (f) correspond to the lane numbers of (e). Quantification was based on four independent experiments.",
    "molecules": "ADP, ADT, ATP"
  },
  {
    "caption": "(b) DMS in vivo probing. After being exposed to DMS, yeast cells were lysed, total RNA collected, and primer-extension analysis carried out (left panel). Lane 1, cells that were not exposed to DMS. Lane 2, wild-type cells exposed to DMS. Lane 3, snR81Δ pus1Δ cells exposed to DMS. Lane 4, snR81Δ pus1Δ cells, transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44, and exposed to DMS. Lane 5, snR81Δ pus1Δ cells, transformed with a plasmid carrying a control guide RNA gene with random guide sequences, and exposed to DMS. The nucleotides in parentheses are DMS-modified C and A residues that are one nucleotide after the actual primer-extension stops (indicated by the arrows). A partial U2 sequence (along with the BSL structure) is shown (right panel). DMS-modified adenosine and cytosine in the BSL are also indicated.",
    "molecules": "cytosine, adenosine, DMS"
  },
  {
    "caption": "(B) Glycemia  one hour after saline  (black) or insulin  (red) i.p. injection [n=15-16 mice  per group]. (C) CPP plasma  levels one hour after saline  or insulin  injection [n=13 mice  per group; mean±SEM for INS: WT 89±5 vs KO 121±13 pg/ml]. (D) GCG plasma  levels one hour after saline  or insulin  injection [n=15-16 mice  per group; mean±SEM for INS: WT 124±19 vs KO 186±21 pg/ml]. (E) Glycemia one hour after saline  (black) or insulin  (red) i.p. injection [n=18-22 mice  per group]. (F) CPP plasma  levels one hour after saline  or insulin  injection [n=18-19 mice  per group; mean±SEM for INS: WT 78±6 vs KO 92±8 pg/ml]. (G) GCG plasma  levels one hour after saline  or insulin  injection [n=17-20 mice  per group; mean±SEM for INS: WT 242±14 vs KO 277±18 pg/ml]. (H-J) Analysis of glycemic  levels and of CPP   and GCG   secretion following insulin  injection in female mice  at each stage of the estrus  cycle [n=3-7 mice  per group]. (H) Glycemic levels. (I) CPP plasma  levels. (J) GCG plasma  levels. Data information: d: day, di: diestrus, est: estrus, INS: insulin, met: metestrus, pro: proestrus, For panels B-G, bars correspond to the mean value per group. For panels H-J, lines correspond to the mean value per group and error bars represent ± SEM. B-G: 2-way ANOVA RM with Bonferroni post-hoc test; H-J: unpaired t-test; *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "INS, insulin, saline"
  },
  {
    "caption": "C57BL6/N male mice  were injected i.p. with saline  or insulin  and c-Fos expression  (green) in AVP   (magenta) neurons  was quantitated by  immunofluorescence microscopy 2 hours later. (A) C-Fos expression  in the PVN  after saline  injection. (B) C-Fos expression  in the PVN  after insulin  injection (C) Quantitation of  c-Fos positive cells in the PVN  after saline  and insulin  injections [n=4 mice  per group]. Data information: 3V: 3rd ventricle, AVP: Vasopressin, INS: insulin, PVN: Paraventricular nucleus, SAL: saline, Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.",
    "molecules": "INS, insulin, SAL, saline"
  },
  {
    "caption": "C57BL6/N male mice  were injected i.p. with saline  or insulin  and  c-Fos expression  (green) in AVP   (magenta) neurons  was quantitated by  immunofluorescence microscopy  2 hours later. (D) C-Fos expression  in the SON  after saline  injection. (E) C-Fos expression in the SON  after insulin  injection (F) Quantitation of c-Fos positive cells in the SON  after saline  and insulin  injections [n=4 mice  per group]. Data information: AVP: Vasopressin, INS: insulin, opt: optic tract, SAL: saline, SON: Supraoptic nucleus. Scale bar=100μm. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.",
    "molecules": "INS, insulin, SAL, saline"
  },
  {
    "caption": "(H) Tmem117  mRNA levels in the PVN  [n=4 mice  per group]. (I)Tmem117  mRNA levels in the SON  [n=4 mice  per group Data information: INS: insulin, PVN: Paraventricular nucleus, SAL: saline, SON: Supraoptic nucleus. Lines correspond to the mean value per group and error bars represent ± SEM. Unpaired t-test; ns: p>0.05, *p<0.05, **p<0.01.",
    "molecules": "INS, insulin, SAL, saline"
  },
  {
    "caption": "(B)  C-Fos expression  in the SON  Data information: AVP: Vasopressin INS: insulin opt: optic tract, SAL: saline, Scale bar=100μm.",
    "molecules": "INS, insulin, SAL, saline"
  },
  {
    "caption": "(G) Membrane potential  was increased comparably in response to 0.1 mM glucose  in both groups [n=7-9 cells per group]. (H) No change in  membrane potential in NR neurons  of both genotypes [n=9 cells per group]. (I) Membrane resistance  was decreased in 6 out of 9 AVPTM117WT and 6 out of 7 AVPTM117KO cells in response to 0.1 mM glucose  [n=7-9 cells per group]. (J) Membrane resistance in NR neurons  was stable for both genotypes [n=9 cells per group]. Data information: AVP: Vasopressin, d: day, GI: glucose  inhibited, NR: non-responding, Lines correspond to the mean value per group and error bars represent ± SEM. G-J: paired t-test for all graphs except Δ membrane potential (unpaired); nsp>0.05, *p<0.05, ***p>0.001.",
    "molecules": "glucose"
  },
  {
    "caption": "(F) Fluorescence microscopy  detection of DHE-derived fluorescence (red) in the SON. (G) Quantification of DHE-derived fluorescence in the SON  [n=8 SON  per group; each group consisted of 4 mice]. Data information: AVP: Vasopressin, d: day, DHE: dihydroethidium  , opt: optic tract, SON: Supraoptic nucleus,. Scale bar=20μm. For panels G data are represented as mean ± SEM. G: unpaired t-test; ; *p<0.05, ***p>0.001.",
    "molecules": "DHE, dihydroethidium"
  },
  {
    "caption": "(J) Calcium  recordings in AVP magnocellular terminals 10 minutes before and one hour after the i.p injection of insulin  (red and orange) or saline  (black). The vertical arrow corresponds to the i.p. injection (time=0). (K) Quantification of mean signal intensity in 10-minute time  bins. [n=6-8 mice  per group]. Data information: AUC: area under the curve, AVP: Vasopressin INS: insulin, SAL: saline  For panels K, data are represented as mean ± SEM. K: 1-way ANOVA with Tukey's post-hoc test; *p<0.05, ***p>0.001.",
    "molecules": "Calcium, INS, insulin, SAL, saline"
  },
  {
    "caption": "(E) Glycemia  one hour after saline  (black) or insulin  (red) i.p. injections in AVPTM117WT and AVPTM117KO male mice  [n=8-9 mice  per group]. (F) CPP plasma  levels one hour after saline  or insulin  injection in AVPTM117WT and AVPTM117KO male mice  [n=8 mice  per group]. (G) GCG plasma  levels one hour after saline  or insulin  injection in AVPTM117WT and AVPTM117KO male mice  [n=8-9 mice  per group]. Data information: AVP: Vasopressin, d: day, INS: insulin, KO: AVPTM117KO, SAL: saline, WT: AVPTM117WT. For panels E-G bars correspond to the mean value per group and error bars represent ± SEM. E-G: 2-way ANOVA for each timepoint in comparison to SAL  baseline with Bonferroni post-hoc test; *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "INS, insulin, SAL, saline"
  },
  {
    "caption": "Green line: Normalized population expression level averaged over all OxyR, and SoxS-regulated promoters; red line: average over all LexA-regulated promoters as measured with a promoter-GFP plasmid library in a plate reader. Oxidative stress promoters clearly precede SOS response promoters in response to NIT stress, when measured at the population level Error bars show standard deviation over all detected oxidative stress and SOS promoters, respectively It is not clear if this temporal order correctly reflects the temporal order in single cells. B. Schematic showing response of two different genes (green and red) in three different cells (solid, dashed, dotted line). The temporal order observed at the population level correctly reflects the temporal order in each individual cell. C. As B. Here the temporal order at the population level is not the same in every single cell: Although in most cells, the green gene responds before the red one, one cell expresses the green gene before the red one (dashed lines).",
    "molecules": "NIT"
  },
  {
    "caption": "Single-cell gene expression time traces from the dnaK promoter in response to TET stress, normalized to the median full response. Response times were determined as the time point at which a threshold expression level was reached This threshold was low enough so that most cells exceed it and high enough to avoid false positives due to the low signal-to-noise ratio at time point zero. Brown line: median of all cells. Time traces are from one microcolony. B. Histogram of the response times for the dnaK promoter with mean µt = 1.87 h and standard deviation σt = 0.37 h, and fit of an Erlang distribution with shape parameter n = 37 Response times are from two microcolonies.",
    "molecules": "TET"
  },
  {
    "caption": "C. Standard deviation σt versus mean response time µt for 23 different promoters in three antibiotic stress conditions (TMP, TET, and NIT). The standard deviation of the response time σt grows with the mean response time µt and does not fall below a 'precision limit' (dashed line) that increases linearly with a slope ~0.165. The dotted line indicates the upper bound to timing variability where σt = µt, see text. The promoter dnaK under TET has low timing variability, whereas the promoters recA, fpr, osmC, and wrbA under TMP stress have high timing variability. The response time mean and standard deviation are from subsampling of descendants of single cells that were present at the time of stress addition Subsampling for each promoter was done from at least 2 microcolonies and the descendants of at least 17 individual cells present at the time of stress addition.",
    "molecules": "NIT, TET, TMP"
  },
  {
    "caption": "E. Standard deviation σt versus mean response time µt for the LlacO-1 promoter induced with different IPTG concentrations.",
    "molecules": "IPTG"
  },
  {
    "caption": "D. Response times of the LlacO-1 promoter (Lutz & Bujard, 1997), induced with 1 mM IPTG at time zero, measured with YFP and CFP constructs in the same cell (r = 0.81 ± 0.04, p = 6 · 10-12).",
    "molecules": "IPTG"
  },
  {
    "caption": "A. Normalized expression from the promoters ybjC and recA in response to NIT stress, measured at the population level using GFP reporters 4 μg/mL NIT was added at time zero. The oxidative stress promoter ybjC clearly precedes the SOS response promoter recA, when measured at the population level Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized ybjC and recA expression over time in response to the addition of 4 μg/mL NIT at time zero in single cells from one microcolony. Dashed line: threshold used to determine response times Lower panels, upper row: Dual-color images of the oxidative stress reporter ybjC (green) and the SOS reporter recA (red) in single cells at 4 different time points after NIT addition in one microcolony. Note that the ybjC signal (green) is stronger at t = 2h, whereas the recA signal (red) is only apparent at t = 4h and 6h. Lower row: Constitutively expressed mCherry used for segmentation. C. Response times for ybjC and recA from individual cells with dual reporters (Figure 3), combined from two microcolonies. All response times for recA are higher than for ybjC and response times for both reporters are strongly correlated (r = 0.74 ± 0.04, p = 3.8 · 10-6). The error of the correlation coefficient is from subsampling of descendants of single cells that were present at the time of NIT addition",
    "molecules": "NIT"
  },
  {
    "caption": "A. Expression from the gadB and recA promoters, measured at the population level using GFP reporters over time; 0.5 μg/mL TMP was added at time zero. The acid stress response (gadB) clearly precedes the SOS response (recA). Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized expression of gadB and recA expression over time in individual cells responding to the addition of 0.5 μg/mL TMP at time zero shown for one microcolony. Dashed line: threshold used to determine response times (Methods). Lower panels, upper row: Dual-color images of gadB (blue) and recA (red) in single cells at 4 different time points. Most cells are predominantly either red or blue, i.e. acid stress and SOS response are typically not both strongly expressed in the same cells. Lower row: Constitutively expressed mCherry used for segmentation. C. Scatter plot of response times for gadB and recA in single cells; there is no clear temporal order and no significant correlation (r = 0.08 ± 0.08, p = 0.68); data is combined from three microcolonies. Error is from subsampling of descendants of single cells present at the time point of TMP addition",
    "molecules": "TMP"
  },
  {
    "caption": "D. Expression of gadB and recA in inosine-supplemented medium, averaged over 13 single cells in a microcolony. Insets: Dual color image of gadB and recA and segmentation image at t = 6h.",
    "molecules": "inosine"
  },
  {
    "caption": "(B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm. Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm.",
    "molecules": "TAM, tamoxifen"
  },
  {
    "caption": "(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.",
    "molecules": "DAB"
  },
  {
    "caption": "(F) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (lower panel). n=6 for each group. GW501516 (10 μM) were added directly into organoid culture medium.",
    "molecules": "GW501516"
  },
  {
    "caption": "(E) Lgr5GFP-CreERT2;Rosa26lsl-lacZ (LRlacZ) were crossed with Rest-/- mice, followed by TAM administration for lineage tracing for intestinal whole-mount staining for β-gal (upper panel) . Mice were sacrificed after one week and typical sections were shown (middle panel). Scale bar, 200 μm. Numbers of traced crypt-villus units were calculated as means± S.D. ** P < 0.01 by two-tailed Student's t-test in right panel. 50 fields (10 fields per mouse) were calculated for each group (n=5) (lower panel).",
    "molecules": "TAM"
  },
  {
    "caption": "(G) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. n=6 for each group. Organoid number per well was counted as means ± S.D. (lower panel). Lentivirus-carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM-treated LRCas9 mice to obtain Prdm16-/- and Ppard-/- ISCs. 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=5 for each group.",
    "molecules": "TAM"
  },
  {
    "caption": "b, SCID mice were infected as in a and liver sections stained for LAMP1 (green), bacteria (red) and DNA (blue). Shown is a LAMP1+ SLAP.",
    "molecules": "DNA"
  },
  {
    "caption": "f, GFP-LC3-transfected macrophages were infected with wild-type or D actA bacteria. Where indicated, chloramphenicol (CM) was added to the media at 3 h post infection. The number of bacteria per SLAP was quantified. Brackets indicate significant differences, and corresponding P values are shown.",
    "molecules": "chloramphenicol"
  },
  {
    "caption": "g, Macrophages were infected with wild-type bacteria for 7 h, pulsed with BrdU for 1 h, and stained for LAMP1 (red), bacteria (blue) and BrdU (green). Magnified images and the arrow indicate SLAPs containing actively replicating bacteria (BrdU+). h, The percentage of BrdU+ bacteria in SLAPs, compared to bacteria and PFA-killed bacteria, was quantified as in c.",
    "molecules": "PFA"
  },
  {
    "caption": "e, The arrow indicates a LAMP1+ SLAP colocalizing with v-ATPase staining in cells infected for 4 h. Scale bar, 5 µm. f, The percentage of v-ATPase+ SLAPs was quantified as in Fig. 2c. Mean ± s.e.m. for three independent experiments. P values for conditions significantly different from PFA-killed bacteria are shown.",
    "molecules": "PFA"
  },
  {
    "caption": "b, Macrophages were infected with IPTG-induced iLLO bacteria. Arrowheads indicate actin- iLLO bacteria within LAMP1+ vacuoles.",
    "molecules": "IPTG"
  },
  {
    "caption": "c, Macrophages were infected with wild-type (triangles) or IPTG-induced iLLO (circles) bacteria, and the percentage of LAMP1+ bacteria was quantified. Mean ± s.e.m. for three independent experiments.",
    "molecules": "IPTG"
  },
  {
    "caption": "d, Macrophages were infected as in c with or without IPTG induction. Intracellular bacterial replication was determined using a gentamicin-protection assay. Shown is fold replication compared to 2 h post infection. Clear triangles, wild type; filled triangles, wild type + IPTG; clear circles, iLLO; filled circles, iLLO + IPTG. Mean ± s.e.m. or range for three (wild type, wild type + IPTG, iLLO + IPTG) or two (iLLO-IPTG) independent experiments, respectively.",
    "molecules": "IPTG"
  },
  {
    "caption": "e, Wild-type or Atg5-/- MEFs were infected with iLLO bacteria, and intracellular bacterial replication was determined as in d. Mean ± s.e.m. for three independent experiments. Clear circles, wild-type MEFs; filled circles, wild-type MEFs + IPTG; clear squares, Atg5-/- MEFs; filled squares, Atg5-/- MEFs + IPTG.",
    "molecules": "IPTG"
  },
  {
    "caption": "f, Wild-type (circles) or Atg5-/- (squares) MEFs were infected as in e with IPTG induction. The percentage of LAMP1+ bacteria was quantified as in c. Mean ± s.e.m. for three independent experiments. g",
    "molecules": "IPTG"
  },
  {
    "caption": "(a) THP-1 cells were transiently infected with BRLF1-expressing or empty control lentivirus. Twenty-four hours later, the control or BRLF1-expressing THP-1 cells were primed with 40 ng/ml TPA overnight and then were either uninfected or infected with HSV-1 (MOI=1) in serum-free medium for 12 h. The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18 and mature IL-18 as indicated.",
    "molecules": "TPA"
  },
  {
    "caption": "(g) After being transduced with control or BZLF1-expressing lentivirus for 48 h, EBV-WT- and EBV-ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry. Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate.",
    "molecules": "PI"
  },
  {
    "caption": "(e) P3HR-1 cells were induced by TPA plus NaB into lytic infection, and the cells were then collected. Immunoprecipitation with anti-mouse IgG or BRLF1 antibody was performed, after which whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.",
    "molecules": "NaB, TPA"
  },
  {
    "caption": "(d) EBER1 5'-pppRNA detection using splint-ligation. EBV-WT- or EBV-ΔBRLF1-harboring HNE1 cells were cotransfected with empty vector, BRLF1 or BRLF1 L578A, BRLF1 plus BZLF1, or BRLF1 L578A plus BZLF1 for 48 h. The total RNAs were extracted, and 5 μg of each RNA was subjected to splint-ligation with FAM-labeled probe to quantify the 5′-monophosphorylated EBER1 and total EBER1 RNA. The ligation products were separated using urea-PAGE, visualized by a fluorescent scanner and analyzed using ImageJ software. The ratio of 5'-pRNA density to total RNA density and the relative 5'-pppRNA level were calculated for three independent experiments.",
    "molecules": "FAM, urea"
  },
  {
    "caption": "(c) Analysis of ASC oligomerization. ASC-expressing plasmid was transfected into 293T cells or HNE1 cells with empty vector or GFP-BRLF1 in the presence of AIM2- or RIG-I-expressing plasmid. Twelve hours after HSV-1 infection (MOI=1) in 293T cells, or 48 h after EBV-ΔBRLF1 infection (MOI=1000) in HNE1 cells, the cell pellets were collected, and cell lysates were treated with DSS to induce chemical cross-linking and analyzed by western blotting for ASC oligomerization.",
    "molecules": "DSS"
  },
  {
    "caption": "(a) Representative image of a Control (left) and Myo1cKO (right) embryo. Top: max projections of embryos stained with Phalloidin (green) and DAPI (gray). Bottom: magnifications at dashed rectangles of Phalloidin (grey) with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "(b-c) Number of fragments without DNA (b) per embryo and mean radius (c) in Control (grey) and Myo1cKO (salmon) embryos (left, Control n = 20, Myo1cKO n = 20). Boxplot shows median, upper and lower quartiles, min and max values. Whisker plot shows median, upper and lower quartiles. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(f) Representative images of embryos treated with DMSO (left) and 37.5 µM Ciliobrevin D (right), stained with Phalloidin. Top: Maximum projection. Bottom: magnifications of a single plane at dashed rectangles of Phalloidin with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.",
    "molecules": "Ciliobrevin D, DMSO, Phalloidin"
  },
  {
    "caption": "(a) Representative images of Control (left) and Myo1cKO (right) embryos labelled with SiRDNA (grey) and expressing a reporter of Cdc42 activity (yellow) in one blastomere, which is undergoing mitosis, shown as max projections. The second blastomere is outlined with a dashed line. White arrows point at the accumulation of active Cdc42 at the cortex in close proximity to DNA. (b) The ratio of active Cdc42 Ic/Iref was compared between < 20 µm and > 20 µm DNA-cortex distances for Control (dark yellow) and Myo1cKO (yellow) embryos (Control embryos n = 10; Myo1cKO embryos n = 10). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests (b Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(c) Representative images of Myo1cKO embryos expressing LifeAct-GFP (green) alone (left) or together with dominant negative Cdc42 (DNCdc42, right) labelled with SiRDNA (grey) shown as max projections. White arrows point at the cortex in close proximity to DNA. (d) LifeAct-GFP Ic/Iref values at DNA-cortex distances < 20 µm and > 20 µm for Myo1cKO (salmon) and Myo1cKO + DNCdc42 (yellow) embryos (Myo1cKO embryos n = 8, cells n = 19; Myo1cKO + DNCdc42 embryos n = 12, cells n = 30). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests d) Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Representative images of a GFP (left) and Ect2-GFP (right) expressing embryo stained with Phalloidin (green) and DAPI (grey) shown as max projections. White arrows indicate fragments. (f) Number of fragments without DNA per embryo in GFP (grey) and Ect2-GFP (blue) embryos (GFP n = 14; Ect2-GFP n = 18). Data information Mann-Whitney test (f) p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.",
    "molecules": "DAPI, DNA, Phalloidin"
  },
  {
    "caption": "F-M Compared with wild-types (F-I), mutants (J-M) completely lost the CM (red) in the gut of 7dpf larva (F, J), as well as in the intestinal bulb (G, K), middle intestine (H, L) and posterior intestine (I, M) of adults. Red, SNAP-CBD stain; blue, DAPI stain; WT, wild-type; MU, chs1-/- mutant.",
    "molecules": "DAPI"
  },
  {
    "caption": "E-J 6/7 dpf larva mutants were fed with 6-μm polystyrene beads. The beads jammed in the intestinal bulb and middle intestine (E, F, H, I). In some cases, the beads escaped the bead cord and stuck between villi (white triangles) (E, G, I, J). Blue rectangle indicates the intestinal bulb. Right panels were magnification of the red rectangle area in left panels.",
    "molecules": "bead, beads, polystyrene beads"
  },
  {
    "caption": "E Immunohistochemical detection of BrdU-labeled cells in the intestine bulb (section S2) of two 3-month-old adult wild-types and two 3-month-old adult mutants to measure the rate of cell proliferation (new cells were stained bright green) and migration. Compared with wild-types (WT), the mutants had more green spots along the villi 2 hours after treatment, and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments. The violin plots (interquartile range with mean line) show the relative numbers of green puncta from tip to base of villi, which were calculated from 3-5 villi from the raw version of these images. Please see Appendix Fig S19 for the images of more animals and time points.",
    "molecules": "BrdU"
  },
  {
    "caption": "A, B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment. Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 seedlings were grown for 6 days on 1/2 MS medium supplemented with 1 μM brassinazole (BRZ) plus different concentrations of epibrassinolide (eBL). Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD; n>20. Scale bars, 2 mm.",
    "molecules": "eBL, epibrassinolide, brassinazole, BRZ, BR"
  },
  {
    "caption": "C RT-qPCR analysis of BZR1-activated gene expression in the bic1bic2 double mutants. The 6-d-old Col-0 and bic1bic2 seedlings grown on 1/2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL (+eBL) or not (-eBL) for 1 hour. The PP2A gene served as internal control. Data are means ± SD (n=3).",
    "molecules": "eBL, BRZ"
  },
  {
    "caption": "C-E BR signaling is enhanced in the 35S:BZR1-MYC/35S:BIC1-YFP plants. Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole (BRZ) plus a gradient of concentrations of epibrassinolide (eBL) under long-day conditions. Images of the representative seedlings when grown with 500 nM eBL (+eBL) or not (-eBL) are shown in (C), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (D) and (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.",
    "molecules": "eBL, epibrassinolide, brassinazole, BRZ, BR"
  },
  {
    "caption": "H BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. The Col-0, 35S:BZR1-MYC, bic1bic2, 35S:BZR1-MYC/bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured. Data are means ± SD (n>20).",
    "molecules": "eBL, BRZ"
  },
  {
    "caption": "A BR enhances the stability of BIC1 proteins. The 5-d-old 35S:BIC1-YFP transgenic plants were treated with 100 μM cycloheximide (CHX) or co-treated with 100 μM CHX and 1 μM epibrassinolide (eBL). Samples were collected at indicated time points and BIC1-YFP protein levels were analyzed by western blots using anti-GFP antibody. Actin was used to verify equal protein loadings. Intensity of the bands was measured using Adobe Photoshop CS3 Extended program. Three independent experiments were performed with similar results.",
    "molecules": "eBL, epibrassinolide, BR, CHX, cycloheximide"
  },
  {
    "caption": "C Transactivation analysis of BIC1 using a yeast assays. The GAL4 DNA-binding domain (BD) alone was used as the negative control. SD-W, synthetic dextrose medium lacking Trp; SD-WH, synthetic dextrose medium lacking both Trp and His.",
    "molecules": "His, dextrose, Trp"
  },
  {
    "caption": "D ChIP-qPCR analysis showing that the enrichment of BZR1 in the promoter regions of its target genes is dependent on BICs. The 10-d-old 35S:BZR1-MYC and 35S:BZR1-MYC/bic1bic2 seedlings were treated with 1 μM epibrassinolide (eBL) for 3 h and then collected for ChIP assays.",
    "molecules": "eBL, epibrassinolide"
  },
  {
    "caption": "B Transcript levels of BIC1 and PIF4 in transgenic plants containing a chemical-inducible construct pERGW-BIC1. 8-d-old pERGW-BIC1 seedlings were treated with 10 μM estradiol for indicated time points before harvest for RNA extraction and RT-qPCR analysis. The PP2A gene served as internal control. Data are means ± SD (n=3).",
    "molecules": "estradiol"
  },
  {
    "caption": "D. RT-qPCR analysis of expression of four upregulated m6A pathway-related genes in response to AMV infection (three biological replicates). Error bars represent the standard error of the mean (SEM). Asterisks indicate a p < 0.05 applying Student's t-test for ΔCt mean values (n = 3).",
    "molecules": "m6A"
  },
  {
    "caption": "A-D. RNA blots were hybridized to AMV-specific probes to reveal accumulation of viral RNA in double (de) and triple (te) ect2/3/4 (A, B) or ect2/3/5 (C, D) mutants compared to WT plants. A and C show local infection at 3 days post-inoculation (dpi); B and D show systemic infection in aerial tissue at 7 dpi. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Dashed lines indicate non-contiguous samples analyzed on the same membrane. Ethidium bromide staining of ribosomal RNAs (EB) was used as RNA loading control. Error bars indicate standard deviations. Asterisks indicate significant differences from the WT (*: p < 0.05; **; p < 0.01) using the Student's t-test (n = 4). AU, arbitrary units.",
    "molecules": "EB, Ethidium bromide"
  },
  {
    "caption": "A, RNA-blot analysis of AMV systemic infection in plants expressing ECT2WT-mCherry (WT) or ECT2W464A-mCherry (W464A) in the de23 background (A) at 7 days post-inoculation. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. In A, upper and lower panels show independent complementation transgenic lines, and different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).",
    "molecules": "EB, Ethidium bromide"
  },
  {
    "caption": "B. RNA-blot analysis of AMV systemic infection in plants expressing in vir-1 plants (B) at 7 days post-inoculation. Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. In B, dashed line indicates non-contiguous samples that are analyzed on the same membrane. Error bars indicate standard deviations. Asterisks indicate significant differences from the WT (**: p < 0.01) applying Student's t-test (n = 4). AU, arbitrary units.",
    "molecules": "EB, Ethidium bromide"
  },
  {
    "caption": "A, Systemic infection of rosettes at 7 days post-inoculation (dpi) (A) Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. #1 and #2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross. Error bars show standard deviations. AU, arbitrary units. Different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).",
    "molecules": "EB, Ethidium bromide"
  },
  {
    "caption": "B. Systemic infection of rosettes and floral stems at 20 dpi (B). Each panel shows a representative RNA blot displaying AMV RNAs 1-4 (left) and its quantification histogram (right). Ethidium bromide staining of rRNAs (EB) was used as RNA loading control. Genotypes are indicated on the top of each northern blot. #1 and #2 correspond to the progeny of two different quadruple homozygous F2 siblings derived from the alkbh9b x te235 cross. Error bars show standard deviations. AU, arbitrary units. Different letters indicate statistical differences according to Fisher's Least Significant Difference (LSD) (p < 0.05).",
    "molecules": "EB, Ethidium bromide"
  },
  {
    "caption": "C. Protein blot developed with mCherry antisera to show accumulation of ECT2WT-mCherry or ECT2ΔN5-mCherry fusion proteins in pools of the same samples used in panel B. Proteins on the membrane were stained with Coomassie-blue as loading control (lower panel).",
    "molecules": "Coomassie-blue"
  },
  {
    "caption": "A) Caspase 3/7 activation ± 16-hour treatment with varying concentrations of BAM15, DNP, or FCCP (N=3 per condition). 5 μM: BAM15 vs. DNP (P=0.030). 10 μM: BAM15 vs. DNP (P=0.013). 20 μM: BAM15 vs. DNP (P=0.0005), BAM15 vs. FCCP (P=0.036). 40 μM: BAM15 vs. DNP (P=0.018), BAM15 vs. FCCP (P=0.021). 80 μM: BAM15 vs. DNP (P=0.018), DNP vs. FCCP (P=0.004).",
    "molecules": "DNP, BAM15, FCCP"
  },
  {
    "caption": "B) The rate of oxygen consumption (OCR), (C) ratio of oxygen consumption to extracellular acidification (OCR/ECAR), ollowing acute injection of 1 μM BAM15, DNP, or FCCP (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001) (N=6 per condition).",
    "molecules": "DNP, BAM15, FCCP"
  },
  {
    "caption": "D) maximal rate of OCR (BAM15 vs. DNP, P<0.0001; DNP vs. FCCP (P<0.0001) (E) time to maximal uncoupling rate (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001), and (F) mitochondrial respiratory half-life following acute injection of 1 μM BAM15, DNP, or FCCP (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001) (N=6 per condition).",
    "molecules": "DNP, BAM15, FCCP"
  },
  {
    "caption": "G) Representative respirometry plot of intact cells ± 16-hour treatment with 20 μM BAM15 sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)",
    "molecules": "antimycin a, BAM15, FCCP, oligomycin, rotenone"
  },
  {
    "caption": "I) Assessment of routine (R) oxygen flux (P=0.030), leak (L) (P=0.010), OXPHOS (P) and ETC (E) capacity in permeabilized C2C12 cells ± 16-hour treatment with BAM15 (N=4 per condition). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine.",
    "molecules": "tetramethyl-p-phenylenediamine, adenosine diphosphate, ADP, BAM15, glutamate, malate, pyruvate, succinate, Tm"
  },
  {
    "caption": "(J) Citrate synthase activity ± 16-hour treatment with 20 μM BAM15 (N=10 per condition). (K) mtDNA (COX2:18S) ± 16-hour treatment with 20 μM BAM15 (N=6 per condition).",
    "molecules": "BAM15, DNA"
  },
  {
    "caption": "(A) Heat map visualization of top 30 differentially regulated mRNA transcripts. Color represents significant down- (blue) or up-regulated (red) mRNA expressed as a log2 fold change relative to vehicle corrected for the false discovery rate.",
    "molecules": "mRNA"
  },
  {
    "caption": "-D (A) Representative immunoblots and (B) quantitative analysis of AKT (1 μM insulin: P=0.038), (C) AS160 (- insulin: P=0.036, 1 μM insulin: P=0.0038), and (D) AMPK (- insulin: P=0.018) phosphorylation ± 16 hour BAM15 treatment and 15 minute insulin stimulation (N=3 per condition) in C2C12 myotubes.",
    "molecules": "BAM15, insulin"
  },
  {
    "caption": ", F (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation (P=0.0139) ± 16-hour BAM15 treatment (N=3 per condition) in C2C12 myotubes.",
    "molecules": "BAM15"
  },
  {
    "caption": "G) [3-3H]glucose uptake ± 16 hour BAM15 treatment and 30 minute insulin stimulation (N=4 per condition; vehicle vs. vehicle + insulin: P=0.0016, vehicle vs. BAM15: P<0.0001, vehicle vs. BAM15 + insulin: P<0.0001, vehicle + insulin vs. BAM15 + insulin: P=0.0018, BAM15 vs. BAM15 + insulin: P=0.0024)",
    "molecules": "BAM15, glucose, insulin"
  },
  {
    "caption": "H) [1-14C]palmitate oxidation ± 16 hour BAM15 treatment and 5 mM glucose and 1 mM pyruvate (N=6 per condition; no glucose/pyruvate: P<0.0001) in C2C12 myotubes.",
    "molecules": "BAM15, glucose, palmitate, pyruvate"
  },
  {
    "caption": "J) Representative respirometry plot of intact cells ± shAMPK and 16 hour BAM15 treatment in C2C12 myotubes cells sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)",
    "molecules": "antimycin a, BAM15, FCCP, oligomycin, rotenone"
  },
  {
    "caption": "K) basal and ATP-linked respiration, proton leak, maximal uncoupling, and spare respiratory capacity (N=5 per condition). Basal: shEV vs. shEV + BAM15 (P=0.0317), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001). ATP: shEV vs. shAMPK + BAM15 (P=0.0026), shEV + BAM15 vs. shAMPK + BAM15 (P=0.0003), shAMPK vs. shAMPK + BAM15 (P=0.040). Proton leak: shEV vs. shEV + BAM15 (P=0.0052), shAMPK vs. shAMPK + BAM15 (P=0.0394). Maximal: shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001). Spare Capacity: shEV vs. shAMPK (P=0.0028), shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001).",
    "molecules": "ATP, BAM15"
  },
  {
    "caption": "L) [3-3H]glucose uptake ± shAMPK, 16 hour BAM15 treatment, and 30 minute insulin stimulation in C2C12 myotubes (N=3 per condition). No insulin: shEV vs. shEV + BAM15 (P=0.0001). Insulin: shEV vs. shEV + BAM15 (P=0.0142).",
    "molecules": "BAM15, glucose, insulin, Insulin"
  },
  {
    "caption": "-O (M) Representative immunoblots and quantitative analysis of (N) AKT (shEV + insulin: P=0.0128, shAMPK - insulin: P=0.0018, shAMPK + insulin, P=0.0124), and (O) AS160 (shEV - insulin: P=0.0283, shEV + insulin: P=0.0137) phosphorylation ± shAMPK, 16 hour BAM15 treatment, and 15 minute insulin stimulation in C2C12 myotubes (N=3 per condition).",
    "molecules": "BAM15, insulin"
  },
  {
    "caption": "(A) Serum concentrations of BAM15 over a 24-hour period (N=3 animals per time point)",
    "molecules": "BAM15"
  },
  {
    "caption": "(B) peak tissue concentrations of BAM15 (N=6 per tissue).",
    "molecules": "BAM15"
  },
  {
    "caption": "C, D (C) Daily body weight (day 8: P=0.0454, day 9: P=0.0353, day 10: P=0.0251, day 11: P=0.0131, day 12: 0.0038, day 13: P=0.0008, day 14: P=0.0005, day 15: P=0.0003, day 16-21: P<0.0001) and (D) cumulative food intake over the 3-week treatment in CTRL and BAM15 treated animals (N=16 per group).",
    "molecules": "BAM15"
  },
  {
    "caption": "(E) Whole body temperature before (pre), one hour (1 hr Post), and 24 hours (24 hrs Post) after intraperitoneal injection of corn oil, vehicle (corn oil and saline), 0.1, 0.5, and, 1 mg BAM15 per kg of body weight (N=11 per group),",
    "molecules": "corn oil, BAM15"
  },
  {
    "caption": "Whole body temperature (F) daily during chronic BAM15 treatment (N=8 per group).",
    "molecules": "BAM15"
  },
  {
    "caption": "G, H (G) Change in fat mass (%) (P<0.0001) and (H) lean mass (%) (P<0.0001) from baseline to 3-weeks in CTRL and BAM15 treated animals (N=8 per group).",
    "molecules": "BAM15"
  },
  {
    "caption": "(I) Fasting plasma glucose (N=8 per group; P=0.007) and (J) insulin (N=7 per group; P=0.0024) after 3 weeks of treatment in CTRL and BAM15 treated animals.",
    "molecules": "BAM15, glucose, insulin"
  },
  {
    "caption": "(K) Plasma glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)",
    "molecules": "glucose"
  },
  {
    "caption": "(L) total area under the curve glucose after 3-weeks in CTRL and BAM15 treated animals (N=8 per group; P=0.0106).",
    "molecules": "BAM15, glucose"
  },
  {
    "caption": "M-O (M) Oxygen consumption rates (N) energy expenditure (daily: P=0.0357, dark phase: P=0.030), and (O) respiratory exchange ratio (daily: P=0.0424, dark phase: P=0.0016) measured by continuous high-resolution indirect calorimetry after 3-weeks of CTRL or BAM15 treatment (N=8 per group).",
    "molecules": "BAM15"
  },
  {
    "caption": "(P) Fecal energy density (% recovered in feces) after 3-weeks of CTRL or BAM15 treatment (N=8 per group).",
    "molecules": "BAM15"
  },
  {
    "caption": "(A) Change in body weight (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P<0.0001), (B) fat mass (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0041, BAM15 vs. CR: P=0.043), and (C) lean mass (CTRL vs. BAM15: P=0.0014, CTRL vs. CR: P<0.0001, BAM15 vs. CR: P=0.0047) from baseline to 2-weeks in CTRL, BAM15, and CR treated animals",
    "molecules": "BAM15"
  },
  {
    "caption": "(D) daily food intake (CTRL vs. CR: P=0.0092, BAM15 vs. CR: P=0.0175) in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).",
    "molecules": "BAM15"
  },
  {
    "caption": "(E) Blood glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)",
    "molecules": "glucose"
  },
  {
    "caption": "(F) total area under the curve glucose (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P=0.003, BAM15 vs. CR: P=0.0261) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).",
    "molecules": "BAM15, glucose"
  },
  {
    "caption": "(G) Plasma insulin concentrations at baseline (PRE), and 0 (CTRL vs. BAM15: P=0.0154, CTRL vs. CR: P=0.0425) and 120 minutes (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0211, BAM15 vs. CR: P=0.0264) after injection of glucose after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8)and",
    "molecules": "BAM15, glucose, insulin"
  },
  {
    "caption": "(H) Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (CTRL vs. BAM15: P=0.0259, CTRL vs. CR: P=0.040) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).",
    "molecules": "BAM15, Insulin"
  },
  {
    "caption": "(A) Representative H&E stained liver, adipose tissue, and skeletal muscle sections from CTRL and BAM15 treated animals. Scale bars (black) = 100 μm.",
    "molecules": "BAM15"
  },
  {
    "caption": "(B) Representative immunohistochemical analysis of pancreatic insulin (red) content counterstained with diaminobenzidine (blue). Scale bars (black) = 100 μm.",
    "molecules": "diaminobenzidine, insulin"
  },
  {
    "caption": "C-F (C) Quantitative analysis of liver steatosis (μm2) (N=6 per group; P<0.0001), (D) gastrocnemius muscle cross sectional area (μm2)(CTRL N=7, BAM15 N=8), (E) pancreatic insulin positive area (% of total area) (N=6 per group; P=0.0420), (F) and β-cell mass (N=4 per group; P=0.0005).",
    "molecules": "BAM15, insulin"
  },
  {
    "caption": "(G-I) Size (area; μm2) and distributional (number) analysis of (G) iWAT, (H) gWAT, and (I) BAT from CTRL and BAM15 treated animals (N=8 per group).",
    "molecules": "BAM15"
  },
  {
    "caption": "A-D (A) Plasma concentrations of non-esterified fatty acids (NEFA) (CTRL N=7, BAM15 N=8, P=0.0073), (B) leptin (N=8 per group, P<0.0001), (C) GDF15 (N=7 per group, P=0.035), and (D) FGF21 (N=7 per group, P=0.0026) after 3-weeks of CTRL or BAM15 treatment.",
    "molecules": "BAM15, NEFA, non-esterified fatty acids, leptin"
  },
  {
    "caption": "E, F (E) Total (basal: P=0.015) and (F) complete (basal: P=0.0137) oxidation of 200 μM [1-14C]palmitate to CO2 from mixed gastrocnemius muscle homogenates (N=7 per group).",
    "molecules": "CO2, palmitate"
  },
  {
    "caption": "(G) Routine (R) oxygen flux, leak (L), OXPHOS (P) and ETC (E) capacity in permeabilized mixed gastrocnemius fibers (N=6 per group). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine, PalM: palmitoylcarnitine and malate, Oct: octanoylcarnitine, and Dur: duroquinol.",
    "molecules": "Dur, tetramethyl-p-phenylenediamine, adenosine diphosphate, ADP, duroquinol, glutamate, malate, Oct, octanoylcarnitine, palmitoylcarnitine, pyruvate, succinate, Tm"
  },
  {
    "caption": "B. HZZ-OTOFop in complex with wild-type or mutant TRC40 was incubated with RM in the presence or absence of ATP and membrane insertion was monitored by opsin-specific immunoblot.",
    "molecules": "ATP"
  },
  {
    "caption": "C. HZZ-OTOFop in complex with wild-type TRC40 was incubated in the presence of RM, trypsin-treated rough microsomes (TRM) or in the presence of WRBcc or CAMLcyt. Membrane insertion was monitored by opsin-specific immunoblot.",
    "molecules": "trypsin"
  },
  {
    "caption": "A. Ca2+ current-voltage relationship of P14-P17 IHCs of Wrbfl/fl:CreA (n = 17 IHCs) and Wrb+/+:CreA (n = 15 IHCs) mice in 2 mM extracellular [Ca2+] showed no change in amplitude or voltage-dependence of the Ca2+ current. Data are represented as mean ± SEM.",
    "molecules": "Ca2+"
  },
  {
    "caption": "B. Representative Ca2+ current (top) and Cm changes (bottom) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 20-ms step depolarizations to -14 mV (the potential eliciting the maximum Ca2+ current).",
    "molecules": "Ca2+"
  },
  {
    "caption": "C. Representative Ca2+ current (top) and Cm changes (bottom; finite impulse response filtered) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 100 ms step depolarizations to -14 mV.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D. Exocytic ΔCm (top) and corresponding Ca2+ current integrals, QCa (bottom) for various depolarization durations. Data are grand averages of the cells' means. Exocytic ΔCm was reduced in the knockout from 20 ms onwards, indicating reduced sustained exocytosis. Data from OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).",
    "molecules": "Ca2+"
  },
  {
    "caption": "E. ΔCm/QCa2+ ratio indicates a lower efficacy of Ca2+ influx in driving exocytosis in Wrbfl/fl:CreA IHCs, for stimuli of 20 ms or longer. Data represents means ± SEM; *p<0.05; **p<0.01; ***p<0.001",
    "molecules": "Ca2+"
  },
  {
    "caption": "B) Left panel: representative elongating replication forks (stable and unstable). Quantification of the average IdU/CldU ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Percentage of highly unstable forks (ratio above 2) are indicated. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. (**** p< 0.0001). At least 450 ratios were recorded from 6 experimental replicates.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "C) The length of both CldU labels surrounding IdU tracks (newly fired origins) was measured before calculating a ratio representing symmetry around new origins. Quantification of the average positive ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. 55 and 75 ratios were recorded in the Mybl2+/+ and Mybl2Δ/Δ respectively from 3 replicates ( **** p< 0.0001).",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "E) Representative images for comets in Mybl2+/+ and Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT (20x magnification). (Scale bar 50μm).",
    "molecules": "CPT"
  },
  {
    "caption": "F) Quantification of the mean and distributions of olive tail moments in Mybl2+/+ and Mybl2Δ/Δ untreated and CPT-treated. Y-axis was cut to change scale and display large values. Statistical analysis using Mann Whitney U tests. At least 300 comets were measured for each group from 5 experimental replicates (**** p< 0.0001).",
    "molecules": "CPT"
  },
  {
    "caption": "G) Frequency of EdU positive cells with over 6 53BP1 foci in Mybl2+/+, Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT. Error bars represent SEM. Statistical analysis using unpaired two-tailed t-test. At least 200 EdU positive nuclei were analysed from 4 experimental repeats.",
    "molecules": "EdU, CPT"
  },
  {
    "caption": "A) Cells were treated for 1.5 hours with 5µM CPT, before sequential addition of IdU and CldU for 20 minutes each. Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with CPT (n=3 independent experiments; Error bars indicate SEM). Statistical analysis using two- tailed unpaired t-test.",
    "molecules": "CldU, IdU, CPT"
  },
  {
    "caption": "(B) Distribution of replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC treated or not with CPT as above. Statistical analysis was carried out using unpaired Mann Whitney U tests. A minimum of 200 forks were quantified for each of the genotypes and conditions shown from 3 independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).",
    "molecules": "CPT"
  },
  {
    "caption": "C) ESCs were treated with or without CPT for 4 hours before 0.1ug/ml colcemid was added during the final 3.5 hours of treatment. Representative image of H3-pS10 positive cells (turquoise). (Scale bar 10μm). Frequency of H3-pS10 positive cells in Mybl2+/+ and Mybl2Δ/Δ ESCs. Data from three independent experiments. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (*p < 0.05; **p< 0.01; ns= no significant).",
    "molecules": "colcemid, CPT"
  },
  {
    "caption": "D) P-CDK1 (Tyr15), CDK1 and Beta-actin expression levels of Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment analyzed by immunoblotting. Bar graph represents average band density of P-CDK1 in Mybl2Δ/Δ ESCs relative to loading control and relative to the MYBL2+/+ from 6 repeats. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (**p< 0.01).",
    "molecules": "CPT"
  },
  {
    "caption": "A) Immunoblot showing the levels of phosphorylated CHK1 (Ser345) and GAPDH in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (2.5µM CPT for 4 hours).",
    "molecules": "CPT"
  },
  {
    "caption": "B) Immunoblot showing the levels of P-CHK1 (Ser345) and CHK1 in irradiated Mybl2+/+, and Mybl2Δ/Δ ESCs with or without 3 hours inhibitor treatment: ATR (AZ20, 5µM) and ATM (Ku60019, 10µM). Cells were also exposed to 5Gy irradiation before the final hour of inhibitor treatment. Bar graph (lower panel) represents average band density of P-CHK1 in Mybl2Δ/Δ ESCs relative to CHK1 and relative to untreated cells. Mybl2+/+ from two independent repeats and Mybl2Δ/Δ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (*p< 0.05).",
    "molecules": "AZ20, Ku60019"
  },
  {
    "caption": "C) Cells were treated for 1.5 hours with 5µM AZ20, before sequential addition of IdU and CldU for 20 minutes each. Dot plot representing the effect of ATR inhibition upon replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC. Statistical analysis of distributions was carried out using unpaired Mann Whitney U tests. A minimum of 240 forks were quantified for each of the genotypes and conditions shown from 2 independent repeats (**** p< 0.0001)",
    "molecules": "CldU, IdU, AZ20"
  },
  {
    "caption": "D) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with ATR inhibitor AZ20. At least 500 replication structures were counted per treatment from two independent repeats. Statistical analysis using two- tailed unpaired t-test.(*p< 0.05; ns= no significant).",
    "molecules": "AZ20"
  },
  {
    "caption": "E) Distribution and average of IdU/CldU fork ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATR inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 150 ratios were calculated per treatment group from two independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "C) Distribution of the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U. At least 260 ratios were calculated per treatment group from 4 separate experiments ( **** p< 0.0001).",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "E) Frequency of anaphases positive for UFB positive cells (based on ERCC/PICH positive immunostaining) for Mybl2+/+, the Mybl2Δ/Δ and the Mybl2+/+ treated for 2 hours with ATM inhibitor (KU60019). Data represents at least 100 anaphases from each group from 2 experimental repeats. Statistical analysis was performed using an unpaired two-tailed t-test.",
    "molecules": "KU60019"
  },
  {
    "caption": "F) Immunoblot showing the levels of phosphorylated ATM (Ser1987) and ATM in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (10µM CPT for 2 hours). Bar graph (lower panel) represents average band density of P-ATM in Mybl2Δ/Δ ESCs treated with CPT relative to ATM and relative to wild type CPT treated cells from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test.",
    "molecules": "CPT"
  },
  {
    "caption": "G) Distribution plot of replication speed in Mybl2+/+and ATM-/- ESC treated or not for 90 minutes with 10µM KU60019 (ATMi), before sequential addition of IdU and CldU for 20 minutes each. Statistical analysis was carried out using the Mann Whitney U test. At least 200 replication forks were counted from 3 separate experiments for wild type and ATM-/- and two separate experiments for ATM-/- treated with ATM inhibitor . ( **** p< 0.0001)",
    "molecules": "CldU, IdU, KU60019"
  },
  {
    "caption": "B and C) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs treated with mirin. Statistical analysis was performed using the Mann Whitney U test. n=3 experimental replicates. The value of the mean fork length is indicated above arrow ( **** p< 0.0001)",
    "molecules": "mirin"
  },
  {
    "caption": "E) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with Mirin. At least 450 replication structures were counted per treatment from 3 independent repeats. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test.",
    "molecules": "Mirin"
  },
  {
    "caption": "F) Distribution and the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without MRE11 inhibitor treatment. The length of both incorporated labels for each elongating fork was measured in µm and the positive ratio was calculated. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 260 ratios were calculated per treatment group from 3 separate experiments.( **** p< 0.0001)",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "C ) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with the indicated inhibitors: CDC7 inhibitor (PHA-767491) and/or ATM inhibitor (Ku60019). At least 300 replication structures were counted per treatment. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test (*p < 0.05).",
    "molecules": "Ku60019, PHA-767491"
  },
  {
    "caption": "E ) Fork stability (average IdU/CldU ratio) of Mybl2+/+ ESC and Mybl2Δ/Δ ESCs treated with the indicated inhibitors alone or in combination. At least 240 ratios were calculated for non-ATM inhibitor treated groups, and a minimm of 130 for ATM inhibitor treated groups. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using Mann Whitney U test (**** p< 0.0001). Data for Mybl2+/+ ESCs treated with ATM inhibitor alone or in combination with CDC7 inhibitor, was collected from two independent experiments. For the rest of the conditions, a minimum of three independent repeats was performed.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "C, Survival assays against substrates of QacA showed loss of activity against monovalent cationic antibacterials (TPP: Tetraphenylphosphonium, Et: Ethidium), while still retaining partial activity against divalent cations (Dq: Dequalinium, Ch:Chlorhexidine). EV: pBAD empty vector. n = 3 for biological replicates. CFU indicates colony forming units. Image color was inverted from black to white background to enhance clarity.",
    "molecules": "Ch, Chlorhexidine, Dequalinium, Dq, Et, Ethidium, Tetraphenylphosphonium, TPP"
  },
  {
    "caption": "G, Colocalization of QacAWT or QacAD411N (tagged with GFP) on E. coli spheroplasts (stained with DAPI for viability) with ICabs (labelled with NHS-Rhodamine) was screened against ICab A4 (left) and ICab B7 (right). n = 10-30 spheroplasts were imaged for each sample.",
    "molecules": "NHS-Rhodamine, DAPI"
  },
  {
    "caption": "C, Whole cell ethidium efflux assay with overexpressed EL7 deletion construct in the background of wild type and D411N mutant. n = 3 for independent replicates, one of which is shown here. Error bars represent S.E.M. from six technical replicates.",
    "molecules": "ethidium"
  },
  {
    "caption": "D, Survival assays of QacAΔEL7 and QacAD411N/ΔEL7 mutants in the presence of 10 μM Tetraphenylphosphonium (TPP) and 16 μM Chlorhexidine (Ch). Data from one representative biological replicate is shown here. Image colors were inverted for improved clarity.",
    "molecules": "Ch, Chlorhexidine, Tetraphenylphosphonium, TPP"
  },
  {
    "caption": "C Whole cell ethidium efflux assay done with E.coli JD838 cells expressing QacAWT or QacA5A. Empty vector (EV) was used as a control for the experiment. A graph of one independent replicate is shown with error bars representing S.E.M. of 6 technical replicates.",
    "molecules": "ethidium"
  },
  {
    "caption": "D A single representative replicate of survival assays shown for the same cells spotted in 10-fold dilution series on 12 μM TPP and 16 μM Ch. Image colors were inverted for clarity in the figure.",
    "molecules": "Ch, TPP"
  },
  {
    "caption": "Whole cell-based ethidium efflux assay done with E.coli JD838 cells expressing QacAWT, its EL7 deletion construct (QacAΔEL7), and the chimeric mutants of LfrA and SmvA. All assays were done in at least 2 independent replicates. One representative replicate shown with data normalized to highest value in each trace. Error bars depict S.E.M. for six technical replicates.",
    "molecules": "ethidium"
  },
  {
    "caption": "I Whole cell-based ethidium efflux assay done with E.coli JD838 cells expressing QacAWT, its EL7 deletion construct (QacAΔEL7), and the chimeric mutants of LfrA and SmvA. All assays were done in at least 2 independent replicates. One representative replicate shown with data normalized to highest value in each trace. Error bars depict S.E.M. for six technical replicates.",
    "molecules": "ethidium"
  },
  {
    "caption": "J Everted vesicles-based assays of chimeric and EL7 deletion constructs of QacA. Traces represent mean values of three replicates performed on vesicles made with a single batch of cells for each construct. Timepoints indicated as 1-3 above the traces indicate addition of 100 μM ATP, 1 mM TPP and 2 μM Nigericin respectively. One representative shown for data obtained from biological triplicates. Error bars represent SEM for technical triplicates from one of three biological replicates.",
    "molecules": "ATP, Nigericin, TPP"
  },
  {
    "caption": "(C) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH-GFP in the one day old flies of the indicated genotypes (n=10).",
    "molecules": "PIP2"
  },
  {
    "caption": "(D) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.",
    "molecules": "PIP2"
  },
  {
    "caption": "(E) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.",
    "molecules": "PIP2"
  },
  {
    "caption": "(F) Quantification of the mean fluorescence intensity of the PIP2 probe PH-GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes (n=10 biological replicates).",
    "molecules": "PIP2"
  },
  {
    "caption": "(B) Confocal images showing the localization of exogenously expressed dEsyt::mCherry protein expressed using the eye specific Rh1-Gal4 in one day old dark reared flies. Rh1-Gal4 is shown as a control. A single ommatidium is shown. Scale bar: 5 µm. Phalloidin marks F-actin staining and highlights rhabdomeres R1-R7.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "(D, E) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are (D) 1 day old-dark reared and (E) 6 days old- exposed to constant illumination. For (D) and (E) RDGB visualized using an antibody against the endogenous protein. Rhabdomeres are outlined using phalloidin which marks F-actin. Scale bar: 5 µm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "D. Blood lactate to glucose ratio at P200 (n=8/group) Bar graphs represent mean±SD The dat were analyzed using Kruskal-Wallis and Mann-Whitney U tests for selected comparison. Significant differences between groups (p value) are indicated on graphs",
    "molecules": "glucose, lactate"
  },
  {
    "caption": "A. Hematoxylin-eosin-stained cross sections of heart and kidney at P200.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "Sirius Red staining (C) for fibrosis in liver, heart and kidney at P200",
    "molecules": "Sirius Red"
  },
  {
    "caption": "H, I. 24 h excretion of (H) albumin (I) creatinine in urine at P200 (n=4/group) Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s tes and Mann-Whitney U tests (for graph I)",
    "molecules": "creatinine"
  },
  {
    "caption": "C-F. Concentrations of the TCA cycle intermediates (C) fumarate and (D) malate, (E) adenylate energy charge and (F) NADH/NAD+ ratio (n=5/group) Bar graph represent mean±S Statistics for graph one-way ANOVA followed by Tukey\"s",
    "molecules": "adenylate, NAD+, fumarate, malate, NADH"
  },
  {
    "caption": "(G) Proline concentratio in heart tissue at P200 (# below detection limit Bar graph represent mean±S",
    "molecules": "Proline"
  },
  {
    "caption": "mRNA expression of proline synthesis-related genes (n=6/group)) (H) Aldh18a1 and (I) Pycr1 at P150 box plots of mRNA expressions represent quartiles, minimum, and maximum value (relative fold change "FC&qu Statistics for graph one-way ANOVA followed by Tukey"s",
    "molecules": "proline"
  },
  {
    "caption": "H. n-Propyl gallate-sensitive AOX mediated cI and cII-linked state 3 respiration (n=7/group, Mann-Whitney U test) Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g",
    "molecules": "n-Propyl gallate"
  },
  {
    "caption": "CI-linked (I state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) We used malate, pyruvate and glutamate to generate NADH for the cI Bar graphs represent mean±SD Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g",
    "molecules": "glutamate, malate, NADH, pyruvate"
  },
  {
    "caption": "cI- and cII-linke state 3 respiration in isolated heart, kidney and liver mitochondria (n=8/group) we added the CII substrate, succinate, to obtain CI&CII-linked respiration in presence of saturating ADP. Bar graphs represent mean±SD. Statistics: one-way ANOVA followed by Tukey"s test. Significant differences between groups (p value) are indicated on g",
    "molecules": "ADP, succinate"
  },
  {
    "caption": "A. Amplex Red-based detection of H2O2 emission, a surrogate for respiratory chain-derived ROS, during cI and cII-linked state 3 respiration (n=6/group) Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey"",
    "molecules": "Amplex Red, H2O2, ROS"
  },
  {
    "caption": "B. Gene expression related to ROS scavenging",
    "molecules": "ROS"
  },
  {
    "caption": "C. Total glutathion in heart and liver tissue (n=5/group Bar graphs represent mean±SD Statistics one-way ANOVA followed by Tukey\"",
    "molecules": "glutathion"
  },
  {
    "caption": "D. Urinary isoprostanes per creatinine as a measure of oxidative stress (n=4/group) Bar graphs represent mean±SD Statistics: Mann-Whitney U test (graph D",
    "molecules": "creatinine, isoprostanes"
  },
  {
    "caption": "E and F. Representative images (D) of tissue sections immunostained with an antibody against 4-hydroxynonenal (4-HNE), a lipid peroxidation marker. (E) Quantification of the 4-HNE immunostaining.",
    "molecules": "4-HNE, 4-hydroxynonenal"
  },
  {
    "caption": "A. Heatmap visualization of the expressions of selected nitric oxide (NO)-related genes.",
    "molecules": "nitric oxide"
  },
  {
    "caption": "B-E. Gene expression of Ca2+ channel gene (B) ryanodin receptor, RYR2, (C) sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2 or ATP2A2), (D) phospholamban (PLN), and (E) endothelial nitric oxide synthase NOS3 (n=6/group) Box plots for mRNA expressio represent quartiles, minimum and maximum value (relative fold change "FC&qu Statistics: one-way ANOVA followed by Tukey"s test for Significant differences between groups (p value) are indicated on graphs",
    "molecules": "Ca2+, ryanodin"
  },
  {
    "caption": "I. Cardia citrulline to arginine ratio (n=5/group) Bar graph represent mean±SD",
    "molecules": "arginine, citrulline"
  },
  {
    "caption": "J. Cardiac protein nitrotyrosine quantified from Western blots",
    "molecules": "nitrotyrosine"
  },
  {
    "caption": "K. Nitrite concentration in heart tissue (n=4/group) Bar graph represent mean±SD",
    "molecules": "Nitrite"
  },
  {
    "caption": "(C) Decreased levels of free carnitine and modified  carnitine species in PKM2  KO cell lines as compared with their parental counterparts (n=4). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.",
    "molecules": "carnitine"
  },
  {
    "caption": "(E) Increased acetyl-CoA  in PKM2 KO  MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=4). (F) Decreased total  cholesterol in PKM2  KO MCF7  and  MDA-MB 231cells as compared with their parental counterparts (n=3). ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.",
    "molecules": "acetyl-CoA, cholesterol"
  },
  {
    "caption": "(B) Western blot  analysis of PKM2, PKM1, NRF1,  TMEM33, RNF5 and SCAP levels in MEF  (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course  experiment.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(H)  RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in  293T cells. 293T cells were stably transfected with shCtrl or shRNF5 constructs, then the indicated plasmids were co-transfected into shCtrl or shRNF5  expressing  293T cells for 18 hours followed by MG-132 treatment for 8 hours. FLAG  M2  beads were used for pull-down.",
    "molecules": "MG-132"
  },
  {
    "caption": "(F, G) Significant decrease of total plasma cholesterol  (F) and  LDL-C  (G) levels in  TMEM33-/- and +/-  mice relative to WT counterparts (n>=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. n.s denotes no significance.",
    "molecules": "cholesterol, LDL-C"
  },
  {
    "caption": "(E) The growth curves of py8119 allografts treated with vehicle and TEPP-46  (50mg/kg) for 17 days in  mice (n=7). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.",
    "molecules": "TEPP-46"
  },
  {
    "caption": "(B) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium.",
    "molecules": "BafA1, Bafilomycin A1, E64d, pepstatin"
  },
  {
    "caption": "(G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.",
    "molecules": "Baf A1, BafA1, E64d, pepstatin"
  },
  {
    "caption": "(A) Proteolysis of long‐lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [3H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid‐soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments.",
    "molecules": "[3H] leucine, Leucine"
  },
  {
    "caption": "(C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3).",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).",
    "molecules": "BafA1, Bafilomycin A1"
  },
  {
    "caption": "(D-I) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F-H), followed by a second transfection overnight with corresponding siRNA and GFP‐WIPI‐1. Cells were incubated for 4 h in full (D, G) or starvation media (E, F, H) in presence (F) or absence (E-H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of WIPI‐1 puncta per cell (I). Bars, s.e.m. (n=3 independent experiments, with an average of 30 cells per experiment). *P0.05, **P0.01 (t‐test).",
    "molecules": "wortmannin"
  },
  {
    "caption": "(L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot.",
    "molecules": "BafA1, rapamycin"
  },
  {
    "caption": "F Distance vs.  time curves  for force-induced H-bond formation between indicated residues within NKG2D-MICA binding  interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.",
    "molecules": "H-bond, H-bonds, residues"
  },
  {
    "caption": "Mapping of open chromatin as well as active and repressive histone modifications at upstream regulatory regions of the Prdm1 locus in pro-B cells and plasmablasts. Open chromatin was determined by ATAC-seq (Buenrostro et al., 2013), and active (H3K4me2, H3K4me3, H3K9ac) and repressive (H3K27me3) histone marks were mapped by ChIP-seq analysis in ex vivo sorted pro-B cells (ATAC-seq and H3K27me3, this study), short-term in vitro cultured pro-B cells (H3K4me2, H3K4me3, H3K9ac) (Revilla-i-Domingo et al., 2012) and in vitro LPS-induced plasmablasts (Minnich et al., 2016) (Table EV4). The indicated upstream regions were previously shown by 3C-analysis to interact with the Prdm1 promoter (Wöhner et al., 2016). The mm9 genomic coordinates of mouse chromosome 10 and the respective positions of two human SNPs (rs658431 and rs548234) are indicated. RPM, reads per million mapped sequence reads",
    "molecules": "LPS"
  },
  {
    "caption": "RT-qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre-B cells and in in vitro differentiated plasmablasts (PB) of the indicated genotypes. Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS. The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild-type pre-B cells (set as 1). Nascent transcripts were PCR-amplified with primers located in the indicated introns (Table EV3). Statistical data are shown as mean value with SEM and were analyzed by the Student"s t-test; *P < 0.05, **P < 0.01. Each dot corresponds to one",
    "molecules": "LPS"
  },
  {
    "caption": "Blimp1 binding and regulation of the commonly repressed target genes Sell (CD62L) and B3gnt5. The ChIP-seq data (left) were obtained with Prdm1ihCd2/+ pro-B cells (this study) and Prdm1Bio/Bio Rosa26BirA/BirA plasmablasts (PB; Minnich et al., 2016). The RNA-seq data (right) were determined in pro-B and pre-B cells of the Prdm1ihCd2/+ (red) and wild-type (gray) genotype and in Blimp1-deficient (Prdm1Gfp/∆, blue) and wild-type (grey) pre-plasmablasts (Pre-PB; Minnich et al., 2016). Cell types expressing Blimp1 (+) are indicated",
    "molecules": "Gfp"
  },
  {
    "caption": "T cell-dependent immune responses. Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2 months were immunized with NP-KLH (in alum) and analyzed at day 14 after immunization by ELISPOT assa The number of anti-NP-IgM antibody-secreting cells (ASCs) in the spleen was determined by ELISPOT assay (C) using NP24-BSA-coated plates for detecting cells secreting total anti-NP-IgM antibodies. ELISPOT results are shown as representative pictures (left) or absolute ASC numbers (right) (C) Dot plots display the serum immunoglobulin titers of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 antibody",
    "molecules": "NP, alum"
  },
  {
    "caption": "T cell-dependent immune responses. Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2 months were immunized with NP-KLH (in alum) and analyzed at day 14 after immunization b ELISA The serum titers of anti-NP-specific IgM, IgG1 and IgG2b antibodies were analyzed by ELISA using NP7-BSA- or NP24-BSA-coated plates for detecting high-affinity IgG1 or total IgM, IgG1 and IgG2b antibodies, respectivel Dot plots display the serum immunoglobulin titers of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice. NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 antibody, whereas IgM and IgG2b amounts are indicated as relative units (RU) by setting the WT data to 1",
    "molecules": "NP, alum"
  },
  {
    "caption": "Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization. Bar graphs display absolute numbers of GC B cells detected in the spleen of both genotype The absence of Bcl6 expression in non-GC B cells (black) is shown as reference",
    "molecules": "NP"
  },
  {
    "caption": "Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Cell surface expression of hCD2 and intracellular staining of Bcl6 are shown for Prdm1ihCd2/+ (red) and wild-type (gray) GC B cell The absence of Bcl6 expression in non-GC B cells (black) is shown as reference",
    "molecules": "NP"
  },
  {
    "caption": "Flow cytometric analysi in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Flow cytometric analysis of splenic TFH cell Bar graphs indicate absolute numbers of TFH cell in hCD2lo (black) and hCD2hi (blue) Prdm1ihCd2/+ as well as wild-type (gray) TFH cells",
    "molecules": "NP"
  },
  {
    "caption": "Flow cytometric analysi in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Flow cytometric analysis of splenic TFH cell histograms display cell surface expression of hCD2 and intracellular staining of Bcl6 (H) in hCD2lo (black) and hCD2hi (blue) Prdm1ihCd2/+ as well as wild-type (gray) TFH cells",
    "molecules": "NP"
  },
  {
    "caption": "Efficient in vitro plasmablast differentiation of immature B cells. Immature B cells sorted from the bone marrow of Prdm1ihCd2/+ (red) and wild-type (gray) mice were stimulated with CpG oligodeoxynucleotides or LPS for 60 hours followed by flow cytometric analysis of CD138+CD22lo plasmablasts (PB). Bar graphs (right) summarize the plasmablast data of the CpG and LPS stimulation experiments",
    "molecules": "CpG, CpG oligodeoxynucleotides, LPS"
  },
  {
    "caption": "ELISA detection of Igκ-containing antibodies in the supernatants of immature B cells at 60 h of CpG or LPS stimulation",
    "molecules": "CpG, LPS"
  },
  {
    "caption": "Presence of IgG antibodies detecting dsDNA, cardiolipin, SSA (Ro-52) and SSB (La) in the serum of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2, 4 and 12 months. The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units (AU). The serum of 6-month-old Faslgld/gld mice was used as positive control",
    "molecules": "cardiolipin, dsDNA"
  },
  {
    "caption": "Representative images of anti-nuclear antibody (ANA) staining obtained with serum from 12-month-old Prdm1ihCd2/+ or wild-type mice (right), as detected by indirect immunofluorescence assay using HEp-2 cells and an Alexa488-conjugated anti-mouse IgG detection antibody. The serum of Faslgld/gld mice was used as positive control. The presence of ANA-IgG antibodies was analyzed for 22 Prdm1ihCd2/+ and 12 wild-type mice, as summarized in the pie-chart (right)",
    "molecules": "Alexa488"
  },
  {
    "caption": "Periodic acid-Schiff (PAS) staining of paraffin-embedded kidney sections of Prdm1ihCd2/+ and wild-type mice at 12 months of age. The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods. Representative pictures of glomeruli of Prdm1ihCd2/+ kidneys with different pathology scores are shown to the left, and dot plots with average scores for male and female mice (Table EV2) are shown to the right. Arrows denote intracapillary hyaline "thrombi", asterisks indicate mesangial sclerosis with obscured capillary loops, and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture. Statistical data (A,C) are shown as mean value with SEM and were analyzed by the Mann-Whitney test (A) or the Student"s t-test (C); *P < 0.05, **P < 0.01, ***P < 0.001. Each dot correspon",
    "molecules": "Periodic acid, Schiff"
  },
  {
    "caption": "a. Lipid droplet time course. Representative photomicrographs  showing lipid droplet abundance in ARPE19 cells treated with DFP starting at 7 h and maximal at 24 h. Neutral lipids  were revealed by  BODIPY 493/503. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in a cell treated with DFP for 24 h. Arrows highlight LD accumulation. Scale bars =5 μm.",
    "molecules": "Hoescht33342, BODIPY 493/503, DFP, Neutral lipids"
  },
  {
    "caption": "b. Mitophagy time course. Representative photomicrographs  showing time course of DFP treatment in mitophagy  reporter cells (human ARPE-19 cells with stable expression of  mito-QC). Mitochondrial networks are visible in yellow and mitolysosomes as red-only puncta signifying mitophagy. Nuclei are counterstained with Hoescht33342. Mitophagy  appears maximal after 24 h of DFP treatment, with little difference between control and 7 h conditions. Inset shows details of mitolysosomes within the mitochondrial network of a highly mitophagic  cell following DFP treatment. Arrows highlight mitolysosomes. Scale bar = 5 μm.",
    "molecules": "Hoescht33342, DFP"
  },
  {
    "caption": "c. Lipid droplets accumulate at mitochondria when cells are treated with DFP. Representative photomicrographs  showing lipid droplet and mitochondria in ARPE19 cells treated with DFP at 7 h and at 24 h. Neutral lipids  were revealed by BODIPY 493/503 and mitochondria by Mitotracker. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in close proximity to mitochondria in cells treated with DFP for 24 h. Scale bar = 5 μm.",
    "molecules": "Hoescht33342, BODIPY 493/503, DFP, Mitotracker, Neutral lipids"
  },
  {
    "caption": "f. Quantification of  lipid droplet and mitochondria proximity over time. n=2 with at least 50 cells quantified per biological replicate for each condition. Ratios between Control and 24h DFP; P<0.05.",
    "molecules": "DFP"
  },
  {
    "caption": "d. DFP-induced Lipid droplet  accumulation is dependent on DGAT1. Representative photomicrographs of ARPE19 cells treated with DFP (1mM) for 7 h followed by treatment with LD inhibitors (5 μM) as indicated. Cells were fixed after 24 h of DFP treatment. Neutral lipid droplets  were stained by BODIPY493/503  and imaged by confocal microscopy after 24 h of DFP treatment ('i' denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Scale bar = 5 μm.",
    "molecules": "BODIPY493/503, DFP, Neutral lipid"
  },
  {
    "caption": "a. ARPE-19 ULK1 CRISPR  KO cells with/without re-introduction of FLAG-ULK1 were treated with DFP  for 24h as indicated. Cells were fixed and stained  with  BODIPY493/503 to visualise  lipid droplets (green) or DAPI to visualise nuclei (blue). Scale bar, 10 μm.",
    "molecules": "BODIPY493/503, DAPI, DFP"
  },
  {
    "caption": "f. Representative   confocal photomicrographs showing the pattern of endogenous mitochondrial NIX  in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Samples were  immunolabelled  with primary antibodies to NIX (green) and ATPB (red). Scale bar = 2 μm. g. Representative  confocal  photomicrograpghs of Cathepsin-reactive endolysosomes in human ARPE-19  cells treated with with DFP ±DGAT1i/2i. Inset shows examples of lysosomal phenotypes. Scale bar = 5 μm. h. Colocalization  quantitation for experiments shown in 6g. One-way ANOVA with Bonferroni's post hoc test. ****P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. i. Colocalization quantitation for ULK1/WIPI2 immunolabelling in  human ARPE-19  cells treated with  DFP ±DGAT1i/2i. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. j. Quantitation of lysosome  positioning in  DFP ±DGAT1i/2i. One-way ANOVA with Bonferroni's post hoc test. ****=P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates.",
    "molecules": "DFP"
  },
  {
    "caption": "C. Demyelinating lesions in chimeric mice at 3 dpl. Ci. Demyelination was induced by injecting LPC in the spinal cord white matter. Cii-Civ. Co-immunolabeling for GFP, MBP, and a mixture of CD11b/CD68, showing graft-derived macrophages in the lesion (identified by lack of MBP staining). White lines indicate lesion borders. Scale bar 20 µm. MBP-myelin basic protein; GFP-green fluorescent protein.",
    "molecules": "LPC"
  },
  {
    "caption": "B-G. Lesions at 7 dpl in GFP (B,E) and Sema3F (C,F) mice. B-C. Co-labeling for PDGFRα, MBP, and DAPI. Arrows indicate OPCs in the lesion. Bi and Ci-insets of white dotted squares in B and C. D. Higher numbers of PDGFRα+ (OPCs) cells in Sema3F mice at 7 dpl (Mann Whitney test: p=0.0031, n=6-8 mice/group). E-F. Co-labeling for Olig2 and MBP. E'-F'. Co-labeling for Olig2 and GFP of the lesions shown in E-F. 3D reconstructions. Proximity of OPCs and Sema 3F-carrying cells. E'i-E'iii and F'i-F'iii-insets of E'-F'. G. Higher numbers of Olig2+ (oligodendroglial) cells in Sema3F-GFP mice at 7 dpl (Mann Whitney test: p=0.0047, n=5-8 mice/group).",
    "molecules": "DAPI"
  },
  {
    "caption": "Impact of MB on cytotoxicity of OT-1 CTLs against EG7 or EG7-L1. Splenocytes from OT-1 mice in culture were stimulated with 10 nM of OVA257-264 (SIINFEKL) for 3 days to generate mature CTLs. CTLs were incubated with CFSE labeled EG7-L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry analysis. Data are representative of three independent experiments. (effector to target ratio = 5:1, unpaired t-test). EG7-L1: EG7 overexpressing PD-L1.   Statistical results of (B).   ELISA measured of LDH release to assess the cytotoxicity of OT-I CTLs on EG7-L1 in the presence of MB. Anti-PD1 antibody (aPD-1) served as positive control.   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.",
    "molecules": "CFSE, MB"
  },
  {
    "caption": "Impact of MB on cytotoxicity of PD-1-/- (PD-1KO) OT-1 CTLs against EG7 or PD-L1 expressing EG7 stable cell line. Data are representative of three independent experiments. (unpaired t-test). Statistical results of (E). Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.",
    "molecules": "MB"
  },
  {
    "caption": "Cytotoxicity of OT-I CTLs against B16-F10-OVA cells (designated B16-OVA) in the presence of 1 μΜ of MB or DMSO. Cytotoxicity was determined by the relative area unoccupied by cells examined under microscope (scale bar = 50 μm). Data are representative of three independent experiments, and were analyzed by unpaired t-test.   Statistical results of (G).   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.",
    "molecules": "DMSO, MB"
  },
  {
    "caption": " Effect of MB on the proliferation of WT CTLs, PD-1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28 coated 96-well plates. 10 μg/ml of PD-L1 were administered in media. Cell proliferation were checked by monitoring dilution of CFSE 48 hours after CD3/CD28 stimulation by gating on CD8 positive population through FACS analysis. WT: splenic cell from wildtype C57BL/J mice. PD-1KO: splenic cell from PD-1 knockout mice.   Statistical results of (A)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 ",
    "molecules": "CFSE, MB"
  },
  {
    "caption": " FACS analysis of the effect of MB on the activation of OT-I CTLs. OT-I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD-L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 hours, surface expression of CD25 and CD69 on OT-1 CTLs were determined through FACS analysis.   Statistical results of (C)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": " Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP-luc by co-culture with Raji-L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE-Loaded Raji (PD-L1 negative) served as positive control). IC50 is calculated to be 117 nM. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1 Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": " Impact of MB on luciferase activity of various engineered Jurkat T cells. J-luc: Jurkat cell harboring NFAT-luciferase transgene; J-luc-sgPD-1: J-luc cells treated with lentivirus expressing sgPD-1/CAS9 simultaneously Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": " qRT-PCR analysis of IL-2 mRNA level in JP-luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD-L1 and MB at indicated concentrations Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": " MB enhancing IL-2 expression by JP-luc stimulated with Raji-L1 for 24-hour quantified through ELISA analysis (aPD1 served as positive control).   MB enhancing production of cytokine and cytolytic granule by OT-I CTLs. CTLs were incubated with EG7-L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule were determined by flow cytometry. aPD1 served as positive control. EG7-L1: EG7 overexpressing PD-L1.   Statistical results of (I)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 ",
    "molecules": "protein transport inhibitor, PTI, MB"
  },
  {
    "caption": " Confocal cell images of Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry. Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry were incubated with 10 μg/ml of human PD-L1 protein in the presence of 100 nM and 1 μM of MB for 2 minutes, fixed with 4% paraformaldehyde and stained with DAPI. Colocalization signal were examined with confocal microscope (scale bar = 2 µm) ",
    "molecules": "DAPI, MB, paraformaldehyde"
  },
  {
    "caption": " Fluorescence complementation analysis of the effect of MB on the interaction between PD-1 with SHP2. Jurkat cells co-expressing PD1-C-Luc or PD1Y248F-C-Luc and SHP2-N-luc were seeded in a 96-well precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD-L1 protein in the presenc of MB at indicated concentrations for 6 hours followed by analysis of luciferase activity Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": " Co-IP analysis of impact of MB on interaction between PD-1 and SHP2. 293T cells co-expressing PD-1 and SHP2-FLAG were treated with MB at indicated concentration for 6 hours ",
    "molecules": "MB"
  },
  {
    "caption": " Ability of MB to block interaction between PD-1 with SHP2 revealed through ELISA. Y248 phosphorylated human PD-1 ITSM peptide was coated at 4 μg/ml in 96 plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 hours. Bound SHP2 were measured by monitoring the activity of horse-peroxidase conjugated on secondary antibody targeting SHP2 Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": " Representative western blot showing the levels of total and phosphorylated ZAP70, PKCθ and PLCγ1 in the lysates of stimulated CTL, inhibited by PD-L1 in the presence of 1μΜ of MB ",
    "molecules": "MB"
  },
  {
    "caption": " Representative western blot showing the levels CD28 phosphorylation. CTL were stimulated with EG7 cells (parental), EG7-L1 and EG7-L1 in the presence 100 nM of MB for 2 hours. Protein immunoprecipitated (IP) with Anti-pY from the lysates of the indicated CTL-EG7 co-culture were subjected to SDS-PAGE separation and blotted with aCD28. β-actin served as loading control. EG7-L1: EG7 overexpressing PD-L1 Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": " Phos-tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7-L1 in the presence of 1μΜ MB. Cell lysate were then separated by SDS-PAGE containing 50 μM of Phos-tag and blotted with aCD2 antibody. Phosphorylated CD28 (slow-moving) species were visualized through exposure",
    "molecules": "MB"
  },
  {
    "caption": " Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat-PD-1 cells were co-transfected with CD28-C-Luc or CD28Y191F-C-Luc and P85-N-Luc. Cells were seeded in a 96-well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD-L1 protein and 100 nM of MB for 6 hours. Luciferase activity was measured as readout for CD28-P85 interaction Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001 ",
    "molecules": "MB"
  },
  {
    "caption": "Schematic of the xenograft mouse model for MB treatment. C57BL/6J mice were inoculated with EG7-L1 cells (2 × 106 cells, s.c.) on the right flank on day 1, followed by injection (2 × 106 cells, i.v.) of CD45.1+ CTL on day 3 and day 6 respectively. The mice were randomized into 3 groups (n = 5) and treated with vehicle, MB (20 mg/kg/day, i.g.), aPD1 (10 mg/kg, every other day, i.p.) served as positive control. Tumor size was measured through calipering every other day. EG7-L1: EG7 overexpressing PD-L1.   Tumor growth curve for (A) was shown as mean ±s.e.m.   Representative image of the tumor on day 10 mentioned in (A).   Tumor weight on day 10 (n=5) mentioned in (A).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.",
    "molecules": "MB"
  },
  {
    "caption": "Schematic of EGFR L858R mice treated with MB.   Tumor burdens recorded through Computed tomography (CT) scanning for EGFR L858R mice (n=5, lung section = 3). Mice were treated with MB (20 mg/kg/day, i.g.) for 1 week, followed by imaged with CT again to record tumor burden.   Statistical results of (J).   Haematoxylin and Eosin staining of lung section of EGFR L858R mice (n = 5, lung section = 3) in (J).   Statistical results of (L).   Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.",
    "molecules": "Eosin, Haematoxylin, MB"
  },
  {
    "caption": "Effect of MB on proliferation of human CD8+ T cells. CFSE labeled human peripheral blood mononuclear cells (PBMC) were pre-incubated with DMSO, MB, 25 μg/ml of pembrolizumab or 20 μg/ml of Nivolumab for 1 h and then seeded in the 96-well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD-L1 Protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.",
    "molecules": "CFSE, DMSO, MB, Nivolumab, pembrolizumab"
  },
  {
    "caption": "Cytokine and cytolytic granule production in CD8+ T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96-well precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD-L1 Protein. Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8+ populations. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.",
    "molecules": "DMSO, MB, nivolumab, Pem, pembrolizumab, phytohaemagglutinin, Niv"
  },
  {
    "caption": "Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP-luc cells were stimulated with Raji-L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25μg/ml of pembrolizumab (Pem) or 20μg/ml of nivolumab (Niv) for 6 hours. Luciferase activity was measured by luminometer. Data are representative of three independent experiments, and were analyzed by unpaired t-test. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001. ",
    "molecules": "MB, nivolumab, Pem, pembrolizumab, Niv"
  },
  {
    "caption": "(A) Bone Marrow Derived Macrophages (BMDMs) were treated with either the AMPK  activator 991, Dex  or a combination for 1 h before analysis by  western blot. (B, C) Phospho-AMPK  (B) and phospho-ACC  (C) were quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 6 (B,C), biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using student's t-test (B, C)",
    "molecules": "991, Dex"
  },
  {
    "caption": "(D) WT and AMPKα1-/- BMDMs  were treated with Dex  for 1 h before analysis by  western blot  and GR phosphorylation  at S211 was quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3 (D) biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests (D,",
    "molecules": "Dex"
  },
  {
    "caption": "(E) WT and AMPKα1-/- BMDMs  were treated with Dex  for 24 h and analyzed by  RNA-seq. Differentially expressed genes between any condition were clustered using c-means clustering.",
    "molecules": "Dex"
  },
  {
    "caption": "(F) WT and AMPKα1-/- BMDMs  were treated with Dex  for 24 h and anti-/pro-inflammatory markers assessed using immunofluorescence. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3-4 (F) biological replicates",
    "molecules": "Dex"
  },
  {
    "caption": "(G WT and AMPKα1-/- BMDMs  were treated with Dex  for 72 h, washed and conditioned medium was recovered after 24 h used to treat  muscle stem cells. Proliferation  (Ki67 immunostaining) (G) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates (G * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests Bar=25 µm (G",
    "molecules": "Dex"
  },
  {
    "caption": "H) WT and AMPKα1-/- BMDMs  were treated with Dex  for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. differentiation  (desmin immunostaining  and counting the percentage of muscle cells  with 2 nuclei or more) (H) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates H). * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests ,H). Bar=25 µm H).",
    "molecules": "Dex"
  },
  {
    "caption": "After cardiotoxin  injection to damage the muscle, AMPKα1fl/fl (WT) and LysM-α1-/- mice  were treated with a single dose of Dexamethasone  (Dex) intra-peritoneal (i.p.) (0.1 mg/kg) at day 3 (D3) and Tibialis Anterior  (TA) muscles  were harvested at day 8 (D8) and 14 (D14) after injury. (A) Representative embryonic myosin heavy chain (EmbMHC) immunostaining  on whole TA muscle  section at D8 and D14 after injury. (B) Quantification of (A), results are expressed as percentage of positive myofibers  expressing the EmbMHC  on the whole muscle section. Data information: results are displayed with box and whiskers in which the central band represents the median (B N= 7-12 muscles  (B * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 500 µm (A),",
    "molecules": "cardiotoxin, Dex, Dexamethasone"
  },
  {
    "caption": "(F) Macrophage  polarization was determined by immunofluorescence  in vehicle, DAMPs  and DAMPs+Dex  treated WT and AMPKα1-/- BMDMs. Data information: results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 4-5 biological replicates (F). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 25 µm (F).",
    "molecules": "Dex"
  },
  {
    "caption": "(F) Macrophage  polarization was determined by immunofluorescence  in vehicle, LPS  and LPS+Dex  treated WT and AMPKα1-/- BMDMs, data are expressed as percentage positive cells. Data information: Results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 3 (F) biological replicates. Statistical analysis by ANOVA F) * p<0.05, ** p<0.01, *** p<0.001. Bar = 25 µm (F).",
    "molecules": "Dex, LPS"
  },
  {
    "caption": "(F) WT and AMPKα1-/- Bone Marrow Derived Macrophages (BMDMs) were treated with either Dex  or the AMPK  activator 991  and protein phosphorylated  on serine were immunoprecipitated. Westen-blot for FOXO3, AMPKα1, actin  and the phosphorylated  form of FOXO3  were done. Quantification of the band intensity is provided normalized to those of actin  and total FOXO3, a representative example is shown. Data information: results are box and whiskers in which the central band represents the median F, N= 4-5 (F biological replicates. * p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001 using ANOVA and Kruskal-Wallis tests",
    "molecules": "991, Dex"
  },
  {
    "caption": "Percentage of the total signal intensity attributed to phosphopeptides (blue bars; 11 unique peptide ions) and non-phosphorylated peptides (grey bars; 25 unique peptide ions) following SAX fractionation at (A) pH 6.0, (B) pH 6.8 and (C) pH 8.0, with later fractions consisting of up to 100% phosphopeptide ion signal. Representative pHis-containing myoglobin peptide (LFTGHPETLEK; solid line), and its non-phosphorylated counterpart (LFTGHPETLEK; dashed line), quantified across all 16 SAX fractions; SAX was performed at pH 6.0 (red), pH 6.8 (blue) or pH 8.0 (green). % of the total peak area of each individual peptide across the gradient is plotted for each fraction. In order to assess pHis stability and effects of SAX separation in a complex mixture (black), SAX was also repeated at pH 6.8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion. Data for the five pHis-containing myoglobin peptides is shown in Fig. EV3.",
    "molecules": "phosphopeptide, myoglobin, phosphopeptides, peptide, peptides"
  },
  {
    "caption": "Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm).",
    "molecules": "trypsin"
  },
  {
    "caption": "Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).",
    "molecules": "Peptides, trypsin"
  },
  {
    "caption": "Comparison of triplet score (≥5% S/N) following HCD of unique phosphopeptides (5% FDR) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides. Site of phosphorylation (ptmRS score ≥ 0.90) is indicated. % of phosphopeptides exhibiting either no neutral loss (Triplet =0), or neutral loss of any 1 (Triplet = 1), 2 (Triplet =2), or 3 ions from the precursor (∆80, ∆98 and/or ∆116 amu). Distribution of triplet score following HCD fragmentation as a function of site localisation confidence (ptmRS score ≥ 0.75, ≥ 0.90, ≥ 0.95, ≥ 0.99) for unique singly phosphorylated peptide as a function of the residue phosphorylated. Numbers of phosphopeptides (ptmRS ≥ 0.90) exhibiting different combinations of the three neutral loss species (∆80, ∆98 and/or ∆116 amu from precursor) for each of the phosphorylated residues.",
    "molecules": "phosphopeptides, peptide"
  },
  {
    "caption": "C-F.  Immunofluorescence images of   esg-GFP and Delta  (Dl) staining  with the midgut  section from the R4 region of 14-day Drosophila (C), 26-day Drosophila (D), 40-day Drosophila (E), and 40-day Drosophila  in response to 0.5 mM  ALA  administration, which started at day 26 after eclosion (F). esg-GFP (green) identifies ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei  are shown in blue.",
    "molecules": "ALA, DAPI"
  },
  {
    "caption": "C. Quantification of the content of  ALA  in midguts of flies with indicated genotypes using  LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA"
  },
  {
    "caption": "D-F.  Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts-GAL4-driven  UAS-lacZ  (D, control), Las RNAi (E), or Las  RNAi in response to ALA  administration (F). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei  are shown in blue. Scale bars represent 10 μm",
    "molecules": "ALA, DAPI"
  },
  {
    "caption": "I-J. Immunofluorescence  images of control (FRT40A, I) and  Las RNAi (J) MARCM clones (green, outlined by white dotted lines) 10 days after clone induction (ACI). Pdm1 staining (red) was used to visualize ECs. K. Quantification of the ratio of Pdm1+ cells per clone of  MARCM clones with indicated genotypes. n is indicated. Each dot corresponds to one clone. L. Quantification of  MARCM clone size of experiments in (I-J). n is indicated. Each dot corresponds to one clone. Data information: DAPI stained nuclei  are shown in blue. Scale bars represent 10 μm (D Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Representative images and quantification of  Drosophila midguts  treated with the pH indicator  Bromophenol Blue. Moreover, quantification of the three categories of Drosophila of indicated conditions. The three categories can be divided into: homeostasis, a well-defined acidic (yellow colored) copper cell region (CCR) is flanked by basic (blue colored) anterior midgut (AM) and posterior midgut (PM). \"Perturbed A\", the acidic region is lost and the whole gut is basic; \"Perturbed B\", the strongly acidic region is lost, and the remainder of the gut also becomes less basic. The numbers of quantified guts  from left to right are 90, 90, and 90. Error bars show the SD of three independent experiments.",
    "molecules": "Bromophenol Blue"
  },
  {
    "caption": "C. Excretion of Drosophila treated with Bromophenol Blue. Images of deposits and quantification of deposit numbers are shown. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments. Data information: The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "Bromophenol Blue"
  },
  {
    "caption": "G. Survival  (percentage) of female W1118 Drosophila with and without supplementation of ALA  as indicated. The numbers of quantified flies: 100 (W1118 + 0.5 mM ALA) and 100 (W1118 + 0 mM  ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan  curves were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA"
  },
  {
    "caption": "I. Survival (percentage) of  female Canton-S Drosophila  with and without supplementation of ALA as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA), 100 (Canton-S + 0.5 mM  ALA). Three independent experiments were conducted. J. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA  at 26-day as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA) and 100 (Canton-S + 0.5 mM ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves ( were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA"
  },
  {
    "caption": "A-F. Immunofluorescence images of midgut  sections from the R4 region in Drosophila carrying esgts-GAL4-driven expression of  CAT  cDNA (A), CAT  cDNA with AD4  administration (B), CAT  cDNA with ALA  administration (C), Keap1 RNAi (D), Keap1  RNAi with AD4 administration (E), or Keap1 RNAi with ALA administration (F). esg-GFP (green) represents ISCs and their differentiating cells. Dl staining (red) was used to visualize  ISCs. G. Quantification of the number  of esg-GFP+ cells, Dl+ cells of Drosophila  carrying esgts-GAL4-driven expression of  lacZ  cDNA, CAT  cDNA, CAT  cDNA with AD4  administration, CAT  cDNA with ALA administration, Keap1  RNAi, Keap1  RNAi with AD4 administration, or Keap1 RNAi with ALA administration. n is indicated. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, 19, 18, 19, 20, 18, 18, 19, and 19. H. Quantification of the number of pH3+ cells and luciferase activity of Drosophila  carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4  administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1  RNAi with AD4 administration, or Keap1 RNAi with ALA administration. The numbers of quantified  guts from left to right are 18, 19, 20, 18, 18, 19, and 19. I- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA, DAPI, AD4"
  },
  {
    "caption": "I-L. Representative images of the  midgut  section from the R4 region of  Drosophila carrying  esgts-GAL4-driven expression of lacZ cDNA (I),  Las RNAi (J), Las  RNAi and CAT cDNA (K), or Las  RNAi and Keap1  RNAi (L). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Volcano plots of differentially expressed genes in pair-wise comparison of 40-day Drosophila treated without ALA administration to 40-day Drosophila treated with 0.5 mM  ALA administration. Blue symbols indicate significantly downregulated mass bins (Log2 FC < -0.6 and p < 0.05), red symbols indicate significantly upregulated mass bins (Log2 FC > 0.6 and p < 0.05), and gray symbols indicate mass bins that were not significantly changed.",
    "molecules": "ALA"
  },
  {
    "caption": "A-C.  Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut  section from the R4 region in 14-day WT flies  (A), 40-day WT flies (B), 40-day WT  flies with ALA  administration (C). esg-GFP (green; outlined by dotted lines), lysotracker  (red). esg-GFP+ cells are outlined by red dotted lines in the images of lysotracker staining channel. D. Quantification of the dot number of Lysotracker in esg positive cells from experiments (A-C). n is indicated. The numbers of quantified guts from left to right are 9, 11, and 8. E. Quantification of the dot size of Lysotracker in esg positive cells from experiments (A-C). n is as indicated. F- Data information:  DAPI stained nuclei are shown in blue. Scale bars represent 5 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA, DAPI, Lysotracker, lysotracker"
  },
  {
    "caption": "F-H. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a  in 14-day Drosophila (F), 40-day Drosophila  (G), 40-day Drosophila with  ALA administration started at the middle age (26 days) (H). GFP  (green) and  mCherry (red). The red arrows indicate the autophagosomes. I. Quantification of the dot number  of mCherry in esg positive cells from experiments (F-H). n is indicated. The numbers  of quantified guts from left to right are 11, 10, and 12. J. Quantification of the dot size of mCherry in esg  positive cells from experiments (F-H). n is as indicated. K- Data information: DAPI stained nuclei  are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA, DAPI"
  },
  {
    "caption": "K-L. Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut  section from the R4 region in 14-day flies carrying esgts-GAL4-driven UAS-lacZ  (K), and 14-day  flies carrying esgts-GAL4-driven  Las RNAi (L). esg-GFP (green; outlined by dotted lines), lysotracker (red). esg-GFP+ cells are outlined by dotted lines. M. Quantification of the dot number of Lysotracker in esg  positive cells from experiments (K-L). n is indicated. The numbers of quantified guts from left to right are 10, and 9. N. Quantification of the dot size of Lysotracker in esg positive cells from experiments (K-L). n is as indicated. O- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 5 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "DAPI, Lysotracker, lysotracker"
  },
  {
    "caption": "O-P. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a in 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ  (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (O), and 14-day  Drosophila  with  Las  depleted in ISCs and EBs (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (P). GFP (green) and mCherry (red). The red arrows indicate the autophagosomes. Data information: DAPI stained nuclei  are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-C.  Immunofluorescence images of midgut  section from the R4 region in 40-day flies (A), 40-day flies with  spermidine administration started at 26th day after  fly  eclosion (B), 40-day  flies  with spermidine  and ALA administration started at 26th day after  fly  eclosion (C). esg-GFP (green) indicates ISCs and their differentiating cells. Dl  (red) staining  was used to visualize  ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm",
    "molecules": "ALA, DAPI, spermidine"
  },
  {
    "caption": "F-I. Representative images of the midgut  section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of  lacZ  cDNA (F, control), Las RNAi (G),  Las RNAi and  Atg5  cDNA (H), or Las  RNAi with spermidine administration (I). GFP  (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm",
    "molecules": "DAPI, spermidine"
  },
  {
    "caption": "A-E. Expressions of Rab5-GFP reporter in ISCs of 14-day WT Drosophila (A), 40-day WT Drosophila (B), 40-day Drosophila with ALA administration (C), 14-day Drosophila carrying  esgts-GAL4>UAS-lacZ (D), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (E). GFP (green). F. Quantification of fluorescence intensity of RAB5-GFP in experiments (A-E). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 13, 11, 12, 10 and 13. G Data information: DAPI stained nuclei  are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA, DAPI"
  },
  {
    "caption": "G-K. Expressions of  Rab7-GFP  reporter in ISCs of 14-day WT Drosophila (G), 40-day WT Drosophila (H), 40-day Drosophila with  ALA administration (I), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (J), and 14-day Drosophila  carrying esgts-GAL4>Las RNAi (K). GFP (green). L. Quantification of fluorescence intensity of  RAB7-GFP  in experiments (G-K). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 10, 11, 13 and 12. M Data information: DAPI stained nuclei  are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA, DAPI"
  },
  {
    "caption": "M-Q. Immunofluorescence images of midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (M, control), constitutively active form of RAB5 (N), constitutively active form of  RAB5 with ALA  administration (O), constitutively active form of RAB7 (P), or constitutively active form of RAB7 with ALA  administration (Q). GFP  (green) and  Dl staining (red) was used to visualize ISCs. Data information:  DAPI stained nuclei  are shown in blue. Scale bars represent 10 μm",
    "molecules": "ALA, DAPI"
  },
  {
    "caption": "A-D. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (A, control), Las  RNAi (B), Las RNAi and RAB5-CA (C), or Las RNAi and RAB7-CA (D). GFP  (green), Dl staining  (red) was used to visualize  ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm",
    "molecules": "DAPI"
  },
  {
    "caption": "F-I. Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40-day Drosophila (F), 40-day Drosophila with ALA administration (G), 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (H), and Drosophila carrying esgts-GAL4-driven Las RNAi (I). dpERK (red). J. Quantification of the fluorescence intensity of dpERK in experiments (F-I). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 12, 12, and 13. K Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.",
    "molecules": "ALA, DAPI"
  },
  {
    "caption": "K-M. Representative images of midgut  sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ  cDNA (K, control), Las RNAi (L), or Las RNAi and dominant negative form of EGFR (EGFR-DN, M). GFP (green) and Dl staining  (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B The oocytes  were exposed to different concentrations of NO3-  in N free MBS for 1.5 h at pH5.5. Net NO3- accumulation was fitted with both Michaelis-Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity. Values are the mean ± SE (biological replicates: n=3) with 3-4 oocytes in each sample.",
    "molecules": "NO3-"
  },
  {
    "caption": "B Expression of  MtNPFs in  roots after transfer to media containing different levels of NO3- for 30 minutes. C=KCl  control, N=KNO3 (Biological replicates: n = 3 (3 pooled root  organs in each replicate), asterisk denotes significance p<0.05, Student's t-test). The means of all samples were compared to that of their concentration-matched KCl controls. Expression is shown relative to  Ubiquitin.",
    "molecules": "NO3-, KCl, KNO3"
  },
  {
    "caption": "A, B  I-V responses in oocytes (subtracted of the water injected controls) exposed to 40 mM Cl  at  pH5.8 (A) and pH7.4 (B). The currents were measured while the oocyte plasma membrane  was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water  injected  oocytes from the MtNPF6.5  injected  oocytes at each Cl- concentration Data information: Value = mean ± SEM, biological replicates: n = 4",
    "molecules": "Cl, Cl-"
  },
  {
    "caption": "C, D  I-V responses in the (C)  water and (D) MtNPF6.5 (subtracted of the water injected controls) injected oocytes exposed to different concentrations of Cl- at pH5.8. The currents were measured while the  oocyte plasma membrane  was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl-  concentration. The  Cl-  -elicited current values in D were obtained by subtracting the currents in water  injected oocytes  from the MtNPF6.5 injected oocytes  at each Cl-  concentration. Data information: Value = mean ± SEM, biological replicates: n = 6",
    "molecules": "Cl-"
  },
  {
    "caption": "C  Oocytes were exposed to 1.17 mM 36Cl- for 2h with 1 and 10 mM NO3-. Biological replicates: n=6 (4  oocytes in each sample). D The same data as in C is shown without AtNPF6.3 to allow for better comparison between MtNPFs. Biological replicates: n=6 (4 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, and black cross represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05.",
    "molecules": "Cl-, NO3-"
  },
  {
    "caption": "A, B Relative  expression of NPFs in M. truncatula roots grown in FP and exposed to different NaCl treatments. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root  organs in each replicate) ±SEM",
    "molecules": "NaCl"
  },
  {
    "caption": "D Relative  expression of NPFs in M. truncatula roots grown in FP  and exposed to 100mM KCl  treatments for different times. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM ",
    "molecules": "KCl"
  },
  {
    "caption": "A  Seedling Cl- content determined by  ion chromatography 2 h after treatment with 50mM NaCl. *p<0.05, Student's t-test; biological replicates: n=4. B Cl-  content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3. *p<0.05, Student's t-test; biological replicates: n=4. ",
    "molecules": "NH4NO3, Cl-, KNO3, NaCl"
  },
  {
    "caption": "C On the left, relative Cl- content determined by XRF of NO3- starved seedlings after KNO3 supplementation. *p<0.05, Student's t-test; biological replicates: n=3-5. On the right, a representative XRF image of Cl-  signal across WT and mutant roots  at the 0h timepoint. Five roots per genotype are shown. The outlines indicate the measurement areas. The color legend indicates relative  Cl- concentration.",
    "molecules": "Cl-, NO3-, KNO3"
  },
  {
    "caption": "A Biomass (fresh weight) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl. *p<0.05, Student's t-test; biological replicates: n=3 (2-5 pooled organs in each replicate).",
    "molecules": "NaCl"
  },
  {
    "caption": "B Image of seedlings  grown on  FP or 50 mM NaCl. Data information: roots were re-arranged to better display their length, they did not exhibit curling/agravitropic growth.",
    "molecules": "NaCl"
  },
  {
    "caption": "B Changes in NO3-  and Cl-  content (mol/g dry weight) of NO3- starved plants after  NO3-  provision Data information: , *p< 0.05, Student's t-test; biological replicates: n=4 ±SEM.",
    "molecules": "Cl-, NO3-"
  },
  {
    "caption": "E Relative MtNPF expression following treatment with 5mM  KNO3 +100mM NaCl Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM.",
    "molecules": "KNO3, NaCl"
  },
  {
    "caption": "A The induction of  MtNPF6.7 and repression of  MtNPF6.5 by  NO3- requires MtNLP1. Plants were grown on FP  plates and then treated with 5 mM KNO3. expression shown is relative to UBQ; *p<0.05, Student's t-test; biological replicates: n= 3 (3 pooled  root organs in each replicate).",
    "molecules": "NO3-, KNO3"
  },
  {
    "caption": "B The switch from Cl-  to NO3-  as the major anion upon NO3- addition is lost in mtnlp1. Plants were grown on FP plates and then treated with 5 mM KNO3 treatment. *p< 0.05, Student's t-test; biological replicates: n=4 (3 pooled root organs in each replicate).C",
    "molecules": "Cl-, NO3-, KNO3"
  },
  {
    "caption": "(b) Ciclesonide escape mutant. Vero cells treated with ciclesonide were infected with parental MERS-CoV or ciclesonide escape mutant. Viral titer was measured as described in panel a.",
    "molecules": "Ciclesonide, ciclesonide"
  },
  {
    "caption": "(b) Culture Medium SARS-CoV-2 RNA (27 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 0.01 in the presence of ciclesonide for 27 h. Viral RNA in culture medium was quantified by real-time PCR using the E gene primer/probe set. Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay.",
    "molecules": "ciclesonide"
  },
  {
    "caption": "(K, L) Representative images of immunofluorescence staining in control and Pfkp knockdown R1 cells for Sox17 following definitive endoderm differentiation (K) or Foxa2 following definitive endoderm differentiation (L). Green staining indicates positive cells with nuclei counterstained with DAPI. Images were representative of three independent experiments. (M, N) Quantitative analysis of Sox17 (M) and Foxa2 (N) in R1 cells from (K) and (L), respectively as determined by flow cytometry. Representative profiles (left panels) and quantitation (right panels) as a percentage of total cells.",
    "molecules": "DAPI"
  },
  {
    "caption": "(J, K) Expression of Lin41 in control versus Pfkp knockdown R1 cells without or with MG132 treatment (J) or with Lactacyctin treatment (K), as determined by Western blot.",
    "molecules": "Lactacyctin, MG132"
  },
  {
    "caption": "(K) Relative stability of mRNAs for the ectodermal genes Fgf5, Otx2, Sox1, and Pax2 R1 cells in control and Pfkp knockdown EBs at Day 6, as determined in actinomycin D chase assays. (L) Relative stability of ectodermal gene mRNAs measured as per (K) in Day 6 Pfkp knockdown EBs transfected with or without Lin41.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "The experimental schematic (upper panel). APLNR+ cells on day 2 were FACS-purified and treated with various dosages of PLB from day 2.5 to 5 during inducing hematopoietic differentiation. On day 5, the generation of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs were assessed. The representative FACS plots showed the frequency of APLNR+ cells, CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment. The bar graphs show the percentage of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment.",
    "molecules": "PLB"
  },
  {
    "caption": "The representative FACS plots show the frequency of CD31+CD34+ EPCs in the GFP+ population at day 5 with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5. The bar graph denotes the percentage of CD31+CD34+ in GFP+ cells as described in (F). P-values were determined by an unpaired two-tailed Student's t-test.",
    "molecules": "PLB"
  },
  {
    "caption": "On day 5 of hematopoietic differentiation, the inclusion or exclusion of NUMB exon 9 were detected by RT-PCR with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5. The bar graph showing the signaling intensity of each specific band from the RT-PCR electropherogram.",
    "molecules": "PLB"
  },
  {
    "caption": "The top panel depicts the location of specific primers targeting NUMB_S and NUMB_L isoform. The bottom bar graph showing the expression of NUMB_S and NUMB_L in cells on day 5 of differentiation with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5 with RT-qPCR.",
    "molecules": "PLB"
  },
  {
    "caption": "The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2-APLNR+ cells, day 5-DMSO treated and day 5-PLB treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO and PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of indicated genes. n=2 technical replicates",
    "molecules": "DMSO, PLB"
  },
  {
    "caption": "The representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression. Statistical analysis of the percentage of CD31+CD34+ EPCs of (H).",
    "molecules": "DOX"
  },
  {
    "caption": "SUMO conjugation assays performed under multiple turnover conditions using Siz2∆CT and different RPA-DNA complexes. SDS-PAGE gels were stained with Coomassie. Bands for Mw marker are fully annotated in Figure 1B.",
    "molecules": "Coomassie"
  },
  {
    "caption": "SUMO conjugation assays performed under multiple turnover conditions using RPA complexes with or without the WH domain of Rfa2 in presence of Siz2∆CT lacking (A) or including (B) its SAP domain. In both cases, reactions were done in technical triplicate and SDS-PAGE gels were stained with SYPRO. Only one representative gel is shown. Bands for Mw marker are fully annotated in Figure 1B.",
    "molecules": "SYPRO"
  },
  {
    "caption": "Electrophoretic mobility shift assays (EMSA) to assess binding of RPA or SUMO-RPA to 25nM single-stranded 6-FAM-labeled 32dT DNA and displacement of SUMO-RPA from 25nM SUMO-RPA bound to single-stranded 6-FAM-labeled 32dT DNA by RPA and vice versa. All titrations were done in technical triplicates. One representative gel is shown.",
    "molecules": "6-FAM"
  },
  {
    "caption": "Electrophoretic mobility shift assays (EMSA) performed with 10 nM 6-FAM-ds20ss32, a 6-FAM-labeled 20-mer dsDNA with a 32 nucleotide 3' overhang and its complexes with RPA, RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2 and serially diluted Siz2. All titrations were done in technical triplicates. One representative gel is shown.",
    "molecules": "6-FAM"
  },
  {
    "caption": "(A) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin-actin in solution. (B) Time-lapse TIRF microscopy of single filament growth from Alexa488phalloidin-stabilized seeds (green) in the presence of 5μM profilin-actin.",
    "molecules": "Alexa488, phalloidin"
  },
  {
    "caption": "(C) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF- and lipid-coated microspheres with profilin-actin, CP and Arp2/3 in solution. (D) Widefield epifluorescence images of indicated network components of bead-attached dendritic networks after kinetic arrest at indicated times.",
    "molecules": "lipid"
  },
  {
    "caption": "(B) TIRF microscopy of actin monomers (blue, 1uM total, 30% Cy5 labeled) in the presence of equimolar profilin recruited to a WAVE1 pattern (magenta).",
    "molecules": "Cy5"
  },
  {
    "caption": "(C) TIRF microscopy of actin polymerization in- (blue arrows) and outside (red arrows) of WAVE1-micropatterns (magenta) in the presence of 1uM profilin-actin visualized by a filament-binding probe (5nM Alexa488-UTRNN).",
    "molecules": "Alexa488"
  },
  {
    "caption": "(B) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of actin polymerization on WAVE1-coated coverslips. Conditions are 1uM actin, 5nM Alexa488-UTRNN.",
    "molecules": "Alexa488"
  },
  {
    "caption": "(B) Widefield epifluorescence images (Alexa488-actin) of bead-attached dendritic networks following kinetic arrest after 10 min of network growth for spike-in experiments (i.e. 60% his10-darkCherry-PWCA, 40% his10-Cherry-W; molar percentage) using indicated WAVE1 variants. Conditions are 5μM profilin-actin, 200nM CP and 50nM Arp2/3. For all conditions, actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction.",
    "molecules": "Alexa488, Latrunculin B, phalloidin"
  },
  {
    "caption": "(D) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of polymerization from profilin-actin on WAVE1-coated coverslips. Conditions are 2µM actin, 3µM profilin, 5nM Alexa488-UTRNN.",
    "molecules": "Alexa488"
  },
  {
    "caption": "(A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone.",
    "molecules": "LatB"
  },
  {
    "caption": "(C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve.",
    "molecules": "Atto540Q, Alexa488, LatB"
  },
  {
    "caption": "(D) WH2 occupancies (left axis, Methods) and FRET efficiencies (right axis) for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P*WCA(null)=cyan, P_CA=grey) as a function of profilin concentration. Concentration of LatB-stabilized, Atto540Q-actin monomers was constant (175nM) at all conditions. Lines are fits to a competitive inhibition model (see Methods).",
    "molecules": "Atto540Q, Alexa488, LatB"
  },
  {
    "caption": "The syntheses of the ligands PR-924, 9, 14, 16, 17 and 18 have been described previously (de Bruin et al, 2014). All ligands share the peptide scaffold shown in the upper left corner with P1, P2 and P3 side chains and an N-cap; they are equipped with a l-Ala (stereochemistry (S)) or d-Ala (stereochemistry (R)) residue at P3 (red) and either an N-acylmorpholine or a 3-methyl-1H-indene group (green). Furthermore, compounds differ in their P2 residue (either methoxytyrosine or tryptophane; blue). Inhibitor 9, also termed LU-015i, is endowed with an exceptional cyclohexyl residue (gray), which is associated with increased selectivity for human β5i.",
    "molecules": "LU-015i, PR-924"
  },
  {
    "caption": "A. Inhibition of human β5c and β5i by ONX 0914 and PR-924. IC50 values are deduced from the fitted data.",
    "molecules": "ONX 0914, PR-924"
  },
  {
    "caption": "A. Complex structures of the human β5c/β6 (left) and β5i/β6 (right) chimeric proteasomes with PR-924. The ligand binds in a linear (l) manner to the β5c/β6 substrate binding channel, but in a kinked (k) conformation to the β5i/β6 active site. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ. The inhibitor and Thr1 have been omitted for phasing.",
    "molecules": "PR-924"
  },
  {
    "caption": "B. Comparison of the conformations of ONX 0914, PR-924, 14, 16, 17 and 18 bound to the chimeric hβ5/hβ6 substrate binding channels. The l amino acid compounds ONX 0914 and 14 display a linear (l) binding mode to the hβ5c/hβ6 (left) and the hβ5i/hβ6 (right) chimeras, whereas the ligands 16 and 17 (P3-d-Ala and morpholine cap) adopt a kinked (k) conformation in both β5 substrate binding channels. Remarkably, the orientation of PR-924 and its analogue 18 (P3-d-Ala and 3-methyl-1H-indene cap) in the hβ5c/hβ6 chimera (and in WT yβ5; see Appendix Figure S5)) differs from that observed with the hβ5i/hβ6 chimera. Note that the inhibitors are rotated by about 45 ° compared to panel A.",
    "molecules": "ONX 0914, PR-924"
  },
  {
    "caption": "A. Complex structures of the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right) with PR-924 visualize the kinked (k) orientation. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A. Steric hindrance of Met31 (green) with the bulky 3-methyl-1H-indene cap is indicated by black double arrows.",
    "molecules": "PR-924"
  },
  {
    "caption": "C. Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3-d-Ala compounds PR-924 and 18. The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45. Residue 31 impacts on the position of the 3-methyl-1H-indene cap of PR-924 and 18. Due to steric hindrance, the N-cap is shifted in the mouse and in the hβ5i-V31M/hβ6 chimera compared to the hβ5i/hβ6 mutant proteasome crystal structures (black double arrow). Note that the structures are rotated by 90° compared to panel B.",
    "molecules": "PR-924"
  },
  {
    "caption": "ONX 0914 binds in a linear mode to all active sites. Changing the stereochemistry at the P3 site from l-Ala (S) to d-Ala (R) leads to a kinked conformation in the β5 substrate binding channels (compound 16). Substitution of the morpholine cap of 16 by a larger 3-metyhl-1H-indene moiety yields PR-924 and results in different inhibitor conformations at the yβ5/hβ5c and the mammalian β5i active sites. Hereby, the selectivity for hβ5i over hβ5c is drastically increased (IC50 ratio hβ5c/hβ5i: 255) compared to ONX 0914 (IC50 ratio hβ5c/hβ5i: 20) and compound 16 (IC50 ratio hβ5c/hβ5i: 5.2, Fig 2). Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR-924, thereby creating 7-fold selectivity for human β5i.",
    "molecules": "ONX 0914, PR-924"
  },
  {
    "caption": "(c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h.",
    "molecules": "z-VAD-fmk, DMSO"
  },
  {
    "caption": "(d) Bax+/+Bak+/+ (WT) and Bax−/−Bak−/− (DKO) MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 6 or 12 h.",
    "molecules": "z-VAD-fmk, DMSO"
  },
  {
    "caption": "(c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).",
    "molecules": "3-MA, 3-methyladenine, DMSO, wortmannin"
  },
  {
    "caption": "(c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Immunoblot analyses were performed to detect LC3 modification and caspase-3 activation.",
    "molecules": "3-MA, 3-methyladenine, DMSO, wortmannin"
  },
  {
    "caption": "(e) Atg5+/+ and Atg5−/− MEFs were cultured in EBSS in the presence of 50 μM of z-VAD-fmk or control DMSO for 0, 6 or 12 h. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).",
    "molecules": "z-VAD-fmk, DMSO"
  },
  {
    "caption": "(b) COS7 cells transfected with Bif-1-GFP were cultured in EBSS for 2 h and the localization of Bif-1-GFP was examined by immunogold electron microscopy using anti-GFP antibodies. Arrows indicate representative gold particles that are detected on autophagosomal membranes. The scale bars represent 10 μm in the main images and 1 μm in the insets in a, and 1 μm in b.",
    "molecules": "gold"
  },
  {
    "caption": "a) FBXL2 binds p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before collection for immunoprecipitation (IP) and immunoblotting as indicated. WCL, whole-cell lysate.",
    "molecules": "MG132"
  },
  {
    "caption": "a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs. 24 h after transfection, cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated.",
    "molecules": "MG132"
  },
  {
    "caption": "(c) p85β(ΔSH2C) is more stable than wild-type p85β. RPE1-hTERT cells were infected with either a retrovirus expressing wild-type p85β or p85β (ΔSH2C). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. (",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(d) p85β (QR/AA) is more stable than wild-type p85β. HEK293T cells were infected with either a retrovirus expressing wild-type p85β or p85β (QR/AA). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(b) Denatured extracts from HEK293T cells stably expressing either FLAG-tagged wild-type p85β or FLAG-tagged p85β(Y655A) were immunoprecipitated (IP) with either an anti-FLAG resin or a 4G10 platinum resin and immunoblotted (IB) as indicated. Immunoblots of whole-cell lysates (WCL) are also shown in the last two lanes.",
    "molecules": "4G10 platinum"
  },
  {
    "caption": "(f) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were transfected with p85β(Y655A) and 16 h after cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "g) During a 48-h serum starvation, U2OS cells stably transfected with a doxycycline-inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently re-stimulated with media containing serum and collected 30 min later. Denatured cell lysates were immunoprecipitated with an anti-FLAG resin and immunoblotted as indicated. The asterisk indicates a non-specific band. Uncropped images of blots are shown in Supplementary Fig. S8.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(c) U2OS cells stably transfected with a doxycycline-inducible p85β construct were serum starved for 48 h. During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.",
    "molecules": "doxycycline"
  },
  {
    "caption": "B. EP300 acetylates H3K14 in PC3 cells. Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A-485 (10 μM). Histone H3 serves as protein loading control.",
    "molecules": "A-485"
  },
  {
    "caption": "B. Cell proliferation curves of PC3 cells treated with siRNA-BAZ2A. Error bars represent the standard deviation from three independent experiments. C. Cell proliferation curves of PC3  cells treated with 5μM BAZ2-ICR  and  GSK2801. Error bars represent the standard deviation from three independent experiments. ",
    "molecules": "BAZ2-ICR, GSK2801"
  },
  {
    "caption": "D. Representative images of tumorspheres  generated from PC3  cells treated with BAZ2-ICR (1μM) or GSK2801  (1μM).",
    "molecules": "BAZ2-ICR, GSK2801"
  },
  {
    "caption": "E. Half-maximal effective concentration (EC50) of BAZ2-ICR. Measurements were performed 5 days after treatment of PC3  cells and initiation of tumorspheres  using the indicated BAZ2-ICR concentration. The amount of tumorspheres was assessed by measurement of cell viability. Error bars represent SD. Values are from three independent experiments.",
    "molecules": "BAZ2-ICR"
  },
  {
    "caption": "A. Representative  bright field images  of organoids derived from mouse prostate cells treated with DMSO, BAZ2-ICR  (5μM), GSK2801 (5μM) or GSK126 (5μM) inhibitors followed by transduction with shRNA-Control or shRNA-Pten.",
    "molecules": "BAZ2-ICR, DMSO, GSK126, GSK2801"
  },
  {
    "caption": "C. Representative images of Ki67-immunostained mouse prostate organoid sections expressing shRNA-control and shRNA-Pten and treated with DMSO or BAZ2-ICR (5μM). D. Quantification of  Ki67+ cells in mouse prostate organoids  shown in C. **< 0.01, ****P < 0.0001, two-tailed Student's t test; ns, not significant. The median values are represented by a line. ",
    "molecules": "BAZ2-ICR, DMSO"
  },
  {
    "caption": "E. Quantification of  mouse prostate organoids  per field expressing shRNA-control and shRNA-Pten and treated with DMSO, GSK126  (5μM), BAZ2-ICR (5μM), or GSK2801  (5μM). Values were normalized to shRNA-control samples. Average values of two independent experiments.",
    "molecules": "BAZ2-ICR, DMSO, GSK126, GSK2801"
  },
  {
    "caption": "(C). Relative miRNA expression of  let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.",
    "molecules": "doxycycline, tetracycline"
  },
  {
    "caption": "(D). Northern blotting for  let-7a and RNU6B in HeLa cells infected with lentiviruses  encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment.",
    "molecules": "doxycycline, tetracycline"
  },
  {
    "caption": "(G). Location of pseudouridine  sites detected by the  CMC primer extension method. Total RNA purified from HEK-293FT  cells were treated with CMC. CMC treated RNA  were reverse-transcribed   with RI-labelled specific primers for tRNAphe or  pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows.",
    "molecules": "ddATP, CMC, RI, pseudouridine, Pseudouridines, tRNAphe"
  },
  {
    "caption": "(A).  RIP analysis of pri-let-7a1 and Flag-TruB1   from HEK-293FT cells. RNA was extracted from  IP material and analyzed by  qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding  tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.",
    "molecules": "doxycycline, tetracycline"
  },
  {
    "caption": "(B). EMSA of 32p-ATP-labelled pri-let-7a1  or  pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes.",
    "molecules": "ATP, 32p"
  },
  {
    "caption": "(A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing  pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). Dicer  KD was used to show the height of pre-let-7a1. (B) RI  intensities of (A) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.",
    "molecules": "RI"
  },
  {
    "caption": "(B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.",
    "molecules": "doxycycline, tetracycline"
  },
  {
    "caption": "(E). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing  tetracycline-inducible TruB1  or GFP, 5 days after doxycycline treatment, with or without KD of  let-7 family members. GFP: GFP  without  let-7 KD. WT: overexpression of wild type TruB1 without  let-7 KD. WT + let7 KD: WT: overexpression of wild type TruB1  with let-7 KD. GFP  + let-7KD: GFP with let-7 KD. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.",
    "molecules": "doxycycline, tetracycline"
  },
  {
    "caption": "A. 3D rendering of the cerebrovascular  tree in brain  slices from male  P90 Ctrl and Poly I:C  MIA offspring stained  with intravascular DiI   (red). Scale bar = 50 µm. B. DiI-positive blood vessels  geometry analysis - quantification of  blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 male  Ctrl and Poly I:C MIA offspring. Vessel  number for Ctrl (20.93 ± 1.73) and Poly I:C (27.41 ± 1.99) MIA offspring, Mann-Whitney U test, **p < .01. Mean diameter for Ctrl (5.58 ± 0.01) and Poly I:C  (4.96 ± 0.01) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel  per image for Ctrl (6.12 ± 0.76) and Poly I:C (9.83 ± 1.48) MIA offspring, Mann-Whitney U test, **p < .01. Number of branches for Ctrl (9.60 ± 0.54) and Poly I:C (15.80 ± 1.21) MIA offspring, Mann-Whitney U test, *p < .05. Average branch length for Ctrl (34.07 ± 1.35) and  Poly I:C (33.47 ± 1.67) MIA offspring. C. 3D rendering of the cerebrovascular  tree in brain  slices from female  P90 Ctrl and  Poly I:C MIA offspring stained  with intravascular DiI  (red). Scale bar = 50 µm. D. DiI-positive blood vessels geometry analysis - quantification of blood vessel  number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 female  Ctrl and Poly I:C MIA offspring. Vessel  number for Ctrl (9.97 ± 1.18) and  Poly I:C (8.61 ± 0.78) MIA offspring. Mean diameter for Ctrl (5.77 ± 0.03) and Poly I:C (6.16 ± 0.02) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel  per image for Ctrl (3.23 ± 0.50) and Poly I:C (2.97 ± 0.30) MIA offspring. Number of branches for Ctrl (10.50 ± 0.87) and Poly I:C (7.24 ± 0.44) MIA offspring. Average branch length for Ctrl (38.06 ± 2.17) and Poly I:C (46.93 ± 1.49) MIA offspring, Mann-Whitney U test, ***p < .001. Data information: Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border. Poly I:C females  = purple bars with purple border. In Fig.1 B and D black dotted lines represent the Ctrl while the blue and purple solid line represent the male Poly I:C  and female Poly I:C offspring respectively. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "DiI, Poly I:C"
  },
  {
    "caption": "E. Quantification of in-vivo brain permeability  at P30 in male Ctrl (0.00 ± 0.00) and Poly I:C (3.68 ± 1.98) treated offspring, Mann-Whitney U test, p=0.060. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "F. Quantification of Evans Blue (EB) analysis of in-vivo brain permeability  at P90 (top) and representative pictures (bottom) of perfused brains  from Male Ctrl (left; 0.00 ± 0.00) and Poly I:C (right; 20.32 ± 10.95) offspring. Mann-Whitney U test, *p < .05. In accompanying pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green. Data information: Ctrl males = white bars with blu border. Poly I:C  males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "EB, Evans Blue, Poly I:C"
  },
  {
    "caption": "G. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized to Calnexin  (CANX) in cortices  from P90 male Ctrl (0.99 ± 0.09) and Poly I:C  (0.58 ± 0.05) offspring. Mann-Whitney U test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "H. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized by α-Tubulin  (αTUB) in cortices  from P90 male Ctrl (1.00 ± 0.04) and Poly I:C (0.75 ± 0.08) offspring. Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "I. Quantification of in-vivo brain permeability  at P30 in female Ctrl (0.58 ± 0.17) and Poly I:C (0.31 ± 0.14) treated offspring. Data information: Ctrl females = white bars with purple border.  Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "J. Quantification of  Evans Blue (EB) analysis of in-vivo brain permeability  at P90 (top) and representative pictures (bottom) of perfused brains  from Female Ctrl (left; 0.70 ± 0.47) and Poly I:C (right; 0.10 ± 0.10) offspring. In accompanying pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "EB, Evans Blue, Poly I:C"
  },
  {
    "caption": "K. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized by CANX  in cortices  from P90 female Ctrl (1.00 ± 0.16) and Poly I:C (1.39 ± 0.13) offspring. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "L. Quantitative analysis of the optical density of ZO-1  immunoreactive bands normalized by αTUB  in cortices  from P90 female Ctrl (1.00 ± 0.11) and Poly I:C (1.19 ± 0.19) offspring. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "M. Representative images of P90 Ctrl and  Poly I:C male offspring cortices stained  for TJs proteins. Claudin-5 (top-green) and ZO-1  (bottom-red) - Scale bar = 10 µm. N. Quantitative analysis of mean intensity of CLDN5  in cortices  from P90 male Ctrl (75.14 ± 1.08) and Poly I:C (67.39 ± 2.00) offspring. Student's t test, *p < .05. O. Quantitative analysis of the CLDN5  signal volume in cortices  from P90 male Ctrl (5.54 ± 0.59) and Poly I:C (3.70 ± 0.48) offspring. Student's t test, *p < .05. P. Quantitative analysis of mean intensity of ZO-1  in cortices  from P90 male Ctrl (73.16 ± 0.59) and  Poly I:C (73.86 ± 1.94) offspring. Q. Quantitative analysis of the ZO-1 signal volume in cortices  from P90 male Ctrl (23.29 ± 1.42) and Poly I:C (16.78 ± 2.18) offspring. Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border.  Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "T. [Left] Quantification of in-vivo cerebrovascular permeability  to EB  of P90 Ctrl (0.16 ± 0.16) and Poly I:C-treated (0.89 ± 0.89) males injected at P20. [Right] Representative pictures of saline  perfused brains  24h after the EB  injection accompanied by pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green - from Ctrl (top) and Poly I:C (bottom) male mice. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "EB, Poly I:C, saline"
  },
  {
    "caption": "U. Quantitative analysis of the optical density of CLDN5  immunoreactive bands normalized to CANX  in cortices  from P90 Ctrl (0.99 ± 0.12) and Poly I:C (0.98 ± 0.10) males treated at P20. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "V. Quantitative analysis of the optical density of ZO-1  immunoreactive bands normalized to CANX in cortices  from P90 Ctrl (1.00 ± 0.11) and Poly I:C  (0.73 ± 0.05) males treated at P20. Data information: Ctrl males = white bars with blu border. Poly I:C  males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "A. qPCR  analyses of GD9+6h male embryos  for: Pecam1  (Ctrl = 1.00 ± 0.17 N=8,  Poly I:C = 0.89 ± 0.23 N=13), CD248  (Ctrl = 1.00 ± 0.20 N=6, Poly I:C = 1.35 ± 0.31 N=11), Vegfa  (Ctrl = 1.00 ± 0.19 N=8, Poly I:C = 0.15 ± 0.04 N=8, Student's t test, ***p < .001), Pdfgb  (Ctrl = 1.00 ± 0.23 N=5, Poly I:C  = 1.10 ± 0.18 N=13), Tgfb1  (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.76 ± 0.22 N=10, Mann-Whitney U test, *p < .05), Tgfb2  (Ctrl = 1.00 ± 0.28 N=6,  Poly I:C = 1.37 ± 0.28 N=13), Tgfb3  (Ctrl = 1.00 ± 0.51 N=6, Poly I:C  = 0.73 ± 0.19 N=10), Foxf2  (Ctrl = 1.00 ± 0.45 N=8, Poly I:C = 0.46 ± 0.18 N=13) mRNAs. B. qPCR  analyses of E17 male cortices  for: Pecam1  (Ctrl = 1.00 ± 0.63 N=10, Poly I:C = 3.07 ± 1.58 N=8; Mann-Whitney U test, *p < .05), CD248  (Ctrl = 1.00 ± 0.23 N=10, Poly I:C = 3.33 ± 0.79 N=9; Mann-Whitney U test, **p < .01), Vegfa  (Ctrl = 1.00 ± 0.10 N=8,  Poly I:C  = 1.70 ± 0.29 N=5; Mann-Whitney U test, *p < .05), Pdfgb  (Ctrl = 1.00 ± 0.75 N=9,  Poly I:C = 2.05 ± 0.96 N=9; Mann-Whitney U test, *p < .05), Tgfb1  (Ctrl = 1.00 ± 0.16 N=7,  Poly I:C = 15.27 ± 5.75 N=7; Mann-Whitney U test, ***p < .001), Tgfb2  (Ctrl = 1.00 ± 0.16 N=8,  Poly I:C = 3.82 ± 1.05 N=6; Mann-Whitney U test, *p < .05), Tgfb3  (Ctrl = 1.00 ± 0.10 N=9, Poly I:C = 10.58 ± 6.73 N=8; Mann-Whitney U test, ***p < .001); Foxf2  (Ctrl = 1.00 ± 0.06 N=5,  Poly I:C = 2.60 ± 0.57 N=8; Mann-Whitney U test, *p < .05) mRNAs. C. qPCR  analyses of P90 male cortices  for: Pecam1  (Ctrl = 1.00 ± 0.18 N=8, Poly I:C = 0.85 ± 0.08 N=8), CD248  (Ctrl = 1.00 ± 0.20 N=9, Poly I:C = 0.93 ± 0.11 N=8), Vegfa  (Ctrl = 1.00 ± 0.07 N=8, Poly I:C  = 0.75 ± 0.06 N=8; Mann-Whitney U test, *p < .05); Pdfgb  (Ctrl = 1.00 ± 0.20 N=8, Poly I:C  = 1.11 ± 0.17 N=4), Tgfb1  (Ctrl = 1.00 ± 0.29 N=5, Poly I:C  = 2.37 ± 0.47 N=7; Mann-Whitney U test, *p < .05); Tgfb2  (Ctrl = 1.00 ± 0.29 N=4, Poly I:C = 1.19 ± 0.26 N=7), Tgfb3  (Ctrl = 1.00 ± 0.18 N=10, Poly I:C = 1.78 ± 0.50 N=11) mRNAs. D. qPCR  analyses of GD9+6h female embryos  for: Pecam1  (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.22 ± 0.50 N=6), CD248  (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.32 ± 0.57 N=6; Mann-Whitney U test, *p < .05), Vegfa  (Ctrl = 1.00 ± 0.38 N=8, Poly I:C = 0.29 ± 0.03 N=5; Mann-Whitney U test, *p < .05), Pdfgb  (Ctrl = 1.00 ± 0.36 N=10, Poly I:C = 1.31 ± 0.47 N=6), Tgfb1  (Ctrl = 1.00 ± 0.16 N=8, Poly I:C = 1.63 ± 0.42 N=7), Tgfb2  (Ctrl = 1.00 ± 0.33 N=9, Poly I:C = 1.75 ± 0.29 N=6; Mann-Whitney U test, *p < .05), Tgfb3  (Ctrl = 1.00 ± 0.29 N=9, Poly I:C = 1.33 ± 0.51 N=6), Foxf2  (Ctrl = 1.00 ± 0.28 N=9, Poly I:C = 1.71 ± 0.52 N=6) mRNAs. E. qPCR  analyses of E17 female cortices  for: Pecam1  (Ctrl = 1.00 ± 0.34 N=6, Poly I:C  = 0.27 ± 0.08 N=8), CD248  (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.04 ± 0.28 N=9), Vegfa  (Ctrl = 1.00 ± 0.70 N=5,  Poly I:C = 0.32 ± 0.13 N=5), Pdfgb  (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 0.19 ± 0.07 N=9), Tgfb1  (Ctrl = 1.00 ± 0.33 N=5, Poly I:C = 0.09 ± 0.03 N=6), Tgfb2  (Ctrl = 1.00 ± 0.32 N=7, Poly I:C = 0.45 ± 0.11 N=8), Tgfb3  (Ctrl = 1.00 ± 0.38 N=7,  Poly I:C = 0.40 ± 0.20 N=9), Foxf2  (Ctrl = 1.00 ± 0.18 N=5, Poly I:C = 1.04 ± 0.28 N=8) mRNAs. F. qPCR  analyses of P90 female cortices  for: Pecam1  (Ctrl = 1.00 ± 0.15 N=5, Poly I:C = 0.69 ± 0.15 N=5), CD248  (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.58 ± 0.09 N=5), Vegfa  (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 1.53 ± 0.38 N=8), Pdfgb  (Ctrl = 1.00 ± 0.16 N=9, Poly I:C = 1.77 ± 0.35 N=17), Tgfb1  (Ctrl = 1.00 ± 0.26 N=13, Poly I:C  = 1.30 ± 0.23 N=14), Tgfb2  (Ctrl = 1.00 ± 0.27 N=11,  Poly I:C = 0.59 ± 0.06 N=4), Tgfb3  (Ctrl = 1.00 ± 0.45 N=11, Poly I:C = 0.51 ± 0.06 N=4) mRNAs. G. qPCR  analyses of GD9+6h male embryos  for Il1α  (Ctrl = 1.00 ± 0.29 N=7, Poly I:C = 2.10 ± 0.57 N=17), Il1β  (Ctrl = 1.00 ± 0.39 N=6, Poly I:C = 10.84 ± 4.70 N=6; Mann-Whitney U test, **p < .01), Il1rn  (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 3.60 ± 1.58 N=11), Il6  (Ctrl = 1.00 ± 0.17 N=6, Poly I:C = 0.82 ± 0.12 N=7) mRNAs. H. qPCR  analyses of E17 male cortices  for: Il1α  (Ctrl = 1.00 ± 0.27 N=9, Poly I:C = 1.77 ± 0.53 N=11), Il1β  (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.33 ± 0.33 N=8), Il1rn  (Ctrl = 1.00 ± 0.25 N=10, Poly I:C  = 0.82 ± 0.40 N=18), Il6  (Ctrl = 1.00 ± 0.24 N=7, Poly I:C = 2.17 ± 0.43 N=8) mRNAs. I. qPCR  analyses of P90 male cortices  for: Il1α  (Ctrl = 1.00 ± 0.24 N=8, Poly I:C = 1.51 ± 0.26 N=10), Il1β  (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.19 ± 0.34 N=8), Il1rn  (Ctrl = 1.00 ± 0.31 N=18, Poly I:C = 1.30 ± 0.50 N=14), Il6  (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 2.40 ± 0.70 N=8) mRNAs. J. qPCR  analyses of GD9+6h female embryos  for: Il1α  (Ctrl = 1.00 ± 0.93 N=6, Poly I:C = 0.03 ± 0.00 N=4), Il1β  (Ctrl = 1.00 ± 0.61 N=5, Poly I:C = 0.17 ± 0.04 N=7), Il1rn  (Ctrl = 1.00 ± 0.25 N=9, Poly I:C = 0.33 ± 0.11 N=7), Il6  (Ctrl = 1.00 ± 0.48 N=7, Poly I:C = 0.59 ± 0.24 N=5) mRNAs. K. qPCR  analyses of E17 female cortices  for: Il1α  (Ctrl = 1.00 ± 0.31 N=5,  Poly I:C = 1.38 ± 0.20 N=12), Il1β  (Ctrl = 1.00 ± 0.30 N=5, Poly I:C = 1.05 ± 0.35 N=7), Il1rn  (Ctrl = 1.00 ± 0.55 N=6, Poly I:C  = 0.45 ± 0.22 N=6), Il6  (Ctrl = 1.00 ± 0.26 N=7, Poly I:C = 0.94 ± 0.14 N=8) mRNAs. L. qPCR  analyses of P90 female cortices  for Il1α  (Ctrl = 1.00 ± 0.08 N=5, Poly I:C = 1.32 ± 0.11 N=9), Il1β  (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.65 ± 0.09 N=8), Il1rn  (Ctrl = 1.00 ± 0.61 N=4, Poly I:C  = 0.85 ± 0.29 N=7), Il6  (Ctrl = 1.00 ± 0.11 N=9, Poly I:C  = 1.28 ± 0.20 N=20) mRNAs. Values are normalized over control (dashed line). Data information: Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border.  Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "M. ELISA  quantification of total TGF-β1  in E17 male embryos brains  as pg/mL (Ctrl = 132.6 ± 18.14, Poly I:C = 129.6 ± 8.62). N. ELISA  quantification of active free TGF-β1 in E17 male embryos brains  as pg/mL (Ctrl = undetectable, Poly I:C = 0.92 ± 0.25). O. ELISA  quantification of total TGF-β1 in E17 female embryos brains  as pg/mL (Ctrl = 177.6 ± 10.88, Poly I:C = 134.4 ± 13.59). P. ELISA  quantification of active free TGF-β1 in E17 female embryos brains  as pg/mL (undetectable). Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C  females = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "Q-S. Western Blotting  quantification of SMAD proteins in E17 male cortices  for (Q) pSMAD1-5 (Ctrl = 0.99 ± 0.21, Poly I:C = 2.67 ± 0.46); Mann-Whitney U test, *p < .05, (R) pSMAD2 (Ctrl = 1.00 ± 0.04, Poly I:C = 0.88 ± 0.24), (S) SMAD4  (Ctrl = 1.00 ± 0.12, Poly I:C = 0.96 ± 0.21). Protein levels were normalized to GAPDH. T-V. Western Blotting quantification of SMAD proteins in E17 female cortices  for (T) pSMAD1-5 (Ctrl = 1.00 ± 0.02, Poly I:C  = 0.70 ± 0.18), (U) pSMAD2 (Ctrl = 1.00 ± 0.01, Poly I:C = 1.09 ± 0.12), (V) SMAD4(Ctrl = 1.00 ± 0.16,  Poly I:C = 1.46 ± 0.57. Protein levels were normalized to GAPDH. Data information: Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border. Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "A. TEER  of the in vitro BBB models composed by a monolayer of ECs  sorted from E17 Male  Ctrl (1.00 ± 0.02), Male Poly I:C (0.92 ± 0.04), Female  Ctrl (1.15 ± 0.04), Female Poly I:C (1.00 ± 0.07) offspring. Data are normalized to male  Ctrl. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex  derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males  = white bars with blu border. Cells derived from Poly I:C males  = blu bars with blu border. Cells derived from Ctrl females  = white bars with purple border. Cells derived from Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "B. Permeability  to 10 kDa Dextran  of the in vitro BBB models composed by a monolayer of sorted ECs  from Male  Ctrl (1.00 ± 0.06), Male Poly I:C (1.13 ± 0.02), Female  Ctrl (0.97 ± 0.05), Female Poly I:C (0.95 ± 0.01) offspring. Mann-Whitney U test, *p < .05. Data are normalized to male  Ctrl. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex  derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males  = white bars with blu border. Cells derived from  Poly I:C males  = blu bars with blu border. Cells derived from Ctrl females  = white bars with purple border. Cells derived from Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.",
    "molecules": "Dextran, Poly I:C"
  },
  {
    "caption": "C. TEER  of barriers composed of bEND.3 alone (1.00 ± 0.00) or co-cultured with sorted PCs  from Male  Ctrl (1.17 ± 0.07), Male Poly I:C (0.95 ± 0.05), Female  Ctrl (1.15 ± 0.08), Female Poly I:C (1.15 ± 0.08) offspring. Mann-Whitney U test, *p < .05. Data are normalized to  bEnd.3. Data information: Embryos prenatally exposed to vehicle (Ctrl) or  Poly I:C  on GD9 were dissected, and cortex   derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males  = white bars with blu border. Cells derived from Poly I:C males  = blu bars with blu border. Cells derived from Ctrl females  = white bars with purple border. Cells derived from Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "D. Permeability  to 10 kDa Dextran  of barriers composed by a monolayer of  bEND.3 alone (1.00 ± 0.06) or co-cultured with sorted PCs  from Male  Ctrl (0.47 ± 0.09), Male Poly I:C (0.46 ± 0.07), Female  Ctrl (0.46 ± 0.11), Female Poly I:C (0.65 ± 0.05) offspring. One sample t test, **p < .01. Data are normalized to bEnd.3. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or  Poly I:C on GD9 were dissected, and cortex  derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males  = white bars with blu border. Cells derived from  Poly I:C males  = blu bars with blu border. Cells derived from Ctrl females  = white bars with purple border. Cells derived from Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.",
    "molecules": "Dextran, Poly I:C"
  },
  {
    "caption": "E. TEER  (Ω.cm2) was monitored over 72h treatment with TGF-β1  and normalized to baseline in BBB models composed by a monolayer of sorted ECs  from Male  (72h TGF-β1  = 82.85 ± 1.56) or Female  (72h TGF-β1  = 85.92 ± 2.03) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). One sample t test, ***p < .001, ****p < .0001. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or  Poly I:C  on GD9 were dissected, and cortex  derived vascular cells were used to build in-vitro barrier models. In Fig.3 E blue and purple dotted lines represent cells derived from male  and female brain  respectively.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "F. TEER  (Ω.cm2) was monitored over 72h treatment with TGF-β1  and normalized to baseline in barrier models composed by a monolayer of bEND.3 alone (72h TGF-β1 = 89.85 ± 2.05) or co-cultured with sorted PCs  from Male  (72h TGF-β1 = 88.83 ± 1.93) or Female  (72h TGF-β1 = 89.02 ± 2.22) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). Wilcoxon signed-rank test, *p < .05. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or Poly I:C  on GD9 were dissected, and cortex   derived vascular cells were used to build in-vitro barrier models. In Fig.3 F blue and purple dotted lines represent cells derived from male  and female  brain respectively.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "G. 10 kDa Dextran permeability  after 72h TGF-β1 treatment of the in vitro BBBs prototypes composed by a monolayer made of sorted ECs  from Male  NT (0.43 ± 0.02), Male TGF-β1 (0.43 ± 0.03); Female  NT (0.45 ± 0.00), Female TGF-β1 (0.47 ± 0.01). Data information: Embryos  prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex  derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males  = white bars with blu border. Cells derived from Ctrl females  = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.",
    "molecules": "Dextran, Poly I:C"
  },
  {
    "caption": "H. 10 kDa Dextran permeability  72h after TGF-β1 treatment of NVU prototypes composed by a monolayer made of bEND.3 alone (NT, solid black bar = 0.45 ± 0.01, TGF-β1, dotted black bar = 0.49 ± 0.00) and co-cultured with sorted PCs  from Male  (NT = 0.45 ± 0.04, TGF-β1  = 0.48 ± 0.00), or Female  (NT = 0.49 ± 0.03, TGF-β1  = 0.53 ± 0.02) embryos. Student's t test, *p < .05. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or  Poly I:C  on GD9 were dissected, and cortex  derived vascular cells were used to build in-vitro barrier models. bEend.3 cells = bars with black border. Cells derived from Ctrl males  = white bars with blu border. Cells derived from Ctrl females  = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.",
    "molecules": "Dextran, Poly I:C"
  },
  {
    "caption": "P. Permeability  to 3kDa dextran  quantified by fluorescence spectrophotometry in NT (1.00 ± 0.10) and  IL-1β  + TGF-β1 (1.92 ± 0.33) treated primary male brain EC  cultures. Student's t test, *p < .05. Q. Permeability  to 10kDa dextran  quantified by  fluorescence spectrophotometry  in NT (1.00 ± 0.11) and IL-1β +  TGF-β1 (1.80 ± 0.26) treated primary male brain EC  cultures. Student's t test, *p < .05. Data information: Cortex-derived-EC  isolated from male mice  at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.",
    "molecules": "dextran"
  },
  {
    "caption": "A. [left] Representative dot plots showing the gating strategy for E17 samples to distinguish NVU cells. The cells were pre-gated on single cells and live cells (Zombie Aqua negative). Live cells were pre-gated on CD45  (CD45-). [right] Brain Pericytes  (PC) were defined as CD146+CD31- cells and brain Endothelial Cells (EC) as CD31+cells. B. [left] Quantification of the ratio between PC  number to EC  numbers for male  (blue) and female  (purple) E17 offspring cortex  samples. Male  Ctrl = 1.10 ± 0.08, Male Poly I:C = 1.34 ± 0.09, Female  Ctrl = 1.02 ± 0.08, Female Poly I:C = 1.12 ± 0.09. Mann-Whitney U test, *p < .05. Bars represent average ratio of individual embryos  ± SEM, numbers in bars indicate the number of embryos. [right up] Percentages of ECs  in Male  Ctrl (0.79 ± 0.05), Male Poly I:C (0.90 ± 0.09), Female  Ctrl (0.65 ± 0.07), Female Poly I:C (0.80 ± 0.07) samples and [right down] PCs  in Male  Ctrl (0.60 ± 0.05), Male Poly I:C (0.57 ± 0.06), Female  Ctrl (0.53 ± 0.07), Female Poly I:C (0.62 ± 0.06) samples. C. [left] Gating strategy used to analyze P30 samples: single cells, live (zombie Aqua negative), CD45- cells. [right] PCs  are identified as CD146+CD13+CD31-; ECs  are CD13-CD31+ D. [left] Quantification of EC/PC  number ratio for male  and female  P30 offspring cortex  samples, obtained by manual gating. Male  Ctrl = 1.78 ± 0.29, Male Poly I:C = 1.74 ± 0.32, Female  Ctrl = 1.49 ± 0.11, Female Poly I:C = 1.60 ± 0.19. [right up] Percentages of ECs  in Male  Ctrl (17.07 ± 1.88), Male Poly I:C  (17.52 ± 1.90), Female  Ctrl (14.58 ± 0.66), Female Poly I:C (15.73 ± 1.07) samples, and [right down] percentage of PCs  in Male  Ctrl (10.55 ± 1.89), Male Poly I:C (10.67 ± 1.04), Female  Ctrl (10.07 ± 0.85), Female Poly I:C (10.20 ± 0.79 Data information: Embryos  prenatally exposed to vehicle (Ctrl) or Poly I:C  on GD9 were dissected, and cortex  single-cell suspension was analyzed by multi-color  flow cytometry. Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border. Poly I:C females  = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "E. Normalized percentage of live(CD45-) EdU-positive cells at E17 in Male  Ctrl (1.00 ± 0.05), Male Poly I:C  (0.62 ± 0.11), Female  Ctrl (0.61 ± 0.04), Female Poly I:C (0.59 ± 0.11) samples. Mann-Whitney U test and Kruskal-Wallis test, *p < .05, **p < .001. F. Percentage of PCs EdU-positive cells at E17 in Male  Ctrl (7.88 ± 1.57), Male Poly I:C (4.55 ± 0.70), Female  Ctrl (7.03 ± 1.04), Female Poly I:C (4.93 ± 0.83) samples. Student's t test, *p < .05. G. Percentage of ECs EdU-positive cells at E17 in Male  Ctrl (4.30 ± 0.71), Male Poly I:C (2.80 ± 0.53), Female  Ctrl (2.84 ± 0.43), Female Poly I:C (2.29 ± 0.77) samples. H. Normalized percentage of live(CD45-) EdU-positive cells at P30 in Male  Ctrl (1.16 ± 0.14), Male Poly I:C (0.66 ± 0.16), Female  Ctrl (1.00 ± 0.08), Female Poly I:C  (0.91 ± 0.13) samples. Mann-Whitney U test, *p < .05. I. Percentage of PCs EdU-positive cells at P30 in Male  Ctrl (0.80 ± 0.24), Male Poly I:C (0.24 ± 0.06), Female  Ctrl (0.26 ± 0.08), Female Poly I:C (0.38 ± 0.13) samples. Mann-Whitney U test, *p < .05. J. Percentage of ECs EdU-positive cells at P30 in Male  Ctrl (0.58 ± 0.10), Male Poly I:C (0.44 ± 0.19), Female  Ctrl (0.38 ± 0.22), Female Poly I:C (0.25 ± 0.06) samples. Data information: Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border.  Poly I:C females  = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "EdU, Poly I:C"
  },
  {
    "caption": "K. [left] CD146  normalized MFI of the EC  cells at P30 in Male  Ctrl (100.00 ± 8.27), Male Poly I:C (65.73 ± 3.63), Female  Ctrl (57.08 ± 14.10), Female Poly I:C (43.07 ± 8.34). Mann-Whitney U test and Ordinary one-way ANOVA, **p < .01. [right] Representative histograms CD146 expression  on ECs  in Male  Ctrl (blue hollow), Male Poly I:C (solid light blue), Female  Ctrl (purple hollow), Female Poly I:C (solid purple) samples. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex  single-cell suspension was analyzed by multi-color flow cytometry. Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border. Poly I:C females  = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "L. Percentages of PC CD146++, CD146+ and CD146- cells respectively at P30 in Male  Ctrl (26.32/36.75/23.48 ± 2.84/3.31/4.62), Male Poly I:C (27.53/42.40/20.60 ± 2.25/0.58/1.94), Female  Ctrl (29.05/40.98/2.19 ± 1.99/1.32/2.19), Female Poly I:C (27.17/46.60/17.10 ± 3.09/3.86/1.96) samples. Data information: CD13  positive pericytes  were represented ad CD146- (dark greed bars), CD146+ (green bars) and CD46++ (light green bars). Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "M. [left] CD105  normalized MFI of the EC  cells at P30 in Male  Ctrl (100.00 ± 7.39), Male Poly I:C (99.58 ± 6.23), Female  Ctrl (100.30 ± 1.61), Female Poly I:C (96.67 ± 3.72). [right] Representative histograms indicating mean fluorescence intensity (MFI) of CD105  on EC  in Male  Ctrl (blue hollow), Male Poly I:C (solid light blue), Female  Ctrl (purple hollow), Female Poly I:C (solid purple) samples. N. [left] Quantitative data of the normalized MFI of CD105  in PCs  at P30 in Male  Ctrl (100.00 ± 3.43), Male Poly I:C  (98.01 ± 5.14), Female  Ctrl (98.57 ± 4.16), Female Poly I:C (87.99 ± 4.21). Mann-Whitney U test, *p < .05. [right] Representative histograms for CD105 expression  on PCs  in Male  Ctrl (blue hollow), Male Poly I:C (solid light blue), Female  Ctrl (purple hollow), Female Poly I:C (solid purple) samples. Data information: Embryos  prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex  single-cell suspension was analyzed by multi-color  flow cytometry. Ctrl males  = white bars with blu border.  Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border. Poly I:C females  = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "O. [top] Representative images of CD13  (red) positive pericytes  covering Lectin-positive vessels  (green) in sections of P90 untreated male brains. [bottom] 3D rendering, Scale bar = 20 µm, enlargement scale bar = 10 µm. P. Percentage of CD13-positive pericyte  coverage at P90 in Male Ctrl (53.42 ± 9.22) and  Poly I:C (27.73 ± 5.29). Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "Q. Percentage of PDGFrB-positive pericyte  coverage at P90 in Male Ctrl (66.15 ± 3.09) and  Poly I:C (55.08 ± 3.10). Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "S. Percentage of CD13-positive pericyte  coverage at P90 in Female Ctrl (71.57 ± 8.37) and Poly I:C  (69.13 ± 6.63). T. Percentage of PDGFrB-positive pericyte  coverage at P90 in Female Ctrl (66.77 ± 11.76) and  Poly I:C (70.56 ± 8.32). Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "A. Representative images of brain  acute bleedings detected through hemoglobin IHC  in P30 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. B. Representative images of brain  acute bleedings  detected through hemoglobin IHC  in P90 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. C. Quantification of hemoglobin   (Hb) positive signal in the brain  of P30 and P90 male MIA offspring, expressed as percentage of the area. P30 Ctrl = 0.00 ± 0.00, P30  Poly I:C  = 0.01 ± 0.00, P90 Ctrl = 0.00 ± 0.00, P90  Poly I:C = 0.01 ± 0.00. Mann-Whitney U test, ***p < .001. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "D. Representative 3D rendering of iron  deposits detected by DAB-enhanced Perls staining  in P30 Ctrl and Poly I:C  MIA male offspring brains. Brain  volume is rendered gold-transparent with positive signal for the staining  color-coded by surface dimension. Scale bar = 100 µm.",
    "molecules": "DAB, Perls, iron, Poly I:C"
  },
  {
    "caption": "E. Quantification of DAB-enhanced histochemical iron stains  in the brain  of P30 Ctrl (0.00 ± 0.00) and Poly I:C (0.01 ± 0.00) male MIA offspring, expressed as Perls-positive percentage of the volume. Mann-Whitney U test, *p < .05. Data information: Ctrl males = white bars with blu border.  Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.",
    "molecules": "DAB, Perls, iron, Poly I:C"
  },
  {
    "caption": "F. Poisson distribution R value of Perls/DAB  positive staining  of P30 male MIA offspring, respectively in the Coronal  (Ctrl = 1.19 ± 0.04, Poly I:C = 0.94 ± 0.01; Mann-Whitney U test, **p < .01), Axial  (Ctrl = 1.16 ± 0.08, Poly I:C = 1.03 ± 0.03), and Sagittal  (Ctrl = 1.09 ± 0.09,  Poly I:C = 1.01 ± 0.05) planes. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.",
    "molecules": "DAB, Perls, Poly I:C"
  },
  {
    "caption": "G. Representative images of iron  deposits detected through Perls staining  in P90 Ctrl and Poly I:C MIA male offspring brains. Scale bar = 500 µm, enlargement scale bar = 100 µm. H. Quantification of Perls-positive staining  in the brain  of P90 Ctrl (0.25 ± 0.05) and Poly I:C (0.59 ± 0.10) male MIA offspring, expressed as percentage of the area. Mann-Whitney U test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.",
    "molecules": "Perls, iron, Poly I:C"
  },
  {
    "caption": "I. Representative image of marble burying test  at 0 and 30 minutes. 15  glass marbles   were evenly spaced on a 5 x 3 grid. Marbles  buried by two-thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by the sawdust. J. Quantification of buried marble  (n°) by P90 Ctrl Male  (3.33 ± 1.67), Poly I:C Male  (5.72 ± 2.10), Ctrl Female  (3.83 ± 1.72) and Poly I:C Female  (3.00 ± 2.00) mice. Student's t test, **p < .01. Data information: Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border. Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "K. Quantification of self-grooming time (sec) by P90 Ctrl Males  (19.34 ± 13.00), Poly I:C Males  (43.38 ± 35.21), Ctrl Females  (19.86 ± 18.06) and Poly I:C Females  (16.93 ± 11.19) mice. Student's t test, *p < .05. Data information: Ctrl males  = white bars with blu border. Poly I:C males  = blu bars with blu border. Ctrl females  = white bars with purple border.  Poly I:C females  = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.",
    "molecules": "Poly I:C"
  },
  {
    "caption": "(A-H) Lateral images of the PHS fixed at 36 hpf (A-B, E-F) and 42 hpf (C-D, G-H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show a decrease in the number of Prox1-positive PHS ECs in the ccbe1 morpholino-injected (B, D) and sFLT4 mRNA-injected embryos (F, H) compared with control morpholino-injected (A, C) and uninjected embryos (E, G). At 42 hpf, ccbe1 morpholino-injected embryos (D) and sFLT4 mRNA-injected embryos (H) lack the PHS-LP domains present in the control embryos (C, G; large arrow heads). (I-J) Quantitation of the percentage of PHS ECs that are lymphatic progenitors relative to the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) (I) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17) (J). (K) Quantitation of the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17) ",
    "molecules": "morpholino"
  },
  {
    "caption": "(L-O) Lateral images of the FLS (L, N; dotted line) or dorsolateral CCV region from where the FLS normally sprouts (M, O; dotted line) fixed at 36 hpf in lyve1b:EGFP embryos that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show no change in the number of Prox1-positive lymphatic progenitors in the ccbe1 morpholino-injected (M) and sFLT4 mRNA-injected (O) embryos compared with control morpholino-injected (L) and uninjected (N) embryos. (P) Quantitation of the percentage of dorsolateral CCV ECs that are lymphatic progenitors relative to the total number of dorsolateral CCV ECs in control morpholino-injected (n = 26), ccbe1 morpholino-injected (n = 27), uninjected (n = 28) and sFLT4 mRNA-injected embryos (n = 32). (Q) Quantitation of the total number of dorsolateral region CCV ECs in 36 hpf embryos that were uninjected or injected with control morpholino (n = 26), ccbe1 morpholino (n = 27), or sFLT4 mRNA (n = 32). ",
    "molecules": "morpholino"
  },
  {
    "caption": "(R-U) Dorsolateral images of the VA-L fixed at 54 hpf in lyve1b:EGFP larva that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show that lymphatic progenitor formation still occurs within the VA-L in ccbe1 morpholino-injected (S) and sFLT4 mRNA-injected embryos (U), with Prox1 expression resembling that of controls (R, T).",
    "molecules": "morpholino"
  },
  {
    "caption": "A, Representative  immunofluorescence staining  of normal, tumor, and residual 3D  organoids grown from primary mammary cells  of a preclinical mouse model of breast cancer (Methods). Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 (red), ZO-1 (green), GM-130  (magenta); DAPI (blue). Right: human MYC  oncoprotein (green) is expressed only in tumor cells,  CDH1 (red). Scale bar: 25 μm. Data information: Number of replicates corresponds to individual mice used to derive organoids.",
    "molecules": "DAPI"
  },
  {
    "caption": ", Selective secreted metabolites  with significant change linked to urea  cycle components from healthy (n=3; blue) and regressed (n=3; green)  mammary glands. Values represent metabolite abundance levels  as quantified by the area under the curve (AUC) of the marker fragment ions/transitions for each metabolite. Values are plotted on the log2 scale. Statistics were calculated using the limma package n R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). ata information: umbers of replicates correspond to individual mice  or  humans. ox plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. umbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).",
    "molecules": "urea"
  },
  {
    "caption": "C, Left, Quantification of cells expressing ARG1  (top), an enzyme converting arginine to urea and  ornithine, and intensity of ARG1  (bottom) in normal cells from healthy (n=5, 2921 cells analyzed; blue) and residual cells from regressed (n=5, 2241 cells analyzed; green) mammary gland tissue sections. Statistical differences were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test). Right, Representative images of  immunofluorescence staining in normal (top) and residual (bottom) duct stained for ARG1 (green), CDH1 (red), and DAPI (blue). Scale bar: 20 μm. Data information: Numbers of replicates correspond to individual mice or  humans. Box plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. Numbers  marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).",
    "molecules": "arginine, DAPI, ornithine, urea"
  },
  {
    "caption": "D,  Nitric oxide synthase (NOS) activity, an enzyme involved in  arginine metabolism, in healthy (n=3; blue) and in residual (n=3; green) mouse mammary glands. The difference is statistically significant by unpaired two-samples t-test. E, Fractional labeling of lactate after cultivation of isolated regressed (n=3; green) and healthy (n=3; blue) mouse mammary glands in cell growth medium supplemented with  [U-13C]  glucose  for 8 h (Methods). The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted and shows enrichment in the residual cells of the regressed  mammary glands. The difference is statistically significant by unpaired two-samples t-test. Data information: Numbers of replicates correspond to individual mice or humans. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).",
    "molecules": "arginine, carbon, [U-13C]  glucose, lactate"
  },
  {
    "caption": "A, (Top) Experimental design and (bottom) quantification of cell death (Methods) of normal, tumor, and residual cells after 72-h treatment with 3-BP  at the indicated doses (n=5 mice per condition). Two-way ANOVA with Tukey's HSD multiple comparison testing was utilized to calculate statistical significance. Dox, doxycycline. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers , minimum to maximum. Number of replicates corresponds to individual  mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).",
    "molecules": "3-BP, Dox, doxycycline"
  },
  {
    "caption": "B, Representative bright-field images of normal (left), tumor (middle), and residual (right) organoids, treated with vehicle (top) or with 50 μM 3-BP (bottom). Scale bar, 100 μm.",
    "molecules": "3-BP"
  },
  {
    "caption": "C, (Top) Experimental design and (bottom) cell-death quantification of passaged residual and normal cells after 48 h of treatment with  3-BP (n=5  mice per condition). Statistics were calculated with multiple t-tests. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers minimum to maximum. Number of replicates corresponds to individual mice  used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).",
    "molecules": "3-BP"
  },
  {
    "caption": "D, (Main) Extracellular glucose abundance changes upon treatment with  3-BP  in all three populations (n=4 mice  per condition). Values represent the ratio of glucose abundance in 3-BP-treated versus untreated cells. Statistics were calculated using the limma package in R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). (Insert) Fractional labeling of lactate  in untreated and 3-BP treated normal (n=2) and residual (n=2) cells following cultivation in growth medium supplemented with [U-13C] glucose. The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted. Statistics were calculated with unpaired two-samples t-tests. Data information: , Box plots: midline, median; box, 25-75th percentile minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).",
    "molecules": "3-BP, carbon, [U-13C] glucose, glucose, lactate"
  },
  {
    "caption": "(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "(c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition).",
    "molecules": "ebselen"
  },
  {
    "caption": "(d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.",
    "molecules": "ebselen, ROS"
  },
  {
    "caption": "(e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.",
    "molecules": "DMSO, ebselen"
  },
  {
    "caption": "PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.",
    "molecules": "bafilomycin A1, ebselen, N-acetylcysteine, rapamycin"
  },
  {
    "caption": "Wild-type (WT) RAD51 SPR curves for ssDNA dN-50 and dsDNA dN-50p. RAD51 was injected at 150 nM in the presence of 2.5 mM ATP (pH 7.5) and 10 mM CaCl2. The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides (N) are plotted versus time, as measured by SPR, following the equation N = S / (L * (MRAD51 / MDNA)), where S is the signal in RU units (1 RU ~ 50 pg/mm2), L is the amount of DNA ligand immobilised onto the experimental flow cell (RU), MRAD51 is the molecular weight (kDa) of RAD51 (~ 37 kDa) and MDNA is the molecular weight (kDa) of the immobilised DNA molecule.",
    "molecules": "ATP, CaCl2, DNA, dsDNA, ssDNA"
  },
  {
    "caption": "Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction. Wild-type (WT) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics.",
    "molecules": "biotin, Biotinylated, ssDNA, streptavidin"
  },
  {
    "caption": "The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides, as measured by SPR (see Fig. 1 for equation) and the ODE model fits (see Fig. 3A for model description), respectively.",
    "molecules": "ssDNA"
  },
  {
    "caption": "As in A, except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics.",
    "molecules": "biotinylated, dsDNA"
  },
  {
    "caption": "As in B, the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides, as measured by SPR and the ODE model fits",
    "molecules": "dsDNA"
  },
  {
    "caption": "Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels C and D, the KD was fixed to the mean value identified in Figs. 3 A and B (i.e. KD = 1.14 nM), and the concentration of WT RAD51 monomer concentration was varied accordingly. In panel D, the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % WT RAD51 = [n-mer] * n / [WT RAD51monomer]). [WT RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. It is important to note that the assumed maximum polymer length in solution (16-mer) leads to an overestimation of the concentration of each n-mer in solution, given that RAD51 is likely to form polymers of length greater than 16 at 150 nM, 3 µM and 30 µM WT RAD51 monomer concentration. However, this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration.",
    "molecules": "coomassie, DNA"
  },
  {
    "caption": "Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction, and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics. The flexibility of respective DNA, the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer-protomer binding energy contribution of WT RAD51 are indicated in gray, green and blue boxes. H and L in each box denote high and low, respectively.",
    "molecules": "biotin, Biotinylated, DNA, streptavidin"
  },
  {
    "caption": "The mean number of WT RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and ODE model fits (solid lines).",
    "molecules": "DNA"
  },
  {
    "caption": "As in Fig. 4B, but F86E RAD51 was injected at the indicated concentration to measure association and dissociation kinetics. The flexibility of respective DNA, the expected relative entropic penalties upon F86E RAD51 binding to each DNA molecule and protomer-protomer binding enegy contribution of F86 RAD51 are indicated in gray, green and blue boxes. H and L shown in each box indicate high and low, respectively.",
    "molecules": "DNA"
  },
  {
    "caption": "The mean number of F86E RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and corresponding model fits (solid lines) using the model shown in panel D. Using ABC-SMC, individual forward rates (kp) and reverse rates (kpr) were fitted to each experimental curve, while a single KD was fitted to all experimental curves.",
    "molecules": "DNA"
  },
  {
    "caption": "Predicted concentrations of F86E RAD51 polymers in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % F86E RAD51 within each polymer state in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels F and G, the KD was fixed to the mean value identified in Fig. 5D (i.e. KD = 10.2 µM), and the concentration of F86E RAD51 monomer concentration was varied accordingly. In panel G, the % F86E RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % F86E RAD51 = [n-mer] * n / [F86E RAD51monomer]). [F86E RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. Bar plots of fitted kpr values for the SPR curves. Mean values of mode particles ± 1 SD derived from the ABC-SMC fits (n=3). Un-paired, one-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.",
    "molecules": "coomassie, DNA"
  },
  {
    "caption": "(B,C) Viability of L929 parental or mCD20-expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH (B) and viability of MCF7 parental or hCD20-expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH (C). Cell viability was measured via an ATP luminescence assay. Each point is the mean of three replicates and error bars are SEM.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Immunohistochemical staining for PECAM-1 (red), ICAM-1 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 7µg wt mTNF or 50µg of mCD13-AFR. Scale bar is 50µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(F) B16Bl6 tumor growth after daily p.l. treatment with indicated doses of mCD13-AFR, Wortmannin (Wm), Birinapant (Bir) or combinations thereof. Wortmannin and Birinapant were given 1 hour before CD13-AFR. Tumor growth is shown as mean TSI + SEM (n = 4 for Wm and Bir, 5 for other groups). The line under the graph represents the treatment period. TSI of individual tumors of the indicated treatment groups at day 17 after tumor inoculation.",
    "molecules": "Bir, Birinapant, Wm, Wortmannin"
  },
  {
    "caption": "(D) Flow cytometric analysis of hCD45+hCD3+eGFP+ CAR T-cells in SKOV3 tumors on day 18 after tumor inoculation. Mice were injected with 6 x 106 CAR T-cells on day 12 and treated with PBS or CD13-AFR",
    "molecules": "PBS"
  },
  {
    "caption": "(E) Viability of B16Bl6 parental or mCD20-expressing cells after 72h stimulation with wt mIFN-γ or del8 mutant fused to a BcII10 or mCD20 VHH, in the presence of 60ng/ml mTNF. Cell viability was measured via an ATP luminescence assay.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Immunohistochemical staining for PECAM-1 (red), cleaved Caspase-3 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 50µg mCD13-AFR and 23.8µg mCD13-AFN-II. Scale bar is 100µm.",
    "molecules": "DNA"
  },
  {
    "caption": "A Activity of an NF-κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 (hTLR4) or an isogenic control cell line that does not express TLR4 (null2) and were stimulated with vehicle (veh), 1 ng/mL LPS, 400 μM nickel chloride, or 25, 50 or 100 μM platinum(II) chloride and platinum(IV) chloride (n=3 independent biological replicates). B As per panel (A) but secreted IL-8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg/mL LPS, 200 μM nickel chloride, 100 μM platinum(II) chloride or 100 μM platinum(IV) chloride (n=4 independent biological replicates). Data Information: For all panels actual individual data from each experiment are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers. Statistical analyses were assessed by 2-way ANOVA: in A) hTLR4 compared to null2 cells; in B) agonist treatment compared to non-treated (nil); ns, not significant ; **, P<.01; ; ****, P<.0001 (Dunnett's test, A, B",
    "molecules": "LPS, nickel chloride, platinum(II) chloride, platinum(IV) chloride"
  },
  {
    "caption": "A IL-8 secretion in HEK cells stably expressing hTLR4 but not MD-2 (HEK-isoTLR4), transfected with empty vector (EV) or MD-2 and left untreated (nil) or treated with 1 ng/mL LPS, 200 μM nickel chloride, 2 μg/ml HMGB1, 100 μM platinum(II) chloride, 100 μM platinum(IV) chloride or 25 μM cisplatin (n=3 or 4 independent biological replicates). C IL-8 secretion in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA and left untreated (nil), or treated with 30 μM cisplatin (n=3 independent biological replicates). Mock cells were not subject to siRNA treatment prior. Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers (A, C) Statistical analyses were determined in comparison to nil treatments using 2-way (A, C) ns, not significant; *, P<.05; ****, P<.0001 (Dunnett's test).",
    "molecules": "cisplatin, LPS, nickel chloride, platinum(II) chloride, platinum(IV) chloride"
  },
  {
    "caption": "A- HEI-OC1 cells containing a Tlr4 deletion (Tlr4-/-) were compared to HEI-OC1 non-targeting (NT) control cells and assessed for cell viability (A), AnnexinV/propidium iodide staining (B), following cisplatin treatment at the indicated concentrations (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to NT at the same cisplatin concentration were assessed by 2-way (A, B) *, P<.05; **, P<.0001(Bonferroni test).",
    "molecules": "cisplatin, propidium iodide"
  },
  {
    "caption": "A IL-6 secretion in HEI-OC1 cells treated with 100 pg/mL LPS or 20μM cisplatin (n=3 or 4 independent biological replicates). B IL-6 secretion in TLR4-/- cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatment with 20μM cisplatin (n=6 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).",
    "molecules": "cisplatin, LPS"
  },
  {
    "caption": "C Il6 and Tlr4 transcript levels in HEI-OC1 cells following treatment with 20μM cisplatin for the indicated times (n=3 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test). ",
    "molecules": "cisplatin"
  },
  {
    "caption": "B Hair cell viability in larval zebrafish pre-treated with control-, tlr4ba- and/or tlr4bb-targeting morpholino oligonucleotides (MO) and subsequently treated with 15 μM cisplatin. Data information: In both panels each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons to control morpholino (B, except as indicated in blue) were assessed by one-way ANOVA. *, P<.05; **, P<.01; ****, P<.0001 (Tukey test).",
    "molecules": "cisplatin, MO, morpholino, morpholino oligonucleotides"
  },
  {
    "caption": "A IL-6 secretion in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 10 ng/mL LPS or 20μM cisplatin (n=5-7 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by 2-way (A) or one-way *, P<.05 ; ****, P<.0001(Dunnett's test).",
    "molecules": "cisplatin, LPS, DMF, TAK, TAK242"
  },
  {
    "caption": "B ROS generation in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 20μM cisplatin (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by one-way (B) ANOVA. ; **, P<.01 P<.0001(Dunnett's test).",
    "molecules": "cisplatin, DMF, ROS, TAK, TAK242"
  },
  {
    "caption": "(L and M) Immunostaining assay  of  GABA (green) in control and V1-PAKI  cells on day 35 of  ventral neural differentiation (L), and quantification of GABA+ cells (M), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).",
    "molecules": "GABA"
  },
  {
    "caption": "(F and G)  Immunostaining assay of GABA (green) in control and SOX1KO cells on day 35 of  ventral neural differentiation (F), and quantification of GABA+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).",
    "molecules": "GABA"
  },
  {
    "caption": "(H and I)  Immunostaining assay of MAP2 (green) after SOX1 overexpression in V1-PAKI cells on day 35 of dorsal neural  differentiation (H), and quantification of MAP2+ cells (I), p=0.0001 (V1-PAKI), p=0.0001 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).",
    "molecules": "dox"
  },
  {
    "caption": "(J and K) Immunostaining assay  of GABA (green) after SOX1 overexpression in V1-PAKI cells on day 35 of  ventral neural differentiation (J), and quantification of GABA+ cells (K), p=0.0001 (V1-PAKI), p=0.0002 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).",
    "molecules": "GABA, dox"
  },
  {
    "caption": "(C  Immunostaining assay of SOX1 (red) after 4  HDAC inhibitors (4i), including RG2833  (2833, 2μM), CAY10683 (CAY, 2μM), PCI-34051 (34051, 5μM) and Bufexamac (Buf, 12.5μM), treatment or remove one inhibitor from these 4 HDAC inhibitors (4i) in V1-PAKI  cells on day 16 of dorsal neural differentiation Scale bar, 100 μm.",
    "molecules": "Buf, Bufexamac, 34051, PCI-34051, 2833, RG2833, CAY, CAY10683"
  },
  {
    "caption": "(G and H) Immunostaining assay of  SOX1  (red) after Bufexamac treatment in V1-PAKI cells on day 16 of  dorsal neural differentiation (G), and quantification of  SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI  group ; (one-way ANOVA).",
    "molecules": "Buf, Bufexamac"
  },
  {
    "caption": "(J and K) Immunostaining assay  of MAP2 (green) after Bufexamac  treatment in V1-PAKI  cells on day 35 of dorsal neural  differentiation (J), and quantification of MAP2+ cells (K), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group (one-way ANOVA).",
    "molecules": "Buf, Bufexamac"
  },
  {
    "caption": "(L) Enrichment of   RNA polymerase Ⅱ   on  SOX1 promoter region after Bufexamac treatment in V1-PAKI  cells on day 16 of  dorsal  (left, p=0.0007 (V1-PAKI), p=0.0002 (+Buf)) and ventral (right, p=0.0083 (V1-PAKI), p=0.0276 (+Buf)) neural  differentiation. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).",
    "molecules": "Buf, Bufexamac"
  },
  {
    "caption": "(G and H)  Immunostaining assay of SOX1  (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of  dorsal neural differentiation (G), and quantification of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of SOX1 (red) after  SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of ventral neural  differentiation (I), and quantification of SOX1+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus -dox group (one-way ANOVA).",
    "molecules": "dox"
  },
  {
    "caption": "(E and F) Immunostaining assay  of ASCL1  (green) after SOX1 overexpression in V1-PAKI cells on day 25 of  dorsal neural differentiation (E), and quantification of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (G and H)  Immunostaining assay  of  ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantification of  ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (I and J)  Immunostaining assay of MAP2 (green) after  ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural  differentiation (I), and quantification of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. (K and L)  Immunostaining assay  of GABA (green) after  ASCL1  overexpression in V1-PAKI  cells on day 35 of ventral neural differentiation (K), and quantification of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox  group (one-way ANOVA).",
    "molecules": "GABA, dox"
  },
  {
    "caption": "(E and F) Immunostaining assay  of SOX1  (red) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal (E) and ventral (F) organoids differentiation. Scale bar, 100 μm. (G)  Immunostaining assay of MAP2  (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of  dorsal organoids differentiation. Scale bar, 100 μm. (H) Immunostaining assay  of GABA (green) after SOX1-OT V1 overexpression in V1-PAKI  cells on day 40 of  ventral organoids differentiation. Scale bar, 100 μm.",
    "molecules": "GABA"
  },
  {
    "caption": "D) SDS-PAGE analysis of purified tubulin from brains of all mouse models used in the current study, stained with Coomassie Brilliant-Blue. Alpha- and β-tubulin are separated using a specific SDS-PAGE protocol",
    "molecules": "Coomassie Brilliant-Blue"
  },
  {
    "caption": "B) Representative IRM and TIRF microscopy images showing microtubule positions and Tau-meGFP intensities for different PTM variants (grey: wild type; blue: Ttll1-/-; yellow: Ttll7-/-; green: Ttll1-/-Ttll7-/-; pink: Atat1‑/‑). In the last column, false-coloured TIRF images highlight differences of Tau-meGFP intensities on different types of microtubules (Movie EV1). C) Quantification of integrated intensity of Tau-meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples. Single assays (Fig EV2A) of one experiment were combined after normalisation to the wild-type microtubule values (A.U. = arbitrary units). Each data point represents the normalised fluorescence intensity value of one microtubule. D) Summary quantification the three independent experiments shown in (C). Each point is a normalised mean of the triplicates, error bars represent standard deviation.",
    "molecules": "GMPCPP"
  },
  {
    "caption": "E) Saturation curve of increasing concentration Tau-mCherry binding to GMPCPP wild-type and Ttll1-/-Ttll7-/- microtubules. Each data point is shown as mean ± standard deviation of the raw integrated intensity values (A.U. ×104) of three experiments with independently purified tubulin samples (Fig EV2D). Dissociation constants Kd and R2 coefficient from non-linear fits of each microtubule type are indicated below the plot.",
    "molecules": "GMPCPP"
  },
  {
    "caption": "Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of tumor progression (average±SD) Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).",
    "molecules": "DC, DC101, Beva"
  },
  {
    "caption": "Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of (C) tumor weight at sacrifice, (D) vessel number, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).",
    "molecules": "DC, DC101, Beva"
  },
  {
    "caption": "Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of percentage of necrotic area by histology, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).",
    "molecules": "DC, DC101, Beva"
  },
  {
    "caption": "Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of survival by Kaplan Meier plot in control and treated animals. Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).",
    "molecules": "DC, DC101, Beva"
  },
  {
    "caption": "G) Representative invasive front profile of 786-O control and short-term DC and Beva treated tumors in vimentin stained sections (4X). Front (red line) and rear (white line) edges of tumor protrusion into kidney determine the depth of invasion (green arrow) perpendicularly connecting red and white lines. Box plot shows the quantification of invasion indicating median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) in treated vs control tumors (7 samples/group; *p < 0.05 by Mann-Whitney test).",
    "molecules": "DC, Beva"
  },
  {
    "caption": "Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of tumor progression average and SD in control (black) compared to DC (red, *p < 0.05) and Beva (blue, # p < 0.05) treatments from 5-13 animals per tumor and treatment group by Mann Whitney test.",
    "molecules": "DC, Beva"
  },
  {
    "caption": " Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor weight in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers. ",
    "molecules": "DC, Beva"
  },
  {
    "caption": " Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of vessel number in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers. ",
    "molecules": "DC, Beva"
  },
  {
    "caption": " Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor necrosis in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers. ",
    "molecules": "DC, Beva"
  },
  {
    "caption": " Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of overall survival comparing Control (black), DC101 (red) and Beva (blue) treated mice from 4-13 mice/tumor/treatment group by Mantel-Cox test where *p < 0.05, **p < 0.01 and ***p < 0.001. ",
    "molecules": "DC, DC101, Beva"
  },
  {
    "caption": "Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on capsular invasion Incidence of Capsular invasion (stripes) in each model and treatment. 5-8 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01.",
    "molecules": "DC, Beva"
  },
  {
    "caption": " C) Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on tumor front of invasion Representative images of Vimentin staining (4X), as described in Figure 1, and box plot quantification of fold-invasion (∆ invasion) of treated vs control tumors indicating median, Q1/Q3 and max/min value whiskers. 6-12 animals per model and treatment group were analyzed by Mann-Whitney test where *p < 0.05, **p < 0.01. ",
    "molecules": "DC, Beva"
  },
  {
    "caption": "D) Effects of sunitinib treatment on Ren13BM and Ren28 on capsular invasion and tumor front invasion. Bar graphs show incidence of capsular invasion (stripes) in control and sunitinib treated tumors. 3-9 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01. Box plots represent median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) of sunitinib-treated vs control tumors. 5-8 animals per model and treatment group were analyzed by Mann-Whitney test. *p < 0.05, **p < 0.01.",
    "molecules": "sunitinib"
  },
  {
    "caption": "Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (C) Representation of ALDH1A3 expression on pro-invasive and non-pro-invasive Ren-PDOX after suntinib treatment (**p < 0.01 by Chi-Square test).",
    "molecules": "sunitinib, suntinib"
  },
  {
    "caption": " D) Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (D) Correlation of pre-treatment ALDH1A3 protein levels by IHC with increased tumor invasion found after sunitinib treatment. n=7, Spearman's non-parametric correlation, p=0.0095. ",
    "molecules": "sunitinib"
  },
  {
    "caption": "(d) LC3 levels in infected MDCK cells. MDCK cells were infected with wild-type or ΔactA2 bacteria for the indicated times. EBSS (Earle's balanced salt solution, a starvation medium) and rapamycin (100 μg ml−1) were used as controls.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(e-f) Intracellular survival of wild-type or ΔactA2 bacteria in MDCK cells (e; n = 8), MDCK cells treated with 10 mM 3-MA (f; n = 4),",
    "molecules": "3-MA"
  },
  {
    "caption": "(g) Immunogold electron micrograph of MDCK/pGFP-LC3 cells infected with ΔactA2. Gold particles of 5 nm (arrowheads) indicate GFP-LC3, and gold particles of 10 nm (arrows) indicate ubiquitin. Scale bars, 0.5 μm.",
    "molecules": "gold particles"
  },
  {
    "caption": "(h) Kinetics of the generation of GFP-LC3-positive or ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). *P 0.001. (i) Quantification of the number of ubiquitin-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3 cells infected with wild-type or ΔactA2 bacteria were untreated or treated with 3-MA (10 mM). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n ≥ 3 experiments). Ub, ubiquitin. WT, wild type.",
    "molecules": "3-MA"
  },
  {
    "caption": "Ala/Phe substitutions of aa 239-244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity. Aβ38, Aβ40, and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA. Aβ(38+40)/42 ratio was calculated to determine GSEC processivity towards APPC99. DKO PSEN1/PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references.",
    "molecules": "Aβ38, Ala, Aβ(38, Aβ40, Aβ42, Phe"
  },
  {
    "caption": "Representative SDS-PAGE/western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits. The presence of mature, glycosylated NCT, N-terminal and C-terminal fragments of the endoproteolysed PSEN1 and PEN-2, compared to NCT knock-out (KO) cells, indicate that WT and mutated NCTs reconstitute GSEC complexes. Arrowheads indicate the position of molecular weight markers.",
    "molecules": "CHAPSO"
  },
  {
    "caption": "Quantification of the effects of the indicated substitutions of aa 241, 242, and 244 in NCT on GSEC processivity of APPC99, estimated as indicated above by the Aβ(38+40)/42 ratio. Figure EV1B shows the corresponding Aβ42/40 ratios.",
    "molecules": "Aβ(38, Aβ42"
  },
  {
    "caption": "Relative GSEC-APP/Aβn stabilities were assessed in cell-based GSEC thermoactivity assays, which evaluate changes in GSEC processivity (Aβ(38+40)/42 ratio) upon incubation at 42°C for 24h, relative to 37°C. Aβ peptides in the conditioned medium collected from the indicated MEF cell lines (transiently expressing APPC99) were quantified and the Aβ(38+40)/42 ratios determined. The data are shown as mean ± 95% CI, as % of the WT GSEC cell line incubated at 37°C for 24h, N = 6 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine the statistical significance (F(DFn, DFd): F(6, 46) = 100); ****p > 0.0001 compared to WT at 37°C, #p > 0.05, ##p>0.01, ####p>0.0001 compared to WT at 42°C. Note: Aβ profiles (including Aβ43 levels) normalized to total Aβ quantified in the cell-based thermoactivity assays with the different mNCT WT/mutants are shown in Figure EV2.",
    "molecules": "Aβ43, Aβ, Aβ(38"
  },
  {
    "caption": "Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.",
    "molecules": "Aβ, Aβ37, Aβ38, Aβ40, Aβ42"
  },
  {
    "caption": "To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions, the ratio of ((Aβ 37/40) + (Aβ 38/42))/2 was determined and used to estimate GSEC processivity of APPC99. All the data are presented as mean ± SD, N ≥ 3, Unpaired, two-tailed Student's t-test was used to determine the statistical significance. **p > 0.01, ****p > 0.0001. Calculated T- and Df values were: T = 11,63; Df = 16 (I241-K28 vs I241E-K28), T = 11,37; Df = 14 (I241-K28 vs I241-K28E), T = 4,572; Df = 8 (I241E-K28 vs I241-K28E), T = 4,497; Df = 5 (I241-K28E vs I241K-K28E), T = 2,561; Df = 7.",
    "molecules": "Aβ 37, Aβ 38, Aβ37, Aβ38, Aβ40, Aβ42"
  },
  {
    "caption": "Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C",
    "molecules": "Aβ, BMPS, Cys"
  },
  {
    "caption": " Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT -APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT- I241C andAPPC99 K28C (mature and immature NCT are indicated with black and white arrows, respectively. The filled orange arrow points at the high molecular weight APPC99/Aβ density (crosslinked NCT-APPC99/Aβ band). Note that peak amplitudes for the cross-linked NCT-APPC99 and the mature NCT band were normalized to similar values for clarity purposes. Full-range molecular weight density profiles are shown in Figure EV3D.",
    "molecules": "Aβ, BME, β-mercaptoethanol"
  },
  {
    "caption": "Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.",
    "molecules": "Aβ, Aβ37, Aβ38, Aβ40, Aβ42, DMSO"
  },
  {
    "caption": "Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).",
    "molecules": "Aβ 37, Aβ 38"
  },
  {
    "caption": "(a) The fate of internalized cells (control MCF10A and MCF10A expressing E7 and Bcl2) was measured with and without autophagy inhibition. Data show the percentage of internalized cell release with autophagy inhibition, compared with each control without autophagy inhibition; mean±s.e.m. from at least three independent experiments (total cell numbers analysed: ATG5 siRNA, 261 cells; Bcl2 ATG5 siRNA, 97 cells; 3-MA, 170 cells; Bcl2 3-MA, 112 cells); *P0.05, **P0.02.",
    "molecules": "3-MA"
  },
  {
    "caption": "(b) Effects of Y27632, 3-MA and VPS34 and ATG5 siRNA on MCF10A cell-in-cell formation in suspension for 7 h; >300 cells per condition were scored for cell-in-cell formation; ±s.e.m.from three independent experiments; **P0.004.",
    "molecules": "3-MA, Y27632"
  },
  {
    "caption": "(B) Chlamydomonas reinhardtii (Cr) UBA5  and UFC1  are active E1  and E2  enzymes. SDS-PAGE analysis showing transfer of UFM1  to CrUBA5  and CrUFC1. The gels are run in non-reducing conditions except where otherwise specified. The presented gel is representative of two independent experiments. BME: β-mercaptoethanol; ATP: Adenosine triphosphate.",
    "molecules": "Adenosine triphosphate, ATP, BME, β-mercaptoethanol"
  },
  {
    "caption": "(C) RPL26  mono- and  di-UFMylation  is lost in Chlamydomonas reinhardtii (Cr) uba5  and ufl1  mutants. Liquid TAP cultures were either left untreated (control) or treated for 24 hours with 200 ng/mL tunicamycin. Protein extracts were analyzed by immunoblotting  with anti-UFM1  antibodies. Total proteins were analyzed by Ponceau S staining. 12 hours and 24 hours treatment replicates are shown in Fig. S3C. Right Panel, Quantification of UFMylated RPL26. RPL26 mono-UFMylated; RPL26-(UFM1)2: RPL26 di-UFMylated.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(D) Chlamydomonas reinhardtii   (Cr) UFMylation  pathway mutants are sensitive to ER  stress triggered by tunicamycin. Liquid TAP cultures of wild type (wt), uba5, ufl1  and ire1  mutants were either left untreated (control) or treated for 3 days with 200 ng/mL of tunicamycin. Left panel, representative images of control and treated liquid cultures taken 3-days after incubation. Middle Panel, optical density (OD) 600  (OD600) quantification of each genetic background under control conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. Right Panel, normalized OD600  quantification of each genetic background under tunicamycin  treatment conditions. Bars represent the mean (± SD) of 5 biological replicates. Two-tailed unpaired t-tests were performed to analyze the differences between wild type and mutants. ns, p-value > 0.05; ***, p-value < 0.001. BR: Biological Replicate.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(F, G) C53  IDR is essential for UFM1 interaction. HsC53 (B) and AtC53 (C) IDRs are necessary to mediate the interaction  with AtUFM1 and HsUFM1 respectively. MBP-AtC53ΔIDR: MBP-AtC53(1-239, (KGSGSTSGSG)2, 373-549); MBP-HsC53ΔIDR: HsC53(1-262, (KGSGSTSGSG), 317-506). F, G) Bacterial lysates containing recombinant protein were mixed and  pulled down with glutathione  magnetic agarose  beads. Input and bound proteins were visualized by immunoblotting  with anti-GST  and anti-MBP  antibodies.",
    "molecules": "agarose, glutathione"
  },
  {
    "caption": "(B) The cAIM peptide cannot outcompete  HsUFM1-HsC53 interaction. Bacterial lysates containing recombinant protein were mixed and  pulled down with glutathione  magnetic agarose  beads. Input and bound proteins were visualized by immunoblotting  with anti-GST  and anti-MBP  antibodies. cAIM peptide was used to a final concentration of 200 µM. HsC53sAIM: HsC53W269A, W294A, W312A.",
    "molecules": "agarose, glutathione"
  },
  {
    "caption": "(C, D) Microscopy-based protein-protein interaction assays  showing unlike GABARAP-C53 interaction, UFM1-C53 interaction  is insensitive to cAIM  peptide competition. Glutathione-sepharose  beads were prepared by incubating them with GST-GABARAP  (C) or GST-HsUFM1  (D). The pre-assembled beads were then washed and mixed with 1 µM of HsC53 containing increasing concentrations of cAIM  peptide (0-100 µM). The beads were then imaged using a confocal microscope. Left Panel, representative confocal  images (inverted grayscale) for each condition are shown. Right panel, normalized fluorescence is shown for each condition with the mean (± SD) of 4 replicates. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and wild type with 100 µM AIM  peptide. ns, not significant, p-value > 0.05, ***, p-value < 0.001.",
    "molecules": "sepharose, Glutathione"
  },
  {
    "caption": "(B, Conversion of sAIM into cAIM leads to reduced UFM1 binding  and stronger ATG8 interaction. Bacterial lysates containing recombinant proteins were mixed and pulled down with glutathione  magnetic agarose  beads. Input and bound proteins were visualized by immunoblotting  with anti-GST  and anti-MBP  antibodies. AtC53sAIM: AtC53 (W276A, W287A, W335A); AtC53cAIM: AtC53(IDWD274WDDI, IDWD285WDDI, IDWD333WDDI); ATG8LDS: ATG8YL50AA.",
    "molecules": "agarose, glutathione"
  },
  {
    "caption": "(G)  AtC53cAIM forms more GFP-ATG8A colocalizing  puncta upon ER  stress. Upper Panel, representative confocal  images of transgenic Arabidopsis  seedlings co-expressing C53-mCherry  (magenta), C53sAIM-mCherry and C53cAIM-mCherry  with GFP-ATG8a  in c53  mutant background under normal condition and after tunicamycin  stress. 6-day old seedlings were incubated in liquid 1/2 MS medium with 1% sucrose  supplemented with DMSO  as control or tunicamycin  (10 μg/ml) for 6 hours before imaging. Scale bars, 30 μm. Inset scale bars, 10 μm. Right Panel, Quantification of the C53-autophagosomes  (C53-APG) per normalized Z-stacks. Bars represent the mean (± SD) of at least twenty roots from 3 biological replicates for each genotype and treatment. Unpaired two-samples Wilcoxon test with continuity correction was performed to analyze the differences between wild type and mutants. ***, p-value < 0.001.",
    "molecules": "DMSO, sucrose, tunicamycin"
  },
  {
    "caption": "D Ratio of toxicity and viability of HCT116,  U87 and BxPC3  cells grown in spheroids  (3D) and treated as indicated for 24h (BxPC3) or 48h (HCT116, U87) (NEN  1.2 µM; Domperidone  (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM). Data information: Data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.",
    "molecules": "Ami, Amitriptyline, Clomi, Clomipramine, Desi, Desipramine, Domp, Domperidone, Imi, Imipramine, NEN"
  },
  {
    "caption": "B  Immunoblot analysis of CHOP, ATF4, phosphorylated  (p-) and t-)  eIF2alpha (eIF2a) of HCT116 cells treated as indicated for 16h (NEN  (N) 1.2 µM; Domperidone (Domp), Amitriptyline (Ami), each 30 µM).",
    "molecules": "Ami, Amitriptyline, Domp, Domperidone, NEN"
  },
  {
    "caption": "F Ratio of toxicity and  viability in HCT116 spheroids  treated as indicated for 48h (NEN  1.2 µM, Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM, ISRIB 1 µM).",
    "molecules": "Ami, Amitriptyline, Clomi, Clomipramine, Desi, Desipramine, Domp, Domperidone, Imi, Imipramine, ISRIB, NEN"
  },
  {
    "caption": "A Relative  mRNA  expression levels of indicated CLEAR network genes in HCT116 cells treated as indicated ((NEN (N) 1.2 µM; Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi) and Amitriptyline  (Ami), each 30 µM; Clomipramine  (Clomi) 20 µM), determined by qPCR. Data information: In (A) (first (TFE3) and third (CD68) graph (N=3); second (TFE3) and fourth (CD68) graph (N=5, besides NEN+Clomi  (N=4) and NEN+Desi  (N=4)) data are presented as mean (SD) and were analyzed by a one-way ANOVA with Tukey post-hoc test. In the second and the fourth graph, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls (Ctrl). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.",
    "molecules": "Ami, Amitriptyline, Clomi, Clomipramine, Desi, Desipramine, Domp, Domperidone, Imi, Imipramine, NEN, TCAs"
  },
  {
    "caption": "B Immunofluorescence (IF) of HCT116 cells treated with DMSO (Control (Ctrl)), NEN, Domperidone (Domp) or NEN+Domp as indicated (NEN, 1.2 µM; Domp, 30 µM) for 16 h and stained with anti-Lamp1 (top panel), Alexa-555 LC3  (middle panel) co-stained with Alexa-647 p62 (bottom panel). Nuclei, Blue (dapi). Scale bar 20 µm. Mean (SEM) fluorescence of Lamp1 (Ctrl, NEN, Domp  (N=20 cells); NEN+Domp (N=24 cells)), LC3-II (Ctrl, NEN+Domp (N=16 cells)); NEN, Domp (N=16 cells)) or P62 (Ctrl (N=38 cells); NEN (N=27 cells); Domp (N=28 cells); NEN+Domp (N=21 cells)) was quantified and analyzed by one-way ANOVA with Tukey post-hoc test (for  LC3-II) or Kruskal-Wallis with Dunn´s post-hoc test (for  Lamp1  and p62). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.",
    "molecules": "Alexa-555, Alexa-647, dapi, DMSO, Domp, Domperidone, NEN"
  },
  {
    "caption": "C Relative UPP1 mRNA expression  levels in  HCT116 cells either transfected with control (siCtrl) or combined  TFE3- and MITF- targeting (siTFE3/siMITF) siRNAs and treated as indicated (NEN  (N) 1.2 µM, Domperidone  (D) 30 µM, ISRIB 1 µM), determined by qPCR. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test",
    "molecules": "Domperidone, ISRIB, NEN"
  },
  {
    "caption": "F  Immunoblot  of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN (low) 0.6 µM or NEN (High) 1.2 µM, Domperidone  (Domp) 30 µM) Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 48h prior to treatment.",
    "molecules": "Domp, Domperidone, NEN"
  },
  {
    "caption": "H Ratio of  toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM). Cells were transfected with control (siCtrl) or  UPP1-targeting (siUPP1) siRNAs 24h prior to spheroid formation. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test",
    "molecules": "Ami, Amitriptyline, Desi, Desipramine, Imi, Imipramine, NEN"
  },
  {
    "caption": "A Relative mRNA expression levels of DHODH  in HCT116  cells treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi), Amitriptyline  (Ami), each 30 µM, Clomipramine (Clomi) 20 µM). Data information: , data are presented as mean (SD) (N=3) and were analyzed by Kruskal-Wallis with Dunn´s post-hoc test (A, left graph), one-way ANOVA with Tukey post-hoc test (A, right graph) In (A), significance is indicated for the comparison of the different treatments to controls. ** p < 0,01; **** p < 0,0001.",
    "molecules": "Ami, Amitriptyline, Clomi, Clomipramine, Desi, Desipramine, Domp, Domperidone, Imi, Imipramine, NEN"
  },
  {
    "caption": "D Caspase activity in HCT116  cells treated as indicated for 24h (NEN 0.6 µM, Imipramine (Imi), Desipramine (Desi) and Amitriptyline  (Ami), each 20 µM, Clomipramine  (Clomi) 10 µM, A77126  (A77) 50 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test",
    "molecules": "Ami, Amitriptyline, Clomi, Clomipramine, Desi, Desipramine, Imi, Imipramine, NEN, A77, A77126"
  },
  {
    "caption": "A Immunofluorescence (IF) images of HCT116 cells treated as indicated for 12h (Imipramine and Domperidone, each 30 µM) and stained for cholesterol  content using filipin III. B Immunofluorescence (IF) images of HCT116 cells treated with  Domperidone  and  co-stained  for cholesterol  and  Lamp1. ",
    "molecules": "cholesterol, Domperidone, filipin III, Imipramine"
  },
  {
    "caption": "E Relative  mRNA  expression  levels of the specified genes in HCT116  cells, treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (D) 30 µM, Cyclodextrin (CyD) 0.75%). F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h (NEN 1.2 µM, Domperidone (Domp) 30 µM, Cyclodextrin (CD) 0.75%). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.",
    "molecules": "CD, Cyclodextrin, CyD, Domp, Domperidone, NEN"
  },
  {
    "caption": "A Ratio of toxicity and  viability  determined in pancreatic cancer-derived  organoids  from two  patients  ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between single and combined drug treatments as indicated (NEN 1.2 µM or 2.5 µM, Amitriptyline (Ami, A), Imipramine  (Imi, I), Desipramine (Desi, D), each 30 µM, Clomipramine (Clomi, C) 20 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.",
    "molecules": "Ami, Amitriptyline, Clomi, Clomipramine, Desi, Desipramine, Imi, Imipramine, NEN"
  },
  {
    "caption": "B Ratio of  toxicity and viability determined in pancreatic cancer-derived   organoids  from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between drug treatments as indicated to the control condition (NEN (N) 1.2 µM or 2.5 µM, Amitriptyline (A),  Imipramine (I) 20 µM, Paclitaxel  (Pacli), 2, 20 or 200nM). As references for sensitization effects to standard chemotherapy, the bars showing single Paclitaxel  treatments using the indicated concentrations are highlighted in blue. Bars for controls (Ctrl), Paclitaxel  (Pacli), NEN (N) alone and combined NEN+Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated. Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.",
    "molecules": "Amitriptyline, Imipramine, NEN, Pacli, Paclitaxel, TCA"
  },
  {
    "caption": "hGS interaction with NF135 (A), NF175 (B) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "hGS interaction with NF54 (C) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "A) Normalized percentage +/- s.d. of intracellular schizont size of NF54 (blue) and NF135 (green) after treatment of GS inhibitors at different concentrations of AIP. Median size of the schizonts were normalized to that of H2O-treated NF135 (which is set at 100%). AIP was added one day post invasion and kept on for 48 hours. Each dot represents a biological replicate where ≥100 schizonts were measured on day 5 p.i.. A two-way ANOVA was performed followed by a Tukey's multiple comparison test with the **** < 0.0001 to compare conditions within the same parasite strain. A two-way ANOVA was performed followed by a Sidak's multiple comparisons test with **** <0.0001 to compare conditions between parasite strains).",
    "molecules": "AIP, H2O"
  },
  {
    "caption": "B) Size difference between the GS positive (closed circle) and negative (open circle) NF135 schizonts after treatment of AIP. Each dot represents the median of a biological replicate where ≥100 schizonts were measured. Three biological replicates were performed with the median from each plotted +/- s.d.. A two-way ANOVA was performed followed by a Dunnett's multiple comparison test with *** = 0.0002 and **** = 0.0001.",
    "molecules": "AIP"
  },
  {
    "caption": "D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX (black), but not in BREX‐containing strain (red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen‐specific DNA are 293, 485, and 1,218 bp, respectively.",
    "molecules": "DNA"
  },
  {
    "caption": "B The system does not interfere with phage adsorption to bacterial cells. Strains either containing (red) or lacking (black) the BREX system were infected with phage Φ3T and then chloroform‐treated 15 min following infection. The culture was plated on Φ3T‐sensitive B. subtilis cells and plaques, representing extracellular, unadsorbed phages, were counted.",
    "molecules": "chloroform"
  },
  {
    "caption": "E Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645-95,416 in the phage genome. Each lane contains 200 ng total DNA.",
    "molecules": "DNA"
  },
  {
    "caption": "A, B   Virus titers in the   blood from  mice (n=5) fed an NSD (0.45% NaCl) or HSD (4% NaCl) for 30 days (A) or 7 days (B) and then intraperitoneally infected with VSV (1x108 PFU per gram body, 48 hrs) were analyzed by the TCID50 assay. Data information: Data represent mean and SEM of five biological replicates For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.",
    "molecules": "NaCl"
  },
  {
    "caption": "H RT-qPCR analysis of viral RNA levels in U937, THP1, HT1080, 2fTGH  and A549 cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). I RAW264.7 cells were infected with VSV/H1N1/SeV (MOI=1.0, 24 hrs) immediately after addition of  NaCl (+34 mM), or were transfected with  HBV-replicon or HCV-replicon and then treated with additional  NaCl  (+34 mM) for 12 hrs. Viral RNA levels were analyzed by  RT-qPCR. Data information: Data show the mean and SD of four or three biological replicates. For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.",
    "molecules": "NaCl"
  },
  {
    "caption": "A Western blot  analysis of VSV-G protein levels in Ifnar1+/+ or Ifnar1-/- MEF cells infected with  VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: are representative of at least two biological replicates",
    "molecules": "NaCl"
  },
  {
    "caption": "C  RAW264.7 cells were treated with mIFNβ (50 IU/ml, 20 hrs) immediately after addition of NaCl  (+17, 34 and 51 mM). After washing, cells were infected with  H1N1 (MOI=1.0, 24 hrs) and viral RNA levels were analyzed by RT-qPCR. Data information: Data represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not significant (p > 0.05), **p < 0.01 and ***p < 0.001.",
    "molecules": "NaCl"
  },
  {
    "caption": "F RT-qPCR analysis of Ifit1,  Isg54 and Isg15 mRNA levels in HEK293T pretreated with additional NaCl  (+34 mM) for 12 hrs and then treated with IFNα (1,000 IU/ml) as indicated. Data information: Data represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not significant (p > 0.05), **p < 0.01 and ***p < 0.001.",
    "molecules": "NaCl"
  },
  {
    "caption": "G RAW264.7  cells were treated with mIFNβ (500 IU/ml) for 12 hrs immediately after addition of  NaCl  (+34 mM) or control  ddH2O (Ctrl). Whole cell lysates were subjected to a quantitative proteomic analysis with a TMT2-plex method to identify the differentially expressed proteins. The upregulated and downregulated proteins were analyzed in the volcano plot. The most dramatically downregulated  ISG protein, Viperin  (Rsad2), was marked in the dotted frame.",
    "molecules": "NaCl"
  },
  {
    "caption": "H Western blot analysis of Viperin  protein in RAW264.7 treated with mIFNβ (100 and 500 IU/ml, 12 hrs) immediately after addition of NaCl  (+34 mM). I Western blot analysis of Viperin  protein in HT1080 treated with IFNα (1,000 IU/ml) and NaCl (+34 mM) as (H). J Western blot analysis of Viperin protein in MEF cells infected with  VSV (MOI=0.5 and 1.0, 24 hrs) immediately after addition of NaCl (+34 mM). K Western blot  analysis of Viperin protein in  THP1 infected with H1N1 (MOI= 1.0) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates",
    "molecules": "NaCl"
  },
  {
    "caption": "B Western blot  analysis of FH-Viperin protein in stable FH-Viperin-expressing  HeLa  cells pretreated with additional NaCl  (+34 mM) and then treated with  CHX (50 μM) for 6 and 12 hrs. Data information: Data are representative of at least two biological replicates.",
    "molecules": "CHX, NaCl"
  },
  {
    "caption": "C RAW264.7 were treated with  MG132 (10 μM) and additional NaCl (+34 mM), followed by mIFNβ (300 IU/ml) treatment for 12 hrs. Viperin  protein levels were analyzed by  immunoblotting. Data information: Data are representative of at least two biological replicates.",
    "molecules": "MG132, NaCl"
  },
  {
    "caption": "O Western blot analysis of  Flag-Viperin in stable Flag-Viperin-expressing HEK293T  cells transfected with shCtrl or shUSP33 and then treated with CHX (50 μM) as indicated. Data information: Data are representative of at least two biological replicates.",
    "molecules": "CHX"
  },
  {
    "caption": "D  Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co-transfected with  Flag-Viperin, HA-Ub  and shUSP33, then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates",
    "molecules": "NaCl, Ub"
  },
  {
    "caption": "E  Western blot  analysis of  Viperin levels in  2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 12 hrs) immediately after addition of  NaCl  (+34 mM). Data information: Data are representative of at least two biological replicates",
    "molecules": "NaCl"
  },
  {
    "caption": "G RT-qPCR analysis of VSV RNA levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: Data (G) represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not significant (p > 0.05). ***p < 0.001.",
    "molecules": "NaCl"
  },
  {
    "caption": "I Survival curves of 8-week-old Usp33-/- mice fed an NSD  (0.5% NaCl) or HSD (4% NaCl) for 7 days and then infected with VSV (1x108 PFU per gram body mouse, n=10). Data information: ; Data (I) show mean and SEM of ten biological replicates, and p value was calculated using logrank (Mantel-Cox) tests. For all statistical testing: NS, not significant (p > 0.05). ***p < 0.001.",
    "molecules": "NaCl"
  },
  {
    "caption": "G Immunoprecipitation analysis of  ubiquitination of Flag-USP33  in p97-WT or p97-KO 2fTGH cells co-transfected with  Flag-USP33 and HA-Ub and then treated with NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.",
    "molecules": "NaCl, Ub"
  },
  {
    "caption": "L Immunoprecipitation  analysis of Viperin ubiquitination in  HT1080 cells transfected with shCtrl or shp97 (1# or 2#) and then treated with IFNα (1,000 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates.",
    "molecules": "NaCl"
  },
  {
    "caption": "N Western blot analysis of  Myc-Viperin in HEK293T cells co-transfected with Myc-Viperin  and shCtrl or shp97, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.",
    "molecules": "NaCl"
  },
  {
    "caption": "E, F Mass spectrometry analysis of acetylation of Flag-p97  (E) in RAW264.7 transfected with Flag-p97 and then treated with additional NaCl  (+34 mM) or control ddH2O (Ctrl) for 12 hrs. The intensity of p97-K663 site was shown in (F).",
    "molecules": "NaCl"
  },
  {
    "caption": "L Western blot analysis of FH-USP33 levels in p97-KO  HEK293T cells co-transfected with Myc-p97 (WT or K663R) and Flag-USP33, and then treated with additional NaCl  (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.",
    "molecules": "NaCl"
  },
  {
    "caption": "M Immunoprecipitation  analysis of  Viperin ubiquitination in  p97-KO 2fTGH cells co-transfected with Myc-p97 (WT or K663R), Flag-Viperin and HA-Ub as indicated, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.",
    "molecules": "NaCl, Ub"
  },
  {
    "caption": "B Western blot analysis of USP33 in RAW264.7 cells cultured in media containing reduced concentration of NaCl  (-17 and -34 mM) for 12 hrs. C  Western blot  analysis of Viperin in HT1080 cells treated with  IFNα (1,000 IU/ml, 12 hrs) in media containing reduced concentration of NaCl  (-17, -34 and -51 mM). Data information: Data are representative of at least two biological replicates",
    "molecules": "NaCl"
  },
  {
    "caption": "D RAW264.7  cells were infected with  VSV (MOI=1.0, 12 hrs) in media containing reduced concentration of NaCl (-17 and -34 mM). Viral RNA  levels were analyzed by RT-qPCR. Data information: Data (D) represent mean and SD of four biological replicates For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test).",
    "molecules": "NaCl"
  },
  {
    "caption": "H Mice (n=5) were intraperitoneally infected with  VSV (1x108 PFU per gram body). 12 hrs post infection, mice were fed an NSD (0.45%  NaCl) or LSD (0.15% NaCl) for 3 days. Then  VSV RNA  levels in mouse spleen tissues were detected by RT-qPCR. I Virus titers in the mouse blood  (n=5) from (H) were analyzed by the TCID50 assay. Data information: Data show mean and SEM of five biological replicates. For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test).",
    "molecules": "NaCl"
  },
  {
    "caption": "Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. Cell supernatants were harvested at indicated time-points and cytokines measured by ELISA (B)",
    "molecules": "h9C12, IVIg"
  },
  {
    "caption": "Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. pro- and cleaved IL-1β was measured in whole cell lysates or in supernatants by western blot (C).",
    "molecules": "h9C12, IVIg"
  },
  {
    "caption": "HMDM were primed with 10 μg/ml pI:C for 2h, then stimulated for 16h with virus +/- antibody. Cytokines in the supernatants were measured by ELISA.",
    "molecules": "pI:C"
  },
  {
    "caption": "AdV-GFP was incubated with 20 ug/ml h9C12 antibody or 20 mg/ml IVIg in PBS. These AdV-Ab complexes were then diluted 1:2 to give final doses of viral particles being added to WT THP-1 cells (with antibody complexed) as indicated. A single experiment representative of 3 independent experiments is shown.",
    "molecules": "h9C12, IVIg"
  },
  {
    "caption": "THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release",
    "molecules": "h9C12, IVIg"
  },
  {
    "caption": "THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay",
    "molecules": "h9C12, PrestoBlue, IVIg"
  },
  {
    "caption": "HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release",
    "molecules": "h9C12, IVIg"
  },
  {
    "caption": "HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay",
    "molecules": "h9C12, PrestoBlue, IVIg"
  },
  {
    "caption": "AdV-mCherry was added to AdV-GFP at a ratio of 1:200 and 50 000 pp/cell of this mix was complexed with h9C12 and IVIg and indicated doses. HMDM were stimulated with these complexes for 16h and infection measured as mCherry fluorescence by flow cytometry. In virus alone the percentage of mCherry positive cells was ~ 20%, the relative infection with antibodies was normalised to virus alone as 100%.",
    "molecules": "h9C12, IVIg"
  },
  {
    "caption": "HMDM were stimulated for 3 h with AdV-Ab complexes as in (A,B) or with LeuLeu at 10μM, and lysosomal damage measured by loss of acridine orange red fluorescence using flow cytometry.",
    "molecules": "acridine orange red, LeuLeu"
  },
  {
    "caption": "(E)The MFI of the AO- red channel (685nM) for the populations determined as e780 positive (dying) or e780 negative (live) for the condition of AdV + IVIg 20 was measured and plotted as mean + SEM of the three donors shown in (D).",
    "molecules": "AO- red, IVIg"
  },
  {
    "caption": "Primed or (C) un- primed HMDM were stimulated with AdV (50 000 pp/cell) +/- h9C12 (20 μg/ml) for 16h.",
    "molecules": "h9C12"
  },
  {
    "caption": "WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and IL-1β measured in the supernatant by ELISA.",
    "molecules": "h9C12, DNA, IVIg, Nigericin"
  },
  {
    "caption": "WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and TNF measured in the supernatant by ELISA.",
    "molecules": "h9C12, DNA, IVIg, Nigericin"
  },
  {
    "caption": "WT mice were injected i.v. with 2.5ug WT or H433A h9C12 antibody, then the next day injected i.v. with 2.5 x 1011 pp AdV-GFP. 4h later spleens were harvested and neutrophil influx measured by flow cytometry.",
    "molecules": "h9C12"
  },
  {
    "caption": "HMDM were stimulated with AdV-GFP (250 pp/cell) in the presence of h9C12 antibodies at indicated doses. Infection was measured after 24h by flow cytometry. Infection relative to AdV alone is shown graphed on the left (n = 4 mean ± s.e.m). Representative plots and a comparison of the different h9C12 mutants at 0.2 ug/ml are also shown in the graph on the right(n = 4,mean ± s.e.m.).",
    "molecules": "h9C12"
  },
  {
    "caption": "HMDM were stimulated with AdV (50 000 pp/cell) and antibody (h9C12; 20 ug/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3h and gene expression was measured by qPCR (n = 4 mean ± s.e.m).",
    "molecules": "h9C12, IVIg, pI:C"
  },
  {
    "caption": "HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro-IL-1β levels in the cytosol measured by western blot. Blot is representative of three independent donors.",
    "molecules": "LPS"
  },
  {
    "caption": "HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data shows average ± s.d. of 2 independent donors",
    "molecules": "LPS"
  },
  {
    "caption": "THP-1s expressing ASC-GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT-DNA or 10 μM Nigericin, in the presence of the pan-caspase inhibitor zVAD-fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, *P-value ≤ 0.05, paired, two-tailed t-test) is shown.",
    "molecules": "DNA, Nigericin, zVAD-fmk"
  },
  {
    "caption": "Primed HMDM were treated with MCC950 (1 μM) for 30 mins before stimulation overnight with AdV (50 000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL-1β (A) or TNF (B) or LDH release (C) were measured in cell supernatants. Data shows average ± s.d. of two independent donors and is representative of three independent experiments.",
    "molecules": "h9C12, IVIg, MCC950, Nigericin"
  },
  {
    "caption": "HMDM were primed with 10 ug/ml pI:C for 2h, treated with 100 μM VX-765 for 30 mins before stimulation for 16 h as in (A). IL-1β or LDH release was measured in cell supernatants. Data shows average ± s.d. of two independent donors",
    "molecules": "pI:C, VX-765"
  },
  {
    "caption": "Primed HMDM were treated with KCl as indicated for 1h, before being stimulated for a further 6h as in (A). IL-1β and TNF were measured in cell supernatants by ELISA. Data is representative of three independent donors.",
    "molecules": "KCl"
  },
  {
    "caption": "Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3h, and then immunostained for ASC or intracellular antibody. Co-localisation was assessed by confocal microscopy. Data is representative of two independent donors.",
    "molecules": "h9C12, Nigericin"
  },
  {
    "caption": "HMDM were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 for 1h, washed 2x with SFM, then whole cell lysates harvested after a further 5h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by western blot. Data is representative of two independent experiments.",
    "molecules": "h9C12"
  },
  {
    "caption": "THP-1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1000 U/ml IFNα for 4 h and protein levels assessed by western blot.",
    "molecules": "RNA"
  },
  {
    "caption": "THP-1s deficient in cGAS and STING were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12, 200 ng/ well HT-DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data shows combined data (mean ± s.e.m) of 3 experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl treated cells (lower panel), *P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).",
    "molecules": "h9C12, DNA, LPS, Nigericin"
  },
  {
    "caption": "WT THP-1s were treated with 5 μM H151 for 30 mins before stimulation as in (D). Data in upper panel is representative of 3 independent experiments. Data in lower panel shows combined data of these 3 experiments (mean ± s.e.m) showing H151 treated cells relative to media treated cells (*P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).",
    "molecules": "H151"
  },
  {
    "caption": "ASC-GFP THP-1s were treated with 5 μM H151 for 30 mins before stimulation with AdV-mCherry (50 000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT-DNA for 8h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three is shown.",
    "molecules": "h9C12, DNA, H151, IVIg"
  },
  {
    "caption": "(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm. (F) Graph showing the percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T n = 312 cells, N = 3 embryos; Sas4cKO n = 296 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT.",
    "molecules": "DAPI"
  },
  {
    "caption": "(K) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 (green), superficial layer marker CUX1 (red) and DAPI (blue). Scale bar = 200 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Cortices from E14.5 brains stained with antibodies against the radial glial cell marker PAX6 (yellow), intermediate progenitor marker TBR2 (red) and DAPI (blue). Scale bar = 50 μm. (B and C) Quantification of the number of (B) radial glial cells (PAX6+) and (C) intermediate progenitors (TBR2+) within a 250 μm-width column of E14.5 cortices. WT N=3, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 3, Sas4cKO;Usp28cKO N = 3; one-way ANOVA with post-hoc analysis.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Cortices from E14.5 embryos stained with antibodies against Cleaved-Caspase 3 (CC3, white), TP53 (red) and DAPI. Scale bar = 50 μm. (E and F) Quantification of the number of CC3+ apoptotic cells (E) and cells with TP53 activation (F) within a 250 μm-width column of E14.5 cortices. WT N = 5, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 4, Sas4cKO;Usp28cKO N = 4; one-way ANOVA with post-hoc analysis.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Representative images of E14.5 cortices stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). White and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) WT and Smc5cKO cortices at E14.5 stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). Insets showing zoomed in view of 2 representative cells. Scale bar: 25 μm. (B and C) Quantification of the number of (B) Centrin foci and (C) γ-Tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Smc5cKO n = 134 cells, N = 3 embryos. #, chi-square test. WT data are from Figures EV1F and EV1G and shown alongside for comparison.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-D)  Skin from healthy young (3-8 years old) and adult  donors ( > 25 years old) and from donors with RDEB with different stages of fibrosis - early  (0-3 years old), mild (6-10 years old) and advanced ( > 15 years old) - stained for CD68 (green), CD3ε (green), periostin (green) and tenascin-C (red). Nuclei were counterstained with DAPI (blue). A and B, scale bar = 50 μm; C and D, scale bar = 100 μm. The bar graphs at the right to the stained sections, represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D, the mean intensity of staining  from 3-6 donors per group. Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction. P values < 0.05 are considered significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A)  Human dermal RDEBF treated daily with 10 μM LY2109761 (LY) (TGF-β Receptor I/II inhibitor‎), 10 μM 1D11 (neutralizing antibody to all TGF-βs) 100 nM Ang-(1-7), or 10 μM  losartan (AT1R antagonist). After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins, THBS-1, thrombospondin-1, β-tubulin was used as loading control. (B) Densitometric  quantification of blots as in A. The abundance was normalized to  β-tubulin and then expressed as % of untreated (-). P values are shown; the data were analyzed with one-way ANOVA with Dunnett's correction (n = 5 biological replicates). ( Data information: P values < 0.05 are considered significant and shown, ns = not significant. individual data points, mean ± S.E.M are shown.",
    "molecules": "Ang-(1-7), losartan, LY, LY2109761"
  },
  {
    "caption": "(B) Kaplan Meier survival curve of mice treated as in A, P values as indicated, the data were tested with log-rank test - PBS treated vs. 0.1 mg/kg Ang-(1-7) treated and PBS treated vs. 1.0 mg/kg Ang-(1-7)  treated; n = 12-15 mice  as indicated in the figure.",
    "molecules": "Ang-(1-7), PBS"
  },
  {
    "caption": "(C) Photos of left and right  forepaws of RDEB mice before and after 7 weeks of daily injections with 0.1, 1.0 mg/kg Ang-(1-7)  or PBS. (D) Plot of numbers of toes  lost during the 7-week observation period, data tested with Kruskal-Wallis test, P values indicated, n= 20-24  paws as indicated in the figure. Median and range are shown. ( ",
    "molecules": "Ang-(1-7), PBS"
  },
  {
    "caption": "(A)  Haemotoxylin and Eosin (H&E) staining  of  toes from forepaws  from WT and  RDEB mice treated with daily injections of 0.1, 1.0 mg/kg  Ang-(1-7) or PBS for seven weeks. Inflammatory cells are elevated in the toes  from the PBS and the two Ang-(1-7) treated RDEB mice compared to the WT counterpart. Scale bar = 100 μm. Data information scale bar = 100 μm.",
    "molecules": "Ang-(1-7), Eosin, Haemotoxylin, PBS"
  },
  {
    "caption": "(F) Staining for  IgG  (red) in forepaws, nuclei counterstained with DAPI. (G) Quantification of fluorescence  staining  intensity of samples as in F. Data information: scale bar = 100 μm. , individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Picrosirius red staining  visualized under polarized light and Elastica van Gieson (EvG)  staining) of  forepaw toes from 12-wk-old WT or RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Note the reduced signal for picrosirus red  in  RDEB mice treated with 0.1 mg/kg Ang-(1-7) indicating less parallel alignment of and/or thinner collagen fibrils, and additionally the more organized and less disrupted appearance of elastic fibers (black in the  EvG staining). (B) Quantification of picrosirius red stained forepaws visualized as in A. A digital threshold was applied to the red channel before quantification. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant; n = 10-15. Individual data points, mean ± S.E.M are shown. ( Data information scale bar = 100 μm",
    "molecules": "Elastica van Gieson, EvG, Ang-(1-7), PBS, Picrosirius red, picrosirius red, picrosirus red"
  },
  {
    "caption": "(D) Dermal β-arrestin-1/2 (red) and NFκB (red) staining  in forepaws from WT and RDEB mice treated as in A. Nuclei counterstained  with DAPI (blue). Data information scale bar = 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(b) Co-IP was performed with 293T whole cell extracts by using ROCK1-crosslinked agarose, and the resulting IP was blotted with indicated antibodies. 293T cells transfected with Flag-Beclin1 for 36 h, cultured in control media or starved (4 h), were immunoprecipitated with anti-Flag beads. Inputs and immune complexes were resolved and detected. Total cell lysates were rerun and examined for LC3 and β-actin expression.",
    "molecules": "agarose"
  },
  {
    "caption": "(a) Inhibition of ROCK1 activity ablates the interaction between Beclin1 and ROCK1. HeLa cells were untreated (H2O) or treated with 10 μM ROCK1 inhibitor, Y27632, for 8 h and then cultured in control media (+) or HBSS (−) for 4 h. Cells were collected, lysed and immunoprecipitated with Beclin1 (left panel) or ROCK1 (right panel) antibody. Left panel: immune complexes were divided into two (ROCK1-MOPS buffer and Beclin1-MES buffer) and probed for ROCK1 and Beclin1. Relative values of ROCK1 versus Beclin1 are shown.",
    "molecules": "H2O, Y27632"
  },
  {
    "caption": "(d) ROCK activity is increased upon starvation. Cell lines (HeLa and EJ) grown in control (+Glu) or starved with HBSS for 1 and 3 h were lysed, extracts normalized and ROCK kinase activity was measured by performing enzyme-linked immunosorbent assay. Graphs represent ROCK activity in starved cells plotted as percent of ROCK1 activity in control (glucose-rich) cells. Data were obtained from triplicates performed at the same time (mean±s.d.). P-value was calculated using Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "(a) The effect of ROCK1 depletion on autophagy marker, LC3II. MEF ROCK1flox/flox cells were generated and treated with Con or Ad-Cre for 72 h. Cells were then incubated in nutrient-rich (+Glucose) or HBSS media for 2 h. Cell extracts were resolved by SDS-PAGE and ROCK1 KO was confirmed by western blotting (left panel). The same blot was used for LC3 and β-actin expression. Cells from the same experiment were fixed with chilled acetone and immunofluroscence was performed against endogenous LC3 (red). Blue represents nuclear DAPI staining. Scale bar, 20 μm.",
    "molecules": "acetone, Glucose"
  },
  {
    "caption": "(b) EJ cells (left) with a stable knockdown using shCont. or shROCK1#1 were incubated in control (+glucose) or nutrient-free (HBSS) medium (−glucose) for 24 h and lysed. Lysates were subjected to western blotting with antibodies indicated. HeLa cells (right) with a transient knockdown using siCont. and siROCK1#2 for 30 h were further incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and analysed for ROCK1, LC3 and β-actin. Relative ratios of LC3II/LC3I are shown.",
    "molecules": "glucose"
  },
  {
    "caption": "(c) HeLa cells were treated with H2O or 10 μM ROCK1 inhibitor, Y27632, for 8 h. Cells were then cultured in high glucose (0 h) or starved for in HBSS nutrient-free media for 6 h. One hour before collection, cells were incubated in dimethylsulphoxide (control) or 0.1 μM bafilomycin-A1. Whole cell lysates were resolved by SDS-PAGE and were analysed by western blotting with indicated antibodies.",
    "molecules": "bafilomycin-A1, dimethylsulphoxide, glucose, H2O, Y27632"
  },
  {
    "caption": "(d) HeLa cells incubated with H2O or 10 μM Y27632 for 8 h were washed with PBS and cultured in control (+Glu) or HBSS media (−Glu) for an additional 8 h. Cells were fixed in acetone and immunofluorescence was performed against endogenous LC3 antibody (green). Scale bar, 20 μm. Graph represents % of LC3 punctae/cell (mean±s.d.), n=25 cells; (Student's t-test) **P=0.00041.",
    "molecules": "acetone, H2O, Y27632"
  },
  {
    "caption": "(a) Inhibition of ROCK1 kinase activity prevents metabolic stress-mediated phosphorylation of Beclin1. HeLa cells left untreated or treated with a ROCK1 inhibitor, Y27632 (10 μM), were starved of glucose (4 h) as indicated, and endogenous Beclin1 was immunoprecipitated and blotted against phospho-Ser/Thr antibody. Whole cell lysates were run as inputs for Beclin1 and β-actin.",
    "molecules": "glucose, Y27632"
  },
  {
    "caption": "(b) Phosphorylation of Beclin1 by ROCK1. Recombinant ROCK1 was used for in vitro kinase assay in the presence or absence of Y27632, using recombinant His-Beclin1 (left) or Flag-Beclin1 immunopurified from transfected HeLa cells (right). Proteins were resolved by SDS-PAGE; phosphorylated protein was visualized with autoradiography, and Beclin1 by Coomassie staining.",
    "molecules": "Y27632"
  },
  {
    "caption": "(c) Identification of ROCK1-mediated phosphorylation domains in Beclin1. The indicated Flag-Beclin1 fragments were purified from HeLa cells and used as substrates for in vitro ROCK1 kinase assay. 32P-autoradiogram (centre) analysed phosphorylation and western blotting (WB; right) determined protein levels. Schematic representation of Beclin1 domain structure and deletion constructs are shown (left).",
    "molecules": "32P"
  },
  {
    "caption": "(d) Identification of the phosphorylation site on Beclin1. Representation of point mutations in the different domains of Beclin1 are shown (left). In vitro kinase assay using Flag-Beclin1 WT and mutants (T38A and T119A), immunoprecipitated from transfected HeLa cells, as substrate and recombinant ROCK1 was performed. Phosphorylation was detected by 32P-autoradiogram, and Flag-Beclin1 levels were examined by WB.",
    "molecules": "32P"
  },
  {
    "caption": "(e) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by WB against phospho-T119 (Beclin) antibody and Beclin1. Input for Beclin1 was run on 7.5% gel and immunoblotted.",
    "molecules": "agarose, glucose, nutrient, Y27632"
  },
  {
    "caption": "(a) Inhibiting ROCK1 activity increases the association between Beclin1 and Bcl-2. HeLa cells, untreated or treated with 10 μM Y27632 (top panel), were cultured in control or glucose-free (4 h) medium, lysed and cell extracts prepared. Endogenous Bcl-2 was immunoprecipitated with cross-linked Bcl-2 agarose and resulting eluted immune complexes were resolved by SDS-PAGE and blotted against indicated antibodies. EJ shCont. and shROCK1#1 cells (bottom panel) were grown in control or starvation media for 4 h and Bcl-2 IP was performed as above. Inputs were rerun to confirm ROCK1 knockdown.",
    "molecules": "agarose, glucose, Y27632"
  },
  {
    "caption": "(b) HeLa cells were transfected with full-length constructs of Flag-Beclin WT, Flag-Beclin T119A or Flag-Beclin T119E. Flag-Beclin WT cells were then treated with Y27632 for 8 h. Post transfection and treatment with Y27632, cells were starved in HBSS medium for 4 h, after which cells were collected and whole cell lysates prepared. IP with Flag-agarose was performed for 2 h and proteins bound to beads were eluted and used for western blotting.",
    "molecules": "agarose, Y27632"
  },
  {
    "caption": "(c) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by western blotting against Bcl-2 and Beclin1. Inputs and IP were run on separate gels and same exposure is shown.",
    "molecules": "agarose, glucose, nutrient, Y27632"
  },
  {
    "caption": "(d) HeLa cells with a transient knockdown using siCont. and siBeclin1-3′-untranslated region for 30 h were further left untreated or treated with 10 μM Y27632. Cells were then incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and were analysed for Beclin1, LC3 and β-actin.",
    "molecules": "Y27632"
  },
  {
    "caption": "(e) Endogenous LC3 staining in WT- or T119A-Beclin1-transfected cells. HeLa cells transfected with Flag-Beclin WT (±Y27632) and T119A were cultured in HBSS media for 6 h, fixed with cold acetone and endogenous LC3 immunofluorescence was performed. Representative images are shown; scale bar, 20 μm. Arrows indicate punctate LC3 staining. Graph represents mean±s.d., n=25, LC3 puncta/cell.",
    "molecules": "acetone, Y27632"
  },
  {
    "caption": "(b) PCR assay using tail genomic DNA from three different genotypes, separated on 0.7% agarose gel (top). Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti-ROCK1 antibody (bottom). β-Actin was used for protein loading control in each lane.",
    "molecules": "agarose"
  },
  {
    "caption": "(e) Hearts from fed and starved ROCK1 WT and KO mice were sectioned, fixed in acetone and immunofluorescence assay was performed against endogenous LC3. Data are representative of two independent experiments from each genotype. Scale bar, 50 μm.",
    "molecules": "acetone"
  },
  {
    "caption": "Super-resolution (gSTED) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained  with DAPI. Bar: 2μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative images of IMR90 overexpressing HMGB1-GFP, immunostained for  p21 and CTCF, and  counterstained  with DAPI. IMR90 transfected with empty vectors provide a control. Bar: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Effects of nutrient starvation on Rubicon, p62 and LC3 in Huh7 cells. Huh7 cells were nutrient-starved for 2 or 6 hours as indicated and lysed for Western-blot analysis. The replicon cells were used as the control for comparison.",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) Colocalization analysis of GFP-LC3 puncta and lysosomes. Stable Huh7 cells that expressed GFP-LC3 were nutrient-starved for 2 hours and stained with Lysotracker-red for lysosomes. The HCV replicon cells were also stained with Lysotracker-red for comparison. (D) Colocalization efficiency of GFP puncta with Lysotracker-red shown in (C). The results represent the average of >30 cells.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) Δ133p53α expression is induced by cycloheximide (CHX). MRC-5 fibroblasts (at population doubling levels (PDLs) 51) were treated with CHX (25 μg ml−1) for indicated time periods and examined in immunoblot for Δ133p53α and full-length p53. β-actin was a loading control for normalization. The relative expression levels of Δ133p53α and full-length p53 are shown below the images.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(b) Δ133p53α expression is induced by phosphoinositide 3-kinase inhibitors. MRC-5 fibroblasts (PDLs 51) were treated for 4 h with LY303511 (10 μM), LY294002 (10 μM), Wortmannin (50 nM) or dimethylsulphoxide alone (as control) and examined in immunoblot for Δ133p53α and β-actin expression.",
    "molecules": "LY303511, dimethylsulphoxide, phosphoinositide 3-kinase inhibitors, LY294002, Wortmannin"
  },
  {
    "caption": "(c) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1. The immunoblot analyses were performed in early-passage (Y) and replicatively senescent (S) human fibroblast strains MRC-5 and WI-38. The examined PDLs were 30 (Y) and 60 (S) for MRC-5; and 30 (Y) and 54 (S) for WI-38. Bafilomycin A 1 treatment was at 100 nM for 4 h. The relative expression levels of Δ133p53α, full-length p53 and p62/SQSTM1 (normalized with β-actin) are shown. Anti-LC3B antibody detected LC3-I and LC3-II, which was used in quantitative analysis shown in d. (d) Replicative senescence is not associated with enhanced autophagy in general. LC3-II expression levels were normalized with β-actin (data in c) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 (S/−; defined as 1). The LC3-II levels in replicatively senescent cells were higher than those in early-passage cells whether in the presence or absence of bafilomycin A1, which may suggest increased synthesis of autophagosomes at replicative senescence. However, the differences in LC3-II levels between the presence and absence of bafilomycin A1 were similar in early-passage and replicatively senescent cells: in MRC-5, 1.9 for Y (2.1−0.2) and 2.0 for S (3.0−1); in WI-38, 1.6 for Y (2.2−0.6) and 1.6 for S (2.6−1). These data indicate that early-passage and replicatively senescent cells have similar rates of autophagic flux, that is, the number of autophagosomes that are delivered to lysosomes for degradation.",
    "molecules": "Bafilomycin A 1, bafilomycin A1"
  },
  {
    "caption": "(d) Knockdown of pro-autophagic proteins and treatment with bafilomycin A1 show similar effects on Δ133p53α and p62/SQSTM1. MRC-5 fibroblasts at late-passage (PDLs 51) were transfected with control siRNA (c), ATG5 siRNA (A5, no. 1 in a), ATG7 siRNA (A7, no. 1 in b) and Beclin-1 siRNA (B1, no. 2 in c) as in a-c, untreated (−) or treated with bafilomycin A1 (+, 100 nM for 4 h), and examined in immunoblots for indicated proteins. The relative expression levels of Δ133p53α and p62/SQSTM1 (normalized with β-actin) are shown below the images.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(a) STUB1 knockdown-induced repression of Δ133p53α is abrogated by bafilomycin A1 treatment. The siRNA transfection in MRC-5 fibroblasts was performed as in Fig. 3e. At 7 days after the initial transfection, the cells were untreated, treated with bafilomycin A1 (100 nM for 4 h) or treated with MG-132 (15 μM for 4 h), and examined in immunoblots as indicated. The results of Δ133p53α, full-length p53 and p62/SQSTM1 in untreated cells replicate those shown in Fig. 3c. The inhibition of autophagy by bafilomycin A1 was confirmed by increased levels of p62/SQSTM1 and LC3B. The inhibition of proteasomal degradation by MG-132 was confirmed by increased full-length p53.",
    "molecules": "bafilomycin A1, MG-132"
  },
  {
    "caption": "(d) Bafilomycin A1 stabilizes LC3B-interacting Δ133p53α in senescent cells. The same set of cells as in c were treated with bafilomycin A1 (+, 100 nM for 4 h) or untreated (−). These cells were used in IP with anti-LC3B antibody, followed by IB with anti-Δ133p53 antibody. Whole-protein lysates without IP were also examined in IB with anti-LC3B antibody. While untreated samples replicate the result shown in c, the differences between early passage and senescent cells become more evident with bafilomycin A1 treatment.",
    "molecules": "Bafilomycin A1, bafilomycin A1"
  },
  {
    "caption": "(a) Δ133p53α protein is polyubiquitinated. FLAG-tagged versions of wild-type Δ133p53α (wt) and mutant Δ133p53α (mut; from c below), along with vector control (−), were retrovirally transduced into MRC-5 fibroblasts. The wild-type Δ133p53α-expressing cells had a pair of STUB1 siRNA-transfected (+, siRNA no. 1 for 4 days) and untransfected (−) counterparts. These cells were maintained for 8 h under amino acid- and serum-starved conditions with bafilomycin A1 (100 nM). Protein lysates were used either in IP with anti-ubiquitin (Ub) antibody, followed by IB with anti-FLAG antibody (top), or directly in IB for FLAG-Δ133p53α, STUB1 and β-actin (lower three panels). Smear signals indicate polyubiquitinated FLAG-Δ133p53α protein (Poly-Ub). Asterisk indicates a nonspecific band in negative control. These experimental conditions resulted in similar amounts of FLAG-Δ133p53α in the presence and absence of STUB1 knockdown, allowing quantitative analysis of ubiquitination. The relative densitometric values of wild-type FLAG-Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown (normalized with total FLAG-Δ133p53α) are shown. (",
    "molecules": "amino acid, bafilomycin A1"
  },
  {
    "caption": "(c) Lysine (K)-to-arginine (R) substitutions at amino-acid residues 248 and 249.",
    "molecules": "arginine, Lysine"
  },
  {
    "caption": "(d) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation. The same three cells as in a were cultured under amino-acid- and serum-starved conditions for 8 h (without bafilomycin A1), transfected with STUB1 siRNA (no. 1 for 4 days) or untreated, and examined in IBs for FLAG-Δ133p53α, STUB1, p62/SQSTM1 and β-actin. The effect of starvation was confirmed by decreased p62/SQSTM1 in each cell. The upregulation of p62/SQSTM1 by wild-type and mutant FLAG-Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression.",
    "molecules": "amino-acid, bafilomycin A1"
  },
  {
    "caption": "(a) Δ133p53α is colocalized with p62/SQSTM1 upon autophagy induction. MRC-5 fibroblasts expressing FLAG-Δ133p53α (wild-type) were cultured under amino-acid- and serum-starved conditions for 4 h, and co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-p62/SQSTM1 antibody (red). Two representative sets of images, including nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and merged images, are shown. Scale bars, 10 μm.",
    "molecules": "amino-acid"
  },
  {
    "caption": "b, Autophagic vacuoles containing glycogen (g), cytosol and cellular constituents in a hepatocyte of an 8-day-old LAMP-2-/- mouse. m, mitochrondria; bc, bile canaliculus. c, Accumulation of numerous early autophagic vacuoles in cultured LAMP-2-/- hepatocytes. Arrow, large late autophagic vacuole containing partially degraded material. Insert, magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents. Scale bars: a, 3.5 µm; b, 0.2 µm; c, 1.3 µm; insert, 0.9 µm.",
    "molecules": "glycogen"
  },
  {
    "caption": "f, Degradation of long-lived proteins followed over 24 h in amino acid and fetal-calf-serum-containing medium. g, Effect of amino-acid (AA) deprivation and 3-methyladenine (MA) on protein degradation during 4 h of chase. The error bars in a, d, e and g represent s.e.m.",
    "molecules": "3-methyladenine, amino-acid"
  },
  {
    "caption": "Representative image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view).Quantifications of CFSE+F4/80+ macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1GFP/GFP CFSE+ macrophages (n = 5/per group (12 h) and n = 6/per group thereafter; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP: 1 day n = 20/group *P < 0.0001; 2 day n = 6/group *P = 0.0317).",
    "molecules": "thioglycollate"
  },
  {
    "caption": "(E) Free energy profiles of VP40-lipid interactions at pH 7.4 and pH 4.5 determined from MD simulations. The plot shows free energy (in kBT) at increasing membrane-VP40 distances (nm) with indicated three states shown in (A).",
    "molecules": "lipid"
  },
  {
    "caption": "(F) EBOV VLP lipid composition showing highly abundant lipids determined by mass spectrometry in mol%. Lipid abbreviations: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lyso-phosphatidylcholine (LPC), sphingomyelin (SM). Prefix \"a\" indicates acyl-linked glycerophospholipids, prefix \"e\" indicates ether-linked (plasmanyl) or the presence of one odd and one even chain fatty acyl. Bars represent mean and error bars represent standard error of the mean (red); n=3 biological replicates.",
    "molecules": "LPC, lyso-phosphatidylcholine, acyl, ether, glycerophospholipids, lipid, Lipid, lipids, phosphatidylserine, PS, PC, phosphatidylcholine, PE, phosphatidylethanolamine, phosphatidylinositol, PI, SM, sphingomyelin"
  },
  {
    "caption": "(D) Plot showing the mean relative fluorescence intensities and standard deviation of VLPs imaged at neutral pH, low pH and in the presence of T-X100 at low pH over time. Data was obtained from 3 independent VLP preparations. Number of technical replicates: n=42 (pH 7.4); n=19 (pH 4.5 and pH 5 + T-X100).",
    "molecules": "T-X100"
  },
  {
    "caption": "(B) Stalk formation energy as a function of VP40-membrane interaction energy. The stalk energy for non-interacting VP40 matrix (u0 = 0) is 65 kBT. VP40 matrix layer Young's modulus legend - red 5.6 MPa, blue 11.2 MPa, black 16.8 MPa, and orange is infinitely rigid. The bending rigidity ratio between the VP40 matrix layer and lipid monolayer are 1, 2, 3, and infinity, respectively. The line represents an infinitely rigid layer.",
    "molecules": "lipid"
  },
  {
    "caption": "(F) Quantification of FACS data showing EBOV VLP entry as measured by a fluorescence shift of infected cells from emission at 510 nm (no entry) to 450 nm (entry). VLPs were treated prior to infection as indicated on the x-axis, with control: uninfected control cells, - : no treatment, T: thermolysin-treatment at neutral pH, Lp: low pH treatment. Target cells were treated with media or ammonium chloride (NH4Cl), n= 3 with 10'000 cells measured per sample.",
    "molecules": "ammonium chloride, NH4Cl"
  },
  {
    "caption": "(D) Genome browser view of  CMS-IP data showing CMS (5hmC) distribution at  Il2ra and Foxp3 locus for CD4+ naïve T cells  and WT Treg cells. Hydroxymethylated regions (hmRs) and DhmRs are shown on the top; conservation (cons) is shown at the bottom.",
    "molecules": "CMS, 5hmC"
  },
  {
    "caption": "(H-J) Box-and-whisker plots of  DNA methylation  levels for DM-CpGs less methylated  in TGFβ + RA + VitC iTregs vs TGFβ iTregs (H), DM-CpGs less methylated  in WT Treg cells vs  CD4+ naïve T cells (I) and DM-CpGs less  methylated in WT Treg cells vs Tet2/3 DKO Treg cells (J). DM-CpGs denotes the CpGs in the respective cell types used for the comparison. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. P-values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey's post-hoc test. Data are from two biological replicates.",
    "molecules": "RA, VitC"
  },
  {
    "caption": "(D) Graphs depicting the percentage of viable cells for TGFβ iTregs (left) and TGFβ+RA+VitC iTregs (right) differentiated with 0 U/ml, 1 U/ml, 5 U/ml and 10 U/ml  IL-2. Error bars show mean ± S.D. from three biological replicates.",
    "molecules": "RA, VitC"
  },
  {
    "caption": "(C)  ChIP-qPCR  showing STAT5 occupancy at Foxp3 CNS2   and  IL2Rα IN1b  regions in TGFβ and TGFβ  + RA  + VitC iTregs  without or with 3 U/ml IL-2 restimulation. GMPR is a negative control region. Data are from two biological replicates.",
    "molecules": "RA, VitC"
  },
  {
    "caption": "(C) Immunofluorescence staining of the spinal cord from GW7, GW8, and GW10 for a dorsal marker (PAX7, green), neuronal precursor marker (ASCL1, red), and nuclei marker (DAPI, blue). Images show the neuronal differentiation process on the dorsal side of the spinal cord. Scale bar: 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Immunofluorescence staining of spinal cord sections at GW7, GW11, GW17, and GW23 for EGFR (green) and nuclei (DAPI, blue). EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord. EGFR expression on the dorsal side was observed in GW11, GW17, and GW23 spinal cord. Dorsal EGFR expression was mainly concentrated within the dorsal horn area. Scale bar: 100 μm. (Top) Higher-magnification view of boxes at bottom. White dashed lines indicate dorsal horn borders.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Colocalization  of LacCer  and LysoTracker  (LyTr) in different CTR  and LSD patient fibroblasts. Mander´s coefficients were calculated using the Fiji JACoP plugin. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's (A, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "LysoTracker, LyTr, LacCer"
  },
  {
    "caption": "(B-C) Confocal  images (B) and statistical analysis (C) showing colocalization  of LacCer  and LyTr  in human  CTR and MLIV fibroblasts, treated with TPC2-A1-P  (30 µM, 16h). (D-E) Confocal  images (D) and statistical analysis (E) showing colocalization   of LacCer  and LyTr  in human  CTR and NPC1 fibroblasts, treated with TPC2-A1-P (30 µM, 48h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's C, E, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "LyTr, LacCer, TPC2-A1-P"
  },
  {
    "caption": "(F-G) Confocal  images and statistical analysis showing LacCer/LyTr colocalization  in MLIV patient fibroblasts  which were mock-electroporated and treated with DMSO  or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry  TOPO 3.1 vector and treated with either DMSO  or TPC2-A1-P (30 µM, 16h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's G, multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO, LyTr, LacCer, TPC2-A1-P"
  },
  {
    "caption": "(H-I) Ca2+ chelation (BAPTA-AM) dose-dependently impairs LacCer  trafficking in CTR fibroblasts. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's I) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "BAPTA-AM, Ca2+, LacCer"
  },
  {
    "caption": "(J-K) Confocal  images (J) and statistical analysis (K) of NPC1 patient fibroblasts  treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2  (siTPC2) for 72 hours. Cells were then treated with DMSO  or  TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, Tukey's (K) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO, TPC2-A1-P"
  },
  {
    "caption": "(A-B) Confocal  images (A) and statistical analysis (B) of cholesterol  accumulation in human  CTR, MLIV, JNCL, and NPC1 fibroblasts. Cholesterol  accumulation was evident for NPC1  and MLIV fibroblasts, but not for JNCL fibroblasts. The images show filipin staining  to visualize cholesterol  accumulation and TO-PRO3 as nuclear staining. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test (B, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "cholesterol, Cholesterol, filipin, TO-PRO3"
  },
  {
    "caption": "(C-D) TPC2-A1-P (30 µM, 48h) rescued NPC1  and MLIV cholesterol  accumulation. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test D, *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "cholesterol, TPC2-A1-P"
  },
  {
    "caption": "Confocal  images (E-F) of MLIV patient fibroblasts  mock-electroporated and treated with DMSO  or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry  TOPO 3.1 vector (white arrow heads) and treated with either DMSO  or TPC2-A1-P (30 µM, 48h).",
    "molecules": "DMSO, TPC2-A1-P"
  },
  {
    "caption": "statistical analysis (G) of MLIV patient fibroblasts  mock-electroporated and treated with DMSO  or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry  TOPO 3.1 vector (white arrow heads) and treated with either DMSO  or TPC2-A1-P  (30 µM, 48h). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's multiple comparisons test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO, TPC2-A1-P"
  },
  {
    "caption": "Confocal  images and statistical analysis of human  CTR patient fibroblasts  treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2  (siTPC2) for 72 hours. Cells were then treated with DMSO  Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells) two-tailed Student's t-test *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO"
  },
  {
    "caption": "Confocal  images and statistical analysis of human NPC1 patient fibroblasts  treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2  (siTPC2) for 72 hours. Cells were then treated with DMSO  or TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); two-tailed Student's t-test K). *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO, TPC2-A1-P"
  },
  {
    "caption": "(A) Confocal  images of CTR and JNCL fibroblasts. Images show LAMP1 staining  and autofluorescence at 405 and 488 nm excitation wavelength, respectively, corresponding to the lipofuscin  autofluorescence spectrum. Cells were treated with DMSO  or TPC2-A1-P  (30 µM) following cell cycle arrest (2h mitomycin C treatment). (B) Confocal  images showing no autofluorescence signal at 594 nm excitation wavelength (used for LAMP1 staining).",
    "molecules": "DMSO, lipofuscin, mitomycin C, TPC2-A1-P"
  },
  {
    "caption": "Mean intensity of shigatoxin   (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO  or TPC2-A1-P (30 µM) after cell cycle arrest. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells; one-way ANOVA, post hoc Bonferroni's multiple comparisons test. **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO, shigatoxin, STX, TPC2-A1-P"
  },
  {
    "caption": "Confocal  images of Gb3  accumulation stained  with shigatoxin   (STX) in CTR and JNCL fibroblasts. Cells were treated with DMSO  or TPC2-A1-P (30 µM) after cell cycle arrest.",
    "molecules": "Gb3, DMSO, shigatoxin, STX, TPC2-A1-P"
  },
  {
    "caption": "Cortical neurons were differentiated from iPSCs, generating  lysosomal storage disease neurons  and isogenic controls. (C-D) Western blot  analysis of  cathepsin B (CtsB) in CTR and MCOLN1IVS3-2A>G neurons  treated with TPC2-A1-P (30 µM) or DMSO. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal  differentiations); two-tailed Student's t-test (C). **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO, TPC2-A1-P"
  },
  {
    "caption": "Cortical neurons  were differentiated from iPSCs, generating  lysosomal storage disease neurons  and isogenic controls. Cortical neurons were treated with compounds and acidic compartments stained  with LysoTracker (LyTr). Endolysosomal  expansion was observed in MCOLN1IVS3-2A>G, CLN3D416G and CLN3ΔEx4-7 neurons, which was ameliorated by TPC2-A1-P  (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal  differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "LysoTracker, LyTr, TPC2-A1-P"
  },
  {
    "caption": "Cortical neurons were differentiated from iPSCs, generating lysosomal storage disease neurons  and isogenic controls. (H-I)  Electron microscopy analysis of neuronal rosettes (neuronal progenitor cells, NPC) treated with DMSO or TPC2-A1-P. TPC2-A1-P treatment significantly decreased the number of inclusion bodies (black arrow heads) in MCOLN1IVS3-2A>G. CLN3D416G and CLN3ΔEx4-7 lacked an appropriate assay window and showed no significant accumulation of  inclusion bodies. However, CLN3ΔEx4-7 NPC  showed significantly more mitochondria  with aberrant cristae  numbers (white arrow heads), a phenotype which was rescued by TPC2-A1-P (30 µM) treatment. Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells, obtained from at least three distinct neuronal  differentiations); one-way ANOVA, post hoc Tukey's multiple comparisons test **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "DMSO, TPC2-A1-P"
  },
  {
    "caption": "(A) Confocal  images of plasma membrane  (PM) LAMP1 immunofluorescence  in CTR fibroblasts. LAMP1  on the PM  is expressed as fold-change relative to DMSO-treated cells. (B) Statistical analysis of lysosomal exocytosis  data as shown in A. (C) Lysosomal exocytosis  in CTR, MLIV, NPC1  and JNCL human fibroblasts. PM-localized LAMP1  was measured by flow cytometry, expressed as % of CTR DMSO-treated cells. Ionomycin  in A-C (4 µM; 10 min treatment) was used as positive control. TPC2-A1-P  and ML-SA1  (30 µM, each; 90 min treatment in A-C). Statistics (B-C): Shown are mean values ± SEM. n > 3 for each tested condition (in B each dot represents an imaged frame containing several cells, in C each dot is the mean FITC  intensity value expressed as percentage obtained from at least 1*104 events); two-way ANOVA, post hoc Dunnett's (B) or Tukey's (C) multiple comparisons test, *p-value < 0.05; ***p-value < 0.001; ****p-value < 0.0001.",
    "molecules": "ML-SA1, DMSO, FITC, Ionomycin, TPC2-A1-P"
  },
  {
    "caption": "Immunoblot  analysis of endogenous LC3  (LC3I-II) following TPC2-A1-P or ML-SA1 (30 µM, each) treatment, alone or with BafA1, under fed  (complete media) or starvation  (HBSS) conditions in CTR, MLIV  and NPC1 patientfibroblasts.",
    "molecules": "ML-SA1, BafA1, TPC2-A1-P"
  },
  {
    "caption": "analysis of endogenous LC3 following  TPC2-A1-P  or ML-SA1  (30 µM, each) treatment, alone or with BafA1, under fed  (complete media) or starvation  (HBSS) conditions in CTR, MLIV and NPC1 patientfibroblasts. Graphs show densitometry  of LC3II  bands normalized to actin. Statistics (G, Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.",
    "molecules": "ML-SA1, BafA1, TPC2-A1-P"
  },
  {
    "caption": "(H) Immunoblot  analysis of endogenous LC3 (LC3I-II) following  TPC2-A1-P (30 µM) or DMSO  treatment, under fed  (complete Neurobasal/B27) or starvation  (DMEM/F12 free) conditions in CTR and MLIV iPSC  derived cortical neurons. Graphs show densitometry  of LC3II  bands normalized to actin. Statistics H, Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.",
    "molecules": "DMSO, TPC2-A1-P"
  },
  {
    "caption": "Immunoblot  analysis of endogenous SQSTM1  (P62) upon  TPC2-A1-P or ML-SA1  (30 µM, each) treatment, under fed  (complete media) or starvation  (HBSS) conditions in CTR, MLIV  and NPC1 patient fibroblasts. Statistics J): Shown are mean values ± SD. n = 3 lysates per condition pooled from 3 independent experiments; two-tailed Student's t-test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.",
    "molecules": "ML-SA1, TPC2-A1-P"
  },
  {
    "caption": "(A) TPC2-A1-P was injected intravenously, and mice  sacrificed at the indicated time. TPC2-A1-P was measured in plasma  and brain  by LC-MS/MS. TPC2-A1-P was rapidly eliminated, being undetectable by 240 min.",
    "molecules": "TPC2-A1-P"
  },
  {
    "caption": "(C) Brain  [TPC2-A1-P] was simulated for various injected doses. 20 mg/kg TPC2-A1-P was chosen to avoid off-target activity while providing a therapeutic dose for >20 min.",
    "molecules": "TPC2-A1-P"
  },
  {
    "caption": "(A) Ookinetes sandwiched between two hydrogel surfaces.",
    "molecules": "hydrogel"
  },
  {
    "caption": "(B) Percentage of ookinetes moving for more than one parasite length within the time of observation (5-10 min) if sandwiched between soft, medium (med) or stiff hydrogel or glass. Bars represent the mean and error bars the standard deviation of at least three independent experiments. (C) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B.",
    "molecules": "glass, hydrogel"
  },
  {
    "caption": "(D) Percentage of motile ookinetes after 20-26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in (B). Shown is the average ± S.D. from at least two independent experiments. (E) Speed of motile ookinetes after 20-26 hours of incubation between hydrogels. Speed on glass was not analyzed (n.a.) due to the low fraction of motile ookinetes at this timepoint.",
    "molecules": "glass, hydrogels"
  },
  {
    "caption": "(F) Migration patterns of ookinetes. Image: Overlay of tracked migration paths (black) and DIC image showing ookinetes at the end of the recorded image sequence. Graph: Quantitative analysis of the indicated different migration patterns on soft, medium (med) and stiff hydrogels.",
    "molecules": "hydrogels"
  },
  {
    "caption": "(G) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints.",
    "molecules": "hydrogels"
  },
  {
    "caption": "(A) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel.",
    "molecules": "glass, hydrogel, PA hydrogel"
  },
  {
    "caption": "(B) Overlay of sporozoite tracks (black) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence. Scale bar, 20 µm.",
    "molecules": "hydrogel"
  },
  {
    "caption": "(C) Speed of WT sporozoites moving in the skin in vivo (Douglas et al, 2018b) and in soft hydrogels with small or large pores.",
    "molecules": "hydrogels"
  },
  {
    "caption": "(D, E) Speed of indicated mutant and control (Ctrl) parasites in hydrogels with small (D) and large (E) pores.",
    "molecules": "hydrogels"
  },
  {
    "caption": "(F, G) Speed of drug-treated and control parasites in hydrogels with small (F) and large (G) pores. Data information: Note the faster speed in gels with larger pores. Data from at least two independent experiments. Numbers above bars indicate the number of sporozoites analyzed. Significance determined by Mann-Whitney test. Box-and-whisker plots (C-G) depict the 25% quantile, median, 75% quantile and nearest observations within 1.5 times the interquartile range (whiskers). Outliers beyond this range are shown as black dots.",
    "molecules": "hydrogels"
  },
  {
    "caption": "(A) Percentage of motile sporozoites on endothelial cells (LSECs) versus glass. Shown is average ± S.D. from three independent experiments. Significance determined using an unpaired t-test.",
    "molecules": "glass"
  },
  {
    "caption": "(B) Left: Merged fluorescence images showing sporozoites on cultured HFF cells. Actin filaments of HFF cells were stained with phalloidin (shown in red), nuclei were stained with Hoechst (blue) and sporozoites expressed GFP (green). Scale bar, 50 µm. Right: Maximum projection of sporozoites moving for 3 min on cultured HFF cells. Scale bar, 50 µm.",
    "molecules": "Hoechst, phalloidin"
  },
  {
    "caption": "(C) Percentage of motile sporozoites on confluent layers of endothelial cells (HUVEC), fibroblasts (HFF) and on glass. Shown is average ± S.D. from two independent experiments. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.",
    "molecules": "glass"
  },
  {
    "caption": "(D) Sporozoite on planar uncoated hydrogels.",
    "molecules": "hydrogels"
  },
  {
    "caption": "(E) Overlay of sporozoite tracks (black) and DIC image showing sporozoites on a hydrogel. Scale bar, 50 µm.(F) Percentage of motile sporozoites on soft, medium (med) or stiff hydrogel or glass 30 min after activation with BSA. Shown is the average ± S.D. from at least three independent experiments. Significance determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.",
    "molecules": "glass, hydrogel"
  },
  {
    "caption": "(G) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA. All sporozoites moving for more than one parasite length within the time of observation were analyzed, even if they stopped moving or detached during imaging. Significance determined by Kruskal-Wallis test with Dunn's Multiple Comparison test.",
    "molecules": "glass, hydrogels"
  },
  {
    "caption": "(H) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses. Note that not all sporozoites analyzed in (G) complied with that requirement. To be able to analyze a high number of sporozoites, movies were taken 10 to 45 min after activation with BSA. Speed of sporozoites on soft hydrogels was not analyzed (n.a.) due to the low fraction of motile sporozoites on these hydrogels. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.",
    "molecules": "hydrogels"
  },
  {
    "caption": "XRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.",
    "molecules": "3′-AB, DMSO"
  },
  {
    "caption": "A, B Percentages of DSBs repaired in increasing time intervals (0.5-2 h, 0.5-6 h, 0.5-24 h) were calculated in XRCC4wt (A) and XRCC4m/m (B) cells from the number of γ-H2AX foci in presence or absence of a PARP-1 inhibitor, 3′-AB. **P < 0.01 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), two-way ANOVA, Bonferroni post hoc test.",
    "molecules": "3′-AB, DMSO"
  },
  {
    "caption": "L) Example images of β-Gal+ traced cells post-irradiation and at homeostasis. Scale bar= 100um. M) The number of fully traced β-Gal+ crypt-villus axes/cm in irradiated and non-irradiated mice. Lineage tracing events were quantified from small intestines divided into four equal segments (S1-4, with S1 being the most proximal), stained with X-Gal, embedded in paraffin, cut into 5um thick sections, and counterstained with neutral red. Data are expressed as mean ± SEM. N=3 mice/group, P-value generated by multiple student's t-tests. N) The number of β-Gal+ fully traced crypt-villus axes (i.e. lineage tracing events) normalized to the number of pre-injury crypt marked β-Gal+ cells (quantified from d0 control mice). S1 + S2 = the two most proximal intestinal segments. Data are expressed as mean ± SEM. N=4 mice/group, P-value generated by two-tailed student's t-test.",
    "molecules": "X-Gal, neutral red"
  },
  {
    "caption": "C) Western blot demonstrating LC3 levels from equal numbers of sorted CytoIDlow and CytoIDhigh cells. 200,000 cells were loaded per lane.",
    "molecules": "CytoID"
  },
  {
    "caption": "E) Representative images of organoids formed from sorted CytoIDlow, CytoIDhigh, and Lgr5-GFPhigh cells. F) Quantification of the organoid-formation efficiency in (E). N=4 mice/group, N=3 wells quantified per mouse. P-Values displayed on graph. Data are expressed as mean ± SEM. P value generated by ratio paired two-tailed t-test.",
    "molecules": "CytoID"
  },
  {
    "caption": "G) Effect of bafilomycin on organoid formation efficiency in CytoIDlow and CytoIDhigh cells. Cells were plated in media containing different concentrations of bafilomycin (0, 2.5, 5, 10, or 25 ng/mL) on day 0, media was changed on day 3 to media that did not contain bafilomycin and organoids were imaged on day 5 after plating. N=4 mice/group, N=4 wells quantified per mouse.",
    "molecules": "CytoID, bafilomycin"
  },
  {
    "caption": "A) Representative plots demonstrating the distribution of cells with and without CytoID puncta within sorted CytoID low, high, Chga+, and c-Kit+ cells.",
    "molecules": "CytoID"
  },
  {
    "caption": "B) Quantification of the percentage of cells with and without puncta within CytoID low, high, Chga+, and c-Kit+ cells. N=3 mice/group (biological replicates).",
    "molecules": "CytoID"
  },
  {
    "caption": "C) Representative images and quantification of organoid-formation efficiency of CytoID low and high cKit+ and Chga+ cells. Scale bars = 500um. N=3 mice/group (biological replicates), N=4 wells quantified per mouse (technical replicates).",
    "molecules": "CytoID"
  },
  {
    "caption": "Immunoblot showing LC3-II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf.",
    "molecules": "Baf"
  },
  {
    "caption": "Immunoblot showing LC3-II formation MB-3-treated HeLa cells in the presence or absence of the lysosome inhibitor Baf.",
    "molecules": "Baf, MB-3"
  },
  {
    "caption": "E Quantification of LC3 puncta in cells The cells were treated with or without Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).",
    "molecules": "Baf"
  },
  {
    "caption": "Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment",
    "molecules": "Baf"
  },
  {
    "caption": "Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bar, 10 µm).",
    "molecules": "Baf"
  },
  {
    "caption": "I GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were cultured with GCN5 siRNA with or without CQ.",
    "molecules": "CQ"
  },
  {
    "caption": "J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP-GCN5 and addition of CQ.",
    "molecules": "CQ"
  },
  {
    "caption": "K Representative images of mCherry-Atg8a (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD) using the pan-fat body driver (cg-GAL4). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, ***p < 0.001, Student's t-test; Scale bars, 10 µm).",
    "molecules": "cg, DAPI"
  },
  {
    "caption": "A LAMP1 puncta (green) and DAPI (blue) in WT and GCN5 KO HEK293 cells (Scale bars, 10 µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "B Fluorescence-activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker. Fluorescence intensity of 10,000 cells per sample was measured.",
    "molecules": "LysoTracker"
  },
  {
    "caption": "G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ.",
    "molecules": "CQ"
  },
  {
    "caption": "H Representative images of LysoTracker staining (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bars, 10 µm).",
    "molecules": "cg, DAPI, LysoTracker"
  },
  {
    "caption": "A Quantification of LC3 puncta in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).",
    "molecules": "Baf"
  },
  {
    "caption": "B Immunoblot showing LC3-II formation in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf.",
    "molecules": "Baf"
  },
  {
    "caption": "E Acetylation of TFEB-Flag in stable TFEB-Flag-expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated. TFEB-Flag was immunoprecipitated with anti-Flag beads and analyzed by immunoblot using anti-acetyl-lysine (Ace-lys).",
    "molecules": "Ace-lys, acetyl-lysine"
  },
  {
    "caption": "G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB-3 or the p300/CBP inhibitor C646. H3, histone H3. Ace-H3 (K9) and Ace-H3 (K27) were used to show the activity of GCN5 and p300, respectively.",
    "molecules": "C646, MB-3"
  },
  {
    "caption": "J Acetylation of Flag-tagged TFEB or TFEB mutants expressed in HEK293 cells. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg.",
    "molecules": "Arg, Lys"
  },
  {
    "caption": "D, E Luciferase activity measured in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (D), and in HEK293 cells with TFEB-WT or TFEB-3KR transfection (E). The cells were transfected or co-transfected with a TFEB-luciferase reporter. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.",
    "molecules": "Arg, Lys"
  },
  {
    "caption": "A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 (Scale bars, 10 µm).",
    "molecules": "Torin1"
  },
  {
    "caption": "B Subcellular localization of Flag-tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1. Cells were stained with anti-Flag antibody (Scale bars, 10 µm).",
    "molecules": "Torin1"
  },
  {
    "caption": "D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA. A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants, and was subjected to electrophoresis. Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non-binding DNA control.",
    "molecules": "DNA"
  },
  {
    "caption": "I TFEB homodimer formation detected by glutaraldehyde (GA) crosslinking. Purified recombinant GST-tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde, then the products were analyzed by immunoblotting using anti-TFEB.",
    "molecules": "GA, glutaraldehyde"
  },
  {
    "caption": "A Acetylation of TFEB-Flag, Myc-GCN5 and histone H3 in HEK293 cells. The cells were either starved of glucose or amino acids, or treated with Torin1 or MB-3.",
    "molecules": "amino acids, glucose, MB-3, Torin1"
  },
  {
    "caption": "E LC3-II formation in HeLa cells transfected with Flag-tagged TFEB or TFEB mutants after TFEB RNAi. The cells were treated with or without amino acid starvation.",
    "molecules": "amino acid"
  },
  {
    "caption": "F GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were treated with or without addition of CQ.",
    "molecules": "CQ"
  },
  {
    "caption": "Representative images of mCherry-Atg8a (red) (G) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).",
    "molecules": "cg, DAPI"
  },
  {
    "caption": "Representative images of LysoTracker (red) staining (H) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).",
    "molecules": "cg, DAPI, LysoTracker"
  },
  {
    "caption": "Quantification of LysoTracker puncta (J) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).",
    "molecules": "LysoTracker"
  },
  {
    "caption": "The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.",
    "molecules": "CQ"
  },
  {
    "caption": "The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.",
    "molecules": "CQ"
  },
  {
    "caption": "C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR-Gal4 driver. The Drosophila were fed with or without CQ. Drosophila (GMR-Gal4/+) was used as the control.",
    "molecules": "CQ"
  },
  {
    "caption": "D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ (30 flies per condition were randomly selected for analyses; *p < 0.05, ***p < 0.001, ꭓ2 test).",
    "molecules": "CQ"
  },
  {
    "caption": "A. 3-OH-C10:0-induced ROS burst is suppressed by A23 and K252a. Two-week-old Col-0 seedlings were treated with 2μM K252a or 40 μM A23. One hour later, 1 μM 3-OH-C10:0 was used to trigger the ROS burst, and the photo count was recorded by a luminometer. Mock is solvent control. RLU, relative light unit. Values are means ± SD (n=3 biological replicates).",
    "molecules": "3-OH-C10:0, K252a, ROS, A23"
  },
  {
    "caption": "B. A23 and K252a suppress 3-OH-C10:0-induced PTI marker gene expression. Treatments with 3-OH-C10:0 and inhibitors were same as in (A), and the plant leaves were collected for RT-qPCR 4 hrs later. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).",
    "molecules": "3-OH-C10:0, K252a, A23"
  },
  {
    "caption": "C. MAPK cascade activation was inhibited by A23. Treatments with 3-OH-C10:0 and A23 were same as in (A). The plant leaves were collected at 0, 5, and 10 min for immunoblotting. Activated MAPKs were detected by immunoblotting with anti-phospho-p44/42 MAPK antibody. The corresponding bands are indicated for MPK3 and MPK6. Ponceau staining of Rubisco was used for estimating equal loading in each lane. The experiment was repeated three times with similar results.",
    "molecules": "3-OH-C10:0, A23"
  },
  {
    "caption": "D. Tyrosine phosphorylation of LORE in vivo. Two-week-old 35S:LORE-FLAG complementation plants were subjected to immunoprecipitation with anti-FLAG agarose beads. A23 (40 μM) was used to inhibit the protein phosphorylation, and the treatments were same as in (A). Protein abundance of precipitated LORE-FLAG was detected by immunoblotting. The tyrosine phosphorylation was probed by anti-pTyrosine (α-pY) antibody. Similar results were observed in three biological replicates.",
    "molecules": "Tyrosine, tyrosine, A23"
  },
  {
    "caption": "E. LORE is an active tyrosine kinase. The α-pY antibody was used to probe tyrosine phosphorylation in recombinant protein MBP-tagged LORE-CD (cytosolic domain). The LORE activation loop conserved K516 was mutated to E, and D613 was mutated to V. The bottom gel showing protein abundance stained by Coomassie Brilliant Blue (CBB).",
    "molecules": "tyrosine"
  },
  {
    "caption": "D. 3-OH-C10:0 induced Y600 phosphorylation. Two-week-old pLORE:LORE-FLAG complementation plants were treated with 1 μM 3-OH-C10:0 at indicated times. Plant leaves were subjected to anti-FLAG IP. Phosphorylation level of Y600 was detected by anti-pY600 antibody. The experiment was repeated three times with similar results.",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": "E. Y600 mutation abolished the ROS burst triggered by 3-OH-C10:0. The transgenic plants were treated with 3-OH-C10:0 and ROS burst was measured. WT(6-1) and WT(10-3) are two independent complementation lines with LORE; Y600F(2-6) and Y600F(7-1) are two independent lines complemented with LOREY600F; and kinase-dead LOREK516E was transformed to lore mutant as a negative control. The 2-3 and 5-2 are two independent lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).",
    "molecules": "3-OH-C10:0, ROS"
  },
  {
    "caption": "F. Y600 is required for growth inhibition by 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. The experiment was repeated three times with similar results. WT(6-1) and Y600 (2-6) lines were used for the assay. Scale bar=0.4 cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": "A. LORE interacts with specific RLCKs (PBL34/35/36) in yeast. BD-LORE-CD and AD-PBLs were co-transformed to yeast cells and were screened on synthetic dextrose media lacking leucine and tryptophan (SD/-Leu-Trp). The single yeast colonies were serially diluted onto SD/-Leu-Trp and SD/-Leu-Trp-His (SD media lacking leucine, tryptophan and histidine) to observe yeast cell growth. 3-AT(20 mM) was used to inhibit the autoactivation of LORE. Yeast co-transformed with pGADT7-T+pGBKT7-53(+) served as a positive control, and the yeast co-transformed with pGADT7-T+pGBKT7-lam(-) and AD-PBL39+BD-LORE served as the negative controls. EV, empty vector.",
    "molecules": "His, 3-AT, histidine, Leu, leucine, Trp, tryptophan"
  },
  {
    "caption": " D. LORE interacts with PBLs, revealed by split-luciferase assays. N. benthamiana leaves were co-infiltrated with Agrobacteria carrying 35S:LORE-nLUC and 35S:cLUC-PBLs. Images of chemiluminescence were obtained by applying 0.5 mM luciferin 36 h post infiltration. Similar results were observed in three biological replicates. ",
    "molecules": "luciferin"
  },
  {
    "caption": " A. ROS burst in pbl34/35/36 triple mutant, induced by 3-OH-C10:0. Two-week-old seedlings were treated with 1 μM 3-OH-C10:0, and the ROS burst was measured. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01). ",
    "molecules": "3-OH-C10:0, ROS"
  },
  {
    "caption": " B. PTI marker gene expression was reduced in pbl34/35/36 triple mutant in response to 3-OH-C10:0. Treatment is same as in A. Samples were collected for RT-qPCR at 4 h post infiltration. Values are means ± SD (n=3 biological replicates). (Student's t-test, **P< 0.01). ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": " C. pbl34/35/36 triple mutant displayed decreased callose depositions in response to 3-OH-C10:0 treatment. Two-week-old seedlings were sprayed with 1 μM 3-OH-C10:0. The leaves were sampled for callose deposition assays 12 hrs later. The stained callose was counted for 1 mm2 of the leaves. Mock is solvent control. Scale bar=100μm.Values are means ± SD (n=6 leaves). (Student's t-test, **P< 0.01). ",
    "molecules": "callose, 3-OH-C10:0"
  },
  {
    "caption": " D. Root length of pbl34/35/36 triple mutant treated with 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. Scale bar=0.4cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01). ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": " A. PBL34 was phosphorylated by LORE in vitro. Purified recombinant proteins were used for in vitro kinase assays. The kinase dead mutant MBP-PBL34* (PBL K180 was substituted to E, predicted by https://www.uniprot.org/uniprot/), was used as the substrate of GST-LORE-CD or its kinase dead variant, GST-LORE-CD* (LOREK516E). MBP served as a negative control. Incorporation of isotope labeled phosphates was visualized by 32P autoradiography. The CBB staining showing protein abundance. ",
    "molecules": "32P"
  },
  {
    "caption": " B. Mutation of PBL34 T306/T310 significantly reduced its phosphorylation by LORE. Recombinant kinase dead PBL34* and PBL34*-2A (T306/T310 was mutated to A) were incubated with LORE-CD. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining. ",
    "molecules": "32P"
  },
  {
    "caption": " C. Mutation at Y600 diminished trans-phosphorylation activity of LORE. Recombinant LORE-CD and LOREY600F-CD were used for the trans-phosphorylation assays. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining. ",
    "molecules": "32P"
  },
  {
    "caption": " D. PBL34 phosphorylation is induced by 3-OH-C10:0. The protoplast of Col-0 was transformed with 35S:PBL34-FLAG and 35S:PBL34-2A-FLAG, respectively. Sixteen hours later, the protoplasts were treated with1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results. ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": " E. 3-OH-C10:0-induced PBL34 phosphorylation depends on LORE. The 35S:PBL34-FLAG was introduced to Col-0 and lore, respectively. Four-week-old homozygous transgenic plants were treated with 1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results. ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": " F. PBL34 dissociated from LORE upon 3-OH-C10:0 activation. The 35S:LORE-FLAG plants were transformed with native promoter driven PBL34 (pPBL34:PBL34-HA). 100 nM flg22 or 1 μM 3-OH-C10:0 was sprayed onto plant leaves, and the leaves were sampled for assays15 min later. The experiment was repeated three times with similar results. ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": " G. Phosphorylation of PBL34K180E did not disassociate from LORE. The 35S:LORE-FLAG protoplasts were transfected with 35S:PBL34K180E-HA. 100nM flg22 or 1 μM 3-OH-C10:0 was used to treat the protoplasts. The protoplasts were sampled 15 min later. The experiment was repeated three times with similar results. ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": " H. PBL34 rescues pbl34/35/36 triple mutant ROS burst in response to 3-OH-C10:0. Treatment was same as in (E). pPBL34:PBL34-HA was introduced to pbl34/35/36 triple mutant, and the homozygous transgenic line was used for ROS burst assays. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01). ",
    "molecules": "3-OH-C10:0, ROS"
  },
  {
    "caption": " I. PTI marker gene expression in PBL34 complementation lines. The PTI marker genes FRK1 and NHL10 expression was examined by RT-qPCR in Col-0, pbl34/35/36, and CM-PBL34-2A lines after 3-OH-C10:0 treatments for 4 hrs. Values are means ± SD (n=6; ANOVA; p < 0.01). ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": " J. PBL34 phosphorylation at T306/T310 is essential for disease resistance. pbl34/35/36 triple mutants were complemented with PBL34 or PBL34-2A driven by its native promoter. The homozygous plants were pretreated with 3-OH-C10:0(C10) 1 d before pathogen inoculation. The plants were inoculated with 2×105 cfu/ml Pst DC3000, and the bacterial titer was analyzed at 3 dpi. Mock is solvent control. Letters indicate the difference from Col-0. Values are means ± SD (n=6; ANOVA; p < 0.01). ",
    "molecules": "3-OH-C10:0"
  },
  {
    "caption": "  E. HopAO1 suppresses 3-OH-C10:0-triggered ROS burst. HopAO1 was ectopically expressed in Col-0 driven by estradiol-inducible promoter. 100μM estradiol was sprayed on Est:HopAO1 plants, and the leaf disc was used for ROS burst assays 12 hrs later. Values are means ± SD (n=6 biological replicates). ",
    "molecules": "3-OH-C10:0, estradiol, ROS"
  },
  {
    "caption": " F. HopAO1 inhibits 3-OH-C10:0-induced disease resistance. The Est:HopAO1 plants were pretreated with 1 μM 3-OH-C10:0 (C10) and100 μM estradiol, and then the plants were inoculated with 5×104 cfu/ml Pst DC3000 24 hrs later. The bacterial titer was analyzed at 3 dpi. Values are means ± SD (n=6; ANOVA; p < 0.01 ",
    "molecules": "3-OH-C10:0, estradiol"
  },
  {
    "caption": "(a) RT-PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). (",
    "molecules": "glutamine, TPG"
  },
  {
    "caption": "(b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate.",
    "molecules": "glutamine, TPG"
  },
  {
    "caption": "(c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine.",
    "molecules": "glutamine"
  },
  {
    "caption": "(d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.",
    "molecules": "glutamine"
  },
  {
    "caption": "(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. *P0.05.",
    "molecules": "BafA1, glutamine, CCI"
  },
  {
    "caption": "(c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins.",
    "molecules": "BafA1, glutamine"
  },
  {
    "caption": "d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.",
    "molecules": "glutamine"
  },
  {
    "caption": "(f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.",
    "molecules": "glutamine"
  },
  {
    "caption": "(a) Colocalization of lysosomes (Lysotracker Green) with phagophores (mCherry-ULK1 and mCherry-glutamine puncta) in glutamine-deprived U2OS cells. Scale bar, 25 μm.",
    "molecules": "glutamine"
  },
  {
    "caption": "(b) Colocalization of LAMP1-positive lysosomes with GFP-LC3-labelled autophagosomes after glutamine deprivation in the absence or presence of 400 nM BafA1 in U2OS cells. Scale bar, 15 μm.",
    "molecules": "BafA1, glutamine"
  },
  {
    "caption": "(c) Immunofluorescence images of U2OS cells stained for p62 expression and LAMP1 following glutamine deprivation and BafA1 treatment. Nuclei were stained with DAPI. Scale bar, 15 μm.",
    "molecules": "BafA1, glutamine"
  },
  {
    "caption": "(d) Immunofluorescence images of cells stained with LAMP1 (lysosomes) and the Golgi marker, RCAS1, following glutamine deprivation. Nuclei were stained with DAPI. Scale bar, 15 μm.",
    "molecules": "glutamine"
  },
  {
    "caption": "(e) U2OS cells were treated for 24 h with cell culture medium containing the indicated amino acids. Cell lysates were immunoblotted to detect changes in S6K phosphorylation and p62. (",
    "molecules": "amino acids"
  },
  {
    "caption": "(f) Immunofluorescence staining for LAMP1 (green) and mTOR (red) in U2OS cells subjected to glutamine deprivation (24 h), in the presence of 100 nM WYE-125132 (WYE-132). −AA denotes cells deprived of all amino acids. Scale bar, 15 μm. Error bars in all figures represent s.d. of three biological replicates. Ess., essential.",
    "molecules": "amino acids, glutamine, WYE-125132, WYE-132"
  },
  {
    "caption": "(b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in b",
    "molecules": "glutamine"
  },
  {
    "caption": "d) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation.",
    "molecules": "glutamine"
  },
  {
    "caption": "(e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.",
    "molecules": "BafA1, glutamine"
  },
  {
    "caption": "(a) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm.",
    "molecules": "BrefA, glutamine, WYE-125132"
  },
  {
    "caption": "(b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132.",
    "molecules": "BrefA, glutamine, WYE-125132"
  },
  {
    "caption": "(c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132.",
    "molecules": "WYE-125132, CCI-779"
  },
  {
    "caption": "(d) RT-PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.",
    "molecules": "glutamine, WYE-125132, CCI-779"
  },
  {
    "caption": "(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.",
    "molecules": "glutamine, CCI-779"
  },
  {
    "caption": "(b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation.",
    "molecules": "glutamine"
  },
  {
    "caption": "e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi).",
    "molecules": "SP600125, glutamine"
  },
  {
    "caption": "(g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h.",
    "molecules": "glutamine"
  },
  {
    "caption": "(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.",
    "molecules": "BPTES, glutamine"
  },
  {
    "caption": "(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.",
    "molecules": "DMαKG, glutamine"
  },
  {
    "caption": "(c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG.",
    "molecules": "DMαKG, BPTES, glutamine"
  },
  {
    "caption": "(d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG.",
    "molecules": "DMαKG, BPTES, glutamine"
  },
  {
    "caption": "(e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.",
    "molecules": "DMαKG, glutamine"
  },
  {
    "caption": "(B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset).",
    "molecules": "nocodazole"
  },
  {
    "caption": "(B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset).",
    "molecules": "nocodazole"
  },
  {
    "caption": "(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown.",
    "molecules": "cycloheximide, glycosylated membrane proteins"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Human osteosarcoma U2OS stably expressing CALR-GFP and H2B-RFP were treated as described above and images were acquired once per hour for 12 h (A). For one representative experiment among three, the mean ± SEM of the average area of high CALR dots (normalized to the control at each timepoint) of quadruplicates is shown (B). Values are depicted as the area under the curve ± SD of triplicates",
    "molecules": "DACT, dactinomycin, mitoxantrone, MTX"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control Treated U2OS cells stably expressing HMGB1-GFP and H2B-RFP images were acquired every hour for 24 h (D). For one representative experiment among three, the mean ± SEM of the green fluorescence intensity in the nucleus (normalized to the control at each timepoint) of quadruplicates is depicted (E). For each cell, the speed of nuclear release (difference of HMGB1 nuclear green fluorescence intensity between two time points) was calculated. Values are depicted as the average speed of the nuclear release ± SD of quadruplicates",
    "molecules": "DACT, dactinomycin, mitoxantrone, MTX"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated for 6, 12 or 24 h and ATP was stained with quinacrine (G). The number of quinacrine negative cells was assessed based on the distribution of cellular green fluorescence intensity in MTX versus control conditions. For one representative experiment among three the mean ± SD of quadruplicate assessments is shown",
    "molecules": "DACT, dactinomycin, ATP, mitoxantrone, MTX, quinacrine"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated with MTX or DACT as described above for 6 h. Then medium was refreshed and 24 h later, type I interferon response was assessed by transferring the supernatant on HT29 MX1-GFP reporter cells lines cells for additional 48 h. Human type 1α interferon (IFNα1) was also added on the cells as an additional positive control. Images were acquired by fluorescence microscopy and the number of positive cells was assessed based on the distribution of cellular green fluorescence intensity in IFNα1 versus control conditions (I). The percentage of MX1 positive cells was calculated and the mean ± SEM of five independent experiments is depicted",
    "molecules": "DACT, dactinomycin, mitoxantrone, MTX"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS wild-type cells were treated as mentioned above for 6 h and then medium was refreshed. Twenty-four hours later, cells were collected and surface-exposed calreticulin (CALR) was stained with an antibody specific for CALR. DAPI was used as an exclusion dye and cells were acquired by flow cytometry (K). The percentage of CALR+ cells among viable (DAPI-) ones are depicted. The mean ± SEM of six independent experiments is depicted",
    "molecules": "DACT, dactinomycin, DAPI, mitoxantrone, MTX"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS cells were treated as described above for 24 h and the concentration of HMGB1 released in the supernatant was quantified with an ELISA kit, then normalized to control. The mean ± SEM of four independent experiments is shown.",
    "molecules": "DACT, dactinomycin, mitoxantrone, MTX"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with dactinomycin (DACT) at 0.5 or 1 µM, or with mitoxantrone (MTX) between 1 and 6 µM as positive control U2OS were treated as described above for 24 h. Concentration of secreted ATP in the supernatant was quantified with a luciferase-based bioluminescence kit. The mean ± SD of quadruplicates from one representative among three experiments is depicted.",
    "molecules": "DACT, dactinomycin, ATP, mitoxantrone, MTX"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin (DACT) (0.25, 0.5 or 1 µM) for 6 h. Thapsigargin (THAPS) at 3 µM was used as a positive control. After fixation, cells were stained with phospho-eIF2α (Ser51) specific antibody followed by an AlexaFluor-647 secondary antibody, nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy. Images were segmented, analyzed and the red cytoplasmic fluorescence intensity was measured. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM",
    "molecules": "Hoechst 33342, DACT, dactinomycin, AlexaFluor-647, Ser, THAPS, Thapsigargin"
  },
  {
    "caption": "U2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM",
    "molecules": "DACT, THAPS"
  },
  {
    "caption": "U2OS stably expressing ATF6-GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM",
    "molecules": "DACT, THAPS"
  },
  {
    "caption": "U2OS cells stably expressing venus in frame with alternatively spliced XBP1 (sXBP1) were treated as described above for 12 h. De novo expressed venus was measured intracellular. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM",
    "molecules": "DACT, THAPS"
  },
  {
    "caption": "U2OS wild-type and knock out for eIF2α kinases 1, 2, 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3-mediated eIF2α phosphorylation or with 1 µM DACT for 6 h. After fixation, cells were stained for peIF2α as described above and cytoplasmic intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM",
    "molecules": "DACT, THAPS"
  },
  {
    "caption": "U2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h",
    "molecules": "Hoechst 33342, DACT, AlexaFluor-647, DAPI, TM, tunicamycin"
  },
  {
    "caption": "Mouse fibrosarcoma MCA205 cells were stained with CellTracker Orange (CMTMR) and treated for 24 h with 1 µM dactinomycin (DACT) or 500 µM oxaliplatin (OXA) as a positive control. Then, untreated or dying MCA205 were co-cultured with differentiated bone marrow-derived dendritic cells (BMDCs) for 4 h at 37 °C or at 4 °C. Cells were collected and dendritic cells were stained with CD11c specific antibody before analysis by flow cytometry The percentage of CMTMR and CD11c double positive cells among all CD11c+ cells are indicated",
    "molecules": "CellTracker Orange, CMTMR, DACT, dactinomycin, OXA, oxaliplatin"
  },
  {
    "caption": "MCA205 cells were treated for 24 h with 500 µM oxaliplatin (OXA) or with 0.25, 0.5 or 1 µM dactinomycin (DACT). Then, untreated or dying MCA205 were co-cultured with BMDCs for 24 h at 37 °C. As a positive control, BMDCs were co-cultured with 1 μg/mL LPS and 100 ng/mL IFNγ. The increase in the percentages of MHCII+ and CD86+ cells among CD11c+ cells was quantified with respect to untreated controls.",
    "molecules": "DACT, dactinomycin, LPS, OXA, oxaliplatin"
  },
  {
    "caption": "1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Next, tumor size was measured regularly and individual tumor growths of DACT-vaccinated versus Ctr mice are depicted",
    "molecules": "DACT, dactinomycin"
  },
  {
    "caption": "1 x 106 mouse fibrosarcoma MCA205 cells were treated in vitro with 1 µM dactinomycin (DACT). Overall survival is depicted and p-values (*** p<0.001.) were calculated with a Log-Rank test",
    "molecules": "DACT, dactinomycin"
  },
  {
    "caption": "3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Tumor size was assessed regularly and individual tumor growth curves of DACT versus Ctr",
    "molecules": "DACT, dactinomycin"
  },
  {
    "caption": "3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-CD4/CD8 versus anti-CD-4/anti-CD-8 Mean tumor area for each group was calculated and significances were tested using a type II ANOVA test Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and hashes indicate the p-values of the DACT + anti-CD-4/anti-CD-8 versus DACT alone",
    "molecules": "DACT, dactinomycin"
  },
  {
    "caption": "3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. DACT + anti-PD-1 versus anti-PD-1 are depicted. Overall survival is depicted and p-values were calculated with a Log-Rank test Stars indicate the p-values of each treatment versus Ctr and of DACT + anti-PD-1 versus anti-PD-1 alone",
    "molecules": "DACT, dactinomycin"
  },
  {
    "caption": "3 x 105 mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c) into the flank of immunocompetent syngeneic Balb/c mice When tumors became palpable, the mice were injected intraperitoneally (i.p.) with injectable solution (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed four days later. Five naïve mice or the eleven mice that were cured by treatment with DACT alone or in combination with PD-1 blockade were (re)challenged with WEHI 164 cells, and individual tumor growths (I, J), as well as overall survival (*** p<0.001, Log-Rank test) (K), were monitored.",
    "molecules": "DACT, dactinomycin"
  },
  {
    "caption": "Mice bearing WEHI 164 sarcomas were treated by systemic (intraperitoneal, i.p.) injections of DACT or PBS as a control (Ctr), and tumor were excised 9 days later for the quantitation of mRNA coding for IFNγ Two independent experiments were conducted with a total of 22 mice in the Ctr group and 23 mice in the DACT-treated group, with each data point indicating one tumor. The cycle threshold of the qRT-PCR of IFNγ was normalized to the one of the housekeeping gene peptidylpropyl isomerase A (Ppia) in each mouse and results are shown normalized with respect to controls as a dot plot depicting mean ± SD . The p-value (* p<0.05) was calculated by means of a Student's t test (B).",
    "molecules": "DACT"
  },
  {
    "caption": "Balb/c mice with palpable WEHI 164 sarcomas (n=7 mice in Ctr and anti-IFNγ groups; n=8 mice in DACT and DACT+anti-IFNγ groups) received two injections of DACT-based chemotherapy (0.5 mg/kg) as well as multiple injections (3 times per week) of neutralizing IFNγ-specific antibody (C). Tumor growth curves are shown for individual mice (D) and as means (E). Statistical difference between DACT-treated tumors and respective controls, which was calculated with a type II ANOVA (* p<0.05), is lost upon IFNγ neutralization (E). Overall survival of mice is also indicated with statistics to respective controls calculated with a Log-Rank test (* p<0.05) (F).",
    "molecules": "DACT"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h and followed by an additional hour of treatment in the presence of 100 mM 5-ethynyl uridine (EU). After fixation cells were permeabilized and EU was stained with an AlexaFluor-488-coupled azide. Representative images are shown for each treatment (A). The EU intensity in the nucleus of each condition was ranked between the untreated control (Ctr, 0 % transcription inhibition) and the control that was not incubated with EU (corresponding to 100 % transcription inhibition) (B). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown",
    "molecules": "5-ethynyl uridine, EU, DACT, dactinomycin, AlexaFluor-488, azide, bortezomib, BTZ, CDDP, cisplatin, CRIZ, crizotinib, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, EPI, epirubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, VB, vinblastine, VC, vincristine"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were treated for 2.5 h and before fixation and permeabilization. Then, cells were stained with a rabbit anti-fibrillarin antibody followed by a staining with an anti-rabbit AlexaFluor-647- or AlexaFluor-546-coupled secondary antibody as well as with a mouse anti-nucleolin antibody followed by a staining with an anti-mouse AlexaFluor-488-coupled secondary antibody. Then images were acquired and colocalization between both signals was assessed (C). The surface overlap coefficient (SOC) was calculated and ranked between the untreated control (Ctr) and the positive control (DACT) Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown",
    "molecules": "DACT, dactinomycin, AlexaFluor-488, AlexaFluor-546, AlexaFluor-647, bortezomib, BTZ, CDDP, cisplatin, CRIZ, crizotinib, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, EPI, epirubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, VB, vinblastine, VC, vincristine"
  },
  {
    "caption": "Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were pre-treated overnight with the aforementioned compounds in complete medium followed by washout and treatment pursued in methionine-free medium for 30 min. Afterwards, the treatments were continued in methionine-free medium supplemented with 25 µM L-azidohomoalanine (AHA) for additional 1.5 h. AHA incorporation was detected after fixation, permeabilization and blocking by the addition of an AlexaFluor-488-coupled azide. Then images were acquired (E) and AHA intensity in the cells was ranked between the untreated control (Ctr, 0 % translation inhibition) and the untreated control without AHA (corresponding to 100 % translation inhibition) (F). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown",
    "molecules": "AHA, L-azidohomoalanine, DACT, dactinomycin, AlexaFluor-488, azide, bortezomib, BTZ, CDDP, cisplatin, CRIZ, crizotinib, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, EPI, epirubicin, methionine, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, VB, vinblastine, VC, vincristine"
  },
  {
    "caption": "Correlation between the transcription measured by EU incorporation and measured by fibrillarin and nucleolin colocalization is depicted with Pearson correlation coefficient (R) and p-value (p) (G). The same parameters are shown for the correlation between transcription measured by EU incorporation and translation measured with AHA incorporation",
    "molecules": "AHA, EU"
  },
  {
    "caption": "U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ICD prediction score Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)",
    "molecules": "EU, DACT, dactinomycin, cisplatin, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, resveratrol, spermidine, VB, vinblastine, VC, vincristine, vinorelbine, VR"
  },
  {
    "caption": "U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and peIF2α expression Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)",
    "molecules": "EU, DACT, dactinomycin, cisplatin, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, resveratrol, spermidine, VB, vinblastine, VC, vincristine, vinorelbine, VR"
  },
  {
    "caption": "U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and CALR exposure Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)",
    "molecules": "EU, DACT, dactinomycin, cisplatin, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, resveratrol, spermidine, VB, vinblastine, VC, vincristine, vinorelbine, VR"
  },
  {
    "caption": "U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and ATP decrease Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)",
    "molecules": "EU, DACT, dactinomycin, ATP, cisplatin, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, resveratrol, spermidine, VB, vinblastine, VC, vincristine, vinorelbine, VR"
  },
  {
    "caption": "U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and HMBG1 exodus Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)",
    "molecules": "EU, DACT, dactinomycin, cisplatin, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, resveratrol, spermidine, VB, vinblastine, VC, vincristine, vinorelbine, VR"
  },
  {
    "caption": "U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and biological ICD score (F) previously measured and expressed as z-scores (except for ICD prediction score)  Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)",
    "molecules": "EU, DACT, dactinomycin, cisplatin, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, resveratrol, spermidine, VB, vinblastine, VC, vincristine, vinorelbine, VR"
  },
  {
    "caption": "U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. For assessing translation, cells were pre-treated with the library for 12 h followed by 30 min in methionine free-medium, before addition of azidohomoalanine (AHA). Percentage of inhibition was calculated and transformed as z-scores. The correlations between transcription and translation inhibitions Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)",
    "molecules": "AHA, azidohomoalanine, EU, DACT, dactinomycin, cisplatin, DAUN, daunorubicin, DOC, docetaxel, DOXO, doxorubicin, methionine, mitoxantrone, MTX, OXA, oxaliplatin, PACL, paclitaxel, resveratrol, spermidine, VB, vinblastine, VC, vincristine, vinorelbine, VR"
  },
  {
    "caption": "The inhibition of transcription was assessed for the negative and positive ICD hits identified with the predictive algorithm (Figure 1). U2OS cells were treated with the agents at concentrations corresponding to their IC60: 1 μM dactinomycin (DACT), 50 μM topotecan, 1 μM becatecarin, 0.5 μM trabectedin, 5 μM UCN-01, 30 μM mycophenolate mofetil, 30 μM nonoxynol-9, 25 μM dactolisib, 2.5 μM β-lapachone, 5 μM 5-fluorodeoxycytidine and 2 μM RH-1 for 1.5 h followed by 1 h with EU. The percentage of transcription inhibition was calculated and the coefficient of correlation (R) and associated p-value (p) between the percentage of inhibition and the theoretical ICD score was calculated using the Pearson method.",
    "molecules": "5-fluorodeoxycytidine, EU, UCN-01, DACT, dactinomycin, becatecarin, β-lapachone, dactolisib, mycophenolate mofetil, nonoxynol-9, RH-1, topotecan, trabectedin"
  },
  {
    "caption": "C. Cresyl violet  and ChAT staining  at the lumbar  level show no medium size (200-299 µm2) cell loss (γMNs) and a decrease of about 50% in the number of large size (>300 µm2) cells (αMNs). Unpaired two-sided t-test. **p<0.005; ****p<0.0001. n=5. Data information: The direct comparison of Cresyl violet and ChAT  in C were performed once. Values are mean ± SD.",
    "molecules": "Cresyl violet"
  },
  {
    "caption": "D. Lumbar  level Cresyl violet staining  shows that, in contrast with small and medium size neurons  (interneurons  and γMNs, left and center panels), large size neurons  (αMNs, right panel) undergo progressive death first measured at 4.5 months. The values represent the average number of cells per  ventral horn. For the small neurons  (100-199 µm2), there was no main effect, two-way ANOVA for repeated measures for treatment group: F(1,43)=0.0017, p=0.968, ns. For the medium sized neurons  (200-299 µm2) two-way ANOVA for repeated measures for treatment group: showed no main effect F(1,43)=2.085, ns. For the large neurons (>300 µm2), two-way ANOVA for repeated measures showed a significant main effect F(1,43)=59.99, p<0.0001. Post-hoc comparisons were performed by unpaired two-sided t-test with equal SD comparing WT with En1-Het at each  time point (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=5 to 6. Data information: The longitudinal studies in D were performed once. Values are mean ± SD.",
    "molecules": "Cresyl violet"
  },
  {
    "caption": "B. Left panel illustrates the use of Alexa Fluor 488-conjugated  α-bungarotoxin (α-BTX, in green) and of neurofilament  and  synaptic vesicle glycoprotein antibodies (2H3  and SV2A, in red) to evaluate the percentage of fully occupied endplates  (>80% occupancy). The right panel shows that the % of fully occupied endplates  decreases progressively in the En1-Het mouse, starting between 3 and 4.5 months of age. Scale bar: 50µm. Two-way ANOVA showed a significant main effect (F(1,47)=45.45, p<0.0001). Unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point  (*p<0.05; **p<0.005). n=4 to 8. See details of analysis in the Methods section. Data information: The longitudinal study in B was performed once. Values are mean ± SD.",
    "molecules": "Alexa Fluor 488"
  },
  {
    "caption": "(C) High-performance thin-layer chromatography (HPTLC) profile of HeLa cells pulsed for 2 hours with [3H]-sphingosine and chased for 24 hours. The peaks corresponding to each SL species is indicated and numbers represent each SL species as percentage of total SL.",
    "molecules": "SL, sphingosine, 3H"
  },
  {
    "caption": "(A) HeLa cells were treated with control or indicated siRNA for 72 hours and SL biosynthesis measured by [3H] - sphingosine pulse-chase assay. GSL levels are expressed as fold changes with respect to control. CERT and FAPP2 knockdowns (blue bars) were used as controls.",
    "molecules": "GSL, SL, sphingosine, 3H"
  },
  {
    "caption": "(D) Control and GRASP55KO cells were processed for Cy3-conjugated Shiga Toxin (ShTxB) and Alexa488-conjugated Cholera Toxin (ChTxB) staining followed by flow cytometry analysis. Mean fluorescence intensity was measured and represented.",
    "molecules": "Alexa488, Cy3"
  },
  {
    "caption": "(C) Control and GRASP55 KO cells were transfected for 16 hours with GCS-HA or LCS-HA and processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles) in case of GCS-HA and anti-HA antibody (15-nm gold particles) and anti-GM130 antibody (10-nm gold particles) in case of LCS-HA. Representative images of the distribution of GCS-HA and LCS-HA are shown. Scale bar 200nm. Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling.",
    "molecules": "gold particles"
  },
  {
    "caption": "(A-C) Control and GRASP55 KO (#2) cells were transfected for 16 hours with the indicated HA-tagged enzymes and processed for cryo-immunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles). Representative images of the distribution of HA-tagged enzymes are shown. Red circles indicate GM130 labelling. Arrow heads represent the peri-Golgi vesicles. Scale bar 150 nm. Quantification of the distribution of enzymes in vesicles represented as normalized linear density",
    "molecules": "gold particles"
  },
  {
    "caption": "(F) HeLa cells co-transfected with HA-tagged GCS and GRASP55-GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GFP antibody (15-nm gold particles). Representative images of the distribution of GCS and GRASP55-GFP are shown. Red arrowhead indicates the clathrin-coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS-HA in TGN. Scale bar 150 nm. Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles.",
    "molecules": "gold particles"
  },
  {
    "caption": "(A Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). Total GSL levels were quantified and expressed as fold changes with respect to control (A).",
    "molecules": "BFA, DMSO, GSL, SL, sphingosine, 3H"
  },
  {
    "caption": "B) Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). The GM/Gb ratio was calculated and represented (B).",
    "molecules": "BFA, DMSO, GM, Gb, SL, sphingosine, 3H"
  },
  {
    "caption": "(F) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO (#2) clone before subjecting to [3H] -sphingosine pulse chase assay. GSLs were quantified and represented as fold change with respect to control.",
    "molecules": "GSLs, sphingosine, 3H"
  },
  {
    "caption": "Immunofluorescent staining of SARS-CoV-2 infected differentiated small airway cultures. Nucleoprotein (NP) stains viral capsid (red), which colocalized with the ciliated cell marker AcTUB Phalloidin was included to stain actin Scale bars indicate 50 μm.",
    "molecules": "Phalloidin"
  },
  {
    "caption": " A) Immunofluorescent staining of fetal lung bud tip progenitor organoids grown in expansion medium co-expressing stem cell markers Sox2 (green) and Sox9 (red). Phalloidin (white) was used to stain actin. Data information: Nuclei are stained with Hoechst ",
    "molecules": "Hoechst, Phalloidin"
  },
  {
    "caption": " B) Immunofluorescent staining of rare HTII-280+ type II pneumocyctes (green) in a subpopulation of fetal lung bud tip progenitor organoids grown in expansion medium. Data information: Nuclei are stained with Hoechst ",
    "molecules": "Hoechst"
  },
  {
    "caption": " Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express alveolar type I (HOPX, green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express HTI-56, green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express type II cells (HTII-280, green markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express LPCAT1, green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express SFTPC (SPC), green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " Differentiated lung bud tip organoids at air-liquid interface in co-culture with donor-specific human fetal lung fibroblasts. After 14 days of differentiation at air-liquid interface, cells express basal cell (TP63 (P63), green, markers in areas containing one cell layer. Dotted lines indicate the barrier between multilayered and single layered epithelium. Data information: Nuclei are stained with Hoechst Scale bars indicate 50 μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " A) pSTAT1 translocation in mock and SARS-CoV-2-infected bronchioalveolar-like cells visualized in green. Scale bars indicate 50 μm. Nucleoprotein (NP) stains viral capsid (red). Nuclei are stained with Hoechst (white). ",
    "molecules": "Hoechst"
  },
  {
    "caption": " C) pSTAT1 translocation in mock and SARS-CoV-2-infected small airway cells visualized in green. Nucleoprotein (NP) stains viral capsid (red). Nuclei are stained with Hoechst (white). Scale bars indicate 50 μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " C) Whole well immunofluorescent staining of representative cultures in (A) and (B) using an anti-NP antibody (red). Nuclei are stained with Hoechst (blue). Scale bar indicates 1mm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point  immunoassay  using the  2C1  monoclonal antibody, in  varying concentrations of compound (shown in panel A). Modification of the phenyl  and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the  2-oxindole GSK716  (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).",
    "molecules": "GSK425, indole, 2-oxindole, phenyl, GSK716"
  },
  {
    "caption": "(C) GSK716 binds to  Z α1-antitrypsin with a high affinity mean pKD  of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.",
    "molecules": "GSK716"
  },
  {
    "caption": "(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin  (Z α1-AT) as reported by  fluorescence polarisation of an Alexa-488-labelled variant of  GSK716.",
    "molecules": "Alexa-488, GSK716"
  },
  {
    "caption": "(E) Representative curves reporting the  interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic  tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1. (F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1. ",
    "molecules": "GSK716, tryptophan"
  },
  {
    "caption": " (G)  CHO-K1 Tet-On cells expressing  Z α1-antitrypsin were induced with  doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716  (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO  were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin  were immunoprecipitated either with a mAb against total α1-antitrypsin  (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE  and detected by autoradiography. (H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2) ",
    "molecules": "35S, Cys, DMSO, doxycycline, Met, NP-40, GSK716, Tet"
  },
  {
    "caption": "(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in  PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.",
    "molecules": "glycerol, PBS, GSK716"
  },
  {
    "caption": "(C) M and Z  α1-antitrypsin  were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.",
    "molecules": "bis-ANS dye, gdnhcl, guanidine hydrochloride, GSK716"
  },
  {
    "caption": "(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the  α1-antitrypsin intrinsic  tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four  α1-antitrypsin  variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.",
    "molecules": "GSK716, tryptophan"
  },
  {
    "caption": "(C) The  inhibitory  activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.",
    "molecules": "GSK716"
  },
  {
    "caption": "Z  α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by  LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the  liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice  and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z  α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant,  dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.",
    "molecules": "GSK716"
  },
  {
    "caption": "Representative calcium signature enriched in P-EMT tumors (comparing ECAD- vs. ECAD+ tumor cells). NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.",
    "molecules": "calcium"
  },
  {
    "caption": "Representative image of tumor cells transduced with GCaMP6 (Left). Yellow arrows (mesenchymal cells), red arrows (epithelial cells), scale bar = 50μm. GCaMP6 mean fluorescent intensity (MFI) in ECADHIGH or ECADLOW cells cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h (Right). Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (*, p<0.05; non-significant (NS); ±SD).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo-1 and cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h. Representative of 2 independent cell lines (n indicates the number of cells analyzed in each group, ECADHIGH 0 Ca2+ n=4963, ECADLOW 0 Ca2+ n=14, ECADHIGH 2 Ca2+ n=6575, ECADLOW 2 Ca2+ n=29). Representative of 2 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (****, p<0.0001; non-significant (NS); ±SD).",
    "molecules": "Ca2+, Indo-1"
  },
  {
    "caption": "Representative 20X brightfield and fluorescent images of murine YFP+ tumor cells treated for 72h with vehicle control (DMSO), 2.5μM Ionomycin, or 10ng/ml TGFβ and co-stained for E-cadherin (Red) and DAPI (Blue). Scale bar is 100μm.",
    "molecules": "DAPI, DMSO, Ionomycin"
  },
  {
    "caption": "Representative flow cytometry analysis of surface ECAD (Left), MFI of surface ECAD (Middle), and percentage of surface ECAD negative cells (Right) in murine PDA cells, after treatment with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 72h. Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by ANOVA (****, p<0.0001; non-significant (NS); ±SD).",
    "molecules": "DMSO, IONO, Ionomycin"
  },
  {
    "caption": "Top: Relative mRNA expression of Ecad after treatment with 2.5μM ionomycin or 10ng/ml TGFβ for denoted time. Bottom: Western blot analysis of total E-cadherin and Vimentin protein for denoted time. Experiment was run in duplicate with three different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; non-significant (NS); ±SEM).",
    "molecules": "ionomycin"
  },
  {
    "caption": "Normalized MFI of surface E-cadherin in PDA cells treated with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 7 days, followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x-axis. Treated cells were analyzed at day 2, 3, and 7. Withdrawn cells were analyzed on days 1, 2, and 7. Experiment was performed in duplicate in two different murine PDA cell lines. Error bars indicate SD.",
    "molecules": "DMSO, IONO, Ionomycin"
  },
  {
    "caption": "Quantification of live cellular migration over 4h after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 48hrs. (n=16 cells per condition. Experiment was run in triplicate in two murine PDA different cell lines) (Right). Quantification of transwell migration after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 72h (n= 2 cell lines, 3 replicates per cell line with 3 20X images taken per transwell) (Left). Statistical analysis by ANOVA (*, p<0.05; ****, p<0.0001; non-significant (NS); ±SD).",
    "molecules": "DMSO, IONO, Ionomycin"
  },
  {
    "caption": "Schematic for RNA sequencing after EMT induction for 48h. Heatmap illustration of EMT related genes in three independent murine PDA cell lines, sequenced in duplicate, treated with DMSO, 2.5μM Ionomycin, or 10ng/ml TGFβ. Scale indicates Z-score.",
    "molecules": "DMSO, Ionomycin"
  },
  {
    "caption": "GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for EMT signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.",
    "molecules": "DMSO, ionomycin"
  },
  {
    "caption": "GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for calcium signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.",
    "molecules": "calcium, DMSO, ionomycin"
  },
  {
    "caption": "Leading edge plots showing enrichment of C-EMT gene signatures in TGFβ treated cells (Left) or P-EMT gene signatures in ionomycin (IONO) treated cells (Right), based on GSEA. Enrichment scores (ES), NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.",
    "molecules": "IONO, ionomycin"
  },
  {
    "caption": "Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD. Blue or red line indicates average of n=22 cells. ±SD. Tracings are representative of experiments performed twice in 2 independent cell lines.",
    "molecules": "ATP, Ca2+, CNO, Fura-2"
  },
  {
    "caption": "Representative 20X brightfield images of Gαq-DREADD-expressing cells 48h after addition of CNO (Left). Scale bar = 100μm. MFI of surface E-cadherin in cells expressing a Gαq-DREADD after treatment with DMSO, 5μM CNO, or 2.5μM ionomycin (Right). Both experiments performed in triplicate in 2 independent cell lines. Statistical analysis by ANOVA (**, p<0.01; ±SD).",
    "molecules": "CNO, DMSO, ionomycin"
  },
  {
    "caption": "MFI of surface E-cadherin in CnB knockout lines after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).",
    "molecules": "DMSO, ionomycin"
  },
  {
    "caption": "MFI of surface E-cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO, 2.5μM ionomycin, or 20μM W7 with 2.5μM ionomycin for 24h. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (**, p<0.01; ***, p<0.001; non-significant (NS); ±SD).",
    "molecules": "W7, DMSO, ionomycin"
  },
  {
    "caption": "MFI of surface E-cadherin of shRNA non-targeting (shNT) (Left) or shCAMK2B (Right) after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).",
    "molecules": "DMSO, ionomycin"
  },
  {
    "caption": "A-C ARR1/10/12 protein levels were examined by western blot when five-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP were treated with or without 200 mM NaCl for the indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-F Quantification analysis of the protein levels of ARR1/10/12 in (A-C). ",
    "molecules": "NaCl"
  },
  {
    "caption": "C In vivo Coimmunoprecipitation assay of MPK3/4/6 and ARR1/10/12. MPK3/4/6-YFP and ARR1/10/12-MYC were co-expressed in Arabidopsis protoplast cells. Protein extracts (Input) were immunoprecipitated with GFP Trap magnetic agarose beads. Immunoblots were performed with anti-MYC antibody to detect ARR1/10/12 and with anti-GFP antibody to detect MPK3/4/6.",
    "molecules": "agarose"
  },
  {
    "caption": "A-C Thiophosphorylation assays of wild type and mutated forms ARR1/10/12 by MPK3/6 in vitro. Potential phosphorylated Ser residues or Thr residues of ARR1, ARR10, and ARR12 were mutated to Ala (ARR1m/ARR10m/ARR12m). Recombinant ARR1/10/12-His and ARR1m/10m/12m-His proteins were incubated with MPK3 and MPK6 which were activated by MKK5DD. Phosphorylated ARR1/10/12, ARR1m/10m/12m, and MPK3/6 were visualized by an anti-thiophosphate ester-specific antibody (α-TPE) (upper panel). Asterisk in (B) indicates non-specific bands. Recombinant MKK5DD, MPK3, MPK6, ARR1/10/12, and ARR1m/10m/12m were detected by comassie brilliant blue (CBB) staining as loading controls (bottom panel).",
    "molecules": "His, Ala, CBB, comassie brilliant blue, Ser, thiophosphate ester, TPE, Thr"
  },
  {
    "caption": "A-C Immunoblot analysis with anti-MYC antibody or anti-GFP antibody showing the upshift of ARR1, ARR10, and ARR12 in a phos-tag gel. The phosphorylated ARR1, ARR10, and ARR12 were abolished after treatment with calf intestinal alkaline phosphatase (CIAP). Seven-day-old seedlings of 35S:ARR1:MYC (A), 35S:ARR10:YFP (B), and 35S:ARR12:YFP (C) in Col and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Total proteins were extracted and subjected to immunoblot analysis. ARR1, ARR10, and ARR12 proteins separated on SDS-PAGE gel without phos-tag were detected as loading controls.",
    "molecules": "NaCl"
  },
  {
    "caption": "A-C Immunoblot analysis the effects of MPK3/6 on salt stress-induced ARR1/10/12 protein degradation. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP in Col-0 and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of MPK3/6 effect on salt stress-induced ARR1/10/12 protein degradation in (A-C). ",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "A-C Immunoblot analysis stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, 35S:ARR12:YFP, 35S:ARR1T553A:MYC, 35S:ARR10S383A:YFP, and 35S:ARR12S323A:YFP were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress in (A-C). ",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "Four-day-old seedlings of 35S:ARR1:MYC, 35S:ARR1T553A:MYC, and 35S:ARR1T553D:MYC were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "Four-day-old seedlings of 35S:ARR10:YFP, 35S:ARR10S383A:YFP, and 35S:ARR10S383D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "Four-day-old seedlings of 35S:ARR12:YFP, 35S:ARR12S323A:YFP, and 35S:ARR12S323D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes were determined.",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later",
    "molecules": "NA-PP1, NaCl"
  },
  {
    "caption": "A: Morphology  (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. B: WT1 immunostaining  (upper panels) and quantification of WT1-positive glomerular cells  (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. C: PAI-1 immunostaining  (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in 28-month-old  p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. Data information: Data are means ± SEM. Statistical analysis: t-student test: AP+ versus AP- mice.",
    "molecules": "AP, AP20187"
  },
  {
    "caption": "E: Immunofluorescence  of  paxilin (green) and  phalloidin (red) in podocytes stimulated with control medium, supernatant (SN) from  glomerular endothelial cells from early (p9) or late (p17) passage and treated either with vehicle or Tiplaxtinin, a  PAI-1 inhibitor (upper panel). Quantification of focal adhesions (lower panel) was performed by counting the number  of  paxilin-positive spots in 30 cells from three independent experiments. Original magnification X1000. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test",
    "molecules": "phalloidin, Tiplaxtinin"
  },
  {
    "caption": "(C-G) Immunofluorescence on TSs caudal spinal cord of E9.5 wildtype littermates and Slc7a5-null embryos; Pax3 and FoxA2 were used as indicators for dorso-ventral organisation in (C, F) wildtype, n=2 (for FoxA2) and n= 4 (For Pax3) and (D, G) Slc7a5-null, n=3 (for FoxA2 ) and n = 3 for Pax3) neural tube. Arrowheads indicate border of Pax3 expression domain. Scale bars 50 µm. (E) Percentage of Pax3 expressing cells was determined by counting these cells and all DAPI labelled nuclei in the neural tube and comparison made between wildtype (4 embryos, 6 sections each) and Slc7a5-null (3 embryos, 6 sections each), each dot represents the average for one embryo, unpaired t-test, *p = 0.018 (see original source data). Error bars indicate SEM. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-D) Proliferation was assessed in the spinal cord (forelimb level) (A, B) using a phospho-Histone3 (phospho-H3) antibody (green) to identify mitotic cells and DAPI (blue) to label nuclei; cells were counted in 3 wildtype (18 sections) and 3 Slc7a5-null embryos (18 sections) and mitotic index calculated; (C, D) Proliferation was also assessed in the forebrain in 3 wildtype embryos (17 sections) and 3 Slc7a5-null embryos (16 sections). White dashed lines indicate midline. Data information All scale bars 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G, H) mTORC1 activity in the spinal cord (hindlimb level) was assessed by immunofluorescence using an antibody against the phospho-ribosomal protein S6 (phosho-S6 Ser 235/236). Data information All scale bars 50 µm.",
    "molecules": "Ser"
  },
  {
    "caption": "(I) Labelling intensity was measured and plotted relative to DAPI intensity; 6 wildtype embryos (18 sections) and 5 Slc7a5-null embryos (19 sections). Each dot represents the average for one embryo, (*p = 0.0457) unpaired t-test with Welch correction Data information: *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "DAPI"
  },
  {
    "caption": "(C, d1) Axin2 transcripts in embryo trunk explant exposed to DMSO (C, c1, n=12/12) were reduced in Wnt-c59 exposed explants (D, d1, n=14/14). Images in (c1, d1) show TS through explants (data from 3 independent embryo explant experiments). (E, F) Slc7a5 transcripts in embryo trunk explant exposed to DMSO (E, e1, n=9/10) or Wnt-c59 (F, f1, n=15/15). Images in (e1, f1) show TS through explants (data from 3 independent embryo explant experiments). Data information: Scale bars = 100 µm",
    "molecules": "DMSO, Wnt-c59"
  },
  {
    "caption": "(E) ATP treatment induces dissociation of IFT54 but not IFT54Δ342-356 mutant from kinesin-II. Immunoprecipitation experiments were performed as shown in (D) in the presence or absence of ATP. The normalized ratios of FLA8 and FLA10 in the immunoprecipitates (IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.37:1.70:1.38 and 1:0.35:1.47:1.45, respectively.",
    "molecules": "ATP"
  },
  {
    "caption": "(F) Analysis of the interaction of IFT54 and IFT54Δ342-356 mutant with kinesin-II by GST-pulldown assay. Bacterial expressed GST, GST-tagged IFT54 or IFT54Δ342-356 mutant was mixed respectively with recombinant kinesin-II purified from insect cells in the presence or absence of ATP followed by GST pulldown and immunoblotting. The normalized ratios of KAP, FLA10 and FLA8 in the pull-down samples (GST-IFT54 without ATP/with ATP/mutant without ATP/with ATP) are 1:0.51:1.31:1.44, 1:0.55:1.29:1.19 and 1:0.50:1.31:1.29, respectively.",
    "molecules": "ATP"
  },
  {
    "caption": "(G) Yeast two hybrid assay for interaction between IFT54 and IFT dynein subunits. Yeast cells that were transformed with each pair of constructs as indicated were grown under selection media lacking leucine, tryptophan, histidine, and adenine (-4) or lacking leucine and tryptophan (-2). DHC1bT, DHC1b tail domain; Empty AD or BD vectors were used as control.",
    "molecules": "adenine, histidine, leucine, tryptophan"
  },
  {
    "caption": "D Lactate dehydrogenase (LDH) activity in cell supernatants. Staurosporine (Stauro, 1 μmol/L) was used to induce cell death by apoptosis. n=4 cell preparations/group.",
    "molecules": "Stauro, Staurosporine"
  },
  {
    "caption": "H Effect of cyclosporin A (CsA, 1 μmol/L) on the percentage of cells with depolarized mitochondria. n=6-8/group.",
    "molecules": "CsA, cyclosporin A"
  },
  {
    "caption": "I Effect of cyclosporin A (CsA, 1 μmol/L) on cell death in serum-starved cardiomyocytes. n=4/group.",
    "molecules": "CsA, cyclosporin A"
  },
  {
    "caption": "J Cell death in serum starved Nox4 knockout MEFs (Nox4KO) and wild-type MEFs (WT). Cat = PEG-catalase (500 U/mL). Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant, Nox4P437H (Mut). n=6-12/group.",
    "molecules": "PEG"
  },
  {
    "caption": "A Basal mitochondrial calcium levels assessed in serum-starved rat cardiomyocytes using a mitochondrial-targeted cameleon CFP/YFP FRET probe. Nox4 was depleted with silencing RNAs (siRNAs) or cells were transfected with a control scrambled siRNA (siScr). Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). The mean changes in Nox4-depleted cells as compared to scrambled control (dotted line) at the bottom. The representative immunoblot shows depletion of Nox4 protein levels. Scale bars: 10 µm. n=3/group (with >50 cells per individual experiment).",
    "molecules": "calcium"
  },
  {
    "caption": "B Basal mitochondrial calcium levels in WT and Nox4KO MEFs after serum starvation. Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). Mean data at the bottom. Scale bars: 10 µm. n=3/group (with >100 cells per individual experiment).",
    "molecules": "calcium"
  },
  {
    "caption": "Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, C) n=3/group (with >30 cells per individual experiment).",
    "molecules": "calcium, histamine"
  },
  {
    "caption": "D Changes in mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, D). n=3/group (with >30 cells per individual experiment).",
    "molecules": "ATP, calcium"
  },
  {
    "caption": "E, Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/L, E) n=3/group (with >30 cells per individual experiment).",
    "molecules": "calcium, histamine"
  },
  {
    "caption": "F Changes in ER calcium levels measured with an ER-targeted cameleon probe in serum-starved WT and Nox4KO MEFs after the addition of ATP (100 μmol/L, F). n=3/group (with >30 cells per individual experiment).",
    "molecules": "ATP, calcium"
  },
  {
    "caption": "G Peak increase in mitochondrial calcium levels response in response to histamine (100 μmol/L) in WT MEFs with or without treatment with PEG-catalase (Cat), and in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4P437H mutant (Mut). n=3/group (with >30 cells per individual experiment).",
    "molecules": "calcium, histamine, PEG"
  },
  {
    "caption": "H Representative time-course of histamine-induced changes in mitochondrial calcium for the experiments shown in (I).",
    "molecules": "calcium, histamine"
  },
  {
    "caption": "B Fractionation of crude mitochondrial fractions (containing mitochondria and MAM) from WT MEFs on a sucrose gradient (5-60%) and immunoblotting for relevant proteins. Fraction size: 200 µL. Representative of 3 independent experiments.",
    "molecules": "sucrose"
  },
  {
    "caption": "C Immunoblotting for phosphorylated and total Akt or InsP3R protein in crude mitochondrial fractions from serum-starved WT and Nox4KO MEFs. FACL4 was used as a loading control for the MAM. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Representative immunoblots are shown at the top and mean data at the bottom. Cat = PEG-catalase. Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant (Mut). n=3/group.",
    "molecules": "PEG"
  },
  {
    "caption": "D ROS levels indexed in MEFs using H2O2 specific HyPer probes targeted to the MAM (FACL4 HyPer) or the cytosol (Cyto HyPer). Imaging was performed after serum starvation for 12h. Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left. The signal obtained using corresponding ROS-insensitive SypHer probes was used to correct for any pH-induced changes in fluorescence; the change in fluorescence ratio between HyPer and corresponding SypHer probe (ΔR) for each condition is reported. n=5 independent cell preparations/group, with at least 20 cells imaged/preparation. Scale bars: 10 µm.",
    "molecules": "H2O2, ROS"
  },
  {
    "caption": "B Basal mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs in the presence or absence of Akti (1 µmol/L) normalized versus WT MEF control. n=6/group with >20 cells imaged per experiment.",
    "molecules": "Akti, calcium"
  },
  {
    "caption": "C Effect of Akti (1 µmol/L) or XeC (1 µmol/L) on histamine-induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n=3/group with >40 cells imaged per experiment.",
    "molecules": "Akti, calcium, histamine, XeC"
  },
  {
    "caption": "D Quantification of cell death in serum-starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n=6/group.",
    "molecules": "Akti, XeC"
  },
  {
    "caption": "E Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia-reperfusion (I/R). XeC was added at a final concentration of 2 µmol/L prior to ischemia. n=6-7/group.",
    "molecules": "XeC"
  },
  {
    "caption": "G Cardiac left ventricular contractile function in isolated Langendorff-perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/L) or vehicle control for 20 min prior to ischemia. RPP, heart rate x left ventricular pressure product; DEVP, left ventricular developed pressure. n=6-7/group.",
    "molecules": "XeC"
  },
  {
    "caption": "Confocal images of mPS-PI stained 6-day-old seedlings of Col-0 (A), bravo-2 (B), wox5-1 (C) and bravo-2 wox5-1 (D) mutants. Left black arrows indicate QC cells and right white arrows indicate CSC. Scale bar: 50 µm.",
    "molecules": "mPS, PI"
  },
  {
    "caption": "Confocal images of PI-stained 6-day-old roots. GFP-tagged expression is shown in yellow. A-C) pBRAVO:GFP in WT (A), bravo-2 (B) and wox5-1 (C) knockout backgrounds. D-G) pWOX5:GFP in the WT (D), bravo-2 (E), wox5-1 (F) and bravo-2 wox5-1 (G) knockout backgrounds. Scale bar: 50 µm. Quantification of the GFP fluorescent signal of the roots in A-C (H) and D-G (I). Boxplot indicating the average pixel intensity of the GFP in the stem cell niche (n>25, 3 biological replicates, *p-value < 0.05 Student´s t- test for each genotype versus the WT in the same condition). Quantification was done by integrating the GFP signal in each root across defined areas that included the whole SCN (Appendix Figure S7). In the boxplot, box width represents the interquartile range (IQR=Q3-Q1), with the horizontal line denoting the median, while whiskers extend from Q1-1.5IQR to Q3+1.5IQR. White dots are the outliers and black dots are the experimental observations.",
    "molecules": "PI"
  },
  {
    "caption": "Yeast two-hybrid assay showing BRAVO interacting with WOX5, BES1-D and TPL; and BES1-D interacting with TPL. In the left column yeast cells were grown on control media, and in the right column yeast cells were grown on control media lacking Leu, Trp and His, indicating an interaction between the proteins.",
    "molecules": "His, Leu, Trp"
  },
  {
    "caption": "(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.",
    "molecules": "glyburide"
  },
  {
    "caption": "(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.",
    "molecules": "glyburide"
  },
  {
    "caption": "(b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(g,h) Lungneutrophil infiltration (g) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(g,h) IL-18 and IFN-γ amounts (h) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(d,e) Analysis of IL-18 production and immunoblotting to analyze caspase-1 (casp-1) p20 activation in IAV-infected BMDCs that had been pretreated with the ROS inhibitor NAC.",
    "molecules": "NAC, ROS"
  },
  {
    "caption": "(f) Immunoblot of casp-1 p20 in cell lysates of IAV-infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO.",
    "molecules": "mitoTEMPO"
  },
  {
    "caption": "(a,b) IL-18 amounts (a), and caspase-1 (casp-1) p20 and LC3-II immunoblots (b) in PR8-infected wild-type (WT) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580.",
    "molecules": "SB203580"
  },
  {
    "caption": "(d) Immunoblots for casp-1 p20 in untreated or NAC-treated WT and Ulk1−/− BMDCs after PR8 infection.",
    "molecules": "NAC"
  },
  {
    "caption": "A. cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 and the wildtype, from left to right on control plates without genotoxic agent 10 days after germination.B. The wildtype, single and double mutants of cycb1 were grown on control plates without genotoxic agent. Root lengths were measured 10 days after germination.C. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 1 mM hydroxy urea (HU) 10 days after germination. The rightmost plant is the wee1 mutant that shows high sensitivity to HU.D. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 1 mM HU. Root lengths were measured 10 days after germination.E. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 0,6 μg/mL bleomycin (BLM) 10 days after germination. The rightmost plant is the ku70 mutant that shows high sensitivity to BLM.F. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.",
    "molecules": "bleomycin, BLM, HU, hydroxy urea"
  },
  {
    "caption": "G. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 15 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.H. cycb1 mutants were germinated on control plates and were transferred to new control plates three days after germination. Root lengths mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.I. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.J. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.",
    "molecules": "cisplatin"
  },
  {
    "caption": "K. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 30 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.L. cycb1 double mutants germinated on control plates and were transferred to new control plates three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.M. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.N. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.",
    "molecules": "cisplatin"
  },
  {
    "caption": "A. Representative examples of comets of 21 days old wild-type plants, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutant seedlings in full spectrum view of the TriTek Comet Score software. Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min (recovery) and plants incubated without cisplatin for one hour (control), respectively.B. Box plot of percentage of tail DNA of wild-type cells, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 under cisplatin treatment. Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications. The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software. The box represents the interquartile range, the line across the box indicates the median values and whiskers represent 5-95 percentile values. Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99.99% calculated with Student's t-test.",
    "molecules": "cisplatin, DNA"
  },
  {
    "caption": "C. Immunostaining of γ-H2AX foci in wild-type plants and mutant cells after two hours of treatment with 50µM cisplatin.D. Counted number of γ-H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the γ-H2AX foci of 100 cells were counted and grouped into six categories: cells with no, 1-2, 3-5, 6-10, 11-20 and more than 20 foci per cell.",
    "molecules": "cisplatin"
  },
  {
    "caption": "B. Wild-type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.C. cycb1;1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.",
    "molecules": "cisplatin"
  },
  {
    "caption": "D. Graphs show numbers of blue sectors per plant grown without drug treatment.E. Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days.F. Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days.",
    "molecules": "cisplatin"
  },
  {
    "caption": "A. The wildtype and CDKA;1-DE mutants were grown on control plates for 10 days. Root lengths were measured 10 days after germination.B. The wildtype and CDKA;1-DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days. The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.C. The wildtype and CDKA;1-DE mutants were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.D. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on control plates for 10 days. Root lengths were measured 10 days after germination.E. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.F. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.",
    "molecules": "bleomycin, hydroxy urea"
  },
  {
    "caption": "G. The wildtype, CDKA;1-DE (grey bar) and the double mutant cdkb1;1cdkb1;2(purple bar) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.H. Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.I. Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.",
    "molecules": "cisplatin"
  },
  {
    "caption": "A. WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin, respectively, three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph. Asterisk indicates significant differences within a 5% confidence interval (Student's t-test).B. Image shows a wild-type plant (left) and rad51 mutant (right) grown on control plates. Images were taken 6 days after germination.C. Image shows a wild-type plant (left) and rad51 mutant (right) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination. Images were taken 6 days after germination, i.e. 3 days after transfer to cisplatin.",
    "molecules": "cisplatin"
  },
  {
    "caption": "D. Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin.",
    "molecules": "cisplatin"
  },
  {
    "caption": "E. Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the RAD51 foci of 100 cells were counted and grouped into six categories: cells with 0, 1, 2, 3, 4 or >5 foci per cell.",
    "molecules": "cisplatin"
  },
  {
    "caption": "F. In vitro kinase assay of purified CDK complexes phosphorylating RAD51. RAD51 and histone H1 kinase assays were performed with [γ-32P]ATP as a phosphate donor. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates (lower panels). Phosphorylated proteins were detected by autoradiograph (upper panels). The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK-cyclin complexes, protein blots indicate the relative amounts of the CDKs in the reaction (bottom panel).Abbreviation: p-RAD51 and p-Histone H1 for [32P]-phosphorylated MBP-RAD51-His6 and recombinant human histone H1, respectively, resulting from kinase assays with radio-labeled ATP. Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure. 1: without kinase, 2: CDKA;1-CYCA2;3, 3: CDKB1;1-CYCA2;3, 4: CDKB1;1-CYCD2;1, 5: CDKA;1-CYCB1;1, 6: CDKB1;1-CYCB1;1.",
    "molecules": "ATP"
  },
  {
    "caption": "A. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box grown on control plates and imaged five days after germination.D. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.",
    "molecules": "cisplatin"
  },
  {
    "caption": "B. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box grown on control plates and imaged five days after germination.E. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.",
    "molecules": "cisplatin"
  },
  {
    "caption": "C. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS grown on control plates, were stained and imaged five days after germination.F. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application.",
    "molecules": "cisplatin"
  },
  {
    "caption": "G. Chromatin immunoprecipitation (ChIP) of wild-type plants and PROSOG1:SOG1-Myc lines and anti-MYC antibody. The promoter region of CYCB1;1 is enriched in SOG1-MYC after cisplatin treatment.H. Structure of genes tested by ChIP with an antibody anti-MYC in PROSOG1:SOG1-Myc and wild-type plants. Red arrowheads indicate the primer binding sites for PCR. A total of four regions were tested as indicated by arrowheads. Asterisks indicates significant differences within a 5% confidence interval (Student's t-test).",
    "molecules": "cisplatin"
  },
  {
    "caption": "A. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on control plates and root lengths were measured 10 days after germination.B. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on plates containing 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.C. Graph represents the ratio of the mean growth rate on 0,6 µg/mL BLM compared to control experiments on plates lacking BLM for the wildtype, ku70, cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70.",
    "molecules": "BLM"
  },
  {
    "caption": "D. Image shows the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) grown on control plates 10 days after germination.E. Images show the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) on plates containing 0,6 μg/mL BLM.",
    "molecules": "BLM"
  },
  {
    "caption": "E Lysates from 293T cells transiently expressing GFP-CbpCT4, GFP-LytR, or GFP in the presence or absence of chloroquine were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.",
    "molecules": "chloroquine"
  },
  {
    "caption": "G p62-KO MEF cells infected with S. pneumoniae R6 ΔcbpF/pCbpFR6-FLAG for 2 h were fixed and stained with DAPI and an anti-FLAG antibody, and representative epifluorescence images are shown. Scale bars, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A Quantification of the band intensities following streptavidin-based pulldown assays using the in vitro-translated biotinylated CbpCT4 and FLAG-tagged autophagy-related proteins shown in Fig EV2A.",
    "molecules": "streptavidin"
  },
  {
    "caption": "A Lysates from MEFs stably expressing HA-Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "B MEFs/HA-Atg14 cells were infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "BafA1, Bafilomycin A1"
  },
  {
    "caption": "(C A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti-Atg14 Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of perinuclear-localizing Atg14 containing cells were quantified.",
    "molecules": "DAPI"
  },
  {
    "caption": "A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and -GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of intact Golgi apparatus-containing cells were quantified.",
    "molecules": "DAPI"
  },
  {
    "caption": "K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti-Atg14 antibody, and percentages of perinuclear-localizing Atg14 containing cells were quantified.",
    "molecules": "DAPI"
  },
  {
    "caption": "L Lysates from MEFs stably expressing GFP-CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS-PAGE and immunoblotted using antibodies against LC3 or p62.",
    "molecules": "chloroquine, rapamycin"
  },
  {
    "caption": "H A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti-Atg14 antibody, and the percentages of perinuclear-localizing Atg14 containing cells were quantified.",
    "molecules": "DAPI"
  },
  {
    "caption": "SH-SY5Y cells with 20 μM NSC87877, 10 μM SSG and 1 μM PHPS-1 were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.",
    "molecules": "NSC87877, PHPS-1, SSG"
  },
  {
    "caption": "SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.",
    "molecules": "saracatinib, sara, SKI-1"
  },
  {
    "caption": ", D. primary cortical neurons were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.",
    "molecules": "saracatinib, sara, SKI-1"
  },
  {
    "caption": "B. A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1, respectively, with 50 μM RA for 5 days.",
    "molecules": "RA, saracatinib, sara, SKI-1"
  },
  {
    "caption": "C. NT or c-src KD #1 or #2 /A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured with 50 μM RA for 5 days. The samples were observed under confocal microscopy, and the number of cells containing double fluorescence labeled puncta were analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to PBS or NT cells, one-way ANOVA.",
    "molecules": "RA"
  },
  {
    "caption": "A. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h.",
    "molecules": "saracatinib, sara, SKI-1"
  },
  {
    "caption": "E. Differentiated A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 5 μM saracatinib (sara) or 10 μM SKI-1 in the presence of DMSO or 50 μM bafilomycin A1 (BafA) for 12 h.",
    "molecules": "BafA, bafilomycin A1, DMSO, saracatinib, sara, SKI-1"
  },
  {
    "caption": "Differentiated NT and c-src KD/A53T α-syn-EGFP OE SH-SY5Y cells were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.",
    "molecules": "BafA"
  },
  {
    "caption": "G. NT and c-src KD primary neurons were incubated with 50 μM BafA for 12 h. Western blot was performed. Data are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05, one-way ANOVA.",
    "molecules": "BafA"
  },
  {
    "caption": "H. SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of DMSO or 50 μM BafA for 12 h. The cells were immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). ** P < 0.01, one-way ANOVA.",
    "molecules": "BafA, DMSO"
  },
  {
    "caption": "SH-SY5Y cells in the presence of 5 μM saracatinib, 10 μM SKI-1 were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.",
    "molecules": "LacCer, BODIPY FL, saracatinib, SKI-1, rhodamine"
  },
  {
    "caption": "B. NT, c-src KD #1 or c-src KD #2 SH-SY5Y cells were incubated with 1 μM α-syn fibrils for 30 min and these cells were further incubated with 50 nM BODIPY FL C5-LacCer and 2.5 μg/ml rhodamine-conjugated transferrin for 20 min. The cells were fixed and observed under confocal microscopy. The intensity was analyzed. Values were derived from five independent experiments (n=5). *** P< 0.001 compared to control, one-way ANOVA.",
    "molecules": "LacCer, BODIPY FL, rhodamine"
  },
  {
    "caption": "Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. B. Fixed brain tissues containing the frontal cortex and the striatum were lysed. Western blot was performed with indicated antibodies. Values were derived from three individual mice (n=3).",
    "molecules": "saracatinib"
  },
  {
    "caption": "Two to three-month-old hemizygous M83 mice were injected into the striatum with sterile PBS or 10 μg of recombinant α-syn fibrils. The mice were treated with saracatinib at 10 mg/kg/day by oral gavage for 4 weeks. C. Immunohistochemistry of pSer129 α-syn was performed. Arrow indicates pSer129 α-syn positive inclusion. Scale bar indicates 50 μm. D. Scoring of pSer129 immunoreactivity in the cortex and the striatum of both regions. Values were derived from eight individual mice (n=8). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS group, unpaired t-test. Data are expressed as mean ± SEM. ",
    "molecules": "Ser, saracatinib"
  },
  {
    "caption": "G. Endogenous MITOL enhances the K48-polyubiquitin chains of Parkin. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for indicated time. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IP-IB assay with the indicated antibodies. K48-polyubiquitin chains were immunoblotted with K48-specific ubiquitin antibody.",
    "molecules": "CCCP, polyubiquitin, MG132, ubiquitin"
  },
  {
    "caption": "E. MITOL regulates mitophagy. MITOL WT HeLa cells, MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0.5µg (+) and 5µg (++) stably expressing HA‐Parkin and mt‐Keima were treated with CCCP (10µM) for 8h. Then, mKeima was measured at 488 (pH 7) and 561 (pH 4) nm lasers using Flow Cytometer. Percentages of mitophagy were calculated from 30,000 cells in each independent experiment. Data represent the mean ± SD of three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.",
    "molecules": "CCCP"
  },
  {
    "caption": "C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.",
    "molecules": "CCCP"
  },
  {
    "caption": "C. Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IB assay with the indicated antibodies. Phospho-Ser65-Parkin is detected by using specific antibody. Right panels are the quantification of IB about Phospho-Ser65-Parkin protein levels of the ER fraction. The data represent the mean ± SD for three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.",
    "molecules": "CCCP, DMSO, Ser"
  },
  {
    "caption": "D. Parkin is detected at the ER in the absence of MITOL. HeLa cells were transfected with vectors for KikGR-Parkin. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel.",
    "molecules": "CCCP"
  },
  {
    "caption": "D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag).",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "E-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.",
    "molecules": "PI, propidium iodide, CCCP, DMSO"
  },
  {
    "caption": "A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. ",
    "molecules": "PI, propidium iodide, BAPTA-AM, calcium, CCCP, DMSO, Z-VAD-FMK"
  },
  {
    "caption": "Combination of IFNγ gene therapy with immuno-oncology drugs. CTRL (Tie2.NGFR) + antibody isotype, n = 6 mice; CTRL +  αCTLA4 antibody, n= 5 mice; CTRL +  αLAG3  antibody, n = 5 mice; CTRL + 1-Methyltryptophan  (1-MT), n = 5  mice; IFNγ (Tie2.IFNγ) + antibody isotype, n = 7 mice; IFNγ  + αCTLA4 antibody, n = 7 mice; IFNγ + αLAG3 antibody, n = 7 mice; IFNγ +1-MT, n = 5 mice. (F) Percentage of MHC II+ macrophages identified by  F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001 as compared to CTRL isotype, ordinary One-Way ANOVA). (G) Percentage of CD8+ T lymphocytes within OFP  negative  BM cells (mean ± SD, each dot represents an individual mouse). S ",
    "molecules": "1-Methyltryptophan, 1-MT"
  },
  {
    "caption": "D Volcano plot showing differentially expressed genes (adj. P.Val < 0.05) between ethanol- and R5020-treated mouse mammary organoids, n=3. Genes with log2(FC) > 2 are highlighted in red, genes with log2FC < -2 in blue. Names of genes with -log10 (adj.P.Val) > 10 and abs(log2(FC)) > 2 are indicated.",
    "molecules": "ethanol, R5020"
  },
  {
    "caption": "H, I Bar graphs showing relative Rankl and Wnt4 transcript levels normalized to 36B4 expression in C57Bl6-derived mammary organoids treated for 6 hours with R5020 (H) or LNG (I) and either 10 μM or 100 μM enzalutamide, n=3 independent experiments.",
    "molecules": "enzalutamide, LNG, R5020"
  },
  {
    "caption": "G Scatter plot showing the genes which are commonly altered upon R5020 stimulation (adj. P-value < 0.05) in both mouse and human organoids relative to their CTRL samples. Color scale as a function of the averaged human and mouse logFC values. The red dashed lines represent an adj. P-value threshold of 0.001.",
    "molecules": "R5020"
  },
  {
    "caption": "Progestin plasma levels in treated mice (n=10, 14, 18, 7, 6, 10) and in women (n=20, 11, 3, 4, 6, 8) on hormonal contraceptives measured by LC-MS, shown as means ± SD, as well as values reported in the literature (Cmax). The active form of DSG, 3-ketodesogestrel was measured.",
    "molecules": "DSG, 3-ketodesogestrel, Progestin"
  },
  {
    "caption": "Progestin plasma levels in treated mice (n=10, 14, 18, 7, 6, 10) and in women (n=20, 11, 3, 4, 6, 8) on hormonal contraceptives measured by LC-MS, shown as means ± SD, as well as values reported in the literature (Cmax). The active form of DSG, 3-ketodesogestrel was measured.",
    "molecules": "DSG, 3-ketodesogestrel, Progestin"
  },
  {
    "caption": "E Representative micrographs showing co-IF with anti-KI67 (red), and anti-hECAD (green) of xenografted milk ducts after 21 days treatment with vehicle or LNG. Scale bar, 50 μm. F Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells, dots represent individual sectors counted in 3 different glands, control (n=30) or LNG (n=28), median (red), statistical significance was assessed by non-parametric Mann-Whitney test. G Bar plot showing relative transcript levels of MKI67 in xenografted glands from control (n=6) and LNG-treated (n=3) mice, Student's t-test. ",
    "molecules": "LNG"
  },
  {
    "caption": "Representative micrographs showing co-IF with anti-KI67 (red) and anti-hECAD (green) of histological sections from glands xenografted with HBECs from mammoplasty M310 exposed to vehicle or progestins. Scale bar, 50 μm. J Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells in M310 derived HBECs, dots represent individual sectors counted in different glands, median (red), CTRL (n=180), P4 (n=134), DSG (n=52), GSN (n=161), LNG (n=32), CMA (n=56), CPA (n=42), DSP (n=100). Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using the CTRL group as reference. ",
    "molecules": "CMA, CPA, DSG, DSP, GSN, LNG, P4"
  },
  {
    "caption": "K Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ HBECs derived from 11 different patients upon 21 days of CTRL (n=360), P4 (n=180), DSG (n=147), GSN (n=298), LNG (n=284), CMA (n=115), CPA (n=156), DSP (n=123) treatment. Red lines show medians. L Violin plot showing percentage of AR+ intraductally-engrafted HBECs derived from 11 different patients upon 21 days of CTRL (n=258), P4 (n=97), DSG (n=173), GSN (n=159), LNG (n=168), CMA (n=68), CPA (n=76), DSP (n=135) pellets. Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions, with batches and patients as random variables and CTRL as reference, median in red. J-L dashed line shows 0%. ",
    "molecules": "CMA, CPA, DSG, DSP, GSN, LNG, P4"
  },
  {
    "caption": "A-F Micrographs of H&E stained tissue sections from mouse mammary glands engrafted with HBECS and exposed to LNG or vehicle for 6 months. In control mice (A, B) most ducts have a narrow lumen and a thin wall due to limited growth of human cells (A); a few mildly dilated ducts exhibit a cellular proliferation made out of multiple small acini burgeoning at the external side of their wall (B, arrows). In LNG-treated mice (C-F) the peripheral acinar proliferation is diffuse, marked in some foci (C), and associated with a variable degree of duct dilatation from mild (C, arrow) up to true cysts formation (D). Proliferating human epithelial cells showing apocrine changes (e.g. abundant eosinophilic cytoplasm and central round nuclei) partly fill the ductal lumen with a complex architecture of micropapillae (E) and bridges (F, arrows). Scalebars, 50 μm.",
    "molecules": "LNG"
  },
  {
    "caption": "A-C Graphs showing in vivo growth of HBECs from different donors as measured by radiance after treatment with LNG, ENZ, or LNG and ENZ, means of radiance in individual glands ± SEM, n= 8-10 per treatment, Wilcoxon matched-pairs test. Right: Dot plot showing plasma levels of progesterone (P), testosterone (T) and LNG in individual xenografted animals.",
    "molecules": "ENZ, LNG, progesterone, testosterone"
  },
  {
    "caption": "D Graph showing in vivo growth of HBECs from a 20-year-old patient transduced with either sh scramble or shAR and treated with LNG. Points show means of radiance in individual glands ± SEM; n= 8-10 per treatment. Wilcoxon matched-pairs test.",
    "molecules": "LNG"
  },
  {
    "caption": "The diffusion times of Scc1-GFP at S phase (B) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.",
    "molecules": "auxin"
  },
  {
    "caption": "The diffusion times of Scc1-GFP at G2 phase (C) and M phase (D) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.",
    "molecules": "auxin"
  },
  {
    "caption": "B. The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP-GFP dimer as the brightness standard. Cells of strain yIO664 (SCC1-GFP) were analyzed throughout the cell cycle by PCH. To examine the effects of Wpl1, Pds5, and Eco1 on cohesin dimerization, the PCH analysis was repeated in strain yEB005 cells (SCC1-GFP WPL1∆), strain yEB002 cells (SCC1-GFP PDS5-AID), and yAM946 (SCC1-GFP ECO1-AID) in which Pds5-AID/Eco1-AID was depleted with auxin.",
    "molecules": "auxin"
  },
  {
    "caption": " B, C CDKL51-352 autophosphorylation is shown. WT CDKL5 uses ATPγS, KD is inactive, F89A uses benzyl-ATPγS with reduced efficiency, F89A/ C152A mutant has rescued activity. Quantification of Thio-P signal is normalized for CDKL5 levels (HA) and KD background is subtracted. Fisher's LSD: n = 4 replicates. bn = benzyl ",
    "molecules": "benzyl-ATPγS, ATPγS"
  },
  {
    "caption": " A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations. 100 µm AP5 was used to block NMDAR activity during KCl treatment. Quantification of phosphorylated EB2 (B) and total CDKL5 (C) are normalized to 0 minutes time point. EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes. CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates",
    "molecules": "AP5, KCl"
  },
  {
    "caption": " D, E Protein lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations. Quantification of phosphorylated EB2 and total CDKL5 is normalized to 0 minutes time point. EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates ",
    "molecules": "NMDA"
  },
  {
    "caption": " E, F Taxol-stabilised microtubule co-sedimentation assay shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5. Quantification of soluble MAP1S in the supernatant (s/n) fraction is normalized to the MT-bound pellet fraction. 3:1 MT:MAP1S LC ratio used here. Tukey's multiple comparison: n = 3 technical repeats ",
    "molecules": "Taxol"
  },
  {
    "caption": "(B) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10% to 80% sucrose gradient. Western blot against anti-V5 is shown.",
    "molecules": "sucrose"
  },
  {
    "caption": "(A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.",
    "molecules": "1HN, 1H, 15N"
  },
  {
    "caption": "(A, B) i. 15N CPMG RD data for residues in the β4 strand (T113) and the β8 strand (S154) in the absence of added peptide. CPMG data are shown for the same residues in the presence of ii. ca. 2% cHSP27-WT peptide complex or iii. ca. 2% of the cHSP27-P182L peptide complex. Uncertainties in R2,eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values. (C) i. The association (kon) and dissociation (koff) rates for WT (red) and P182L (blue) IxI/V peptide binding to the ACD, calculated directly from the CPMG RD data. Residues that are impacted by the presence of a small amount of peptide are indicated by red (ii., WT) and blue (iii., P182L) spheres. In both cases, residues in similar ACD regions show enhanced Rex, indicative of chemical exchange at similar sites.",
    "molecules": "15N"
  },
  {
    "caption": "(C-D) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) using anti-V5 beads. Co-immunoprecipitation was quantified and presented as a percentage relative to the P182L variant (n=3). The mean ± one standard deviation (n=3) is shown. Right: the location of Pro182 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the P182L mutation (blue). The peptide is shown in white sticks. P182L specifically weakens binding of the HSP27 IxI/V motif.",
    "molecules": "Pro"
  },
  {
    "caption": "(F) Co-immunoprecipitation from HeLa cells transiently transfected with V5-epitope-tagged HSP27 (P182L or S155Q/P182L mutant) using anti-V5 beads (n=2). Right: the location of Ser155 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the S155Q mutation (purple) showing a clash with the bound peptide. The peptide is shown in white sticks.S155Q disrupts binding of all IxI/V motifs.",
    "molecules": "Ser"
  },
  {
    "caption": "Montages of representative DAPI-stained nuclei are shown for gene knockdowns that led to reduced nuclear size. Cell-to-cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability. Ten nuclei per column, organized by maximum nuclear size z-score. Scale bar, 25 µm. Median nuclear size z-scores are plotted for gene knockdowns that led to reduced nuclear size. Data are based on three different siRNA oligo sequences for each gene and two biological replicates. Error bars represent the SEM for biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "MCF-10AT1k.cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1. Representative images are shown.",
    "molecules": "DMSO, importazole"
  },
  {
    "caption": "MCF-10AT1k.cl2 cells were transfected with control siRNA or with siRNA against XPO1. Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high-throughput imaging as previously described [123] (see Methods). The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. For the bottom row of images, cells transfected with control siRNA were treated with 20 ng/ml leptomycin B for 24 hours. Cells were fixed and stained with 2 µg/ml FITC. Representative images are shown. Scale bar, 20 µm. For each experiment, nuclear cross-sectional areas and nuclear FITC staining intensities were quantified for 105-620 nuclei per condition (267 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.",
    "molecules": "FITC, leptomycin B"
  },
  {
    "caption": "A. Chemical structure of Nec-1. The schematic diagram was drawn by S. Lee using ChemDraw Professional 15.0. software.",
    "molecules": "Nec-1"
  },
  {
    "caption": "B, C. MTT cytotoxicity assay. (B) HT22 cell line was treated with various concentrations of Nec-1 (0 - 200 μM). Aggregated Aβ(1-42) was used as the positive control for the induction of neural cell death. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.",
    "molecules": "Nec-1"
  },
  {
    "caption": "B, C. MTT cytotoxicity assay. (C) HT22, BV2cell lines and primary astrocytes were pre-treated with Nec-1 (50 μM) 15 minutes prior to application of 10 μM of pre-aggregated Aβ(1-42) for indicated times. Cell proliferation was then measured by MTT assay. All data represented the mean ± standard deviation of three independent experiments performed in triplicate.",
    "molecules": "Nec-1"
  },
  {
    "caption": "D, E. Neural cell death levels measured by staining with the Live/Dead Viability/Cytotoxicity Assay Kit (Molecular Probes). Pre-treatment of cells with 50 μM Nec-1 preceded the application of 10 μM pre-aggregated Aβ(1-42) by 15 minutes. The assay was performed 24 hours after Aβ(1-42) application. (D) Representation of the stained cells. Scale bar = 500 μm (E) Quantification of dead cells stained by EthD-1.",
    "molecules": "Nec-1"
  },
  {
    "caption": "F. Internalization of Aβ(1-42) into HT22 and BV2 cell lines in the presence or the absence of Nec-1 pre-treatment for indicated periods.",
    "molecules": "Nec-1"
  },
  {
    "caption": "G. Cellular expression levels of indicated proteins in HT22, BV2 cell lines, and primary astrocyte stimulated with Aβ(1-42) aggregates in the presence or the absence of Nec-1 pre-treatment for 24 hours.",
    "molecules": "Nec-1"
  },
  {
    "caption": "A-E. Behavioural tests of APP/PS1mice. (B-D) Y-maze and passive avoidance tests on 7-month-old APP/PS1mice after Nec-1 administration for 12 weeks. Average alternation (%) for each test group (B) and total entry number into each arm (C) on Y-maze. (D) Average latency time in seconds for each test group on passive avoidance test.",
    "molecules": "Nec-1"
  },
  {
    "caption": "A-E. Behavioural tests of APP/PS1mice. (E) Survival of Wt and APP/PS1mice after injection of Nec-1 (6.25 mg/kg).",
    "molecules": "Nec-1"
  },
  {
    "caption": "F-I. Behavrioural tests of Aβ(1-42) infusion mice. (H, I) Y-maze tests on Aβ(1-42) infusion mice model injected with Nec-1. Average alternation (%) for each test group (H) and total entry number into each arm (I) on Y-maze.",
    "molecules": "Nec-1"
  },
  {
    "caption": "B. A close-up view of the interactions between Nec-1 and surrounding residues in RIPK1. Nec-1 is shown in green. The hydrophobic residues around Nec-1 in RIPK1 kinase domain are shown in sticks. Hydrogen bonds in this and all other figures are represented by yellow dashed lines. Docking score of Nec-1 in RIPK1 was -9.550.",
    "molecules": "Nec-1"
  },
  {
    "caption": "C. Surface treated view of 12-mer Aβ fibrils (2LMO, VDW radius = 0.55 Å) having hydrophobic pockets plausible for Nec-1 binding.",
    "molecules": "Nec-1"
  },
  {
    "caption": "D. Docked structure of 12-mer Aβ fibrils (2LMO) with 4 molecules of Nec-1. Docking scores for four top-ranked Glide docking poses of Nec-1 in Aβ plaques; -8.338, -4.755, -8.493, and -6.754 in clockwise order starting from the top-left corner.",
    "molecules": "Nec-1"
  },
  {
    "caption": "E. Surface plasmon resonance sensorgrams of Nec-1 targeting Aβ(1-42) aggregates (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.",
    "molecules": "Nec-1"
  },
  {
    "caption": "4-month-old APP/PS1 (n = 13, male) and age-matched wild-type (Wt, n = 14, male) mice were intravenously injected with Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for 12 weeks. After the completion of behavioural tests, the brains of each group of mice were subjected to analysis.A. ThS-stained insoluble Aβplaques in whole brain and the hippocampal region of each group. Scale bars = 1 mM (upper), 200 μM (lower).B-G. Statistics of ThS-positive Aβplaques in the brains of APP/PS1mice. Total numbers and areas of ThS-positive Aβplaques in the whole brains (B and C), cortex (D and E) and hippocampus (F and G).",
    "molecules": "Nec-1, DMSO"
  },
  {
    "caption": "A. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). Diffuse plaques in the brain sections were stained by anti-Aβ antibody (6E10 clone, green colour) and anti-GFAP antibody (red colour). Hoechst 33342 (blue colour) was applied for nuclear counterstaining. Scale bars = 200 μM.",
    "molecules": "Nec-1, DMSO"
  },
  {
    "caption": "A. Western blot analysis of phosphorylation levels of tau and expression levels of indicated proteins in the cortical and hippocampal regions of the brain in APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS). The membranes analysed in (A) are identical to those in Fig 5F.",
    "molecules": "Nec-1, DMSO"
  },
  {
    "caption": "B. Immunohistochemical analysis of cortical and hippocampal regions of the brains in wild-type (Wt) and APP/PS1mice after administration of Nec-1 (6.25 mg/kg) or vehicle (2.5% DMSO in PBS) for phosphorylated tau expression levels. Anti-tau (phospho Ser199) antibody (red colour) was applied for staining tau phosphorylation on Serine-199 and Hoechst 33342 (blue colour) for nuclear counterstaining in the brain sections. Scale bars = 200 μM.",
    "molecules": "Nec-1, DMSO"
  },
  {
    "caption": "C. Western blot analyses of phosphorylated RIPK3 expressions in cortex and hippocampus of APP/PS1mice injected with Nec-1 (upper). The membranes analysed in (C) are identical to those in Fig 1G and 5F.",
    "molecules": "Nec-1"
  },
  {
    "caption": "C. Western blot analyses of phosphorylated RIPK3 expressions in primary cultured astrocytes treated with Nec-1 (lower). The membranes analysed in (C) are identical to those in Fig 1G and 5F.",
    "molecules": "Nec-1"
  },
  {
    "caption": "F. Surface plasmon resonance sensorgrams of Nec-1 targeting tau (left), and the corresponding dissociation fitting curve from the saturated region (right) under various concentrations of Nec-1.",
    "molecules": "Nec-1"
  },
  {
    "caption": "A) Differential interference contrast (DIC) image of cortical neurons in culture. The arrowhead points to a neuron expressing Ub-R-GFP. B) GFP fluorescence before and after exposure to lactacystin (10µM).",
    "molecules": "lactacystin"
  },
  {
    "caption": "C) A neuron coexpressing Ub-R-GFP and CFP before (left) and 10 hours after exposure to lactacystin (right). Top row - GFP fluorescence; bottom row - CFP fluorescence. Bars: 50µm (A,B), 20 µm (C). D) Changes in GFP and CFP fluorescence following exposure to lactacystin at t=1 hour (vertical dashed line). Changes normalized to initial fluorescence in same cells. 9 neurons from 5 experiments; averages and SEM. E) Same as in D, except that here neurons were exposed only to carrier solution. 11 neurons from 5 experiments; averages and SEM (barely observable). F) Comparison of GFP fluorescence accumulation rates. Linear fits shown as black lines; fit parameters shown next to fits. Same data as in D, and E.",
    "molecules": "lactacystin"
  },
  {
    "caption": "A) Hippocampal neurons in primary culture immunolabeled against three synaptic proteins (the postsynaptic scaffold molecule Dlg4/PSD-95, the presynaptic active zone molecule RIM and the presynaptic vesicle protein SV2A) following 10 hour incubations in carrier solution (control, upper panels) or Lactacystin (10 μM; bottom panels). Scale bar, 10μm",
    "molecules": "Lactacystin"
  },
  {
    "caption": "B) Average synaptic immunofluorescence following 10 hour incubations in Lactacystin or carrier solution. Numbers within bars represent the number of fields of view analyzed for each condition (~170 synapses/field of view, on average). Error bars, SEM; P values - two tailed unpaired t-test. Note that a statistically significant accumulation was observed only for RIM, whereas immunolabeling of all other proteins was either unchanged or reduced.",
    "molecules": "Lactacystin"
  },
  {
    "caption": "C) Hippocampal neurons in primary culture were grown in the presence of Lactacystin (10 μM), fixed at two hour intervals and immunolabeled against Shank3/ProSAP2. Two representative fields of view at t=0 and t=24h. Scale bar, 10μm. D) Average synaptic Shank3/ProSAP2 immunofluorescence at increasing times in the presence of Lactacystin. Fluorescence was normalized to mean fluorescence at t=0. Numbers under data points reflect the numbers of fields of view analyzed at each time point. Error bars, SEM.",
    "molecules": "Lactacystin"
  },
  {
    "caption": "B) Comparisons of H/L ratios obtained for 1409 proteins in lactacystin and untreated cells. H/L ratios are averages of 2 experiments.",
    "molecules": "lactacystin"
  },
  {
    "caption": "A) Average log2(H/M) ratios for 1,750 proteins at four time points in three separate experiments. Each time point shows the average and standard deviation of three mean log2(H/M) values obtained in three separate experiments. Each mean, in turn, represents the mean log2(H/M) value measured in the population of 1,750 proteins in that experiment and time point. B) Distribution of log2(H/M) values for 1,750 proteins at t=0. The log2(H/M) value of each protein represents the average value for that protein in the three experiments. In principle, log2(H/M) for all proteins at t=0 should be 0, but in practice they are distributed around 0 as shown here. The H/M ratios for 3 standard deviation limits are shown beneath the X axis. C, D) The same as in A and B but for two experiments in which Epoxomicin was used (1,823 proteins). E, F) The same as in A and B but for the combined data from all experiments (3 experiments in which lactacystin was used and 2 experiments in which epoxomicin was used; 1,469 proteins).",
    "molecules": "Epoxomicin, epoxomicin, lactacystin"
  },
  {
    "caption": "A) log2(H/M) values for 5 synaptic proteins for which degradation rates were significantly slowed down in the presence of lactacystin. Each point represents average log2(H/M) from 3 experiments. B) log2(H/M) values for the same 5 synaptic proteins in experiments in which epoxomicin was used. Each point represents average log2(H/M) from 2 experiments. C) Average (± SEM) log2(H/M) ratios for the same 5 synaptic proteins, based on all five experiments (3 lactacystin, 2 epoxomicin). P values - two-tailed Welch's t-test, comparing the five log2(H/M) measurements at t=24 to those measured for the same proteins at t=0.",
    "molecules": "epoxomicin, lactacystin"
  },
  {
    "caption": "Measured log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 1,416 proteins for which half-life estimates were available.",
    "molecules": "epoxomicin, lactacystin"
  },
  {
    "caption": "Measured Log2(H/M) values at four time points as a function of their half-lives estimated here and elsewhere (Cohen et al, 2013). All log2(H/M) values shown here are averages of values obtained in 5 experiments (3 using lactacystin, 2 using epoxomicin). Proteins for which statistically significant differences at t=24 hours were observed (P<0.05, two-tailed Welch's t-test) are shown in red. Expected log2(H/M) values based on equation 4 are plotted as light blue lines. To minimize the masking of potential dependencies by the slight imperfections in the H/M normalization process (which introduces small offsets along the Y scale), average population log2(H/M) values of each time point (Fig. 6E) were subtracted from all measured Log2(H/M) values. Data for 174 synaptic proteins for which half-life estimates were available.",
    "molecules": "epoxomicin, lactacystin"
  },
  {
    "caption": "(D) Time-dependent survival of armyworm larvae injected with 0.8 µmol/larva N-acetyl-L-tyrosine (NAT): post NAT-injection 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. zero time.",
    "molecules": "N-acetyl-L-tyrosine, NAT"
  },
  {
    "caption": "(E) Dose-dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min (data are means ± SEM; n=12). Injection of more than 10 nmol NAT/larva caused a significant difference compared with zero (log rank test using R version 3.2.2).",
    "molecules": "NAT"
  },
  {
    "caption": "(F) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. PBS. Each chemical (0.5 µmol/larva) was dissolved in PBS; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine.",
    "molecules": "dimethyl sulfoxide, tyrosine"
  },
  {
    "caption": "(A) HPLC chromatogram and L-MS spectrogram of N-acetyltyrosine purified from pooled human serum (#12181201, Cosmo Bio Co., Japan). The peak fraction purified by HPLC was confirmed to be N-acetyltyrosine by LC-MS (inserted spectrogram).",
    "molecules": "N-acetyltyrosine"
  },
  {
    "caption": "(B) NAT concentrations in the serum of mice (Slc:ddY, 18 wks old, male) exposed to heat (40oC ) and restraint stress (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. zero time.",
    "molecules": "NAT"
  },
  {
    "caption": "(C) Effects of NAT on corticosterone concentrations in the serum of mice (Slc:ddY, 9-10 wks old, male) exposed to restraint stress for 60 min (data are means ± SEM; n=6). ** P<0.01 vs. without stress. Test mice drank 1.5 mg/ml NAT solution for 24 h before exposure to stress.",
    "molecules": "corticosterone, NAT"
  },
  {
    "caption": "(D) Effect of NAT on stress-induced peroxidized lipid in the serum of mice (Slc:ddY, 9 wks old, male) forced to swim for 60 min (data are means ± SEM; n=10). *P<0.05 vs. PBS.",
    "molecules": "lipid, NAT"
  },
  {
    "caption": "(A) Survival of Drosophila melanogaster (W1118) larvae fed with 2 µmol NAT/g-diet 2 d after heat stress at 38.5oC for 60 min (data are means ± SEM; n=10). **P<0.01 vs. zero time.",
    "molecules": "NAT"
  },
  {
    "caption": "(B) Effects of NAT feeding on expression of antioxidant enzymes, catalase, SOD1, and SOD2 (data are means ± SEM; n=10). *P<0.05 vs. zero time.",
    "molecules": "NAT"
  },
  {
    "caption": "(C) Representative images of nuclei (left) and FoxO (middle) in the fat body of Drosophila larvae before and after NAT feeding for 12 h. White signals in the merged image (right) show the overlap of both signals. Scale bar: 50 µm. (D) Relative whiteness intensities before and after NAT feeding (data are means ± SEM; n=15). **P<0.01 vs. zero time. ",
    "molecules": "NAT"
  },
  {
    "caption": "(E) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae (data are means ± SEM; n=8). *P<0.05, **P<0.01 vs. zero time.",
    "molecules": "NAT"
  },
  {
    "caption": "(F) Effects of N-acetyltransferase (Dat) knockdown in gut enteroendocrine cells (TK-Gut-Gal4>UAS-dsDat) on survival of Drosophila larvae (data are means ± SEM; n=15). **P<0.01 vs. without NAT.",
    "molecules": "NAT"
  },
  {
    "caption": "(A) Survival of Drosophila S2 cells treated with preheating, 100 µM NAT, and 100 µM NAT with 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, those were removed by changing the medium. *P<0.05, **P<0.01 vs. PBS.",
    "molecules": "2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, BCH, NAT"
  },
  {
    "caption": "(B) Survival of Drosophila S2 cells pretreated with 100 µM NAT, 100 µM NAT with 30 µg/ml antimycin A, and preheating with or without 30 µg/ml antimycin A 48 h after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, they were removed by changing the medium. *P<0.05 vs. PBS.",
    "molecules": "antimycin A, NAT"
  },
  {
    "caption": "(C) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells. Upper: representative images of time-dependent changes in MitoRed localization in mitochondria after adding NAT. Scale bar: 20 µm. Lower: relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader. Different letters above bars represent significant differences [P <0.05 (data are means ± SEM, n=12)].",
    "molecules": "MitoRed, NAT"
  },
  {
    "caption": "(D) ROS concentrations in S2 cells after adding 100 µM NAT (data are means ± SEM; n=12). *P<0.05 vs. zero time.",
    "molecules": "NAT, ROS"
  },
  {
    "caption": "(F) Effects of dsRNA targeting of FoxO on NAT-dependent expression of Keap1 (data are means ± SEM; n=12). *P<0.05, **P<0.01 vs. without NAT. Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies.",
    "molecules": "NAT"
  },
  {
    "caption": "(G) Effect of NAT on growth of HCT116 colon cancer cells in mice (BALB/cSlc-nu/nu, 6 wks old, female) (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. without NAT. Test mice began to freely drink 5 mg/ml NAT solution for 7 d before transplantation of the tumor cells on day 0: the average amount of NAT consumed by each mouse was about 8.8 mg/day (0.4 - 0.5 g/kg weight/day). HCT116 cells (5.0 x 106 cells) were transplanted into each test mouse using a 1 ml syringe with a 27G needle.",
    "molecules": "NAT"
  },
  {
    "caption": "I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J)  IP3R activation with  ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.",
    "molecules": "ATP, caffeine, Ca2+, histamine, Rhod2"
  },
  {
    "caption": "WT and Mfn2 KO cells were transfected with plasmids encoding  GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (S)  ATP levels of GFP+ cells was analyzed after 24 h treatment with  2DG (10 mM) (n= 3 independent experiments).",
    "molecules": "2DG, ATP"
  },
  {
    "caption": "WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: J-N) GFP and  ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG  (10 mM) for 6 h, and  ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.",
    "molecules": "2-DG, ATP, Oxygen"
  },
  {
    "caption": "WT and Mfn1/2 double KO were transfected with: C) GFP  and the indicated plasmids. After 48 h,  mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.",
    "molecules": "Ca2+"
  },
  {
    "caption": "WT and Mfn1/2 double KO were transfected with: D) LAR-GECO1 and the indicated plasmids. After 48 h  ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.",
    "molecules": "caffeine, Ca2+"
  },
  {
    "caption": "WT and Mfn1/2 double KO were transfected with: E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.",
    "molecules": "caffeine, Ca2+"
  },
  {
    "caption": "D) Primary neurons of tamoxifen-inducible Mfn2 KO  mice  were transfected at DIV3 with  ER-GFP,  mt-RFP  plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.",
    "molecules": "tamoxifen, Tamoxifen"
  },
  {
    "caption": "(B) The inherent elastic response of a lipid membrane (POPC) was studied by enforcing indentations via the isolated SNARE C-termini in the presence or absence of a short linker peptide, or the linker plus a fluorescent protein tag (FP). We define \"relative indentation\" as the change in C-terminus (Nyv1) - C-terminus (Vam3) distance that occurs when the SNARE complex performs mechanical work. It reflects the depth of the formed well upon squeezing. The slope of the obtained work profile estimates the required force (thin black line). Perforation (red arrow) requires a force of 80 pN for the wild-type C-termini. The plateau within the work profile indicates that the membrane gives up its elastic response (perforation transition). C-terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime. Furthermore, if the unstructured peptide-spacer would be alpha-helical - and thus much shorter - indentation forces would still be only slightly affected.",
    "molecules": "POPC"
  },
  {
    "caption": "(E) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8-EGFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Scale bar: 5 µm. (F) Fusion activity: vacuole fusion was assayed by measuring the percentage of co-localization of the two Pho8-EGFP and Pho8-mCherry signals. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "(B) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [65] but ensures that trans-SNARE complexes can only form in one orientation, between the Q-SNARE in DKY6281 and the R-SNARE in BJ3505. The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either incubated on ice, which prevents fusion, or in the presence of 0.5% Triton X-100, which allows fusion-independent access of the maturase Pep4 to pro-ALP and controls for the levels of these two reporter enzymes in the samples.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(D) Using strains from C, lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti-Vam3 antibody (3 µg) was added to some reactions in order to inhibit trans-SNARE pairing and fusion. Means and the s.d. are shown for three independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (D) Fusion activity: vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8-mCFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments. Scale bar: 5 µm. (E) Vacuole fusion was assayed by measuring the percentage of co-localisation of the two Pho8-mCFP and Pho8-mCherry signals.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Yeast strain expressing a plasmid with Vph1-GFP was grown in SC-URA medium for 16 h at 30°C. The mobility of Vph1-GFP in the absence (upper panels) or presence (lower panel) of CPZ (150 µM) was assessed by FRAP. (B) The histogram shows the GFP signals in the bleached area during the FRAP procedure. (C) The half-time is shown. Means and s.d. are shown for 20 cells of 3 independent experiments.",
    "molecules": "CPZ"
  },
  {
    "caption": "(D) Yeast cells were grown in SC medium for 16 h at 30°C. After staining with FM4-64, cells were treated with CPZ (150 µM). Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition. Scale bar: 5 µm.",
    "molecules": "CPZ"
  },
  {
    "caption": "(E) Fusion activity. Vacuoles were isolated from BJ cells carrying Nyv1-S9-EGFP and DKY cells deleted for Nyv1 and carrying Vam3-S9-mCitrine or from wild-type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine (CPZ, 150 µM) and anti-Vam3 (3 µg). Content mixing was assayed via alkaline phosphatase activity. Means and s.d. are shown for three independent experiments.",
    "molecules": "chlorpromazine, CPZ"
  },
  {
    "caption": " (B) TLC analysis of ganglioside species in human serum. (C) Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum. Data expressed as mean ± SD, n = 6. ",
    "molecules": "GD1a, ganglioside, GM3"
  },
  {
    "caption": " (E) Quantification by LC-MS/MS analysis of serum GM3 species with differing acyl chain structures. Total abundance of 10 species was defined as 1. Data expressed as mean ± SD, n = 6. ",
    "molecules": "GM3"
  },
  {
    "caption": " (A) Heat map analysis of serum GM3 species in various pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Colors indicate fold change average of each species relative to control (defined as 1), as shown in key at right. Order of pathological phases corresponds to increments of HOMA-IR and serum CRP. ",
    "molecules": "GM3"
  },
  {
    "caption": " (B-D) Properties of various GM3 species as a function of pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Data shown are relative abundances of total LCFA species (16:0, 18:0, 20:0) (B), total VLCFA species (22:0, 23:0, 24:0, h24:0) (C), and total unsaturated VLCFA species (22:1, 24:1, h24:1) (D) relative to total of ten major GM3 species (defined as 1) in each subject. ",
    "molecules": "22:0, 22:1, GM3, 16:0, 20:0, LCFA, 18:0, 24:0, 24:1, 23:0, unsaturated VLCFA, VLCFA"
  },
  {
    "caption": " (E-H) Properties of various GM3 species as a function of BMI: LCFA-GM3 (E), VLCFA-GM3 (F), unsaturated VLCFA-GM3 (G), and α-hydroxyVLCFA-GM3 (h24:0) (H). Colors indicate disease severity: light blue, no abnormal scores (n=25); orange, early-phase obesity (n=74); purple, severe obesity (n=23). ",
    "molecules": "α-hydroxyVLCFA, GM3, LCFA, unsaturated VLCFA, VLCFA"
  },
  {
    "caption": " (I, J) Spearman's correlations for GM3 h24:0 vs. ALT (I) and vs. HOMA-IR (J). ",
    "molecules": "GM3"
  },
  {
    "caption": " (K) Plots of α-hydroxylation rate (h24:0/24:0) vs. serum CRP. Colors indicate range of CRP value (mg/dL): light blue, 0.01-0.02 (n=21); orange, 0.03-0.09 (n=56); gray, 0.10-0.29 (n=29); red, 0.3-1.0 (diagnostically abnormal; n=15). ",
    "molecules": "24:0"
  },
  {
    "caption": " (B, C) GM3-mediated enhancement and inhibition of proinflammatory cytokine production from LPS-stimulated monocytes (LPS: 0.06, 0.13, 0.25 ng/mL). TNF-α (B) and IL-6 (C) production in culture supernatant was measured by ELISA. ",
    "molecules": "GM3, LPS"
  },
  {
    "caption": " (D, E) Co-stimulation of monocytes by LPS plus GM3 species or complex ganglioside species (1.5, 3.0, 4.5 µM). TNF-α (D) and IL-6 (E) production were shown in heat maps. ",
    "molecules": "ganglioside, GM3, LPS"
  },
  {
    "caption": " (F) Co-stimulation of monocytes by LPS plus GM3 species or precursor GSL species. TNF-α, IL-6, and IL-12/23 production were shown in heat maps. ",
    "molecules": "GM3, GSL, LPS"
  },
  {
    "caption": " (G) Inhibitory effect of LCFA and unsaturated VLCFA-GM3 on LVCFA-GM3 species. IL-6 production in culture supernatant was measured by ELISA. ",
    "molecules": "GM3, LCFA, unsaturated VLCFA, LVCFA"
  },
  {
    "caption": " (A) Co-stimulation of human monocytes by GM3 species plus various TLR ligands: LPS (0.13 ng/mL), TLR4/MD-2, Pam3CSK4 (0.5 µg/mL), TLR1/2, Flagellin (50 ng/mL), TLR5, R848 (0.5 µg/mL), TLR7/8, MALP-2 (1.0 ng/mL), TLR2/6. IL-6 production in culture supernatant was quantified by ELISA (shown in a heat map). ",
    "molecules": "GM3, LPS, Pam3CSK4, R848, MALP-2"
  },
  {
    "caption": " (B) Co-stimulation of monocytes by GM3 species (16:0, 24:0, 24:1) plus synthetic TLR4 ligands LA506 (15 ng/mL), LA505 (150 ng/mL), or LA504 (150 ng/mL). ",
    "molecules": "GM3, 16:0, 24:0, 24:1"
  },
  {
    "caption": " (C, D) Production of IL-6 (C) and TNF-α (D) in culture supernatant following co-stimulation of monocytes by GM3 species plus human HMGB1. ",
    "molecules": "GM3"
  },
  {
    "caption": " (E) Overexpression of hTLR4, hTLR4/hMD-2, hTLR4 (P714H) /MD-2 in HEK293T cells, and co-stimulation by GM3 22:0 with LPS (5 ng/mL). TLR4 activation was monitored by NF-κB luciferase reporter assay (termed "Relative Luc Activity" on Y-axis). ",
    "molecules": "22:0, GM3, LPS"
  },
  {
    "caption": " (F, Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) ",
    "molecules": "GM3, LPS"
  },
  {
    "caption": " G) Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) and further addition of soluble human CD14 (1 µg/mL) ",
    "molecules": "GM3, LPS"
  },
  {
    "caption": " (H) Stimulation of Mal-overexpressing HEK293T cells by GM3 species. ",
    "molecules": "GM3"
  },
  {
    "caption": " (I) Co-stimulation of hTLR4/hMD-2 by LPS (5 ng/mL) plus various mixtures of GM3 species. ",
    "molecules": "GM3, LPS"
  },
  {
    "caption": " (A) Co-stimulation of RAW macrophages by GM3 species plus various TLR ligands: LPS (0.5, 1.0 ng/mL), bovine thymus HMGB1 (0.25 µg/mL), Pam3CSK4 (50 ng/mL), Poly I:C (10 µg/mL), R848 (4 ng/mL), and CpG type-B (20 nM). TNF-α production in culture supernatant was quantified by ELISA. ",
    "molecules": "CpG type-B, GM3, LPS, Pam3CSK4, Poly I:C, R848"
  },
  {
    "caption": " (B) Co-stimulation of RAW macrophages by low- and high-dose LPS (0, 5, 50 ng/mL) plus GM3 22:0 (5 µM). Time course of TNF-α production in culture supernatant was quantified by ELISA. ",
    "molecules": "22:0, GM3, LPS"
  },
  {
    "caption": " (C) Co-stimulation of BMDMs from C3H/HeN or C3H/HeJ mice by GM3 species plus LPS (0.5 ng/mL), and TNF-α production in culture supernatant. ",
    "molecules": "GM3, LPS"
  },
  {
    "caption": " (d) Co-stimulation of mTLR4/mMD-2-expressing HEK293T cells by GM3 species plus LPS (2.5 ng/mL), and further addition of soluble mouse CD14-Fc fusion protein (1 µg/mL). ",
    "molecules": "GM3, LPS"
  },
  {
    "caption": " (E Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and complex ganglioside species (E; 2.5, 5.0, 10 µM), (shown in heat maps). ",
    "molecules": "ganglioside, GM3, LPS"
  },
  {
    "caption": " F) Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and precursor GSL species (F; 2.5, 5.0, 10 µM) (shown in heat maps). ",
    "molecules": "GM3, GSL, LPS"
  },
  {
    "caption": " (A) GM3 molecular species of 6-wk-old control C57/BL6 mice and ob/ob mice were analyzed respectively by LC-MS/MS (n=3). ",
    "molecules": "GM3"
  },
  {
    "caption": " (B) GM3 molecular species of normal-diet (ND) and high-fat-diet (HFD) C57/BL6 mice were analyzed by LC-MS/MS (n=4). ",
    "molecules": "GM3"
  },
  {
    "caption": " (C) TLC analysis of acidic GSL fraction (equivalent to 0.1 mg protein) from epididymal fat pads of C3H/HeN (A) and C3H/HeJ mice (B) on ND or HFD for 8 wk. ",
    "molecules": "GSL"
  },
  {
    "caption": " (D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4). ",
    "molecules": "GM3"
  },
  {
    "caption": " (C, D) Alanine scanning for basic residues involved in signal transduction via VLCFA-GM3 (n=5) (C), and combinations of effective mutations (n=6) (D). Signal transduction was monitored by NF-κB reporter assay. ",
    "molecules": "GM3, Alanine, VLCFA"
  },
  {
    "caption": " (E-G) Comparative inhibitory effects of GM3 16:0 on mTLR4/mMD-2 (E), mTLR4/hMD-2 (domain-swapped complex) (F), and hTLR4/hMD-2 (G) (n=3). ",
    "molecules": "GM3, 16:0"
  },
  {
    "caption": " (H) Cross-linked SDS-PAGE analysis of recombinant mTLR4 (extracellular domain)/mMD-2 complexed with GM3 18:0, GM3 species enhancing mTLR4 activation. ",
    "molecules": "GM3, 18:0"
  },
  {
    "caption": "(F) Representative images of germ cells immunostained for SYCP3 (green), DMC1 (red), and DDX4 (magenta) at each meiotic sub-stage. Nuclear DAPI staining is shown in white. The sub-stages of meiosis were defined based on the staining patterns of SYCP3, DMC1, and DAPI as described in Materials and Methods. (G) Percentages of germ cells at each meiotic sub-stage as defined in Fig 1F. The average percentages in the outer or inner ovarian cortex on each meiotic sub-stage were calculated from at least three fetal ovaries [8 wpf (n=4), 10/12/14/16/18 wpf (n=3)].",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) IF for oogonia (POU5F1, TFAP2C), meiotic (SYCP3, SYCP1), and oogenic (ZP3, TP63) markers in the transplanted cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of each marker (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate weeks after transplantation (w-ptp). Scale bar = 20 μm. (D) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3−15 w-ptp [3 w-ptp (n=3), 6/9/12 w-ptp (n=4), 15 w-ptp (n=6)]. The average values with SDs are shown. (E) H&E staining of transplanted cy rOvaries at 21 w-ptp. Dashed lines indicate the transplanted cy rOvaries beneath the kidney capsule of KSN/Slc mice. K, mouse kidney. Scale bars = 200 μm (left) and 100 μm (right).",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) IF analyses of key markers as described in Fig 1E in the cultured cy rOvaries. Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper-right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower-right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white). The numbers written in the upper-left corner indicate the period of IVC (w-ivc). Scale bar = 20 μm. (F) Percentages of cells positive for individual markers among DDX4+ germ cells from the IF of cy rOvaries at 3-15 w-ivc [3/6/9/12 w-ivc (n=3), 15 w-ivc (n=3 or 4)].",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Gross appearance (a), H&E staining (b), and IF (c) for FOXL2/DDX4/DAPI of the human in vivo female gonads (11W, left column) and the cultured rOvaries at 7 and 14 w-ivc. FOXL2, granulosa cell marker; DDX4, germ cell marker; DAPI, nucleus. Scale bars = 1 mm (a, left), 200 μm (a, middle/right), 50 μm (b), and 20 μm (c). g.a., gestational age.",
    "molecules": "DAPI"
  },
  {
    "caption": "IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers for human in vivo female gonad (11W, B) Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).",
    "molecules": "DAPI"
  },
  {
    "caption": "IF for oogonia (POU5F1), cell proliferation (Ki67), meiotic (SYCP3, DMC1, γH2AX, SYCP1), and oogenic (ZP3, TP63) markers human in vitro cultured rOvaries at 14 w-ivc (C). Germ cells and nuclei were marked with DDX4 (magenta) and DAPI (white), respectively. The upper right magnified images of each panel show the expression of key markers (green) co-stained with DDX4 (magenta). The lower right magnified images of each panel show DDX4 expression (magenta) co-stained with DAPI (white).",
    "molecules": "DAPI"
  },
  {
    "caption": "(I) IF analyses of CLGN with TP63 (oogenic marker) and DDX4 (germ cell marker) in a 13-year-old human oocyte of a primordial follicle. Nuclear DAPI staining is shown in white.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "(D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "Strumpellin and VPS35 are in close proximity to RAB5 and RAB21. (A) Anti-biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35. Lysates correspond to 1% of input, n=3 independent experiments. RAB21 can be seen interacting with various WASH and retromer complexes subunits.",
    "molecules": "biotin"
  },
  {
    "caption": "RAB21 actively pull-downs WASH and retromer complex subunits (D) Bacterially-purified and GTP-loaded GST:RAB21 pull-down of HeLa cell lysates followed by Strumpellin, FAM21, CAPZα and VPS35 immunoblots. Ponceau staining reveals the purity of the GST and GST:RAB21 used for the pull-downs. n=3 independent experiments.",
    "molecules": "GTP"
  },
  {
    "caption": "(I-J) RAB21 knockout reduces endosomal F-actin levels. (I) Immunofluorescence of endogenous F-actin using Alexa-488 conjugated phalloidin and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (J) Per cell Pearson Correlation between internal F-actin and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.",
    "molecules": "Alexa-488, phalloidin"
  },
  {
    "caption": "(G-H) RAB21 is required for appropriate SLC3A2, Basigin and CD44 trafficking. (G) Antibody-uptake assays in parental or knockout RAB21 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Note that non-internalized antibodies were removed by an acid wash, which was not fully efficient for CD147 and CD44, explaining plasma membrane labeling. (H) Percentage of cells with tubules in parental or RAB21 knockout cells; SEM, n=3 independent experiments. Data information scale bars represent 20µm or 5µm in enlarged views. Statistical tests used Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats",
    "molecules": "Alexa488"
  },
  {
    "caption": "(B) Antibody-uptake assays in parental or knockout FAM21, VARP and VPS29 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Data information: (B) scale bars represent 20µm",
    "molecules": "Alexa488"
  },
  {
    "caption": "(B) Lanes 1 and 2: negatively supercoiled plasmid (YEp24) and its equilibrium distribution of Lk topoisomers upon its relaxation with Topo I. Lanes 3 and 4: distribution of Lk topoisomers of YEp24 upon its relaxation in lysates of ∆top1 TOP2 yeast cells in absence and after the addition of ICRF-193. Plots compare the relative intensity of individual topoisomers of the Lk distributions in lanes 2, 3 and 4.",
    "molecules": "ICRF-193"
  },
  {
    "caption": "(C) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome present in the lysates of ∆top1 TOP2 yeast cells before and after the addition of ICRF-193 (see details in Fig EV3). Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots (see details in Fig EV4). The graph shows Pkn of YEp13 (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.",
    "molecules": "DNA, ICRF-193"
  },
  {
    "caption": "(F) Top: 2D gel electrophoresis of the Lk distributions of the YEp13 minichromosome produced in the presence of TOP2 or Top2-∆83. Plots compare the relative intensity of the Lk distributions (divided into ten sections). Bottom: 2D gel electrophoresis of the same samples upon nicking the DNA in order to reveal the occurrence of knots. Graph: Pkn of YEp13 (mean ± SD from three independent experiments).",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Top, DNA content (n/2n) of exponentially growing (OD600 = 0.6-0.8) smc2-8 yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). Graph: Pkn of YEp13 before and after the inactivation of condensin.",
    "molecules": "DNA"
  },
  {
    "caption": "Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (B) Experiments conducted as in (A) but in scc1-73 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of cohesin.",
    "molecules": "DNA"
  },
  {
    "caption": "Top, DNA content (n/2n) of exponentially growing yeast cells. First blot: 2D gel electrophoresis of the distribution of Lk topoisomers (Lk) of the YEp13 DNA in cells quenched at 26oC and after shifting the culture to 35oC for 60 min. Second blot: 2D gel electrophoresis of the same samples upon nicking the YEp13 DNA in order to reveal the occurrence of knots (kn). (C) Experiments conducted as in (A) but in smc6-9 yeast cells. Graph: Pkn of YEp13 before and after the inactivation of the Smc5/6 complex.",
    "molecules": "DNA"
  },
  {
    "caption": "(D, E) Experiments conducted as in (A), but in cells arrested in G1 with alfa-factor (D) or in metaphase with nocodazole (E) for 2 h at 26oC and for one additional hour at 26oC or 35oC.",
    "molecules": "alfa-factor, nocodazole"
  },
  {
    "caption": "(F, G) Experiments conducted as in (B), but in cells arrested in G1 with alfa-factor for 2 h at 26oC (F) or in metaphase with nocodazole (G) and for one additional hour at 26oC or 35oC.",
    "molecules": "alfa-factor, nocodazole"
  },
  {
    "caption": "(H, I) Experiments conducted as in (C), but in cells arrested in G1 with alfa-factor (H) or in metaphase with nocodazole (I) for 2 h at 26oC and for one additional hour at 26oC or 35oC.",
    "molecules": "alfa-factor, nocodazole"
  },
  {
    "caption": "(A-E) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of condensin (35oC) in smc2-8 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of condensin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05; **p < 0.01; ***p < 0.001. ",
    "molecules": "DNA"
  },
  {
    "caption": "(F) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of condensin.",
    "molecules": "DNA"
  },
  {
    "caption": "(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of cohesin (35oC) in scc1-73 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of cohesin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05.",
    "molecules": "DNA"
  },
  {
    "caption": "(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.",
    "molecules": "DNA"
  },
  {
    "caption": "(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of Smc5/6 complex (35oC) in smc6-9 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of Smc5/6 complex (mean ± SD from two independent experiments).",
    "molecules": "DNA"
  },
  {
    "caption": "(E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of Smc5/6 complex.",
    "molecules": "DNA"
  },
  {
    "caption": "(D) smFISH images showing LAT  RNA in  Ntrk1-positive neurons. SCG-derived cultures were infected with  HSV-1 in the presence of ACV and allowed to establish latency over 7 days before being processed for smFISH  using probes for HSV-1 LAT (green) and  neuronal  marker Ntrk1  (TrkA) (red) or fibroblast  marker Col3a1  (red). Signal intensities for the LAT  probe were quantified in neurons and fibroblasts and plotted by ImageJ. The percentage of LAT-positive cells were quantified and displayed as bar graphs with mean ± SEM. Left: 3 biological replicates, signal intensities of 50 cells were quantified for each replicate. Middle: 3 biological replicates, signal intensities of 30 cells were quantified for each replicate. Right: the blue and red dots represent the number of biological replicates done for quantification of each cell type. Scale bar, 10 μm. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).",
    "molecules": "ACV"
  },
  {
    "caption": "(A) smFISH images showing selected viral mRNAs in reactivating  neurons probed for  ICP27 (immediate-early), UL30 (early),  UL36 (true-late) or cellular markers, Ntrk (neurons), or  Col3a1 (fibroblasts). Nuclear  DNA was visualized using DAPI  (blue). To induce reactivation, latently-infected SCG-derived cultures were simultaneously treated with LY294002 (20 μM) and WAY-150138 (20 μg/mL) and cultured for 48 hrs prior to processing for smFISH. Scale bar, 10 μm.",
    "molecules": "DAPI, LY294002, WAY-150138"
  },
  {
    "caption": "(B) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM. Latently-infected cultures were untreated (latent) or treated with  LY294002 and WAY-150138  for 18, 48, or 72 hrs. Each dot corresponds to the value from each biological replicate. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.",
    "molecules": "LY294002, WAY-150138"
  },
  {
    "caption": "(A)  smFISH images showing nuclear Gadd45b mRNA in latent (UL36-negative) neurons and nuclear/cytoplasmic Gadd45b  mRNA in viral  UL36-positive neurons. Latently-infected SCG neurons treated with  LY294002 and WAY-150138 for 48 hrs were processed using smFISH to detect Gadd45b (green) and viral UL36 (red) mRNA levels and colocalization. Nuclei  were stained with DAPI (blue). Scale bar, 10 μm. (B) smFISH images processed as in (A) showing  nuclear Gadd45b mRNA in uninfected neurons  at 48 hrs post treatment with LY294002. Scale bar, 10 μm.",
    "molecules": "DAPI, LY294002, WAY-150138"
  },
  {
    "caption": "(E) The percentage of HSV-1  latently-infected neurons or reactivating neurons  at 48 hrs post treatment with LY294002 and WAY-150138  were scored for either predominantly nuclear or nuclear/cytoplasmic Gadd45b mRNA  localization and displayed as mean ± SEM. Each purple dot represents a biological replicate experiment. (F) Quantification of nuclear or nuclear/cytoplasmic Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO. Each light blue dot represents a biological replicate experiment. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ****, p<0.0001).",
    "molecules": "DMSO, LY294002, WAY-150138"
  },
  {
    "caption": "(H) Combined  smFISH and  immunofluorescence  (IF) images showing high GFP-expressing neurons (GFP-positive) or low  GFP-expressing (GFP absent) neurons simultaneously probed for   Gadd45b  mRNA. Reactivation was induced by treatment with  LY294002 and WAY-150138 for 60 hrs and processed for simultaneous smFISH and IF staining. Right panel shows quantification (as in G) of the smFISH signal intensity of Gadd45b mRNA and IF  signal intensity of GFP protein at 48 hrs post treatment with LY294002 and WAY-150138 (n=3 biological replicates). The signal intensity was quantified, plotted, and analysed by ImageJ. Bar, 10 μm. Data information: : R and R square values were calculated using Correlation analysis by Prism 8.",
    "molecules": "LY294002, WAY-150138"
  },
  {
    "caption": "(A) Viral DNA polymerase inhibitor  phosphonoacetic acid (PAA, 300 μg/ml) treatment suppressed Gadd45b mRNA expression. Latently-infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA  until the termination time-points  indicated. RNA was then collected and levels of Gadd45b mRNA quantified by RT-qPCR (n=3 biological replicates, the bars and error bars are mean ± SD). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.",
    "molecules": "LY294002, PAA, phosphonoacetic acid"
  },
  {
    "caption": "(C)  Immunofluorescence images showing nuclear-enriched Gadd45b in uninfected  neurons at 72 hrs post treatment with  LY294002. Scale bar, 20 μm. (D) The signal intensity quantification showing the ratio of nuclear/cytoplasmic Gadd45b at 72 hrs post treatment with LY294002  and WAY-150138 neurons. The signal intensity was quantified by ImageJ. 3 biological replicates, 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate. The horizonal line and error bars are mean ± SD. (E) The percentage of Gadd45b nuclear puncta-localized neurons were quantified from latent  neurons treated with LY294002  and WAY-150138 for 72 hrs. Each dot represents each biological replicate; 20 cells were quantified for each replicate. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.",
    "molecules": "EdU, LY294002, WAY-150138"
  },
  {
    "caption": "(G) Latent SCG neurons  treated with  LY294002 and WAY-150138 for 72 hrs, the Gadd45b nuclear  puncta-presenting neurons  were ICP4  negative (none observed in over 60   Gadd45b nuclear  puncta-positive  neurons), but a small population (~5%) in Gadd45b nuclear-enriched neurons were  ICP4 positive. Scale bar, 20 μm.",
    "molecules": "LY294002, WAY-150138"
  },
  {
    "caption": "(C) Ectopic expression of Gadd45b-Myc-Flag  inhibits viral mRNA  expression. Lentivirus-delivered ectopic expression of Gadd45b-Myc-Flag in SCG neurons  was confirmed by immunoblotting. Latently-infected SCG neurons  were transduced with Gadd45b-Myc-Flag for 3 days and then treated with LY294002 and WAY-150138 to induce reactivation. RNA was collected at the indicated time-points (LY treatment for 18 or 72 hours) and levels of viral  UL36 mRNA quantified by RT-qPCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).",
    "molecules": "LY, LY294002, WAY-150138"
  },
  {
    "caption": "(D) Ectopic expression of Gadd45b-Myc-Flag antagonizes LY294002-induced HSV-1 reactivation. Latently-infected SCG neurons were either transduced with vector control (Ctrl) or Gadd45b-Myc-Flag  from  lentivirus for 3 days prior to addition of LY294002. Reactivation was quantified by scoring the percentage of wells expressing  GFP fluorescence at the indicated days. The percentage of wells expressing GFP-positive was scored as the mean ± SEM (n=3 biological replicates). Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates.",
    "molecules": "LY294002"
  },
  {
    "caption": "(F) Ectopic expression of Gadd45b-Myc-Flag  suppressed viral  ICP4 mRNA expression. Latently-infected SCG neurons were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY-150138 for 72 hrs. Viral ICP4 mRNA was quantified by qRT-PCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).",
    "molecules": "LY294002, WAY-150138"
  },
  {
    "caption": "(G)  Immunoblots for viral  ICP4  protein in latent SCG neurons treated with LY294002  and WAY-150138 in the presence or absence of  Gadd45b-Myc-Flag was quantified by ImageJ (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).",
    "molecules": "LY294002, WAY-150138"
  },
  {
    "caption": "b, Local expression of oncogenic Ras  in the eye disc  decreases the amount of carnitine  in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.",
    "molecules": "carnitine"
  },
  {
    "caption": "g, Feeding of  acetyl-CoA precursor (acetate) makes GMR-RasV12 flies survive  even with TMLHE  knockdown in the fat body. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by two-tailed unpaired t-test NS, not significant, **P <0.01, ***P <0.001, ****P <0.0001.",
    "molecules": "acetate, acetyl-CoA"
  },
  {
    "caption": "D. Control and OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0 (normoxia), 24, or 48 hours. Cell lysates were then assessed by Western blotting with antibodies against OPA1, OMA1, or β-Actin. β-Actin was used as a loading control. Representative immunoblots were from n = 3 independent experiments.",
    "molecules": "O2"
  },
  {
    "caption": "E. OPA1-null mouse embryonic fibroblasts (MEFs) expressing control (empty vector) or OPA1 isoform 1 were cultured in normoxia or hypoxia (1% O2) for 24 hours. Cell lysates were analyzed by Western blotting with anti-OPA1 or anti-Tubulin antibody. Representative immunoblots were from n = 3 independent experiments.",
    "molecules": "O2"
  },
  {
    "caption": "F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were cultured in hypoxia (1% O2) for 0, 24, or 48 hours, and the cell lysates were assessed by western blot with antibodies against OMA1, and GAPDH. β-Actin was used as a loading control (representative data from three independent experiments). The asterisk indicates a nonspecific band. G. The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1-Flag/β-Actin or E324Q-Flag/β-Actin in hypoxia for 0, 24 or 48 hours (n = 3 independent experiments). The data are presented as mean ± SD. ",
    "molecules": "O2"
  },
  {
    "caption": "B. Body weight changes of WT (n=11) and OMA1-/- (n=12) mice during AOM/DSS treatment. Data are presented as mean ± SEM. Statistical significance was assessed by a two-way ANOVA, **p < 0.01.",
    "molecules": "DSS, AOM"
  },
  {
    "caption": "J. Relative mRNA expression levels of pro-inflammatory genes (TNFα, IL-6, COX-2, CCL2) in colorectal homogenates of WT and OMA1_KO mice treated with AOM/DSS (n =4-5 per genotype). The data represent the mean ± SEM., n = 3 independent experiments, and statistical significance was determined by a two-tailed student's t-test. *p < 0.05, **p < 0.01.",
    "molecules": "DSS, AOM"
  },
  {
    "caption": "A, B. Control or OMA1 KO HCT116 cells expressing control (empty vector) or OMA1-Flag were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (A) and lactate production (B) were measured by using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD of three independent experiments, statistical significance was assessed by a two-way ANOVA, *P < 0.05. Cells were lysed and assessed by western blot with antibodies against OMA1 and GAPDH (A). The asterisk indicates a nonspecific band.",
    "molecules": "glucose, lactate, O2"
  },
  {
    "caption": "C. Control and OMA1 KO HCT116 cells were treated with DMSO or 1mM DMOG in fresh medium for 12 h. Subsequent injections of glucose, oligomycin (Oligo), and 2-deoxy-glucose (2-DG) were performed as indicated. The extracellular acidification rates (ECAR) were measured with a Seahorse Extracellular Flux Analyzer XF96. The data are presented as mean ± SD (n = 3 independent experiments).",
    "molecules": "2-deoxy-glucose, 2-DG, DMSO, DMOG, glucose, Oligo, oligomycin"
  },
  {
    "caption": "E, F. Lactate production (E) and ATP production (F) were measured in control or OPA1 KO HCT116 cells upon DMSO or 2-DG treatment. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, **p < 0.01, ***p < 0.001.",
    "molecules": "2-DG, ATP, DMSO, Lactate"
  },
  {
    "caption": "G, H. Control or OPA1 KO HCT116 cells were maintained in normoxia or hypoxia (1% O2) for 24 hours. The glucose uptake (G) and lactate production (H) were measured, and the values were normalized to the protein concentration. Statistical significance was assessed by a two-way ANOVA; error bars are presented as mean ± SD of three independent experiments; N.S., not significant, *p < 0.05, **p < 0.01.",
    "molecules": "glucose, lactate, O2"
  },
  {
    "caption": "A. Control and OMA1 KO HCT116 cells were cultured in normoxia or in hypoxia (1% O2) for 24 or 48 hours. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, HK2, LDHA, PKM2, GPI, OMA1, or β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "O2"
  },
  {
    "caption": "B. Control or OMA1 KO Hela cells were untreated (-) or CoCl2-treated (+) for 24 hours with 10 mM MG132 or vehicle added for the last 4 hours. Cell lysates were assessed by Western blotting with antibodies against HIF-1α or β-Actin. Relative protein levels were further evaluated by densitometry analysis. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01.",
    "molecules": "CoCl2, MG132"
  },
  {
    "caption": "C. Dihydroethidium (DHE) staining of optimum cutting temperature (OCT) covered tissues from the colons of WT and OMA1-/- mice; DAPI was used as counterstain. DHE was shown in red color, while DAPI in blue color (representative data from three independent experiments). D. Quantification of DHE staining. Error bars are presented as mean ± SD (n=3 biological replicates), statistical significance was assessed by a two-way ANOVA, **p < 0.01, ***P< 0.001. ",
    "molecules": "DHE, Dihydroethidium, DAPI"
  },
  {
    "caption": "E. Control or OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours were stained with mitoSOX, and then analyzed by confocal microscopy (representative data from three independent experiments). F. The fluorescence intensity of mitoSOX was further analyzed by ImageJ software. Bars indicate the mean ± SD of three independent experiments by a two-way ANOVA, ***p < 0.001. ",
    "molecules": "mitoSOX, O2"
  },
  {
    "caption": "G-I. Control or OMA1_KO HCT116 cells were cultured in normoxia or hypoxia for 12 hours, and were treated with DMSO, NAC (10 mM) or Rotenone (1 μM) for 12 hours at the same time. The whole cell lysates were analyzed by Western blotting with antibodies against HIF-1α, and Tubulin (G). The glucose uptake (H) and lactate production (I) were measured using microplate reader, and the values were normalized to the protein concentration. Error bars indicate the mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-way ANOVA. N.S., not significant, *P < 0.05, ***P< 0.001.",
    "molecules": "DMSO, glucose, lactate, NAC, Rotenone"
  },
  {
    "caption": "E. HCT116 cells stably expressing control (empty vector) or OMA1-Flag, and HCT116 cells infected with control or shOMA1 (OMA1 knockdown) lentiviral particles and further cultured for 5 days, were then treated with DMSO or 20 μm CCCP for 4 hours. Cell lysates were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, MT-CO2, OMA1 or Tubulin. The black arrowhead indicated the full-length COX4L1 or NDUFB6, and the red arrowhead pointed to the cleaved band. The asterisk indicates a nonspecific band.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "F. WT, OMA1 KO and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were treated with CHX (100 µg/ml) plus CCCP (20 µM) for 0, 2, 4, 6 hours. The cell lysates were then assessed by western blot with antibodies against NDUFA4, COX4L1, NDUFB5, NDUFB6, OMA1 or HSPD1 (representative data from three independent experiments). The asterisk indicates a nonspecific band.",
    "molecules": "CCCP, CHX"
  },
  {
    "caption": "G. Control or OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0, 24, or 48 hours. Cell lysates were assessed by Western blotting with anti-NDUFB5, anti-NDUFB6, anti-COX4L1, anti-NDUFA4 antibodies, or anti-β-Actin. The relative protein levels were further evaluated by densitometry analysis using ImageJ software and were presented as bar graphs in the right panels. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "O2"
  },
  {
    "caption": "A. Mitochondria isolated from tumors (T) or adjacent normal (N) tissues of WT and OMA1-/- mice were subjected to blue native PAGE (BN-PAGE), and respiratory chain complexes were analyzed by Western blotting with anti-NDUFB8 (Complex I), anti-SDHA (Complex II), anti-COX4L1 (Complex IV), and anti-ATP5A1 (Complex V, ATP synthase) antibodies. SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "SDS"
  },
  {
    "caption": "B. Digitonin-solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours, and then analyzed by BN-PAGE and immunoblotting using the indicated antibodies against NDUFB8 (Complex I), SDHA (Complex II), NDUFA4 (Complex IV), and ATP5A1 (Complex V). SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, *p<0.05.",
    "molecules": "Digitonin, O2, SDS"
  },
  {
    "caption": "D. A549-ACE2  cells were treated for 6 hours with different concentrations of indicated inhibitors and then infected with SARS-CoV-2-GFP. GFP-fluorescence relative to DMSO-treated control cells was determined 48 hpi as described in (C). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO  (0 µM). Asterisks indicate significance compared to DMSO  (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "DMSO"
  },
  {
    "caption": "E. A549-ACE2  cells were treated for 30 min with PMA  at indicated concentrations and then infected with SARS-CoV-2-GFP. Infection  of cells was monitored as in C. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Asterisks indicate significance compared to the DMSO  control of each time point  (N = 9). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "DMSO, PMA"
  },
  {
    "caption": "F, A549-ACE2  cells were treated with apratastat, DPC-333 at indicated concentrations. Experiment was conducted and analyzed as in (D). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO  (0 µM). Asterisks indicate significance compared to DMSO  (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "apratastat, DPC-333, DMSO"
  },
  {
    "caption": "G. A549-ACE2  cells were treated with apratastat, DPC-333 or BB94  at indicated concentrations. Experiment was conducted and analyzed as in (D). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed for every indicated concentration compared to DMSO (0 µM). Asterisks indicate significance compared to DMSO  (N = 3-6). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "apratastat, BB94, DPC-333, DMSO"
  },
  {
    "caption": "H, I. A549-ACE2  cells were treated for 1 h with indicated inhibitors (10 µM) or DMSO  before inoculation with GFP-encoding spike  pseudoparticles (VSV  pseudotyped with  SARS-CoV-2 spike  protein (VSV-S)) (H) or as a control LCMV glycoprotein  (VSV-GP) (I). At 16 hpi, infection  was analyzed by counting GFP-positive cells. Data are normalized to the number of infected cells in virus-only control wells without DMSO  (N = 3-5). One-way ANOVA with Tukey's post-hoc multiple comparison test. Asterisks indicate significance compared to DMSO. Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "DMSO"
  },
  {
    "caption": "A, RT-qPCR quantification of  viral RNA (SARS-CoV-2 N gene  relative to RPLP0) upon infection  of A549-ACE2  cells with indicated SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94  (10 µM) or DMSO  for 6 h. Data are normalized to DMSO  (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three (A, biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "BB94, DMSO"
  },
  {
    "caption": "B.  RT-qPCR  quantification of  viral RNA  (SARS-CoV-2 N gene relative to RPLP0) upon infection  of A549-ACE2  cells with indicated  SARS-CoV-2 strains at 24 hpi. Cells were pretreated with BB94  (10 µM), DPC-333 (10 µM) or DMSO  for 6 h. Data are normalized to DMSO (N = 3-7). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI from at least three , B) biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "BB94, DPC-333, DMSO"
  },
  {
    "caption": "C. Normal Human Bronchial Epithelial cells (NHBE) from ten donors were infected with SARS-CoV-2. NHBE   cells were pre-treated with camostat mesylate (10µM), BB94  (1 µM or 10 µM) or DMSO  for 6 hours. RNA was isolated at 24 hpi and levels of SARS-CoV-2 N relative to RPLP0  from total cellular RNA was measured by RT-qPCR. Data are normalized to the DMSO  control (N = 5-10). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "BB94, camostat mesylate, DMSO"
  },
  {
    "caption": "D, NHBE  cells from five donors were infected with GFP-expressing  SARS-CoV-2. Infection  of cells was monitored microscopically  as green fluorescence through  live-cell imaging at 72 hours post-infection  (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. In (D) NHBE  cells were pre-treated with the indicated drugs for 6 hours before infection  Data are normalized to the DMSO  control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "DMSO"
  },
  {
    "caption": "E. NHBE  cells from five donors were infected with GFP-expressing SARS-CoV-2. Infection of cells was monitored microscopically  as green fluorescence through live-cell imaging at 72 hours post-infection  (hpi) and is shown as the mean of the GFP-positive area relative to the whole area covered by cells in the same well. NHBE cells were were treated in (E) with the indicated drugs 4 hours after infection. Data are normalized to the DMSO  control (N = 5). Two-sided independent Student's t-test with Benjamini-Hochberg FDR correction. Data information: Data are represented as means ± 95% CI from at least five/ten biological replicates *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "DMSO"
  },
  {
    "caption": "C. RT-qPCR  quantification of viral RNA (SARS-CoV-2 N gene relative to RPLP0) upon infection  of A549-ACE2  NTC or KO cells with SARS-CoV-2. Cells were treated with BB94  (10 µM) or DMSO  for 6 h prior to infection  and RNA was isolated at 24 hpi. Two-way ANOVA with Tukey's post-hoc multiple comparison test. Data are normalized to NTC DMSO  (N = 3). Data information: Data are represented as means ± 95% CI from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "BB94, DMSO"
  },
  {
    "caption": "B. Representative images of syncytia  formation assay. Large syncytia  were observed for NTC and ADAM17  KO cells, but were strongly reduced when ADAM10  was knocked-out, either alone or together with ADAM17  or upon inhibition with BB94  (10 µM). For both ADAMs, the cell lines obtained with gRNA sequence 1 were used. Images show GFP  fluorescence and Hoechst staining. Scale bars, 50 µm. C. Quantification of images from fluorescence microscopy (B). Left plot: the area of the fused cells in green (GFP) was quantified and normalized to the Hoechst  signal (blue) of the entire image to account for differences in cell density. Right plot: The nuclei  within syncytia  were determined by calculating the ratio between the Hoechst  signal within syncytia  and the total Hoechst  signal in the entire image. The data are normalized to NTC DMSO  (N >= 8). Two-way ANOVA with Tukey's correction for multiple comparisons. All data are represented as means ± 95% CI of at least 3 independent experiments. **p < 0.01, ***p < 0.001.",
    "molecules": "Hoechst, BB94, DMSO"
  },
  {
    "caption": "In vitro cleavage assay  of SARS-CoV-2 spike  protein. Recombinant spike  protein (1 µg) was digested with ADAM10, TMPRSS2  (A) for 24 hours at a 1:1 ratio and analyzed by Western Blot. Camostat mesylate or BB94  (10 µM) were pre-incubated with TMPRSS2  or ADAM proteases, respectively, for 15 min before addition of spike  protein. The spike  protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.",
    "molecules": "BB94, Camostat mesylate"
  },
  {
    "caption": "B. In vitro cleavage assay  of SARS-CoV-2 spike  protein. Recombinant spike  protein (1 µg) was digested with ADAM17  (B) for 24 hours at a 1:1 ratio and analyzed by Western Blot. BB94  (10 µM) were pre-incubated with ADAM proteases, respectively, for 15 min before addition of  spike  protein. The  spike  protein and fragments were detected using an antibody against its C-terminal 6xHIS tag.",
    "molecules": "BB94"
  },
  {
    "caption": "C. Representative images and quantification of a proximity ligation assay  (PLA) between the SARS-CoV-2 Spike  and ADAM17  in A549-ACE2  cells. Cells were infected with SARS-CoV-2 and placed on ice (4°C). After 2 h, cells were shifted to 37°C for 15 min or left on ice. Images are presented as overlay between the Nuclei  (Hoechst, blue) and the PLA  (pseudo-color: red) channel. Scale bar, 20 µm. The number of PLA  foci per cell was determined for the acquired images (N = 28-30). A two-sided independent Student's t-test with Benjamini-Hochberg FDR correction was performed. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "Hoechst"
  },
  {
    "caption": "D. A549-ACE2  cells were treated for 48 h with BB94  (10 µM), DPC-333 (5 µM) or DMSO. PMA  (25 ng/ml) was added for 3 h before harvest to stimulate ACE2   shedding. Concentration of the soluble ACE2  fragment was determined in the conditioned medium by ELISA  and is shown relative to the concentration in the DMSO  control (N = 6). One-way ANOVA with Tukey's post-hoc multiple comparison test. Data information: Data are represented as means ± 95% CI of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "BB94, DPC-333, DMSO, PMA"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG  or TFEB-S401A-FLAG  treated with 200 μM NaAsO2  or 250 nM Torin-1  for 1 h. Two different rabbit polyclonal antibodies raised against a TFEB-S401 phospho-specific peptide were tested.",
    "molecules": "NaAsO2, Torin-1"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from  ARPE-19 cells expressing TFEB-WT-FLAG, TFEB-S3A/R4A-FLAG  or TFEB-R245-247A-FLAG  treated with 200 μM NaAsO2  for 1 h. Immunoblots  are representative of at least three independent experiments.",
    "molecules": "NaAsO2"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from HeLa  cells stably expressing TFEB-WT-FLAG  incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2  for 1 h. Quantification of immunoblot  data shown in (A). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, and (****)p<0.0001 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).",
    "molecules": "NaAsO2"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from HeLa   cells stably expressing TFEB-WT-FLAG  incubated with either 200 μM NaAsO2  for 1 h, EBSS  for 4 h, 100 ng/ml EGF   for the indicated times  or 37 μM Anisomycin  for 1 h. Before the addition of EGF  or Anisomycin, cells were serum  starved for 8 h.",
    "molecules": "Anisomycin, NaAsO2"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from HeLa  cells stably expressing TFEB-WT-FLAG  exposed to 30J/m2 of UV-C and allowed to recover in complete medium for the indicated times. Cells were incubated with p38 MAPK inhibitor (20 μM, SB203580) for 1 h before  UV-C irradiation  and allowed to recover for 30 min in the presence of the inhibitor.",
    "molecules": "SB203580"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from HeLa  cells stably expressing TFEB-WT-FLAG  depleted of either p38 MAPK (α+β) or JNK1  or JNK2  or JNK(1+2) and incubated with 200 μM NaAsO2  for 1 h. Immunoblots  are representative of at least three independent experiments.",
    "molecules": "NaAsO2"
  },
  {
    "caption": "Immunoblot  analysis of in vitro p38 MAPK kinase assay  using GST-TFEB-PRD as substrate in the presence or absence of either recombinant human  active p38α MAPK  or ATP. Quantification of immunoblot  data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).",
    "molecules": "ATP"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from Raw 264.7  cells stably expressing TFEB-WT-FLAG  incubated with 1μg/ml LPS  for the indicated times.",
    "molecules": "LPS"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from  Raw 264.7 cells stably expressing TFEB-WT-FLAG  incubated with either 20 μM SB203580  or 250 nM Torin-1 for 1 h prior to the addition of 1 μg/ml LPS  for 30 min.",
    "molecules": "LPS, SB203580, Torin-1"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from THP1  cells incubated with 1 μg/ml LPS  for the indicated times.",
    "molecules": "LPS"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from THP1  cells incubated with either vehicle (DMSO) or 20 μM SB203580  for 1 h prior to the addition of 1μg/ml LPS  for the indicated times.",
    "molecules": "DMSO, LPS, SB203580"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from THP1  cells depleted of  p38 MAPK (α), p38 MAPK (β) or p38 MAPK (α+β) and incubated with 1 μg/ml LPS  for 1 h. Immunoblots  are representative of at least three independent experiments. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).",
    "molecules": "LPS"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates from naïve THP1-WT or TFEB-S401A knock-in (clone M17) cells treated with 50 ng/ml PMA  for the indicated times, PMA-differentiated THP1  (Rested) cells treated without or with 250 nM Torin-1  for 1 h.",
    "molecules": "PMA, Torin-1"
  },
  {
    "caption": "Immunoblot  analysis of proteins from nuclear  and cytosolic  fractions from naïve THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells treated with 50 ng/ml PMA  for the indicated times  and PMA-differentiated THP1  (Rested) cells.",
    "molecules": "PMA"
  },
  {
    "caption": "Immunoblot  analysis of proteins from nuclear  and cytosolic  fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA  for the indicated times.",
    "molecules": "PMA"
  },
  {
    "caption": "Immunoblot  analysis of proteins from nuclear  fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA or 250 nM  Torin-1 for 1 h. Immunoblots  are representative of at least three independent experiments.",
    "molecules": "PMA, Torin-1"
  },
  {
    "caption": "Immunoblot  analysis of proteins from nuclear  and cytosolic  fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA  for 6 h.",
    "molecules": "PMA"
  },
  {
    "caption": "Immunoblot  analysis of protein lysates   and cell culture medium from PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 0.1 μg/ml LPS  for 4 h prior to the addition of 15 μM Nigericin  for 45 min. C-F. Quantification of immunoblot  data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05, (**)p<0.01, (***)p< 0.001 and (****)p< 0.0001 from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).",
    "molecules": "LPS, Nigericin, PMA"
  },
  {
    "caption": "Fluorescence confocal microscopy of PMA-differentiated (Rested) THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells incubated with 1 μg/ml LPS  for 6 h prior to the detection of dead cells with LIVE/DEAD fixable blue dead cell stain. Scale bars, 10 μm. Quantification of the  percentage of dead cells shown in (G). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant and (****)p< 0.0001 as compared to their corresponding untreated (Control) cells, with >200 cells counted per trial from three independent experiments. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).",
    "molecules": "LPS, PMA"
  },
  {
    "caption": "Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread cultured wild-type control (WT Control), and okadaic acid treated wild-type (WT + OA) and Smc1β-/- (Smc1β-/- + OA) spermatocytes.A-B WT control cultured spermatocytes in (A) late zygotene (L. zyg.) and (B) metaphase I (M I). Arrows indicate the cell poles.C-D WT cultured spermatocytes treated with okadaic acid in (C) late zygotene (L. zyg.) and (D) metaphase I-like (M I-like).E-F Smc1β-/- cultured spermatocytes treated with okadaic acid in (E) pachytene-like (Pac.-like) and (F) metaphase I-like (M I-like). Arrowheads indicate nucleolar-associated round bodies. The unsynapsed axial elements of the sex chromosomes (X,Y) in prophase I, as well as the sex bivalent (XY) in metaphases I are indicated. Scale bar: 10 µm.",
    "molecules": "OA, okadaic acid"
  },
  {
    "caption": "EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band.",
    "molecules": "DNA"
  },
  {
    "caption": "Electron microscopy revealed a number of demyelinated or degenerated axons in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment at 3 months of age for 2 months improved myelination in PLP-150Q mice. Scale bars: 2μm (Low magnification) and 0.5 μm (High magnification).",
    "molecules": "LAQ"
  },
  {
    "caption": "G-ratios, which were calculated and plotted against axon diameter with linear regression, were shown beneath the micrographs and were significantly decreased in PLP-150Q mice after LAQ treatment (G=0.6418±0.0191), compared with age-matched vehicle group (G=0.7727±0.0203). One-way ANOVA with Tukey's test. *P=0.0166. At least 182 axons per genotype were examined from 3 mice in each group.",
    "molecules": "LAQ"
  },
  {
    "caption": "Western blotting showing that LAQ (5 or 25 mg/kg) treatment for 2 months up-regulates multiple myelin proteins (MBP, MOBP and MOG) in the corpus callosum in PLP-150Q mice, which were treated from 3 months of age. Ratios of MBP, MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right. One-way ANNOVA followed with Tukey's test. MBP: ***P=0.0009; MOG: ***P=0.0003; MOBP: **P=0.0067. Data are mean ±SEM",
    "molecules": "LAQ"
  },
  {
    "caption": "Quantitative PCR of the transcript expression of myelin associated genes (MBP and MOG) in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 or 25 mg/kg) treatment for 2 months. Student's t-test. MBP: ***P=0.0007; MOG: ***P=0.0009. Data are mean ±SEM (n=3).",
    "molecules": "LAQ"
  },
  {
    "caption": "The MBP DNA promoter was inserted into the pGL4.1 luciferase report vector and was co-expressed with N-terminal MYRF (nMYRF) and mHTT to assess its transcription activity via the luciferase assay. MYRF markedly enhanced the MBP promoter activity. N-terminal mutant HTT significantly inhibited the reporter activity, ***P< 0.001; which was reversed by LAQ (5 μM) treatment, **P< 0.01. The ratios were obtained from 3 independent experiments. One-way ANNOVA followed with Tukey's test. Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "Immunoprecipitation of transgenic mHTT from 3-month-old PLP-150Q mouse brains revealed that 5 or 25 mg/kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF. fMYRF: full-length MYRF; nMYRF: N-terminal MYRF. The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right. One-way ANNOVA followed with Tukey's test. **P=0.009. Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "Western blotting of the phosphorylated- NF-kB, -Akt, -JNK in PLP-150Q mouse corpus callosum at 3 months of age showing that LAQ (5 or 25 mg/kg) treatment for 2 months could dephosphorylate these proteins. Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. NF-kB: ***P=0.00067(left) and 0.00086(right); JNK: ***P=0.00013(left) and 0.00047(right); Akt: **P=0.0014 and ***P=0.00012. Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "In vitro phosphorylation assay of N-terminal MYRF (nMYRF). GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP-23Q or PLP-150Q mice that were treated with vehicle or 5 mg/kg LAQ (left panel). GST fusion proteins containing wild type N-terminal MYRF, S259A, or S261A were incubated with PLP-150Q mouse tissue lysates (right panel). The beads were then centrifuged and analyzed by western blotting with anti-GST (lower panels) and anti-phosphor-serine (upper panels). Note that LAQ treatment could eliminate mutant HTT (PLP-150Q)-mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation.",
    "molecules": "LAQ, phosphor, serine"
  },
  {
    "caption": "Co-immunoprecipitation of nMYRF and N-terminal HTT (1-212 aa) containing 150Q in HEK293 cells. Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co-immunoprecipitated HTT. Λ-PPase served as the positive control for the dephosphorylation of MYRF. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots, which were obtained from 3 independent western blotting experiments. One-way ANNOVA with Tukey's test. ***P=6.51x10-5. Data are mean ±SEM.",
    "molecules": "LAQ, pho, serine"
  },
  {
    "caption": "Co-transfection of nMYRF with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ (5 μM) treatment. Coomassie blue staining confirms the presence of immunoprecipitated N-terminal MYRF bands (arrow).",
    "molecules": "LAQ"
  },
  {
    "caption": "Co-transfection of wide type N-terminal MYRF, S259A, or S261A with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF. Compared to wide type MYRF and S261A, less S259A (non-phosphorylated) was precipitated with mutant HTT. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P=2.30x10-5 . Data are mean ±SEM.",
    "molecules": "pho, serine"
  },
  {
    "caption": "Western blotting analysis of the corpus callosum in PLP-23Q or PLP-150Q mice with anti-pS259 and an antibody to total MYRF. Note that LAQ (5 mg/kg) treatment for 2 months decreased the phosphorylation in both full length (fMYRF) and N-terminal MYRF (nMYRF) in PLP-150Q mice.",
    "molecules": "LAQ"
  },
  {
    "caption": "Ratios of phosphor-S259-MYRF (pMYRF) and total MYRF to GAPDH. The ratios were obtained from 3 independent western blotting experiments. Student's t-test; ***P=0.0007. Data are mean ±SEM.",
    "molecules": "phosphor"
  },
  {
    "caption": "Anti-Ser259-MYRF immunohistochemical staining of the corpus callosum (CC, indicated between two dotted lines) of PLP-23Q and PLP-150Q mice showing decreased expression of Ser259-MYRF by LAQ (5 mg/kg) treatment for 2 months. Micrographs at 10X (upper) and 40X (lower) are shown. Scale bars: 40 μm.",
    "molecules": "LAQ"
  },
  {
    "caption": "Heatmaps of gene expression in PLP-150Q mouse corpus callosum at 5 months after LAQ (5 mg/kg) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes.",
    "molecules": "LAQ"
  },
  {
    "caption": "Western blotting showing down-regulation of PRKG2 in PLP-150Q mouse corpus callosum at 3 months of age by 5 mg/kg LAQ. Ratios of PRKG2 or phosphorylated VASP (p-VASP) to GAPDH obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. PRKG2: **P=0.0076(left) and ***P=0.0015(right); p-VASP: ***P=3.86x10-5(left) and 1.01x10-5(right). Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 mg/kg) treatment for 2 months. Only PRKG2 was decreased by LAQ. n=3 mice in each group. Student's t-test; **P=0.0073; ***P=0.00041. Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "The treatment of LAQ (5 or 10 μM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P<0.001. Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP-150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n=3 mice in each group. Student's t-test; Cyp1a1: ***P=0.00042; Ugt1a6a; ***P=0.00057. Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.",
    "molecules": "LAQ"
  },
  {
    "caption": "The semi-PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The results were obtained from three independent experiments. One-way ANNOVA with Tukey's test. LAQ: ***P=0.00065; 2,3,7,8-TCDD: ***P=0.00014. Data are mean ±SEM",
    "molecules": "2,3,7,8-TCDD, DMSO, LAQ"
  },
  {
    "caption": "Western blotting showing that the phosphorylation of transfected N-terminal MYRF (pMYRF) was promoted by treatment with the PKG activator of 8-Br-cGMP (10 or 100 μM) for 1 hr or co-transfected 150Q-HTT for 24 hr, which was blocked by treatment of the PKG inhibitors of RKRARKE (50 μM) or KT5823 (5 μM) for 12 hr. trans-nMYRF: transfected N-terminal MYRF.",
    "molecules": "RKRARKE, 8-Br-cGMP, KT5823"
  },
  {
    "caption": "Localization of FUS-DDIT3 and YAP1 in MLS 1765-92. Nuclei were counterstained with DAPI. The original magnification was x63, and images were zoomed in four times for co-localization analysis. The correlation between red and green fluorescence was determined by Pearson coefficient analysis (square, area for signal acquisition).",
    "molecules": "DAPI"
  },
  {
    "caption": "Viability and proliferation of MLS 402‑91, MLS 1765‑92, and MLS 2645-94 cells cultured in the presence of verteporfin. Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate.",
    "molecules": "verteporfin"
  },
  {
    "caption": "Flow cytometric analysis of apoptosis (cleaved PARP) and mitotic fraction (phosphorylated histone H3S10) in MLS cell lines cultured in the presence of 0.25 µM Verteporfin. One of two independent experiments with similar results is shown.",
    "molecules": "Verteporfin"
  },
  {
    "caption": "Expression of total YAP1 and downstream effectors (FOXM1 and PLK1) in MLS cell lines treated with 0.5 or 1 µM verteporfin for 15 hours. One of at least three independent experiments with similar results is shown.",
    "molecules": "verteporfin"
  },
  {
    "caption": "YAP1-responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin. Relative luciferase activity is displayed relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.",
    "molecules": "verteporfin"
  },
  {
    "caption": "Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin. Shown are tumor volumes and representative photographs of tumors.",
    "molecules": "verteporfin"
  },
  {
    "caption": "(G). Two tailed Pearson's correlation coefficient analysis of plasma AKG level and body mass index (BMI) in Chinese adults (10 males and 35 females).",
    "molecules": "AKG"
  },
  {
    "caption": "(H). Serum AKG concentration-time profile obtained before and after 40-min resistant exercise. At 8 weeks of age, male C57BL/6 mice were switched to HFD and continuously fed with HFD for 12 weeks. At 20 weeks of age, mice received resistance exercise for 40 min. Another group of chow fed male C57BL/6 mice (10 weeks) received resistance exercise for 40 min. The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after exercise (n = 8-10 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(I). Serum AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1 day or resistance exercise for 40 min.(n = 8-10 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(J). Muscles AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after AKG gavage (10 mg/kg). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after gavage (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(B-D). Water intake (B), cumulative food intake (C) and body weight gain (D) of male C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).",
    "molecules": "AKG, water"
  },
  {
    "caption": "Fat and lean mass (E) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "representative images (F) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(G-I). Weight index of BAT (G), gWAT (H) and iWAT (I) from male mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(J). Serum AKG level of male mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(K-L). Representative images (K) and quantification (L) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(M-O). Water intake (M), cumulative food intake (N) and body weight gain (O) of female C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).",
    "molecules": "AKG, water"
  },
  {
    "caption": "(P-Q). Fat and lean mass index (P) and representative image (Q) of body composition from female mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(R-T). Weight index of BAT (R), gWAT (S) and iWAT (T) from female mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(U-V). Representative images (U) and quantification (V) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(A-B). Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(C). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(D-E). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "The mRNA expression of thermogenic genes (F) in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(H-I). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 9 per group).",
    "molecules": "AKG, DAB"
  },
  {
    "caption": "(J). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks (n = 9 per group).",
    "molecules": "NEFA, AKG"
  },
  {
    "caption": "(K). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(L-M). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(P-Q). Representative images (P) and quantification (Q) of p-HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group).",
    "molecules": "AKG, DAB"
  },
  {
    "caption": "(R). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(S-V). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group).",
    "molecules": "AKG, T3, NE, T4"
  },
  {
    "caption": "(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hrs cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Oxygen consumption (F-G) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.",
    "molecules": "SR59230A, AKG"
  },
  {
    "caption": "RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.",
    "molecules": "SR59230A, AKG"
  },
  {
    "caption": "(J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).",
    "molecules": "AKG, water"
  },
  {
    "caption": "serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).",
    "molecules": "NEFA, AKG, water"
  },
  {
    "caption": "(O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(Q). The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(R-S). Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(A). The mRNA expression of AKG-sensing genes in the adrenal gland tissue of 12-weeks male C57BL/6 mice (n = 4 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(D). The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i.p. injection of saline or 10 mg/kg AKG, or immediately after 40-mins resistance exercise (n=8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(F). E level in the medium from adrenal chromaffin cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 30 mins (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(G). Intracellular calcium ion [Ca2+] changes in adrenal medulla cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 (n = 30 per group).",
    "molecules": "AKG, calcium ion, Ca2+"
  },
  {
    "caption": "(H-I). Body weight gain (H) and cumulative food intake (I) of male WT control (littermates) or OXGR1 global knock out (OXGR1KO) mice. At 12 weeks of age, both control and KO mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group).",
    "molecules": "AKG, water"
  },
  {
    "caption": "(J-K). Representative images of body composition (J) and fat and lean mass index (K) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(L-M). Weight index of iWAT (L) and gWAT (M) in male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Oxygen consumption (P-Q) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "RER (R-S) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Body weight gain and cumulative food intake of male OXGR1 adrenal-specific reexpression mice (OXGR1REAG). Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP (OXGR1KO control) or HBAAV2/9-OXGR1 (OXGR1REAG). Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group).",
    "molecules": "AKG, water"
  },
  {
    "caption": "Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Immunoblots and quantification of p-HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "RER of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise.",
    "molecules": "AKG"
  },
  {
    "caption": "Ingenuity Pathway Analysis (IPA) of AKG-induced transcriptome signature in adrenal chromaffin cell treated with AKG. The mRAN was extracted from adrenal chromaffin cells after 3-hrs incubation of vehicle or AKG (100 μM). (n = 3 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "Immunoblots and quantification of p-IKK/IKK, p65, p-IκB/IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group).",
    "molecules": "AKG"
  },
  {
    "caption": "p65 translocation in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group). Scale bars, 100 μm.",
    "molecules": "AKG"
  },
  {
    "caption": "Immunoblots and quantification of p-IKK/IKK, p65 and p-IκB/IκB protein in adrenal chromaffin cells cultured with vehicle, AKG (100 μM), IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs.",
    "molecules": "IKK16, AKG"
  },
  {
    "caption": "E level in the medium from adrenal chromaffin cell cultured with vehicle, AKG (100 μM), IKK16 or AKG + IKK16 for 3 hrs (n = 10 per group).",
    "molecules": "IKK16, AKG"
  },
  {
    "caption": "A) HeLa cells transfected with indicated plasmids expressing mouse (m) or human (h) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag (red) and Dapi staining (blue). ∆C1-hDGKk construct bears deletion of phorbol ester C1 domain (328-449).",
    "molecules": "Dapi, phorbol ester"
  },
  {
    "caption": "B) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-HA-∆N-DGKk, untransfected (NT), or plasmid pCI control, with the indicated amount of plasmid (µg) and after 24h, incubated with puromycin (20µg/ml, 30 min) to measure rates of protein synthesis. GAPDH was used as a loading control. 0 indicates no puromycin treatment, -/+ indicates treatment without or with DGK inhibitor (DGKi) 3 µM R59022 and 0.2 µM R59949 at 6 µM, 15 min. Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions.",
    "molecules": "R59022, R59949, puromycin"
  },
  {
    "caption": "C) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV (days in vitro) with 10e9 VG/ml of AAVRh10-GFP or AAVRh10-∆N-DGKk and assessed after 5 days using anti-MAP2 and anti-HA for ∆N-DGKk or direct 488 nm excitation for GFP. DAPI was used to visualize nuclei on merged images.",
    "molecules": "DAPI"
  },
  {
    "caption": "B) Viral titers (viral genome copy VG per cell) determined by qPCR in cortical, hippocampal and rest of brain areas of Fmr1-KO mice treated with saline solution (Vehicle), AAVPHP.eB-∆N-DGKk (PHP.eB), AAVRh10-∆N-DGKk (Rh10) 8 weeks after injections. Data are mean ± SEM. Each dot represents an individual mouse.",
    "molecules": "saline"
  },
  {
    "caption": "D) Representative coronal brain sections processed for detection of ∆N-DGKk using immunohistochemistry on Fmr1-KO mice treated with indicated treatment, 8 weeks post-injections, counter stained with eosin hematoxylin. Adjacent sections were immunolabelled with NeuN. 3 mice per genotype were processed. The sections shown are between Bregma levels -1.50 mm and -1.80 mm. Scale bar is 2 mm. Magnifications of regions of cortex (c), hippocampus (h), and striatum (s) are shown in side panels, scale bar 200 µm.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "E) Total phosphatidic acid (PA) level measure by mass spectrometry in cortex of WT mice treated with saline solution (WT-S) and Fmr1-KO mice treated with saline (Fmr1-S), AAVPHP.eB-∆N-DGKk (Fmr1-PHP.eB), AAVRh10-∆N-DGKk (Fmr1-Rh10) 8 weeks after injections. Data are expressed as mol % of total lipids and represented as median with interquartile range with minimum and maximum values. Statistics: one-way ANOVA and Tukey's multiple comparisons test, n=8 individual animals, except for WT-S and Fmr1-S n=11. **P < 0.01, ***P < 0.001.",
    "molecules": "lipids, PA, phosphatidic acid, saline"
  },
  {
    "caption": "F) Total diacylglycerol (DAG) level in cortex measured as in E).",
    "molecules": "DAG, diacylglycerol"
  },
  {
    "caption": "B. CYP26C1protein representation and luciferase assay. Variants found in CYP26C1 are indicated in green. Cignal-RARE system luciferase assays to test the impact of CYP26C1 variants on its RA degradation activity were performed in U2OS cells treated with 250 nM all-trans retinoic acid (ATRA) for 24 hours (n = 4). Experiments were performed in triplicates.pIRES2-EGFP empty vector was used as control. The residue C459 represents the Iron binding residue (Q6VOL0, UniProtKB) and was mutated to Ala and used as a positive control. TM, Transmembrane helix; P450, cytochrome p450 domain. RLU, Relative Light Units.",
    "molecules": "all-trans retinoic acid, ATRA"
  },
  {
    "caption": "B. Relative expression of SHOX mRNA normalized to the reference genes SDHA and HPRT in human primary chondrocytes treated with ATRA 100 nM for 6h (n = 5). One outlier with high relative expression (2.3 fold) was excluded from ATRA 100 nM treatment (two-sided Grubb's test, Z value 2.34, p-value < 0.05).",
    "molecules": "ATRA"
  },
  {
    "caption": "C. SHOX promoter was cloned in pGL3basic for luciferase reporter experiments as previously described (Verdin et al, 2015). In silico analysis of SHOX promoter identified three putative RXRa binding sites which were mutated to test their direct effect on SHOX expression upon treatment with ATRA 250 nM in U2OS cells (n = 4). Experiments were performed in triplicates. RLU, Relative Light Units.",
    "molecules": "ATRA"
  },
  {
    "caption": "F. Cignal-RARE system luciferase assay testing cyp26c1 MO on RA acid levels in zebrafishembryos (n = 5 replicates). Embryos were separated in groups of 20-30 and luciferase assayed. RLU, Relative Light Units.",
    "molecules": "RA"
  },
  {
    "caption": "H. Relative shox mRNA expression normalized to reference genes eef1a and b-actin in zebrafishembryos treated with 100 nM ATRA (n = 4). Embryos were collected at 24 hpf and treated with mock control or ATRA. RNA was extracted from 10-15 embryos after 6 hours treatment.",
    "molecules": "ATRA"
  },
  {
    "caption": "(D) atg1Δ and atg1Δatg13Δ cells expressing HA‐tagged Atg1 and wild‐type (wt) cells containing an empty plasmid were grown to mid log phase (rich), and then treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1 was immunoprecipitated and its association with Atg13 analysed by immunoblotting. Note that phosphorylated Atg13 isolated from cells grown in rich medium migrates slightly slower on SDS-PAGE, and the extent corresponds to the shift typically observed under our gel conditions.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(E) Yeast cells containing endogenously tagged Atg1‐TAP with and without endogenously tagged Atg13‐GFP, or conversely cells containing endogenously tagged Atg13‐TAP and Atg1‐GFP expressed from a centromeric plasmid were grown to mid log phase (rich) and treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1‐TAP and Atg13‐TAP were affinity purified and their association with GFP‐tagged proteins was analysed by western blotting.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(F) Histone3‐HA tagged Atg1 was expressed together with wild‐type (wt) or the Atg13‐FV mutant fused to Suv methylase (Suv) in atg1Δatg13Δ cells, or for control together with Pbs2‐Suv in atg1Δ cells. Logarithmically growing cultures were treated with rapamycin (rapa) for 0 or 120 min, and methylation was monitored by preparing cell extracts and western blotting with an anti‐trimethylation‐specific antibody. Note that Suv‐, GFP‐ or TAP‐tagged Atg13 are fully functional, however, do not show the characteristic phosphorylation‐induced reduced mobility observed with the untagged protein under our gel conditions.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Exponentially growing ypt7Δ cells were starved in SD‐N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX‐100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti‐Atg1, anti‐Pep12 and anti‐Pgk1 antibodies.",
    "molecules": "TX‐100"
  },
  {
    "caption": "E. Total RNAs from naïve CD4+ T cells, PHA-stimulated CD4+ T cells and resting memory CD4+ T cells were extracted and proceeded to RT-qPCR to quantitate the relative expression of CBX4. The expression of CBX4 in PHA-stimulated and resting memory CD4+ T cells was normalized to naïve CD4+ T cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "PHA"
  },
  {
    "caption": "B. Super-resolution SIM image of CBX4 in HEK293T cells. DAPI was used to dye DNA which was colored into blue. FITC-tagged antibodies were used to label endogenous CBX4 which was colored into green. Data information: Scale bars in (B) 5 μm. All the samples were imaged to obtain at least three sets of images.",
    "molecules": "DAPI, FITC"
  },
  {
    "caption": "F-L. RFP-tagged EZH2, RING1B, SUMO4, SUZ12, CHAF1A, DNMT1 and HP1α were co-overexpressed with GFP-tagged CBX4 in HEK293T cells, respectively. SIM imaging was performed for each combination. The line scan profiles of SIM images which showed co-localization and non-co-localization between CBX4 and its partners were shown on the right of each panel. The red arrows in merged images indicated the positions where the line scans profiled. M-O. GFP-tagged CBX4 was overexpressed in HEK293T cells, followed by treating with AF568-tagged antibodies against H3K4me3, H3K9me3 and H3K27me3, respectively. CBX4 and each histone modification were imaged with SIM. Line scan profiles were shown on the right of each panel. Red arrows in merged images indicated the positions where line scans profiled. Data information: Scale bars 5 μm. All the samples were imaged to obtain at least three sets of images.",
    "molecules": "AF568"
  },
  {
    "caption": "D. The endogenous EZH2 in TZM-bl cells within 6 cm dishes was knocked down by siRNAs targeting 3'UTR of EZH2 mRNA, followed by the overexpression of 2 μg of EZH2, 4 μg of Flag-tagged SUMO4, 250 ng of Flag-tagged UBC9 or 250 ng of Flag-tagged CBX4. EZH2 was IP with anti-EZH2 antibodies. IP samples were IB with anti-EZH2 antibodies. Total samples were IB with anti-EZH2, anti-H3K27me3, anti-Histone H3 and anti-GAPDH antibodies. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.",
    "molecules": "H3K27me3"
  },
  {
    "caption": "E. In the first group, HEK293T cells within 6 cm dishes were transfected with empty vectors. In the second group, 3 μg of EZH2 was overexpressed. In the third group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED and 2 μg of SUZ12. In the fourth and sixth group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. In the fifth group, 3 μg of EZH2mut (Y731D) was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. Forty-eight hours post transfection, EZH2 and EZH2mut were IP with anti-EZH2, followed by the incubation with 2 μg of mononucleosomes (H3.1) and 20 μM of the cofactor S-adenosyl-L-methionine (SAM) to measure the methyltransferase activity of EZH2. The IP reaction in the sixth group was incubated with 2 μg of mononucleosomes (H3.1 K27M) and 20 μM of SAM. Both total and IP samples were IB with anti-EZH2, GAPDH, H3K27me3 and H3.1 antibodies. The purities of recombinant mononucleosomes which included H3.1, H2B, H2A and H4 were verified by Coomassie blue staining. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.",
    "molecules": "H3K27me3, cofactor S-adenosyl-L-methionine, SAM"
  },
  {
    "caption": "A. PHA-activated primary CD4+ T cells were infected with wildtype HIV-1 viruses which harbored a GFP ORF after Nef gene, followed by infecting with shluc and shCBX4 lentiviruses respectively. The percentages of GFP-positive cells, which were HIV-1-expressing cells, were monitored every three days. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "PHA"
  },
  {
    "caption": "D. PHA-activated CD4+ T cells were infected with wildtype HIV-1 viruses as in (A). One group of HIV-1-infected CD4+ T cells were left untreated. One group of HIV-1-infected cells were infected with shluc. Another group of HIV-1-infected cells were infected with shCBX4. Two weeks later, one part of HIV-1-infected CD4+ T cells were reactivated by αCD3/αCD28/IL-2. One part of shluc- and shCBX4-infected cells were co-stimulated with LRA JQ-1. GFP-positive cells in each group were measured by flow cytometry to indicate reactivated HIV-1-infected cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "JQ-1, PHA"
  },
  {
    "caption": "(d) Western blot analysis of HeLa cells treated (48 h) with an inhibitor of mTORC1 (RAP, 20 nM) or control (DMSO).",
    "molecules": "DMSO, RAP"
  },
  {
    "caption": "(f) Western blot analysis of MDA-231, T47D and MDA-435 cells treated (48 h) with inhibitors of mTORC1 (RAP, 20 nM), mTORC1 and 2 (PP242, 100 nM), or DMSO (control).",
    "molecules": "DMSO, RAP, PP242"
  },
  {
    "caption": "(g) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h.",
    "molecules": "CQ, DMSO"
  },
  {
    "caption": "(h) Western blot analysis of HeLa cells treated with BAF (200 nM, 12 h), RAP (20 nM, 24 h), or RAP and BAF. The results in a-h are representative of three experiments and use α-tubulin (TUBA) as a loading control.",
    "molecules": "BAF, RAP"
  },
  {
    "caption": "(k,l) RT-qPCR analysis of AGO1, AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO, BAF or CQ (k) or siRNA for 84 h (l). Error bars show s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.",
    "molecules": "BAF, CQ, DMSO"
  },
  {
    "caption": "(a) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and NDP52 in HeLa cells treated with RAP or control. The arrows highlight some examples of co-localization. The lower left insets show higher magnifications of the areas outlined in the main panel. Scale bars, 5 μm. (b) Quantification of DICER co-localization with NDP52. Total fluorescence intensity of DICER co-localized with punctae (>0.2 μm3) of NDP52 was quantified with Volocity software in RAP-treated cells/control-treated cells over several Z-stacked microscope fields per experiment (n = 4, error bars s.e.m.).",
    "molecules": "RAP"
  },
  {
    "caption": "(c) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and HcRed-LC3 in HeLa cells treated with BAF or control. The arrows highlight some examples of co-localization. Insets as in a. Scale bar, 5 μm. (d) Quantification of DICER co-localization with HcRed-LC3 was performed as for NDP52 in b.",
    "molecules": "BAF"
  },
  {
    "caption": "(f-i) Localization of DICER (monoclonal antibody 13D6) detected with anti-mouse IgG (10 nm gold beads) by electron microscopy in CQ- (20 μM, 12 h) treated HeLa cells. Mito, mitochondria; MVB, multivesicular body. The black arrows highlight all gold beads.",
    "molecules": "gold beads, CQ"
  },
  {
    "caption": "(j) Western blot analysis of fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV; ref. ). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body and lysosomal markers are indicated. Material that was pelleted by centrifugation at 100,000g (100 K pellet) or that remained in solution after the 100,000g spin (soluble) was not added to the gradient.",
    "molecules": "CQ, Histodenz"
  },
  {
    "caption": "(k) Western blot analysis of UB and p62 in discontinuous Histodenz gradients (as in j) of HEK293T cells treated with control (DMSO) or CQ. Uncropped images of blots are shown in Supplementary Fig. S3.",
    "molecules": "CQ, DMSO, Histodenz"
  },
  {
    "caption": "(a) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body (MVB) and lysosomal markers are indicated.",
    "molecules": "CQ, Histodenz"
  },
  {
    "caption": "(c) Western blot analysis of GEMIN4 or control (TUBA) in HeLa cells cultured in DMEM 10% FBS (Serum), Hank's buffered salt solution (Starve) for 2 h, or treated for 16 h with control (DMSO), BAF (200 nM), RAP (20 nM), RAP+BAF, or PP242 (100 nM).",
    "molecules": "BAF, DMSO, RAP, PP242"
  },
  {
    "caption": "(i) Western blot analysis with anti-UB monoclonal antibody (mono- and polyubiquitylated proteins, FK2 clone) in Flag immunoprecipitates performed under stringent denaturing conditions. Flag-AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet-inducible Flag-AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(k) Top, RT-qPCR analysis of miR-16 or let-7a levels in AGO2 immunoprecipitates from DMSO- and BAF-treated HeLa cells. Error bars represent s.e.m. Bottom, a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments. Uncropped images of blots are shown in Supplementary Fig. S3.",
    "molecules": "BAF, DMSO"
  },
  {
    "caption": "(e) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let-7a-let-7a* labelled on the 3′ end of either let-7 or let-7a* with FITC. Cells were treated 16 h before with DMSO (control), BAF (200 nm) or geldanamycin (10 μM). Representative results of one experiment are shown.",
    "molecules": "BAF, DMSO, geldanamycin"
  },
  {
    "caption": "(B) A fragment of genomic DNA (2.2 kb) was amplified by  PCR and in vitro sgRNA-guided  Cas9-mediated cleavage assay was performed with each of the sgRNAs. sgRNA-targeting sites are indicated by arrows, genomic DNA  size is indicated by asterisk.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) CD45.2 frequency among GCs induced by   eOD-GT8 60 mer at different  time points. Adoptively transferred  mice with 1 in 104 of precursor frequency were immunized with eOD-GT8 60mer. On days 8, 15 and 36, the  splenic B cells were isolated to measure the CD45.2  cell frequency in GC B cells by  FACS using the markers mentioned above by  FACS. Gated plots represent the CD45.2 frequency among GC B cells. (B) Quantification of CD45.2  frequency among  GCs at three time points. X-axis represents adoptively transferred mice  groups with different precursor frequency (1 in 104, 2 in 105, 3 in 106) at three  time point, y axis represents CD45.2 frequency among GC. Each circle represents one mouse. For CLK21 and  CLK09  panel, n=5 mice in each group. For  CLK19 panel, n=3 mice in each group. Bars indicate mean ± SD from mice in each group.",
    "molecules": "eOD-GT8 60 mer, eOD-GT8 60mer"
  },
  {
    "caption": "(C) IgG titers detected by ELISA.  eOD-GT8  or eOD-GT8 KO soluble proteins were used to coat ELISA plates. Sera from adoptively transferred mice at different  time points  were used to detect by  ELISA. Bars indicate geometric mean and geometric SD from mice in each group. For  CLK21 and  CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. X-axis represents precursor frequency, and Y-axis represents the change of area under curve (AUC coated eOD-GT8   -AUC coated eOD-GT8  KO). Each circle represents one mouse.",
    "molecules": "eOD-GT8"
  },
  {
    "caption": "C Maximal cytochrome c  release for all  DX/REL pairs with ≥3 biological replicates); box-whisker plots summarize data (box 25%-75%; belt=median; dot=mean; whiskers=minimum and maximum values). Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend p<0.10).  Cyto c, cytochrome c",
    "molecules": "Cyto c, cytochrome c"
  },
  {
    "caption": "A In vitro  viability  curves after 72hr exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated   cytochrome c  release (CHLA15/CHLA20 and SKNBE1/SKNBE2C) and one without (CHLA122/CHLA136). Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates dotted-line represents 50% viability.",
    "molecules": "cytochrome c, doxorubicin, mafosfamide"
  },
  {
    "caption": "F Cytochrome c  release from  mitochondria after exposure to  tBid  or BimBH3  peptide for parental  NB1643  and SY5Y  cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643-ALKR and SY5Y-ALKR). Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.",
    "molecules": "BimBH3, crizotinib, Cytochrome c"
  },
  {
    "caption": "H In vitro viability of CHLA15-ctrl, CHLA15-shMFN2, CHLA15-shPACS2, and CHLA20 cells following 72hr exposure to ABT-737; dotted-line represents 50% viability. Data information: data points are mean and SD from triplicate wells, experiments are representative of at least two biological replicates.",
    "molecules": "ABT-737"
  },
  {
    "caption": "I Mitochondrial cytochrome c  release in response to tBid  and BimBH3 peptide in CHLA15-Ctrl, CHLA15-shMFN2 and  CHLA15-shPACS2 cells. Data information: , data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates",
    "molecules": "BimBH3, cytochrome c"
  },
  {
    "caption": "A, B Cytosolic  (black-tracing) and mitochondrial (colored-tracing) Ca2+ concentration measured using fluorescent reporters in the  CHLA15 (n=29 cells)/CHLA20 (n=65 cells) and SKNBE1 (n=37 cells)/SKNBE2C (n=52 cells) pairs. ER Ca2+ release was induced by 100μM carbachol, an  IP3R-agonist;  time  added indicated by arrow; tracings are mean +/- SD error bars. Coupling time (time  between achieving 50% of maximal cytosolic and mitochondrial  concentrations) is an index of  ER-mitochondrial proximity and transfer efficiency; plotted below. Data information: data is derived from at least 3 separate cell transfections (biological replicates). statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05.",
    "molecules": "Ca2+, carbachol"
  },
  {
    "caption": "F Cytochrome c  release in response to  tBid and BimBH3 assessed. Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.",
    "molecules": "BimBH3, Cytochrome c"
  },
  {
    "caption": "A, B, C Concentration of ceramide  species as measured by LC/MS from DX and REL pair whole cell pellets. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each",
    "molecules": "ceramide"
  },
  {
    "caption": "D, E Cumulative ceramides  and sphingomyelins  (of C32-C36 chain length) for DX/REL pairs. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each error bars are +/- SD.",
    "molecules": "ceramides, sphingomyelins"
  },
  {
    "caption": "H Summary data for CHLA15/CHLA20  and  SKNBE1/SKNBE2C  showing relative  cytochrome c release when pre-incubated with  GW4869 to inhibit ceramide  generation, compared with untreated cells. Data information: All data points from two (CHLA15) or three (SKNBE1) biological replicates are depicted in H. Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend, p<0.10).",
    "molecules": "ceramide, cytochrome c, GW4869"
  },
  {
    "caption": "(A) Anti-acetyllysine Western blot analyses and total protein stains of E. coli cell extracts. Cellular protein extracts before and after induction of His6-MBP-GNAT expression were separated on 12 % acrylamide gels and immunoblotted by using an anti-acetyllysine antibody or stained with Coomassie Dye. GNAT1, 2, 5, 6, 7 and 10 as well as His6-MBP control were expressed in E. coli BL21(DE3)pLysS. GNAT3 and 4 were expressed in Rosetta(DE3). In the total protein stains, recombinant GNAT protein constructs were highlighted by blue arrowheads. Protein expression before (ni) and after (i) induction with IPTG is indicated. The luminescence signal, indicating acetylated proteins, was usually recorded after 40-120 seconds. Since MBP-GNAT4 expression resulted in a saturated signal, the luminescence was additionally recorded for 10 seconds (indicated by an asterisk).",
    "molecules": "His6, Coomassie Dye, IPTG, acetyllysine"
  },
  {
    "caption": "(C) Comparison of NTA yield of retrieved N-termini of proteins starting at position 1 or 2. The majority of these proteins, corresponding mostly to cytosolic components, undergoing or not to N-terminal methionine excision, were not affected by inactivation of GNAT2. For statistical analyses nsi-1 and nsi-2 were pooled and compared to the wild type. Two independent technical replicates of four biological replicates for each of the WT, nsi-1 and nsi-2 samples were analysed. Error bars are ± SD. (D) Comparison of NTA yield of retrieved N-termini of proteins starting at positions > 2. Clear alteration of NTA yield was observed in nsi mutant lines in the pool of nuclear-encoded plastid proteins. nsi-1 and nsi-2 samples were treated as in D. Error bars are ± SD (E) Comparison of NTA yields of retrieved N-termini in plastid proteins. Similar variation as in panel D was observed when NTA of only plastid proteins was analyzed.",
    "molecules": "methionine"
  },
  {
    "caption": "P. falciparum symptomatic  (n=30) and asymptomatic  (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. A-F. Clinical parameters determined in the study include age  (A), gender (B), parasitemia  (C), hemoglobin  (g/dL blood) (D), hematocrit   (E), and platelet  count (F). Boxes represent the 25th to 75th percentiles, whiskers show the range (minimum to maximum), and lines represent the median. Significance was determined by the Kruskal-Wallis test with Dunn's multiple comparisons (A, D-F), the Chi-square test (B), and the Mann-Whitney test (C) using 30 (symptomatic), 40 (asymptomatic) and 31 (healthy immune controls) biological replicates. ****p<0.0001.",
    "molecules": "hemoglobin"
  },
  {
    "caption": "A,E Mean ABR thresholds (± SD) for clicks and tone pips are plotted for wt (green), het (blue) and hom (red) mice at ages A, P14 (wt, n=3; het, n=8; hom, n=6); B, 4 weeks (wt, n=38; het, n=26; hom, n=37); C, 14 weeks (wt, n=10; hom, n=14); D, 28 weeks (wt, n=15; het, n=5; hom, n=25); E, 44 weeks (wt, n=9; het, n=2; hom, n=11). Grey symbols and lines indicate thresholds of individual mutants. In B, open symbols represent thresholds under urethane anaesthesia (see methods), showing no difference compared with ketamine/xylazine used for all other thresholds.F, Mean thresholds for mutants aged 2 weeks (yellow), 4 weeks (purple), 14 weeks (cyan), 28 weeks (grey) and 44 weeks (black).",
    "molecules": "ketamine, xylazine, urethane"
  },
  {
    "caption": "(A-C) Telophase GSCs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 show non-overlapping patterns (A). Telophase GSCs expressing old (green) and new (red) histone H4 show moderate levels of overlap (B). GSCs expressing old (green) and new (red) histone H2A show high levels of overlap (C). (D) A control where the old and new fluorescent tags have been switched shows the non-overlapping patterns of old (red) versus new (green) H3Rev histone in mitotic telophase GSCs marked by H3S10p (gray). (E) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev GSCs, H2A GSCs, and H4 GSCs. Data gathered over 16 separate experiments for H3 GSCs. Values are mean + 95% CI. (F-H) Telophase CBs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 also show non-overlapping patterns. (F) Telophase CBs expressing old (green) and new (red) histone H4 (G) and H2A (H), which depict more overlap like GSCs. (I) A control with switched old and new fluorescent tags shows the non-overlapping old old (red) versus new (green) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p (gray).",
    "molecules": "histone"
  },
  {
    "caption": "C. Representative ATPase activity assay (ATP  regenerating system). At the indicated time, 200 nM  ConA is added to the assay.",
    "molecules": "ATP, ConA"
  },
  {
    "caption": "C. Growth phenotypes of wild type and c″-mutant  yeast  strains probed on (1) rich media buffered to pH 5 (YPD pH 5); (2) synthetic dropout (SD) plus all amino acids (SD + all a.a.); (3) SD minus uracil  (SD -  ura); (4,5)  YPD buffered to pH 7 plus 4 mM Zn2+ or 60 mM Ca2+, respectively; (6) YPD buffered to  pH 7; (7) YPD plus 60 mM Ca2+.",
    "molecules": "a.a, amino acids, Ca2+, ura, uracil, Zn2+"
  },
  {
    "caption": "F. Oxr1  prevents binding  of V1Hchim to VoND as probed by  BLI. Streptavidin coated BLI sensors were loaded with Vo in lipid  nanodiscs containing biotinylated MSP, and sensors were then dipped into wells containing recombinant C and V1Hchim in absence and presence of  Oxr1. Only the V1Hchim association  and  dissociation phase from one representative of two experiments using two different preparations is shown.",
    "molecules": "lipid  nanodiscs, Streptavidin"
  },
  {
    "caption": "G.  Oxr1  mediated  dissociation of holo  V-ATPase as analyzed by  negative stain electron microscopy. V1HchimVoND was incubated without and with a 3-fold molar ratio of Oxr1  for 16 h. Samples were then spotted on carbon coated copper grids, stained  with 1% uranyl acetate and observed by  transmission electron microscopy. Number of assembled V1Vo and disassembled V1 and Vo particles were counted on a total of 20 micrographs each. The horizontal dotted lines indicate the mean. A few holoenzyme and free V1 and VoND particles are highlighted by white circles and boxes, respectively. Bar = 50 nm. The data from one representative of two experiments using two different preparations are shown.",
    "molecules": "uranyl acetate, carbon, copper"
  },
  {
    "caption": "Representative density plots of Ca2+ flux from conjugated OptoCAR‑T cells over time at 37ºC. Conjugated cells were stimulated with dimerizer addition for 0 (F), 5 (G), 10 (H), 15 (I) or 30 (J) minutes prior to disrupting signaling by illuminating cells.",
    "molecules": "Ca2+, dimerizer"
  },
  {
    "caption": "Representative fluorescent Western blot showing the dynamics of FOS phosphorylation (p-FOSS32) on light-modulated control. Conjugated OptoCAR‑T cells were stimulated for a defined period after dimerizer addition (denoted above blots) before either being left in the dark (dark line) or illuminated (blue line). Full blot image with total protein normalization control shown in Figure EV3A. Representative fluorescent Western blot showing the dynamics of FOS expression (FOSTotal) on light-modulated control, from the same dataset as in (A). Full blot image with total protein normalization control shown in Figure EV3B.",
    "molecules": "dimerizer"
  },
  {
    "caption": "C Volcano plots derived from LC-MS/MS analyses of ALFA-tagged CNX, immunoprecipitated in 1% NP-40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector (EV) control co-IPs. Depicted is an enlarged section of the associated complete volcano plot in the upper right. Among the significantly enriched proteins, annotations are Uniprot gene names. CNX is shown in green. ER proteins with the GO-term annotation \"endoplasmic reticulum\", which are localized to the ER lumen, are highlighted in blue and among those several known CNX interaction partners are indicated with their names. Membrane proteins are shown in red and those which are non-glycosylated are additionally labeled with their gene names.",
    "molecules": "DSSO, NP-40"
  },
  {
    "caption": "F Immunofluorescence images of Cx32 and its mutants. COS-7 cells were transiently transfected with the indicated FLAG-tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). All three channels are overlayed. Images are representative of cells from at least three different biological replicates. GJ denotes gap junctions observed at cell-cell junctions of cells transfected with WT Cx32.",
    "molecules": "DAPI"
  },
  {
    "caption": "C Interaction analysis of Calnexin with the first transmembrane domain of Cx32. Intracellular BMH crosslinking of transiently transfected V5-tagged CNX with the transiently transfected R26C mutant of Cx32-TMD1. In the AA mutant of CNX, the two cysteines within the CNX TMD were replaced by alanines. The species at approx. 250 kDa are possibly CNX dimers.",
    "molecules": "BMH"
  },
  {
    "caption": "E COS-7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of minCNXTMD and both control constructs was carried out by mScarlet-I fluorescence (red). Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "C COS-7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry (red) as an ER marker. ConMem, detected by sfGFP fluorescence (green), localizes to the plasma membrane. Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Analysis of Rhodopsin degradation and its dependency on CNX. CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints. CNX WT and CNX YTL immunoblots are from the same blot (dashed line), showing similar expression levels. Hsc70 was used as a loading control (also from the same blot, dashed line). Quantifications are shown below representative immunoblots",
    "molecules": "cycloheximide"
  },
  {
    "caption": "Representative images of hematoxylin and eosin-stained testicular sections from the patient (V:3) and a man diagnosed with obstructive azoospermia, serving as the control. The magnified view of the boxed area is shown in the lower left corner of the image from the patient. The blue arrow indicates unaligned chromosomes in the representative metaphase cells. Scale bars, 50 μm.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Immunofluorescence staining of testicular sections from control and patient V:3 with antibodies against H3S10p (red), a marker of metaphase cells. The nuclei were stained with Hoechst 33342 (blue). The magnified view of the red box is shown in the lower left corner of the image from the patient.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "Meiotic metaphase I (MMI) spermatocytes stained with Giemsa. The arrows indicate univalent chromosomes.",
    "molecules": "Giemsa"
  },
  {
    "caption": "Representative haemotoxylin-stained ovarian sections of 5-dpp-old control and M1apKI/KI mice. Follicle counts and total oocyte counts per ovary at 5 dpp.",
    "molecules": "haemotoxylin"
  },
  {
    "caption": "B. Western blot analysis of the symmetric dimethylarginine (sDMA), monomethylarginine (mMA) and asymmetric dimethylarginine (aDMA) levels in MTAP-overexpressing and MTAP-knockout cells. V5-MTAP wild-type and D220A mutant were restored in H1650 MTAP-knockout cells. Data shown are representative of three independent experiments.",
    "molecules": "dimethylarginine, DMA, mMA, monomethylarginine"
  },
  {
    "caption": "C. In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti-sDMA antibody. Arrow marks dimethyl-vimentin.",
    "molecules": "DMA, dimethyl"
  },
  {
    "caption": "D. Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells. Arrow marks dimethyl-vimentin. Data shown are representative of three independent experiments.",
    "molecules": "dimethyl"
  },
  {
    "caption": "F. Validation of the PRMT5-catalyzed methylation sites on V5-vimentin. HEK293T cells were transfected with indicated constructs and subjected to immunoprecipitation analysis. The intensity of sDMA signals was quantified by ImageJ software, normalized to the intensity of corresponding immunoprecipitated V5-vimentin, then relative to the V5-vimentin wild-type group and represented as percentage. Data were the averaged percentage from three independent experiments.",
    "molecules": "DMA"
  },
  {
    "caption": "D. Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.",
    "molecules": "MTA"
  },
  {
    "caption": "A. Measurement of V5-vimentin wild-type, 4RK and 4RF mutant protein levels in CL1-5 vimentin-knockout cells treated with 10 μM MG132 by Western blots. Data shown are representative of three independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "B. Western blot analysis of vimentin protein levels in control and MTAP-knockout cells treated with 10 μM MG132. Data shown are representative of three independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "F-G. Western blot analysis of V5-vimentin wild-type, 4RK and 4RF mutant levels in CL1-0 cells (F) or endogenous vimentin levels in control and MTAP-knockout cells (G) treated with 50 μg/ml cycloheximide (CHX). Bottom: the intensity of V5-vimentin and vimentin signals was quantified by ImageJ software, normalized to the internal control β-actin, then normalized to zero time point, and plotted against time points (mean ± SE, n=3, biological replicates).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(A - D) Color fundus photographs and SW-AF examinations on four patients of β-thalassemia subject to chelation therapy by DFO for at least 16 years. Fundus phenotypes, including RPE mottling and depigmentation in the macula (Case Ⅰ) (Gelman et al, 2014), choroidal sclerosis in the perimacular areas (Case Ⅱ), peripapillary (Case Ⅲ and Ⅳ), and subretinal pigmentation (Case Ⅳ) were evaluated. RPE lesions including concentric distribution of stippled hyper-autofluorescence at the macula (Case Ⅰ), large areas of hypo-autofluorescent regions at parapapillary or perimacular area (Case Ⅱ and III), and extensive RPE loss in both eyes (Case Ⅳ) were observed by SW-AF.",
    "molecules": "DFO"
  },
  {
    "caption": "(A - C) Adult C57BL/6J mice (four months old) were intraperitoneally injected with DFO at 100 mg/kg three times a week for three months in a row. SW-AF images were acquired longitudinally at one, two and three months post injection.",
    "molecules": "DFO"
  },
  {
    "caption": "(I, J) Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death. Hoechst was used for nuclear staining. Untreated mice of similar age were used as the healthy controls. Flat mount images were captured by 10× confocal microscopy and stitched together.",
    "molecules": "Hoechst, DFO"
  },
  {
    "caption": "(K - N) The RPE was dissected from the mice injected with DFO for three and nine months respectively and stained with phalloidin. Age-matched untreated mice were included as the controls. The images were taken at 40× magnification.",
    "molecules": "DFO, phalloidin"
  },
  {
    "caption": "(I - L) Cell death among the iRPE population was examined by TUNEL assay 24 hours and 48 hours post treatment. Untreated iRPE cells were included as the control. The cells were counterstained with Hoechst.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) iRPE cells treated with DFO were collected and lysed to measure the expression of HIF1α and HIF2α 24 and 48 hours after the treatment. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the control. Actin was used as the loading control. HIF1α (post-translationally modified) was predicted to be ~100 kDa; HIF2α (post translationally modified) was predicted to be ~ 120 kDa. (B) iRPE cells treated with DFO were lysed and fractionated by centrifugation to measure the expression of HIF1α and HIF2α in the nuclei 24 and 48 hours after the treatment. Untreated iRPE cells were used as the normal control. Histone H3 was used as the loading control. (",
    "molecules": "DFO, DMSO"
  },
  {
    "caption": "(A) Ferrous iron was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05.",
    "molecules": "Ferrous iron"
  },
  {
    "caption": "(B) The transcripts of ferroptosis-associated markers were determined by qPCR using iRPE cells as the template. The RNA extract from untreated iRPE was included as the control. The level of each transcript was normalized to ACTB. The statistics are analyzed by ratio paired student's t-test for each target gene. The results are presented as mean ± S.E.M., n = 3 iRPE lines for each group. * p < 0.05; ** p < 0.01. Round dots: untreated iRPE; square dots: DFO-treated iRPE.",
    "molecules": "DFO"
  },
  {
    "caption": "(C) The level of ROS was measured. The statistics are analyzed by one-way ANOVA with the Tukey test. The results are presented as mean ± S.E.M., n = 6 iRPE lines for each group. * p < 0.05, ** p < 0.01. DHE: Dihydroethidium.",
    "molecules": "DHE, Dihydroethidium, ROS"
  },
  {
    "caption": "(D) Mitochondrial respiration of the iRPE cells with DFO treatment was determined by seahorse extracellular flux assay. OCR was determined over a course of time. Each data point is shown as mean ± S.E.M., n = 4 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.",
    "molecules": "AA, antimycin A, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, FCCP, DFO, Rot, rotenone"
  },
  {
    "caption": "(A - C) Light microscopy of morphological protection of AKG against DFO toxicity on iRPE. The cells were treated with DFO at 100 mM for 48 hours (B). AKG was supplemented at 10 mM simultaneously (C). The control group was supplemented with 0.1% DMSO (A). Scale bar: 100 µm. (D - F) The DFO-treated iRPE cells with or without AKG supplementation were subject to phalloidin staining (E and F). The iRPE cells treated with 0.1% DMSO-containing media were included as the controls (D). The images were captured at 40× magnification. Scale bar: 30 µm. (G - I) Detection of cell death in the DFO-treated iRPE cells with or without AKG supplementation by TUNEL staining. Hoechst was used for nuclear staining. The images were captured at 40× magnification. Scale bar: 30 µm. (J",
    "molecules": "Hoechst, AKG, DFO, DMSO, phalloidin"
  },
  {
    "caption": "(J - L) SW-AF was performed longitudinally on the mice treated with DFO for up to 10 months in conjunction with supplementation of AKG for seven months.",
    "molecules": "AKG, DFO"
  },
  {
    "caption": "(A) Mitochondrial respiration of the DFO-treated (100 mM) iRPE cells with AKG supplementation (10 mM) for 24 hours was determined by seahorse extracellular flux assay. OCR was measured over a course of time. Each dot is shown as mean ± S.E.M., n = 5 iRPE lines for each group. OCR: oxygen consumption rate; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot: rotenone; AA: antimycin A.",
    "molecules": "AKG, AA, antimycin A, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, FCCP, DFO, Rot, rotenone"
  },
  {
    "caption": "(G) The DFO-treated (100 mM) iRPE cells supplemented with AKG (10 mM) for 48 hours were lysed for immunoblotting against HIF2α. The iRPE cells supplemented with 0.1% DMSO in the culture media were used as the controls. HIF2α (post translationally modified) was predicted to be ~ 120 kDa. Actin was included as the loading control. (H) RPE lysate from the one-year-old mice subject to 10-month DFO treatment with or without concomitant supplementation of AKG was used for immunoblotting against HIF2α. The RPE of the age-matched untreated mice was included as the control. HIF2α (degraded) was predicted to be 40 - 80 kDa. Actin was used as the loading control.",
    "molecules": "AKG, DFO, DMSO"
  },
  {
    "caption": "(C) Lactate levels in MEF cells Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in KRASWT MEF. ***p<0.001.",
    "molecules": "Lactate"
  },
  {
    "caption": "(C) Effect of lactate on phosphorylated S6K (T389), 4E-BP1 (T37/46), ULK1 (S757) in COUP-TFII-silenced HCT116 cells.",
    "molecules": "lactate"
  },
  {
    "caption": "(E) Inhibition of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cell proliferation and its recovery by treatment with lactate. Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control. *p<0.05, **p<0.01, and ***p<0.001.",
    "molecules": "lactate"
  },
  {
    "caption": "(F) Clonogenic assay of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cells in the presence or absence of lactate.",
    "molecules": "lactate"
  },
  {
    "caption": "(A) Effect of lactate on the ERK/TSC2 and AKT/TSC2 signaling pathway in serum-starved HCT116 cells.",
    "molecules": "lactate"
  },
  {
    "caption": "(B) Effect of lactate on phosphorylated S6K (T389) inhibited by TSC2 overexpression in HCT116 cells.",
    "molecules": "lactate"
  },
  {
    "caption": "(C) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb-overexpressing HCT116 cells after serum deprivation overnight.",
    "molecules": "lactate"
  },
  {
    "caption": "(D) Molecular interaction between TSC2 and recombinant GDP-loaded Rheb in the presence or absence of lactate in vitro.",
    "molecules": "lactate"
  },
  {
    "caption": "(E) Effect of lactate on Rheb activation in serum-starved HCT116 cells.",
    "molecules": "lactate"
  },
  {
    "caption": "(F) Interaction between endogenous Rheb and mTOR in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.",
    "molecules": "lactate"
  },
  {
    "caption": "(G) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.",
    "molecules": "lactate"
  },
  {
    "caption": "(A) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP-TFII-silenced HCT116 cells.",
    "molecules": "lactate"
  },
  {
    "caption": "(B) Effect of lactate on Rheb activation in COUP-TFII-silenced HCT 116 cells.",
    "molecules": "lactate"
  },
  {
    "caption": "(D) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb- and/or COUP-TFII-overexpressing HCT116 cells treated with 25 nM trametinib for 24 h.",
    "molecules": "lactate, trametinib"
  },
  {
    "caption": "(E) Effects of lactate and COUP-TFII overexpression on Rheb activation in HCT116 cells treated with 25 nM trametinib for 24 h.",
    "molecules": "lactate, trametinib"
  },
  {
    "caption": "Effects of COUP-TFII inhibition and lactate treatment on tumor growth in vivo. (A) Growth curve in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).",
    "molecules": "lactate, sodium chloride, sodium L-lactate"
  },
  {
    "caption": "(B) tumor volume at the experimental endpoint, in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).",
    "molecules": "sodium chloride, sodium L-lactate"
  },
  {
    "caption": "Effects of COUP-TFII inhibition and lactate treatment on tumor growth in vivo. (C) Western blot analysis of the indicated proteins in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).",
    "molecules": "lactate, sodium chloride, sodium L-lactate"
  },
  {
    "caption": "(D) Effects of trametinib treatment and overexpression of COUP-TFII on tumor growth in vivo. Tumors were derived from xenografted HCT116 expressing HA-COUP-TFII or carrying a control vector and treated with trametinib (3mg/kg).",
    "molecules": "trametinib"
  },
  {
    "caption": "(E) Western blot analysis of indicated proteins in tumors derived from xenografted HCT116 treated with trametinib with or without COUP-TFII overexpression. Asterisk indicates exogenous HA-COUP-TFII.",
    "molecules": "trametinib"
  },
  {
    "caption": "(F-G) Thermal shift assay traces of human Teneurin4WT (F) and human Teneurin4Mut (G) in the presence of physiological calcium concentrations (+ Ca2+; black), low calcium concentration (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey).",
    "molecules": "Ca2+, calcium"
  },
  {
    "caption": "(A) Dimensionless Kratky plots for Teneurin4WT (left) and Teneurin4Mut (right) in physiological calcium concentrations (+ Ca2+; black), low calcium concentrations (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey). I(0) and Rg were obtained from Guinier analysis (Fig EV5A, B). Crosshairs indicate the maximum position for a fully folded globular protein.",
    "molecules": "Ca2+, calcium"
  },
  {
    "caption": "(A) Representative images of HEK293-T cells transfected with GFP (top), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP- Teneurin4Mut (GFP-Ten4Mut; bottom) for 24h and stained with Alexa 568 Phalloidin (magenta) and Dapi (blue). Images are maximum intensity projections of 14-16 stacks. Scale bar, 10 µm. (B) Zoom shows the regions indicated in A. White arrows indicate the tip of the filopodium from the transfected cell. Scale bar, 2 µm. (",
    "molecules": "Alexa 568, Dapi, Phalloidin"
  },
  {
    "caption": "B Lamin B1 distribution in human putamen was analysed by immunohistochemistry. Antibody against lamin B1 (red) was combined with DAPI-Fluoromount G (blue) to label nuclei. Representative images show the distribution of lamin B1 in the putamen of non-affected individuals (CTL) and HD patients at different stages of the disease (VS II-IV: Vonsattel grades). Yellow and white arrowheads indicate MSNs and glial cells, respectively. Scale bar 50 μm and 20 μm for low and high magnification, respectively.",
    "molecules": "DAPI"
  },
  {
    "caption": " C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry. Anti-lamin B1 antibody (red) was combined with anti-GFAP antibody (green) and nuclei were labelled with DAPI-Fluoromount G (blue). Representative images show the distribution of lamin B1 at Vonsattel grade III. Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP-positive cells. Scale bar 10 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "A Representative images showing the distribution of lamin B1. On the left, images showing maximal Z-projection. Scale bar 150 μm. Small images correspond to CA1 and DG magnified images showing independent DAPI, GFAP and Lamin B1 channels, and merge, of representative confocal Z-stack images. Scale bar 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": " A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3. B Representative images showing primary striatal neurons transfected with mApple-C1 or with mApple-LB1, both in red. Neuronal nuclei were stained with DAPI-Fluoromount G (blue). Lamin B1 is shown in white. Scale bar 10 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "B,C Graphs show the percentage of time exploring each object with respect to the total exploration time in the (B) NOLT and (C) NORT, 5 weeks after treatment (Veh, vehicle; BA, betulinic acid; WT, wild-type). ****P < 0.0001 compared to the corresponding old location/object. WT veh N = 13; R6/1 veh N = 10; R6/1+BA N = 10.",
    "molecules": "BA, betulinic acid"
  },
  {
    "caption": "D Accelerating rotarod was assessed after 7 weeks of treatment. **P < 0.01 and ***P < 0.001 compared to vehicle-treated wild-type (WT) mice; #P < 0.05 compared to vehicle-treated R6/1 mice. WT veh N = 13; R6/1 veh N = 9; R6/1+BA N = 9.",
    "molecules": "BA"
  },
  {
    "caption": "E Lamin B1 levels were analysed by Western blot in the striatum (WT veh N = 10; R6/1 veh N = 6; R6/1+BA N = 7), hippocampus (WT veh N = 12; R6/1 veh N = 8; R6/1+BA N = 7) and cortex (WT veh N = 11; R6/1 veh N = 6; R6/1+BA N = 7) after 12 weeks of treatment. *P < 0.05, **P < 0.05 compared to vehicle-treated wild-type (WT) mice and #P < 0.05 compared to vehicle-treated R6/1 mice. Representative immunoblots of lamin B1 and α-tubulin (as loading control) for each treatment group are shown.",
    "molecules": "BA"
  },
  {
    "caption": " F Lamin B1 intensity and nuclear morphology were analysed by FANSI in hippocampal CA1 neurons nuclei from wild-type (WT) and R6/1 mice after 12 weeks of treatment. *P < 0.05 compared to vehicle-treated WT mice. *P < 0.05 as compared to vehicle-treated WT mice. Lamin B1 intensity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 4; Lamin B1 circularity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 5. ",
    "molecules": "BA"
  },
  {
    "caption": " G Nuclear permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 weeks of treatment. *p < 0.05 compared to vehicle-treated wild-type (WT) mice. WT veh N = 4; R6/1 veh N = 5; R6/1+BA N = 6. ",
    "molecules": "BA"
  },
  {
    "caption": "(A) Confocal microscopy image analysis of sEV fusion to MGC-803 cells. The MGC-803 (left side) and MKN-45 (right side) cells were treated with sEVs derived from MGC-803 cells (Con sEVs) or LSD1 knockout (KO) MGC-803 cells (KO sEVs) and stained with PKH26 for 12 h. Additionally, the cell membrane was stained with Dio, while the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 100 µm.",
    "molecules": "Dio, PKH26, 4',6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "(B) Immunofluorescence confocal microscopy analysis of LSD1 (green) in LSD1 KO MGC-803 cells (left panel) and LSD1 KO MKN-45 cells (right panel) incubated with 20 μg/mL Con sEVs and KO sEVs for 12 h. The cell membrane was stained with PKH26, while the nuclei were stained with DAPI. Scale bar = 100 µm.",
    "molecules": "PKH26, DAPI"
  },
  {
    "caption": "(N) Immunoprecipitation of Kme1/2 on SOX2 (WT indicates HEK293T cells co-transfected with LSD1-WT and SOX2-WT; Mut indicates HEK293T cells co-transfected with LSD1-WT and SOX2-Mut; WT, wild type; Mut, mutant).",
    "molecules": "Kme1"
  },
  {
    "caption": "Proliferation assay results of MGC-803 (A), MKN-45 (B), NCI-N87 (C) cells treated with oxaliplatin along with indicated treatments (n = 3 biological replicates, mean ± standard error of mean (SEM); *P = 0.0204 (MGC-803), *P = 0.0498 (MKN-45), *P = 0.0473 (NCI-N87), and *P = 0.0019 (LSD1 KO MGC-803); two-tailed unpaired Student's t-test).",
    "molecules": "oxaliplatin"
  },
  {
    "caption": "Representative images of tumors (E) and the tumor weight (F) of mice treated with oxaliplatin in the presence or absence of sEVs (n = 3 biological replicates; mean ± SEM; *P = 0.0488; two-tailed unpaired Student's t-test).",
    "molecules": "oxaliplatin"
  },
  {
    "caption": "(B) Electrophoretic mobility shift assay (EMSA) with increasing concentration (0, 15nM, 30nM, 60nM, 1.25μM and 2.5μM) of GST-FOXD3 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) from Sox15 promoter (positive control), MSR, MERVL-LTR, MERVL-int, L1MdA and Hprt promoter (negative control). The experiment was repeated 3 times. (D) ChIP-qPCR enrichment of FOXD3 in mESCs using primers specific for MSR, MERVL-LTR, MERVL-int (including and excluding the FOXD3 cognate motif), L1MdA promoter sequences. Sox15 promoter and Actb promoter primers are used as positive and negative control respectively. Data is represented as percentage of input and the average of 3 biological replicates is plotted. Error bar indicates standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to no antibody control levels (*: p< 0.05, paired t-test).",
    "molecules": "5'-Cy5"
  },
  {
    "caption": "(C) Immunofluorescence analysis of the MERVL-gag protein in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting immunofluorescence signal is indicated. Scale bar represents 10 μm, n= 3 biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting the immunofluorescence signal is indicated. Scale bar represents 10 μm, n=3 biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Electrophoretic mobility shift assay (EMSA) with increasing concentrations (0, 30nM, 60nM, 1.25μM, 2.5μM and 5μM) of recombinant GST-FOXD3, DNA binding mutants GST-FOXD3 M1 and GST- FOXD3 M2 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) representing MERVL and MSR. The experiment was repeated 3 times.",
    "molecules": "5'-Cy5"
  },
  {
    "caption": "(B) RT-qPCR analysis of MERVL, MSR and L1MdA in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(D) RT-qPCR analysis of, 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(G) Immunofluorescence analysis of control and Foxd3i GFP-SUV39H1 expressing Suv39h dn mESCs stained with DAPI, FOXD3 and H3K9me3. The percentage of counted cells (~150 cells per experiment) exhibiting fluorescence signal is indicated. Scale bar=2μm. n= 3 biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": " A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+ lymphocytes respectively. B Mean absolute numbers of LAG-3+CD49b+Foxp3-or CD25+Foxp3+ CD4 T cells in LNs as described in (A). Bars are mean + SEM (n=3, 4 or 5) *p<0.05 by one-way ANOVA with Tukey post hoc test.",
    "molecules": "DMSO, KLH, PBS, RA"
  },
  {
    "caption": "D IL-10, IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + RA, KLH + IL-2, KLH + RA + IL-2 or KLH + LP-GMP (LP1569 + c-di-GMP) and cultured for 5 days with increasing concentrations of KLH. Bars are mean + SEM (n=4 or 5). **p<0.01, ***p<0.001, KLH + IL-2 + RA vs KLH, ##p<0.01, ###p <0.001 KLH + IL-2 + RA vs KLH + RA, †††p<0.001 KLH + IL-2 + RA vs KLH + IL-2 by two-way ANOVA and Tukey post hoc test.",
    "molecules": "DMSO, GMP, KLH, PBS, RA"
  },
  {
    "caption": " A Flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), MOG + DMSO (MOG), MOG + RA + IL-2. Cells were gated on live single CD3+ lymphocytes. FMO: Fluorescence Minus One. B Mean percentages of IL-10+, LAG-3+, PD1+ or Foxp3+ CD25+ CD4 T cells as described in A. Cells were gated on live single CD3+ lymphocytes (top left panel) or on live single CD3+ CD4+ lymphocytes. Bars are mean + SEM (n=5). *p <0.05, ***p <0.01 by one-way ANOVA and Tukey post hoc test. ",
    "molecules": "MOG, DMSO, PBS, RA"
  },
  {
    "caption": " C EAE clinical scores in mice immunized s.c. with MOG + RA, MOG + IL-2, MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=12, except for MOG + IL-2, where n=6; combined from two experiments). *p<0.05, **p<0.01, ***p <0.001, MOG + IL-2 + RA vs Veh, #p<0.05, ##p<0.01, ###p<0.001, MOG + IL-2 + RA vs MOG + RA, †p<0.05, ††p<0.01, †††p <0.001, MOG + IL-2 + RA vs MOG + IL-2 by two-way ANOVA and Tukey post hoc test. ",
    "molecules": "MOG, DMSO, PBS, RA"
  },
  {
    "caption": " D Representative FACS plots of MOG dextramer+ CD4 T cells in LNs of naïve mice or 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+. ",
    "molecules": "MOG"
  },
  {
    "caption": " E Mean percentages of MOG dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+. Bars are mean + SEM (n=5 or 6). *p <0.05 by unpaired two-tailed t test. ",
    "molecules": "MOG"
  },
  {
    "caption": "F Mean fluorescence intensity (MFI) of CD49b, ICOS and PD-1 by MOG-dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+ dextramer+. Bars are mean + SEM (n=5 or 6). *p <0.05, **p <0.01 by unpaired two-tailed t test.",
    "molecules": "MOG"
  },
  {
    "caption": " G MOG-specific IL-17 and IFN-γ production by spleen cells from day 7 of EAE, quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG. Bars are mean + SEM (n=3 or 4). *p<0.05, **p<0.01 and ***p<0.001 by two-ANOVA and Tukey post hoc test. ",
    "molecules": "MOG"
  },
  {
    "caption": " A Representative FACS plots of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2 or vehicle (Veh) only. Cells were gated as single live CD45+ CD3+ CD4+. B Mean absolute numbers of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2. Bars are mean + SEM (n=4 or 5). *p<0.05 by unpaired two-tailed t test. ",
    "molecules": "MOG, RA"
  },
  {
    "caption": " A IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice with EAE (EAE) co-cultured for 3 days with MOG-specific Tr1 cells (from mice immunized twice with MOG, RA and IL-2 and amplified in vitro with MOG, RA and IL-2 for 8 days) or control T cells from LNs of mice injected with PBS + DMSO (Veh). Bars are mean + SD of one experiment. *p<0.05, **p<0.01 and ***p<0.001 by unpaired two-tailed t test. ",
    "molecules": "MOG, DMSO, PBS, RA"
  },
  {
    "caption": " B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG-specific Tr1 cells amplified in vitro with MOG, RA and IL-2. Results are mean + SEM (n=4 or 6). *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni post hoc test. ",
    "molecules": "MOG, RA"
  },
  {
    "caption": " G EAE clinical scores of WT and IL-10-/- mice immunized s.c. with MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=6 or 7). ***p <0.001, WT MOG + IL-2 + RA vs WT Veh, ##p<0.01, ###p<0.001, IL-10-/- MOG + IL-2 + RA vs IL-10-/- Veh by two-way ANOVA and Tukey post hoc test. ",
    "molecules": "MOG, DMSO, PBS, RA"
  },
  {
    "caption": " Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test. ",
    "molecules": "DMSO, PBS, RA"
  },
  {
    "caption": " Retinal inflammation scores in HEL-TCR transgenic mice were s.c. injected with PBS + DMSO (Veh) or HEL + RA + IL-2 on pn 18 (1x) or on pn 18 and 25 (2x). Results are mean + SEM (n=3, 4 or 5). *p<0.05 and ***p<0.001, HEL + IL-2 + RA 1x vs Veh, ##p<0.01 HEL + IL-2 + RA 2x vs Veh by two-way ANOVA with Tukey post hoc test. ",
    "molecules": "DMSO, PBS, RA"
  },
  {
    "caption": "(e) Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib-/- tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50μm",
    "molecules": "Lipid Tox"
  },
  {
    "caption": "(h) Coomassie feeding assay to asses food intake of RasV12; scrib-/- over time, three repeated measurements of an average food intake of 20 larvae.",
    "molecules": "Coomassie"
  },
  {
    "caption": "(a-e) Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild type cells in light brown and atg13 mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labelled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus) and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (a) RasV12; ctrl tumor bearing animal at day 6. (b) RasV12; scrib-/- tumor bearing animal at day 6. (c) RasV12; scrib-/- tumor bearing animal at day 8. (d) RasV12, scrib-/-, atg13-/-//atg13-/- at day 8. (e) RasV12, scrib-/-, atg13-/-//atg13-/- animal complemented with transgenic atg13, rescuing the tumor growth, at day 8.",
    "molecules": "Lipid Tox, Hoechst, Phalloidin"
  },
  {
    "caption": "(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6.",
    "molecules": "amino acids, carbohydrates, fatty acids"
  },
  {
    "caption": "(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae.",
    "molecules": "glycogen"
  },
  {
    "caption": "(b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8.",
    "molecules": "glycogen"
  },
  {
    "caption": "(g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.",
    "molecules": "carbon"
  },
  {
    "caption": "(A) Representative images and percentage of HeLa cells with more than 5 H3S10-P foci after transfection with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). Mitotic cells were excluded for the analysis by DAPI staining. More than 300 total cells were considered. Data represent mean ± SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test). Black stars denote significant increases whereas red stars denote significant decreases.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Left column: Flow cytometry profiles showing DNA content of the indicated RNase III-treated cells. G1 (red), S (blue) and G2 (green) phases were calculated from the profile. Right column: Flow cytometry histograms depicting intensity of S9.6 signals in each phase of the cell cycle for the indicated cells. (B) Quantification of panel B. The mean ± SD of the S9.6 mean intensity of five experiments is shown. ***p<0.001, **p<0.01, *p<0.05 (repeated measures ANOVA test with Bonferroni's post-test). (C) Graph represents fold change of S9.6 mean signal in G2 with respect to G1 cells. Values for five independent experiments together with the mean are shown. *p<0.05 (one-tailed paired t-test).",
    "molecules": "DNA"
  },
  {
    "caption": "(A) DNA-RNA hybrids, DNA damage and fork stalling in 9-1-1/ATR/CHK1, ATM/CHK2 and PRR deficient cells. DNA-RNA hybrids represent the mean ± SEM of all the DRIP data ***p<0.001, **p<0.01, *p<0.05 (Paired t-test). DNA damage represents the mean of medians ± SEM of all the comet tail moment data ***p<0.001, **p<0.01, *p<0.05 (paired t-test). Fork stalling represents the median ± SEM of all the fork asymmetry data ***p<0.001, **p<0.01, *p<0.05 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.",
    "molecules": "DNA, RNA"
  },
  {
    "caption": "Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in total vs. poly(A)+ RNA fractions (F) were determined by qRT-PCR.",
    "molecules": "poly(A)+ RNA"
  },
  {
    "caption": "FISH analysis of the spatial distribution of HIF-1A mRNA (red) and HIF1A-AS2 transcripts (white) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs. Nuclei were counterstained by DAPI. Scale bars, 10 μm.",
    "molecules": "CoCl2, DAPI"
  },
  {
    "caption": "Expression of HIF1A-AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides (ODN)-based knockdown. Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition, and subjected to gene expression analyses: The RNA abundance of HIF-1A and HIF1A-AS2 was analyzed by real-time PCR and normalized by 18s rRNA (A).",
    "molecules": "ODN, oligo phosphodiester nucleotides, CoCl2"
  },
  {
    "caption": "Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes under mock vs. CoCl2 treatment (C)",
    "molecules": "CoCl2"
  },
  {
    "caption": "HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment (E), The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.",
    "molecules": "CoCl2"
  },
  {
    "caption": "HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. ChIP analysis of RNA polII binding on chromatin region of the HIF1A-AS2 and HIF-1A promoters. The extent of occupancy was compared between control and ADAR1-knockdown cells. The precipitated DNA fragments were quantitatively analyzed by real-time PCR and normalized by the values of IgG.",
    "molecules": "DNA"
  },
  {
    "caption": "Chromatin binding of ADAR1 to HIF1A-AS2 and HIF-1A promoters under nomoxia (20% O2) vs. hypoxia (1% O2, 5 hrs) conditions, as assessed by ChIP.",
    "molecules": "O2"
  },
  {
    "caption": "Transcriptome-wide changes in the cells subjected to CoCl2 treatment, HIF1A-AS2 or ADAR1 knockdown were profiled by RNA-Sequencing. For genes that undergo up-regulation upon CoCl2 treatment (n = 505), overall expression profiles of the different cell groups are represented by the heatmap. The expression values (represented by normalized read counts) were displayed in shades of red or blue (linear scale) relative to the means of all corresponding values within individual experimental groups. Clusters of genes (based on unsupervised hierarchical clustering) are indicated and denoted by their experimental types/conditions.",
    "molecules": "CoCl2"
  },
  {
    "caption": "GSEA comparing the gene expression profile by gene up-regulated upon CoCl2 treatment and those down-regulated in ADAR1 knockdown. The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets. The bar‐code plot below enrichment score plot denote the rank of each member in the CoCl2 up-regulated gene list.",
    "molecules": "CoCl2"
  },
  {
    "caption": "HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. The expression levels of ADAR1 and HIF-1α protein (E) were analyzed respectively by immunoblotting.",
    "molecules": "ODN, CoCl2"
  },
  {
    "caption": "HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. For cells in (E), RNA expression of heat shock protein-encoding genes (HSPA1A, HSPA1B, and HSPA6) with reported roles in angiogenesis were profiled by qRT-PCR.",
    "molecules": "ODN, CoCl2"
  },
  {
    "caption": "To inspect HIF-1α protein abundance and modification with ubiquitin, anti- HIF-1α, anti-ubiquitylation (Ub), and anti-VHL immunoprecipitation experiments were performed, coupled with immunoblotting detection of ubiquitylation and HIF-1α, as indicated. MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used. Protein signals were quantified using the ImageJ software. The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input (In), with the control (siGFP) group being represented as 1.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "A, B: HeLa  cells were transfected with empty vector (e.v., control) or Myc-tagged wild type  PRMT1-containing plasmid.  Immunofluorescence (IF) staining was performed using α-p14ARF (red, endogenous p14ARF), α-Myc (green, exogenous PRMT1) antibodies, DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin). In (A), representative IF results are shown, with asterisks indicating cells with exclusive nucleolar and arrowheads indicating cells with predominantly nucleo-/cytoplasmic p14ARF localization. The Merge displays the combination of p14ARF and PRMT1 staining. Scale bars: 15 μm. The quantification of exclusive nucleolar p14ARF was performed by cell counting (percentage of cells) and is shown for three independent experiments in (B) (mean ± SD).",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "C, D:  U2OS cells were transfected with EGFP-tagged p14ARF alone or in combination with Myc-tagged wild type PRMT1-containing plasmids. p14ARF-EGFP (green), DAPI (blue, nuclei/DNA) and PRMT1 (red, using α-Myc  antibody) were visualized by fluorescence microscopy, for which representative results are shown in (C), with the lower images displaying a magnification as indicated in the upper images by the rectangles. Scale bars: 10 μm. The subcellular distribution of  p14ARF-EGFP (exclusively nucleolar or not-exclusively nucleolar but predominantly nucleo-/cytoplasmic) was quantified in  p14ARF-positive and p14ARF/Myc-PRMT1-double positive cells by cell counting (percentage of cells) for three independent experiments in (D) (mean ± SD, ***: p-value ≤ 0.001 using Welch´s t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "HeLa cells were  transfected with the indicated siRNAs (two control/non-targeting siRNAs and three PRMT1-specific siRNAs). The cellular p14ARF distribution was determined by   immunofluorescence staining (IF) using α-p14ARF antibody. Representative IF images for the siControl_1 and siPRMT1_1 condition are shown in (G) (endogenous p14ARF in red and the merge additionally with DAPI in blue for nuclei/DNA). Scale bars: 15 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, U2OS cells were  transfected  with  EGFP-tagged wild type (wt) or mutant (RG, RF, RK) p14ARF-containing plasmids. p14ARF-EGFP (green), endogenous  NPM  as a nucleolar marker (red, using α-NPM antibody), DAPI (blue, nuclei/DNA) and Phalloidin (grey, cytoplasm/F-actin) were visualized by fluorescence microscopy, for which representative results are shown in (A). Scale bars: 10 μm.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "PRMT1-proficient (-Dox) as well as PRMT1-depleted cells (+ Dox) were further treated with the protein synthesis inhibitor cycloheximide (CHX) and harvested after 0, 0.5, 1, 2, 3, 4, and 6 hours. Cell lysates were subjected to immunoblotting using α-p14ARF and α-β-TUBULIN (loading control) antibodies and conventional ECL detection (F). For precise quantification of the p14ARF protein stability in the -/+ Dox conditions during the CHX time course, immunoblotting was performed with independent cell lysates and primary antibodies, as in (F), which were then detected using fluorescence dye-coupled secondary antibodies and the LI-COR Odyssey system. The quantitative  p14ARF signals were normalized to the corresponding quantitative β-TUBULIN signals and displayed in (G). The CHX-untreated samples (0 hour) were set to 100% for each condition.",
    "molecules": "CHX, cycloheximide, Dox"
  },
  {
    "caption": "HeLa   cells were transfected with the indicated siRNAs (two control/non-targeting siRNAs and two PRMT1-specific siRNAs) and irradiated at 150 J/cm2 UVC or not irradiated. After 24 hours, the cellular distribution of endogenous p14ARF was determined by immunofluorescence (IF) staining using α-p14ARF antibody (red, endogenous p14ARF) and in the merge additionally DAPI  (blue, nuclei/DNA). Representative  IF  results are shown for two conditions (siControl_4 and siPRMT1_1) in (A), with asterisks indicating exclusively nucleolar and arrowheads indicating not-exclusively nucleolar but additionally or predominantly nucleo-/cytoplasmic p14ARF localization. Scale bars: 15 μm. The subcellular distribution of p14ARF, as determined by IF, was quantified by cell counting (percentage of cells) for all conditions (B).",
    "molecules": "DAPI"
  },
  {
    "caption": "E: Immunofluorescence (IF) stainings of the 75  PDAC specimens were performed using α-p14ARF (red), α-CK8/18 (green, staining ductal cells in normal pancreas and neoplastic cells in PDAC) antibodies and DAPI (blue, nuclei/DNA). Representative  IF images are shown for normal human pancreatic tissue, two PDAC specimens displaying no  p14ARF expression  and three PDAC specimens displaying strong p14ARF expression  in tumor cells. Left and corresponding right images  show the same tissue section. Asterisks indicate nucleolar p14ARF localization in  neoplastic cells of PDAC. The last  image pair shows a magnification of the  image pair above (indicated by the rectangle). Scale bars: 35 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B: PaTu8988t cells were treated with 0, 1, 3, and 10 μM gemcitabine (GEM). After 48 hours, cell lysates were analyzed by  immunoblotting using the indicated antibodies. The p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (- GEM) set to 1. The percentage of PARP cleavage was also densitometrically quantified and is indicated below the  blot. C: PaTu8988t cells were treated with 3 μM gemcitabine (GEM) or left untreated (0 hour). After 0, 6, 12, 24 and 48 hours, methylation of endogenous p14ARF was analyzed by immunoblotting using α-me-p14ARF, α-p14ARF and α-GAPDH (loading control) antibodies. The methylated p14ARF and the p14ARF signals were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blots, with the untreated condition (0 hour) set to 1.",
    "molecules": "GEM, gemcitabine"
  },
  {
    "caption": "D, PaTu8988t and MiaPaCa-2 cells were treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed to 0, 1, or 3 μM gemcitabine (GEM). Subsequently, cell death was quantified by flow cytometry (in (D)) using  FITC-labeled AnnexinV and propidium iodide (PI) for four independent experiments (mean ± SD, *: p-value ≤ 0.05 using Welch´s t-test).",
    "molecules": "FITC, GEM, gemcitabine, MS023, PI, propidium iodide"
  },
  {
    "caption": "G: Apoptosis was quantified by flow cytometry (G) using FITC-labeled AnnexinV  and propidium iodide (PI) for six independent experiments (mean ± SD, **: p-value ≤ 0.005, *: p-value ≤ 0.05, ns: not significant using Welch´s t-test).",
    "molecules": "FITC, PI, propidium iodide"
  },
  {
    "caption": "A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (A) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine (CQ). Cell lysates were evaluated by immunoblotting. ",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (B) Cells were infected, for 1 and 4 hours, with K. pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01. ",
    "molecules": "gentamycin"
  },
  {
    "caption": "A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (D-E) Cells were infected, for 1-4 hours, with WT (D) or ΔpscD P. aeruginosa (E) at MOI of 10. All infections were followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: , the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01. ",
    "molecules": "gentamycin"
  },
  {
    "caption": "(B) A549 cells infected with WT or ΔpscD P. aeruginosa for 4 hours or left uninfected (NI) in the presence of lysosomal inhibitor CQ (50 nM). The cell lysates were evaluated by immunoblotting.",
    "molecules": "CQ"
  },
  {
    "caption": "(C) A549 cells stably expressing GFP-LC3 were left non-infected or infected, for 4 hours, with WT or ΔpscD P. aeruginosa, and evaluated by fluorescence microscopy. Cell nuclei were counterstained with DAPI (blue) and autophagosome were visualized as green puncta. Graph represents quantitative analysis of GFP-LC3 positive autophagosome per cell, scale bars: 10 µm. Each bar represents mean value of three independent experiments, error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. **p ≤ 0.01.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D-E) A549 cells were treated for 16 hours with rapamycin 0.8 μg/ml or DMSO, then infected with either WT (D) or ΔpscD P. aeruginosa and followed by 1 hour of gentamycin treatment (E). The circles and squares represent the individual test (three technical replicates per individual test) with DMSO and rapamycin treatment. The bars represent the means of CFU from three independent experiments, error bars represent standard deviation. The significance of differences between different drug treatment was determined using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.",
    "molecules": "DMSO, gentamycin, rapamycin"
  },
  {
    "caption": "(G) A549 cells, stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA, were infected with WT or P. aeruginosa mutants (ΔSTY, ΔTY and ΔS) at MOI of 10. All infections were followed by 1 hours of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: The triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells from three biological replicates. The bars represent the means of CFU of each group (three biological replicates each group), error bars represent standard deviation. The significance of differences between different assay was determined using two-tailed student's t test with Welch's correction, NS: not significant; **p ≤ 0.01.",
    "molecules": "gentamycin"
  },
  {
    "caption": "(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.",
    "molecules": "ADP"
  },
  {
    "caption": "(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting.",
    "molecules": "MIBG"
  },
  {
    "caption": "F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.",
    "molecules": "ADP"
  },
  {
    "caption": "(B, The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.",
    "molecules": "phosphatidylinositol, PI3P"
  },
  {
    "caption": "D) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.",
    "molecules": "phosphatidylinositol, PI3P"
  },
  {
    "caption": "(A) Amylose pulldown from  benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated   cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree  images of the gels before transfer were used as loading control",
    "molecules": "Amylose, benzonase"
  },
  {
    "caption": "(B) Immunoprecipitation  (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse  IgG was used as negative control. Immunoblot  of DDX5  and BRCA2. StainFree image  of the gels before transfer were used as loading control (cropped  image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5  antibody.",
    "molecules": "benzonase"
  },
  {
    "caption": "(C) Left: Representative  images of in situ proximity ligation assay (PLA) between BRCA2  and  DDX5  antibodies in  U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with  Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the  number of PLA  spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.",
    "molecules": "Cordy, Cordycepin, DAPI"
  },
  {
    "caption": "(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control",
    "molecules": "amylose"
  },
  {
    "caption": "(A) Left: Representative  images  of S9.6  immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2),  DDX5  (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing  DDX5. The merged  images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of  BRCA2 (siBRCA2), DDX5  (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference. The data represent at least 235 cells per condition from three independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Top: Representative  images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS  cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the  DAPI image  (Image J) our outlined in yellow. Bottom: Quantification of  PLA spots per nucleus  in each condition as indicated. At least 300 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single  PLA  spot.",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "(A) Left: Representative images of in situ PLA  between S9.6 and γH2AX antibodies in  U2OS  cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin  (Cordy) for 2h at 37°C before fixation. Single antibody controls from non-irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei  as defined by auto threshold plugin on the  DAPI image (Image J) our outlined in yellow. Right: Quantification of PLA  spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single  PLA spot.",
    "molecules": "Cordy, Cordycepin, DAPI"
  },
  {
    "caption": "(B) DRIP-qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS  DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1  (RH) pre-immunoprecipitation where indicated. The experiment was performed in both untreated cells (-OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant difference.",
    "molecules": "OHT, tamoxifen"
  },
  {
    "caption": "(B) DDX5 ChIP-qPCR signal values at  RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells (-OHT) or after tamoxifen addition (+OHT). The data represent the mean ± SEM from three independent experiments. The green line represents the background levels of DDX5 signal. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant differences between untreated cells (-OHT) and after tamoxifen addition (+OHT).",
    "molecules": "OHT, tamoxifen"
  },
  {
    "caption": "(C) Top: Representative images of immunofluorescence  of U2OS DIvA cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) and either untreated cells (-OHT) or after tamoxifen addition (+OHT), as indicated. Scale bar indicates 10 µm. Bottom: Quantification of the number of γH2AX foci per nucleus  (left) and DDX5 nuclear intensity (right). The data represent at least 800 cells per condition from three independent experiments. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference.",
    "molecules": "OHT, tamoxifen"
  },
  {
    "caption": "(A) Top: PAGE  gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 2 nM purified EGFP-MBP-BRCA2 (top gel left) or 10 nM purified  BRCA2LT3 (top gel right) or 50 nM purified BRCA2T1 (bottom gel). Bottom: Quantification of the unwinding experiments showing the percentage of free RNA  relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2  (red) or BRCA2LT3 (blue) or BRCA2T1 (pink). The data represent the mean ± SD of at least three independent experiments. Right: SDS-PAGE  showing 500 ng of purified 2XMBP-BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay.",
    "molecules": "32P"
  },
  {
    "caption": "(B) Left: Thin layer chromatography (TLC) plate showing a representative ATP hydrolysis assay  in which purified MBP-DDX5-GST (50-200 nM) was incubated with R-loop synthetic substrate and [γ32P] ATP in presence or absence of or 6 nM purified BRCA2LT3. Right: Quantification of the ATP hydrolysed in each condition. No protein control was used as background. The data represent the mean ± SD from three independent experiments.",
    "molecules": "[γ32P] ATP, ATP"
  },
  {
    "caption": "(B) Left: Representative  images  of S9.6  immunofluorescence  of DLD1 cells bearing BRCA2 (WT) or BRCA2-T207A (T207A). The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of the average nuclear intensity of S9.6 antibody in  DLD1 BRCA2-WT (WT) or BRCA2-T207A (T207A) cells transfected with either a plasmid expressing GFP alone (-RH) or GFP-RNase H1 (+RH), as indicated. The red line in the plot indicates the median and each symbol represents the value of a single cell. Data correspond to at least 170 cells per condition from two independent experiments. The statistical significance of the difference was calculated with Mann-Whitney U-test; p-values show the significant difference.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Top: Representative images of in situ PLA performed between S9.6 (DNA-RNA hybrids) and anti-γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2  (WT) or BRCA2-T207A (T207A). When indicated, cells were transfected/treated with RNase H1 (RH) or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls in non-irradiated BRCA2  WT cells are shown. Scale bar indicates 10 µm. Nuclei  as defined by auto threshold plugin on the  DAPI image (Image J) our outlined in yellow. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA  spot.",
    "molecules": "Cordy, Cordycepin, DAPI"
  },
  {
    "caption": "(E) Top: PAGE gel showing a representative  unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with  32P-labelled synthetic R-loop substrate in presence or absence of 50 nM purified BRCA2T1-T207A (bottom right). Bottom left: Quantification of the unwinding experiments showing the percentage of free RNA relative to the  R-loop substrate (unwound product) as a function of DDX5  concentration alone (black) or in presence of BRCA2T1-T207A (green). The data represent the mean ± SD of at least three independent experiments. Bottom right: SDS-PAGE showing 650 ng of purified 2XMBP-BRCA2T1-T207A used in the unwinding assay.",
    "molecules": "32P"
  },
  {
    "caption": "(B) Representative immunohistochemical staining for calreticulin in tumor masses of control (PBS) and BoxA-treated mice. Scale bar 50 μm. The distribution of  calreticulin positive pixels describes the location of calreticulin (intracellular vs ecto-calreticulin). Statistics: Mann-Whitney test, n=4.",
    "molecules": "BoxA, PBS"
  },
  {
    "caption": "(C) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours. Statistics: One-way Anova with Dunnett's post-test; n=3. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.",
    "molecules": "BoxA, MTX"
  },
  {
    "caption": "(D) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours; β-actin is shown as loading control. Statistics: One-way Anova with Dunnett's post-test; n=2. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.",
    "molecules": "BoxA, MTX"
  },
  {
    "caption": "(G) Confocal immunofluorescence microscopy  of cleaved caspase 3  (red) in MM cells  treated for 24 hours with MTX (1 μM) or increasing concentrations of BoxA. Nuclei  were stained with Hoechst (blue) and cytosol with phalloidin (green). Scale bar 50 μm.",
    "molecules": "BoxA, Hoechst, MTX, phalloidin"
  },
  {
    "caption": "(I) Kaplan-Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H (Log rank Gehan Breslow Wilcoxon test, p=0.59).",
    "molecules": "BoxA, PBS"
  },
  {
    "caption": "(E) Representative immunofluorescence staining  of CD47 (red) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours. Nuclei are  counterstained with DAPI. Scale bar 50 μm.",
    "molecules": "BoxA, DAPI"
  },
  {
    "caption": "(A) Ecto-calreticulin exposure (expressed as MFI) in CT26 cells treated with 800 nM BoxA  or 1 μM MTX  for 2 and 4 hours. Error bars indicate standard deviation. Data were pooled from two different experiments. Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.",
    "molecules": "BoxA, MTX"
  },
  {
    "caption": "(A) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4,  CD47 and RAGE, CD47 and TLR4. Representative images from one experiment out of 3 performed are shown. MM cells were incubated overnight with 400 nM BoxA or  PBS.  Nuclei are in blue (DAPI), phalloidin is in green. Red dots represent physical contact of CD47  with CXCR4 or RAGE or TLR4. Scale bar, 20 μm. The intensity of red signal in  PLA was quantified as described in Materials and Methods in individual cells (n as indicated). Insets are enlargements of the y axis. Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.",
    "molecules": "BoxA, DAPI, phalloidin, PBS"
  },
  {
    "caption": "(C) Surface CD47  on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12. The experiment shown is representative of 2 performed, in biological triplicate. Data information: bars and error bars represent mean ± SD; statistics: one-way Anova plus Dunnett's post-test. In all panels, *p<0.05, **p<0.01, ****p<0.0001.",
    "molecules": "CXCL12"
  },
  {
    "caption": "(A) STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid, Flag-control (ctrl = Sirt3) plasmid or without DNA. 24h later, cells were incubated with Okadaic Acid (OA), (final concentration = 1µM) for 1 hour or with DMSO. Flag fusions were pulled-down and co-immunoprecipited proteins were analyzed by western blotting (WB). Upper panel displays whole cell lysates (WCL) and lower panel represents immunoprecipitated proteins.",
    "molecules": "DMSO, OA, Okadaic Acid"
  },
  {
    "caption": "STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or DMSO for 4 hours. Cells were then fixed and stained for myc-tag. Representative images are shown and scale bars are 40 µm.",
    "molecules": "DMSO, KPT-185, KPT-330"
  },
  {
    "caption": "XPO1 activity is required for nutrient starvation-induced autophagy. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe [26] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 (final concentration = 1 µM) or DMSO for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ns, not significant, Mann-Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process",
    "molecules": "DMSO, KPT-185, KPT-330"
  },
  {
    "caption": "(A-B) STK38 is required for XPO1_S1055 phosphorylation. (A) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag-XPO1(wt) transient transfection the following day. 48 hours later, cells were incubated with Okadaic Acid (OA, final concentration = 1 µM) for 1 hour or with DMSO. Immunoblotting was performed on whole cell lysates with indicated antibodies. (B) Graphical representation of the phospho S1055-XPO1 signal on total XPO1 (n = 3 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test). As expected, XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced",
    "molecules": "DMSO, OA, Okadaic Acid, Ser"
  },
  {
    "caption": "(C) STK38 kinase activity is required for XPO1_S1055 phosphorylation. HekRasV12 cells silenced (or not) for endogenous STK38 were transfected with Flag-XPO1(wt) expression plasmid in addition to myc-STK38(wt) or myc-STK38(K118R) (STK38 kinase-dead version) plasmids. The next day, cells were treated with 1 µM OA or with vehicle (DMSO) for 1 hour. Flag fusions were immunoprecipited and pulled-down proteins were analyzed by western blotting. Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates.",
    "molecules": "DMSO, OA"
  },
  {
    "caption": "(A) Graphical representation of XPO1 protein. C528 to S amino acid mutation confers XPO1 resistance to both KPT-185 and KPT-330 chemical inhibitors [24]. S1055 amino acid correspond to the phosphorylation target of the STK38. Finally, the ΔCter region is also highlighted corresponding to a XPO1 construct lacking the 39 C-terminal residues (B-C) XPO1_S1055 phosphorylation is required and sufficient for STK38 nuclear exit upon nutrient starvation. (B) HeLa cells were transiently transfected with myc-STK38(wt) in addition to indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity. Cells were then fixed and stained for Flag and myc tags. Only cells positives for both Flag-XPO1 and myc-STK38(wt) were captured. Representative images are shown and scale bars are 40 µm. (C) Graphical representation of myc-STK38(wt) nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Mann-Whitney test). Expression of wt XPO1 failed to induce cytoplasmic localization of STK38 upon nutrient starvation in presence of KPT-185 where C528S mutant recapitulates results described in Figure 2B-C. Expression of phosphonegative XPO1 (S1055A) failed to induces STK38 cytoplasmic localization upon EBSS treatment while phosphomimetics XPO1 (S1055D and S1055E) were sufficient to promote STK38 cytoplasmic localization without autophagic stimuli. Finally, expression of XPO1 lacking in its 39 C-terminal residues mimicked phosphomimetics variants.",
    "molecules": "KPT-185, KPT-330"
  },
  {
    "caption": "(E) Hek-HT-iRFP-LC3 cells were transiently transfected with siRNA targeting endogenous STK38 (or with non-targeting siRNA (siNT)). Two days after, cells were transiently transfected with indicated Flag-XPO1 mutant plasmids. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours both supplemented with KPT-185 (final concentration = 1 µM) in order to inhibit endogenous XPO1 activity and with chloroquine (final concentration = 10 µM). Cells were then fixed and the number of iRFP-LC3 dots per cell was recorded only in cells positive for Flag-XPO1 mutants As expected, silencing of endogenous STK38 prevented the formation of LC3 dots upon starvation where introduction of phosphonegative XPO1 (S1055A) failed also to increase LC3 dots upon starvation. Interestingly, phosphomimetic XPO1 (S1055D) was sufficient to increase the number of LC3 dots even in complete and also STK38-depleted conditions.",
    "molecules": "chloroquine, KPT-185"
  },
  {
    "caption": "(A) XPO1 and STK38 are required for Beclin1 cytoplasmic accumulation upon nutrient starvation-induced autophagy. HeLa cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT). 72 hours later, cells were incubated with XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or with DMSO for all other conditions for 2 hours prior incubation with DMEM or EBSS supplemented (or not) with inhibitors for 2 hours. Cells were fixed and stained for endogenous Beclin1. Graphical representation of endogenous Beclin1 nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce Beclin1 cytoplasmic accumulation upon nutrient starvation",
    "molecules": "DMSO, KPT-185, KPT-330"
  },
  {
    "caption": "(A) XPO1 and STK38 are required for YAP1 nuclear export at high confluence. A549 cells were transiently transfected with the indicated siRNA (control and KPT conditions = siNT) at low or high confluency. 48 hours later, cells were incubated overnight in the presence of XPO1 inhibitors KPT-185 and KPT-330 (final concentration = 1 µM) or with DMSO for all other conditions. The next day, cells were fixed and stained for endogenous YAP1. Quantitative representation of YAP1 nuclear fluorescence intensity (n > 300 cells from 3 independent experiments, mean ± SEM; ***p<0.001, Mann-Whitney test). XPO1 activity inhibition by both KPT-185 and KPT-330 as well as STK38 silencing failed to induce YAP1 nuclear exit at high confluence",
    "molecules": "DMSO, KPT-185, KPT-330"
  },
  {
    "caption": "E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).",
    "molecules": "H3, Vorinostat"
  },
  {
    "caption": "Planarians were injected with Smed-exoc3 dsRNA = exoc3(RNAi) or with control gfp(RNAi) injections. (A) The representative photographs show staining for phosphorylation of 10th serine residue of Histone 3 (H3S10p, a stem cell marker in planarians) at days-38 of the injection protocol (n=3 experimental replicates with 8-10 biological replicates per experiment, scale bar: 0.5 mm).",
    "molecules": "serine"
  },
  {
    "caption": "FACS-based time-course analysis on changes in the fraction of somatic stem cells (X1-population) at indicated time points. Cells were obtained from whole body-trypsinized planarians of gfp(RNAi)-injected controls and exoc3(RNAi)-treated planarians and stained with the cytoplasmic dye Calcein-AM and the nuclear (DNA) dye Hoechst 33342: (C) Representative FACS profiles",
    "molecules": "Hoechst 33342, Calcein-AM, DNA"
  },
  {
    "caption": "FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (D) Representative FACS blot of BODIPY staining. Unstained WT EBs served as a negative control (grey).",
    "molecules": "BODIPY, BODIPY493/503"
  },
  {
    "caption": "FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (E) Quantification of staining intensity [n=3 independent cultures per genotype; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].",
    "molecules": "BODIPY493/503"
  },
  {
    "caption": "(H) UPLC-MS/MS analysis revealed a reduction in triacylglycerol (TAG) content in Tnfaip2-/- EBs compared to WT EBs at the indicated time points after differentiation induction [n=3 independent cultures of EBs per time point; data of EBs on day-0 to day-3 of differentiation were statistically analyzed; mean centered group data were normally distributed and (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm Sidak correction for multiple testing].",
    "molecules": "TAG, triacylglycerol"
  },
  {
    "caption": "Differentiation-induced WT EBs and Tnfaip2-/- EBs were treated with palmitic acid (PA) and palmitoyl-L-carnitine (PC) (8μM each) or a vehicle control (C,D) The diameters of the rosette structure of differentiated WT EBs and Tnfaip2-/- EBs were measured on day-3 after differentiation induction. (n=3 repeat experiments with 7-62 EBs per experiment per group): (C) Representative photographs (scale bar: 0.02 mm) and (D) quantification of the diameter of the rosette structure of the indicated groups [data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and thus analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].",
    "molecules": "PA, palmitic acid, palmitoyl-L-carnitine, PC"
  },
  {
    "caption": "RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (E) "stem cell maintenance: positive and negative regulators",",
    "molecules": "PA, PC"
  },
  {
    "caption": "RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets ;, (F) "mesoderm development"",
    "molecules": "PA, PC"
  },
  {
    "caption": "RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (G) "ectoderm development". Five other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2-/- EBs versus WT EBs The color scale represents the gene-wise z-score calculated from normalized gene expression levels.",
    "molecules": "PA, PC"
  },
  {
    "caption": "(H) Representative image of immunofluorescence staining against ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) on day-3 of differentiation induction of Tnfaip2-/- EBs +/- co-treatment with PA/PC. Three repeat experiments were conducted with a total number of 9 EBs per group",
    "molecules": "PA, PC"
  },
  {
    "caption": "Oct4-eGFP reporter MEFs or WT MEFs were (E,F) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A. (E) Representative FACS profiles and (F) quantification of the percentage of SSEA1+ iPSCs in MEF cultures on day-18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [n=5 biological replicates; data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].",
    "molecules": "Etomoxir"
  },
  {
    "caption": "(A) TAG levels in exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated controls as determined by UPLC-MS/MS, [n=4 experimental replicates on pools of 25 planarians per group per experiment; data were normally distributed",
    "molecules": "TAG"
  },
  {
    "caption": "(D) Representative images of regenerating planarians that were injected with gfp-RNAi, Smed-β-catenin-1-RNAi or Smed-β-catenin-1-RNAi in combination with PA/PC injection Heads and tails were cut on day-10, images were taken on day-17 after amputation. Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.",
    "molecules": "PA, PC"
  },
  {
    "caption": "(E) Images of body plan maintenance in non-injured planarians that were injected with gfp-RNAi, exoc3-RNAi or exoc3-RNAi in combination with PA/PC injection Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.",
    "molecules": "PA, PC"
  },
  {
    "caption": "E. Left: representative immunofluorescence  of the MN  marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining  in blue. Scale bar=100 mm.. Right: box plot shows the distribution of Isl1+ nuclei  with respect to the total  cell number marked with DAPI staining  in WT and in lncMN2   KO mutants (clone 1 and clone 2). Data are expressed as mean and error bars represent SD. The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Scale bar=100 mm. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Left: representative immunofluorescence  of the MN  marker Isl1  (red signals) in cells maintained in vitro for 14 days after  EB6 dissociation (DIV14). DAPI counterstaining  in blue. Scale bar=100 mm. Right: box plot shows the distribution of Isl1+ nuclei  with respect to the total cell number marked with DAPI staining  in WT and in ∆SP mutants (clone 1 and clone 2). The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Data are expressed as mean and error bars represent SD. Scale bar=100 mm. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures infected with HSV-1 in the presence or absence of IFNα (600 IU/ml), then treated with an α-ΙFNAR1 neutralizing antibody. n=9 biological replicates. RT-qPCR for viral mRNA transcripts at 3 days post- LY294002-induced reactivation of SCGs infected with HSV-1 in the presence or absence of IFNα. n=9 biological replicates. RT-qPCR for viral mRNA transcripts at 20 hours post-LY294002-induced reactivation in SCGs infected with HSV-1 in the presence of absence of IFNα. n=9 biological replicates.",
    "molecules": "LY294002"
  },
  {
    "caption": "
Representative images of vDNA foci detected by click chemistry to PML at 1, 3, 6 and 8 dpi in P6 SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). Z-stack images of individual vDNA foci were acquired. Scale bar, 20μm. Zoom scale bar, 1 μm. Percent colocalization of vDNA foci detected by click chemistry to PML at 1, 3, 6 and 8 dpi in SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.
",
    "molecules": "EdC"
  },
  {
    "caption": "(C) 3D reconstruction of a high-resolution Z-series confocal image showing PML entrapment of a HSV-1EdC vDNA foci. Scale bar, 2μm. Enlargement of PML entrapped vDNA outlined by white dashed box. Scale bar, 0.5μm. Quantification of PML-NB volume in SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). n=64 biological replicates.",
    "molecules": "EdC"
  },
  {
    "caption": "Percent colocalization of vDNA foci detected by click chemistry to PML at 8 dpi in SCG neurons infected with HSV-1EdC following IFNα treatment for 18 hours prior to infection or for 18 hours at 3 days prior to infection. Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.",
    "molecules": "EdC"
  },
  {
    "caption": "Percent colocalization of vDNA foci detected by click chemistry to PML at 3 dpi in SCG neurons with HSV-1EdC infected with ICP0-null mutant HSV-1, n212, or a rescued HSV-1 virus, n212R, in P6 SCG neurons treated with IFNα for 18 hours prior to infection or for 18hours at 3 days prior to infection. Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.",
    "molecules": "EdC"
  },
  {
    "caption": "Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures transduced with shRNA targeting PML for 3 days prior to infection with HSV-1 in the presence or absence of IFNα (150 IU/ml). n=9 biological replicates.",
    "molecules": "LY294002"
  },
  {
    "caption": "Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures transduced with shRNA targeting PML at 1 day post-infection with HSV-1 in the presence or absence of IFNα (150 IU/ml). n=9 biological replicates. Statistical comparisons were made using a Mann-Whitney test (ns not significant, * P<0.05).",
    "molecules": "LY294002"
  },
  {
    "caption": "(B) Picrosirius red staining of the femoral growth plates of a control and the ATD R05-365A fetus. The control growth plate shows normal architecture with clearly distinguishable reserve (R), proliferating (P) and hypertrophic (H) zones. A profound disruption of growth plate architecture is obvious in the GRK2-/- growth plate, which shows irregularly-shaped reserve zone chondrocytes, a lack of discernable proliferating chondrocytes and a significantly under-developed hypertrophic zone. Scale bars, 100 μm.",
    "molecules": "Picrosirius red"
  },
  {
    "caption": "(C) Shortened long bones in the chicken wings injected with CMPD101. The right limb bud was injected with CMPD101, the left one was left as a control. Note the shortened humerus (H), ulna (U) and radius (R) in the CMPD101-injected wing. Mean ± SEM. Mann-Whitney U test; number of animals is indicated. Scale bar, 150 μm.",
    "molecules": "CMPD101"
  },
  {
    "caption": "(D) Alcian blue staining of the femoral growth plates of a control and the ATD R05-365A fetus. Note the significantly lesser blue staining of the R05-365A growth plate stained by alcian blue pH 1.0, suggesting impaired sulfation of the extracellular matrix. Scale bars, 200 μm.",
    "molecules": "Alcian blue, alcian blue"
  },
  {
    "caption": "(E) Alcian blue staining of the chicken limb bud micromasses treated with GRK2 inhibitor CMPD101. Note the gradual inhibition of the cartilage nodules with increasing concentration of CMPD101, quantified below. Mean ± SEM. Mann-Whitney U test; number of micromasses is indicated. A representative experiment of four (pH 2.5) and three (pH 1.0) is shown. Scale bar, 100 μm. (F) Alcian blue staining of the micromasses generated from Grk2+/+ and Grk2-/- NIH3T3 cells. Note the mildly weaker staining by alcian blue pH 2.5 and the nearly absent sulfation stained by alcian blue pH 1.0 in the Grk2-/- micromasses. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of micromasses is indicated. Scale bar, 1 mm. ",
    "molecules": "Alcian blue, alcian blue, CMPD101"
  },
  {
    "caption": "(A,B) Defective GLI3 processing in GRK2-/- fibroblasts. Cells were serum-starved to induce cilia before treatment with the Hh pathway activator SAG (Smoothened agonist), and the whole cell lysates were immunoblotted for GLI3. Note the SAG-mediated downregulation of both full-length (FL) and repressor (R) GLI3 in control fibroblasts, demonstrating normal processing. There was no effect of SAG on GLI3 processing in GRK2-/- cells. A representative experiment of four is shown. (B) Quantitation of the GLI3FL to GLI3R ratio using densitometry. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.",
    "molecules": "SAG, Smoothened agonist"
  },
  {
    "caption": "(C) Defective SAG response in GRK2-/- fibroblasts. Cells were treated with SAG for 24 hours. Transcript levels of GLI3 targets GLI1 and PTCH1 were determined by qRT-PCR; GAPDH was used for normalization. Note the SAG-mediated induction of GLI1 and PTCH1 expression in controls but not in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.",
    "molecules": "SAG"
  },
  {
    "caption": "Defective SMO accumulation in cilia of GRK2-/- fibroblasts. Cells were treated with SAG and immunostained for SMO, ARL13B and pericentrin. Note the failure in SMO accumulation in cilia of GRK2-/- cells (arrow). Scale bar, 2 µm (E).",
    "molecules": "SAG"
  },
  {
    "caption": "Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 12 (A, B) (A,B) Cilia were stained by ARL13B and SMO antibodies, and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO accumulation in cilia caused by GRK2 inhibition (black arrow). Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.",
    "molecules": "SAG, CMPD101, paroxetine"
  },
  {
    "caption": "Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 24 hours (C D). (C) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation; actin levels served as a loading control. GLI3FL and GLI3R, full-length and repressor GLI3 variants, respectively. (D) Quantification of GLI3 and GLI1 levels by densitometry. Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine. Mean ± SEM. Mann-Whitney U test (GLI3 ratio) and Welch´s t-test (GLI1 levels); number of biological experiments is indicated.",
    "molecules": "SAG, CMPD101, paroxetine"
  },
  {
    "caption": "Defective SMO cilia accumulation in chondrocytes with downregulated GRK2. (E) Doxycycline (DOX)-inducible GRK2 downregulation in control human R00-082 chondrocytes, tested by western blot, normalized to actin and plotted below. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. shScr, scramble shRNA.",
    "molecules": "DOX, Doxycycline"
  },
  {
    "caption": "(F) Cells pre-treated with DOX for three days were serum starved, SAG treated, stained and analyzed as in (A,B), and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO translocation to cilia caused by GRK2 loss. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.",
    "molecules": "SAG, DOX"
  },
  {
    "caption": "Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. Control human R00-082 chondrocytes (C) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.",
    "molecules": "CMPD101"
  },
  {
    "caption": "Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. rat chondrosarcoma (RCS) cells (D) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.",
    "molecules": "CMPD101"
  },
  {
    "caption": "(D) Grouping E. rectale individuals from the Danish (DK) and Swedish (SE) studies, based on this deletion shows a significant (*p < 0.05, **p < 0.01, ***p < 0.001) increase in BMI and blood fasting insulin levels as well as a decrease in overall community diversity in individuals who are predominantly colonised by the flagellum carrying subspecies (see Methods). The same trend is observed for E. eligens subspecies in the same studies, though not statistically significant (n.s.).",
    "molecules": "insulin"
  },
  {
    "caption": "A. Localization of TRAPP complex components after sodium arsenite (SA) treatment in HeLa cells treated with 300 µM SA for 30 min. Fluorescence microscopy of fixed cells using antibodies against TRAPPC2 and eIF3 (to label SGs) at steady state and after SA treatment (top two rows). Other panels show localization of GFP-TRAPPC3 and endogenous TRAPPC1 to eIF3-labeled SGs and of endogenous TRAPPC9 and TRAPPC8 to G3BP-labeled SGs. DAPI (blue). Scale bar, 10 µm.",
    "molecules": "DAPI, SA, sodium arsenite"
  },
  {
    "caption": "A. HeLa cells, untreated or treated with SA, were fixed and visualized by fluorescence microscopy using anti-Sec24C Ab, anti-eIF3 Ab, and DAPI (blue). Data information: Scale bars, 10 µm.",
    "molecules": "DAPI, SA"
  },
  {
    "caption": "B. Quantification of Sec24C redistribution to SGs over time after SA treatment (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± s.e.m. n = 50 cells per experiment, N = 3.",
    "molecules": "SA"
  },
  {
    "caption": "C. Quantification of residual Sec24C in ERES after SA treatment. The data are expressed as percentage of steady state values (CTRL). Mean ± s.e.m. n = 100 cells per experiment, N = 3.",
    "molecules": "SA"
  },
  {
    "caption": "D. Representative images of TRAPPC2 localization in Sec23AB-KD and Sec24ABCD-KD cells treated with SA. Cells were fixed and visualized by fluorescence microscopy using anti-TRAPPC2 Ab, anti-G3BP Ab (to label SGs) and DAPI (blue). Data information: Scale bars, 10 µm.",
    "molecules": "DAPI, SA"
  },
  {
    "caption": "F. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC3-KD and TRAPPC2-KD cells treated with SA. Depletion of the entire TRAPP complex (via TRAPPC3 depletion) or of only TRAPPC2 reduces Sec24C recruitment. Graph, quantification of Sec24C at SGs, calculated as in (E). Mean ± s.e.m., n = 40-60 cells per experiment, N = 3. ****p< 0.0001, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "G. Representative images of Sec24C localization at SGs (stained for G3BP) in TRAPPC2-KO cells treated with SA. Data information: Scale bars, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "A. TRAPP and COPII associate more stably with SGs than with ERES. The membrane­­ association of Sec24C was evaluated in non-permeabilized or permeabilized cells with or without SA treatment, as indicated. G3BP was used as an SG marker. Left panel insets, G3BP staining in non-permeabilized cells. Dashed white lines show the outline of permeabilized SA-untreated cells. Blue, DAPI. Data information; Scale bars, 10 µm.",
    "molecules": "DAPI, SA"
  },
  {
    "caption": "B. Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs. HeLa cells overexpressing GFP-Sar1H79G were treated with SA and immunostained for Sec24C, TRAPPC2, and eIF3, as indicated. Blue, DAPI. Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP-Sar1H79G-expressing cells. Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES, expressed as a percentage of the non-transfected (NT) cells. Mean ± s.e.m. n = 60-70, three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.",
    "molecules": "DAPI, SA"
  },
  {
    "caption": "C. Effect of Sec31A depletion on TRAPPC2 recruitment to SGs. Cells were mock-treated or KD for Sec31, treated with SA, and then immunostained for TRAPPC2 and eIF3 as indicated. Graphs, quantification of TRAPPC2 at ERES and SGs after SA treatment. Mean ± s.e.m., one representative experiment; n=60-80. ** p<0.001, ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "D. Effect of Sec31A depletion on Sec24C recruitment to SGs. Cells were mock-treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 (to stain ERES, top panels) and for eIF3 (bottom panels). Insets show eIF3. Graphs, quantification of Sec24C at ERES and in SGs after SA treatment. n=80. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "A. Cells were treated with 273 kinase inhibitors (10 µM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. B. Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio. ",
    "molecules": "SA"
  },
  {
    "caption": "C. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates.",
    "molecules": "Ponceau, Dinaciclib, SA"
  },
  {
    "caption": "F. Representative images of HeLa cells treated with the three best hits (Flavopiridol, SNS-032, Dinaciclib; 1µM 150 min) and then exposed to SA (300 µM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10 µm.",
    "molecules": "Dinaciclib, Flavopiridol, SNS-032, SA"
  },
  {
    "caption": "G. HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 µM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%), N=12.",
    "molecules": "BS-181, Dinaciclib, Flavopiridol, SNS-032, SA"
  },
  {
    "caption": "H. Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 µm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "Dinaciclib, Flavopiridol, SNS-032, SA"
  },
  {
    "caption": "A. Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 µM, 3 h) cells. Graphs show the quantification of Sec24C and TRAPPC2 at ERES (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001, Student's unpaired two-tailed t-test. Data information: Scale bars, 10 µm.",
    "molecules": "SNS-032"
  },
  {
    "caption": "B. HeLa cells were treated with Dinaciclib (1 µM, 3 h) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Data information: Scale bars, 10 µm.",
    "molecules": "digitonin, Dinaciclib"
  },
  {
    "caption": "C. Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 µM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "Digitonin"
  },
  {
    "caption": "D. HeLa cells transfected with HA-hnRNPK were left untreated (Steady state) or treated with SA (300µM, 30 min) or SA+Dinaciclib (10 µM, 150 min) and then immunostained with anti-HA and anti-G3BP antibodies. Data information Scale bars, 10 µm.",
    "molecules": "Dinaciclib, SA"
  },
  {
    "caption": "F. Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3. Data information: Scale bars, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "G. Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP. Data information: Scale bars, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "A. HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 µM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "C. HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 µm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta (normalized for the cytosolic Sec24C) at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of 5 biological replicates. n= 50-80. ns: not significant; ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "SA"
  },
  {
    "caption": "E. Growing and differentiated podocytes were treated with SA (300 µM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10 µm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments. ****p<0.0001, Student's unpaired two-tailed t-test.",
    "molecules": "SA"
  },
  {
    "caption": "A. SG area in mock, TRAPPC2 TRAPPC3, TRAPPC8, and TRAPPC9-depleted cells treated with SA. Data are shown in box-and-whisker plots: Box-plot central line, median; box limits, upper and lower quartiles; whiskers show the minimum to maximum. N=3. ***p<0.001, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "SA"
  },
  {
    "caption": "B. HeLa cells microinjected with control IgG or a TRAPPC3-specific antibody (right panels in green) were treated with SA. The TRAPPC3 Ab disrupts the Golgi (Yu et al, 2006), monitored using an anti-TGN46 Ab. Anti-G3BP was used to stain SGs. The graph shows quantification of the SG area. Mean ± s.e.m. three independent experiments; n>80. ****p<0.0001, Student's unpaired two-tailed t-test. Scale bar, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "Localization of Raptor (D) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. three independent replicates. *p<0.02; **p<0.009 in (D) , One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "SA"
  },
  {
    "caption": "Localization of RACK1 (E) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. independent replicates. , *p<0.05; ****p<0.0001 in (E), One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "SA"
  },
  {
    "caption": "Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with ant-Raptor (F) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for Raptor).",
    "molecules": "SA"
  },
  {
    "caption": "Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with anti-RACK1 (G) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for RACK1",
    "molecules": "SA"
  },
  {
    "caption": "Localization of Raptor (H) untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "SA"
  },
  {
    "caption": "Localization of RACK1 (I) in untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "SA"
  },
  {
    "caption": "J. Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi (SNS-032; Flavopiridol or Dinaciclib, 1 µM, 150 min) and then treated or not with SA (500 µM, 3 h). Images were acquired automatically by OPERETTA microscope. Values indicate the percentage of the total number of nuclei (stained with DAPI) positive for BoBo-3 staining. Mean ± s.d. of one representative experiment out of three independent replicates. ns: not significant, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "BoBo-3, DAPI, Dinaciclib, Flavopiridol, SNS-032, SA"
  },
  {
    "caption": "K. Analysis of cell death in HeLa cells that were left untreated, treated with non-targeting siRNAs or siRNAs against TRAPPC2, and in two cell clones knocked out for TRAPPC2, with and without SA treatment. Mean ± s.d. of three independent replicates.",
    "molecules": "SA"
  },
  {
    "caption": "A. Left, control or SA-treated (500 µM, 120 min) human fibroblasts (HFs) were incubated at 40°C for 180 min and stained for eIF3, PCI, and Sec24C. Right, the cells were imaged 10 min after shifting the temperature from 40°C to 32°C and stained for eIF3, PCI, and Giantin (to label the Golgi). Data information Scale bars, 10 µm.",
    "molecules": "SA"
  },
  {
    "caption": "B. HeLa cells were exposed to SA (200 µM) for the indicated time, alone or in combination with ISRIB and SGs were analyzed. Quantification of SG area (µm) of three independent experiments. Mean ± s.e.m. n = 60-80 cells per experiment, N = 3.",
    "molecules": "ISRIB, SA"
  },
  {
    "caption": "D. HeLa cells expressing VSV-G-UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB (1 µM) (120 min ISRIB pre-treatment and 30 min SA+ISRIB) and then pulsed with blue light. Images were taken 10 min after the UV pulse and processed for staining with the indicated markers. E. Cells were treated with SA for 30 min, the SA was washed out (WO), and cells were left to recover for 120 min in the presence of cycloheximide, followed by a blue light pulse and processing for staining as described in (D). F. Quantification of VSV-G (mean intensity) in the Golgi area to the total VSV-G per cell under the conditions described in (D,E). Data are expressed as percentage of the control. Mean ± s.e.m. of three independent experiment. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.",
    "molecules": "cycloheximide, ISRIB, SA"
  },
  {
    "caption": "A. AiryScan images of HeLa cells treated with SA for the indicated times and stained with GM130 (gray). Scale bar, 10 µm. The graph shows quantification of Golgi objects in SA-treated cells. Mean ± s.e.m. of three independent experiments. n=90-100. ****p<0.0001; ns: not significant, Student's unpaired two-tailed t-test.",
    "molecules": "SA"
  },
  {
    "caption": "B. Control cells and cells treated with Dinaciclib (CDKi) were treated with SA and stained for GM130 (in gray). Insets, eIF3. Blue, nuclear DAPI staining. The graph shows quantification of Golgi objects in HeLa cells untreated or treated with SNS-032, Flavopiridol, or Dinaciclib for 150 min and then treated with SA (300 µM, 30 min). Mean ± s.e.m. of three independent experiments. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "DAPI, Dinaciclib, Flavopiridol, SNS-032, SA"
  },
  {
    "caption": "C. Quantification of Golgi objects in cells plated at different percentage of confluency and treated or untreated with SA. Mean ± s.e.m. of three independent experiments. **p<0.001; ***p<0.0002; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.",
    "molecules": "SA"
  },
  {
    "caption": "D. Quantification of Golgi objects in cells pre-treated with ISRIB (1 µM) for 3 h and then exposed to SA (30 min) in the presence of ISRIB. ***p<0.002, Student's unpaired two-tailed t-test.",
    "molecules": "ISRIB, SA"
  },
  {
    "caption": "E. Electron microscopy images of cells treated (300 µM, 30 min) or untreated with SA. The arrow indicates the Golgi complex. Scale bar, 200 nm.",
    "molecules": "SA"
  },
  {
    "caption": "F. AiryScan images of HeLa cells at steady state or exposed to SA for the indicated times. A Rab1-GTP specific antibody was used to monitor the pool of active Rab1, and GM130 to stain the Golgi complex. Insets, eIF3. Scale bar, 10 µm. The graph shows quantification of Rab1-GTP at the Golgi complex expressed as a percentage of the control. Mean ± s.e.m. of three independent experiments. *p<0.011, **p<0.002, Student's unpaired two-tailed t-test.",
    "molecules": "GTP, SA"
  },
  {
    "caption": "G. HeLa cells transfected or not with WT GFP-Rab1B were left untreated or treated with SA and stained for GM130 and G3BP to monitor SGs. Dashed white line, WT GFP-Rab1B transfected cells. Scale bar, 10 µm. The graph shows quantification of Golgi objects in the different conditions. NT: non-transfected. Mean ± s.e.m. of three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test.",
    "molecules": "SA"
  },
  {
    "caption": "C Immunoblotting  of ACE2  and CD63  proteins in serum exosomes  isolated from the sera of 4 healthy controls (HC) and 7 COVID-19 patients  from NHRI Biobank for COVID-19 patients  in Taiwan (Cohort #3). Serum  collection days from the onset are shown; treatment of oxygen  therapy on the patient  is also indicated. Exosomes  were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2  proteins; asterisk denotes non-glycosylated ACE2  proteins.",
    "molecules": "oxygen"
  },
  {
    "caption": "C In situ PLA  assays of the interaction  between ACE2  and Flag-tagged GLK proteins in HEK293T  cells. Nuclei  were stained  with DAPI. Red dots represent direct interaction  signals. Original magnification, x200. Scale bars, 50 μm. Data information: Data shown are representative results of three C, independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "G Cycloheximide pulse-chase experiments in HEK293T  cells. Immunoblotting  of Flag-tagged ACE2  (anti-Flag), Myc-tagged GLK  (anti-GLK), and tubulin  proteins from HEK293T  cells co-transfected with Flag-ACE2  and Myc-GLK. Transfected cells were treated with 100 μg/ml cycloheximide  (CHX) for up to 24 h. Data information: Data shown are representative results of three G, independent experiments.",
    "molecules": "CHX, Cycloheximide, cycloheximide"
  },
  {
    "caption": "H Flag-tagged ACE2  proteins were immunoprecipitated from lysates of HEK293T  cells co-transfected with Flag-ACE2  and Myc-GLK, followed by immunoblotting with anti-Lsy48-linked ubiquitination  or anti-Flag  antibody. Cells were treated with 25 μM MG132  for 2 h before being harvested. NS, normal serum. Data information: Data shown are representative results of three H, independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "H Flag-tagged ACE2   proteins were immunoprecipitated  from lysates of HEK293T  cells transfected with Flag-ACE2   wild-type or a phospho-mimetic ACE2   (S776/783E) mutant, followed by immunoblotting  with anti-Lsy48-linked ubiquitination  or anti-Flag  antibody. Cells were treated with 25 μM MG132  for 2 h before being harvested. Data information: Data shown are representative results of three H) independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "C Immunoblotting  of Flag-tagged ACE2  (anti-Flag), Myc-tagged UBR4  (anti-Myc), and tubulin  proteins from HEK293T  cells co-transfected with Flag-ACE2  and Myc-UBR4. The co-transfected cells were treated with 25 μM MG132  for 2 h before being harvested. Data information: Data shown are representative results of three C, independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "D Flag-tagged ACE2  proteins were immunoprecipitated  from lysates of HEK293T  cells co-transfected with Flag-ACE2  and Myc-UBR4, followed by immunoblotting  with anti-Lsy48-linked ubiquitination, anti-Myc, or anti-Flag  antibody. Cells were treated with 25 μM MG132  for 2 h before being harvested. Data information: Data shown are representative results of three D, independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "E In situ PLA  assays of the interaction  between Flag-tagged ACE2  (wild-type or mutant) and Myc-tagged UBR4  proteins in HEK293T  cells treated with 25 μM MG132  for 2 h. Nuclei  were stained  with DAPI. Red dots represent direct interaction  signals. Original magnification, x200. Scale bars, 20 μm. Data information: Data shown are representative results of three E, independent experiments.",
    "molecules": "DAPI, MG132"
  },
  {
    "caption": "F Mass spectrometry analysis of the ACE2  peptides containing ubiquitination  residues from Flag-ACE2-transfected HEK293T  cells treated with 25 μM MG132  for 2 h.",
    "molecules": "MG132"
  },
  {
    "caption": "H Flag-tagged ACE2  proteins were immunoprecipitated  from lysates of HEK293T  cells co-transfected with Flag-ACE2  wild-type or Flag-ACE2  (K26/112/114R) mutant plus Myc-UBR4  plasmids, followed by immunoblotting  with anti-Lsy48-linked ubiquitination  or anti-Flag  antibody. Cells were treated with 25 μM MG132  for 2 h before being harvested. Data information: Data shown are representative results of three H) independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "Human ACE2  transgenic (EF1α-hACE2 Tg) mice  were intranasally infected with 2x105 pfu of live SARS-CoV-2. The lung  tissues of infected mice  at day 3 days post infection were collected and then analyzed by immunohistochemistry  (IHC) and PLA  assays. Representative IHC  data of hACE2 (in red) and GLK  (in green) in the lung  tissues of the infected wild-type and EF1α-hACE2 Tg mice  were shown (I). Cell nuclei  were stained  with DAPI. Original magnification, ×630. Scale bars, 10 μm. Data information: WT, wild-type mice; Tg, transgenic mice.",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) Immunofluorescence images showing anti-RPA-1-positive foci in response to 5-FU after depleting the indicated genes by RNAi or empty vector control RNAi (L4440; scale bars, 5 μm).",
    "molecules": "5-FU"
  },
  {
    "caption": "b) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci.",
    "molecules": "5-FU"
  },
  {
    "caption": "(c) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci in N2, mlh-1(ok1917) and exo-1(tm1842) mutants fed control (L4440) or RNAi targeting APN-1 (apn-1).",
    "molecules": "5-FU"
  },
  {
    "caption": "(d) Immunofluorescence showing CHK-1 phosphorylation at Ser139 in response to 5-FU in N2 but not in msh-6(pk2504) mutants or in N2 after apn-1(RNAi) (scale bars, 5 μm). (e) Quantification of the fraction (%) of 5-FU-treated embryos with phospho-CHK-1 foci.",
    "molecules": "5-FU"
  },
  {
    "caption": "(f) Western blottings showing H1X.101 levels in N2 and msh-6 mutants, treated (+) or not (−) with 5-FU. Actin was used as loading control. (g) Quantification of H1X.101 levels relative to actin in control versus 5-FU-treated worms. (b,c,e,g) Bar graphs show the mean±s.d. from three independent experiments.",
    "molecules": "5-FU"
  },
  {
    "caption": "a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm).",
    "molecules": "5-FU"
  },
  {
    "caption": "(b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm).",
    "molecules": "5-FU"
  },
  {
    "caption": "(c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product.",
    "molecules": "3-MA, 5-FU"
  },
  {
    "caption": "(f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm).",
    "molecules": "5-FU"
  },
  {
    "caption": "(g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment.",
    "molecules": "5-FU"
  },
  {
    "caption": "a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm).",
    "molecules": "5-FU"
  },
  {
    "caption": "(b) The fraction (as % of untreated control) of hermaphrodites having embryos with excessive GFP expression following 5-FU treatment. Autophagy induction in independent experiments (circles) and the mean (line) are presented.",
    "molecules": "5-FU"
  },
  {
    "caption": "(a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm). (b) The number of animals harbouring embryos with excessive ATF-2::GFP expression was scored in at least 150 animals and given as the fraction (%) of the total.",
    "molecules": "5-FU"
  },
  {
    "caption": "(c) N2 or msh-6(pk2504) embryos were continuously monitored under differential interference contrast microscopy (scale bars, 5 μm) from the three-cell stage until completion of the four-cell stage. Magnified images of representative nuclei are shown. (d) The fraction of embryos with visible, enlarged nucleoli (arrows) after 5-FU treatment was scored. Bar graphs represent mean±s.d. from three independent experiments.",
    "molecules": "5-FU"
  },
  {
    "caption": "(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin.",
    "molecules": "5-FU, Mx, Staurosporin"
  },
  {
    "caption": ". (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200-1,600 cells were scored per condition.",
    "molecules": "5-FU, Mx"
  },
  {
    "caption": "(i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase-PCR.",
    "molecules": "5-FU"
  },
  {
    "caption": "IL-1β ELISA of supernatants from LPS-primed (1 μg/mL, 4hrs.) MDMs pre-incubated with 0.1% DMSO or P22077 (2.5 µM) 15 mins. before treatment with either nigericin (10 μM, 45 mins.) or CPPD crystals (250 μg/mL, 2 hrs.). Bars represent the mean, ± S.D. n = 13 and 11 independent blood donors for nigericin and CPPD, respectively. IL-18 ELISA of supernatants from MDMs treated as in A. Bars represent the mean, ± S.D. n = 11 independent blood donors.",
    "molecules": "CPPD, P22077, DMSO, LPS, nigericin"
  },
  {
    "caption": "LDH-release from MDMs treated as in A. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 10 and 11 independent blood donors for nigericin and CPPD, respectively.",
    "molecules": "CPPD, nigericin"
  },
  {
    "caption": "Quantitative RT-PCR showing the mRNA expression levels of IL-1β, IL-18, ASC, CASP1 and NLRP3 in MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL; 4 hrs.), as indicated. Bars represent the mean fold increase from an untreated (UT) control",
    "molecules": "P22077, LPS"
  },
  {
    "caption": "Western blots showing the relative abundance of NLRP3, ASC, pro-caspase-1, pro- IL-1β, pro-IL-18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL) for 4 hrs., as indicated. β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.",
    "molecules": "P22077, LPS"
  },
  {
    "caption": "IL-18 ELISA of supernatants from LPS-primed (1 μg/mL, 10 mins.) MDMs pre-incubated with either vehicle (0.1% DMSO) or P22077 (2.5 µM) for 15 mins. before treatment with nigericin (10 μM, 45 mins.). Bars represent the mean, ± S.D. n = 9 independent blood donors.",
    "molecules": "P22077, DMSO, LPS, nigericin"
  },
  {
    "caption": "Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in insoluble fractions from THP1 cell lysates. PMA differentiated THP1 cells were LPS-primed (1 μg/mL, 4 hrs.) and pre-incubated for 15 min with either 0.1% DMSO or P22077 (2.5 µM). followed by treatment with nigericin (10 μM, 45 mins.). The 'input' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction. Blots show membranes that were probed with an anti-ASC antibody. Data is representative of at least three independent experiments.",
    "molecules": "DSS, PMA, P22077, DMSO, LPS, nigericin"
  },
  {
    "caption": "Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in supernatants from THP1s treated as in A. Blots show membranes that were probed with an anti-ASC antibody. The input represents non-crosslinked supernatant. Data is representative of at least three independent experiments.",
    "molecules": "DSS"
  },
  {
    "caption": "NLRP3 oligomerisation analysed by native PAGE. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 min.). Digitonin-solubilised cell lysates were analysed by native-PAGE or SDS-PAGE and NLRP3 western-blotting. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inflammasome inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120 kDa). β-actin is shown as a loading control. Blots are representative of 4 independent murine donors.",
    "molecules": "P22077, ATP, Digitonin, DMSO, LPS"
  },
  {
    "caption": "NLRP3 ubiquitination analysed by western blot. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 mins.). Endogenous NLRP3 was immunoprecipitated (IP) with anti-NLRP3 and probed with either anti-ubiquitin (Poly-Ub), anti-K63-ubiquitin (Poly-K63-Ub) or anti-NLRP3. As a negative control, cell lysate made from LPS-primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG. Total levels of NLRP3 in cell lysates (input) were also probed. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120kDa). β-actin is shown as a loading control. Blots are representative of 2 independent murine donors for 2.5µM P22077 and 4 independent murine donors for 10 µM.",
    "molecules": "P22077, ATP, DMSO, LPS, Ub, ubiquitin"
  },
  {
    "caption": "Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then treated with nigericin (10 μM) for differing lengths of time, as indicated. Relative, activity-based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs. Higher panels show lysates incubated with ABPs (labelled), with the active DUB bound to ABP indicated by the suffix '-VME'. Lower panels, without probe labelling, show total USP7 or USP47 levels. (Unlabelled). Western blots are representative of 3 independent MDM donors.",
    "molecules": "LPS, nigericin, VME"
  },
  {
    "caption": "Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then pre-incubated with either 0.1% DMSO or P22077 (2.5 μM) prior to incubation with CPPD crystals (250 µg/mL, 3 hrs.). Blots are labelled as in B and are representative of 3 independent MDM donors.",
    "molecules": "CPPD, P22077, DMSO, LPS"
  },
  {
    "caption": "Western blots showing activity changes in both USP7 and USP47 in LPS-primed (1 μg/mL, 4 hrs.) MDMs pre-incubated with different P22077 concentrations, as indicated, prior to activation by nigericin treatment (10 μM; 45 mins.). Blots are labelled as in B and are representative of 3 independent MDM donors.",
    "molecules": "P22077, LPS, nigericin"
  },
  {
    "caption": "Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium (High K+) conditions. MDMs were LPS-primed (1 μg/mL, 4 hrs.) and then changed to media that either had normal or high concentrations of K+, and subsequently treated with nigericin (10 μM; 45 mins.). Blots are labelled as in B and are representative of at least 2 independent MDM donors. Pro-IL-1β (31 kDa) and mature IL-1β (mIL-1β, 17 kDa) in the supernatants of these cells are shown as a control. Non-specific bands are highlighted with an asterisk.",
    "molecules": "LPS, nigericin, potassium"
  },
  {
    "caption": "Western blots of supernatants and cell lysates from THP-1 cells engineered by CRISPR/Cas9 to induce USP7 and/or USP47 KOs. Deficiency of both USP7 and USP47 was induced by doxycycline (Doxy) treatment (1 µg/mL; 3 days), as indicated. All cells were differentiated with PMA and either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and treated with nigericin (10 μM, 45 mins.), as indicated. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-caspase-1 (pro-Casp-1; 45 kDa); mature caspase-1 (mCasp-1; 20 kDa); USP7 and USP47. β-actin is shown as a loading control. Blots are representative of at least 3 independent experiments.",
    "molecules": "PMA, Doxy, doxycycline, LPS, nigericin"
  },
  {
    "caption": "IL-1β release measured from doxycycline treated (1 µg/mL; 3 days) PMA-differentiated and LPS-primed (1 μg/mL, 4 hrs.), nigericin activated (10 μM, 45 mins.) THP-1 cells, which contain the indicated sgRNA. Bars represent the mean percentage of IL-1β release relative to their respective non-doxycycline treated control cells, ± S.D. LDH-release measured from cells as treated in B. Bars represent the mean percentage of LDH release relative to their respective non-doxycycline treated control cells, ± S.D.",
    "molecules": "PMA, doxycycline, LPS, nigericin"
  },
  {
    "caption": "LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15 nm dots indicate immunogold-labeled LC3 clusters and 5nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b-d) are presented. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.",
    "molecules": "gold"
  },
  {
    "caption": "Western blot analysis of MsrB2 and LC3 I/II in Meg-01 cell after shMsrB2 transfection (72 hrs). Cells were then treated with H2O2 (1 mM for 1 hr) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control. (shMsrB2 vs. shCon; *p=0.024, shMsrB2/H2O2 vs. shCon/H2O2; *p=0.0295, shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC **p=0.0100, n=3)",
    "molecules": "H2O2, NAC"
  },
  {
    "caption": "Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA. (ROS; *p=0.0139, MsrB2 fl/fl n=5, MsrB2 -/- n=6)",
    "molecules": "DCFH-DA, reactive oxygen species, ROS"
  },
  {
    "caption": "C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice. Results are expressed as % apoptotic cells. (TMRE; *p=0.0118, MsrB2 fl/fl n=5, MsrB2 -/- n=5)",
    "molecules": "TMRE"
  },
  {
    "caption": "Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n=6 samples per group. Values expressed as Mean ± SEM. (caspase-3 activity ; *p=0.0157)",
    "molecules": "Ac-DEVD-pNA"
  },
  {
    "caption": "Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin, was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer.",
    "molecules": "MetO, β-mercaptoethanol, SDS"
  },
  {
    "caption": "In vitro assay. Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs. Western blot analysis of Parkin and MsrB2 was performed. (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.",
    "molecules": "H2O2"
  },
  {
    "caption": " ROS levels were measured in platelet with FACS using specific oxidative stress detection dyes (CellROX, Molecular Probes, USA) in HC (n=8) and DM (n=10) platelets. (ROS; **p<0.0001 vs. HC).",
    "molecules": "CellROX, ROS"
  },
  {
    "caption": "Western blot analysis of Methionine Sulfoxidation (MetO), Methionine sulfide reductase B2 (MsrB2) and Parkin in human Healthy Control (HC) and Diabetic Mellitus (DM) platelets. P.C. is a BSA-MetO positive control (Cayman Chemical Co.). Quantitation of western blot analysis shown in Fig 1C. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (MetO; **p=0.0003, MsrB2; *p=0.0186, Parkin; **p=0.0003 vs. HC).",
    "molecules": "MetO"
  },
  {
    "caption": "Parkin IP was performed in HC, HC plus H2O2, and DM patients.",
    "molecules": "H2O2"
  },
  {
    "caption": "MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.",
    "molecules": "MetO"
  },
  {
    "caption": "Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets. Western blot analysis of precipitated MsrB2, Parkin and ubiquitinated MsrB2 (UB).",
    "molecules": "UB, ubiquitinated"
  },
  {
    "caption": "Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV (mitochondria marker), ubiquitin and MsrB2. Colocalization is demomstrated by arrows and quantitated. (colocalization ubiquitin, MsrB2; **p=0.0003 vs. HC).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "MsrB2 ubiquitination assay after transient transfection of MsrB2-GFP (1µg) with RFP empty vector (E.V.) or RFP-Parkin (3µg) in HEK293. After transfection (48hrs), an IP was performed using GFP-Trap bead, followed by Western blot analysis using UB and MsrB2 and Parkin antibodies. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.",
    "molecules": "UB"
  },
  {
    "caption": "Western blot analysis of MsrB2 and Cytochrome C in isolated mitochondria after incubation w/o Cyclosporin A (CsA; 2 μM for 2 hrs.) or w/ CsA. ATPB served as the loading and negative control (mitochondrial matrix protein).",
    "molecules": "CsA, Cyclosporin A"
  },
  {
    "caption": "Western blot analysis of Parkin, LC3I/II, ubiquitinated MsrB2 in chow (n=4) and HFD (n=6) mouse platelets.   Quantification of individual band intensity. (Parkin; *p=0.0153, Modified Parkin; **p=0.0055, LC3II; *p=0.0153, Modified MsrB2; *p=0.02245 vs. HC). Actin served as the loading control.  ",
    "molecules": "ubiquitinated"
  },
  {
    "caption": "A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI  (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative  image is shown. Scale bar=100 um.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunoblot analysis of LC3B and GABARAP lipidation in wild-type (WT) and ATG16L1-KO HEK293A cells, with or without stimulation for 2h as indicated. BafA1 stands for Bafilomycin A1. Gamma-Tubulin (γ-Tubulin) is loading control. Densitometry analysis of lipidated LC3B (LC3B-II) to γ-tubulin (normalized to untreated WT cells) from immunoblots in (A). Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.",
    "molecules": "BafA1, Bafilomycin A1"
  },
  {
    "caption": "LC3B/GABARAP lipidation in ATG16L1- and ATG5-KO HEK293A cells treated for 2h as indicated. Vinculin is loading control. Cell lysates were immunoblotted for the specified proteins. Ratio of LC3B-II to Vinculin quantified from immunoblots in (C) and normalized to MC3 treated ATG16L1 KO cells. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.",
    "molecules": "MC3"
  },
  {
    "caption": "Immunoblot analysis of LC3B and GABARAP lipidation from WT, ATG16L1-KO (L1-KO), ATG16L2-KO (L2-KO), TECPR1-KO (TEC-KO), ATG16L1/L2-DKO (L1/L2-DKO), ATG16L1/TECPR1-DKO (L1/TEC DKO), and ATG16L1/L2/TECPR1-TKO (L1/L2/TEC-TKO) PC-3 cells, treated as indicated for 2h, were immunoblotted for the indicated proteins. Vinculin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.",
    "molecules": "MRT68921, SAR405"
  },
  {
    "caption": "LC3B/GABARAP lipidation in ATG16L1-KO and ATG16L1/TECPR1-DKO HEK293A cells with or without TECPR1 rescue, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins. Vinculin is loading control. Ratio of LC3B-II to Vinculin quantified from immunoblots in (G) and normalized to ATG16L1-KO cells treated with MC3. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.",
    "molecules": "MC3"
  },
  {
    "caption": "Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing mScarlet-LC3B, with or without EGFP-TECPR1, treated for 2h with Cy5-labeled MC3 particles (left) or LLOMe (right) as indicated. Scale bars: 10 µm.",
    "molecules": "Cy5, MC3, LLOMe"
  },
  {
    "caption": "Confocal time lapse images of ATG16L1/TECPR1-DKO HeLa cells, showing localization of mScarlet-LC3B and EGFP-TECPR1, after treatment with 400 μM LLOMe. Scale bar: 10 µm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO HEK293A cells, treated with 400µM LLOMe for indicated periods of time. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (E) and normalized to 2hr (120min) LLOMe treatment. (Data presented as mean + SEM, n = 4 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test).",
    "molecules": "LLOMe"
  },
  {
    "caption": "Time series of confocal images showing redistribution of EqtSM-mCherry (sphingomyelin (SM) probe) and EGFP-TECPR1 in ATG16L1/TECPR1-DKO cells with doxycycline-inducible (dox, 2μg/ml) expression of EqtSM-mCherry cells following treatment with 400µM LLOMe. Scale bar: 10 µm.",
    "molecules": "dox, doxycycline, LLOMe, SM, sphingomyelin"
  },
  {
    "caption": "LC3B/GABARAP lipidation in ATG16L1- and TECPR1-KO HEK293A cells with or without dox-inducible (2μg/ml) expression of nSMase2-mCherry, treated for 2h as indicated. Cell lysates were immunoblotted against the indicated proteins. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (B) and normalized to untreated ATG16L1-KO cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).",
    "molecules": "dox"
  },
  {
    "caption": "Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing EGFP-TECPR1, with or without dox-inducible (2μg/ml) nSMase2 overexpression, treated for 2h as indicated. Scale bars: 10 µm. Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they originate from. Total cells per condition/images per condition: 952/30 (LLOMe treated - nSMase2) and 1056/30 (LLOMe treated + nSMase2) were analyzed. (P-values determined using Student's unpaired t test).",
    "molecules": "dox, LLOMe"
  },
  {
    "caption": "Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S: supernatant; P: pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).",
    "molecules": "Liposome, liposomes, SM"
  },
  {
    "caption": "Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO or ATG16L1/TECPR1-DKO HEK293A cells with or without rescue with TECPR1 (WT), TECPR1 W77A, TECPR1 W154A, TECPR1 W829A or TECPR1 F908A, and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (C) and normalized to ATG16L1-KO cells. Data information: All data presented as mean + SEM (in (D) n = 3 independent experiments. P-values determined using one-way ANOVA followed by Tukey's multiple comparisons test in D and H; ns= not significant.",
    "molecules": "LLOMe"
  },
  {
    "caption": "LC3B/GABARAP lipidation in ATG16L1/TECPR1-DKO HEK293A cells rescued with TECPR1 (WT), TECPR1 W77A, TECPR1 W829A or TECPR1 W77A/W829A (WAWA), and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control.",
    "molecules": "LLOMe"
  },
  {
    "caption": "Maximum intensity projection of confocal images of HeLaK cells expressing EGFP-TECPR1 (WT) or W77A/W829A (WAWA) mutant, with or without 400µM LLOMe for 30min. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (G) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they came from. Total cells per condition/images per condition: 2502/59 (WT untreated), 2549/48 (WT LLOMe), 2256/59 (WAWA untreated) and 2816/59 (WAWA LLOMe) were analyzed.",
    "molecules": "Hoechst 33342, LLOMe"
  },
  {
    "caption": "Coomassie-stained gel depicting in vitro LC3B lipidation reactions using the indicated combinations of proteins and liposomes (with or without SM), after being subjected to a liposome co-sedimentation assay. LC3B-AMP is denoted with an asterix (*). The extent of LC3B-I to -II conversion in (A) was determined and plotted as percentage of total LC3B. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).",
    "molecules": "liposome, liposomes, SM"
  },
  {
    "caption": "Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S, supernatant; P, pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).",
    "molecules": "Liposome, liposomes, SM"
  },
  {
    "caption": "Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S, supernatant; P, pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).",
    "molecules": "Liposome, liposomes, SM"
  },
  {
    "caption": "LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).",
    "molecules": "MRT68921, dox, monensin, SAR405"
  },
  {
    "caption": "LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, Cell lysates were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).",
    "molecules": "MRT68921, dox, SAR405"
  },
  {
    "caption": "Confocal images of WT and TECPR1-KO cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying number of LC3 puncta per cell in the images represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single cells are color-coded and superimposed according to the biological replicate they originated from. Total cells per condition/images per condition: 1169/30 (WT LLOMe), 1064/29 (WT-mcherrySopF LLOMe), 692/23 (TECKO LLOMe) and 983/30 (TECKO-mcherrySopF untreated) were analyzed. (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).",
    "molecules": "Hoechst 33342, dox, LLOMe"
  },
  {
    "caption": "Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each).",
    "molecules": "IRDye700, dsDNA, Ub"
  },
  {
    "caption": "Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub­) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.",
    "molecules": "IRDye700, DNA, dsDNA, Ub"
  },
  {
    "caption": "Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape.",
    "molecules": "DNA, dsDNA, Ub"
  },
  {
    "caption": "Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.",
    "molecules": "RED-tris-NTA"
  },
  {
    "caption": "FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE.",
    "molecules": "dsDNA"
  },
  {
    "caption": "FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison.",
    "molecules": "IRD700, DNA, dsDNA"
  },
  {
    "caption": "FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.",
    "molecules": "dsDNA, DNA, His, IRDye700"
  },
  {
    "caption": "USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody.",
    "molecules": "dsDNA, DNA, His, Ub"
  },
  {
    "caption": "USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining.",
    "molecules": "Ub"
  },
  {
    "caption": "Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.",
    "molecules": "DNA, dsDNA, Ub, ubiquitin"
  },
  {
    "caption": "B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control.",
    "molecules": "histones"
  },
  {
    "caption": "F. BAZ2A is required for the differentiation of ESC+2i but not of ESC+serum. Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10% serum and in the absence of LIF.",
    "molecules": "alkaline phosphatase"
  },
  {
    "caption": "K. Active histone marks increase in the B compartment upon BAZ2A knockdown. Boxplots indicating the log2 fold-changes of normalized H3K27ac and H3K4me3 read counts in ESC+2i treated with siRNA-Baz2a and siRNA-control at boundaries and centers of A and B compartments. ). Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, * < 0.05). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.",
    "molecules": "histone"
  },
  {
    "caption": "E. Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A-regulated genes in ESC+2i observed upon TOP2A or BAZ2A knockdown. ESCs were treated with the TOP2 inhibitor ICF-193 for 24 hours. mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, ** < 0.01, *** < 0.001, **** < 0.0001).",
    "molecules": "DMSO, ICF-193"
  },
  {
    "caption": "B Infiltration of increasing concentrations of GRIp65-84 into Col‐0leaves, electrolyte leakage was measured after 12 h. Background (red line) shows ion leakage from infiltration of leaves with increasing concentrations of (inactive) GRIp80-96.",
    "molecules": "electrolyte, ion"
  },
  {
    "caption": "C Infiltration of leaves with 37 nM GRIp31-96 induced elevated ion leakage in Col‐0 and prk4, but not in prk5‐1 or prk5‐2. Infiltration with GST caused the same background effect for all lines.",
    "molecules": "ion"
  },
  {
    "caption": "D Genomic complementation of prk5 rescues the insensitivity to induction of elevated ion leakage by GRIp31-96.",
    "molecules": "ion"
  },
  {
    "caption": "E Enzymatic superoxide production from xanthine/xanthine oxidase (XXO) induced more electrolyte leakage in gri compared to Col‐0 or prk5‐1, prk5‐2 and prk4 after infiltration into leaves. Infiltration with xanthine buffer (X) was used as a control.",
    "molecules": "electrolyte, xanthine"
  },
  {
    "caption": "H Kinase activity of PRK5 in in vitro phosphorylation assays using γ32P‐ATP and myelin‐basic protein (MBP) as a substrate in the presence of 10 mM MnCl2. GST‐PRK5 and GST‐PRK4 did not show kinase activity. Mutation of conserved residues in kinase subdomains VIb and VII to reconstitute the consensus kinase domain motif restored GST‐PRK5H500DA520G kinase activity. GST‐CRK7 was used as a positive control. Upper panel shows autoradiograph, and lower panel shows the Coomassie‐stained 15% SDS-polyacrylamide gel.",
    "molecules": "γ32P‐ATP, MnCl2"
  },
  {
    "caption": "A In vitro‐produced 35S‐labeled ectodomains of PRK5 (PRK540-281) or PRK4 (PRK440-279) were incubated with bacterially produced GST, GST‐GRI or GST‐GRI31-96 and purified. GST‐GRI31-96 but not GST directly bound to the ectodomain of PRK5. Binding of GST‐GRI to PRK5 ectodomain and binding of GST‐GRI31-96 and GST‐GRI to the ectodomain of PRK4 were strongly reduced. Upper part: autoradiograph, lower part: Coomassie‐stained 12% SDS-polyacrylamide gel. Asterisks in the Coomassie‐stained gel indicate GST, GST‐GRI and GST‐GRI31-96, respectively. Supplementary Fig S12 shows a Western blot of the GST‐tagged proteins.",
    "molecules": "35S"
  },
  {
    "caption": "B Infiltration of 37 nM GST, GRIp31-96, GRIp65-84 or Y‐GRIp65-84 into Col‐0 or prk5‐2 leaves. Tyrosine‐labeled GRIp65-84 still induced cell death in Col‐0 but not in prk5‐2 plants.",
    "molecules": "Tyrosine"
  },
  {
    "caption": "C 125I‐labeled Y‐GRIp65-84 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of three samples, triangles show individual data points).",
    "molecules": "I"
  },
  {
    "caption": "E Binding of 125I‐Y‐GRIp65-84 (0.46 nM) to immunoprecipitates (using anti‐c‐myc antibodies) from prk5‐2 protoplasts transfected with PRK5‐c‐myc, PRK4‐c‐myc or YFP, respectively. Binding was competed out with 10 μM unlabeled Y‐GRIp65-84. Bars show the average of two samples, and triangles show individual data points. Western blot is shown in panel (D).",
    "molecules": "I"
  },
  {
    "caption": "G Excess of GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp65-84 to membrane extracts from Col‐0 (all bars show the average of two samples, triangles show individual data points).",
    "molecules": "I"
  },
  {
    "caption": "B Infiltration of wild‐type, prk5‐1 and atmc9‐1 leaves with 37 nM GRIp31-96, GRIp65-84, GRIp68-78 or GST. GRIp65-84 and GRIp68-78 but not GRIp31-96 were able to induce elevated ion leakage in the atmc9‐1 mutant.",
    "molecules": "ion"
  },
  {
    "caption": "E 125I‐labeled Y‐GRIp68-78 (0.46 nM) bound specifically to Col‐0membrane fractions (light gray bars), the binding was significantly reduced in prk5‐1 and prk5‐2 plants. Excess of non‐radioactive Y‐GRIp65-84 (10 μM) reduced binding to background levels (dark gray bars; all bars show the average of four samples, triangles show individual data points).",
    "molecules": "I"
  },
  {
    "caption": "F Excess of GRIp68-78 and GRIp65-84 (10 μM) but not of GRIp31-96 or other peptides (GRIp31-51, GRIp47-68, GRIp80-96) competed the binding of 0.46 nM 125I‐Y‐GRIp68-78 to membrane extracts from Col‐0 (all bars show average of four samples, triangles show individual data points).",
    "molecules": "I"
  },
  {
    "caption": "G Saturation binding curve for 125I‐Y‐GRIp68-78 to Col‐0membrane extracts. Specific binding was calculated by subtracting non‐specific binding from the total binding. The affinity of 125I‐Y‐GRIp68-78 to the receptor (Kd = 1.9 nM) was calculated by non‐linear regression analysis. Scatchard plot is shown in Supplementary Fig SI7.",
    "molecules": "I"
  },
  {
    "caption": "(A) Histogram (mean, 95 %CI) of fold induction of PXR activity reporter assay (luc, luciferase) in LS180 cells transiently transfected with wild-type PXR and p3A4-luc reporter plasmids. Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.",
    "molecules": "FKK5, FKK6, FKK999, DMSO"
  },
  {
    "caption": "(B), same as (A), in HepG2 cells stably expressing pCYP1A1 luciferase plasmid (AhR reporter). Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. Indole is colored blue. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.",
    "molecules": "FKK5, FKK6, FKK999, DMSO"
  },
  {
    "caption": "D, Chromatin Immunoprecipitation (ChIP) assay in LS174T cells. Top panel, PCR products cells exposed to vehicle or FKK6 and run on a 2% agarose gel. DNA 100 base pair marker; Vehicle,10% DMSO; FKK6 (10 μM); 1/10 Input - 0.2 million cells before IP; IgG - IP with polyclonal rabbit IgG; PXR - IP with PXR antibody. Bottom panel, quantitative PCR from the ChIP assay for compounds tested with the gene specific PCR amplicon normalized to GAPDH (fold expression). Dash line, 2-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biological replicates, n = 4 technical replicates). Mean fold expression is expressed above each histogram (mean + SD).",
    "molecules": "FKK6, DMSO"
  },
  {
    "caption": "F, Microcalorimetric titrations of PXR ligand binding domain (LBD) with FKK5 and FKK6. A single-site binding model was used for fitting the data. The upper panel shows the output signal, dQ/dt, as a function of time. The middle panel shows the integrated heats as a function of the ligand/PXR molar ratio in the cell. The solid line represents the best non-linear least-squares fit of the data. The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain.",
    "molecules": "FKK5, FKK6"
  },
  {
    "caption": "B, Histogram represents fold change in mRNA expression, normalized to GAPDH, by RT-qPCR from Caco-2 cells exposed to compounds. DMSO, 0.1% DMSO vehicle; 9.3 is 9.3-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biologic replicates, n = 4 technical replicates).",
    "molecules": "DMSO"
  },
  {
    "caption": "Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK5 (n = 9 paired samples). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.",
    "molecules": "FKK5"
  },
  {
    "caption": "Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK6 (n = 3 paired samples; one removed due to poor inflammatory response). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.",
    "molecules": "FKK6"
  },
  {
    "caption": "Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK9 (n = 5; 3 removed due to poor response and tissue integrity). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.",
    "molecules": "FKK9"
  },
  {
    "caption": "Fold induction (mRNA) of genes in C57BL/6 mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expression",
    "molecules": "FKK6, DMSO"
  },
  {
    "caption": "Fold induction (mRNA) of genes in pxr -/- mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expression",
    "molecules": "FKK6, DMSO"
  },
  {
    "caption": "Fold induction (mRNA) of genes in hPXR mice (mice expressing the human PXR gene) gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks 2-fold gene induction.",
    "molecules": "FKK6, DMSO"
  },
  {
    "caption": "After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. A, % weight loss from baseline (day 1 vs day 10)",
    "molecules": "FKK6, DSS, DMSO"
  },
  {
    "caption": "After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. B, colon length (cm)",
    "molecules": "FKK6, DSS, DMSO"
  },
  {
    "caption": "After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. C, Inflammation score",
    "molecules": "FKK6, DSS, DMSO"
  },
  {
    "caption": "After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. D, Fecal lipocalin 2 (pg/ml)",
    "molecules": "FKK6, DSS, DMSO"
  },
  {
    "caption": "After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. E, serum FITC-dextran (μg/ml).",
    "molecules": "FITC, FKK6, DSS, dextran, DMSO"
  },
  {
    "caption": "After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. F, fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS (n = 3/group; each PCR performed in quadruplicate).",
    "molecules": "FKK6, DSS, DMSO"
  },
  {
    "caption": "A) oxLDL plasma concentration in ACM patients and HC (n=36; Mann-Whitney test). B) oxLDL plasma concentration in mutated ACM (n=7) and NON ACM relatives, carriers of the same causative mutation (n=9; Mann-Whitney test). C)  oxLDL plasma concentration in ACM patients carriers of a  PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations, or gene elusive (n=10 vs. n=26; One-Way ANOVA). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.",
    "molecules": "oxLDL"
  },
  {
    "caption": "D) Representative images of  MDA immunostaining (green) on ACM and HC ventricular  tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). E) Representative images of CD36 immunostaining  on  ACM  and HC ventricular  tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are  counterstained  with Hoechst33342  (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. ",
    "molecules": "Hoechst33342, MDA"
  },
  {
    "caption": "B) Left panels: representative images of three cases of ACM in Steady State Free Procession sequences at cardiac MRI. On the left a case with RV dilation, regional bulging of the  RV  wall without fat infiltration, and  oxLDL levels below the cut-off; in the center and on the right, two cases with fat infiltration in the right or  left ventricle wall (white arrow), respectively, and oxLDL levels above the cut-off. Right panel: quantification of the myocardial fat mass  of the two  ACM subpopulations (above or below the cut-off) whose  MRI  was available for re-analysis (n=14 oxLDL<86ng/ml, n=25 oxLDL>86ng/ml; Two-tailed Student's t-test).",
    "molecules": "fat, oxLDL"
  },
  {
    "caption": "G) Kaplan-Meier  analysis of actual MAE free  survival  of patient belonging to the two ACM  cohort subgroups in the first 5-year follow-up (n=26 oxLDL<86ng/ml vs. n=41oxLDL>86ng/ml; Log-Rank p<0.0001; HR=0.223[0.116-0.428]).",
    "molecules": "oxLDL"
  },
  {
    "caption": "B) Left panels: representative images of MDA immunostaining (green) on ACM  and HC C-MSC  in GM. Nuclei  are counterstained  with Hoechst33342 (blue). Right panel: image quantification (n=4 biological replicates ACM, n=5 biological replicates HC; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05, ** p<0.01.",
    "molecules": "Hoechst33342, MDA"
  },
  {
    "caption": "F) Top panels: representative images of internalization of oxLDL (red) in HC and ACM cells, cultured either in GM or in AM, and subjected to 10μg/ml  DiI-oxLDL treatment. Nuclei are  counterstained with Hoechst33342 (blue). Bottom panel: quantification of the relative mean DiI fluorescence for each sample, measured by FACS analysis (n=3 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01. ",
    "molecules": "Hoechst33342, DiI, oxLDL"
  },
  {
    "caption": "A) Left panels: representative images of ORO staining   on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ  and GAPDH protein expression  of ACM  and HC C-MSC protein extracts in  AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "ORO, oxLDL"
  },
  {
    "caption": "B) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "13HODE, NAC, ORO"
  },
  {
    "caption": "C) Left panel: representative images of Western Blot of  CD36, PPARγ and GAPDH protein expression of ACM  and HC C-MSC protein extracts in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L  NAC or both (n=8 biological replicates). Right panels: d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001. ",
    "molecules": "13HODE, NAC"
  },
  {
    "caption": "D) Left panel: representative images of internalization  of  oxLDL (red) in ACM C-MSC  treated with scramble siRNA or CD36 siRNA, cultured in  AM  and subjected to 10μg/ml  DiI oxLDL treatment. Right panel: quantification of the  DiI fluorescence normalized on  nuclei number (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.",
    "molecules": "DiI, oxLDL"
  },
  {
    "caption": "E) Left panel: representative images of internalization  of oxLDL (red) in ACM C-MSC cultured in AM or  AM+5μM GW9662 and subjected to 10μg/ml DiI oxLDL treatment.  Nuclei  are  counterstained with  Hoechst33342  (blue). Right panel: quantification of the DiI fluorescence  normalized on nuclei number for each sample (n=3 biological replicates, arbitrary units; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01.",
    "molecules": "Hoechst33342, DiI, GW9662, oxLDL"
  },
  {
    "caption": "F) Left panel: representative images of Western Blot of CD36  and GAPDH expression of protein extracts of ACM C-MSC   cultured in AM or  AM+5μM GW9662. Right panels: d.a. normalized on the house-keeping GAPDH  (n=7 biological replicates; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05; ** p<0.01. ",
    "molecules": "GW9662"
  },
  {
    "caption": "C) Left panels: representative images of ORO staining  of  HFD-fed WT and Pkp2+/-   cardiac sections. Right panel: quantification of ORO positive area percentage (n=10). For comparison, quantification of ORO positive area of cardiac sections of  CD-fed WT and Pkp2+/- mice (n=9; Two-Way Anova) is shown (Appendix Figure S3). D) Representative images of  PPARγ  (green) immunostaining on  HFD-fed WT and  Pkp2+/- mice cardiac sections (n=10; Two-Way Anova). Quantification of the PPARγ staining in HFD is shown relative to the values of CD (n=9). E) Representative images of MDA (green)  immunostaining  on  HFD-fed WT and Pkp2+/- mice cardiac  sections (n=10; Two-Way Anova). Quantification of the  MDA staining  in HFD is shown relative to the values of  CD  (n=9). F) Representative images of CD36 immunostaining (green) on HFD-fed WT and Pkp2+/-  mice cardiac sections (n=10; Two-Way Anova). Nuclei  are counterstained with Hoechst33342 (blue). Quantification of the staining  in HFD  is shown relative to the values of CD  (n=9). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.",
    "molecules": "Hoechst33342, MDA, ORO"
  },
  {
    "caption": "C) Left panel: representative images of ORO staining of cardiac sections  of Pkp2+/- mice, fed a 3-month HFD plus atorvastatin. Right panel: quantification of the percentage of ORO positive area (n=9) is compared to that in HFD (as in Figure 6C; Two-tailed Student's t-test). D) Representative images of  PPARγ immunostaining (green) on  cardiac  sections of Pkp2+/- mice fed a 3-month  HFD plus atorvastatin  (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6D; Two-tailed Student's t-test). E) Representative images of MDA immunostaining (green) on  cardiac  sections  of Pkp2+/- mice fed a 3-month HFD  plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in  HFD  and relative to WT in CD (as in Figure 6E; Two-tailed Student's t-test). F) Representative images of CD36 immunostaining (green) on cardiac sections of Pkp2+/-  mice  fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- HFD  and relative to WT in  CD (as in Figure 6F; Two-tailed Student's t-test). Nuclei  are counterstained  with  Hoechst33342  (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.",
    "molecules": "Hoechst33342, atorvastatin, MDA, ORO"
  },
  {
    "caption": "RT-qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR+/+ and LXR−/− primary BMMs. Fold changes are shown relative to LXR+/+ 0 h. Error bars represent ± SEM for n = 4-8 (**P = 0.001 at 8 h and **P = 0.00004 at 16 h versus LXR−/−; **P = 0.0003 at 0 h versus LXR+/+); bottom, proposed model explaining the ligand‐dependent induction of Abca1. Following ligand stimulation, LXR-corepressor complexes are exchanged for LXR-coactivator complexes, which promote gene expression. The LXR-cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown.",
    "molecules": "T0901317"
  },
  {
    "caption": "Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length SND1, SART1, or HMBOX1. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. A diagram of the reporter construct is shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 9 (**P = 0.001, *P = 0.011 for SND1, *P = 0.046 for SART1).",
    "molecules": "T0901317"
  },
  {
    "caption": "Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length NCOA5 or an NCOA5 mutant lacking the NH2‐terminus. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. Diagrams of the reporter constructs are shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 6-12 (**P 0.001, *P = 0.035).",
    "molecules": "T0901317"
  },
  {
    "caption": "RT-qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus. LXR ligand stimulations were performed with 1 μM T0901317 for 18 h. Fold changes are shown relative to vehicle‐stimulated control. Error bars represent ± SEM for n = 4 (*P = 0.029).",
    "molecules": "T0901317"
  },
  {
    "caption": "In vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs. Assays were performed for 2 h in the presence or absence of 2 μM T0901317. Samples were immunoblotted as indicated. Note the requirement of the NCOA5 NH2‐terminus for interacting with LXRα.",
    "molecules": "GST, T0901317"
  },
  {
    "caption": "Immunoprecipitation of Protein C‐tagged LXRα from stable RAW 264.7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h, followed by immunoblotting for endogenous NCOA5 or over‐expressed LXRα. Protein immunoblots of nuclear extracts are shown below.",
    "molecules": "T0901317"
  },
  {
    "caption": "NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the ligand‐stimulated association of NCOA5. Error bars represent ± SEM for n = 4-9 (*P = 0.02).",
    "molecules": "T0901317"
  },
  {
    "caption": "NCOA5 ChIP assays from LXR−/− BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6 (**P = 0.0003 for vehicle and **P = 0.002 for T0901317).",
    "molecules": "T0901317"
  },
  {
    "caption": "RT-qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non‐silencing or Ncoa5‐specific shRNA. Fold changes are shown relative to shControl 0 h. Note the elevated Abca1 expression at 24-30 h following loss of NCOA5. Error bars represent ± SEM for n = 4-6 (**P = 0.0007 at 24 h and **P = 0.004 at 30 h, *P = 0.02).",
    "molecules": "T0901317"
  },
  {
    "caption": "Quantification of ABCA1 immunoblots from primary BMMs infected as in (A). Representative immunoblot is shown in Supplementary Fig S7E. LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h. Error bars represent ± SEM for n = 2 (**P = 0.007).",
    "molecules": "T0901317"
  },
  {
    "caption": "A, B RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317 alone or together with 6 μg/ml PolyIC (A) or 10 ng/ml LPS (B). Fold changes are shown relative to vehicle‐stimulated shControl. Note only the loss of TLR3‐mediated repression following Ncoa5 silencing. Error bars represent ± SEM for n = 4-10 (**P = 0.0003 for A, **P = 0.0004 for shControl in B, **P = 0.0002 for shNcoa5 in B versus T0901317).",
    "molecules": "LPS, PolyIC, T0901317"
  },
  {
    "caption": "C, D RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h unstimulated, or with 6 μg/ml PolyIC (C) or 10 ng/ml LPS (D). Fold changes are shown relative to unstimulated shControl. Error bars represent ± SEM for n = 4-6 (**P = 0.0006 for C, **P = 0.00000001 for shControl in D, **P = 0.000001 for shNcoa5 in D, *P = 0.013 versus T0901317).",
    "molecules": "LPS, PolyIC, T0901317"
  },
  {
    "caption": "E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the increased occupancy only in the presence of both lipid and inflammatory ligands. Error bars represent ± SEM for n = 8-12 (**P = 0.0007 versus T0901317).",
    "molecules": "lipid, PolyIC, T0901317"
  },
  {
    "caption": "F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg/ml PolyIC or left unstimulated. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6.",
    "molecules": "PolyIC"
  },
  {
    "caption": "G Cholesterol efflux assays to APOA1 from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Agonist stimulations were performed for 6 h. Error bars represent ± SEM for n = 3 (*P = 0.02 versus T0901317).",
    "molecules": "Cholesterol, T0901317"
  },
  {
    "caption": "A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 TSS. Error bars represent ± SEM for n = 6-9 (**P = 0.0001 in A, **P = 0.002 in B versus T0901317).",
    "molecules": "PolyIC, T0901317"
  },
  {
    "caption": "C, D RNAPII ChIP assays as in (A, B) but performed from LXR−/− BMMs. Error bars represent ± SEM for n = 5-9 (**P = 0.000000001, *P = 0.02 versus T0901317).",
    "molecules": "T0901317"
  },
  {
    "caption": "E RT-qPCR of Abca1 expression from LXR+/+ versus LXR−/− BMMs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 together with 6 μg/ml PolyIC. Fold changes are shown relative to vehicle‐stimulated LXR+/+. Error bars represent ± SEM for n = 4 (**P = 0.004, *P = 0.012 versus T0901317).",
    "molecules": "PolyIC, T0901317"
  },
  {
    "caption": "F RNAPII pSer2 ChIP assays from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 + 6 μg/ml PolyIC. qPCR was performed for the Abca1 TSS. Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs (**P = 0.004 versus baseline). Error bars represent ± SEM for n = 3 from 11 mice.",
    "molecules": "PolyIC, T0901317"
  },
  {
    "caption": "D) Western blot of LRP6 and TBLR1 in the miR-cluster-Dox-MCF-10A model upon upregulation of miR-cluster with 100 ng/ml of doxycycline for the specified number of days. The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β-actin. The expression level of miR-424 and miR-503 is indicated in the bar graph below the blots. UN indicates samples untreated with doxycycline. The error bars show the standard deviation.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation.",
    "molecules": "Dox, dox"
  },
  {
    "caption": "B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection.",
    "molecules": "Dox"
  },
  {
    "caption": "C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.",
    "molecules": "Dox"
  },
  {
    "caption": "(D, E) Immunoblot analysis of indicated proteins from d2HEK cells treated with different concentrations of MG132 for 5 h (D) or transfected with indicated amounts of BAG1S plasmid for 24 h (E).",
    "molecules": "MG132"
  },
  {
    "caption": "(A) 293 cells were transfected with nonsense (nons), bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 (both 10 μg/ml; Pep.A/E64) or DMSO as control, followed by immunoblot analysis of the indicated proteins.",
    "molecules": "DMSO, E64, Pep.A, pepstatinA"
  },
  {
    "caption": "(C) Diagram shows the autophagic flux of 293 cells with differently modulated BAG1 and BAG3 levels as described in (A) and (B). Autophagic flux was determined by the strength of LC3‐II accumulation in a 2‐h treatment period with Pep.A/E64. Therefore, normalised LC3‐II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep.A/E64. Values are expressed as mean±s.e.m. *P0.05 versus control‐transfected cells, n=3.",
    "molecules": "E64, Pep.A"
  },
  {
    "caption": "(D) Young and old I90 cells were treated for 2 h with bafilomycin A1 (BafA1, 2 μM) or DMSO as control followed by immunoblot analysis of indicated proteins.",
    "molecules": "bafilomycin A1, DMSO"
  },
  {
    "caption": "(F) Indirect immunofluorescence staining of endogenous LC3 (green), SQSTM1 (red) and WIPI1 (white) in I90 cells of young and old age. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells with indicated characteristics counted as in Figure 3E.",
    "molecules": "DNA"
  },
  {
    "caption": "(G) Old and young I90 cells were treated for 1 h with DMSO as control (C), lactacystin (L, 2 μM) or NH4Cl (20 mM) plus leupeptin (Leu, 5 μM) (N, NH4Cl/Leu). Western‐blot analyses were performed for detection of indicated proteins. In the diagram (right panel), levels of polyUb‐proteins and SQSTM1 are depicted after normalisation to corresponding Tubulin levels. Values are expressed as mean±s.e.m. *P0.05 versus old control, #P0.05 versus young control, n=3.",
    "molecules": "NH4Cl, DMSO, lactacystin, Leu, leupeptin"
  },
  {
    "caption": "(H) I90 cells of old (upper panel) and young (lower panel) age were transfected with atg7 or nonsense (nons) siRNA. After transfection for 4 days , the cells were treated with NH4Cl/Leu or DMSO for 1 h followed by fractioning of cell lysates in TritonX‐100 (TX‐100) soluble and insoluble material. Equal protein amounts of both fractions were directed to immunoblot analysis for analysis of indicated proteins. Gapdh and Histone H3 were used as loading controls of soluble and insoluble fractions, respectively.",
    "molecules": "NH4Cl, DMSO, Leu, TritonX‐100"
  },
  {
    "caption": "(F) Same analysis as in Figure 5G but old I90 cells transfected as in (A) were used. C, control; L, lactacystin, N, NH4Cl/Leu. Values are expressed as mean±s.e.m. *P0.05 versus nons control, or as indicated, n=3.",
    "molecules": "NH4Cl, lactacystin, Leu"
  },
  {
    "caption": "(A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm.",
    "molecules": "Tg"
  },
  {
    "caption": "(B) EM images of Tg‐treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm.",
    "molecules": "Tg"
  },
  {
    "caption": "(C) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β‐GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm.",
    "molecules": "Tg"
  },
  {
    "caption": "(D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A‐bodipy to stain ER and Golgi. Representative flow‐cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P0.05 versus WT group.",
    "molecules": "Tg"
  },
  {
    "caption": "(A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.",
    "molecules": "Tg"
  },
  {
    "caption": "(B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.",
    "molecules": "Bref, brefeldin A, Tg, Tm, tunicamycin"
  },
  {
    "caption": "(D) Flow‐cytometry analysis of the Sub G1 DNA fragmentation in methanol‐fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.",
    "molecules": "Tg"
  },
  {
    "caption": "(E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3‐L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.",
    "molecules": "Tg"
  },
  {
    "caption": "(F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.",
    "molecules": "Tg"
  },
  {
    "caption": "(G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z‐VAD‐fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P0.05 versus WT+Tg group.",
    "molecules": "z‐VAD‐fmk, Tg"
  },
  {
    "caption": "(H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm.",
    "molecules": "CHX, Tg"
  },
  {
    "caption": "(I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.",
    "molecules": "Tg"
  },
  {
    "caption": "(A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P0.05 versus WT group; #P0.05 versus WT non‐Tg‐treated group.",
    "molecules": "Tg"
  },
  {
    "caption": "(B, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b‐I and LC3b‐II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b‐II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P0.05 versus WT+Baf+Tg group.",
    "molecules": "Baf, Bafilomycin, Tg"
  },
  {
    "caption": "(C) WT or Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm.",
    "molecules": "Bref, Brefeldin, Tg, Tm, tunicamycin"
  },
  {
    "caption": "(G) Expression of Beclin‐1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.",
    "molecules": "Tg"
  },
  {
    "caption": "(B) Immunodetection of p‐PERK,PERK, GADD34, p‐eIF2α, eIF2α, ATF4, CHOP, and XBP‐1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated.",
    "molecules": "Tg"
  },
  {
    "caption": "(D) Immunodetection of p‐eIF2α, eIF2α, and ATF4 in WT and Mfn2 KO cells treated with 0.5 μg/ml Tunicamycin (Tm) for the times indicated.",
    "molecules": "Tm, Tunicamycin"
  },
  {
    "caption": "(E) Transcriptional activity driven by ATF6. WT and Mfn2 KO cells were co‐transfected with the 5xATF6‐GL3 and TK‐Renilla plasmids and treated with 1 μM Tg for 24 h after transfection. Data are mean±s.e.m. (n=4). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.",
    "molecules": "Tg"
  },
  {
    "caption": "(B) Caspase activity was detected by measurement of DEVD-AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.",
    "molecules": "Tg"
  },
  {
    "caption": "(E, F) Immunodetection of LC3b‐I and ‐II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.",
    "molecules": "Baf, Tg"
  },
  {
    "caption": "(A) Immunodetection of XBP‐1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed to XBP‐1 (XBP‐1 KD), and treated with Tg for 6 h.",
    "molecules": "Tg"
  },
  {
    "caption": "(C, D) Total and cleaved caspase 3 levels were detected by western blot. WT and Mfn2 KO cells subjected or not to XBP‐1 silencing (Scr, and KD) and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.",
    "molecules": "Tg"
  },
  {
    "caption": "(E, F) Immunodetection of LC3b‐I and ‐II in Scr and XBP‐1 KD WT or Mfn2 KO cells treated as indicated (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). #P0.05 versus Scr group.",
    "molecules": "Baf, Tg"
  },
  {
    "caption": "(G) Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b and subjected or not to XBP‐1 silencing (Scr or KD) were treated with 1 μM Tg, 100 ng/ml Bref, or 0.5 μg/ml Tm for 24 h and examined by confocal microscopy. Scale bar: 10 μm.",
    "molecules": "Bref, Tg, Tm"
  },
  {
    "caption": "(E) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4‐phenyl butyric acid for 6 h and stained with MitoTracker Green.",
    "molecules": "4‐phenyl butyric acid, TUDCA"
  },
  {
    "caption": "(F) Flow‐cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO cells",
    "molecules": "ROS"
  },
  {
    "caption": "(A) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells. Cells were loaded with Rhod‐2 and then treated with 2.5 mM CaCl2 (left). Calcium uptake was monitored by confocal microscopy. Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod‐2 and incubated with 2.5 mM CaCl2 for 5 min (right). Scale bars: 10 μm.",
    "molecules": "CaCl2"
  },
  {
    "caption": "(B-E) Mitochondrial oxygen consumption (OCR) was measured in Scr and PERK KD Mfn2 KO cells (B), Scr and PERK KD WT cells (C), Scr and PERK KD Mfn1 KO cells (D), and WT cells stably expressing PERK‐myc (E). Data are mean of three independent experiments. The following parameters were measured: oxygen consumption under routine conditions (DMEM with 5.5 mM glucose), maximal respiratory capacity reached after uncoupling with FCCP, and respiratory leak, measured after inhibition of ATP synthase with oligomycin. Data are mean±s.e.m. *P0.05 versus Scr group or versus the empty plasmid group.",
    "molecules": "FCCP, glucose, oligomycin"
  },
  {
    "caption": "(e) Percent survival of Col-0 plants with and without ACC under HS, HS+ACC, LAT and LAT+ACC conditions. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).",
    "molecules": "ACC"
  },
  {
    "caption": "F-T. Representative images of 2 dpf zebrafish which displayed the rescue of the vascular integrity defects in the progeny of an outcross of veal2gib005Δ8/+ zebrafish upon complementing with the wild type (WT) veal2 RNA. (F-J) Control zebrafish embryos. (K-O) veal2gib005Δ8/+ embryos injected with vehicle control. (P-T) veal2gib005Δ8/+ embryos complemented with veal2 RNA. (F,K,P) bright field. (G,L,Q) mRFP. (H,M,R) Animals stained with O-dianisidine stain. (I,N,S) eGFP. (J,O,T) merged eGFP and mRFP filters. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (F-H,K-M,P-R) magnification-5X, scale bar-100μm (I-J,N-O,S-T) magnification-20X, Scale bar-50μm.",
    "molecules": "O-dianisidine"
  },
  {
    "caption": "F-K. Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8/+ zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8/+. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure. (F-I) Magnification-5X, Scale bar-100μm, (J-K) Magnification-20X, Scale bars-50μm.",
    "molecules": "Enzastaurin"
  },
  {
    "caption": "R-T. Co IF for PRKCB and smFISH of VEAL2 highlight their colocalization. (R) PRKCB in the GFP channel. (S) VEAL2 in CAL-fluor red (610nM). (T) Merged image for PRKCB and VEAL2. Magnification-100X, scale bar-20μm.",
    "molecules": "CAL-fluor red"
  },
  {
    "caption": "B. Bright-field picture of a 0B K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=3). C. Bright-field picture of EB K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=4). D. Bright-field pictures of K8-eYFP LB embryos (n=30), and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas. K8-eYFP and DAPI appear in yellow and cyan, respectively. Da",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Immunostaining for Cdx2 (n=20). F. Immunostaining for DAPI (cyan, left), Sox2 (middle), and Gata2 (right). Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm. G. Immunostaining for Cebpb, detectable in most cells with variable intensity. H. Immunostaining for Fn1 (n=22), Dlk1 (n=8), and Acta2 (n=12), primarily expressed in Chorion Mesenchyme. I. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Dlk-1 (n=8 and 4, respectively) and Acta2 (n=6 and 3). Data",
    "molecules": "DAPI"
  },
  {
    "caption": "(G and H) Real-time PCR analysis of TNF-α, IL-6 and IL-1β (G) and of IFN-β, ISG54 and ISG56 (H) mRNA production in RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times (average mRNA production in si-NC cells at 0 hour = 1).",
    "molecules": "LPS"
  },
  {
    "caption": "(I) ELISA analysis of TNF-α, IL-6, IL-1β and IFN-β production in the culture supernatants of human PBMCs transfected with si-NC and si-p38IP and then stimulated with 1μg/mL LPS for 24 hours.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Statistical analysis of p65 nuclear translocation of sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated with 50 ng/ml PMA plus 1µM ionomycin for the indicated times. p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(C) Luciferase reporter assay of sh-NC and sh-p38IP Jurkat E6.1 cells transfected with NF-κB luc plus an internal control Renilla luciferase reporter for 36 hours, followed by stimulation with ( + ) or without ( - ) 50ng/ml PMA plus 1 µM ionomycin for 8 hours. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(D,E) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) sh-NC or sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times and lysed, followed byTAK1 immunoprecipitation. TAK1 beads were washed three times with lysis buffer and two times with kinase buffer, then resuspended in kinase buffer containing 100 μM ATP and 1μg recombinant GST-IKKβ(111-250aa) protein as substrate to perform in vitro kinase assay, and then immunoblot analysis with p-IKKβ antibody.",
    "molecules": "ATP"
  },
  {
    "caption": "(H) Jurkat TAg T cells transfected with cMyc vector or cMyc-p38IP were stimulated with 10μg/ml anti-CD3 for the indicated times, and then were lysed and immunoprecipitated with anti-TAK1 antibody and immunoblotted with anti-K63Ub, anti-cMyc and anti-TAK1 antibodies. Lysates were blotted with the indicated antibodies, and actin served as the loading control.",
    "molecules": "Ub"
  },
  {
    "caption": "(I) sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-K63Ub and anti-TAK1. Lysates were immunoblotted with the indicated antibodies.",
    "molecules": "Ub"
  },
  {
    "caption": "(J) Jurkat TAg T cells transfected and stimulated as in (H) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2.",
    "molecules": "Ub"
  },
  {
    "caption": "(K) sh-NC and sh-p38IP Jurkat E6.1 cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2. Data (B-F, H-K) are representatives of at least three independent experiments.",
    "molecules": "Ub"
  },
  {
    "caption": "(B) HEK293T cells transfected with Flag-tagged TAB2 (open arrow) or TAB2-ub (solid arrow) were lysed and immunoprecipitated with anti-Flag, and then immunoblotted with the indicated antibodies. The asterisk indicates a non-specific band. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.",
    "molecules": "ub"
  },
  {
    "caption": "(C) HEK293T cells co-transfected with HA-tagged p38IP together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow) were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. Asterisk indicates non-specific bands. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.",
    "molecules": "ub"
  },
  {
    "caption": "(D) HEK293T cells co-transfected with HA-tagged p38IP, Flag-tagged TAB2-ub, together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow), were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.",
    "molecules": "ub"
  },
  {
    "caption": "(A) GSH-Sepharose beads-immobilized full length or truncated GST-TAK1 proteins were incubated with K63-linked polyUb chains in 300 μl binding buffer, followed by washing and resolving in SDS-PAGE, and then immunoblotted with anti-ub. GST-fused NEMO serve as positive control and NEMO L329P mutant as negative control. The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST-fused proteins (lower panel).",
    "molecules": "Sepharose, GSH, Ponceau S, polyUb, ub"
  },
  {
    "caption": "(C and D) Immobilized GST-TAB2 was pre-incubated with K63-linked polyUb chains and then eluted from beads, followed by incubation with eluted GST-TAK1 and GST-p38IP as shown in (B). Incubation mixture were immunoprecipitated with anti-TAB2 or anti-TAK1, and then immunoblotted with anti-ub, anti-TAB2, anti-TAK1 and anti-p38IP. (E) Statistical analysis of polyUb binding to TAB2 or TAK1 in (C and D). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).",
    "molecules": "polyUb, ub"
  },
  {
    "caption": "(F) Immobilized GST-TAB2 and GST-TAK1 were respectively pre-incubated with K63-linked polyUb chains or not as indicated, then eluted from beads, followed by coincubation as indicated. Incubation mixture were immunoprecipitated with anti-TAB2, and then immunoblotted with anti-TAB2 and anti-TAK1. The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).",
    "molecules": "polyUb"
  },
  {
    "caption": "(G) Immobilized GST-TAK1 was pre-incubated with K63-linked polyUb chains or not, then eluted from beads, followed by incubation with eluted GST-p38IP. Incubation mixture were immunoprecipitated with anti-TAK1, and then immunoblotted with anti-TAK1 and anti-p38IP. The relative intensity of immunoprecipitated p38IP was calculated and shown in the right. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).",
    "molecules": "polyUb"
  },
  {
    "caption": "(D) GST pull down assay. 3μg recombinant GST-T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg/μl, after 4 hrs incubation, washed beads with cell lysis buffer five times and subjected to SDS-PAGE for immunoblotting with anti-TAK1.",
    "molecules": "Sepharose, glutathione"
  },
  {
    "caption": "(E) sh-NC and sh-p38IP Jurkat E6.1 cells transfected with si-NC, si-p38IP, Flag or Flag-USP4 as indicated were stimulated with anti-CD3/CD28 for 0 and 15 min at 48 hours post transfection, and then lysed and immunoprecipitated with anti-TAK1 under stringent conditions and immunoblotted with anti-ub. Statistical analysis of p-p38 levels was shown below the blot. **P < 0.01; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).",
    "molecules": "ub"
  },
  {
    "caption": "j, GFP-LC3 punctae per crypt after treatment with tamoxifen, and vehicle or salubrinal (n = 10; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).",
    "molecules": "salubrinal, tamoxifen"
  },
  {
    "caption": "k, Enteritis histology score after salubrinal and tamoxifen co-treatment (n = 12/14/13; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P 0.05, ***P 0.001.",
    "molecules": "salubrinal, tamoxifen"
  },
  {
    "caption": "d, TUNEL+ intestinal epithelial cells (IECs) per 100 crypts after BAY11-7082 or vehicle treatment (n = 3/4/4; one-way ANOVA with post-hoc Holm's-corrected unpaired Student's t-test; mean ± s.e.m.). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n = 10/10/9; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).",
    "molecules": "BAY11-7082"
  },
  {
    "caption": "a, Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days (n = 3). b, Enteritis histology score for experiment as in a (n = 4; median shown; Mann-Whitney U-test).",
    "molecules": "rapamycin"
  },
  {
    "caption": "Concentration of total cell-free DNA (cfDNA) in 13 CSF samples. Samples in which SCNAs were detected are shown in dark blue, and tended to have higher levels of total cfDNA",
    "molecules": "cfDNA, DNA"
  },
  {
    "caption": "Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples. Shared detection in tissue and CSF is indicated in green, detection of the alteration only in tissue in orange, and non-detection in blue. The top bars indicate the cfDNA concentration (copies/mL; in a range of purples), the size of the tumors (in a range of browns), the type of glioma (in a range of blues), and whether the tumor was in direct contact with the CSF or not (based on MRI, green or red, respectively). Samples are ranked from the left to right by decreasing concentration of cfDNA (copies/mL)",
    "molecules": "cfDNA"
  },
  {
    "caption": "A) Fragment size distribution, determined by sWGS, in CSF (blue line), plasma (red dashed line) and urine (black dashed line) from patient G1. The 3 samples were collected simultaneously before initiation of treatment. B) Fragment size distribution of cfDNA, determined by sWGS, in CSF from 13 glioma patients. The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~145 bp and ~167 bp. The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red. All samples were collected simultaneously before initiation of treatment C) Cumulative frequency analysis of the average density of fragment size in CSF (blue) and plasma (red). The vertical dashed line represents 167 bp",
    "molecules": "cfDNA, DNA"
  },
  {
    "caption": "Log2 ratio of the difference in cfDNA fragment sizes between CSF samples with detected SCNAs and those samples in which no SCNAs were detected",
    "molecules": "cfDNA"
  },
  {
    "caption": "The amplitude of the 10 bp periodic peaks in fragment size is a signature for non-tumor DNA in the CSF. The amplitude of the 10 bp periodic peaks in fragment length distribution (in the range between 75 and 150 bp, see Methods) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation (Pearson -0.85; p=0.0002)",
    "molecules": "DNA"
  },
  {
    "caption": "E. Substrate-binding pocket mutations Y90A and Y74A/Y90A/T105A/S107K affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-262 cells to varying degrees. Five-fold serial dilutions of mis18-262 cells transformed with plasmids harbouring the indicated spMis18fl constructs, spotted on PMG - uracil + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.",
    "molecules": "thiamine, uracil"
  },
  {
    "caption": "B & C. Dimer-II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-818 and mis18-262 cells, while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization-dependent manner. Five-fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome, spotted on complete PMG + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.",
    "molecules": "thiamine"
  },
  {
    "caption": "D. Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres. qChIP analyses of spMis18fl-GFP association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates.",
    "molecules": "thiamine"
  },
  {
    "caption": "C DNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM pyridostatin (PDS) for 16 hours and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm. D Quantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; NS, P > 0.5. E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5.",
    "molecules": "DNA, PDS, pyridostatin"
  },
  {
    "caption": "F BRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM pyridostatin (PDS) for 24 hours. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.",
    "molecules": "NU-7441, NU, PDS, pyridostatin"
  },
  {
    "caption": "G Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for six days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates.",
    "molecules": "NU-7441, PDS, pyridostatin"
  },
  {
    "caption": "Quantitative RT-PCR of cells grown as in (A) and treated with 10 µM pyridostatin (PDS) for 3 days was performed using primers specific for the indicated genes. mRNA levels are expressed relative to GAPDH and to -DOX (+BRCA2) cells. Error bars represent the SEM of n = 3 independent experiments, each performed in technical triplicate. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; ***, P ≤ 0.001.",
    "molecules": "DOX, PDS, pyridostatin"
  },
  {
    "caption": "BRCA2+/+ and BRCA2-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. Tubulin and GAPDH were used as loading controls.",
    "molecules": "PDS, pyridostatin"
  },
  {
    "caption": "H1299+shBRCA2DOX cells treated as in (B) were fixed and prepared for immunofluorescence with antibody against cGAS. DNA was counterstained with DAPI. Scale bar represents 20 µm. Quantification of cGAS-positive micronuclei per cells shown in (E). Graph and error bars represent the mean and SEM of of n = 3 independent experiments. A minimum of 250 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; **, P ≤ 0.01; NS, P > 0.5.",
    "molecules": "DAPI, DOX"
  },
  {
    "caption": "FVB female mice were injected intramuscularly with (A) BRCA1-proficient KP3.33 (Brca1+/+) or (B) BRCA1/53BP1-deficient KB1PM5 (Brca1-/-, Tp53bp1-/-) mouse mammary tumour cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 16, using an unpaired two-tailed t-test. ****, P ≤ 0.0001; NS, P > 0.5.",
    "molecules": "PDS, Pyridostatin, talazoparib"
  },
  {
    "caption": "BRCA1+/+, BRCA1-/- and BRCA1-/-/TP53BP1-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) or 2 μM olaparib for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.",
    "molecules": "olaparib, PDS, pyridostatin"
  },
  {
    "caption": "B VHIO179 PDTXs were grafted into CB17-SCID female mice. Pyridostatin was administered intravenously (7.5 mg/kg/day) over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 7 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 24, using an unpaired two-tailed t-test. ****, P ≤ 0.0001.",
    "molecules": "Pyridostatin"
  },
  {
    "caption": "(F) PARN detection by Western blot in the indicated conditions. Expression was induced by incubating cells with 10 ng/ml doxycycline for 72 h. Actin is used as loading control. PARN/actin ratios, normalized to doxycycline-treated WT/PARN cells, are shown below.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(G) In vitro deadenylation activity assay using protein extracts from doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(H) qRT-PCR analysis of the indicated gene transcripts in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to WT/empty. Three independent doxycycline inductions were performed for each cell line. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(K) qRT-PCR analysis of TERRA from the indicated chromosome ends in doxycycline-treated KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to KO/empty. Three independent doxycycline inductions were performed for each cell line. Error bars indicate s.e.m..",
    "molecules": "TERRA, doxycycline"
  },
  {
    "caption": "(A) Analysis of telomeric aberrations by Telo-FISH in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Two independent experiments were performed. Counted chromatids: WT/empty: n=1824; WT/PARN: n= 1744; KO/empty: n=1452; KO/PARN: n=1920. Averages are shown and χ2-tests were applied to compare KO/empty with either WT/empty or KO/PARN.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) TRF analysis of telomere length (kb) in KO/empty and KO/PARN cells treated with 10 ng/ml Dox for 72h. A control Western blot with PARN and Actin is shown below for the corresponding samples.",
    "molecules": "Dox"
  },
  {
    "caption": "Immunofluorescence staining for neuronal marker Tuj1 in neuronal cultures grown on poly-ornithine/laminin at 21 days of differentiation.",
    "molecules": "laminin, poly-ornithine"
  },
  {
    "caption": "B. LN229 cells treated with or without Wnt3a (50 ng/ml) were pulsed with [35S] methionine for 30 min and chased for the times indicated. Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS-PAGE and autoradiography. The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software, and the densities of the FoxM1 bands at time 0 were set as 100%. Values are mean ± SD from two independent experiments.",
    "molecules": "methionine"
  },
  {
    "caption": "D. FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl (10 mM) and CHX (100 µg/ml) for indicated times.",
    "molecules": "CHX, LiCl"
  },
  {
    "caption": "E. GSK3 wild-type (WT) and knockout (KO) cells were treated with CHX (100 µg/ml) for indicated times, and FoxM1 and GSK3α/β expression levels were determined by Western blotting.",
    "molecules": "CHX"
  },
  {
    "caption": "F. Flag-FoxM1 and GSK3βKA or GSK3βKD plasmids were co-transfected into 293T cells for 48 hr. The cells were then treated with CHX (100 µg/ml) for indicated times. Exogenous Flag-FoxM1 expression level was determined by Western blotting.",
    "molecules": "CHX"
  },
  {
    "caption": "D. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmid were co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody, followed by IB with Flag and HA antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "E. HA-ubiquitin was co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 phospho-S474 antibody, followed by IB with FoxM1 and HA antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "F. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 antibody, followed by IB with FoxM1 and HA antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "E. Axin siRNA or control siRNA with HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "F. Wild type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "G. HA-Ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells, after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "B. 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6-well plates. After 36 hr of transfection, cells were treated with 100 µg/ml CHX for 0, 2, 4, or 6 hr. FoxM1 protein levels in the cells were then determined by Western blotting.",
    "molecules": "CHX"
  },
  {
    "caption": "G. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "H. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without siUSP5 into U87 cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a and 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "A,B) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to (A) hypercontraction and (B) increased contraction force in FLNAΔECS aortae in myograph chambers. Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae. For each condition, 10-12 aortic rings from at least 4 wild-type (wt) and 4 FLNAΔECS mice were used. Data shown as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001 (Student's t test)",
    "molecules": "U46619"
  },
  {
    "caption": "(C) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt (red) and FLNAΔECS (green) primary vascular smooth muscle cells (vSMC) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619. Data shown as mean ± SD from three independent experiments.",
    "molecules": "U46619"
  },
  {
    "caption": "(D) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619. Data shown as mean ± SD from three independent experiments. P value <0.05 (Student's t test) was considered significant. The difference between wt and FLNAΔECS was significant at every concentration of U46619.",
    "molecules": "U46619"
  },
  {
    "caption": "(B) Rhotekin Pull down activation assay was performed on wt and FLNAΔECS vSMC lysates and activated (GTP) bound RhoA was compared between the 2 genotypes. FLNAΔECS vSMCs show more GTP-loaded RhoA than wt vSMCs. Tubulin was used as a loading control. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)",
    "molecules": "GTP"
  },
  {
    "caption": "(C) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin light chain phosphatase (pMYPT1) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor-specific MYPT1 antibody. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)",
    "molecules": "U46619"
  },
  {
    "caption": "(A) Dorsal aortae were fixed, cross-sectioned and then stained with Elastica van Gieson stain to visualize collagen around the blood vessel. 6 independent mice (5-6 months) of each genotype were used for the analysis. Scale bar: 50 μm. (B) Plot shows individual data points (black-wt, red-mutant) of adventitial area in both the genotypes. Measurements show increased adventitial area in FLNAΔECS aortae. P value <0.05 was considered significant (Student's t test) ",
    "molecules": "Elastica van Gieson, collagen"
  },
  {
    "caption": "(C) Representative heart sections of a 5-6 months old mice stained with Masson Trichrome shows increased collagen (blue) around coronary vessels (marked by arrowheads) Scale bar: 50 μm. (D) Quantification shows a significant increase in perivascular fibrosis in FLNAΔECS hearts. Four sections from each heart were measured. Four mice analyzed for each genotype. P value <0.05 considered significant. (mixed model approach) ",
    "molecules": "collagen"
  },
  {
    "caption": "(G) Heart cross-sections were stained with FITC-WGA (Wheat germ agglutinin) to visualize cell membranes of cardiomyocytes. Scale bar: 20 μm. (H) Respective cell areas measured by Image J software and represented by box plots for each animal (six per genotype). Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value. P value <0.05 (mixed model approach) was considered significant. ",
    "molecules": "FITC"
  },
  {
    "caption": " (D) Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to sample cultured in the presence of control DMSO (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test). ",
    "molecules": "DAPT, DMSO"
  },
  {
    "caption": " Quantitative RT-PCR expression analysis of miR-17 in human CD34+ thymocyte progenitors after 5 and 2 days, respectively, of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the sample cultured in the presence of control DMSO (F) (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown). ",
    "molecules": "DAPT, DMSO"
  },
  {
    "caption": "(F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments.",
    "molecules": "pifithrin α, rapamycin"
  },
  {
    "caption": "(A, B) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells were co‐transfected with a GFP-LC3‐encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1WT) or the DN TAK1K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by immunoblotting for the detection of TAK1 and endogenous LC3 (A) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP-LC3 puncta (GFP-LC3VAC cells) (B) (mean values±s.d., n=3; *P0.01 versus control cells). GAPDH levels were monitored to ensure equal loading.",
    "molecules": "pifithrin α, rapamycin"
  },
  {
    "caption": "(A-D) Impact of bafilomycin A1 (BafA1) on the induction of GFP-LC3+ puncta by TAB2 and TAB3 depletion. HeLa cells stably expressing GFP-LC3 were transfected with a control siRNA (siUNR) or with siRNAs targeting TAB2 and TAB3 for 24 h. During the last 12 h of this period, BafA1 was optionally added. After fixation and permeabilization, LAMP2 was detected by immunofluorescence. Representative confocal microphotographs for the TAB2 siRNA are shown (A), together with the profiles of colocalization of fluorescent signals (B) along the indicated direction (α-ω). Columns in (C) represent the percentage of colocalization of GFP-LC3 and LAMP2 (mean values±s.d.; *P0.01 versus siUNR‐transfected cells), as quantified in at least 50 cells for each condition. The frequency (mean±s.d.) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VACcells) is plotted in (D).",
    "molecules": "BafA1, bafilomycin A1"
  },
  {
    "caption": "(E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3‐I/LC3‐II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading.",
    "molecules": "BafA1"
  },
  {
    "caption": "(A) Effects of full‐length TAB2 and TAB3 or their deletion mutants (as in Figure 1C) on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h, then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα) for 4 h. Finally, the frequency (mean±s.d., n=3) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (*P0.01 versus control cells transfected with the same plasmid; #P0.01 versus pcDNA3.1‐transfected cells treated with the same pro‐autophagic trigger).",
    "molecules": "pifithrin α, rapamycin"
  },
  {
    "caption": "E. Naïve  MCF7  cells and  MCF7aTT cells were exposed to the indicated stress conditions for 3 h, and cell viability was assessed by  staining dead cells with  trypan blue. The bar graphs represent the percentage of  trypan blue excluding cells. (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).",
    "molecules": "trypan blue"
  },
  {
    "caption": "A. Whole lysates of MCF7  and  MCF7aTT cells exposed to heat shock and/or proteasome  (MG132, 20 μM, 1h) and HSP70 (VER-155008, 50 μM, 1h) inhibitors were prepared as described in Materials and Methods. Total lysate, soluble fractions and insoluble fractions were stained by SYPRO Ruby (left) and quantified (right). (means +/- SD, n = 3, numbers above the bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).",
    "molecules": "SYPRO Ruby, VER-155008, MG132"
  },
  {
    "caption": "C. The soluble and insoluble fractions shown in (A) were assayed for the  level  of  K48-linked  polyubiquitin, and polyubiquitylated proteins were quantified (blue line graph, means +/- SEM, n = 4, numbers above the bars indicate p values). The data for bulk protein obtained in (A) were overlaid onto the graph for comparison (red line, means +/- SEM, n = 3).",
    "molecules": "K48"
  },
  {
    "caption": "B. The same experiment as described in (A) but with MCF7 cells exposed to  MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min). Means +/- SEM, n = 2. C. Lysate from MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min) as indicated were subjected to immunoprecipitation with anti-puromycin antibodies, followed by assaying for co-precipitation of  K48-ubiquitylated  proteins. Actin is shown as a reference. ",
    "molecules": "K48, MG132, puromycin"
  },
  {
    "caption": "B. Same experiment as in (A) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER-155008 (50 μM, 1h) or MG132 (20 μM, 1h). The signal obtained for ribosomal protein RPS19 and RPL7 are shown for reference, documenting residual  polysomes despite the flat UV trace. Right panels are quantifications of K48-ub (means +/- SEM, n = 2), black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The bar graphs represent the P/M ratios (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). C. Sucrose density gradient  fractions described in (B) were assayed for partitioning of  HSPA1 and HSPH1 by immunoblotting. HSPA1 data were quantified in a line graph (means +/- SEM, n = 4). D ",
    "molecules": "VER-155008, K48, MG132"
  },
  {
    "caption": "D. Cell lysate was spun through a 30% sucrose cushion to pellet polysomes. Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA (see Appendix Figures S3). Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48-linked polyubiquitin. The levels of K48-linked polyubiquitin were quantified (means +/- SD, n = 3). Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines.",
    "molecules": "EDTA, K48, sucrose"
  },
  {
    "caption": "D. Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells, and puromycin labeled peptides were pull down as illustrated in the scheme. Precipitates were assayed for the presence of K48-linked polyubiquitin, chaperones, and proteasome subunits by immunoblotting.",
    "molecules": "K48, puromycin"
  },
  {
    "caption": "E. Whole lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were fractionated into  soluble and  insoluble fractions as described in Materials and Methods. Insoluble fractions were assayed for the level of K48-linked polyubiquitin  (left panel). Total levels of  HSPA1 and HSPH1 are shown for reference. K48 ubiquitin signals were quantified and displayed in a bar graph (means +/- SD, n = 4).",
    "molecules": "K48"
  },
  {
    "caption": "F. Lysates of cells KYSE150 and  KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were obtained and polysomes were isolated by centrifugation through a 30% sucrose cushion. Whole cell lysates (left panel) and the polysomal pellets (right panel) were assayed by immunoblotting for the levels of HSPH1, HSPA1 and proteasome subunits.",
    "molecules": "sucrose"
  },
  {
    "caption": "(B) NanoSightquantification of the amount of particles in the CSF 0, 1, 2, 4 and 6 hours after i.p. LPS injection (n=3-5).",
    "molecules": "LPS"
  },
  {
    "caption": "(C) Size distribution of the EVsin vivo in the CSF before (black; n=5) and 6 h after (grey; n=3) LPS treatment determined by NanoSight analysis.",
    "molecules": "LPS"
  },
  {
    "caption": "(A-B) In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence (black) or presence (grey) of LPS (n=5) determined by NanoSight analysis.",
    "molecules": "LPS"
  },
  {
    "caption": "(C-E) TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D) and miR-155 (E) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n=3).",
    "molecules": "LPS"
  },
  {
    "caption": "(F-I) Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H) and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n=3). Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3).",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "(B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit.",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "(E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "(A) Representative confocal images of CD63, RAB5 and ANXA2 (red) in the choroid plexus (CP) 0, 4 and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scalebar = 100 µm.",
    "molecules": "LPS"
  },
  {
    "caption": "(B-C) Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 hours after (C) LPS administration in vivo. White arrow heads point to exosomes present in MVBs (n=3). Scale bar = 9 µm.",
    "molecules": "LPS"
  },
  {
    "caption": "(K) NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8).",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "(L) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (grey), a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 4).",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "(M) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.",
    "molecules": "LPS"
  },
  {
    "caption": "(A-B) Representative confocal images of choroid plexus (CP) on brain sections from naive mice and four hours after LPS injection (n=3). Brain sections were stained for TLR4 (A) and TNFR1 (B) (red), pan-cytokeratine (panCK; green) and the nuclei were stained with Hoechst (blue). The ependymal cells aligning the ventricles are marked with a dotted line. Scalebar = 100 µm.",
    "molecules": "LPS"
  },
  {
    "caption": "(A-B) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice (A) and in brain tissue (B) before (black) and 8 h after LPS injection (grey) (n=3).",
    "molecules": "LPS"
  },
  {
    "caption": "(C) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα by qPCR in mixed cortical cultures (left; in vitro) incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice and in brain tissue (right; in vivo) before (black) and 8 h after LPS injection (grey) (n=3).",
    "molecules": "LPS"
  },
  {
    "caption": "(D) Cytokine analysis (IL-6, IL-1β and TNF) of supernatant of mixed cortical cultures (left; in vitro) incubated with EVs isolated from untreated (black; n=3) or LPS treated (grey; n=3) mice and of CSF (right; in vivo) from untreated (black; n=3) and LPS treated mice (grey; n=6).",
    "molecules": "LPS"
  },
  {
    "caption": "(E-F) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (E) and miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (F) in mixed cortical cultures incubated with CD63-depleted EVs isolated from untreated mice (black) and mice treated with LPS for 6 h (grey) (n=3).",
    "molecules": "LPS"
  },
  {
    "caption": "(G-H) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (G) and inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (H) in brain tissue from LPS-injected mice icv injected with vehicle (black) or GW4869 (grey; n=7). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05.",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "(F) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta) and microtubules (α-tubulin, grey). n=21, 28, 24 oocytes. A representative oocyte fixed at 5 hours after NEBD is shown. Arrowheads indicate central MTOCs.",
    "molecules": "Hoechst33342"
  },
  {
    "caption": "(A) K-fibers are connected to MTOCs and extend to spindle poles. Oocytes 6 hours after NEBD were fixed following cold treatment. Microtubules (α-tubulin, green), MTOCs (pericentrin, magenta), kinetochores (ACA, red) and DNA (Hoechst333342) are shown. Five oocytes from 2 independent experiments were analyzed. Scale bar, 10 μm.",
    "molecules": "Hoechst333342"
  },
  {
    "caption": "H. Knockdown efficiency was analyzed by real-time RT-PCR in MN1 cells transfected with control or Lrig1 shRNA vectors. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs. Data are shown as individual values of a representative assay measured in triplicates. n=2 independent experiments were performed.",
    "molecules": "puromycin"
  },
  {
    "caption": "H. TrkB ubiquitination was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag-Lrig1 vector into MN1 cells. After 36 h, cells were serum-starved, pre-treated with the cell-permeable proteasome inhibitor MG-132 (20 M) and stimulated with BDNF for 15 min. Total lysates were immunoprecipitated with anti-HA antibodies followed by immunoblot (IB) with antibodies against ubiquitin. Reprobing of the same blot with anti-HA antibodies is also shown. TrkB activation (p-TrkB) was evaluated in cell lysates. Reprobing of the same blot with anti-TrkB and anti-Flag antibodies is also shown. Fold of p-TrkB (p-Y705) change relative to total TrkB is indicated. Data represent n=3 independent assays.",
    "molecules": "MG-132"
  },
  {
    "caption": "D. Percentage of IFNγ+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. E. Percentage of IL10+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. F. Total cell counts following in vitro Th1 polarisation (6 days) in the presence or absence of phenylephrine (PE). For (D-E), cells were purified from 3 mice per group and 6 replicates performed. Data information: statistical significance was determined with 1-way ANOVA followed by Bonferroni's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.",
    "molecules": "PE, phenylephrine"
  },
  {
    "caption": "(C) EC were seeded on gelatin-coated Transwell inserts. RFP-labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***, P<0.001 (two-tailed Mann-Whitney U test).",
    "molecules": "gelatin"
  },
  {
    "caption": "Distribution of 25-35 mer reads mapping to the mouse genome. Proportion of 30-35 nt genomic reads matching mouse tRNA sequences. The sequencing was done in duplicates. Error bars represent SEM, significance was calculated using unpaired t-test.",
    "molecules": "tRNA"
  },
  {
    "caption": "Perbase sequence coverage of 30-35 nt reads across parental tRNA sequence. Anticodon positions are demarcated by discontinuous lines (Blue = LIF-treated, Grey = Wnt3+LIF- treated, Red = RA- treated mESCs). Perbase coverage of 30-35 nt reads across the 468 individual parental tRNAs plotted as heatmaps.",
    "molecules": "RA, tRNA, tRNAs"
  },
  {
    "caption": "Northern blot of candidate tRNAs and tsRNAs tsGlnCTG, tsGlyGCC, tsGluTTC, tsValCAC and tsLysTTT. The black arrows represent the tRNAs and the red arrows mark the tsRNAs.",
    "molecules": "tsGlnCTG, tsGluTTC, tsGlyGCC, tsLysTTT, tsValCAC, tRNAs"
  },
  {
    "caption": "Northern Blot of 5'-tsRNAs; tsGlyGCC, tsGlnCTG, tsGluTTC, tsLysTTT, tsValCAC at various timepoints of RA-induced mESCs differentiation showing dynamic expression of these 5'-tsRNAs. Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates. The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs.",
    "molecules": "tsGlnCTG, tsGluTTC, tsGlyGCC, tsLysTTT, tsValCAC, RA, tRNAs"
  },
  {
    "caption": "Effect of ASO-mediated inhibition of 5'-tsRNAs on alkaline phosphatase activity in RA-treated mESCs. (n = 3), Error bars represent SEM, significance was calculated using unpaired t-test.",
    "molecules": "RA"
  },
  {
    "caption": "Relative expression of stemness markers in RA-induced differentiation mESCs blocked for 5'-tsRNA function with ASOs. (n = 2) Error bars represent SEM, significance was calculated using unpaired t-test.",
    "molecules": "RA"
  },
  {
    "caption": "Scatter plot showing Log2 fold-change enrichment of protein interactome between LIF- and RA-treated mESCs. All peptides identified by LC-MS/MS had < 1% FDR (Fig EV4E). LC-MS/MS was conducted twice (R2 = 0.85 between LIF duplicates and R2 = 0.82 between RA-treated mESCs duplicates).",
    "molecules": "RA"
  },
  {
    "caption": "Scatter plot showing Log2 fold-change enrichment of mRNAs associated with tsGlnCTG over Scramble (Scr1) in LIF- versus RA-treated mESCs.",
    "molecules": "tsGlnCTG, RA"
  },
  {
    "caption": "Bar plots and representative gene lists depicting association of tsGlnCTG with 'pluripotency-associated' (D) and 'differentiation-responsive' (D') genes in LIF- or RA-treated mESCs.",
    "molecules": "tsGlnCTG, RA"
  },
  {
    "caption": "In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. (n = 2), Error bars represent S.E.M.",
    "molecules": "tsGlnCTG"
  },
  {
    "caption": "Quantification of the Igf2bp1 bound c-Myc mRNA between LIF- versus RA-treated mESCs (n = 2; see Fig. EV5F for duplicate data).",
    "molecules": "RA"
  },
  {
    "caption": "Association of c-Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition. (n = 2), Error bars represent SD, significance was calculated by one-tailed unpaired t-test.",
    "molecules": "RA"
  },
  {
    "caption": "Relative enrichment of c-Myc mRNA in translating (80S and Polysome) and non-translating (mRNPs) pools (fractionated from polysome profiling) between ASO-treated and Mock-treated RA-induced differentiating mESCs. (n =2) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.",
    "molecules": "RA"
  },
  {
    "caption": "Relative levels of c-Myc mRNA in ASO-treated and Mock-treated RA-induced differentiating mESCs compared to LIF treated mESCs. (n = 3) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.",
    "molecules": "RA"
  },
  {
    "caption": "A Image sequence from confocal time lapse of a neuron  that does not extend a nascent axon during nocodazole  treatment labelled  with a membrane  marker and  lifeact-Ruby  before (-10m) and during (0m to 170m) nocodazole  treatment. Protrusions present before nocodazole  addition are retracted upon nocodazole  treatment (20m). Short, transient non-axonal  protrusions are extended during nocodazole  treatment. Images are transverse reconstructions from confocal  z-stacks. B Image sequence from confocal time lapse of a neuron  that extends a nascent axon  during nocodazole  treatment labelled  with a membrane  marker and  lifeact-Ruby  before (-10m) and during (0h to 170m) nocodazole  treatment. Small protrusions present before nocodazole  addition (-10m) are retracted upon nocodazole  treatment (0m). A nascent axon-like protrusion  (long, broad, long-lived) is extended during nocodazole  treatment (110m to 170m). Transient non-axonal  protrusions  are also present. Images are transverse reconstructions from confocal  z-stacks. Data information: All scale bars = 10 µm.",
    "molecules": "nocodazole"
  },
  {
    "caption": "C Graph showing duration (minutes) of nascent axon-like protrusions in nocodazole  treated cells and non-axonal  protrusions in control and nocodazole  treated cells. Control nascent axons  were not analysed as they do not retract. Bars show mean and standard deviation. Nocodazole nascent axons: n = 14 protrusions from 14 cells from three experiments; mean = 65.17 minutes, s.d. = 20.33. Control non-axons: n = 40 protrusions from 10 cells from three experiments; mean = 17.15 minutes, s.d. = 8.804. Nocodazole non-axons: n = 41 protrusions from 16 cells from three experiments; mean = 16.182 minutes, s.d. = 7.147. One-way ANOVA with multiple comparisons: P < 0.0001; nocodazole nascent axon vs nocodazole  non-axon P < 0.0001; control non-axon  vs nocodazole non-axon P = 0.9132. D Graph showing length (µm) of  nascent axon-like protrusions  and non-axonal  protrusions  in control and nocodazole  treated cells. Bars show mean and standard deviation. Control nascent axons were analysed after 60 minutes: n = 18 protrusions from 18 cells; mean = 14.092 µm, s.d. = 5.149. Nocodazole nascent axons: n = 14 protrusions from 14 cells; mean = 9.596 µm, s.d. = 2.371. Control non-axons: n = 40 protrusions from 10 cells; mean = 5.802 µm, s.d. = 2.553. Nocodazole non-axons: n = 41 protrusion from 16 cells; mean = 4.407 µm, s.d. = 2.024. One-way ANOVA with multiple comparisons: P < 0.0001; control nascent axon vs nocodazole nascent axon P = 0.0002; control nascent axon vs control non-axon P < 0.0001; nocodazole nascent axon vs nocodazole non-axon P < 0.0001; control non-axon  vs nocodazole non-axon P = 0.1485. E Graph showing width (µm) at the base of  nascent axon-like protrusions  and non-axonal  protrusions in control and nocodazole  treated cells. Bars show mean and standard deviation. Control nascent axons: n = 19 protrusions from 19 cells from six experiments; mean = 7.806 µm, s.d. = 3.163. Nocodazole nascent axons: n = 14 protrusions from 14 cells from three experiments; mean = 4.188 µm, s.d. = 1.637. Control non-axons   from three experiments: n = 40 protrusions from 10 cells; mean = 1.061 µm, s.d. = 0.652. Nocodazole non-axons  from three experiments: n = 41 protrusions from 16 cells; mean = 0.984 µm, s.d. = 0.757. One-way ANOVA with multiple comparisons: P < 0.0001; control nascent axon vs nocodazole nascent axon P < 0.0001; control nascent axon  vs control non-axon P < 0.0001; nocodazole nascent axon  vs nocodazole non-axon P < 0.0001; control non-axon  vs nocodazole non-axon P = 0.9958. Data information: *** P < 0.001. **** P < 0.0001.",
    "molecules": "nocodazole, Nocodazole"
  },
  {
    "caption": "F Plots showing nascent axon position on the soma  of control cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 19 cells from six experiments). Numbers show total count of axons  in each quadrant. G Plots showing nascent axon position on the soma  of nocodazole  treated cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 14 cells from three experiments). Numbers show total count of axons  in each quadrant. H Plots showing the position of non-axonal protrusions on the soma  of control cells relative to the  cell centroid  at 0,0 for dorsal and transverse view (n = 40 protrusions from 10 cells from three experiments). Numbers show total count of protrusions in each quadrant. I Plots showing the position of non-axonal protrusions on the soma  of nocodazole  treated cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 41 protrusions from 16 cells from three experiments). Numbers show total count of protrusions originating in each quadrant.",
    "molecules": "nocodazole"
  },
  {
    "caption": "J Graph showing the ratio of kif5c560-YFP  fluorescence intensity in the soma  compared to the axon  with or without nocodazole. Bars show mean and standard deviation. Control: n = 6 cells from four experiments; mean = 1.81, s.d. = 1.59. Nocodazole: n = 7 cells from four experiments; mean = 0.75, s.d. = 0.19. Data information: *** P < 0.001. **** P < 0.0001.",
    "molecules": "nocodazole, Nocodazole"
  },
  {
    "caption": "K Image sequence from confocal time lapse of a neuron  that extends a nascent axon  during nocodazole  treatment labelled  with a membrane  marker and kif5c560-YFP  before (-10m) and during (0h to 144m) nocodazole  treatment. A nascent axon-like protrusion is extended during nocodazole  treatment (92m to 144m) but kif5c560-YFP  does not accumulate there. Images are transverse reconstructions from confocal  z-stacks. Data information: All scale bars = 10 µm.",
    "molecules": "nocodazole"
  },
  {
    "caption": "C Transverse sections from a confocal  z-stack of a neuron  at the time  of nascent axon initiation labelled with a membrane  marker in a utr-mCherry embryo  to identify the basal surface  of the spinal cord. Three different sections show the middle of the soma, the axon  initiation site, and the  axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. D Transverse sections from confocal  z-stacks of a neuron  at the time  of nascent axon initiation labelled with a membrane  marker in a utr-mCherry embryo  injected with  lamMO. Three different sections show the middle of the soma, the axon  initiation site, and the axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. E Graphs showing distance (µm) between the basal edge of the soma, the axon  initiation site or the axon tip and the  basal surface of the spinal cord. Measurements were performed by measuring between basal-most green and magenta peaks in graphs of relative grey values (C, D). Bars show mean and standard deviation. WT: n = 5 cells from one experiment; lamMO: n = 11 cells from two experiments. Soma: WT mean = 2.007 µm, s.d. = 1.055;  lamMO  mean = 1.871 µm, s.d. = 1.830. Student's two-tailed test, P = 0.880. Axon  initiation site: WT mean = 1.366 µm, s.d. = 3.13; lamMO mean = 2.353 µm, s.d. = 2.125. Student's two-tailed test, P = 0.468. Axon tip: WT mean = 0.637 µm, s.d. = 1.078; lamMO mean = 2.903 µm, s.d. = 2.0433. Student's two-tailed test, P = 0.037. Data information: All scale bars = 5 µm. * P < 0.05.",
    "molecules": "MO"
  },
  {
    "caption": "ASGP-R labeling in FP+ and endosomes. Cells exposed for 40 min to 5-nm gold tracer. Section immunolabeled for ASGP-R with 10-nm gold. (a) Low ASGP-R labeling in tracer-carrying endocytic vesicles (arrowheads), adjacent to (top) or fusing with (bottom) an AVi/d. (b) ASGP-R labeling at the plasma membrane and in endosomal elements near the cell surface (ex, extra cellular space). Vesicles of the MVE (asterisk) and VE (arrowhead) type, containing endocytosed gold, are present in the same area. (c) As b. A VE-like vesicle with endocytosed gold and low ASGP-R labeling (arrowhead) is shown connected to an ASGP-R rich element (arrow), possibly a recycling endosome. Bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "Comparison of structural preservation in cryosections picked up with sucrose (a) and MU (b). In isolated hepatocytes, autophagy was induced for 30 min in suspension buffer before fixation. (a) Cryosection picked up with 2.3 M sucrose and labeled for SOD. Note the presence of large, \"empty\" gaps between sequestered contents of the AV and the surrounding cytoplasm. (b) A similar section as a, but picked up with an MU mixture. The AV are no longer distorted and the overall membranous structures are sharply delineated. The SOD-labeling density of AVi is as that of the cytosol, whereas AVd show much more intense labeling. (Inset) An AVi/d that is distinct from AVi by its single limiting membrane. The highly pleiomorphic AVd are also bound by a distinctive single membrane, but in contrast to AVi/d, their contents are characterized by debris of digested material. Bars, 250 nm.",
    "molecules": "sucrose"
  },
  {
    "caption": "Labeling patterns of cathepsin D and CAIII in MU picked-up cryosections. (a) Cells treated as in Fig. 1 b, but the section was immunolabeled for cathepsin D, which is not detectable in the AVi, moderately present in the AVi/d, and abundant in AVd. Cells in b and c had endocytosed 5-nm gold tracer for 20 and 40 min, respectively, before fixation and CAIII labeling. (b) Complex fusion profile of a CAIII-positive AVi, an AVd which is without CAIII labeling, and a VE that contains an endocytosed gold probe (arrow). Close to the AVd is another tracer-bearing VE. (c) Fusion profile of a tracer-bearing MVE and an AVi/d that exhibits a single limiting membrane and a CAIII-labeling concentration similar to that of the cytoplasm. Bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "Fusion profiles. Cells exposed to 5-nm gold tracers for 20 (a and c) and 25 min (b) before fixation. Sections were picked up by MU and air-dried (c) or underwent immunolabeling with anti-SOD antibodies (a and b). (a) Fusion between a small VE (arrow) and an AVi. A cluster of two gold particles (arrowhead) is also seen at another location. (b) Fusion between an electron lucent MVE (arrow) and an AVd. (c) Fusion between an MVE (arrow) and an AVd. Bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": " (A) Alizarin red staining of hBMSC cells for 11 days. ",
    "molecules": "Alizarin red"
  },
  {
    "caption": "(B) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.",
    "molecules": "Alizarin Red"
  },
  {
    "caption": " (D) Alizarin red staining of hBMSC cells. ",
    "molecules": "Alizarin red"
  },
  {
    "caption": "(E) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.",
    "molecules": "Alizarin Red"
  },
  {
    "caption": " (H) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells treated with SR and NPS-2143. ",
    "molecules": "NPS-2143, SR"
  },
  {
    "caption": "PI(3)P expression levels detected by a 2xFYVE-GFP reporter in a third instar larval wing disc (A) and eye disc (B) containing mtm mutant clones (marked by the absence of RFP). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.",
    "molecules": "PI(3)P"
  },
  {
    "caption": "Quantification of the mean FYVE-GFP fluorescence intensity of mtm mutant clones and surrounding wild-type (WT) cells are shown (C). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(D) Electron microscopy images from purified 40S and  60S ribosomal complexes (1 pmol each) assembled in vitro in the presence of MgCl2, and in the presence or absence of 5 pmol of (PR)20. Assembled 80S particles are indicated (red arrows). Scale bar (white) represents 10 nm. (E) Quantification of 80S particles identified in (D) (n=1000) in non-assembly (1 mM MgCl2) or assembly (5 mM MgCl2) conditions. ( ",
    "molecules": "(PR)20, MgCl2"
  },
  {
    "caption": "(C, D) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h.  EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative  images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.",
    "molecules": "(PR)20, polyA, 5-ethynyl-uridine, EU"
  },
  {
    "caption": "(E) Effect of  (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.",
    "molecules": "(PR)20"
  },
  {
    "caption": "(B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by  qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.",
    "molecules": "(PR)20"
  },
  {
    "caption": "(D) HTM-mediated analysis of  5-ethynyl-deoxyuridine (EdU) levels per  nucleus found in  U2OS cells exposed to 10 μM (PR)20 for 4 h.  EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.",
    "molecules": "PR)20, 5-ethynyl-deoxyuridine, EdU"
  },
  {
    "caption": "(E)  HTM-mediated analysis of  γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated  times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after  IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.",
    "molecules": "(PR)20"
  },
  {
    "caption": "(D) Immunofluorescence of  Cy3-labelled  salmon protamine (red) and the nucleolar factor  UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.",
    "molecules": "Cy3, protamine"
  },
  {
    "caption": "(E, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin  (OPP) levels per cell (F) in U2OS cells exposed to 30 μM  protamine for 16 (E) or 24 (F) h. EU  and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).",
    "molecules": "O-propargyl-puromycin, OPP, protamine, EU"
  },
  {
    "caption": "(D) Immunofluorescence of HA-(PR)20 (green) and the  nucleolar factor UBF1  (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA  oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.",
    "molecules": "(PR)20, ssDNA  oligonucleotide"
  },
  {
    "caption": "(E)  Immunofluorescence of  HA-(PR)20 (green) and Cy5 (red) in  U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.",
    "molecules": "(PR)20, Cy5, ssDNA, ssRNA oligonucleotides"
  },
  {
    "caption": "(F) Clonogenic survival assay of  U2OS cells exposed to 7.5 μM with the indicated peptides or protamine  (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.",
    "molecules": "Heparin, PROT, protamine, ssDNA oligonucleotide"
  },
  {
    "caption": "(G) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor  UBF1 (red) in U2OS cells treated with 7.5 μM  HA-(PR)20 alone or together with  heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.",
    "molecules": "(PR)20, heparin"
  },
  {
    "caption": "(A) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei  were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide. (B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM. ( Data information: **, p<0.01; ***, p<0.001.",
    "molecules": "(PR)20, DAPI"
  },
  {
    "caption": "(D) Immunofluorescence of the neuronal marker TUBB3 (red) in  mouse motor neurons differentiated from mESC (mMN) or from  NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. (E, F)  Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001. S ",
    "molecules": "(PR)20, DAPI, heparin"
  },
  {
    "caption": "(A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel).",
    "molecules": "galactose"
  },
  {
    "caption": "(B) Cells were pulse-labelled with [35S]methionine and chased on non-radioactive starvation medium. Aliquots were taken at the indicated times and acid-soluble small peptides generated by proteolysis were determined.",
    "molecules": "methionine, small peptides"
  },
  {
    "caption": "(A) Atg8WT-HA, Atg8F77A-HA, Atg8F79A-HA or Atg8F77/79A-HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) containing 6 M urea, followed by immunoblot analysis with anti-Atg8 antibodies.",
    "molecules": "urea"
  },
  {
    "caption": "(A) Cell extracts were subjected to 13.5% SDS-polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies.",
    "molecules": "urea"
  },
  {
    "caption": "(A) Δatg8 cells transformed with GFP-Atg8WT, GFP-Atg8L50A or GFP-Atg8Y49A constructs were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. API maturation was tested using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies.",
    "molecules": "galactose"
  },
  {
    "caption": "(D) Atg8WT, Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h. Cell extracts were subjected to 13.5% SDS-PAGE containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐N15 antibodies. GFP, green fluorescent protein; WT, wild type.",
    "molecules": "N15, urea"
  },
  {
    "caption": "F-H RT-qPCR analysis of interferon-related genes and TA-ERVs (F), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (G), and ELISA of IFNβ and CXCL10 in culture supernatant (H) in U2OS cells ectopically expressing TERT-WT or TERT-K626A using Dox-inducible system. GFP was used as negative control. Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH, as indicated at the bottom of the blot (G).",
    "molecules": "Dox"
  },
  {
    "caption": "A Expression of murine ERVs in blood of WT and G1 Tert-/- mice. n = 5 mice per group. B Expression of MMERGLN_LTR and MMERGLN-int in livers of WT and G1 Tert-/- mice treated with ENU or saline (control). n = 3 mice per group. C",
    "molecules": "ENU"
  },
  {
    "caption": "D Heat map showing log2 fold change differences of interferon-related genes expression between ENU treatment and control (saline) in WT and G1 Tert-/- mice by RNA-seq. n = 3 mice per group. E Heat map showing log2 fold change differences of cytokine/chemokine between ENU treatment and control (saline) in WT and G1 Tert-/- mice by cytokine/chemokine array. n = 5 mice per group. F",
    "molecules": "ENU"
  },
  {
    "caption": "Changes in HDX behavior for dynamin analyzed (B) in solution the presence GMPPCP, (C) in solution in the presence of GDP•AlF4-, (D) membrane-bound, or (E) membrane-bound in the presence of GMPPCP and mapped onto the structures of dynamin monomers. Color coding (see scale below) shows the extent of solvent exchange relative to apo dynamin in solution. All HDX MS data shown represents an average of 3 replicates and are color coded from red to blue with warm colors representing increased conformational dynamics (red being the highest D2O uptake) and cool colors representing decreased conformational dynamics (blue being the lowest D2O uptake). Grey indicates no statistically significant change between the two states. Quantitative data on the individual peptides used to generate this map are provided in Appendix (Figure S1-S4).",
    "molecules": "D2O, H, DX, GDP•AlF4-, GMPPCP"
  },
  {
    "caption": "(A) Changes in solvent exchange superimposed onto the structure (PDB ID: 2SNH) of the G domain or (B) the stalk/PHD of dynamin in the GMPPCP or lipid bound states, respectively. Key structural elements are identified and the color-coding shows the magnitude of solvent exchange relative to the apo state (see scale below).",
    "molecules": "GMPPCP, lipid"
  },
  {
    "caption": "(C) Differential kinetics of solvent exchange of selected peptides that were identified as altered in both GMPPCP and GDP•AlF4--bound states. The peptides are mapped to secondary structure elements on the polypeptide sequence. Differences in kinetics of solvent exchange of select peptides measured (D) for dynamin bound to GMPPCP in the presence or absence of lipid nanotubes (E) for dynamin bound to lipid nanotubes in the presence or absence of GMPPCP. HDX data are presented as an average of three independent triplicates and color coded as in Figure 1.",
    "molecules": "H, DX, GDP•AlF4-, GMPPCP, lipid"
  },
  {
    "caption": "(A) Design of a functional dynamin mutant to probe PHD stalk interactions. Minimal structure of the PHD and the PHD-Stalk interface showing the location of important residues discussed in this study. The unresolved loop L1NS in the stalk hosts the IAEDANS label and is sensitive to the two tryptophans (W525 and W542) in the PHD. The distance between two introduced cysteines (S357C and native C602) used to construct a double cysteine Dyn1 is also shown.",
    "molecules": "IAEDANS, tryptophans"
  },
  {
    "caption": "(B) Comparison of emission spectra of various IAEDANS-labeled Dyn1 RCL variants (D352C, Y354C, S357C, I365C, H367C), upon Trp excitation at 295 nm.",
    "molecules": "IAEDANS, Trp"
  },
  {
    "caption": "(C) FRET transfer upon excitation is contributed by two neighboring Trps (W525 and W542); Substitution by Phe resulted in significant loss of FRET.",
    "molecules": "Trps"
  },
  {
    "caption": "(D) IAEDANS emission kinetics upon addition of lipid nanotubes with or without PI4,5P2 (ex=295). The arrow indicates addition of lipids.",
    "molecules": "IAEDANS, PI4,5P2, lipid, lipids"
  },
  {
    "caption": "(B) Histogram shows basal and assembly-stimulated GTPase activities of non-crosslinked Dyn1CC vs crosslinked Dyn1Closed and Dyn1WT. Inset shows crosslinking of the PHD and stalk domains in Dyn1Y354C/S607C (Dyn1CC) with the bifunctional crosslinker, MTS-2-MTS, to yield Dyn1Closed, which migrates more slowly on SDS-PAGE, as well as liposome binding of Dyn1CC compared to Dyn1Closed (Bound, B; Unbound, UB).",
    "molecules": "MTS-2-MTS"
  },
  {
    "caption": "(F) Negative-stain electron micrographs of Dyn1WT, Dyn1ΔΔ and Dyn1Closed assembled onto lipid nanotubes. Scale bar = 200 nm",
    "molecules": "lipid"
  },
  {
    "caption": "(A) Temperature dependence of basal and lipid stimulated GTPase activity of Dyn1WT and Dyn2S619L.",
    "molecules": "lipid"
  },
  {
    "caption": "(C) Detection of Dyn2 PHD-stalk interactions by FRET using IAEDANS-labeled Dyn2L354C.",
    "molecules": "IAEDANS"
  },
  {
    "caption": "(D) Curvature dependent membrane binding and consequent opening of PHD in Dyn1Y354C-IAEDANS and Dyn2L354C-IAEDANS.",
    "molecules": "IAEDANS"
  },
  {
    "caption": "(E) Differential temperature dependence of PHD-stalk FRET for WT and S619L mutant Dyn2IAEDANS. Data are presented as relative FRET distances assuming a single donor/acceptor pair.",
    "molecules": "IAEDANS"
  },
  {
    "caption": "(F) Differential solvent exchange kinetics of Dyn1S619L at ambient temperature compared to Dyn1WT under identical conditions. Yellow coloring indicates a significant increase in HDX. Quantitative data on the individual peptides used to generate this map are provided in (Appendix Fig. S5).",
    "molecules": "H, DX"
  },
  {
    "caption": "B. The in vitro synthesized [35S]Met-labeled single-cysteine Bax proteins were activated and targeted to the mitochondria that were pretreated with NEM to block the sulfhydryls of mitochondrial proteins. The resulting mitochondria were isolated and oxidized by CuPhe for 30 min. NEM and EDTA were then added to stop the oxidation. For the \"0 min\" controls, NEM and EDTA were added before the addition of CuPhe. The resulting samples were analyzed by phosphor-imaging after non-reducing or reducing SDS-PAGE (see Appendix Figure S2A).",
    "molecules": "Met, EDTA, NEM, CuPhe"
  },
  {
    "caption": "D. Oxidized mitochondria with the radioactive single-cysteine Bax protein pair or double-cysteine Bax protein were prepared as in (B). After an aliquot was withdrawn as input, another aliquot was extracted by Na2CO3 (pH 11.5) and centrifuged through a sucrose cushion to separate the integral proteins in the membrane pellet from the soluble and peripheral proteins in the supernatant. The input, pellet and supernatant were analyzed by non-reducing SDS-PAGE and phosphor-imaging. In a parallel control experiment, the pellet and supernatant were analyzed by reducing SDS-PAGE and immunoblotting with an antibody specific to PDHE1α, a soluble mitochondrial matrix protein.",
    "molecules": "Na2CO3"
  },
  {
    "caption": "B. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in helix α2, α3, α4 or α5, were activated and targeted to the mitochondria. The resulting mitochondria were isolated and treated with IASD in the absence or presence of CHAPS, urea, or both. After 30 min, the labeling reactions were stopped by mercaptoethanol. For the \"0 min\" controls, the samples were pretreated with mercaptoethanol before addition of IASD. The IASD-labeled radioactive Bax proteins were resolved from the unlabeled ones using either isoelectric focusing (IEF; as indicated) or gradient SDS-PAGE and detected by phosphor-imaging. Triangles and arrows indicate the unlabeled and IASD-labeled Bax proteins, respectively. n = 3 for V111C, and K119C; 4 for W107C, and A117C; 2 for other mutants.C-D. The phosphor-imaging data for IASD labeling of Bax mutants in (B) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices as described and shown in Appendix Figure S3. The membrane burial indices were normalized by that of G179C in α9, a reference for the membrane-buried residues. The resulting relative membrane burial indices are shown in (C). The residues with the relative membrane burial index ≥ 40% of that of Gly179 are considered as buried in the MOM. Similarly, the relative protein burial indices shown in (D) were obtained by using Y115C in α5 as a reference for the protein-buried residues. The residues with the relative protein burial index ≥ 40% of that of Tyr115 are considered as buried in the protein or its complex.",
    "molecules": "IASD, mercaptoethanol, urea"
  },
  {
    "caption": "C. Oxidized mitochondria with the radioactive double-cysteine Bax protein were prepared, subjected to Na2CO3 extraction and analyzed as in Figure 1D.",
    "molecules": "Na2CO3"
  },
  {
    "caption": "B and D. The radioactive single- or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated (mito) or pretreated with NEM (NEM-mito), then oxidized and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.",
    "molecules": "NEM"
  },
  {
    "caption": "C. The in vitro synthesized [35S]Met-labeled Bax E69C protein with or without the additional mutations (G108E and/or S184V) were activated by the in vitro synthesized [35S]Met-labeled tBid L105C protein and targeted to the Bax-/-/Bak-/- mitochondria that were pretreated with NEM. The resulting mitochondria were isolated and oxidized by CuPhe for 0 or 30 min. The resulting \"0 min\" samples (1 equivalent each) and \"30 min\" samples (1 equivalent each) were analyzed by non-reducing SDS-PAGE and phosphor-imaging. The remaining \"30 min\" samples (4 equivalent each) were immunoprecipitated (IP) by either Bax- or tBid-specific antibody, and then analyzed by non-reducing SDS-PAGE and phosphor-imaging. The identities of the four major products, indicated on the right side of the image, were based on their Mr and recognition by the respective antibody. n = 2.",
    "molecules": "Met, NEM, CuPhe"
  },
  {
    "caption": "B and D. The radioactive single- or double-cysteine Bax proteins with or without the indicated mutations were activated and targeted to the mitochondria that were either untreated (mito) or pretreated with NEM (NEM-mito), then oxidized and analyzed as in Figure 1B. Protein standards, and monomers and disulfide-linked dimers of the Bax proteins are indicated as in Figure 1B. n = 2 for all mutants.",
    "molecules": "NEM"
  },
  {
    "caption": "A. The in vitro synthesized radioactive Bax proteins, each with a single cysteine positioned in α9, were activated and targeted to the mitochondria, and labeled with IASD as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using gradient SDS-PAGE, detected by phosphor-imaging and indicated by triangles and arrows, respectively. For all mutants, n = 2, except for A183C, n = 3.B-C. The IASD labeling data in (A) and the similar data from the independent replicates were quantified to derive the membrane or protein burial indices shown in Appendix Figure S5, from which the relative membrane (B) or protein (C) burial indices were obtained as described in Figure 2C-D, and shown.D. The structure of α9 (extracted from the NMR structure of Bax monomer, PDB entry 1F16) is shown with the residues that are buried in the MOM or exposed to the aqueous milieu, as the IASD labeling data in (A-C) suggested, presented in yellow or cyan stick form, respectively.",
    "molecules": "IASD"
  },
  {
    "caption": "C and E. The oxidized mitochondria with the radioactive single-cysteine Bax proteins were prepared, subjected to Na2CO3 extraction, and analyzed as in Figure 1D.",
    "molecules": "Na2CO3"
  },
  {
    "caption": "B. The BMH-treated mitochondria with the radioactive single- and double-cysteine Bax proteins were prepared and analyzed as in Figure EV3G, except that closed triangles indicate the BMH-linked Bax oligomers.",
    "molecules": "BMH"
  },
  {
    "caption": "D. The in vitro synthesized radioactive Bax proteins with indicated mutations were targeted to the mitochondria in the absence or presence of purified tBid protein, and then labeled with IASD, as in Figure 2B. The IASD-labeled and unlabeled Bax proteins were resolved using either gradient SDS-PAGE or IEF, detected by phosphor-imaging, and indicated by triangles and arrows, respectively. n = 3 for A178C,G179I; 2 for other mutants.E. The IASD labeling data in (D) and the similar data from the independent replicates were quantified to derive the membrane burial indices shown in Appendix Figure S7A, from which the relative membrane burial indices were obtained as described in Figure 2C, and shown.",
    "molecules": "IASD"
  },
  {
    "caption": "C-F. Mice pre-treated with DSS were inoculated with no organisms (sham), E1 only, or E1 competed with N1 WT or N1 ΔtssC. Five days post-inoculation, the ceca were weighed (C) and fixed for histopathological examination after sham (D), E1 only (E) and E1-N1 WT (F) colonizations. Scale bars denote 100μm (main image) and 200μm (inset).",
    "molecules": "DSS"
  },
  {
    "caption": "(a) Western blot analysis of myosin-VI-depleted HeLa cells untreated or treated with 100 nM bafilomycin A1. (b) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01, ***P0.001",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(e) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA-resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "(f) Western blot analysis of MEFs cultured from wild-type and Snell's waltzer (SV) mice treated with 1 μM MG132 or 100 nM bafilomycin A1.",
    "molecules": "bafilomycin A1, MG132"
  },
  {
    "caption": "(g) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01. (h) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control. (±s.d.; n = 3).",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1. (j) Quantification of western blot LC3-II intensity was performed (n = 2). (k) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control (n = 2).",
    "molecules": "bafilomycin A1, MG132"
  },
  {
    "caption": "(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment (zero time point) and 8 h post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was used to identify nuclei (blue).",
    "molecules": "MG132"
  },
  {
    "caption": "(b) Top, western blot analysis confirming siRNA depletion. Bottom, quantification of immunofluorescent p62-positive aggregates was evaluated using an automated Cellomics VTi microscope. The results were calculated as the average p62 fluorescence intensity at 8 h post-MG132 washout normalized to the zero time point and are represented as the percentage of the control (±s.d.; n = 3). *P0.05, ***P0.001. See Methods and Supplementary Table S1 for details of the SMARTpool (SP) and single (07) oligonucleotides.",
    "molecules": "MG132"
  },
  {
    "caption": "(c) Parental HeLa cells or HeLa cells stable expressing siRNA-resistant GFP-myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy at 16 h post-MG132 treatment (t = 0) or allowed to recover following washout of MG132 for 8 h (t = 8). Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity. The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout (t = 8; ±s.d.; n = 3).",
    "molecules": "MG132"
  },
  {
    "caption": "(d) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). (e) Quantification of HttQ72-GFP aggregates was performed on myosin-VI-siRNA-transfected HeLa cells. The results were calculated as the percentage of GFP-expressing cells with greater than 15 GFP-positive spots per cell. The results represent the mean (±s.d.) from n = 3independent experiments, ***P0.001. The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.",
    "molecules": "saponin"
  },
  {
    "caption": "(a) RPE cells transiently co-transfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 h (bottom row) were processed for immunofluorescence microscopy. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.",
    "molecules": "Torin1"
  },
  {
    "caption": "(b) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co-localization. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein-interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 h to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction. The arrowheads indicate areas of co-localization. Scale bar, 20 μm. (c) A Pearson's coefficient was calculated on the basis of the degree of co-localization between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence micrographs. The graph represents data from more than 20 transfected cells from n = 2 independent experiments.",
    "molecules": "saponin, Torin1"
  },
  {
    "caption": "(a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h. Immunolabelling was performed against the indicated endogenous proteins optineurin, T6BP and NDP52 (red), and LC3 (green). The insets show higher magnifications of the areas outlined in the main images. Right, from the confocal images, a Pearson's coefficient was calculated to estimate the degree of co-localization of the different autophagy adaptors with LC3. As a negative control, a Pearson's coefficient was calculated after rotation of one colour image by 90°. The results represent >20 cells from n = 2 independent experiments.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(b) Left, stable cherry-LC3-expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 h to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of co-localization are white in the merged three-colour images and the insets show higher magnifications of the areas outlined in the main images. Right, a Pearson's coefficient was calculated for the degree of co-localization between the three colours. As a negative control, a Pearson's coefficient was calculated after rotating one colour image by 90°. Results represent >10 cells from n = 2 independent experiments.",
    "molecules": "Torin1"
  },
  {
    "caption": "(c) HeLa cells were either mock transfected or co-transfected with siRNA targeted against TNO. Cells were either left untreated or treated with100 nM bafilomycin A1 (BfnA1) for 4 h. Western blot analysis was performed against the indicated proteins. (d) Quantification of LC3-II intensity (±s.d.) was performed from western blot data of TNO-siRNA treated HeLa cells. The results represent n = 3independent experiments. **P0.01, ***P0.001",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis (left) and immunofluorescence microscopy (middle) to evaluate GFP-myosin VI tail localization. Cells were either fixed directly or saponin-extracted before fixation. Quantification (right) was performed on RPE cells stably expressing GFP-myosin VI tail and transfected with mock, Tom1 or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP-myosin VI tail punctae per cell using an automated Cellomics VTi microscope. More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP-myosin VI tail punctae per cell (±s.d.). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.",
    "molecules": "saponin"
  },
  {
    "caption": "(a) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h. Western blot analysis was performed on whole-cell lysates against the indicated proteins. (b) Quantification of LC3-II intensity of western blots was performed by an infrared imaging system. (±s.d.; n = 3). *P0.05, **P0.01.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(g) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with Tom1 siRNA (top right), saponin-extracted, and processed for immunofluorescence microscopy (left) to quantify HttQ72-GFP punctae (bottom right). Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labelled with Hoechst. The results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots per cell (±s.d.; n = 3). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.",
    "molecules": "saponin"
  },
  {
    "caption": "(b) GFP-LC3-expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labelled with Texas-red-dextran (red) for 4 h, before chase into fresh medium containing 1 μM MG132 for 2 h. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labelled with Hoechst (blue). The insets represent higher magnifications of the areas outlined in the main panels. (c) Mander's overlap coefficients for the degree of LC3 signal co-localizing with dextran were calculated using Volocity software. The results represent >20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum.",
    "molecules": "MG132"
  },
  {
    "caption": "(d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-red (TR)-dextran for 16 h followed by a chase period of 4 h. Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas-red-dextran. Scale bars, 20 μm (a,b); 200 nm (d).",
    "molecules": "gold"
  },
  {
    "caption": "(A) MYB knockdown (MiaPaCa-shMYB and Panc1-shMYB) and forced MYB-overexpressing (BxPC3-MYB) PC cells along with their respective control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were subjected to hypoxia (1% O2) treatment for different time intervals (0-96 h). Their growth was measured by viable cell counting using a trypan blue exclusion assay.",
    "molecules": "O2, trypan blue"
  },
  {
    "caption": "(E, F) ECAR (E) and OCR (F) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 (100 μM for 4 h). Thereafter, ECAR was measured in PC cells with sequential injection of glucose, oligomycin, and 2-DG, while OCR was measured with the serial addition of oligomycin, FCCP, and antimycin A/rotenone.",
    "molecules": "2-DG, antimycin A, FCCP, CoCl2, glucose, oligomycin, rotenone"
  },
  {
    "caption": "(F) Cells were treated with either vehicle or CoCl2 (100µM) for 6 h, and total protein and RNA were isolated. The expression of HIF1α and MYB was examined. β-actin was used as an internal control.",
    "molecules": "CoCl2"
  },
  {
    "caption": "(F) MYB-silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h. Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels. The data was normalized with the number of cells and presented as relative fold changes.",
    "molecules": "glucose, lactate"
  },
  {
    "caption": "(G) Tumor xenograft serial sections were immunostained for pimonidazole (PDZ) adduct, MYB, HK2, GLUT3, and MCT4 using specific antibodies. Images were taken at 40X magnification. Scale bar, 60 μm (lower left corner). The dashed lines demarcate the hypoxic region based on the PDZ staining pattern.",
    "molecules": "PDZ, pimonidazole"
  },
  {
    "caption": "A. Validation of the top candidate genes in the C11 cell line. C11 cells were infected by lentiCRISPR v2.0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg/ml puromycin. The percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 reactivation.",
    "molecules": "puromycin"
  },
  {
    "caption": "D. The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h.",
    "molecules": "EGCG, DHA"
  },
  {
    "caption": "E. Induction of PEBP1 by EGCG or DHA in HIV-1 infected primary CD4+ T cells. Primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. Seventy-two hours post-treatment with 10 µM EGCG or DHA, the expression of PEBP1 was detected by qPCR.",
    "molecules": "EGCG, DHA"
  },
  {
    "caption": "F. The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4+ T cells. The primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B and E, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. The supernatants from HIV-1-infected CD4+ T cell were collected 1, 2, 3 or 4 days post-infection. Replication of HIV-1 was analyzed by p24 ELISA.",
    "molecules": "EGCG, DHA"
  },
  {
    "caption": "A. EGCG inhibited α-CD3/CD28 reactivation of latent HIV-1. Primary CD4+ T cells were isolated from HIV-1 positive patients on antiretroviral therapy (n=5, P1-P5). Primary CD4+ T cells were treated with α-CD3/CD28 alone or α-CD3/CD28 and 10 µM EGCG. Cell-associated RNA was extracted 7 days post-treatment. The transcription of HIV-1 was determined by real-time qPCR.",
    "molecules": "EGCG, RNA"
  },
  {
    "caption": "B. PEBP1 mRNA was induced by EGCG in patient primary CD4+ T cells. Primary CD4+ T cells were treated with 10 μM EGCG. The expression of PEBP1 in the cells was measured by qPCR.",
    "molecules": "EGCG"
  },
  {
    "caption": "C. PEBP1 protein was induced by EGCG in patient primary CD4+ T cells, which was determined by Western blot of whole cell lysate from primary CD4+ T cells.",
    "molecules": "EGCG"
  },
  {
    "caption": "D. EGCG-induced PEBP1 suppressed nuclear entry of NF-κB/p65. This was measured by Western blot of nuclear protein of primary CD4+ T cells treated with α-CD3/CD28 or α-CD3/CD28 plus 10 μM EGCG.",
    "molecules": "EGCG"
  },
  {
    "caption": "(A) 3.8 Å resolution mFo-DFc omit map density (green, calculated with sharpened B-factors and contoured at 2.0) reveals the presence of ADP and a short segment of RNA bound to FRH.",
    "molecules": "ADP, RNA"
  },
  {
    "caption": "(B) 3.8 Å resolution 2mFo-DFc calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6orange density shown for surrounding protein (0.6.",
    "molecules": "ADP, RNA"
  },
  {
    "caption": "(C) Comparison of Mtr4 and FRHADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds). (c) Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains.",
    "molecules": "ADP, RNA"
  },
  {
    "caption": "(A) Key residues for FRH function shown on the large-cell structure: K208 and S324 compose the ATP binding site, E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712 and K766 contribute to a positively charged surface on the KOW domain. R806H disrupts the clock. Q131, G132 and V142 in the N-terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig1, ADP and RNA are in orange bonds). (B) Same as (A) after rotation about a horizontal axis.",
    "molecules": "ADP, RNA"
  },
  {
    "caption": "(C) Close-up of the ADP-binding pocket and N-terminal interaction for the large-cell structure (cyan ribbons) and the small-cell structure (blue ribbons).",
    "molecules": "ADP"
  },
  {
    "caption": "B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into tail vein of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) every 3 days. On day 21, lung metastasis was analyzed by hematoxylin and eosin (H&E) staining F), scale bars = 500µM. Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "+/- inhibitors: anti-MyD88 peptide inhibitor, anti-mouse TLR2 antibody, or lipopolysaccharide (LPS)-RS, an antagonist of TLR4 in (D)",
    "molecules": "(LPS)-RS, lipopolysaccharide"
  },
  {
    "caption": "+/- inhibitors: anti-mouse IL-6 or JAK1/2 inhibitor (Ruxolitinib) in (E).",
    "molecules": "Ruxolitinib"
  },
  {
    "caption": "TRF2-overexpressing or -knockdown BJcl2 cells were co-cultured with MSC2 cells for 18h +/- mouse IL6 blocking antibody or Ruxolitinib. After FACS sorting, MSC2 cells were co-cultured for 18h with primed and purified NK cells before a 4h challenge with YAK-1. NK cell degranulation and IFN-γ production were analyzed by FACS (G). Data information: Panels show all points with mean ± SEM corresponding to the percentages of each cell population among NK cells (G) (n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001; Student's t-test).",
    "molecules": "Ruxolitinib"
  },
  {
    "caption": "TRF2-overexpressing or -knockdown BJcl2 cells were co-cultured with MSC2 cells for 18h +/- mouse IL6 blocking antibody or Ruxolitinib. Alternatively, a 4h challenge with NIH3T3 cells was performed and the ability of NK cells to kill NIH3T3 cells were determined by colorimetric analysis (AlamarBlue) (H). Data information: Panels show all points with mean ± SEM corresponding to the percentage of specific target lysis (H) (n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001; Student's t-test).",
    "molecules": "AlamarBlue, Ruxolitinib"
  },
  {
    "caption": "B, C Heatmap of H3K27ac (B) or H3K23me3 (C) peaks on the 4 kilobase (Kb) region around genomic ITSs (ENCODE data for the K562 cell line). The mean intensity of the region is depicted in the histograms. ITSs are clustered depending of the peak mean intensity obtained from H3K27ac or H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) of the 4 Kb region. ITSs detected by ChIP-seq using an anti-TRF2 antibody are shown on the left.",
    "molecules": "H3K23me3, H3K27ac, H3K27me3"
  },
  {
    "caption": "D Specific qPCR for HS3SH4, GPC6, and VCAN ITSs after ChIP using an anti-H3K27ac antibody in TRF2-overexpressing or -knockdown BJcl2 cells. Data represent fold enrichment of the H3K27ac level compared with the total H3 level. Data information: data represent n = 3 independent experiments with mean ± SEM",
    "molecules": "H3K27ac"
  },
  {
    "caption": "A Analysis of Heparan Sulfate (HS) expression by FACS using the 10E4 antibody. Data information histograms representative (left panels) and MFIs from three independent experiments (*p < 0.05 and **p < 0.01; Student's t-test) are shown +/- SEM.",
    "molecules": "Heparan Sulfate, HS"
  },
  {
    "caption": "D Analysis of pSTAT3 levels in MDSCs after co-culture with GFP-expressing or TRF2-overexpressing cancer cells treated with or without sodium chlorate (NaClO3). Data information: In (D), data represents mean of n = 3 independent experiments +/- SEM (***p < 0.001; Student's t-test).",
    "molecules": "NaClO3, sodium chlorate"
  },
  {
    "caption": "Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. The tumor volume was determined every 2 days using a caliper, and growth curves over time (C) Data information: , data represent mean values +/- SEM (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test).",
    "molecules": "5-FU"
  },
  {
    "caption": "Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. tumor volume were determined on day 19 and represented using Box plots Min to Max showing all points with median (D). Data information: (D), data are represented by Box plots Min to Max showing all points with median (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test)",
    "molecules": "5-FU"
  },
  {
    "caption": "Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. Overall survival was also determined (E).",
    "molecules": "5-FU"
  },
  {
    "caption": "Mice were analysed 2 days after TAC (a-e). a, Electron microscopic analysis. Images of mitochondria at higher magnification are shown in subsets. Scale bar, 1 µm. b, Autolysosome after incubation with anti-DNA antibody and 10 nm gold staining. Scale bar, 200 nm. Arrows indicate labelled DNA.",
    "molecules": "DNA, gold"
  },
  {
    "caption": "b-d, Four days after TAC. c, Echocardiographic parameters. Open and closed bars represent ODN2088 control- and ODN2088-treated groups, respectively (n = 5-8 per group).",
    "molecules": "ODN2088"
  },
  {
    "caption": "(D) Western blot  analysis of BBOX1  in CRIP1-overexpressing Huh7  cells treated with CHX  (100 μg/ml) for the indicated durations (lfte). The graph shows quantification of relative BBOX1  levels (right). Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01. Differences were tested using an unpaired 2-tailed Student's t-test D).",
    "molecules": "CHX"
  },
  {
    "caption": "(E) Western blot  analysis of BBOX1  protein levels in CRIP1-overexpressing Huh7  and Hep3B  cells treated with MG132  (10 μM) for 10 h. D).",
    "molecules": "MG132"
  },
  {
    "caption": "Representative images of  tumor sphere formation and quantification of spheres (C) in CRIP1-overexpressing Huh7  cells with or without iCRT3  (50 μM) for 24 h. Scale bar: 50 μm. Data information: Data represent the mean ± SD of at least three independent experiments. **P <0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test",
    "molecules": "iCRT3"
  },
  {
    "caption": "(E) Western blot  analysis of HCC  cancer stem cell  markers in CRIP1-overexpressing Huh7  cells with or without iCRT3  (50 μM) for 24 h.",
    "molecules": "iCRT3"
  },
  {
    "caption": "(F) Coimmunoprecipitation and immunoblot  analyses of β-catenin acetylation in CRIP1  knockdown MHCC-97H cells treated with mildronate  (5 mM) for 24 h.",
    "molecules": "mildronate"
  },
  {
    "caption": "(G) Western blot analysis of cytoplasmic  and nuclear  fractions of  β-catenin in CRIP1  knockdown MHCC-97H cells treated with mildronate  (5 mM) for 24h.",
    "molecules": "mildronate"
  },
  {
    "caption": "(I)  Co-immunoprecipitation and immunoblot  analyses of β-catenin ubiquitination  in CRIP1  knockdown MHCC-97H cells treated with mildronate  (5 mM) and EX527  (20 μM) for 24 h.",
    "molecules": "EX527, mildronate"
  },
  {
    "caption": "A. CAF and WI-38 cells pretreated with EVs from MDA-MB-231 or MCF10A cells, or PBS for 48 h were unstarved (U) or starved (S) for 2 h in EBSS prior to addition of a mixture of leucine, arginine, and lysine (LRK) or a cocktail of essential AAs (EAA). Cells were analyzed by Western blot analysis in a time course as indicated.",
    "molecules": "arginine, leucine, lysine"
  },
  {
    "caption": "B. Western blot analysis of puromycin (Puro)-labeled fibroblasts (CAF, normal human mammary fibroblasts, WI-38, and NIH3T3) after 2-h starvation and EAA add-back in the presence or absence of cycloheximide (CHX). C. Western blot analysis of Puro-labeled fibroblasts cultured in growth medium. ",
    "molecules": "CHX, cycloheximide, Puro, puromycin"
  },
  {
    "caption": "A,B. Decreases of leucine, arginine, and lysine (A) as well as production of ammonium (B) in the conditioned medium (CM) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS (n=3 biological replicates for each group).",
    "molecules": "ammonium, arginine, leucine, lysine"
  },
  {
    "caption": "C. Intracellular levels of leucine, arginine, and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells (n=3 biological replicates for each group).",
    "molecules": "arginine, leucine, lysine"
  },
  {
    "caption": "C. Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX.",
    "molecules": "CHX, Puro"
  },
  {
    "caption": "D. Western blot analysis of NIH3T3 cells that had been transfected with a pRK5-RAGC-HA-GST expression plasmid or control vector and treated with EVs or PBS for 48 h, and then labeled with Puro.",
    "molecules": "Puro"
  },
  {
    "caption": "D. A heatmap showing the comparison of log2 fold change (FC) of the abundance of each identified 13C6-labeled protein and its corresponding mRNA level determined by RNA-seq.",
    "molecules": "13C6"
  },
  {
    "caption": "C. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. Apc1-loop500 WT or its derivatives (∆11 or 3A) were incubated with the 35S-labelled Flag-B56γ in interphase extract (Inter) or anaphase extract (Ana) supplemented with CycB∆167 at 23˚C for 1 hr. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or coomassie brilliant blue (CBB) staining. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.",
    "molecules": "amylose, coomassie brilliant blue, 35S"
  },
  {
    "caption": "D. Cdk-dependent in vitro kinase assay of Apc1-loop500. MBP fused WT or 3A Apc1-loop500 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 23˚C for 10 min, separated by SDS-PAGE and detected by autoradiography.",
    "molecules": "ATP, 32P"
  },
  {
    "caption": "E. Cdk can promote Apc1-loop500 and B56 interaction. MBP fused WT or 3A Apc1-loop500 fragment was incubated in the presence or absence of Cdk2-cyclin A at 30˚C for 60 min. MBP-fused peptides (-/+ kinase) were isolated and incubated with purified Flag-B56γ at 23˚C for 30 min. The bound proteins were recovered by amylose beads and analysed by SDS-PAGE and immunoblotting with indicated antibodies. F. Quantification of E. Bound B56γ to WT Apc1-loop500 control (-Cdk) was arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.",
    "molecules": "amylose"
  },
  {
    "caption": "C. B56 binding site mutant APC/C (1-L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1-L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay using 35S-labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS-PAGE and autoradiography. D. Quantification of C. Error bars, s.e.m. from three independent experiments.",
    "molecules": "35S"
  },
  {
    "caption": "A. (left panel) Cdk-dependent in vitro kinase assay of Apc1-loop300. 3xFlag-tagged WT or 7T Apc1-loop300 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 30˚C for 30 min, and separated by SDS-PAGE and detected by autoradiography. (right panel) 32P-phosphorylated WT or 7T Apc1-loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS-PAGE followed by autoradiography. B. Quantification of removal of radioactivity in A. Error bars, s.e.m. from three independent experiments. ",
    "molecules": "ATP, 32P"
  },
  {
    "caption": "(E) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non‐covalent Atg12‐Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild‐type.",
    "molecules": "phosphatidylethanolamine, urea"
  },
  {
    "caption": "(C) Re-localization of Apl5 upon Vps41 re-localization. Vps41 was tagged with a C-terminal FRBGFP tag in a strain, where Apl5 carries a C-terminal mCherry and Pma1 a FKBP-tag Cells were grown in either the absence (control) or presence of 10 μM rapamycin, and localization of Vps41 and Apl5 was analysed by fluorescence microscopy. Scale bar, 5 μm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(D) Effect of clathrin deletion on the AP-3 pathway. GFP-tagged Nyv1 with a C-terminal Snc1 transmembrane domain (GNS) was localized relative to FM4-64 stained vacuoles in the indicated strains. Labeling of the cell surface by GFP indicates a defect in the AP-3 pathway (E) Quantification of AP-3 defect from (D). Linear intensity plots were laid over the cells, the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 30 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(C) Tethering of AP-3 vesicles to the mitochondrial surface. Cells were grown at 30°C in YPG and kept in logarithmic phase. Just prior to imaging, the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC (difference interference contrast) image of cells. Scale bar, 5 µm. The inset shows the indicated magnification. Scale bar in inset, 2 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects. First column shows DIC image of cells. Scale bar, 5 µm. (B) Quantification of the AP-3 defect from (A). Linear intensity plots were laid over cells grown in medium containing glucose (D) or galactose (G), the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 15 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "(C) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots,",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(E) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells in a GAL1pr-Vps41-GFP-Fis1 background, from a label-free proteomics analysis of streptavidin-biotin pulldowns with cells grown in YPG. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.001) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(B) AP-3 sorting defects in GAP deletions. The AP-3 reporter was localized relative to FM4-64 stained vacuoles in the indicated strains. First column shows the DIC image of cells. Scale bar, 5 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(D) Localization of AP-3 in ArfGAP deletion strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. First column shows DIC image of cells. Scale bar, 5 µm. (E) Quantification of Apl5-dots in (D). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(A, B) AP-3 sorting defects in strains overexpressing Gcs1 (A) or Age2 (B). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(C) Localization of AP-3 in ArfGAP overexpression strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (D) Quantification of Apl5 dots in (C). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean. ",
    "molecules": "FM4-64"
  },
  {
    "caption": "(E, F) AP-3 sorting defects in GAP-dead mutants of Gcs1 (E) and Age2 (F). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(G) Localization of AP-3 in GAP-dead mutants of Gcs1 and Age2. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (H) Quantification of Apl5 dots in (G). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean. ",
    "molecules": "FM4-64"
  },
  {
    "caption": "(A-C) The relative levels of (A) canalicular membrane (BA) transporter Bsep, Mrp2, Mdr1b, Mdr2, Abcg5 and Abcg8 mRNA transcripts, (B) basolateral membrane BA transporter Mrp3, Mrp4, Ostβ, Ntcp and Oatp1a1 mRNA transcripts, and (C) BA synthetic enzyme Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and Cyp2c70 mRNA transcripts. Wild type, wild type mice, n=6; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=7. Data are shown as means ± SD. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.",
    "molecules": "BA"
  },
  {
    "caption": "(G) Multiplex IF analyses revealed that transfection with the SEMA7A_WT or SEMA7A_R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose-dependent manner (0, 0.2, 1.0 μg per well of 6-well plate; 1.5 ml culture medium per well). Scale bar, 50 µm. Together, the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis (Appendix Table S16). Subsequently, cholestasis triggered an adaptive response in the liver by down-regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up-regulating the expression of BA efflux transporters Mrp3, Mrp4 and Ostα/β.",
    "molecules": "BA, collagen"
  },
  {
    "caption": "Treatment with UDCA (13 mg/kg/d) and GSH (40 mg/kg/d) for 2 weeks restored normal liver function in the child. However, when the treatment was ceased the levels of serum ALT, AST and TBA returned to abnormally high levels in the patient. Interestingly, treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST, and reduced serum TBA in the child patient.",
    "molecules": "TBA, GSH, UDCA"
  },
  {
    "caption": "A Immunostaining of HeLa cells probed for FITC‐labeled streptavidin and DNA and the merge streptavidin (red) and DNA (blue). Cell surface was labeled using amine biotinylation. Scale bar, 10 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "A Tubulin and PCDH7immunostaining of unsynchronized HeLa cells: tubulin, green; PCDH7, red; DNA, blue. PCDH7 localizes to the mitotic cell surface (arrows). PCDH7 staining is barely detectable in the surrounding interphase cells. Scale bar, 5 μm.B Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis (n = 6, red) and interphase (n = 7, blue). Bars show mean ± SEM.",
    "molecules": "DNA"
  },
  {
    "caption": "C PCDH7‐EGFP‐transfected unsynchronized U2OS cells, stably expressing mCherry‐tubulin, at mitosis (top) and interphase (bottom). PCDH7, green; tubulin, red; DNA, blue. Scale bar, 10 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "D Immunostaining of HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes (green) probed for tubulin (red) and DNA (blue). Top panel shows mitotic cell those were treated with 5 μM STC for 10 h, and bottom panel shows interphase cells. Scale bar, 10 μm.",
    "molecules": "DNA, STC"
  },
  {
    "caption": "A Confocal microscopy images representing the localization of PCDH7. U2OS cells expressing full‐length (WT) PCDH7 at mitosis (top) and interphase (middle). Cytoplasmic deleted (CytΔ) PCDH7‐expressing cells at mitosis (middle) and interphase (bottom). PCDH7, green; DNA, blue. Scale bar, 20 μm.B Quantification of PCDH7::GFP (WT and CytΔ) protein localization on the cell surface in mitosis [WT (n = 11), CytΔ (n = 6), red] and interphase [WT (n = 25), CytΔ (n = 20), blue]. Interphase cytΔ PCDH7 localizes to the cell surface more than WT (full length) PCDH7 (insets). Bars show mean ± SEM.",
    "molecules": "DNA"
  },
  {
    "caption": "B Flow cytometry analysis of HeLa cells treated with anti‐PCDH7 antibody, fixed and stained for phospho‐Histone H3 and DNA. Forty‐two percent of unsorted HeLa cella are in G2/M phase. When cells are sorted based on their phospho‐Histone H3 (middle) expression, phospho‐Histone H3+ (high expression) cells are preferentially mitotic (87.3% in G2/M phase) and 42% of those are PCDH7+. Similarly, PCDH7+ (high expression) cells are preferentially mitotic (82.1% in G2/M phase) and 79% of those are phospho‐Histone H3+ (bottom).",
    "molecules": "DNA"
  },
  {
    "caption": "(e) DASH mutants increase histone-humanization in the scc4D65Y background. Humanization rates of the scc4D65Y mutant with and without DAD1E50D or DAM1N80Y. Significance of the mean difference in 5-FOAR frequency was determined with the Mann-Whitney test. Each dot represents one biological replicate (n = 8), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.",
    "molecules": "5-FOA"
  },
  {
    "caption": "(b) Flow cytometry analysis of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at the permissive (24˚C) and non-permissive temperature (30ºC). Yeast cultures were grown to logarithmic phase at 24˚C and divided and grown at 24˚C and 30˚C for 6 hours before cells were collected and processed for DNA content analysis using Sytox Green stain.",
    "molecules": "Sytox Green"
  },
  {
    "caption": "(c) Growth assays of wild type, mad3∆ mutants, and mad3∆ DAD1E50D double mutants on YPD with and without 30 µg/ml benomyl. Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto the indicated solid media and grown at 30˚C for three days.",
    "molecules": "benomyl"
  },
  {
    "caption": "(b) Histone-humanization assay for various dam1N80X mutants. 5-FOA is used to counter-select the yeast histone plasmid, forcing growth with the human histone plasmid. Yeast were serially diluted from a starting culture of 1.0 OD600.",
    "molecules": "5-FOA"
  },
  {
    "caption": "RT-PCR  analysis of p53  3' flanking region using the same primers employed in the data panel above (n=3). Lane 1 is a control PCR  amplification of cDNA derived from the reverse transcription  of a control mRNA using  oligo (dT). Lanes 2-7 are PCR  amplification of reverse transcribed p53 chromatin-associated pre-mRNA using oligo oligo (dT).",
    "molecules": "oligo (dT)"
  },
  {
    "caption": "Enzyme activity assay of fetal organoids for H Lactase, I sucrase, J maltase, K trehalase and L arginase. Activity is given in µM glucose/µg protein·min-1 (experiment was generated from single organoid culture (see material and methods) and repeated in 3 independent organoid cultures with similar results).",
    "molecules": "glucose"
  },
  {
    "caption": "Enzyme activity assay of fetal ( ) and adult ( ) organoids. H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28, while I sucrase, J maltase, K trehalase and L arginase increase to adult levels. Activity is given in µM glucose/µg protein·min-1 (n=3 independent organoid cultures).",
    "molecules": "glucose"
  },
  {
    "caption": "Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. Decrease in relative expression of neonatal marker A Blimp-1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated organoids",
    "molecules": "dexamethasone"
  },
  {
    "caption": "Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).",
    "molecules": "Dexamethasone, glucose"
  },
  {
    "caption": "Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. expression of adult marker E Treh and F Arg2 remains unchanged (n=3 individual wells from single organoid culture",
    "molecules": "dexamethasone"
  },
  {
    "caption": "Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).",
    "molecules": "Dexamethasone, glucose"
  },
  {
    "caption": "(C) Live-cell recording as in (A). In addition, mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy. Arrows mark sites where nucleoids but no lamellar cristae are present.",
    "molecules": "PicoGreen, DNA"
  },
  {
    "caption": "(D) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA. Arrows mark sites where nucleoids are present.",
    "molecules": "dsDNA"
  },
  {
    "caption": "(D) 3D SIM of living WT and Mic10-KO cells. Cells were labeled with Mitotracker Green. Images show maximum intensity projections.",
    "molecules": "Mitotracker Green"
  },
  {
    "caption": "(F) 2D STED nanoscopy of mitochondria. Mic19-KO cells were transfected with a plasmid for Mic10-FLAG expression under the control of a tetracycline-inducible promoter. Left panel: Mitochondria from noninduced cell. Right: Mitochondria from induced cell expressing Mic10-FLAG.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(C) Recovery of mitochondrial networks upon Mic60 re-expression. Cells were induced with doxycycline hyclate for 72 h, immunolabeled for TOM20 and visualized by confocal microscopy.",
    "molecules": "doxycycline hyclate"
  },
  {
    "caption": "(A,B) Live-cell nanoscopy of Mic10-TO cells. Cells expressing COX8A-SNAP were labeled with SNAP-cell SiR and recorded with STED nanoscopy. (A) Cristae architecture in noninduced cells. (B) Cristae architecture after Mic10 re-expression (24 h induction with doxycycline). STED image data were deconvolved.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C) Quantification of Yki reporter genes expression upon overexpression of Yki isoforms in the wing posterior domain (hh-Gal4 driver). For each target gene, relative expression between posterior and anterior domain in the wing discs was quantified by immunostaining using anti-ßgal antibody (for ex-lacZ and diap1-lacZ) or direct visualization of GFP (ban-sensor, in this case posterior domain was visualized by immunostaining against V5-tag present in UAS-Yki transgenes). In the scatter dot plot, each symbol (circle, square and triangle) represents a single wing disc, bars represent mean with SEM. ** p-value<0.01; **** p-value<0.0001 (unpaired two-tailed t-tests).",
    "molecules": "ßgal"
  },
  {
    "caption": "(A-C) Atg7+/+ and Atg7-/- MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.",
    "molecules": "LLOMe, silica"
  },
  {
    "caption": "(A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(F-H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B‐mutant (H) cells was quantified by G‐Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.",
    "molecules": "E64d, LLOMe, Pepstatin A"
  },
  {
    "caption": "(A-F) HeLa cells stably expressing GFP‐LC3 were transfected with mStrawberry‐Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B-F). Green: LC3; magenta: Gal3; blue: DAPI.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(G-I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(A) Plasmacreatinine and ureanitrogen in Atg5F/F and Atg5F/F;KAPmice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/Fmice; F/F;KAP: Atg5F/F;KAPmice.",
    "molecules": "nitrogen, creatinine, OA, urea, UA"
  },
  {
    "caption": "(B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4-7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C).",
    "molecules": "OA, UA"
  },
  {
    "caption": "(D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm.",
    "molecules": "OA, UA"
  },
  {
    "caption": "(E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm.",
    "molecules": "OA, UA"
  },
  {
    "caption": "C. Confocal images of U2OS cells transiently expressing lifeAct-GFP wild type and mutants of lifeAct residues interacting with F-actin, cells were additionally stained with SiR-actin to confirm the actin filaments. The line scan as indicated with yellow line on the cells shows the extent of lifeAct (green) and SiR-actin (magenta) co-staining of actin structures. Scale bar = 5µm.",
    "molecules": "lifeAct"
  },
  {
    "caption": "D. Binding affinities calculated from titration data of co-sedimentation assays of lifeAct-14 (1.2µM) and lifeAct-17 (2.2µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.",
    "molecules": "lifeAct-14, lifeAct-17"
  },
  {
    "caption": "C. Representative TIRF images of lifeAct binding experiments as indicated. The remaining lifeAct concentration images are shown in Supplement Figure 4. Scale bar = 5µm.",
    "molecules": "lifeAct"
  },
  {
    "caption": "D. Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F-actin (mean and s.e.m; n = 2 or 3 independent experiments with >50 actin filament for each set).",
    "molecules": "lifeAct, jasplakinolide, phalloidin"
  },
  {
    "caption": "(D,E) Increased phosphorylation of β-catenin at Ser-675 but not at Ser-552 in the nucleus-enriched P1 fraction but not in whole-brain lysates of PtenΔC/ΔC mice (3 months). Note that total levels of β-catenin were not changed in the P1 fraction or whole-brain lysates. (n = 4 mice for WT-WB/P1 and ΔC-WB/P1, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.",
    "molecules": "Ser"
  },
  {
    "caption": "(B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.",
    "molecules": "glycine, proline"
  },
  {
    "caption": "(B) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by DCS treatment (20 μM), as shown by fEPSP slopes. (n = 15 slices from 9 mice for WT-V/vehicle, 11 (5) for WT-D/DCS, 16 (10) for ΔC-V, 13 (7) for ΔC-D, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.",
    "molecules": "DCS"
  },
  {
    "caption": "(F) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by sarcosine treatment (750 µM), as shown by fEPSP slopes. Note that the data for WT-V and ΔC-V are identical to those shown in Fig. 4B because the whole experiments were performed together; we generated independent figures for DCS and sarcosine results for the clear presentation of the data. (n = 15 slices from 9 mice for WT-V/vehicle, 9 (4) for WT-Sarc/Sarcosine, 16 (10) for ΔC-V/Vehicle, 18 (4) for ΔC-S, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.",
    "molecules": "DCS, Sarcosine, sarcosine"
  },
  {
    "caption": "(D) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display partially normalized levels of whole-brain glycine, as shown by the lack of difference between saline-treated WT and ASO-treated PtenΔC/ΔC mice. Note that the glycine levels observed here are ~4 times lower than those measured in Fig. 3A, which could be attributable to different mouse ages (P21 in Fig. 3A vs. 2-4 months in Fig. 5D), absence and presence of ASO injection, or lot-to-lot variation of the ELISA kits. (n = 8 mice for WT-saline, 5 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The error bars represent SEM.",
    "molecules": "ASO, glycine, saline"
  },
  {
    "caption": "(E) PtenΔC/ΔC mice (P20-37) treated with Slc6a20a-ASO display an NMDA/AMPA ratio at hippocampal SC-CA1 synapses that is comparable to that in ASO-treated WT mice. (n = 11 neurons from 5 mice for WT-ASO, 10 (4) for ΔC-ASO, ns, not significant, Mann-Whitney's U test). The error bars represent SEM.",
    "molecules": "ASO"
  },
  {
    "caption": "(G) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, *P < 0.05, **P <0.01, ns, not significant, two-way ANOVA with Bonferroni's test. The error bars represent SEM.",
    "molecules": "ASO, saline"
  },
  {
    "caption": "(D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM.",
    "molecules": "glycine, proline"
  },
  {
    "caption": "(B) Glycine/proline-induced currents for mouse SLC6A20A are dependent on sodium chloride, as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate. Each experimental condition was sequentially applied to HEK293T cells, as shown in Appendix Fig. S7B. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 21 cells for untransfected/blank, 17 for human SLC6A20-V1, 25 for human SLC6A20-V2, 33 for mSLC6A20A, 28 for mSLC6A20B ***P < 0.001 (relative to buffer not containing glycine), two-way ANOVA with Bonferroni's test; proline, n = 17 cells for untransfected, 23 for human SLC6A20-V1, 47 for SLC6A20-V2, 31 for mSLC6A20A, and 31 for mSLC6A20B, ***P < 0.001, two-way ANOVA with Bonferroni's test). The error bars represent SEM.",
    "molecules": "choline chloride, glycine, Glycine, proline, sodium chloride, sodium gluconate"
  },
  {
    "caption": "Representative CT images of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on an NCD or HFD, showing the whole-body fat composition.",
    "molecules": "fat"
  },
  {
    "caption": "Glucose tolerance tests (GTT) and Insulin tolerance tests (ITT) were performed on WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. Blood glucose concentrations during GTT and ITT were monitored. (n= 10, per genotype)",
    "molecules": "glucose"
  },
  {
    "caption": "Blood glucose and plasma insulin levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)",
    "molecules": "glucose"
  },
  {
    "caption": "Plasma Free Fatty Acid (FFA) and Glycerol levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)",
    "molecules": "FFA, Free Fatty Acid, Glycerol"
  },
  {
    "caption": "H&E and Oil Red O staining, and liver triglyceride (TG) content in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)",
    "molecules": "Oil Red O, TG, triglyceride"
  },
  {
    "caption": "Photograph of WT fl/fl and QKI KO fl/fl:AP2-Cre classical BAT in 4% paraformaldehyde solution, representative transmission electron micrographs, and UCP1 staining of BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice.",
    "molecules": "paraformaldehyde"
  },
  {
    "caption": "Representative recording of Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from BAT of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on NCD; Oxygen consumption was performed under basal conditions, following the addition of 0. 25 mg mitochondria (Mit), 5 mM substrate glycerol-3-phosphate (G3P), 2 mM GDP, and 450 mM ADP, 2 μg/ml oligomycin, and 1.5 μM FCCP. Quantification of experimental data. (n=3 biological replicates, per group)",
    "molecules": "ADP, FCCP, GDP, oligomycin, G3P, glycerol-3-phosphate"
  },
  {
    "caption": "UCP1-dependent oxygen consumption rates estimated as GDP-inhabitable rates, based on the data shown in (K). (n=3 biological replicates)",
    "molecules": "GDP"
  },
  {
    "caption": "Representative pictures of oil red O staining in three adipocyte types: ShScramble+ovCon cells, QKI knockdown cells (ShQKI+ovCon) and QKI knockdown with QKI-5 re-expression cells (ShQKI+ovQKI).",
    "molecules": "oil red O"
  },
  {
    "caption": "The mRNA stability in adipocytes was measured by incubating cells with actinomycin D (5 μg/ml). Actinomycin D was added to stop transcription, and RNAs were harvested at the indicated times (x-axis) after transcription inhibition. Real-time PCR was used to determine remaining UCP1, PGC1α and Actin mRNA levels compared with the starting time. Increasing PGC1α mRNA half-life by depletion of QKI (ShScramble T1/2 = 4.73 h, ShQKI T1/2 = 8.04 h). (n=4 biological replicates)",
    "molecules": "actinomycin D, Actinomycin D"
  },
  {
    "caption": "Red Nile staining in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).",
    "molecules": "Red Nile"
  },
  {
    "caption": "TG content in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).",
    "molecules": "TG"
  },
  {
    "caption": "The relative luminescence of a QKI promoter reporter under cAMP (Forskolin and IBMX) stimulus. (n=3 biological replicates)",
    "molecules": "cAMP, IBMX, Forskolin"
  },
  {
    "caption": "The relative mRNA levels of genes (UCP1, PGC1α, Dio2) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus, following cAMP (Forskolin and IBMX) stimulation. (n=3 biological replicates)",
    "molecules": "cAMP, IBMX, Forskolin"
  },
  {
    "caption": "b, c Phospho-sites identified after TiO2 enrichment b and phospho-tyrosine immunoprecipitation (pY-IP) c. Left panel: pie chart presenting the proportion of identified phosphorylated serines (pS), threonines (pT) and tyrosines (pY) in the two data sets. Right panel: cumulative number of unique identified phospho-sites across the biological replicates (3 and 4 independent experiments for the TiO2 and pY-IP, respectively). Phosphorylated serines, threonines and tyrosines are presented individually next to the total number of identified sites (pSTY). The phospho-sites that were not previously reported in any species in the PhosphoSitePlus database (www.phosphosite.org) are reported in isolation in the graph on the right.",
    "molecules": "serines, threonines, tyrosine, tyrosines, TiO2"
  },
  {
    "caption": " a, b Output of the statistical analyses of the TiO2-fractionated a and the pY-IP-fractionated b data sets. Left panel: number of phospho-sites significantly regulated upon TCR stimulation presented in bar plots for each time point, next to the total number of regulated sites across the entire time course (total). The number of phosphorylated threonines (pT) and serines (pS) regulated in the TiO2 data set are indicated in yellow and green, respectively. Right panel: representative volcano plot presenting the statistical significance distribution against the log2-transformed fold change between 300 seconds and the unstimulated control. For each condition, phospho-sites were considered significantly up- (red) or down- (blue) regulated when displaying a corrected P-value ≤ 0.05 (Anova test) and an absolute fold change ≥ 1.75 (see Materials and Methods for more detailed information). ",
    "molecules": "serines, threonines, TiO2"
  },
  {
    "caption": " d Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control. ",
    "molecules": "AKTi VIII, DMSO, U0126"
  },
  {
    "caption": " b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers (0.1 nM) in presence or absence of soluble anti-CD28 antibody or with IL-7 as control. Cell surface expression of CD69 and CD5 were analyzed by flow cytometry (Z-score normalization of the Geometric Means of fluorescence MFI per experiment) in four independent experiments. Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test. Boxplot elements: center line corresponds to median, box limits correspond to the first and third quartiles, whiskers indicate variability from Q1-1.5. IQR to Q3+1.5 IQR. Outliers are shown as black dots.",
    "molecules": "N4"
  },
  {
    "caption": " c Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments. ",
    "molecules": "N4, Iono, ionomycin, PMA"
  },
  {
    "caption": "e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.",
    "molecules": "N4"
  },
  {
    "caption": " a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP, sgITSN2-1 or sgITSN2-2 and left unstimulated (-) or stimulated for 2 or 5 min with N4 tetramers (20 nM). Upper panel: membrane probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT (loading control). Lower panel: membrane probed with antibodies to anti-CBL-pY774, anti-ZAP70-pY319/352, anti-AKT-pT308, anti-ERK1/2-pY204/T202 or anti-ERK1/2 (loading control). ",
    "molecules": "N4, Tyr, tyrosine"
  },
  {
    "caption": " b Heat map showing normalized levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 left unstimulated (-) or stimulated for 1, 3 or 10 min with N4 tetramers (1 or 10 nM) and analyzed by mass cytometry. Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. ",
    "molecules": "N4"
  },
  {
    "caption": " c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points (left panel) or stimulated with increased doses of N4 peptides for 4 h (right panel), were analyzed by FACS for surface TCR expression. Data are presented as mean ± SEM and comparisons were performed using a paired t-test (*, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001). A representative analysis of three independent experiments is shown. ",
    "molecules": "N4"
  },
  {
    "caption": " d Heatmap showing scaled expression levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 and stimulated for 6 h with increasing doses of N4 peptides or PMA/Ionomycin (PI) analyzed by single-cell mass cytometry (left). Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. Signal difference (mean arcsinh difference) between sgEGFP and sgITSN2-1 transfected cells is depicted on the right panel. ",
    "molecules": "N4, Ionomycin, PMA"
  },
  {
    "caption": "(D) Substrate-dependent oxygen consumption rates in isolated heartmitochondria. Complex I substrates (pyruvate, malate, glutamate); complex II substrate (succinate). State 3 - ADP stimulated oxygen consumption rate; State 4 - respiration rate after addition of oligomycin; Uncoupled - oxygen consumption rate in the presence of FCCP. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=7).",
    "molecules": "ADP, FCCP, glutamate, malate, oligomycin, oxygen, pyruvate, succinate"
  },
  {
    "caption": "(E) In organello transcription analysis upon 60 minutes labelling with -32P-UTP.",
    "molecules": "UTP"
  },
  {
    "caption": "(A) De novo synthesized mitochondrial proteins were isolated after labelling with 35S-methionine for 1 hour. Positions of individual mitochondrial encoded proteins are indicated.",
    "molecules": "methionine"
  },
  {
    "caption": "(B) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin, doxycycline and chloramphenicol (+ antibiotic), followed by a 48 hours recovery period (- antibiotic). During both periods, proteins are isolated at 12h, 24h and 48h. Antibodies against subunits of C I (NDUFB6), C III (UQCRC1), C IV (COXI), MRPS35 and MRPL37 were used as indicated.",
    "molecules": "actinonin, chloramphenicol, doxycycline"
  },
  {
    "caption": "(C) Quantitative analysis of the COXI recovery upon treatment with antibiotics (Actinonin, DOXY - doxycycline and CAP - chloramphenicol) as in B. The values represent ratios of recovered (48h, - antibiotics) versus untreated (0h) COXI levels. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4). (Actinonin and CAP n=3, DOXY n=4).",
    "molecules": "Actinonin, chloramphenicol, doxycycline"
  },
  {
    "caption": "(D) Cycloheximide (CHX) chase assay to determine the stability of ERAL1 and EFG1. Proteins are visualized by Western blot with respective antibodies. Proteins are isolated after 2, 4, 6, 8,10 and 12 hours after beginning of chase (0). The arrowhead indicates positions of ERAL1.",
    "molecules": "CHX, Cycloheximide"
  },
  {
    "caption": "(C) De novo synthesis of mitochondrial proteins in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) MEFs upon Eral1 siRNA. Proteins were isolated after labeling with 35S-methionine for 1 hour from cells on control siRNA (Ctrl) or cells on Eral1 siRNA (Eral1). Positions of individual mitochondrial encoded proteins are indicated. Coomassie blue staining was used to ensure equal loading.",
    "molecules": "methionine"
  },
  {
    "caption": "(E) Western blot analysis of RC subunits upon Eral1 knockdown in MEFs. Cells (0) were treated with doxycycline for 48 hours (T) and upon doxycycline removal allowed to recover for 24 hours (R).(F) Quantification of the relative recovery of COXI and NDUFB6 upon Eral1 siRNA. The values are calculated as ratio between recovered (R) to doxycycline untreated (0) protein levels in Eral1-knockdown cells (Eral1) and compared to the same results obtained in control cells (Ctrl). Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (COXI n=4; NDUFB6 n=3). Numbers within bars represent statistical difference between control/Eral1 siRNA treated cells and numbers above bars represent difference between +/+; Eral1 siRNA and -/-; Eral1 siRNA.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A) Sirt5 deficiency leads to higher ROS levels in MEFs. ROS levels were determined in the indicated MEFs as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).",
    "molecules": "ROS"
  },
  {
    "caption": "(B) Sirt5 deficiency suppresses GSH production in MEFs. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).",
    "molecules": "GSH, GSSG"
  },
  {
    "caption": "(C) Sirt5 deficiency inhibits NADPH production in MEFs. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).",
    "molecules": "NADP+, NADPH"
  },
  {
    "caption": "(E) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat. The levels of cleaved PARP and Caspase-3 were determined by western blot analysis.",
    "molecules": "Paraquat"
  },
  {
    "caption": "(F) Sirt5 protects MEFs from ROS-induced cell death. Wild-type and Sirt5 KO MEFs were treated with Paraquat (500 μM) for 24 hrs, and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).",
    "molecules": "Paraquat, ROS"
  },
  {
    "caption": "(G-I) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection. Female Sirt5 KO and WT littermates (n=3~5 per group) were intraperitoneally injected with saline or paraquat (10 mg/kg) once a week for 3 consecutive weeks. The ratios of [NADPH/NADP+] and [GSH/GSSG] and the level of lipid peroxide were determined in tissue extracts. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).",
    "molecules": "lipid peroxide, GSH, GSSG, NADP+, NADPH, Paraquat, paraquat"
  },
  {
    "caption": "(J-K) Sirt5 KO mice are more sensitive to Paraquat-induced nigrostriatal dopaminergic degeneration. Female Sirt5 KO and WT littermates were treated with paraquat as in (G-I). The brain sections were stained to detect tyrosine hydroxylase (TH) positive dopaminergic neurons in the SNc region, as described in 'Materials and Methods'. Representative immunohistochemistry images (original magnification, 100×; scale bar, 100 μm) and the corresponding quantifications are shown in (J) and (K), respectively. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).",
    "molecules": "Paraquat, paraquat"
  },
  {
    "caption": "(A)Knockdown of SIRT5 inhibits NADPH production in HEK293T cells. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).",
    "molecules": "NADP+, NADPH"
  },
  {
    "caption": "(B) Knockdown of SIRT5 suppresses GSH production in HEK293T cells. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).",
    "molecules": "GSH, GSSG"
  },
  {
    "caption": "(C) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).",
    "molecules": "ROS"
  },
  {
    "caption": "(D) Knockdown of SIRT5 leads to higher sensitivity to Paraquat. The expression levels of PARP and Caspase-3 were determined by western blot analysis.",
    "molecules": "Paraquat"
  },
  {
    "caption": "(E) SIRT5 protects HEK293T cells from ROS-induced cell death. The indicated stable HEK293T cells were treated as mentioned above in (D), and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).",
    "molecules": "ROS"
  },
  {
    "caption": "(A) G6PD activity is not regulated by lysine succinylation. Purified Flag-tagged G6PD was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of G6PD enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).",
    "molecules": "succinyl-CoA"
  },
  {
    "caption": "(B) IDH2 activity is suppressed by lysine succinylation. Purified Flag-tagged IDH2 was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of IDH2 enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).",
    "molecules": "succinyl-CoA"
  },
  {
    "caption": "(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).",
    "molecules": "H2O2, Paraquat"
  },
  {
    "caption": "(D) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli. Flag-SIRT5 was ectopically expressed in HEK293T cells. The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).",
    "molecules": "H2O2, Paraquat"
  },
  {
    "caption": "(E) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5. Flag-tagged SIRT5 was ectopically expressed in HEK293T cells, and then purified by immunoprecipitation with Flag-beads and eluted with Flag peptide. The purified Flag-SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).",
    "molecules": "H2O2, Paraquat"
  },
  {
    "caption": "(F) Chemical oxidants enhance the ability of SIRT5 to activate IDH2. Flag-SIRT5 was ectopically expressed in HEK293T cells, and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then incubated with purified Flag-IDH2 in vitro. IDH2 enzyme activity was measured as described in 'Materials and Methods', normalized by its protein level. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).",
    "molecules": "H2O2, Paraquat"
  },
  {
    "caption": "Western blot and quantification for Coasy from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as a loading control. n=8, 4 for WT and n=8, 6 for KO (vehicle and pPanSH groups, respectively).",
    "molecules": "4'-phosphopantetheine, pPanSH, Ponceau S"
  },
  {
    "caption": "Total iron quantity in cytosol and mitochondria from GP using ICP-MS. The data distribution is presented using a box plot, including minimum, first quartile, median, third quartile, and maximum. The magenta-colored line represent mean. n=6 for both genotypes",
    "molecules": "iron"
  },
  {
    "caption": "Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase (D) and complex I (E) in GP by genotype and treatment status (veh = vehicle; pPanSH = 4'-phosphopantetheine). n=5 for both genotypes and treatment groups.",
    "molecules": "4'-phosphopantetheine, pPanSH"
  },
  {
    "caption": "Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control.",
    "molecules": "4'-phosphopantetheine, pPanSH, Ponceau S"
  },
  {
    "caption": "Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype, brain region and treatment status (pPanSH = 4'-phosphopantetheine). n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) QRT-PCR. n=5 for Western blot for all groups",
    "molecules": "4'-phosphopantetheine, pPanSH"
  },
  {
    "caption": "Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; Oligomycin, FCCP; Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy-glucose. Quantification of baseline OCR and ECAR is shown, as well. n=3 per genotype with four technical replicates.",
    "molecules": "2-deoxy-glucose, 2-DG, Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, FCCP, Oligom, Oligomycin"
  },
  {
    "caption": "Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n=4 for both genotypes and treatment groups.",
    "molecules": "pPanSH"
  },
  {
    "caption": "Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate (0.8 mg/g body weight) or gemfibrozil (1.2 mg/g body weight). n=8 for both genotypes treated with vehicle, n=5 for both KO groups treated with either bezafibrate or gemfibrozil.",
    "molecules": "bezafibrate, gemfibrozil"
  },
  {
    "caption": "Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds: 4'-phosphopantetheine (pPanSH), pantetheine, vitamin B5, and CoA. n=8 (WT-veh), 5 (KO-veh), 5 (KO-pPanSH), 4 (KO-all other intermediates). Correction of Coasy, Tfrc, Ireb2, and Drd1 expression in GP by 4'-phosphopantetheine over a range of doses. n=8 (WT-veh), 5 (KO-veh), 5 (KO-0.82), 4 (WT-5), 5 (KO-5), 5 (KO-8.2), 5 (KO-10). 5 (WT-20), 5 (KO-20)",
    "molecules": "CoA, pantetheine, 4'-phosphopantetheine, pPanSH, vitamin B5"
  },
  {
    "caption": "Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH. n=3 for both genotypes.",
    "molecules": "pPanSH"
  },
  {
    "caption": "ECAR measured in cultured human fibroblasts by genotype and treatment status. Cells were treated for three days with 4'-phosphopantetheine (pPanSH) or vehicle (veh). n=3 for both genotypes.",
    "molecules": "4'-phosphopantetheine, pPanSH"
  },
  {
    "caption": "Relative quantification of Coasy, Tfrc and Drd1 mRNA from GP by number of days post-cessation of treatment with 5μg of 4'-phosphopantetheine per g of body weight. n= 11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-pPanSH-D7), 4 (KO-pPanSH-D7),",
    "molecules": "4'-phosphopantetheine, pPanSH"
  },
  {
    "caption": "(A) ANAC032expression in WT plants sprayed with Pst DC3000, 6 and 24 hpi, compared to control (sprayed with 10 mM MgCl2 (Mock)). Error bars represent means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). ANAC032expression in WT treated with (B) SA or MeJA or (C) COR for 3 and 6 h compared to non-treated controls. In B and C, means ± SD are given (n = 3; 'n' represents independently performed experiments, each including at least 20 seedlings). FCh, fold change. Asterisks indicate a significant difference from their respective controls (p < 0.01; Student´s t-test).",
    "molecules": "MeJA, COR, MgCl2, SA"
  },
  {
    "caption": "(C) Confocal microscopy image showing nuclear localization of ANAC032-GFP fusion protein expressed from the ANAC032 promoter in ANAC032prom:ANAC032-GFP/anac032-1 seedlings treated with Pst at 6 hpi. Left, bright field; right, chlorophyll auto-fluorescence (red) and GFP fluorescence (green) under bright field.",
    "molecules": "chlorophyll"
  },
  {
    "caption": "Transcript levels of (A) NIMIN1 and (B) PR1 in WT, anac032-1 and 35S:ANAC032 plants after 3 and 6 h of treatment with SA compared to their respective controls.",
    "molecules": "SA"
  },
  {
    "caption": "(C), Expression of PDF1.2A in WT and anac032-1 plants after 3 and 6 h of treatment with SA compared to their respective controls. Transcript levels were measured using qRT-PCR and numbers on the y-axis indicate fold change (FCh; log2 basis). Means are shown ± SD (n = 3; 'n' represents independently performed experiments). Asterisks indicate a significant difference from wild type (p < 0.05; Student's t test).",
    "molecules": "SA"
  },
  {
    "caption": "(D), Stomatal aperture 1 h after treatment with COR, ABA, or COR plus ABA, compared to mock. In (A), (B), and (D), data are means ± SD of 12 to16 measurements; in each measurement the rosette leaves from at least 6 - 8 plants were used). Asterisks indicate a significant difference from mock treatment (p < 0.0001; Student´s t-test).",
    "molecules": "ABA, COR"
  },
  {
    "caption": "(A Representative images of HUVECs (A; n=6) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. (D Quantification of tube surface area of micrographs shown in (A, (D: *P=0.0119 (- vs COCO 30ng/ml); *P=0.0108 (- vs COCO 60ng/ml)",
    "molecules": "fibrin"
  },
  {
    "caption": "B) Representative images of HRMECs (B; n=4) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. E) Quantification of tube surface area of micrographs shown in B). (E: **P=0.0013 (- vs COCO 30ng/ml); ***P=0.00026 (- vs COCO 60ng/ml)).",
    "molecules": "fibrin"
  },
  {
    "caption": "(B) Retinal flat mounts of P5 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0059 (PBS vs COCO); *P=0.0169 (PBS vs Flt1Fc). Vessel junctions: **P=0.0063 (PBS vs COCO); *P=0.0159 (PBS vs Flt1Fc); (n=4 mice/group).",
    "molecules": "PBS"
  },
  {
    "caption": "(F) Visualization of pericyte coverage (NG2:green; IsoB4:red) in PBS- or COCO-injected retinas. Scale bar, 50 μm. (G) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining (% overlay) (n=8).",
    "molecules": "PBS"
  },
  {
    "caption": "(B) Retinal flat mounts of P28 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0019 (PBS vs COCO); *P=0.0128 (PBS vs Flt1Fc). Vessel junctions: **P=0.0073 (PBS vs COCO); *P=0.0302 (PBS vs Flt1Fc); (n=4).",
    "molecules": "PBS"
  },
  {
    "caption": "(D) COCO injections (P1, P7 and P14) do not result in ONL thinning in P28 retinas. Scale bar, 40 μm. (E) Quantification of apoptotic cells (cleaved caspase-3; CC3) in control and COCO-injected mice (p-values are versus PBS treatment). (n=4).",
    "molecules": "PBS"
  },
  {
    "caption": "(B) Retinal flat mounts of adult mice injected with PBS or COCO for 5 days are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints; (n=4).",
    "molecules": "PBS"
  },
  {
    "caption": "(E) Retinal flat mounts of adult mice injected with PBS or COCO for 28 days are stained with IB4. Scale bar, 90 μm. (F) Quantification of vascular length and number of branchpoints; (n=4).",
    "molecules": "PBS"
  },
  {
    "caption": "(B) Retinal flat mounts of P17 OIR mice injected with PBS, COCO or Flt1Fc are stained with IB4. Red areas highlight vascular tufts.",
    "molecules": "PBS"
  },
  {
    "caption": "(C) Quantification of neovascular tuft area in P17 OIR. **P=0.0021 (PBS vs COCO); *P=0.0122 (PBS vs Flt1Fc); (n=6). (D) Quantification of central avascular area in P17 OIR; (n=6).",
    "molecules": "PBS"
  },
  {
    "caption": "(F) Choroidal flat mounts of adult mice subjected two weeks prior to Laser-CNV injected with PBS, COCO or Flt1Fc stained with IB4 (green) and phalloidin (red). Scale bar, 100 μm. (G) Quantification of CNV surface area. *P=0.0210 (PBS vs COCO); *P=0.0113(PBS vs Flt1Fc); (n=12-14 burns from 5 animals/group).",
    "molecules": "phalloidin, PBS"
  },
  {
    "caption": "(B) Differential gene expression heatmap generated by DESeq2 of top-altered genes of HUVECs treated with COCO compared to PBS-treated control cells (n = 4 independent samples/group).",
    "molecules": "PBS"
  },
  {
    "caption": "(B) Media glucose levels (BioNova analysis) in HUVECs cultured with COCO for 1 or 6 hours, expressed as % of initial levels *P=0.0459; (n=4).",
    "molecules": "glucose"
  },
  {
    "caption": "(C) Evaluation of ATP content in HUVECs stimulated with COCO for 1, 6 or 24 hours. *P=0.0120 (- vs 1hour); *P=0.0109 (- vs 6hours); *P=0.0219 (- vs 2-DG); (n=4).",
    "molecules": "2-DG, ATP"
  },
  {
    "caption": "(D) Determination of NAD+/NADH ratio in NADH for 6 or 24 hours with COCO. **P=0.0028 (6hours); *P=0.0207 (24hours); (n=3).",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "E) Evaluation of pyruvate content in HUVECs stimulated with COCO for 1, 3, 6, 12 or 24 hours. *P=0.0244; (n=4).",
    "molecules": "pyruvate"
  },
  {
    "caption": "(F) Determination of cellular ROS using DCFDA fluorescence in HUVECs treated with COCO and/or N-acetylcystein. *P=0.0343 (- vs 48hours-NAC); *P=0.0114 (- vs H2O2-NAC); *P=0.0252 (48hours-NAC vs 48hours+NAC); **P=0.0067 (H2O2-NAC vs H2O2+NAC); (n=4).",
    "molecules": "DCFDA, H2O2, N-acetylcystein, NAC, ROS"
  },
  {
    "caption": "(G) Determination of mitochondrial superoxide using MitoSox fluorescence in HUVECs treated with COCO. *P=0.0372 (- vs 24hours); *P=0.0137 (- vs 3-NP); (n=3).",
    "molecules": "MitoSox, superoxide"
  },
  {
    "caption": "(H) Representative images of choroidal explants cultured for 4 days in the presence or absence of COCO and/or N-acetylcystein. Scale bar, 500 μm. (I) Quantification of sprouting surface area of micrographs shown in (H). *P=0.0126 (- vs COCO-NAC); *P=0.0200 (COCO-NAC vs COCO+NAC); (n=6).",
    "molecules": "N-acetylcystein, NAC"
  },
  {
    "caption": " A, B Core histones were prepared from NSPCs treated with various concentrations of crotonate (A) and 1 μM MS-275 (B) for 24 h and subjected to western blotting analysis using anti-Kcr antibody, and the experiments were repeated three times. ",
    "molecules": "crotonate, MS-275, histones"
  },
  {
    "caption": " C NSPCs were treated with 20 μM C646, 10 mM acetate (Ac), 20 μM C646 and 10 mM acetate (Ac+C646), 10 mM crotonate (Cr), and 20 μM C646 and 10 mM crotonate (Cr+C646) for 24 h, then core histones were extracted and subjected to western blotting analysis using anti-Kcr antibody, the experiments were repeated three times. ",
    "molecules": "Ac, acetate, C646, Cr, crotonate, histones"
  },
  {
    "caption": " E Quantification analysis of changes in histone Kcr levels in NSPCs treated with DMSO (Control), 5 μM triptolide (FP), 5 μM flavopiridol and 10 mM crotonate (FP+CR), and 10 mM crotonate (CR) for 1 h. Data are presented as mean ± SEM of three independent biological replicates, n = 3. One-way ANOVA with post hoc Tukey's test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001. ",
    "molecules": "flavopiridol, CR, crotonate, DMSO, histone, FP, triptolide"
  },
  {
    "caption": " A, B Representative images (A) and quantification analysis (B) of changes in cell proliferation ability measured by BrdU incorporation rate among different groups of NSPCs. Scale bar, 50 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.01. ",
    "molecules": "BrdU"
  },
  {
    "caption": " A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment. C, D Quantification analysis of changes in H3K9cr (C) and H3-Kcr (D) levels by western blotting among different groups of explants. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001. ",
    "molecules": "H3K9cr, crotonate, histones"
  },
  {
    "caption": "(B) Representative images of GFAP+Sox2+BrdU+ RGLs, GFAP-Sox2+BrdU+ progenitors, Tbr2+BrdU+ IPCs  in the DG. Scale bar = 20 μm. (C) Quantification of GFAP+Sox2+BrdU+ RGLs  (WT vs. KO, Student's t-test, p = 0.022), GFAP-Sox2+BrdU+ progenitors  (WT vs. KO, Student's t-test, p = 0.0046), Tbr2+BrdU+ IPCs  (WT vs. KO, Student's t-test, p = 0.003) in the DG  of WT and KO mice. WT, n = 4 mice; KO, n = 6 mice. (D) Representative images of GFAP+Mcm2+ RGLs, Tbr2+Mcm2+ IPCs, DCX+Mcm2+ NBs  in the DG. Scale bar = 20 μm. (E) Quantification of GFAP+Mcm2+ RGLs  (WT vs. KO, Student's t-test, p = 0.016), Tbr2+Mcm2+ IPCs  (WT vs. KO, Student's t-test, p = 0.003), DCX+Mcm2+ NBs  (WT vs. KO, Student's t-test, p = 0.003) in the DG  of WT and KO mice. WT, n = 3 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.",
    "molecules": "BrdU"
  },
  {
    "caption": "(H) Representative images of BrdU-retaining RGLs  (tdT+BrdU+) in the DG. Scale bar = 20 μm. (I) Quantification of BrdU-retaining RGLs  (BrdU+tdT+GFAP+, WT vs. cKO, Student's t-test, p = 0.009) in the DG  of WT and cKO mice. WT, n = 4 mice; cKO, n = 3 mice.",
    "molecules": "BrdU"
  },
  {
    "caption": "(I) Representative images of NeuN+BrdU+ adult-born neurons  in the DG  of 6 month-old mice. Scale bar = 50 μm. (J) Quantification of NeuN+BrdU+ adult-born neurons  (WT vs. KO, Student's t-test, p = 0.0052) in the DG  of WT and KO mice  at 7 months of age. WT, n = 4 mice; KO, n = 3 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.",
    "molecules": "BrdU"
  },
  {
    "caption": "(A) Representative images of aNPCs in vitro isolated from WT and KO mice  and followed by a BrdU pulse labeling. Scale bar = 50 μm. (B) Percentage of BrdU+ aNPCs  among total aNPCs  isolated from WT or KO mice  (WT vs. KO, Student's t-test, p = 0.0075). WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; **, p < 0.01, ***, p < 0.001.",
    "molecules": "BrdU"
  },
  {
    "caption": "(A) DARTS analysis  indicated HK1  as a molecular target of L-arginine in aNPCs. The aNPC  lysates treated with  L-arginine, digested with pronase  and then analyzed by  Western blot with anti-HK1  and GAPDH  antibodies. (B) Quantification of HK1  expression level in aNPCs  lysates treated with  L-arginine  and followed by digesting with pronase  (One-way ANOVA, F(3, 8)= 3.61, p = 0.043). n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.",
    "molecules": "L-arginine, pronase"
  },
  {
    "caption": "(I) Co-IP of HK1  with VDAC1  in vehicle or  L-arginine-treated cells. (J) Quantification of the  co-IP efficacy of HK1  and VDAC interaction  in vehicle or L-arginine-treated cells (IP: HK1, vehicle vs.  L-arginine, Student's t-test, p < 0.001; IP: VDAC1, vehicle vs.  L-arginine, Student's t-test, p = 0.0078). Vehicle, n = 3 independent experiments;  L-arginine, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.",
    "molecules": "L-arginine"
  },
  {
    "caption": "(C-E) Quantification of glucose  (C, WT vs. KO, Student's t-test, p < 0.001) and glutamine  (E, WT vs. KO, Student's t-test, p = 0.94) consumption and lactate  production (D, WT vs. KO, Student's t-test, p = 0.019) in WT- and KO-aNPCs. WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05 ***, p < 0.001.",
    "molecules": "glucose, glutamine, lactate"
  },
  {
    "caption": "(C) Quantification of Nestin-GFP+GFAP+ RGLs  in the DG  of WT and KO mice  after vehicle or IACS  treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.99). WT + Vehicle, n = 4 mice; WT + IACS, n = 4 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 3 mice. (D) Quantification of Nestin-GFP+GFAP+BrdU+ activated RGLs  in the DG  of WT and KO mice  after vehicle or IACS  treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (E) Quantification of Nestin-GFP+GFAP-BrdU+ progenitors  (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.028) in the DG  of WT and KO mice  after vehicle or IACS  treatment. WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (F) Quantification of Nestin-GFP+Tbr2+BrdU+ IPCs  (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.02) in the DG  of WT and KO mice  after vehicle or IACS  treatment. WT + Vehicle, n = 4 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.",
    "molecules": "IACS, BrdU"
  },
  {
    "caption": "(H) Representative images of NeuN+BrdU+ adult-born neurons  in the DG. Scale bar = 20 μm. (I) Quantification of NeuN+BrdU+ adult-born neurons  in the DG  of WT and KO mice  at P90 after vehicle or IACS  treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 5 mice; WT + IACS, n = 5 mice; KO + Vehicle, n = 5 mice, KO + IACS, n = 5 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.",
    "molecules": "IACS, BrdU"
  },
  {
    "caption": "(b) Experimental validation of predicted synergistic SR-based combinational therapies in head and neck cancer: A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer-targeting treatments in 5 HNSC cell lines, based on drug treatment experiments. Synergism was estimated using standard Fa-CI analysis. The table displays the average combination index (CI; synergism CI < 1, additivity effect CI = 1, antagonism CI > 1, NAN indeterminate CI) at 50% growth inhibition (Fraction affected). Combinations that are synergistic are colored blue (black otherwise) for each cell lines tested. The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix (number indicates % cell viability at 48h, n = 3), and Fa-CI curve.",
    "molecules": "BYL719, Dasatinib"
  },
  {
    "caption": "(a) Experimental testing of the predicted SR (DU) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine. The matrix displays drug interactions between Decitabine, a DNMT1 inhibitor, and inhibitors of its predicted rescuer genes (X-axis) across 18 NSCLC cell lines (Y-axis) that are insensitive to Decitabine. Row labels present rescuer genes and their inhibitors. Colors in the matrix show whether the interactions found are significantly synergistic (red), antagonistic (green) or non-significant (in gray). Values in the matrix show average synergism (<1 synergism and >1 antagonism Thirteen predicted DU-SR rescuers (red lines), two predicted DD SR rescuers (green lines) of DNMT1 and one random control (JAK3i) were tested.",
    "molecules": "Decitabine"
  },
  {
    "caption": "I. U2OS cell lines conditionally expressing indicated GFP-FAM111A alleles were fixed at the indicated times after treatment with Doxycycline (DOX) to induce expression of the transgenes and stained with crystal violet.",
    "molecules": "crystal violet, DOX, Doxycycline"
  },
  {
    "caption": "A. Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP-FAM111A.",
    "molecules": "DOX"
  },
  {
    "caption": "B. DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse-labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image-based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6A.",
    "molecules": "EdU, DAPI, DNA, DOX"
  },
  {
    "caption": "D-F. Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Figure S6B-D.",
    "molecules": "EdU"
  },
  {
    "caption": "G. Analysis of FAM111A interactors. U2OS/GFP-FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP) and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/-DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).",
    "molecules": "DOX"
  },
  {
    "caption": "K. Quantification of data in (J) for S phase (EdU-positive) cells (red bars, mean).",
    "molecules": "EdU"
  },
  {
    "caption": "A. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated with DOX for the indicated times.",
    "molecules": "DOX"
  },
  {
    "caption": "B. Representative images of U2OS/GFP-FAM111A cell lines that were treated or not with DOX, fixed and stained with γ-H2AX antibody and DAPI.",
    "molecules": "DAPI, DOX"
  },
  {
    "caption": "D. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated or not with DOX and the pan-Caspase inhibitor Z-VAD-FMK as indicated.",
    "molecules": "DOX, Z-VAD-FMK"
  },
  {
    "caption": "Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX for the indicated times were analyzed by QIBC (red bars, mean; n>2000 cells per condition).",
    "molecules": "DOX"
  },
  {
    "caption": "Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU were analyzed by QIBC (red bars, mean; n>2000 cells per condition).",
    "molecules": "EU, DOX"
  },
  {
    "caption": "Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU and stained with DAPI were analyzed by QIBC (red bars, mean; n>2000 cells per condition).",
    "molecules": "EU, DAPI, DOX"
  },
  {
    "caption": "H. U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, and analyzed by QIBC (n>2000 cells per condition). I. Quantification of data in (H) (red bars, mean). ",
    "molecules": "DOX"
  },
  {
    "caption": "U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, J. Representative images from the experiment in (H). Scale bars, 10 μm.",
    "molecules": "DOX"
  },
  {
    "caption": "B. Immunoblot analysis of U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of the indicated GFP-FAM111A alleles. U2OS/GFP-FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP-FAM111A WT cells used in Figure 1 and 2 (see Figure EV4A).",
    "molecules": "DOX"
  },
  {
    "caption": "C. U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC.",
    "molecules": "EdU, DAPI, DOX"
  },
  {
    "caption": "U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. D. Quantification of data in (C) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). ",
    "molecules": "EdU, DAPI, DOX"
  },
  {
    "caption": "U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. cells were pulse-labeled with EU.",
    "molecules": "EU, DAPI, DOX"
  },
  {
    "caption": "U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. Quantification of EU incorporation in cells in (E) (red bars, mean; n>2000 cells per condition).",
    "molecules": "EU, DAPI, DOX"
  },
  {
    "caption": "G. U2OS/GFP-FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre-extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC.",
    "molecules": "DAPI, DOX"
  },
  {
    "caption": "U2OS/GFP-FAM111A cell lines that were treated or not with DOX were pre-extracted and fixed, and stained with DAPI. DAPI signal intensities were analyzed by QIBC. H. cells were stained with RPB1 antibody.",
    "molecules": "DAPI, DOX"
  },
  {
    "caption": "I. Immunoblot analysis of U2OS/GFP-FAM111A cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.",
    "molecules": "DOX, Z-VAD-FMK"
  },
  {
    "caption": "Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK. J. using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles.",
    "molecules": "DOX, Z-VAD-FMK"
  },
  {
    "caption": "B. U2OS/GFP-FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies. C. As in (B), using U2OS/GFP-FAM111B WT cells.",
    "molecules": "DOX"
  },
  {
    "caption": "E. Immunoblot analysis of U2OS/GFP-FAM111B cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.",
    "molecules": "DOX, Z-VAD-FMK"
  },
  {
    "caption": "F. Cells in (E) were pulse-labeled with EdU, stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC.",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "Cells analyzed using QIBC. G. Quantification of data in (F) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).",
    "molecules": "EdU"
  },
  {
    "caption": "Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC. H. cells were pulse-labeled with EU.",
    "molecules": "EU, DAPI"
  },
  {
    "caption": "Cells analyzed using QIBC. I. Quantification of EU incorporation in cells in (H) (red bars, mean; n>2000 cells per condition).",
    "molecules": "EU"
  },
  {
    "caption": "J. U2OS/GFP-FAM111B cell lines (Figure 5E) were fixed at the indicated times after DOX treatment and stained with crystal violet.",
    "molecules": "crystal violet, DOX"
  },
  {
    "caption": "(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (D) LLO pre-incubated with cholesterol (LLOCHT)",
    "molecules": "cholesterol"
  },
  {
    "caption": "(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (E) LLO in medium supplemented with 140 mM K+(HighK+) and LLO in Ca2+-free medium Ca2+ (Ca2+free)",
    "molecules": "Ca2+, K+"
  },
  {
    "caption": "(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (F) LLO in the presence of 25 μM blebbistatin (BB).",
    "molecules": "BB, blebbistatin"
  },
  {
    "caption": "(E-F) Number of (E) blebs per cell or (F) retracting blebs per cell evaluated by time-lapse microscopy analysis of LLO-treated shCtrl or shGp96 cells. shCtrl n=32 cells and shGp96 n=40 cells, p-values were calculated using two-tailed un-paired Student's t-test *p<0.5. (G) Sequential frames of time-lapse microscopy analysis of LLO-treated HeLa cells expressing GFP-NMHCIIA (shCtrl, shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl-BB). LLO was added to culture medium 10 seconds before t0.",
    "molecules": "Blebbistatin"
  },
  {
    "caption": "(A-B) Epifluorecence microscopy images of HeLa cells (A) left untreated or treated with LLO (0.5 nM, 15 min). Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.",
    "molecules": "PS"
  },
  {
    "caption": "(A-B) Epifluorecence microscopy images of HeLa cells (B) infected with GFP-expressing wt Lm for 6 h. Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.",
    "molecules": "PS"
  },
  {
    "caption": "(C) Quantification of the % of infected cells with exofacial-PS sites in HeLa cells infected with wt Lm for 6 h. Data are the mean ±SEM (n=3) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5.",
    "molecules": "PS"
  },
  {
    "caption": "(C) From top to bottom: experimental design, fluorescence pictures of the SVZ of a 4D- (left) and 4D+ (right and insets magnified) mice and quantification of the proportion of BrdU+ among B1, C and A cells (identified as in B). Note that in 4D+ mice quantification was restricted to the RFP+ subpopulation (red bars).",
    "molecules": "BrdU"
  },
  {
    "caption": "(C) Line graph indicating the proportion of correct responses (performance) for bins of 100 trials each during testing with binary mixtures of concentrated octanols. Discontinuous line indicates the similar 95% plateau performance of 4D- (black) and 4D+ (red) mice. (D) Whiskers box plots of performance (left) and lick frequency (right) during a probe of 200 trials with 1:10 diluted octanols after testing as in (C).",
    "molecules": "octanols"
  },
  {
    "caption": "A Phenotypes of WT (Col-0), two gso1  mutant alleles (gso1-1, gso1-3) and two independent complementation lines (com-1, com-2). Seedlings  were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl  for 10 days. B,C Primary root length and seedling  fresh weight  of seedlings  depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)",
    "molecules": "NaCl"
  },
  {
    "caption": "C High resolution localization  analyses of SOS2  and GSO1  expressed under their respective native promoter at 1.5 mm from the  quiescent center. Propidium iodide  (PI) was used to stain  the cell wall. Scale bars: 100 µm. (BF: brightfield)",
    "molecules": "PI, Propidium iodide"
  },
  {
    "caption": "D Phenotypes of WT (Col-0), gso1-3, sos2-2 and gso1-3 sos2-2. Seedlings  were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS supplemented with or without extra NaCl  for 10 days. E,F  Primary root length and fresh weight  of seedlings  depicted in (D) were measured at day 7 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)",
    "molecules": "NaCl"
  },
  {
    "caption": "A Yeast  strain JP837  cells transformed with empty vectors (control) or the indicated combinations of Arabidopsis  genes were grown on AP medium supplemented with or without 100 mM NaCl  for 3 days at 28℃. (M: myristoylation motif, KD: kinase domain)",
    "molecules": "NaCl"
  },
  {
    "caption": "G-L Phenotypes, primary root length and seedling  fresh weight  of indicated plant lines grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with 100 mM NaCl  for 7 days. (mean ± SEM, n = 10, P < 0.05, one-way ANOVA)",
    "molecules": "NaCl"
  },
  {
    "caption": "A  Coomassie Brilliant Blue stain (CBB) and autoradiograph  of in vitro kinase assay combining the indicated combinations of SOS2/SOS2K40N and GSO1KD/GSO1KDK979N.",
    "molecules": "CBB"
  },
  {
    "caption": "B Immunoblot  using anti-Myc  antibody and CBB  and autoradiograph  from kinase assays combining Myc-SOS2  with SOS1C300 as substrate. 10-day-old seedlings  stably expressing Pro35S:6Myc-SOS2  in Col-0 or gso1-3 were treated with or without 100 mM NaCl  for 12 h, and Myc-SOS2  protein immunoprecipitated  with anti-C-Myc  antibody-conjugated agarose  from roots  was used in the assays. Signal strength of the phosphorylation  bands has been calculated relative to the left lane.",
    "molecules": "agarose, CBB, NaCl"
  },
  {
    "caption": "C CBB  and autoradiograph  of in vitro kinase assays  combining GSO1KD (kinase domain) with SOS2K40N or SOS2T16AK40N. D CBB  and autoradiograph  of in vitro kinase assays  using SOS2   p-site variants addressing autophosphorylation  activity.",
    "molecules": "CBB"
  },
  {
    "caption": "A Phenotypes of WT (Col-0), gso1-3, esb1, and casp1 casp3. Seedlings  were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium with or without 100 mM NaCl  for 10 days. B, C Primary root length and seedling  fresh weight  of seedlings  depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 12, two-way ANOVA, P < 0.05)",
    "molecules": "NaCl"
  },
  {
    "caption": "G Phenotypes of WT (Col-0), cif1 cif2, and gso1-3. Seedlings  were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl  for 10 days.",
    "molecules": "NaCl"
  },
  {
    "caption": "J Quantification of Fluorol Yellow fluorescence intensity indicating amounts of suberin  accumulation in maximum projections of z-stacks in the first mm of the continuously suberized endodermis  in the indicated genotypes. Seedlings  were grown on 1/2 MS for 5 days and then treated with the indicated NaCl  concentrations for 2 days. (mean ± SEM, n = 3-5, P < 0.05, one-way ANOVA and post hoc Tukey Kramer test, different letters indicate significant difference)",
    "molecules": "Fluorol Yellow, NaCl, suberin"
  },
  {
    "caption": "A-D Protein accumulation (A, C, D) or  gene expression  (B) of SOS2, CIF2, GSO1, and SGN1 displayed as false color fluorescence intensity. Seedlings  from the indicated plant lines were grown on 1/2 MS for 4-5 days and then transferred to 1/2 MS with or without 100 mM NaCl  for 1 day. Scale bars: 200 µm.",
    "molecules": "NaCl"
  },
  {
    "caption": "C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for WB (C)",
    "molecules": "OHT"
  },
  {
    "caption": "C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for growth curves and RT-qPCR (D,E).",
    "molecules": "OHT"
  },
  {
    "caption": "F-G. 2a and 2b ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves and RT-qPCR analyses.",
    "molecules": "OHT"
  },
  {
    "caption": "A-D. WB and growth curves of 2ac (A), 2abc (B), 2ab (C) and 2bc (D) ESCs cultured in 2i medium and exposed to OHT as indicated.",
    "molecules": "OHT"
  },
  {
    "caption": "E. Flow cytometry profiles of 2ac ESCs pulsed with BrdU in 2i medium. The upper table shows the percentages of living cells (the subG1 fraction is omitted from the analysis), which can be classified as being in S (BrdU+), G1 (2N DNA content, BrdU-) or G2/M phase (4N DNA content, BrdU-). The percentages of dead cells (subG1, events with a <2N DNA content) is shown in red.",
    "molecules": "DNA"
  },
  {
    "caption": "G-J. 2ac and 2abc ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves (G-H) and RT-qPCR analyses (I-J). The RT-qPCR data are also presented in Appendix Fig S2C-D with a zoom-in on the lower region of the y-axis.",
    "molecules": "OHT"
  },
  {
    "caption": "A-B. 2ac ESCs expressing GFP were cultured in the absence or presence of OHT and subsequently used for morula injections. (A) Average volume of the GFP-positive area in chimeric embryos at 24 and 72 hrs after morula injection. Number of embryos used for quantification: Ctrl 24h n=18; +OHT 24h n=15; Ctrl 72h n=5; +OHT 72h n=5. Data are presented as mean ± SD. (B) Representative images of chimeric embryos. The white bars indicate a length of 32 µm. Relative intensities of the GFP signal are indicated for each image.",
    "molecules": "OHT"
  },
  {
    "caption": "B. Heat map showing H3K9me3 ChIP-seq data for Jmjd2a/c bound TSSs (+/- 5kb) sorted according to read number in 2abc #13+OHT.",
    "molecules": "OHT"
  },
  {
    "caption": "E. Heat map presenting array data in the form of log2(fold change) values comparing OHT with control treated ESCs. Genes are shown for which TSS regions (+/- 1kb) show the most substantial increase in H3K9me3 levels in OHT treated cells according to the ChIP-seq analyses (defined in Fig EV4).",
    "molecules": "OHT"
  },
  {
    "caption": "A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (A) WB.",
    "molecules": "OHT"
  },
  {
    "caption": "A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (B) RT-qPCR analyses",
    "molecules": "OHT"
  },
  {
    "caption": "A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (C) serial plating for the generation of growth curves.",
    "molecules": "OHT"
  },
  {
    "caption": "(A) HeLa cells were infected with S. Typhimurium (Salmonella) for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and transferrin receptor (TfR). Bar, 10 µm.",
    "molecules": "Effectene"
  },
  {
    "caption": "(B) HeLa cells were infected with Salmonella for 1 h or transfected with Effectene-coated latex beads for 3 h and then subjected to immunocytochemistry for LC3 and galectin3. Bar, 5 µm.",
    "molecules": "Effectene"
  },
  {
    "caption": "(C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student's unpaired t test. *, P < 0.01.",
    "molecules": "Effectene"
  },
  {
    "caption": "(D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead-autophagosomes were fractionated as described in Materials and methods. The bead-autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized.",
    "molecules": "Effectene"
  },
  {
    "caption": "(G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.",
    "molecules": "Effectene"
  },
  {
    "caption": "(A and B) NIH3T3 cells stably expressing mStr-Ub and GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, or ULK1 were transfected with Effectene-coated latex beads for 30 min. Then, live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after Ub localization was measured for at least 30 cases for each combination. Each value represents the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.",
    "molecules": "Effectene"
  },
  {
    "caption": "(C) NIH3T3 cells stably expressing GFP-tagged Ub, LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated beads for 3 h in the presence or absence (mock) of 30 µM UBEI-41 (a ubiquitin E1-specific inhibitor) and subjected to immunocytochemistry for galectin3. The percentages of GFP-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05.",
    "molecules": "Effectene, UBEI-41"
  },
  {
    "caption": "(D and E) Parent NIH3T3 cells, Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.",
    "molecules": "Effectene"
  },
  {
    "caption": "(E) wild-type MEFs, and Atg5-KO MEFs stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.",
    "molecules": "Effectene"
  },
  {
    "caption": "(A) HeLa cells were lysed with 1% Triton X-100 lysis buffer and subjected to immunoprecipitation with FK2- or control IgG-immobilized beads. The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(B-K') The Drosophila posterior midguts containing Su(H)-Gal4>UAS-CD8:GFP (Su(H)>GFP) with sucrose (Suc, control) (B-C'), P.e for 36 h (D-F'), Ecc15 for 36 h (G-I') and DSS for 36 h (J-K') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (C-C', E-E', H-H' and K-K') Magnification of selected areas containing PH3+GFP─ cells from B, D, G and J. The EB cell close to the PH3+ cell (white arrow) in C-C' is out of focus. (F-F' and I-I') Magnification of selected areas containing PH3+ EBs (GFP+) from D and G. White arrowheads and red arrows indicate PH3 in GFP─ and GFP+ cells, respectively. (L and M) Quantification of PH3 GFP─ ISCs (L) and PH3+GFP+ EBs (M) with given treatments. n = 21 guts for each treatment.",
    "molecules": "DAPI, DSS, Suc, sucrose"
  },
  {
    "caption": "(D-F'') When the ISCs were marked by esgGal4, su(H)-Gal80>UAS-GFP, PH3 expression was found in GFP+ ISCs with feeding of Suc (control) (D-D''), P.e (E-E'') and Dss (F-F''). However, PH3 expression is present in EBs with GFP─Pros─ and small nuclei upon P.e infection (E-E'', red arrows). The white arrows indicate GFP+ cells with PH3 expression. (G) Quantification of PH3+GFP─Pros─ EBs with given treatments. n = 11 guts for each treatment. Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (G).",
    "molecules": "Dss, Suc"
  },
  {
    "caption": "(D-N') The Drosophila midguts expressing Su(H)ts>GFP with control (D-E'), UAS-RasV12 for 6 days (F-H'), EGFRA887T for 4 days (I-K') and UAS−λTop for 5 days (L-N') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (E-E', G-G', J-J' and M-M') Magnification of selected areas containing PH3+GFP─ cells from D, F, I and L. (H-H', K-K' and N-N') Magnification of selected areas containing PH3+GFP+ cells from F, I and L. White arrowheads and red arrows indicate PH3 in GFP─ ISCs and GFP+ EBs, respectively. (O, P) Quantification of PH3+GFP─ (P) and PH3+GFP+ (O) in midguts with indicated genotypes. n = 13 guts for each genotype. (Q) Quantification of PH3+ EBs (GFP+) with the given genotypes in response to P. e infection. n = 11 guts for each genotype.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B-C''') Flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ were fed with Suc (B-B''') or P.e (C-C''') for 36h and their midguts were dissected and immunostained with antibodies against GFP, β-gal and Dl. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control, P. e infected midguts, n=50. (E) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control with Suc or P. e, or knockdown of EGFR with Suc or P. e infection, n=50.",
    "molecules": "Suc"
  },
  {
    "caption": "A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments.",
    "molecules": "α-satellite DNA, DNA"
  },
  {
    "caption": "B. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes. C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes. ",
    "molecules": "Commassie blue"
  },
  {
    "caption": "A. Graph showing the relative abundance of undigested DNA (145 bp) as a function of time during digestion with micrococcal nuclease (MN-ase) for the CENP-A nucleosome (red), the CENP-A/CENP-CCR complex (blue) and the CENP-A/CENP-Cmotif complex (black).",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Protein occupancy over the idn  operon for each condition (colors are denoted in (A)). The quantile normalized robust z scores of the  protein occupancy at each 5 bp are represented by the  IPOD-HR occupancy. There is a large loss in protein occupancy when cells are shifted to 5KDG, leading to the loss of the call ed EPOD. Protein occupancy is restored once cells are grown in the second glucose  condition. The 500bp normalized average of previously published H-NS ChIP-seq [20] exhibits high  H-NS binding  on the idnD  promoter region.",
    "molecules": "glucose"
  },
  {
    "caption": "(B) Difference in Log2 ratio of fitnesses of the KDG-exposed cells with unexposed lacZ::cat  relative to that observed prior to KDG  exposure, given as a function of time  since exposure to KDG. Day -1 corresponds to the day before KDG  exposure, Day 0 is cells taken immediately after growth  in KDG, and subsequent timepoints  reflect different durations of growth  in M9 Min Glu  prior to competition (See (A)). Points show medians across replicates (3 biological replicates for 0.5 days, 6 biological replicates for all other timepoints); error bars show 95% confidence intervals for the difference relative to the Day -1 timepoint, calculated using the R wilcox.test function. Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint  to the -1 day (naive) timepoint: *, p<0.05; **, p<0.005.",
    "molecules": "Glu, KDG"
  },
  {
    "caption": "(G) P1 vir transduction  experiment to test the viability  of ∆fis  and ∆hfq. -Hfq  indicates deleting Hfq  and -CspE  indicates deleting -CspE  as a control. Strain identities are indicated in the box. Number of transductions  were counted on LB + Kan  plates; all efficiencies are relative to WT, and thus the log-scaled relative transduction rate for the WT itself is 0 by definition. -R1 indicates that the prophage region in (A) was deleted to test whether the loss of prophages silenced by Fis  and Hfq  restored viability  of a ∆fis  ∆hfq  genotype. R2 and R3 were other regions in the genome that contained prophages that appeared to have Fis/Hfq  dependent EPODs. Plotted values are mean efficiencies across replicates, with error bars showing a 95% credible interval obtained via Bayesian inference, assuming the replicate-level colony counts are Poisson distributed with a (conjugate) Gamma(0,0) prior; all log ratios (including error bounds) are plotted relative to the mean WT value. Data obtained from 5 biological replicates for hfq::kan  transductions  and 4 biological replicates for cspE::kan  transductions.",
    "molecules": "kan, Kan"
  },
  {
    "caption": "(A, Neutrophils  isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice  were incubated with LPS  (3 hr) before culture media were analysed for secreted IL-1β (A) Cytokine values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA and P-values were corrected for multiple comparisons (Bonferroni). **, p ≤ 0.01.",
    "molecules": "LPS, tamoxifen"
  },
  {
    "caption": "B). Neutrophils  isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice  were incubated with LPS  and cell lysates  and culture media were immunoblotted  for β-actin, caspase-1  and IL-1β (B).",
    "molecules": "LPS, tamoxifen"
  },
  {
    "caption": "LPS-primed neutrophils  of indicated genotypes were left untreated (Mock) or stimulated with LeTx  (3 hr) (A, B) IL-1β secretion levels were determined in culture media (A, and combined  cell lysates and culture media (Lys+Sup) were immunoblotted  for cleavage of pro-IL-1β (~39 kDa) into mature  IL-1β (p17) (B, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots  are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of indicated genotypes were left untreated (Mock) or stimulated with TcdA  (3 hr) (C, D) IL-1β secretion levels were determined in culture media C, and combined cell lysates and culture media (Lys+Sup) were immunoblotted  for cleavage of pro-IL-1β  (~39 kDa) into mature  IL-1β (p17) D, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots  are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of indicated genotypes were left untreated (Mock) or infected with F. tularensis (18 hr) (E, F) IL-1β secretion levels were determined in culture media E, and combined  cell lysates  and culture media (Lys+Sup) were immunoblotted  for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) F, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots  are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of indicated genotypes were left untreated (Mock) or stimulated with Flatox  (3 hr) (G, H). IL-1β secretion levels were determined in culture media G); and combined cell lysates and culture media (Lys+Sup) were immunoblotted  for cleavage of pro-IL-1β  (~39 kDa) into mature IL-1β (p17) H). Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots  are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with LeTx (A), PMP  was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.",
    "molecules": "SYTOX Green, LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (B) PMP  was assessed by measurement of  SYTOX Green  incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.",
    "molecules": "SYTOX Green, LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with ATP (C) PMP  was assessed by measurement of SYTOX Green  incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.",
    "molecules": "SYTOX Green, ATP, LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (D). PMP  was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.",
    "molecules": "SYTOX Green, LPS"
  },
  {
    "caption": "(E) LPS-primed B6 BMNs  were left untreated (LPS) or subsequently stimulated with FlaTox  (LPS+FlaTox) for 3 hr. Data are representative of n=3 biological repeats. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted  for release of cytosolic GAPDH  into culture media. Data are representative of n=3 biological repeats.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with TcdA  (3 hr) (A Whole  cell lysates (Lys) and culture media (Sup) were immunoblotted  separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD  (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox  (3 hr) (B, Whole cell lysates  (Lys) and culture media (Sup) were immunoblotted  separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β  (p17) and cleaved GSDMD  (p30). * with  Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with nigericin  (100 mins) (C, Whole  cell lysates  (Lys) and culture media (Sup) were immunoblotted  separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD  (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with TcdA  (3 hr) D, PMP  was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "SYTOX Green, LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox  (3 hr) E, PMP  was assessed by measurement of SYTOX Green  incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "SYTOX Green, LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with nigericin  (100 mins) F, PMP  was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "SYTOX Green, LPS, nigericin"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with TcdA  (3 hr) G), IL-1β secretion levels were determined by  Luminex assay. Values represent mean ± SEM of n=3 (G, biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox  (3 hr) , H) IL-1β secretion levels were determined by  Luminex assay. Values represent mean ± SEM of n=4 (H) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock) or stimulated with nigericin  (100 mins) I). IL-1β  secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 I) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with PMA  (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted  for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD  (p30) (A) * with Western blots indicates non-specific bands.",
    "molecules": "LPS, PMA"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with PMA  (3 hr) (B, PMP  was assessed by measurement of  SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "SYTOX Green, LPS, PMA"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with PMA  (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted  for extracellular release of β-actin and GAPDH",
    "molecules": "LPS, PMA"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with PMA  (3 hr) Whole cell lysates  (Lys) and culture media (Sup) were immunoblotted  for extracellular release of  β-actin and GAPDH",
    "molecules": "LPS, PMA"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with nigericin  (100 mins) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted  for extracellular release of β-actin and GAPDH",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with nigericin  (100 mins) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted  for extracellular release of  β-actin and GAPDH",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with nigericin  (100 mins) G) PMP  was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "SYTOX Green, LPS, nigericin"
  },
  {
    "caption": "LPS-primed neutrophils  of the indicated genotypes were left untreated (Mock), stimulated with TcDA  (3 hr) (H). Whole cell lysates (Lys) and culture media (Sup) were immunoblotted  for extracellular release of β-actin and GAPDH  H).",
    "molecules": "LPS"
  },
  {
    "caption": "Neutrophils  of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (A) Representative confocal micrographs  taken at different time points were stained  with DAPI  (dark blue nuclei) and SYTOX Green (light blue). Scale bar: 20 μM.",
    "molecules": "SYTOX Green, DAPI"
  },
  {
    "caption": "(B) Representative confocal micrographs  of untreated (Mock), S. Typhimurium-infected or PMA-stimulated wildtype BMNs stained  for neutrophil elastase  (Red) and SYTOX Green (Green) (Scale bar: 10 μM).",
    "molecules": "SYTOX Green, PMA"
  },
  {
    "caption": "Neutrophils  of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (G) Representative  confocal micrographs of neutrophils stained  with DAPI  (dark blue nuclei) and SYTOX Green (light blue) taken at different time points post-infection (scale bar: 20 μM). (H) PMP  was assessed by measurement of SYTOX Green incorporation over time. Data information: In H, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).",
    "molecules": "SYTOX Green, DAPI"
  },
  {
    "caption": "(C) Newly synthesized claudin 1 and occludin proteins in cells treated as described in A. After cells were exposed to L-azidohomoalaine (AHA), cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne-azide-modified protein complex was pulled down by paramagnetic streptavidin-conjugated dynabeads. (D) Quantitative data of immunoblots shown in C. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.",
    "molecules": "AHA, L-azidohomoalaine, alkyne, azide, biotin, streptavidin"
  },
  {
    "caption": "(A) Association of vtRNA2-1 with HuR in Caco-2 cells as measured by biotin-labeled RNA pull-down assays. After cytoplasmic lysates were incubated with biotinylated vtRNA2-1, levels of HuR and other proteins in the pull-down material were assessed. Three separate experiments showed similar results.",
    "molecules": "biotin, biotinylated"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with the indicated antibodies (n = 3). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.",
    "molecules": "amyloid-β, Aβ"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with the indicated antibodies (n = 3). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. Aβ, amyloid-β; EPO, erythropoietin.",
    "molecules": "amyloid-β, Aβ"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "PBS"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages  were PMs  isolated from EPOR-MKO mice.",
    "molecules": "PBS"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "PBS"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages  were PMs  isolated from EPOR-MKO mice.",
    "molecules": "PBS"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with indicated antibodies (n = 3). Data information: vehicles were PBS. Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "PBS"
  },
  {
    "caption": "Macrophages  were detected by immunoblotting  with indicated antibodies (n = 3). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. EPOR-KO macrophages  were PMs  isolated from EPOR-MKO mice.",
    "molecules": "PBS"
  },
  {
    "caption": "Flag-Aβ42  was injected into the  lateral ventricles of Lyz2-Cre+/+/Epor+/+ mice  (EPOR-C) (8 w) mice  pretreated with rhEPO  10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42  in spleens  and brains  was detected by immunoblotting  (A, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "Aβ42"
  },
  {
    "caption": "Flag-Aβ42  was injected into the  lateral ventricles  of Lyz2-Cre+/+/Epor+/+ mice  (EPOR-C) (8 w) mice  pretreated with ARA290  (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42  in spleens  and brains  was detected by immunoblotting  B, Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290",
    "molecules": "Aβ42, ARA, ARA290"
  },
  {
    "caption": "Flag-Aβ42  was injected into the  lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice  (EPOR-MKO) (8 w) mice  pretreated with rhEPO 10000 IU/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42  in spleens  and brains  was detected by immunoblotting  Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "Aβ42"
  },
  {
    "caption": "Flag-Aβ42  was injected into the  lateral ventricles of Lyz2-Cre+/+/Eporloxp/loxp mice  (EPOR-MKO) (8 w) mice  pretreated with ARA290  (0.03 mg/kg/d, i.p.) for 2 days (twice per day) (n = 3). One day later, Flag-Aβ42  in spleens  and brains  was detected by immunoblotting  E). Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290",
    "molecules": "Aβ42, ARA, ARA290"
  },
  {
    "caption": "APP/PS1-21 mice  (5 m) treated with rhEPO (10000 IU/kg/d) or ARA290  (0.03 mg/kg/d) (i.p., once a day, for 14 days) (n=10), and Aβ plaques   in cortex  and hippocampus  (n = 5) were detected by IHC  (I). Data information: For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. ARA, ARA290; IHC, Immunohistochemical staining.",
    "molecules": "Aβ, ARA, ARA290"
  },
  {
    "caption": "11-month-old APP/PS-1-21+/+/Lyz2-Cre+/+/Eporloxp/loxp mice  (APP/PS1-21+EPOR-MKO) or APP/PS-1-21+/+/Lyz2-Cre+/+/Epor+/+ mice  (APP/PS1-21+EPOR-C), Aβ plaques  in the cortex  and hippocampus  of mice  of 11-month-old (M) (n = 5) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.",
    "molecules": "Aβ"
  },
  {
    "caption": "R Aβ plaques  in the cortex  and hippocampus  (n = 4) were detected by IHC. Data information: Unpaired t-tests were used for the comparison of two groups. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IHC, Immunohistochemical staining.",
    "molecules": "Aβ"
  },
  {
    "caption": "Old APP/PS1-21 mice  (11 m) and WT mice  at the same age were treated with rhEPO (10000 IU/kg/d), ARA290  (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Splenic macrophages  were isolated for detection by immunoblotting  with the indicated antibodies (n = 3) (G). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "ARA290, PBS"
  },
  {
    "caption": "Old APP/PS1-21 mice  (11 m) and WT mice  at the same age were treated with rhEPO (10000 IU/kg/d), ARA290  (0.03 mg/kg/d) or vehicle (i.p., once a day, for 14 days). Protein levels of IDE  and NEP  were detected by immunoblotting  with indicated antibodies (n = 3) (I). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.",
    "molecules": "ARA290, PBS"
  },
  {
    "caption": "PMs  were treated with Aβ42  (0, 5, 7.5 or 10 μM) for 12 hr, and cell lysates were detected by immunoblotting  with the indicated antibodies (n = 3) (K). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "Aβ42, PBS"
  },
  {
    "caption": "PMs  were treated with Aβ42  (0, 5, 10 μM) for 12 hr, and Protein levels of IDE  and NEP  were detected by immunoblotting  with indicated antibodies (n = 3) (M). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IDE, insulin degrading enzyme; NEP, neprilysin.",
    "molecules": "Aβ42, PBS"
  },
  {
    "caption": "E Aβ plaques   in the cortex  and hippocampus  (n = 5) were detected by IHC. Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance.",
    "molecules": "Aβ, PBS"
  },
  {
    "caption": "G Expression of MAP-2 or the apoptotic neurons  of hippocampus  were detected by IF staining  and then statistically quantified (n = 5). Data information: vehicles were PBS. For multiple groups, one-way ANOVA was used. Results are presented as mean ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; ns denotes no statistical significance. IF, Immunofluorescent; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclei; TUNEL, Terminal dUTP Nick End Labeling.",
    "molecules": "PBS"
  },
  {
    "caption": "Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.",
    "molecules": "ETO, etoposide"
  },
  {
    "caption": "MDM were treated with increasing concentrations of ETO and CTH for 66h. Cells were stained to distinguish between live and dead cells using LIVE/DEAD fixable Dead cell stain protocol. Percentages of live/dead cells were determined using automated cell‐imaging system Hermes WiScan and ImageJ. Addition of 20% ethanol for 10min into cells treated with ETO was used as positive control. (n = 3, mean ± s.e.m).",
    "molecules": "CTH, ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP viruses wild type (wt) HIV-1 capsid mutants (N74D or P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent Adenovirus type 5 AdV (AdV), Semliki Forest Virus (SFV) and HSV-1. The percentages of infected cells were determined using an automated cell‐imaging system Hermes WiScan and ImageJ and normalized to untreated control ~100%. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; ***P‐value ≤ 0.001; (ns) non‐significant, paired t‐test). Cells from a representative donor were used for immunoblotting.",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP. 104 cells were recorded and analysed for GFP expression 48h post-infection using an automated cell‐imaging system Hermes WiScan and ImageJ. (n = 4, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full-length replication competent macrophage tropic HIV-1 virus BaL. Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": " G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.  Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).  ",
    "molecules": "ETO, AZT"
  },
  {
    "caption": "G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.2LTR circles. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; ***P‐value ≤ 0.001, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ** P‐value ≤ 0.01; ***P‐value ≤ 0.001, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL (3, 6, 12 ffu/cell) and DNA isolated 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL and DNA isolated 4, 6, and 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV‐G pseudotyped HIV‐1 GFP 18h later. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP) or deleted for Vpx (SIV VLP del Vpx) or deleted for Vpr (SIV VLP del Vpr). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 4, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).",
    "molecules": "ETO, AZT"
  },
  {
    "caption": "D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in Human serum instead of FCS.",
    "molecules": "ETO"
  },
  {
    "caption": "A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in FCS. A standard culture condition used in all experiments. See Materials and Methods.",
    "molecules": "ETO"
  },
  {
    "caption": " C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.  The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. Cells from a representative donor were used for immunoblotting. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).  ",
    "molecules": "ETO"
  },
  {
    "caption": "C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).",
    "molecules": "ETO, AZT"
  },
  {
    "caption": "C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO for 18h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalised to GAPDH. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; (ns) non‐significant, paired t‐test).",
    "molecules": "cGAMP, ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm.",
    "molecules": "LPS, cGAMP, ETO"
  },
  {
    "caption": "MDM were treated with increasing concentrations of ETO and CTH. Cells were stained for MCM2 expression, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired. (n = 3, mean ± s.e.m.).",
    "molecules": "CTH, ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.",
    "molecules": "ETO"
  },
  {
    "caption": "MDM were treated with 5μM ETO or 0.01μM CTH  for 18h, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.",
    "molecules": "ETO"
  },
  {
    "caption": "A GFP‐Atg11 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h, and autophagy was induced by treating cells for 1 h with rapamycin. Scale bar, 5 μm.",
    "molecules": "copper sulfate, rapamycin"
  },
  {
    "caption": "B atg34Δ yeast cells containing HTB‐Atg34 were grown to mid‐log phase and treated with rapamycin. Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; Atg11 binding region: green; lysine substitution: gray.",
    "molecules": "rapamycin"
  },
  {
    "caption": "D GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg19Δ cells containing BFP‐Ape1 and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h. coloc.: Cells with GFP‐Atg11/BFP‐Ape1 co‐localization. n = 50 (wt), 84 (hrr25‐ts), 127 (atg19Δ). Scale bar, 3 μm.",
    "molecules": "CuSO4"
  },
  {
    "caption": "Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4)P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle.",
    "molecules": "PI(3,4)P2"
  },
  {
    "caption": "Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.",
    "molecules": "FM4-64"
  },
  {
    "caption": "Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin.",
    "molecules": "Wortmannin"
  },
  {
    "caption": "Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A.",
    "molecules": "Latrunculin A"
  },
  {
    "caption": "Time-lapse confocal images showing dynamic localization of PI(3,4)P2. Nocodazole was added at time 0 during the live cell imaging.   Quantification of vesicle number, vesicle number/min, and vesicle lifetime (s) from experiment in E. T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus After Nocodazole group; mean±SEM (n = 10).   ",
    "molecules": "PI(3,4)P2, Nocodazole"
  },
  {
    "caption": "(L) Representative confocal images of tPH-CynA-KikGR in wild-type Dictyostelium AX2 or RacH− cells treated with 5 μM latrunculin A Scale bar: 5 μm. (M) Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wild-type Dictyostelium AX2 and RacH− cells. T-test was carried out by GraphPad Prism, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 33).",
    "molecules": "latrunculin A"
  },
  {
    "caption": "Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. The macropinosome at the front of the cell was photoconverted at time 0. White arrow points to PI(3,4)P2 localization at the back of the membrane. Cell front and back are shown, box showing the photoconverted region.",
    "molecules": "PI(3,4)P2"
  },
  {
    "caption": "F Allele-specific expression of enhancer RNA in heterozygous CD4 T cells. DNA used for technical control (n=6; paired t-test; two-tailed).",
    "molecules": "RNA"
  },
  {
    "caption": "D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination.",
    "molecules": "EU"
  },
  {
    "caption": "E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells.",
    "molecules": "ATTO-550"
  },
  {
    "caption": "(C-F) C. Total fresh weight, D. Total dry weight, E. Primary root length and F. Lateral root density of 21-day-old, non-colonized (Mock) and SA190-colonized plants upon growth  for 16 days on ½ MS or ½ MS + 25% PEG. Boxplot shows data from three independent biological replicates (technical replicates n = 18-24) where whiskers represents minimum and maximum values, \"+\" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (*** p < 0.001).",
    "molecules": "PEG"
  },
  {
    "caption": "J. Confocal microscopy  showing the colonization  of SA190:GFP  on five days-old seedlings  grown on ½ MS and 25% PEG. Green signal corresponds to GFP-tagged SA190  while in magenta are plant cell wall stained  with propidium iodide (PI). Bar indicates 100 nm. K. SA190 colonization  levels in Arabidopsis  under normal (½ MS) and drought proxy (½ MS + 25% PEG) conditions as quantified by colony forming units  of bacteria per mg of plant fresh weight. Boxplot shows data from four independent biological replicates where whiskers represents minimum and maximum values, \"+\" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).",
    "molecules": "PEG, PI, propidium iodide"
  },
  {
    "caption": "F-G.  Primary root length and  Lateral root  density of 21-day-old, mock- and SA190-colonized Col-0, aba2-1 and qpyr/pyl  plants upon growth  for 16 days on ½ MS + 25% PEG. Data information: Boxplot shows data from three independent biological replicates (technical replicates n = 36) where whiskers represents minimum and maximum values, \"+\" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001).",
    "molecules": "PEG"
  },
  {
    "caption": "B. Relative  gene expression  of aquaporin PIP  and TIP genes by qRT-PCR analysis normalized to tubulin  levels in 21-days old mock- and SA190-colonized roots of Arabidopsis  grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).",
    "molecules": "PEG"
  },
  {
    "caption": "C. Relative  gene expression of aquaporin PIP and TIP genes by  qRT-PCR analysis in Col-0, aba2-1 and qpyr/pyl mutants normalized to tubulin  levels in 21-days old mock- and SA190-colonized roots  of Arabidopsis  grown for 16 days on ½ MS with or without 25% PEG. Data information: Values represent the means of three biological experiments. Error bars indicate SE. All plots represent the mean of 3 biological replicates (biological replicates n= 3). Asterisks indicate a statistical difference based on the Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).",
    "molecules": "PEG"
  },
  {
    "caption": "A-B. Total fresh weight  of 21 days old non-colonized- (Mock) and SA190-colonized (SA190) Col-0, tip2;1  and qpip  plants grown for 16 days under normal (½ MS) or drought stress proxy conditions (½ MS + 25% PEG). Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (A) 16-20, (B) 8-20 where whiskers represents minimum and maximum values, \"+\" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).",
    "molecules": "PEG"
  },
  {
    "caption": "G. Transpiration rate of mock- or SA190- colonized 21 days old plants after transfer of 5-day-old seedlings  from ½ MS to ½ MS +/- 25 % PEG. Transpiration  was quantified as rate of water loss by calculating the changes in fresh weight  for 30 min with reading at every 5 min interval.",
    "molecules": "PEG, water"
  },
  {
    "caption": "H-I. Comparison of  stomatal  aperture of SA190- and non-colonized 21-days old plants grown for 16 days under non-stress (½ MS) or drought stress proxy (½ MS + 25% PEG) conditions. Scale bar = 20 µm. Data information: All plots represent the mean of 3 biological replicates. Error bars represent SE. Boxplot shows data from three independent biological replicates (technical replicates n = (I) 94-104, where whiskers represents minimum and maximum values, \"+\" indicates mean and horizontal line represents median. Asterisks indicate the statistical differences based on the Student's t-test (* p < 0.05, *** p < 0.001). Asterisks indicate a statistical difference based on the Student's t-test *** p < 0.001).",
    "molecules": "PEG"
  },
  {
    "caption": "(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm.",
    "molecules": "glucose"
  },
  {
    "caption": "(C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II.",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm.",
    "molecules": "glucose"
  },
  {
    "caption": "(E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB.",
    "molecules": "amino acid, glucose, Rapa, rapamycin"
  },
  {
    "caption": "(B) HeLa cells were transiently transfected with the plasmids as indicated. 24 h after transfection, the cells were treated with or without glucose starvation (GS) for 4 h. The cell lysates were used in IP and IB.",
    "molecules": "glucose"
  },
  {
    "caption": "(A) HeLa cells were cultured in normal medium (NM) or glucose starvation (GS) for 4 h. The starved lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate groups and analyzed by immunoblotting (IB) with Phos-tag gel or regular SDS-PAGE.",
    "molecules": "glucose"
  },
  {
    "caption": "(D) HeLa cells were incubated with compound C (20 mM, 30 min) with or without GS, followed by IB with Phos-tag gel or regular SDS-PAGE.",
    "molecules": "compound C"
  },
  {
    "caption": "(B) PAQR3-deficient HeLa cells were transfected with WT or T32A mutants of PAQR3 plasmids. 24 h later, the cells were incubated with normal medium (NM) or glucose starvation (GS) for 4 h, followed by immunoblotting (IB) in Phos-tag gel or regular SDS-PAGE.",
    "molecules": "glucose"
  },
  {
    "caption": "(C) HeLa Cells were incubated with NM or GS for 1 h or treated with compound C (C.C, 20 mM, 30 min) prior to GS for 1 h. In parallel, 25 mM 2-DG or 1 mM AICAR was added in NM for 1 h. Cell lysates were analyzed by IB.",
    "molecules": "2-DG, AICAR, C.C, compound C"
  },
  {
    "caption": "(E) WT, phosphorylation defective (T32A), or phospho-mimetic (T32E) PAQR3 were transfected in the presence or absence of constitutively active AMPK (CA-AMPK) into PAQR3-deficient HeLa cells as indicated. 24 h later, the cells without AMPK transfection were treated with GS for 4 h. 10% of the cell lysates were subjected to IB to detect PAQR3 expression level (shown in Appendix Fig S4A), the remaining cell lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody, followed by VPS34 activity measurement by a quantitative PI(3)P ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; *p < 0.05; **p < 0.01 as compared to the first group).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(I) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI(3)P ELISA assay (n = 5; **p < 0.01).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals. Mice were injected at P17 and sacrificed at P20 (n = 3 for each genotype). Scale bars, 50 μm.",
    "molecules": "BrdU"
  },
  {
    "caption": "Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids after 5 days of treatment with 50 μM Pioglitazone or vehicle (DMSO). Scale bars, 100 μm.",
    "molecules": "DMSO, Pioglitazone"
  },
  {
    "caption": "qPCR analysis of Lgr5, Pcna and Angptl4 expression in organoid cultures after 5 days of treatment with 50 μM Pioglitazone. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Pioglitazone-treated organoids relative to the DMSO vehicle (dashed line). * P = 0.02, Student`s t test.",
    "molecules": "DMSO, Pioglitazone"
  },
  {
    "caption": "Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at day 1 after passaging Pioglitazone- or vehicle (DMSO)-treated organoids. Scale bars, 100 μm.",
    "molecules": "DMSO, Pioglitazone"
  },
  {
    "caption": "Transposon screen validation of UBX5. ubx5∆ restores the growth of tdp1wss1. Cells were grown in YEPD and spotted on a medium supplemented with 1 mM auxin (for tdp1-degron degradation) and 5 μg/mL Camptothecin (CPT). Plates were incubated for 2 days at 30°C.",
    "molecules": "auxin, Camptothecin, CPT"
  },
  {
    "caption": "Ubx5 levels are decreased in tdp1wss1. (E) HA-Ubx5 protein levels at steady state are compared by immunoblotting in different mutants. Mutant strains carrying the additional ubx5∆ mutation were complemented with a plasmid expressing HA-tagged Ubx5 from the endogenous pUBX5 promoter. 1mM auxin was added where indicated for 6 h to degrade tdp1-degron, when present. Pgk1 was used as a loading control.",
    "molecules": "auxin"
  },
  {
    "caption": "The UBX∆ mutant of Ubx5 is sufficient to suppress FA and HU sensitivities of wss1∆. ubx5∆wss1∆ mutants were complemented with plasmids encoding HA-Ubx5-WT or HA-Ubx5 mutants overexpressed from the pGAL promoter; cells were grown in SC-trp and either treated with 40 mM FA for 15 min or spotted directly on 20 mM HU. Plates were prepared either with glucose (no HA-Ubx5 expression) or galactose (HA-Ubx5 expression) as the source of sugar. Plates were incubated for 2.5 days at 30°C. o/e, overexpression.",
    "molecules": "FA, galactose, glucose, HU"
  },
  {
    "caption": "(B) The ubx5∆ mutation rescues growth defects caused by Flp-nick galactose-induction in the tdp1∆ wss1∆ top1∆ mutant. Indicated strains were grown in YEP-2% raffinose prior to plating on YEP-2% glucose or YEP-2% galactose plates. Plates were incubated for 3 days at 30°C.",
    "molecules": "galactose, glucose, raffinose"
  },
  {
    "caption": "(E) Ubx5 occupancy at the Flp-bound FRT site. Levels of Ubx5-TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in cells grown as described in (C). The graph shows enrichment of qPCR signals over the unrelated intergenic region. Data are presented as means ± SDs for n = 5 independent biological replicates. p-values for +200 bp were defined by 2-way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; *p < 0.05; ** p < 0.01; **** p < 0.0001). In addition to the indicated mutations, all strains are bar1∆ Ubx5-TAP. No FRT strain and raffinose (raf) samples were used as negative controls.",
    "molecules": "formaldehyde, raf, raffinose"
  },
  {
    "caption": "Cdc48 occupancy at the Flp-bound FRT site. Levels of Cdc48-TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in asynchronous cells before and after Flp-cc induction with galactose. The graph shows enrichment of qPCR signal over the unrelated intergenic region. Data are presented as means ± SDs of 3 to 4 independent biological replicates. p-values for +200 bp were defined by 2‑way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; **** p < 0.0001). In addition to the indicated mutations, all strains are Cdc48-TAP. No FRT strain was used as a negative control.",
    "molecules": "formaldehyde, galactose"
  },
  {
    "caption": "Ubiquitination of the FRT locus. Asynchronous cultures were grown in 2% raffinose (raf) or additionally supplemented with 3% galactose (gal) to induce flp-H305L expression. Cells were also collected during glucose repression of flp-H305L expression at time points 30 min (glu 30') and 120 min (glu 120'). Ubiquitin antibody was used for ChIP-qPCR analysis following formaldehyde crosslinking. Data are shown as the mean ± SDs of three to six independent replicates. p-values for +200 bp were defined by 2‑way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; *p < 0.05; *** p < 0.001; **** p < 0.0001). In addition to all indicated mutations, all strains are bar1∆ Ddi1-TAP.",
    "molecules": "formaldehyde, gal, galactose, glu, glucose, raf, raffinose, Ubiquitin"
  },
  {
    "caption": "ubx5∆ restores wss1∆ resistance to HU and FA but not upon additional deletion of DDI1. Strains were grown in YEPD and either treated with 40 mM FA for 15 min or directly spotted on plates containing 100 mM of HU or 5 μg/mL CPT. Plates were incubated for 2 days at 30°C.",
    "molecules": "CPT, FA, HU"
  },
  {
    "caption": "Catalytic activity of the protease Ddi1 is essential for survival of the ubx5∆ wss1∆ yeast mutant. Mutants carrying wild-type (WT) Ddi1, D220A catalytic mutant or ddi1∆ in combination with ubx5∆, wss1∆ or double ubx5∆ wss1∆ were grown in YEPD. Cells were treated with 40 mM formaldehyde (FA) for 15 min or directly spotted on plates supplemented with 100 mM hydroxyurea (HU). Plates were incubated for 2 days at 30°C.",
    "molecules": "FA, formaldehyde, HU, hydroxyurea"
  },
  {
    "caption": "Ubx5 loss restores HU-induced Rpb1 degradation in wss1∆ and requires Ddi1. Rpb1 levels were defined in cells growing in the presence of 200 mM HU and 100 μg/mL cycloheximide (CHX) at the indicated time points. Rpb1 and Pgk1 levels in total cell extracts were probed by immunoblotting and quantified using fluorescent secondary antibodies. See (B) for quantifications. Images show immunoblotting using chemiluminescence.",
    "molecules": "CHX, cycloheximide, HU"
  },
  {
    "caption": "Rpb1 chromatin enrichment following HU treatment. (C) Chromatin fractions were isolated from cells treated for 2 h with 200 mM HU and 100 μg/mL CHX. Pgk1 and H3 were used as controls to monitor the fractionation. Quantifications of 8 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparison test and WT as a control (n.s., non-significant; ** p < 0.01).",
    "molecules": "CHX, HU"
  },
  {
    "caption": "Rpb1 chromatin enrichment following HU treatment. (D) Quantifications of Rpb1 and H3 levels in chromatin fractions. Relative Rpb1 to H3 levels were set to 1 in the respective WT samples. Quantifications of 8 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparison test and WT as a control (n.s., non-significant; ** p < 0.01).",
    "molecules": "HU"
  },
  {
    "caption": " B. Analysis of NP137 binding on netrin-2L, netrin-1; netrin-4 or netrin-G2 by bio-layer interferometry assays. Kd rhNTN1: 0.96 ± 0.30nM; Kd for rchNTN2: 1.53 ± 0.96nM ",
    "molecules": "NP137"
  },
  {
    "caption": " C. NP137 inhibits NTN1/UNC5B or NTN-2L/UNC5B interaction, detected by Elisa assays. Here the recombinant proteins are the murine forms (Control IgG= mouse IgG2A, mNP137= murine Fc, UNCB=rat UN5H2) bars indicate SD, n=3. ",
    "molecules": "NP137"
  },
  {
    "caption": " D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.). E. NP137 enhances survival of mice engrafted with NCI-H82 cell line (see D). Analysis of Kaplan-Meier survival curves of mice treated or not with NP137. Mantel Cox test; n=10 animals/group. ",
    "molecules": "NP001, NP137, PBS"
  },
  {
    "caption": "A, B In-gel kinase assay of OST1 activity in ABA receptor mutants under cold stress. Ten-day-old wild type, ost1-3, pyr1 pyl1,4, pyr1 pyl1,2,4, pyr1 pyl4,5,8 and pyr1 pyl1,4,5,8 mutants were treated at 4°C for 2 h. Total protein extracts were prepared and separated on SDS-PAGE gel containing 0.1 mg/mL GST-∆ABF2 as a substrate, and incubated with 60 μCi of [γ-32P]ATP. Representative pictures are shown in (A) and relative kinase activity is shown in (B). Data information: In (A , Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (B , each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).",
    "molecules": "ATP, CBB, Coomassie Brilliant Blue"
  },
  {
    "caption": "C, D In-gel kinase assay of OST1 activity in ABI1-OE14 and abi1 abi2 hab1 mutants under cold stress. Representative pictures are shown in (C) and relative kinase activity is shown in (D). Data information: In C), Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In D), each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).",
    "molecules": "CBB, Coomassie Brilliant Blue"
  },
  {
    "caption": "C Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively.",
    "molecules": "agarose"
  },
  {
    "caption": "E EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ-32P]ATP in kinase reaction buffer for 30 min at 30°C, and then separated by SDS-PAGE. Top, autoradiograph; bottom, CBB staining.",
    "molecules": "ATP, CBB"
  },
  {
    "caption": "F, G In-gel assay of OST1 activity in 10-d-old wild type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress. Data information: the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).",
    "molecules": "CBB"
  },
  {
    "caption": "H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress. Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).",
    "molecules": "CBB"
  },
  {
    "caption": "Freezing phenotype (A) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA).",
    "molecules": "agar"
  },
  {
    "caption": "survival rate (B) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).",
    "molecules": "agar"
  },
  {
    "caption": "ion leakage (C) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).",
    "molecules": "agar"
  },
  {
    "caption": "N-myristoylation of EGR2 and EGR2G2A in N. benthamiana leaves. Protein extracts prepared from N. benthamiana leaves expressing EGR2-Myc or EGR2G2A-Myc were immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies.",
    "molecules": "agarose, myristic acid"
  },
  {
    "caption": "D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants. Total proteins were prepared from two-week-old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22°C for 72 h and then subjected to click-reaction with the biotin azide. The reaction was protected from light at room temperature for 3 h. The products were detected by anti-streptavidin-HRP antibody.",
    "molecules": "biotin azide, myristic acid alkyne, streptavidin"
  },
  {
    "caption": "E N-myristoylation of EGR2 in plants. Total proteins were prepared from two-week-old EGR2-Myc seedlings The products were immunoprecipated by anti-Myc agarose beads and detected by anti-streptavidin-HRP antibody. EGR2-Myc detected by anti-Myc antibody was used as a loading control.",
    "molecules": "agarose, streptavidin"
  },
  {
    "caption": "F Immunoblot assay of myristoylated EGR2 under cold stress. Total proteins were prepared form EGR2-Myc plants treated with 4°C for the indicated period, and immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies. The ratio of band intensity detected by anti-myristic acid antibody to anti-Myc antibody without cold treatment was set to 1.00. Representative pictures are shown (left) and quantitative analysis is shown (right). Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).",
    "molecules": "agarose, myristic acid"
  },
  {
    "caption": "Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). Representative photographs (-5oC, 0.5 h; -8oC, 0.5 h) are shown in (H)",
    "molecules": "agar"
  },
  {
    "caption": "Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). survival rate is shown in (I). Data shown in (I) are mean values ± SEM of three biological replicates, each of which has three technical replicates. Asterisks indicate significant differences compared with wild type for the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test); n. s., not significant.",
    "molecules": "agar"
  },
  {
    "caption": "C, D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co-IP assay. Arabidopsis protoplasts expressing HF/NMT1-Myc and HF-EGR2/NMT1-Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc and anti-HA were used to detect NMT1-Myc and HF-EGR2, respectively. In (C), relative band intensity is shown below the co-IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two-tailed t-test).",
    "molecules": "agarose"
  },
  {
    "caption": "E In vitro pull-down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from two-week-old EGR2-Myc plants grown at 4°C for 0 h and 6 h were immunoprecipated by anti-Myc agarose beads and incubated with GST-NMT1 in pull down buffer for 2 h at 4°C. Anti-Myc and anti-GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot.",
    "molecules": "agarose"
  },
  {
    "caption": "OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I) In (I): the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00.",
    "molecules": "CBB"
  },
  {
    "caption": "A, B Pull-down assay. Total proteins prepared from EGR2-Myc or EGR2G2A-Myc transgenic plants was immunoprecipated with anti-Myc agarose beads, and then incubated with GST-OST1. Anti-Myc and anti-GST antibody were used to detect GST-OST1 and EGE2-Myc. Representative pictures are shown in (A). Relative interaction is shown in (B). Data information: In (A Relative band intensities are shown. In (B each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).",
    "molecules": "agarose"
  },
  {
    "caption": "D, E Co-IP assay. EGR2-Myc/HF-OST1, EGR2G2A-Myc/HF-OST1, HF/EGR2-Myc and HF/EGR2G2A-Myc were expressed in N. benthamiana leaves. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-HA and anti-Myc antibodies were used to detect HF-OST1, EGR2-Myc and EGR2G2A-Myc, respectively. Representative pictures are shown in (D), and relative interaction is shown in (E). Data information: In : Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).",
    "molecules": "agarose"
  },
  {
    "caption": "F, G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF-OST1/EGR2-GFP/EGR2-Myc, HF-OST1/EGR2-GFP/EGR2G2A-Myc, or HF-OST1/EGR2-GFP. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc, anti-GFP, and anti-HA antibodies were used to detect EGR2-Myc, EGR2-GFP, and HF-OST1, respectively. Representative pictures are shown in (F), and relative interaction is shown in (G). Data information In Relative band intensities are shown. In each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).",
    "molecules": "agarose"
  },
  {
    "caption": "H, I Effect of cold stress on the interaction of OST1 and EGR2. Arabidopsis protoplasts expressing EGR2-Myc/HF-OST1 and EGR2-Myc/HF were treated at 4°C for 0 and 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. The co-immunoprecipitated products were detected by anti-HA and anti-Myc antibodies, respectively. Representative pictures are shown in (H), and relative interaction is shown in (I). Data information: In H) Relative band intensities are shown. In I): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).",
    "molecules": "agarose"
  },
  {
    "caption": "(c) HEK293 cells were treated as in b, in the presence of leupeptin and stained by anti-AMBRA1 (red) and anti-ULK1 (green) antibodies. The merge of the two signals and DAPI (blue) for nuclear staining is shown in the right panels. Scale bar, 6 μm. The outlined areas are shown at a higher magnification in the lower panels. White arrows point to the co-localization puncta. Measurements by Metamorph indicate a co-localization ratio of 46% between ULK1 and AMBRA1 on autophagy induction.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(e) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide (CHX) for 6 and 8 h before extraction. ULK1, actin and AMBRA1 were analysed by western blotting. Quantification is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.049 and P = 0.02.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(e,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels, n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.003 and P = 0.0015, respectively.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a) HeLa cells were transfected with FLAG-TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.017 for LC3 and P = 0.01 and P = 0.04 for p62.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB).",
    "molecules": "ATP"
  },
  {
    "caption": "(c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies.",
    "molecules": "Torin1"
  },
  {
    "caption": "(d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038.",
    "molecules": "chloroquine"
  },
  {
    "caption": "B. Growth of DiCre-3D7 transgenic lines following LDH1, HAD4, and  Lipin depletion with 100 nM  rapamycin and 2.5 mM  glucosamine was assessed relative to uninduced parasite cultures. The untransfected DiCre-3D7 parasite line was used as a negative control. Data are presented as the mean ± standard error of the mean (SEM) from three independent replicates.",
    "molecules": "glucosamine, rapamycin"
  },
  {
    "caption": "C. Untargeted   LC-MS analysis of LDH1 depletion (one cycle of rapamycin and glucosamine treatment) indicated minimal but selective metabolic changes. m/z feature intensities are plotted as the log2 ratio of treated/untreated and the Benjamini-Hochberg corrected P values across six biological replicates plotted as -log10(P). Below,  LDH1 substrate and product (pyruvate and lactate respectively) abundance plotted as the ratio of treated/untreated from biological replicates (mean±SEM) and the parental DiCre line presented as the negative control.",
    "molecules": "glucosamine, lactate, pyruvate, rapamycin"
  },
  {
    "caption": "D. Loss of Lipin (PF3D7_0303200) leads to accumulation of various lipid species (three cycles of  rapamycin and glucosamine treatment). Data are presented as the mean log2 fold change  Lipin depleted (treated) enriched trophozoite-stage  iRBCs to untreated controls from three to six biological replicates (± SD). The schematic depicts the proposed  lipid phosphatase activity of Lipin.",
    "molecules": "glucosamine, lipid, rapamycin"
  },
  {
    "caption": "E. Loss of HAD4 leads to increases in intracellular levels of several nucleotides and intermediates in lower glycolysis. Data are represented as the mean log2 fold change of enriched trophozoite-stage iRBCs treated for three cycles compared to untreated controls from three/four biological replicates. Three nucleotide-nucleoside pairs are depicted in the top inset panels, HAD4 disruption leads to accumulation of the nucleotide-monophosphate and depletion of the  nucleoside. The schematic depicts lower glycolysis and triose-phosphate interconversion, with the corresponding  metabolite levels following HAD4 disruption depicted in the lower right inset.",
    "molecules": "triose-phosphate, metabolite, monophosphate, nucleoside, nucleotide, nucleotides"
  },
  {
    "caption": "B. Relative  parasitemia was assessed for the untransfected DiCre 3D7 parental line and transfectant parasite lines with AMR1 under inducible disruption. Rapamycin/glucosamine treatment began on cycle zero and parasitemia determined by  flow cytometry  and compared to identical lines that were left untreated. Data represents the mean relative parasitemia ± SEM from three biological replicates (y-axis) across seven replication cycles (x-axis).",
    "molecules": "glucosamine, Rapamycin"
  },
  {
    "caption": "C. Metabolite profile following three cycles of rapamycin and glucosamine treatment. The abundance of isoprenoid biosynthestic intermediates (1-deoxy-xylulose-5-P (DOXP), methyl-erithritol-4-P (MEP), methyl-erithritol-cyclo-2,4-PP (MEcPP), and isopentyl-PP), and hemoglobin-like peptides are presented as the mean log2 fold change from four biological replicates (±SD).",
    "molecules": "1-deoxy-xylulose-5-P, DOXP, MEcPP, methyl-erithritol-cyclo-2,4-PP, MEP, methyl-erithritol-4-P, glucosamine, hemoglobin, isopentyl-PP, isoprenoid, rapamycin"
  },
  {
    "caption": "C.  HA-tagged SHMT-M  detection via immunofluorescence microscopy. DAPI  was used to visualise the nucleus, and MitoTracker CMXros used to illuminate the  mitochondrion. Anti-HA signal partially colocalises with the mitochondrion.",
    "molecules": "HA, DAPI, MitoTracker CMXros"
  },
  {
    "caption": "D.  Pyrimidine biosynthetic intermediates accumulate when SHMT-M is disrupted compared to the DiCre parental line (C) following rapamycin/glucosamine treatment (mean ±SEM). Pyrimidine biosynthetic disruption islinked to  dihydroorotate dehydrogenase (DHODH) inhibition and blockage of the mitochondrial electron transport chain (mETC). Ubiquinone (Q) is the necessary electron carrier for the  mETC.",
    "molecules": "dihydroorotate dehydrogenase, glucosamine, Pyrimidine, rapamycin, Ubiquinone"
  },
  {
    "caption": "E. Asexual growth when SHMT-M is depleted. Parasitemia was determined across seven replication cycles (x-axis) via flow cytometry and presented as the mean ± SEM from three independent experiments. Decylubiquinone (Dub) was added at 10 µM to determine if the growth defect observed was due to impaired ubiquinone synthesis or recycling.",
    "molecules": "Decylubiquinone, Dub, ubiquinone"
  },
  {
    "caption": "Representative immunofluorescence images of lung sections of KrasG12D mice (6 weeks post Ad-Cre inhalation) co-stained for ADAM17 and markers for club cells (CC10) (A), alveolar type II (surfactant protein-C, SP-C) (B) and type I (Podoplanin, Pdpn) (C) cells, total immune cells (CD45) (D) and endothelial cells (CD31) (E). DAPI nuclear staining is blue. Scale bars, 100μm. Arrowheads in merged images indicate dual-positive ADAM17-expressing cells. Quantification of immunofluorescence staining from A-E (n = 5 mice per stain).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative images of pADAM17-stained lung sections from KrasG12D mice injected with a single dose of vehicle (control), SB203580 p38 MAPK or U0126 ERK1/2 inhibitors. Scale bar, 100μm.",
    "molecules": "SB203580, U0126"
  },
  {
    "caption": "Immunoblots with the indicated antibodies on lysates from A549 and NIH-H23 LAC cell lines treated with SB203580 for 24 hours.",
    "molecules": "SB203580"
  },
  {
    "caption": "MTT viability assays of A549 and NCI-H23 LAC cells treated with SB203580.",
    "molecules": "MTT, SB203580"
  },
  {
    "caption": "ATP proliferation (K) assays of A549 and NCI-H23 LAC cells treated with SB203580.",
    "molecules": "SB203580"
  },
  {
    "caption": "Representative immunofluorescence staining of KrasG12D mouse lung sections (n = 5 mice) for ADAM17 and either (panels from left to right) IL-6R, TGFα, Notch1, Nrg1 and EGF. Sections were counterstained with DAPI (blue). Scale bars, 100μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative immunoblots with the indicated antibodies on whole cell lysates from primary immune and epithelial cells isolated from the lungs of mice (n = 3) harbouring the oncogenic KrasG12D allele that were stimulated with either PBS (-) or 100ng/ml Hyper-IL-6 (+). Graphs depict semi-quantitative densitometry of pERK1/2 MAPK protein levels in stimulated immune and epithelial cells (n = 2 experiments).",
    "molecules": "PBS"
  },
  {
    "caption": "MTT viability assays of A549 cells transduced with nontargeted control sgRNA (Ctl) and ADAM17 sgRNA (knockout, KO).",
    "molecules": "MTT"
  },
  {
    "caption": "Representative images of H&E-stained lung sections from KrasG12D mice treated with either vehicle (control) or the ADAM17 prodomain inhibitor, A17pro (1mg/kg, 3 times a week), for 6 weeks starting on the day after Cre inhalation. Scale bars, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated groups (n = 6 per group)",
    "molecules": "A17pro"
  },
  {
    "caption": "Tumor volume (mm3) of a KRAS mutant PDX treated with vehicle control (Ctl) or A17pro (1mg/kg) every second day. Treatment started when tumors reached a volume of 100-150mm3 after injection of 2 × 106 cells in NSG mice. n = 5 per group.",
    "molecules": "A17pro"
  },
  {
    "caption": "Immunoblots with the indicated antibodies of representative lysates from individual PDX tumors treated with vehicle control or A17pro.",
    "molecules": "A17pro"
  },
  {
    "caption": "D. In contrast to controls (i), Tau AT180 antibody (magenta) that specifically recognizes the phosphorylated Threonine 231 of Tau protein distinctly localizes at the somas of SARS-CoV-2-positive neurons (AB4, green) (ii). At least four organoids from two different batches (n=2) are tested. Figures display scale bars.",
    "molecules": "Threonine"
  },
  {
    "caption": "A. Cells from mock-organoids display a healthy nucleus labeled by DAPI (blue) (i). SARS-CoV-2-positive cells (green) display condensed (middle panel, ii) and fragmented DNA (bottom panel, iii, arrows). At least 75 cells from two (n=2) independent batches of organoids were examined. Figures display scale bars.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "A-G Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes on (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) prometaphase I, (F) metaphase I and (G) telophase I. Chromatin has been stained with DAPI (blue). Scale bar in G represents 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-F Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) interkinesis, (B) early prophase II, (C) late prophase II, (D) prometaphase II, (E) metaphase II and (F) telophase II. Chromatin has been stained with DAPI (blue). Scale bar in F represents 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-L Double immunolabelling of CETN3 (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) leptotene, (B) zygotene, (C) pachytene, (D) metaphase I, (E) telophase I, (F) early interkinesis (F), late interkinesis (G), prophase II (H), metaphase II (I), telophase II (J), round spermatid (K), and elongated spermatid (L). Amplifications at 300% magnification for the centrosomic CETN3 signals are presented for each image. Chromatin has been stained with DAPI (blue). Scale bar in L represents 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-J Double immunolabelling of PLK1S137P (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) pachytene, (C) diplotene, (E) diakinesis, (G) metaphase I and (I) metaphase II. Chromatin has been stained with DAPI (blue). Fourth column shows the double Immunolabelling of PLK1-S137P (green) and γ-Tubulin (magenta) on squashed WT mouse spermatocytes at (B) pachytene, (D) diplotene, (F) diakinesis, (H) metaphase I and (J) metaphase II. Insets correspond to centrosomes at 200% magnification. White arrows indicate the location of centrosomes. Scale bar in J represents 10µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-L First column shows the triple immunolabelling of CETN3 (green), PCNT (red) and SYCP3 (grey) on squashed WT mouse spermatocytes. Second column shows the correspondent labellings for Plk1(∆/∆). Third column shows signals for BI2536-treated spermatocytes. Images are shown for (A, E, I) zygotene, (B, F, J) pachytene, (C, G, K) diplotene and (D, H, L) diakinesis. Amplifications at 300% magnification for all labellings are displayed for each image. M-O Quantification of the percentage of cells without PCNT at centrosomes (PCNT-) in (M) zygotene, (N) pachytene and (O) diplotene in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules. Data are presented as mean ± SD for three biological replicates for each condition. ",
    "molecules": "BI2536"
  },
  {
    "caption": "A-C Immunolabelling of γ-Tubulin and SYCP3 during prophase I in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Images are show for pachytene, diplotene and diakinesis in Control (A), Plk1(∆/∆) (B) and BI2536-treated spermatocytes (C). First columns show the double immunolabelling of PCM -PCNT- (green) and SYCP3 (red), and second columns shows γ-Tubulin (γ-Tub, magenta) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Amplifications at 300% magnification for all labellings are displayed for each image. Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules",
    "molecules": "BI2536, DAPI"
  },
  {
    "caption": "(D) Immunolabelling of CETN3 and γ-Tubulin in control and Plk1(∆/∆). Double immunolabelling of CETN3 (red) and γ-Tubulin (green) in pachytene (a-b) and diakinesis (c-d). Chromatin has been stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Quantification of the number of cells without γ-Tubulin at centrosomes (γ-Tub -) in pachytene and diplotene spermatocytes in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data represent average of two biological replicates per condition.",
    "molecules": "BI2536"
  },
  {
    "caption": "A-D Control: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I (A) and metaphase II (B). Double immunolabelling of PCNT (green) and α-Tubulin (red) on control squashed spermatocytes at metaphase I (C) and metaphase II (D). Chromatin has been stained with DAPI (pseudo coloured in grey). E-H Plk1 (∆/∆) mouse model: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) in monopolar spindle I (E) and monopolar spindle II (F). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (G) and monopolar spindle II (H). Chromatin has been stained with DAPI (pseudo coloured in grey). I-L BI2536-treated spermatocytes: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) at monopolar spindle I (I) and monopolar spindle II (J). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (K) and monopolar spindle II (L). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules. M-P MLN8237-treated spermatocytes: Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (M) and monopolar spindle II (N). Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (O) and monopolar spindle II (P). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules. ",
    "molecules": "MLN8237, BI2536, DAPI"
  },
  {
    "caption": "Q-R Quantitative analysis of the incidence of monopolar spindles during the first (Q) and second (R) meiotic divisions for each experimental condition. Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Experiments were conducted in three different biological replicates.",
    "molecules": "MLN8237, BI2536"
  },
  {
    "caption": "A-B AURKP distribution in control, Plk1(Δ/Δ), 100µM 8h BI2536-treated and 10µM 8h MLN8237-treated spermatocytes. (A) Double immunolabelling of AURKP (grey/green) and α-Tubulin (red) in control, Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Images are shown for bipolar spindles I and II for each condition. (B) Monopolar spindles I and II are shown for Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Chromatin has been stained with DAPI (blue)..",
    "molecules": "MLN8237, BI2536, DAPI"
  },
  {
    "caption": "(C) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting (blue bars) or lacking (green bars) centrosomic AURKP signals for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD. (D) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting (blue bars) or lacking (green bars) centrosomic AURKP signal for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD. ",
    "molecules": "MLN8237, BI2536"
  },
  {
    "caption": "(E) Quantitative analysis of AURKP signal at cell poles. Experiments were conducted for two biological replicates. Number of cells analysed: control (n=32), Plk1(Δ/Δ) (n=27), BI2536-treated spermatocytes (n= 27). Data are mean ± SD; (****) p<0.0001, Student's t-test.",
    "molecules": "BI2536"
  },
  {
    "caption": "(B) Double immunolabelling of KIF11 (grey/green), and SYCP3 (red) in (a) control spindle, (b) Plk1(Δ/Δ) monopolar spindle, (c) BI2536 monopolar spindle and (d) MLN8237 monopolar spindle. Chromatin has been stained with DAPI (bue). Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules.",
    "molecules": "MLN8237, BI2536, DAPI"
  },
  {
    "caption": "(C) MALT1 deficiency inhibits CARD14-induced gene expression in HaCaT keratinocytes. HaCaT cells were transfected with scrambled (scr) or MALT1-targeting siRNA prior to doxycycline-induced CARD14 expression as described in materials and methods. IL-8 and MCP-1 concentration in the cell supernatants was measured 8 h (IL-8) or 24 h (MCP-1) later by ELISA. Values are the mean of triplicates ± s.e. Doxycycline-induced CARD14 expression and MALT1 knock-down were verified by immunoblotting (right panel). Data are representative of two independent experiments.",
    "molecules": "doxycycline, Doxycycline"
  },
  {
    "caption": "(E) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14-induced gene expression in HaCaT keratinocytes. CARD14 expression was induced by doxycycline (dox) treatment in the absence or presence of different concentrations of mepazine as indicated. The next morning, cell culture medium was refreshed and IL-8 and MCP-1 concentration in the cell supernatant was measured 8 h later by ELISA. TNF stimulation (MALT1-independent) was used as a negative control. Values are the mean of triplicates ± s.e. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.",
    "molecules": "dox, doxycycline, mepazine"
  },
  {
    "caption": "(D) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14-induced IL-8 production and A20 processing in human primary keratinocytes. Cells were transfected with different CARD14 variants in the presence or absence of mepazine as indicated. TNF treatment was used as a control. 24 h later, IL-8 levels in the cell supernatant were measured by ELISA. Values are the mean of triplicates ± s.e. Expression of different CARD14 variants and CARD14-induced processing of endogenous A20 were analyzed by immunoblotting. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.",
    "molecules": "mepazine"
  },
  {
    "caption": "RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A), or labeled with a fluorogenic substrate for proteases, DQ-Green (C). The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B) or DQ-Green labeled (D) lysosomes was quantified, respectively. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with lysosomes; scale bar = 5 µm. Quantification of data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(E and F) RAW264.7 cells transfected with miR-155 mimic (E) or inhibitor (F) was incubated with DMSO or bafilomycin A1 (BafA.) at a concentration of 100 nM for 2 h, and then LC3 levels were detected by Western-blot. The ratios of LC3-II/β-actin were calculated as shown below the representative blot. ***, p<0.001.",
    "molecules": "bafilomycin A1, DMSO"
  },
  {
    "caption": "(E and F) RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with a specific fluorescent dye MDC (50 µM). The MDC-labeled autophagic vacuoles were detected by confocal microscopy (E) and cells with MDC-positive autophagic vacuoles were quantified (F). Cells treated with rapamycin were used as a positive control. Arrows indicate the GFP-LC3 puncta or autophagic vacuoles labeled with MDC; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 300 cells). **, p<0.01; ***, p<0.001.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(E) After transient transfection with control or miR-155 mimic, RAW264.7 cells were infected with Texas Red-labeled BCG for 1 h, and then were labeled with a specific fluorescent dye MDC (50 µM) for autophagic vacuoles. The co-localization of BCG with MDC-positive autophagic vacuoles was detected by confocal microscopy. (F) Quantification of the co-localization of BCG with MDC-positive autophagosomes is shown. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with autophagosomes; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01; ***, p<0.001.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A and B) RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with DMSO or 3-MA for 2 h. Protein levels of LC3 were detected by Western-blot in uninfected cells (A)",
    "molecules": "3-MA, DMSO"
  },
  {
    "caption": "(A) RAW264.7 cells were transfected with a plasmid expressing Rheb for 24 h, and then incubated with DMSO or BafA. for 2 h. The expression levels of Rheb and LC3 were detected by Western-blot. Values of LC3-II/β-actin ratios are indicated below the representative blot.",
    "molecules": "BafA, DMSO"
  },
  {
    "caption": "Vero and Caco-2 cells were infected with SARS-CoV-2 at MOIs of 0.2 for 8 h. Infected cells were then permeabilized and immunostained for the intracellular SARS-CoV-2 nucleoprotein (NP, red). Nuclei were stained with Hoechst (blue) before imaging by fluorescence confocal microscopy. Scale bars: 100 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Cells were pretreated with the indicated concentrations of aprotinin (A) and SB412515 (B), which are inhibitors of TMPRSS2 and cathepsin L, respectively. Infection of Calu-3, Caco-2, A549*, and Vero cells with SARS-CoV-2 at MOIs of 0.3, 0.4, 0.2, and 0.3, respectively, was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 2-4 biological replicates.",
    "molecules": "aprotinin, SB412515"
  },
  {
    "caption": "SARS-CoV-2 particles were bound to A549* and Vero cells (MOIs 0.2 and 0.3, respectively) (C) or Calu-3 and Caco-2 cells (0.6 and 0.5, respectively) (D) on ice for 90 min and subsequently warmed rapidly to 37°C to allow infectious penetration. SB412515 (10 µM, C) or aprotinin (30 µM, D) was added at different times post warming to block further proteolytic activation. Infection was analyzed by flow cytometry, and data were normalized to samples where protease inhibitors had been omitted. n = 2.",
    "molecules": "aprotinin, SB412515"
  },
  {
    "caption": "Cells were pretreated with endosomal-pH interfering drugs at the indicated concentrations and subsequently infected with SARS-CoV-2 as described in Fig. 2A and 2B in the continuous presence of NH4Cl (A), chloroquine (B), bafilomycin A1 (C), or concanamycin B (D). The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to control samples that were not treated with inhibitors. n = 2-6 biological replicates.",
    "molecules": "NH4Cl, bafilomycin A1, chloroquine, concanamycin"
  },
  {
    "caption": "A549* cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate (B), SB412515 (C), and bafilomycin A1 (D). Infection with SARS-CoV-2 (MOI ~0.2) was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 5-6 biological replicates.",
    "molecules": "bafilomycin A1, camostat mesylate, SB412515"
  },
  {
    "caption": "The timing of the MG-132-sensitive step during SARS-CoV-2 infectious entry into Calu-3 and Caco-2 cells was assayed as detailed in D but using 60 µM MG-132 for Caco-2 cells and MOIs of 0.5 and 0.6, respectively. n = 2.",
    "molecules": "MG-132"
  },
  {
    "caption": "A549* cells expressing or lacking TMPRSS2 were pretreated with MG-132 at the indicated concentrations and subsequently infected with SARS-CoV-2 (MOI ~0.2) in the continuous presence of the drug. The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to that from control samples for which MG-132 had been omitted. n = 6 biological replicates.",
    "molecules": "MG-132"
  },
  {
    "caption": "SARS-CoV-2 was subjected to pretreatment with trypsin and furin for 15 min at 37°C prior to infection of Caco-2 and Vero cells (MOIs of 0.4 and 0.3, respectively). Infected cells were quantified by flow cytometry as described in Fig. 1D. Data were normalized to samples not pretreated with trypsin. n = 4-6 biological replicates.",
    "molecules": "furin, trypsin"
  },
  {
    "caption": "A549* expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS-CoV-2 at MOIs of 0.1 and 0.2, respectively, for 24 h and then cocultured for 5 h along with target cells, not infected but prestained with CMFDA, a cytosolic green dye. Cells were subsequently treated with trypsin or furin for 5 min at 37°C and left to coculture for an additional hour at 37°C. After fixation, nuclei were stained with Hoechst (blue), and infected cells were subjected to immunofluorescence staining against SARS-CoV-2 nucleoprotein (magenta). Samples were ultimately imaged by confocal fluorescence microscopy. White stars indicate syncytia with at least four nuclei. Scale bars: 100 µm. Images of microscope fields obtained in (E) were quantified [A549*: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 80, and n (furin) = 60; A549* TMPRSS2: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 79, and n (furin) = 60; Vero: n (no virus) = 40, n (no protease) = 112, n (trypsin) = 114, and n (furin) = 60]. The fusion index is given as f = 1 - [(number of cells in a field after fusion)/(number of nuclei)].",
    "molecules": "Hoechst, CMFDA, furin, trypsin"
  },
  {
    "caption": "SARS-CoV-2 particles (MOI of 0.3) were first subjected to trypsin treatment for 15 min at 37°C and then exposed to buffers at the indicated pH for 10 min at 37°C, and vice versa. Calu-3 and Vero cells were then infected at a MOI of 0.3 and analyzed by flow cytometry as described in Fig. 1D. n = 4-8 biological replicates.",
    "molecules": "trypsin"
  },
  {
    "caption": "SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.",
    "molecules": "SB412515, thermolysin, trypsin"
  },
  {
    "caption": "D Kaplan-Meier (non-parametric) survival curve showing significantly extended tumor-free survival of KPC mice treated with LOX antibody (1 mg/kg i.p., twice weekly) + gemcitabine (100 mg/kg i.p., twice weekly) (green line, n = 17), compared with gemcitabine ± isotype control antibody-treated mice (blue line, n = 22, P = 0.014) or LOX antibody alone (red line, n = 9). Treatment was initiated when mice were 70 days old (randomization was not used when recruiting the mice) and the mice were treated twice weekly. Censored mice did not develop PDAC.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "E H&E-stained section of PDAC harvested from LOX + gemcitabine-treated KPC mouse. Note necrosis.F Boxplot showing quantification of necrosis in H&E sections of PDACs harvested from these mice, as indicated. At least 30 fields of view from at least five mice per cohort were scored.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "G-H H&E-stained sections of PDAC harvested from LOX + gemcitabine-treated KPC mice. Note stromal collapse (G) and marked dilation of blood vessels (H).I Representative H&E image showing a section of PDAC harvested from a control-treated KPC mouse.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "A Outer left panels: Immunohistochemistry analysis of macrophage infiltration (by F4/80 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner left panels: Immunohistochemistry analysis of neutrophil infiltration (by MPO staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner right panels: Immunohistochemistry analysis of tumorvasculature (by CD31 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Outer right panels: Immunohistochemistry analysis of tenascin C expression in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice.B-D Plots showing quantification of macrophage infiltration (B), neutrophil infiltration (C) and tumorvasculature (D) in tumors from KPC mice treated as indicated. At least 30 fields of view from at least four mice per cohort were scored, and scoring was conducted blind. P-values were calculated using Mann-Whitney U-test and median is indicated by horizontal lines.E Correlation of LOX protein with tenascin C expression in 47 cases of PDAC (Spearman's rho correlation coefficient = 0.61; P < 0.0001).",
    "molecules": "gemcitabine"
  },
  {
    "caption": "(A) Representative  confocal photomicrographs  showing coronal  slices from 8W hGFAP‐tTA;H2B‐GFP mice  with and without doxycycline  treatment, upon immunofluorescent staining  with the stem cell  marker Nestin  (red). DAPI  was used for nuclear counterstain. Cross indicates slice directions: M = medial, D = dorsal, V = ventral, L = lateral. Scale bar = 20 µm. (B) Quantification of Nestin expression  in apical  and basal cells in doxycycline-treated and untreated animals. N = 3 (- Doxy), N = 4 (+ Doxy). Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical  and basal  (*) and between doxycycline  treatments (#); */#p<0.05, **p<0.01, by one-way ANOVA with Tukey's multiple comparisons test (B)",
    "molecules": "DAPI, Doxy, doxycycline"
  },
  {
    "caption": "(A) Repesentative confocal photomicrographs of coronal  sections of the SVZ  of 8W hGFAP-H2BGFP mice, which had been administered doxycycline  (Doxy) as indicated. Nuclear GFP expression  and Ki67  immunoreactivity are shown in green and red, respectively. DAPI counterstaining  of the nuclei  is blue. Scale bar indicates 20 µm.",
    "molecules": "DAPI, Doxy, doxycycline"
  },
  {
    "caption": "(B, C) Quantitative analyses of the number of apical  and basal cells expressing GFP  (G+) or not (G-); N = 8 (B), and Ki67  immunoreactivity in these populations; N ≥ 4 (C). Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. * indicates significance between doxycycline  treated and nontreated specimens with apical or basal cell populations. * p<0.05, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) Representative micrographs  of the OB  of AAV-GFP-injected animals, at 14 days (14D) and 6 weeks (6W) after virus injection. Scale bar indicates 20 µm. (C) Quantification of GFP+ and/or DCX+ cells in the OB, as percentage of total DAPI  cells N ≥ 5. Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A, B) Representative micrographs  of the  V-SVZ of mice  injected with an AAV  overexpressing GFP  and a shRNA against Hes1  (AAV Hes1-sh) or a scrambled sequence (AAV  sc-sh). Coronal  sections were stained  with either notch intracellular domain (NICD, A) or Ki67  to visualize cycling cells (B). DAPI  was used for nuclear counterstain, scale bars indicate 20 µm. Arrows indicate apical GFP+ (white) and basal GFP- (yellow) cells displaying NICD staining. Quantification of fold change in NICD-levels; N = 3 (C), Ki67+ cells; N = 5 (D) Data information: N number refers to biological replicates; for each biological replicate at least three technical replicates were performed. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test",
    "molecules": "DAPI"
  },
  {
    "caption": "Reverse Transcriptase PCR showing that bd1971, and control gene dnaK, are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus. RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E. coli cells. Primers were designed to anneal specifically to the gene of interest. L = 100bp DNA ladder, AP = Attack Phase cells, 15-45 = 15-45 minutes respectively since infection, 1h-4h = hours respectively since infection. Ec = E. coli strain S17-1 RNA (negative control: no Bdellovibrio RNA); NT = control with no template RNA; gen = B. bacteriovorus HD100 genomic DNA (positive control). The cartoon above represents each stage in the predatory cycle.",
    "molecules": "RNA"
  },
  {
    "caption": "Relative levels of c-di-GMP in matched cell biomass host-independent (HI) cultures of bd1971::kan compared with fliC1 merodiploid KnR HI control strain (Wild-Type for bd1971). The bd1971 mutant strain contains significantly more c-di-GMP than the control. Data are from 3 independent experiments.",
    "molecules": "c-di-GMP, kan, Kn"
  },
  {
    "caption": "Relative levels of c-di-GMP in matched cell biomass wild-type predatory cultures of HD100 strain, silent Bd1971 deletion strain (Δ1971), Bd1971D306307A strain (bd1971DD) and Bd1971R67D strain (bd1971R). All mutant strains had higher levels of c-di-GMP compared to the wild type strain. Error bars show SEM. Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants.",
    "molecules": "c-di-GMP"
  },
  {
    "caption": "Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation.",
    "molecules": "cAMP, cGMP, PNPP, CaCl2, MgCl2, nucleotide"
  },
  {
    "caption": "HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.",
    "molecules": "cAMP, cGMP, c-di-GMP"
  },
  {
    "caption": "ITC titration curves and relative heats of binding for (A) cAMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.",
    "molecules": "cAMP"
  },
  {
    "caption": "ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.",
    "molecules": "cGMP"
  },
  {
    "caption": "Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments.",
    "molecules": "X-Gal, c-di-GMP"
  },
  {
    "caption": "A Gene expression analysis of myokines (table EV3) during a five day time course of myogenic differentiation going from cycling myoblasts in growth media (Ham's F10 supplemented with 20% Fetal Bovine Serum, 1% penicillin/streptomycin, 2.5 ng/ml basic Fibroblast Growth Factor) to terminally differentiated myocytes (2 days in differentiation media, DMEM supplemented with 5% horse serum) to post mitotic multinucleated myotubes (5 days in differentiation media). Gene expression was assayed in biological triplicate by microarray (Soleimani et al., 2018; Soleimani et al., 2012a). Expression of each gene was averaged across three replicates and normalized to mean zero and standard deviation of one. Expression values were then truncated at +/- 3 for color display. Blue indicates lower than average expression, while red indicates higher than average expression. B Distribution of Myf6 peaks within 100kb of the TSS of the myokine genes (table EV3), similar to the analysis shown in (A).",
    "molecules": "penicillin, streptomycin"
  },
  {
    "caption": "C-E Snapshots of the UCSC genome browser showing enrichment of Myf6 ChIP-Seq reads compared to control around EGF (C), LIF and OSM (D), and VEGFA (E) loci superimposed on reads for Histone H3 lysine 4 mono methyl (H3K4me1), Histone H3 lysine 4 tri methyl (H3K4me3) and total H3 in myoblasts (pink) and myotubes (green).",
    "molecules": "lysine 4 mono methyl, lysine 4 tri methyl"
  },
  {
    "caption": "Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 3 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm J Quantification of the number of MuSCs per fiber between 3 month-old Myf6-KO (n=41 fibers from n=3 mice) and WT (n=31 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD K Immunofluorescent analysis of myofiber-associated MuSCs isolated from the EDL muscles of 10 month-old Myf6-KO and WT counterparts. Fibers were stained with PAX7 antibody and counterstained with DAPI at time T=0 hours post fiber isolation. Scale bar=25μm L Quantification of the number of MuSCs per fiber between 10 month-old Myf6-KO (n=38 fibers from n=2 mice) and WT (n=67 fibers from n=3 mice) counterparts. Two-tailed t-test, error bars = +/- SD",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescent analysis of WT and Myf6-KO primary myotubes cultured in differentiation media for 7 days and stained with MF20 and DAPI. Scale bar=200μm J Quantification of the fusion index between Myf6-KO (n=54 fields of view from n=3 mice) and WT (n=81 fields of view from n=4 mice) 7DM myotubes. Fusion index is calculated as the percentage of myonuclei out of the total number of nuclei per field of view. Two-tailed t-test, error bars = +/- SD",
    "molecules": "DAPI"
  },
  {
    "caption": "EDL myofibers isolated from WT and Myf6-KO mice fixed at time T=0 and stained with PAX7, MYOD and DAPI. Scale bar=30μm L Quantification of the percentage of MYOD+/PAX7+ satellite cells per fiber in Myf6-KO (n= 88 myofibers) vs WT (n= 86 myofibers) myofibers. Two-tailed t-test, error bars = +/- SD M Quantification of the average percentage of MYOD+/PAX7+ satellite cells per mouse in Myf6-KO and WT myofibers (n=3 mice). Two-tailed t-test, error bars = +/- SD",
    "molecules": "DAPI"
  },
  {
    "caption": "E-F Immunostaining of TA cross-sections of injured muscle from 3 month-old WT (E) and Myf6-KO (F) mice stained with laminin (LAMA1) and PAX7 antibodies. Acute muscle injury was performed by injection of Cardiotoxin (CTX) into the TA muscle of Myf6-KO and WT mice (n=5 animals per group). Arrowheads indicate PAX7+ MuSCs. Scale bar=100μm G Quantification of the number of PAX7+ cells per unit area of TA muscle cross sections between Myf6-KO and WT mice (n= 5 animals per group) two-tailed t-test, bars represent mean +/- SD",
    "molecules": "Cardiotoxin, CTX"
  },
  {
    "caption": "EdU staining of cultured primary myoblasts isolated by FACS (see materials and methods) from Myf6-KO and WT mice. Staining was analyzed after 6, 12 and 24 hours of EdU incorporation. Scale bar=100μm M Quantification of the percentage of EdU+ myoblasts after 6 hours (WT n=75 fields of view from n=3 mice, Myf6-KO n=72 fields of view from n=3 mice), 12 hours (WT n=82 fields of view from n=3 mice, Myf6-KOn=82 fields of view from n=3 mice), and 24 hours (WT n=70 fields of view from n=3 mice, Myf6-KO n=65 fields of view from n=3 mice) of EdU incorporation. Scale bar=100μm",
    "molecules": "EdU"
  },
  {
    "caption": "Representative images of a co-culture assay between WT myotubes and either WT or Myf6-KO myoblasts. Briefly, WT myoblasts were differentiated for 2 days before adding equal numbers of either WT or Myf6-KO myoblasts for an additional 2 days, followed by immunostaining for MF20 and DAPI. Scale bar=400μm O Quantification of the average number of myoblasts per mm2 that have remained mononuclear after their addition to 2DM WT myotubes, for 2 additional days (n=3 biological replicates). Two-tailed t-test, bars represent mean +/- SD.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Fold-change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n=47), baricitinib 1-mg (n=23), 2-mg (n=24), 4-mg (n=23), and 8-mg (n=23) in the phase 2b randomized, placebo-controlled study NCT01469013. p values are for comparisons of baricitinib 1-mg, 2-mg, 4-mg, and 8-mg change from baseline compared to the placebo change from baseline. Treatment effects were estimated using a mixed effects repeated measures model (SAS Proc Mixed) using log10 transformed IL-6 levels with an unstructured covariance matrix.",
    "molecules": "baricitinib"
  },
  {
    "caption": "(A and B) Binding affinities in Kd are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration response curve with fixed top parameters and a four-parameter logistic model. Kd, equilibrium affinity constant (n=3)",
    "molecules": "AZD7762, baricitinib, erlotinib, sunitinib, tofacitinib, upadacitinib"
  },
  {
    "caption": "(C) Protein and mRNA expression of relevant NAKs in primary human liver organoids (n=3) (p<0.05). The immunofluorescence shows the juxtaposition of 1A9 (signal from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n=1). Colocalization of 1A9 and ACE2 is depicted with an arrow.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n=10).",
    "molecules": "ATP"
  },
  {
    "caption": "(H) Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection (*p<0.05; Student's t-test). Data are mean ± standard error of mean (n=3).",
    "molecules": "baricitinib"
  },
  {
    "caption": "(A) Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH were simultaneously amplified. Here the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cut-off. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the X-axis. Fever and cough symptoms are indicated. The bars in grey indicate that cough has improved but has not resolved.",
    "molecules": "baricitinib"
  },
  {
    "caption": "Detection of IgG antibodies against the S1/S2 antigens of SARS-CoV-2. Time of baricitinib treatment is indicated for all patients (patients A, B, and D for 10 days, patient C for 12 days).",
    "molecules": "baricitinib"
  },
  {
    "caption": "(A) Levels of D-dimer, CRP, ferritin, white blood cells, and neutrophils are shown for all patients.",
    "molecules": "D-dimer"
  },
  {
    "caption": "(B) Levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin are shown for all patients. ALT, alanine aminotransferase; AST, aspartate aminotransferase",
    "molecules": "bilirubin"
  },
  {
    "caption": "SARSCoV-2 MA30  infection in C57BL/6 mice  7 days follow up. PEGSerp-1 treatment significantly improved Mean (C) weights  (N = 20 mice, C mean ± SD)) Data analysis information: Mean ± SD ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; Unpaired T tests; indicated by line at top with subgroup analyses in brackets below; Prophylactic treatment black - Saline, red -PEGSerp-1 treatments.",
    "molecules": "PEG"
  },
  {
    "caption": "E Micrographs illustrate lung  consolidation in Saline  and PEGSerp-1 treated lungs. Large blue arrows indicate areas of consolidation in H&E stained lung  sections (Mag 2X).",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "G, Combined weights  for the 4 and 7 day follow up studies demonstrate consistently improved mean weights  (G; p < 0.0153- 0.0001) with PEGSerp-1 treatments in MA30  infected mice. (N = 39mice, mean ±SD) Data analysis information: black - Saline, red -PEGSerp-1 treatments.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "I Micrographs illustrate lung  consolidation divided by total section analyzed in Saline  and PEGSerp-1  treated MA30  infected mice  at 4 days follow up.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "PEGSerp-1 delayed treatment at 10ng/gm starting 2 days after infection reduced weight  loss (L; p < 0.001-0.05). (N = 24 mice, mean ± SE) Data analysis information: Mean ± SE ; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; blue - Saline, green and blue - PEGSerp-1.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "A, B Micrographs illustrating iNOS  positive  M1 macrophage cell infiltrates on IHC stained lung  sections in Saline  and PEGSerp-1 (A) treated mice  with significantly reduced IHC  positive cell counts at 4 days follow up after PEGSerp-1 prophylactic treatment (B; *p < 0.045). IHC stained lung  sections (Mag 40X). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back circles Saline, red circles PEGSerp-1 treatments.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "C, D iNOS  positive infiltrates in IHC stained lung  sections with Saline  or  PEGSerp-1 (C) treatment with significantly reduced IHC  positive cell counts at 7 days follow up with prophylactic PEGSerp-1 treatment (D; **p < 0.0052). IHC stained lung  sections (Mag 40X). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back circles Saline, red circles PEGSerp-1 treatments.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "E Delayed PEGSerp-1  10ng/gm dose treatment given 2 days after infection also reduced iNOS  positive cell counts (*p < 0.0368). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; blue circles Saline, red and green circles  PEGSerp-1  treatments.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "G CD3 + T cell counts were not significantly altered at 4 days (p = 0.1058). H CD4 + T cell counts are not reduced at 4 days (p =0.5031) but are reduced at 7 days (*p < 0.0454) with PEGSerp-1  treatments. I CD8 positive T cell  counts on IHC stained  sections have a trend toward an increase at 4 days (p = 0.1503) and a significant reduction at 7 days (*p < 0.0303) with PEG Serp-1 treatments. J IHC  for Neutrophil  marker Ly6G  did not detect significant changes in PEGSerp-1 treatment at days 4 (p = 0.0860) and 7 (p = 0.6570). Data information: Mean ± S.E.; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatments.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "iNOS M1 stained macrophage  invasion in Saline  control treated mice  (A) Black arrows point to brown iNOS  positive macrophage  on IHC stained lung  sections (Mag 40X).",
    "molecules": "Saline"
  },
  {
    "caption": "PEGSerp-1 treatment reduced iNOS  + macrophage  infiltration. iNOS M1 stained macrophage  invasion is significantly reduced with  PEGSerp-1  treatment (B). Black arrows point to brown iNOS  positive macrophage  on IHC stained lung  sections (Mag 40X).",
    "molecules": "PEG"
  },
  {
    "caption": "H & E stained histology  sections of lung  illustrating consolidation in Saline  treated mice  (E) saline  treated uninfected mouse  tissues had similar levels of consolidation Black arrows point to brown iNOS  positive macrophage  on IHC stained lung  sections (Mag 40X).",
    "molecules": "Saline, saline"
  },
  {
    "caption": "PEGSerp-1  treatment reduced lung  consolidation H & E stained histology  sections of lung  illustrating consolidation is reduced by  PEGSerp-1 treatment (F). Black arrows point to brown iNOS  positive macrophage  on IHC stained lung  sections (Mag 40X).",
    "molecules": "PEG"
  },
  {
    "caption": "A, B Micrographs illustrating iNOS  positive cells in Saline  (A) and PEGSerp-1 (B) treated mice  at 4 days follow up. IHC stained  sections 40X.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "PEGSerp-1 treated mouse lung   sections from MA10   at day 2 follow up have significant reduction in uPAR  positive stained   areas (A) with reduced positively stained hemorrhagic  areas in lungs   at 2 days in MA10   infected BALB/c (B; p < 0.041) and reduced uPAR  + stained   cell counts at 4 days (C; p < 0.0202) and 7 days (C in alveoli   (p < 0.0148) in MA30   infected C57BL/6 mice. With delayed treatments uPAR  is reduced at 7 days follow up (D; ANOVA p< 0.0001, p < 0.0002 for PEGSerp-1 at 10µg/kg). FX staining   was reduced with prophylactic  PEGSerp-1 at day 7 but not at day 4 follow up (E; ANOVA p < 0.0008, p < 0.0001 for day 7 and p = 0.5148 for day 4). Data information: Mean ±SE. * p < 0.05, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1  treatments",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "C5b-9 staining  is reduced in prophylactic PEGSerp-1 treatment in MA30 infected C57BL/6 mice  on IHC stained  sections (H) with significant reductions at day 4 (I; p <0.0114), and a nonsignificant trend at day 7 follow up (p = 0.5694). With delayed PEGSerp-1  treatment C5b/9 detection on IHC  is significantly reduced by both PEGSerp-1 doses (J; p < 0.0131). Data information: Mean ±SE. * p < 0.05, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below; back and blue circles Saline, red and green circles PEGSerp-1 treatments",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "D-G In MA30  infected C57BL/6 mice  increased S protein is seen on IHC staining  seen in lung  sections from Saline  treated mice  (D) is reduced with  PEGSerp-1 treatment at 7 days follow up (E, F; p < 0.001) and in the small vessels (G; p < 0.0364). Data information: Mean ±SE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ANOVA indicated by line at top with subgroup analyses in brackets below.",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "A, B Relative gene expression is significantly increased for SARS-CoV-2 MA30 viral E gene (A; p < 0.0017) and interferon response ISG15  (B; p < 0.0044) in Saline  treated infected lung  extracts at 4 days follow up, when compared to uninfected mouse lungs. Data information:  Gene expression is normalized to GAPDH expression. Y axes vary dependent upon the detected relative expression  levels after normalizing data to the internal control. (ANOVA indicated by line at top with subgroup analyses below; infected mice  - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "Relative changes in gene expression for SARS-CoV-2  infections and associated modulation in inflammatory and coagulation responses are increased after   SARS-CoV-2 MA30 infection in lung  and heart  tissues (SARSCoV-2 MA30 infection - N = 18 mice  4 days follow up; N = 20 mice  7 days follow up; Uninfected mice  - N = 2). PEGSerp-1  treatment significantly modified gene expression for uPAR and complement pathways. C-L Inflammatory cytokines and growth factors are significantly increased with infection; IL-1 (C; p < 0.0003), IL-6 (D p < 0.0001),  IL-10 (E; p < 0.0001) ), TNF  (F; p < 0.0044), TGFb  (G; p < 0.0022) are increased with SARS-CoV-2 infection at 4 days follow up; with VEGF  the exception (H; p = 0.4695 ) Factors in the coagulation pathway, fX  (I; p < 0.0001), tPA  (J; p < 0.0382), and neuroserpin  (K; p = 0.1883 ) are also increased. None of these markers are modified by PEGSerp-1 treatments. Data information: Gene expression  is normalized to GAPDH expression. Y axes vary dependent upon the detected relative expression  levels after normalizing data to the internal control. (ANOVA indicated by line at top with subgroup analyses below; infected mice  - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "Relative changes in  gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after  SARS-CoV-2 MA30 infection in lung  tissues (SARSCoV-2 MA30 infection - N = 18 mice  4 days follow up; N = 20 mice  7 days follow up; Uninfected mice  - N = 2). PEGSerp-1 treatment significantly modified gene expression  for uPAR  A-C Gene expression for  PEGserp-1 targets are increased after infection and modified by PEGSerp-1 treatments. uPA  (A; p = 0.3263), PAI-1 (B; p < 0.0001) and uPAR  (C; p < 0.0001) were increased in infected mouse lung  extracts at 4 days follow up when compared to uninfected lungs  with SARS-CoV-2  MA30 infection. PEG Serp-1 treatment produced a significant increase in uPAR  (C; p < 0.0157). Data information: N = 19 mice  at 4 days follow up; N = 20 mice  at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice  - blue circles Saline, red circle PEGSerp-1  treatments, green circle uninfected mice).",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after SARS-CoV-2 MA30  infection in lung  tissues (SARSCoV-2 MA30 infection - N = 18 mice  4 days follow up; N = 20 mice  7 days follow up; Uninfected mice  - N = 2). PEGSerp-1  treatment significantly modified gene expression  for complement pathways. D-F Complement pathways genes encoding C3  (D; p < 0.0021), C5  (E; p < 0.001) and C1INh  (F; p < 0.0008) were also significantly increased with infection. PEG Serp-1 treatment produced a borderline decrease in C5  (E; p = 0.0623) and a significant increase in C1Inh  (F; p < 0.0309). Data information: N = 19 mice  at 4 days follow up; N = 20 mice  at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice  - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "Relative changes in  gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after  SARS-CoV-2 MA30 infection in lung and heart  tissues (SARSCoV-2  MA30 infection - N = 18 mice  4 days follow up; N = 20 mice  7 days follow up; Uninfected mice  - N = 2). PEGSerp-1 treatment significantly modified gene expression  for complement pathways. G-I At 7 days follow up, relative gene expression  is no longer increased for C1Inh  with PEGSerp-1  treatment in MA30  infected mouse lungs  (G; p = 0.5956), but C1Inh  was significantly increased in myocardial  extracts with PEGSerp-1 treatment at 7 days (H; p < 0.0419). C5  was not significantly increased with infection (I; p = 0.1601) Data information: N = 19 mice  at 4 days follow up; N = 20 mice  at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice  - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "Relative changes in gene expression for SARS-CoV-2 infections and associated modulation in coagulation and immune responses are increased after SARS-CoV-2  MA30 infection in lung  and heart  tissues (SARSCoV-2 MA30  infection - N = 18 mice  4 days follow up; N = 20 mice  7 days follow up; Uninfected mice  - N = 2). PEGSerp-1  treatment significantly modified  gene expression for uPAR  J-L PAI-1 gene expression  was not increased in lung  extracts with  PEGSerp-1 treatment at 7 days follow up in MA30  infected mouse lungs  (J; p = 0.2403), but PAI-1 expression  was significantly increased at 7 days follow up in heart  extracts (K; p < 0.0454). uPAR  was not significantly increased in lung  extracts at 7 days follow up (p = 0.1514). A time course for  gene expression illustrates a peak time for changes in C1Inh gene expression demonstrates greater changes at 4 days when compared to day2 and day15 (L). Data information: N = 19 mice  at 4 days follow up; N = 20 mice  at 7 days follow up. ANOVA indicated by line at top with subgroup analyses below; infected mice  - blue circles Saline, red circle PEGSerp-1 treatments, green circle uninfected mice).",
    "molecules": "PEG, Saline"
  },
  {
    "caption": "(C) Samples were stained  with antibodies for β4-integrin or laminin  (green; yellow arrows). The actin  and nucleus  were counterstained  with phalloidin (red) and DAPI  (blue), respectively. Scale bar, 10 μm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(D) HMT-3522 S-1 MECs plated as monolayers  on a rigid rBM  (2D) or as spheroids  within rBM  (3D) were treated with increasing doses of Paclitaxel  (Pac), TRAIL, Doxorubicin  (Doxo) and irradiation  (IR). Percent cell death    was quantified by immunofluorescence  as percentage of cells stained  positive for cleaved caspase-3 at 48h post-treatment (mean ± SEM; n=3 independent biological replicates). ****P < 0.0001 (Student's t test).",
    "molecules": "Doxo, Doxorubicin, Pac, Paclitaxel"
  },
  {
    "caption": "(F) Representative immunofluorescence  microscopy images of MCF10A MECs  plated in the indicated conditions for 18h and stained  with phalloidin  to reveal F-actin organization. Cells were plated as single cells on rigid  glass coverslips (2D  glass; top left) or fully embedded within rBM  (3D  fully embedded; bottom left). Cell spreading was inhibited by plating cells on either  laminin-111 conjugated, 10-μm micropatterned glass (2D/micropatterned, rigid substrate; top middle) or on compliant 75 Pa rBM-laminated polyacrylamide  (PA) gels (2D/PA, soft substrate; top right). The single 2D  cells were overlaid with either purified laminin-111 (bottom middle) or rBM  (bottom right) to create a 3D ECM  microenvironment. Images show maximum intensity z-projections of confocal  stacks for F-actin phalloidin staining. Scale bar, 10 μ",
    "molecules": "phalloidin, PA, polyacrylamide"
  },
  {
    "caption": "(G) Representative immunofluorescence microscopy  images of MCF10A MECs  stably expressing recombinant mCherry-tagged golgi  marker (mCherry-GalT; red; yellow arrows) ligated with rBM  in either 2D   or 3D  for 18h. The  actin cortex  and nucleus  were counterstained  with phalloidin (green) and DAPI  (blue), respectively. Images show the cross-sectional view of each cell compartment (dashed lines; xy plane) and side view of confocal  stacks (xz plane) in individual MCF10A MECs. Scale bar, 10 μm. (H) Golgi staining  was assessed within non-spread MCF10A MECs  ligated with rBM  in 2D  and 3D  for 18h and values were plotted as a function of subcellular localization   (2D, n=8; 3D, n=9 cells from two independent experiments).",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(D) Representative line graph showing the changes in cytosolic  [Ca2+] levels of MCF10A MECs  under different treatment.  Fura-2-loaded MECs  ligated to 2D  (blue) or 3D rBM  (red) were pre-incubated with EGTA  to chelate extracellular  Ca2+ in the bath solution, challenged with 2μM thapsigargin  to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values for each cell were quantified over the course of imaging (mean ± SD; 2D, n=7; 3D, n=7 cells from one experiment). (E) Quantification of the amplitude of the Ca2+ response induced by thapsigargin  (∆F=F-F0), where F0 is the basal fluorescence before thapsigargin  treatment (mean ± SEM (2D, n=20; 3D, n=21 cells from three independent experiments). ***P = 0.0007 (Student's t test).",
    "molecules": "Ca2+, EGTA, Fura-2, thapsigargin"
  },
  {
    "caption": "(A) Representative immunofluorescence microscopy images of MCF10A MECs  ligated with either a rBM  in 2D  or 3D  for 18h and stained  with antibody targeting filamin  (green).  F-actin was counterstained  with phalloidin  (red). Images show maximum intensity z-projections of xy confocal  stacks (left) and middle focal plane of confocal  stacks from MECs  (right). Yellow arrows indicate filamin  aggregates in individual MECs  ligated to rBM  in 2D. Yellow blunt-end lines highlight the extent of filamin  distribution away from the cell edge. Scale bar, 10 μm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "(G) Representative immunoblots  of filamin, phosphorylated EIF2a  (pEIF2a), total EIF2a  and alpha-tubulin in cell lysate from MCF10A MECs  ligated to rBM  in 3D and treated with ethanol  (mock) or doxycycline  (FLMN) for 18h to induce filamin   expression and corresponding quantification data (mean ± SEM; n=5 independent biological replicates). **P = 0.0018 (Student's t test).",
    "molecules": "doxycycline, ethanol"
  },
  {
    "caption": "(A) Schematic showing the principles behind  Atomic Force Microscopy  (AFM) (left). A cantilever at the end of the microscope probe is deflected when it is in contact with the cell surface. Cell cortex-mediated resistance to indentation alters the path of the laser beam focused on the cantilever that is then reflected onto a photodetector to enable measurement of cellular  cortical actin tension. AFM  was used to measure the  cortical actin tension in MCF10A MECs  ligated to a laminin-111  substrate in 2D  or 3D  (right) and treated with blebbistatin  (Bleb) to reduce cortical  actin  tension, induced to overexpress filamin  expression (FLMN), or activated ROCK  (ROCK) to increase  cortical actin tension. MECs  were indented using a 2-µm beaded tip on the AFM  cantilever and the Hertz model was used to fit each indentation curve to extract the Young's modulus of the cell cortex (mean ± SEM; 2D, n=123; 3D, n=184; 3D+FLMN, n=83; 2D+Bleb, n=66; 3D  + Bleb, n= 65; 3D+ROCK, n=212; n=AFM  indentation from >30 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Tukey's multiple comparisons test. 2D  versus 3D, ****P < 0.0001; 3D  versus 3D  + FLMN, ****P < 0.0001; 3D  versus 3D  + ROCK, ****P < 0.0001; 2D  versus 2D  + Bleb, ***P=0.0004.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(B) Schematic of the principles behind traction force microscopy (left). MECs   are plated on compliant polyacrylamide  gels containing 100 nm fluorescent beads (close to the cell-polyacrylamide  gel interface). Traction stresses are calculated based on the bead displacement induced by substrate deformation and relaxation. Quantification of the traction stresses in individual MCF10A MECs  ligated with rBM  in 2D  and 3D  and treated in the absence and presence of blebbistatin  (Bleb) for 18h (right; mean ± SEM; 2D, n=139; 3D, n=124; 2D+Bleb, n=42; 3D  + Bleb, n= 43 cells from three independent experiments). The background bead displacement was measured from gel areas that lacked ligated cells (n=8 fields from one experiment). Statistical analysis by one-way ANOVA followed by Tukey's multiple comparisons test. ****P < 0.0001.",
    "molecules": "Bleb, blebbistatin, polyacrylamide"
  },
  {
    "caption": "(C) Schematic of the principle of active microrheology (left). MCF10A MECs  endocytosed 0.5 μm polystyrene  particles, which were trapped and oscillated using laser optical tweezers to measure the cytoplasmic  modulus. The cytoplasmic  modulus  was measured for MECs  ligated to a rBM  in 2D  or 3D  and treated in the absence or presence of blebbistatin  (Bleb) to reduce cortical actin tension (right); individual modulus values were calculated based on the slope in the linear range of the normalized force-displacement curve (mean ± SEM; 2D, n= 66; 3D, n=69; 2D+Bleb, n=46; 3D+Bleb, n=60 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D  versus 3D, **P = 0.0028. 2D  versus 2D  + Bleb, *P = 0.0182.",
    "molecules": "Bleb, blebbistatin, polystyrene"
  },
  {
    "caption": "(D) Schematic depicting strategy used to measure cortical  tension using  laser ablation. (Top) Cells with high cortical  tension exhibit plasma membrane blebbing  when cortical actin is severed by a pulsed laser, whereas cells with lower cortical  tension do not. (Bottom left) Bar graph of the laser ablation response of MCF10A MECs  ligated to a rBM  in 2D  or 3D  (mean ± SD; 2D, n= 35; 3D, n=40 cells from three independent experiments). (Bottom right) Bar graph showing the laser ablation response of MCF10A MECs  ligated to a rBM  in 2D or 3D  and treated in the absence or presence of blebbistatin  (2D+Bleb  and 3D+Bleb) or expressing constitutively active ROCK  (3D+ROCK) (mean; 2D, n= 14; 3D, n=15; 2D+Bleb, n=17; 3D+Bleb, n=14; 3D+ ROCK, n=10 cells from one experiment).",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(E) Representative fluorescence  and brightfield  images of bleb  formation induced by  laser ablation in MECs  stably expressing LifeAct-RFP. Arrowhead: the site of laser ablation. Scale bar, 10 μm.",
    "molecules": "LifeAct"
  },
  {
    "caption": "(A) Representative line graph showing the changes in cytosolic  [Ca2+] levels of MCF10A MECs  in response to treatment. Fura-2-loaded MECs  ligated with rBM  in 2D were treated with DMSO  (2D; blue) or with blebbistatin  (2D+Bleb; red) and preincubated with EGTA  to chelate extracellular Ca2+ in the bath solution, challenged with 2 μM thapsigargin  to induce ER Ca2+ release, and replenished with 4 mM  Ca2+ in bath solution at the indicated times. F340/F380 values for each cell were quantified over the course of imaging and plotted (mean ± SD; 2D, n=7; 2D + Bleb, n=7 cells from one experiment). (B) Quantification of the amplitude of the  Ca2+ response (∆F=F-F0) induced by thapsigargin, where F0 is the basal fluorescence before thapsigargin  treatment. The data shown indicated mean ± SEM (2D, n=13; 2D + Bleb, n=12 cells from two independent experiments). ****P < 0.0001 (Student's t test).",
    "molecules": "Bleb, blebbistatin, Ca2+, DMSO, EGTA, Fura-2, thapsigargin"
  },
  {
    "caption": "(C) Representative immunoblots  of phosphorylated EIF2a  (pEIF2a), total EIF2a  and  alpha-tubulin in cell lysate from MCF10A MECs  ligated to a rBM  in 2D  or 3D  and treated in the absence or presence of blebbistatin  (Bleb). Corresponding quantification data are shown at bottom (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D  versus 3D, **P = 0.0022. 2D  versus 2D  + Bleb, *P = 0.0248.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(D) Bar graph of qPCR  data measuring the relative levels of ATF3  mRNA in MECs  ligated with rBM  in 2D and treated in the absence or presence of blebbistatin  (Bleb) (mean ± SEM; n=4 independent biological replicates). *P = 0.0158 (Student's t test).",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(E) Graph of the levels of GFP-MAPPER  at the  plasma membrane relative total cellular GFP-MAPPER  fluorescence in MCF10A MECs  ligated with rBM  in 2D and treated in the absence or presence of blebbistatin  (Bleb) (mean ± SEM; 2D, n=39; 2D + Bleb, n=39 cells from three independent experiments). ***P = 0.0002 (Student's t test).",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(G) Scatter plot of the number of proximity ligation assay (PLA) puncta within individual cells expressing myc-PERK  that were ligated to rBM  in 2D  or 3D, treated in the absence or presence of blebbistatin  (Bleb) for 25h and stained  with PLA  antibody probes specific for endogenous filamin  and myc-PERK. Representative images of PLA staining  from these experiments can be found in Panel H. Background PLA  signal was measured from cells stained  in the absence of primary antibodies (mean ± SEM; 2D, n=47; 3D, n=32 cells; 2D  + Bleb, n=39; background, n=14 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(H) Representative fluorescence microscopy  images of cells expressing myc-PERK  that were ligated to rBM  in 2D  or 3D, treated in the absence or presence of blebbistatin (Bleb) for 25h and stained  with PLA  antibody probes specific for endogenous filamin  and myc-PERK  (red puncta). F-actin  was stained  with phalloidin",
    "molecules": "Bleb, blebbistatin, phalloidin"
  },
  {
    "caption": "(I) Representative line graph showing the changes in cytosolic  [Ca2+] levels of MCF10A MECs  in response to treatment. Fura-2-loaded MCF10A MECs  ligated with rBM  in 2D and expressing filamin  repeats 21-23 (2D+FLMNIg21-Ig23) or vector control (2D) were pre-incubated with EGTA  to chelate extracellular Ca2+ in the bath solution, challenged with 2 μM thapsigargin  to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values of individual cells were quantified over the course of imaging (mean ± SD (2D, n=8; 2D + FLMNIg21-Ig23, n=7 cells from one experiment). (J) Quantification of the amplitude of the Ca2+ response (∆F=F-F0) induced by thapsigargin, where F0 is the basal fluorescence before thapsigargin  treatment. The data shown indicated mean ± SEM (2D, n=21; 2D + FLMNIg21-Ig23, n=19 cells from three independent experiments). ****P < 0.0001 (Student's t test).",
    "molecules": "Ca2+, EGTA, Fura-2, thapsigargin"
  },
  {
    "caption": "(A) Bar graph of qPCR  data measuring the levels of SEC61B  mRNA in MECs  ligated with rBM  in 2D and treated in the absence and presence of blebbistatin  (Bleb) (mean ± SEM; n=3 independent biological replicates). **P = 0.0046 (Student's t test).",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(D) Representative time-lapse confocal microscopy  images of MECs  stably expressing LifeAct-RFP  that were ligated to a rBM  in 2D  (2D) or 3D  (3D). Time  in seconds (s) is indicated in each inset; scale bar, 10 μm.",
    "molecules": "LifeAct"
  },
  {
    "caption": "(E) Bar graph of  protrusion length  measurements in MECs  ligated to rBM  in 2D, 3D, or in 2D  treated with blebbistatin  (2D  + Bleb) (mean ± SEM; 2D, n= 139; 3D, n=106; 2D  + Bleb, n=88 protrusions  from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001. (F) Bar graph of protrusion residence time measurements in MECs ligated to rBM  in 2D (2D), 3D  (3D), or in 2D  treated with blebbistatin  (2D+ Bleb) (mean ± SEM; 2D, n= 30; 3D, n=30; 2D  + Bleb, n=38 protrusions from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(G) 30-nm thick slice through a cellular tomogram  focusing on protrusions  emanating from a vitrified no-spread MECs  cultured on a rBM  in the absence (left, 2D) or in the presence of blebbistatin  (right, 2D+ Bleb). The position of the cell body is towards the upper right-hand corner (marked with white asterisk). Scale bars, 200 nm. Note: under conditions of low cortical actin tension (blebbistatin  treatment) the protrusions  visualized in these MECs  appeared to be highly interdigitated with sharp kinks, consistent with a compliant phenotype. By contrast, the protrusions  observed in the 2D samples (higher  cortical actin tension) were predominantly straight and outwardly projected, suggesting they were stiffer than the protrusions  formed in the MECs  with lower cortical actin tension (e.g. blebbistatin  treated). (H) Quantification of membrane   protrusion width in tomograms  from non-spread MECs  interacting with rBM  treated with (2D+Bleb) and without blebbistatin  (2D). n > 2000 protrusions. Statistical significance of differences between the distributions was assessed using Mann-Whitney rank tests. ****P < 0.0001.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(C) (Top) Model of how exocyst  components (including Exo70) tether vesicles  during exocytosis. Representative fluorescence microscopy images of MECs  stably co-expressing EGFP-Exo70  (Exo70) and the plasma membrane reporter farnesylated mCherry  (PM) ligated to rBM  in 2D, 3D, and treated with blebbistatin  (2D+Bleb). Fluorescent images of whole cells are shown (left) and cell-edge protrusions are magnified within inset (right). Colocalization  of Exo70  and membrane   protrusions  are highlighted with yellow arrows. Scale bar (whole cell), 5 μm; Scale bar (magnified inset), 1 μm.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(D) Bar graph of the relative level of EGFP-Exo70  at the plasma membrane versus cytoplasm  in MECs  ligated to rBM in 2D  or 3D  in the absence and presence of blebbistatin  (Bleb). Membrane localization  of EGFP-Exo70  was quantified as a ratio of plasma membrane  to cytoplasmic  fluorescence (mean ± SEM; 2D, n= 59; 3D, n=60; 2D  + Bleb, n=57; 3D  + Bleb, n=54 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(E) 30-nm thick slices through a cellular tomogram  focusing on subplasmalemmal  regions enriched with membrane-encased compartments of vitrified MECs  cultured on rBM  treated with (2D, right panel) or without (2D+Bleb, left panel) blebbistatin. The position of the cell body  is towards the upper right-hand corners (marked with white asterisk). Scale bars, 200 nm.",
    "molecules": "Bleb, blebbistatin"
  },
  {
    "caption": "(F) Quantification of the number of membrane-enclosed compartments (vesicles) proximal to the plasma membrane  that were classified as either 'filled' (black) or 'empty' (white), based on the absence or presence of encapsulated macromolecules. Membrane-enclosed compartments were independently classified by three  cryo-EM experts using 28 tomograms  per condition. The resulting standard deviation for assigning 'empty' versus 'filled' was 10.3% (n=3 independent expert classifications). Statistical significance of differences between the distributions was assessed using Mann-Whitney rank tests. ****P < 0.0001.",
    "molecules": "macromolecules"
  },
  {
    "caption": "(H) Volcano plot showing differentially associated proteins in MECs  ligated to rBM  in 2D treated with vehicle (DMSO; blue; log 2> 0.5; high  cortical actin  tension) versus blebbistatin  (Bleb; red; log 2> 0.5; low  cortical actin tension). Differentially expressed genes with an adjusted p-value<0.1 and log2 (fold change) > 0.5 are shown (n=2 biological replicates).",
    "molecules": "Bleb, blebbistatin, DMSO"
  },
  {
    "caption": "(J) Representative  immunofluorescence microscopy images of MECs  ligated to rBM  in 2D, 3D, or 2D  treated with blebbistatin  (2D+ Bleb) and immunostained  with antibodies targeting ANXA2   or S100A14  (green). Cellular  F-actin was counterstained  using phalloidin  (red) and actin-rich protrusions  are highlighted with yellow arrows. Fluorescent images of whole cells (left) and actin-rich protrusions  containing ANXA2  or S100A14  are magnified in the inset (right). The edge of the actin cortex in MECs  ligated to rBM  in 2D  was marked with a yellow dashed line. Scale bar (whole cell), 5 μm; Scale bar (magnified insets), 1 μm.",
    "molecules": "Bleb, blebbistatin, phalloidin"
  },
  {
    "caption": "(A) Graph of the percent cell death  measured in MCF10A MECs  ligated to rBM  in 2D  and 3D  (left) and in 2D  treated with blebbistatin  (2D+Bleb) 48h post-plating (right) that was quantified using calcein AM  and ethidium homodimer staining  (mean ± SEM; 2D, n= 3; 3D, n=3 independent experiments; 2D  + DMSO, n = 5; 2D  + Bleb, n=5 independent experiments). 2D  versus 3D, ***P = 0.0010, 2D  versus 2D  + Bleb, **P = 0.0248 (Student's t test).",
    "molecules": "Bleb, blebbistatin, calcein AM, DMSO, ethidium homodimer"
  },
  {
    "caption": "(B) Bar graph of the percent cell death measured in MCF10A MECs  stably expressing shRNAs targeting Luciferase  (Luc), filamin (FLMN) or actinin  (ACTN) and ligated to rBM  in 2D. Cell death  was assessed at 48h post-plating via calcein AM  and ethidium homodimer staining  (mean ± SEM; n =3 independent biological replicates). **P = 0.0034; ns= not significant (Student's t test).",
    "molecules": "calcein AM, ethidium homodimer"
  },
  {
    "caption": "(C) Bar graph of the percent cell death in MCF10A MECs  expressing luciferase  control or filamin  repeats 21-23 (FLMNIg21-Ig23) that were ligated with rBM  in 2D. Cell death was assessed at 48h post-plating via calcein AM and ethidium homodimer staining  (mean ± SEM; n =3 independent biological replicates). *P = 0.0489 (Student's t test).",
    "molecules": "calcein AM, ethidium homodimer"
  },
  {
    "caption": "(E) Bar graph of the percent cell death measured in MECs  expressing shRNA targeting Exo70  (shExo70) or luciferase  (control) and ligated with rBM  in 2D  or 3D. Cell death was assessed at 48h post-plating via calcein AM  and ethidium homodimer staining  (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D  versus 3D, ***P = 0.0004. 3D + shLuc versus 3D  + shExo70, **P = 0.0091.",
    "molecules": "calcein AM, ethidium homodimer"
  },
  {
    "caption": "(F) Spheroids were induced to express constitutively active ROCK  or left uninduced (control) and co-stained with antibodies for laminin  or S100A14  (green), phalloidin  (red) and DAPI  (blue). Scale bar, 10 μm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(H) Spheroids were induced to express constitutively active ROCK  or left uninduced (control), treated with Paclitaxel  (2nM), lysed and immunoblotted  for cleaved caspase-3  and alpha-tubulin  (top). In parallel, spheroids were stained  with antibody and phalloidin  to evaluate the levels of caspase-3 (green) and actin  organization (red), respectively. Scale bar, 10 μm.",
    "molecules": "Paclitaxel, phalloidin"
  },
  {
    "caption": "G, Benzidine staining  of whole spleen  [top] or marrow  [bottom] from 3 month old mice  [left] or 3 year old naked mole-rats  [right]. Scale bar 250µm, arrows indicate  nucleated erythroid progenitors (NEPs).",
    "molecules": "Benzidine"
  },
  {
    "caption": "H, Relative counts of Benzidine-stained  cytospins  from whole spleen  or WBM. p-value determined by Sidak's Two-way ANOVA comparing BM  vs spleen  between mouse  (n=5) and naked mole-rat  (n=8). I, Relative counts of Benzidine-stained  cytospins from naked mole-rat   sorted spleen  fractions; p-value determined by Sidak's Two-way ANOVA comparing BM  vs spleen; n=4.",
    "molecules": "Benzidine"
  },
  {
    "caption": "C,  Mitotracker Red staining  in mouse  (n=4), human  (n=4) and  naked mole-rat  (n=5) BM, histogram of merged per-species data. unstained, BM  from mouse  [solid], human  [dotted] or NMR  [area under curve (AUC)]; yLSK, LIN-/Sca-1+/Kit+, 3 month old; oLSK, 24 month; yLTC, LIN-/Thy1.1int/CD34+, 3 year old; hHSC, human CD34+/CD38lo;",
    "molecules": "Mitotracker Red"
  },
  {
    "caption": "A Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10/300 GL preparative grade column (GE Healthcare) in buffer containing 0.15 M NaCl, and every second fraction denatured and analysed by immunoblotting with the indicated antibodies. The white dotted lines indicate that the samples were run on different polyacrylamide gels.",
    "molecules": "NaCl"
  },
  {
    "caption": "C Yeast two‐hybrid (Y2H) assays were performed with a GAL4 DNA‐binding domain fusion and/or activation domain for each protein indicated in the table to detect interaction between these proteins. Cells grown on media lacking LEU, TRP and HIS (to select for bait and prey plasmids) or tested for lacZ reporter gene activity.",
    "molecules": "HIS, LEU, TRP"
  },
  {
    "caption": "A HEK293 cells were treated with the proteasome inhibitor lactacystin (5 μM) or bortezomib (10 μM) for the times indicated. Cells were lysed in the presence of 0.1 M N‐ethylmaleimide to inhibit deubiquitinylation and lysates were subjected to immunoblotting with indicated antibodies.",
    "molecules": "bortezomib, lactacystin, N‐ethylmaleimide"
  },
  {
    "caption": "C USP45 KO U2OS cells were transiently transfected with a construct encoding FLAG‐ERCC1. Thirty‐six hours post‐transfection cells were treated with lactacystin (5 μM) for 6 h and lysed with a buffer containing 0.1 M N‐ethylmaleimide. Flag‐ERCC1 was immunoprecipitated and treated in the absence or presence of 0.1 μg of recombinant USP45 wild‐type (WT), non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] (AA) or catalytically inactive USP45 [Cys199Ala] (CA) for 1 h. The reactions were terminated by addition of sample buffer and analysed by immunoblot and Coomassie staining. Similar results were obtained in at least two separate experiments.",
    "molecules": "lactacystin, N‐ethylmaleimide"
  },
  {
    "caption": "A Clonogenic survival assays were carried out in wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA). The cells were treated with the indicated doses of mitomycin C (MMC) 16 h. Medium was changed and number of colonies quantified after 10 days. Each data point is the average of 3 experiments undertaken with 6 replicates ± SD.",
    "molecules": "mitomycin C"
  },
  {
    "caption": "E, F As in (A), except that DNA damage is induced using UV‐C irradiation (E) or 16 h hydroxyurea treatment (F). Similar results were obtained in at two separate experiments.",
    "molecules": "hydroxyurea"
  },
  {
    "caption": "A Wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA) were used to analyse ERCC1 foci formation. Staining and analysis of endogenous ERCC1 foci formation was undertaken before (left panel), or after DNA damage induction by mitomycin C (40 ng/ml, 16 h, middle panel) or UV (20 J/m2 followed by 3‐h recovery, right panel). Three independent experiments were performed in which 500 cells per experiment were analysed. Scale bar, 10 μm.B Proportion of cells displaying more than 10 endogenous ERCC1 foci were quantified. Three independent experiments were performed in which 500 cells per experiment were analysed. Results are the mean of 3 experiments ± SD.C The wild‐type and USP45 knockout U2OS cells were treated with mitomycin C (40 ng/ml, 16 h) and total number of ERCC1 staining foci per cell was quantified in at least 50 independent cells at the indicated times. Similar results were obtained in two separate experiments. The data are presented as the average number of ERCC1 staining foci per cell.",
    "molecules": "mitomycin C"
  },
  {
    "caption": "A Representative immunofluorescence microscopy images of endogenous wild‐type (WT) and USP45 knockout (KO) U2OS cells treated with no drug, BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h). Cells were fixed 10 min after laser micro‐irradiation and stained for localisation of endogenous USP45, phosphorylated γ‐H2AX and DAPI. The right‐hand panel shows magnification of one micro‐irradiated cell. Similar results were obtained in at least three independent experiments. Scale bar, 10 μm.",
    "molecules": "angelicin, psoralen"
  },
  {
    "caption": "A U2OS cells were transiently transfected with the GFP-USP45 or GFP alone. Thirty‐six hours post‐transfection cells were irradiated with UV‐C (150 J/m2) through a micropore filter (lower panels) or left untreated (upper panels). Five minutes post‐irradiation, cells were fixed and co‐immunostained with antibodies recognising cyclobutane pyrimidine dimer (CPD) DNA lesions and GFP. The white arrows indicate co‐localisation between GFP-USP45 and CPD. Scale bar, 10 μm.",
    "molecules": "CPD, DNA, cyclobutane pyrimidine dimer"
  },
  {
    "caption": "B U2OS wild‐type (WT) and USP45 knockout (KO) cells were irradiated with UV‐C (20 J/m2). At the indicated post‐irradiation times, genomic DNA was extracted and subjected to Southern dot blot analysis using antibodies recognising CPD and dsDNA total antibody. Data are shown from a triplicate experiment in which each blot is derived from independent cells.",
    "molecules": "CPD, DNA, dsDNA"
  },
  {
    "caption": "C Same as (B) but using a High Sensitivity CPD/Cyclobutane Pyrimidine Dimer Elisa kit (NM‐MA‐K001) from Cosmo Bio. For the DNA damage detection, the manufacturer protocol was followed. Absorbance at 492 was measured that represent amount of CPDs in each sample at indicated times. Calf thymus DNA, UV‐Cirradiated (10 J/m2) and not irradiated was used as positive and negative samples, respectively.",
    "molecules": "CPD, CPDs, Cyclobutane Pyrimidine Dimer, DNA"
  },
  {
    "caption": "A Live‐cell fluorescence microscopy analysis of U2OS cells stably expressing either GFPNLS empty, wild‐type (WT) USP45 or catalytically inactive USP45 [Cys199Ala]. Cells were treated with BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h) and images captured at the indicated times after laser micro‐irradiation. The location of the micro‐irradiation stripe in each cell is indicated with a yellow line. Similar results were obtained in at least two independent experiments. Scale bar, 10 μm.",
    "molecules": "angelicin, psoralen"
  },
  {
    "caption": "(D) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 (n=3) or WT EAAT2 (n=3). Cells were intracellularly dialyzed with a 115mM KGluc-based solution and held at 0 mV.",
    "molecules": "CholGluc, KGluc"
  },
  {
    "caption": "(B) Representative WT and mutant whole-cell current responses to voltage jumps before and after removal of external K+. Cells were dialyzed with a KNO3-based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3.",
    "molecules": "CholineNO3, KNO3, K+"
  },
  {
    "caption": "(C) Relative changes in fluorescence for WT, D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [K+]. Solid lines represent non-parametric fits, and shaded area show bootstrapped 99% confidence intervals for those fits. Non-overlapping confidence intervals indicate significant difference between WT and mutant binding curves. Values are given as mean ± SD (n≥3).",
    "molecules": "K+"
  },
  {
    "caption": "(D) Representative whole-cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline-NO3-based external solutions or in NaNO3-based solutions supplemented with 1 mM L-glutamate. Glutamate-induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 (GltPh D312 and D405) are K+-independent, but are functionally expressed in the plasma membrane.",
    "molecules": "choline-NO3, glutamate, Glutamate, L-glutamate, NaNO3"
  },
  {
    "caption": "(E) Current-voltage relationships of steady-state currents from cells dialyzed with a KNO3-based pipette solution. Currents were measured at external 140 mM KNO3 and normalized to the current at -170 mV from a consecutive recording of the same cell in K+-free external solutions. Values are given as mean ± SD (n≥3)",
    "molecules": "KNO3, K+"
  },
  {
    "caption": "(E) Representative transport current recordings from cells expressing L448A or L448T (GltPh A360) or WT EAAT1 upon voltage jumps to ˗140 mV before and after superfusion with 1 mM l-glutamate. Cells were intracellularly dialyzed with a K+-free solution without permeating anions to isolate K+-independent transport currents. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.",
    "molecules": "l-glutamate"
  },
  {
    "caption": "(F) Current-voltage relationship for net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for choline-based pipette solutions. Values are given as mean ± SD with indicated numbers of experiments.",
    "molecules": "choline"
  },
  {
    "caption": "(G) Time-dependent changes in L448A EAAT1 (GltPh A360) uptake currents during two repetitive glutamate applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus the time. Glutamate application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.",
    "molecules": "glutamate, Glutamate"
  },
  {
    "caption": "(E) Representative transport current recordings from a cell expressing R478A EAAT2 (GltPh R397) in the absence of internal K+. Transport currents were elicited by voltage jumps to -140mV before and after superfusion with 5 mM l-serine. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.",
    "molecules": "l-serine, K+"
  },
  {
    "caption": "(F) Time-dependent changes in steady-state R478A EAAT2 (GltPh R397) uptake currents during two consecutive serine applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus time. Serine application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.",
    "molecules": "serine, Serine"
  },
  {
    "caption": "(G) Current-voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.",
    "molecules": "l-serine"
  },
  {
    "caption": "(H) Mean net transport currents for WT and R478A EAAT2 (GltPh R397) induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.",
    "molecules": "l-serine"
  },
  {
    "caption": "High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type.",
    "molecules": "10-N-nonyl acridine orange, NAO, cardiolipin, phospholipids"
  },
  {
    "caption": "High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. H1975 cells transduced with matrix-targeted-DsRed and stained with NAO. Matrix-targeted Ds-Red differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment.",
    "molecules": "NAO"
  },
  {
    "caption": "High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel A, labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments.",
    "molecules": "NAO"
  },
  {
    "caption": "High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency. A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.",
    "molecules": "NAO, cardiolipin"
  },
  {
    "caption": "High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria from HeLa cell, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows the red and green intensities of TMRE and NAO colocalized. Scale bars = 500 nm. N = 3 independent experiments. Mitochondria in L6 myoblast, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows colocalizing NAO and TMRE at the cristae membrane (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.",
    "molecules": "NAO, TMRE"
  },
  {
    "caption": "High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria in HeLa cells, stained with NAO alone (top row) vs. NAO + TMRE (bottom row), and simultaneously excited with 488- and 561-nm lasers. Note that mitochondria stained with NAO alone do not emit noticeable fluorescence in the red channel; only after adding TMRE does strong signal appear in the red channel, showing negligible bleed-through. Scale bars = 500 nm. N = 2 independent experiments.",
    "molecules": "NAO, TMRE"
  },
  {
    "caption": "Time-lapse Airyscan imaging of ΔΨm in living HeLa cell, co-stained with MTG (ΔΨm-insensitive after loading) and TMRE (ΔΨm sensitive). Arrowhead points to a flickering event where a mitochondrion depolarizes (~9 sec) and repolarizes (~16 sec), showing that heterogeneous signal from TMRE (but not MTG) disappears and reappears. Scale bar = 500 nm. N = 4 independent experiments. Quantification of A. Plot shows average TMRE fluorescence intensity (FI) of cristae (dark red line) vs. matrix (light red line) during the time series. The drop in TMRE FI during the depolarization phase of the flickering is the ΔΨm-sensitive component of the TMRE signal. The remaining TMRE FI during depolarization can be considered as the ΔΨm-insensitive portion of TMRE signal. Note that the remaining TMRE FI after depolarization at the cristae and matrix are approximately identical, indicating that differences in TMRE FI between the cristae and matrix prior to depolarization are derived from differences in ΔΨm. N = 4 independent experiments.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "Live-cell Airyscan image of mitochondrion in HeLa cell, showing different membrane potentials in different mitochondrial regions. Membrane potentials were calculated based on TMRE FI differences between compartments (FIcomp). Regions of interest from cristae and IBM used for ΔΨm calculations are labeled with arrows on the right-hand side of the mitochondrion, using the cytosol as the reference compartment. Labeled with forks on the left-hand side of the mitochondrion are the calculations of ΔΨm between individual cristae and their neighboring IBM. Nernst equation used to calculate different voltages. Scale bar = 500 nm. LUT of mitochondrion in HeLa cell shown in A, color-coding TMRE FIs on scale of white (most intense) to blue (least intense): LUT scale shown in lower left-hand corner. Arrowheads indicate cristae. Scale bar = 500 nm. Note that most-intense pixels (white) only associate with cristae. Quantification of ΔΨm (mV) at cristae and IBM relative to the average ΔΨm of the whole mitochondrion, calculated as in A. Note: ΔΨm at cristae is significantly higher than ΔΨm at IBM, indicating electrochemical boundaries separate these two regions of the IMM. N = 3 independent experiments.",
    "molecules": "TMRE"
  },
  {
    "caption": "Live-cell Airyscan imaging of mitochondria in cultured primary mouse hepatocytes stained with TMRE. Arrowheads denote cristae. D. Image showing TMRE-labeled mitochondria. E. LUT color-coding of TMRE FI. F. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 2 independent experiments.",
    "molecules": "TMRE"
  },
  {
    "caption": "Live-cell STED imaging of mitochondria in HeLa cell stained with TMRM. Arrowheads show cristae. Scale bar = 500 nm. G. Image showing TMRM-labeled mitochondrion. H. LUT color-coding of TMRM FI. I. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 4 independent experiments. Note that calculation of ΔΨm differences between the compartments based on images captured by STED and by Airyscan were very similar.",
    "molecules": "TMRM"
  },
  {
    "caption": "Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Quantification of total mitochondrial TMRE FI. N = 3 independent experiments. Quantification of cristae ΔΨm relative to IBM (ΔΨCr-IBM) in response to oligomycin compared to FCCP. F1Fo ATP Synthase is exclusively localized to the cristae and significant increase in ΔΨCr-IBM in response to blocking of F1Fo ATP Synthase with oligomycin indicates that differences in TMRE FI between cristae and IBM are driven by oxidative phosphorylation. N = 3 independent experiments.",
    "molecules": "FCCP, MTG, oligomycin, TMRE"
  },
  {
    "caption": "Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Representative Airyscan images showing mitochondria from living HeLa cells stained with MTG and TMRE. Cells were first stained for one hour and only then treated with oligomycin or FCCP. Images show hyperpolarization of cristae under oligomycin (middle row; arrowheads) and depolarization of cristae under FCCP (bottom row; arrowheads) compared to control (top row; arrowheads). Scale bar = 500 nm. N = 3 independent experiments. Note that the ΔΨm-independent fraction of TMRE staining is less than 5% of the signal in control conditions.",
    "molecules": "FCCP, MTG, oligomycin, TMRE"
  },
  {
    "caption": "Zoomed-in region of FCCP-treated mitochondria in C. Increased contrast was used to visualize TMRE to compensate for TMRE loss induced by FCCP. These images show diminished heterogeneity of ΔΨCr-IBM values.",
    "molecules": "FCCP, TMRE"
  },
  {
    "caption": "The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that ΔΨCr-IBM is decreased. Scale bar = 500 nm. N = 3 independent experiments. Quantification of A, showing deletion of Mic13 in HeLa cells results in significant decrease in ΔΨCr-IBM. While Mic13-KO cells show a substantial decrease in cristae number, the loss of cristae does not appear absolute, making it feasible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.",
    "molecules": "TMRE"
  },
  {
    "caption": "The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied by disrupting cristae using Mic10- and Mic60-deficient cells. Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models. Representative images of Hap1 control (top row) vs. Mic60 KO (middle row) and Mic10 KO (bottom row); Mic60-KO mitochondria show very few cristae structures. TMRE staining along the IMM is relatively homogeneous, indicating decreased ΔΨCr-IBM. In general, Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM (arrowheads) as compared to control cells. Deletion of Mic10 induces detachment of cristae from IBM (see Figure 8). Except for the detached cristae vesicles, the TMRE staining is homogeneously distributed n Mic10-KO cells, indicating decreased ΔΨCr-IBM. Scale bar = 500 nm. N = 3 independent experiments. Quantification of C, showing Mic60 KO in Hap1 cells results in significant decrease in ΔΨCr-IBM. While Mic60-KO cells show a marked loss of normal cristae structures, depletion of cristae does not appear absolute, making it possible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments. Quantification of C, showing Mic10 KO in Hap1 cells results in a significant decrease in ΔΨCr-IBM. Note: quantification of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM. N = 3 independent experiments.",
    "molecules": "TMRE"
  },
  {
    "caption": "Studies show that Opa1 interacts with MICOS complex, promoting closure of CJs. Thus, we tested whether Opa1 was required to maintain the difference in ΔΨm between cristae and IBM. Live-cell Airyscan images of MEF control (top row) vs. Opa1 KO (bottom row), co-stained with NAO and TMRE. Arrowheads indicate decreased intensity of TMRE FI at cristae compared to IBM. Note the more even distribution of TMRE staining, indicating the cristae and IBM are relatively equipotential. Scale bar = 500 nm. Quantification of G, showing Opa1-KO MEFs have significantly decreased ΔΨCr-IBM. N = 3 independent experiments.",
    "molecules": "NAO, TMRE"
  },
  {
    "caption": "Live-cell Airyscan images of laser-induced depolarization time series of Mic10 KO Hap1 cell stained with TMRE. Mitochondrion exposed to rapid, high 2-photon laser power in region of white box, arrow (~0.3 sec); large, hyperpolarized vesicle (arrowhead) remains polarized after the rest of the mitochondrion loses ΔΨm, indicating electrochemical discontinuity between hyperpolarized vesicles and the rest of the mitochondrion, including the IBM. Scale bar = 500 nm. N = 2 independent experiments.",
    "molecules": "TMRE"
  },
  {
    "caption": "Laser-induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM, visualized using live-cell Airyscan microscopy. Representative images of laser-induced depolarization time series from L6 myoblast, stained with ΔΨm-dependent dye, Rho123. Images show LUT color-coded for Rho123 FI. White and blue pixels represent most and least intense ΔΨm, respectively. See legend of LUT colors on the right. Note that at the first time point (~0.15 sec) white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Ensuing frames show wavelike depolarization away from site of perturbation (arrowheads). Scale bar = 500 nm. N = 3 independent experiments. (See Movie EV 2 for time series.) Profile plot showing Rho123 pixel intensity as function of distance along mitochondrion in A. Note that zero value in the X axis corresponds to top area of the mitochondrion shown in A, near site of 2-photon laser exposure (box). Red line corresponds to frame immediately before depolarization (~0.15 sec); green, blue, purple, and dark purple lines correspond to ~0.30, ~0.60, ~1.05, and ~2.10 sec, respectively, after initial depolarization, showing wavelike dissipation of ΔΨm over time.",
    "molecules": "Rho123"
  },
  {
    "caption": "Laser-induced mitochondrial membrane depolarization in living human skin fibroblasts co-stained with TMRE and MTG and imaged with Airyscan, showing gradual depolarization of elements along the IMM. Time-lapse images of merged green (MTG) and red (TMRE) channels following laser-induced mitochondrial membrane depolarization. Note that, at the first time point (0.3 sec), white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Depolarization is marked by the dissipation of the ΔΨm-dependent dye (TMRE) while the green (MTG) signal persists. Scale bar = 1 µm. N = 4 independent experiments. TMRE channel from C, showing gradual of the loss of TMRE along the IMM. Note that although depolarization has largely completed at the area near the phototoxic stimulus already at 0.9 sec, some specific regions at the very top maintain ΔΨm while others depolarize. Quantification of ΔΨm using TMRE FI from C-D. Measurement of TMRE pixel intensities immediately after laser-induced depolarization at site distal (≥ 10 µm) vs. proximal (≤ 1 µm) to box. N = 4 independent experiments; see specific p values in panel. Quantification of C-D. Percentage of mitochondria that depolarize in a wavelike (i.e., gradual) vs. instantaneous manner after laser-induced depolarization. Note: imaging at high temporal resolution (~100-500 msec/frame) reveals wavelike depolarizations predominate, suggesting the ΔΨm is composed of multiple, disparate electrochemical domains along the IMM. The time scale of propagation of depolarization is slower than the propagation of electrical phenomena. N = 4 independent experiments.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Stability of cristae ΔΨCr-IBM over time. Representative images from time series of HeLa cells co-stained with MTG and TMRE. Top sequence shows merged green (MTG) and red (TMRE) channels. Bottom sequence shows color-coded LUT of TMRE signal of the same sequence shown above. White arrowheads in the image point to specific cristae that were measured over time. Scale bar = 500 nm. N = 5 independent experiments. Quantification of A, showing average ΔΨCr-IBM of Cristae \"A-D\" over time. Note that Cristae C and D maintain more than double the ΔΨCr-IBM of Cristae A and B. Error bars indicate STDEV. Quantification of A, showing standard deviation in ΔΨCr-IBM (mV) of individual cristae over time compared to the standard deviation in ΔΨCr-IBM (mV) among different cristae along a single mitochondrion. N = 5 independent experiments. Note that there is less variability in ΔΨCr-IBM of a single crista over time within the same mitochondrion compared to the variability of ΔΨCr-IBM among cristae over time, indicating cristae possess unique bioenergetic properties.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Representative live-cell Airyscan image from time series showing merged green (MTG) and red (TMRE) channels. Area marked by dashed box is shown in the time series in E. Scale bar = 500 nm. Time series of cropped region from D, showing tip of mitochondrion (arrowhead) maintaining ΔΨm while neighboring region of same mitochondrion depolarizes. The capacity of some cristae to maintain polarity, while adjacent cristae transiently depolarize, indicates cristae can exhibit a significant amount of electrochemical autonomy. N = 3 independent experiments. (See Movies EV 3, 4 for time series.) Zoomed-in LUT of mitochondrion from E (~2 sec), showing some cristae remaining polarized (red) while adjacent cristae depolarize (blue). (See Movie EV 5 for time series.) Quantification of D-F, showing that during a flickering event some cristae maintain their membrane potentials while other cristae depolarize, resulting in large differences in ΔΨCr-IBM along the individual mitochondrion. N = 3 independent experiments.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "A. Mutations in the kinase motif abolished the yeast toxicity of MavQ. S. cerevisiae was transformed with plasmids expressing Flag-tagged wild-type MavQ or the indicated MavQ mutants under the galactose-inducible promoter. Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A. Biochemical assays for the kinase activity of MavQ. Purified MavQ was incubated with or without specific PI substrates, the conversion of ATP to ADP was measured by the ADP-Glo assay. RLU, relative luminescence units.",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "B. H149, N152 and D160 in MavQ are critical for its kinase activity. Purified wild-type and mutant MavQ were reacted with PtdIns and ATP. The kinase activity was determined by the ADP-Glo assay.",
    "molecules": "ATP, PtdIns"
  },
  {
    "caption": "C. The PI product produced by MavQ determined by TLC assays. di-C8-Bodipy-FL-PtdIns was used to react with MavQ (lane 4) and samples were further incubated with the 3-phosphatase MTM which hydrolyzes both PtdIns3P and PtdIns(3,5)P2 (lane 5) or with the PI4K LepB_NTD which phosphorylates PtdIns3P into PtdIns (3,4)P2 (lane 6). A schematic illustration of the enzymatic reactions is shown in the lower panel.",
    "molecules": "Bodipy-FL, PtdIns (3,4)P2, PtdIns(3,5)P2, PI, PtdIns, PtdIns3P"
  },
  {
    "caption": "D. Mutations in H149, N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P.",
    "molecules": "PtdIns, PtdIns3P"
  },
  {
    "caption": "A. Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP-2xFYVEHrs and MavQ with or without wortmannin treatment. Scale bars, 5 μm.",
    "molecules": "PtdIns3P, wortmannin"
  },
  {
    "caption": "B. Percentage of HeLa cells transfected with the indicated plasmids in which GFP-2xFYVEHrs localizes to vesicle-like structures. At least 100 cells (n≥100) were counted for each sample. Wort, wortmannin.",
    "molecules": "Wort, wortmannin"
  },
  {
    "caption": "C. Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin. Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP. The wortamannin-sensitive PI3K p110α was used as a control. The ADP-Glo assay was used to measure the kinase activity.",
    "molecules": "ATP, PtdIns, wortamannin, wortmannin"
  },
  {
    "caption": "D. Wortmannin does not affect the production of PtdIns3P by MavQ. Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature. di-C8-Bodipy-FL-PtdIns and ATP were then added to the reaction mixtures. The TLC assay was employed to detect the generation of PtdIns3P by MavQ. The wortamannin-sensitive PI3K p110α was included as a control.",
    "molecules": "ATP, Bodipy-FL, PtdIns, PtdIns3P, wortamannin, Wortmannin, wortmannin"
  },
  {
    "caption": "D. Intracellular growth of L. pneumophila in macrophages. BMDMs were infected with indicated L. pneumophila strains at an MOI of 0.05. At the indicated time points, CFUs were determined by plating the saponin-solubilized lysates of infected cells on CYE plates.",
    "molecules": "saponin"
  },
  {
    "caption": "A. Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ, LepB_NTD and SidF. MavQ was reacted with di-C8-Bodipy-FL-PtdIns, resulting in the production of PtdIns3P (lane 6). The PtdIns3P-specific PI4K LepB-NTD was then added into the reaction, yielding PtdIns(3,4)P2 (lane 7). The product of the sample receiving LepB_NTD was further incubated with the 3-phosphatase SidF to yield PtdIns4P (lane 8). The lipid products were detected and visualized by TLC. A schematic illustration of the enzymatic reactions is shown in the lower panel.",
    "molecules": "Bodipy-FL, PtdIns(3,4)P2, PtdIns, PtdIns3P, PtdIns4P"
  },
  {
    "caption": "D. Expression and translocation of SidC. The lysates of L. pneumophila strains used for infection were probed for protein expression, ICDH was detected as a loading control (top panel). SidC translocation was detected by probing the saponin-soluble fractions of infected U937 cells with SidC-specific antibodies. Tubulin was probed as a loading control (bottom panel).",
    "molecules": "saponin"
  },
  {
    "caption": "D. Co-expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ. The association of the fluorescence probe GFP-2xFYVEHrs with vesicle-like structures was used to indicate the distribution of PtdIns3P in cells. At least 100 cells were scored in each sample (n≥100) (left panel). Wort, wortmannin. Expression of MavQ and SidP in the samples were shown in the right panel. EV represents empty vector.",
    "molecules": "PtdIns3P, Wort, wortmannin"
  },
  {
    "caption": "b, Representative confocal images of NeuN  (green) and Npas4  (red)  immunostaining performed 1h after the last injection of cocaine or saline (scale bar: 50 µm).",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "c, Quantifications of Npas4-positive cells (fold change normalized to the 30min saline control group) in the  NAc and dStr. Mean + SEM; n = 4 mice per group. 10 images (5 per hemisphere) per mouse for the NAc  and 10 images (5 per hemisphere) for the  dStr. p** < 0.01, unpaired Student's t-test.",
    "molecules": "saline"
  },
  {
    "caption": "c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos  and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates.",
    "molecules": "SCH, SCH-23390, SKF, SKF-38393"
  },
  {
    "caption": "d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the  D1-agonist SKF-38393  (10 µM) for 1h, 2h or 4h. Subsequently, qPCR  was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates.",
    "molecules": "HiK, potassium chloride, SKF-38393"
  },
  {
    "caption": "g, Differential induction of Npas4 through HiK and not forskolin in hiPSC-derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX  (Fsk, 10 µM; TTX  1 µM) for 1h before  qPCR analysis was performed to assess mRNA levels of Npas4  and Arc. Mean + SEM; n = 7  organoids in control condition, n = 5 organoids for  HiK, n = 5 organoids for  Fsk/TTX. One-way ANOVA with Tukey's post-hoc test, ns = not significant, p* < 0.05, p***<0.001.",
    "molecules": "forskolin, Fsk, HiK, TTX"
  },
  {
    "caption": "MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the  synapse-to-nucleus cascades mediating transcription of Npas4, Fosb  and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. a, co-incubation with the NMDA receptor blocker APV  (100 mM, applied 5min before) reveals that all IEG inductions through HiK in  MSNs depend on NDMA receptor   activity. Mean + SEM; n = 4 independent cultures as biological replicates. b, blockade of voltage-dependent calcium channels through verapamil (30 µM, 5min before) and  TTX  (1 µM, 5min, to prevent verapamil-induced bursting) leads to significant reduction in gene-inductions for Npas4 and Fosb. Mean + SEM; n =4 independent cultures as biological replicates. c, inhibition of ERK1/2 through  UO126 (10 µM, 20min before) leads to significant blockade of mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. d, inhibition of p38alpha and  p38beta through SB203580 or SB202190 respectively (both 10 µM and 20min before) leads to significant reductions in mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. e, blockade of PKA activity through H-89  (10 µM, 20min before) leads to reduced induction of Arc, unaffected induction of Fosb  and a superinduction of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. f, inhibition of calcineurin through co-application of Cyclosporin and FK506 (1 µM each, 20min before) leads to unaffected induction of mRNAs for Arc and Fosb, but significantly reduced levels of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates.",
    "molecules": "UO126, calcium, Cyclosporin, APV, H-89, HiK, SB203580, SB202190, FK506, TTX, verapamil"
  },
  {
    "caption": "MSN  cultures were stimulated with  HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of  Npas4, Fosb  and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. g, blockade of  CaMKII/IV through application of KN-93  (1 µM, 20min before) leads to significantly reduced mRNA inductions of Arc, Fosb and Npas4. Mean + SEM; n = 4 independent cultures as biological replicates. h, co-application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc  or Npas4, Mean + SEM; n = 6 independent cultures as biological replicates. ",
    "molecules": "KN-93, HiK, SKF"
  },
  {
    "caption": "investigating the role of  nuclear calcium signaling in mRNA induction of Arc, Fosb and Npas4. k,  immunohistochemical analysis  of MSNs infected with mCherry-NLS as control or the competitive  nuclear Ca2+/CaM antagonist CaMBP4-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. scale bar = 10 µm. l, MSNs expressing rAAV-mCherry-control or CaMBP4   (BP4) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, ns = not significant, p****<0.0001. n = 5 independent cultures as biological replicates. m, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the nuclear Ca2+-buffer Parvalbumin-NLS-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. n, MSNs expressing rAAV-mCherry-control or PV-NLS were treated with 55mM HiK  for 1h and  qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p** < 0.01, p****<0.0001. n = 3 independent cultures as biological replicates.",
    "molecules": "Ca2+, calcium, HiK"
  },
  {
    "caption": "e, Npas4  controls activity-dependent upregulation of genes for glutamatergic synapse  formation and  Ca2+ channels. MSNs were infected with rAAVs expressing shRNA-scrambled (control) or shNpas4 (Npas4 knockdown) on DIV5. On DIV 12, cultures were stimulated with HiK (55 mM) for 1h or 6h and mRNA levels were analysed by qPCR. mRNA level = fold change in mRNA compared to shscrambled unstimulated control. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p***<0.001. n = 5 independent cultures as biological replicates.",
    "molecules": "Ca2+, HiK"
  },
  {
    "caption": "b, cocaine-induced hyperlocomotion is attenuated by Npas4-knockdown while locomotor behaviour after saline injection is unaffected on all days. Mean + SEM; two-way ANOVA followed by Bonferroni post hoc test. p* < 0.05, p** < 0.01, ns = not significant. n = 12 - 16 animals per condition.",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "(H-I) Time-course of DCLK1  induction in response to oxLDL in mouse primary peritoneal macrophages (MPMs). MPMs  were exposed to oxLDL (50 μg/mL) for indicated time. Western blot  analysis (H) and densitometric  quantification (I) of DCLK1  were shown. GAPDH  was used as the loading control (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.",
    "molecules": "LDL"
  },
  {
    "caption": "Representative en face Oil Red O staining  (A) of Oil Red O-positive plaque lesion   area in aortas. Plaque  area was defined as percentage of total surface area of the aorta  (n=10 biological replicates).",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(E-F) Representative images of Oil Red O staining  (E) and quantification (F) of  atherosclerotic lesion in aortic roots (scale bar=250 μm, n=10 biological replicates). Plaque  area was quantified by the proportion of plaque  area to aortic root area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(G-H) Representative images of Masson's Trichrome staining  (G) and quantification (H) for collagen  deposition in aortic roots (scale bar=25 μm, n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.",
    "molecules": "Masson's Trichrome, collagen"
  },
  {
    "caption": "Representative immunofluorescence staining  images (A) of F4/80 (green) in aortic roots. Tissues were counterstained  with DAPI  (blue). Scale bar=250 μm, n=10 biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-B) Mouse primary peritoneal macrophages (MPMs) isolated from DCLK1f/f and  DCLK1MCKO mice  were challenged with oxLDL  (50 μg/mL) for 24 h. Protein levels of TNF-α  (A) and  IL-6 (B) were analyzed using ELISA  (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.",
    "molecules": "LDL"
  },
  {
    "caption": "(C-D) MPMs  isolated from DCLK1f/f and DCLK1MCKO mice  were challenged with oxLDL (50 μg/mL) for 6 h. mRNA levels of Tnf-α  (C) and  Il-6 (D) were determined via RT-qPCR (n=3 biological replicates). The values were normalized to β-actin. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.",
    "molecules": "LDL"
  },
  {
    "caption": "(N-O) MPMs  were treated as described in panel (J). Representative immunofluorescence staining  images (N) and quantification (O) of NF-κB p65  (red) translocating  into nucleus  in MPMs. Cells were counterstained  with DAPI  (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "(P-Q) Representative immunofluorescence staining  images (P) and quantification (Q) of p-p65  (red) in aortic roots. Tissues were counterstained  with DAPI  (blue). Scale bar=100 μm, n=10 biological replicates. p-p65  area was quantified by the proportion of p-p65  positive area to plaque  area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Co-immunoprecipitation of DCLK1  and IKKβ  in 293T  cells transfected with Flag-DCLK1. Flag-DCLK1  was immunoprecipitated  by anti-Flag  antibody. IgG, immunoglobulin G. (E) Co-immunoprecipitation of DCLK1  and IKKβ  in MPMs  challenged with oxLDL  (50 μg/mL) for 1 h. DCLK1  was immunoprecipitated  by anti-DCLK1  antibody.",
    "molecules": "LDL"
  },
  {
    "caption": "(B) MPMs  were pretreated with DCLK1-IN-1  (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 24 h. Protein levels of TNF-α and IL-6 were analyzed using ELISA  (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.",
    "molecules": "DCLK1-IN-1, DMSO, LDL"
  },
  {
    "caption": "(D-E) MPMs  were pretreated with DCLK1-IN-1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 1 h. Western blot  analysis (D) and densitometric  quantification (E) of IκBα, p-IKKβ  and p-p65. GAPDH, IKKβ  and p65 were used as loading controls (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.",
    "molecules": "DCLK1-IN-1, DMSO, LDL"
  },
  {
    "caption": "(H-I) MPMs  were treated as described in panel (D). Representative immunofluorescence staining  images (H) and quantification (I) of NF-κB p65  (red) translocating  into nucleus  in MPMs. Cells were counterstained  with DAPI  (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after serum removal (a)",
    "molecules": "protein"
  },
  {
    "caption": "(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after treatment with rapamycin (rapam) or thapsigargin (thapsig) (b).",
    "molecules": "protein, rapamycin, thapsigargin"
  },
  {
    "caption": "(c) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl.",
    "molecules": "NH4Cl, protein"
  },
  {
    "caption": "(d) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA).",
    "molecules": "3-MA, 3-methyladenine, protein"
  },
  {
    "caption": "(f,g) Rates of protein degradation in wild-type and HD MEFs (f) and striatal cells (g) in the absence (control) or presence (Atg7−) of Atg7 RNA interference.",
    "molecules": "protein"
  },
  {
    "caption": "(b) Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "(a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503.",
    "molecules": "lipids"
  },
  {
    "caption": "(c-e) Neutral lipids in striatal cells from 7Q-htt and 111Q-httmice (c), grown on a monolayer of their own astrocytes",
    "molecules": "lipids"
  },
  {
    "caption": "(c-e) Neutral lipids in primary striatal neurons from 18Q-htt and 111Q-httmice grown on a monolayer of their own astrocytes (d)",
    "molecules": "lipids"
  },
  {
    "caption": "(c-e) Neutral lipids in lymphoblasts from a control (UC) and HD-affected human (e) stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b.",
    "molecules": "lipids"
  },
  {
    "caption": "MEFs from 18Q-htt and 111Q-httmice (e) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e. CCCP was added to control cells in e as a positive control for depolarization. *P 0.05.",
    "molecules": "CCCP"
  },
  {
    "caption": "(f,g) Striatal cells from 7Q-htt and 111Q-htt mice untreated or treated with vinblastine were co-stained for LC3 and Mitotracker (f). Arrows, colocalization events. Extended study in Supplementary Figure 16.",
    "molecules": "vinblastine"
  },
  {
    "caption": "(f,g) Striatal cells from 7Q-htt and 111Q-httmice untreated or treated with vinblastine were co-stained for LC3 and BODIPY 493/503 (g). Arrows, colocalization events. Extended study in Supplementary Figure 16.",
    "molecules": "vinblastine"
  },
  {
    "caption": "(B) Met-Phe formation monitored upon rapidly mixing initiation complexes (80S IC) with ternary complexes eEF1A-GTP-[14C]Phe-tRNAPhe in the absence (orange, 0.13±0.02 s-1) or presence (cyan, 0.15±0.02 s-1) of eEF3 (2 µM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1. Data are presented as mean ± SEM of n = 3 biological replicates. (C) Met-Phe-Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green, 0.15±0.01 s-1), eEF2 (red) or eEF3 (blue). Data presented as mean ± SEM of n = 3 biological replicates. (D) Met-Phe-Val formation monitored with 80S 2C incubated with eEF2 and eEF3 (green, 0.4±0.14 s-1), eEF2 (red, 1.0±0.5 s-1) or eEF3 (blue), or in the absence of eEF2 and eEF3 (brown) for 15 min before adding with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal and monitoring the kinetics of Val incorporation. Data presented as mean ± SEM of n = 3. Data information: Datasets in (C) and (D) are normalized to Met-Phe-Val formation in presence of eEF2 and eEF3 with the maximum value set to 1. ",
    "molecules": "14C, GTP, Met, Phe, tRNAPhe, tRNAVal"
  },
  {
    "caption": "(B) mRNA-tRNA translocation monitored in a time-resolved Pmn assay upon mixing 80S 2C with Pmn in the absence (brown triangles) or presence of eEF2 and eEF3 (green, 0.13±0.01 min-1), eEF2 (red, 0.030±0.005 min-1) or eEF3 (blue, 0.10±0.01 min-1). Data are normalized to Met-Phe-Pmn formation in presence of eEF2 with the maximum value set to 1. Data presented as mean ± SEM of n = 3.",
    "molecules": "Met, Phe, Pmn"
  },
  {
    "caption": "(C) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet(Flu) upon rapidly mixing of 80S 2C with buffer (grey), or with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green), eEF2 (red), eEF3 (blue), or eEF2 and eEF3 without the ternary complex (magenta) in a stopped-flow apparatus. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.",
    "molecules": "14C, GTP, tRNA, tRNAfMet, tRNAVal"
  },
  {
    "caption": "(D) tRNAfMet(Flu) co-eluting with 80S 2C incubated without factors (orange), with eEF2 (red), with eEF2 and eEF3 (magenta), or with eEF2, eEF3 and TC-Val (green) for 15 min before loading on a BioSuite 450 size-exclusion column to separate ribosome-bound and -unbound tRNA. Data are normalized to tRNAfMet(Flu) bound to 80S 2C in the absence of eEF2/eEF3 with the maximum value set to 100%. Data presented as mean ± SEM of n = 3.",
    "molecules": "tRNA, tRNAfMet"
  },
  {
    "caption": "(E) Dissociation of tRNAfMet(Flu) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 (green) or eEF2 (red) for 15 min before adding with ternary complexes. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.",
    "molecules": "tRNA, tRNAfMet"
  },
  {
    "caption": "(F) Codon-specific ribosome occupancies for 21 nt RPFs from WT and eEF3d cells (two biological replicates). CGA and AGG codons are shown in red and purple, respectively. Variance for each dataset is indicated.",
    "molecules": "AGG, CGA, Codon, codons"
  },
  {
    "caption": "(G) Scatter plot of codon-specific occupancies for 28 nt RPFs comparing eEF3d to WT cells from libraries prepared with CHX+ANS. Trend line (blue) and its slope are shown. Codons that encode proline and glycine are colored in purple and green, respectively.",
    "molecules": "Codons, ANS, CHX, glycine, proline"
  },
  {
    "caption": "B. Representative E-cadherin (E-cad) and DAPI immunofluorescence images of AECs  treated apically with TGFβ1  or vehicle control after 72 h in the co-culture system (scale bars 25 µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "E.  ChIP-qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5-OZ (mean + s.d., n = 3 biological replicates). Unspecific  IgG was used as negative control.",
    "molecules": "5-OZ"
  },
  {
    "caption": "B. Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows abolition of TGFβ1-induced profibrotic transcriptional complex of EZH2/POL2/actin upon the convergent treatment of TGFβ1 and  Y27632. Unspecific IgG binding was used as a negative control. Representative from 3 biological replicates is shown.",
    "molecules": "Y27632"
  },
  {
    "caption": "A. Simple western analysis  (Peggy Sue) and quantifications (right panels) of mouse lung epithelial cells shows increased ph-EZH2 (T311), ph-TAK1, myosin activity (ph-MLC2) and POL2-K7m levels in AAV-mediated TGFβ1  over-expression. Note, increased ph-EZH2 and POL2-K7m levels are attenuated by the EZH2 inhibitor  GSK126, whereas ph-TAK1 and ph-MLC2 levels cannot be rescued by GSK126. Quantifications (right panels) show violin plots, *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant, Kruskal-Wallis/Dunn's. Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12  AAV-TGFβ1  and 13 GSK126-treated AAV-TGFβ1 mice.",
    "molecules": "GSK126"
  },
  {
    "caption": "B. Representative of 3D computed tomography (CT) reconstruction of the lung from control, AAV-TGFβ1 and  GSK126-treated AAV-TGFβ1 mice (green: lung tissue, red: airways, and region-of-interest (ROI): blue). Insets show µCT slices in the middle of the  lung from respective mice. Note, GSK126 attenuates TGFβ1-induced lung injury. Quantification (right panel) shows mean  intensity of ROIs from the whole lung (violin plots, *p = 0.0385, **p = 0.0012, ANOVA/Holm-Sidak's). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12  AAV-TGFβ1 and 13 GSK126-treated AAV-TGFβ1 mice.",
    "molecules": "GSK126"
  },
  {
    "caption": "D. Immunofluorescence analysis of KRT5 as a marker for alveolar metaplastic basal cells and ph-EZH2 (scale bars 100 µm), pro-SFTPC as a marker for alveolar type 2 epithelial cells (scale bars 50 µm) and quantifications (right panels) show percentage of KRT5+ pods area per 10X field and percentage of pro-SFTPC+ cells per 20X field (*p < 0.05, unpaired t-test). Data information: All violin plots display minimum, first quartile, median, third quartile and maximum; n = 5 control, 12 AAV-TGFβ1 and 13 GSK126-treated  AAV-TGFβ1 mice. ",
    "molecules": "GSK126"
  },
  {
    "caption": "(F) Brain sections of WT and KO  mice at E16.5 were immunostained  with the  mitotic marker pH3 and DAPI. Scale bar represents 20 μm. (G) Statistics of pH3+ cells of the cortex. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Representative images of E16.5 coronal brain sections immune stained for Tbr2 and  BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2h of  pulse labeling. Scale bar represents 20 μm. (B) Statistics of TBR2+ cells per 100μm2 surface of  VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared to WT  mice. n=6  mice, independent replicates. (C) Statistics of BrdU+ cells per 100μm2 surface of  VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.",
    "molecules": "BrdU"
  },
  {
    "caption": "(E) Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained  with anti-BrdU and anti-Ki67 in WT mice and KO mice  that were administered BrdU (100 mg/kg) for 24 h and harvested at E16.5. Scale bar represents 20 μm. (F) Percentage of  cell cycle exit (BrdU+Ki67−/ BrdU+) in WT  mice and KO mice. n=7 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.",
    "molecules": "BrdU"
  },
  {
    "caption": "(D) Graph bars of the normalized clearance in A.U. of iOVCAR8 (left) and iSKOV3 (right) spheroids after the indicated treatments. The two cell lines show different clearance capacity. For each cell line, three independent experiments were performed. For iOVCAR8, we analyzed 47 spheroids in DMSO and 56 spheroids in centrinone. For iSKOV3 we analyzed 56 spheroids in DMSO and 48 spheroids in centrinone. Dots represent the mean and the shadow±SEM. Statistical significance was assessed with an ANCOVA test. Dots represent the mean and the shadow±SEM. Statistical significance was assessed with an ANOVA test.",
    "molecules": "centrinone, DMSO"
  },
  {
    "caption": "(F) Representative images of DMSO (left) and centrinone (right) spheroids. Mean±SD. Red nuclei represent false colored invading nuclei. (G) Dot plot showing the quantification of the number of nuclei detected on the bottom side of the BM. Three or four positions from three BM inserts were analyzed in each condition. Mean±SD. Statistical significance was assessed with the Mann-Whitney test. For all the experiments, the centrosome number was verified in parallel to confirm the decreased centrosome conditions compared to DMSO.",
    "molecules": "centrinone, DMSO"
  },
  {
    "caption": "C. Western blot for POT1 and DDR markers upon POT1 deletion induced with 0.5 μM 4-OHT. Cl75 - parental cell line. gRNA1 - POT1 knock-out in the population by transient transfection with pSpCas9(BB)-2A-puro containing gRNA1 sequence. EV - corresponding empty vector.",
    "molecules": "4-OHT, puro"
  },
  {
    "caption": "D. Time course of telomere length changes upon POT1 removal in clone 35. d (days) TP0 - time point 0, no 4OHT.",
    "molecules": "4OHT"
  },
  {
    "caption": "F. Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35. The bars show percentage of cells containing more than 5 TIFs ± SD. Experiments were performed in triplicate. At least 120 cells were analyzed per condition per replicate. Significance was determined using two-way ANOVA. p-values are indicated on the graph.",
    "molecules": "EdU"
  },
  {
    "caption": "A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate.",
    "molecules": "DNA"
  },
  {
    "caption": "B. 2D gels for telomeric DNA from clone 35 cells with POT1 (NT) and upon POT1 removal for 7 days (7d 4OHT).",
    "molecules": "4OHT, DNA"
  },
  {
    "caption": "C. Examples of metaphase spreads for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. White arrowheads indicate fragile telomeres, the red arrowhead indicates a sister-chromatid fusion. Scale bar equals 6 µm. D. Quantification of fragile telomeres (left graph), sister chromatid fusions (middle graph) and chromosome fusions (right graph) as a percentage of events per metaphase. Experiments were performed in triplicate. At least 25 metaphases were examined per condition per replicate. The mean is displayed and error bars represent ± SD. Significance was determined using one-way ANOVA. p-values are indicated on the graph. ",
    "molecules": "4-OHT"
  },
  {
    "caption": "E. Quantification and representative images of anaphases with bridges and micronuclei for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Scale bar equals 6 µm. Experiments were performed in triplicate. The bars show percentage of cells containing anaphases, anaphases with bridges or micronuclei + SD. At least 650 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT.",
    "molecules": "4-OHT"
  },
  {
    "caption": "B. Quantification of precipitated telomeric DNA in 2-step QTIP (% of input) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA (based on dot blot analyses). Bars represent data from three independent experiments ± SD.",
    "molecules": "DNA"
  },
  {
    "caption": "C. Scatter plots representing immunoprecipitation specificity (2-step QTIP/IgG ratio) versus difference between non treated cells (NT) and cells at day 4 upon 4-OHT induction (4d 4OHT/NT ratio) or difference between non treated cells (NT) and cells at day 7 upon 4-OHT induction (7d 4OHT/NT ratio) respectively (data from Table EV1). MRN complex in red, 9-1-1 complex in blue, shelterin complex in orange except for POT1 in black. Values are the average of the 3 biological replicates.",
    "molecules": "4-OHT, 4OHT"
  },
  {
    "caption": "D. Heat map representing changes in levels of selected proteins involved in DNA damage (MRN complex in red, 9-1-1 complex in blue) and HDR proteins (purple) after POT1 knockout induction. Proteins marked with * pass t-test, but not t-test with Benjamini-Hochberg correction (p < 0.05, q < 0.09). Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.",
    "molecules": "4-hydroxytamoxifen, 4-OHT"
  },
  {
    "caption": "B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L (pulsed-field gel electrophoresis).",
    "molecules": "GRN163L"
  },
  {
    "caption": "C. DRIP assay for DNA-RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9.6 antibody. Dot blots represent two replicates. Bars represent data from three independent experiments ± SD. The IgG signal was subtracted. Significance was determined using one-way ANOVA, p-values are indicated on the graph.",
    "molecules": "DNA, RNA"
  },
  {
    "caption": "D. C-circle (CC) assay for clone 35 upon POT1 removal. Bars represent data from three independent experiments ± SD. DNA from ALT positive cell line U2OS was used as positive control. Significance was determined using one-way ANOVA, p-values are indicated on the graph. In all experiments, POT1 deletions was induced with 0.5 μM 4-OHT for 4 and 7 days.",
    "molecules": "4-OHT"
  },
  {
    "caption": "F. Quantification and representative pictures for visualization of the PML protein (IF, green) and telomeres (FISH, red) in non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Arrows indicate co-localizations. Scale bar equals 10 µm. Bars represent data from three independent experiments ± SD. At least 150 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT. Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells, GRN163L - telomerase inhibitor Imitelstat, siNT -non-tagreting siRNA, negative control.",
    "molecules": "4-hydroxytamoxifen, 4-OHT, GRN163L, Imitelstat"
  },
  {
    "caption": "(A) Time series of TIRF microscopyimages showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).",
    "molecules": "Alexa568"
  },
  {
    "caption": "(B) individual and dual colour kymographs showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).",
    "molecules": "Alexa568"
  },
  {
    "caption": "(C) Merged triple colour and single fluorescence channel kymographs showing microtubule end tracking of GFP-dynein (green in merge) and Alexa647-EB3 (red in merge) on dynamic Alexa568-microtubules (blue in merge). Concentrations were 10 nM GFP-dynein, 10 nM human dynactin, 10 nM Alexa647-EB3, and 17.5 µM tubulin.",
    "molecules": "Alexa568, Alexa647"
  },
  {
    "caption": "(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of either dynactin (D) . Concentrations of the proteins present as in A.",
    "molecules": "Alexa568"
  },
  {
    "caption": "(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of EB proteins (E). Concentrations of the proteins present as in A.",
    "molecules": "Alexa568"
  },
  {
    "caption": "(G) Kymographs of GFP-dyneinmicrotubule end tracking in the absence of ATP (in contrast to all other experiments that contain ATP). Other conditions as in A. For merged kymographs, microtubule plus and minus ends are labelled by (+) and (−). Experiments were performed at 30°C.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Merged triple colour and single fluorescence channel TIRF microscopy kymographs of GFP-dynein showing end tracking, processive motility and diffusive and static binding in the presence of all DDB components and EB1. Protein concentrations are 10 nM GFP-dynein (green in merge), 20 nM human dynactin, 20 nM EB1, 200 nM Alexa647-BicD2-N (magenta in merge), and 17.5 µM Alexa568-tubulin (blue in merge). Alexa647-BicD2-N often co-localises with processively moving and occasionally with statically bound GFP-dynein, but not with plus end-tracking GFP-dynein.",
    "molecules": "Alexa568, Alexa647"
  },
  {
    "caption": "(B) Averaged fluorescence intensity profiles of GFP-dynein (green squares) and Alexa647-BicD2-N (magenta circles) at growing microtubule plus ends. Mean values from three separate experiments per condition (with a total of at least 75 kymographs) are shown; error bars are s. e.",
    "molecules": "Alexa647"
  },
  {
    "caption": "(C, D) Dual and single colour kymographs of an Atto565-microtubule (magenta) in the presence of GFP-dynein (green), human dynactin and EB1, and either (C) in the absence or (D) presence of 5 µM BicD2-N; other conditions as in A. The increased BicD2-N concentration in D compared to A strongly reduces microtubule plus end tracking of GFP-dynein.",
    "molecules": "Atto565"
  },
  {
    "caption": "(A) Example dual colour kymograph and a schematic showing how the distance of initiation of processive runs was measured. The protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 5 µM BicD2-N, 20 nM EB1 and 17.5 µM Atto647-tubulin.",
    "molecules": "Atto647"
  },
  {
    "caption": "(D) Histogram of spatial initiation probabilities of DDB on static GMPCPP-microtubules. 294 initiation positions were detemined (from three experiments). Concentrations of DDB components as in B.",
    "molecules": "GMPCPP"
  },
  {
    "caption": "A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (A) 10 nM GFP-dynein (green) tracking the growing end of a Atto565-microtubule (magenta) in the presence of pig dynactin at an elevated concentration of 80 nM. Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).",
    "molecules": "Atto565"
  },
  {
    "caption": "(A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (B) 10 nM GFP-dynein (green) tracking the plus end of an Atto565-microtubule (blue) in the presence of 10 nM Alexa647-p150 (a fragment containing the first 517 amino acids of p150; called p150-N in the text) (red). Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).",
    "molecules": "Alexa647, Atto565"
  },
  {
    "caption": "(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (C) In standard assay buffer, 10 nM Alexa647-p150 (green) accumulates at the plus end and on the GMPCPP-stabilised segment of a growing Atto565-microtubule (magenta).",
    "molecules": "Alexa647, Atto565, GMPCPP"
  },
  {
    "caption": "(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (D) At higher ionic strength (BRB80 supplemented with 60 mM KCl, see Methods), Alexa647-p150 (green) binds only weakly to the microtubule without a detectable plus end preference. Protein concentrations as in C. The Atto565-tubulin concentration was always 17.5 µM tubulin. The temperature was 30°C.",
    "molecules": "Alexa647, Atto565, KCl"
  },
  {
    "caption": "(A-C) Kymographs showing GFP-dynein (green) on dynamic Atto647N-microtubules (magenta) in the presence of all the regulators at different Lis1 concentrations. Protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 20 nM EB1, 5 µM BicD2-N, and (A) either no Lis1, (B) 1 μM mCherry-Lis1, or (C) 5 μM mCherry-Lis1. Together with BicD2, Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus-end localisation of dynein in the presence of BicD2-N. Microtubule orientation as indicated.",
    "molecules": "Atto647N"
  },
  {
    "caption": "C Size determinations of mCFU-E and BaF3-EpoR cells. Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective. The bar respresents 10 µm distance (upper panel). Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of mCFU-E and BaF3EpoR cells. All cells were growth-factor deprived and unstimulated.",
    "molecules": "Calcein, DRAQ5"
  },
  {
    "caption": "GTP-Ras (F) and ppERK were determined upon stimulation with indicated, color-coded Epo doses. pEpoR was analyzed by immunoprecipitation followed by immunoblotting, GTP-Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting. For pAkt and ppERK, cellular lysates were subjected to quantitative immunoblotting. Calibrator proteins were used for EpoR, AKT, GTP-Ras and ERK to facilitate the conversion to nM concentrations. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.",
    "molecules": "GTP"
  },
  {
    "caption": "A Epo-induced signaling upon AKT or MEK inhibitor treatment. Growth-factor deprived cells were pretreated for half an hour with AKT VIII or U0126, respectively, and subsequently stimulated with 5 U/ml Epo. PDI served as loading control.",
    "molecules": "AKT VIII, U0126"
  },
  {
    "caption": "C Analysis of cell-cycle indicator genes upon AKT VIII and U0126 treatment. Growth-factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor, followed by stimulation with 5 U/ml Epo for 0 h and 3 h. The expression of cyclinD2, cyclinG2 and p27 was measured by quantitative RT-PCR and normalized to the Rpl32 gene. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, n.s. not significant, * p<0.05, ** p<0.01, *** p<0.005.",
    "molecules": "AKT VIII, U0126"
  },
  {
    "caption": "B Size determinations of hCFU-E compared to mCFU-E cells. Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of hCFU-E and mCFU-E cells. All cells were growth-factor deprived and unstimulated.",
    "molecules": "Calcein, DRAQ5"
  },
  {
    "caption": "F Model prediction and experimental validation of proliferation upon single or combined inhibitor treatment with AKT VIII and U0126 upon 5 U/ml Epo stimulation. Maximum proliferation was scaled to 1. Proliferation was measured as cell numbers, counted with a hemocytometer after 96 hours by trypan blue exclusion assay. Solid lines represent model trajectories. Experimental data is represented as mean ± standard deviation. N=3",
    "molecules": "AKT VIII, U0126"
  },
  {
    "caption": " D-F Validation of TMEM41B. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non-targeting (NT) control, treated with 500 nM AZD8055, 50nM Bafilomycin A1 (BafA1) or DMSO vehicle control for 24 h, and analyzed by immunoblotting. (E, F) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells (E), or as flux by calculating the ratio in BafA1-treated cells versus vehicle control (F). Data are presented as mean ± SD (n=6 independent experiments) with paired t-test values ",
    "molecules": "AZD8055, BafA1, Bafilomycin A1, DMSO"
  },
  {
    "caption": " A H4 Cas9 cells were infected with sgRNAs targeting ATG7, TMEM41B or NT control, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting. B Ratio of LC3-II to LC3-I band intensities are depicted as mean ± SD (n=6 independent experiments) with Wilcoxon test values ",
    "molecules": "AZD8055"
  },
  {
    "caption": " J mCherry-GFP-LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h, fixed and imaged. Scale bar: 20 μm. The number of mCherry- and GFP-positive puncta per cell was quantified using YAS and are depicted as mean ± SD (n=3 technical replicates) ",
    "molecules": "BafA1"
  },
  {
    "caption": " A H4 Cas9 cells stably expressing NT, TMEM41B or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope. Puncta area per cell was quantified using YAS and depicted as mean ± SD (n=4 technical replicates). Scale bar: 20 μm ",
    "molecules": "BODIPY, BODIPY 493, FL C12, cholesterol"
  },
  {
    "caption": " B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA-conjugated oleic acid or 0.1% BSA as vehicle control, stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope. Scale bar: 20 μm ",
    "molecules": "LipidTox, Lipid, oleic acid"
  },
  {
    "caption": " C Lipid droplet size, number and mean fluorescence intensity was quantified using Harmony software. Data are presented as mean ± SD (n=3 technical replicates). An average of 1500 cells was analyzed per replicate ",
    "molecules": "Lipid droplet"
  },
  {
    "caption": " A H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12, chased in complete medium or upon serum-deprivation, and stained with MitoTracker. Cells were imaged live with an automated CV7000 confocal microscope. Scale bar: 20 μm ",
    "molecules": "Red C12, fatty acid, MitoTracker"
  },
  {
    "caption": " C-E H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96. (C) Basal oxygen consumption rates (OCR) and extra cellular acidification rates (ECAR) were measured in cells grown in complete medium. OCR/ECAR ratio is also reported. Data are presented as mean ± SEM (n=30-32 technical replicates) from one representative experiment ",
    "molecules": "oxygen"
  },
  {
    "caption": "(D) OCR was measured in cells grown in substrate-limited medium and treated in the absence or presence of Etomoxir (Eto). Oligomycin (OA), FCCP (FC) and a mixture of rotenone and antimycin A (RA) was added as indicated. Data are shown as mean ± SEM (n= 69 technical replicates)",
    "molecules": "antimycin, FCCP, Etomoxir, Oligomycin, rotenone"
  },
  {
    "caption": "(E) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto-treated cells from untreated cells. Data are shown as mean ± SEM (n= 69 technical replicates)",
    "molecules": "Eto, FA"
  },
  {
    "caption": "Typical images of Hematoxylin&Eosin (H&E - upper images) and Ki67 expression (Ki67 - bottom images) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in H. 4X and 20X magnification are shown. Right graph reports the percentage of Ki67 positive cells. Each dot represents one tumor. Data are expressed as mean (±SD) and unpaired t-test was used to verify the statistical significance.",
    "molecules": "Eosin, Hematoxylin"
  },
  {
    "caption": "qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in control (shCTR) and antimiR-9 FaDu cells treated with Cetuximab (CTX) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA test was used to verify the statistical significance.",
    "molecules": "Cetuximab, CTX"
  },
  {
    "caption": "Graph reporting the cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of Gefitinib as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and unpaired t-test was used to verify the statistical significance per each dose.",
    "molecules": "Gefitinib"
  },
  {
    "caption": "Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib (GEFI - left panel) or Cetuximab (CTX - right panel) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.",
    "molecules": "Cetuximab, CTX, GEFI, Gefitinib"
  },
  {
    "caption": "Typical images of Hematoxylin&Eosin (H&E) (C) and Ki67 expression (Ki67) (D) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in A/B.",
    "molecules": "Eosin, Hematoxylin"
  },
  {
    "caption": "Kaplan-Mayer curve evaluating the overall survival of HNSCC patients treated with RT+Cetuximab (CTX) combination included in the TCGA dataset, segregated on the expression of miR-9 in the primary tumor (Low expression <75819 reads n=8; High expression ≥75819 reads n=23). Number of evaluated samples (n) and p value are reported in the graph. Statistical significance was calculated with long-rank test.",
    "molecules": "Cetuximab, CTX"
  },
  {
    "caption": "Kaplan-Mayer curve evaluating the progression free survival of HNSCC patients treated with RT+Cetuximab (CTX) combination at the CRO-Aviano National Cancer Institute and at the University Cattolica segregated based on miR-9 expression in primary tumors, defined as the expression in above (high expression n=18) or below (low expression n=17) the median expression, as defined by ddPCR. Hazard Ratio (HR) and statistical significance were calculated with long-rank (Mentel-Cox) test and are reported in the graph.",
    "molecules": "Cetuximab, CTX"
  },
  {
    "caption": "E. Immunoblot  (IB) analysis of stable Ub   replacement cell lines treated or not with DOX  for 72 h. Data information: Data are representative of three (E independent experiments with similar outcome.",
    "molecules": "DOX"
  },
  {
    "caption": "D. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines were transfected with shUb-resistant Ub(G76V)-GFP construct and treated or not with  p97i for 4 h. Cell extracts were subjected to GFP IP followed by  immunoblotting  with indicated antibodies. Data information: Data are representative of three independent experiments with similar outcome.",
    "molecules": "DOX"
  },
  {
    "caption": "E. Immunoblot  analysis of DOX-treated U2OS/shUb/HA-Ub cell lines treated or not with MG132 for 4 h. Data information: Data are representative of three independent experiments with similar outcome.",
    "molecules": "DOX, MG132"
  },
  {
    "caption": "G. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines transfected with indicated Ub(G76V)-GFP expression constructs were subjected to  GFP IP under denaturing conditions followed by immunoblotting  with indicated antibodies. Data information: Data are representative of four (G) independent experiments with similar outcome.",
    "molecules": "DOX"
  },
  {
    "caption": "A. FLAG IPs from U2OS  cells transfected with indicated FLAG- tagged expression constructs were incubated with purified K27 di-Ub and analyzed by immunoblotting. Data information: Data are representative of three (A independent experiments with similar outcome.",
    "molecules": "di-Ub"
  },
  {
    "caption": "I. U2OS/Ub(G76V)-GFP and U2OS/Ub(G76V)-20AA-GFP  cell lines were transfected or not with FLAG-UCHL3  C95S expression construct, treated with MG132  for 2 h and subjected to GFP IP  followed by immunoblotting. Data information: Data are representative of three independent experiments with similar outcome.",
    "molecules": "MG132"
  },
  {
    "caption": "H. Scatter diagram of lncRNA-seq in HCT116/L-OHP and HCT116 cells.",
    "molecules": "L-OHP"
  },
  {
    "caption": "B. PCR analysis of  lnc-AP in serum samples from  oxaliplatin-resistant (PD, n = 15) and -sensitive (PR, n = 9) CRC patients (mean±SEM, t test).",
    "molecules": "oxaliplatin"
  },
  {
    "caption": "E. PCR  and WB analysis of  HEK293T cells transfected with either  lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). F. PCR  and WB analysis of HCT116/L-OHP  and SW480/L-OHP cells transfected with either lnc-AP-Flag OE plasmid (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.",
    "molecules": "L-OHP"
  },
  {
    "caption": "J.  IF detection of  pep-AP in HCT116/L-OHP and  SW480/L-OHP cells transfected with either HA-lnc-AP OE plasmid. Scale bar, 10 µm.",
    "molecules": "L-OHP"
  },
  {
    "caption": "B. CCK-8 assay of inhibition ratio by L-OHP in two groups of  CRC cells with down- or up-regulation of  lnc-AP  (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.",
    "molecules": "L-OHP"
  },
  {
    "caption": "C.  IF detection of H2AFX for  DNA damage by L-OHP in two groups of CRC cells with down- or up-regulation of   lnc-AP, and nuclei were stained with DAPI (n = 3, biological replicates, mean±SEM, Dunnett-t test).",
    "molecules": "DAPI, L-OHP"
  },
  {
    "caption": "J. CCK-8 assay of inhibition ratio by L-OHP in  HCT116 and  SW480 cells with  lnc-AP/pep-AP attenuation and restoration (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.",
    "molecules": "L-OHP"
  },
  {
    "caption": "F.  IF detection of the colocalization of pep-AP-Flag and  TALDO1 in  HCT116/L-OHP and SW480/L-OHP cells. Scale bar, 10 µm.",
    "molecules": "L-OHP"
  },
  {
    "caption": "G. WB confirmation of the  interaction  between HA-pep-AP and TALDO1 in HCT116/L-OHP and SW480/L-OHP cells.",
    "molecules": "L-OHP"
  },
  {
    "caption": "D. Cellular ROS was measured by flow cytometry and shown in both curve and bar charts (n = 3, biological replicates, mean±SEM, t test) Data information: * for P < 0.05; ** for P < 0.01. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.",
    "molecules": "ROS"
  },
  {
    "caption": "E. MMP detection. Scale bar, 10 µm. F. IF detection of H2AFX for DNA damage by L-OHP. Scale bar, 10 µm. ",
    "molecules": "L-OHP"
  },
  {
    "caption": "E. Cellular  ROS was measured by flow cytometry and shown in both curve (left) and bar (right) charts (n = 3, biological replicates, mean±SEM, Dunnett-t test) Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.",
    "molecules": "ROS"
  },
  {
    "caption": "C. Flow cytometry of the cell apoptosis ratio under  L-OHP with down- or up-regulation of  lnc-AP/pep-AP in two groups of CRC cells, which was quantified in bar charts on the right (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA.",
    "molecules": "L-OHP"
  },
  {
    "caption": "E. CCK-8 assay of the inhibition ratio by L-OHP  with inhibitors of apoptosis Z-VAD-FMK or ferroptosis Fer-1 in HCT116/L-OHP and SW480/L-OHP cells (n = 3, biological replicates, mean±SEM). Data information: * Control referred to the corresponding plasmid vector",
    "molecules": "Z-VAD-FMK, Fer-1, L-OHP"
  },
  {
    "caption": "G. Relative NADPH/NADP+  ratio within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). H. GSH contents within the tumors in C(a) (n = 6, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector.",
    "molecules": "GSH, NADP+, NADPH"
  },
  {
    "caption": "Representative composite immune-fluorescent images of whole hair follicle units from non-lesional and lesional scalp psoriasis patients. K15 (green), c-JUN (red A, C) and JUNB (red B, D) and DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "Confocal images of the bulge region of human psoriatic hair follicles from non-lesional and lesional regions of the scalp. K15 (green), c-JUN (red E, G) and JUNB (red F, H) and DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "Intravital confocal time-lapse imaging of non-mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO*-mT/mG at day 7 after first tamoxifen injection. Dotted circles emphasize areas in which mutant GFP+ keratinocytes are replaced by non-mutant Tomato+ keratinocytes.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Fluorescence imaging of DKO*-mT/mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP+ keratinocytes reside along the hair follicles. White dotted line separates epidermis and dermis. White arrows represent GFP+ hair follicles.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Intravital confocal time-lapse imaging of DKO*-mT/mG at day 15 after first tamoxifen injection. Mutant GFP+ Keratinocytes (white arrows) are maintained around hair follicles.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative images of hematoxylin and eosin (H&E) staining of ear sections from control, DKO* and DKO*K15 mice.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Representative immunofluorescence images of ear sections from CoK15-mT/mG and DKO*K15-mT/mG mouse models after 15 days of induction. Red (Tomato), green (GFP) and blue (DAPI). Hyperplasia of Tomato+ keratinocytes in psoriatic-like DKO*K15-mT/mG mouse with clusters of GFP+ keratinocytes are derived from hair follicle K15+ GFP+ stem cells. n=3 per group. Dotted lines separate epidermis and dermis. Quantification analysis of cleaved Caspase-3 (cCas3) and Ki67 in ear skin of DKO*K15-mT/mG mice at different time points during psoriasis-like disease progression.",
    "molecules": "DAPI"
  },
  {
    "caption": "Average number of live and dead (TOPRO-3+ Dapi+) primary keratinocytes derived from HF-SCGFP at different time points of time lapse capture during 48 hours. There is a slight increase over time of live HF-SCs from DKO* mice when compared to control cells, and no difference in average of dead cells. n = 4 fields from two independent experiments. Average number of live and dead (TOPRO-3+ DAPI+) primary keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO* basal live keratinocytes at 12 hours, followed by a significant increase in cell death at 48 hours.",
    "molecules": "Dapi, DAPI, TOPRO-3"
  },
  {
    "caption": "Percentage of EdU+ Tomato+ keratinocytes after blocking with anti-TSLP. Anti-TSLP neutralizes the hyper-proliferation of non-mutantTom KCs in vitro after infection with Ad-cre.",
    "molecules": "EdU"
  },
  {
    "caption": "Representative images of treated ears after 9 days of anti-TSLP or IgG treatment.",
    "molecules": "IgG"
  },
  {
    "caption": "Percentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 mice",
    "molecules": "EdU"
  },
  {
    "caption": "Representative immune-fluorescence images of ear sections from control or DKO* mice treated with IgG or anti-TSLP) stained for Ki67. Ki67 expression was reduced in mice treated with anti-TSLP (n =3).",
    "molecules": "IgG"
  },
  {
    "caption": "Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients. c-JUN (purple), K-15 (green), TSLP (red) and DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients (D) and magnification images of specific regions from the HFs (E). CD200 (green), c-JUN (white), TSLP (red), DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Dose response curves displaying the percentage of G1-arrested p53-WT (blue) or KO (green) RPE1-FUCCI cells following 24hr incubation with palbociclib (dark solid lines) or 24h after subsequent washout (light dotted lines). Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment.",
    "molecules": "palbociclib"
  },
  {
    "caption": "(C) Representative images and quantifications of colony forming assays in p53-WT or KO RPE1 cells treated with palbociclib (1.25μM) for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Each bar displays mean data + SEM from 3 experiments.",
    "molecules": "palbociclib"
  },
  {
    "caption": "(A) Representative immunofluorescence images of p21 levels in p53-WT or KO RPE1 cells, 48 hours after release from 1, 4 or 7 days palbociclib (1.25μM) treatment. Zoom inserts are 3x magnification of the indicated regions. Scale bars = 250 μM. (B) Quantification of p21 intensities in cells treated as in panel A. At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats. Violin plots display the variation in intensities between individual cells. Horizontal lines display the median, and error bars show 95% confidence intervals. (",
    "molecules": "palbociclib"
  },
  {
    "caption": "(D-E) Quantification of nuclear morphologies (D) and γH2AX-positive DNA damage foci (E) following palbociclib (1.25μM) treatment in p53 WT and KO RPE1 cells. Cells were treated for 1, 4 or 7 days and then analysed before or after drug washout for 48 hours. 100 cells (nuclear morphology) or 50 cells (γH2AX foci) were scored per condition per experiment, and bar graphs represent mean data +SEM from 6 experiments.",
    "molecules": "palbociclib"
  },
  {
    "caption": "(J-K) Representative immunofluorescence images (J) and quantifications (K) of mitotic DNA replication assays (MiDAS) in p53-KO RPE1 cells released from 7 days of palbociclib (1.25μM) treatment or following 0.4uM aphidicolin treatment for 40Hrs. EdU foci were quantified in nocodazole-arrested cells. Scale bar= 5 μM, zoom inserts = 3x magnification of highlighted areas. 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats.",
    "molecules": "EdU, aphidicolin, nocodazole, palbociclib"
  },
  {
    "caption": "(A) Volcano plot of proteins up or downregulated following prolonged palbociclib (1.25μM) treatment in RPE1 cells. The top 10 significantly upregulated and downregulated proteins are shown in blue and red, respectively.",
    "molecules": "palbociclib"
  },
  {
    "caption": "(D) Representative western blots of whole cell lysates from RPE1-WT cells treated with palbociclib (1.25μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times to reflect when the majority of cells are in S-phase (see Figure 1C).",
    "molecules": "palbociclib"
  },
  {
    "caption": "(F-G) Representative images (F) and quantifications (G) of colony forming assays with RPE1 cells treated with palbociclib (1.25μM) for indicated times, and then grown at low density without palbociclib for 10 days. DMSO (control) or indicated genotoxic drugs were applied for the first 24 h after palbociclib washout. Each bar displays mean data + SD from 4 experiments.",
    "molecules": "DMSO, palbociclib"
  },
  {
    "caption": "(A) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib (1 μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times (see Figure 1C). (B) Analysis of adjusted relative density from 3 independent western blot experiments. Bars display mean values -/+ SD. Significance determined by unpaired Student's T test comparing treated target protein to asynchronous target control. (* < 0.01, ** < 0.001, *** < 0.0001).",
    "molecules": "palbociclib"
  },
  {
    "caption": "(C,D) Quantification of the nuclear morphologies (C) or γH2AX-positive DNA damage foci (D) from MCF7, MCF7 p53 KO or T47D cells that were treated with palbociclib (1μM) for 0, 1, 4, or 7 days and then analysed 48hr after drug washout. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.",
    "molecules": "palbociclib"
  },
  {
    "caption": "(F) Quantification of colony forming assays of MCF7, MCF7 p53 KO and T47D cells treated with CDK4/6 inhibitor for 0, 1, 4 or 7 days and then grown at a low density without palbociclib for 14-21 days. Bar graphs displays mean data + SEM from 4-5 experiments.",
    "molecules": "palbociclib"
  },
  {
    "caption": "(B,C) Quantification of the nuclear morphologies (B) or γH2AX-positive DNA damage foci (C) from different cancer lines, as indicated, treated continuously with palbociclib (1μM) for 0, 1, 2, or 3 weeks. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.",
    "molecules": "palbociclib"
  },
  {
    "caption": "(D) Quantification of weekly proliferation rate in cells treated as in B. A total of 60,000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks. Every 7 days, cells were trypsinised and total cell counts were determined. The 0-week timepoint shows the 7-day fold increase of untreated cells. The data points show fold increase in cell count over each 7-day period, and bars represent the mean from 2 experiments.",
    "molecules": "palbociclib"
  },
  {
    "caption": "E. Aliquots of the supernatants were treated with or without Endo-H and electrophoresed under reducing conditions. The blot was decorated with anti-µ antibodies. N563-glycans acquire Endo-H resistance in µsL566A and µsC575A, but remain largely sensitive in wild type µs.",
    "molecules": "glycans"
  },
  {
    "caption": "A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively.",
    "molecules": "N-glycans"
  },
  {
    "caption": "A. I.29µ+  B lymphoma  cells  were treated with  LPS to induce differentiation towards  IgM secretion. At the indicated  times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the  blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to  µs2L2C575OX, the intensity of which increases upon LPS stimulation.",
    "molecules": "LPS, TritonX114"
  },
  {
    "caption": "C. µ-chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the  time of lysis and hence alkylated by  NEM.",
    "molecules": "AMS, NEM"
  },
  {
    "caption": "D. The bar graphs show the levels of C575 and C414 oxidation, expressed as the ratio between IAA and NEM modified peptides.",
    "molecules": "IAA, NEM"
  },
  {
    "caption": "ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant  µs  chains and Halo-ERp44, or  Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were  blotted  under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs  or L566A chains . Expression of  HaloERp44 restores C575-dependent retention, and wild type  µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and  µs  or L566A. The slightly faster mobility  of µs2L2 and µsL complexes released by cells expressing  ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi  enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.",
    "molecules": "glycan"
  },
  {
    "caption": "C. Lysates of the  ERp44KO transfectants were  precipitated with NP-sepharose, resolved under non reducing conditions and the  blot  decorated sequentially with anti-µ, anti-ERp44  or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.",
    "molecules": "sepharose, NP"
  },
  {
    "caption": "B. To selectively label intracellular  HC575A, cells were treated with 1 nM  Halo-tag TMR Direct Ligand for 24 hours. The left panel shows aliquots of the lysates of the indicated transfectants, resolved under non-reducing conditions and decorated with anti-λ antibodies. In the two right panels, samples were precipitated with  NP-Sepharose and electrophoresed under non reducing conditions. The signal for the Halo-tag TMR was checked directly in the gel. The arrows point at the various assemblies between µ,  HC575A and λ chains.",
    "molecules": "Sepharose, NP"
  },
  {
    "caption": "C. The experiment shown aims to determine whether ERp44 can bind also to a single C575, or rather to non-native bonds. Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP-Sepharose, resolved under non reducing conditions and the  blot  decorated with anti-λ antibodies or anti-ERp44  as indicated. Black asterisks point to covalent complexes of Halo-ERp44 with µs2L2.",
    "molecules": "Sepharose, NP"
  },
  {
    "caption": "A-B Proteins of GS and GS2KR (A) and GDH and GDHK128R (B) were expressed from Salmonella in LB in the presence or absence of 50 mM NAM. : The acetylation levels of GS and GDH were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.",
    "molecules": "NAM"
  },
  {
    "caption": "GS (C) were expressed from Salmonella in minimal media with 50 mM glucose and various concentrations of nitrate (left) and ammonium (right), as indicated. The acetylation levels of GS were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.",
    "molecules": "ammonium, glucose, nitrate"
  },
  {
    "caption": "E-F GS (E) or GDH (F) were expressed from Salmonella under LB and minimal media with 50 mM glucose, 50 mM 2-DG, and 25 mM lactose as indicated. : The acetylation levels of GS and GDH were assayed by immunoblotting. Quantitation of signals is shown. Immunoblots represent at least two independent experiments For all immunoblotting data, molecular weights in kD are indicated to the right.",
    "molecules": "2-DG, glucose, lactose"
  },
  {
    "caption": "A-B GS (A) or GDH (B) were expressed from Salmonella in minimal media with 100 mM ammonium and various glucose concentrations as indicated. The specific activity of GS and GDH from 2 mM glucose was set as a control for statistical analysis. : Acetylation levels were determined by immunoblotting. The specific activities of GS and GDH were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experiments",
    "molecules": "ammonium, glucose"
  },
  {
    "caption": "C In vivo GS-specific activity of Salmonella cultured in LB with or without NAM. The specific activity of GS from cells untreated by NAM was set as the statistical control. : Acetylation levels were determined by immunoblotting. The specific activities of GS were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experiments",
    "molecules": "NAM"
  },
  {
    "caption": "GSs (F) were expressed and purified from Salmonella cultured in minimal media with high (100 mM) or low (2 mM) ammonium supplemented with high (50 mM) or low (2 mM) glucose. The specific activity of GS from 2 mM glucose was set as a control for statistical analysis. Acetylation levels were determined by immunoblotting. The specific activities of GS were measured spectrophotometrically and compared. The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Immunoblots represent at least two independent experiments Each symbol represents an independent biological replicate Data are from three independent biological replicates and expressed as the mean ± SD",
    "molecules": "ammonium, glucose"
  },
  {
    "caption": "K GDH-His was inserted into the chromosome of Salmonella to replace GDH-coding gene gdhA named as gdhA::gdhAHis and cultured in minimal media with glucose levels as indicated. The specific activity of GDH from 5 mM glucose was set as a statistical control. Acetylation levels were determined by immunoblotting. The specific activities GDH were measured spectrophotometrically and compared. Immunoblots represent at least two independent experiments K). Each symbol represents an independent biological replicate Data are from three independent biological replicates and expressed as the mean ± SD",
    "molecules": "glucose"
  },
  {
    "caption": "A-B GS and GDH, purified from Salmonella cultured in LB media, were treated with Pat/Ac-CoA in vitro. Specific activities and acetylation levels of GS (A) and GDH (B) were measured. Specific activities of GS and GDH without any treatment were set as controls. : Immunoblots represent at least two independent experiments. The degrees of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Each symbol represents an independent biological replicate and data are from three independent biological replicates and expressed as the mean ± SD",
    "molecules": "Ac-CoA"
  },
  {
    "caption": "GS (C) proteins were expressed from Salmonella wild-type, Δpat and Δpta mutant strains cultured in LB with or without 50 mM glucose.",
    "molecules": "glucose"
  },
  {
    "caption": "G-H GS and GDH were expressed from the Salmonella strain cultured in LB media supplemented with 100 mM ammonium. GS (G) or GDH (H) were treated with CobB, PatWT and its catalytically inactive mutants PatA810V and PatD592N in vitro. Specific activities of GS and GDH without any treatment were set as controls. Immunoblots represent at least two independent experiments. The degrees of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity. Each symbol represents an independent biological replicate and data are from three independent biological replicates and expressed as the mean ± SD",
    "molecules": "ammonium"
  },
  {
    "caption": "B-C GSK164K353 and GDHK128 were purified from LB, LB+20 mM glucose, LB+20 mM citrate, and LB+50 mM NAM for 4 hr. The acetylation levels of GSK164K353 (B) and GDHK128 (C) were detected by western blotting by normalization to their fully acetylated endogenous proteins GSK164AcK353Ac and GDHK128Ac. The specific activities of GS and GDH were determined. The correlation between the individual acetylation ratio of GSK164K353 (B) or GDHK128 (C) and their specific activity was plotted. The acetylation levels of GSK164K353 or GDHK128 were set as the X axis, and the specific activities of GS or GDH were set as the Y axis. Immunoblots represent at least two independent experiments Specific activities of GS and GDH without any treatment were set as control. Data are from one experiment",
    "molecules": "citrate, glucose, NAM"
  },
  {
    "caption": "B GSs were expressed and purified from Salmonella wild-type and ΔglnE knockout mutants in minimal media with or without 50 mM NAM supplemented with 2 mM glucose and 2 mM ammonium. The specific activity of GS from ΔglnE with NAM treatment was set as the control. Immunoblots represent at least two independent experiments (B). The states of adenylylation GS were determined by spectral analysis of total glutamyltransferase activity (B).",
    "molecules": "ammonium, glucose, NAM"
  },
  {
    "caption": "F The specific activities of GSWT, GS2KR and GS2KQ were compared. Each enzyme was expressed in Salmonella wild-type, Δpat and ΔcobB strains cultured in minimal media with 20 mM glucose and either 2 mM or 100 mM ammonium. Specific activities of GS from high ammonium or low ammonium were set as controls. Each symbol represents an independent biological replicate",
    "molecules": "ammonium, glucose"
  },
  {
    "caption": "A-B Specific activities of endogenous GS in Salmonella wild-type, Δpat, ΔcobB strains (A) and ΔglnA::glnAC, ΔglnA::glnACΔpat, ΔglnA::glnACΔcobB strains (B) were compared before and after glucose shock. : Immunoblots represent at least two independent experiments (A and B). The specific activities of GS before shock were set as controls. Each symbol represents an independent biological replicates and data are from three independent biological replicates and expressed as the mean ± SD",
    "molecules": "glucose"
  },
  {
    "caption": "D GSWT, GSY398S and GSY398F were expressed from the Salmonella wild-type, Δpat and ΔcobB strains, respectively. The specific activities of GSWT, GSY398S and GSY398F were compared before and after glucose shock. The specific activities of GS before shock were set as controls. Each symbol represents an independent biological replicates and data are from three independent biological replicates and expressed as the mean ± SD",
    "molecules": "glucose"
  },
  {
    "caption": "E The cellular activities of GDH were determined in Salmonella ΔgltB, ΔgltBΔpat and ΔgltBΔcobB strains, as well as in the ΔgltBΔpat strain transformed by a pat-containing plasmid and the ΔgltBΔcobB strain transformed by a cobB-containing plasmid before and after glucose shock. The specific activities of GDH before shock were set as controls. Each symbol represents an independent biological replicates and data are from three independent biological replicates and expressed as the mean ± SD",
    "molecules": "glucose"
  },
  {
    "caption": "A-D Growth curves of ΔgdhA, ΔgdhAΔpat and ΔgdhAΔcobB Salmonella strains were measured in minimal medium with 100 mM ammonium and 50 mM glucose in the absence (A) and presence (B) of 20 mM glutamine in the media. in these strains were also measured at different growth stages.",
    "molecules": "ammonium, glucose, glutamine"
  },
  {
    "caption": "E Growth curves of Salmonella ΔgdhA strain complemented with GDH (blue), with GDHK128Q (brown), with GDHK128R (red), with GDHK128R supplemented with 50 mM glutamate in culture media (green), with GDHK128R induced with 1 mM IPTG (purple), with GDHDead (orange) and with GDHK128R induced with 1 mM IPTG (black) in the minimal medium with 50 mM glucose and 100 mM ammonium were compared. Growth curves of total protein levels were normalized by Coomassie Brilliant Blue (CBB) staining. Induction and acetylation of GDH was monitored by western blot. Immunoblots represent at least two independent experiments",
    "molecules": "ammonium, Coomassie Brilliant Blue, glucose, glutamate, IPTG"
  },
  {
    "caption": "G-H Wild-type, ΔcobB and Δpat Salmonella strains were grown under minimal media supplemented with different ratios of glucose and ammonium as indicated. The specific activities of GDH in the cell lysate and steady-state growth rates were determined for each bacterium. Relative GDH activities were normalized to total protein (G) or GDH mRNA (H). Each symbol represents an independent biological replicates",
    "molecules": "ammonium, glucose"
  },
  {
    "caption": "A-B Growth curves of Salmonella glnA::glnAWT, glnA::glnAY398S, glnA::glnAY398F, glnA::glnA2KQY398F, glnA::glnA2KQY398S, glnA::glnA2KQY398S-gdhA::gdhAK128Q, and glnA::glnA2KQY398S-gdhA::gdhAK128R strains were measured in minimal medium with 100 mM ammonium and either 5 mM (A) or 50 mM glucose (B) in the media. data are pooled from the three independent experiments",
    "molecules": "ammonium, glucose"
  },
  {
    "caption": "qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in different cell types. Cells were stimulated with ecGAMP (5 μg/ml) for 4 h.",
    "molecules": "cGAMP"
  },
  {
    "caption": "ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular 2'3'-cGAMP at indicated concentrations.",
    "molecules": "2'3'-cGAMP"
  },
  {
    "caption": "ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular c-di-AMP at indicated concentrations.",
    "molecules": "c-di-AMP"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 mRNA abundance in THP-1 cells treated with ecGAMP and icGAMP at indicated concentrations for 4 h.",
    "molecules": "cGAMP"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 mRNA abundance in THP-1 cells treated with ec-di-AMP and ic-di-AMP (B) at indicated concentrations for 4 h.",
    "molecules": "c-di-AMP"
  },
  {
    "caption": "ELISA detection of IFNβ release from THP-1 cells stimulated with ecGAMP and icGAMP (C) at indicated concentrations for 4 h.",
    "molecules": "cGAMP"
  },
  {
    "caption": "ELISA detection of IFNβ release from THP-1 cells stimulated with ec-di-AMP and ic-di-AMP (D) at indicated concentrations for 4 h.",
    "molecules": "c-di-AMP"
  },
  {
    "caption": "qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ecGAMP and icGAMP (E) at indicated concentrations for 4 h.",
    "molecules": "cGAMP"
  },
  {
    "caption": "qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ec-di-AMP and ic-di-AMP (F) at indicated concentrations for 4 h.",
    "molecules": "c-di-AMP"
  },
  {
    "caption": "ELISA detection of IFNβ release from mBMDMs stimulated with ecGAMP and icGAMP at indicated concentrations for 4 h.",
    "molecules": "cGAMP"
  },
  {
    "caption": "ELISA detection of IFNβ release from mBMDMs stimulated with ec-di-AMP and ic-di-AMP (H) at indicated concentrations for 4 h.",
    "molecules": "c-di-AMP"
  },
  {
    "caption": "Frequencies of FITC+ THP-1 cells stimulated with FITC-ecGAMP for 1 h in presence of DMSO or indicated inhibitors including Dynasore (10 μM), chlorpromazine (CPZ, 10 μM), dimethylamiloride (DMA, 100 μM), polyinosinic acid (Poly I, 50 μg/ml) or mannans from Sacharomyces cerevesiae (Mannans, 1 mg/ml).",
    "molecules": "cGAMP, chlorpromazine, CPZ, DMSO, dimethylamiloride, DMA, Dynasore, FITC, mannans, Mannans, Poly I, polyinosinic acid"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 (B) and IL6 (C) mRNA in THP-1 cells stimulated with ecGAMP (5 μg/ml) or intracellular 2′3′-cGAMP (icGAMP) (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM). qRT-PCR detection of Ifnb1 (D) and Il6 (E) mRNA in mBMDMs stimulated with ecGAMP (5 μg/ml) or icGAMP (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM).",
    "molecules": "2′3′-cGAMP, cGAMP, DMSO, dynasore"
  },
  {
    "caption": "Immunostaining for EEA1 (red) (F) and LAMP2 (red) (G) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 30 min, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.",
    "molecules": "cGAMP, DAPI, FITC"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 and IL6 mRNA in THP-1 cells (H, I) and mBMDMs (J, K) stimulated with ecGAMP (5 μg/ml) and icGAMP (0.1 μg/ml), respectively, for 4 h in presence of DMSO or bafilomycin A1 (BafA) (1 μM).",
    "molecules": "cGAMP, BafA, bafilomycin A1, DMSO"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 mRNA in scrambled (Scramble) or STING shRNA stably transfected (STING KD) THP-1 cells stimulated with ec-di-AMP (5 μg/ml) and ecGAMP (5 μg/ml) or transfected with ISD or poly(I:C).",
    "molecules": "ISD, cGAMP, c-di-AMP, poly(I:C)"
  },
  {
    "caption": "ELISA detection of IFNβ protein in supernatants of Scramble or STING KD THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C).",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "qRT-PCR detection of Ifnb1 mRNA in WT and Sting-/- (Sting KO) mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.",
    "molecules": "ISD"
  },
  {
    "caption": "ELISA detection of IFNβ protein secretion in supernatants of mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C).",
    "molecules": "ISD, poly (I:C)"
  },
  {
    "caption": "qRT-PCR detection of Ifnb1 mRNA in WT, Sting KO or Sting KO complemented with mouse STING (Sting KO+mSTING) RAW264.7 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.",
    "molecules": "ISD"
  },
  {
    "caption": "Western blot detection of indicated proteins in lysates of WT and Sting-/- (KO) mBMDMs stimulated with ecGAMP (5 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.",
    "molecules": "ISD, cGAMP"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 mRNA levels in STING stable HEK293T cells (HA-STING-HEK293T) stimulated with ecGAMP or icGAMP at 5 μg/ml for indicated times.",
    "molecules": "cGAMP"
  },
  {
    "caption": "Western blot detection of indicated proteins in HA-STING-HEK293T cells stimulated with increasing amounts of ecGAMP and icGAMP at 5 μg/ml for 24 h. Data are representative of 3 independent experiments.",
    "molecules": "cGAMP"
  },
  {
    "caption": "qRT-PCR detection of the induction of IFNB1 mRNA in HEK293T cells stably transfected with pcDNA3.1-HA (HA), HA-STING and HA-STING+HA-cGAS stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD.",
    "molecules": "ISD, cGAMP"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C) for 4 h.",
    "molecules": "ISD, poly (I:C)"
  },
  {
    "caption": "ELISA detection of IFNβ in supernatants of WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas-/- (Cgas KO) mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "ELISA detection of Ifnb1 in supernatants of mBMDMs from WT and Cgas KO mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.",
    "molecules": "ISD, poly(I:C)"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.",
    "molecules": "ISD, cGAMP"
  },
  {
    "caption": "qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas KO stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.",
    "molecules": "ISD, cGAMP, cGMAP"
  },
  {
    "caption": "Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.",
    "molecules": "ISD, cGAMP"
  },
  {
    "caption": "qRT-PCR detection of IFNB1 mRNA of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).",
    "molecules": "cGAMP"
  },
  {
    "caption": "ELISA detection of IFNβ in supernatants (I) of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).",
    "molecules": "cGAMP"
  },
  {
    "caption": "Purified h-cGAS protein was precipitated with Ctrl beads or cGAMP beads and then immunoblotted.",
    "molecules": "cGAMP"
  },
  {
    "caption": "THP-1 cells were stimulated with biotin-cGAMP for indicated times and then whole cell lysates (WCL) were precipitated with streptavidin beads followed by immunoblotting.",
    "molecules": "cGAMP, biotin, streptavidin"
  },
  {
    "caption": "Immunofluorescent staining of cGAS (red) in THP-1 cells treated with FITC-ecGAMP (5 μg/ml) (green) or FITC-icGAMP (0.1 μg/ml) (green) for 4 h, nucleus in blue (DAPI). Scale bar, 10 μm.",
    "molecules": "cGAMP, DAPI, FITC"
  },
  {
    "caption": "Purified h-cGAS protein was precipitated with beads coupled with cGAMP, c-di-GMP or c-di-AMP followed by immunoblotting.",
    "molecules": "cGAMP, c-di-GMP, c-di-AMP"
  },
  {
    "caption": "Binding curves of surface-immobilized 2'3'-cGAMP with His-cGAS at indicated concentrations. Vertical lines mark the start of association and dissociation phases of the binding events. The dashed lines are global fits to a Langmuir reaction model; global fitting parameters are listed in the table below the plot. (n=4 independent experiment).",
    "molecules": "2'3'-cGAMP, His"
  },
  {
    "caption": "The small-angle X-ray scattering analysis of the full-length cGAS with cGAMP. The EOM fit of the measured SAXS data. The goodness-of-the fit χ2 = 1.1.",
    "molecules": "cGAMP"
  },
  {
    "caption": "Immunostaining of STING (anti-STING, red) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.",
    "molecules": "cGAMP, DAPI, FITC"
  },
  {
    "caption": "Cellular localization of ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are presentative of 5 independent experiments. Scale bar, 10 μm.",
    "molecules": "cGAMP, DAPI, FITC"
  },
  {
    "caption": "Frequency of perinuclear accumulation of FITC-ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml) for 2 h. Data are means+SD averaged from 5 independent experiments and approximately 100 cells were imaged and counted in each experiment. Each symbol represents the percentage of THP-1 cells with perinuclear cGAMP aggregates in every independent experiment. Mann-Whitney U test was used for statistical analysis. **p<0.01.",
    "molecules": "cGAMP, FITC"
  },
  {
    "caption": "Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of cGAMP agarose. Purified GST-cGAS and His-STING were incubated separately or together with cGAMP agarose. The input and immunoprecipitates were immunoblotted.",
    "molecules": "cGAMP, agarose, His"
  },
  {
    "caption": "Western blot detection of the presence of His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in IP lysis buffer overnight at 4°C.",
    "molecules": "Glutathione, His"
  },
  {
    "caption": "Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in the absence or presence of increasing 2′3′-cGAMP.",
    "molecules": "2′3′-cGAMP, Glutathione, His"
  },
  {
    "caption": "Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads. THP-1 cells were stimulated with biotin-ecGAMP (5 μg/ml) or biotin-icGAMP (1 μg/ml) for indicated time and cell lysates were harvested for IP with streptavidin beads. Data (A-B, D-G) are representative of 3 independent experiments.",
    "molecules": "cGAMP, biotin, streptavidin"
  },
  {
    "caption": "qRT-PCR detection of Ifnb1 mRNA (A) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.",
    "molecules": "cGAMP"
  },
  {
    "caption": "ELISA detection of IFNβ in supernatants (B) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.",
    "molecules": "cGAMP"
  },
  {
    "caption": "Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are representative of 3 independent experiments.  ",
    "molecules": "cGAMP"
  },
  {
    "caption": "A. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT), ΔtssM cells or ΔtssM cells carrying the AHT-inducible FLAG-tagged tssM-borne plasmid (tssM+) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA and FLTssM were immunodetected using anti-TolB (middle panel), anti-HA (lower panel) and anti-FLAG (upper panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.",
    "molecules": "AHT, bulgecin"
  },
  {
    "caption": "B. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.",
    "molecules": "bulgecin A"
  },
  {
    "caption": "A. Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 WT, ΔmltE cells or ΔmltE cells producing wild-type (mltE+) or E64Q mutant (mltEE64Q) VSV-G-tagged MltE (MltEV) from arabinose-inducible plasmids. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB, HcpFL and MltEV were immunodetected using anti-TolB (upper panel), anti-FLAG (middle panel) and anti-VSV-G (lower panel) antibodies, respectively. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.",
    "molecules": "arabinose"
  },
  {
    "caption": "B. Anti-bacterial activity. E. coli K-12 prey cells (W3110 gfp+, kanR) were mixed with the indicated attacker cells, spotted onto Sci-1 inducing medium (SIM) agar plates and incubated for 4 hours at 37°C. The image of a representative bacterial spot and the average and standard deviation (n=3) of the relative fluorescence of the bacterial mixture (in arbitrary unit, AU) are shown in the upper graph. The number of recovered E. coli prey cells (counted on selective kanamycin medium) is indicated in the lower graph (in log10 of colony-forming unit (cfu)). The black, dark grey and light grey circles indicate values from three independent assays, and the average is indicated by the bar. The experiment was performed in triplicate and a representative result is shown. Asterisks indicate significant differences compared to the wild-type attacker strain (NS, non significant; ***, p < 0.001).",
    "molecules": "kanamycin"
  },
  {
    "caption": "A. Remazol brilliant blue assay. The absorbance of supernatants from the reaction containing purified and RBB-labelled E. coli peptidoglycan and the indicated protein (50 µg) were measured at =595 nm after incubation for 0.5 or 4 hours at 37°C. The results shown are the average and standard deviation from triplicate reactions (n=3). Asterisks indicate significant differences compared to the buffer (NS, non significant; **, p < 0.01; ***, p < 0.001). The supernatant of the reaction after 4 hours of incubation is shown on bottom.",
    "molecules": "peptidoglycan"
  },
  {
    "caption": "B. Peptidoglycan hydrolysis. The decrease of the absorbance of the M. luteus peptidoglycan suspension in presence of the indicated protein (50 µg) was measured at =600 nm at 37°C over time. The experiment was performed in triplicate and a representative result is shown.",
    "molecules": "peptidoglycan"
  },
  {
    "caption": "C. BS3 cross-linking assay. 2×109 cells of the indicated strain producing FLAG-tagged TssM (with the exception of ∆tssM cells) were treated (+) or not (-) with the BS3 cross-linker agent. After the cross-linking reaction, cells were boiled in Laemmli buffer and total proteins were subjected to 7%-acrylamide SDS-PAGE and immunodetected with anti-FLAG antibodies. The TssM protein (FLTssM) and its complexes (*, TssM-TssJ; **, TssM-TssL) are indicated on the right as well as the TssM dimer (arrow). Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.",
    "molecules": "BS3"
  },
  {
    "caption": " B The intracellular levels of c-di-GMPin PAO1, ΔsiaC, ΔsiaB and the corresponding complemented strains were detected by LC-MS/MS ",
    "molecules": "c-di-GMP"
  },
  {
    "caption": " E Bacterial two-hybrid assay reveals an interaction between SiaC and SiaD. The recombinant strains harboring different vectors were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P was used as positive controls ",
    "molecules": "3-amino-1, 2, 4- triazole, streptomycin"
  },
  {
    "caption": " F Pull down assays confirmed the interaction between SiaC and SiaD. Cell lysates of P. aeruginosa containing pMM67EH-siaD-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads ",
    "molecules": "glutathione"
  },
  {
    "caption": " H Production of c-di-GMP by either SiaD or SiaD with SiaC at the indicated time-point was determined by HPLC ",
    "molecules": "c-di-GMP"
  },
  {
    "caption": " A Bacterial two-hybrid assay reveals an interaction between SiaB and SiaC. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P were used as positive controls. ",
    "molecules": "3-amino-1, 2, 4- triazole, streptomycin"
  },
  {
    "caption": " B Pull-down assays confirmed the interaction between SiaB and SiaC. Cell lysates of P. aeruginosa containing pMM67EH-siaC-Flag were incubated with GST or GST-SiaB individually, and protein complexes were captured by glutathione beads. ",
    "molecules": "glutathione"
  },
  {
    "caption": " E Bacterial two-hybrid assayreveals an interaction between SiaC and SiaA PP2C-like domain. NA represents the empty vector pBT or pTRG. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). The strain expressing LGF2 and Gal11P were used as positive controls. ",
    "molecules": "3-amino-1, 2, 4- triazole, streptomycin"
  },
  {
    "caption": " F Pull-down assays confirmed the interaction between SiaC and SiaA. Cell lysates of P. aeruginosa containing pMM67EH-siaA-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads. ",
    "molecules": "glutathione"
  },
  {
    "caption": " G Pull-down assays confirmed the interaction between SiaC and SiaA PP2C-like domain. Cell lysates of P. aeruginosa containing pMMB67EH-siaA386-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads. ",
    "molecules": "glutathione"
  },
  {
    "caption": " A The relative ATP hydrolysis activity of indicated sample was determined. After ATP hydrolysis reaction, the residual ATP concentration in the reaction buffer was determined by mixing the buffer with luciferase reagent. The emitted light was measured using a microplate luminometer. The amount of ATP hydrolyzed during reaction for each sample represents the ATP hydrolysis activity of each sample. The relative activity of each sample was normalized to that of the sample using SiaB and SiaC. (-) represents no substrate protein was added to reaction mixture.",
    "molecules": "ATP"
  },
  {
    "caption": " B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate. Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides. ",
    "molecules": "phosphate"
  },
  {
    "caption": " C Determination of modification sites by collision induced dissociation (CID) analysis. The MS/MS spectrum of modified L63LYLNTSSIK72 is shown. The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation, suggesting phosphorylation of Thr68. ",
    "molecules": "Thr"
  },
  {
    "caption": " D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro. SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP. Phosphorylated products were separated by SDS-PAGE. ",
    "molecules": "ATP, 32P"
  },
  {
    "caption": " G Production of c-di-GMP at the indicated time-point by SiaD with SiaC or SiaCT68A was determined by HPLC. To evaluate the effect of SiaB-mediated phosphorylation on SiaD DGC activity, SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation. ",
    "molecules": "ATP, c-di-GMP"
  },
  {
    "caption": " E Leu110 and Phe174 residues are important for SiaB function. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). Data represent the means and SDs of three biological replicates. **, P<0.01 based on one-way ANOVA test. ",
    "molecules": "Leu, Phe"
  },
  {
    "caption": "(C-D) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 and immunogold stained with anti-GFP (15 nm gold) to detect ORP5 or ORP8 and anti-PDI (10 nm gold) to label the ER lumen. ORP5 and ORP8 localize at ER-PM (arrows) and ER-mitochondria contact sites (arrowheads). m, mitochondria; cER, cortical ER. Scale bar 200nm. (E-F) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 (E) and GFP-Sec61β (F) in transfected HeLa cells. Results are presented as the percentage of the total number of gold particles (800 per condition, n=35-45 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (non-cER, non-cortical ER; ER-PM, ER-PM contact sites; ER-mito, ER-mitochondria contact sites. % gold particles ±SEM. Data are the mean of three independent replicates. *P<0,001 compared to non-cER and ER-mito [EGFP-ORP5] and to ER-PM and ER-mito [EGFP-ORP8 and GFP-Sec61β], #P<0,001 compared to ER-PM and ER-mito [GFP-Sec61β]).",
    "molecules": "gold"
  },
  {
    "caption": "(B) Electron microscopy of ultrathin cryosections of HeLa cells co-transfected with EGFP-ORP5 and PTPIP51-HA and immunogold stained with anti-GFP (15 nm gold) and anti-HA (10 nm gold) showing EGFP-ORP5 localization at ER-mitochondria contacts (arrows). Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria. Scale bar 200 nm. (C) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 and GFP-Sec61β in HeLa cells co-transfected with PTPIP51-HA. Results are presented as the percentage of the total number of gold particles (800 per condition, n=30-35 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (ER, reticular ER; ER-mito MCS, ER-mitochondria membrane contact sites; ER-PM MCS, ER-PM membrane contact sites). % gold particles ±SEM. Data are representative of three independent replicates. **P<0,001.",
    "molecules": "gold"
  },
  {
    "caption": "(E) Mitochondrial oxygen consumption rate (OCR) measured in Ctrl and ORP5 siRNA transfected cells. OCR trace was obtained by sequential measurement of basal OCR (OCRBAS), OCR after the addition of oligomycin (OM) and OCR after addition of antimycin A (AA). Note the reduced basal OCR (OCRBAS) compared to ctrl siRNA cells. Error bars denote ±SEM. Data are the mean of 4 independent repeats (n=4), *P<0,05.",
    "molecules": "AA, antimycin A, oxygen, oligomycin, OM"
  },
  {
    "caption": "A-B. Scheme of engineered Spc and Ccsp loci after integration of split-Cre and split-tTA effectors (red mark = stop codon) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections. Blue: DAPI. Scale bar: 100 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of adult BASC viewer animals. Arrow highlights labeled BASC at BADJ (magnified in box). Blue: DAPI. Scale bars: 100 µm (overview) and 20 µm (boxed magnification).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Doxycycline-dependent reversible labeling of BASCs in BASC viewer mice. Scale bar: 200 µm.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "A. β-galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v-race animals 21 days (bleomycin, naphthalene) or 35 days (influenza) following injury. Scale bar: 5 mm (whole lungs) and 1 mm (magnification).",
    "molecules": "bleomycin, naphthalene"
  },
  {
    "caption": "B. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of control and injured BASC v-race mice. Clusters of BASC-derived AT2 (YFP+) and Club cells (mCherry+) are highlighted. Blue: DAPI. Scale bar: 200 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Contribution of BASCs to different cellular compartments. Bronchiolar and alveolar accumulations of lineage-traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β-gal+ cells. Data depicted as mean±SD, n=4 (bleomycin, naphthalene) or n=5 (influenza).",
    "molecules": "bleomycin, naphthalene"
  },
  {
    "caption": "C. Club cell depletion/recovery after naphthalene treatment. Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3, 7 and 21 days post naphthalene (dpn). Blue: DAPI. Scale bar: 200 µm.",
    "molecules": "DAPI, naphthalene"
  },
  {
    "caption": "D. β-galactosidase staining of lung sections from control and injured BASC viewer animals 3, 7 and 21 days post naphthalene (dpn). Scale bar: 200 µm.",
    "molecules": "naphthalene"
  },
  {
    "caption": "C. Combined β-galactosidase (blue) and acetylated tubulin staining (cilia, brown color) of lung sections from BASC v-race animals at 21 dpn. Red: DAPI (pseudocolor). Examples of lineage-traced ciliated cells (arrowheads) and Club cells (asterisks) are highlighted. Scale bar: 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and quantification of β-gal+ bronchioles in lung sections from adult BASC v-race (ctrl.) and BASC-ablated mice (DTA). Data depicted as mean±SD, n=3. E. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and fluorescence microscopy of lung sections from uninjured and naphthalene-exposed BASC v-race (ctrl.) and BASC-ablated (DTA) mice 12 days post naphthalene (dpn). Dotted lines depict bronchiolar boundaries. Scale bar: 100 µm. F. Quantification of Club cell recovery in injured BASC v-race (ctrl.) and BASC-ablated (DTA) animals, expressed as percentage of bronchiolar circumference re-populated by mCherry+ cells at 12 dpn. Data depicted as mean±SD, n=5, **p = 0.0079 (Mann-Whitney test). DTA - diphtheria toxin fragment A.",
    "molecules": "naphthalene"
  },
  {
    "caption": "(B) Diffusion pseudotime, (C) p21, (D) cyclin D1, (E) DNA  content of unperturbed (left panels) or serum-starved  cells (right panels) are plotted on the map.",
    "molecules": "DNA"
  },
  {
    "caption": "Unified cell cycle map of unperturbed (gray) and etoposide-treated cells (1 μM; 1d: light green, 2d: green, 3d: dark green, 4d: darker green - see inset, N = 4315 cells). The unperturbed cell cycle trajectory (dotted gray line) and two arrest trajectories (into  G02C and G04C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).",
    "molecules": "etoposide"
  },
  {
    "caption": "B-E. (B) Diffusion pseudotime, (C) DNA  content, (D) phospho/total RB  and (E) p21  of unperturbed (left panels) or etoposide-treated cells (right panels) are plotted on the arrest architecture. Median nuclear  values are shown.",
    "molecules": "DNA, etoposide"
  },
  {
    "caption": "F. Time-lapse imaging of CDK2  activity (DHB-mCherry, gray) and p21-YPet  (green) intensity in etoposide-treated cells. Schematic shows the two arrest trajectories observed following etoposide  treatment. Cells that successfully complete G2 (\"Mothers\", N = 32 cells) but arrest following cell division (\"Daughters\", N = 45 cells) are shown in the two leftmost panels. Cells that arrest in G2  (N = 40 cells) are shown in the rightmost panel. The solid lines represent population medians and the shaded area indicates the 95% confidence interval.",
    "molecules": "etoposide"
  },
  {
    "caption": "B. Progression of arrest states over time  following etoposide  treatment. Etoposide-treated cells (upper panel: colored by condition, lower panel: colored by DNA   content) populating 2C, 4C, 8C  and senescent  arrest states at each day of treatment (1-4 d).",
    "molecules": "DNA, etoposide, Etoposide"
  },
  {
    "caption": "C. Distribution of  senescence-associated β-galactosidase  (SA-βgal) activity in individual cells following etoposide  treatment (1 μM, 0-4 d).",
    "molecules": "etoposide"
  },
  {
    "caption": "D-K. (D) GSK3β, (E) phospho(Thr157)-p27, (F) p27, (G) CDK4, (H) cyclin D1, (I) cyclin E, (J) cell area and (K) DNA:cytoplasm ratio of etoposide-treated cells are plotted on the map. Median nuclear  values are shown in D-I. Area indicated with a dotted line represents the senescent region of the map.",
    "molecules": "DNA, etoposide"
  },
  {
    "caption": "L-O. (L)  Cyclin A, (M) cyclin B1, (N) CDH1  and (O) SKP2  of etoposide-treated cells are plotted on the map. Median nuclear  values are shown. Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence.",
    "molecules": "etoposide"
  },
  {
    "caption": "A. Distribution of senescence-associated β-galactosidase  (SA-βgal) activity in individual cells following etoposide  or palbociclib  treatment (1 μM, 4/8 d). Control: N = 6050 cells, etop  4d: N = 409 cells, etop  8d: N = 732 cells, palbo  4d: N = 1837 cells, palbo  8d: N = 1677 cells. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.0001).",
    "molecules": "etop, etoposide, palbo, palbociclib"
  },
  {
    "caption": "B. Unified  cell cycle map of the unperturbed (gray), palbociclib- (palbo, 1 μM, 4/8 d, blue) and etoposide- (etop,1 μM, 4/8 d, green) treated cells. Control: N = 10499 cells, etop: N = 2692 cells, palbo: N = 4931 cells",
    "molecules": "etop, etoposide, palbo, palbociclib"
  },
  {
    "caption": "C-K. (C) DNA  content, (D) phospho/total RB, (E) p16, (F) TGFβ1, (G) IL-6, (H) IL-8, (I) phospho-p38, (J) phospho-STAT3  and (K) phospho-STAT5  are plotted on the map. Median nuclear  values are shown.",
    "molecules": "DNA"
  },
  {
    "caption": "D. Representative single-cell trace demonstrating mitotic skipping and endoreduplication following etoposide  treatment (1 μM) by time-lapse imaging. CDK2  activity (DHB-mCherry, blue), cell cycle phase (PCNA-mTq2, S phase  shown as gray shaded area) and p21-YPet  intensity (green) are plotted versus time  of etoposide  treatment.",
    "molecules": "etoposide"
  },
  {
    "caption": "E. Cyclin D1  abundance (left) and the proportion of polyploid  cells (right), as measured by immunofluorescence  and Hoechst staining, respectively, following siRNA-mediated knockdown of  cyclin D in control and etoposide-treated cells. Boxplots show data from four independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.",
    "molecules": "Hoechst, etoposide"
  },
  {
    "caption": "F. Cyclin D1  abundance (left) and the proportion of polyploid  cells (right), as measured by immunofluorescence  and Hoechst staining, respectively, following doxycycline  (dox)-induced upregulation of  cyclin D1 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.",
    "molecules": "Hoechst, dox, doxycycline, etoposide"
  },
  {
    "caption": "G. Cyclin A  abundance (left) and the proportion of polyploid  cells (right), as measured by immunofluorescence  and Hoechst staining, respectively, following doxycycline  (dox)-induced upregulation of cyclin A2  in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.",
    "molecules": "Hoechst, dox, doxycycline, etoposide"
  },
  {
    "caption": "A Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 10 weeks of HFD. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=9 for GFP and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.",
    "molecules": "glucose"
  },
  {
    "caption": "B Insulin tolerance test of mice in (A) at 10 weeks of HFD. Mice were fasted for four hours and administered insulin (0.75 U/kg body weight). n=9 for GFP control and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.",
    "molecules": "insulin"
  },
  {
    "caption": "C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test.",
    "molecules": "insulin"
  },
  {
    "caption": "D Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 6 weeks of HFD. Note that no difference was found in body weight between the groups by two-way ANOVA repeated measures followed by Bonferroni's test.. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=7 for GFP control and n=10 for PLTP. AUC of glucose is shown in the right graph. *P<0.05 by Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "E 18F-FDG PET/CT scan images of mice that received adenovirus GFP (control) or PLTP at 14 weeks of HFD. Mice received CL316,243 at dose of 1 mg/kg of body weight 30 min before the PET/CT images. Representative images of n=5 for both groups. Yellow arrows indicate anatomical references as bladder, heart and interscapular BAT.",
    "molecules": "18F-FDG"
  },
  {
    "caption": "F Quantification of 18F-FDG uptake in indicated tissues from mice in (E). After the 18F-FDG PET/CT imaging, indicated tissues were harvested and the radioactivity was measured by scintillation counter. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.",
    "molecules": "18F-FDG"
  },
  {
    "caption": "A Lipidomics-based quantification of indicated lipid species in mice that received tail-vein injection of adenovirus expressing PLTP or GFP (control). Blood and tissues were harvested after 10 weeks of HFD. n=4 for GFP and n=5 for PLTP in mice on HFD. LPC = (lyso)phosphatidylcholine, LPE = (lyso)phosphatidylethalonamine, PA = phosphatidic acid, SM = sphingomyelin, TAG = triglycerides, FFA = free fatty acid. Statistical analysis is demonstrated (P value) relative to GFP by Student's t-test.",
    "molecules": "FFA, free fatty acid, lipid, (lyso)phosphatidylcholine, LPC, (lyso)phosphatidylethalonamine, LPE, PA, phosphatidic acid, SM, sphingomyelin, TAG, triglycerides"
  },
  {
    "caption": "B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test.",
    "molecules": "cholesterol"
  },
  {
    "caption": "D Bile acid levels in the liver of mice in (B). n=8 in mice on RD and n=6 in mice on HFD. *P<0.05, **P<0.01 relative to GFP by Student's t-test.",
    "molecules": "Bile acid"
  },
  {
    "caption": "E Bile acid levels in the plasma of mice in (B). n=8 in mice on RD and n=7 in mice on HFD. ***P<0.001 relative to GFP by Student's t-test. F Bile acid levels in the feces of mice in (B). Feces were collected from mice on RD and HFD after seven days of treatment. n=7 in mice on RD and HFD. **P<0.01, ***P<0.001 relative to GFP by Student's t-test. ",
    "molecules": "Bile acid"
  },
  {
    "caption": "H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid (CA). Cells were treated with CA at indicated concentrations for 24 h. 0.1% ETOH was used as vehicle control (Ctr). n=3. Ucp1 mRNA expression was normalized by Tbp as an internal control. **P<0.01 relative to Ctr by one-way ANOVA followed by Tukey's test. N.S., not significant.",
    "molecules": "CA, cholic acid, ETOH"
  },
  {
    "caption": "G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD. n=5 for GFP and n=6 for both PLTP and PLTPM159E. *P<0.05, **P<0.01, ***P<0.001 PLTP relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. AUC of glucose, shown in the right graph, was analyzed by one-way ANOVA followed by Tukey's test. N.S., not significant.",
    "molecules": "glucose"
  },
  {
    "caption": "H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD. n=7 for all groups. *P=0.05 by ANOVA one-way followed by Fisher's LSD test. N.S., not significant.",
    "molecules": "cholesterol"
  },
  {
    "caption": "I Plasma bile acids in (H). n=7 for all groups. ***P<0.001 PLTP relative to GFP and PLTPM159E by ANOVA one-way followed by Tukey's test.",
    "molecules": "bile acids"
  },
  {
    "caption": "J Plasma phospholipid in (H). n=7 for all groups. ***P<0.001 PLTP and PLTPM159E relative to GFP; *P<0.05 PLTP relative to PLTPM159E by ANOVA one-way followed by Fisher's LSD test.",
    "molecules": "phospholipid"
  },
  {
    "caption": "EMSA showing that BZR1 binds to the E-box of the KTN80.4 promoter regions in vitro. Nonlabelled (free) native probe in 10- to 100-fold molar excess relative to the biotin-labeled native probe was used as a cold competitor. The mutant probe was used as a negative control.",
    "molecules": "biotin"
  },
  {
    "caption": "(A) GAPC enzymatic activity was measured in wild-type crude extracts treated with H2O (mock), and 100 µM of Tyr-Asp, Tyr, Asp, and  Ile-Glu. (B-D) The enzymatic activities  of GAPC (B), GAPA/B (C), and GAPN  (D) were measured in the wild type and double k.o. mutant gapc1gapc2 treated with  H2O (mock), and 100 µM of Tyr-Asp. Data information: Data are mean ± SEM of n=4 (A-D; four technical replicates) , significance was assessed using unpaired two-tailed Student t-test.",
    "molecules": "Asp, Glu, H2O, Ile, Tyr"
  },
  {
    "caption": "(A)  Fresh  weight   quantification of Arabidopsis and tobacco seedlings. Seedlings were pretreated with mock (water), Tyr-Asp (100 μM), Tyr  and Asp (100 μM), Ser-Leu (100 μM), or Gly-Pro (100 μM) for 1 h prior an oxidative stress (catechin or H2O2). Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm.",
    "molecules": "Asp, catechin, Gly, H2O2, Pro, Ser, Leu, Tyr, water"
  },
  {
    "caption": "(B) Catechin- (left upper panel) and (catechin-) Tyr-Asp-treated tobacco  plants (right upper panel) grown for four days in liquid media. H2O2- (left upper panel) and (H2O2-) Tyr-Asp-treated tobacco  plants (right upper panel) grown for two days in liquid media. All plants were 2-weeks-old before starting the stress treatment. Data information: Data are mean ± SEM of n=10-12 (seedlings).",
    "molecules": "Asp, Catechin, catechin, H2O2, Tyr"
  },
  {
    "caption": "(A-C) Fresh weight  measurements of Arabidopsis wild-type and gapc1/gapc2  double k.o. plants subjected to the oxidative stress (e.g.  catechin) and Tyr-Asp treatments. Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). (D-F) Representative Arabidopsis wild type and gapc1/gapc2  double k.o. seedlings from the different treatments. All plants were 10-day-old at the stress onset. The seedlings were exposed to  catechin for three days. Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm. ",
    "molecules": "Asp, catechin, Tyr"
  },
  {
    "caption": "(L) Representative immunofluorescent images of the toe pad for filaggrin (Flg). K14 marks basal keratinocytes and DAPI marks nuclei. (M) Quantification of the average size of filaggrin+ deposits at 52 DPI as shown in (L). n=3 biological replicates per genotype.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C-D) Representative H/E images (C) and quantitative analysis (D) of epidermal cell rounding, in control and iDKO mice at 4 MPI. n=4 biological replicates for PBS-iDKO; n=5 biological replicates for all else.",
    "molecules": "PBS"
  },
  {
    "caption": "(E) Representative immunofluorescent images of back skin for Flg, K14, and DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Quantification of LN weight. Control-PBS: n=10 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=9 biological replicates; iDKO-Dex: n=7 biological replicates. (H-J) Flow cytometry analysis of LNs at 4 MPI. Control-PBS: n=8 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=5 biological replicates; iDKO-Dex: n=4 biological replicates for (H,J) and Control-PBS: n=7 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=5 biological replicates; iDKO-Dex: n=3 biological replicates for (I).",
    "molecules": "Dex, PBS"
  },
  {
    "caption": "(L) Body weight over time represented as a percentage of the starting weight. Control-PBS: n=13 biological replicates; iDKO-PBS: n=4 biological replicates; Control-Dex: n=9 biological replicates; iDKO-Dex: n=5 biological replicates.",
    "molecules": "Dex, PBS"
  },
  {
    "caption": "A. Top row: MG132 arrested HeLa cells stably expressing tubulin-GFP (green) and immunostained for endogenous PRC1 (AlexaFluor555 shown in magenta) in 5 individual z-images. Enlargements of the boxed region (top: merge, middle: GFP, bottom: AlexaFluor555) below the first image show the bridging fiber determined as the line connecting two ends of the sister k-fibers in green channel and localization of endogenous PRC1 (magenta). Corresponding maximum projections are shown to the right (max z, left: merge, middle: GFP, right: AlexaFluor555).B. Quantification of tubulin-GFP bridging fibers (green bar) immunostained for endogenous PRC1 (magenta bar).",
    "molecules": "MG132"
  },
  {
    "caption": "B Negative stain image of 2D crystals containing the FERM-kinase region of FAK bound to a PI(4,5)P2 monolayer. Top right insert: Fourier transform of the 2D crystal image. Bottom right insert: 2.5x zoom of the main image.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": " A Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles (PC; phosphatidylcholine) or PI(4,5)P2 containing vesicles. Plotted are mean values from two independent autophosphorylation reactions, each determined in duplicates (n=4). Mutant nomenclature is as shown in Table 2. ",
    "molecules": "PC, phosphatidylcholine, PI(4,5)P2"
  },
  {
    "caption": " C To determine whether autophosphorylation occurs in cis or trans, autophosphorylation was measured for WT or a kinase dead form of FAK (KD, contains K454R mutation) in presence or absence of a substrate deficient FAK mutant (Y397F) and/or PI(4,5)P2 vesicles. PI(4,5)P2 negative experiments contain control PC vesicles instead. Note that the scales of the two plots are different, i.e. autophosphorylation is increased in presence of PI(4,5)P2. Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4). Left: Schematic of possible cis- and trans-autophosphorylation (red arrows) in mixtures of FAK-WT/FAK-Y397F and FAK-KD/FAK-Y397F. ",
    "molecules": "PC, PI(4,5)P2"
  },
  {
    "caption": " D Src phosphorylation of Y577 in FAK in presence of PC or PI(4,5)P2 vesicles as determined by an ELISA method. The plot on the left is obtained in presence Src containing only the kinase domain (Src254-536) and the plot on the right with Src containing additionally the SH3 and SH2 domains (Src84-536). Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4). ",
    "molecules": "PC, PI(4,5)P2"
  },
  {
    "caption": " Interactions between WT or interface mutant FAK with a PI(4,5)P2 containing membrane was probed using a pulldown assay with membrane coated silica beads. Plotted are bound protein relative to the binding for WT at the highest PI(4,5)P2 concentration. Mutant naming is as in Table 2. For WT the non-specific binding to a PC membrane is shown for comparison. Plotted are mean values from at least three independent experiments. Error bars: s.d. ",
    "molecules": "PC, PI(4,5)P2"
  },
  {
    "caption": "FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-FAK anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": " B FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-pSrc Y416 , anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm. The graph (B) shows the quantification of internalized active Src from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.d. * = p < 0.01. ",
    "molecules": "DAPI"
  },
  {
    "caption": " B FAK-WT, interface mutant FAK or FAK -/- SCC cells were seeded on growth factor-reduced Matrigel in serum-free media. After 72 h, invasion towards a serum gradient was analyzed by staining the cells with calcein and taking images at 10 μm increments through the matrigel. Representative images of the invasion assay are shown. Scale bars, 200 μm. The graph shows relative invasion from five independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.e.m. * = p < 0.01, # = p < 0.05. ",
    "molecules": "calcein"
  },
  {
    "caption": "Changes of OCT4 half-life upon SOX2-SNAP or YPET-SOX2-delDBD overexpression (n=20 cells each). p-values: Mann-Whitney U test. SBROS cells harbouring inducible SOX2-SNAP or YPET-SOX2-delDBD were treated with 0 or 500 ng/ml dox for 6-8 hours followed by pulse-labelling of OCT4-HALO and live-cell imaging.",
    "molecules": "dox"
  },
  {
    "caption": "Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 3μM CHIR (n=7). p-values: two-sided t-test with unequal variance.",
    "molecules": "CHIR"
  },
  {
    "caption": "Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 1 μM SB431542 and 25 ng/ml bFGF (n=7). p-values: two-sided t-test with unequal variance.",
    "molecules": "SB431542"
  },
  {
    "caption": "Percentage of regions in each group showing an overlap with OCT4, SOX2 and H3K4me3 ChIP-seq peaks.",
    "molecules": "H3K4me3"
  },
  {
    "caption": "C. cGAMP-VLPs induce an IFN-I response in target cells. HEK293 cells were infected with decreasing amounts of cGAMP-VLPs and Empty-VLPs (1/5 serial dilutions starting at 2μL of VLP stocks per well) and the infection was monitored 24 hours later by quantifying GFP+ cells by flow cytometry. Supernatants from the same infected cells were then transferred to a reporter cell line expressing firefly luciferase under a promoter induced by IFN-I (ISRE). Luciferase activity measured 24 hours later indicated the presence of IFN-I in the supernatants.",
    "molecules": "VLP, VLPs, cGAMP"
  },
  {
    "caption": "Immunisation with cGAMP-VLPs facilitates induction of HIV-1 Gag-specific polyfunctional CD8 T cell responses. Alternatively, cells were analysed by ICS six hours after stimulation with peptide. CD8 T cells were gated as live, CD90.2+, CD8+. CD8 T cells expressing CD107a, IFNγ, TNFα or IL2 were analysed as shown in (E).",
    "molecules": "VLPs, cGAMP"
  },
  {
    "caption": "A, B. cGAMP loading enhances IgG responses specific to VLP proteins, including VSV-G. ELISA plates were coated with lysate from cGAMP-VLPs (A) or recombinant VSV-G protein (B). Antibodies of different isotypes specific for these proteins were measured in sera from immunised mice by ELISA. The optical density at increasing serum dilutions is shown in the first three graphs from the left and the EC50 is on the right.",
    "molecules": "VLP, VLPs, cGAMP"
  },
  {
    "caption": "C. Enhanced antibody production following immunisation with cGAMP-VLPs relies on STING signalling. WT or Sting-/- mice were immunised with PBS, cGAMP-VLPs, Empty-VLPs or Empty-VLPs + poly(I:C). IgG2b antibodies recognising VLP proteins were assessed by ELISA. The optical density at increasing serum dilutions is shown in the first two graphs from the left and the EC50 is on the right.",
    "molecules": "VLP, VLPs, cGAMP, PBS, poly(I:C)"
  },
  {
    "caption": "Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B).",
    "molecules": "VLPs, cGAMP"
  },
  {
    "caption": "Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. the percentage of germinal centre B cells amongst B220+ cells is shown in (E).",
    "molecules": "VLPs"
  },
  {
    "caption": "C, D. Low doses of cGAMP-HA-VLPs confer protection following IAV challenge. One month after immunisation with the indicated doses of VLPs, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (C, upper graph shows mean and lower graphs show individual mice for each condition). Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (D).",
    "molecules": "VLPs, cGAMP"
  },
  {
    "caption": "B. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (B).",
    "molecules": "VLPs, cGAMP, AddaVax"
  },
  {
    "caption": "A. cGAMP-S-VLPs and Empty-S-VLPs incorporate SARS-CoV-2 S. Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS-CoV-2 S, Gag-GFP and actin using the indicated antibodies.",
    "molecules": "VLP, VLPs, cGAMP"
  },
  {
    "caption": "B, C. Immunisation with cGAMP-S-VLPs augments anti-Spike and anti-RBD antibody titres. Mice were immunised via the intra-muscular route with 5x105 IU of cGAMP-S-VLPs, Empty-S-VLPs, or PBS as a control. Three weeks after immunisation, sera were collected, heat-inactivated, and titres of antibodies capable of binding to SARS-CoV-2 S (B) or its RBD (C) were determined by ELISA. The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10SD). The dotted line shows the limit of detection (LOD).",
    "molecules": "VLPs, cGAMP, PBS"
  },
  {
    "caption": "D. Immunisation with cGAMP-S-VLPs induces neutralising antibodies. Using serum samples from (B), antibody titres capable of neutralising SARS-CoV-2 were determined by microneutralisation (MN) assay. Calculated IC50 doses from multiple serum dilutions are shown. The dotted line shows the LOD.",
    "molecules": "VLPs, cGAMP"
  },
  {
    "caption": "siPool mediated LINC00941 knockdown (B) in calcium-induced keratinocyte differentiation (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).",
    "molecules": "calcium"
  },
  {
    "caption": "siPool mediated LINC00941 knockdown in calcium-induced keratinocyte differentiation, results in increased abundance of early and late differentiation marker transcripts on day 3 of differentiation (C) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).",
    "molecules": "calcium"
  },
  {
    "caption": "B LINC00941 knockdown on day 3 in calcium-induced keratinocyte differentiation cultures results in increased expression of SPRR5 (FC = fold change) (n = 3 biological replicates/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).",
    "molecules": "calcium"
  },
  {
    "caption": "siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation (A) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).",
    "molecules": "calcium"
  },
  {
    "caption": "siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown by qRT-PCR (B) (FC = fold change) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).",
    "molecules": "calcium"
  },
  {
    "caption": "siPool-mediated SPRR5 knockdown on day 4 to 6 (D4-D6) of calcium-induced keratinocyte differentiation leads to decreased expression of key differentiation markers as shown western blot analysis (C) (n = 3-5 cultures/knockdown group). Data is presented as mean ± SD. Statistical significance was tested by an unpaired t-test and corrected for multiple testing after Bonferroni (* = adj. p-value < 0.05, ** = adj. p-value < 0.01, *** = adj. p‑value < 0.001, and n.s. = not significant).",
    "molecules": "calcium"
  },
  {
    "caption": "(g) An increase in UVRAG stability induced by Beclin1. After transfection with Flag-UVRAG and empty vector or Flag-UVRAG and Beclin1-V5, WCLs were treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time, followed by immunoblotting with anti-Flag.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(d) After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Autophagome was quantified as the mean ± s.d. of combined results from three independent experiments.",
    "molecules": "3-methyladenine, rapamycin"
  },
  {
    "caption": "(a) HCT116.vector and HCT116.UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis. HCT116.vector, HCT116.UVRAG, and HCT116.UVRAG ΔCCD cells were treated with or without wortmannin (1 μM) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity. Data represents mean ± s.d. of three experiments.",
    "molecules": "wortmannin"
  },
  {
    "caption": "(b) UVRAG induces autophagosome formation in NIH3T3 cells. After transfection with GFP-LC3 vector, NIH3T3.vector and NIH3T3.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Data represents mean ± s.d. of three experiments.",
    "molecules": "3-methyladenine, rapamycin"
  },
  {
    "caption": "(a) Increased Beclin1-PI(3)KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells were used for immunoprecipitation with anti-PI(3)KC3 followed by immunoblotting with anti-Beclin1. WCLs were used for immunoblotting with anti-Beclin1 and anti-PI(3)KC3. HCT116.UVRAG cells expressing Flag-tagged UVRAG were incubated under nutrient-rich conditions (lanes 1 and 2), starvation for 4 h (lane 3), or 2 μM rapamycin treatment for 2 h (lane 4). WCLs were then used for immunoprecipitation with anti-Beclin1 (lanes 2, 3 and 4) or preimmune antibody (lane 1) followed by immunoblotting with anti-Flag. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.",
    "molecules": "rapamycin"
  },
  {
    "caption": "B Observed dsDNA processing patterns of Sgs1, including an overview over consecutive bursts and pauses (upper panel) as well as detailed views into individual bursts containing multiple unwinding events (lower panels). A typical unwinding event of Sgs1 starts with slow, gradual unwinding of the dsDNA followed by DNA rehybridization, that can be almost instant (76% of events, see blue sections in lower right panel) or contain slow rewinding sections (24% of events, see orange sections in lower right panel).",
    "molecules": "DNA, dsDNA"
  },
  {
    "caption": "B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses (upper panel) and detailed views of single bursts (lower panels). Unwinding events are always followed by slow rewinding events (orange sections).",
    "molecules": "dsDNA"
  },
  {
    "caption": "D Force-dependence of the Sgs1 unwinding (black squares) (N = 2139) and rewinding rates (black diamonds) (N = 2091) in the presence of RPA (errors given as standard deviations). Red circles represent the force-dependent DNA opening and closure rates measured for RPA alone",
    "molecules": "DNA"
  },
  {
    "caption": "B Typical dsDNA processing event. The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease-dead Dna2 E675A and RPA.",
    "molecules": "DNA, dsDNA"
  },
  {
    "caption": "B dsDNA processing events observed for Sgs1 in the presence of Top3-Rmi1. Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone.",
    "molecules": "dsDNA, DNA"
  },
  {
    "caption": "E dsDNA processing by Sgs1 in the presence of Top3-Rmi1 and RPA. In contrast to the absence of RPA, DNA closure is seen as a slow rewinding.",
    "molecules": "dsDNA, DNA"
  },
  {
    "caption": "C Mean processivity of unidirectional DNA unwinding (N > 350). Error bars represent standard error of the mean.",
    "molecules": "DNA"
  },
  {
    "caption": "200 µg of yeast protein extract in different buffer compositions was processed by R2-P2 followed by DDA-MS peptide analysis. Sample preparation was performed in triplicate. Mean count of unique phosphopeptides are shown for the different conditions (n=3).   Fraction of phosphopeptides with 0, 1 or 2 missed cleavage sites. The mean is displayed (n=3).   Boxplot depicting phosphopeptide MS1 signal intensity distributions.   Boxplot depicting coefficients of variation (CVs) distributions for phosphopeptide MS1 signal intensities (n=3).   ",
    "molecules": "phosphopeptide, phosphopeptides, peptide"
  },
  {
    "caption": "Different amounts (25 µg, 50 µg, 100 µg, 200 µg and 400 µg) of yeast protein extract were processed by R2-P2 or by in-solution digestion and reversed phase C18 SPE desalting, phospho-enriched by robotic Fe3+-IMAC followed by peptide analysis with DDA-MS. Sample preparation was performed in triplicate. Boxplot depicting MS1 signal intensity distributions for phosphopeptides detected in all samples.",
    "molecules": "Fe3+, phosphopeptides, peptide"
  },
  {
    "caption": "Line plot of log2 phosphopeptide intensity versus protein input amounts with each line representing an individual phosphopeptide. For every phosphopeptide a linear regression was performed and lines are colored according to the slope of the linear regression. Black line represents the linear regression of the median phosphopeptide intensities and the corresponding function is shown on top.   Boxplot depicting the distribution of slope values resulting from linear regressions of individual phosphopeptides shown in B.  Boxplot depicting the distribution of r squared values resulting from linear regressions of individual phosphopeptides shown in B.   ",
    "molecules": "phosphopeptide, phosphopeptides"
  },
  {
    "caption": "Average number of unique peptides identified for R2-P1 performed on 5 different days (n=5).   Average number of unique phosphopeptides identified for R2-P2 performed on 5 different days (n=5).   Boxplot depicting CVs of peptide MS1 signal intensities for reproducibility analysis of R2-P1 within the same batch (n=5) and between different batches conducted on different days (n=5). For each peptide, one replicate per batch was randomly chosen for inter-batch comparisons.   Boxplot depicting CVs of phosphopeptide MS1 signal intensities for reproducibility analysis of R2-P2 within the same batch (n=5) and between different batches conducted on different days (n=5). For each peptide, one replicate per batch was randomly chosen for inter-batch comparisons.   ",
    "molecules": "phosphopeptide, phosphopeptides, peptide, peptides"
  },
  {
    "caption": "250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate.   Average number of unique phosphopeptides identified by the different enrichments (n=3).   Phosphopeptide enrichment efficiency shown as the fraction of phosphorylated peptides over total peptides (n=3).   Fraction of phosphopeptides with 1, 2 or 3 phosphorylation sites (n=3).   ",
    "molecules": "Fe3+, Phosphopeptide, phosphopeptides, peptide, peptides, TiO2, Ti4+, Zr4+"
  },
  {
    "caption": "250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate. Boxplot depicting distributions of phosphopeptide MS1 signals (n=3).   Boxplot depicting distributions of phosphopeptide MS1 signal CVs (n=3).   ",
    "molecules": "Fe3+, Phosphopeptide, phosphopeptide, peptide, TiO2, Ti4+, Zr4+"
  },
  {
    "caption": "Unique phosphopeptides present in at least two (out of three) replicates identified in the DIA-MS experiment.   Boxplot depicting distributions of Pearson correlations for biological replicates and different treatments for different time points. P-values are calculated by Wilcoxon rank-sum test.   Significantly regulated phosphopeptide isoforms for the different stimulation and time points compared to the corresponding untreated control as determined by a two sample t-test (permutation-based FDR < 0.05, and fold change > 1.5). Only phosphopeptide isoforms with all phosphosites localized were considered.   ",
    "molecules": "phosphopeptide, phosphopeptides"
  },
  {
    "caption": "Principal component analysis (PCA) of the DIA-MS phosphoproteomic data for the different stimuli separated by time points. Hierarchical clustering analysis of z-scored intensity values of significantly regulated (ANOVA, permutation-based FDR <0.05) phosphopeptides of the different time points. Column clustering hierarchy is indicated at the top. Row clusters for term enrichment analysis are color coded and indicated on the right.",
    "molecules": "phosphopeptides"
  },
  {
    "caption": "(A) Representative images showing the localization  of AID  (green), AIDR190X (green), and endogenous IgH loci (red) in nucleofected Aicda-/- CH12F3  cells. The IgH   loci are visualized using Sμ-gRNA and indicated by white arrows. An illustration of CRISPR-Sirius IgH  locus visualization is shown on top. Scale bar, 5 µm.",
    "molecules": "Sirius"
  },
  {
    "caption": "(B) OptoDroplet  formation in CH12F3  cells. Schematic illustration of light-induced optoDroplet  system is depicted on top. AID  variants were tagged to the N terminus of the optoDroplet  construct and blue light was employed to promote the formation of puncta three times followed by imaging  each time. Representative images after the third blue light activation in Aicda-/- CH12F3  cells with the indicated AID  variants are shown at the bottom. White dashed line and green line depict the shapes of nucleus  and nucleolus  based on Hoechst staining  or GFP-FBL, respectively. Scale bar, 5 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Representative immunofluorescence  images of endogenous AID  in the presence of ectopically-expressed EV, AID, AIDR190X, or AIDcry in cytokine-stimulated CH12F3  cells. EV, empty vector control. An antibody against AID   C terminus (185-198 aa) detected both endogenous and ectopically-expressed full-length AID. The nucleolus  is indicated by co-nucleofected mCherry-FBL. The white dashed line depicts the shape of the nucleus  based on Hoechst staining. Arrows indicate the location  of puncta formed by endogenous AID. The images were taken by the N-SIM microscope and processed via N-SIM software. A middle slice of the images was shown. Scale bar, 5 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(B)  Co-condensation  of AID  and AIDR190X in the nucleus  of CH12F3  cells in the early G1 phase. Top: experimental layout. GFP-tagged wild-type AID  (green) and mCherry-tagged AID  variants (red) were co-nucleofected in Aicda-/- CH12F3  cells. The resulting cells were arrested with nocodazole  for 12 hours after transfection. Six hours later, nocodazole  was removed from the system for 1.5 hours before imaging. Bottom left: representative images showing subcellular localization  of indicated AID  variants. The white dashed line depicts the shape of the nucleus  based on Hoechst staining. The white arrow indicates the location  of the co-condensed puncta. Scale bar, 5 µm. Bottom right: plot profiles of the images. Cyto: cytoplasm, Nuc: nucleus.",
    "molecules": "Hoechst, nocodazole"
  },
  {
    "caption": "(A) Left, representative images of MBP-GFP-AID  in the presence of 10% PEG8000. Scale bar, 10 µm and 2 µm, respectively. Right, the schematic protein sequences.",
    "molecules": "PEG8000"
  },
  {
    "caption": "(B) Left, representative images of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry in the presence 10% PEG8000  at the indicated protein concentrations. Scale bar, 10 µm. Right, sizes of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry protein condensates are plotted as box plots with three technical replicates. The box represents values between the lower quartile and the upper quartile, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. All points are listed and mean is indicated by \"+\". Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.",
    "molecules": "PEG8000"
  },
  {
    "caption": "C. Pull-down experiments of the assembled mElp123456 complexes carrying patients-derived mutations. The mElp123WT subcomplexes were immobilized on the Dynabeads (strep) via Elp3  protein and incubated with the Elp456 variants. The inputs and pull downs were loaded on the SDS-PAGE  gels and visualized by Coomassie staining. The molecular mass marker is indicated on the left (M).",
    "molecules": "strep"
  },
  {
    "caption": "A. High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) used to quantify the Elongator-dependent tRNA  modification  5-carbamoylmethyluridine (ncm5U) in human fibroblasts. Pseudouridine  (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. B. HPLC-MS  quantification of Elongator-dependent tRNA  modification  5-methoxy-carbonylmethyluridine (mcm5U). Pseudouridine  (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. C. HPLC-MS quantification of Elongator-dependent tRNA  modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Pseudouridine  (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.",
    "molecules": "5-carbamoylmethyluridine, ncm5U, 5-methoxycarbonylmethyl-2-thiouridine, mcm5s2U, 5-methoxy-carbonylmethyluridine, mcm5U, Pseudouridine, tRNA"
  },
  {
    "caption": "D. HPLC-MS  quantification of Elongator-independent tRNA  modification 1-methyladenosine  (m1A). Pseudouridine  (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. E. HPLC-MS  quantification of Elongator-independent tRNA  modification 7-methylguanosine (m7G). Pseudouridine  (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.",
    "molecules": "1-methyladenosine, m1A, 7-methylguanosine, m7G, Pseudouridine, tRNA"
  },
  {
    "caption": "Glycolytic flux of wildtype (WT) and TM6 strains strongly correlates with fructose-1,6-bisphosphate (FBP) concentration. The glycolytic flux is reported here as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were obtained on the basis of physiological and metabolome data, and via a novel method to estimate intracellular fluxes (Niebel et al, 2019). While on high glucose, the WT strain accomplishes a high glucose uptake rate (and thus glycolytic flux), the mutant strain (TM6) only generates a low glucose uptake (and thus glycolytic flux). On maltose, also the mutant strain achieves a high glycolytic flux, since maltose is transported by a separate transporter (Chang et al, 1989). To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the model-derived glycolytic flux (A) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.",
    "molecules": "carbon, FBP, fructose-1,6-bisphosphate, F6P, fructose 6-phosphate, glucose, maltose"
  },
  {
    "caption": "FBP concentration as a function of cellular growth rate shows weaker correlation. For metabolite levels and growth rates, error bars correspond to the standard deviation between three independent experiments; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space (cf. Methods). The carbon sources were used at a final concentration of 10 gL-1 and are indicated: Glucose (GLU); Galactose (GAL); Maltose (MAL) and Pyruvate (PYR). To assess the linear correlation between the FBP concentration and the growth rate (B) across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the growth rate (B) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.",
    "molecules": "carbon, FBP, GAL, Galactose, GLU, Glucose, MAL, Maltose, PYR, Pyruvate"
  },
  {
    "caption": "CggR intracellular abundance in the wildtype (WT) strain on glucose strongly varies with the promoter used. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL-1. Error bars represent the standard deviation of at least three replicate experiments.",
    "molecules": "carbon, glucose"
  },
  {
    "caption": "The relative abundance of CggR (normalized to the abundance measured on glucose and the same promoter) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells, but not with PTEFmut2 and PCMV. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose, galactose, maltose or pyruvate as carbon sources at a final concentration of 10 gL-1. WT data includes all three promoters, whereas TM6 only includes the PTEFmut2 and PTEFmut7 data. Error bars represent the standard deviation of at least three replicate experiments.",
    "molecules": "carbon, galactose, glucose, maltose, pyruvate"
  },
  {
    "caption": "CggR-FBP affinity constants (KD's) of the wildtype and mutant variants, determined by fitting a simple cooperative binding model to the melting curves data. Error bars indicate the 95% confidence intervals.",
    "molecules": "FBP"
  },
  {
    "caption": "Quantification of CggR binding to DNA. The CggR bound to DNA fraction was calculated by dividing the intensity of the protein-DNA complex band by the total DNA. The background-subtracted total intensities of the CggR-DNA complex and the free-DNA bands were assessed with ImageJ. Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype.",
    "molecules": "DNA"
  },
  {
    "caption": "The four reporter plasmids were transferred to the wildtype strain containing the CggR (R250A) under the control of the PTEFmut7. The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l-1 glucose. The FL1 and FL3 fluorescence shown is the non-background corrected median of 100 000 cell events. The non-FL control is the signal from a wildtype strain grown under the same conditions. The background fluorescence, assessed by the wildtype harboring the YCplac33 plasmid, was substracted from FL1 and FL3. The final reporter activity is the ratio of the background-corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments.",
    "molecules": "glucose"
  },
  {
    "caption": "Reporter activity of the sensor across (D) multiple FBP levels Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.",
    "molecules": "FBP"
  },
  {
    "caption": "Reporter activity of the sensor across glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.",
    "molecules": "FBP, fructose-1,6-bisphosphate, F6P, fructose 6-phosphate"
  },
  {
    "caption": "Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound/bound state using the Tm at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one-site specific binding model in GraphPad. The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.",
    "molecules": "FBP"
  },
  {
    "caption": "Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019).",
    "molecules": "FBP, F6P, fructose 6-phosphate"
  },
  {
    "caption": "YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose.",
    "molecules": "glucose"
  },
  {
    "caption": "Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium.",
    "molecules": "glucose"
  },
  {
    "caption": "The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.",
    "molecules": "FBP, glucose"
  },
  {
    "caption": "Effects of ActA and BMP4 on the expression of selected pluripotency, PGC, and mesodermal genes during PGC‐precursor induction. DM: dorsomorphin, SB: SB431542. Samples were calibrated with iPSC values, and iPSC values depict 1. Y‐axes are in log2 scale.",
    "molecules": "dorsomorphin, SB, SB431542"
  },
  {
    "caption": "Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (white) on days 0, 1, and 2. Nuclei were stained with Hoechst (blue). The culture contained BMP4 (5 ng/ml), Activin A (50 ng/ml), and bFGF (20 ng/ml). Scale bar: 100 μm.",
    "molecules": "Activin A"
  },
  {
    "caption": "Bisulfite sequence analysis of DMRs of the imprinted genes (PEG1, KvDMR1, PEG10, and NESP55) in iPSCs (top) and d6 TRA‐1‐81+/cKIT+ PGCLCs (bottom). White and black circles represent unmethylated and methylated CpG sequences, respectively.",
    "molecules": "CpG"
  },
  {
    "caption": "Immunofluorescence analysis for 5mC in d6 PGCLCs.Nuclei were stained with Hoechst (blue). Scale bar: 50 μm.",
    "molecules": "5mC"
  },
  {
    "caption": "Effects of KSR and WNT3A during PGC‐precursor induction on the expression of selected pluripotency, PGC, and a mesodermal gene of d2 cultures. The value for iPSCs is set as 1, and values are on log2 scale. Data are presented as means ± SD (n = 3).",
    "molecules": "KSR"
  },
  {
    "caption": "Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA‐1‐81 and cKIT.",
    "molecules": "KSR"
  },
  {
    "caption": "Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0% (control) or 20% KSR (KSR) condition during PGC‐precursor induction. Samples were calibrated with iPSC values, and iPSC values depict 1. Data are presented as means ± SD (n = 3).",
    "molecules": "KSR"
  },
  {
    "caption": "Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (yellow) in d2 precursors cultured in increasing KSR concentrations. Nuclei were stained with Hoechst (blue). Scale bar: 100 μm.",
    "molecules": "KSR"
  },
  {
    "caption": "Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d (PBS57) tetramer.",
    "molecules": "PBS57"
  },
  {
    "caption": "Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18-/- or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n=3 biological replicates.",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells.",
    "molecules": "PBS57"
  },
  {
    "caption": "Lymphocytes from the Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer. Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among the Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=22; Vα14 n=16; Jα18-/- n=8.",
    "molecules": "PBS57"
  },
  {
    "caption": "CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample.",
    "molecules": "PBS57"
  },
  {
    "caption": "Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD.",
    "molecules": "PBS57, ionomycin, PMA"
  },
  {
    "caption": "Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.",
    "molecules": "PBS57, α-GalCer, Brefeldin A"
  },
  {
    "caption": "Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants.",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. IL-4 were measured by ELISA of culture supernatants.",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1 (D-E) were measured by ELISA of culture supernatants.",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of coculture",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ class switched B cells were enumerated by flow cytometry after 4 days of coculture",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with the indicated antibodies. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. Quantification of data from E, n=3 mice per group. Mann-Whitney test. Error bars are SEM. *p<0.05.",
    "molecules": "PBS57"
  },
  {
    "caption": "Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies, analyzed by flow cytometry and gated on CD1d (PBS57) tetramer+CD3+ cells.",
    "molecules": "PBS57"
  },
  {
    "caption": "(A) Immunoblot  showing the activation of PI3K/AKT  pathway (by assessing the levels of p-AKT Ser473) of ECs 24h after induction of Pik3caH1047R expression.",
    "molecules": "Ser"
  },
  {
    "caption": "(A, B) Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice  treated with decreasing doses of 4-OHT on P1. Retinas  were stained  for  blood vessels  (IB4) and GFP  as indicated. Scale bars: 150 μm.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(D) Quantification of IB4-positive area per retina  in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each  4-OHT dose. Error bars are s.e.m. n = 4 retinas  per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(A) Representative images of EC-Pik3caH1047R/WT P6 retinas  isolated from mice  treated with 0.125 mg/kg of  4-OHT  on P1. Retinas  were immunostained  for blood vessels (IB4). Red asterisks show arteries  and arterioles  and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels). (B) Representative images of P6 retinas  from control and EC-Pik3caH1047R/WT mice  treated with 0.125 mg/kg 4-OHT on P1, following immunostaining  for p-S6  (S235/236) and blood vessels  (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained  for blood vessels (IB4), EC nuclei  (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (D-G) Quantification of (D) p-S6  (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC  number by Erg  positive cells and (G) retinal vascularity  by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas  per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.",
    "molecules": "EdU, 4-OHT"
  },
  {
    "caption": "(B, C) Representative images of P6 retinas  isolated from control and EC-Pik3caH1047R/WT mouse  littermates. Blood vessels  were stained  with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei  (Erg), EdU  incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity  by IB4 staining, (E) EC  number by Erg immunostaining, (F) EC proliferation  by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas  per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.",
    "molecules": "EdU"
  },
  {
    "caption": "(B, C) Representative confocal  images of P6 retinas  isolated from control and EC-Pik3caH1047R/WT mouse  littermates. Blood vessels were stained  with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei  (Erg), EdU  incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity  by IB4 staining, (E) EC  number by Erg immunostaining, (F) EC proliferation  by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 3 retinas  per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.",
    "molecules": "EdU"
  },
  {
    "caption": "(B, C) Representative confocal  images of P6 retinas  isolated from control and EC-Pik3caH1047R/WT mouse  littermates. Blood vessels were stained  with IB4. Lower panels showing high magnification images of the representative areas showing (B)  blood vessels (IB4), EC nuclei  (Erg), EdU  incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity  by IB4 staining, (E) EC  number by Erg immunostaining, (F) EC proliferation  by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas  per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.",
    "molecules": "EdU"
  },
  {
    "caption": "(C) Representative confocal  images of PIK3CA  and TEK patient-derived ECs immunostained  for VE-cadherin (EC-specific junctional protein) and ERG  (EC-specific transcription factor). Cell nuclei  were visualised with DAPI. Scale bars: 30 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E-F) Immunoblot  showing the impact of miransertib  at increasing doses on PI3K/AKT/mTORC1  pathway (by assessing p-AKT  and p-S6 levels) in (E) PIK3CA  and (F) TEK  mutant patient-derived ECs.",
    "molecules": "miransertib"
  },
  {
    "caption": "(G) Wild-type (HUVECs), PIK3CA  and TEK  mutant EC viability  upon the treatment with miransertib  for 72h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50  values for wild-type (HUVECs), PIK3CA  and TEK ECs  are shown. For PIK3CA  and TEK-mutant ECs  n = 3 biological replicates (patients); for wild-type ECs  (HUVECs) n = 4 technical replicates PIK3CA-mutant vs. TEK-mutant p = 0.501; PIK3CA-mutant vs. HUVECs  p = 0.0815; TEK-mutant vs. HUVECs: p = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test.",
    "molecules": "miransertib"
  },
  {
    "caption": "(I) TAP-SLD5 cells were synchronised as indicated DNA content was monitored throughout the experiment by flow cytometry.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Cells were treated with 5 µM CB-5083 as indicated (p97i = p97 inhibitor). Extracts were then incubated with IgG beads to isolate the GINS complex via TAP-SLD5 and the indicated factors were monitored by immunoblotting. Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2-7.",
    "molecules": "CB-5083"
  },
  {
    "caption": "(B) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA (examples marked by arrows; colocalization of HP1 and condensed DNA in 83 % of cells, n = 103).",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": "(D) GFP-SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst. Arrows indicate accumulation of GFP-SLD5 on constitutive heterochromatin (53 % cells, n = 125).",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D) The activity of CUL2LRR1 was inhibited in GFP-SLD5 cells , by treatment with a combination of LRR1 siRNA and MLN4924 Fixed cells were analysed Arrows indicate the accumulation of GFP-SLD5 on constitutive heterochromatin (49 % cells, n = 125). (E) Cells were treated as in (D) and the localisation of GFP-SLD5 was compared with CLASPIN by immunofluorescence. Arrows indicate the co-localisation of GFP-SLD5 and CLASPIN on constitutive heterochromatin (in 90 % cells with GFP-SLD5 patches, n = 103).",
    "molecules": "MLN4924"
  },
  {
    "caption": "A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233 respectively. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test).",
    "molecules": "233, 69A, 963, 966, RGFP966"
  },
  {
    "caption": "B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24h in culture, Tuj1 positive neurons) on PDL-laminin or Myelin substrates. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)",
    "molecules": "PDL, 966"
  },
  {
    "caption": "D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)",
    "molecules": "966"
  },
  {
    "caption": "F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj-1/V5 double positive neurons) after 36h in culture, which is repressed by wildtype HDAC3 overexpression. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus V5 vs AAV-GFP ANOVA followed by Bonferroni test).",
    "molecules": "966"
  },
  {
    "caption": "B-C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counter staining was used to label nuclei N= 9-10 biological replicates. (***p<0.005) indicate significant difference versus sham or laminectomy (lam) (ANOVA followed by Bonferroni test).",
    "molecules": "DAPI"
  },
  {
    "caption": "PP4 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).",
    "molecules": "calcium, EGTA"
  },
  {
    "caption": "PP2 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).",
    "molecules": "calcium, EGTA"
  },
  {
    "caption": "C. SNA but not DCA significantly enhances calcium levels in DRG. Data is expressed as mean ± s.e.m. N= 4 biological replicates. (*p<0.05) indicate significant difference versus sham or lam (ANOVA followed by Bonferroni test).",
    "molecules": "calcium"
  },
  {
    "caption": "D-F. Immunoblotting of DRG homogenates shows that nerve injury-dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve (Axo=Axotomy). Cytoplasmic pERK levels are used as control of axotomy-dependent signalling. (E-F). Data is expressed as mean fold change of band intensity levels ± s.e.m. N= 3 biological replicates. (**p<0.01, ***p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).",
    "molecules": "calcium, EGTA"
  },
  {
    "caption": "Significant Pearson correlations between calcium levels (Fluo4AM) and H3K9ac, r2: 0.7802 (G) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin (calcium and H3K9ac, r2: 0.0007 Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.",
    "molecules": "calcium, Fluo4AM, Fostriecin, KCl"
  },
  {
    "caption": "Significant Pearson correlations between calcium levels (Fluo4AM) ; or pHDAC3, r2: 0.6366 (H) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3, r2: 0.3025). Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.",
    "molecules": "calcium, Fluo4AM, Fostriecin, KCl"
  },
  {
    "caption": "J-L. Immunofluorescence (J) in DRG tissue sections 24h after injury in vivo shows that the PP2a/PP4 inhibitor Fostriecin (F) inhibits the injury-induced decrease in pHDAC3 (K) and the increase in H3K9ac (L). Scale bar, 10 μm. (K-L). Data is expressed as mean fluorescence intensity in arbitrary units ± s.e.m. N= 3 (L) or 4 (K) biological replicates (*p<0.05; **p<0.01) indicate significant difference versus sham (ANOVA followed by Bonferroni test).",
    "molecules": "Fostriecin"
  },
  {
    "caption": "M-N. Pharmacological inhibition of PP2a/PP4 impairs conditioning-induced DRG neurite outgrowth ex vivo (12h in culture). M. DRG neurite outgrowth (TUJ1 positive cells) 24h after sham or sciatic nerve axotomy (SNA) prior to i.t. administration of vehicle (V) or Fostriecin (F) (240µM). Scale bar, 100µm. (N) Data is expressed as average neurite length per neuron ± s.e.m. N= 3 biological replicates per condition, technical triplicate. *** p<0.005 indicate significant difference versus sham/vehicle (One way ANOVA followed by Bonferroni).",
    "molecules": "Fostriecin"
  },
  {
    "caption": "A-E. PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl (A-C, arrows), both in the nucleus and the cytoplasm (D). Scale bar, 20 μm. (C,D) Data is expressed as mean fold change vs scrambled (scr) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s.e.m. N=3 biological replicates. (**p<0.01; ***p<0.005) indicate significant difference versus scrambled siRNA (Student's t-test). (E) PCR against PP4c demonstrates silencing of PP4c mRNA. F-G. PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 (F). (G) Data is expressed as mean fold change of immunoblot band intensity ± s.e.m. N= 3 biological replicates (*p<0.05; ****p<0.001) indicate significant difference versus control siRNA (Student's t-test). ",
    "molecules": "KCl"
  },
  {
    "caption": "H-I. Genetic modification of activity-dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth. (H) DRG neurite outgrowth after transfection of control (V5 control), WT HDAC3-V5, phospho-dead HDAC3 (S424A)-V5 or phospho-mimetic HDAC3 (S424D)-V5 and after 24h in culture. Scale bar, 50µm. (I) Data is expressed as mean ± s.e.m. N= 8-9 biological replicates, approximately 35 V5 positive cells each. * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference (One way ANOVA followed by Bonferroni).",
    "molecules": "serine"
  },
  {
    "caption": "C. Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA (RNAseq) with an increased or decreased H3K9ac occupancy (ChIPseq), respectively. D. Odds ratio analysis of the enrichment/depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in (C); the numbers in red represent the p value given by Fisher's exact test (ns= not significant). ",
    "molecules": "H3K9ac"
  },
  {
    "caption": "A, B. Co-immunofluorescence of anti-neuronal and anti-regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle. Shown is increased protein expression for regeneration-associated targets in NF200 positive DRG neurons (arrows) after 966 treatment (5 weeks following SCI). Scale bar, 50 μm. (B) Data is expressed as fold change of fluorescence intensity in NF200+ cells, 966 versus vehicle ± s.e.m. N=4-7 biological replicates * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference versus vehicle (Student's t-test).",
    "molecules": "966"
  },
  {
    "caption": "A-B. AAV-HDAC3 mutant (Y298H; dominant negative deacetylase inactive) promoted DRG regenerative growth after spinal cord injury vs AAV-V5 control (injected 5 weeks prior to SCI in the sciatic nerve bilaterally) as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, (A-B) 200 μm.",
    "molecules": "dextran"
  },
  {
    "caption": "C-F. Immunofluorescence for H3K9ac. AAV-HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons, but not in surrounding nuclei of satellite cells. Scale bar, (C-F) 50 μm.",
    "molecules": "H3K9ac"
  },
  {
    "caption": "G-J. New synaptic formations (VGlut1+) from regrowing axons are shown beyond the lesion site after injection of AAV-HDAC3mut. (G) Scale bar 200 μm, (H-J) High magnification insets depict co-localization of Dextran and vGlut1 staining. Scale bar 50 μm.",
    "molecules": "Dextran"
  },
  {
    "caption": "K-L. Quantification of dextran positive axons shows that AAV-HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site. Data is expressed as percentage of dextran+ axons at each distance vs dextran+ axons at -700μm from the lesion margin (K) or distance from the caudal margin of the lesion to the most rostral dextran+ axon tip for each animal (L). N= 10 animals per condition, ± s.e.m. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 indicate a significant difference (ANOVA followed by Bonferroni test).",
    "molecules": "dextran"
  },
  {
    "caption": "A-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm.",
    "molecules": "dextran, 966, RGFP966"
  },
  {
    "caption": "C-D. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).",
    "molecules": "dextran"
  },
  {
    "caption": "E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm.",
    "molecules": "H3K9ac, 966"
  },
  {
    "caption": "F-G. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test).",
    "molecules": "H3K9ac, DAPI"
  },
  {
    "caption": "(A) Overlay of 15N-1H HSQC spectra of 15N-labeled Ca2+/CaM by itself (red peaks) and after addition of saturating, unlabeled PSD-95 (1-21) (blue peaks) or saturating, unlabeled PSD-95_pT19 (green peaks). All NMR experiments were recorded at 600 MHz 1H frequency.",
    "molecules": "15N"
  },
  {
    "caption": "(B) Chemical shift perturbation of CaM amide resonances in the absence vs. presence of PSD-95_pT19. Chemical shift difference (CSD) plotted on the vertical axis was defined as CSD = {(HNA - HNB)2 + (15NA - 15NB)2}1/2 , where \"A\" and \"B\" designate free and bound states of CaM. HN and 15N represent amide 1H and 15N chemical shifts, respectively. Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation (see Methods).",
    "molecules": "1H, 15N"
  },
  {
    "caption": "(C) Representative data from IPAP [15N-1H]-HSQC spectra of 15N-labeled Ca2+/CaM bound to PSD-95_pT19 in the absence (blue) and presence of 17 mg/ml filamentous bacteriophage Pf1 (red). Residual dipolar couplings (RDCs) for backbone HN resonances are calculated (in Hertz) as the difference in splitting for the 15N-{1H} doublet components (red peaks) relative to the isotropic 1JNH coupling (blue peaks). RDC values are calculated to be +40, +30, +20, +20, and -20 Hz for I100, I27, N137, D64, and I130, respectively.",
    "molecules": "15N"
  },
  {
    "caption": "(A) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres.",
    "molecules": "Ca2+"
  },
  {
    "caption": " Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads. (A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down.",
    "molecules": "BIC, NH2OH"
  },
  {
    "caption": " Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD-95 (sh95) and sh-resistant PSD-95-EGFP. Thus endogenous PSD-95 was replaced with either wild-type (WT), E17R, or T19K PSD-95. Cultures were treated with or without BIC (50 μM) at 17 DIV for 24-48 h or left untreated before fixation and staining for surface GluA1. (A) Representative confocal microscopic images of PSD-95-EGFP fluorescence (green) and surface GluA1 immunostaining (red) at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm). ",
    "molecules": "BIC"
  },
  {
    "caption": " Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as \"fill\" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale. ",
    "molecules": "BIC"
  },
  {
    "caption": "(B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct.",
    "molecules": "BIC"
  },
  {
    "caption": "(C) mCherry showed no enrichment in spines and BIC had no effect on its localization.",
    "molecules": "BIC"
  },
  {
    "caption": " Cultured hippocampal neurons were transfected at 10-11 DIV with the indicated CaM-IRES-ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. Cultures were treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence, and surface GluA1 immunostaining at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm). ",
    "molecules": "BIC"
  },
  {
    "caption": "Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom).",
    "molecules": "BIC"
  },
  {
    "caption": "(B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right).",
    "molecules": "BIC"
  },
  {
    "caption": "A. Western blot of total pol II, Ser2P (Abcam or Cell Signaling), Ser5P (Abcam or Cell Signaling), and histone H3 as a loading control, on the chromatin fraction of CDK9as or wild-type HEK293 cells treated with DMSO or 15 µM 1-NA-PP1 for 15 or 30 minutes. B. Quantification of the western blots shown in A. n=2 biological replicates, except for Ser2P / pol II (Abcam) CDK9as NA 30 min, Ser5P / pol II (Abcam) CDK9as NA 15 min and NA 30 min, and Ser5P / pol II (Cell Signaling) CDK9as NA 30 min, where n=3 biological replicates.",
    "molecules": "1-NA-PP1, NA, DMSO"
  },
  {
    "caption": "B. Western blot of total SF3B1, SF3B1 T142P, and histone H3 as a loading control, on the chromatin fraction of CDK9as cells treated for 30 minutes with DMSO, NA, tautomycetin (TT), calyculin A (CA), NA+TT, or NA+CA. The histone H3 loading control (CA samples) is the same as the loading control shown in Appendix Figure S3B as the same western blot experiment is shown in two different figures.",
    "molecules": "NA, CA, calyculin A, DMSO, tautomycetin, TT"
  },
  {
    "caption": "C. Co-immunoprecipitation of CPSF2 in the CDK9as cell treated for 30 minutes with DMSO or NA followed by western blot with total pol II, CPSF2, CPSF3, SF3B1, SF3B3 and β-actin (negative control) antibodies.",
    "molecules": "NA, DMSO"
  },
  {
    "caption": "D. Co-immunoprecipitation of SF3B1 in the CDK9as cell treated for 30 minutes with DMSO, NA, or DRB followed by western blot with total pol II, SF3B1, SF3B2, and GAPDH (negative control) antibodies.",
    "molecules": "NA, DRB, DMSO"
  },
  {
    "caption": "B. qRT-PCR of nuclear polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.",
    "molecules": "DRB, CA, DMSO"
  },
  {
    "caption": "C. qRT-PCR of cytoplasmic polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.",
    "molecules": "DRB, CA, DMSO"
  },
  {
    "caption": "D. ChIP-qPCR of Ser2P, CPSF73, or CPSF2 ratioed to total pol II after 30 minutes treatment with DMSO, DRB, CA, or DRB+CA on KPNB1.",
    "molecules": "DRB, CA, DMSO"
  },
  {
    "caption": "E. Co-immunoprecipitation of total pol II from HeLa cells treated for 30 minutes with DMSO, DRB, CA, or DRB+CA followed by western blot with total pol II, Ser2P, SF3B1, CPSF2, Xrn2, and GAPDH antibodies.",
    "molecules": "DRB, CA, DMSO"
  },
  {
    "caption": "a, HeLa cells were treated with biotin-conjugated peptides (30 μM, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1. b-T-B, biotin-Tat-beclin 1; b-T-S, biotin-Tat-scrambled.",
    "molecules": "biotin"
  },
  {
    "caption": "c, GFP-LC3-positive dots in GAPR-1 siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (20 &mgr;M, 3 h) with or without 100 nM bafilomycin A1. Bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. NC, non-silencing control.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "a, Percentage of cells with small htt103Q aggregates (left) and number of aggregates per cell (right) in HeLa cells expressing doxycycline (Dox)-repressible CFP-htt103Q after daily treatment with doxycycline or peptide (20 μM, 4 h per day) for 2 days. Bars represent mean ± s.e.m. of triplicate samples (60-120 cells per sample). Similar results were observed in three independent experiments.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(G) Fold change in total RNA modification levels upon loss of m5C. Fold changes were calculated by dividing the peak area ratio of noNSUN samples by the one of wild type samples. n = 3 independent biological replicates. Multiple t-tests. Data information: In G), data are presented as mean ± SEM.",
    "molecules": "m5C"
  },
  {
    "caption": "(A, B) Site-specific methylation analysis by whole-transcriptome bisulfite sequencing. Scatter plots show individual cytosines and their respective non-conversion rates in rRNAs (A) and tRNAs (B) of wild type and noNSUN strains; pie chart showing most frequently methylated tRNA isoacceptors. Data information: In (A, B n = 3 independent biological replicates.",
    "molecules": "cytosines, tRNA, tRNAs"
  },
  {
    "caption": "(A) RNA bisulfite sequencing map for mitochondrial tRNA Met-CAU in wild type (top) and noNSUN (bottom) strains. Each row represents one sequence read and each column one cytosine. Data information: In (A), a representative map of the replicates is shown, n = 3 independent biological replicates. Similar effects were observed in all replicates analysed.",
    "molecules": "cytosine, Met, tRNA"
  },
  {
    "caption": "(D) Translation efficiency of UUG-enriched, leucine-enriched, proline-enriched and random genes in each sample. A gene was considered enriched in a certain codon when the proportion of this codon in the gene was at least 3-fold higher than the proportion of the same codon across the transcriptome. Data information: In n = 3 biological replicates. In (D), boxplots show the median (central band) and IQR (boxes) ± 1.5 x IQR (whiskers), Welch's t-test, p-value < 0.05.",
    "molecules": "leucine, proline"
  },
  {
    "caption": "(d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA-agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates.",
    "molecules": "agarose"
  },
  {
    "caption": "(f) Endogenous USP33 interacts with RALB. Flag-RALB was immunoprecipitated using anti-Flag (M2) agarose and analysed for co-precipitation with endogenous USP33 using anti-USP33 antibody. Cells expressing USP33 shRNA were used as a negative control.",
    "molecules": "agarose"
  },
  {
    "caption": "(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The GST-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.",
    "molecules": "GTPγS, agarose, GDP, glutathione, GTP"
  },
  {
    "caption": "(h) USP33 affects ubiquitylation of RALB, but not RALA. 6xHis-tagged ubiquitin and Flag-RAL-G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALA or RALB. Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "Co2+"
  },
  {
    "caption": "(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The USP33-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "(a) Tandem affinity purification of ubiquitylated RALB. 6xHis-ubiquitin and Flag-RALB-G23V were co-transfected into HEK293T cells, and ubiquitylated RALB was purified using anti-Flag resin followed by Co2+ metal affinity chromatography. Isolated RALB was visualized by immunoblotting using anti-RALB antibody.",
    "molecules": "Co2+"
  },
  {
    "caption": "(d) USP33 regulates RALB ubiquitylation at Lys 47. (e) The GTP-GDP status of RALB affects RALB ubiquitylation at Lys 47. For c-e, 6xHis-tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA. Ubiquitylated RALB was purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALB. WT, wild type.",
    "molecules": "Co2+, GDP, GTP"
  },
  {
    "caption": "(f) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5. The indicated Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-SEC5 antibody.",
    "molecules": "agarose"
  },
  {
    "caption": "g) Ubiquitylation at Lys 47 impairs RALB binding to EXO84. Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-EXO84 antibody.",
    "molecules": "agarose"
  },
  {
    "caption": "(b) USP33 modulates interaction between RALB and SEC5. Flag-tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA, USP33 shRNA, empty vector (V) or HA-tagged USP33. The indicated proteins were then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-SEC5 antibodies.",
    "molecules": "agarose"
  },
  {
    "caption": "(c) USP33 regulates RALB binding to EXO84. Flag-tagged RALB was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-HA or anti-EXO84 antibody.",
    "molecules": "agarose"
  },
  {
    "caption": "(e) RALB ubiquitylation controls interaction between EXO84 and SEC5. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing RALB-G23V or RALB-G23V-K47R. The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti-EXO84 antibody.",
    "molecules": "agarose"
  },
  {
    "caption": "(f) USP33 regulates EXO84-SEC5 complex formation. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti-EXO84 antibody.",
    "molecules": "agarose"
  },
  {
    "caption": "(a) Suppression of USP33 facilitates TBK1-SEC5 complex formation. Flag-tagged SEC5 was introduced into HEK293T cells expressing empty vector (V) or HA-tagged RALB-G23V together with GFP shRNA or USP33 shRNA as indicated, and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using antibody specific for TBK1.",
    "molecules": "agarose"
  },
  {
    "caption": "(c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(c) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(d) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3. At 48 h after transfection with empty vector (V), Flag-RALB-G23V and Flag-RALB-G23V-K47R, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(f,g) RALB but not RALA undergoes deubiquitylation under nutrient deprivation. 6xHis-tagged ubiquitin and the indicated RAL mutants were overexpressed in HEK293T cells. At 48 h after transfection cells were deprived of nutrients and incubated in Hank's buffered salt solution (HBSS) medium for 90 min. Ubiquitylated RALproteins were purified by Co2+ metal affinity chromatography and analysed by immunoblotting using anti-RALA or anti-RALB antibodies.",
    "molecules": "Co2+, nutrient, nutrients"
  },
  {
    "caption": "(h) Nutrient starvation results in the accumulation of USP33. HEK TE cells were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using anti-USP33 antibody.",
    "molecules": "Nutrient"
  },
  {
    "caption": "(i) USP33 co-localizes with RALB-positive vesicles in the absence of nutrients. At 48 h after co-transfection with RALB-G23V-EGFP and USP33-mCherry, HeLa cells were incubated in HBSS medium for 90 min. Cells were imaged for EGFP and mCherry fluorescence. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels. Arrows indicate USP33 co-localization to RALB-positive vesicles.",
    "molecules": "nutrients"
  },
  {
    "caption": "(a) USP33 depletion inhibits accumulation of LC3-lipid conjugates. HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h, in the presence or absence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting analysis using anti-LC3 antibody. LC3-II/GAPDH ratios have been calculated using ImageJ densitometric analysis.",
    "molecules": "chloroquine, lipid"
  },
  {
    "caption": "(f) Overexpression of the RALB-G23V-K47R mutant leads to accumulation of LC3-lipid conjugates. HEK293T cells overexpressing the RALB-G23V or RALB-G23V-K47R mutants were assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting using anti-LC3 antibody.",
    "molecules": "lipid"
  },
  {
    "caption": "(a) Fluorescence microscopy of GFP-LC3-transduced HeLa cells left unstimulated (Unstim) or stimulated for 4 h with C12-iEDAP (2.5 μg/ml) or for 2 h with rapamycin (Rapa; 50 μg/ml). Green, GFP-LC3; blue, DAPI (DNA-intercalating dye). Scale bar, 5 μm. (b) Quantification of dot- or ring-shaped GFP-LC3 signals (representing autophagosomes) in HeLa cells stimulated as described in a.",
    "molecules": "C12-iEDAP, rapamycin"
  },
  {
    "caption": "(c) Confocal microscopy of wild-type and Nod2-deficient BMDMs transduced with GFP-LC3 (green) and left unstimulated or stimulated for 4 h with LPS (1 μg/ml) or MDP (100 μg/ml) or for 2 h with rapamycin (50 μg/ml). Scale bar, 15 μm. (d) Quantification of dot- or ring-shaped GFP-LC3 (green) in BMDMs stimulated as described in c.",
    "molecules": "LPS, MDP, rapamycin"
  },
  {
    "caption": "(e) Immunofluorescence microscopy of macrophages recovered from the peritoneal cavities of thioglycolate-injected wild-type, Nod1- and Nod2-deficient mice 4 h after intraperitoneal injection of MDP (300 μg/ml), FK565 (300 μg/ml) or rapamycin (300 μg/ml); after centrifugation by cytospin, cells were stained for endogenous LC3 (red) and with DAPI (blue). Scale bar, 10 μm. (f) Quantification of dot- or ring-shaped LC3 signals in macrophages recovered from the peritoneal cavities of mice treated as described in c. Data are representative of four (a-d) or two (e,f) independent experiments with at least 100 cells per condition (error bars (b,d,f), s.d.).",
    "molecules": "FK565, MDP, rapamycin, thioglycolate"
  },
  {
    "caption": "(e,f) Plate assay of bacteria in wild-type or Nod1-deficient MEFs infected for 0.5 h with S. flexneri ΔIcsB in the presence or absence of the autophagy blocker LY294002 (LY) or protease inhibitors (PI), followed by 5.5 h of incubation in the presence of gentamicin.",
    "molecules": "gentamicin, LY294002"
  },
  {
    "caption": "(k) Enzyme-linked immunosorbent assay of KC secretion by wild-type and RIP2-deficient MEFs infected for 0.5 h with M90T and incubated for 5.5 h in the presence of gentamicin.",
    "molecules": "gentamicin"
  },
  {
    "caption": "(l,m) Confocal microscopy (l) and quantification of the colocalization of GFP-LC3 signals (green) with M90T and ΔIcsB (m) of wild-type and NEMO-deficient (Ikbkg−/−) GFP-LC3-expressing MEFs infected for 0.5 h with wild-type S. flexneri (M90T) or S. flexneri ΔIcsB and incubated for 1.5 h in the presence of gentamicin. Red, RFP. Arrows indicate colocalization of GFP-LC3 signals with S. flexneri. Scale bars, 10 μm. *P 0.05, and **P 0.01 (t-test). Data represent three independent experiments with at least 100 cells each (error bars, s.e.m.).",
    "molecules": "gentamicin"
  },
  {
    "caption": "(a) Fluorescence microscopy of immortalized GFP-LC3-transduced lymphoblasts from donors homozygous for the normal ATG16L1*300T allele (300T) or risk allele ATG16L1*300A (300A), left unstimulated or treated for 2 h with rapamycin (50 μg/ml) or for 4 h with MDP (20 μg/ml). Green, GFP-LC3. Scale bar, 5 μm. (b) Induction of GFP-LC3 signals by rapamycin, MDP or Gram-positive peptidoglycan (PG+; 20 μg/ml) in cells treated as described in a, presented relative to GFP-LC3 signals in unstimulated control cells. *P 0.05 (t-test). Data represent three independent experiments (error bars (b), s.d.).",
    "molecules": "MDP, peptidoglycan, rapamycin"
  },
  {
    "caption": "(e) Immunofluorescence microscopy of wild-type and Nod2fs-KI BMDMs transduced with GFP-LC3 (green) and infected for 0.5 h with S. flexneri M90T-RFP (red), followed by incubation for 1 h with gentamicin. Arrows indicate no colocalization of Nod2 with ΔN85-ATG16L1 or FL-ATG16L1. Scale bar, 15 μm. (f) Quantification of the colocalization of GFP-LC3 signals with S. flexneri M90T-RFP in experiments as described in e. Data are representative of one of three independent experiments (error bars (f), s.d.).",
    "molecules": "gentamicin"
  },
  {
    "caption": "(D) α-Synuclein inclusions in fibril-introduced HEK293 cells were detected immunocytochemically with anti-phosphorylated α-synuclein (P-αSyn), anti-ubiquitin (Ub), and anti-p62 (p62) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Autophagic clearance of α-synuclein inclusions was verified by immunocytochemical analysis with anti-phosphorylated α-synuclein (P-αSyn) and anti-LC3 (LC3) antibodies. Confocal images reconstructed in the z-axis along the white lines are shown in the right panel (Z-axis). Blue, DAPI. Scale bar, 10 µm. Phosphorylated α-synuclein-positive inclusions were sequestrated into LC3-positive autophagosomes.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Similarly, this sequestration was confirmed in HEK293 cells stably expressing DsRed-LC3. The upper and lower panels show α-synuclein fibrils-and mock-introduced cells, respectively. Blue, DAPI. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) HEK293 cells stably expressing GFP-LC3 were transfected with control (upper) or Atg-5 siRNA (lower). After 36 h, α-synuclein fibrils were introduced into them for 4 h, followed by immunostaining with anti-phospho-α-synuclein antibody (P-αSyn). Blue, DAPI. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Autophagy flux was analyzed by LC3 protein monitoring. α-Synuclein fibrils-introduced HEK293 cells were treated with (+)/without (−) bafilomycin A1 (BafA1) for 2 h. Cell lysates were subjected to immunoblotting analysis using anti-LC3 (upper panel) or anti-actin (lower panel) antibodies. Quantification of the relative levels of LC3-II/Actin or autophagic flux (Baf+/Baf-) is shown with the ratio. Relative level of LC3-II/Actin was represented by mean ± s.d. as a graph. Statistical analysis was performed with one-way ANOVA (post-hoc Tukey's test). *p<0.05. This experiment was repeated three times.",
    "molecules": "Baf, BafA1, bafilomycin A1"
  },
  {
    "caption": "(B) α-Synuclein fibrils were introduced into HEK293 cells, followed by addition of DMSO, 100 nM bafilomycin (BafA1), or 200 nM rapamycin (Rapa). Cells were stained by anti-phosphorylated α-synuclein antibody (green) and DAPI (blue) 1 h (left panels) or 24 h (right panels) after introduction.",
    "molecules": "BafA1, bafilomycin, DAPI, Rapa, rapamycin"
  },
  {
    "caption": "(D) Colocalization of α-synuclein inclusions and lysosomes. α-Synuclein inclusions (P-αSyn) and lysosomes (LAMP1) were immunostained with anti-phospho-α-synuclein polyclonal antibody (green) and anti-LAMP1 antibody (red) 1 h (left panel) or 4 h (middle panel) after fibril introduction. Fibril-unintroduced cells were used as mock control (right panel). Arrowheads and arrows indicate localized or unlocalized α-synuclein inclusions to lysosomes, respectively. Blue, DAPI. Scale bars, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Mitochondrial clearance was confirmed in mock- (upper panels) or α-synuclein fibrils-introduced cells (lower panels). After introduction, EGFP-parkin cells were treated with DMSO (left) or 10 µM CCCP for 4 h (middle) or 16 h (right). Cells were examined immunocytochemically with anti-Tom20 (red) and anti-phosphorylated α-synuclein antibody (P-αSyn; aqua). Figures were presented as merged images. Individual images were provided in supplemental figure (Fig. S8). Scale bar, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A-C) qRT-PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages (A), THP1 cells (B) and human peripheral blood mononuclear cells  (PBMC) (C) that were left unstimulated or stimulated with 2'3' cGAMP  (0.1μg/mL) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib (0.1, 1, 10 μM). The data are presented as fold induction of Ifnb1/Gapdh transcripts. The data shown are mean±SEM of n=3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. *, P<0.05; ****, P<0.0001.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "(D) MTS detection of the viability of mouse peritoneal macrophages (PM), THP1 and PBMC cells left untreated (DMSO) or treated with Palbociclib (1 μM, 10 μM) for 4 h. The data shown are mean ± SEM from n=3 biological replicates. Statistical significance was determined by one-way ANOVA. ns, not significant.",
    "molecules": "DMSO, Palbociclib"
  },
  {
    "caption": "(E-F) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (E) and THP1 cells (F) stimulated with 2'3' cGAMP  (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "(G-H)  qRT-PCR measurement of Il1b (G) and Il6 (H) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant. (I-J) qRT-PCR  measurement of Il1b (I) and Il6 (J) transcripts in THP1 cells left unstimulated or stimulated with 2'3' cGAMP  (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "(K-L) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (K) and THP1 (L) cells stimulated with  2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "(M-N) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (M) and THP1 (N) cells stimulated with  2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "(A) A real-time OR-ID assay showing the association-dissociation curves of surface-immobilized Palbociclib with recombinant STING protein. The data shown are representative of n=3 biological replicates.",
    "molecules": "Palbociclib"
  },
  {
    "caption": "Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with Flag-STING (C) The data shown are representative of n=3 biological replicates.",
    "molecules": "biotin, Palbociclib, streptavidin"
  },
  {
    "caption": "Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) mouse peritoneal macrophages (D) The data shown are representative of n=3 biological replicates.",
    "molecules": "biotin, Palbociclib, streptavidin"
  },
  {
    "caption": "Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) THP1 cells (E). The data shown are representative of n=3 biological replicates.",
    "molecules": "biotin, Palbociclib, streptavidin"
  },
  {
    "caption": "(F) Immunoblotting of the streptavidin precipitates of the mixture of biotin-c-diAMP (2 μM) and recombinant SUNO-tagged STING protein (10 μg/mL) in the presence of DMSO and increasing concentrations of Palbociclib (0, 1, 10 μM). The data shown are representative of n=3 biological replicates.",
    "molecules": "biotin, c-diAMP, DMSO, Palbociclib, streptavidin"
  },
  {
    "caption": "(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-STING in the presence of DMSO and indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.",
    "molecules": "DMSO, Palbociclib"
  },
  {
    "caption": "Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages  stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (H). Scale bar, 25 μm.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "STING and ERGIC in mouse peritoneal macrophages stimulated with  2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (I). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ****, P<0.0001.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "(J) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STEEP and HA-STING in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.",
    "molecules": "DMSO, Palbociclib"
  },
  {
    "caption": "(K) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-TBK1 in the presence of DMSO or indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.",
    "molecules": "DMSO, Palbociclib"
  },
  {
    "caption": "(C) Immunoblotting of the streptavidin precipitates of the mixture of biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with corresponding Flag-STING  mutants. The data shown are representative of n=3 biological replicates.",
    "molecules": "biotin, Palbociclib, streptavidin"
  },
  {
    "caption": "(D) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING or Flag-STING Y167A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.",
    "molecules": "DMSO, Palbociclib"
  },
  {
    "caption": "Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (E). Scale bars, 25 μm.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP  (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (F). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ***, P<0.001; ****, P<0.0001; ns, not significant.",
    "molecules": "2'3' cGAMP, DMSO, Palbociclib"
  },
  {
    "caption": "(H) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING C91A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.",
    "molecules": "DMSO, Palbociclib"
  },
  {
    "caption": "(B) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib. Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; ns, not significant.",
    "molecules": "H151, DSS, Palbociclib"
  },
  {
    "caption": "A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).",
    "molecules": "ROS"
  },
  {
    "caption": "B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.",
    "molecules": "ROS"
  },
  {
    "caption": "E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.",
    "molecules": "PS"
  },
  {
    "caption": "D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures.",
    "molecules": "gold"
  },
  {
    "caption": "B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control.",
    "molecules": "H2O2"
  },
  {
    "caption": "C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC.",
    "molecules": "H2O2, ROS"
  },
  {
    "caption": "D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control.",
    "molecules": "H2O2, NAC"
  },
  {
    "caption": "E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets.",
    "molecules": "H2O2"
  },
  {
    "caption": "F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group).",
    "molecules": "H2O2, SP600125"
  },
  {
    "caption": "G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).",
    "molecules": "H2O2, SP600125"
  },
  {
    "caption": "A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53, p53, and LAMP1 in HC platelets. Platelets were treated with high glucose (25mM), with or without CCCP (10µM). This Western blot is representative of three independent experiments.B Quantification analysis of phosphorylated p53 and LAMP1 (HG; *p=0.018 vs DMSO group, HG/CCCP; **p=0.001 vs HG group, n=4 for each group).",
    "molecules": "CCCP, DMSO, glucose"
  },
  {
    "caption": "C The recognized autophagy inhibitor (2mM 3-Methyadenine; 3MA for 1h) was used to treat DM platelets compared to HC assessing for phosphorylated p53, p53, and LC3I/IID Quantification analysis of phosphorylated p53/p53 was performed. Each value indicated the average band density from a total of three independent samples (DM/3MA; *p=0.026 vs. DM group).",
    "molecules": "3-Methyadenine, 3MA"
  },
  {
    "caption": "E Western blot analysis of PLPs transfected with control or ATG3 siRNA, with or without treatment of 1 mM H2O2. Each lane displayed a different transfected group.F Quantification analysis of ATG3, phosphorylated p53 (ser15), p53, and GAPDH in each group. (ATG3 in ATG3 siRNA; **p=0.0003 vs control group, pp53 in ATG3 siRNA/ H2O2; *p=0.0145 vs. H2O2 group).",
    "molecules": "H2O2"
  },
  {
    "caption": "A Western blot analysis of phosphorylated p53 (ser15), p53, LC3I/II, and GAPDH in WT mouse platelets. Mouse platelets were treated high glucose (25mM) with or without CCCP (10µM).B Quantification analysis of phosphorylated p53 (ser15) and LC3 II (pp53 in HG; **p=0.009 vs WT group, pp53 in HG/CCCP ; *p=0.022 vs HG group, LC3 II in HG/CCCP ; *p=0.028 vs HG group, n=3 for each group).",
    "molecules": "CCCP, glucose"
  },
  {
    "caption": "C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 or WT_ H2O2/3MA in mouse platelets. ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin-V (PS externalization). Representative of n=3.D Graph indicates the percentage of TMRM positive cells in total cell population (H2O2; **p=0.002 vs. not treated group, H2O2/3MA; **p=0.008 vs. not treated group, NS means no significance, n=3).E Graph indicates the percentage of Annexin V positive cell in total cell population (H2O2; NS, p=0.058 vs. not treated group, H2O2/3MA; **p=0.004 vs. not treated group, **p=0.008 vs. H2O2 group, NS means no significance, n=3).",
    "molecules": "H2O2, 3MA, PS"
  },
  {
    "caption": "F Representative western blot analysis of LC3 I/II in analysis in WT or WT/H2O2 or WT/H2O2/3MA in mouse platelets (n=3).",
    "molecules": "H2O2, 3MA"
  },
  {
    "caption": "H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 and Parkin -/- or Parkin -/-_H2O2 mouse platelets. ΔΨm was detected using TMRM (WT_ H2O2; **p= 0.002 vs. WT group, Parkin-/-_ H2O2; **p= 1.61525E-06 vs. Parkin-/- group, n=3)I Apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of Annexin V positive cell in total cell population (Parkin-/- : *p=0.010 vs. WT group, Parkin-/-_ H2O2 ;**p= 0.008 vs. WT_ H2O2; group, Parkin-/-_ H2O2; **p=0.001 vs. Parkin-/- group, NS means no significance n=3)",
    "molecules": "H2O2, PS"
  },
  {
    "caption": "J Western blot analysis in WT and Parkin -/- demonstrating the inability to induce LC3 despite the addition of H2O2. Shown graphically are the LC3II to LC3I ratios (WT_ H2O2;*p=0.028 vs. WT group, n=3)",
    "molecules": "H2O2"
  },
  {
    "caption": "D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury, indicative of occlusive thrombosis. Flow was plotted as percentage of baseline flow before injury (DM: n=3, PINK1-/- DM; n=3).",
    "molecules": "FeCl3"
  },
  {
    "caption": "(B)  Kaplan-Meier survival curve based on n=17 144DG11-treated and n=9 vehicle-treated animals. 144DG11 significantly increased survival (log-rank test p-value<0.000692).",
    "molecules": "144DG11"
  },
  {
    "caption": "(M) average duration in movement in an open field as a function of time after treating  Gbeys/ys mice with vehicle (n=8) or 144DG11 (n=9) at 6 months of age (at onset). Data information: Two way ANOVA with repeated measures show that, throughout the period, treatment values were not different than vehicle (p<0.15).",
    "molecules": "144DG11"
  },
  {
    "caption": "(A) Left panel, Mice, treated as indicated, were sacrificed and the indicated tissues were collected and stained for PG (arrows) with  PAS following diastase treatment. Scale bars, 200 µm (liver, muscle), 50 µm (cortex, heart, peripheral nerve). Right panel, PAS staining  was quantified based on analysis of 4 sections from each tissue in n=3 wt, n=7 Gbeys/ys vehicle-treated, and n=9 144DG11-treated mice.",
    "molecules": "PAS, diastase, PG, 144DG11"
  },
  {
    "caption": "(C) 144DG11  Pharmacokinetics. Gbeys/ys mice injected with 144DG1 were sacrificed 30, 60, 90, and 210 min post injection and the indicated tissues were removed, as well as 200 uL of serum drawn. Graph shows means (+/- SEM) of 144DG11 levels in the different tissues determined by  LC-MS/MS Results obtained from n = 3 mice at each  time point.",
    "molecules": "144DG1, 144DG11"
  },
  {
    "caption": "A-F Mice were monitored over a 24 hr period. Effective  mass was calculated by ANCOVA Data are mean±SEM from nine month old mice  (n=11, wt vehicle-treated, n=6 Gbeys/ys vehicle-treated, and n=7 Gbeys/ys 144DG11-treated). Vehicle-treated Gbeys/ys mice  demonstrate lower respiratory quotient (in the  light) (A), total energy expenditure (TEE) (B), and  fat oxidation (C) compared to wt controls. 144DG11  treatment increased these parameters (for fat oxidation only in the dark  and total time). Carbohydrate oxidation and ambulatory activity, not significantly affected by the diseased state, were increased by  144DG11 even beyond wt control levels (D and E) (note, while 144DG11 increased carbohydrate oxidation in the light (D), p was only <0.06). 144DG11 also reversed the decrease in meal size and water  sip volume observed in Gbeys/ys mice as compared to wt control (F). Data information: *p<0.05 v wt controls, #p<0.05 v Gbeys/ys vehicle treated mice. Statistical differences were determined by two-tailed t-tests.",
    "molecules": "Carbohydrate, carbohydrate, fat, 144DG11, water"
  },
  {
    "caption": "(G) Blood metabolic panel based on n=3, 9.5 month old mice treated as indicated. Blood glucose was increased and blood triglycerides decreased in Gbeys/ys cells by 144DG11 (p<0.05).",
    "molecules": "glucose, 144DG11, triglycerides"
  },
  {
    "caption": "(A) PAS staining for total glycogen in  skin  fibroblasts  from different APBD patients. Staining  fluorescence was quantified by InCell2200 (see Methods). Means (+/- SEM) are statistically different from each other (p<0.0001, One Way ANOVA). n=3 technical replicates from each patient  indicated at the x-axis were used.",
    "molecules": "PAS, glycogen"
  },
  {
    "caption": "(B) PAS staining for total glycogen in APBD87 fibroblasts glucose-starved for 48 h (left), or glucose starved and then replenished for the last 24 h to induce  glycogen burden (right). Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3  filter. Scale bar, 100 µm.",
    "molecules": "PAS, CY3, glucose, glycogen"
  },
  {
    "caption": "(D) Glycolytic (red) and mitochondrial  (blue)  ATP production determined by Agilent's Seahorse machine and real-time ATP rate assay kit. HC and APBD patient fibroblasts were serum/glucose-starved for 48 h and then full medium was replenished for 24 h without (untreated), or with (chronic) 50 µM 144DG11. Acute, 50 µM 144DG11  was added on assay for 20 min after 24 h of serum/glucose  replenishment. Readings were normalized to cell number as determined by Crystal Violet staining. Shown are mean and SEM values based on n=3-6 technical repeats. For all experiments (HC, p<0.01; APBD1, p<0.02; APBD2, p<0.09; APBD3, p<0.11; One Way ANOVA with Sidak's post-hoc correction for multiple comparisons) glycolytic ATP production was increased, as compared to untreated control, by acute, but not chronic, supplementation of 144DG11. Mitochondrial ATP  production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts (p<0.06).",
    "molecules": "ATP, Crystal Violet, glucose, 144DG11"
  },
  {
    "caption": "(C) Cellular thermal shift assay  (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1  was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D.",
    "molecules": "144DG11"
  },
  {
    "caption": "(A) Left panel, Autophagic flux, determined by the extent of lysosomal inhibitors-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II/LC3I), is increased by  144DG11 (50µM, 24h). Right panel, 144DG11-mediated increase in  autophagic flux, demonstrated by enhanced degradation of the autophagy substrate p62. Shown are representative immunoblots (grouped from different gels) and densitometric quantifications +/-s.d. (for LC3II/LC3I, n=4 biological replicates, *p<0.0023; for p62, n=5 biological replicates, *p<0.0099; two-tailed t-test).",
    "molecules": "144DG11"
  },
  {
    "caption": "(B) Left panel, TEM images of liver tissue from vehicle or  144DG11-treated Gbeys/ys mice. High-magnification (right, scale bars=200 nm) and low-magnification (left, scale bars=1 µm) images show higher levels of  glycogen/polyglucosan in  lysosomes and cytosol, respectively. Right panel, quantification (+/- s.d) of lysosomal glycogen particles (n=3 biological replicates, *p<0.03, two-tailed t-test). G, Glycogen/polyglucosan; L, Lysosomes; M, Mitochondria.",
    "molecules": "polyglucosan, glycogen, Glycogen, 144DG11"
  },
  {
    "caption": "(C) 144DG11 reduces LC3 and  p62  in mouse liver, but not muscle (n=3 biological replicates, *p<0.04 for  LC3, **p<0.0007 for  p62; two-tailed t-test, SEM). Scale bars, 10 µm",
    "molecules": "144DG11"
  },
  {
    "caption": "(E)  LAMP1-KD and 144DG11 treatment cause  lysosomal acidification. Upper panel, flow cytometry results showing that 144DG11 slightly increased acidification (yellow to blue median fluorescence ratio (Y/B)) in control, GFP-transduced, APBD fibroblasts (Y/B(GFP/144DG11)>Y/B(GFP), p<0.12), but significantly acidified  LAMP1-KD, GFP-shLAMP1-transduced, APBD fibroblasts (Y/B(LAMP1-KD/144DG11)>(Y/B(LAMP1-KD), p<0.03). LAMP1-KD itself led to the most significant acidification (Y/B (LAMP1-KD)>Y/B(GFP), p<0.007). n=3, two-tailed t-tests. Middle panel Lysosensor staining of the corresponding cells. Yellow fluorescence intensity correlates with acidification. Lower panel, PAS  (glycogen)  staining  of the corresponding cells. Scale bars, 50 µm.",
    "molecules": "Lysosensor, glycogen, PAS, 144DG11"
  },
  {
    "caption": "(G) 144DG11  reduces  lysosomal  (LAMP1 positive) area in liver, but not muscle, of Gbeys/ys mice  (n=3 biological replicates, *p<0.01, two-tailed t-test , SEM).Scale bars, 5 µm for liver (upper left panel), and 10 µm for muscle (lower left panel). Right panel shows quantification of the left panel.",
    "molecules": "144DG11"
  },
  {
    "caption": "(B) Morphological  characterization of lysosomes based on  lysotracker staining  of n=4 HC and n=4 (biological replicates) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h. Values are deviations from untreated HC. 144DG11 has reduced lysotracker  mean intensity, integrated intensity (DXA), and area in both HC and APBD fibroblasts  (***p<0.0001, One-Way ANOVA with Sidak's multi-comparison post-hoc test).",
    "molecules": "lysotracker, 144DG11"
  },
  {
    "caption": "(C) Functional (mitochondrial membrane potential (TMRE parameters)) and biomass (mitotracker) characterization of mitochondria in HC and APBD  cells treated and analyzed as in (B). Mean (***, p<0.0001) and integrated (***, p<0.0006) TMRE  intensities, and mitochondrial  biomass (***, p<0.0001), were reduced in APBD v HC. 144DG11 increased TMRE mean (**, p<0.009, **, p<0.006) and integrated (*, p<0.01, **, p<0.001) intensities in APBD  and HC cells, respectively and  mitotracker  intensity in APBD cells only (***, p<0.0001). Data represent n=3 biological replicates. Statistical differences analyzed by One-way ANOVA with multiple comparisons.",
    "molecules": "mitotracker, 144DG11, TMRE"
  },
  {
    "caption": "E. Blockade of permeation through the K+ channel altered the speed of deciliation. After serum starvation, medium containing serum and Astemizole (10 µM) was added, and cells were fixed and stained at he indicated time points (N=12).",
    "molecules": "Astemizole"
  },
  {
    "caption": "A. Actively proliferating hTERT-RPE1 cells were transfected with scrambled or KV10.1 siRNA, and cell cycle distribution was determined using acridine orange staining. Knockdown of KV10.1 induces an increase in cells with DNA/RNA content compatible with G0 (rectangle). However, the quantitative increase (to approximately 10%) was insufficient to explain the abundance of ciliated cells (over 40%)",
    "molecules": "DNA, RNA"
  },
  {
    "caption": "D. Primary cilia were present in cells actively synthesizing DNA (measured by EdU incorporation). Cells were transfected with siRNA against KV10.1, and 24 hours later were incubated in the presence of EdU for 4h before fixation. KV10.1 knockdown resulted in the presence of abundant EdU-positive and simultaneously ciliated cells, suggesting a decoupling between deciliation and cell cycle progression. Scale bar: 10µm",
    "molecules": "DNA"
  },
  {
    "caption": "Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100nM 1 hour; **p = 0.0041; control n=21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n=21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "Latrunculin, Latrunculin B"
  },
  {
    "caption": "Latrunculin B treatment statistically significantly reduced kugel diameter (*p = 0.0164; control n=169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n=361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).",
    "molecules": "Latrunculin, Latrunculin B"
  },
  {
    "caption": "Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 µM 1h; ****p <0.0001; control n=22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n=24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "Blebbistatin"
  },
  {
    "caption": "Blebbistatin treatment had no effect on kugel diameter (p = 0.3731; control n=83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n=26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "Blebbistatin"
  },
  {
    "caption": "Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow).",
    "molecules": "Tricaine"
  },
  {
    "caption": "Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead).",
    "molecules": "Dextran, dextran"
  },
  {
    "caption": "Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead).",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "MO"
  },
  {
    "caption": "Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).",
    "molecules": "Alexa 647, IgG"
  },
  {
    "caption": "VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number (****p<0.0001; DMSO control n=30 embryos 8.53 ± 2.62 (mean ± s.e.m.); AV951 n=31 embryos 21.10 ± 2.71 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Mann-Whitney U test).",
    "molecules": "AV951, DMSO"
  },
  {
    "caption": "Mean kugel diameter was not statistically significantly different after AV951 treatment (p = 0.7890; DMSO control n=243 kugeln from 30 embryos 9.94 ± 0.76 (mean ± s.e.m.); AV951 n=652 kugeln from 31 embryos 9.73 ± 0.32 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Student's t-test).",
    "molecules": "AV951, DMSO"
  },
  {
    "caption": "DAF-FM staining, a vital dye for nitric oxide (NO), showed that 57.56% of kugeln were positive for nitric oxide reactivity (2.5µM incubation from 96-102hpf; n=22 4dpf embryos; 118 of 205 kugeln filled; 3 experimental repeats). Images show representative "filled" (DAF-FM positive) and "unfilled" (DAF-FM negative) kugeln.",
    "molecules": "DAF-FM, nitric oxide, NO"
  },
  {
    "caption": "Time-lapse acquisition with DAF-FM revealed that kugeln contained NO early in their biogenesis (grey LUT; inverted).",
    "molecules": "DAF-FM, NO"
  },
  {
    "caption": "Application of LysoTracker, a vital dye that stains lysosomes or acidic compartments, showed that 17.08% of kugeln contained acidic contents (8.33µM; 96-101hpf; n=22 4dpf embryos; 62 of 363 kugeln filled; 3 experimental repeats). Images show representative \"filled\" (Lysotracker positive) and \"unfilled\" (Lysotracker negative) kugeln.",
    "molecules": "Lysotracker, LysoTracker"
  },
  {
    "caption": "The nitric oxide synthase (NOS) inhibitor L-NAME had no statistically significant effect on kugel number per embryo (0.5mM L-NAME 18h; p = 0.4870; control n=22 embryos 14.27 ± 3.35 (mean ± s.e.m.), L-NAME n=24 embryos 7.46 ± 1.61 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "L-NAME"
  },
  {
    "caption": "Diameter of kugeln was not affected by L-NAME (p = 0.4161; control n=315 kugeln from 22 embryos 8.77 ± 0.49 (mean ± s.e.m.), L-NAME n=179 kugeln from 24 8.66 ± 0.71 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "L-NAME"
  },
  {
    "caption": "Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "DAPT"
  },
  {
    "caption": "Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).",
    "molecules": "DAPT"
  },
  {
    "caption": "Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "XAV-939"
  },
  {
    "caption": "Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test)",
    "molecules": "XAV-939"
  },
  {
    "caption": "Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).",
    "molecules": "XV"
  },
  {
    "caption": "(D) Subpopulations of the 80S*HCVIRES complex containing P-site tRNA. The 80S is in the classical configuration and the head tilt is not present. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink),40S subunit (yellow),60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts, respectively.28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray),HCVIRES (pink), tRNA (green).",
    "molecules": "tRNA"
  },
  {
    "caption": "(E) The cryo-EM map was filtered according to resolution. 40S*HCV IRES. Left column shows an overview of the final cryo-EM reconstructions with the HCV IRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCV IRES (pink), tRNA (green).",
    "molecules": "18S, 28S, 5.8S, 5S"
  },
  {
    "caption": "(A) Comparison of 40S subunit positions in Rolled, Rotated, Classical/no head tilt (orange) and Classical 80S*HCVIRES complex (yellow) in common 60S alignment. The cryo-EM map of the Classical 80S*HCVIRES was filtered to 6 Å. Arrows indicate the direction of movement during transition between two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units.(B) Comparison of the HCVIRES in Classical 80S*HCVIRES complex (gray) and upon P-tRNA binding (Classical, no head tilt; purple) in a common 40S body alignment. The distance of the movement of domain II was indicated with an arrow and Å units",
    "molecules": "tRNA"
  },
  {
    "caption": "(A) The secondary structure of the HCVIRES domain III (hIII1, hIII2, IIIe and IIId) and helix 26 (h26) from the 18S rRNA. Colors of HCVIRES Domains are the same as in Fig 3. Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line.(B) The interaction core between IIId, IIIe, hIII1 of HCVIRES (colored as in (A)) and Expansion segment 7 (ES7) of h26 (dark gray).(C) The rearrangement of h26 (red) upon binding of HCVIRES. The 80S*HCVIRES complex (yellow) is compared to the elongating POST state (gray; PDB 5Aj0) (Behrmann et al, 2015). The loop closing the helix is highlighted in red and dark gray.",
    "molecules": "18S"
  },
  {
    "caption": "(A, B) Comparative solvent side views of the small subunit with the bacterial SD-chamber on the 30S from the 70S POST state (PDB 2HGR) (Yusupova et al, 2006) and the corresponding region of the eukaryotic 40S in the classical 80S*HCV IRES complex. Analogous proteins and rRNA helices are highlighted in the same colours, respectively. (A) Crystal structure of the 30S subunit with mRNA. The SD-helix is established between the mRNA (orange) and the 3' end of the 16S rRNA (blue).(B) Atomic model of the 40S subunit with the HCV IRES model (pink). The mRNA-part and S2 of the IRES are shown in orange and cyan, the 3'-end of the 18S rRNA is shown in blue.",
    "molecules": "16S, 18S"
  },
  {
    "caption": "(C) Overview and zoomed view of Classical 80S*HCVIRES. The cryo-EM map of the Classical 80S*HCVIRES was filtered according to resolution.(D and E) mRNA exit channel from in Classical 80S*HCVIRES and POST state (PDB 5AJ0) (Behrmann et al, 2015). The two structures are aligned at the 40S body. We note eS28 as part of the 40S head is differently located because of the head tilt. The surface representation was created using the pdb models. Shown are the 18S rRNA (yellow), ribosomal proteins (gray), eS26 (cyan) eS28 (purple), HCVIRES (pink).",
    "molecules": "18S"
  },
  {
    "caption": "(A) Comparison of HCV IRES from 80S*HCV IRES Classical (pink) and tRNAs from POST state (gray)(PDB 5AJ0) (Behrmann et al, 2015) in a common 40S body alignment of the cryo-EM maps.(B) Same as above but in a common 40S head alignment.",
    "molecules": "tRNAs"
  },
  {
    "caption": "(C) Comparison of the 40S subunit and bound ligands of the classical 80S*HCVIRES complex (orange) and 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (yellow)(EMD 2683) (Yamamoto et al, 2014) in common 60S alignment.(D) The models of the 80S*HCVIRES Classical complex (gray), 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (green)(PDB 4UPZ) (Yamamoto et al, 2014) and 80S*HCVIRES Classical/no head tilt complex (pink) are rigid body fitted into the corresponding densities in a common 40S body alignment. The Helix 68 from the 28S rRNA is depicted as well (blue).",
    "molecules": "tRNA, GMPPNP, 28S"
  },
  {
    "caption": "(A and B) Comparison of the 80S*HCV IRES Classical complex and Classical/no head tilt complex (40S yellow, 60S blue, IRES pink, P-tRNA green) at intersubunit view. Inter subunit bridge 1b/c and 1a are indicated as B1b/c and B1a, respectively. The cryo-EM map of classical 80S*HCV IRES classical complex was filtered to 6 Å.(C and D) Close up of the mRNA tunnel. The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å units",
    "molecules": "tRNA, 18S"
  },
  {
    "caption": " C) Oxygen consumption rate of ES (blue line) and 2-cell-like cells (green line). Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Basal, maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates are indicated. A representative graph of 3 independent biological replicates performed on the Seahorse extracellular flux analyser is shown. Due to the low number of 2-cell-like cells available, compared to ESCs, one technical replicate of the former was analysed per biological replicate, while three or more technical replicates were performed for the latter. Accordingly, mean ± S.D. of technical replicates is shown for ESCs. ",
    "molecules": "Antimycin-A, FCCP, glucose, L-glutamine, pyruvate, Rotenone"
  },
  {
    "caption": " D) Basal oxygen consumption rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser. Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Boxes indicate the range between the first and third quantile, the band specifies the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each of the individual technical replicates. ",
    "molecules": "glucose, L-glutamine, pyruvate"
  },
  {
    "caption": " E) Oxygen consumption rate of ES (blue line), Zscan4+ (red line) and 2-cell-like cells (green line) in glucose-free media and upon acute injection of sodium pyruvate or sodium L-lactate. Note that L-glutamine - but not glucose or pyruvate - was initially present in the assay medium. Maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates following pyruvate or lactate treatment are also are indicated. A graph including data from 3 independent biological replicates is presented, and the mean ± S.D. of technical replicates is shown. ",
    "molecules": "Antimycin-A, FCCP, glucose, L-glutamine, lactate, pyruvate, Rotenone, sodium L-lactate, sodium pyruvate"
  },
  {
    "caption": " G) Representative single section of CellROX-DeepRed fluorescence in ES and 2-cell-like cells (green arrow) obtained using live cell microscopy. Scale bar, 10 μm. n, indicates the number of independent replicates. Representative images from 3 independent biological replicates are shown. ",
    "molecules": "CellROX-DeepRed"
  },
  {
    "caption": " H) FACS-assisted quantification of CellROX-DeepRed fluorescence intensity in ES and 2-cell-like cells. Measurements were obtained from 2 independent biological replicates. *** P < 0.005; Mann-Whitney U test. ",
    "molecules": "CellROX-DeepRed"
  },
  {
    "caption": " A) ATP content in ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 4 independent biological replicates. ",
    "molecules": "ATP"
  },
  {
    "caption": " C) Glucose uptake rates in Zscan4+ (red) and 2-cell-like cells (green) were measured using a luciferase-based assay across 4 independent biological replicates and are represented relative to those of control ES cells (blue). ",
    "molecules": "Glucose"
  },
  {
    "caption": " F) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4+ and 2-cell-like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each. ",
    "molecules": "glucose, Glucose"
  },
  {
    "caption": " Percentage of 2-cell-like cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. ",
    "molecules": "D-ribose, sodium acetate, sodium L-lactate"
  },
  {
    "caption": " Percentage of Zscan4+ cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. ",
    "molecules": "D-ribose, sodium acetate, sodium L-lactate"
  },
  {
    "caption": " Percentage of Zscan4+ cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. ",
    "molecules": "D-ribose, sodium acetate, sodium L-lactate"
  },
  {
    "caption": " Percentage of 2-cell-like cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. ",
    "molecules": "D-ribose, sodium acetate, sodium L-lactate"
  },
  {
    "caption": " A) Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate treated ESC cultures. Green arrows indicate 2-cell-like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm. ",
    "molecules": "acetate, DAPI"
  },
  {
    "caption": " B) RT-qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h. Shown are the mean ± s.d. of 3 independent cell cultures, performed in 2 technical replicates. ",
    "molecules": "sodium acetate"
  },
  {
    "caption": " C) Percentage of 2-cell-like cells obtained upon transfection of control or Dux-targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from 2 independent cell cultures, performed across 3 independent biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate. ",
    "molecules": "sodium acetate"
  },
  {
    "caption": " D) Percentage of 2-cell-like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of 4 independent cell cultures, performed across 2 independent biological replicates. ",
    "molecules": "sodium acetate"
  },
  {
    "caption": "(C) Reaction progress was measured for the Twist COVID-19 synthetic RNA (MT007544.1) from 1 million molecules of virus (106), then titrated down by log10 dilutions. The colorimetric findings of the LAMP assay are based on a yellow to pink gradient with higher copies of SARS-CoV-2 RNA corresponding to a yellow color. The limit of detection (LoD) range is shown with a gradient after 30 minutes between 10 and 100 viral copies (lower right).",
    "molecules": "RNA"
  },
  {
    "caption": "(E) The sensitivity and specificity of the LAMP assay from 201 patients (132 negative and 69 positive for SARS-CoV-2, as measured by qRT-PCR). Thresholds are DNA quantified by the Quantifluor.",
    "molecules": "DNA"
  },
  {
    "caption": "(e)Survival curves for requiring mechanical respiration (identified by intubation procedure notes) (e) and mortality (f). Patients with a history of ACE inhibitor exposure were more likely to require intubation (HR=2.63 95%CI 2.01-3.43, p=1.22E-12; (e) and less likely to survive (HR=1.68 95%CI: 1.22-2.31, p=1.42E-03; (f). Because several individuals were intubated shortly before they first tested positive for SARS-CoV-2 infection, each first positive test was set back by seven days to account for testing delays.",
    "molecules": "ACE inhibitor"
  },
  {
    "caption": "Untreated or Ruxolitinib  (10 µM)-treated PBMCs  from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs  inoculated with supernatant from Vero E6 cell cultures mixed with PBS  and OptiPro serum-free medium supplemented with 0.5% gelatine  were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated  time points  post-inoculation and analyzed for: Infectivity  in cell culture supernatants by plaque titration assay Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure Ruxo. = Ruxolitinib.",
    "molecules": "gelatine, PBS, Ruxo, Ruxolitinib"
  },
  {
    "caption": "PBMCs  from four individual donors were exposed to SARS-CoV-2 (MOI 0.5). PBMCs  inoculated with supernatant from Vero E6 cell cultures mixed with PBS  and OptiPro serum-free medium supplemented with 0.5% gelatine  were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Infectivity  in cell culture supernatants by  plaque titration assay  Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV  = Remdesivir",
    "molecules": "gelatine, PBS, Remdesivir, RMV"
  },
  {
    "caption": "Untreated or Ruxolitinib  (10 µM)-treated PBMCs  from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs  inoculated with supernatant from Vero E6 cell cultures mixed with PBS  and OptiPro serum-free medium supplemented with 0.5% gelatine  were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points  post-inoculation and analyzed for: Viral RNA (genome  equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; Ruxo. = Ruxolitinib.",
    "molecules": "gelatine, PBS, Ruxo, Ruxolitinib"
  },
  {
    "caption": "PBMCs  from four individual donors were exposed to SARS-CoV-2  (MOI 0.5). PBMCs  inoculated with supernatant from Vero E6 cell cultures mixed with PBS  and OptiPro serum-free medium supplemented with 0.5% gelatine  were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Viral RNA (genome  equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR  Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV  = Remdesivir",
    "molecules": "gelatine, PBS, Remdesivir, RMV"
  },
  {
    "caption": "Untreated or Ruxolitinib  (10 µM)-treated PBMCs  from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs  inoculated with supernatant from Vero E6 cell cultures mixed with PBS  and OptiPro serum-free medium supplemented with 0.5% gelatine  were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points  post-inoculation and analyzed for: (E) Relative changes of cell-associated  viral genomic RNA quantities by Q-RT-PCR  and normalized to RNASEP  levels. Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. h.p.e. = hours post-exposure Ruxo. = Ruxolitinib.",
    "molecules": "gelatine, PBS, Ruxo, Ruxolitinib"
  },
  {
    "caption": "RNA extracted from Ruxolitinib-treated or mock-treated, and SARS-CoV-, SARS-CoV-2- PBMCs  was analyzed for (A) IFIT1, (B) IFNA1  and IFNB1  mRNA expression by  Q-RT-PCR at indicated time points. Suspension and adherent cell fractions were analyzed separately, except at the four hours  time point. Values were normalized to cellular RNASEP  expression and are shown as fold change over mock-inoculated conditions. The dotted line indicates the expression level of mock-inoculated cell cultures and is set to 1. Data information: Data were generated in four individual experiments using PBMCs  from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M (A-B Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor and time points. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).",
    "molecules": "Ruxolitinib"
  },
  {
    "caption": "(C) Supernatants from Ruxolitinib- or mock-treated and  SARS-CoV-, SARS-CoV-2  or mock-inoculated PBMCs  were collected 48 hours post exposure and  cytokine expression of IFN-α2, IP-10/CXCL10, MCP-1/CCL2  and MCP-3/CCL7  were quantified using a Luminex-based immunoassay. PHA- or LPS-treated PBMCs  were used as a positive control. Bars represent the results of a pool of four individual samples per condition. Data information: Data were generated in four individual experiments using PBMCs  from four or more individual donors represented by different symbols, bars represent the mean, Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing  SARS-CoV to  SARS-CoV-2-treated samples from the same donor P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown). n.d. = not detectable.",
    "molecules": "LPS, PHA, Ruxolitinib"
  },
  {
    "caption": "(G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.",
    "molecules": "CHX, cycloheximide, DMSO, MG132"
  },
  {
    "caption": "(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "(B and C) H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53).",
    "molecules": "thapsigargin"
  },
  {
    "caption": "(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control (\"Th + si ctl\" or \"-\" ) or as indicated).",
    "molecules": "etoposide"
  },
  {
    "caption": "(D-E) A431 (D) and HT29 (E) cells endogenously expressing mutant R273Hp53 were treated with control siRNA (si Ctl) or control MO (MO Ctl-2 or MO Ctl-1) or siRNA targeting exon 7 of p53 (si) or MO targeting Δ160p53's translation initiation site (MO), as indicated, submitted to endoplasmic reticulum stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. wtp53-expressing A549 cells were similarly control-treated and used as comparison. Data was normalized against A549 cell condition, which was set to 100.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "(A) Kinetics of cell numbers in Lineage-EPCR+CD150+CD48- (L-ESLAM) HSCs, Lineage-EPCR+CD150+CD48+ (CD150+CD48+) progenitor cells, and Lineage-EPCR+CD150-CD48+ (CD150-CD48+) progenitor cells after the administration of 5-FU (250 mg/kg, i.v.). The small graph is an enlargement of 0 to 8 days after the administration of 5-FU. (* or **) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days, respectively.",
    "molecules": "5-FU"
  },
  {
    "caption": "(A) A principal component analysis (PCA) using the ATAC-seq data of HSCs in the steady state (untreated), early phase (5-FU 6d), or late phase (5-FU 10d).",
    "molecules": "5-FU"
  },
  {
    "caption": "mRNA (C) in Acly in HSCs after the administration of 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "protein (D), and phosphorylation levels of Ser455 (E) in Acly in HSCs after the administration of 5-FU.",
    "molecules": "5-FU, Ser"
  },
  {
    "caption": "The levels of intracellular Glu (G) in HSCs obtained from untreated or 5-FU-treated mice.",
    "molecules": "5-FU, Glu"
  },
  {
    "caption": "total acetylated lysine (I) in HSCs obtained from untreated or 5-FU-treated mice.",
    "molecules": "5-FU, lysine"
  },
  {
    "caption": "(B-C) The effects of CTPI-2 (B) or BMS303141 (BMS) (C) on H3K27ac in HSCs in the early phase after the administration of 5-FU.",
    "molecules": "5-FU, BMS, BMS303141, CTPI-2"
  },
  {
    "caption": "(D-E) The effects of CTPI-2 (D), BMS303141 (BMS) (E), A-485 (D), and TV-3664 (E) on HSC expansion in the early phase after the administration of 5-FU. Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells.",
    "molecules": "5-FU, BMS, BMS303141, A-485, CTPI-2, TV-3664"
  },
  {
    "caption": "Global H3K27ac (A), Acly levels (B) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.",
    "molecules": "5-FU"
  },
  {
    "caption": "acetylated lysine (C), and ΔΨm (D) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.",
    "molecules": "5-FU, lysine"
  },
  {
    "caption": "(C) The frequencies of the CD150+CD48- and CD48+ fractions within the lineage-EPCR+ fraction after a 4-day culture of HSCs in the late phase with or without BMS303141 (BMS; 100 μM), SB204990 (SB; 100 μM), an alternative Acly inhibitor, or CTPI-2 (50 μM). (D and E) Total cell number (D) or CD48+ cell number (E) after the 4-day culture of HSCs in the late phase with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment).",
    "molecules": "SB, SB204990, BMS, BMS303141, CTPI-2"
  },
  {
    "caption": "Western blotting of samples after ectopic overexpression of HBP1 (B) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Western blotting of samples after ectopic overexpression of ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2)",
    "molecules": "ubiquitin"
  },
  {
    "caption": "D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "B LNCaP cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h before being transfected with the indicated siRNAs. The cells were then cultured for the indicated times, and cell growth was measured by Cell Counting Kit-8. n = 3, *P < 0.0001. Error bars indicate SD.",
    "molecules": "R1881"
  },
  {
    "caption": "D LNCaP cells stably expressing shRNA against STAMP2 or control shRNA were treated with or without 1 nM R1881. The cells were then collected, and membrane fractions were prepared followed by Western blot analysis.",
    "molecules": "R1881"
  },
  {
    "caption": "D LNCaP cells were transfected with either control or STAMP2-specific siRNA. Three days after transfection, cells were treated with either 50 ng/ml TRAIL or 20 μmol/l LY294002 (LY) for 24 h, or both agents for 6 h, and then subjected to TUNEL/FACS analysis. Representative histograms of TUNEL-stained cells are shown. FL1-H refers to the gating of the cells for the TUNEL staining with fluorescence measurement.E The extent of apoptosis from the experiment in (D) is presented. Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.027; **P = 0.0058; ***P = 0.0016. Error bars indicate SD.",
    "molecules": "LY, LY294002"
  },
  {
    "caption": "A 22Rv1 cells were cultured in RPMI 1640 medium containing 10% CT-FBS and treated with or without 1 nM R1881 for 24 h and were then transfected with the indicated siRNAs. The cells were then cultured for 10 days. The colonies formed were stained and photographed.B Quantification of data from (A). Student's t-test was performed to analyze the statistical significance, n = 3. *P = 0.0003; **P < 0.0001. Error bars indicate SD.",
    "molecules": "R1881"
  },
  {
    "caption": "A LNCaP cells were transfected with either control or STAMP2-specific siRNA in the presence of 10−8 M R1881. RNA was isolated, and qPCR was used to determine ATF4mRNA levels. Student's t-test was used to analyze the statistical significance, n = 3. *P = 0.002. Error bars indicate SD.",
    "molecules": "R1881"
  },
  {
    "caption": "E 293T cell line with Dox-inducible STAMP2 expression or vector control was generated. Western blot analysis confirmed STAMP2 expression in a Dox-inducible manner.",
    "molecules": "Dox"
  },
  {
    "caption": "F The cells from (E) were either left untreated or treated with increasing amounts of Dox, and ferrireductase activity was determined. *P < 0.0001.",
    "molecules": "Dox"
  },
  {
    "caption": "G The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Prior to ferrireductase activity measurement, the cells were treated with or without DPI as indicated for 1 h. *P < 0.0001; **P = 0.0012; ***P = 0.048.",
    "molecules": "DPI, Dox"
  },
  {
    "caption": "H The cells from (E) were treated with 100 ng/ml Dox for 2 days. Oxidative stress was then measured by NBT staining, and the stained cells were photographed. There were equal numbers of cells on the plates for −/+ Dox as shown in Supplementary Fig S4.",
    "molecules": "Dox"
  },
  {
    "caption": "I The cells with Dox-inducible STAMP2 expression from (E) were either left untreated or treated with Dox for 48 h. Then, the cells were treated with or without 10 μM DPI before being subjected to NBT staining. *P < 0.0001.",
    "molecules": "DPI, Dox"
  },
  {
    "caption": "D LNCaP cells were transfected with either scrambled siRNA (siCtrl) or siRNA against STAMP2 (siST2) and were cultured in the presence of 10 nM R1881 or vehicle for 2 days. Intracellular ROS levels were then measured by CellROX reagent staining. *P = 0.002. ns, not significant.",
    "molecules": "R1881, ROS"
  },
  {
    "caption": "E LNCaP cells stably expressing an empty vector (Vec), a vector expressing wild-type STAMP2 (WT), or a STAMP2 mutant (dGSR) were cultured, and the NADPH/NADP+ ratio was determined as described in Materials and Methods. *P = 0.015.",
    "molecules": "NADP+, NADPH"
  },
  {
    "caption": "F LNCaP cells were transfected with either control siRNA (siCtrl) or STAMP2 siRNA (siST2). Cells were then cultured for 2 days and harvested, and the NADPH/NADP+ ratio was determined as above. *P = 0.026.",
    "molecules": "NADP+, NADPH"
  },
  {
    "caption": "G LNCaP cells stably expressing wild-type STAMP2 were treated with or without DPI (1 μM) for 4 h, harvested, and used in the NADPH/NADP+ assay. *P = 0.018.",
    "molecules": "DPI, NADP+, NADPH"
  },
  {
    "caption": "A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type, shh mRNA and dnPKA mRNA injected wild type and cyclopamine (Cyc(A)) treated wild type 20hpf embryos. Gli2a-FL levels are low relative to Gli2aR levels in wild type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a-FL is undetectable in CycA treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti-GRK3 (which recognizes the zebrafish Grk2 protein) reveals a complete loss of full length Grk2 in MZgrk2 embryos. Probing with anti-γ-tubulin was performed as a loading control. Three biological replicates of this analysis were performed.",
    "molecules": "Cyc(A), CycA, cyclopamine"
  },
  {
    "caption": "A Western blot analysis of GRK2, GLI1, PTCH1, and GLI3 protein levels in Flp-In-3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG. α-tubulin was used as loading control. Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments. GLI3 panel shows both full-length (GLI3FL) and repressor form of GLI3 (GLI3R). (n=3)",
    "molecules": "SAG"
  },
  {
    "caption": "B, C Hh reporter activity assay for wild type and Grk2-/- cells. Cells were treated with PKA peptide inhibitor (PKI) and mutant form of PKA peptide inhibitor (PKI-M) respectively, in presence or absence of SHH (B) Cells were transfected with mSmoA1-GFP expressing constructs in the presence of SHH or SAG (C). Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).",
    "molecules": "PKI, SAG"
  },
  {
    "caption": "E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo-/- MEFs in response to Shh or SAG stimulation. Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not; all mutants affecting phosphorylation failed to restore the response to Shh or SAG. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; and n.s. (not significant).",
    "molecules": "SAG"
  },
  {
    "caption": "C Hh reporter activity assay in wild type and Grk2-/- 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2, Grk2K220R, Grk2K220M and Grk2D335N after 24h Shh-N and SAG treatment. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; *P<0.05; and n.s. (not significant).",
    "molecules": "SAG"
  },
  {
    "caption": "B C99-Bpa constructs remain essentially cleavage-competent. Numbers denote the relative γ-secretase cleavage efficiencies of C99-Bpa substrates as calculated from the AICD/C99-Bpa ratios relative to that of WT C99, which was set to 1. Inefficiently cleaved substrates (cleavage efficiency below 20%) were mostly found for Bpa substitutions in extramembraneous domains. Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L-685,458. Residues of the TMD are highlighted in orange here and elsewhere where appropriate.C Values obtained in (B) were additionally plotted. Yellow line indicates the cleavage efficiency of WT C99, which was set to 1. ECD, extracellular domain; ICD, intracellular domain.",
    "molecules": "Bpa, L-685,458"
  },
  {
    "caption": "B Identification of C99 interaction sites with γ-secretase. C99-Bpa substrates were irradiated with UV light in the presence of CHAPSO-solubilized γ-secretase. Crosslinked substrates were captured by Ni-NTA affinity pulldown and bound subunits were identified by a ~10 kDa molecular weight increase compared to the input. Results are shown for the major substrate crosslinking residues. Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X-100, which dissociates the γ-secretase complex, proving crosslink specificity. Bracket indicates the molecular weight range of putative C99-APH-1 crosslink bands. APH-1aL, long splice variant of APH-1a.",
    "molecules": "Bpa, Triton X-100"
  },
  {
    "caption": "C Excess amounts of parental C99 WT substrate compete crosslinking of C99-Bpa substrates to γ-secretase. Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer, respectively.",
    "molecules": "Bpa"
  },
  {
    "caption": "A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site. L, L-685,458; D, DAPT.",
    "molecules": "DAPT, L-685,458"
  },
  {
    "caption": "B Substrates bound to exosites in the PS1 NTF at 4°C can be cleaved in the 37°C chase incubation in a L-685,458-dependent manner. Note that, although many of these substrates crosslinked only weakly at the exosites, their cleavage in the 37°C chase proved that these reflected functional substrate-binding states.C Substrates crosslinking in the exosites to NCT and PEN-2 cannot be cleaved.",
    "molecules": "L-685,458"
  },
  {
    "caption": "A bar-plot showing percentage of down- and up-regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation.",
    "molecules": "H3K27me3"
  },
  {
    "caption": "C Up, Representative western blot from kidney whole cell extracts, showing H2AX protein levels in 24 months old wt and junD-/- mice, either untreated (-) or treated with the anti-oxidant agent, N-acetyl-cysteine (NAC). Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 4 mice per treatment and per genotype.",
    "molecules": "N-acetyl-cysteine, NAC"
  },
  {
    "caption": "D,E Left, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and Kap1 phosphorylation (P-Kap1) in wt and junD-/- fibroblasts (D) or in wt and Nfe2l2-/- fibroblasts (E), after H2O2 exposure for the indicated times (hours, h). Right, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown on the left). n = 3 independent experiments.",
    "molecules": "H2O2"
  },
  {
    "caption": "F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, after camptothecin (CPT) treatment (H) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.",
    "molecules": "camptothecin, CPT"
  },
  {
    "caption": "G,I Up, Representative γ-H2AXimmunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after after camptothecin (CPT) treatment for 1h (I).. Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.",
    "molecules": "camptothecin, CPT"
  },
  {
    "caption": "B Up, Representative western blot from chromatin-free fraction extracts showing H2AX protein levels in wt and junD-/- fibroblasts either untreated (-) or after 8h treatment (+) with proteasome inhibitor MG132. Jun is used as positive control for treatment efficiency. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).C Bar plot showing stabilization of H2AX protein in wt and junD-/- fibroblasts, as assessed by densitrometry analysis of western blots (as shown in B). Data are expressed as percentage (%) of H2AX protein levels after treatment with MG132 with respect to untreated conditions.",
    "molecules": "MG132"
  },
  {
    "caption": "E Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels (revealed using Flag-specific antibody) from whole cell extracts in wt and junD-/- fibroblasts. Cells were first transfected with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and next treated with cycloheximide (CHX) for the indicated times (hours, h). Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in wt and junD-/- fibroblasts, as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "F Left, Representative western blots showing H2AX-WT protein levels (revealed using Flag-specific antibody) from whole cell extracts in junD-/- fibroblasts co-transfected with Flag-tagged H2AX-WT construct and either non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), RNF8 (siRNF8) or BMI1 (siBMI1). Efficiency of each siRNA has been verified and reached 60% of inhibition, in average, as shown for RNF168 (Up). Protein extracts were processed following cycloheximide (CHX) treatment for the indicated times (hours, h). Right, Bar graph showing the percentage (%) of decrease of H2AX-WT protein levels after 4h treatment with CHX (t=4) with respect to untreated conditions (t=0) in junD-/- fibroblasts transfected with specific siRNA, as indicated.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "G Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels from whole cell extracts in junD-/- fibroblasts transfected either with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and co-transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168). Cells were treated with cycloheximide (CHX) for the indicated times (hours, h) before protein extraction. Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in junD-/- fibroblasts transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "C Correlation plot between Cisplatin IC50 (half maximal inhibitory concentration) and H2AX protein level defined by western blots in BC cell lines (MCF7, BT474, MDA361, SKBr3, MDA453, MDA231, HCC70). Correlation coefficients σ and P-value are based on Spearman's rank correlation test.",
    "molecules": "Cisplatin"
  },
  {
    "caption": "E Bar plot showing the percentage (%) of apoptotic cells as assessed by flow-cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX), and next treated with cisplatin for 72 hrs.",
    "molecules": "cisplatin"
  },
  {
    "caption": "B. Statistical analysis of Parkin localization in cells subjected to AICD or to 10μM CCCP for the indicated times is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).",
    "molecules": "CCCP"
  },
  {
    "caption": "C. Cells were treated as in (A). Where indicated, lysosomal proteases inhibitors E64D and PepA were added. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected cells per condition. Data represent mean ± SE of three independent experiments. Scale bar, 5μm",
    "molecules": "E64D, PepA"
  },
  {
    "caption": "H. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated, lysosomal proteases inhibitors E64D and PepA were added. WB are representative of at least three independent experiments.",
    "molecules": "E64D, PepA"
  },
  {
    "caption": "H-I. Cells where treated as in (A) and where indicated 10μM H89 was added. Quantitative analysis of P-AMPK Ser485-491 phosphorylation (H) and of LC3-II/LC3-I ratio (I) during AICD are shown.",
    "molecules": "H89"
  },
  {
    "caption": "J-K. AICD was induced in Jurkat cells (J) and in hPBT cells (K) in presence of 10μM H89. At indicated time points (hours) after AICD induction, apoptotic cells were detected by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown.",
    "molecules": "H89"
  },
  {
    "caption": "A. Apoptotic cells were detected at indicated time points after AICD induction in hPBT cells by flow cytometry as AnnexinV/PI double positive cells. Where indicated hPBT cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Data represent mean ± SE of six independent experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "B. AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of six independent experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "C. Morphometric analysis of mitochondrial shape 24h after AICD induction. Jurkat cells were transfected with mtYFP 24h before AICD induction and, where indicated, 100nM rapamycin was added. Data represent mean ± SE of four independent experiments (n = 30 cells per condition in each experiment).",
    "molecules": "rapamycin"
  },
  {
    "caption": "D. Representative electron micrographs of cells subjected to AICD untreated (upper panel) or treated with 100nM rapamycin (lower panel). 24h after AICD induction cells were fixed and processed for electron microscopy, Scale bar, 2μm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "D. Jurkat cells were co-transfected with GFP-LC3 and Parkin-Cherry, 32h after AICD induction cells were fixed and immunostained with an anti-TOM20 antibody. Representative images for each condition are presented. Scale bar, 5μm.E. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments. Data referring to Ctrl and AICD-stimulated cells without rapamycin correspond to the ones presented in Fig.3C.",
    "molecules": "rapamycin"
  },
  {
    "caption": "B. Splenocytes were isolated from ATG7 fl/fl and wt C57/BL6mice. Activation of splenocytes and AICD were performed as previously described. At day 5 after activation, cells were infected with retroviral constructs expressing CRE recombinase. Where indicated, cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Apoptotic cells were detected 2h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.",
    "molecules": "rapamycin"
  },
  {
    "caption": "C. Jurkat cells were transfected with scramble or ATG7 siRNA 24h before AICD induction. AICD was induced as described in materials and methods. Where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of five independent experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": " Quantification of piRNA sensor expression. Representative DIC and fluorescence microscopy images of piRNA sensor expression in wild type (mjIs144) (D) and rpb‑9 (mj261) animals (two images with varying GFP expression are shown, scale bar = 20 µm) (E) ",
    "molecules": "piRNA"
  },
  {
    "caption": "
 representative DIC and fluorescence microscopy images of piRNA sensor expression in the rpb-9 rescue (mj261; mjSi70) animals (two images with varying GFP expression are shown). Scale bar = 20 µm (G). 
",
    "molecules": "piRNA"
  },
  {
    "caption": " A) Differential expression analysis of transposable elements in rpb-9 (mj261) mutants versus wild type (polyA-selected RNA-seq libraries). [p<=0.01, log2(fold change)>=1, DESeq2 analysis] ",
    "molecules": "polyA"
  },
  {
    "caption": " C) Genome-wide differential gene expression analysis of rpb-9 (mj261) mutants versus wild-type (polyA-selected RNA-seq libraries). [p<=0.01, log2(fold change)>1 or <-1, DESeq2 analysis]. ",
    "molecules": "polyA"
  },
  {
    "caption": " D) Differential expression analysis of germline-specific and soma-specific genes, as classified in (Reinke et al, 2004). \"All genes\" is shown for comparison. (PolyA-selected RNA-seq libraries, n=3). ",
    "molecules": "PolyA"
  },
  {
    "caption": " Pairwise correlation plots of rpb-9 (mj261) and prg-1 (n4357) (E), hrde-1 (tm1200) transcriptomes. Differentially expressed genes in rpb-9 (mj261) are shown in black, shared upregulated genes in red, shared downregulated genes in blue. The piRNA sensor transcript is highlighted in green. (Total RNA \"Ribo-Zero\" RNA-seq libraries). ",
    "molecules": "piRNA"
  },
  {
    "caption": " Pairwise correlation plots of gonad-dissected prg-1 (n4357) transcriptomes. Differentially expressed genes in rpb-9 (mj261) are shown in black, shared upregulated genes in red, shared downregulated genes in blue. The piRNA sensor transcript is highlighted in green. (Total RNA \"Ribo-Zero\" RNA-seq libraries). ",
    "molecules": "piRNA"
  },
  {
    "caption": " A-C) Analysis of RNA Pol II binding (RPB-1/AMA-1 ChIP-seq) at upregulated genes in rpb-9 (mj261) mutants, as defined in Figure 3C. Class I: upregulated genes with increased RNA Pol II binding (A); Class II: upregulated genes with invariant RNA Pol II binding (B); Class III: upregulated genes with reduced RNA Pol II binding (C). n=3 biological replicates are shown per each genotype. (PolyA-selected RNA-seq libraries). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. ",
    "molecules": "PolyA"
  },
  {
    "caption": " D) RNA Pol II binding profile over the piRNA sensor locus. ",
    "molecules": "piRNA"
  },
  {
    "caption": "  C) Transcriptome binning according to increasing 22G siRNA density in wild-type animals (grey, n=3). Mean normalized rpb-9 (mj261) mRNA reads (red, n=3) are overlaid with mean normalized wild-type mRNA reads (grey). (PolyA-selected RNA-seq libraries). The piRNA sensor transcript is indicated with red (rpb-9 (mj261)) and grey (wild type) dots. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. ",
    "molecules": "piRNA, PolyA"
  },
  {
    "caption": " D) Distribution of piRNA targets (as defined in (Bagijn et al, 2012) and of HDRE-1- and CSR-1-dependent 22G siRNAs across bins as defined in (C)).",
    "molecules": "piRNA"
  },
  {
    "caption": " E) TE binning according to increasing 22G siRNA density in wild-type animals (grey, n=3). Mean normalized rpb-9 (mj261) mRNA reads (red, n=3) are overlaid with mean normalized wild-type mRNA reads (grey). (PolyA-selected RNA-seq libraries). The CEMUDR1 and Chapaev-2 transcripts are indicated with red (rpb-9 (mj261)) and grey (wild type) box plots. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. ",
    "molecules": "PolyA"
  },
  {
    "caption": "  G) Distribution of normalized 22G siRNA reads mapping over the piRNA sensor. ",
    "molecules": "piRNA"
  },
  {
    "caption": "A) Expression levels of mature piRNAs in wild-type, rpb-9 (mj261), rpb-9 rescue (mj261; mjSi70) and prg-1 (n4357) animals (n=3, hyper-geometric test). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.",
    "molecules": "piRNAs"
  },
  {
    "caption": " B) Mature piRNA expression along chromosome IV coordinates (motif-dependent piRNA clusters I and II) in wild-type and rpb-9 (mj261) animals. ",
    "molecules": "piRNA"
  },
  {
    "caption": "
  C) Northern blot quantification of mature piRNA 21UR-1 levels in wild-type, prg-1 (n4357), mut-7 (mj253) and rpb-9 (mj261) animals. 
",
    "molecules": "piRNA"
  },
  {
    "caption": " A) Median precursor length shift of nascent (left) and nucleoplasmic (right) piRNA precursors in rpb-9 (mj261) mutants and rescue (mj261; mjSi70) animals compared to wild-type (n=2). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. B) Distribution of nascent piRNA precursor lengths in rpb-9 (mj261) and rescue (mj261; mjSi70) animals compared to wild type. C) Peak positions of nascent piRNA precursor length distributions in 2000 subsamples of 5000 precursor sequences sampled without replacement. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. D) Distributions of nucleoplasmic piRNA precursor lengths in rpb-9 (mj261) and rescue (mj261; mjSi70) animals compared to wild type. ",
    "molecules": "piRNA"
  },
  {
    "caption": " E) Distribution of 5′ ends of short capped RNA reads mapping to motif-dependent piRNA loci. Total counts of read 5′ ends mapping within a 50bp window of motif-dependent piRNA loci, aggregated by position relative to 21U-RNA 5′ U sites. A comparable enrichment of reads initiating 2 nt upstream of 21U-RNA 5′ U sites is observed across replicates and genotypes. ",
    "molecules": "piRNA"
  },
  {
    "caption": " F) rpb-9 recruits Integrator at piRNA loci. Representative single-plan immunofluorescence images of Integrator (ints-6::GFP ) and PRDE-1 (prde-1::mCherry) localization in wild-type and rpb-9 (mj261) animals. Scale bar = 5 µm. G) Co-localization analysis of PRDE-1::mCHERRY and INTS-6::GFP foci in wild-type and rpb-9 (mj261) animals. Central bands represent the median, notches represent the confidence interval around the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. n (nuclei/genotype) > 16. (Welch's t-test, p = 1.7e-17).",
    "molecules": "piRNA"
  },
  {
    "caption": " H) Positions of unique 5′ monophosphate small RNA reads mapping at piRNA promoters after removal of reads >15 nt initiating at annotated piRNA 5′ U sites. The average signal is normalized to sequences per million of mapped reads. ",
    "molecules": "piRNA"
  },
  {
    "caption": "(E) The number of binucleated hepatocytes per field was counted on H&E stained paraffin sections of PBS treated wt and Pidd1-/- mice at t(0h) and at t(48h) after PBS or DEN treatment. N-numbers refer to biological replicates.",
    "molecules": "DEN, paraffin, PBS"
  },
  {
    "caption": "(F) Immunofluorescence of the same livers was perfomed for the distal centriolar protein CP110 to stain centrioles, β-catenin for cell borders, and DAPI to stain DNA. Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles is shown.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) After knockdown of Tollip, SH-SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM Antimycin A (AA) alone for 2 hours, then fixed and stained for PDH E2/E3bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4x magnification and arrowheads indicate TOM20-positive MDVs.",
    "molecules": "antimycin A, Antimycin A, oligomycin B"
  },
  {
    "caption": "(E) SH-SY5Y cells were transfected with GFP-labelled Tollip for 24 hours then treated with AO, AO/100 μM bafilomycin A1 (AO/BfnA1) or AA alone for 2 hours prior to fixation. Cells were then immunostained for GFP (green), PDH E2/E3bp (red) and TOM20 (blue) and imaged by widefield immunofluorescence microscopy. GFP-Tollip was observed on TOM20+ve/PDH-ve MDVs (arrowheads). Zoom insets represent 7x magnification.",
    "molecules": "bafilomycin A1, BfnA1"
  },
  {
    "caption": "(B) SH-SY5Y cells were transfected with GFP-Tollip for 24 hours, then treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 hours prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used.",
    "molecules": "antimycin A, bafilomycin A1, BfnA1, oligomycin B"
  },
  {
    "caption": "C) GFP-Tollip/Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3-6 GFP-transfected cells/condition, per experiment (n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin.",
    "molecules": "BfnA1"
  },
  {
    "caption": "(E-F) SH-SY5Y cells were transfected with GFP-Tollip R78A or CUE mutant for 24 hours, then treated with AO or AO/BfnA1 for 2 hours prior to fixation and immunostaining",
    "molecules": "BfnA1"
  },
  {
    "caption": "(A) HeLa cells expressing mycBioID-Tollip and HA-Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 hours in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS-PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID-Tollip were used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions.",
    "molecules": "antimycin A, bafilomycin A, BfnA1, biotin, biotinylated, Biotinylation, oligomycin B, streptavidin"
  },
  {
    "caption": "(D) HeLa cells expressing mycBioID-Tollip containing the CUE domain mutation (CUEmut) and HA-Parkin were subjected to biotinylation and streptavidin pulldowns, followed by western blot analysis.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(A) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours, then fixed and stained with antibodies specific to TOM20 (green) and PDH E2/E3bp (red). Nuclei are labelled with Hoechst (blue). Zoom insets represent 5x magnification and arrowheads indicate TOM20-positive MDVs.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(H) HeLa cells transiently transfected with HA-Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A (AA) for 2 hours, then fixed and stained with antibodies specific to HA, PDH and TOM20. TOM20+ve/PDH-ve MDVs were quantified in cells that were either expressing or not expressing HA-Parkin (n = 12-14 cells per condition from 3 independent experiments).",
    "molecules": "antimycin A"
  },
  {
    "caption": "(I) Hela cells stably coexpressing BioID-Tollip and either HA-Parkin WT or Δ1-76 (ΔUBL) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns. Western blot analysis was performed on lysate inputs and streptavidin pulldowns (Streptavidin PD) using antibodies against the indicated proteins or epitope tags.",
    "molecules": "biotin, streptavidin, Streptavidin"
  },
  {
    "caption": "(A-B) siRNA knockdown of Tollip or Parkin in SH-SY5Y cells was performed over a 72 hour period, then 18 hours prior to treatment cells were transfected with GFP-2xFYVE to label endocytic membranes. Cells were treated with 25 μM antimycin A (AA) for 2 hours then fixed and immunostained with antibodies specific to PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs that colocalise with GFP-2xFYVE and arrows denote TOM20+ve/PDH-ve MDVs that do not.",
    "molecules": "antimycin A"
  },
  {
    "caption": "(F) SH-SY5Y cells were transfected with GFP-Tollip and treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3bp (blue). (G) Quantification of GFP-Tollip/Rab7a colocalisation using Pearson's correlation.",
    "molecules": "antimycin A, bafilomycin A, BfnA1, oligomycin B"
  },
  {
    "caption": "(A) Quantitation of Tollip vesicular clusters in SH-SY5Ys treated with 5 μM antimycin A/10 μM oligomycin B (AO) for 2 hours. The percentage of cells with vesicular clusters were quantified from 7-23 cells for each condition from 4 independent experiments.",
    "molecules": "antimycin A, oligomycin B"
  },
  {
    "caption": "(A) A subset of TOM20+ve/PDH-ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH-SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM antimycin A (AA) for 2 hours. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3bp (red) and TOM20 (blue). Arrows denote TOM20+ve/PDH-ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. (B) From immunofluorescence images, the total number of TOM20+ve/PDH-ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell (n = 25-32 cells per condition from 3 independent experiments).",
    "molecules": "antimycin A, oligomycin B"
  },
  {
    "caption": "C and D Defective mTORC2  signaling upon Elp3  deficiency in myeloid cells.  Peritoneal macrophages from Elp3Control and Elp3∆Mye mice  were isolated and cultured ex-vivo. They were untreated or stimulated with a combination of LPS  (100 ng/ml) and IFNγ  (50 ng/ml) for the indicated periods of time  and the resulting cell extracts were subjected to WB  analyses.",
    "molecules": "LPS"
  },
  {
    "caption": "F mTORC2  activation by M1 polarization signals relies on Ctu2. Control and Ctu2-depleted bone marrow-derived macrophages  (BMDMs) (SiRNA-mediated depletions) were treated or not with IFNγ  (50 ng/ml) and LPS  (100 ng/ml) and the resulting cell extracts were subjected to WB  analyses.",
    "molecules": "LPS"
  },
  {
    "caption": "C Elp3  deficiency in myeloid cells  exacerbates the decrease of colon  length upon DSS  administration (n = 7 mice; mean +/- SD, **= p< 0.01).",
    "molecules": "DSS"
  },
  {
    "caption": "E More severe histological  score in mice  lacking Elp3  in myeloid cells  upon DSS  administration. Representative intestinal crypts from the indicated genotypes are illustrated. The histological  score was established as described in methods (n = 5 mice  per genotype; mean +/- SD, Student T test, *= p< 0.05).",
    "molecules": "DSS"
  },
  {
    "caption": "F Increased infiltration of iNOS+/CD68+ cells upon DSS  administration in  Elp3∆Mye mice. The percentage of iNOS+ in CD68+ cells was established in both genotypes treated with DSS  (n = 7 mice  per genotype; mean +/- SD, Student T test, **= p< 0.01). Anti-iNOS  and -CD68 immunofluorescence  analyses carried out in  intestinal crypts from both genotypes are illustrated.",
    "molecules": "DSS"
  },
  {
    "caption": "A IL-4  and IL-13 induce the expression of tRNA-modifying enzymes in macrophages. Peritoneal macrophages were untreated (« Control ») or stimulated with  IL-4 (10 ng/ml), IL-13 (10 ng/ml) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB  analyses.",
    "molecules": "tRNA"
  },
  {
    "caption": "B The pool of thiolated tRNAs  in macrophages  is enhanced by IL-4/IL-13 but decreased by LPS/IFNγ. On the left, Northern blot  analysis assessing t:Q(UUG) tRNA thiolation  in Thioglycollate-elicited peritoneal macrophages subjected to the indicated treatments for 24 hours. On the right, quantification of t:Q(UUG) tRNA thiolation, calculated as the ration of thiolated  over nonthiolated t:Q(UUG), in the indicated experimental conditions.",
    "molecules": "LPS, Thioglycollate, tRNA, tRNAs"
  },
  {
    "caption": "C Elp3  induction by  IL-4/IL-13 occurs through a PI3K-dependent pathway. Peritoneal macrophages were pretreated with DMSO  (control vehicle) or with Wortmannin  (200 nM) for one hour and subsequently left untreated or stimulated with IL-4  (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB  analyses.",
    "molecules": "DMSO, Wortmannin"
  },
  {
    "caption": "F  IL-4/IL-13  rely on Stat6  to induce Elp3  expression. Peritoneal macrophages  were pretreated with the control vehicle (DMSO) or with the Stat6  pharmacological inhibitor AS1517499  (5 μM) for one hour and subsequently left unstimulated or treated with IL-4 (10 ng/ml)/IL-13  (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB  analyses.",
    "molecules": "AS1517499, DMSO"
  },
  {
    "caption": "B Elp3  promotes Ric8B   mRNA translation. BMDMs  from Elp3Control and Elp3∆Mye mice  were treated by  IL-4  for 24 hours, followed by a treatment with 10 μg/ml puromycin  for 5 minutes. To detect newly synthesized Ric8b  proteins, a Puro-PLA  assay was conducted. Representative images detecting Ric8b (Red) and α-Tubulin+ microtubules  (green) in BMDMs  are illustrated. On the right, the graph shows a quantification of signals for Ric8b  in α-Tubulin+ areas. A random of six different areas were counted (6 technical replicates, mean +/- SD, Student's t-test, *= p< 0.05).",
    "molecules": "Puro, puromycin"
  },
  {
    "caption": "Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).",
    "molecules": "LysoTracker"
  },
  {
    "caption": "Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm. Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).",
    "molecules": "LysoTracker"
  },
  {
    "caption": "LysoTracker signal colocalizes with the astroglial reporter slc1a2b:citrine and does not appear to overlap with the pan-neuronal reporter elavl3:GCaMP5G.",
    "molecules": "GCaMP5G, LysoTracker"
  },
  {
    "caption": "Identification of the ENU-induced hearing loss pedigree MPC169, subsequently named clarinet. ABR phenotyping of pedigree Muta-Ped-C3PDE-169 at 3-months of age identified 8 mice with elevated hearing thresholds (red triangles) compared to their normal hearing colony mates (n=61, black triangles). Indeed, all eight affected mice were found to not respond to the highest intensity stimulus (90 dB SPL) at the three frequencies tested, or the click stimulus, and so their thresholds are shown as 95 dB SPL.",
    "molecules": "ENU"
  },
  {
    "caption": "Saturating MET currents recorded from P6 Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) apical-coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at −121 mV and +99 mV. The driver voltage (DV) signal of ± 40 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The holding potential was −81 mV. Extracellular Ca2+ concentration was 1.3 mM. Arrows and arrowheads indicate the closure of the MET currents (i.e. resting MET current) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials, respectively. Dashed lines indicate the holding current, which is the current at the holding membrane potential.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Potassium currents recorded from mature Clrn2clarinet/+ (A, P15) and Clrn2clarinet/clarinet (B, P16) apical-coil OHCs. Currents were elicited by depolarizing voltage steps (10 mV nominal increments) from -144 mV to more depolarized values from the holding potential of -84 mV. Note that the current characteristic of mature OHCs, The size of IK,n, measured in isolation as the deactivating tail currents (difference between instantaneous and steady-state inward currents) for voltage steps from the holding potential to −124 mV, was 545 ± 115 pA, (n=5) in Clrn2clarinet/+ and 595 ± 67 pA, (n=7) in Clrn2clarinet/clarinet OHCs.",
    "molecules": "Potassium"
  },
  {
    "caption": "Average peak current-voltage relation for the total K+ current recorded from Clrn2clarinet/+ (P15-16, n=7) and Clrn2clarinet/clarinet (P15-16, n=5) OHCs.",
    "molecules": "K+"
  },
  {
    "caption": "Potassium currents recorded from P22 mature Clrn2clarinet/+ and Clrn2clarinet/clarinet apical-coil IHCs, respectively, using the same voltage protocol described above. Holding potential of -64 mV.",
    "molecules": "Potassium"
  },
  {
    "caption": "Peak current-voltage relation for the total K+ current recorded from the IHCs",
    "molecules": "K+"
  },
  {
    "caption": "Voltage responses from P22 Clrn2clarinet/+ (G) and Clrn2clarinet/clarinet (H,I) IHCs. Note that Ca2+-dependent action potentials could be induced in mature IHCs (I).",
    "molecules": "Ca2+"
  },
  {
    "caption": "TBH cytotoxicity assay in vivo of the ScAhp1 lysine mutants in yeast. Growth  of yap1Δahp1Δ cells carrying empty vector (ev), wild type peroxiredoxin   gene (AHP1) or indicated cysteine or lysine substitutions was monitored without or with 1 mM TBH. ScAhp14KR: ScAhp1C31S K47/48R K124R K156R; ScAhp15KR: ScAhp1C31S K32R K47/48R K124R K156R.",
    "molecules": "TBH"
  },
  {
    "caption": "TBH cytotoxicity assay in vivo. Basic growth and the synthetic effect in wild type, ahp1Δ, urm1Δ,  tum1Δ, ncs6Δ, cys3Δ, cys4Δ, cys3Δ urm1Δ, and cys4Δ urm1Δ strains was monitored without or with 1 mM TBH.",
    "molecules": "TBH"
  },
  {
    "caption": "H2S  production using Biggy agar plates- Wild type, ahp1Δ, urm1Δ, tum1Δ, ncs6Δ, cys3Δ,  cys4Δ,  cys3Δ urm1Δ, and cys4Δ urm1Δ strains were grown on Biggy medium, and H2S  production was recorded",
    "molecules": "H2S"
  },
  {
    "caption": "Northern-blot  analysis for thiolation  levels of tRNA<sub>UUC</sub><sup>Glu</sup>  in the genetic background of the indicated yeast  strains. Total tRNA  was resolved on  denaturing PAGE  supplemented with APM  to retard the migration of thiolated tRNAUUCGlu. APM: ([N-Acryloyl-amino] phenyl) mercuric chloride. (bottom) Quantified thiolation  levels from 3 independent biological replicates.",
    "molecules": "([N-Acryloyl-amino] phenyl) mercuric chloride, APM, thiolated, tRNA, tRNAUUCGlu"
  },
  {
    "caption": "Three independent samples (Exp 1 to 3) of EV secreted by labeled 3T3-F442A cells were analyzed by mass spectrometry (in duplicate injections, Inj1/2). The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated.",
    "molecules": "amino acid"
  },
  {
    "caption": "Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n=5).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6).",
    "molecules": "FA, Lipids"
  },
  {
    "caption": "Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy.",
    "molecules": "BODIPY FL C16, FL C16, DAPI"
  },
  {
    "caption": "Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n=3).",
    "molecules": "BODIPY FL C16, Etomoxir, DAPI"
  },
  {
    "caption": "Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n=5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n=15).",
    "molecules": "fatty acid, Lipids"
  },
  {
    "caption": "Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24h. Then, cells were fixed, stained with BODIPY and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n=5 for SKMEL28 and n=6 for 1205Lu).",
    "molecules": "BODIPY, DAPI"
  },
  {
    "caption": "Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). Scale bar represents 1µm. A zoomed crop of the area with autophagic structures containing lipids (indicated by an arrow) is shown (scale bar: 0.5µm).",
    "molecules": "lipids"
  },
  {
    "caption": "1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization.",
    "molecules": "BODIPY FL C16, LysoTracker, Lalistat 2"
  },
  {
    "caption": "1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BOBIPY FL C16 staining per cell is shown beside (n=4).",
    "molecules": "BOBIPY FL C16, BODIPY FL C16, DAPI, Lalistat 2"
  },
  {
    "caption": "1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). Cell migration was then evaluated in Boyden chamber assays (left panel; n=5) or cell motility was tracked by video microscopy (right panel; n=40 cells per group).",
    "molecules": "Lal, Lalistat 2"
  },
  {
    "caption": "1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY and counterstain with DAPI before observation by confocal microscopy. Quantification of BODIPY staining per cell is shown on the right (n=5).",
    "molecules": "BODIPY, DAPI, Lal, Lalistat 2"
  },
  {
    "caption": "1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy (n=40 cells per group).",
    "molecules": "Lal, Lalistat 2"
  },
  {
    "caption": "1205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15).",
    "molecules": "Mdivi-1, MitoTracker"
  },
  {
    "caption": "1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel).",
    "molecules": "Mdivi-1"
  },
  {
    "caption": "1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).",
    "molecules": "Mdivi-1"
  },
  {
    "caption": "Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=6).",
    "molecules": "BODIPY, Phalloidin"
  },
  {
    "caption": "Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=5 for SKMEL28 and n=6 for 1205Lu).",
    "molecules": "BODIPY, Phalloidin"
  },
  {
    "caption": "1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown (scale bar: 5µm).",
    "molecules": "BODIPY, MitoTracker"
  },
  {
    "caption": "Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions (n=3).",
    "molecules": "BODIPY, LysoTracker"
  },
  {
    "caption": "BODIPY staining of neutral lipid stores in the indicated melanoma cell lines, which present increasing degrees of aggressiveness (from left to right). DAPI was used to counterstain nuclei. Scale bar: 20µm.",
    "molecules": "BODIPY, DAPI, lipid"
  },
  {
    "caption": "(B) Immunoblot analysis of pro- (p31) and cleaved (p17) IL-1β, and pro- (p45) and cleaved (p20) caspase-1 in cytosolic fractions of ASC deficient (Pycard-/-) immortalized murine macrophages that were primed with LPS (200 ng ml−1, for 3h). Fractions were incubated with recombinant ASC specks in the presence of 2, 20 or 200 µg ml−1 of VHHASC or mutVHHASC. One representative of three independent experiments is shown. (C) Quantitative analysis of band densitometry of three independent experiments as shown in B. Data represents the fold change from all conditions (lanes 1- 9) .vs Pycard−/− lysates incubated with ASC specks alone (lane 2). ns, p > 0.05; *, p<0.05; **, p<0.005; ***, p<0.0002, One-way ANOVA, multiple comparison (Dunnet test). Data is displayed as floating bars with the max/min values and mean (thicker band).",
    "molecules": "LPS"
  },
  {
    "caption": "(C) WES capillary electrophoresis and immunoblotting of DSS cross-linked in-vitro-generated human ASC-mTurquoise (ASC) specks that were incubated for 1h (RT) with 2, 20 or 200 µg ml−1 of VHHASC, mutVHHASC or with 20 µg ml−1 of a polyclonal anti-ASC Ab (a-ASC pAb). Data is from one representative of four independent experiments.",
    "molecules": "DSS"
  },
  {
    "caption": "A-D (A-B) Human IL-1β (hIL-1β) concentrations in cell-free supernatants of LPS-primed (10 ng ml−1, 150 min) primary human macrophages that were left untreated, or pre-incubated with VHHASC or mutVHHASC (100 µg ml−1), CRID3 (50 µM) or VX-765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml−1) for 2h. (C-D) Mouse IL-1β (mIL-1β) concentrations in cell-free supernatants of LPS-primed mouse BMDMs (200 ng ml−1, 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml−1). Data is combined from two independent experiments, each performed with two donors (A-B) or mice (C-D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band).",
    "molecules": "LPS, CRID3, nigericin, VX-765, VX765"
  },
  {
    "caption": "E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC-mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml−1, 150 min), pre-treated with VX-765 (50 µM, 30 min), then treated with VHHASC, VHHmASC (100 µg ml−1) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2h and finally fixed with 4% PFA and nuclei stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are representative of one out of three independent experiments that were quantified in F. Data is displayed as floating bars with the max/min values and mean (thicker band).",
    "molecules": "DRAQ5, LPS, CRID3, nigericin, PFA, VX-765"
  },
  {
    "caption": "THP-1 cells expressing a Dox-inducible CRISPR-Cas9 cassette targeting GSDMD gene were treated with 1 µg ml-1 Dox for one or two cycles of 72 h (1x, or 2x respectively). As control, cells were left untreated and cultured in parallel (-). (A) Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA-differentiation, where indicated. Data is from one representative of two independent experiments.",
    "molecules": "Dox, PMA"
  },
  {
    "caption": "GFP+ specks detected in culture supernatants by (D) immunoblotting analysis of ASC-GFP oligomeric state (DSS cross-linked) of extracellular specks recovered from supernatants of cells treated similarly to (A-B). (D) is from one out of at least two independent experiments.",
    "molecules": "DSS"
  },
  {
    "caption": "(A) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 µm.",
    "molecules": "PMA"
  },
  {
    "caption": "(B) Representative confocal microscopy images of pPMNs that were treated without (control) or with 50 nM PMA for 3 h, and stained for lamin B and DNA as described in panel B. The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope. Scale bar 10 µm.",
    "molecules": "DNA, PMA"
  },
  {
    "caption": "(E) Summary analysis of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, then fixed by 2% PFA and stained by Sytox Green, following by detection with fluorescent microplate reader.",
    "molecules": "Sytox Green, PFA, PAF"
  },
  {
    "caption": "(F) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.",
    "molecules": "Sytox Green dye, DNA, PAF, Triton-X-100"
  },
  {
    "caption": "(G,H) Representative images (G) and summary analysis (H) of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red and cell impermeable Sytox Green, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (G) Scale bars, 40 μm.",
    "molecules": "SYTO Red, Sytox Green, PAF"
  },
  {
    "caption": "(A,B) Representative and summary of immunoblots of full-length lanes of lamin B in human dPMNs that were treated with PMA for 0, 0.5, 1, 2, 3 h during NET formation, or apoptotic dPMNs that were induced by PMA for longer term (12 h) treatment (A). β-actin served as loading control",
    "molecules": "PMA"
  },
  {
    "caption": "(C,D) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0, 0.5, 1, 2, 3 h during NET formation. β-actin served as loading control",
    "molecules": "PAF"
  },
  {
    "caption": "(E) Representative confocal microscopy image of a group of pPMNs with NET formation (enclosed by a light blue trapezoid) and their extracellular NETs (indicated by a purple rectangle square) released by these cells, as well as the immunoblot analysis of un-cleaved lamin B and cleaved histone H3 with the whole cell lysates, as well as immunoblot analysis of un-cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment. The anti-human lamin B was used in all immunoblots confocal image (E), and the latter was further detected by FITC-labeled 2nd Ab, scale bar, 20 μm.",
    "molecules": "FITC, PMA"
  },
  {
    "caption": "(F) Representative immunoblots with full-length lanes for lamin B analysis, and pro-caspase-3 and its activated form caspase-3 in human dPMNs with NET formation that were treated with PMA for 0, 0.5, 1, 2, 3 h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control",
    "molecules": "PMA"
  },
  {
    "caption": "(G) Representative immunoblots display full-length lamin B (69 kDa) and their cleaved fragments (45 and 25 kDa), and pro-caspase-3 and its activated form caspase-3 in the apoptotic human dPMNs that were treated with PMA for 12, 24h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control",
    "molecules": "PMA"
  },
  {
    "caption": "(A) Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min, then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), following by confocal fluorescent microscopy analysis. White-empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining.",
    "molecules": "DAPI, DNA, FITC, PE, PMA"
  },
  {
    "caption": "(B) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti-lamin B, and FITC-labeled secondary antibody.",
    "molecules": "FITC"
  },
  {
    "caption": "(C Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PMA (C) for 0, 0.5, 1, 2, 3 h.",
    "molecules": "PMA"
  },
  {
    "caption": "D) Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PAF (D) for 0, 0.5, 1, 2, 3 h.",
    "molecules": "PAF"
  },
  {
    "caption": "(A) Representative immunoblots of p-PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h.",
    "molecules": "PMA"
  },
  {
    "caption": "(B) Representative sectional images of nuclei of two neutrophils treated by PMA, stained for lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), p-PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody), and nuclear DNA (DAPI). The accumulation of p-PKCα (red arrow pointed) at the site of ruptured/interrupted (green arrow) nuclear lamin B, and swollen/de-condensed DNA (blue arrow) were indicated (b1). A neutrophil (white empty arrow) with NET formation that contained the mixture of DNA with p-PKCα and lamin B (white arrow) was also demonstrated (b2). Scale bars, 10 µm.",
    "molecules": "DAPI, DNA, FITC, PE, PMA"
  },
  {
    "caption": "(C) Immunoblot images of lamin B, then stripped and probed for p-PKCα, of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA.",
    "molecules": "PMA"
  },
  {
    "caption": "(D,E) Representative and summary immunoblot (IB) detection of phospho-lamin B and total lamin B with the lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were treated either by PMA (D,E) for 0 or 3 h. Data represent mean ± SD (n=3-5 biological replicates) for E. P*<0.05 between groups as indicated. Comparison between two groups was analyzed by student t test.",
    "molecules": "PMA"
  },
  {
    "caption": "(A) Summary and representative immunoblots of total PKCα and p-PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated without or with PMA (A) for 3 h. Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit (A)",
    "molecules": "Go6976, PMA"
  },
  {
    "caption": "(B) Immunoblot (IB) detection of the phospho-lamin B and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated by PMA (B) for 3 h.",
    "molecules": "Go6976, PMA"
  },
  {
    "caption": "(C) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h, and then treated without or with PMA for 3 h, followed by staining of lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and phosphorylated PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody). The green/white arrows indicate the site of nuclear envelope rupture. Scale bars, 10 μm (C).",
    "molecules": "FITC, Go6976, PE, PMA"
  },
  {
    "caption": "(E) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B (WT control), or mutants with single or multiple defined point mutations at PKCα-consensus-phosphorylation sites (S395A, S405A, S408A) of lamin B, and treated for 3 h by 10 μM PAF in medium containing cell-permeable dye SYTO Red (500 nM) for the total cell count, while the cells with NET formation were stained by cell impermeable dye SYTOX Blue (5 μM). Then the images were taken with Olympus confocal microscopy, followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation. percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ (E)",
    "molecules": "SYTO Red, SYTOX Blue, PAF"
  },
  {
    "caption": "(B) Representative immunoblot (IB) detection of the phospho-lamin B (Ser) and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from mouse peritoneal mPMNs from WT vs PKCα-/- mice, stimulated without or with PAF for 3 h.",
    "molecules": "Ser, PAF"
  },
  {
    "caption": "(C) Summary analyses of NET formation in peritoneal mPMNs, from WT, PKCα+/-, or PKCα-/- mice, which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2% PFA fixation and fluorometric microplate analysis.",
    "molecules": "PFA, PAF"
  },
  {
    "caption": "(D) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα-/- mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.",
    "molecules": "Sytox Green dye, DNA, PAF, Triton-X-100"
  },
  {
    "caption": "(E, F) (E) Representative and (F) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα-/- mice stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (E) Scale bars, 50 μm. % cells with NET formation by image analysis using ImageJ (F)",
    "molecules": "SYTO Red dye, Sytox Green dye, PAF"
  },
  {
    "caption": "(A,B) representative analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.",
    "molecules": "Alexa Fluor-488, Alexa Fluor-647, cit, citrullinated, DAPI, DNA"
  },
  {
    "caption": "(C) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.",
    "molecules": "Alexa Fluor-488, Alexa Fluor-647, cit, citrullinated, DAPI, DNA"
  },
  {
    "caption": "(D,E) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL-17A (D) or TNFα (E), in the skin tissue of WT vs Lmnb1TG mice without (sham) or with UVB exposure, by co-staining of Ly6B (stained as described in panels A-C) with IL-17A (D) or TNFα (E) using FITC-conjugated rat anti-mouse IL-17A, or FITC conjugated rat anti-mouse TNFα Abs.",
    "molecules": "FITC"
  },
  {
    "caption": "(A) Growth curve of recombinant BL21 (DE3) cells upon IPTG mediated induction of proteins, represented as colored line graphs.",
    "molecules": "IPTG"
  },
  {
    "caption": "(E, F) Graph showing the effect of native TsiTBg and HTH or Imm52 domain deleted variant of TsiTBg proteins on Gus reporter gene expression (quantified using 4-methylumbelliferyl ß-D-glucuronide, MUG, as substrate) under the external (pExtTseTBg1/pExtTseTBg2) and internal (pIntTseTBg1/pIntTseTBg2) promoters in E. coli.",
    "molecules": "4-methylumbelliferyl ß-D-glucuronide, MUG"
  },
  {
    "caption": "(A) Interaction of external (pExtTseTBg1) and internal (pIntTseTBg1) promoter DNA fragments of TseTBg1 operon with the TsiTBg1 protein. (B) Interaction of pExtTseTBg1 and pIntTseTBg1 promoter DNA fragments of TseTBg1 operon with the HTH domain deleted variant of TseTBg1 protein (TsiTBg1ΔHTH). (C) Interaction of pExtTseTBg2 and pIntTseTBg2 promoter DNA of TseTBg2 operon with the TsiTBg1 protein. (D) Interaction of control (random) DNA with the TsiTBg1 protein. ",
    "molecules": "DNA"
  },
  {
    "caption": "Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS (4.5 mg/kg BW) or PBS injection (i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM",
    "molecules": "LPS"
  },
  {
    "caption": "Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection. (B) Cxcl1 levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (C) TNF-α levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (D) IL-1β levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. Each dot (B-D) represents an individual mouse. Horizontal lines indicate mean.",
    "molecules": "LPS"
  },
  {
    "caption": "Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL-1 receptor antagonist (IL-1RA) Anakinra (200 µg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n=5 mice, vertical lines indicate SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4 WT and KO mice injected with LPS, LPS and IL-1RA or control. Horizontal lines indicate mean.",
    "molecules": "LPS"
  },
  {
    "caption": "Percent body weight of male Prdx4-flox and Prdx4-∆LysMCre mice over the 48 h course of 4.5 mg/kg BW LPS ( i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4-flox and Prdx4-∆LysMCre mice injected with LPS. Each dot (B, D) represents an individual mouse. Horizontal lines indicate mean.",
    "molecules": "LPS"
  },
  {
    "caption": "Concentration of Cxcl1, TNF-α and IL-1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation (100 ng/ml LPS, time points indicated).",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β release of Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for indicated time points with ATP (5 mM).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "Western blot analysis of IL-1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs, primed with LPS (100 ng/ml) and pulsed with ATP (5 mM) for 4 h or left untreated. Dashed line indicates vertical slice.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 45 min of LPS-primed cells. Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI. Scale bar indicates 20 µm. ASC speck-positive cells were counted and expressed as percentage of total cells. Bars represent a mean of n=4 mice, vertical lines indicate SD. n.s. not significant (two-tailed t-test).",
    "molecules": "DAPI, LPS, Nigericin"
  },
  {
    "caption": "Western blot analysis of caspase-1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 1 h after priming with LPS (100 ng/ml), LPS-priming alone or without stimulation. Whole cell lysates were analyzed for pro-caspase-1 and Gapdh levels.",
    "molecules": "LPS, Nigericin"
  },
  {
    "caption": "IL-1β release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or Flagellin (1 µg/ml each) or treated with transfection agent only.",
    "molecules": "poly (dA:dT), ATP, LPS, Nigericin"
  },
  {
    "caption": "Quantification of cell death by LDH-release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or flagellin (1 µg/ml each) or treated with transfection agent only.",
    "molecules": "poly (dA:dT), ATP, LPS, Nigericin"
  },
  {
    "caption": "Rate of caspase-1 activity in the presence of non-reduced decameric rPrdx4, reduced dimeric and monomeric rPRDX4, YVAD or control.",
    "molecules": "YVAD"
  },
  {
    "caption": "Foldchange in IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 WT or Cys-to-Ser mutants C362S or C397S and co-transfected with Prdx4 or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.",
    "molecules": "ATP, Cys, Ser"
  },
  {
    "caption": "IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.",
    "molecules": "Ala, ATP, Cys"
  },
  {
    "caption": "Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT or control and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A.",
    "molecules": "Ala, Cys"
  },
  {
    "caption": "Western blot analysis of Prdx4, pro-caspase‑1, Gapdh and E-Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP-stimulated BMDMs or untreated controls.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs, primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP. Each circle represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "Concentration of Prdx4 in the serum of WT mice, injected with LPS (4.5 mg/kg BW) for the time points indicated. Each dot represents an individual mouse. Horizontal lines indicate mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).",
    "molecules": "LPS"
  },
  {
    "caption": "Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs. Cells were primed with LPS (100 ng/ml) for 6 h, followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation. Each bar represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).",
    "molecules": "YVAD, ATP, DMSO, LPS"
  },
  {
    "caption": "Relative levels of Prdx4 secretion in response to LPS+ATP stimulation and pretreatment with either Glycine, NSA or GW4869 and relative levels of caspase-1 secretion in response to LPS+ATP stimulation and pretreatment with in response to LPS+ATP stimulation and pretreatment with GW4869. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; n.s. not significant (two-tailed t-test).",
    "molecules": "ATP, Glycine, GW4869, LPS, NSA"
  },
  {
    "caption": "Western blot analysis of subcellular organelle fractions. OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS (100 ng/ml) for 12 h and stimulated with 5 mM ATP for 4 h.",
    "molecules": "LPS"
  },
  {
    "caption": "Cxcl1 concentration in supernatants of caspase-1-deficient BMDMs stimulated with EVs from LPS, LPS and ATP or control-treated Prdx4 WT or KO BMDMs, as well as caspase-1-deficient BMDMs pre-treated with Anakinra and stimulated with EVs from LPS and ATP-treated Prdx4 WT or KO BMDMs. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD.",
    "molecules": "Anakinra, ATP, LPS"
  },
  {
    "caption": "Serum Cxcl1 in C57Bl6/N mice injected with either PBS or EVs from LPS and ATP or control-treated Prdx4 WT or KO or ASC KO BMDMs. Each dot represents an individual mouse. Horizontal lines indicate mean.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(B) Alternative splicing of sams-3, sams-4 and sams-5 in unfed and fed worms without or with a protein synthesis inhibitor emetine. Synchronized L1 larvae of an NMD-deficient strain KH1668: smg-2 (yb979) (lanes 1-3) and a wild-type strain N2 (lanes 4-6) were incubated in S-complete medium alone (lanes 1, 4), with a standard E. coli strain OP50 (OD600=10.0) (lanes 2, 5) or OP50 supplemented with 10 mg/ml emetine (lanes 3, 6) for 3 hours at 20°C. Total RNAs were extracted from whole animals and subjected to semi-quantitative RT-PCR, whose products were analyzed by capillary electrophoresis. Representative gel-like presentation is indicated (n=3). Schematic structure of each PCR product is indicated on the right. Open reading frames (ORFs) for full-length and truncated proteins are in orange and cyan, respectively. rpl-12 was used as an unaffected control.",
    "molecules": "emetine"
  },
  {
    "caption": "(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3).",
    "molecules": "cLeu, cycloleucine, L-Met"
  },
  {
    "caption": "(B, C) Western blot analysis of SAMS-1, SAMS-3 and SAMS-4 during larval development in the smg-2 (yb979) (B) and wild-type (C) backgrounds. Genotypes of the worms are smg-2 (w) and smg-2; sams-5; sams-1 (s) in (B) and wild-type (w) and sams-1 (s) in (C). Synchronized L1 larvae of each strain were incubated with OP50 at 20°C and subjected to Western blot analysis at indicated time points. Anti-β-tubulin (B) or coomassie brilliant blue (CBB) staining (C) was used as a loading control. Specificity of the antibodies is confirmed in Appendix Figure S12. Note that upregulation of SAMS-1 protein in the wild type during larval development in (C) is consistent with feeding-induced upregulation of sams-1 mRNA in Figure EV3D.",
    "molecules": "coomassie brilliant blue"
  },
  {
    "caption": "(A) Mass chromatograms of RNA fragments to detect m6A (top) or unmodified A (bottom) after in vitro incubation of 127-nt sams-3/sams-4 pre-mRNA (sequence available in Fig EV2) with recombinant full-length METT-10 protein in the presence (left) or absence (right) of 1 mM SAM. The sequence, m/z value, and charge state for each fragment are indicated on the right. Asterisks indicate non-specific signals.",
    "molecules": "m6A, SAM"
  },
  {
    "caption": "(B) The negatively-charged ions of RNase T1 fragment is decomposed in the instrument by collision-induced dissociation (CID) using helium gas. The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel. Nomenclature of the product ions are described in the literature (McLuckey et al, 1992). Product ions of c and y series derive from the 5' and 3' termini of the fragment, respectively.",
    "molecules": "helium"
  },
  {
    "caption": "A, B) m6A-IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal/productive 3'SS. (A) Representative gel-like images (n=3) of semi-quantitative RT-PCR analysis of sams-3, sams-4 and sams-5 in input and immunoprecipitated (IP) RNAs from the smg-2 (yb979) mutant. The splicing patterns were analyzed and presented as in Figure 2B. (B) Relative enrichment of total and each of mRNA isoforms compared to eef-1A.1 mRNA by RT-qPCR. Error bars indicate standard error of mean (n=3). ( ",
    "molecules": "m6A"
  },
  {
    "caption": "C) Examples of normalized Nanopore currents for unmodified and m6A-modified in-vitro transcribed sams-3/sams-4 RNAs at nucleotide positions -4 through +4 relevant to the m6A site. One hundred reads are lotted for each. Color codes are indicated. (D) Distribution of mean (top) and standard deviation (bottom) of normalized Nanopore currents at nucleotide positions -4 through +4 relevant to the m6A site for 5,000 of the unmodified and 2,680 of the m6A-modified sams-3/sams-4 RNAs and 85 of an endogenous sams-3 mRNA isoform E2/E3L. Color codes are indicated. Central bands represent the median, boxes represent the 25th and 75th percentiles, and whiskers represent the lowest and highest values. A red line at position 0 in the top panel indicates a cut-off line (mean current = 1.7557) that discriminates between the unmodified and m6A-modified sams-3/sams-4 RNAs with accuracies of 64.58% for both. ( ",
    "molecules": "m6A"
  },
  {
    "caption": "C, D Two types of TNTs were detected in  H1299  (C) and  A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and  F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin  (red) were used for fluorescent staining. Scale bar: 20 μm. Data information: nuclei were  stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.",
    "molecules": "Hoechst 33342, phalloidin"
  },
  {
    "caption": "H,I H1299 cells stably expressing the Ctr or GFP-M2 were cultured in the presence of 20 μM 2-DG for 24 h to induce TNT formation. TNTs were imaged by confocal microscopy (A) and the percentage of cells forming TNTs was quantified (B). n=21, 20 and 19 images were captured from Ctr, GFP-M2#1 and GFP-M2#2, respectively. Three independent experiments were performed. Scale bar: 60 μm. Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed",
    "molecules": "2-DG"
  },
  {
    "caption": "A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in  collagen-coated dishes and cultured for 24 h before  time-lapse life cell  images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel. B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining  using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm. C Data information: nuclei were stained with Hoechst 33342 (blue).",
    "molecules": "Hoechst 33342, collagen, phalloidin"
  },
  {
    "caption": "E Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in  galactose medium or co-cultured through a filter.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining  with anti-Tom20  antibody (green) and staining using  phalloidin (red). Scale bar: 25 μm. Data information:  nuclei were stained with Hoechst 33342 (blue).",
    "molecules": "Hoechst 33342, phalloidin"
  },
  {
    "caption": "A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting  Miro2. Following transfection, cells were stained with anti-Tom20 antibody (green) and  phalloidin (red). Confocal microscopic images are shown. Scale bar: 20 μm. Data information: nuclei were stained  with Hoechst 33342 (blue).",
    "molecules": "Hoechst 33342, phalloidin"
  },
  {
    "caption": "A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B  (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted. Data information: data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.",
    "molecules": "5-FU, Cyto B"
  },
  {
    "caption": "D MICAL2PV mutants containing the MO domain are active in depolymerizing F-actin. Following transfection of plasmids expressing different HA-MICAL2PV mutants (#1, #2, or #4) into shM2 cells, confocal microscopy was performed following staining with phalloidin (red) and anti-HA antibody (green).  F-actin signals were reduced in cells expressing different mutants (HA-positive green cells marked by dashed lines). Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).",
    "molecules": "Hoechst 33342, phalloidin"
  },
  {
    "caption": "C. IF  for Cas9  and CENP-C of metaphases  of RPE1-dCas9-Kin14VIb  cells transduced with Chr1-telo  sgRNA in the presence or absence of 500 nM rapalog. The Chr1-telo  loci (Cas9  foci, white arrowheads) are aligned on the metaphase  plate in control condition (- rapalog), while the loci are facing either a single or opposite poles  after rapalog  addition to induce Kin14VIb binding  to dCas9. Scale bar = 5 µm. For clarity, the maximum intensity projection of a subset of z stacks (18-50/100) is shown. Data information: (C for optimal visibility of the Chr1-telo  foci, the brightness and contrast of all channels were linearly adjusted for individual cells.",
    "molecules": "rapalog"
  },
  {
    "caption": "D,  Live cell microscopy   of asynchronously growing RPE1-dCas9-Kin14VIb  cells transduced with Chr1-telo  sgRNA in the presence or absence of 500 nM rapalog  to induce kinesin binding  to the subtelomeric  locus. (D) Stills showing the most frequently observed metaphase localization  and daughter cell  distribution (white arrowheads) of the duplicated Chr1-telo  loci. SiR-DNA  was used to visualize the DNA, GFP  depicts the Chr1-telo  loci. Time  (h:min). Scale bar = 5 µm. Note that in the upper panel, the cell was already in mitosis when imaging started (t =0:00). Data information: (D, Cells were derived from three independent imaging experiments. N = 23 cells (- rapalog), n = 41 cells (+ rapalog). Sp = same pole, op = opposite pole, ss = same sisters. D): for optimal visibility of the Chr1-telo  foci, the brightness and contrast of all channels were linearly adjusted for individual cells.",
    "molecules": "rapalog, SiR-DNA, DNA"
  },
  {
    "caption": "B,  Live cell microscopy  of asynchronously growing RPE1-dCas9-Kin14VIb  cells transduced with Chr9-cen  sgRNA and plus/minus rapalog  to induce kinesin binding  to the pericentromeric  locus (Movies S4-S8). (B) Stills of the most frequently observed metaphase  orientations and daughter cell distributions of the duplicated Chr9-cen  loci (white arrowheads) are shown.  SiR-DNA was used to visualize the DNA, GFP  depicts the Chr9-cen  loci. Time  (h:min). Scale bar = 5 µm. Data information: Cells were derived from two independent imaging experiments.. In (B), for optimal visibility of the Chr9-cen  foci, the brightness and contrast of all channels were linearly adjusted for individual cells. See Movies EV4- S8 for equally adjusted corresponding examples.",
    "molecules": "rapalog, SiR-DNA, DNA"
  },
  {
    "caption": "D. Time  between  nuclear envelop break down (NEB) and anaphase  onset in the presence (Kin14VIb  bound to dCas9) or absence (Kin14VIb  expressed, but not bound to dCas9) of rapalog. Data information: Cells were derived from two independent imaging  experiments.. (D): Mean and S.D. are shown.",
    "molecules": "rapalog"
  },
  {
    "caption": "A. Representative IF  images of RPE1-dCas9-Kin14VIb  cells, showing the localization  of the Chr9-cen  loci in metaphase  in the absence or presence or rapalog. Magnifications of the white boxed regions (each region showing one Chr9-cen  locus) are shown in the corners (scale bars = 2 µm). The fraction of cells with Chr9  mis-aligned and CENP-C near the locus (Cas9) is indicated below the image. For clarity, the maximum intensity projection of a subset of z stacks in which the Chr9-cen  loci are in focus (2-10/100) is shown. B. Frequency of the observed metaphase localizations  of the Chr9-cen  loci as shown in (A). Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. (B): **** P<0.0001, Fisher's exact test. N ≥ 31 cells per condition, per experiment. (A, For optimal visibility of the Chr9-cen  foci the brightness and contrast of all channels were linearly adjusted for individual cells.",
    "molecules": "rapalog"
  },
  {
    "caption": "(C) GST-LC3B but not GST-p62 or GST alone binds to FYCO1 in a GST pull-down assay. GST, GST-LC3B, or GST-p62 bound to glutathione-Sepharose beads was incubated with [35S]methionine-labeled myc-FYCO1. Formed protein complexes were isolated and visualized by autoradiography (top) or Coomassie blue staining (bottom).",
    "molecules": "methionine"
  },
  {
    "caption": "(B) The region of FYCO1 between aa 1,276 and 1,294 is essential and sufficient for the interaction with LC3B. GST or GST-LC3B were incubated with [S35]methionine-labeled deletion mutants of FYCO1 and processed as in Fig. 1 C.",
    "molecules": "methionine"
  },
  {
    "caption": "(D) Both N- and C-terminal domains of LC3B are needed for efficient interaction with FYCO1. GST, GST-LC3B, or its deletion mutants were incubated with [S35]methionine-labeled myc-FYCO1 and processed as in Fig. 1 C. ARG, autoradiography; CB, Coomassie blue.",
    "molecules": "methionine"
  },
  {
    "caption": "(C) FYCO1 partially colocalizes with LC3B, LAMP1, ATG5, ATG16L, p62, and HuntingtinQ64 and does not colocalize with EEA1. HeLa cells or mouse embryonic fibroblasts (for ATG16L staining) were transfected with the indicated vectors or stained with the indicated antibodies and imaged 24 h after transfection. For p62 staining, HeLa cells were incubated with 0.2 µM BafA1 for 12 h before fixation.",
    "molecules": "BafA1"
  },
  {
    "caption": "(B-D) Immunoelectron micrographs of HeLa cells transfected with GFP-FYCO1 and stained with anti-GFP polyclonal antibody (10-nm gold particle).",
    "molecules": "gold"
  },
  {
    "caption": "(C) FYCO1 can self-interact in a CC-dependent manner. Full-length GFP-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of myc-tagged FYCO1 in rabbit reticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.",
    "molecules": "S35"
  },
  {
    "caption": "(D) Dimers but not a monomer of the FYVE domain from FYCO1 can bind PI3P in protein-lipid overlay assay. PIP Strips were incubated with 1-µg/ml solutions of MBP-FYVEFYCO1, MBP-2xFYVEFYCO1, or MBP-FYCO1863-1,233 for 1 h, and bound proteins were detected by immunostaining with anti-MBP antibody. LPA, lysophosphatic acid; LPC, lysophosphocholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.",
    "molecules": "phosphatidylserine, PS, LPA, lysophosphatic acid, LPC, lysophosphocholine, PA, phosphatidic acid, PC, phosphatidylcholine, PE, phosphatidylethanolamine, phosphatidylinositol, PI, PI3P, S1P, sphingosine-1-phosphate"
  },
  {
    "caption": "(B) FYCO1990-1,233 is the smallest FYVE domain-containing deletion mutant of FYCO1 that can efficiently interact with the full-length FYCO1. Full-length myc-FYCO1 was cotranscribed and cotranslated with the indicated deletion mutants of GFP-tagged FYCO1 in rabbitreticulocyte lysate. S35-labeled FYCO1 complexes were immunoprecipitated with anti-SDS-PAGE antibody, separated by SDS-PAGE, and visualized by autoradiography. IP, immunoprecipitation.",
    "molecules": "S35"
  },
  {
    "caption": "(B) Peripheral enrichment of FYCO1 is dependent on intact MTs. HeLa cells were transfected with GFP-FYCO1 and cultured in normal medium (left) or treated with 5 µM colcemid (middle) or 5 µM latrunculin A (right) for 2 h.",
    "molecules": "colcemid, latrunculin A"
  },
  {
    "caption": "(A) Raji cells stably expressing HIV-1 YU-2 ENV (Raji-Env) were stained with bNAbs (10-1074 or 3BNC117) or isotype control (mGO53) and analyzed by flow cytometry. The numbers indicate the % of positive cells. One representative experiment is shown.",
    "molecules": "10-1074, mGO53, 3BNC117"
  },
  {
    "caption": "(B) Binding of the 24 indicated antibodies to the surface of Raji-Env cells. Results are expressed as the median fluorescence intensity (MFI) of staining. n=6 independent experiments. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.",
    "molecules": "mGO53"
  },
  {
    "caption": "(D) C3 deposition by the 24 indicated antibodies. For each antibody, a normalized MFI is calculated by subtracting the MFI of the "no antibody" condition. (nd. not done). n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.",
    "molecules": "mGO53"
  },
  {
    "caption": "(F) CDC induction by the 24 indicated antibodies. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.",
    "molecules": "mGO53"
  },
  {
    "caption": "(G) Raji-Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs (10-1074, 3BNC117 or PGDM1400) or isotype control (mGO53). Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the "no antibody" condition. n=3 donors of serum.",
    "molecules": "10-1074, mGO53, 3BNC117, PGDM1400"
  },
  {
    "caption": "(A) Raji-Env cells were subcloned to obtain cell lines expressing increasing amounts of Env. Env surface levels were determined by the binding of 10-1074 and measured by flow cytometry. One representative experiment is shown.",
    "molecules": "10-1074"
  },
  {
    "caption": "(B) Parental Raji cells and the different Raji-Env clones were cultured with 10-1074 or an isotype control (mGO53) and NHS for 24h. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum.",
    "molecules": "10-1074, mGO53"
  },
  {
    "caption": "(C) Non-infected (NI) and HIV-1-infected primary CD4 T cells (strain NLAD8) as well as three Raji-Env clones (D1, D2 and D4) were incubated with 10-1074 and antibody surface levels were analyzed by flow cytometry. Results are expressed as median fluorescence (MFI) of staining in total cells (Raji, n=3 independent experiments) or in infected (Gag+) cells (CD4 T cells, n=5 donors).",
    "molecules": "10-1074"
  },
  {
    "caption": "(D) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry (left panel). The staining obtained with mGO53 was similar to the background signal observed on unstained cells. Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected (Gag+) cells were determined by flow cytometry (right panel). One representative experiment is shown.",
    "molecules": "mGO53"
  },
  {
    "caption": "(E) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were incubated with NHS and either 10-1074 (left panel) or 3BNC117 (right panel). Both WT or E430G-mutated antibodies were used. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. The MFI of C3 staining on Gag- cells was included as a control. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.",
    "molecules": "10-1074, 3BNC117"
  },
  {
    "caption": "(C) CD4 T cells not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with the serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown on the left. On the right, results are expressed as MFI of staining. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Error bars indicate SEM. *P<0.05, Wilcoxon test. Only significant comparisons are depicted.",
    "molecules": "sKB104, sKB105"
  },
  {
    "caption": "(D) Primary CD4 T cells either not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with 50% NHS from a healthy donor and serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:100). The levels of C3 deposition on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown (left panel). Results are expressed as MFI of staining (right panel). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.",
    "molecules": "sKB104, sKB105"
  },
  {
    "caption": "(E) Correlation between patients' sera (sK104 and sKB105) antibody binding and C3 deposition on CD4 T cells either mock-infected or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058). Each dot is the mean of 6 donors of CD4 T cells. Correlation was analyzed by Spearman correlation. Correlation coefficient (r) and p-value are indicated.",
    "molecules": "sK104, sKB105"
  },
  {
    "caption": "(A) In vitro pull-downs between strep(II)-tagged DDB1-DCAF12 and untagged wild type (WT) CCT5 or CCT5 (1-529) (Δ(Ct)) seen on a Coomassie-stained SDS-PAGE gel.",
    "molecules": "strep(II)"
  },
  {
    "caption": "(B) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1-DCAF12 or terbium-coupled streptavidin (Tb-SA) (n=3). Signal originating in the absence of TbDDB1-DCAF12 is unspecific and was subtracted in subsequent experiments.",
    "molecules": "ATTO488, SA, streptavidin, Tb, terbium"
  },
  {
    "caption": "(C) TR-FRET counter-titration of unlabeled wild type (WT) CCT5 or an unlabeled CCT520 peptide into pre-assembled TbDDB1-DCAF12488 (n=3).",
    "molecules": "Tb"
  },
  {
    "caption": "(D) TR-FRET counter-titrations of label-free CCT5 C-terminal peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position and corresponding CCT5 amino acid number. (E) Counter-titration of unlabeled CCT510 mutant peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position. D",
    "molecules": "Tb"
  },
  {
    "caption": "(D) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type (WT) or mutant TbDDB1-DCAF12 complexes (n=3). Unspecific signal arising from the Tb-SA label was subtracted.",
    "molecules": "ATTO488, SA, Tb"
  },
  {
    "caption": "(E) In vitro ubiquitination of CCT5 by wild type (WT) or mutant CRL4DCAF12 complexes in the presence of ubiquitin, ATP, E1 and E2 enzymes.",
    "molecules": "ATP, ubiquitin"
  },
  {
    "caption": "(A) Counter-titration of unlabeled CCT5 or unlabeled TRiC into pre-assembled TbDDB1-DCAF12488 (n=3).",
    "molecules": "Tb"
  },
  {
    "caption": "(B) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin, ATP, E1 and E2 enzymes. CCT5, but not TRiC, is modified with poly-ubiquitin (Ubn) chains by CRL4DCAF12. Free CCT5 protein carries an N-terminal strep(II) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC.",
    "molecules": "ATP, poly-ubiquitin, Ubn, strep(II), ubiquitin"
  },
  {
    "caption": "(A,B) Effect of MPC inhibition on cellular lipid droplet content. (A) Live-cell super-resolution confocal imaging of primary brown adipocytes pre-stained overnight with the fatty acid tracer BODIPY C12 558/568 (Bodipy C12, red). Cells were stained with mitotracker green (MTG, green) prior to imaging. Cells were imaged before and 120 minutes after treatment with either vehicle (DMSO) or 100 nM UK5099. As a positive control, cells were imaged 30 min after treatment with 1 µM norepinephrine. LD, lipid droplet; N, nucleus. Scale bar=10 µm. (B) Quantification of changes in lipid droplet cross-sectional area with indicated treatments from images shown in (A). Data are represented as percentage change in LD area at time=120 min compared to time=0 min.",
    "molecules": "Bodipy C12, BODIPY C12 558/568, UK5099, DMSO, fatty acid, mitotracker green, MTG, norepinephrine"
  },
  {
    "caption": "(C,D) Effect of MPC inhibition on fatty acid uptake and incorporation into triacylglycerides (TAGs) (C) Representative thin-layer chromatography (TLC) plate of lipids extracted from primary brown adipocytes and cell culture media. Cells were incubated with Bodipy C12 in presence of vehicle (V), 100 nM UK5099 (UK) or 1 µM norepinephrine (NE) for 24 h and triacylglyceride (TAG) synthesis (Lipids in cells) and fatty acid uptake (Lipids in media) were detected. The relative polarity of the lipid species determines the motility, with nonpolar TAG migrating the furthest. (D) Quantification of C12 uptake from media, TAG in cells and TAG per amount of free fatty acid uptake shown in (C), n=5 individual experiments. UK5099 treatment decreases the amount of synthesized TAG per free fatty acid uptake, similarly to NE.",
    "molecules": "Bodipy C12, C12, UK, UK5099, fatty acid, lipid, lipids, Lipids, NE, norepinephrine, TAG, TAGs, triacylglyceride, triacylglycerides"
  },
  {
    "caption": "(E-G) Effect of UK5099 treatment on cellular energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or UK5099 at indicated concentrations for 2 h. Oxygen consumption rates (OCR) were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK-5099. (E) Quantification of non-stimulated OCR from n=3-7 individual experiments. Data were normalized to vehicle for each individual experiment. UK5099 increases OCR in non-stimulated brown adipocytes. vehicle. (F) Brown adipocytes were treated with 100 nM UK5099 or vehicle. Etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. Representative OCR traces averaging 6 technical replicates. (G) Quantification of non-stimulated OCR, etomoxir sensitive, and etomoxir insensitive OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. *",
    "molecules": "UK-5099, UK5099, AA, antimycin A, DMSO, Eto, Etomoxir, etomoxir, glucose, glutamine"
  },
  {
    "caption": "(H,I) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid. (H) Schematic representation of metabolite tracing using [U-13C16] palmitate. (I) [U-13C16] palmitate tracing in fully differentiated primary brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h. Data shows mole percent enrichment (MPE) of isotope labeled substrate into respective metabolite.",
    "molecules": "UK5099, fatty acid, [U-13C16] palmitate"
  },
  {
    "caption": "(A,B) Effect of UK5099 treatment on norepinephrine-stimulated energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 4 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and Eto-sensitive OCR as shown in (A) from n=9 individual experiments. The effects of UK5099 treatment were normalized to vehicle for each experiment.",
    "molecules": "UK5099, AA, antimycin A, ATP, DMSO, Eto, etomoxir, glucose, glutamine, NE, norepinephrine, Norepinephrine, Oligo, oligomycin A"
  },
  {
    "caption": "Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (F) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (G) Quantification of basal, NE-stimulated and Eto-sensitive OCR as shown in (F) from n=7 individual experiments. Data were normalized to Scramble RNA for each individual experiment. (H) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n=5 individual experiments. Data were normalized to vehicle for each experiment.",
    "molecules": "UK5099, AA, antimycin A, Eto, etomoxir, glucose, glutamine, NE, Norepinephrine, Oligo, oligomycin A"
  },
  {
    "caption": "(A,B) Requirement for glutamine in UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in presence of either 5 mM glucose and 3 mM glutamine or 5 mM glucose alone. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and etomoxir-sensitive OCR after vehicle or UK5099 treatment as shown in (A) from n=4 individual experiments.",
    "molecules": "UK5099, AA, antimycin A, ATP, DMSO, Eto, etomoxir, glucose, glutamine, NE, Norepinephrine, Oligo, oligomycin A"
  },
  {
    "caption": "(C) Effect of glutaminase inhibitor on UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, or 100 nM UK5099 + 2.5 µM CB839 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099 or UK5099 + CB839 from n=5 individual experiments. Data were normalized to vehicle for each experiment.",
    "molecules": "UK5099, CB839, DMSO, glucose, glutamine"
  },
  {
    "caption": "(D) Effect of MPC inhibition on glutamate/aspartate ratio measurement. Glutamate/aspartate ratio was quantified using GC-MS as a measure of glutamine catabolism. Primary brown adipocytes were treated for 24 h with vehicle (DMSO) or 10 µM UK5099 from n=6 individual experiments. **",
    "molecules": "UK5099, aspartate, DMSO, glutamate, Glutamate, glutamine"
  },
  {
    "caption": "(B) Effect of transaminase inhibition on UK5099-induced energy expenditure. Brown adipocytes were pre-treated with vehicle (DMSO), 50-100 nM UK5099, 1 mM aminooxyacetic acid (AOA), or a combination of UK5099 and AOA for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of the tested compounds. Data show quantification of non-stimulated OCR from n=5 individual experiments.",
    "molecules": "aminooxyacetic acid, AOA, UK5099, DMSO, glucose, glutamine"
  },
  {
    "caption": "(C-E) Effect OGC1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transduced with Scramble RNA (Scramble) or shOGC1 (OGC1 KD) adenovirus. Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM,) oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (C) Representative OCR traces averaging 4 technical replicates. (D) Quantification of non-stimulated OCR as measured in (C) from n=6 individual experiments. (E) Quantification of Eto-sensitive OCR as measured in (C) from n=6 individual experiments. Data were normalized to vehicle for each experiment.",
    "molecules": "UK5099, AA, antimycin A, DMSO, Eto, etomoxir, glucose, glutamine, NE, Norepinephrine, Oligo, oligomycin A"
  },
  {
    "caption": "(F-H) Effect of Aralar1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transfected with Scramble RNA (Scramble) or siRNA for Aralar1 (Aralar1 KD). Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin a (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin a (AA; 4 µM) were injected where indicated. (F) Representative OCR traces averaging 4 technical replicates. (G) Quantification of non-stimulated OCR as measured (G) from n=4 individual experiments. Data were normalized to vehicle for each experiment. (H) Quantification of Eto-sensitive OCR as measured in (F) from n=4 individual experiments.",
    "molecules": "UK5099, AA, antimycin a, DMSO, Eto, etomoxir, glucose, glutamine, NE, Norepinephrine, Oligo, oligomycin a"
  },
  {
    "caption": "(I) Brown adipocytes transfected with Scramble RNA or Aralar1 siRNA were treated for 24 hours with vehicle (DMSO) or 10 µM UK5099. Data shows quantification of the ratio of aspartate to glutamate abundance as measured by GC-MS from n=3 individual experiments.",
    "molecules": "UK5099, aspartate, DMSO, glutamate"
  },
  {
    "caption": "(A,B) Effect of UK5099 on energy expenditure contributed by ATP demand and by mitochondrial proton leak in non-stimulated brown adipocytes. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 hours. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Oligomycin A (Oligo; 4 µM), mitochondrial uncoupler FCCP (2 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of mitochondrial proton leak and ATP-linked OCR (oligomycin sensitive) as measured in (A) from n=9 individual experiments. Data were normalized to vehicle for each individual experiment.",
    "molecules": "UK5099, AA, antimycin A, ATP, FCCP, DMSO, glucose, glutamine, Oligo, oligomycin, Oligomycin A"
  },
  {
    "caption": "(C) To determine the contribution of ATP demand to increased energy expenditure under UK5099 cells were permeabilized and cytosolic ADP concentrations were clamped. OCR were measured in the presence of 5 mM pyruvate, 3 mM malate, 0.1 mM palmitoyl-CoA, 0.5 mM carnitine and 5 mM ADP. Cells were treated with either 100 nM, 1 µM or 10 µM UK5099. Data shows maximal state 3 OCR normalized to maximal complex 4 activity from n=3 individual experiments.",
    "molecules": "UK5099, ADP, ATP, carnitine, malate, palmitoyl-CoA, pyruvate"
  },
  {
    "caption": "(E) Effect of MPC inhibition on glycerol 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) generation. G3P/DHAP is used as a measure of acylglycerol synthesis. Brown adipocytes were treated with either vehicle, 100 nM UK5099 or 1 µM norepinephrine (NE) for 24 h from n=4 individual experiments.",
    "molecules": "UK5099, DHAP, dihydroxyacetone phosphate, acylglycerol, NE, norepinephrine, G3P, glycerol 3-phosphate"
  },
  {
    "caption": "(F,G) Contribution of lipolysis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, or Atglistatin. (F) Quantification of non-stimulated OCR from n=4 individual experiments. (G) Quantification of ATP-linked respiration from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.",
    "molecules": "UK5099, Atglistatin, ATP, DMSO, glucose, glutamine"
  },
  {
    "caption": "(H,I) Contribution of TAG synthesis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 1 µM DGAT1 and 1 µM DGAT2 or a combination of DGAT1/2 inhibitors with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, DGAT1/2 inhibitors, or UK5099 + DGAT1/2 inhibitors. (H) Quantification of non-stimulated OCR from n=6 individual experiments. (I) Quantification of ATP-linked OCR from n=6 individual experiments. Data were normalized to vehicle for each individual experiment.",
    "molecules": "UK5099, ATP, DMSO, glucose, glutamine, TAG"
  },
  {
    "caption": "(J,K) Contribution of ACS to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, Triacsin C, or UK5099 + Triacsin C. (J) Quantification of non-stimulated OCR from n=4 individual experiments. (K) Quantification of ATP-linked OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.",
    "molecules": "UK5099, ATP, DMSO, glucose, glutamine, Triacsin C"
  },
  {
    "caption": "B. DIC microscopic image of WTSOD12SH diluted to 100 µM in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated 37 °C, 180 RPM for 30 min shows no droplet formation; scale bar: 100 µm; inset shows Alexa Fluor 488 maleimide labelled WTSOD12SH;",
    "molecules": "Alexa Fluor 488, maleimide, NaCl"
  },
  {
    "caption": "C. Solution turbidity plot (absorbance at 600 nm) shows that WTSOD12SH (at concentrations ranging from 25 to 200 µM does not undergo LLPS in presence or absence of heparin, LLPS inducer; data is represented as mean ± SD (from 3 biological replicates).",
    "molecules": "heparin"
  },
  {
    "caption": "D. DIC microscopic image of ApoSOD12SH under condensate inducing conditions (37 C, 180 RPM) at a concentration of 100 µM incubated 100 mM NaCl; scale bar:100 µm (inset on bottom left shows Alexa Fluor 488 maleimide labeled protein condensates;",
    "molecules": "Alexa Fluor 488, maleimide, NaCl"
  },
  {
    "caption": "E. Fluorescent microscopic images of Alexa Fluor 488 maleimide labeled ApoSOD12SH condensates diluted in HEPES buffer, pH 7.4 and 100 mM NaCl and incubated (180 RPM, 37 C) in absence (left) and presence (right) of heparin at 30 min time-point",
    "molecules": "Alexa Fluor 488, heparin, maleimide, NaCl"
  },
  {
    "caption": "F. Solution turbidity measurements with ApoSOD12SH (absorbance at 600 nm) shows that while in the absence of heparin the turbidity increases with time, the presence of heparin results in faster LLPS;",
    "molecules": "heparin"
  },
  {
    "caption": "G. Comparison of ApoSOD12SH droplet numbers per microscopic field of view (view area 0.001963 mm2) calculated from 5 different images of droplets incubated with and without heparin at 2 h timepoint.",
    "molecules": "heparin"
  },
  {
    "caption": "J. Scheme depicting that presence of Zn and not Cu in pre-incubation mixture inhibits LLPS. K. DIC microscopic images of ApoSOD12SH in presence of Zn (top) showing no droplet formation and Cu (below) showing the presence of droplets",
    "molecules": "Cu, Zn"
  },
  {
    "caption": "L. Solution turbidity plot (absorbance at 600 nm) of ApoSOD12SH subjected to increasing concentrations of Cu and Zn; turbidity decreases in a dose dependent manner for Zn while no significant change is observed in presence of Cu;",
    "molecules": "Cu, Zn"
  },
  {
    "caption": "A. DIC microscopic images of I113T SOD12SH (top) and G85R SOD12SH (bottom) droplets incubated in the absence and presence of Zn and Cu respectively (scale bar: 100 µm); insets on bottom left show Alexa Fluor 488 maleimide labeled protein condensates for I113T SOD12SH and G85R SOD12SH respectively; scale bar: 5 µm. Droplet dissolution was observed for I113T SOD12SH on addition of Zn while droplets persisted on addition of Cu. Red arrowhead indicates fusion of liquid droplets.",
    "molecules": "Alexa Fluor 488, Cu, maleimide, Zn"
  },
  {
    "caption": "C. On metal cofactor removal, ApoG37R SOD12SH formed condensates which ceased to exist when protein was incubated with Zn, while condensates persisted on Cu addition (left to right);",
    "molecules": "Cu, Zn"
  },
  {
    "caption": "B. Plot of the loge of the hydrodynamic radius (rH) versus the log e of the number of residues in the polypeptide chain. The line fitted to these data for the native folded proteins (yellow dashed) has a slope of 0.29 ± 0.02 and a y-axis intercept of 1.56 ± 0.1, while the other fitted to the chemically denatured protein (grey dashed) data has a slope of 0.57 ± 0.02 and a y-axis intercept of 0.79 ± 0.07. Literature data have been used for folded and chemically denatured proteins while we employed FCS to calculate the rH of all SOD12SH variants with (solid shapes) and without Zn (hollow shapes). The hollow grey circle of G85R SOD12SH is directly under hollow green I113T SOD12SH in the log-log plot.",
    "molecules": "Zn"
  },
  {
    "caption": "C. Variation of diffusion coefficients of de-metalated protein variants determined from FCS measurement with increasing Zn concentration (inset shows a comparison of binding constants between mutants and Zn; ApoSOD12SH and ApoG37R SOD12SH has a higher binding affinity to Zn than ApoI113T SOD12SH and ApoG85R SOD12SH).",
    "molecules": "Zn"
  },
  {
    "caption": "D. Disordered/ extended conformation content decreases in ApoSOD12SH and ApoI113T SOD12SH with the addition of Zn calculated from FTIR spectra;",
    "molecules": "Zn"
  },
  {
    "caption": "C. Upper panel shows fluorescence images of the maturation of Alexa Fluor 488 labeled ApoSOD12SH droplets with time, samples were incubated at 37 C up to ~25 hours. Images are taken at indicated time point: 0* hour (the time after incubation required for droplet formation), 12 hours and >24 hours; images below show magnified scale bar: 5 µm. The middle panel shows magnified view of the marked region from corresponding images on the upper panel. Red arrowhead indicates matured (solid) portion inside the droplet. Lower panel shows coexistence of liquid and solid phase inside droplet; scale bar: 10 µm. Intensity plot (bottom right) shows the distribution of high intensity region within matured droplet.",
    "molecules": "Alexa Fluor 488"
  },
  {
    "caption": "E. ThT fluorescence assay plot showing aggregation kinetics of all SOD12SH variants. ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show high ThT fluorescence.",
    "molecules": "ThT"
  },
  {
    "caption": "F. AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH show fibrillar aggregates after prolonged incubation at 37 ℃, 180 RPM in absence of Zn (left panel); AFM micrographs of ApoSOD12SH, I113T SOD12SH and G85R SOD12SH incubated with Zn (at 1:5 protein: Zn molar ratio) show presence of oligomers for ApoSOD12SH, I113T SOD12SH and short fibrils for G85R SOD12SH (right panel);",
    "molecules": "Zn"
  },
  {
    "caption": "H. Dot plots showing flow cytometric analysis of annexin V and propidium iodide (PI) staining of apoptotic SHSY5Y neuroblastoma cells following a 12 h treatment with 5 µM ApoSOD12SH condensates, I113T SOD12SH condensates, G85R SOD12SH condensates, ApoSOD12SH fibrils, I113T SOD12SH fibrils and G85R SOD12SH fibrils respectively (see Methods for details). High cytotoxicity was observed for SOD12SH condensates.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "C, D The 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.",
    "molecules": "LPS"
  },
  {
    "caption": "E CMT-93 cell lines stably expressing Smad6 amino acids 400-441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "D, E The SBE-Luc or 5xNF-κB-Luc reporter plasmids were co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 6 h or LPS for 2 h, and luciferase activities were measured and normalized.",
    "molecules": "LPS"
  },
  {
    "caption": "A Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by qRT-PCR.",
    "molecules": "LPS"
  },
  {
    "caption": "B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.",
    "molecules": "LPS"
  },
  {
    "caption": "B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h.",
    "molecules": "LPS"
  },
  {
    "caption": "D Peli1 knockdown or wild-type humanTHP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by qRT-PCR. Data show the mean ± SD of three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin-agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.",
    "molecules": "LPS"
  },
  {
    "caption": "G-I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "The survival rates of severe CLP-induced sepsis mice by subcutaneously injected Smaducin-6 were compared with those by intraperitoneally injected antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin). Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. n = 10 mice per group per experiment.",
    "molecules": "cephalosporin, gentamycin"
  },
  {
    "caption": "Therapeutic effects of Smaducin-6 alone, or antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin), or different amounts of Smaducin-6 plus antibiotics were observed in CLP mice. Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. Smaducin-6 was subcutaneously injected at 2 h post-CLP followed by three injections at 12-h intervals. n = 10 mice per group.",
    "molecules": "cephalosporin, gentamycin"
  },
  {
    "caption": "Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).",
    "molecules": "LPS"
  },
  {
    "caption": "CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.",
    "molecules": "LPS"
  },
  {
    "caption": "Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.",
    "molecules": "LPS"
  },
  {
    "caption": "(F) Relative mRNA expression  of Foxc1  and Foxc2  in isolated CD45-CD31+ ECs  from distal jejuna 12d after Tm  treatment. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~5, *P<0.05. Data information: The box-and-whisker plots in (F) display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.",
    "molecules": "Tm"
  },
  {
    "caption": "(G) Representative immunostaining images of intestinal crypts labeled with BrdU (proliferative marker, injection performed 2h before tissue collection) and EpCAM (epithelial marker) show the proliferation of epithelial cells in crypts. Paraffin sections (4 µm), (H) Quantification of the number of BrdU+ epithelial cells per crypt based on Figure 2G. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 3~6, *P<0.05, n.s. = not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.",
    "molecules": "BrdU"
  },
  {
    "caption": "(A) Representative BrdU/CD31/LYVE1/DAPI immunostaining images of intestinal paraffin sections (15 µm) from mice injected with BrdU 18.5h before euthanasia for the analysis of proliferative BECs (B in 1, 3, 5, 7) and LECs (L in 2, 4, 6, 8) in intestines. Arrow heads show BrdU+ BECs or LECs. White/yellow bars = 100 or 10 µm, respectively. (B) The numbers of BrdU+ BECs and LECs per 0.1 mm2 blood vessel (CD31+LYVE1-) and lymphatic vessel (CD31+LYVE1+) area in intestinal mucosa were quantified respectively based on Figure 5A. Data are box-and-whisker plots, Kruskal-Wallis One-way ANOVA test, each symbol represents one mouse, N = 3~8, *P<0.05, **P<0.01, n.s. = not significant.",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": "(C) Representative TUNEL/CD31/LYVE1/DAPI immunostaining images of distal jejunums for the analysis of apoptotic BECs/LECs in intestinal paraffin sections (15 µm). High magnification images are from the dotted line boxes (1~4). Arrow heads show the apoptotic BECs (B in 1, 2) and LECs (L in 3, 4) at villus (1, 3) or submucosa (2, 4). White/yellow scale bars = 100 or 20 μm, respectively. (D) The numbers of apoptotic BECs and LECs per 0.1 mm2 blood vessel (CD31+LYVE1-) and lymphatic vessel (CD31+LYVE1+) area in intestinal mucosa were quantified respectively based on Figure 5C. Data are box-and-whisker plots, Mann Whitney U test, each symbol represents one mouse, N = 3~6, **P<0.01, n.s. = not significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (C) Representative images of crypts immunostained with OLFM4 and β-catenin in PBS/RSPO3 treated EC-Foxc-DKO mice 24h after I/R. The accumulation of β-catenin in the nuclei of ISCs (dotted circles) was found in RSPO3 rescued mice. Paraffin sections (4 µm), (D) Quantification of relative fluorescent intensity (FI) of β-catenin immunostaining within ISC and (E) quantification of the number of OLFM4+ ISCs were performed based on Figure 9C. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, *P<0.05.",
    "molecules": "PBS"
  },
  {
    "caption": "In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (F) Representative images of immunostaining of CCND1 in intestines in PBS/RSPO3 treated EC-Foxc-DKO mice at I/R-24h. Scale bars = 100 µm. (G) Quantification of the number of CCND1+ epithelial cells per crypt at I/R-24h are based on Figure 9F. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 7, **P<0.01.",
    "molecules": "PBS"
  },
  {
    "caption": "In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (H) Relative mRNA expression of Rspo3 in sorted LECs and Cxcl12 in sorted BECs from intestines of PBS/RSPO3 treated EC-Foxc-DKO mice at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, *P<0.05.",
    "molecules": "PBS"
  },
  {
    "caption": "In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. Representative images of H&E staining show the rescue effects of RSPO3 in intestinal mucosa LEC-Foxc-DKO (I) mice as well as their control mice 24h after I/R. Red numbers indicate the Chiu scores. Quantification of Chiu Score for the intestines at I/R-24h are based H&E staining as shown in Figure 9A and 9I. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 6~13, **P<0.01.",
    "molecules": "PBS"
  },
  {
    "caption": "In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (K) Representative images of CD31/LYVE1 immunostaining in the intestines of PBS/RSPO3 treated LEC-Foxc-DKO mice at I/R-24h. (L, M) Quantification of the vessel density (L) (= vessel area/total intestinal tissue area x 100%) for the blood (B) and/or lymphatic (L) vessels (markers listed below the graph were used to identify B and L) as well as the measurement of lacteal length (M) was performed based on Figure 9K. The Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 7, **P<0.01, n.s.=not significant.",
    "molecules": "PBS"
  },
  {
    "caption": "In CXCL12 rescue experiments, mice were treated with 50μg/kg CXCL12 in PBS by retro-orbital injection 30 min before ischemia. Mice treated with PBS were used as control. (H) Representative confocal images of CD31 immunostaining of distal jejuna in PBS- and CXCL12- treated EC-Foxc-DKO mice after I/R at 24h. Paraffin sections (15 µm), scale bars = 100 μm. L represents the lacteal length measured in Figure 10J. (I, J) (I) Quantification of CD31+ vessel density (% = total CD31+ vessel area/total intestinal tissue area x 100%) and (J) quantification of lacteal length were performed based on Figure 10H. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, **P<0.01.",
    "molecules": "PBS"
  },
  {
    "caption": "In CXCL12 rescue experiments, mice were treated with 50μg/kg CXCL12 in PBS by retro-orbital injection 30 min before ischemia. Mice treated with PBS were used as control. (K) Relative mRNA expression of Rspo3 in sorted intestinal LECs from PBS/CXCL12 treated EC-Foxc-DKO mice at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 5~6, n.s.=not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.",
    "molecules": "PBS"
  },
  {
    "caption": "In vivo 18F-GE180 TSPO μPET imaging. Two female Grn-/-/Tmem106b-/- and five female wild type mice underwent longitudinal TSPO μPET at 2.0 and 3.5 months of age. All analyses were performed by PMOD (V3.5, PMOD technologies). Normalization of injected activity was performed by the previously validated myocardium correction method.",
    "molecules": "18F-GE180"
  },
  {
    "caption": "Immunofluorescence analysis of lipofuscin (red) and IBA1 (green) in sagittal brain sections. Representative images of the Thalamus (TH). Scale bar indicates 25 µm.",
    "molecules": "lipofuscin"
  },
  {
    "caption": "Western blot analysis of ubiquitin and the autophagy marker LC3 in RIPA, and p62 in RIPA and urea total brain lysates from 4.5-month-old mice with the indicated genotype. Calnexin used as a loading control. (n = 3 biological replicates per genotype). Quantification of B. Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.",
    "molecules": "ubiquitin, urea"
  },
  {
    "caption": "Western blot analysis of TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sequential high salt (HS)/RIPA/Urea fractionations of total brain lysates from 4.5-month-old mice with the indicated genotype. Analysis of C-terminal fragments of TDP-43 (CTF) in RIPA fraction. p-TDP-43 analyzed in Urea fraction. Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.",
    "molecules": "salt, Urea"
  },
  {
    "caption": "Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm. Passage number between P14-P15. Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. Passages number between P10-15. Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented (OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubulated (DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented); ; ****p < 0.0001, ns; not significant..",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative confocal images of wild type (WT) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant.",
    "molecules": "mitoYFP, NucBlue"
  },
  {
    "caption": "Representative confocal images of WT, Opa1Crispr MEFs complemented with pLenti-Opa1, Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant..",
    "molecules": "mitoYFP, NucBlue"
  },
  {
    "caption": "Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for the indicated time points. Images were captured every hour for 18 hours. Scale bar=10μm. Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of three independent experiments, (131-426 cells per cell line), One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.",
    "molecules": "carbonyl cyanide m-chlorophenyl hydrazine, CCCP, CHX"
  },
  {
    "caption": "Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion (SiMH) with 10μM cycloheximide (CHX) for the indicated time points. Images were captured every hour for 9 hours. Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of four independent experiments, (155-745 cells per cell line), One-way ANOVA.",
    "molecules": "CCCP, CHX, cycloheximide"
  },
  {
    "caption": "Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular (Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented); *** p < 0.001, ****p < 0.0001, ns; not significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative confocal micrographs of MEFs WT, Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubulated (Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented) ; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "I Cell lysates of 293T cells transfected with Myc-UHRF1 expression vector were incubated with 2 of purified GST, GST-PWWP2B-N, or GST-PWWP2B-C recombinant proteins for 1 h at 4 °C. After pull-down with GST-beads, the bound complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using the indicated antibodies (upper panel). The Coomassie blue stain shows the purified GST fusion proteins.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "D-G. Quantification of radiation-induced RAD51, BRCA1, and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs. All data were obtained from over cells (open circles), and the mean values are shown as red bars (upper panel). Immunofluorescence analysis of irradiation-induced RAD51, BRCA1, and 53BP1 foci formation. HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (D and E), BRCA1 (F), or 53BP1 (G) antibodies. The cells in S phase were identified by Cyclin A staining. (E) The siPWWP2B-resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA-mediated PWWP2B depletion. IR-induced RAD51 foci formation was examined by immunofluorescence.",
    "molecules": "thymidine"
  },
  {
    "caption": "(A) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1,5 g/L of D-glucose. Glucose was added either at the beginning of growth (+ glucose, left panel) or after 3h of growth (right panel).",
    "molecules": "D-glucose, Glucose, glucose"
  },
  {
    "caption": "(B) Northern blot experiment shows the expression of RsaI during growth phase in the HG001, ∆ccpA mutant strain, and ∆codY mutant strain, in MHB medium with (+) or without (-) the addition of 1,5 g/L of D-glucose.",
    "molecules": "D-glucose"
  },
  {
    "caption": "(D) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g/L of glucose, fructose or xylose. For all the experiments, loading controls were done using the expression of 5S rRNA (5S) as revealed after hybridization of the membranes with a specific probe. However for these controls, we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes.",
    "molecules": "glucose, fructose, xylose"
  },
  {
    "caption": "(A) Toe-print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU_2 and fn3K mRNAs, respectively. Lane 1 : incubation control of mRNA alone; lane 2 : incubation control of mRNA with 30S subunits; lane 3: incubation control of mRNA with RsaI; lane 4 : formation of the ribosomal initiation complex containing mRNA, 30S and the initiator tRNAfMet (tRNAi); lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI, respectively : 50 nM (lane 5), 100 nM (lane 6), 150 nM (lane 7), 300 nM (lane 8), and 400 nM (lane 9). Lanes T, A, C, G: sequencing ladders. The Shine and Dalgarno (SD) sequence, the start site of translation (START) and the toe-printing signals (+16) are indicated. At the bottom of the gels are shown the predicted interactions between RsaI and its targets. Translation start codons are in green, and the Shine and Dalgarno (SD) sequence is underlined, the arrowheads depict the toe-printing signals.",
    "molecules": "tRNA"
  },
  {
    "caption": "(C) In vivo effect of RsaI on PIA-PNAG synthesis in the S. aureus wild-type (WT) 132 strain, the ∆rsaI mutant, and the strain carrying a deletion of icaR 3'UTR (∆3'UTR). This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5. The PIA-PNAG exopolysaccharide biosynthesis was quantified using dot-blot assays. Serial dilutions (1/5) of the samples were spotted onto nitrocellulose membranes and PIA-PNAG production was detected with specific anti-PIA-PNAG antibodies. RsaI was detected in the same samples by Northern blot using a probe directed against RsaI. Ethidium bromide staining of rRNA was used as loading controls of the same gel.",
    "molecules": "PIA-PNAG, rRNA, Ethidium bromide, nitrocellulose"
  },
  {
    "caption": "(C) Measurements of the half-lives of RsaI and RsaG in HG001-∆rsaG or HG001-∆rsaI mutant strains using Northern blot experiments. The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2, 4, 8, 15, 30 and 60 min at 37°C in BHI. 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples, which were however run on two different gels. Calculated half-lives are shown beneath the autoradiographies and are the average of 2 experiments. The data were normalized to 5S rRNA.",
    "molecules": "rifampicin"
  },
  {
    "caption": "B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting.",
    "molecules": "rapamycin"
  },
  {
    "caption": "Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition. LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. for 6 h treatment with Nutlin-3.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test.",
    "molecules": "Ganetespib, Geldanamycin"
  },
  {
    "caption": "F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.",
    "molecules": "SDS"
  },
  {
    "caption": "E. Ectopic expression of CG14044   in the  wing pouch (GFP  area indicated by the dotted line) induces caspase  activation and DNA  fragmentation (TUNEL) in the  larval disc, melanization in the  pupal wing    (arrow head) and a structure defect in the adult wing. Data information: Scale bars, 100 μm in E (cleaved caspase) and 20 μm in E (TUNEL) and 500 μm in E (pupae and adult wing).",
    "molecules": "DNA"
  },
  {
    "caption": "H-I. Lysates from HEK293T  cells that express Flag-Debcl  or Flag-Buffy  were incubated with GST  or GST-Synr   bound to glutathione-Sepharose. Flag-Debcl  or Flag-Buffy  is pulled down  with GST-Synr   but not with GST.",
    "molecules": "Sepharose, glutathione"
  },
  {
    "caption": "O. Debcl  can be photo-crosslinked with Synr  at BPA  that corresponds to Y124 and F141. BPA  at Y340 was used as a negative control. Red arrowheads indicate covalently crosslinked products. P. Buffy  is also photo-crosslinked with Synr  at BPA  that corresponds to Y124 and F141. Red arrowheads indicate covalently crosslinked products.",
    "molecules": "BPA"
  },
  {
    "caption": "A. Ectopic expression of Synr   induces autolysosome  accumulation, which was detected by Lysotracker, in the wing disc. B. Ectopic expression of Synr   also induces autophagosome  accumulation, which was detected by mCherry-atg8a. The dotted square regions are magnified in the right pictures. Data information: Scale bars, 100 μm in A and H, 50 μm in B",
    "molecules": "Lysotracker"
  },
  {
    "caption": "(b) THPN-induced cell death is prevented by the autophagic inhibitors wortmannin (100 nM) and chloroquine (20 μM) but not by the apoptotic inhibitor zVAD (20 μM) or the necrotic inhibitors butylated hydroxyanisole (100 μM) and necrostatin-1 (30 μM). The cells were pretreated with each inhibitor for 1 h separately before the THPN treatment (20 μM, 48 h). Ctrl, control.",
    "molecules": "necrostatin-1, butylated hydroxyanisole, chloroquine, wortmannin, zVAD"
  },
  {
    "caption": "(c) THPN induces autophagy in melanoma cells. Top, THPN (20 μM, 24 h) induces a change in the localization of GFP-LC3 fusion proteins from a ubiquitous, diffuse pattern to cytoplasmic puncta. Serum starvation (SS, 24 h) was used as a positive control (scale bars, 10 μm). Middle, the GFP-LC3-positive cells with puncta were calculated from five randomly picked regions. Bottom, the conversion of LC3-I to LC3-II induced by THPN was determined by western blotting. Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.",
    "molecules": "THPN"
  },
  {
    "caption": "(a) THPN induces TR3 translocation from the cytoplasm to the mitochondria, detected by western blotting for the various cell fractionations (top) and by confocal microscopy (bottom). The cells were treated with THPN at the times indicated and detected by TR3 (red) and Hsp60 (green). The nuclei were stained with DAPI (blue) (scale bars, 10 μm). PARP, Tubulin and Hsp60 were used as loading controls for nuclear extracts (NE), cytoplasmic extracts (CE) and mitochondrial fractions (Mito), respectively. IB, immunoblotting.",
    "molecules": "THPN"
  },
  {
    "caption": "(b) TR3 mutant Flag-ΔNLS but not Flag-NLS promotes the induction of autophagy in response to THPN treatment. The siRNA-resistant versions of Flag-ΔNLS and Flag-NLS were transfected into TR3-knockdown A375 cells. Top, the cells cotransfected with GFP-LC3 were further treated with THPN for 24 h before microscopic analysis (scale bars, 10 μm). Middle, the conversion of LC3-I to LC3-II was analyzed by western blotting. The rate of cell survival was determined after THPN treatment for 48 h (bottom). '-' represents vehicle treatment only.",
    "molecules": "THPN"
  },
  {
    "caption": "(c) Nix assists the TR3 mitochondrial targeting. Cells with Nix, Pink1 and Bnip3 knockdown were treated with THPN (20 μM, 6 h). Top, mitochondrial TR3 was assayed by western blotting. Nix knockdown abolishes the THPN-induced (20 μM, 24 h) formation of LC3 foci (middle and bottom; scale bars, 10 μm). Images of full blots are shown in Supplementary Figure 7. All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01; NS, not significant; ctrl, control.",
    "molecules": "THPN"
  },
  {
    "caption": "(b) Site C of LBD is critical for Nix binding, as determined by the in vitro pull-down assay. The purified bead-bound GST or GST-Nix proteins were incubated with different mutants as indicated and THPN (20 μM) for 3 h; the proteins were then analyzed by western blotting. IB, immunoblotting.",
    "molecules": "THPN"
  },
  {
    "caption": "(e) DPDO cannot facilitate Nix binding to TR3. Top, the structure of DPDO. Middle, DPDO binding could not form the interacting surface for Nix, unlike THPN. The structures of Arg563 and Ser553 are the same as those in the native structure. Bottom, DPDO does not enhance the interaction between TR3 and Nix. Different plasmids were transfected into A375 cells as indicated, and cells were treated with DPDO (20 μM, 3 h). The TR3 interaction with Nix was determined by co-immunoprecipitation. Images of full blots are shown in Supplementary Figure 7. IP, immunoprecipitation; HA, hemagglutinin.",
    "molecules": "THPN, DPDO"
  },
  {
    "caption": "(a) THPN induces the dissipation of ΔΨm in both time- and dose-dependent manners. The compromised ΔΨm is shifted to the right upon THPN treatment in flow cytometry. '-' represents vehicle treatment only.",
    "molecules": "THPN"
  },
  {
    "caption": "(b) Effects of different plasmids on the THPN-induced dissipation of ΔΨm. Top, endogenous TR3 was knocked down by shRNA, and the siRNA-resistant version of TR3, TR3-NLS, TR3-ΔNLS or TAD (Supplementary Fig. 2c) were separately transfected into the cells as indicated. Bottom, similarly, the siRNA-resistant version of Nix or ΔTM were separately transfected into Nix knockdown cells. Transfected cells were treated with THPN (20 μM, 12 h), and their ΔΨm was assessed by flow cytometry. Ctrl, control.",
    "molecules": "THPN"
  },
  {
    "caption": "(c) ANT1 and VDAC1 are required for the THPN-induced autophagy. The ANT1 and VDAC1 shRNA-expressing plasmids, which also encoded EGFP for evaluating the transfection efficiency, were separately transfected into cells; mCherry-LC3 was cotransfected to display autophagy. Green cells are those transfected with ANT1 or VDAC1 shRNA, and the cells with red cytoplasmic puncta are autophagic cells (top; scale bars, 10 μm).",
    "molecules": "THPN"
  },
  {
    "caption": "(d) ANT1 and VDAC1 are required for the THPH-induced dissipation of ΔΨm and cell death. Endogenous ANT1 or VDAC1 was separately knocked down by shRNA, and the dissipation of ΔΨm was determined after THPN (20 μM, 12 h) treatment (left). ANT1 and VDAC1 knockdown abolishes the THPN-induced (20 μM, 48 h) cell death (right). All of the data are presented as the mean ± s.e.m. of three independent experiments. **P 0.01.",
    "molecules": "THPN"
  },
  {
    "caption": "(a) Endogenous TR3 is localized in the MIM in response to THPN stimulation. Left, isolated mitochondria from cells treated with THPN (20 μM, 6 h) were incubated with proteinase K (20 μM and 40 μM) for 30 min. TR3 expression was determined. Tom20 in MOM and Tim23 in MIM were used as controls. Right, the mitochondria were further subjected to submitochondrial fractionation. VDAC1, Tim23 and Hsp60 were used to represent MOM, MIM and mitochondrial matrix protein, respectively. '-' represents vehicle treatment only.",
    "molecules": "THPN"
  },
  {
    "caption": "(c) Tom40 and Tom70 are required for the THPN-associated dissipation of ΔΨm (left), conversion of LC3-I to LC3-II (right and top) and cell death (right and bottom). Endogenous Toms were separately knocked down by the corresponding shRNAs. ΔΨm, LC3-II and cell death were detected as described above. Images of full blots are shown in Supplementary Figure 7.",
    "molecules": "THPN"
  },
  {
    "caption": "(d) The mitochondria were markedly eliminated after treatment with THPN for 24 h. Right, endogenous Tom40 and Tom70 were knocked down, and the amount of mitochondria was measured by the fluorescence intensity of Tom20 (green). Left, the volume of mitochondria from five randomly picked regions was quantified by ImageJ software. All of the data are presented as the mean ± s.e.m. of three independent experiments. *P 0.05, **P 0.01, ***P 0.001; NS, not significant.",
    "molecules": "THPN"
  },
  {
    "caption": "(a) THPN inhibits the formation of tumors in the liver and lung in a TR3-dependent manner. B16 cells transfected with either control shRNA, TR3 shRNA or ATG7 shRNA (5 × 105) were separately injected into the lateral tail vein of 6-week-old C57BL/6J mice (n = 7). THPN were administered 3 d after inoculation. The tumors formed in the lung and liver are indicated (top; scale bars, 5 mm) and were counted (bottom). Ctrl, control.",
    "molecules": "THPN"
  },
  {
    "caption": "(b) Crossed Tyr:NRasQ61K;Ink4a/Arf−/−;TR3+/+ and Tyr:NRasQ61K;Ink4a/Arf−/−;TR3−/− mice (n = 12) were treated topically with DMBA to initiate cutaneous melanocytic neoplasms, and mice were treated with THPN as described in Online Methods. The representative images of dorsal melanomas (top) and the tumor numbers (bottom) are presented.",
    "molecules": "THPN, DMBA"
  },
  {
    "caption": "(a) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3, left untransfected or transfected for 6 h with poly(dA:dT) (1.5 μg/ml), then exposed to control antibody (Control Ab), antibody to neutralize IL-1β (Anti-IL-1β), dimethyl sulfoxide (DMSO) or 3-MA (5 mM); results are presented as quantification of GFP-LC3+ dots (top) and images of individual cells (middle). Scale bars, 10 μm.",
    "molecules": "3-MA, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(e) Immunoblot analysis of LC3-I and LC3-II in or wild-type, ASC-deficient (Pycard-/-; called 'Asc-/-' here) or caspase-1-deficient (Casp1-/-) BMDMs primed with LPS (20 ng/ml) and then left untransfected or transfected with poly(dA:dT) or left untreated or treated with nigericin for 4 h; numbers below lanes as in d. Actin serves as a loading control throughout. NS, not significant; *P 0.05 and **P 0.01 (unpaired t-test). Data are from three individual experiments",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(e) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for ASC (red): left image in each pair, three-dimensional volume renderings reconstructed from z-stack images; right image in each pair, surface-segmentation models (2× enlargement). Scale bars, 10 μm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(f) Electron microscopy of differentiated THP-1 cells stimulated with LPS and ATP (left) or transfected with poly(dA:dT) (right) for 2 h and immunostained for ASC, visualized with 15-nm gold-conjugated protein A. Red arrows indicate ASC immunostaining. Right image in each pair, 4.5× enlargement of outlined area at left. Scale bars, 500 nm. Data are representative of three experiments (a,c) or two experiments (b,d-f).",
    "molecules": "gold, ATP, LPS"
  },
  {
    "caption": "(c) Confocal microscopy of differentiated THP-1 cells left untreated or treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for endogenous LC3 and NLRP3. Scale bars, 10 μm. Data are representative of at least three experiments.",
    "molecules": "LPS, ATP"
  },
  {
    "caption": "(a) Immunoblot analysis of AIM2, ASC and LC3-II in lysates of differentiated THP-1 cells left untransfected or transfected with poly(dA:dT) (1.5 μg/ml) and left untreated or treated with 3-MA, rapamycin or amino-acid starvation, then separated into an inflammasome-enriched fraction (top) or an autophagosome-enriched fraction (middle). Bottom, immunoblot analysis of total cell lysates.",
    "molecules": "3-MA, amino-acid, rapamycin"
  },
  {
    "caption": "(c) Immunoblot analysis of LC3-I and LC3-II (top) in lysates of mouse BMDMs primed with LPS (20 ng/ml) and left untreated or treated with alum, then left unexposed or exposed for 5 h to live (Live TB) or dead (Dead TB) M. tuberculosis (numbers below lanes as in Fig. 1d). Below, immunoblot analysis of IL-1β in supernatants of LPS-primed mouse BMDMs infected overnight with live M. tuberculosis in the presence or absence of 3-MA (2 mM); treatment with LPS and ATP serves as a positive control. Data are representative of at least two experiments.",
    "molecules": "3-MA, alum, ATP, LPS"
  },
  {
    "caption": "(c) Confocal microscopy of differentiated THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3mM), immunostained for p62 (green) and ASC (red): left two images, merged channel volume and surface rendering of a three-dimensional reconstruction; right four images, enlargement of images at left (yellow arrow), with arrows indicating removal of part of the signal to reveal the red signal (ASC) inside the green-stained area (p62).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(a) Immunoblot analysis of lysates of human monocytes-macrophages primed with LPS (10 ng) and then transfected with poly(dA:dT) (1.5 μg/ml) or treated with nigericin (4 μM) for 6 h (numbers below lanes as in Fig. 1d).",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(b) Immunoblot analysis of processed caspase-1 and IL-1β (top) and enzyme-linked immunosorbent assay of IL-1β (middle) in supernatants of monocytes-macrophages treated for 6 h with various combinations (bottom) of poly(dA:dT) (b) or nigericin (c), plus 3-MA and amino-acid starvation. *P 0.01 (unpaired t test).",
    "molecules": "3-MA, amino-acid, nigericin"
  },
  {
    "caption": "(d) Confocal microscopy of LPS-primed human monocytes-macrophages transfected for 3 h with poly(dA:dT) (top), treated for 3 h with nigericin (middle) or left unstimulated (bottom), immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins. Second row, three-dimensional volume rendering reconstructed from confocal z-stack images. Scale bars, 10 μm. Data are representative of at least two experiments.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(a) Immunoblot analysis of lysates of LPS (20 ng/ml)-primed mouse BMDMs exposed to LPS (500 ng/ml) and ATP (3 μM), poly(dA:dT) (1.5 μg/ml) or uric acid crystals (50 μg/ml), detecting GTP-bound RalB (collected by RaBP1-agarose affinity purification) and RalB in total lysates.",
    "molecules": "LPS, ATP, GTP, uric acid"
  },
  {
    "caption": "(b) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB-specific siRNA on 2 sequential days, then primed with LPS and exposed for 6 h to poly(dA:dT) (1.5 μg/ml), probed for RalB, LC3 and IL-1β; supernatants were collected after overnight culture in serum-free medium (numbers below lanes as in Fig. 1d). Nonsp, nonspecific band.",
    "molecules": "LPS"
  },
  {
    "caption": "(c) Immunoblot analysis of lysates of wild-type and Aim2−/− mouse BMDMs transfected with control or AIM2-specific siRNA on 2 sequential days, primed for 2 h with LPS and then exposed for 1 h to poly(dA:dT) (numbers below lanes as in Fig. 1d).",
    "molecules": "LPS"
  },
  {
    "caption": "(d) Immunoblot analysis of ASC immunoprecipitates from LPS-primed mouse BMDMs left untreated or transfected for 1 h with poly(dA:dT) and treated for 30 min with LPS and ATP. Mock, immunoprecipitation with control antibody.",
    "molecules": "LPS, ATP"
  },
  {
    "caption": "(e) Confocal microscopy of LPS-primed mouse BMDMs left untransfected or transfected for 30 min with poly(dA:dT), immunostained for RalB and AIM2 (left three columns); RalB and ASC (middle columns); and beclin-1 and AIM2 (right two columns). Bottom row, 3× electronic enlargement of areas outlined above. Scale bars, 10 μm. Data are representative of at least two experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Adult Swiss mice (n = 11 Veh; 15 AβOs) received a single i.c.v. injection of vehicle or 10 pmol AβOs and were assessed in a glucose tolerance test (2 g glucose/kg body weight, i.p.) 7 days after injection. Blood levels of glucose were measured at several time points following glucose administration. Bar graph represents areas under the curves in the time course plot. Data are representative of three independent experiments with similar results. Left panel: ***P = 0.0006, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0207, Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "Insulin tolerance test (1 IU insulin/kg body weight, i.p.) (n = 7 Veh; 8 AβOs). Blood levels of glucose were measured at several time points following insulin administration. Bar graph represents the kinetic constants for glucose disappearance (Kitt) calculated from the time course plot. Data are representative of two independent experiments with similar results. Left panel: *P = 0.0456 and ***P = 0.0007, two-way ANOVA followed by Bonferroni post hoc test; right panel: **P = 0.0033, Student's t-test.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice 7 days after a single intracaudal (C; n = 8 animals/group) or intraperitoneal (D; n = 13 animals/group) injection of AβOs (10 pmol) or vehicle.",
    "molecules": "glucose"
  },
  {
    "caption": "Glucose tolerance test (2 g glucose/kg body weight, i.p.) in 8- to 13-month-old APP/PS1 mice (E; n = 9 animals/group) or 6-month-old 3xTg-AD male mice (F; n = 10 WT; 9 3xTg), or their corresponding wild-type littermates. Bar graph represents areas under the curves in the time course plots. In (E), left panel: *P = 0.0466, two-way ANOVA followed by Bonferroni post hoc test; right panel: &P = 0.072, Student's t-test. In (F), left panel: *P = 0.0171 and #P = 0.0781, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0101, Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "Representative images of GLUT-4 immunofluorescence in insulin-stimulated skeletal muscle from mice that were i.c.v.-injected with vehicle (Veh) or 10 pmol AβOs. Bar graphs show quantification of GLUT-4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin (1 IU/kg body weight) 7 days after i.c.v. injection of vehicle or AβOs, as indicated (n = 5 animals/group). Scale bar = 25 μm. *P = 0.0144, one-way ANOVA followed by Bonferroni post hoc test.",
    "molecules": "insulin"
  },
  {
    "caption": "Plasma levels of insulin (K; n = 12 animals/group), leptin (L; n = 11 Veh; 12 AβOs), cholesterol (M; n = 8 Veh; 6 AβOs), triglycerides (N; n = 8 Veh; 6 AβOs) or noradrenaline (O; n = 7 Veh; 8 AβOs) measured 7 days after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. In (O), *P = 0.0361, Student's t-test.",
    "molecules": "cholesterol, insulin, leptin, noradrenaline, triglycerides"
  },
  {
    "caption": "Representative immunofluorescence images of eIF2α-P in hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Scale bar = 30 μm. Graph represents integrated immunofluorescence intensities of eIF2α-P levels from three independent hypothalamic cultures (three coverslips/experimental condition per experiment, 20 images per coverslip). Bars represent means ± SEM. *P = 0.0489, one-way ANOVA followed by Bonferroni post hoc test comparing AβO-treated versus vehicle-treated cultures.",
    "molecules": "infliximab"
  },
  {
    "caption": "Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Similar patterns of AβO binding were observed in both conditions. Scale bar = 20 μm.",
    "molecules": "infliximab"
  },
  {
    "caption": "Twelve-hour food intake after a single i.c.v. infusion of insulin (200 mU) in mice. Experiment was performed 7 days after i.c.v. injection of vehicle or AβOs (n = 5 PBS; 5 Veh + Insulin; 9 AβOs + Insulin), data are representative of two independent experiments with similar results. ***P < 0.0001, one-way ANOVA followed by Bonferroni post hoc test comparing Veh-Insulin versus PBS groups.",
    "molecules": "food, insulin, Insulin"
  },
  {
    "caption": "Accumulated chow intake (normalized by body weight) measured during 7 days following a single i.c.v. injection of vehicle or 10 pmol AβOs in mice (n = 13 Veh; 10 AβOs; data are representative of two independent experiments with similar results). ***P < 0.0001; Student's t-test.",
    "molecules": "chow"
  },
  {
    "caption": "Swiss mice received a single i.c.v. injection of vehicle (Veh) or 10 pmol AβOs, and their brains were analyzed by Fluorojade staining of degenerating cells 7 days after the injection. Representative images of Fluorojade staining in the hypothalamus of vehicle- or AβO-injected mice (n = 4/group). Scale bar = 100 μm in left panels (top and bottom) and 20 μm in right panels (top and bottom). Positive control (bottom left panel) was the hippocampus of a mouse that received one i.c.v. injection of quinolinic acid (36.8 nmol) and was analyzed 24 h after.",
    "molecules": "quinolinic acid"
  },
  {
    "caption": "Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice that received i.c.v. injections of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA (when used, TUDCA was administered in 5 i.c.v. injections of 5 μg TUDCA each, before and after oligomer injection; see Materials and Methods. Control groups received injections of saline). Glucose tolerance test (GTT) was performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 15 Veh; 15 AβOs; 10 Veh + TUDCA; 16 AβOs + TUDCA). Data are representative of two independent experiments with similar results. Left panel: **P = 0.0048, **P = 0.003, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0384, one-way ANOVA followed by Bonferroni post hoc test.",
    "molecules": "glucose, TUDCA"
  },
  {
    "caption": "Plasma noradrenaline (NA) levels measured 7 days after i.c.v. injection of vehicle, vehicle + TUDCA, AβOs or AβOs + TUDCA in mice (n = 7 animals/group). Data are representative of two independent experiments with similar results. *P = 0.0071, one-way ANOVA followed by Bonferroni post hoc test.",
    "molecules": "noradrenaline, TUDCA"
  },
  {
    "caption": "Glucose tolerance test (2 g glucose/kg body weight, i.p.) in TNFR1−/− mice or wild-type littermates performed 7 days after i.c.v. injection of vehicle or AβOs. Bar graph represents areas under the curves (AUC) in the time course plots (n = 8 WT + Veh; 7 WT + AβOs; 7 TNFR−/− + Veh; 8 TNFR−/− + AβOs). Left panel: **P = 0.0049, ***P < 0.0001, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0001, one-way ANOVA followed by Bonferroni post hoc test.",
    "molecules": "glucose"
  },
  {
    "caption": "Adult Swiss mice pre-treated with minocycline or PBS received a single i.c.v. injection of vehicle or 10 pmol AβOs, and hypothalamic levels of mRNA for AgRP (F; n = 5 Veh; 6 AβOs; 5 Veh + Mino; 4 AβOs + Mino) and NPY (G; n = 14 Veh; 13 AβOs; 8 Veh + Mino; 9 AβOs + Mino) were analyzed 7 days after injection. In (F), *P = 0.0097, one-way ANOVA followed by Bonferroni post hoc test; in (G), *P = 0.0219, one-way ANOVA followed by Bonferroni post hoc test.",
    "molecules": "Mino, minocycline"
  },
  {
    "caption": "Glucose tolerance test (GTT) in APP/PS1 mice before and after i.c.v. injection of infliximab (0.2 μg daily for 4 days). Bar graph represents areas under the curves (AUC) in the time course plots (n = 9 animals/group). Left panel: *P = 0.0177, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0327, paired t-test.",
    "molecules": "infliximab"
  },
  {
    "caption": "C. RADX interacts with ssDNA. Extracts of U2OS cells stably expressing GFP-RADX WT or mutants were incubated with biotin-coupled ssDNA oligo immobilized on Streptavidin beads, washed extensively and immunoblotted with GFP and RPA1 antibodies.",
    "molecules": "biotin, ssDNA"
  },
  {
    "caption": "D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting.",
    "molecules": "ssDNA"
  },
  {
    "caption": "E. Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in (C) and immunoblotted with RADX and RPA1 antibodies.",
    "molecules": "dsDNA, ssDNA"
  },
  {
    "caption": "G. Representative images from in situ proximity ligation assays (PLAs) in BrdU-labeled U2OS and GFP/GFP-RADX cell lines (Fig EV1E,F) using GFP and BrdU antibodies under native conditions. Scale bar, 10 μm.",
    "molecules": "BrdU"
  },
  {
    "caption": "A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU.",
    "molecules": "HU"
  },
  {
    "caption": "B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A.",
    "molecules": "HU"
  },
  {
    "caption": "E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation.",
    "molecules": "CPT, EdU, HU"
  },
  {
    "caption": "C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).",
    "molecules": "IdU, CldU"
  },
  {
    "caption": "E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).",
    "molecules": "CldU"
  },
  {
    "caption": "F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).",
    "molecules": "IdU"
  },
  {
    "caption": "H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).",
    "molecules": "CldU"
  },
  {
    "caption": "I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).",
    "molecules": "IdU, CldU"
  },
  {
    "caption": "A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).",
    "molecules": "IdU, CldU"
  },
  {
    "caption": "C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).",
    "molecules": "CldU"
  },
  {
    "caption": "D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).",
    "molecules": "IdU"
  },
  {
    "caption": "F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).",
    "molecules": "IdU, CldU"
  },
  {
    "caption": "B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition).",
    "molecules": "IdU, CldU"
  },
  {
    "caption": "D. U2OS or U2OS/GFP-RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co-immunostained with RPA1 and γ-H2AX antibodies and analyzed by QIBC. Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation (Fig EV5A), reflecting replication fork catastrophe [17], is indicated (bars, mean; n=5 independent experiments (≥3000 cells analyzed per condition); *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, unpaired t-test).",
    "molecules": "HU"
  },
  {
    "caption": "E. As in (D), but using U2OS or U2OS/Super-RPA cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B.",
    "molecules": "HU"
  },
  {
    "caption": "F. Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h (mean±SEM;n=3 independent experiments). LQ (Linear Quadratic) model was fitted to the fractional survival data, using non-linear least square method. Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment (*No overlap of 95% confidence interval with siCTRL).",
    "molecules": "CPT"
  },
  {
    "caption": "G. As in (F), except cells were treated with indicated doses of HU (mean±SEM;n=3 independent experiments).",
    "molecules": "HU"
  },
  {
    "caption": "(D) Mean and SEM of mRNA levels of Xbp1s, Bip/Hspa5 and Chop/Ddit3 were determined in dissected hippocampus of i.p. treated mice by quantitative RT-PCR. (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups).",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(E) Animals received bilateral intra-hippocampal injections of 2 μl tunicamcyin (5 mg/mL) or vehicle for 24 h. Graphs indicate mean and s.e.m. of Xbp1s mRNA levels in the hippocampus (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups.",
    "molecules": "tunicamcyin, tunicamycin"
  },
  {
    "caption": "E) Localization analysis of GFP-fused CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 (green) on the mitotic chromosomes in chicken DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 10 μm.",
    "molecules": "4',6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "F) Localization of CENP-C in CENP-A knockout DT40 cells stably expressing GFP-fused gCENP-A WT, RG-AA, or ∆RG. Scale bar indicates 10 μm. CENP-C was stained by an anti-CENP-C antibody (red) and DNA was stained by DAPI (blue). CENP-C signals on kinetochores in mitotic cells were quantified in each cell. Bar graph indicates mean ± SD (n = 7; ****, P < 0.0001, unpaired t test, two tailed).",
    "molecules": "DAPI"
  },
  {
    "caption": "E) Localization analysis of GFP-fused CENPC-CT and CENPC643-864 lacking the CM upstream region (green) on mitotic chromosomes in CENP-C knockout DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A) Competitive pull-down assays for the CENP-A nucleosome binding of CENPC-CT and CENPN-NT. The left panel displays a complex of MBP-CENPN-NT (MBP-CN-NT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of phosphorylated CENPC-CT (CC-CT) and pulled down by MBP affinity. The right panel displays a complex of phosphorylated MBP-CENPC-CT (MBP-CC-CT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of CENPN-NT (CN-NT) and pulled down by MBP affinity. The signal intensities of all histones in CENP-A nucleosomes, which were precipitated with MBP proteins in indicated lanes (*), were quantified. The signal intensities were normalized to MBP-CN-NT or MBP-CC-CT signals. Bar graph indicates mean with SD (n = 3).",
    "molecules": "histones"
  },
  {
    "caption": "The cells were grown in the growth medium and stained for progerin (red) and F-actin with phalloidin (green). The cell with high or low progerin expression was defined by the progerin fluorescence intensity above or below 3.5 x 106 (A.U.), respectively. The cell spreading area was measured according to the phalloidin staining (n ≥ 30). Scale bars, 50 m.",
    "molecules": "phalloidin"
  },
  {
    "caption": "D The cells were serum-starved for 32 h to allow cilia formation and then treated with (+) or without (-) 250 nM cytochalasin D (Cyto D) for another 16 h before fixation. The cells were stained for progerin (red), acetylated tubulin (green), F-actin (white), and DNA (blue). The numbers of actin filaments within 60 μm2 around the cilia (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (n ≥ 55). Scale bars, 20 μm or 2 μm (in the magnified insets).",
    "molecules": "Cyto D, cytochalasin D"
  },
  {
    "caption": "D-G The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D (250 nM) for another 16 h. The cells were stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (D). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of actin filaments within 60 μm2 around the basal body (as illustrated by the circle) from the cells under serum-starvation without Cyto D treatment were measured (E, n ≥ 132). The percentage of the cells with cilia in the total counted cells (F, n ≥ 357) and the length of cilia (G, n ≥ 126) were measured.",
    "molecules": "Cyto D"
  },
  {
    "caption": "E,F RPE cells expressing shNesprine 2 or shLuc were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The percentage of the cells with cilia in the total counted cells (E, n ≥ 197) and the length of cilia (F, n ≥ 52) were measured.",
    "molecules": "Cyto D"
  },
  {
    "caption": "G-I RPE cells and those expressing shRNAs to importin 9 were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h and then stained for acetylated tubulin (white), pericentrin (green) and F-actin (red). Representative images are shown (G). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The percentage of the cells with cilia (H, n ≥ 385) and the length of cilia (I, n ≥ 111) were measured.",
    "molecules": "Cyto D"
  },
  {
    "caption": "B The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The cells were stained for acetylated tubulin (Ac-tubulin, green) and TTBK2 (red). The graphs shown are the quantifications of TTBK2 intensity at cilia-negative basal bodies and cilia-positive basal bodies.",
    "molecules": "Cyto D"
  },
  {
    "caption": ". e) Southern Blot analysis to detect integration events in LV- and NILV-S/MAR-transduced single cell clones with DNA probed against the viral genome. A 6.8 kb positive control from a linearized plasmid (lane 3) was included for indication of the size of DNA circles formed by non-integrated NILV-S/MAR vector.",
    "molecules": "DNA"
  },
  {
    "caption": "f-h) A padlock probe assay, as illustrated in Appendix Figure S4, was used to demonstrate persistence of proviral DNA either in a circular or integrated form. Representative images show DNA persistence in (f) LV-, (g) NILV- or (h) NILV-S/MAR-transduced cells. Each red dot (Cy3) represents a single DNA molecule and cell nuclei were stained with Hoechst 33342.",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Complex I in gel activity assays (IGA) after Blue-native gel electrophoresis (BNGE) of WT and ∆4-CYB samples solubilized either with 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 hours (lighter signals in DDM gels) or were left to continue the reaction for 24 hours to obtain darker signals (DDM and Digitonin gels).",
    "molecules": "digitonin, Digitonin, DDM"
  },
  {
    "caption": "(B) Labeling of the thirteen mtDNA-encoded MRC structural subunits. Cells were incubated with [35S]-L-Met for one hour in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation.",
    "molecules": "mtDNA, emetine, L-Met, 35S"
  },
  {
    "caption": "(B) Second-dimension BNGE of digitonin-solubilized samples from WT and ∆4-CYB cells, western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles.",
    "molecules": "digitonin"
  },
  {
    "caption": "(B) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM.",
    "molecules": "digitonin, DDM"
  },
  {
    "caption": "(C) BNGE, Western blot and immunodetection, with the monoclonal (M) anti-UQCRQ antibody (Abcam ab110255), of non-transduced ∆4-CYB and WT cells. The mitoplast samples were solubilized with DDM",
    "molecules": "DDM"
  },
  {
    "caption": "(F) SDS-PAGE, Western blot and immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing an HA-tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).",
    "molecules": "cDNA"
  },
  {
    "caption": "(G) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the cell lines solubilized either with digitonin.",
    "molecules": "digitonin"
  },
  {
    "caption": "(A) SDS-PAGE resolving the radioactively labeled mitochondrial translation products after a 2-hour pulse (P). The 35S-Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times (2, 5 and 24 hours).",
    "molecules": "Met, cycloheximide, 35S"
  },
  {
    "caption": "(B) First dimension (1D) BNGE analysis of the same cells prepared with digitonin.",
    "molecules": "digitonin"
  },
  {
    "caption": "(D) Complex I IGA (cI-IGA) analysis of digitonin-solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0h). After removing the drug and restoring synthesis of the mtDNA-encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 hours. SS = steady state. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature",
    "molecules": "mtDNA, digitonin, doxycycline"
  },
  {
    "caption": "(E) 2D BNGE, Western blot and immunodetection analysis of WT and ∆4-CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear-encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 sec. (low exposures) or 160 sec. (high exposures) in order to visualize the qualitative signals in the ∆4-CYB samples.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every six hours after substituting the medium in two replicate 24-well plates, one plate with medium without uridine (Uridine-), and the second plate with medium supplemented with 50 µg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n=6 wells per cell line).",
    "molecules": "uridine, Uridine"
  },
  {
    "caption": "(D) 1D BNGE, Western blot and immunodetection analyses of digitonin-solubilized mitochondria from WT and ∆4-CYB cybrids expressing AOXHA and their corresponding EV controls.",
    "molecules": "digitonin"
  },
  {
    "caption": "(G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.",
    "molecules": "antimycin A, CCCP, oligomycin, ROTENONE, rotenone"
  },
  {
    "caption": "(H) Respirometry analyses , in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.",
    "molecules": "antimycin A"
  },
  {
    "caption": "(I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.",
    "molecules": "antimycin A, digitonin"
  },
  {
    "caption": "(D) 1D BNGE, Western blot and immunodetection of two cIV subunits (MT-CO1 and COX7B) in samples from WT and ∆4-CYB cells solubilized with DDM and digitonin (Dig).",
    "molecules": "Dig, digitonin, DDM"
  },
  {
    "caption": "(F) Percentage of cells infected with WT Listeria, in the presence (black bars) or absence (white bars) of rapamycin, displaying one or several GFP‐LC3+ Listeria autophagosomes. Values are means±s.e.m. n=2. Scale bars: 5 μm.Source data for this figure is available on the online supplementary information page.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(B) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected with Listeria WT for 4 h were analysed by IF using an antibody against ubiquitinated proteins (Ubi).",
    "molecules": "proteins"
  },
  {
    "caption": "(E) HeLa cells infected with Listeria WT for 4 h, in the absence or presence of Wortmannin added during the last hour of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. Scale bars: 5 μm for all panels except (D) (1.5 μm).",
    "molecules": "Wortmannin"
  },
  {
    "caption": "(B) HeLa cells left unstimulated (CTR) or infected with Listeria WT or PlcA/B− for 4 h were lysed and PI3P levels measured by competitive ELISA.",
    "molecules": "PI3P"
  },
  {
    "caption": "(D) HeLa cells infected with Listeria PlcA/B− for 3 h, in the absence or presence of Wortmannin added during the last 10 min of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. The arrow indicates an NDP52+ granule. Scale bars: 5 μm.",
    "molecules": "Wortmannin"
  },
  {
    "caption": "A. Cryo-sections of  human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect  immunofluorescence. Nuclei  are  stained with DAPI. Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Human intestinal organoids were incubated with media (mock) or infected with  HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei  (DAPI, blue). Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "C.  Human intestinal  organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained  for actin (magenta) and Laminin (red) respectively. Nuclei are  stained  with  DAPI  (blue). Scale bar is 20 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. One  image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show  co-localization of FABP6 and  CCL2 with HAstV1 signal. Scale bar 100µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Michaelis-Menten analysis of the reduction of  2-hydroxyacetophenone (2-HAP). The initial velocities (V0/[Etot]) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays  were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH  and 100 nM SCR  or 1 µM SCR-his6. Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.",
    "molecules": "2-HAP, 2-hydroxyacetophenone, NADPH, sodium phosphate"
  },
  {
    "caption": "Comparison of kcat values obtained from Michaelis-Menten analyses of the reduction of four different substrates. The turnover rates  (kcat) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays  were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH  and 100 nM SCR  or 1 µM SCR-his6. The data points show the average values for these technical replicates, while the error bars show the standard deviations.",
    "molecules": "NADPH, sodium phosphate"
  },
  {
    "caption": "(A) Analytical gel-filtration  of purified his8-MBP-SCR2  (black), strep-SCR  (yellow), and his8-MBP-SCR2/strep-SCR  on a Superdex 200 Increase column at 20 ˚C in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0.",
    "molecules": "Tris-HCl, NaCl, strep"
  },
  {
    "caption": "(B) Michaelis-Menten kinetic analyses of NADPH-dependent reduction of 2-hydroxyacetophenone and methyl acetoacetate  at 30 ˚C in 100 mM sodium phosphate, pH 6.0. The  turnover rate (V0/[Etot]) is given for the individual subunits  Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.",
    "molecules": "2-hydroxyacetophenone, methyl acetoacetate, NADPH, sodium phosphate"
  },
  {
    "caption": "A, Cell surface biotinylation assay performed in HEK293T cells transfected as indicated and treated with 10 μM MG132 to prevent proteasomal degradation. Consecutive immunoprecipitations were carried out as indicated in the scheme. The Avidin pulldown was carried under denaturing conditions (1st IP). The unrelated receptor CD147 was used as loading control for the avidin pull down, and CUL1 for the total input of cell lysate.",
    "molecules": "Avidin, avidin, MG132"
  },
  {
    "caption": "E-H, Immunofluorescence microscopy showing endogenous E-cadherin levels in wt and WLS (Evi) KO HCT116 cells upon transfection with siUSP42 (E), or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs(G). Scale bar = 20 μm. In (F,G), quantification of the fluorescent signal (Alexa-488) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown.",
    "molecules": "Alexa-488"
  },
  {
    "caption": "I-L, Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 (I-K) or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs (L). Scale bar = 500 μm. Where indicated, cells were treated with 5 μM IWP-2 or 20 μM LGK-974 (PORCN inhibitors). The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown.",
    "molecules": "IWP-2, LGK-974"
  },
  {
    "caption": "A, Representative images of mouse small intestinal organoids generated with empty (Non-edited) or USP42gRNA containing CRISPR-concatemers, and cultured for 3 days in matrigel with the indicated growth factors, or the Wnt secretion inhibitor IWP-2. EN, EGF and noggin. Note that knockout of Usp42 (Usp42 gRNAs) rendered the organoids insensitive to RSPO1 withdrawal. Scale bar = 1 mm.",
    "molecules": "IWP-2"
  },
  {
    "caption": "F) In vitro competition assay  of sgCtrl- and sgTsc2-expressing D10  cells co-cultured with MART-1 T cells  in the presence of necroptosis (Nec1-s, 20uM) or apoptosis inhibitors (Q-VD-Oph, 50uM), normalized to inhibitor plus Ctrl  T cell-treated groups, showing the net MART-1 T cell  effect. Statistical analysis was performed by one-way ANOVA with Holm-Sidak's multiple comparisons test. Error bars indicate SD of three biological replicates with different T cell  donors (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.",
    "molecules": "Q-VD-Oph, Nec1-s"
  },
  {
    "caption": "total protein in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.",
    "molecules": "protein"
  },
  {
    "caption": "total eFluor780+ events (E) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.",
    "molecules": "eFluor780"
  },
  {
    "caption": "Total protein in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.",
    "molecules": "protein"
  },
  {
    "caption": "total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.",
    "molecules": "eFluor780"
  },
  {
    "caption": "Total protein in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.",
    "molecules": "protein"
  },
  {
    "caption": "total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.",
    "molecules": "eFluor780"
  },
  {
    "caption": "Total eFluor780+ events in the BAL of influenza-infected mice described in A, B 10 days p.i. (n = 3 mice from each group).",
    "molecules": "eFluor780"
  },
  {
    "caption": "Oxygen consumption rate (OCR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with oligomycin (Oligo), FCCP or rotenone (Rot) and antimycin A (AA).",
    "molecules": "AA, antimycin A, FCCP, Oligo, oligomycin, Rot, rotenone"
  },
  {
    "caption": "Extracellular acidification rate (ECAR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with glucose, Oligo, or 2-Deoxy-D-glucose (2-DG).",
    "molecules": "2-Deoxy-D-glucose, 2-DG, glucose, Oligo"
  },
  {
    "caption": "D-E. Two orthogonal views of the partially open SARS-CoV-2 S trimer cryoEM map (one SB domain is open). F. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel D. The glycans were omitted for clarity.",
    "molecules": "glycans"
  },
  {
    "caption": "a, Internalization of beads with PAM3CSK, LPS, or Alexa-Fluor-555-labelled BSA (Red-BSA; yellow arrows) and association with GFP-LC3 in RAW cells was followed by time-lapse video for 3 h, 1 frame/2 min (representative frames are shown).",
    "molecules": "PAM3CSK, LPS"
  },
  {
    "caption": "GFP-LC3 in RAW cells translocates to phagosomes containing zymosan (red), assessed as in a",
    "molecules": "zymosan"
  },
  {
    "caption": "c, Time course of translocation of GFP-LC3 to a phagosome containing zymosan (red) (see Supplementary Movie 2). Time (minutes) is indicated in the top left of each panel.",
    "molecules": "zymosan"
  },
  {
    "caption": "d, LC3 translocation to the phagosome is induced by TLR signalling. GFP-LC3 was transiently transfected into wild-type or Tlr2-/- macrophages, which were exposed to zymosan for 45 min.",
    "molecules": "zymosan"
  },
  {
    "caption": "a, RAW cells expressing GFP-LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides. At 24 h after transfection, cells were fed with zymosan (yellow arrows) and the internalization process was followed by time-lapse video microscopy. Images obtained at 5 and 30 min after zymosan engulfment are shown. b, The percentage of GFP-LC3-associated phagosomes (n ≥ 100 per /group) was obtained from three independent time-lapse videos (2 h each) of macrophages transfected with control or Atg5 siRNA (means and ranges are shown).",
    "molecules": "zymosan"
  },
  {
    "caption": "e, Ly294002 (Ly29, 10 µM) was added 10 min before zymosan, and wortmannin was added after engulfment but before GFP-LC3 translocation in RAW-GFP-LC3 cells. Representative images are shown. f, Percentage of GFP-LC3-associated phagosomes (n ≥ 50 per group) with P(I)3K inhibitors assessed by time-lapse video (means and ranges are shown; three independent 3 h videos).",
    "molecules": "zymosan, P(I)3K inhibitor, Ly294002, wortmannin"
  },
  {
    "caption": "g, RAW cells expressing GFP-LC3 and the PX domain of p40(phox) fused to mCherry (red) were fed with zymosan (yellow arrows) or with beads (Supplementary Fig. 6). Internalization was followed at 1-min intervals (Supplementary Movie 5), representative images are shown. Single cells were followed for at least 2 h (n ≥ 20 per group) (Supplementary Movies 6, 7 and Supplementary Fig. 6). Mean ± s.d. duration of zymosan-PX translocation to the zymosan for beads (B) and zymosan (Z) are shown.",
    "molecules": "zymosan"
  },
  {
    "caption": "h, Internalization of zymosan (yellow arrows) was followed at 1.5-min intervals for at least 2 h in RAW cells transiently transfected with GFP-BECN1. Representative images are shown. Translocation of GFP-BECN1 to the phagosome for beads (B) and zymosan (Z) was quantified (mean ± s.d.) using time-lapse movies (Supplementary Movie 8 and 9; n ≥ 20 cells per group). For g and h, time (in minutes) is indicated at the top left of each panel.",
    "molecules": "zymosan"
  },
  {
    "caption": "a, Internalization of control or PAM3CSK4-covered beads was followed in RAW-GFP-LC3 cells pre-loaded with lysotracker (red) by time-lapse video for 3 h. Time (in minutes) is indicated at the top left of each panel.",
    "molecules": "PAM3CSK4"
  },
  {
    "caption": "b, RAW cells pre-loaded with lysotracker were exposed to control or PAM3CSK-covered beads for 6 h. Acidification of the beads (n ≥ 20 per group), measured as maximum lysotracker relative light units (RLU) on a horizontal cross-section of each bead represented in the x axis (mean ± s.d.; Wilcoxon-Mann-Whitney test, P 0.0005), is shown.",
    "molecules": "PAM3CSK"
  },
  {
    "caption": "c, d, Knockdown of ATG5 reduced zymosan-induced GFP-LC3 translocation and lysosomal fusion.",
    "molecules": "zymosan"
  },
  {
    "caption": "Phenotypic changes of HSCs by citral. Phase-contrast images (left), oil red O-stained images (middle), and autofluorescence images (right) are shown for HSCs after passage 1 with or without the treatment of citral (100 μM) for 24 h. Scale bar = 10 μm.",
    "molecules": "citral"
  },
  {
    "caption": "Western blot analysis of cell lysates from the citral-treated HSCs in (A). α-tubulin was used as a loading control. The quantitative densitometric data are expressed as the percentage of untreated control and represent the means (SD) for three independent experiments. **P-value = 0.002, paired t-test (compared to control).",
    "molecules": "citral"
  },
  {
    "caption": "Reverse-phase HPLC analysis of whole-cell lysates from the siRNA-ALDH1A-treated HSCs in (E). Typical chromatograms of all-trans retinoic acid and 13-cis retinoic acid. The retention time is 14 min for 13-cis-RA and 23 min for all-trans RA.",
    "molecules": "RA, retinoic acid"
  },
  {
    "caption": "Phenotypic changes of HSCs by RAR antagonist. HSCs-d3 were incubated with AGN193109 (1 μM) ± all-trans RA (10 μM) for 3 days and subjected to Oil red O staining. Scale bar = 10 μm.",
    "molecules": "AGN193109, RA"
  },
  {
    "caption": "Western blot analysis of cell lysates from AGN193109 ± RA-treated HSCs in (G). **P = 0.003, paired t-test (n = 3) (AGN193109 compared to control).",
    "molecules": "AGN193109, RA"
  },
  {
    "caption": "Albumin and R-III downregulate RA signaling. HSCs-P1 were either transfected with RARE Cignal reporter and expression plasmid containing vehicle or albumin (left panel) or treated with R-III (25-250 nM) after transfection with RARE Cignal reporter (right panel). After 36 h, the transfected cells were harvested for luciferase assay. Renilla luciferase activity was normalized to firefly luciferase activity. Ratios were normalized against the cells transfected with vehicle plasmid. **P-value, two-sample t-test (n = 9) (compared to control), albumin: 0.0037, R-III 125 nM: 0.0032.",
    "molecules": "RA"
  },
  {
    "caption": "Phenotypic changes of HSCs by the expression of albumin and/or ALDH1A. HSCs-P1 were transfected with plasmids encoding albumin, ALDH1A1, and ALDH1A2, either individually or in combination, and incubated in the presence of all-trans retinoic acid (50 nM) for 18 h. Cells were analyzed by oil red O staining. Scale bar, 20 μm.",
    "molecules": "retinoic acid"
  },
  {
    "caption": "RAR agonist counteracts R-III action. HSCs-P1 were treated with His-tagged R-III (final concentration, 0.15 μM) in the presence or absence of RAR agonist (AGN191183, 1 μM) for 18 h and analyzed by Western blotting. **P = 0.0024, paired t-test (n = 3) (R-III compared to control). The separating lines demarcate bands that come from non-consecutive lanes of the same gel.",
    "molecules": "AGN191183"
  },
  {
    "caption": "Representative macroscopic pictures of livers from control and CCl4-, CCl4/albumin-, CCl4/RBP-, and CCl4/R-III-treated mice.",
    "molecules": "CCl4"
  },
  {
    "caption": "Hydroxyproline content in the livers from control and CCl4-, CCl4/albumin-, and CCl4/R-III-treated mice (μg/g liver). *P = 0.037, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).",
    "molecules": "CCl4, Hydroxyproline"
  },
  {
    "caption": "Liver sections from control and CCl4- and CCl4/R-III-treated mice were immunohistochemically stained for TGF-β and F4/80. Scale bar, 50 μm.",
    "molecules": "CCl4"
  },
  {
    "caption": "Liver sections from control and CCl4- and CCl4/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm.",
    "molecules": "CCl4"
  },
  {
    "caption": "Hydroxyproline content in the livers (μg/g liver). *P = 0.034, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).",
    "molecules": "CCl4, Hydroxyproline"
  },
  {
    "caption": "Liver sections from control, BDL, and BDL/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm..",
    "molecules": "BDL"
  },
  {
    "caption": "Hydroxyproline content in the livers (μg/g liver). *P-value, two-sample t-test (n = 10) (compared to BDL-treated mice); BDL + R-III (10 μg): 0.031, BDL+R-III (5 μg): 0.026.",
    "molecules": "BDL, Hydroxyproline"
  },
  {
    "caption": "Plasma from healthy individuals receiving (B) placebo (n=9) or (C) sitagliptin (n=27) were analyzed by Simoa. Blood samples were collected at screening visit (SV) and day 0 (D0) before treatment, at day 3 (D3), day 14 (W2) and day 28 (W4) under treatment and 5 weeks after treatment interruption (W9). (C) Plasma from chronic HCVpatients receiving sitagliptin (n=3) were collected before (D0) and weekly during sitagliptin treatment (W1 & W3). Antagonist CXCL103-77 (short CXCL10, in blue), agonist CXCL101-77 (long CXCL10, in red) and total CXCL10 (in black) levels are shown. Each dot represents a donor and bars are at the median. Grey areas highlight the period under placebo or sitagliptin treatment. Statistical analysis of (B) and (C) was performed using non parametric Friedman's test, ns: non significant, **: P<0.01, ****: P<0.001. For (C), additional size effect analysis was performed and Cohen's d values are reported. No statistical analysis was performed in (D) due to sample size, nd: non-determined.",
    "molecules": "sitagliptin"
  },
  {
    "caption": "(D) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel (top-down view); wing disc resting on a stretching device (anchor) and 10 min after stretch; M=microchannel.",
    "molecules": "PDMS"
  },
  {
    "caption": "(F) Sqh::GFP polarity in enGal4> dia iR, UAS-Act5C::RFP discs labeled with Phalloidin-647; Inset (Phall-647) zooms in on cell shape in anterior and posterior disc compartments.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "(G) Discs expressing nubGal4 and Cd8::GFP (control) or dia iR (KK) labeled with DAPI; ellipse is fitted into the pouch region (red dotted line) and aspect ratio determined as long ellipse axis (b) divided by short ellipse axis (a) (white lines).",
    "molecules": "DAPI"
  },
  {
    "caption": "SMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. NIPBL was depleted in G2 phase after completion of DNA  replication to avoid potential effects of NIPBL depletion on cohesion establishment. B, Representative images from wild type prometaphase cells labelled on one or two sister chromatids as indicated. C, Representative images from wild type G2 cells labelled on one or two sister chromatids as indicated. D, Quantification of sister chromatid separation in one-sister labelled prometaphase cells for experimental conditions as indicated. Dots represent individual cells; red bars indicate the mean. Wild type (n = 83 cells), ΔSMC4 (n = 75 cells) and ΔNIPBL (n = 48 cells) were analysed. Significance was tested using a two-tailed Mann Whitney U test; P = 2.33 x 10-27. Two-sister labelled wild type prometaphase (n = 4), one-sister labelled wild type prometaphase (n = 6), ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3), wild type G2 (n = 4), ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates Two-sister labelled wild type prometaphase (n = 9), one sister-labelled wild type prometaphase (n = 14), ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6), wild type G2 (n = 8), ΔSMC4 G2 (n = 5), ΔNIPBL G2  (n = 8). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 μm, insets: 1 μ",
    "molecules": "5-Ph-IAA, DNA"
  },
  {
    "caption": "SMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. NIPBL was depleted in G2 phase after completion of DNA replication to avoid potential effects of NIPBL depletion on cohesion establishment. F, Representative images from ΔSMC4 prometaphase cells labelled on one sister chromatid. G, Representative images from ΔSMC4 G2 cells labelled on one sister chromatid. H, Representative images from ΔNIPBL prometaphase cells labelled on one sister chromatid. I, Representative images from ΔNIPBL G2 cells labelled on one sister chromatid. Two-sister labelled , ΔSMC4 prometaphase (n = 4), ΔNIPBL prometaphase (n = 3) , ΔSMC4 G2 (n = 3), ΔNIPBL G2 (n = 5). Technical replicates: Two-sister labelled , ΔSMC4 prometaphase (n = 9), ΔNIPBL prometaphase (n = 6) , ΔSMC4 G2 (n = 5), ΔNIPBL G2 (n = 8).",
    "molecules": "5-Ph-IAA"
  },
  {
    "caption": "WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. One sister chromatid was labelled per chromosome as in Fig. 1 SCC1 (WAPL-dTAG cells) was visualised by immunofluorescence. A, Representative images of control WAPL-dTAG cells synchronised to G2 by RO-3306. B, Representative images of ΔWAPL cells synchronised to G2 by RO-3306. C, Representative images of ΔSororin cells synchronised to G2 by RO-3306. D, Quantification of sister chromatid separation as in Fig. 1D Control WAPL-dTAG G2 (n = 36 cells), ΔWAPL G2 (n = 53 cells) and ΔSororin G2 (n = 45 cells) cells were analysed. Dots represent individual cells; red bars indicate the mean. Significance was tested using a two-tailed Mann Whitney U test; P = 3.89 x 10-11 (Control G2, ΔWAPL G2), P = 5.90 x 10-7 (Control G2, ΔSororin G2), P = 2.75 x 10-3 (ΔWAPL G2, ΔSororin G2). Biological replicates: Control WAPL-dTAG G2 (n = 3), ΔWAPL G2 (n = 3), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3), wild type late prophase (n = 4). Technical replicates Control WAPL-dTAG G2 (n = 6), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 6), ΔWAPL ΔSororin G2 (n = 6), wild type late prophase (n = 7). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. To aid visualisation, the SCC1 channel is not contrast matched. Scale bars large panels: 5 μm, insets: 2 μ",
    "molecules": "RO-3306"
  },
  {
    "caption": "WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. One sister chromatid was labelled per chromosome as in Fig. 1 and SMC4 (wild type late prophase cells) or SCC1 (WAPL-dTAG cells) was visualised by immunofluorescence. E, Representative images of ΔWAPL ΔSororin cells synchronised to G2 by RO-3306. F, Representative images of wild type cells synchronised to late prophase by release from a RO-3306-mediated G2 arrest. G, Quantification of sister chromatid separation as in Fig. 1D, E. ΔWAPL ΔSororin G2 cells (n = 51 cells) and wild type late prophase (n = 55 cells) cells were analysed. Dots represent individual cells; red bars indicate the mean. Biological replicates: Control WAPL-dTAG G2 (n = 3), ΔWAPL G2 (n = 3), ΔSororin G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3), wild type late prophase (n = 4). Technical replicates Control WAPL-dTAG G2 (n = 6), ΔWAPL G2 (n = 6), ΔSororin G2 (n = 6), ΔWAPL ΔSororin G2 (n = 6), wild type late prophase (n = 7). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. To aid visualisation, the SCC1 channel is not contrast matched. Scale bars large panels: 5 μm, insets: 2 μm.",
    "molecules": "DNA, RO-3306"
  },
  {
    "caption": "A-C, Analysis of sister DNA and SMC protein complex distribution in cross sections perpendicular to the long chromosome axis, as shown in Fig. 2B, E, F. Density of Hoechst minus F-ara-EdU was calculated by subtracting F-ara-EdU fluorescence from Hoechst fluorescence. Line profiles indicate the mean curves of individual line profile measurements aligned to the midpoint between two SMC4 peaks (wild type late prophase), the SCC1 peak (ΔWAPL G2 cells), or the midpoint between two SCC1 peaks (ΔWAPL ΔSororin G2 cells). A, Lines from wild type late prophase (n = 157 lines from 31 cells). B, ΔWAPL G2 cells (n = 110 lines from 18 cells). C, ΔWAPL ΔSororin G2 cells (n = 138 lines from 18 cells) were analysed. Wild type late prophase (n = 4), ΔWAPL G2 (n = 3), ΔWAPL ΔSororin G2 (n = 3). Technical replicates: Wild type late prophase (n = 7), ΔWAPL G2 (n = 6), ΔWAPL ΔSororin G2 (n = 5). Scale bars: 2μm.",
    "molecules": "Hoechst, F-ara-EdU, DNA"
  },
  {
    "caption": "A, Representative images of one-sister labelled sister chromatids from ΔWAPL, ΔSMC4 ΔWAPL, or ΔSMC4 ΔWAPL ΔSororin prometaphase cells, as indicated. SMC4 was depleted in G1 through the addition of 5-Ph-IAA 1 h before the final release into S phase such that cells progressed through S and G2 phase in the absence of condensins. WAPL was depleted in G2 phase after completion of DNA replication to avoid potential effects of WAPL depletion on cohesion establishment. B, Quantification of sister chromatid separation as in Fig. 1D, E. ΔWAPL (n = 21 cells), ΔWAPL ΔSMC4 (n = 25 cells), and ΔWAPL ΔSMC4 ΔSororin (n = 28 cells) prometaphase cells were analysed. Dots represent individual cells; red bars indicate the mean. Significance was tested using a two-tailed Mann Whitney U test; P = 6.57 x 10-8 (ΔWAPL); P = 3.64 x 10-7 (ΔWAPL ΔSMC4 ΔSororin). Biological replicates , ΔWAPL prometaphase (n = 3), ΔWAPL ΔSMC4 prometaphase (n = 3), ΔWAPL ΔSMC4 ΔSororin prometaphase (n = 3).",
    "molecules": "5-Ph-IAA, DNA"
  },
  {
    "caption": "C, Representative images of control prometaphase cells labelled on one sister chromatid and depleted of SMC4 for different amounts of time as indicated. SMC4 was visualised by immunofluorescence using an anti-SMC4 antibody. Cells were synchronised to G2 phase by RO-3306, before being arrested in prometaphase by STLC for 60 min. SMC4 was then depleted through the addition of 1 μM 5-Ph-IAA for 120 or 240 minutes, as indicated. Control (n = 2), ΔSMC4 120 min (n = 2), ΔSMC4 240 min (n = 2). All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 μm, insets: 1 μm.",
    "molecules": "5-Ph-IAA, RO-3306, STLC"
  },
  {
    "caption": "A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6).",
    "molecules": "LPS"
  },
  {
    "caption": ". MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 hours as indicated. Cells were fixed and permeabilized and stained for LC3. Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter. B. Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy. LC3 puncta were identified using spot function software to locate puncta ranging from 0.5-1.0mm diameter. LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter. ",
    "molecules": "chloroquine, monensin"
  },
  {
    "caption": "MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1, δWD and LC3 as indicated. Control MEFS express α and β isoforms of ATG16L1 at 70 kDa, and MEFs from δWD mice express a truncated ATG16L1 at 30kDa. (D) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean (±SD) of three replicate blots.",
    "molecules": "chloroquine, monensin"
  },
  {
    "caption": "E) fluorescence images of phagosomes following engulfment of Zymosan (red) by bone marrow-derived macrophages (BMDM) from control and δWD mice. White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center. F shows examples of line profile analysis G, H. (G) shows the percentage of LC3 positive phagosomes per cell and (H) shows line profile analysis of data from 10 cells, bars represent the mean ± SD and were compared by Student's t-test (****P<0.0001). ",
    "molecules": "Zymosan"
  },
  {
    "caption": "B). IAV binding is similar between control and δWD MEFs. Cells were bound with IAV X31 labeled with AF488 on ice for 1 h, and fixed immediately. The cells were stained with anti-HA (H3SKE, red) without permeabilisation to visualise extracellular IAV particles, counter stained with Hoechst (nuclei) and WGA-AF647 to label the cell membrane. Cells were imaged by automated confocal microscopy using a 40x objective, and the maximum intensity projection images were analysed to quantify the number of bound IAV particles per cell. The results and means (horizontal line) of n = 3 independent experiments (50-100 cells per experiment) are shown.",
    "molecules": "Hoechst, AF488, AF647"
  },
  {
    "caption": "C) IAV infection following acid bypass is comparable between control and δWD MEFs. IAV was bound on ice for 1 h, and warmed at either neutral pH (6.8) or low pH (pH 5.0) for 2 min, followed by incubation in STOP medium containing NH4Cl for 18 hours. Cells were fixed and stained for NP and Hoechst, imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity. The results from N=3 independent experiments (5,000-10,000 cells quantified per experiment), and the means are shown. Scale bar; 500 µm.",
    "molecules": "Hoechst, NH4Cl"
  },
  {
    "caption": "D). IAV fusion with endosomes is increased in δWD MEFs. Cells were infected with dual labeled (R18/SP-DiOC18) IAV for 1 h, fixed, counterstained with Hoechst (nuclei blue) and WGA-AF647 (cell perimeter in white). Cells were imaged by automated confocal microscopy and the number of fusion events per cell (represented by number of SP-DiOC18 puncta) was quantified. Fused and non-fused viral particles are shown as green and red spots respectively. The results and means (horizontal lines) of n = 3 independent experiments (30-60 cells per experiment) are shown. Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right.",
    "molecules": "Hoechst, SP-DiOC18, AF647, R18"
  },
  {
    "caption": "E, F) Numbers of fusion positive cells is increased for δWD MEFs. MEFs from δWD (white circles) or littermate control mice (black circles) were incubated with dual-labelled (SP-DiOC18/R18) IAV at 40C for 45 min and warmed to 37°C for 30 ad 60 minutes. Cells were harvested by trypsinisation, fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion (E) and median fluorescence intensity (MFI) of dequenched SP-DiOC18 signa (F). Graphs show individual replicates (n = 3) with a horizontal line at the mean and were compared using two-way ANOVA with Bonferroni post-tests **P<0.01, ***P<0.001).",
    "molecules": "SP-DiOC18, PFA, R18"
  },
  {
    "caption": "(G, H) Endocytosis of IAV increases in δWD MEFs. MEFs from δWD or littermate control mice were incubated with AlexFluor 488-labelled IAV at 40C for 1hr and warmed to 37°C for 15 Cells were fixed and permeabilised and stained with antibody against HA (red). The blue line indicates the plasma membrane of the cell. Green arrows indicate examples of viruses only labelled with green fluorescence. Double-labelled virus particles (green-red) represent non-endocytosed particles bound at the cell surface, whereas green particles represent endocytosed particles. The results and means of n = 3 independent experiments (50-100 cells per experiment) are shown.",
    "molecules": "AlexFluor 488"
  },
  {
    "caption": "E Cruciform unfolding  assays as in (D) with WRN, WRN  K577M, and WRN  E84A (all used at 10 nM), carried out either in the presence or absence of ATP, or with ATP-ƔS. Averages shown; n ≥ 3 technical replicates; error bars, SEM.",
    "molecules": "ATP-ƔS, ATP"
  },
  {
    "caption": "B Mapping of TA repeats cruciform  structure. T7 Endonuclease I activity, followed by SspI, cleaves the cruciform  within negative  supercoiled DNA, producing two bands of the expected size (607 bp and 2119 bp, indicated by the green arrows, lane 2). Simultaneous incubation of the substrate with SspI  and EcoRI  allows EcoRI  cutting at the TA repeats sequence (lane 5), while the extruded cruciform  is refractory to EcoRI  digestion (lane 4, the * indicates scDNA).",
    "molecules": "scDNA, supercoiled DNA"
  },
  {
    "caption": "C Representative cruciform  detection assays. Plasmid DNA was subjected to Cruciform  extrusion procedure, where indicated. Supercoiled DNA (cruciform, refractory to EcoRI) and  linear DNA (no cruciform, cleaved by EcoRI) were resolved on a 1% agarose  gel, stained  with GelRed. Top, quantitation of extruded cruciform  in % with respect to the amount of total DNA that is present in the lane. Averages shown; n = 4 technical replicates; error bars, SEM. D Representative cruciform unfolding  assays with increasing WRN   concentrations, using pUC19 with either random inverted repeats or TA repeats as a substrate. Top, quantitation of cruciform unfolding. The amount of linear DNA  from the \"No protein\" lane was subtracted from all other samples. Averages shown; n = 3 technical replicates; error bars, SEM.",
    "molecules": "agarose, GelRed, linear DNA, Supercoiled DNA"
  },
  {
    "caption": "A Recombinant BLM  and S. cerevisiae Sgs1  used in this study. The gel was stained  with Coomassie Brilliant blue.",
    "molecules": "Coomassie Brilliant blue"
  },
  {
    "caption": "B Recombinant full-length and truncation WRN   variants used in this study are shown. The gel was stained  with Coomassie Brilliant blue.",
    "molecules": "Coomassie Brilliant blue"
  },
  {
    "caption": "F Representative cruciform unfolding  assays with MutSβ and MutS⍺, carried out without or with ATP, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM. G Representative cruciform unfolding  assays with MutSβ and MutS⍺, carried out without or with hRPA, using the random-IR cruciform as a substrate. Top, quantitation of cruciform unfolding. Averages shown; n = 3 technical replicates; error bars, SEM.",
    "molecules": "ATP"
  },
  {
    "caption": "A Representative polyacrylamide  gel showing recombinant MutSβ, MutSβ-3K255A (MSH3  with mutation K255A, deficient in mismatch recognition) and MutSβ-3K902A (MSH3  with mutation K902A, ATPase-deficient). The gel was stained  with Coomassie Brilliant blue.",
    "molecules": "Coomassie Brilliant blue, polyacrylamide"
  },
  {
    "caption": "B Electrophoretic mobility shift assay  with MutSβ, MutSβ-3K255A and MutSβ-3K902A, using either dsDNA  (50 bp) or dsDNA  bearing one extrahelical T, as a substrate. 6% native acrylamide gel was used.",
    "molecules": "dsDNA"
  },
  {
    "caption": "B.  Western blot  analysis of untreated (Ctrl) and Ribavirin  treated (Rib) Vector and eIF4E NOMO-1 cell lines. Myc, and Mcl1  served as positive controls, and β-actin as a loading control. Each β-actin blot  corresponds to the above WBs. Experiments were carried out at least three independent times, and one representative experiment is shown. Ribavirin  dose was 10uM which is clinically achievable",
    "molecules": "Rib, Ribavirin"
  },
  {
    "caption": "C. Validation of splicing targets identified by rMATs in U2OS (upper panel) and  NOMO-1 (lower panel) cell lines. RT-qPCR analysis using specific primers for each splicing event normalized to the corresponding total levels of that given transcript Data were normalized to Vector control to calculate fold change. The mean and standard deviation, as well as p-values were derived from three biological replicates. P-values were calculated between eIF4E  overexpressing and Vector cells (when the asterisk is over eIF4E), and eIF4E  cells treated with Ribavirin  vs eIF4E  overexpressing untreated cells (for the asterisk over Ribavirin). Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001",
    "molecules": "Ribavirin"
  },
  {
    "caption": "(C) Percentage of conservation along the nucleotide positions in the ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a. Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71-366 nt of ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. Box plots display median (line), first and third quartiles (box), and 5th /95th percentile value (whiskers). Each dot represents the outliers. Two-tailed P values were calculated using the student's t-test.",
    "molecules": "nucleotide"
  },
  {
    "caption": "a, MEFs of RagA+/+, RagAGTP/+ and RagAGTP/GTP genotypes were deprived of amino acids for 1 h and re-stimulated for 10 min. Whole-cell protein lysates were immunoblotted for indicated proteins.",
    "molecules": "amino acids"
  },
  {
    "caption": "g, Survival curve of fasted neonates. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 26; G/G: n = 10). h, Survival curve of fasted neonates treated with rapamycin. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 13; G/G: n = 6). Numbers indicate the median survival for each curve. *P<0.05; **P<0.01; ***P<0.005.",
    "molecules": "rapamycin"
  },
  {
    "caption": "b, Rapamycin significantly increases glycaemia in RagAGTP/GTP fasted for 6 and 10 h (mean ± s.e.m.). NS, not significant.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "c, Extension of survival by glucose injections in fasted RagAGTP/GTP neonates (untreated: n = 10; glucose: n = 5).",
    "molecules": "glucose"
  },
  {
    "caption": "d, Normal expression of genes involved in glucose metabolism in neonatal liver (+/+: n = 2, G/+: n = 5; G/G: n = 4; mean ± s.e.m.).",
    "molecules": "glucose"
  },
  {
    "caption": "e, Left: representative electron microscopy images showing abundant glycogen content in RagA+/+ and RagAGTP/GTP hepatocytes before fasting (0 h, upper panels) and more pronounced glycogen depletion after 10 h of fasting (lower panels) in RagAGTP/GTP neonates. Right: quantification of hepatic glycogen content (+/+: n = 5, 3, 4, 4; G/+: n = 11, 7, 14, 15; G/G: n = 6, 4, 4, 6; for 0, 3, 6 and 10 h, respectively; mean ± s.e.m.; AU, arbitrary units).",
    "molecules": "glycogen"
  },
  {
    "caption": ". f, Partial rescue of hepatic glycogen content in RagAGTP/GTP neonates fasted for 10 h and treated with rapamycin (rapa) (mean ± s.e.m.).",
    "molecules": "glycogen, rapamycin"
  },
  {
    "caption": "g, Quantification of neonatal plasma amounts of branched-chain (BCAA) and essential amino acids at birth (left), after 10 h fasting (middle) and after 10 h fasting with rapamycin treatment (right) (n for +/+, G/+ and G/G, respectively: n = 4, 5 and 4 for 0 h; n = 4, 4 and 3 for 10 h; n = 2, 5 and 3 for rapamycin; mean ± s.d.). Values are expressed relative to RagA+/+ amounts at each time point.",
    "molecules": "amino acids, BCAA, rapamycin"
  },
  {
    "caption": "h, Extension of survival by injection of a combination of gluconeogenic amino acids (a.a.) in fasted RagAGTP/GTP neonates (untreated: n = 10; amino acids: n = 8). *P  0.05; **P  0.01; ns, P > 0.0",
    "molecules": "amino acids"
  },
  {
    "caption": "d, Triggering of autophagy by amino-acid withdrawal in MEFs. MEFs were deprived of amino acids for the indicated time points, whole-cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting.",
    "molecules": "amino-acid"
  },
  {
    "caption": "e, Recombinant LC3B-GFP was expressed in RagA+/+ and RagAGTP/GTPMEFs and LC3B localization, in the presence and absence of amino acids, monitored by fluorescence microscopy. LC3B-GFP (green fluorescent protein) clustering, indicative of autophagy, was observed in amino acid-starved RagA+/+ but not RagAGTP/GTPMEFs. Scale bar, 10 µm.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "f, Localization of recombinant TFEB-GFP was determined in MEFs as in e. Nuclear (active) TFEB was observed in RagA+/+ MEFs upon amino-acid withdrawal.",
    "molecules": "amino-acid"
  },
  {
    "caption": "a) AMPKa1/a2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min. Wholecell extracts were immunoblotted for the indicated proteins.",
    "molecules": "glucose"
  },
  {
    "caption": "b, Immortalized MEFs of the indicated genotypes were deprived of growth factors, glucose, amino acids or glucose and amino acids for 1 h and re-stimulated with glucose and/or amino acids for 10 min. Whole-cell lysates were immunoblotted for the indicated proteins.",
    "molecules": "amino acids, glucose"
  },
  {
    "caption": "c, RagA+/+ and RagAGTP/GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h. Cell number is indicated relative to cell number at the start of the treatment; mean ± s.d.; ***P  0.005",
    "molecules": "amino acids, glucose"
  },
  {
    "caption": "d, mTOR localization as detected by immunofluorescence. In HEK-293T cells, glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm. Re-addition of glucose leads to mTOR shuttling to the lysosomal surface, co-localizing with the lysosomal protein Lamp2. HEK-293T-RagBGTP cells show mTOR localized at the lysosomal surface, regardless of glucose concentrations. Scale bars, 10 μm.",
    "molecules": "glucose"
  },
  {
    "caption": "e, Glucose and amino acids affect the binding of the v-ATPase to the Ragulator complex. HEK-293T expressing Flag-p14 was deprived of glucose or amino acids for 90 min and re-stimulated for 20 min. Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins.",
    "molecules": "amino acids, Glucose, glucose"
  },
  {
    "caption": "f, RagA+/+ and RagAGTP/GTP primary MEFs were cultured for 1 h in media with the glucose and amino-acid concentrations measured in neonates at birth (0 h) or after fasting for 1 h (1 h) and whole-cell protein extracts were analysed by immunoblotting.",
    "molecules": "amino-acid, glucose"
  },
  {
    "caption": "B) Summary table of experimental binding affinities of the parental components targeting VEGF-A (ranibizumab) and ANG-2 (LC10) as well as of RG7716 measured by isothermal titration calorimetry. RG7716 binds VEGF-A121 and -165 with an affinity comparable to ranibizumab (3.3 vs. 3.1 nM). ANG-2 binding of RG7716 is slightly decreased to 22 nM compared to α-ANG-2 IgG1 (LC10) with 4.1 nM. No binding to ANG-1 was detectable for RG7716 or α-ANG-2 IgG1 (LC10).",
    "molecules": "LC10, RG7716, ranibizumab"
  },
  {
    "caption": "D) The ability of RG7716 to bind to ANG-2 and VEGF-A simultaneously and independent is demonstrated by a surface plasmon resonance experiment. RG7716 is captured via an anti-Fc antibody on the chip surface. The green curve depicts the experiment with ANG-2 in the first and VEGF-A in the second binding event. The red curve shows the binding events in the other sequence.",
    "molecules": "RG7716"
  },
  {
    "caption": "Time-dependent binding of RG7716 is shown in pink, anti-VEGF-A/ANG-2 without modification of the Fc- part in red (wild-type), anti-VEGF-A/ANG-2 with modification of the FcRn binding site only in yellow (FcRn-), and anti-VEGF-A/ANG-2 with modification of the FcγR binding in grey (FcγR-).A) Shows SPR binding profile over time to immobilized FcγR-I, B) to immobilized FcγR-II, C) to immobilized FcγR-III, D) to immobilized FcRn. E) Summary data table of binding data for RG7716 in comparison to anti-VEGF-A/ANG-2 CrossMAbs without modifications in the Fc-part.",
    "molecules": "RG7716"
  },
  {
    "caption": "A) Bar graph demonstrating platelet aggregation induced by complexes of heparin, VEGF-A165, and anti-VEGF-A (IgG1) in vitro using washed human platelets. Introduction of modification of the Fc-region in RG7716 does not allow platelet aggregation to happen as compared to anti-VEGF-A as wild type IgG1. All three components are needed as heparin/anti-VEGF-A alone does not activate platelet aggregation. Error bars show SEM of three independent experiments and **** denotes significance using ANOVA (P < 0.0001) and Dunnett's multiple t-test comparing to IgG control with P < 0.0001 of Heparin/VEGF-A/anti-VEGF-A against IgG control.",
    "molecules": "heparin, Heparin"
  },
  {
    "caption": "B) Inhibition of neovascularization measured in severity grades, change of severity from baseline is shown for each treatment; all treatment significantly reduced the severity grade compared to IgG control. In addition, efficacy of RG7716 (150 kDa molecule at 90 µg) was significantly better at equal molar concentration of binding sites than anti-VEGF-A (ranibizumab, 50 kDa molecule at 30 µg), anti-ANG-2 and the low dose of RG7716 (30 µg).C-G) Representative figures of fluorescence fundusangiograms at day 28 and H-L) of cross sections of a hematoxylin staining of a spot at the end of the experiment from eyes treated with IgG control, anti-VEGF-A, anti-ANG-2 and RG7716 at 30 and 90 μg, respectively.",
    "molecules": "ranibizumab"
  },
  {
    "caption": "(A) Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3 in H1299 cells in normal medium (HBSS ''-'') or after 1 hr HBSS starvation (HBSS ''+'') pretreated with or without NAC (3mM).",
    "molecules": "NAC"
  },
  {
    "caption": "(B) Western blot detection of p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment.",
    "molecules": "H2O2"
  },
  {
    "caption": "(C) Western blot detection of p62 and LC3 in H1299 cells cotransfected with the indicated shRNA and the indicated plasmid and cultured in normal medium or after H2O2 (500μM) treatment. CHK2 NTm, a shRNA nontargetable mutant CHK2 rescue plasmid.",
    "molecules": "H2O2"
  },
  {
    "caption": "(D) Autophagic flux is shown by representative confocal microscopic images of 293 cells stably-expressing GFP-mCherry-LC3 transfected with the indicated shRNA following HBSS starvation and H2O2 (500μM) treatment for 3h. Scale bar, 10 μm.",
    "molecules": "H2O2"
  },
  {
    "caption": "(E) Quantitation of autophagosomal (yellow) and autolysosomal (red) LC3 puncta following HBSS starvation and H2O2 (500μM) treatment for 1h (n=30). Data are presented as mean±s.e.m. from 3 independent experiments; **P < 0.01 (Student's t-test).",
    "molecules": "H2O2"
  },
  {
    "caption": "(F) Representative electron microscopic image of autophagic vesicles or autophagosomes in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Scale bars, 500 nm. The red arrows indicate double-membraned autophagic structures.",
    "molecules": "H2O2"
  },
  {
    "caption": "(G) Electron microscopic quantification of autophagy vacuole in H1299 cells transfected with the indicated shRNA in normal medium or after 3 hr HBSS starvation or treated with H2O2 (500μM). Data are presented as mean±s.e.m. from 3 independent experiments; *P < 0.05; **P < 0.01 (Student's t-test).",
    "molecules": "H2O2"
  },
  {
    "caption": "(C) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after H2O2 (500μM) stimulation in the cytoplasmic fractions.",
    "molecules": "H2O2"
  },
  {
    "caption": "(D) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation. The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.",
    "molecules": "NAC"
  },
  {
    "caption": "HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation (E) The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.",
    "molecules": "CHK2 inhibitorⅡ"
  },
  {
    "caption": "HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 (500μM) stimulation (F). The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.",
    "molecules": "CHK2 inhibitorⅡ, H2O2"
  },
  {
    "caption": "HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with H2O2 (500μM) stimulation (I) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.",
    "molecules": "H2O2"
  },
  {
    "caption": "(J) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 (500 μM) cultured for 3 hr.",
    "molecules": "H2O2"
  },
  {
    "caption": "(K) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 (500μM) stimulation. Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3.",
    "molecules": "CHK2 inhibitorⅡ, H2O2"
  },
  {
    "caption": "(A) The indicated recombinant fragments of GST-Beclin1 were incubated with recombinant CHK2 in the presence of 32P-labeled ATP for in vitro kinase assays. MBP was used as a positive control. Ponceau staining indicates the expression of GST-Beclin 1 fragments for in vitro kinase assays. The asterisks in the blot indicate the domain of phosphorylated Beclin 1.",
    "molecules": "ATP, 32P, Ponceau"
  },
  {
    "caption": "Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 (500μM) stimulation (E).",
    "molecules": "H2O2"
  },
  {
    "caption": "Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 (500μM) stimulation (G).",
    "molecules": "H2O2"
  },
  {
    "caption": "(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation.",
    "molecules": "NAC"
  },
  {
    "caption": "(A) The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl-2. GST-Beclin1 WT, AA mutant and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag-Bcl-2 (preys).",
    "molecules": "ATP"
  },
  {
    "caption": "(B) Immunoprecipitation of Bcl-2 with Flag-Beclin 1 WT, AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 (500μM) treatment for 1h.",
    "molecules": "H2O2"
  },
  {
    "caption": "(C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2.",
    "molecules": "H2O2"
  },
  {
    "caption": "(E) Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H2O2 (500μM) for 6 hr. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 (500μM) treatment. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.001 compared to Beclin 1 WT; #P < 0.001 compared to Beclin 1 WT treated with H2O2; &P < 0.001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2.",
    "molecules": "H2O2"
  },
  {
    "caption": "(F) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93, Beclin 1, p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment for 3h.",
    "molecules": "H2O2"
  },
  {
    "caption": "(A) Western blot detection of p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.",
    "molecules": "glucose"
  },
  {
    "caption": "(C) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.",
    "molecules": "glucose"
  },
  {
    "caption": "(E) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to hypoxia with or without pretreatment with NAC.",
    "molecules": "NAC"
  },
  {
    "caption": "(F) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to glucose starvation with or without pretreatment with NAC.",
    "molecules": "glucose, NAC"
  },
  {
    "caption": "(G) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, and AMPKα in H1299 cells in normal medium or after H2O2 (25μM) stimulation.",
    "molecules": "H2O2"
  },
  {
    "caption": "(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG (5mM) and oligomycin (2.5μM) treatment.",
    "molecules": "2DG, oligomycin"
  },
  {
    "caption": "Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation (Glu-)",
    "molecules": "Glu, glucose, ROS"
  },
  {
    "caption": "Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia (B).",
    "molecules": "ROS"
  },
  {
    "caption": "(C) Western blot detection of CHK2, HSP60, TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation (Glu-) or 36 hr hypoxia.",
    "molecules": "Glu, glucose"
  },
  {
    "caption": "(D-E) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation (Glu-) medium or hypoxia. The yellow asterisks mark mitochondria. The red arrows indicate autophagic structures. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ***P < 0.001 compared to shNC treated with H2O2 or hypoxia (Mann-Whitney test); Scale bar, 1 μm.",
    "molecules": "Glu, glucose, H2O2"
  },
  {
    "caption": "The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).",
    "molecules": "glucose"
  },
  {
    "caption": "H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation (A) Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments; *p < 0.05 compared to NC glucose starvation (A) (Student's t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation (C) Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).",
    "molecules": "glucose, H2O2"
  },
  {
    "caption": "H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation (D). Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).",
    "molecules": "H2O2"
  },
  {
    "caption": "(E) CHK2+/+ and CHK2-/- mice were subjected to MCAO for 1 hr and reperfusion for 12 h. Contralateral (C) and ipsilateral (I) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC.",
    "molecules": "TTC"
  },
  {
    "caption": "(B) Digitonized mitochondria (digitonin-to-protein ratio of 2:1) were analyzed by BN-PAGE, followed by CI in gel activity (IGA) assays and Coomassie staining. The SC I+III2+IV1 band (b1) was excised from the control (143B) lane and the SC I+III2 bands (b2 to b4) were excised from the COX1∆ and COX2∆ lanes. Their protein compositions were subsequently analyzed by nano-LC/ESI-MS (see also Figure EV2). The relative position of the molecular weight marker is indicated on the left.",
    "molecules": "Coomassie, digitonin, Digitonized"
  },
  {
    "caption": "(A) Digitonin-solubilized mitochondria from doxycycline-treated 143B cells, COX1∆ and COX2∆ cybrids were analyzed by BN-PAGE followed by CI-IGA assays (upper panels) or immunoblotting (medium and lower panels) with antibodies against CI subunits NDUFA9 and NDUFS1. The identities of MRC complexes and SCs are as in Figure 1.",
    "molecules": "Digitonin, doxycycline"
  },
  {
    "caption": "(A) Digitonized mitochondria from doxycycline-treated 143B cells and COX2∆ cybrids were analyzed by 2D-BN/SDS-PAGE and western blot with the indicated antibodies.",
    "molecules": "Digitonized, doxycycline"
  },
  {
    "caption": "Mitochondrial translation products were pulse-labelled in control 143B cells (left panel), COX1∆ (middle panel) and COX2∆ (right panel) cybrids with [35S]-methionine for 30 minutes in the presence of anisomycin to inhibit cytosolic translation. For chase, cells were washed and incubated with fresh culture medium for the indicated time points (T0-T24 hours). Radiolabeled mitochondrial proteins (indicated on the right) were separated by 2D-BN/SDS-PAGE and visualized by autoradiography. A enlarged section of the SCs region at T24 (delimited by a square) shows the co-migration of COX1 with SC I+III2plus (black arrow) in the COX2∆ cybrids relative to the position of canonical SC I+III2 (gray arrow). The identities of MRC complexes and SCs (bottom) are as in Figure 1. Molecular weight markers are indicated on the left.",
    "molecules": "35S, anisomycin, methionine"
  },
  {
    "caption": "Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. Histogram of DCV fusion events per stimulation per time point. Stimulations include 1 action potential (AP) (grey), 16 bursts of 50 AP at 50 Hz (light-green), superfusion with 60 mM KCl Tyrode solution for 30 s. (dark-green) and superfusion with 5 μM Ionomycin for 30 s. (blue). Inset: Typical ∆F/F0 trace of rise in intracellular calcium (Fluo5-AM) upon different stimulations. Traces are corrected for baseline (first 10 frames) and normalized.",
    "molecules": "Tyrode, calcium, Fluo5, Ionomycin, KCl"
  },
  {
    "caption": "Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. DCV fusion events per neuron per stimulation. Kruskal-Wallis with Dunn's correction: *p < 0.05, **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p > 0.05. DCV release probability per neuron per stimulation. Kruskal-Wallis with Dunn's correction: **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p>0.05.",
    "molecules": "Ionomycin, KCl"
  },
  {
    "caption": "C. Representative western blot  showing changes in various ER  stress markers and O-GlcNAcylation-related  protein expression  in THP-1  cells treated with  Stx2a  (10 ng/mL) for 3 h in the presence or absence of O-linked N-acetylglucosamine transferase (OGT) inhibitor OSMI-1 (10 µM final) or serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 a (IRE1a) inhibitor MKC-3946 (10 µM final). D. Quantification of the band intensities in (C). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against  β-actin, which was used as a loading control. Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "MKC-3946, OSMI-1"
  },
  {
    "caption": "E. Representative  western blot of  O-GlcNAcylation and pro-caspase-3  cleavage in  THP-1 cells treated with Stx2a (10 ng/mL) in the presence or absence of OGT inhibitor OSMI-1 (10 µM final). F. Quantification of the band intensities for  O-GlcNAcylation (RL-2) in (E). Data are presented as mean ± S.E.M. (n = 3 biological replicates) normalized against β-actin, which was used as a loading control. The effects of Stx2a-mediated induction for  O-GlcNAc levels at each time-point  were compared to 0 h (left panel), and OSMI-1 treatment was compared with that of the vehicle (DMSO) control at each time-point (right panel). Data information: Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "B. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on cytokine and chemokine production from  THP-1 cells exposed to  Stx2a (10 ng/mL) for 9 h (n = 3 biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "C. Real-time qRT-PCR analyses of fold changes in transcript levels for each cytokine/chemokine gene in D-THP-1  cells following   Stx2a (10 ng/mL) intoxication for various time-points in the presence or absence of OSMI-1 (10 µM, final). Data are normalized using GAPDH  as a loading control (n = 3, biological replicates). Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001. The effects of OSMI-1 were compared with those of the vehicle (DMSO) control at each time-point.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "A. Representative  western blot showing changes of overall O-GlcNAcylation  (upper panel) or  phosphorylation status of each target protein (lower panels) in THP-1 cells treated with Stx2a  (10 ng/mL) for 9 h in the presence or absence of  OSMI-1 (10 µM, final). B. Quantification of the relative band intensities for O-GlcNAcylation (RL-2) or each of the phosphorylated- and total-protein bands in (A). Data are normalized against  β-actin, which was used as a loading control (n = 3 biological replicates). The effects of Stx2a  in vehicle controls were compared with lysates prepared from cells maintained in the absence of  Stx2a, and  OSMI-1  treatment was compared with that of the vehicle (DMSO) controls in the presence of  Stx2a. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "B.  LDH cytotoxicity assay following Stx2a (10 ng/mL) treatment of HRPTEpi (adult) cells for 48 h in the presence or absence OSMI-1  (10 µM, final) (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with culture medium prepared from HRPTEpi cells maintained in the absence of Stx2a, and OSMI-1  treatment was compared with that of the vehicle (DMSO) controls in the presence of  Stx2a. Data information: For graphs error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on cytokine/chemokine production from HRPTEpi  cells (adult) exposed to  Stx2a  (10 ng/mL) for 48 h (n = 3 biological replicates). The effects of OSMI-1  were compared with those of the vehicle (DMSO) control. Data information: For graphs , error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of HRPTEpi cells exposed to Stx2a  (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of  OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "F. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8  and CCL-2 production from 3D cultures of human mini-kidneys exposed to Stx2a  (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "G, H. Human apoptosis antibody array  analysis  of multiple proteins using pooled lysate from 3D-human  mini-kidney spheroids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (G) Antibody spots representing signal differences are indicated in red boxes. (H) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of  Stx2a   exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Raw values of fluorescence intensities are provided in the Source Data. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "OSMI-1"
  },
  {
    "caption": "C, D.  ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on Kidney injury molecule-1  (KIM-1) secretion (C) and IL-8 and CCL-2 production (D) in culture supernatants of  iPSC-derived human kidney organoids exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "DMSO, OSMI-1"
  },
  {
    "caption": "E, F. Human apoptosis antibody array  analysis of multiple proteins using pooled lysates from iPSC-derived human kidney organoids following  Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (E) Antibody spots representing signal differences are indicated in red boxes. (F) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.",
    "molecules": "OSMI-1"
  },
  {
    "caption": "B.  Survival rate of mice challenged once with Stx2a  (132.5 ng/kg) and injected daily with two doses (300 or 1000 µg/mouse) of OSMI-1 (n = 10, 5 per dose) or vehicle (n = 10). The effect of OSMI-1 (1000 µg/mouse) was compared with that of the vehicle control (a log-rank test with GraphPad Prism software).",
    "molecules": "OSMI-1"
  },
  {
    "caption": "C. Chemical and hematological analyses of blood pooled from three mice at day 3 after Stx2a  challenge. Data are presented as mean ± S.E.M. (n = 3 biological replicates from nine mice, two-tailed Student's t-test). The effects of OSMI-1  (1000 µg/mouse) were compared with those of the vehicle control. *P < 0.05; **P < 0.01; and ***P < 0.001",
    "molecules": "OSMI-1"
  },
  {
    "caption": "(A) Schematic representation of the SILAC-based quantitative proteomics experiment in B. subtilis. Cells overexpressing active YqgP (BS50, Table EV2) or its catalytically dead mutant YqgP.S288A (BS51), both auxotrophic for lysine, were grown in parallel in \"heavy\" (containing 13C615N2- Lysine isotope) or \"light\" (containing stable 12C614N2- Lysine) M9 minimal medium, respectively. After mixing the cell cultures in the 1:1 ratio (based on OD600), cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC-MS/MS experiment. Using bioinformatic analysis of the MS data, highest-scoring candidate substrates were further evaluated.",
    "molecules": "lysine, Lysine"
  },
  {
    "caption": "(A) Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B. subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 µM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE)",
    "molecules": "magnesium"
  },
  {
    "caption": "(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b(). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.",
    "molecules": "STS736"
  },
  {
    "caption": "(A) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B. subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalized to enzyme expression level (lower panel).",
    "molecules": "glucose, magnesium, MgSO4, manganese, MnCl2"
  },
  {
    "caption": "(B) Growth curves of wild-type (BTM843, Table EV2), yqgP deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 µM MnSO4 in mid-exponential phase (stress-phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain, and that reintroduction of YqgP rescues fitness during manganese stress to above-wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time-point, which illustrates the reproducibility of the assay.",
    "molecules": "magnesium, MgSO4, manganese, MnSO4"
  },
  {
    "caption": "(C) Top panel: growth curves of wild-type (BTM843), yqgP deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 µM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions.",
    "molecules": "magnesium, MgSO4, manganese, MnSO4"
  },
  {
    "caption": "(D) Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al., 2017b()) abolishes the YqgP induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B).",
    "molecules": "STS736, manganese"
  },
  {
    "caption": "(E) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels C - E, representative experiments of 2-3 independent biological replicates are shown.",
    "molecules": "MnSO4"
  },
  {
    "caption": "(C) Similar analysis of the same strains but grown in minimal M9 medium (with 10 µM MgSO4) supplemented with increasing concentrations of MnCl­­2. Black arrows denote full-length substrate (MgtE), red arrows denote the cleavage products generated by YqgP. (D) Western blots of four independent experiments shown in panel (C) were quantified by near-infrared fluorescence detection, quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl­­2 relative to 1 µM MnCl­­2 activity. Average values and all fours datapoints are plotted for each indicated YqgP variant.",
    "molecules": "MgSO4, MnCl­­2"
  },
  {
    "caption": "(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series.",
    "molecules": "CaCl2, CoCl2, MgSO4, MnCl2, NiCl2, ZnCl2"
  },
  {
    "caption": "(C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.",
    "molecules": "CaCl2, cobalt, CoCl2, CoSO4, MgSO4, manganese, MnCl2, nickel, NiCl2, zinc, ZnCl2"
  },
  {
    "caption": "(E Detection of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP.",
    "molecules": "Mn, MnCl2"
  },
  {
    "caption": "F) quantification of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2.",
    "molecules": "Mn, MnCl2"
  },
  {
    "caption": "(E) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products. B. subtilis strains expressing YqgP or its S288A mutant (strains BTM78; 501 and 502, Table EV2) in the presence or absence of endogenous FtsH protease (strains BTM795, BTM796 and BTM797, Table EV2) were cultured in LB medium and at early exponential phase, after having been expressing YqgP variants for 30 min, were either left grown untreated (as a control with ongoing translation) or treated with 20 μg/mL tetracycline to stop proteosynthesis (translation shut-off). At given time intervals afterwards, all cultures were analysed by α-MgtE western blotting with near infrared detection. Equal cell number (judged by OD600) was loaded into each lane. Black arrow marks full-length MgtE, red full arrows mark YqgP-dependent and red open arrows mark YqgP.S288A-dependent cleavage product of MgtE.",
    "molecules": "tetracycline"
  },
  {
    "caption": "Assays were performed in HEK293 cells for A) F508del-CFTR For the F508del-CFTR measurements since this mutant does not respond well to Fsk alone (Fsk + VX-770 potentiator is required to obtain a detectable signal) we compared the CFTR inh172 (inhibitor) response (F1/'F-CFTRinh172'; where F1 represents peak RFU immediately before inhibitor addition and 'F-CFTRinh172' is the minimum RFU reached at the end of the CFTR inh172 measurement cycle). For each of F508del-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.",
    "molecules": "CFTR inh172, CFTRinh172, Fsk, VX-770"
  },
  {
    "caption": "Assays were performed in HEK293 cells for B) wt-CFTR. For the wildtype measurements, we compared the maximum Fsk response (Maximum F /F0; where F0 represents baseline RFU right before the stimulation). For each of wt-CFTR data for three proteins not detected as interactors (first three bars following CFTR only) are shown as negative controls. Fluorescence signals were normalized to the CFTR-only control and the statistical significance of differences assed by unpaired t-test (*p<0.05, **p<0.01 and ***p<0.001). Samples run in n=3 replicates, and all values are mean ± s.d.",
    "molecules": "Fsk"
  },
  {
    "caption": "B) Blot quantification showing relative fold change of band B and band C versus treatment with a non-targeting siRNA (Neg1). One-way Anova followed by Dunnett's post hoc test was performed to determine significance versus baseline (siNeg1 + DMSO treatment, *p<0.05) and 5 µM VX-661 (#p<0.05). Measurements were obtained from biological triplicates (n=3) and all values are mean ± s.d.",
    "molecules": "DMSO, VX-661"
  },
  {
    "caption": "C) Representative confocal microscopy images of calcein green labelled FIS organoids of a healthy individual and a CF patient, before and 60 min after stimulation with Fsk (5 uM). Scale bar, 200 μm.",
    "molecules": "calcein, Fsk"
  },
  {
    "caption": "(h) Representative WB of IGF1Rβ and IGF1Rβ immunoprecipitation products immunoblotted for p-Tyr in untreated, IGF2-treated and IGF2+anti-IGF2R-treated (1:500, 1:1000 and 1:2000 dilutions of anti-IGF2R) C2C12 cells (cells were treated for 2 and 24 h). Densitometric analysis of data are expressed as the ratio of pTYR/IGF1Rβ in arbitrary units in the lower panel. Each experiment was performed in triplicate wells. All values are expressed as the mean±SEM.",
    "molecules": "Tyr"
  },
  {
    "caption": "(d) Over-expressing CD20 in C2C12 myoblasts that co-expressed CD20 (in green) and IGF2R (in red). DAPI-labelled nuclei are shown in blue. Scale bars=75 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs (E, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation reactions using reticulocyte  lysates and the indicated constructs F, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "J In vitro translation  (IVT)/biotinylation  reactions using Hela  cell lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated protein. Bottom, the indicated proteins were prepared by in vitro translation  using Hela  cell lysates and analyzed by αFlag western blotting.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "B In vitro translation/biotinylation  reactions using reticulocyte  lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated substrate. Bottom, the indicated proteins were prepared by in vitro translation  using reticulocyte  lysates and analyzed by αFlag western blotting.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "E In vitro translation/biotinylation  reactions using reticulocyte  lysates and construct 1 (left) or construct 7 (right). Streptavidin far western  (FW) assesses biotinylation. Anti-HA western blot  (WB) measures translated substrate. Bottom, the indicated proteins were prepared by  in vitro translation  using reticulocyte  lysates and analyzed by αFlag western blotting.",
    "molecules": "Streptavidin"
  },
  {
    "caption": " (A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. \"Nor\" = normal medium. Actin served as the loading control. The red text \"-Cys\" indicates that cystine deficiency most significantly activates AMPK and its substrates. ",
    "molecules": "amino acid, Cys, cystine"
  },
  {
    "caption": "(F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control.",
    "molecules": "cystine"
  },
  {
    "caption": " (I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2. ",
    "molecules": "cystine"
  },
  {
    "caption": " (E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control. ",
    "molecules": "cystine"
  },
  {
    "caption": " (J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control. ",
    "molecules": "DMSO, STO-609"
  },
  {
    "caption": " (D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control. ",
    "molecules": "cystine"
  },
  {
    "caption": "(F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.",
    "molecules": "cystine"
  },
  {
    "caption": "(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.",
    "molecules": "AICAR, crystal violet, cystine"
  },
  {
    "caption": " (F WB analysis of p-AMPK, total AMPK, CARS, AMPKγ2, and CaMKK2 protein expression with 786-O cells transfected with Flag-EV, Flag-CARS, Flag-CaMKK2 or Flag-AMPKγ2 and further treated with cystine-deficient medium for 8 h. Actin served as the loading control. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups. ",
    "molecules": "cystine"
  },
  {
    "caption": "D H&E (hematoxylin-eosin) staining (left) and oil red O staining (right) of livers. Scale bar, 50μm.",
    "molecules": "eosin, hematoxylin, oil red O"
  },
  {
    "caption": "E Serum cholesterol concentrations of each group. Liver cholesterol concentrations (F) of each group were examined.",
    "molecules": "cholesterol"
  },
  {
    "caption": "H and I Cholesterol levels in p53+/+ and p53-/- HepG2 cells (H) or HCT116 cells (I) were examined. Protein expression was shown by western blotting (bottom panel).",
    "molecules": "Cholesterol"
  },
  {
    "caption": "F and G p53+/+ and p53-/- HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum (Serum) or lipoprotein-depleted FBS (LPDS). Protein expression was shown by western blotting (F). mRNA levels of SQLE were examined by qRT-PCR (G). P: premature SREBP2; M: mature SREBP2.",
    "molecules": "lipoprotein"
  },
  {
    "caption": "A-C Squalene (A), lanosterol (B) and cholesterol (C) abundance in p53+/+ and p53-/- HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS). Protein levels were analyzed by western blotting with specific antibodies (A bottom panel).",
    "molecules": "cholesterol, lanosterol, Squalene"
  },
  {
    "caption": "J - M p53+/+ HCT116 cells (J and K) and p53+/+ HepG2 cells (L and M) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours. Cholesterol concentration (upper panel) and protein expression (bottom panel) were determined (J and L). Cell proliferation is shown (K and M).",
    "molecules": "Cholesterol"
  },
  {
    "caption": "G and H Cholesterol levels, protein expression (G) and cell growth (H) of SK-HEP-1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol.",
    "molecules": "Cholesterol, cholesterol"
  },
  {
    "caption": "p53-/- C57BL/6N male mice (n=7) were injected with a single dose of diethylnitrosamine (DEN) (25mg/kg) at two weeks and then fed with HFD diet at 4 weeks. Oral administration of terbinafine were given at 13 weeks old as indicated. Shown are liver tumor incidence (L) and tumor numbers (M).",
    "molecules": "DEN, diethylnitrosamine, terbinafine"
  },
  {
    "caption": "(N) H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(O) Total liver cholesterol concentrations of mice were examined.",
    "molecules": "cholesterol"
  },
  {
    "caption": "E H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice",
    "molecules": "Oil Red O"
  },
  {
    "caption": "A Time-course analysis of cytosolic free calcium concentration ([Ca2+]cyt) dynamics in 10-d-old wild type (Col-0), atann1, and atann1 AtANN1 complementation line (#10) after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment. ",
    "molecules": "calcium, Ca2+, LaCl3, water"
  },
  {
    "caption": "C GST pull-down assay showing that OST1 interacts with AtANN1 in vitro. Purified recombinant GST-OST1, GST-OST1G33R, or GST proteins from E. coli were immunoprecipitated with GST beads and then incubated with MBP-His-AtANN1. Precipitated proteins were detected with anti-GST and anti-His antibodies.",
    "molecules": "His"
  },
  {
    "caption": "E,F Interaction of OST1 and AtANN1 detected by Co-IP assays in Arabidopsis. Twelve-day-old OST1-Myc overexpressing plants grown on MS medium at 22°C were placed at 4°C for 0, 0.5, 2 h, and total proteins were extracted and immunoprecipitated with anti-Myc agarose beads. The wild type (Col-0) treated with 4°C for 2 h was used as control. The OST1-Myc protein was detected with anti-Myc antibody and the AtANN1 protein was detected with anti-AtANN1 antibody. Representative pictures are shown in (E) and relative protein level in (F).",
    "molecules": "agarose"
  },
  {
    "caption": "G Co-IP assay showing the interaction between OST1 and AtANN1 in vivo. The construct combinations were expressed in N. benthamiana leaves. Total proteins were extracted after the N. benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti-Myc agarose beads. The proteins were detected with anti-Myc and anti-GFP antibodies.",
    "molecules": "agarose"
  },
  {
    "caption": "A,B OST1 phosphorylates AtANN1 (A) and AtANN4 (B) in vitro. Purified recombinant MBP-His-OST1 was incubated with GST-AtANN1, GST-AtANN4 or GST in kinase reaction buffer with 1 µCi [γ-32P] ATP for 30 min at 30°C, followed by separation with SDS-PAGE. Phosphorylated AtANN1 and AtANN4 were detected by autoradiography. Recombinant OST1, AtANN1 and AtANN4 were stained by Coomassie brilliant blue (CBB).",
    "molecules": "32P, CBB, Coomassie brilliant blue, His, ATP, SDS"
  },
  {
    "caption": "C In-gel kinase assays of OST1 in Col-0 and ost1-3 mutant under cold stress. Twelve-day-old Col-0 and ost1-3 mutant were treated at 4°C for 2 h. Total protein extracts were prepared and separated on a SDS-PAGE gel containing 0.2 mg/mL GST-AtANN1 as a substrate, and incubated with 70 µCi [γ-32P] ATP. Top, autoradiograph; bottom, CBB staining.",
    "molecules": "32P, ATP, SDS"
  },
  {
    "caption": "D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1. After phosphorylation, the proteins were separated by SDS-PAGE and subjected to autoradiography. Top, autoradiograph; bottom, CBB staining.",
    "molecules": "SDS"
  },
  {
    "caption": "E OST1 phosphorylates AtANN1 at S289 in vivo in LC/MS analysis. Total proteins were extracted from 12-d-old AtANN1-Myc overexpressing plants treated at 4°C for 0, 10, 30 and 120 min, followed by trypsin digestion. Phosphopeptides were enriched for mass spectrometry analysis. Representative picture for 10 min was shown.",
    "molecules": "Phosphopeptides, trypsin"
  },
  {
    "caption": "A AtANN1 current recordings in Xenopus oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, OST1, or AtANN1 + OST1 cRNA. Bath solution was described in Materials and Methods. The voltage protocol as well as time and current scale bars for the recordings are shown. B Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (A). Negative current is influx of cations into the oocytes. ",
    "molecules": "ddH2O"
  },
  {
    "caption": "D AtANN1 current recordings in oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, AtANN1S289A, OST1, AtANN1 + OST1, and AtANN1S289A + OST1 cRNA. E Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (D). ",
    "molecules": "ddH2O"
  },
  {
    "caption": "A Time-course analysis of [Ca2+]cyt dynamics between 10-day-old wild type (Col-0) and ost1-3 mutant after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment. ",
    "molecules": "Ca2+, LaCl3"
  },
  {
    "caption": "C MST assays of the calcium-binding affinity of GST-AtANN1, GST-AtANN1S289A, and GST-AtANN1S289E. GST was used as a control.",
    "molecules": "calcium"
  },
  {
    "caption": "Micrographs of maximum intensity projections of forebrain sections from 3 mo old GLASTCreERT2/eGFP mice  21 days after tamoxifen treatment Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 20 µm (B), 75 µm (C), 30 µm (D), Histograms showing quantifications (B, n = 6 animals; 5 region of interest (ROI) covering the diencephalon on a total of 3 slides analyzed per animal Data information: data are presented as mean ± SEM. Each data point represents one animal.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Micrographs of maximum intensity projections of forebrain  sections from 3 mo old C57BL/6J mice after EdU  application for 4 weeks in drinking water (F) immunostained as indicated on top of the panels. Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 10 µm (F).",
    "molecules": "EdU, water"
  },
  {
    "caption": "A)  Time course  of biomass concentration. B) Time course of  glucose concentration. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.. Note: although no  nitrogen source was present in the medium, some minor growth (especially of the control and wild type strains) remained (A), which is a known phenomenon within the first hours of cultivation after  nitrogen depletion",
    "molecules": "glucose, nitrogen"
  },
  {
    "caption": "C) Specific glucose  (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). Data information: The reaction rates in C were calculated from the beginning of cultivation until the last sampling time point where  glucose was still present in the medium. The means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD..",
    "molecules": "Ace, acetate, Eth, ethanol, For, formate, Glc, glucose, Lac, lactate, Suc, succinate"
  },
  {
    "caption": "For all growth   regimes tested, the glucose uptake rate shows a biphasic response curve to increasing   ATPase levels. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.",
    "molecules": "glucose"
  },
  {
    "caption": "Simulation of the PFK  rate with varying ADP/(ATP+ADP) ratios. Data information: The PFK flux was simulated as single (isolated) reaction with the following fixed metabolite concentrations  relevant for the kinetic rate  law of the PFK PEP: 0.27 µmol/gDW; F6P: 0.91 µmol/gDW; FBP: 9.74 µmol/gDW ; total concentration ATP+ADP: 2.67 µmol/gDW.",
    "molecules": "ADP, ATP, F6P, FBP, PEP"
  },
  {
    "caption": "A) Time course  of biomass concentration. B) Time course of glucose  concentration. C Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD. ",
    "molecules": "glucose"
  },
  {
    "caption": "C) Specific  glucose (Glc) uptake rate  and specific productivity for  ethanol  (Eth), acetate (Ace), formate  (For), lactate (Lac), and succinate (Suc). D) Yield of Eth, Ace,  For, Lac, and Suc. Data information: The reaction rates in C were calculated for the exponential phase under assumption of quasi-steady state. The means ( and the means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD. ",
    "molecules": "Ace, acetate, Eth, ethanol, For, formate, Glc, glucose, Lac, lactate, Suc, succinate"
  },
  {
    "caption": "(B, C) Partial cytochrome c release by FL-BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID. DKO MEFs re-expressing human BAK or TKO MEFs were permeabilised, and then the cells incubated with the indicated concentrations of Bim BH3 peptide, cBID or FL-BID, alone or in the indicated combinations, at 30oC for 30 min. Cells were then co-stained for cytochrome c and TOM20 and analysed by flow cytometry. Left hand panels show median fluorescence data from replicate experiments (squares denote experiment 1, circles denote experiment 2). Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 (treatments are as listed in the left-hand panels).",
    "molecules": "cytochrome c"
  },
  {
    "caption": "A. Representative  bright field micrographs  of netrin-1 immunohistochemistry  in formalin-fixed paraffin-embedded human ovarian, endometrial  and  hepatocellular carcinoma  (HCC) tumor sections. Scale bar 50 µm. B. Quantification by immunoblots  of netrin-1  in the ECM  of cancer cells of human  tumors(n=3, error bars are SD). The vertical line corresponds to the intercellular zone",
    "molecules": "formalin, paraffin"
  },
  {
    "caption": "D. Bio-layer interferometry  analysis of netrin-1/NP137   and derivates after NODAGA bioconjugation. Numbers indicate the concentration in nM of NP137   and derivates.",
    "molecules": "NODAGA"
  },
  {
    "caption": "D. Biodistribution properties of NP137-NODAGA-111In in a Balb/cJ mouse  bearing 4T1 xenografts  at 48 h (4 mice), 72 h (4 mice) and 96 h (3 mice). Radioactivity  incorporation was quantified by the percentage of the injected dose per gram of organ. Error bars indicate s.d..",
    "molecules": "111In, NODAGA"
  },
  {
    "caption": "G. Whole body autoradiography  of NP137-NODAGA-111In in Balb/cJ mice  bearing a 4T1  (top) or an EMT6  (bottom) tumor using a beta imager system.",
    "molecules": "111In, NODAGA"
  },
  {
    "caption": "H. Analysis of the autoradiography  ratio of NP137-NODAGA-111In in tumor versus bone  in Balb/cJ mice  bearing EMT6  (n= 14 relative values) or 4T1  (n=24 relative values) tumors. Whiskers indicate min to max, Central band of the box indicates the median, bottom of the box indicates Q1 and top of the box indicates Q3.",
    "molecules": "111In, NODAGA"
  },
  {
    "caption": "A. (Top) Balb/cJ mice  were engrafted with EMT6  cells by subcutaneous injection of 1 million cells. After 5 days, animals were treated by IV injection of PBS, DOTA-NP137  or NP137-177Lu. n = 9 animals/group for PBS  and DOTA-NP137; n = 12 animals/group for NP137-177Lu; p < 0.001 between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu  (Two-way ANOVA) between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu. Error bars indicate s.e.m.. (Bottom) NP137-177Lu survival  of mice  engrafted with EMT6  cells. Kaplan-Meier survival curves of mice  treated or not with DOTA-NP137. Mantel Cox test; n = 9 animals/group for PBS and DOTA-NP137; n = 12 animals/group considered as dead at a tumor volume of 2,000mm3 for NP137-177Lu; p < 0.001 between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu. B. (Top) Balb/c mice  were engrafted with 4T1  cells by subcutaneous injection of 1 million cells. After 8 days, animals received IV injections of PBS, DOTA-NP137 or NP137-177Lu; n = 10 animals/group for PBS  and DOTA-NP137; n = 9 animals/group for NP137-177Lu; p < 0.001 between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu  (Two-way ANOVA). Error bars indicate s.e.m.. (Bottom) NP137-177Lu survival  of mice  engrafted with 4T1  cells.  Kaplan-Meier survival curves of mice treated or not with DOTA-NP137. Mantel Cox test; n = 10 animals/group for PBS  and DOTA-NP137; n = 9 animals/group considered as dead at a  tumor volume  of 2,000 mm3 for NP137-177Lu; p = 0.002 between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu. C. (Top) NMRI nude mice  were engrafted with H358  cells by subcutaneous injection of 5 million cells and considered as dead when  tumor volume reached 2,000mm3. After 15 days, animals were treated by IV injection of PBS, DOTA-NP137  or NP137-177Lu; n = 8 animals/group for PBS  and DOTA-NP137; n = 9 animals/group for NP137-177Lu; p < 0.001 between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu  (2-way ANOVA) between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu. Error bars indicate s.e.m. (Bottom) NP137-177Lu survival  of mice  engrafted with H358  cells.. Kaplan-Meier survival curves of mice  treated or not with DOTA-NP137. Mantel Cox test; n = 8 animals/group for PBS  and DOTA-NP137; n = 9 animals/group for NP137-177Lu; p = 0.025 between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu.",
    "molecules": "DOTA, 177Lu, PBS"
  },
  {
    "caption": "D. (Left) Quantification of Ki67 expression  in EMT6  tumors harvested in Balb/cJ mice  7 days post injection of PBS, DOTA-NP137  or NP137-177Lu. (Right) Representative bright field micrographs of Ki67 immunohistochemistry  in formalin-fixed paraffin-embedded EMT6  tumors harvested in Balb/cJ mice  7 days post injection of PBS, DOTA-NP137  or NP137-177Lu; n = 4 mice  per groups; One-way ANOVA. Error bars indicate s.e.m. Scale bar: 50μm. E. (Left) Quantification of cleaved caspase-3 expression  in EMT6  tumors harvested in Balb/cJ mice  7 days post injection of PBS, DOTA-NP137  or NP137-177Lu. (Right) Representative bright field micrographs  of cleaved caspase-3 immunohistochemistry  in formalin-fixed paraffin-embedded EMT6  tumor harvested in Balb/cJ mice  7 days post injection of PBS, DOTA-NP137  or NP137-177Lu; n = 4 mice per groups; One-way ANOVA. Error bars indicate s.e.m. Scale bar:.50μm.",
    "molecules": "DOTA, formalin, 177Lu, paraffin, PBS"
  },
  {
    "caption": "E. Biodistribution properties of NP137-NODAGA-111In  in MMTV/neuT mice  at 96 h (3 mice) and measured for all organs. Radioactivity  incorporation was quantified by the percentage of the injected dose per gram of organ. Error bars indicate s.d.",
    "molecules": "111In, NODAGA"
  },
  {
    "caption": "F. NP137-177Lu  enhances MMTV/neuT mouse survival. Mice  were treated at 3.5 months with PBS, DOTA-NP137  or NP137-177Lu. Kaplan-Meier survival curves  of mice  treated or not with NP137  and considered as dead when cumulative tumors volume reached 2,000 mm3. Mantel Cox test; n = 10 animals/group for PBS; n = 14 animals/group for DOTA-NP137; n = 8 animals/group for NP137-177Lu; p < 0.001 between PBS  and NP137-177Lu  and between DOTA-NP137  and NP137-177Lu.",
    "molecules": "DOTA, 177Lu, PBS"
  },
  {
    "caption": "(E) qPCR  and immunofluorescence  analysis displayed an enhanced expression of ZBTB22  in MPHs  after FSK  treatment for 24h and a reduction of ZBTB22  in MPHs  after insulin  treatment for 15min (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.",
    "molecules": "FSK, insulin"
  },
  {
    "caption": "(D-E) ZBTB22  overexpression increase lipid  deposition in primary hepatocytes, as shown by the enhanced lipid TOX probes and TG  content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.",
    "molecules": "lipid TOX, lipid, TG"
  },
  {
    "caption": "(I-J) ZBTB22  knockdown suppress lipid  deposition in  primary hepatocytes, as shown by the weakened lipid TOX probes and TG  content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.",
    "molecules": "lipid TOX, lipid, TG"
  },
  {
    "caption": "(K) qPCR  analysis of mRNA levels of Pgc1α, PCK1  in  primary hepatocytes infected with Ad-shCtrl or Ad-shZBTB22 with or without forskolin  treatment (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.",
    "molecules": "forskolin"
  },
  {
    "caption": "(E) ZBTB22  deletion increased the  heat production, CO2  generation and O2  consumption in mice  fed on HFD (n=3 mice).",
    "molecules": "CO2, O2"
  },
  {
    "caption": "ZBTB22  deletion effectively decrease the fasting blood glucose  of mice  fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.",
    "molecules": "glucose"
  },
  {
    "caption": "(J) Hepatic ZBTB22  overexpression decreased the insulin-induced phosphorylation  of AKT  and GSK3β  in the liver  of ZBTB22  deficiency mice  fed on HFD.",
    "molecules": "insulin"
  },
  {
    "caption": "(D) Hepatic ZBTB22  overexpression significantly decreased insulin-induced phosphorylation  of hepatic AKT  and GSK3β  in normal C57 mice.",
    "molecules": "insulin"
  },
  {
    "caption": "PCK1  silencing , as well as the stimulatory effects of ZBTB22  on SREBP1C expression  (J) (n=4 biological replicates) and cellular glucose  output (K) (n=4 biological replicates) Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.",
    "molecules": "glucose"
  },
  {
    "caption": "(G) Hepatic ZBTB22  overexpression failed to change serum  and hepatic TG  and TC  levels in PCK1-silencing C57 mice  (n=6 mice). Data information: Data are means ± SEM. *p < 0.05, **p< 0.01, ***p< 0.001 by Student's t test.",
    "molecules": "TG"
  },
  {
    "caption": "(a) Western blotting was performed to determine FoxO1 expression, p62 degradation and LC3-II accumulation in H1299, HCT116 and HeLa cells in response to serum starvation (SS) for 24 h or 0.5 mM H2O2 for 6 h in the presence or absence of E64 and pepstatin A (PEPS A).",
    "molecules": "E64, H2O2, pepstatin A"
  },
  {
    "caption": "(b, c) Stable FoxO1 knockdown HCT116 cells, or cells with an empty RNAi plasmid, were incubated in a serum-free medium for up to 36 h (b) or treated with 0.5 mM H2O2 for up to 12 h (c); western blotting was performed to detect changes in FoxO1, p62, LC3-II or β-actin.",
    "molecules": "H2O2"
  },
  {
    "caption": "(d) HCT116 cells were labelled with [3H]leucine for 48 h, washed and incubated for 24 h, before being subjected to serum starvation for 24 h or 0.5 mM H2O2 for 12 h in the presence or absence of 3-MA. At 12 or 24 h after treatment, the relative degradation of long-lived proteins was measured by counting radioactivity. Data in d are means ± s.d. (n = 3). Two asterisks, P 0.01; asterisk, P 0.05; NS, not significant, P > 0.05; Student's t-test.",
    "molecules": "[3H]leucine, 3-MA, H2O2"
  },
  {
    "caption": "(f) LC3-immunopositive punctate signals were observed in HCT116 or in stable FoxO1-RNAi HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 treatment. (g) Quantification of LC3-positive punctate cells in f. Data in g are means ± s. d. (n = 3).",
    "molecules": "H2O2"
  },
  {
    "caption": "(h) A rescue experiment using a specific mutated FoxO1 plasmid to validate the activity of FoxO1 in the induction of autophagy in HCT116 cells in the presence or absence of serum, with or without E64 and pepstatin A.",
    "molecules": "E64, pepstatin A"
  },
  {
    "caption": "(d) H1299 cells were labelled with [3H]leucine for 48 h, then transfected with a control plasmid or various Flag-FoxO1 plasmids, in the presence or absence of 3-MA or E64 and pepstatin A. Degradation of long-lived proteins was measured at the indicated time, 24 h after transfection. Data in d are means ± s.d. (n = 3).",
    "molecules": "[3H]leucine, 3-MA, E64, pepstatin A"
  },
  {
    "caption": "(f) A set of RNAi-resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1-RNAi HCT116 cells in the presence or absence of E64 and pepstatin A. Western blotting was performed to detect p62 degradation and LC3-II accumulation.",
    "molecules": "E64, pepstatin A"
  },
  {
    "caption": "(b) Quantitative PCR was performed to measure the transactivity of autophagy-related genes in transfected H1299 cells with different FoxO1-expressing plasmids (upper panel) or in stable FoxO1-RNAi or non-RNAi-treated HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 for 12 h (lower panel). All data are means ± s.d. (n = 3).",
    "molecules": "H2O2"
  },
  {
    "caption": "(a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.5 mM H2O2 for 12 h (lower panels); cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 and probed with anti-acetylated lysine (Ace-lyn). Both bottom panels display cytosolic FoxO1. IP, immunoprecipitation.",
    "molecules": "H2O2, lysine"
  },
  {
    "caption": "(c) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid. At 48 h after transfection, the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 (upper panels), or for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine (lower panels). IB, immunoblot.",
    "molecules": "lysine"
  },
  {
    "caption": "(d) HCT116 cells were treated with AGK2 for 24 h at different doses, as indicated. The cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine.",
    "molecules": "AGK2, lysine"
  },
  {
    "caption": "HCT116 cells weretreated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7",
    "molecules": "AGK2"
  },
  {
    "caption": "(a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A.",
    "molecules": "E64, pepstatin A"
  },
  {
    "caption": "(b) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h, in the presence or absence of E64 and pepstatin A. Changes in FoxO1, p62 and LC3-II were determined by western blotting (upper panels), or the formation of LC3 punctate signals was observed (lower panels). HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed. Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel. Data are means ± s.d. (n = 3).",
    "molecules": "AGK2, E64, pepstatin A"
  },
  {
    "caption": "(f) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(K262R), Flag-FoxO1(K265R), Flag-FoxO1(K274R) or Flag-FoxO1(KR). At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted, the cell lysate was extracted and immunoblotted with anti-Flag, anti-LC3, anti-p62 or anti-β-actin.",
    "molecules": "E64, pepstatin A"
  },
  {
    "caption": "(a) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(3A), Flag-FoxO1(ΔDB) or Flag-FoxO1(ΔDB3A), in the presence or absence of 10 mM 3-MA (upper panel) or in the presence or absence of 50 μM Z-VAD-fmk (lower panel); cells were stained with propidium iodide (PI) 48 h after transfection. PI-stained (dead) cells were counted by flow cytometry and the percentage cell loss was calculated.",
    "molecules": "3-MA, Z-VAD-fmk"
  },
  {
    "caption": "FP competition binding of C5 and Tat-C5 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer. Saturation binding curve is shown in EV1B.",
    "molecules": "5FAM"
  },
  {
    "caption": "1H-15N-HSQC 2D spectra of PICK1-PDZ-C10 (orange) following addition of Tat-C5 (green) and subsequent addition of protease (red) to allow for exchange between C10 and Tat-C5.",
    "molecules": "15N"
  },
  {
    "caption": "FP competition binding of C5 (also used in C) and Tat-P4-(C5)2 to PICK1 with 5FAM-Tat-C5 (20 nM) as tracer.",
    "molecules": "5FAM"
  },
  {
    "caption": "SEC profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of TMR-Tat-P4-(C5)2 (blue (abs280 nm), red (abs544 nm), Absmax=10.7 ml).",
    "molecules": "TMR"
  },
  {
    "caption": "Representative confocal images of hippocampal neurons showing membrane penetration of TMR-Tat-P4-(C5)2 and TMR-Tat-C5 (5µM) (magenta), but not the control TMR-C5 (all 20µM). The cell membrane was stained with DiO (grey). Scale bars: 10µm and 5µm on the zooms/orthogonal views.",
    "molecules": "DiO, TMR"
  },
  {
    "caption": "Representative confocal images of hippocampal neurons transduced with the viral vector encoding GFP-PICK1 and incubated with 5nM TMR-Tat-P4-(C5)2. Partial co-localization between TMR-Tat-P4-(C5)2 (red), GFP-PICK1 (green) and GluA2 (blue) is seen in insert zooms. Experiment was done three times. Scale bars: 10µm and 1µm on the zooms.",
    "molecules": "TMR"
  },
  {
    "caption": "Representative confocal images of hippocampal neurons stained for surface GluA2 (sGluA2, light blue) and internalized GluA2 (iGluA2, red). Treatment with Tat-P4-(C5)2 (20 µM) significantly increases the constitutive, but not PMA induced internalization. Scale bars 10µm. Quantification of iGluA2/sGluA2 within the region of interest for individual neuron (see I, top right corner). Tat-C5 did not increase GluA2 internalization (see Appendix Fig S9D-F).",
    "molecules": "PMA"
  },
  {
    "caption": "Representative coronal slices from lumbar spinal cord show the presence of both TMR-Tat-P4-(C5)2 and TMR-Tat-C5 peptides (magenta) 1 hour after i.t. administration in naïve mice. Scale bar 200µm. DH dorsal horn, VH ventral horn. Insert zooms of merged channels (right panels), scale bar 50µm.",
    "molecules": "TMR"
  },
  {
    "caption": "Immunoblot of PICK1 following pull-down from lumbar spinal cord with Biotin-Tat-P4-(C5)2 or Biotin following 20 µM i.t. administration demonstrating in vivo target engagement.",
    "molecules": "Biotin"
  },
  {
    "caption": "Images of lumbar spinal cord sagittal sections showing the time course of TMR-Tat-P4-(C5)2 (magenta) after i.t. administration (20 µM) in SNI mice showing maximal accumulation in cells after 60 min. Scale bar 1000 µm and 100 µm in Zooms.",
    "molecules": "TMR"
  },
  {
    "caption": "Overlay with immunohistochemical staining for the neuronal marker NeuN (yellow) highlights strong neuronal tropism of TMR-Tat-P4-(C5)2 (magenta). Scale bar 100 µm",
    "molecules": "TMR"
  },
  {
    "caption": "Representative images of kainate-induced cobalt uptake in spinal cord slices from p14 mouse pups treated with i) saline, ii) TNFα and iii) TNFα + Tat-P4-(C5)2. Scale Bar: 100 µm. Quantification of cobalt positive (black soma) cells. Each data point represents the average 4-6 25 µm sections from a single 400 µm slice. Bars show means of 4-6 slices and error bars represent SEM. (one-way ANOVA followed by Bonferroni's multiple comparisons test, *P <0.05, **P<0.01).",
    "molecules": "cobalt, kainate"
  },
  {
    "caption": "(F， G)  RNA fluorescence in situ hybridization   of circBtnl1 in small intestine tissues (F) and organoid (G). Green region shows the distribution of circBtnl1  using antisense probe, gray region shows the nuclei staining  by DAPI. For (F)，scale bars，5um; for (G)，scale bars，20 um.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G)  Electrophoretic mobility shift assay (EMSA) using biotin-labeled circBtnl1 and recombinant Ddx3y.",
    "molecules": "biotin"
  },
  {
    "caption": "Cse4-K49Ac and -R37Me are increased in S-phase of the cell cycle. Cells carrying 3xHA-tagged Cse4 were used to immunoprecipitate Cse4 with an α-Cse4-K49Ac (left, top) and an α-HA antibody (left, bottom). To detect Cse4-R37 methylation, 3xHA-tagged Cse4 was immunoprecipitated with an α-Cse4-R37Me2a (right, top) (Samel et al., 2012) and an α-HA antibody (right, bottom). Western blots were probed for the presence of Cse4 with the α-HA antibody. G1, treatment with α-factor; S, arrest with hydroxyurea; G2/ M, arrest in nocodazole; asynch, asynchronous culture.",
    "molecules": "α-factor, hydroxyurea, nocodazole"
  },
  {
    "caption": " WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI. Cilia in the magenta box are shown enlarged on the right picture ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI Percent of ciliated cells, as well as the length of cilia, was quantified from A. Cilia length was measured by a semi-automated ImageJ macro as described in methods. Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition. Error bars represent the mean ± SD. **** P≤ 0.0001. High means 80-90% and low 40-50% confluency of RPE1 cells",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " hCDC14A-GFP and hCDC14AC278S-GFP under control of the TetON promoter were stably integrated into RPE1 cells. hCDC14A-GFP and hCDC14AC278S-GFP localized to the C-Nap1 marked basal body upon Dox induction. In addition, expression of hCDC14A-GFP but not the phosphatase dead hCDC14AC278S-GFP mutant inhibited cilia formation. Ciliated cells were quantified 48 h after serum starvation with and without Dox addition. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. *** P≤ 0.001; ns: not significant ",
    "molecules": "Dox"
  },
  {
    "caption": "TetON-hCDC14A-GFP RPE1 cells were serum starved for 48 h prior to fixation and immunofluorescence microscopy. hCDC14A-GFP colocalizes with Phalloidin 555 conjugated dye that marks F-actin fibers. The boxed area was 10-fold enlarged and displayed below the corresponding images to indicate co-localization of hCDC14A-GFP and F-actin. Line scan inside the magenta colored box showed in (A)",
    "molecules": "Phalloidin 555"
  },
  {
    "caption": "hCDC14A, not the inactive hCDC14AC278S, dephosphorylates the serine residue 142 of DBN1 in vivo. RPE1 cells expressing GFP, hCDC14A-GFP, and hCDC14AC278S-GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence. DBN1pS142 was detected with a phospho-specific antibody. DNA was stained with DAPI. Scale Bar = 10 μm",
    "molecules": "DAPI, DNA, Dox, serine"
  },
  {
    "caption": "Immunoblot analysis of samples from (A). The DBN1pS142/DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot. In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001",
    "molecules": "Dox"
  },
  {
    "caption": "C Characteristics of 2a2iL-RH6 cells represented by dome-shaped colonies, ALP activity, and a normal male karyotype (46, XY). 2a2iL-RH6 expressed the common pluripotency markers OCT4, NANOG, SOX2, and TRA-1-81, but not SSEA1. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI). Scale bars: 200, 100, and 50 µm in the inset.",
    "molecules": "4′, 6-diamidino-2-phenylindole, DAPI, PI, Propidium iodide"
  },
  {
    "caption": "G Immunofluorescence staining against pRH5 and 2a2iL-RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Reactivation of inactive X-chromosome in female 2a2iL-RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 µm.",
    "molecules": "4′, 6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "C Immunofluorescence images of differentiated 2a2iL-RH6 cells that depict expression of markers for neuron-like cells (TUJ-1), cardiac-muscle-like cells (CTNT), and hepatocyte-like cells (SOX17). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.",
    "molecules": "4′, 6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "E Expression of the common pluripotency markers OCT4, SOX2, and TRA-1-81, and naïve-related pluripotency markers NANOG, KLF17, and TFCP2L1 in nRH13. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.",
    "molecules": "4′, 6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "C Inhibition of the TGF-β pathway by two small molecules, SB43 and A83. Left panel: morphology of naïve-like cells three days after treatment. Right panel: morphology of treated cells after passaging. Scale bar: 100 µm.",
    "molecules": "A83, SB43"
  },
  {
    "caption": "B Inhibition of TGF-β pathway using SB43 during conversion of primed to naïve pluripotency. Scale bar: 100 µm.",
    "molecules": "SB43"
  },
  {
    "caption": "G Immunofluorescence images show the expression of naïve pluripotency markers in TGF-β2iL-RH6. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.",
    "molecules": "4′, 6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "A) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col-0 and mkkk7.",
    "molecules": "ROS"
  },
  {
    "caption": "B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines.",
    "molecules": "ROS"
  },
  {
    "caption": "C) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col-0 and two independent inducible MKKK7DD transgenic lines. Graphs represent means with error bars ± SEM (n=24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results.",
    "molecules": "ROS"
  },
  {
    "caption": "Immunofluorescence analysis of p-c-Jun in micrometastatic (day 7) and macrometastatic (day 21) nodules in a xenograft mouse model injected intravenously with MDA231-LM2 cells. DAPI was used to stain cell nuclei, and cancer cells express GFP as a marker. Scale bar, 50 μm. Quantification of p-c-Jun+ cancer cells",
    "molecules": "DAPI"
  },
  {
    "caption": "Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment.",
    "molecules": "paclitaxel, PAX"
  },
  {
    "caption": "GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm.",
    "molecules": "DAPI, PAX"
  },
  {
    "caption": "Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.",
    "molecules": "PAX"
  },
  {
    "caption": "SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD.",
    "molecules": "PAX"
  },
  {
    "caption": "SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD.",
    "molecules": "DOXO, doxorubicin"
  },
  {
    "caption": "SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD.",
    "molecules": "5-Fluorouracil, methotrexate"
  },
  {
    "caption": "SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells.",
    "molecules": "paclitaxel, PAX"
  },
  {
    "caption": "Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors.",
    "molecules": "PAX"
  },
  {
    "caption": "Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice.",
    "molecules": "PAX"
  },
  {
    "caption": "Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors.",
    "molecules": "cyclophosphamide, Adriamycin, doxorubicin"
  },
  {
    "caption": "Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P < 0.0001. Lung metastases quantified in tumor bearing mice",
    "molecules": "PAX"
  },
  {
    "caption": "Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001.",
    "molecules": "paclitaxel, PAX"
  },
  {
    "caption": "Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel. Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm.",
    "molecules": "paclitaxel, PAX"
  },
  {
    "caption": "Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM.",
    "molecules": "PAX"
  },
  {
    "caption": "(b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA-Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a ΔΨm dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag-Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively.",
    "molecules": "CCCP"
  },
  {
    "caption": "(d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type.",
    "molecules": "CCCP"
  },
  {
    "caption": "Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control.",
    "molecules": "CCCP, doxycycline"
  },
  {
    "caption": "(c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not.",
    "molecules": "CCCP"
  },
  {
    "caption": "(e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies.",
    "molecules": "CCCP"
  },
  {
    "caption": "d-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (g) Quantification of results from cells in e.",
    "molecules": "CCCP"
  },
  {
    "caption": "(f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.",
    "molecules": "CCCP"
  },
  {
    "caption": "a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. (",
    "molecules": "CCCP"
  },
  {
    "caption": "(c-f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c-e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) Time-course experiment of HeLa cells transfected with Flag-Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag-Parkin.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag-Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. (",
    "molecules": "CCCP, Ni, NTA, urea"
  },
  {
    "caption": "(c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag-Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.",
    "molecules": "CCCP, Ni, NTA"
  },
  {
    "caption": "(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a-c. WT, wild-type; Ub, ubiquitin.",
    "molecules": "CCCP"
  },
  {
    "caption": "VDAC1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. Both effects can be rescued by re-transfection with wild-type VDAC1. (a) Immunofluorescence and (b) western blot of siRNA-transfected HeLa cells used in c-g demonstrate partial silencing of endogenous VDAC1. (a) Silenced cells were stained with anti-HtrA2/Omi, a mitochondrial marker (pseudo coloured; blue), and anti-VDAC1 (red) antibodies. (b) Lysates from silenced cells were subjected to western blot analysis and probed with an anti-VDAC1 antibody, while TOM20 and β-actin probing served as loading controls.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c-e) Control siRNA- or VDAC1 siRNA-silenced HeLa cells were transfected with the indicated combinations of EGFP-Parkin wild-type, VDAC1-Flag wild-type or appropriate empty vector controls. Cells were treated with CCCP for 2 h and 24 h and stained with anti-HtrA2/Omi (pseudo coloured; blue) and anti-Flag (red) to visualize mitochondrial morphology and re-transfected VDAC1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative pictures of three independent experiments are shown. Scale bars, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(f, g) Quantification of d (f) and e (g). Parkin wild-type colocalization with impaired mitochondria after 2 h (f) and uncleared mitochondria after 24 h (g) of CCCP treatment (n = 3). Data represent the mean ± s.d., *P ≤ 0.05 compared with control siRNA-transfected or with VDAC1-Flag wild-type-transfected cells.",
    "molecules": "CCCP"
  },
  {
    "caption": "(G) Chs3 GFP GPD-CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain. Plates were incubated at 30°C for 2-3 days. A representative drop test of three independent biological experiments is shown.",
    "molecules": "calcofluor"
  },
  {
    "caption": "(D) Chitin production is reduced upon Bch1 overexpression. Fluorescence images of chitin stained with calcofluor in Chs3-GFP WT, chs5 and GPD-BCH1 strains.",
    "molecules": "calcofluor, chitin"
  },
  {
    "caption": "(B) THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS HSV-1 and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.",
    "molecules": "ACV, acyclovir"
  },
  {
    "caption": "(E) THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 µg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(A) MDMs were infected for 18 h with HSV-1 (KOS), MOI 1. Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown.",
    "molecules": "iTRAQ"
  },
  {
    "caption": "(E) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity",
    "molecules": "ACV, acyclovir"
  },
  {
    "caption": "(G-I) HEK293T cells were ((H) transfected with STING and stimulated for 16 h with cGAMP (4 μg/ml) together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(G) THP1 cells were infected with HSV-1 KOS (MOI 10) for 8 h in the presence or absence of the TBK1 inhibitor BX795 (BX, 200 nM). Total lysates were generated and STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.",
    "molecules": "BX795"
  },
  {
    "caption": "C, Cell viability of BT308NS, irradiated in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM), and analyzed at the indicated time points. Vehicle: non-irradiated cells (fold vs. vehicle-treated cells at time 0, mock). *: t-test, p<0.01.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "E, Flow-cytometry analysis of Annexin V/DAPI incorporation in BT308NS irradiated (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Histograms show the percentage of Annexin V-positive cells 36 h after IR. Ctrl: non-irradiated cells. *: t-test (5 Gy + JNJ) vs. (5 Gy), P=0.01.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "F, Western blot of BT308NS showing caspase 3 activation (cleaved casp-3), at the indicated time points after IR in the absence (-) or in the presence (+) of JNJ38877605. Vinculin was used as loading control.",
    "molecules": "JNJ38877605"
  },
  {
    "caption": "G, Clonogenic assay with METneg and METhigh subpopulations sorted from BT308NS, irradiated (5 Gy) in the absence (vehicle) or in the presence of MET inhibitors (JNJ38877605 and Crizotinib, 500 nM). Ctrl: non-irradiated cells. *: t-test, p<0.0004.",
    "molecules": "JNJ38877605, Crizotinib"
  },
  {
    "caption": "H, LDA (sphere forming assay) with METneg and METhigh subpopulations sorted from BT308NS, measuring GSC frequency after irradiation in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. *: 2 test, P=0.0002.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "I, Flow cytometry analysis of phosphorylated histone H2AX (H2AX) in the whole BT308NS at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605 (fold vs. non-irradiated cells at time 0, mock). *: t-test, p<0.05.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "J, Neutral comet assay with BT308NS performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. Comet tail's length is proportional to DSB extent. Data are represented as mean ± SEM or ± CI in H.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "B, Western blot of BT308NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM). Total ATM is also shown. β-actin was used as loading control. Veh: non-irradiated vehicle-treated cells. C, Densitometric analysis of ATM phosphorylation shown in (B). *: t-test, p<0.01.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "D, Western blot of BT308NS showing phosphorylation of ATM, Chk2, and AKT (pAKT), and accumulation of RAD51 24 h after IR (5 Gy) in the absence (vehicle, veh) or in the presence of MAPK (PD98059, 20 M), AKT (Ly294002, 20 M), MET (JNJ38877605), or ATM (CGK733, 10 M) inhibitors. Total ATM and AKT are also shown. β-actin was used as loading control. Ctrl: non-irradiated cells.",
    "molecules": "CGK733, PD98059, JNJ38877605, Ly294002"
  },
  {
    "caption": "E, Western blot of BT463NS showing phosphorylation of ATM, Chk2, and Aurora Kinase A (pAurora A) at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + Ly) of the AKT inhibitor Ly294002. Total ATM and Aurora Kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.",
    "molecules": "Ly294002"
  },
  {
    "caption": "F, Western blot of BT308NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Total Aurora kinase A is also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "H, Western blot of BT308NS showing phosphorylation of ATM and Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + MLN) of the Aurora Kinase A inhibitor MLN8237 (20 M). Total ATM and Aurora kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.",
    "molecules": "MLN8237"
  },
  {
    "caption": "I, Cell viability of BT308NS, measured 72 h after IR (5 Gy) in the absence (vehicle) or in the presence of MET (JNJ38877605), Aurora Kinase A (MLN8237), or AKT (Ly294002) inhibitors (fold vs. non-irradiated vehicle-treated cells, mock). *: t-test (5 Gy + inhibitor) vs. (5 Gy + vehicle), p<0.01.",
    "molecules": "MLN8237, JNJ38877605, Ly294002"
  },
  {
    "caption": "B, Western blot of BT463NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM).",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "C, Western blot of BT308NS showing p21 localization in cytoplasmic/nuclear extracts at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vinculin and lamin B were used as loading controls for cytoplasmic and nuclear fractions, respectively.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "D, Representative immunofluorescence staining of p21 (red) on BT308NS cells 3 h after IR (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Ctrl: non-irradiated cells. Nuclei are counterstained with DAPI (blue). Scale bar, 10 µm (63× magnification). E, Quantification of the percentage of cells showing p21 cytoplasmic or nuclear localization in BT308NS represented in (D) (n = 10 HPF/group). HPF: high-power field. *: t-test (5 Gy + JNJ) vs. (ctrl or 5 Gy), p<0.0001.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "F, Cell cycle analysis of BT314NS, performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated cells. *: one-way ANOVA, P=0.008.",
    "molecules": "JNJ, JNJ38877605"
  },
  {
    "caption": "C, Growth curves of tumors, generated by subcutaneous injection of BT308NS, irradiated in the absence (2 Gy × 3 days) or in the presence (combo) of JNJ38877605, which was administered for 15 days as indicated (n = 7/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=0.0006.",
    "molecules": "JNJ38877605"
  },
  {
    "caption": "D. Relative levels of mtDNA of Fbxl4 knockout (-/-) embryos and the corresponding wild type (+/+) embryos at E13.5. Data represent mean ± SEM, n=5. Student's t-test; *, p<0.05.",
    "molecules": "DNA"
  },
  {
    "caption": "A. Relative levels of mtDNA in tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR (normalized to controls). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; **, p<0.01; ***, p<0.001.",
    "molecules": "DNA"
  },
  {
    "caption": "E. Quantitative TMT-based proteomic analysis of wild-type and Fbxl4 knock-out liver tissue protein extracts. Proteins from three control and three Fbxl4 knock-out liver protein extracts were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and knock-out animals were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to mouse MitoCarta 2.0(Calvo et al, 2016) and highlighted in red; lysosomal proteins were selected according to hLGDB database(Brozzi et al, 2013) and highlighted in yellow.",
    "molecules": "TMT"
  },
  {
    "caption": "B. Relative mtDNA content in fibroblasts from patients and controls as determined by qRT-PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe. Individual values are presented for each sample, as well as their mean ± SEM values.",
    "molecules": "DNA"
  },
  {
    "caption": "F. Quantitative TMT-based proteomic analysis of control and patient fibroblasts. Proteins from three control and three patient fibroblast cell lines were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and patients were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to human MitoCarta 2.0 (Calvo et al, 2016) and highlighted in blue; lysosomal proteins were selected according to hLGDB database (Brozzi et al, 2013) and highlighted in yellow.",
    "molecules": "TMT"
  },
  {
    "caption": "C. (Upper panel) Pulse-chase labeling of mitochondrial translation products in wild type (WT) and FBXL4 knockout (KO) cells. The cells were incubated for 1 hour with a 35S methionine-cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation (pulse). After removal of the medium, the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours (chase). A representative experiment of 4 biological replicates is shown. A Coomassie-stained part of the gel is shown as loading control. (Lower panel) Quantification of the MTCO1-ND4 signal presented as mean ± SEM; n=4 biological replicates; Student's t-test; *, p<0.05.",
    "molecules": "anisomycin, cysteine, methionine, 35S"
  },
  {
    "caption": "D. (Upper panel) Mitochondrial translation products were labeled in wild type (WT) and FBXL4 knock-out (KO) RKO cells for 60 minutes as in C, followed by 24 h chase in the presence of 5 μM epoxomicin (epoxo) or 20 mM ammonium chloride (NH4Cl). A representative experiment of n≥3 biological replicates is shown. A fragment of the Coomassie-stained gel is shown as loading control. (Lower panel) Quantification of the ND4-MTCO1 signal. n≥3 biological replicates, data are presented as mean ± SEM. Student's t-test; *, p<0.05.",
    "molecules": "ammonium chloride, NH4Cl, epoxo, epoxomicin"
  },
  {
    "caption": "E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4Cl",
    "molecules": "NH4Cl"
  },
  {
    "caption": "E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin (Epoxo, F).",
    "molecules": "Epoxo, epoxomicin"
  },
  {
    "caption": "A. Western blot analysis of mTORC1 activity in C2C12-Vevtor (VC) and Aster-C KO (KO) cells in complete medium (CM) and in response to nutrient starvation or stimulation with amino acids for 30 min. B and C. Statistical analysis of phosphorylated level of S6K (B) and 4E-BP1 (C) in C2C12-Vector and Aster-C KO cells in CM, under nutrient starvation (-AA) and in response to stimulation with amino acids (+AA) using ImageJ software. Data are represented as mean ± SD (n=3 biological replicates). *p < 0.05, **p<0.01 by Student's t test.",
    "molecules": "AA, amino acids"
  },
  {
    "caption": "D. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under nutrient starvation (-AA) and in response to AA stimulation (+AA). LAMP1 was immunostained as a lysosome marker. Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).",
    "molecules": "AA"
  },
  {
    "caption": "F. Western blot analysis of mTORC1 activity in Aster-C KO cells stably re-expressing exogenous GFP-Aster-C under nutrient starvation and AA stimulation. Aster-C KO cells were transfected with GFP-Aster-C and selected with G418 for 2 weeks.",
    "molecules": "AA, G418"
  },
  {
    "caption": "G. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under cholesterol depletion by treatment with 0.5% MCD for 2 hrs (-CHOL) or cholesterol (50 µM) re-stimulation for 1 hr (+CHOL). Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).",
    "molecules": "CHOL, cholesterol, MCD"
  },
  {
    "caption": "H. Western blot analysis of mTORC1 activity in C2C12-Vector and Aster-C KO cells in CM, under starvation, cholesterol depletion by treatment with 0.5% MCD for 2 hrs or cholesterol (50 µM) re-stimulation for the indicated time.",
    "molecules": "cholesterol, MCD"
  },
  {
    "caption": "A. Confocal image analysis depicting the co-localization of Aster-C with the ER in C2C12 cells transiently expressing GFP-Aster-C and DsRed-ER under starvation and in response to AA stimulation. Scale bar, 5 μm.",
    "molecules": "AA"
  },
  {
    "caption": "B. Subcellular fractionation analysis of mTOR, GATOR2 complex and Aster-C localization in HEK293T cells transiently expressing FLAG-Aster-C under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively. Microsomal fraction (Micro) was also used in the blot.",
    "molecules": "AA"
  },
  {
    "caption": "C. Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12-Vector and Aster-C KO cells under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively.",
    "molecules": "AA"
  },
  {
    "caption": "D. Co-IP analysis of the interaction of Aster-C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins. Anti-Myc antibody was used for immunoprecipitation.",
    "molecules": "AA, insulin"
  },
  {
    "caption": "A. Co-IP analysis of the interaction of Aster-C with COPA and COPG in response to starvation and AA stimulation using FLAG-DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.",
    "molecules": "AA"
  },
  {
    "caption": "B. Co-IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP-COPA in response to starvation and AA stimulation. Anti-GFP antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.",
    "molecules": "AA"
  },
  {
    "caption": "C. Co-IP analysis of the interaction of COPA with mTOR in C2C12-Vector and Aster-C KO cells in response to starvation and AA stimulation. Anti-COPA antibody was used for immunoprecipitation.",
    "molecules": "AA"
  },
  {
    "caption": "D. Confocal imaging analysis depicting the co-localization of YFP-mTOR (yellow) with GFP-COPA (green) and lysosomes (red) in live C2C12-Vector and Aster-C KO cells in response to nutrient starvation and re-stimulation by AA. Arrows highlight co-localization of mTOR, COPA and lysosomes. Scale bar, 20 μm. E and F. Statistical analysis of the Pearson's correlation coefficient of mTOR/Lysosome (E) and COPA/Lysosome (F) in Figure 3D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.",
    "molecules": "AA"
  },
  {
    "caption": "Time-lapse confocal imaging analysis of the co-localization of YFP-mTOR (red) with GFP-COPA (green) and lysosomes (blue) in live C2C12 cells under nutrient starvation (0 min), and in response to AA re-stimulation for indicated time (0.5 min, 2 min, 6 min and 8 min). Arrows highlight COPA puncta that were co-localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes.. Scale bar, 5 μm.",
    "molecules": "AA"
  },
  {
    "caption": "A. Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM) in live C2C12 cells transiently expressing YFP-mTOR and stained with Lysotracker Red. Scale bar, 20 μm.",
    "molecules": "AA, BFA, Lysotracker Red, Exo-2"
  },
  {
    "caption": "Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) (B)",
    "molecules": "AA, BFA"
  },
  {
    "caption": "C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of Exo-2 (10 μM) (C).",
    "molecules": "AA, Exo-2"
  },
  {
    "caption": "D. Western blot analysis of mTORC1 activity in C2C12-Vector and ARF1 KO cells in CM, in response to nutrient starvation and AA stimulation.",
    "molecules": "AA"
  },
  {
    "caption": "E. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with lysosomes (red) in live C2C12 cells transiently expressing GFP-COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM). Arrows highlight co-localization of COPA with lysosomes. Scale bar, 5 μm.",
    "molecules": "AA, BFA, Lysotracker Red, Exo-2"
  },
  {
    "caption": "A. Co-IP analysis of the interaction of Aster-C with COPA, MYH10, WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A (BFA, 10 μM) and/or blebbistatin (BBS, 10 μM), inhibitors of ARFs and MYH10, respectively. Anti-FLAG antibody was used for immunoprecipitation.",
    "molecules": "BBS, blebbistatin, BFA, brefeldin A"
  },
  {
    "caption": "B. Western blot analysis of mTORC1 activity in wild type (WT) and MYH10 KO MEF cells cultured in CM, or in response to starvation and AA stimulation.",
    "molecules": "AA"
  },
  {
    "caption": "C. Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re-expressed exogenous mCherry-MYH10 in response to starvation and AA stimulation. MYH10 KO MEF cells were transfected with mCherry-MYH10 and selected with G418 for 2 weeks.",
    "molecules": "AA, G418"
  },
  {
    "caption": "D. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with mTOR (red) in live C2C12 cells transiently expression GFP-COPA and YFP-mTOR in response to nutrient starvation in the presence or absence of BBS (10 µM). Arrows highlight the co-localization of mTOR with COPA. Scale bar, 20 μm.",
    "molecules": "BBS"
  },
  {
    "caption": "A. Confocal imaging analysis depicting mTOR puncta (green) formation on actin filaments (red) in COS-7 cells transiently expressing YFP-mTOR in response to AA stimulation in the presence or absence of latrunculin B (LA-B, 100 nM). Actin filaments were stained using BODIPY™ 558/568-Phalloidin. Arrows indicated the association of mTOR with actin filaments. Scale bar, 5 μm.",
    "molecules": "AA, LA-B, latrunculin B, 558/568-Phalloidin"
  },
  {
    "caption": "B. Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation (-AA), or in response to AA stimulation (+AA) in the presence or absence of LA-B (100 nM). Scale bar, 20 μm.",
    "molecules": "AA, amino acids, LA-B"
  },
  {
    "caption": "C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of LA-B (100 nM).",
    "molecules": "AA, LA-B"
  },
  {
    "caption": "D. Co-IP analysis of the interaction of Aster-C with COPA and MYH10 in the presence or absence of LA-B (100 nM) or cytochalasin D (Cyto-D, 250 nM) in HEK293T cells transiently expressing FLAG-Aster-C. Anti-FLAG antibody was used for immunoprecipitation.",
    "molecules": "Cyto-D, cytochalasin D, LA-B"
  },
  {
    "caption": "CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in miR-CTL and miR-195 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.",
    "molecules": "Ca2+, calcium, FCCP"
  },
  {
    "caption": "CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in control siRNA (siCTL) and siMICU1 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.",
    "molecules": "Ca2+, calcium, FCCP"
  },
  {
    "caption": "(E) Bar graph illustrating the number of Ca2+ pulses handled by control miR, miR-195, siCTL and siMICU1 transfected cells. Mean ±SEM; n=3-4 (Biological repeats). *P<0.05, Student's t-test.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(F) Representative [Ca2+](m) traces after addition of FCCP (10 µM) in permeabilized CP20 cells transfected with non-target miR-CTL or miR-195. (G) Quantification of resting matrix [Ca2+](m) after addition of FCCP. n=5 (Biological repeats) *P<0.05, Student's t-test, ",
    "molecules": "Ca2+, FCCP"
  },
  {
    "caption": "(J) Intracellular lactate was measured in miR-CTL, miR-195, siCTL, and siMICU1 expressing OVCAR4, CP20, and OVSAHO cells. n=3, biological repeats *P<0.05, Student's t-test.",
    "molecules": "lactate"
  },
  {
    "caption": "(K&L) miR-CTL, miR-195, or miR-195 + pLYS1-MICU1-Flag transfected cells were stained with MitoSOX Red, and mitochondrial ROS levels were determined by flow cytometry. The histogram shows representative staining and bar graph (right) shows results of three independent experiments. Mean ± SEM; . n=3, biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.",
    "molecules": "MitoSOX Red, ROS"
  },
  {
    "caption": "(A&B) Stable cell lines for CP20 and OVCAR4 expressing GFP-miR-195 or non-target miR-GFP were generated using lentiviral mediated transduction and were selected using puromycin; representative images are shown. Scale bars, 50 µm.",
    "molecules": "puromycin"
  },
  {
    "caption": "A) Binding and endocytosis studies with Alexa Fluor 647-conjugated S-RBD or transferrin in Calu1-ACE2-RFP cells deficient in PLAC8 and SPNS1. Left: scatterplots of flow cytometry data showing the cutoffs used to define ACE2-RFP and Alexa Fluor 647 positive cells. Right: bar plots showing the ratio of cells positive for Alexa Fluor 647 in either ACE2-RFP positive (red) or negative (blue) cells in binding and endocytosis experiments with S-RBD or transferrin. Bars represent the average and SEM of three biological replicates (three different CRISPR KO cell lines). The significance above each bar represents the t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.",
    "molecules": "Alexa Fluor 647"
  },
  {
    "caption": "B-E) Endocytosis experiments with S-RBD in PLAC8- or SPNS1-KO Calu1-ACE2-RFP cells with overexpression of GFP-PLAC8 or GFP-SPNS1, respectively. Cells were incubated with S-RBD for 1 h at 37 ºC and fixed with 4% p-formaldehyde after an acid wash to remove non-internalized S-RBD. Representative wide-field fluorescence images (B) and quantification of mean fluorescence intensities of each fluorophore (C) in each cell show that S-RBD signal correlates with ACE2-RFP expression but not with GFP-PLAC8 or GFP-SPNS1. The Pearson correlation coefficient (r) and correlation test p-value (p) is indicated within each scatterplot. Each dot represents a cell measurement from different microphotographs of a single replicate. Scale bar: 20 µm. Representative fluorescence confocal microphotographs (D) and spot colocalization analyses using ComDet plugin (E) demonstrate colocalization of S-RBD endosomes with ACE2-RFP and GFP-PLAC8 or GFP-SPNS1. (D) Red ovals defining the detected S-RBD dots are overlaid in each channel to assess the colocalization with ACE2-RFP, GFP-PLAC8 or GFP-SPNS1. Inlets show a magnification of the region defined by the white squares. Scale bar: 20 µm. (E) Boxplots representing the percentage of internalized S-RBD that colocalize with ACE2-RFP, GFP-PLAC8, GFP-SPNS1 or their combination (ACE2-RFP and GFP-PLAC8 or ACE2-RFP and GFP-SPNS1). The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate.",
    "molecules": "p-formaldehyde"
  },
  {
    "caption": "C) Representative images (left) and quantification (right) of the number of acidic particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using live-cell lysotracker staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used.",
    "molecules": "lysotracker"
  },
  {
    "caption": "D) Lysosomal degradation studies in Calu1ACE2 cells using the fluorogenic substrate DQ-Red-BSA. Left: density plots representing the shift in fluorescence intensity in NT596 cells untreated or treated with DQ-Red-BSA under growth or serum starvation conditions. Right: bar plot representing the mean and SEM of the median fluorescence intensity values (MFI) of three biological replicates of PLAC8- and SPNS1-KO and CRISPR non-targeting cells in growth and serum starvation conditions.",
    "molecules": "DQ-Red"
  },
  {
    "caption": "(A) HeLa cells were incubated for 4 h in either EBSS or media without glucose/pyruvate. Lysates were then analysed by western blot for the presence of MCL‐1, as well as actin as a loading control.",
    "molecules": "glucose, pyruvate"
  },
  {
    "caption": "(C) H1299 cells stably expressing GFP-LC3 were incubated for 2 h in EBSS, and expression of various BCL‐2 homologues was analysed by western blot. Alternatively, primary cortical neurons were incubated for 4 h in media without glucose/pyruvate.",
    "molecules": "glucose, pyruvate"
  },
  {
    "caption": "(D) H1299 cells stably expressing GFP-LC3 were incubated for 3 h in media without glucose/pyruvate, followed by re‐addition of glucose and pyruvate for the indicated times.",
    "molecules": "glucose, pyruvate"
  },
  {
    "caption": "(E) HeLa cells were infected with 5 p.f.u. per cell of either Ad HA‐tBID or Ad LacZ for 7 h, the media being changed to EBSS for the last 3 h where indicated. Alternatively, cells were treated with 50 μM camptothecin for 3 h.",
    "molecules": "camptothecin"
  },
  {
    "caption": "(F, G) HeLa cells were treated as in E in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence microscopy for active BAX (6A7 epitope; F) or cyt c (G). Data are expressed as the average of three experiments±s.d. *P0.01.",
    "molecules": "zVAD‐FMK"
  },
  {
    "caption": "(A) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence for cyt c or active BAX (6A7 epitope). As a positive control, cells were infected with 5 p.f.u. per cell Ad HA‐tBID for 7 h. Data are expressed as the average of three experiments±s.d.",
    "molecules": "zVAD‐FMK"
  },
  {
    "caption": "(B) H1299 cells stably expressing GFP-LC3 were incubated for 4 h in either EBSS or media without glucose/pyruvate. Cells were then fixed, stained with an antibody against cyt c and analysed by immunofluorescence. Scale bars=50 μm.",
    "molecules": "glucose, pyruvate"
  },
  {
    "caption": "(D) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in either EBSS or media without glucose/pyruvate and analysed by western blot for the presence of MCL‐1 and autophagic markers.",
    "molecules": "glucose, pyruvate"
  },
  {
    "caption": "(E, F) H1299 cells stably expressing GFP-LC3 were pre‐incubated for 1 h with 5 mM 3MA (E) or 200 nM bafilomycin (F), and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin. Lysates were then analysed for the presence of MCL‐1, the autophagy marker p62 and actin as a loading control.",
    "molecules": "3MA, bafilomycin"
  },
  {
    "caption": "(C, D) H1299 cells stably expressing GFP-LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated. The levels of endogenous LC3‐II relative to actin were quantified by western blot in C. Data are expressed as the average of three experiments±s.d, *P0.05.",
    "molecules": "bafilomycin"
  },
  {
    "caption": "(A) Cortices from the indicated genotypes and ages were analysed by western blot for the presence of active caspase‐3, p62 and MCL‐1. Primary cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase‐3.",
    "molecules": "camptothecin"
  },
  {
    "caption": "Alternatively, they were analysed by immunofluorescence for c release (G) in the presence of 50 μM zVAD‐FMK to prevent apoptotic cells from detaching from the coverslip. Data are expressed as the average of 3-7 embryos±s.d. *P0.001.",
    "molecules": "zVAD‐FMK"
  },
  {
    "caption": "(A) Quality of the cell fractionation assays was controlled by western blot by assessing the distribution of nuclear and cytosolic markers in the whole cell extract (WCE), the cytosol and the nucleus. Protein lysates were isolated from mouse primary neocortical cells (14 days in culture) treated for 3hrs with the transcription inhibitor triptolide (1µM). (four independent cultures)",
    "molecules": "triptolide"
  },
  {
    "caption": "Efficiencies of bicuculline (A) stimulation were assessed by controlling the induction of ERK phosphorylation by immunostaining. Mouse primary neocortical cells (14 days in culture) were stimulated with bicuculline (20µM) to elevate the neuronal network excitation Cells were stained with anti-phospho-ERK (green, bottom) and anti-MAP2 (white, top) antibodies and counterstained with Hoechst (blue, top). (four independent cultures)",
    "molecules": "Hoechst, bicuculline"
  },
  {
    "caption": "RT-PCR validations of regulated IR-transcripts through splicing upon bicuculline stimulation (C) Expression of the IR-isoforms and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs (standard deviations) of PIR values are shown beneath each panel. In addition, fold changes in the expression of the IR-transcripts (red and orange) and spliced (dark grey) isoforms were assessed with real-time qPCR using three different primer sets, as represented in the top scheme. Note that the Gria3 spliced transcript (C) does not correspond to the canonical mRNAs and presumably arise from a first step of recursive splicing and thereby likely require splicing completion to generate fully mature Gria3 transcripts (Sibley et al, 2015).",
    "molecules": "bicuculline"
  },
  {
    "caption": "(A) Heatmap of the IR-transcript expression values in control, bicuculline-stimulated and BDNF-stimulated conditions. IR-transcripts regulated in at least one condition are displayed (fold change of intron-retaining transcript expression ≥ 20% and |z-score| ≥ 1.5).",
    "molecules": "bicuculline"
  },
  {
    "caption": "RT-PCR validations of IR-transcripts , specifically regulated upon bicuculline stimulation (D), specifically regulated upon BDNF stimulation (E), and bi-directionally regulated (F). Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR.",
    "molecules": "bicuculline"
  },
  {
    "caption": "Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm.",
    "molecules": "MMS"
  },
  {
    "caption": "Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD.",
    "molecules": "PARP inhibitor, MMS"
  },
  {
    "caption": "Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.",
    "molecules": "MMS"
  },
  {
    "caption": "Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.",
    "molecules": "DNA"
  },
  {
    "caption": "IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.",
    "molecules": "MMS"
  },
  {
    "caption": "NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control.",
    "molecules": "4‐OHT"
  },
  {
    "caption": "Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet.",
    "molecules": "4‐OHT"
  },
  {
    "caption": "NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm.",
    "molecules": "4‐OHT, MMS"
  },
  {
    "caption": "SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.",
    "molecules": "4‐OHT"
  },
  {
    "caption": "SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.",
    "molecules": "4‐OHT"
  },
  {
    "caption": "Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h.",
    "molecules": "4‐OHT"
  },
  {
    "caption": "Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.",
    "molecules": "4‐OHT"
  },
  {
    "caption": "Intrauterine dwarfism observed upon NSMCE2 deletion on 17.5 dpc embryos. Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal injections of 4‐OHT. The image represents the difference in size observed between Nsmce2+/+ and Nsmce2lox/lox embryos carrying the UQ.CreERT2 transgene (Nsmce2+/+ and Nsmce2lox/lox, from now on).",
    "molecules": "4‐OHT"
  },
  {
    "caption": "Representative picture of Nsmce2+/+ and Nsmce2lox/lox animals fed for 40 weeks with a 4‐OHT‐containing diet (see also Movies EV1 and EV2).",
    "molecules": "4‐OHT"
  },
  {
    "caption": "A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control.",
    "molecules": "lipopolysaccharide, LPS"
  },
  {
    "caption": "B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells.",
    "molecules": "DNA"
  },
  {
    "caption": "E Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.",
    "molecules": "IdU, DNA, CldU"
  },
  {
    "caption": "SCE events per metaphase on B cells obtained from CD19+/Cre animals of the indicated genotypes, 72 h after being stimulated in vitro with LPS. **P 0.01, ***P 0.001.",
    "molecules": "LPS"
  },
  {
    "caption": "Analysis of forward (FSC) and side (SSC) scatter parameters of B cells obtained from CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with LPS. The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2.",
    "molecules": "LPS"
  },
  {
    "caption": "Representative images of B‐cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS. Insets provide a magnified view of an individual cell (arrowhead) for better comparison of the observed differences in cell size. Scale bar, 100 μm.",
    "molecules": "LPS"
  },
  {
    "caption": "Examples of the types of nuclei found on Blm and Nsmce2 double‐mutant B cells (compared with wild‐type B cells), in cells obtained from (E). DAPI was used to stain DNA. Whereas wt nuclei are regular in size, double‐mutant cells invariably presented enlarged, multilobulated, and irregular nuclei. Scale bar, 2.5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "E Confocal images showing that SPT5, NELFA, Pol II-S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1% 1,6-hexanediol (abbreviation as 1,6-hex) for 30 minutes. F Violin plot showing the number of cells containing nuclear puncta in Fig 1E. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. For SPT5, p = 0.1301. For NELFA, p = 0.2756. For Pol II-S5P, p = 0.8179. For AFF4, ***p < 0.0001. G",
    "molecules": "1,6-hex, 1,6-hexanediol"
  },
  {
    "caption": "B Confocal imaging of nascent RNA labeled by EU in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion.",
    "molecules": "EU, nascent RNA"
  },
  {
    "caption": "B Fluorescence microscopy images showing the phase-separated AFF4-IDR-eGFP and SPT5-CTR-mCherry droplets formed in 50 mM NaCl containing buffer in the presence of 10% PEG-8000. The concentration ratios are indicated as CAFF4-IDR/CSPT5-CTR. C Dot plot showing the droplet area in Fig 4B. Fields per condition n = 5. D",
    "molecules": "PEG, NaCl"
  },
  {
    "caption": "H Confocal images showing SPT5 in nuclear puncta in WT or ENL KO cells treatment with KL-1. I Violin plot showing the number of cells containing nuclear puncta in Fig 5H. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.",
    "molecules": "KL-1"
  },
  {
    "caption": "J-M (J) Confocal images showing co-localization of Pol II-S5P with SPT5 in nuclear puncta in KL-1 treated HCT 116 cells. Results shown are representative images from three independent experiments. (K) Boxplots showing the mean values of the Pearson correlation coefficient of co-localization ratios of SPT5 and Pol II-S5P in Fig 5J. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, **p = 0.0050. (L) Same as Fig 5J. but for SPT5 and NELFA. (M) Same as Fig 5K. but for SPT5 and NELFA. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, *p = 0.0100.",
    "molecules": "DMSO, KL-1"
  },
  {
    "caption": "B Fluorescence microscopy images showing that 2 μM SPT5-CTR-eGFP formed phase-separated droplets in the NaCl containing buffer. C Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. For droplet number, SPT5-CTR + P-TEFb (- ATP) vs. SPT5-CTR, p = **0.0015. SPT5-CTR + P-TEFb (+ vs.- ATP), **p = 0.0032. For droplet area, SPT5-CTR + P-TEFb (+ vs. - ATP)",
    "molecules": "ATP, NaCl"
  },
  {
    "caption": "E Fluorescence microscopy images showing the phase-separated droplets formed in 50 mM NaCl containing buffer with 10 μM AFF4-IDR-eGFP and 10 μM SPT5-CTR-mCherry or SPT5-CTR-TE-mCherry in the presence of 10% PEG-8000. F Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. AFF4-IDR + SPT5-CTR-TE vs. AFF4-IDR + SPT5-CTR, **p = 0.0029.",
    "molecules": "PEG, NaCl"
  },
  {
    "caption": "I. Total number of MB puncta normalized to axon length (µm). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 928 puncta, 61 axons (WT), 226 puncta, 15 axons (co-MO), 208 puncta, 35 axons (MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site.",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "M. Frequency (in percentage) of puncta colocalization between MB and cy5-pre-miR-181a-1 (#MB+/pre-miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 354 puncta, 32 axons (MB), 337 puncta, 32 axons (cy5-pre-miR-181a-1). n=5 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon.",
    "molecules": "cy5"
  },
  {
    "caption": "D. Frequency distribution (in percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta along the RGC axon shaft. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.",
    "molecules": "cy3"
  },
  {
    "caption": "E. Average velocity of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; antero, anterograde movement; retro, retrograde movement.",
    "molecules": "cy3"
  },
  {
    "caption": "F. MSD data for MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles were fitted with an anomalous diffusion model and α thus calculated (red). Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiment. Values are mean ± SEM. Abbreviations: MB, molecular beacon.",
    "molecules": "cy3"
  },
  {
    "caption": "G. MSD alpha coefficient distribution for each single MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particle. Each data point corresponds to one particle. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range.",
    "molecules": "cy3"
  },
  {
    "caption": "H. Relative frequency distribution (percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles. Each data point corresponds to one independent experiment. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.",
    "molecules": "cy3"
  },
  {
    "caption": "J. Representative kymographs before Nocodazole (top panel; - Noco) and 30 min after 2.4μM Noco bath-application (bottom panel; +Noco). Stationary puncta in both panels are indicated with black arrows. Scale bars: 5µm.",
    "molecules": "Noco, Nocodazole"
  },
  {
    "caption": "K. Frequency distribution (in percentage) of MB punctum speed. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco); 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.",
    "molecules": "Noco, nocodazole"
  },
  {
    "caption": "L. Frequency distribution of MB punctum speed. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco), 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.",
    "molecules": "Noco, nocodazole"
  },
  {
    "caption": "D. Frequency (in percentage) of CD63 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons, and independent experiments analyzed: 540 puncta, 23 axons, n=4 (CD63/Rab7a), 416 puncta, 18 axons, n=3 (CD63/LysoTracker), 584 puncta, 17 axons, n=2 (CD63/Lamp1), 649 puncta, 19 axons, n=3 (CD63/Rab5a). Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP.",
    "molecules": "LysoTracker"
  },
  {
    "caption": "E. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: cyan for pre-miR-181a-1 and yellow for Lamp1 or Rab5a. Abbreviations: Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bars: 5μm.",
    "molecules": "cy3, cy5"
  },
  {
    "caption": "F. Frequency (in percentage) of pre-miR-181a-1 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 825 puncta, 41 axons, n=8 (pre-miR-181a-1/CD63), 732 puncta, 33 axons, n=7 (pre-miR-181a-1/Rab7a), 681 puncta, 29 axons, n=5 (pre-miR-181a-1/LysoTracker), 501 puncta, 17 axons, n=2 (pre-miR-181a-1/Lamp1), 791 puncta, 25 axons, n=3 (pre-miR-181a-1/Rab5a). Values are median with interquartile range. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP, Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.",
    "molecules": "cy3, cy5, LysoTracker"
  },
  {
    "caption": "G. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63, cyan for pre-miR-181a-1, yellow for Rab7a and blue for LysoTracker. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bar: 5μm.",
    "molecules": "cy3, cy5, LysoTracker"
  },
  {
    "caption": "H. Frequency (in percentage) of pre-miR-181a-1 colocalization with vesicles double positive for organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 745 puncta, 23 axons, n=5 (CD63/pre-miR-181a-1/Rab7a), 577 puncta, 18 axons, n=3 (CD63/pre-miR-181a-1/LysoTracker). Values are median with interquartile range. Abbreviations: ns, not significant; CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.",
    "molecules": "cy3, cy5, LysoTracker"
  },
  {
    "caption": "I. Representative 3D-STED super-resolution image. Total number of puncta, axons and growth cones analyzed: 99 puncta, 16 axons and 10 growth cones (MB), 291 puncta, 36 axons and 15 growth cones (cy3-pre-miR-181a-1). n=2 (MB), n=3 (cy3-pre-miR-181a-1) independent experiments. Abbreviations: CD63, CD63-eGFP; pre-miR-181a-1, cy3-pre-miR-181a-1; MB; molecular beacon.",
    "molecules": "cy3"
  },
  {
    "caption": "Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-5p) (E) Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.",
    "molecules": "MO, morpholino, MOs"
  },
  {
    "caption": "Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-3p) (F). Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.",
    "molecules": "MO, morpholino, MOs"
  },
  {
    "caption": "B. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. C. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 45 brains (co-MO), 52 brains (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino. ",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "D. Schematic representation of the experimental paradigm. Concentrations used: 0.5μg/μL pCS2-mCherry or pCS2-eGFP plasmids; 250μM miR-181-MO or control MO cocktail; 50μM miR-181 or control mimics (top). Representative images of RGC axons within the optic tectum. White arrows indicate axons targeted both by MO (green) and miRNA mimics (red) (bottom). Abbreviations: co-MO, control morpholino. Scale bars: 20µm. E. Quantification of misprojecting axons. The number reported on the bars are the total number of co-electroporated axons. Note how miR-181 mimics rescued aberrant misprojection of morphant axons in vivo. Each data point corresponds to one independent experiment. Total number of axons and brains analyzed: 96 axons, 4 brains (co-MO+ctrl mimics), 101 axons, 4 brains (miR-181-MO + ctrl mimics), 134 axons, 4 brains (miR-181-MO+miR-181 mimics). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino; ns, not significant. ",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "F. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. G. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 21 brains (co-MO), 31 brains (miR-181-MO). n=3 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino. ",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "I. Frequency (in percentage) of the amount of time embryos spent on black background. Each data point corresponds to one embryo. Total number of embryos analyzed: 43 embryos (co-MO), 45 embryos (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 100µM cycloheximide Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). (B) 200ng/mL Sema3A was bath applied with or without 100µM cycloheximide (CHX, a translational blocker). Mutating TUBB3 3'UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 2µM co-MO or MOs-3p. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). isolated transfected axons (D) were used for FRAP analysis.",
    "molecules": "MO, MOs"
  },
  {
    "caption": "H,I. Representative growth cones depicting Venus fluorescence intensity as a heatmap. Dashed white lines delineate the growth cones. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III. Scale bars: 10µm.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "B. Schematic representation of the experimental paradigm. Representative images of local translation events of TUBB3 in the growth cone. Dashed white line delineates the growth cone. Each white arrow points to a puro-PLA puncta. Abbreviations: TUBB3, tubulin beta 3 class III; Puro, puromycin. Scale bars: 5 μm. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 30 (PBS), 36 (Sema3A). n= 2 independent experiments. Values are median with interquartile range.",
    "molecules": "puro, Puro, puromycin"
  },
  {
    "caption": "D. Schematic representation of the experimental paradigm. Concentrations used: 2 μM co-MO; 2 μM MOs-3p; 200ng/mL Sema3A. Representative images of local translation events of TUBB3 in the growth cone of transfected axons. Dashed white line delineates the growth cone. White arrows represent number of puro-PLA puncta per growth cone. Abbreviations: co-MO, control morpholino. Scale bars: 5 μm. E. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 32 (co-MO + PBS), 45 (co-MO + Sema3A), 27 (MOs-3p + Sema3A). n=2 independent experiments. Values are median with interquartile range. Abbreviations: co-MO, control morpholino; ns, not significant.",
    "molecules": "MO, morpholino, MOs, puro"
  },
  {
    "caption": "G. Percentage of collapsed growth cones in axons transfected with co-MO, MOs-3p + co-MO and MOs-3p + MOs-TUBB3. Each data point corresponds to one independent experiment. Total number of growth cones counted: 152 (co-MO), 155 (MOs-3p + coMO), 179 (MOs-3p + MOs-TUBB3). n= 3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; TUBB3, tubulin beta 3 class III.",
    "molecules": "MO, morpholino, MOs"
  },
  {
    "caption": "B, Immunofluorescence staining of single nuclei for H3K79me3 or H3K79me2 (green) and the pericentromeric heterochromatin marker HMGA1 (red). Upper panels, prometaphase; lower panels, interphase. DNA is stained with DAPI (blue). Scale bar, 2µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, Immunofluorescence staining of HP1β (green) in wild type and Dot1l KO mESCs. DNA is stained with DAPI (blue). Quantification of signal is shown at right. Each point represents one nucleus (Dot1l KO n=83 and control n=104 nuclei). Scale bar, 10µm. ***p<0.0001, Mann-Whitney U test.",
    "molecules": "DAPI"
  },
  {
    "caption": "H, Immunofluorescence staining of SMARCA5 (green) and HMGA1 (red) in mESCs, indicating localization of SMARCA5 to heterochromatic regions. Arrowheads indicate an example of SMARCA5 enrichment at PCH. DNA is stained with DAPI (blue). Scale bar, 4µm. Bottom, quantitation of fluorescence intensity in each channel along a line bisecting the nucleus.",
    "molecules": "DAPI"
  },
  {
    "caption": "L, Immunofluorescence staining of H3K79me3 (red) in mouse 3T3 fibroblasts transduced with a vector expressing GFP and a short hairpin against Smarca5 (shSmarca5) compared to untransduced control. Arrowheads indicate DAPI-dense chromocenters. Merge includes GFP channel for transduced cells. Scale bar, 10µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, Immunofluorescence staining of H3K79me3 (green) and H3S10P (red) in a single nucleus of control and Dot1l KO mESCs. DNA is stained with DAPI (blue). Scale bar, 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, Immunofluorescence staining of H3K79me3 (green) and H3K9me3 (red) in wild type mouse embryos during the first 48 hours of preimplantation development. Bottom images show an embryo treated with DOT1L inhibitor at 48 hours post fertilization (hpf). DNA is stained with DAPI (blue). Scale bars, 10 µm (main image) or 4 µm (inset). Quantitation of pixel-by-pixel fluorescence intensity for H3K9me3 and H3K79me3 for a representative embryo is shown at right. AU, arbitrary units.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, Quantitation of numbers of total, H3K9me3-positive, and H3K79me3-positive chromocenters in control and inhibitor-treated embryos at 48 hpf. Chromocenters were defined as DAPI-dense foci. Each point represents one nucleus. N=69 (control) or n=20 nuclei (inhibitor treated) in n=19 (control) or n=5 (inhibitor treated) embryos *p<0.05, ***p<0.001, two-tailed unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "(d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61β (green) and TagRFP-DFCP1 (red) were treated with 10 μM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61β) and impaired mitochondria (parkin) overlap. Scale bar, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin.",
    "molecules": "CCCP"
  },
  {
    "caption": "(f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each '+' marks the times measured in individual HeLa cells (the red '□' (square) denotes the average from five cells). P=0.0172 (Student's t-test).",
    "molecules": "Wortmannin"
  },
  {
    "caption": "(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β).",
    "molecules": "CCCP"
  },
  {
    "caption": "(d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.",
    "molecules": "MG-132"
  },
  {
    "caption": "Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted.",
    "molecules": "hemoglobin"
  },
  {
    "caption": " Protein abundance distribution of CSF showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled. ",
    "molecules": "glucose"
  },
  {
    "caption": " Protein abundance distribution of brain showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled. ",
    "molecules": "glucose"
  },
  {
    "caption": "(B) Immunoblot analysis of membrane fractions of HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) = 0.0011; p (DMSO vs isotype) = 0.992; p (isotype vs 4D9) = 0.0005; n.s., not significant.",
    "molecules": "DMSO, GM6001"
  },
  {
    "caption": "(C) Immunoblot analysis of conditioned media from HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) < 0.0001; p (DMSO vs isotype) = 0.6372; p (isotype vs 4D9) < 0.0001; n.s., not significant.",
    "molecules": "DMSO, GM6001"
  },
  {
    "caption": "(B) In vitro ADAM17 sheddase activity is blocked by 4D9-effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab and isotype control. Data represent the mean ± SEM (n=6). One-way ANOVA, Tukey's post hoc test; p (4D9 Fab vs 4D9 mAb) = 0.8855; p (4D9 Fab vs uncleaved) < 0.0001; p (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant.",
    "molecules": "FAM"
  },
  {
    "caption": "(E) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg/ml POPC/POPS liposomes and 20µg/ml 4D9 mAb, 4D9 Fab or isotype for 5 minutes. pSYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM (n=3). Two-way ANOVA, Tukey's post hoc test (cell line effect: F1,16=365.7, p=<0.0001; treatment effect: F3,16=39.35, p<0.0001; cell line x treatment effect: F3,16=38.75, p<0.0001); p (no Ab vs isotype) = 0.6218; p (no Ab vs 4D9 mAb) < 0.0001; p (no Ab vs 4D9 Fab) = 0.7301; p (isotype vs 4D9 mAb) < 0.0001; p (isotype vs 4D9 Fab) > 0.9999; p (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant.",
    "molecules": "POPC, POPS"
  },
  {
    "caption": "(D) Wildtype, Trem2+/-, and Trem2-/- BMDMs were plated in low concentration of M-CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM (n=2 and n=1, respectively).",
    "molecules": "ATP"
  },
  {
    "caption": "(B) Uptake assay for fluorescently labelled myelin, Aβ(1-42) and inactivated E.coli particles. The top row shows that myelin and Aβ (pseudocoloured in white) accumulate within the plasma membrane of primary microglia (labeled with dylight-649 isolectin and pseudocoloured in red). Overnight pre-treatment with antibody 4D9 significantly increased the percentage of substrate positive cells (middle row). Pre-treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E.coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar= 20µm. (C, D, E) Quantification of the change in uptake of myelin (C), Aβ(1-42) (D), and E.coli (E) upon antibody treatment relative to the uptake of the respective substrate in the absence of antibody. The number of substrate positive cells relative to the total number of cells was quantified in each condition. Data represent the mean ± SEM (n=3). Two-way ANOVA (myelin), Sidak's multiple comparisons test (time effect: F1,4=0.01123, p=0.9207; treatment effect: F1,4=27.98, p=0.0061; time x treatment effect: F1,4=0.5948, p=0.4836); p (1h) = 0.1362; p (2h) = 0.0185; unpaired t-test (two-tailed) with Welch's correction (Aβ(1-42) and E.coli); p (Aβ(1-42)) = 0.0151; p (E.coli) = 0.5751; n.s., not significant. ",
    "molecules": "dylight-649, Aβ, Aβ(1-42)"
  },
  {
    "caption": "(E) Confocal images of P2RY12 (red), IBA1 (green), and Aβ (gray) costainings from cortex. Top panel: Dotted white boxes indicate the area in P2RY12 and IBA1 costainings that are magnified as inset. Bottom panel: Insets show P2RY12, IBA1 and Aβ null at a larger magnification. Of note, we did not observe a complete co-localization of P2RY12 and null suggesting different microglial populations. Scale bar= 10µm; scale bar (insets) = 5µm.",
    "molecules": "Aβ"
  },
  {
    "caption": "(G) MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in (F). Mann-Whitney U test, p=0.026. (H) MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in (F). Unpaired t-test (two-tailed) with Welch's correction; p=0.0877. ",
    "molecules": "Aβ42"
  },
  {
    "caption": "A, Schematic representation of signaling events. Left: time course of changes in cAMP (dotted line) and cGMP (solid line); right: time course of changes in Vm (green), pHi (red); [Na+]i (orange), and [Ca2+]i (pink). Vertical dotted lines indicate time of stimulation.",
    "molecules": "cAMP, cGMP, Ca2+, Na+"
  },
  {
    "caption": "A, Time course of the change in voltage Vm evoked by a 2-ms light flash in sperm that was loaded with caged cGMP (15 μM) and recorded in 100KASW. Inset: extended timescale showing the rise of Vm. Fit of data (black) using two exponentials with time constants τrise = 17.4 ms and τrelax = 354 ms. Light flash in all panels is depicted as dashed magenta line.",
    "molecules": "cGMP"
  },
  {
    "caption": "B, Time course of the changes in Vm evoked by 100-ms flash in 100KASW using caged cGMP (blue) and caged 8-Br-cGMP (black). The flashes released 260 nM cGMP or 2,600 nM 8-Br-cGMP.",
    "molecules": "cGMP, 8-Br-cGMP"
  },
  {
    "caption": "C, Time course of changes in Vm in 100KASW evoked by 2-s flashes at different light energy that correspond to 440, 220, 75, and 8 nM s-1 of cGMP. Data was fitted using a heuristic model described by equation 3 (Methods section) (black lines). D, Amplitude (red) and decay time τrelax (black) for different light energies and release rates for experiments as shown in panel C. τrelax was determined using a single exponential fit. The linear fit of the Vm amplitude was restricted to low light levels; at higher levels, the Vm response saturates (see panel C). Points indicate mean ± s.d. from 5 experiments.",
    "molecules": "cGMP"
  },
  {
    "caption": "A, Time course of cGMP-evoked changes in Vm in 100KASW (red, 500 nM cGMP; pink, 9 nM cGMP) and normal ASW (blue, 500 nM cGMP; navy, 9 nM cGMP ). The time constants of recovery after cGMP release (9 nM) were 289 ms (depolarization) and 303 ms (hyperpolarization). Signals in panels were detected using the potentiometric probe FluoVolt; dashed magenta lines indicate the timing of the photolysis flash.",
    "molecules": "cGMP"
  },
  {
    "caption": ", Representative measurement of the dependence of recovery-time constant on the amount of released cGMP. Inset: decay time was constant for low stimulation levels equivalent to ≲ 30 cGMP molecules or ≤ 3 resact molecules.",
    "molecules": "cGMP"
  },
  {
    "caption": ", Voltage response evoked by photolysis of DEACM-caged cGMP (blue) or 8-Br-cGMP (red). The light energy was adjusted such that Vm response amplitudes became similar; for 8-Br-cGMP, about 5-times more light energy was required. Light pulse 10 ms.",
    "molecules": "DEACM, cGMP, 8-Br-cGMP"
  },
  {
    "caption": ", Vm responses evoked by cGMP in the presence (blue) and absence (red) of the HCN channel blocker ZD7288 (50 μM).",
    "molecules": "cGMP, ZD7288"
  },
  {
    "caption": "E, cGMP-evoked Vm response in ASW (black), in the presence of IBMX (1 mM) (red), and in Ca2+-free ASW ([Ca2+] ≤ 500 nM) in the presence of IBMX (1 mM) (blue). Signals were detected using the potentiometric probe Di-8-ANEPPS in the ratio mode. F, Normalized responses from panel E.",
    "molecules": "cGMP, IBMX, Ca2+"
  },
  {
    "caption": "A, B, Ca2+ signals elicited by cGMP release and recorded with FluoForte. The UV flash released 28 (A) and 215 B) cGMP molecules. Light flashes are indicated by dashed magenta lines.",
    "molecules": "cGMP, Ca2+"
  },
  {
    "caption": "C, pHi signals, elicited by the release of approximately 1,400 cGMP molecules, were recorded with pHrodo Red. An increase of pHi is indicated by a decrease of ∆F/F. Exemplary recording from a single cell. D, Average of pHi signals recorded from n = 25 cells. Error bars represent s.d.",
    "molecules": "cGMP"
  },
  {
    "caption": "B, Relationship between cGMP synthesis rate and free cGMP concentration. We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset: expanded scale.",
    "molecules": "cGMP"
  },
  {
    "caption": "DA uptakes assessed by nanoScanPET/MRI analysis using [18F]FP-CIT. n=9 (WT), 8 (cKO). *p<0.05, ***p<0.001.",
    "molecules": "[18F]FP-CIT, DA"
  },
  {
    "caption": "Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. WT and cKO mice were injected with MPTP (20mg/kg, i.p) for 5 days and behaviors of the animals were assessed for 1 month after the MPTP injection",
    "molecules": "MPTP, Parkinsonian toxin"
  },
  {
    "caption": "Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. The mouse brains at 1 month after initial MPTP exposure were subjected to immunohistochemical analyses for TH+ mDA neuron counts at SN (L) and DAT MFI at striatum (M).",
    "molecules": "MPTP, Parkinsonian toxin"
  },
  {
    "caption": "The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. General cell survival (calcein-AM)/death (EthD-1). (B) Cleaved caspase-3+ apoptotic cell death. (C) Cellular senescence (β-gal staining). (D) DNA damage estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (E) Mitochondrial ROS (MitoSox). (F) Mitochondrial membrane potential (MitoID).",
    "molecules": "MitoSox, calcein-AM, EthD-1"
  },
  {
    "caption": "Representative in vitro phase-contrast (A), TH/DAPI or TH (insets) (B), and TH/SYNAPSIN-stained (C) images in the cultures terminally differentiated from mutant (c13, c19) and corrected hiPSCs (c22, c59).",
    "molecules": "DAPI"
  },
  {
    "caption": "DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA levels were measured in the media conditioned in the differentiated cultures for 24 h (D15) and evoked by KCl-mediated depolarization for 30 min.",
    "molecules": "DA, KCl"
  },
  {
    "caption": "Mitochondrial ROS (MitoSox) were analyzed after terminal differentiation.",
    "molecules": "MitoSox"
  },
  {
    "caption": "Accumulation of α-synuclein oligomers analyzed by western blotting. The mutant (c13) and corrected (c22) neural stem cells (NSCs) were transduced with α-synuclein-expressing lentiviruses. At 6 days after differentiation, cells were extracted and fractionated to Triton X-100 (1%)-soluble and insoluble fractions. Shown is the representative WB image of three independent experiments. α-Synuclein oligomers are indicated with a bracket, while monomer bands are marked with an arrowhead.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Amphetamine-induced rotation scores. The ipsilateral net rotation values of individual animals transplanted with differentiated mutant (n=7) and corrected (n=6) pt-hiPSCs are depicted. Significant differences from the mutant cell-transplanted at each post-transplantation time point are at *p<0.05 or **p<0.001. Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05.",
    "molecules": "Amphetamine, DiR"
  },
  {
    "caption": "Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05. Total number of TH+ cells in the graft (TH+ numbers/mm3) per animal.",
    "molecules": "DiR"
  },
  {
    "caption": "LIN28A protein stability assays. HEK293 cells were transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G. Protein samples were prepared during 24 h of cycloheximide (50 μg/ml) treatment. Representative western blot data for LIN28A protein level changes normalized to β-actin protein levels (upper) or with normalization based on initial (0 h) LIN28A protein levels (lower). Band intensities were quantified using ImageJ, and normalized values are depicted (lower graph) (n=5).",
    "molecules": "cycloheximide"
  },
  {
    "caption": "The proteasome inhibitor MG132 treatment increases LIN28A(R192G) protein level. After serum starvation for 3 hours, HEK293T cells expressing LIN28A(R912G) were treated with or without MG132 (0.1-10 uM) for 30 minutes, and then followed by WB analyses.",
    "molecules": "MG132"
  },
  {
    "caption": "LC-MS/MS analysis. Immunoprecipitated FLAG-LIN28A WT was loaded on an SDS gel (left upper). Bold red sequences indicate peptides matching LIN28A (left lower). Right, LC-MS/MS spectra of WT LIN28A.",
    "molecules": "SDS"
  },
  {
    "caption": "Cytoplasmic localization of WT LIN28A and LIN28A(R192G) proteins. HeLa cells transfected with pFLAG-WT-LIN28 and pFLAG-LIN28A(R192G) were stained with FLAG antibody and counterstained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Macrophages were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Immunoblot of co-immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β-actin as loading control.",
    "molecules": "CL097, rapamycin"
  },
  {
    "caption": "(B) Cells were incubated with 10 µg/mL pepstatin A for 4 h prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.",
    "molecules": "pepstatin A"
  },
  {
    "caption": "Macrophages transduced with non-specific scrambled shRNA (shNS) or TLR8 shRNA (shTLR8) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Cells were lysed and immunoblot performed with antibodies to TLR8 and β-actin.",
    "molecules": "CL097, rapamycin"
  },
  {
    "caption": "A-B) Macrophages were treated for 24 h with CL097, ssRNA40 or ssRNA41. (A) Top, qRT-PCR for CYP27B1 after 6 h. Bottom, immunoblots of CYP27B1 using antibody to CYP27B1 or β-actin.",
    "molecules": "CL097"
  },
  {
    "caption": "(C-D) Macrophages transduced with non-specific scrambled shRNA (shNS), CYP27B1 shRNA (shCYP27B1) or VDR shRNA (shVDR) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (C) Left, immunoblots performed with antibodies to CYP27B1, VDR and β-actin. Right, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.",
    "molecules": "CL097, rapamycin"
  },
  {
    "caption": "(E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or β-actin. Bar charts are reported as mean ± s.e.m., n = 4. * p<0.05.",
    "molecules": "25D3, CL097, rapamycin"
  },
  {
    "caption": "Macrophages transduced with non-specific scrambled shRNA (shNS) or CEBPB shRNA (shCEBPB) were treated for 24 h with CL097, ssRNA40, or ssRNA41. (A) Cells were lysed and immunoblot performed with antibodies to CEBPB and β-actin.",
    "molecules": "CL097"
  },
  {
    "caption": "TLR8 agonists inhibit HIV replication.Macrophages were incubated with (A) ssRNA40 or (B) CL097 for 24 h before infection with HIV for 3 h. Cells were then washed and incubated with ssRNA40 or CL097 for 10 d. Extracellular release of HIV p24 antigen into the cell supernatant at days 0, 3, 5, 7, 10 was detected by ELISA. Results are reported as mean ± s.e.m., n = 5. * p<0.05.",
    "molecules": "CL097"
  },
  {
    "caption": "(A) Macrophages transduced with non-specific scrambled shRNA (shNS), TLR8 shRNA (shTLR8), CYP27B1 shRNA (shCYP27B1), or VDR shRNA (shVDR) were treated with CL097 for 24 h before infection with HIV with continuous CL097 treatment. Top, immunoblot analysis performed using antibodies raised to TLR8, CYP27B1, VDR and β-actin. Bottom, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4.",
    "molecules": "CL097"
  },
  {
    "caption": "(B) Macrophages were incubated with ssRNA40 or CL097 in the presence of 45 or 100 nmol/L 25D3 for 24 h before infection with HIV. Cells were then washed and incubated with 25D3 and ssRNA40 or CL097 for 10 d Extracellular release of HIV p24 antigen was detected by ELISA. Results are reported as mean ± s.e.m., n = 6. * p<0.05.",
    "molecules": "25D3, CL097"
  },
  {
    "caption": "(B) were incubated with CL097 for 24 h before infection with HIV with continuous CL097 treatment for 10 d. Right, immunoblot analysis performed using antibodies raised to BECN1, ATG5 and β-actin. Left, ELISA performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 4.",
    "molecules": "CL097"
  },
  {
    "caption": "(C) Macrophages were pretreated with bafilomycin A1 (Baf A1), SID 26681509 (SID) or vehicle control before incubation with CL097 or ssRNA40 for 24 h before infection with HIV. Cells were then washed and incubated with TLR8 agonists and inhibitors for 10 d and ELISA performed for extracellular HIV p24 antigen release over 10 d. Results are reported as mean ± s.e.m., n = 5. * p<0.05; # p<0.001.",
    "molecules": "bafilomycin A1, SID 26681509, CL097"
  },
  {
    "caption": "(A) Macrophages were treated with CL097, ssRNA40, ssRNA41 for 6 h after which qRT-PCR for CAMP was performed. Results are reported as mean ± s.e.m., n = 4.",
    "molecules": "CL097"
  },
  {
    "caption": "(B) Macrophages were treated for 24 h with CL097 or ssRNA40 in the presence of 45 nmol/L or 100 nmol/L 25D3. Left, qRT-PCR for CAMP performed after 6 h. Results are reported as mean ± s.e.m., n = 4. Right, after 24 h cells were stained with anti-CAMP antibodies and analyzed by flow cytometry. Representative histograms from three donors are shown.",
    "molecules": "25D3, CL097"
  },
  {
    "caption": "(C) Macrophages transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) were incubated with CL097, rapamycin (Rapa) or vehicle control for 24 h. Top, immunoblot analysis performed using antibodies raised to CAMP and β-actin. Bottom, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.",
    "molecules": "CL097, rapamycin"
  },
  {
    "caption": "(D) After 24 h treatment with CL097 or ssRNA40, macrophages from part C were also washed and infected with HIV and incubated with CL097, ssRNA40 or vehicle control for 10 d. ELISA was performed for extracellular release of HIV p24 antigen over 10 d. Results are reported as mean ± s.e.m., n = 6. * p<0.05.",
    "molecules": "CL097"
  },
  {
    "caption": "Coomassie Blue staining of recombinant human DDX5 purified in bacteria. M denotes the molecular mass markers in kDa.",
    "molecules": "Coomassie Blue"
  },
  {
    "caption": "U2OS cells transfected with siCTL, or siDDX5 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The gene location and genomic qPCR amplification region are shown at the top of each panel. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI. The identified R-loop peaks were extracted from the R-loop database (R-loop DB) for each region. The bar graphs are the average and S.E.M. from 3 independent experiments. Statistical significance was assessed using t-test. *: p < 0.05.",
    "molecules": "IgG"
  },
  {
    "caption": "U2OS cells were lysed and the lysates were incubated with anti-PRMT5 antibody or immunoglobulin G (IgG) as control followed by protein A Sepharose bead addition. The immunoprecipitated (IP) proteins or whole cell extract (WCE) were separated by SDS-PAGE and Western blotted (WB) with the indicated antibodies. Same as panel (A) except anti-DDX5 antibody was used instead of anti-PRMT5 antibody for immunoprecipitation.",
    "molecules": "IgG, immunoglobulin G"
  },
  {
    "caption": "HEK293 cells transfected with Flag-DDX5 were lysed and immunoprecipitated with anti-Flag antibodies. The bound proteins were along with WCE from untransfected (-) and Flag-DDX5 (+) transfected cells were Western blotted with anti-FLAG, anti-methylarginine antibodies (MeR).",
    "molecules": "MeR, methylarginine"
  },
  {
    "caption": "Representative images of S9.6 staining in siCTL, siDDX5, or siPRMT5 knockdown at transcription on (TA-KR) or off (TetR-KR) genomic loci in U2OS TRE cells. The nuclei are visualized by DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies.",
    "molecules": "IgG"
  },
  {
    "caption": "Coomassie blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti-XRN2 antibodies. The bound proteins were separated by SDS-PAGE followed by Western blotting with anti-DDX5 or anti-XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow.",
    "molecules": "IgG, immunoglobulin G"
  },
  {
    "caption": "Untransfected and U2OS stably expressing Flag-DDX5 WT or Flag-DDX5-RK were subjected to immunoprecipitation with anti-Flag antibody. The WCE and the anti-Flag immunoprecipitated proteins were Western blotted with anti-monomethylarginine (MeR), anti-Flag and anti-XRN2 antibodies, respectively. M denotes molecular mass markers in kDa.",
    "molecules": "MeR, monomethylarginine"
  },
  {
    "caption": "U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.",
    "molecules": "IgG"
  },
  {
    "caption": "HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.",
    "molecules": "IgG"
  },
  {
    "caption": "D Immunofluorescence  images of Cdk5rap2  WT, HET and null erythroid progenitors  isolated from fetal livers. Progenitors  were stained  for CDK5RAP2 (grey), γ-tubulin (magenta) and DNA   (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 3 μm. Insets show higher magnification of centrosomes. Scale bar, 1 μm. E Quantification of mean centrosomal  signal intensities of CDK5RAP2  and γ-tubulin from (D). Numbers in brackets correspond to number of embryos  analyzed with a total number of 470 (WT), 406 (HET) and 379 (null) progenitors. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos  E, Statistical significances were determined by One-way ANOVA test with Tukey's E, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "Hoechst"
  },
  {
    "caption": "I ImageStream images of ex vivo cultured enucleating EBs    and reticulocytes. Cells were stained  for TER119  (erythroid  marker, green) and DNA  (Hoechst, blue). BF: brightfield. Scale bar, 5 μm. J Quantification of enucleating EBs    and reticulocytes  after 48 hours (T48) in ex vivo culture. Genotypes are as indicated. The numbers in brackets correspond to the number of embryos  analyzed. K Quantification of enucleating EB   size from (I). The numbers in brackets refer to the number of embryos  analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos  I and J) Statistical significances were determined by One-way ANOVA test with Tukey's J) or Kruskal Wallis test with Dunn's (K) multiple comparisons.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": "E ImageStream images showing EBs  enucleating with 2N or 4N DNA    content at different stages from (B). EBs  were stained  for DNA  (Hoechst, blue) and TER119  (erythroid  marker, green). Scale bar, 5 μm. BF: brightfield. F Quantification of  enucleation stages  according to DNA  content from (B). 4 (WT), 14 (HET) and 8 (null) embryos  were analyzed. Data information: Bar graphs display mean ± s.d. All statistical analysis were based on the number of embryos. Statistical significance was determined by Two-way ANOVA with Tukey's (F) multiple comparisons. **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": "B Immunofluorescence  images of untreated (untx) or CB-treated  erythroid progenitors at stated differentiation stages after 24 (T24) and 48 (T48) hours in ex vivo culture. Cells were stained  for  γ-tubulin  (magenta), TER119  (erythroid  marker, green) and DNA  (Hoechst, blue). Scale bar, 3 μm.",
    "molecules": "Hoechst, CB, DNA"
  },
  {
    "caption": "C Quantification of centrosome  numbers (marked by  γ-tubulin) upon CB  treatment from (B). For the left panel, in total 632 (untx, T24), 637 (CB, T24), 544 (untx, T48) and 467 (CB, T48) cells from two litters were analyzed. T24 = 24 hours. T48 = 48 hours. For the right panel, in total 799 (untx, non-enucl), 142 (untx, enucl), 235 (untx, RetiC), 839 (CB, non-enucl), 127 (CB, enucl) and 131 (CB, RetiC) cells from two litters were analysed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "CB"
  },
  {
    "caption": "A Immunofluorescence  images of untreated (untx) or centrinone-B (CB)-treated cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained  for α-tubulin  (magenta), γ-tubulin (grey), phospho-Histone H3 (pHH3, green) and DNA  (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. B Quantification of mitotic spindle morphologies in untreated (untx) or centrinone-B (CB)-treated cells at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle  phenotypes. In total 315 (untx, T24), 367 (CB, T24), 295 (untx, T36), 282 (CB, T36) cells were analyzed from 3 litters. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (B) Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test (B, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "Hoechst, CB, centrinone-B, DNA"
  },
  {
    "caption": "C Immunofluorescence  images of  Cdk5rap2HET and Cdk5rap2null cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained  for α-tubulin (magenta),  γ-tubulin (grey), pHH3 (green) and DNA  (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. D Quantification of  mitotic spindle morphology at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle  phenotypes. At T24, 3  Cdk5rap2WT (323 cells), 3 Cdk5rap2HET (296 cells) and 2  Cdk5rap2null (197 cells) embryos  were analyzed. At T36, 5 Cdk5rap2WT (387 cells), 6 Cdk5rap2HET (384 cells) and 3 Cdk5rap2null (202 cells) embryos were analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos  (D, Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test D, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": "E Schematic (left) shows experimental outline for nocodazole  treatment of ex vivo cultured erythroid progenitors at indicated time points. Representative ImageStream images of ex vivo cultured cells at different mitotic stages (right). Cells were stained  for phospho-Histone H3  (pHH3, green), TER119  (erythroid  marker, magenta) and DNA   (Hoechst, blue). BF: brightfield. Scale bar, 10 μm. F and G Quantification of pHH3pos, TER119pos cells and mitotic stages (see text for details) after 36 hours (T36) upon DMSO  or nocodazole  treatment according to (E) using ImageStream. Number of embryos  analyzed is shown in brackets. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos  F and G). Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test F and G).*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "Hoechst, DNA, DMSO, nocodazole"
  },
  {
    "caption": "B Immunofluorescence  images of cells in interphase  (I) and mitosis  (M) at 24 (T24) and 36 (T36) hours of ex vivo culture. Cells were stained  for  γ-tubulin  (magenta), protein of interest (POI, CDK5RAP2, PCNT  or CEP192  in grey) and DNA   (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. C Quantification of mean centrosomal  signal intensities of PCM  proteins from (B). Three litters were analyzed with the following number of cells for I, T24; M, T24; I, T36 and M, T36: 290, 157, 259 and 174 (CDK5RAP2); 291, 190, 203 and 181 (PCNT); 314, 122, 283 and 152 (CEP192); 895, 465, 720 and 493 (γ-tubulin). Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": "G Quantification of total cholesterol levels in cell lysates from WT, P301L and Tau KO cells. Data information: (G Data are presented as mean ±SEM of n = 12-18 replicates per group (4 independent experiments) in percentage of the WT cells. * P<0.05, ***P<0.001; One-way ANOVA + Tukey's post hoc test.",
    "molecules": "cholesterol"
  },
  {
    "caption": "K-N Mitochondrial cholesterol levels (K, L) and pregnenolone levels (M, N) in WT, P301L and Tau KO cells (K, M) and in brain preparations from WT and pR5 mice (L, N). Data information: K, M) Data are presented as mean ±SEM of n = 12-18 replicates per group (4 independent experiments) in percentage of the WT cells. * P<0.05, ***P<0.001; One-way ANOVA + Tukey's post hoc test. L, N) Data are presented as mean ±SEM of n = 6 WT and n = 5 pR5 mice with 2 technical replicates per animal. * P<0.05, ** P<0.01, Student unpaired t-test.",
    "molecules": "cholesterol, pregnenolone"
  },
  {
    "caption": "O-Q Quantification of total cholesterol levels (O), mitochondrial cholesterol levels (P) and pregnenolone levels (Q) in patient-derived induced pluripotent stem cells (iPSC) bearing a P301L tau mutation (iPSC-P301L) and the corresponding isogenic wild-type cells (iPSC-WT). (O-Q) Data are presented as mean ±SEM of n = 27 replicates/group (O), n = 9 replicates/group (P) and n = 18 replicates/group (Q). * P<0.05, *** P<0.001, Student unpaired t-test.",
    "molecules": "cholesterol, pregnenolone"
  },
  {
    "caption": "(A Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green and (A) TOMM20 (mitochondria), in red with xy- and xz-axis orthogonal views framing the corresponding image. (B-C Manders' coefficients M1 representing the proportion of Filipin III overlapping with (B) TOMM20 M2 representing the proportion of (C) TOMM20 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm TOMM20: translocase of the outer mitochondrial membrane complex subunit 20.",
    "molecules": "cholesterol, Filipin III"
  },
  {
    "caption": "D, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (D) ER-RFP in red with xy- and xz-axis orthogonal views framing the corresponding image. E-F Manders' coefficients M1 representing the proportion of Filipin III overlapping with (E) ER-RFP M2 representing the proportion of (F) ER-RFP overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm ER-RFP: endoplasmic reticulum red fluorescent protein",
    "molecules": "cholesterol, Filipin III"
  },
  {
    "caption": "G, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (G) GM130 (Golgi apparatus) in red with xy- and xz-axis orthogonal views framing the corresponding image. H-I, Manders' coefficients M1 representing the proportion of Filipin III overlapping with (H) GM130 M2 representing the proportion of (I) GM130 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm GM130: Golgi matrix protein 130",
    "molecules": "cholesterol, Filipin III"
  },
  {
    "caption": "J, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green , (J) EEA1 (endosomes) in red with xy- and xz-axis orthogonal views framing the corresponding image. Manders' coefficients M1 representing the proportion of Filipin III overlapping with (K) EEA1 M2 representing the proportion of (L) EEA1 overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm EEA1. early endosome antigen 1",
    "molecules": "cholesterol, Filipin III"
  },
  {
    "caption": "M, Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (M) LipidSpot (lipid droplets) in red with xy- and xz-axis orthogonal views framing the corresponding image. N-O (N) LipidSpot M2 representing the proportion of (O) LipidSpot overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm",
    "molecules": "LipidSpot, cholesterol, Filipin III"
  },
  {
    "caption": "P) Representative microscopy images (z-projections) of co-staining with Filipin III (cholesterol) in green (P) M6PR (lysosomes) in red with xy- and xz-axis orthogonal views framing the corresponding image. Q-R) Manders' coefficients M1 representing the proportion of Filipin III overlapping with (Q) M6PR, and M2 representing the proportion of (R) M6PR overlapping with Filipin III in wild-type (WT) versus P301L cells. Data information: Data are presented as mean ±SEM (n=40-90 images per group, 3-4 independent experiments). * P<0.05, ** P<0.01, *** P<0.001; Student unpaired t-test. White scale bars: 10 µm",
    "molecules": "cholesterol, Filipin III"
  },
  {
    "caption": "C-D Representative microscopy images (z-projections) of co-staining with Filipin III in green and (C) TOMM20 (mitochondria) or (D) ER-RFP (endoplasmic reticulum) in red with xy- and xz-axis orthogonal views framing the corresponding image. White scale bars: 10 µm Data information: * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20",
    "molecules": "Filipin III"
  },
  {
    "caption": "E-H Manders' coefficient M1 representing the proportion of Filipin III overlapping with (E) TOMM20 or (G) ER-RFP, and M2 representing the proportion of (F) TOMM20 or (H) ER-RFP, overlapping with Filipin III. In total, 20-60 cells were analyzed per group (4 independent experiments). Data information: E-J) Data are presented as mean ± SEM. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein; TOMM20: translocase of the outer mitochondrial membrane complex subunit 20;",
    "molecules": "Filipin III"
  },
  {
    "caption": "A-D (A, C) Representative microscopy images (z-projections) of tau phosphorylation using (A) AT8 and (C) AT180 antibodies (in red) in GFP-tagged P301L cells (GFP signal in gray) treated for 24 h with CHIR99021 (CHIR) (right) and untreated cells (left). White scale bars: 25 µm. (B, D) Quantification of (B) AT8 and (D) AT180 staining fluorescence intensity. Data are presented as mean ±SEM of n = 50-60 cells per group (4 independent experiments). *** P<0.001; Student unpaired t-test. Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells.",
    "molecules": "CHIR, CHIR99021"
  },
  {
    "caption": "E Visualization of the VAPB-PTPIP51 interactions by in situ proximity ligation assay (PLA) in untreated wild-type (WT) cells and P301L cells as well as in CHIR-treated cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. White scale bars: 20 µm F Quantitative analysis of the PLA signal represented as number of contact points between VAPB-PTPIP51 per cell in percentage of the WT cells. In total, 60 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B.",
    "molecules": "CHIR"
  },
  {
    "caption": "G-H Representative microscopy images (z-projections) of co-staining with Filipin III in green and (G) TOMM20 (mitochondria) or (H) ER-RFP (endoplasmic reticulum) in red with xy- and xz-axis orthogonal views framing the corresponding image. White scale bars: 10 µm Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20",
    "molecules": "Filipin III"
  },
  {
    "caption": "I-L Manders' coefficients M1 representing the proportion of Filipin III overlapping with (I) TOMM20 or (K) ER-RFP, and M2 representing the proportion of (J) TOMM20 or (L) ER-RFP, overlapping with Filipin III in untreated WT cells and P301L cells as well as in CHIR-treated cells. In total, 35-75 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20;",
    "molecules": "CHIR, Filipin III"
  },
  {
    "caption": "A Scheme of the lentiviral vector with inducible FlagOmomyc (top) and immunoblotting (bottom) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0-48 hours.",
    "molecules": "Dox"
  },
  {
    "caption": "B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0-96 h (n = 3; mean ± SD). Viable cells were counted using a haemocytometer.",
    "molecules": "Dox"
  },
  {
    "caption": "C-D Self-renewal assay upon Dox treatment. C: histograms showing the percentage of cells capable of re-forming a neurosphere seven days after dissociation (n = 3; mean ± SD). D: representative micrographs of BT168FO cell neurospheres.",
    "molecules": "Dox"
  },
  {
    "caption": "E qRT-PCR of proliferation, stem cell and differentiation markers (CCND1, PTEN, SOX2, NOTCH1, NESTIN, MYC) in BT168FO cells after 48 h Dox treatment, compared to uninduced cells (n = 3; mean ± SD). Expression levels in non-induced cells were set as 1.",
    "molecules": "Dox"
  },
  {
    "caption": "F. Transwell migration assay of BT168FO cells after 3 days with or without Dox (n = 3; mean ± SD). 10 fields were counted per assay.",
    "molecules": "Dox"
  },
  {
    "caption": "G. Immunofluorescence analyses of GSC differentiation. To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days. The upper panel displays immunofluorescence images of NESTIN, GFAP, ß-tubulin III, SOX2, and FlagOmomyc expression. FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels, while inducing the onset of ß-tubulin III. The lower panel shows the percentage of positive cells for each cell marker evaluated (n = 4; mean ± SD). 16 fields for each assay were examined; scale bar = 100 μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in BT168FO (A) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.",
    "molecules": "Dox"
  },
  {
    "caption": "A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in U87FO (B) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.",
    "molecules": "Dox"
  },
  {
    "caption": "A Heatmap of RNA Pol II levels around TSSs of MYC promoter-target genes in BT168FO cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in tags/50bp.",
    "molecules": "Dox"
  },
  {
    "caption": "B Tracks of Pol II binding signals (ChIP-seq) at NCL, miR-17-92, OLIG2, HDAC1, and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h. The y-axis shows Pol II binding signals as tags/500bp/million reads. The x-axis displays genomic positions; introns as lines, exons as boxes. Arrowheads denote direction of transcription. Blue and green bars denote the presence of MYC or Omomyc peaks. Grey boxes are TSS regions.",
    "molecules": "Dox"
  },
  {
    "caption": "E RNA Polymerase II traveling ratio distribution from the Pol II ChIP-seqs, in the presence or absence of Dox.",
    "molecules": "Dox"
  },
  {
    "caption": "A Promoter occupancy by MYC correlates with transcript levels in cells untreated with doxycycline (black line). In doxycycline treated cells promoter occupancy by MYC is lower, and no longer increases together with transcript levels. The scatter plot displays MYCChIP-seq reads at promoters (-1000, +100 regions with respect to TSS) in BT168FO cells - untreated (black) or upon 48 h Dox treatment (blue) - versus transcript levels (FPKM from RNA-seq data).",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "B Distribution of transcript level differences (Δ FPKM) of single genes classified as promoter-targets (green), intragenic-targets (blue), intergenic-targets (red) in Dox treated (24 h) versus untreated cells, plotted against expression in untreated cells (n=3). Δ FPKM = FPKM_24h Dox - FPKM_no Dox.",
    "molecules": "Dox"
  },
  {
    "caption": "C The table first row shows the number of genes significantly up or down regulated at 24 h of Dox treatment, subdivided in promoter-, intragenic-, intergenic-targets (± 50 kbp from TSS), and the rest. The following rows depict - for each gene class - mean (average) values of FPKM in cells treated or not with Dox for 24 h and the corresponding Δ FPKM and Fold change (Fc). Data are from 3 independent biological replicates.",
    "molecules": "Dox"
  },
  {
    "caption": "D Transcript level distribution of MYC promoter-targets significantly down (red dots) or up (green dots) modulated in cells treated with Dox for 24 h vs. untreated cells. Transcript levels, expressed as FPKM, represent averages of three independent experiments.",
    "molecules": "Dox"
  },
  {
    "caption": "A RNA-seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction (n = 3 independent biological replicates). The block on the left represents DUSP (dual specificity phosphatase) family genes. The middle block contains transcription factors (TFs): the upper thirteen (ASCL1-SALL1) are GSC-specific and the remaining ones are oncogenic. The right block contains genes, including well-known MYC targets, with functions in proliferation, neurodifferentiation, and gliomagenesis. The first column of each block represents the average expression (log2 FPKM) in untreated cells (0 h) in the colour scale illustrated by the lower bar: violet indicates low and blue high expression. The other columns depict relative expression versus untreated cells (average log2 FC) at different times (8-48h) of Dox treatment, according to the scale shown by the upper bar: red indicating low and green high expression. The table in the upper left summarizes the GSEA score of the set 19 GSC-specific transcription factors [40], showing a significant downregulation by Omomyc.",
    "molecules": "Dox"
  },
  {
    "caption": "B Immunoblots of EGFR, ZEB1 and GAPDH loading control expression in BT168FO and BT275FO after 2 days with (+) or without (-) Dox. Representative images are shown.",
    "molecules": "Dox"
  },
  {
    "caption": "C RT-qPCR expression analysis of selected miRs in U87FO cells grown 48 h in the presence (+) or absence (-) of Dox (n = 3; mean ± SD). Expression levels were normalized using U6RNA as control. Expression levels in non-induced cells were set as 1.",
    "molecules": "Dox"
  },
  {
    "caption": "E EGFR, ZEB1, and GAPDHimmunoblots in uninduced U87FO cells (-) and in cells transfected with LNA oligos targeting miR-200a-200b-429 (LNA 200) and miR-146b (LNA 146b), or with a non-targeting LNA (C), and induced with Dox for 48 h (+Dox). Representative images are shown.",
    "molecules": "Dox, LNA"
  },
  {
    "caption": "F MTS cell proliferation assay of uninduced or Dox induced U87FO cells, transfected with either a non-targeting LNA (LNA C), or LNA oligos targeting the miR-200a-200b-429 cluster (LNA 200), miR-146b (LNA 146b), or both (LNAmix) and analysed at 24, 48 and 72h (n = 3; mean ± SD).",
    "molecules": "Dox, LNA"
  },
  {
    "caption": "G Migration assay of U87FO cells transfected with non-targeting control LNA oligos (LNA C) or with LNA oligos targeting miR-200a-200b-429 (LNA 200); cells were either untreated (-) or treated with Dox for 24 h (+Dox); (n = 3; mean ± SD).",
    "molecules": "Dox, LNA"
  },
  {
    "caption": "B C10M reduces binding of pCRP to immobilized PC. pCRP was incubated with p-aminophenyl phosphoryl choline agarose beads under binding conditions with and without C10M. Porous solid column chromatography was then used to evaluate the binding capacity of pCRP to PC. Biological replicates, n=3, mean ± SEM. P value was calculated with Student's t-test.",
    "molecules": "p-aminophenyl phosphoryl choline, agarose, PC, C10M"
  },
  {
    "caption": "A Flow cytometry was used to determine binding of pCRP to activated platelets. P-selectin positive platelets were analyzed for CRP-Atto 594 fluorescence. Platelets were incubated without labeled pCRP (control), and with labeled pCRP (50 μg/ml) ± C10M (1:100 molar ratio). Results are displayed as mean ± SEM. P values were calculated with ANOVA and Tukey's post hoc test. Biological replicate, n=3.",
    "molecules": "Atto 594, C10M"
  },
  {
    "caption": "B Confocal fluorescence microscopy of ADP-activated human platelets in the presence of pCRP. Depicted are 3D reconstructions of monolayers of ADP (20 μM) activated platelets. Washed platelets were detected by anti-CD62P (green). 50 μg/ml pCRP-Atto 594 (red) was incubated with isolated platelets with (bottom) or without (top) C10M. pCRP localizes on platelets, which can be inhibited by C10M.",
    "molecules": "Atto 594, ADP, C10M"
  },
  {
    "caption": "C Western blot of pCRP binding to activated platelets. Platelets bind less pCRP when incubated with C10M as detected with the anti-CRP antibody, compared to the anti-GAPDH antibody as a control. Washed human platelets were incubated with pCRP and pCRP+C10M, respectively, lysed and separated on SDS-PAGE. D Densitometric quantification of protein bands of Western blots (biological replicates, n=3) from the experiment described in (C). P value was calculated by Student's t-test. Graph shows mean ± SEM.",
    "molecules": "C10M"
  },
  {
    "caption": "A CRP-coated platelets visualized by confocal microscopy bound to whole blood leukocytes. Washed platelets (green) were incubated in serum supplemented with 50 μg/ml pCRP-Atto 594 (red), without (left) and with C10M (right, 1:100 molar ratio). Platelets were then added to whole blood samples and imaged after RBC lysis. Shown are platelet-leukocyte-aggregates with and without CRP attached.",
    "molecules": "Atto 594, C10M"
  },
  {
    "caption": "D Static adhesion of monocytes to 20 μg/ml fibrinogen was tested as described before. Purified monocytes (3 x 106 per ml) were stimulated with 50 μg/ml pCRP and 20 μM ADP and allowed to adhere for 35 min. ADP was used to activate supplemented platelets. Monocyte adhesion was quantified by calorimetric method and absorbance measured after 30 min.",
    "molecules": "ADP"
  },
  {
    "caption": "E Expression of TNF, IL1β and IL6 (from left to right) in monocytes analyzed by flow cytometry. Addition of C10M to the whole blood samples inhibits the CRP-dependent expression of pro-inflammatory cytokines. P values were calculated with ANOVA and Tukey's post-hoc test. Biological replicates, n=6 (IL6), n=5 (TNF) and n=4 (IL1β), respectively,",
    "molecules": "C10M"
  },
  {
    "caption": "F Representative examples of whole blood samples treated with pCRP and C10M visualized by confocal fluorescence microscopy. Depicted are 3D reconstructions multiple focal planes at 20x magnification. The leukocytes were stained with DAPI (blue) and anti-TNF-FITC (green).",
    "molecules": "DAPI, FITC, C10M"
  },
  {
    "caption": "A Confocal fluorescence microscopy of ADP-activated platelets bound to HUVEC mono cell layers. pCRP-Atto 594 (depicted in red) was added and incubated with and without C10M. Anti-CD62P-FITC antibody was used to detect the platelets (green). HUVEC nuclei were counterstained with DAPI (blue). pCRP colocalizes with platelets on the endothelial cells. C10M inhibits CRP binding to activated platelets. Scale bar 50 µm.",
    "molecules": "Atto 594, ADP, DAPI, FITC, C10M"
  },
  {
    "caption": "B,C Quantification of ICAM-1 (B) and VCAM-1 (C) expression on pCRP*/mCRP-activated HUVECs. ICAM-1 and VCAM-1 expression were measured by flow cytometry. ADP-stimulated platelets were added to each sample (except for "Control" and "pCRP") and served as activated cell membranes for pCRP dissociation to pCRP*/mCRP. C10M inhibits the generation of pCRP*/mCRP and thereby reduces the expression of ICAM-1 and VCAM-1. Mean fluorescence intensity (MFI) results in flow cytometry are shown with results normalized to control,",
    "molecules": "ADP, C10M"
  },
  {
    "caption": "D,E Expression of integrin subunit αM (CD11b) in neutrophils (D) and CD14+ monocytes (E) was accessed by flow cytometry as described previously (Kiefer et al, 2021). Human whole blood was incubated with 25 µg/ml pCRP, 20 µM ADP and C10M (molar ratio 1:100, pCRP:C10M), respectively. CD11b expression was analyzed by flow cytometry in neutrophils (CD16+, SSC high) and monocytes (CD14+, SSC low). Shown are scatter plots of MFI results in flow cytometry with results normalized to control,",
    "molecules": "ADP, C10M"
  },
  {
    "caption": "F,G ROS generation in whole blood detected in CD14+ monocytes (F) and neutrophils (G) by redox-indicator dihydroethidium (DHE; 10 µg/ml). Blood samples incubated for 3 hours at 37 °C, 5% CO2 with 50 µg/ml pCRP and mCRP, 20 µM ADP and C10M (molar ratio 1:100, pCRP:C10M), respectively. Control was left unstimulated and mCRP served as positive control (Thiele et al, 2018). Cells were washed after red blood cell lysis and analyzed by flow cytometry. Shown are MFI results with results normalized to control, mean ± SEM. P values were calculated with ANOVA and Tukey's post-hoc test. Biological replicates, n=6.",
    "molecules": "ADP, CO2, DHE, dihydroethidium, ROS, C10M"
  },
  {
    "caption": "H pCRP*/mCRP dependent NETosis in isolated human neutrophils detected by confocal immunofluorescence microscopy. Isolated neutrophils incubated for 3 hours at 37 °C, 5% CO2 with 100 µg/ml pCRP with and without PC:LPC liposomes (LP) and C10M (molar ratio 1:100, pCRP:C10M), respectively. Control was left unstimulated and 100 nM phorbol 12-myristate 13-acetate (PMA) served as positive control. Cells were washed, fixed and stained, and analyzed by confocal microscopy. Results are given as a ratio of NETing cells/all cells per ROI,",
    "molecules": "liposomes, CO2, LP, LPC, phorbol 12-myristate 13-acetate, PMA, PC, C10M"
  },
  {
    "caption": "B Detection of C10M by mass spectrometry in rat urine. Renal excretion of C10M detected by mass spectrometry in three rats (biological replicates, n=3) intravenously injected with C10M. Urine samples were collected at the indicated timepoints. 80% of the applied C10M mass was excreted after 90 min",
    "molecules": "C10M"
  },
  {
    "caption": "C Immunohistochemistry of rat kidneys subjected to IRI and i.p. pCRP application revealed distinct staining by anti-pCRP*/mCRP-9C9 antibody (green, arrows). C10M reduces the deposition of total CRP in the impaired tissue. No deposits in the non-ischemic tissue (sham). Exemplary stainings out of at least three are shown.",
    "molecules": "C10M"
  },
  {
    "caption": "D Tissue lysates of rat kidney were separated on SDS-PAGE and total CRP was identified with anti-CRP antibody. A band at the size of mCRP (~23 kDa) was detected in kidneys subjected to IRI and pCRP, but not in animals treated additionally with C10M. The household gene protein GAPDH served as a control for loading equal amounts of protein. 50 and 100 ng human pCRP, respectively, served as a positive control. Representative results are shown for replicated assays (n=3).",
    "molecules": "C10M"
  },
  {
    "caption": "E Renal excretion is impaired by pCRP*/mCRP-driven tissue damage. Blood urea nitrogen (BUN) was utilized as surrogate marker for the excretion function of the kidney. Blood samples were taken before the surgical procedure (preoperative) and 24 hours after the procedure (harvest). Graph shows mean ± SEM.",
    "molecules": "nitrogen, urea"
  },
  {
    "caption": "F Immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. Quantification of immunohistochemical results is shown as mean ± SEM. pCRP (25 µg/ml) increased the number of CD68+ cells transmigrated into injured renal tissue significantly, while C10M abolished these effects. Presented are mean cell counts per ROI in each animal.",
    "molecules": "C10M"
  },
  {
    "caption": "G Periodic acid-Schiff (PAS) stained kidney sections show increased damage after renal IRI in rats when pCRP (25 µg/ml) was injected i.v. The tubulointerstitial injury was quantified by the loss of tubular brush border and by cast formation following an established protocol (Megyesi et al, 1998, 2001). Quantification of immunohistochemical results is shown as mean ± SEM.",
    "molecules": "PAS, Periodic acid-Schiff"
  },
  {
    "caption": "H Representative results for the immunohistochemical detection of transmigrated CD68+ cells in IRI kidneys. CD68+ cells are stained with HistoGreen substrate (green). Scale bars indicate 100 µm.",
    "molecules": "HistoGreen"
  },
  {
    "caption": "I Representative results for the periodic acid-Schiff (PAS) stained kidney sections quantified in (G). Scale bars indicate 100 µm.",
    "molecules": "PAS, periodic acid-Schiff"
  },
  {
    "caption": "B Kaplan-Meier plots for control, pCRP, and pCRP+C10M (above) and C10M vs control (bottom) treatment for total allograft survival with and without CRP ± C10M. Kaplan-Meier curves for different treatments were compared by Mantel-Cox log-rank test. Allograft survival was significantly reduced by pCRP administration (P=0.0005, median survival control vs pCRP, 7.8 vs 4.8 days). C10M masks the CRP-accelerated hindlimb rejection (median survival 7.2 days). Biological replicates, n=4 per group.",
    "molecules": "C10M"
  },
  {
    "caption": "C Representative photographic examples of transplanted VCA hindlimb allografts. Shown are Lew hindlimb transplants in orthotopic situ on BN recipients three days after transplantation. Rats receiving i.p. pCRP presented VCA with massive edema (left). C10M treatment inhibits pCRP-aggravated early graft rejection (middle). The depicted C10M control without CRP shows no clinical signs of rejection after five days (right).",
    "molecules": "C10M"
  },
  {
    "caption": "D,E Representative immunofluorescence images from transplanted grafts reveal distinct signals (green) using anti-CD68 and anti-CRP antibodies, respectively. In C10M treated animals, less CD68+ cells infiltrate the transplanted tissue. Scale bars, 100 μm. (D) Immunofluorescence of transplanted muscle and (E) skin tissue revealed distinct CRP-deposition (green) in pCRP treated animals, but only minimal staining in rats treated additionally with C10M.",
    "molecules": "C10M"
  },
  {
    "caption": "A Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled S. pneumoniae by human monocytes serves as exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index (percentage of target positive cells of subtype / all cells of subtype) of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. B Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled E. coli by human monocytes serves as a second exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index as described above of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. C Phagocytosis of pCRP-opsonized, heat-treated and FITC-labeled zymosan by human monocytes serves as a third exemplary flow cytometry-based phagocytosis assay. Bar chart shows phagocytic index as described above of un-opsonized (control, dark-grey) and CRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively.",
    "molecules": "zymosan, FITC"
  },
  {
    "caption": "D Experiments described in (A) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of S. pneumoniae-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. E Experiments described in (B) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of E. coli-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. F Experiments described in (C) were repeated but with targets incubated with pCRP (100 µg/ml) and C10M (1:100 molar ratio) for 30 min, 37 °C. Bar chart shows phagocytic index of zymosan-positive monocytes for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively.",
    "molecules": "zymosan, C10M"
  },
  {
    "caption": "G Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled S. pneumoniae by human neutrophils. The same blood samples described in (A) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. H Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled E. coli by human neutrophils. The same blood samples described in (B) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively. I Phagocytosis of pCRP-opsonized, heat-killed and FITC-labeled zymosan by human neutrophils. The same blood samples described in (C) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils. Bar chart shows phagocytic index of un-opsonized (control, dark-grey) and pCRP-opsonized targets (red) after 5, 10, 15, and 20 min, respectively.",
    "molecules": "zymosan, FITC"
  },
  {
    "caption": "J Phagocytosis of pCRP+C10M-treated S. pneumoniae by human neutrophils. Results from experiments described in (D) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of S. pneumoniae-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. K Phagocytosis of pCRP+C10M-treated E. coli by human neutrophils. Results from experiments described in (E) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of E. coli-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively. L Phagocytosis of pCRP+C10M-treated zymosan by human neutrophils. Results from experiments described in (F) were analyzed for the phagocytic index of S. pneumoniae by human neutrophils by flow cytometry. Bar chart shows phagocytic index of zymosan-positive neutrophils for un-opsonized (control, dark-grey) and targets incubated with pCRP and C10M (green) after 5, 10, 15, and 20 min, respectively.",
    "molecules": "zymosan, C10M"
  },
  {
    "caption": "(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown.",
    "molecules": "DNA, protein"
  },
  {
    "caption": "(C) In vivo degradation assays of FtsA, FtsZ and DnaB. ΔIndLon mutants expressing the different N-terminal FLAG-tagged derivatives were grown in depleting conditions for 36h. Then, Lon expression was transiently induced for 3h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti-FLAG antibodies. LC, loading control. As controls, non-induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments.",
    "molecules": "gentamicin, Gm"
  },
  {
    "caption": "(I) Similar to panel C, but for FtsH candidate substrates. In this case, C-terminal FLAG-tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment.",
    "molecules": "gentamicin"
  },
  {
    "caption": "(B) Lon surveillance of truncated protein variants. As an example, an N-terminal FLAG-tagged derivative of MPN304, which encodes a truncated variant of ArcA (ArcA-Nt), was expressed in the ΔIndLon mutant, and its accumulation monitored over time under non-inducing conditions using anti-FLAG antibodies. Anti-Lon antibodies were used to monitor Lon depletion along the time course experiment (western blot figure on left). MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants (36h of depletion), in which protein expression was blocked with gentamicin (Gm) during the indicated time points after transient induction of Lon for 3h. As control, samples after Gm treatment were also taken from non-induced samples. LC, loading control (western blot figure on right). Immunoblots are representative of two independent experiments. A schematic representation of the arginine deiminase pathway, which is inactive in M. pneumoniae, and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing (+) or depleting (-) conditions.",
    "molecules": "gentamicin, Gm"
  },
  {
    "caption": "E, IGV visualization showing m6A and YTHDF2 binding at Nlrc3, Nfkbie, and Traf3 loci in CD4+ T cells (GSE188853).",
    "molecules": "m6A"
  },
  {
    "caption": "F, WT or Ythdf2 cKO Treg cells from the spleen were stimulated with 10 ng/mL TNFα in vitro. 24 hours post TNFα stimulation, cells were treated with Actinomycin D. Decay of Nlrc3, Nfkbie, and Traf3 was monitored by RT-qPCR (n=3, biological replicates). Data are presented as means ± s.d. and analyzed two-way ANOVA.",
    "molecules": "Actinomycin D"
  },
  {
    "caption": "A Dose-response binary food choice assay of saponin (n ≥ 4).",
    "molecules": "saponin"
  },
  {
    "caption": "B Dose response curve of tip recordings using the indicated concentrations of saponin on S6 sensilla (n ≥ 8).",
    "molecules": "saponin"
  },
  {
    "caption": "C Sample traces of saponin-induced action potentials in (B).",
    "molecules": "saponin"
  },
  {
    "caption": "D Dose response curve for sugar inhibition using the indicated concentrations of saponin with 100 mM sucrose on L4 sensilla (n ≥ 8). E Sample traces from (D). The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (**P < 0.01). The error bars represent SEMs. ",
    "molecules": "saponin, sucrose"
  },
  {
    "caption": "A Binary food choice assay with 26 Gr mutants for 5% saponin (n ≥ 4). The error bars represent SEMs.",
    "molecules": "saponin"
  },
  {
    "caption": "B Concentration-dependent avoidance of saponin in control, Gr28bMi and Gr28b Df/ Gr28bMi (n ≥ 4).",
    "molecules": "saponin"
  },
  {
    "caption": "C Amount of ingestion calculated at the indicated time points (15, 30, 60, and 240 min) using spectrophotogram analysis for control and 5% saponin-containing food (n = 8).",
    "molecules": "saponin"
  },
  {
    "caption": "D, E PER assay for control and Gr28bMi flies in the (D) legs or (E) proboscis. Flies were initially given 2% sucrose, then 2% sucrose in combination with 5% saponin (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (*P < 0.05, **P < 0.01). ",
    "molecules": "saponin, sucrose"
  },
  {
    "caption": "A Mapping analysis of all the sensilla following stimulation with 5% saponin in control and Gr28bMi flies (we followed Tanimura's nomenclature) (n ≥ 8). The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).",
    "molecules": "saponin"
  },
  {
    "caption": "B Sample traces of saponin-induced action potentials on S6 sensilla.",
    "molecules": "saponin"
  },
  {
    "caption": "C Tip recording analysis of S6 sensilla from control and Gr28bMi flies. Stimuli used are 0.2% DEET, 1.0 mM coumarin, 0.5 mM quinine, 10 mM umbelliferone, 10 mM caffeine, 0.3 mM strychnine, 0.3 mM lobeline, 0.1 mM chloroquine, 0.2 mM denatonium, 0.5 mM papaverine, 0.1 mM berberine and 0.1% SDS (n ≥ 7). The error bars represent SEMs.",
    "molecules": "berberine, caffeine, chloroquine, coumarin, denatonium, lobeline, DEET, papaverine, quinine, SDS, strychnine, umbelliferone"
  },
  {
    "caption": "D Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01). D Saponin-induced action potential frequencies from the indicated transgenic flies on S6 sensilla (n = 7).",
    "molecules": "Saponin"
  },
  {
    "caption": "A, B Survival rate of (A) control and (B) Gr28bMi adult raised on control and the indicated concentrations of saponin-containing cornmeal diet (n = 4).",
    "molecules": "saponin"
  },
  {
    "caption": "C, D Survival rate based on binary food choice. control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM (C) or 200 mM fructose (D) mixed with 5% or 7.5% saponin.",
    "molecules": "fructose, saponin"
  },
  {
    "caption": "E, F Cumulative pupation rate of (E) control and (F) Gr28bMi larvae raised on the indicated saponin-containing food as well as control food (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from normal diet fed which were detected by a single-factor ANOVA with Scheffe's analysis (*P < 0.05, **P < 0.01).",
    "molecules": "saponin"
  },
  {
    "caption": "D. A549  cells were treated with 125nM dBRD9-A  (+) or DMSO (-) for 6h prior stimulation with 1000 IU/mL of IFN-α2 for 16h. Following lysis, western blot was used to detect levels of the indicated proteins. Data are representative of at least two biological replicates.",
    "molecules": "dBRD9-A, DMSO"
  },
  {
    "caption": "C. Impact of dBRD9-A pre-treatment on the induction of ISGs identified in (B). Colors indicate IFN-induced transcripts significantly affected (blue) or unaffected (gray) by dBRD9-A  pretreatment (FC>2, p-value ≤ 0.001).",
    "molecules": "dBRD9-A"
  },
  {
    "caption": "F.  Western blot  analysis of samples from independent small-scale  streptavidin purifications (termed 'Eluates') following the proximity labeling approach outlined in (B). The indicated proteins were detected with specific antibodies. M indicates the marker lane. Data are representative of at least two biological replicates.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(a,b) E1-charging assay for Uba1 and Atg7 with either Atg8a (a) or ubiquitin (Ub; b), n = 2 experiments with independently isolated proteins and analyses. Flag-tagged baculoviral-expressed E1s were incubated with either His-tagged Atg8a or Ub in the presence or absence of β-mercaptoethanol, separated by electrophoresis and blotted. Band shift was detected with anti-Flag antibody. Relative molecular mass (Mr K) ladders are indicated. Representative images are shown. Relative molecular masses: Flag-Uba1, 135K; Flag-Atg7, 81K; His-Atg8a, 29K; Ub, 9K.",
    "molecules": "β-mercaptoethanol"
  },
  {
    "caption": "(d-f) Representative immuno-TEM images of Uba1IRclone cells at puparium formation. Uba1IR-expressing cells possess gold particles; control cells lack gold particles (d). Control cells possess numerous autolysosomes in the cytoplasm (arrowheads) and few mitochondria (asterisks); Uba1IR-expressing cells possess numerous mitochondria and few autophagic structures. Scale bars, 20 μm (a-c) and 1 μm (d-f).",
    "molecules": "gold"
  },
  {
    "caption": "Human, mouse and total tau protein levels in cortical TBS-extracts of rTg4510, rTg4510-Mapt0/0, and control mice: The amount of human tau (Tau13 antibody) was comparable in rTg4510 and rTg4510-Mapt0/0, moTau (Tau/5) was comparable in WT and rTg4510, and total tau levels (hu+moTau, DAKO antibody) were (expected) highest in rTg4510 mice. Mean±SEM, n=3 mice/group, non-significant.",
    "molecules": "TBS"
  },
  {
    "caption": "Extraction of cortices revealed similar human tau (Tau13) in TBS-extracts (not significant) but significantly more human tau in TritonX-100 (TX-100) extracts of rTg4510-Mapt0/0 compared to rTg4510 mice. Mean±SEM, n =3 mice/group, two-tailed Student's T test, ns non significant.",
    "molecules": "TBS, TritonX-100, TX-100"
  },
  {
    "caption": "Native gel electrophoresis of cortical TBS-extracts showed small differences in HMW (oligomeric) human tau between rTg4510 and rTg4510-Mapt0/0 brains. Western blot lanes were averaged across ~2/3 of the width (black rectangular and arrow in Tau13 blot). The mean±SEM (n=3 mice/group) of these averages was plotted as longitudinal lane profiles. Differences in HMW tau are indicated by red and black arrows.",
    "molecules": "TBS"
  },
  {
    "caption": "TBS-brain extracts were applied to a HEK293 cell tau aggregation seeding assay(Holmes et al, 2014, Sanders et al, 2014), in which TauRDP301S-CFP and TauRDP301S-YFP are co-expressed intracellularly. The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer (FRET) activity between co-aggregated CFP and YFP-tags and correlates with the tau aggregation seeding activity of the applied brain extracts. After 24 hours, cells treated with extract (0.5 µg and 1.0 µg total protein per 96 well) from 9-month-old rTg4510 had significantly more intracellular YFP-positive (white arrows) aggregates compared to rTg4510-Mapt0/0 extracts; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510-Mapt0/0. WT and Mapt0/0 extracts never showed seeding activity. Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510-Mapt0/0 mice. Two-tailed Student's T-test, mean±SEM ns, not significant, Insets 100 µm, Scale bars 50 µm.",
    "molecules": "TBS"
  },
  {
    "caption": " A MMS-induced checkpoint activation is not altered in irc5-Δ1 cells. The level of Rad53 phosphorylation was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody ",
    "molecules": "MMS"
  },
  {
    "caption": " B Prolonged checkpoint activation in irc5-Δ1 cells during recovery from MMS treatment. Rad53 phosphorylation status was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody; M, MMS ",
    "molecules": "MMS"
  },
  {
    "caption": " D Disruption of IRC5 leads to accumulation of ssDNA-containing lesions. ChEC analysis of wild type (BYR52MN) and irc5-Δ1 (TB040) cells cultured in the presence or absence of 0.03% MMS for 2 hours (n=3) ",
    "molecules": "ssDNA, MMS"
  },
  {
    "caption": " A The irc5-Δ1 mutant exhibits S-phase delay in the presence of MMS. DNA content of wild type (W303-1a) and irc5-Δ1 (IL012) cells was measured by FACS and plotted as histograms ",
    "molecules": "DNA, MMS"
  },
  {
    "caption": " B Completion of DNA replication is delayed in the irc5-Δ1 mutant after MMS treatment. Intact chromosomes were isolated from wild type (W303-1a) and irc5-Δ1 (IL012) cells and processed for PFGE. M, 0.03% MMS ",
    "molecules": "MMS"
  },
  {
    "caption": "C Translocase activity of Irc5 is important for completion of DNA replication after MMS treatment. PFGE was performed as in Fig 2B using wild type (EMD09) and irc5DAEA (EMD10) strains",
    "molecules": "MMS"
  },
  {
    "caption": "E Cohesin loading complex is required for completion of chromosome replication after MMS treatment. Intact chromosomes were isolated from wild type (Y39) and scc2-4 (Y4366) cells cultivated at permissive temperature (25°C) and processed for PFGE. M, MMS",
    "molecules": "MMS"
  },
  {
    "caption": "B, C Levels of DNA repair foci in scc2-4 and scc2-4 irc5-Δ1 mutants. Logarithmically growing cultures of scc2-4 (TB071 or TB073) and scc2-4 irc5-Δ1 (TB072 or TB074) were treated with MMS for 1 hour at 25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3)",
    "molecules": "MMS"
  },
  {
    "caption": "E, F Levels of Rfa1-YFP and Rad52-YFP foci in scc1-73 and irc5-Δ1 scc1-73 mutants. Logarithmically growing cultures of scc1-73 (TB076 or TB078) and irc5-Δ1 scc1-73 (TB077 or TB079) were treated with MMS for 1 hour at  25°C. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=3)",
    "molecules": "MMS"
  },
  {
    "caption": "G Replication completion defect of scc1-73 is not exacerbated by IRC5 disruption. Intact chromosomes were isolated from scc1-73 (JC1339) and irc5-Δ1 scc1-73 cells (TB075), cultured at 25°C, and processed for PFGE. M, 0.03% MMS",
    "molecules": "MMS"
  },
  {
    "caption": "(A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.",
    "molecules": "Am80"
  },
  {
    "caption": "(A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.",
    "molecules": "Am80"
  },
  {
    "caption": "(A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.",
    "molecules": "ROS, Am80"
  },
  {
    "caption": "(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii).",
    "molecules": "ROS, Am80"
  },
  {
    "caption": "(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii).",
    "molecules": "Am80"
  },
  {
    "caption": "(E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies.",
    "molecules": "ROS"
  },
  {
    "caption": "(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days.",
    "molecules": "ROS"
  },
  {
    "caption": "(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).",
    "molecules": "ROS"
  },
  {
    "caption": "(A) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment.",
    "molecules": "Am80"
  },
  {
    "caption": "(B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens.",
    "molecules": "Am80"
  },
  {
    "caption": "(C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples.",
    "molecules": "Am80"
  },
  {
    "caption": "(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i).",
    "molecules": "Am80"
  },
  {
    "caption": "(B) Fresh BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. ROS production was assessed at day 12 (section ii).",
    "molecules": "ROS, Am80"
  },
  {
    "caption": "(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Bacterial killing was assessed at day 12 (sections iii).",
    "molecules": "Am80"
  },
  {
    "caption": "(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Morphologic differentiation was assessed at day 12 (section iv). White arrows indicate neutrophil nuclear segmentation.",
    "molecules": "Am80"
  },
  {
    "caption": "(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).",
    "molecules": "Am80"
  },
  {
    "caption": "(A) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide (CPA) consists of both neutrophil decrease and neutrophil recovery stages. ANC, absolute neutrophil count.",
    "molecules": "CPA, cyclophosphamide"
  },
  {
    "caption": "(D & E) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and/or GCSF in BM (panel D) and PB (panel E). White arrows indicate neutrophilnuclear segmentation.",
    "molecules": "Am80"
  },
  {
    "caption": "(B) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 3 independent experiments with similar results.",
    "molecules": "CPA"
  },
  {
    "caption": "(E) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 2 independent experiments with similar results.",
    "molecules": "CPA"
  },
  {
    "caption": "(G) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii).",
    "molecules": "CPA"
  },
  {
    "caption": "(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section i).",
    "molecules": "Am80"
  },
  {
    "caption": "(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section ii).",
    "molecules": "Am80"
  },
  {
    "caption": "(C) The increased spleen size on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (section iii).",
    "molecules": "Am80"
  },
  {
    "caption": "(C) The increased spleen weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv).",
    "molecules": "Am80"
  },
  {
    "caption": "B The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d.",
    "molecules": "K+, NaCl, Na+"
  },
  {
    "caption": "C Linear regression of root Na+/K+ ratio under salt stress (150 mM NaCl for 1 d) and fruit weight in 195 accessions from three groups. R2, coefficient of determination. n is the accession number of the three groups.",
    "molecules": "K+, NaCl, Na+"
  },
  {
    "caption": "D The Na+/K+ ratios in roots of TS-21, TS-422, TS-577, and TS-670 after treatment with 150 mM NaCl for the indicated time (n = 3 biological repeats).",
    "molecules": "K+, NaCl, Na+"
  },
  {
    "caption": "E, F Salt-resistance assay of two PIM accessions (TS-21 and TS-422) and two BIG (TS-577 and TS-670). 20-day-old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks (E). The survival rates were obtained from at least 12 plants in three repeated experiments (F).",
    "molecules": "NaCl"
  },
  {
    "caption": "A Manhattan plot displaying the GWAS results of Na+/K+ ratio in root. The red-dashed line indicates the Bonferroni-adjusted significance threshold (P = 1.0 × 10-7). Red arrow indicates the significant SNP signal of Na+/K+ ratio associated with SlHAK20.",
    "molecules": "K+, Na+"
  },
  {
    "caption": "B The nucleotide diversity ratios between PIM and CER, and between CER and BIG on chromosome 4. The black-dashed horizontal lines indicate top 10% threshold for entire chromosome 4 (1.82 πPIM/πCER for domestication and 4.27 πCER/πBIG for improvement). The red arrows indicate the position of SlHAK20 in the sweeps. π, nucleotide diversity.",
    "molecules": "nucleotide"
  },
  {
    "caption": "D Haplotypes of SlHAK20 among tomato natural variations. The distribution of Na+/K+ ratio in each haplotype group is exhibited by a box plot. The n indicates the number of accessions belonging to each haplotype. In the box plots, the middle line indicates the median, the box indicates the range of the percentiles of the total data using Turkey method, the whiskers indicate the interquartile range and the outer dots are outliers. Significant difference was determined by Student's t-test.",
    "molecules": "K+, Na+"
  },
  {
    "caption": "E Subcellular localization of SlHAK20-YFP in tomato. The SlHAK20Hap1-YFP (left) and SlHAK20Hap2-YFP (right) were localized in the plasma membrane. YFP, yellow florescence protein. FM4-64, a lipophilic styryl compound as a red fluorescent marker of plasma membrane. Bar, 20 μm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "F SlHAK20 complements the K+ uptake-defective yeast mutant assayed using a dose-response method. The K+ uptake-defective yeast mutant R5421 transformed with SlHAK20Hap1, SlHAK20Hap2, and vector (p416-GPD) were cultured in AP medium containing indicated concentrations of K+ for 24 h. The data points represent three replicates.",
    "molecules": "K+"
  },
  {
    "caption": "G Na+ uptake analysis of yeast cells expressing SlHAK20. Shown are the Na+ concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1, SlHAK20Hap2, or empty vector. The initial NaCl concentration in the medium was 60 μM. The data are shown as the means ± SD (n = 4).",
    "molecules": "NaCl, Na+"
  },
  {
    "caption": "H Na+ uptake kinetic analysis of SlHAK20 in yeast cells. The curves show concentration- dependent Na+ influx in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0, 5, 10, 20, 30, 40, 50, 60, 100, 150 or 200 μM NaCl in the medium. The data are expressed as mean ± SD (n = 3).",
    "molecules": "NaCl, Na+"
  },
  {
    "caption": "Na+/K+ ratio contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).",
    "molecules": "K+, Na+"
  },
  {
    "caption": "Na+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).",
    "molecules": "Na+"
  },
  {
    "caption": "K+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).",
    "molecules": "K+"
  },
  {
    "caption": "Na+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).",
    "molecules": "NaCl, Na+"
  },
  {
    "caption": "K+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).",
    "molecules": "K+, NaCl"
  },
  {
    "caption": "H Salt resistance of TS-21 wild type and slhak20 mutants. TS-21, slhak20-1, and slhak20-2 grown under normal growth conditions for 24 days, followed by a treatment with 200 mM NaCl for 14 days, and then recovered for 10 days. Control plants grown under normal growth conditions for 38 days. Scale bars, 5 cm.",
    "molecules": "NaCl"
  },
  {
    "caption": " Salt-tolerance assay of TS-670 and slhak20 mutant plants. TS-670, slhak20-3, and slhak20-4 grown under normal growth conditions for 23 days, and then treated with 150 mM NaCl for 5 days. Control plants grown under normal growth conditions for 28 days. ",
    "molecules": "NaCl"
  },
  {
    "caption": " Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. Shoot height f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).",
    "molecules": "NaCl, salt"
  },
  {
    "caption": " Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. resh weight f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype). ",
    "molecules": "NaCl, salt"
  },
  {
    "caption": " Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative plants ere analyzed among these genotypes. ontrol plants were grown under normal growth conditions for 28 days ",
    "molecules": "NaCl"
  },
  {
    "caption": " Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative fresh weight of shoots were analyzed among these genotypes. Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured. Values are means ± SD (n = 12 plants of each genotype for fresh weight ",
    "molecules": "NaCl"
  },
  {
    "caption": " Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative survival rate were analyzed among these genotypes. ",
    "molecules": "NaCl"
  },
  {
    "caption": "38-day-old plants grown in soil as control. 20-day-old TS-670 and Hap1OE-1 to -3 were treated with 175 mM NaCl for 3 weeks and recovered for 2 weeks",
    "molecules": "NaCl"
  },
  {
    "caption": "The Na+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.",
    "molecules": "NaCl, Na+"
  },
  {
    "caption": "The K+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.",
    "molecules": "K+, NaCl"
  },
  {
    "caption": "The Na+/K+ ratio in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.",
    "molecules": "K+, NaCl, Na+"
  },
  {
    "caption": "A. Serum, plasma or urine levels of vitamin D, vitamin A, folate, zinc, iron and iodine status biomarkers in diet groups. Solid lines indicate cut-offs for deficiency, and dashed lines indicate cut-offs for insufficiency. The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1.17 µmol/l and 0.83 µmol/l, respectively. Vitamin A status was calculated based on RBP, transthyretin, and CRP.(Talsma et al, 2015).",
    "molecules": "folate, iodine, iron, vitamin A, Vitamin A, zinc"
  },
  {
    "caption": "C. Amino acid concentrations in untargeted metabolomics. Numeric means and standard deviations in this figure can be found in Appendix Table S4. #Valine and betaine are two major components under the same m/z-peak, causing uncertainty in the interpretation of valine levels.",
    "molecules": "valine, Valine"
  },
  {
    "caption": "A. Fatty acid analysis from untargeted MS metabolomics including free alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA).",
    "molecules": "ALA, alpha-linolenic acid, DHA, docosahexaenoic acid"
  },
  {
    "caption": "B. Carnitine-bound fatty acids commonly found in serum that were detected in mass spectrometry.",
    "molecules": "Carnitine"
  },
  {
    "caption": "C. Lysophosphatidylcholine and lysophosphatidylethanolamine with C16:0 (palmitic acid) and C18:1 (oleic acid).",
    "molecules": "C16:0, palmitic acid, Lysophosphatidylcholine, lysophosphatidylethanolamine, C18:1, oleic acid"
  },
  {
    "caption": "Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI. Representative pictures at 40x are shown (n=30). Scale bars, 100 μM.",
    "molecules": "DAPI, Formalin, paraffin"
  },
  {
    "caption": "FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin (H&E) or immunostained with antibodies to CK19 or Amylase and revealed by IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C).",
    "molecules": "IgG"
  },
  {
    "caption": "Protein extracts from control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin.",
    "molecules": "IgG"
  },
  {
    "caption": "Protein extracts from MIAPaCa-2 cells (left) and control were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C, cell lysates were treated with DNase I before coimmunopreciptation.",
    "molecules": "IgG"
  },
  {
    "caption": "PL45 stably expressing Dox-inducible control or sh-RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1.",
    "molecules": "Dox"
  },
  {
    "caption": "PL45 stably expressing Dox-inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.",
    "molecules": "agarose, Dox"
  },
  {
    "caption": "Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.",
    "molecules": "agarose"
  },
  {
    "caption": "PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6).",
    "molecules": "Dox, Tam"
  },
  {
    "caption": "MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Expression of Twist1 was examined by qRT-PCR (D).",
    "molecules": "G418"
  },
  {
    "caption": "MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel.",
    "molecules": "agarose, G418"
  },
  {
    "caption": "MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cells were selected with neomycin for 2-3 weeks, and counted using an automatic cell counter (D) (n=6).",
    "molecules": "neomycin"
  },
  {
    "caption": "(e-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "E, Chromosome  congression is affected by the absence of GCN2. E Immunofluorescence  images of HeLa  cells stained  for a kinetochore  marker, CREST  (grey) and DAPI  for DNA (blue). Arrowheads indicate misaligned chromosomes. Scale bars represent 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B There is no significant difference in global translation rates in mitosis  after GCN2  knock-down. A HeLa  cells were pulse labeled with OPP  and fixed with 70% ethanol. OPP  was detected by Click-IT (AF 647) and mitotic  cells were identified by staining  for phospho-histone H3. 2D  flow cytometry plots are shown, where AF647  intensity corresponds to OPP  incorporation. Translation levels normalized to siCtr G1. The graph shows averages and SEM from three independent experiments. Paired two-tailed t-tests.",
    "molecules": "AF 647, AF647, ethanol, OPP"
  },
  {
    "caption": "D, Mitotic translation of selected proteins. Hela  cells were incubated with 5 µM Eg5i  overnight and mitotic cells were shaken off. MG132  was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express  HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting  every 10 minutes (D) Representative immunoblots  of three biological replicates are shown.",
    "molecules": "Gcn2-IN-1, HY-100877, MG132"
  },
  {
    "caption": "E Mitotic translation of selected proteins. Hela  cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off. MG132  was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting  every 10 minutes Representative (E) quantifications of three biological replicates; averages and SEM are shown. Values are normalized to t=0.",
    "molecules": "Gcn2-IN-1, HY-100877, MG132"
  },
  {
    "caption": "A Verification of the mutants. HeLa  cells transduced with the indicated GCN2-siR constructs were transfected with control (Ctr) or GCN2-targeting siRNA. Halofuginone  (HFG) was added to 40 nM for 4 hours, and GCN2 autophosphorylation, ATF4  induction and eIF2α phosphorylation  were assessed by immunoblotting. γ-tubulin is shown as loading control. The line in the GCN2  blot indicates that a longer exposure of the same blot  is shown on the left than on the right.",
    "molecules": "Halofuginone, HFG"
  },
  {
    "caption": "C, GCN2 kinase activity  is regulated in the cell cycle. HeLa  cells were arrested at the G1/S transition using thymidine  and released from the cell cycle at time  0 h. Samples were taken at the indicated time-points for immunoblotting Aurora A and Aurora A-P are shown to indicate the timing of mitosis, GCN2-P is shown to assess GCN2 activity. Representative immunoblot  of GCN2 phosphorylation  normalized to GCN2  (total), three biological repeats, three technical repeats each.",
    "molecules": "thymidine"
  },
  {
    "caption": "D GCN2   can phosphorylate PP1α  and γ in vitro. HeLa  cells carrying GFP-PP1α  or GFP-PP1γ  were arrested in prometaphase  with an Eg5  inhibitor (Lane 3), then GFP-tagged PP1α  or PP1γ  were immunoprecipitated  using GFP-trap (input, Lane 2). The immunoprecipitates  were incubated in kinase  buffer and ATP  with constitutively active, recombinant GCN2. The samples were run on phos-tag gels and immunoblotted  with PP1γ  or PP1α  antibody.",
    "molecules": "ATP"
  },
  {
    "caption": "HeLa  cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine  block-and-release and samples were collected for  flow cytometry  at the indicated time points. A Cell-cycle progression in the cells synchronized by thymidine  block-and-release, three independent experiments, averages and SEM are shown.",
    "molecules": "thymidine"
  },
  {
    "caption": "HeLa  cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine  block-and-release and samples were collected for immunoblotting  at the indicated time points. B Representative immunoblots  of TACC3-S558 phosphorylation  and  Aurora A-T288 phosphorylation",
    "molecules": "thymidine"
  },
  {
    "caption": "F Dephosphorylation of PP1 substrates after  Aurora A inhibition in the presence and absence of GCN2  activity. HeLa cells were treated with Aurora Ai (MLN8237, MedChemTronica HY-10971), and with both Aurora Ai and GCN2i. Cell suspensions were incubated at 37oC and at the indicated times  samples were removed, mixed with Laemmli sample buffer and boiled. F The immunoblots  were probed with the indicated antibodies.",
    "molecules": "HY-10971, MLN8237"
  },
  {
    "caption": "B HeLa  cells were transfected with control or GCN2-targeting siRNA or treated with the indicated concentrations of GCN2  inhibitor, cells were fixed after 72 h incubation, and the number of cells containing micronuclei  was determined by DAPI staining. Four independent experiments, averages with SD are shown. n= 187, 72, 162, 531 for siCtr; n= 159, 112, 200, 656 for siGCN2; p= 0.009; n=721, 821, 682 for 0 µM; n= 607, 585, 635 for 1 µM, n= 414, 510, 723 for 2 µM; n= 123, 147, 280 for 3 µM; p=0.004 for 1 µM, **p=0.005 for 2 µM, **p=0.005 for 3 µM (paired two-tailed t-tests).",
    "molecules": "DAPI"
  },
  {
    "caption": "E PP1α  and PP1γ  bandshifts are diminished in cells arrested in mitosis upon pharmacological inhibition of GCN2. HeLa  or hTert-RPE1  cells were arrested in mitosis using a PLK1  inhibitor and GCN2-IN1  was added to 2 µM for 3 h before harvesting the cells. Representative E quantifications of the ratios of the phospho-PP1/PP1 signal (average and SEM) from four independent experiments are shown. HeLa: ***p=0.0007 (PP1α); **p=0.0023 (PP1γ);  htert-RPE1: *p=0.0111 (PP1α) and ***p=0.0007(PP1γ), paired two-tailed t-tests.",
    "molecules": "GCN2-IN1"
  },
  {
    "caption": "C HeLa  and hTert-RPE1 cells were seeded in triplicate and incubated in the presence of the GCN2  and Aurora A inhibitors for 7 (HeLa) or 10 (RPE1) days, then fixed and colonies were counted. Shown are averages and SEM from three biological replicates. For HeLa  cells *p=0.0267 for A versus G2A; *p= 0.0432 A versus G3A (paired two-tailed t-tests). For hTert-RPE1  cells *p=0.0391 A versus G2A; **p=0.021 A versus G3A (paired two-tailed t-tests). D HeLa  and Tert-RPE1  cells were treated with the indicated inhibitors for 72 h, stained  with Hoechst  and analyzed by  flow cytometry. The graph shows the percentage of Hoechst-positive (dead) cells in four independent experiments. Averages and SEM are shown. For HeLa  cells **p=0.0087 A versus AG; **p=0.0027 Ctr versus AG; *p= 0.0237 Ctr versus G; p= 0.0809 Ctr versus A (paired two-tailed t-test). For hTert-RPE1 cells ***p=0.0005 A versus AG; **p=0.0036 Ctr versus AG; *p=0.0210 Ctr versus G; p= 0.29184 Ctr versus A (paired two-tailed t-tests).",
    "molecules": "Hoechst"
  },
  {
    "caption": "E, F HeLa  and Tert-RPE1  cells were treated with the indicated inhibitors for 72 h, stained  with the CellEvent™ Caspase-3/7 green detection reagent and Hoechst  and analyzed by flow cytometry. Representative 2D plots of Alexa-Fluor 488 (a measure of Caspase-3/7 activity) and Hoechst intensity are shown for E HeLa  and F RPE1  cells. All single cells are plotted in magenta, Hoechst-negative cells are overlaid in grey. Note that all Hoechst-positive (dead) cells are also stained  with the  Caspase-3/7 green reagent, indicating apoptosis.",
    "molecules": "Hoechst, Alexa-Fluor 488"
  },
  {
    "caption": "A C57BL/6 WT or Rag2−/− mice were immunosuppressed with hydrocortisone +/− FK506 and infected with 105 AF CEA10 conidia intranasally. Mortality was assessed using a 20% weight loss endpoint. Mortality was significantly increased in FK506 immunosuppressed mice reaching 100% by day 10 p.i. (P = 0.003 for HC versus HC+FK506 in Rag2−/− using log-rank test, n = 10 per group).",
    "molecules": "FK506, hydrocortisone"
  },
  {
    "caption": "B, C PAS-stained lung sections of Rag2−/− mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ. FK506-treated animals had increased inflammation at day 2 p.i. compared to control. P = 0.0126 (using Student's t-test), n = 3-5 per group. (C) Close-up of an infectious focus showing increased inflammation and fungal growth in the FK506-treated group. Scale bar: 400 μm.",
    "molecules": "FK506"
  },
  {
    "caption": "D, E Murine macrophages (J774A.1) were pre-treated with hydrocortisone, and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy. The nuclei are indicated in red, and the GR is indicated in green. Scale bar: 50 μm. FK506 did not cause increased translocation of the glucocorticoid receptor. Data represent mean + SEM calculated from 10 fields of vision. One out of 2 experiments is shown.",
    "molecules": "FK506, hydrocortisone"
  },
  {
    "caption": "Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.A Weight was monitored daily for 7 days p.i. Immunosuppressed mice showed increased weight loss through days 1-3 p.i. Data points represent mean ± SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. B BALs were performed over a 3-day period p.i. and fungal killing assessed by enumerating CFUs. Fungal killing was significantly impaired in immunosuppressed mice. Error bars indicate SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n. C, D Flow cytometry analysis of the CD45+leucocyte populations in BAL. Representative dot plots for CD45+ cells (C) and percentages of Ly-6G-positive neutrophils infiltrating the infected lungs over a 48-h p.i. time course (D). The bars indicate the mean percentage (± SEM) of neutrophils of total CD45+ leucocytes (n = 5 mice per group). Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p.i.",
    "molecules": "FK506"
  },
  {
    "caption": "Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.E PAS-stained lung sections of immunocompetent and FK506-treated animals at 6 h p.i. Scale bar: 50 μm. IC animals showed inflammatory cell infiltrates around conidia (black arrows), whereas in FK506-treated animals, only uptake of conidia by the resident macrophage population could be seen.",
    "molecules": "FK506"
  },
  {
    "caption": "Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ. Bars represent mean percentages (± SEM), n = 3.",
    "molecules": "FK506"
  },
  {
    "caption": "Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. G Luminex multiplex cytokine analysis of BAL SNs at 6 h p.i. Bars represent mean ± SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.A, B Absolute neutrophil numbers in treated and untreated mpx:GFPlarvae were quantified by direct counting of fluorescent neutrophils in non-infected whole fish (n = 11-13 per group). There were no significant differences.",
    "molecules": "FK506, DMSO"
  },
  {
    "caption": "At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.C For survival analysis, lyz:dsRedlarvae were microinjected with ˜10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan-Meier analysis for survival over 6 days post-infection (n = 16 per group). Immunosuppression led to a significant increase in mortality.",
    "molecules": "FK506, DMSO"
  },
  {
    "caption": "At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. D TNF-α mRNA levels were measured in whole non-infected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNF-α expression during infection.",
    "molecules": "FK506, DMSO"
  },
  {
    "caption": "At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. E-G Lyz:dsRedlarvae were infected with ˜50 eGFP-expressing conidia of A. fumigatus and were imaged daily from 24 h p.i. (E) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ. A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown (60 h p.i.). The arrows indicate infectious foci. (F) Neutrophil recruitment was minimal throughout the first 60 h p.i. in immunosuppressed larvae (n = 8 per group). (G) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements (n = 8 per group).",
    "molecules": "FK506, DMSO"
  },
  {
    "caption": "A J774A.1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. FK506 significantly reduced TNF-α levels at 5 and 10 ng/ml for AF. A significant reduction in TNF-α was also seen for zymosan, but not LPS.",
    "molecules": "FK506, zymosan, LPS"
  },
  {
    "caption": "B J774A.1 cells were treated with FK506 (10 ng/ml) and either left non-stimulated (NS) or stimulated with AF SC (MOI=1) for 6 h. TNF-α mRNA levels were quantified from whole-cell RNA extracts by RT-PCR. FK506 significantly reduced TNF-α expression.",
    "molecules": "FK506"
  },
  {
    "caption": "C J774A.1 macrophages were treated with control (Ctrl.) or calcineurin A (CnA)-targeting siRNA (50 nM) and stimulated with either AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. CnA siRNA led to significant reductions in both AF- and zymosan-dependent TNF-α levels, but had no significant effect on LPS responses.",
    "molecules": "zymosan, LPS"
  },
  {
    "caption": "B J774A.1 macrophages were stimulated with live or fixed swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFAT translocation was assessed by confocal microscopy. NFAT is indicated in green, the nuclei are indicated in red, and AF conidia can be seen in pink. Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan. Data represent mean ± SEM. Scale bar: 50 μm. Arrows indicate A. fumigatus or zymosan-positive cells.",
    "molecules": "zymosan"
  },
  {
    "caption": "C J774A.1 macrophages were stimulated with zymosan after FK506 (10 ng/ml) or control treatment, and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF-κB translocation. NFATc2 and NF-κB nuclear translocation was observed, which was maximal at 1 h p.i., in stimulated macrophages. FK506 specifically inhibited NFATc2 translocation, but had no effect on NF-κB.",
    "molecules": "FK506, zymosan"
  },
  {
    "caption": "D J774A.1 macrophages were pre-treated with FK506 (10 ng/ml) or vehicle and stimulated with live swollen conidia or zymosan. NFATc2 translocation was evaluated 30 min after stimulation. Box and whisker plot shows median ± maximum/minimum. Scale bar: 50 μm. FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan. Statistical analysis was carried out by Student's t-test. NS, non-stimulated. Arrows indicate A. fumigatus or zymosan-positive cells.",
    "molecules": "FK506, zymosan"
  },
  {
    "caption": "B BMDMs were stimulated with swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation. Whilst almost complete abrogation of NFAT translocation was seen in Dectin1−/−macrophages stimulated with zymosan, NFAT responses to AF were unaffected.",
    "molecules": "zymosan"
  },
  {
    "caption": "C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (C) TNF-α was measured in the SN after overnight incubation. Syk inhibition significantly reduced swollen conidia-dependent or zymosan-dependent TNF-α levels.",
    "molecules": "zymosan, piceatannol"
  },
  {
    "caption": "C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (D) NFATc2 translocation was quantified by confocal microscopy. Syk inhibition impaired zymosan-dependent NFAT translocation, but did not affect AF-dependent NFAT translocation.",
    "molecules": "zymosan, piceatannol"
  },
  {
    "caption": "C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (E) Whole-cell lysates of Syk or Ctrl. siRNA-treated cells were separated by SDS-PAGE, followed by Western blotting, and membranes were probed with anti-Syk and anti-β-actin antibodies.",
    "molecules": "zymosan"
  },
  {
    "caption": "F J774A.1 macrophages were pre-treated with piceatannol and infected with swollen conidia (MOI = 5). Phagocytosis was quantified by FACS 30 min after stimulation. Syk inhibition led to impaired phagocytosis of zymosan, but had no effect on phagocytosis of SC.",
    "molecules": "zymosan, piceatannol"
  },
  {
    "caption": "G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (G) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy.",
    "molecules": "zymosan, cytochalasin D, CytoD"
  },
  {
    "caption": "G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (H) For quantification of NF-κB translocation, nuclear extracts were separated by SDS-PAGE, followed by Western blotting. Membranes were probed with anti-NF-κB p65 and anti-HDAC1 antibodies.",
    "molecules": "zymosan, cytochalasin D, CytoD"
  },
  {
    "caption": "G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (I) After overnight stimulation, TNF-α was measured in the SN by ELISA. Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF, but not ionomycin (G). In addition, inhibition of phagocytosis lead to increased translocation of NF-κB (H) and increased levels of TNF-α in response to either zymosan or AF.",
    "molecules": "zymosan, cytochalasin D, CytoD, ionomycin"
  },
  {
    "caption": "A J774A.1 macrophages were pre-treated with bafilomycin A1 (100 nM) and stimulated with swollen conidia (MOI = 3) for 30 min. NFATc2 translocation was assessed by confocal microscopy. Acidification was required for NFATc2 translocation.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "G BMDMs from WT or MyD88−/−mice were pre-treated with ODN2088 (10 μM) and infected with swollen conidia (MOI = 5) for 30 min. NFATc2 translocation was quantified by confocal microscopy. In both, WT and MyD88−/− BMDMs, blocking TLR9 significantly impaired NFATc2 translocation.",
    "molecules": "ODN2088"
  },
  {
    "caption": "A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (A) After overnight stimulation, TNF-α was measured in the SN by ELISA.",
    "molecules": "LFM-A13"
  },
  {
    "caption": "A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (B) After 30 min of stimulation, NFATc2 translocation was quantified by confocal microscopy. Both TNF-α levels and NFATc2 translocation were significantly reduced by BTK blockade.",
    "molecules": "LFM-A13"
  },
  {
    "caption": "D, E J774A.1 macrophages were treated with a control, BTK (25 nM)- or TLR9 (75 nM)-targeting siRNA and additionally pre-treated with either ODN2088 (10 μM) or the BTK inhibitor LFM-A13 (12.5 μM). The cells were infected with swollen conidia (MOI = 5), and NFATc2 translocation was measured by confocal microscopy. Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade.",
    "molecules": "ODN2088, LFM-A13"
  },
  {
    "caption": "Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.A Representative FACS-phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples.",
    "molecules": "MCV1, FK506, SC514"
  },
  {
    "caption": "Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.B Inhibition of alveolar macrophage responses to AF with FK506, MCV1 or SC514 led to a significant reduction in TNF-α levels.",
    "molecules": "MCV1, FK506, SC514"
  },
  {
    "caption": "A, B Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52Δtap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1.",
    "molecules": "His"
  },
  {
    "caption": "C Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52Δtap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1.",
    "molecules": "HIS"
  },
  {
    "caption": " (B-E) FT-peptide competition ELISA to map the epitopes of the indicated Fabs. Peptides that strongly inhibit the binding of the Fabs to recPrP23‑231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes. Positive control: recPrP23‑231; negative controls: Neu, BSA, and the secondary antibody (2° Ab). ",
    "molecules": "Neu"
  },
  {
    "caption": " (C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity. ",
    "molecules": "Neu"
  },
  {
    "caption": "(A) Examples of sororin (dotted arrows) and SMC3ChIP-seq and BrdU DIP-seq data from early S-phase as compared to G2-phase using the Integrated Genome Browser (IGB; Nicol et al, 2009).(B) Alignment of sororin, BrdU and BrdU-negative control sequencing bins in early S-phase to humanchromosome 18 (GRCh37/hg19). Bars correspond to called peaks, curves to read enrichments after smoothing (k=25 kbp).(C) Quantification of the co-occupancy of BrdU incorporation and sororin localization in early S-phase and sororin in G2-phase depicted as p-value distributions calculated with IntervalStats (Chikina & Troyanskaya, 2012) and compared to randomized controls. Sororin peaks were identified as overlapping peaks of 4 samples in early S-phase and 2 samples in G2-phase, using only the common peaks.(D) Quantification of the co-occupancy of SMC3 in early S-phase and in G2-phase with BrdU incorporation in early S-phase.",
    "molecules": "BrdU"
  },
  {
    "caption": "(D) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen-induced Cre expression. Extracts were prepared either one or two days after release from the arrest as indicated. Chr, fractionated chromatin; sol, soluble proteins.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(E) Proliferation curve of cells with the indicated genotype after tamoxifen treatment.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(F) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(I) Western blot showing protein depletion efficiency. Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(J) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment. Representative images from every category are shown; size bar, 10 µm; error bars denote s.e.m.; n > 500 per genotype.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(A) Schematic illustration of the LAP-AID-tag integrated into the bacterial artificial chromosome containing the mouse Cdca5 locus.(B) Immunofluorescence staining of cells expressing sor-LAP-AID and the F-box helper protein after treatment with auxin. Antibodies against GFP mark the fusion protein and Aurora B antibody stains cells in G2/M-phase. Scale bar, 10 µm.(C) Quantification of immunofluorescence after treating cells with auxin. Error bars denote s.e.m.; a.u., arbitrary units.(D) Time course quantification of GFP immunofluorescence signal decrease after auxin addition.",
    "molecules": "auxin"
  },
  {
    "caption": "(E) Analysis of chromosome spreads after auxin treatment.",
    "molecules": "auxin"
  },
  {
    "caption": "(F) Western blot showing cell extracts after treatment with auxin.",
    "molecules": "auxin"
  },
  {
    "caption": "(A) Cell cycle distribution of immortalized Cdca5 Δ/Δ sor-LAP-AID fibroblasts after 24-hour incubation with aphidicolin and release.",
    "molecules": "aphidicolin"
  },
  {
    "caption": "(B) Western blot showing cell extracts after incubation with aphidicolin and release.",
    "molecules": "aphidicolin"
  },
  {
    "caption": "(B) Quantification of EdU-labeled prometaphase cells after treating cells with auxin and EdU at indicated time points. Error bars denote s.e.m.; n > 200 cells per condition.",
    "molecules": "EdU, auxin"
  },
  {
    "caption": "(C) Analysis of chromosome spreads after auxin and EdU treatment and Giemsa staining.",
    "molecules": "auxin"
  },
  {
    "caption": "(D) Representative picture of the most prominent phenotype class upon auxin and EdU treatment and immunofluorescence microscopy. Scale bar, 10 µm.(E) Analysis of chromosome spreads after auxin treatment and EdU and DAPI labeling. Error bars denote s.e.m.; n > 200 cells per condition.",
    "molecules": "EdU, auxin"
  },
  {
    "caption": "(A) Pancreatic cancer cells (PANC-1) and normal human fibroblasts (NHFs) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points, cells were incubated with WST-1 cell viability dye for 4 hours. Absorbance was read at 450 nm and results were expressed as a percent of the control. Values are expressed as mean ± SD from quadruplicates; (i) PANC-1 treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (ii) NHF treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (iii) PANC-1 treated with Green 1 (Interaction between each concentration: p = 0.9955; Control versus column factor: ****p<0.0001); (iv) NHF treated with Green 1 (Interaction between each concentration: p = 0.3642; Control versus column factor: *p = 0.0370)",
    "molecules": "Green 1, NSC 51046"
  },
  {
    "caption": "Following treatment with either Green 1(A) Pancreatic cancer cells (BxPC-3 and PANC-1) and normal human and fetal fibroblasts (NHFs and NFFs) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis. Brightly stained, condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis.",
    "molecules": "Green 1"
  },
  {
    "caption": "(B) PANC-1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046. Cells were counterstained with Hoechst 33342 dye and propidium iodide (PI) to visualize nuclear morphology and cell death. Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine.",
    "molecules": "NSC 51046, colchicine"
  },
  {
    "caption": "(A) Pancreatic cancer cells, human leukemia cells (Jurkat) and NHFs were incubated with Monodansylcadaverine (MDC) to stain autophagic vacuoles and propidium iodide (PI) for cell death to determine if autophagic induction led to cell death. Bright, punctate staining is indicative of autophagy, as seen in our positive control (Tamoxifen treated cells). Images were taken at 400X magnification on a fluorescent microscope.",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "(B) Western blot analyses of Beclin-1 expression and LC3 conversion were conducted on PANC-1 cells treated with 5.0 µM Green 1 at various time points. β-actin was measured as an internal control. Samples treated for 3 and 6 hours were compared to the early control, while the sample treated for 48 hours was compared to the 48-hour control. Results are expressed as mean ± SD from two independent experiments for both Western blots.",
    "molecules": "Green 1"
  },
  {
    "caption": "(A) Colchicine derivatives (Green 1 and NSC 51046) were incubated with porcine tubulin for an hour in a 96-well plate. The absorbance (OD 340 nm) was obtained every minute during the one hour incubation. Colchicine was used for comparison to the derivatives.",
    "molecules": "Green 1, Colchicine, NSC 51046"
  },
  {
    "caption": "(B) Isolated mitochondria from PANC-1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase (HRP). Results were compared to control untreated mitochondria, colchicine treated mitochondria and positive control, paraquat (PQ). Fluorescence readings were taken in 5 min intervals for 4 hours at Ex. 560 nm and Em.590 nm and expressed as relative fluorescence units (RFU). Results are a representative of three independent experiments.",
    "molecules": "Green 1, colchicine, paraquat, ROS"
  },
  {
    "caption": "Following acclimatization, CD-1 nu/nu mice were separated into two groups, one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS (10 µL in 200 µL PBS), while the second group received subcutaneous injections of Green 1 (10 mg/kg/day for a total volume of 10 µL Green 1/200 µL PBS) three times a week for a period of one month. (A) Mice weights were recorded twice a week for the duration of the study.",
    "molecules": "Green 1, Me2SO"
  },
  {
    "caption": "(K-L) Co-staining of phalloidin (F-actin reporter; magenta) and βH-spectrin (green) of the apical surface of a mesodermal cell at the onset of ventral furrow formation (K) and at a later time point (L) visualized by confocal microscopy. White dashed lines indicate the cell boundaries segmented based on the phalloidin-staining of sub-apical confocal sections. Scale bars: 5 μm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "(S-T) Two-photon microscopy images of apical βH-spectrin (green) 1.5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water (S) or the ROCK-inhibitor Y-27634 (T). Dashed boxes indicate photo-activated cells. Y-27634-injected embryos did not undergo apical constriction and did not accumulate βH-spectrin upon photo-activation, while water-injected embryos constricted and showed increased βH-spectrin levels. Scale bars: 10 µm.",
    "molecules": "water, Y-27634"
  },
  {
    "caption": "A HeLa-H2B/tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.",
    "molecules": "thymidine"
  },
  {
    "caption": "HeLadox-YFP-TIARr cells were transfected with control or TIAR siRNAs, and 24h hours later cell were cultured in the abscence or presence of 1 μg/ml doxocycline for 48 hours. The expression of p(S10)-H3, total H3 and TIAR was measured by Westen blot analysis. The graph shows the quantification of p-H3 levels normalized to total H3 (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.",
    "molecules": "dox, doxocycline"
  },
  {
    "caption": "F HeLadox-YFP-TIARr-RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.",
    "molecules": "dox"
  },
  {
    "caption": "G HeLadox-YFP-TIARr-dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.",
    "molecules": "dox"
  },
  {
    "caption": "C HeLa cells were transfected with control or TIAR siRNAs for 4 days, and subjected to colcemid for 2 hours prior to preparation of metaphase spreads. Chromosomes were stained with Hoechst (red) and anti-CENP-A antibody (green); the yellow arrowhead marks a chromosome break.",
    "molecules": "colcemid, Hoechst"
  },
  {
    "caption": "F HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA, and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads. The frequency of cells with scattered chromatids was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). G Metaphase spreads were prepared as in (F), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3). Data information , statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.",
    "molecules": "colcemid"
  },
  {
    "caption": "A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0.4 µM APH. Cells were fixed at regular time intervals, and p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SE, n = 3).",
    "molecules": "APH"
  },
  {
    "caption": "B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 2000 cells examined per experiment and condition). Each dot represents the signal from one cell, horizontal lines indicate mean values, and the blue area delineates cells above an arbitrarily chosen threshold. Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.",
    "molecules": "APH"
  },
  {
    "caption": "D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi (5 μM.) alone or together with Ro3306 (5 μM) for 12 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 1000 cells examined per experiment and condition). Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.",
    "molecules": "Ro3306"
  },
  {
    "caption": "E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours, and the sub-G1 population was quantified by flow cytometry following propidium iodide staining (mean ± SD, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.",
    "molecules": "propidium iodide"
  },
  {
    "caption": "F HeLa cells transfected with control or TIAR siRNAs were treated with APH, alone or in combination with Gö6976 (1µg/ml) or UCN-01 (300 nM). After 24 hours, p(S10)-H3-positive cells were quantified by flow cytometry, and expressed relative to the values in the untreated condition (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.",
    "molecules": "UCN-01, APH, Gö6976"
  },
  {
    "caption": "A HeLa cells, untreated or treated for 24 hours with 0.4 µM APH, were fixed with methanol and analyzed by IF microscopy after staining with anti-p(S10)-H3 antibody, anti-TIAR antibody and DAPI. Yellow arrows mark focal accumulation of TIAR in GMGs.",
    "molecules": "APH, DAPI, methanol"
  },
  {
    "caption": "HeLa cell were treated for 16 hours with (F) the CDK1 inhibitor Ro3306 (9 µM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.",
    "molecules": "DAPI, methanol, Ro3306"
  },
  {
    "caption": "HeLa cell were treated for 16 hours with (G) ICFR-193 (1 μM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition).",
    "molecules": "DAPI, ICFR-193, methanol"
  },
  {
    "caption": "A-G HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR antibody, DAPI in combination with (A) anti-PCNA-1 antibody, (B) anti-FANCD2 antibody, (C) anti-BRCA1 antibody, (D) anti-PRP19 antibody, (E) anti-Sm(Y12) antibody, (F) anti-HuR antibody and (G) pS2-RPOL2 (H5) antibody. Intensity profiles along the white line in the merged image are presented on the right side; yellow and green arrows mark GMGs.",
    "molecules": "APH, DAPI"
  },
  {
    "caption": "A HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR and anti-CDK1 antibodies. Intensity profiles along the white line in the merged image are presented on the right side; yellow arrows mark GMGs.",
    "molecules": "APH"
  },
  {
    "caption": "C HeLa cells transfected with control or TIAR siRNAs were treated with 0.4 µM APH 24 hours prior to fixation. Cells were processed for IF microscopy after staining with anti-PRP19 and anti-CDK1 antibodies. Intensity profiles from staining of the same cells with anti-TIAR and anti-CDK1 antibodies are depicted on the right side.",
    "molecules": "APH"
  },
  {
    "caption": "A) Representative specimen of colorectal (CRC) primary tumour stained with haematoxylin/eosin/saffron (HES), or antibodies against E-cadherin or Vimentin. i) The blue, orange and pink dotted lines highlight the normal mucosa, the submucosa and the muscularis propria respectively. Red dotted line highlights the neoplastic tissue. Black arrowheads indicate the direction of invasion. Boxed regions ii and iii show high magnification of normal colonic glands (ii) and the CRC invasive front (iii). Scale bar: 2 mm and 500 μm.",
    "molecules": "eosin, haematoxylin, saffron"
  },
  {
    "caption": "A) Representative confocal images of non-invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii, iii and iv are displayed at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars show off-centered nuclei. Scale bars: 20 μm.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "D) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel monitored by DIC microscopy over 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.",
    "molecules": "collagen-I"
  },
  {
    "caption": "A) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm.",
    "molecules": "Phalloidin, Hoechst"
  },
  {
    "caption": "B) Representative confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm.",
    "molecules": "Y27632, DAPI, H1152, Phalloidin"
  },
  {
    "caption": "C) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01).",
    "molecules": "Y27632, H1152"
  },
  {
    "caption": "E) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01).",
    "molecules": "Y27632, H1152"
  },
  {
    "caption": "F) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001). ",
    "molecules": "Y27632, collagen-I, DAPI, H1152, Phalloidin"
  },
  {
    "caption": "C) Representative images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. D Explants displaying (D) lumens from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).",
    "molecules": "Y27632, collagen-I, DAPI, H1152, Phalloidin"
  },
  {
    "caption": "F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.",
    "molecules": "Y27632, collagen-I"
  },
  {
    "caption": "A) Caco-2 cysts expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnifications. Arrowheads point to non-protruding cells. Arrow points to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The number of protrusive Caco-2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts (Means ± SEM of at least 3 independent experiments, paired t-test, ***p<0.001, *p<0.05 n.s: non significant). ",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "C) Caco-2 cysts stably expressing GFP alone or GFP-ROCK2DN#2, were submitted to a 2 days period invasion assay, then fixed and stained for F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White and black stars show nuclei that engage in protrusions. Scale bars: 20μm. D) Bargraph representing the percentage of protrusive cysts transduced with GFP (CTRL, with or without Y27632) or with ROCK2DN#2 (Means ± SEM of at least 3 independent experiments, paired t-test, **p<0.01 n.s: non significant). ",
    "molecules": "Y27632, DAPI, Phalloidin"
  },
  {
    "caption": "E) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole (arrows) reminiscent of collective invasion. T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus-ROCK2-DA and treated with doxycycline 1μg/ml. Scale bars: 20μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "F) Bar graph representing the percentage of protrusive T84 cysts in control (WT), treated with ROCK inhibitor (Y27632) or transduced with lentiviruses expressing GFP or GFP-ROCK2-DA after induction by Doxycycline (Means ± SEM of at least 3 independent experiments) (paired t-test, ***p<0.001, *p<0.05).",
    "molecules": "Y27632, Doxycycline"
  },
  {
    "caption": "C) Bar graph representing the percentage of mosaic Caco-2 cysts transduced with GFP or GFP-ROCK2DN#, with or without Y27632, harbouring a protruding morphology (Means ± SEM of at least 3 independent experiments, unpaired t-test, ***p<0.001, *p<0.05).",
    "molecules": "Y27632"
  },
  {
    "caption": "D) Quantification of the fate of the GFP positive cells, leader or follower, in mosaic cysts transduced with GFP-alone and treated with Y27632 (GFP+Y27632) or GFP-ROCK2DN#2 from 3 independent experiments (Means ± SEM, unpaired t-test, ***p<0.001).",
    "molecules": "Y27632"
  },
  {
    "caption": "E) Representative pull-down of RAC1-GTP by PAK-Crib, in Caco-2 cells treated with ROCK inhibitor Y27632, H1152 or blebbistatin and lysed at 12 or 16 hours. Immunoblot analyses were performed with anti-Rac1 and anti-GAPDH antibody from three independent experiments.",
    "molecules": "Y27632, blebbistatin, GTP, H1152"
  },
  {
    "caption": "F) DIC acquisition of fixed Caco-2 cysts treated with ROCK inhibitors alone (Y27632 or H1152), or in combination with RAC1 inhibitor NSC23766. Arrowheads point to non-protruding cells and arrow point to protruding cell. Scale bar 20 μm.",
    "molecules": "Y27632, H1152, NSC23766"
  },
  {
    "caption": "G) Bar graph representing the percentage of protruding Caco-2 cysts in control conditions (non-treated, NT) or treated with NSC23766, Y27632 or H1152 (Means ± SEM of at least 3 independent experiments, unpaired t-test, ****p<0.0001, ***p<0.001, *p<0.05",
    "molecules": "Y27632, H1152, NSC23766"
  },
  {
    "caption": "A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) .",
    "molecules": "Y27632, Hoechst"
  },
  {
    "caption": "B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01). ",
    "molecules": "Y27632, Phalloidin"
  },
  {
    "caption": "D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm. E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented corresponding to non-treated, Y27632 or H1152 treatments)",
    "molecules": "Y27632, H1152"
  },
  {
    "caption": "A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm. B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant). ",
    "molecules": "Y27632"
  },
  {
    "caption": "C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to \"dendritic\" protrusions. D) Bar graph representing the percentage of cysts with the \"invasive\" or \"dendritic\" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant). ",
    "molecules": "Y27632, Blebbistatin, DAPI, Phalloidin"
  },
  {
    "caption": "E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm. F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant). ",
    "molecules": "Blebbistatin, blebbistatin"
  },
  {
    "caption": "B, C Aβ40 and Aβ42 (pmol/g wet brain) in the FA-soluble brain extract from the same APP23mice showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 56) = 221.114, P < 0.001 and F(1, 56) = 370.947, P < 0.001, respectively).",
    "molecules": "FA"
  },
  {
    "caption": "Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 micesAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group.Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend (F(1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig S2 for details).Swedish sAPPβ showed an age-dependent increase in APP24mice following a linear trend (F(1, 84) = 11.264, P = 0.001).Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend (F(1, 18) = 68.980, P < 0.001).Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. For absolute values, see Supplementary Fig S2.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(i) Purified 6x-His-FMRP and various regions of β-catenin proteins fused to GST were incubated with glutathione-agarose beads. Purified GST or GST-fusion proteins were visualized by Western blotting using the GST antibody, and sizes of corresponding GST-fusion proteins were confirmed. Pulled-down FMRP proteins by GST fused β-catenin were detected by immunoblotting with the FMRP antibody. (ii) Interaction between FMRP and β-catenin was assessed in the presence or absence of RNA. HEK 293T cells were subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody, in the presence or absence of RNase A (10 µg/mL). The corresponding protein was detected in the precipitated immunocomplex by Western blot, and non-programmed rabbit IgG was used as a control. RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A",
    "molecules": "agarose, glutathione, His"
  },
  {
    "caption": "A. Co-sedimentation of protein/RNA complexes on a 10-50% sucrose gradient. Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients; peaks corresponding to the 40S and 60S subunits, 80S monosome, and polysomes were detected by UV absorbance at 254 nm, and indicated proteins in these fractions were detected by Western blot. Corresponding total cell lysate was used as input",
    "molecules": "sucrose"
  },
  {
    "caption": "(i) Precipitated proteins in A10 cell lysates with m7GTP-agarose beads were identified by Western blot analysis. eIF4E and tubulin were shown as positive and negative controls, respectively. Total lysates were used as input. (ii) A10 cell lysates were incubated with GTP-agarose beads, and none of the proteins tested were precipitated with the beads. Total lysates were used as input control",
    "molecules": "m7GTP, agarose, GTP"
  },
  {
    "caption": "(i) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to m7GTP-agarose pull-down as in (C)",
    "molecules": "m7GTP, agarose"
  },
  {
    "caption": "(ii) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to GTP-agarose pull-down as in (i). E. HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP-agarose pull-downs as in (C)",
    "molecules": "m7GTP, agarose, GTP"
  },
  {
    "caption": "F. HEK 293T cells lysates were subjected to m7GTP-agarose pull-downs as in (C) in the presence or absence of RNase A (10 µg/mL). RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis.",
    "molecules": "m7GTP, agarose"
  },
  {
    "caption": "Primary VSMCs (left panel), A10 (middle panel), and HEK 293T cells (right panel) were transfected with siRNAs targeting ctnnb1 (β-catenin) or scrambled RNA. After 36 hr, the cells were pulsed with 0.5 µM puromycin for 15 min and then harvested. The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody. β-catenin blots indicated efficacy of siRNA and actin was used as a loading control. The average puromycin/actin ratio (n=3) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation. A one-way ANOVA and Tukey post-hoc test was performed, with p-values indicated above the error bars",
    "molecules": "puromycin"
  },
  {
    "caption": "A10 cells were transfected with Flag-FMRP then treated with either cycloheximide or its solvent (DMSO) for 4 hr prior to fixation. β-catenin (green), Flag (FMRP, red) were visualized by immunofluorescence. Nucleus (blue) was identified by DNA staining with Hoechst 33342. Arrows indicate a loss of β-catenin signal from the perinuclear region that was seen in the control following cycloheximide treatment",
    "molecules": "DNA, Hoechst 33342, cycloheximide, DMSO"
  },
  {
    "caption": "A10 cells were transfected with Flag-FMRP as above and then pre-treated with rapamycin or solvent (DMSO) for 1 hr in serum-free media. Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β-catenin antibody. Non-programmed rabbit IgG was used as a control. Existence of Flag-FMRP in the immunocomplex was detected by Flag antibody and β-catenin by GFP antibody",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "A10 cells were transfected with Flag-FMRP followed by rapamycin treatment similar to (D). Cells were fixed and stained for β-catenin (green), Flag (FMRP, red), and nucleus (blue). A loss of β-catenin signal from the perinuclear region that was seen in the control is indicated by the arrows.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(Top panel) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were treated with 0.5 µM puromycin for 15 min and harvested for Western blot analysis. Total β-catenin levels are indicated, and actin was used as a loading control. The average puromycin/actin ratio (n=3) was graphed. Error bars indicate standard deviation. An independent samples t-test was performed (2-tailed), with the p-value indicated above the error bar. (Bottom panel) Cells were treated similarly to above and fixed and stained for β-catenin (green) and nucleus (blue) The experiment in (A) was repeated using A10 cells",
    "molecules": "puromycin"
  },
  {
    "caption": "(i) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were harvested and subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody. The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis. Non-programed rabbit IgG was used as a control. Efficacy of Wnt-3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β-catenin (S33/37/T41) by Wnt-3a. (ii) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. Cell lysates were then subjected to m7GTP-agarose pull-down analysis. eIF4E and tubulin were used as positive and negative controls, respectively, and phospho-β-catenin (S33/37/T41) Western blots were used as controls for Wnt-3a stimulation.",
    "molecules": "m7GTP, agarose"
  },
  {
    "caption": "Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5-/- or ATG16L1-/-. Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.",
    "molecules": "amino acid"
  },
  {
    "caption": "Immunofluorescence analyses of ATG16L1-/- and WIPI2-/- stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 hr in the presence or absence of 3'MA (and additional pretreatment for 30 min) followed by fixation and immuno-staining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.",
    "molecules": "3'MA, AA, amino acid"
  },
  {
    "caption": "Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm.",
    "molecules": "amino acid"
  },
  {
    "caption": "Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm.",
    "molecules": "phosphoinositides, rhodamine"
  },
  {
    "caption": "Microscopy-based protein-liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values.",
    "molecules": "PI3P, rhodamine"
  },
  {
    "caption": "ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell.",
    "molecules": "amino acid"
  },
  {
    "caption": "ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2 positive dots per cell.",
    "molecules": "amino acid"
  },
  {
    "caption": "WIPI2-/- cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag positive dots per puncta-positive cell.",
    "molecules": "amino acid"
  },
  {
    "caption": "ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Cells were amino acid starved (AA starved) to induce mTORC1-dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis.",
    "molecules": "amino acid, BafA1"
  },
  {
    "caption": "ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Mitophagy was induced by CCCP (10 μM) treatment for 6 hr prior to lysis.",
    "molecules": "CCCP"
  },
  {
    "caption": "ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Glucose starvation (Glu starved) for 20 hr was used to induce mTORC1-independent autophagy. BafA1 was added to all conditions 2 hr prior to lysis.",
    "molecules": "BafA1, Glu, Glucose"
  },
  {
    "caption": "ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LC3-associated phagocytosis (LAP)-like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 hr prior to lysis. Right panel shows quantification of LC3-II/LC3-I ratio.",
    "molecules": "CCCP"
  },
  {
    "caption": "ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LAP-like LC3 lipidation induced by monensin or ammonium chloride (NH4Cl) for 2 hr prior to lysis.",
    "molecules": "ammonium chloride, NH4Cl, monensin"
  },
  {
    "caption": "Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial protein",
    "molecules": "Coomassie"
  },
  {
    "caption": "Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values.",
    "molecules": "PI3P"
  },
  {
    "caption": "ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.",
    "molecules": "3'MA, AA, amino acid"
  },
  {
    "caption": "Cells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1.­­ Scale bar: 10 μm.",
    "molecules": "amino acid"
  },
  {
    "caption": "Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies.",
    "molecules": "amino acid, BafA1"
  },
  {
    "caption": "WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel.",
    "molecules": "amino acid, BafA1"
  },
  {
    "caption": "Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.",
    "molecules": "AA, amino acid, PI"
  },
  {
    "caption": "(D and E)  qRT-PCR  analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence (D) or presence (E) of translation-blocking GFP MO. Bar charts show the average of three independent biological replicates. Error bars show standard deviation (SD). Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.",
    "molecules": "MO"
  },
  {
    "caption": "(B-E) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the codon effects measured by PACE (y-axis). Each dot represents a single codon. Graphs for all codons (B), codons for polar amino acids (C), nonpolar amino acids (D), and charged amino acids (E) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.",
    "molecules": "charged amino acids, nonpolar amino acids, polar amino acids"
  },
  {
    "caption": "(F-I) Scatter plots showing a correlation between relative  ribosome density (28-30-nt footprints; x-axis) and the   tRNA  levels (y-axis, Each dot represents a single codon. Graphs for all  codons (F), codons for  polar amino acids  (G), nonpolar amino acids (H), and charged amino acids (I) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.",
    "molecules": "charged amino acids, nonpolar amino acids, polar amino acids"
  },
  {
    "caption": "(D)   Poly(A) tail analysis of  Asn AAC codon tag reporter mRNA at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a   poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).",
    "molecules": "poly(A), Poly(A)"
  },
  {
    "caption": "(F) Poly(A) tail analysis of  Leu CUG codon tag reporter mRNA  at 2 and 6 hpf. (G) Quantification of the  PAT assay  at 6 hpf in (F).",
    "molecules": "Poly(A)"
  },
  {
    "caption": "(D)  Poly(A) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a  poly(A)  tail. (E) Quantification of the PAT assay  at 6 hpf in (D).",
    "molecules": "Poly(A), poly(A)"
  },
  {
    "caption": "(B) Results of the tandem  ORF assay  with Lys AAG×8 and  Lys AAA×8 sequences. (C) Results of the  tandem ORF assay with  Gly×8 and Pro×8 sequences. (D) Results of the tandem  ORF assay  with Asn AAC and Asn AAU  codon tags. (E) Results of the tandem  ORF assay using EGFP ORFs with different codon optimalities. Data information: normalized Fluc activity with no insert was set to one. All experiments were repeated three times, and the average Fluc activity is shown as bar charts. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.",
    "molecules": "Gly, Pro"
  },
  {
    "caption": "(A) Localization  of endogenous SHP-1 in hematopoietic  and non-hematopoietic cells was determined by confocal imaging after staining with SHP-1  antibody. DAPI  was used for nuclear staining, Scale bar:5µm. Fluorescence intensities along the line drawn in a representative cell were obtained using Zen software and the co-localization plots were made using GraphPad (Red: SHP-1, Blue: DAPI).",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) SFB  empty vector, SFB SHP-1 or SFB PTPN7  was co-transfected with Myc  tagged H2B  in 293T  cells. The interaction  of H2B  with  SHP-1 was detected through immunoblotting  using Myc  antibody after  pull down  with streptavidin sepharose  beads.",
    "molecules": "sepharose, streptavidin"
  },
  {
    "caption": "(G) Glutathione Sepharose  beads immobilized with bacterially expressed recombinant GST  or GST-H2B  proteins were incubated with bacterially purified recombinant MBP-SHP-1. The association of SHP-1  with H2B  was detected by immunoblotting  with MBP  antibody. Expression of GST, GST-H2B  and MBP SHP-1 was shown by Coomassie staining.",
    "molecules": "Glutathione"
  },
  {
    "caption": "(A) Nuclear  extracts of WT and SHP-1  knockout cells (derived from two independent guide RNAs) were made and the levels of H2BUb  were detected.",
    "molecules": "Ub"
  },
  {
    "caption": "(B) Wild type (WT) 293T  cells along with SHP-1 KO cells transfected with either Myc  vector, Myc SHP-1 ∆SH2 or Myc SHP-1 NLS mutant were lysed.  H2BUb levels were determined using specific antibody.",
    "molecules": "Ub"
  },
  {
    "caption": "(C) WT and SHP-1 KO 293T  cells transfected with Myc  vector, Myc SHP-1 or Myc SHP-1  C/S (C453S) mutant were lysed. H2BUb levels were determined using specific antibody.",
    "molecules": "Ub"
  },
  {
    "caption": "(D) H2B  WT along with the indicated Y/F and Y/D mutants were transfected into 293T  cells. Histone extract was prepared from cells transfected with each construct and subjected to western blotting  with anti-H2B  and anti-H2BUb  antibodies.",
    "molecules": "Ub"
  },
  {
    "caption": "(E) In vitro ubiquitination assay was performed using bacterially purified GST, GST  tagged H2B  and its mutants as substrates. Recombinant E1, E2(UBE2A) and ubiquitin  were included in each reaction. Ubiquitination  was detected by H2BUb antibody.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "(F) In vitro ubiquitination  was performed using biotin  tagged non-phospho  and phospho  (pY121) peptide of H2B  as substrates in the presence of E1  and E2. H2BUb was detected by immunoblotting  with specific antibody.",
    "molecules": "biotin, Ub"
  },
  {
    "caption": "(A) In vitro phosphorylated GST H2B  by Syk  kinase was incubated with bacterially purified recombinant wild type SHP-1 or elution buffer and the amount of released phosphate  was assayed colorimetrically  using the malachite green reagent (A620 nm). Data represent mean absorbance from four independent experiments. Error bar indicates SD; *p<0.05, by Student's t test.",
    "molecules": "malachite green, phosphate"
  },
  {
    "caption": "(C) Phospho   (pH2B) Y121 and non-phospho   peptide of H2B  was incubated with constitutively active (SHP-1 ΔSH2) or catalytically inactive mutant (C/S) of SHP-1  and the released phosphate  was assayed colorimetrically  using the malachite green reagent (A620nm). n=3 independent experiments, Error bars indicate SD; ***p<0.001 (one-way ANOVA, Tukey's multiple comparison test).",
    "molecules": "malachite green, phosphate"
  },
  {
    "caption": "(F) Wild type (WT) along with SHP-1 KO 293T  cells transfected with either Myc  vector, Myc SHP-1 or Myc SHP-1 C/S mutant were lysed. Levels of pY121-H2B  and H2BUb were determined using specific antibodies.",
    "molecules": "Ub"
  },
  {
    "caption": "(G) 293T  Cells were treated with  SHP-1 inhibitor TPI  (50nm) for 1hr and levels of pY121-H2B  was determined using pY121H2B antibody.",
    "molecules": "TPI"
  },
  {
    "caption": "(A) Cell lysates derived from 293T  cells transfected with empty vector, SFB-tagged H2B, H2B  Y121F or H2B  Y121D constructs were pulled down with S-protein agarose  beads. RNF20 interaction with H2B   was detected by immunoblotting  with specific antibody.",
    "molecules": "agarose"
  },
  {
    "caption": "(C) Glutathione Sepharose  beads bound with bacterially expressed recombinant GST-H2B, GST H2B  Y121F and GST-H2B  Y121D proteins were incubated with bacterially purified recombinant His UBE2A, and interaction  of UBE2A  with H2B  was detected by immunoblotting  with His  antibody. Recombinant protein expression and pulldown  was shown by Coomassie staining.",
    "molecules": "Glutathione"
  },
  {
    "caption": "(F) HEK293T  cells were transfected with SFB  vector or SFB H2B  along with Myc UBE2A.Cells were treated with SHP-1  inhibitor TPI  (50nm) for 1hr after 24hrs of transfection. Cells lysates were subjected to pull-down  with S-protein beads, and western blotting was performed using specific antibodies.",
    "molecules": "TPI"
  },
  {
    "caption": "(E) Nuclear  extracts of cells treated with either DMSO  or  SHP-1 inhibitor TPI  (50nm) were used to detect total RNA Polymerase II and its modifications by immunoblotting  using specific antibodies.",
    "molecules": "DMSO, TPI"
  },
  {
    "caption": "(G) Immunofluorescence  of WT or SHP-1  knockout MCF7  cells labelled with the  5-Ethynyl Uridine (EU) for 60 minutes to capture nascent RNA  synthesis. Scale bars 10μm. (H) The EU incorporation  in MCF-7  WT and SHP-1 KO cells was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).",
    "molecules": "5-Ethynyl Uridine, EU, RNA"
  },
  {
    "caption": "(I) Representative images of immunofluorescence  of DMSO  or TPI  treated (50nm) MCF7  cells labelled with EU  for 60 minutes was shown. Scale bar: 40µm. (J) The EU incorporation  in MCF7  cells treated with DMSO  or TPI  was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).",
    "molecules": "EU, DMSO, TPI"
  },
  {
    "caption": "(C, D) MCF-7  WT cells along with SHP-1  KO cells transfected with either vector, H2B  Y121F or H2B  Y121D mutant were used for ChIP q-PCR  analysis of the H2BUb at indicated genes. The data shown is derived from two independent experiments. Error bars indicate the mean ± SD.",
    "molecules": "Ub"
  },
  {
    "caption": "Expression of Mpc proteins in mitochondria assessed by SDS-PAGE and immunoblot analysis of crude mitochondrial preparations. The individual untagged Mpc1 (Mpc1), histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc3 (Mpc3his) or the Mpc1/Mpc3 hetero-complex (Mpc1/Mpc3his) were detected with antibodies raised against Mpc1 (left panel) or Mpc3 (right panel) and are shown with dashed arrows.",
    "molecules": "his, histidine"
  },
  {
    "caption": "The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM-adduct formation. The thermal denaturation profiles (left) were used to calculate the first derivative (right), which provides the apparent melting temperature, indicated with the same colour coding.",
    "molecules": "CPM"
  },
  {
    "caption": "Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers.",
    "molecules": "Nickel"
  },
  {
    "caption": "SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein-detergent-lipid complex (PDL, green), the detergent-lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS-PAGE and visualised by Coomassie Blue staining (B, inset).",
    "molecules": "lipid"
  },
  {
    "caption": "Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis.",
    "molecules": "Nickel"
  },
  {
    "caption": "Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n=8) in comparison to empty liposomes (n=6) at a ΔpH of 1.6.",
    "molecules": "pyruvate"
  },
  {
    "caption": "Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n=3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25-600 μM. The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats.",
    "molecules": "pyruvate"
  },
  {
    "caption": "Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n=4) compared to empty liposomes (n=4).",
    "molecules": "pyruvate"
  },
  {
    "caption": "In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n=4).",
    "molecules": "pyruvate"
  },
  {
    "caption": "Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1-100 μM), Zaprinast (1-1000 μM), lonidamine (10-10000 μM) and 7ACC2 (5-500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099 Zaprinast and 7ACC2 (average IC50 9 ± 7 μM, 18 ± 8 μM and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50, 118 ± 24 μM).",
    "molecules": "7ACC2, 14C, lonidamine, pyruvate, UK5099, Zaprinast"
  },
  {
    "caption": "[14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n=6).",
    "molecules": "14C, pioglitazone, pyruvate, rosiglitazone"
  },
  {
    "caption": "Time course of pyruvate homo-exchange in proteoliposomes at a ΔpH of 1.6 was compared for Mpc3 (n=4), the Mpc1/Mpc3 hetero-complex (n=6) and empty liposomes (n=4).",
    "molecules": "pyruvate"
  },
  {
    "caption": "Time course of pyruvate homo-exchange at a ΔpH of 1.6 was compared for Mpc1 (n=4), Mpc1/Mpc3 hetero-complex (n=4) and empty liposomes (n=4).",
    "molecules": "pyruvate"
  },
  {
    "caption": "Thermostability analysis via cpm. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc3 (blue lines) (n=3).",
    "molecules": "cpm, Zaprinast"
  },
  {
    "caption": "Thermostability analysis via nanoDSF. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc1 (orange lines) (n=3).",
    "molecules": "Zaprinast"
  },
  {
    "caption": "Example of a 384-well plate treated with compounds. The panel shows the staining with filipin (free cholesterol in green pseudo-colour) and anti-LBPA antibodies (red). The leftmost column shows control wells treated with DMSO only, and the right penultimate column shows wells treated with U18666A.",
    "molecules": "free cholesterol, DMSO, filipin, LBPA, U18666A"
  },
  {
    "caption": "Effects of U18666A, thioperamide and trimeprazine, compared with controls. Cells treated with the indicated compounds or with DMSO alone for 18h at 37°C, were processed for immunofluorescence microscopy. Low magnification views are shown after staining with anti-LBPA antibodies (red), filipin (green pseudo-color) and DAPI (blue). The scale bar is 20 µm.",
    "molecules": "DAPI, DMSO, filipin, LBPA, thioperamide, trimeprazine, U18666A"
  },
  {
    "caption": "Screen data plot. The plot shows the integrated intensity of LBPA vs. filipin (cholesterol) staining in all HeLa cells of each well analyzed in the screen (each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound). Only compounds that showed less than 20% toxicity are plotted. The cells treated with U18666A, thioperamide maleate and trimeprazine tartrate are indicated, as well as the DMSO controls.",
    "molecules": "cholesterol, DMSO, filipin, LBPA, thioperamide maleate, trimeprazine tartrate, U18666A"
  },
  {
    "caption": "Integrated intensity of cholesterol and LBPA staining. The integrated intensity of cholesterol (filipin in green) and LBPA (red) staining in all cells of the indicated samples is quantified after normalization to the DMSO controls. (n=3 independent experiments, 64 images analysed per experiments, error bars = SD, two ways ANOVA; **=p<0.01; ***=p<0.005).",
    "molecules": "cholesterol, DMSO, filipin, LBPA"
  },
  {
    "caption": "Thioperamide treatment of tissue culture cells. BHK, CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C. Low magnification views are shown after staining with anti-LBPA antibodies (green) and DAPI (blue). n=3 independent experiments with > 200 images acquired and analysed automatically.",
    "molecules": "DAPI, LBPA, Thioperamide, thioperamide"
  },
  {
    "caption": "Cholesterol quantification by mass spectrometry. A431 cells were treated with DMSO alone, pitolisant 10µM (Pito), thioperamide 10µM (thio) or U18666A 10µM (U18) for 18h at 37°C. After extraction, free cholesterol, cholesteryl esters and total cholesterol were quantified by mass spectrometry, and normalized to the DMSO controls. (n=3 independent experiments, error bars = SD, one-way ANOVA, ****=p<0.0001",
    "molecules": "Cholesterol, cholesterol, free cholesterol, cholesteryl esters, DMSO, Pito, pitolisant, thio, thioperamide, U18, U18666A"
  },
  {
    "caption": "Screen data plot of the cholesterol content of LBPA-endosomes. Automated unbiased quantification of the filipin integrated fluorescence signal (cholesterol content) in LBPA-containing endosomes, after treatment with each compound of the Prestwick library. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The fluorescence signal of LBPA-endosomes was used to segment the imaged and generate a mask, which was then applied on the micrographs to quantify the integrated intensity of filipin staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated.",
    "molecules": "cholesterol, DMSO, filipin, LBPA, thioperamide, trimeprazine, U18666A"
  },
  {
    "caption": "Immunogold labelling with anti-LBPA antibodies. HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy. Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A-gold. Multivesicular endosomes are pseudocoloured (see Fig EV1 for uncoloured images). Bars, 200nm. The data in (A) were quantified in a double-blind analysis of two sets of 16 micrographs for each condition; the number of gold particles per endosome is shown in a scatter plot (B) for each endosome identified without any bias in each micrograph of the two control (DMSO) and thioperamide-treated (THIO) samples.",
    "molecules": "protein A-gold, DMSO, gold, LBPA, THIO, thioperamide"
  },
  {
    "caption": "Distribution of LBPA-containing endosomes in the perinuclear region. Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells, after treatment with each compound of the Prestwick library as in Fig 2C. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell, calculated from the nuclei DAPI-staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated. Number of perinuclear LBPA endosomes. The number of individual LBPA-positive structures was measured in the perinuclear region as in (C). The z-factors are shown to evaluate the distribution of endosomes containing LBPA; color code as in (C).",
    "molecules": "DAPI, DMSO, LBPA, thioperamide, trimeprazine, U18666A"
  },
  {
    "caption": "Effect of Pitolisant on LBPA. HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37°C were labeled with DAPI and anti-LBPA antibodies and analysed by fluorescence microscopy (quantification in fig EV3A).",
    "molecules": "DAPI, LBPA, Pitolisant, pitolisant"
  },
  {
    "caption": "LBPA intensity in a mixed population of cells expressing HRH3-GFP. After transfection with HRH3-GFP, uncloned stably expressing cells (C) were labelled with anti-LBPA antibodies and analysed by automated microscopy (C). Unbiased quantification (D) shows the inverse correlation between HRH3-GFP expression (high expressing cells in green) and the endosome integrated intensity of LBPA staining (high LBPA labelling in red). (n=2 independent experiments with 500,000 cells analysed automatically, error bars = SD).",
    "molecules": "LBPA"
  },
  {
    "caption": "LBPA intensity in cells expressing or not HRH3-GFP. HeLa MZ cells stably expressing HRH3-GFP grown in 96-well plates were separately treated, with 4 different siRNAs that target HRH3 (siHRH3#1; siHRH3#2; siHRH3#3; siHRH3#4) or with control non-target (siNT) siRNA (E). HRH3-GFP was analysed by fluorescence microscopy in 4 rows of cells per condition; micrographs are stitched together in the montage. Panel F shows an example of cells treated with siHRH3#2 or siNT, and then labelled with DAPI and antibodies against LBPA. Cells treated as in (E) and labelled with DAPI and antibodies against LBPA as in (F), were analysed by automated microscopy and the integrated intensities of LBPA (left panel) and HRH3-GFP (right panel) signals are compared (G). (n=2 independent experiments with 192 images acquired and analysed automatically, error bars = SD).",
    "molecules": "DAPI, LBPA"
  },
  {
    "caption": "Treatment of NPC fibroblasts and NPC null mice with thioperamide. Fibroblast lines obtained from patients with well-established heterozygote mutations in the NPC1 (GM17912 line: NPC1 P1007A / T1036M; GM17911 line: NPC1 I1061T / T1036M) or with a homozygote mutation in the NPC2 gene (GM17910 line: C93F / C93F) were treated or not for 72h with thioperamide 10µM, stained with filipin (cholesterol) and analysed by fluorescence automated microscopy.",
    "molecules": "cholesterol, filipin, thioperamide"
  },
  {
    "caption": "Quantification of cholesterol and LBPA staining in NPC fibroblasts. Cells as in (A) were treated or not with thioperamide for 48h or 72h, labelled with filipin (cholesterol) and anti-LBPA antibodies, and analysed by fluorescence automated microscopy. The panels show the integrated intensity of the filipin (left) and LBPA (right) signals were quantified after 48 (top) and 72h (bottom). The colour code of each fibroblast cell line is as in (A). (n=3 independent experiments with 144 images acquired and analysed automatically, >2000 cells per experiment, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001).",
    "molecules": "cholesterol, filipin, LBPA, thioperamide"
  },
  {
    "caption": "Quantification of cholesterol in NPC fibroblasts by mass spectrometry. NPC cell lines (A) were treated or not with thioperamide or 0.1% (2-Hydroxypropyl)-ß-cyclodextrin for 72h. After extraction, lipids were analysed and quantified by mass spectrometry. (n=3; error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001)",
    "molecules": "2-Hydroxypropyl)-ß-cyclodextrin, cholesterol, lipids, thioperamide"
  },
  {
    "caption": "Quantification of cell counts in the 3 NPC cell lines treated with thioperamide. The experiment was as in (A-C) except that nuclei were labelled with DAPI and quantified by automated microscopy. Data are normalized to DMSO control (n=2 independent experiments, 75 images per experiments)",
    "molecules": "DAPI, DMSO, thioperamide"
  },
  {
    "caption": "Effects of thioperamide on cholesterol-dependent transcriptional regulation. The parental HeLa MZ cells, NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18h. Total mRNA was extracted and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT-PCR. (n=3 independent experiments, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005)",
    "molecules": "cholesterol, thioperamide"
  },
  {
    "caption": "Quantification of cholesterol in NPC null mice by enzymatic analysis. WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed. The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue. The total content of LBPA in the same liver extracts was quantified by mass spectrometry (see Appendix Figure S1), and is therefore expressed as a percentage of total phospholipids. The total LBPA content of WT mice was only marginally increased by the drug, either because the doses were low, or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level (despite much effort, we were unable to visualize LBPA in liver samples by immunocytochemistry). The relative ratio of LBPA vs. cholesterol values is shown.",
    "molecules": "cholesterol, LBPA, phospholipids, thioperamide"
  },
  {
    "caption": "(C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the \"natural dynamics\" Nutlin-3 dosing regimen.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(E-F) p53-Venus expression in response to the \"natural dynamics\" Nutlin-3 dosing regimen. Single cell traces (gray; E) and the mean (red; E) are shown for p53-Venus expression in response to each Nutlin-3 regimen. Heat map (F) shows an alternative representation of all traces as shown in (E). N = 51 cells.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the \"natural dynamics\" Nutlin-3 dosing regimen.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "Dose-response curves representing the rate of mCherry expression from the MDM2 (pink dots) and CDKN1A (purple dots) promoters as a function of total p53 levels. Values of expression levels were averaged across all cells in the first 15-h response to the long duration Nutlin-3 dosing regimen. Solid black lines represent the best fit to a Hill function model of promoter activation. Dashed lines indicate the half-maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(H) Mean nuclear MDM2-YFP levels in single cells at times corresponding to the first pulse (5.5 h) or second pulse (11 h) of p53 expression in response to natural and long duration Nutlin-3 dosing regimens. N = at least 40 cells per condition, error bars = SEM.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "Percentage of cells undergoing cell division (D) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "Percentage of cells positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "B. Stoichiometry measurement. Fluorescence scan of an SDS-PAGE gel analysis of the T4SS3-10+D4 complex where cysteine residues were reacted with AlexaFluor 633 C5 maleimide. Signals corresponding to the proteins coupled to AlexaFluor 633 were detected at a wavelength of 633 nm. Fluorescent Trw protein bands are labelled and the derived stoichiometry for TrwB/VirD4 is indicated.",
    "molecules": "AlexaFluor 633, cysteine, maleimide"
  },
  {
    "caption": "(B) BMDMs were infected with Mtb-Δeis in the absence or presence of 3-methyladenine (3-MA; 10 mM) and subjected to confocal analysis as described in Figure 1A. LC3-punctated cells were counted manually. Each condition was assayed in triplicate, and at least 250 cells were counted in each well. ***p<0.001, vs. SC.",
    "molecules": "3-methyladenine"
  },
  {
    "caption": "(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs were infected with Mtb-Δeis in the presence or absence of 3-MA (10 mM) or bafilomycin A1 (Baf-A1; 100 nM). Gel images representative of three experiments are shown. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane (C and E). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (E)).",
    "molecules": "3-MA, bafilomycin A1"
  },
  {
    "caption": "(C and D) BMDMs were stimulated with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 30 min. Cells were then incubated with 10 µM DHE or 5 µM DCFH-DA for 15 min, washed thoroughly, and immediately analyzed for superoxide or H2O2 production by flow cytometry (C, Left). Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry (D, top). Quantitative analysis of ROS generation (C, right; D, bottom). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.",
    "molecules": "H2O2, ROS"
  },
  {
    "caption": "(A-C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom)",
    "molecules": "catalase, DPI, NAC, tiron"
  },
  {
    "caption": "(D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis-infected condition without inhibitors (D). UI, uninfected.",
    "molecules": "3-MA, Staurosporine, zVAD-fmk"
  },
  {
    "caption": "(C and D) BMDMs were pretreated with U0126 (5, 10, 20 µM), SB203580 (SB; 1, 5, 10 µM), or SP600125 (SP; 5, 10, 20 µM) for 45 min, and then infected with Mtb-Δeis for 30 min (C) or 36 h (D). (C) Cells were then incubated with DHE for 15 min, washed rapidly and thoroughly, and analyzed immediately for superoxide levels by flow cytometry. Quantitative DHE fluorescence data represent the mean±SD of four experiments. (D) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy. (",
    "molecules": "U0126, SP600125, SB203580, superoxide"
  },
  {
    "caption": "(E) Raw264.7 cells were transfected with siRNA specific for JNK1 (siJNK) or a non-specific control siRNA (siNS). At 24 h after transfection, cells were infected with Mtb-Δeis for 36 h. Cell death was then assessed by PI staining, and then examined by flow cytometry. Transfection efficiency was assessed by RT-PCR (inset). Data represent the mean±SD of five random fields and are representative of three independent experiments (D and E). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition (B); SC (C and D); siNS (E). UI, uninfected; SC, solvent control (0.1% DMSO).",
    "molecules": "DMSO"
  },
  {
    "caption": "(A) Intracellular superoxide production was analyzed by flow cytometric analysis. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of recombinant Eis protein (rEis; 5, 10, 20 µg/mL) or 30 kDa Mtb antigen (30 k; 5, 10, 20 µg/mL). Upper, representative flow cytometric analysis; lower, quantitation of superoxide generation.",
    "molecules": "superoxide"
  },
  {
    "caption": "(B) THP-1 cells were transfected with mock, Eis-WT, or Eis-ΔAT constructs, and infected with Mtb-Δeis for 30 min. Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometric analysis. Inset, transfection efficiency.",
    "molecules": "H2O2, superoxide"
  },
  {
    "caption": "(E and F) THP-1 cells transfected with mock, Eis-WT, or Eis-ΔAT constructs were pretreated with SP600125 (SP; 20 µM) or SB203580 (SB; 5 µM) for 45 min before infection with Mtb-Δeis for 30 min (E) or 18 h (F). E, Intracellular superoxide production was analyzed by flow cytometric analysis. F, ELISA analysis for TNF-α and IL-6 levels. Data are presented as the mean±SD of three experiments. **p<0.01, ***p<0.001, vs. SC (A, C, E, and F); mock control (B and D). UI, uninfected; SC, solvent control (0.1% DMSO).",
    "molecules": "SP600125, DMSO, SB203580, superoxide"
  },
  {
    "caption": "E. 4sUDRB mapping of transcription kinetics through IGVL. Representative locations of qPCR amplicons (blue) are shown relative to the TSS of IGVL (distance is shown in parenthesis). qPCR signals were normalised to the enrichment at time zero (before release). Error bars represent 1SD of qPCR triplicates.",
    "molecules": "4sU, DRB"
  },
  {
    "caption": "A. Location of ssDNA tracts ≥ 8nt in IGVL in wild type (WT) and h3.3 cells detected by bisulphite sequencing. Data is from three independent experiments, the results from each being shown as black, red and green bars respectively. ssDNA on the coding strand is shown above the marker line (numbered relative to the TSS at 0) and on the template strand below. In the centre, the percentage of G and C bases on the coding strand in a 30 bp rolling average window is shown.B. Quantitation of the bisulphite detection of single stranded DNA in wild type, h3.3 and h3.3 complemented cells, normalised to wild type. p-values calculated with a  test. Sequences analysed: Wild type n = 322, h3.3n = 320 and h3.3 + H3.3 n = 95.",
    "molecules": "single stranded DNA, ssDNA"
  },
  {
    "caption": "C. Bisulphite detection of single stranded DNA in wild type cells with and without RNAseHI overexpression. p-value calculated with a chi‐square test. Sequences analysed: RNAseH1+ve n = 94, RNAseH1-ve n = 90.",
    "molecules": "single stranded DNA"
  },
  {
    "caption": "A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP-positive cells was determined 16 h later by flow cytometry.",
    "molecules": "doxycycline"
  },
  {
    "caption": "B LDH release from HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. At 24 h post transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells.",
    "molecules": "doxycycline"
  },
  {
    "caption": "C-D. PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng/ml doxycycline for 8 h and the level of PI staining (C) or LDH release (D) was determined.",
    "molecules": "doxycycline, PEGs"
  },
  {
    "caption": "E-F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight (numbers on the X-axis, 30 mM final concentration).* p<0.05 as determined by student's t test. Graphs show mean and s.d. of quadruplicate wells. Data are representative of at least three independent expeirments.",
    "molecules": "LPS, PEGs"
  },
  {
    "caption": "A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10-40 minutes with log phase S. typhimurium (MOI=50).",
    "molecules": "LPS"
  },
  {
    "caption": "B. Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked.",
    "molecules": "DSS"
  },
  {
    "caption": "C. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4C for 1 h at 120,000g.",
    "molecules": "DMPC, polar lipid extract"
  },
  {
    "caption": "A-I. Dye release time courses from liposomes as a percentage of maximal release. A. Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli polar lipid extract were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line.",
    "molecules": "6-carboxifluorescein, polar lipid extract"
  },
  {
    "caption": "A-I. Dye release time courses from liposomes as a percentage of maximal release. B. Five different reactions, where 130 nM of GSDMD and 400μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli extract polar lipid were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line.",
    "molecules": "6-carboxifluorescein, polar lipid"
  },
  {
    "caption": "A-I. Dye release time courses from liposomes as a percentage of maximal release. C. Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of (nM): 520, 260,100 (colored dark to light green).",
    "molecules": "6-carboxifluorescein, lipid"
  },
  {
    "caption": "A-I. Dye release time courses from liposomes as a percentage of maximal release. E. Two different sets of reactions, where wild type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130 and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes. The time point of 60 minutes is highlighted by a vertical dashed line.",
    "molecules": "6-carboxifluorescein"
  },
  {
    "caption": "A-I. Dye release time courses from liposomes as a percentage of maximal release. G-I. Dye release from 400 μM liposomes loaded with the 6-carboxifluorescein-derivates FD-20, FD-40 and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes.",
    "molecules": "6-carboxifluorescein, FD-150, FD-20, FD-40"
  },
  {
    "caption": "A-C. Cryo-EM micrographs of GSDMDNterm pores in E. coli extract polar lipids liposomes. The micrographs were acquired at protein/lipid molar ratios of 1/1000, 1/500 and 1/100, respectively. Black arrows indicate ring-shaped structures corresponding to oligomeric GSDMDNterm pore forms. Scale bars = 80 nm.",
    "molecules": "polar lipids"
  },
  {
    "caption": "A. AFM topograph of GSDMDNterm bound to lipid membranes of E. coli extract polar lipids. Overview topograph showing arc-, slit- and ring-like GSDMD oligomers.",
    "molecules": "polar lipids"
  },
  {
    "caption": "F-H. Analysis of GSDMDNterm oligomers bound to lipid membranes of E. coli extract polar lipids. F. GSDMDNterm oligomers assembled into arcs, slits and rings leading to transmembrane (TM) pores or not transmembrane aggregate (n = 218). G. Height of GSDMDNterm oligomers protruding from the lipid membrane. Bars represent mean and error bars represent s.e.m. (n = 218). H. Distribution of the diameters of rings formed by GSDMDNterm oligomers. The average distribution was 21.2 ± 5.6 nm (n = 164; average ± s.d.) and the bin size was 3 nm.",
    "molecules": "polar lipids"
  },
  {
    "caption": "(D) The nuclei with condensed, intermediate, or decondensed heterochromatin status, as marked by DAPI staining and H3K27me signals. (E) Percentages of nuclei with condensed, intermediate, or decondensed heterochromatin signals in the wild type and the arid2/3/4 mutant. n=113.",
    "molecules": "DAPI"
  },
  {
    "caption": "Knockdown of components of the BRM complex reduces Su(H) recruitment both in Notch-OFF (B) and Notch-ON (C) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with brm, Snr1 or GFP RNAi as a control. Notch-ON conditions (C) were induced by 30 minutes of EGTA treatment. Mean +/- SEM, n = 3 (B); Mean, n = 2 (C); * p<0.05 with one-tailed student's t-test compared to GFP RNAi control.",
    "molecules": "EGTA"
  },
  {
    "caption": "Effect of brm RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) induction by Notch activation (EGTA treatment) measured by reverse transcription-qPCR; shown as fold difference to lacZ RNAi control. Mean +/- SEM; n = 3.",
    "molecules": "EGTA"
  },
  {
    "caption": "Effect of Brm dominant-negative on expression of E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) measured by reverse transcription-qPCR. Expression was analyzed in stable cell lines containing pMT-inducible BrmWT or BrmK804R in the absence (left, uninduced) or presence of copper sulfate (right, Cu2+ induced). The response of E(spl)mβ-HLH and E(spl)m3-HLH to Notch activation ("N-On" = EGTA treatment vs. "N-Off" = PBS control) was reduced in the BrmK804R-expressing cells compared to BrmWT-expressing cells, only when induced with copper (right graph). Mean, n = 2 (left); Mean +/- SEM, n = 3 (right); * p<0.05 with one-tailed student's t-test comparing BrmWT and BrmK804R.",
    "molecules": "copper, Cu2+, copper sulfate, EGTA"
  },
  {
    "caption": "Knockdown of actin-related subunit, BAP55, reduces Su(H) recruitment in both Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with Bap55 or lacZ RNAi as a control. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.",
    "molecules": "EGTA"
  },
  {
    "caption": "Effect of Bap55 RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) expression levels measured by reverse transcription-qPCR in Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Expression level of RpII215 is shown as a control gene. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.",
    "molecules": "EGTA"
  },
  {
    "caption": "Nucleosome turnover measured by CATCH-IT-qPCR; fold enrichment over input samples compared to Sec15 tr control region. Su(H)-bound enhancers show increased nucleosome turnover in response to Notch signaling. Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT-NICD with copper excluded in the control. Positions of E(spl)-C primers are shown in Fig 3A; the remaining primers are control non-Notch-responsive regions. Mean, n = 2.",
    "molecules": "copper"
  },
  {
    "caption": "brm RNAi reduces incorporation of histone H3.3. V5 ChIP-qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3-V5 or H3.3-V5 expression induced from the pMT promoter by 3 hours of copper treatment, shown as fold enrichment over input samples. Mean +/- SEM; n = 3. * p<0.05 with two-tailed Welch's t-test compared to lacZ RNAi control.",
    "molecules": "copper"
  },
  {
    "caption": "C Cold response of cytosolic  free Ca2+  concentration in root cells expressing NES-YC3.6. Superimposition of representative normalized ratio variations for wild-type, oscrt3-1, and the complemented transgenic pOsCRT3 root  cells. The rectangle represents the regions of interest (ROIs) for the ratio measurements. The blue background indicates duration of cold treatment. Bar: 50 μm.",
    "molecules": "Ca2+"
  },
  {
    "caption": "A OsCIPK7 showed autophosphorylation  activity but could not phosphorylate OsCRT3 in vitro. The loaded protein is indicated by red arrows. Autoradiograph  of  γ-32P-labeled OsCIPK7 indicates autophosphorylation  of  OsCIPK7.",
    "molecules": "γ-32P"
  },
  {
    "caption": "B OsCIPK7 didn't phosphorylate OsCBL7 or OsCBL8 in vitro. Loaded proteins stained  with Coomassie brilliant blue  (CBB) are shown in the top panel and indicated by red arrows. Autoradiograph  of γ-32P-labeled myelin basic protein  and OsCIPK7 are shown in the bottom panel.",
    "molecules": "CBB, Coomassie brilliant blue, γ-32P"
  },
  {
    "caption": "C The  kinase activity OsCIPK7  was enhanced with increasing concentrations of OsCRT3 protein. SDS-PAGE gel showing CBB-stained OsCRT3, myelin basic protein, and OsCIPK7  (top panel). γ-32P-labeled myelin basic protein and OsCIPK7 substrate phosphorylation  (bottom panel). The amount of OsCRT3 added was 0 μg, 0.2 μg, 0.4 μg, 0.6 μg, 0.8 μg in the SDS-PAGE gel lanes, respectively.",
    "molecules": "γ-32P"
  },
  {
    "caption": "(E) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings.",
    "molecules": "Auxin"
  },
  {
    "caption": "(E) Sensitivity of makr5 mutant roots to continuous brassinolide (BL) treatment (7-day-old seedlings).",
    "molecules": "brassinolide"
  },
  {
    "caption": "(B) Cells were treated for 30 min with 100 µM cycloheximide or 100 µM MG132. Expression of tagged proteins was analyzed by anti-GFP or anti-Flag blotting of whole cell extracts. Five times less extracts were analyzed for ltn1-ΔRing-Flag cells. A nonspecific band served as a loading control. White lanes indicate that intervening lanes have been spliced out. WT, wild type.",
    "molecules": "cycloheximide, MG132"
  },
  {
    "caption": "E Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).",
    "molecules": "DAPI"
  },
  {
    "caption": "A Localization on the lnc-SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc-SMaRT pull-down experiment.",
    "molecules": "biotinylated"
  },
  {
    "caption": "A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.",
    "molecules": "lnc-SMaRT, Mlx, SMaRT, SYBR, SYBR GOLD, γ-oligo, N-methyl mesoporphyrin IX, NMM, RNA"
  },
  {
    "caption": "B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel. Oligos were treated as described. °C = oligos were heated (+) or were not heated (-) at 100 °C for 10 min and loaded after slow cooling. KCl= oligos were incubated (+) or were not (-) in a buffer containing 100mM KCl (for G-quadruplex stabilization). The gel was stained with total RNA staining (SYBR GOLD). Data information: Representative gels from at least three independent experiments are shown.",
    "molecules": "SYBR GOLD, KCl, RNA"
  },
  {
    "caption": "C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.",
    "molecules": "SMaRT, γ-oligo, N-methyl mesoporphyrin IX, NMM"
  },
  {
    "caption": "V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.",
    "molecules": "L-arabinose"
  },
  {
    "caption": "V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.",
    "molecules": "ethanol"
  },
  {
    "caption": "V. cholerae wild-type and ΔrpoE strains carrying PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.",
    "molecules": "ethanol"
  },
  {
    "caption": "V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.",
    "molecules": "ethanol"
  },
  {
    "caption": "V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).",
    "molecules": "L-arabinose, RNA"
  },
  {
    "caption": "V. cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD-rpoE or an empty vector control (pCtr) were grown to OD600 of 1.5 and L-arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT-PCR.",
    "molecules": "L-arabinose, RNA"
  },
  {
    "caption": "V. cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to an OD600 of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.",
    "molecules": "RNA"
  },
  {
    "caption": "E. coli wild-type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS-PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kanR and ompC::kanR for specific assignment of OmpA and OmpC bands.",
    "molecules": "kan, RNA"
  },
  {
    "caption": "V. cholerae wild-type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.",
    "molecules": "ethanol"
  },
  {
    "caption": "V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.",
    "molecules": "ethanol"
  },
  {
    "caption": "V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, Sel3) were cultivated in LB to OD600 of 2.0. Membrane fractions were analyzed by SDS-PAGE. The indicated bands were analyzed by mass spectrometry.",
    "molecules": "ethanol"
  },
  {
    "caption": "V. cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1-15) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with RNAPα and 5S rRNA as loading controls).",
    "molecules": "RNA"
  },
  {
    "caption": "V. cholerae wild-type, ΔrpoE, ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.",
    "molecules": "ethanol"
  },
  {
    "caption": "(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody.",
    "molecules": "lopinavir, LPV"
  },
  {
    "caption": "Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 cells. Mean ± SD, n=3.",
    "molecules": "LPV, PMA"
  },
  {
    "caption": "Titration of LPV-treated HIV-1 GFP viruses on PMA-treated U87 cells. Mean ± SD, n=3.",
    "molecules": "LPV, PMA"
  },
  {
    "caption": " ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). ",
    "molecules": "LPV, PMA"
  },
  {
    "caption": " ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). ",
    "molecules": "LPV, PMA"
  },
  {
    "caption": " ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). ",
    "molecules": "LPV, PMA"
  },
  {
    "caption": "PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G) CXCL-10 protein in cell supernatants from D-F (ELISA).",
    "molecules": "LPV, PMA"
  },
  {
    "caption": "(H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ.",
    "molecules": "DMSO, LPV, PMA, ruxolitinib"
  },
  {
    "caption": "(I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses.",
    "molecules": "LPV, PMA"
  },
  {
    "caption": "(J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24.",
    "molecules": "LPV"
  },
  {
    "caption": "(K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.",
    "molecules": "LPV"
  },
  {
    "caption": "(C ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).",
    "molecules": "PMA"
  },
  {
    "caption": "D) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).",
    "molecules": "PMA"
  },
  {
    "caption": "PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). E) CXCL-10 protein in supernatants from (C, D) (ELISA).",
    "molecules": "PMA"
  },
  {
    "caption": "(H) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib.",
    "molecules": "cGAMP, DMSO, ruxolitinib"
  },
  {
    "caption": "(J) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib.",
    "molecules": "DMSO, ruxolitinib"
  },
  {
    "caption": "primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib. K) CXCL-10 protein in supernatants from (J) (ELISA).",
    "molecules": "DMSO, ruxolitinib"
  },
  {
    "caption": "(A) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.",
    "molecules": "DMSO, neviripine, raltegravir"
  },
  {
    "caption": "THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir. B) CXCL-10 protein in supernatants from (A) (ELISA).",
    "molecules": "DMSO, neviripine, raltegravir"
  },
  {
    "caption": "ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.",
    "molecules": "DMSO, neviripine, raltegravir"
  },
  {
    "caption": "ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.",
    "molecules": "DMSO, neviripine, raltegravir"
  },
  {
    "caption": "(A) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING or MAVS, or a gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP 75 % ΔCA-SP1 (0.4 U RT/ml) or stimulated by transfection with either HT-DNA (0.1 μg/ml) or poly I:C (0.5 μg/ml).",
    "molecules": "HT-DNA, PMA, poly I:C"
  },
  {
    "caption": "IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line stimulated for 24 h with poly I:C (transfection, 0.5 μg/ml), HT-DNA (transfection, 0.1 μg/ml), LPS (50 ng/ml) or cGAMP (transfection, 0.5 μg/ml)",
    "molecules": "HT-DNA, cGAMP, LPS, PMA, poly I:C"
  },
  {
    "caption": "IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml)",
    "molecules": "PMA"
  },
  {
    "caption": "PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml) (C). D) CXCL-10 protein in supernatants from (C) (ELISA). Triangles indicate CXCL-10 not detected.",
    "molecules": "PMA"
  },
  {
    "caption": "(E) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).",
    "molecules": "DRV, PMA"
  },
  {
    "caption": "(F) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).",
    "molecules": "DRV, PMA"
  },
  {
    "caption": "PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). G) CXCL-10 protein in supernatants from (E).",
    "molecules": "DRV, PMA"
  },
  {
    "caption": "PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). (H) CXCL-10 protein in supernatants from (F) (ELISA).",
    "molecules": "DRV, PMA"
  },
  {
    "caption": "(I) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 control cells transduced for 48 h with WT, DRV-treated (DRV, 12.5 nM) or HIV-1 GFP containing 90 % ΔCA-SP1 (1×1010 genomes/ml) in the presence of DMSO vehicle, 2 μM ruxolitinib or 0.5 μg/ml H151.",
    "molecules": "H151, DRV, DMSO, PMA, ruxolitinib"
  },
  {
    "caption": "Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with LPV-treated HIV-LUC viruses Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.",
    "molecules": "LPV"
  },
  {
    "caption": "(A) Infection data for THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).",
    "molecules": "DMSO, PF-74"
  },
  {
    "caption": "THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM). B) IFIT-1 reporter activity in supernatant from (A).",
    "molecules": "DMSO, PF-74"
  },
  {
    "caption": "ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).",
    "molecules": "DMSO, PF-74"
  },
  {
    "caption": "ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).",
    "molecules": "DMSO, PF-74"
  },
  {
    "caption": "(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).",
    "molecules": "DMSO, PF-74"
  },
  {
    "caption": "(F) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM) and ruxolitinib (Rux, 2 μM) as indicated.",
    "molecules": "DMSO, PF-74, Rux, ruxolitinib"
  },
  {
    "caption": "(G) IRF reporter activity from THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).",
    "molecules": "DMSO, PF-74"
  },
  {
    "caption": "(H) IFIT-1 reporter activity from THP-1-IFIT-1 cells lacking MAVS or a matching gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).",
    "molecules": "DMSO, PF-74"
  },
  {
    "caption": "D. Representative time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (ER) (green) and SiR-lysosome (orange). Zoom insets highlight regions of interest containing a static (upper panels) or mobile endosome (lower panels) as denoted by the white arrows. First row shows ER (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Scale bars: full size 10µm, zoom inset 3µm. Images relate to movies EV2 and 3. E. Kymograph of the mobile vesicle in panel (D). F. MDA quantification of (D). Mobility of static and mobile endosomes was quantified and normalized to the static endosomes. Graph represents mean ± s.d. of analyses at multiple locations within the same cell(n=20 cells per condition from 3 independent experiments). All images acquired for 90 frames at 4Hz. Significance two-tailed student t-test, **** P<0.0001. ",
    "molecules": "SiR"
  },
  {
    "caption": "C. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) marked ER and SiR-lysosome-stained LE/Lys (orange) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either DMSO or 2µm paclitaxel. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz.Images related to movie EV7 and 8 D. Kymograph of panel (C). E. MDA quantification of panel (C) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n = 20 cells per condition from 3 independent experiments). Significance two-tailed student t-test **** P<0.0001; ns = not significant. ",
    "molecules": "DMSO, SiR, paclitaxel"
  },
  {
    "caption": "F. Representative images of n = 3 of COS7 cells stably expressing GFP-TMD (green) to mark the ER and SiR-lysosome stained LE/Lys (orange) merged. Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either siControl or siKTN1. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV9 and 10 G. Kymograph of panel (F). H. MDA quantification of panel (F) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions, respectively. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=20 cells per condition from 3 independent experiments). Significance two-tailed student t-test. **** P<0.0001; ns = not significant ",
    "molecules": "SiR"
  },
  {
    "caption": "E. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) and in lower panels transiently expressing mScarlet-RILP (magenta) and SiR-lysosome-stained LE/Lys (orange). Zoom insets shows region of interest containing ER (GFP-TMD). First row represents ER (GFP-TMD) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels. Scale bar: 10µm, zoom insets 3µm. Images related to movie EV19 and movie EV20. Images were acquired for 90 frames at 1Hz. F. MDA quantification of panel (E) and figure EV3A normalized to 1 showing total average ER movement. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired 90 frames at 1Hz. Significance two-tailed student t-test. **** P<0.0001 ns = not significant. ",
    "molecules": "SiR"
  },
  {
    "caption": "B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange). COS7 cells in the middle panels overexpress mCherry-RILP (magenta). The COS7 cells in the lower panels overexpress mCherry-TMEM55B (magenta). Zoom insets show region of interest containing the ER (TMD-GFP) (green) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average. Scale bars: 15µm. zoom insets: 3µm. C. Quantification of the number of ER junctions per µm2 from panel (B) as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell (n=15 from 3 independent experiments). Significance two-tailed student t-test. ***P<0.001. ",
    "molecules": "SiR"
  },
  {
    "caption": "A. Representative images of time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (green) and SiR-lysosome-stained LE/Lys (orange) treated with either siControl or co-treated with siVAPA, siVAPB and siMOSPD2. Zoom insets show regions of interest containing mobile LE/Lys as denoted by the white arrow. First row represents ER (TMD-GFP) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and SiR-lysosome stained LE/Lys (orange). Scale bar: 5µm, zoom insets 3µm. Images related to movie EV40 and 47. Images were acquired for 90 frames at 1Hz.",
    "molecules": "SiR"
  },
  {
    "caption": "B. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange) treated with siControl or with siVAPA, siVAPB and siMOSPD2. Zoom insets show region of interest containing ER (TMD-GFP) (green) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average. Scale bars: 10µm. zoom insets: 3µm.",
    "molecules": "SiR"
  },
  {
    "caption": "C. MDA quantification of ER of panels (A, B) and fig. EV4B-G normalized to control cells showing average ER movement surrounding mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=30 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. **P<0.01, ****P< 0.0001, ns=not significant D. MDA quantification of SiR-lysosome-stained LE/Lys of (A, B) and fig. EV4B - G normalized to 1 showing total average endosomal movement. Graph represents mean ±s.d. of n = 15 cells per condition from 3 independent experiments. Significance two tailed student t-test. ns= not significant. E. Quantification of number of ER junctions per µm2 from (B) and fig. H - M as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell of n = 15 from 3 independent experiments. Significance two-tailed student t-test. *P<0.05, **P<0.01 ***P<0.001, ****P< 0.0001, ns=not significant. ",
    "molecules": "SiR"
  },
  {
    "caption": "A, Hierarchical clustering analysis of RNA-sequencing data from cells treated with DMSO, 20 Gy IR, 250nM AZD6738, or AZD6738 + IR (n=2 biological replicates).",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "C, Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR.",
    "molecules": "AZD6738"
  },
  {
    "caption": "D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies. h, hours; pSTAT1, pSTAT1(Y701); pIRF3, pIRF3(S396); pTBK1, pTBK1(S172).",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "E, MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with indicated dose. 72 hours later, cells were harvested for immunoblotting with indicated antibodies.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "A, MCF10A WT cells were pretreated with AZD6738(250 nM) and RO-3306 (9 μM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.",
    "molecules": "AZD6738, RO-3306"
  },
  {
    "caption": "B, Same condition except that RO-3306 is replaced by PD-0332991 (1µM).",
    "molecules": "PD-0332991"
  },
  {
    "caption": "C, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cell cycle distribution was measured by flow cytometry. EdU (10 μM) was added into medium 2 hours before harvesting for flow cytometry.",
    "molecules": "EdU, AZD6738, DMSO"
  },
  {
    "caption": "D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, and cells were harvested at indicated time points for immunoblotting.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "E, MCF10A control cells and CDC25C-WT/constitutively active mutant CDC25C-S216A over-expressed cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cells were harvested for immunoblotting with indicated antibodies.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "A, MCF10A Wild-type (WT), cGAS knockout (KO), and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours, and cells were harvested for immunoblotting.",
    "molecules": "cGAMP"
  },
  {
    "caption": "B, MCF10A WT, cGAS KO, and STING KO cells were transfected with herring testis DNA (HT-DNA, 2 µg/ml) for 6 hours with lipofectamine 3000. Immunoblotting was performed with indicated antibodies.",
    "molecules": "lipofectamine 3000, DNA"
  },
  {
    "caption": "MCF10A WT or two independent clones for cGAS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "MCF10A WT or two independent clones for STING KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "MCF10A WT or two independent clones for cGAS, STING, and IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, except that cells were harvested for real-time quantitative PCR. IFNB1 mRNA level was measured and statistical analyzed by two-way ANOVA (*** means p value < 0.001). Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "MCF10A WT or two independent clones for IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "(A MCF10A WT or two independent clones for MAVS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "MCF10A WT or two independent clones for MAVS, RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, (B cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "C, MCF10A WT or two independent clones for RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "D, MCF10A WT or two independent clones for MDA5, KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "MCF10A WT or two independent clones for MDA5 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later E) cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "F) MCF10A WT or two independent clones for IRF7 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.",
    "molecules": "AZD6738, DMSO"
  },
  {
    "caption": "A, 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 10 Gy. 4 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738"
  },
  {
    "caption": "C, MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 20 Gy. 3 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.",
    "molecules": "AZD6738"
  },
  {
    "caption": "A, MCF10A WT, cGAS KO, STING KO, and MAVS KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.",
    "molecules": "poly(dA-dT)/LyoVec"
  },
  {
    "caption": "B, 4T1 WT, Sting KO, Mavs KO, and Irf3 KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.",
    "molecules": "poly(dA-dT)/LyoVec"
  },
  {
    "caption": "C, END-seq was used to map double-strand breaks (DSBs) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments. ATR inhibitor (ATRi) AZD6738 ( 250nM) was used with ionizing radiation (IR, 10 Gy) for 6 hours. NT, not treated.",
    "molecules": "AZD6738, TA dinucleotide repeats"
  },
  {
    "caption": "D, MCF10A WT, MDA5 KO, RIG-I KO, and MAVS KO cells were transfected with AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.",
    "molecules": "AT-rich oligos, LyoVec"
  },
  {
    "caption": "E, MCF10A WT and STING KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with lipofectamine 3000 for 4 hours. Immunoblotting was performed with indicated antibodies.",
    "molecules": "AT-rich, lipofectamine 3000, non-AT-rich oligos"
  },
  {
    "caption": "F, 4T1 WT, Sting KO, and Irf3 KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.",
    "molecules": "AT-rich, LyoVec, non-AT-rich oligos"
  },
  {
    "caption": "(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red.",
    "molecules": "LPS"
  },
  {
    "caption": "(b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm.",
    "molecules": "BafA1, bafimolycin A1, LPS"
  },
  {
    "caption": "(c) Immunoblot analysis of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in LPS-stimulated B cells with (+NH4Cl) or without treatment with NH4Cl (left). Actin serves as a loading control throughout. Right, quantification of LC3-II band intensity in the absence of NH4Cl, presented relative to that of actin.",
    "molecules": "NH4Cl, LPS"
  },
  {
    "caption": "(d) Quantitative RT-PCR analysis of transcripts encoding autophagy-related molecules in ASCs 3 d after stimulation with LPS, presented relative to that in unstimulated B cells.",
    "molecules": "LPS"
  },
  {
    "caption": "(e) Immunoblot analysis of p62 in LPS-stimulated B cells (bottom). Above, quantification of band intensity (as in c).",
    "molecules": "LPS"
  },
  {
    "caption": "(a) Proliferation of B cells purified from Atg5f/fCD19-Cre and Atg5f/f control mice and activated for 3 d with LPS, assessed by flow cytometry analysis of dilution of the membrane dye CFSE. (b) Size of B cells purified as in a and activated for various times (horizontal axis) with LPS, assessed by the intensity of forward scatter (FSC) by flow cytometry. (c) CD138 expression of B cells purified and activated as in a. APC, allophycocyanin; PE, phycoerythrin.",
    "molecules": "LPS"
  },
  {
    "caption": "(d) Immunoblot analysis of ER proteins in lysates of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left), and quantification of band intensity (right; as in Fig. 1c). IgM-H, IgM heavy chain. NS, not significant; *P 0.05 and **P 0.01 (Student's t-test).",
    "molecules": "LPS"
  },
  {
    "caption": "(a) Electron microscopy of Atg5f/f and Atg5f/fCD19-Cre B cells 3 d after stimulation with LPS (left and middle), and quantification of the ER by stereology (right). Scale bars (left and middle), 1 μm.",
    "molecules": "LPS"
  },
  {
    "caption": "(b) G6Pase electron-microscopy cytochemistry of differentiating PCs 3 d after stimulation with LPS (left), and quantification of G6Pase+ (ER) area, relative to total cell area (right). Scale bars (left), 1 μm.",
    "molecules": "LPS"
  },
  {
    "caption": "(c) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1 (sXBP-1), total XBP-1 (tXBP-1) and BiP in differentiating PCs at 0 d and 3 d after stimulation with LPS, presented relative to that of unstimulated Atg5f/f B cells at day 3.",
    "molecules": "LPS"
  },
  {
    "caption": "(d) Quantitative RT-PCR analysis of CHOP mRNA in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS (left and middle), and in Atg5f/f B cells stimulated for 3 d with LPS and treated for the final 24 h with tunicamycin (Atg5f/f 24 h tun; 0.5 μg/ml; positive control), presented relative to that in Atg5f/f B cells stimulated for 3 d with LPS.",
    "molecules": "LPS, tunicamycin"
  },
  {
    "caption": "(e) Immunoblot analysis (left) of ER proteins in Atg5f/f B cells left untreated (UT) or treated with lysosomal protease inhibitors (10 μM leupeptin and E-64d) for the final 8 h of the third day of LPS-induced differentiation (Leu + E-64d (8 h)). Right, quantification of band intensity (presented as in Fig. 1c). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).",
    "molecules": "E-64d, leupeptin, LPS"
  },
  {
    "caption": "(a) Enzyme-linked immunosorbent assay (ELISA) of IgM in supernatants of Atg5f/fCD19-Cre and Atg5f/f B cells stimulated for 3 d with LPS and plated for 4 h (as trypan blue-negative cells at a density of 1 × 106 cells per ml in dedicated media), presented relative to results obtained for Atg5f/f cells.",
    "molecules": "LPS"
  },
  {
    "caption": "(b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units.",
    "molecules": "LPS"
  },
  {
    "caption": "(b,c) Immunoglobulin assembly (b) and total intracellular (IC) and secreted (Sec) IgM heavy chain (IgM-H, c) in Atg5f/f and Atg5f/fCD19-CreB cells stimulated for 3 d with LPS, then pulse-labeled and chased (time, above lanes), followed by immunoprecipitation of the μ-chain from lysates and SDS-PAGE (left) in nonreducing conditions (b) or reducing conditions (c). Right, quantification of intracellular IgM polymers by densitometry (b) and of secretion efficiency, estimated as the amount of radioactive secreted μ-chain at the end of the chase relative to intracellular μ-chain at the end of the pulse (c). AU, arbitrary units.",
    "molecules": "LPS"
  },
  {
    "caption": "(d) Quantitative RT-PCR analysis of mRNA encoding Blimp-1 and the secreted μ-chain in Atg5f/f and Atg5f/fCD19-Cre B cells stimulated for 3 d with LPS, presented relative to that of Atg5f/f cells.",
    "molecules": "LPS"
  },
  {
    "caption": "(e) Quantitative RT-PCR analysis of transcripts encoding spliced XBP-1, BiP, Blimp-1 and secreted μ-chain in Atg5f/f B cells stimulated for 3 d with LPS and given no further treatment (−tun) or treated for the final 24 h with tunicamycin (tun 24 h; 0.5 μg/ml), presented relative to results obtained with LPS-stimulated cells given no further treatment. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).",
    "molecules": "LPS, tunicamycin"
  },
  {
    "caption": "(a) Intracellular ATP in fresh lysates of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for 3 d with LPS (3) or left untreated (0).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(b) Death of Atg5f/f and Atg5f/fCD19-Cre splenic B cells activated for various times (horizontal axis) with LPS, quantified by flow cytometry by propidium iodide (PI) staining.",
    "molecules": "LPS"
  },
  {
    "caption": "(d) Quantitative RT-PCR analysis of Atg5 mRNA in differentiating Atg5f/f and Atg5f/fCD19-Cre B cells at 0 and 3 d after stimulation with LPS, presented relative to that in untreated Atg5f/fCD19-Cre B cells. *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).",
    "molecules": "LPS"
  },
  {
    "caption": "(a) ELISA of anti-pneumovax IgM titers in diluted serum 1 day before and 4, 10 and 14 d after intraperitoneal immunization of Atg5f/f and Atg5f/fCD19-Cre mice with 1 μg pneumovax.",
    "molecules": "pneumovax"
  },
  {
    "caption": "(b) ELISA of anti-pneumovax IgG3 titers in serum 14 d after injection as in a. Results are presented as absorbance (A). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).",
    "molecules": "pneumovax"
  },
  {
    "caption": "Flow cytometry analysis of Rex1-GFPd2 cells following addition of 10mM 2-DG during the 48h EpiLC induction. Representative GFP intensity distributions are depicted. Average proportions of Rex1-GFPd2 positive (GFP+) cells are quantified from two independent biological replicates.",
    "molecules": "2-DG"
  },
  {
    "caption": "Expression analysis by qRT-PCR of naïve pluripotency and epiblast marker genes in bulk 48h cells after 10mM 2-DG treatment. Relative expression levels, normalized to control culture conditions, are shown. Graphs represent averages from triplicate (duplicate for Klf4 and Tfcp2l1) independent biological experiments.",
    "molecules": "2-DG"
  },
  {
    "caption": "Colony-forming ability following 10mM 2-DG supplementation during EpiLC stimulation. Representative images of alkaline phosphatase (AP)-stained colonies are displayed. Scale bar, 250 um. The average colony formation, normalized to control culture conditions, quantified from two independent biological replicates, is shown.",
    "molecules": "2-DG"
  },
  {
    "caption": "Pseudotime expression profiles for the αKG-regulating enzymes Idh2 and Dlst during the transition from naïve to primed pluripotency. Tricarboxylic acid (TCA) cycle enzymes and metabolites produced within the TCA cycle are illustrated.",
    "molecules": "αKG, TCA, Tricarboxylic acid"
  },
  {
    "caption": "Representative flow cytometry profiles of Rex1-GFPd2 cells following 4mM dm-αKG supplementation during the EpiLC induction are depicted. Graphs show average fractions of Rex1-GFPd2 positive (GFP+) cells from six independent biological assays.",
    "molecules": "dm-αKG"
  },
  {
    "caption": "qRT-PCR analysis of naïve pluripotency regulators and epiblast marker genes following EpiLC stimulation in the presence of 4mM dm-αKG. Expression data are normalized to control culture conditions and represent averages from five biological replicates in bulk 48h cells.",
    "molecules": "dm-αKG"
  },
  {
    "caption": "Colony forming ability succeeding 4mM dm-αKG treatment during the 48h EpiLC induction. Characteristic images of AP-stained colonies are shown. Scale bar, 250 um. Colony formation is normalized to control-treated cells and quantified from quadruplicate experiments.",
    "molecules": "dm-αKG"
  },
  {
    "caption": "Representative super-resolution images of TOM-20 immune-labelled mitochondria in ESCs following 48h culture in 2i/Lif/KSR media, and EpiLC-inducing conditions in the presence of 4mM dm-αKG and DMSO, respectively, are displayed. Scale bar, 3um.",
    "molecules": "DMSO, dm-αKG"
  },
  {
    "caption": "10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. (F) Characteristic bright field images of Rex1-GFPd2 cells after 10 days of culture in 2i/Lif/KSR and N2B27/Lif/KSR, in the presence of dm-αKG and DMSO, respectively. Scale bar, 10um.",
    "molecules": "DMSO, dm-αKG"
  },
  {
    "caption": "10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. Flow cytometer-based quantification of Rex1-GFPd2 positive (GFP+) cells. Representative GFP intensity distributions are displayed. The average fractions of GFP+ cells are measured from duplicate experiments.",
    "molecules": "DMSO, dm-αKG"
  },
  {
    "caption": "10-day culture of Rex1-GFPd2 cells in N2B27/Lif/KSR with 4mM dm-αKG and DMSO, respectively, with passaging every 2.5-days. qRT-PCR analysis of naïve pluripotency and differentiation markers in bulk cells harvested at 2.5 day-intervals during the 10-day culture in N2B27/Lif/KSR with dm-αKG or DMSO. Expression data are normalized to time-matched ESCs in 2i/Lif/KSR-culture conditions and are averaged over two independent biological experiments.",
    "molecules": "DMSO, dm-αKG"
  },
  {
    "caption": "FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 embryoids specified in the presence of 4mM dm-αKG and PGC cytokines. Representative flow cytometer profiles are depicted. Graphs show the average fractions of GFP+ cells from duplicate experiments. P1-GFP, Prdm1-GFP.",
    "molecules": "dm-αKG"
  },
  {
    "caption": "FACS analysis of Prdm1-GFP positive (GFP+) cells in day-2 embryoids aggregated under addition of LIF (10ng ml-1) and BMP4 (500ng ml-1), dm-αKG (4mM), or DMSO. Representative flow cytometer profiles are displayed. Average fractions of GFP+ cells are calculated from duplicate assays. P1-GFP, Prdm1-GFP.",
    "molecules": "DMSO, dm-αKG"
  },
  {
    "caption": "FACS analysis of Prdm1-GFP positive (GFP+) cells in day-4 PGCLC aggregates specified from 4mM dm-αKG-treated (t=48h to t=72h) EpiLCs. Representative flow cytometer profiles are depicted. The average fractions of GFP+ cells, quantified from triplicate experiments, are shown. P1-GFP, Prdm1-GFP.",
    "molecules": "dm-αKG"
  },
  {
    "caption": "Transcript analysis by qRT-PCR of PGC specifiers, demethylating enzymes, mesoderm, endoderm, and ESC regulators in FACS-sorted day-4 Prdm1-GFP embryoids induced from 4mM dm-αKG-treated 72h EpiLCs. Expression levels are normalized to Prdm1-GFP negative cells from control embryoids. Graphs represent averages from triplicate experiments. +, Prdm1-GFP positive cells; -, Prdm1-GFP negative cells.",
    "molecules": "dm-αKG"
  },
  {
    "caption": "Western blot analysis for H3K9me2, H3K27me3, and DNMT3b in 4mM dm-αKG-treated EpiLCs. H3 is used as a loading control. 48h, dm-αKG and DMSO, respectively, supplementation from t=24h to t=48h during the EpiLC differentiation. 72h, dm-αKG and DMSO, respectively, supplementation from t=48h to t=72h during the EpiLC differentiation.",
    "molecules": "DMSO, H3K27me3, dm-αKG"
  },
  {
    "caption": "ChIP-qPCR analysis of H3K9me2 and H3K27me3 in putative enhancer regions of genes associated with the naïve pluripotent state (Esrrb, Arid5b) and PGC fate (Tfap2c), respectively, in naïve ESCs and at t=48h following EpiLC induction in the presence of 4mM dm-αKG and DMSO, respectively. Graphs show enrichment of H3K9me2, H3K27me3, and IgG control, respectively, relative to DMSO-treated EpiLCs.",
    "molecules": "H3K9me2, DMSO, H3K27me3, dm-αKG"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by CellTiter-Glo (A) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cell viability was assessed by Crystal Violet (B) after 16 hours. Data are means ± SEM of three independent experiments. *P< 0.1, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Crystal Violet, Nec-1s, necrostatin-1, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 nd Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with Annexin V (A5). Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Flow cytometric analysis of cells stained with TO-PRO-3 Histograms show means ± SEM of three independent experiments. ***P<0.001, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with TO-PRO-3 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with TO-PRO-3 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 hours of treatment, and stained with propidium iodide (PI) and A5 Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, PI, propidium iodide, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). Cells were collected after 4 and 24 hours of treatment, and stained with propidium iodide (PI) Data are means ± SEM of three independent experiments. **P<0.01, ***P< 0.001, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, PI, propidium iodide, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). J Measurement of lactate deshydrogenase (LDH) released from necroptotic cells, as indicated (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with siRNA for MLKL or scramble non-specific (NS) siRNA for 72 hours. Cells pre-treated with 10 μM QVD-OPh (Q), 5 μM Birinapant (S), 20 μM necrostatin-1 (Nec-1s) were stimulated with 1-10 ng.mL-1 TNFα (T). K Time lapsed microscopy analysis of A5 and TO-PRO-3 staining upon induction of necroptosis. The presented data are representative of at least three independent experiments.",
    "molecules": "Birinapant, Nec-1s, necrostatin-1, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "A HT-29 cells were transfected with two individual siRNA for PANX1, or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) together with 5 μM Birinapant (S), prior stimulation with 10 ng.mL-1 of TNFα (T), as indicated. Western blotting for hallmarks of necroptosis, as indicated. The arrowhead indicates MLKL cleaved fragment. Molecular weight markers (Mr) are shown.",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "B Cells were exposed to TQS for 5 hours. Necrostatin-1 (Nec-1s, 20 μM) was also used. MLKL oligomers (MLKLn) were resolved by non-reducing SDS-PAGE after cross-linking.",
    "molecules": "Nec-1s, Necrostatin-1"
  },
  {
    "caption": "Flow cytometric analysis of cells treated with TQS for 4 hours and stained with TO-PRO-3 (E and F). Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": "HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death, and analyzed by Western blotting (G).",
    "molecules": "cycloheximide"
  },
  {
    "caption": "HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death Cell survival was assessed by CellTiter-Glo after 24 hours of treatment (H). Histogram shows means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated as in (A) with the addition of 1 μM cycloheximide (C) to enhance cell death Annexin V staining was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death TO-PRO-3 uptake was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA.",
    "molecules": "cycloheximide, TO-PRO-3"
  },
  {
    "caption": "PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. TO-PRO-3 uptake was analyzed by flow cytometry Shown are means ± SEM, n=3 biological replicates, ****P<0.0001 ANOVA.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": "PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. TO-PRO-3 uptake was analyzed by flow cytometry Shown are means ± SEM, n=3 biological replicates, ****P<0.0001 ANOVA.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": "NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated Cell survival was evaluated by CellTiter-Glo after 24 hours (N). Data are means ± SEM, n=4 biological replicates, ***P<0.001, ****P<0.0001, ANOVA.",
    "molecules": "carbenoxolone, CBX, Probe, Probenecid, Trova, Trovafloxacin"
  },
  {
    "caption": "NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated TO-PRO-3 uptake was analyzed by flow cytometry (O). Data are means ± SEM, n=3 biological replicates, ***P<0.001, ****P<0.0001, ANOVA.",
    "molecules": "carbenoxolone, CBX, Probe, Probenecid, TO-PRO-3, Trova, Trovafloxacin"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) were visualized by non-reducing SDS-PAGE after cross-linking proteins (A). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA). Data information Arrowhead, MLKL cleaved fragment.",
    "molecules": "Birinapant, necrosulfonamide, NSA, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). NS- and MLKL-silenced cells were treated with 5 μM necrosulfonamide (NSA). MLKL oligomers (MLKLn) Cells were exposed to TQS for 5 hours. Molecular weight markers (Mr) are shown. TO-PRO-3 uptake was analyzed by flow cytometry in cells treated with TQS for 4 hours (B). Data are means ± SEM of three independent experiments. ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, necrosulfonamide, NSA, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). C Cell lysates from NS-, and ITPK1-silenced cells were analyzed by Western blotting as indicated. Data information: Arrowhead, MLKL cleaved fragment.",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). D Western blotting analysis of MLKL oligomerization",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). E Cell survival was evaluated by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA).",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). F Measurement of TO-PRO-3 uptake as in (B). Dead TO-PRO-3high cells were discarded. Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA).",
    "molecules": "Birinapant, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). G Small EVs fractions were purified from the culture medium by ultracentrifugation. Shown is a representative diagram of particle count obtained with tunable resistive pulse sensing analysis (TRPS, qNano, IZON).",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). H Cells were subjected to Western blotting analysis Data information: Arrowhead, MLKL cleaved fragment.",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). I MLKL oligomerization was determined Data information Arrowhead, MLKL cleaved fragment.",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). J Cell survival by CellTiter-Glo after 24 hours (mean ± SEM, n=4 biological replicates, **P<0.01, ***P<0.001, ****P<0.0001, ANOVA).",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "HT-29 cells were transfected with a nonspecific (NS) siRNA or with the indicated siRNAs for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T). K TO-PRO-3 uptake Data are means ± SEM, n=3 biological replicates, ****P<0.0001 (ANOVA). The presented data are representative of at least three independent experiments.",
    "molecules": "Birinapant, QVD-OPh, TO-PRO-3"
  },
  {
    "caption": "A HT-29 cells were transfected with a siRNA for PANX1 (#3), or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) plus 5 μM Birinapant (S), and exposed to 10 ng.mL-1 of TNFα (T) for 6 hours. Shown is a normalized densitometric analysis for the presence of 120 cytokines with an antibody array. The color scale (0-10) represents the means of normalized densitometry values (n=2 technical replicates).",
    "molecules": "Birinapant, QVD-OPh"
  },
  {
    "caption": "IFNα dose-dependency of STAT1 and STAT2 phosphorylation in Huh7.5 cells. Cells were seeded 24 hours prior to the start of the experiment. Three hours before stimulation, cells were growth factor-depleted and were subsequently stimulated with the indicated concentrations of IFNα. Cytoplasmic protein lysates were collected one hour after the stimulation and phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=1 to N=38) using a combined scaling and error model. Data is approximated with a sigmoidal function and signals corresponding to a low dose (2.8 pM IFNα), a medium dose (28 pM IFNα) and a high dose (1400 pM IFNα) are displayed with dashed lines.",
    "molecules": "tyrosine"
  },
  {
    "caption": "Representative immunoblot of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 cells prestimulated for 24 hours with 2.8 pM IFNα, 1400 pM IFNα or without prestimulation. Time points after presimulation are indicated. 20 µg of cytoplasmic lysates were analyzed using antibodies for the indicated targets. Phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. An asterisk indicates pSTAT1β. Calnexin served as loading control. Molecular weights are indicated on the left. Immunoblot detection was performed with chemiluminescence employing a CCD camera device (ImageQuant).   Quantification of immunoblots of IFNα-induced phosphorylation of cytoplasmic and nuclear STAT1 and STAT2 in Huh7.5 cells prestimulated with 2.8 pM IFNα, 1400 pM IFNα or without pretreatment. Time points after presimulation are displayed. Data is displayed as fold change relative to untreated cells. Errors were estimated with a combined scaling and error model, comprising 1σ confidence interval estimated from biological replicates (N=3). Dashed lines indicate smoothing splines.   ",
    "molecules": "tyrosine"
  },
  {
    "caption": "Growth factor-depleted Huh7.5 cells were prestimulated with 2.8 pM, 28 pM, 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later. IFNα-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD-based camera. Data was normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.   Model calibration with time-resolved IFNα-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.   ",
    "molecules": "Tyr"
  },
  {
    "caption": "Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) (left panel), of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers (middle panel) and of the pSTAT1:pSTAT2:IRF9 trimers (right panel) in untreated Huh7.5 cells and in Huh7.5 cells prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Lines with shading represent model predictions with 68% confidence intervals using the prediction profile likelihood method. For experimental validation of the dynamics of OccGASbs, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were incubated with radioactively-labeled oligonucleotides probes harboring the GAS-binding region of the human IRF1 promoter. Samples were resolved on a native polyacrylamide gel and radioactivity was visualized and quantified from three independent experiments (left panel). For experimental validation of the dynamics of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations (IP) were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (middle panel). For experimental validation of the dynamics of the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation (IP) with antibodies recognizing IRF9 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (right panel). Antibodies and the corresponding proteins in the complexes are underlined. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3) using a combined scaling and error model.",
    "molecules": "oligonucleotides, polyacrylamide"
  },
  {
    "caption": "Dose-dependent sensitization of signal transduction induced by the therapeutic interferon Roferon. Growth factor-depleted Huh7.5 were prestimulated with 0, with 1.2 or 608 pM Roferon and stimulated with 608 pM Roferon 24 hours later. Concentrations of Roferon correspond to equipotent concentrations of IFNα. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescense utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=8) using a combined scaling and error model. Model trajectories are represented by lines.   Dose-dependent sensitization of signal transduction induced by Roferon in HepG2-hNTCP. Growth factor-depleted HepG2-hNTCP were prestimulated with 0, 1.8 or 887 pM Roferon and stimulated with 887 pM Roferon 24 hours later. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescence utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3) using a combined scaling and error model. Model trajectories are represented by lines.   ",
    "molecules": "Roferon"
  },
  {
    "caption": "Overexpression of Huh7.5 is not sufficient to explain negative memory. Induced expression of USP18 after treatment of Huh7.5-TetON-USP18 and Huh7.5-TetON-control cells with doxycycline for 24 hours in comparison with parental Huh7.5 cells treated with 1400 pM IFNα for 24 hours. Analysis was performed by quantitative immunoblotting. Error bars represent 1σ confidence intervals estimated from biological replicates (N=3 to N=5) (left). Model fits and experimental data of Huh7.5-TetON-USP18 treated with doxycycline for 24 hours and stimulated with 1400 pM IFNα or parental Huh7.5 cells prestimulated with 0 or 1400 pM IFNα and stimulated with 1400 pM IFNα after 24 hours are shown. The dynamics of IFNα-induced phosphorylation of STAT1 and STAT2 and induction of USP18 are depicted. Experimental data was obtained by quantitative immunoblotting using chemiluminescence and CCD camera device (ImageQuant). For modeling purposes data from Huh7.5-TetON empty vector control and untransduced Huh7.5 are combined to one condition. Data is displayed as filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=4) using a combined scaling and error model. Line represents model trajectories.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Experimental data and model fits of IFNα- and Roferon-induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor-depleted primary human hepatocytes prestimulated with 0, 2.8 or 1400 pM IFNα (patients 1-3) or 0, 1.2 or 608 pM Roferon (patients 4-6). Primary human hepatocytes from the same patients 1-6 as in (B) were used. Experimental data is represented by filled circles (N=1 per patient). Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα. Lines indicate model fits.",
    "molecules": "Roferon"
  },
  {
    "caption": "(A) ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 hrs) THP-1 cells then infected with ZIKV (MOI=1) for 36 hrs. Uninfected cells serves as mock control.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated or primed by LPS (500 ng/ml, 3 hrs), after ZIKV infection (MOI=1) for 36 hrs.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1 or AIM2 KO#1 THP-1 cells left untreated (UT), or pretreated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) stimulation, or stimulated with poly (dA:dT) (1mg/ml, 6 hrs), or infected with ZIKV (MOI=1, 36 hrs).",
    "molecules": "ATP, LPS, poly (dA:dT)"
  },
  {
    "caption": "(G) ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 hrs or mock control. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) treatment for 6 hrs.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(H) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI=1) or mock control for 24 hrs. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) stimulation for 6 hrs.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(J) Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 hrs.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(K) ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs), or pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.",
    "molecules": "ATP, LPS, poly (I:C)"
  },
  {
    "caption": "(L) Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.",
    "molecules": "ATP, LPS, poly (I:C)"
  },
  {
    "caption": "(A) Relative qRT-PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.",
    "molecules": "ATP, LPS, RNA"
  },
  {
    "caption": "(B) Relative qRT-PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.",
    "molecules": "RNA"
  },
  {
    "caption": "(D) Relative qRT-PCR analysis of ZIKV RNA in WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.",
    "molecules": "RNA"
  },
  {
    "caption": "(G) Relative qRT-PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.",
    "molecules": "RNA"
  },
  {
    "caption": "(H) Relative qRT-PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μΜ) for 3 hrs followed by ZIKV infection (MOI=1) for the indicated time points.",
    "molecules": "Ac-YVAD-cmk, Dox, doxycycline, RNA"
  },
  {
    "caption": "(J) Relative qRT-PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.",
    "molecules": "RNA"
  },
  {
    "caption": "(K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.",
    "molecules": "Ac-YVAD-cmk, DMSO, RNA"
  },
  {
    "caption": "(B) Co-immunoprecipitation and immunoblot analysis of extracts of NS1-inducible THP-1 cells, which expressed Flag-tagged NS1, treated with or without doxycycline (Dox) (100 ng/ml).",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(C) Immunoblot analysis of extracts of THP-1 cells treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs) followed by increasing doses of Dox treatment.",
    "molecules": "ATP, Dox, LPS"
  },
  {
    "caption": "(F) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 followed by cycloheximide (CHX) treatment (100 μg/ml) for the indicated time points.",
    "molecules": "cycloheximide, CHX"
  },
  {
    "caption": "(G) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), Lactacystin (5 μM), Carfilzomib (100 mM) or CQ (50 mM) for 6 hrs.",
    "molecules": "Carfilzomib, CQ, Lactacystin, MG132"
  },
  {
    "caption": "(H) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml).",
    "molecules": "Dox"
  },
  {
    "caption": "(I) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-NS1, followed by MG132 (10 mM) treatment for 6 hrs.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) Immunoblot analysis of extracts of transfected with Flag-caspase-1 together with empty vector or Myc-USP8 then treated with or without MG132 (10 mM) for 6 hrs.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml) then transfected with or without poly (I:C) (2 mg/ml, 6 hrs).",
    "molecules": "Dox, poly (I:C)"
  },
  {
    "caption": "(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 and Myc-USP8 together with HA-NS1 followed by MG132 treatment (10 mM) for 6 hrs.",
    "molecules": "MG132"
  },
  {
    "caption": "(G) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin (Ub) or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-USP8, followed by MG132 (10 mM) treatment for 6 hrs.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) Relative qRT-PCR analysis of IFNB mRNA levels in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(D) Relative qRT-PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag-NS1-inducible THP-1 cells treated with or without Dox then left untreated or treated with YVAD (20 μM) for 3 hrs, followed by ZIKV infection (MOI=1) for the indicated time points.",
    "molecules": "YVAD, Dox"
  },
  {
    "caption": "(F) Relative qRT-PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.",
    "molecules": "Ac-YVAD-cmk, DMSO"
  },
  {
    "caption": "(G) ELISA of IFN-β in the supernatants of PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.",
    "molecules": "Ac-YVAD-cmk, DMSO"
  },
  {
    "caption": "(K) Relative qRT-PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection",
    "molecules": "RNA"
  },
  {
    "caption": "(A) Relative qRT-PCR analysis of ZIKV RNA levels in wild type (WT) or cGAS knockout (KO) THP-1 cells after ZIKV infection (MOI=1) for indicated time points.",
    "molecules": "RNA"
  },
  {
    "caption": "(C) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP, cGAS or NLRP3 and infected with ZIKV (MOI=1) for 36 hrs.",
    "molecules": "RNA"
  },
  {
    "caption": "(D) Relative qRT-PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP-1 cells treated with or without YVAD (20 μM), followed by ZIKV infection (MOI=1) for indicated times points.",
    "molecules": "YVAD, RNA"
  },
  {
    "caption": "(E) Immunoblot analysis of extracts of Flag-cGAS-overexpressed THP-1 cells treated with or without YVAD (20 μM) then infected with ZIKV (MOI=5) for 24 hrs or infected with HSV-1 (MOI=1) for 24 hrs.",
    "molecules": "YVAD"
  },
  {
    "caption": "(K) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in WT, cGAS KO THP-1 cells as well as cGAS KO THP-1 cells reconstituted with WT or D140A/D157A mutant of cGAS, followed by ZIKV infection (MOI=1) for 24 hrs.",
    "molecules": "RNA"
  },
  {
    "caption": "B Relative mRNA expression of NFATc1‐ and EMT‐related marker genes on administration of CsA (1 μM) in KPNC cells. Data represents means ± SD from at least three independent experiments. Asterisks show significance (*P 0.05).",
    "molecules": "CsA"
  },
  {
    "caption": "E, F NFATc1 and Sox2 protein expression after (E) NFATc1 depletion or (F) upon CsA (1 μM, 24 h) treatment in Panc1 cells.",
    "molecules": "CsA"
  },
  {
    "caption": "I, J ChIP assays show RNA polymerase II binding to Sox2 promoter upon CsA (1 μM, 24 h) treatment in L3.6 cells (J).",
    "molecules": "CsA"
  },
  {
    "caption": "D Sox2 mRNA expression upon CsA (1 μM) treatment in L3.6 cells (adherent versus spheres) (*P 0.05).",
    "molecules": "CsA"
  },
  {
    "caption": "Mixed WT:DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days. EdU incorporation was analyzed one day after the last injection. Steady-state WT and DKO mice were injected with EdU and analyzed as in A. Representative flow cytometry plots of EdU incorporation by AMs (C) and their quantification (results pooled from two independent experiments, 7 WT and 6 DKO mice were analyzed in total) (B) is shown.",
    "molecules": "EdU"
  },
  {
    "caption": "WT (CD45.1) and DKO (CD45.2) AMs were sorted, mixed in a 1:1 ratio, labeled with CellTrace Violet and cultured in the presence of GM-CSF. The ratio of CD45.1 and CD45.2 cells and CellTrace Violet dilution was analyzed prior to culture (day 0) and after 7 days of culture as described in Materials and Methods.",
    "molecules": "CellTrace Violet"
  },
  {
    "caption": "Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O. Quantification of Oil Red O-positive cells in WT and DKO mice for individual animals. Results pooled from two independent experiments; 4 WT and 5 DKO mice were analyzed in total.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493/503. Representative results of two independent experiments with three mice per group. Analysis as in C performed with WT:DKO mixed BM chimeras. Gating on CD45.1 and CD45.2 was applied to identify cells of WT and DKO donor origin. Representative results of two independent experiments with at least three chimeras.",
    "molecules": "BODIPY 493/503"
  },
  {
    "caption": "Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2-kb regions around the Bhlhe40 peak summits (right - for all Bhlhe40 peaks called with P value of < 10−10; center - for top 3,000 Bhlhe40 peaks (ranked by P value)) and around WT AM ATAC-seq peaks (Immgen) that did not have a Bhlhe40 peak in their close proximity (filtered as described in Materials and Methods) (left). Volcano plots show adjusted P values (vertical axes) and log2 fold change (horizontal axes) for H3K27ac pairwise comparison in WT vs DKO AMs; genes assigned to some of the Bhlhe40-binding regions are labeled on the left plot.",
    "molecules": "H3K27ac"
  },
  {
    "caption": "C 17K5D, but not 17K or 17K5A, showed binding to BYDV vsiRNA. The GST fusions of 17K, 17K5D, and 17K5A, as well as free GST, were used in the RNA EMSA assays with biotin-labeled vsiRNA899 or vsiRNA899 duplex as probes.",
    "molecules": "biotin"
  },
  {
    "caption": "Quantitative analysis of HvSDN1 cleavage activities, with the fluorescence recorded using Typhoon FLA9500 (F and G) Compared with free GST or GST-17K, the GST fusions of 17K5D or three derivative mutants (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) inhibited HvSDN1-catalzyed cleavage of fluorogenic vsiRNA899, which was revealed by a fluorospot assay (F), relative comparison of fluorescence values (G)",
    "molecules": "Typhoon FLA9500"
  },
  {
    "caption": "Luciferase sequestration as an assay for in vitro uptake. (A) Luciferase cofractionating with vacuoles is proteinase K protected. In vitro uptake reactions of three times the standard volume were performed (60 min, 27°C) in the presence or absence of the ATP-regenerating system. The vacuoles were reisolated, washed, resuspended in 150 mM KCl in PS buffer to a concentration of 0.23 mg/ml, and split into aliquots. After addition of 1% Triton X-100 or buffer, the mixture was digested with the indicated concentrations of proteinase K (15 min, 0°C). Proteinase treatment was stopped by adding one volume of 0.5 mM PMSF in 150 mM KCl in PS buffer. Then, luciferase activity was assayed.",
    "molecules": "Luciferase, luciferase, ATP, PMSF, KCl, Triton X-100"
  },
  {
    "caption": "(B) Luciferase can be released from the vacuolar lumen by freeze-thaw treatment. An uptake reaction was performed (60 min, 27°C). The vacuoles were reisolated and washed as described in the Materials and Methods section. The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots. One was frozen at −80°C and thawed again slowly, whereas the other remained on ice. The samples were centrifuged (15 min, 145,000 g, 4°C). The supernatants (Sup)were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl. Luciferase activity in both fractions was determined. The reaction tubes for this experiment had been coated with BSA (0.5 mg/ml, 15 min at room temperature) to reduce unspecific binding of luciferase to the surface.",
    "molecules": "Luciferase, luciferase, KCl"
  },
  {
    "caption": "time course and temperature dependence of uptake. (A) Standard uptake reactions were performed in the presence or absence of the ATP-regenerating system. After the indicated times at 27°C, the vacuoles were reisolated, washed, treated with protease, and luciferaseuptake activity was assayed.",
    "molecules": "luciferase, ATP, protease"
  },
  {
    "caption": "(B) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.",
    "molecules": "luciferase, protease"
  },
  {
    "caption": "Requirements for in vitro uptake. Standard uptake reactions were performed (60 min) (A) at different temperatures, or in the presence of different concentrations of (B) cytosol, (C) ATP, or (D) MgCl2. Luciferase uptake was determined as described in the legend to Fig. 3 A. In B, titration curves for four independent cytosol preparations are shown. The maximal uptake signal of each titration curve was set at 100%. In D, the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction.",
    "molecules": "ATP, EDTA, MgCl2"
  },
  {
    "caption": "(B) Vacuoles and cytosols were prepared from sec18-1 and from corresponding wild-type (Wt) cells grown and spheroplasted at 23°C. The isolated vacuoles and the cytosols were exposed to heat treatment (10 min, 37°C). Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations. Control reactions were performed in the absence of ATP. Five independent experiments were averaged; the sample with vacuoles and cytosol from wild-type cells was used as the 100% reference. Error bars indicate SD. Mut, mutant.",
    "molecules": "ATP"
  },
  {
    "caption": "In vitro uptake depends on GTP hydrolysis and on the V-ATPase. (A) Standard uptake reactions were performed in the presence of different concentrations of GTPγS. Luciferase uptake was assayed as in the legend to Fig. 3 A. Four independent experiments were averaged, using the sample without inhibitor as the 100% reference. Error bars indicate SD. GTPγS did not influence luciferase activity by itself (not shown).",
    "molecules": "luciferase, GTPγS"
  },
  {
    "caption": "(B) Standard uptake reactions were performed in the presence of FCCP (CMA, 20 μM) or concanamycin A (20 μM), or in the absence of ATP.",
    "molecules": "CMA, ATP, FCCP, concanamycin"
  },
  {
    "caption": "(B) Relative TAG levels in different genetic backgrounds were measured by glyceride  assay kit. For quantification, TAG  levels were normalized to protein. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant.",
    "molecules": "TAG"
  },
  {
    "caption": "(B  3rd instar larval fat bodies  were  stained  by BODIPY (green) for  lipid droplets  and  DAPI (blue) for  nuclei. Scale bars represent 25 µm. (B) RDH/CG2064 RNAi suppresses the enlarged  lipid droplet   phenotype in CSN2  RNAi under  DGAT1  overexpression (ppl>DGAT1) or high-fat diet conditions. (C) Quantification of the   lipid droplet  size in (B). HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each  fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.",
    "molecules": "BODIPY, DAPI"
  },
  {
    "caption": ", F 3rd instar larval fat bodies were stained  by  BODIPY  (green) for   lipid droplets and DAPI (blue) for  nuclei. Scale bars represent 25 µm. (F) RDH/CG2064 overexpression increases  lipid droplet  size in ppl>DGAT1 larvae. (G) Quantification of the  lipid droplet size in (F). data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.",
    "molecules": "BODIPY, DAPI"
  },
  {
    "caption": "3rd instar larval fat bodies were stained by BODIPY (green) for  lipid droplets   and DAPI  (blue) for nuclei. Scale bars represent 25 µm. (H) Relative TAG  levels in different genetic backgrounds were measured by glyceride assay  kit. For quantification, TAG levels were normalized to protein. (I) RDH/CG2064 and  CG2065 deletion does not affect lipid droplet   size under normal or high-fat diet conditions. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.",
    "molecules": "BODIPY, DAPI, TAG"
  },
  {
    "caption": "(B) 3rd instar larval fat bodies  were  stained  by BODIPY (green) for lipid droplets and DAPI  (blue) for  nuclei. Overexpressing wild-type or site-mutated RDH/CG2064 increases the lipid droplet size in larvae with DGAT1  overexpression (ppl>DGAT1). Scale bar represents 25 µm.",
    "molecules": "BODIPY, DAPI"
  },
  {
    "caption": "(F) 3rd instar larval fat bodies  were  stained by BODIPY  (green) for lipid droplets  and  DAPI (blue) for nuclei. Under  high-fat conditions, RDH/CG2064 overexpression increases the lipid droplet size, and Plin2 RNAi suppresses the enlargement induced by  RDH/CG2064 overexpression. Scale bar represents 25 µm.",
    "molecules": "BODIPY, DAPI"
  },
  {
    "caption": "(A, B Images of  Plin2-GFP in 3rd instar larval fat body  cells  of different genotypes. Scale bar represents 25 µm. (A) CSN2 RNAi or RDH/CG2064 overexpression reduces the peripheral localization of  Plin2-GFP and increases the localization  of Plin2-GFP on small peri-nuclear lipid droplets (boxed).  DAPI (blue)  stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. (B) The peripheral localization  of Plin2-GFP is greatly reduced in  fat body  cells from  larvae with  CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) The  peripheral lipid droplets in larval fat body cells  from different genetic backgrounds were  stained by BODIPY (green). CSN3 RNAi or RDH/CG2064 overexpression reduces the number of peripheral lipid droplets in both wild-type and ppl>DGAT1 larvae.",
    "molecules": "BODIPY"
  },
  {
    "caption": "(A, Confocal  images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (A) Knocking down  CSN2 or overexpressing  RDH/CG2064  in animals with DGAT1 overexpression (ppl>DGAT1) decreases the level and  lipid droplet localization  of Bmm-GFP. (B) The GFP intensity along the line across a  lipid droplet  in (A) was measured by ImageJ. The  lipid droplet localization  of Bmm-GFP, represented by two peaks, is clearly visible in fat cells from ppl>DGAT1 larvae, but it is lost in  fat cells from ppl>DGAT1 larvae  with CSN2  RNAi or overexpression of RDH/CG2064. More than 30  lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.",
    "molecules": "DAPI"
  },
  {
    "caption": "F)  Confocal  images of  Bmm-GFP  (green) in 3rd instar larval fat bodies of different genotypes. DAPI  (blue)  stains nuclei. Scale bar represents 25 µm. (F) Overexpression of Plin2 decreases the level and  lipid droplet localization of Bmm-GFP in  fat cells  from  larvae  with DGAT1  overexpression. (G) GFP intensity along the line across a  lipid droplet  from (F) was measured by ImageJ. The  lipid droplet localization  of Bmm-GFP, represented by two peaks, was evident in fat cells from ppl>DGAT1 larvae, but is lost in ppl>DGAT1 larvae with overexpression of  Plin2. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) 3rd instar larval fat bodies were stained  by BODIPY (green) for   lipid droplets and  DAPI (blue) for nuclei. bmm RNAi increases the lipid droplet size in fat cells  from  larvae with  DGAT1 overexpression. Scale bar represents 25 µm.",
    "molecules": "BODIPY, DAPI"
  },
  {
    "caption": "(A) S2  cells expressing  RDH/CH2064-GFP  were treated with the protein synthesis inhibitor cycloheximide  (CHX) for the indicated time (h: hours). The level of RDH/CH2064-GFP was determined by  western blotting using anti-GFP antibody. Tubulin was used as loading control. (B) Quantification of the relative RDH/CH2064-GFP protein levels in (A). Error bars represent ± SEM.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(C) S2 cells expressing Bmm-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of Bmm-GFP was determined by western blotting  using anti-GFP antibody. Tubulin was used as loading control. (D) Quantification of the relative Bmm-GFP protein levels in (C). Error bars represent ± SEM.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(F) Oil red staining of 3rd instar larval oenocytes in different genetic backgrounds under fed  or starvation conditions.",
    "molecules": "Oil red"
  },
  {
    "caption": "HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+. Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was -80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps). Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).",
    "molecules": "Ba2+"
  },
  {
    "caption": "HEK293 cells were transfected with α11.2, α2δ-1, and β2A before whole cell patch recording in 20 mM Ba2+. Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of -80 mV to the indicated potentials (the voltage protocol is schematized in the upper left corner). Representative current traces upon step depolarizations to the reversal potential (Erev) for 20 ms to obtain movement of the ON-gating charges (Qon), and subsequent to -50 mV for 10 ms to obtain tail currents (Itail). Insets: magnifications of exemplary Qon for CaV1.2 WT, K1647A, Y1649A, I1654A, F1658A and K1662E. Plots of Itail (in this panel corrected for variations in cell capacitance) versus total detectable charge transfer for Qon. Slopes of regression curves are strongly reduced for CaV1.2 K1647A, Y1649A and I1654A versus WT (see Table 7 for more details). Dot plots and means + SEM of Qon (in this panel corrected for variations in cell capacitance; *p<0.05; **p<0.01; one-way ANOVA with Bonferroni post-hoc test; n = 13-18; see Table 7 for more details). The reduction in slope of the regression curve of combined population data for K1647A, Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α-actinin binding to the IQ motif is diminished.",
    "molecules": "Ba2+"
  },
  {
    "caption": "(D) Anti-Flag co-IP of DAXXMycHis, RNF2 and H3K27me3 with CBX23×Flag from extracts of HEK293 cells in presence or absence of benzonase, followed by immuno-blot analyses for indicated antigens.",
    "molecules": "benzonase"
  },
  {
    "caption": "(F) Ni-pulldown of 6xHisSUMO1/2/3-conjugated proteins from HEK293 extracts expressing 6xHis-tagged SUMO1, SUMO2 or SUMO3 in combination with CBX2-EGFP. CBX2 and SUMO-conjugated CBX2 were detected with an anti-GFP antibody.",
    "molecules": "Ni"
  },
  {
    "caption": "(G) Ni-pulldown of 6xHis-SUMO2 conjugated proteins from HEK293 extracts transiently co-expressing 6xHis-SUMO2 with CBX2-EGFP or CBX24KR-EGFP. Detection of CBX2",
    "molecules": "Ni"
  },
  {
    "caption": "(F) IF of 5-methyl and 5-hydroxy-methyl cytosine staining of wild-type (n=42), Daxxm-z+ (n=14) and Ring1m-z+; Rnf2m-z+ (n=6) zygotes with lagging chromosomes. Yellow arrowheads indicate lagging chromosomes. Data information: All scale bars 10µm.",
    "molecules": "5-hydroxy-methyl cytosine, 5-methyl"
  },
  {
    "caption": "(K) IP of DAXX-EGFP and DAXXR244A-EGFP proteins with an anti-GFP antibody from nuclear extract of HEK293 cells. Immuno-blot detection was performed with antibodies recognizing GFP, histone variant H3.3 and CBX2.",
    "molecules": "histone"
  },
  {
    "caption": "(B) Violin plots showing the expression  of prostaglandin  (PG) receptors in pre-adipocytes and mature adipocytes from human  deep-neck BAT(E-MTAB-8564).",
    "molecules": "PG, prostaglandin"
  },
  {
    "caption": "(A)   Oil Red O staining   of mouse BAs differentiated  from normal or EP3-deficient brown pre-adipocytes. Scale bar: 50 μm. (B) Mean  lipid droplet area of differentiated BAs  from normal or EP3-deficient brown  pre-adipocytes. *P < 0.05 vs WT (n = 6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests (B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(D) Representative image of hematoxylin  and eosin  (H&E) staining  for iBAT  from EP3F/F and  EP3F/FMyf5Cre mice. Scale bar: 50 μm. (E) Mean adipocyte area of BAs  in iBAT  from  EP3F/F and EP3F/FMyf5Cre mice  (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(A) N6-methyladenosine (m6A) modification levels of mRNAs in normal and EP3-deficient brown pre-adipocytes  with or without brown adipogenesis induction, detected by m6A RNA Methylation  Quantification Kit (Colorimetric) (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test (A *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).",
    "molecules": "m6A, N6-methyladenosine"
  },
  {
    "caption": "(B)  qRT-PCR analysis of the relative mRNA levels of m6A  writers (METTL3, METTL4, METTL14  and WTAP) and erasers (FTO  and ALKBH5) in normal and EP3-deficient brown pre-adipocytes  with or without brown adipogenesis induction (n = 6-9). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).",
    "molecules": "m6A"
  },
  {
    "caption": "(C) Western blotting analysis of m6A  writers and erasers in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction.",
    "molecules": "m6A"
  },
  {
    "caption": "(J) Representative picture of  oil red O-stained BAs differentiated  from EP3γ-expressed EP3-/- brown pre-adipocytes with WTAP  siRNA treatment. Scale bar: 50 μm.",
    "molecules": "oil red O"
  },
  {
    "caption": "(A) Western blotting analysis of ubiquitination  of WTAP  in the control and EP3-deficient brown pre-adipocytes with or without sulprostone  treatment. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "sulprostone"
  },
  {
    "caption": "(C) Effect of ERK1/2 inhibitor, ravoxertinib, on ubiquitin-dependent degradation of WTAP  in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "ravoxertinib, sulprostone, ubiquitin"
  },
  {
    "caption": "(D) Effect of wortmannin  (Wor), pertussis toxin (PTX), U73122, barbadin, and melittin  on ubiquitin-dependent degradation  of WTAP  in sulprostone-treated  brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "barbadin, melittin, sulprostone, U73122, ubiquitin, Wor, wortmannin"
  },
  {
    "caption": "(F) Effect of PKA  inhibitor, H89, on ubiquitin-dependent degradation of WTAP  in EP3-/- brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "H89, ubiquitin"
  },
  {
    "caption": "(G) Effect of ERK1/2 phosphorylation  site mutation (HA-WTAP-2A) on EP3-mediated ubiquitin-dependent degradation  of WTAP  in brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(H) Representative picture of oil red O-stained BAs differentiated  from sulprostone-treated brown pre-adipocyte  with treatment of PTX, dbcAMP, or ravoxertinib. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "dbcAMP, oil red O, ravoxertinib, sulprostone"
  },
  {
    "caption": "(I) Western blotting assay of ERK1/2, WTAP, ZFP410, PRDM16, and UCP1  in BAs  differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "dbcAMP, ravoxertinib, sulprostone"
  },
  {
    "caption": "(B)Representative pictures of nile red-stained human BAs differentiated  from hESC-derived brown progenitors. Scale bar: 50 μm.",
    "molecules": "nile red"
  },
  {
    "caption": "(D) Representative pictures of hESC-derived BAs  treated with L798106  alone or combined with H89. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates",
    "molecules": "L798106, H89"
  },
  {
    "caption": "(E) Effect of H89  on the relative mRNA level of brown marker gene in hESC-derived BAs  treated with L798106  detected by RT-qPCR (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons (E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).",
    "molecules": "L798106, H89"
  },
  {
    "caption": "(F) Effect of H89  on WTAP  mRNA expression  in hESC-derived BAs  treated with L798106  (n = 6-7). (G) Effect of H89  on ZNF410  mRNA expression  in hESC-derived BAs  treated with L798106  (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two-way ANOVA followed by Tukey's test for multiple comparisons F, G). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).",
    "molecules": "L798106, H89"
  },
  {
    "caption": "(H) Western blotting  assay of ERK1/2, WTAP, ZNF410, PRDM16, and UCP1  in hESC-derived BAs  treated with L798106 alone or combined with H89.",
    "molecules": "L798106, H89"
  },
  {
    "caption": "D. Expression of Ldb2 in the cerebral cortex. DAPI-stained nuclei visualized (right) in the cerebral cortex.",
    "molecules": "DAPI"
  },
  {
    "caption": "Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with methylphenidate (MPD: 3 mg/kg, A) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.",
    "molecules": "methylphenidate, MPD"
  },
  {
    "caption": "Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with MK-801 (0.2 mg/kg, B) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.",
    "molecules": "MK-801"
  },
  {
    "caption": "C. Popping behavior induced by the injection of MK-801 (0.5 mg/kg) was manually assessed in WT (n = 4) and KO (n = 6) animals. Typically, each episode is constituted by blocks of 5-15 successive jumping behaviors. Episode numbers in 20 min (left) and total duration (right) are shown in a box-whisker plot.",
    "molecules": "MK-801"
  },
  {
    "caption": "A. Fear-conditioning test. Freezing (%) was monitored in the cued (top) and contextual (bottom) tests before (pre) and after (post) administration of clozapine (left) or haloperidol (right). Data are presented as means ± S.E.M. (n = 7-9/group). *, p < 0.05; ***, p < 0.001 (Tukey test); #, p < 0.05 (t-test, KO/saline vs. KO/clozapine).",
    "molecules": "clozapine, haloperidol, saline"
  },
  {
    "caption": "B. Home cage-activity test. Spontaneous locomotor activity of mice chronically fed chow with or without 0.24% (w/w) lithium carbonate for two months was monitored for 6 days in the home cage.",
    "molecules": "lithium carbonate"
  },
  {
    "caption": "A. Hematoxylin and eosin (H&E)-stained cross section of a human liver slice. Scale bar is representative of A-F. Image is also used in Fig. S3A. B. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from A. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant) which is representative of B, D, and F. C. H&E-stained cross section of a human lung slice. Image is also used in Fig. S3D. D. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from C. E.H&E-stained cross section of frontal cortical region of a human brain tissue. Image is also used in Fig. S3G. F. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from E. G. Quantification of relative abundance of total glycogen calculated from the sum of CL3-CL18 in human liver, lung, and brain. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison for total glycogen. H. Glycogen structure represented by normalized chain length distribution (ratio to CL3) of each human liver, lung and brain from G. Values are presented as mean +/- standard error (n=3 technical replicates per region).",
    "molecules": "eosin, glycogen, Glycogen, Hematoxylin"
  },
  {
    "caption": "A. Hematoxylin and eosin (H&E)-stained cross section of adenocarcinoma of the prostate, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and Ewing sarcoma (ES) of the abdomen tumor. Annotated regions are T-tumor, S-stroma, D/G-duct/glandular, and A-adipose tissue. Scale bar is representative of A and B. Images are also used in Fig. S4A, 5D and S6G. B. Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Images are also used in Fig. S4E, 5D, and S6G. C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from tumor regions shown in A. Values are presented as mean +/- standard error (n=3 technical replicates per region).",
    "molecules": "eosin, glucose, glycogen, Glycogen, Hematoxylin"
  },
  {
    "caption": "A. (Left) Hematoxylin and eosin (H&E)-stained cross section of an Ewing sarcoma (ES) of the shoulder tumor. Annotated regions are annotated as T-tumor and M-muscle. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A-E. B. (Left) H&E stained cross section of an ES of the chest wall tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. C. (Left) H&E stained cross section of an ES of the rib tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. D. (Left) H&E stained cross section of an ES of the abdomen tumor. Annotated regions are T-tumor, S-stroma, and A-adipose tissue. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. E. (Left) H&E stained cross section of an ES of the bladder tumor. Annotated regions are T-tumor and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. F. Magnified H&E cross section of (top, left) tumor, and (top, right) muscle tissue from ES rib with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, and (bottom, right) muscle tissues. G. Magnified H&E cross section of (top, left) tumor, (top, middle) stroma, and (top, right) adipose tissues from ES abdomen with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, (bottom, middle) stroma and (bottom, right) adipose tissues.",
    "molecules": "eosin, glycogen, Hematoxylin"
  },
  {
    "caption": "A. (Left) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A. B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.",
    "molecules": "eosin, glucose polymers, glycogen, Glycogen, Hematoxylin"
  },
  {
    "caption": "G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.",
    "molecules": "guaiacol"
  },
  {
    "caption": "J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant). K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.",
    "molecules": "glycogen"
  },
  {
    "caption": "(A) Representative images of 5-day-old wt,   gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals  stained  with Oil Red O (ORO) (scale bar= full image-100 μm;  hindgut  magnification-20 μm). (B) Quantification of hindgut (left panel), foregut  (center panel) and whole body (right panel) of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2  O/E mutant  nematodes stained with  ORO (one-way ANOVA with Tukey's multiple comparison test, n=18-26 animals). (C) Quantification of mean intensity of ORO staining   in the hindgut of wt, gas-1(fc21) and gas-1(fc21); cest-2.2  O/E mutant nematodes  at different time points, starting from L4 stage (one-way ANOVA with Dunnett's post hoc correction, n=18-26 animals). (D) Mean intensity of ORO staining in the whole body  of 5-day-old wt, gas-1(fc21) and gas-1(fc21);  cest-2.2 O/E mutant nematodes  plotted against body area. Data information: , bars represent mean ±SEM points represent mean ±SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.",
    "molecules": "Oil Red O, ORO"
  },
  {
    "caption": "(E) Representative images of wt,  cest-2.2 O/E and cest-2.2 (lf) animals  stained  with  ORO. Arrows indicate mislocalized lipid droplets. (F) Quantification (mean intensity ± SEM) of  ORO staining in hindgut  (left panel), foregut (center panel) and whole body (right panel) of 5-day-old  nematodes (one-way ANOVA with Dunnett's post hoc correction, n=11-17 animals). Data information: , bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.",
    "molecules": "ORO"
  },
  {
    "caption": "(G) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with  LipidTOX (scale bar= 100 μm). (H) Statistical analysis of whole-body LipidTOX intensity of 5-day-old nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=25-37 animals). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.",
    "molecules": "LipidTOX"
  },
  {
    "caption": "(I-J) Representative images of  nematodes stained with LipidTOX (I) (scale bar= 100 μm), with quantification (J) (mean intensity ± SEM) of LipidTOX staining. Data information: bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.",
    "molecules": "LipidTOX"
  },
  {
    "caption": "(K) Representative  thin layer chromatography (TLC) plate of samples from 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes. (L) Densitometry of  TLC-separated free fatty acids (FFA) across three biological replicates in 5-day-old mutant animals. Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.",
    "molecules": "FFA, free fatty acids"
  },
  {
    "caption": "(N) Sum of TLC-separated  FFA of 5-day old animals (n=5 biological replicates). (O) Quantification of individual FFA detected after  TLC  separation in 5-day-old gas-1(fc21) and  gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.",
    "molecules": "FFA"
  },
  {
    "caption": "Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), or bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Representative flow cytometry histogram of BTV-VP7 expression in inhibitor-treated cells.",
    "molecules": "3-MA, BAF-A1, bafilomycin-A1, chloroquine, CQ, lactacystin, LC, MG, MG132"
  },
  {
    "caption": "Vero cells were infected at MOI 1 (or mock infected) and treated with proteasome inhibitors MG132 (MG 20 µM), lactacystin (LC 20 µM), chloroquine (CQ 50 µM), 3-MA (5 mM), for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.",
    "molecules": "3-MA, chloroquine, CQ, lactacystin, LC, MG, MG132"
  },
  {
    "caption": "Vero cells were infected at MOI 1 (or mock infected) and treated with bafilomycin-A1 (BAF-A1 0.2 µM) for the final 6h. Immunoblots probed for STAT1, STAT2, LC3 and GAPDH expression.",
    "molecules": "BAF-A1, bafilomycin-A1"
  },
  {
    "caption": "Immunofluorescence of BTV-8 or mock-infected Vero cells (treated for the last 6h with BAF-A1) stained for BTV proteins, STAT2 and p62. Inset shows detail of STAT2 and p62 colocalization. Nuclei were counterstained with DAPI. Arrowheads in inset indicate p62 and STAT2 colocalization. Scale bar = 20 μm. Inset scale bar = 3 μm.",
    "molecules": "BAF-A1, DAPI"
  },
  {
    "caption": "Immunoblots of BTV-NS3-transfected 293T cells treated with proteasome inhibitors MG or LC. Membranes were probed for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH. (B-D) Immunoblots of BTV-NS3-transfected 293T cells treated with increasing concentration of lysosome acidification inhibitors (B) CQ or (C) NH4Cl or with increasing concentration of the (D) autophagosome formation inhibitor 3-MA, and probed (IB) for STAT1, STAT2, BTV-NS3 (FLAG) and GAPDH expression.",
    "molecules": "3-MA, NH4Cl, CQ, LC, MG"
  },
  {
    "caption": "Immunofluorescence confocal images of Vero cells infected with rgBTV-8, BTV-8 ΔNS3 or mock-infected and treated with IFN-U for 30min prior to fixation and staining. Cells were stained for BTV-VP7 and (I) P-STAT1 or (J) STAT2. Nuclei were counterstained with DAPI. (Scale bar = 30 μm). (I) Arrowhead in merge image indicate partial inhibition of STAT1 phosphorylation in BTV-8 ΔNS3 infected cells. (J) Arrowhead in merge image shows STAT2 translocation to nucleus in BTV-8 ΔNS3-infected cells.",
    "molecules": "DAPI"
  },
  {
    "caption": "Fluorescence intensity profile of cell section indicated by the white line (blue: DAPI, green: NS3; red: Ub). Arrowheads on image and arrows on intensity profile indicate matching fluorescence signal for NS3 and Ub.",
    "molecules": "DAPI"
  },
  {
    "caption": "FLAG-tag IP assay of BTV-NS3 and WCE immunoblot of 293T co-transfected FLAG-BTV-NS3 plasmid and HA-UB-WT or HA-UB lysine (K to R) mutants. IP were probed (IB) for ubiquitin (HA) and BTV-NS3 (FLAG) expression. WCE were probed for STAT1, STAT2, ubiquitin (HA), BTV-NS3 (FLAG), and GAPDH expression.",
    "molecules": "lysine"
  },
  {
    "caption": "B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids (or 120 µg ssODN) per mouse.",
    "molecules": "ODN"
  },
  {
    "caption": "D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector treated group) or by TA-clone sequencing (Cas9/ssODN group).",
    "molecules": "ODN"
  },
  {
    "caption": "B: Consequences of ROS on the SUMO proteome vary amongst cell lines. HeLa, MCF7, U2OS or RPE cells were treated with increasing H2O2 concentration for 15 min and lysed in Laemmli buffer supplemented with 20 mM N-Ethylmaleimide (NEM). Samples were analyzed by immunoblotting with SUMO1 antibodies.",
    "molecules": "N-Ethylmaleimide, NEM, H2O2, ROS"
  },
  {
    "caption": "C: H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells. HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min. Cells were lysed with 1% SDS in PBS buffer and 20 mM NEM. Upon immunoprecipitation of the SUMO1 proteome, samples were analyzed by immunoblotting with TRIM28 antibodies.",
    "molecules": "NEM, H2O2, SDS"
  },
  {
    "caption": "D: Induction of the Uba2~Ubc9 disulfide is a conserved mechanism. HeLa, MCF7, U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min, lysed in non reducing buffer supplemented with 20 mM NEM, and analyzed by immunoblotting with Uba2 antibodies.",
    "molecules": "H2O2, NEM"
  },
  {
    "caption": "A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described.",
    "molecules": "H2O2, ATP"
  },
  {
    "caption": "B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP.",
    "molecules": "H2O2, ATP"
  },
  {
    "caption": "B: In FRET-based assay, Ubc9 D100A is active upon H2O2 treatment. Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig. 2B.",
    "molecules": "H2O2"
  },
  {
    "caption": "A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration.",
    "molecules": "DTT, ATP, H2O2"
  },
  {
    "caption": "B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2",
    "molecules": "glutathione, H2O2"
  },
  {
    "caption": "B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1.",
    "molecules": "H2O2"
  },
  {
    "caption": "C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p < 0.05",
    "molecules": "H2O2"
  },
  {
    "caption": "D: Constitutive expression of Ubc9 D100A affects cellgrowth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between \"Ubc9 wt\"and \"Ubc9 DA\" cells. The average size of \"Ubc9 wt\" was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M.",
    "molecules": "O2"
  },
  {
    "caption": "A: Phosphorylation of ATM is impaired in Ubc9 D100A cells. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.",
    "molecules": "H2O2"
  },
  {
    "caption": "B: Phosphorylation of gamma-H2AX is impaired in Ubc9 D100A. Experiment was performed as in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.",
    "molecules": "H2O2"
  },
  {
    "caption": "C: Phosphorylation of Chk2 is impaired in Ubc9 D100A. Experiment was performed as described in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.",
    "molecules": "H2O2"
  },
  {
    "caption": "D: Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure. Stable U2OS cell populations were exposed to 2.5 mM hydroxyurea or 250 µM H2O2. Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies.",
    "molecules": "H2O2, hydroxyurea"
  },
  {
    "caption": "E: Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells. Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2.5 mM hydroxyurea. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.",
    "molecules": "hydroxyurea"
  },
  {
    "caption": "A: Impaired y-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05.",
    "molecules": "H2O2"
  },
  {
    "caption": "B: H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells. Cells were treated as described in A. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. An alkaline comet assay was performed to evaluate the DNA damage. Representative images are shown. Error intervals represent S.E.M, n=4 independent experiments.",
    "molecules": "DNA, H2O2"
  },
  {
    "caption": "A: Diamide is inducing the SUMO E1~E2 disulfide but not ATM phosphorylation. U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2. Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer. The lysates were resolved by SDS-PAGE.",
    "molecules": "NEM, Diamide, diamide, H2O2"
  },
  {
    "caption": "B: Left panel: The PP2A inhibitor okadaic acid rescues the y-H2AX defect of the \"Ubc9 DA\" cells. \"Ubc9 wt\" or \"Ubc9 DA\" cells were treated with 0.25 µM okadaic acid for 30 min. After addition of 250 µM H2O2 in fresh medium for 3 hours, immunofluorescence microscopy was performed as described in Fig. 7A. Error bars represent S.E.M, n=3 independent experiments. Right panel: Representative immunofluorescence images.",
    "molecules": "H2O2, okadaic acid"
  },
  {
    "caption": "C: Left panel: Ubc9 D100A enhances sensitivity to Ara-C or etoposide. Cell survival assays were performed as described in Fig. 5C. Cells were exposed to Ara-C or etoposide (VP16) for 1 h and evaluated 10 days later. Right panel: N-acetylcysteine treatment rescues the higher toxicity of Ara-C or etoposide on \"Ubc9 DA\" cells. Cells were treated with 0.5 mM NAC 16 h prior to addition of etoposide or Ara-C. Graphs represent means of three to four independent experiments, error bars represent S.E.M. * p < 0.05.",
    "molecules": "Ara-C, etoposide, N-acetylcysteine"
  },
  {
    "caption": "(A) BI‐1 WT and KO MEFs were treated with EBSS (left panel) or glucose/serum‐free RPMI media (right panel) for the indicated time points. Then, levels of LC3 were determined by western blot analysis. LC3‐I and LC3‐II forms are indicated. Hsp90 levels were assessed as loading control.",
    "molecules": "glucose"
  },
  {
    "caption": "(C)Cells were pre‐treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3‐methyladenine (3‐MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3‐II levels relative to Hsp90 were performed in the experimental conditions described in (C).",
    "molecules": "3‐methyladenine, bafilomycin A1, E64d, pepstatin"
  },
  {
    "caption": "(A) Left panel: BI‐1 WT and KO MEFs cells were incubated in EBSS, and then cell viability was monitored using the MTS assay. Right panel: a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h. Mean and standard deviation are presented of triplicates representative of three independent experiments.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(B) BI‐1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h. Cell death was determined after propidium iodide (PI) staining and FACS analysis. In addition, cells were treated with 100 ng/ml tunicamycin (Tm) for 24 h. Mean and standard deviation are presented of one experiment performed in triplicates.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(B) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125. Mean and standard deviation are presented (N=3). Student's t‐test was used to analyse statistical significance, *P0.001 and **P0.0001.",
    "molecules": "SP600125"
  },
  {
    "caption": "(C) BI‐1 WT and KO cells were treated with EBSS for indicated time points, in the presence or absence of 10 μM of the JNK inhibitor SP600125. Levels of phosphorylation of JNK (pJNK) and LC3‐II were determined by western blot. The levels of total JNK and Hsp90 are shown as control (N=4). Image was assembled from cropped lanes of the same western blot analysis of the same gel.",
    "molecules": "SP600125"
  },
  {
    "caption": "(D) BI‐1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125, and the electrophoretic shift associated with BCL‐2 phosphorylation was monitored by western blot.",
    "molecules": "SP600125"
  },
  {
    "caption": "(B) (B) LC3 levels were monitored in control (Da‐Gal4>huLC3:GFP) or dBI‐1 RNAi larvae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) under basal or fasting conditions. Then, huLC3-GFP levels were analysed by western blot. In addition, dBI‐1i larvae were treated with 100 μM SP600125 (added in the growing media).",
    "molecules": "SP600125"
  },
  {
    "caption": "(E) Control or dBI‐1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days. In all, 100 individuals were monitored in each condition. Data represent mean and standard error (N=3). Two‐way ANOVA was used to analyse statistical significance between groups.",
    "molecules": "nutrient"
  },
  {
    "caption": "(F) Second instar dBI‐1 RNAi or control larvae were grown in food supplemented with 25 μg/ml Tm dissolved in DMSO or 0.5% DMSO as control. The number of individual reaching the adult fly stage was evaluated. Mean and standard error are presented (N=3), **P0.01.",
    "molecules": "DMSO"
  },
  {
    "caption": "(C) bi‐1+/+ and bi‐1−/− mice were injected with 50 ng/ml of tunicamycin by intraperitoneal injection (N=3) and then LC3 levels were monitored in liver protein extracts by western blot analysis. Right panel: quantification of relative LC3‐II levels, **P0.005.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "G Parental and SMCR8 KO cells were subjected to a cycloheximide (CHX) chase in the absence or presence of DMSO or Btz. * indicates unspecific protein band detected by C9orf72 antibody. α‑tubulin served as loading control. NRF2 confirmed proteasomal inhibition by Btz.",
    "molecules": "Btz, CHX, cycloheximide, DMSO"
  },
  {
    "caption": "C SMCR8 KO cells were treated with DMSO or MLN7243 prior to fixation and immunostaining with an anti-C9orf72 antibody. Untreated parental and C9orf72 KO cells served as controls. Scale bar, 10 µm.",
    "molecules": "DMSO, MLN7243"
  },
  {
    "caption": "D Empty or stably C9orf72-HA expressing SMCR8 KO cells were treated with Btz or DMSO followed by lysis and immunoblotting. Untreated parental cells served as control. Calnexin served as loading control. NRF2 confirmed proteasomal inhibition by Btz.",
    "molecules": "Btz, DMSO"
  },
  {
    "caption": "E Parental and SMCR8 KO cells stably expressing C9orf72-HA were subjected to CHX chase in absence and presence of DMSO or Btz. NRF2 confirmed proteasomal inhibition by Btz.",
    "molecules": "Btz, CHX, DMSO"
  },
  {
    "caption": "F Lysates from empty or stably C9orf72-HA expressing SMCR8 KO cells treated with DMSO or Btz were subjected to HA-IP under denaturing conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2.",
    "molecules": "Btz, DMSO"
  },
  {
    "caption": "D Lysates from C9orf72-HA expressing parental and SMCR8 KO cells treated with DMSO or Btz were subjected to HA-IP followed by immunoblot analysis. Empty parental and SMCR8 KO served as controls. : NRF2 confirmed proteasomal inhibition by Btz.",
    "molecules": "Btz, DMSO"
  },
  {
    "caption": "E SMCR8 KO cells expressing C9orf72-HA were transiently transfected with GFP-UBR5 or GFP and treated with DMSO or Btz. Lysates were incubated with Streptavidin agarose coupled to biotinylated GFP VHH nanobodies and bound proteins were analyzed by immunoblotting.",
    "molecules": "nanobodies, agarose, Btz, DMSO, Streptavidin"
  },
  {
    "caption": "A SMCR8 KO cells stably overexpressing C9orf72-HA were treated with Btz and lysates were subjected to HA-IP under mild conditions in the absence and presence of the catalytic domain of the deubiquitinase USP2. FK2 confirmed deubiquitination by USP2.",
    "molecules": "Btz"
  },
  {
    "caption": "D SMCR8 KO cells transiently overexpressing GFP, GFP-C9orf72 WT or GFP-C9orf72 mutant K156R, K388R, K414R, K422R, K424R, K425R, K428R (KR7) were treated with Btz followed by streptavidin pulldown with biotinylated GFP VHH nanobodies under denaturing conditions.",
    "molecules": "nanobodies, Btz, streptavidin"
  },
  {
    "caption": "B SMCR8 KO cells stably expressing GFP-C9orf72 were treated with DMSO or Btz followed by denaturing lysis and differential USP2 treatment. Lysates were incubated with biotinylated GFP VHH nanobodies coupled to Streptavidin agarose and enriched proteins were analyzed by immunoblotting.",
    "molecules": "nanobodies, agarose, Btz, DMSO, Streptavidin"
  },
  {
    "caption": "D SMCR8 KO cells were transiently transfected with siUBR4/5, siCTRL or left untreated but grown in the presence of Btz. Lysates were subjected to immunoblotting. Quantification of C9orf72 levels in SMCR8 KO cells treated with siUBR4/5 or siCTRL. n = 3 biological replicates. Data represent mean ± SD. Statistical analysis of C9orf72/α‑tubulin ratio was performed using one-tailed, unpaired Student's t-test. * p < 0.05. Representative Western blot is shown beside. α‑tubulin served as loading control.",
    "molecules": "Btz"
  },
  {
    "caption": "(a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm.",
    "molecules": "3-MA"
  },
  {
    "caption": "(c) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).",
    "molecules": "ammonium chloride, chloroquine"
  },
  {
    "caption": "(b) The degree of MR persistence depends on the time and not on cell-cycle duration. HeLa cells were either left untreated or were arrested in S-phase with hydroxyurea (HU, 5 mM). Cells were fixed and stained as indicated. Scale bars are 20 μm. The right panel shows quantification of MR counting (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).",
    "molecules": "hydroxyurea"
  },
  {
    "caption": "(a) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels). The alleles are indicated. Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA. Arrowheads point to the accumulated MRs. Scale bars are 20 μm. (b) Quantification of a (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).",
    "molecules": "DNA"
  },
  {
    "caption": "(G) RT-qPCR analyses of selected ERS UP and LIVER-ID TF genes monitoring expression changes induced by 4h ERS in MPH pre-treated or not for 30min with 5mM PBA (3 independent experiments). Mean Log2 FC ERS/Control are shown. The bar graph shows means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.",
    "molecules": "PBA"
  },
  {
    "caption": "(B) Left panel, BRD4 mRNA (4 independent experiments) or protein expression levels (5 independent experiments; densitometric quantification in MPH subjected to acute ERS. The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance. Right panel, Total protein extracts from MPH pre-treated for 3h with 0.01µM MZ1 followed by addition of 1µM thapsigargin (ERS) for 4h were subjected to Western blot with an antibody against BRD4. LMNA was used as loading control.",
    "molecules": "MZ1, thapsigargin"
  },
  {
    "caption": "(C) Heatmaps showing Log2 FC (relative to the DMSO/Control condition) for 5 ERS UP and LIVER-ID genes issued from RT-qPCR analyses of MPH pre-treated with 500nM JQ1 (left) or 0.01µM MZ1 (right) followed by addition of 1µM thapsigargin (ERS) for 4h (3 to 6 independent experiments). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance.",
    "molecules": "JQ1, DMSO, MZ1, thapsigargin"
  },
  {
    "caption": "(A) RT-qPCR analyses of 21 LIVER-ID genes in MPH which were treated with 1µM thapsigargin (ERS) for 4h, 8h, 16h or 24h (4 independent experiments). For each gene, the timepoint showing maximal repression by ERS was recorded. The heatmap depicts the percentage of genes maximally repressed by ERS at each timepoint.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "(B) RT-qPCR analyses of Hnf4a, Nr1h4 and Foxa2 expression in MPH treated with vehicle (Control) or 1µM thapsigargin (ERS) for 1h or 4h (4 independent experiments). The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "(D) Nuclear extracts from MPH pre-treated for 30min with 1µM ISRIB followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or DDIT3. LMNA was used as loading control. Results obtained from 3 independent biological replicates are shown. (E) Densitometric quantification of the protein expression data shown in panel (D). Repression by ERS in the ISRIB condition (average of 3 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.",
    "molecules": "ISRIB, thapsigargin"
  },
  {
    "caption": "(F) Nuclear extracts from MPH pre-treated for 30min with 10µM MG132 or 10µM PP2 followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. LMNA was used as loading control. Results obtained from 2 independent biological replicates are shown. (G) Densitometric quantification of the protein expression data shown in panel (F) Repression by ERS in MG132 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.",
    "molecules": "MG132, PP2, thapsigargin"
  },
  {
    "caption": "(I) Densitometric quantification of the protein expression data Repression by ERS in the PP2 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%.",
    "molecules": "PP2"
  },
  {
    "caption": "(J) RT-qPCR analyses of selected ERS UP and LIVER-ID TF genes monitoring expression changes induced by 4h ERS in MPH pre-treated or not for 30min with 10µM PP2 (3 independent experiments). Mean Log2 FC ERS/Control are shown. The bar graph shows means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.",
    "molecules": "PP2"
  },
  {
    "caption": "(H) Nuclear extracts from livers of sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. TFIIB was used as loading control. (I) Densitometric quantification of the protein expression data from 6 mice precondition shown in panel (H) (3 mice per condition) The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC TUDCA/Vehicle is statistically different from 0, *P < 0.05.",
    "molecules": "TUDCA"
  },
  {
    "caption": "(J) Serum alanine aminotransferase (ALT) (left) and aspartate aminotransferase (AST) activities (right) from sepsisBIM mice pre-treated for 4 consecutive days with vehicle or 500 mpk TUDCA (10 mice per group). The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance, *P < 0.05.",
    "molecules": "TUDCA"
  },
  {
    "caption": "(B) Box plots showing Log2 FC ERS/Control in mouse liver for ERS UP genes 8h and 34h after tunicamycin injection in WT or ATF6 KO mice (3 mice per experimental condition). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05. NS, not significant. (C) Similar analyses to panel (B) for ERS DOWN and LIVER-ID genes (3 mice per experimental condition). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05. ",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(F) Expression of LIVER-ID TF encoding genes was analyzed in the livers of deceased critically-ill patients with sepsis displaying agonal bilirubin levels below (Bil<2; n=34) or above (Bil>2; n=28) 2mg/dL. Median fold-change expression level of each group for the different genes has been used to generate the box plots. Expression levels in the critically-ill groups are expressed relative to those in the control group (n=18). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-tailed t-test was used to assess whether expression of LIVER-ID TF encoding genes in the Bil>2 group is significantly greater than in the Bil<2 group, *P < 0.05.",
    "molecules": "Bil, bilirubin"
  },
  {
    "caption": "(G) RT-qPCR analyses of indicated LIVER-ID TF encoding genes monitoring expression in the livers of Bil<2 (n=34) or Bil>2 (n=28) groups of deceased critically-ill patients with sepsis vs control donors (n=18). Data are shown as box plots, with mRNA levels of the critically-ill groups expressed relative to those of the control group. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Wilcoxon test was used to assess statistically significant differences with the Bil>2 group, *P < 0.05.",
    "molecules": "Bil"
  },
  {
    "caption": "(H) RT-qPCR analyses of indicated ERS UP genes performed Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. Wilcoxon test was used to assess statistically significant differences with the Bil>2 group, *P < 0.05.",
    "molecules": "Bil"
  },
  {
    "caption": "F - Left: relative mRNA expression of Chat in ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 9). Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: quantification by LC/MS-MS of Ach concentration in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 6).",
    "molecules": "Ach"
  },
  {
    "caption": "A - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). B - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). C - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). D - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). E - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice treated with vehicle (veh) or 1 mg/kg NE for 2 h (n = 6). F - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice treated with veh or 1 mg/kg NE for 2 h (n = 6). ",
    "molecules": "NE"
  },
  {
    "caption": "D-E - Percentage of (D) total IWAT SVF cells and (E) IWAT MΦ expressing ChAT-eGFP in reporter mice with genetic deletion combinations of β-ARs 1, 2 and 3 following treatment with veh (white bar: n = 7) or 1 mg/kg NE (black bars: from left to right, n = 7, 5, 4, 5, 4, 7) for 2 h.",
    "molecules": "NE"
  },
  {
    "caption": "F - LC/MS-MS quantification of acetylcholine levels secreted by SVF cells derived from IWAT of β2WT and β2KO mice treated with veh or 1 mg/kg Form for 2 h (n = 3).",
    "molecules": "acetylcholine, Form"
  },
  {
    "caption": "M - Left: BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2 antagonist (Buto) (n = 6). Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: Total number of ChAT-eGFP+ BMDMs. ChAT-eGFP BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2-AR antagonist (Buto) (n = 4). An equal number of events (50,000) were analyzed by flow cytometry.",
    "molecules": "Buto, butoxamine, NE"
  },
  {
    "caption": "N - Left: Bicompartmental co-culture system with media alone (Ctrl) or WT SVF cells isolated from IWAT in the upper compartment (transwell insert) and freshly isolated IWAT explants from β-less mice in the lower compartment. Cells were co-cultured for 4 h in the presence or absence of β2-AR agonist (2.5 μM Form). 150 μM rivastigmine was added to the media to prevent degradation of Ach. Right: qPCR analyses of Chat and Ucp1 mRNA levels in β‑less explants following co-culture with media (n = 4), vehicle (n = 10) or β2-AR agonist (n = 10) treated SVF cells. mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.",
    "molecules": "Ach, Form, rivastigmine"
  },
  {
    "caption": " A Western blot of SDS-PAGE of different fractions from 143B cells expressing APOPT1HA. Tot: total lysate. Cyto: post-mitochondrial fraction (cytoplasm). Mt: isolated mitochondria. Mt sol: Soluble mitochondrial fraction. Mt memb: mitochondrial membranes. CO32- pellet: Pellet after carbonate extraction with 0.1 M Na2CO3, pH 10.5 for 30 minutes. CO32- sol: soluble fraction after the carbonate extraction ",
    "molecules": "carbonate, CO32-, Na2CO3"
  },
  {
    "caption": " B Western blot of SDS-PAGE of mitochondria used for protease protection assay after digitonin treatment. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA exposed to increasing amounts of digitonin (expressed in µg) and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity. TOM20: Translocase of the outer membrane (OM) 20 kDa. ACO2: Aconitase 2 (mitochondrial isoform). AIF: Apoptosis inducing factor. AK2: Adenylate kinase 2. OM: outer mitochondrial membrane. IM: inner mitochondrial membrane. IMS: intermembrane space ",
    "molecules": "digitonin, Triton X-100, trypsin"
  },
  {
    "caption": " C Western blot of SDS-PAGE of mitochondria used for protease protection assay after hypotonic shock. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA incubated either in isotonic (Iso) or hypotonic buffers for 5 minutes (Hypo 5') or 15 minutes (Hypo 15') and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity ",
    "molecules": "Triton X-100, trypsin"
  },
  {
    "caption": " E L-[35S]-Methionine pulse-chase labeling of mtDNA-encoded proteins. After a two-hour exposure with the radioactive label (pulse), cells were cultured in cold medium for the indicated chase times. The graphs show the densitometric quantification of the bands corresponding to MT-CO1 (left graph) and MT-CO2+MT-CO3 (right graph) normalized to the ATP6 band over the indicated time points. Graphs represent the values of three biological replicas for each cell line. Data are presented as mean ± SEM (n = 4 for controls, n = 2 for S2/S6/S2 APOPT1GFP/S6 APOPT1GFP). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: MT-CO1 - **P = 0.0039 ( 3.5 hours, controls vs S6), *P = 0.0202 (3.5 hours, controls vs S2), *P = 0.0163 (5 hours, S6vs S6 APOPT1GFP), *P = 0.0118 (5 hours, controls vs S2), **P = 0.0011 (20 hours, controls vs S6), *P = 0.0343 (20 hours, S2 vs S2 APOPT1GFP), ***P = 0.0007 (20 hours, S6 vs S6 APOPT1GFP ), ***P = 0.0002 (20 hours, controls vs S2), *****P < 0.0001 (20 hours, controls vs S6), MT-CO2/MT-CO3 - *P = 0.0194 (3.5 hours, S6 vs S6 APOPTGFP), ***P = 0.0003 (3.5 hours, controls vs S6), *P = 0.0375 (5 hours, S6 vs S6 APOPT1GFP), *P = 0.0234 (20 hours, S2 vs S2 APOPT1GFP), ****P < 0.0001 (20 hours, S6 vs S6 APOPT1GFP), **P = 0.0013 (20 hours, controls vs S2), ****P < 0.0001 (20 hours, controls vs S6)",
    "molecules": "L-[35S]-Methionine"
  },
  {
    "caption": " A Western blot analysis of SDS-PAGE of MGM132-treated 143B APOPT1HA cells. The graph represents the densitometric quantification of the signals for the precursor and mature protein ",
    "molecules": "MGM132"
  },
  {
    "caption": " B The upper part of the panel (Input) shows Western blot analyses of APOPT1HA in the MGM132-treated cells. Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor. The bottom part of the panel (Purified fractions) shows the analysis of fractions from the same cells immunoprecipitated with anti-HA. Note that the higher molecular weight species are cross-reacting with both anti-HA and anti-ubiquitin ",
    "molecules": "MGM132"
  },
  {
    "caption": " C Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 100 µM H2O2, as illustrated by the scheme (H2O2 treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2 ",
    "molecules": "H2O2"
  },
  {
    "caption": " D Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 5 µM MitoParaquat (MitoPQ), as illustrated by the scheme (MitoPQ treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ ",
    "molecules": "MitoParaquat, MitoPQ"
  },
  {
    "caption": " A Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times. UT: untreated cells. B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas. C Densitometric quantification of MT-CO1 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas (n=3) were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%. The levels of MT-CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells (*P = 0.0202, two-tailed unpaired Student's t-test). D Densitometric quantification of MT-CO2 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100% ",
    "molecules": "MitoPQ"
  },
  {
    "caption": "D Western blot  analysis of exogenous   Hwa in HEK293T cells. Treatment of cells with lysosome inhibitors  bafilomycin A1 (Baf-A1) and NH4Cl for 24 hours, DMSO  was used as control. Dosage of Baf-A1: 10 nM, 50 nM, 100 nM; Dosage of NH4Cl: 2.5 mM, 5 mM, 10 mM. Hwa with modification is indicated by white arrow; Degraded Hwa is indicated by black arrow. Blots are representative of 3 independent experiments. E Densitometry analysis  of Hwa bands from the western blot in (D) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, **p < 0.01 by unpaired t test. ",
    "molecules": "NH4Cl, Baf-A1, bafilomycin A1, DMSO"
  },
  {
    "caption": "E Colocalization of Hwa-Flag (red) and  ZNRF3-HA (green) or ZNRF3ΔRING-HA (green) in Hela cells is indicated by yellow dots. Baf-A1, 50 nM, 12 hrs. Magnification: 100 ×; scale bar: 10 μm.",
    "molecules": "Baf-A1"
  },
  {
    "caption": "B Whole mount  in situ hybridization of Spemann organizer markers  chrd, gsc, and xnr3 in gastrula embryos (stage 10.5). Oocytes were injected with AS or mRNAs as indicated, Dosage: znrf3 AS (3 ng AS4 + 3 ng AS9), znrf3  mRNA 100 pg, znrf3ΔRING mRNA 50 pg. embryos were obtained through Host transfer technology. Scale bars: 1 mm.",
    "molecules": "AS"
  },
  {
    "caption": "I Representative phenotypes  of  tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl (gfp mRNA), znrf3FL, and znrf3 truncations as indicated at 4-cell stage. Duplicated axes (red arrowhead). znrf3FL, full-length znrf3; znrf3ΔRING, znrf3 lacking the RING domain; znrf31-320, amino acids  1-320 of  znrf3; znrf3320-606, amino acids 320-606 of znrf3; znrf3606-854, amino acids 606-854 of znrf3. Scale bars: 1 mm.",
    "molecules": "amino acids"
  },
  {
    "caption": "B Hwa ubiquitination was analyzed under denaturing conditions. HEK293T cells co-transfected with wildtype His-HA-Ubiquitin (His-HA-Ub WT) or lysine-less mutant (K0), Hwa-Flag, and  ZNRF3-Myc. The lysates were immunoblotted with Myc,  Flag, and α-tub (α-tubulin). Lysates immunoprecipitated  with anti-Flag were immunoblotted  with HA and Flag  antibodies. K0, all lysine sites on ubiquitin were mutated. Blots  are representative of 3 independent experiments.",
    "molecules": "lysine, Ub, Ubiquitin, ubiquitin"
  },
  {
    "caption": "D Screen of ZNRF3-mediated Hwa ubiquitination  sites under denaturing conditions. HEK293T cells were co-transfected with His-HA-Ub, Hwa-Flag or  Hwa10KR-Flag, and  ZNRF3 or ZNRF3ΔRING. The lysate was  immunoblotted  with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were  immunoblotted  with HA and Flag antibodies. Hwa10KR-Flag mutant, all lysine sites in Hwa  sequence were replaced with arginine. Denaturing conditions: before  immunoprecipitation, lysates were denatured by adding SDS to a final concentration at 1% and boiling at 95℃ for 15 min. Blots  are representative of 3 independent experiments.",
    "molecules": "arginine, lysine, SDS, Ub"
  },
  {
    "caption": "C Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag and ZNRF3-HA. Cells were treated with or without Baf-A1 (50 nM) or MG132 (20 μM) for about 12 hours. Lysates were immunoblotted with  Flag, HA, and  α-tub (α-tubulin) antibodies.  Blots are representative of 3 independent experiments. D Densitometry analysis of Hwa bands from the western blot in (C) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01, ****p < 0.0001 by unpaired t test. ",
    "molecules": "Baf-A1, MG132"
  },
  {
    "caption": "I Surface biotinylation and streptavidin pulldown  of Hwa-Myc-Flag. HEK293T cells were co-transfected with Hwa-MF (Hwa-Myc-Flag), and ZNRF3-Myc or  ZNRF3ΔRING-Myc. Lysate was immunoblotted  with Myc, Flag, and GAPDH  antibodies. Lysates immunoprecipitated  with anti-Flag (2nd elution) were immunoblotted with Flag and HA  antibodies.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(D) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp (without ATG) to the sequence from 5' cap of pri-miR-31 to ATG site or which mutated to ATT of the sORF. Cells were selected by G418 for 2 days. Analysis of the GFP expression by confocal microscopy or flow cytometry, scale bar 100 μm.",
    "molecules": "G418"
  },
  {
    "caption": "(A) CD4+ T cells were activated with anti-CD3/28 and treated with different concentration of FAM-miPEP31 for 24 hours, the fluorescein was detected by Flow Cytometry.",
    "molecules": "FAM, fluorescein"
  },
  {
    "caption": "(B) Confocal Microscopy was used to analyze the fluorescence of FAM-miPEP31 in anti-CD3/28 activated CD4+ T cells treated with 10 μM FAM-miPEP31. Scale bar 5 μm.",
    "molecules": "FAM"
  },
  {
    "caption": "(E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, FAM-labeled miPEP31 was added. Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4+ T cells. Scale bar 7 μm.",
    "molecules": "FAM"
  },
  {
    "caption": "(F, G) C57BL/6 mice were injected intravenously with 100 μg FAM-labeled miPEP31. Splenocytes were isolated and stained for CD4 and DAPI. Imaging Flow Cytometry analysis of miPEP31 in CD4+ T cells (F) and non-CD4 splenocytes (G) , scale bar 10 μm.",
    "molecules": "DAPI, FAM"
  },
  {
    "caption": "(D, E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, 10 μM FAM-labeled miPEP31 was added for 20 min at 4℃ or 37℃. Confocal Microscopy analysis of the fluorescence. Scale bar 10 μm. The localization of miPEP31 with DAPI was shown in (E). Data are representative nuclear co-localization of miPEP31 in four representative images from four biological experiments. (mean ± SEM). ****p < 0.0001, unpaired two-tailed Student's t-test.",
    "molecules": "DAPI, FAM"
  },
  {
    "caption": "scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization. (D, E) H&E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization. Histopathology scores of inflammation and demyelination were shown in (E). Scale bars, 70 μm. Data in (C, E) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each. ****",
    "molecules": "Fast blue"
  },
  {
    "caption": "(A) Coomassie‐stained gels of liposome co‐sedimentation assays using either Atg5-Atg12/Atg16 (upper panel) or Atg5/Atg16 (lower panel) and liposomes with the indicated lipid composition (Folch: Folch lipids, PE/PI3P: 40% POPC, 35% POPS, 20% POPE, 5% PI3P, PE/PI: 40% POPC, 35% POPS, 20% POPE, 5% PI, DAG/PI3P: 40% POPC, 35% POPS, 20% DAG, 5% PI3P, DAG/PI: 40% POPC, 35% POPS, 20% DAG, 5% PI). Note that the Atg5-Atg12/Atg16 and the Atg5/Atg16 complexes show almost identical binding behaviours.",
    "molecules": "POPC, POPE, POPS, DAG, PE, PI, PI3P"
  },
  {
    "caption": "(C) Coomassie‐stained gel of a liposome co‐sedimentation assay showing the lipid requirements of the Atg5-Atg12/Atg16 complex. Liposomes were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. In the liposomes containing no POPE or POPS, these lipids were replaced with POPC.",
    "molecules": "POPE, POPS, PI3P"
  },
  {
    "caption": "(C) Coomassie‐stained gels showing the results of Atg8-PE conjugation assays using the indicated proteins and E. coli lipids derived liposomes. Atg8-PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea‐SDS gels. The numbers above the gels indicate the time in minutes. Note that at least under these conditions almost no Atg8-PE conjugation was detected in the absence of the Atg5-Atg12 conjugate or the Atg5-Atg12/Atg16 complex. The numbers next to the gels indicate the molecular weight in kDa. The averages and the error bars representing the standard deviations are shown. P‐values were calculated using Student's t‐test. NS, non‐significant; P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.",
    "molecules": "PE"
  },
  {
    "caption": "(A) Confocal microscopy images showing eGFP-Atg8 recruitment to GUVs is dependent on the Atg5-Atg12/Atg16 complex and PE. The membrane was labelled by incorporation of rhodamine‐PE.",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(E) Widefield images showing individual frames of a time series of GUVs incubated with Atg3, Atg7, Atg8 and the Atg5-Atg12/Atg16 complex (see Materials and methods for details). The membrane was labelled by incorporation of rhodamine‐PE. The time is indicated in minutes.",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(F) Widefield images of GUVs incubated with Atg3, Atg7, eGFP-Atg8 and the Atg5-Atg12/Atg16 complex for 35 min (left) or 70 min (right). The membrane was labelled by incorporation of rhodamine‐PE. Note the massive accumulation of eGFP-Atg8 at the interface between individual GUVs. The arrows point to some of these interfaces. For the quantifications, the averages and standard deviations are shown. P‐values were calculated using Student's t‐test. Scale bars: 10 μm. Figure source data can be found with the Supplementary data.",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(A) Frames of widefield images taken from a time series of rhodamine‐PE labelled GUVs in the presence of the Atg5-Atg12/Atg16 complex. For the control condition buffer but no protein was added. Note the increase in clustered GUVs over time. The numbers indicate the time in minutes.",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(B) Confocal images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP complex. Note the accumulation of the complex at the interface between GUVs (arrows).",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(C) Individual confocal microscopy frames of a time series of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the GUV-GUV interface where the protein initially accumulated.",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(D) Confocal microscopy images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the concentrations of Atg5-Atg12/Atg16 on the GUV membrane. Scale bars: 10 μm (A-C) and 3 μm (D).",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(A) Widefield images of rhodamine‐PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5-Atg12/Atg16 and Atg5/Atg16 complexes.",
    "molecules": "PE, rhodamine"
  },
  {
    "caption": "(C) Yeast cells were transformed with the indicated expression constructs and Atg5-2 × Myc was immunoprecipitated with an anti‐Myc antibody. The precipitated protein was subjected to anti‐Atg5 and anti‐Atg12western blotting. The substitution of R171 and K160 to glutamate caused the mutant protein to run higher. The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3).",
    "molecules": "glutamate"
  },
  {
    "caption": "(D) Anti‐Atg12 western blot of cell fractions prepared from rapamycin‐treated yeast cells. The anti‐Pgk1 and anti‐Pex30 served as control for the cytosolic and membrane fractions, respectively.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(I) Ape1 protease protection assay with the indicated yeast strains. The percentages below the blots indicate the percent of protected ApeI in the +protease K, −Triton X‐100 lanes and are the average of three independent experiments±the standard deviation. The numbers next to the blots indicate the molecular weight in kDa. Figure source data can be found with the Supplementary data.",
    "molecules": "Triton X‐100"
  },
  {
    "caption": "(A) Yeast cells of the indicated genotype expressing Atg5-3 × mCherry or Atg5 K160E, R171E-3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope. Maximum projections of z‐stacks are shown.",
    "molecules": "rapamycin"
  },
  {
    "caption": "F. Magnified (60x) dorsal regions from organoids fusions at day 60; Co-immunostaining of GFP with markers of interneuronal (GABA), GABAergic (DLX2) and mature neuronal (NeuN) identity. Scale bars, 10μm. White arrows indicate GFP+ cells with positive labeling. G. Quantification of co-expression of GFP with markers mentioned in 1F in dorsal and ventral regions of organoid fusions at day 60. Quantification was performed on a total of 3 organoid fusions (technical replicates) from two independent differentiations each (biological replicates), with 2 regions of interest (ROI; 10x field of view) in the dorsal and ventral regions of each fusion analyzed. Each dot represents the quantification of co-expression for both ROI analyzed per organoid fusion. Significance values, * = <0.05, ** = <0.01, *** = <0.001. Statistical analysis was performed using an unpaired two-tailed Student's t-test. The central band displays the mean and error bars depict the SD.",
    "molecules": "GABA"
  },
  {
    "caption": "C. Heatmap depicting the control-normalized values (log2-scaled) for selected parameters (from Figure S9A) for the 8 receptors analyzed in 5B. Grey values indicated non-significant values. In total 3979 moving cell tracks with the following number of tracks per group: Control - 696, Bicuculline-Methiodide - 1189, Saclofen - 527, TPMPA - 138, AP-5 - 429, CNQX - 500, Strychnine - 98, Serotonin - 180, and SR57227 - 222, were analyzed.",
    "molecules": "Methiodide, CNQX, AP-5, Bicuculline, Saclofen, Serotonin, SR57227, Strychnine, TPMPA"
  },
  {
    "caption": "A, B, C. K18-hACE2-transgenic mice treated with C6G25S by 1.48 mg/L of AI for 30 min (A), 50 μL of saline containing 50 μg of C6G25S by IN (B), or PBS (C) (n = 5 per group). C6G25S distribution in lungs was visualized by in situ hybridization (ISH) staining with C6G25S-specific probe (red color). Bronchi (i) and bronchioles (ii) marked with the boxes are enlarged on the right.",
    "molecules": "PBS, saline"
  },
  {
    "caption": "E, F. C57/B6 mice (n = 3 per group) were treated by AI with C6G25S 1.48 mg/L for 30 min (E) or 50 μL of saline containing 50 μg of C6G25S by IN administration (F). The C6G25S deposited in whole lungs and nasal cavities was quantified after whole tissue homogenization followed by stem-loop RT-qPCR. Quantification data represent mean ± SD. P value by Student t test.",
    "molecules": "saline"
  },
  {
    "caption": "A. K18-hACE2-transgenic mice (Winkler et al., 2020) were treated once daily for 3 days before intranasal challenge with 104 plaque-forming units (PFU) of the original virus. Prophylactic treatment consists of 30 min of AI (1.48 mg/l of C6G25S), followed by IN of 50 μg C6G25S. Mice receiving vehicle control (saline) for both AI and IN are annotated as control. Viral RNA (left) and infectious virions (right) in lungs were quantified with RT-qPCR and plaque forming assay, respectively, at 2 days post-infection (dpi). B. Mice were challenged intranasally with 104 PFU of virus and co-treatment with 1.48 mg/L of C6G25S or vehicle control (saline) by AI for 30 min on day 0 (right after infection) and day 1. Viral RNA and infectious virions were quantified at 2 dpi. C",
    "molecules": "saline"
  },
  {
    "caption": " (F) Cell growth assay in BT549 cells with GFP or MIG-6 knockdown in the absence and presence of gefitinib for 72 hours. ",
    "molecules": "gefitinib"
  },
  {
    "caption": " (B) Extracellular acidification rate (ECAR) in BT549 cells with GFP or MIG-6 knockdown. The ECAR flux profile of GFP and MIG-6 knockdown BT549 cells upon stimulation of glucose, oligomycin, and 2-DG is shown on the left. Quantitative results for the basal glycolysis and maximal glycolysis capacities in GFP and MIG-6 knockdown BT549 cells are indicated by orange and green boxes, graphed on the right. Results are presented as mean ± SEM (n=15; biological replicates). ",
    "molecules": "2-DG, glucose, oligomycin"
  },
  {
    "caption": " (C) A heatmap diagram shows the expression profile of genes involved in glycolysis, the TCA cycle, the pentose phosphate pathway, pyruvate oxidation, gluconeogenesis, and glycogen metabolism obtained from two sets of GFP and MIG-6 knockdown BT549 cells. ",
    "molecules": "glycogen"
  },
  {
    "caption": " (H) Lactate production assay in BT549 cells with GFP or MIG-6 knockdown (n=3). The medium was collected to measure lactate concentration using lactate test strips and an Accutrend Lactate analyzer. The rate of lactate production was determined (lactate production rate = lactate concentration/cells/time) and normalized to the rate detected in the control group. The quantified results are presented as mean ± SEM (n=3; biological replicates). ",
    "molecules": "lactate, Lactate"
  },
  {
    "caption": " (E) Representative flow cytometry images (upper) and quantitative results (lower) of measuring glucose uptake in GFP and MIG-6 knockdown BT549 cells (n=3). Cells were grown in the presence of the fluorescent analog 2-NBDG for various time periods, and glucose uptake was quantified using flow cytometric analysis. The red box indicates the gating for 2-NBDG+ cells and the number indicates the percentage of 2-NBDG+ cells. ",
    "molecules": "2-NBDG, glucose"
  },
  {
    "caption": " (F) Immunoblotting analysis for GLUT1 protein expression in GFP and MIG-6 knockdown BT549 cells with or without MG132 treatment. ",
    "molecules": "MG132"
  },
  {
    "caption": " (E) Immunoblotting analysis for HIF1α protein expression in GFP and MIG-6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132. ",
    "molecules": "MG132"
  },
  {
    "caption": " (F) GFP and MIG-6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132, subjected to immunoprecipitation (IP) with Flag-tag or HIF1α antibody, followed by immunoblotting analysis. MG132 was used to rescue HIF1α protein degradation mediated by MIG-6 knockdown, leading to similar HIF1α protein levels in control and MIG-6 knockdown BT549 cells, which were used as the input to examine the role of MIG-6 in the interaction between HIF1α and HAUSP. ",
    "molecules": "MG132"
  },
  {
    "caption": " (G) Luciferase and MIG-6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment. Afterward, cells were harvested for IP with HA antibody, followed by immunoblotting analysis to determine the level of K48-linked ubiquitination of HIF1α. MG132 was used to preserve the degradative K48-linked ubiquitination signals. ",
    "molecules": "MG132"
  },
  {
    "caption": " (H) Immunoblotting analysis for MIG-6 and HIF1α expression in Luciferase and MIG-6 knockdown 293 without MG132 treatment. ",
    "molecules": "MG132"
  },
  {
    "caption": " (E) Immunoblotting analysis for MIG-6 expression in BT549 cells with MIG-6 inducible knockdown (iMIG-6-shRNA) and the non-targeting shRNA control (iNT-shRNA) upon doxycycline treatment. ",
    "molecules": "doxycycline"
  },
  {
    "caption": "TCF-reporter TOP-flash assay performed on the indicated cell lines treated with 10 μM CHIR, for 24 hrs. The control FOP-flash reporter is not activated upon CHIR administration. The data represent the mean ± s.e.m. of averages of nine independent experiments (N = 9). dBcat, β-catenin knockout cells; d4TCF, TCF/LEF quadruple knockout cells Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond to Wnt3 as measured in a TOP-flash assa",
    "molecules": "CHIR"
  },
  {
    "caption": "Quantitative RT-PCR analyses of Axin2 transcripts, performed on the different cell lines treated with 10 μM CHIR or DMSO (control) for 24 hrs. Error bars show that standard deviation obtained from 3 independent experiments Transfection of individual TCF-expressing plasmids could restore the ability of d4TCF cells to respond t CHI as measured by Axin2 mRNA abundance",
    "molecules": "CHI, CHIR, DMSO"
  },
  {
    "caption": "a Heat map showing the upregulated genes in wild-type (WT) parental HEK 293T cells upon CHIR treatment, ranked based on the log2 fold change. Increased and decreased transcription is depicted in red and blue, respectively. Gene names are indicated along the y-axis. Canonical Wnt targets are among the most upregulated genes in WT cells (red boxes). The genotypes of the cell clones used is indicated on the bottom of the heat map. Triplicates of each cell line are included. b Hierarchical clustering of differentially expressed genes with at least a 2-fold change in any comparison. Dendrogram clustering indicates the relationships between the different experimental conditions. c Volcano plot displays differentially regulated genes in d4TCF (C) and dBcat (C") compared to WT parental cells. Red dots indicate significantly regulated genes based on adjusted p-value and log-fold-change (logFC). d Smear plots for every cell line indicating the behaviour of every gene after CHIR treatment. Differential gene expression is indicated by the log2 fold change (logFC), and the average logCPM (logarithm of counts per million reads) was used to estimate the accuracy of the expression",
    "molecules": "CHIR"
  },
  {
    "caption": "e RT-qPCR-based validation of three differentially expressed genes, upon CHIR stimulation in WT, dBcat or d4TCF cell lines. The data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student\"s t-test. Asterisks (*) indicate a p-value<0.05, and (**) a p-value<",
    "molecules": "CHIR"
  },
  {
    "caption": "b, c RT-qPCR-based validation of selected TCF-independent β-catenin-dependent target genes (B) upregulated or (C) downregulated in d4TCF cells (TCF-independent) treated with 10μM CHIR or DMSO for 24hr. Data represent the mean ± s.e.m. of averages of three independent experiments (N = 3). Samples were compared using Student\"s t-test. Asterisks (*) indicate a p-value<",
    "molecules": "CHIR, DMSO"
  },
  {
    "caption": "b RT-qPCR validation of the β-catenin-GHOST response. A selection of 7 genes (colour coded) that are activated upon CHIR treatment in d4TCF, or in wild-type (WT) in combination with ICAT overexpression, but not in pentaKO cells - indicating that their regulation is dependent on β-catenin but can only occur in the absence of TCF/LEF-β-catenin interaction. The genotype and the treatment for each condition is indicated in the x-axis. Error bars show that standard deviation obtained from 3 independent experiments",
    "molecules": "CHIR"
  },
  {
    "caption": "c FOXO4 but not FOXO3 overexpression upregulates the β-catenin-GHOST target GADD45 both in CHIR-treated WT (black bars) or d4TCF (grey bars) cells. Samples were compared using Student\"s t-test. Asterisks (*) indicate a p-value<",
    "molecules": "CHIR"
  },
  {
    "caption": "f GADD45 is upregulated by CHIR in d4TCF cell; this positive transcriptional regulation is blocked by FOXO4-specific siRNA but not control scrambled siRNA. All experiments were done at least three times (N=3). Samples were compared using Student\"s t-test. Asterisks (**) indicate a p-value<0.02; ns, non-statistically significant change was obs",
    "molecules": "CHIR"
  },
  {
    "caption": "(C) miR-155 and miR-181b reduced cell viability in GDC0941-sensitive breast cancer cell lines. Error bars indicate mean ± SD (n=3).",
    "molecules": "GDC0941"
  },
  {
    "caption": "(E) Stimulation of PEO4 cells with 10 ng/mL TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X. Each point represents the mean of 3 experiments ± SD and * denotes p-value < 0.05 by Student's T-test.",
    "molecules": "AKT Inhibitor X"
  },
  {
    "caption": "(D, E) HTM-mediated quantification of MPM-2 (D) and H3S10P (E) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 (USP7i) for 4 hrs. Nuclei were stained with DAPI (blue). Scale bar, 30 μm. The quantification is shown to the right of representative images for each analysis. (*** p<0.05; t-test). Grey lines indicate mean values.",
    "molecules": "DAPI, DMSO, P22077"
  },
  {
    "caption": "(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band.",
    "molecules": "P22077"
  },
  {
    "caption": "(B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.",
    "molecules": "DAPI, P22077"
  },
  {
    "caption": "(B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control.",
    "molecules": "BAY 11-7082, DMSO, Okadaic acid, P22077, Ponceau"
  },
  {
    "caption": "(D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.",
    "molecules": "DAPI, DMSO, Okadaic acid, P22077"
  },
  {
    "caption": "(E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.",
    "molecules": "DMSO, DT-061, P22077, Ponceau"
  },
  {
    "caption": "(F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H)pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control.",
    "molecules": "BAY 11-7082, Calyculin A, DMSO, Okadaic acid, P22077, Ponceau"
  },
  {
    "caption": "(E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.",
    "molecules": "DMSO, P22077, Ponceau, RO3306"
  },
  {
    "caption": "(B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM.",
    "molecules": "Hoechst, DMSO, DT-061, P22077, RO3306, TMRE"
  },
  {
    "caption": "(C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).",
    "molecules": "HBX 19818, BAY 11-7082, P22077, RO3306"
  },
  {
    "caption": "Speed distribution profile of PMN treated with 0.5 µM E4 or DMSO for 30 min. Graph shows an increase in the rate of axonal transport upon E4 treatment (DMSO, 153 endosomes, 13 axons; E4, 165 endosomes, 13 axons; N=4 independent experiments, data shown are mean ± SEM).",
    "molecules": "DMSO"
  },
  {
    "caption": "PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example images of DIV6 PMN treated with 1 µM PPP or 50 ng/ml IGF1. PMN were stained with 30 nM AlexaFluor555-HcT. The scale bar is 50 µm.",
    "molecules": "AlexaFluor555, PPP"
  },
  {
    "caption": "PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example of HcT-containing organelle tracking in the axon of PMN. Each colour denotes a single endosome tracked over time. The scale bar is 10 µm.",
    "molecules": "AlexaFluor555, PPP"
  },
  {
    "caption": "PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles, after PPP treatment compared to controls (DMSO: 138 endosomes, 24 axons, 6,433 movements; PPP: 130 endosomes, 23 axons, 5,222 movements) (** P= 0.0061, Student's t-test, N=4 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. There was no difference in pausing between PPP-treated neurons and controls (P=0.45, Student's T-test, N=4 independent experiments data shown are mean ± SEM).",
    "molecules": "AlexaFluor555, DMSO, PPP"
  },
  {
    "caption": "PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles after IGF1 treatment compared to control (water: 138 endosomes, 22 axons, 5,119 movements; IGF1: 106 endosomes, 20 axons, 5,202 movements; *** P=2.83x10-5, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles spent pausing per axon. IGF1 significantly increased the pausing time of HcT-containing organelles (* P=0.011, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).",
    "molecules": "AlexaFluor555"
  },
  {
    "caption": "PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of PPP-treated neurons and controls. PPP caused an increase in instantaneous velocities of HcT-containing organelles (N=3 independent experiments, data shown are mean ± SEM).",
    "molecules": "AlexaFluor555, PPP"
  },
  {
    "caption": "PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of IGF1-treated neurons and controls. IGF1 caused a decrease in instantaneous velocities of HcT-containing organelles compared to controls (N=3 independent experiments , data shown are mean ± SEM).",
    "molecules": "AlexaFluor555"
  },
  {
    "caption": "Western blot showing the levels of pAKT (Ser473), total Akt, pErk1/2 (T202/Y204) and total Erk1/2 after treatment with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Protein was normalised to GAPDH. Western blot quantification. PMN treated with PPP showed a significant decrease in Akt activation (* P=0.049, N=5 independent experiments, Student's T-test), but no significant difference was seen on Erk1/2 activation (P=0.07, Student's T-test, N=5 independent experiments). 50 ng/ml IGF1 treated neurons showed a significant increase in Akt activation (** P=0.008, Student's T-test, N=3 independent experiments). In contrast, no change in Erk1/2 activation (P=0.88, Student's T-test, N=3 independent experiments) was observed (data shown are mean ± SEM).",
    "molecules": "PPP"
  },
  {
    "caption": "Graph shows the average velocity of HcT-containing organelles treated with Capivasertib or Ipatasertib (10 nM) for 30 min compared to controls (DMSO: 483 endosomes, 34 axons, 41,897 movements; Capivasertib: 658 endosomes, 43 axons, 49,841 movements; Ipatasertib: 605 endosomes, 32 axons, 41,485 movements) (** P=2 x10-16, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib and DMSO-Ipatasertib *** P <0.0001; N=6 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing (* P=1.14 x10-7, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib P= 9.37x10-5, DMSO-Ipatasertib P= 2 x10-7; data shown are mean ± SEM, N=6 independent experiments).",
    "molecules": "Capivasertib, DMSO, Ipatasertib"
  },
  {
    "caption": "PMN were treated with either an shRNA targeting IGF1R (1 µl) or a scrambled control 6 h after plating. At DIV 6-7, retrograde axonal transport was analysed using 30 nM AlexaFluor555-HcT. Knockdown of IGF1R caused a significant increase in the average velocity of signalling endosomes compared to controls (IGF1R shRNA: 108 endosomes, 21 axons, 801 movements; scrambled shRNA: 94 endosomes, 20 axons, 777 movements, * P=0.011, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control. IGF1R knockdown caused an increase in instantaneous velocities of HcT-containing organelles and decreased the amount of pausing (N=3 independent experiments, data shown are mean ± SEM).",
    "molecules": "AlexaFluor555"
  },
  {
    "caption": "Western blot of PMN treated with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins, all proteins were assessed from the same blot. Hook3 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 102.16 ± 4.3%, P=0.67, Student's T-test, N=3 independent experiments; water, 100%; IGF1, 93.48 ± 4.2%, P=0.26, N=3 independent experiments, data shown are mean ± SEM). However, BICD1 expression was significantly increased in PMN treated with PPP compared to control (DMSO, 100%; PPP, 137.1 ± 7.3%, **P=0.002, Student's T-test, N=7 independent experiments). In contrast, IGF1R stimulation by IGF1 had no effect on BICD1 expression (water, 100%; IGF1, 88.1 ± 14.0%, P=0.49, Student's T-test, N=3 independent experiments, data shown are mean ± SEM). BICD2 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 104.44 ± 6.5%, P=0.51, N=3 independent experiments: water, 100%; IGF1, 106 ± 7.5%, P=0.51, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).",
    "molecules": "DMSO, PPP"
  },
  {
    "caption": "Western blot of PMN pre-treatment with cycloheximide (50 µg/ml) or MG-132 (10 µM) for 30 min before incubation with 1 µM PPP for 60 min. Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells. (DMSO, grey: 100%, N=7 independent experiments; PPP, blue: 137.1 ± 7.3%, N=7 independent experiments; CHX, pink: 89.5 ± 12.7%, N=5 independent experiments; MG-132, teal: 124 ± 28.7, N=3 independent experiments; P=0.009, one-way ANOVA; Tukey's post-hoc test: DMSO-PPP * P=0.04, PPP-PPP+CHX * P=0.01; data shown are mean ± SEM).",
    "molecules": "CHX, cycloheximide, Cycloheximide, DMSO, MG-132, PPP"
  },
  {
    "caption": "Puromycin-PLA signal in PMN grown in microfluidics devices. Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment. The scale bars are 10 µm. White arrows point to puromycin-PLA puncta within the β3-tubulin mask; the empty arrow shows a PLA puncta not associated with neurons.",
    "molecules": "Puromycin, puromycin"
  },
  {
    "caption": "Quantification of puromycin-PLA signal. PPP causes an increase in PLA puncta in the axon of PMN, which is abolished by treatment with 40 µM anisomycin (DMSO, grey: 100%; PPP, blue: 139.3 ± 7.7%; PPP + anisomycin, purple: 86.3 ± 10.0; P=0.005, one-way ANOVA, Tukey's post-hoc test; DMSO vs PPP, * P=0.02; PPP vs PPP + anisomycin, ** 0.005, N=3 independent experiments, 27-57 images, data shown are mean ± SEM).",
    "molecules": "anisomycin, DMSO, PPP, puromycin"
  },
  {
    "caption": "The graph shows the average velocity of HcT-containing organelles in vivo in D72/73 SOD1G93A mice and wild type littermate controls. PPP (blue) treatment increased the transport rate of signalling endosomes in wild type and SOD1G93A mice compared to animals treated with vehicle control (grey) (WT control: cargo 371, movements 15,272, N=4 independent experiments; WT + PPP: cargo 388, movements 14,512, N=4 independent experiments; SOD1G93A control: cargo 322, movements 15,425, N=4 independent experiments; SOD1G93A + PPP: cargo 404, movements 17,643, N=4 independent experiments; P=2.24x10-12 (treatment), P=2.22 x10-16 (genotype), two-way ANOVA, Tukey's post-hoc test: all conditions *** P<0.005, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). PPP treatment does not alter the pausing of signalling endosomes (WT: 16.1 ± 1.5%, WT + PPP: 13.1 ± 1.5%; SOD1G93A: 16.6 ± 1.9%, SOD1G93A + PPP: 16.2 ± 2.1%; P=0.36 (treatment), P=0.32 (genotype), N=4 independent experiments for each condition, data shown are mean ± SEM.",
    "molecules": "PPP"
  },
  {
    "caption": "Treatment with PPP had no effect on Akt levels in the sciatic nerve (WT: Control-100 ± 8.6, PPP-91.5 ± 14.6%; SOD1: Control-101.4 ± 13.5%, PPP-117.6 ± 32.9%, P=0.81 (treatment), P=0.53 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D). Erk1/2 activation was down-regulated after treatment with PPP in the sciatic nerve of wild type and SOD1G93A mice (WT: control-100 ± 15.4%, PPP-64.4 ± 3.2%; SOD1: Control-78.0 ± 11.9 %, PPP-51.2 ± 8.2%, * P=0.013 (treatment), P=0.13 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D). Protein was normalised to GAPDH protein levels.",
    "molecules": "PPP"
  },
  {
    "caption": "a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. a, Samples were processed for western blot using anti-Rad53, Srs2 and Ddc2 antibodies.",
    "molecules": "galactose, VPA"
  },
  {
    "caption": "a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and −VPA. b, A schematic diagram showing probe locations with respect to the HO cut site. The resection rate was calculated as the rate of HO cut band disappearance.",
    "molecules": "galactose, VPA"
  },
  {
    "caption": "a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. c, Fold enrichment of the 0.2-kb fragment was calculated after ChIP of Rfa1-Flag, Ddc2-Myc.",
    "molecules": "galactose, VPA"
  },
  {
    "caption": "d, VPA effect on ectopic recombination (rec.) in wild-type (WT) cells. Error bars represent standard deviation (s.d.) calculated from four independent experiments.",
    "molecules": "VPA"
  },
  {
    "caption": "a, MRE11::MYC cells were treated as in Fig. 1a. Cell samples were processed for ChIP analysis and the fold enrichment of the 0.2-kb fragment after ChIP of Mre11-Myc without (-VPA) or with (+VPA) VPA was calculated.",
    "molecules": "VPA"
  },
  {
    "caption": "a, Cherry::APE1GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h. Samples were processed for microscopy. The table shows numbers corresponding to the experiment. Percentage of fluorescence signals is presented and error bars represent the s.d. obtained from three independent experiments. DIC, differential interference contrast. Scale bars, 3 µm.",
    "molecules": "nitrogen, VPA"
  },
  {
    "caption": "a, 4HA-Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine. Eluate was analysed using anti-HA. Lane 1: input Sae2; 2: input Sae2-HA -VPA; 3: as in 2 but +VPA; 4: 3 µl elution Sae2-HA -VPA after anti-HAimmunoprecipitation (IP; input AcK-IP); 5: double amount of 4; 6: IP anti-AcK elution from anti-HA IP of Sae2-HA -VPA; 7: as in 6 but +VPA.",
    "molecules": "VPA"
  },
  {
    "caption": "b, SAE2::PKerg6Δ cells were treated as in Fig. 2a. After 30 min induction, VPA and PMSF were added or not. Samples were processed for western blot using anti-PK.",
    "molecules": "PMSF, VPA"
  },
  {
    "caption": "c, Wild-type SAE2::PK, SAE2::PKatg1Δ and SAE2::PKatg19Δ cells were grown as in b. After 30 min induction, VPA was added or not and samples treated as in b.",
    "molecules": "VPA"
  },
  {
    "caption": "d, Wild-type SAE2::PK cells were grown as in b. After 120 min induction, rapamycin (200 ng ml-1) was added or not and samples were treated as in b.",
    "molecules": "rapamycin"
  },
  {
    "caption": "a, Survival of wild-type, rpd3Δ, hda1Δ and rpd3Δ hda1Δ strains after 4NQO, MMS and HU treatment. Error bars represent s.d. calculated from seven independent experiments.",
    "molecules": "4NQO, HU, MMS"
  },
  {
    "caption": "d, e, Wild-type SAE2::PK and gcn5Δ SAE2::PK strains were grown as in b and after 30 min of HO induction either VPA (d) or rapamycin (e) was added or not. Western blot was performed.",
    "molecules": "rapamycin, VPA"
  },
  {
    "caption": "(C) Virion stability assay using TIRF microscopy to quantify the number of viral particles in presence of SLO and IP6 for the indicated mutant after 1 hour. Mean data represent n=2 technical replicates. Scale bar 10 μm.",
    "molecules": "SLO, IP6"
  },
  {
    "caption": "(F) Quantification of CXCL10 and IFIT1 transcripts by qPCR of the indicated THP-1 cell lines stimulated for 24 hours with LPS (1 ng/ml) normalised to mock. Mean data n=2 biological replicates.",
    "molecules": "LPS"
  },
  {
    "caption": "(H) Quantification of the indicated chemokine transcripts by qPCR from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants normalised to mock infected. Mean data represent n=3 biological replicates.",
    "molecules": "PMA"
  },
  {
    "caption": "(I-J) Protein quantification of CXCL10 (I) and TNFa (J) by Luminex™ from supernatant of PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants. Data were normalised to mock. Mean data represent n=2 biological replicates.",
    "molecules": "PMA"
  },
  {
    "caption": "(D) qPCR quantification of the indicated chemokine transcripts from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with the indicated VLPs mutants. (E) qPCR quantification of the indicated chemokine transcripts level from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants.",
    "molecules": "PMA"
  },
  {
    "caption": "(F) Protein quantification of IL-6 by Luminex™ from PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants.",
    "molecules": "PMA"
  },
  {
    "caption": "IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with either poly I:C (0.5 μg/ml) (B) mean data represent n=2 biological replicates",
    "molecules": "PMA, poly I:C"
  },
  {
    "caption": "IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with HT-DNA (0.1 μg/ml) (C) mean data represent n=2 biological replicates",
    "molecules": "PMA"
  },
  {
    "caption": "IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection infected for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants (D).",
    "molecules": "PMA"
  },
  {
    "caption": "IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO infected with for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants in the presence of DMSO or 0.5 μg/ml H151 (E)",
    "molecules": "H151, DMSO, PMA"
  },
  {
    "caption": "A. Detection of Sufu and Skp1 after immunoprecipitation of the indicated Flag-tagged F-Box Proteins (FBPs). An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.",
    "molecules": "MLN4924"
  },
  {
    "caption": "B, C. Immunoprecipitation of endogenous Sufu from PC3 (B) and DAOY cells (C) transfected with either an empty vector (EV) or Myc-tagged Fbxl17. Non-specific rabbit immunoglobulin G (IgG) was used as a negative control. Detection of light chains of immunoglobulin (IgG) was used to assess the amount of IgG used for each immunoprecipitation reaction. Treatment with MLN4924 (2 M) was started 5 hours before cell collection.",
    "molecules": "MLN4924"
  },
  {
    "caption": "D. Quantification of Fbxl17 mRNA levels in PC3 and DAOY transfected with a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2). Cells were serum starved for 24 hours in serum-reduced medium and treated with SAG (100 nM) for 24 hours prior collection.",
    "molecules": "SAG"
  },
  {
    "caption": "G. Detection of Sufu protein levels following cycloheximide (CHX) treatment for the indicated hours, and Fbxl17 depletion using two different siRNAs. (Hrs=hours). Representative image of two independent experiments is shown",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "A. Detection of polyubiquitylated species of endogenous Sufu co-immunopurified from DAOY cells transfected with Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.",
    "molecules": "MG132"
  },
  {
    "caption": "B. Detection of polyubiquitylated species of Sufu upon co-transfection of HEK293T cells with HA-tagged Sufu and Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.",
    "molecules": "MG132"
  },
  {
    "caption": "D. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with HA-tagged Sufu, Myc-ubiquitin (Ub) along with either Flag-tagged Fbxl17 wild type (WT) or a mutant lacking the F-box domain (Fbxl17ΔF). MG132 (10µM) was added in all samples.",
    "molecules": "MG132"
  },
  {
    "caption": "E. Detection of ubiquitylated Sufuco-immunoprecipitated from HEK293T cells co-transfected with Myc-tagged ubiquitin (Ub), Flag-tagged Fbxl17 and HA-tagged Sufu WT or Sufu mutant K257R. MG132 (10µM) was added in all samples.",
    "molecules": "MG132"
  },
  {
    "caption": "A. Detection of Myc-tagged Fbxl17 after immunoprecipitation of HA-tagged Sufu WT or Sufu S342/6A and S342/6D, as indicated. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.",
    "molecules": "MLN4924"
  },
  {
    "caption": "B. Detection of Flag-tagged Gli1 and Myc-tagged Fbxl17 binding to immunoprecipitated HA-tagged Sufu Wild-Type (WT) or to Sufu S352F. An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.",
    "molecules": "MLN4924"
  },
  {
    "caption": "F. Detection of ubiquitylated Sufu immunoprecipitated from HEK293T cells co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged Fbxl17 and Flag-tagged Gli1. MG132 (10µM) was added in all samples.",
    "molecules": "MG132"
  },
  {
    "caption": "E. Detection of HA-tagged Sufu wild type (WT) and HA-tagged Sufu S352F protein levels in NIH3T3 cells after cycloheximide (CHX) treatment. Where indicated, cells were transfected with a non- targeting siRNA (Control) or two siRNA to Fbxl17 (1) and (2).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": " Compound 13b in the substrate-binding cleft located between domains I and II of the Mpro, in the monoclinic crystal form (space group C2). 2Fo-Fc electron density is shown for the inhibitor (contouring level: 1σ). Carbon atoms of the inhibitor are magenta, oxygens red, nitrogens blue, and sulfur yellow. Note the interaction between the N-terminal residue of chain B, S1*, and E166 of chain A. ",
    "molecules": "Compound 13b"
  },
  {
    "caption": "Effect of MG132 on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.",
    "molecules": "MG132"
  },
  {
    "caption": "E Effect of MG132 and Leu on LT-induced NLRP1B degradation. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and MG132 for 8 h. 10 mM Leu was added to cells 30 min prior to LT treatment. Shown are the anti-HA and anti-tubulin immunoblots of the total cell lysates.",
    "molecules": "Leu, MG132"
  },
  {
    "caption": "F LT-induced ubiquitination of NLRP1B. 293T cells expressing NLRP1B (1-983)-HA were treated with LT and/or MG132 for 6 h. Cells were then harvested for anti-HA immunoprecipitation and subjected to immunoblotting analyses using indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Daily 10 μg 4-AP enhanced recovery of sciatic nerve motor function as compared with treatment with vehicle at 3 days post injury (dpi) through 8 dpi. (*: Day3 (D3), p= 0.0131; D5, p = 0.0475; D8, p =0.0472; n=6; ANOVA with post-hoc comparisons using two-tailed unpaired t-test)",
    "molecules": "4-AP"
  },
  {
    "caption": "(B) Daily 10 μg 4-AP administration also enhanced recovery of nerve conduction velocity (NCV) as observed beginning at 21 days post-injury, eventually restoring NCV to near-normal values while NCV in vehicle-treated mice remained less than half that of uninjured animals (*: D21, p= 0.0454; D28, p = 0.0487; D35, p =0.0475; n=5; ANOVA with post-hoc comparisons using two-tailed unpaired t-test).",
    "molecules": "4-AP"
  },
  {
    "caption": "(C, D) Analysis of withdrawal latency in response to thermal (C) or mechanical (D) stimuli revealed that 4-AP treatment did not worsen these diagnostics of neuropathic pain syndromes. In the case of response to thermal hyperalgesis, 4-AP treated mice showed a significantly more rapid return to baseline levels. (*: (C) D3, D5, D8 : p<0.001 saline vs. baseline; D3 p= 0.006; 4-AP vs. baseline; (D) D3, p= 0.014; D5, p = 0.008; saline versus baseline; D3, p = 0.006 4-AP vs. baseline; D8: p =0.0472; n=10 ; ANOVA with post-hoc comparisons using two-tailed unpaired t-test.) In this and all other figures data is mean + SEM and shows a representative experiment from 2-3 repetitions.",
    "molecules": "4-AP"
  },
  {
    "caption": "(A) Local 4-AP treated crushed sciatic nerve (black: vehicle PLGA films; red: (4-AP)-PLGA films) regained partial walking ability as early as 3 days post-injury compared to vehicle treated group. (*: D5, p= 0.0004; D8, p = 0.0012; D11, p =0.003; D14, p=0.0089; n=6; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).",
    "molecules": "4-AP, PLGA"
  },
  {
    "caption": "(B) Local 4-AP treated crushed sciatic nerve (black: vehicle PLGA films; red: (4-AP)-PLGA films) showed faster improvement in NCV restoration compared with vehicle-treated mice, beginning at 21 days post-injury.",
    "molecules": "4-AP, PLGA"
  },
  {
    "caption": "(A) Electron microscopy images of healthy, crushed, and 4-AP treated crushed sciatic nerve at 21 days post-injury. Scale bar at 3000x = 5m; scale bar at 8000x = 2m) (B) Comparison of the axonal area of randomly chosen individual axons of vehicle treated and 4-AP treated mice (n=4 for each experimental group; 40 axons analyzed per mouse). 4-AP treated sciatic nerve showed statistically greater axonal area compared to the vehicle-treated group (p<0.05; ANOVA; restricted-maximum-likelihood). Moreover, 4-AP treated mice had a greater proportion of axons with areas greater than the mean value for uninjured mice (shown in the green line), with inset figure displaying all axons with values above this mean. This experiment represents a single group of mice of one of three replicates on NCV recovery.",
    "molecules": "4-AP"
  },
  {
    "caption": "(A, B) Sustained local 4-AP administration was associated with increased myelin area and close-to-normal Garea-ratio compared to untreated group, as determined by analysis of 40 randomly chosen myelinated axons from sections of 4 nerves for each group. (**: p<0.0001; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). 4-AP treated mice had a greater proportion of axons for which the associated myelin area was greater than the mean value for uninjured mice (shown in the green line), with inset figure displaying all axons with values above this mean.",
    "molecules": "4-AP"
  },
  {
    "caption": "(C, D) 4-AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis (**:p<0.01; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).",
    "molecules": "4-AP"
  },
  {
    "caption": "(C, D) 4-AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis (**:p<0.01; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).",
    "molecules": "4-AP"
  },
  {
    "caption": "(E) Increases in P0 protein over time also were observed by immunofluorescence analysis at different time points, with P0 protein expression increasing to a greater extent in nerves of mice treated with 4-AP. Scale bar = 200 m.",
    "molecules": "4-AP"
  },
  {
    "caption": "(F, G) 4-AP treatment increased the number of myelinated axons (**: p=0.002, n=4; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). Even though the 4-AP treated group also exhibited a greater number of total axons, this difference was not statistically significant. All myelinated and total axons were counted in 5 randomly chosen grids from each of 4 nerves for each group. This experiment represents a single group of mice of one of three replicates on NCV recovery.",
    "molecules": "4-AP"
  },
  {
    "caption": "(A) At 1 day post-injury, a single dose of 10 μg 4-AP significantly improved walking function as determined by SFI analysis (*: p=0.001, n=5). (B) In contrast, even higher doses of 4-AP administration (50 μg, ip) had no effect on SFI in mice with transected nerves (n=10). (C) In contrast with effects of 4-AP, treatment with neostigmine did not cause improvements in SFI. (p = 0.0024 for 4-AP vs. saline, n=8, 1-way ANOVA with Tukey's multiple comparison test).",
    "molecules": "4-AP, neostigmine"
  },
  {
    "caption": "F Left, confocal images of PHcrac (red) and PAK1-GBD (green) in the same cells treated with LatA. Scale bars represent 10 μm. Right, temporal profiles of normalized mean cytosolic intensities of PHcrac (red) and PAK1-GBD (green) (mean±s.e.m., n=18 cells) in response to cAMP.",
    "molecules": "cAMP, LatA"
  },
  {
    "caption": "E Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits Inp54p to membrane induced by rapamycin at time 0. The white arrow (-45 s) points to a nascent wave before Inp54p recruitment, while the red (60 s) arrow after.",
    "molecules": "rapamycin"
  },
  {
    "caption": "J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment, where rapamycin is added at time 0 to induce Inp54p recruitment. White arrows point to wave patches right before rapamycin addition.",
    "molecules": "LatA, rapamycin"
  },
  {
    "caption": "A Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.",
    "molecules": "rapamycin"
  },
  {
    "caption": "D Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.",
    "molecules": "rapamycin"
  },
  {
    "caption": "G Time lapse confocal images of PHcrac at the bottom of a giant cell treated with LatA, where rapamycin is added at time 0 to induce PKBA recruitment. All scale bars above represent 20 μm.",
    "molecules": "LatA, rapamycin"
  },
  {
    "caption": "A Left, time lapse confocal images of LimE in single cells following RacGEF1ΔN recruitment, induced by rapamycin addition at time 0. Right, temporal profile of normalized mean LimE intensity at cell cortex (mean±s.e.m., n=20 cells).",
    "molecules": "rapamycin"
  },
  {
    "caption": "B Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane induced by rapamycin at time 0.",
    "molecules": "rapamycin"
  },
  {
    "caption": "A Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin.",
    "molecules": "rapamycin"
  },
  {
    "caption": "E Time lapse confocal images of PHcrac in single cells, which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0. White arrow points to PHcrac patches at cell cortex.",
    "molecules": "rapamycin"
  },
  {
    "caption": "F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment. Red dashed lines in F indicate rapamycin addition at time 0.",
    "molecules": "rapamycin"
  },
  {
    "caption": "G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment (mean±s.e.m., n=20 cells). Red dashed lines in F and G indicate rapamycin addition at time 0.",
    "molecules": "rapamycin"
  },
  {
    "caption": "H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment, with (solid) or without (hollow) RacGEF1ΔN recruitment (~6000 cells from 3 independent experiments).",
    "molecules": "LatA"
  },
  {
    "caption": "I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0, with (red) or without (blue) RacGEF1ΔN recruitment (mean±s.e.m., n=25 cells each).",
    "molecules": "cAMP"
  },
  {
    "caption": "D Phalloidin staining of cells after perturbations. Maximal intensity projections are shown.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "(A) TNF concentrations in supernatants of WT and Tnf-/- BMDMs unstimulated or stimulated with 10 ng/ml Pam3CSK4, quantified by ELISA. (Left) 0 h, 4 h and 8 h time points. (Right) Close-up of 0 h time point. Dashed line indicates mean of blank measurements. Data are represented as mean ± SD of 3 (4 h, 8 h) or 5 (0 h) biological replicates using cells from 2 (4 h, 8 h) or 3 (0 h) mice. Statistical significance was determined by two-tailed paired-sample t-test.",
    "molecules": "Pam3CSK4"
  },
  {
    "caption": "(E - J) Nuclear localization of mVenus-RelA upon stimulation with 10 ng/ml TNF (E - G) or 10 ng/ml Pam3CSK4 (H - J), measured over time in WT and Tnf-/- BMDMs by fluorescence microscopy and quantified by automated image analysis. (E, H) Each row of the heatmap represents the NFκB signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from a single representative experiment is depicted. (F, I) Indicated dynamic features were calculated from signaling trajectories. Violin plots depict pooled data from 8 (F) or 6 (I) biological replicates (total # of cells: 5769/5238 (F), 3843/3899 (I)). Marker indicates median of distribution. Statistical significance was determined by Wilcoxon rank sum test. Number indicates log10(p-value). (G, J) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment. Data are represented as mean ± SD of 8 (G) or 6 (J) biological replicates. Statistical significance was determined by two-tailed paired-sample t-test. (F, G) Some replicates for Tnf-/- BMDMs overlap with data",
    "molecules": "Pam3CSK4"
  },
  {
    "caption": "(E) Levels of nuclear NFκB DNA binding activity in Tnf-/- and Tnf-/-p100-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by EMSA. Quantification of the NFκB-DNA complex was normalized to the quantified NFY-DNA complex, and then to maximum WT value also obtained with each replicate. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Data for Tnf-/- BMDMs is also shown",
    "molecules": "DNA"
  },
  {
    "caption": "(E) Volcano plot of single-cell gene expression in Tnf-/- vs. WT BMDMs upon TNF stimulation. Dashed lines indicate p-value threshold of 0.01 and absolute log2(FC) threshold of 0.5. Only genes induced by P3C4 or TNF stimulation over mock in WT cells are included (p-value threshold of 0.05 and log2(FC) threshold of 0.25).",
    "molecules": "P3C4"
  },
  {
    "caption": "(F) Violin plots of single-cell gene expression in WT and Tnf-/- BMDMs upon mock, TNF, or Pam3CSK4 stimulation. The 10 genes shown are significantly upregulated in Tnf-/- vs. WT cells upon TNF stimulation (see (E)).",
    "molecules": "Pam3CSK4"
  },
  {
    "caption": "(D) Violin plot of log­2(FC) of normalized H3K4me1 ChIP-seq signal in TNF- or Pam3CSK4-stimulated over unstimulated WT or Tnf-/- BMDMs within Cluster 1 regions (533 regions). Statistical significance was determined by Kolmogorov-Smirnov test. Data are represented as mean of two biological replicates for each genomic region. In the boxplot, central band indicates the median, boxes the interquartile range (IQR), whiskers values within 1.5 IQR from the median.",
    "molecules": "Pam3CSK4"
  },
  {
    "caption": "(E) Examples of genomic regions with differential H3K4me1 signal in TNF stimulation in Cluster 1. Tracks for WT and Tnf-/- BMDMs unstimulated or stimulated with TNF or Pam3CSK4 in two biological replicates (R1, R2) are shown.",
    "molecules": "Pam3CSK4"
  },
  {
    "caption": "(H) Ubiquitin Chain Restriction (UbiCRest) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs. UT = untreated.",
    "molecules": "polyubiquitin"
  },
  {
    "caption": "(C) Immunofluorescence micrographs of VAMP8 (green) and Syntaxin-7 (STX7; red) show strong colocation around 3 µm silica bead phagosomes (white) in BMA macrophages. Nuclei are stained with Dapi. Scale bar is 5 µm. To account for people with red-green colour-blindness, (D) Colocation of VAMP8 (green) and STX7 (red) on individual phagosomes between WT and RNF115 KO cells represented by intersection, Manders' M1 and overlap coefficients. Data is represented as a box and whisker plot of total values across biological duplicate experiments, where the whiskers represent the minimum to maximum values and the central band indicates the median.",
    "molecules": "Dapi"
  },
  {
    "caption": "(D) Box and whisker plot represents the quantification of the percentage (%) of the hepatic lesion area in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Haematoxylin and eosin (H&E)-stained liver sections of one representative animal per genotype are shown on the right, dotted line depicts the damaged area.",
    "molecules": "eosin, Haematoxylin"
  },
  {
    "caption": "B Haematoxylin and eosin (H&E) staining of skin biopsies from patient 1 and healthy controls. Scale bars: 200 µm.",
    "molecules": "eosin, Haematoxylin"
  },
  {
    "caption": "A LC-MS/MS analysis of S1P-binding proteins. Total protein lysate was subjected to coimmunoprecipitation (Co-IP) with normal IgG or S1P antibody. The purified protein complex was separated by SDS-PAGE and then subjected to silver staining. The arrows indicate the bands containing S1P, ETFA and ETFB. The differential bands were then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).",
    "molecules": "silver"
  },
  {
    "caption": "E Cycloheximide (CHX) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells. Left panel: Representative western blotting images of the ETFA and ETFB protein levels during CHX chase. Right panel: Quantification of the immunoblotting results corresponding to the left panel (n = 3 biological replicates).",
    "molecules": "CHX, Cycloheximide"
  },
  {
    "caption": "H Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), enhanced the incorporation of FAD into the ETF complex. The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm, indicating the incorporation of FAD in the ETF complex. In the presence of the wild-type S1P protein, the two peaks were shifted by 0.02 OD units, whereas the additional mutant S1P (p.Val355Gly and p.Ter1053Arg) weakly shifted the two peaks by 0.005-0.01 OD units.",
    "molecules": "FAD, flavin"
  },
  {
    "caption": "Mitochondrial respiration (OCR) in control (Ctrl) and MBTPS1-knockout (KO) HaCaT cells was quantified in real time using a Seahorse extracellular flux analyser. Right subpanels: Quantification of basal respiration, maximal respiration, and OCR-coupled ATP production in mitochondrial respiration (n = 5 biological replicates).",
    "molecules": "ATP"
  },
  {
    "caption": "D MBTPS1 knockout led to a decrease in global ATP production in HaCaT cells. Control and MBTPS1-KO HaCaT cells were cultured in 6-well dishes. A standard curve was generated to calculate the sample ATP concentrations using an ATP Lite Luminescence Assay Kit (n = 4 biological replicates).",
    "molecules": "ATP"
  },
  {
    "caption": "E MBTPS1 knockout significantly increased the generation of mitochondrial reactive oxygen species (Mito SOX) in HaCaT cells. Left panel: Representative immunofluorescence images of Mito SOX in HaCaT cells. Scale bars: 100 µm. Right panel: The immunofluorescence intensity was quantified to calculate the relative Mito ROS level in HaCaT cells (n = 4 biological replicates).",
    "molecules": "Mito SOX, ROS, reactive oxygen species"
  },
  {
    "caption": "A The S1P deficiency-induced decrease in ETFA and ETFB was significantly reversed by riboflavin (Rib) in a concentration-dependent manner in MBTPS1-KO HaCaT cells. GAPDH was used as a loading control. MBTPS1-KO cells were initially cultured in DMEM for 24 h and supplemented with 0, 2.5, 5, or 10 µM riboflavin for 3 days. The expression of ETFA and ETFB was then detected by immunoblotting.",
    "molecules": "Rib, riboflavin"
  },
  {
    "caption": "B The S1P deficiency-induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells. Left panel: Mitochondrial respiration (OCR) was quantified in real time using a Seahorse extracellular flux analyser.",
    "molecules": "riboflavin"
  },
  {
    "caption": "C The decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells (n = 4 biological replicates).",
    "molecules": "ATP, riboflavin"
  },
  {
    "caption": "D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation. The representative head image of patient 1 before therapy is shown in Figure 1A (panel 1).",
    "molecules": "riboflavin"
  },
  {
    "caption": "(B) GST-β-catenin-1-200 was incubated with purified FLAG-CK1α, FLAG-MDMX and ATP at indicated ratios. Phosphorylation of β-catenin S45 was determined by western blot. MDMXS289A contains mutation of CK1α phosphorylation site.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Kinetic characterization of CK1α inhibition by MDMX. Reaction velocity as a function of β-catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. The resulting parameter values are shown in the table.",
    "molecules": "ATP"
  },
  {
    "caption": "(D) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation.",
    "molecules": "ATP"
  },
  {
    "caption": "(B GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.",
    "molecules": "ATP"
  },
  {
    "caption": "C, D) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.",
    "molecules": "ATP"
  },
  {
    "caption": "(B) Purified FLAG-CK1α was incubated with ATP, GST-MDMX, GST-β-catenin-1-200, pDI peptide (LTFEHYWAQLTS, 5 µM), and pDI3A peptide (LTAEHYAAQATS, 5 µM). β-catenin pS45 level was determined by western blot.",
    "molecules": "LTAEHYAAQATS, LTFEHYWAQLTS, pDI peptide, pDI3A peptide, ATP"
  },
  {
    "caption": "(B) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX. The level of pS45 was determined by western blot.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) GST-β-catenin-1-200 was incubated with identical amounts of purified FLAG-CK1α mutants and ATP. The level of pS45 was determined by western blot.",
    "molecules": "ATP"
  },
  {
    "caption": "(D). ELISA plates coated with GST-β-catenin-1-200 was incubated with purified FLAG-CK1α mutants, ATP, and MDMX. The level of pS45 was determined by ELISA assay. The results are average of 3 experiments (mean +/- SD).",
    "molecules": "ATP"
  },
  {
    "caption": "D. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and stat6-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. E. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.",
    "molecules": "cresyl violet"
  },
  {
    "caption": "L. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and il10-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. M. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course in wild-type (WT) and il10-/- macrophages. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.",
    "molecules": "cresyl violet"
  },
  {
    "caption": "A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the LPS O-polysaccharide mutant, strain 52145-Δglf, for 60 or 120 min.",
    "molecules": "LPS, O-polysaccharide"
  },
  {
    "caption": "C. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.",
    "molecules": "CPS"
  },
  {
    "caption": "E. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 for 60 or 120 min",
    "molecules": "PMA"
  },
  {
    "caption": "O. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.",
    "molecules": "CPS, PMA"
  },
  {
    "caption": "F) Dephosphorylation by the PP2A-B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted. Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. Mean and standard deviation from 3 experiments is shown.",
    "molecules": "ATP, phosphate"
  },
  {
    "caption": "D) Dephosphorylation by the PP2A-B56α holoenzyme complex of the indicated phosphorylated GST-ADAM17 (V724-C827) fragments. The GST-ADAM17 (V724-C827) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. The mean and Standard deviation of 4 independent experiments are shown.",
    "molecules": "ATP, phosphate"
  },
  {
    "caption": "F) Amphiregulin (AREG) shedding measured by ELISA of conditioned media from untreated, H2O2 treated or irradiated with x-ray DLD-1 Adam17-/- cells (clone #1) expressing full-length ADAM17 variants (wt, I762A or LEE). Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01, ***p<0.001. Mean and standard deviation indicated from at least three independent experiments.",
    "molecules": "H2O2"
  },
  {
    "caption": "G) Exogenous wt and I762A full length ADAM17 was immuno-purified from Adam17-/- cells treated with H2O2 and subjected to label-free LC-MS/MS to determine differential phosphorylation status of T735 and S808 (S811 in murine ADAM17).",
    "molecules": "H2O2"
  },
  {
    "caption": "A) EGFR activation in the ADAM17 variant expressing DLD-1 Adam17-/- cells upon H2O2 treatment, determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.",
    "molecules": "H2O2, Tyr"
  },
  {
    "caption": "a, GFP-MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells. Scale bar, 20 µm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "I ISRE reporter activity in Myc-SIRT5 (0, 100 ng or 200 ng)-transfected HEK293T cells with or without poly(I:C) transfection for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "A Confocal microscopy image of endogenous SIRT5 co-localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti-SIRT5 and anti-MAVS antibodies. Mito, Mitotracker; Scale bar = 8 µm.",
    "molecules": "Mitotracker"
  },
  {
    "caption": "F GST pull-down assay for GST-tagged MAVS and His-tagged SIRT5. GST-tagged MAVS and His-tagged SIRT5 were expressed in Escherichia coli (BL21), respectively. The association of GST-MAVS with His-SIRT5 was detected by immunoblotting with anti-SIRT5 antibody. GST and GST-MAVS proteins were stained with Coomassie blue.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "A-B MAVS could be succinylated in vitro. GST-MAVS (A) extracted from E.coli or Flag-tagged MAVS (B) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; GST-MAVS or Flag-MAVS was stained by Coomassie blue.",
    "molecules": "Coomassie blue, lysine, succinyl-CoA"
  },
  {
    "caption": "GST-MAVS (F) extracted from E.coli cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.",
    "molecules": "lysine, succinyl-CoA"
  },
  {
    "caption": "Flag-MAVS (G) purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.",
    "molecules": "lysine, succinyl-CoA"
  },
  {
    "caption": "H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5-intact H1299 cells (SIRT5 +/+) (3.2 vs. 1.0). The cell lysates from SIRT5-deficient H1299 cells or the SIRT5-intact H1299 cells were immunoprecipitated with anti-MAVS antibody or mouse IgG control, followed by immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.",
    "molecules": "Lys"
  },
  {
    "caption": "I Reconstitution of WT SIRT5 in SIRT5-deficient H1299 cells (SIRT5 -/-) caused a significant reduction in succinylation of MAVS at Lys 7 (1.0 vs. 0.2); but overexpression of SIRT5-H158Y in SIRT5 -/- H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 (1.0 vs. 1.2). The SIRT5 -/- H1299 cells were transfected with Flag-SIRT5 or Flag-SIRT5-H158Y, followed by immunoprecipitating with anti-MAVS antibody or mouse IgG control, and immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.",
    "molecules": "Lys"
  },
  {
    "caption": "J-K Knockout of Sirt5 increased Mavs succinylation in mouse livers (J) and lungs (K). Proteins extracted from livers (J) and lungs (K) of Sirt5 KO and the wildtype littermates (n = 3 per group) were detected by the indicated antibodies. MAVS succinylation was determined by anti-succ-K7-MAVS antibody, and anti-pan-succinyl-lysine antibody was used as positive controls.",
    "molecules": "lysine"
  },
  {
    "caption": "J qPCR analysis of IFNβ mRNA in HEK293T cells transfected with the control plasmid (Myc empty), or the plasmid expressing Myc-SIRT5 (Myc-SIRT5), or its enzyme-deficient mutant H158Y (Myc-SIRT5-H158Y) for 24 h, followed by stimulation with or without poly(I:C) for 8 h. Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "G Hematoxylin-and-eosin-stained images of lung sections from mice in F. Scale bar = 50 µm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "Representative immunofluorescence (IF) staining of Ki67 and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; upper panel in blue) or neural marker NeuN (lower panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.",
    "molecules": "4',6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "Representative image of comet assay for DNA fragmentation and the quantification of cells with comet tails in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Scale bar: 50 μm. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, *p = 0.0114 by t-test.",
    "molecules": "DNA, tails"
  },
  {
    "caption": "Representative IF staining of γH2AX and TDP-43 in the regions of frontal cortices from 6-mon-old WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel in blue) or NeuN (middle panel in green). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX immunoreactivity and TDP-43 proteinopathies from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0008 by t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF staining of γH2AX and Ki67 in the regions of frontal cortices from WT and FTLD-TDP Tg mice. Nuclei were stained with DAPI (upper panel) or NeuN (middle panel). Scale bar: 50 μm. Subregions, scale bar: 15 μm. Lower panel: quantification of cells or neurons with γH2AX and Ki67 immunoreactivity from each view of microscope. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM, ***p = 0.0004 by t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "Nuclear HDAC1 activity assay from frontal cortices and hippocampus in 6-mon-old of FTLD-TDP and WT mice. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. N = 5 mice per group, data are presented as mean ± SEM (%), *p = 0.0415 by t-test.",
    "molecules": "Trichostatin A, TSA"
  },
  {
    "caption": "Left, representative western blot data of nuclear acetylated-histone H3 (Lys 9/14) and total-histone H3 in 6-mon-old of WT and FTLD-TDP Tg mice. Right, quantification of nuclear acetylated-histone H3/total-histone H3 ratio. N = 5 mice per group, data are presented as mean ± SEM (%), * p = 0.0147 by t-test.",
    "molecules": "Lys"
  },
  {
    "caption": "Left graph: IF staining of TDP-43 and HDAC1 during progression of TDP-43 proteinopathies in the frontal cortices from the FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm. Right histogram: Quantification of co-localized TDP-43 and HDAC1 immunoreactivity in the cytosol or nucleus in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 9 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. *Nucleus: Tg 1m vs. Tg 6m or 12m; #Cytosol: Tg 1m vs. Tg 6m or 12m; @ Tg 6m vs Tg 12m. ****/####/@@@@ p < 0.0001 by multiple comparison.",
    "molecules": "DAPI"
  },
  {
    "caption": "Left graph: IF staining of γH2AX and HDAC1 in the frontal cortices from the 12-month-old FTLD-TDP Tg and WT mice. Nuclei were stained with DAPI (in blue). Arrow head: γH2AX+ nucleus; arrow: mislocalized HDAC1. Scale bar: 50 μm. Right histogram: Quantification of γH2AX+ and HDAC1 mislocalized cells in the WT or 1-, 6-, and 12-month-old FTLD-TDP Tg mice. n = 4 sections per mouse, N = 5 mice per group, data are presented as mean ± SEM. ** p = 0.0079, **** p < 0.0001 by multiple comparison.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western-blot of HDAC1 and TDP-43 in urea-soluble fractions. N = 5 mice per group.",
    "molecules": "urea"
  },
  {
    "caption": "Escape latency of mice in Morris water maze task. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by two-way ANOVA with Bonferroni's multiple comparisons. #P < 0.05, ***P ≤ 0.001 and ****/####P ≤ 0.0001 Data information: N = 12 mice per group All data are presented as mean ± SEM, **** p < 0.0001 by multiple comparison.",
    "molecules": "5104434"
  },
  {
    "caption": "Nuclear HDAC1 activity in mice after 2 months of compound 5104434 treatment. The nuclear extracts of the frontal cortices and hippocampus from 8-mon-old of mice were separated from total tissue lysates and were immunoprecipitated for HDAC1, and were then conducted to HDAC1 activity assay. N = 6 mice per group. TSA: nuclear extracts that were treated with Trichostatin A (TSA, an HDAC inhibitor) as a negative control for HDAC1-transferred fluorescent activity during the HDAC1 activity assay. * p = 0.0203 (WT vs Tg-Veh) or 0.0408 (Tg-Veh vs Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%",
    "molecules": "5104434, Trichostatin A, TSA"
  },
  {
    "caption": "Representative data of western blot for nuclear acetylated-histone 3 (Lys 9/14), total histone H3, and quantification of protein levels. N = 6 mice per group. * p = 0.0105 or *** p = 0.0008 by multiple comparison. Data information: All data are presented as mean ± SEM (%)",
    "molecules": "Lys"
  },
  {
    "caption": "Representative data of western blot for nuclear HDAC1 levels and semi-quantification of the expression levels. N = 6 mice per group. ** p = 0.0022 (Tg)/ 0.0012 (Tg+5104434) by multiple comparison. Data information: All data are presented as mean ± SEM (%)",
    "molecules": "5104434"
  },
  {
    "caption": "Left panel: representative data of western blot for cell cycle- and DNA damage-related proteins. Right histogram: semi-quantification of protein levels. N = 6 per group. *WT+Vehicle mice vs. Tg+Vehicle mice, #Tg+5104434 mice vs. Tg+Vehicle mice. Statistical analysis by multiple t-test with FDR correction, Q = 1%. **P ≤ 0.01; ###P ≤ 0.001 and ****/####P ≤ 0.0001. Data information: All data are presented as mean ± SEM (%)",
    "molecules": "5104434"
  },
  {
    "caption": "IF staining of γH2AX in the frontal cortices and quantification of the immunoreactive cells from 8-mon-old of WT+Vehicle, Tg+Vehicle, Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. * p = 0.0421 or **** p < 0.0001 by multiple comparison. Data information: number of cells in each view is presented.",
    "molecules": "5104434"
  },
  {
    "caption": "Immunohistochemistry of NeuN and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 8-mon-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm.* p = 0.0414 or *** p = 0.0006 by multiple comparison. Data information: All data are presented as mean ± SEM (%)",
    "molecules": "5104434"
  },
  {
    "caption": "IF staining of GFAP and the quantification of the immunoreactive cells in the hippocampus of three groups of mice, 12-month-old WT+Vehicle, Tg+Vehicle and Tg+5104434. n = 9 sections per mouse, N = 6 mice per group. Scale bar: 50 μm. ** p = 0.002 and **** p < 0.0001 by multiple comparison. Data information: where number of cells in each view is presented.",
    "molecules": "5104434"
  },
  {
    "caption": "Dot-blot of HDAC1 and TDP-43 in urea-soluble fractions from the frontal cortex of normal individuals and patients with FTLD-TDP.",
    "molecules": "urea"
  },
  {
    "caption": "Quantification of HDAC1 and TDP-43 expression levels in urea-soluble fractions. N = 5 per group. Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.",
    "molecules": "urea"
  },
  {
    "caption": "F. Left panel: Schematic of the A70.2 INV-4 cell line strategy to induce RAG1/2-mediated recombination using imatinib. Right panel: A70.2 INV-4 cell lines were transduced with lentiviruses encoding the lentiCRISPRv2 expressing sgRNAs against the 53bp1, Shld1, Shld2, Shld3, and Lig4 genes. Guide RNA targeting chicken AID was used as a negative control (Ctrl). Cells were selected with puromycin and treated with 3 μM imatinib for 4 days after which GFP frequency was measured (mean ± SD of 3 biological replicates). The insertion-deletion (indel) penetrance as measured by TIDE analysis [39] of sequence for each of these sgRNA constructs is shown in Figure EV1E, and the baseline GFP frequency prior to imatinib stimulation is shown in Figure EV1F. sgRNA sequences used are shown in Table EV2.",
    "molecules": "imatinib, puromycin"
  },
  {
    "caption": "C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates.",
    "molecules": "NP-CGG"
  },
  {
    "caption": "D. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG, spleens were isolated and anti-IgG secreting cells were enumerated by the ELISPOT assay. Values are mean frequency ± SD of 4 biological replicates, except for Shld2-/- (n=3); * P ≤ 0.05, ** P ≤ 0.01, unpaired two-tailed t test.",
    "molecules": "NP-CGG"
  },
  {
    "caption": "A. WT and Shld2−/− B cells were purified from spleens and stimulated with LPS + IL4 and examined for IgM and IgG1 expression 3, 6, and 9 days post-stimulation by flow cytometry. Representative plots are shown for both WT and Shld2−/− B cells 6-days post stimulation. The graph plots show proportion of IgG1+ and Iglo cells, mean ± SD from 6 biological replicates; ** P ≤ 0.01, *** P ≤ 0.001, two-way ANOVA with post hoc Dunnett's test.",
    "molecules": "LPS"
  },
  {
    "caption": " A. HEK293 cells were treated for 8 and 24 h with 100 μM H2O2. GSNOR was evaluated by Western blot. Densitometry of each lane is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01 calculated with regard to H2O2-untreated cells. ",
    "molecules": "H2O2"
  },
  {
    "caption": " B. HEK293 cells were treated for 8 h with 100 or 200 μM H2O2. Nrf2 (left panel) and GSNOR (right panel) were evaluated by Western blot performed in nuclear and cytosol-enriched fractions. GAPDH and Lamin A/C were used as loading and purity controls of cytosol and nuclei, respectively. Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05. ",
    "molecules": "H2O2"
  },
  {
    "caption": " C. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline (Trig). Heme oxygenase 1 (HMOX-1), glutamate:cysteine ligase (GCL) and GSNOR expressions were evaluated RT-qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate. *p<0.05; n.s., not significant. ",
    "molecules": "H2O2, Trig, trigonelline"
  },
  {
    "caption": " D. RT-qPCR analyses of GSNOR mRNA after 2-to-24 h incubation with 100 μM H2O2. Results shown are the means ± SEM of n = 3 experiments run in triplicate, analyzed using ANOVA with Dunnett multiple comparisons test. n.s., not significant. ",
    "molecules": "H2O2"
  },
  {
    "caption": " E. Western blot analysis of GSNOR in HEK293 cells treated for 8 h with 100 μM H2O2 in the presence or absence of trigonelline (Trig). Densitometry of GSNOR immunoreactive bands is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01. ",
    "molecules": "H2O2, Trig, trigonelline"
  },
  {
    "caption": " HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of cycloheximide (CHX, F), GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant. ",
    "molecules": "CHX, cycloheximide, H2O2, PBS"
  },
  {
    "caption": " G. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of the proteasome inhibitor MG132 (G). GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant. ",
    "molecules": "H2O2, MG132, PBS"
  },
  {
    "caption": " H. HEK293 cells were treated for 8 h (blue) or 24 h (red) with 100 μM H2O2. Polysome profile showing monosomes and polysomes was obtained from control (Vehicle) and H2O2 treated lysates (for either 8 or 24 h) by separation on 5-50% sucrose linear density gradient and collection using a gradient fractionation system. ",
    "molecules": "H2O2, sucrose"
  },
  {
    "caption": " I. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2-treated samples (for either 8 or 24 h). H3A mRNA expression level was used as housekeeping gene control. Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A. ***, p<0.001 Vehicle: PBS. ",
    "molecules": "H2O2, PBS"
  },
  {
    "caption": " J. HEK293 cells treated for 8 h or 24 h with 100 μM H2O2 in the presence or absence of puromycin (tyrosyl-tRNA mimic used to label nascent proteins), or cycloheximide (CHX, used to block protein translation). Nascent polypeptides were assessed by Western blot performed with an anti-puromycin (anti-PMC) antibody. Vehicle: PBS. β-actin was used as loading control. ",
    "molecules": "CHX, cycloheximide, H2O2, PBS, polypeptides, PMC, puromycin, tyrosyl-tRNA"
  },
  {
    "caption": " A. HEK293 cells treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2 were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 immunoreactive bands are normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 4 independent experiments. *, p<0.05; **, p<0.01. ",
    "molecules": "H2O2"
  },
  {
    "caption": " B. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 (KU). Basal and phospho-active forms of ATM, CHK2, and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05. ",
    "molecules": "H2O2, KU, KU55933"
  },
  {
    "caption": " C. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the CHK2 inhibitor AZD7762 (AZD). Basal and phospho-active forms of CHK2, together with GSNOR were assesses by Western blot. Phospho:basal level ratios of CHK2 and densitometry of GSNOR immunoreactive bands are normalized to GAPDH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. ",
    "molecules": "AZD, AZD7762, H2O2"
  },
  {
    "caption": " D. HEK293 cells were transfected with a pool of siRNA against ATM (siATM) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM, together with GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01. ",
    "molecules": "H2O2"
  },
  {
    "caption": " E. HEK293 cells were transfected with a pool of siRNA against CHK2 (siCHK2) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of CHK2, together with GSNOR were assessed by Western blot. GAPDH was used as loading control. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05. ",
    "molecules": "H2O2"
  },
  {
    "caption": " F. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 (KU). Lysates were subjected to biotin-switch assay, and S-nitrosylated proteins (PSNOs) revealed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin. Results obtained in the absence of ascorbate (- ASC) are shown as negative controls. Densitometry of each lane intensity is normalized to LDH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01. ",
    "molecules": "ASC, ascorbate, H2O2, KU, KU55933"
  },
  {
    "caption": " G. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2 treated samples in the absence or in the presence of the ATM inhibitor KU55933 (KU). H3A mRNA expression level was used as housekeeping gene control. Results are the means ± SEM of n = 3 independent experiments run in duplicate and shown as %, normalized by H3A. *, p<0.05. ",
    "molecules": "H2O2, KU, KU55933"
  },
  {
    "caption": " H. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of CHK1 phosphorylation at Ser317. Phospho-CHK1Ser317 was assessed by Western blot; densitometry normalized to Vinculin, selected as loading control, and expressed as arbitrary units (with HU arbitrarily set to 1). Values shown represent the means ± SD of n = 3 independent experiments. n.s., not significant. ",
    "molecules": "H2O2, HU, hydroxyurea"
  },
  {
    "caption": " I. HEK293 cells were transfected with a pool of siRNA against CHK1 (siCHK1) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. CHK1 and GSNOR were assessed by Western blot analysis of. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05. ",
    "molecules": "H2O2"
  },
  {
    "caption": " A. HEK293 cells treated for 4 h with 100 μM H2O2. Phospho-histone H2A.X (γH2A.X) was assessed by immunofluorescence analysis. Nuclei (blue) were stained with Hoechst 33342. scale bar: 10μM. 4X digital magnification is shown at the bottom left of each image. ",
    "molecules": "Hoechst 33342, histone, H2O2"
  },
  {
    "caption": " B. Additionally, γH2A.X was also assessed by Western blot at the indicated time points. GAPDH was used as loading control (top panel). Densitometry of each lane intensity is normalized to GAPDH, selected as loading control, and expressed as % of γH2A.X in H2O2-treated versus untreated cells. Values represent the means ± SD of two independent experiments (bottom panel). ",
    "molecules": "H2O2"
  },
  {
    "caption": " C. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of ATR/CHK1 axis activation. Basal and phospho-active forms of ATR and CHK1 (at Ser345) were assessed by Western blot. GAPDH was used as loading control. ",
    "molecules": "H2O2, HU, hydroxyurea"
  },
  {
    "caption": " D. HEK293 cells were treated for 8 h with 20 μM neocarzinostatin (NCS). (left) Western blot analysis of basal and phospho-active forms of ATM, CHK2, ATR, CHK1, p53, and GSNOR in HEK293 cells treated for 8 h with 20 μM neocarzinostatin (NCS). Vinculin and GAPDH were used as loading controls. (right) Phospho:basal level ratios of ATM, CHK2, ATR, CHK1, along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.",
    "molecules": "NCS, neocarzinostatin"
  },
  {
    "caption": " E. HEK293 cells were treated for 2, 4 and 8 h with 20 μM neocarzinostatin (NCS). After treatment, cells were incubated with 2',7'-H2DCF-DA to cytofluorometrically assess the intracellular production of H2O2. Values are shown as units of DCF fluorescence relative to untreated cells. Values are shown as units of fluorescence relative to NCS-untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. n.s., not significant. ",
    "molecules": "2',7'-H2DCF-DA, DCF, H2O2, NCS, neocarzinostatin"
  },
  {
    "caption": " F. HEK293 cells were transfected with plasmids coding for the wild-type (WT), C2991L redox mutant (CL) of ATM, or with an empty vector (Empty) (used as negative control) for 40 h and treated for additional 8 h with 100 μM H2O2. Basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot analysis. Vinculin and GAPDH were used as loading controls. Two different exposures (low and high) were selected to highlight differences in P-ATM levels in different experimental settings. ",
    "molecules": "H2O2"
  },
  {
    "caption": " A. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of 20 μM pifithrin-α (Pft). Basal and phospho-active forms of ATM, CHK2, p53, and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM, CHK2 and p53 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. ",
    "molecules": "H2O2, Pft, pifithrin-α"
  },
  {
    "caption": " B. HEK293 cells were transfected with a pool of siRNA against p53 (sip53) or with control siRNA (siScr) for 24 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, as well as p53 and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. ",
    "molecules": "H2O2"
  },
  {
    "caption": " C. HCT116 cells expressing the wild type form of p53 (p53wt), or an empty vector (Empty, selected as negative control), were treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, p53 and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2, along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. ",
    "molecules": "H2O2"
  },
  {
    "caption": " A, B. HEK293 cells treated for 2, 4 and 8 h with 100 μM H2O2. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001. ",
    "molecules": "MitoSox, 2',7'-H2DCF-DA, DCF, H2O2, superoxide"
  },
  {
    "caption": " C. HEK293 were transfected for 48 h with siRNA against ATM (siATM), GSNOR (siGSNOR), or control siRNA (scramble, siScr). Afterwards, they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [i.e., NDUFB8 (complex I), SDHB (complex II), MTCO2 (complex IV)]. Basal and phospho ATM, as well as GSNOR were used to check the efficiency of siRNA-mediated knock down. Vinculin was used as loading control. ",
    "molecules": "H2O2"
  },
  {
    "caption": " D. Densitometry of mitochondrial protein immunoreactive bands of panel C (normalized to Vinculin) is indicated as H2O2-treated versus untreated cells (CTR) and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant. ",
    "molecules": "H2O2"
  },
  {
    "caption": " F. U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 4 h with 100 μM H2O2. Mitophagy was assessed by RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 6 experiments. *p<0.05; ***p<0.001. ",
    "molecules": "doxycycline, H2O2"
  },
  {
    "caption": " In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 7 different cells analyzed. *p<0.05; **p<0.01; ***p<0.001. ",
    "molecules": "chloroquine, CLQ"
  },
  {
    "caption": " H. In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes. ",
    "molecules": "chloroquine, CLQ"
  },
  {
    "caption": " A, B. ATMWT U2OS cells were treated with 10 μM CCCP for 8 h. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. ***p< 0.001. ",
    "molecules": "MitoSox, 2',7'-H2DCF-DA, DCF, CCCP, H2O2, superoxide"
  },
  {
    "caption": " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. Mitophagy was evaluated by: (D) RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 4 experiments. *p<0.05; **p<0.01; ***p<0.001. ",
    "molecules": "CCCP, doxycycline"
  },
  {
    "caption": " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (E fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes. ",
    "molecules": "CCCP, chloroquine, CLQ, doxycycline"
  },
  {
    "caption": " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. F) fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 9 different cells. **p<0.01; ***p<0.001; n.s., not significant. ",
    "molecules": "CCCP, chloroquine, CLQ, doxycycline"
  },
  {
    "caption": " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (G Western blot of different subunits of mitochondrial proteins, i.e., NDUFB8 (complex I), SDHA and SDHB (complex II), MTCO2 (complex IV) and voltage-dependent anion channel (VDAC). Tubulin, LDH and Vinculin were used as loading controls. ",
    "molecules": "CCCP, doxycycline"
  },
  {
    "caption": " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. H) Densitometry of mitochondrial protein immunoreactive bands and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant. ",
    "molecules": "CCCP, doxycycline"
  },
  {
    "caption": " B. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) in the presence or absence of the ATM inhibitor KU55933 (KU). Analysis of dead cells were performed with Trypan blue exclusion assay. Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency. Data, shown as fold change of dead cells relative to untreated cells (arbitrarily set to 1), represent the mean count ± SEM of n = 4 experiments done in duplicate *p<0.05; with respect to Empty cells. ",
    "molecules": "DPTA, H2O2, KU, KU55933, Trypan blue"
  },
  {
    "caption": " C. Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU. ",
    "molecules": "DPTA, H2O2, KU"
  },
  {
    "caption": " E. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were transfected with siRNA against Pakin (siParkin) or control siRNA (scramble, siScr). Afterwards, they were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) and viability assessed by Alamar blue (AB) fluorescent assay. Data, shown as fold change of viable cells, refer to AB fluorescence (relative to untreated cells, arbitrarily set to 1) and represent the means ± SD of n = 6 independent experiments. **, p<0.01; n.s., not significant. ",
    "molecules": "DPTA, H2O2, AB, Alamar blue"
  },
  {
    "caption": " F. Western blot of Gsnor was assessed in mouse adult fibroblasts (MAFs), obtained from wild type (WT) and Gsnor-null (KO) mice treated with 200 or 400 μM H2O2 for 24 h. ",
    "molecules": "H2O2"
  },
  {
    "caption": " WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. ",
    "molecules": "DPTA, H2O2"
  },
  {
    "caption": " H. WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. Scale bar = 50 µm. Data, shown as % of dead (red) cells, represent the mean count ± SD of n = 3 different fields of three independent experiments. *p<0.05; n.s., not significant with respect to WT MAFs. ",
    "molecules": "DPTA, H2O2"
  },
  {
    "caption": " A, B. Jurkat cells were treated for 10 and 30 min with PMA (200 ng/ml) and ionomycin (iono) (300 ng/ml). After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or DAF-FM-DA (B) to evaluate the production of H2O2 or NO, respectively. Values are expressed as units of DCF or DAF-FM fluorescence relative to untreated cells (arbitrarily set as 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 (A) and n = 6 (B) independent experiments. *p<0.01; **p<0.001; ***p<0.001. ",
    "molecules": "2',7'-H2DCF-DA, DCF, DAF-FM, DAF-FM-DA, H2O2, iono, ionomycin, NO, PMA"
  },
  {
    "caption": " C. (left) Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml). Basal and phospho-active form of ATM, and GSNOR were assessed by Western blot. Vinculin was used as loading control. (right) Phospho:basal level ratios of ATM along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05. ",
    "molecules": "H2O2, ionomycin, PMA"
  },
  {
    "caption": " D. Jurkat cells were treated for 24 h with H2O2 (50 μM) or PMA/ionomycin (200 + 300 ng/ml), in the presence or absence of ATM inhibitor (KU55933) (5 μM) or GSNOR inhibitor (N6022) (25 μM). Cell death was assessed cytofluorimetrically upon staining with propidium iodide (PI). Values are expressed as % of sub-G1 population of PI-stained cells and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 5 independent experiments. *p<0.05; **p<0.01. ",
    "molecules": "H2O2, ionomycin, KU55933, N6022, PMA, PI, propidium iodide"
  },
  {
    "caption": " E, F. CD4+ T cells were incubated for 30 min with anti-CD3, anti-CD28 and anti-CD49d (stimulation). After stimulation, cells were incubated with 5 μM 2',7'-H2DCF-DA (E) or DAF-FM-DA (F) to fluorometrically evaluate the production of H2O2 or NO, respectively. Values are shown as units of DCF or DAF-FM fluorescence relative to non-stimulated cells (arbitrarily set as 1). Values are shown as fold change and represent the median plus range with all the experimental points of n = 3 independent experiments. **p < 0.01. ",
    "molecules": "2',7'-H2DCF-DA, DCF, DAF-FM, DAF-FM-DA, H2O2, NO"
  },
  {
    "caption": " G. CD4+ T cells were stimulated for 96 h with anti-CD3, anti-CD28 and anti-CD49d in the presence or absence of ATM inhibitor (KU, upper panel) or GSNOR inhibitor (N6022, bottom panel). Cell death was assessed cytofluorometrically upon staining with Annexin V (AnV) and PI. Values are expressed as fold change of AnV+/PI+ cells relative to control (CD4+ without inhibitor, vehicle, arbitrarily set to 1) and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n = 4 independent experiments. *p<0.05; ***p<0.001. ",
    "molecules": "KU, N6022, PI"
  },
  {
    "caption": "A, B. HeLa cells treated with non-targeting siRNA (Ctrl) or C9orf72 siRNA and transfected with mCherry-EGFP-LC3 were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), bafilomycin A1 (100 nM; 6 h - BafA1) or combinations thereof as indicated. Autophagosomes (green+red) and autolysosomes (red only) were quantified per cell (Mean ± SEM from 3 independent experiments; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N (cells) = Ctrl/Ctrl: 120; Ctrl/Torin1: 101; C9orf72/Ctrl: 99; C9orf72/Torin1: 106; Ctrl/BafA1: 116; Ctrl/Torin1/BafA1: 118; C9orf72/BafA1: 109; C9orf72/Torin1/BafA1: 106). Scale bar = 20 µm. C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2).",
    "molecules": "BafA1, bafilomycin A1, Torin1"
  },
  {
    "caption": "C, D. HEK293 cells treated with non-targeting (Ctrl) or C9orf72 siRNA were incubated with BafA1, BafA1+Torin1 (C) or BafA1+rapamycin (D) and levels of LC3-I and II were determined on immunoblots. Levels of LC3-II were normalized against α-tubulin and are shown relative to the BafA1-treated sample (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N = 3 experiments).",
    "molecules": "BafA1, rapamycin, Torin1"
  },
  {
    "caption": "E, F. Primary cortical neurons (DIV5/6) were transfected with ECFP non-targeting (Ctrl) or C9orf72 miRNA (cyan) and EGFP-LC3 (green). 3 days post transfection neurons were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), BafA1 (100 nM; 5 h) or combinations thereof as indicated. Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 77; Ctrl miRNA/Torin1: 68; C9orf72 miRNA/Ctrl: 57; C9orf72 miRNA/Torin1: 69; Ctrl miRNA/BafA1: 35; Ctrl miRNA/Torin1/BafA1: 57; C9orf72 miRNA/BafA1: 66; C9orf72 miRNA/Torin1/BafA1: 64). Scale bar = 5 µm.",
    "molecules": "BafA1, Torin1"
  },
  {
    "caption": "A. HEK293 cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with EGFP-LC3 and either empty vector control (EV), Myc-C9orf72S or Myc-C9orf72L. 24 h post transfection cells were treated with vehicle or 100 nM BafA1 for 6 h. Samples were lysed and subjected to SDS-PAGE and immunoblot. Autophagy levels were determined by immunoblot for EGFP-LC3-I and II. Expression of Myc-C9orf72 was confirmed using anti-Myc (* indicates a nonspecific band). FIP200 knockdown was confirmed using anti-FIP200 antibodies. α-tubulin was used as loading control.",
    "molecules": "BafA1"
  },
  {
    "caption": "B. HeLa cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with empty vector (EV), Myc-C9orf72S or Myc-C9orf72L (red) and EGFP-LC3 (green) to label autophagosomes. As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagy was quantified as the number of EGFP-LC3 positive autophagosomes per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: **** P ≤ 0.0001, N (cells) = Ctrl/EV: 73; FIP200/EV: 76; Ctrl/EV/Torin1: 73, FIP200/EV/Torin1: 70; Ctrl/C9orf72L: 71; FIP200/C9orf72L: 74; Ctrl/C9orf72S: 72; FIP200/C9orf72S: 72). Scale bar = 20 µm. FIP200 knockdown was confirmed by immunoblot (Appendix Fig S2).",
    "molecules": "Torin1"
  },
  {
    "caption": "I, J, K. 35S-radiolabeled recombinant FIP200-6xHis segments (I) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).",
    "molecules": "35S"
  },
  {
    "caption": "I, J, K. 35S-radiolabeled recombinant Myc-ATG13 (J) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).",
    "molecules": "35S"
  },
  {
    "caption": "I, J, K. 35S-radiolabeled recombinant HA-ULK1 (K) were added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant proteins were visualized by phosphoimager (top panels). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panels). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S2).",
    "molecules": "35S"
  },
  {
    "caption": "A. HEK293 cells were transfected with non-targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho-ULK1 (Ser757), total ULK1 and GAPDH Abs (loading control).",
    "molecules": "rapamycin"
  },
  {
    "caption": "B. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry-FIP200. 24 h post transfection cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, **** P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 65; Ctrl/Torin1: 60; C9orf72/Ctrl: 54; C9orf72/Torin1: 49). C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.",
    "molecules": "Torin1"
  },
  {
    "caption": "C. Primary cortical neurons (DIV5/6) were transfected with EmGFP non-targeting (Ctrl) or C9orf72 miRNA (green) and mCherry-FIP200 (red); for rescue experiments the cells were additionally transfected with mCerulean-tagged C9orf72s and C9orf72L (cyan). 3 days post transfection neurons were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 134; Ctrl miRNA/Torin1: 125; C9orf72 miRNA/Ctrl: 101; C9orf72 miRNA/Torin1: 78; C9orf72 miRNA+C9orf72L+C9orf72S: 41; C9orf72 miRNA+C9orf72L+C9orf72S/Torin1: 39). Scale bar = 5 µm.",
    "molecules": "Torin1"
  },
  {
    "caption": "D. HeLa cells were co-transfected with mCherry-FIP200 (red) and empty vector (EV), FLAG-C9orf72L or FLAG-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, *** P ≤ 0.001, **** P ≤ 0.0001); N (cells) = EV: 47, EV + Torin1: 31, C9orf72L: 46, C9orf72S: 45). Scale bar = 10 µm.",
    "molecules": "Torin1"
  },
  {
    "caption": "A, B. HeLa cells (A) or SH-SY5Y neuroblastoma cells (B) treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with mCherry-FIP200 (red) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell (A, HeLa, Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; **** P ≤ 0.0001; N (cells from 3 independent experiments) = Ctrl/EV: 81; Ctrl/EV/Torin1: 48; Ctrl/C9orf72L: 86; Ctrl/C9orf72S: 48; Rab1a/EV: 79; Rab1a/EV/Torin1: 68; Rab1a/C9orf72L: 78; Rab1a/C9orf72S: 52; B, SH-SY5Y: Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; ** P ≤ 0.01; **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 70; Ctrl/EV/Torin1: 56; Ctrl/C9orf72L: 45; Ctrl/C9orf72S: 43; Rab1a/EV: 63; Rab1a/EV/Torin1: 55; Rab1a/C9orf72L: 44; Rab1a/C9orf72S: 37). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.",
    "molecules": "Torin1"
  },
  {
    "caption": "C. SH-SY5Y neuroblastoma cells treated with non-targeting (Ctrl) or Rab1a siRNA were co-transfected with EGFP-LC3 (green) and empty vector (EV), Myc-C9orf72L or Myc-C9orf72S (red). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells from 2 independent experiments) = Ctrl/EV: 102; Ctrl/EV/Torin1: 92; Ctrl/C9orf72L: 97; Ctrl/C9orf72S: 76; Rab1a/EV: 102; Rab1a/EV/Torin1: 107; Rab1a/C9orf72L: 101; Rab1a/C9orf72S: 87). Rab1a knockdown was confirmed by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm.",
    "molecules": "Torin1"
  },
  {
    "caption": "D. 35S-radiolabelled recombinant Myc-Rab1a protein loaded with vehicle, GDP or GMP-PNP was added to GST, GST-C9orf72S and GST-C9orf72L immobilized on glutathione-coated beads. 35S-radiolabelled recombinant Myc-Rab1a protein was visualized by autoradiography (top panel). Coomassie-stained GST, GST-C9orf72S and GST-C9orf72L in the pull-down samples are shown (bottom panel). The identity of the Coomassie protein bands was confirmed by mass spectrometry (# = E. coli DnaK Chaperonin; * = E. coli 60kD Chaperonin; Appendix Fig S3). Relative binding of Rab1a to C9orf72 was quantified from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).",
    "molecules": "35S, GDP, GMP-PNP"
  },
  {
    "caption": "E. Increasing volumes of 35S-radiolabelled recombinant Myc-C9orf72L protein were incubated with equal amounts of GST-Rab1a immobilized on glutathione-coated beads in an equilibrium binding experiment. 8 µl of 35S-radiolabelled recombinant Myc-C9orf72L protein was incubated with GST as a negative control. Bound 35S-radiolabelled Myc-C9orf72L protein was visualized by phosphoimager. Coomassie stained GST-Rab1a and GST in the pull down samples are shown. Densitometry analysis of the amount of 35S-radiolabelled recombinant Myc-C9orf72L protein bound to GST-Rab1a in the different binding reactions was used to fit an equilibrium binding hyperbola (R2=0.94).",
    "molecules": "35S"
  },
  {
    "caption": "B. Primary cortical neurons (DIV5) were transduced with 4TU/cell EmGFP non-targeting control miRNA (Ctrl) or C9orf72 miRNA (green); for rescue experiments the cells were additionally transduced with 4TU/cell mVenus-tagged C9orf72s and C9orf72L (verified by immunoblot, Appendix Fig S2). Neurons were immunostained for endogenous p62 3 days post transduction. Accumulation of p62 was quantified by counting p62 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ** P ≤ 0.01; N (cells) = Ctrl miRNA: 80; C9orf72 miRNA: 80; C9orf72 miRNA+C9orf72L+C9orf72S: 75). Scale bar = 10 µm.",
    "molecules": "4TU"
  },
  {
    "caption": "A, B. Two C9ALS/FTD iNeuron cultures (183 and 201) and their matching controls (170 and 155) were treated with vehicle (Ctrl) or bafilomycin A1 (100 nM; 6 h - BafA1) and processed for immunoblot detection of LC3. LC3-II levels were normalized to α-Tubulin (Mean ± SEM; one-way ANOVA with Fisher's LSD test; ns, not significant; * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001; Ctrl 209/Pat 201, n=4; Ctrl 155/Pat 183, n=6).",
    "molecules": "BafA1, bafilomycin A1"
  },
  {
    "caption": "A Heatmap depiction of the 3,731 DNA hypomethylation loci in ago4‐6. Each hypomethylated region corresponds to a colored horizontal bar, and the bars are clustered numerically into a column (y‐axis). Cytosines were examined as CG, CHG, and CHH. The color‐scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions.",
    "molecules": "Cytosines, cytosines, DNA"
  },
  {
    "caption": "B Heatmap depiction of the 3,678 DNA hypomethylation loci in ago6‐2.",
    "molecules": "DNA"
  },
  {
    "caption": "B DNA methylation levels in different mutants. The 2,174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered (y‐axis). Cytosines were examined as CG, CHG, and CHH.",
    "molecules": "Cytosines, DNA"
  },
  {
    "caption": "C DNA methylation levels at TAS1a, TAS1c, and TAS3a loci. Snapshots from whole‐genome bisulfite sequencing results are shown.",
    "molecules": "DNA"
  },
  {
    "caption": "A AGO4 and AGO6 were visualized by immunofluorescence microscopy using their specific antibodies. Yellow signals would be expected in the merged images if the two proteins co‐localize, as a result of the overlap of red and green channels. DNA (blue) was stained with DAPI.",
    "molecules": "DNA"
  },
  {
    "caption": "B NRPE1 (red) was visualized by its specific antibody in cells expressing FLAG‐tagged AGO6 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co‐localization. DNA (blue) was stained with DAPI. AGO6‐NRPE1 co‐localized to nucleoplasmic foci (yellow dots, marked with white arrow).",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Exemplary images of COS-7 cells expressing PEX-mRFP-FKBP before (upper panels) and 30 minutes after (middle panels) rapalog addition in the presence of a translocating motor - KIF1C-MDC-FRB and a non-translocating motor - KIF3B-MDC-FRB. Yellow lines indicate COS7 cell outline. Lower panel represents the overlay of sequential binarized images color coded by time (see also Appendix Figure S1). Scale bar = 20 µm",
    "molecules": "rapalog"
  },
  {
    "caption": "E) Quantitative representation of average peroxisome displacement 30 min after addition of rapalog for COS-7 cells transfected with kinesin constructs summarized in (A). The number of analyzed cells is summarized in Appendix Table S1. Kinesins were considered translocators, if they displaced peroxisomes ≥ 5 µm (marked with a blue dashed line) in 30 minutes after rapalog addition. Error bars indicate SEM.",
    "molecules": "rapalog"
  },
  {
    "caption": "(A) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV14 hippocampal neurons expressing PEX-mRFP-FKBP, KIF1C-MDC-FRB, KIF21A-MDC-FRB or KIF5B-MDC-FRB. The morphology of transfected cell is visualized using a BFP fill. Axons are marked with a blue line. Arrows mark peroxisome targeting to dendrites (gray) and axon (blue) 30 min after rapalog addition. Scale bar = 20 µm.",
    "molecules": "rapalog"
  },
  {
    "caption": "(B) Plots representing intensity time traces of dendrites and axons before and after rapalog addition for KIF1C-MDC-FRB (n = 6 neurons), KIF21A-MDC-FRB (n = 4) and KIF5B-MDC-FRB (n = 8). Traces were normalized to the average intensity before rapalog addition, and to the background.",
    "molecules": "rapalog"
  },
  {
    "caption": "C) Quantitative representation of percentage of DIV14 neurons transfected as in (A) with constructs showed in Figure 1A, in which peroxisomes after addition of rapalog redistributed to either axons (\"a\", blue bars) or to axons and at least 2 dendrites (\"a+d\", red bars). Kinesins were considered \"dendrite targeting\" if dendrites were targeted in ≥ 50% of cells and are highlighted in red. Kinesin constructs are transfected as indicated; the number of analyzed cells is summarized in Appendix Table S1. Error bars indicate SEM",
    "molecules": "rapalog"
  },
  {
    "caption": "(D) Representative maximum intensity projections of peroxisome distribution before and after rapalog addition in DIV2 hippocampal neurons transfected as in (A). Scale bars: 20 µm",
    "molecules": "rapalog"
  },
  {
    "caption": "(E) Percentage of DIV2 neurons transfected with KIF1C-MDC-FRB (n=9 neurons), KIF21A-MDC-FRB (n=14) and KIF5B-MDC-FRB (n=14) in which peroxisomes redistributed into at least 2 neurites after addition of rapalog and (F) Percentage of neurites of DIV2 neurons transfected as in (E) targeted with peroxisomes after addition of rapalog. Error bars indicate SEM; Scale bars: 20 µm.",
    "molecules": "rapalog"
  },
  {
    "caption": "(G) Representative maximum projections of peroxisome distribution in DIV14 neurons transfected with PEX-mRFP-FKBP, KIF1C (MDC)-FRB and the indicated shRNA before and 30 minutes after rapalog addition. BFP fill was co-transected to visualize the morphology of transfected neuron. Axons are marked with a blue line. Arrows mark dendrite (gray) and axon (blue) targeting of peroxisomes after rapalog addition. Scale bar = 20 µm",
    "molecules": "rapalog"
  },
  {
    "caption": "(H) Quantitative representation of the percentage of DIV14 neurons transfected with PEX-RFP together with either KIF1C, KIF21A or KIF5B and an indicated shRNA. as in (A) in which after addition of rapalog peroxisomes redistributed to axons (\"a\", blue bars), to both axons and dendrites (\"a+d\", red bars) or did not redistribute into any neuronal compartment (\"nt\"-no targeting, gray bars). Per condition 19-44 neurons were analyzed; N = 2.",
    "molecules": "rapalog"
  },
  {
    "caption": "(J, K) Biotin pull-downs (PD) from extracts of HEK293 transfected with biotin-tagged DCLK1 and (J) KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB, KIF5B-MDC-HA-FRB constructs and probed for HA/DCLK1, (K) different GFP-tagged KIF1A truncation constructs and probed for GFP/DCLK1. The ratio input/pellet is 2% for all pull-down experiments.",
    "molecules": "biotin"
  },
  {
    "caption": "A) Fibrosis was analyzed in the diaphragm via Masson Trichrome staining. Scale bars, 100 μm.",
    "molecules": "Masson Trichrome"
  },
  {
    "caption": "(A, B) Representative images (A) and quantification (B) of calcium-stable  k-fiber  intensity levels by immunofluorescence  analysis of n≥41 tubulin-stained mitotic cells of neonatal (N) and elderly (87y) human dermal fibroblasts  (HDF) at prometaphase  and metaphase  stages. Intensity levels were normalized to neonatal samples. Scale bar, 5µm. Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed Mann-Whitney test",
    "molecules": "calcium"
  },
  {
    "caption": "(E, Live cell imaging  of neonatal (N) and elderly (87y) fibroblasts  expressing H2B-GFP/α-Tubulin-mCherry  treated with kinesin-5 inhibitor (STLC) to induce monopolar spindles, followed by washout (WO) into medium with DMSO  (-) or ZM447439  (+; 500nM). (E) Quantification of anaphases  with lagging chromosomes  in n=cells scored",
    "molecules": "DMSO, STLC, ZM447439"
  },
  {
    "caption": "F) Live cell imaging of neonatal (N) and elderly (87y) fibroblasts  expressing H2B-GFP/α-Tubulin-mCherry treated with kinesin-5 inhibitor (STLC) to induce monopolar spindles, followed by washout (WO) into medium with DMSO  (-) or ZM447439  (+; 500nM). (F) representative movie frame series of a young and an elderly dividing cell that underwent correct (Normal) and incorrect (Lagging) chromosome  segregation, respectively. Time, min:sec. Scale bars: 5µm (images) or 2µm (insets).",
    "molecules": "DMSO, STLC, ZM447439"
  },
  {
    "caption": "(C, D) Representative images (C) and quantification (D) of calcium-stable k-fiber intensity levels by immunofluorescence  analysis of n≥26 tubulin-stained metaphase  cells transduced as indicated. Levels were normalized to the control neonatal sample. Scale bar, 5μm. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed D) Mann-Whitney test.",
    "molecules": "calcium"
  },
  {
    "caption": "(E) Representative images of cytochalasin D-induced binucleated (BN) cells without (top) and with (bottom) mis-segregation (MS), FISH-stained  for three chromosome   pairs (7, 12 and 18). Scale bar, 10µm. (F) Percentage of BN cells with MS  in n=cells scored. (G) Percentage of micronuclei  in n=cells analyzed. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by (F,G) chi-square test.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(A) Mitotic  duration of neonatal (N) and elderly (87y) human dermal fibroblasts  (HDF) treated for 24 hrs with different concentrations of UMK57  (MCAK  agonist). n≥58 cells were analyzed per condition. For all subsequent experiments UMK57  was used at 1µM for 24 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed (A Mann-Whitney tests.",
    "molecules": "UMK57"
  },
  {
    "caption": "(B, Representative images (B) of calcium-stable k-fiber  intensity levels in metaphase, scored by immunofluorescence  analysis of n≥34 tubulin-stained mitotic cells of neonatal and elderly samples treated with DMSO  (-) and UMK57  (+). Levels were normalized to neonatal DMSO-treated condition. Scale bar, 5µm.",
    "molecules": "calcium, DMSO, UMK57"
  },
  {
    "caption": "C) quantification (C) of calcium-stable k-fiber intensity levels in metaphase, of neonatal and elderly samples treated with DMSO  (-) and UMK57  (+). Levels were normalized to neonatal DMSO-treated condition. Scale bar, 5µm. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed C) Mann-Whitney tests.",
    "molecules": "calcium, DMSO, UMK57"
  },
  {
    "caption": "(E) Percentage of cytochalasin D-induced binucleated (BN) n=cells with chromosomes  7, 12, and 18 mis-segregation (MS). Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(F) Percentage of micronuclei  in n=cells scored when treated with DMSO  or UMK57. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.",
    "molecules": "DMSO, UMK57"
  },
  {
    "caption": "(A) Mitotic  duration of n=100 neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) treated with DMSO  (-) or UMK57  (+) for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed (A Mann-Whitney tests.",
    "molecules": "DMSO, UMK57"
  },
  {
    "caption": "(B) Relative calcium-stable k-fiber  intensity levels scored by immunofluorescence analysis of n≥29 tubulin-stained metaphase  cells of neonatal and elderly samples treated with DMSO  and UMK57  for 96 hrs. Levels were normalized to neonatal DMSO-treated condition. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed B, Mann-Whitney tests.",
    "molecules": "calcium, DMSO, UMK57"
  },
  {
    "caption": "(D) Cytochalasin D-induced  binucleated (BN) n=cells with mis-segregation  (MS) of  chromosomes  7, 12 and 18 in neonatal and elderly samples treated for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.",
    "molecules": "Cytochalasin D"
  },
  {
    "caption": "(E) Percentage of n=cells staining  positive for double immunostaining  of Cdkn1a/p21  (cell cycle inhibitor) and 53BP1  (≥1 foci; DNA damage) senescence  biomarkers after 96hrs of treatment. (F) Percentage of n=cells staining  positive for SA-β-gal  activity when treated for 96 hrs with DMSO or UMK57. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.",
    "molecules": "DMSO, UMK57"
  },
  {
    "caption": "(G, Representative images of phospho-Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres in n≥75 neonatal and elderly metaphase  cells treated with UMK57  for 24 hrs or 96 hrs. Intensity levels were normalized to ACA. Scale bar, 5µm.",
    "molecules": "Thr, UMK57"
  },
  {
    "caption": "H) quantification (H) of phospho-Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres  in n≥75 neonatal and elderly metaphase  cells treated with UMK57  for 24 hrs or 96 hrs. Intensity levels were normalized to ACA. Scale bar, 5µm. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed H) Mann-Whitney tests.",
    "molecules": "Thr, UMK57"
  },
  {
    "caption": "H. Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC>GFP (green) stained with anti-Ct (red) and 4´,6-diamidino-2-phenylindole (DAPI, blue). Full genotype y-, w-/w-; UAS-GFP/+; SPARC-GAL4/+.",
    "molecules": "4´,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "F. Confocal single plane image of third instar larval wing disc stained with anti-Nub, anti-Wg, anti-Svp, anti-Hairy (red), reporting expression of dpp-lacZ, Dad-lacZ (anti-β-gal, red) and counterstained with DAPI (cyan). Scale bars: 100 μm. Full genotypes: P{CaryP}attP2 (top and bottom panels), P{PZ}dpp[10638];+, and y- w-;+; P{lacW}Dad[j1E4].",
    "molecules": "DAPI"
  },
  {
    "caption": "Confocal single plane images of third instar larval wing discs stained with: K. Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red), and fluorescent in situ hybridization of Ama-RNA probe (white). L. Confocal single plane images of adult DFM of Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red) stained with DAPI (white) dorsal right, anterior up. DFM are numbered in white following (Lawrence, 1982). ",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Confocal single plane images of third instar larval wing discs from Mef2>mCherry-RNAi, (left panel) and Mef2>chinmo[HM04048]-RNAi (right panel) stained with anti-Zfh1 (red) and DAPI (cyan). Scale bars: 50 μm. F. Quantification of phenotype displayed in (E). Stacked bars showing the percentage of wing discs scored as either normal or as abnormal. Data are expressed as mean ± sem, N = 32 discs per genotype, n = 3 independent experiments. ",
    "molecules": "DAPI"
  },
  {
    "caption": "G-H. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Zfh1 (green) and (G) anti-Nrt (white) at wandering third instar larval stage (110 - 135 h AEL) or (H) DAPI (red) at early third instar larval wing discs (72 - 110 h AEL).",
    "molecules": "DAPI"
  },
  {
    "caption": "Confocal single plane images of 1151>Ama-RNAi animals and 1151>mCherry-RNAi. A. Forming DFM at 40 h APF stained with anti-Zfh1 (green), anti-Futsch (22c10, red) and anti-Kettin (white). White arrows point to the wing hinge, wings pointing left, anterior up. B. Forming IFM (DLM) at 16 h APF, stained with anti-Futsch (22c10, red), anti-Zfh1 (green) and DAPI (cyan). Yellow-dashed box indicates magnified area (bottom panel). Anterior up.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. DFM at 96 h APF stained with Phalloidin (red), anti-Kettin (green) and DAPI (blue). Anterior up, dorsal right. DFM are numbered in white as in (Lawrence, 1982). D. IFM at 96 h APF, transverse section (top) stained with anti-PS-integrin (green), Phalloidin (red) and DAPI (blue), dorsal up; sagittal section (bottom) stained with anti-Kettin (green), Phalloidin (red) and DAPI (blue), anterior up, dorsal right. ",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "A Intracellular localization of total (HA) and polymeric ATZ (2C1) in WT MEF mock-treated, Confocal Laser Scanning Microscopy (CLSM). B Same as A for MEF exposed to 50 nM BafA1 for 12 h. C Same as A, 4 h after BafA1 wash-out. D Quantification of ATZ-positive, LAMP1-positive endolysosomes (EL) (n=13, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.",
    "molecules": "BafA1"
  },
  {
    "caption": "E ATZ polymers immunoisolated from lysates of WT MEF mock treated (lane 1), incubated for 12 h with BafA1 (lane 2) and 4 h after BafA1 wash-out (lane 3). Immunoprecipitation (IP) of ATZ polymers with polymer-specific 2C1 antibody, transfer on PVDF membrane, revealed with anti-HA antibody on western blot (WB). F Quantification of E, n=3, mean ± SEM. Unpaired two-tailed t-test, ns P>0.05, * P<0.05. Data information: Scale bars: 10 μm.",
    "molecules": "BafA1"
  },
  {
    "caption": "G-K Same as B in WT MEF, in cells exposed to 20 mM CST and in Cnx-, Crt- and ERp57-KO MEF. L Quantification of ATZ-positive EL (n=10, 9, 11, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm. ",
    "molecules": "CST"
  },
  {
    "caption": "A HEK293 cells transfected with empty vector (lanes 1, 6), ATZ-HA (2, 7), FAM134B-V5 (3 and 8), FAM134B-V5 and ATZ-HA (4, 9), or FAM134BLIR-V5 and ATZ-HA (5, 10), incubated for 12 h with 50 nM BafA1 and then treated with the cross-linker DSP before lysis as described in Methods. Lanes 1-5, WB of the total cell extract (TCE); lanes 6-10, WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR. The membranes were probed with antiV5 (upper panels), antiCNX, antiHA and antiLC3 antibodies. B Quantification of LC3 co-precipitating with FAM134B-V5 (A, lanes 8 and 9). Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05. ",
    "molecules": "DSP, BafA1"
  },
  {
    "caption": "E, F Distribution of gold-labeled ATZ-HA by IEM in BafA1 treated WT MEF and WT MEF overexpressing FAM134BLIR, respectively. EV, ER-derived vesicles, red arrowheads; EL, endolysosome. G Quantification of ATZ-gold density of E, F (n=75 and 79 EL, respectively). Unpaired two-tailed t-test, **** P <0.0001. ",
    "molecules": "BafA1, gold"
  },
  {
    "caption": "I Decay of ATZ polymers (CHX chase, upper panel) immunoisolated with the polymer-specific 2C1 antibody (visualized with antiHA in WB) in HEK293 cells mock-transfected (lanes 1-4) or expressing FAM134BLIR-V5 (lanes 5-8). Middle panel, expression of FAM134BLIR-V5 assessed by WB; lower panel, loading control. (J) Quantification of (I) (Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05). ",
    "molecules": "CHX"
  },
  {
    "caption": "K Flow cytometry analysis of ATZ-HA polymer levels in MEFs mock treated, exposed to BafA1, and co-expressing FAM134LIR. MFI: mean fluorescence intensity (Mean ± SEM, n=5, unpaired two-tailed t-test, ns P>0.05, ** P<0.01, *** P <0.001).",
    "molecules": "BafA1"
  },
  {
    "caption": "A-H Intracellular localization of LAMP1-positive EL, total (HA) and polymeric (2C1) ATZ in WT MEF exposed to BafA1 for 12 h (A); in Atg4BKO MEF (B); in Atg7KO MEF (C); in Fip200KO MEF (D); in Ulk1/2 double-KO MEF (E); in Atg13KO MEF (F); in Atg9KO MEF (G); in RubiconKO MEF (H). Scale bars: 10 μm. I Quantifications of A-H (n=10, 9, 9, 10, 7, 15, 9, 12 cells). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. ",
    "molecules": "BafA1"
  },
  {
    "caption": "A Distribution of gold-labeled ATZ-HA by IEM in WT MEF exposed to BafA1 (the cell on the right is not transfected, as specificity control for gold labeling). ER, endoplasmic reticulum; EV, ER-derived vesicle; EL, endolysosomes. Scale bar: 1 μm. B Inset of A. Red asterisks show site of EV:EL membranes fusion ",
    "molecules": "BafA1, gold"
  },
  {
    "caption": "C-E Distribution of gold-labeled ATZ-HA by IEM in WT MEF. ES, ER subdomains. Red asterisk shows site of EV:EL membranes fusion. Scale bar: 1 μm.",
    "molecules": "gold"
  },
  {
    "caption": "F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ-HA (visualized as white dots ES, ER subdomains. Scale bar: 500 nm.",
    "molecules": "HA, gold"
  },
  {
    "caption": "G CLEM showing ATZ (red), LAMP1-positive EL (cyan) and GFP-derived fluorescence (green) in correspondence to heavily labeled vesicles, which were identified as autophagosomes for their double membrane (insets 1-3). GFP-LC3 was also stained by immunogold. Scale bar: 1 μm.",
    "molecules": "gold"
  },
  {
    "caption": "C-E CLSM analysis of ATZ delivery to LAMP1-positive endolysosomes in BafA1-treated CRISPRWT MEF (C); in CRISPR17 MEF(D); in CRISPR8 MEF (E). Scale bars: 10 μm.",
    "molecules": "BafA1"
  },
  {
    "caption": "G-I Distribution of gold-labelled ATZ-HA by IEM in CRISPRWT MEF exposed to BafA1 (G); in CRISPR17 (H); in CRISPR8. (I). ER, endoplasmic reticulum; EV, ER-derived vesicles; EL, endolysosomes. Scale bars: 1 μm.",
    "molecules": "BafA1, gold"
  },
  {
    "caption": "GFP reconstitution in ATZ-HA transfected CRISPR17 MEF expressing FAM134B-T11 and GFP1-10-LC3. Scale bar: 10 μm. K Same as J by CLEM. ATZ-HA staining in red, LAMP1-positive endolysosomes in cyan. FAM134B:LC-induce GFP reconstitution in green. FAM134B in gold is shown. Scale bar: 1 μm.",
    "molecules": "gold"
  },
  {
    "caption": "(d-g) Rapamycin treatment of wild-type mice led to downregulation of red/green opsin ventrally (e) but not dorsally (red, d). Ventral blue opsin (red, f) remained unaffected, as did PNA (d-g) (green) and P-mTOR itself (red, g).",
    "molecules": "Rapamycin"
  },
  {
    "caption": "(a-c) Retinal flat mounts of Pde6b−/− mutants at PW7 stained for LacZ to detect cones (see Methods and Supplementary Fig. 9). An example of untreated control is shown in a, an example of a mouse injected with streptozotocin is shown in b and an example of a mouse injected daily with insulin is shown in c. (d) Quantification of cone survival after four weeks of treatment. Data represents an average of at least eight retinae. The y axis represents the percentage of the cone surface area versus the surface area of entire retina (see Supplementary Figs. 8 and 10).",
    "molecules": "insulin, streptozotocin"
  },
  {
    "caption": "(e, f) Measurements of blood glucose levels (e) and body weight (f) performed weekly over the time span of the experiment.",
    "molecules": "glucose"
  },
  {
    "caption": "(g, h) Immunofluorescence microscopy staining on retinal flat mounts for HIF-1α (red) and PNA (green) in untreated control Pde6b−/− (g) and Pde6b−/− mice treated for 4 weeks with insulin (h). DAPI is shown in blue. Error bars in d-f represent s.d.",
    "molecules": "insulin"
  },
  {
    "caption": "Real-time qRT-PCR analysis of C/EBPβ mRNA in CD4+ naïve T cells inactivated (naive) or cultured for 24 hr under conditions as indicated with DMSO or ATRA (all-trans retinoic acid). Data are representative of two independent experiments with consistent results and normalized with β-actin (mean and s.e.m. of quadruplicates)",
    "molecules": "all-trans retinoic acid, ATRA, DMSO"
  },
  {
    "caption": "EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl- or methyl-CRE sequence in the TSDR",
    "molecules": "methyl"
  },
  {
    "caption": "Flow cytometry of CFSE-labeled CD45.1+CD4+ T cells stimulated 3 d alone or together with CD45.2+GFP(Foxp3)+NGFR+-sorted CD4+ naïve T cells from Foxp3EGFP mice transduced with control retrovirus (Min) or retrovirus encoding C/EBPβ and cultured for 3 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. Histograms are gated for CD45.1+. The ratios shown are responder to suppressor. Data are representative of two independent experiments with consistent results",
    "molecules": "CFSE"
  },
  {
    "caption": "Flow cytometry of CFSE-labeled human CD4+CD25- T cells (responder) stimulated 4 d alone or together with NGFR+-sorted human CD4+CD25-CD45RA+ naïve T cells (suppressor) transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 5 d in the presence of hTGF-β, anti-hIFN-γ and anti-hIL-4 Abs. Histograms are gated for CFSE+. The ratio of responder to suppressor is 1:0.2 (top) or 1:0.4(bottom). Data are representative of two independent experiments with consistent results",
    "molecules": "CFSE"
  },
  {
    "caption": "(g) Basal lipid peroxidation levels increased with the loss of function of YAP/TAZ. HLE-shLuc and HLE-shY/T cells were stained with C11-BODIPY 581/591. Reduced-Bodipy  was measured by flow cytometry  using a 488  nm laser, and oxidized-Bodipy was measured with a  561 nm laser. A significant shift of oxidized-Bodipy occurred upon depletion of YAP/TAZ. Results represent three independent experiments.",
    "molecules": "Bodipy, C11-BODIPY 581/591"
  },
  {
    "caption": "(b) Quantitative RT-PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP/TAZ. HLE  cells were transfected with control siRNA (siCtrl) or siRNA against  YAP/TAZ (siY/T) and cultured with DMSO  or 6μM Sorafenib for 18 hours. RNA was extracted and analyzed by quantitative RT-PCR. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.",
    "molecules": "DMSO, Sorafenib"
  },
  {
    "caption": "(a) SLC7A11  protein levels decreased upon siRNA-mediated depletion of ATF4 but not of NRF2  either with or without Sorafenib treatment, and Sorafenib promoted the expression of ATF4. HLE cells were transfected with siCtrl, siATF4 or siNRF2 and treated with or without 6μM Sorafenib for 18 hours. The expression  of SLC7A11, ATF4  and NRF2 was determined by  immunoblotting. GAPDH served as loading control. Results represent three independent experiments.",
    "molecules": "Sorafenib"
  },
  {
    "caption": "(f) Colony formation assay demonstrating that the ferroptosis  inhibitor Ferrostatin-1 (Fer) reversed Sorafenib-induced cell death in ATF4-deficient HCC cells. HLE cells transfected with siCtrl or siATF4 were treated with Sorafenib (8μM) or DMSO plus either DMSO or Ferrostatin-1 (Fer; 5μM) for 2 weeks. Results represent three independent experiments.",
    "molecules": "DMSO, Fer, Ferrostatin-1, Sorafenib"
  },
  {
    "caption": "(g) The forced expression of SLC7A11 rescued cell death induced by  ATF4 ablation. HLE cells were transfected with a construct coding for SLC7A11 (SLC7A11 OE) or empty vector (EV) and with siCtrl or siATF4 every other day. Cells were then treated with either DMSO or 6μM Sorafenib for 2 weeks. Colony formation  was visualized by  crystal violet staining. Results represent three independent experiments.",
    "molecules": "crystal violet, DMSO, Sorafenib"
  },
  {
    "caption": "(a) YAP/TAZ deficiency repressed Sorafenib-induced expression of ATF4, whereas ATF4  depletion had no effect on YAP/TAZ protein levels. HLE  cells were transfected with siCtrl or siATF4 or siY/T and treated with 6μM Sorafenib or not for 18 hours. ATF4 and YAP/TAZ protein levels were analyzed by immunoblotting. GAPDH served as loading control. Results represent three independent experiments.",
    "molecules": "Sorafenib"
  },
  {
    "caption": "(b) Quantitative RT-PCR analysis verified that depletion of YAP/TAZ  or ATF4 declined the expression  levels of SLC7A11 and CHAC1. HLE cells were transfected with siCtrl, siY/T or siATF4 and cultured with 6μM Sorafenib  for 18 hours.  Quantitative RT-PCR was conducted to determine SLC7A11 and CHAC1  mRNA levels. Data are shown as mean ± standard deviation (SD).Statistical significance was calculated using Two-way ANOVA. Results represent 3 independent experiments.",
    "molecules": "Sorafenib"
  },
  {
    "caption": "(g) YAP/TAZ bind to DNA fragments containing the AARE binding motif within the SLC7A11  promoter via ATF4. HLE cells were cultured with 6μM Sorafenib for 18 hours before harvest. In a 1st round ChIP ATF4 was  immunoprecipitated  with antibody against  ATF4, rabbit IgG was used as control. DNA-protein immunocomplexes were eluted and in a 2nd round ChIP antibody against  YAP/TAZ was used to precipitate DNA fragments which were then amplified and analyzed by quantitative PCR for the  AARE motif in the SLC7A11 promoter. NC10  served as negative PCR control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.",
    "molecules": "Sorafenib"
  },
  {
    "caption": "Combination of  YAP/TAZ deficiency and Sorafenib treatment suppressed tumor growth in a HCC xenograft  model. SNU398-shLuc or SNU398-shYAP/TAZ (shY/T) cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with 20mg/kg Sorafenib  or vehicle control, and tumor sizes were measured twice a week (b).  Tumor weights were also recorded after sacrifice of the mice Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers of shLuc + Vehicle, shY/T + Vechicle and shY/T + Srf  were 4 per cohort, mouse numbers of shLuc + Srf  were 3 per cohort.",
    "molecules": "Sorafenib, Srf"
  },
  {
    "caption": "(e) Pharmacological inhibition of glutathione synthesis and function sensitizes  HCC tumors to Sorafenib therapy. SNU398 cells were transplanted into the flanks of immunodeficient NSG mice. Once the tumors were palpable, mice were treated with vehicle control or with 20mg/kg Sorafenib alone, or with 20mg/kg Sorafenib and 20mM BSO in the drinking water or 120mg/kg SSA per os. Tumor sizes were measured twice a week. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using two-way ANOVA analysis. Mouse numbers were 8 for the experimental cohorts Vehicle, Srf, and  Srf + SSA and 7 for  Srf + BSO.",
    "molecules": "glutathione, BSO, Sorafenib, Srf, SSA"
  },
  {
    "caption": "(g) Quantification of 4-HNE-positive cells of the  immunohistochemical stainings described in (f). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Each data point represents the average of 3 technical replicates of one histological section, the numbers of tumor samples are given in (e).",
    "molecules": "4-HNE"
  },
  {
    "caption": "(A) ELISA showing the dose-dependent binding of plate-bound MRC-1 peptides P2, P3, and the control peptides CP1 and CP2 to PLY (0-0.5 μM). BSA was used as negative control to show the binding specificity. Data are mean ± s.e.m. of two independent experiments, each containing three replicates per condition.",
    "molecules": "P3, peptides P2"
  },
  {
    "caption": "(B) Hemolysis assay (n=4) of 1 μg/ml purified PLY in the presence of increasing concentrations of MRC-1 peptides, P2, scrambled P2, P3 and control peptide CP2(1-1000 μM). Data represent mean ± s.e.m. * denotes P < 0.05 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4.",
    "molecules": "P2, P3"
  },
  {
    "caption": "(C) LDH cytotoxicity assay in human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, scrambled P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from 4 independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.",
    "molecules": "P2, P3, peptides P2, Cholesterol"
  },
  {
    "caption": "(D) Binding of FITC-labelled peptides P2 and CP2 to wild-type pneumococci, TIGR4 (T4) and isogenic PLY mutant (T4Δply) was visualized by fluorescence microscopy. Scale bars, 10 μm. In magnified images, scale bars, 1 μm. Images are representative of three independent experiments.",
    "molecules": "FITC, peptides P2"
  },
  {
    "caption": "(E) The hemolytic activity of wild-type pneumococci, TIGR4 (T4) and PLY mutant, T4Δply in the presence of 100 μM peptide P2 and CP2. Data are the mean ± s.e.m. of three independent experiments. *** denotes P < 0.001 by one-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.",
    "molecules": "peptide P2"
  },
  {
    "caption": "(A) IL-8 released by human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from three independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.",
    "molecules": "P3, peptides P2, Cholesterol"
  },
  {
    "caption": "(C) 3D volume images of the GFP-lung epithelial models at 1 h and 3 h post stimulation with 1 μg/ml PLY in the presence or absence of 100 μM peptide P2 or the control peptide CP2. Images are representative of two independent experiments with n=3 models/condition.",
    "molecules": "peptide P2"
  },
  {
    "caption": "(D) Invasion of wild-type pneumococci T4 (TIGR4) or its isogenic PLY mutant T4Δply into the lung epithelial models (n=3/condition) in the presence or absence of 100 μM peptide P2 or the control peptide CP2 at 2h post infection was measured using CFU viability assay following gentamicin killing of extracellular bacteria. Anti-PLY was used as control to test the effect of blocking PLY. Data in d and e are mean ± s.e.m. of n=3 models/condition from two independent experiments. % bacterial entry = (bacteria uptaken/input) x100. ** denotes P < 0.01 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.",
    "molecules": "peptide P2, gentamicin"
  },
  {
    "caption": "(E) Human DCs were infected with type 4 and type 2 pneumococci, T4 and D39, respectively, at MOI of 10 in the presence or absence of 100 μM peptides, P2 or CP2, and intracellular bacteria were counted at 3 h post infection following gentamicin killing of extracellular bacteria. Cytochalasin D (0.5 mM) was used as negative control to inhibit phagocytosis. Anti-PLY was used as control to test the effect of blocking PLY. Data are mean ± s.e.m. of three independent experiments. **** denotes P < 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.",
    "molecules": "P2, Cytochalasin D, gentamicin"
  },
  {
    "caption": "(F) DCs were infected with the unencapsulated pneumococcal strain T4R in the presence of 100 μM peptides, P2 or CP2 at MOI of 10 for 2 h. Immunofluorescence microscopy images show that in DCs treated with peptide P2 (but not the control peptide CP2), intracellular T4R (green) do not co-localize with MRC-1 (red), but with the autophagy protein LC3B (cyan). Images are representative of three independent experiments. Scale bars, 10 μm. In magnified images, scale bars, 5 μm. Arrows indicate regions of co-localization of intracellular T4R with MRC-1 and LC3B. (G) Quantification of percentage of intracellular S. pneumoniae (n=50) in infected DCs that co-localize with MRC-1 and LC3B. Data are mean±s.e.m from two independent experiments. **** denotes P <0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. P values are shown in Appendix Table S4. ",
    "molecules": "P2, peptide P2"
  },
  {
    "caption": "(B) Zebrafish survival percentage upon infection with T4 alone or together with peptide P2 or CP2 or P2-conjugated CaP NPs (P2-NPs). *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4.",
    "molecules": "P2, peptide P2, CaP, NPs"
  },
  {
    "caption": "(C) Survival of mice (n=10) upon intranasal infection with 2x106 CFU of S. pneumoniae T4 together with peptide P2 or CP2 or P2-NPs over 3 days post infection. Infected mice were checked twice daily in the morning at 9 am (early check) and in evening at 7 pm (late check) for clinical symptoms. Unloaded NPs and the isogenic PLY mutant strain (T4Δply) served as negative controls. * denotes P < 0.05 and ** denotes P < 0.005 by Mantel Cox test. Exact P values are shown in Appendix Table S4.",
    "molecules": "P2, peptide P2, NPs"
  },
  {
    "caption": "(B) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Percipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(G) USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.",
    "molecules": "polyubiquitin"
  },
  {
    "caption": "(H) USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S) , HA-Akt(K8R) or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).",
    "molecules": "polyubiquitin"
  },
  {
    "caption": "(E) USP1 inhibition significantly increases glucose tolerance in mice during fasting. Left: mice injected i.p. with specific USP1 Inhibitor (12ug/gr body weight) or saline during fasting (2d) were subjected to glucose tolerance test. Blood glucose levels were measured at the indicated time points following glucose injection (1 mg/gr body weight). Data is depicted in a graph as mg/dL glucose (n=3). * p<0.05, ** p<0.005, *** p<0.0005 vs. mice injected with saline by one-tailed t-test. Data are represented as mean ±SEM. Right: soluble fractions of muscles from injected mice were analyzed by SDS-PAGE and immunoblot.",
    "molecules": "glucose"
  },
  {
    "caption": "(B) PHLPP1 downregulation increases rates of protein synthesis during fasting. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Analysis of right (R) and left (L) limbs for each mouse is shown.",
    "molecules": "puromycin"
  },
  {
    "caption": "(E) During fasting, inhibition of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.",
    "molecules": "puromycin"
  },
  {
    "caption": "(G) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308. However, a simultaneous downregulation of USP1 is required to promote TSC1 accumulation. Soluble fraction of muscles expressing shLacz or gUSP1 (in vivo CRISPR) from fed and fasted mice injected i.p. with rapamycin (6mg/kg body weight) or saline were analyzed by SDS-PAGE and immunoblot.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(C( Dab2 promotes cleavage of K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.",
    "molecules": "polyubiquitin"
  },
  {
    "caption": "J - Box plot showing length of the poly(A) tail in RPB1-52R and RPB1-25R cells measured using Nanopore sequening data. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.",
    "molecules": "poly(A)"
  },
  {
    "caption": "A- Changes in RNA synthesis in response to 15 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 15 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes ). B- Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold change >=1). P value =0.02257 (Mann-Whitney test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Changes in RNA synthesis in response to 30 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 30 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes) D- Box plots showing log2 fold change in RNA synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold chage >=1). P value =0.001749 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. ",
    "molecules": "DMSO, TPA"
  },
  {
    "caption": "A- Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells upon 15 and 30 min TPA treatment (200 nM) as well as in the DMSO controls.",
    "molecules": "DMSO, TPA"
  },
  {
    "caption": "B- Box plots showing RNA synthesis levels (RPK) of putative eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 15 min of TPA treatment (200 nM). p value = 0.000732 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Box plots showing RNA synthesis levels (RPK) of putative enhancer eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 30 min of TPA treatment (200 nM). p value = 2.77e-5 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. ",
    "molecules": "TPA"
  },
  {
    "caption": "C-F. Polysome profiling of yeast cells with (C-E) or without (F) cycloheximide (CHX) treatment, immunoblots for Stm1 and Rpl3 in polysome fractions, and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient-rich condition (C, F), rapamycin treatment for 1 hour (D), or nitrogen starvation for 1 hour (E). Individual polysome profiles were fractionated on a 5-50% sucrose density gradient containing 150 mM KCl into 9 fractions, precipitated and immunoblotted for Stm1 and Rpl3. Rpl3 is used as a loading control. Each experiment was repeated at least three times.",
    "molecules": "CHX, cycloheximide, nitrogen, KCl, rapamycin"
  },
  {
    "caption": "G. Polysome profiles of stm1∆ cells containing either full-length or N-terminally deleted or C-terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour.",
    "molecules": "nitrogen"
  },
  {
    "caption": "A-B. In vitro kinase assay (A) and its quantitation (B) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P-γ-ATP. Immunoblots for Stm1 and Raptor are shown. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used as an inhibitor of mTOR. Minimum of three replicates are used for statistical analysis by unpaired t-test. Data are presented as the mean ± SD.",
    "molecules": "ATP, 32P, INK128"
  },
  {
    "caption": "C. Polysome profiles of wild-type and stm1∆ cells under nitrogen starvation for 1 hour. D. Polysome profiles of wild-type and STM1AAA cells under nitrogen starvation for 1 hour. E. Polysome profiles of wild-type and STM1EEE cells under nitrogen starvation for 1 hour. F-",
    "molecules": "nitrogen"
  },
  {
    "caption": "A-B. Ribosomal subunit profiles (A) and quantitation of ribosome content (B) of stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. For the ribosomal subunit profile analyses, the cell extracts were treated with 50 mM EDTA and separated on 5-25% sucrose density gradients. At least five biological replicates are used for calculation of p-values from multiple unpaired t-tests are indicated in the graphs.",
    "molecules": "EDTA, rapamycin"
  },
  {
    "caption": "E-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).",
    "molecules": "rapamycin"
  },
  {
    "caption": "J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.",
    "molecules": "bortezomib, rapamycin"
  },
  {
    "caption": "A-B. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.",
    "molecules": "AAA, EEE, nitrogen, puromycin"
  },
  {
    "caption": "A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.",
    "molecules": "DMSO, harringtonine, KCl, INK128"
  },
  {
    "caption": "I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.",
    "molecules": "INK128"
  },
  {
    "caption": "A. SF-1, FATE1 and β-tubulin protein levels shown in basal condition and after Dox treatment in H295R/TR, H295R/TRSF-1, H295R/TRFATE1 and H295R/TR N-FlagFATE1cells.",
    "molecules": "Dox"
  },
  {
    "caption": "B. FATE1 mRNA expression is increased in H295R/TRSF-1 cells by Dox treatment. The efficiency of FATE1 knockdown by specific (siFATE1) vs. control (siC) siRNA nucleofection is shown (mean ± SEM; n=4 with 3 replicates/experiment).",
    "molecules": "Dox"
  },
  {
    "caption": "C. Immunofluorescence microscopy showing endogenous FATE1 protein (red) induction of expression by Dox-treatment of H295R/TRSF-1 cells. SF-1 (green), DAPI (blue). Scale bars, 10 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "D. Subcellular localization of endogenous FATE1 (red) and transfected fluorescent markers for Golgi, ER and mitochondria, respectively (green) in Dox-treated H295R/TRSF-1 cells. DAPI (blue). Scale bars, 10 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "E. Subcellular localization of the endogenous FATE1 protein (green) in Dox-treated H295R/TRSF-1 cells costained with an antibody against the mitochondrial marker HSP60 (red). SF-1 (blue). Scale bar, 10 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "F. Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox-treated H295R/TR N-FlagFATE1 cells. Scale bar, 500 nm.",
    "molecules": "Dox"
  },
  {
    "caption": "G. Dox-treated H295R/TR N-FlagFATE1 cells were fractioned into nuclear (N), heavy membranes (HM), light membranes (LM) and cytosolic (C) fractions and localization of SF-1, FATE1, ribosomal protein RPL7, VDAC1 and GAPDH was revealed by immunoblot.",
    "molecules": "Dox"
  },
  {
    "caption": "H. Effect of increasing concentrations (0, 1, 10 and 100 μg/ml) of proteinase K and 0.1% Triton X-100 (TX-100) treatment of the mitochondrial fraction from Dox-treated H295R/TR N-FlagFATE1 cells on VDAC1, FATE1, TOM20 and cytochrome c.",
    "molecules": "Dox, Triton X-100, TX-100"
  },
  {
    "caption": "I. FATE1 and VDAC1 are associated with the pellet (membrane; P) fraction after high-speed centrifugation of the alkaline-extracted mitochondrial fraction of Dox-treated H295R/TR SF-1 cells, while cytochrome c is found in the supernatant (S).",
    "molecules": "Dox"
  },
  {
    "caption": "B. Subcellular localization of EGFP-fusions of full-length (FL) FATE1 and its mutants: N-terminal (aa. 1-124), C-terminal (aa.125-183), 1-162, transmembrane domain (aa. 155-183) and L151D (green) transfected in H295R/TRSF-1 cells. Mitochondria were stained by anti-TOM20 antibody (red) and DNA by DAPI (blue). Scale bars, 10 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "E. Mic60/mitofilin and Flag-tagged FATE1 were coimmunoprecipitated from Dox-treated H295R/TR N-FlagFATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) antibody.",
    "molecules": "Dox"
  },
  {
    "caption": "F. Mitochondrial localization of Mic60/mitofilin (green) and FATE1 (red) in Dox-treated H295R/TRSF-1 cells. Scale bar, 10 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "G. Left, triple immunofluorescence microscopy labelling of endogenous FATE1 (green), ER labelled by calreticulin (red) and mitochondria labelled by HSP60 staining (blue) in Dox-treated H295R/TRSF-1 cells. One area showing close apposition of red, green and blue staining (white signals) is shown at higher magnification. Scale bar, 10 μm. Right, graph showing quantification of FATE1 signal in total mitochondria (white histogram) vs. ER-mitochondria contact sites (red histogram) (mean ± SEM; n=13). **p<0.01.",
    "molecules": "Dox"
  },
  {
    "caption": "A. H295R/TR N-FlagFATE1 cells were transfected with D1 ER marker for ER (green) and mtRFP marker for mitochondria (red). Manders' coefficient for green-red signals colocalization in basal (white histograms) and Dox-treated (black histograms) cells is shown on the right (mean ± SEM; n=12). ***p<0.001. Scale bars, 10 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "B. Effect of FATE1 expression on ER-mitochondria distance in H295R/TR N-FlagFATE1 cells measured using a split-GFP probe [29]. Quantification is shown in the graph. White histogram, number of fluorescent objects/cell in basal conditions; black histogram, number of fluorescent objects/cell in Dox-treated cells (mean ± SEM; n=6 with 90 cells analyzed in total). **p<0.01. Scale bars, 5 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "C. Transmission electron microscopy images of H295R/TR N-FlagFATE1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=38 for basal and 47 for Dox-treated cells). ***p<0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "D. Transmission electron microscopy images of H295R/TRSF-1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=41 for basal and 42 for Dox-treated cells). ***p<0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "E. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-FlagFATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm.F. Mitochondrial fragmentation index in H295R/TR N-FlagFATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n=111 for basal and 101 for Dox-treated cells). ***p<0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "G. Mitochondrial fragmentation index in H295R/TRSF-1 cells after Dox treatment (green histogram) compared to cells cultured in basal conditions (white histogram). (mean ± SEM; n=37 for basal and 49 for Dox-treated cells). ***p<0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "H. Mitochondrial membrane potential (∆Ψ) measured by TMRM fluorescence after Dox treatment of H295R/TR N-FlagFATE1 cells (red histogram) compared to cells cultured in basal conditions (white histogram). Mean ± SEM is shown. (mean ± SEM; n=3 with 12 replicates/experiment). ns, not significant.",
    "molecules": "Dox"
  },
  {
    "caption": "A. MitochondrialCa2+ uptake. H295R/TR parental clone (white/grey histograms) H295R/TRSF-1 (white/green histograms), H295R/TR N-FlagFATE1 (white/red histograms) and H295R/TRFATE1 cells (white/violet histograms) (mean ± SEM; n=3-5 with 12 replicates/condition). **p<0.01; ***p<0.001; ns, not significant.B. CytosolicCa2+ concentration. Histograms as in A (mean ± SEM; n=3-5 with 12 replicates/condition). ns, not significant.",
    "molecules": "Ca2+"
  },
  {
    "caption": "C. Mitochondrial (left) and cytosolic (right) Ca2+ concentrations in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs (mean ± SEM; n=3-5 with 12 replicates/condition). ***p<0.001; ns, not significant.",
    "molecules": "Ca2+, Dox"
  },
  {
    "caption": "D. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) treated with H2O2 (500 μM) C2-ceramide (50 μM) or staurosporine (STS) (1 μM). (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; **p<0.01; ns, not significant.",
    "molecules": "Dox, H2O2, C2-ceramide, staurosporine, STS"
  },
  {
    "caption": "E. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with H2O2 or STS (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; ns, not significant.",
    "molecules": "Dox, H2O2, STS"
  },
  {
    "caption": "F. Flow cytometry analysis of TUNEL staining in H295R/TR N-Flag FATE1 cells cultured in basal conditions or in the presence of Dox treated with vehicle (control) H2O2 (500 μM), C2-ceramide (50 μM) or staurosporine (STS) (1 μM).",
    "molecules": "Dox, H2O2, C2-ceramide, staurosporine, STS"
  },
  {
    "caption": "G. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) and treated with mitotane (50 μM) (mean ± SEM; n=5-8 with 3 replicates/condition). **p<0.01.",
    "molecules": "Dox, mitotane"
  },
  {
    "caption": "H. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with mitotane (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05.",
    "molecules": "Dox, mitotane"
  },
  {
    "caption": "(B) CHD1 is recruited to an I-SceI-induced DSB site and is co-localized with γH2AX. Immunofluorescence studies using U2OS19 ptight13 GFP-LacR cells containing a stably integrated I-SceI cleavage site flanked by 256 copies of lac operator (lacO) on one side and 96 copies of the tetracycline response element on the other side (tetO). The localization of the GFP-lac repressor protein (GFP-LacR) at the lac-operator DNA sequences in the nucleus before (− I-SceI) and 16 h after I-SceI-induced (+ I-SceI) DSB. After 16 h of doxy treatment, CHD1 and γH2AX co-localized at I-SceI cleavage site, along with DNA-bound GFP-LacR but not in uninduced cells (−I-SceI). Scale bar 10 µm.",
    "molecules": "DNA, tetracycline"
  },
  {
    "caption": "(D) PC3 cells were treated with NCS (100 ng/ml) for 2 h and with EdU for 45 min. Cells were stained with EdU first and then proximity ligation assay (PLA) was performed using γH2AX and CHD1 antibodies. Scale bar 10 µm.",
    "molecules": "NCS"
  },
  {
    "caption": "(F) CHD1 is recruited to the chromatin upon DNA double strand break induction. PC3 cells with stable control (shCont) or CHD1 shRNA (shCHD1) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies. H2B was used as a loading control. See also Figure EV1A-EV1F.",
    "molecules": "NCS"
  },
  {
    "caption": "(A) PLA assay with CtIP and CHD1 in PC3 cells after 2 h of NCS treatment. Scale bar 5 µm.",
    "molecules": "NCS"
  },
  {
    "caption": "(B) shCont and shCHD1 PC3 cells were treated with NCS for the indicated times and chromatin fractions were immunoblotted for CtIP, RPA1 and RAD51. H2B is shown as a loading control.",
    "molecules": "NCS"
  },
  {
    "caption": "CHD1 depletion leads to decreased (C) CtIP and (D) RPA1 recruitment to I-SceI-induced DSB in U2OS19 ptight13 GFP-LacR cells. CHD1 was depleted in U2OS19 ptight13 GFP-LacR cells by siRNA and 48 h after transfection cells were treated with doxy for 16 h and co-immunostained for γH2AX and CtIP or RPA1. The number of cells with CtIP or RPA1 foci from (C) and (D) were counted and represented the mean in the graph as percentage of foci positive cells as ± SD (n=3), more than 50 cells were counted. Scale bar 5 µm.",
    "molecules": "doxy"
  },
  {
    "caption": "(E) PC3 cells were transfected either with mock or siCHD1 followed by empty vector, wt mChd1 (Wt) or ATPase-mutant (Mt) mChd1. After 48 h of transfection cells were treated with of 4-OH tamoxifen (4-OHT) for 24 h and processed for PLA with γH2AX and CtIP antibodies. Cells demonstrating positive focal interactions indicative of DNA repair hubs (punctate staining) are present only in control and (Wt) CHD1-rescued cells but not following knockdown or reconstitution of an ATPase-mutated CHD1 (Mt). Scale bar 20 µm.",
    "molecules": "4-OH tamoxifen, 4-OHT"
  },
  {
    "caption": "(F) PC3 cells which stably express HA-mChd1-ERT2, were transfected with either mock or siCHD1. After 24 h of transfection cells were treated with 4-OHT for 24 h to induce HA-mChd1-ERT2 nuclear translocation. Western blot analysis of chromatin fractions for CHD1, ERT2 (mChd1), CtIP and RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ. The relative quantification for the indicated proteins is shown in the graph. See also Figure EV4A-EV4P.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(A) CHD1 recruitment is dependent on MRE11 activity. PC3 cells were transfected with mock or siMRE11 (SmartPool) and 48 h after transfection cells were treated with NCS and chromatin fractions were isolated and analyzed by Western blot.",
    "molecules": "NCS"
  },
  {
    "caption": "(B) CHD1 is upstream of CtIP. PLA with γH2AX and CHD1 antibodies in mock or CtIP depleted cells after 2 h of NCS. Scale bar is 50 µm. Verification of knockdown efficiency is shown by Western blot on the right. HSC70 is shown as a loading control. Scale bar 20 µm. (C) Quantification of PLA signal from (B) represented as mean values from three independent experiments as ± SD, more than 100 cells were counted per condition.",
    "molecules": "NCS"
  },
  {
    "caption": "(D) qPCR analyses for chromatin accessibility at two HR-repaired sites (DSB-I and DSB-II) was analyzed by FAIRE in AsiSI-ER-U20S cells transfected with either mock or CHD1 siRNA. After 48 h of transfection cells were treated with 4-OHT for the indicated time points and processed for FAIRE. The data are represented as mean ± SD (n=3) p-values were calculated using ANOVA (0.02 and 0.03, *p ≤ 0.1).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(E) Native BrdU staining of PC3 cells transfected either with mock or siCHD1 (SmartPool) were grown for 48 h and then treated with NCS for 2 h prior to staining with anti-BrdU and γH2AX antibodies. Scale bar 5 µm.",
    "molecules": "NCS"
  },
  {
    "caption": "(A) CHD1-depleted cells show hypersensitivity to Mitomycin (MMC) treatment. For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented (n=3, ± SD, p-value=0.0002, ANOVA, ***p ≤ 0.001).",
    "molecules": "Mitomycin, MMC"
  },
  {
    "caption": "(B) CHD1-depleted cells show increased hypersensitivity to Irinotecan. For cell proliferation analysis, shCont and shCHD1 cells were treated with 1 µM Irinotecan and proliferation was measured by Celigo and the relative confluency (mean) was plotted in the graph (n=3, ± SD, p-value=0.0004, ANOVA, ***p ≤ 0.001).",
    "molecules": "Irinotecan"
  },
  {
    "caption": "(C) Loss of CHD1 leads to increased sensitivity to PARP inhibition. Control or CHD1-depleted BPH1 cells were treated with the indicated concentrations of the PARP inhibitor olaparib and surviving fractions were measured by counting colonies after 3 weeks. Data represented as mean values (n=3, ± SD, p-value=0.0003, ANOVA, ***p ≤ 0.001).",
    "molecules": "olaparib"
  },
  {
    "caption": "(D) CHD1 depletion render cells sensitive to PARP inhibition in combination with irradiation. shCont- or shCHD1-expressing BHP1 cells were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X-rays and surviving fractions (normalized to the unirradiated condition) were measured by counting colonies after 3 weeks. Data is represented as mean ± SD (n=3, p-value=0.07, ANOVA, *p ≤ 0.1).",
    "molecules": "olaparib"
  },
  {
    "caption": "B. The in vitro helicase loading assay demonstrates that an Mcm2 C-terminal deletion mutant supports complex assembly (lanes 6 and 7) and blocks formation of the final helicase loading product (lanes 8 and 9).",
    "molecules": "helicase"
  },
  {
    "caption": " (B) Automatic cluster-mapping of the temperature-dependent splicing changes in CHX, based on changes in PSI (percent spliced in). Categories (right) were manually assigned. ",
    "molecules": "CHX"
  },
  {
    "caption": " (A) Temperature-dependent poison events in Hnrnpdl (left, see also Figure 1C), Hnrnph3 (middle) and Cirbp (right). For each target, on top a simplified exon-intron structure is given and below Sashimi plots show the distribution of raw sequencing reads. Exon-Exon junction reads are indicated by the numbers connecting the exons. Below, sequence conservation across placental species is indicated. Hnrnpdl exhibits a heat-included exon that leads to a PTC, Hnrnph3 a cold-skipped exon that leads to a frameshift, and in Cirbp, heat leads to inclusion of an alternative transcript end, which leads to formation of a PTC. (B) For each gene from A, the triplicate normalized read counts in DMSO (left y-axis, green) and the percentage of the poison isoform in CHX (right y-axis, orange) are plotted at the two temperatures. Line represents mean PSI. ",
    "molecules": "DMSO, CHX"
  },
  {
    "caption": " (C) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature-dependent AS-decay skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform between the CHX samples. Top shows the GE change between the DMSO samples and bottom between the CHX samples. R² and P (deviation from zero slope) are indicated. N=24. ",
    "molecules": "DMSO, CHX"
  },
  {
    "caption": " (B) Poison exon inclusion for Srsf2 (left) and Srsf10 (right) in a 24h temperature-rhythm in human cells. Hek293 cells were pre-entrained with square-wave temperature cycles (12h 34°C/ 12h 38°C) for 48h. For the last 24h, cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT-PCR (n=3, mean ± SD). White area: 34°C; Red area: 38°C. In Hek293 cells, inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target. ",
    "molecules": "DMSO, CHX"
  },
  {
    "caption": " (E) Correlation of poison isoform inclusion and GE levels for temperature-dependent poison skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform. Decay targets were identified by using data from CHX-treated plants. R² and P(deviation from zero slope) are indicated. N=80 ",
    "molecules": "CHX"
  },
  {
    "caption": "B Relative (left) and absolute (right) amount of 13C-Lys-labeled and unlabeled Aβ  in APPtg mouse brains  after labeling from 75-125 d (n = 1), 75-175 d (n = 4), 50-100 d (n = 5), 50-125 d (n = 2), 50-150 (n = 4) or 50-175 d (n = 9) of age. Error bars show mean ± SD.",
    "molecules": "Aβ, 13C, Lys"
  },
  {
    "caption": "C Representative chromatogram  and mass spectra of doubly charged ions of unlabeled Aβ17-28 (663.3 m/z) and 13C-Lys Aβ17-28 (666.3 m/z) in an  APPtg brain  after labeling from 75-175 d. %- ion intensity, m/z- mass-to-charge. Data were acquired in data-independent analysis mode (MSE). D Representative chromatograms  of unlabeled  Aβ17-28  (left) and 13C-Lys Aβ17-28 (right) in an APPtg brain  after labeling from 50-175 d. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed. Data were acquired by MRM  in nanoflow mode.",
    "molecules": "Aβ17-28, 13C, Lys"
  },
  {
    "caption": "F Representative chromatograms  of unlabeled Aβ17-28  (left) and 13C-Lys Aβ17-28 (right) in a non-APPtg brain  after labeling  from 90-100 d. Data were acquired by MRM  in microflow mode.",
    "molecules": "Aβ17-28, 13C, Lys"
  },
  {
    "caption": "A MALDI-TOF mass spectrum of trypsin-digested, synthetic Aβ1-42 (10 pmol). The  Aβ17-28 [MH]+ fragment was detected with the highest intensity. Measurements in reflectron positive mode. The sequence of Aβ1-42  with the tryptic  cleavage sites is indicated at the top",
    "molecules": "Aβ17-28, Aβ1-42, trypsin, tryptic"
  },
  {
    "caption": "B MS/MS spectrum of unlabeled Aβ17-28 and 13C15N-Lys Aβ17-28 synthetic peptides in the 50 fmol range. m/z of precursors ions (m/z 663.34 for Aβ17-28 [MH]2+, m/z 667.36 for 13C15N-Lys Aβ17-28 [MH]2+) and their derived fragments are indicated as a series of y ions (y4-y10). The peptides were analyzed in data independent acquisition MSE mode",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "C Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were trypsin-digested and analyzed by MRM. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed for  13C15N-Lys Aβ17-28, showing a detection limit level of 25 amol. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD",
    "molecules": "Aβ17-28, Aβ1-42, 13C, Lys, 15N, trypsin"
  },
  {
    "caption": "D Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were spiked into the insoluble fraction of APPtg brains, immunoprecipitated, trypsin-digested, and analyzed by MRM. Assessment of the intensities of y10, y9, y8 and y7 showed a detection limit level of 50 amol for 13C15N-Lys Aβ17-28 and detected an excess of brain-derived unlabeled Aβ17-28. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD",
    "molecules": "Aβ17-28, Aβ1-42, 13C, Lys, 15N, trypsin"
  },
  {
    "caption": "B Temporal MRM  analysis of 13C-Lys Aβ17-28 and unlabeled Aβ17-28 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after 13C-Lys-APPtg brain  extract i.p. injection (n = 3 mice per  time point  and tissue at 1-10 d, except for n = 2 mice  for  peritoneal cells at 3 d; n = 4 mice per time point and tissue at 25-100 d, except for n = 3 mice  for spleen  at 100 d). Measurements in technical triplicates, normalization to  13C15N-Lys Aβ17-28  intensity as a spiked internal standard and additionally to organ weight  (mg) for  lymph nodes. Error bars show median ± interquartile range.",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "C Temporal 4G8 immunoassay  quantification of Aβ42  in the liver, spleen,  mesenteric lymph nodes, and peritoneal cells after unlabeled APPtg (n = 8 mice  at 5 h - 7 d, except for n = 5 mice  for  peritoneal lavage, n = 5 mice  at 10-14 d, n = 4 mice  at 21 d) or non-tg brain  extract injection (n = 2 mice  at 5 h and 3 d, n = 3 mice  at 1 d, n = 1 mouse  at 5 and 7 d, except for peritoneal cells where n = 1 mouse  at 5 h and 3 d, and n = 2 mice  at 1 d). Error bars show median ± interquartile range. D Normalization of APPtg extract group data presented in (C) to organ weight  (mg) and injected  Aβ amount (range 1,027-1,426 ng). Error bars show median ± interquartile range.",
    "molecules": "Aβ, Aβ42"
  },
  {
    "caption": "A Temporal MRM  analysis of unlabeled Aβ17-28 and 13C-Lys Aβ17-28 in the soluble brain  fraction after 13C-Lys APPtg brain  extract i.p. injection (n = 3 mice  per  time point  at 1-10 d, n = 4 mice per time point  at 25-100 d). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight  (mg). All error bars show mean ± SD.",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "Comparative MRM  analysis of 13C-Lys Aβ17-28 (B) in the insoluble brain  fraction after 13C-Lys-APPtg brain  extract (donors labeled  for 50-125 d) and 13C-Lys-non-tg brain  extract (donors labeled  for 10 d) i.p. injection (n = 3 mice  per group and time point at 1-10 d, n = 4 mice  per group and time point at 25, 50, 100 d, n = 3 mice  in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points  1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys  non-tg extract-injected for each  time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "C Comparative MRM analysis of unlabeled Aβ17-28 (C) in the insoluble brain  fraction after  13C-Lys-APPtg brain extract (donors labeled  for 50-125 d) and  13C-Lys-non-tg brain  extract (donors labeled  for 10 d) i.p. injection (n = 3 mice  per group and  time point at 1-10 d, n = 4 mice  per group and  time point  at 25, 50, 100 d, n = 3 mice  in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for  time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys  non-tg extract-injected for each  time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight  (mg). All error bars show mean ± SD.",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "D Comparative 4G8 immunoassay   quantification of Aβ42  in the insoluble brain  fraction after  13C-Lys APPtg or  13C-Lys  non-tg brain  extract i.p. injection (n = 3 mice  per group and  time point at 1-10 d, n = 4 mice  per group and time point  at 25-100 d), right side zoom for time points 1-10 d. Normalization to protein concentration.",
    "molecules": "Aβ42, 13C, Lys"
  },
  {
    "caption": "E Representative chromatograms  of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in the brain  of an unlabeled 50-day-old APPtg mouse  not injected with labeled brain  extract. Note the absence of 13C-Lys Aβ17-28.",
    "molecules": "Aβ17-28, 13C, Lys"
  },
  {
    "caption": "F MRM  analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in  APPtg mouse brains  after 13C-Lys-labeled  diet for 1 or 3 d (n = 3 mice  per time point). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28  intensity as a spiked internal standard and brain weight  (mg). All error bars show mean ± SD.",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "G  Comparative MRM analysis  of  13C-Lys Aβ17-28  in the insoluble brain  fraction after 13C-Lys APPtg and 13C-Lys  non-tg brain  extract (all donors labeled  for 100 d) i.p. injection (n = 6 mice  per group). Data information: measurements in technical triplicates, normalization to  13C15N-Lys Aβ17-28  intensity as a spiked internal standard and brain weight  (mg). All error bars show mean ± SD.",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "H MRM  analysis of unlabeled and  13C-Lys-labeled Aβ17-28 in non-APPtg mouse brains  after 13C-Lys-labeled diet for 1 d (n = 3 mice) or at 1 d after 13C-Lys-APPtg brain  extract i.p. injection (n = 3 mice). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight  (mg). All error bars show mean ± SD.",
    "molecules": "Aβ17-28, 13C, Lys, 15N"
  },
  {
    "caption": "Growth rate trajectories for duplicate experiments (n=2 evolution experiments per substrate condition) (green and purple) for the example case of D-lyxose. Population growth rates are plotted against cumulative cell divisions. Clones were isolated for whole genome sequencing at notable growth-rate plateaus as indicated by the arrows. Mutations gained at each plateau are highlighted beside the arrows (mutations arising earlier along the trajectory persisted in later sequenced clones).",
    "molecules": "D-lyxose"
  },
  {
    "caption": "YihS V314L + R315S mutant enzyme activity on D-mannose and D-lyxose. LC-MS was used to analyze YihS activity at saturating substrate concentrations to compare turnover rates on each substrate. Product formation was followed over time at a constant enzyme concentration. Turnover rates were calculated using linear regression (n=3 replicates for each enzyme, Dataset EV4). The error bars represent standard deviation (n=3) of the peak area.",
    "molecules": "D-lyxose, D-mannose"
  },
  {
    "caption": "Turnover ratios of substrate conversion of D-lyxose / D-mannose are shown for the wild type YihS and mutant YihS enzymes. A ratio < 1 indicates a higher turnover rate on D-mannose compared to D-lyxose. Error bars represent standard error (n=3) calculated from the linear regression analysis.",
    "molecules": "D-lyxose, D-mannose"
  },
  {
    "caption": "Expression data (RNAseq) for significantly differentially expressed genes (q-value < 0.05, FDR-adjusted p-value, n=2 biological replicates for each condition). Scatter plot shows log2(fold change) of gene expression data comparing endpoint to initial populations for Exp. 1 and Exp. 2 (grey dots) with the location of the gene in the reference genome as the x-axis. Those genes that are associated with AraC transcription units are highlighted (red dots for Exp. 1 and blue dots for Exp. 2). Above the plot, the transcription units are labeled green if AraC activates expression (in the presence of arabinose) or red if AraC represses expression of those genes.",
    "molecules": "arabinose"
  },
  {
    "caption": "Growth rate analysis of various weaned (starting point of static phase) and optimized (endpoint of static phase) strains with or without fucK or araB genes knocked out. Strains were grown in triplicate (n=3) on M9 minimal media with D-arabinose as the sole carbon source. The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation. The p-values reported were calculated using a two-sided Welch's t-test.",
    "molecules": "carbon, D-arabinose"
  },
  {
    "caption": "Tumor neutrophil (left panel) and monocyte (right panel) content after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=10) treatments.",
    "molecules": "DMSO"
  },
  {
    "caption": "Average tumor volume per mouse at the end of αGr1 + CXCR2i (n=9) or IgG + DMSO (n=12) treatments as indicated in B.",
    "molecules": "DMSO"
  },
  {
    "caption": "FACS analysis of CD45+ TCRβ+ cells, CD45+ TCRβ+ CD8+ cells and CD45+ TCRβ+ CD4+ Foxp3+ cells in extracted tumors after αGr1 + CXCR2i (n=8) or IgG + DMSO (n=7) treatments.",
    "molecules": "DMSO"
  },
  {
    "caption": "FACS analysis of IFNγ (left panel) and IL17A (right panel) expression in tumor extracted T cells after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=7) treatments.",
    "molecules": "DMSO"
  },
  {
    "caption": "Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.",
    "molecules": "Galunisertib"
  },
  {
    "caption": "Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) Protein levels of free active TGFβ1 in cell culture supernatants of indicated conditions determined by ELISA.",
    "molecules": "GM6001"
  },
  {
    "caption": "Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.",
    "molecules": "GM6001"
  },
  {
    "caption": "Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) or with 10µm Galunisertib plus 10µm GM6001 (T + Ne + TGFBRi + MMPi). T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.",
    "molecules": "GM6001, Galunisertib"
  },
  {
    "caption": "Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). , Co-immunostaining for pSMAD3 (green) and IGFBP7 (magenta) on representative tumor-containing colon sections derived from Apcfl/fl-Cdx2CreERT2 mice treated with vehicle (A) MMPi (B).",
    "molecules": "SB3-CT"
  },
  {
    "caption": "Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). Quantification of pSMAD3+ cells (left panel) and pSMAD3 staining intensity per pSMAD3+ cell (right panel) on at least five arbitrarily selected tumor areas per section of MMPi (n=6) or vehicle (n=6) treated mice.",
    "molecules": "SB3-CT"
  },
  {
    "caption": "effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation. Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, one day post treatment, injected with either MMPi (n=10) or the TGFBRi SB431542 (n=10) at 40mg/kg for two weeks with five injections per week. Vehicle treated mice: n=11. Average tumor size per mouse was assessed during colon resection one week after the end of treatments (E).",
    "molecules": "SB431542, Tamoxifen"
  },
  {
    "caption": "Proliferation of activated blood T cells in vitro in co-culture with autologous blood neutrophils. Cells were derived from both CRC patients (n=2) and healthy volunteers (n=5). T cells and neutrophils were cultured at a 1:1 ratio and treated with vehicle (T + Ne) or 10µm Galunisertib (T + Ne + TGFBRi). Each line represents a neutrophil sample from an individual healthy volunteer.",
    "molecules": "Galunisertib"
  },
  {
    "caption": "A. Northern blots showing levels of β-globin reporter mRNAs in wild-type HeLa Tet-off cells and UPF3B knockout HeLa Tet-off cells. β39 is a tetracycline (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. β-GAP is a stable, constitutively-expressed, longer β-globin mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t1/2) along with standard errors of means are on the bottom.",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "C. Northern blot showing steady-state levels of β39 NMD reporter and β-GAP control in HeLa Tet-off UPF3B knockout cells upon transient overexpression of wild-type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to β-GAP control) as compared to the normalized β39 reporter levels in UPF3B expressing cells.",
    "molecules": "Tet"
  },
  {
    "caption": "A, B. Western blots showing levels of EJC proteins or HNRNPA1 (control) in input, IgG IP or EIF4A3 IP following overexpression (OE) of CASC3 wild-type (WT) and EJC binding deficient (HDAA) mutant proteins in (A) HeLa Tet-off cells, and (B) 3BKO#3 HeLa Tet-off cells. Ramps above lanes indicate expression levels of the CASC3 proteins.",
    "molecules": "Tet"
  },
  {
    "caption": "(A) HMGB1 translocates from the nucleus to the cytosol during autophagy, but not apoptosis. Mouse embryonic fibroblasts (MEFs) and the human Panc2.03 tumor cell line were treated with 1 µM rapamycin (Rap) for 12 h, starvation (HBSS) for 3 h, or UV irradiation at 50 mJ/cm2 for 5 min before a 12-h recovery and then were immunostained with HMGB1-specific antibody (green) and Hoechst 33342 (blue). The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was determined by imaging cytometric analysis as described in Materials and methods. *, P < 0.05 and **, P < 0.005 versus untreated (UT) group; n = 3. Representative images are depicted (right).",
    "molecules": "Rap, rapamycin"
  },
  {
    "caption": "(B) Inhibition of autophagy blocks HMGB1 translocation. Cells were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5-specific shRNA for 48 h and were stimulated with starvation (HBSS) for 3 h and immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted (right). In parallel, the indicated cells were transfected with GFP-LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP-LC3 punctae. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus HBSS group; n = 3. Ctrl, control.",
    "molecules": "Ly294002, wortmannin"
  },
  {
    "caption": "(E) mETC inhibitors promote ROS production, autophagy, and HMGB1 translocation. Panc2.03 cells were stimulated with rotenone (Rot), thenoyltrifluoroacetone (TTFA), and antimycin A (AA) at indicated doses for 12 h, and ROS production was assessed by measuring the fluorescent intensity of CM-H2DCFDA in a fluorescent plate reader. In parallel experiments, cells were then immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus untreated group; n = 3.",
    "molecules": "thenoyltrifluoroacetone, TTFA, AA, antimycin A, ROS, Rot, rotenone"
  },
  {
    "caption": "(G) Antioxidant and SOD RNAi limit starvation-induced autophagy and HMGB1 translocation. Panc2.03 cells were pretreated with the antioxidant (NAC) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h. Then cells were starved (HBSS) for 3 h and analyzed by imaging cytometry to determine the mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus HBSS group; n = 3. A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here.",
    "molecules": "NAC"
  },
  {
    "caption": "(C) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (pepA) at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.05 versus Hmgb1+/+ group; n = 3. Actin was used as a loading control. AU, arbitrary units.",
    "molecules": "E64D, pepstatin A"
  },
  {
    "caption": "(E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. Representative images are depicted (left).",
    "molecules": "E64D, pepstatin A"
  },
  {
    "caption": "Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1−/− and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1−/− and Hmgb1+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.",
    "molecules": "cytochalasin B"
  },
  {
    "caption": "(A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP or Western blotting.",
    "molecules": "U0126, PD98059"
  },
  {
    "caption": "(B) Knockout of HMGB1 limits the disassociation of the Bcl-2-Beclin1 complex during treatment with autophagic stimuli. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or Western blotting.",
    "molecules": "U0126"
  },
  {
    "caption": "(A) The C106 mutation (C106S) of HMGB1 impairs its nuclear localization. Hmgb1−/− MEFs were transfected with wild-type and cysteine mutant HMGB1-GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved (HBSS) for 3 h. The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was analyzed by imaging cytometric analysis. *, P < 0.05 versus HMGB1+/+ group; n = 3. Representative images of HMGB1 location are shown on the left (green, HMGB1; blue, nucleus). The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C-terminal domain, with the cysteine mutation locations identified.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+ MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. LC3 punctae formation was assayed by imaging cytometric analysis (B)",
    "molecules": "ethyl pyruvate"
  },
  {
    "caption": "(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/−MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. Apoptosis was assayed by FACS (C) (C) as described in Materials and methods. *, P < 0.05 and **, P < 0.005; n = 3. UT, untreated. Non, nontransfection.",
    "molecules": "ethyl pyruvate"
  },
  {
    "caption": "(E) Reducing reagents disrupt the interaction between wild-type/C106 HMGB1 and Beclin1. As a control, before IP, samples were incubated with 50 mM DTT (+DTT) and assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of two independent experiments with similar results.",
    "molecules": "DTT"
  },
  {
    "caption": "(F) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol. Cells were stimulated with HBSS, rapamycin (Rap), rotenone (Rot), or thenoyltrifluoroacetone (TTFA) for 3 h or 12 h, and LC3 punctae formation was assayed as indicated. **, P < 0.005 versus Beclin1 shRNA group; n = 3. Ctrl, control. Data are means ± SEM.",
    "molecules": "thenoyltrifluoroacetone, TTFA, Rot, rotenone, Rap, rapamycin"
  },
  {
    "caption": "Expression of HMGB1 or the C106S mutant, but not C23S and C45S mutants, restore autophagic flux in Hmgb1−/− MEFs. (A) HMGB1−/− MEFs were transfected with wild-type (WT) or the cysteine mutant HMGB1-GFP plasmids as indicated, starved (HBSS) for 3 h in the presence or absence of 100 nM bafilomycin A1, and then immunostained with LAMP2-specific antibody/Alexa Fluor 594 secondary antibody (shown in red), LC3-specific antibody/Alexa Fluor 647 secondary antibody (shown in green), and Hoechst 33342 (shown in blue). Images were acquired digitally from a randomly selected pool of 10-15 fields under each condition. (B) Quantitative analysis of the percentage of LAMP2/LC3 colocalization was detected by Image-Pro Plus 5.1 software. Data are means ± SEM.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT-qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.",
    "molecules": "E2"
  },
  {
    "caption": "Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs",
    "molecules": "4-OHT, doxycycline, Shield-1"
  },
  {
    "caption": "ChIP for telomeric DNA associated with RAD51. DNA dot blot probed with DIG labeled telomere probe or Alu probe. HuO9 cells were transfected with 20nM siRAD54 #2, or 15µg myc-TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody (Mock, siRAD54 conditions) or myc antibody (myc-TRF2 positive control). Quantification of (CCCTAA)4 dot blot performed in C. IP values were normalized to loading (IP/input) and then normalized to mock control. Values shown are mean±sd of n=3 biological replicates. Values were compared using two-tailed unpaired student's t-test.",
    "molecules": "DNA"
  },
  {
    "caption": "Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres. HuO9 or SaOS2 cells were transfected with 20nM siRAD54#2 (representative images for siRAD54#1 not shown) for 48 hours followed by a 1.5 hr pulse of EdU. Pan-nuclear EdU stain represents S-phase cells. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in A. HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. Cells with pan-nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S-phase cells. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.",
    "molecules": "EdU"
  },
  {
    "caption": "Representative DNA dot blot from C-circle assay on HuO9 cells, either mock transfected or transfected with 20nM siRAD54#2 for 48 hours. Quantification of C-circle assay in C. Signal was quantified with densitometry, background (- Φ29) was subtracted and signal was normalized to mock. N=5 biological replicates values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.",
    "molecules": "DNA"
  },
  {
    "caption": "Representative images of combined EdU staining (Click-it) and DNA FISH for telomeric DNA. HuO9 cells were forward transfected with 2µg empty vector (EV), RAD54-WT, RAD54-K189R, or S49E construct. After 24 hours, cells were transfected with 20 nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in G. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to EV-Mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=4 for EV, WT, S49E, n=3 for K189R. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Sidak's multiple comparison test.",
    "molecules": "EdU, DNA"
  },
  {
    "caption": "Quantification of telomere sister chromatid exchange events. Cells were transfected with 20nM of siRNA for 48 hours prior to the addition of BrdU/BrdC. Metaphases were imaged by confocal microscopy and each data point represents a metaphase. For each condition, there were 3 biological repeats with a total of at least 1300 chromosome ends quantified. Values shown are mean±sem. Conditions were analyzed using two-way ANOVA followed by Sidak's test.",
    "molecules": "BrdC, BrdU"
  },
  {
    "caption": "Quantification of data shown in G. Anaphases were scored from 4 independent experiments (mock), or 3 independent experiments (remaining conditions). Anaphases were considered positive for UFB if there was PICH staining between DAPI bodies. Single knockdown conditions had >50 anaphases scored. Combined knockdown conditions had >88 anaphases scored. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of data shown in G. Number of PICH stained UFBs counted per anaphase. Values shown are total counts over 3 independent experiments, shown mean±sem. Total number of anaphases per condition were as follows: Mock n=134, siBLM/siSLX4 n=90, siRAD54/siSLX4 n=96, siRAD54/siBLM/siSLX4 n=88. Conditions were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test.",
    "molecules": "DAPI"
  },
  {
    "caption": "Quantification of data shown in J. Anaphases were scored from 3 independent experiments. A total of 90 anaphases were scored per condition. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of micronuclei from HuO9 cells transfected with siRAD54#2 alone or siRAD54#2 cotransfected with siSLX4 for 48 hours. Micronuclei containing telomeric DNA were identified using DAPI and telomere FISH. Micronuclei were normalized to the number of nuclei counted. Values shown are mean±sd of n=3 biological replicates. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(A) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.",
    "molecules": "Baf A"
  },
  {
    "caption": "(A) HeLa cells stably expressing GFP‐GATE‐16 (left panel) or GFP‐LC3B (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE‐16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.",
    "molecules": "Baf A"
  },
  {
    "caption": "(A) Bulk protein degradation was tested in HeLa cells that were transfected with non‐targeting siRNA (control siRNA), a pool of GABARAP/GATE‐16 siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either αMEM (control) or EBSS (starvation) medium, in the presence or absence of 10 mM of 3‐MA, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(F) Representative circle charts of relative abundance of bacterial phyla, class, order and family (from central to peripheral), in gut microbiota of RD-fed mice with vehicle, HFD-fed mice with, HFD-fed mice with neomycin and RD-fed mice with neomycin and (G) relative proportion per group of different phyla; n= 7(RD), 8(HFD), 6(HFD+AB), 7(RD+AB); Bacteroidetes; CI 99.9%, Firmicutes; CI 99%, Proteobacteria; CI 99%, Actinobacteria and Spirochaetes.",
    "molecules": "neomycin"
  },
  {
    "caption": "(L) Representative confocal images of IBA-1-stained MPs on choroidal flat mounts from RD- and HFD-fed mice, treated with vehicle or with neomycin. Examples of labeled macrophages (white dots) are presented in side-panels. (M) Total number of MPs around laser impact area; n= 7(RD), 9(HFD), 4(HFD+AB), 7(RD+AB), with 23(RD), 29(HFD), 13(HFD+AB), 23(RD+AB) burns total; CI 99%.",
    "molecules": "neomycin"
  },
  {
    "caption": "(B) Activation of PRRs induced by stimulation with serum isolated from mice fed RD or HFD for 7 weeks and receiving vehicle or neomycin for 3 weeks; fold RD; n=3 for all groups; CI 99%.",
    "molecules": "neomycin"
  },
  {
    "caption": "Representative immunoblot  of HeLa  wildtype (WT) cells transfected with scrambled siRNA (control) or siRNA targeting TMBIM5  and treated with the indicated drugs for 16 h. Control (0.1% DMSO), actinomycin D  (1 µM), venetoclax  (1 µM), A-1155463 (1 µM), staurosporine  (1 µM), thapsigargin  (2 µM). n=3 independent experiments.",
    "molecules": "A-1155463, actinomycin D, DMSO, staurosporine, thapsigargin, venetoclax"
  },
  {
    "caption": "Representative immunoblot  of HeLa  WT and TMBIM5-/- cells transfected with scrambled siRNA (control) or siRNA targeting MCU  for 72 h. Where indicated samples were treated with staurosporine  (STS; 1 µM) for 16 h. (n=3 independent experiments).",
    "molecules": "staurosporine, STS"
  },
  {
    "caption": "Viability  of WT and TMBIM5-/- cells (depleted of MCU  when indicated) after incubation with staurosporine  (STS; 0.1 µM) for 16 h. Cell viability was assessed cytofluorimetrically  monitoring NucView488 and RedDot2 staining  and expressed as percentage of all cells. n=3 independent experiments; two-tailed t-test; mean ±SD. A p-value of <0.05 was considered statistically significant.",
    "molecules": "NucView488, RedDot2, staurosporine, STS"
  },
  {
    "caption": "WT and TMBIM5-/- HeLa  cells were transfected with the indicated scrambled siRNA (control) and siRNA targeting MCU  for 48 h and incubated with staurosporine  (0.1 µM) for 16 h in the presence of Z-VAD-FMK  (50 µM) and epoxomicin  (1 µM) to prevent apoptosis. Cells were subjected to immunofluorescence  analysis with antibodies directed against cytochrome c (green) and TOMM20  (magenta). *Cells with cytosolic cytochrome c. (n=3 independent experiments; number of cells > 150 each experiment).",
    "molecules": "epoxomicin, staurosporine, Z-VAD-FMK"
  },
  {
    "caption": "Calcium retention capacity (CRC) was assessed in isolated mitochondria  of HEK293T  WT cells transfected with the indicated siRNA. The experiment was performed in sucrose-based medium containing respiratory substrates and the membrane-impermeable Ca2+  sensor Ca2+  Green-5N. Ca2+ Green-5N fluorescence was monitored following repeated addition of Ca2+ pulses (10 µM). Data show representative traces (2 independent experiments run in triplicate each condition).",
    "molecules": "Ca2+  Green-5N, Ca2+ Green-5N, Ca2+, sucrose"
  },
  {
    "caption": "Fluorescence (F) F/F0 ratio quantifications, where F0 is the average of the first ten seconds and F is the fluorescence peak measured after thapsigargin  (2 µM) administration (left panel). The right shows mtRED-GECO  ratio traces of HeLa  cells transfected with scrambled siRNA (control) and siRNA targeting TMBIM5. Each measurement was performed in at least 10 cells from 6 different preparations. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "Mitochondrial membrane potential was monitored with TMRM   in WT and TMBIM5-/- HeLa  cells. Fluorescent intensity was calculated as ratio to the value upon CCCP  addition (15 µM). Values are expressed as mean ±SD relative to control. Each measurement was performed in triplicate from 4 different preparations.",
    "molecules": "CCCP, TMRM"
  },
  {
    "caption": "Liposome assay. Recombinant TMBIM5  was reconstituted in liposomes   containing 100 µM Fura-2. CaCl2  was added later at the concentration of 100 µM. Rmax was obtained at the end of the experiment after the addition of ionomycin  (10 µM). Data are expressed as mean of R/Rmax, where R is the peak after CaCl2  addition. Where indicated GdCl3   (100 µM) was added 10 min before the experiment. n=3 independent experiments, shown as black, white and grey dots. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant. Liposome assay (as in E). Slope was calculated in the 100 s after 100 µM CaCl2  addition as expressed as mean ±SD. n=3 independent experiments, shown as black, white and grey dots. Two-tailed t-test. A p-value of <0.05 was considered statistically significant.",
    "molecules": "CaCl2, Fura-2, GdCl3, ionomycin"
  },
  {
    "caption": "Representative immunoblot  of TMBIM5-/- HeLa  cells transfected with scrambled siRNA (control) or siRNA targeting AFG3L2  for 48 h. Samples were treated with the protein synthesis inhibitor cycloheximide  (CHX; 10 μg/ml) and collected at the indicated  time points (n=3 independent experiments).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Volcano plot of mitochondrial  protein (MitoCarta 3.0) changes in WT HeLa  cells treated with oligomycin  (10 μM for 16 h) when compared to untreated cells. Before MitoCarta 3.0-filtering, significantly altered proteins were identified using a two-sided t-test followed by permutation-based FDR estimation to 0.05 and are colored in blue (n=5 independent experiments).",
    "molecules": "oligomycin"
  },
  {
    "caption": "Representative immunoblot  of WT HeLa  cells treated with the indicated drugs for 16 h. Antimycin A (AA; 10 μM), piericidin A  (10 μM) and oligomycin  (10 μM).",
    "molecules": "AA, Antimycin A, oligomycin, piericidin A"
  },
  {
    "caption": "Representative immunoblot  of WT HeLa  cells transfected with scrambled siRNA (control) and siRNA targeting AFG3L2  for 48 h, treated when indicated with oligomycin  (10 μM) for 16 h. Quantification of TMBIM5  levels is shown on the right panel. TMBIM5  levels in untreated WT cells was set to 1 (n=3 independent experiments; mean ±SD).",
    "molecules": "oligomycin"
  },
  {
    "caption": "TMBIM5  abundance determined by mass spectrometry  in WT HeLa  cells treated with oligomycin  (10 µM; 16 h) and depleted of AFG3L2  when indicated. n=5 independent experiments. P-value was calculated using a two-sided unpaired t-test. The central band of each box is the median value, and the box defines the 25th (lower) and 75th (higher) quantile. The whiskers represent the minimum and maximum value in the data excluding outliers (greater than 1.5 * inter quantile range distance to median - no outliers found here).",
    "molecules": "oligomycin"
  },
  {
    "caption": "Zoom in to selected clusters of hierarchical clustering of Z-Score normalized log2 LFQ intensities. Proteins that were significantly (FDR<0.05) accumulated between HeLa  WT and TMBIM5-/- cells after oligomycin  treatment (10 µM) for 16 h were clustered using Euclidean distance and the complete method on protein features. The figure shows identified cluster C4 and C5 with gene names and MitoCarta 3.0 localization  annotations. The row dendrogram is omitted.",
    "molecules": "oligomycin"
  },
  {
    "caption": "C. HEK293 cells  transfected  with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control. D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0). E ",
    "molecules": "cycloheximide, FSK"
  },
  {
    "caption": "H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with  FSK as in C. I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0). ",
    "molecules": "FSK"
  },
  {
    "caption": "A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5. B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05. C ",
    "molecules": "DRAQ5"
  },
  {
    "caption": "C. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected  with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were  stained with anti-ARL13B and anti-flag antibodies. D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001. F ",
    "molecules": "DRAQ5"
  },
  {
    "caption": "F. HEK293 cells  transiently transfected  with flag-OFD1 or flag-S735A were serum  deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5. G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for  flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01. H ",
    "molecules": "DRAQ5, FSK"
  },
  {
    "caption": "B. Confocal images of cilia  of the  neural tube cells in the wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31  KD + hOFD1S735D and Ol-TBC1D31  KD + hOFD1S735D + hpraja2rm stained  with anti-acetylated α-tubulin antibody (green) and DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "C, D, Currents from IHCs of control (C, P9) and Kir2.1-OE (D, P10) pre-hearing mice. Currents were elicited by using depolarizing and hyperpolarizing voltage steps, with a nominal increment of 10 mV, from a holding potential of −84 mV. Test potentials are shown next to some of the traces. Note that the large inward rectifier Kir2.1 current is only present in the IHC of the Kir2.1-OE mouse (D). The outward current is primarily carried by a delayed rectifier current IK. IK1 identifies the small inwardly rectifying K+ current normally expressed in IHCs. E, Steady-state current-voltage curves obtained from IHCs of control (P9-P11) and Kir2.1-OE (P9-P11) mice.",
    "molecules": "K+"
  },
  {
    "caption": "F, G, Representative ∆F/F0 traces from the IHCs and GER of P7-P9 control (F) and Kir2.1-OE (G) mice in the presence of 0.3 mM Ca2+. Spontaneous ATP-dependent Ca2+ waves from the GER (green traces) were eliciting coordinated Ca2+ signals in the IHCs from both controls and Kir2.1-OE mice. For each genotype, two separate sets of recordings from 2 mice are shown (top and bottom right), with the top traces being linked to the images on the left: before [1], during [2] and after [3]) the generation of a large Ca2+ wave from the GER. For details about the frequency and duration of the Ca2+ waves, and the number of mice and recordings see Fig EV3. All recordings were obtained at body temperature. Traces are computed as pixel averages of regions of interest centred on IHCs.",
    "molecules": "ATP, Ca2+"
  },
  {
    "caption": "J, Example of FM1-43 uptake by IHCs from P11 control (top) and Kir2.1-OE (bottom) mice, showing the lack of fluorescence labelling in the latter, which is an indication of the lack of MET channels open at rest at this stage in the IHCs overexpressing the Kir2.1 channels. At least 3 mice for each genotype were used.",
    "molecules": "FM1-43"
  },
  {
    "caption": "A,B, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P6 and P11 control (A) and Kir2.1-OE (B) mice immunostained with antibodies against MYOSIN VI (blue) and EPS8 (magenta). At least 3 mice for each genotype were used. In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.",
    "molecules": "phalloidin"
  },
  {
    "caption": "C,D, Confocal images of the hair bundles of P11 IHCs from control (C) and Kir2.1-OE (D) mice immunostained with antibodies against MYOSIN XV isoform 1 (blue) and WHIRLIN (magenta). In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.",
    "molecules": "phalloidin"
  },
  {
    "caption": "A,B, Maximum intensity projections of confocal z-stacks showing the IHCs of the apical cochlear region from control (A) and Kir2.1-OE (B) pups with the females being in the continuous presence of DOX in the drinking water from conception up to when the pups were P5 (upper panels). Middle and bottom panels show IHCs at P7 and P14 following the removal of DOX at P5 for both control (A) and Kir2.1-OE mice (B). IHCs were stained with antibodies against the K+ channel Kir2.1 (green) and Myosin 7a (Myo7a, blue: cell marker). Note that after 2 days following the removal of DOX, Kir2.1overexpression was already largely downregulated. At least 3 mice for each genotype were used.",
    "molecules": "DOX, water"
  },
  {
    "caption": "H,I, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P14 control (H) and Kir2.1-OE (I) mice stained with phalloidin. DOX was removed from the mother's drinking water when the pups were P5. At least 3 mice for each genotype were used.",
    "molecules": "DOX, phalloidin, water"
  },
  {
    "caption": "J,K, SEM images showing the normal structure of the hair bundles of the IHCs in the apical coil of the cochlea of P14 control (J) and P14 Kir2.1-OE (K) mice. DOX was removed from the mother's drinking water when the pups were P5. Note that the morphological profile of the hair bundles in IHCs is comparable between control and Kir2.1-OE mice, indicating that the removal of the intrinsic Ca2+ action potentials prior to the second postnatal week has no effect on the mechanoelectrical transduction apparatus. At least 3 mice for each genotype were used.",
    "molecules": "Ca2+, DOX, water"
  },
  {
    "caption": "Representative pictures of mitotic NSC marked by P-Vimentin and PH3 revealed by DAPI at the apical surface of the E14.5 mouse cortex after IUE. A representative vertically dividing cell was shown in upper panel, and a representative horizontally dividing cell was shown in lower panel. White dash line indicates the apical surface and splitting plane. Scale bar: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Primary skin fibroblasts from a normal individual, SLOS patient, ZS patients, X-ALD patient, and NPC patient incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 5 μm. The intensity of Filipin III signal at primary cilia from (a) was remarkably reduced in SLOS and ZS patient cells (**p<0.01: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "Cholesterol, Filipin III"
  },
  {
    "caption": "Primary fibroblasts from a normal individual, and SLOS and ZS patients were treated with 1.5% methyl-β-cyclodextrin for 45 min to remove cellular cholesterol, and then incubated with or without cholesterol (cholesterol/methyl-β-cyclodextrin complex) for 1 h. After washing out the exogenous cholesterol, the fibroblasts were stimulated with 50 nM Shh-N for 24 h in the presence of pravastatin, and then immunostained with anti-Smo (green), anti-acetylated-tubulin (blue), and anti-pericentrin (red) antibodies. For the alternative cholesterol complementation, LDL (0.06 mg/ml) was co-incubated with Shh-N and pravastatin for 24 h after methyl-β-cyclodextrin-mediated cholesterol depletion. Scale bar, 2.5 μm. Quantification of the Smo intensity at primary cilia in the fibroblasts from (F). The ciliary accumulation of Smo upon the Shh-N stimulation of fibroblasts from an SLO patient was restored by the complementation of both exogenous cholesterol/methyl-β-cyclodextrin complex and LDL, while that of cells from ZS patients was efficiently rescued by not LDL but cholesterol/methyl-β-cyclodextrin complex (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 90-100 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "cholesterol, LDL, methyl-β-cyclodextrin, pravastatin"
  },
  {
    "caption": "Quiescent G0-phase wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells transfected with AcGFP1-tagged D4 as a cholesterol probe were immunostained with anti-pericentrin (white) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (red). Arrows and arrowheads indicate primary cilia and cytosolic accumulations of AcGFP1-tagged D4, respectively. Scale bar, 5 μm. Quantification of the Filipin III intensity at primary cilia from (E). PEX1−/− and PEX14−/− hTERT-RPE1 cells had significant reductions in the ciliary signal of Filipin III (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively. Quantification of the AcGFP1-tagged D4 intensity at primary cilia from (E). The ciliary signal of AcGFP1-tagged D4 in PEX1−/− and PEX14−/− hTERT-RPE1 cells was significantly diminished (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 25-30 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "cholesterol, Cholesterol, Filipin III"
  },
  {
    "caption": "Quiescent G0-phase wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells were treated with or without 1.5% methyl-β-cyclodextrin for 45 min, and then incubated with or without cholesterol (cholesterol/methyl-β-cyclodextrin complex) for 1 h. After removing exogenous cholesterol, they were stimulated with 50 nM Shh-N for 24 h in the presence of pravastatin, and then immunostained with anti-Smo (green), anti-acetylated-tubulin (blue), and anti-γ-tubulin (red) antibodies. For the alternative cholesterol complementation, LDL (0.06 mg/ml) was co-incubated with Shh-N and pravastatin for 24 h after methyl-β-cyclodextrin-mediated cholesterol depletion. Scale bar, 2.5 μm. Quantification of the Smo intensity at primary cilia in wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells from (H). PEX1−/− and PEX14−/− hTERT-RPE1 cells exhibited the dampened Shh-N ligand-induced ciliary accumulation of Smo. The complementation of exogenous cholesterol (cholesterol/methyl-β-cyclodextrin complex) restored the ciliary accumulation of Smo in both PEX-knockout cells, while the LDL complementation did not rescue the ciliary phenotypes efficiently (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 90-100 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "cholesterol, LDL, methyl-β-cyclodextrin, pravastatin"
  },
  {
    "caption": "Quiescent G0-phase hTERT-RPE1 cells were immunostained with anti-PEX14 (red), anti-ninein (white), and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (Green). Magnified images of the boxed regions showing peroxisome accompanied by cholesterol (arrows). Three-dimensional reconstitution of the same cell indicates that Filipin III stains the membrane regions of ciliary axonemes and peroxisomes. The scale bars indicate 2.5 μm and 1.25 μm in lower- and higher-magnified images, respectively.",
    "molecules": "Cholesterol, cholesterol, Filipin III"
  },
  {
    "caption": "Quiescent G0-phase wild-type hTERT-RPE1 cells treated with Cytochalasin-D (200 nM), colcemid (50 nM), or Ciliobrevin-D (10 μM) for 6 h were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrows indicate the peroxisomes interacting with the ciliary pocket. Scale bar, 5 μm. Quantification of proportion of primary cilia interacting with peroxisomes from (B). Colcemid (50 nM) and Ciliobrevin-D (10 μM) significantly inhibited the spatial interaction between peroxisomes and primary cilia (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment).",
    "molecules": "colcemid, Colcemid, Ciliobrevin-D, Cytochalasin-D"
  },
  {
    "caption": "Quiescent G0-phase wild-type hTERT-RPE1 cells were treated with Cytochalasin-D (200 nM), Colcemid (50 nM), or Ciliobrevin-D (10 μM) for 6 h and then immunostained with anti-acetylated tubulin (blue) and anti-pericentrin (green) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 2.5 μm. Quantification of the Filipin III intensity at primary cilia from (D). Colcemid and Ciliobrevin-D interfered with the distribution of cholesterol in the ciliary membrane (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "Colcemid, Cholesterol, cholesterol, Ciliobrevin-D, Cytochalasin-D, Filipin III"
  },
  {
    "caption": "Recombinant GST (0.2 μg) or GST fused to PEX14 proteins (1 μg) and 6×His-EHD3 protein (1 μg) were pulled down using glutathione-Sepharose beads. GST-tagged PEX14 and 6×His-tagged EHD3 proteins in the pull-down fractions and inputs were detected by western blotting.",
    "molecules": "Sepharose, glutathione"
  },
  {
    "caption": "3D reconstitution of the quiescent G0-phase hTERT-RPE1 cells transfected with AcGFP1-tagged EHD3 (white) immunostained with anti-acetylated-tubulin (blue) and anti-PEX14 (red) antibodies. Cholesterol was stained with Filipin III (green). Cholesterol-containing peroxisome interacted with the ciliary pocket (arrow). The scale bars indicate 2 μm.",
    "molecules": "Cholesterol, Filipin III"
  },
  {
    "caption": "ORP3+/+ and ORP3−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 5 μm. Quantification of the Filipin III intensity at primary cilia from (C). ORP3−/− hTERT-RPE1 cells exhibited a significant reduction of ciliary cholesterol (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "Cholesterol, cholesterol, Filipin III"
  },
  {
    "caption": "ORP3−/− hTERT-RPE1 cells were transfected with AcGFP1-tagged ORP3, PH domain deleted-ORP3 mutant (ΔPH), FFAT motif-deleted-ORP3 mutant (ΔFFAT), or OHD domain deleted-ORP3 mutant (ΔOHD) and cultured without serum for 24 h before Filipin-III (green)-mediated cholesterol staining and immunostaining with anti-GFP (red), anti-pericentrin (white), and anti-acetylated-tubulin (blue) antibodies. Arrows represent AcGFP1-tagged ORP3 or the mutants localized to the ciliary pocket. AcGFP1-tagged ORP3ΔPH mutant mis-localized to the ciliary pocket. The scale bars indicate 5 μm. Quantification of the Filipin III intensity at primary cilia from (H). AcGFP1-tagged ORP3 deletion mutants did not restore the ciliary cholesterol insufficiency in the ORP3−/− hTERT-RPE1 cells (*p<0.05, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "cholesterol, Filipin III, Filipin-III"
  },
  {
    "caption": "Rabin8+/+ and Rabin8−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Scale bar, 5 μm. Quantification of the Filipin III intensity at primary cilia from (A). Rabin8−/− hTERT-RPE1 cells exhibited a significant reduction of ciliary cholesterol (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "Cholesterol, cholesterol, Filipin III"
  },
  {
    "caption": "Quiescent G0-phase hTERT-RPE1 cells transiently expressing PEX3-GFP-FRB (green) and tdTomato-BicD2-FKBP (red) treated with rapamycin were imaged live for 10 min by confocal microscopy (Movie EV10). Scale bar, 5 μm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "Quiescent G0-phase Rabin8+/+ and Rabin8−/− hTERT-RPE1 cells transfected with PEX3-GFP-FRB (red) and tdTomato-BicD2-FKBP (white) were treated with or without 100 μM rapamycin for 30 min, and then immunostained with anti-acetylated-tubulin (blue) antibody. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. Quantification of the Filipin III intensity at primary cilia from (G). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the reduction of ciliary cholesterol in the Rabin8−/− hTERT-RPE1 cells (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "Cholesterol, cholesterol, Filipin III, rapamycin, Rapamycin"
  },
  {
    "caption": "Rabin8−/− hTERT-RPE1 cells were transfected with AcGFP1-tagged Rabin8, GFP-tagged Rab8A, GFP-tagged Rab8A-Q67L (constitutively active form), GFP-Rab8B, GFP-Rab8B-Q67L (constitutively active form), AcGFP1-tagged Rab10, or AcGFP1-tagged Rab10-Q68L (constitutively active form), and then immunostained with anti-GFP (red), anti-acetylated-tubulin (blue), and anti-ninein (white) antibodies. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm. Quantification of (I) showing that AcGFP1-tagged Rabin8 and AcGFP1-tagged Rab10-Q68L effectively restored the defect in ciliary enrichment of cholesterol in Rabin8−/− hTERT-RPE1 cells (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "Cholesterol, cholesterol, Filipin III"
  },
  {
    "caption": "Western blot analysis of the ciliary cholesterol trafficking-associated components in Pex1+/+ and Pex1−/− hTERT-RPE1 cells. Total cell lysates were separated to crude peroxisomal (Crude Pex), lysosomal and mitochondrial (Lyso/Mito), and peroxisomal (PEX) fractions. CYPOR, a lysosomal and mitochondrial protein, served as a positive control for the Lyso/Mito fractionation. Total cell lysates were gradually injected at 20 µg and 5 µg into a gel for SDS-PAGE, while equal amounts (5 µg) of protein from each fraction were loaded. Rabin8, Rab10, and KIFC3 were concentrated in the PEX fraction in Pex1+/+ hTERT-RPE1 cells. CYPOR (cytochrome P450 reductase) is a mitochondrial protein.",
    "molecules": "cholesterol"
  },
  {
    "caption": "KIFC3+/+ and KIFC3−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Scale bar, 5 μm. Quantification of (G) indicating that KIFC3−/− hTERT-RPE1 cells significantly reduced the ciliary accumulation of cholesterol (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "Cholesterol, cholesterol, Filipin III"
  },
  {
    "caption": "KIFC3−/− hTERT-RPE1 cells were transfected with AcGFP1, AcGFP1-tagged KIFC3, rod domain-deleted KIFC3 mutant (ΔRod), or motor domain-deleted KIFC3 mutant (ΔMotor) and cultured without serum for 24 h before Filipin-III (green)-mediated cholesterol staining and immunostaining with anti-GFP (red), anti-pericentrin (white), and anti-acetylated-tubulin (blue) antibodies. Arrows represent ciliary localization of cholesterol. The scale bars indicate 5 μm. Quantification of the Filipin III intensity at primary cilia from (K). AcGFP1-tagged KIFC3 deletion mutants did not restore the ciliary cholesterol insufficiency in the KIFC3−/− hTERT-RPE1 cells (mean +/- s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "cholesterol, Filipin III, Filipin-III"
  },
  {
    "caption": "Quiescent G0-phase wild-type, Rab10−/−, KIFC3−/−, ORP3−/−, and EHD3−/− hTERT-RPE1 cells transfected with PEX3-GFP-FRB (red) and tdTomato-BicD2-FKBP (white) were treated with or without 100 μM rapamycin for 30 min, and then immunostained with anti-acetylated-tubulin (blue) antibody. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 μm.",
    "molecules": "Cholesterol, cholesterol, Filipin III, rapamycin"
  },
  {
    "caption": "Quantification of the Filipin III intensity at primary cilia from (A). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10−/−and KIFC3−/−hTERT-RPE1 cells, but not in ORP3−/−and EHD3−/− hTERT-RPE1 cells (*p<0.05, **p<0.01, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 35-40 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.",
    "molecules": "cholesterol, Filipin III, Rapamycin"
  },
  {
    "caption": "(A) PolyP accumulation in vivo. The polyP content of wild-type cells was set to 100%. Knockout of Vtc1, Vtc2, Vtc3 or Vtc4 impacts cellular polyP levels. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "PolyP, polyP"
  },
  {
    "caption": "PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and Vtc4/Vtc2/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro.",
    "molecules": "ATP, InsP6, PolyP"
  },
  {
    "caption": "PolyP synthesis by isolated vacuoles carrying VTC complexes in the absence or presence of 1 µM 5-InsP7 in vitro.",
    "molecules": "5-InsP7, PolyP"
  },
  {
    "caption": "Cellular polyP content of Vtc4p point mutants expressed under the control of their native promoters in the vtc4Δ background. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "polyP"
  },
  {
    "caption": "Cellular polyP content of VTC4 and VTC3 point mutants expressed under the control of their native promoters in the vtc4Δ and vtc3Δ(vtc2Δ) backgrounds, respectively. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "polyP"
  },
  {
    "caption": "Cellular polyP content of VTC4 point mutants expressed under the control of their native promoters in the vtc4Δ backgrounds. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "polyP"
  },
  {
    "caption": "PolyP synthesis by isolated vacuoles carrying Vtc4(ΔSPX)/Vtc3/Vtc1 complex, Vtc4/Vtc3(ΔSPX)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "5-InsP7, 1,5-InsP8, PolyP"
  },
  {
    "caption": "PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(R223E)/Vtc1 complex, Vtc4/Vtc3(R226E)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "5-InsP7, 1,5-InsP8, PolyP"
  },
  {
    "caption": "PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and truncated Vtc4/Vtc3(Δlp)/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3). (D) PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(CL2-Ala6)/Vtc1 complex, Vtc4/Vtc3(CL2-Asp6)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "5-InsP7, 1,5-InsP8, ATP, InsP6, PolyP"
  },
  {
    "caption": "Cellular polyP content of VTC4, VTC3 and VTC1 point mutants expressed under the control of their native promoters in the vtc4Δ, vtc3Δ(vtc2Δ) and vtc1Δ backgrounds, respectively. Δ indicates that the entire subunit was knocked out. FL indicates full length, indicating that the subunit has not been modified in any way. Data show the mean±s.d (biological replicates n=3).",
    "molecules": "polyP"
  },
  {
    "caption": "(C Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P8 (C) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.",
    "molecules": "Et-OH, Ser, rapamycin"
  },
  {
    "caption": "D) Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P34 (D) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.",
    "molecules": "Et-OH, Ser, rapamycin"
  },
  {
    "caption": "(A) Immunoblot analysis with anti-LC3 antibody shows that NO donors reduced LC3-II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1-treated rat primary cortical neurons and HeLa cells.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "C) Immunoblot analysis with anti-phospho-Bcl-2 antibody shows that DETA NONOate could not reduce phospho-Bcl-2 in Flag-CA JNK1-transfected HeLa cells, where phospho-Bcl-2 was higher compared to mock-transfected cells.",
    "molecules": "DETA NONOate"
  },
  {
    "caption": "(G) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that AAA Myc-Bcl-2 decreased Flag-Beclin 1-hVps34 interaction in the presence (right panel) or absence (left panel) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1, and with pcDNA3.1 or AAA Myc-Bcl-2. Graphical data denote mean ± SEM.",
    "molecules": "DETA NONOate"
  },
  {
    "caption": "(A) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Bcl-2 and AAA Bcl-2 MEFs.",
    "molecules": "DETA NONOate, bafilomycin A1"
  },
  {
    "caption": "(E) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Jnk, Jnk1−/−, and Jnk2−/− MEFs.",
    "molecules": "DETA NONOate, bafilomycin A1"
  },
  {
    "caption": "(G) Confocal microscope images of immunofluorescence with anti-phospho-S6 antibody show that DETA NONOate increased S6 phosphorylation; arrowhead shows a cell where this effect was not observed.",
    "molecules": "DETA NONOate"
  },
  {
    "caption": "(I) Confocal microscope images of immunofluorescence with anti-phospho-mTOR and anti-LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho-mTOR but did not alter its distribution with lysosomes. Graphical data denote mean ± SEM.",
    "molecules": "DETA NONOate"
  },
  {
    "caption": "(D) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated Tsc2+/+ and Tsc2−/− MEFs.",
    "molecules": "DETA NONOate, bafilomycin A1"
  },
  {
    "caption": "(E) DETA NONOate decreased EGFP-LC3 vesicles in Tsc2+/+ and Tsc2−/−MEFs transfected with EGFP-LC3, along with either pcDNA3.1 or AAA Myc-Bcl-2. Although it further reduced EGFP-LC3 vesicles in Tsc2+/+MEFs expressing AAA Myc-Bcl-2 compared to mock-transfected Tsc2+/+MEFs, this effect was not seen in Tsc2−/−MEFs. Tsc2+/+ and Tsc2−/−MEFs were analyzed separately. Graphical data denote mean ± SEM.",
    "molecules": "DETA NONOate"
  },
  {
    "caption": "(A-D) Immunoblot analyses with anti-LC3, along with anti-nNOS, anti-iNOS, and anti-eNOS antibodies, show decreased LC3-II levels in stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and D), or eNOS (C and D) compared to HEK293 control cells in the presence or absence of bafilomycin A1.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(E and F) mCherry-LC3 vesicles were reduced in mCherry-LC3-transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS, an effect that was restored in the NOS cells by L-NAME (F). Images were acquired by a confocal microscope (E).",
    "molecules": "L-NAME"
  },
  {
    "caption": "(A) Immunoblot analyses with anti-LC3 antibody show that rapamycin (8 hr) and L-NAME (24 hr) increased autophagosome synthesis in bafilomycin A1-treated mouse primary cortical neurons and HeLa cells.",
    "molecules": "bafilomycin A1, L-NAME, rapamycin"
  },
  {
    "caption": "(B and C) Confocal microscopy images (B) and analysis of autophagic flux by automated Cellomics microscope (C) show that L-NAME increased autolysosomes in mRFP-GFP-LC3 HeLa cells.",
    "molecules": "L-NAME"
  },
  {
    "caption": "(D) L-NAME reduced EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs.",
    "molecules": "L-NAME"
  },
  {
    "caption": "(E) Drosophila expressing mutant huntingtin exon 1 (Q120) shows a significant decrease in neurodegeneration (p < 0.001, paired t test) upon L-NAME treatment compared to DMSO.",
    "molecules": "DMSO, L-NAME"
  },
  {
    "caption": "(G-I) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates (arrows) (G). Treatment with rapamycin or L-NAME reduced the number of aggregates (H). L-NAME did not reduce aggregates in the presence of NH4Cl, which increased aggregate count (I).",
    "molecules": "NH4Cl, L-NAME, rapamycin"
  },
  {
    "caption": "(B) Detection of  S-acylation  of BSKs in plant cells. 12 BSK proteins were fused with GFP  at their C terminus and expressed in Arabidopsis protoplasts  for 24 hours and the cells were collected for Biotin-switch assay. Cell lysates are shown as input, and the  S-acylated proteins enriched on the resin which is dependent on hydroxylamine  (NH2OH) are indicated as pulldown  samples. All images of anti-GFP immunoblots  are representative of three biological independent experiments.",
    "molecules": "hydroxylamine, NH2OH"
  },
  {
    "caption": "(D) Effect of  2-BP on subcellular localization  of BSK1  in  root cells of intact transgenic plants. Six-day-old seedlings of 35S:BSK1-GFP  were treated with 80 μM 2-BP for 2 h; DMSO  was supplemented in the control samples. Representative GFP  (green) and merged (with bright field) signals from three biologically independent experiments are shown. Bars, 10 µm.",
    "molecules": "2-BP, DMSO"
  },
  {
    "caption": "(E) Effect of 2-BP on the localization  of BSK1-GFP  in transgenic plants using a  cell fractionation assay. Six-day-old seedlings  were treated with 80 μM  2-BP (DMSO  was used in the control sample) for 3 h before protein extraction. Total proteins (T) were divided into soluble  (S) and pellet  (P) fractions using ultra-centrifugation. The representative immunoblot  using an anti-GFP  antibody is shown.",
    "molecules": "2-BP, DMSO"
  },
  {
    "caption": "(E, F) The effect of S-acylation on the function of BSK3   in BR responses. The WT or 3M (C5, 10, 11S) version of BSK3  driven by a native promoter was introduced into the bsk3-1 mutant to generate complementation lines. The phenotype of 8-day-old seedlings  with and without 100 nM of BL  treatment is shown in (E). Bars, 1 cm. Quantitative data of hypocotyl  and root  length under the control or BL  treatment condition are shown in (F). Data are means ± SD from 20 seedlings  in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "BL"
  },
  {
    "caption": "(C) Detection of the regulation of BSK1 S-acylation mediated by SA. BSK1-GFP  was expressed in protoplasts  with 0-, 20-, 40-, or 60-min treatment with 100 µM of SA. Cells were collected for S-acylation  detection in a biotin-switch assay. Representative immunoblots  are shown in the top graph. Immunoblot  signals were quantified by ImageJ and the  S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the 0 min sample was set to 1. The data in the bottom graph are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "SA"
  },
  {
    "caption": "(D, E) Effect of SA  on the physiological function of BSK1. The indicated seeds  were germinated on the MS medium with or without 40 µM of SA. The photographs were taken 8 days after germination. Representative images are shown in (D). Bars, 1 cm. Quantitative data of fresh weight  (per 10 seedlings) of different genotypes are shown in (E). Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "SA"
  },
  {
    "caption": "(A, B) Expression of ABAPT11  during SA  treatment. (A) Seven-day-old wild-type (WT) seedlings  were treated with 100 µM SA  for 0, 10, 20, 30, 40, 50, and 60 min before collection for RNA preparation and RT-qPCR to detect the transcript level of ABAPT11. (B) Transcript levels of ABAPT11  in wild-type (WT) and npr1  mutant 7-day-old seedlings  with or without 100 µM SA  for 20 min. ACTIN2  was used as an internal control. Data are means ± SD from triplicated replications in an experiment; the pattern was consistent in three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "SA"
  },
  {
    "caption": "(C) Accumulation of ABAPT11  proteins in the SA  response. Seven-day-old seedlings  expressing ABAPT11-YFP  under its own promoter were treated with 100 µM SA  for 0, 20, 40, and 60 min before collection for immunoblotting  with an anti-GFP  antibody. Coomassie blue staining of total proteins was used as the loading control. Quantitative relative protein levels (YFP  / loading) from three biologically independent experiments are shown in the bottom graph. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "SA"
  },
  {
    "caption": "(D) The  S-acylation level of BSK1  in the ABAPT11  mutant during SA  treatment. BSK1-GFP  was expressed in WT or abapt11-1 mutant cells (with or without 100 µM SA  treatment for 40 min) for S-acylation  measurement in a biotin-switch assay. Representative immunoblots  are shown in the top graph. The immunoblotting  signals were quantified by ImageJ and the  S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the wild-type sample without SA  treatment was set to 1. Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "SA"
  },
  {
    "caption": "(G, Confirmation of the SA-induced BSK1  translocation which is mediated by ABAPT11. BSK1-GFP  was expressed in WT or abapt11-1 mutant cells, with or without 100 µM SA  treatment for 40 min. Total proteins (T) were separated into soluble  (S) and pellet  (P) fractions using ultra-centrifugation. Representative immunoblots  using an anti-GFP  antibody are shown in (G).",
    "molecules": "SA"
  },
  {
    "caption": "(A, B) Effect of BL  and SA on hypocotyl  development in ABAPT11  mutants. Seeds  were germinated on regular medium (Control), medium containing 80 nM BL, medium containing 40 µM SA, or medium containing both 80 nM BL  and 40 µM SA. Hypocotyl  length was measured 8 days after germination. Representative images from four independent experiments are shown in (A). Quantitative data of hypocotyl  lengths are shown in (B). Data are means ± SD from 20 seedlings  in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "BL, SA"
  },
  {
    "caption": "D) Effect of BL  and SA  on root  development of ABAPT11  mutants. Four days after germination, seedlings  of WT, abapt11-1, and abapt11-2 were transferred onto a regular medium (Control), medium containing 15 nM BL, medium containing 20 µM SA, or medium containing both 15 nM BL  and 20 µM SA. Quantitative data of root  elongation 5 days after transferring are shown in (D). Data are means ± SD from 20 seedlings  in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).",
    "molecules": "BL, SA"
  },
  {
    "caption": "Ttyh1-expressing cells in E15.5 embryonic brains that were intraventricularly electroporated at E13.5 were immunostained using an anti-GFP primary (reporter gene) and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. Quantification of (E)",
    "molecules": "Alexa Fluor 488, DAPI"
  },
  {
    "caption": "Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm.",
    "molecules": "Alexa Fluor 488, 555"
  },
  {
    "caption": "Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm. Quantification of (G) (n = 3).",
    "molecules": "Alexa Fluor 488, 555"
  },
  {
    "caption": "Immunolabeling of E14.5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1-resistant Ttyh1 (Ttyh1res) expression vector at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. The DAPI nuclear counterstain is shown in blue. Scale bar: 100 μm. Quantification of (I)",
    "molecules": "Alexa Fluor 488, DAPI"
  },
  {
    "caption": "The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. E Quantification of GFP+ cell positions in (D) (n = 3). ",
    "molecules": "DAPI"
  },
  {
    "caption": "Ttyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3).",
    "molecules": "DAPT"
  },
  {
    "caption": "Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibody",
    "molecules": "DAPT"
  },
  {
    "caption": "Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for qPCR for Notch target genes (I)",
    "molecules": "DAPT"
  },
  {
    "caption": "The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. Quantification of GFP+ cell localization in (J) dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunolabeling of E15.5 brain sections electroporated in utero with indicated plasmids at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). (D) Quantification of GFP+ cell localization in (C) (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.",
    "molecules": "Alexa Fluor 488, DAPI"
  },
  {
    "caption": "Cell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin (10 μM), were immunoblotted with an antibody to Rer1.",
    "molecules": "DMSO, lactacystin"
  },
  {
    "caption": "HEK293T cells were transfected with Ttyh1-Myc and Rer1-HA expression plasmids, and after 24 h, cells were treated with cycloheximide (CHX) (100 μg/ml) and then harvested at the indicated time intervals. Cell lysates were analyzed by Western blotting using anti-HA antibody. (J) Quantification of band intensities in (I) (n = 3).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13.5 brains and subsequent GFP immunofluorescence of E15.5 brain sections. GFP and DAPI signals are shown in green and blue, respectively. (M) Distribution of GFP+ cells in each brain layer in (L) was quantified (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "M-O: Schematic describing the measurement of a cell's approach angle to the nearest point on the AER (M). Merged, immunofluorescent images of WT (top), slit3sq49 (centre) and s1pr2te273 (below) mesenchymal cells in sqet37Et background stained for EGFP (green), Gamma tubulin (red) and DAPI (blue). White lines run from the centre of nucleus to the nearest point on the AER, through the MTOC (N). Graph depicting the approach angles to AER of leading (Tier 1), following (Tier 2) and trailing (Tier 3) cells of WT, slit3sq49 and s1pr2te273 embryos (O). A minimum of 30 cells were measured for each tier of each genotype. *p<0.05; **p<0.005; ***p<0.001; ANOVA with Fisher's post-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).",
    "molecules": "BrdU, Brdu"
  },
  {
    "caption": "Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.",
    "molecules": "BrdU"
  },
  {
    "caption": "Western blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samples",
    "molecules": "Ponceau red"
  },
  {
    "caption": "Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-miR-neg vector (CTRL) and GFP-miRNA-Cstb (KD), analyzed 3 dpe and immunostained with GFP. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-empty vector control and GFP-miRNA-Cstb, analyzed 3 dpe and immunostained with GFP and Ki67. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricular structures transfected with GFP-miR-neg vector or GFP-miRNA- Cstb in (C). Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-Cstb or R68X, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of GAD65-GFP interneurons/ cortical area (µm2) in (N). Data shown as Z-scores relative to the mean of GFP control vector. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Results of the ELISAs used to determine IFN-β (A) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.",
    "molecules": "PMA"
  },
  {
    "caption": "Results of the ELISAs used to determine IL6 (B) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.",
    "molecules": "PMA"
  },
  {
    "caption": "(G) Immunoblot showing the levels of the phosphorylated (p) and total IRF3, TBK1, and VSV-G proteins in PMA-differentiated THP-1 cells transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.",
    "molecules": "PMA"
  },
  {
    "caption": "(B) Y2H analysis in the AH109 yeast strain co-transformed with the indicated plasmids. A positive RNF34-MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan, leucine, adenine, and histidine containing X-gal. pGBKT7-TP53+pGADT7-T and pGBKT7-lam+pGADT7-T were used as positive and negative controls, respectively. AH109 co-transfected with pGBKT7-RNF34+pACT-2 was used to exclude the self-activation of RNF34.",
    "molecules": "X-gal, adenine, histidine, leucine, tryptophan"
  },
  {
    "caption": "(C) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-V. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody.",
    "molecules": "agarose"
  },
  {
    "caption": "(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (wild-type, WT; Lys-to-Arg mutants of all Ub lysines, KO; K27/29R). Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.",
    "molecules": "Arg, Lys, lysines"
  },
  {
    "caption": "(D) Immunoprecipitation analysis of K27 ubiquitination of MAVS in HEK293T cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.",
    "molecules": "Ub"
  },
  {
    "caption": "Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or Lys to Arg mutants together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.",
    "molecules": "Arg, Lys"
  },
  {
    "caption": "(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or K311R mutant together with HA-RNF34 and/or Myc-TRIM31 following a 3MA (0.2 mM) treatment. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Flag, anti-Ub-K63 or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. ell-based studies were performed independently at least three times with comparable results.",
    "molecules": "3MA"
  },
  {
    "caption": "(B) Immunoblot showing the degradation of MAVS in HEK293T cells infected with VSV (MOI = 1.0) for the indicated times in the presence or absence of NH4Cl (3 mM). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.",
    "molecules": "NH4Cl"
  },
  {
    "caption": "(F) SDD-AGE analysis of MAVS aggregates in HEK293T cells expressing Flag-MAVS together with WT Myc-RNF34 or its ligase-dead mutant and cultured in the presence or absence of MG132 (10 μM), 3-MA (1 mM) or NH4Cl (3 mM). SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.",
    "molecules": "MA, NH4Cl, MG132"
  },
  {
    "caption": "(A) Immunoprecipitation analysis of HEK293T cells transfected with Flag-SQSTM1 or its I431A mutant together with HA-MAVS. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.",
    "molecules": "agarose"
  },
  {
    "caption": "(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.",
    "molecules": "Z-VAD"
  },
  {
    "caption": "(E) (left) Western blot of NANOG, OCT4, and SOX2. (right) Relative NANOG  levels as estimated by densitometry  (n=8). NANOG  was nearly 7-fold more in PD, which is twice that of 2iL/SL2i. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).",
    "molecules": "PD"
  },
  {
    "caption": "(G)  Western blot of pERK and ERK in SLPD, SLCHIR, 2iL, and SL2i after 8, 12, 16, and 24 hrs of culture relative to SL, where the cells in SL were harvested 24 hrs after the media change (n>3).",
    "molecules": "CHIR, PD"
  },
  {
    "caption": "(H) (left) Western blot  of pERK and ERK in 1μM PD  and increasing concentrations of CHIR  in serum-free N2B27   media. (right) Relative pERK levels (n=3). (I) (left) Western blot  of pERK and ERK in 3μM CHIR  and increasing concentrations of PD  in serum-free N2B27  media. (right) Relative pERK levels (n=6). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in H, and I. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).",
    "molecules": "CHIR, PD"
  },
  {
    "caption": "(G) (left) Western blot of pERK in TNERT, TNERTFgfr1-/-, TNERTFgfr2-/-, TNERTFgf4-/-, and TNERTFgfbp1-/- with or no OHT. (Right) relative pERK levels estimated by densitometry  (n=4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in G, *P < 0.05, **P < 0.01, ***P < 0.001, and ns = not significant (paired two-tailed student's t-test).",
    "molecules": "OHT"
  },
  {
    "caption": "(E) Western blot of FGFR1, FGFR2, and pERK in TNERT  at one-hour intervals at indicated time points  after OHT  treatment (n>3).",
    "molecules": "OHT"
  },
  {
    "caption": "(C) Western blot of pERK and ERK in TNERT  and  TNERTNono-/- cells treated with or no OHT  (n=3).",
    "molecules": "OHT"
  },
  {
    "caption": "(D) Immunoprecipitation  analysis showing interactions between ERK and NONO  in the presence or absence of Nanog  induction by Doxycycline  in TDiN  cells.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "(E) (left) Western blot of pERK and ERK in E14Tg2a  cells treated with PD  or FGF4. (Middle and right) Relative levels of pERK and NONO  in E14Tg2a  cells treated with PD  or FGF4  (n>4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in E *P < 0.05, **P < 0.01, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).",
    "molecules": "PD"
  },
  {
    "caption": "(L) Cycloheximide  chase assay of NANOG  in SL, SLPD, and SLFGF4 in E14Tg2a  cells.",
    "molecules": "PD"
  },
  {
    "caption": "(B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654-828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm.",
    "molecules": "metformin, puromycin"
  },
  {
    "caption": "  293 cells stably expressing HA-ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti-HA or control anti-FLAG antibodies. The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies. ",
    "molecules": "AICAR"
  },
  {
    "caption": "(A) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm.",
    "molecules": "AICAR"
  },
  {
    "caption": "(B) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1.",
    "molecules": "AICAR"
  },
  {
    "caption": "A. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine. In this and subsequent similar figures, the number of mice is indicated.",
    "molecules": "iodine"
  },
  {
    "caption": "B. Averaged intracellular Ca2+ signals in cultured mouse DRG neurons in response to consecutive applications of 0.25 ppm iodine, 30 μM AITC and 2 μM capsaicin. All of the iodine responsive neurons (n = 332) from 6 adult mice were included in the analysis.",
    "molecules": "Ca2+, iodine, AITC, capsaicin"
  },
  {
    "caption": "C. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of HC030031 (HC) (C) or vehicle.",
    "molecules": "iodine, HC030031"
  },
  {
    "caption": "D. Quantification of the nociceptive responses in mice within 5 min after intraplantar injection of control saline or 500 ppm iodine, following the intraperitoneal injection of AMG517 (D) or vehicle.",
    "molecules": "iodine, AMG517"
  },
  {
    "caption": "E. Quantification of the nociceptive responses within 5 min after intraplantar injection of control saline in WT mice or 500 ppm iodine in WT and TRPA1-/-mice.",
    "molecules": "iodine"
  },
  {
    "caption": "F. Quantification of the nociceptive responses in TRPA1-/- mice within 5 min after intraplantar injection of control saline or 1,000 ppm iodine, following intraperitoneal injection of saline or vehicle.",
    "molecules": "iodine"
  },
  {
    "caption": "A. Left, time course of ear swelling elicited by topical application of 5% PVP-I solution in 0.15% Oxa-challenged WT and TRPA1-/-mice, or 5% povidone solution in Oxa-challenged WT mice. In this and subsequent similar figures, the number of mice is indicated. Right, bar graph highlighting the response at 24 h shown at left.",
    "molecules": "Oxa, povidone, PVP-I"
  },
  {
    "caption": "B. Time course of ear swelling elicited by topical application of the indicated concentrations of iodine in 0.15% Oxa-challenged mice, and a bar graph highlighting the response at 24 h.",
    "molecules": "iodine, Oxa"
  },
  {
    "caption": "C. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged WT and TRPA1-/-mice, and bar graph highlighting the response at 24 h.",
    "molecules": "iodine, Oxa"
  },
  {
    "caption": "D. Hematoxylin and eosin stained tissue sections of iodine treated ear in 0.15% Oxa-challenged WT and TRPA1-/-mice (n ≥ 3).",
    "molecules": "iodine, Oxa"
  },
  {
    "caption": "E. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged TRPA1-/-mice, following intraperitoneal injection of AMG517 or vehicle, and bar graph highlighting the response at 24 h.",
    "molecules": "iodine, Oxa, AMG517"
  },
  {
    "caption": "F. Time course of ear swelling elicited by topical application of 3% iodine in 0.15% Oxa-challenged mice, following intraperitoneal injection of RP67580 or vehicle, and bar graph highlighting the response at 24 h.",
    "molecules": "iodine, Oxa, RP67580"
  },
  {
    "caption": "A. Representative intracellular Ca2+ signals in hTRPA1-expressing HEK293 cells in response to different concentrations of iodine. 2-APB, a TRPA1 agonist, was subsequently applied to fully activate TRPA1. RFU: relative fluorescence unit.",
    "molecules": "Ca2+, 2-APB"
  },
  {
    "caption": "B. Concentration-response relationships of iodine-induced intracellular Ca2+ increase in HEK293 cells expressing WT or mutant TRPA1 channels. Data are presented as mean ± s.e.m. n ≥ 8 for each construct at each concentration. The smooth curves are fits to the Hill equation.",
    "molecules": "Ca2+, iodine"
  },
  {
    "caption": "C. Time course of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. HC: HC030031",
    "molecules": "iodine, HC030031"
  },
  {
    "caption": "D. Concentration-response relationship of iodine-induced currents in hTRPA1-expressing Xenopus oocytes. Data are presented as mean ± s.e.m. n ≥ 8 for each concentration. The smooth curve is a fit to the Hill equation.",
    "molecules": "iodine"
  },
  {
    "caption": "E. Time course of intracellular iodine-induced macroscopic currents in an inside-out patch from hTRPA1-expressing HEK293 cell (n = 3).",
    "molecules": "iodine"
  },
  {
    "caption": "F. Time course of iodine-induced currents in a hTRPA1-expressing Xenopus oocyte, which presents the current reduction upon DTT treatment (n = 3).",
    "molecules": "iodine, DTT"
  },
  {
    "caption": "I. Representative whole-cell currents in HEK293 cells expressing human TRPV1 in response to iodine and subsequently applied capsaicin (n = 3).",
    "molecules": "iodine, capsaicin"
  },
  {
    "caption": "Alkaline comet assay revealed that HO-1-/- LT-HSCs possess (L) higher oil tail moment and (M) more DNA in the comet tail. 1068-1189 cells analyzed from 6 mice/group.",
    "molecules": "DNA"
  },
  {
    "caption": "A: Brightfield and fluorescence images of a NTH/GFP zygote at 4h post-fertilization, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. B: Image of an NTH/GFP ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "C: Brightfield and fluorescence images of oocysts of parasite lines NTH/GFP and NTH-KO at 15 days post-infection, showing lack of sporulation in the NTH null mutant. Hoechst DNA staining (artificially depicted red) labels parasite nuclei. The large nuclei of the epithelial midgut cells also show. Scale bar = 20μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "F: Image of group of NTH/GFP sporozoites stained with MitoTracker Red CMXRos, showing distinct subcellular positions of apicoplasts (green) and mitochondria (red). Scale bar = 10μm.",
    "molecules": "MitoTracker Red CMXRos"
  },
  {
    "caption": "UBE2D3˜ubiquitin-release assays analysed by non-reducing SDS-PAGE and immunodetection. His-tagged UBE2D3 was charged with ubiquitin by incubation with ATP and UAE, and charging was stopped by ATP depletion with apyrase. No CUL3 (negative control, lane 1), CUL3WT or CUL3Δ403-459 was then added to the charged E2 and the activity of the RING domain, RBX1, monitored by the hydrolysis of ubiquitin from UBE2D3, visualised by immunodetection with anti-His antibody (upper panel, \"˜\" denotes thioester bond). The lower panel is an immunoblot for CUL3, showing equivalent amount of both CUL3WT and CUL3Δ403-459 was added to the reaction mix; over time, the appearance of higher molecular weight bands represents the modification of CUL3 by covalent attachment of ubiquitin. The right two lanes are control lanes incubated for 10 min with E2˜Ubi but then treated with reducing agent prior to SDS-PAGE.",
    "molecules": "ATP"
  },
  {
    "caption": "HEK-293 cells over-expressing either FLAG-CUL3WT or FLAG-CUL3Δ403-459 were immunoprecipitated with M2 Flag-binding sepharose, in the presence of a deneddylase inhibitor (OPT). Extract, cells were lysed in the presence of OPT and clarified by centrifugation. IP:M2, clarified supernatant samples were incubated for 1 h at 4°C with M2 (anti-Flag) sepharose, then washed with PBS and eluted with SDS-Laemmli buffer, prior to SDS-PAGE and immunodetection with anti-FLAG antibody. Bands are labelled as follows: 1. CUL3WT, 2. CUL3WT-N8, 3. CUL3Δ403-459, 4. CUL3Δ403-459-N8, 5. CUL3Δ403-459-2(N8).",
    "molecules": "OPT"
  },
  {
    "caption": "Neddylation in HEK-293 cells over-expressing FLAG-CUL3WT or FLAG-CUL3Δ403-459 was blocked by treatment with a Nedd8-E1-enzyme inhibitor, 3 μM MLN4924 (Millennium Pharmaceuticals), in cells over-expressing FLAG-CUL3WT and FLAG-CUL3Δ403-459 respectively. Cell extracts were sampled over time in the presence of OPT to prevent de-neddylation and subjected to SDS-PAGE and immunodetection of the FLAG tag.",
    "molecules": "OPT, MLN4924"
  },
  {
    "caption": "FLAG-CUL3WT or FLAG-CUL3Δ403-459 was immunoprecipitated with M2-sepharose as described in (F). +MLN4924 indicates cell culture media supplementation with 3 μM MLN4924 for 5 h prior to cell lysis to achieve complete deneddylation of CUL3. Following SDS-PAGE, immunoblotting with COP9 Signalosome (CSN) or CAND1 antibodies determined the interaction between these Cullin regulators and FLAG-CUL3WT and FLAG-CUL3Δ403-459, respectively.",
    "molecules": "MLN4924"
  },
  {
    "caption": "A-D In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. (A) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403-459, KLHL3 was included into the reaction at 2× molar concentration over CUL3WT and CUL3Δ403-459. (B, C) Reactions serve to determine basal auto-ubiquitylation of CUL3WT or CUL3Δ403-459 and do not contain KLHL3 or other potential substrate proteins. Lysine residues on CUL3WT or CUL3Δ403-459 act as the substrate. (B) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono-ubiquitylation of a number of CUL3 lysine residues. (C) Activity assays contain methylated ubiquitin, a form of ubiquitin incapable of forming ubiquitin chains; as such, higher band shifts reflect the attachment of mono-ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403-459, respectively. (D) Activity assay performed as in (C) with methyl-ubiquitin, the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in (F).",
    "molecules": "ATP, MgCl2, NaCl"
  },
  {
    "caption": "A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitatedWNK1 in (A). In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.",
    "molecules": "ATP, MgCl2, NaCl"
  },
  {
    "caption": "A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitated over-expressed FLAG-WNK4 in (B, C) (see Materials and Methods). The WNK kinases are modified by CUL3WT-KLHL3, with the higher molecular weight smear observed in anti-WNK1 and anti-FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein. CUL3Δ403-459 is unable to modify WNKs. Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction. (C) CUL3WT, CUL3Δ403-459 and an equimolar solution CUL3WT:CUL3Δ403-459 (1:1 Mix) were incubated with KLHL3 and immunoprecipitatedFLAG-WNK4 in ubiquitylation reactions to determine the influence of CUL3Δ403-459 on the ubiquitylation activity of CUL3WT. Notably, the presence of CUL3Δ403-459 does not inhibit WNK ubiquitylation by CUL3WT.In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.",
    "molecules": "ATP, MgCl2, NaCl"
  },
  {
    "caption": "Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3WT/Δ403-459 versus CUL3WT mice (*P = 0.0245). Two-tailed unpaired Student's t-test; data are mean ± SEM.",
    "molecules": "aldosterone"
  },
  {
    "caption": "Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTATbloodgas and electrolyte measurements. CUL3WT/Δ403-459mice present with abnormal electrolyte homoeostasis compared to CUL3WTmice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10−5) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10−5), base excess (BE) (***P = 9.1 × 10−5) and partial pressure of carbon dioxide (pCO2) (***P = 0.0038). Two-tailed unpaired Student's t-test; data are mean ± SEM.",
    "molecules": "electrolyte, carbon dioxide, CO2, bicarbonate"
  },
  {
    "caption": "In vivo dose-responses to phenylephrine and angiotensin II. After baseline measurements were obtained from mice in (A), the right external jugular vein was cannulated for administration of bolus doses of vasopressors in increasing half-log steps. Dose-response curves were generated and data analysed using a logistical function to determine the estimated dose producing half-maximal response (ED50) and maximum response (Emax). The fitted Emax for phenylephrine was increased in CUL3WT/Δ403-459 versus CUL3WT (183.9 ± 2.5 versus 164.9 ± 1.4 mmHg) (***P = 1 × 10−6) indicating an increased vasoconstrictor response to adrenergic stimulation elevating systolic blood pressure substantially above the CUL3WT maximum. Similarly, the fitted Emax for angiotensin II stimulation was higher in CUL3WT/Δ403-459 versus CUL3WT (155.5 ± 1.8 versus 138.3 ± 2.2 mmHg) (***P = 1.7 × 10−6). However, there was no change between CUL3WT/Δ403-459 versus CUL3WT sensitivity (as measured by ED50) to phenylephrine (36.6 ± 5.1 versus 31.4 ± 2.8 μg/kg bw) (P = 0.3778) or angiotensin II (0.77 ± 0.16 versus 0.93 ± 0.13 μg/kg bw) (P = 0.4401). Two-tailed unpaired Student's t-test; data are mean ± SEM.",
    "molecules": "phenylephrine"
  },
  {
    "caption": "B Correlation of serum SCT  levels and serum triglycerides  (TG), total cholesterol  (TC) and nonesterified fatty acids (NEFA) levels in pregnant women in the  middle trimester to the  early third trimester (n = 15). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001.",
    "molecules": "NEFA, nonesterified fatty acids, TC, total cholesterol, TG, triglycerides"
  },
  {
    "caption": "D Random blood glucose  levels in M-SCT  floxed and M-SCT  KO mice  with HFD  (n = 8 per group). E Insulin  levels in M-SCT  floxed and M-SCT  KO mice  with HFD  (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.",
    "molecules": "glucose, Insulin"
  },
  {
    "caption": "F Intraperitoneal glucose tolerance test  (GTT) (1.0 g glucose/ kg body weight) in M-SCT  floxed and M-SCT  KO mice  with HFD  (n = 5 per group) and AUCs were calculated. Mice  were fasted for 18h before assay. G Intraperitoneal insulin tolerance test (ITT) (0.75 U insulin/kg body weight) in M-SCT  floxed and M-SCT  KO mice  with HFD  (n = 5 per group) and AUCs were calculated. mice  were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "H Serum TG, TC  and NEFA  levels in M-SCT  floxed and M-SCT  KO mice  with HFD  (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ns, not statistically significant.",
    "molecules": "NEFA, TC, TG"
  },
  {
    "caption": "I, J OCR  of Oligomycin, FCCP  and Antimycin A/Rotenone-treated matured adipocytes  derived from iWAT-SVF  of M-SCT  KO and M-SCT floxed mice  with HFD, and the AUC of OCR  as indicated were calculated (n = 5 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.",
    "molecules": "Antimycin A, FCCP, Oligomycin, Rotenone"
  },
  {
    "caption": "K Glucose  oxidation in adipocytes  derived from iWAT-SVF  of M-SCT  KO and M-SCT  floxed mice  with HFD  (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.",
    "molecules": "Glucose"
  },
  {
    "caption": "E Western blot analysis of p-AMPKα, AMPKα, p-ACC, ACC, DNMT1  and HSP90  in MEF  cells treated with 10 nM SCT  or 20 μM compound C (C.C.) for 24 h.",
    "molecules": "C.C, compound C"
  },
  {
    "caption": "F Western blot  analysis of p-AMPKα, AMPKα, DNMT1, SCTR  and HSP90. MEF  cells were transfected with indicated siRNA and then treated with 1 mM AMPK  agonist (AICAR) for 24 h.",
    "molecules": "AICAR"
  },
  {
    "caption": "H, I Western blot analysis of Flag-DNMT1  in Flag-DNMT1-transfected HEK293T  cells treated with AICAR  with indicated dose or time  periods.",
    "molecules": "AICAR"
  },
  {
    "caption": "E GTT  (1 g glucose/kg body weight) in M-HFD  and M-HFD+SCT mice  with HFD  (n = 5 per group) and AUCs were calculated in right part. Mice  were fasted for 18h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.",
    "molecules": "glucose"
  },
  {
    "caption": "F ITT  (0.75 U insulin/kg body weight) in M-HFD  and M-HFD+SCT mice  with HFD, and AUCs were calculated in bottom (n = 5 per group). Mice  were fasted for 6h before assays. Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.",
    "molecules": "insulin"
  },
  {
    "caption": "G Serum TG, TC  and NEFA  levels in M-HFD  and M-HFD+SCT mice  with HFD. (n = 8 per group). Data information: Statistical significance was determined using Two-tailed Student's t test. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.",
    "molecules": "NEFA, TC, TG"
  },
  {
    "caption": "(D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm.",
    "molecules": "BafA1, bafilomycin"
  },
  {
    "caption": "(F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).",
    "molecules": "BafA1"
  },
  {
    "caption": "(I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.",
    "molecules": "DQ"
  },
  {
    "caption": "(B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3).",
    "molecules": "DMKG, ROS"
  },
  {
    "caption": "Representative confocal micrographs (scale bars are 20 µm) of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3).",
    "molecules": "DMKG"
  },
  {
    "caption": "(H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).",
    "molecules": "BafA1, DMKG"
  },
  {
    "caption": "(E), Proteomic analysis of CTNS-/- cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, GLUD1; GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation-independent mannose-6-phosphate receptor, GSTK1; Glutathione S-transferase kappa 1, COX6B1; Cytochrome c oxidase subunit 6B1, ACACA; Acetyl-CoA carboxylase 1-Biotin carboxylase, GLS; Glutaminase kidney isoform, mitochondrial, CASP3; Caspase-3, NPC1; Niemann-Pick C1 protein.",
    "molecules": "bicalutamide, cysteamine"
  },
  {
    "caption": "(D, E) Representative confocal micrographs and quantification of DQ-BSA in CTNSWT, and CTNS-/- cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8-14 quantified images).",
    "molecules": "bicalutamide, DQ"
  },
  {
    "caption": "(F) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNS-/- in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6). (G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNSPatient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).",
    "molecules": "bicalutamide, cysteamine, cystine"
  },
  {
    "caption": "(C, D) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in two different patient-derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)-bicalutamide (35 μM) combination treatment (n = 3). (E) αKG levels measured in patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine-bicalutamide combination treatment using metabolomics (n = 3). ",
    "molecules": "αKG, bicalutamide, cysteamine, cystine"
  },
  {
    "caption": "(I) Survival rates in ctns-/- zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).",
    "molecules": "bicalutamide, cysteamine"
  },
  {
    "caption": "(K) Hatching rates in surviving ctns-/- zebrafish evaluated at 72 and 96 hrs post fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).",
    "molecules": "bicalutamide, cysteamine"
  },
  {
    "caption": "(A-B) THP1 over-expressing IFITM3, IFITM3-Y20F, IFITM3-∆21 or control were pre-treated or not with Amphotericin B for 1 hour and then transduced with a VSV-gp (A) or Measles-gp (B) pseudotyped LV. Transduction efficiencies were evaluated at FACS five days later. Fold rescue of transduction over the DMSO control was calculated. Data are shown as mean ± SEM (n=5-8 biological replicates run in technical duplicate). P values are for one sample t test versus Oe-Luc DMSO =1, ** for p<0.01, *** for p<0.001.",
    "molecules": "Amphotericin B, DMSO"
  },
  {
    "caption": "(E-F) THP1 over-expressing IFITM3, IFITM3 lysin-less mutant (4KR) (E), IFITM3 CIL-lysin mutant (3KR) (F) or control were transduced with VSV-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=9-6 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01. **** for p<0.0001 and ns for not significant.",
    "molecules": "DMSO"
  },
  {
    "caption": "(G-H) THP1 over-expressing IFITM1, IFITM1-3KR (G), IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR (H) or control were transduced with Measles-gp pseudotyped LV. Transduction efficiencies were evaluated by flow cytometry five days post-TD. Data represent the mean ± SEM (n=4-9 biological replicates run in technical duplicate). p values are for one sample t test verso Oe-Luc DMSO= 1. * for p<0.05 ** for p<0.01.**** for p<0.0001.",
    "molecules": "DMSO"
  },
  {
    "caption": "K562 over-expressing IFITM3, IFITM3-Y20F, IFITM3-Y20F-3KR or control were pre-exposed or not to Ampho B and then transduced with Measles-gp (G), BaEV-gp (H) pseudotyped LV. Transduction efficiencies were measured by flow cytometry five days after TD (mean ± SEM, n=5-4-4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1 or unpaired t test for Ampho B versus DMSO. * for p<0.05, ** for p<0.01, *** for p<0.001, **** for p<0.0001.",
    "molecules": "Ampho B, DMSO"
  },
  {
    "caption": "(A) Interaction between IFITM3 wild-type or 3KR and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay. Images were acquired using TCS SP5 Leica confocal microscope 60x with oil in THP1 knock out for endogenous IFITM3 and expressing either IFITM3 wild-type or IFITM3-3KR. Number of foci were counted on imageJ and normalized over the number of nuclei (mean ± SEM, n=5 biological replicates). White arrows indicate foci, scale bar 40μm.",
    "molecules": "Phosphatydinositol-3 phosphate, PIP3"
  },
  {
    "caption": "(B) Phosphatydinositol-3 phosphate (PIP3) levels were measured by immunofluorescence in THP1 over-expressing the His-IFITM3/HA-IFITM3 and His-3KR/HA-3KR. PIP3 was quantified via Imagej (mean ± SEM, n=5 biological replicates).",
    "molecules": "Phosphatydinositol-3 phosphate, PIP3"
  },
  {
    "caption": "(C) THP1 over-expressing IFITM3 or control were transduced with VSV-gp pseudotyped LV in presence or absence of Rapamycin (Rapa). Transduction efficiency was measured at FACS five days post-TD (mean ± SEM, n=6 biological replicates run in technical duplicate. p values are for one sample t test versus Oe-Luc DMSO=1 and Mann Whitney test for Rapa vs DMSO. ** for p<0.01, **** for p<0.0001.",
    "molecules": "DMSO, Rapa, Rapamycin"
  },
  {
    "caption": "(D) THP1 over-expressing IFITM3-Y20F-3KR or control were exposed or not to Rapamycin and transduced with Measles-gp pseudotyped LV. Transduction efficiency was evaluated by flow cytometry five days post-TD (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for one sample t test versus Oe-Luc DMSO=1. ** for p<0.0.",
    "molecules": "DMSO, Rapamycin"
  },
  {
    "caption": "(E) Hematopoietic stem and progenitor cells (HSPC) were transduced with VSV-gp or Measles-gp pseudotyped LV in presence or absence of Rapamycin. Transduction was evaluated at FACS five days later measuring the % of BFP expression (mean ± SEM, n=4 biological replicates run in technical duplicate). p values are for Mann Whitney test. * for p<0.05.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "Levels of phosphatidylinositol (PI) precursors of PIP3 and PIP2 were measured through lipidomic analysis in HSPC and K562. The results are presented as log plots of the PI ratio in K562 over HSPC (D).",
    "molecules": "phosphatidylinositol, PI, PIP3, PIP2"
  },
  {
    "caption": "(E) PIP3 levels were measured by immunofluorescence (scale bar 40μm) and quantified as integrated density with ImageJ software in THP1 and K562 over-expressing IFITM3 (mean ± SEM, n=3 biological replicates). p values are for Mann Whitney test, * for p<0.05.",
    "molecules": "PIP3"
  },
  {
    "caption": "(F) Interaction between IFITM3 and Phosphatydinositol-3 phosphate (PIP3) (red dots) was evaluated by proximity ligation assay using TCS SP5 Leica confocal microscope 60x with oil in THP1 or K562 expressing same levels of IFITM3. Number of foci and PLA foci intensity were measured on imageJ and normalized over the number of nuclei (mean ± SEM, n=4 biological replicates run in technical duplicate), scale bar 40μm. p values are for Mann Whitney test, ** for p<0.01, *** for p<0.001.).",
    "molecules": "Phosphatydinositol-3 phosphate, PIP3"
  },
  {
    "caption": "PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in THP1 and K562 (G) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.",
    "molecules": "PIP3"
  },
  {
    "caption": "PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in HSPC (H) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.",
    "molecules": "PIP3"
  },
  {
    "caption": "PIP3 levels were measured by immunofluorescence and quantified as integrated density with ImageJ software in MDM (I) pre-stimulated or not with IFN-α (mean ± SEM, n=3 biological replicates. p values are for Mann Whitney test, *** for p<0.001.",
    "molecules": "PIP3"
  },
  {
    "caption": "(I) IFITM3 and PIP3 level were measured by immunofluorescence (scale bar 100µm) and quantified as integrated density with ImageJ software in Calu3 infected or not with Omicron (n=3 biological replicates). p values are for Mann Whitney test, ** for p<0.01. (J) PIP3 coupled to a specific PIP carrier (PIP3+Carr Ctrl) or Carr Ctrl were provided to Calu3 over-expressing IFITM3, control or KO for IFITM3 prior to infection with SARS-CoV2 Omicron. Infectious supernatants were collected after three days and tittered in Vero E6 cells (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Wilcoxon signed ranked test, * for p<0.05. The inhibitory effect of exogenous PIP3 on infection is represented as Fold versus Carr Ctrl (mean ± SEM, n=4 biological replicates run in technical duplicate ). p values are for Mann Whitney test, * for p<0.05.",
    "molecules": "PIP, PIP3"
  },
  {
    "caption": "D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells. E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. ",
    "molecules": "benomyl, latrunculin A"
  },
  {
    "caption": "A, Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.",
    "molecules": "Chloro, chloroform, CHX, Cycloheximide, VerruA, Verrucarin A"
  },
  {
    "caption": "FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.",
    "molecules": "Chloro, chloroform, CHX, VerruA"
  },
  {
    "caption": "C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given.",
    "molecules": "CHX"
  },
  {
    "caption": "B The effect of ∆scp160 on HXT2 mRNA localization is independent of its increased ploidy. Scp160 truncations that lack either the last two (∆C2) or the last four (∆C4) KH domains or a Tet-off SCP160 construct, in which expression is blocked by the addition of doxycycline (Doxy)",
    "molecules": "Doxy, doxycycline"
  },
  {
    "caption": "D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud. Treatment of cells with forskolin (+Forsko) or using a ∆bcy1 strain, causes HXT2 mRNA to be bud-enriched.",
    "molecules": "Forsko, forskolin"
  },
  {
    "caption": "A Table of the glucose affinity of the four most important hexose transporters. B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT. C Quantification of (B). ",
    "molecules": "glucose, hexose"
  },
  {
    "caption": "B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud. Cells were treated upon glucose addition with either 120 mg/ml benomyl for 15 min, 30 mg/ml latrunculin A for 30 min or 200 µM CK-666 for 30 min. HXT2 mRNA was detect by FISH.",
    "molecules": "benomyl, CK-666, glucose, latrunculin A"
  },
  {
    "caption": "A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted. B Quantification of (A). ",
    "molecules": "glucose"
  },
  {
    "caption": "C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope.",
    "molecules": "glucose"
  },
  {
    "caption": "(a) MEFs stably expressing EGFP-Parkin were exposed to CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. The asterisk indicates a nonspecific band.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) SH-SY5Y cells were exposed to CCCP (10 μM) for the indicated times and then subjected to immunoblot analysis.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) MEFs stably expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c and with a confocal microscope. The fluorescence of EGFP-Parkin was monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a,c) MEFs (a) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a,c) HeLa cells (c) stably expressing FLAG-Parkin and either EGFP-VAP-A or HA-VAP-A were treated with CCCP (30 and 10 μM, respectively) for the indicated times, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to HA or to FLAG (M2). The fluorescence of EGFP-VAP-A was also monitored directly. (b,d) Higher-magnification views of the boxed areas in a and c, respectively.",
    "molecules": "CCCP"
  },
  {
    "caption": "(e) NIH 3T3 cells stably expressing EGFP-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, after which whole-cell homogenates as well as cytosolic, ER and mitochondrial fractions were prepared and subjected to immunoblot analysis. Scale bar (a-d), 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) MEFs stably expressing EGFP-tagged wild-type (WT) or T240R mutant forms of Parkin as well as FLAG-tagged VAP-A were incubated in the absence or presence of CCCP (30 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP was also monitored directly.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) MEFs as in a were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) for 24 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38, to FLAG (M2) and to cytochrome c. The fluorescence of EGFP-Parkin was also monitored directly.",
    "molecules": "CCCP, lactacystin"
  },
  {
    "caption": "(d) MEFs stably expressing EGFP-Parkin were incubated for 12 h in the absence or presence of CCCP (30 μM) and lactacystin (10 μM) as indicated and then subjected to immunoblot analysis. Scale bar (a,c), 10 μm.",
    "molecules": "CCCP, lactacystin"
  },
  {
    "caption": "(b) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were treated with CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. Scale bar, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) MEFs stably expressing mCherry-tagged Parkin and EGFP-tagged forms of FKBP38, Omp25, Omp25/FKBP38 or FKBP38/Omp25 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) MEFs stably expressing FLAG-Parkin and either EGFP-Bcl-2 or EGFP-Bcl-xL were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) MEFs stably expressing FLAG-Parkin and EGFP-tagged forms of Bcl-2, Bcl-xL, Bcl-2/xL or Bcl-xL/2 were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP was also monitored directly. (d) Higher-magnification views of the boxed areas in c. Scale bar (c,d), 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) MEFs stably expressing EGFP-FKBP38(N402K) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-FKBP38(N402K) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) MEFs stably expressing EGFP-FKBP38(N402K), mCherry-FKBP38 and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FLAG (M2). The fluorescence of EGFP-FKBP38(N402K) and mCherry-FKBP38 was also monitored directly.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) MEFs stably expressing FLAG-Parkin and either EGFP-FKBP38 or EGFP-FKBP38(N402K) were incubated in the absence or presence of CCCP (30 μM) for 24 h and then subjected to immunoblot analysis. (d) Densitometric quantitation of EGFP fusion protein abundance in immunoblots similar to those in (c). Data are expressed relative to the corresponding control value (CCCP 0 h) and are means±s.d., from three independent experiments. **P0.01 (Student's t test).",
    "molecules": "CCCP"
  },
  {
    "caption": "(e) MEFs stably expressing EGFP-Bcl-2(H235R) and FLAG-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c and to FLAG (rabbit polyclonal). The fluorescence of EGFP-Bcl-2(H235R) was also monitored directly. Higher-magnification views of the boxed areas are shown in the right panels of each set.",
    "molecules": "CCCP"
  },
  {
    "caption": "(g) HeLa cells stably expressing FLAG-Parkin were transiently transfected with vectors for KikGR-FKBP38ΔN47 or KikGR-FKBP38ΔN47(N402K), exposed to UV, incubated in the absence or presence of CCCP (10 μM) for 4 h, and then monitored for red and green KikGR fluorescence. Scale bar (a,b,e,g), 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) Wild-type (Mfn2+/+) and Mfn2 knockout (Mfn2-/-) MEFs stably expressing mCherry-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c. The fluorescence of mCherry-Parkin was also monitored directly.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) MEFs as in a but expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis. The asterisk indicates a nonspecific band.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) Wild-type MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) or nocodazole (10 μM) for 6 h as indicated, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c, to FLAG and to FKBP38. The fluorescence of EGFP-Parkin was also monitored directly. (d) Higher-magnification views of the boxed areas in (c,e). Wild-type MEFs stably expressing FLAG-Parkin and EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 10 h and then subjected to immuno-electron microscopic analysis with antibodies to GFP. Black arrowheads indicate the mitochondria. White arrowheads indicate small vesicle.",
    "molecules": "CCCP, nocodazole"
  },
  {
    "caption": "(f) Wild-type (Fkbp38+/+) and Fkbp38 knockout (Fkbp38−/−) MEFs stably expressing FLAG-Parkin and either EGFP or EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then stained with allophycocyanin (APC)-labelled annexin V and propidium iodide (PI) for flow cytometric analysis of the percentage of apoptotic cells (annexin V+ and PI+ cells). Data are means±s.d., from three independent experiments.",
    "molecules": "CCCP"
  },
  {
    "caption": "Ultrastructural localization of PTPδ-tdTomato protein (red arrows; DAB staining) at axon terminals juxtaposed to electron-dense postsynaptic densities (purple arrows; PSDs) and also at vGlut2-positive excitatory synaptic axon terminals (green arrows; immunogold staining) but not at GAD67/65-positive inhibitory synaptic axon terminals (blue arrows; immunogold staining) in the stratum lacunosum-moleculare (SLM) region of the CA1 hippocampal region (P21). Note that ~85% of PTPδ-tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs, ~10% at presynaptic terminals not apposed to PSDs, and ~5% in neuropils other than presynaptic terminals. Axon terminals are indicated by pink shades. Scale bar, 500 nm. (n = 4 areas from 2 mice [WT and KO]).",
    "molecules": "DAB"
  },
  {
    "caption": "Strong PTPδ-tdTomato (red) signals in the SLM layer of the CA1 region and the MO layer of the DG in the hippocampus (2 months), revealed by quantitative analysis of PTPδ-tdTomato fluorescence across hippocampal lamina. DAPI was used for nuclear staining. CC, corpus callosum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare; MO, molecular layer; SG, granule cell layer; PO, polymorph layer. Scale bar 500 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A volcano plot of proteins that showed significant up- or downregulation of pTyr levels (absolute fold change > 1.5, p < 0.05) in the whole brain of Ptprd-/- mice (P21). Note that IL1RAPL1 is the protein with the strongest decrease in pTyr levels. (n = 3 for WT and 3 for KO). See Dataset EV1, 'Phosphoscan Results' tab for a full list of significantly changed proteins.",
    "molecules": "Tyr"
  },
  {
    "caption": "A volcano plot of presynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).",
    "molecules": "Tyr"
  },
  {
    "caption": "A volcano plot of postsynaptic proteins included in the SynGO list to show that they are significantly changed in pTyr levels (p < 0.05).",
    "molecules": "Tyr"
  },
  {
    "caption": "Decreased pTyr levels, but normal total levels of IL1RAPL1 protein, in the Ptprd-/- brain (P21-27), as revealed by immunoprecipitation of IL1RAPL1 from whole-brain lysates followed by immunoblotting for total IL1RAPL1 protein and pTyr levels (4G10 antibody).",
    "molecules": "Tyr"
  },
  {
    "caption": "Strongly decreased levels of tyrosine-phosphorylated PTPδ protein in whole-brain lysates of Ptprd-meA-/- mice (P21-27), as shown by the immunoprecipitation of IL1RAPL1 protein from whole-brain lysates followed by immunoblotting for total and tyrosine-phosphorylated PTPδ protein.",
    "molecules": "tyrosine"
  },
  {
    "caption": "(F) Immunoblot  analysis of  autophagic flux in mWAT  (upper panel) and gWAT  (lower panel) adipose tissue stimulated ex vivo with lysosomal  inhibitors 100nM Bafilomycin A1  and 20mM NH4Cl  for 4 hours or DMSO  (Vehicle) (n = 3-4/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.",
    "molecules": "NH4Cl, Bafilomycin A1, DMSO"
  },
  {
    "caption": "(G) Representative  transmission electron microscopy  images from mesenteric adipose tissue 7 days post DSS-induced colitis  induction. Lower panel is showing magnification of selected area. White arrows show autophagosomal  structures.",
    "molecules": "DSS"
  },
  {
    "caption": "(F) Colon  length after two weeks post-deletion (steady state; n = 14/group) and after DSS  at day 7 (n = 18-22/group). Data are represented as mean ± s.e.m. , F, Unpaired Student's t-test.",
    "molecules": "DSS"
  },
  {
    "caption": "(G) Representative H&E staining  images (10x magnification) of colon  sections and quantification of histological  score at steady state  (n = 9/group) and DSS colitis  (n = 18-22/group). Data are represented as mean ± s.e.m. G, Unpaired Student's t-test.",
    "molecules": "DSS"
  },
  {
    "caption": "(I) Absolute number CD45+ immune cells  from colons  at  steady state (n = 13-14/group) or at 7 days post-DSS  induction (n = 18-22/group). (J) Frequency of  myeloid cell  population in colon  at day 7 post-DSS  induction (n = 18-25/group). (K) Absolute number of Ly6C+ monocytes  discriminated by the absence or presence of MHCII  for infiltrating  and inflammatory monocytes respectively (n = 18-25/group). Data are represented as mean ± s.e.m. I) Unpaired Student's t-test. (J, K) Two-Way ANOVA.",
    "molecules": "DSS"
  },
  {
    "caption": "(A) Ex vivo lipolysis  measured by released  free fatty acid (left, n = 4-5/group) and glycerol  (right, n = 7-8/group) in culture supernatant of adipose tissue explants simulated with isoproterenol  (10µM) for 1-2h. (B) Ex vivo lipolysis  measured by released  free fatty acid  (left, n = 4/group) and glycerol  (right, n = 7/group) adipose tissue  explants simulated with TNFα  (100ng/mL) for 24h before replacing with fresh medium in the absence of TNFα  for 3h. Data are represented as mean ± s.e.m. (A, B, Two-Way ANOVA. (B, Unpaired Student's t-test.",
    "molecules": "free fatty acid, glycerol, isoproterenol"
  },
  {
    "caption": "(E) Concentration of individual FFA species in serum  in water-treated and DSS-treated mice  as measured by  FID-GC   (n = 12-14/group). Data are represented as mean ± s.e.m. E) Two-Way ANOVA.",
    "molecules": "DSS, water"
  },
  {
    "caption": "(C) Ex vivo lipolysis  assays on Atg7-deficient adipose tissue  explants simulated with isoproterenol  (10µM) for 1-2h (n = 5-6/group). Data are represented as mean ± s.e.m. (C, Two-Way ANOVA.",
    "molecules": "isoproterenol"
  },
  {
    "caption": "(H) Expression   of pro-inflammatory cytokines in colon  tissues at 7 days post-DSS  induction (n = 8/group). Dotted line represents non-inflamed controls. Data are represented as mean ± s.e.m. Unpaired Student's t-test.",
    "molecules": "DSS"
  },
  {
    "caption": "(E) Fold change expression of NRF2-target genes in primary visceral adipocytes  at day 7 after DSS  induction from normalized counts of RNAseq dataset (n = 6/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.",
    "molecules": "DSS"
  },
  {
    "caption": "(H) Representative immunoblot  of EPHX1  and EPHX2  in  gonadal adipose tissues  at day 7 after DSS  induction. Asterix indicating non-specific band (n = 14-18/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.",
    "molecules": "DSS"
  },
  {
    "caption": "(F) Serum  cytokines upon DSS-induced colitis  at day 7 post-induction (n = 17-23/group). Data are represented as mean ± s.e.m. Two-Way ANOVA.",
    "molecules": "DSS"
  },
  {
    "caption": "(h) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of  GPR15 in human, mouse, Japanese  quail, painted  turtle. YM.254890 inhibits  Ca2+  ion levels in cells transfected with GPR15 receptors of vertebrates. The fold-change in  calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).",
    "molecules": "Ca2+, calcium, YM.254890"
  },
  {
    "caption": "(i, j) The cellular levels of  Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in coelacanth, Asian bony tongue, and their mutants. (k, l) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulating  GPR15 in Japanese quail and its mutants. The fold-change in  calcium  levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).",
    "molecules": "Ca2+, calcium"
  },
  {
    "caption": "The expression levels of GPR15,  C10ORF99,  FOXP3 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food  chow and a positive control (propionate  added to  commercial food  chow) at different times (weanling  mice were defined as w-1) in colonic  tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three  mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).",
    "molecules": "propionate"
  },
  {
    "caption": "The expression levels of IL-10  in mice fed with corn, chicken, and mixture of corn plus  chicken,  commercial food chow and a positive control (propionate added to  commercial food chow) at different times  (weanling mice were defined as w-1) in  colonic tissue were determined by  q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three  mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).",
    "molecules": "propionate"
  },
  {
    "caption": "(f, g) SCFAs and  acetic acid content in dietary experiments. Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).",
    "molecules": "acetic acid, SCFAs"
  },
  {
    "caption": "Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IOSCA (A) Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acid",
    "molecules": "CDCA, chenodeoxycholic acid"
  },
  {
    "caption": "Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 CDCA, chenodeoxycholic acid",
    "molecules": "CDCA, chenodeoxycholic acid"
  },
  {
    "caption": "Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of PEO (C) Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; GABA, γ-aminobutyric acid; OH-Kyn, 3-hydroxy-DL-kynurenine",
    "molecules": "3-hydroxy-DL-kynurenine, OH-Kyn, CDCA, chenodeoxycholic acid, GABA, γ-aminobutyric acid"
  },
  {
    "caption": "A - D Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MELAS/MIDD (D). Data information: Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3 HIAA, 5-Hydroxyindole-3-acetic acid; SDMA, symmetric dimethylarginine; TCA, taurocholic acid.",
    "molecules": "SDMA, symmetric dimethylarginine, 5-Hydroxyindole-3-acetic acid, HIAA, taurocholic acid, TCA"
  },
  {
    "caption": "Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of IBM (A) Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid; OH-Kyn, 3-hydroxy-DL-kynurenine SDMA, symmetric dimethylarginine.",
    "molecules": "SDMA, symmetric dimethylarginine, 3-hydroxy-DL-kynurenine, OH-Kyn, CDCA, chenodeoxycholic acid"
  },
  {
    "caption": "Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of NMD patients (B) Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. C3, component 3; CDCA, chenodeoxycholic acid",
    "molecules": "CDCA, chenodeoxycholic acid"
  },
  {
    "caption": "Clustering of metabolome data in patients and controls; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites in blood of MIRAS carriers (C). Data information Colours in VIP score and volcano plots indicate the same most relevant and/or significantly changed metabolites among all patients groups. cAMP, cyclic AMP; C3, component 3; GABA, γ-aminobutyric acid HIAA, 5-Hydroxyindole-3-acetic acid; IMP, inosine monophosphate; OH-Kyn, 3-hydroxy-DL-kynurenine; OH-Trp, hydroxytryptophan; SDMA, symmetric dimethylarginine.",
    "molecules": "cAMP, cyclic AMP, SDMA, symmetric dimethylarginine, 5-Hydroxyindole-3-acetic acid, HIAA, 3-hydroxy-DL-kynurenine, OH-Kyn, GABA, γ-aminobutyric acid, hydroxytryptophan, OH-Trp, IMP, inosine monophosphate"
  },
  {
    "caption": "A Relative values of single metabolites and creatine/creatinine ratios in blood of primary MD, IBM and NMD patients, and MIRAS carriers compared to controls. Data information: All data represent mean ± SD. For individual metabolites: *P < 0.05, **P < 0.01, ***P < 0.001 (two sample T-test). For creatine/creatinine (cr/crn) ratio: *P = 0.022, **P = 0.005, ***P = 0.0001 (Kruskal-Wallis test with Dunn´s multiple comparisons test or Mann-Whitney test).",
    "molecules": "cr, creatine, creatinine, crn"
  },
  {
    "caption": "Metabolomes of muscle of MIRAS (A) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. AMP, adenosine monophosphate NAD, nicotinamide adenine dinucleotide TCA, taurocholic acid",
    "molecules": "adenosine monophosphate, AMP, NAD, nicotinamide adenine dinucleotide, taurocholic acid, TCA"
  },
  {
    "caption": "Metabolomes of muscle of PEO (B) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. car., carnitine NAD, nicotinamide adenine dinucleotide",
    "molecules": "car, carnitine, NAD, nicotinamide adenine dinucleotide"
  },
  {
    "caption": "D Relative values of metabolites in muscle of MIRAS, PEO and MELAS/MIDD patients compared to controls. All data represent mean ± SD. *P = 0.031, **P = 0.008 (two sample T-test). UDP, uridine diphosphate.",
    "molecules": "UDP, uridine diphosphate"
  },
  {
    "caption": "A Separate specificity and sensitivity (ROC) of sorbitol, alanine, myoinositol and cystathionine (left) and conventional blood biomarkers lactate and pyruvate, and cytokine FGF21 (right) in blood of MIRAS, PEO and MELAS/MIDD patients (n = 20) compared to controls (n = 30). Data information: ROC analysis: AUC of sorbitol 0.81 (95% CI 0.68 - 0.94, P = 0.0003), alanine 0.81 (95% CI 0.66 - 0.94, P = 0.0003), myoinositol 0.79 (95% CI 0.66 - 0.91, P = 0.0007) and cystathionine 0.78 (95% CI 0.65 - 0.91, P = 0.001). AUC of conventional biomarkers: FGF21 0.87 (95% CI 0.74 - 0.99, P = 0.0001), lactate 0.86 (95% CI 0.76 - 0.97, P = 0.0001) and pyruvate 0.78 (95% CI 0.64 - 0.93, P = 0.0017).",
    "molecules": "sorbitol, alanine, cystathionine, lactate, myoinositol, pyruvate"
  },
  {
    "caption": "B Combined 'multi-biomarker' value of sorbitol/alanine/myoinositol/cystathionine; ROC-analysis (left); mean centroids MD, IBM and NMD patients, and MIRAS carriers compared to controls (right). Data information: ROC analysis AUC of 'multi-biomarker' 0.94 (95% CI 0.89 - 0.995, P = 0.0001). Mean centroid data represent mean ± SD. **P < 0.01, ***P < 0.001 (1-way ANOVA with Dunnett´s multiple comparison test).",
    "molecules": "sorbitol, alanine, cystathionine, myoinositol"
  },
  {
    "caption": "A FACS analysis of the percentages of CD44+/CD24−/ESA+ breast CSC-like populations in CDK12-overexpressing ZR-75-30 and HCC-1419 cells and CDK12-siRNA-transfected BT474 and HCC-1954 cells. Top, representative dot plot of CD44-APC versus CD24-PE expression for viable cells (red) and ESA-positive cells (blue) from the viable cells. Bottom, the percentage of CD44+/CD24-/ESA+ cell population (blue dots at lower right quadrant in the plots) was quantified in the indicated cell lines. Mean ± s.d. of three independent samples. P-values were based on two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) and one-way ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). Amp, amplification; non-amp, non-amplification; Trz, trastuzumab.",
    "molecules": "Trz, trastuzumab"
  },
  {
    "caption": "B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines. Cells either treated with ALDH inhibitor DEAB (with DEAB; negative control) or untreated (without DEAB) were incubated with ALDEFLURO substrate (BAAA). In the FACS analysis, the percentage of ALDEFLUOR-positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB. Mean ± s.d. . P-values were calculated based on a two-tailed Student's t-test cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.",
    "molecules": "BAAA, DEAB, trastuzumab"
  },
  {
    "caption": "C The self-renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere-formation assay. The number of tumorsphere cells (> 100 μm in diameter) was counted after 3, 5, and 7 days. Data represent the mean ± s.d. (n = 3). P-value was calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "D Cell growth of the indicated stable cell lines treated with trastuzumab (Trz) or vehicle (veh) as measured by SRB assay. Trastuzumab-sensitive HER2+ breast cancer cell lines (S) and trastuzumab-insensitive HER2+ breast cancer cell lines (partial responsive, PR; resistant, R) were treated with 1 µg/mL or 100 µg/mL trastuzumab, respectively. Data represent the mean ± s.d. P-values were calculated using RM ANOVA with a post-hoc LSD test. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.",
    "molecules": "Trastuzumab, Trz, trastuzumab"
  },
  {
    "caption": "E Analysis of the effect of CDK12 deficiency on the in vivo trastuzumab response of HER2+ breast cancer. Mice were orthotopically xenografted with the indicated HER2+ breast cancer cells and treated with 20 mg/kg trastuzumab. The growth curve of each group was analysed twice weekly for 5 to 6 weeks (n = 8/group; mean ± s.e.m.). P-values were calculated using one-way ANOVA with a post-hoc LSD test for the last day of tumor measurement. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "E ChIP-qPCR analysis displaying the fold-enrichment of the indicated proteins and histone H3 acetylation at the TSS of CDK12-target genes in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "F Effect of CDK12 kinase inhibition on the expression of WNT1, WNT3, and IRS1. Cells were treated with different doses of dinaciclib for 12 h, and the expression of candidate targets of CDK12 were analysed by immunoblot and qRT-PCR Mean ± s.d. (n = 3). P-values by ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells ; R, trastuzumab-resistant; S, trastuzumab-sensitive.",
    "molecules": "dinaciclib, trastuzumab"
  },
  {
    "caption": "G Cell viability was measured by SRB assay in trastuzumab-sensitive BT474 cells and trastuzumab-resistant HCC-1954 cells treated with 1 µg/mL or 100 µg/mL trastuzumab (Trz) and/or 5 nM or 10 nM dinaciclib (Dina), respectively. Mean ± s.d. (n = 3). P-values, RM ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.",
    "molecules": "Trz, Dina, dinaciclib, trastuzumab"
  },
  {
    "caption": "H Tumor growth curves for mice xenografted with the indicated HER2+ breast cancer cells treated with 20 mg/kg trastuzumab and/or 20 mg/kg dinaciclib (n = 8/group; mean ± s.e.m.). P-values, one-way ANOVA with a post-hoc LSD (BT474) or Dunnett T3 (HCC-1954) test for the last day of tumor measurement. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.",
    "molecules": "dinaciclib, trastuzumab"
  },
  {
    "caption": "A Western blot and qRT-PCR results showing phosphorylation levels of GSK-3β and WNT1 and WNT3 expression, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive",
    "molecules": "trastuzumab"
  },
  {
    "caption": "Subcellular localization of β-catenin in the indicated stable cell lines were analysed by immunofluorescence staining (C). Nuclear translocation of β-catenin in control, CDK12-overexpressing, and CDK12-knockdown stable cell lines was quantified by counting the cells harboring exclusively nuclear β-catenin (N) from the immunofluorescence image. Mean ± s.d. (n = 3). P-value was calculated using a two-tailed Student's t-test. M+C, cells containing membrane and/or cytoplasmic β-catenin; N, cells dominantly expressing nuclear β-catenin. Green, β-catenin staining; Blue, DAPI staining for visualization of nucleus; Scale bar = 10 μ. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive.",
    "molecules": "DAPI, trastuzumab"
  },
  {
    "caption": "F Effect of WNT/ β-catenin/TCF pathway on the CDK12 regulation of breast CSCs. Cells were treated with DMSO (vehicle) or 10 nM FH535, a β-catenin/TCF inhibitor, for 24 h, and changes in the CD44+/CD24-/ESA+ cell population were measured using FACS analysis. Cells seen at lower right quadrant in the dot plots indicate CD44+/CD24- population (red) and CD44+/CD24-/ESA+ population (blue). Data represent the mean ± s.d. ofP-value was calculated using one-way ANOVA with a post-hoc LSD test.",
    "molecules": "DMSO, FH535"
  },
  {
    "caption": "A Human phospho-RTK array displaying the activation of phosphokinase-related proteins in CDK12-knockdown BT474 cells. The bar graph shows the fold change of spot intensity. Cell line information: a/a, CDK12-amplified/HER2-amplified cells S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "B Immunoblots showing the levels of the indicated proteins in different types of HER2+ breast cancer stably expressing CDK12 or with CDK12 knocked down. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "Co-IP analysis showing the amounts of bindings between ErbB receptors. Cell lysates from CDK12-overexpressing ZR-75-30 cells were immunoprecipitated with anti-HER2 antibody and immunoblotted using the indicated antibodies. IgG, negative control. Cell line information: ; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "D Western blot and qRT-PCR analysis of the phosphorylation levels and expression of IRS1, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "E Co-IP analysis indicating the amounts of IRS1 protein interaction with ErbB receptors and p85 in CDK12-overexpressing ZR-75-30 cells. Cell line information n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "F IRS1 effect on CDK12-induced phosphorylation of ErbB receptors. Lysates from cells treated with either control siRNA or IRS1 siRNA (siIRS1) for 24 h were subjected to immunoblotting with the indicated antibodies. Cell line information: n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "B-E. Left: At least 150 cells were counted per time point and the number of LD per cell was inspected visually and plotted. The median value of each timepoint is indicated by an orange bar and number. Each panel corresponds to a representative time-course experiment in response to each indicated DNA-damaging agent (out of at least 3 per agent). \"unt\" = untreated condition. \"mock\" refers to the culture being treated with DMSO, the dissolving agent for CPT. Used concentrations were 100 µg/mL zeocin, 100 µM CPT, 100 mM HU and 15 µg/mL nocodazole. Right: the same cultures were analysed by cytometry to assess DNA content (1C = unreplicated DNA, 2C = fully replicated DNA) during the time-course to confirm that the used agents were causing cell arrest at the expected phases of the cell cycle.",
    "molecules": "CPT, DNA, DMSO, HU, nocodazole, zeocin"
  },
  {
    "caption": "H-K. Cells as described in (G) in which the non-induced condition varied between glycerol (H) or raffinose (I-K), and the cells harboured either a plasmid driving 59 cuts in the genome (H-J) or none (K).",
    "molecules": "glycerol, raffinose"
  },
  {
    "caption": "B. Exponentially growing S. cerevisiae WT, ste∆ and tag∆ cells were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each point by dividing the raw signal of the upper band by the total signal in that lane, thus plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented by the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. C. Identical to (B) but comparing unloaded WT and WT pre-loaded for 2 h with 0.05% oleate in order to inhibit sterol esterification.",
    "molecules": "oleate, zeocin, sterol"
  },
  {
    "caption": "D. Non-confluent RPE-1 and Huh-7 human cell lines were either left untreated, or treated for 2 h with the DNA-damaging agents MMS (0.005%) or zeocin (10 µg/mL). Prior to that, cells were pre-loaded with oleate (4 h at 60 µM) to inhibit sterol esterification or treated with 30 µM BSA (mock) as a control. The downstream effector kinase of the DDR CHK2 was monitored by western blot for its activation using a specific antibody against its phosphorylation at Thr68 (P-CHK2). The western blot signal for total CHK2, calnexin and the Ponceau staining are used as loading controls. The bars in the graph show the mean value of the P-CHK2 to CHK2 signals ratio for genotoxins-treated cells while the dots correspond to the individual values out of 3 independent experiments. E. Identical to (D) though only in response to zeocin and by inhibiting sterols with the specific inhibitor avasimibe (5 µM for 2 hours). Plot details as in (D).",
    "molecules": "avasimibe, MMS, oleate, zeocin, sterol, sterols"
  },
  {
    "caption": "A. Exponentially growing S. cerevisiae WT and yeh2∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for Western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each timepoint and plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented as the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated.",
    "molecules": "zeocin"
  },
  {
    "caption": "C. Exponentially growing S. cerevisiae WT and ste∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for inspection by fluorescence microscopy. The establishment of DNA resection factories was assessed by counting the number of Rfa1-CFP foci per cell. Scale bar is 4 µm. Left: the mean values of each timepoint of three independent experiments were used to obtain this graph (mean and SEM). Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. Right: representative images of both strains at timepoint 120 min. Scale bar is 4 µm.",
    "molecules": "zeocin"
  },
  {
    "caption": "D. WT and yeh2∆ cells carrying a plasmid bearing the HO nuclease gene under a galactose-inducible promoter were exposed to 2% galactose to trigger the expression of the HO nuclease to cut at the MAT locus, and samples were retrieved at the indicated time points for genomic DNA extraction. Subsequent SspI  digestion, in combination with the use of a probe targeted to MAT, allows defining the fate of the cut fragment by Southern blot analysis. As resection progresses on DNA, SspI  restriction sites are lost, leading to progressively longer ssDNA fragments (whose sizes are indicated at the right of the gel) that can be separated on an agarose gel under denaturing conditions. Given the much stronger signal of the \"cut\" fragments with respect to the \"resected\" fragments, the gel has been split into low and high contrast halves, respectively. The ssDNA over cut yet unresected DSB molecules was calculated for each shown resection intermediate. The graph on the right plots the sum of all these values (cumulative ssDNA/DSB) for a given resected intermediate during the interval from 90 to 240 min. The error bar is the SEM of three independent experiments. * derives from applying a t-test that compares the two populations of values.",
    "molecules": "agarose, galactose, ssDNA"
  },
  {
    "caption": "E. Cells were pre-loaded with 60 µM oleate 30 µM BSA (mock) for 4 h, and then exposed to 0.005% MMS for the indicated times. Total genomic DNA was extracted (left panel) and 1500 ng of each condition were spotted onto a nylon membrane directly. The DNA, after being crosslinked to the membrane, was subjected to detection using an anti-ssDNA antibody (middle panel). The raw ssDNA signals obtained this way were quantified for three independent kinetics. The graph (right panel) shows the mean raw ssDNA signal value and the error bars are the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. As a control for the specificity of the anti-ssDNA antibody, we used the sample "MMS 3 h" for treatment with Mung Bean Nuclease (MBN), in order to digest single stranded DNA (ssDNA), or for denaturation, by addition of NaOH to a final 0.4 N concentration. These three samples are shown in the top part of the membrane.",
    "molecules": "MMS, oleate, single stranded DNA, ssDNA, NaOH"
  },
  {
    "caption": "C, D. Pulsed Field Gel Electrophoresis (PFGE) performed on DNA samples prepared from RPE-1 or Huh-7 cells that had been treated (or not) with zeocin and/or oleate (2 hours at 10 µg/mL zeocin; pre-treatments for 4 hours with 60 µM oleate) to evaluate the presence of DSBs under such treatments. Intact DNA molecules remain in the well, while broken molecules migrate into the gel. Given the action of zeocin, in-gel molecules can be subdivided in high-molecular weight fragments (indicated by \"HMW\") and low-molecular weight ones (LMW). Plugs were prepared at 37°C to prevent artefactual breaking of DNA molecules The bottom graphs provide the percentage of total broken molecules per lane with respect to all molecules in that lane. 3-4 independent experiments are plotted, and their median value indicated by an orange bar. The lateral graphs provide the relative intensity (arbitrary units) of signals associated with in-gel broken molecules. The values corresponding to zeocin-treated samples are displayed in pink, those corresponding to oleate + zeocin-treated samples in orange. For pink and orange curves, the mean value of 3-4 independent experiments is displayed in dark colour, while the SEM is indicated in light colours. Statistical analyses of the difference between treatments was assessed using a t-test.",
    "molecules": "DNA, oleate, zeocin"
  },
  {
    "caption": "A. Exponentially growing S. cerevisiae WT, ste∆, tag∆ and yeh2∆ cells were treated with 100 µg/mL zeocin for 90 minutes. This treatment duration recurrently results in more cells accumulating in the G1 phase of the cell cycle After 90 min, cells were washed to remove zeocin and resuspended in fresh, zeocin-free medium, and samples were retrieved every hour, as indicated. All samples were processed for western blot analysis and cytometry. The de-activation of the DDR was monitored following the progressive de-phosphorylation of Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. Top: samples were migrated using commercial 3-8% gradient gels. Bottom: samples were migrated in home-made resolutive gels (see M&M).",
    "molecules": "zeocin"
  },
  {
    "caption": "C. Left: Representative images obtained after immunofluorescence to detect P-ATM (ATM phosphorylation at Serine 1981) signal in RPE-1 and Huh-7 fixed cells that had been treated (or not) with 0.005% MMS or 10 µg/mL zeocin for 2 hours; pre-treatments were done for 4 hours with 60 µM oleate. Size bars are 10 µm. Right: Quantification of the percentage of P-ATM-positive cells. The values obtained from 3 to 4 independent experiments are plotted as black spots, and the mean of them is shown as an orange bar. At least 200 cells were counted per condition and experiment. The p-values obtained after performing an unpaired t-test are shown.",
    "molecules": "MMS, oleate, zeocin"
  },
  {
    "caption": "D. RPE-1 and Huh-7 cells were treated or not with MMS 0.005% for 2 hours. Thereafter, the ATM inhibitor AZD0156 was added at a final concentration of 1, 10 or 50 nM for 1 hour. Cells were collected and phosphorylation on Thr68 of CHK2 and tubulin levels were analyzed by western blot.",
    "molecules": "AZD0156, MMS"
  },
  {
    "caption": "B. A hydrophobic membrane on which 100 pmol of the indicated lipid species were spotted was incubated with immunopurified FLAG-Tel1 and further developed using an anti-FLAG antibody. The bar lengths in the graph represent the mean value of 4 independent experiments and the error bars, the associated SEM. To assess whether the mean values differed significantly, a t-test was performed and p-values are shown. Additionally, an identical lipid-containing membrane was incubated with 3xFLAG peptide and subsequently developed using an anti-FLAG antibody to control that the observed signals were not due to FLAG peptide binding alone. PI; phosphatidylinositol; PI(4)P, phosphatidylinositol-4-phosphate; PI(4,5)P, phosphatidylinositol-4,5-bisphosphate; PI(3,4,5)P, phosphatidylinositol-3,4,5-triphosphate.",
    "molecules": "phosphatidylinositol-3,4,5-triphosphate, PI(3,4,5)P, phosphatidylinositol-4,5-bisphosphate, PI(4,5)P, phosphatidylinositol, PI, phosphatidylinositol-4-phosphate, PI(4)P"
  },
  {
    "caption": "D. Proximity ligation assay (PLA) to assess whether ATM and PI(4)P are in close proximity (< 40 nm). Top: representative pictures of RPE-1 cells, in which PLA signals are pictured in green. Size bar is 10 µm. Bottom left: the graph shows the mean PLA signal area occupied per cell when doing the experiment with both antibodies (experiment) or by omitting one primary antibody at a time (technical controls). In practice, each point is the value of having measured all the PLA signals present in one photo (20 to 40 cells) and divided this value by the number of nuclei. To account for reproducibility, we used SuperPlots to draw the graphs each independent experiment is plotted in a different colour, where the mean value of each independent experiment is highlighted by a more intense colour than the individual values of that experiment, for which the colour is more translucid. Last, the solid horizontal line marks the mean of the means. A one-way ANOVA for multiple comparisons was applied to evaluate whether there were significant differences between the means. Bottom right: the graph shows the mean PLA signal area occupied per cell when doing the experiment either in cells transfected with an siRNA Control (experiment) or with three different siRNAs targeted against ATM (biological controls). The meaning of each plotted point, of the horizontal line and the statistical analysis is as explained above.",
    "molecules": "PI(4)P"
  },
  {
    "caption": "B. Left: RPE-1 cells untreated or exposed to 10 µg/mL zeocin for 2 h were used either for whole-cell protein extraction (W) or for fractionation into cytoplasmic (C) and nuclear (N) fractions in order to assess relative ATM distribution. Fractionation quality was assessed by checking for histone 3 (H3) (nucleus) and GOLPH3 (cytoplasm) signals. Right: To quantify the relative distribution of total ATM between nucleus and cytoplasm, total ATM signals were normalized to total Ponceau signals from the corresponding fraction. Each dot of a given color belongs to an independent experiment, the bar height shows the mean of those experiments, and the error bars represent the SEM. The p-value is derived from an unpaired t-test.",
    "molecules": "zeocin, Ponceau"
  },
  {
    "caption": "D. Exponentially growing S. cerevisiae WT cells transformed with an empty vector ("control") or with a plasmid overexpressing OSH4 were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for inspection by fluorescence microscopy to evaluate the percentage of cells displaying GFP-Tel1 foci. Top: illustrative images of GFP channel (nuclear Tel1 signals) in the indicated conditions. Note that we systematically monitor mCherry-Pus1 to ascertain whether signals are nuclear or not. Scale bar is 6 µm. Bottom: graph displaying the mean value of the percentage of cells forming GFP-Tel1 foci and the SEM of 3 independent experiments for the indicated conditions. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated.",
    "molecules": "zeocin"
  },
  {
    "caption": "A. Western blot showing P-ATM (ATM phosphorylation at Serine 1981) detection. Calnexin is used as a loading control The experiment assesses the ability of the ATM inhibitor AZD0156 (ATMi) or the OSBP1 inhibitor schweinfurthin G (SWG) to extinguish P-ATM signals triggered by the previous exposure to different genotoxins. Thus, RPE-1 cells were treated either with 0.005% MMS, or 10 µg/mL zeocin, or 120 ng/mL neocarzinostatin (NCZ), or 200 µM camptothecin for 2 h, then 500 nM AZD156 (ATMi) or 10 nM SWG were added in the sustained presence of the genotoxins for 2 additional hours. The images are illustrative of three to four independent experiments whose quantifications are shown in the bottom. Each point illustrates the observed raw P-ATM signal for each condition in each experiment, and similarly coloured dots belong to a same experiment. All the signals belonging to a same experiment were obtained from simultaneously run and transferred gels and simultaneously hybridized and exposed membranes. Thick dashed black lines represent the median of the experiments, and their specific value is shown as a red number on top of each violin plot, while thin dashed black lines indicate first and third quartiles.",
    "molecules": "AZD0156, AZD156, camptothecin, MMS, NCZ, neocarzinostatin, zeocin, schweinfurthin G, SWG"
  },
  {
    "caption": "B. Top: RPE-1 cells transfected after 3 days with siRNA control or against OSBP1 (3 different siOSBP1 sequences were assessed independently) were exposed to 0.005% MMS for the indicated times and the phosphorylation status of KAP-1 on Ser 824 was assessed by western blot. Total KAP-1 signals and the Ponceau staining are used as loading controls. Bottom: graph displaying the P-KAP-1 / KAP-1 ratio. Each plotted value is the mean out of three independent experiments, and the error bars account for the SEM. The displayed p-value highlights the significant difference of the concerned means after applying an unpaired t-test.",
    "molecules": "MMS"
  },
  {
    "caption": "D. RPE-1 cells were grown with 10 µM BrdU in the culture medium for the last 16 h prior to further processing. Cells were then either mock-treated, or pre-treated for 1 h with 500 nM of the ATM inhibitor (ATMi) AZD0156 or 10 nM of the specific OSBP1 inhibitor schweinfurthin G (SWG). Then, 200 µM CPT was added and cells were processed for BrdU detection at the indicated time points. Left: an illustrative image of BrdU-positive nuclei, indicated by yellow arrowheads. Scale bar is 15 µm. Right: the percentage of cells in the population whose nuclei were positive for BrdU was established by visual inspection of the acquired images. The graph shows the mean percentage out of three independent experiments and the error bars indicate their associated SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value for the one being significantly different is indicated.",
    "molecules": "BrdU, AZD0156, CPT, schweinfurthin G, SWG"
  },
  {
    "caption": "C, Microscale thermophoresis (MST) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711. D, dDATmfc did not show any interactions  with NO711  in the MST experiments. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.",
    "molecules": "NO711"
  },
  {
    "caption": "E, SKF89976a interacts  with purified ts5 dDATGAT construct with an affinity of 34 µM. F, MST  experiments display no interactions  of purified dDATmfc with SKF89976a. The MST  profiles shown are one of two independent measurements. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.",
    "molecules": "SKF89976a"
  },
  {
    "caption": "G, Michaelis-Menten uptake kinetics  of 3H-GABA by the GAT1WT used in the study displaying a KM  value of 11.4 μM.",
    "molecules": "GABA, 3H"
  },
  {
    "caption": "H, Inhibition of 3H-GABA uptake  by the substrate analogue nipecotic acid displaying a Ki  value of 14.4 μM. Data information: All measurements of inhibition potency  were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.",
    "molecules": "GABA, nipecotic acid, 3H"
  },
  {
    "caption": "I, Inhibition  of  3H-GABA uptake  through  GAT1WT by tiagabine  (Ki= 725 nM), NO711  (1.07 μM) and SKF89976a  (7.3 μM). Data information: All measurements of  inhibition potency  were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.",
    "molecules": "GABA, NO711, SKF89976a, tiagabine, 3H"
  },
  {
    "caption": "Changes to Michaelis-Menten kinetics  for L300F mutant that displays weakened 3H-GABA uptake   activity with KM  = 20.9 μM. A near 10-fold loss of  nipecotic acid inhibition potency  was observed at a value of 134.3 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows;  nipecotic acid,",
    "molecules": "GABA, nipecotic acid, 3H"
  },
  {
    "caption": "Changes to Michaelis-Menten kinetics  for L300F mutant that displays weakened 3H-GABA uptake  activity with KM  = 20.9 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of  inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows panel E-0.0001; tiagabine, panel F-0.0017; NO711, panel G-0.018; SKF89976a",
    "molecules": "GABA, NO711, SKF89976a, tiagabine, 3H"
  },
  {
    "caption": "E, F, Eadie-Hofstee plots of E, tiagabine  and F, NO711  display competitive mode of inhibition of 3H GABA uptake  in GAT1WT. G, Eadie-Hofstee plot of SKF89976a  displaying mixed (competitive+non-competitive) inhibition  of 3H-GABA uptake  in  GAT1WT. Data information: (E-G) Concentrations of the inhibitors were decided based on the Ki  values calculated for individual inhibitors. Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates. Error bars represent s.e.m.",
    "molecules": "GABA, NO711, SKF89976a, tiagabine, 3H"
  },
  {
    "caption": "D, 3H-GABA uptake inhibition  by the substrate analogue nipecotic acid for GAT1  S359E displaying a Ki  value of 30.1 μM Data information: The kinetic  plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.",
    "molecules": "GABA, nipecotic acid, 3H"
  },
  {
    "caption": "F) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.",
    "molecules": "PI"
  },
  {
    "caption": "G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition.",
    "molecules": "PI"
  },
  {
    "caption": "H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.",
    "molecules": "Ferrostatin-1, NSA, ZVAD"
  },
  {
    "caption": "A) Representative confocal immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm. B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition.",
    "molecules": "S63586, ABT737"
  },
  {
    "caption": "E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition.",
    "molecules": "TRAIL"
  },
  {
    "caption": "I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.",
    "molecules": "TRAIL"
  },
  {
    "caption": "D) Effect of tBID wt and α6-mutants overexpression on HCT AKO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. n=3 independent experiments, ** p<0,025 and * p<0,05 with respect to tBID-GFP wt condition. E) Effect of tBID wt and α6-mutants overexpression on MEF BID KO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification. n=3 independent experiments *** p<0,001 with respect to tBID-GFP wt condition.",
    "molecules": "DRAQ7"
  },
  {
    "caption": "A) representative western blot of cytosolic cyt c and SMAC in HeLa cells. B,C) Western Blot analysis of active BAX (B) and BAK (C), in HeLa cells treated with STS or infected with Shigella flexneri (MOI 30) at different time points after infection.",
    "molecules": "STS"
  },
  {
    "caption": "A) Caspase-3/7 activity induced by ABT199 and etoposide on Nalm6 (venetoclax-sensitive) and Nalm6 199R (venetoclax-resistant) cells, normalized to untreated cells. Unpaired Student's t-test *** p<0,001 and ** p<0,025 (Nalm6 with respect to Nalm6 199R at different conditions). Dots correspond to technical replicates of n=3 independent experiments.",
    "molecules": "etoposide, ABT199, venetoclax"
  },
  {
    "caption": "D) Representative kinetics of cell death, represented as DRAQ7+ cells in Nalm6 199R cells (grey), Nalm6 199R BID KO #1 cells (blue) and Nalm6 199R BID KO #5 cells (purple) upon 250 ng/ml TRAIL treatment. Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 5 technical replicates. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.E",
    "molecules": "DRAQ7, TRAIL"
  },
  {
    "caption": "H) Cell death induced by GFP-BAK and GFP-BAK R127H on HCT AKO, measured as DRAQ7+/GFP+ cells. Unpaired Student's t-test. *** p<0,001 with respect to GFP BAK wt.",
    "molecules": "DRAQ7"
  },
  {
    "caption": "I) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BAK KO and Nalm6 199R BOK KO, measured as DRAQ7+ cells with respect of total cell surface and normalized to untreated conditions. Each dot corresponds to the average normalized value of 3-4 technical replicates in n=3 independent experiments.",
    "molecules": "DRAQ7, TRAIL"
  },
  {
    "caption": "(B) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex facing (outer) side. Scale Bar = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.",
    "molecules": "cellulosic, Basic Fuchsin, Calcofluor White, Lignin"
  },
  {
    "caption": "(C,D) Localization of SGN1-Citrine and lignin deposition patterns in pCASP1::SGN1-Citrine lines in wild-type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm-SGN1-Citrine localization and lignin deposition patterns in pCASP1::myrpalm-SGN1-Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcoflour White) are shown in magenta and white respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle-facing (inner) side. Scale bars = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.",
    "molecules": "Basic Fuchsin, Calcoflour White, lignin, Lignin"
  },
  {
    "caption": "(B) Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition).",
    "molecules": "PI, Propidium iodide"
  },
  {
    "caption": "(C) [γ-32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase-dead SGN1 variant was used to avoid auto-phosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF-SGN1. Representative result of three independent experiments is shown.",
    "molecules": "ATP, γ-32P"
  },
  {
    "caption": "(B) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2-hour 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White) respectively. Representative pictures are shown, 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 µm (lignin overviews), 5 µm (median sections). "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.",
    "molecules": "Basic Fuchsin, Calcofluor White, Lignin, lignin"
  },
  {
    "caption": "(B,C) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours. Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in (sky blue and creme boxes in C and black boxes in D). Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment. Scale bars = 500 nm.",
    "molecules": "ROS"
  },
  {
    "caption": "(D) In situ H2O2 detection at Casparian strips in WT, sgn3, sgn1, rbohF, rbohD and rbohDF with or without 24-hour treatment of 1 µM CIF2. Brackets and arrowheads indicate Casparian strips (seen as uniformly whitish cell wall areas) and H2O2 production sites (black area) respectively. Scale bar = 1000 nm.",
    "molecules": "H2O2"
  },
  {
    "caption": "(E) Quantification of ROS production as a number of dark pixels area (n = 23 - 44 sites from 5 roots of each condition in one experiment.). In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters mean significant statistical differences. (p<0.01, One-way ANOVA and Tukey's test.)",
    "molecules": "ROS"
  },
  {
    "caption": "(A) [γ-32P]ATP radioactive in vitro kinase assay of TF-SGN1 against GST-N-terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results.",
    "molecules": "ATP, γ-32P"
  },
  {
    "caption": "(B) HEK293Tcell based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel, bars indicate S.D.. Experiments were repeated three times",
    "molecules": "Calyculin A"
  },
  {
    "caption": "(A) CASP1-GFP and lignin deposition in WT, sgn3, rbohD, rbohF and rbohDF. CASP1-GFP and lignin (fuchsin) are presented in green and magenta respectively. Pictures were obtained from more than 10 roots from each background with similar results. Scale bar = 10 µm.",
    "molecules": "fuchsin, lignin"
  },
  {
    "caption": "(B) Time lapse imaging of single- or cotreatment of 10 nM CIF2 with 25 µM cycloheximide (CHX) on CASP1-GFP in cif1 cif2. Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium. Scale bar = 10 µm (See also movie EV1). (C) Quantification of (B). Relative numbers of holes in CASP1-GFP domain after single- or co-treatment with CIF2 or CHX from the pictures in (B). Bars are S.D. * indicates statistical significance from all other conditions (p<0.01) after one-way ANOVA and Tukey test. 6 roots in total for each condition were observed during two independent tests. ",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "A Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls.",
    "molecules": "glucose, glycerol"
  },
  {
    "caption": "A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids.",
    "molecules": "amino acids, glucose"
  },
  {
    "caption": "B Plasmid‐encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co‐expressed with either Mpc2 or Mpc3. Cells were grown in selective medium containing glycerol as a carbon source. MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti‐HA or anti‐Flag antibodies. Mitochondrial enzymes aconitase (Aco1) and malate dehydrogenase (Mdh1) were used as loading controls.",
    "molecules": "glycerol"
  },
  {
    "caption": "D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated.",
    "molecules": "digitonin, glycerol"
  },
  {
    "caption": "E, F Stoichiometry of Mpc1 complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).",
    "molecules": "disuccimidyl glutarate, DSG"
  },
  {
    "caption": "A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B). Mitochondria were treated with IASD as indicated. Negative [quenching by pretreatment with DTT (pre‐DTT)] and positive [lysis with 0.5% Triton X‐100 (T‐X100)] controls for the labeling reaction are shown. As a further control, also translocase of the inner membrane subunit Tim23 is detected by Western blot, which contains three endogenous cysteines, all of which are located in membrane‐embedded regions. Labeling of an IMS‐exposed cysteine with IASD is indicated by a mobility shift on SDS-PAGE corresponding to the molecular weight of IASD (˜500 Da).",
    "molecules": "DTT, T‐X100, Triton X‐100"
  },
  {
    "caption": "C Protease protection assay in mitochondria expressing Mpc1‐GFP, Mpc2‐GFP, or Mpc3‐GFP. Intact mitochondria (M), mitoplasts with a ruptured outer membrane after hypo‐osmotic swelling (Sw), or mitochondrial lysates with 0.5% Triton X‐100 (Tr) were treated with proteinase K (PK). Loading controls are Tom70 (outer membrane), Tim23 (inner membrane), and Mdh1 (matrix). GFP‐fused Mpc2 and Mpc3 are degraded in mitoplasts, whereas Mpc1‐GFP is not.",
    "molecules": "Triton X‐100"
  },
  {
    "caption": "A MPCOX has higher transport activity than MPCFERM. The uptake of 14C‐labeled pyruvate into intact mitochondria was measured in vitro. Mitochondria had been isolated from cells grown in glycerol‐containing medium and expressing no subunit (vectors), MPCFERM, MPCOX, Mpc2, or Mpc3. Imported pyruvate was quantified by re‐isolation of mitochondria and subsequent scintillation counting after 1, 2, or 5 min of incubation with [14C]‐pyruvate.B Imported [14C]‐pyruvate as in (A) after 5‐min incubation. The difference between MPCOX and MPCFERM (**P = 0.0037) was significant (unpaired t‐test).",
    "molecules": "glycerol, pyruvate"
  },
  {
    "caption": "Pyruvate transport activity of the Mpc1/Mpc2C3 complex reaches ˜85% of MPCOX activity. Uptake of [14C]‐pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro. Difference between MPCFERM and Mpc1/Mpc2C3 is significant (unpaired t‐test; *P = 0.02). Means of n = 4 experiments are shown, with error bars representing SEM.",
    "molecules": "pyruvate"
  },
  {
    "caption": "Heatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 28) and glioblastomas (n = 217) from the Rembrandt dataset. Gene expression values are z-transformed and colored red for high expression and blue for low expression, as indicated in the scale bar. Volcano plot showing the fold-change (log2) in cholesterol-related gene levels based on GBM vs normal brain tissue. Data were obtained from the Rembrandt dataset.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Growth curves for GBM cells in vitro infected with lenti-Ctrl or lenti-CYP46A1 derived from trypan blue staining. Shown are means and SEM (n = 3). LN229: **P = 0.003; GBM#P3: **P = 0.002. Statistical significance was determined by two-sided Student's t‐test.",
    "molecules": "trypan blue"
  },
  {
    "caption": "24OHC levels in spent media from LN229 (left) and GBM#P3 (right) cells transduced with lenti-Ctrl or lenti-CYP46A1 measured using targeted LC-MS/MS",
    "molecules": "24OHC"
  },
  {
    "caption": "IC50 curves for 24OHC in LN229 and GBM#P3 cells using the CCK-8 assay.",
    "molecules": "24OHC"
  },
  {
    "caption": "Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3).",
    "molecules": "24OHC, FITC, PI"
  },
  {
    "caption": "Colony forming ability of GBM cell lines treated with 24OHC (0 - 20 μM) for 14 days.",
    "molecules": "24OHC"
  },
  {
    "caption": "Western blot analysis of the apoptosis marker c-PARP, c-caspase 3 and PCNA in lysates (20 µg) from GBM cells treated with 24OHC (0 - 20 μM) for 72 h.",
    "molecules": "24OHC"
  },
  {
    "caption": "Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation. Data are shown as the mean ± SEM (n = 3). GBM#P3: *P = 0.0192, ***P = 0.0006, ***P = 0.0002; GBM#BG7: **P = 0.0017, ***P < 0.0001, ***P < 0.0001; GBM#BG5: **P = 0.0027, ***P = 0.0001, ***P < 0.0001.",
    "molecules": "24OHC"
  },
  {
    "caption": "Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen™ Amplex™ Red Cholesterol Assay Kit and normalized to total protein. Shown are means and SEM (n = 3). LN229: **P = 0.002; GBM#P3: **P = 0.011. Statistical significance was determined by two-sided Student's t‐test.",
    "molecules": "24OHC, cholesterol, DMSO"
  },
  {
    "caption": "Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC (0 - 20 μM) for 72 h. Scale bar = 30 μm.",
    "molecules": "24OHC, filipin"
  },
  {
    "caption": "Growth curves for LN229 cells treated with DMSO, 0.5 μg/mL cholesterol or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol using trypan blue staining.",
    "molecules": "24OHC, cholesterol, DMSO, trypan blue"
  },
  {
    "caption": "Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0021, **P = 0.0046; GBM#P3: **P = 0.0012, *P = 0.0193.",
    "molecules": "24OHC, cholesterol, DMSO, FITC, PI"
  },
  {
    "caption": "Western blot analysis of c-PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h.",
    "molecules": "24OHC, cholesterol, DMSO"
  },
  {
    "caption": "Tumoursphere formation assays for GBM#P3 and GBM#BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol. Scale bar = 100 μm. Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0032, *P = 0.031; GBM#BG7: ***P = 0.0008, *P = 0.0242.",
    "molecules": "24OHC, cholesterol, DMSO"
  },
  {
    "caption": "CCK-8 assay to determine relative cell survival in LN229 or GBM#P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0051, ***P = 0.0002; GBM#P3: **P = 0.0018, ***P < 0.0001.",
    "molecules": "MβCD"
  },
  {
    "caption": "Heatmap of differentially expressed transcripts in RNA-seq data from GBM#P3 cells treated with DMSO or 20 μM 24OHC for 72 h. Gene expression data was mean z-transformed for display, and colored red for high expression and blue for low expression.",
    "molecules": "24OHC, DMSO"
  },
  {
    "caption": "GSEA plot showing normalized enrichment scores (NES) for cholesterol homeostasis, positive regulation of cholesterol efflux, SREBP targets and stem cell proliferation signatures using RNA-seq data from 24OHC and DMSO treated cells.",
    "molecules": "24OHC, cholesterol, DMSO"
  },
  {
    "caption": "Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included.",
    "molecules": "24OHC"
  },
  {
    "caption": "qRT-PCR analysis of 24OHC-regulated genes in GBM#P3 cells after treatment with 20 μM 24OHC for 72 h. GAPDH was used as an internal control. Data are shown as the mean ± SEM (n = 3).",
    "molecules": "24OHC"
  },
  {
    "caption": "Cell viability assay performed on GBM#P3 cells after 3 days of treatment with DMSO or 5 -10 μM LXR-623 in the presence or absence of 10 μM 24OHC. Data are shown as the mean ± SEM (n = 3).",
    "molecules": "24OHC, LXR-623, DMSO"
  },
  {
    "caption": "Western blot analysis of cholesterol metabolism-related proteins in LN229 or GBM#P3 after treatment with 24OHC (0 - 20 μM).",
    "molecules": "24OHC, cholesterol"
  },
  {
    "caption": "Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC (0 - 40 μM) as assessed by a trypan blue test. Data are shown as the mean ± SEM (n = 3).",
    "molecules": "24OHC, trypan blue"
  },
  {
    "caption": "Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days.",
    "molecules": "DMSO, fatostatin"
  },
  {
    "caption": "Representative images of filipin staining in LN229 (up) or LN18 (down) cells treated with different concentration of fatostatin (0 - 20 μM). Scale bar = 30 μm.",
    "molecules": "fatostatin, filipin"
  },
  {
    "caption": "Intracellular concentrations of cholesterol in GBM cells after treatment with 20 μM EFV or DMSO and normalized to total protein. Data are shown as the mean ± SEM (n = 3). LN229: *P = 0.013; GBM#P3: **P = 0.0023.",
    "molecules": "cholesterol, DMSO, EFV"
  },
  {
    "caption": "Filipin staining of GBM cells after treatment with EFV (0 - 20 μM). Scale bar = 30 μm.",
    "molecules": "EFV, Filipin"
  },
  {
    "caption": "IC50 curves for EFV in GBM cells determined using OD readings (450nm) from the CCK-8 assay.",
    "molecules": "EFV"
  },
  {
    "caption": "24OHC levels in spent media from LN229 (left) or GBM#P3 (right) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC-MS/MS.",
    "molecules": "24OHC, DMSO, EFV"
  },
  {
    "caption": "Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV (0 - 20 μM). Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0056, ***P < 0.0001; LN18: ***P < 0.0001.",
    "molecules": "EFV"
  },
  {
    "caption": "Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV (0 - 20 μM) for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0006, ***P < 0.0001; GBM#P3: ***P < 0.0001.",
    "molecules": "EFV, FITC, PI"
  },
  {
    "caption": "Tumoursphere formation assays for GSCs treated with different concentrations of EFV (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation (right). Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0011, ***P = 0.0003, *** P < 0.0001; GBM#BG7: ***P < 0.0001; GBM#BG5: **P = 0.0037, **P = 0.0015, ***P = 0.0002.",
    "molecules": "EFV"
  },
  {
    "caption": "Western blot analysis of cholesterol homeostasis related proteins (LDLR, ABCA1, P-SREBP1 and N-SREBP1), c-PARP, PCNA and SOX2 in GBM cells treated with EFV (0 - 20 μM) for 72 h.",
    "molecules": "cholesterol, EFV"
  },
  {
    "caption": "The survival curves of tumour-bearing mice implanted with LN229 or GBM#P3 cells after EFV or PBS treatment (n = 5 per group). A log-rank test was used to assess statistical significance.",
    "molecules": "EFV"
  },
  {
    "caption": "Bioluminescent images and the corresponding quantification of tumours in mice implanted with GBM#P3 cells at days 7 (n = 5 per group, P = 0.58) and 21 (n = 5 per group, P = 0.005). Statistical significance was determined by two-sided Student's t‐test. Body weight of tumour-bearing mice after 3 weeks of EFV or PBS treatment. Data are shown as the mean ± SEM. Statistical significance was determined by two-sided Student's t‐test.",
    "molecules": "EFV"
  },
  {
    "caption": "(A) HeLa cells expressing HA-Parkin were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "(B) HeLa cells stably expressing HA-Parkin or intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments. I, C, and M indicate input, cytosol-rich supernatant, and mitochondria-rich membrane pellet, respectively.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "(E) HeLa cells expressing HA-Parkin with various pathogenic mutations were treated with CCCP, followed by immunocytochemistry. (A, D, and E) Higher magnification views of the boxed areas are shown in the insets. (F) Parkin colocalization with mitochondria was analyzed in >100 cells per mutation. Example figures indicative of robust colocalization (counted as 1), weak colocalization (counted as 0.5), and no colocalization (counted as 0) are shown. Error bars represent the mean ± SD values of at least three experiments.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A) HeLa cells expressing wild-type Parkin or E3-inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies. When E3-inactivating mutations were introduced into Parkin, the mitochondrial ubiquitylation signal disappeared.",
    "molecules": "CCCP"
  },
  {
    "caption": "(B and C) HeLa cells expressing HA-Parkin (B) or expressing both Mt-GFP and HA-Parkin (C) were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "(D) Localization of GFP-Parkin to the mitochondria after CCCP treatment. (A-D) Higher magnification views of the boxed areas are shown in the insets.",
    "molecules": "CCCP"
  },
  {
    "caption": "(F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane.",
    "molecules": "CCCP"
  },
  {
    "caption": "(G) GFP-Parkin-expressing HeLa cells with various pathogenic mutations (Fig. 1 C) were treated with CCCP and subjected to immunoblotting. Asterisks show ubiquitylation of GFP-Parkin. Vertical black lines indicate that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.",
    "molecules": "CCCP"
  },
  {
    "caption": "(E) Endogenous PINK1 gradually accumulated after CCCP treatment. The first through the fifth lanes show endogenous PINK1, and the sixth lane shows overexpressed untagged PINK1. Note that the asterisk indicates a cross-reacting band because it was not affected by overproduction of untagged PINK1.",
    "molecules": "CCCP"
  },
  {
    "caption": "(F) Subcellular fractionation of endogenous PINK1. Intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments (same sample as Fig. 1 B). I, C, and M indicate input, cytosol-rich supernatant, and the mitochondria-rich membrane pellet, respectively.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "(G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band.",
    "molecules": "CCCP, cycloheximide"
  },
  {
    "caption": "(H) N-terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP. The top panel shows control HeLa cells expressing only GFP. (B, C, and H) Higher magnification views of the boxed areas are shown in the insets. Bars, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C)",
    "molecules": "CCCP"
  },
  {
    "caption": "(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study. MTS, mitochondria-targeting sequence; TMD, transmembrane domain. (B) Subcellular localization of Parkin in PINK1−/− cells complemented by various pathogenic and deletion mutants of PINK1-Flag. Cells were treated with CCCP. Higher magnification views of the boxed areas are shown in the insets. (C) The number of cells with Parkin-positive mitochondria was counted as in Fig. 3 A. Error bars represent the mean ± SD values of least three experiments.",
    "molecules": "CCCP"
  },
  {
    "caption": "(D) PINK1−/− MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti-Parkin or anti-Flag (tag of PINK1) antibodies. IB, immunoblot; Ub, ubiquitylation.",
    "molecules": "CCCP"
  },
  {
    "caption": "Biotinylated full-length CALIC or antisense CALIC (negative control) generated in vitro were incubated with lysates from DLD-1 colon cancer cells and precipitated with streptavidin beads. Precipitated proteins were resolved by SDS-PAGE followed by silver staining. The protein band indicated by the arrow was excised and subjected to mass spectrometry.",
    "molecules": "Biotin, streptavidin"
  },
  {
    "caption": "(Top) schematic of the full-length and mutants of CALIC used for the precipitation of hnRNP-L from HCT116 cell lysates. The mutated elements are indicated by the cross marks. Se, sense transcript; AS, antisense transcript. +, detectable binding activity; -, no detectable activity. (Bottom) precipitated hnRNP-L and biotin-labeled fragments of CALIC are shown.",
    "molecules": "biotin"
  },
  {
    "caption": "RIP analysis was performed using control rabbit IgG or anti-hnRNP-L antibody. HPRT1 and the lncRNA MALAT1 were used as negative controls.",
    "molecules": "IgG"
  },
  {
    "caption": "ChIP assays were performed using anti-hnRNP-L antibody or control mouse IgG. The promoter regions of GAPDH and Egr1 were used as negative and positive controls, respectively.",
    "molecules": "IgG"
  },
  {
    "caption": "B. MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.",
    "molecules": "NEO2734, MTS, CPI-637, JQ1"
  },
  {
    "caption": "Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells. The survival curve showed IC50 for SPOP Q165P (0.69 µM) and EV cells (1.08 µM) (C).",
    "molecules": "NEO2734"
  },
  {
    "caption": "DU145 cells expressing lenti-EV or lenti-HA-SPOP-Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantitative data shown in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01.",
    "molecules": "NEO2734"
  },
  {
    "caption": "B, C. SCID male mice with PDX tumors were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), combination of JQ1 (50 mg/kg) and CPI-637 (30 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of drug treatment were photographed (B) and tumor growth are shown in (C). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; *** P < 0.001.",
    "molecules": "NEO2734, CPI-637, JQ1, polyethylene glycol"
  },
  {
    "caption": "Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for IFC with p-AKT-S473 antibody (B). E-cadherin antibody was used to indicate the cell membrane (red) and DAPI for nucleus (blue). Scale bars: 25 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. The representative images of organoids after the treatment are shown in (C)",
    "molecules": "NEO2734, CPI-637, JQ1"
  },
  {
    "caption": "Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. the quantified data of the organoid diameter are shown in (D). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.",
    "molecules": "NEO2734, CPI-637, JQ1"
  },
  {
    "caption": "E, F. 313HR (SPOP WT) and 573R (Q165P) organoids were cultured for 5 days, followed by the treatment with the indicated inhibitors for 5 days. The organoids were stained with Caspase-3 antibody (green) to detect apoptotic cells. E-cadherin antibody staining was used to define the cell membrane (red) and the nucleus was counterstained with DAPI (blue). The representative images of cleaved Caspase-3 IFC staining are shown in (E). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.",
    "molecules": "DAPI"
  },
  {
    "caption": "B MTS was performed to compare the cell growth between EV, Q165P, F133V C4-2 groups after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.",
    "molecules": "NEO2734, MTS, CPI-637, JQ1"
  },
  {
    "caption": "C. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4-2 cells. The survival curve showed IC50 for EV (1.01 µM), SPOP Q165P (0.621 µM) and SPOP F133V cells (0.395 µM).",
    "molecules": "NEO2734"
  },
  {
    "caption": "D, E. C4-2 cells expressing EV, Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantification data in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.",
    "molecules": "NEO2734"
  },
  {
    "caption": "F - H. A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration (F). When the tumor reached 100 mm3, mice were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of treatment were photographed (G) and tumor growth are shown in (H). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01; *** P < 0.001.",
    "molecules": "NEO2734, JQ1, polyethylene glycol"
  },
  {
    "caption": "Pulldowns of pointed end site 1-4 mutants using Strep-Hook3 (D) or Strep-BICD2 (E). Binding is shown relative to the binding of the wildtype pointed end construct (dashed lines). * shows p<0.05. For BICD2, p=0.06 for Site 1 mutant, and p=0.1 for Site 4 mutant. Data presented as mean ±SEM, n=3. Statistical significance calculated using ANOVA with Tukey's test for multiple comparisons.",
    "molecules": "Strep"
  },
  {
    "caption": "Human tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands. Positive signals (in blue) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts, as well as in pancreatic tissues.",
    "molecules": "sialic acid"
  },
  {
    "caption": "Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green). The co‐localized signals for the Siglec‐Fc probes with an anti‐Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid‐independent ligands of Siglec‐5 and Siglec‐14 in the lung bronchiole. White dashes mark the lung bronchiole epithelium. All images are representative of at least 2-3 replicates.",
    "molecules": "sialic acid"
  },
  {
    "caption": "Using flow cytometry, undifferentiated or PMA‐differentiated THP1 cells with empty vector (EV), Siglec‐5 (Sig5), or Siglec‐14 (Sig14) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface. The mean fluorescence intensity (MFI) of signals from an anti‐Hsp70 antibody on unpermeabilized THP1 cells is shown.",
    "molecules": "PMA"
  },
  {
    "caption": "Secretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone. Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone.",
    "molecules": "LPS"
  },
  {
    "caption": "Similarly, IL‐8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70. Secretion of IL‐8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone.",
    "molecules": "LPS"
  },
  {
    "caption": "Western blots demonstrate phosphorylated p38 (p‐p38) was reduced in THP1Siglec‐5cells when exposed to both LPS and Hsp70 in comparison with LPS alone. This reduction was also greater in THP1Siglec‐5cells in comparison with the control THP1cells. Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control (total p38).",
    "molecules": "LPS"
  },
  {
    "caption": "Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng/ml LPS with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was increased in only THP1 Sig5 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this increase was not replicated in THP1 EV cells.",
    "molecules": "LPS, PMA"
  },
  {
    "caption": "Secretion of TNFα by THP1 cells transfected with a Siglec‐5 or Siglec‐14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin‐neutralizing agent polymyxin B. Only THP1 Siglec‐14 cells exhibited significant secretion of TNFα in an Hsp70 dose‐dependent manner.",
    "molecules": "polymyxin B"
  },
  {
    "caption": "Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this change was not observed in THP1 EV cells.",
    "molecules": "LPS, PMA"
  },
  {
    "caption": "TNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B. Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B, suggesting a mechanism independent of endotoxin contaminants. DnaK, the E. coli homolog of human Hsp70, did not induce significant secretion of TNFα above background levels.",
    "molecules": "LPS, polymyxin B"
  },
  {
    "caption": "Human monocytes isolated from SIGLEC14+/+, SIGLEC14+/−, and SIGLEC14−/− individuals were exposed to varying concentrations of Hsp70, and TNFα secretion was evaluated. The amount of TNFα secreted was normalized to the respective cells' response to 1 ng/ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals.",
    "molecules": "LPS"
  },
  {
    "caption": "TNFα secretion from SIGLEC14−/− monocytes stimulated with 15 ng/ml LPS and various concentrations of Hsp70. TNFα secretion was reduced with increasing concentrations of Hsp70.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells. The label was provided simultaneously to all the samples at 6 hpi.",
    "molecules": "RNA"
  },
  {
    "caption": " IEM detection of viral markers in MERS-CoV-infected cells.(A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.",
    "molecules": "dsRNA"
  },
  {
    "caption": "D HERC3 and HERC3-CA extend the half-life of TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 4, 8 or 12 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "F Depletion of HERC3 decreases the half-life of YAP/TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 2, 4 or 6 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "A HERC3 interacts with β-TrCP, but not YAP or TAZ. HEK293T cells were transfected with expression plasmids for FLAG-HERC3, HA-YAP or HA-TAZ or HA-β-TrCP. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA and FLAG antibodies. B HERC3 interacts with endogenous β-TrCP. MDA-MB-231 cells stably expressing HERC3 or HERC3-CA were harvested and subjected to IP with IgG or HA antibodies. Protein levels were detected by Western blotting with β-TRCP, HA and β-Actin antibodies. C",
    "molecules": "agarose"
  },
  {
    "caption": "H HERC3 blocks the interaction between β-TrCP and YAP. HEK293T cells were transfected with expression plasmids for FLAG-YAP, HA-β-TRCP, Myc-HERC3 and Myc-HERC3-CA as indicated. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA, FLAG and Myc antibodies.",
    "molecules": "agarose"
  },
  {
    "caption": "Depletion of HERC3 attenuates cell migration. Control or HERC3-deficient MDA-MB-231 cells (upper panel) and SUM159 cells (lower panel) were analyzed by transwell migration assay. E, migrated were fixed in 4% PFA and stained with 0.1% crystal violet and photographed. data quantification of migrated MDA-MB-231 cells (left panel) and SUM159 (right panel) cells counts by Image J.",
    "molecules": "crystal violet, PFA"
  },
  {
    "caption": "(A) Representative images of a PDGFB tumour: H&E on the right to identify the tumour, an adjacent section stained for Iba1 (red), p-S6 (green) and DAPI (blue) at the centre and the region selection for quantification on the left (non-tumour in light blue, tumour edge in orange and tumour core in dark blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Representative images of core, edge and adjacent non-tumour brain tissue for Iba1, p-S6 and DAPI staining in Pten-/-p53-/- (right) and GL261 (left) tumours. Percentage of Iba1+ cells co-expressing p-S6 in the three defined regions. Five high grade glioma models were analysed - GL261 (n=3), PDGFB (n=3), Ntv-a;PDGFB+shp53 (n=2), Pten-/-p53-/- (n=3), and Pten-/-p53-/-Idh1mut (n=3) (mean ±SEM; Two-Way ANOVA Tukey test).",
    "molecules": "DAPI"
  },
  {
    "caption": "Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr (B and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "Torin"
  },
  {
    "caption": "Flow cytometry analysis was carried out in microglia for (C) p-S6 S240/244 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "Torin"
  },
  {
    "caption": "Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "Torin"
  },
  {
    "caption": "Flow cytometry analysis was carried out in microglia for (E) p-4EBP1 T37/46 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "Torin"
  },
  {
    "caption": "Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "Torin"
  },
  {
    "caption": "Flow cytometry analysis was carried out in microglia for (G) p-AKT S473; Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin, was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "Torin"
  },
  {
    "caption": "Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 0.5hr (H) and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM +LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "LY"
  },
  {
    "caption": "Flow cytometry analysis was carried out in microglia for (I) p-AKT T308. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+ LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.",
    "molecules": "LY"
  },
  {
    "caption": "(D) Tumour tissues from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=4) were stained for CD8, CD4 and FoxP3. DAB staining was quantified as % of positive cells using Definiens software. Representative images of the stained tissues are shown. (mean ±SEM; unpaired parametric t test). Scale bar is 100µm.",
    "molecules": "DAB"
  },
  {
    "caption": "(E) Microglia were pre-treated with Torin, or medium as indicated and then stimulated with mNSC-CM, mGICPten-/-;p53-/--CM, or GL261-CM. p-NF-ĸB (p-P65) and p-STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr.",
    "molecules": "Torin"
  },
  {
    "caption": "(F) Immunofluorescence images of NF-ĸB (P65) in conditioned microglia. Quantification of nuclear translocation of NF-κB from staining. Units represent the % of voxels in the NF-κB channel colocalised with DAPI (n=3; mean ±SEM; One-Way ANOVA Tukey test - right). Scale bar is 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(I-K) (I) P-S6, (J) p-STAT3, and (K) p-NF-ĸB (p-P65) were analysed by flow cytometry in iMGL, pre-treated with Torin, as indicated and then stimulated with matched patient EPSC- or hGIC-CM. (n=3; mean ±SEM, Two-Way ANOVA Tukey test).",
    "molecules": "Torin"
  },
  {
    "caption": "B-C, Changes in lipid content (total amount) observed in THEM6  KO CRPC cells when compared to CTL. Data information: Data are presented as mean values +/- SD. Statistical analysis : *p-value < 0.05 using a 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent biological experiments. DAG: diacylglycerol; TG: triglyceride;  PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine;  Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester.",
    "molecules": "Cer, Ceramide, CE, Cholesteryl ester, DAG, diacylglycerol, LysoPC, lysophosphatidylcholine, LysoPE, lysophosphatidylethanolamine, PC, phosphatidylcholine, PE, phosphatidylethanolamine, phosphatidylinositol, PI, SM, sphingomyelin, TG, triglyceride"
  },
  {
    "caption": "F, Labelled cholesterol fraction derived from  13C-glucose and 13C-glutamine  in CTL and THEM6 KO CWR22res cells after 72 hours of incubation. G, Labelled cholesterol fraction derived from 13C-glucose  and 13C-glutamine  in CTL and THEM6 KO MCF-7 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: , F, : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. G: *p-value < 0.05 using a two-tailed Student t-test. Data reproducibility: : n = 3 independent wells from the same cell culture.",
    "molecules": "13C, cholesterol, glucose, glutamine"
  },
  {
    "caption": "J, Labelled palmitic, oleic and stearic acid  fractions derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO  22rv1  cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent wells from the same cell culture.",
    "molecules": "13C, glucose, glutamine, palmitic, stearic acid, oleic"
  },
  {
    "caption": "J, Western blot  analysis of ATF4 and CHOP expression  in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid (200 µM) or hexadecylglycerol (50 µM) for 48 hours. Data information: HSC70  was used as a sample loading control. Data reproducibility: representative image from 3 independent biological experiments.",
    "molecules": "palmitic acid, hexadecylglycerol"
  },
  {
    "caption": "(A) Isolation of FTCD from rat liver extract. The p47(1-170) fragment, which does not contain the p97-binding site, was immobilized on beads and incubated with rat liver extract. Bound proteins were fractionated by SDS-PAGE, and stained with CBB.",
    "molecules": "CBB"
  },
  {
    "caption": "(B) FTCD binds directly to p47. GST-tagged p47 (0.54 μg) or GST-tagged p37 (0.45 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.",
    "molecules": "His, glutathione"
  },
  {
    "caption": "(E) p47 binds to the CD domain of FTCD. GST-tagged p47 (0.50 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.63 μg). The blots were probed with antibodies to His-tag and GST.",
    "molecules": "His"
  },
  {
    "caption": "(G) FTCD(R382A), which lacks binding ability to polyglutamates, does not bind to p47. GST-tagged p47 (0.35 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.",
    "molecules": "His, polyglutamates"
  },
  {
    "caption": "(H) p47 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were first incubated with either GST-tagged p47wt, p47(E86K, E88K) or p47(F253S). The isolated membranes were solubilized, and then GST-tagged p47wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.",
    "molecules": "glutathione"
  },
  {
    "caption": "(A) FTCD directly binds to p97. GST-tagged p97 (2.1 μg) was incubated with His-tagged FTCD (0.80 μg), and then isolated on glutathione beads. The blots were probed with antibodies to FTCD and GST.",
    "molecules": "His, glutathione"
  },
  {
    "caption": "(D) p97 also binds to the CD domain of FTCD. GST-tagged p97 (2 μg) was incubated with either His-tagged FTCD(1-326) (FT, 0.88 μg) or His-tagged FTCD(327-541) (CD, 0.62 μg). The blots were probed with antibodies to His-tag and GST.",
    "molecules": "His"
  },
  {
    "caption": "(F) FTCD(R382A), which lacks binding ability to polyglutamates, shows a very low binding affinity to p97. GST-tagged p97 (1.4 μg) was incubated with His-tagged FTCDwt/mutant (0.53 μg). The blots were probed with antibodies to His-tag and GST.",
    "molecules": "His, polyglutamates"
  },
  {
    "caption": "(G) p97 binds to FTCD in Golgi membranes. Salt-washed Golgi membranes were incubated with either GST-tagged p97wt or p97(V722k, E724K). The isolated membranes were solubilized, and then GST-tagged p97wt/mutants and their binding proteins were recovered with glutathione-beads. The bound proteins were assayed by Western blotting with antibodies to FTCD and GST.",
    "molecules": "glutathione"
  },
  {
    "caption": "(A) Immunofluorescence staining of FTCD and GM130, a Golgi marker. HepG2 cells were fixed, permeabilized, stained with a monoclonal antibody to FTCD, a polyclonal antibody to GM130 and DAPI, and observed by confocal microscopy. Scale bar = 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Two sets of FTCD molecules and a p97/p47 complex form a quaternary complex. His-tagged FTCD was biotinylated and immobilized on streptavidin beads (His-FTCD-beads). The beads (1.0 μg) were incubated with Flag-tagged FTCD (2.0 μg) in the absence or presence of p47 (1.0 μg) and p97 (2.0 μg). The blots were probed with HRP-conjugated avidin and antibodies to Flag-tag, p97 and p47.",
    "molecules": "biotinylated"
  },
  {
    "caption": "(C) Mutants that lack binding affinity disrupt the formation of the quaternary complex. His-FTCD-beads (1.0 μg) and Flag-tagged FTCD (2.0 μg) were incubated in the presence of AMP-PNP together with p97wt/mutant (2.0 μg) and p47wt/mutant (1.0 μg). The blots were probed as described in (A).",
    "molecules": "AMP-PNP"
  },
  {
    "caption": "(E) FTCDwt-beads are aggregated in the presence of both p97 and p47. Biotinylated FTCDwt and FTCD(R382A) were immobilized on streptavidin magnetic beads to form FTCDwt-beads and FTCD(R382A)-beads, respectively. The beads were incubated with the indicated proteins (p97, 0.1 μg/μl; p47, 0.1 μg/μl; p37, 0.1 μg/μl) in the presence of AMP-PNP (1 mM), and observed under a bright-field microscope. Scale bar = 50 μm. (F) The results of quantification of (E). The percentages of beads in the aggregates were calculated. Results are expressed as the mean+SD of ten sets of independent experiments. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). ",
    "molecules": "AMP-PNP, Biotinylated"
  },
  {
    "caption": "(H) p47 and p97 mutants that lack binding affinity inhibit p97/p47-mediated aggregation of FTCDwt-beads. FTCDwt-beads were incubated with p97wt/mutant (0.1 μg/μl) and p47wt/mutants (0.1 μg/μl) in the presence of AMP-PNP. The representative images are presented in Figure EV1F. Data are shown as the mean+SD of nine sets of independent experiments. Asterisks indicate a significant difference at P<0.01 compared with the positive control ('p97wt+p47wt') (Bonferroni method).",
    "molecules": "AMP-PNP"
  },
  {
    "caption": "(A) The expression of FTCDwt-HA-MAO causes the aggregation of mitochondria. HeLa Tet-off cells inducibly expressing either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO were cultured in DOX-free medium for 48 hrs. Cells which were cultured in the DOX-containing medium were used as a control. The cells were fixed and stained with a monoclonal antibody to mitochondria and a polyclonal antibody to HA, followed by observation by confocal microscopy. Panels a-j display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). ",
    "molecules": "DOX, Tet"
  },
  {
    "caption": "(C) Representative EM images of a cell expressing FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were cultured for 48 hrs in the absence of DOX, and used for EM observation. Panel b displays the inset in panel a. Arrowheads show narrow gaps between mitochondria. Scale bar = 0.2 μm. The low magnification image is presented in Figure EV2C.",
    "molecules": "DOX, Tet"
  },
  {
    "caption": "(D) Endogenous p47 and p97 are necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with either mock, p47 siRNA or p97 siRNA duplexes and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCDwt-HA-MAO, and then analyzed as in (A). Panels a-l display representative images. Scale bar = 10 μm. (E) The results of quantification of (D). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). ",
    "molecules": "DOX, Tet"
  },
  {
    "caption": "(F) Binding between FTCD and p47 and between p47 and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with mammalian expression constructs of siRNA-insensitive Flag-tagged p47wt/mutants at the same time as the treatment of p47 siRNA, and cultured for 24 hrs. The cells were further cultured in DOX-free medium for 48 hrs for the induction of FTCD-HA-MAO. After fixation, the cells were visualized with a monoclonal antibody to mitochondria and polyclonal antibodies to HA and Flag. Panels a-l display representative images. Scale bar = 10 μm. (G) Binding between FTCD and p97 is necessary for mitochondria aggregation mediated by FTCDwt-HA-MAO. The HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO were transfected with the mammalian expression construct of siRNA-insensitive Flag-tagged p97wt/mutant at the same time as treatment with p97 siRNA. The following procedures were the same as in (F). Panels a-i display representative images. Scale bar = 10 μm. (H) The results of quantification of (F) and (G). Results are shown as the mean±SD of five sets of independent experiments, with 100 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with siRNA treatment alone ('none') and compared with mutant expression (Bonferroni method). ",
    "molecules": "DOX, Tet"
  },
  {
    "caption": "(A) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method). ",
    "molecules": "Tet"
  },
  {
    "caption": "(C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group. ",
    "molecules": "MitoTracker, DOX, Tet"
  },
  {
    "caption": "(E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.",
    "molecules": "MitoTracker, DOX, Tet"
  },
  {
    "caption": "(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).",
    "molecules": "DOX"
  },
  {
    "caption": "C) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U/mL IFNγ for 24 h. Following stimulation, HFFs were infected with either RH (MOI=1) or Pru (MOI=3) for another 24 h. Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei. The number of PVs in 5-6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field. Experiments were performed with RH (n=3), Pru (n=3). Representative images of percentage infected cells for each strain and condition are provided. Scale bar is 100 µm.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (A) Total ubiquitin (n=3 for RH, n=5 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.",
    "molecules": "Hoechst 33258, ubiquitin"
  },
  {
    "caption": "HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (B) K63-linked ubiquitin (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.",
    "molecules": "Hoechst 33258, ubiquitin"
  },
  {
    "caption": "HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (C) p62 (n=3 for RH, n=10 for Pru and n=7 for PruΔgra15, n=4 for Pru Δgra15+GRA15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (D) NDP52 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (E) LC3B (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15, n=3 for Pru Δgra15+GRA15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (F) GABARAP (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15) is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (G) LAMP1 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15) is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "H) The number of PVs per 20X objective field was counted and compared between IFNγ-stimulated and IFNγ + bafilomycin A1 (100 nM) treated HFFs 24 h p.i. with Pru strain. Images from at least 6 fields were taken for each condition (n=3).",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "A) HFFs were stimulated with IFNγ (10 U/mL for 24 h) or left unstimulated and subsequently treated with PYR41 (1 µM) for 2 h prior to infection. Cells were washed and subsequently infected with Pru parasites (MOI=3) for 3 h. The percentage of vacuoles that stained positive for total ubiquitin, p62 or LC3B was determined (n=3).",
    "molecules": "PYR41, ubiquitin"
  },
  {
    "caption": "For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, p62 was calculated.",
    "molecules": "Ub"
  },
  {
    "caption": "For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, LC3B was calculated.",
    "molecules": "Ub"
  },
  {
    "caption": "For all the co-staining experiments IFNγ-stimulated HFFs infected for 3 h with the Pru strain were used. For each staining at least 50 vacuoles were scored (n=3). On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain. Scale bar is 20 µm. The yellow box inside each representative image is shown as inset pictures with higher magnification. The total number of coated vacuoles was set at 100% and the percentage of vacuoles positive for Ub, GABARAP was calculated.",
    "molecules": "Ub"
  },
  {
    "caption": "A) HFFs were stimulated with IFNγ for 24 h (10 U/ml) or left unstimulated. The NF-κB inhibitor BAY11-7082 (1µM) was added 2 h pre-infection and HFFs were subsequently infected with Pru parasites. Parasite growth (using luciferase assay) was measured 24 h p.i. Means from unstimulated cells were set at 100%. Experiments were performed three times.",
    "molecules": "BAY11-7082"
  },
  {
    "caption": "B) Nuclear translocation of the NF-κB p65 subunit was quantified in HFFs or HFFs treated with BAY11-7082 (1µM, added 2 h pre-infection) 24 h p.i. with Pru parasites. Experiments were done 4 times where each dot represents one experimental mean of at least 15 nuclei. In the right panel, representative images are shown. Parasites were expressing GFP, nuclei are stained with Hoechst 33258. Scale bar is 20 µm.",
    "molecules": "BAY11-7082, Hoechst 33258"
  },
  {
    "caption": "E) Immunofluorescence analysis of TRAF6 recruitment to the PVM of HFFs infected for 3 h with RH, Pru and PruΔgra15 strains. On the right-hand side, a representative fluorescent image is shown of TRAF6 recruitment to the PVM where Toxoplasma Pru strain expresses GFP, DNA was stained with Hoechst 33258. Scale bar is 10 µm. n=3 for all the strains. The antibody against TRAF6 was purchased from Abnova (Appendix Table S2).",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (A) IRGB6 For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (B) GBPs, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (C) total ubiquitin, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.",
    "molecules": "Hoechst 33258, ubiquitin"
  },
  {
    "caption": "MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (D) K63-linked ubiquitin, For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.",
    "molecules": "Hoechst 33258, ubiquitin"
  },
  {
    "caption": "MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (E) K48-linked ubiquitin For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.",
    "molecules": "Hoechst 33258, ubiquitin"
  },
  {
    "caption": "MEFs were stimulated with IFNγ for 24 h (100 U/ml). IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 or RH for 3 h and subsequently fixed, permeabilized and stained for (F) LC3B. For analysis, at least 100 vacuoles were scored. All experiments were performed 3 times. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. All experiments were performed 3 independent times.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "B) IFNγ-stimulated MEFs were infected with Pru, PruΔgra15 and RH for 3 h and subsequently stained for TRAF6. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as an inset picture with magnification. Experiments were performed 3 times.",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "Immunofluorescence analysis of ubiquitin was done in IFNγ-stimulated MEFs infected with RH+GRA15WT, RH+GRA15TRAF2mut or RH+GRA15TRAF2/6mut (n=3).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "B. Airyscan confocal analysis of endogenous FIP200/γTUBULIN and ATG13/γTUBULIN colocalization with lentiviral delivered HA-OFD1 in HK2 cells. Cells were cultured in HBSS (90min). Green, FIP200 and ATG13; red, HA-OFD1; Grey, γTUBULIN; blue, Hoechst for nuclei. Insets show magnifications and single-color channels of selected areas. Scale bar=3,5μm. C. Quantification of HA-OFD1/ATG13 and HA-OFD1/FIP200 colocalization, expressed as % of total OFD1 fluorescence (mean ± SEM). n=3 independent experiments; n=53 cells for ATG13 and n=39 for FIP200 were analyzed. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "B. Top, wild-type and KO-OFD1 cells were incubated with 50µg/ml CHX. Immunoblots were probed with indicated antibodies. Bottom, quantification of βTUBULIN and ACTIN-normalized protein levels versus untreated conditions (-), data are expressed as mean ± SEM; n=4 independent experiments. CHX=Cycloheximide, ns=not significative.",
    "molecules": "CHX, Cycloheximide"
  },
  {
    "caption": "C. Immunofluorescence of ULK1 and ATG13 puncta in wt and KO-OFD1 cells cultured in HBSS (90min). Green, ATG13 and ULK1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) Graphs display quantification of puncta/cell, data are expressed as mean ± SEM; n=5 independent experiments, n≥200 cells/antibody.",
    "molecules": "Hoechst"
  },
  {
    "caption": "E. Western Blot of ULK1-complex components in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or empty vector (Empty) and treated (+) or not (-) with SAR405 (10μM, 6h). ACTIN is the loading control. On the right, ATG13 levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.",
    "molecules": "SAR405"
  },
  {
    "caption": "F. Representative confocal images of co-staining of 3xFLAG-OFD1 with ATG13 in KO-OFD1 cells incubated in HBSS, treated (+) or not (-) with Baf-A1(100nM, 90min). Green, ATG13; red, 3xFLAG-OFD1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=3 independent experiments, n≥100 cells.",
    "molecules": "Hoechst, Baf-A1"
  },
  {
    "caption": "B. Representative confocal images of endogenous OFD1 (green) co-staining with LC3B (red), and of 3xFLAG-OFD1 (red) with GABARAP (green) in HK2 cells (HBSS,90min). Hoechst labels nuclei (blue). Insets show magnifications and single-color channels of selected areas. Scale bar=10μm; n≥50 cells/sample.",
    "molecules": "Hoechst"
  },
  {
    "caption": "E. Representative confocal images of OFD1∆LIR co-staining with LC3B (left) and GABARAP (right) in HK2 cells (HBSS,90min). Green, LC3B and GABARAP; red, 3xFLAG-OFD1∆LIR; blue, Hoechst for nuclei. Scale bar=5μm. n≥50 cells/sample.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A. Representative confocal images of 3xFLAG-OFD1 (OFD1) and 3xFLAG-OFD1∆LIR (OFD1∆LIR) co-staining with ATG13 in KO-OFD1 cells (HBSS,90min). Green, ATG13; red, FLAG; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=4 independent experiments, n≥100 cells.",
    "molecules": "Hoechst"
  },
  {
    "caption": "D. Scanning confocal microscopy analysis of wt and KO-OFD1 cells incubated in HBSS (8h), treated (+) or not (-) with Baf-A1(50nM, 8h). Green, LAMP1; red, ATG13; blue, Hoechst for nuclei. Scale bar=6μm. Insets show higher magnification and single colour channels of the boxed area. (Bottom) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1/cell (mean ± SEM). n=30 WT cells, n=28 KO-OFD1 cells counted; three independent experiments.",
    "molecules": "Hoechst, Baf-A1"
  },
  {
    "caption": "E. Time course of OFD1 levels in HBSS-starved wt HK2 cells treated with Baf-A1 (100nM) and collected at different time points. LC3B is the control for Baf-A1 treatment. (Right) Graphs show ACTIN-normalized OFD1 levels versus FM condition (-) expressed as mean ± SEM; n=4 independent experiments. STV=starvation, Baf-A1=bafilomycin, Ctrl=control.",
    "molecules": "Baf-A1, bafilomycin"
  },
  {
    "caption": "F. Western blot of FLAG in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) and treated or not (-) with Baf-A1 (100nM,2h). (Right) Quantification of GAPDH-normalized FLAG levels versus untreated (-) is expressed as mean ± SEM; n=3 independent experiments. Baf-A1=bafilomycin.",
    "molecules": "Baf-A1, bafilomycin"
  },
  {
    "caption": "B. Wild-type and KO-OFD1 cells were starved in HBSS with/without Baf-A1 (100nM, 2h) and chloroquine (CQ) (50μM, 2h) and total lysates were analyzed by immunoblotting using anti-OFD1, -LC3B (LC3B-I 18 kDa and LC3B-II 16 kDa) and -ACTIN antibodies. (Right) ACTIN-normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of wt cells. n=5 independent experiments.",
    "molecules": "Baf-A1, chloroquine"
  },
  {
    "caption": "C. Representative images of LC3B and WIPI2 puncta in wt and KO-OFD1 cells (HBSS, 90min). Green, LC3B; red, WIPI2; blue, Hoechst, nuclei. Scale bar=10μm. (Right) Quantification of LC3B and WIPI2 puncta is shown. ≥100 cells analyzed/sample, n=5 independent experiments.",
    "molecules": "Hoechst"
  },
  {
    "caption": "F. Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP-OFD1 (HBSS,90min). Green, eGFP-OFD1, red, WIPI2; blue, Hoechst labels nuclei. On the right, quantification of WIPI2 puncta in eGFP-OFD1 transfected cells compared with non-transfected cells (Ctrl) is shown. Scale bar=10μm. n≥90 cells analyzed/sample, n=3 independent experiments.",
    "molecules": "Hoechst"
  },
  {
    "caption": "G. Representative images of WIPI2 and OFD1 co-staining in KO-OFD1 cells transfected with empty vector (Empty), 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) (HBSS,90min). Red, WIPI2; green, OFD1; blue, Hoechst labels nuclei. Scale bars: 10μm. On the right, quantification of WIPI2 puncta. ≥100 cells analyzed/sample, n=4 independent experiments.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A. Western blot of LC3B in lymphoblasts of OFD type I patients and controls, either untreated (-) or exposed to Baf-A1 (10nM, 2h)(+). Cells were cultured in complete medium (FM, top) or under serum starvation for 4h (STV, bottom). ACTIN and GAPDH were used as loading controls.",
    "molecules": "Baf-A1"
  },
  {
    "caption": "A. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1fl/y;creKsp and control mice. (Right) LC3B-II protein levels normalized to Actin (loading control) are expressed as fold increase versus control mice, n=4 mice/group.",
    "molecules": "leupeptin"
  },
  {
    "caption": "B. Representative images of GFP-LC3 puncta (autophagosomes, green) in aquaporin2-positive (AQP2, red) renal distal tubules from P8 Ofd1fl/y;creKsp;GFP-LC3 and Ofd1fl/y;GFP-LC3 mice. Scale bar=10μm. Hoechst labels nuclei. Bar graphs on the left show quantitative analysis of GFP-LC3 puncta; n=4 mice/group for a total of 300 AQP2 positive nuclei/group analyzed.",
    "molecules": "Hoechst"
  },
  {
    "caption": "C. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1-IND and control mice. (Right) LC3B-II protein levels relative to βTubulin (loading control) are expressed as fold increase compared with control mice, n=5 mutant/8 control mice .",
    "molecules": "leupeptin"
  },
  {
    "caption": "F. Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl/y;Atg7fl/fl;creKsp, Ofd1fl/y;Atg7+/+;creKsp and control mice at P45. n=8 mice per group.",
    "molecules": "Nitrogen, Urea"
  },
  {
    "caption": "Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgAkt1E17K. EditR (Kluesner et al., 2018) was used to calculate the frequency (%) of C-to-T conversion at C7 of the protospacer targeted by sgAkt1E17K in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.",
    "molecules": "cytosines"
  },
  {
    "caption": "Sanger-sequencing chromatograms showing the target region of sgAkt1E17K in 3 independent tumors from WB1P-BE3 females injected with Lenti-sgAkt1E17K-Myc. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at C7 of the protospacer in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgAkt1E17K-Myc.",
    "molecules": "cytosines"
  },
  {
    "caption": "Sanger-sequencing chromatograms showing the target regions of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacers that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPik3caE542K, Lenti-sgPik3caE545K and Lenti-sgPik3caE453K.",
    "molecules": "cytosines"
  },
  {
    "caption": "EditR was used to calculate the frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.",
    "molecules": "cytosines"
  },
  {
    "caption": "Sanger-sequencing chromatograms showing the target region of sgPik3caE542K, sgPik3caE545K and sgPik3caE453K in 3 independent tumors from WB1P-BE3 females injected with the corresponding Lenti-sgPik3ca-Myc vectors. Arrowheads highlight cytosines of the protospacer that show base editing. EditR was used to calculate the average frequency (%) of C-to-T conversion at the indicated target cytosines of the protospacers in tumors from WB1P-BE3 females injected with Lenti-sgNT-Myc or Lenti-sgPik3caE542K-Myc, Lenti-sgPik3caE545K-Myc and Lenti-sgPik3caE453K-Myc.",
    "molecules": "cytosines"
  },
  {
    "caption": "Sanger-sequencing chromatograms showing the target region of sgPtenQ245* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgPtenQ245*. EditR was used to calculate the frequency (%) of C-to-T conversion at C4 of the protospacer targeted by sgPtenQ245* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.",
    "molecules": "cytosines"
  },
  {
    "caption": "BE Analyzer (Hwang et al., 2018) was used to assess from next-generation sequencing data the fraction of wild-type Pten alleles, base edited alleles or alleles with insertions/deletions (indels) in tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc or Lenti-sgPtenQ245*-Myc. TIDE analysis showing the spectrum of indels of the targeted Pten alleles in two independent representative tumors from WB1P-BE3 mice injected with Lenti-sgPtenQ245*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgPtenQ245* (PCR products were subcloned for clarity). Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by indels at both alleles, while in the upper one by Q245* base editing in one allele and a deletion at the second copy of the gene.",
    "molecules": "cytosines"
  },
  {
    "caption": "Sanger-sequencing chromatograms showing the target region of sgTrp53Q97* in wild-type (WT) and base edited (BE) cells. Arrowheads highlight cytosines of the protospacer that show base editing 5 days after transduction of BE3-expressing NIH3T3 cells with Lenti-sgTrp53Q97 EditR was used to calculate the frequency (%) of C-to-T conversion at C8 of the protospacer targeted by sgTrp53Q97* in BE3-expressing NIH3T3 cells 5 days after transduction with the indicated sgRNA vectors.",
    "molecules": "cytosines"
  },
  {
    "caption": "BE Analyzer was used to assess from next-generation sequencing data the fraction of wild-type Trp53 alleles, base edited alleles or alleles with indels in tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ animals injected with Lenti-sgTrp53Q97*-Myc. Tumors from WB1P-BE3 animals injected with Lenti-sgNT-Myc mice were used as control. TIDE analysis showing the spectrum of indels of the targeted Trp53 alleles in two independent representative tumors from WapCre;Brca1F/F;Trp53F/+;Col1a1invCAG-BE3/+ mice injected with Lenti-sgTrp53Q97*-Myc. For the two tumors shown in (F), Sanger-sequencing chromatograms showing the target region of sgTrp53Q97*. Arrowheads highlight cytosines of the protospacer that show base editing. In the lower example the gene was inactivated by a deletion, while in the upper one by Q97* base editing. Of note, the allele with the indel also displays base editing at C6 of the protospacer.",
    "molecules": "cytosines"
  },
  {
    "caption": "I. α-Gal A specific activity in Fabry patient-derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P.",
    "molecules": "M6P"
  },
  {
    "caption": "A, B. Levels of globotriaosylceramide (Gb3) were measured by LC/MS in plasma and extracts of liver, spleen, heart, and kidneys of mice engrafted with healthy donor (HDo)-derived (A) or Fabry donor (FDo)-derived (B) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.",
    "molecules": "Gb3, globotriaosylceramide"
  },
  {
    "caption": "C, D. Levels of lyso-Gb3 were measured by LC/MS in plasma and extracts of tissues of mice engrafted with HDo-derived (C) or FDo-derived (D) TRaMs.",
    "molecules": "lyso-Gb3"
  },
  {
    "caption": "b, Accumulation of Triton-X-100-solublepolyubiquitinated proteins in the brains of Atg5flox/flox; nestin-Cre mice. Brain homogenate prepared at the indicated times from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice were separated into Triton-X-100-soluble (S) and -insoluble (P) fractions and analysed by immunoblotting using anti-ubiquitin antibodies. Arrowhead indicates the stacking gel.",
    "molecules": "Triton-X-100"
  },
  {
    "caption": "d, Immunohistochemistry of liver sections from six-week-old Atg5flox/flox; Mx1-Cre mice at the indicated time points after pIpC injection, using anti-ubiquitin antibodies. Arrowheads indicate ubiquitin-positive inclusion bodies. Scale bar, 25 µm.",
    "molecules": "pIpC"
  },
  {
    "caption": "Soft agar colony formation assay for HFF (A-B) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in HFF cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.",
    "molecules": "soft agar, Soft agar"
  },
  {
    "caption": "Soft agar colony formation assay for MKN28 (C-D) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in MKN28 cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.",
    "molecules": "Soft agar, soft agar"
  },
  {
    "caption": "(F)αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41.",
    "molecules": "P32, GDP"
  },
  {
    "caption": "(G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3).",
    "molecules": "GDP"
  },
  {
    "caption": "(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta.",
    "molecules": "Torin"
  },
  {
    "caption": "(B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA.",
    "molecules": "Torin"
  },
  {
    "caption": "(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).",
    "molecules": "P32, ATP"
  },
  {
    "caption": "(B) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.",
    "molecules": "tetrazolium"
  },
  {
    "caption": "(A-B)  Human fibroblasts (BJ) were treated with vehicle (water for 8 times 24 hours) or abemaciclib (1 μM for 8 times 24 hours). The population doubling over the 8 days treatment is plotted (A). 1 or 8 dpt, cells were incubated with  EdU for 20 hours and stained and quantified (n=9 samples from 3 independent experiments; scale bar, 150 μm) (B). Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001. dpt, days post treatment.",
    "molecules": "EdU, abemaciclib"
  },
  {
    "caption": "(C) Chromatin was extracted from BJ  cells treated with vehicle or  abemaciclib (1 μM for 8 times 24 hours) or nutlin-3a (10 μM for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) and ChIP  assays using an antibody against  p53 were performed. qRT-PCR was performed using primers amplifying promoter regions of CDKN1A, GADD45A and MDM2 genes containing  p53 binding sites. Values indicate fold enrichment relative to the vehicle group (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "abemaciclib, doxorubicin, nutlin-3a"
  },
  {
    "caption": "(E) 3*103 BJ cells of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (1 μM) and stained with 0.2%  crystal violet 8 dpt (n=3 independent experiments). (F) 8 dpt, treated  BJ cells were incubated with EdU for 20 hours and stained (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "EdU, abemaciclib, crystal violet"
  },
  {
    "caption": "(G) 3*103 mouse dermal fibroblasts (MDFs) of the indicated genotypes were re-plated after vehicle or abemaciclib treatment (4 μM for 8 times 24 hours) and stained with 0.2% crystal violet 8 dpt (n=3 independent experiments). (H) 8 dpt, treated MDFs were incubated with EdU for 20 hours and stained  (n=9 samples from 3 independent experiments). ( Data information: Data are means ±SD. Unpaired student t-test *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "EdU, abemaciclib, crystal violet"
  },
  {
    "caption": "(I) RNA was isolated from vehicle or abemaciclib (1 μM) treated scramble/shp53 BJ cells and mRNA for the indicated genes quantified by qRT-PCR relative to  tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "abemaciclib"
  },
  {
    "caption": "p16-3MR  mice were treated with vehicle (PBS, 7 consecutive days),  doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). n=6  mice/group. 14 dpt, bioluminescence was visualized and quantified by the IVIS spectrum in vivo imaging system, as shown by representative bioluminescence images (A) and quantification (B). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.",
    "molecules": "abemaciclib, doxorubicin, PBS"
  },
  {
    "caption": "p16-3MR  mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). n=6 mice/group. RNA isolated from  kidneys and mRNA encoding p16 quantified by qRT-PCR (C). SA-β-gal activities in vehicle-, doxorubicin- or  abemaciclib-treated mouse kidney sections at 15 dpt quantified (E). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.",
    "molecules": "abemaciclib, doxorubicin, PBS"
  },
  {
    "caption": "(G) RNA was isolated from abemaciclib ±  pifithrin-α  treated  kidneys  and mRNA encoding p16  and p21 genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.",
    "molecules": "abemaciclib, pifithrin-α"
  },
  {
    "caption": "For the  doxorubicin or abemaciclib  treated  mice (n=6 mice/group), Percentage of T cells, B cells, granulocytes and macrophages were determined by flow cytometry  analysis (N). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.",
    "molecules": "abemaciclib, doxorubicin"
  },
  {
    "caption": "(A) 8 dpt, cells were  stained for γ-H2AX  (scale bar, 60 μm) and quantified (vehicle=288 cells, doxorubicin=280 cells,  palbociclib=276 cells, abemaciclib=266 cells from 3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant. Doxo, doxorubicin.  Abe, abemaciclib. dpt, days post treatment.",
    "molecules": "Abe, abemaciclib, Doxo, doxorubicin, palbociclib"
  },
  {
    "caption": "(B) BJ cells transduced with a NF-κB reporter were treated with vehicle, doxorubicin, paclitaxel (50 nM for 24 hours),  palbociclib  (1 μM for 8 times 24 hours) or abemaciclib and luciferase activity measured 8 dpt.  TNFα treatment was used as positive control (n=3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.",
    "molecules": "abemaciclib, doxorubicin, paclitaxel, palbociclib"
  },
  {
    "caption": "(G) Proteins in the serum-free  conditioned media (CM) collected from doxorubicin- or abemaciclib-treated  BJ fibroblasts  (8 dpt) were measured by cytokine array. Purple boxes indicated internal positive controls from each membrane; Red boxes highlighted the most typical SASP factors.",
    "molecules": "abemaciclib, doxorubicin"
  },
  {
    "caption": "p16-3MR mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or  abemaciclib (50 mg/kg, 7 consecutive days). RNA isolated from kidneys and mRNA encoding indicated NF-κB  associated genes (J) quantified by qRT-PCR. n=6 mice/group. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.= Not significant.",
    "molecules": "abemaciclib, doxorubicin, PBS"
  },
  {
    "caption": "p16-3MR  mice were treated with vehicle (PBS, 7 consecutive days), doxorubicin (5 mg/kg, 3 consecutive days) or abemaciclib (50 mg/kg, 7 consecutive days). From a subset of the mice, RFP+ cells were sorted from the  renal cortex using  FACS and mRNA encoding indicated NF-κB associated pro-inflammatory genes quantified by qRT-PCR. The values of fold change over vehicle were plotted in the heatmap. n=4 mice/group (K).",
    "molecules": "abemaciclib, doxorubicin, PBS"
  },
  {
    "caption": "(B)  Human BJ fibroblasts,  hTERT-RPE1 epithelial cells  and lung mesenchymal stem cells were treated with vehicle (DMSO for 8 times 24 hours) or doxorubicin (250 nM for 24 hours) or paclitaxel (50 nM for 24 hours) or palbociclib (1 μM for 8 times 24 hours) or abemaciclib  (1 μM for 8 times 24 hours). At 8 dpt, RNA was isolated and mRNA for the indicated  p53-associated SASP genes quantified by qRT-PCR relative to  tubulin (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "abemaciclib, DMSO, doxorubicin, paclitaxel, palbociclib"
  },
  {
    "caption": "(D and E) RNA was isolated from vehicle, abemaciclib (1 μM) (D) or palbociclib (1 μM) (E) treated scramble/shp53 BJ cells and quantified by qRT-PCR for  IGFBP3 or LIF genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "abemaciclib, palbociclib"
  },
  {
    "caption": "(F) RNA isolated from  kidneys treated with  doxorubicin or abemaciclib and mRNA encoding indicated  p53-associated SASP genes quantified by qRT-PCR (n=6 mice/group). (G) RNA was isolated from kidneys of  p16-3MR mice treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days), and mRNA encoding  p53 associated SASP genes was quantified by qRT-PCR and normalized on  tubulin (n=5  mice/group). ( Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "abemaciclib, doxorubicin, pifithrin-α"
  },
  {
    "caption": "(I) RNA was isolated from vehicle or nutlin-3a (1 μM for 8 times 24 hours) treated BJ  cells at 8 dpt and quantified by qRT-PCR for p53 target cell cycle genes, SASP genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "nutlin-3a"
  },
  {
    "caption": "(J)  Mouse dermal fibroblasts  (MDF) or MDF-p53-/- fibroblasts were transfected with empty vector (EV) or p53-overexpressing vector (p53OE), then the cells were treated with vehicle (water) or doxorubicin (250 nM for 24 hours) and qRT-PCR  was performed for Igfbp3  and Lif  genes (n=3 independent experiments). Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001. EV, empty vector",
    "molecules": "doxorubicin"
  },
  {
    "caption": "(A) MCF7  cells were incubated with serum-free  CM collected from treated BJ fibroblasts (8 dpt) containing  EdU for 45 hours and EdU+ cells quantified (n=9 samples from 3 independent experiments). Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. CM, conditioned media.",
    "molecules": "EdU"
  },
  {
    "caption": "(B)  MCF7 cells migrated through the pores of trans-well after 24 hours incubation with CM (8 dpt; scale bar, 1 mm; n=9 samples from 3 independent experiments) were  stained with 0.2% crystal violet and quantified. Data information: Data are means ±SD. One-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant. CM, conditioned media.",
    "molecules": "crystal violet"
  },
  {
    "caption": "(H and I) 2*105 cells of vehicle-treated (water for 8 times 24 hours) mouse dermal fibroblasts  (MDF) were injected into the  left flank  of the p16-3MR  mice. Doxorubicin-induced (250 nM for 24 hours) (H) or abemaciclib-induced (4 μM for 8 times 24 hours) (I) MDFs were injected into the  right  flank  of the same animal. 7 dpt, the above-mentioned mice  were injected with coelenterazine and bioluminescence from the p16-3MR  mouse was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified. Lower panel, scheme of experimental design (n=4 mice/group). Data information: Data are means ±SD. Paired student t-test *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.",
    "molecules": "abemaciclib, Doxorubicin, coelenterazine"
  },
  {
    "caption": "(J) Equal amount (2*105 cells) of doxorubicin-induced (250 nM for 24 hours, right  flank) and abemaciclib-induced (4 μM for 8 times 24 hours, left flank) senescent MDF-3MR cells were subcutaneously injected into the same wild-type  mice. At 1 dpt, 7 dpt and 15 dpt, the above-mentioned mice were injected with coelenterazine and bioluminescence from the injected MDF-3MR cells was visualized/quantified by the IVIS spectrum in vivo imaging system and quantified (n=4 mice/group). Upper panel, scheme of experimental design. Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.",
    "molecules": "abemaciclib, doxorubicin, coelenterazine"
  },
  {
    "caption": "C-D. ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination (B), whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination (C). Protein complexes were assembled in vitro on ice in the presence of dsDNA (32 bp) and their in vitro ubiquitination at 30o C was subsequently monitored in a time-course: at indicative time-points, aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies (Top). For each protein complex, data-points from three replicate experiments (three technical replicates) were used in fitting to a one-phase association model (Bottom).",
    "molecules": "dsDNA"
  },
  {
    "caption": "A. USP1-UAF1-mediated deubiquitination of V5-FANCI and FANCD2 was assessed in the absence or presence of DNA (51 bp), when ubiquitinated versions of these proteins were in isolation (IUb and D2Ub) or within singly/doubly ubiquitinated ID2 complexes (IUbD2, IUbD2Ub and ID2Ub). At indicated time-points, aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies.",
    "molecules": "DNA, Ub"
  },
  {
    "caption": "Alanine mutagenesis of key FANCD2's residues (H209, V243 and P244) participating in the extended FANCD2-ubiquitin interface (IUbD2-DNA and IUbD2Ub-DNA structures), results in enhanced FANCI deubiquitination by USP1-UAF1. C. FANCI deubiquitination by USP1-UAF1 (incubation with 50 nM USP1-UAF1 for 30 minutes at room temperature) in the presence of DNA and, in the absence or presence of wild-type (D2WT) or mutant (D2H209A, VP243AA) FANCD2. Replicate residual ubiquitination values (three technical replicates) and statistically significant changes (t-test and one-way ANOVA test with Bonferroni correction) are shown.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "D. FANCI was ubiquitinated with DyLight-680 labelled ubiquitin (IUb-DL680) and its deubiquitination by USP1-UAF1 (incubation with 100 nM USP1-UAF1 for 20 minutes at room temperature), was assessed at increasing concentrations (20, 200 or 200 nM) of, either wild-type (D2WT), or mutant (D2H209A, VP243AA) FANCD2. Experiment was repeated three times (technical replicates), and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb-DL680 at 20 nM FANCD2 (average from six values, for both D2WT and D2H209A, VP243AA). Mean (with range) fold-increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration (t-test and two-way ANOVA test with Bonferroni correction) are shown.",
    "molecules": "DL680, DyLight-680, Ub, ubiquitin"
  },
  {
    "caption": "B Relative ATX gene expression in the MCD-diet-fed (n=9) compared to control mice  (n=9) (***P < 0.001); CCl4  (n=8) compared to control mice  (n=5) (*P < 0.05). Bars represent the mean ± SEM, statistical analysis was performed by two-tailed student's t-test. *P < 0.05, ***P < 0.001 denotes significance versus respective healthy controls.",
    "molecules": "CCl4"
  },
  {
    "caption": "D Representative images (scale=50μm) of ATX stained liver  sections (using AEC  chromogen denoted by red color staining  or DAB  chromogen denoted by brown color staining; nuclei  were stained  blue with hematoxylin) from MCD-diet-fed NASH  (n=9) and CCl4-induced  liver  fibrosis (n=8) mouse  models compared to respective controls (n=9 or n=5).",
    "molecules": "DAB, AEC, hematoxylin, CCl4"
  },
  {
    "caption": "A Structural binding  of type I ATX  inhibitor PF8380   to ATX; Potency (IC50) of PF8380  for inhibiting  the catalysis of LPC to LPA. Bars represent the mean + SEM, n=3. B Structural binding  of type IV ATX  inhibitor Cpd17  to ATX; Potency (IC50) of Cpd17  for inhibiting  the catalysis  of LPC to LPA. Bars represent the mean + SEM, n=3.",
    "molecules": "Cpd17, LPA, PF8380"
  },
  {
    "caption": "A Representative images (scale=10µm) and quantification of HepG2 cells stained  with  oil-red-O after 48 hours of incubation with or without 200μM palmitate  with or without 1µM of PF8380  or Cpd17. Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. ##P < 0.01 denotes significance versus control (non-palmitate) and *P < 0.05 denotes significance versus vehicle (palmitate); ns=non-significant.",
    "molecules": "Cpd17, palmitate, oil-red-O, PF8380"
  },
  {
    "caption": "B Representative images (at 0h and 24h) (scale=50µm), and quantitative analysis (after 24h) of migration  by control (M0) and LPS- and IFNγ-induced M1 RAW264.7 macrophages  treated with medium alone, PF8380  (1μM) or Cpd17  (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus M0  (non-stimulated macrophages) and *P < 0.05 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.",
    "molecules": "Cpd17, LPS, PF8380"
  },
  {
    "caption": "C Relative gene expression (normalized with GAPDH) for CCL2   and iNOS  in control (M0) and LPS- and IFNγ-induced M1 RAW 264.7 macrophages  treated with medium alone, PF8380  (1μM) or Cpd17  (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus M0 (non-stimulated macrophages) and *P < 0.05, **P < 0.01 denotes significance versus vehicle (LPS- and IFNγ-induced  M1 macrophages); ns=non-significant.",
    "molecules": "Cpd17, LPS, PF8380"
  },
  {
    "caption": "D Relative gene expression (normalized with 18s RNA) for CCL2  and iNOS  in control (M0) and LPS-induced PMA-differentiated human THP1 macrophages  treated with medium alone, PF8380  (1μM) or Cpd17  (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ##P < 0.01, ###P < 0.001 denotes significance versus M0 (non-stimulated macrophages) and **P < 0.01, ***P < 0.001 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.",
    "molecules": "Cpd17, LPS, PF8380, PMA"
  },
  {
    "caption": "A  Collagen I  and α-SMA stained images (scale=50µm) of control and TGFβ-activated LX2  cells with or without PF8380  (1μM) or Cpd17  (1μM) (n=3).",
    "molecules": "Cpd17, PF8380"
  },
  {
    "caption": "B Relative Collagen I, α-SMA and PDGFβR gene expression (normalized with GAPDH) in control and TGFβ-activated LX2  cells with or without PF8380  (1μM) or Cpd17  (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by two-tailed student's t-test. #P < 0.05, ###P < 0.001 denotes significance versus control (non-TGFβ  cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.",
    "molecules": "Cpd17, PF8380"
  },
  {
    "caption": "C,D Representative images (at 0h and 24h) (scale=100µm) (C) and quantitative analysis (after 24h) (D) of migration  by control and TGFβ-activated LX2  cells with or without PF8380  (1μM) or Cpd17  (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ   cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.",
    "molecules": "Cpd17, PF8380"
  },
  {
    "caption": "E,F Representative images (E) and quantitative analysis (F) showing 3D collagen  gel contractility  of control and TGFβ-activated LX2  cells with or without PF8380  (1μM) or Cpd17  (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ  cells) and **P < 0.01, ****P < 0.0001 denotes significance versus vehicle (TGFβ-activated cells).",
    "molecules": "Cpd17, PF8380"
  },
  {
    "caption": "B Top panel shows  time-course  stimulation to LPA  and uninhibited or inhibited LPC-treated ATX followed by RhoA activation, measured as a YFP/CFP  fluorescent ratio. Bottom panel depicts quantitation of the burst response to the stimulants. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05, ***P <0.001 denotes significance versus ATX+LPC  treated cells.",
    "molecules": "LPA, LPC"
  },
  {
    "caption": "C Representative images and quantification showing  Western-blot analysis for p-Akt and p-ERK  normalized with β-actin in control, LPC, LPA, ATX, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17  stimulated LX2  cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC  treated cells.",
    "molecules": "Cpd17, LPA, LPC, PF8380"
  },
  {
    "caption": "D Representative images and quantification showing Western-blot analysis for LPAR1  on the cell surface and in lysate, normalized with β-actin in lysate, in LPC, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17  stimulated LX2  cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC  treated cells.",
    "molecules": "Cpd17, LPC, PF8380"
  },
  {
    "caption": "B Liver weight  to total body weight ratio from control (n=5), CCl4  (n=5) and CCl4+Cpd17  (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (healthy mice); ***P < 0.001 denotes significance versus CCl4 mice.",
    "molecules": "Cpd17, CCl4"
  },
  {
    "caption": "C Serum ALT  levels from control (n=5), CCl4  (n=5) and CCl4+Cpd17  (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ###P < 0.001 denotes significance versus control (healthy mice); P = 0.26 denotes significance versus CCl4 mice.",
    "molecules": "Cpd17, CCl4"
  },
  {
    "caption": "D,E Representative images (scale=100μm) (D) and quantitative analysis (E) of liver  sections from control (n=5), CCl4  (n=5) and CCl4+Cpd17  (n=4) mice  and stained  with H&E, collagen-I and F4/80. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); P = 0.098 or P = 0.08 denotes significance versus CCl4 mice.",
    "molecules": "Cpd17, CCl4"
  },
  {
    "caption": "D, Representative images (scale=100μm) (D) of liver  sections from control (n=5), MCD  (n=6) and MCD+Cpd17 (n=6) mice  and stained  with H&E, oil-red-O and Collagen I.",
    "molecules": "Cpd17, oil-red-O"
  },
  {
    "caption": "quantitative analysis (E) of liver  sections from control (n=5), MCD  (n=6) and MCD+Cpd17  (n=6) mice stained  with oil-red-O  and Collagen I. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); *P < 0.05, **P < 0.01, ***P < 0.001 denotes significance versus MCD-diet fed mice.",
    "molecules": "Cpd17, oil-red-O"
  },
  {
    "caption": "(A) Tamoxifen schedule: pulse of the pregnant dam at E9.5, harvest at E10.5. Representative fluorescent immune-staining of E10.5 Sox7iECKO mutants and sibling control embryos. The endothelium is delineated by endomucin (EMCN) (blue) and endothelial cells by ETS-related gene (ERG) (red), SOX7 positive cells are shown in white. (Top right graph) The percentage of SOX7 positive endothelial cells in different vascular beds, quantified from 10 sequential transverse sections in 2 control and 3 Sox7iECKO mutants. (Bottom right panel) Graph indicating the levels of Sox7 transcript in sibling controls and Sox7iECKO mutants. Mean ± SEM; scored sibling control, n=9; Sox7iECKO mutants, n=7; Mann-Whitney U-test. P<0.0005 (***).",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "(A-D) SOX7 transcriptionally activates Notch effector, HEY1, to repress Vegfc. (A) qPCR on FAC-sorted PECAM+CD45-endothelial cells of Sox7iECKO mutants and sibling controls at E14.5 after Cre induction with tamoxifen at E11.5 and E12.5. Expression is normalised to the endothelial marker Pecam and Cdh5. Scored sibling controls, n=9; Sox7iECKO mutants, n=8. (B-C) qPCR on (B) human arterial endothelial cells (HUAECs) and (C) human venous endothelial cells (HUVECs) transfected with SiSOX7 or SiCTRL for 17 h. In addition to HEY1, DLL4 levels were also downregulated in the human cell line experiments. Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. (D) HEY1 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc and either EV (empty vector) or HEY1 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3 independent repeats of the same experiment. Mean ± SEM; t-test. P<0.05 (*); P<0.005 (**); P<0.0005 (***).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(a-c) HeLa cells were either left untreated, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunostained, or immunoblotted using antibodies as shown. Co-localization panels show an overlap between mTOR and LAMP1 signals. Note that changes in the positioning of lysosomal mTOR (quantified as the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles; a,c) correlate with mTORC1 activity (levels of phosphorylated S6K relative to the total S6K; b).",
    "molecules": "amino-acid"
  },
  {
    "caption": "(d) Visualization of Akt activated in response to recovery after serum starvation. After nutrient recovery, LAMP1-positive vesicles localize to peripheral regions with higher concentrations of phospho-Akt. DAPI, 4,6−diamidino−2−phenylindole. For all panels, values are means ± s.e.m. of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons are not significant (n.s.). Representative maximum-intensity projections of serial confocal optical sections are shown. Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a,b) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability. Cells were treated as in Fig. 1a followed by incubation with dimethylsulphoxide (vehicle) or with nocodazole during the last 2 h before fixation/lysis. Samples were analysed by immunofluorescence (a) or immunoblotting (b). Quantification of phopho-S6K levels is shown in (b).",
    "molecules": "dimethylsulphoxide, Nocodazole, nocodazole, nutrient"
  },
  {
    "caption": "(a-c) HeLa cells transfected with ARL8B or KIF2 siRNA, or with ARL8B overexpression construct (non-targeting siRNA and empty pcDNA vector used as transfection controls), were either left untreated, serum/amino-acid starved for 5 h, or starved and then recovered in amino-acid- and FBS-containing medium for 30 min. Cells were immunostained using LAMP1 antibody (a) and the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles was quantified (b) or subjected to immunoblotting (c) using antibodies as shown. Quantification of phospho-S6K levels relative to the total S6K is shown in (c). Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "amino-acid"
  },
  {
    "caption": "(b-d) Changing pHi from 7.1 to 7.7 is sufficient to affect localization of lysosomes and mTORC1 activity. pHi was titrated in full tissue-culture medium containing nigericin, which enables changes in pHi to be forced by altering pH in the medium, followed by immunostaining (b,c) or western blotting (d) using antibodies as shown.",
    "molecules": "nigericin"
  },
  {
    "caption": "(f,g) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions. Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation-recovery (f) or to 1 h changes of pHi in full tissue-culture medium containing nigericin (g) are shown.",
    "molecules": "nigericin, nutrient"
  },
  {
    "caption": "(h,i) Effect of nutrients and pHi on binding of ARL8 and KIF2 to polymerized microtubules. HeLa cells treated as in (f) and (g), followed by isolation of polymerized microtubule fraction and western blotting. The asterisk indicates a nonspecific band. For all panels values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "nutrients"
  },
  {
    "caption": "(b) Serum and amino-acid starvation of HeLa cells for 5 h reduces LC3-II levels (above), but increases autolysosome numbers detected using tfLC3 (below). With tfLC3, GFP- and RFP-positive puncta represent autophagosomes before lysosomal fusion, whereas RFP-positive/GFP-negative puncta represent autolysosomes-GFP is more rapidly quenched by low lysosomal pH (see Methods). Increased autolysosomes indicate enhanced starvation-induced flux of LC3 to lysosomes.",
    "molecules": "amino-acid"
  },
  {
    "caption": "(c) ARL8B knockdown increases autophagosomal synthesis. siRNA-transfected HeLa cells were incubated for 48 h, then left untreated or incubated with bafilomycin A1. LC3-II levels versus actin were quantified (bottom graphs). Asterisk: nonspecific band.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(e) HeLa cells were co-transfected with either ARL8B overexpression construct or siRNA (non-targeting siRNA and empty peGFP vector were transfection controls) together with mCherry-LC3 for 48 h. After fixation, cells were stained for endogenous LAMP1 and DNA (DAPI). Representative maximum-intensity projections of serial confocal optical sections are shown. Co-localization panels show overlapping mCherry-LC3 and LAMP1 signals. (f-h) Quantification of autophagosome-lysosome fusion in HeLa cells. Percentages of autolysosomes (positive for both mCherry-LC3 and LAMP1) to autophagosomes (positive for mCherry-LC3 and negative for LAMP1) were quantified. In (h), we analysed cells treated for 2 h with nocodazole before fixation, which dispersed the perinuclear lysosomal cluster (see Fig. 2a). (i) Autophagosomal and autolysosomal numbers in tfLC3-expressing cells after ARL8B overexpression and knockdown (Supplementary Fig. S5o shows representative cells.). In (f-i) we analysed 20 cells per group in three independent experiments. Values are means ± s.e.m of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons not significant (n.s.). Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "DNA, nocodazole"
  },
  {
    "caption": "D. [35S]Jac1 was imported into wild-type (WT) and Tim50-depleted mitochondria for indicated times and samples were treated with Proteinase K. p, precursor; m, mature.",
    "molecules": "35S"
  },
  {
    "caption": "E. Quantification of [35S]Jac1 import into wild-type (WT) and Tim50-depleted mitochondria. The amount of imported protease-protected protein in WT mitochondria at 15 min was set to 100%; error bars indicate SEM (n=3).",
    "molecules": "35S"
  },
  {
    "caption": "C. Jac1488 and pAmJac1 conjugated to DyLight 488, 680, and 800 were imported into wild-type mitochondria for indicated times and samples were treated with Proteinase K. Prec., purified precursor protein; p, precursor; m, mature.",
    "molecules": "DyLight 488"
  },
  {
    "caption": "E. Jac1488 and pImJac1 conjugated to DyLight 488, 680 and 800 were imported into wild-type mitochondria for indicated times and samples treated with Proteinase K. Prec., purified precursor protein; p, precursor; m, mature.",
    "molecules": "DyLight 488"
  },
  {
    "caption": "A. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were separated by SDS-Page and analyzed by fluorescence scanning or digital autoradiography. Prec., purified precursor protein; p, precursor; m, mature protein.",
    "molecules": "35S"
  },
  {
    "caption": "B. Import of Atp5488 with increasing concentrations of CCCP to study dependency of import on membrane potential (∆ψ). P, precursor; m, mature protein.",
    "molecules": "CCCP"
  },
  {
    "caption": "D. [35S]Atp5 was imported with increasing CCCP concentrations as described in B. Samples were separated by SDS-PAGE and analyzed by digital autoradiography. E. Quantification of import of [35S]Atp5 with increasing CCCP concentrations. The amount of imported protease-protected protein in the absence of CCCP was set to 100%; error bars indicate SEM (n=3).",
    "molecules": "CCCP, 35S"
  },
  {
    "caption": "G. Atp5488 (left panel) or [35S]Atp5 (right panel) were imported into wild-type and atp21∆ mitochondria for indicated times and in the presence or absence of a membrane potential (∆ψ). After proteinase K treatment, samples were solubilized in digitonin buffer and separated by BN-PAGE. Aliquots of the samples was analyzed by SDS-PAGE (lower panels). Subsequently proteins were visualized by fluorescence scanning or digital autoradiography. Complex V dimer, (CVD) and monomer (CVM). Prec., purified precursor ; p, precursor; m, mature protein.",
    "molecules": "digitonin, 35S"
  },
  {
    "caption": "Aci2  was grown with alanine  and glutamate  supplied at 25 different ratios from 1:16 to 16:1 with the total supply kept constant at 0.1%w/ Data Information: Data were collected at 600nm.",
    "molecules": "alanine, glutamate"
  },
  {
    "caption": "The population size at which Aci2's  diauxic shift occurred is linearly correlated to the alanine  supply, indicating that Aci2  initially consumes almost entirely alanine. Data Information: Data were collected at 600nm.",
    "molecules": "alanine"
  },
  {
    "caption": "Diauxic  lag times were fit from the monoculture data and are plotted as circles.  Lag times  could not be fit for conditions with too little growth  on the remaining glutamate. The  lag times  used in the modeling are shown as lines through the data.",
    "molecules": "glutamate"
  },
  {
    "caption": "A) Ten-fold serial dilutions from overnight cultures of wild-type and cdc14-1 cells dropped and grown on solid rich media or media containing MMS at 25, 28, 30 or 33ºC. Note that cdc14-1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild-type cells.",
    "molecules": "MMS"
  },
  {
    "caption": "B) Ten-fold serial dilution from mid-log phase cultures of wild-type and cdc14-1 cells grown on solid rich media or media containing mock DMSO (as non-treated control), 4NQO, HU, phleomycin and benomyl at 30ºC. Note that cdc14-1 cells present a market sensitivity to all DNA damaged agents tested.",
    "molecules": "4NQO, benomyl, DMSO, HU, phleomycin"
  },
  {
    "caption": "C) Left panel: Schematic representation showing relevant genomic structure of the strain used to assess intra-chromosomal repair. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of intra-chromosomal repair in wild-type and cdc14-1 cultures at the semipermisive temperature. After DSB formation by the expression of the HO, glucose was added to repress it, thus allowing repair with HM donor sequences. Genomic DNA was digested with StyI, separated on agarose gel and blotted. Blots were hybridized with a probe corresponding to the MATa-distal sequence. A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time-point. Immunoblot analysis of Rad53 during mating type switching experiments is shown. Coomassie blue staining is depicted as loading control. Graphs show quantification of gene conversion (GC) leading to the re-establishment of MATa or switching to MAT and DSB formation. The data was normalized with the actin loading control. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.",
    "molecules": "glucose"
  },
  {
    "caption": "A) Left panel: Schematic diagram displaying relevant genomic structure of the strains used to measure inter-chromosomal DNA repair efficiency. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that crossover and non-crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 mutant strains carrying the inter-chromosomal gene conversion assay. Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression, thus producing a DSB on chromosome V. Samples were taken at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were hybridized with a MATa-only DNA sequence and an actin probe was used as loading control. Asterisk denotes an overexposed film to visualize crossover formation. Immunoblot of Rad53 was performed as previously described. Coomassie staining is shown as a control for loading. Graphs show the mean ± S.D. of gene conversion, DSB induction and crossover vs non-crossover ratio from three independent experiments. All data was normalized using actin as loading control. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.",
    "molecules": "galactose"
  },
  {
    "caption": "B) Left panel: Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 cells harbouring the repair pathway choice assay. Cells were grown overnight and HO induction was attained through addition of galactose for 1.5 hour at semipermisive temperature. After induction of the HO, glucose was added to repress the HO expression and samples were taken to analyse repair efficiency. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Blots were hybridized with a probe corresponding to the MATa-only DNA sequence. A second probe to the HIS3gene was used to control the amount of genomic DNA loaded at each time-point. Graphs show quantification of mating type switching by gene conversion (HR), restoration of MATa (NHEJ) and the kinetics of the DSB induction. All data was normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A) Live-cell imaging of Cdc14-YFP and Cnm67-RFP asynchronous cells treated with the DNA damage agent phleomycin. Cells were grown overnight and phleomycin was added to a final concentration of 1μM. Samples were taken at different intervals to determine Cdc14 localization. Cnm67 was used as SPB reporter to determine spindle length. Scale bar: 3μm. Graphs represent the average percentage ± SD from three independent experiments of cells arrested in metaphase (left graph) and with Cdc14 signal at the nucleoplasm (right graph).",
    "molecules": "phleomycin"
  },
  {
    "caption": "A) Western blot analysis to analyse Rad53 and Net1 phosphorylation levels under these conditions are shown. Note that Rad53 is rapidly phosphorylated in response to phleomycin and maintained throughout the entire experiment. Detection of Net1 phosphorylation levels was achieved by using Phos-tag polyacrylamide gels. Coomassie staining is showed as loading control.",
    "molecules": "phleomycin"
  },
  {
    "caption": "C) Cells expressing Cdc14-YFP and Cnm67-RFP were grown overnight and blocked in G2/M by using nocodazole prior phleomycin treatment (1μM). Samples were taken every 30 minutes to follow Cdc14 re-localization. The efficiency of the arrest was determined as in Fig 3A. Scale bar: 3μm. Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion (left graph) and cells arrested in metaphase (right graph) from three independent experiments.",
    "molecules": "nocodazole, phleomycin"
  },
  {
    "caption": "D) Live-cell imaging of Cdc14 localization in the presence of a non-reparable DSB at the MAT locus. Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media, thus producing a DSB on chromosome III. Cdc14, the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 (used as DSB reporter) were labelled with the green, red and cyan fluorescent proteins respectively. Micrographs display the maximum projection of 9 planes showing Cdc14 co-localizing with the DSB and the SPBs in response to DNA damage (arrows). Scale bar: 3μm. Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction (left graph) and the percentage of Cdc14-SPB-DSB co-localization after 5h in galactose (right graph).",
    "molecules": "galactose"
  },
  {
    "caption": "E) Western blot showing Net1-6HA phosphorylation state before and after the generation of a DSB induced by the HO expression. A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control. The phospho-bands were resolved by using Phos-tag polyacrylamide gels as in Fig 3A and Fig 3B. Coomassie staining is depicted as loading control.",
    "molecules": "galactose"
  },
  {
    "caption": "C) An RFP-marked I-SceI recognition site is re-localized to the SPBs after expressing the endonuclease. Cells harbouring a TetO array adjacent to the I-SceI site and a plasmid containing the endonuclease under the control of the galactose promoter were grown overnight in SC-Ade prior galactose induction. To check reproducibility with previous results a Rad52-CFP was used. Cnm67 was labelled with the GFP to be used as SPB reporter. D1, D2 and D3 distances were measured in wild-type and cdc14-1 mutants before and after endonuclease expression by using both TetI-RFP and Rad52-CFP foci using the maximum projection of nine z-planes images. Graphs represent the average of three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. A representative picture of a wild-type strain depicting TetI-Rad52-SPB interaction is shown. Scale bar: 3μm.",
    "molecules": "galactose"
  },
  {
    "caption": "A) Identification of Cdc14 phospho-targets during the DNA damage response by mass spectrometry analysis. Wild-type and cdc14-1 cells were grown overnight and blocked in G2/M by using nocodazole to avoid cell cycle-dependent changes in protein phosphorylation between both strains. After the block was attained, cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h. Differential phosphorylation of phospho-peptides detected between the wild-type and cdc14-1 were grouped into broad categories depending on the molecular function of the proteins. The table includes the DNA damage and checkpoint-related proteins with Cdc14-dependent hyper-phosphorylated status and the relative ratio between the wild-type and cdc14-1 during the DNA damage response. Red and blue indicate relative amount of the residue phosphorylation between both strains (Red, high; Blue, low).",
    "molecules": "nocodazole"
  },
  {
    "caption": "B) Cdc14 dephosphorylates Spc110 in response to a DSB. Spc110 was tagged with the 6HA epitope in wild-type, cdc14-1 and spc110S36-91A backgrounds. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as loading control.",
    "molecules": "galactose, raffinose"
  },
  {
    "caption": "C) Lambda-phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response. Cells were grown overnight in raffinose containing media (line 1) and incubated with galactose for 2.5 hours to induce HO expression (line 2). Extracts from raffinose-containing cultures were treated with mock (line 3) or λ-PPase (line 4). All samples were separated in a Phos-Tag containing gel and blotted. Coomassie staining is shown as loading control.",
    "molecules": "galactose, raffinose"
  },
  {
    "caption": "D) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response. Spc110 was labelled with the 3HA tag in both wild-type and esp1-1 background strains. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce the HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as a control for loading.",
    "molecules": "galactose, raffinose"
  },
  {
    "caption": "A) Cells treated with nocodazole are affected in DSB-SPB interaction. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. Diagram depicts the genomic background used. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide (DMSO) were scored and plotted. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture is shown. Scale bar: 3μm.",
    "molecules": "DMSO, nocodazole"
  },
  {
    "caption": "B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa-distal sequence and the HIS3 gene (as loading control) were used. Graphs show the quantification of MATa restoration (NHEJ), mating type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test.",
    "molecules": "DMSO, galactose, glucose, nocodazole"
  },
  {
    "caption": "B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. FACS analyses are shown.",
    "molecules": "DMSO, galactose, glucose, nocodazole"
  },
  {
    "caption": "A) Growth sensitivity to MMS and phleomycin of a Cdk phospho-deficient mutant of Spc110. Ten-fold serial dilutions from overnight cultures of wild-type, spc110S36-91A and a cdc14-1 cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.",
    "molecules": "DMSO, MMS, phleomycin, Phleomycin"
  },
  {
    "caption": "D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.",
    "molecules": "galactose"
  },
  {
    "caption": "D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. FACS profiles for DNA content are included. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.",
    "molecules": "DNA, galactose"
  },
  {
    "caption": "A) DNA damage sensitivity to MMS and phleomycin of a Cdk phospho-mimic mutant of Spc110. Ten-fold serial dilutions from mid-log phase cultures of wild-type, spc110S36-91A and spc110S36-91D cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.",
    "molecules": "DMSO, MMS, Phleomycin"
  },
  {
    "caption": "D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.",
    "molecules": "galactose"
  },
  {
    "caption": "D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. FACS profiles for DNA content are displayed. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.",
    "molecules": "DNA, galactose"
  },
  {
    "caption": "PI(5)P Regulates Autophagosome Biogenesis(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with carrier alone, or in combination with PI(5)P di-C16 at indicated concentrations for 1 hr, in the absence and presence of 400 nM BAF (treated in combination with lipids). Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we used (see Figure S1C). However, this was not a problem because one should relate LC3-II to tubulin (mean ± SEM).",
    "molecules": "BAF, PI(5)P di-C16"
  },
  {
    "caption": "(B and C) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells treated with DMSO or increasing concentrations of YM-201636 for 2 hr in the presence or absence of BAF (mean ± SEM).",
    "molecules": "BAF, DMSO, YM-201636"
  },
  {
    "caption": "(D and E) HeLa cells transiently transfected with Strawberry-ATG16L1 were treated with YM-201636 (100 nM, 2 hr) in HBSS, then fixed and stained for endogenous WIPI-2. Bar, 10 μm. (E) Quantification of ATG16L1 and WIPI2 structures per cell, n = 20 cells (mean ± SEM; n = 3 independent experiments; ∗∗∗p < 0.001, t test).",
    "molecules": "YM-201636"
  },
  {
    "caption": "(F) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells transfected with HA-ATG12 and ATG5 and treated with YM-201636 (100 nM, 2 hr) (mean ± SEM).",
    "molecules": "YM-201636"
  },
  {
    "caption": "(H and I) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in cells incubated with YM-201636 (100 nM, 2 hr) and loaded with exogenous PI(5)P for the last 1 hr in the presence or absence of BAF (mean ± SEM). See also Figure S2.",
    "molecules": "BAF, PI(5)P, YM-201636"
  },
  {
    "caption": "(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected for 5 days with two rounds of control, PI5P4K2A, 2B, or 2C siRNA either left untreated or treated with BAF (200 nM, 16 hr) (mean ± SEM).",
    "molecules": "BAF"
  },
  {
    "caption": "(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in SKNSH and MEFs treated for 2 hr with carrier alone or in combination with PI(5)P di-C16 (10 μM) in the absence and presence of BAF (mean ± SEM).",
    "molecules": "BAF, PI(5)P di-C16"
  },
  {
    "caption": "PI(5)P Triggers Autophagy in the Absence of VPS34 Activation(A and B) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells pretreated with Wm (200 nM, 2 hr), then loaded with 10 μM PI(5)P for 1 hr (in the presence of Wm and BAF) (mean ± SEM).",
    "molecules": "BAF, PI(5)P, Wm"
  },
  {
    "caption": "(C) Quantification of numbers of GFP-LC3 vesicles in HeLa cells stably expressing GFP-LC3 treated as in (A) and then shifted to starvation media (HBSS) or complete media (Basal) for 2 hr (in the presence of Wm) (mean ± SEM).",
    "molecules": "Wm"
  },
  {
    "caption": "(D) HeLa cells transfected with HA-ATG12 and ATG5 treated with 200 nM Wm as in (A) and subjected to western blot analysis with anti-HA antibody to detect free ATG12 and the ATG5-ATG12 complex (mean ± SEM). ATG5-ATG12 conjugation was checked for endogenous proteins in the same conditions using an anti-ATG12 antibody (bottom).",
    "molecules": "Wm"
  },
  {
    "caption": "(E and F) Western blot analysis of LC3-II and tubulin levels and quantification of LC3-II/tubulin ratio in HeLa cells transfected with control, PI5P4K2A, 2B, and 2C siRNA and treated with Wm (200 nM, 2 hr in the presence of BAF) (mean ± SEM).",
    "molecules": "BAF, Wm"
  },
  {
    "caption": "(G and H) Western blot analysis of free ATG12 and ATG5-ATG12 complex levels with anti-HA antibody in HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA, transfected with HA-ATG12 and ATG5 for last 16 hr and treated with Wm for 2 hr (mean ± SEM).",
    "molecules": "Wm"
  },
  {
    "caption": "(I) HeLa cells stably expressing GFP-LC3 treated with control, PI5P4K2A, 2B, and 2C siRNA, were pretreated with Wm for 2 hr in complete medium and then shifted in HBSS media for 2 hr (in the presence of Wm) (mean ± SEM).",
    "molecules": "Wm"
  },
  {
    "caption": "(J and K) HeLa cells stably expressing GFP-LC3 were transfected for 30 hr with myc-tagged empty vector, myc-MTMR3WT, and myc-MTMR3C413S, incubated for 4 hr in HBSS in the presence of BAF and in the presence or absence of Wm. Cells were fixed, stained with anti-myc antibodies, and imaged by confocal microscope. Asterisks indicate transfected cells. Quantification of numbers of GFP-LC3vesicles per cell is shown in (K) (mean ± SEM).",
    "molecules": "BAF, Wm"
  },
  {
    "caption": "(L and M) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, and RFP-LC3 for 30 hr were loaded with 10 μM PI(3)P for 1 hr in complete medium, and then fixed and imaged on confocal microscope. Bar, 10 μm. Quantification of cells (percentage of total) showing more than 10 RFP-LC3 vesicles in the different conditions from (J) is shown in (M); n = 200 cells (mean ± SEM). See also Figure S5.",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(A) HeLa cells transfected with GFP-WIPI2B or GFP-DFCP1 preloaded with indicated concentrations of PI(5)P for 1 hr, starved in HBSS for 1 hr, and then incubated with Wm in HBSS were tracked by time-lapse microscopy for 10 min after the addition of Wm. Quantification of WIPI2B or DFCP1vesicles (percentage of those at the starting time [T0]) during the treatments are shown in the graphs.",
    "molecules": "PI(5)P, Wm"
  },
  {
    "caption": "(B-D) HeLa cells treated with control, PI5P4K2A, 2B, and 2C siRNA were transfected with GFP-WIPI2B (B), GFP-WIPI2D (C), or GFP-DFCP1 (D); starved in HBSS (1 hr); and then incubated with Wm in HBSS. WIPI2 or DFCP1 structures were tracked and quantified as in (A).",
    "molecules": "Wm"
  },
  {
    "caption": "(E) Lysates from HeLa cells stably expressing GFP-WIPI2B were incubated with agarose beads coated with PI, PI(5)P, and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. Uncoated beads and lysates from HeLa cells stably expressing GFP alone were used as internal controls.",
    "molecules": "PI, PI(3)P, PI(5)P"
  },
  {
    "caption": "(F-H) Cell extracts from HeLa cells stably expressing GFP-WIPI2B were incubated for 3 hr with PI(5)P-containing liposomes (F and H) or PI(3)P-containing liposomes (G and H) before a pull-down experiment using the indicated beads. PS-containing liposomes were used as internal controls for both competition assays in (F and G).",
    "molecules": "PI(3)P, PI(5)P"
  },
  {
    "caption": "(I) Lysates from HeLa cells stably expressing GFP-WIPI2B WT and GFP-WIPI2B FTTG mutant were incubated with agarose beads coated with PI(5)P and PI(3)P, eluted with SDS-PAGE sample buffer, and recovered proteins were assessed by western blotting using antibodies against GFP. See also Figure S6.",
    "molecules": "PI(3)P, PI(5)P"
  },
  {
    "caption": "(A) HeLa cells stably expressing GFP-LC3 were shifted to starvation media (HBSS or glucose-free DMEM) for 4 hr (in the presence of 200 nM Wm or 100 nM YM-201636).(B) Quantification of numbers of GFP-LC3 vesicles per cell in HeLa cells stably expressing GFP-LC3 treated as in (A) is shown in the graph (mean ± SEM).",
    "molecules": "glucose, Wm, YM-201636"
  },
  {
    "caption": "(C) Western blot analysis of LC3-II and tubulin levels in MEF and SKNSH cells were treated as in (A) in complete media or glucose-free media in the presence of 100 nM YM-201636, 200 nM Wm, and 400 nM BAF for 4 hr.",
    "molecules": "BAF, glucose, Wm, YM-201636"
  },
  {
    "caption": "(D) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in complete media (basal) or glucose-free media (glucose free) for 4 hr in the presence or absence of 100 nM YM-201636, then fixed and imaged on a confocal microscope.",
    "molecules": "glucose, YM-201636"
  },
  {
    "caption": "(E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or PI5P4K2C catalytic dead and RFP-LC3 for 30 hr (E) were starved for glucose for 4 hr, then fixed and imaged on a confocal microscope. Bar, 10 μm. (F) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM).",
    "molecules": "glucose"
  },
  {
    "caption": "(G) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP-PHD3X, Strb-ATG16L1, and myc-MTMR3 WT. Cells were starved for glucose for 4 hr, then fixed, stained with anti-myc antibodies, and imaged on a confocal microscope. Bar, 10 μm.",
    "molecules": "glucose"
  },
  {
    "caption": "(H) HeLa cells transfected for 16 hr with GFP-PHD3X and Strb-ATG16L1 were starved for 4 hr in HBSS or glucose-free media, then fixed, stained for PI(3)P antibodies, and imaged on a confocal microscope. Bar, 10 μm. See also Figure S7.",
    "molecules": "glucose, PI(3)P"
  },
  {
    "caption": "(A and B) Tau increased β-catenin acetylation (Ace-β-cat) measured by immunoprecipitation using anti-β-cat, or Western blotting using anti-β-cat, anti-acetylated lysine (Ace-lys) and HT7 (probes specifically human total Tau). HEK293 cells transiently transfected with wildtype Tau40 (Tau) or the empty vector (Vec) (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec Data were analyzed by Student's t test",
    "molecules": "lys, lysine"
  },
  {
    "caption": "(C and D) β-cat mutation at K49 (K49R) but not K19 (K19R) abolished Tau-induced acetylation. HEK293 cells were co-transfected with eGFP-β-cat (WT, or K19R, or K49R, or K19R/K49R) and Tau or the empty vector for 48 h, and then immunoprecipitated using anti-GFP and Western blotting using anti-β-cat, anti-Ace-lys and HT7 (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT (D) ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat WT plus Tau (D) ; &&, P<0.01, &&&, P<0.001 vs β-cat K19R plus Tau (D); the absence of asterisk indicates that the difference is not significant. Data were analyzed by one-way ANOVA",
    "molecules": "lys"
  },
  {
    "caption": "(G and H) K18 deletion of Tau (Tau-k18(-)) attenuated Tau-induced β-cat acetylation. HEK293 cells were transfected with empty Vec, or Tau40 or Tau-k18(-) for 48 h, and the ace-β-cat was measured by immunoprecipitation using anti-β-cat and Western blotting using anti-ace-lys, anti-β-cat, and Tau5 (detecting total Tau; n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau (H) Data were analyzed by one-way ANOVA",
    "molecules": "lys"
  },
  {
    "caption": "(O and P) Inhibiting acetyltransferases CBP/P300 by TPOP146 (134 nM) or PCAF by L-45 (126 nM) for 24 h did not significantly decrease the K49-acetylated β-cat level in Tau-overexpressing N2a cells (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec Data were analyzed by one-way ANOVA",
    "molecules": "L-45, TPOP146"
  },
  {
    "caption": "(A) Tau directly acetylates β-cat in test-tube. Both Tau40 and β-cat were purified from E. coli using Ni-NTA resin (n = 3 from three independent experiments).",
    "molecules": "Ni-NTA resin"
  },
  {
    "caption": "(A and B) Expressing Tau inhibited β-cat ubiquitination in HEK293 cells, measured by immunoprecipitation using antibody against β-cat and Western blotting using anti- ubiquitination (anti-Ub) and anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Vec (B) Data were analyzed by Student's t test.",
    "molecules": "Ub"
  },
  {
    "caption": "(C and D) Overexpressing Tau inhibited β-cat ubiquitination in hippocampi of Tau transgenic mice, measured by immunoprecipitation using anti-Ub and Western blotting using anti-β-cat, respectively (n = 3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau- (D) Data were analyzed by Student's t test.",
    "molecules": "Ub"
  },
  {
    "caption": "(E and F) Expressing Tau-k18(-) attenuated Tau-induced β-cat elevation. HEK293 cells were transfected with Vec, or Tau-40, or Tau-k18(-) for 24 h, and then were treated with Chx for 12 h or 24 h, followed by Western blotting. β-cat protein level was normalized to β-actin (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau (F) Data were analyzed by Two-way repeated-measures ANOVA",
    "molecules": "Chx"
  },
  {
    "caption": "(G and H) K49 acetylation of β-cat inhibited its ubiquitination. HEK293 cells were co-transfected with HA-ubiquitin and GFP-β-cat or pseudo-acetylated K49-β-catenin (K49Q), and then β-cat was immunoprecipitated by anti-GFP and blotted by anti-Ub and anti-β-cat (n=3 from three independent experiments). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Student's t test.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "(I and J) K49 acetylation of β-cat inhibited its degradation. HEK293 cells were transfected with β-cat WT or K49Q for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT Data were analyzed by Two-way repeated-measures ANOVA",
    "molecules": "Chx, cycloheximide"
  },
  {
    "caption": "β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group).",
    "molecules": "Chx, cycloheximide"
  },
  {
    "caption": "β-cat mutation at K49 (K49R) restored the degradation of β-catenin. HEK293 cells were transfected with β-cat WT or co-transfected with β-cat WT and Tau40, or co-transfected with β-cat K49R and Tau40 for 24 h, and then treated with cycloheximide (Chx, 100 μg/ml) for 12 h or 24 h (n=4 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs Tau+β-cat WT (L) Data were analyzed by Two-way repeated-measures ANOVA",
    "molecules": "Chx, cycloheximide"
  },
  {
    "caption": "(M and N) K49 acetylation of β-cat inhibited its phosphorylation at N-terminal domain. HEK293 cells were transfected with GFP-vec, or eGFP-β-cat (WT) or K49Q for 48 h, then treated with wortmannin (WO, 1 μM) plus GF109203X (GFX, 1 μM) to activate GSK-3β for 1 h followed by Western blotting (n=3 biological replicates each group). Data information: Data are presented as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001 vs β-cat WT plus WO/GFX (N). data in (N) was analyzed by one-way ANOVA.",
    "molecules": "GF109203X, GFX, WO, wortmannin"
  },
  {
    "caption": "(A and B) Expressing Tau-k18(-) abolished its effect on β-cat K49-acetylation and c-cas-3 (cleaved caspase-3). N2a cells were transfected with Vec, or Tau40, or Tau-k18(-) for 48 h, and then treated with 0.5 μM staurosporine (STP, an apoptotic inducer) for 4 h, followed by Western blotting (n=3 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs vec (B) #, P<0.05, ##, P<0.01, ###, P<0.001 vs Vec plus STP (B) ; $$$, P<0.001 vs Tau plus STP (B) Data were analyzed by one-way ANOVA.",
    "molecules": "staurosporine, STP"
  },
  {
    "caption": "Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. ; N2a cells, transfected with myc-Vec, or myc-Tau40, or myc-Tau-k18(-), or myc-Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by flow cytometry using Annexin-FITC (C) Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP Data were analyzed by one-way ANOVA.",
    "molecules": "FITC, STP"
  },
  {
    "caption": "Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) (D In panel D (middle row), the Tau expression (white arrowheads) was largely not co-localized with c-casp-3 (yellow arrowheads) in the same visual field (n=4~8 biological replicates each group; scale bar, 10 μm).",
    "molecules": "STP"
  },
  {
    "caption": "Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with eGFP-Vec, or eGFP-Tau40, or eGFP-Tau-k18(-), or Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by co-staining with c-cas-3 (red) The apoptotic rate (E) was presented as the ratio of yellow (c-casp-3 and GFP co-stained) to green (the total GFP-expressing cells) staining. Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP Data were analyzed by one-way ANOVA.",
    "molecules": "STP"
  },
  {
    "caption": "Expressing Tau-k18(-) or Tau-2CA abolished its anti-apoptotic function. N2a cells, transfected with myc-Vec, or myc-Tau40, or myc-Tau-k18(-), or myc-Tau-2CA for 48 h, were then treated with 0.5 μM STP for 4 h followed by CCK8 assay (F). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Tau plus STP ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.",
    "molecules": "STP"
  },
  {
    "caption": "(G) Non-acetylation modifiable mutation of β-cat at K49 (K49R), not K19 (K19R), abolished the anti-apoptotic effect of Tau. N2a cells were co-transfected with Tau40 and β-cat WT, or K49R, or K19R for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs vs β-cat + Vec plus STP (G) ; $$$, P<0.001 vs β-cat + Tau plus STP (G) ; &&&, P<0.001 vs β-cat K49R+ Tau plus STP (G). ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.",
    "molecules": "STP"
  },
  {
    "caption": "(H) Expressing β-cat K49Q most significantly increased cell viability. N2a cells were transfected with β-cat WT or K49R or K49Q or the empty Vec for 48 h, and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs β-cat plus STP (H), ; $$$, P<0.001 vs β-cat K49Q plus STP (H) ♧♧♧, P<0.001 vs vec Data were analyzed by one-way ANOVA.",
    "molecules": "STP"
  },
  {
    "caption": "(I) Overexpressing Ser199-phosphorylation mimic Tau (TauS199E) further augmented cell viability. N2a cells were transfected with empty vec, Tau40 or TauS199E for 48h and then treated with 0.5 μM STP for 4 h. The cell viability was measured by CCK8 assay (n=8 biological replicates each group). Data information: Data are expressed as mean ± SEM, *, P<0.05; **, P<0.01, ***, P<0.001 vs Vec plus STP ; #, P<0.05, ##, P<0.01, ###, P<0.001 vs vs Tau plus STP (I) Data were analyzed by one-way ANOVA.",
    "molecules": "Ser, STP"
  },
  {
    "caption": "(A) Co-expressing myc-Tau and eGFP-β-catenin increased nuclear translocation of β-catenin, while co-expressing myc-Tau and eGFP-β-cat K49R abolished the nuclear translocation of β-catenin in HEK293 cells. The nuclei were stained using DAPI (n=3 from three independent experiments, scale bar, 5 μm).",
    "molecules": "DAPI"
  },
  {
    "caption": "Expressing β-cat K49Q increased its nuclear translocation. HEK293 cells were transfected with eGFP-β-cat WT or K49Q for 48 h and immunofluorescent labeling using a laser scanning confocal microscope (D). The nuclei (blue) was stained by DAPI (n=3 from three independent experiments, scale bar, 2 μm).",
    "molecules": "DAPI"
  },
  {
    "caption": "Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.",
    "molecules": "creatinine, uric acid"
  },
  {
    "caption": "B) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine, cysteine, carnitine and nicotinamide. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM)",
    "molecules": "carnitine, cysteine, nicotinamide, serine"
  },
  {
    "caption": "Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , B) plasma level of 10 most significant inflammation related proteins",
    "molecules": "creatinine, fat, uric acid"
  },
  {
    "caption": "Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , C) the abundance of the species in gut microbiome",
    "molecules": "creatinine, fat, uric acid"
  },
  {
    "caption": "Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with D) the abundance of the species in oral microbiome. AST, Aspartate aminotransferase; ALT, Alanine transaminase. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient.",
    "molecules": "creatinine, fat, uric acid"
  },
  {
    "caption": "Proliferation inhibition analysis of ara-C and RNRi combination treatment in SAMHD1+/+ or -/- THP-1 cells. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.",
    "molecules": "3-AP, dF-dC, HU, ara-C"
  },
  {
    "caption": "Ara-C EC50 values plotted as a function of RNRi concentration in SAMHD1+/+, -/- and rescue (WT, H233A) THP-1 cell line pairs. EC50 values in the absence of RNRi are indicated by the black and red dotted line. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.",
    "molecules": "3-AP, dF-dC, HU, Ara-C"
  },
  {
    "caption": "Drug synergy plots for ara-C and the indicated RNRi in SAMHD1+/+, -/- and rescue (WT, H233A) cell line pairs. Each data point indicates an average delta score from a single dose-response matrix experiment performed in duplicate. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. The horizontal line and the error bars indicate the mean and s.d., respectively, statistical significance was determined using a two-tailed unpaired t-test: ns, not significant, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.",
    "molecules": "ara-C"
  },
  {
    "caption": "Spearman correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C versus HU or dF-dC in a panel (n = 9) of haematological cancer cell lines. Error bars indicate s.e.m. Each data point corresponds to SAMHD1 protein levels determined by immunoblot analysis (n = 4 for each cell line, representative blot shown in Appendix Fig S2) and an average delta score from repeated dose-response matrix experiments each performed in triplicate: THP-1, n =4; HuT-78, n = 2; HL-60/iva, n = 1; KBM-7, n = 2 (HU) and 3 (dF-dC); K562, n = 3 (HU) and 4 (dF-dC); CCRF-CEM, n =3 (HU) and 4 (dF-dC); MV-4-11, n = 2 (HU) and 3 (dF-dC); Jurkat, n = 2 (HU) and 3 (dF-dC); MOLT-4, n = 2 (HU) an 3 (dF-dC).",
    "molecules": "dF-dC, HU, ara-C"
  },
  {
    "caption": "Immunoblot analysis of lysates prepared from SAMHD1+/+ or -/- THP-1 cells treated for 24 h with ara-C and HU, as indicated. Representative of 3 independent experiments.",
    "molecules": "HU, ara-C"
  },
  {
    "caption": "Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- THP-1 cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.",
    "molecules": "HU, ara-C"
  },
  {
    "caption": "Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- HL-60/iva cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group.",
    "molecules": "HU, ara-C"
  },
  {
    "caption": "Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ THP-1 cell clone (day 0) and treated with ara-C and/or dF-dC as indicated (day 6). n = 7 per treatment group.",
    "molecules": "dF-dC, ara-C"
  },
  {
    "caption": "Kaplan-Meier analysis of CD45.2 C57BL/6J mice injected i.v. with murine MLL-AF9-transformed AML blasts (day 0) and treated with ara-C and/or HU days 20-24. n = 5 per treatment group, except for vehicle (n = 4).",
    "molecules": "HU, ara-C"
  },
  {
    "caption": "Drug synergy plots for ara-C and HU or dF-dC in primary patient-derived AML blasts. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate, n = 16 for HU and n = 9 for dF-dC. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. Median, upper and lower quartiles, and range of delta scores are indicated by box-and-whisker plots.",
    "molecules": "dF-dC, HU, ara-C"
  },
  {
    "caption": "Pearson correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C and HU or dF-dC in primary patient-derived AML blasts (n = 23).",
    "molecules": "dF-dC, HU, ara-C"
  },
  {
    "caption": "Immunoblot of primary patient-derived AML blasts treated with control (dX) or Vpx-containing (X) virus-like particles (VLPs): patient A2953 (C), ALG17_001 (E). Accompanying proliferation inhibition analysis of ara-C and indicated RNRi combination in these samples: patient A2953 (D), ALG17_001 (D). Error bars indicate s.d. of single experiment performed in triplicate.",
    "molecules": "ara-C"
  },
  {
    "caption": "Paired drug synergy plot for ara-C and RNRi (HU, n = 7; dF-dC, n = 5) in primary patient-derived AML blasts pre-treated with control (dX) or Vpx-containing (X) VLPs. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy and <5 indicates strong antagonism. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate. Statistical testing was performed using two-way ANOVA.",
    "molecules": "dF-dC, HU, ara-C"
  },
  {
    "caption": "Intracellular dNTP measurements using a primer-extension assay in SAMHD1+/+ THP-1 cells treated for 4 or 24 h with either 50 µM HU (middle panel) or 2.5 nM 3-AP (right panel), ratios of dCTP-to-dATP were calculated. Bars indicate mean values of three independent experiments, error bars indicate s.e.m. Statistical analyses were done using unpaired two-tailed t-tests: **, P < 0.01.",
    "molecules": "3-AP, dNTP, HU, dATP, dCTP"
  },
  {
    "caption": "Intracellular ara-CTP (upper panel) and dCTP:dATP ratio (lower panel) in the indicated cell lines following the indicated treatment determined using HPLC-MS/MS. SAMHD1-/- THP-1 cells were treated with 500 nM ara-C and SAMHD1+/+ THP-1 cells were treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h prior. Values relative to mean ara-CTP amounts in ara-C treated SAMHD1+/+ THP-1 cells shown (indicated by dashed line). Circles, columns and error bars correspond to individual values, means and s.e.m. of at least three experiments performed independently.",
    "molecules": "3-AP, dF-dC, HU, ara-CTP, ara-C, dATP, dCTP"
  },
  {
    "caption": "Quantificaiton of dCK phosphorylation at serine-74 (S74) with respect to total dCK in SAMHD1+/+ THP-1 cells treated with either solvent, 500 nM ara-C or a combination of 500 nM ara-C and an RNRi (HU, 50 µM; dF-dC, 10 nM; 3-AP, 150 nM) for 24 h. Circles and squares, columns and error bars correspond to individual measurements, means and s.e.m. of one representative out of two independent experiments performed in triplicates.",
    "molecules": "3-AP, dF-dC, HU, ara-C, serine"
  },
  {
    "caption": "Measurement of released inorganic triphosphate (PPPi) from hydrolysis of ara-CTP (200 µM) by recombinant SAMHD1 (0.35 µM) in the presence of GTP (200 µM) and a titration of different non-hydrolysable dNTP analogues (dNTPαS) in the enzyme-coupled malachite green assay. Error bars indicate s.e.m. of two independent experiments performed in triplicate and quadruplet.",
    "molecules": "dNTP, ara-CTP, GTP, inorganic triphosphate, PPPi"
  },
  {
    "caption": "(A, B) Knockdown of hnRNPA3 increases poly-GA expression while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control (\"x0\") vector lacks the G4C2 repeats but still contains the 5' flanking region and 3x TAG. ANOVA, Dunnett.",
    "molecules": "G4C2, poly-GA"
  },
  {
    "caption": "(C, D) Overexpression of hnRNPA3 suppresses poly-GA expression. n=2 experiments performed in duplicates. ANOVA, Tukey.",
    "molecules": "poly-GA"
  },
  {
    "caption": "(A-C) Rescue of repression of poly-GA and repeat RNA by wild type (wt) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD. (B) n=3 experiments performed in duplicates; (C) n= 2 experiments performed in duplicates. ANOVA, Tukey.",
    "molecules": "poly-GA"
  },
  {
    "caption": "(D-F) Rescue of repression of poly-GA and repeat RNA by hnRNPA3WT but not the M9-NLS deletion mutant. (E) n=3 experiments performed in duplicates; (F) n=4 experiments performed in duplicates. ANOVA, Tukey. All Graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (See also Figure EV1).",
    "molecules": "poly-GA"
  },
  {
    "caption": "(A) poly-GA aggregates co-localize with p62-positive deposits.",
    "molecules": "poly-GA"
  },
  {
    "caption": "(B) Redistribution of nuclear TDP-43 to the cytoplasm in a poly-GA/poly-GR double-positive cell. Note that nuclei of poly-GA/poly-GR double positive cells are frequently disrupted.",
    "molecules": "poly-GA, poly-GR"
  },
  {
    "caption": "(D) Filter trap assay probed with anti-FLAG, anti-myc or anti-HA antibodies reveal abundant DPR production. (E) Relative expression level of poly-GA, poly-GP and poly-GR normalized to GR. n=3 experiments, ANOVA, Tukey.",
    "molecules": "poly-GA, poly-GP, poly-GR"
  },
  {
    "caption": "Rathippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (A) Neurons were fixed, immunostained and imaged by confocal microscopy. Double immunofluorescence for poly-GA aggregates (green) and tag RFP (red). Nuclei were labeled with DAPI. Scale bar represent 20 μm.",
    "molecules": "poly-GA"
  },
  {
    "caption": "Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tag RFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (C) Poly-GA aggregates were detected in a filter trap assay using an anti-Flag antibody. (D) The amounts of poly-GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct. Means ± SD of three independent experiments are shown. * p<0.05 by a Student's t-test. n= 3 replicates.",
    "molecules": "Poly-GA, poly-GA"
  },
  {
    "caption": "(C) Knockdown of hnRNPA3 increases RNA foci in cells derived from C9orf72 carriers. No G4C2 repeat RNA foci are detected in fibroblast without C9orf72 repeat expansions (WT). Nucleoli were stained with anti nucleolin antibodies (green) and nuclei were stained with DAPI (blue). (D) Quantification of the relative frequency of RNA foci positive cell (RNA foci positivity) (fold change). n=3 experiments for each case. (E) Quantification of the number of RNA foci per RNA foci-positive cell (fold change). n=3 experiments for each case. (D, E) The average foci number of non-treated (N/T) fibroblast was normalized to 1 in each case. Bars indicate mean. Individual points indicate mean values obtained from a single experiment (49-214 cells were counted in single experiment). Error bars indicate 95%CI, ANOVA, Tukey-Kramer HSD, *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "G4C2"
  },
  {
    "caption": "(A) Double immunofluorescence staining with anti-GA (green) and anti-hnRNPA3 (red) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases. In C9 mutation cases with low nuclear hnRNPA3 expression (#8 & #1) more poly-GA aggregates were observed as in cases with high nuclear hnRNPA3 (#7). Inserts show examples of co-localization of poly-GA and hnRNPA3 aggregates.",
    "molecules": "poly-GA"
  },
  {
    "caption": "(B) Granular layer neurons of a C9orf72 mutation case with (white arrows) or without (orange arrows) nuclear clearance of hnRNPA3. (C) poly-GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels (divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups). Bar graph indicates mean value. Error bars indicate 95% CI. Single points indicate mean from three micrographs per case. Note that the difference in GA positivity between both groups remains significant (p = 0.0086) after removal of the highest outlier in the low nuclear A3 group. Two-tailed t-test. Scale bar = 10 μm.",
    "molecules": "poly-GA"
  },
  {
    "caption": "C) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 15-120 min. After labeling, proteins were separated on SDS-PAGE and newly synthesized proteins in SN labeled with [35S] were visualized by autoradiography (left panel). To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol (CAP, 50 μg/ml) or with puromycin (PURO, 3mM) respectively (right panel). After 1 hour after the stimulation, a significant increase in 35S-labelled proteins was observed (n=6 biological replicates, *** p<0.001; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test) and the CAP treatment did not affect the overall levels of de novo synthetized proteins (n=6 biological replicates, ns; p=0.220). In contrast, PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes (n=6 biological replicates, ** p=0.0091, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.",
    "molecules": "35S, 35S-methionine, CAP, chloramphenicol, cysteine, PURO, puromycin"
  },
  {
    "caption": "(A-D) Results of high-resolution quantitative mass spectrometry analysis identifying proteins with increased abundance in stimulated synaptoneurosomes. Volcano plots (Whitney-Mann test, Benjamini Hochberg correction, n=5 per group) showing abundance of identified proteins in NMDAR-stimulated (20 min) synaptoneurosomes as compared to unstimulated using iTRAQ8-plex (A) and TMT10-plex (B) labelling methods. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. For nine mitochondrial proteins their mRNA abundance on polyribosomes was quantified, the data is presented on Figure EV2. (C) Biological functions of proteins identified as significantly upregulated after NMDAR stimulation (assembled from all three proteomic analyzes: LFQ, iTRAQ8, TMT10",
    "molecules": "iTRAQ8, iTRAQ8-plex, TMT10, TMT10-plex"
  },
  {
    "caption": "(A) Respiration activity of permeabilized synaptoneurosomes (upper panel) as compared to permeabilized HEK293T (lower panel). Oxygen concentration (blue line) and oxygen consumption (red line) measured using O2k oxygraph are shown.",
    "molecules": "Oxygen"
  },
  {
    "caption": "(A) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 1h. Samples were separated using blue-native electrophoresis (BN-PAGE). Protein complexes were visualized by Coomassie staining (left) and autoradiography (right). Autoradiography of the BN-PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes.",
    "molecules": "35S-methionine, Coomassie, cysteine"
  },
  {
    "caption": "To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m-chlorophenyl hydrazone (10 μM CCCP) (B) Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed (B, n=4 biological replicates, * , ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n=4 biological replicates, * p=0.02, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test)",
    "molecules": "35S, 35S-methionine, Coomassie, carbonyl cyanide m-chlorophenyl hydrazone, CCCP"
  },
  {
    "caption": "To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture (containing 1 μM valinomycin, 20 μM oligomycin, 8 μM antimycin) (C). Additionaly, in order to block protein synthesis, SN were pretreated with puromycin (PURO, 3mM) (C). Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed C, n=5 biological replicates, ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of VOA (C; n=5 biological replicates, ** p=0.0044, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Also, significant inhibition of 35S-methionine/cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin (C, n=5 biological replicates, * p=0.0236, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.",
    "molecules": "35S, 35S-methionine, antimycin, Coomassie, cysteine, oligomycin, PURO, puromycin, valinomycin"
  },
  {
    "caption": "(B) Equal amounts of synaptoneurosomal samples isolated from mouse brains (Fmr1 KO and wild-type littermates) were permeabilized by saponin and assayed on MitoPlates for 2 hours. Results are presented as the average rate/min/µg of protein, +/- SEM (n=3 per genotype; two-way ANOVA, post-hoc Sidak's multiple comparisons test; *** p< 0.0001).",
    "molecules": "saponin"
  },
  {
    "caption": "(C) Mitochondrial O2 consumption rates in SN were measured by high-resolution respirometry in the presence of following respiratory substrates: malate, pyruvate, and glutamate for Complexes I-IV, succinate for Complexes II-IV, glycerol 3-phosphate for mGpDH-IV and ascorbate and TMPD for complex IV, and normalized to protein content. Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD (*p=0.0483 and *p=0.0360 respectively, n=9 per genotype, paired two-tailed t-test). Data are presented as a box-and-whiskers graph (the box extends from 25th to 75th percentiles, central horizontal line is plotted at the median, whiskers show 5th - 95th percentile).",
    "molecules": "ascorbate, glutamate, malate, pyruvate, glycerol 3-phosphate, succinate, TMPD"
  },
  {
    "caption": "(D) At 48h after transfection of H7   plasmid, the transfectants were incubated with TPCK-trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease  inhibitor AEBSF for 30 min and then applied to examine HA cleavage.",
    "molecules": "AEBSF, TPCK, PBS, trypsin"
  },
  {
    "caption": "(A) At 48 hours post transfection of the indicated protease plasmids in triplicate, BHK21  cells were incubated with a fluorogenic substrate and wtSPINK6 or loss-of-function mutSPINK6  or PBS for 2 hours and then applied to fluorescence assay. Data represent mean and SD of triplicated wells in a representative experiment performed 3 times. **, P<0.01. Student's t test.",
    "molecules": "PBS"
  },
  {
    "caption": "(B) At 24h after transfection of HAT or  KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay  (black bars), the other was pre-treated with  TPCK-trypsin for 1 hour prior to plaque assay  (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed 3 times. *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.",
    "molecules": "TPCK, trypsin"
  },
  {
    "caption": "(D) Mouse lung  tissues are applied to immunofluorescence staining to identify the viral NP positive cells (green). Nuclei  are counterstained with DAPI (blue). Representative confocal images of virus-infected cells in the indicated mice. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B) Analysis of puromycin incorporation in MelJuSo cells that were left untreated (- HS), exposed to 43°C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.). The blots were probed with antibodies against puromycin, and GAPDH as loading control.",
    "molecules": "puromycin"
  },
  {
    "caption": "A) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. B) Quantification of the nucleus/cytoplasm ratios of puromycin intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).",
    "molecules": "puromycin"
  },
  {
    "caption": "E) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) U2OS cells. Cells were subjected to 43°C heat shock F) Quantification of the numbers of puromycin positive TIA dots per cell in images from E). The numbers of positive foci per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).",
    "molecules": "puromycin"
  },
  {
    "caption": "G) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. H) Quantification of the nucleolus/nucleoplasm ratio of puromycin intensities in images from G). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).",
    "molecules": "puromycin"
  },
  {
    "caption": "E) Analysis of SUMO2/3 conjugates in parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), exposed to a heat shock (+ HS), and followed for 2 hours (2h Rec.) with or without 100 nM proteasome inhibitor epoxomicin (EPX).",
    "molecules": "epoxomicin, EPX"
  },
  {
    "caption": "D) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1/2 knock-out U2OS cells that were left untreated (-HS), exposed to 43°C (+HS) followed by 2 hours recovery (2h Rec.) in absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). E) Quantification of the TDP43 band densities in the SDS-soluble fraction. Data represent the mean ± SD (n=3 independent experiments, Student's unpaired t-test, * P<0.05).",
    "molecules": "epoxomicin, EPX, NP40, SDS"
  },
  {
    "caption": "A) Analysis of SUMO2/3 conjugates in control (siControl) or RNF4-depleted (siRNF4) G3BP1/2 knock-out U2OS cells. Cells were left untreated (-HS), exposed to 43°C (+ HS), followed by 2 hours recovery (HS + 2h Rec.) in the absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). B) Quantification of the total SUMO2/3 band densities. Data represent the mean ± SD (n=3 independent experiments, *P<0.05).",
    "molecules": "epoxomicin, EPX"
  },
  {
    "caption": "E) Representative confocal images of control (siControl), RNF4-depleted (siRNF4), G3BP1/2-depleted (siG3BP1/2) and G3BP1/2+RNF4-depleted (siG3BP1/2 + siRNF4) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1. Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin (EPX). The nucleolar marker nucleolin was stained by immunofluorescence.",
    "molecules": "epoxomicin, EPX"
  },
  {
    "caption": "Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGE",
    "molecules": "digitonin"
  },
  {
    "caption": "Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGE",
    "molecules": "digitonin"
  },
  {
    "caption": "Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates).",
    "molecules": "digitonin"
  },
  {
    "caption": "BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. Immunodetection of the indicated proteins",
    "molecules": "digitonin"
  },
  {
    "caption": "BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates).",
    "molecules": "digitonin"
  },
  {
    "caption": "I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.",
    "molecules": "Dig, digitonin, SDS"
  },
  {
    "caption": "G-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines).",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "D, E, Mitochondrial DNA copy number per nuclear copy number in muscle in females (D) and males (E) (Δ1 n=6, Δ2 n=6 and same number for their respective controls).",
    "molecules": "DNA"
  },
  {
    "caption": "I, Maximum uncoupled (FCCP) OCR in isolated mitochondria from adult fish (male and females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with the indicated site I [pyruvate (Pyr), glutamate (Glu), malate (Mal)] or site II [succinate (Succ)] substrates.",
    "molecules": "FCCP, Glu, glutamate, Mal, malate, Pyr, pyruvate, Succ, succinate"
  },
  {
    "caption": "D-F, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (D) Adipose tissue area per total section area (average of three sections/biological replicate) and (E) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) (Δ1 +/+ n=3, Δ2 n=8). scaf1Δ1 and scaf1Δ2 are represented with circles and squares, respectively.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (F) Representative images of ventral fat deposits (dotted lines).",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "G, number of eggs per clutch (standard diet: Δ1 +/+ n=4, Δ1 -/- n=3, Δ2 +/+ n=3, Δ2 -/- n=2, double diet: Δ1 +/+ n=5, Δ1 -/-, n=5 Δ2 +/+ n=1, Δ2 -/- n=2). H, Quantification of mature ovary follicles per ovary section in haematoxylin-eosin (H&E) histological sections (average of three sections/biological sample) (Δ1 n=3, Δ2 n=3). ",
    "molecules": "eosin, haematoxylin"
  },
  {
    "caption": "B, Maximum uncoupled (FCCP) oxygen consumption rate in whole zebrafish mitochondria (females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with glutamate (Glu), malate (Mal) and succinate (Succ).",
    "molecules": "FCCP, Glu, glutamate, Mal, malate, Succ, succinate"
  },
  {
    "caption": "A, B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG-A for 16 h at 37°C. Anti-BDCA2-mediated IFNα inhibition in whole-blood assays (A). IFNα levels were detected using human IFNα ELISA. Graph depicts average of duplicate wells of one representative donor (n = 10). IC50 of 24F4A-mediated IFNα inhibition (B) in whole blood (circles) (n = 10) and PBMC (n = 18) from healthy human donors (squares) or SLE patients (triangles) (n = 11). Horizontal bar represents the mean IC50 for each sample type. Error bars represent SD of IC50 between donors.",
    "molecules": "24F4A, CpG-A"
  },
  {
    "caption": "C Anti-BDCA2-mediated internalization in whole-blood assays. Whole blood was treated with increasing concentrations of 24F4A for 16 h. Mean fluorescence intensity (MFI) of BDCA2 was determined with a non-cross-blocking anti-BDCA2 mAb (2D6). Shown is a representative plot of 10 experiments conducted.",
    "molecules": "24F4A"
  },
  {
    "caption": "A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments.",
    "molecules": "24F4A"
  },
  {
    "caption": "B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted.",
    "molecules": "24F4A"
  },
  {
    "caption": "C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.",
    "molecules": "24F4A, CpG-A"
  },
  {
    "caption": "E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.",
    "molecules": "24F4, CpG-A"
  },
  {
    "caption": "Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A-C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).",
    "molecules": "24F4A"
  },
  {
    "caption": "Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.D PK/PD relationship between 24F4Aserum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i-iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.",
    "molecules": "24F4A"
  },
  {
    "caption": "Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).",
    "molecules": "24F4A, CpG-A"
  },
  {
    "caption": "A-F Isolated human pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (black squares), or 10 μg/ml of the isotype control (black triangle) and stimulated with 5 μM of R848 (A and B), 1 μM of CpG-A (C and D), or immune complexes (E and F) for 16 h at 37°C. IFNα concentrations in cultured supernatants were determined using ELISA. Representative plots for each stimulation (A, C and E) and the average IC50 of 24F4A and 24F4A-ef for each stimulation condition (B, D and F) are shown (n = 3). Horizontal bars represent mean IC50, and error bars represent SD of the mean from three independent experiments.",
    "molecules": "24F4A, CpG-A, R848"
  },
  {
    "caption": "G Isolated human pDCs were treated with 10 μg/ml of 24F4A (black line), 24F4A-ef (gray shaded histogram), or the isotype control (gray dotted line) for 10 min at 37°C. Flow cytometry was used to evaluate pSYK and pPLCγ2. Shown is a representative plot of two independent experiments.",
    "molecules": "24F4A"
  },
  {
    "caption": "H Isolated pDCs treated with 10 μg/ml of anti-CD32a (AT10) and stimulated with CpG-A, R848, or immune complexes. Error bars represent SD of percent inhibition of IFNα from three independent experiments.",
    "molecules": "CpG-A, R848"
  },
  {
    "caption": "A-F Isolated pDCs were treated with increasing concentrations of 24F4A (red circles), 24F4A-ef (squares), Chi-6G6 (triangle), or isotype control (diamond) for 16 h at 37°C and stimulated with CpG-A. Flow cytometry was performed to evaluate the MFI of (A) BDCA2 and (C) CD32a on the surface of pDCs. Percent internalization of BDCA2 (B) and CD32a (D) on pDCs treated with 10 μg/ml of 24F4A, 24F4A-ef, Chi-6G6, or isotype control. Error bars represent SD of percent internalization of five experiments conducted. (E) Percent CD32a downmodulation on isolated pDCs treated with 10 μg/ml of an anti-CD40 mAb. Error bars represent SD of percent downmodulation of two independent experiments. FMO represents background staining of CD32a (inverted triangle). (F) Percent of maximal expression of BDCA2 or CD32a in the presence of increasing concentrations of 24F4A or Chi-6G6. Maximal expression was defined by the MFI of both BDCA2 and CD32a on untreated cells. Shown is a representative plot of five independent experiments conducted.",
    "molecules": "24F4A, CpG-A"
  },
  {
    "caption": "A. Nascent protein synthesis revealed by HpG incorporation (green) in wt and mdi1 egg chambers. Arrowheads point to the HpG signal on the ER in the perinuclear region and cell periphery. Arrows point to HpG signal associated with mitochondria. Mitochondria are marked by a Tom20-mCherry (red) created by inserting mCherry at the endogenous Tom20 locus.",
    "molecules": "HpG, protein"
  },
  {
    "caption": "Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. A. Nascent protein synthesis was monitored by AHA incorporation, and detected by anti-biotin antibody. Tom20 served as a loading control. Note that the synthesis of nuclear-encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20-Larp (mdi1/TL).",
    "molecules": "AHA, biotin, protein"
  },
  {
    "caption": "Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. C. Nascent protein synthesis of Tamas and TFAM was decreased in mdi1 ovary, whereas mtSSB was not affected. Tfamgfp, Tamasgfp and mtSSBgfp were expressed in wt or mdi1 background. Nascent proteins were labeled by AHA incorporation and then the GFP-tagged protein was immunopurified with a GFP antibody and the nascent protein synthesis was detected by anti-biotin antibody.",
    "molecules": "AHA, biotin, protein"
  },
  {
    "caption": "Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. D. Representative profile of 254 nm absorbance of wt, mdi1 and mdi1/TL ovary extracts.",
    "molecules": "protein"
  },
  {
    "caption": "Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. F. The nascent protein synthesis of COX4 and mRPL19 were decreased in mdi1 ovary and were restored mdi1/TL flies. The proteins were immunopurified with antibodies against the endogenous proteins.",
    "molecules": "protein"
  },
  {
    "caption": "(F) Ca2+ influx in Fura-2 loaded PBMC of P1, P2, the mother and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca2+ followed by addition of 1 mM extracellular Ca2+. Bar graphs show the integrated Ca2+ influx response (AUC, area under the curve) from 400-800 sec and the peak Ca2+ influx normalized to baseline Ca2+ levels (F340/380) at 400 sec. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t-test. **p<0.01, ***p<0.001.",
    "molecules": "Ca2+, Fura-2, TG, thapsigargin"
  },
  {
    "caption": "GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (D,E) Representative TIRF microscopy images from one of two repeat experiments. Scale bars, 10 μm.",
    "molecules": "Ca2+, TG"
  },
  {
    "caption": "GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (F) Percentages of WT or mutant mCherry-STIM1 present at the plasma membrane (i.e. visible in the TIRFM evanescent field) before TG stimulation. Data are the mean ± SEM calculated from 550 (WT) and 830 (L374P) cells from two independent experiments. (G) Change of mCherry-STIM1 (WT or L374P mutant) fluorescence [F(t)/F(0)] in the TIRFM evanescence field. Fluorescence normalized to values at the time of TG addition in Ca2+-free Ringer's buffer at 0 sec. Traces show mean ± SEM of 40-50 cells from two independent experiments. (H) Colocalization of mCherry-STIM1 (WT or L374P mutant) and GFP-ORAI1 from the time of TG addition at 0 sec. Colocalization measured using the Pearson's coefficient for GFP and mCherry.",
    "molecules": "Ca2+, TG"
  },
  {
    "caption": "Cell size (A) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (A) Representative histograms of FSC (left panel) and percentages of T cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel).",
    "molecules": "FK506"
  },
  {
    "caption": "proliferation (B) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments.",
    "molecules": "CFSE, FK506"
  },
  {
    "caption": "(C,D) Cytokine production by PBMC from P1, P2, the mother and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 hours. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4 and CD45RO, permeabilization and intracellular cytokine staining for GM-CSF, IL-22 and IL-17A. Representative flow cytometry plots (C) and quantification of Th17 (GM-CSF, IL-22, IL-17A), Th1 (TNF-α, IFN-γ) and Th2 (IL-4) cytokines (D). Data represent the mean ± SEM from two independent experiments.",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "B) PBMC of a healthy donor (HD1) cultured for 6 days in the presence of 6.5 mg/ml C. albicans protein. At day 6, PBMC were restimulated with PMA and ionomycin in the presence of 10 μM GSK-7975A or DMSO for 6 h and CD4+ and CD8+ T cells were analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.",
    "molecules": "DMSO, GSK-7975A, ionomycin, PMA"
  },
  {
    "caption": "Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (C) T cells were restimulated with PMA and ionomycin for 6 h in the presence or absence of 10 μM GSK-7975A and analyzed for cytokine production by flow cytometry. Bar graphs represent the mean ± SEM from two independent experiments.",
    "molecules": "GSK-7975A, ionomycin, PMA"
  },
  {
    "caption": "Human T cells from two healthy donors, P1 and his mother were expanded and cultured in the presence of C. albicans and IL-2 for 2-4 weeks. (D) Fura-2 loaded T cells were stimulated with thapsigargin (TG) in Ca2+-free buffer followed by readdition of 2 mM Ca2+ to induce SOCE. Bar graphs represent the mean ± SEM from two independent experiments.",
    "molecules": "Ca2+, Fura-2, TG, thapsigargin"
  },
  {
    "caption": "ROS production of neutrophils depend on SOCE. (I) ROS production by Cd11b+Gr-1+ neutrophils from WT, Stim1fl/flMx1Cre and Stim1fl/flStim2fl/flMx1Cre mice was measured after loading of cells with dihydrorhodamine 123 and stimulation with 10 nM PMA for 30 minutes. Representative histogram plots and bar graphs indicating the percentages of ROS+ neutrophils normalized to WT neutrophils stimulated with PMA.",
    "molecules": "dihydrorhodamine 123, PMA, ROS"
  },
  {
    "caption": "Systemic C. albicans infection. WT and Stim1fl/fl Cd4Cre mice were injected i.v. with 2x105 CFU C.albicans or PBS as control. (J) Frequencies of CD4+ T cells isolated from the spleens of mice at day 6 p.i. producing the indicated cytokines after restimulation with 20 nM PMA and 1 μM ionomycin for 6 hours and analyzed by flow cytometry.",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(A) Heat map of metabolism-associated DEGs (p (adj.) < 0.10) in npTh17 cells of WT and Stim1fl/flCd4Cre mice determined by RNA-Seq as described in Figure 7. Heatmap shows relative minimum and maximum values per gene (row min/max). Numerical values are relative gene expression. TCA, tricarboxylic acid.",
    "molecules": "tricarboxylic acid, TCA"
  },
  {
    "caption": "Glucose and mitochondrial metabolism in npTh17 cells from WT (grey or black) and Stim1fl/flCd4Cre (blue)mice differentiated for 3 days in vitro. (B) Glucose uptake by npTh17 cells were loaded with 2-NBDG (+) or not (-) and analyzed by flow cytometry 90 min later. Bar graphs show the delta MFI of 2-NBDG fluorescence normalized to unlabeled cells. Data represent the mean ± SEM from 3 independent experiments.",
    "molecules": "2-NBDG, Glucose"
  },
  {
    "caption": "(C) Extracellular acidification rate (ECAR) of npTh17 cells before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-deoxy-D-glucose (2-DG). Seahorse graphs and bar graphs depicting glycolysis, glycolytic capacity and reserve the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments.",
    "molecules": "2-deoxy-D-glucose, 2-DG, glucose, oligomycin"
  },
  {
    "caption": "Glucose and mitochondrial metabolism in npTh17 cells from WT (grey or black) and Stim1fl/flCd4Cre (blue)mice differentiated for 3 days in vitro. (D) Oxygen consumption rate (OCR) of npTh17 cells before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs and bar graphs depicting basal and maximal respiration, coupling efficiency and ATP production represent the mean ± SEM from of 2-3 mice per genotype from one representative out of three independent experiments.",
    "molecules": "antimycin, ATP, FCCP, Glucose, oligomycin, rotenone"
  },
  {
    "caption": "Glucose and mitochondrial metabolism in human CD4+ T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 hours before analysis. Some HD T cells were stimulated in the presence of 1μM FK506. (E,F) ECAR before and after addition of 25 mM glucose, 5 μM oligomycin and 100 mM 2-DG.",
    "molecules": "2-DG, Glucose, glucose, oligomycin, FK506"
  },
  {
    "caption": "Glucose and mitochondrial metabolism in human CD4+ T cells isolated from PBMC of a HD and P1 and stimulated with 5 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 24 hours before analysis. Some HD T cells were stimulated in the presence of 1μM FK506. (G,H) OCR before and after addition of 1 μM oligomycin, 1.5 μM FCCP and 100 nM rotenone / 1 μM antimycin. Seahorse graphs (G) and bar graphs (H) depicting basal and maximal respiration, SRC and ATP production (right) represent the mean ± SEM of 2 technical replicates from isolated T cells from one experiment.",
    "molecules": "antimycin, ATP, FCCP, Glucose, oligomycin, rotenone, FK506"
  },
  {
    "caption": "Hoxa5 expression upon miR-27 knockdown, with exposure to RAhi for 72 h, or with exposure to RAhi for 48 h, and then switching to RAlow plus RAR inhibitor (1 µM) for a further 24 h, or with exposure to RAmed for 72 h.",
    "molecules": "RA"
  },
  {
    "caption": "Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8-depleting antibody and PD-1 antibody or PD-1/GVAX as indicated. Tumor volume was measured over time points. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.",
    "molecules": "GVAX"
  },
  {
    "caption": "F. Representative genes with m6A sites generated by integrative genomics viewer. Data is representative of duplicates with similar results. Red represents reads coverage of IP sample and blue represents reads coverage of input sample. Rectangular cyan shade represents the m6A peaks located on transcripts.",
    "molecules": "m6A"
  },
  {
    "caption": "G. m6A enrichment of Stat1 and Irf1 was examined by m6A RIP-qPCR in control, Mettl3, or Mettl14 depleted CT26 tumors as indicated. Ctla4 functioned as a m6A negative control (Wang et al., 2019) Data are mean ± s.d. * * P < 0.01 by Student's t-tests.",
    "molecules": "m6A"
  },
  {
    "caption": "D, E. mRNA stability of Stat1 and Irf1 were measured by qRT-PCR in tumor cells treated with IFN-γ and actinomycin D. Mean ± SD of n=3. * * P < 0.01; * * * P < 0.001 by Student's t-tests.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "I, J. qPCR analysis of the mRNA stability of Stat1 and Irf1 in the indicated CT26 cells treated with IFNγ and actinomycin D. Mean ± SD of n=3. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "(A) Human osteosarcoma U2OS GFP-LC3 cells were treated with a library of polyphenols and polyamines for 6 h And then GFP-LC3 dots were counted to measure autophagy activity. Top 20 hits were shown in the right frame.",
    "molecules": "polyamines, polyphenols"
  },
  {
    "caption": "Human osteosarcoma U2OS cells (B or neuroglioma H4 cells (D stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated , or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Representative images are shown in (B,D). Scale bar equals 10 µm. (C,E) Acetylation: U2OS and H4 cells were treated as described above, followed by incubation with specific antibodies to block acetylated tubulin; Thereafter immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor conjugated secondary antibodies before the assessment of cytoplasmic fluorescence intensities. Representative images of acetylation are shown in (C,E). Scale bar equals 10 µm.",
    "molecules": "AlexaFluor, lysine"
  },
  {
    "caption": "Human osteosarcoma U2OS cells F) or neuroglioma H4 cells H) stably expressing GFP-LC3 were treated with a library of chalcones (30 µM) as indicated or rapamycin (10 µM), or were left untreated for 6 h. The cells were then fixed and GFP-LC3 dots were counted as an indicator for autophagy. Data are means ± SD (* = p < 0.05; ** = p < 0.01;*** = p < 0.001). Viability: U2OS and H4 cells were treated with 30 µM of the indicated chalcones for 24 h and then nuclei were stained with Hoechst 33342 and the number of cells harboring normal nuclei (i.e. non pyknotic, "healthy cells") was determined. All chalcones were hierarchically clustered upon z-scoring the following phenotypes: autophagy (number of GFP-LC3 dots), viability (number of healthy cells) and acetylation (cytoplasmic fluorescence intensity of acetylated lysine residues) in U2OS and H4 cells. Results are reported as a heatmap (G, I).",
    "molecules": "Hoechst 33342, lysine, rapamycin"
  },
  {
    "caption": "(J, K) U2OS cells were treated with 3,4-DC in the presence or absence of dichloroacetate (DCA), α-ketoisocaproic acid (KIC), or L-leucine (Leu). GFP-LC3 dots were quantified in (J). Acetylation staining was performed as previously described, and acetylation intensity in the cytoplasm was measured in (K). Data are means ± SD (*** = p < 0.001 versus DMSO/Ctr; ##=p<0.01, ###=p<0.001 versus 3,4-DC/Ctr).",
    "molecules": "3,4-DC, KIC, α-ketoisocaproic acid, DCA, dichloroacetate, DMSO, L-leucine, Leu"
  },
  {
    "caption": "Hepatoma HepG2 cells were treated with the indicated concentrations of 3,4-DC for 8 h (A,B) GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in (B Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05",
    "molecules": "3,4-DC"
  },
  {
    "caption": "Hepatoma HepG2 cells were treated with 30 µM 3,4-DC for the indicated time points (C,D). Then, cells were processed to measure LC3 and p62 protein levels by SDS-PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of p62, GAPDH, LC3-I and LC3-II were measured and ratios of p62 or LC3-II versus GAPDH (LC3-II/GAPDH, p62/GAPDH) and LC3-II versus LC3-I (LC3-II/LC3-I) were calculated in D). Data are means ± SEM of at least three independent experiments (LC3-II/GAPDH: * = p < 0.05, ** = p < 0.01; p62/GAPDH: # = p < 0.05, ### = p < 0.001; LC3-II/LC3-I: $=p<0.05).",
    "molecules": "3,4-DC"
  },
  {
    "caption": "(E,F) HepG2 were treated with 30 µM 3,4-DC for 8 h in the presence or absence of chloroquine (CQ, 50µM) for 4 h. SDS-PAGE and immunoblot were performed Band intensities of LC3-II and GAPDH were assessed and their ratio (LC3-II/GAPDH) was calculated in (F). Data are means ± SEM of at least three independent experiments (* = p < 0.05, ** = p < 0.01 versus untreated control; ##=p<0.01 versus CQ).",
    "molecules": "3,4-DC, chloroquine, CQ"
  },
  {
    "caption": "(G,H) U2OS-GFP-LC3 cells were treated with 30 µM 3,4-DC, 10 µM rapamycin, or left untreated for 16 h in the presence or absence of CQ for 4 h. GFP-LC3 dots were measured. Data are means ± SD (*** = p < 0.001 versus untreated; ###=p<0.001 versus CQ).",
    "molecules": "3,4-DC, CQ, rapamycin"
  },
  {
    "caption": "(I,J) U2OS cells stably expressing LC3 fused with tandem fluorescent GFP-RFP proteins (GFP-RFP-LC3) were treated with the indicated raising doses of 3,4-DC or 50 µM chloroquine (CQ) for 16 h. After fixation, GFP-LC3 and RFP-LC3 dots were measured by automated image acquisition and analysis. GFP and RFP double positive LC3 dots indicating autophagosomes (GFP+), and GFP negative but RFP positive LC3 dots indicating autolysosomes (GFP-) were counted in (J). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001). Representative images are shown in I. Scale bar equals 10 µm.",
    "molecules": "3,4-DC, chloroquine, CQ"
  },
  {
    "caption": "A, B) HepG2 cells were treated for the indicated time with 30 µM 3,4-DC (A) or indicated dose of 3,4-DC for 8 h (B). Thereafter, cells were collected and SDS-PAGE and immunoblots were performed as described before. Total p70S6K, phosphorylated p70S6K at T389 (P-p70S6K), Atg14, Lamp1, LC3, p62, and GAPDH protein levels were measured with specific antibodies. Samples for immunoblots in A and B were run in parallel instances, then cut into horizontal stripes and probed separately. Blots probed for P-p70S6K were stripped and reprobed to detect p70 independently from its phosphorylation status. The same applies for LAMP1 and GAPDH.",
    "molecules": "3,4-DC"
  },
  {
    "caption": "(C, D) H4-GFP-LC3 cells were treated with 30 µM 3,4-DC in the presence or absence of CHX or AMD or with CQ for 16 h as controls, as indicated. GFP-LC3 dots were quantified in (D). Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus untreated control; # = p < 0.05 versus untreated with CQ).",
    "molecules": "3,4-DC, AMD, CQ, CHX"
  },
  {
    "caption": "(E) RNA expression levels 16 h after exposure to 3,4-DC of the indicated genes in HepG2 cells were measured by RT-PCR and normalized to the expression of the housekeeping gene (GAPDH). Data are means ± SD (* = p < 0.05;** = p < 0.01;*** = p < 0.001).",
    "molecules": "3,4-DC"
  },
  {
    "caption": ",G) U2OS GFP-LC3 cells were enucleated to obtain cytoplasts, which were further treated with 3,4-DC and torin1. GFP-LC3 dots in cytoplasts were counted as shown in (G). Data are means ± SD (** = p < 0.01). Representative images with cytoplast marked by a white square are shown in (F). Scale bar equals 10 µm.",
    "molecules": "3,4-DC, torin1"
  },
  {
    "caption": "(C, D) U2OS cells were treated with 30 µM 3,4-DC for 6 h, and then endogenous TFEB translocation was assessed by immunostaining (C). TFEB intensities in nuclei and cytoplasm were measured and the ratio of TFEB intensities in nuclei and cytoplasm were calculated to indicate TFEB translocation to nuclei (D). Data are means ± SD (*** = p < 0.001). Representative images are shown in (C). Scale bar equals 10 µm.",
    "molecules": "3,4-DC"
  },
  {
    "caption": "(E, F) U2OS cells were treated with 30 µM 3,4-DC for 6 h or were left untreated. Cytoplasmic and nuclear fractions were assessed for nuclear translocation of the transcription factors TFEB and TFE3 by SDS page.",
    "molecules": "3,4-DC"
  },
  {
    "caption": "MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. The cells were fixed and immunofluorescence was performed to detect endogenous TFEB. The ratio of TFEB intensities between nuclei and cytoplasm was calculated in (H). Data are means ± SD (*** = p < 0.001 versus Ctr; ###=p<0.001 versus WT/3,4-DC). Representative images are shown in (G). Scale bar equals 10 µm.",
    "molecules": "3,4-DC"
  },
  {
    "caption": "MEF wild type and TSC2 knockout (TSC2 KO) cells were treated with 3,4-DC for 6 h. (I) After the treatment, nuclei were isolated and western blot was performed to detect nuclear and cytosol TFEB protein levels.",
    "molecules": "3,4-DC"
  },
  {
    "caption": "(J-L) MEF wild type and TSC2 KO cells were collected and lysed for western blot after the treatment with 3,4-DC. Antibodies against LC3, p62, TSC2, or GAPDH were administrated to detect protein levels, and phosphorylation of mTOR substrate p70 S6K at Threonine 389 (T389) was measured with the phosphorylation specific antibody (P-p70(T389)) and anti-p70 antibody. The bands intensities of LC3-II, p62, and GAPDH were measured with Image J, and the ratios of LC3-II/GAPDH and p62/GAPDH were calculated in (K) and (L). Data are means ± SEM of at least three independent experiments (** = p < 0.01, *** = p < 0.001 vs Ctr; #=p<0.05, ##=p<0.01 vs WT/3,4-DC)",
    "molecules": "3,4-DC, Threonine"
  },
  {
    "caption": "(M,N) U2OS cells treated with 3,4-DC or Torin for 6 h were collected and processed for western blot. TFEB phosphorylation at Serine 211 was checked with the phosphorylation specific antibody (P-TFEB(S211)) and total TFEB protein level was also detected with anti-TFEB. The bands intensities of P-TFEB and total TFEB were measured with Image J, and their ratio was calculated in (N). Data are means ± SEM of at least three independent experiments (* = p < 0.05 vs Ctr).",
    "molecules": "3,4-DC, Serine, Torin"
  },
  {
    "caption": "U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot",
    "molecules": "3,4-DC"
  },
  {
    "caption": "U2OS-GFP-LC3 cells transfected with 3 individual siRNAs specifically targeting TFEB (siTFEB-#1, siTFEB-#2, siTFEB-#3) or scrambled siRNA (siCtr) were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy Data are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown Scale bar equals 10 µm.",
    "molecules": "3,4-DC"
  },
  {
    "caption": "U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. TFEB knockdown efficiency by TFEB siRNAs and TFEB deficiency by knockout were checked by SDS-PAGE and immunoblot",
    "molecules": "3,4-DC"
  },
  {
    "caption": "U2OS wild type (wt) and TFEB knockout (TFEB KO) GFP-LC3 cells were treated with 3,4-DC as indicated for 16 h. GFP-LC3 dots were quantified as indicator of autophagy ata are means ± SD (* = p < 0.05, ** = p < 0.01 versus siCtr or WT cells treated with 3,4-DC). Representative images are shown cale bar equals 10 µm.",
    "molecules": "3,4-DC"
  },
  {
    "caption": "U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. Following, cells were collected and SDS-PAGE and immunoblots were performed as described before. TFEB, TFE3, LC3, p62, and GAPDH protein levels were measured with specific antibodies (G).",
    "molecules": "3,4-DC"
  },
  {
    "caption": "U2OS wild type (WT), TFEB or TFE3 knockout, or double knockout (TF DKO) cells were treated with 30 µM 3,4-DC for 16 h. GFP-LC3 dots were quantified as indicator of autophagy (H). Data are means ± SD (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 versus Ctr, respectively).",
    "molecules": "3,4-DC"
  },
  {
    "caption": "(I) U2OS WT, knockout for TFEB (TFEB-/-) or TFE3 (TFE3-/-), double knockout (DKO) were treated with 3,4-DC for 24 h and stained with LysoTracker Red for 30 min. Thereafter, the red (positive) dots were measured. Data are means ± SD (* = p < 0.05; ** = p < 0.01; *** = p < 0.001).",
    "molecules": "3,4-DC, LysoTracker Red"
  },
  {
    "caption": "(J) Relative mRNA expression levels of key autophagy genes such as Lamp1, Lc3b and p62 were detected in controls and 3,4-DC treated U2OS cells. Data is depicted as a heatmap showing means of at least three independent experiments (*** = p < 0.001 versus WT/Ctr and # = p < 0.05, ## = p < 0.01, as compared to WT treated with 3,4-DC).",
    "molecules": "3,4-DC"
  },
  {
    "caption": "(A-D) Female C57/BL6 animals were treated with 3,4-DC for 24 h. Following, liver and heart tissue was excised and cells were subjected to subcellular fractionation. Thereafter proteins were separated by SDS PAGE and immunoblots were performed to detect the nuclear translocation of TFEB and TFE3 (A, C). GAPDH and H3 were used as controls for cytosolic and nuclear fractions, respectively. Band intensities of nuclear TFEB, TFE3 and H3 were measured and ratios of nuclear TFs versus H3 (TFsnuc/H3) were calculated in (B, D). Data are means ± SEM of at least three mice (*** = p < 0.001 versus Ctr)",
    "molecules": "3,4-DC"
  },
  {
    "caption": "GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in liver tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).",
    "molecules": "3,4-DC, Leu, Leupeptin"
  },
  {
    "caption": "C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following liver tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).",
    "molecules": "3,4-DC"
  },
  {
    "caption": "GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in heart (I, J) tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).",
    "molecules": "3,4-DC, Leu, Leupeptin"
  },
  {
    "caption": "C57/BL6 mice were i.p. injected with 3,4-DC or were starved for 24 hours. Following heart tissue was excised and subjected to SDS PAGE and immunoblot GAPDH was measured as a loading control. Band intensities of GAPDH and LC3-II were measured and ratios LC3-II versus GAPDH (LC3-II/GAPDH) were calculated Data are means ± SEM of at least three mice (* = p < 0.05, ** = p < 0.01).",
    "molecules": "3,4-DC"
  },
  {
    "caption": "(A) Twelve weeks-old wild-type and cardiac specific Atg7 knockout (Atg7cKO) mice were injected with corn oil (vehicle control, Ctr) or 3,4-DC 24 h before surgery and subjected to 3 h prolonged ischemia. Representative images of left ventricular (LV) myocardial sections after alcian blue and triphenyltetrazolium chloride (TTC) staining are depicted. Scale bar equals 1 mm.",
    "molecules": "corn oil, 3,4-DC, alcian blue, triphenyltetrazolium chloride, TTC"
  },
  {
    "caption": "A. Induction of autophagy in murine MCA205 fibrosarcomas. Cells were treated with 3,4 DC alone or in combination with chloroquine, and were harvested 6 hours later for immunoblot detection of LC3 lipidation.",
    "molecules": "3,4 DC, chloroquine"
  },
  {
    "caption": "(J-L) Immunocompetent C57Bl/6 mice were subcutaneously inoculated with TFEB/TFE3 double knockdown MCA205 cells or its scramble control cells (K). When tumors became palpable, mice were treated as indicated Tumor growth curves from mice subjected to 3,4-DC administration alone or in combination with MTX are shown (J,L). Asterisks indicate significant effect of MTX with respect to untreated controls (mean value ±SEM, *p<0.05, **p<0.01, ***p<0.001), while hash symbols refer to the comparison of the effects of MTX plus 3,4-DC to MTX alone (#p<0.05, ##p<0.01, ###p<0.001)(n=6-14).",
    "molecules": "3,4-DC, MTX"
  },
  {
    "caption": "WB analysis of smc5-AID clones in the absence or presence of Auxin using FLAG. Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag.",
    "molecules": "Auxin"
  },
  {
    "caption": "Proliferation curve of wt and smc5-AID cells in the presence or absence of Auxin. The data represents means ± SD of 3 independent experiments.",
    "molecules": "Auxin"
  },
  {
    "caption": "Survival curve of smc5 cells treated with different concentrations of cisplatin (CDDP). Data represents means ± SD of 3 experiments.",
    "molecules": "CDDP, cisplatin"
  },
  {
    "caption": "Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin. The experiment is carried out at 39.5ºC. Data represents means ± SD of at least 3 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Survival curve of smc5 fancc cells treated with different concentrations of cisplatin. Data represents means ± SD of 3 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "WB analysis of CHK1-P levels at steady state or after treatment with 1 μM cisplatin for 3 hours. CHK1 total protein is used as loading control. Quantitative analysis of CHK1-P levels at steady state versus total CHK1 is shown in the bottom panel. Data represents means ± SD of 4 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours. Analysis of the ratio between FANCD2 ubiquitinated/non-ubiquitinated forms is shown on the bottom panel. Data represents means ± SD of 3 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Left panel: Growth curve was carried out to test smc5 fancm cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 fancm cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Clonogenic assay to test cisplatin sensitivity of smc5 fancm cells in long term assays. Data represents means ± SD of 2 independent experiments with two independent clones analyzed for smc5 fancm.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Left panel: Growth curve was carried out to test smc5 rad17 (C) cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 rad17 (C) cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Left panel: Growth curve was carried out to test smc5 ddx11 (D) cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 ddx11 (D) cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Top. Representative Western blots illustrating the expression of SMC6, FANCJ and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Bottom. Survival curves of cells transfected with indicated siRNA and treated with different concentrations of mitomycin C (MMC). Data represents the means and SEM (standard error of the mean) of 3 independent experiments.",
    "molecules": "mitomycin C, MMC"
  },
  {
    "caption": "Representative Western blots illustrating the expression of SMC6 and of monoubiquitylated FANCD2 in protein extracts from HeLa cells transfected with the indicated siRNAs and treated 48 hours after transfection with HU (5mM, 18 hours) or Mitomycin C (200 ng/ml, 18 hours). Vinculin is used as loading control.",
    "molecules": "HU, Mitomycin C"
  },
  {
    "caption": "Top. Representative images of nuclei (DAPI stained, blue) with RAD51 (green) foci in HeLa cells transfected with the indicated siRNAs and treated with HU or MMC, as indicated in panel A. The scale bar represents 10 μm. Bottom. Histograms presenting the percentage of RAD51 foci-positive cells as evaluated in the different conditions of above. Data represent the means+/-S.E.M. and were analyzed by unpaired t-test. siSMC6 vs siLacZ: n=3, NT, MMC or HU p=n.s.; siFANCD2 vs siLacZ: n=4, NT p=n.s., MMC p=0.0003, HU p=0.0479; siSMC6+siFANCD2 vs siLacZ: n=3, NT p=n.s., MMC p=0.0439, HU p=0.0458.",
    "molecules": "DAPI, HU, MMC"
  },
  {
    "caption": "Left panel: Representative images of anaphase HeLa cells with micronuclei (white arrows). 48 hours after transfection with indicated siRNAs cells were incubated 18 hours with 2 μg/ml cytochalasyn B to block cytokinesis. The scale bar represents 20 μm. Right panel: Histograms presenting the percentage of binucleate cells with micronuclei. Data represent the mean +/- SEM of 3 independent experiments, t-test, unpaired. siSMC6 vs siLacZ, p=0.0085; siFANCD2 vs siLacZ, p=0.0057; siSMC6+siFANCD2 vs siLacZ, p=0.0019.",
    "molecules": "cytochalasyn B"
  },
  {
    "caption": "Immunoprecipitation of FLAG-SMC6 and FLAG-NSMCE2 with FANCI in HEK293T cells overexpressing the SMC6 or NSMCE2, respectively, in the presence or absence of Ethidium bromide (EtBr). The experiment was performed 3 times, and the blot shows a representative experiment. The band under the one indicated as FANCI may represent a degradation product.",
    "molecules": "EtBr, Ethidium bromide"
  },
  {
    "caption": "Co-immunoprecipitation of endogenous SMC5-mAID-EGFP and FANCD2 in TK6 cells in the presence of benzonase. Both experiments were performed twice independently, with the note that the cisplatin condition was performed only in one of the experiments, and the FANCD2 pull down in the experiment not shown was performed without benzonase.",
    "molecules": "benzonase, cisplatin"
  },
  {
    "caption": "F. Top and bottom panels depict a representative nucleus of BJH2B-2FPs ESC after 15 min of ATP depletion by GFP intensity (i.e., nucleosome concentration) and FRET efficiency, respectively. Yellow arrows indicate nucleosome-rich foci associated with high levels of FRET. Scale bars,10 µm. The FRET efficiencies (%) from nucleosome-rich foci in untreated and ATP-depleted cells are depicted as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET efficiency (%) values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range. ****, p < 0.0001 (Mann-Whitney test, n = 61 cells). Mean distribution of the FRET efficiency of BJH2B-2FPs from nucleosome-rich foci in untreated (black, n = 162) and ATP-depleted cells (red, n = 61 cells). ****, p = 2.2e-16; K-S test.",
    "molecules": "ATP"
  },
  {
    "caption": "D. Representative image of the FRET efficiency map of interphase and mitotic ESCs (top and bottom panels, respectively) after fixation with 4% PFA. The mean FRET efficiency is displayed using a continuous pseudo-colour scale from 0 to 80%.",
    "molecules": "PFA"
  },
  {
    "caption": "E. Left panel, representative images of immunostaining for H4K20me3 in control ESCs (top row) and ESCs treated with A-196 during 2 days (bottom row). Scale bars, 10 µm. Right panel, Box-and-Whisker plots represent the normalized fluorescence intensity of immunostaining for H4K20me3. The box plots indicate median values (middle lines), mean values (middle crosses), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the fluorescence intensity. Data are means of n=2 biological replicates. Number of nuclei = 76/60 for control ESCs/A-196 treated ESCs.",
    "molecules": "A-196"
  },
  {
    "caption": "F. Total cell extracts from untreated, DMSO treated or A-196 treated BJH2B-2FPs during 24h or 48h, analysed by western blotting with antibody against H4K20me3. Arrowhead indicates the H4K20me3-specific bands. The asterisk indicates unspecific bands.",
    "molecules": "A-196, DMSO"
  },
  {
    "caption": "F. Left panel, mean distribution of the FRET efficiency related to the pixel fraction in nucleosome-rich foci from untreated EpiLCs (black, n = 124 cells) and A-196 EpiLCs treated cells (red, n = 113 cells). ****, p = 2.2e-16; K-S test. Right panel, Box-and-Whisker plots indicate the FRET efficiency (%) from untreated EpiLCs (black, n = 124 cells) and treated with A-196 during 2 days (red, n = 113 cells). The box plots indicate median values (middle lines), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the FRET efficiency.",
    "molecules": "A-196"
  },
  {
    "caption": "Strep-Tactin pull down  of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding  of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt.",
    "molecules": "Strep-Tactin, strep"
  },
  {
    "caption": "Lysates from yeast  strains expressing endogenously tagged 3xFLAG-Gid4  or 3x-FLAG-Gid10  that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol  treatment (EtOH  rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl  for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE  gel and immunoblotted  with αFLAG (two exposures from the same gel are shown) and αPGK.",
    "molecules": "ethanol, EtOH, NaCl"
  },
  {
    "caption": "In vitro competition assay  probing the ability of N-terminally 2xStrep-tagged Gid4   (2xS-Gid4) to exchange with Gid10  in the GIDSR10 complex. Proteins after  Strep-Tactin pull-down  were visualized by Coomassie-stained SDS-PAGE.",
    "molecules": "Strep-Tactin, Strep"
  },
  {
    "caption": "In vitro ubiquitination  assay probing the ability of Gid10  and Gid4  to promote ubiquitination  of Art22-29 N-terminus and its P2S mutant. Progress of the reaction was monitored by fluorescent scan of the gel visualizing the  Art2 peptide with FAM appended to its C-terminus (pep*).",
    "molecules": "FAM"
  },
  {
    "caption": "E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.",
    "molecules": "NE, 2APB, Ca2+, ionomycin"
  },
  {
    "caption": "F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.",
    "molecules": "SKF, 2APB"
  },
  {
    "caption": "B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.",
    "molecules": "2APB, dimethyl sulfoxide, DMSO, LPC"
  },
  {
    "caption": "C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.",
    "molecules": "triglyceride"
  },
  {
    "caption": "B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.",
    "molecules": "DMSO, ISO, isoproterenol"
  },
  {
    "caption": "D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.",
    "molecules": "DMSO, fatty acid, forskolin"
  },
  {
    "caption": "F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.",
    "molecules": "BAPTA-AM, ISO"
  },
  {
    "caption": "B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).",
    "molecules": "food, water"
  },
  {
    "caption": "A, B and C. Changes in mRNA expression of Ucp1 (A), Pgc1α (B) and Pparγ(C) from WT and TRPV2KO iBAT 4 h after intraperitoneal administration (i.p., arrows) of saline or a selective β3-adrenergic receptor agonist, BRL37344 (600 µg/kg body weight). Mean ± SEM, n = 5 - 7; * P < 0.05 vs. saline group; # P < 0.01 vs. WT BRL37344 administration group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.",
    "molecules": "BRL37344"
  },
  {
    "caption": "D and E. Average traces of changes in iBAT temperature (D) or rectal body temperature (E) from WT and TRPV2KO mice after BRL37344 (600 µg/kg body weight) administration (i.p., arrows). Mean ± SEM, n = 5. * P < 0.05 vs. WT group. Unpaired Student's t-test.",
    "molecules": "BRL37344"
  },
  {
    "caption": "F and G. iBAT NE turnover rates (F) and rate constants k (G) in WT and TRPV2KO mice upon cold exposure (4oC) for 4 h. Data are presented as mean ± SEM, n = 8.",
    "molecules": "NE"
  },
  {
    "caption": "B and C. Blood glucose level (B) and plasma insulin level (C) of WT and TRPV2KO mice after 8-week HFD treatment.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells.",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) Compared with control siRNA, Atg14L siRNA decreased long-lived protein degradation in NIH 3T3 cells under normal (*P = 0.007) and starvation (*P = 5 × 10−6) conditions (one-tailed Student's t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA, 10 mM), a PI(3)K inhibitor.",
    "molecules": "3-methyladenine"
  },
  {
    "caption": "(c) Vps34 kinase assay. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Atg14L or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-Myc antibody for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by thin-layer chromatography (TLC), quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34 as measured by western blot (upper panel). The quantified results (lower panel) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2.5-fold, but only when Beclin 1 was also overexpressed (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 5).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(a) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient-starvation conditions.",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient-starved conditions in HEK 293 cells either stably expressing (upper rows) or transiently transfected with (lower rows) Rubicon-EGFP. The control cells were either stably expressing or transiently transfected with the EGFP-N3 vector.",
    "molecules": "nutrient"
  },
  {
    "caption": "(c) Vps34 kinase activity. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Rubicon or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by an anti-Myc antibody and used for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by TLC, quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34, as measured by western blotting (upper panel). The quantified results (lower panel) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0.58-fold, but only without Beclin 1 overexpression (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 4).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(b) Partial colocalization of Rubicon-EGFP-associated structures with the MVB marker LBPA (arrows) in HeLa cells transfected with Rubicon-EGFP. Scale bar, 10 μm.",
    "molecules": "LBPA"
  },
  {
    "caption": "(d) Representative ultrastructural images show late endosome/lysosome-like structures that are labelled with anti-GFP gold particles (panels 3, 4) in HEK 293T cells transiently transfected with Rubicon-EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labelled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5-7) antibodies. Note that mitochondria are mostly negative for Rubicon-EGFP (panel 4). The negative controls are without antibody (panels 1-2). M, mitochondria. Scale bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "(b) Colocalization of the PI(3)P-enriched lipid domain marker p40 (phox)-PX-EGFP (green) and Rubicon-AsRed (red) on large punctate structures (arrows) in the co-transfected HeLa cells (upper panels). Treatment with the PI(3)K inhibitor wortmannin (75 nM) for 1 h caused disappearance of the PI(3)P-enriched lipid domains, whereas the Rubicon-AsRed-positive structures were maintained (lower panels). Scale bars, 10 μm.",
    "molecules": "PI(3)P, wortmannin"
  },
  {
    "caption": "Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C. elegans hypodermal mitochondria. Scale bar 10 µm.",
    "molecules": "MitoTrackerTM Red CMXRos"
  },
  {
    "caption": "Representative TMRE fluorescence traces in arbitrary units (a.u.) before and after Δψm dissipation with FCCP. Dashed lines indicate where FCCP was added. +/- ATR traces were performed in the absence of succinate. mtON activation was continuous throughout the traces. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in fluorescence (ΔF) normalized to the maximum change in fluorescence given by succinate respiration (ΔFmax) for each mitochondrial preparation. Data are from the maximum light dose in Fig. EV 3A. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0469, +ATR succinate vs. +ATR +light p = 0.9978, n = 6 mitochondrial isolations. Data show mean ± SEM.",
    "molecules": "ATR, FCCP, succinate, TMRE"
  },
  {
    "caption": "Representative BCECF-AM 490/440 nm ratio trace. Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation. Dashed lines are where light or FCCP treatment occurred. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in BCECF-AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization. Two-way ANOVA with Sidak's test for multiple comparisons, -ATR succinate vs. -ATR, +light *p = 0.0212, +ATR succinate vs. +ATR light p = 0.999, +ATR succinate vs. +ATR,-light p = 0.0237, +ATR, +light vs, +ATR, -light p = 0.0474, n = 3 mitochondrial isolations. Some individual points overlap. Data show mean ± SEM.",
    "molecules": "ATR, BCECF-AM, FCCP, succinate"
  },
  {
    "caption": "ATP levels normalized to total ATP synthesis given by succinate respiration. Data from Fig. EV 3B. Succinate data shown for comparison after normalization. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0011, +ATR succinate vs +ATR light p = 0.5680, n = 3,7,3,7, biological replicates for each bar from left to right. Some individual points overlap. Data show mean + SEM.",
    "molecules": "ATR, ATP, succinate, Succinate"
  },
  {
    "caption": "O2 required to consume 50 nmoles of ADP after mtON activation. Data are the maximum illumination from Fig. EV 4D using time-matched dark conditions, One-way ANOVA *p = 0.013 (-ATR, -light vs. +ATR, +light p = 0.010. -ATR, +light vs. +ATR, +light p = 0.013.) -ATR, -light n = 5, rest n = 6 mitochondrial preparations. Data show mean + SEM.",
    "molecules": "ADP, ATR, O2"
  },
  {
    "caption": "Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.",
    "molecules": "ATR, antimycin A, azide, oligomycin A, rotenone"
  },
  {
    "caption": "mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.",
    "molecules": "ATR"
  },
  {
    "caption": "Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.",
    "molecules": "AICAR, ATR"
  },
  {
    "caption": "Protection (%) is percent survival minus percent survival of control condition. PC data calculated from panel B. FCCP final concentrations 0.001, 0.01, 0.1, 1 nM. One-way ANOVA with Tukey's post hoc test, PC vs. 0.001 nM FCCP, *p = 0.0012, 0.001 nM FCCP vs 0.01 FCCP, *p = 0.0142, n = 4,3,3,3,3 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.",
    "molecules": "FCCP"
  },
  {
    "caption": "Illumination was continuous throughout PC alone; control illumination was for the same duration under normoxic conditions (see methods & panel A). Two-way ANOVA comparing +ATR vs -ATR in each group. *p = 0.016, n = 11,11,4,4,6,6 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.",
    "molecules": "ATR"
  },
  {
    "caption": "CSF extract was treated with DMSO, CsA, buffer or His-CnA420-508 at the indicated concentrations. Meiotic exit was induced by calcium addition and samples were taken at the indicated time points. Samples were immunoblotted for cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin.",
    "molecules": "buffer, calcium, CsA, DMSO"
  },
  {
    "caption": "CSF extract was treated with DMSO or CsA. Both reactions were divided and supplemented with the indicated amounts of calcium or H2O. After 20min, samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown. The cyclin B2 membrane was stripped and reprobed for α-tubulin.",
    "molecules": "calcium, CsA, DMSO, H2O"
  },
  {
    "caption": "CSF extract was supplemented with Myc-XErp1 wt IVT at the indicated dilutions. An empty IVT reaction not expressing Myc-XErp1 served as control. All reactions were divided and treated with DMSO or CsA. Calcium was added to all reactions and samples were taken at the indicated time points, treated with λ-phosphatase and immunoblotted for XErp1, the Myc-tag, cyclin B2 and α-tubulin. Asterisks indicate unspecific bands.",
    "molecules": "Calcium, CsA, DMSO"
  },
  {
    "caption": "CSF extract was supplemented with Myc-XErp1 wt IVT. The reaction was divided and treated with DMSO or CsA. Both reactions were treated with calcium, samples were taken at the indicated time points and as indicated incubated with λ-phosphatase. Samples were immunoblotted for XErp1 and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.",
    "molecules": "calcium, CsA, DMSO"
  },
  {
    "caption": "CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands.",
    "molecules": "calcium, CsA, DMSO"
  },
  {
    "caption": "CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.",
    "molecules": "alanine, Calcium, CsA, DMSO"
  },
  {
    "caption": "CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands.",
    "molecules": "Calcium, CsA, DMSO"
  },
  {
    "caption": "Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.",
    "molecules": "calcium, calmodulin"
  },
  {
    "caption": "CSF extract was supplemented with mRNA encoding Flag-Cdc20 that was either wild-type or mutated to serine at Thr64, Thr68 and Thr79 (3TS). After expression, calcium was added, samples were taken at the indicated time points and resolved by either conventional or Phos-tagTM SDS-PAGE. Samples were immunoblotted for the Flag-tag, Cdc27 and cyclin B2.",
    "molecules": "calcium, serine, Thr"
  },
  {
    "caption": "CSF extract was supplemented with Myc-XErp1 wt IVT and treated with DMSO or CsA. Calcium was added, samples were taken at the indicated time points, resolved by either conventional or Phos-tagTM SDS-PAGE and immunoblotted for Cdc20, Cdc27 and cyclin B2. A control sample was treated with λ-phosphatase. A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig. EV4A. The cyclin B2 membrane was stripped and reprobed for α-tubulin.",
    "molecules": "Calcium, CsA, DMSO"
  },
  {
    "caption": "CSF extract was supplemented with Flag-Cdc20 wt IVT coupled to α-Flag beads. Calcium was added and Flag-Cdc20 was isolated by α-Flag immunoprecipitation at the indicated time points. Input and pellet (IP: Flag) samples were immunoblotted for the Flag-tag, pThr68 Cdc20 and p150(Glued). The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2. Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain. Asterisks in other immunoblots indicate unspecific bands.",
    "molecules": "Calcium, Thr"
  },
  {
    "caption": "CSF extract was supplemented with Flag-Cdc20 wt IVT. Flag-Cdc20 was re-isolated by α-Flag immunoprecipitation, supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the Flag-tag and pThr68 Cdc20. The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA. Asterisk indicates the IgG heavy chain.",
    "molecules": "calcium, calmodulin, Thr"
  },
  {
    "caption": "A, Gait analysis using the CatWalk XT system reveals motor defects in 6-OHDA lesion model. Both dCAM and AAV-dCAS  animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread. Naive vs. 6-OHDA P=0.0063. AAV-dCAS: GFP  vs.  ALN P=0.015 multiple comparison ANOVA F(2,17)=12.81) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP  vs ALN, GFP  vs  ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice  per condition.",
    "molecules": "6-OHDA"
  },
  {
    "caption": "the stride length of  front paws: B, Naive vs. 6-OHDA P=0.0164. AAV-dCAS: GFP  vs. ALN  P=0.005, ALN  vs. ALNe-218  P=0.0042, multiple comparison ANOVA F(4,22)=9.9) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP  vs ALN, GFP  vs  ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice  per condition.",
    "molecules": "6-OHDA"
  },
  {
    "caption": "the duty cycle of left front paws. C, Naive vs. 6-OHDA P=0.0096. dCAM: GFP  vs. ALN P=0.036 and ALN vs. ALNe-218 P=0.0252, multiple comparison ANOVA F(2,20)=5.199). Data information: Statistics: naive vs. 6-OHDA  unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP  vs ALN, GFP  vs ALNe-218 and  ALN  vs ALNe-218  Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice  per condition.",
    "molecules": "6-OHDA"
  },
  {
    "caption": "D, In contrast to this, dopamine  dependent drug-induced behavior does not show rescue effects: amphetamine-induced  rotation analysis: change in rotational behavior in lesioned animals upon treatment with dopamine  releaser substance. Net rotation = ipsilateral rotation-contralateral rotation (naïve vs. 6-OHDA P=0.09). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP  vs ALN, GFP  vs  ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice  per condition. CatWalk error bars represent mean ± SD. Rotation analysis error bars represent mean ± SEM.",
    "molecules": "amphetamine, dopamine, 6-OHDA"
  },
  {
    "caption": "WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. A Cells were incubated under unstimulated conditions or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies for 0 to 5 days (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).",
    "molecules": "CFSE"
  },
  {
    "caption": "WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. B Dose dependence of EGR4-dependent differences in proliferation were also determined in both CD4+ and CD8+ cells aft er treatment with anti-TCRβ (E,F) or anti-CD3/CD28 (G,H) at the indicated concentrations before FACS analysis (Appendix Fig S2) (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).",
    "molecules": "CFSE"
  },
  {
    "caption": "WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. C,D Generation analysis was then assessed in WT, EGR1-/- and EGR4-/- CD4+ (C) or CD8+ (D) T cells after a 4 day incubation, under unstimulated conditions, or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies. Generation analysis was determined using FlowJo as depicted in Appendix figure S3 (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).. EGR4 deletion significantly shifted the number of generations as determined by two-way ANOVA; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions (p < 0.0001).",
    "molecules": "CFSE"
  },
  {
    "caption": "(A) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4+ and CD8+ T cells isolated by negative selection from WT, EGR1KO and EGR4KO mice. Cells were stimulated with anti-CD3/CD28 antibodies for the indicated time periods before fixation and staining. Nuclear localization of NFATc1 in CD4+ and CD8+ T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis (mean±SEM; data are based upon 13 to 20 cells/data point from one experiment; experiment was repeated 3 times). EGR4-mediated differences in NFATc1 localization were determined by two-way ANOVA. NFAT localization was dependent upon EGR4 expression in both CD4+ (p=0.025) and CD8+ (p=0.039) T cells. Post-hoc analysis revealed EGR4-dependent differences at specific timepoints * p < 0.05; *** p < 0.001.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) WT, EGR1KO and EGR4KO CD4+ T cells were isolated from the spleen by negative selection before plating on poly-lysine (control) or CD3/CD28 and loading with fura2. Ca2+ levels shown are from representative single cells measured from multiple experiments. (C) Scatter plots showing area under the curve (AUC) and variance for each cell measured under all conditions.",
    "molecules": "Ca2+, fura2, poly-lysine"
  },
  {
    "caption": "CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (A) CD4+ and CD8+ T cells were loaded with Fura-2AM and plated on poly-D-lysine (2 hrs) before treating with biotinylated anti-CD3ε, followed by streptavidin where indicated. The average response of the majority of CD4+ (WT 74.2±3.2%; EGR4-/- 77.8±2.9%) and CD8+ (WT 79.4±3.5%; EGR4-/- 78.2±2.5%) cells that responded to streptavidin crosslinking of CD3 (mean±SEM; 40-60 cells/coverslip; 7 to 8 separate experiments).",
    "molecules": "biotinylated, Fura-2AM, poly-D-lysine, streptavidin"
  },
  {
    "caption": "CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (B) Cells were plated on anti-CD3/CD28 antibodies for 2 hrs; BTP2 (10 μM) was added where marked by the arrow.",
    "molecules": "BTP2"
  },
  {
    "caption": "CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (A) WT or EGR4-/- cells were loaded with Fura-2AM and plated on anti-CD3/CD28 antibodies for 2 hrs. Senicapoc (4 μM) or Tram-34 (5 μM) were added where marked by the arrow.",
    "molecules": "Fura-2AM, Senicapoc, Tram-34"
  },
  {
    "caption": "CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (B) CD4+ T cells were isolated by negative selection from WT or EGR4-/- mice and incubated with anti-CD3/CD28 antibodies for 6 hrs in the presence or absence of either senicapoc, TRAM-34 or Shk-Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates. *** p < 0.001; **** p < 0.0001.",
    "molecules": "senicapoc, Shk-Dap22, TRAM-34"
  },
  {
    "caption": "CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (C) To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4-/- CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti-TCRβ antibodies in the presence of vehicle, Senicapoc (5 µM) or TRAM-34 (5 µM) for 4 days and then collected for FACS analysis. (D) Generation analysis was completed for all data using FlowJo software. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates.",
    "molecules": "CFSE, Senicapoc, TRAM-34"
  },
  {
    "caption": "B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (A) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections. ",
    "molecules": "luciferin"
  },
  {
    "caption": "Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (A) CD4+ and CD8+ T cells were stimulated in vitro with ionomycin/PMA before intracellular staining for IFNγ, IL-17 and IL-9 and FACS analysis.",
    "molecules": "Ficoll-Hypaque, ionomycin, PMA"
  },
  {
    "caption": "Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (B,C) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to D",
    "molecules": "Ficoll-Hypaque"
  },
  {
    "caption": "Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (D) Percentages of each population.",
    "molecules": "Ficoll-Hypaque"
  },
  {
    "caption": "Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (E) Gating strategies.",
    "molecules": "Ficoll-Hypaque"
  },
  {
    "caption": "Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (F) FR4 and PD1 levels in WT vs. EGR4-/- T cells.",
    "molecules": "Ficoll-Hypaque"
  },
  {
    "caption": "(A)  Acyl-RAC assay of HEK293T  cells transfected with the indicated plasmids for 24 h. Data information: EtOH: ethanol; HAM: hydroxylamine; 2-BP:  2-bromopalmitate",
    "molecules": "2-BP, 2-bromopalmitate, ethanol, EtOH, HAM, hydroxylamine"
  },
  {
    "caption": "(C) Click chemistry was applied to detect endogenous cGAS palmitoylation  in RAW264.7  cells. Data information: PA: palmitic acid; 17-ODYA: 17-octadecanoic acid.",
    "molecules": "PA, palmitic acid, 17-octadecanoic acid, 17-ODYA"
  },
  {
    "caption": "(D and E) In vitro palmitoylation assay for the indicated proteins. NBD-Palm-CoA: (N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] amino) palmitoyl-CoA. The recombinant cGAS  protein used in the assay was indicated by  Coomassie blue staining.",
    "molecules": "(N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] amino) palmitoyl-CoA, NBD-Palm-CoA"
  },
  {
    "caption": "(G) Acyl-RAC assay of HEK293T  cells transfected with the indicated plasmids for 24 h. Data information: HAM: hydroxylamine",
    "molecules": "HAM, hydroxylamine"
  },
  {
    "caption": "(H-I) THP-1 cells were treated with DMSO  or 2-BP (50 μM) (H) or palmitic acid (100 μM) (I) for 12 h. Six hours after transfection with HT-DNA (2 μg/mL),  cGAMP was extracted and quantified by  cGAMP ELISA. Data information: 2-BP: 2-bromopalmitate Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001",
    "molecules": "HT-DNA, cGAMP, 2-BP, 2-bromopalmitate, DMSO, palmitic acid"
  },
  {
    "caption": "(J) THP-1  cells were treated with  palmitic acid  (0, 50 μM, or 100 μM) for 12 h before a cGAMP  bioassay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001",
    "molecules": "cGAMP, palmitic acid"
  },
  {
    "caption": "BMDMs  were treated with  palmitic acid  (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA  (2 μg/mL) for 6 h before  RT-qPCR analysis of Ifna4  (K), IFNb1  (L) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001",
    "molecules": "HT-DNA, palmitic acid"
  },
  {
    "caption": "BMDMs  were treated with  palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of , Cxcl10  (M) and Rantes  (N) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001",
    "molecules": "HT-DNA, palmitic acid"
  },
  {
    "caption": "(O) L929  cells were treated with BSA  and palmitic acid (100 μM) and transfected with HT-DNA (2 μg/mL) for the indicated times  before immunoblotting  analysis with the indicated antibodies.",
    "molecules": "HT-DNA, palmitic acid"
  },
  {
    "caption": "(C) Acyl-RAC assay  of HEK293T  cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine.",
    "molecules": "HAM, hydroxylamine"
  },
  {
    "caption": "(K) Immunofluorescence  analysis of HA-cGAS  (green) and Flag-ZDHHC18 (red) in HT-DNA-stimulated (or not) HeLa  cells. Scale bars: 7 μm. (L) Colocalization  (merged volume of total cGAS  signal) of cGAS  and ZDHHC18  in (K). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 L).",
    "molecules": "HT-DNA"
  },
  {
    "caption": "(A) Immunofluorescence  analysis of GFP-cGAS, the nucleus (DAPI), the ER  (calnexin), the Golgi apparatus (GM130), and early endosomes  (EEA1) in HeLa cells. 2-BP: 2-bromopalmitate; ISD: IFN-stimulatory DNA. Arrows indicate cGAS  puncta. Scale bars: 10 μm.",
    "molecules": "IFN-stimulatory DNA, ISD, 2-BP, 2-bromopalmitate, DAPI"
  },
  {
    "caption": "(F HEK293T  cells were transfected with the indicated plasmids. Biotin-conjugated ISD  was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin  beads and then assessed by immunoblot  analysis as shown. Data information: WCL: whole-cell lysate; IP: immunoprecipitation. Data are representative of at least two independent experiments.",
    "molecules": "ISD, Biotin, streptavidin"
  },
  {
    "caption": "G) HEK293T  cells were transfected with the indicated plasmids. Biotin-conjugated ISD  was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin  beads and then assessed by immunoblot  analysis as shown. Data information: WCL: whole-cell lysate; Data are representative of at least two independent experiments.",
    "molecules": "ISD, Biotin, streptavidin"
  },
  {
    "caption": "(H)  Confocal microscopy of BMDMs  (Zdhhc18+/+ or Zdhhc18−/−) transfected with FITC-conjugated ISD  for 6 h (left). Scale bar: 20 μm.",
    "molecules": "ISD, FITC"
  },
  {
    "caption": "(C) Cross-correlated motions of the Cα  atoms of the WT and palmitoylated cGAS  proteins.",
    "molecules": "Cα  atoms"
  },
  {
    "caption": "(E) Immunoprecipitation  (with an anti-Flag  antibody) and immunoblot  analysis of HEK293T  cells transfected with the indicated plasmids followed by the addition of DMSO,  2-BP  (50 μM) or palmitic acid  (100 μM). Data information: WCL: whole-cell lysate; IP: immunoprecipitation; 2-BP:  2-bromopalmitate. Data are representative of at least two independent experiments.",
    "molecules": "2-BP, 2-bromopalmitate, DMSO, palmitic acid"
  },
  {
    "caption": "(F) FRET  assay of HeLa  cells transfected with the indicated plasmids with the addition of DMSO,  2-BP (50 μM) or palmitic acid  (100 μM). Scale bar: 8 μm. (G) FRET  efficiency of cGAS-GFP  and cGAS-mCherry  in (F). Data information:  2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 (G",
    "molecules": "2-BP, 2-bromopalmitate, DMSO, palmitic acid"
  },
  {
    "caption": "(A) HEK293T  cells (5×105) were transfected with HA-cGAS  (200 ng), Flag-STING  (200 ng) and Myc-ZDHHC18  (WT or CS mutant) expression plasmids (0, 50, 100, or 200 ng) for 24 h before  luciferase reporter assays were conducted. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine  substitution in the DHHC motif of ZDHHC18  All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 (A",
    "molecules": "cysteine, serine"
  },
  {
    "caption": "(B) HEK293T  cells (1×106) were transfected with HA-cGAS  (500 ng), Flag-STING  (500 ng) and Myc-ZDHHC18  (500 ng) expression plasmids for 24 h. TBK1 phosphorylation  was detected by immunoblotting. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine  substitution in the DHHC motif of ZDHHC18",
    "molecules": "cysteine, serine"
  },
  {
    "caption": "(C and D) HEK293T  cells were transfected with the indicated plasmids, and the amount of cGAMP  in the lysates was quantified by  LC-MS/MS. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine  substitution in the DHHC motif of ZDHHC18; LC-MS/MS: liquid chromatography-tandem mass spectrometry;",
    "molecules": "cGAMP, cysteine, serine"
  },
  {
    "caption": "L929  cells stably transfected with control shRNA or ZDHHC18  shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times  before RT-qPCR  analysis of Ifna4  (G), expression. Data information:  HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001",
    "molecules": "herring testis DNA, HT-DNA"
  },
  {
    "caption": "L929  cells stably transfected with control shRNA or ZDHHC18  shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times  before RT-qPCR analysis of IFNb1  (H), and Cxcl10  (I) expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001",
    "molecules": "herring testis DNA, HT-DNA"
  },
  {
    "caption": "(J) L929  cells stably transfected with control shRNA or ZDHHC18  shRNA were transfected with HT-DNA  (2 μg/mL) for the indicated times   before immunoblotting  analysis with the indicated antibodies. Data information: HT-DNA: herring testis DNA; NC: negative control.",
    "molecules": "herring testis DNA, HT-DNA"
  },
  {
    "caption": "(B) Representative photomicrographs  of H&E-stained liver  and kidney  tissue sections from mice  (Zdhhc18+/+ or Zdhhc18-/-) intravenously injected with HSV-1-FS (5 × 107 PFU per mouse) or not after 2 days. Scale bars: 100 μm (liver), 200 μm (kidney). Data information: PFU: plaque-forming units; H&E: hematoxylin-eosin. Data are representative of at least two independent experiments.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "A. Western blot of nuclear and cytoplasmic protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured with anti-IgM (α-IgM) for 4h, followed by the indicated treatment for 2h. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to Sam68 or Tubulin and compared to the DMSO-treated sample. Representative data of n = 3 independent experiments.",
    "molecules": "DMSO"
  },
  {
    "caption": "B. qPCR analysis of Cyp1a1 expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 24h as indicated. Cyp1a1 expression was normalized to Hprt1. Cyp1a1 expression among groups was normalized to Medium DMSO. n = 2 independent experiments; mean ± range.",
    "molecules": "DMSO"
  },
  {
    "caption": "A. Flow cytometry analysis of CTV dilution in splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, solid grey) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, black) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. Representative data of n = 4-5 independent experiments.B-D. Flow cytometry analysis of expansion index (B), % of divided cells (C) and replication index (D) of splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. n = 4-5 independent experiments; mean ± SEM; 2-way ANOVA, Sidak's multiple comparison test.",
    "molecules": "CH223191"
  },
  {
    "caption": "A, B. Tables showing top 20 down-regulated (A) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.",
    "molecules": "FICZ"
  },
  {
    "caption": "A, B. Tables showing top up-regulated (B) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.",
    "molecules": "FICZ"
  },
  {
    "caption": "E. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM in presence or absence of 250 nM FICZ or 1 μM 3-MC. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.",
    "molecules": "3-MC, FICZ"
  },
  {
    "caption": "F. Chromatin Immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter and an irrelevant region (-3.3 Kb from Ccno transcription starting site) in Ahr+/+ (black) and Ahr-/- (white) B cells 5h after activation with 10 µg/ml α-IgM + 20 ng/ml IL-4. 250 nM FICZ was added in the last hour of culture. Representative data of n = 2 independent experiments; mean ± SEM; two-way ANOVA, Sidak's multiple comparison test.",
    "molecules": "FICZ"
  },
  {
    "caption": "(A, B) Representative images of the expression  of NEDD4L in the skins from IMQ-treated mice (A) or psoriasis patients (B) as detected by  IHC on the left. The  NEDD4L expression level was calculated by multiplying the staining intensity score and the extent score and is shown on the right. (A, n=5, B, normal, n=15, psoriasis, n=36). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.",
    "molecules": "IMQ"
  },
  {
    "caption": "(C, D) Representative images of the expression of EZH2 in the skin epidermis from the IMQ-treated  mice (C) and the  psoriasis patients (D). The number  of  EZH2-positive cells per HPF (high power field) in each mouse and patient is shown on the right. (C, control, n=3, IMQ, n=6. D, normal, n=13, psoriasis, n=27). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.",
    "molecules": "IMQ"
  },
  {
    "caption": "(E) WB analysis of NEDD4L, EZH2  and EZH1 expression in the EZH2-specific siRNA (siEZH2)- or negative control siRNA (siNC)-transfected NHEKs with  IMQ stimulation. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×).",
    "molecules": "IMQ"
  },
  {
    "caption": "(D) Representative images of HE staining of the back tissues are shown on the left. The epidermal thickness measured using the scale in the microscope is shown on the right. (E) Representative images of Ki67 staining in back tissues are shown on the left. The number of Ki67-positive cells per HPF in each mouse is shown on the right. (F) Representative images of HE staining of the ear tissues are shown on the left. The epidermis thickness measured using the scale in the microscope is shown on the right. (G) Representative images of Ki67 staining in ear tissues (same specimen as in F) are shown on the left. The number of Ki67 positive cells per HPF in each mouse is shown on the right. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Student's t test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance.",
    "molecules": "IMQ"
  },
  {
    "caption": "Six- to eight-weeks old WT and Nedd4l  cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected  intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative images of Ki67 staining in back tissues are on the left. The number of Ki67-positive cells per HPF is shown on the right. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test C, control, n=5, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.",
    "molecules": "IMQ"
  },
  {
    "caption": "Six- to eight-weeks old WT and Nedd4l  cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Keratinocyte proliferation associated genes in the skin tissues  are detected using real-time PCR analysis. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.",
    "molecules": "IMQ"
  },
  {
    "caption": "(C) WT or  NEDD4L KO NHEK  cells was infected with  lentivirus that overexpress NEDD4L-WT, NEDD4L WW domain truncated mutant (NEDD4L-ΔWW) or its enzymatic activity mutant (NEDD4L-C943S), the cells were followed by denaturation-IP with anti-GP130 antibody and WB analysis with anti-Ub antibodies.",
    "molecules": "Ub"
  },
  {
    "caption": "(E) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with full length Myc-NEDD4L, HA-Ub and Flag-GP130 or its lysine mutants (K-R) in the intracellular domain, followed by denaturation-IP with ANTI-FLAG M2 magnetic beads and WB analysis with anti-HA antibodies.",
    "molecules": "lysine"
  },
  {
    "caption": "Polyubiquitination assays of MT-HCT116 cells cotransfected with two independent β-catenin siRNAs and HA-ubiquitin (Ub) for 72 hr in the presence of the proteasomal inhibitor MG132, and WCLs were immunoprecipitated with an anti-pan-RAS antibody",
    "molecules": "MG132"
  },
  {
    "caption": "Effects of KYA1797K or XAV939 on degradation of β-catenin and RAS in isogenic HCT116 cells harboring WT- or MT-CTNNB1. IB analyses of β-catenin and RAS levels in WT- or MT-HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hr",
    "molecules": "KYA1797K, XAV939"
  },
  {
    "caption": "ICC analyses of WT- or MT-HCT116 cells treated with 20 µM of KYA1797K for 24 hr. Scale bars, 10 µm",
    "molecules": "KYA1797K"
  },
  {
    "caption": "For polyubiquitination assays, WT- or MT-HCT116 cells were transfected with FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132. WCLs were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses to detect each protein",
    "molecules": "KYA1797K, MG132"
  },
  {
    "caption": "The 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assays were performed for measurements of growth of WT- or MT-HCT116 cells treated with the indicated concentrations of KYA1797K. Data are presented as the mean ± SD (n = 3)",
    "molecules": "3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide, KYA1797K"
  },
  {
    "caption": "Nuclear magnetic resonance (NMR) titration for 15N-labeled KRAS (full-length, 1-188) with β-catenin (C-terminal fragment, 661-781). The titration points are indicated in red (1:0), green (1:5), and blue (1:10). A gradual perturbation in chemical shift is indicated by the arrow",
    "molecules": "15N"
  },
  {
    "caption": "Effects of KYA1797K on the interaction and phosphorylation of β-catenin and pan-RAS. WT- or MT-HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses",
    "molecules": "KYA1797K, MG132"
  },
  {
    "caption": "Effects of Wnt3a-CM treatment on the formation of β-catenin destruction complex and phosphorylation of β-catenin and RAS in HEK293 cells treated with L-CM or Wnt3a-CM in the presence of MG132 for 8 hr. WCLs were immunoprecipitated with β-catenin or pan-RAS antibodies and subjected to IB analyses",
    "molecules": "MG132"
  },
  {
    "caption": "IB analyses were performed to detect β-catenin and pan-RAS in DLD1 cells cotransfected with FLAG-β-catenin-WT or -MT and Myc-KRAS-WT or -2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA-GSK3β for 24 hr, respectively",
    "molecules": "KYA1797K"
  },
  {
    "caption": "IB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG-β-catenin and GFP-BBR or -Mock in the presence of proteasome inhibitor, ALLN, for 24 hr",
    "molecules": "ALLN"
  },
  {
    "caption": "Effects of BBR overexpression on KYA1797K-induced RAS degradation. I analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr",
    "molecules": "KYA1797K"
  },
  {
    "caption": "Effects of BBR overexpression on KYA1797K-induced RAS degradation ICC (D; Scale bars, 10 µm) analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr",
    "molecules": "KYA1797K"
  },
  {
    "caption": "MT-HCT116 cells were cotransfected with GFP-Mock or -BBR and FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr, and WCLs were immunoprecipitated with pan-RAS antibody",
    "molecules": "KYA1797K, MG132"
  },
  {
    "caption": "IB analyses of IP with β-catenin antibody in MT-HCT116 cells transfected with GFP-BBR-WT, -2E, or GFP-Mock in the presence of ALLN for 24 hr",
    "molecules": "ALLN"
  },
  {
    "caption": "IB analyses of MT-HCT116 cells transfected with GFP-Mock, -BBR-WT, or -BBR-2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hr",
    "molecules": "KYA1797K"
  },
  {
    "caption": "MT-HCT116 cells were transfected with GFP-Mock, -BBR-WT, or -BBR-2E together with FLAG-Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses",
    "molecules": "KYA1797K, MG132"
  },
  {
    "caption": "Effects of KYA1797K and PTD-BBR on RAS degradation IB assay of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively",
    "molecules": "KYA1797K"
  },
  {
    "caption": "Effects of KYA1797K and PTD-BBR o cell growth MTT assays of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively. Data are presented as the mean ± SD (n = 3)",
    "molecules": "KYA1797K"
  },
  {
    "caption": "Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growth",
    "molecules": "KYA1797K"
  },
  {
    "caption": "Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformation",
    "molecules": "KYA1797K"
  },
  {
    "caption": "Effects of cotreatments with KYA1797K and PTD-BBR on xenografted tumor growth. MT-HCT116 cells were subcutaneously injected in nude mice with subsequent i.p. injection of vehicle, 20 mg/kg of KYA1797K, or a cotreatment of PTD-BBR (25 mg/kg) and KYA1797K (20 mg/kg) for 20 d. Tumor volumes (upper panel) were measured every 4 days, and tumor weights (lower panel) were measured at the times of sacrifice. Data are presented as the mean ± SD (four mice per group)",
    "molecules": "KYA1797K"
  },
  {
    "caption": "IHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI. Scale bar, 20 µm",
    "molecules": "DAPI"
  },
  {
    "caption": "Viability of single and double mutants of RNAPII and rad52Δ, mre11Δ and cdc44‐8. Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0.005% MMS.",
    "molecules": "HU, MMS"
  },
  {
    "caption": "HU and MMS sensitivity of different mutant combinations of rpb1‐1, rpb1‐S751F, pol32Δ and rad51Δ.",
    "molecules": "HU, MMS"
  },
  {
    "caption": "Western blot against the phosphorylated form of histone H2A in the presence of 100 mM HU in asynchronously growing WT and RNAPII mutant cells. Ponceau staining is shown as a loading control.",
    "molecules": "HU"
  },
  {
    "caption": "Recombination frequencies using the direct‐repeat chromosomal system leu2‐k::ADE2‐URA3::leu2‐k in WT and RNAPII mutants. Deletion events were detected and quantified in media containing 5‐fluorotic acid (FOA) and could be directly visualized as red sectors.",
    "molecules": "5‐fluorotic acid, FOA"
  },
  {
    "caption": "Recombination frequencies between plasmid‐born direct repeats in WT and RNAPII mutants. The frequency of Leu+ recombination was determined in the systems L‐lacZ and GL‐lacZ. Recombination frequencies are plotted as a function of the transcription levels. Low transcription refers to the GL‐lacZ systems in strains cultured in 2% glucose; medium refers to L‐lacZ in 2% glucose, and high to GL‐lacZ in 2% galactose.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "Mitotic stability of centromeric plasmid pGAL‐lacZ in RNAPII mutants. Stability was analyzed in galactose after 23 generations. A small diagram of each system (not drawn to scale) is shown.",
    "molecules": "galactose"
  },
  {
    "caption": "Viability after different times in 10 mM HU. The same cultures without HU were used as a control. The average values and standard deviations from three independent experiments are shown.",
    "molecules": "HU"
  },
  {
    "caption": "FACS analysis of cell cycle progression in rpb mutants. Cells were synchronized in G1 with α‐factor and monitored at different times after release in the presence of 40 mM HU.",
    "molecules": "HU"
  },
  {
    "caption": "Slowdown of RF progression through a chromosomal region. 2D gel electrophoresis of replication intermediates of 4.3‐kb PstI DNA fragments from WT and RNAPII mutants in the presence of 40 mM HU at different times after G1 release. A black arrow indicates accumulation of long Y molecules, and a white one indicates the specific accumulation of X molecules.",
    "molecules": "HU"
  },
  {
    "caption": "DNA combing analysis of replication. Graphs indicate the distribution of BrdU track length (kb) at 60 and 120 min after α‐factor release in the presence of 40 mM HU, the replication speed (kb/min) determined with the two time points analyzed and the inter‐origin distance (kb) at 60 min after release. The median test was applied.",
    "molecules": "HU"
  },
  {
    "caption": "Dephosphorylation of Rad53 after exposure to HU. WT, rpb1‐1 and rpb1‐S751F cells were synchronized in G1 with α‐factor. After G1 release, cells were exposed to 100 mM HU for 1 h, washed to remove HU and cultured in HU‐free medium for the indicated periods. Western blot against Rad53, signal quantification (ratio Rad53‐P/total Rad53 protein) and FACS profiles are shown.",
    "molecules": "HU"
  },
  {
    "caption": "DNA repair of MMS damage. Chromosome VII species revealed by hybridization with ADE5,7 probes in pulsed‐field gel electrophoresis (PFGE). Asynchronously growing WT and rpb1 cells were exposed to 0.1% MMS for 30 min, washed to remove MMS and cultured in MMS‐free medium for the indicated periods to allow repair. Repair was calculated as the percentage of FLC appearing after MMS treatment. The FLC signal was quantified with respect to the total signal of each lane. Data show the mean and SD of 3 independent experiments. NLC, nonlinear chromosome. FLC, full‐length linear chromosome. CF, chromosome fragment resulting from DNA breaks.",
    "molecules": "MMS"
  },
  {
    "caption": "Northern blot analysis of the expression of PMA1 gene. RNA was isolated from mid‐log phase cultures grown in YPD‐rich medium. As 32P‐labeled DNA probes, an internal 776‐bp PMA1 fragment and an internal 513‐bp SCR1 fragment obtained by PCR were used. Bars indicate the SD of 2 independent experiments.",
    "molecules": "32P"
  },
  {
    "caption": "(A) Drosophila S2 cells stably expressing GFP-RFP-Atg8a showed an increase in the percentage of cells containing two or more autolysosomes after starvation, which was blocked after Bafilomycin A1 (BafA1) treatment. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01). (B) Representative images of S2-GFP-RFP-Atg8a cells after the indicated treatments.",
    "molecules": "BafA1, Bafilomycin A1"
  },
  {
    "caption": "(C) Western blot from l(2)mbn cells subjected to nutrient-rich or starvation conditions for 6 h. Cells were separated into cytosolic (C) and mitochondrial enriched (M) fractions.",
    "molecules": "nutrient"
  },
  {
    "caption": "(G) Control and Dcp-1 RNAi-treated cells were subjected to nutrient-rich or starvation conditions and stained with NAO. Mean fluorescence was measured by flow cytometry. Graph represents ± SEM of three independent experiments (n = 3).",
    "molecules": "nutrient"
  },
  {
    "caption": "(A) l(2)mbn cells were labeled with the ATPsyn-α, and mitochondrial morphology was scored as fragmented, normal, or elongated. (B) Cells were treated with control or Dcp-1 dsRNA and subjected to nutrient-rich media or 1 h of starvation. Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined. At least 100 cells were examined manually in three independent experiments (n = 3). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA with a Bonferroni post test (*, P < 0.05; **, P < 0.01).",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) Mitochondrial targeted GFP (mitoGFP) was expressed in the germline using the nosGAL4 driver. Staining shows mitoGFP, Armadillo, and DNA. (D) Mitochondria were scored as healthy (H), clustered (C), or elongated and overly connected (E). All of mitochondria from fed UASp-mitoGFP/+;nosGAL4/+ flies were scored as healthy. n = 15 egg chambers manually scored. (E) mitoGFP was expressed in Dcp-1Prev1 flies using the nosGAL4 driver. (F) 54% of egg chambers from UASp-mitoGFP/+;Dcp-1Prev1/Dcp-1Prev1;nosGAL4/+ flies contained elongated mitochondria, 39% contained mitochondria that were scored as healthy, and 7% contained clustered mitochondria. n = 28 egg chambers manually scored.",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp-1Prev1 flies. Data represent ± SEM of five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test (**, P = 0.014; ***, P < 0.001).",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Fed or starved w1118 and Dcp-1Prev1 flies were treated with DMSO or 25 µg/ml oligomycin A, and Ref(2)P levels were assessed by immunoblot analysis. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graph represents ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P = 0.02).",
    "molecules": "DMSO, oligomycin A"
  },
  {
    "caption": "(D and E) Control UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies (D) and Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies (E) were subjected to starvation conditions supplemented with DMSO or 25 µg/ml oligomycin A. Bars, 25 µm. Quantifications show percentage of autolysosomes (autolysosomes/total autophagic structures). At least eight egg chambers were manually quantitated per genotype per condition (n = 8). Statistical testing was determined using a two-tailed Student's t test. ***, P = 0.0002.",
    "molecules": "DMSO, oligomycin A"
  },
  {
    "caption": "(D) V5-tagged Dcp-1C<A or V5 vector only control was expressed in l(2)mbn cells and subjected to nutrient-full or starvation conditions for 2 h. After IP with anti-V5 antibodies, lysates were separated by SDS-PAGE. Proteins were visualized with colloidal Coomassie stain.",
    "molecules": "nutrient"
  },
  {
    "caption": "(B) NIH3T3 cells expressing the WT or mutant forms of vBcl-2 as indicated were transfected with GFP-LC3 and treated with 2 µM rapamycin for 6 h. GFP-LC3 was detected using an inverted fluorescence microscope (top). Autophagy was quantified as mean±SEM of the combined results from three independent experiments. Scale bar, 5 µm; **, P<0.01.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(C) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2, as indicated, were treated with 2 µM rapamycin. LC3-I and LC3-II levels were then determined by immunoblotting with an antibody against LC3 (top). Densitometric quantification of the LC3-II/LC3-I ratios under normal and rapamycin treatment conditions is shown at the bottom. Similar results were obtained from three independent experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A),",
    "molecules": "cycloheximide, CHX"
  },
  {
    "caption": "NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B)",
    "molecules": "cycloheximide, CHX"
  },
  {
    "caption": "NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) or for the caspase 3 activation using flow cytometry (C). Apoptotic cells in (B) were counted under high power magnification (60×magnification). Mock, untreated condition. Data represents mean±SEM of combined results from three independent experiments. Scale bars, 100 µm (A), 5 µm (B). **, P<0.0001 versus vector cells.",
    "molecules": "cycloheximide, CHX"
  },
  {
    "caption": "Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.",
    "molecules": "thymidine"
  },
  {
    "caption": "Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.",
    "molecules": "DMSO, nocodazole"
  },
  {
    "caption": "Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.",
    "molecules": "DMSO, nocodazole, MLN4924"
  },
  {
    "caption": "Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.",
    "molecules": "CHX, cycloheximide, MLN4924"
  },
  {
    "caption": "C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.",
    "molecules": "alanine, alanines, MG132, Nocodazole"
  },
  {
    "caption": "Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.",
    "molecules": "Nocodazole"
  },
  {
    "caption": "Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.",
    "molecules": "thymidine"
  },
  {
    "caption": "Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.",
    "molecules": "MG132"
  },
  {
    "caption": "Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.",
    "molecules": "HU"
  },
  {
    "caption": "Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.",
    "molecules": "HU"
  },
  {
    "caption": "Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.",
    "molecules": "doxycycline, HU"
  },
  {
    "caption": "Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.",
    "molecules": "doxycycline, HU"
  },
  {
    "caption": "Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.",
    "molecules": "nocodazole"
  },
  {
    "caption": "qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm.",
    "molecules": "DAPI, nocodazole"
  },
  {
    "caption": "Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01).",
    "molecules": "HU"
  },
  {
    "caption": "(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth for 10 hours and then moved to room temperature for 3-day recovery or (B) sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.",
    "molecules": "methyl viologen"
  },
  {
    "caption": "(A) Electrolyte leakage.",
    "molecules": "Electrolyte"
  },
  {
    "caption": "(B) Seven days-old seedlings Col-0 WT, atg5, atg7 and nbr1 grown on solid MS medium were transferred to the same medium (control) or the same medium containing 0.16 M NaCl and photographed 5 days later. The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter.",
    "molecules": "NaCl"
  },
  {
    "caption": "(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT), atg7 and nbr1 mutants collected at indicated hours (h) under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated. (B) Profiles of soluble and insoluble proteins. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and stained with Coomassie brilliant blue. Major proteins accumulated in the pellets from heat-stressed atg7 mutant plants were indicated by arrows.",
    "molecules": "proteins"
  },
  {
    "caption": "(E) COUP-TFII expression increased significantly in organoids treated with TGFβ1 (10ng/ml),  cobalt chloride (100 μM), IL-1β (10ng/ml), but not PDGF (50ng/ml). Quantification by confocal micrographs  in 400x hpf. *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, mean ± SD. The higher magnification of the insert from IL-1β φησwed co-localization of COUP-TFII (green) and αSMA (red) staining.",
    "molecules": "cobalt chloride"
  },
  {
    "caption": "(D) COUP-TFII+ cells decrease significantly in the UUO kidney in TAM-treated homozygous (F/F;Cre/+) mice (KO group, n=6) compared to wild-type littermates (WT group, n=4). Expression of αSMA (red) and collagen1 (yellow) are also markedly reduced. Masson Trichrome staining  shows less kidney fibrosis in KO compared to WT group. Quantification by confocal micrographs in 200x hpf. **p<0.01 by unpaired t test, mean ± SD.",
    "molecules": "TAM"
  },
  {
    "caption": "(D) WT, COUP-TFII overexpression (OE) and COUP-TFII knockout (KO) cells were seeded in Seahorse XF-24 cell culture microplates. The cells were rendered quiescent in 0.5% FBS DMEM overnight and then treated with or without 10 ng/ml TGFβ1 for 24 hours. All the cells were incubated in the glycolysis stress test medium without glucose and pyruvate, followed by sequential treatments with glucose (10mM), oligomycin (5μg/ml) and 2-DG (50mM). Real-time extracellular acidification rate (ECAR) was recorded as the baseline (before  glucose), the rate of glycolysis (after glucose), glycolytic capacity (after oligomycin), and glycolytic reserve (after 2-DG) (n = 10).",
    "molecules": "2-DG, glucose, oligomycin, pyruvate"
  },
  {
    "caption": "G, H. Representative images of tumor (G) or liver (H) sections from the Founder5 model stained with CD3 (red), Endomucin (white), and DAPI (blue). Right panels: quantification of CD3+ cell counts in Founder5 whole tumor (G) or liver (H) sections normalized to tissue areas. All samples were analyzed 24 h post treatment, n = 8/9 mice/treatment.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Mitochondria from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ), expressing Pex13-V5 (under its endogenous promotor) were either left untreated (lane 1,2,3) or exposed to Na2CO3 and separated into the membrane pellet (4,5,6) or supernatant fraction (7,8,9). After separation by SDS-PAGE the fractions were immunoblotted for Pex13-V5 (α-V5) and porin (α-porin).",
    "molecules": "Na2CO3"
  },
  {
    "caption": "(B) Digitonin-solubilized (1 or 4 g/g digitonin/protein ratio, as indicated) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by BN-PAGE with a 3-18% gradient and stained with Coomassie brilliant blue. BHM, bovine heart mitochondria; V2, complex V-dimer; SL,supercomplex III2IV2; Ss, supercomplex III2IV1; V1, complex V-monomer.",
    "molecules": "Coomassie brilliant blue, Digitonin, digitonin"
  },
  {
    "caption": "(A) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients-cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1-expressing PEX13-GFP and stained with Mitotracker Deep Red FM. Representative images are shown, scale bar, 10 μm.",
    "molecules": "Mitotracker Deep Red FM"
  },
  {
    "caption": "(A) Bioenergetic profile of human fibroblast cell lines. OCR (pmol/min/norm.unit) for cWT, cWT+ATAD1, PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1-/-, and PEX3-/- atad1-/- cells plotted against time (XF96e Extracellular Flux Analyzer, Mito-Stress-Test). 1 μM Oligomycin A, 0.15 μM FCCP, and 1 μM rotenone + 1 μM antimycin A (final concentrations) were sequentially delivered to the XF96e assay medium through injection ports in the sensor cartridge. The graph is the compilation of two independent assays, error bars show mean +/- s.d. (n=4, biological replicates).",
    "molecules": "antimycin A, FCCP, Oligomycin A, rotenone"
  },
  {
    "caption": "(E) Scatter plot representation of cardiolipin lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 11 detected cardiolipin species, log10 pareto-scaled is visualized. The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line. ­­(F) Scatter plot representation of a subpopulation of the phosphoethanolamine (PE) lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 20 phosphoethanolamine (PE) species (decreased in PEX3-/-), log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line. (G) Scatter plot representation of ether-phospholipid lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 21 detected ether-phospholipid (plasmalogen) species, log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line.",
    "molecules": "plasmalogen, cardiolipin, caridolipin, ether-phospholipid, PE, phosphoethanolamine"
  },
  {
    "caption": "B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.",
    "molecules": "Mito-C, DAPI, DMSO"
  },
  {
    "caption": "C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).",
    "molecules": "Mito-C, DMSO"
  },
  {
    "caption": "E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.",
    "molecules": "Mito-C, fluo"
  },
  {
    "caption": "A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20). B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates). C",
    "molecules": "Mito-C"
  },
  {
    "caption": "C, Western blot analysis of DRP1, GAPDH (cytosolic marker) and TOM20 (mitochondrial marker) protein in cytosolic and mitochondrial fractions treated or not, with 2µM Mito-C as indicated. D, Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions. Errors bars show the standard deviation (SD) of 5 independent experiments. E",
    "molecules": "Mito-C"
  },
  {
    "caption": "E, HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not, with Mito-C (T for transfected, NT for not transfected). F, Quantification of the mitochondrial phenotypes observed in E. Errors bars show the standard error of the mean (SEM). (30-35 images each with an average of 15-20 cells from triplicate independent experiments were analyzed). G",
    "molecules": "Mito-C"
  },
  {
    "caption": "G, Electron micrographs (EM) from HeLa cells treated with 2µM Mito-C or DMSO for 120 minutes. Empty arrowheads indicate ER-mitochondria contact sites. H, Quantification of ER-mitochondria membrane contact sites events (reported in 50µm² section; 50 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). I, Quantification of ER-mitochondria membrane contact sites density (in µm; 30 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). Da",
    "molecules": "Mito-C, DMSO"
  },
  {
    "caption": "B, Huh7 cells were treated with or without (DMSO) Mito-C, infected with dengue virus and fixed after 48h of infection. Cells were then immunostained for endogenous TOM20 (green channel), viral non-structural protein NS5 (red) and DAPI (Blue). Cropped areas illustrate the mitochondrial morphology in described conditions.",
    "molecules": "Mito-C, DAPI, DMSO"
  },
  {
    "caption": "(A) Measurement of maximum glycolytic capacity of MOTS-c in dystrophic muscle cells after oligomycin and 2-DG treatments (n=6; *p<0.05).",
    "molecules": "MOTS-c, oligomycin, 2-DG"
  },
  {
    "caption": " (B) Quantification of ECAR changes in relative to basal ECAR in dystrophic muscle cells treated with MOTS-c (n=6; *p<0.05). ",
    "molecules": "MOTS-c"
  },
  {
    "caption": " (C) ATP assay for dystrophic muscle cells in the presence of MOTS-c (n=6; *p<0.05). NC refers to untreated H2K or H2K mdx cells. ",
    "molecules": "MOTS-c"
  },
  {
    "caption": " (D) Tissue distribution and quantitative analysis of Rhodamine B-labeled MOTS-c in mdx mice after single intravenous injection (500 μg) (n=6; *p<0.05, *p<0.001). Tissues were harvested and examined 2 h after injection. This experiment was repeated twice. NC refers to untreated mdx mice. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle, D-diaphragm, H-heart, Li-liver, S-spleen, Lu-lung and K-kidney. ",
    "molecules": "MOTS-c, Rhodamine B"
  },
  {
    "caption": " (E) Measurement of ATP levels in peripheral muscles of mdx mice after single intravenous injection of MOTS-c (500 μg) (n=6; *p<0.05). ",
    "molecules": "MOTS-c, ATP"
  },
  {
    "caption": " (A) Measurement of PMO uptake in dystrophic muscle cells (H2K mdx cells) in the presence of MOTS-c (scale bar: 10 μm) (n=6; **p<0.001). PMO-M refers to PMO and MOTS-c (the same is for the rest unless otherwise specified). ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " (B) Tissue distribution of FITC-labeled PMO (50 mg/kg) mixed with MOTS-c (500 μg) in mdx mice 48 h after single intravenous injection. NC represents untreated mdx control. The abbreviation is the same as Figure 1D. Color scale represents the fluorescence intensity. (C) Quantification of fluorescence intensity in body-wide tissues of mdx mice treated with PMO-M or PMO alone (n=3; *p<0.05). The comparison was conducted between PMO-M and PMO. ",
    "molecules": "MOTS-c, PMO, FITC"
  },
  {
    "caption": " (D, E) Immunohistochemistry (D) and quantitative analysis (E) of dystrophin-positive fibers in mdx TA muscles treated with single intramuscular injection of 0.5 μg PMO mixed with MOTS-c (20 μg) (PMO-M) or PMO alone (scale bar: 100 μm) (n=3; **p<0.001). C57 means wild-type control C57BL/6. ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " (F) Representative Western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO-M or PMO alone (n=3; *p<0.05). 0.5 µg and 2.5 µg total protein from C57BL/6 and 50 µg from muscle samples of untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA muscles from C57BL/6 were used as normal controls (the same is for all Western blots unless otherwise specified). ",
    "molecules": "PMO"
  },
  {
    "caption": " (G) Systemic evaluation of MOTS-c (500 μg) and PMO at the dose of 50 mg/kg/week for 3 weeks in adult mdx mice intravenously. Tissues were examined two weeks after last injection. Immunohistochemistry for dystrophin- positive fibers in body-wide muscles of treated mdx mice (scale bar: 100 μm). C57 means wild-type control C57BL/6. ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (scale bar: 100 μm). TA refers to tibialis anterior. ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " (C, D) Western blot (C) and quantitative analysis (D) for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO-M (+MOTS-c) (n=3). 5 µg, 10 µg and 20 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " (E Immunohistochemistry of dystrophin-positive fibres in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) (PMO-M) intravenously (scale bar: 100 μm) (n=3; *p<0.05). PMO represents PMO in saline. ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " F) quantitative analysis of dystrophin-positive fibres in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) (PMO-M) intravenously (scale bar: 100 μm) (n=3; *p<0.05). PMO represents PMO in saline. ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " (G, H) Western blot (G) and quantitative analysis (H) of dystrophin expression in body-wide muscles from mdx mice treated with PMO at 12.5 mg/kg/week for 3 weeks mixed with MOTS-c (500 μg) intravenously (n=3; *p<0.05). 0.5 µg, 1 µg, 2.5 µg, 5 µg and 10 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. Fold change refers to PMO-M relative to PMO alone. ",
    "molecules": "MOTS-c, PMO"
  },
  {
    "caption": " Immunohistochemistry of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test). ",
    "molecules": "PMO"
  },
  {
    "caption": " quantitative analysis of dystrophin-positive fibres in body-wide muscles from mdx mice treated with repeated doses of PMO-M (n=4) or PMO alone (n=3) (scale bar: 100 μm) (*p<0.05, **p<0.001; two-tailed t test). ",
    "molecules": "PMO"
  },
  {
    "caption": " (D) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-M or PMO alone. 0.5 µg, 2.5 µg, 5 µg, and 15 µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. ",
    "molecules": "PMO"
  },
  {
    "caption": " (E) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05; two-tailed t test). ",
    "molecules": "PMO"
  },
  {
    "caption": " (F) Muscle function was assessed to determine the physical improvement with grip strength test for mdx mice treated with PMO-M (n=4) or PMO alone (n=3), untreated mdx controls (n=3) and C57BL/6 (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test). ",
    "molecules": "PMO"
  },
  {
    "caption": " (G) Measurement of muscle endurance with the running wheel test for wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). Muscle functional tests were conducted two weeks after last injection. ",
    "molecules": "PMO"
  },
  {
    "caption": " (A) Measurement of serum creatine kinase (CK) levels in wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). ",
    "molecules": "PMO"
  },
  {
    "caption": " (C) Measurement of body-weight changes of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3). ",
    "molecules": "PMO"
  },
  {
    "caption": " (D) Quantitative analysis of centrally nucleated fibres (CNFs) in triceps and quadriceps of wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman- Keuls test). ",
    "molecules": "PMO"
  },
  {
    "caption": " (E) Measurement of cross-sectional area (CSA) of muscle fibres from mdx mice treated with repeated doses of PMO-M (n=4) or PMO (n=3) (*p<0.05, two-tailed t test). ",
    "molecules": "PMO"
  },
  {
    "caption": " (A Measurement of serum indices including liver enzymes (AST, ALT and GGT) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). ",
    "molecules": "PMO"
  },
  {
    "caption": " B) Measurement of kidney markers (CREA and UA) from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO alone (n=3) to reflect liver and kidney functions (*p<0.05, One way-ANOVA post hoc Student-Newman-Keuls test). ",
    "molecules": "PMO, CREA"
  },
  {
    "caption": " (D Immunohistochemistry of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test). ",
    "molecules": "PMO"
  },
  {
    "caption": " E) quantification of macrophages, T cells and monocytes in quadriceps and diaphragm from wild-type C57BL/6 (C57) (n=3), untreated mdx controls (mdx) (n=3) and mdx mice treated with PMO-M (n=4) or PMO (n=3) (scale bar: 100 μm). The arrowheads point to CD68+ macrophages, CD3+ T cells or CD11b+ monocytes (*p<0.05, **p<0.001, One way-ANOVA post hoc Student-Newman-Keuls test). ",
    "molecules": "PMO"
  },
  {
    "caption": "Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP.Fluorescence t 1/2 values of centromeric or telomeric Swi6 obtained from FRAP experiments performed with cells expressing Swi6‐EGFP.",
    "molecules": "RNA"
  },
  {
    "caption": "Fluorescence t 1/2 values of centromeric Swi6 obtained from FRAP experiments performed after release of cells expressing NLS‐Swi6‐EGFP (black dots) or the RNA‐binding mutant NLS‐Swi6‐KR25A‐EGFP (red dots) from G1/S cell cycle arrest. Each dot represents one FRAP experiment at the respective time after release from cell cycle arrest. Swi6 is dispersed in M phase due to H3S10 phosphorylation, and M phase was therefore not included in the FRAP analysis.",
    "molecules": "RNA"
  },
  {
    "caption": "sRNA reads mapping to centromere 1 in swi6+ (green) and swi6∆ (blue) cells. The dashed red box highlights the loss of brdrRNAs at the IRC1R in the absence of Swi6. Centromeric repeat elements and the central core are indicated. Counts were normalized to the library size. Asterisks denote tRNA fragments. Note that tRNA genes flank IRC3 (Fig EV4) but not IRC1R.",
    "molecules": "tRNA"
  },
  {
    "caption": "(A-E) GFP-pho8Δ60-expressing wild-type (WT), atg5Δ, atg7Δ, and atg5Δpep4Δ cells were starved (A) or treated with AmphoB (B-E) and subjected to western blotting. Cells were treated with 2.5 µg/ml AmphoB for the indicated time (B) or with the indicated doses for 24 hr (C-E). Generation of free GFP was a marker of proteolysis.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(F) GFP-pho8Δ60- and Pep4-mRFP-expressing atg5Δ cells were cultured with (AmphoB) or without (NT) AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-pho8Δ60 and Pep4-mRFP was observed by confocal microscopy. Arrowheads indicate the cells in which GFP-pho8Δ60 was delivered to the vacuoles. Scale bars = 2 µm.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(G, H) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(I) AB-like structures accumulated in the vacuole. Scale bar = 0.5 µm. (J) AB-like structures containing lipid particles and mitochondria accumulated in the cross-section of the vacuole. An autophagosome (AP)-like double-membrane compartment containing a ribosome and Golgigranule is present in the cytosol. Scale bar = 0.5 µm. A magnified photo is available in Appendix Fig. S4B. (K) The cells containing AB-like structures were counted under electron microscopy [no treatment (NT): n = 20, AmphoB: n = 192]. * p < 0.01 vs the value of NT.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(A-D) Generation of Golgi stacks by AmphoB. Stacked Golgicisternae are observed in quick-frozen replicas (A-C) and substituted thin section (D) of atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml) for the indicated time. (A) Scale bar = 1 µm. (B) The magnified image shows the area indicated by the square in (A). Scale bar = 0.5 µm. (C, D) A representative Golgi stack with four cisternae is shown. Scale bar = 0.5 µm.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(H, I) The scene that AP-like structure was fused with vacuole. A magnified image of (H) is shown in (I). In atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr), outer membrane of AP-like structure (red arrows) was fused with vacuolar membrane (blue arrows). Inner membranes of AP-like structure (yellow arrowheads) were entered into the vacuoles to become AB-like structure. Green arrows indicate the membrane fusion site. Scale bars = 0.5 µm (H) and 0.2 µm (I).",
    "molecules": "AmphoB"
  },
  {
    "caption": "(K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP-Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (L, M) Representative electron microscopy images of grh1Δatg5Δpep4Δ (L) and gvp36Δatg5Δpep4Δ cells (M) treated with AmphoB (2.5 µg/ml, 24 hr). AP-like and AB-like structures were absent. (L) A unilamellar Golgi cisterna was observed in the cytoplasm (dotted square). Scale bar = 1 µm. (M) Formation of a Golgi stack, but not Golgi curvature, was observed (dotted square). Bar = 0.5 µm.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (N) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB-like structures was counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (O) GFP-pho8Δ60-expressing atg5Δ, grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. An unidentified band at 32kD (asterisk) is non-specific band because it was present in Pep4-lacking grh1Δatg5Δ cells.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(A) Localization of cellular PI(4)P. GFP-2xPHOsh2-expressing atg5Δ, sac1-23/atg5Δ, and pik1-83/atg5Δ cells were incubated with or without AmphoB (2.5 µg/ml) for 6 hr at 37 °C, and their localization was observed by confocal microscopy. Scale bars = 2 µm.",
    "molecules": "AmphoB, PI(4)P"
  },
  {
    "caption": "(B) The same experiments were performed using GFP-GOLPH3 (a GolgiPI(4)P-monitoring protein) instead of GFP-2xPHOsh2. Scale bars = 2 µm.",
    "molecules": "PI(4)P"
  },
  {
    "caption": "(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (C) Indicated cells expressing GFP-pho8Δ60 were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (D) The number of AP-like and AB-like structures was counted using EM images (Fig. EV2B, C). (E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase.",
    "molecules": "AmphoB"
  },
  {
    "caption": "(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (H) Sec7-HA-expressing pik1-83/atg5Δ cells were incubated at 37 °C (temperature shift) for 3 hr, and freeze replica immunolabelling was performed as described in Methods. Sec7-HA-positive signals (labelled with 15 nm gold particle) were observed on the surface membrane of AP-like structure and Golgi membrane. Scale bar = 0.1 µm.",
    "molecules": "gold"
  },
  {
    "caption": "Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr), and cells containing AB-like structures were counted by phase-contrast microscopy (K). * p < 0.01 vs the value of NT.",
    "molecules": "CBM"
  },
  {
    "caption": "Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr). Cells were also observed using EM (L). AB-like structures were observed. Scale bar = 0.5 µm.",
    "molecules": "CBM"
  },
  {
    "caption": "(M−O) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED. Indicated cells expressing Hsp150-HA(3×) were treated as follows: NT, no treatment; starve, starvation for 6 hr; AmphoB, 2.5 µg/ml for 24 hr (M); CBM, 1 mM for 24 hr (N); temperature shift, from 25 °C to 37 °C for 3 hr (O). The cells were then lysed and examined for Hsp150-HA(3×) expression by western blotting. Accumulation of O-glycosylated Hsp150-HA(3×) indicated the trafficking failure from the Golgi to the PM.",
    "molecules": "CBM, AmphoB"
  },
  {
    "caption": "(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (A, B) EM analysis revealed the formation of AP-like and AL-like structures. Atg5 KO MEFs were treated with CBM (2 mM) for 3 hr. A representative low-magnification image (A) and high-magnification images (B) are shown. Scale bar = 1 µm (A) and 0.2 µm (B). The arrowheads and arrows indicate AL-like and AP-like structures, respectively.",
    "molecules": "CBM"
  },
  {
    "caption": "(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (C) CLEM analysis of mCherry-syntaxin 6-expressing Atg5 KO MEFs. Cells were treated with CBM (2 mM) for 24 hr and observed using fluorescence and electron microscopy. mCherry-syntaxin 6puncta merged with the AL-like structures. Scale bar = 5 µm. \"N\" indicates nucleus. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.",
    "molecules": "CBM"
  },
  {
    "caption": "(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (D, E) Requirement of Grasp65 in the generation of AL-like structures. Atg5 KO and Grasp65-silenced Atg5 KO MEFs were treated without (NT) or with CBM (2 mM) for the indicated times followed by immunostaining with an anti-Lamp2 antibody. Representative images (at 24 h) are shown in (D). Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 1 µm. CBM induced large ring-like Lamp2 fluorescence. (E) Percentages of cells with large ring-like Lamp2 immunofluorescence (mean ± s.e.m., n = 4). *p < 0.01 vs the value of Atg5 KO MEF.",
    "molecules": "CBM"
  },
  {
    "caption": "(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (F) Colocalization of Lamp2 and GFP-syntaxin 6 in CBM-treated Atg5 KO MEFs. GFP-syntaxin 6-expressing Atg5 KO MEFs were treated with (CBM) or without (NT) CBM (2 mM) for 6 hr and immunostained with an anti-Lamp2 antibody. The ring-like Lamp2 fluorescence merged with the signal for syntaxin 6. Scale bar = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.",
    "molecules": "CBM"
  },
  {
    "caption": "(G) The monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) tandem protein assay revealed the induction of GOMED. Atg5 KO MEFs stably expressing tandem mRFP-GFP were incubated without (no treatment) or with CBM (2 mM) for 24 hr. Red signals indicate acidic compartments. Lysosomes were counterstained with an anti-Lamp2 antibody (cyan). Scale bars = 5 µm. Regions of interest (ROI) are indicated by dashed squares. Arrowheads indicate GOMED structure. Scale bars = 2 µm.",
    "molecules": "CBM"
  },
  {
    "caption": "(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (H, I) Degradation of GFP-fused proteins indicated the induction of GOMED. Atg5 KO MEFs stably expressing VSVG-GFP (H) or M6PR-GFP (I) were incubated with CBM (5 mM) for the indicated times. Cell lysates were subjected to immunoblotting with the anti-GFP antibody.",
    "molecules": "CBM"
  },
  {
    "caption": "(A) Suppression of insulin secretion in Atg5 knockout (KO) MIN6 cells after glucose deprivation. Atg5 KO MIN6 cells were incubated with normal culture medium (NT) or glucose-depleted medium for 1 hr. The amount of secreted insulin is expressed as the percentage of that of the untreated control (NT) (mean ± s.e.m, n = 3). *p < 0.01 vs the value of NT.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(B, C) EM analysis revealed the induction of GOMED structures. Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr. Representative images are shown. The white arrow and black arrows indicate the AP-like structure and insulinvesicles, respectively. Scale bars = 0.2 µm. (D) The number of AP-like and AL-like structures was calculated from the EM images of cells left untreated (NT) or subjected to glucose deprivation. Error bars indicate s.e.m. (NT: n = 17, glucose deprivation: n = 84). *p < 0.01 and #p < 0.05 vs the value of NT.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(E) Involvement of Golgi membrane in the generation of GOMED. Lamp1-GFP/mCherry-Syntaxin 6-expressing Atg5 KO MIN6 cells were subjected to glucose deprivation, and analysed by TEM and confocal microscopy. CLEM analysis revealed that AL-like structures (arrows) merged with Lamp1-GFP fluorescence and Syntaxin 6 fluorescence. Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.",
    "molecules": "glucose"
  },
  {
    "caption": "(F) GOMED-mediated degradation of (pro)insulin by glucose deprivation of Atg5 KO MIN6 cells. Atg5 KO MIN6 cells were incubated in glucose-depleted medium in the absence or presence of bafilomycin A1 (10 nM) for the indicated times. The cell lysates were then subjected to immunoblot analysis using an anti-(pro)insulin antibody.",
    "molecules": "bafilomycin A1, glucose"
  },
  {
    "caption": "(G) Immunofluorescence analysis of (pro)insulin and Lamp2. Atg5 KO MIN6 cells were left untreated (NT) or subjected to glucose deprivation for 1 hr in the presence of E64d (25 µM) or bafilomycin A1 (10 nM), and were stained with anti-(pro)insulin and anti-Lamp2 (lysosomal marker) antibodies and observed by fluorescence microscopy. Scale bar = 1 µm. Arrowheads indicate the colocalization of (pro)insulin with GOMED structures.",
    "molecules": "bafilomycin A1, E64d, glucose"
  },
  {
    "caption": "(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. A representative AP-like structure containing insulingranule is shown in (A). An AL-like structure containing insulingranules is shown in (B). Scale bars = 0.2 µm.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. (C, D) GOMED structures containing Golgi vesicles. An AP-like structure containing multiple Golgi vesicles (arrows) (C) and an AL-like structure fusing with a lysosome (arrowhead) (D) are shown. Scale bar = 0.5 µm. A magnified image of the dashed rectangle is shown in the inset. Scale bar = 0.2 µm. AP-like structure contained double-membrane.",
    "molecules": "glucose"
  },
  {
    "caption": "(E) The total number of AL-like structure was calculated from the EM images of WT and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.",
    "molecules": "glucose"
  },
  {
    "caption": "(F) The total number of GOMED structure was calculated from the EM images of Atg7 KO β-cells after glucose deprivation in the absence or presence of bafilomycin A1 (10 nM). Error bars indicate s.e.m. (n = 109−116). *p < 0.01 vs the value of 60 min.",
    "molecules": "bafilomycin A1, glucose"
  },
  {
    "caption": "(G−J) EM analysis showed the induction of mitophagy. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing mitophagy in Atg7 KO β-cells are shown in (G). Arrowhead (G) indicates mitophagy. Scale bars = 0.5 µm. (H, J) The number of structures representative of mitophagy (H) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.",
    "molecules": "glucose"
  },
  {
    "caption": "(G−J) EM analysis showed the induction of crinophagy/SINGD. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing crinophagy/SINGD in WT β-cells are shown in (I). White arrow (I) indicates crinophagy/SINGD. Arrows indicate insulingranules (I). Scale bars = 0.5 µm. (H, J) The number of structures representative of crinophagy/SINGD (J) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(K) The number of (pro)insulingranule-containing AL-like structure was calculated from the EM images of wild-type (WT) and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.",
    "molecules": "glucose"
  },
  {
    "caption": "Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. OGT protein levels were examined by immunoblotting.   Quantification of (G).   ",
    "molecules": "O-GlcNAc"
  },
  {
    "caption": "Yeast cells expressing the indicated constructs were induced in galactose-containing medium, and representative images were shown using fluorescence microscopy to visualize TDP-43 aggregates. DIC: Differential interference contrast. TDP-43 aggregate foci numbers from individual cell of (B) were quantified from over 50 cells each of the indicated yeast strains. The foci numbers in individual cell > 3 were labelled with yellow, and the foci numbers in individual cell < 3 are labelled with blue.",
    "molecules": "galactose"
  },
  {
    "caption": "SH-SY5Y cells treated with the indicated compounds were immunostained with TDP-43 or pTDP-43 and observed by immunofluorescence. Quantification of signal intensities of pTDP-43 staining (left) or foci numbers per cell (right). Cell number counted: left panel, "Mock" n = 58, "GlcNAc" n = 35, "EA" n = 45, "GlcNAc+EA" n = 52; right panel, "Mock" n = 1167, "GlcNAc" n = 1051, "EA" n = 542, "GlcNAc+EA" n = 815.",
    "molecules": "EA, GlcNAc"
  },
  {
    "caption": "SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT or catalytic-inactive mutant OGT treated with 20 μM EA, and the fractions were analyzed by immunoblotting. Quantification of (F).",
    "molecules": "EA, SDS"
  },
  {
    "caption": "   .Adult flies were fed with regular sucrose diet (RS) or high sucrose diet (HS), and survival percentage of adult flies of the indicated genotypes were examined. Kaplan-Meier survival analysis, n ≥ 40 for each group.   ",
    "molecules": "sucrose"
  },
  {
    "caption": "E Northern blot  analysis of globin  reporter and xrFrag. Ethidium bromide stained 28S and 18S rRNAs are shown as controls. Quantification results are shown as data points and mean (n=3).",
    "molecules": "Ethidium bromide"
  },
  {
    "caption": "Immunohistochemistry performed on 12 month-old THY-Tau22 mice showing AT100-positive neurons (green), CBP-positive nuclei (red) and DAPI-stained nuclei (blue) in the CA1 region of the dorsal hippocampus. A representative image is shown with a focus. Scale bars: 40µm and 20µm as noted. (n=5 mice). Arrows depict neurons bearing neurofibrillary tangles and do not display CBP immunostaining. The star depicts a ghost tangle (AT100-positive neuron and no nucleus)",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blot analyses perfomed in the dorsal hippocampus of 12 month-old THY-Tau22 mice compared to age-matched controls. NeuN, actin, tubulin and total H2B levels are not changed. Phosphorylated tau on serine 404 (Tau-Ph404) attest for samples from tauopathic mice. Quantification represents the ration of the protein level detected on the total amount of proteins on the membranes, with WT arbitrarily set at 1 (fold induction). Bar graphs are mean ± SEM. n=5-7/group as noted, multiple t tests, CBP * p=0.003 and GFAP **p=0.0001 for THY-Tau vs. WT mice",
    "molecules": "serine"
  },
  {
    "caption": "Long-term spatial memory testing: Mice were injected 3 times (1 per week) with Vehicle (WT mice saline, WT VEH, n=17), Vehicle (THY-Tau22 mice CSP, 500 μg /mouse, TAU VEH, n=10) or Molecule (THY-Tau22 mice CSP-TTK21, 500 μg/mice TAU MOL, n=13) before training of spatial memory in the Morris water maze (MWM); retention (Probe test) was tested 10 days after the last training session. Acquisition (Escape latencies, seconds) and retention performances (Time in target quadrant, seconds) are shown for the 3 groups of mice. All groups of mice displayed significant acquisition of the platform location (Day effect, F(4,148)= 26.45, p<0.001; Group and Group X Day effects, ns), but only TAU VEH exhibited impaired retention. CSP-TTK21 treatment fully restored the ability of THY-Tau22 mice to remember the platform location. Bar graphs are mean ± SEM. Student's t test to a constant value, # when compared to random (dotted line, 15sec) WT VEH, t(16)=4.6323, #p=0.0002; TAU VEH, t(9)=0.3606, p=0.7267; TAU MOL, t(12)=3.945, #p=0.0019. One-way ANOVA; F(2,37)= 3.55; p= 0.03; * in the different comparisons: TAU MOL vs. TAU VEH, *p=0.0166; WT VEH vs. TAU VEH, *p= 0.040; TAU MOL vs WT VEH, non significant p=0.437). TQ, Target Quadrant; O, Other, corresponds to the mean of the 3 other quadrants",
    "molecules": "TTK21, saline"
  },
  {
    "caption": "The timeline of GFP-lentivirus and CSP or CSP-TTK21 (one injection, 500 μg/mouse) injection is shown. (Left) The total number of spines was significantly decreased in TAU VEH compared to WT VEH mice. TAU MOL hippocampi showed a significant increase in the total number of spines compared to TAU VEH (One-way ANOVA p=0.0011, post hoc Holm Sidak's multiple comparisons test F(2,243) = 7.03; WT VEH vs. TAU VEH, p < 0.0009; TAU MOL vs. TAU VEH, p=0.0455). (Middle) Based on spine type, the number of head spines (Mushrooms, thins) was significantly lower in both TAU VEH and TAU MOL than in WT VEH controls (One-way ANOVA p=0.0018, post hoc Holm Sidak's multiple comparisons test F(2,243) = 6.467; WT VEH vs. TAU VEH, p=0.0013; WT VEH vs. TAU MOL, p=0.0272. Stubby spine density was significantly increased in TAU MOL compared to TAU VEH mice (One-way ANOVA F(2,243) = 4.311, p = 0.0145, post hoc Holm Sidak's multiple comparisons test: TAU MOL vs. TAU VEH, p=0.0109), as the number of filopodia (One-way ANOVA F(2,243) = 3.845, p = 0.0227, post hoc Holm Sidak's multiple comparisons test TAU MOL vs. TAU VEH, p=0.0179). (Right) Typical images are presented showing a dendrite fragment for each condition. White arrowhead depicts stubby spines. Scale bar, 2 μm. Number of dendritc segments: WT VEH, n=67; TAU VEH, n=93, TAU MOL, n=87; Number of neurons: WT VEH, n=20; TAU VEH, n=28, TAU MOL, n=16; Number of mice: WT VEH, n=2; TAU VEH, n=3, TAU MOL, n=3 ",
    "molecules": "TTK21"
  },
  {
    "caption": "CSP-TTK21 injection into THY-Tau22 mice rescues mature dendritic spines formation in response to learning. (Top left) The time line of injections is shown: Mice were injected 3 times (1 per week) with Vehicle (WT mice, WT VEH, NaCl 0.9%), Vehicle (THY-Tau22 mice (TAU VEH), CSP 500 μg /mouse) or Molecule (THY-Tau22 mice (TAU MOL), 500 μg/mice) and either trained over a 4-day acquisition period in the MWM ("learning" group) or left in their home cage ("basal" group). Mushroom-shaped spines were counted in dorsal CA1, four days post-training. (Bottom left) The number of mature spines was significantly increased by learning in WT VEH and TAU MOL mice. Bar graphs are mean ± SEM. (Two-way ANOVA; Learning effect, F(1, 139)=54.18; p< 0.0001; ### post hoc Holm Sidak's multiple comparisons test: Learning vs. Basal in WT VEH (p=0.0001) and in TAU MOL mice; (p=0.0001). After learning, WT VEH and TAU MOL mice displayed significantly higher number of mature spines than TAU VEH mice (Genotype X Treatment effect, F(2,139)= 9.704; p=0.0001; *** post hoc Holm Sidak's multiple comparisons test: TAU MOL vs. TAU VEH (p=0.0001), WT VEH vs. TAU VEH (p=0.0001). (Right) Typical examples of a dendritic fragment bearing mushroom spines (arrows) are shown for each sub-group in response to learning. Number of dendritc segments: Learning: WT VEH, n=27; TAU VEH, n=27, TAU MOL, n=30; WT VEH_HC, n=27; Number of mice: WT VEH, n=3; TAU VEH, n=3, TAU MOL, n=3. Basal: WT VEH, n=27; TAU VEH, n=7, TAU MOL, n=15; Number of mice: WT VEH, n=3; TAU VEH, n=3, TAU MOL, n=2 ",
    "molecules": "TTK21, NaCl"
  },
  {
    "caption": "Mice were injected 3 times (1 per week) with saline (WT VEH), CSP (Vehicle, VEH) or CSP-TTK21 (Molecule, MOL) (500 μg/mice; THY-Tau22 mice (TAU) before euthanasia. Long Term Depression measurements were performed on hippocampal slices. (Top left) Examples of analog traces recorded 10 min before (a) and 55 min after LTD induction (b; dotted line) in the three groups of mice. (Bottom left) Time course of LTD; LTD is expressed as a percent change in fEPSP (field excitatory postsynaptic potentials) slope over time. After the 20-min baseline recording, a low-frequency stimulation (LFS, 2Hz for 10 min) was applied (arrow). Recording was stopped during the 10-min conditioning stimulation and resumed after completion of LFS. LFS induced a strong depression of the fEPSP slope, which recovered partially to reach a stable level of depression about 20 min after stimulation. (Right) Average depression measured in the last 10 min of LTD. LTD was significantly different in TAU VEH (88.4 ± 4.1% of the baseline, n=10) compared to controls (WT VEH, 71.1 ± 4.4%, n=9) (F(1,17)=8.8, p=0.008). CSP-TTK21 treatment restored LTD to control levels (64.9 ± 5.2%, n=10) (WT VEH vs. TAU MOL: F(1,17)=0.83, p=0.37, ns; TAU VEH vs. TAU MOL: F(1,18)=13.2. p=0.0019). Bar graphs are mean ± SEM",
    "molecules": "TTK21, saline"
  },
  {
    "caption": "RNA-seq data showing expression of several immediate early genes (IEGs) : Nr4a1, Arc, Egr1, Dusp1, Fos and Junb. Most of the down-regulated IEGs in tauopathic compared to WT mice present a significant induction in THY-Tau22 mice after CSP-TTK21 injection. Adjusted p-values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH",
    "molecules": "TTK21"
  },
  {
    "caption": "RNA-seq data showing that CSP-TTK21 treatment of tauopathic mice restored the expression of plasticity/memory-relevant target genes : Klotho (Kl), and Neurotensin (Nts). Adjusted p values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH. F RT-qPCR validations performed in a different cohort of mice from the RNA-seq study (n=4-5/group). One-way ANOVA with uncorrected Fisher"s test. Klotho, Kl: F(3,16)=2.949, p=0.0643; * learning p=0.0145 for WT VEH vs. WT VEH_HC, $ pathology p=0.0395 for WT VEH vs. TAU VEH. Nts: F(3,14)=4.290, p=0.0241; $ pathology p=0.0036 for WT VEH vs. TAU VEH, # molecule p=0.0081 for TAU MOL vs. TA",
    "molecules": "TTK21"
  },
  {
    "caption": "Western blot for Klotho and Neurotensin expression in the different experimental conditions. Immunoblot results are shown (n=5/group). Bar graphs represent the quantification of the protein levels as percentage of the control group WT VEH_HC, arbitrarily set at 100%. Each detected protein was normalized to the corresponding amount of total proteins in the gels or the nitrocellulose membrane. Klotho and Neurotensin were further normalized to the level of actin. One-way ANOVA with uncorrected Fisher"s test. Klotho: F(3,16)=5.192, p=0.0107, * learning p=0.0117 for WT VEH vs. WT VEH_HC, $ pathology p=0.0027 for WT VEH vs. TAU VEH, # molecule p=0.0282 for TAU MOL vs. TAU VEH. Nts: F(3,16)=3.748, p=0.0326, $ pathology p=0.0200 for WT VEH vs. TAU VEH, # molecule p=0.0079 for TAU MOL vs. TA",
    "molecules": "nitrocellulose"
  },
  {
    "caption": "A Functional annotation charts (Benjamini, p<0.05) using using DAVID (GOTERM_Cellular Component) perfomed on the 82 significantly down-regulated genes by CSP-TTK21 treatment in tauopathic mice (TAU MOL vs. TAU VEH), showing a significant association with neuronal terms. Significance is indicated as -log10(Benjamini p_value). B Heatmap representing expression z-score of the 82 significantly down-regulated genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s dis",
    "molecules": "TTK21"
  },
  {
    "caption": "C RNA-seq data showing expression of several genes belonging to « synapse » annotation (DAVID): Grik4, Homer3 and Sv2b, which are significantly down-regulated by CSPTTK21 treatment in THY-Tau22 mice. Adjusted p values corresponding to p<0.05 are indicated by * for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH",
    "molecules": "TTK21"
  },
  {
    "caption": "A Mean enrichment profiles performed with SeqMiner presenting the genomic distribution of H2Bac and H3K27ac reads obtained in ChIP-seq experiments in the different experimental groups, established along putative neuronal CBP enhancers (± 4Kb) (Kim et al., 2010). In the dorsal hippocampus, H2Bac levels are enriched at CBP enhancers in WT WEH (blue), decreased in tauopathic mice (TAU VEH, pink) and induced by CSP-TTK21 treatment (TAU MOL, green). B Mean profiles were separated in two sets of loci, including 17817 peaks highly enriched in H2Bac and showing a differential regulation in the experimental groups and 23331 peaks that were poorly enriched in H2Bac and not regulated",
    "molecules": "TTK21"
  },
  {
    "caption": "UCSC genome browser view of the c-fos genomic locus with H3K27ac ChIP-seq (blue), H2Bac ChIP-seq (green) and Input (black) signals. Blue shading indicates locations of the c-fos enhancers (Joo et al., 2016); gray shading indicates location of the promoter. Differential peaks were analyzed with SICER. This set of data shows that H2Bac levels were significantly decreased at e1 (FDR=1.95.10-08), e2 (FDR=0.0005) and e5 (FDR= 2.44.10-05) enhancers in THY-Tau22 compared to WT mice, and significantly enriched at e1 (FDR= 4.01.10-08) the regions encompassing e3, e4 and TSS (FDR=7.53.10-07) and e5 (FDR= 0.001) after CSP-TTK21 treatment. Expanded View Figure Legend",
    "molecules": "TTK21"
  },
  {
    "caption": "Haematoxylin and eosin staining of sections representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors. Necrotic areas are highlighted with an asterisk (*). Quantification of TUNEL+ tumor area (representing the fraction of dying/dead cell population) of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors (n= 5 per condition). Quantification of the Ki67+ cells within the tumor area (representing the fraction of proliferative cell populations in non-necrotic areas) of CntlKD (n=7), BNIP3KD (n=6) and ATG5KD (n=8) B16-F10 tumors.",
    "molecules": "eosin, Haematoxylin"
  },
  {
    "caption": "Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm.",
    "molecules": "FerroOrange, Fe2+"
  },
  {
    "caption": "D. Relative FCR activities in the roots of wild-type plants and photoreceptor mutants grown under white light with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.",
    "molecules": "Fe"
  },
  {
    "caption": "D-F. Fe contents of whole plants, FER expression in roots and relative FCR activities in the roots of plants grown under Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d. Plants were exposed to high (H-, 2.0) or low (L-, 0.5) red (R) to far red (FR) ratios.",
    "molecules": "Fe"
  },
  {
    "caption": "D. Fe content of whole plants. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.",
    "molecules": "Fe"
  },
  {
    "caption": "E. FER expression in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.",
    "molecules": "Fe"
  },
  {
    "caption": "E. Direct binding of HY5 to the FER promoter analyzed using ChIP-qPCR in 35S-HY5-3HA-overexpressing (HY5-OE) tomato plants. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d. Input chromatin was isolated from root samples at 7 d. The epitope-tagged HY5-chromatin complex was immunoprecipitated with an anti-HA antibody. A control reaction was processed side-by-side using mouse IgG. Input- and ChIP-DNA samples were quantified by RT-qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in (D). The ChIP results are presented as percentage of the input DNA.",
    "molecules": "Fe, 35S"
  },
  {
    "caption": "A-C. Fe content of whole plants (A) and the photochemical efficiencies of PSI and PSII [Y(I) and Y(II)] (B and C) in young leaves of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.",
    "molecules": "Fe"
  },
  {
    "caption": "B. The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room.",
    "molecules": "EDTA, Fe"
  },
  {
    "caption": "D. Fe content in the fruits of WT and phyB plants. Plants were grown in soil in a greenhouse.",
    "molecules": "Fe"
  },
  {
    "caption": "(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 11-15 pigmented or grey HFs from 4 different donors. (B) Representative images of p-S6 immunofluorescence. Only anagen VI HFs were investigated and analyzed immediately after surgery. Analyses were performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Quantitative analysis of TSC2 protein levels. N = 11-14 pigmented or grey HFs from 4 different donors. (D) Representative images of TSC2 immunofluorescence. Only anagen VI HFs were investigated and analyzed immediately after surgery. Analyses were performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 23-30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (B) Representative images of p-S6 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(C) Hair cycle staging was performed using Ki-67 and Masson-Fontana histochemistry Mean ± SEM; N = 36-39 HFs per group from 6 different donors treated with Rapamycin 20 ng/ml or vehicle (control) for 7 days. (D) Representative fluorescence images of Ki-67 and bright field microscopic images of Masson-Fontana. for C-D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.",
    "molecules": "Masson-Fontana, DAPI, Rapamycin"
  },
  {
    "caption": "(E) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 28-30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (F) Representative bright field microscopic images of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test",
    "molecules": "Masson-Fontana, melanin, Rapamycin"
  },
  {
    "caption": "(G) Quantitative analysis of tyrosinase activity. N = 18-21 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (H) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(I) Quantitative analysis of gp100 expression. N = 24-26 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (J) Representative images of gp100 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(K) Quantitative analysis of melanocyte dendricity. N = 22-24 anagen VI HFs from 6 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (L) Representative images of gp100 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(M) Quantitative analysis of α-MSH expression. N = 17-20 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (N) Representative images of α-MSH immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(O) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 8-10 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng/ml, 2ug/ml Agouti, Rapamycin+Agouti or untreated (vehicle) for 7 days. (P) Representative images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and One-Way ANOVA Nuclei stained with DAPI.",
    "molecules": "Agouti, DAPI, Rapamycin"
  },
  {
    "caption": "(Q) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 8-11 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng/ml, 2 ug/ml Agouti, Rapamycin+Agouti or untreated (vehicle) for 7 days. (R) Representative bright field microscopic images of Masson-Fontana histochemistry. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and One-Way ANOVA",
    "molecules": "Masson-Fontana, Agouti, melanin, Rapamycin"
  },
  {
    "caption": "Quantitative analysis of TSC2 protein expression (B). N = 8-10 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos (NTO) for 6 days. (C) Representative images of TSC2 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 2-3 anagen VI HFs from 1 donor treated with siTSC2 or non-targeting oligos for 6 days. (E) Representative images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Quantitative histomorphometry of melanin production. N = 9-10 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (G) Representative bright field microscopic images of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color.",
    "molecules": "Masson-Fontana, melanin"
  },
  {
    "caption": "(H) Quantitative analysis of tyrosinase activity. N = 8-9 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (I) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(J) Quantitative analysis of α-MSH expression. N = 8-9 anagen VI HFs from 4 different donors treated with siTSC2 or non-targeting oligos for 6 days. (K) Representative images of α-MSH immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Samples from each donor are represented by a different color. Nuclei stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 9-10 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (B) Representative fluorescence images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(C) Hair cycle staging was performed using Ki-67 and Masson-Fontana histochemistry. Mean ± SEM; N = 16-17 grey HFs per groups from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (D) Representative fluorescence images of Ki-67 and bright field microscopic images of Masson-Fontana. for C-D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.",
    "molecules": "Masson-Fontana, DAPI, Rapamycin"
  },
  {
    "caption": "(E) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 7-9 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (F) Representative pictures of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test",
    "molecules": "Masson-Fontana, melanin, Rapamycin"
  },
  {
    "caption": "(G) Quantitative analysis of tyrosinase activity. N = 7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (H) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(I) Quantitative analysis of α-MSH expression in defined reference area within the bulb. N = 5-7 grey anagen VI HFs from 3 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (J) Representative images of α-MSH immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mann-Whitney U-test Nuclei stained with DAPI.",
    "molecules": "DAPI, Rapamycin"
  },
  {
    "caption": "(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.",
    "molecules": "CPT"
  },
  {
    "caption": "(C) Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values ± SD are plotted. Four biological replicates have been performed.",
    "molecules": "CPT"
  },
  {
    "caption": "(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.",
    "molecules": "CPT"
  },
  {
    "caption": "(A) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad52∆, rad51∆, mus81∆ and pol32∆ cells and further released into S phase. Cells were synchronized in G1 with α -factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Three biological replicates have been performed.",
    "molecules": "α -factor, CPT, DNA, DMSO"
  },
  {
    "caption": "(B) (C) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad3-102, rad3-102 rad51∆, rad3-102 mus81 and rad3-102 mus81-dd cells and further released into S phase. Cells were synchronized in G1 with α-factor, untreated or irradiated with 20 J/m2 UV-C, let in G1 for 2h, and released into S phase. Three biological replicates have been performed.",
    "molecules": "α-factor, DNA"
  },
  {
    "caption": "(A) Analysis of replicated DNA tracks length by single-molecule DNA combing in WT, rad52∆, rad51∆, mus81∆ and pol32∆ cells exposed to CPT. Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse-labeled with 50 µM EdU for 20min. DNA fibers were combed on silanized coverslips and EdU-labeled DNA was detected by Click chemistry. Graph depicts the distribution of EdU tracks length in kb. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively. Median EdU tracks length is indicated in kb (values from two biological replicates were pooled). Asterisks indicate the P-value of the Mann-Whitney unpaired t test, **: p=0.0015. The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain. Representative images of DNA fibers are shown. Red and white: EdU, green: DNA.",
    "molecules": "EdU, CPT, DNA, DMSO"
  },
  {
    "caption": "(C) Analysis of replication intermediates by 2D gel electrophoresis. Replication intermediates were monitored at early origin ARS305 and region C in WT, rad3-102, mus81∆ and rad3-102 mus81∆ cells. Cells were synchronized in G1 with α-factor and collected at the indicated time points after release into S phase. A scheme of the studied chromosomal region is shown (drawn to scale). Relevant probes are indicated by gray bars, and coordinates of ARS and restriction sites are indicated in bp. Two biological replicates have been performed.",
    "molecules": "α-factor"
  },
  {
    "caption": "(B) (C) Kinetic analysis of Rad52 foci formation. Wild-type and mus81∆ cells containing Rad52-YFP and mCHERRY-Pus1 were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean ± SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed.",
    "molecules": "α-factor, CPT, DMSO"
  },
  {
    "caption": "(D) Mms4-Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT. Wild-type cells were synchronized in G1 with α-factor, treated with DMSO or 50 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points and Mms4 was immunodetected with anti-Flag antibodies. Clb2 immunodetection serves as a marker for G2 phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed.",
    "molecules": "α-factor, CPT, DMSO"
  },
  {
    "caption": "(E) CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles compared to the wild type, mus81, mms4∆ and cdc5-2 mutants. Three biological replicates have been performed.",
    "molecules": "CPT"
  },
  {
    "caption": "(A) (B) Pulsed-field gel electrophoresis (PFGE) analysis of wild type, mus81∆, cdc5-2 and G2-MUS81 cells in response to CPT. Cells were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points. DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE. Upper panel: agarose gel stained with ethidium bromide; lower panel: Southern blot using a chromosome IV specific probe. JMs, joint molecules accumulated in the gel wells. The mean value of JMs relative to the total amount of DNA is indicated for each time point. Two to three biological replicates have been performed.",
    "molecules": "agarose, α-factor, CPT, DNA, DMSO, ethidium bromide"
  },
  {
    "caption": "(C) (D) PFGE analysis of rad52∆ and rad52∆ mus81∆ cells in response to CPT performed as in (A) (B). The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the +CPT 140 min time points in rad52∆ and rad52∆ mus81∆ cells are shown. Two biological replicates have been performed.",
    "molecules": "CPT, Ethidium bromide"
  },
  {
    "caption": "Six-month-old hTau-A152T(L1) mice and NTG controls were or were not treated with DOX for 2 months and analyzed at 8 months of age by immunohistochemistry. (A-I) Untreated hTau-A152T(L1) mice had increased neuronal labeling with the hTau antibody HT7 (A), the phosphorylation-dependent tau antibody PHF1 (D), and the conformation-dependent tau antibody MC1 (G). HT7 (A) and PHF1 (D) staining patterns were similar. MC1 (G) specifically stained hippocampal mossy fibers. In hTau-A152T(L1) mice, DOX reduced hippocampal staining with HT7, PHF1, and MC1 (B, E, H) to the levels in DOX-treated NTG controls (C, F, I).",
    "molecules": "DOX"
  },
  {
    "caption": "(J-M) DOX also normalized CA1 levels of GFAP immunoreactivity (IR) in hTau-A152T(L1) mice, as shown by representative photomicrographs (J-L) and quantitation of GFAP immunoreactivity (M). Scale bars: 300 µm (A-I), 25 µm (J-L). n = 3-4 mice per genotype. *P < 0.05 by two-tailed Welch's t test without Holm adjustment. Values are means ± SEM. a.u., arbitrary units.",
    "molecules": "DOX"
  },
  {
    "caption": "(A-C) Subdural EEG recordings from freely behaving mice at 4-9 months of age before (A top trace, B) and after (A bottom trace, C) injection of PTZ at a dose (30 mg/kg) that did not produce convulsions. (A) Representative EEG traces from an hTau-A152T(L1) mouse. Arrows indicate epileptic discharges. (B, C) Quantitation of spikes per hour at baseline (B) and of spikes per 20-min intervals after PTZ injection (C). n = 31 (B) or 23 (C) NTG; 13 (B) or 11 (C) CaMKII-tTA; 12 (B) or 10 (C) hTau-WT(L32); and 22 (B) or 21 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch's one-way ANOVA and two-tailed Welch's t test with Holm adjustment (B) or by two-way repeated-measures ANOVA and Tukey test (C). Values are means ± SEM.",
    "molecules": "PTZ"
  },
  {
    "caption": "(A-D) Numbers of offspring from crosses of hAPP-J20 mice with hTau-A152T(L1) (A, B) or hTau-WT(L32) (C, D) mice that were (A, C) or were not (B, D) treated with DOX. (E) Numbers of offspring from crosses of lower expresser hAPP-J9 mice with hTau-A152T(L1) mice that were not treated with DOX. n = 141 (A), 112 (B), 94 (C), 83 (D), and 282 (E) mice per cohort. Yields from two crosses indicated significant deviations from Mendelian inheritance: hAPP-J20 x TRE-hTau-A152T(L1) x CaMKII-tTA without DOX (Holm-adjusted P = 0.003, unadjusted P = 0.0008) and hAPP-J9 x TRE-hTau-WT(L32) x CaMKII-tTA without DOX (adjusted and unadjusted P < 0.0001) by chi-square goodness-of-fit test.",
    "molecules": "DOX"
  },
  {
    "caption": "B. Images of rosettes and seedlings (15 DAG) of Col-0, arf7/arf19, slr, and gLBD16-SRDX as well as the inducible lateral-root-less lines pGATA23::shy2-2-GR and pGATA23::slr1-GR grown on DMSO control medium or 30 µM Dexamethasone (DEX), Scale bar 1 cm. The rosettes were stained with a Lugol's Iodine solution for starch accumulation at the end of the dark period (representative images of n=3 biological replicates). C. Box plots of starch quantification in rosette tissues at 15 DAG (n=4 biological replicates). Comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.",
    "molecules": "DEX, Dexamethasone, DMSO, Lugol's Iodine, starch"
  },
  {
    "caption": "E. Representative confocal sections of calcofluor counterstained (E, n=5 biological replicates) and differential interference contrast (DIC) images of GUS-stained root bends (F, n=16 biological replicates) in DR5:GUS seedlings treated as indicated 48h after gravistimulation. In E, orange arrows indicate the pericycle (marked in yellow). F. Fraction of root bends forming an LRP and showing DR5 GUS staining after treatment with either control or 2D containing agar blocks (n=16 biological replicates, ± SE).",
    "molecules": "2D, agar, calcofluor"
  },
  {
    "caption": "A. Representative western blot of root tissues of pUB10:S6K1-3xHA treated by the indicated combination of auxin (IAA, 10µM), sucrose (Suc, 110mM), 2-deoxy-d-glucose (2D, 20mM) and AZD8055 (AZD, 10µM) and probed with anti-S6K1/2, anti-HA or anti-S6K1-T449P. Blot is one of three biological replicates. B. Quantification of the relative S6K activation. Box plots show three biological replicates, and comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.",
    "molecules": "2-deoxy-d-glucose, 2D, AZD, AZD8055, IAA, Suc, sucrose"
  },
  {
    "caption": "G, H. Relative expression levels (normalized to ACTIN) of ARF19 (G) and LBD16 (H) measured by RT-qPCR upon TOR activity inhibition with AZD8055. Comparison between samples was performed by one-way ANOVA. Different letters indicate significant differences based on a post-hoc Tukey HSD Test (n=5 biological replicates, α = 0.05).",
    "molecules": "AZD8055"
  },
  {
    "caption": "L, M. Total lysates prepared from lateral roots treated or not with IAA and AZD were fractionated through sucrose gradients, and the relative redistribution (percentage of total) of ACTIN, ARF7, ARF19, and LARP1 mRNAs in every 8 fractions were studied by RT-qPCR analysis. (L) Polysome profiles. 40S, small ribosomal subunit; 60S, large ribosomal subunit; 80S, mono-ribosome; polysomes, polyribosomes. AU is arbitrary units of RNA absorbance at A260 nanometers. (M) RT-qPCR analysis of mRNA redistribution through a sucrose gradient (8 fractions collected). Translation efficiency was computed as a percentage of mRNA in non-polysome fractions (40/60/80S; fractions 1-3) against both lights (fractions 4-5) and heavy polysomes (fractions 6-8). The plot is representative of three independently performed experiments with similar results.",
    "molecules": "AZD, IAA, sucrose"
  },
  {
    "caption": "B. Distribution of GUS-staining in UB10pro>>amiR-TOR/WOX11pro::GUS seedlings 6 days after transfer to 10µM Est. The proportion of root bends with the depicted phenotype is indicated.",
    "molecules": "Est"
  },
  {
    "caption": "C. The arf7/arf19 mutant shows defective LR initiation. D. Formation of roots (arrows) close to the wound site of arf7/arf19 primary roots 6 days after excision (DAE). E. Blocking TOR-activity via exposure to 10µM AZD8055 (AZD) resulted in the loss of rooting capability on the wound site of arf7/arf19 primary roots at 6 DAE. S",
    "molecules": "AZD, AZD8055"
  },
  {
    "caption": "F, G. Close-ups indicate root formation location or absence of root formation on ctrl media and AZD-containing media, respectively, in arf7/arf19 primary roots at 6 DAE. The proportion of root wound sites with the depicted phenotype is indicated.",
    "molecules": "AZD"
  },
  {
    "caption": "Detection of PUAR in the nuclear and non-nuclear fractions by RT-qPCR. U6 and tRNA were used as RNA markers for the nuclear and non-nuclear fractions, respectively. The seedlings were grown under white light for 6 d, and then maintained under white light, or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 4, n refers to technical replicates).",
    "molecules": "tRNA"
  },
  {
    "caption": "Remaining levels of PHYA and GAPDH at different time points post 1 mM cordycepin in Col-0 and PUAR-S1ox lines. 6 d-old seedlings were shade-treated for 4 h prior to cordycepin treatment. SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Data are presented as means ± SD (n = 3, n refers to biological replicates).",
    "molecules": "cordycepin"
  },
  {
    "caption": "PIF7 specifically binds to PUAR in vitro. Purified TF-His-PIF7 from E. coli was incubated with the biotinylated PUAR at 4 °C for 1 h, and then non-biotinylated PUAR was added according to the dose as shown in the figure at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate RNA complex at 4 °C for 2 h.",
    "molecules": "agarose, biotinylated, streptavidin"
  },
  {
    "caption": "Interaction between PUAR and PIF7 was determined using an in vivo pull-down assay. RNA-protein extracts were from plants that overexpressed PIF7-Flash*Col-0 and PIF7-Flash*PUAR-S1ox, respectively. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Streptavidin-coupled agarose beads were added in the reaction mix to isolate RNA complex for 2 h.",
    "molecules": "agarose, Streptavidin"
  },
  {
    "caption": "PIF7 specifically binds to 5′ UTR of the PHYA in vitro. Purified His-PIF7 from E. coli was incubated with the biotinylated 5′ UTR of the PHYA fragment at 4 °C for 1 h, and then non-biotinylated 5′ UTR of the PHYA fragment was added according to the dose, as shown in the figure, at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate DNA complex for at 4 °C for 1 h.",
    "molecules": "agarose, biotinylated, streptavidin"
  },
  {
    "caption": "PUAR specifically blocks the binding of PIF7 to the 5′ UTR of PHYA in vitro. Purified TF-His-PIF7 from E. coli was incubated with the biotinylated 5′ UTR of the PHYA fragment or fragment of the PIL1 promoter at 4 °C for 1 h, and then PUAR or anti-PUAR was added according to the dose as shown in the figure, at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate DNA complex for at 4 °C for 1 h.",
    "molecules": "agarose, biotinylated, streptavidin"
  },
  {
    "caption": "Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin (A, C) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict lysine acetylation site ratios (A, B)for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.",
    "molecules": "apicidin"
  },
  {
    "caption": "Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A (B, D) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict lysine acetylation site ratios (A, B)for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.",
    "molecules": "trichostatin A"
  },
  {
    "caption": "Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors apicidin (A, C)versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict protein ratios (C, D) for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points. cancel ",
    "molecules": "apicidin"
  },
  {
    "caption": "Figure 3. Differential lysine acetylation and protein expression in Arabidopsis leaves after inhibitor treatment. Vacuum-infiltration of leaf strips with solutions containing either of the two deacetylase inhibitors trichostatin A (B, D) versus a buffer control for 4 hours leads to differential accumulation of lysine acetylation sites. Volcano plots depict protein ratios (C, D) for inhibitor treatment vs. control, with p-values determined using the LIMMA package. Orange, protein with nuclear localization according to SUBA4 database. Blue, proteins with lysine acetylation sites identified. Dashed lines indicate significance thresholds of either uncorrected p-values < 5 % or Benjamini-Hochberg corrected FDR < 5 %. A missing line indicates that the significance threshold was not reached by any of the data points.",
    "molecules": "trichostatin A"
  },
  {
    "caption": "Figure 4. HDA14 protein localizes to the chloroplasts and mitochondria in Arabidopsis, its activity is dependent on cofactors and can be inhibited by deacetylase inhibitors. (A) GFP localization (green) of the HDA14-GFP fusion construct in Arabidopsis protoplasts (35S:HDA14:GFP) from stable transformants. The mitochondrial marker TMRM is depicted in purple. GFP+TMRM shows the overlay image of 35S:HDA14:GFP and TMRM, AF indicates the chlorophyll autofluorescence and BF the",
    "molecules": "TMRM"
  },
  {
    "caption": "(B) Deacetylase activity of the recombinant 6xHis-HDA14 protein using a colorimetric assay. Co2+ and Zn2+ were used as cofactors, apicidin (100 µM) and trichostatin A (5 µM) as deacetylase-inhibitors (n = 5, ±SD).",
    "molecules": "Zn2+, apicidin, Co2+, trichostatin A"
  },
  {
    "caption": "Figure 6. RuBisCO activity and RuBisCO activation state is increased in the hda14 mutant under low light conditions. (A) RuBisCO initial and total activity in WT and hda14 in low light treated plants. Initial activity was measured directly upon extraction. For the total activity samples were incubated with H2CO3 for 3 minutes to fully carbamylate the active site of RuBisCO (n = 10, *t-test p-value < 0.05).",
    "molecules": "H2CO3"
  },
  {
    "caption": "(C) ATPase activity of recombinant 6x-HisRCAß1 WT, K438Q and K438R with ATP and ADP/ATP= 0.11, respectively (n = 3, *t-test p-value < 0.05, +t-test p-value< 0.1).",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21% O2 vs. 85% O2, at post-natal day (P)5 and P14.",
    "molecules": "O2"
  },
  {
    "caption": "B Quantitative RT-PCR detection of microRNA-34a/b/c-5p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization. C Quantitative RT-PCR detection of microRNA-34a/b/c-3p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization.",
    "molecules": "O2"
  },
  {
    "caption": "C Quantitative RT-PCR detection of miR-34a/b/c-5p levels in MLg cells in vitro, maintained under 21% O2 or 85% O2 (n = 3 separate cell cultures for each group).",
    "molecules": "O2"
  },
  {
    "caption": "D Immunoblot detection of PDGFRα levels in the lungs of mouse pups (n = 6 animals for each group) at post-natal day (P)5, during normal (21% O2) and aberrant (85% O2) alveolarization.",
    "molecules": "O2"
  },
  {
    "caption": "E Immunoblot detection of PDGFRα levels in MLg cells in vitro, maintained under 21% O2 or 85% O2, where cells had been transfected wither with a scrambled (SCR) antimiR, or an antimiR directed against miR-34a (A34a) (n = 3 separate cell cultures for each group).",
    "molecules": "O2"
  },
  {
    "caption": "F Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of mouse pups (n = 4 animals for each group; data from an independent repetition are provided in Appendix Fig S5) at P5, maintained under 21% O2 or 85% O2 from birth.",
    "molecules": "O2"
  },
  {
    "caption": "I Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14 during aberrant (85% O2) alveolarization, compared with 34ai∆PC/i∆PC during normal (21% O2) alveolarization (scale bar, 50 µm). Data are representative of observations made in four other experiments. J Quantification of total number of alveoli by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group). K Quantification of mean septal thickness by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group). ",
    "molecules": "O2"
  },
  {
    "caption": "B Quantitative RT-PCR detection of miR-34a-5p levels in wild-type mouse pups at post-natal day (P)14 that had been treated either with a scrambled antimiR (S), or antimiR-34a (A34a), during normal (21% O2) and aberrant (85% O2) alveolarization (n = 4 animals for each group).",
    "molecules": "O2"
  },
  {
    "caption": "A Mice expressing nuclear‑localized GFP under the control of the Pdgfra promoter were maintained under normoxic (21% O2) or hyperoxic (85% O2) conditions, and lungs were harvested, processed and immunostained for Ki67 to determine proliferation status. DAPI staining revealed nuclei of all cells present in the section. Low-magnification images from individual channels are presented to the right of the merged (large) image first row of images. The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image, as well as visualization of a single Ki67+,GFP+ cell (white arrowhead) in all three channels separately.",
    "molecules": "O2, DAPI"
  },
  {
    "caption": "C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild-type mice that were treated with an isotype-matched control IgG used for the Ki67 staining experiments. Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining. Scale bar: 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.",
    "molecules": "4′,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "The impact of administration of scrambled antimiR (S) or an antimiR directed against miR-34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro-surfactant protein C, Sftpc) was assessed in 3-µm sections of paraffin‑embedded lung tissue from P5 mice undergoing normal (21% O2) or aberrant (85% O2) lung alveolarization. DAPI, 4′,6-diamidino-2-phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O2 groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 µm.",
    "molecules": "O2, 4′,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "H Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male C57Bl/6J mice and fluorescence double labeling to visualize bone formation (insets) after 4-weeks of treatment with anti-miR-19a/b (n=12), scr control oligonucleotides (n=11) or veh (n=8). Scale bars indicate 1 mm (black) and 50 μm (white). I Bone histomorphometric analysis of the proximal tibiae of the same animals as in (H) Abbreviations: BV/TV, bone volume/tissue volume; MS/BS, mineralizing surface/bone surface; BFR/BS, bone formation rate/bone surface; MAR, mineral apposition rate; OS/BS, osteoid surface/bone surface; Ob.S/BS, osteoblast surface/bone surface; N.Ob/BS, number of osteoblasts/bone surface; ES/BS, eroded surface/bone surface; Oc.S/BS, osteoclast surface/bone surface; N.Oc/BS, number of osteoclasts/bone surface.",
    "molecules": "von Kossa, oligonucleotides"
  },
  {
    "caption": "J Images of von Kossa-stained histological sections of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b, scr or veh. Scale bar indicates 1 mm. K Quantification of bone mass (BV/TV) of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b (n=12), scr (n=12) or veh (n=8).",
    "molecules": "von Kossa"
  },
  {
    "caption": "A Images of von Kossa-stained histological sections of the bone and bone marrow (upper panel) and of hematoxylin-eosin-stained liver sections (lower panel) of 12-week-old mice after treatment with anti-miR-19a/b (n=12), scrambled (scr) control oligonucleotides (n=11) or vehicle (veh) (n=8). Scale bars indicate 100 μm (upper panel) and 50 μm (lower panel). B, C Quantification of the femur length (B) and width (C) after treatment with anti-miR-19a/b (n=8), scr (n=7) or veh (n=8).",
    "molecules": "von Kossa, eosin, hematoxylin, oligonucleotides"
  },
  {
    "caption": "I, J CTG assay-based quantification of luminescence signal intensity as surrogate parameter of metabolically active osteoblasts of the MC3T3-E1 cell line (I) and of hepatocytes of the HepG2 cell line (J) treated with anti-miR-19a/b, scr or veh and after stimulation with staurosporine to induce cell death as positive control. n= 6.",
    "molecules": "staurosporine"
  },
  {
    "caption": "C Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male mice of the genotypes Dmp1-Cre-;Tgif1fl/fl treated weekly with scr (n=8) or anti-miR-19a/b (n=10) and Dmp1-CreTg;Tgif1fl/fl treated weekly with scr (n=7) or anti-miR-19a/b (n=14) for 4 weeks. Scale bar indicates 1 mm. D Bone histomorphometric analysis of the proximal tibiae of the same animals as in (C). For abbreviations see legend to figure 1.",
    "molecules": "von Kossa"
  },
  {
    "caption": "E Images of von Kossa-stained histological sections of the fourth lumbar vertebral bodies of the same animals as in (C) and (F) quantification of the BV/TV. Scale bar represents 1 mm.",
    "molecules": "von Kossa"
  },
  {
    "caption": "B Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male wild type mice after treatment with scr oligonucleotides and vehicle (veh) (n=8), anti-miR-19a/b and veh (n=10), scr and intermittent PTH (n=12) or a co-treatment with anti-miR-19a/b and PTH (n=10) for 4 weeks. Scale bar indicates 1 mm. C Histomorphometric analysis of the proximal tibiae of the same animals as in (B) after treatment with intermittent PTH or veh and/or weekly injections of anti-miR-19a/b or scr control for 4 weeks. For abbreviations see legend to figure 1.",
    "molecules": "von Kossa, oligonucleotides"
  },
  {
    "caption": "D Images of von Kossa-stained histological sections of the fourth lumbar vertebral bodies of the same animals as in (B). Scale bars indicate 1 mm. E Histomorphometric analysis of the fourth lumbar vertebral body. For abbreviations see legend to figure 1, scr, veh n=8; anti-miR-19a/b, veh n= 10; scr, PTH n=12; anti-miR-19a/b, veh n=10. F Analysis of serum carboxy-terminal collagen crosslinks (CTX) in the same animals as in (B).",
    "molecules": "von Kossa"
  },
  {
    "caption": "C Representative images of TRAP-stained osteoclast cultures of bone marrow cells transfected with scrambled (scr) control oligonucleotide or anti-miR-19a/b (representative images of 3 independent experiments). Scale bar indicates 200 μm. D Quantification of multinucleated TRAP-positive multi-nucleated cells (MNCs) after 4 days of osteoclast differentiation of bone marrow cells transfected with scr (n=4) or anti-miR-19a/b (n=8).",
    "molecules": "oligonucleotide"
  },
  {
    "caption": "D, E Relative expression of miR-19a (D) and miR-19b (E) in Ocy454 cells upon estrogen deficiency (ED) and control (C) treatment (n=6).",
    "molecules": "estrogen"
  },
  {
    "caption": "H Expression of Rankl mRNA in Ocy454 cells transfected with scrambled (scr) control oligonucleotide or anti-miR-19a/b in the context of ED (n=9).",
    "molecules": "oligonucleotide"
  },
  {
    "caption": "I Representative images of von Kossa-stained human bone samples obtained from non-fractured femoral heads of postmenopausal women after assignment to groups of relatively higher bone mass (HBM, n=4) and relatively lower bone mass (LBM, n=4). J Histomorphometric analysis of bone mass (BV/TV, bone volume/tissue volume), Trabecular number (Tb.N/mm), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of the same human bone samples as in (I).",
    "molecules": "von Kossa"
  },
  {
    "caption": "(A) Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 µg/ml tetracycline for 48 h. 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti-CD24 SWA11 mAb. The membrane was reprobed with anti-tubulin antibody to assess the uniformity of sample loading.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(D) Western blot analysis of EXO-CD24 exosomes. Tetracycline (Tet) was used to induce CD24 expression in the cell line from which exosomes are derived. HSP70 was used as an exosomal marker.",
    "molecules": "Tet, Tetracycline"
  },
  {
    "caption": "(B) respiratory rate and (C) Blood oxygen (SpO2) and before treatment (Day 1, measured before administration of first dose) and after treatment (Day 7). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for Blood oxygen (SpO2) and respiratory rate. these are presented graphically via box plots before and after treatment.",
    "molecules": "O2, oxygen"
  },
  {
    "caption": "(B) Representative images of cells released from mitosis for 1, 2 or 3h in EdU-containing medium. Top, DNA staining  with DAPI; Bottom, EdU immunofluorescence. L, M and H indicate cells with low, medium or high levels of EdU  incorporation. The barplot shows the quantification of the proportion of replicating cells at each time point.",
    "molecules": "EdU, DAPI, DNA"
  },
  {
    "caption": "Y-axis, z-score; X-axis, timepoints. A indicates asynchronous  cells, M mitotic  cells and the numbers represent the minutes after release from the mitotic  block). Dots show individual replicates and lines averages (n=3 for A and M; n=2 for each post-mitotic   sample). Pre-mRNA levels are shown for pluripotency genes (Oct4, Klf4, Esrrb) and cell cycle regulators (E2f1, Cdk2, Rb); mRNA levels for individual histone   genes (only averages are shown).",
    "molecules": "histone"
  },
  {
    "caption": "(B) Heatmaps presented for untreated (-IAA; left) or CTCF-depleted (+IAA; right) CTCF-AID cells.",
    "molecules": "IAA"
  },
  {
    "caption": "(A) Gene expression changes (z-score) in asynchronous  cells and during  post-mitotic  release of CTCF-AID cells treated or not with IAA  to deplete CTCF  (blue, presence of CTCF; red, absence). Samples are indicated on the X-axis (A: asynchronous  cells; M: mitotic  cells; numbers: minutes since release from mitotic  block). Six gene groups are shown, based on the time  of their response to CTCF  depletion (asy, late, early) and the direction of the transcriptional change (up, down). The number of genes of each group is indicated. The yellow shadow areas indicate times  showing expression  changes. The boxplots show the median (bar), 25-75% percentiles (box) and 1.5-fold inter-quartile range (whiskers).",
    "molecules": "IAA"
  },
  {
    "caption": "Presence of TOC proteins in the  central vacuole of protoplasts. Arabidopsis protoplasts  expressing the YFP-tagged chloroplast  proteins were analyzed by confocal microscopy following ConA  treatment for four hours. Representative images are shown (A). Chlorophyll   autofluorescence was used to determine the localization  of the YFP  fluorescence signals relative to the chloroplasts. Signals for SP1-YFP  and FAX1-YFP, which here are control proteins, were only present at the chloroplast envelope. Accumulation of YFP  puncta in the  central vacuole  is indicated by the arrowheads. Scale bar, 10 μm.",
    "molecules": "Chlorophyll, ConA"
  },
  {
    "caption": "Presence of TOC proteins in the  central vacuole of Arabidopsis leaf  and hypocotyl  cells. Eight-day-old transgenic Arabidopsis  seedlings stably expressing pToc33:YFP-Toc33  or pToc75:YFP-Toc75  were treated with ConA  for two days, and then analyzed by confocal microscopy. Accumulation of YFP  puncta in the central vacuole is indicated by the arrowheads. Scale bar, 10 μm.",
    "molecules": "ConA"
  },
  {
    "caption": "Lack of TOC proteins in the  central vacuole  of atg7-2 mutant hypocotyl  cells. Eight-day-old transgenic Arabidopsis  seedlings stably expressing pToc33:YFP-Toc33  and pToc75:YFP-Toc75  in the atg7-2 mutant background were treated with ConA  for two days, and then analyzed by confocal microscopy. Note that YFP-Toc33  and YFP-Toc75  bodies (arrowheads) were retained at  plastid  bridges and extensions, and not released into the vacuole, as occurs in the WT background Scale bar, 10 μm.",
    "molecules": "ConA"
  },
  {
    "caption": "Autophagy inhibition affected accumulation of TOC proteins. Eight-day-old wild-type (WT) and atg7-2 plants were treated respectively with proteasome  inhibitor, bortezomib  (Btz), cysteine protease  inhibitor, E64d, or DMSO  (Mock) for one day. Total protein extracts from these plants were analyzed by immunoblotting  using antibodies as indicated. Actin  acted as a loading control. Typical immunoblotting  results are shown (B). In B, the arrowhead indicates full-length Toc159, and the hollow arrow indicates a truncated form of Toc159. Molecular weight markers (sizes in kD) are indicated to the right of the images.",
    "molecules": "E64d, bortezomib, Btz, DMSO"
  },
  {
    "caption": "Autophagy inhibition affected accumulation of TOC proteins. Eight-day-old wild-type (WT) and atg7-2 plants were treated respectively with proteasome  inhibitor, bortezomib  (Btz), cysteine protease  inhibitor, E64d, or DMSO  (Mock) for one day. Band intensities were quantified and normalized to equivalent data for Actin  (C). Data are means ± SEM from three biological replicates. Asterisks in C indicate significance according to an unpaired two-tailed Student's t-test (*, significant at P < 0.05; **, significant at P < 0.01).",
    "molecules": "E64d, bortezomib, Btz, DMSO"
  },
  {
    "caption": "Presence of autophagosome  in the chloroplast envelope. Arabidopsis protoplasts  expressing free YFP  and YFP-ATG8  (autophagosome  marker) were analyzed by confocal microscopy. Corresponding chlorophyll  autofluorescence images were employed to orientate the YFP  signal in relation to the chloroplasts. For eliminating interference from cytosolic  signals, intact protoplasts  (upper panels) were ruptured using 10-μm nylon mesh to isolate the chloroplasts. Retention of YFP  signals in association with chloroplasts   upon rupturing the cell (lower panels) clearly indicated that the YFP-ATG8  was associated with chloroplasts. Representative images are shown (A).",
    "molecules": "chlorophyll"
  },
  {
    "caption": "Phenotypes of seedlings  under UVB  stress. Briefly, eight-day-old WT, atg7-2, nbr1-2 and NBR1-OX (overexpressor) seedlings  grown under normal condition were subjected to UVB  stress, with UVB  treatment for 3 hours and recovery  for 7 days. Representative plant images (A) and total chlorophyll  content values (B) are shown; the latter provide a convenient metric for biomass. The chlorophyll  values for the mutants and overexpressor plants were statistically significantly different from that for the WT (Student's t test; *, significant at P < 0.05; ***, significant at P < 0.001).",
    "molecules": "chlorophyll"
  },
  {
    "caption": "Toc159  was modified by both K48 and K63 ubiquitin  linkage types. Eight-day-old WT plants were grown under normal growth condition (-), or subjected to UVB  stress (+), with UVB   treatment for four hours. The protein extract from the plants was analyzed by IP  using native Toc159  antibody, or its preimmune serum  as a negative control, and analyzed by immunoblotting  using antibodies as indicated to the left of the images (E). Tic40  acted as a negative control. The asterisk indicates a truncated Toc159  form without A domain Molecular weight markers (sizes in kD) are indicated to the right of the images. Please note that ubiquitin  antibodies tend to produce stronger signals for hyper-ubiquitinated  species than for hypo-ubiquitinated  or mono-ubiquitinated  species (based on the number of epitopes), which contrasts with the behaviour of the Toc159  antibody. Thus, the signals of the two antibodies are not expected to fully overlap. Poly-ubiquitinated Toc159  may not be detected by the Toc159  antibody due to the much lower abundance of these species compared with the mono-ubiquitinated  or unmodified forms. This is why the  poly-Ub K48 and K63 signals appear at higher molecular weight positions than the Toc159  signals. Relative Toc159  amounts modified by K48 or K63 ubiquitin  linkage in plants with or without UVB  stress were assessed by quantifying the smeary band intensity of the IP  samples in α-K48 or α-K63 blots  (F). All data are means ± SEM from three experiments.",
    "molecules": "poly-Ub, ubiquitin"
  },
  {
    "caption": "Analysis of  protein import efficiency  in vitro. Chloroplasts  were isolated from 12-day-old WT, nbr1-2 and NBR1-OX seedlings  grown in vitro under normal condition, and were subjected to in vitro protein import assay by using 35S-labeled Rubisco small subunit  (SSU) precursor protein as the import substrate, followed by corresponding quantification of the maturation of the radiolabeled    precursor protein. A representative phosphor  screen image is shown (upper left), and Coomassie staining  of Rubisco large subunit (LSU) indicated equal loading (lower left); Times  indicate minutes after the start of each import reaction. IVT, in vitro translation product used as a control. Together with similar images from two additional experiments, this was used to conduct the quantitative analysis shown (right); the amount of imported, mature protein in each sample was expressed as a percentage of that present at the final  time point for the WT. Data are means ± SEM from three biological replicates.",
    "molecules": "35S"
  },
  {
    "caption": "Phenotypes of two-week-old WT, nbr1-2, ppi1  and ppi1 nbr1-2 seedlings  grown on soil. Representative plant images (A) and chlorophyll  contents (B) are shown. The chlorophyll  values for the ppi1  and ppi1 nbr1-2 plants were statistically significantly different (Student's t test; ****, significant at P < 0.0001). Values are means ± SEM from 20 plants.",
    "molecules": "chlorophyll"
  },
  {
    "caption": "(a) Plasma ornithine  concentrations in Oatrhg mice  following intravenous injections of AAV-OAT  at the doses of 1x1013 (n=9) and 3x1013 genome copies (gc)/Kg (n=6), or AAV-GFP  at the dose of 1x1013 gc/Kg (n=9). Normal range was established on wild-type mice  (n=7) and average ± 2SD was calculated. Data are shown as averages ± SEM. ***p<0.001, ****p<0.0001 (Two-way ANOVA test).",
    "molecules": "ornithine"
  },
  {
    "caption": "(c) Plasma ornithine  and lysine  concentrations at 12 months of age (10-months post-injection) in  Oatrhg mice injected with AAV-OAT  (n=3) or AAV-GFP  (n=3); WT mouse  controls (n=5) are also shown; each sample was analyzed as triplicates, data are shown as averages ± SEM. One-way ANOVA test with Tukey correction; *p <0.05; ****p<0.0001.",
    "molecules": "lysine, ornithine"
  },
  {
    "caption": "(a) Plasma ornithine  concentrations in AAV-GFP- (n=3) and AAV-OAT-injected (n=3) OatΔ mice. Normal range of wild-type mice  is also shown. Each value plotted represents mean ± SEM; Two-way ANOVA test, *p <0.05, **p<0.01.",
    "molecules": "ornithine"
  },
  {
    "caption": "(C,D) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 (F1 probe or mut F1 probe) ,the confirmed NRF1 binding site in the promoter of VSNL1 (positive probe). The probes were incubated with HeLa nuclear extracts, then run on a gel (C). A super-shifted band (asterisk) is detected in the presence of an anti-NRF1 antibody (D). Labeled probe: biotin labeled wild type Fundc1 probe; Cold probe: wild type Fundc1 probe; Mut probe: NRF1 binding site mutant Fundc1 probe; asterisk indicates the supershifted band generated with antibody to NRF1.",
    "molecules": "biotin"
  },
  {
    "caption": "mitochondrial superoxide (I) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.",
    "molecules": "superoxide"
  },
  {
    "caption": "Tumor growth effects of treatment with doxorubicin, sorafenib, rapamycin and combinations thereof in the five MPNST xenograft models. Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value. Statistically significant differences are shown as *P < 0.05 and **P < 0.001 versus control group by the Bonferroni test.",
    "molecules": "doxorubicin, rapamycin, sorafenib"
  },
  {
    "caption": "(B) Representative images of FITC-labelled TPA  and AAVP particle diffusion in Matrigel  at 5 and 30 minutes post-inoculation (n = 3). Scale bar, 100 μm. (C)  Particle diffusion  using a vertical  trans-well system partitioned by Matrigel. Samples were collected from the wells below the Matrigel at days 1 to 7, n = 3, technical replicates. Data are expressed as mean + SEM, independent t-test was used for data analysis.",
    "molecules": "FITC, Matrigel"
  },
  {
    "caption": "(C) Phagocytosis of 1 µM HiLyte™ Fluor 488 Aβ1-42 (fAβ42) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non-binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.61, genotype P<0.0001, treatment P=0.0001; post hoc tests wt vs. mu for the following conditions: fAβ42 P=0.0043, fAβ42-2D8 P=0.0436).",
    "molecules": "Cytochalasin D, CytoD"
  },
  {
    "caption": "(B) mAb11 but not the IC co-localized with methoxy-X04.",
    "molecules": "methoxy-X04"
  },
  {
    "caption": "BMDM (C) or primary microglia (D) were cultured on cryosections pre-incubated with mAb11 (1 µg/ml). After 24 h, sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy-X04 staining to visualize Aβ. Note that both cell types internalize Aβ into intracellular vesicles (right panels show enlargement of insets). Scale bar: 10µm.",
    "molecules": "methoxy-X04"
  },
  {
    "caption": "(A) BMDM from wt or Trem2 ko mice were cultured on APP/PS1micebrain cryosections incubated with or without mAb11 (1 µg/ml) or an isotype control (IC; 1 µg/ml) for 24 h. Sections were then probed with methoxy-X04. Scale bar, 500 µm.(B) The amyloid plaque load was quantified from the entire sagittal section. Sections incubated with medium (no cell) were set as baseline. (n = 6, +/- SEM; 2 way ANOVA, interaction P<0.0001, genotype P<0.0001, treatment P<0.0001; Tukey's multiple comparisons tests; wt vs. ko for the following conditions: no antibody P=0.0304, IC P=0.0049, mAb11 P=0.0212; wt : IC vs. wt : mAb11 P=0.0008; ko : IC vs. ko : mAb11 P=0.0001).",
    "molecules": "methoxy-X04"
  },
  {
    "caption": "(A) Cryosections from unfixed brain of 6-months old APP/PS1mice were pre-incubated with increasing concentrations of mAb11 (0.001, 0.01, 0.1, 1, 5 µg/ml). BMDM from wt or Trem2 ko mice were added for 24 h. Sections were stained with methoxy-X04. Scale bar, 500 µm.(B) Methoxy-X04 signals were quantified from the entire sagittal section. (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.0082, genotype P<0.0001, treatment P<0.0001. Fisher's LSD post hoc comparisons; * show statistics between wt and ko under the same experimental condition. # in black show wt compares to no antibody stimulation; # in grey show ko compares to no antibody stimulation; wt vs. ko for the following conditions: no antibody P=0.0053, mAb11 0.001 µg/ml P=0.0003, mAb11 0.01 µg/ml P<0.0001, mAb11 0.1 µg/ml P=0.0001, mAb11 1 µg/ml P=0.0007, mAb11 5 µg/ml P=0.0011; Following conditions compare to wt / no antibody: wt / mAb11 0.01 µg/ml P=0.0166, wt / mAb11 0.1 µg/ml P=0.0002, wt / mAb11 1 µg/ml P<0.0001, wt / mAb11 5 µg/ml P<0.0001; Following conditions compare to ko / no antibody: ko / mAb11 0.1 µg/ml P=0.0099, ko / mAb11 1 µg/ml P<0.0001, ko / mAb11 5 µg/ml P<0.0001.",
    "molecules": "Methoxy-X04, methoxy-X04"
  },
  {
    "caption": "Each plot shows the relative mass isotopomer distribution (MID) values for metabolite species detected from cell extracts in each of the five independent experiments shown in Figure 1. Each value was divided by the average value of the entire time series (x-axis) for that species from the same experiment, and Log2-transformed (y-axis). Loess curves and the std errors at a 0.95 level are shown. The Log2(relative abundance) values of pyruvate(M1) and leucine(M6) showed the most significant changes",
    "molecules": "leucine, pyruvate"
  },
  {
    "caption": "C, DNA content profiles of BAT1 and bat1Δ cells from asynchronous cultures, in SMM medium. Where indicated, exogenous amino acids were added at 1mM final concentration. On the x-axis of the histograms is fluorescence per cell, while on the y-axis is the cell number. Peaks corresponding to cells in G1 with unreplicated (1N) and cells in G2 and M phases with fully replicated (2N) DNA are indicated. The percentage of cells with G1 DNA content (%G1) from 3 independent measurements is shown in each case (mean and sd).",
    "molecules": "DNA"
  },
  {
    "caption": "A, The indicated strains were spotted at 10-fold serial dilutions on solid Synthetic Minimal Medium (SMM) agar plates. Rapamycin was added at 30 ng/mL, as shown in each case. The plates were incubated at 30 °C and photographed after 5 and 7 days, as indicated.",
    "molecules": "agar, Rapamycin"
  },
  {
    "caption": "D, Exogenous addition of valine leads to sustained activation of TORC1 and phosphorylation of Rps6 in cells lacking Bat1. Wild type (BAT1+) and bat1∆ (two independent isolates, #4, and #5) strains were grown overnight in minimal (SMM) medium, diluted to 1E+06 cells/mL in fresh SMM media containing the indicated amino acid (at 1mM), and harvested when they reached 5E+06 cells/mL. The levels of phosphorylated Rps6 (α-Rps6-P) and Pgk1 (α-Pgk1) are shown in immunoblots from total cell extracts in each case. The relative levels of Rps6-P/Pgk1 are shown in each case at the bottom. The boxplot graphs were generated with R language functions. Each box is drawn from the first to the third quartile, with a horizontal line drawn in the middle to denote the median. The whiskers show the interquartile range (IQR), and they were drawn at 1.5xIQR. The replicates were all biological ones.",
    "molecules": "valine"
  },
  {
    "caption": "A, Immunoblots of total cell extracts from synchronous, elutriated wild-type (CEN.PK) cells in minimal (SMM) medium with exogenous Leu or Val added at 1mM immediately after elutriation. At the top, the percent of budded cells (%B), cell size (in fL), and time (in min) are indicated. The levels of phosphorylated Rps6 (α-Rps6-P) and Pgk1 (α-Pgk1) are shown in each case.",
    "molecules": "Leu, Val"
  },
  {
    "caption": "J MTM‐3 but not MTM‐3(C459S) exhibits phosphatase activity toward PtdIns3P and PtdIns(3,5)P2 in vitro.",
    "molecules": "PtdIns(3,5)P2 , PtdIns3P"
  },
  {
    "caption": "Same as in (D) but in the presence of an isotype control antibody or an anti-PD-1 antibody with the equivalent sequence to pembrolizumab. The effects on CD4 T cells from a G1 and a G2 patient are shown, divided in CD28 high or low/negative subsets as indicated. Relevant statistical comparisons are shown with paired Student´s t test.",
    "molecules": "pembrolizumab"
  },
  {
    "caption": "Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. but in the presence of an isotype control antibody or an anti-PD-1 antibody molecularly equivalent to pembrolizumab. Relevant statistical comparisons are shown with comparisons carried out with paired Student´s t test.",
    "molecules": "pembrolizumab"
  },
  {
    "caption": "E H2A.B interacts with RPA194 in live cells. Western blot analysis was performed to detect  RPA194  in the BioID  samples from L1236  cells (left panel), and L428  cells (right panel). The samples were prepared using  pull-down with streptavidin beads from the cell lysate of the empty vector, BirA*-H2A.B  and H2A.B-BirA* transduced cells, which were preincubated with 50 µM biotin  for 24 h. The positive control was loaded as 1% input.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "MITOL-KO MEFs was vulnerable to ER stress. Control MEFs (MITOLF/F) and MITOL-KO MEFs (MITOL-/-) were treated with DMSO as control, 0.8 µM thapsigargin (Tg), 0.7 µg/mL tunicamycin (Tu) or 1.2 µg/mL brefeldin A (Br) for 24 hours. All experiments using Tu below were performed at concentration of 0.7 µg/mL. The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers. Viable cells were detected by cell viability assay using cell counting kit-8.",
    "molecules": "Br, brefeldin A, DMSO, Tg, thapsigargin, Tu, tunicamycin"
  },
  {
    "caption": "MITOL KO increased ER stress-induced apoptosis. MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V-FITC for the detection of apoptosis using flow cytometry.",
    "molecules": "FITC"
  },
  {
    "caption": "MITOL re-expression in MITOL-KO MEFs rescued ER stress vulnerability. MEFs were transfected with MITOL-coding vector (WT), MITOL C65/68S-coding vector (CS), or empty vector (Vec) 24 hours prior to Tu treatment for 18 hours, followed by immunoblotting with indicated antibodies. Cleaved caspase-3 (cC3) and cleaved PARP (cPARP) were used as apoptosis markers.",
    "molecules": "Tu"
  },
  {
    "caption": "MITOL KO enhanced ER stress-induced Bax translocation and cytochrome c release. Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours, followed by immunoblotting with indicated antibodies.",
    "molecules": "Tu"
  },
  {
    "caption": "MITOL KO induced excessive mitochondrial depolarization by ER stress. MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM, followed by flow cytometric analysis.",
    "molecules": "TMRM, Tu"
  },
  {
    "caption": "IRE1α inhibition compensated MITOL-KO MEFs for vulnerability to ER stress. MEFs were transfected with scramble or siIRE1α (#1, #2). After 24 hours, these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).",
    "molecules": "Tu"
  },
  {
    "caption": "MEFs were treated with 4µ8C (25 µM) for 2 hours prior to Tu treatment for 24 hours (B), followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).",
    "molecules": "4µ8C, Tu"
  },
  {
    "caption": "MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE.",
    "molecules": "Tu"
  },
  {
    "caption": "MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR",
    "molecules": "Tu"
  },
  {
    "caption": "MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. MEFs were transfected with either miR-17 or miR-34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours, followed by luciferase reporter assay (F). Error bars represent SD (n=3).",
    "molecules": "Tu"
  },
  {
    "caption": "MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR",
    "molecules": "Tu"
  },
  {
    "caption": "IRE1α-mediated JNK phosphorylation was prolonged by MITOL KO. MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies. Error bars represent SD (n=3).",
    "molecules": "Tu"
  },
  {
    "caption": "JNK inhibition reduced ER stress-induced apoptosis of MITOL-KO MEFs. MEFs were treated with 20 µM JNK inhibitor-II (JNK inh) 2 hours prior to Tu treatment for 24 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).",
    "molecules": "(JNK inh) 2, JNK inhibitor-II, Tu"
  },
  {
    "caption": "MITOL KO facilitated IRE1α oligomerization. MEFs were transfected with IRE1α-GFP expression vector for 24 hours prior to Tu treatment for indicated periods (A). The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment (A). To evaluate the oligomerization level of endogenous IRE1α, cells without any transfection were solubilized and separated by sucrose density-gradient centrifugation, followed by immunoblotting with anti-IRE1α antibody (B).",
    "molecules": "Tu"
  },
  {
    "caption": "IRE1α oligomers were stabilized by MITOL KO. MEFs transfected with IRE1α-GFP were treated with Tu. After 4 hours, these cells were washed with PBS and subsequently re-fed with fresh media for indicated periods. The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment. Scale bar represents 10 µm.",
    "molecules": "PBS, Tu"
  },
  {
    "caption": "Sustained IRE1α autophosphorylation after wash out of Tu in MITOL-KO MEFs. MEFs were treated with Tu for 4 hours and washed with PBS and re-fed with fresh media for indicated periods, following by immunoblotting anti-IRE1α antibody after Phos-tag SDS-PAGE. Error bars represent SD (n=3).",
    "molecules": "PBS, Tu"
  },
  {
    "caption": "MITOL deletion promoted the interaction between IRE1α and BIM under ER stress. MITOL-depleted HEK293 cells (KO) were generated using CRISPR-Cas9 system. Cells were co-transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours. Cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.",
    "molecules": "Tu"
  },
  {
    "caption": "MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected",
    "molecules": "lysine"
  },
  {
    "caption": "MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected",
    "molecules": "lysine"
  },
  {
    "caption": "MITOL added polyubiquitin chains to K481 of IRE1α. Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. 481: K481R, 545: K545R, 568: K568R.",
    "molecules": "lysine"
  },
  {
    "caption": "Overexpression of IRE1α K481R induced apoptosis. MEFs were transfected with indicated vectors. After 24 hours, these cells were stained with Annexin V-FITC",
    "molecules": "FITC"
  },
  {
    "caption": "Hyper-oligomerization of IRE1α K481R. MEFs were transfected with IRE1α-GFP or IRE1α K481R-GFP. After 24 hours, these cells were exposed with Tu for indicated periods and GFP signals were observed. The right panels show 5.5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment.",
    "molecules": "Tu"
  },
  {
    "caption": "High solubility of MITOL and IRE1α in the low dose digitonin. Raft-like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin. S: supernatant, P: pellet.",
    "molecules": "digitonin"
  },
  {
    "caption": "Loss of MITOL led to hyper-oligomerization of IRE1α in the spinal cord under ER stress. 3 month-old WT or MITOLnestin mice were treated with 1 mg/kg Tu for 24 hours. Spinal cord was solubilized with NP-40 lysis buffer and separated by sucrose-density gradient centrifugation.",
    "molecules": "NP-40, Tu"
  },
  {
    "caption": "MITOL deletion resulted in hyper-activation of IRE1α RNase in ER stress. WT or MITOLnestin mice were injected with Tu for 24 hours and spinal cord was analyzed by qRT-PCR.",
    "molecules": "Tu"
  },
  {
    "caption": "Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using TUNEL and hoechst (F) Arrowheads indicate the representative neurons.",
    "molecules": "hoechst, Tu"
  },
  {
    "caption": "Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using cresyl violet (G). Arrowheads indicate the representative neurons.",
    "molecules": "cresyl violet, Tu"
  },
  {
    "caption": "(C) Upper and middle panels: immunoblots showing precipitation of ACTN4 and β-actin from RPE-1 nuclear lysates in early-G1 in the presence or absence of biotin-phalloidin, as indicated. Lower panel: Immunoblot of cytosolic (cyt) and nuclear (nuc) fractions. Proteins were detected with antibodies against (α) ACTN4, β-Actin, α-Tubulin or Histone 3 as indicated.",
    "molecules": "biotin, phalloidin"
  },
  {
    "caption": "(D) Immmunofluorescence images of endogenous ACTN4 in non-dividing and post-mitotic NIH3T3 and RPE-1 cells in as indicated. Shown are maximum intensity projections. Cells were fixed in 4% PFA and stained for ACTN4 and nuclei (DAPI) as indicated. Scale bars 10 μm.",
    "molecules": "DAPI, PFA"
  },
  {
    "caption": "(A) Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a timeseries on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. Scale bar overview (nAC) represents 10 µm. Scale bar time series and magnification 5 µm.",
    "molecules": "doxycycline"
  },
  {
    "caption": " Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. B) Linescan of an actin filament with associated ACTN4 from square 3 in A. ",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) Upper panel shows expression of ACTN4 constructs (green, confocal image) and phalloidin-AF647 staining in the same cells (orange, dSTORM image) scale bar 10 µm; lower panel represents zoom of the indicated areas (white boxes); scale bars 5 µm.",
    "molecules": "AF647, phalloidin"
  },
  {
    "caption": "(C) Phalloidin-AF647 localizations per post-mitotic nuclei; data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei and 40 nuclei of non-dividing cells (ACTN4-wt) from 5 biological replicates. ***p(ACTN4ΔCH1-NLS post-mitotic) < 0.001, ****p(ACTN4-wt non-dividing) < 0.0001, *p (ACTN4ΔCH1-NLS post-mitotic vs. ACTN4-wt non-dividing) < 0.05 by One Way ANOVA.",
    "molecules": "AF647, Phalloidin"
  },
  {
    "caption": "F Immunoblot analysis of FLAG-METTL14, LC3-II, and β-actin in HEK293T cells exposed to the autophagy inhibitor Baf-A1 and activator Torin 1, respectively. The FLAG-METTL14 or LC3-II /β-actin densitometric ratio was recorded by ImageJ.",
    "molecules": "Baf-A1, Torin 1"
  },
  {
    "caption": "G Dot blots of m6A levels in the presence of MG132 in 293T, RBE, SK-Cha-1, and SK-N-SH cells.",
    "molecules": "MG132, m6A"
  },
  {
    "caption": "L Immunoblots showing CHX assay in HEK293T cells. Negative control (NC; no transfected cells) and STUB1-FLAG plasmid-transfected HEK293T cells were exposed to 100 μg/ml CHX for 0, 4, 8, or 12 hours. β-actin was used as the loading control. The METTL14/β-actin densitometric ratio was recorded by ImageJ.",
    "molecules": "CHX"
  },
  {
    "caption": "M, N Dot blot assays showing that enforced expression of wild-type STUB1, but not the STUB1H260Q mutant, significantly decreases total m6A levels.",
    "molecules": "m6A"
  },
  {
    "caption": "B Immunoblots showing the abundance of METTL14 truncated mutants with or without 10 µM MG132 treatment. Histone H4 was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ.",
    "molecules": "MG132"
  },
  {
    "caption": "F Immunoblots showing the levels of METTL14 protein after transfection with METTL14-3KR mutant plasmids with or without 10 µM MG132. β-actin was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ. G Immunoblots showing the protein level of METTL14 after transfection with METTL14-3KR mutant plasmids under STUB1 knockdown. β-actin was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ.",
    "molecules": "MG132"
  },
  {
    "caption": "B METTL14 ubiquitination, as detected by IP of METTL3 truncated mutants with anti-HA antibody. The accumulation of Ub and METTL14 was confirmed in whole-cell lysates. β-actin was used as the loading control.",
    "molecules": "Ub"
  },
  {
    "caption": "(b) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript (N=3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm",
    "molecules": "DSS, Dapi"
  },
  {
    "caption": "(c) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged. Crypts are dramatically injured by d5. At d8 through d12, regenerating crypts are observed. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm",
    "molecules": "DSS, Dapi, Eosin, Hematoxylin"
  },
  {
    "caption": "(d-e) Compared to untreated controls, DSS treatment ablates Lgr5-GFP+ stem cells and results in functional loss of tdTomato+ lineage tracing as early as d5. Lgr5-GFP+ stem cells reappear by d12 (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm Data are represented as mean ± SD (e) analyzed using Student's t-test. *P≤0.05, ** P≤0.01.",
    "molecules": "DSS, Dapi"
  },
  {
    "caption": "(f) Effects of DSS colitis injury on RNA expression levels of stem (Lgr5 and Krt19) and progenitor (Atoh1 and Notch1) cell markers. Lgr5 mRNA expression is significantly decreased after exposure to DSS (N=6-7 per condition). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.",
    "molecules": "DSS"
  },
  {
    "caption": "Mice body weight (h) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.",
    "molecules": "DSS"
  },
  {
    "caption": "Mice survival (i) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.",
    "molecules": "DSS"
  },
  {
    "caption": "(j) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS+DT treated mice. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bar (j) =500µm.",
    "molecules": "DSS, Dapi, Eosin, Hematoxylin"
  },
  {
    "caption": "(k) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS+DT (to ablate Lgr5+ stem cells) (N=3 DSS; N=6 DSS+DT). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.",
    "molecules": "DSS"
  },
  {
    "caption": "(l) RNAscope for Lgr5 in Lgr5DTR-GFP mice showing loss of Lgr5 transcripts after DT treatment (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm",
    "molecules": "Dapi"
  },
  {
    "caption": "(b-c) Krt19+ cells lineage trace entire colonic crypts during both normal homeostasis and injury (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. Scale bars (b) =45µm Data are represented as mean ± SEM *P≤0.05, **P≤0.01.",
    "molecules": "Dapi"
  },
  {
    "caption": "DT ablation of Krt19+ cells (N≥3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (e) =30µm",
    "molecules": "Dapi"
  },
  {
    "caption": "(g) Mice body weight and survival remain unchanged between DSS and DSS+DT treated groups (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test P≤0.05, **P≤0.01.",
    "molecules": "DSS"
  },
  {
    "caption": "Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS+DT showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (h) =1mm.",
    "molecules": "DSS, Dapi, Eosin, Hematoxylin"
  },
  {
    "caption": "quantification of damaged epithelial area (i), showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.",
    "molecules": "DSS"
  },
  {
    "caption": "(b-c) Notch1+ progenitors are able to generate Lgr5+ stem cells and contribute to epithelial renewal in homeostasis; however, do not contribute to regeneration after colitis (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test. ****P≤0.0001.",
    "molecules": "Dapi"
  },
  {
    "caption": "Images of whole mount showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.",
    "molecules": "Dapi"
  },
  {
    "caption": "Images of tissue sections Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice (f) showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.",
    "molecules": "tdTomato, Dapi"
  },
  {
    "caption": "(a-b) Atoh1+ progenitors show stem cell capacity during normal homeostasis and regeneration. At early stages of recovery, d10-12, Atoh1+ cells lineage trace the colonic crypt (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.",
    "molecules": "Dapi"
  },
  {
    "caption": "(c-d) By d30 after DSS, Atoh1 derived stem cells continue to lineage trace complete colonic crypts (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars first two panels of c =50μm; scale bars (last panel of c =100μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.",
    "molecules": "DSS, Dapi"
  },
  {
    "caption": "Whole mount (f) images of Atoh1 lineage labeled crypts. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm",
    "molecules": "Dapi"
  },
  {
    "caption": "section (g) images of Atoh1 lineage labeled crypts. Majority of crypts are labeled after Lgr5+ stem cell ablation and remain label with the appearance of Lgr5+ stem cells (inset, arrowheads). Atoh1 lineage labeled crypts remain by d30. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bars =100μm.",
    "molecules": "Dapi"
  },
  {
    "caption": "(j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.",
    "molecules": "DSS, Dapi"
  },
  {
    "caption": "(b-c) Sporadic labeling of Atoh1+ cells after 4-OHT induction. Following irradiation, entire colonic organoids exhibit full lineage labeling of Atoh1+ cells, characteristic of multipotent stem cells (N=3 per condition). Data information: Scale bars =100μm Data are represented as mean ± SD analyzed using Student's t-test (c)",
    "molecules": "4-OHT"
  },
  {
    "caption": "(b) Atoh1+ lineage labelling post-DSS and DSS+DT. Data information: Scale bars (b)=200μm",
    "molecules": "DSS"
  },
  {
    "caption": " (c) DSS+DT treated-mice exhibit significant weight loss and lower survival compared to DSS alone (N=4-7 control; N=3-4 DSS). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.",
    "molecules": "DSS"
  },
  {
    "caption": "(d-e) Hematoxylin and Eosin staining of colonic epithelium after DSS versus DSS+DT (d). Extensive epithelial damage was observed (d, arrow) and quantified of in DSS+DT treated mice (N=7 control; N=4 DSS). Data information scale bars (d)=400μ Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.",
    "molecules": "DSS, Eosin, Hematoxylin"
  },
  {
    "caption": "B: Western Blot of PSA-treated uninfected and infected GT-1/7  cells. Increasing concentration of PSA  leads to a more prominent reduction of  PrPSc  in mouse  cells. PK-western blot is quantified relative to DMSO.",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "C: mRNA levels of Hnrnpk  and Prnp  following siRNA and PSA  treatment. FPKM: Fragments per kilobase of transcript per million mapped reads. Hnrnpk  siRNAs lead to a decrease in Hnrnpk  mRNA levels as well as an increase in Prnp  mRNA levels. No difference is seen between DMSO  treated and PSA  treated cells for either Hnrnpk  levels of Prnp  levels. n = 2 per treatment group.",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "D: Quantification of PrPSc  levels in RML GT-1/7  cells following treatment with 1 µM PSA  in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "F: Quantification of PrPSc levels in PG127  hovS  cells following treatment with 1 µM PSA  in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "H: Quantification of PrPSc  levels in PG127 hovS  cells following treatment with shRNA against HNRNPK  and 1 µM PSA  in comparison to DMSO  and NT. Each dot represents an experiment. *= p-value < 0.027 (Student's t-test). Shown are mean ± SD.",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "I: Gene set overrepresentation analysis of differentially expressed genes (log2FC -0.25 ≥ or 0.25 ≤ and FDR ≤ 0.05) for siRNA mediated Hnrnpk  downregulation or PSA  treatment in RML GT-1/7 cells analyzed by  RNAseq. Differentially regulated genes (up in siRNA treatment and down in PSA  or vice versa) were overlapped and used for pathway analysis. No significantly enriched pathways are detected for upregulated genes in Hnrnpk  and downregulated in PSA  treatment. For the opposing direction, an enrichment of genes involved in glucose  metabolism was detected.",
    "molecules": "glucose, PSA"
  },
  {
    "caption": "A: Western Blot  of RML prion  infected COCS  treated with 1 µM PSA  or DMSO. PSA  treatment was started two weeks after infection and continued until lysis. PSA  reduced the amount of PrPSc. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values: mean ± SD. * p = 0.0211 (Student's t-test).",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "B:  Western Blot  analysis of NBH-treated COCS. PSA  treatment was identical to the samples of A. PSA  did not affect PrPC  expression. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values represent mean ± SD. n.s. = not significant (Student's t-test). ⍺: anti",
    "molecules": "PSA"
  },
  {
    "caption": "C:  Negative geotaxis climbing assay in prion  infected Drosophila. Flies were treated with DMSO, 0.5 mM PSA, 0.75 mM PSA, or 1 mM PSA  at the larval stage and during adulthood for the duration of the assay. Climbing ability was assessed on groups of flies (n=3 x 15) three times  a week and expressed as a performance index. Statistical analysis on the difference between PG127 prion  infected versus control prion-free treatment group data in each graph was performed by an unpaired t test: DMSO: p = 0.0002; PSA: 0.5 mM: p = 0.0186; 0.75 mM: p = n.s.; 1 mM: p = n.s. n.s. = not significant. Shown are mean ± SD",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "D: RT-QuIC analysis of whole-head  homogenates of prion  infected Drosophila. For each sample, 10 male  and 10 female heads from the same treatment group were homogenized, 1:20 diluted and applied to the  RT-QuIC. Shown are the RT-QuIC reactions of three independent homogenates per treatment group, each assessed in quadruplicates. For quantification, the lag-time of each reaction was calculated and plotted in a graph. Shown are mean ± SD. The assays were performed for 100 hours, samples not yielding a positive reaction are considered negative. *** = p ≤ 0.0006, n.s. = not significant, flies fed with NBH and treated with DMSO  were used as a negative control, a standard prion-free and a prion-containing sample were used as assay controls. To control for potential interference of DMSO  or PSA  with the RT-QuIC reaction, prion  positive sample was spiked with 1 µM PSA.",
    "molecules": "DMSO, PSA"
  },
  {
    "caption": "(F) Stills from time-lapse  sequence of representative  Hela cells expressing  mNeon-Green Aurora B  and Cell Mask to label cell membrane, exiting mitosis, showing the dynamic  re-localization of Aurora B  from DNA  to the overlapping  microtubules and cleavage furrow with control siRNA treatment (arrows top). With knock-down of RACGAP1, Aurora B still re-localises from the DNA to the microtubules and furrow  in the midzone in early anaphase (arrows bottom), but the microtubule localization is lost at later stages (asterisk bottom). Scale bar - 10 µm.",
    "molecules": "Cell Mask"
  },
  {
    "caption": "(E) Stills of representative Hela  cells expressing  Lifeact-GFP and  H2B-mCherry   at 10 mins following forced mitotic exit with siControl, siRACGAP1, DMSO  and 2µM ZM447439 treatment. Control siRNA, RACGAP1  siRNA and DMSO  treated cells show clearance  of  actin from the cortex  close to the DNA, while ZM447439 treated cells fail to clear  actin, quantified in (F). Note, failure in  actin  accumulation in RACGAP1 depleted cell, asterisk. Scale bar - 10 µm",
    "molecules": "DMSO, ZM447439"
  },
  {
    "caption": "(F) Quantification of  actin    intensity across the perimeter  of the cells based on distance from DNA, with 5 and 6 being closest to DNA and 1 and 10 furthest away, as in cartoon (B). Graph shows decrease in  actin intensity closer to the DNA in siControl (n=26), siRACGAP1 (n=37) and Control DMSO  treated cells (n=38 cells), seen by levels lower than 1 (dotted red line), while this decrease is absent in  ZM447439 treated cells (n=42 cells). Data presented as mean ± SD.",
    "molecules": "DMSO, ZM447439"
  },
  {
    "caption": "(B) Stills from time-lapse  sequence of the basal most region of Hela  cells expressing Lifeact-GFP and H2B-mCherry forced to undergo flat monopolar mitotic exit. While actin  is cleared underneath the DNA in control DMSO treated cells, it fails to be cleared around the DNA when cells are treated with Aurora B  inhibitor  ZM447439  during mitotic exit. Dotted mask shows position of DNA in actin  channel. Scale bar -10µm.",
    "molecules": "DMSO, ZM447439"
  },
  {
    "caption": "(D) Still images of representative  Hela  cells expressing Lifeact-GFP and H2B-mCherry captured at early anaphase  under different conditions i.e. Control siRNA, knock-down of centralspindlin  protein RACGAP1, in the presence and absence of  Aurora B kinase inhibitor (2µM ZM). Scale bar - 10 µm (E) Quantification of aspect ratio of cells treated with different conditions as in (D) at 6 mins after anaphase onset, before furrow formation. Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio, while together have a stronger impact during mitotic exit. In combined treatment, mitotic cells hardly undergo any elongation and remain spherical before re-spreading. Ordinary one-way ANOVA with multiple comparison. Each treatment is compared with siControl, ****p<0.0001 for all comparisons. Data represented as box-whisker plot, with box showing 25th-75th percentile values, whiskers-min to max value and line representing median. Mean±SD for different treatments- siControl (n=38) - 1.455±0.19, ZM  (n=32) - 1.219±0.16, siRACGAP1  (n=25) -1.144±0.078 and siRACGAP1+ZM (n=16) - 1.013±0.03. Cells for different treatment pooled from n>3 independent experiments. ",
    "molecules": "ZM"
  },
  {
    "caption": "(C) Quantification of actin  intensity at the poles of cells at 6 mins post anaphase onset, before furrow  formation under different conditions as in (A). Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin  and Aurora B kinase  activity are affected, while it is cleared upon RACGAP1 depletion. Data represented as mean±SD for treatments - siControl- 0.98±0.05, siRACGAP1  - 0.99±0.04, ZM - 1±0.039, siRACGAP1+ZM - 1±0.037. Ordinary one-way ANOVA with multiple comparison- siControl vs siRACGAP1 - n.s., p=0.896, siControl vs  ZM - **p=0.0018, siControl vs siRACGAP1+ZM - ****p<0.0001 and ZM  vs siRACGAP1+ZM - p=0.1528.",
    "molecules": "ZM"
  },
  {
    "caption": "(G) Quantification of shortest distance between centroid of DNA and cortical actomyosin  network. While chromatin is able to reach close to the cortex in both siControl and siRACGAP1  treated cells, the distance of chromatin  from cortex  increases upon  ZM treatment and is further enhanced in the double treatment condition. Data represented as mean±SD. siControl - 4.7±0.87, siRACGAP1 - 4.4±0.64,  ZM - 5.47±0.87, siRACGAP1+ZM - 6.35±1.14 microns. Ordinary one-way ANOVA with multiple comparison, siControl vs ZM, ***p=0.002, siControl vs siRACGAP1, p=0.2823, siControl vs siRACGAP1+ZM, ****p<0.0001, siRACGAP1 vs siRACGAP1+ZM, ****p <0.0001, ZM  vs siRACGAP1, ****p<0.0001, ZM  vs siRACGAP1+ZM, ***p=0.0004 .",
    "molecules": "DNA, ZM"
  },
  {
    "caption": "(A) β-Catenin is hyperphosphorylated during the mitotic phase. HeLa CCL2 cells were synchronized at the G1/S phase using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.). Cells were harvested at the indicated time points and treated with (+) or without (-) λ-phosphatase (λ-PPase). The resulting cell lysates were used for immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE. Asterisk indicates the phosphorylated form of Cdc25C.",
    "molecules": "Noc, nocodazole, thymidine"
  },
  {
    "caption": "(F) Total cell lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were immunoprecipitated with two different Plk1 antibodies. Each antibody (gifted from Kyung S. Lee, NIH/NCI) recognized either Plk1 N-terminus (N-term. Ab.) or C-terminus (C-term. Ab.). The resulting precipitates were subjected to immunoblot analysis. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.",
    "molecules": "Noc, nocodazole"
  },
  {
    "caption": "(G) Lysates from nocodazole (Noc.)-treated HeLa CCL2 cells were mixed with either control (phosphate-buffered saline only, +PBS), 10 μg/ml of high-affinity Plk1 PBD binding phospho-peptide (PLHSpT, +P), or 10 μg/ml of non-phospho-peptide (PLHST, +NP). The resulting cell lysates were immunoprecipitated with anti-Plk1 antibody. Precipitates were subjected to immunoblot analysis. Immunoblotting with anti-Dvl2 antibody represents a positive control of phospho-peptide (PLHSpT, +P) competition for Plk1 binding. Hyperphosphorylated β-catenin (red arrowhead) was distinguished from non-/less-phosphorylated β-catenin (blue arrowhead) by slower migration during SDS-PAGE.",
    "molecules": "PBS, phosphate-buffered saline, Noc, nocodazole"
  },
  {
    "caption": "(C) Bacterially produced β-catenin T1 WT, T3 WT, and corresponding alanine ("A") mutants were purified and reacted with Flag-Plk1 WT in the presence of [γ-32P]ATP. Incorporation of 32P onto each "A" mutant of β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.",
    "molecules": "alanine, ATP, 32P"
  },
  {
    "caption": "(H) HeLa CCL2 cells were treated for 18 h with 200 ng/mL nocodazole followed by treatment with either DMSO (control) or BI 2536 for an additional 1 h. The resulting cells were lysed with 2× Laemmli SDS sample buffer and used for immunoblot analysis with the indicated antibodies.",
    "molecules": "BI 2536, DMSO, nocodazole"
  },
  {
    "caption": "(E, F) HeLa CCL2 cells infected with the indicated lentiviruses were co-transfected with HA-Ect2 and Flag-Plk1. Cells were then treated with nocodazole (200 ng/mL) for 18 h and released from cell cycle arrest by incubation in fresh medium for 6 h. The resulting cells were used for immunoprecipitation analysis using anti-Flag (E) or anti-HA (F) antibodies. Precipitated beads were used for immunoblot analysis using the indicated antibodies.",
    "molecules": "nocodazole"
  },
  {
    "caption": "HeLa CCL2 cells treated with DMSO (control) or the Plk1 inhibitor, BI 2536 (1 μM) for 20 min were immunostained with a combination of either anti-total β-catenin and anti-Ect2 antibodies or anti-β-catenin p-S60 and anti-Ect2 antibodies (A). Scale bars, 10 μm.",
    "molecules": "BI 2536, DMSO"
  },
  {
    "caption": "(C) After 18 h of nocodazole (200 ng/mL)-induced cell cycle arrest, HeLa CCL2 cells transfected with HA-Ect2 were released by incubation in fresh medium for 2 h and treated with either DMSO (control) or Plk1 inhibitor (BI 2536) for the indicated times. The resulting cells were used for immunoprecipitation analysis using anti-HA antibody, and immunoprecipitates were separated by SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The band intensity of β-catenin bound to HA-Ect2 was quantified with ImageJ and normalized to the signal intensity of immunoprecipitated HA-Ect2, and the relative values are shown below the bands.",
    "molecules": "BI 2536, DMSO, nocodazole"
  },
  {
    "caption": "(G) HeLa CCL2 cells were transfected with either wild type Flag−β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D), then treated with 200 ng/mL nocodazole for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were used for pull-down analysis with rhotekin-RBD beads, and the pulled down beads were separated by 12 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The membrane was then stained with Coomassie (CBB) to determine the amount of RBD precipitated.",
    "molecules": "CBB, Coomassie, nocodazole"
  },
  {
    "caption": "(A) 786-0 or 769-P cells stably expressing shNT or shGDH1  were treated with or without aa starvation for the indicated  times, and then  cell viability was assessed. (B) 786-0  or 769-P  cells stably expressing shNT or shGDH1  were reconstituted with or without various amounts of rGDH1 WT (3*WT indicates 3-fold expression WT) and then treated with aa  starvation for the indicated  time. Cell viability  was subsequently assessed. ( Data information: The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.)",
    "molecules": "aa"
  },
  {
    "caption": "(D)  Immunofluorescence (IF) assay was performed to determine the subcellular location of GDH1 with or without  aa  starvation. GDH1 localization was indicated by GDH1 antibody and  MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.",
    "molecules": "aa, MitoTracker"
  },
  {
    "caption": "(E)  Cytoplasm-mitochondrial fractionation assay  was performed to determine the subcellular location of GDH1 with or without aa  starvation. The fractionation  efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.",
    "molecules": "aa"
  },
  {
    "caption": "(G) Immunofluorescence (IF) assay was performed to determine the subcellular location of WT or   mitochondrial  signal peptide deleted  (dMSP) GDH1. GDH1 localization was indicated by GDH1 antibody and MitoTracker  (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.",
    "molecules": "MitoTracker"
  },
  {
    "caption": "(D) FLAG-tagged GDH1 (including WT and mutants with different  lysine mutations to  arginine) was co-transfected with  HA-tagged ubiquitin for 48 hours, and then cells were subjected to  aa  starvation for 12 hours before collecting cells.  WB and qPCR separately tested the levels of GDH1  protein and mRNA, respectively. Data information: The data are represented as the mean ± SEM of three independent experiments. The one-way ANOVA test (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).",
    "molecules": "aa, arginine, lysine"
  },
  {
    "caption": "(H) 786-0 or 769-P cells stably expressing shGDH1  were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time. Cell viability was determined. Unpaired two-tailed t-test was used. Data information: The data are represented as the mean ± SEM of three independent experiments. The unpaired t-test were used (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).",
    "molecules": "aa"
  },
  {
    "caption": "786-0 cells were treated with MG132  (10 μM) under aa  starvation. Antibodies against GDH1  (C) were used to enrich  protein complexes in cells with or without GDH1. IgG was used as a control. WB was performed using the indicated antibodies.",
    "molecules": "aa, MG132"
  },
  {
    "caption": "(H) 786-0  or 769-P cells (WT or stably expressing shGDH1) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times. Cell viability  was determined. Unpaired t-test was used with ***p < 0.001.",
    "molecules": "aa"
  },
  {
    "caption": "786-0  cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. In the above cells in (A), qPCR was used to measure the mRNA of the indicated RP genes (C).",
    "molecules": "aa"
  },
  {
    "caption": "(J) KIRC clinical samples were collected for  αKG  measurement, and we assessed overall survival (left panel) with log-rank test according to the dividing groups of high and low intracellular levels of   αKG with the best separation (right panel). Unpaired t-test was used in the right panel with ***p < 0.001.",
    "molecules": "αKG"
  },
  {
    "caption": "(G) An in vitro demethylation assay was performed with recombinant  KDM4A (0.05 mM) or  KDM6A  (0.05 mM), and the indicated histone H3 lysine methylation  peptides under 50-μM  αKG conditions (no αKG was used as a blank control). Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system.",
    "molecules": "2HG, αKG, lysine"
  },
  {
    "caption": "(H) 786-0  cells were supplemented with or without 2HG (500 μM) for 48 hours, and the cells were fixed with 1%  formaldehyde. ChIP assays were performed using antibodies against H3K9me3  or H3K27me3. qPCR was used to test the enrichment of the above  histone H3 lysine  modifications on the promoters of the indicated RP genes.",
    "molecules": "2HG, formaldehyde, lysine"
  },
  {
    "caption": "(A) WT, Irgm1-/-, Irgm2-/- and Irgm3-/- mice (n = 4 mice/genotype) were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hours post injection (hpi) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel. Relative concentration (fold change relative mean of WT) is shown for the indicated cytokines (absolute cytokine concentrations of same experiment are shown in Fig EV1).",
    "molecules": "LPS"
  },
  {
    "caption": "(B) WT (n = 9), Irgm1-/- (n = 7), Irgm2-/- (n = 9) and Casp1-/-Casp11-/- (n = 7) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, IL-18, and TNFα was measured via ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) qPCR measurement of IL-1β, and TNF-α mRNA levels in WT and Irgm2-/- BMMs following 8-hour stimulation with LPS (1 µg/ml).",
    "molecules": "LPS"
  },
  {
    "caption": "(B) WT, Irgm2-/- and Casp1-/-Casp11-/- BMMs were treated for 24 hours with LPS at indicated doses and supernatant TNFα, IL-1β, and IL-18 was measured by ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) IFNγ-primed WT and Irgm2-/- BMMs were treated with LPS, Pam3CSK4, poly(I:C) or a combination of Pam3CSK4 and poly(I:C) (1 µg/ml for all treatments) for 24 hours and cell supernatant IL-1β and IL-18 concentrations were assessed by ELISA.",
    "molecules": "LPS, Pam3CSK4, poly(I:C)"
  },
  {
    "caption": "(D) WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and IL-1β, IL-18, and LDH release were assessed at 24 hours post treatment (hpt).",
    "molecules": "LPS"
  },
  {
    "caption": "(E) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 hours and subsequently stained with anti-ASC antibody and Hoechst stain (DNA/nuclei). Representative images are shown with white arrows pointing at ASC specks. Number of ASC specks per nuclei was quantified. Scale bars: 20 µm.",
    "molecules": "Hoechst stain, LPS"
  },
  {
    "caption": "(F) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) for 24 h and cell lysates and supernatants collected. Protein levels in cell lysates (caspase-1, and actin) and supernatants (caspase-1 p20) were visualized via immunoblotting.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) IFNγ primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 h. Following treatment, cells were stained with anti-ASC antibody and Hoechst (DNA/nuclei). Representative images of ASC specks (white arrows point at specks) are shown and number of ASC specks per nuclei quantified. Scale bars: 20 µm.",
    "molecules": "Hoechst, LPS"
  },
  {
    "caption": "(B) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hours-post treatment (hpt).",
    "molecules": "LPS"
  },
  {
    "caption": " (C) WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hpt. ",
    "molecules": "LPS"
  },
  {
    "caption": " (D) IFNγ-primed WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml) for 24 hours and cell lysates and supernatants collected. Protein levels in cell lysates (Caspase-1, and actin) and supernatants (Caspase-1 p20) were visualized via immunoblotting. ",
    "molecules": "LPS"
  },
  {
    "caption": " (A) WT, Irgm2-/-, and Nlrp3-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin for 1 h and IL-1β/LDH release was measured. ",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": " (B) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin and/or MCC950 for 1 h and IL-1β/LDH release was measured. ",
    "molecules": "LPS, MCC950, nigericin"
  },
  {
    "caption": " (C) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and/or MCC950 for 24 h and IL-1β/LDH release was measured. ",
    "molecules": "LPS, MCC950"
  },
  {
    "caption": " (D) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt. ",
    "molecules": "lipofectamine, LPS"
  },
  {
    "caption": " (E) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were co-treated with LPS (indicated doses) and Listeria monocytogenes (MOI 10) and LDH release measured at 4 hpt. ",
    "molecules": "LPS"
  },
  {
    "caption": " (A) WT (n = 9), Irgm2-/- (n = 10), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 8) mice were injected i.p. with LPS (8 mg/kg). Morbidity and mortality were observed for 48 h at 3 hour intervals. ",
    "molecules": "LPS"
  },
  {
    "caption": " (B) WT (n = 9), Irgm2-/- (n = 8), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 10) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, and IL-18 was measured by ELISA. Data shown are from 2 pooled experiments.",
    "molecules": "LPS"
  },
  {
    "caption": " (A) WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 24 hpt (n = 3 independent experiments). (B) IFNγ-primed WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 8 hpt/ hpi (n = 3 independent experiments). ",
    "molecules": "LPS"
  },
  {
    "caption": " (C) WT (n = 15), Irgm2-/- (n = 12), and panIrgm-/- (n = 13) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β and TNFα was measured via ELISA. Data shown are from 3 pooled experiments. ",
    "molecules": "LPS"
  },
  {
    "caption": " (D) WT, Irgm2-/-, panIrgm-/- and Casp11-/- mice (n = 8 mice/genotype) were injected i.p. with LPS (8 mg/kg body weight). Morbidity and mortality were observed for 48 h at 3-h intervals. ",
    "molecules": "LPS"
  },
  {
    "caption": "A,B Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wildtype and 6 KO mice (B).",
    "molecules": "FCCP, glucose, oligomycin"
  },
  {
    "caption": "D,E Seahorse analysis of basal and maximal ECAR (D) or OCR (E) using wildtype or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right).",
    "molecules": "DMSO, Rapa, rapamycin, Torin 1"
  },
  {
    "caption": "F,G Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (-) of rapamycin (10 nM) and Torin 1 (100 nM).",
    "molecules": "rapamycin, Torin 1"
  },
  {
    "caption": "H qRT-PCR analysis of Ifng gene expression in naïve wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (-) or rapamycin.",
    "molecules": "rapamycin"
  },
  {
    "caption": "I-K Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wildtype or Peli1-KO OT-I CD8 T cells (4 x 105).",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with insulin (B).",
    "molecules": "insulin"
  },
  {
    "caption": "Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with insulin (D)",
    "molecules": "insulin"
  },
  {
    "caption": "Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype and Peli1-KO CD8 T cells that were activated for 1 h with anti-CD3 and anti-CD28 and then treated with cycloheximide (CHX) for the indicated time periods. Data are presented as a representative blot (B)",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1-KO CD8 T cells stimulated with anti-CD3 plus anti-CD28 for 1 h and then incubated with (+) or without (-) cycloheximide (CHX) and/or MG132 for 2 h. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.",
    "molecules": "CHX, cycloheximide, MG132"
  },
  {
    "caption": "Ubiquitination analysis of immunoprecipitated TSC2 (E) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "Ubiquitination analysis of immunoprecipitated TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "Analysis of ubiquitin conjugation to TSC1 (G, H) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "Analysis of ubiquitin conjugation to TSC1 TSC2 (I, J) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "A Analysis of ubiquitin conjugation to wildtype (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag-tagged TSC1 and its mutants along with (+) or without (-) the indicated expression vectors.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) TSC1 WT or K30A and treated with MG132 for 2 h before lysis.",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "F Immunoblot analysis of endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with Myc-TSC1 wildtype or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "C. Profile of Ca2+ responses induced by oocyte injection with spermatozoa from WT and Iqcn-/- male mice (n = 5). Spermatozoa from WT male mice induced 8-9 Ca2+ spikes over a 3-h period (blue line), while spermatozoa from Iqcn-/- male mice failed to induce spikes (red line).",
    "molecules": "Ca2+"
  },
  {
    "caption": "C. Immunostaining of calmodulin (red) and α-tubulin (green) in HeLa cells. The normal (WT) and mutant (c.910C>T and c.2453_2454del) plasmids of IQCN were transfected into HeLa cells. HeLa cells were treated with an antagonist of calmodulin W-7. Multipolar spindles were observed in HeLa cells transfected with mutant plasmids and W-7 treatment.",
    "molecules": "W-7"
  },
  {
    "caption": "(B-F) Basal views through living SCs, with dashed white circles approximating the outline of a single SC, and acidic compartments marked by the vital dye LysoTracker® Red (magenta). In merge images, a single non-acidic (C, D, F) and acidic (E) compartment containing intraluminal vesicles (ILVs) is boxed and magnified in the right panel (Zoom). ILVs appear as membrane-delineated vesicles, using super-resolution 3D-structured illumination (3D-SIM) microscopy for the brighter overexpressed GFP-tagged constructs (yellow; C and F). However, ILVs appear only as puncta, using lower resolution wide-field microscopy for the fainter endogenously expressed YFP-tagged Rab GTPases (yellow; D and E). (B) Wide-field fluorescence image, including differential interference contrast (DIC), of SC expressing a GFP-tagged version of human CD63 (CD63-GFP). CD63-GFP expression is apparent on the limiting membranes of non-acidic compartments and their ILVs, and also on the limiting membranes of the enlarged acidic compartments. Most large non-acidic compartments are Rab11-positive (D) and contain dense-core granules, which have a 'fried egg' appearance with DIC (arrowheads) (Corrigan et al., 2014; Redhai et al., 2016). (C) 3D-SIM image of CD63-GFP-expressing SC. Arrow highlights CD63-GFP-marked ILVs (Zoom). Many more ILVs are apparent in non-acidic compartments in a complete Z-stack of a non-acidic compartment (Movie EV1). (D) Wide-field fluorescence image of an SC expressing a YFP-Rab11 gene trap. YFP-Rab11 marks the limiting membranes of most non-acidic compartments and internal puncta (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1B). (E) Wide-field fluorescence image of SC expressing a YFP-Rab7 gene trap. YFP-Rab7 marks the limiting membranes of acidic compartments and internal puncta (arrow in Zoom). Enlarged acidic compartments are also present in adjacent main cells. (F) 3D-SIM image of SC expressing a GFP-tagged version of Breathless (Btl-GFP). Btl-GFP marks the limiting membranes of non-acidic compartments and their ILVs (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1C). Images from six-day-old male flies shifted to 29°C at eclosion. This induces GAL4/UAS-dependent SC transgene expression in (B), (C) and (F). The genotypes of flies carrying multiple transgenes are: w; P[w+, UAS-CD63-GFP] P[w+, tub-GAL80ts]/+; dsx-GAL4/+ (B, C); w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] (F).",
    "molecules": "LysoTracker® Red"
  },
  {
    "caption": "(A-D) Wide-field fluorescence images of basal views through living SCs expressing a GFP-tagged form of Btl-GFP (yellow) in non-acidic compartments, with SC outline approximated by dashed white circles. Acidic compartments are marked by LysoTracker Red® (magenta). Boxed non-acidic compartments in merge images are magnified in A-D, Zoom. On the right, lower magnification confocal transverse images of fixed accessory gland (AG) lumens are shown with dotted lines indicating the basal side of the AG epithelial layer, which contains several fluorescent SCs (highlighted by arrows). (A) SC with no RNAi construct expressed (control) and AG lumen from same genotype. Btl-GFP-positive compartments containing fluorescent ILVs (in Btl-GFP panel), ILV membranes inside compartments (in Zoom panel) and secreted fluorescent puncta (AG lumen panel) are marked by arrowheads. (B) SC also expressing RNAi construct targeting ESCRT-0 subunit, Stam, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (C) SC also expressing RNAi construct targeting ESCRT-I subunit, Vps28, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (D) SC also expressing RNAi construct targeting ESCRT-III subunit, shrb, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta are strongly reduced. Genotypes are: w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] with no knockdown construct (A), UAS-Stam-RNAi (HMS01429; B), UAS-Vps28-RNAi (v31894; C) or UAS-shrb-RNAi (v106823; D).",
    "molecules": "LysoTracker Red"
  },
  {
    "caption": "(A-D) Confocal images of fixed HCT116 cells, with boxed regions enlarged to the right. DAPI (grey) marks nucleus. Rab11a antibody is isoform-specific except in (F). (A) Rab11a (yellow) is located in compartments distinct from the late endosomal and lysosomal marker, LAMP2 (magenta). (B) CD63 (magenta) predominantly co-localises with the late endosomal and lysosomal marker, LAMP1 (yellow). (C) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-replete conditions. (D) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-depleted conditions.",
    "molecules": "DAPI, glutamine"
  },
  {
    "caption": "(E) Super-resolution 3D-SIM image of fixed HCT116 cell expressing GFP-Rab11a (yellow), and stained with CD63 (magenta). DAPI (grey) marks nucleus. Boxed Rab11a-positive compartments, which frequently cluster, are magnified in Merge Zoom. This panel is further magnified in Merge Zoom x 2, revealing GFP-Rab11a (arrows in right panel) inside compartments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Wide-field fluorescence image of fixed HCT116 cells, stained with Rab11a (yellow) and Rab7 (magenta), expressing constitutively active GFP-tagged Rab5 (GFP-Rab5CA; cyan), which stalls endosomal maturation and produces enlarged Rab5-positive endosomes. One of these is boxed in the Merge and magnified in Merge Zoom, revealing internal puncta marked by Rab11a (arrows) and Rab7 (arrowheads) and limiting membrane subdomains of Rab11a (yellow arrowhead) and Rab7 (magenta arrowhead). DAPI (grey) marks nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Western blot analysis of putative exosome markers in lysates from HCT116 cells cultured in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) medium for 24 h. Gel was loaded with equal amounts of protein (total cell lysate protein was reduced by 19 ± 4 % after glutamine depletion). The activity of mTORC1 was assessed via phosphorylation of S6 and 4E-BP1, using phospho-specific antibodies and a pan-4E-BP1 antibody, where the hyper-phosphorylated form of 4E-BP1 produced by mTORC1 gives the slowest migrating γ band. Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates.",
    "molecules": "glutamine"
  },
  {
    "caption": "Western blot analyses of EV preparations. (B) EVs isolated by ultracentrifugation (UC) of medium from HCT116 cells cultured in glutamine-replete and -depleted conditions for 24 h, and loaded according to cell lysate protein levels to compare secretion on a per cell basis, as shown in bar chart from four biological replicates.",
    "molecules": "glutamine"
  },
  {
    "caption": "Western blot analyses of EV preparations. (C) EV preparation isolated by size-exclusion chromatography (SEC) from glutamine-depleted HCT116 cells and then separated by high-resolution iodixanol-PBS density centrifugation. Note that exosomes (exos) and microvesicles (MVs) are found in only partially overlapping fractions and the pattern of Rab11a separation mirrors that of exosome markers (primarily 1.064 - 1.085 g/ml).",
    "molecules": "glutamine, iodixanol"
  },
  {
    "caption": "Western blot analyses of EV preparations. (D) EVs isolated by UC under glutamine-depleted conditions as in (B) and then subjected to Proteinase K (Prot K) digestion in the presence or absence of Triton® X-100 (Triton-X). Only membrane-associated CD81 is digested in the absence of Triton® X-100, while CD63 is resistant to digestion, even in the presence of Triton® X-100.",
    "molecules": "glutamine, Triton-X, Triton® X-100"
  },
  {
    "caption": "Western blot analyses of EV preparations. EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD63 antibodies coupled to magnetic beads. This method only pulls down a fraction of CD63, so the protein from pull-down of eight times the input is loaded for comparison. Approximately the same low levels of Rab11a are captured by control IgG and anti-CD63 beads.",
    "molecules": "glutamine"
  },
  {
    "caption": "Western blot analyses of EV preparations. (F) EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD81 antibodies coupled to magnetic beads. All or most CD63 and CD81 appears to be pulled down, but less than 10% of Rab11a and Cav-1 is captured. NTA analysis suggests about 10% of all particles are pulled down with this approach.",
    "molecules": "glutamine"
  },
  {
    "caption": "Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (A) EVs isolated by UC of medium from HeLa cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24 h (see Appendix Fig S7A'' for analysis of SEC-isolated EVs).",
    "molecules": "glutamine"
  },
  {
    "caption": "Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (B) EVs isolated by size-exclusion chromatography (SEC; fractions two to seven) from LNCaP cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24",
    "molecules": "glutamine"
  },
  {
    "caption": "Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (C) EVs isolated by SEC from LNCaP cells cultured in the presence or absence of 120 nM Torin 1 for 24 h.",
    "molecules": "Torin 1"
  },
  {
    "caption": "Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (E) EVs isolated by SEC from HCT116 cells cultured in the presence or absence of 3 µM Akt inhibitor AZD5363 for 24 h.",
    "molecules": "AZD5363"
  },
  {
    "caption": "Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. (F) EVs isolated by SEC from HCT116 cells cultured under glutamine-replete conditions in the presence or absence of 10 µM KRAS inhibitor BI-2852 (or control compound Bl2853) for 24 h.",
    "molecules": "Bl2853, BI-2852, glutamine"
  },
  {
    "caption": "(A) Growth curves for HCT116 recipient cells in reduced (1%) serum conditions following 30 min pre-incubation with EVs isolated by UC (104 per cell; top) or by SEC (4 x 103 per cell; bottom) from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Fold change in confluency is a measure of cell area occupying well relative to zero time, as measured by IncuCyte software.",
    "molecules": "glutamine"
  },
  {
    "caption": "(B) Cumulative tube length for HUVEC recipient cells following treatment with 104 EVs isolated by UC from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Both EV preparations promote tubulation, but the network is more stable with EVs from glutamine-depleted HCT116 cells.",
    "molecules": "glutamine"
  },
  {
    "caption": "(C) Western blot analysis of EVs isolated by UC from HCT116 cells cultured in glutamine-replete and -depleted medium for 24 h, following transduction with a Rab11a or control non-targeting (NT) shRNA knockdown construct over previous two days. Bar chart shows change in putative exosome proteins in EVs secreted from Rab11a knockdown cells relative to NT-treated cells in glutamine-depleted conditions, following initial normalisation to cell lysate protein. (C') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (C) or with vehicle (PBS). ****P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and Rab11a knockdown cells (red).",
    "molecules": "glutamine"
  },
  {
    "caption": "(D) Left hand group of four images show cells grown in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) conditions for 24 h, then incubated with an anti-CD63 antibody (yellow) for 30 min at 4oC, washed with PBS, chased at 37oC for 30 min, then fixed, immunostained and imaged. Right hand group of four images show cells grown in glutamine-replete and glutamine-depleted conditions for 24 h, incubated with Tfn-Alexa Fluor® 488 (yellow) for 30 min at 4oC, shifted to 37oC for 30 min, then washed, immediately fixed and imaged.",
    "molecules": "Alexa Fluor® 488, glutamine"
  },
  {
    "caption": "(E) Western blot showing levels of anti-CD63 heavy chain and biotin-conjugated Tfn in HCT116 cells cultured for 24 h in glutamine-replete or -depleted conditions, incubated for 30 min in medium containing these molecules at 4oC, then chased at 37oC for 15 min (the chase step was not performed for Tfn in D, to reduce loss due to rapid recycling of Tfn).",
    "molecules": "biotin, glutamine"
  },
  {
    "caption": "(F) Western blot analysis of EVs isolated by UC from HCT116 cells in glutamine-replete medium, transduced with a Rab7 or non-targeting (NT) control shRNA knockdown construct. Bar charts show changes in putative exosome proteins in EVs isolated by ultracentrifugation, following normalisation to cell lysate protein. (F') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (F) or with vehicle (PBS).",
    "molecules": "glutamine"
  },
  {
    "caption": "(A) Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete or -depleted HCT116 cells or vehicle (PBS). Bar chart shows ratio of p-ERK to ERK levels from triplicate independent experiments.",
    "molecules": "glutamine"
  },
  {
    "caption": "(B) Bar chart shows HCT116 recipient cell growth over 120 h, after pre-treatment with EV preparations isolated as in (A) or with PBS, and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 (1.00 µM).",
    "molecules": "SCH772984"
  },
  {
    "caption": "(C) Western blot analysis showing levels of the EGFR ligand, Amphiregulin (AREG) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine-replete (2.00 mM) or -depleted (0.15 mM) conditions for 24 h. Gel loading was normalised to cell lysate protein levels. AREG's molecular weight (approximately 26-28 kDa) suggests it is in its membrane-associated form (see Appendix Fig S8D). In the bar chart, the levels of putative exosome proteins were normalised to cell lysate protein levels. Significantly decreased levels are in blue and increased levels are in red.(C') Growth curves in low (1%) serum are for HCT116 recipient cells pre-treated with the EV preparations [isolated as in (C)], which had themselves been pre-treated with and without anti-AREG neutralising antibody or with a control IgG. Solid red line shows growth-promoting effect of EVs isolated under glutamine depletion, which is blocked by anti-AREG antibody (red dashed line). ***P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and anti-AREG-treated cells (red). (C'') Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete (2.00 mM) or -depleted (0.15 mM) HCT116 cells or vehicle (PBS), which were pre-treated with anti-AREG (AREGab) or a control immunoglobulin (Ig). Note increase in ERK phosphorylation using EVs from glutamine-depleted cells, which is blunted by the addition of the anti-AREG antibody.",
    "molecules": "glutamine"
  },
  {
    "caption": "HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (A) Sections immunostained for CD31, which labels endothelial cells and blood vessels, with blood vessel number (upper) and total area (lower) represented in bar charts.",
    "molecules": "glutamine"
  },
  {
    "caption": "HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (B) Sections immunostained for Ki67, which stains proliferative cells. Proportion of tumour cells with Ki69 staining is represented in bar chart.",
    "molecules": "glutamine"
  },
  {
    "caption": "HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (C) Sections stained with haematoxylin and eosin, which highlights necrotic regions (pale staining). Proportion of tumour area that is necrotic is represented in bar chart.",
    "molecules": "eosin, glutamine, haematoxylin"
  },
  {
    "caption": "HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (D) Sections immunostained for CA9, which is expressed in hypoxic regions. Proportion of tumour cells with CA9 staining is represented in bar chart.",
    "molecules": "glutamine"
  },
  {
    "caption": "(A) Heatmap of tRNA profiling of MCF10A cells at 8 and 24 hours post exposure to oxidative stress (200μM H2O2). Biological triplicate data is depicted at each time point relative to control cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "(B) MCF10A cells were exposed to oxidative stress (200μM H2O2) and processed for small RNA-sequencing. The log2-fold induction levels for tRFs derived from distinct tRNA isoacceptor is plotted.",
    "molecules": "H2O2"
  },
  {
    "caption": "(A) A northern blot depicting a time course experiment ranging from five minutes to 24 hours of MCF10A cells in response to oxidative stress. A single probe complementary to pre-tRNATyrGUA, mature tRNATyrGUA, and tRFTyrGUA expression was 32P-labeled and used for detection. (B) Quantification of pre-tRNATyrGUA (left) and mature tRNATyrGUA levels (right) by northern blot analysis from multiple independent experiments (normalized to U6 levels) are shown (n=6). ",
    "molecules": "32P, tRFTyrGUA, tRNATyrGUA"
  },
  {
    "caption": "(C) MCF10A cells were exposed to oxidative stress (200μM H2O2) once daily for five continuous days. (D) Quantification of mature tRNATyrGUA bands by northern blot after cells were treated once daily for five continuous days (normalized to U6) from multiple independent experiments (n=12). ",
    "molecules": "H2O2, tRNATyrGUA"
  },
  {
    "caption": "(E) Quantification of northern blot analysis for pre-tRNATyrGUA (left) and tRNATyrGUA (right) after one hour and 24 hours respectively in HBEC30 cells upon exposure to oxidative stress (200μM H2O2) as in (A) (n=6).",
    "molecules": "H2O2, tRNATyrGUA"
  },
  {
    "caption": "(A) Growth curves of MCF10A cells exposed to oxidative stress (200μM H2O2) relative to control cells (n=3). Two-way ANOVA was used to test for significance.",
    "molecules": "H2O2"
  },
  {
    "caption": "(B) Northern blot of MCF10A cells expressing a control short-hairpin RNA or a hairpin targeting tRNATyrGUA.",
    "molecules": "tRNATyrGUA"
  },
  {
    "caption": "(D) Growth curves of MCF10A cells expressing RNAi targeting mature tRNATyrGUA or YARS relative to cells expressing a control hairpin (n=3). Two-way ANOVA was used to test for significance.",
    "molecules": "tRNATyrGUA"
  },
  {
    "caption": "(E) Cell growth of MCF10A cells transiently transfected with a tRNATyrGUA overexpression vector relative to an empty control vector (n=3). A one-tailed Mann-Whitney test was used to test for significance at day 3.",
    "molecules": "tRNATyrGUA"
  },
  {
    "caption": "(A) Cells depleted of tRNATyrGUA or YARS were processed for label free quantitation by mass spectrometry to identify proteins that were reduced by a log2-fold change of 0.5 or more. This set was overlapped with proteins containing a higher than median abundance of Tyr codon content to identify candidate mediators of the pleiotropic effects of tRNATyrGUA depletion.",
    "molecules": "tRNATyrGUA, Tyr"
  },
  {
    "caption": "(C) Quantitative western blot validation depicting abundances of protein targets (EPCAM, SCD, and USP3) identified from (A). HSC70 was used as a loading control and is not modulated upon molecular perturbation of tRNATyrGUA. (D) Quantification of western blot analysis in (C) (n=4). A one-tailed Mann-Whitney test was used to test for statistical significance. ",
    "molecules": "tRNATyrGUA"
  },
  {
    "caption": "(A) Quantitative western blot EPCAM, SCD, and USP3 in MCF10A cells 24 hours after treatment with H2O2 (200μM). HSC70 was used as a loading control. (B) Quantification of western results in (A) (n=9). A one-tailed Mann-Whitney test was used to test for statistical significance. ",
    "molecules": "H2O2"
  },
  {
    "caption": "(E) Ribosome occupancy of 20-22nt RPFs in tRNATyrGUA-depleted cells compared to control cells reveal greater occupancy at both tyrosine codons in tRNATyrGUA-depleted cells using a Wilcoxon test.",
    "molecules": "tRNATyrGUA, tyrosine"
  },
  {
    "caption": "(A-B) Quantification of tRFTyrGUA induction in response to oxidative stress as a function of time in MCF10A (A) (n=4) and in HBEC30 (B) (n=6) with mean ± s.e.m shown for each cell line.",
    "molecules": "tRFTyrGUA"
  },
  {
    "caption": "(C) Volcano plot of mass spectrometry results from a synthetic 5'-biotinylated tRFTyrGUA co-precipitation experiment with cell lysate. Log2 fold enrichment values of proteins identified from tRFTyrGUA relative to scrambled tRF control samples.",
    "molecules": "tRFTyrGUA"
  },
  {
    "caption": "(D) Western blot validation of mass spectrometry results for three of the top hits in (A), showing co-precipitation of endogenous proteins with transfected tRFTyrGUA relative to a scrambled control sequence (Scr).",
    "molecules": "tRFTyrGUA"
  },
  {
    "caption": "(B) IGV plots from an hnRNPA1 HITS-CLIP (Huelga et al., 2012) reveals interactions with tRFTyrGUA.",
    "molecules": "tRFTyrGUA"
  },
  {
    "caption": "(C) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion. SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA. (D) Similar to the IGV plots shown in (C), but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion. ",
    "molecules": "tRFTyrGUA, tRNATyrGUA"
  },
  {
    "caption": "(E) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3' UTR hnRNPA1 CLIP binding (Huelga et al., 2012). Transfection of tRFTyrGUA led to a significant right-shift in the expression levels of 3' UTR bound hnRNPA1 transcripts. Statistical significance was measured using the Kolmogorov-Smirnov test. (F) Cumulative distribution as in (E). Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left-shift in mRNA stability of 3'UTR bound hnRNPA1 transcripts. Statistical significance was assessed using the Kolmogorov-Smirnov test. ",
    "molecules": "tRFTyrGUA, H2O2"
  },
  {
    "caption": "(G, H) Metabolic rate of O2 consumption (G) and CO2 production (H) normalized to lean mass for a 48-hour cycle of 4-6-month-old WT (n=8) and Prmt5MKO (n=7) mice measured by an indirect calorimetry.",
    "molecules": "CO2, O2"
  },
  {
    "caption": "(A, B) Representative images of ORO staining (A) and immunofluorescence (B) in TA muscle from WT and Prmt5MKO mice. Scale bar: 100 μm.",
    "molecules": "ORO"
  },
  {
    "caption": "(F) OCR was measured at basal state and after sequential addition of Oligomycin, FCCP, and Rotenone/Antimycin A to determine basal respiration (BS), proton leak (PL), ATP respiration (ATP R), maximal respiration (Max R), spare capacity (SC), and non-mitochondrial respiration (NR); (n=4, technical replicates).",
    "molecules": "Antimycin A, FCCP, Oligomycin, Rotenone"
  },
  {
    "caption": "(E) HEK293 cells were transfected with PRMT5-GFP alone or PRMT5-GFP + SREBP1a-Flag for 24 hours, followed by cycloheximide (30 μg/ml) and protein analysis at 0, 4, 8 hours. Lysates were immunoblotted with Flag, GFP, and tubulin antibodies.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(I) Representative images of ORO staining in TA muscles under brightfield (upper panel) and fluorescent imaging (bottom panel) from WT, Prmt5MKO, and Prmt5/Pnpla2MKO mice. Scale bar: 50 μm.",
    "molecules": "ORO"
  },
  {
    "caption": "(b) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28).",
    "molecules": "SMER18, SMER10, SMER28, DMSO"
  },
  {
    "caption": "(c) COS-7 cells transfected with EGFP-HDQ74 construct were treated with DMSO (control), 0.2 μM rapamycin (Rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, and we assessed the proportions of apoptotic or aggregate-containing transfected cells. Error bars: 95% confidence interval. P 0.0001 (Rap and SMER28), P = 0.013 (SMER10), P = 0.019 (SMER18) (aggregation); P 0.0001 (Rap, SMER18 and SMER28), P = 0.004 (SMER10) (cell death).",
    "molecules": "SMER18, SMER10, SMER28, DMSO, rapamycin"
  },
  {
    "caption": "(d) Wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs were transfected with EGFP-HDQ74 and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h, and we assessed the percentages of aggregate-containing transfected cells. The control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95% confidence interval. P = 0.039 (SMER10), P 0.0001 (SMER18 and SMER28) (in Atg5+/+ cells); P = 0.092 (SMER10), P = 0.271 (SMER18), P = 0.358 (SMER28) (in Atg5−/− cells). Note that EGFP-HDQ74 aggregation was higher in Atg5−/− than in Atg5+/+ cells (Supplementary Fig. 3a).",
    "molecules": "SMER18, SMER10, SMER28, DMSO"
  },
  {
    "caption": "e) The percentages of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor) or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, expressed as odds ratios. Error bars: 95% confidence interval. P 0.0001 (control versus Lact), P = 0.014 (SMER10 versus Lact), P 0.0001 (SMER18 versus Lact), P = 0.001 (SMER28 versus Lact). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.",
    "molecules": "SMER18, SMER10, SMER28, DMSO, lactacystin"
  },
  {
    "caption": "(a) COS-7 cells transfected with EGFP-LC3 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (positive control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 16 h, and analyzed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. Error bars denote s.e.m. P 0.0001 (all SMERs).",
    "molecules": "SMER18, SMER10, SMER28, DMSO, rapamycin"
  },
  {
    "caption": "(b) HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM.",
    "molecules": "SMER18, SMER10, SMER28, DMSO"
  },
  {
    "caption": "(c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (Baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (above) and densitometry analysis relative to actin (below). Error bars denote s.e.m. P = 0.0259 (baf), P 0.0001 (SMER10), P = 0.0003 (SMER18 and SMER28) versus control; P = 0.0025 (SMER10), P = 0.0218 (SMER18), P = 0.0195 (SMER28) versus bafilomycin A1. ***P 0.001; **P 0.01; *P 0.05.",
    "molecules": "SMER18, SMER10, SMER28, bafilomycin A1, DMSO"
  },
  {
    "caption": "(a-c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO (control) alone (2 d after eclosion). Rhabdomere counts from all three independent experiments are included. n = 600 ommatidia (SMER10), n = 1,500 ommatidia (SMER18) and n = 600 ommatidia (SMER28). Mann-Whitney test values P 0.0001 (all SMERs). Student's t-test (1-tailed) P = 0.005 (SMER10), P = 0.004 (SMER18), P = 0.03 (SMER28) comparing means of distributions from independent experiments. These SMER concentrations cause no overt toxicity to flies (see Supplementary Methods). Distributions of DMSO-treated flies may vary when SMERs are treated in different experiments at different times. For instance, an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time-dependent.",
    "molecules": "SMER18, SMER10, SMER28, DMSO"
  },
  {
    "caption": "(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (a), 43 μM SMER18 (b) or 47 μM SMER28 (c)) or both for the 8 h switch-off period-was analyzed by immunoblotting with antibody against HA (above) and densitometry analysis relative to actin (below). The concentration of rapamycin is saturating for its effect on the clearance of A53T α-synuclein. Error bars denote s.e.m. P = 0.0025 (Rap), P = 0.0018 (SMER10), P 0.0001 (SMER10 + Rap), P 0.0001 (Rap or SMER10 versus SMER10 + Rap) (a); P = 0.0069 (Rap), P = 0.0498 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.0038 (Rap versus SMER18 + Rap), P = 0.0007 (SMER18 versus SMER18 + Rap) (b); P 0.0001 (Rap, SMER28, Rap versus SMER28 + Rap, SMER28 versus SMER28 + Rap) (c).",
    "molecules": "SMER18, SMER10, SMER28, DMSO, rapamycin"
  },
  {
    "caption": "(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (above) and cell death (below) in COS-7 cells, as in Figure 1c-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)) or both for 24 h-were expressed as odds ratios. Error bars: 95% confidence interval. Aggregation (top panels): P = 0.248 (Rap), P = 0.217 (SMER10), P 0.0001 (SMER10 + Rap), P 0.001 (Rap or SMER10 versus SMER10 + Rap) (d); P = 0.248 (Rap), P = 0.543 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.008 (Rap versus SMER18 + Rap), P = 0.002 (SMER18 versus SMER18 + Rap) (e); P = 0.248 (Rap), P = 0.002 (SMER28), P 0.0001 (SMER28 + Rap), P 0.0001 (Rap versus SMER28 + Rap), P = 0.012 (SMER28 versus SMER28 + Rap) (f). Cell death (bottom panels): P = 0.002 (Rap), P 0.0001 (SMER10, SMER10 + Rap, Rap or SMER10 versus SMER10 + Rap) (d); P = 0.002 (Rap), P = 0.948 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.015 (Rap versus SMER18 + Rap), P 0.0001 (SMER18 versus SMER18 + Rap) (e); P = 0.002 (Rap), P 0.0001 (SMER28, SMER28 + Rap, Rap or SMER28 versus SMER28 + Rap) (f). Note that we have treated cells for a shorter time in this experiment (24 h), compared with Figure 1c (48 h). This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.",
    "molecules": "SMER18, SMER10, SMER28, DMSO, rapamycin"
  },
  {
    "caption": "(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c)-was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below). All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars denote s.e.m. P = 0.0058 (SMER10a), P = 0.6736 (SMER10b), P = 0.9507 (SMER10c), P = 0.0481 (SMER10) (a); P = 0.0006 (SMER18a), P = 0.0249 (SMER18b), P = 0.0167 (SMER18c), P = 0.0117 (SMER18d), P = 0.0269 (SMER18e), P = 0.0165 (SMER18f), P = 0.0148 (SMER18g), P = 0.0011 (SMER18h), P = 0.7369 (SMER18i), P = 0.0012 (SMER18j), P = 0.1531 (SMER18k), P = 0.0006 (SMER18l), P = 0.0001 (SMER18) (b); P = 0.0014 (SMER28a), P = 0.0002 (SMER28b), P = 0.0001 (SMER28d), P = 0.0048 (SMER28h), P = 0.0002 (SMER28i), P = 0.0162 (SMER28j), P 0.0001 (SMER28c, SMER28e-g, SMER28k, SMER28) (c).",
    "molecules": "SMER28j, SMER28k, SMER18j, SMER18f, SMER18e, SMER18, SMER18l, SMER18g, SMER18i, SMER18b, SMER18k, SMER18a, SMER18h, SMER18c, SMER10b, SMER10, SMER28h, SMER18d, SMER28a, SMER28, SMER28e, SMER10c, DMSO, SMER28b, SMER28c, SMER28d, SMER28i, SMER10a"
  },
  {
    "caption": "(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.",
    "molecules": "SMER28i, SMER18j, SMER18f, SMER18e, SMER18, SMER18l, SMER18g, SMER18a, SMER18h, SMER18c, SMER10, SMER28h, SMER18d, SMER28a, SMER28, SMER28e, DMSO, SMER28b, SMER28c, SMER28d, SMER10a"
  },
  {
    "caption": "(B) xTF change from baseline during the first 29 days since treatment start for each PDX patient line. xTFs were normalised to baseline (day 1) xTF values for each mouse (dashed lines). Carboplatin-treated mice are shown in purple, control mice are shown in teal. Bold lines show the linear-model fitted line across all mice within the same treatment and patient group. Horizontal dashed lines at y=1 indicate normalised baseline.",
    "molecules": "Carboplatin"
  },
  {
    "caption": "(C) Fraction of blood spot samples in which putative driver amplifications were detected over time. The fraction of samples with detected gene amplifications decreases in carboplatin treated group, while increasing in the control group over time.",
    "molecules": "carboplatin"
  },
  {
    "caption": "(D) Correlation between xTF values and tumour volumes of the nearest matched time-point for both untreated (Pearson's R = 0.45, p = 0.00018), and carboplatin-treated (Pearson's R = 0.056, p = 0.78) PDX mice.",
    "molecules": "carboplatin"
  },
  {
    "caption": "(C)  Western blot analysis of the indicated proteins in Huh7  cells, transiently transfected with HERV-R  expression plasmid, infected with the  SARS-CoV-2 and treated with U0126. (D) Densitometry  quantification of P-ERK  and N-protein band intensities (normalized to that of total-ERK  and GAPDH, respectively) from two independent blots, representative image shown in panel (C). Data information: In D, P-ERK  and N-protein band intensities were normalized to that of GAPDH  [as shown in panel (C)] and represented as mean of two independent blots. Individual values are shown as circles.",
    "molecules": "U0126"
  },
  {
    "caption": "D. Co-IP  assay of the interaction  between NLRP3  and TRIM50  after stimulation with nigericin  for 0, 15, 30 and 60 min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "nigericin"
  },
  {
    "caption": "E. Co-IP  assay of the interaction  between NLRP3  and TRIM50  in mouse peritoneal macrophages primed with LPS  for 6h and treated with ATP  for 30min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "F. Confocal microscopy  assay of the interaction  between TRIM50  and NLRP3  in THP1  cells with stimulation by LPS and ATP. Scale bars: 10μm. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "B.  qRT-PCR analysis of Nlrp3  and IL-1β mRNA levels in LPS-primed Si-Trim50  transfected mouse peritoneal macrophages. Data information: P-values are shown, two-way analysis of variance (ANOVA) (B, Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "LPS"
  },
  {
    "caption": "C, D. Immunoblot  analysis of Caspase1-P20 (C) and  Gasdermin D (D) of WT or Trim50-/- mouse peritoneal macrophages after LPS  stimulation for 6h and ATP  treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "E, Immunoblot  analysis of  Caspase1-P20 (E) of WT or Trim50-/- BMDM  cells after LPS  stimulation for 6h and ATP  treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "F. Immunoblot  analysis of Gasdermin D  (F) of WT or Trim50-/- BMDM  cells after LPS  stimulation for 6h and ATP  treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "D. Immunoblot  analysis of the ASC oligomerization  in cross-linked cytosolic  pellets (insoluble) and the whole  cell lysate (soluble) in HEK293T  cells with nigericin  treatment for 30 min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.",
    "molecules": "nigericin"
  },
  {
    "caption": "E. Immunoblot  analysis of NLRP3  protein levels in cycloheximide  (CHX) treated HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "F. Immunoblot  analysis of NLRP3  in HEK293T  cells transfected with NLRP3  plasmid and TRIM50  or TRIM50   (ΔRING) mutant, followed with the further treatment with CHX  for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "CHX"
  },
  {
    "caption": "G. Immunoblot  analysis of NLRP3  in mouse peritoneal macrophages, followed with the further treatment with CHX  for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "CHX"
  },
  {
    "caption": "B.  SDD-AGE  analysis of NLRP3 oligomerization  in NLRP3  and TRIM50   transfected HEK293T  cells followed with nigericin  treatment for 30min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "nigericin"
  },
  {
    "caption": "C. Immunoblot  analysis of ASC oligomerization  in cross-linked cytosolic  pellets (insoluble) and whole cell lysate (soluble) in HEK293T  cells transfected with NLRP3, ASC  and TRIM50   plasmids followed with nigericin   treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "nigericin"
  },
  {
    "caption": "D. SDD-AGE analysis of NLRP3 oligomerization  in WT or Trim50-/- peritoneal macrophages  with LPS  treatment for 6h and ATP  treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "E. Immunoblot  analysis of ASC oligomerization  in cross-linked cytosolic  pellets (insoluble) and whole  cell lysate (soluble) in WT or Trim50-/- peritoneal macrophages by LPS  stimulation for 6h and ATP  treatment for 30min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "F. SDD-AGE  analysis of NLRP3 oligomerization  in WT or Trim50-/- BMDM  cells with LPS  treatment for 6h and ATP  treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "G. Immunoblot  analysis of ASC oligomerization  in cross-linked cytosolic  pellets (insoluble) and whole  cell lysate (soluble) in WT or Trim50-/- BMDM  cells by LPS  stimulation for 6h and ATP  treatment for 30min. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "J. Immunoprecipitation  analysis of NLRP3 ubiquitination  in HA-ubiquitin, His-NLRP3  and Myc-TRIM50  transfected HEK293T  cells with further treatment with tranilast  or MCC950  for 24h. Data information: Data are representative of three biological replicates with similar results.",
    "molecules": "MCC950, tranilast"
  },
  {
    "caption": "E. Immunoblot  analysis of NLRP3  in HEK293T  cells transfected with NLRP3  plasmid and TRIM50   coiled-coil domain-deleted mutants, followed with the cycloheximide  (CHX) treatment for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA(E). Data are representative of three biological replicates with similar results. (mean ± SD in E).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "A. Survival  status of WT and Trim50-/- mice  after intraperitoneal injection with LPS  (n=10 mice/group). Data information: P-values are shown, two-way ANOVA (A) Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "LPS"
  },
  {
    "caption": "B. ELISA  analysis of  IL-1β, TNF-α and IL-6 in serum  of WT or Trim50-/- mice  after i.p. injection with LPS  for 6h (n=4-6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "LPS"
  },
  {
    "caption": "C. Histopathological  analysis of the lung  tissues by hematoxylin  and eosin  (H&E) staining. The quantified  lung  injury  was depicted by defined clinical parameters in ALI score (n=6 mice/group). Scale bars: 50μm. Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "D. Flow cytometry analysis of the neutrophils  (left) and  Ly6C+ monocytes  (right) in peritoneum  of WT or Trim50-/- mice  after i.p. injection with Alum. (n=6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "Alum"
  },
  {
    "caption": "E. WT or Trim50-/- mice  were i.p. injected with FA  for 36h, followed with immunoblot  analysis of NLRP3, pro-IL-1β, Caspase1 p20, IL-1β in the kidney  tissue samples. Data information: P-values are shown, two-way ANOVA (A) or two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD",
    "molecules": "FA"
  },
  {
    "caption": "G. Periodic-acid-Schiff (PAS) staining  of kidney  tissue sections. Scale bars: 20μm.",
    "molecules": "PAS, Periodic-acid-Schiff"
  },
  {
    "caption": "D-P) Immunofluorescence analysis showed amelioration of Rhodopsin localization (D-F'; white arrowheads); C-arrestin expression (G-I') and PR outer segment (OS) and inner segment (IS) structures, as determined, respectively, by PNA (J-L'); and Phalloidin staining (M-O'') in P347S/miR-181a/b+/- vs P347S eyes at p30. M''-O'' show higher magnification of M-O. Scale bars 25 μm in D-O'; 5μm in M''-O''. Fluorescence densitometry quantification of each staining is reported in (P), N=3 eye/genotype for each staining. WT vs P347S p values are reported in red, P347S vs P347S/miR-181a/b+/- p values are reported in black.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "D-L) Immunofluorescence analysis of Rhodopsin (D-E'), C-arrestin (F-G'), PNA (H-I'); and Phalloidin staining (J-K'') in P347S and P347S/miR-181a/b+/- retinas at p90. J''-K'' show higher magnification of J-K. Scale bars 25 μm in D-K'; 10μm in J''-K''.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "Immunofluorescence analysis of Rhodopsin C-arrestin PNA and Phalloidin staining in P347S and P347S/miR-181a/b+/- retinas at p90. Fluorescence densitometry quantification of each staining is reported in (L), N=3 eye/genotype/staining.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "A) WB analysis of Drp1, one of the key proteins involved in the mitochondrial fission pathway, in the optic cup of WT, P347S DMSO-treated, P347S/miR-181a/b+/- DMSO-treated and of P347S/miR-181a/b+/- FEDRATINIB-treated (an inhibitor of the JAK2/STAT3 pathway) mice at p30.",
    "molecules": "DMSO, FEDRATINIB"
  },
  {
    "caption": "B) Quantification revealed that Drp1 protein levels are increased in P347S with respect to WT, and partially rescued in P347S/miR-181a/b+/-. Data are normalized to Gapdh. N ≥ 4 eye/genotype. Please note that all compared bands from WT, P347S, P347S/miR-181a/b+/- and P347S/miR-181a/b+/- with FEDRATINIB samples are from the same blots, which were cropped and shown organized in the panel for the sake of data presentation clarity (see source data).",
    "molecules": "FEDRATINIB"
  },
  {
    "caption": "F) Quantification of WB in A reveals decreased levels of Drp1 in P347S/miR-181a/b+/- DMSO-treated vs P347S and increased levels in P347S/miR-181a/b+/- FEDRATINIB-treated ex-vivo retinas. N≥4 eyes/genotype.",
    "molecules": "DMSO, FEDRATINIB"
  },
  {
    "caption": "(C) Western blot analysis of STAT2 protein levels in mock-, WT-RCMV-E-, ΔE27-RCMV-E- or UV-inactivated RCMV-E-infected REF. 24 h p.i., cells were either left untreated or treated for 5.5 h with DMSO, 2.5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition. IE1 and β-tubulin served as infection and loading controls, respectively.",
    "molecules": "DMSO, MG132, MLN4924"
  },
  {
    "caption": "(A, B) GF analysis of in vitro reconstitutions containing the indicated protein combinations. Coomassie blue-stained SDS-PAGE analyses of fractions collected during the GF runs are shown next to the chromatograms. The red * indicates a contaminant, which we identified by cryo-EM analysis as E. coli ArnA, a notorious contaminant in E. coli Ni-NTA protein purifications (Andersen et al., 2013) (Appendix Fig S2, S3). Note that ArnA is present in GF fraction 6 (Ve ~ 10.2 ml) of all runs containing E27, and migrates similarly to E27 on the SDS-PAGE. * - additional contaminant from E27 preparation; ** - contaminant from STAT2 preparation.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.",
    "molecules": "Carbenicillin, chloramphenicol"
  },
  {
    "caption": "G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.",
    "molecules": "CAT"
  },
  {
    "caption": "G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.",
    "molecules": "Carbenicillin, chloramphenicol"
  },
  {
    "caption": "(G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.",
    "molecules": "Carbenicillin"
  },
  {
    "caption": "Time courses of swarmbots treated with varying concentrations of carbenicillin and glucose. Solid lines indicate cell densities inside swarmbots; dotted lines indicate those in the chamber. Safeguard was established for lower nutrient levels (0.004% or 0.04% glucose) along with carbenicillin treatment. At a high nutrient level (0.4% glucose), the safeguard was lost due to either growth in the chamber (panels 3 and 6) or gradual killing of the population inside swarmbot (solid line in panel 9). These results are qualitatively consistent with the model predictions (Fig 4).",
    "molecules": "carbenicillin, glucose, nutrient"
  },
  {
    "caption": "Snapshots of safeguard performance at the end time point of (A). The red arrow in panel 6 indicates significant growth in the chamber; the dimmer fluorescence of the swarmbot in panel 9 indicates killing of the population.",
    "molecules": "glucose, nutrient"
  },
  {
    "caption": "Figure 6. Experimental control of microbial swarmbot safeguard by other circuits.Demonstration of modular safeguard performance of swarmbots with different collective survival systems. For the swarmbots containing cells carrying the QS-CAT circuit, pulsing flow of medium containing 100µg/ml chloramphenicol established safeguard control but not for the static condition. For the swarmbots with the QS-BlaM circuit, the static condition of 100 µg/ml carbenicillin was sufficient for safeguard control. For all the conditions, safeguard did not occur in the absence of antibiotic (top panels from each condition). All the images were from the swarmbots cultured at 37˚C for 16 hours. The scale bar is 250 µm.",
    "molecules": "chloramphenicol"
  },
  {
    "caption": "(B) B-RafE586K signals independently from Ras-GTP, but requires an intact AL. The MEK-ERK activation potential of the indicated HA-tagged B-Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat-E cells.",
    "molecules": "GTP"
  },
  {
    "caption": "(C) B-RafCAAX but not the B-RafV600E oncoprotein signals independently from Ras-GTP, but requires an intact AL. Same experimental set-up as in (B). (D) Quantification of experiments shown in (B) and (C). The signal elicited by the individual reference proteins (B-Rafwt, B-RafE586K, B-RafV600E and B-RafCAAX) was set in each analysis to 100%. n = 3, mean + SEM, t-test, * p < 0.05, ** p < 0.01",
    "molecules": "GTP"
  },
  {
    "caption": "(A) Braf-/-;ERTmHRasG12V MEFs were reconstituted with HA-tagged hBRAF constructs or empty vector (EV). Infected cells were either treated with 4-HT (1µM) to induce oncogenic Ras activation or with ethanol (OH) as a control. (B) Quantifications of pMEK (left) and pERK (right) normed to WT level. n = 3, mean + SEM, t-test, ** p < 0.01.",
    "molecules": "ethanol, OH, 4-HT"
  },
  {
    "caption": "(C) The AVKA mutation has no discernible influence on Ras-induced paradoxical ERK activation triggered by the D594A mutation. Experimental set-up as in (A), shown are lysates from 4-HT treated MEFs. (D) Quantification of pMEK and pERK levels normed to HA expression. n = 3, mean + SEM, t-test, ** p < 0.01.",
    "molecules": "4-HT"
  },
  {
    "caption": "(F) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti-IgM stimulation. n = 3, mean + SEM, test, ** p < 0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) IP of EGF stimulated MEFs expressing B-RafWT or B-RafAVKA. Sorafenib was used to increase complex build up.",
    "molecules": "Sorafenib"
  },
  {
    "caption": "(F) Decay of TAg expression in MEFs following dox withdrawal.",
    "molecules": "dox"
  },
  {
    "caption": "Growth curves of the Tet-on Crispr-Cas9-MCF7 (left) and Tet-on Crispr-Cas9-T47D cells (right). Expression of Cas9 was induced with 5uM doxycycline after transfection of the sgRNAs. sgNC: non-targeting sgRNA as a negative control, sgHN-1: sgRNA sequence 1 for HNRNPC, sgHN-2: sgRNA sequence 2 for HNRNPC, sgLMNA: sgRNA for LMNA as another negative control. Each sample has 3 replicates. Error bars represent mean ± SD.",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "Growth curves of the MCF7 (left) or T47D (right) cells upon HNRNPC knock-down and JAK-STAT inhibition with Ruxolitinib (5uM). Error bars represent mean ± SD. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. Each sample has 3 replicates. Error bars represent mean ± SD.",
    "molecules": "Ruxolitinib"
  },
  {
    "caption": "Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC, with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green). Cells transfected with poly I:C was included as a positive control of dsRNA, and the cells treated with RNase III was used as a negative control. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. The size of scale bar is 10um.",
    "molecules": "4′,6-diamidino-2-phenylindole, DAPI, dsRNA, poly I:C"
  },
  {
    "caption": "Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC, identified in the dsRNA-enriched libraries with anti-dsRNA J2 pull-down or in the control libraries including the IgG control and the input control for dsRNA pull-down. Each bar represents the overlapping dsRNA species from two replicate experiments. Among each set of the dsRNA regions, the dsRNAs from Alus were marked in grey.",
    "molecules": "dsRNA, dsRNAs"
  },
  {
    "caption": "MA plot showing all the dsRNA regions identified in the dsRNA-enriched libraries. For each dsRNA region, mean of the read counts (log2) for two replicates of siHNRNPC and siNC cells was shown on the x-axis, and the average fold change (log2) in siHNRNPC vs. siNC on the y-axis. The up-regulated dsRNA regions were marked in red and down-regulated regions in black.",
    "molecules": "dsRNA"
  },
  {
    "caption": "A. Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p-HA in ring (R), trophozoite (T) and schizont (S) stages of P. falciparum asexual growth. PfH3p-HA was detected using anti-HA antibodies (green) and endogenous histone H3 with anti-histone H3 N-terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Nuclei isolated from wild-type (WT) or PfH3p-HA-expressing (WT + PfH3p-HA) schizont stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue (C.B.) or visualized by immunoblotting with anti-HA (α-HA) or anti-C-terminal histone H3 (α-H3c) antibodies.",
    "molecules": "agarose, DNA, ethidium bromide"
  },
  {
    "caption": ". (b) Conjugation of LC3 and two other mammalian homologues (GL1 and GL2) with phosphatidylethanolamine (PE) was performed as described in ref. . Complete reactions contain 1.5 μM Atg7, 2.5 μM Atg3, 8-12 μM LC3/GL1/GL2, 2 mM lipid (30 nm liposomes composed of 10 mol% bl-PI, 55 mol% DOPE, 0.15 mol% Rh-DOPE and 34.85 mol% POPC; see Methods for full definitions), 1 mM dithiothreitol and 1 mM ATP and were run at 30 °C for 90 min. Each reaction was run on a 12% SDS-PAGE gel and visualized by Coomassie blue stain. The mobility of the lipidated protein is faster than the corresponding non-lipidated forms. Intermediates in the reaction are also detectable, including the Atg7-GRL1 conjugate, the Atg3-GRL1 conjugate and a modified form of GRL1, GRL2 and LC3 that is labelled simply as GRL1/GRL2/LC3-intermediate and is probably the adenylated form of each LC3-related protein.",
    "molecules": "dithiothreitol, POPC, ATP, DOPE, lipid, PE, phosphatidylethanolamine, PI"
  },
  {
    "caption": "(c) The lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes. Reactions are as in b, but run on 400 nm liposomes. Liposomes are composed of the indicated amount of DOPE, 10 mol% bl-PI, 0.15 mol% Rh-DOPE and POPC.",
    "molecules": "POPC, DOPE, PI"
  },
  {
    "caption": "(b) Filling the defects with inverted cone-shaped lipids inhibits lipidation. In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol% DOPE (2 mM total lipid) as in Fig. 1. To determine whether inverted cones influenced the reaction, increasing amounts of stearoyl-CoA were added immediately before initiating the reaction with ATP. Quantification is of n = 3 samples; error bars represent standard deviation. P values represent a comparison with samples without stearoyl-CoA. **P 0.1; *P 0.05.",
    "molecules": "ATP, DOPE, lipid, stearoyl-CoA"
  },
  {
    "caption": "(c) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction. In vitro coupling reactions of GL1 were run on liposomes of three different sizes (400 nm and 50 nm extruded liposomes and sonicated liposomes) and of three different lipid compositions. Lipid compositions include 30 mol% DPPE, DSPE or DOPE (see Methods for full definitions), 10% bl-PI, and POPC. Total lipid = 2 mM. Quantification is of n = 3 samples; error bars represent standard deviation. The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry. P values represent comparison with most active liposomes (sonicated DOPE). **P 0.1; *P 0.05.",
    "molecules": "DPPE, DSPE, POPC, DOPE, lipid, PE, PI"
  },
  {
    "caption": "(b) To determine the impact of curvature on lipidation, we compared liposomes with unphysiologically high surface densities of PE (55 mol%) as have been used in other Atg8 or LC3 lipidation publications (for example, refs , , ), with liposomes having the highest densities of liposomes observed in mammalian organelle membranes (30 mol%). At physiologically relevant concentrations of liposomes (30 mol%), lipidation becomes membrane curvature dependent. In vitro coupling reactions were performed as in Fig. 1, except that liposomes were made by extrusion through membranes with different pore diameters (as indicated). Actual diameters of these liposomes were determined by dynamic light scattering (Supplementary Table 1). Top: the lipidation of GL2, GL2 and PE proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55% PE. However, when the liposomes is reduced to 30%, flat liposomes are no longer suitable for lipidation, while highly curved liposomes (100 nm diameter or less) continue to couple effectively. Bottom: to confirm that this curvature dependence reveals the lipidated product and not the intermediate, the 30% reactions were each run on Nycodenz flotation gradients. Only the lipidated product is recovered at the top of the gradient.",
    "molecules": "PE"
  },
  {
    "caption": "(c) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes. Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes (400 nm, black squares; sonicated, red circles) and varying molar percentage of DOPE. The extent of lipidation is determined as in Fig. 2. (n = 2 for 30 and 55% PE, n = 4 for all the other points, and error bars represent standard deviation.) P values were determined by comparing sonicated with 400 nm samples at each PE concentration. **P 0.1; *P 0.01. Uncropped versions of all gels are included in Supplementary Fig. 7.",
    "molecules": "DOPE, PE"
  },
  {
    "caption": "(b) Atg3 binding to liposomes. Atg3 (10 μM) was incubated at 30 °C for 90 min with liposomes containing 0, 30 or 55 mol% DOPE (5 mM lipids) and of varying sizes (extrusion membrane dimensions are shown on the figure; actual final sizes were determined by dynamic light scattering (Supplementary Table 1)). The liposome-associated Atg3 was recovered by Nycodenz density gradient centrifugation and analysed by SDS-PAGE.",
    "molecules": "DOPE"
  },
  {
    "caption": "(c) Quantification of liposome-associated Atg3. Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b. Error bars indicate s.e.m. from three independent experiments. P values calculated as compared with putative negative control (0% PE, 400 nm). *P 0.05. Uncropped versions of all gels are included in Supplementary Fig. 7.",
    "molecules": "PE"
  },
  {
    "caption": "(b) Analysis of reaction efficiency for point mutants of the amphipathic helix. GL1 lipidation reactions were run on 400 nm and sonicated liposomes (30 mol% DOPE) with the indicated Atg3 mutants. The extent of GL1-PE formation (top gel) was determined from densitometry and is plotted as percentage of total GL1 (bar graph). The ability to form the Atg3-GL1 conjugate was also assessed for each mutant (bottom immunoblot). Note that this conjugate forms in every case, indicating that no mutant inhibited a step upstream in the reaction. Further, this conjugate specifically accumulates in reactions where lipidation is impaired. In both panels, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the lines.",
    "molecules": "DOPE, PE"
  },
  {
    "caption": "(c) Curvature dependence of K11L mutant binding to liposomes compared with wild-type Atg3. The binding reaction was performed as in Fig. 4b on 30 DOPE% liposomes. F, float-up; T, total input.",
    "molecules": "DOPE"
  },
  {
    "caption": "(d) Lys 11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled. Lipidation assays were run as in b. Total GL1 lipidation was assessed from reactions with wild-type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated. Lipidation efficiency on low PE/low curvature liposomes (30% PE, 400 nm) increases with increasing hydrophobicity of the new amino acid, while liposomes with high PE (55%) or high curvature (sonicated) are good substrates for all active forms of Atg3. Left: Coomassie gels of individual lipidation reactions. Right: densitometry of Coomassie-stained gels revealed the ratio of GL1-PE to the sum of all three GL1 species as in Fig. 2. Light grey bars are sonicated liposomes; dark grey bars are 400 nm extruded liposomes. For b,c, P values were calculated between sonicated and 400 nm samples of the same mutant. For d, P values represent comparison with WT protein on the same liposome size and composition. For all panels, n = 3 independent experiments, error bars represent standard deviation and **P 0.05; *P 0.01. Uncropped gels in Supplementary Fig. 7.",
    "molecules": "Lys, PE"
  },
  {
    "caption": "Atg3-knockout MEFs (Atg3−/−) are incapable of forming LC3-II and do not accumulate LC3 puncta. Instead, they accumulate autophagic intermediates that are positive for Atg16. To determine whether the amphipathic helix is critical for in vivo function, Atg3 with wild-type or mutant forms of the amphipathic helix was introduced into Atg3−/− MEFs by lentiviral infection and rescue of the three phenotypes was tested. (a) Immunoblot analysis of LC3-II, GL2-II and GABARAP-II formation. GABARAP lipidation is significantly observed only in the presence of bafilomycin A1 (BafA1: 100 nM). Asterisk indicates a nonspecific band recognized by the Atg3 antibody.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(b) Cells were infected as in a and transduced cells were selected with puromycin for 1-2 days before starvation and immunolabelling with the anti-LC3B antibody. Representative images are shown in b",
    "molecules": "puromycin"
  },
  {
    "caption": "B, Co-immunoprecipitation (Co-Ip) of TSN and RBP47 in protein extracts prepared from 10-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47 and grown under no stress (NS), HS (39ºC for 60 min) or salt (NaCl) stress (150 mM NaCl for 60 min) conditions. The GFP-expressing line was used as a negative control. Endogenous TSN (107 kD) was detected in total fractions (Input) and fractions co-immunoprecipitated (Co-Ip) with RBP47 but not with free GFP in all three conditions. Input and Co-Ip fractions were analyzed by immunoblotting using α-TSN and α-GFP.",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "C, Localization of GFP-tagged proteins in root cells of 5-d-old Arabidopsis seedlings expressing Pro35S:GFP-RBP47, Pro35S:GFP-UBP1, Pro35S:GFP-TCTP and ProUBQ:GFP-SnRK1α2. The seedlings were grown under 23°C (NS), incubated at 39°C for 60 min (HS) or treated with 200 mM NaCl at 23°C for 60 min (NaCl). Scale bars = 10 μm",
    "molecules": "NaCl"
  },
  {
    "caption": "J, Colocalization of GFP-SnRK1α1, GFP-SnRK1α1CD, or GFP-SnRK1α1RD with RFP-RBP47 in N. benthamiana protoplasts subjected to HS (40 min at 39°C). For colocalization analysis under NS conditions see Fig. EV5B. For CHX treatment, protoplasts were incubated with 200 ng μL-1 CHX for 30 min at 23ºC before HS. GFP and RFP fusion proteins were expressed under the control of the UBQ and 35S promoter, respectively. Scale bars = 5 μm.",
    "molecules": "CHX"
  },
  {
    "caption": "G, Expression levels of DIN2 and DIN6 in Arabidopsis WT, tsn1 tsn2, tsn1 tsn2;TSN2 and snrk1α1-/- snrk1α2-/+ 10-d-old heat-stressed seedlings relative to unstressed controls. For CHX treatment, the WT seedlings were pre-treated with 200 ng μL-1 CHX for 30 min before HS. Upper and lower box boundaries represent the first and third quantiles, respectively. Horizontal lines mark the median of five replicate measurements, and whiskers mark the highest and lowest values. Means with different letters are significantly different at P < 0.05 (one-way ANOVA).",
    "molecules": "CHX"
  },
  {
    "caption": "(D, E) Imaging of luciferase activity in the liver of  HIF1aAlbKO (D) and  HIF2aAlbKO (E) mice after a hydrodynamic tail vein injection with a HRE-luc reporter plasmid or control (PBS) according to body weight followed by 6h normoxia or   hypoxia. (F, G) Bioluminescent photon counts normalized to the  PBS control group in liver of (F) HIF1aAlbKO and (G) HIF2aALbKO mice and their wild-type littermates 6h after normoxia  of  hypoxia. Data information: All bars represent mean ± SEM. Each individual data point represent individual mice. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. *** P<0.001; ** P≤ 0.01; * P≤ 0.05. N =  normoxia, H =  hypoxia. Signal around the perimeter of the Petri dishes represent aspecific luciferase signal. ",
    "molecules": "PBS"
  },
  {
    "caption": "(E) Scatter plot showing log fold change (LFC) of all DEX-upregulated genes (LFC > 1 and P ≤ 0.05) and DEX-downregulated genes (LFC < -1 and P ≤ 0.05) in normoxia   vs  hypoxia after 24h. The black line represents the diagonal, and the red line represents the real slope ± standard error of the data as analysed by linear regression.",
    "molecules": "DEX"
  },
  {
    "caption": "(F, G) Examples of  GR-responsive genes based on the RNA-seq data 6h (F) and 24h (G) after  normoxia or hypoxia  and DEX  stimulation. Data information: All bars represent mean ± SEM. N=3 per group. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01.",
    "molecules": "DEX"
  },
  {
    "caption": "(H ChIP-seq on liver derived from mice which were subjected to  hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver  isolation. (H) Box plots showing the LFC of genes responsive to DEX in both  normoxia  or  hypoxia  (6h and 24h). The central band represents the median. The box ranges from the first quartile (Q1) to the third quartile (Q3) which represents the interquartile range (IQR = Q3 - Q1) and covers the central 50% of the data. The whiskers illustrate the minimum (Q1 - 1.5*IQR) and maximum (Q3 + 1.5*IQR) of the data. Outliers are shown as dots. N=3 biological replicates per condition.",
    "molecules": "DEX"
  },
  {
    "caption": "Female C57BL/6J  mice were put in  normoxia or hypoxia for 6h or 24h, injected i.p. with PBS or DEX (10 mg/kg) and livers were isolated for genome-wide transcriptomics via RNA-seq. N=3 per group for a single  RNA-seq. (A, B) Examples of DEX-responsive genes in  hypoxia 6h (A) and 24h (B) which were not induced by  DEX  in  normoxia. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "DEX, PBS"
  },
  {
    "caption": "ChIP-seq on liver  derived from  mice which were subjected to hypoxia (6h and 24h), followed by DEX  (10 mg/kg) injection and 2h later sacrificed for liver  isolation. (E) Examples of specific GR  DNA-binding peaks, associated with the DEX-induced gene Slc22a5 in hypoxia (6h and 24h).",
    "molecules": "DEX"
  },
  {
    "caption": "(F) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice  were put in   hypoxia for 6h and stimulated with DEX. Slc25a30 expression was measured in the liver via RT-qPCR. N=3 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "DEX"
  },
  {
    "caption": "(C) Heatmap representing log2 values of shared stress GRE genes (counts) induced by  hypoxia 6h and 24h and induced by DEX in  normoxia  (LFC > 1 and P ≤ 0.05).",
    "molecules": "DEX"
  },
  {
    "caption": "(D-G) Female C57BL/6J and ADX mice were put in  normoxia or hypoxia for 6h or 24h, n=6/group, two independent experiments. (D, E) Confirmation of RNA-seq data via RT-qPCR [6h (D) or 24h (E)]. (F, G) Plasma GC concentration [6h (F) or 24h (G)]. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "GC"
  },
  {
    "caption": "(N) Expression levels of DEX  responsive genes in  normoxia and unique DEX responsive genes induced by hypoxia were measured in the liver of ADX mice after DEX stimulation during  hypoxia (24h) via RT-qPCR. N=3-4 per group, one experiment. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "DEX"
  },
  {
    "caption": "(D-G)  FFA levels and stress GRE genes were determined in the  plasma  and  iWAT  of female C57BL/6J mice injected with 5 mg  RU486 or vehicle (DMSO) (D, F), and in  GRdim/dim mice  and their wild-type littermates (E, G) after 6h of   normoxia   and hypoxia. N=4-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "DMSO, FFA, RU486"
  },
  {
    "caption": "(H-J)  Blood ketone body  levels 6h after   normoxia and hypoxia  in female C57BL/6J and ADX mice (H), in female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (I), and in GRdim/dim mice  and their wild-type littermates (J). N=5-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "DMSO, ketone body, RU486"
  },
  {
    "caption": "(A) Female C57BL/6J mice (n=3 per group) were put in normoxia  or hypoxia  for 24h, injected with PBS or  DEX  (10 mg/kg) and 2h later, indicated organs were isolated and gene expression  was measured via RT-qPCR. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "DEX, PBS"
  },
  {
    "caption": "(B) LPS LD100 dose-response in female C57BL/6J mice injected with indicated LPS doses after 24h  hypoxia. During the follow-up of lethality,  mice remained under hypoxic  conditions. N-values are indicated in the legend.",
    "molecules": "LPS"
  },
  {
    "caption": "(E) Female C57BL/6J  mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg  LPS (hypoxia) after 24h  normoxia   or hypoxia. Liver was isolated 6h after injected and typical GRE genes were measured via RT-qPCR. N=4-5 per group. (F) Female ADX mice were put in normoxia or hypoxia for 24h, injected i.p. with  PBS or DEX  (10 mg/kg) and 2h later, liver was isolated for RT-qPCR analyses of typical GRE genes. N=3-4 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. Survival curves were analysed with Fisher's exact test. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.",
    "molecules": "DEX, LPS, PBS"
  },
  {
    "caption": "(G) Female C57BL/6J mice were injected with 14.5 mg/kg LPS (normoxia) or 5 mg/kg LPS (hypoxia) after 24h normoxia  or hypoxia, with or without pre-treatment with 10 mg/kg  DEX 1h before  LPS injection. During the follow-up of lethality, mice remained under normoxic  or hypoxic conditions. Mice in  normoxia: black circles (LPS), black squares (DEX-LPS); mice in  hypoxia: white circles (LPS), white squares (DEX-LPS).",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "(H) Female ADX mice were injected with 0.05 mg/kg  LPS after 24h normoxia or hypoxia, , with or without 10 mg/kg DEX pre-treatment 1h before  LPS injection. During the follow-up of lethality, mice remained under normoxic or  hypoxic conditions. ADX mice in  normoxia: black circles (LPS), black squares (DEX-LPS); ADX mice in hypoxia: white circles (LPS), white squares (DEX-LPS).",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "(D) HEK293T cells were cultured in regular or starvation medium for 2 h. Cells were harvested and treated with DSP.",
    "molecules": "DSP"
  },
  {
    "caption": "(E) HEK293T cells were harvested and treated with DSP.",
    "molecules": "DSP"
  },
  {
    "caption": "(F) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h. After treatment with DSP, cell lysates were subjected to IP analysis.",
    "molecules": "DSP, wortmannin"
  },
  {
    "caption": "(G) Atg14F/F (undeleted) or Atg14Δ/Δ (deleted) MEFs were harvested and treated with DSP. * (E) and ** (G) indicate the positions of the immunoglobulin light and heavy chains, respectively. aa, amino acid; DSP, dithiobis(succinimidyl propionate); IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts.",
    "molecules": "dithiobis(succinimidyl propionate, DSP"
  },
  {
    "caption": "(B) m5‐7 cells were cultured in regular medium containing 10 ng/ml Dox for 8 days, and then cultured in the absence of Dox for a further 2 days.",
    "molecules": "Dox"
  },
  {
    "caption": "(C, D) m5‐7 cells were cultured in regular medium with or without 10 ng/ml Dox for 4 days, and then cultured in the presence of 50 μg/ml CHX for the indicated time periods (C). mRNA levels of the indicated genes were measured by quantitative PCR following the 4‐day Dox treatment. Data represent mean±s.e. (*P0.05) (D). CHX, cycloheximide; Dox, doxycycline; MEFs, mouse embryonic fibroblasts; mRNA, messenger RNA; WT, wild‐type.",
    "molecules": "CHX, Dox"
  },
  {
    "caption": "(A) WT MEFs, Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h. * indicates nonspecific immunoreactive bands.",
    "molecules": "BafA1"
  },
  {
    "caption": "(D, E) Cells stably expressing GFP-LC3 were cultured in regular DMEM, starvation medium or starvation medium in the presence of 0.2 μM WM or 100 nM BafA1 for 6 h. Total cellular GFP-LC3 signals were analysed by flow cytometry. Representative FACS data were shown (D). The geometric mean of fluorescence intensity was determined. Values are expressed as a percentage of the mean of control cells cultured in regular DMEM. Data represent mean±s.e. (*P0.05) (E). BafA1, bafilomycin A1; GFP, green fluorescent protein; KO, knockout; MEFs, mouse embryonic fibroblasts; NS, not significant; WM, wortmannin; WT, wild‐type.",
    "molecules": "BafA1, WM, wortmannin"
  },
  {
    "caption": "Immunofluorescence shows that the Vret signals (green) co-localize with the Ago3 signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the image on the left-hand side. This panel arrangement applies also for panels B−H. The bar chart shows the percentage of Vret-positive nuage in Ago3-positive nuage. Immunofluorescence shows that the Vret signals (green) partially co-localize with the Siwi signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The bar chart shows the percentage of Vret-positive nuage in Siwi-positive nuage.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence showing the Ago3 signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence showing the Vret signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) expressed in naïve BmN4 cells (Control). DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) in Ago3-depleted cells (Ago3 KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence showing the Ago3 signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This panel arrangement applies also for panels B, H and I. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 106; Small: IntDen value < 106). Immunofluorescence showing the Vret signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105).",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence showing the Flag-Ago3 signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence showing the Flag-Ago3 8SA and 8SE mutant signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). 32P-labeling of RNAs isolated from Ago3 (a mixture of unphosphorylated and phosphorylated) immunopurified from Siwi-depleted cells. 15-20-nt RNAs (indicated by a black line) were isolated and sequenced.",
    "molecules": "32P"
  },
  {
    "caption": "32P-labeling of RNAs isolated from Ago3. Target-Ss are isolated from Siwi-depleted BmN4 cells but are not isolated from Siwi/Vret double knockdown cells.",
    "molecules": "32P"
  },
  {
    "caption": "Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. The panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This arrangement applies also for panel C. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi KA mutant (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC concentration in the same conditions as in (B). Bars represent mean ± SD. n = 6 biological replicates/group except for Ch25fl/fl IL-1β were n = 5 biological replicates. The experiment was performed once. (D) 25-OHC, 7-keto-25-OHC, and 24(S)-OHC levels in spinal cord tissue extracted from Ch25hfl/fl (n = 8), Ch25hECKO (n = 3) and Ch25hBBBKO (n = 6) at the peak of EAE.",
    "molecules": "24(S)-OHC, 25-OHC, 7-keto-25-OHC"
  },
  {
    "caption": "(C) Impact of CNS PMN-MDSC and BM-PMN on CD4+ T cell proliferation assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the CNS (right panel) and BM (left panel) of WT mice at the peak of EAE. NS = non stimulated. n = 4 biological replicates/group. Bars indicate mean ± SD. CNS PMN-MDSC/CD4+ T cells: representative results of 2 independent experiments; BM-PMN: 1 experiment.",
    "molecules": "CFSE"
  },
  {
    "caption": "B Immunoblot analysis with anti-H3K122succ AB #2 on indicated histone octamers. Octamers with H3K122succ are specifically recognized.",
    "molecules": "H3K122succ"
  },
  {
    "caption": "C Pairwise scatterplots of H3K122succ read counts within peaks from ChIP experiments using all three H3K122succ antibodies (AB #1, AB #2 and AB #3).",
    "molecules": "H3K122succ"
  },
  {
    "caption": "G Boxplots visualizing expression values of genes (reads per kb; RPK) with peaks for indicated different combinations of histone PTMs at their TSS. Histone modifications are (from left to right): none (no PTM, black); only H3K122succ (green); H3K122succ plus one tail modification (H3K4me3, or H3K9ac, or H3K27ac, green and light orange); H3K122succ plus two tail modifications (green, yellow and light orange); H3K4me3 and H3K9ac and H3K27ac without H3K122succ (yellow, light orange and dark orange); H3K122succ and all three tail modifications (green, yellow, light orange, and dark orange). The number of genes within the groups is indicated below each boxplot. Boxes indicate the range between the first and third quartile, the central line depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Each distribution was compared to the \"no PTM\" distribution.",
    "molecules": "H3K122succ, H3K9ac, H3K27ac, H3K4me3"
  },
  {
    "caption": "B In vitro succinyltransferase assay on recombinant histone octamers as substrate. After incubation with p300 and suc-CoA, the H3K122succ levels were assessed by immunoblot. Ponceau staining of membrane is shown as a loading control.",
    "molecules": "suc-CoA"
  },
  {
    "caption": "C In vitro succinyltransferase assay on unmodified peptides spanning H3K122. Samples were incubated with increasing amounts of p300 (0 to 160 ng) in the presence of radiolabeled suc-CoA. The radioactivity incorporated was quantified by liquid scintillation counting. The plot displays average counts per minute (CPMs), ± SD and individual datapoints of two independent replicates.",
    "molecules": "suc-CoA"
  },
  {
    "caption": "In vitro desuccinylation assay on H3K122succ peptides with recombinant SIRT7 (D) Unmodified H3K122 peptide was used as control (left dot). Peptides were probed with H3K122succ AB #2 after incubation with sirtuins.",
    "molecules": "H3K122succ"
  },
  {
    "caption": "In vitro desuccinylation assay on H3K122succ peptides with recombinant SIRT5 (E). Unmodified H3K122 peptide was used as control (left dot). Peptides were probed with H3K122succ AB #2 after incubation with sirtuins.",
    "molecules": "H3K122succ"
  },
  {
    "caption": "F H3K122succ levels in Sirt5-KO cells. Histones were acid extracted from wild type (WT) and Sirt5-KO MEFs and the levels of H3K122succ determined by immunoblot. The level of H3K122succ was normalized to histones extracted from WT MEFs.",
    "molecules": "H3K122succ"
  },
  {
    "caption": "B In vitro transcription in the presence of GAL4-VP16 and p300, in the presence of no CoA, ac-CoA or suc-CoA. Shown is a representative autoradiogram of the IVT product. C Quantification of IVT reactions. Expression relative to no CoA is plotted.",
    "molecules": "ac-CoA, CoA, suc-CoA"
  },
  {
    "caption": "A Schematic representation of the smFRET assay (top), as well as merged smFRET efficiency histograms of unmodified (middle) and H3K122succ mono-nucleosomes (bottom) measured under indicated NaCl conditions (color coded). The nucleosome population is highlighted by the dashed box.",
    "molecules": "NaCl"
  },
  {
    "caption": "B Fraction of bursts in the nucleosome population (FRET efficiency, E ≥ 0.45) for unmodified (red, open circles) and H3K122succ nucleosomes (turquoise, open circles) plotted against NaCl concentrations and normalized to the respective mean at 0 M NaCl. Mean values (filled circles) were fitted with the Boltzmann sigmoid function (solid lines) to determine the NaCl concentration where half of the nucleosomes have disassembled (x0) as 0.61 M (± 0.04) for unmodified and 0.49 M (± 0.03) for H3K122succ nucleosomes.",
    "molecules": "H3K122succ, NaCl"
  },
  {
    "caption": "(D) Western Blot for pUb on extracts from day 12 iNeuron control and FBXO7-/- cells after treatment with BafA or AO for indicated times.",
    "molecules": "BafA"
  },
  {
    "caption": "(A) 3D-SIM images of HeLa control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interested are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) 3D-SIM images of iN day 12 control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interest are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Confocal images of iNeuron d12 Control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (Hoechst33342), mitochondria (HSP60) and pUb. Scale bar = 10 µm and 5 µm. (H) Evaluation of pUb volume after mitophagy induction. Error bars depict S.D. from three biological replicates. One-way ANOVA with multiple comparisons; p(****)<0.0001.",
    "molecules": "Hoechst33342"
  },
  {
    "caption": "(B) Mean Acidic:Neutral mtKeima per-cell ratios measured by flow cytometry for HeLa cells expressing Parkin ndicating the number of hours treated with AO (Antimycin A (5 µM) and Oligomycin (10 µM)) or three hours with 25 nM BafilomycinA (BafA). Error bars depict S.D. from biological triplicate measurements from three independent clones. Two-way ANOVA with multiple comparisons; p(****)<0.0001.",
    "molecules": "Antimycin A, BafA, BafilomycinA, Oligomycin"
  },
  {
    "caption": "(F) mtKeima mitophagy flux readout of indicated iNeuron genotypes. Cells were treated with AO for either 1 or 6 h and mitophagic flux measured by flow cytometry (>10,000 cells). Pooled data from biological replicates is shown, normalized to 6h BafA treated cells.",
    "molecules": "BafA"
  },
  {
    "caption": "(A) Workflow for analysis of total protein abundance in HeLa cells expressing Parkin with and without depolarization with AO (16 h). Cell extracts from triplicates were digested with trypsin prior to 18-plex TMT labelling and analysis by mass spectrometry.",
    "molecules": "TMT, trypsin"
  },
  {
    "caption": "(F) PINK1 levels in control cells and in two FBXO7-/- clones were measured by TMT-proteomics in fed cells, from biological triplicates from two independent clones.",
    "molecules": "TMT"
  },
  {
    "caption": "(F,G) Mitochondrial morphology in iNeurons (iN) was assessed using confocal imaging after staining cells with HSP60 to detect mitochondria and DAPI to identify nuclei, in either fed cells or cells treated with AO (3 h) (left panel). The number of median # of mitochondria/cell, median mitochondrial length, and median mitochondrial circularity is shown. Error bars depict S.D. from triplicates from three differentiations (9-12 image stacks each), as shown in panel I left, center, and right, respectively. Two-way ANOVA with multiple comparisons; p(*)=0.0416, p(****)<0.0001. Scale bar = 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A panel of 42 breast cancer cell lines was challenged with increasing concentrations of ATRA (11 nM-10 μM) for 3, 6, and 9 days, and cell growth was determined.The graphs show the growth-inhibitory effect exerted by the indicated concentrations of ATRA in SKBR3, HCC-1954, and MDA-MB436 cells which are representative of lines characterized by a high, intermediate and low ATRA score, respectively. Each point is the mean ± SD of three replicate cultures. **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).",
    "molecules": "ATRA"
  },
  {
    "caption": "Cell lines are ranked in ascending order according to the ATRA score. The plot distinguishes four separate groups of cell lines (D-A) with increasing ATRA scores, as indicated by the colored scale on the right. The cell lines are also grouped in tertiles, T1-T3, according to an ascending ATRA score. Each calculated value is representative of at least two independent experiments.",
    "molecules": "ATRA"
  },
  {
    "caption": "SCID mice were xenografted subcutaneously with 10 × 106HCC-1599 cells on both sides. One week after transplantation 10 animals/experimental group were treated intraperitoneally with vehicle (DMSO) or two doses of ATRA (7.5 and 15.0 mg/kg) once/day, 5 days a week for a total of 24 days. At the end of this period, treatment was discontinued until sacrifice.The size of the tumors was determined with a caliper and the volume plotted. Each point is the mean ± SE of 20 tumors. *Significantly lower than the corresponding vehicle-treated group (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).",
    "molecules": "ATRA, DMSO"
  },
  {
    "caption": "Magnetic resonance imaging (MRI) analysis was performed on five animals/experimental group on day 24. The picture shows representative 2D images of tumor sections from one animal treated with vehicle and one animal treated with 15.0 mg/kg ATRA. The bar graph shows the volume of the tumors calculated after 3D reconstruction of the MRI images. Each point is the mean ± SE of five tumors. The P-value of the comparisons of ATRA versus vehicle is shown.",
    "molecules": "ATRA"
  },
  {
    "caption": "ATRA-dependent anti-tumor activity in short-term cultures of primary breast tumorsTissue slices deriving from surgical specimens were challenged with vehicle (DMSO) or ATRA (0.1 μM) for 48 h.The panel illustrates examples of the immuno-histochemical data obtained in four representative cases: (i) Luminal-A (Lum A); (ii) Luminal-B (Lum B); (iii) Her2+ with RARA coamplification (Her2+/RARA+) and (iv) triple negative (TN).",
    "molecules": "ATRA, DMSO"
  },
  {
    "caption": "The gene-expression microarray and RNA-seq data associated with 40 of the breast cancer cell lines were used for the analyses. The panels illustrate the associations of RARα and PPARβ/δ with ATRA sensitivity. The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups (13 cell lines in each of the T1 and T3 groups) defined by ascending ATRA scores. The intermediate and right panels indicate the same results after stratification for the Luminal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) and the Basal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) phenotype, respectively. fpkm = fragments per kilobase of exon per million fragments mapped.",
    "molecules": "ATRA"
  },
  {
    "caption": "The basal expression levels of the indicated RAR-isoform variants were determined with the use of specific Taqman assays. The results are associated with ATRA sensitivity before (TOTAL) and after stratification of the cell lines for the Luminal and Basal phenotype as in (A).Data information: Significant P-values (Student's t-test) are indicated in red.",
    "molecules": "ATRA"
  },
  {
    "caption": "The plots illustrate the average expression levels of the indicated mRNAs (mean ± SD of two replicates) in tumor samples classified as ATRA-sensitive (Sens) and ATRA-resistant (Res) according to the response of Ki67. *Significantly different (P-value < 0.05, Student's t-test).",
    "molecules": "ATRA"
  },
  {
    "caption": "The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A-D groups and T1-T3 tertiles).",
    "molecules": "ATRA"
  },
  {
    "caption": "Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell linesThe indicated Luminal and Basal cell lines were challenged with increasing concentrations of ATRA, the RARα agonist, AM580, the RARβ agonist, UVI2003, and the RARγ agonist, BMS961, for 6 days. The complement of RAR-variant transcripts expressed in each cell line is shown in the left bar graphs (mean ± SD of two replicate measurements). The growth curves (sulforhodamine assay) of the cell lines are illustrated by the right linear plots. The results are expressed in % values relative to the corresponding control dishes treated with vehicle alone (right graphs). Each result is the mean ± SD of five replicate wells. ATRA sc = ATRA score.",
    "molecules": "AM580, ATRA, BMS961, UVI2003"
  },
  {
    "caption": "A RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistantMDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 andRARA-C7) and two appropriate control clones (Vect-C1 andVect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitiveSKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 andRARA-sh19) and two appropriate control clones (Vect-C6 andVect-C8) were isolated.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blotanalysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.",
    "molecules": "ATRA, DMSO"
  },
  {
    "caption": "C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.",
    "molecules": "ATRA, DMSO"
  },
  {
    "caption": "A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARαprotein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.",
    "molecules": "ATRA, DMSO"
  },
  {
    "caption": "A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARαprotein measured in the indicated clones by Western blotanalysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.",
    "molecules": "ATRA, DMSO, retinoid"
  },
  {
    "caption": "F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.",
    "molecules": "ATRA, DMSO"
  },
  {
    "caption": "G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.",
    "molecules": "ATRA"
  },
  {
    "caption": "A. Representative immunofluorescence images and quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67+ (red) acinar cell nuclei co-staining with DAPI (blue) and Amylase (green). Arrows point to KI67+ acinar cells in Hnf1apKO mouse. Scale bar is 200 μm. Acinar proliferation is represented as the average of the KI67+/Amylase+ cell ratio. Quantifications were performed on 3 random fields from 3 Pdx1Cre and 3 Hnf1apKO mice. P-values are from two-tailed Student's t test.",
    "molecules": "DAPI"
  },
  {
    "caption": "F. KDM6A-bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas.",
    "molecules": "H3K27ac"
  },
  {
    "caption": "G. Genes that were downregulated in Kdm6apKO pancreas (KDM6A-dependent genes) showed greatest changes in histone marks. Box plots show fold-changes of H3K27me3 (left) and H3K27ac (right) signals in Kdm6apKO compared to control. Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent (grey; n=420 regions) and independent (white; n=8035 regions). The signals are average values from ChIP-seq experiments in two biological replicates. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Mann-Whitney U test.",
    "molecules": "H3K27ac, H3K27me3"
  },
  {
    "caption": "H. Changes in KDM6A, H3K27me3 and H3K27ac ChIP-seq profiles in the Ppp4r4-Serpina10 locus in Kdm6apKO pancreas.",
    "molecules": "H3K27ac"
  },
  {
    "caption": "I. qPCR of H3K27me3, H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in (H) (R1, R2, R3), and Serpin10 mRNA. Error bars show +/-SD and P-values are from two-tailed Student's t test, n=3.",
    "molecules": "H3K27ac, H3K4me1"
  },
  {
    "caption": "J. KDM6A-bound genes that showed decreased expression in Kdm6apKO pancreas (functional KDM6A binding sites) showed simultaneous gain of H3K27me3 and loss and of H3K27ac.",
    "molecules": "H3K27ac"
  },
  {
    "caption": "C Representative H&E (scale bar 50 µm) and fluorescent images of cl-Caspase 3 staining in B16 WT and XIAPKO tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (right panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5).",
    "molecules": "DAPI"
  },
  {
    "caption": "H Human cytokines array analysis of HEK293T cells transfected with myc-XIAP or myc-XIAPΔRING. Medium was changed after 16 h and the supernatant was collected after 48 h. Pre-spotted nitrocellulose membranes were incubated with the supernatants overnight at 4°C.",
    "molecules": "nitrocellulose"
  },
  {
    "caption": "B Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "C Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "D Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "F IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h.",
    "molecules": "d89, DMSO"
  },
  {
    "caption": "G IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h.",
    "molecules": "Bir, birinapant, DMSO, BV6"
  },
  {
    "caption": "The dose-response curves of arteries from wild type, Tek-Cre::Lama5-/- (B) and Lama4-/- mice (C) and wild type littermates stimulated with methacholine do not show significant differences (n.s.). Data are expressed as percent relaxation of the maximum force developed in presence of 0.3 µM U46619, and are mean values ± s.e.m from 7 experiments with 1 wild type and 1 KO artery in each experiment.",
    "molecules": "methacholine, U46619"
  },
  {
    "caption": "EVs were purified by SEC from supernatants of Jurkat cells. (C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81.",
    "molecules": "APC"
  },
  {
    "caption": "Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (C) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation. Six fractions were recovered and analyzed by Western blot for the presence of SPN, SERINC3 and p24. (T = top; B = bottom). (D) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations (exp 1: open symbols, exp 2: closed symbols). AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). ",
    "molecules": "iodixanol"
  },
  {
    "caption": "Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (G) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with EV samples. Captured EVs were detected with a mixture of APC-labeled anti-CD9, anti-CD63, and anti-CD81. Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction. Results were relativized to the values obtained with control sample for each experiment. Dotted line represents values obtained for EVs isolated from control cell CM.",
    "molecules": "APC"
  },
  {
    "caption": " Combining the ABA signaling components ABF2, OST1, the PP2C ABI1, and RCAR11 provides ABA-controlled LUC expression. The reporter is under the control of a minimal promoter that contains ABA-responsive cis elements (ABREs). Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor (PP2C) in the presence of ABA and allows the activation of the protein kinase SnRK2. SnRK2 phosphorylates and activates the key transcription factor ABF, which targets promoters containing ABRE. Reporter activity was determined 16 h after exogenous ABA (0.1 mM) administration and referred to samples expressing ABF2 alone. ",
    "molecules": "ABA"
  },
  {
    "caption": " In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1-effector in combination with 5 µg ABA receptor DNA (± 10 µM ABA) per sample containing 105 protoplasts. Data information: In (A) each bar represents the mean ± s.e.m; n = 12, biological replicates (derived from four independent yeast transformants); for RCAR1/RCAR5 and RCAR3/RCAR13 18 and 15 biological replicates were used, respectively. Outliers detected by Grubbs-test were removed from the analysis. In (B) each bar represents the mean ± s.e.m of three independent transformations normalized to the activity of a control samples without RCAR and ABA.",
    "molecules": "ABA"
  },
  {
    "caption": " The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I). A-C) The analysis in yeast was conducted as mentioned in Fig. 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated. D-F) Analysis of protoplasts was performed in the ABA-deficient background aba2-1 in the presence of different exogenous ABA concentrations. The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs (Tischer et al, 2017). The induction of the reporter gene is related to protoplasts without ectopic RCAR expression, and in the absence of exogenous ABA. G -I) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations. Maximum inhibition of PP2C activity by ABA was set to 100%. Data information: In (A-C) each data point represents the mean ± s.d.; n = 9 biological replicates derived from three independent yeast transformants. In (D-F) data represent the mean ± s.d. of three independent transfections. In (G-I) data represent the mean ± s.d. of three replicates. ",
    "molecules": "ABA"
  },
  {
    "caption": " Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast. Phosphorylation occurred at serine 175 or threonine 176, and at threonine 179. The relative abundance is given as the ratio of signal intensity of phosphorylated versus non-phosphorylated peptide for OST1, SnRK2.3, and SnRK2.4 in the mass spectrometric analysis. The numbering of phosphosites corresponds to OST1. ",
    "molecules": "serine, threonine"
  },
  {
    "caption": "A,B) ABF2-mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1, and varying exogenous ABA concentrations. Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. represent Replicates were normalized to controls without SnRK2 in (A, B)",
    "molecules": "ABA"
  },
  {
    "caption": " C) Maximum ABA response recovery of samples shown in (A,B) relative to the reporter activity of 0.1 mM ABA-treated yeast cells expressing SnRK2s but no ABI1. Data information: Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in (C). ",
    "molecules": "ABA"
  },
  {
    "caption": " D-F) Analysis of the ABA response mediated by OST1 and SnRK2.4 using the receptors RCAR1 (D), RCAR8 (E), and RCAR14 (F). Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. The data in (D-F) are the mean ± s.e.m; n = 12, biological replicates from four independent yeast transformants for OST1 + RCAR1/RCAR8, and SnRK2.4 + RCAR8. ",
    "molecules": "ABA"
  },
  {
    "caption": "FRET-FLIM analysis of GFP:OST1 or GFP:SnRK2.4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA. GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana.   Representative false colour images of GFP:SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2.4 in combination with mCherry:ABI1 and RCAR1 without exogenous ABA. The bar marks 20 µm and the nucleus (n) and cytosol (c) are indicated.   ",
    "molecules": "ABA"
  },
  {
    "caption": " Analysis of SnRK2-ABI1 interaction as mentioned in (A) but in the presence of RCAR14 and different exogenous ABA levels. RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1.   Representative GFP lifetime false colour image of the analysis shown in C) 2h after 30 µM exogenous ABA administration. Details are as described in (B).   ",
    "molecules": "ABA"
  },
  {
    "caption": "The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants, as shown by propidium iodide (PI) staining of five-day-old roots. Arrowhead, oblique cell wall. Insets, magnifications of areas showing cell wall stubs. Scale bars, 50 µm.",
    "molecules": "propidium iodide"
  },
  {
    "caption": "In vivo localization of mCherry-PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five-day-old complemented pi4kβ1 pi4kβ2 plants. The mCherry-PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry-PI4Kβ1. Scale bar, 20 µm. Whole-mount immunostaining of five-day-old seedlings expressing mCherry-PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti-tubulin (red) and anti-mCherry (green) antibodies, and DAPI (blue). (І), (ІІ), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "The effects of reduced association of PtdIns(4)P with the cell plate on the recruitment of CLC2-GFP were analyzed in root meristem cells of five-day-old plants. Top, Cell plate-associated fluorescence of the PtdIns(4)P reporter, 2×mCherryFAPP1-PH, was monitored at the end of cytokinesis in five-day-old wild type controls or pi4kβ1 pi4kβ2 double mutants, as indicated. Scale bars, 10 µm. Bottom, The intensity of 2×mCherryFAPP1-PH signals was quantified at the end of cytokinesis at the cell plate (left plot) and at the TGN (right plot). Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane (wild type, n = 37 cells, 24 roots; pi4kβ1 pi4kβ2, n = 55 cells, 41 roots). TGN intensities were recorded using the Airyscan VP mode, and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity (wild type, n=30 cells, 22 roots; pi4kβ1 pi4kβ2, n=37 cells, 31 roots). ***, significant differences (p<0.0001) according to two-tailed Mann-Whitney U-tests.",
    "molecules": "PtdIns(4)P"
  },
  {
    "caption": "Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of wild type seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.",
    "molecules": "FM4-64"
  },
  {
    "caption": "Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.",
    "molecules": "FM4-64"
  },
  {
    "caption": "Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Five-day-old seedlings were immunostained with anti-KNOLLE (green) and counterstained with DAPI (blue). In the pi4kβ1 pi4kβ2 double mutant, diffusion of KNOLLE occurred at the plasma membrane (arrowheads). Scale bars, 10 μm. Quantitative analysis of lateral diffusion of KNOLLE at the plasma membrane (PM) from (A). Data are mean ± SD from four independent experiments. **, a significant difference (p<0.01) according to a two-tailed Student's t test (untreated wild type, n= 97 cells, 25 roots; untreated pi4kβ1 pi4kβ2, n = 142 cells, 44 roots; BFA treated wild type, n = 126 cells, 28 roots; BFA treated pi4kβ1 pi4kβ2, n = 183 cells, 43 roots).",
    "molecules": "BFA, DAPI"
  },
  {
    "caption": "Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of KNOLLE at the cell plate at the end of cytokinesis in the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were immunostained with anti-KNOLLE antibodies (green) and counterstained with DAPI (blue). Scale bars, 10 μm. Quantification of KNOLLE intensity at the cell plate from (C), normalized to the signal intensity of intracellular compartments. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 26 cells, 20 roots; pi4kβ1 pi4kβ2, n = 41 cells, 37 roots).",
    "molecules": "DAPI"
  },
  {
    "caption": "Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of PIN2-GFP at the cell plate at the end of cytokinesis in the the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were stained with 2 μM FM 4-64 for 3 minutes at room temperature. Scale bars, 10 μm. Quantification of PIN2-GFP signal at the cell plate from (E), normalized to the intensity at the apical plasma membrane intensity, as indicated by the dashed lines. ***, a significant difference (p<0.001) according to a two-tailed Mann-Whitney U-test (wild type, n = 28 cells, 16 roots; pi4kβ1 pi4kβ2, n = 45 cells, 26 roots).",
    "molecules": "FM 4-64"
  },
  {
    "caption": "Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Internalization of PIN2-GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min, then washed and incubated with 50 μM CHX and 25 μM BFA. Scale bars, 10 μm. Quantification of punctate signals from (G) induced by BFA in wild type and pi4kβ1 pi4kβ2 double mutants. Data are mean ± SD. *, a significant difference (p<0.05) according to a two-tailed Student's t test; (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 110 cells, 7 roots).",
    "molecules": "BFA, CHX"
  },
  {
    "caption": "Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Delayed internalization of FM 4-64 from the plasma membrane in the pi4kβ1 pi4kβ2 double mutant. Four-day-old seedlings were pulsed with 2 μM FM 4-64 for 3 minutes on ice and then fluorescence was recorded for 20 min. Scale bars, 10 μm. Quantification of intracellular FM 4-64 signal from (i), normalized to apical and basal plasma membrane intensities. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 66 cells, 9 roots).",
    "molecules": "FM 4-64"
  },
  {
    "caption": "pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Immunostaining of meristem cells of five-day-old plants using anti-tubulin (green) counterstained with DAPI (false colour red). Asterisks, multiple nuclei in one cell; arrowheads, multiple phragmoplasts in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Whole-mount immunostaining of root tips of five-day-old seedlings. Green, anti-KNOLLE; red, anti-tubulin; blue, DAPI. (І), (ІІ), magnifications of areas highlighted in dashed boxes. Arrowheads, solid phragmoplasts with thicker cell plates. Scale bars, 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "The dynamics of the microtubule bundling protein, MAP65-3, were recorded in live cell time lapse analyses of cytokinetic cells from root tips of wild type controls and pi4kβ1 pi4kβ2 double mutants. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old wild type seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 8 cells from 5 roots. Scale bar, 10 µm. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old pi4kβ1 pi4kβ2 seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 11 cells from 10 roots. Arrowheads, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Scale bars, 10 µm. Plot profiles were obtained from medial confocal planes at time points displaying the transition from disc to ring phragmoplasts, marked by dashed lines in (A) and (B). Arrowhead, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Times in (A-C) are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0.",
    "molecules": "FM 4-64"
  },
  {
    "caption": "Medial confocal planes from z-stacks from (a) and (b) demonstrating attachment of the FM 4-64-stained cell plate with the peripheral plasma membrane, defining t0. Scale bars, 10 µm.",
    "molecules": "FM 4-64"
  },
  {
    "caption": "3D reconstruction of ring and disc-shaped immunofluorescence patterns for GFP-MAP65-3 and microtubules, based on super resolution structured illumination microscopy (SIM), costained with DAPI. Top panels, wild type; bottom panels, pi4kβ1 pi4kβ2 double mutants. Scale bars, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants. Crosses of pi4kβ1 pi4kβ2 and mpk4-2 mutants resulted in combined genotypes with increased growth defects, as indicated. 14-day-old seedlings are shown. Scale bars, 1 cm. Top, The severe growth defects of the combined genotypes were accompanied by increased tissue distortion, as shown by 3D projections after propidium iodide staining of root tips of 14-day-old seedlings. Scale bars, 100 µm. Bottom, Multinucleation of cells was increased in the combined genotypes, as evident from immunofluorescence of microtubules (red) counterstained with DAPI (blue). Asterisks, multiple nuclei in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "In vitro pull-down  assays. GST  or GST-PSKR1JK  fusion proteins coupled with glutathione  beads were used to pull down His-CPK28. The amount of fusion proteins loaded on the gel was indicated by Coomassie brilliant blue (CBB) staining. Asterisks indicate the specific bands of fusion proteins.",
    "molecules": "glutathione"
  },
  {
    "caption": "Co-IP  assays. PSKR1-HA  was co-expressed with CPK28-GFP  or GFP  in N. benthamiana leaves. PSK  (10 μM) or H2O  was injected 2 hours before leaves  sampling. The protein was precipitated using GFP-Trap. Both immunoprecipitated  and input samples were subjected to western blotting  using an anti-HA  or anti-GFP  antibody.",
    "molecules": "H2O, PSK"
  },
  {
    "caption": "A. Co-IP  assay showing that PSK  promotes CPK28-GS2 interaction. GS2-HA  was co-expressed with CPK28-GFP  or GFP  in  N. benthamiana leaves. PSK  (10 μM) or H2O  was injected 2 hours before leaves  sampling. The protein was precipitated by GFP-Trap. Immunoprecipitated  and input samples were subjected to western blotting using an anti-HA  and anti-GFP  antibody. Data information: Experiments were repeated twice with similar results.",
    "molecules": "H2O, PSK"
  },
  {
    "caption": "A. PSK  induces the phosphorylation  of GS2   by CPK28 in vivo. GS2-GFP  was co-expressed with CPK28-HA  (empty vector as a negative control) in N. benthamiana leaves. PSK  (10 μM) or H2O  was injected 2 hours before leaves  sampling. The phosphorylated GS2   protein was precipitated by GFP-Trap, and then immunoblotted  with anti-pSer/pThr and anti-pSer antibodies.",
    "molecules": "H2O, PSK"
  },
  {
    "caption": "D,E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5μM) or Reversine (500nM), either before (D) or after (E) mitotic entry.",
    "molecules": "AZ-3146, nocodazole, Reversine"
  },
  {
    "caption": "H,I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 or C24) treated with nocodazole.",
    "molecules": "nocodazole"
  },
  {
    "caption": "J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells).",
    "molecules": "dox, doxycycline, nocodazole"
  },
  {
    "caption": "L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).",
    "molecules": "nocodazole"
  },
  {
    "caption": "A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days.",
    "molecules": "doxycycline"
  },
  {
    "caption": "B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation.",
    "molecules": "nocodazole"
  },
  {
    "caption": "A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM. D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment. ",
    "molecules": "AZ-3146, nocodazole"
  },
  {
    "caption": "A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM.",
    "molecules": "AZ-3146, nocodazole"
  },
  {
    "caption": "C. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.",
    "molecules": "AZ-3146, MG132, nocodazole"
  },
  {
    "caption": "D. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.",
    "molecules": "AZ-3146, nocodazole"
  },
  {
    "caption": "a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX. Only liposomes containing PtdIns(3)P were labelled intensely. GST-PXR58A, a PX-domain mutant that does not bind PtdIns(3)P, yielded virtually no labelling in PtdIns(3)P-containing liposomes. Scale bar, 0.2 μm.",
    "molecules": "phosphatidylcholine, PtdIns(3)P, phosphoinositide"
  },
  {
    "caption": "(b) QF-FRL of unilamellar liposomes containing 0-5 mol % of PtdIns(3)P. Scale bar, 0.2 μm. The labelling density increased roughly in proportion to the PtdIns(3)P content of the liposomes. The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes. Values for all concentrations are statistically different from those in the adjacent conditions (for example, 1 versus 2 mol%; mean±s.e.m.; Student's t-test; P0.05).",
    "molecules": "gold, PtdIns(3)P"
  },
  {
    "caption": "(b) QF-FRL in wild-type yeast in log phase. PtdIns(3)P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm.",
    "molecules": "gold, PtdIns(3)P"
  },
  {
    "caption": "c) Quantification of the labelling intensity of PtdIns(3)P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.",
    "molecules": "gold, PtdIns(3)P"
  },
  {
    "caption": "(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagosomal membranes. A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane. Scale bar, 0.5 μm.",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": "(b) Autophagosomes in wild-type yeast cultured in S(-NC) for 4 h. The autophagosome was identified as a smooth-surfaced double-membrane structure, and seen in two different patterns, as shown in the diagrams in c. Upper panel: convex structures displaying the outer membrane EF and the inner membrane PF. Lower panel: concave structures displaying the outer membrane PF and the inner membrane EF. In both cases, PtdIns(3)P was labelled more intensely in the EF than in the PF. Scale bars, 0.2 μm.",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": "(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen. A lower amount of labelling was also seen in the PF of autophagic body membranes. Scale bar, 0.5 μm. (",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": "(c) Labelling intensity of PtdIns(3)P in wild-type yeast cultured in S(-NC) for 4 h (average±s.e.m.). PtdIns(3)P was found in both leaflets of the autophagosome and autophagic body membrane, and the labelling was much stronger in the EF than in the PF. In the vacuolar membrane, significant labelling was found in the EF, albeit at much lower levels than in the PF. The experiment was repeated three times, and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.",
    "molecules": "C, gold, N, PtdIns(3)P"
  },
  {
    "caption": "(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3)P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. (",
    "molecules": "bafilomycin A1, gold, PtdIns(3)P, Torin1"
  },
  {
    "caption": "(b) NIH3T3 cells expressing Atg4C78A were incubated in Earle's balanced salt solution for 1 h. The isolation membrane was observed as parallel membranes with few IMPs. PtdIns(3)P was labelled only in the PF. Scale bar, 0.1 μm. Other examples are presented in Supplementary Fig. 5.",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": "(c) Labelling density of PtdIns(3)P in mammalian autophagosomal membrane (mean±s.e.m.). The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images. Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF-PF boundary (white circles; see Supplementary Fig. 6c for more examples), suggesting that many of them actually represent PtdIns(3)P in the PF.",
    "molecules": "gold, PtdIns(3)P"
  },
  {
    "caption": "(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3)P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm.",
    "molecules": "C, N, PtdIns(3)P"
  },
  {
    "caption": "(b) The PF/EF ratio of the PtdIns(3)P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images.",
    "molecules": "gold, PtdIns(3)P"
  },
  {
    "caption": "(b) Diagrams of the deletion-mutation constructs of p62 (left) and the corresponding input (upper right) and pull-down assay (lower right). The MBP-tagged mouse p62 deletion mutants conjugated to amylose (AM) resins were incubated with purified GST-tagged mouse Keap1-DC. The pulled-down complexes with the MBP-p62 mutants were subjected to SDS-PAGE and revealed by staining with Coomassie brilliant blue. The bands corresponding to MBP-p62 and its mutants are indicated by black dots. Red arrowheads indicate the band corresponding to GST-Keap1-DC. For details of construct 14 see Supplementary Information, Fig. S3.",
    "molecules": "amylose"
  },
  {
    "caption": "(a) Insolubilization of Keap1 in Atg7-deficient hepatocytes. Liver homogenates from Atg7F/F:Mx1 mice on various days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. The data displayed are representative of three separate experiments.",
    "molecules": "poly(I)•poly(C), Triton X-100"
  },
  {
    "caption": "(b) Immunoblot analysis of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 p62-deficient (Atg7F/F:Mx1:p62−/−) livers. Liver homogenates from mice of the stated genotypes at 12 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Atg7F/F mice in which Atg7 is efficiently expressed at a level similar to that in the wild-type mice were used as control. Data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(c) Quantitative real-time PCR analyses of Nqo1 and Gstm1 in mouse livers. Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in Atg7F/F liver. Data are means ± s.d. for three experiments.",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(d) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (top) and anti-p62 (middle) antibodies. Bottom: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(a) Immunoblotting of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 Nrf2-deficient (Atg7F/F:Mx1:Nrf2−/−) livers. Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1, Gstm1 and actin (bottom left section). Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C), subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B (as control) (bottom right section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(b) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (left) and anti-p62 (middle) antibodies. Right: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient and Atg7 Nrf2-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(c) Quantitative real-time PCR analyses of Nqo1, Gstm1 and Cyp2a5 in mouselivers. Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in the Atg7F/F liver. Data are means ± s.d. for three experiments.",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(d) Liver weight. The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly(I)•poly(C) were measured. Data are means ± s.d. for five mice from each group. Three asterisks, P 0.001 (Student's t-test).",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(e) Histological analysis of the mouse liver of the indicated genotypes. At 28 days after injection of poly(I)•poly(C), the livers were processed for haematoxylin/eosin staining. Higher-magnification views are shown in the bottom panels. CV, central vein; P, portal vein. Scale bars, 100 μm.",
    "molecules": "poly(I)•poly(C)"
  },
  {
    "caption": "(A, B) BMDCs were infected with Pru parasites  (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected  (NI) conditions.  IRE1α/XBP1s pathway: Ern1  (IRE1α), Xbp1s,  Dnajb9 (ERdj4), Sec24D, Bloc1s1  and Sec61a1; PERK/ATF4  pathway: Atf4, Ddit3 (CHOP) and  Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1  (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice  / independent experiment).",
    "molecules": "TN, tunicamycin"
  },
  {
    "caption": "(C, D)  Immunoblot analysis of  IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel),  Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the  indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.",
    "molecules": "TN"
  },
  {
    "caption": "(I) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of H-2Kb by Pru Tomato SAG1‐OVA infected XBP1fl/fl IRE1fl/fl treated or not with 4μ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns: p>0.05.",
    "molecules": "4μ8C"
  },
  {
    "caption": "Representative confocal images of CAD cells exposed for 4 h to the indicated treatments. Cells were fixed and then stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments.",
    "molecules": "DAPI, WGA"
  },
  {
    "caption": "Top: Schematic of the co-culture system used to assess vesicle transfer from donor (labeled with DiD) to acceptor (labeled with CTG) cells. After labeling, cells were detached, replated in 1:1 ratio and cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Middle: Representative confocal images of co-cultured donor and acceptor CAD cells. Each image corresponds to the projection of the entire Z-stack. Bottom: The insets correspond to the projection of selected Z-stacks of the area marked with a yellow square in each condition to better illustrate the transferred vesicles in acceptor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.",
    "molecules": "CTG, DiD, H2O2"
  },
  {
    "caption": "Top: Schematic of the culture system used to assess vesicle transfer by secretion. Donor cells (labeled with DiD) were cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Then acceptor cells (labeled with CTG) were treated for 4 h with the conditioned media from donor cells. Bottom: Representative confocal images of acceptor CAD cells treated with the conditioned media from donor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor cells.",
    "molecules": "CTG, DiD, H2O2"
  },
  {
    "caption": "Representative confocal images of CAD cells cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Cells were fixed and stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments.",
    "molecules": "TAT, DAPI, DMSO, H2O2, KN-93, WGA"
  },
  {
    "caption": "Representative confocal images of co-cultured donor and acceptor CAD cells. Cells were co-cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.",
    "molecules": "TAT, DiD, DMSO, H2O2, KN-93"
  },
  {
    "caption": "Representative confocal images of CAD cells stained for vinculin (red) and HCS CellMask™ Blue after being treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. The ring circumscribed between the green lines mark the area where the vinculin puncta were counted for each cell. This area is defined by the board of the HCS CellMask™ Blue staining and the vinculin staining around it. Average number of vinculin puncta per cell with the indicated treatments.",
    "molecules": "CellMask™ Blue, TAT, DMSO, H2O2, KN-93"
  },
  {
    "caption": "Representative confocal images of CAD cells expressing the indicated GFP-fusion proteins. Top: Cells showing WGA (white) and DAPI (blue) staining. Bottom: Cells showing phalloidin (red) and DAPI (blue) staining. Note that the expression pattern of βCaMKII WT and the mutants K43R and A303R, is rather punctate than diffused.",
    "molecules": "DAPI, phalloidin, WGA"
  },
  {
    "caption": "Percentage of acceptor cells containing DiD-labeled vesicles when acceptor cells were co-cultured with donor cells expressing the indicated GFP-fusion proteins.",
    "molecules": "DiD"
  },
  {
    "caption": "Top: Schematic of the co-culture system used to assess vesicle transfer in transfected cells. Donor cell were transfected with the indicated GFP-fusion proteins and then labeled with DiD. Acceptor cells were transfected with a H2B-mCherry plasmid. After 24 h, the cells were detached, replated in 1:1 ratio and cultured for 4 h. Cells were then fixed and stained with HCS CellMask™ Blue. Bottom: Representative confocal images of co-cultured donor and acceptor CAD cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.",
    "molecules": "CellMask™ Blue, DiD"
  },
  {
    "caption": "Time lapses showing a TNT connecting two transfected cells. Top: GFP-vector transfected cells. Middle: GFP-βCaMKII WT transfected cells. Bottom: GFP-βCaMKII T287D transfected cells. Cells were stained with WGA (white). Yellow arrowheads point out a TNT in each condition. Scale bars represent 10 μm.",
    "molecules": "WGA"
  },
  {
    "caption": "CODIM super-resolution images (3 right side panels) of TNTs shown in the confocal expanded field images (left panels) of transfected cells showing expression of indicated GFP-tagged proteins (green). Cells were stained for F-actin with phalloidin (red). Scale bars in the expanded fields represent 5 µm and 1 µm in the insets.",
    "molecules": "phalloidin"
  },
  {
    "caption": "Confocal images and 3D reconstruction showing the intracellular localization of endogenous βCaMKII (green) in CAD cells. Cells were stained with DAPI (blue) and phalloidin (red). Yellow arrows point to βCaMKII-positive puncta inside a TNT. Scale bars in the expanded fields represent 10 µm in the micrographs and 1 µm in the 3D projection.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "Confocal images of untransfected and GFP-βCaMKII WT transfected CAD cells (green), treated or not with Wnt7a. Top: Cells were stained with phalloidin (red) and DAPI (blue). Middle: Phalloidin staining (white) of the cells is shown. Bottom: Pseudocolor images of cells stained with phalloidin highlight the differences in F-actin levels among the treatments. Scale bars represent 5 μm. Anti-actin immunoblot and bar graph showing changes in the ratio between G-actin (in supernatant fraction, s) and F-actin (in the pellet fraction, p) of CAD cells untransfected or transfected with GFP-βCaMKII WT plasmid and treated or not with Wnt7a.",
    "molecules": "DAPI, phalloidin, Phalloidin"
  },
  {
    "caption": "Confocal images of CAD cells transfected with GFP-βCaMKII WT plasmid (green) and treated or not with Wnt7a. Cells were fixed and then stained with phalloidin (red) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field images (left). 3D reconstruction shows co-localization of GFP/phalloidin signals. Scale bars represent 10 µm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "Top: Schematic of the culture systems used to assess cell-to-cell contact-dependent or secretion-mediated transfer of vesicles in primary neurons. After dissection, neurons were labeled in suspension with DiI (donor, D) or CTG (acceptor, A). In the co-culture system, neurons were plated in 1:1 ratio and cultured for 20 h and then treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h. In the secretion-based system, donor neurons cultured alone for 20 h were treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h, and the medium was then collected and replaced for the medium of acceptor neurons cultured separately. Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h. Bottom: Representative confocal images of co-cultured donor and acceptor neurons. The insets (right panels) show a magnification of the area depicted in the expanded field images (left). Yellow arrows point to DiI-labeled vesicles inside acceptor neurons.",
    "molecules": "CTG, DiI"
  },
  {
    "caption": "Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments. Percentage of acceptor neurons containing DiI-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons.",
    "molecules": "DiI"
  },
  {
    "caption": "Confocal image showing a donor (D) and an acceptor (A) neuron connected by TNT-like structures (yellow arrowhead in the merged panel). Neurons were stained with WGA (white) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field (left).",
    "molecules": "DAPI, WGA"
  },
  {
    "caption": "Representative confocal images of co-cultured donor (D, labeled with DiI) and acceptor (A, labeled with CTG) neurons treated or not with 200 ng/mL of Wnt5a, 200 ng/mL sFRP-2 or 200 ng/mL Wnt5a plus 200 ng/mL sFRP-2 for 4 h. Yellow arrows point to DiI-labeled vesicles inside acceptor neurons. Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments.",
    "molecules": "CTG, DiI"
  },
  {
    "caption": "Primary cortical neurons at 1 DIV were fixed and labeled with MAP-2 (green), β-III-tubulin (red) and WGA (white). The inset shows a magnified image of the area depicted in the merged panel. Yellow arrowheads point out TNT-like structures.",
    "molecules": "WGA"
  },
  {
    "caption": "Transfer of α-syn fibrils is shown in neurons at 1 DIV. Cells were loaded in suspension with either Alexa-568 α-syn fibrils (red) or Alexa-488 α-syn fibrils (green) and cultured together. The insets (right panels) show a magnification of the area depicted in the expanded field image (left). Yellow arrows point to red and green α-syn puncta contained in the soma of a neuron. Yellow arrowhead points to a TNT-like connection.",
    "molecules": "Alexa-488, Alexa-568"
  },
  {
    "caption": "A TNT-like connection containing Alexa-488 α-syn positive puncta is found in neurons at 1 DIV stained with phalloidin (white) and DAPI (blue). The insets (right panels) show the 3D reconstruction of the area depicted in the expanded field image (left).",
    "molecules": "Alexa-488, DAPI, phalloidin"
  },
  {
    "caption": "Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. After 20 h post-plating, neurons were treated with 200 ng/mL Wnt5a for 4 h. The insets (right panels) show a magnification of the area depicted in the expanded field (left). Yellow arrows point to α-syn puncta inside acceptor neurons.",
    "molecules": "CTG, Alexa-568"
  },
  {
    "caption": "Time lapses of neurons loaded with Alexa-568 α-syn fibrils and cultured in control conditions or treated with 200 ng/mL Wnt5a. Neuron-to-neuron transfer of α-syn puncta (yellow arrows) through a TNT-like structure (yellow arrowheads) is shown upon Wnt5a treatment.",
    "molecules": "Alexa-568"
  },
  {
    "caption": "Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. Neurons were obtained from WT or βCaMKII K.O. mice. Yellow arrows point to α-syn puncta inside acceptor neurons in both conditions.",
    "molecules": "CTG, Alexa-568"
  },
  {
    "caption": "Growth curves of Hdac1-proficient and -deficient cell lines that were left untreated or were infected with empty virus (pLKO) or shRNAs against Dot1L and selected with puromycin. Growth curves were determined by a series of resazurin assays, which measure metabolic activity, starting four days post-infection. Error bars indicate the range of two replicates from independent cell lines.",
    "molecules": "puromycin, resazurin"
  },
  {
    "caption": "Inhibitor dose-response curves of the two DOT1L inhibitors EPZ-5676 (Pinometostat) and SGC-0946 in Hdac1-proficient and -deficient cell lines. Cell viability was measured by a resazurin assay after three days of treatment and measurements were normalized to DMSO-treated cells. Two independent cell lines are plotted separately; error bars indicate the range of two biological replicates.",
    "molecules": "DMSO, EPZ-5676, Pinometostat, resazurin, SGC-0946"
  },
  {
    "caption": "Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC-0946 for two days. Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live (Annexin V low; DAPI low), apoptotic (Annexin V high; DAPI low) and dead (Annexin V high; DAPI high) cells. Quantification of several independent FACS experiments, including the experiment shown in panel (A). Mean with individual data points of 2-4 replicates each of two independent lines per genotype. p<0.001 (***) by two-way ANOVA, comparison to corresponding DMSO control.",
    "molecules": "DAPI, DMSO, SGC-0946"
  },
  {
    "caption": "F In vitro kinase assay. Purified recombinant p62  (268-440 aa), p62  (320-440 aa), or mCherry-p62  was incubated for 20 min at 30 °C with purified SNAP-Atg1  or SNAP-ULK1  in the presence or absence of ATP. Reactions were then terminated by adding LDS sample buffer containing reducing agent, followed by immunoblot  analysis with the indicated antibodies. As positive and negative controls, Huh-1 cell lysates treated with or without lambda protein phosphatase (λPP) were used. Data were obtained from three independent experiments. Asterisks show possible dimeric structures of p62  (268-440 aa), p62  (320-440 aa) and mCherry-p62.",
    "molecules": "ATP"
  },
  {
    "caption": "A In vitro formation of p62-8xUb condensates. 10 μM SNAP-8xUb labelled with Alexa Fluor 649  was mixed with 10 μM mCherry-p62  wild-type, mCherry-p62S403E S407E, or mCherry-p62S349E and observed by fluorescence microscopy. Scale bars: 20 μm. The graph indicates the quantified area of p62  condensates formed by wild-type or mutant p62. Data are means ± s.d of wild-type p62, p62S403E, S407E and p62S349E condensates (n = 4). Statistical analysis was performed by Šidák's test after one-way ANOVA.",
    "molecules": "Alexa Fluor 649"
  },
  {
    "caption": "A Immunoblot  analysis. Huh-1 cells were treated with or without 2.5 µM MRT68921  for 6 h, and the cell lysates were subjected to immunoblot  analysis with indicated antibodies. The asterisk indicates non-specific bands. Data shown are representative of three separate experiments. Bar graphs show the results of quantitative densitometric  analysis of Ser349- or Ser403-phosphorylated p62  forms relative to total p62  (n = 3), and of Ser318-phosphorylated ATG13  relative to total ATG13  (n = 3). Data are means ± s.e. Statistical analysis was performed by Welch's t-test.",
    "molecules": "MRT68921"
  },
  {
    "caption": "B  Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 µM MRT68921  for 6 h and immunostained  with the indicated antibodies. The ratio of p62  (p-S403) to p62  on p62  bodies and the size and number of p62  bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).",
    "molecules": "MRT68921"
  },
  {
    "caption": "C  Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 µM MRT68921  for 6 h and immunostained  with the indicated antibodies. The ratio of p62  (p-S349) to p62  and the signal intensity of KEAP1  on p62  bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).",
    "molecules": "MRT68921"
  },
  {
    "caption": "D Gene expression of NRF2  targets. Total RNAs were prepared from Huh-1 cells treated with or without 2.5 µM MRT68921  for 6 h. Values were normalized against the amount of mRNA in non-treated Huh-1 cells.  qRT-PCR   analyses were performed as technical replicates on each biological sample. Data are means ± s.e. Statistical analysis was performed by two-sided Welch's t-test.",
    "molecules": "MRT68921"
  },
  {
    "caption": "G, H Hematoxylin  and eosin  (HE) staining  (G) and immunohistochemical  analysis of NQO1  (H) of livers  from  p62+/+ and p62S351E/+ mice  at P19. Scale bars, 100 μm (low magnification panels), and 10 μm (high magnification panels).",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "i Serum  levels of  aspartate aminotransferase (AST), alanine aminotransferase  (ALT), glucose, total cholesterol, blood urea nitrogen  (BUN), and creatinine  from p62+/+ (n = 4 at P12, n = 10 at P15) and p62S351E/+ (n = 4) mice  at P12 and P15 were measured. IU/l, international units/liter. Data are means ± s.e. Statistical analysis was performed by Tukey's test after one-way ANOVA.",
    "molecules": "blood urea nitrogen, BUN, cholesterol, creatinine, glucose"
  },
  {
    "caption": "(E) A. thalianaleaves were inoculated with wild-type, XccbphP, pXccBphP and pC13S strains, stained for callose deposits and observed by fluorescence microscopy. MgCl2 buffer (untreated) and flg22 peptide were used as negative and positive controls, respectively. Top panel: representative pictures of three independent experiments. Scale bar represents 200 µm. Bottom panel: the number of callose deposits per field of view (0.45 mm2) were determined (n = 8 replicates). Data are representative of two independent experiments. (A-E) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a two-tailed Mann-Whitney test (*P < 0.05, **P < 0.01, ***P < 0.001)",
    "molecules": "callose, MgCl2"
  },
  {
    "caption": "(A) Five µL supernatants from wild-type, XccbphP, pXccBphP and pC13 strains bacterial cultures (OD600 = 1) grown under light or dark conditions were plated onto PYM-carboxymethyl cellulose (CMC) agar plates and revealed with Congo redstaining (n = 2 replicates). The extracellular β-1,4-endoglucanase production levels correlated with CMC degradation halo radiuses (top panel). Halo measurements are presented in the bottom panel. Data derive from 10 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).",
    "molecules": "carboxymethyl cellulose, CMC"
  },
  {
    "caption": "(A) Five µL from wild-type, XccbphP, pXccBphP and pC13S bacterial cultures (OD600 = 1) were plated onto PYM-glucose agar plates under light or dark conditions. Xanthan production was determined measuring colony diameters (n = 4 replicates) for 20 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).",
    "molecules": "Xanthan, glucose"
  },
  {
    "caption": "(B) Wild-type, XccbphP, pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions. Extracellular xanthan was purified by KCl addition and ethanol precipitation, then dried and weighted (n = 2 replicates) for 4 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).",
    "molecules": "xanthan, ethanol, KCl"
  },
  {
    "caption": "A Analysis of MNase-digested chromatin DNA fragments from wild-type (WT, sizes indicated in black) and nap1Δ (N, sizes indicated in red) cells by agarose gel electrophoresis. Diagram illustrating reduction of array length in nap1Δ cells.",
    "molecules": "MNase, DNA"
  },
  {
    "caption": "B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq.",
    "molecules": "DNA"
  },
  {
    "caption": "D In vitro GST-pulldown assay showing the role of residues in the HBR interfaces in H2A-H2B binding. The mutant labeled HBR1 (lane 6) contains the triple mutation D201R, D205R, E310R and HBR2 (lane 12) the triple mutations E332, D333, E336. HBR1+2 (lane 13) contains all six mutations. The mutant labeled HBR1+2A (lane 14) has Alanine replacements in the six HBR residues. Top panel: Bound. Bottom panel: Input.",
    "molecules": "Alanine"
  },
  {
    "caption": "A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488- (left) or AF647-labeled DNA (right). 31 nM - 4 µM of yNap1-H2A-H2B (lanes 6-13) were incubated with a mixture of 0.8 µM AF488-labeled H3-H4-DNA tetrasome complexes and 0.8 µM AF647-labeled DNA (lane 5). Lanes 1-3 show migration of tetrasomes (tetra), nucleosomes (nucl) or free H2A-H2B obtained by salt deposition onto AF647-labeled DNA.",
    "molecules": "DNA"
  },
  {
    "caption": "B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.",
    "molecules": "DNA"
  },
  {
    "caption": "C Analysis of MNase-digested chromatin DNA fragments from wild-type (BY4741), nap1Δ, and nap1Δ cells complemented with different yNap1 expression plasmids. Total MNase units per 50 ml of culture are indicated on the bottom of the lanes. Kb+: DNA Marker.",
    "molecules": "MNase, DNA"
  },
  {
    "caption": "Purified GST-tagged CARM1 was pulled down with the Flag-LSD1 purified from HEK-293T cells. The amounts of GST and GST-tagged CARM1 were visualized by Coomassie Blue staining (asterisk: GST-CARM1).",
    "molecules": "Coomassie Blue"
  },
  {
    "caption": "E In vitro methylation assays. Purified fusion protein of GST-tagged LSD1 WT or the four methylated arginine mutants (R-A), or four methylated arginine mutants in combination (4RA), were incubated with GST-CARM1 in the presence of 3H-SAM. Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining (arrows: the position of LSD1; arrowheads: the position of CARM1).",
    "molecules": "arginine, Coomassie Blue, SAM, 3H"
  },
  {
    "caption": "F Increasing amounts of GST-CARM1 incubated with GST-LSD1 in presence of SAM at 30°C for 1 h. And methylation of LSD1 was analyzed by immunoblotting with anti-LSD1 R838me2a antibody.",
    "molecules": "SAM"
  },
  {
    "caption": "G GST-CARM1 incubated with GST-LSD1 WT or GST-LSD1 R838A/R838K in presence of SAM at 30°C for 1 h, followed by immunoblotting with anti-LSD1 R838me2a to detect LSD1 methylation.",
    "molecules": "SAM"
  },
  {
    "caption": "D MDA-MB-231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies.",
    "molecules": "MS049"
  },
  {
    "caption": "E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.",
    "molecules": "DMSO, MS049, MG132"
  },
  {
    "caption": "F MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by western blotting.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "G MDA-MB-231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by western blotting.",
    "molecules": "CHX"
  },
  {
    "caption": "H MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-LSD1 antibody and then analysed by immunoblotting.",
    "molecules": "MG132"
  },
  {
    "caption": "I MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti-LSD1 antibody.",
    "molecules": "MS049, MG132"
  },
  {
    "caption": "J HEK-293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time. LSD1 were examined by western blotting analysis.",
    "molecules": "CHX"
  },
  {
    "caption": "K HEK-293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-Flag antibody and then subjected to immunoblotting.",
    "molecules": "MG132"
  },
  {
    "caption": "L HEK-293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co-IP assay was performed using anti-Flag and then subjected to western blotting.",
    "molecules": "MS049, MG132"
  },
  {
    "caption": "B MDA-MB-231 cells were treated with the indicated amounts of P5091 for 48 h, LSD1 protein levels were assessed by immunoblotting.",
    "molecules": "P5091"
  },
  {
    "caption": "C MDA-MB-231 cells were treated with P5091 (10 μM) for 48 h and MG132 for 10 h. The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti-LSD1 antibody.",
    "molecules": "P5091, MG132"
  },
  {
    "caption": "D HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-LSD1 antibody followed by immunoblotting analysis.",
    "molecules": "MG132"
  },
  {
    "caption": "E MDA-MB-231 cells overexpressing Flag-tagged LSD1 WT/R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated by anti-Flag antibody and then analysed by immunoblotting.",
    "molecules": "P5091, MG132"
  },
  {
    "caption": "F MDA-MB-231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h. And cell lysates were immunoprecipitated with anti-LSD1 then subjected to immunoblotting.",
    "molecules": "MG132"
  },
  {
    "caption": "G MDA-MB-231 cells were treated with or without MS049 for 48 h and MG132 for 10 h, Cell lysates were immunoprecipitated using anti-LSD1 antibody and then analysed by immunoblotting.",
    "molecules": "MS049, MG132"
  },
  {
    "caption": "H HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with or without MS049 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated with anti-LSD1 antibody followed by immunoblotting analysis.",
    "molecules": "MS049, MG132"
  },
  {
    "caption": "Migration assays (24 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (A) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).",
    "molecules": "MS049"
  },
  {
    "caption": "invasion assays (48 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (B) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).",
    "molecules": "MS049"
  },
  {
    "caption": "Migration assays (24 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (C) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).",
    "molecules": "MS049"
  },
  {
    "caption": "D invasion assays (48 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (D) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).",
    "molecules": "MS049"
  },
  {
    "caption": "E MM-231-shLSD1-WT/R838A/R838K/R838F cells (1×106) were injected into the tail veins of 5-week-old female nude mice, and 7 weeks later, mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions. The number of visible surface metastatic lesions in lungs was counted (mean of 7 mice) (Data are mean ± SD, n = 7 mice for each group, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).",
    "molecules": "paraformaldehyde"
  },
  {
    "caption": "F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.",
    "molecules": "eosin, haematoxylin"
  },
  {
    "caption": "MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (A) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).",
    "molecules": "P5091, DNA, MS049"
  },
  {
    "caption": "B MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (B) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).",
    "molecules": "P5091, DNA, MS049"
  },
  {
    "caption": "MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the E-cadherin (C) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).",
    "molecules": "P5091, MS049"
  },
  {
    "caption": "D MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the vimentin (D) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).",
    "molecules": "P5091, MS049"
  },
  {
    "caption": "MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (E) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).",
    "molecules": "H3K9me2, P5091, DNA, MS049"
  },
  {
    "caption": "F MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (F) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).",
    "molecules": "H3K9me2, P5091, DNA, MS049"
  },
  {
    "caption": "C RNA-decay rates of WT and 402C>G FOXL2 mRNAs in KGN cells were determined after treatment with 5 µg/mL ActD for the indicated times. The estimated half-lives of each transcript are presented. The data are presented as the mean ± SEM from three independent experiments.",
    "molecules": "ActD"
  },
  {
    "caption": "G In vitro annealing kinetics of miR-1236 with 230 nt-long transcripts of WT or variant FOXL2. 32P-labeled miR-1236 (0.5 nM) was incubated with increasing concentrations of synthetic FOXL2 transcripts (0, 2.5, 12.5, 25, or 50 nM). FOXL2 mRNA-miR-1236 complexes were resolved on a 6% native gel and detected by autoradiography (left). The predicted Kds for the WT and 402C>G FOXL2 transcripts are presented in the right graph.",
    "molecules": "32P"
  },
  {
    "caption": "D Association of endogenous miR-1236 with RISC components in KGN cells was determined via pulldown assays using immobilized 2′-O-methylated oligonucleotides (2′-O-Me oligos) complementary to miR-1236 followed with a pulldown using streptavidin-coupled Dynabeads and western blot analyses (top). Relative quantification of bound proteins compared with proteins from the input is presented as fold enrichment (bottom). Efficient pulldown of endogenous miR-1236 using the 2′-O-Me oligos was confirmed with depleted miR-1236 in the discarded supernatant following the pulldown (Appendix Fig S8). As a control, 2′-O-Me oligos not complementary to miR-1236 were used. The data are the means ± SEM from three independent experiments. The p values were analyzed by unpaired, two-tailed Student's t-test (*p < 0.05, **p < 0.01).",
    "molecules": "streptavidin"
  },
  {
    "caption": "F We examined whether DHX9 affected the association between miR-1236 and AGOs. After transfecting KGN cells with control siRNA or siDHX9, the total RNA and AGOs-mediated RISC-associated RNAs were isolated following immunoprecipitations using anti-AGO3 or anti-AGO2 antibodies. The AGOs-immunoprecipitated RNAs were extracted using an acidic phenol:chloroform mixture (5:1, pH 4.3) and precipitated with isopropanol using 10% of 3 M NaOAc (pH 5.2). The enrichment of miR-1236 within miRISCs was detected using the TaqMan® microRNA assay in the immunoprecipitated RNAs and normalized using the level of total miR-1236. The data (means ± SEM) are presented as the fold enrichment calculated from three independent experiments. Different letters denote statistically significant differences (p < 0.05; Student-Newman-Keuls test).",
    "molecules": "chloroform, phenol, isopropanol, NaOAc"
  },
  {
    "caption": "A-C. The cross-presentation ability after incubation with A) soluble OVA, B) OVA/BSA-coated beads and C) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS-II DCs was evaluated with the B3Z hybridoma. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. A) ***P = 0.0001 and B) P = 0.1432 (ns); **P = 0.0044. The two-tailed Student's unpaired t test was performed.",
    "molecules": "SIINFEKL"
  },
  {
    "caption": "H-I. The cross-presentation capacity after the incubation with I) the SIINFEKL control peptide at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t test was performed.",
    "molecules": "SIINFEKL"
  },
  {
    "caption": "J. Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS-II DCs and T cell activation was determined 2 h later with the B3Z hibridoma. To control endogenous MHC-I antigen presentation specificity, DCs were also treated with brefreldin A (BFA). The use of this drug markedly reduced CD8+ T cell response to similar levels obtained by DCs without any antigen (∅). Data represent mean ± SEM of triplicate values and are representative of three independent experiments.",
    "molecules": "befreldin A, BFA"
  },
  {
    "caption": "K. Immunoblotting showing the amount of OVA incorporated by Scramble (a) and Rab22a KD (b) JAWS-II DCs after electroporation and BFA treatment.",
    "molecules": "BFA"
  },
  {
    "caption": "B. Confocal microscopy images of HeLa, RPE, and U2OS cell lines that were pre-extracted with Triton X-100 before fixed with formaldehyde and co-stained with DAPI and antibodies against PCNA and FAM111A. Scale bars: 10 μm",
    "molecules": "DAPI, formaldehyde, Triton X-100"
  },
  {
    "caption": "HEK293, RPE, or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293), 48 h (RPE), or 72h (U2OS). The colonies were fixed then stained with crystal violet.",
    "molecules": "crystal violet, doxycycline"
  },
  {
    "caption": "Cell-cycle profiles of HEK293, RPE and U2OS FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.",
    "molecules": "doxycycline"
  },
  {
    "caption": "C. HEK293 cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293) Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3). *p<0.05, **p<0.01, ****p<0.0001 (two-tailed unpaired t-test).",
    "molecules": "doxycycline"
  },
  {
    "caption": "D. Cell-cycle profiles of HEK293 FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A. HEK293 or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from 105 cells was resolved by SDS-PAGE, and probed in western blots for both endogenous FAM111A (71 kDa) and MycBioID-FAM111A (110 kDa) as well as full length and cleaved PARP (116 and 89 kDa, respectively). The Tubulin western blot is a sample loading control.",
    "molecules": "doxycycline, SDS"
  },
  {
    "caption": "B. HEK293/MycBioID-FAM111A cell lines with no treatment (- Dox), or treated with doxycycline, along with DMSO or 100 μM Z-VAD-FMK for 24 h (total inhibition of PARP cleavage was also observed when 50 μM Z-VAD-FMK was used). Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots with Myc, PARP, or Tubulin antibodies.",
    "molecules": "DMSO, doxycycline, Dox, SDS, Z-VAD-FMK"
  },
  {
    "caption": "C. HEK293 cell lines were treated with doxycycline and Z-VAD-FMK for 24 h. The colonies were fixed then stained with crystal violet. Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3).",
    "molecules": "crystal violet, doxycycline, Z-VAD-FMK"
  },
  {
    "caption": "C. Cells were treated with doxycycline along with DMSO or Z-VAD-FMK for 24 h. They were fixed with formaldehyde, and stained with NPC antibody Mab414 and DAPI. The images were captured by confocal microscopy. Scale bar: 10 μm.",
    "molecules": "DAPI, DMSO, doxycycline, formaldehyde, Z-VAD-FMK"
  },
  {
    "caption": "D. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from an equal number of cells was incubated with magnetic streptavidin beads, and pull downs were analyzed by western blotting using GANP and Myc antibodies.",
    "molecules": "DMSO, doxycycline, Z-VAD-FMK"
  },
  {
    "caption": "E. HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h, while growing in the presence or absence of 50 mM supplemental biotin. Total protein from whole cell lysates was incubated with magnetic streptavidin beads. Pulldowns were analyzed by western blotting using GANP and Myc antibodies.",
    "molecules": "biotin, doxycycline"
  },
  {
    "caption": "F. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots using antibodies against NPC (Mab414), PARP, cleaved Caspase 3, Myc, or Tubulin.",
    "molecules": "DMSO, doxycycline, SDS, Z-VAD-FMK"
  },
  {
    "caption": "A. Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA. Images were taken with a Zeiss Axio Imager. LT is shown in green, and DAPI blue. Scale bar: 100 μm. B. Quantification of LT positive cells described in A and fixed at indicated times post transfection. Five hundred or more cells were counted for each condition. C. Quantification of RFP marker distribution upon co-transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA. Cells were fixed at the indicated times. Five hundred or more cells were counted for each condition. In B & C, values are mean ± s.d. of independent experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired t-test). ",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Hermaphrodites sterilized via FUdR treatment produce no exophers or only a few per animal. n = 118 and 112 animals; N = 3.",
    "molecules": "FUdR"
  },
  {
    "caption": "(E) Males and sterile hermaphrodites (via FUdR treatment) show the formation of spherical structures in the BWM that resemble mature exophers. MOM - mitochondrial outer membrane.",
    "molecules": "FUdR"
  },
  {
    "caption": "A. Bclaf1  wild type (WT) and two Bclaf1 knockout HeLa cell lines (KO-1 and -2) were pre-treated with  DMSO or cycloheximide  (CHX, 1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) together with CHX for 12 hours. Afterwards, the cells were subjected to Annexin V/7AAD staining  followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis, or protein extractions followed by western blotting analysis. B, C. HeLa (B) or primary MEFs (C) were transfected with control (siCtrl) or two siRNAs against Bclaf1 (siBclaf1-1 and -2) and then treated and analyzed as described in A. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.",
    "molecules": "7AAD, CHX, cycloheximide, DMSO"
  },
  {
    "caption": "D, E. MEFs transfected with control (siCtrl) or siBclaf1-1 and/or siBclaf1-2 siRNAs were pre-treated with  CHX (1 μg/ml)/z-VAD (20 μM) (D) or SM-164 (100 nM)/z-VAD (20 μM) (E) for 30 min, and then added TNF (10 ng/ml) for indicated times. The cells were subjected to  Annexin V/7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and  Annexin V  negative cells (up), or protein extractions and western blotting analysis at indicated time periods (down). Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test. ",
    "molecules": "7AAD, CHX, SM-164, z-VAD"
  },
  {
    "caption": "G, H. HeLa  cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with  TNF (10 ng/ml) in the presence of  CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H). Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test.",
    "molecules": "7AAD, CHX"
  },
  {
    "caption": "E, HeLa  cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX  (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by  western blotting",
    "molecules": "CHX, DMSO"
  },
  {
    "caption": "siRNAs against Bclaf1  and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B) and Hematoxylin-and-eosin staining (C) Bclaf1 knockdown efficiency is detected by  immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, 100 μm in C.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "D. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "E. Localization of  P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "C. Super resolution (STED) imaging of PhiL1-GFP and CSP as well as concavin-GFP and CSP. Cells were stained with an anti-GFP antibody in combination with Atto-594  (green) in addition to an anti-CSP staining in combination with Atto-647. Images were deconvolved using the Richardson-Lucy algorithm. The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji. Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. Scale bar: 1 µm.",
    "molecules": "Atto-594, Atto-647"
  },
  {
    "caption": "D. Localization of PhiL1-GFP (green) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. E. Localization of SiR-Tubulin (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. F. Localization of CSP (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. Data information: Asterisks in D-F indicate apical part of the sporozoite.",
    "molecules": "Hoechst, SiR"
  },
  {
    "caption": "F, G. Only normally shaped concavin(-) sporozoites  released by salivary glands  move on helical paths (arrows) through polyacrylamide gels that mimic the skin (F). Quantification of two individual experiments Scale bar 10 µm. (G): only normal shaped sporozoites were able to migrate through the gel.",
    "molecules": "polyacrylamide"
  },
  {
    "caption": "b, Peptide hydrolysis activity of 20S and 26Sproteasomes. Homogenates from P28Atg7flox/+; nestin-Cre (blue) and Atg7flox/flox; nestin-Cre (pink) brains were fractionated by glyceroldensity gradient centrifugation (10-40% glycerol from fraction 1 to fraction 30). Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.05% SDS. The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads. Note that whereas 26Sproteasomes exist in active forms in tissues, 20Sproteasomes are latent and are activated artificially by a low concentration of SDS.",
    "molecules": "glycerol, SDS"
  },
  {
    "caption": "c, ATP-dependent degradation of [35S]-labelled ODC. Degradation of [35S]-labelled ODC was assayed using crude extracts from P28 Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains. The experiment was repeated three times, and values represent mean ± s.d. In the above assays, there were no significant differences between Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice.",
    "molecules": "ATP"
  },
  {
    "caption": "(A-D) The posterior midguts of 10-day-old control (A), HP1c knockdown (B), HP1c null mutant (C) and HP1c overexpression flies (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-EB, EB-EB or ISC-ISC nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(E-I) Confocal images of the posterior midgut clones of the indicated genotypes, marked by GFP (dotted lines). Arrows mark ISCs/EBs, arrowheads mark ee cells, and asterisks mark ECs. DNA was visualized with DAPI and ee cells were stained with Pros. Scale bars, 10 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(A-D) Compared with the control (A) flies, posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells (B), while HP1c OE flies show increased ISCs and ee cells (C), and the phenotype of Notch OE can be rescued by HP1c OE (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-ISC, ISC-EB or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "Bright-field image of depot-derived cell lines after in vitro adipogenic differentiation and Oil Red O staining (Lee et al., 2017a). Original photographs were taken at 10x magnification (left panel). Scale bar = 100 μm.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods. Data are shown as mean ± SEM of 3-7 samples, and the entire experiment was repeated twice.",
    "molecules": "Triglyceride"
  },
  {
    "caption": "[3H]-labeled 2-deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples and repeated twice. [3H]-labeled 2-deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.",
    "molecules": "2-deoxyglucose, 3H"
  },
  {
    "caption": "[14C] D-glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. [14C] D-glucose incorporation was measured after three hours, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.",
    "molecules": "lipids, 14C, D-glucose"
  },
  {
    "caption": "Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation. Data are shown as mean ± SEM of 3-7 replicates and normalized by lipid content of the cells. The entire experiment was repeated twice.",
    "molecules": "glycerol, isoproterenol, lipid"
  },
  {
    "caption": "Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.",
    "molecules": "oxygen"
  },
  {
    "caption": "Bright-field image of immortalized mGFP and mTomato positive preadipocytes after in vitro adipogenic differentiation. Neutral lipoids were stained with Oil Red O and original photographs were at 10x magnification (left panel). Scale bar = 100 μm.",
    "molecules": "lipoids, Oil Red O"
  },
  {
    "caption": "Triglyceride content of immortalized mGFP and mTomato positive preadipocytes after six days of differentiation. Data are shown as mean ± SEM of 5-8 replicates, and the entire experiment was repeated with three independent cell lines.",
    "molecules": "Triglyceride"
  },
  {
    "caption": "Basal respiration of preadipocytes was of immortalized mGFP and mTomato positive preadipocytes determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated twice. Data are shown as mean ± SEM of 3 cell lines per group.",
    "molecules": "oxygen"
  },
  {
    "caption": "(d) Wild-type and pep4Δ cells expressing RPL25-GFP were analysed as in b; 0.1 mM of PMSF was added to pep4Δ cells at the time of starvation to block all vacuolar proteases completely. The LI-COR imaging system was used to quantify the ratio between cleaved GFP and full-length protein as described in Methods. The last time point of wild-type cells containing RPL25-GFP was set to 1 arbitrary unit (a.u.). One of two independent experiments is shown.",
    "molecules": "PMSF"
  },
  {
    "caption": "(a) The vacuolar localization of GFP-Atg8p was analysed in more than 100 wild-type, ubp3Δ, bre5Δ and atg7Δ cells containing GFP-ATG8 after 10-15 h of nitrogen starvation (right panel). Corresponding GFP and phase contrast images of representative cells are shown in the left panel.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(c) Wild-type, ubp3Δ and bre5Δ strains containing APE1-RFP were analysed by RFP microscopy (left) and phase contrast (right) 15 h after nitrogen starvation. Scale bars, 5 μm.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(a) Wild-type, ubp3Δ or bre5Δ strains containing either a control plasmid (pRS315) or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with (left) or without (right) 5 nM rapamycin. The plates were photographed after 2 days at 30 °C.",
    "molecules": "YPD, rapamycin"
  },
  {
    "caption": "(b) Induction of a GCN4-LacZ reporter was measured 0, 2 and 4 h after amino-acid starvation, and is plotted as Miller units.",
    "molecules": "amino-acid"
  },
  {
    "caption": "(c) Polysome profiles of exponentially growing wild-type and ubp3Δ cells were determined in rich medium or 3 h after nitrogen starvation. The arrows mark the 60S peak.",
    "molecules": "nitrogen"
  },
  {
    "caption": "Localization of R-LPS (green) and S-LPS (red) in exponential phase bacteria. Scale bars: 2 μm. Distribution of R-LPS and S-LPS in exponential phase culture analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events). Numbers in each corner corresponds to % of relative frequencies.",
    "molecules": "LPS"
  },
  {
    "caption": "Roughness measurements on R-LPS labeled (green) WT and Δgmd cells. AFM images of whole bacteria and of the separated areas (coloured squares in first images, 0.4 x 0.4 μm2) are shown. The arithmetic roughness Ra is indicated below each area. Scale bars: 1 μm. Quantitative roughness measurement of R-LPS labeled WT and Δgmd cells. The areas with the lower roughness (more regular surfaces) of Δgmd cells were assigned as areas 1. mAb: monoclonal antibody. Ab: antibody. nWT = 13 bacteria. nΔgmd = 7 bacteria. Differences were statistically analyzed by t-test. **P < 0.01. ns: not significant.",
    "molecules": "LPS"
  },
  {
    "caption": "Detection of forces between AFM tip functionalized with mAb against R-LPS and unlabeled WT cells. Height image and the corresponding adhesion image are shown on top and zooms of depicted areas (black squares, 0.4 x 0.4 μm2) below. The arithmetic roughness Ra and percentage of adhesion Padh are indicated below each area. Scale bar: 1 μm. Correlation between roughness and detection of R-LPS on unlabeled WT cells. The areas with the lower roughness (more regular surfaces) were assigned as areas 1. The boxplot represents the mean values (squares), the median (horizontal line), the 25% and 75% quartiles (box limits) and the standard deviations (whiskers). n = 9 bacteria. Error bars: s.d. Statistical differences were analyzed by t-test. **P < 0.01. *P < 0.05.",
    "molecules": "LPS"
  },
  {
    "caption": "Co-localization of Omp2b (red) and R-LPS (green) in exponential phase bacteria. Scale bars: 2 μm.",
    "molecules": "LPS"
  },
  {
    "caption": "Co-detection of Omp2b (red) and R-LPS by AFM. R-LPS was detected using AFM tips functionalized with mAb against R-LPS. AFM images of whole bacteria and of the separated areas (red squares in first images, 0.4 x 0.4 μm2) are shown. The percentages of adhesion Padh are indicated below each area. Scale bars: 1 μm.",
    "molecules": "LPS"
  },
  {
    "caption": "Localization of initially labeled Omp25 (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). As a control, Omp25 localization after pulse-chase labeling with TRSE is shown at the bottom panel. Scale bars: 2 μm.",
    "molecules": "TRSE"
  },
  {
    "caption": "Localization of initially labeled Omp2b (green) on TRSE-labeled (red) bacteria before (0 h) and after 2 h of growth in the absence of both labelings (2 h). Scale bars: 2 μm.",
    "molecules": "TRSE"
  },
  {
    "caption": "Co-localization of Omp25 and Omp2b positive pixels with TRSE positive or negative pixels, respectively. Error bars correspond to s.d. from independent experiments. Differences were statistically analyzed by t-test. ***P < 0.001. nOmp25 = 279 bacteria, 85666 pixels (5 biological replicates). nOmp2b = 252 bacteria, 74630 pixels (3 biological replicates).",
    "molecules": "TRSE"
  },
  {
    "caption": "Short pulse labeling of exponential phase bacteria with the fluorescent D-amino acid HADA. Merge is showing old pole marker PdhS-mCherry (red) and PG insertion sites (white). Scale bars: 2 μm.",
    "molecules": "HADA, D-amino acid, PG"
  },
  {
    "caption": "Demographic representation of HADA labeling. Bacteria were sorted according to their cell length and oriented with the old pole at the bottom of the demograph by PdhS-mCherry fluorescence signal. NFI: normalized fluorescence intensity. n = 393 bacteria (3 biological replicates).",
    "molecules": "HADA"
  },
  {
    "caption": "Localization of S-LPS (green) in the eFluor-labeled (magenta) inducible rough strain Δgmd plac-gmd possessing the old pole marker PdhS-mCherry (red). The strains Δgmd and Δgmd plac-gmd -IPTG were used as negative controls. Scale bars: 2 μm.",
    "molecules": "eFluor, IPTG, LPS"
  },
  {
    "caption": "Demograph represents fluorescence profile of S-LPS labeling from (A). Cells were oriented with the old pole at the bottom of the graph by PdhS-mCherry and sorted by cell length. NFI: normalized fluorescence intensity. n = 468 bacteria (3 biological replicates).",
    "molecules": "LPS"
  },
  {
    "caption": "Short pulse labeling of LPS (2 h) by Kdo-N3 (green) on eFluor-labeled (magenta) bacteria expressing the old pole marker PdhS-mCherry (red). Scale bars: 2 μm.",
    "molecules": "eFluor, Kdo-N3, LPS"
  },
  {
    "caption": "Demographs represent fluorescence intensities of eFluor (OM, left) and Kdo-N3 (LPS, right) labeling. Cells were sorted by cell length and aligned with the old pole at the bottom of the graph by PdhS-mCherry localization. NFI: normalized fluorescence intensity. n = 566 bacteria (3 biological replicates).",
    "molecules": "eFluor, Kdo-N3, LPS"
  },
  {
    "caption": "Distribution of R-LPS and S-LPS in Δgmd plac-gmd after 3, 6, 9 and 24 h post-induction analyzed by flow cytometry (one representative example among 3 biological replicates, n = 20,000 events).",
    "molecules": "LPS"
  },
  {
    "caption": "Quantification of S-LPS labeling from (A) as relative frequencies. Error bars correspond to s.d. from 3 independent experiments. n0h = 77 bacteria, n3h = 254 bacteria, n6h = 616 bacteria, n9h = 937 bacteria, n24h = 1059 bacteria.",
    "molecules": "LPS"
  },
  {
    "caption": "SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 (A,B)independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4 for A,B)",
    "molecules": "nutrient"
  },
  {
    "caption": "SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 (A,B) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4 for A,B)",
    "molecules": "nutrient"
  },
  {
    "caption": "SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).",
    "molecules": "nutrient"
  },
  {
    "caption": "SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).",
    "molecules": "nutrient"
  },
  {
    "caption": "SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with silver, to detect all proteins (A),. A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.",
    "molecules": "nutrient"
  },
  {
    "caption": "SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with Pro-Q® Diamond, which stains phosphorylated proteins (B). A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.",
    "molecules": "nutrient"
  },
  {
    "caption": "(J) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or shifted to different starvation media for 30 minutes. These include both the removal of the nitrogen source, ammonium chloride (NH4Cl), and the reduction of the carbon source, glucose, from a concentration of 2% to 0.5%. Alternatively, cells were either only deprived of NH4Cl or only exposed to reduced glucose levels (2% to 0.5%).",
    "molecules": "ammonium chloride, NH4Cl, glucose"
  },
  {
    "caption": "(K) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or deprived of NH4Cl for one hour. Then, NH4Cl was added back to the medium for 30 or 60 minutes. Both short and long exposures of the FLAG IBs are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. Anti-tubulin IBs are shown as controls for loading between samples. Shown are star that are representative of at least 3 independent experiments. The number sign (#) symbol identifies antibody heavy chains and the star (*) symbol labels an unspecific band detected by the anti-FLAG antibody in S. pombe.",
    "molecules": "NH4Cl"
  },
  {
    "caption": "(E) FLAG-tagged Taf12 and TAP-tagged Pab1 were affinity purified separately from cells grown in rich conditions (upper panels). TAP-tagged Pab1 was cleaved off from beads using the Tobacco Etch Virus (TEV) protease, releasing a shorter form of Pab1 (CBP-Pab1) in the eluate (E). CBP-Pab1 was then mixed with beads containing FLAG-Taf12 IPs and incubated in a phosphatase buffer, with and without 0.5 µM microcystin. Each reaction was then analyzed by IB and simultaneously probed with anti-FLAG or anti-CBP antibodies (lower panel).",
    "molecules": "microcystin"
  },
  {
    "caption": "(F) Exponentially growing cells were treated for 1 hour with Torin1, which was added at increasing concentrations, 7 and 21 µM, to rich medium. Dimethylsulphoxide (DMSO) was used as the vehicle and added as a negative control.",
    "molecules": "Torin1"
  },
  {
    "caption": "Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments.",
    "molecules": "17β‐estradiol, R5020"
  },
  {
    "caption": "A Whole‐mount microscopy of X‐gal‐stained Axin2+/lacZ in E12.5 embryo showing β‐galactosidase expression in the mammary buds (arrows) (n = 8). Scale bar: 1 mm. Arrowheads mark mammary buds.",
    "molecules": "β‐galactosidase"
  },
  {
    "caption": "B-F Whole‐mount microscopy of X‐gal (blue)‐ and carmine alum (red)‐stained mammary glands harvested from Axin2+/lacZ mice at distinct developmental stages. (B) At postnatal day 1, β‐galactosidase activity detected in the nipple area (n = 6). Scale bar: 1 mm. (C, D) In 5‐week‐old mammary glands, reporter activity was detected around the ducts (small arrows) and in the neck region of the terminal end buds (TEBs) (large arrows) (C) (n = 8). Scale bars: 400 μm (C) and 100 μm (D). (E) At 8.5 day of pregnancy, reporter expression was detected in the ducts. Higher magnification (inset) suggests myoepithelial expression (n = 10). Scale bar: 1 mm. (F) Whole‐mount at day 14.5 of pregnancy: reporter activity is limited to ducts (n = 5). Scale bar: 200 μm.",
    "molecules": "β‐galactosidase"
  },
  {
    "caption": "G Histology section of Axin2::LacZ mammary gland at day 8.5 of pregnancy counterstained with nuclear red; luminal epithelial cells show no detectable β‐galactosidase activity but myoepithelial cells do. Scale bar: 200 μm.",
    "molecules": "β‐galactosidase"
  },
  {
    "caption": "H β‐galactosidase activity (blue) colocalizes with the myoepithelial marker p63 (green) detected by immunofluorescence microscopy. Arrows point to myoepithelial cells. L, lumen. Scale bar: 100 μm.",
    "molecules": "β‐galactosidase"
  },
  {
    "caption": "K Stereo microscopy of X‐gal‐ and carmine alum‐stained mammary glands from ovariectomized Axin2::LacZ females treated for 72 h with vehicle (n = 4) (left), 17‐β‐estradiol (E2) (n = 6) (center), 17‐β‐estradiol and progesterone (E2 and P) (n = 8) (right). Scale bar: 200 μm.",
    "molecules": "17‐β‐estradiol, progesterone"
  },
  {
    "caption": "L, M Stereo microscopy of contralateral glands whole‐mounted and X‐gal stained after engraftment with mammary buds from Axin2::LacZ transgenic and Wnt4+/+ or Wnt4−/− female E12.5 and E13.5 embryos (L) or Axin2::LacZ transgenic and either PR+/− or PR−/− 8‐week‐old females (M), at day 8.5 of pregnancy. β‐galactosidase expression reflecting Axin2 transcription is readily detected in PR+/− as well as Wnt4+/+ mammary epithelia but not in the PR−/− (n = 6) and Wnt4−/− counterparts (n = 6). Blue: X‐gal staining; magenta: carmine alum counterstain. Scale bars: 200 μm.",
    "molecules": "β‐galactosidase"
  },
  {
    "caption": "I-K. Time-lapse of SKIP-mediated transport of late endosomes. I. Schematic representation of tamoxifen-induced activation of SKIP onto endosomal membranes. J/K. Live HeLa cells co-expressing GFP-ER-SKIP (green) and mCherry-Rab7 (magenta) together with HA-RILP (unstained) expressed at low levels (cells transfected at 1:5 RILP:SKIP ratio) were imaged in the J. absence or K. presence of tamoxifen, allowing on-demand association of SKIP with endosomal membranes. Confocal frames from time-lapses taken at the indicated time-points following treatment are shown. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10µm",
    "molecules": "tamoxifen"
  },
  {
    "caption": "A/B. In situ SKIP/TBC1D15 complex formation assayed using proximity-based biotin ligation (BioID). A. Neutravidin precipitates (PD) from biotin-treated HEK293T cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with HA-BioID-SKIP or HA-BioID-EV. Representative immunoblots against GFP, HA and VPS18 , TL: total lysate. B. Quantification of biotinylation of GFP-TBC1D15 (top graph) and endogenous VPS18 (eVPS18, bottom graph) by BioID-SKIP, expressed relative to BioID-EV (1.0), n=4 independent experiments.",
    "molecules": "biotin, Neutravidin"
  },
  {
    "caption": "F. Neutravidin precipitates (PD) from biotin-treated HeLa cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with BioID-SKIP or BioID-EV. Representative immunoblots against GFP, HA and VPS18 are shown, TL: total lysate. G. Quantification of GFP-TBC1D15 biotinylation by BioID-SKIP over BioID-EV in response to VPS18 depletion expressed as % of control (siC), n=4 independent experiments performed in HeLa or HEK293T cells. H. Quantification of VPS18 protein abundance from experiments in G, n=4.",
    "molecules": "biotin, Neutravidin"
  },
  {
    "caption": "(E) H&E staining  of colon cancer tumours from GDH1IEC+/+ and GDH1IEC-/- mice  (scale bar, 200 μm). F F4/80 staining  in a noninflamed portion of the colon  after AMO/DSS  treatment (scale bar, 50 μm). G ZO-1 staining  in colon  sections of mice  treated with sequential AMO/DSS  treatment (scale bar, 200 μm).",
    "molecules": "AMO, DSS"
  },
  {
    "caption": "K Both HIF1α  protein levels and AMPK  activity were assessed in the intestine  of  GDH1IEC-/- mice  after AOM/DSS  treatment using the indicated antibodies.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "D The K503Q mutation reduces GDH1  activity leading to αKG  production while enhancing the  enzymatic activity leading to Glu  production. His-tagged GDH1  was purified from E. coli. (n=3). Data information: Data are mean ± SD from the biological replicates (C-G Statistics: one-way ANOVA with Tukey's HSD post hoc test",
    "molecules": "αKG, Glu"
  },
  {
    "caption": "F HCT116  or SW480  cells were depleted of endogenous GDH1  and rescued with SFB-tagged rGDH1 WT, K503Q or K503R. rGDH1-SFB  proteins were enriched using M2-FLAG  beads to determine the Vmax  of αKG  production (F). (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc test",
    "molecules": "αKG"
  },
  {
    "caption": "ITC  assays were performed with precipitated His-GDH1  proteins and αKG (I). (n=3). Data information: Data are mean ± SD from the biological replicates I). Statistics: one-way ANOVA with Tukey's HSD post hoc test I).",
    "molecules": "αKG"
  },
  {
    "caption": "A, B Immunoblot  of the HIF1α   protein level under hypoxia  for 6 hours when enzymes related to the αKG  level were overexpressed in cells, including GDH1  (A). Relative αKG  levels in contrast to the control (B). (n=3). Data information: Data are mean ± SD from the biological replicates (B Statistics: one-way ANOVA with Tukey's HSD post hoc test (B",
    "molecules": "αKG"
  },
  {
    "caption": "I EGLN1  activity test. His-GDH1  with K503 or K527 acetylation  (AcK503 or AcK527) and mimic acetylation  at K503 or K527 (K503Q or K527Q) were purified from  E. coli. Then, His-GDH1  was incubated for 1 hour with HA-EGLN1(I). EGLN1  activity was tested by converting αKG  to succinate. (n=3). Data information: Data are mean ± SD from the biological replicates Statistics: one-way ANOVA with Tukey's HSD post hoc test",
    "molecules": "αKG, succinate"
  },
  {
    "caption": "P HIF1α  transactivation was examined in the indicated cell lines under hypoxia  with or without treatment with the HIF1α  transactivation inhibitor 2-MeOE2 (1 μM) for 12 hours. (n=3). Data information: Data are mean ± SD from the biological replicates P). Statistics: one-way ANOVA with Tukey's HSD post hoc test , P)",
    "molecules": "2-MeOE2"
  },
  {
    "caption": "A, Cell viability in the indicated cell lines under hypoxia  were analysed by trypan blue (A) (n=3). Data information: Data are mean ± SD from the biological replicates (A, Statistics: two-way ANOVA with Tukey's HSD post hoc test (A",
    "molecules": "trypan blue"
  },
  {
    "caption": "A NTL-2-foci localize in close proximity to mitochondria in young adult animals; hypodermis is imaged (green: NTL-2, red: TMRE (Tetramethylrhodamine, ethyl ester, perchlorate), a mitochondrial membrane potential-dependent dye) (n=3 independent experiments). B Localization of NTL-2-foci relative to mitochondria in young adult animals; body wall muscle cells are imaged (green: NTL-2, red: TOMM-20, an outer mitochondrial membrane (OMM) protein-marker of mitochondria) (n=3 independent experiments). C EDC-3-foci localize in close proximity to mitochondria in young adult animals; body wall muscle cells are imaged (green: EDC-3, red: TMRE) under control conditions (n=3 independent experiments). D Localization of EDC-3-foci relative to mitochondria in young adult animals; body wall muscle cells are imaged (red: EDC-3, green: mitochondrial matrix targeted by GFP) (n=3 independent experiments).",
    "molecules": "Tetramethylrhodamine, ethyl ester, perchlorate, TMRE"
  },
  {
    "caption": "I EDC-3- and NTL-2-foci lose their specific localization close to mitochondria upon paraquat treatment of transgenic animals expressing mitochondria-targeted GFP , top: EDC-3-foci, bottom: NTL-2-foci (n=3 independent experiments).",
    "molecules": "paraquat"
  },
  {
    "caption": "L Quantification of the percentage of fluorescence recovery after photobleaching (FRAP) to measure the rates of de novo protein synthesis upon genetic inhibition of either ntl-2 or dcap-2 in 6-day-old-adult animals; the protein synthesis inhibitor cycloheximide (500μM) and let-363 genetic inhibition where used as controls. Measurements started after animals have recovered for 2hrs post photobleaching (n=3 independent experiments with at least 50 animals/experiment).",
    "molecules": "cycloheximide"
  },
  {
    "caption": "B Representative images showing the localization of NTL-2/storage bodies relative to mitochondria upon genetic inhibition of either tomm-20 or akap-1 (green: NTL-2, red: TMRE, a mitochondrial-membrane potential-dependent dye; n=3 independent experiments). Scale bars, 20 µm. Images were acquired using a X63 objective lens. C Quantification of the distances NTL-2/storage bodies acquire from mitochondria upon genetic inhibition of either tomm-20 or akap-1 (n=3 independent experiments with at least 45 animals/experiment; ***P< 0.001; one-way analysis of variance (ANOVA)).",
    "molecules": "TMRE"
  },
  {
    "caption": "A Percent survival of wild-type, DCAP-2- and NTL-2- depleted animals subjected to heat stress for 5 hrs at 37oC and then counted every 24 hours (n=4 independent experiments with at least 294 animals/experiment). B Percent survival of wild-type, DCAP-2- and NTL-2- depleted animals following paraquat (8mM) administration and counted every 24 hours (n=4 independent experiments with at least 144 animals/experiment). C Percent survival of wild-type, DCAP-2- and NTL-2- depleted animals counted every 24 hours post CCCP (15μM) treatment (n=4 independent experiments with at least 116 animals/experiment).",
    "molecules": "CCCP, paraquat"
  },
  {
    "caption": "(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml).",
    "molecules": "kanamycin"
  },
  {
    "caption": "(C, D Confocal  micrographs of HeLa  cells expressing GFP-TECPR1  and treated with osmotic shock assay or LLOMe  (C), or infected with the bacteria indicated (D) and fixed at 1 h (S. Typhimurium and L. monocytogenes) or 0.5 h (S. flexneri) post-infection. Cells were stained  for Galectin-8 and DNA (DAPI). Scale bar, 20 μm.",
    "molecules": "DAPI, LLOMe"
  },
  {
    "caption": "(F) Percentage of bacteria positive for mCherry-TECPR1 in ATG5  knockout (KO) or ATG5-complemented MEF  cells at 1 h (S. Typhimurium) or 0.5 h (S. flexneri) post-infection. Mean +/- SD of four (S. Typhimurium) or two (S. flexneri) independent experiments. n > 100 bacteria per coverslip. (G) Percentage of S. Typhimurium  positive for GFP-TECPR1  at 1 h post-infection  in HeLa  cells pretreated or not with 100 nM Wortmannin. Mean +/- SD of two independent experiments. n > 100 bacteria per coverslip.",
    "molecules": "Wortmannin"
  },
  {
    "caption": "H)  Confocal  micrographs of HeLa  cells expressing GFP-TECPR1  either alone or together with FLAG-tagged neutral sphingomyelinase 2  (nSMase2; (H)) Cells were stained  for Galectin-8  and DNA (DAPI). Scale bar, 20 μm. (I) Percentage of S. Typhimurium positive for GFP-TECPR1  or endogenous Galectin-8  at 1 h post-infection  in HeLa  cells expressing FLAG-nSMase2  or not. Mean +/- SD of two (TECPR1) or three (Gal8) independent experiments. n > 100 bacteria per coverslip.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Confocal  micrographs  of HeLa  cells expressing TECPR1  N' DysF-GFP  with or without FLAG-nSMase2, infected with S. Typhimurium, fixed at 30 min post-infection and stained  with anti-Galectin 8 antibody. DNA stained  with DAPI. Scale bar, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Confocal  micrographs of HeLa  cells expressing either GFP-TECPR1  WT or W154A and treated with LLOMe  or DMSO  vehicle control for 10 min and fixed. Scale bar, 20 μm. (G) Number of GFP-positive puncta per cell were enumerated in samples depicted in (F). Data from 50 cells from a single experiment, representative of two. p value from one-way ANOVA with Tukey's multiple comparisons test.",
    "molecules": "DMSO, LLOMe"
  },
  {
    "caption": "(C-E) Liposome sedimentation assay. The indicated proteins were incubated with either liposomes   (containing sphingomyelin  or not) or buffer only, pelleted by centrifugation and equal portions of supernatant  and pellet  fractions analysed by SDS-PAGE  and Coomassie staining. Representative gels depicted (C). Fraction of TECPR1  (D) or DysF  WT or W154A (E) proteins present in pellet  under the indicated conditions, expressed as % binding. Note pelleting in the absence of liposomes   likely due to affinity of proteins for tube walls. Mean +/- SD of three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.",
    "molecules": "Liposome, liposomes, sphingomyelin"
  },
  {
    "caption": "(F) In vitro LC3 lipid conjugation assay. Liposomes  containing sphingomyelin  or not were incubated with LC3 lipid conjugation machinery: ATG7  (E1), ATG3  (E2), ATG5-ATG12  (E3), LC3B, MgCl2, ATP  and the indicated TECPR1   proteins. Control reactions without ATG3  or ATP  were included. Mixes were incubated at 37 C for 3 h, centrifuged and equal fractions of both supernatant  and pellet  analysed by SDS-PAGE  and Coomassie staining. Lipidated LC3B-II  appears in pellet  fraction whereas both soluble  LC3B-I  and AMPylated LC3B  are in the supernatant, the latter of the same apparent molecular weight as  LC3B-II.",
    "molecules": "Liposomes, ATP, MgCl2, sphingomyelin"
  },
  {
    "caption": "(G) Fraction of LC3B  protein present in pellet from complete in vitro LC3 lipid conjugation assay with the indicated TECPR1  proteins in either absence or presence of sphingomyelin. Mean +/- SD from three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.",
    "molecules": "sphingomyelin"
  },
  {
    "caption": "(E) Percentage of S. Typhimurium positive for endogenous LC3  in indicated MEF  cells treated with 100 nM Wortmannin  or DMSO  vehicle control, fixed at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. p value from one-way ANOVA with Tukey's multiple comparisons test.",
    "molecules": "DMSO, Wortmannin"
  },
  {
    "caption": "(A) Percentage of S. Typhimurium  positive for  Galectin-8 and/or GFP-TECPR1  in either control Hela  cells or those expressing GFP-TECPR1  at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. (B) Confocal  micrographs  of HeLa  cells expressing the indicated GFP-TECPR1  constructs or not (control), infected with S. Typhimurium  for 30 min, fixed and stained  with anti-Galectin-8 and DAPI. Scale bar, 20 μm.­­ Arrowheads indicate bacteria positive for Galectin 8, of which more are present in cells expressing GFP-TECPR1  WT but not W154A. Arrow indicates bacterium to which GFP-TECPR1  but not Galectin 8 has been recruited.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type (HSD-wt) and Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. **p<0.005; ***p<0.0005. 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.",
    "molecules": "ATP, ROS"
  },
  {
    "caption": "(D) SCC13 cells were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with Zymosterol (2.5uM) or ethanol vehicle alone for 48hours. Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.",
    "molecules": "ATP, ethanol, ROS, Zymosterol"
  },
  {
    "caption": "Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).",
    "molecules": "ISO, isoproterenol, water"
  },
  {
    "caption": "Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).(E,F,K,L) For quantification of coIP, α11.2 and GluA1 signals were normalized to β2AR (***p<0.001, One Way ANOVA).",
    "molecules": "ISO, isoproterenol, water"
  },
  {
    "caption": "Hippocampal cultures were transfected with FLAG-β2AR and α11.2-HA at 6 days in vitro (DIV), treated with vehicle (water) or 1 M isoproterenol (ISO) for 5 min at 18 DIV, fixed and surface labeled for HA and FLAG.(A,B) Representative immunofluorescence microscopy images obtained by wide-field microscopy at lower and higher resolutions (scale bar in A is 5 m).(C) Quantification of distance between centers of HA and FLAG puncta (**p<0.001, Mann-Whitney Rank Sum Test).",
    "molecules": "ISO, isoproterenol, water"
  },
  {
    "caption": "Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated.(B,C) For quantification, α11.2 and GluA1 IB signals were normalized to β2AR (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (A, lanes 2-6) lasted at least 3 min but not 20 min (compare lanes 6 and 7).",
    "molecules": "ISO, water"
  },
  {
    "caption": "Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).",
    "molecules": "ISO, water"
  },
  {
    "caption": "Forebrain slices from S1928A KI mice were treated with vehicle (lanes 1,5) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) α11.2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 and middle part for pS845, pS831, and total GluA1. In S1928A KI mice, S1700 re-phosphorylation after a 3 min wash out of ISO was not blunted (lane 6) in contrast to WT mice but rather augmented (compare to lane 5).(B-E) For quantification, pS1928 (virtually absent) and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ***p<0.001, ANOVA).",
    "molecules": "ISO"
  },
  {
    "caption": "(A) Representative single channel recordings from hippocampal neurons at 7-14 DIV with 500 nM BayK-8644, 1 µM ω-conotoxin GVIA, and 1 µM MVIIC in patch pipette upon depolarization from -80 to -30, -20, -10, and 0 mV. The right panel depicts the single-channel current-voltage relationship. Mean amplitude of unitary currents for different membrane potential studied are -2.07 pA (-30 mV), -1.75 (-20 mV), -1.53 (-10 mV) and -1.25 (0 mV; n=5 patches per test potential). Solid line represents best-fit of data using a linear equation (R2=0.92) revealing a slope conductance for these channels of 27 ± 2 pS.",
    "molecules": "BayK-8644"
  },
  {
    "caption": "(B-D) Representative single channel traces and summary plot upon depolarization from -80 to 0 mV under control conditions and in the presence of ISO in the patch pipette. Cultures were pre-incubated for 15 min with 10 M 11R-PKI if indicated. The patch pipette contained either vehicle for control, nifedipine (nif; 1M), ISO (1M), or ISO plus nifedipine, which blocked all currents. The ISO-induced increase in NPo was prevented by 11R-PKI.",
    "molecules": "ISO, nif, nifedipine"
  },
  {
    "caption": "(E-H) Representative single channel currents upon depolarization from -80 to 0 mV and summary plot after pretreatment of whole cultures with ISO. Cultures were pre-incubated with vehicle (H2O, mock wash, E) or 1 M ISO for 5 min (F,G) and washed for 3 (E,F) or 10 min (G) before forming the cell-attached patch with ISO present in the patch pipette. The upregulation of NPo to ~0.4 (cf. C,D) occurred only if neurons were pretreated with vehicle instead of ISO (E,H; mock wash) or if ISO wash out duration was 10 min (G,H) but not if wash out was only 3 min (F,H).",
    "molecules": "ISO, H2O"
  },
  {
    "caption": "(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(A) Myr-Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr-DSPL (lane 1 vs. 3).(B,C) For quantification, α11.2 and GluA1 immunosignals were normalized to β2AR signals (**p<0.01, ***p<0.001, One Way ANOVA).",
    "molecules": "ISO, water"
  },
  {
    "caption": "(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(D) Myr-Pep2 blunted ISO-induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr-DSPL (lane 2 vs. 4).(E,F) For quantification, pS1700 and pS845 immunosignals were normalized to α11.2 and GluA1 signals, respectively (***p<0.001, One Way ANOVA).",
    "molecules": "ISO, water"
  },
  {
    "caption": "(G) Representative cell-attached recordings from hippocampal neurons as in Figure 7. In interleafed experiments cultures were pre-incubated for 30 min with 10 M Myr-Pep2 or scrambled Myr-Pep2 (Myr-Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 µM ISO. The ISO induced increase in NPo was prevented by Myr-Pep2 but not Myr-Pep2scr. Arrows indicate the 0-current level (i.e. closed channel).(H) Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison (n=10-12 patches; *p<0.05, One Way ANOVA with Tukey post-hoc test).",
    "molecules": "ISO, H2O"
  },
  {
    "caption": "Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(A-C) Litter-matched WT but not conditional Cav1.2 KO mice showed PTT-LTP (**p < 0.001, one-way ANOVA).(D-F) WT as well as Cav1.2 KO mice showed NMDAR-dependent 100 Hz LTP.",
    "molecules": "ISO"
  },
  {
    "caption": "Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C, G-I) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr-Pep2 or Myr-Pep2scr. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(G-I) Myr-Pep2 but not Myr-Pep2scr blocked PTT-LTP (**p < 0.001, one-way ANOVA).",
    "molecules": "ISO"
  },
  {
    "caption": "(A, B) Poly(I:C) treatment inhibits axonal growth at 3 DIV (A) and dendritic growth at 6 DIV (B) in WT neurons. Neurons were transfected with GFP at 1 or 4 DIV. One day later, neurons were treated with poly(I:C) for 24 h before harvest. (C) Axon and dendrite morphology of Tlr3-/- neurons after poly(I:C) treatment. Data in A,B,C were analyzed by unpaired t-test. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. ** P < 0.001, *** P < 0.0001. Scale bar, 20μm in A and B.",
    "molecules": "Poly(I:C), poly(I:C)"
  },
  {
    "caption": "(D, E) Neuronal morphology in saline- and poly(I:C)-treated Tlr3+/+;Thy1-Yfp (D) and Tlr3-/-;Thy1-Yfp (E) mouse brains. Total dendrite length, dendritic tip number and Sholl analysis of dendritic processes were used to examine the dendrite phenotype. Data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons and mice are presented in each column. Mean values SEM are shown. * P < 0.05, ** P < 0.001, *** P < 0.0001. Scale bar, 50 μm in D and E.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(A, B) Quantitation of dendrite morphology of Trif mutant neurons (A) and Myd88-/- neurons (B) after poly(I:C) stimulation. Trif mutant or Myd88-/- neurons were transfected with a GFP construct at 4 DIV and treated with poly(I:C) one day later. Neuron morphology was analyzed at 6 DIV according to the GFP signal. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(D) MYD88 knockdown, but not TRIF knockdown, robustly increases dendritic arbors and loses the response to poly(I:C). Three DIV neurons were transfected with control shRNA (shCtrl), MYD88 shRNA (shMYD88) or TRIF shRNA (shTRIF) and poly(I:C) was applied into the culture at 5 DIV for 24 h before harvest. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(B) Poly(I:C) treatment increases the interaction between TLR3 and MYD88. One day after transfection, cells were treated with saline or poly(I:C) for 30 min before harvesting.",
    "molecules": "Poly(I:C), poly(I:C)"
  },
  {
    "caption": "(D) Full-length and N-terminal MYD88 inhibit dendrite outgrowth. Myd88-/- neurons were transfected with control vector (Ctrl), HA-tagged MYD88 (MYD88), N-terminal MYD88 (MYD88-N), or C-terminal MYD88 (MYD88-C) with a GFP construct. Poly(I:C) was applied into the culture at 5 DIV and neuronal morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm.",
    "molecules": "Poly(I:C)"
  },
  {
    "caption": "(B, C) Poly(I:C) treatment inhibits dendrite outgrowth of Il-6-/- neurons (B) and Tnfα-/- neurons (C). Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test.",
    "molecules": "Poly(I:C)"
  },
  {
    "caption": "At 4 DIV, cultured cortical neurons were transfected with indicated plasmids. Three days later, neurons were treated with 10 μg/ml poly(I:C) for 24 h. Neuronal morphology was monitored by GFP signals at 8 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001, ns: non-significant.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(A) Expression of 12 neuropsychiatric disorder-related genes in saline- and poly(I:C)-treated WT neurons.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(B) Expression of 9 neuropsychiatric disorder-related genes in poly(I:C)-treated Tlr3-/- neurons. Relative expression levels compared with vehicle control are shown. In (B-D), dashed lines indicate the level of saline control.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(C, D) Expression of Auts2, Disc1, Fmr1, Pten and Ube3a in poly(I:C)-treated Myd88-/- neurons (C) and poly(I:C)-treated P5 WT mouse brains (D). In (B-D), dashed lines indicate the level of saline control.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(E) DIV 5 WT neurons were treated with poly(I:C) for 24 h. The total neuronal lysates were subjected to immunoblotting with the indicated antibodies. VCP and β-actin were used as loading controls. The protein levels were normalized with β-actin. Data were analyzed by unpaired t-test. Numbers of experimental repeats (N) are shown in each column. * P < 0.05, ** P < 0.001, *** P < 0.0001.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(B-C) Overexpression of DISC1 in cortical neurons of mouse brain is resistant to poly(I:C)-triggered reduction of dendritic arborization. Vector control (Ctrl) (B) or Myc-DISC1 (C) was co-expressed with GFP in cortical layer 2/3 neurons in mouse brain. After poly(I:C) injection at P4 and P5, the neuronal morphology was analyzed at P7 by tracing the GFP signals. The data of dendrite length and tip number were analyzed by unpaired t-test. The Sholl data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. The numbers of analyzed neurons collected from 3-4 mice of each group are indicated. Mean values SEM are shown. Scale bar, 30 μm. * P < 0.05, *** P < 0.0001.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(A) Cortical and hippocampal mixed neuronal cultures were transfected with a GFP construct at 12 DIV and treated with poly(I:C) (pIC) at 17 DIV for 24 h. Spine morphology was examined at 18 DIV. Three secondary dendrites of each neuron were selected to analyze the spine density, the width of the spine head and the spine length. Mean values SEM of representatives of three independent experiments are shown. For the saline group, 15 neurons/45 dendrites were examined; for the poly(I:C) group, 16 neurons/48 dendrites were examined. Data were analyzed by unpaired t-test. Scale bar, 20 μm in right upper panel and 5 µm in lower left panel. *** P < 0.0001.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(C, D) Spine morphology of P21 Thy1-Yfp (C) and Tlr3-/-; Thy1-Yfp mice (D) after poly(I:C) stimulation at P4 and P5. One secondary dendrite of an apical dendrite of each somatosensory layer 5 cortical neuron was selected to examine the spine morphology. Three mice were analyzed for each group. Saline-treated Thy1-Yfp, 44 neurons; poly(I:C)-treated Thy1-Yfp, 43 neurons; saline-treated Tlr3-/-;Thy1-Yfp, 46 neurons; poly(I:C)-treated Tlr3-/-;Thy1-Yfp, 50 neurons. Mean values SEM are shown. Data were analyzed by unpaired t-test. Scale bar, 5 μm. *** P < 0.0001.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": " Effects of the QKAA, KKEE and L348R mutations on MUN-catalyzed transition detected by FRET assay. Schematic diagram is shown on the top of the chart. FRET between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored. ",
    "molecules": "BODIPY FL, TMR"
  },
  {
    "caption": " F Effects of the QKAA, KKEE and L348R mutations on lipid mixing between liposomes with Munc18-1/Syx1 and liposomes containing Syb2 in the presence of Munc13 (M13), SN25 and Syt1/Ca2+. Illustration of the reactions is shown on the top of the chart. ",
    "molecules": "liposomes, Ca2+"
  },
  {
    "caption": " A Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21). B Sample traces (left), summary graphs of normalized amplitude (middle) and normalized charge transfer (right) of evoked IPSCs recorded from neurons as described in panel A (Control, n = 19; None, n = 19; WT, n = 19; QKAA, n = 13; KKEE, n = 18; P335A, n = 17; L348R, n = 21). C Sample traces (left) and quantification of the normalized charge transfer evoked by hypertonic sucrose (right) recorded from the neurons described in panel A (Control, n = 17; None, n = 13; WT, n = 17; QKAA, n = 17; KKEE, n = 18; P335A, n = 15; L348R, n = 14). ",
    "molecules": "sucrose"
  },
  {
    "caption": " A Representative images of HEK293T cells transfected with mCherry2-Syx1 or EGFP-Munc18-1. Lipophilic dye DiD (1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) was used as the plasma membrane marker. B Representative images of HEK293T cells co-transfected with mCherry2-Syx1 and EGFP labeled WT or mutants of Munc18-1. ",
    "molecules": "1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine, DiD"
  },
  {
    "caption": " C,D Summary graphs of the membrane distribution of Syx1 (C) and Munc18-1 WT or mutants (D) quantified by the ratio of fluorescence at the plasma membrane (PM) to the total fluorescence in the whole cell (total). DiD is the PM marker. Numbers of pictures analyzed are shown as follows: no Syx1, n = 14; no M18, n = 15; WT, n = 24; QKAA, n = 23; KKEE, n = 12; L348R, n = 22. ",
    "molecules": "DiD"
  },
  {
    "caption": " A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored. B Quantification of the FRET results in A. ",
    "molecules": "BODIPY FL, TMR"
  },
  {
    "caption": "CRTH2 subcellular localization in GFP-fused CRTH2 expressing plasmid-transfected cells. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative images of CRTH2 subcellular localization in NIH 3T3 cells in response to TGF-β1 (10 ng/mL) treatment. Red arrows indicate CRTH2 localized in the ER and white arrows indicate CRTH2 localized in the plasma membrane. Green, GFP-CRTH2; blue, DAPI; red, Calnexin; Scale bar, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": ", Representative images of Masson′s trichrome staining of lung sections from CRTH2flox/floxCol1a2-CreERT (F-CRTH2 KO) and CRTH2flox/flox (Control) mice 14 days after bleomycin challenge. Blue indicates the collagen deposition regions. Scale bar, 100 μm. Quantification of collagen content in mouse lung sections after bleomycin challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each; bleomycin groups, n = 8 each.",
    "molecules": "bleomycin, collagen, saline"
  },
  {
    "caption": "Representative images of Masson′s trichrome staining of liver sections from control and F-CRTH2 KO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm.",
    "molecules": "CCl4"
  },
  {
    "caption": "Representative images of Masson′s trichrome staining of heart sections from control and F-CRTH2 KO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. *P < 0.05 vs control, #P < 0.05 vs saline group (two-way ANOVA); PBS groups, n = 3 each, ISO groups, n = 7-8.",
    "molecules": "collagen, ISO, PBS, saline"
  },
  {
    "caption": "Representative images of Masson's trichrome staining of lung sections from F-LARP6 KO and F-LARP6/F-CRTH2 double KO (F-DKO) mice 14 days after bleomycin challenge; Scale bar, 100 μm. , Quantification of collagen content in mouse lung sections after bleomycin challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 7-8. Hydroxyproline content in lungs from F-LARP6 KO and F-DKO mice treated with bleomycin. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, bleomycin groups, n = 8 each.",
    "molecules": "bleomycin, collagen, Hydroxyproline, saline"
  },
  {
    "caption": "Representative images of Masson's trichrome staining of liver sections from F-LARP6 KO and F-DKO mice 4 weeks after CCl4 challenge; Scale bar, 100 μm. Quantification of collagen content in mouse liver sections after CCl4 challenge. #P < 0.05 vs mineral oil group (two-way ANOVA); mineral oil groups, n = 4 each, CCl4 groups, n = 8 each. Hydroxyproline content in livers from F-LARP6 KO and F-DKO mice treated with CCl4. #P < 0.05 vs mineral oil group (two-way ANOVA); saline groups, n = 4 each, CCl4 groups, n = 8 each.",
    "molecules": "collagen, Hydroxyproline, mineral oil, saline, CCl4"
  },
  {
    "caption": "Representative images of Masson's trichrome staining of heart sections from F-LARP6 KO and F-DKO mice 21 days after ISO challenge; Scale bar, 50 μm. Quantification of collagen content in mouse heart sections after ISO challenge. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 3 each, ISO groups, n = 7 each. , Hydroxyproline content in hearts from F-LARP6 KO and F-DKO mice treated with ISO. #P < 0.05 vs saline group (two-way ANOVA); saline groups, n = 4 each, ISO groups, n = 8 each.",
    "molecules": "collagen, Hydroxyproline, ISO, saline"
  },
  {
    "caption": "Microscale thermophoresis (MST) analysis of the binding affinity of Bumetanide with MBP-LARP6",
    "molecules": "Bumetanide"
  },
  {
    "caption": "Quantification of collagen content in lung sections from Bumetanide-treated control (CRTH2flox/flox) and F-CRTH2 KO mice. *P < 0.05 vs control, #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups. Effect of Bumetanide treatment on pulmonary hydroxyproline levels in control and F-CRTH2 KO mice. *P < 0.05 vs control; #P < 0.05 vs Vehicle (two-way ANOVA); n = 7 for all groups.",
    "molecules": "Bumetanide, collagen, hydroxyproline"
  },
  {
    "caption": "Bumetanide interferes with the interaction of CRTH2 with LARP6 in MRC-5 human lung fibroblasts MRC-5. MRC-5 cells were co-transfected with myc-LARP6 and flag-CRTH2 expressing plasmids for 24 h, and then treated with Bumetanide (0.1, 2 μM) for an additional 24 h. Whole cell lysates from MRC-5 cells were immunoprecipitated and immunoblotted with the indicated antibodies.",
    "molecules": "Bumetanide"
  },
  {
    "caption": "Effect of Bumetanide on Collagen (Col) I and III protein expression in MRC-5 cells.",
    "molecules": "Bumetanide"
  },
  {
    "caption": "LGG-1::GFP-positive puncta labeling autophagic membranes [22] were counted in wild-type or in food-limited animals.(A) Micrographs of eat-2(ad1116) L3 larvae expressing GFP-tagged lgg-1/LC3. Arrow indicates autophagic focus. Magnification is indicated.(B) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) mutants and N2 wild-type animals (WT), p < 0.0001.(C) Average number of LGG-1::GFP-containing puncta in N2 wild-type, food-restricted animals grown in liquid media (WT, food limited) and N2 wild-type animals grown in liquid with a higher concentration of bacteria (WT, fully fed), p < 0.0001; see Methods.Between three and ten seam cells were counted in each of 20-40 animals using high-power microscopy and averaged. n, total number of seam cells observed. Error bars: ±SEM. p-Values were calculated as unpaired, two-tailed t-test. Animals were raised at 20 °C. Please see Table S1 for quantification of all data.",
    "molecules": "food"
  },
  {
    "caption": "(C) HeLa cells were treated with 5 Gy γIR and 2 h post treatment fixed and stained with the indicated antibodies. (D) HeLa cells were irradiated with 5 Gy and 2 h post treatment stained for RASSF1A (R1A) and NUCLEOLIN (NCL). (E) HeLa cells were treated with 50 ng/ml NCS for 30 min, washed and 2 h later fixed and stained with the indicated antibodies. The distance of RASSF1A+/γH2AX+ foci or RASSF1A-/γH2AX+ foci to the closest nucleolus (based on Fibrillarin (Fb) staining) was measured. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values.",
    "molecules": "Fb, Fibrillarin, NCS"
  },
  {
    "caption": "(F) AsiSI expression in U2OS cells was induced by OHT and cells were stained for RASSF1A and γH2AX. Boxed areas are shown in higher magnification. Nucleolar boundaries are marked with dashed lines. (G) Quantification of the number of cells with RASSF1A foci in (F). Error bars represent standard deviation and derive from 3 independent experiments.",
    "molecules": "OHT"
  },
  {
    "caption": "(C) Assessment of RASSF1A localisation after induction of rDNA DSBs using the I-PpoI endonuclease in methanol fixed HeLa cells. Fluorescence intensity profile of RASSF1A (green) and LAMIN A/C (red) signals across the HeLa nuclei in the presence and absence of rDNA DSBs in control and I-PpoI treated cells. Position of line scan indicated by the yellow line.",
    "molecules": "methanol"
  },
  {
    "caption": "(J) U2OS cells were treated with siLUCIFERASE (siLUC), siRASSF1A (siR1A) or si53BP1, treated with 10 μM BrdU for 24 h prior to I-PpoI mRNA transfection. 6 h post I-PpoI treated cells were pre-extracted, fixed and stained for BrdU under non denaturing conditions to visualize ssDNA. (K) Quantification of (J). Fold change of nuclear BrdU signal relative to siLUC is shown. Error bars represent standard deviation and derive from 3 independent experiments.",
    "molecules": "BrdU, ssDNA"
  },
  {
    "caption": "(N) HeLa cells treated with siLUCIFERASE (siLUC) or siMST2 and 48 h later transfected with I-PpoI mRNA. Cells were treated with 5-EU for 30 min prior to fixation and assessed for incorporation. (O) Quantification of 5-EU intensity in siLUCIFERASE (siLUC) or siMST2 treated cells transfected with I-PpoI mRNA. Middle line represents the median and the boxes 25th and 75th percentiles. The whiskers mark the smallest and largest values. 100 values were analyzed in each condition.",
    "molecules": "5-EU"
  },
  {
    "caption": "C Peak density distribution of YTHDC1 binding  sites near different poly(A) sites. Genes with shortened 3' UTR shows a higher density of YTHDC1 binding  peaks near proximal APA sites compared to background genes. The density of YTHDC1 binding  site peak were calculated as the number of peak in a 10-nt interval divided by the total number of mRNAs that contained this position.",
    "molecules": "poly(A)"
  },
  {
    "caption": "G Scatter plot of  qRT-PCR for APA validation. Mutation of the m6A binding  site of YTHDC1  loses its inhibitory effect on proximal APA sites. Extended/Common ratios of ten genes were obtained by qRT-PCR in HEK293T  cells overexpressing YTHDC1-WT and YTHDC1-Mut. The ratios were normalized to those of control cells. Data are presented as mean ± SEM of three biological replicates. *p<0.05, **p<0.01, ns: no significance. the p values were obtained from unpaired two-tailed Student's t-test.",
    "molecules": "m6A"
  },
  {
    "caption": "F Endogenous YTHDC1  and FIP1L1  were stained  with antibodies labeled with Alexa Fluor 488  and Alexa Fluor 568 fluorophores, respectively. Immunofluorescence  results show that YTHDC1  and FIP1L1  have a high spatial association in the nucleus. Scale bars 15µm.",
    "molecules": "Alexa Fluor 488, Alexa Fluor 568"
  },
  {
    "caption": "(A) Chromatin immunoprecipitation (ChIP) analysis of receptor binding to the ER8 element (region 1) of the nrf2 promoter. Immunoprecipitation with nonspecific IgG and PCR amplification of an adjacent region (2) served as controls. ChIP assays were also carried out to analyse binding to ER8s in the il10, ddit3 and tyrobp genes. RAR, retinoic acid receptor; VDR, vitamin D receptor.",
    "molecules": "ER8"
  },
  {
    "caption": "(B) Reverse transcription-PCR analysis of 1,25‐dihydroxyvitamin D3 (1,25D3)‐ or retinoic acid (RA)‐regulated expression of ddit3, il10, and tyrobp in U937, Calu‐3 and MCF‐7 cells.",
    "molecules": "1,25‐dihydroxyvitamin D3, 1,25D3, RA, retinoic acid"
  },
  {
    "caption": "(A) Electrophoretic mobility shift assay (EMSA) of binding of vitamin D receptors (VDRs)/retinoid X receptors (RXRs) to thep19ink4dER8, with binding to the mouseosteopontin (mop) DR3vitamin D responsive element as a control, along with a comparison of binding to ER6 and ER8 motifs (right‐hand panel).",
    "molecules": "ER6, ER8, vitamin D"
  },
  {
    "caption": "(B) EMSA of retinoic acid receptor (RAR)/RXR binding to the p19ink4d ER8, and the rarβ DR5 retinoic acid response element, as a control.",
    "molecules": "ER8"
  },
  {
    "caption": "(C) Chromatin immunoprecipitation (ChIP) analysis of binding of VDRs and RARs to the p19ink4d ER8 (region 1) and RNA polymerase II (POLII) binding to the transcription start site.",
    "molecules": "ER8"
  },
  {
    "caption": "(D) ReChIP analysis confirms the presence of VDR/RXRs and RAR/RXRs but not of VDR/RARs on the p19ink4d ER8.",
    "molecules": "ER8"
  },
  {
    "caption": "(E) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter. 3 (1,25D3)‐ (F) and retinoic acid (RA)‐regulated (G) luciferase expression is dependent on the ER8 element in the p19ink4d promoter. Luciferase expression driven by a thymidine kinase (tk) control promoter is shown.",
    "molecules": "1,25‐Dihydroxyvitamin D3, ER8, retinoic acid"
  },
  {
    "caption": "3 and retinoic acid induce p19ink4d expression. Expression of p19ink4d messenger RNA by reverse transcription-PCR (A) or protein by western blotting (B) was carried out on extracts of cells treated with 3 (1,25D3) or retinoic acid (RA), as indicated.",
    "molecules": "1,25‐dihydroxyvitamin D3, retinoic acid"
  },
  {
    "caption": "(C) Treatment with 1,25D3 or RA transiently enhances co‐immunoprecipitation (IP) of p19INK4D with cyclin‐dependent kinase CDK4 in U937 cells. Similar results were obtained in SCC25 cells (not shown). A control western blot (W) of CDK4 levels in ligand‐treated cells is shown below.",
    "molecules": "1,25D3, RA"
  },
  {
    "caption": "(D) Treatment with 1,25D3 enhances co‐immunoprecipitation of p27KIP1 with CDK4 in U937 cells. Extracts of U937 cells were probed for p27KIP1 and CDK4, and immunoprecipitated with an anti‐CDK antibody and probed for p27KIP1 co‐immunoprecipitation.",
    "molecules": "1,25D3"
  },
  {
    "caption": "(C,D) Fluorescence‐activated cell sorting analysis of cell‐cycle distribution of control SCC25 cells, or cells transfected with scrambled (Scr), p19ink4d‐ or p27kip1‐directed siRNAs, individually or together (Dbl). (C) Cells were treated with vehicle or 1,25‐dihydroxyvitamin D3 (1,25D3), as indicated. (D) Cells were treated with DMSO vehicle, retinoic acid (RA) or 1,25D3, as indicated.",
    "molecules": "1,25‐dihydroxyvitamin D3, 1,25D3, DMSO, RA, retinoic acid"
  },
  {
    "caption": "(E) Loss of p19INK4D enhances lysosomal β‐galactosidase activity in SCC25 cells. (Left) Histogram of numbers of cells staining for β‐galactosidase activity in the absence (−) or presence of 1,25D3 in control cells (−) or in cells transfected with scrambled (Scr) or p19INK4D siRNAs. (Right) Bright field images of untransfected cells (−/−) or p19INK4D‐depleted cells treated with 1,25D3 (p19/D3).",
    "molecules": "1,25D3"
  },
  {
    "caption": "Media from soluble TREM2-Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times",
    "molecules": "agarose"
  },
  {
    "caption": "Media from soluble TREM-Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5",
    "molecules": "agarose"
  },
  {
    "caption": "E In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs conditioned in vitro with TGF-β (or medium) for 24 h in the presence or absence (Ctrl) of catalytic inhibitors as in C. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).",
    "molecules": "HY"
  },
  {
    "caption": " G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads). ",
    "molecules": "HY"
  },
  {
    "caption": "C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.",
    "molecules": "tyrosine"
  },
  {
    "caption": "E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D.",
    "molecules": "Tyrosine"
  },
  {
    "caption": "A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT or IDO1.Y149F and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": " A Pull-down assay of lysates from untransfected P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated (pYENM) IDO1 peptides, analyzed by immunoblotting with anti-p85, -p110α, -p110β, -p110γ, and -p110δ antibodies. Whole cell lysates (WCL) were used as control of lysate amount. ",
    "molecules": "tyrosine"
  },
  {
    "caption": "B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT incubated with class IA PI3K p110 isoform inhibitors (indicated) or medium alone (Ctrl) for 24 h and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from P1.HTR cells transfected with IDO1.WT cells and treated with PI3K catalytic inhibitors (indicated) or medium alone (ctrl). Data represent the mean ± SD (n = 3 technical replicates).",
    "molecules": "Tyrosine"
  },
  {
    "caption": "A Confocal immunofluorescence microscopy images of freshly purified pDCs co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF-β or IFN-γ and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). Scale bar, 5 µm. C Pearson's coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF-β or IFN-γ (as in B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from three independent experiments). ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Growth curves of S. pombe cultures in minimal medium without (EMM) or with antimycin A (EMM + antA). Cultures were supplemented with either the complete amino-acid mix (all AA mix, black), without any amino acids (no AA, red), with arginine only (R, blue), or with individual amino acids other than arginine (individual AAs, grey).",
    "molecules": "antA, antimycin A, arginine"
  },
  {
    "caption": "(B) Growth curves as in (A) for cultures supplemented with complete amino-acid mix (black), without any amino acids (red), or amino-acid mix without arginine (green).",
    "molecules": "arginine"
  },
  {
    "caption": "(C) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine (EMM+R) (Dataset file 1). Mutants that passed our filtering (Methods) and whose colony-size ratios were 30% bigger when supplemented with arginine are shown in red; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles.",
    "molecules": "arginine"
  },
  {
    "caption": "(E) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants (grey) and respiratory deficient mutants (orange, based on previous screens (Malecki & Bähler, 2016) (Welch's t-test pval** < 0.002). The same analysis was performed for mutants grown on EMM (left; 4 biological repeats, Dataset file 1) or on EMM supplemented with antimycin A (right; 2 biological repeats, Dataset file 2). Boxplots were created using the R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.",
    "molecules": "antimycin A, arginine"
  },
  {
    "caption": "(A) Normalised concentration values of 16 amino acids in cells grown in EMM without (grey) or with (blue) antimycin A. For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. Boxplots were created using R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.",
    "molecules": "antimycin A, Orn, Ornithine"
  },
  {
    "caption": "(B) Amino-acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM. Left: heat map showing concentration changes (log2 fold change) of each measured amino acid during the time course relative to time point 0 (before antimycin A addition). Right: simplified scheme of amino-acid biosynthetic pathways. Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow. *Ornithine (Orn) is abbreviated as O.",
    "molecules": "antimycin A, Orn, Ornithine"
  },
  {
    "caption": "(C) Left panels: cell growth in EMM without (red) or with (blue) arginine supplementation for five selected respiratory mutants as indicated (graphs selected from Fig EV3). Right panels: normalised intracellular amino-acid concentrations as in (A) for the same 5 respiratory mutants (blue) and wild-type control cells (grey). For each mutant and the wild type strain, four independent samples were analysed. *Ornithine (Orn) is abbreviated as O. Boxplot features as in (A).",
    "molecules": "arginine, Orn, Ornithine"
  },
  {
    "caption": "(A) Normalised amino-acid concentrations in cells grown in EMM without (grey) or with arginine (red). For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.",
    "molecules": "arginine, Orn, Ornithine"
  },
  {
    "caption": "(C) Growth curves of strains deleted for car1, car2 or car3, and a wild-type control. Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine (light blue) or EMM with both arginine and antimycin A (dark blue) as indicated.",
    "molecules": "antimycin A, arginine"
  },
  {
    "caption": "(D) Growth curves of strains deleted for car2 (top) or car3 (bottom). Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine and antimycin A (dark blue), EMM with mix of all amino acids and antimycin A (bright blue), and EMM with antimycin A and each individual amino acid from the mix (grey).",
    "molecules": "antimycin A, arginine"
  },
  {
    "caption": "(E) Growth curves of wild-type control (WT) and car2 deletion strain (Δcar2). Strains were grown in EMM media with antimycin A and with either a mix of all amino acids (+AA mix) or different combinations of lysine (K), glutamine (Q), glutamic acid (E), or arginine (R) as indicated. Dashed line: time point where wild-type strain saturates in EMM with amino-acid mix.",
    "molecules": "antimycin A, arginine, glutamic acid, glutamine, lysine"
  },
  {
    "caption": "(A) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without (EMM) or with arginine (EMM+R). Transcripts annotated as up- or down-regulated Core Environmental Stress Response (CESR) are indicated in red and blue, respectively. All data is normalised to EMM time point 0.",
    "molecules": "antimycin A, arginine"
  },
  {
    "caption": "(B) Graphs as in (A) showing expression profiles of transcripts encoding enzymes for arginine catabolism (blue) or arginine biosynthesis (red).",
    "molecules": "arginine"
  },
  {
    "caption": "(C) Heat map of expression changes for transcripts repressed over 2-fold at 30 min in cells supplemented with arginine (EMM +R) relative to cells without supplementation (EMM). Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM, EMM with arginine and ssp1 deletion mutant grown in EMM. The data are normalised to EMM timepoint 0.",
    "molecules": "antimycin A, arginine"
  },
  {
    "caption": "(D) Heat map of expression changes of the same genes as in (B) after one hour of inhibiting TOR kinase with torin1 or caffeine/rapamycin treatment relative to cell before treatment; cells were grown in rich media - YES (data from (Rodríguez-López et al., 2019))",
    "molecules": "caffeine, rapamycin, torin1"
  },
  {
    "caption": "D. Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.",
    "molecules": "glucose, Glucose"
  },
  {
    "caption": "E. The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum-free medium for 4 h with DMSO or Bafilomycin A1 (100 nM). Cells were lysed and the cell lysates were analyzed by immunoblotting.",
    "molecules": "Bafilomycin A1, DMSO"
  },
  {
    "caption": "H. Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum-free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non-significant and *P < 0.05. Student's t-test was used for statistical analysis.",
    "molecules": "BSA, Lipid"
  },
  {
    "caption": "H. Knockdown of LRP6 induced cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP or LRP6 and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear-localized YAP per image (n=8). Error bars indicate standard deviation. ***P < 0.001. Student's t-test was used for statistical analysis.",
    "molecules": "DAPI"
  },
  {
    "caption": "I. Overexpression of LRP6 induced nuclear localization of YAP under starvation conditions. HEK293A cells were transfected with LRP6-EGFP and 1 d after transfected cells were incubated with or without serum-containing medium for 4 h, and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear localized YAP per image (n=6). Error bars indicate standard deviation. *P < 0.05. Student's t-test was used for statistical analysis.",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Merlin and LATS1/2 were necessary for the LRP6 knockdown-mediated-cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag-Merlin and LRP6-EGFP. After transfection, cells were incubated in serum-free medium for overnight with Bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-LRP6 antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "B. LRP6 is O-GlcNAcylated. LRP6-EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI-1 (50 μM) for 6 h with Bafiolmycin A1 (100nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.",
    "molecules": "Bafiolmycin A1, Thiamet G, OSMI-1"
  },
  {
    "caption": "C. LRP6 is O-GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.",
    "molecules": "GlcNAc"
  },
  {
    "caption": "D. Blocking of O-GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI-1 (50 μM) and Bafilomycin A1 (100 nM) for 6 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.",
    "molecules": "Bafilomycin A1, OSMI-1"
  },
  {
    "caption": "E. The O-GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6-EGFP. Cells were incubated in serum-free medium with Bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. The ratio of O-GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.",
    "molecules": "Bafilomycin A1, GlcNAc"
  },
  {
    "caption": "F. Increase of O-GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6-EGFP and Flag-Merlin transfected HEK293T cells were incubated in serum-free medium with Bafilomycin A1 (20 nM) and Thiamet G (30μM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.",
    "molecules": "Bafilomycin A1, Thiamet G"
  },
  {
    "caption": "G. Enhancement of global O-GlcNAcylation blocked the serum starvation-mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc-LATS1 and Flag-Merlin. After transfection, cells were incubated in serum-free medium with Thiamet G (30μM) and Bafilomycin A1 (20nM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.",
    "molecules": "Bafilomycin A1, Thiamet G"
  },
  {
    "caption": "D. LRP6 was O-GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.",
    "molecules": "GlcNAc"
  },
  {
    "caption": "qRT-PCR for INHBA relative to RPL27 using RNA from SCC13 cells transduced with a lentiviral vector allowing expression of INHBA in a doxycycline (DOX)-inducible manner (SCC13 Act clone 1 and 2) or empty vector (EV) (N=3). SCC13 Act and EV cell lines were generated from lentivirally transduced cells upon single cell clonal expansion.",
    "molecules": "DOX, doxycycline"
  },
  {
    "caption": "Western blot of cell lysate and conditioned media (CM; bottom) of transduced SCC13 cells for the activin βA subunit and GAPDH (loading control for cell lysate) under reducing conditions. The higher molecular weight of recombinant activin βA results from the HA-epitope tag. Ponceau S staining of the membrane was used as a loading control for the CM.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "Representative images of sections from tumors formed by SCC13 EV and SCC13 Act (clone 2) stained for Ki67 (red), PDGFR-α (green) and counterstained with Hoechst (blue). Quantification of Ki67/PDGFR-α positive cells is shown in the graph. N=3 tumors, n=3-4 histological sections.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Representative images of fibroblasts treated with activin A, TGF-β1 or vehicle (control) as in (C), stained for αSMA (green), counterstained with rhodamine-coupled phalloidin (red) and Hoechst (blue). Bar graph shows percentage of fibroblasts with stress fibers. N=3.",
    "molecules": "Hoechst, rhodamine, phalloidin"
  },
  {
    "caption": "epresentative images of control and activin A-treated fibroblasts stained with rhodamine-coupled phalloidin; scatter plot shows quantification of filopodium length in control, activin A- and TGF-β1-treated fibroblasts. N=27-70 filopodia.",
    "molecules": "rhodamine, phalloidin"
  },
  {
    "caption": "qRT-PCR for INHBA relative to RPL27 using RNA from primary human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h (Fb Act, clones 1 and 2) or with empty vector (Fb EV) (N=3). Fb Act and Fb EV cultures were generated from lentivirally-transduced cells upon clonal expansion of resistant single cells.",
    "molecules": "DOX"
  },
  {
    "caption": "Western blot of conditioned media (CM) of transduced fibroblasts showing mature activin βA. Ponceau S staining of the membrane was used as a loading control.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX-inducible dominant-negative ActRIB mutant (dnActRIB) or an empty lentivirus (EV), treated with activin A for 2.5 h and analyzed by Western blot for mDia2 and GAPDH.",
    "molecules": "DOX"
  },
  {
    "caption": "Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells (clone 2) stained for mDia2 or K14 (green) (top), mDia2 (green) or MECA32/LYVE1 (red, bottom) and counterstained with Hoechst (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "Representative Western blots for p53, mDia2 and GAPDH using total lysates of mDia2 knock-down and control mouse fibroblasts treated for 24 h with pifithrin-α (PFT-α) (15 μM).",
    "molecules": "PFT-α, pifithrin-α"
  },
  {
    "caption": "Representative p53 immunofluorescence images of mDia2 knock-down mouse fibroblasts treated with PFT-α or vehicle (DMSO) (top) and quantification of cells showing accumulation of p53 in the nucleus (bottom). N=3.",
    "molecules": "DMSO, PFT-α"
  },
  {
    "caption": "qRT-PCR for mDia2, Postn, Mmp13, Fn1, Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock-down and control fibroblasts treated with PFT-α or vehicle. N=3.",
    "molecules": "PFT-α"
  },
  {
    "caption": "Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act (clone 2) co-injected with sh-EV or sh-mDia2 fibroblasts, stained for E-cadherin (green) and periostin or p53 (red), counterstained with Hoechst (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co-injected with EV or dnActRIB fibroblasts stained for K14 (left) or PDGFRα (right) (green) and mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin (FST) (50 ng/ml) and/or SMIFH2 (10 μM), or with activin A (20 ng/ml) and stained for Ki67. N=3 tumor explants, n=4 histological sections.",
    "molecules": "SMIFH2"
  },
  {
    "caption": "Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 (green) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3. Spheroids were either treated with FST (50 ng/ml) or SMIFH2 (10 μM) or left untreated. N=6-8 tumor spheroids.",
    "molecules": "PKH67, collagen, SMIFH2"
  },
  {
    "caption": "Increase in A431 tumor volume (mm3) compared to day 0 (time when the tumors were first injected with FST, approximately 3-4 weeks after injection of the cells) upon treatment of the tumors with FST (100 ng per injection) or vehicle (PBS). N=3-5 tumors per group. Representative immunofluorescence images the FST or vehicle-treated tumors, stained for mDia2 (green) and K14 (red), counterstained with Hoechst (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "(V) Survival of hydrogen peroxide-treated animals. (In all three repeats, P = 0.000 for epg-7 vs. sqst-1;epg-7; P > 0.2 for wild type vs. sqst-1;epg-7; log-rank test).",
    "molecules": "hydrogen peroxide"
  },
  {
    "caption": "E) BDNF leads to an increase in the levels of TRBP (p=0.03) and PACT (p=0.004) in the cytoplasmic fraction. Cortical neurons (7 DIV) were control- or BDNF-stimulated and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each experiment. The position of the star corresponds to the TRBP band used for analysis (~43kDa) (t-test type 3, n=4, error bars; s.d.).",
    "molecules": "digitonin"
  },
  {
    "caption": "F) The BDNF-induced increase in cytoplasmic TRBP is dependent on intracellular calcium. Cortical neurons (7 DIV) were treated with vehicle control, BDNF and BAPTA-AM as indicated, and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each independent experiment (p=0.03, n=3, error bars; s.d.).",
    "molecules": "BAPTA-AM, digitonin"
  },
  {
    "caption": "A Phenotypes of WT, dja6, dja5, dja6 dja5 and RNAi seedlings on MS medium with 2%  sucrose. Scale bars = 0.5 cm.",
    "molecules": "sucrose"
  },
  {
    "caption": "B,C  Phenotypes and chlorophyll fluorescence images of 6-week-old WT, dja6 dja5 and complemented plants (B) on MS medium with 2% sucrose, scale bars = 0.5 cm; WT, dja6, dja5 and RNAi mutants (C) grown on soil,­­­ scale bars = 1 cm. Fluorescences in (B) and (C) were measured with the FluorCam700MF and visualized using a pseudocolor index as indicated on the right.",
    "molecules": "chlorophyll, sucrose"
  },
  {
    "caption": "D Chlorophyll content of total leaves in 6-week-old WT, dja6, dja5, dja6 dja5  and RNAi seedlings. FW, fresh weight. Data are the means ± SEM (n = 7 biological replicates).",
    "molecules": "Chlorophyll"
  },
  {
    "caption": "A, B Total protein was extracted from 4-week-old WT,  dja6, dja5, dja6 dja5 and RNAi  seedlings, respectively. WT and dja6 dja5 used in (A) were grown on MS medium with 2% sucrose, while WT, dja6, dja5 and RNAi  mutants used in (B) were grown on soil. 10 μg protein was loaded, except for detection with anti-SUFE1 and anti-SUFA antiserum, for which 25 μg were loaded. Specific bands were identified by immunoblotting and by their molecular weight, and are indicated by asterisks. Designations of photosynthetic protein complexes and their diagnostic components are labeled on the left and right, respectively. The Fe-S types of Fe-S proteins are shown in parentheses. Actin  served as controls to normalize protein levels. For each protein, three independent biological replicates were performed and a representative one is shown.",
    "molecules": "sucrose"
  },
  {
    "caption": "A-C Metal concentrations in the WT,  dja6 dja5 and RNAi mutants. Fe concentrations in total  leaves (A and B) and total chloroplast (C) from 4-week-old WT, dja6 dja5 and RNAi mutants were quantified by inductively coupled plasma mass spectrometry, and are shown as means ± SEM (n = 3 biological replicates) of μg g-1 dry weight  (DW). WT and RNAi mutants used in (A and C) were grown on soil, while WT and dja6 dja5 seedlings used in (B) were grown on MS medium with 2% sucrose. Similar results were obtained in two additional independent biological experiments. Asterisks indicate significant differences from the value of WT (two-sample Student's t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).",
    "molecules": "Fe, sucrose"
  },
  {
    "caption": "D Volcano plot of the result from quantitative transcriptome profiling in WT and dja6 dja5 mutant. Statistical analysis was performed using Student's t-test. Data on the X-axis represent the Log2-ratio of FC (fold change) between the absolute abundances of transcripts identified from dja6 dja5 against WT. The Y-axis represents the Log10 of FDR (false discovery rate). All DEGs (differentially expressed genes) with FDR < 0.01 and FC > 2 are marked in grey, while all iron-related DEGs with FDR < 0.01 and FC > 2 are marked in red. The iron-related DEGs labeled with red dots within the black boxes are referred to in the text. All data are based on three biological replicates.",
    "molecules": "iron"
  },
  {
    "caption": "D DJA6 can bind  Fe2+ in chloroplast. Arabidopsis mesophyll protoplasts  were transformed with plasmids that express the indicated gene constructs, respectively. The untransformed protoplasts and protoplasts  transformed with non-fused EBFP proteins were used as controls. The protoplasts were further stained by FeRhoNoxTM-1 (red) to visualize the cellular distribution of Fe2+. Green, EBFP fluorescence signals; magenta, chloroplast autofluorescence signals; yellow, regions where the EBFP and  FeRhoNoxTM-1 fluorescence signals merged. The speckles  which DJA6-EBFP co-localizes with FeRhoNoxTM-1  are indicated by white arrows. Scale bars = 5 μm.",
    "molecules": "FeRhoNoxTM-1, Fe2+"
  },
  {
    "caption": "C Phenotypes and chlorophyll fluorescence images of 6-week-old WT and 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants  grown on soil. Scale bars = 1 cm.",
    "molecules": "chlorophyll"
  },
  {
    "caption": "A, B Detection of γb palmitoylation through the  biotin-switch assay in  N. benthamiana. The γb-3xFlag and γb3CS-3xFlag proteins were treated with (Hyd+) or without (Hyd-) hydroxylamine, the thioester  cleavage reagent. Lanes labelled ' Palmitoylation ' show γb-3xFlag or  γb3CS-3xFlag  amounts recovered from the neutravidin  beads. The loading controls indicate sample loading onto the neutravidin  beads. Data information: In (A, representative data are shown and three biological replicates had similar results.",
    "molecules": "Hyd, hydroxylamine, neutravidin, thioester"
  },
  {
    "caption": "A Effects of γb palmitoylation on BSMV  replication. Movement-deficient BSMV  (RNAα  + RNAγ/RNAγ3CS) was agroinfiltrated into N. benthamiana leaves. At 3 dpi, total protein and RNA were extracted for Western and Northern blot analyses. Antibodies and probes used for molecular analyses are indicated on the right of each panel. Actin  was used to monitor equal protein loading, and methylene blue-stained rRNAs  served as RNA loading controls. Data information: representative data are shown and three biological replicates had similar results.",
    "molecules": "methylene blue"
  },
  {
    "caption": "D Subcellular localization  of γb-GFP  or γb3CS-GFP  in isolated N. benthamiana protoplasts   harvested at 3 dpi. The fifth and sixth columns show γb localization  in BSMVγb-GFP- (movement-deficient) N. benthamiana protoplasts  after treatment with 200 μM 2-BP inhibitor. The released protoplasts  were collected after 48 h incubation in DMSO  or 2-BP followed by confocal microscopy visualization. Figures at the bottom indicate the normalized fluorescence intensities of the GFP  (green) and the chloroplast   autofluorescence (red) channels along the dashed white lines in the merged images above. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.",
    "molecules": "2-BP, DMSO"
  },
  {
    "caption": "D Biotin-switch assay  to access palmitoylation of γb  in BSMV-infected N. benthamiana leaves  at different timepoints. BSMVγb-3xFlag was treated with the hydroxylamine  (Hyd+) thioester  cleavage reagent. Equal sample amounts used for palmitoylation analyses were monitored by Western blot  analysis with an anti-Flag  antibody. Data information: In representative data are shown and three biological replicates had similar results.",
    "molecules": "Hyd, hydroxylamine, thioester"
  },
  {
    "caption": "F, G Western blot analysis of subcellular fractions extracted from N. benthamiana leaf  tissues at 3 dpi. S3 indicates the supernatant separated by centrifugation at 3,000 g; and S30 is the 30,000 g supernatant; P30 is the 30,000 g pellet. The association of  γb with membranes was determined by 1%  Triton X-100 extraction.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "D Confocal microscopy  analyses showing RFP-TGB1 localization  at the  chloroplast periphery  in N. benthamiana leaves  agroinfiltrated with αa-GFP  and CFP-γb plasmids. CFP-γb  or CFP-γb3CS were co-expressed with RFP-TGB1  and αa-GFP  in  N. benthamiana leaves. Chloroplast  autofluorescence was displayed in false pink color. The third and fourth rows show leaves treated with 400 μM 2-BP inhibitor or the DMSO  control at 1 dpi. Two days later, confocal microscopy  observations of RFP-TGB1, αa-GFP, and CFP-γb  were performed. Figures on the right indicate the normalized fluorescence intensity of GFP, RFP, and CFP  channels along the white dashed line in the merged confocal images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.",
    "molecules": "2-BP, DMSO"
  },
  {
    "caption": "D Co-IP assays of interactions between γb  and TGB1  in NbPAT15- and NbPAT21-silenced N. benthamiana leaves, TRV:GUS  and TRV:GUS combined with the  2-BP inhibitor provided positive and negative controls, respectively. The silenced leaves  were inoculated with BSMV3xFlag-TGB1/γb-GFP mixtures (RNAα + RNAβ3xFlag-TGB1 + RNAγγb-GFP) and harvested at 3 dpi. Total proteins were immunoprecipitated  with anti-Flag  beads. Input and IP  proteins were analyzed by Western blotting with anti-GFP or anti-Flag  antibodies. E Average amounts of RFP-TGB1  foci co-localizing with αa-GFP and CFP-γb  at the  chloroplast peripheries  shown in panel C (n = 12). Data information: In E), representative data are shown and three biological replicates had similar results. In (E), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).",
    "molecules": "2-BP"
  },
  {
    "caption": "E Western blot  analysis of subcellular fractions of BSMV  infected N. benthamiana leaves  at 3 dpi. The S3 and S30 fractions are the 3000 and 30000 g supernatants, and P30 is the 30000 g pellet. Membrane  associated γb  was released by 1%  Triton X-100 treatment.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "D Analysis of the cleaved 5(6)-carboxyfluorescein (CF) movement in N. benthamiana leaves. RNAα  + RNAγ/RNAγ3CS were co-infiltrated into transiently overexpressed  NbREM1 N. benthamiana leaves, and 1 μL CFDA was loaded onto the adaxial leaf  blade surface and incubated for 5 min followed by confocal  analysis at 3 dpi. The areas of dye diffusion are marked by dashed white circles. Scale bars, 100 μm. Data information: In D, representative data are shown and three biological replicates had similar results.",
    "molecules": "5(6)-carboxyfluorescein, CF, CFDA"
  },
  {
    "caption": "E Quantification of CF  movement. Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software (n = 6). Data information: In (E, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).",
    "molecules": "CF"
  },
  {
    "caption": "F Aniline blue staining  of callose  in N. benthamiana leaf  tissues. Leaves  were infiltrated with mixtures of different A. tumefaciens  constructs indicated beside the panels. At 3 dpi, the 0.1% aniline blue fluorochrome was infiltrated into N. benthamiana leaves. After incubation at 5 min in the dark, the leaves  were observed by confocal microscopy at 405 nm. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.",
    "molecules": "callose, Aniline blue, aniline blue"
  },
  {
    "caption": "G Quantification of callose staining intensity as shown in panel F. The relative callose staining intensity was set to 1 in the REM samples (n = 10). Data information: In , the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).",
    "molecules": "callose"
  },
  {
    "caption": "H Aniline blue staining  of callose  in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants. Scale bars, 20 μm. Data information: In H), representative data are shown and three biological replicates had similar results.",
    "molecules": "callose, Aniline blue"
  },
  {
    "caption": "I Quantification of callose staining intensity as shown in panel H. The relative callose staining intensity was set as 1 in the samples of EV (n = 8). Data information: In I), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).",
    "molecules": "callose"
  },
  {
    "caption": "(F) Effects of 4 weeks of oral administration of gliadin on IL-15, IL-17A and IFN-γ protein levels in small intestine homogenates from CftrF508del/F508del and CftrWT mice (n =10 mice per group of treatment). Mean ± SD of triplicates of independent pooled samples. **p<0.01, ***p<0.001 (CftrF508del/F508del vs CftrWT mice prior gliadin challenge), °°°p<0.001 (CftrF508del/F508del mice vs CftrF508del/F508del mice after gliadin challenge), (ANOVA, Bonferroni post hoc test).",
    "molecules": "gliadin"
  },
  {
    "caption": "(G-I) BALB/c mice (G) fed with a gluten-free diet for at least 3 generations, or (H) NOD or (I) NOD-DQ8 mice orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks). Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=3 independent experiments). Mean±SD of samples assayed; **p<0.01, ***p<0.001 vs challenged with gliadin (Student's t test).",
    "molecules": "CFTR inhibitor 172, CFTRinh172, chloride, Cl-, forskolin, Fsk, gliadin, gluten"
  },
  {
    "caption": "(A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-inducible chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers after 3 h of incubation with P57-68 or P33-mer or P31-43 peptides (20 µg/ml) and of P31-43 in the presence of up to 30 minutes of pre-treatment of VX-770 (10 µM); quantification of the peak CFTR Inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. ***p<0.001 vs P31-43 challenge, ##p<0.01 vs Caco-2 challenged with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).",
    "molecules": "CFTR Inhibitor 172, CFTRinh172, chloride, Cl-, forskolin, Fsk, VX-770"
  },
  {
    "caption": "(B) Incubation of Caco-2 cells with P31-43 or control peptides (p57-68 or PGAV) for 1 h that were preceded or not by 20 min of pre-treatment with VX-770. Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody. Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "VX-770, streptavidin"
  },
  {
    "caption": "(F) Surface plasmon resonance (SPR) analysis of rhNBD1 binding to P31-43 and P57-68 biotinylated peptides immobilized on SA sensor chip.",
    "molecules": "biotinylated"
  },
  {
    "caption": "Blue native polyacrylamide gel electrophoresis (PAGE) western blotting of P31-43 and P57-68 biotinylated peptides in the presence of rhNBD1 (G) All the recombinant proteins and the indicated peptides were pre-incubated in an appropriate buffer at 4°C for 30 min and then resolved in native conditions to preserve the formation of peptide/protein complexes. Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "biotinylated"
  },
  {
    "caption": "Blue native polyacrylamide gel electrophoresis (PAGE) western blotting of WT and double NBD1 mutants in the presence of biotinylated P31-43 (H). All the recombinant proteins and the indicated peptides were pre-incubated in an appropriate buffer at 4°C for 30 min and then resolved in native conditions to preserve the formation of peptide/protein complexes. Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "biotinylated"
  },
  {
    "caption": "(I) P31-43 induced modifications on NBD1 ATP binding site using the intrinsic W401 fluorescence.",
    "molecules": "ATP"
  },
  {
    "caption": "(K) P31-43 effect on NBD1 ATPase activity, applied at its IC50, compared to that obtained with 5µM of the non-hydrolyzable ATP analogue (P-ATP) as a positive control of inhibition. Mean ± SD of triplicates of independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "Incubation of Caco-2 cells with P31-43 or with modified P31-43 (P31-43-2QA 4-5th or P31-43-2QA 10-11th or P31-43/4QA mutant (4QA) peptides) for 1 h. (C) Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody (n=3 independent experiments). Mean ± SD of samples assayed. °°p<0.01 vs P31-43 challenge, ***p<0.001 vs Caco-2 challenged with 4QA (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "streptavidin"
  },
  {
    "caption": "Incubation of Caco-2 cells with P31-43 or with modified P31-43 (P31-43-2QA 4-5th or P31-43-2QA 10-11th or P31-43/4QA mutant (4QA) peptides) for 1 h. (D) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. °°p<0.01 vs P31-43 challenge, ***p<0.001 vs Caco-2 challenged with 4QA (ANOVA, Bonferroni post hoc test).",
    "molecules": "CFTR inhibitor 172, CFTRinh172, chloride, Cl-, forskolin, Fsk"
  },
  {
    "caption": "Incubation of Caco-2 cells with P31-43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON, TG2-siRNA Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunoblot with anti-isopeptide glutamime-lysine and CFTR",
    "molecules": "Z-DON, glutamime, lysine"
  },
  {
    "caption": "Incubation of Caco-2 cells with P31-43 in the presence or absence of pre-treatment with TG2 inhibitor Z-DON BAPTA-AM immunoprecipitation of CFTR and then immunoblot with anti-TG2 or anti-CFTR antibodies",
    "molecules": "Z-DON, BAPTA-AM"
  },
  {
    "caption": "Incubation of Caco-2 cells with P31-43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON or VX-770. Effects of treatment with VX-770 and Z-DON on TG2 transamidating activity in situ. (H) In situ detection of TG2 activity in Caco-2 cells pulsed with Ca2+ by immunoblotting of the TG-catalyzed incorporation of 5-biotinamidopentylamine (BAP) and blotting with anti-biotin antibody (n=3 independent experiments). Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment. ",
    "molecules": "Z-DON, 5-biotinamidopentylamine, BAP, biotin, Ca2, VX-770"
  },
  {
    "caption": "Incubation of Caco-2 cells with P31-43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON VX-770. Effects of treatment with VX-770 and Z-DON on TG2 transamidating activity in situ. (I) Assay of TG2 activity by immunostaining of the TG-catalysed incorporation of monodansylcadaverin in Caco-2 cells pre-treated with VX-770 or Z-DON and then pulsed with P31-43. Scale bar, 50µm. Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "Z-DON, VX-770, monodansylcadaverin"
  },
  {
    "caption": "Incubation of Caco-2 cells with P57-68 or P31-43, in the presence or absence of pre-treatment with VX-770. (J) Immunoprecipitation of CFTR and immunoblot with anti-CFTR and anti-TG2 antibodies. Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "VX-770"
  },
  {
    "caption": "Incubation of Caco-2 cells with P57-68 or P31-43, in the presence or absence of pre-treatment with VX-770. (K) Immunoprecipitation in non-reducing and non-denaturing conditions of PKAr2α protein and immunoblot with isopeptide glutamine-lysine and PKAr2α antibodies (n=3 independent experiments). Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "glutamine, VX-770, lysine"
  },
  {
    "caption": "Incubation of Caco-2 cells with P57-68 or P31-43, in the presence or absence of pre-treatment with VX-770. (L) Immunoblot of phospho-PKA (phPKA) protein; densitometric analysis of protein levels relative to β-actin. Mean±SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (A) Immunoblot of PPARγ or phospho-ERK1/2 (phERK1/2) and densitometric analysis of protein levels relative to β-actin. Mean ± SD of triplicates of independent experiments. **p<0.01 vs P31-43, °°p<0.001 vs VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P57-68 or PGAV or with P31-43 in the presence or absence of VX-770 Immunoblotting with specific antibodies in Caco-2 cells challenged for 2 or 4 h in the presence or absence of VX-770. NF-κB p65 in cytoplasmic and nuclear extracts (B) Densitometric analysis of protein levels relative to loading control. Mean ± SD of triplicates of independent experiments. **p<0.01 and °°p<0.001 vs 2h or 4h of culture with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).",
    "molecules": "VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P57-68 or PGAV or with P31-43 in the presence or absence of VX-770 Immunoblotting with specific antibodies in Caco-2 cells challenged for 2 or 4 h in the presence or absence of VX-770. NLRP3 expression (C) Densitometric analysis of protein levels relative to loading control. Mean ± SD of triplicates of independent experiments. **p<0.01 and °°p<0.001 vs 2h or 4h of culture with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P57-68 or PGAV or with P31-43 in the presence or absence of VX-770 Immunoblotting with specific antibodies in Caco-2 cells challenged for 2 or 4 h in the presence or absence of VX-770. caspase-1 cleavage (D). Densitometric analysis of protein levels relative to loading control. Mean ± SD of triplicates of independent experiments. **p<0.01 and °°p<0.001 vs 2h or 4h of culture with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P57-68 or with P31-43 in the presence or absence of VX-770 or Vrx-532. (E) Immunoblot of membrane protein fractions with anti-NHERF-1, anti-ezrin Abs and anti-flotillin as a control. Densitometric analysis of immunoblot (mean ± SD of triplicates of independent experiments); *p<0.01 vs treatment with VX-770 or °°p<0.01 vs treatment with Vrx-532 (ANOVA, Bonferroni post hoc test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "Vrx-532, VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (F) Confocal image staining with anti-F-actin. DAPI (blue), nuclear counterstaining. Scale bar, 50 µm.",
    "molecules": "DAPI, VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (G) IL-15 production (quantified by specific ELISA) in CFTR-WT Caco-2 cells treated or not with P31-43 (left) or in Caco-2CFTR-KO cells (right), in the presence or absence of VX-770. Mean ± SD of triplicates of independent experiments. ***p<0.001 vs untreated and °°°p<0.001 vs CFTR-WT Caco-2 cells treated with P31-43+VX-770 (ANOVA, Bonferroni post hoc test). **p<0.01 vs Caco-2 CFTR WT (Student's t-test).",
    "molecules": "VX-770"
  },
  {
    "caption": "Caco-2 cells incubated with P31-43 in the presence or absence of VX-770 (H) Caco-2 or Caco-2CFTR-KO cells in the presence or absence of pre-treatment with VX-770. Immunoblot of phERK1\2 or β-actin (left); densitometric analysis of protein levels (right). Mean ± SD of triplicates of independent experiments. *p<0.05 (Student's t-test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "VX-770"
  },
  {
    "caption": "BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=5). Mean ± SD of samples assayed. ***p<0.001 gliadin vs gliadin+VX-770 (Student's t-test).",
    "molecules": "CFTR inhibitor 172, CFTRinh172, chloride, Cl-, forskolin, Fsk, gliadin, gluten, VX-770"
  },
  {
    "caption": "BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (B) Immunoblot with anti-CFTR antibody and β-actin loading control. Densitometric analysis are mean ± SD of triplicates of independent pooled samples. **p<0.01 vehicle vs gliadin; °p<0.05, gliadin vs VX-770+gliadin (ANOVA, Bonferroni post test). Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "gliadin, gluten, VX-770"
  },
  {
    "caption": "BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (C) Plasma markers of intestinal permeability in mice. Plasma concentration of FITC-dextran 4000 (FITC-D4000) measured 1 h after gavage of a single dose of 600 mg FITCD4000 per kg body weight. Quantification of plasma concentration from n=10 mice per group of treatment expressed as mean ± SD of triplicates of independent pooled samples. ***p<0.001 vehicle vs gliadin; °°°p<0.01, gliadin vs VX-770+gliadin (ANOVA, Bonferroni post test).",
    "molecules": "D4000, dextran 4000, FITC, gliadin, gluten, VX-770"
  },
  {
    "caption": "BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (D) NLRP3 expression by immunoblotting with specific antibodies in small intestines from 3 mice. Densitometric analysis are mean ± SD of triplicates of independent pooled samples. **p<0.01 vehicle vs gliadin; °p<0.05, gliadin vs VX-770+gliadin. Data information: The blots are representative of one experiment for group of treatment.",
    "molecules": "gliadin, gluten, VX-770"
  },
  {
    "caption": "BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (E-F) mRNA (E) or protein (by specific ELISA) (F) levels of IL-15, IL-21, IL-17A and IFN-γ. Mean±SD of triplicates of independent pooled samples. *p<0.05, **p<0.01, ***p<0.001 vehicle vs gliadin; °p<0.05, °°p<0.01, °°°p<0.001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).",
    "molecules": "gliadin, gluten, VX-770"
  },
  {
    "caption": "BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). Transcript level of Rorc and Tbet (G) from small intestine homogenates. Mean ± SD of triplicates of independent pooled samples. ***p<0.001 or ****p<0.0001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).",
    "molecules": "gliadin, gluten, VX-770"
  },
  {
    "caption": "BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). protein levels (by specific ELISA) of IL-10 and TGF-β (H) from small intestine homogenates. Mean ± SD of triplicates of independent pooled samples. ***p<0.001 or ****p<0.0001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).",
    "molecules": "gliadin, gluten, VX-770"
  },
  {
    "caption": "IFN-γ release (ELISA) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co-culture model upon 24 h challenge of confluent CaCo-2 cells in the upper compartment with PT-gliadin (I) in presence or absence of VX-770. Mean ± SD of triplicates of independent pooled samples. ***p<0.001, PT-gliadin vs medium; °°°p<0.001, PT-gliadin vs PT-gliadin + VX-770 (n=4) (ANOVA, Bonferroni post hoc test).",
    "molecules": "gliadin, VX-770"
  },
  {
    "caption": "IFN-γ release (ELISA) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co-culture model upon 24 h challenge of confluent CaCo-2 cells in the upper compartment with combination of P31-43 and P57-68 (J) in presence or absence of VX-770. Mean ± SD of triplicates of independent pooled samples. ; ***p<0.001, P57-68 or P31-43 vs P31-43/P57-68 combination (n=6); °°°p<0.001, P57-68/P31-43 combination vs VX-770 + P57-68/P31-43 (n=3), (ANOVA, Bonferroni post hoc test).",
    "molecules": "VX-770"
  },
  {
    "caption": "(K) IL-10 release (ELISA) in culture supernatants by PBMC from 4 celiac patients cultured as in (J). Mean ± SD of triplicates of independent pooled samples. ****p<0.0001, P57-68/P31-43 combination vs P57-68/P31-43+VX-770 treatment (n=3) (ANOVA, Bonferroni post hoc test).",
    "molecules": "VX-770"
  },
  {
    "caption": "(L) IFN-γ release (ELISA) into culture supernatants by PBMC from 3 celiac patients cultured in the lower compartment of a bidimensional co-culture model upon a 24 h challenge of confluent Caco-2CFTR-KO cells in the upper compartment with a combination of P31-43 and P57-68 in the presence or absence of VX-770, as in J and K. *p<0.05, medium vs P57-68/P31-43 combination (n=3) (ANOVA, Bonferroni post hoc test).",
    "molecules": "VX-770"
  },
  {
    "caption": " (B) 35S labeling of de novo mitochondrial protein synthesis of HeLa cells transfected as in (A). Polypeptide assignments flank the gel images. Coomassie stained gels are used as loading controls and immunoblots indicate the efficiency of LETM1 knockdown ",
    "molecules": "35S, Coomassie"
  },
  {
    "caption": " (E) Mitochondrial oxygen consumption rate (OCR) measured using a Seahorse flux analyzer before (basal) and after the addition (maximal) of the uncoupler FCCP, in HeLa cells treated with NT or siLETM1 (siR1 or siR2). Data are means ± SEM of n=5 experiments ",
    "molecules": "FCCP, oxygen"
  },
  {
    "caption": " (A) LETM1 expression was suppressed in HeLa cells by transfection with targeted siRNAs (siR1, siR2 or siR3). A non-target dsRNA (NT) served as control. Cells were fixed and immunolabeled with anti-DNA antibody (green). A higher magnification of selected mtDNA foci is shown beside each picture. (B) Quantification of cells in (A) displaying mtDNA abnormalities. At least 50 cells per siRNA were counted from 4 (siR2) and 5 (siR1 or siR3) independent experiments. Data are expressed as means ± SEM. ***P<0.001 (One-way ANOVA) ",
    "molecules": "DNA"
  },
  {
    "caption": " (C) HeLa cells were labeled with bromouridine (BrU) for 60 mins, 144 h after transfection with NT or siR2 and stained with anti-BrdU (green) and anti-GRSF1 (red) antibodies. Cell nuclei were stained with DAPI (blue) ",
    "molecules": "BrdU, bromouridine, BrU, DAPI"
  },
  {
    "caption": " (D) HeLa cells were labeled with anti-DNA (green) and anti-GRSF1 (red) antibodies 144 h after transfection with NT or siR3. Cell nuclei were stained blue with DAPI ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " (A) Representative immunoblots of mitochondrial lysates of HEK cells separated on a 20-42.5% iodixanol gradients probed for LETM1, PDH and TFAM ",
    "molecules": "iodixanol"
  },
  {
    "caption": " (C) Left panel: change in cell confluence of control (C1-C3), WHS LETM1+/- (S1-S4) and WHS LETM1+/+ (S5) fibroblasts grown in BHB over the course of 188 h. Data are expressed as means ± SEM of n=3 independent experiments. Right panel: images of control and WHS LETM1+/- S4 cells growing either in the presence of 25 mM glucose (HG) or BHB (KB) for 6 days. Scale bar 300 µm ",
    "molecules": "BHB, glucose"
  },
  {
    "caption": " (D) Confocal images of control cells (C1) grown for 24 h in the presence of BHB and immunolabeled with anti-TOM20 (red) and anti-DNA (green), and DAPI stained (blue) ",
    "molecules": "DAPI, BHB, DNA"
  },
  {
    "caption": "eATP levels in APF from S. indica inoculated and mock-treated barley root samples collected at the predominantly biotrophic phase (3 and 5 dpi) as well as the predominantly cell death associated phase (7 and 10 dpi). Error bars show the standard error of the mean obtained from three independent biological replicates at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.",
    "molecules": "eATP"
  },
  {
    "caption": "eATP levels measured from culture medium collected from mock treated and S. indica colonized Arabidopsis seedlings at 3, 5, 7 and 10 dpi. Error bars represent ±SE of the mean from three independent biological replicates. RLU: relative light units. Asterisks indicate significance at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.",
    "molecules": "eATP"
  },
  {
    "caption": "S. indica colonization of Arabidopsis dorn1-3 mutant and the parental Col-0aq lines quantified by qPCR at 3, 5, 7 and 10 dpi. The ratio of fungal (SiTEF) to plant (AtUBI) amplicons representing fungal colonization levels in plant root tissue was calculated using gDNA as template and the 2-ΔCT method. Error bars represent standard error of the mean of three technical replicates. The experiment was repeated 3 times for 3, 5 and 7 dpi with similar outcomes.",
    "molecules": "gDNA"
  },
  {
    "caption": "Ecto-5′-nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S::E5′NT (#303), Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) or Pro35S::mCherry (#305). E5′NT activity was measured after incubation with 100 µM of either ATP, ADP or AMP. In the membrane protein preparations from Pro35S::E5′NT (#303) lines phosphate release was specifically increased upon incubation with purines. Error bars represent the standard error of the mean from three technical repetitions. The coomassie stained SDS-PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines. Equal volumes were loaded. The experiment was repeated two times with similar results.",
    "molecules": "AMP, ADP, ATP, phosphate, purines"
  },
  {
    "caption": "S. indica induced eATP release in different Arabidopsis transgenic lines. Culture medium was collected from mock treated or S. indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured. RLU: relative light units. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).",
    "molecules": "eATP"
  },
  {
    "caption": "Expression analysis of the eATP responsive gene At1g58420 measured by qRT-PCR. Error bars represent ±SE of the mean from three independent biological replicates (independent from those shown in Fig. 2C). Letters indicate significant groups (ANOVA, p<0.01, for the line 305 p<0.05).",
    "molecules": "eATP"
  },
  {
    "caption": "Relative fluxes of phosphates (Pi) and sugars (C) across the fungal plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of phosphate via the proton-coupled phosphate (H/P) transporter from the fungus to the apoplast and a constant flux of sugar from the apoplast to the fungus. For better comparison these fluxes were normalized to 1 and -1, respectively, and all other fluxes were calculated relative to these control values. With increasing ATP-release and Pi-production the Pi-efflux gets smaller and is zero at a relative Pi-production rate of 1 (grey triangles). At higher ATP-release and Pi-production rates the fungus imports Pi, i.e. the transport direction of phosphate has been inverted in comparison to the control condition (white square). The C-flux via the proton-coupled sugar (H/C) transporter is not affected by the additional Pi-source (horizontal grey line).",
    "molecules": "sugar, sugars, ATP, phosphate, phosphates, Pi"
  },
  {
    "caption": "Relative fluxes of Pi and C across the plant plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of sugar via the H/C transporter from the plant to the apoplast (and thereafter to the fungus) and a constant flux of phosphate (coming from the fungus) via the H/P transporter from the apoplast to the plant. With increasing ATP-release and Pi-production the Pi-influx via the H/P transporter increases while the C-flux via the H/C transporter is unaffected by the additional Pi-source (horizontal grey line).",
    "molecules": "sugar, ATP, phosphate, Pi"
  },
  {
    "caption": "Phosphate fluxes across the plant plasma membrane. Besides the Pi-uptake via the H/P transporter (B, black line, for clarity not shown in C), the plant loses Pi due to the ATP-release (light grey line). The difference between Pi-uptake and Pi-loss is the net Pi-balance of the plant (dark grey line). Without ATP-release (value 0 at the x-axis, black dots) the plant gains Pi. At a relative ATP-release and Pi-production of 1 (grey triangles) the plant release as much phosphate as it takes up, while at higher ATP-release values, the plant loses Pi.",
    "molecules": "ATP, Phosphate, phosphate, Pi"
  },
  {
    "caption": "Direct fluorescence microscopic visualization for YFP (green), the macrophage marker Iba1 (red) and DAPI for the nuclei (blue) at P0. YFP+Iba1+ double-positive cells are marked by arrows. YFP-Iba1+ single-positive cells are labelled by asterisks. Representative images out of five examined animals are shown. Scale bars represents 25 µm.   Quantitative analysis of regional YFP expression in Iba1+ macrophages in TAM-induced and untreated Cx3cr1CreERT2:Rosa26-YFP mice. Bars represent means ± s.e.m. Quantification was done from three (untreated) or five (TAM) mice obtained from one (untreated) or two (TAM) independent experiments. Level of significance determined by Mann-Whitney test between TAM and untreated revealed *p < 0.05 and Kruskal-Wallis test between retina, ciliary body and cornea revealed *p = 0.0204.  ",
    "molecules": "DAPI, TAM"
  },
  {
    "caption": "Above: Representative funduscopic pictures from living healthy Cx3cr1CreERT2:Rosa26-tdTomato mice on d0. Funduscopy and red fluorescence visualize the fundus and regular distribution of tomato+ microglia before the laser-induced lesion formation. Below: Corresponding immunofluorescence pictures. Non-lesioned retina show a regular pattern of Iba1+ (green) tomato+ (red) retinal microglia while macrophages are absent on the retinal pigment epithelium (RPE) under native conditions. Pictures are representative for six mice analyzed in one experiment. Scale bars represents 50 µm. c) Above: In vivo funduscopy on d7 post lesion. Funduscopic and red fluorescence image depict the lesions (encircled with dashed white lines) and accumulation of tomato+ microglia in Cx3cr1CreERT2:Rosa26-tdTomato mice. Intraperitoneal fluorescein application was performed to label retinal vessels and areas of choroidal neovascularization. Below: Representative immunofluorescence for Iba1 (green) in Cx3cr1CreERT2:Rosa26-tdTomato mice. Resident retinal microglia are Iba1+tomato+ (asterisks) whereas blood-derived myeloid cells are Iba1+tomato- (arrows) and accumulate at sites of laser-induced CNV. Overlay is shown left. Typical pictures from six mice obtained from one independent experiment are shown. Scale bars represents 50 µm.",
    "molecules": "fluorescein"
  },
  {
    "caption": "c) HLA-DRA mRNA expression examined by RNA-Seq via Massive analysis of cDNA ends (MACE) analysis of human formalin-fixed and paraffin-embedded (FFPE) membranes of choroidal neovascularization (CNV). Bars represent means ± s.e.m. of four investigated human samples of age-related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE. Level of significance was calculated using DESeq (p = 3.61x10-21).",
    "molecules": "formalin, paraffin"
  },
  {
    "caption": "(D) Western blot analysis of a strain expressing TurboID-HA-Emc6, Bpl1-AID*-9myc and OsTIR1 which was grown overnight in SD media with or without auxin. The cells were then diluted into fresh SD media and grown to mid logarithmic phase (~4hours) with or without auxin (respectively). When required, biotin was added either 30min before harvesting, or for the entire 4hours. H3 (histone H3) is used as a loading control.",
    "molecules": "auxin, biotin"
  },
  {
    "caption": "(B) Volcano plots for each of the four distinct LC-MS/MS experiments highlighted in Figure 2A showing -log(p-value) against log2(fold-change) for all proteins identified in the appropriate Emc6/Sbh1 samples. Proteins enriched in the Emc6 samples are colour/pattern-coded as follows: dotted outline for HA-IP of BioID2-HA-Emc6; blue fill for streptavidin-AP of BioID2-HA-Emc6; black, solid outline for streptavidin-AP of TurboID-HA-Emc6; and yellow fill for streptavidin-AP of TurboID-HA-Emc6/ABOLISH. Grey dots represent proteins that passed the p-value and fold-change criteria, but were only identified by one peptide. Only EMC complex components or proteins with EMC client features are named.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(C) Streptavidin blots of diploid strains expressing either AviTag-Fks1, -Gnp1, -Pdr5, -Pdr12 or -Stv1 (as negative control) together with BirA-Emc6, grown in the conditions described in Figure 1E. For each strain, blots showing both the 'lower' (L) and 'higher' (H) contrast settings are shown side-by-side. The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "D U2OS cells were transfected with control or NAT10 siRNAs. Seventy-two hours later, cells were treated with cycloheximide (CHX) and harvested at the indicated time points. Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 (upper panels). Beta-actin was evaluated as a loading control (lower panel). p53 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "E U2OS cells were treated with MG132 (10μM) for 4 hours after transfected with the indicated siRNAs. Proteins from cell extracts were detected by western blot for the indicated proteins.",
    "molecules": "MG132"
  },
  {
    "caption": "F HCT116 p53+/+ cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibody and immunoblotted with anti-Ub antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "B HCT116 cells were transfected with Flag-NAT10 and harvested at indicated time points after treated with CHX. Proteins from cell lysates were immunoblotted with the antibodies as indicated (left panel). Relative Mdm2 protein levels standardized by β-actin are shown in the right. Mdm2 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.",
    "molecules": "CHX"
  },
  {
    "caption": "C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours. The Mdm2 protein was immunoprecipitated with anti-Mdm2 antibody and immunoblotted with anti-HA antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "I HCT116 cells were transfected with the indicated constructs. Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared. Mdm2 protein levels were evaluated by western blot. Relative Mdm2 protein levels standardized by β-actin are shown in the right.",
    "molecules": "CHX"
  },
  {
    "caption": "A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points. Proteins from extracts were analyzed by western blot using the indicated antibodies.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "B U2OS and H1299 cells were treated with actinomycin D for 12 hours. Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Proteins from cell lysates were subjected to western blot.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin (Dox) for 12 hours. Cells were harvested and total proteins were immunoprecipitated with anti-NAT10 antibody. Immunoprecipitates were analyzed by western blot for the indicated proteins.",
    "molecules": "actinomycin D, Dox, doxorubicin"
  },
  {
    "caption": "E HCT116 cells were treated with actinomycin D for the indicated times. Cells were fixed and immuno-stained as indicated. Immunofluorescence images were taken by confocal microscopy. Scale bar represents 10 μM.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "F H1299 cells were transfected with the indicated vectors. After treated with actinomycin D, cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "G H1299 cells were transfected with GFP-NAT10 vectors. After treated with actinomycin D for 12 hours, cells were fixed and nuclei were stained with DAPI. Bar represents 10μM.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin. Western blot was performed with total cell extracts and Mdm2-specific immunoprecipitates for the indicated proteins.",
    "molecules": "actinomycin D, doxorubicin"
  },
  {
    "caption": "I HCT116 cells were transfected with the indicated constructs. Cells were harvested after treatment with actinomycin D. p53 protein was immunoprecipitated with an anti-p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody (DO-1).",
    "molecules": "actinomycin D"
  },
  {
    "caption": "B U2OS cells were transfected with siRNAs as indicated. Seventy-two hours later, cells were harvested after treated with actinomycin D for 12 hours. Relative mRNA levels for the indicated genes were analyzed by RT-qPCR.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "C-E U2OS cells (C and D) and H1299 cells (D) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)",
    "molecules": "actinomycin D"
  },
  {
    "caption": "E HCT116 Ctrl or c2-4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)",
    "molecules": "actinomycin D"
  },
  {
    "caption": "F HCT116 Ctrl cells or c2-4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed. The total proteins were analyzed by western blot using the indicated antibodies.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "(C) The quantification of labeled-m6A in mRNA and labeled-6mdA in genomic DNA.",
    "molecules": "m6A, 6mdA"
  },
  {
    "caption": "F) the quantitative results (F) of mRNA [D3]-m6A and genomic [D3]-6mdA of mES, HEK293T, C2C12, and NIH3T3 cells. The cells were treated with [D3]-L-methionine for 7 days, and [15N5]-6mdA nucleoside (50 amol) was spiked in digested DNA samples as external isotopic standard.",
    "molecules": "[D3]-L-methionine, D3, m6A, 6mdA, 15N"
  },
  {
    "caption": "(D) The determination of kinetic constants for recombinant ADAL catabolizing m6rAMP (red) and 6mdAMP (blue). The kinetic data were fitted with the Michaelis-Menten equation.",
    "molecules": "m6rAMP, 6mdAMP"
  },
  {
    "caption": "(E) The catalytic efficiency (Kcat/ Km) of ADAL to 6mdAMP and m6rAMP. Error bars = s.d., three technical replicates.",
    "molecules": "m6rAMP, 6mdAMP"
  },
  {
    "caption": "B) quantitative results (B) of DNA 6mdA in [15N5]-rA-treated mES cells after Adal knockdown for 7 days. Two independent biological replicates.",
    "molecules": "rA, 6mdA, 15N"
  },
  {
    "caption": "D) quantification (D) of genomic 6mdA in Adal-/- mES cells treated with [15N5]-rA for 3 days. Error bars = s.d., three independent biological replicates.",
    "molecules": "rA, 6mdA, 15N"
  },
  {
    "caption": "C) the quantitation (C) of intracellular free [D3]-m6rA-related nucleotides in WT and Adal-/- mES cells. Total free [D3]-m6rA = Free [D3]-m6rA + [D3]-m6rAMP + [D3]-m6rADP + [D3]-m6rATP. Error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope.",
    "molecules": "D3, m6rADP, m6rAMP, m6rA, m6rATP"
  },
  {
    "caption": "E) quantitation (E) of intracellular free [D3]-6mdA-related nucleotides in WT and Adal-/- mES cells. The cells were treated with [D3]-methionine for 7 days. Total free [D3]-6mdA = Free [D3]-6mdA + [D3]-6mdAMP + [D3]-6mdADP + [D3]-6mdATP, Error bars = s.d., three independent biological replicates. *The standard was not labeled with heavy stable isotope. ND, not detected.",
    "molecules": "6mdADP, 6mdATP, [D3]-methionine, D3, 6mdA, 6mdAMP"
  },
  {
    "caption": "(C) The quantification of genomic 6mdA in [15N5]-rA-traced Adal-/- mES cells overexpressing flag-tagged ADK, APRT, AK1 or NDPK gene for 7 days. (D) The quantification of genomic 6mdA in NIH3T3 cells overexpressing ADK, APRT, AK1 or NDPK gene. [15N5]-rA treatment began at 24 h after plasmid transfection and lasted for 72 hours. Two technical replicates.",
    "molecules": "rA, 6mdA, 15N"
  },
  {
    "caption": "The knockdown of Ak1 leads to a significant decreased i6mdA in NIH3T3 cells. western blot (F) were applied to determine the knockdown of Ak1 in NIH3T3 cells. Two independent biological replicates. (G) The quantitation of misincorporated 6mdA in Ak1-knockdown cells. [15N5]-rA treatment began at 24 h after siRNA transfection and lasted for 48 hours. Error bars = s.d., three technical replicates.",
    "molecules": "rA, 6mdA, 15N"
  },
  {
    "caption": "(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson's coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.",
    "molecules": "DMSO, Dynasore"
  },
  {
    "caption": "(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.",
    "molecules": "Dynasore"
  },
  {
    "caption": "(H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm.",
    "molecules": "Dynasore"
  },
  {
    "caption": "(I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1-3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.",
    "molecules": "gold"
  },
  {
    "caption": "(C) HeLa cells transfected with mStrawberry-ATG16L1 (20 hr) and starved (4 hr) were processed for immunogold labeling on cryosections. Double labeling was performed with rabbit anti-ATG16L1 (10 nm gold) and rabbit anti-RAB11 (15 nm gold). Scale bar, 150 nm. Schematic diagram shows membrane outlines from EM. Green lines indicate structures positive for both ATG16L1 and RAB11, whereas red lines are only positive for RAB11. Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11. 281 tubulovesiclular structures from different experiments were analyzed. Error bars, SD.",
    "molecules": "gold"
  },
  {
    "caption": "(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B).",
    "molecules": "NH4Cl"
  },
  {
    "caption": "(I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector.",
    "molecules": "Baf, Bafilomycin A1, DMSO"
  },
  {
    "caption": "(A) HeLa cells were transfected with mStrawberry-ATG16 for 20 hr and treated for 10 min with N-ethylmaleimide (NEM; 100 μM) in full medium, fixed, and stained for mATG9. Histogram shows correlation between mATG9 and ATG16L1 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.",
    "molecules": "N-ethylmaleimide, NEM"
  },
  {
    "caption": "(B) HeLa cells transfected with control or VAMP3 siRNA (oligo A) for 4 days were treated during the last 16 hr with Bafilomycin A1 (Baf A1) or DMSO (basal) and processed for LC3-II western blots.",
    "molecules": "Baf A1, Bafilomycin A1, DMSO"
  },
  {
    "caption": "E) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF   PLD into hnRNPA2 LCRK droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LCCD,R) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2  LCRK does not phase separate much as  hnRNPA2  LC at these conditions, see Figure S1J for quantification of  phase separation  of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.",
    "molecules": "arginine, arginines, lysine, MES, NaCl, urea"
  },
  {
    "caption": "A) AlexaFluor488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2  LC  droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1%  fluorescently tagged), 20 mM MES pH 5.5, 50 mM  NaCl, 150 mM urea. Scale bar: 10 µm.  hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently. B) Similar to hnRNPF PLD, AlexaFluor555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM  urea. Scale bar: 10 µm. ",
    "molecules": "AlexaFluor488, AlexaFluor555, MES, NaCl, urea"
  },
  {
    "caption": "E) AlexaFluor555-tagged  hnRNPF  PLD or  FL and AlexaFluor405-tagged TOG D1 partition simultaneously into AlexaFluor488-tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm.",
    "molecules": "AlexaFluor405, AlexaFluor488, AlexaFluor555, MES, NaCl, urea"
  },
  {
    "caption": "C) Fluorescence micrographs  showing that although  TOG D1 and hnRNPF  PLD partition into hnRNPA2 LC droplets  (rows 1 and 3), they are unable to partition into  tyrosine phosphorylated hnRNPA2 LC droplets (rows 2 and 4). Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES  pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. D) Phosphomimic  variants containing 5 or 12 serine to glutamate substitutions (hnRNPA2 LC5E and  hnRNPA2 LC12E, respectively) both allow partitioning of  TOG D1  and hnRNPF PLD, even though hnRNPA2 LC12E barely phase separates at these conditions, indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and  hnRNPF PLD. See Appendix Figure S7B for quantification of LLPS  of hnRNPA2 LC5E and hnRNPA2 LC12E. Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. ",
    "molecules": "glutamate, MES, serine, NaCl, tyrosine, urea"
  },
  {
    "caption": "A) After cleavage of a C-terminal maltose binding protein solubility tag, hnRNPA2 FL WT undergoes LLPS  and disease mutants D290V and P298L aggregate. In contrast, phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested. Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested. Conditions: 20 µM proteins, 20 mM Tris  pH 7.4, 50 mM NaCl. Scale bar: 10 µm",
    "molecules": "NaCl, Tris"
  },
  {
    "caption": "D) After 22 hours of paraquat induced oxidative  stress, both  HRPA-1mScarlet  and HRPA-1HsLCWTmScarlet assemble more than when  unstressed (ANOVA p<0.0001). No significant differences were found within the no stress (p=0.8)  stress groups (p=0.45) by ANOVA, indicating that there is no difference in  assembly between  HRPA-1mScarlet and   HRPA-1HsLCWTmScarlet. N = 12 animals per genotype.",
    "molecules": "paraquat"
  },
  {
    "caption": "F) There is no significant difference in number of spots in HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing  Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype. G) After exposure to 22 hours  paraquat induced oxidative stress there is no significant difference between HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet  lines in animals also expressing  Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype. ",
    "molecules": "paraquat"
  },
  {
    "caption": "D) Loss of  C. elegans TDP-43 ortholog,  tdp-1, rescues  glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not  hrpa-1(∆). N=4-12 animals/genotype/trial, significance from  two tailed t-test; tdp-1∆=tdp-1(tgx58). E) Loss of C. elegans TDP-43 ortholog,  tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(ok803). ",
    "molecules": "paraquat"
  },
  {
    "caption": "G) Expression of Fyn* in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) reduces glutamatergic tail/phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours  paraquat induced oxidative stress. N=9-12 animals/genotype/trial, significance from two tailed t-test. H) Expression of an empty Fyn control (Fynempty) in sensory glutamatergic  neurons (osm-10  promoter, unc-54 3'UTR) does not alter glutamatergic phasmid  neuron degeneration in hrpa-1HsLCD290V animals  after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial. ",
    "molecules": "paraquat"
  },
  {
    "caption": "B N2a cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control. Treatment with CCCP (8 µM, 17 h) induces mitophagy in control cells, as indicated by a decrease in the GFP/mCherry fluorescence ratio, but not in hTau or hP301L cells. Deconvolution (Classic Maximum Likelihood Estimation) was applied to images for display only. C Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing significant main effects of CCCP treatment, F (1, 314) = 15.66, p < 0.0001, and tau expression, F (2, 314) = 12.26, p < 0.0001, and a significant interaction effect, F (2, 314) = 6.137, p = 0.0024, n = 40-73 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm",
    "molecules": "CCCP"
  },
  {
    "caption": "D Cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control, treated with a mixture of antimycin and oligomycin (AO, 15 µM, 17 h). E Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing no significant main effects of AO treatment, F (1, 212) = 0.564, p = 0.4534, or tau expression F (2, 212) = 1.579, p = 0.2087, but a significant interaction effect, F (2, 212) = 9.854, p < 0.0001, n = 16-50 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm",
    "molecules": "antimycin, oligomycin"
  },
  {
    "caption": "B Representative images of N2a cells expressing GFP-Parkin and hTau, hP301L or empty vector (Control) for 24 h, that were then treated with 10 μM CCCP for 4 h. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "E To assess colocalisation of Parkin clusters and mitochondria at 24 h post-transfection, Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated. Control r = 0.616, p = 0.0001; hTau r = 0.217, p = 0.2771; hP301L r = 0.548, p = 0.0014 (n = 27-34 cells/ group). F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post-transfection. Control r = 0.434, p = 0.0167; hTau r = 0.128, p = 0.4941; hP301L r = -0.299, p = 0.2794 (n = 15-31 cells/ group). Note the different intensity scales between (E) and (F) reflect different bit depths of the images. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "A FACS analysis of cells loaded with Rhodamine 123. The geometric mean fluorescence was calculated and expressed as % of the empty vector control. A CCCP-treated cohort was included as a positive control, but not included in the statistical analysis. Data were analysed by one-way ANOVA, F (2, 14) = 0.196, p = 0.8245. B FACS analysis of cells treated with CCCP after 24 h of tau expression. Data were analysed by one-way ANOVA, F (2, 15) = 0.434, p = 0.6555. The DMSO negative control is shown but was not analysed statistically. Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).",
    "molecules": "Rhodamine 123, CCCP, DMSO"
  },
  {
    "caption": "C Representative dot plots of cells loaded with Rhodamine 123. The gates (black line) were set in unstained cells to distinguish Rhodamine-positive cells (right half) from Rhodamine-negative cells (left half). Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).",
    "molecules": "Rhodamine, Rhodamine 123"
  },
  {
    "caption": "B Taxol induces microtubule bundling to a similar extent to tau expression. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.",
    "molecules": "Taxol"
  },
  {
    "caption": "C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment (Mann-Whitney test, U = 22448, p < 0.0001, n = 224, 273 cells/group for CCCP only and CCCP + Taxol, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.",
    "molecules": "CCCP, Taxol"
  },
  {
    "caption": "F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide (JPL) (Mann-Whitney test, U = 16069, p <0.0001, n = 224, 199 cells/group for CCCP only and CCCP + JPL, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.",
    "molecules": "CCCP, Jasplakinolide, JPL"
  },
  {
    "caption": "J, K Cells expressing tau constructs together with GFP-Parkin (24 h) with and without CCCP treatment. Parkin clusters were counted, illustrating that Parkin translocation was impaired by ∆Tau, but not MTBD. Data were analysed with a Kruskal-Wallis test χ2 (2) = 10.4, p = 0.0056, followed by Dunn's post-hoc, n = 67, 42, 53 cells/ group for for hTau, ∆Tau, and MTBD, respectively. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.",
    "molecules": "CCCP"
  },
  {
    "caption": "M Cells were transfected with GFP-Parkin and hTau-V5, hP301L-V5 or an empty vector control, and treated with CCCP. PLAs were performed for tau and Parkin. Notably, at Parkin clusters (arrow), no interactions between tau and Parkin were evident. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.",
    "molecules": "CCCP"
  },
  {
    "caption": "C, D Analysis of mitophagy in worms transgenic for mito-Rosella but not tau (WT), that were treated with NaN3 (8 mM, 1 h) or CCCP (15 µM, 2 h). Data were analysed by unpaired t test, NaN3 t = 5.56, p < 0.0001; CCCP t = 3.92 p = 0.0002. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord",
    "molecules": "CCCP, NaN3"
  },
  {
    "caption": "E, F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment (unpaired t test, t = 1.62, p = 0.1126) and CCCP treatment (Mann-Whitney test, U = 113, p = 0.002). Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm.­­­­ Images are high-magnification of a small number of mitochondria in the ventral cord",
    "molecules": "CCCP, NaN3"
  },
  {
    "caption": "G, H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 (Mann-Whitney test, U = 126, p = 0.2711) or CCCP (Mann-Whitney test, U = 375, p = 0.3697) treatment. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm.­­­­ Images are high-magnification of a small number of mitochondria in the ventral cord",
    "molecules": "CCCP, NaN3"
  },
  {
    "caption": "(A) MCF10A-eGFP-LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown.",
    "molecules": "3‐MA, rapamycin"
  },
  {
    "caption": "(C) MCF10A cells left untreated (Control) or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa) for 24 h were analysed for LC3‐I, LC3‐II and tubulin by immunoblotting. Pepstatin A and E64d were added to the cells 4 h before harvesting. Similar results were obtained in two independent experiments.",
    "molecules": "E64d, Pepstatin A, rapamycin"
  },
  {
    "caption": "(D) MCF10A cells were treated with 5 μM rapamycin (Rapa) or indicated concentrations of TRAIL for 24 h, and the increase in the degradation of long‐lived proteins as compared with the untreated cultures (Control) was measured.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Protein lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa, positive control) for 24 h were analysed by immunoblotting for phosphorylated p70S6K (P‐p70S6K), total p70S6K and GAPDH (loading control).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(B) Immunoblot analysis of the levels of P‐ACC and tubulin (loading control) in lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL for 24 h, 1 mM AICAR for 4 h (positive control) or 5 μM rapamycin for 24 h (negative control).",
    "molecules": "AICAR, rapamycin"
  },
  {
    "caption": "(B) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h, and left untreated or treated with 500 ng/ml TRAIL for 2 h, 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h (starvation) were analysed for P‐ACC and tubulin (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.",
    "molecules": "amino acids, glucose, ionomycin"
  },
  {
    "caption": "(C) Cells transfected as in (B) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. The values represent mean±s.d. of three independent experiments. ***P‐value 0.001 as compared with cells treated in a same way, but transfected without siRNA.",
    "molecules": "amino acids, glucose, ionomycin"
  },
  {
    "caption": "(D) Cells transfected as described in (C) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation.",
    "molecules": "amino acids, glucose, ionomycin"
  },
  {
    "caption": "A. KRI1 localized to the nucleoli in MCF-7 cells (HPA043574). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D. FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "G. METTL5 localized to the nucleoli in U-2 OS cells (HPA038223). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.",
    "molecules": "Hoechst, DAPI"
  },
  {
    "caption": "C. IF staining of GNL3 shows localization to nucleoli rim in U-2 OS cells (HPA036743). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.",
    "molecules": "Hoechst, DAPI"
  },
  {
    "caption": "D. HEK 293T cells expressing endogenous levels of N-terminus mNG-MKI67 show a faint nucleoli rim localization, although still visible. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.",
    "molecules": "Hoechst, DAPI"
  },
  {
    "caption": "E. Fixed HEK 293T cells expressing mNG-tagged MKI67 show a clear nucleoli rim localization. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.",
    "molecules": "Hoechst, DAPI"
  },
  {
    "caption": "F. HEK 293T cells expressing endogenous levels of C-terminus tagged GNL3 show nucleoli rim localization. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.",
    "molecules": "Hoechst, DAPI"
  },
  {
    "caption": "A. MKI67 (HPA000451). B. NOC2L (HPA044258). C. EMG1 (HPA039304). D. BMS1 (HPA043081). E. BRIX1 (HPA039614). F. RSL1D1 (HPA043483). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "C-E. IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered. Protein of interest shown in green and DAPI in blue. Scale bar 10 µm. C. CCDC86 (HPA041117), 100% predicted disorder level. D. RRP15 (HPA024639), 87% predicted disorder level. E. PRR19 (HPA070350), 67% predicted disorder level.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category. B-F. IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase, showing a typical late recruitment expression pattern. B. NOL12 (HPA003547). C. TIGD1 (HPA041717). D. BYSL (HPA031219). E. ACTL6B (HPA045126). F. ZNF202 (HPA059229). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "A-B. GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells. A. GNL3 (HPA036743). Median overlapKO = 0.539, median overlapWT = 0.795 (P = 9.34x10-9) and median Pearson correlationKO = -0.335, median Pearson correlationWT = 0.134 (P = 3.58x10-6). B. PES1 (HPA040210). Median overlapKO = 0.516, median overlapWT = 0.755 (P = 1.77x10-13) and median Pearson correlationKO = -0.293, median Pearson correlationWT = 0.129 (P = 4.06x10-13). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. No protein aggregates were observed in MKI67 KO cells for PWP1 (HPA038708), despite being localized to mitotic chromosomes in WT cells. Median overlapKO = 0.523, median overlapWT = 0.861 (P = 1.05x10-13) and median Pearson correlationKO = -0.190, median Pearson correlationWT = 0.515 (P = 8.71x10-15). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.",
    "molecules": "DAPI"
  },
  {
    "caption": "D-E. Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells. White arrows indicate where protein aggregates can be seen. D. DENND4A (HPA065343). Median overlapKO = 0.671, median overlapWT = 0.803 (P = 1.02x10-4) and median Pearson correlationKO = -0.224, median Pearson correlationWT = 0.236 (P = 1.91x10-5). E. NOL12 (HPA003547). Median overlapKO = 0.468, median overlapWT = 0.820 (P = 1.01x10-17) and median Pearson correlationKO = -0.318, median Pearson correlationWT = 0.381 (P = 1.81x10-10). White arrow indicates metaphase cell. Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Relative share of the three PIKKs in NCS-responsive phosphorylations.",
    "molecules": "NCS"
  },
  {
    "caption": "(C) The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin (NCS) addition and by ATR 1 hr after hydroxyurea (HU) addition. Box plots depict 23 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).",
    "molecules": "HU, hydroxyurea, NCS, neocarzinostatin"
  },
  {
    "caption": "(D) Western blotting analysis confirming that the ATM substrate, pS824/KAP1 is targeted by ATR in response to HU. pS345/CHK1 - an established ATR substrate - served as a positive control for ATR activation.",
    "molecules": "HU"
  },
  {
    "caption": "(A) A cluster of 71 neocarzinostatin (NCS)-induced phosphorylations that were enhanced upon continuous inhibition of DNA-PK. Untreated cells are marked UT. This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition. Clusters were obtained using K-means algorithm implemented in Perseus on Z-scored intensities. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).",
    "molecules": "NCS, neocarzinostatin"
  },
  {
    "caption": "(B) Depicted are phosphopeptides that responded to NCS treatment in A-T cells. No significant elevation was observed in these phosphorylations in A-T cells following continuous inhibition of DNA-PK. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 time the interquartile range (IQR).",
    "molecules": "NCS"
  },
  {
    "caption": "(C) The first time points at which 'compensated sites' responded to NCS in WT and A-T cells are presented.",
    "molecules": "NCS"
  },
  {
    "caption": "(A) A group of exclusively ATM-dependent sites that were ATM-dependent one hr after NCS addition, were still phosphorylated or dephosphorylated 4 hr after treatment, but were not affected by ATMi at that time point. Box plots depict 18 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).",
    "molecules": "NCS"
  },
  {
    "caption": "(A-F) Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.",
    "molecules": "NCS, neocarzinostatin"
  },
  {
    "caption": "G, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm.",
    "molecules": "fat, water"
  },
  {
    "caption": "hIGFREO glucose intolerance measured by glucose tolerance test (n=5-7 mice per group).",
    "molecules": "glucose"
  },
  {
    "caption": "G, H, Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve (n=10-12 mice per group).",
    "molecules": "triglycerides"
  },
  {
    "caption": "D, E, There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n=7-9 mice per group).",
    "molecules": "glucose"
  },
  {
    "caption": "(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N or SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours, and dead cells stained by phloxine B were observed by fluorescent microscopy. Scale bar, 25 µm.",
    "molecules": "MES-KOH"
  },
  {
    "caption": "(B-C) WT and atg1Δ cells grown nitrogen starved as (A) for the indicated times. Cell viability (B) and medium pH (C) were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N +50 mM MES-KOH (pH 6.2). After 120 hours, cultures were diluted 5.0×105 fold and plated onto YEPD agar. The plates were incubated at 30°C for four days. Insets indicate the plate overlaid with TTC agar to examine the respiratory competency of formed colonies.",
    "molecules": "MES-KOH"
  },
  {
    "caption": "(B) WT, and atg1Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(C) WT, atg1Δ, atg2Δ, atg7Δ, atg11Δ, atg15Δ, and atg32Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm.",
    "molecules": "MES-KOH"
  },
  {
    "caption": "(B-C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells).",
    "molecules": "MES-KOH"
  },
  {
    "caption": "(D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations",
    "molecules": "NAC, MES-KOH"
  },
  {
    "caption": "(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control.",
    "molecules": "MES-KOH"
  },
  {
    "caption": "(B) WT, atg1Δ, rho0, and rho0 atg1Δ cells grown in YEPD medium were transferred to SD-N with or without 10 mM NAC for the indicated time. In the presence of NAC, medium pH was adjusted by using KOH. Cell viability and medium pH were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.",
    "molecules": "NAC, KOH"
  },
  {
    "caption": "A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown, and the quantitative analyses are shown in B-D. Scale bar: 10 µm. B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test. C, D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated. Violin plot for SETDB1's foci numbers from all the cells analysed in three independent experiments is shown in c. The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D. Over 100 cells were analysed per sample per experiment. *P < 0.05 by unpaired Student's t-test. ",
    "molecules": "DAPI"
  },
  {
    "caption": "E, IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown. Arrows indicate the region used for line-plot analysis. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis. Scale bar: 10 µm. F The line-plot analysis for E. Relative intensities of SETDB1 signal were measured by Image J. G The quantitative analysis for E is shown. SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. NS: P > 0.05, **P < 0.01 versus "Mock, WT" group by Dunnett's test. ",
    "molecules": "DAPI, LMB"
  },
  {
    "caption": "C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells, but no significant impact for ATF7IP KO HEK293T cells. Cells were treated with 10ng/ml of LMB for 5 h and then analysed with immunofluorescent staining. The representative data of three independent experiments is shown. Scale bar: 10 µm. D SETDB1 signal in the nucleus shown in C was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. **P < 0.01 by Student's t-test (two-sided test). ",
    "molecules": "LMB"
  },
  {
    "caption": "E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells. The representative data of three independent experiments is shown. Scale bar: 10 µm. F SETDB1 signal in the nucleus shown in E was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells. *P < 0.05 and **P < 0.01 by Dunnett's method. ",
    "molecules": "bortezomib"
  },
  {
    "caption": "C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis.",
    "molecules": "LMB"
  },
  {
    "caption": "A. Sanger screen IC50  drug sensitivity data (IC50  values) for  DDR  inhibitors targeting ATR, CHK1/2 and  Wee1 in T-ALL, PTCL (DEL,  KARPAS-299, SUP-M2) and pan-cancer  cell lines. ANOVA p=0.413 (AZD6738, ATR  inhibitor), p=0.750 (AZD772, CHK1/2 inhibitor), p=0.119 (MK-8776, ATR  inhibitor) and 0.282 (AZD1775, Wee1  inhibitor). Bars represent SEM of biological replicates.",
    "molecules": "AZD772, MK-8776, AZD6738, AZD1775"
  },
  {
    "caption": "B.  PTCL (red) and T-ALL (yellow) human  cell lines were dosed in vitro with AZD6738  for 72h and  viable cell numbers assayed by luminescence  readout using Cell TiterGlo. Average IC50  values from three experiments are plotted. Bars represent SEM.",
    "molecules": "AZD6738"
  },
  {
    "caption": "F. Western blot for γH2AX  relative to total H2AX levels of spleen lysates from mice  dosed for 71 days with AZD6738  (ATR; n=4) relative to untreated mPTCL spleens  taken at endpoint (n=6). T-test p=2.70E-06. PC9  cells dosed in vitro 24h with 1 µM aphidicolin  (Aph; an inhibitor of DNA replication) or with AZD6738  (ATR) are positive controls for DNA damage. Bars represent SEM. ***p<0.001",
    "molecules": "Aph, aphidicolin, AZD6738"
  },
  {
    "caption": "I MDA‐MB‐231 cells stably expressing Flag‐LAPTM4B were stained with LAPTM4B anti‐sera followed by silver‐enhanced immuno‐electron microscopy. The early and late MVEs were defined by the number of intraluminal vesicles. N, nucleus; M, mitochondria; MVE, multi‐vesicular endosome; and PM, plasma membrane. Scale bars: 2 μm (left); 200 nm (middle and right).",
    "molecules": "silver"
  },
  {
    "caption": "A-D Control or LAPTM4B siRNA‐transfected MDA‐MB‐231 cells were starved, pretreated or not with chloroquine for 2 h, stimulated with 100 ng/ml EGF for 15 min, washed, and chased for indicated time periods before fixation for costaining of EGFR (red) with EEA1 (A, green) or LAMP1 (C, green). Quantification of the average percentages of EGFR signals colocalized with EEA1 (B) and LAMP1 (D) at indicated time points; mean + SD; n = 3; *P 0.05, **P 0.01, ***P 0.001, one‐tailed t‐test.",
    "molecules": "chloroquine"
  },
  {
    "caption": "C Control or LAPTM4B knockdown MDA‐MB‐231 cells were starved overnight, cell surface EGFR was labeled with immuno‐gold on ice. Cells were then stimulated with EGF for 1 h at 37°C and fixed for the EM study. Scale bar, 200 nm. See Materials and Methods for details.D Relative amounts of immuno‐gold‐labeled EGFR in the MVE lumen versus MVE limiting membrane were quantified. Over 80 endosomes for each siRNA treatment from three independent experiments were used for quantification (mean + SD; ***P 0.001, one‐tailed t‐test).",
    "molecules": "gold"
  },
  {
    "caption": "F Purified LAPTM4B N‐termini from (E) were used in PIP strips assay. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine 1‐phosphate; PI3,4,5P3, PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)‐trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.",
    "molecules": "phosphatidylserine, LPA, lysophosphatidic acid, LPC, lysophosphocholine, PA, phosphatidic acid, PS, PC, phosphatidylcholine, PE, phosphatidylethanolamine, phosphatidylinositol (3,4,5)‐trisphosphate, PI3,4,5P3, PtdIns(3,4,5)P3, S1P, sphingosine 1‐phosphate"
  },
  {
    "caption": "I Top: GSTpull‐down assay of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with increased concentration of PtdIns(4,5)P2 addition. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean ± SD; n = 4).",
    "molecules": "PtdIns(4,5)P2"
  },
  {
    "caption": "J LAPTM4B N‐terminus binds multiple phosphoinositides including PtdIns(4,5)P2 in liposome‐binding assay.",
    "molecules": "PtdIns(4,5)P2"
  },
  {
    "caption": "L Top: GSTpull‐down assay of wild‐type or 6RQ mutant of LAPTM4B N‐terminus and PIPKIγi5 C‐terminus with 0.5 μM addition of PtdIns(4,5)P2. Bottom: Quantification of the relative amounts of PIPKIγi5 C‐terminus bound to LAPTM4B N‐terminus in top panel (mean + SD; n = 4).",
    "molecules": "PtdIns(4,5)P2"
  },
  {
    "caption": "Plotting of the position of the 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem hippocampal tissues (PM hippo) from patients from the CBB. Each row in the heatmap depicts the distribution of counts of the 5'ends of reads mapped across the Alu metagene for each patient. The x-axis represents a metagene combining all unique Alu RNA sequences (ALUome) aligned at the start site of their consensus sequence with numbers representing the distance from the Transcriptional Start Site (TSS) area. Heatmap density corresponds to normalized counts of the 5'end of the reads with red corresponding to higher density of these 5'ends at a specific position. XL1, XR1 and XR2 denote the Alu processing areas defined by the high-density areas in the heatmap at specific positions of the Alu metagene, with the middle letter corresponding to the arm of the Alu RNA (see Figure 1C, L=Left, R=Right), in which the area is located.",
    "molecules": "Alu"
  },
  {
    "caption": "Normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed (rows) between AD and no AD for each CBB patient (columns) (Dataset EV1). Red corresponds to higher normalized counts of processed Alu RNA fragments.",
    "molecules": "Alu"
  },
  {
    "caption": "Boxplots depict differences in hippocampi of AD and no AD patients regarding SINE Alu RNA processing ratio (left panel) (a p value of 0.05 was considered as threshold for statistical significance with p=0.01 , n=24, unpaired non-directional t-test) and in total Alu RNA levels (right panel) (ns=not significant). In the boxplots, the central band (the line that divides the box into 2 parts) represents the median of the data, the ends of the box shows the upper (Q3) and lower (Q1) quartiles, the extreme line shows Q3+1.5xIQR to Q1-1.5xIQR (the highest and lowest value excluding potential outliers), while dots beyond the extreme line show potential outliers.",
    "molecules": "Alu"
  },
  {
    "caption": "Plotting of the position of the 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem cortex tissues from patients from the MAP cohort. As in Figure 1B each row represents the distribution of normalized counts of the 5'ends of reads mapped across the Alu metagene for each patient. As in Figure 1B x-axis numbers represent the distance from the Transcriptional Start Site (TSS) area and heatmap density corresponds to normalized counts of the 5'end of the reads with red for higher density of these 5'ends at a specific position. XR1 as in Figure 1B.",
    "molecules": "Alu"
  },
  {
    "caption": "As in Figure 2B, normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed (rows) between AD and no AD for each MAP patient (columns) (Dataset EV2). Red corresponds to higher normalized counts of processed Alu RNA fragments.",
    "molecules": "Alu"
  },
  {
    "caption": "Boxplots depict differences in cortex of AD and no AD patients regarding SINE Alu RNA processing ratio (left panel) (a p value of 0.05 was considered as threshold for statistical significance with p<0.001, n=118, unpaired non-directional t-test) and in full length Alu RNAs (p=0.005). Boxplot representation as in subfigure 2D.",
    "molecules": "Alu"
  },
  {
    "caption": "Boxplots depict full length Alu RNA levels in cortex of AD and no AD patients separated into three age groups. No significant difference observed between the different age groups of either AD or no AD patients or for the comparisons between no AD and AD of each age group (unpaired non-directional t-test, with p<0.05 considered the threshold for statistical significance, n for no AD=19 (below 85), 19 (between 85 to 90), 13 (above 90); n for AD=8 (below 85), 21 (between 85 to 90), 38 (above 90)). Boxplot representation as in subfigure 2D.",
    "molecules": "Alu"
  },
  {
    "caption": "Boxplots depict differences in SINE Alu RNA processing ratio in cortex of AD and no AD patients separated into three age groups. No significant difference observed between the different age groups of either AD or no AD patients. A p value 0.05 was considered as threshold for statistical significance for the comparisons between no AD and AD of each age group (unpaired non-directional t-test, with p=0.003 for the comparison below 85, and p < 0.001 for the other two comparisons and n numbers as in (E)). Boxplot representation as in subfigure 2D.",
    "molecules": "Alu"
  },
  {
    "caption": "Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients with regard to final clinical diagnosis (p < 0.01 for the comparison between NCI and MCI, between NCI and AD, and between MCI and AD, unpaired, non directional t-test). NCI=no cognitive impairment (n=71), MCI=mild cognitive impairment (n=67), AD=Alzheimer's disease (n=87). Boxplot representation as in subfigure 2D. Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients (n=241) with regard to Braak stage classified into three categories (p = 0.004 for the comparison between 0-2 and 3-4, and p < 0.001 for the comparisons between 3-4 and 5-6 and between 0-2 and 5-6, unpaired, non directional t-test, n=39[0-2],n=148[3-4] and n=54[5-6]). Boxplot representation as in subfigure 2D.",
    "molecules": "Alu"
  },
  {
    "caption": "Scatterplot depicting the positive correlation between TP53 expression values and Alu RNA processing ratio in AD patients (r=0.65, p < 0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting no correlation between TP53 expression values and full length Alu RNA levels in AD patients (r=0.06, no correlation, ns=non-significant). Statistical test as in subfigure 5C.",
    "molecules": "Alu"
  },
  {
    "caption": "Association between SINE Alu RNA ratio (upper panel) and gene expression levels (lower panel) of genes up-regulated in AD (Dataset EV3, |log2FoldChange|> 0.5). Every column in both panels corresponds to the same MAP patient of either the no AD or AD group. Patients in each group are sorted from left to right in an ascending order with regard to Alu RNA processing ratio. Every row in the heatmap corresponds to one gene, with color density representing normalized FPKM values from RNA-seq for this gene for each patient (columns) (red represents high and blue low expression levels). Association between full length Alu RNA levels (upper panel) and gene expression levels (lower panel) of genes down-regulated in AD (Dataset EV4, |log2FoldChange|> 0.5). Every column in both panels corresponds to the same MAP patient of either the no AD or AD group. Patients in each group are sorted from left to right in an ascending order with regard to full length Alu RNA levels. Every row in the heatmap corresponds to one gene, with color density representing normalized FPKM values from RNA-seq for this gene for each patient (columns) (red represents high and blue low expression levels).",
    "molecules": "Alu"
  },
  {
    "caption": "Scatterplot depicting the positive correlation between average gene expression values of upregulated genes in Figure 6B and Alu RNA processing ratio in AD patients (r = 0.88, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting the negative correlation between average gene expression values of down-regulated genes in Figure 6C and full length Alu RNA levels in AD patients (r = -0.42, p<0.001). Statistical test as in subfigure 6D.",
    "molecules": "Alu"
  },
  {
    "caption": "Correlation between gene expression and Alu RNA processing ratio for AD upregulated genes (Dataset EV3) (upper panel) and a random set of non-differentially expressed genes (Dataset EV5) (lower panel). For every gene a correlation coefficient was calculated (Pearson correlation) together with the respective p-value. Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Based on the r and p value genes were classified into four categories: i)NS, non-significant for p value > 0.05, ii) no correlation or weak correlation (r≤0.05), iii) significant (p < 0.05) and strong correlation (0.5",
    "molecules": "Alu"
  },
  {
    "caption": "Expression levels of full length Alu RNA (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for the comparison between anti-Alu LNA treated samples (anti-Alu) and samples treated with a scramble control LNA (ctrl) with p=0.049 and n=3, unpaired non-directional t-test. Error bars represent standard deviation from the mean. Expression levels of P53 (RT-qPCR) in the Alu RNA KD experiment. Statistical significance (p value threshold 0.05) for levels in anti-Alu LNA treated samples (anti-Alu) greater than samples treated with a scramble control LNA (ctrl) with p=0.046 and n=3, unpaired directional t-test. Error bars represent standard deviation from the mean.",
    "molecules": "Alu"
  },
  {
    "caption": "Expression levels (RT-qPCR)_of selected genes from panel A that are either strongly correlated with Alu RNA processing (left, r> 0.5) or only weakly/not correlated (right, r <0.5). Statistical significance (p value threshold 0.05) for anti-Alu greater than control (depicted as asterisk, n=3/group, unpaired directional t-test, error bars represent standard deviation from the mean). Pearson correlation co-efficient for each gene is depicted below the name of each gene.",
    "molecules": "Alu"
  },
  {
    "caption": "In vitro incubation of one of the Alu RNA consensus sequences for different incubation periods. In vitro transcribed Alu RNA (67nM) incubated at 37oC with 250nM HSF1 in the course of 6 hours with time intervals of 1 hour.",
    "molecules": "Alu"
  },
  {
    "caption": "Comparison among Hsf1 (~60KDa), denatured HSF1, Poly A polymerase (~56KDa) and no protein (just TAP buffer) with regard to Alu RNA processing (estimating relative full length RNA remaining) (two replicates). The full gels are available as Source Data For Figure 8. Relative full length RNA remaining was calculated using ImageJ area under the curve software over time. Error bars represent standard deviation from the mean.",
    "molecules": "Alu"
  },
  {
    "caption": "Comparison among Alu RNA (three replicates), and two control RNAs (two replicates) regarding the full-length RNA levels remaining after in vitro incubation for 90 minutes at 37C with HSF1. Sizes of control RNAs are control for RNA #1, 143nt and for control RNA #2, 432nt. Incubation in the absence of HSF1 but presence of the same buffer (TAP) was used as control to take into account any non HSF1 specific RNA destabilization due to non-specific degradation. Threshold for statistical significance was a p value of 0.05 with p=0.03 (unpaired, non-directional t-test) for the comparison between HSF1 and no protein incubation (n=3, for Alu RNA). The full gels are available as Source Data For Figure 8. Error bars represent standard deviation from the mean.",
    "molecules": "Alu"
  },
  {
    "caption": "Plotting of the position of the first base (5' end) of Alu RNA fragments across the Alu consensus sequence produced by Alu RNA that has been processed in vitro for 90 minutes at 37C in the presence of HSF1 (upper panel) and compared with one of the in vivo samples of Figure 1 (lower panel). The x axis represents an Alu RNA metagene aligned at the start site of the Alu consensus sequence and the y axis shows the 5' end count for Alu RNA fragments aligning to any position downstream of position +1. The in vivo sample x axis depicts a 11nt sift compared to the in vitro one.",
    "molecules": "Alu"
  },
  {
    "caption": "Scatterplot depicting the positive correlation between HSF1 mRNA expression values and Alu RNA processing ratio in MAP AD patients (r=0.72, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting lack of correlation between HSF1 mRNA expression values and full length Alu RNA levels in MAP AD patients (ns=non-significant, with p value 0.05 as the significance threshold). Statistical test as in subfigure 8E.",
    "molecules": "Alu"
  },
  {
    "caption": "A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.",
    "molecules": "DNA, HU"
  },
  {
    "caption": "B FACS profiles of DNA content for each cell population assayed.",
    "molecules": "DNA"
  },
  {
    "caption": "A Genome‐wide analysis of Mcm2‐7 localization at the G1/S transition by ChIP‐chip. Mcm2‐7 enrichment from HU‐arrested cells is depicted for a 5‐Mb section of chromosome 2L. Inset: transcribed (green) and non‐transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.B Bimodal distribution of Mcm2‐7 enrichment over transcribed and non‐transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non‐transcribed (red) genes.C \"Meta\"‐gene analysis of Mcm2‐7 enrichment for different deciles of gene expression and their aggregated probe intensities.",
    "molecules": "HU"
  },
  {
    "caption": "D Mcm2‐7 is displaced from chromatin by DNA replication. Genome‐wide analysis of Mcm2‐7 localization in late S‐phase (6 h post HU release) by ChIP‐chip. Mcm2‐7 enrichment is depicted for a 10‐Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.E Box‐plots representing late S‐phase Mcm2‐7 ChIP signal found within early (246) or late (167) replication domains.",
    "molecules": "HU"
  },
  {
    "caption": " Decreasing ∆ψm from ∆III to ρ0 cells. WT and mutant yeast cells were cultured to log phase and stained with TMRM for FACS analysis. ",
    "molecules": "TMRM"
  },
  {
    "caption": "Polysome fractionation analysis of OXPHOS mutants. Polysomes were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.",
    "molecules": "sucrose"
  },
  {
    "caption": " Western blot analysis of copper-inducible overexpression of Inh1. A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus. Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours. ",
    "molecules": "copper, CuSO4"
  },
  {
    "caption": " Overexpression of Inh1 decreases ∆ψm in ρ0 cells. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Cells were then stained with 125 nM TMRM for FACS analysis. ",
    "molecules": "CuSO4, TMRM"
  },
  {
    "caption": " Overexpression of Inh1 represses the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30℃ for 2 days. ",
    "molecules": "CuSO4"
  },
  {
    "caption": " Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins (Nfs1 and Yah1) and nuclear ISC-containing protein Pol3. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Nfs1, Yah1 were endogenously tagged with FLAG, and Pol3 was endogenously tagged with HA. ",
    "molecules": "CuSO4"
  },
  {
    "caption": " F. Deletion of PUF3 decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis ",
    "molecules": "TMRM"
  },
  {
    "caption": " I. Mutating the Puf3-binding motif of MIA40 mRNA decreases ∆ψm of ρ0 cells. J. Mutating the Puf3-binding motif of MIA40 mRNA impairs the proliferation of ρ0 cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis",
    "molecules": "TMRM"
  },
  {
    "caption": " K. Overexpression of MIA40 rescues ∆ψm of ρ0 puf3∆ cells. Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains. L. Overexpression of MIA40 rescues the proliferation of ρ0 puf3∆ cells. Strains were prepared as in (K). Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis",
    "molecules": "TMRM"
  },
  {
    "caption": " Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Treatments and strains with Puf3 hyper-phosphorylation was highlighted in red. Glucose depletion was used as a positive control. Asterisk indicates a non-specific band. ",
    "molecules": "antimycin, Glucose, oligomycin, KCN"
  },
  {
    "caption": " Puf3-15A mutation inhibits Puf3 hyper-phosphorylation upon oligomycin treatment and in ∆V and ρ0 cells. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Asterisk indicates a non-specific band. ",
    "molecules": "antimycin, oligomycin, KCN"
  },
  {
    "caption": " Puf3-15A mutation decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison. ",
    "molecules": "TMRM"
  },
  {
    "caption": " Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells, and represses ribosome and Ribi gene expression in ρ0 cells. ρ0 snf1∆ cells were prepared by acute EtBr (25 µg/ml) treatment of snf1∆ cells in SCD for 2 days. RNAseq was performed with three biological replicates per genotype (Dataset EV8). All the transcripts and two categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated. ",
    "molecules": "EtBr, glucose"
  },
  {
    "caption": " Snf1 represses ribosome biogenesis in WT and ρ0 cells. Polysomes of the indicated strains were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254. ",
    "molecules": "sucrose"
  },
  {
    "caption": " Deletion of SNF1 decreases ∆ψm in both ∆V and ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis ",
    "molecules": "TMRM"
  },
  {
    "caption": " Deletion of MIG1 in ρ0 snf1∆ cells rescues ∆ψm. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis ",
    "molecules": "TMRM"
  },
  {
    "caption": " Heat map plots of the mRNA level changes of chaperones, ubiquitin-proteasome pathway, and autophagy genes (gene list in Table EV1F) in the indicated strains (data from Dataset EV1 and Dataset EV10). Selected genes for growth analysis in B are highlighted in red. ",
    "molecules": "ubiquitin"
  },
  {
    "caption": " Deletion of HSC82 selectively decreases ∆ψm in ρ0 puf3∆ cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison. ",
    "molecules": "TMRM"
  },
  {
    "caption": "(A) incorporation of 14C-labeled  glycerol 3-phosphate  (G3P) into various lipids in the presence of  palmitoyl-CoA  (10 µM) in microsomal membranes   prepared from HEK293-AT1  cells. After incubations at room temperature for 30 min, lipids were extracted and separated by TLC  before quantification using autoradiography and phosphorimaging. Lipid species corresponding to the major spots visualized are indicated. Note the appearance of phosphatidylcholine (PC) and cytidine diphosphate-diacylglycerol  (CDP-DAG) in the presence of  cytidine trisphosphate (CTP), as well as the production of phosphatidylinositol  (PI) only when both CTP  and myo-inositol  are present. Also, there is a relatively small effect of the DAG kinase inhibitor (DGKi) on CDP-DAG and PI  formation in spite of the fact that plenty of phosphatidic acid (PA) is available from de novo synthesis (although it should be noted that no ATP  was added during these reactions). A representative experiment is shown, but these results have been reproduced in other replicates using different treatment combinations. The insert shows the effect of the DGK  inhibitor on PI  labeling.",
    "molecules": "ATP, CDP-DAG, cytidine diphosphate-diacylglycerol, CTP, cytidine trisphosphate, myo-inositol, palmitoyl-CoA, PA, phosphatidic acid, PC, phosphatidylcholine, phosphatidylinositol, PI, G3P, glycerol 3-phosphate, 14C"
  },
  {
    "caption": "(B) Effect of lipin-1γ overexpression on the metabolic fate  of de novo synthesized PA. Experiments were performed as in panel A, except that the membranes were also prepared from cells expressing a GFP-tagged lipin 1γ, as indicated. Note the large amount of TG  and increased production of PC  under these conditions. Results of a representative experiment are shown, and these experiments were repeated with identical results.",
    "molecules": "PA, PC, TG"
  },
  {
    "caption": "Effect of different fatty-acyl-CoA   analogs (each used in 10 µM) on the  metabolic fate of de novo synthesized PA. Note the preferential conversion of  oleoyl-CoA containing PA  into TG  and the very poor incorporation of  stearoyl-CoA into TG. Arachidonoyl-CoA  is a relatively poor substrate for the generation of de novo synthesized  PA; however, this substrate has the highest conversion rate into PI, which was calculated as the ratio of the PI  and PA  mass shown in panel D. , while quantified values (means ± range) from two such experiments are shown in (D) and (E).",
    "molecules": "Arachidonoyl-CoA, fatty-acyl-CoA, oleoyl-CoA, PA, PI, stearoyl-CoA, TG"
  },
  {
    "caption": "Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice  were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse), Ki67  as percentage of total DAPI-stained  cells (n = 3-4 biological replicates per group, n = 20 glands  per mouse), GIF+ area (n = 4-5 biological replicates per group, n = 10 glands  per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands  per mouse). Data information: For (one-way ANOVA + Tukey´s multiple comparison test for C,",
    "molecules": "DAPI"
  },
  {
    "caption": "Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice  were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 2-4 biological replicates per group, n = 10 glands  per mouse), Ki67  as percentage of total DAPI-stained  cells (n = 2-4 biological replicates per group, n = 20 glands  per mouse), GIF+ area (n = 2-4 biological replicates per group, n = 10 glands  per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands  per mouse). Data information: For C, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for C,",
    "molecules": "DAPI"
  },
  {
    "caption": "F Quantification of p65+ area as percentage of total DAPI-stained  cells of uninfected and infected Lgr4-/- and control mice  (n = 4 biological replicates per group, n = 20 glands  per mouse). Data information: For data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).",
    "molecules": "DAPI"
  },
  {
    "caption": "G Immunofluorescence co-localization of p65 acetyl  K310 (activated form of NF-κB) in the mouse antrum  tissue of infected Lgr4-/- and in control mice. Insets show magnified images. Scale bars: 50µm. Data information: For G images represent findings that were reproduced at least twice in the laboratory in independent biological replicates.",
    "molecules": "acetyl"
  },
  {
    "caption": "B Quantification of p65+ area as percentage of total DAPI-stained  cells of infected Rspo3-/- and control mice  (n = 3 biological replicates per group, n = 20 glands  per mouse). Data information: For B, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for B",
    "molecules": "DAPI"
  },
  {
    "caption": "E Myh11CreERT2/Rosa26Sor6(CAG-Rspo3) (Rspo3 KI) mice  were infected for 6 weeks with H. pylori. Immunofluorescence  labeling for p65, GSII  and KI67  in uninfected and infected Rspo3 KI and control mice. Scale bars: 50 µm. F Quantification of p65+ area as percentage of total DAPI-stained  cells of uninfected and infected Rspo3 KI and control mice  (n = 2-3 biological replicates per group, n = 20 glands  per mouse). G Quantification of GSII+ area of uninfected and infected Rspo3 KI and control mice  (n = 2-3 biological replicates per group, n = 20 glands  per mouse). H Quantification of Ki67  as percentage of total DAPI-stained  cells of uninfected and infected Rspo3  KI and control mice  (n = 2-3 biological replicates per group, n = 20 glands  per mouse). Data information: For , F, G and H data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant one-way ANOVA + Tukey´s multiple comparison test for F, G and H).",
    "molecules": "DAPI"
  },
  {
    "caption": "I Immunofluorescence  labeling for p65 acetyl  K310 in infected Rspo3 KI and control mice. Insets show magnified images. Scale bars: 50 µm.",
    "molecules": "acetyl"
  },
  {
    "caption": "Antrum organoids  from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α was added for 2 h to all organoid  conditions prior to analysis. A Bright-field  images of organoids  in respective conditions before treatment with ADP heptose  or TNF-α. Scale bars: 200 µm.",
    "molecules": "ADP heptose"
  },
  {
    "caption": "Antrum organoids  from wildtype mice  were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α  was added for 2 h to all organoid  conditions prior to analysis. B Immunofluorescence labeling for GSII  and Muc5ac  of organoids  in respective conditions before treatment with  ADP heptose  or  TNF-α. Scale bars: 50 µm.",
    "molecules": "ADP heptose"
  },
  {
    "caption": "Antrum organoids  from wildtype mice  were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose or 10 ng/ml TNF-α  was added for 2 h to all organoid  conditions prior to analysis. E qPCR  expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2  in organoids  after treatment with ADP heptose compared to untreated controls within the respective medium conditions (n = 6 biological replicates per condition). F qPCR  expression level of Cxcl1, Cxcl2, Cxcl10, Ccl2 in organoids  after treatment with TNF-α compared to untreated controls within the respective medium conditions (n = 2-3 biological replicates per condition). Data information: For E and F data are mean + SD. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001 ; t-test for E and F). Genes without asterisks are not significantly altered.",
    "molecules": "ADP heptose"
  },
  {
    "caption": "Antrum organoids  from wildtype mice  were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 µM ADP heptose was added for 2 h to all organoid  conditions prior to analysis. A Electrophoretic mobility shift assay (EMSA) and SDS-PAGE western blot from whole cell protein (WCL) lysates from gastric organoids  untreated or treated with ADP heptose, and maintained either in FM in medium without Rspo, without Wnt  and Rspo, or +BMP2. Representative images from n = 3 biological replicates. B Quantification of EMSA  based on n = 3 biological replicates using ImageJ with normalization to loading (ɑ-Tubulin). Data information: For B data are mean + SD. Significance is calculated by one-way ANOVA, F = 9.21.",
    "molecules": "ADP heptose"
  },
  {
    "caption": "C EMSA  supershift from FM + ADP heptose organoids  with the antibodies indicated. Blue arrow indicates NF-κB binding. Red arrow points to gel expected locations of supershifts from antibodies against p65, cRel, or RelB. Grey arrow points to supershift location on the gel from antibodies against p52  or p50. IgG lane serves as a negative control.",
    "molecules": "ADP heptose"
  },
  {
    "caption": "D Representative H&E staining  and immunofluorescence  labeling for GSII, Ki67  and MUC5AC  in uninfected ΔN and control mice. Scale bars: 50 µm. E Quantification of GSII  and Ki67  as percentage of total DAPI-stained  cells in ΔN and control mice  (n = 5 biological replicates per group, n = 20 glands  per mouse). Data information: For E data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.",
    "molecules": "iodixanol"
  },
  {
    "caption": "(B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.",
    "molecules": "iodixanol, pepstatin A"
  },
  {
    "caption": "(C) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h. Cells were then harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.",
    "molecules": "AT-2, iodixanol"
  },
  {
    "caption": "(B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.",
    "molecules": "AT-2"
  },
  {
    "caption": "(E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.",
    "molecules": "sirolimus"
  },
  {
    "caption": "(F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.",
    "molecules": "AT-2, bafilomycin A1, LyoVec, sirolimus"
  },
  {
    "caption": "(B) Cells were harvested then subjected to flow cytometry analysis of saponin-resistant LC3B-II in macrophages. Representative histograms of cells displaying saponin-resistant LC3B-II are shown.",
    "molecules": "saponin"
  },
  {
    "caption": "(D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.",
    "molecules": "AT-2, iodixanol"
  },
  {
    "caption": "(F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.",
    "molecules": "AT-2"
  },
  {
    "caption": "(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4.",
    "molecules": "AT-2"
  },
  {
    "caption": "(C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.",
    "molecules": "AT-2"
  },
  {
    "caption": "Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7). [Correction added after publication on 4 July 2013: The y axis of the graph shown in Fig. 1A has been corrected from \"concentration (mM)\" to \"concentration (nM)\"]",
    "molecules": "DMSO, Imatinib, Bosutinib, Nilotinib"
  },
  {
    "caption": "In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6mice (N = 5) and (H) parkin−/− as control.PLA in Tg-APPmice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5).",
    "molecules": "DMSO, Bosutinib, Nilotinib"
  },
  {
    "caption": "Graph represents human Aβ1-42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1-42 (or LacZ) or Beclin-1 shRNA for 24 hr, and then treated with 1 μM Bosutinib for an additional 24 hr (N = 12). *Significantly different to control or as indicated, Mean ± SEM, ANOVA with NeumannKeuls multiple comparison.",
    "molecules": "Bosutinib"
  },
  {
    "caption": "Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different (A,B) Tg-APP + DMSO and (C) thioflavin-S staining in Tg-APP treated with DMSO (N = 7). (D,E) Tg-APP treated with 5 mg/kg Bosutinib for 3-weeks and (F) thioflavin-s staining.",
    "molecules": "DMSO, Bosutinib"
  },
  {
    "caption": "Staining of 20 μm thick brain sections shows (G) parkin, (H) Aβ1-42 and (I) merged figure in cortex of Tg-APPmice after 3 weeks of DMSO treatment, and (J) parkin, (K) Aβ1-42 and (L) merged figure in cortex of Tg-APPmice after 3 weeks of 5 mg/kg Bosutinib treatment. (M) Parkin, (N) Aβ1-42 and (O) merged figure in hippocampus of Tg-APPmice after 3 weeks of DMSO treatment, and (P) parkin, (Q) Aβ1-42 and (R) merged figure in cortex of Tg-APPmice after 3 weeks of Bosutinib treatment (N = 7).",
    "molecules": "DMSO, Bosutinib"
  },
  {
    "caption": "(S) Graphs represent quantification of amyloidplaques in Tg-APP with and without Bosutinib.",
    "molecules": "Bosutinib"
  },
  {
    "caption": "Graphs represent ELISA of human Aβ1-42 in (A) brain levels and (B) bloodAβ1-42 levels in 8-month old Tg-APPmice injected I.P. every other day for 6 weeks with Bosutinib or Nilotinib (N = 10).",
    "molecules": "Bosutinib, Nilotinib"
  },
  {
    "caption": "ELISA of human soluble and insoluble brain (C) Aβ1-42 and (D) Aβ1-40 levels in 8-month old Tg-APPmice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).",
    "molecules": "Bosutinib"
  },
  {
    "caption": "ELISA of (E) mouse p-Tau levels in 8-month old Tg-APP mice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).",
    "molecules": "Bosutinib"
  },
  {
    "caption": "Staining of 20μm brain sections shows intracellular Aβ1-42 within the (A) hippocampus of lentiviralAβ1-42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1-42. Staining of 20μm brain sections shows intracellular Aβ1-42 within the (C) cortex of WT mice with the lentiviralAβ1-42, and (D) Bosutinib clearance of intracellular Aβ1-42 (N = 7).",
    "molecules": "Bosutinib"
  },
  {
    "caption": "ELISA of human soluble and insoluble Aβ1-42 at 6 weeks post-injection of lentiviralAβ1-42 and daily treatment with 5 mg/kg Bosutinib (N = 9) for 3 weeks. * Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.",
    "molecules": "Bosutinib"
  },
  {
    "caption": "WB on 4-12% NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin−/−mice treated with 5 mg/kg Bosutinib for 3 weeks on 4-12% NuPAGE SDS gel (Invitrogen) showing parkin (1st blot)total Abl (2nd blot), T412 Abl (3rd blot), Beclin-1 (4th blot) and LC3 (5th blot) relative to actin (N = 7).",
    "molecules": "Bosutinib"
  },
  {
    "caption": "Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post-injection with (G) lentiviralAβ1-42 + 3 weeks DMSO treatment and (H) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) clears plaques in WT mice (N = 7).(I) Lentiviral Aβ1-42 + DMSO and (J) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) in parkin−/−mice (N = 7).",
    "molecules": "DMSO, Bosutinib"
  },
  {
    "caption": "Graphs represent ELISA (N = 5) in autophagic vacuoles (AVs) in the brain of 4 and 8 months old Tg-APPmice treated with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib (N = 5) for 3 weeks showing (A)humanAβ1-42 (Insert is WB analysis of AVs showing LC3-B in AV10 and AV20 (1st blot) and LAMP2a in Lys (2nd blot) fraction) and (B)Aβ1-40,(C) p-Tau and (D) parkin.",
    "molecules": "Bosutinib, Nilotinib"
  },
  {
    "caption": "ELISA in autophagic vacuoles of 1 year old WT and parkin−/−mice (N = 5) injected with lentiviralAβ1-42 for 3 weeks and treated with 10 mg/kg Nilotinib and 5 mg/kg Bosutinib for 3 additional weeks showing (E) humanAβ1-42 and (F) p-Tau levels.",
    "molecules": "Bosutinib, Nilotinib"
  },
  {
    "caption": "(A) Represents results of Morris water maze test in lentiviralAβ1-42-injected ± Bosutinib WT (N = 12) and parkin−/− (N = 10) mice, and (B) heat maps for each group.Graphs represent total number of entry into platform area and distance travelled.",
    "molecules": "Bosutinib"
  },
  {
    "caption": "Represents results of Morris water maze test in Tg-APP ± Bosutinib (N = 12) mice.Graphs represent total number of entry into platform area and distance travelled.",
    "molecules": "Bosutinib"
  },
  {
    "caption": "(B-C) Representative confocal images and their quantification of   Gb3  accumulation within the  lysosome, detected by fluorescent-conjugated Shiga toxin, from WT, CLN3-KO and CLN7-KO ARPE-19 cells treated with  PDMP or silenced for Gb3 synthase (siGb3S) (*** vs WT, °°° vs DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "PDMP, Gb3, DMSO"
  },
  {
    "caption": "(A-B) Representative confocal images of Gb3   accumulation, revealed by STX staining, in  brain sections from CLN7Δex2 mice at 3 and 7.5 months of age compared with CLN7 WT mice. Scale bars: 80 µm.",
    "molecules": "Gb3"
  },
  {
    "caption": "(D) Representative  confocal images and quantification of  STX staining within the  lysosome of ARPE-19 WT and  CLN7  KO in DMSO or treated 48h with Tamoxifen. (*** vs WT, °°° vs CLN3  KO  DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "DMSO, Tamoxifen"
  },
  {
    "caption": "(E) Representative confocal images and quantification of  STX staining within the  lysosome  of NPCs WT and derived from CLN7Pa474 patient IPSCs in  DMSO or treated with  Tamoxifen for 48h. (*** vs WT, °°° vs CLN7Pa474 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "DMSO, Tamoxifen"
  },
  {
    "caption": "(A) Representative  confocal images  and Quantification of STX  within the  lysosome  in U2-OS  and HeLa  cells after acute silencing of CLN3 (siCLN3) in  DMSO  or treated 48h with Tamoxifen. (*** vs siSCR DMSO, °°° vs siCLN3 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "DMSO, Tamoxifen"
  },
  {
    "caption": "(B) Representative  confocal image and quantification of Gb3 in ARPE-19 CLN3 KO cells silenced with siRNA against scramble sequence and TFEB (siTFEB) for 72h and treated for the last 48h with DMSO or  Tamoxifen. Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "Gb3, DMSO, Tamoxifen"
  },
  {
    "caption": "(C) Representative confocal image and quantification of TFEB  in U2-OS cells silenced with siRNA against scramble sequence and CLN3  (siCLN3) for 72h and treated for the last 48h with DMSO or Tamoxifen (5 µM and 10 µM). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "DMSO, Tamoxifen"
  },
  {
    "caption": "(C) Representative confocal image and quantification of TFEB localization in HeLa TFEB-GFP transfected with RagC for 48h and treated for the last 3h with DMSO, Torin1, Tamoxifen  or Ospemifene. Ratios of nuclear to  cytosolic TFEB localization in RagC non-expressing (RagC-) and RagC-expressing cells (RagC+) are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "DMSO, Ospemifene, Tamoxifen, Torin1"
  },
  {
    "caption": "D) Representative confocal images and quantification of STX in ARPE-19 CLN3 KO cells transfected with empty vector or HA-RagC for 48h and treated for 48h with DMSO or Tamoxifen. STX average spot area presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.",
    "molecules": "DMSO, Tamoxifen"
  },
  {
    "caption": "Representative confocal images of STX  in the  Cortex, Hippocampus, and Cerebellum brain  section derived from 7.5 months-old mouse  WT or CLN7Δex2 injected with vehicle or Tamoxifen (Tamox). Quantification of confocal images, the plot shows the quantification of the STX  average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°°/°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, **/oo: P ≤ 0.001, ***/ooo: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 60 µm.",
    "molecules": "Hoechst, Tamox, Tamoxifen"
  },
  {
    "caption": "Representative  confocal images  of SCMAS in the Cortex, Hippocampus, and Cerebellum brain  section derived from mouse  WT or CLN7Δex2 injected with vehicle or Tamoxifen. Quantification of  confocal images, the plot shows the quantification of the SCMAS average spot area normalized for the number of  Hoechst  positive cells. (*** vs WT, °°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological). Scale bars: 60 µm.",
    "molecules": "Hoechst, Tamoxifen"
  },
  {
    "caption": "(A-B) Representative confocal images  and quantification of  IBA-1  in the  Cortex, Hippocampus, and Cerebellum brain  section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen. (***/**/* vs WT, °°°/°°/° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, */o: P ≤ 0.01, **/oo: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 50 µm.",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "(D) Representative Images of  hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen.",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "G. Using high power, high resolution laser scanning confocal microscopy, no human LAG3 signal could be detected in human neurons (Auto-hLAG3 transduced, DOX OFF) by two different anti-human LAG3 antibodies (17B4 and D2G40; left panel and zoomed-in insets) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 (DOX ON; right panel and zoomed-in insets). Scale bars 25 µm.",
    "molecules": "DOX"
  },
  {
    "caption": "D. Using high power, high resolution laser scanning confocal microscopy, no mouse LAG3 signal could be detected in murine primary neuronal cultures (Auto-mLAG3 transduced, DOX OFF and non-transduced neurons) using anti-mouse LAG3 antibody (MABF954) whereas LAG3 was clearly detected in primary neurons induced to express mLAG3 (Auto-mLAG3 transduced, DOX ON). Scale bars 20 µm.",
    "molecules": "DOX"
  },
  {
    "caption": "Human neural cultures transduced by Auto-hLAG3 were treated by α-synuclein PFFs 4 days post LAG3 expression induction by DOX and kept in culture for 2 or 4 weeks. Both transgenic (hLAG3 D2G40-positive in (A) and 17B4-positive in (B)) and non-transduced, wild-type neurons (selected neurons in zoomed-in insets) propagated α-synuclein and developed characteristic pS129-positive (81A-positive in (A) and EP1536Y-positive in (B)) α-synuclein aggregates. Scale bars 25 µm. Trained ilastik algorithms were used to segment pixels of 81A, EP1536Y and MAP2 stainings imaged by high-content wide-field microscope, which were used to quantify the signal of 81A-positive (C) and EP1536Y-positive (D) α-synuclein aggregates expressed as % of MAP2-positive area. Almost the entire wells (182 fields per well) were imaged for every condition and replicate and each datapoint in the plot represents the entire well. Error bars indicate mean ± SD of biological replicates (duplicates). For few conditions, unicates were used, shown as one dot. One-way ANOVA followed by Tukey's multiple comparison test demonstrated that neurons that did not express LAG3 (DOX OFF) showed no difference in α-synuclein propagation when compared to LAG3-expressing neurons (DOX ON) as demonstrated by two different pS129 α-synuclein antibodies (p=0.9998 for 81A at 2 weeks and p=0.2522 at 4 weeks; p=0.9986 for EP1536Y at 2 weeks and p=0.3042 at 4 weeks).",
    "molecules": "DOX"
  },
  {
    "caption": "B. Immunostaining for hyperphosphorylated α-synuclein aggregates (pS129) and aggregated protein (Thioflavin S) in sections of end-stage symptomatic mice; no obvious difference in terms of staining pattern or number of stained cells were apparent in midbrain or brain stem. Scale bars represent 1 mm (sagittal section) and 100 µm (panel).",
    "molecules": "Thioflavin S"
  },
  {
    "caption": "(b) SAR405 inhibits the function of Vps34 in the GFP-FYVE cellular assay. HeLa cells stably transfected with GFP-FYVE were incubated with DMSO (left) or with 1 μM SAR405 (right) for 2 h. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). Fluorescence was analyzed using an imaging cytometer (X40). One representative image out of three independent experiments is depicted. Scale bars, 20 μm.",
    "molecules": "DMSO, SAR405"
  },
  {
    "caption": "(c) SAR405 does not prevent phosphorylation of Akt and S6. U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations. The effect of compounds on phosphorylated Akt (p-Akt) Ser473 and Thr308 residues, total Akt and on phosphorylated S6 (Ser240 and Ser244 residues) and total S6 protein was measured using western blotting. One representative experiment from at least two independent experiments is shown. Full-length blots are shown in Supplementary Figure 6.",
    "molecules": "PI103, SAR405"
  },
  {
    "caption": "(a) SAR405 induces late endosome-lysosome swelling. RKO cells were treated with DMSO (upper panels) or with 10 μM SAR405 for 16 h (lower panels). The late endosomes-lysosomes compartment was assessed using Lysotracker green (left) or Alexa 488-conjugated anti-Lamp1 antibodies (right), as indicated. Nuclei were stained using Hoechst 33342 (blue). One representative image chosen from three independent experiments, showing fluorescence imaged using a Zeiss microscope (X40), is shown for each condition. Arrows indicate accumulation of swollen late endosome-lysosomes. Scale bars, 20 μm.",
    "molecules": "DMSO, SAR405"
  },
  {
    "caption": "(b) SAR405 triggers accumulation of p62. RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h. The effect on the p62 biomarker was analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 14.",
    "molecules": "SAR405"
  },
  {
    "caption": "(c) SAR405 affects the maturation of cathepsin D (cat D). RKO cells were treated at the indicated concentrations of SAR405 for 24 h. The different forms of cathepsin D (PPCD, pre-pro-catD) were analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 16.",
    "molecules": "SAR405"
  },
  {
    "caption": "(a) GFP-LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image, acquired with an iCyte imaging cytometer, for three independent experiments is depicted (X40). White arrows indicate GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells and GFP intensity, measured using an imaging cytometer for different compound concentrations on GFP-LC3 HeLa cells. Black and gray histograms correspond to fed and starved conditions, respectively. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.",
    "molecules": "hydroxychloroquine"
  },
  {
    "caption": "(c) Wild-type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine (HCQ), as indicated. Cells were treated at the same time with SAR405 at different concentrations. LC3-II was measured by western blotting, and GAPDH was used as a loading control. One representative experiment for three independent experiments is shown. Full-length blots are shown in Supplementary Figure 18. (d) Histograms show the LC3-II/GAPDH ratio for each condition in the two cell lines. Black and gray histograms correspond to fed condition and EBSS conditions, respectively. The addition of hydroxychloroquine at 10 μM is indicated. Values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.",
    "molecules": "hydroxychloroquine, SAR405"
  },
  {
    "caption": "(a) GFP-LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO (left) or with 1 μM SAR405 (right). Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image of cells out of three independent experiments, acquired using an iCyte imaging cytometer, is shown (X40). White arrows indicate the GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells (top) and the GFP intensity (bottom), as measured using an imaging cytometer. Treatment of GFP-LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.",
    "molecules": "DMSO, SAR405, AZD8055"
  },
  {
    "caption": "(c) The concentration needed to reach 40% inhibition (IC40) of cell proliferation is indicated. The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines. The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405. The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼0.5, corresponding to compounds that are in equipotent proportion in the mixture (Supplementary Fig. 20). The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments.",
    "molecules": "everolimus, SAR405"
  },
  {
    "caption": " B. Tumors formed with WT MC38 cells were treated with PBS or 5-FU. Tumor volumes were quantified at the indicated days. N=5. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " C. STING-KO or sgRNA control (Ctrl) MC38 cells were treated in vitro with the indicated concentrations of 5-FU. Cell viability were determined using the CellTiter-Glo assay after two days, with normalized luminescence levels shown. N=3. ",
    "molecules": "5-FU"
  },
  {
    "caption": " Mice were injected with Ctrl or STING-KO MC38 cells, and treated with PBS or 5-FU. (D) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": "  Mice were injected with Ctrl or STING-KO MC38 cells, and treated with PBS or 5-FU. (E) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5 as shown in (D).  ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with Ctrl or STING-KO MC38 cells, and treated with PBS or 5-FU. (F) Tumor and spleen weights at the endpoint for (E), with each dot representing a mouse. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " WT MC38 cells were injected into STING+/+ or STING-/- mice, and treated with 5-FU or PBS following the schematics in (A). (G) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": "  WT MC38 cells were injected into STING+/+ or STING-/- mice, and treated with 5-FU or PBS following the schematics in (A). (H) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5.  ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " WT MC38 cells were injected into STING+/+ or STING-/- mice, and treated with 5-FU or PBS following the schematics in (A). (I) Tumor and spleen weights at the endpoint for (H), with each dot representing a mouse. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " C. STING-KO or sgRNA control (Ctrl) YUMM1.7 cells were treated in vitro with the indicated concentrations of DTIC. Cell viability were determined using the CellTiter-Glo assay after two days, with relative cell viability shown. N=3. ",
    "molecules": "DTIC"
  },
  {
    "caption": " YUMM1.7 Ctrl or STING KO cells were injected into mice. 5-FU and DTIC responses were followed according to the schematics in (A). (E) Representative images of tumors and spleens. Image panels were cropped from the same picture. ",
    "molecules": "5-FU, DTIC"
  },
  {
    "caption": " YUMM1.7 Ctrl or STING KO cells were injected into mice. 5-FU and DTIC responses were followed according to the schematics in (A). (F) Mean tumor volumes of Ctrl or STING KO YUMM1.7 with DMSO, DTIC or 5-FU treatment were followed during the experiment. N=3 to N=5, as shown in (E). ",
    "molecules": "5-FU, DTIC, DMSO"
  },
  {
    "caption": " YUMM1.7 Ctrl or STING KO cells were injected into mice. 5-FU and DTIC responses were followed according to the schematics in (A). (G) Tumor (left panel) and spleen (right panel) weights were determined at the endpoint for the experiment followed in (F). Each dot represents data from a mouse. ",
    "molecules": "5-FU, DTIC"
  },
  {
    "caption": " A. Control (Ctrl) or STING-KO MC38 cells were treated in vitro with STING agonist cGAMP (left) for 6 hours or 5-FU (right) or relevant vehicle controls for 24 hours. The expression of indicated genes was determined by qRT-PCR. N=3. ",
    "molecules": "cGAMP, 5-FU"
  },
  {
    "caption": " B. Ctrl or STING-KO CT26 cells were treated with 5-FU or vehicle control for 24 hours. The RNA expression levels of indicated genes were determined by qRT-PCR. N=3. ",
    "molecules": "5-FU"
  },
  {
    "caption": " C. Ctrl or STING-KO YUMM1.7 cells were treated with (left panel) vehicle control (DMSO) or DTIC, or (right panel) PBS or 5-FU for 24 hours. The expression of indicated genes was determined by qRT-PCR. N=3. ",
    "molecules": "5-FU, DTIC, DMSO, PBS"
  },
  {
    "caption": " E. MC38 Ctrl or Ifn-DKO cells were treated with (left panel) control or the STING-agonist DMXAA for 4 hours, or (right panel) control or 5-FU for 16 hours. Ifnβ levels were determined in harvested culture media by ELISA. N=2. ",
    "molecules": "5-FU, DMXAA"
  },
  {
    "caption": " Mice were injected with Ctrl MC38 cells or Ifn-DKO cells and treated with 5-FU or PBS. (F) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with Ctrl MC38 cells or Ifn-DKO cells and treated with 5-FU or PBS. (G) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with Ctrl MC38 cells or Ifn-DKO cells and treated with 5-FU or PBS. (H) Tumor and spleen weights at the endpoint for (G), with each dot representing a mouse. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with Ctrl or cGAS-KO MC38 cells and treated with PBS or 5-FU. (B) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with Ctrl or cGAS-KO MC38 cells and treated with PBS or 5-FU. (C) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5, as shown in (B). ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with Ctrl or cGAS-KO MC38 cells and treated with PBS or 5-FU. (D) Tumor and spleen weights at the endpoint for (C), with each dot representing a mouse. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " B. Ctrl or Ifnar1-KO MC38 cells were treated with PBS or recombinant Ifnβ for 4 hours. The RNA expression levels of indicated ISGs were analyzed using qRT-PCR. N=3. ",
    "molecules": "PBS"
  },
  {
    "caption": " Mice were injected with control (Ctrl) or Ifnar1-KO MC38 cells, and treated with 5-FU or PBS. (C) Pictures of tumors and spleens from a representative experiment. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with control (Ctrl) or Ifnar1-KO MC38 cells, and treated with 5-FU or PBS. (D) Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with control (Ctrl) or Ifnar1-KO MC38 cells, and treated with 5-FU or PBS. (E) Tumor and spleen weights at the endpoint for (D), with each dot representing a mouse. Ctrl tumor data in (C-E) are the same as those in Figure 4B-4D. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": "  experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS. (G) Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": "
 experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS. (H) Tumor volumes were quantified at the indicated days post cancer cell injection. N=4 to N=5, as shown in (G). 
",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": "
 experiment to test the function of Ifnar1 in bone marrow (BM) derived cells. WT C57BL/6 mice were transplanted with either Ifnar1+/+ or Ifnar1-/- BM cells. Recipient mice were allowed to recover followed by the injection of WT MC38 cells, before treatment with 5-FU or PBS. (I) Tumor and spleen weights at the endpoint for (H), with each dot representing a mouse. 
",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with control (Ctrl) or STING-KO MC38 cells and treated with PBS or 5-FU. Tumors were harvested 2 weeks after cancer cell injection and intratumoral immune cells were examined by flow cytometry. A. The counts of CD45+ cells per gram of tumor. B. The percentages of CD45+ cells among forward scatter (FSC) and side scatter (SSC) gated live cell population. C. The counts of CD3+, CD3-NK1.1+, CD3-CD19+, CD11b+ and CD11c+CD103+ cells per gram of tumor were quantified. D. The percentages of CD3+, CD3-NK1.1+, CD3-CD19+, CD11b+ and CD11c+CD103+ cells among CD45+ cells were quantified. E. The counts of CD4+ and CD8+ T cells per gram of tumor. F. The percentages of CD4+ and CD8+ cells among CD3+ T cells were quantified. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " effect of T cell depletion on 5-FU response, during which anti-CD4/anti-CD8 antibodies (Ab), control IgG Ab, or anti-NK1.1 Ab were administrated on day 5 and day 9. (B) Pictures of tumors and spleens. Image panels were cropped from the same picture. N=10. ",
    "molecules": "5-FU"
  },
  {
    "caption": " effect of T cell depletion on 5-FU response, during which anti-CD4/anti-CD8 antibodies (Ab), control IgG Ab, or anti-NK1.1 Ab were administrated on day 5 and day 9. (C) Tumor volumes were quantified at the indicated days post cancer cell injection. N=10. ",
    "molecules": "5-FU"
  },
  {
    "caption": " effect of T cell depletion on 5-FU response, during which anti-CD4/anti-CD8 antibodies (Ab), control IgG Ab, or anti-NK1.1 Ab were administrated on day 5 and day 9. (D) Tumor and spleen weights at the endpoint for (C), with each dot representing a mouse. ",
    "molecules": "5-FU"
  },
  {
    "caption": " Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS. A. Pictures of tumors and spleens from a representative experiment. Image panels were cropped from the same picture. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS. B. Tumor volumes were quantified at the indicated days post cancer cell injection. N=5. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": " Mice were injected with control (Ctrl) or STING-KO MC38 cells, and subjected to treatments, with each dose consisting of 25 mg/kg, 50 mg/kg or 75 mg/kg 5-FU, or PBS. C. Tumor and spleen weights at the endpoint for (B), with each dot representing a mouse. ",
    "molecules": "5-FU, PBS"
  },
  {
    "caption": "B Phospholipid transport/synthesis, as measured by 3H-serine incorporation, in human fibroblasts (mean±SE; n=9, with 5 replicates/experiment) and in primary mouse hippocampal neurons (mean±SE; n=3, with 4 or 5 replicates/experiment). *, p<0.05 vs E3.",
    "molecules": "serine"
  },
  {
    "caption": "C Comparison of 3H-serine incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 3 replicates/experiment).",
    "molecules": "serine"
  },
  {
    "caption": "D Duramycin sensitivity in ApoE ACM-treated fibroblasts. Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4-treated cells, which was blocked upon the addition of 15 μM exogenous PtdEtn (mean±SD; n=3, with 3 replicates/experiment).",
    "molecules": "Duramycin, duramycin, PtdEtn"
  },
  {
    "caption": "C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).",
    "molecules": "lipoprotein"
  },
  {
    "caption": "A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.",
    "molecules": "oleate, CE, TAG, triglyceride"
  },
  {
    "caption": "B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment).",
    "molecules": "oleate"
  },
  {
    "caption": "(B)Pull-down analysis for the hexameric IFT-B2 complex. A mixture of IFT172ΔC, IFT80, and the IFT57/38 complex ('input') shows only weak non-specific binding to GSH-resin ('empty'), but all proteins are pulled down by IFT54/20-GST complex. Note that the IFT20-GST and IFT38 proteins co-migrate on the gel and cannot be properly resolved. However, the presence of the IFT57 protein in the pull-down sample implies that its binding partner IFT38 is also there.",
    "molecules": "GSH"
  },
  {
    "caption": "(D) GSH resin pull-down of a mixture of IFT80 and IFT172ΔC (input, lane 1) with GST-tagged CH-domains of IFT57 or IFT38 (IFT57CH or IFT38CH) or with the GST-tagged coiled-coil (CC) region of IFT57 mixed with His-tagged coiled-coil region of IFT38. The SDS-PAGE gel shows interaction between IFT57CH-IFT172ΔC, IFT38CH-IFT80 and IFT57CC-IFT38CC.",
    "molecules": "His, GSH"
  },
  {
    "caption": "(E) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT38/38 (both subunits His-tagged). The two bands for IFT38 on the SDS PAGE gel correspond to His-tagged (upper band) and proteolytically cleaved untagged IFT38 (lower band).",
    "molecules": "His, Ni, NTA"
  },
  {
    "caption": "(F) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT80.",
    "molecules": "His, Ni, NTA"
  },
  {
    "caption": "(A) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with His-tagged IFT-B2 (sub)-complexes. A Coomassie stained SDS PAGE gel shows the IFT-B2 complexes used for pull-downs (top) and a western blot for anti-α-tubulin shows co-precipitated αβ-tubulin (bottom).",
    "molecules": "His, Ni, NTA"
  },
  {
    "caption": "(F) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with WT and mutant MmIFT54CH. (top) Coomassie stained SDS PAGE gel of input (I) and pulled down (PD) proteins and the corresponding western blot to visualize αβ-tubulin.",
    "molecules": "Ni, NTA"
  },
  {
    "caption": "(A) Coomassie-stained SDS-PAGE gel showing GST-pulldown experiments between the IFT-B2 complex and an IFT88/52N subcomplex. The pre-assembled IFT-B2 complex (lane 1) shows no non-specific interaction with empty GSH-beads (lane 2), but is efficiently pulled down by IFT88/52N-GST (with an IFT52(1-335) construct comprising the N-terminal domain as well as the IFT88-binding region) (lane 3). Both IFT88 and IFT52N are required for the interaction with IFT-B2 because neither the N-terminal IFT52 domain (residues 1-275; lane 4) alone, nor an IFT88/52 complex lacking the N-terminal IFT52 domain (with an IFT52(281-335) construct containing only the IFT88-binding region) (lane 5) show efficient pull-down of IFT-B2. Contaminants from the purification of the complex shown in lane 5 are marked with asterisks.",
    "molecules": "GSH"
  },
  {
    "caption": "(B) Coomassie-stained SDS-PAGE gel showing a salt-stability test of the (IFT88/52N)-(IFT-B2) interaction. Pre-assembled IFT-B2 complex was bound to IFT88/52N-GST immobilized on GSH-beads. The beads were then divided into five aliquots which were washed with buffers containing increasing concentrations of NaCl. After these washes the bound material was eluted and analyzed. IFT172ΔC is the only salt-labile component in this complex and is already washed off at a NaCl-concentration of 150-200 mM, whereas all the other IFT-B2 proteins remain associated even after washing with 1 M NaCl.",
    "molecules": "GSH, NaCl, salt"
  },
  {
    "caption": "(C) Coomassie-stained SDS-PAGE gels showing GST-pulldown experiments between IFT88/52N-GST complex immobilized on GSH beads and various combinations of IFT-B2 proteins. The left gel shows the IFT-B2 input mixtures, the right gel shows the bound material after washing and elution from the beads. A pentameric IFT-B2 complex (IFT80/57/54/38/20) is efficiently pulled down by IFT88/52N-GST, but not by empty beads (compare lanes 1 and 2). Omission of IFT54/20 (lane 4), or IFT80 (lane 5) does not influence the binding of any of the other components, but a lack of IFT57/38 in the mixture abolishes detectable pull-downs of IFT54/20 and IFT80 (lane 4), indicating that IFT57/38 is the direct interaction partner of IFT88/52N. Indeed, IFT57/38 alone (lane 7), but not IFT54/20 (lane 6) or IFT80 (lane 8), is sufficient to be pulled down in this assay. Contaminating bands from the IFT88/52N sample are marked with asterisks.",
    "molecules": "GSH"
  },
  {
    "caption": "(C) Flow cytometric analysis of violet-labeled LLC-BrM cells phagocytosed by microglia in the brains of LLC-BrM cell-allografted BALB/c nude mice on day 1 after intracardiac injection (1×106 control or IFITM1-knockout cells). Representative flow cytometry pseudocolor density plots and the gating strategy are shown, and the bar graph shows the percentage of violet+ microglia. (The n-values denote the number of mice per group.)",
    "molecules": "violet"
  },
  {
    "caption": "(G) Results of ELISA for mouse IFNγ in the brain on day 1 after the intracardiac injection of control or IFITM1-knockout LLC-BrM cells (1×106) into BALB/c nude mice. PLX5622 (1,200 PPM) was administered in chow from day -3 to day 1. BLZ945 (200 mg/kg) was administered by oral gavage from day -2 to day 1. Minocycline (33 mg/kg) was administered intraperitoneally on days -1, 0, and 1. Clodronate liposomes (100 μl/10 g) were administered intraperitoneally on day -1. Normal brain tissue was used as the control. (The n-values denote the number of mice per group.)",
    "molecules": "BLZ945, liposomes, Clodronate, Minocycline, PLX5622"
  },
  {
    "caption": "(G) Bioluminescence imaging and quantification of brain metastases on day 20 in C57BL/6 mice isografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (3×105 cells). Mice were systemically primed-boosted with homologous LLC-BrM cell lysates (1×106 cells for day -21 and 1×105 cells for day -7) before injection of cancer cells. Then, IgG or an anti-CD8α antibody (2 μg/mouse) was injected intravenously on day 3 alone or in combination with minocycline treatment (33 mg/kg) on day 1. Minocycline was administered intraperitoneally. (The n-values denote the number of mice per group.) one-way (G)",
    "molecules": "minocycline, Minocycline"
  },
  {
    "caption": "A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations. Error bars represent mean ± SEM; n = 10 replicates.",
    "molecules": "potassium acetate"
  },
  {
    "caption": "B Confocal microscopy images of recombinant, BODIPY‐labeled TFG in the presence of varying potassium acetate concentrations. Scale bar, 2 μm.",
    "molecules": "BODIPY, potassium acetate"
  },
  {
    "caption": "C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain‐EM. Large arrowheads highlight TFG polymers (only found in the presence of elevated potassium acetate). Small arrowheads highlight individual 11 nm cup‐like TFG octamers. Scale bar, 30 nm.",
    "molecules": "potassium acetate"
  },
  {
    "caption": "D Circular dichroism spectroscopy was used to characterize the carboxyl‐terminus of C. elegans TFG (amino acids 195‐486). Samples were analyzed at different concentrations, and the data were normalized relative to one another. CD spectra were collected at 25°C in 25 mM sodium phosphate (pH 7.2) using a 1 mm path length quartz cell. The spectra are characteristic of an intrinsically disordered protein.",
    "molecules": "sodium phosphate"
  },
  {
    "caption": "E The distributions of endogenous TFG and Sec31A were examined using immunogold EM. Large arrowheads highlight 15 nm gold particles bound to α‐TFG antibodies. The small arrowhead highlights 5 nm gold particles bound to α‐Sec31A antibodies. At least 15 different cells were examined, and representative images are shown. Scale bar, 100 nm.",
    "molecules": "gold"
  },
  {
    "caption": "B A recombinant polyhistidine‐ and SUMO‐tagged TFG fragment (Sumo‐TFGc, amino acids 194‐400; total molecular mass of 42 kDa) was expressed and purified from Escherichia coli extracts using nickel affinity resin. Coomassie‐stained gels of the peak elution fractions after separation of the recombinant protein on a S200 gel filtration column (top) or a 10-30% glycerol gradient (bottom) are shown. To determine the native molecular weight of the protein, the following equation was used: M = 6πηNas/(1 − υρ), where M is the native molecular weight, η is the viscosity of the medium, N is Avogadro's number, a is the Stokes radius, s is the sedimentation value, υ is the partial specific volume, and ρ is the density of the medium (Siegel Monty, ). These data suggest that this fragment of TFG is capable of forming dimers in solution.",
    "molecules": "polyhistidine, SUMO"
  },
  {
    "caption": "A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple (ManII‐mApple) were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and ManII in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.B Human RPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and GM130 in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.",
    "molecules": "BFA, brefeldin A, DMSO"
  },
  {
    "caption": "A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.",
    "molecules": "BFA, brefeldin A"
  },
  {
    "caption": "B HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α‐Sec16A and α‐Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.C Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII‐positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A‐labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A‐labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.",
    "molecules": "BFA"
  },
  {
    "caption": "E. As in (D), except that cells were treated with Cisplatin for 24 h (mean±SEM; n=3 independent experiments). F. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (9 nM) for 48 h, fixed and co-stained with PCNA antibody and DAPI. Cell cycle distribution was analyzed by quantitative image-based cytometry (QIBC) (≥2000 cells analyzed per condition). Data from a representative experiment are shown.",
    "molecules": "Cisplatin, DAPI, MMC, Strep"
  },
  {
    "caption": "G. U2OS WT, U2OS/SCAI KO and U2OS/SCAI KO/Strep-HA-SCAI cells were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). H. As in (G), except that cells were co-stained with γH2AX antibody and DAPI (≥3000 cells analyzed per condition; mean± SD; n=3 independent experiments; *, p<0.05; **, p<0.01; ns, not significant, two-tailed paired t-test).",
    "molecules": "DAPI, MMC, Strep"
  },
  {
    "caption": "I. Experimental workflow for metaphase chromosome morphology analysis (top) and representative images of metaphase spreads from indicated cell lines treated or not with MMC (bottom). DNA was stained with DAPI. Scale bars, 10 µm. J. Quantification of radial chromosomes in (I) (mean±SD; 180 cells analyzed per condition; n=3 independent experiments; *, p<0.05; **, p<0.01, ns, not significant, two-tailed t-test). K. Quantification of chromosomal breaks/gaps in (I) (mean±SEM; 99 metaphase cells analyzed for each condition pooled from 3 independent experiments; **, p<0.01; ****, p<0.0001, ns, not significant, Mann-Whitney U test).",
    "molecules": "DAPI, MMC"
  },
  {
    "caption": "F. U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later and co-stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; *, p<0.05; ns, not significant, two-tailed paired t-test). G. As in (F), except that cells were stained with γH2AX antibody (≥3000 cells analyzed per condition; mean±SD; n=3 independent experiments; **, p<0.01; ns, not significant, two-tailed paired t-test).",
    "molecules": "DAPI, MMC"
  },
  {
    "caption": "I. Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments). J. Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI DKO cells subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).",
    "molecules": "MMC"
  },
  {
    "caption": "K. Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.",
    "molecules": "MMC"
  },
  {
    "caption": "F. pICLpt was replicated in mock- or SCAI-depleted egg extract in the presence of [α-32P]dATP for the indicated times, and reactions were analyzed by native agarose gel electrophoresis. ∆SCAI-C and ∆SCAI-N denote SCAI immunodepletion with an antibody raised against the C- or N-terminus of SCAI, respectively; RI, replication intermediates; OC, open circular; SC, supercoiled.",
    "molecules": "dATP, 32P"
  },
  {
    "caption": "H. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non-targeting control (CTRL) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h (mean±SEM; n=3 independent experiments).",
    "molecules": "MMC"
  },
  {
    "caption": "A.  Fura2 340/380  time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised  macrophage precursor cell lines (M-MØP) of Plcg2P522 mice (P522, blue) and Plcg2R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA  and 2µM Ionomycin as indicated. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; **** = p< 0.0001). B. Fura2 340/380 time traces from primary  microglia derived from the cortex  of Plcg2R522 mice (blue: P522) and Plcg2R522 mice (red: R522) with or without pre-exposure for 2 hours with  Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM  Ionomycin. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; *** = p<0.001; **** = p< 0.0001). C ",
    "molecules": "Edelfosine, EGTA, Fura2, Ionomycin"
  },
  {
    "caption": "C-E. M-MOPS (C), primary  mouse microglia (D) and hIPSC (E) (blue: P522 and red: R522) were loaded with  Fluo-8 Ca2+ indicator and examined for peak changes in  fluoresce after exposure to DOPS-liposomes (25 μg/ml). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001). ",
    "molecules": "DOPS, liposomes, Ca2+, Fluo-8"
  },
  {
    "caption": "A-F. Phagocytotic activity of R522 and P522 M-MOP and microglia  was assessed using  pHrodo Red E. coli   and  zymosan  BioParticles. Phagocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. E.coli  uptake in M-MOP (A), primary mouse microglia (B), hIPSC-derived microglia (C).  Zymosan  uptake in  M-MOP (D), primary mouse microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia  and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)",
    "molecules": "pHrodo Red, zymosan, Zymosan"
  },
  {
    "caption": "A-F.  Endocytic activity of R522 and P522 M-MOP and microglia was assessed using FITC soluble  Aβ1-42 oligomers and pHrodo Red Dexran (10,000 MW). Endocytosis  arbitrary units (A.U) describes the amount of bioparticle cellular  fluorescence emission at each time point.  Aβ1-42 oligomer uptake in M-MOP (A), primary microglia (B), and hIPSC-derived microglia (C). Dextran uptake in  M-MOP (D), primary  microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak's multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001. (blue: P522; red: R522)",
    "molecules": "pHrodo Red, Aβ1-42, Dexran, Dextran, FITC"
  },
  {
    "caption": "level of   PIP2 in M-MOPS was examined by measuring immunofluorescence from images  at set  time points after exposure after exposure to 5µg/ml anti-FcγRII/III (2.4G2), 50 ng/ml LPS  or oligomers of 40 µM  Aβ1-42 Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522).",
    "molecules": "PIP2, Aβ1-42, LPS"
  },
  {
    "caption": "A-C. A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In  M-MOP cells (left panel) and primary microglia (right panel) PI(4,5)P2 (A), PI(3,4)P2 (B) and PIP3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak's multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)",
    "molecules": "PI(3,4)P2, PIP3, PI(4,5)P2, 3-a-aminocholestane, LY294002, SF1670"
  },
  {
    "caption": "A-C. Iba1 was used as a marker for  microglia and PIP2 levels were detected by measuring the  intensity of florescence  per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data shows the mean±SD of 3 independent experiments analysed using 2 way ANOVA with Sidak's multiple comparison tests performed **=p<0.01, ****=p<0.0001.",
    "molecules": "PIP2"
  },
  {
    "caption": "A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.",
    "molecules": "DAPI"
  },
  {
    "caption": " A) His6-tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho-tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt ",
    "molecules": "tyrosine"
  },
  {
    "caption": "(A) Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP or lacking expression of any Polo transgene. Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre-anaphase figures. Insets display magnifications of the outlined regions, which highlight both low-tension misaligned (inset 1 and 2) and high-tension aligned KTs (inset 3). (B) Graph represents the percentage of cells in each indicated mitotic state, as shown in (A) (n≥ 532 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Asterisk highlights either an aligned KT pair attached to MTs in an end-on fashion (PoloWT-EGFP) or an aligned KT pair in which a sister KT is laterally attached to the end of a MT fiber (PoloT182D-EGFP). Plotted profiles show the overlap between Polo-EGFP and tubulin signals for the highlighted KT. (D) Graph represents the percentage of metaphase cells showing at least 1 KT with a lateral interaction, as shown in (C) (asterisk) (n≥58 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.",
    "molecules": "calcium"
  },
  {
    "caption": "(A) Representative immunofluorescence images of Spindly localization to unaligned KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Cells were treated with MG132 prior to fixation to increase the number of late prometaphase figures and allow better identification of late congressing KTs. CID was used as a KT reference. (B) Graph represents the percentage of Spindly levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in cells expressing PoloWT-EGFP, which was set to 100% (n≥121 KTs from at least 39 cells for each condition, n=3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.",
    "molecules": "MG132"
  },
  {
    "caption": "(F) Representative immunofluorescence images of Spindly accumulation at unattached KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference. Data information: Scale bar: 5 μm.",
    "molecules": "colchicine"
  },
  {
    "caption": "(A) In vitro kinase assays with the indicated recombinant protein in the presence of [γ-32P]-ATP for 30 min. Phosphorylation was detected by autoradiography and levels of proteins were visualized by coomassie blue staining. Data information: Scale bar: 5 μm.",
    "molecules": "ATP, coomassie blue, 32P"
  },
  {
    "caption": "(D) Representative immunofluorescence images of Spindly-phospho(ph)Ser499 levels at unattached KTs in control Drosophila S2 cells and in Polo-depleted or BI2536-treated cells. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference. Data information: Scale bar: 5 μm.",
    "molecules": "BI2536, colchicine, Ser"
  },
  {
    "caption": "(D) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing SpindlyWT-, SpindlyS499A-, SpindlyS499D- or SpindlyS499D-EGFP in a ZW10-depleted background. Insets display magnifications of the outlined regions which highlight different attachment configurations (E - end on; L - lateral; M - merotelic). Cartoon depicts the attachment configuration of the respective KT pair. Asterisk highlights an aligned KT pair in which a sister KT appears to be laterally attached to the end of a MT fiber. Plotted profiles show the overlap between CENP-C and tubulin signals for the highlighted KT. CENP-C was used as a KT reference. (E) Graph represents the percentage of metaphase cells showing only end-on attachments or at least 1 KT with lateral or merotelic attachment, as shown in D (n≥44 cells for each condition, n≥2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.",
    "molecules": "calcium"
  },
  {
    "caption": "CSF p3-Alcβ37 values in subjects who were categorized along the AD continuum (n = 131). Cut-off values are 359.6 pg/mL for Aβ42 (A+ indicates <359.6 pg/mL), 30.6 pg/mL for p-tau (T+ indicates >30.6 pg/mL), and 105.3 pg/mL for t-tau (N+ indicates >105.3 pg/mL) respectively. Statistical significance was determined by a one-way ANOVA followed by Tukey's multiple comparisons test (means ± SEM; A-: n = 36, A+T-N-: n = 51, A+T+N-: n = 30, A+T+N+: n = 14), and significant p-values (p < 0.01, p < 0.0001) are indicated on the graph.",
    "molecules": "Aβ42"
  },
  {
    "caption": "(D, E, F) Effect of p3-Alcβ on the neuronal toxicity of Aβ42 oligomers. WT neurons (div 15-20) were incubated for 24 h in the presence (10 μM) or absence (-) of p3-Alcβ9-19 and p3-Alcβ1−37 with (+) or without (-) Aβ42 oligomers (Aβo, 2.5 μM). Neuronal viability was evaluated by MTT (D), ATP generation (E), and LDH release (F) assays and expressed relative to neurons cultured in the absence of p3-Alcβ and Aβo (assigned a value of 1.0). Statistical significance was determined by Dunnett's multiple comparisons tests (means ± SEM; MTT: n = 36, ATP: n = 40, LDH: n = 60). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) versus cells incubated in the absence (-) of p3-Alcβ and Aβo are indicated in the graphs. The significant p-values (#p < 0.05, #p < 0.01) versus cells incubated in the presence (+) of Aβo and in the absence (-) of p3-Alcβ) are indicated in the graphs.",
    "molecules": "Aβ, Aβ42, ATP"
  },
  {
    "caption": "(G) Effect of Aβ42 oligomers (Aβo) and p3-Alcβ peptides on the generation of reactive oxygen species (ROS). WT neurons (div 15-17) were incubated for 24 h with (+) or without (-) Aβo (2.5 μM) and in the presence (+) or absence (-) of p3-Alcβ (10 μM). ROS generation was assessed and expressed relative to that of neurons incubated in the absence (-) of Αβo, which was assigned a value of 1.0. Statistical significance between samples with or without Aβo was evaluated with a Student's t-test (means ± SEM; n = 10), and the significant p-value (p < 0.05) is indicated on the graph.",
    "molecules": "Aβ, Aβ42, Αβ, reactive oxygen species, ROS"
  },
  {
    "caption": "(A, B) Suppression of Ca2+ influx induced by Aβo in neurons treated with p3-Alcβ9-19 (A) and p3-Alcβ37 (B). Mouse neurons (div 11-13) pretreated with Fluo 4-AM were stimulated at 5 min (arrow) with (+) or without (-) Aβo (5.2μM) in the presence (+) or absence (-) of p3-Alcβ (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 18 min (5-23 min at time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence (-) of p3-Alcβ and Aβo (assigned a value of 1.0).",
    "molecules": "Aβ, Ca2+, Fluo 4-AM"
  },
  {
    "caption": "(C, D) Suppression of Ca2+ influx into neurons followed by Ca2+ administration in the presence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). Mouse neurons (div 11-13) pretreated with Fluo 4-AM in calcium-depleted medium were administered Ca2+ (2 mM) at 5 min (arrow) in the presence or absence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 7.5 or 9 min (5 to 12.5 min in panel C and 5 to 14 min in panel D at the indicated time points) is shown (right). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparison test (means ± SE; n = 14 (A), n = 15 (B), n = 10 (C)). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graphs. Statistical significance was determined with a Student's t-test (means ± SEM; n = 11 (D)), and the significant p-value (p < 0.001) is indicated on the graph.",
    "molecules": "Ca2+, calcium, Fluo 4-AM"
  },
  {
    "caption": "(A) Suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons treated with the NMDA receptor antagonist D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence or absence of D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 24 min (5-29 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence of D-AP5 and Aβo (assigned a value of 1.0). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test (means ± SE; n = 4), and significant p-values (p < 0.05, p < 0.01) are indicated on the graph.",
    "molecules": "Aβ, Aβ42, Ca2+, D-AP5, Fluo 4-AM"
  },
  {
    "caption": "(D) Non-synergistic suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons by p3-Alcβ and D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence (+) or absence (-) of p3-Alcβ9-19 (50 μM) and D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 23 min (5-28 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in experiment No.1 (assigned a value of 1.0). Statistical significance was determined with a one-way ANOVA with Tukey's multiple comparison test (means ± SEM; n = 12), and significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graph.",
    "molecules": "Aβ, Aβ42, Ca2+, D-AP5, Fluo 4-AM"
  },
  {
    "caption": "Quantitative RT-PCR of stx after OA treatment in w1118 control and Octβ1R, Octβ2R mutants, Octβ3R mutants. (From left to right mean±SEM: 1.000±0.0288, 0.781±0.0176, 1.477±0.043，1.403±0.0578, 1.157±0.0617, 0.881±0.0689, 1.149±0.0398, 0.953±0.0543, 1.017±0.0600, 1.090±0.0378, N=3).",
    "molecules": "OA"
  },
  {
    "caption": "Ellipsoid body knock down of Octβ2R results in sleep rebound increase. In response to OA, ellipsoid knock down of Octβ2R rescues control phenotype. Data represent mean±SEM: 13.80±2.439, n=31; 5.465±2.036, n=25; 19.55±2.275, n=27; 12.47±2.684, n=24; 30.03±3.428, n=25; 43.67±4.616, n=25.",
    "molecules": "OA"
  },
  {
    "caption": "Sleep profile of PcXT109 mutant with or without OA treatment. In both control and PcXT109 mutant flies, the treatment of OA results in sleep decrease, n=16 (G). Quantifications of the decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies (From left to right mean±SEM: 13.90±1.397; 21.25±1.493, N=4) (H).",
    "molecules": "OA"
  },
  {
    "caption": "H-K) Representative images showing that Hh overexpression using pan-glial (repo-GAL4) and cortex-glial (NP2222-GAL4) drivers both result in a decrease in NB EdU index, quantified in (J) (n=16, 20 brain lobes) and (K) (n=11, 14 brain lobes), respectively.",
    "molecules": "EdU"
  },
  {
    "caption": "N-P) Overexpression of ciACT in NBs (dnab-GAL4) reduces EdU index, quantified in (P) (n=12, 10 brain lobes).",
    "molecules": "EdU"
  },
  {
    "caption": "E-G) Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed effectively reduces LD number in CB (outlined in yellow dashed lines). H-K) Representative images showing that NB EdU index is rescued upon Lsd-2 knockdown in cortex glial cells (NP2222-GAL4) where hh is overexpressed, quantified in (K) (n=11, 14; 16, 14; 6, 14 brain lobes). The NP2222-GAL4>w1118 vs hhOE columns depict the same data as Fig 2K. EdU+ NBs are circled with yellow, dashed lines. ",
    "molecules": "EdU"
  },
  {
    "caption": "A-E) Representative images showing that both pan-glial (repo-GAL4>) and cortex glial (NP2222-GAL4>) htlACT overexpression significantly reduce NB EdU index, quantified in (E) (n=15 ,14; 10, 16 brain lobes). (A, B, C, D) are single sections with Mira and EdU staining, and (A', B' C', D') are Z-projection of the EdU staining.",
    "molecules": "EdU"
  },
  {
    "caption": "Representative still images from ex vivo CNS live imaging at 72ALH showing that pan-glial (repo-GAL4) htlACT overexpression lengthens NB cell cycle, (n= 25, 6 NBs imaged from 3 brains per genotype). NBs (Dpn::GFP, red; Histone RFP, grey) are circled with blue dashed lines.",
    "molecules": "Histone"
  },
  {
    "caption": "M-O) Representative images showing that the number of EdU+ neurons generated per NB is significantly reduced upon pan-glial overexpression of FGF (repo-LexA > LexAop-htlACT; yellow arrows), quantified in (O) n= 94, 127 NB lineages imaged from 5 and 7 brain lobes, respectively). NB lineages are marked with dnab-gal4 > GFP.",
    "molecules": "EdU"
  },
  {
    "caption": "H) Cortex glial (NP2222-GAL4>) overexpression of two independent hh RNAis significantly rescue EdU incorporation defects caused by htlACT overexpression (n=10, 16; 7, 12; 14, 11 brain lobes).",
    "molecules": "EdU"
  },
  {
    "caption": "B) Inhibition of palmitoylation (via two independent rasp RNAis) rescues NB EdU incorporation defects induced by cortex glial (NP2222-GAL4>) htlACT overexpression, while knockdown of Rasp in cortex glial cells alone does not alter NB EdU index (n=10, 15; 10, 16; 18, 21; 26, 29 brain lobes).",
    "molecules": "EdU"
  },
  {
    "caption": "C-C') Representative images showing that Hh.N.EGFP (which cannot undergo cholesterol modification) are found as puncta on the surface of NBs (yellow arrows) when overexpressed in neighbouring cortex glial cells (NP2222-GAL4>).",
    "molecules": "cholesterol"
  },
  {
    "caption": "d, Binding of galectin 8 to bacteria and HeLa cells. The indicated bacteria and HeLa cells were incubated with His-GST-ubiquitin (Ub), His-GST-galectin 8 or buffer as indicated, followed by murine anti-His antibody and PE-labelled anti-mouse serum.",
    "molecules": "PE"
  },
  {
    "caption": "f, Confocal images of HeLa cells expressing the indicated YFP-tagged galectins. Cells were left untreated or were exposed to hypertonic conditions, with or without (w/o) PEG as indicated, followed by hypotonic shock.",
    "molecules": "PEG"
  },
  {
    "caption": "F, G Validation of Gadd45α-regulated levels of Grin2a mRNA by qPCR with primers covering exons 3-4. Grin2a mRNA levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT=7 and Gadd45a-KO=7) and 1 hour after PA (Gadd45a-WT=5, black bars; Gadd45a-KO=6, blue bars). Random primed cDNA levels (F), were very similar in all the experimental groups. Oligo(dT)-primed cDNA (G) revealed a significant increase of Grin2a 1h after PA, which was only observed in Gadd45a-WT mice. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01, *** = p<0.001. H, I mRNA levels of proximal vs. distal parts of the 3´UTR analyzed in the same set of oligo(dT)-primed cDNA. (H) Significant increase in Grin2a mRNA levels was found for the proximal part only in Gadd45a-WT. (I) Distal parts of the 3´UTR remained unchanged (n=6 for all groups). Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: * = p<0.05. ",
    "molecules": "cDNA"
  },
  {
    "caption": " (B, C) Representative immunoblots (B) and quantification of Tau degradation (C) from 14 DIV primary neurons transduced with EGFP or shTSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shTSG101-expressing neurons exhibit markedly decreased Tau degradation compared to EGFP-expressing controls (n=18-19 per condition, unpaired student's t-test, *p=0.0108) ",
    "molecules": "cycloheximide, DMSO"
  },
  {
    "caption": " (D, E) Representative immunoblots (D) and quantification of Tau degradation (E) from 14 DIV neurons transduced with mCh or mCh-TSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. Overexpression of TSG101 increases Tau degradation (n=17/condition, unpaired student's t-test, *p=0.0115) ",
    "molecules": "cycloheximide, DMSO"
  },
  {
    "caption": " (F, G) Representative immunoblots (F) and quantification of Tau degradation (G) from 14 DIV neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shRab35-expressing neurons exhibit markedly decreased Tau degradation compared to mCh-expressing controls (n=23-26 per condition, unpaired student's t-test, ***p=0.0008)",
    "molecules": "CHX, cycloheximide, DMSO"
  },
  {
    "caption": " (H, I) Representative immunoblots (H) and quantification of Tau degradation (I) from 14 DIV neurons transduced with mCh or mCh-Rab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau or tubulin. Rab35 overexpression increases Tau degradation (n=18 per condition, unpaired student's t-test, *p=0.0126)",
    "molecules": "CHX, cycloheximide, DMSO"
  },
  {
    "caption": " (A, B) Representative immunoblots (A) and quantification of Tau degradation (B) from 14 DIV primary neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for pSer396/404-Tau (PHF1), pSer262-Tau, or pSer202-Tau (CP13) and tubulin. shRab35-expressing neurons exhibit markedly decreased pSer262- and p396/404-Tau degradation compared to mCh-expressing controls, while pSer202-Tau degradation is unaffected (n=4 per condition for pSer396/404-Tau and pSer202-Tau, n=3 for pSer262-Tau, unpaired student's t-test, **p=0.0027, *p=0.0120) ",
    "molecules": "CHX, cycloheximide, DMSO"
  },
  {
    "caption": " (A, B) Representative immunoblots (A) and quantification of Hrs, TSG101, CHMP2b, and Rab35 protein levels (B) from 14 DIV neurons treated with either DMSO (CON) or glucocorticoids (GC). GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs, TSG101, or CHMP2b (n=9-10/condition, unpaired student's t-test, *p=0.0221) ",
    "molecules": "DMSO, GC, glucocorticoids"
  },
  {
    "caption": " (C) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells (for neurons, n=9/condition, unpaired student's t-test, **p=0.0036; for N2a cells, n=6/condition, unpaired student's t-test, *p=0.0138) ",
    "molecules": "GC"
  },
  {
    "caption": " (E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257) ",
    "molecules": "GC"
  },
  {
    "caption": " (G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003). Data information: All numeric data represent mean ± SEM ",
    "molecules": "DAPI, GC"
  },
  {
    "caption": " (A, B) Representative immunoblots (A) and quantification of Tau degradation (B) in 14 DIV neurons expressing mCh-Rab35 or mCh, treated for 24 h with cycloheximide (CHX) or DMSO (CON) under GC conditions. GC significantly decreases Tau degradation, whereas Rab35 overexpression blocks this effect (n=12-14/condition; 1-way ANOVA, Dunnet post-hoc analysis, *pmCh vs mCh GC=0,0319, *pmCh vs Rab35 GC=0,047) ",
    "molecules": "CHX, cycloheximide, DMSO, GC"
  },
  {
    "caption": " (D, E) Representative immunoblots (D of Tau levels (E) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP, but not Rab35 (n=5-6 animals/group, 2 replicates, 2-way ANOVA, GC x Rab35 interaction F1, 39=10,51 p=0.002; Sidak posthoc analysis *p=0.0168)",
    "molecules": "GC"
  },
  {
    "caption": " (G) GC treatment reduces the length of apical dendrites in animals expressing EGFP, but not EGFP-Rab35 (n=6-7 animals/group; 6-8 neurons/animal, 2-way ANOVA , GC x Rab35 interaction F1, 155=3.969 p=0.0481, overall GC effect F1, 155=8.998 p=0.0031, Sidak posthoc analysis **p=0.0021) ",
    "molecules": "GC"
  },
  {
    "caption": " (H) GC treatment reduces mature spine density in animals expressing EGFP, but not EGFP-Rab35 (2-way ANOVA, GC x Rab35 interaction F1, 499=12.33 p=0.0005, overall GC effect F1, 499=4.373 p=0.0370, Sidak posthoc analysis *** p=0.0003; n=6-7 animals/group; 6-8 neurons per animal) ",
    "molecules": "GC"
  },
  {
    "caption": " (I, J) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP-expressing animals; however, this effect is not seen in EGFP-Rab35-expressing animals (3-way ANOVA, GC x Rab35 interaction F1, 4212= 14,926 p<0.0001, Simple effect analysis, Sidak test for multiple comparisons *p120=0.012, **p140=0.001, **p220=0.002, ***p240<0.001, ***p260<0.001, **p280=0.001, **p300=0.001, *p320=0.021, *p340=0.047, n=6-7 animals/group, 6-8 neurons per animal) ",
    "molecules": "GC"
  },
  {
    "caption": "H Western blots  of de novo global   protein synthesis in PFC  slices of WT and HET mice, measured by SUnSET.  α-tubulin was used as a loading control. I Quantification analysis of puromycin-labeled protein levels. WT, n=5 mice, HET, n=4 mice. Unpaired t tests. **p=0.0011. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.",
    "molecules": "puromycin"
  },
  {
    "caption": "J Representative images of AHA incorporation  (green) with CaMKⅡα+ staining  (red) in mPFC  of WT and HET mice. Arrows indicate CaMKⅡα+ cells. Scale bar, 20 μm. K Quantification of total AHA incorporation. 12 slices from 3 mice  for each genotype. Unpaired t test. *p=0.0268. L Quantification of AHA incorporation  in CaMKⅡα+ cells. WT, n=276 cells from 3 WT mice, n=293 cells from 3 HET mice. Mann-Whitney test. **** p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.",
    "molecules": "AHA"
  },
  {
    "caption": "G Representative images of Golgi‐stained dendrite spines in the mPFC  from WT and HET mice. Scale bar, 5 μm. H Quantification of spine  density per 10 μm of dendrites. n=18 dendrites  from 3 WT mice; n=23 dendrites  from 3 HET mice. Unpaired t test. ****p<0.0001. Data information: All data are shown as means ± s.e.m. All the tests are two-tailed.",
    "molecules": "Golgi"
  },
  {
    "caption": "A PF-4778574 (PF) directly potentiates EPSC amplitude, dashed line represents baseline value of normalized EPSC amplitude. Bottom: representative EPSC traces from baseline (1), maximal potentiation (2), and recovery (3). B, C Quantifications of EPSC peak amplitude (B) and decay time (C) after PF (6 cells from 3 HET mice). (B) Wilcoxon signed-rank test, *p=0.0313 (PF v.s. baseline) and p=0.1600 (Wash out v.s. baseline); (C) Paired t test, ****p<0.0001 (PF v.s. baseline) and p=0.4423 (Wash out v.s. baseline). Data information: Data are shown as means ± s.e.m., data",
    "molecules": "PF-4778574"
  },
  {
    "caption": "D PF rescued AMPAR-EPSC in L5 neurons from HET mice. 25-30% increase at strongest stimulation (7 cells from 3 HET mice), paired t test, **p=0.0014. Bottom: representative traces of the effects of PF on AMPAR-EPSC evoked by different stimulus intensities. Data information: Data are shown as means ± s.e.m.",
    "molecules": "PF"
  },
  {
    "caption": "E Representative heatmaps of the two social tasks (left, social approach; right, social novelty) in the Three-chamber test performed by Eef2 HET mice intraperitoneal administered with PF-4778574 or DMSO (vehicle control). E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. F Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.7335. WT, n=9 mice; HET, n=8 mice. G Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0492. WT, n=9 mice; HET, n=8 mice. Data information: Data F, G, are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.",
    "molecules": "PF-4778574, DMSO"
  },
  {
    "caption": "I Representative heatmaps of the two social tasks (left, social approach; right, social novelty) performed by Eef2 HET mice with mPFC infusion of PF-4778574 or DMSO (vehicle control) in the Three-chamber test. E: empty cup; S1: stranger #1 contained cup; S2: stranger #2 contained cup. J Discrimination score of social approach performed by two groups of mice, calculated by the difference of time spent in sniffing S1 and E. Unpaired t test. p=0.3984. n=11 mice for each group. K Discrimination score of social novelty performed by two groups of mice, calculated by the difference of time spent in sniffing S2 and S1. Unpaired t test. *p=0.0489. n=11 mice for each group. Data information: Data of J and K are shown as interquartile range, with line across the box indicating median, whiskers show the highest and lowest values. n.s., not significant. All the t tests are two-tailed.",
    "molecules": "PF-4778574, DMSO"
  },
  {
    "caption": "(B, C) (B) Growth inhibition  assay of  E. coli DH10B  cells harbouring pTrc200 vector or each of its derivatives expressing Tde1  variants with IPTG  induction. (C) Growth inhibition assay of E. coli  DH10B   cells co-expressing the Tde1 variants expressed from pTrc200 plasmid and Tdi1 immunity gene expressed from pRL662 plasmid. Growth  curve was determined at OD600. Graphs of panels B and C show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 4.6x10-5 and 5.19x10-8 for panels B and C respectively).",
    "molecules": "IPTG"
  },
  {
    "caption": "(A) Growth inhibition assay of E. coli DH10B  cells harbouring pTrc200 vector or each of its derivatives expressing Tde1  variants with IPTG  inducible expression. The growth  of  E. coli  was monitored by CFU counting every 1 hr. Data information: Graphs of panels A, show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison.",
    "molecules": "IPTG"
  },
  {
    "caption": "For  membrane permeabilization  assays, BW25113  WT alone or ∆lacY(pYTA-lacZ) cells harbouring pTrc200 vector or each of its derivatives expressing Tde1  variants were carried out for (C) propidium iodide permeability   with cells treated with Propidium iodide  and Hoechst  for detection by fluorescence microscope (Scale bar = 5 μm). For quantification of cells with PI  signals, a total of 6 randomly selected images obtained from two biological repeats were used to quantify the number of PI-stained  cells / number of Hoechst-stained  cells as indicated.",
    "molecules": "Hoechst, PI, Propidium iodide, propidium iodide"
  },
  {
    "caption": "(D) Bacteriostatic activity assay. E. coli DH10B  cells harbouring pTrc200 vector or each of its derivatives expressing Tde1  variants were cultured with or without IPTG  induction for 1 hr. The IPTG-induced cells were further centrifuged and resuspended in the fresh medium with or without IPTG. Cell density was measured again before continuous growth  for additional 1 hr. Data information: Graphs of panels show mean ± SD of three biological replicates (n=3), each averaged with 3 technical repeats. One way ANOVA was used for the analysis of statistical significance followed by the Tukey's multiple comparison. Different letters indicate statistically different groups of strains (p value, 1×10-16 and 2×10-16 for panels D respectively).",
    "molecules": "IPTG"
  },
  {
    "caption": "(B) Cells were stimulated with 10 μg/mL of MDP for 24 h, and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis. The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported.",
    "molecules": "MDP"
  },
  {
    "caption": "(D) Subcellular localization of NOD2 was ascertained in unstimulated (left, ×80,000) and MDP‐stimulated cells (right, ×63,000) using an anti‐NOD2 antibody and detected with a secondary antibody coupled to 5‐nm gold particles, indicated with arrowheads. Bar represents 150 nm. Micrographs were obtained from one experiment.",
    "molecules": "MDP"
  },
  {
    "caption": "NOD2 ligation in alveolar macrophages induces pro‐inflammatory cytokine release. Cells were incubated for 24 h in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS. Production of IL‐1β, IL‐6, TNF‐α, and IL‐10 was measured in culture supernatants using Milliplex technology. Depicted are box plots with median values and quartiles for each cytokine. The data are representative of two independent experiments (n = 11); *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.",
    "molecules": "LPS, MDP"
  },
  {
    "caption": "LL37 and IRGM gene expression is upregulated following NOD2 activation (A‐D). Alveolar macrophages were incubated in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS for 24 h. Upregulation of (A) LL37 and (D) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. The fold change in gene expression relative to unstimulated cells is depicted. Data are representative of two independent experiments (n = 7-12). Bold lines indicate median values.",
    "molecules": "LPS, MDP"
  },
  {
    "caption": "(A, B, D, E, G, H) The subcellular localization of autophagy proteins was ascertained in untreated and MDP‐treated cells using (A, B) anti‐IRGM, (D, E) anti‐LC3, and (G, H) anti‐ATG16L1 antibodies and detected with a secondary antibody coupled to 5‐nm gold particles indicated with arrowheads; bar represents 150 nm.",
    "molecules": "MDP"
  },
  {
    "caption": "(C, F, I) Gold particles co‐localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p 0.05 using a two‐tailed paired t‐test. (B) Transmission electron microscopy (TEM) magnification ×50,000; the rest of the micrographs are TEM magnification ×80,000. Data were generated from one experiment.",
    "molecules": "Gold"
  },
  {
    "caption": "Macrophages treated with MDP after M. tuberculosis infection develop autophagy vacuoles. Alveolar macrophages were infected with M. tuberculosis H37Rv at an MOI of 5 for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour. The cells were then treated with 10 μg/mL of MDP for 24 h. Autophagy indicators (arrowheads) such as (A) vesicles containing organelles (TEM magnification ×20,000) and (B) onion skin‐like lamellar multivesicular bodies (TEM magnification ×16,000) were identified by ultrastructural analysis..",
    "molecules": "MDP"
  },
  {
    "caption": "Uptake of radio-labeled 3H-2-deoxyglucose in siRNA-transfected Capan-1 cells (48 h post-transfection); n = 6 cell culture wells per group; statistically different (P < 0.05) from control (siNC #2) $P = 0.03; Welch's t-test.",
    "molecules": "3H, 2-deoxyglucose"
  },
  {
    "caption": "Extracellular flux measurement of siRNA-transfected Capan-1 cells with glycolysis stress test assay (48 h post-transfection); 2-DG: 2-deoxyglucose; ECAR: extracellular acidification rate. n = 6-8 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2) $. Upper symbols: statistics for siTBL#1, P-values: 2.18 × 10−3 (*) time-point 8, 9.83 × 10−3 (*) time-point 9, 0.0102 (*) time-point 10, 0.044 (*) time-point 11; 2.42 × 10−6 (#) time-point 8, 1.47 × 10−4 (#) time-point 9, 3.17 × 10−4 (#) time-point 10, 0.0277 (#) time-point 11; 9.44 × 10−7 ($) time-point 8, 1.23 × 10−6 ($) time-point 9, 1.33 × 10−6 ($) time-point 10, 7.14 × 10−5 ($) time-point 11. Lower symbols: statistics for siTBL#2, P-values: 3.94 × 10−3 (*) time-point 5, 0.0143 (*) time-point 6, 0.0342 (*) time-point 7, 1.45 × 10−3 (*) time-point 8, 1.27 × 10−3 (*) time-point 9, 2.38 × 10−3 (*) time-point 10; 0.01 (#) time-point 5, 0.0362 (#) time-point 6, 1.22 × 10−6 (#) time-point 8, 1.28 × 10−5 (#) time-point 9, 5.84 × 10−5 (#) time-point 10; 0.0118 ($) time-point 5, 0.0208 ($) time-point 6, 0.0291 ($) time-point 7, 4.52 × 10−7 ($) time-point 8, 6.04 × 10−8 ($) time-point 9, 1.43 × 10−7 ($) time-point 10, 4.83 × 10−3 ($) time-point 11. Two-way ANOVA with Bonferroni post-test.",
    "molecules": "2-deoxyglucose, 2-DG"
  },
  {
    "caption": "TBL1-depletion leads to reduction in PI3 kinase p110α and cell cycle-associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection. After 21 days, mice were sacrificed, tumors were removed and proteins were extracted and immunoblotted.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.",
    "molecules": "BodiPy 493/503, lipid, neutral lipid"
  },
  {
    "caption": "C) Ubiquitination of GFPAKIRIN2. D) Ubiquitination of GFPUbAKIRIN2. E) Ubiquitination activity of wild-type and mutant UBR5ΔUBA on GFPUbAKIRIN2 substrate. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions C were performed at 37°C. Reactions in D performed at 25°C. The final concentration of E3 enzyme in C, D: 0.3µM",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "F) Sucrose gradient sedimentation profiles of GFPUbAKIRIN2 in the absence (upper panel) or in the presence of UBR5wt (middle panel), and UBR5ΔUBA (lower panel). Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels of assays are a representative of at least three experimental replicates.",
    "molecules": "Ub"
  },
  {
    "caption": "G) Ubiquitination activity of wild-type and oligomerisation-defective mutants on GFPUbAKIRIN2. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions in G were performed at 25°C. The final concentration of E3 enzyme , in G: 0.2µM.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "F) Ubiquitination of GFPUbAKIRIN2 by UBR5 and UBR5ΔPL and G) isolated UBR5HECT. Gels were imaged as described in Figure 3. Gels of assays are a representative of at least two experimental replicates. All reactions were performed using a final concentration of 0.3µM E3.",
    "molecules": "Ub"
  },
  {
    "caption": "(A) After randomized, 8-12 weeks old and sex-matched Phb1F/F mice  and Phb1MyeKO mice  were administered with PBS  or LPS  (30 mg/kg) for 12 hours via intraperitoneal injection. Serum  was collected and ELISA  assays were used to detect the level of  IL-1β (n = 6, 10, 6, and 12 mice  for each group, respectively). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p< 0.0001 (Student's t-test).",
    "molecules": "LPS, PBS"
  },
  {
    "caption": "(B) BMDMs  were extracted from  Phb1F/F or Phb1MyeKO mice  and stimulated by 20% culture supernatant  of L929  cells for 7 days. Differentiated BMDMs  were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes, the cells were lysed and the culture medium was collected. Western blotting (WB) assays were used to detect protein levels in whole cell lysates (Lys) and  culture medium (Sup).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(C) BMDMs  were extracted from  Phb1F/F or Phb1MyeKO mice and stimulated with 20% culture supernatant of L929  cells for 7 days. Differentiated BMDMs  were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. ELISA  assays were used to detect protein levels in the culture medium. Data of BMDMs  from the separate mice  (n = 6 mice  for each group) and are presented as mean ± SEM. *** p < 0.001 (Student's t-test).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(D) After randomized, 4 weeks old and sex-matched Phb1F/F mice  and Phb1MyeKO mice  were administered with PBS  or LPS  (300 mg/kg) to induce septic  shock via intraperitoneal injection. The life span of  Phb1F/F mice  (n = 6) and Phb1MyeKO mice  (n = 7) was recorded.",
    "molecules": "LPS, PBS"
  },
  {
    "caption": "J774A.1  cells were stimulated with LPS  (200 ng/ml) for 6 hours Intracellular mRNA levels were detected by RT-qPCR assays. Data are from four independent experiments for (G) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).",
    "molecules": "LPS"
  },
  {
    "caption": "(I) After  J774A.1 cells were stimulated with LPS  (200 ng/ml) for 0, 3, 6, 12 hours and ATP  (4 mM) for 45 minutes, the cells were lysed, and the  culture medium  was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB  assays.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(J) After  J774A.1 cells with Phb1  or Phb2  knockdown were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB  assays.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(K) After J774A.1 cells with Phb1  or Phb2  knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes, the culture medium was collected. ELISA  assays were used to detect IL-1β  levels in the culture medium. Data are from three independent experiments (mean ± SEM). n.s., no significance, **** p < 0.0001 (Student's t-test).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(A-B) GFP-expressing HeLa  cells with stable Phb1  knockdown were fixed and subjected to immunofluorescence  (IF) analysis to detect mitochondria  (MitoTracker, red; A) or ΔΨm  (TMRM, red; B). Images shown are representative of at least three independent experiments. White boxed regions in the panels in (A) are enlarged. Scale bar: 10 μm.",
    "molecules": "TMRM, MitoTracker"
  },
  {
    "caption": "(C)  Flow cytometric  analysis of ΔΨm  (TMRM). HeLa  cells with stable Phb1  knockdown were treated with CsA  (2 μM) for 30 minutes or FCCP  (4 μM) for 5 minutes.",
    "molecules": "TMRM, FCCP, CsA"
  },
  {
    "caption": "(E) HeLa  cells with stable PHB1  ablation were incubated with H2O2 (2 μM) and CsA  (2 μM) for 30 minutes. Flow cytometric analysis of intracellular ROS  levels (DHE).",
    "molecules": "CsA, DHE, H2O2, ROS"
  },
  {
    "caption": "(F-G) Confocal microscopy analyses of mitochondrial Ca2+ in time-series mode. HeLa  cells stably expressing 4mt-RCaMPh with or without Phb1  knockdown were treated with CsA  (2 μM) for 30 minutes. During acquisition of fluorescence images (F), cells were treated with histamine  (200 μM). Traces of mitochondrial Ca2+ (4mt-RCaMPh) dynamics are shown in (G) (mean ± SEM). n = 16, 16, 15, 16 (from the top to the bottom). Scale bar: 10 μm. * p < 0.05 relative to Scramble group, # p < 0.05 relative to shPHB1 group (Student's t-test).",
    "molecules": "4mt-RCaMPh, Ca2+, CsA, histamine"
  },
  {
    "caption": "(A) The  co-localization between mtDNA  (SG-ALK, green) and mitochondria  (MitoTracker, red) was examined in J774A.1 cells with or without knockdown of Phb1  or Phb2. White boxed regions in the panels are enlarged. The white arrows indicate mtDNA  outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from ten, eight, and seven images from three independent experiments (left to right, respectively) (mean ± SEM). n.s., no significance, *** p < 0.001 (Student's t-test).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "(D) BMDMs  isolated from Phb1F/F mice  and Phb1MyeKO mice  were fixed and subjected to IF  analysis to detect the co-localization between mitochondria  (MitoTracker, red) and mtDNA (SG-ALK, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA  outside mitochondria. Scale bar: 10 μm. (E) Quantitative analysis of data from (D). Data are from ten and eight images from three independent experiments (mean ± SEM). ** p < 0.01 (Student's t-test).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "(G) HeLa  cells expressing TFAM-FLAG  with or without Phb1  knockdown were incubated with CsA  (2 μM) for 30 minutes. Cells were fixed and subjected to IF  analysis to detect the  co-localization between mitochondria  (anti-TOMM20, red) and TFAM-FLAG  (anti-FLAG, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA  outside mitochondria. The yellow arrows indicate mtDNA  inside mitochondria. Scale bar: 10 μm.",
    "molecules": "CsA"
  },
  {
    "caption": "(I) HeLa  cells with or without Phb1  knockdown were incubated with H2O2 (5 mM) for 20 minutes and CsA  (4 μM) for 12 hours. Cells were lysed, and DNA was extracted. qPCR assays were used to detect mtDNA  levels (ND1) in the cytosol  and nuclear  DNA levels (18S) in the whole cell lysates. Data are from four independent experiments (mean ± SEM). ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).",
    "molecules": "CsA, H2O2"
  },
  {
    "caption": "HeLa  cells with or without Phb1  knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR  assays were used to detect mtDNA  levels (ND1  in HeLa  cells in the cytosol  relative to nuclear  DNA levels (TERT  in HeLa  cells in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).",
    "molecules": "VBIT-4"
  },
  {
    "caption": "J774A.1 cells with or without Phb1  knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR  assays were used to detect mtDNA  levels D-loop  in J774A.1 cells) in the cytosol  relative to nuclear  DNA levels Tert  in J774A.1  cells) in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).",
    "molecules": "VBIT-4"
  },
  {
    "caption": "(M) BMDMs  isolated from  Phb1F/F mice  and  Phb1MyeKO mice  were incubated with MCC950  (50 nM) for 30 mins and were subsequently stimulated by LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. Protein levels in whole  cell lysates and culture medium  were detected by WB  assay.",
    "molecules": "MCC950, ATP, LPS"
  },
  {
    "caption": "(A) BMDMs  isolated from  Phb1F/F mice  and Phb1MyeKO mice  were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. Cells were fixed and subjected to IF  analysis to detect the co-localization between NLRP3  (anti-NLRP3, red) and mtDNA  (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized with NLRP3. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from at least 14 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).",
    "molecules": "ATP, LPS, PicoGreen"
  },
  {
    "caption": "(C) J774A.1  cells with or without Phb1  knockdown were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. Cells were fixed and subjected to IF  analysis to detect the co-localization  between NLRP3  (anti-NLRP3, red) and mtDNA  (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA co-localized  with NLRP3. Scale bar: 10 μm.",
    "molecules": "ATP, LPS, PicoGreen"
  },
  {
    "caption": "(E) J774A.1 cells with or without Phb1  knockdown were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. After cells were lysed, NLRP3  was immunoprecipitated  by anti-NLRP3  antibody. mtDNA  levels (D-loop) from immunoprecipitants were detected by qPCR in J774A.1 cells with or without Phb1  knockdown. Data are from five independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(F) J774A.1 cells with Phb1  knockdown were incubated with EtBr  (150 ng/ml) for 3 days, followed by LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. WB  was used to detect the protein levels in whole cell lysates  and culture medium.",
    "molecules": "ATP, EtBr, LPS"
  },
  {
    "caption": "(G) BMDMs  isolated from Phb1F/F mice  and Phb1MyeKO mice were treated with NAC  (2 mM) for 8 hours, LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. WB  assay was used to detect the protein levels in whole cell lysates and in the culture medium.",
    "molecules": "ATP, LPS, NAC"
  },
  {
    "caption": "(A) HeLa  cells were incubated with H2O2  (5 mM) for 20 minutes and then lysed. The whole cell lysate was immunoprecipitated  by IgG, anti-AFG3L2  antibody or anti-SPG7  antibody. WB  was used to detect the SPG7  or AFG3L2  levels, respectively, in the precipitated products.",
    "molecules": "H2O2"
  },
  {
    "caption": "(B) J774A.1  cells were stimulated with LPS  (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated  by IgG, anti-AFG3L2  antibody or anti-SPG7  antibody. The respective levels of precipitated SPG7  or AFG3L2  were examined by WB.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) J774A.1 cells with knockdown of Phb1  alone, Afg3l2  alone, Spg7  alone, Phb1   + Afg3l2, or Phb1  + Spg7  were fixed and subjected to IF  analysis to assess the  co-localization  between mitochondria  (MitoTracker, red) and mtDNA  (PicoGreen, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA  outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of the data from (A). Data are from at least 12 images from three independent experiments (mean ± SEM). ** p < 0.01, **** p < 0.0001 (Student's t-test).",
    "molecules": "MitoTracker, PicoGreen"
  },
  {
    "caption": "(D)  J774A.1 cells with knockdown of Phb1  alone, Afg3l2  alone, Spg7  alone, Phb1 + Afg3l2, or Phb1  + Spg7  were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. The culture medium was collected and ELISA  assays were used to detect IL-1β levels in culture medium. Data are from four independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(E)  J774A.1 cells with Phb1  knockdown, Spg7  knockdown, or combined Phb1  and Spg7  knockdowns were stimulated with LPS  (200 ng/ml) for 6 hours and ATP  (4 mM) for 45 minutes. WB  assay was used to detect the protein levels in whole cell lysates and culture medium.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(F) J774A.1   cells with Phb1   knockdown, Afg3l2   knockdown, and combined Phb1   and Afg3l2   knockdowns were stimulated with LPS   (200 ng/ml) for 6 hours and ATP   (4 mM) for 45 minutes. WB   assay was used to detect the protein levels in whole  cell lysates  and culture medium.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(G) J774A.1 cells were stimulated with LPS  (200 ng/ml) for 6 hours and lysed. The whole cell lysate was immunoprecipitated  by IgG or anti-SPG7  antibody. WB  was used to detect the CYPD  and VDAC  levels in the precipitated products.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) Discharge and K63 poly-ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved.",
    "molecules": "glutamate"
  },
  {
    "caption": "A. SDS-PAGE analysis of the D391N DIS3L2-RNA complexes. RNAs were radioactively labeled at the 5' end and DIS3L2-bound RNAs were detected by autoradiography. The negative control was performed with HEK293T-Rex cells (Control)",
    "molecules": "RNA"
  },
  {
    "caption": "G. D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs. Scatter plot of transcript abundances estimated from RNA-seq from the cell line overexpressing D391N DIS3L2 and control HEK293T-Rex cells. CLIPed transcripts are marked in white.",
    "molecules": "RNAs"
  },
  {
    "caption": "B. U+ read coverage in downstream regions of mature U12 snRNA, Vault1 and 7SL ncRNAs. The full dark box is representing mature ncRNA, the thin line indicates the region downstream of the mature 3' end (extension).",
    "molecules": "7SL ncRNAs, Vault1"
  },
  {
    "caption": "B. Uridylated tRNA reads overlap mainly with 3' trailers, T-loop and anticodon loop regions. Heat map showing the coverage of uridylated reads on pre-tRNAs. The pre-tRNA sub-regions (as indicated in (A)) are in small letters on the x-axis.",
    "molecules": "tRNAs"
  },
  {
    "caption": "C. Northern blot analysis of RNAs immunoprecipitated with Flag-tagged WT (DIS3L2) and D391N (MUT) DIS3L2, AGO2 and DIS3, respectively. The blots were probed with radioactively labeled probes for 5' and 3' ends of mature tRNAVal, respectively.",
    "molecules": "RNAs, tRNAVal"
  },
  {
    "caption": "D. Sequencing of MUT DIS3L2-bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP-seq. Genomic sequence for each tRNA is indicated on top with the 3' trailer region separated by a vertical line. Non-templated uridine residues are in red.",
    "molecules": "tRNA, tRNALeu, tRNAVal"
  },
  {
    "caption": "E. Identification of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) mapping to tRNAPhe(GAA) and tRNAAsn(GTT) genes. To the right: the schematic representation of selected hybrid reads with corresponding color-code.",
    "molecules": "tRNAAsn, tRNAPhe"
  },
  {
    "caption": "A. DIS3L2-bound uridylated mRNA reads map to the 5' and 3' UTRs.",
    "molecules": "mRNA"
  },
  {
    "caption": "D. Representative traces for E and F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from hippocampal slices treated with DMSO (vehicle control), AR-18 (10 µM), CHIR (1 µM) or SC79 (20 µM). E, F. As B, C, for DMSO, AR-18, CHIR and SC79. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. ",
    "molecules": "AR-18, CHIR, DMSO, SC79"
  },
  {
    "caption": "C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (inactive control), GSK3α (blue) and GSK3β (orange) inhibitor, and AR-18-treated neurons (yellow). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. ",
    "molecules": "AR-18"
  },
  {
    "caption": "A. Representative confocal images from EGFP-GSK3α FRAP experiments. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but for spines expressing EGFP-GSK3β. Scale bars = 1 µm. ",
    "molecules": "NMDA"
  },
  {
    "caption": "G. Representative western blots from whole extracts of organotypic slices incubated in ACSF (untreated; 'baseline'), treated with 20 µM NMDA for 5 minutes ('cLTD') or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ('washout'). Blots were probed for pSer21/9 GSK3α/β, total GSK3α/β, pSer845 GluA1, and total GluA1. H. Quantification of experiments as those shown in G. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 4 independent experiments for all conditions. Asterisks (**) denote P < 0.01. ",
    "molecules": "NMDA, Ser"
  },
  {
    "caption": "A. Representative confocal images from EGFP-GSK3α FRAP experiments in organotypic slices cultured from C57 wild-type mice. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in wild-type mouse slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout ') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but in slices cultured from C57 tauKO mice. Scale bars = 1 µm. ",
    "molecules": "NMDA"
  },
  {
    "caption": "A) Upper panels: Representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of AC16 cells kept at 37°C or exposed to different cold temperatures for 24h. Lower panels: DAPI signal segmented into chromatin and interchromatin regions according to relative intensity. Scale bar: 5 μm. Arrows mark chromatin concentrated at the nuclear periphery (P) and around nucleoli (N). B) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions. Median (central line), interquartile range (IQR, box edges), most extreme data point no more than 1.5x IQR from box edges (whiskers). Number of nuclei: 37°C (31), 28°C 24h (26), 18°C 5h (19), 18°C 24h (22), 18°C 24h then 37°C 2h (25), 8°C 24h (23). C) Left and central panels: representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of cells kept at 37°C or exposed to 18°C for 24h showing the spatial distribution of the immunofluorescence (IF) signal of hnRNPC (green) and nuclear pore complexes (NPCs, red). Right panel: DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z-plane shown. Scale bar whole nuclei: 5 μm. Scale bar enlarged section: 1 μm. D) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition. Number of distances: 37°C (112511), 28°C 24h (94521), 18°C 5h (72340), 18°C 24h (74998), 18°C 24h then 37°C 2h (92896), 8°C 24h (80570).",
    "molecules": "DAPI"
  },
  {
    "caption": "C) Nuclear and cytoplasmic full-length RNA-Seq data from AC16 cells kept at 37°C or exposed to 18°C for 24h for REV-ERBα and CRY2. Data for each track have been normalized to show counts per million aligned counts (CPM) for each nucleotide. Number of 3' end RNA-seq sample replicates: 37°C (C: 6, N: 7), 18°C 5h (4), 10h (4), 24h (6), 18°C 24h then 37°C 2h (4), 5h (2), 10h (2), 24h (2). Number of cytoplasmic (C) and nuclear (N) samples are the same for each condition except 37°C",
    "molecules": "nucleotide"
  },
  {
    "caption": "Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for CRY2 (B)",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for TP53 (C)",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative maximum intensity z-stack-projected images of DAPI-stained (blue) U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα",
    "molecules": "DAPI"
  },
  {
    "caption": "F Representative BLI  and histology  of organotypic cultures with H2030-BrM cancer  cells treated with  DMSO or decreasing concentrations of DEBIO-0932. Scale bar: 100 µm; high magnification: 50 µm. G Quantification of the bioluminescence signal emitted by each condition shown in (F) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with  DMSO. Day 0 is considered 12-16h after the addition of cancer cells and treatment or DMSO. Values are shown in box-and-whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 DMSO, n=8 BKM120 and n=7 per concentration of DEBIO-0932-treated organotypic cultures, 2 independent experiments). P value was calculated using two-tailed t-test.",
    "molecules": "DEBIO-0932, BKM120, DMSO"
  },
  {
    "caption": "A-C Immunofluorescence against HSP90  in mouse brains  with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the  medial habenula, where  neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained  with HSP90. Dotted lines delineate the  metastasis  (cc: cancer cells). Scale bars: 75 µm. (C)  Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification).",
    "molecules": "BB, bisbenzamide"
  },
  {
    "caption": "B Representative in vivo and ex vivo images of vehicle and  DEBIO-0932  treated mice  5 weeks (experimental endpoint) after intracardiac inoculation of H2030-BrM cells.",
    "molecules": "DEBIO-0932"
  },
  {
    "caption": "E Representative sections of  brains from vehicle and DEBIO-0932 treated mice in (B-D). The dotted lines surround the metastases (GFP+). Representative field of view of metastasis stained  with  GFP  and cleaved caspase 3. Scale bars: slices,1mm; cleaved caspase 3, 50 µm. F Quantification of established metastases  found in vehicle and DEBIO-0932 treated brains  from panel (E). Values are shown in box-and-whisker plots where every dot represents a different  brain  and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (vehicle: n=10 brains; DEBIO-0932: n=14 brains). P value was calculated using two-tailed t-test. G Quantification of number of cleaved caspase 3  (CC3+) in cancer cells found in vehicle and  DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 metastatic lesions from 4  brains per condition). P value was calculated using two-tailed t-test.",
    "molecules": "DEBIO-0932"
  },
  {
    "caption": "I Representative BrM-PDOC stained with proliferation markers (BrdU) and markers of the microenvironment (GFAP for  astrocytes, Iba1 for microglia/  macrophages). Scale bar: 50 µm.",
    "molecules": "BrdU"
  },
  {
    "caption": "H Representative images of vehicle and DEBIO-0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO-0932 treated mice, respectively.",
    "molecules": "DEBIO-0932"
  },
  {
    "caption": "C Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO-0932 compared to DMSO (n=3 biological replicates (mice) per condition, n≥12 brain metastases per mouse were pooled together). Proteins with a P value <0.05 and a log2 ratio >1 or <-1 were defined as deregulated. Gray dotted lines indicate P value and log2 ratio cut offs. The names of the top deregulated proteins are shown.",
    "molecules": "DEBIO-0932, DMSO"
  },
  {
    "caption": "D Representative organotypic cultures from the experiment in panel (C). RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP-LC3+ puncta. The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP-LC3 accumulation. Dotted lines in lower panel surround individual cancer cells. Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP-LC3+ puncta. Scale bar: low magnificantion, 25 µm; high magnification (cells), 10 µm; high magnification (puncta), 2.5 µm. E Quantification of GFP-LC3+ vesicles per cell of the experiment in panel (C). Values are shown in box-and-whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (DMSO: n=15 fields of view, 2232 cancer cells from 3 organotypic cultures; DEBIO-0932: n=20 fields of view, 3260 cancer cells from 4 organotypic cultures). P value was calculated using two-tailed t-test.",
    "molecules": "DEBIO-0932, DMSO"
  },
  {
    "caption": "C E139R, E143R, and the combination ER/ER mutant substitutions in UBE2S increase the amount of automodification, monitored by the presence of *Ub (* denotes fluorescently labeled protein) and using SDS-PAGE and fluorescent scanning.",
    "molecules": "Ub"
  },
  {
    "caption": "D ER/ER substitutions increase Ub chain formation in multiple UBE2S constructs (WT, UBC only, and UBC-UBD fusion), monitored by Coomassie-staining of non-reduced SDS-PAGE gels.",
    "molecules": "Coomassie"
  },
  {
    "caption": "C The E139R substitution decreases the stability of UBE2S~Ub compared to WT, revealed by fluorescent scanning of a lysine discharge assay (top) and its quantification (bottom). Statistical comparison was performed using one-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, n=4 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.",
    "molecules": "Ub"
  },
  {
    "caption": "D UBE2R2 R149E has a similar E2~Ub stability as WT, monitored by fluorescent scanning (top) of the lysine discharge assay and its quantification through an unpaired Welch's t-test (bottom) (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.",
    "molecules": "Ub"
  },
  {
    "caption": "E Fluorescent scans (top) and quantification (bottom) of a *LRLRGG peptide transfer assay comparing UBE2S WT and ER/ER reveals no significant difference through an unpaired Welch's t-test (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2S.",
    "molecules": "Ub"
  },
  {
    "caption": "F UBE2R2 R149E is still defective in the transfer of the *LRLRGG peptide to an acceptor Ub compared to WT, probed by fluorescent scanning (top) and its quantitation (bottom). Statistical comparison was performed using an unpaired Welch's t-test (** p ≤ 0.01, n=5 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2R2.",
    "molecules": "Ub"
  },
  {
    "caption": "Role of the UBE2RHTH in diUb formation is determined from kinetic parameters (G-H) for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub Apparent Km and Vmax were determined using the Michaelis-Menten equation (n≥3 independent, technical replicates). Error bars: SEM. Data normalized to the WT apparent Vmax.",
    "molecules": "Ub"
  },
  {
    "caption": "Role of the UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub monitored by fluorescent scanning of nonreducing SDS-PAGE gels",
    "molecules": "Ub"
  },
  {
    "caption": "B Reducing the positive charge of *Ub reduces the autoubiquitination of the E139R-substituted UBE2S variant, monitored by fluorescent scanning of an SDS-PAGE gel.",
    "molecules": "Ub"
  },
  {
    "caption": "D Plot of 15N-labeled UBE2SUBC WT and ER/ER 1H linewidth changes in the presence of Ub revealing the ER/ER variant has a more dynamic active-site cysteine and HTH, but a more stabilized crossover helix, indicative of the "closed" state. Shown are the best-fit values and standard deviations (SD) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension.",
    "molecules": "cysteine, 1H, 15N, Ub"
  },
  {
    "caption": "F C118A substitutions decrease UBE2S autoubiquitination, measured by fluorescent scanning of SDS-PAGE gels, indicating the significance of the \"closed\" formation of the UBE2S~Ub for activity.",
    "molecules": "Ub"
  },
  {
    "caption": "C Securin ubiquitination by APC/C and either UBE2C WT or K157E, resolved by SDS-PAGE and fluorescent scanning. Quantification of 4 or more ubiquitins added on the substrate (right) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch's t-test (**** p ≤ 0.0001, n=5 independent, technical replicates). Error bars: SEM. D Similar to C, Ub-securin ubiquitination by APC/C and either UBE2D2 WT or charge-swap variants. Quantification of 4 or more ubiquitins (right) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one-way ANOVA (**** p ≤ 0.0001, n=4 independent, technical replicates). Error bars: SEM.",
    "molecules": "Ub, ubiquitins"
  },
  {
    "caption": "E Similar to B, the role of the \"closed\" conformation in UBE2S-dependent Ub chain elongation of the APC/C substrate Ub-CycB*, monitored by the incorporation of C118A-substituted UBE2S variants (top). Quantification of ubiquitinated Ub-CycB* was assessed (bottom) and statistical significance by one-way ANOVA between C118A and WT, C118A/E139R, and C118A/E143R (** p ≤ 0.01, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT UBE2S 4+ Ub additions.",
    "molecules": "Ub"
  },
  {
    "caption": "(A) PBMCs were stimulated with PMA and ionomycin for 3 hrs and then intracellular AREG was detected within the different subsets of NK cells and ILCs by flow cytometry.",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(B) PBMCs were cultured in MACS NK medium at 37 °C in 5% CO2, 50 ng/ml IL-2, 50 ng/ml IL-15, 10 ng/ml IL-12 or 50 ng/ml IL-18 for 5 days, then AREG, IFN-γ or TNF-α from different subsets of NK cells and ILCs were detected.",
    "molecules": "CO2"
  },
  {
    "caption": "(F PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of AREG+NK cells (F) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.",
    "molecules": "CO2, CHIR99021, ionomycin, PMA"
  },
  {
    "caption": "G) PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of ILCs (G) (n=15) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.",
    "molecules": "CO2, CHIR99021, ionomycin, PMA"
  },
  {
    "caption": "(H) As in (F), PBMCs were additionally treated with CHIR99021 and TGF-β1 (50 ng/ml) before AREG+NK cells were detected by flow cytometry (n=8). two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.",
    "molecules": "CHIR99021"
  },
  {
    "caption": "(I) AREG+NK cells from control or RUNX3 knockout groups were detected after IL-12+IL-15+IL-18 stimulation in MACS NK medium at 37°C in 5% CO2 for 16 hrs (n= two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.",
    "molecules": "CO2"
  },
  {
    "caption": "(A PBMCs from HIV-1- (n=20), HIV-1+ viremic (n=20), HIV-1+ ART suppressed (n=19), and HIV-1+ spontaneous controllers (n=20) were stimulated with PMA and ionomycin in RPMI 1640 at 37 °C in 5% CO2 for 3 hrs. The percentage (A) of AREG+ cells from are shown for the indicated populations. Mann-Whitney test. ns, not significant.",
    "molecules": "CO2, ionomycin, PMA"
  },
  {
    "caption": "(E) Percentage of CD56-NK cells among total NK cells from PBMCs and CD4-PBMCs after culture in RPMI 1640 at 37°C in 5% CO2 for 5 days (n=7).",
    "molecules": "CO2"
  },
  {
    "caption": "(A) PBMCs were cultured at 37 °C in 5% CO2 with or without IL-2 (50 ng/ml) for 5 days, the phosphorylation of mTOR on Ser2448, AKT on Ser473, 4EBP1 on Thr36 and Thr45, and S6 on Ser235 and Ser236, and the surface CD71 from NK cells (Lin-TBX21+) were detected by flow cytometry (n=8). (B) As in (A), PBMCs or CD4-PBMCs were cultured for 5 days, the indicated targets from NK cells were detected (n=8). (C) As in (A), PBMCs were cultured with or without IL-15 (50 ng/ml) for 5 days, the indicated targets were detected (n=8). All data were generated using blood from healthy HIV-1-negative donors.",
    "molecules": "CO2"
  },
  {
    "caption": "(E, F) PBMCs or CD4-PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F, n=8) or Torin 1 (250 nM) (G, n=8), with or without IL-2 (10 ng/ml) or IL-15 (10 ng/ml), for 5 days. IFN-γ was detected in Lin-TBX21+ cells. All data were generated using blood from healthy HIV-1-negative donors.",
    "molecules": "rapamycin, Torin 1"
  },
  {
    "caption": "(G, H) PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F) or Torin 1 (250 nM) combined with or without IL-2 (10 ng/ml) (G, n=8) or IL-15 (10 ng/ml) (H, n=8) for 5 days. The indicated targets from the Lin-TBX21+ population were detected as in (A). All data were generated using blood from healthy HIV-1-negative donors.",
    "molecules": "rapamycin, Torin 1"
  },
  {
    "caption": "B P10 plasma total bilirubin levels (n=6 per group, unpaired t test, **P=0.0091). C P10 plasma thrombin antithrombin levels (n=6 per group, unpaired t test, ns, P=0.2788).",
    "molecules": "bilirubin"
  },
  {
    "caption": "L Adult brain Evans blue assay (Jag1CTRL n=7, Jag1Ndr/Ndr n=8, unpaired t test, ns, P=0.3807).",
    "molecules": "Evans blue"
  },
  {
    "caption": "(O) Blood pressure increase in Jag1CTRL and Jag1Ndr/Ndr mice treated with vehicle or AngII for two weeks (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparisons).",
    "molecules": "AngII"
  },
  {
    "caption": "(P) Detection of Evans blue leakage in brain in Jag1CTRL and Jag1Ndr/Ndr mice with or without AngII treatment. (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P=0.9687, Treatment **P=0.007, Genotype P=0.5058).",
    "molecules": "AngII, Evans blue"
  },
  {
    "caption": "(Q) One Jag1Ndr/Ndr mouse (male) displayed a macroscopic EB leakage, upon AngII treatment.",
    "molecules": "AngII, EB"
  },
  {
    "caption": "® Retinal arteriolar αSMA coverage in vehicle or AngII-treated mice. Green arrowheads denote stenosis, blue arrowheads label αSMA-negative gap. (S) αSMA-negative gaps in arterioles per retina (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=3-5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P = 0.1054, treatment P=0.0815; Genotype P=0.0747. Ší'ák's multiple comparisons test: AngII treated Jag1+/+ vs AngII treated Jag1Ndr/Ndr *P= 0.0315).).",
    "molecules": "AngII"
  },
  {
    "caption": "(E) Cell death (% LDH) evaluation in IFNγ-primed WT, Irgm2-/- and Casp11-/- BMDMs 4 hours after electroporation or not with 1µg of E. coli LPS.",
    "molecules": "LPS"
  },
  {
    "caption": "(F-H) Survival of WT, Casp11-/- and Irgm2-/- mice primed with 100µg poly(I:C) for 6 hours and injected (i.p.) with 5mg/k-1 LPS or 5 and 25 µg of OMVs (n=6 animals per condition).",
    "molecules": "LPS, poly(I:C)"
  },
  {
    "caption": "(C) Florescence microscopy and associated quantifications of GBP-2 (green) recruitments to intracellular S. Tm orgA--mCherry (MOI 10, red) in IFNγ-primed WT and Irgm2-/-BMDMs. Nucleus was stained with Hoechst (blue). Confocal images shown are from one experiment and are representative of n=3 independent experiments; scale bars 5 µm. For quantifications, the percentage of GBP-associated bacteria was quantified and 'n=' refers to the number of intracellular bacteria counted in each experiment; quantifications from n=3 independent experiments were then plotted and expressed as mean ± SEM.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D) Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red) in IFNγ-primed iWT, iIrgm2-/-, iGBPChr3-/- and iGBPChr3-/-/sgIrgm2 BMDMs after 8 hours of infection. Nucleus (blue) was stained with Hoescht; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction.",
    "molecules": "Hoescht"
  },
  {
    "caption": "(F) Representative confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red, MOI 10) in IFNγ-primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection. Nucleus (blue) was stained with Hoechst; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs) infected with S. Typhimurium orgA- (MOI25) for 16 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.",
    "molecules": "Z-LEVD, MCC950"
  },
  {
    "caption": "(B) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs), primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then stimulated with Nigericin (20µM) for 4 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.",
    "molecules": "Z-LEVD, MCC950, PAM3CSK4, Nigericin"
  },
  {
    "caption": "(C, D) LDH and IL-1B release from siRNA-treated WT or GBP1/2/5-/- U937 Monocytic cell line, primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then infected with (C) with S. Typhimurium orgA- (MOI25) for 10 hours or (D) electroporated with 1µg of E. coli LPS for 4 hours. Φ indicates that no LPS electroporation was performed.",
    "molecules": "LPS, PAM3CSK4"
  },
  {
    "caption": "C) Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X-Axis shows sample concentration determined by antibody molarity. Y-Axis shows FcγR activation determined by reporter cell mouse IL-2 production (OD 450nm). Two independent experiments performed in technical duplicates. Error bars = SD.",
    "molecules": "Rituximab, Rtx"
  },
  {
    "caption": "Ultra-pure antigen (Ag, TNF-α) mixed with therapy-grade mAb (infliximab, Ifx) was used to generate sICs. X-Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR-specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL-2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL-2 was measured via anti-IL-2 ELISA (A450nm) and IL-2 concentrations were calculated from an IL-2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. MFI were normalized to untreated cells (ctrl.) and are presented as fold-change increase.",
    "molecules": "Ifx, infliximab, Rtx"
  },
  {
    "caption": "A) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry. NK cells were incubated with immobilized IgG (mepolizumab, Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 106 cells). Incubation with PMA and Ionomycin (Iono) served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one donor measured in technical duplicates. Error bars = SD. One-way ANOVA (Tukey); *p<0.05, ****p<0.0001.",
    "molecules": "Iono, Ionomycin, mepolizumab, Mpz, PMA"
  },
  {
    "caption": "B) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry. Neutrophils were incubated with immobilized IgG (Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 2*105 cells). Incubation with PMA or immobilized rituximab served as positive controls. Incubation with medium served as a negative control. Immobilized FcγRII and FcγRIII specific mAbs served as functional controls. Mean florescence intensity (MFI) values at t=30 minutes of incubation are presented as increase over t=0 min. Each dot represents one donor measured in technical duplicates. Error bars = SD. Two-way ANOVA compared to medium (Dunnett); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.",
    "molecules": "Mpz, PMA, rituximab"
  },
  {
    "caption": "B) sICs of different size were generated by cross-titration according to the AF4 measurement. Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNF-α, right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either antigen or antibody, respectively (TNF-α calculated as monomer). Two independent experiments performed in technical duplicates. Error bars = SD.",
    "molecules": "infliximab"
  },
  {
    "caption": "C) Purified primary NK cells from three different donors were incubated with cross-titrated sICs as in B. NK cells were measured for MIP-1β expression (% positivity). Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one experimental replicate, the dashed line represents the mean values.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right).",
    "molecules": "PEG"
  },
  {
    "caption": "C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.",
    "molecules": "dsDNA"
  },
  {
    "caption": "Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.",
    "molecules": "nitrogen, PMSF"
  },
  {
    "caption": "c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.",
    "molecules": "nitrogen"
  },
  {
    "caption": "d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.",
    "molecules": "nitrogen"
  },
  {
    "caption": "c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of perimCherry/cytoGFP E. coli bacteria exposed to (different concentrations of) human serum. Inner membrane damage correlates with killing (samples where bacteria are killed are indicated with gray shadings and a cross, see CFU data in Fig EV1b). Data information: (C-E) Data represent mean ± SD of 3 independent experiments.",
    "molecules": "Sytox"
  },
  {
    "caption": "(D) Serum-induced inner membrane damage (% Sytox positive) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.",
    "molecules": "Sytox, sytox"
  },
  {
    "caption": ", E Serum-induced killing (CFU/ml) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.",
    "molecules": "sytox"
  },
  {
    "caption": "(D) Bacterial viability of convertase labeled E. coli MG1655 exposed to C5-C9 or conditions lacking one MAC component. As an extra control, convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin. Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.",
    "molecules": "compstatin"
  },
  {
    "caption": "(A) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli cells incubated with a concentration range of C5 and fixed concentrations of C6-C9. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.",
    "molecules": "Sytox"
  },
  {
    "caption": "(B) Inner membrane damage of perimCherry/cytoGFP E. coli exposed to a concentration range of ΔC5 serum and, after washing, to C5-C9. As controls, bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.",
    "molecules": "compstatin"
  },
  {
    "caption": "(D) Confocal microscopy images of convertase-labeled perimCherry/cytoGFP E. coli exposed to buffer (Conv) or C5-C9 (Conv-MAC). Unlabeled bacteria exposed to 1% serum served as control. Green = GFP, Red = To-pro-3 DNA dye. Scale bars = 3 µm.",
    "molecules": "DNA, To-pro-3"
  },
  {
    "caption": "(C) Inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.",
    "molecules": "Sytox"
  },
  {
    "caption": "A, B Step-wise assembly of MAC on convertase-labeled bacteria. Convertase-labeled bacteria were incubated with C5/C6 or C5/C6/C7 for 15 minutes, and subsequently washed (@) or treated with 10 μg/ml Eculizumab (Ecu). Then, the remaining MAC components (C7-9 for C5/C6 or C8-9 for C5/C6/C7 respectively) were added to the incubation mixture. In the control conditions (Conv-MAC), the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor. (A) Outer membrane (mCherry) and (B) inner membrane damage (% Sytox positive) were determined. Data information: (A-B) Data represent mean ± SD of 3 independent experiments. Statistical analysis was done using a ratio paired two-tailed t-test in which the test conditions were compared to Conv-MAC and displayed only when significant as * p≤0.05. ",
    "molecules": "Ecu, Sytox, Eculizumab"
  },
  {
    "caption": "(A) Convertase-labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9-Cy3 was added. Controls at 0 nM C5b6 or C5-C6 confirm that the detected C9-Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, 2007). Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg/ml trypsin for 15 min at 37°C (dotted lines). Data information: (A) Data represent mean ± SD of 3 independent experiments.",
    "molecules": "Cy3"
  },
  {
    "caption": "(B) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm.",
    "molecules": "Cy5"
  },
  {
    "caption": "A. Binding of ubiquitin chains to immobilized GST-fusion proteins. Indicated proteins were purified from E. coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains, which were either K48- or K63-linked. The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS‑PAGE and fluorescence scan. Total fluorescence was quantified per lane and normalized to the first lane (rel. fluo.). Asterisk (*) indicates lane from the same gel that was cropped and moved.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "B. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.). D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate.",
    "molecules": "amino acid, ubiquitin"
  },
  {
    "caption": "b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determination",
    "molecules": "amino acid, monoubiquitin, Ub"
  },
  {
    "caption": "c. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified.",
    "molecules": "monoubiquitin"
  },
  {
    "caption": "d. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination",
    "molecules": "amino acid"
  },
  {
    "caption": "a. The activity of Ubc1 was compared with the K48-linked chain building enzyme Ubc7/Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin, K48‑Ub2 or K63-Ub2. Initial rates were determined",
    "molecules": "monoubiquitin"
  },
  {
    "caption": "D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.",
    "molecules": "Alexa488, monoubiquitin, Ub"
  },
  {
    "caption": "F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.",
    "molecules": "HU, hydroxyurea"
  },
  {
    "caption": "(e) Distribution of KFERQ-PA-mCherry-1 in TH1 cells transiently transfected with a CMA reporter vector, photoactivated, and left unstimulated (Rest) or stimulated with anti-CD3 and anti-CD28 in the presence (3-MA) or absence (vehicle (veh)) of 3-methyladenine (left); arrows indicate mCherry+ puncta. Original magnification, ×630. Right, quantification of the results for the cells at left. *P 0.05 (analysis of variance with Tukey's post-test).",
    "molecules": "3-MA, 3-methyladenine"
  },
  {
    "caption": "(g) Immunoblot analysis of LAMP-2A in resting TH1 cells and TH1 cells activated in the presence (NAc) or absence (Veh) of N-acetylcysteine. (h) Quantitative PCR analysis of Lamp2a mRNA in cells as in g (results presented as in b). *P = 0.007 (t test).",
    "molecules": "N-acetylcysteine, NAc"
  },
  {
    "caption": "(j) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated (control (Ctrl)) or stimulated for 16 h with 50 μM paraquat (Pq) or 1 mM H2O2; results are presented in relative light units (RLU).",
    "molecules": "H2O2, paraquat"
  },
  {
    "caption": "(l) Immunoblot analysis of LAMP-2A in lysates of resting or activated TH1 cells in the presence (right half) or absence (left half) of cyclosporine A (CsA).",
    "molecules": "CsA, cyclosporine A"
  },
  {
    "caption": "(b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control.",
    "molecules": "NH4Cl, leupeptin"
  },
  {
    "caption": "(c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine.",
    "molecules": "N-acetylcysteine, NAc"
  },
  {
    "caption": "(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).",
    "molecules": "NH4Cl, leupeptin"
  },
  {
    "caption": "(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).",
    "molecules": "ROS"
  },
  {
    "caption": "A Primary mousecardiomyocytes were treated with H2O2 (1 mM) for 3 hours and cardiomyocyteviability was determined by flow cytometry. Representative results of flow cytometry analysis.",
    "molecules": "H2O2"
  },
  {
    "caption": "B Quantitative analysis of PI-positive cells treated with H2O2 was shown (42.9 ± 3.1% in wild-type group vs. 10.1 ± 4.4% in Cuedc2-/- group, * p = 0.0004, n = 5 wells per group and repeated for three times).",
    "molecules": "H2O2"
  },
  {
    "caption": "C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours and then reoxygenation for 30 minutes. Cardiomyocytes were then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. Representative results of flow cytometry analysis are shown, and all experiments were repeated for three times.D ROS levels were quantitated and summarized. * p = 0.0108, n = 3 wells per group and repeated for three times.",
    "molecules": "ROS"
  },
  {
    "caption": "E HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in sham and I/R injury heart tissue (30-min ischemia followed by 30-min reperfusion), 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. *p < 0.0001, n = 5 in each group.F In the same samples of (E), HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. *p = 0.0001, n = 5 in each group.",
    "molecules": "DHE, dihydroethidium, 2-hydroxyethidium, EOH, ethidium, H2O2, ONOO"
  },
  {
    "caption": "D Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 (1 mM) for 3 hours. Cell viability was detected by FACS. * p = 0.0001; ** p = 0.0275, n = 3 wells per group. NS, not significant.",
    "molecules": "H2O2"
  },
  {
    "caption": "F Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 (1 mM) for 30 minutes and then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. * p = 0.0056; ** p = 0.0218, n = 3 wells per group, n = 3 wells per group. NS, not significant.",
    "molecules": "H2O2"
  },
  {
    "caption": "A Lysates from MEF-WT or MEF-Cuedc2-/- cells treated with 20μM cycloheximide (CHX) for the indicated times were subjected to immunoblotting (left). Relative GPX1 levels were quantified by densitometry (right).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "B MEF-Cuedc2-/- cells were transfected with increasing amounts of FLAG-CUEDC2 (mouse) plasmids (1 μg and 2 μg). At 24 hours after transfection, the cells were treated with the proteasome inhibitor MG-132 (10 μM). Cells were cultured for additional 6 hours and subjected to immunoblotting.",
    "molecules": "MG-132"
  },
  {
    "caption": "C HEK293T cells were transfected with the FLAG-GPX1, HA-CUEDC2, and Myc-ubiquitin plasmids as indicated and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-FLAG (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-FLAG or anti-HA antibody to determine the protein of Flag-GPX1 and HA-CUEDC2.",
    "molecules": "MG-132"
  },
  {
    "caption": "E HEK293T cells were transfected with the Flag-CUEDC2 or Flag-CUEDC2△CUE (deletion of the CUE domain of CUEDC2), and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-Flag (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-Flag or anti-GPX1 antibody to determine the protein of Flag-CUEDC2 and GPX1. v: vector.",
    "molecules": "MG-132"
  },
  {
    "caption": "C HEK293T cells were transfected as indicated, at 18 h after transfection, the cells were treated with the proteasome inhibitor MG132 (20μM) for 6 hours and then harvested. Cell lysates were immunoprecipitated by Flag-antibody (M2 beads) and Ubiquitin-conjugated GPX1 was detected by Western blotting with anti-Myc antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "A HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in young (8-week-old) and old mouse (20-month-old) heart tissue, 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. * p = 0.0087, n = 5 in each group.",
    "molecules": "DHE, dihydroethidium, 2-hydroxyethidium, EOH"
  },
  {
    "caption": "B HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. * p = 0.0021, n = 5 in each group.",
    "molecules": "ethidium, H2O2, ONOO"
  },
  {
    "caption": "C Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in young and old mice. * p = 0.0164, n = 6 mice per group.",
    "molecules": "8-hydroxy-2’-deoxyguanosine, 8-OHdG"
  },
  {
    "caption": "A, B: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.",
    "molecules": "S1P"
  },
  {
    "caption": "Pearson correlation between S1P and apoM (C) Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.",
    "molecules": "S1P"
  },
  {
    "caption": "Pearson correlation between S1P and RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.",
    "molecules": "HGB, S1P"
  },
  {
    "caption": "A, B: correlations between S1P and HDL-C (A), and albumin (B). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.",
    "molecules": "HDL-C, S1P"
  },
  {
    "caption": "C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.",
    "molecules": "HDL-C"
  },
  {
    "caption": "(A-D) The serum concentrations of S1P (A), apoM (B), albumin (C) and HDL-C (D) in noICU (n=89) and ICU (n= 22) patients are shown. The box-plots represent the interquartile range with median (central line); the whiskers represent the measured range of noICU and ICU patients. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.",
    "molecules": "HDL-C, S1P"
  },
  {
    "caption": "Pearson correlation between S1P (A-C) PSI days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.",
    "molecules": "S1P"
  },
  {
    "caption": "(A,B) Cumulative risk for ICU admission (A) and cumulative survival (B) in COV patients (n=111), grouped for cut-off value of S1P serum level of 0.60 μM. Statistical analysis was performed by Cox regression. Exact p values are reported.",
    "molecules": "S1P"
  },
  {
    "caption": "(D) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.",
    "molecules": "Digitonin"
  },
  {
    "caption": "(A) UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag-tagged UBQLN4 protein was affinity-purified from extracts of HeLa cells that were treated with (+) or without (-) 20 μM MG-132 for 4 h before harvesting, and the Flag-precipitates (IP:Flag) were blotted with anti-polyubiquitin (FK2), anti-BAG6, and anti-Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4-BAG6 co-precipitation can be observed exclusively in the presence of polyubiquitinated substrates.",
    "molecules": "MG-132"
  },
  {
    "caption": "(B) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 μM MG-132 as well as CHX (either 10 or 25 μg/mL) for 4 h as indicated, and then subjected to immunoprecipitation with an anti-Flag antibody as in (A).",
    "molecules": "CHX, cycloheximide, MG-132"
  },
  {
    "caption": "(A) UBQLN4 co-localizes with cytoplasmic aggregates after puromycin treatment. HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 µg/mL puromycin for 2 h and then followed by 1% Triton X-100 extraction and 4% paraformaldehyde fixation. Fixed cells were stained with anti-polyubiquitin FK2 (shown in red to detect ubiquitin-positive cytoplasmic aggregates, ALIS) and anti-Flag antibodies (shown in green).",
    "molecules": "puromycin, Triton X-100"
  },
  {
    "caption": "(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch's t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).",
    "molecules": "Puromycin, puromycin"
  },
  {
    "caption": "(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch's t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).",
    "molecules": "Puromycin, puromycin"
  },
  {
    "caption": "(D) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin-induced cell death. After 48 h of UBQLN4 siRNA treatment, the cells were treated with 5 µg/mL puromycin for 15.5 h and then viability was measured. The data represent mean ± SD calculated from 3 independent experiments (n=3). P‐values were calculated by Student's t-test compared with puromycin-treated control siRNA cells (*P < 0.01).",
    "molecules": "puromycin"
  },
  {
    "caption": "(D) The glycosylated form of IL-2Rα WT is a stable protein, while mislocalized IL-2Rα ΔSS is degraded rapidly via its TMD. A series of IL-2Ra deletion proteins were expressed in HeLa cells and then chased with 20 μg/mL CHX for the indicated periods. Actin was used as a loading control. The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments (n=4).",
    "molecules": "CHX"
  },
  {
    "caption": "(E) IL-2Rα ΔSS is degraded in a proteasome-dependent manner. HeLa cells expressing Flag-tagged IL-2Rα ΔSS were treated with 20 μg/mL CHX as well as 0.1% DMSO (DM.), 10 μM MG-132 (MG.), 2 μM Bortezomib (Bor.), 10 μM Leupeptin (Leu.), 0.1 μM Bafilomycin A1 (Baf.), or 10 μg/ml Pepstatin A (pep. A)/E64d for the indicated periods. The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments (n=3).",
    "molecules": "Baf, Bafilomycin A1, Bor, Bortezomib, CHX, DM, DMSO, E64d, Leu, Leupeptin, MG, MG-132, pep. A, Pepstatin A"
  },
  {
    "caption": "(F) The Flag-tagged IL-2Rα ΔSS mutant and T7-tagged ubiquitin were co-expressed in HeLa cells and the cells were treated with (+) or without (-) 10 μM MG-132. After 4 h, Flag-precipitates were blotted with anti-T7 and anti-Flag antibodies.",
    "molecules": "MG-132"
  },
  {
    "caption": "(G) HeLa cells expressing Flag-tagged IL-2RaWT, ΔSS, and ΔSSΔTM proteins were treated with (+) or without (-) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed by immunoblotting using the indicated antibodies. Note that a higher migrating form of non-glycosylated (and thus defective) IL-2Ra WT (indicated by the arrowhead) is detected strongly in the presence of MG-132.",
    "molecules": "MG-132"
  },
  {
    "caption": "(A, B) IL-2Rα ΔSS is stabilized in UBQLN4-knockdown cells. HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1~#3) or control siRNA. At 48 h after siRNA transfection, Flag tagged-IL-2Rα ΔSS was expressed in the cells. At 24 h after IL-2RaSS transfection, the cells were chased with 20 μg/mL CHX and harvested at the indicated time after CHX addition. Anti-Flag signals in the control or UBQLN4 siRNA-treated cells (siRNA#1~#3) were quantified at the indicated time points. The data represent mean ± SEM calculated from 4 independent experiments (n=4). The UBQLN4 signal is marked by an arrowhead, while the band just below it (indicated by an asterisk) is a non-specific band, since this signal was never affected by any of the UBQLN4 siRNAs. Actin was used as a loading control.",
    "molecules": "CHX"
  },
  {
    "caption": "(C) Polyubiquitin modification of IL-2Rα ΔSS was not diminished, but rather strengthened, in UBQLN4 knockdown cells. At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6, Flag-tagged IL-2Rα ΔSS and T7-tagged ubiquitin were expressed with (+) or without (-) 10 mM MG-132. From these cells, Flag-tagged IL-2Rα ΔSS was affinity-purified using Hot lysis analysis, and Flag-precipitates were blotted with an anti-T7 antibody to detect polyubiquitination of IL-2Rα ΔSS.",
    "molecules": "MG-132"
  },
  {
    "caption": "(A) A series of Flag-tagged truncated mutants of IL-2Ra substrates were expressed in HeLa cells with T7-tagged UBQLN4 and treated with (+) or without (-) 10 µM MG-132 for 4 h. Flag-IL-2Ra substrates were immunoprecipitated and their co-precipitation with UBQLN4 was analyzed.",
    "molecules": "MG-132"
  },
  {
    "caption": "(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2RαΔSS (B and C). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.",
    "molecules": "MG-132"
  },
  {
    "caption": "(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2Rα ΔSS (B and C) and T7-tagged ubiquitin (D). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.",
    "molecules": "MG-132"
  },
  {
    "caption": "(A) UBQLN4 interacts with non-glycosylated (and thus defective) IL-2Ra WT in SRP54-knockdown cells. After treatment of HeLa cells with SRP54 siRNA (10 nM) for 48 h, Flag-tagged IL-2RaWT and T7-tagged UBQLN4 proteins were expressed, and the cells were treated with (+) or without (-) 10 µM MG-132 at 4 h before harvesting. UBQLN4 was affinity-purified from cell extracts and probed with an anti-Flag antibody. Note that the levels of non-glycosylated IL-2RaWT (indicated as Defective) increased, while the glycosylated form of this protein (indicated as Assembled) decreased in SRP54 knockdown cells.",
    "molecules": "MG-132"
  },
  {
    "caption": "(B, C) SRP54 knockdown stimulates polyubiquitinated proteins co-precipitation of UBQLN4. Flag-tagged UBQLN4 and T7-tagged ubiquitin were expressed in SRP54 siRNA-treated cells, and Flag precipitates were probed with an anti-T7 antibody to detect polyubiquitinated clients co-precipitation with UBQLN4 under no addition of MG-132 (B). Note that the interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown cells treated with MG-132 for 4 h before harvesting (C).",
    "molecules": "MG-132"
  },
  {
    "caption": "(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8 were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A),",
    "molecules": "cerulenin, DMSO, palmitic, N, nitrogen, stearic, myristic"
  },
  {
    "caption": "(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A), or were visualized by fluorescence microscopy (B)",
    "molecules": "cerulenin, DMSO, palmitic, N, nitrogen, stearic, myristic"
  },
  {
    "caption": "(C) Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm.",
    "molecules": "cerulenin, DMSO, N"
  },
  {
    "caption": "(D) WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS‒PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. (",
    "molecules": "cysteine, cerulenin, DMSO, methionine"
  },
  {
    "caption": "(E) Cells of the fas1∆ (TOS029) strain expressing GFP‒Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies.",
    "molecules": "fatty acids, palmitic, N, stearic, myristic"
  },
  {
    "caption": "(F) WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin (25, 50) or with DMSO (-) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (-). Cells were lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type YPD, complete medium.",
    "molecules": "cerulenin, DMSO, N, nitrogen"
  },
  {
    "caption": "(A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).",
    "molecules": "cerulenin, DMSO, N"
  },
  {
    "caption": "(C) WT (SCY62) and tagΔsteΔ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm.",
    "molecules": "N"
  },
  {
    "caption": "(D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies.",
    "molecules": "N"
  },
  {
    "caption": "(E) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.",
    "molecules": "N, nitrogen"
  },
  {
    "caption": "(A) GAL-DGA1GAL-ARE2lro1Δ are1Δ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinoseovernight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescencemicroscopy. Scale bar, 5 μm.",
    "molecules": "dextrose, galactose, glucose, N, raffinose"
  },
  {
    "caption": "(C, D) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 (C) or GFP−Atg1 (D) were grown as in A and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild-type.",
    "molecules": "N, nitrogen"
  },
  {
    "caption": "(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm.",
    "molecules": "N"
  },
  {
    "caption": ". (C) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. (",
    "molecules": "N"
  },
  {
    "caption": "(D) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells were grown as in (A). Lysates were subjected to SDS−PAGE in urea gel, followed by western blot analysis using anti-Atg8 and anti-Pgk1 antibodies. Atg8 I, non-lipidated Atg8; Atg8 II, lipidated Atg8. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.",
    "molecules": "N, nitrogen, urea"
  },
  {
    "caption": "Fig. 6 Lack of lipid droplets inhibits starvation-induced formation of autophagosomes. (A, H) WT pep4∆ (pep4∆) (A,B), tag∆ste∆ pep4∆ (tag∆ste∆pep4∆)(C,D), ste∆ pep4∆ (ste∆pep4∆)(E,F) and tag∆ pep4∆ (tag∆pep4∆) (G,H) cells were grown to an exponential phase in YPD before being starved in SD-N for 2 h, and were then processed for electron microscopy. H is a magnification of the box in panel G. Magnification of proliferating ER in a tag∆ste∆ (H1246) cell is shown in (I) Asterisks indicate autophagic bodies. N, nucleus; PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; #, trehalose; F, ER proliferation. Scale bars: 1 µm (A-H) and 200 nm (I). (J) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles. Error bars represent standard deviations fromcounting of the three grids. ***P < 0.001 (Student's t-test). SD-N,nitrogen-starvation medium; WT, wild type YPD, complete medium.",
    "molecules": "trehalose"
  },
  {
    "caption": "(A) WT (BY4741), tgl1∆, yeh1∆ ,and yeh2∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).",
    "molecules": "N"
  },
  {
    "caption": "(F) WT (BY4741), ice2∆ and yeh1∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 4 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.",
    "molecules": "N, nitrogen"
  },
  {
    "caption": "E. ELISA of IFN-β (left) and IL-6 (right) in the serum of mice described in (d) (Student's t-test).",
    "molecules": "mice"
  },
  {
    "caption": "F. ELISA of serum IFN-β (left) and IL-6 (right) after poly(I:C) injection. HDAC6+/+ and HDAC6-/- mice were intravenously injected with poly(I:C). Serum was collected at the indicated time points and analyzed (200 μg/mouse; n = 3 per group; Student's t-test).",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "E. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in HDAC6+/+ and HDAC6-/- BMDMs treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).",
    "molecules": "5’ppp-dsRNA, poly(I:C)"
  },
  {
    "caption": "G. Induction of mRNA for type I IFN, IL-6, and other IFN-related antiviral genes in HDAC6+/+ and HDAC6-/- BMDMs in response to a RIG-I agonist stimulation at 6 h. HDAC6+/+ and HDAC6-/- BMDMs were stimulated with 5'ppp-dsRNA (0.5 µg/ml) for 6 h.",
    "molecules": "5’ppp-dsRNA"
  },
  {
    "caption": "C. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in control and HDAC6 knockdown RAW264.7 cells treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).",
    "molecules": "5’ppp-dsRNA, poly(I:C)"
  },
  {
    "caption": "F. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (D) and (E), and in HDAC6+/+ and HDAC6-/- MEFs transfected with poly(I:C) (1 µg/ml).",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "C. ELISA of IFN-b (upper) and IL-6 (lower) levels in the supernatant of (A), (B), and in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).",
    "molecules": "5’ppp-dsRNA, poly(I:C)"
  },
  {
    "caption": "D, E. Luciferase assay in 293T cells transfected with an IFN-β luciferase promoter and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng), followed by PR8-GFP (MOI = 2) infection (D) or by poly(I:C) (E) (1 μg/ml) transfection for another 12 h.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "A. 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "B. BMDMs were isolated from HDAC6+/+ and HDAC6-/- mice and were transfected with 5ˊppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed.",
    "molecules": "5ˊppp-dsRNA"
  },
  {
    "caption": "C. HeLa cells were transfected with 5ˊppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (bar, 20µm).",
    "molecules": "5ˊppp-dsRNA"
  },
  {
    "caption": "C. A luciferase assay was performed using 293T cells transfected with the IFN-βluciferase promoter and TK-Renilla together with 2 ng of RIG-I wild-type, RIG-I 858Q, RIG-I 858R, RIG-I K909Q, or RIG-I K909R mutant, followed by transfection with poly(I:C) (1 μg/ml) for another 12 h.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "A. The structural modeling result of RIG-I/dsRNA complex and positions of the lysine residue were presented. RIG-I helicase A and B are shown as green and pale blue ribbons. The dsRNA strands were presented as purple and yellow rods, respectively. Two lysine residues, K907 and K909, on RIG-I that interact with dsRNA are indicated as violet sticks. K907 forms hydrogen bonding with neither 5´-ppp dsRNA nor K907.B, C. Acetylated K909 and K907 on RIG-I are indicated as green sticks. There were hydrogen bonding between NH2 of K907 and oxygen of acetyl-K909.",
    "molecules": "5´-ppp dsRNA"
  },
  {
    "caption": "(a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b-/- mice and Becn1+/- mice and the corresponding wild-type littermate mice, then left unstimulated (-) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A). p10, mature form of caspase-1. Right margin, molecular size in kilodaltons (kDa).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(b) Enzyme-linked immunosorbent assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(c) Immunoblot analysis of caspase-1 and IL-1β in lysates (left) and ELISA of cytokines in supernatants (right) of Map1lc3b+/+ and Map1lc3b-/- BMDMs incubated with LPS, followed by 30 min (immunoblot) or 1 h (ELISA) of ATP treatment.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(d) ELISA of IL-1β secretion by LPS-primed Map1lc3b-/- mice and Becn1+/- BMDMs incubated for 1 h with z-YVAD-fmk (z-YVAD; 10 μM) or vehicle (dimethylsulfoxide (DMSO)), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three or four experiments (mean and s.d. in b-d).",
    "molecules": "ATP, dimethylsulfoxide, DMSO, LPS, z-YVAD, z-YVAD-fmk"
  },
  {
    "caption": "(a,b) Transmission electron microscopy of morphological changes in mitochondria (arrows) in Map1lc3b−/− BMDMs (a) or Becn1+/− BMDMs (b) left untreated or incubated for 6 h with LPS (10 ng/ml) and then stimulated for 30 min with ATP (1 mM). Outlined areas (middle) enlarged at right. Scale bars, 500 nm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(d) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment. Data are representative of three experiments.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(a) ELISA of cytokines in supernatants (left) and immunoblot analysis of caspase-1 and IL-1β in lysates (right) of J774A.1 macrophages exposed to EtBr (0, 10, 100 or 500 ng/ml; wedges) and left unstimulated or stimulated with LPS alone or with LPS and ATP.",
    "molecules": "ATP, EtBr, LPS"
  },
  {
    "caption": "(b) Immunoblot analysis of caspase-1 and ASC in lysates of J774A.1 or ρ0 J774A.1 macrophages (treated with 100 ng/ml of EtBr) incubated with LPS, followed by stimulation for 0, 15 or 30 min with ATP.",
    "molecules": "ATP, EtBr, LPS"
  },
  {
    "caption": "(c) Cytokine secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with various combinations of LPS and ATP (below graph).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(d) Cytokine secretion (right) and immunoblot analysis of caspase-1 and IL-1β in lysates (left) of LPS-primed macrophages incubated for 1 h with rotenone (+; 5 μM) or DMSO (−) before ATP treatment.",
    "molecules": "ATP, DMSO, LPS, rotenone"
  },
  {
    "caption": "(e) ELISA of IL-1β secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with LPS and rotenone (or DMSO), followed by ATP.",
    "molecules": "ATP, DMSO, LPS, rotenone"
  },
  {
    "caption": "(f) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito-TEMPO and then stimulated with various combinations of LPS and ATP.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(g,h) ELISA of IL-1β secretion (left) and immunoblot analysis for caspase-1 in lysates (right) of Map1lc3b+/+ and Map1lc3b−/− (g) or Becn1+/+ and Becn1+/− (h) peritoneal macrophages incubated for 1 h with Mito-TEMPO (500 μM), followed by LPS and ATP. *P 0.05 (Student's t-test). Data represent three experiments (mean and s.d. in a,c,d-h).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(a) ELISA of cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− BMDMs incubated with cyclosporine A or DMSO, followed by stimulation for 1 h with ATP.",
    "molecules": "ATP, cyclosporine A, DMSO, LPS"
  },
  {
    "caption": "(b) ELISA of cytokine secretion by LPS-primed Becn1+/+ or Becn1+/− BMDMs incubated with cyclosporine A (10 μM) or DMSO, followed by ATP stimulation.",
    "molecules": "ATP, cyclosporine A, DMSO, LPS"
  },
  {
    "caption": "(c) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDMs incubated for 1 h with rotenone (5 μM) or vehicle (DMSO) before ATP stimulation.",
    "molecules": "ATP, mtDNA, DMSO, LPS, rotenone"
  },
  {
    "caption": "(d) Quantitative PCR analysis of cytosolic mtDNA in macrophages preincubated for 1 h with Mito-TEMPO (500 μM) and then incubated with LPS, followed by ATP stimulation.",
    "molecules": "ATP, mtDNA, LPS"
  },
  {
    "caption": "(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDM incubated with cyclosporine A (10 μM), plus either rotenone (5 μM) or DMSO, followed by ATP stimulation.",
    "molecules": "ATP, cyclosporine A, mtDNA, DMSO, LPS, rotenone"
  },
  {
    "caption": "(f) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed Map1lc3b−/− and Becn1+/− BMDMs stimulated for 30 min with ATP. The amount of cytosolic mtDNA is presented relative to the amount in untreated control cells, set as 1. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).",
    "molecules": "ATP, mtDNA, LPS"
  },
  {
    "caption": "(a) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I, lactate dehydrogenase LDH (Control) or heat-inactivated DNase I (HI DNase I) and then stimulated with LPS, followed by stimulation for 1 h with ATP.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(b) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS, followed by stimulation for 15 min with ATP.",
    "molecules": "ATP, mtDNA, LPS"
  },
  {
    "caption": "(c) ELISA of IL-1β secretion by Aim2+/+ and Aim2−/− BMDMs primed for 4 h with LPS (200 ng/ml), then transfected for 6 h with 1 μg mtDNA (through the use of liposomes as the vehicle), followed by stimulation for 1 h with ATP. *P 0.05, versus Aim2−/− cells treated with LPS and ATP (Student's t-test).",
    "molecules": "ATP, mtDNA, LPS"
  },
  {
    "caption": "(d) ELISA of cytokine secretion by LPS-primed Aim2−/− BMDMs transfected for 6 h with mtDNA, poly(dA:dT) or mtDNA predigested by DNase I (+ DNase; far left), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).",
    "molecules": "ATP, mtDNA, LPS"
  },
  {
    "caption": "(a) Cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− (a) or Becn1+/+ and Becn1+/− (b) peritoneal macrophages incubated for 15 min with glybenclamide (Glyb; 100 μM (a) or 50-100 μM (b)), Bay 11-7082 (Bay; 12 μM) or DMSO (-), followed by ATP.",
    "molecules": "Bay 11-7082, ATP, DMSO, glybenclamide, LPS"
  },
  {
    "caption": "(c) Cytokine secretion by LPS-primed wild-type (WT) and NALP3-deficient (NALP3-KO) macrophages incubated with rotenone, followed by stimulation for 1 h with ATP.",
    "molecules": "ATP, LPS, rotenone"
  },
  {
    "caption": "(d) Quantitative PCR analysis of cytosolic mtDNA in wild-type and NALP3-deficient BMDMs left unstimulated (-) or stimulated with LPS, followed by treatment for 15 or 30 min with ATP (left), and immunoblot analysis of cytosolic cytochrome c and activation of caspase-1 in the cells at left (right).",
    "molecules": "ATP, mtDNA, LPS"
  },
  {
    "caption": "(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed wild-type, NALP3-deficient or ASC-deficient (ASC-KO) BMDMs stimulated for 15 min with ATP. *P 0.05, NALP3-deficient or ASC-deficient versus wild-type treated with LPS and ATP (Student's t-test).",
    "molecules": "ATP, mtDNA, LPS"
  },
  {
    "caption": "(f) Flow cytometry of untreated macrophages (control) or of LPS-primed macrophages left unstained or stained with MitoSOX, then stimulated for 15 min with ATP.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(g) Flow cytometry of untreated macrophages (control) or of macrophages stimulated with LPS and ATP, stained with MitoTracker Deep Red. Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(h) Flow cytometry of LPS-primed wild-type, ASC-deficient and NALP3-deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V. Data represent three experiments (mean and s.d. in a-e).",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(a) ELISA of serum IL-1β and IL-18 at 24 h after intraperitoneal injection of LPS (12 mg per kg body weight) into male Map1lc3b+/+ and Map1lc3b−/− mice.",
    "molecules": "LPS"
  },
  {
    "caption": "(b) Survival of Map1lc3b+/+ mice (n = 11) and Map1lc3b−/− mice (n = 10) after LPS challenge (100 mg per kg body weight), assessed over a period of 2 weeks.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) Anti-ubiquitin western blot in control and patient fibroblasts and total amount of proteins loaded (stainfree) showing increased ubiquitination in patient cells (lane 4).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(B) Histogram showing the quantification of ubiquitin with western blot assays. The data shown correspond to the sum of all bands detected by the anti-ubiquitin antibody expressed as a percentage of the amount of ubiquitin in "Control 1" cells. Bars show mean of ten independent experiments +/- SD (n=10, t-test *: p<0.01, **: p<0.05).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(B) Ten micrograms of control and patient cell lysates were tested for their chymotrypsin-like activity by incubating them with 0.1 mM of the Suc-LLVY-AMC substrate at 37°C over a 180-h period of time in quadruplicates on a 96-well plate. Indicated on the y-axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide. Bars show the mean of 4 independent experiments +/- SD.",
    "molecules": "AMC"
  },
  {
    "caption": "(D) Proteins extracted from control and CI PSMC3 were separated by 10 or 12.5% SDS-PAGE prior to western-blotting using primary antibodies directed against ubiquitin and several proteasome subunits and/or components including α6, β1, β; β5, β5i, Rpt2 (PSMC1), Rpt5 (PSMC3), Rpt3 (PSMC4), Rpt4 (PSMC6) and PA28-α, as indicated. For the PSMC3 staining, two exposure times are shown. Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant. Equal protein loading between samples was ensured by probing the membrane with an anti-α-Tubulin antibody.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "Control and patient (index case, IC PSMC3) fibroblasts were exposed to a 16-h treatment with 30 nM of the proteasome inhibitor carfilzomib or left untreated (as a negative control). Following treatment, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, β1i, PA28-α and α-Tubulin (loading control) as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.",
    "molecules": "carfilzomib, ubiquitin"
  },
  {
    "caption": "(A) Control and/or patient (index case, IC PSMC3) fibroblasts were subjected to a 24-h transfection with pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 prior to RIPA-mediated protein extraction and subsequent western-blotting using antibodies specific for ubiquitin, PSMC3 (i.e. Rpt5) and α-Tubulin (loading control).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(B) Densitometry analysis showing the relative ubiquitin contents detected by western blotting in patient fibroblasts exposed to either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3, as indicated. The y-axis represents the percent changes in densitometry measurements (of pixel intensities using Image J) which are set as 100 % for cells transfected with the pcDNA3.1/empty vector (mock) at 24 post-transfection (n=4, *: p<0.05, t-test). Bars show the mean +/- SEM.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(C) Patient (index case, IC PSMC3) fibroblasts transfected with either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 were exposed to a 30-nM treatment of carfilzomib or left untreated (as a negative control). After 16 hours, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, PA28-α and α-Tubulin (loading control), as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.",
    "molecules": "carfilzomib, ubiquitin"
  },
  {
    "caption": "(B-B'). Cataract detection revealed abnormal lens reflection in psmc3 morpholino (MO)-mediated knockdown but not in controls (uninj, ctrl-mo). Similarly, abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA injected embryos without Cas9 (sgRNA2). Co-injection of wt psmc3 mRNA with either psmc3-mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection. Scale bar = 50 µm. (B') Quantification of embryos with abnormal lens reflection.",
    "molecules": "MO, morpholino"
  },
  {
    "caption": "(D-D') bright field images of inner ear development (lateral position). (D) Epithelial projections were fused and formed canal pillars in 4-day-old uninjected and control injected fish (ctrl-mo, sgRNA2) but not in morphants (mo) and crispants (sgRNA2+Cas9). Co-injection of wt psmc3 mRNA with psmc3-mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype. Black asterisks indicate fused pillars. Red arrowheads mark unfused projections. Scale bar = 100 µm. (D') Quantification of embryos with abnormal projection outgrowth.",
    "molecules": "mo, morphants"
  },
  {
    "caption": "(E-E') An anti-acetylated tubulin antibody (green) staining revealed an abnormal amount of kinocilia in psmc3 crispants (sgRNA2+Cas9) compared to uninjected and control injected embryos (sgRNA2). Nuclei are stained in blue with DAPI. Representative images show kinocilia of the lateral cristae. Scale bar = 20 µm. (E') Quantification of embryos with an abnormal amount of kinocilia.",
    "molecules": "DAPI"
  },
  {
    "caption": "B: Double-IHC of coronal brain sections (40 μm) of a littermate (LM) and a NexCre cTKO mouse (age: 5 months) stained for Ctip2 (green, expressed in layer V/VI) and Calbindin (red, expressed in layer II/III and to some extent in layer V). Note that no gross abnormalities in cortical lamination were observed. Layers of DAPI stained cell nuclei are denoted on the right (I-VI), scale bar: 100 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C: Giemsa-stained, glycolmethacrylate-embedded coronal brain sections (bregma -1.9) displaying the hippocampus and adjacent callosal fiber tracts of a wildtype (WT, top), littermate control (LM, middle) and a NexCre cTKO (cTKO, bottom; age: 5-6 months). Note the agenesis of the corpus callosum (no CC) in NexCre cTKO mice, which should always be present at this bregma and in sections in which the medial habenula (mHb) is present (CC in WT and LM). While WTs and LMs show a compact layer of CA1 cells, NexCre cTKOs display a bilaminar cytoarchitecture (boxed regions marking higher magnification to the right) with scattered ectopic cells in the stratum radiatum (white arrowheads). In addition, the CA3 appears less compact (red arrowheads). Layer identity visualized via Calbindin staining (right panel, scale bar: 50 µm).",
    "molecules": "Giemsa, glycolmethacrylate"
  },
  {
    "caption": "(A) Annealed Fo−Fc electron density map for the C‐terminal region (residues 119-122) of LC3(1-124). Left, LC3(1-124) bound to the HsAtg4B H280A mutant; right, LC3(1-124) bound to the HsAtg4B C74S mutant. Structure of the C‐terminal region of LC3 is also shown as a stick model, in which carbon, nitrogen and oxygen atoms are coloured green, blue and red, respectively.",
    "molecules": "carbon, nitrogen, oxygen"
  },
  {
    "caption": "(B) Structure of the C‐terminal tail of LC3(1-124) and the catalytic site of HsAtg4B. Left, LC3(1-124) bound to HsAtg4B H280A mutant, right, LC3(1-124) bound to the HsAtg4B C74S mutant. HsAtg4B is shown as a ribbon model, whereas LC3 is shown as a stick model. The side chains of the residues comprising the catalytic triad and Tyr54 of HsAtg4B are also shown as a stick model. Carbon, nitrogen, oxygen and sulphur atoms are coloured green, blue, red and yellow, respectively. Mutated catalytic residues are coloured cyan.",
    "molecules": "Carbon, nitrogen, oxygen, sulphur"
  },
  {
    "caption": "(D) Chemical shift perturbations of the LC3 backbone amide groups upon complex formation with the N‐terminal‐tail peptide. The combined 1H and 15N chemical shift differences, calculated using the equation Δp.p.m.=[(ΔδHN)2+(ΔδN/5)2]1/2 were plotted against residue numbers.",
    "molecules": "15N"
  },
  {
    "caption": "(A) Inducible GFP‐S‐Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP‐S‐Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C) 293 cells were transfected with GFP‐S, GFP‐S‐Ubqln1 or GFP‐S‐Ubqln4 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(E) 293 cells were transfected with GFP‐S‐Ubqln4 WT, GFP‐S‐Ubqln4 ΔSTI1 12, GFP‐S‐Ubqln4 Δ between STI1 or GFP‐S‐Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.",
    "molecules": "chloroquine"
  },
  {
    "caption": "Relative cellular levels of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. Each colored datum shows the mean from three independent cultures.",
    "molecules": "fatty acids"
  },
  {
    "caption": "G. Relative cellular levels of lipid-containing fatty acids in MDA-MB-231 cells with knockout of KAR or TECR. The top curve plot indicates the fraction of the corresponding fatty acid. Each colored datum shows the mean from three independent cultures.",
    "molecules": "fatty acid, fatty acids, lipid"
  },
  {
    "caption": "H. Mass isotopomer analysis of fatty acids in MDA-MB-231 cells with knockout of KAR or TECR cultured with 10 mM of 13C6-glucose for 48 h. Error bars represent mean ± SD. Data are from three independent cultures.",
    "molecules": "13C6-glucose, fatty acids"
  },
  {
    "caption": "MDA-MB-231 Wt cells or ACC-DKO cells were pretreated for 24 h and then cultured in FBS medium or DFBS medium with the indicated treatment for 5 days. Treatments were as follows: z-VAD-FMK (z-VAD, 10 μM), necrostain-1 (Nec-1, 2 μM), Disulfiram (0.4 μM), ferrostain-1 (Ferr-1, 10 μM), Trolox (100 μM), N-Acetyl-L-cysteine (NAC, 3 mM), and deferoxamine mesylate (DFOM, 10 μM). Data are presented relative to the values obtained for the control cells cultured in FBS medium. Dotted line shows the survival level of cells in DFBS medium without treatment (control).",
    "molecules": "deferoxamine mesylate, DFOM, Disulfiram, Ferr-1, ferrostain-1, N-Acetyl-L-cysteine, NAC, Nec-1, necrostain-1, Trolox, z-VAD, z-VAD-FMK"
  },
  {
    "caption": "Relative cellular levels of ROS in MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement.",
    "molecules": "ROS, VLCFA"
  },
  {
    "caption": "The inhibitory effects of H2O2 and PMS on MDA-MB-231 ACC-DKO or Wt cells cultured in FBS medium for 24 h with indicated concentrations.",
    "molecules": "PMS, H2O2"
  },
  {
    "caption": "Annexin-V & Dead cell (7-AAD) flow cytometry analysis of MDA-MB-231/ACC-DKO and Wt cultured in DFBS medium. The right panel shows the quantification of annexin V-positive cells in the left panels.",
    "molecules": "7-AAD"
  },
  {
    "caption": "The transmembrane potential in mitochondria of MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis. The bar graphs show the percentage of JC-1 monomer.",
    "molecules": "JC-1"
  },
  {
    "caption": "MDA-MB-231/ACC-DKO and Wt cells were stained for Mito Tracker to visualize mitochondria. Scale bar, 10 μm. Mitochondrial morphology was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type.",
    "molecules": "Mito Tracker"
  },
  {
    "caption": "MDA-MB-231/ACC-DKO cells were in FBS medium with or without exogenous LCFA or VLCFA supplementation, and were stained for MitoTracker to visualize mitochondria. Scale bar, 10 μm.   Mitochondrial morphology in (D) was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type. Error bars represent mean ± SD, and data are from triplicate experiments.  ",
    "molecules": "LCFA, MitoTracker, VLCFA"
  },
  {
    "caption": "(A-C'') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-luc) and UAS-dpn and UAS-D; UAS-dpn are expressed in hs flp clones (72 hr after clone induction). Miranda (Mira, magenta) and EdU (grey). Despite increased number of Mira+ cells in UAS-dpn compared to control, each NB underwent slower cell cycle progression, as indicated by EdU+ incorporation. Quantified in E. This slow cell cycle speed was not rescued in UAS-D; UAS-dpn clones. Quantified with UAS-luc; UAS-dpn in F.",
    "molecules": "EdU"
  },
  {
    "caption": "F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.",
    "molecules": "Met, methionine"
  },
  {
    "caption": "D. Anthocyanin accumulation in seedlings Average and SEM are shown (n = 3).",
    "molecules": "Anthocyanin"
  },
  {
    "caption": "F. Yeast 3-hybrid analysis of the COP1-HY5 interaction in the presence of the UVR8HY5C44 chimera. (Left pane) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Right panel) Representative filter lift assays of the yeast analyzed in left panel. AD, activation domain; BD, DNA binding domain; Met, methionine.",
    "molecules": "Met, methionine"
  },
  {
    "caption": "E-H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected (E), 2 months after intraductal injection with MCF7, arrows point to undilated ductal tips (F), MM134, dotted line highlight the subtending duct (G), or SUM44 cells (H), (n>6) scale bar, 1 mm.",
    "molecules": "carmine alum"
  },
  {
    "caption": "E, F Representative micrographs of  IHC for Ki67, ER, and PR on histological  sections of MM134 (E) and SUM44  (F) xenografts 5 months after intraductal injection counter  stained  with hematoxylin. Scale bars, 200 μm. Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for >1000 cells counted on 3 glands.",
    "molecules": "hematoxylin"
  },
  {
    "caption": "D Representative RNAscope image of sections from patient ILCs (n=5) stained with anti-LOXL1  probes (red) and  counterstained with DAPI (blue). Scale bars; 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E Representative immunofluorescence image of primary ILC (n=3) stained  with anti-LOXL1 (green) and anti-ER (red) antibodies  counterstained with DAPI (blue). Scale bars; 50 μm and right inset 30 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E Radiance-based tumor growth curves (mean ± SE) for T47D treated with vehicle (n=8) or BAPN (n=12) 8 days after intraductal injections. n.s not significant.",
    "molecules": "BAPN"
  },
  {
    "caption": "F Radiance-based tumor growth curves for (mean ± SE) ILC ER+ PDXs T125 and T137 treated with vehicle (n=6, 7) or BAPN (n=7, 8) 66 days after engraftment. ** p < 0.01.",
    "molecules": "BAPN"
  },
  {
    "caption": "G Radiance-based tumor growth  curves (mean ± SE) for non-ILC ER+ PDXs T99 and T157 treated with vehicle (n=7, 7) or BAPN  (n=8, 12), 8 days after intraductal injection 3 weeks after intraductal injections. n.s not significant.",
    "molecules": "BAPN"
  },
  {
    "caption": "J, K Representative photomicrographs of picrosirius red stained  histological sections for sh-scramble or sh-LOXL1 MM134 (J) and SUM44 (K) xenografts. Scale bars, 100 μm.",
    "molecules": "picrosirius red"
  },
  {
    "caption": "(C-I) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control.",
    "molecules": "ATP, Coomassie Blue, LicoB, LPS, nigericin"
  },
  {
    "caption": "(A-C) BMDMs were primed with LPS and then treated with LicoB (20 μΜ) for 1 h, followed by stimulation with nigericin, ATP, poly (I:C), or MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then transfected with LPS. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs. (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured.",
    "molecules": "ATP, LicoB, LPS, MSU, Pam3CSK4, nigericin, poly (I:C)"
  },
  {
    "caption": "(D-F) LPS-primed BMDMs were treated with LicoB (20 μM) for 1 h and then stimulated with nigericin for 45 min, or poly(dA:dT)/Salmonella for 6 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (D). Caspase-1 activity (E) and IL-1β secretion (F) in the SN were measured.",
    "molecules": "LicoB, LPS, nigericin, poly(dA:dT)"
  },
  {
    "caption": "(A-C) LPS-primed BMDMs were pre-treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h. Western blot analysis was used to detect cross-linked ASC in the Triton X-insoluble pellet, the arrow indicates ASC (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured again to verify NLRP3 activation.",
    "molecules": "ATP, LicoB, LPS, Triton X"
  },
  {
    "caption": "(E) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, poly (dA:dT), or Salmonella.",
    "molecules": "LicoB, LPS, nigericin, poly (dA:dT), Triton X"
  },
  {
    "caption": "(A) Western blot analysis of protein in whole cell lysates (WCL) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h (LicoB after LPS), or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h (LicoB before LPS).",
    "molecules": "LicoB, LPS"
  },
  {
    "caption": "(I) Cell lysates of LPS-primed BMDMs treated with/without nigericin were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed using Western blot analysis. (J) Cell lysates of LPS-primed BMDMs were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 mM and 1 mM). The pull-down samples and input were analysed using Western blot.",
    "molecules": "sepharose, LicoB, LPS, nigericin"
  },
  {
    "caption": "(K) HEK-293T cells were transfected with Flag-NLRP3 or Flag-vector and then treated with LicoB (40 μM). Immunoprecipitation was performed with anti-DYKDDDDK (Flag) affinity gel agarose beads; Western blot analysis has been shown.",
    "molecules": "LicoB"
  },
  {
    "caption": "(A) Survival of WT mice pre-treated with vehicle, LicoB (20 mg/kg), LicoB (40 mg/kg), MCC950 (40 mg/kg), or LicoB (40 mg/kg) + MCC950 (40 mg/kg), followed by i.p. injection with LPS (20 mg/kg); mice survival was monitored for 72 h (n=10 mice per group).",
    "molecules": "LicoB, LPS, MCC950"
  },
  {
    "caption": "C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of ALT (H) (n=8 mice per group).",
    "molecules": "LicoB"
  },
  {
    "caption": "C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of AST (I), and CRE (J) were measured, (n=8 mice per group).",
    "molecules": "CRE, LicoB"
  },
  {
    "caption": "(A-D) Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with MSU (50 mg/kg) and treated for 6 h (n=6 mice per group). The levels of IL-1β in the peritoneal lavage fluid (A) and serum (B) were measured using ELISA. Quantification of peritoneal exudate cells (PECs) (C) and neutrophils (Ly6G and CD11b) (D) using flow cytometry.",
    "molecules": "LicoB, MCC950, MSU"
  },
  {
    "caption": "Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950, or a combination of LicoB and MCC950 (n=8 mice per group (B, C (A) Representative liver section images stained with H&E, Sirius Red, and Masson's trichrome stains. Scale bar: 0.5 cm (top row) and 200 μm (3 bottom ows). (B, C) The serum levels of ALT and AST (n=8 mice per group) were measured using ELISA.",
    "molecules": "Masson's trichrome, Sirius Red, choline, LicoB, MCC950, methionine"
  },
  {
    "caption": "Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of Col1a1 (E), TNF-α (F) in the mice livers, as described in (A) (n=6 mice per group).",
    "molecules": "choline, LicoB, MCC950, methionine"
  },
  {
    "caption": "Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of IL-1β (G), and IL-18 (H) in the mice livers, as described in (A) (n=6 mice per group).",
    "molecules": "choline, LicoB, MCC950, methionine"
  },
  {
    "caption": "B GFP+ EBs derived from CT and RA cultures at day 10; scale bar: 100 μm.",
    "molecules": "RA"
  },
  {
    "caption": "C Representative FC plots depicting percentage of GFP+ cells obtained at day 15, from CT and RA cultures in a typical experiment.",
    "molecules": "RA"
  },
  {
    "caption": "D Heat map demonstrating enrichment of cardiac genes in GFP+ fractions (CT+, RA+) compared to GFP− fractions (CT−, RA−) at day 31.",
    "molecules": "RA"
  },
  {
    "caption": "E, F Heat map of a select list of genes (E) upregulated and (F) downregulated in RA+ compared to CT+ at day 31. Fold change > 2.",
    "molecules": "RA"
  },
  {
    "caption": "A-C qPCR of selected transcripts at day 31 to validate (A) enrichment of cardiac markers in GFP+ fractions against GFP− fractions, (B) upregulation of atrial and (C) downregulation of ventricular genes in RA+ compared to CT+ (n = 3).Data information: Data are presented as mean ± SEM. In (A-C), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), for TNNT2, P = 0.0001 for CT− against CT+ and P = 0.0002 for RA− against RA+; for NKX2.5, P = 0.00005 for CT− against CT+ and P = 0.00007 for RA− against RA+. In (B), P = 0.0006 for NPPA and P = 0.0002 for PITX2. In (C), P = 0.02 for HEY2 and P = 0.007 for IRX4.",
    "molecules": "RA"
  },
  {
    "caption": "B Representative APs of day 31 CMs from control (CT) and RA-treated (RA) groups at 1 Hz.",
    "molecules": "RA"
  },
  {
    "caption": "C-E RMP, APAmax and APAplat (C), dV/dtmax (D) and APD20, APD50 and APD90 of CT and RA CMs (E).F Plot showing all measured APAplat values of CT and RA CMs.",
    "molecules": "RA"
  },
  {
    "caption": "G Representative APs of CT and RA CMs at 0.5-4 Hz.H Average APAplat at 0.5-4 Hz. Please note that the AP differences in morphology are present at all measured frequencies.",
    "molecules": "RA"
  },
  {
    "caption": "B, C Typical examples (left) and current-voltage relationships (right) of (B) IKur and (C) IK,ACh in VM and AMCMs.Data information: Data are presented as mean ± SEM. In (A), *P < 0.05 by unpaired t-test. In (B, C), *P < 0.05 by two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (B) and Mann-Whitney rank-sum test for (C). In (B), P = 0.778, 0.350, 0.03, 0.02, 0.002, 0.001, < 0.001 and < 0.001, respectively, for comparison between VM and AM within membrane potentials of −20, −10, 0, 10, 20, 30, 40 and 50 mV. In (C), P = 0.01, 0.01, 0.01, 0.01, 0.03, 0.397, 0.397, 0.397, 0.671, 0.207, 0.207, 0.09, 0.01, 0.039 and 0.015, respectively, for comparison between VM and AM within membrane potentials of −120, −110, −100, −90, −80, −70, −60, −50, −40, −30, −20, −10, 0, 10 and 20 mV. CMs = cardiomyocytes; hESC-atrial (AM) and hESC-ventricular (VM) CMs; IK,ACh = acetylcholine-activated potassium current; IKur = potassium ultra-rapid delayed rectifier current. 4-AP = 4-aminopyridine; CCh = carbachol.",
    "molecules": "4-aminopyridine, 4-AP, carbachol, CCh"
  },
  {
    "caption": "D, E Representative APs of VM and AM at 1 Hz in response to (D) IKur block by 4-AP and (E) IK,ACh activation by CCh. AP parameters are shown in Supplementary Table S5.",
    "molecules": "4-AP, CCh"
  },
  {
    "caption": "A Representative APs at 1 Hz of VM and AM CMs in response to vernakalant. Inset shows dV/dtmax.B, C Average APAplat (B) and dV/dtmax (C)in the absence and presence of vernakalant at 1-4 Hz. AP parameters are shown in Supplementary Table S6. Abbreviations as in Figs3 and 6.Data information: Data are presented as mean ± SEM. *P < 0.05 by Mann-Whitney rank-sum test for (B). P = 0.01, 0.008, 0.008 and 0.007, respectively, for comparison of APAplat between VM and AM groups at frequencies of 1.0, 2.0, 3.0 and 4.0 Hz. Two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (C). P = 0.686 between VM and AM groups and hence not statistically significant. For VM, P = 0.06 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.001 for 2 versus 3 Hz; P = 0.002 for 2 versus 4 Hz; and P = 0.857 for 3 versus 4 Hz. For AM, P = 0.03 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.02 for 2 versus 3 Hz; P = 0.02 for 2 versus 4 Hz; and P = 0.621 for 3 versus 4 Hz.",
    "molecules": "vernakalant"
  },
  {
    "caption": "A Representative APs of VM and AM CMs in the absence, presence and following washout of 3 μmol/l XEN-D0101. AP parameters are shown in Supplementary Table S7.",
    "molecules": "XEN-D0101"
  },
  {
    "caption": "B Representative APs (1 Hz) of VM and AM in the CCh, to activate IK,ACh and subsequent addition of XEN-R0703. AP parameters are shown in Supplementary Table S9.",
    "molecules": "XEN-R0703, CCh"
  },
  {
    "caption": "C, D Experiments performed in RAP conscious dogs in the presence of vehicle or following 1, 3 and 10 mg/kg XEN-R0703 show (C) mean right AERP values (left), mean Van de Water's QTc (right) and (D) AF inducibility plotted as a function of dose.Data information: For RAP dog experiments, n = 5; statistical significance tested with paired t-test. Data are presented as mean ± SEM. AERP = atrial effective refractory period; AF = atrial fibrillation; RAP = rapid atrial pacing; N.S. = not significant. Other abbreviations as in Figs3, 6 and 7.",
    "molecules": "XEN-R0703"
  },
  {
    "caption": "(A) Schematic indicating the imaging area in the tail  at 72hpf, as indicated by the black box; fluorescence imaging of the CHT  in double transgenic cd45:CFP-NTR/runx1:mcherry embryos  in DMSO  and after treatment with MTZ  and PGH2  or PGE2. (B) Quantification of runx1:mcherry  positive-cells (C) Quantification of cd45:CFP  positive-cells in double transgenic cd45:CFP-NTR/runx1:mcherry embryos  in DMSO  and after treatment with MTZ  and PGH2 or PGE2. Data information: Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001 ****P<.0001. Scale bar 200μm (A).",
    "molecules": "DMSO, MTZ, PGE2, PGH2"
  },
  {
    "caption": "(A) Anti-GFP  and pH3 immunostainings  of either controls (Ctrl-Mo) or slco2b1-morphants (slco2b1-MO) cmyb:GFP embryos. (B) Quantification of the number of the pH3+ HSCs  in controls or slco2b1-morphants. Centre values denote the mean, and error values denote s.e.m, statistical analysis was completed using an unpaired two tailed t test. ***P < .001. Data information: Scale bar 50μm (A)",
    "molecules": "Mo, MO"
  },
  {
    "caption": "(D) Quantification of PGE2   concentration in control and slco2b1   morphants. The statistical analysis was completed using an unpaired two tailed t test *P < .01. Centre values denote the mean, and error bars denote s.e.m.",
    "molecules": "PGE2"
  },
  {
    "caption": "(E) Fluorescence imaging in the CHT  of cmyb:GFP embryos  injected with control- and slco2b1-MOs  and treated with PGE2. (F) Quantification of GFP  positive cells. Statistical analysis: one-way ANOVA, multiple comparison, *P < .01; ****P < .0001. Centre values denote the mean, and error bars denote s.e.m. Data information: Scale bar 200μm (E).",
    "molecules": "MOs, PGE2"
  },
  {
    "caption": "(A) Fluorescence imaging of the CHT  of double transgenic cd45:CFP-NTR/runx1:mcherry embryos  injected with control- or slco2b1-morpholinos, after treatment with DMSO. (B) Fluorescence imaging of the CHT  of double transgenic cd45:CFP-NTR/runx1:mcherry embryos  injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ. (C) Fluorescence imaging of the CHT  of double transgenic cd45:CFP-NTR/runx1:mcherry embryos  injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ+PGH2. (D) Quantification of runx1:mcherry  positive-cells. Each dot represents the number of red cells for each embryo  (biological replicates). Each experiment has been repeated three independent times. (E) Quantification of cd45:CFP  positive-cells. Each dot represents the number of blue cells for each embryo  (biological replicates). Each experiment has been repeated three independent times.Data information for (D) and (E): Statistical analysis was completed using one-way ANOVA, multiple comparison test. ****P<.0001. Centre values denote the mean, and error bars denote s.e.m Scale bar 200μm (A-C).",
    "molecules": "DMSO, MTZ, PGH2"
  },
  {
    "caption": "(A) Immunohistochemistry  on paraffin  sections for cKit  (HSPCs  marker) and CD31  (endothelial cell  marker) at E13 and E16 stages of mouse fetal liver. (B) Distance distribution between cKithigh cells and endothelial cells (n=79 from 3 E13  fetal livers; n=92 from 3 E16  fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test. *P<.01; **P<.001; ***P<.0001. Centre values denote the mean, and error values denote s.e.m All nuclei  marked with DAPI. Scale bar 20μm",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Immunohistochemistry  on paraffin  sections for cKit  (HSPCs  marker) and F4/80 (macrophage cell marker) at E13 and E16 stages of mouse fetal liver. (D) Distance distribution between cKithigh cells and macrophages (n=54 from 3 E13 fetal livers; n=64 from 3 E16 fetal livers), binned into 5μm intervals. Statistical analysis was completed using t-test. All nuclei  marked with DAPI. Scale bar 20μm",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Immunohistochemistry  on paraffin  sections for cKit  (HSPCs  marker) and MBP (neutrophil  cell marker) at E13 and E16 stages of mouse fetal liver. (F) Distance distribution between  cKithigh cells and neutrophils  (n=45 from 3 E13 fetal livers; n=53 from 3 E16  fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test ***P<.001. All nuclei  marked with DAPI. Scale bar 20μm",
    "molecules": "DAPI"
  },
  {
    "caption": "Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Fluorescence images of tissue after microinjection with dextran-A555 (magenta). The tissue was stained for Tbr2 (green) and TuJI (white). Microinjected cell at 0 h; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2. Two-daughter cell progeny 24 h after microinjection; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2; the bottom cell is a newborn BP positive for Tbr2. Both cells are negative for the neuronal marker TuJI (white). Four-daughter cell progeny 48 h after microinjection; note that daughter 1 and 2 reside in the VZ, while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ. Distribution of microinjected cells and their progeny in the VZ, SVZ and CP (cortical plate). n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface (0 µm) and expressed as % of total. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.",
    "molecules": "A555, Dextran, dextran"
  },
  {
    "caption": "Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Expression of Tbr2 in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Expression of TuJI in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.",
    "molecules": "A555, Dextran"
  },
  {
    "caption": "Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dx-A488 along with mRNA for RFP Organotypic slices of mouse E14.5 dorsal telencephalon injected with Dextran-A488 (green) and RFP poly-A+-mRNA. After 24h in culture the progeny expresses RFP (magenta; blue: DAPI, n = 17 cells injected, 8 RFP positive). Percentage of RFP-positive and RFP-negative cells (n = 17 cells injected).",
    "molecules": "A488, DAPI, Dextran, Dx"
  },
  {
    "caption": "Automated microinjection was performed on organotypic slices of mouse E14.5 - E15.5 dorsal telencephalon using a solution containing Dextran-A555 (not shown) and Alexa488 (green). Slices were fixed after microinjection (n = 71 cells total) and were stained for Tbr2 (white, H-J). (A-C) Representative examples of a non-coupled cell (A, cartoon on the right), a 2-cell cluster (B, cartoon on the right) and a 5-cell cluster (C, cartoon on the right). The asterisks indicate the Dx-A555-positive, microinjected cell. Percentage of microinjected cells found in a coupled cluster (coupled, green). Cluster size, expressed as % of total. Distribution of coupled cells expressed as the distance from the ventricular surface (0 µm = ventricle surface. Distribution of coupled cells divided into 11 bins and expressed as % of total. the images on the left are maximum intensity projections (MIP) of 18, 17, 23, 12 and 30-focal planes, respectively",
    "molecules": "Alexa488, A555, Dextran, Dx"
  },
  {
    "caption": "A representative picture of a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2. Microinjected cells are stained for Tbr2. A representative picture of (H) a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2) and (I) a 7-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 7), the latter containing one Tbr2 positive cell. Microinjected cells are stained for Tbr2. The asterisks indicate the Dx-A555-positive, microinjected cell. comparison of Tbr2-positive cells (expressed as % of total) among all the cells in the VZ (VZ; n=1003 from 4 different confocal images from 3 independent experiments; error bar represents standard deviation) and in coupled clusters in the VZ (coupled clusters, VZ; n= 104 from two independent experiments). Coupled cells were scored based one presence/absence of apical and/or basal polarity cues, and results are expressed as % of total. the images on the right are single optical sections corresponding to the nucleus (white dotted line); scale bars are 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.",
    "molecules": "A555, Dx"
  },
  {
    "caption": "Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 dorsal telencephalon using Dx-A555 Phase contrast image of automated microinjection into mouse neurons. Scale bar is 100 µm.",
    "molecules": "A555, Dx"
  },
  {
    "caption": "Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection and stained for Reelin (white, C) Left: overview image of a mouse microinjected area (arrow indicates a pyramidal neuron, arrowhead indicates a Cajal-Retzius neuron; image is MIP of 20 focal planes). Microinjected neurons show different morphology and positivity for the Cajal-Retzius marker Reelin (white). Right, top row: the pyramidal neuron indicated with an arrow in the overview image is negative for Reelin. Right, bottom row: the Cajal-Retzius neuron indicated with an arrowhead in the overview image is positive for Reelin. Images on the top and bottom row left (LY MIP) are a MIP of 2 and 3 focal planes, respectively. The three images on the top and bottom right (LY, Reelin and DAPI) are single optical sections. Scale bar is 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.",
    "molecules": "A555, DAPI, Dx, LY"
  },
  {
    "caption": "Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green Slices were fixed after microinjection and stained for SatB2 (white, Overview image of a mouse microinjected area. The arrow indicates a pyramidal neuron; the image is MIP of 30 focal planes, scale bar: 50 µm. Row on the right: the microinjected neuron with pyramidal morphology indicated with an arrow in the overview is positive for the upper-layers marker SatB2 (white). Images are single focal planes. Scale bar is 10 µm. Proportion of neurons positive or negative for Reelin (n = 12 cells total). Proportion of neurons positive or negative for SatB2 (n = 39 cells in total). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.",
    "molecules": "A555, Dx, LY"
  },
  {
    "caption": "Automated microinjection of human (G,H) neurons. Automated microinjection was performed on organotypic slices of human 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection Microinjected neurons in the developing human telencephalon. Top, MIP of 20 focal planes (scale bar: 50µm). Bottom: high magnification (scale bar: 10 µm). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.",
    "molecules": "A555, Dx, LY"
  },
  {
    "caption": "(A) PKH26 (PE)-labelled RM1 (V500/eCFP) cells were injected intracardiac (IC) into C57BL/6 mice and FACS-isolated from bones with evident tumor burden (8 individual mice across 5 independent experiments) from day 16 onward and individual dormant and proliferating cells were isolated for scRNA-seq. Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH+ (PE/V500) and PKH- (V500) RM1 cells and bone marrow cells (grey).",
    "molecules": "PE, PKH, PKH26, V500"
  },
  {
    "caption": "(B) goana gene ontology (GO) analysis (limma) of all DE genes enriched in proliferating (PKH-; n = 32) cells compared to dormant cells (PKH+; n = 28). Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.",
    "molecules": "PKH"
  },
  {
    "caption": "(C) goana GO analysis (limma) of all DE genes uniquely enriched in PKH+ compared to PKH- cells. Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.",
    "molecules": "PKH"
  },
  {
    "caption": "(B) ELISA of IFN-α production by RM1 parental (n = 3), RM1 bone-derived Irf low (RM1 BD Irf-; n = 4) and RM1 BD REV cells (n = 3) subsequent to poly I:C stimulation.",
    "molecules": "poly I:C"
  },
  {
    "caption": "(C) qRT-PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf- cells ± 48 h treatment with MS275 (1 μM) (n = 7-9).",
    "molecules": "MS275"
  },
  {
    "caption": "(G) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co-culture with naïve BM ± MS275 (1 μM) (n = 3-6 per condition).",
    "molecules": "MS275"
  },
  {
    "caption": "(C) ELISA of IFN-α production by RM1 parental, RM1 BD Irf- base vector (BV) and RM1 BD Irf7 over-expressing (OE) with 24 h poly I:C stimulation (n = 4).",
    "molecules": "poly I:C"
  },
  {
    "caption": "(D) Bone metastasis-free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf- BV (n = 9) and RM1 BD Irf7 OE (n = 7) cells (**p = 0.0028 by log-rank (Mantel-Cox) test).",
    "molecules": "IC"
  },
  {
    "caption": "(F) Immunohistochemical (IHC) staining for cerulean (anti-eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE tumor-bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor-bearing bones derived from Ifnar1 -/- animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G-I) FACS analysis of dormant (claret+) RM1 BD Irf- and RM1 BD Irf7 OE cells from bone at (G) day 11 post-IC injection (n = 4 per group) and (h) day 17 (n = 4-6 mice group/time point) with (I) D17 active (claret-) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation (n = 9 bones from 3 mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits.",
    "molecules": "IC"
  },
  {
    "caption": "(A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6).",
    "molecules": "MS275, poly I:C"
  },
  {
    "caption": "(B) Mean fluorescence intensity of H2-Kb staining on RM1 BD Irf- cells by FACS ± MS275, poly I:C or combination treatment (n = 3).",
    "molecules": "MS275, poly I:C"
  },
  {
    "caption": "(C) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1)",
    "molecules": "MS275, poly I:C"
  },
  {
    "caption": "FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown.",
    "molecules": "MS275, poly I:C"
  },
  {
    "caption": "(E) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 BD Irf- cells ± MS275, poly I:C or combination treatment (n = 6 per group; log-rank (Mantel-Cox) test).",
    "molecules": "MS275, poly I:C"
  },
  {
    "caption": "B) FACS analysis of FoxP3+ CD4+ and CD4+ effector memory T cell status at days 4 and 10 post-IC tumor cell inoculation across treatment settings (n = 4-6 per group).",
    "molecules": "IC"
  },
  {
    "caption": "D) post-IC inoculation with associated specific CD8+ memory T cell response. This is represented by FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation upon re-stimulation with RM1 BD Irf- cells and MS275, poly I:C or combination treatments in vitro, with NAC and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1). Representative FACS plots shown on right.",
    "molecules": "MS275, IC, poly I:C"
  },
  {
    "caption": "(E) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated. IRF9 indicated by brown (DAB) staining. Scale bar represents 100 μm. Full-face slides shown in Appendix Fig S7C.",
    "molecules": "DAB"
  },
  {
    "caption": "(G) CIBERSORT analysis of absolute leukocyte proportions (arbitrary units, AU) in mRNA samples of bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). p values by Mann-Whitney U.",
    "molecules": "mRNA"
  },
  {
    "caption": "(H) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.",
    "molecules": "mRNA"
  },
  {
    "caption": "(C). Time course analysis of Xist RNA expression by RT-qPCR during EB differentiation of Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) cells at the indicated timepoints. Rif1+/+ (solid line) and Rif1-/- (dashed line), female (black) and male (grey). Data are presented as mean ± standard deviation from three (female lines) or two (male lines) independent experiments. Statistical significance was determined using 2-way ANOVA comparing female Rif1+/+ to female Rif1-/- cell lines (****p ≤ 0.0001). Xist RT-primers Xist ex7 F and R were used. Values are normalised to a geometric mean consisting of the expression of Gapdh, Ubiquitin and β-Actin.",
    "molecules": "OHT, Ubiquitin"
  },
  {
    "caption": "RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, in ESCs (A) P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant.",
    "molecules": "OHT"
  },
  {
    "caption": "(F). Quantification by ChIP-qPCR of RIF1 association with the indicated regions in the Fa2L cell line, following treatment with DMSO only (black) or flavopiridol (grey). Primers as in (E). (G). Same as in (F) but for a wild type female mESC line. All enrichments are presented relative to input DNA. Mean ±standard deviation from three (F) and two (G) independent experiments are presented. Statistical significance was determined using Student's two-tailed, paired t test (*p ≤ 0.05, **p ≤ 0.01 and ns=not significant).",
    "molecules": "flavopiridol, DMSO"
  },
  {
    "caption": "(A). Time course analysis of Xist expression by RT-qPCR during EB differentiation of female mESCs following knock down of Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Data are presented as mean ±standard deviation from three independent experiments. Statistical significance was determined using 2-way ANOVA. Xist primers Xist ex3 F and Xist ex4 R were used. Normalisation was performed using a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (*p ≤ 0.05). (B). RT-qPCR analysis of Xist expression levels during differentiation of the Fa2L cells, following expression of shRNA against Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Mean ±standard deviation from a minimum of three independent experiments is presented. 2-way ANOVA was used to determine statistical significance. ns=not significant. (",
    "molecules": "Ubiquitin"
  },
  {
    "caption": "(C). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha.",
    "molecules": "Ubiquitin"
  },
  {
    "caption": "(A). ChIP-qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs (Rif1+/+, same as used in Fig. 3B but without OHT) and during early differentiation. ZFP629 is a well-characterised KAP1 associated region (positive control). Xite A and C indicate two regions within the Tsix enhancer Xite, Tsix region 1 indicates Tsix major promoter, Tsix region 2 indicates the Dxpas34 region, Tsix region 3 indicates a region slightly downstream of the Dxpas34 region. P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 is an intergenic region. See Appendix Fig. S2C for the positions of the primers within Xist and Tsix. The data are presented as mean ±standard deviation from three (2d EB and 1d EB) and two (ESCs) independent experiments. Statistical significance was calculated by Student's two-tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB (*p ≤ 0.05 and ns=not significant). In the inset, Tsix RNA levels were quantified by RT-qPCR during the differentiation of wild type female ESCs shown in Fig. 4F. The average of two experiments is shown. Tsix values are normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. Error bars indicate standard deviations.",
    "molecules": "OHT, Ubiquitin"
  },
  {
    "caption": "(C). KAP1 association with Xist promoter in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female mESC cell lines. Ezr is an additional region known to be associated with KAP1 in mESCs. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments per cell line are displayed. Statistical significance was determined using Student's two-tailed, unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1+/+ versus Rif1-/- cells (**p ≤ 0.01).",
    "molecules": "OHT"
  },
  {
    "caption": "(D) Immunoblots of Aurkb and Aurka in total lung lysates of normal (CCSP/-) and fibrotic (CCSP/TGFα) mice fed with Dox for 6wks. Quantification was performed using phosphor imager software and normalization was done using loading control GAPDH. *P < 0.05, unpaired t-test, (n=4).",
    "molecules": "Dox"
  },
  {
    "caption": "(E) Immunostaining was performed using AURKB antibody in lung sections of control (CCSP/-) and TGFα (CCSP/TGFα) mice on Dox for 6wks (n=6). Representative images were obtained at 20X (low; Scale bar: 100µm) and 63X (high; Scale bar: 30µm) magnification. Dashed box indicates area of the section showing in high magnification.",
    "molecules": "Dox"
  },
  {
    "caption": "(F) Western blot analysis of Aurkb in total lung lysates from saline and bleomycin treated mice. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. **P < 0.005, unpaired t-test, (n=4-5).",
    "molecules": "bleomycin"
  },
  {
    "caption": "(B) Lung-resident fibroblasts from TGFα mice on Dox for 4 wks were transiently transfected with control or WT1 siRNA for 72hrs and AURKB transcripts were quantified. ****P < 0.00005, unpaired t-test, (n=4).",
    "molecules": "Dox"
  },
  {
    "caption": "(E) Primary lung-resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 wks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter-specific PCR primers. Non-immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t-test, (n=2).",
    "molecules": "Dox"
  },
  {
    "caption": "(C) Lung sections from control and TGFα mice fed with Dox food for 4 wks were stained for AURKB (green) and Ki-67(red). Merged image shows cells that co-express AURKB and Ki-67 (yellow).",
    "molecules": "Dox"
  },
  {
    "caption": "(F) Proliferation was measured in primary lung-resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 wks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20ng/mL) for 24hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).",
    "molecules": "Dox"
  },
  {
    "caption": "(H) Lung sections from control and TGFα mice fed with Dox food for 6 wks were stained for AURKB (green) and αSMA (red). Images were obtained at 40X magnification. Scale bar: 50µm. (n=4).",
    "molecules": "Dox"
  },
  {
    "caption": "(I) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in lung-resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 wks and treated with control or AURKB siRNA for 72h. **P < 0.005, ****P < 0.00005, 2-way ANOVA, (n=4).",
    "molecules": "Dox"
  },
  {
    "caption": "(A) Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48hrs. ****P < 0.00005, 1-way ANOVA, (n=9-11).",
    "molecules": "barasertib"
  },
  {
    "caption": "(B) Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48hrs. ***P < 0.0005, 1-way ANOVA, (n=9-11).",
    "molecules": "barasertib"
  },
  {
    "caption": "(C) Primary lung resident fibroblasts isolated from TGFα mice on Dox for 4 wks were treated with vehicle or 5µM barasertib for 48hrs and immunostained using PCNA antibody. Images were obtained at 40X magnification. Scale bar: 50µm. The number of PCNA-positive cells and total DAPI-positive cells were quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI-positive cells. ****P < 0.00005, unpaired t-test, (n=3-4).",
    "molecules": "barasertib, DAPI, Dox"
  },
  {
    "caption": "(D) Proliferation was measured in primary fibroblasts treated with TGFα (20ng/mL) and indicated doses of barasertib for 48hrs.. **P<0.005, 1-way ANOVA, (n=9-11).",
    "molecules": "barasertib"
  },
  {
    "caption": "(E) Quantification of apoptotic cells using Incucyte ZOOM (Caspase-3/7- positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5µM Barasertib. ****P < 0.00005, 2-way ANOVA, (n=4).",
    "molecules": "Barasertib"
  },
  {
    "caption": "(B) Representative images of Masson's trichrome-stained lung sections from the vehicle and barasertib treated mice. Images were obtained at 10X magnification. Scale bar: 200μm.",
    "molecules": "barasertib"
  },
  {
    "caption": "(C) Quantification of right lung weight of mice treated with vehicle or barasertib. ***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8-10 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(D) Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=8-10 mice/group).",
    "molecules": "barasertib, hydroxyproline"
  },
  {
    "caption": "(E) Quantification of Col1α, Col5α and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib.***P< 0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(A) Lung sections from vehicle and barasertib treated mice were immunostained using Ki-67 antibody. Representative images were obtained at 20X magnification. Scale bar: 50μm.",
    "molecules": "barasertib"
  },
  {
    "caption": "(B) Immunoblot analysis in lung lysates from vehicle and barasertib treated mice using PCNA antibody. Gapdh is used as loading control. **P< 0.005, 1-way ANOVA, (n=4-5 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(C) Quantification of Aurkb, Plk1 and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=8 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(B) Quantification of right lung weight of mice treated with vehicle and barasertib. **P< 0.005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(C) Western blot analysis in lung lysates from vehicle and barasertib treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P< 0.05, **P<0.005, ***P < 0.0005, 1-way ANOVA, (n=4-5 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(D) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.",
    "molecules": "barasertib"
  },
  {
    "caption": "(E) Quantification of lung mechanics in mice treated with vehicle and barasertib. *P<0.05, **P< 0.005, ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=9-10 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(B) Representative images of Masson's trichrome stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10X magnification. Scale bar: 200μm.",
    "molecules": "barasertib"
  },
  {
    "caption": "(C) Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P< 0.05, ***P < 0.0005, 1-way ANOVA, (n=7-8 mice/group).",
    "molecules": "barasertib, hydroxyproline"
  },
  {
    "caption": "(D) Quantification of Col1α1, Col3α, Col5α, Col14, Col15, αSma, Ccna2, and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib. *P< 0.05, **P<0.005 ***P<0.0005, ****P < 0.00005, 1-way ANOVA, (n=6 mice/group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(E) Quantification of αSma protein levels in lung lysates from vehicle and barasertib treated mice. Gapdh is used as loading control. Data are presented as mean ± SEM. **P<0.005, unpaired t-test (n=5mice /group).",
    "molecules": "barasertib"
  },
  {
    "caption": "(A) Biotinylation of SMO by SAR1B-BirA* (asterisk denotes the R118G mutation in BirA) in a proximity-dependent biotinylation assay. The cells were transfected with plasmids encoding SMO and SAR1B-BirA*-FLAG. After 48 h, cells were treated with different doses of biotin for 4 h. Cells were harvested and lysates were dialyzed to remove free biotin. Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis. (B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.",
    "molecules": "biotin"
  },
  {
    "caption": "(C) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A.",
    "molecules": "biotin, GDP, GTP"
  },
  {
    "caption": "(C) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A. (D) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A. (E) The Shh increased the binding of SAR1B to SMO. After transfection for 24 h, cells were incubated with the ShhN conditioned medium for an additional 24 h. Then 15 μM biotin was added into the culture medium for 4 h. The cells were harvested and analyzed as described in Fig. 2A.",
    "molecules": "biotin, GDP, GTP"
  },
  {
    "caption": "(D) In vitro budding of SMO. Membranes were prepared and incubated in vitro in the presence of cytosol, ATP, GTP, and an ATP-regenerating system as described in experimental procedures. Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis.",
    "molecules": "ATP, GTP"
  },
  {
    "caption": "(C) The CP-modified SMO was mainly resistant to Endo H. The CP-modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃. Then proteins were eluted with SDS-PAGE sample buffer and were subjected to immunoblotting analysis. Results shown are representative of two independent experiments.",
    "molecules": "streptavidin"
  },
  {
    "caption": "ALOD4 inhibited the cholesterol modification of SMO Cells were treated with ALOD4 for 1 h, and then incubated with a medium containing ShhN and CP for 2 and 3 h. Cells were harvested and analyzed",
    "molecules": "cholesterol"
  },
  {
    "caption": "ALOD4 inhibited the cholesterol modification of SMO(4A). Cells were treated with ALOD4 for 1 h In C, ShhN and CP added for 2 h. Cells were harvested and analyzed",
    "molecules": "cholesterol"
  },
  {
    "caption": "(A, B) GRAMD1B increased cholesterylation of SMO. HEK293T cells were co-transfected with the SMO or SMO (4A) and GRAMD1B protein expression plasmids for 24 h, and then incubated with a cholesterol depletion medium (DMEM supplemented with 5% lipoprotein-deficient serum, 1 μM lovastatin and 10 μM mevalonate) for 12 h, followed by 2 μg/mL of CP treatment. The cells were harvested and analyzed as Results shown are representative of two independent experiments.",
    "molecules": "cholesterol, lipoprotein, lovastatin, mevalonate"
  },
  {
    "caption": "(C, D) GRAMD1B promoted SMO maturation. The expression plasmids of SMO or SMO(D95E) and GRAMD1B were transfected into SMO-KO HEK293T cells. After 12 h, cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD-cholesterol for 8 h. Black bracket indicates mature glycosylated form.",
    "molecules": "cholesterol, MβCD"
  },
  {
    "caption": "(E) Expression of Gli1 mRNAs measured by qRT-PCR in NIH3T3 cells stably expressing GRAMD1B. After incubating in serum starvation medium for 16 h, cells were depleted of cholesterol by treatment with 1.5% HPCD for 0.5 h and then incubated in serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 10 μM MβCD-cholesterol, ShhN as indicated for 24 h.",
    "molecules": "cholesterol, HPCD, lovastatin, MβCD, mevalonate"
  },
  {
    "caption": "(F) Confocal images of NIH3T3 cells stably expressing mCherry-GRAMD1B. PM was labeled with wheat germ agglutinin (WGA)-CF®488A conjugate. Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD-cholesterol for 1 h. Cells were then treated with or without ShhN conditioned medium for another 2 h. Results shown are representative of three independent experiments.",
    "molecules": "cholesterol, MβCD"
  },
  {
    "caption": "(A) The cholesterol modification of endogenous SMO was decreased in NIH3T3 cells lacking Gramd1a, 1b, 1c, 2, and 3 (Gramd-5KO). The cells were incubated in a cholesterol depletion medium for 12 h, followed by 2 μg/mL of CP and ShhN for an additional 12h.",
    "molecules": "cholesterol"
  },
  {
    "caption": "(I) The GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 cells. The cells were incubated in cholesterol depletion medium for 16 h and then treated with 3 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 2 μg/mL of CP for an additional 2 h.",
    "molecules": "AI3d, cholesterol"
  },
  {
    "caption": "(J) The GRAMD inhibitor AI3d decreased the mRNA levels of Gli1 in NIH3T3 cells. Cells were incubated in serum starvation medium for 16 h, depleted of cholesterol by 1.5% HPCD for 0.5 h, and then treated with serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 1 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 10 μM MβCD-cholesterol for an additional 16",
    "molecules": "AI3d, cholesterol, HPCD, lovastatin, MβCD, mevalonate"
  },
  {
    "caption": "(A‑B) BMDCs were plated at different densities and stimulated with poly(I:C) for 24h or left unstimulated. DC activation was evaluated based on surface expression of CD86 and CD40 molecules as measured by flow cytometry. (A) Representative dot plots showing CD86 and CD40 expression. (B) Quantification of CD86 and CD40 upregulation as a fold change in geometric mean fluorescence intensity (gMFI) over unstimulated controls. Data are pooled from 3 independent experiments.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(A) IFN‑α (left) and IFN‑β (right) production by WT and Irf3-/-Irf7-/- BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) (blue dots) or left unstimulated (grey dots). Dotted lines represent the ELISA threshold of detection. Data are representative of 2 independent experiments.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(B) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3-/-Irf7-/- (orange dots) BMDCs plated at high density (2x105 cells/cm2) and stimulated with poly(I:C) or left unstimulated. Data are pooled from 2 independent experiments. (C) CD86 and CD40 expression on Irf3-/-Irf7-/- BMDCs plated at 2x105 cells/cm2 and stimulated with poly(I:C) (dark blue dots), IFN-α (light blue dots) or a combination of both (green dots). Data are representative of 2 independent experiments.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(A) CD86 and CD40 expression was quantified on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs in non‑draining (ndLN) and draining (dLN) popliteal lymph nodes of WT and Irf3‑/‑Irf7‑/‑ mice injected with poly(I:C) or PBS. Data are pooled data from 2 independent experiments.",
    "molecules": "PBS, poly(I:C)"
  },
  {
    "caption": "(D) Representative dot plots of CD86 and CD40 expression on WT (blue dots) and Irf3‑/‑Irf7‑/‑ (orange dots) DCs of chimeras injected with PBS or poly(I:C).",
    "molecules": "PBS, poly(I:C)"
  },
  {
    "caption": "(B) Representative dot plots showing CD86 and CD40 expression by low‑density BMDCs stimulated with poly(I:C) for different periods of IFN‑α exposure.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "Monocyte recruitment in the draining lymph node requires collective DC activation (A) Representative dot plots showing the percentage of monocytes (CD11b+ Ly6Chigh cells, black box) present in popliteal dLN of WT:Irf3‑/‑Irf7‑/‑ chimeric mice injected with poly(I:C) (middle and right) or with PBS (left). CD11b+ Ly6Clow/int cells corresponded to neutrophils and were not included in the analysis.",
    "molecules": "PBS, poly(I:C)"
  },
  {
    "caption": "Collective DC activation is essential for the generation of T cell responses. (G) Representative dot plots showing the percentage of CD44high tetramer H‑2Kb‑OVA+ CD8+ T cells in the draining lymph node of chimeric mice injected with PBS or a mixture of OVA+poly(I:C).",
    "molecules": "PBS, poly(I:C)"
  },
  {
    "caption": "Collective DC activation is essential for the generation of T cell responses. (H) Flow cytometric quantification of CD44high H‑2Kb‑OVA tetramer+ CD8+ T cells in the draining lymph node of 90:10 and 5:95 (WT:Irf3‑/‑Irf7‑/‑) chimeric mice injected with PBS or OVA+poly(I:C). Data are pooled from 2 independent experiments.",
    "molecules": "PBS, poly(I:C)"
  },
  {
    "caption": "b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3)P and PtdIns(4)P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.",
    "molecules": "lysophosphatidic acid, lysophosphatidylcholine, phosphatidic acid, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns, sphingosine 1-phosphate"
  },
  {
    "caption": "(c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units.",
    "molecules": "phosphoinositides"
  },
  {
    "caption": "(d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left).",
    "molecules": "PtdIns"
  },
  {
    "caption": "(g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST-Hrs-FYVE and GST-FAPP1-PH are also shown.",
    "molecules": "phosphoinositides"
  },
  {
    "caption": "(a-k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP-Man II (green, f, medial-Golgi), endogenous TGN46 (red, g, trans-Golgi), and overexpressed PtdIns(4)P probe, GFP-FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d., n = 150 cells obtained by gathering data from three independent experiments); **P0.01; ***P0.001.",
    "molecules": "PtdIns(4)P"
  },
  {
    "caption": "(b) Wortmannin treatment releases UVRAG from the ER. HeLa cells were treated with 100 nM wortmannin for 1h, and processed for confocal microscopy using anti-UVRAG and anti-PDI antibodies (top panel). UT, untreated. Insets highlight the relative localization of UVRAG at the ER. Confocal co-localization is indicated (bottom left panel). Data are mean±s.d.; n = 75 cells for each group from three independent experiments; ***P0.001. Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting (bottom right panel).",
    "molecules": "Wortmannin, wortmannin"
  },
  {
    "caption": "(c) UVRAG interaction with bothPtdIns(3)P and RINT-1 is required for COPI-dependent retrograde transport, as shown by the redistribution of VSVG-KDELR from the Golgi to the ER. HeLa cells were transfected with control shRNA or UVRAG shRNA. The UVRAG-depleted cells were then complemented with empty vector, Flag-UVRAG, UVRAGΔ270−442, UVRAGK78A/R82A or UVRAGΔC2, along with the transfection of VSVG-KDELR-YFP. The ER pattern for the chimaeric KDELR was quantified (left panel). Data are the mean±s.d.; n = 200 cells obtained from three independent experiments; *P0.05; **P0.01. Endogenous and reconstituted UVRAG expression was confirmed by immunoblotting (right panel).",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": "(d) PtdIns(3)Pdepletion inhibits Golgi-to-ER retrograde transport of VSVG-KDELR. HeLa cells were transfected with empty vector, wild-type MTMR3, the C413S mutant of MTMR3, or treated with wortmannin or rapamycin, and then the ER pattern of VSVG-KDELR was quantified. UT, untreated. Data are the mean±s.d.; n = 100 cells for each group obtained from three independent experiments; **P0.01.",
    "molecules": "PtdIns(3)P, rapamycin, wortmannin"
  },
  {
    "caption": "e) Interaction between endogenous UVRAG and the ER tethering complex under wortmannin treatment. WCLs of HEK293T cells treated with wortmannin were used for immunoprecipitation with anti-UVRAG or anti-RINT-1 antibody, followed by immunoblotting with the indicated antibodies. The input panel shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.",
    "molecules": "wortmannin"
  },
  {
    "caption": "(d,e) UVRAG interaction with PtdIns(3)P and RINT-1 is required or Golgi integrity. (d) HeLa cells were transfected with control shRNA (first row) or UVRAG shRNA (second to sixth rows). The UVRAG-depleted cells, as indicated by Flag expression, were then transfected with empty vector (second row), UVRAGK78A/R82A-UVRAG (third row), UVRAGΔ270−442mutant (fourth row), UVRAGK78A/R82A mutant (fifth row), or UVRAGΔCCD mutant (sixth row), followed by confocal microscopy using anti-GM130 (red) for cis-Golgi staining, anti-Flag (purple) for the ectopically expressed UVRAG protein expression, and UVRAG for nuclei (blue). Asterisks indicate shRNA-transfected cells and arrows denote the cells reconstituted with GM130 (wild-type or mutant) expression. (e) The dispersed distribution of GM130 was quantified, and data represent the mean±s.d.; n = 200 cells from four independent experiments; *P0.05; **P0.01.",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": "(a,b) Differential interaction of UVRAG with the RINT-1-ZW10-NAG tethering complex and the beclin-1-Bif-1-PI(3)KC3 autophagy complex after treatment with rapamycin (a) and HBSS (b). Cells (HEK293T) treated with rapamycin (50 nM) in a or with HBSS in b were subjected to immunoprecipitation with anti-UVRAG (first panel), anti-RINT-1 (second panel) or anti-PI(3)KC3 (third panel), followed by immunoblotting with the indicated antibodies. Input (10%) shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(c) Gel filtration analysis of UVRAG complex formation under normal conditions (untreated, UT) and after rapamycin-induced autophagy (50 nM). Affinity-purified UVRAG complexes from HCT116 cells stably expressing Flag-UVRAG were fractionated by Superose-6 gel filtration column and the eluates were analysed by western blotting for UVRAG, RINT-1, ZW10, beclin 1, PI(3)KC3 and Bif-1 proteins. Whole-cell lysates (2.5%) were used as the input (lane-1). The elution profile of each protein was quantified by densitometry analysis and normalized. Relative densitometry units were plotted against fraction number. Black arrows indicate the positions of the molecular weight size markers. Red arrows indicate the peak shift of the UVRAG eluates.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a-c) UVRAG interaction with beclin 1 and PtdIns(3)P, but not with RINT-1, is required for autophagy-induced Atg9 dispersal. (a) HeLa cells were stably transfected with control shRNA (first row) or UVRAG shRNA (second-sixth rows). The UVRAG-depleted cells were then transfected with empty vector (second row), Flag-UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row) or UVRAGΔCCD mutant (sixth row), and incubated in complete medium (untreated, UT, first panel), starvation medium (HBSS 2 h, third panel), or treated with rapamycin (50 nM 2 h, second panel) or SMER28 (50 μM, 2 h, fourth panel). The distribution pattern of endogenous Atg9 (green) in the UVRAG (wild-type or mutant)-transduced cells (red) was analysed by confocal microscopy (a). Asterisks denote condensed (non-dispersed) Atg9 in UVRAG-depleted HeLa cells and arrows denote the cells complemented with wild-type or mutant UVRAG.",
    "molecules": "SMER28, PtdIns(3)P, rapamycin"
  },
  {
    "caption": "(d) Effect of knockdown of UVRAG, beclin 1 andRINT-1 on the translocation of Atg9 to the LC3-labelled autophagsomes during autophagy. Control-, UVRAG-, BECN1- or RINT-1-knockdown HeLa cells were incubated in complete medium (normal condition), starvation medium (HBSS, 2 h), or treated with rapamycin (100 nM, 2 h) or SMER28 (50 μM, 2 h). The cells were then stained for endogenous Atg9 (green) and LC3 (red) followed by confocal microscopy analysis (top and bottom left panels). The percentage of Atg9 vesicles co-localized with LC3+-autophagic puncta was quantified (bottom middle panel). Endogenous UVRAG, beclin 1 and RINT-1 protein expression are shown (bottom right). Data represent mean±s.d.; n = 200 cells obtained from three independent experiments. NS, not significant; *P0.05; **P0.01; ***P0.001. Scale bars, 10 μm.",
    "molecules": "SMER28, rapamycin"
  },
  {
    "caption": "H-J. Hematoxylin and eosin staining from tail sections of wounded Fbln7 WT vs. KO mice (H) and quantitation of the re-epithelialization length (I) and thickness at the healing front (J). Scale bar: 200 μm. N=3 WT and 5 KO mice.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "(C) Immunoblots  analysis of TOMM34  expression in parental and metformin  adaptive HCC   cells.",
    "molecules": "metformin"
  },
  {
    "caption": "(G) The morphology  (Left), migration  (Middle) and invasion  (Right) of metformin  adaptive Huh7  cells with (shTOM) or without (shNC) knockdown of TOMM34  are shown (5×104 cells for  Transwell assay). Scale bars, (Left) 25 μm, (Middle) and (Right) 100 μm. (n=3 biological replicates, Two-way ANOVA). Data information: Data are presented as means ± S.D.",
    "molecules": "metformin"
  },
  {
    "caption": "(F-G) Western blot  showing protein level of ATP5B  in Huh7  (F) or PLC/PRF/5 (G) cells treated with or without 10 mg/mL CHX  for 12 hours or 25 μM MG132  for 8 hours. Relative grey value of ATP5B  was calculated using Image J (shTOM: shTOMM34, TOMM34  knockdown cells; OE-TOM: OE-TOMM34, TOMM34  overexpressed cells; CHX, cyclohexane). (n=3 technical replicates, Student's t-test). Data information: Data are presented as means ± S.D.",
    "molecules": "CHX, cyclohexane, MG132"
  },
  {
    "caption": "(A-B) Co-IP  assay was performed to detect the interaction  between TOMM34  and ATP5B  treated with or without Gboxin  for 24 hours in parental or adaptive PLC/PRF/5 cells.",
    "molecules": "Gboxin"
  },
  {
    "caption": "(C-D) Immunoblotting  of EMT markers of HCC  cells treated with or without 200 nM Gboxin  for 24 hours (shTOM: shTOMM34, TOMM34  knockdown cells; OE-TOM: OE-TOMM34, TOMM34  overexpressed cells).",
    "molecules": "Gboxin"
  },
  {
    "caption": "(E-F) Transwell assay  showing the migration  and invasion  of HCC  cells treated with or without 200 nM Gboxin  for 36 hours (5×104 cells; shTOM: shTOMM34, TOMM34  knockdown cells). Scale bars, 100 μm. (n=3 biological replicates, two-way ANOVA). Data information: Data are presented as means ± S.D.",
    "molecules": "Gboxin"
  },
  {
    "caption": "(B) Two-color 3D  DNA-FISH  analysis of ex vivo sorted pro-B cells of the indicated genotypes with the RP23-340K14 (red) and RP24-275L15  (green) BAC probes (Fig 1A). Representative images are show above. Dot plots (below) show the distances measured between the two DNA  signals in individual Igh alleles (2,123 for Rag2-/-, 1,514 for Igh∆890/∆890 Rag2-/- and 502 for Vav-Cre Pax5fl/fl (Pax5∆/∆) Rag2-/-pro-B cells) together with the mean distance (red bar) and SEM determined for each genotype. In total, four independent DNA-FISH experiments were performed. Data information: Statistical data were analyzed by multiple t-tests (unpaired one-way ANOVA (Tukey post-hoc test; B); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Representative fluorescence micrographs from the indicated cell lines co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(F) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co-stained with indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(B) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(E) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(A) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h. A fraction of DHCB treated cells were released into fresh medium for other 24h (release). Samples were subjected to immunoblotting; n = 3 independent experiments.",
    "molecules": "DHCB, DMSO"
  },
  {
    "caption": "(B) Fluorescence micrographs of RPE1 cells of the indicated genotypes, either treated for 24h with DHCB or with vehicle alone (DMSO). Blow-up without Hoechst 33342 is magnified 2.5X. Scale bar: 5 μm. (C) Immunofluorescence micrographs of RPE1 as in (B) were used to visually assess the percentage of cells presenting one or two nuclei. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of binucleated cells between the wild type sample and all the other genotypes was assessed (ANOVA test; *P < 0.05). (D) Immunofluorescence micrographs of RPE1 as in (B) were used to visual score the number of centrosomes per cell by counting the number of γ-tubulin-positive centrioles. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of cells with > 2 centrosomes between the wild type sample and all the other genotypes was assessed (ANOVA test; **P < 0.01). ",
    "molecules": "Hoechst 33342, DHCB, DMSO"
  },
  {
    "caption": "(E) Quantitative assessment of the fraction of RPE1 cells undergoing cytokinesis failure upon DHCB treatment, inferred on the basis of the increase of ploidies ≥ 4C (upper panel), and of the fraction of the abovementioned cells undergoing genome reduplication upon release after DHCB treatment (lower panel). Individual values of biological replicates, their mean and standard deviations are reported.",
    "molecules": "DHCB"
  },
  {
    "caption": "(F) RPE1 cells were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated Myc-ANKRD26 constructs. Subsequently, cells were treated either with DMSO or ZM447439 for 24h and subjected to immunoblotting; n = 3 independent experiments.",
    "molecules": "DMSO, ZM447439"
  },
  {
    "caption": "(C) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced (mock), or transduced with PIDD1-V5 lentiviral vectors and co- stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(F) Representative fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors expressing the PIDD1 non-cleavable derivative (PIDD1S446A-S588A) or truncations thereof. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(A) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1-V5 in its wild type form. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.",
    "molecules": "DMSO, ZM447439"
  },
  {
    "caption": "(B,C) A549 cells of the indicated genotypes were either left untransduced (mock), or transduced with lentiviral vectors expressing PIDD1-V5 in its wild type form or carrying the indicated point mutations. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.",
    "molecules": "DMSO, ZM447439"
  },
  {
    "caption": "(D) Fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors carrying the indicated point mutations, and stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(A) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1-GFP. Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(C) Fluorescence micrographs of RPE1 cells stably expressing CETN1-GFP treated either with DMSO or with DHCB for 24h. Blow-ups 33342 are magnified 2.5X. Scale bar: 5 μm. (D) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in (C). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition, a.u. = arbitrary units. ",
    "molecules": "33342, DHCB, DMSO"
  },
  {
    "caption": "(E) Movie stills of a representative RPE1 cell stably expressing CETN1-GFP treated with DHCB and subjected to time-lapse video microscopy in the presence of SiR-DNA. Time is expressed in minutes, relative to anaphase onset. The dashed line indicates the plasma membrane of the cell of interest, arrowheads indicate the centrosomes' position.",
    "molecules": "SiR-DNA, DHCB"
  },
  {
    "caption": "(F) Centrosomal distance over time in RPE1 cells stably expressing CETN1-GFP and treated with DHCB. Time zero corresponds to the frame preceding anaphase onset. Coloured dots (lower panel) summarize the clustering time for each cell, mean ± standard deviation in black. Data calculated from four-dimensional imaging as in (E). N = 10 cells.",
    "molecules": "DHCB"
  },
  {
    "caption": "(A) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments (ZM = ZM447439; REV = reversine). Nocodazole concentrations are 0.03 μM, 0.1 μM, 0.33 μM, 1 μM, 3.3 μM. Median (red) and 95% confidence interval thereof (black) are shown. N > 50 cells were analysed. (B) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in (A). N = 3 independent experiments. (C) DNA content analysis of A549 cells subjected to the indicated treatments as in (A) either for 24h (left panels) or 48h (right panels). N = 2 independent experiments. ",
    "molecules": "Nocodazole, REV, reversine, ZM, ZM447439"
  },
  {
    "caption": "(F) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2X. Scale bar: 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(A) A549 cells of the indicated genotypes were treated for 24h as indicated (CPT = camptothecin, ZM = ZM447439). Samples were subjected to immunoblotting; n = 3 independent experiments. (B) A549 cells treated as in (A) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ-tubulin-positive centrioles per cell. N = 3, ≥ 50 cells from each independent experiment.",
    "molecules": "camptothecin, CPT, ZM, ZM447439"
  },
  {
    "caption": "(C) RT-qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. The average fold of induction ± standard deviation is shown. N = 3 biological replicates with two technical replicates each. Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type (WT) cells",
    "molecules": "Nutlin 3a"
  },
  {
    "caption": "with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. (D) Immunoblot analysis of samples treated N = 3 independent experiments.",
    "molecules": "Nutlin 3a"
  },
  {
    "caption": "C) ThT binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.",
    "molecules": "ThT"
  },
  {
    "caption": "D) pFTAA binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.",
    "molecules": "pFTAA"
  },
  {
    "caption": "D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.",
    "molecules": "NaCl"
  },
  {
    "caption": "A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots.",
    "molecules": "cellulose"
  },
  {
    "caption": "Morphological changes of vacuolar membranes and peroxisomal GFP-SKL during microautophagic degradation of peroxisomes after glucose adaptation. Double fluorescence of methanol-grown cells of P. pastoris cells at various times after the switch to glucose medium. (a) 0 h, (b) 30 min, (c) 2 h.",
    "molecules": "glucose, methanol"
  },
  {
    "caption": "Kinetics of morphological changes in vacuoles and peroxisomalGFP-SKL during microautophagic degradation of P. pastoris in glucose adaptation. At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point. Cells were counted in three randomly selected fields, and standard deviations are shown as vertical bars.",
    "molecules": "glucose"
  },
  {
    "caption": "Macroautophagic degradation of peroxisomes after transfer of methanol-grown P. pastoris cells to ethanol medium. (a-e) Electron micrographs of intermediates seen during macroautophagy. (a) After 1 h of ethanol adaptation, peroxisomes (P) were often surrounded by autophagosomal membranes, higher magnifications of which are in d and e. (b) After 1 h of ethanol adaptation, the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane. (c) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole. Magnified pictures of samples obtained at the (d) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes, and at the (e) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes. (f) Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL. Other conditions are described in Materials and Methods, and are the same as in Fig. 4. (g) Fluorescence images taken after 2 h of ethanol adaptation of wild-type P. pastoris cells.",
    "molecules": "ethanol, methanol"
  },
  {
    "caption": "Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation. Wortmannin and PMSF were dissolved in DMSO, and added to the glucose medium. (Filled circle) Control (2% DMSO); (open circle) 1 mM wortmannin (with 2% DMSO); (filled square) 3 mM PMSF (with 2% DMSO); (filled triangle) 10 mg/ml cycloheximide.",
    "molecules": "cycloheximide, DMSO, ethanol, glucose, PMSF, Wortmannin, wortmannin"
  },
  {
    "caption": "Microautophagic degradation of peroxisomes is not observed in a vacuolar protease-negative strain (STK7) of P. pastoris. Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL during (a) glucose adaptation and (b) ethanol adaptation. (c) Fluorescence images taken after 2 h of glucose adaptation. Arrows show GFP-SKL in vacuolar matrix. Some cells showed diffusion of GFP-SKL into the vacuolar matrix.",
    "molecules": "ethanol, glucose"
  },
  {
    "caption": "(a) Visualization of alcohol oxidase activity in colonies after (a) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild-type (strain PPY12) and pag mutant cells (strains STK1-STK6, Materials and Methods) of P. pastoris. (b) Western-blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time. Antibodies used were AOX, alcohol oxidase; F1, mitochondrial F1β subunit; G6PDH, glucose-6-phosphate dehydrogenase. Growth curve of the wild-type strain PPY12 and the pag4 mutant (strain STK4) after shift from methanol to glucose.",
    "molecules": "ethanol, glucose, methanol"
  },
  {
    "caption": "Fluorescence images of (a) pag2 (strain STK2) and (b) pag3 (strain STK3) mutants of P. pastoris after a 3 h shift to glucose. (b) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant.",
    "molecules": "glucose"
  },
  {
    "caption": "Fluorescence images of the pag4 (strain STK4) mutant of P. pastoris after (a) 0, (b) 3, or (c) 6 h of glucose adaptation. The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation.",
    "molecules": "glucose"
  },
  {
    "caption": "Microautophagy of peroxisomes in a pex1-ts mutant (strain SJH242) of P. pastoris expressing GFP-SKL from the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter at the restrictive temperature for peroxisome assembly (34°C). (a) Kinetics of peroxisome degradation at 34°C. Strain SJH242 was induced on methanol at 23°C, and glucose adaptation was performed at 34°C. (b) Changes in vacuole morphology were seen even in the absence of normal peroxisomes. At 0 h, SJH242 cells were grown at 34°C when GFP-SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies. After 2 h, ∼20-30% vacuoles showed complicated membrane structures representative of stage 2 cells.",
    "molecules": "glucose, methanol"
  },
  {
    "caption": "The central axial sections of the reconstructions. Note the C-terminal domain of gp104 and other low-density features that do not visualize well in the isosurface representation (panels (A-C)). Letters G, H and I indicate the position of the cross-sections shown in the homonymously labeled panels below. A 69 Å-thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis. The proximal and distal parts of one of the three fibers that run at 82° to the sixfold axis are indicated with a semitransparent cyan and magenta lines, respectively. Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate/proximal part of the fiber plane, whose position in the reconstructions is indicated with a red line in panels (E and F). The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol.",
    "molecules": "urea"
  },
  {
    "caption": "(A) Left quadriceps muscle biopsy performed at age 10 months (i-iii) demonstrating a dystrophic process with variation in fiber size (black arrow), increased connective tissue (blue arrow) and rare internalized nuclei (white arrow) on hematoxylin and eosin (H&E) staining (i) and Gömöritrichrome staining (ii) and type 1 fiber atrophy on nicotinamide adenine dinucleotide (NADH) staining (iii). Muscle biopsy in an unaffected (control) individual (iv-vi). Scale bar: 100μm.",
    "molecules": "Gömöritrichrome, eosin, hematoxylin, NADH, nicotinamide adenine dinucleotide"
  },
  {
    "caption": "(D) HeLa-cells stably expressing the pC4-reporter were transfected with control siRNA (ctrl) or two independent siRNAs (#1 and #2) directed against BET1 (siRNA #1 is shown in figure EV 2B). 2d post-transfection the pC4-secretion assay was performed for 0 and 10 min post induction of secretion. Representative images are shown (upper panel ctrl and lower panel #2 siRNA, respectively. Scale bar: 20 µm. Right panels represent magnifications of dashed areas.",
    "molecules": "pC4"
  },
  {
    "caption": "(B) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas. Parameter occupancy could be more than 100% if several stable hydrogen bonds could be established by residue. Bars represent average fraction of the simulated time the residue is involved in a hydrogen bond(s). Data represented are mean ± SD.",
    "molecules": "hydrogen bond, hydrogen bonds, Asp68, Asp72"
  },
  {
    "caption": "(A) Volcano plot of AP-MS data using HA-BET1 or Ile51Ser.",
    "molecules": "Ile51, Ser"
  },
  {
    "caption": "MCF-10A cells incubated in the absence or presence of puromycin (200μM) were fractionated on sucrose gradients, and distribution of CIP2A-BP transcripts was quantified by qPCR.",
    "molecules": "puromycin, sucrose"
  },
  {
    "caption": "TNBC cells were pretreated with specific antagonist against Smad3 (SIS3, 3μM) for 1h and then cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.",
    "molecules": "SIS3"
  },
  {
    "caption": "Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A. Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245).",
    "molecules": "amino acid"
  },
  {
    "caption": "The indicated cells were pretreated with specific antagonist against AKT (MK-2206，3μM) for 1h. Then whole lysates of these cells were subjected to immunoblot analysis.",
    "molecules": "MK-2206"
  },
  {
    "caption": "(a) HeLa.Vec and HeLa.UVRAG cells transfected with HA-Vps16 and GFP-LC3 were treated with 2 μM rapamycin for 2 h, and processed for confocal microscopy (left panel; scale bars, 5 μm). The percentage of GFP-LC3 punctae positive for HA-Vps16 staining was quantified (right panel; data are mean ± s.e.m., n = 60, *P 0.01).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a) UVRAG enhances the colocalization efficiency of GFP-LC3 with LAMP1. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-LC3 were either untreated or treated with rapamycin (2 μM) in the absence or presence of bafilomycin A1 (0.1 μM) and stained for LAMP1. Insets highlight the colocalization. The percentage of LAMP1-positive autophagosomes was calculated in each setting (data are mean ± s.e.m., n = 200; 5 independent experiments, **P 0.01; *P 0.05).",
    "molecules": "bafilomycin A1, rapamycin"
  },
  {
    "caption": "(c) UVRAG promotes autophagic degradation of long-lived proteins. HeLa.Vec and HeLa.UVRAG cells were incubated for 16 h with L-3H-Leu (1 μCi ml-1). The degradation of long-lived proteins was measured at the indicated time points in complete medium (NC), EBSS alone (Starv.), or EBSS + 0.1 μM bafilomycin A1 (Starv.+BalfA1). Results (data are mean ± s.e.m. of triplicates) are representative of 2 independent experiments. Scale bars, 5 μm.",
    "molecules": "L-3H-Leu, bafilomycin A1, proteins"
  },
  {
    "caption": "(a) Colocalization of GFP-LC3 and LAMP1. HeLa cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 and incubated under normal conditions or treated with rapamycin (2 μM). Autophagosome fusion with LAMP+ structures was analysed by confocal microscopy (left panel; rapamycin-treated) and quantified (right upper panel; data are mean ± s.e.m., n = 100). Arrows and insets show the LAMP1+ autophagosomes. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope (right bottom panel; data are mean ± s.e.m., n = 60, *P 0.05; **P 0.0001).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-Rab7 and RFP-LC3 were maintained either under normal conditions (NC) or treated with rapamycin, followed by confocal microscopy. Insets highlight the Rab7 acquisition of autophagosomes. The percentage of Rab7+-autophagosomes was quantified (right panel; data are mean ± s.e.m., n = 60, ***P 0.001; **P 0.01). Scale bars, 5 μm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b) Rab7 GTPase activity is increased with UVRAG expression. At 48 h post-transfection with GFP-Rab7 together with vector, UVRAG or UVRAGΔN(1-147) vector, 293T WCLs were used for immunoprecipitation with anti-GFP, followed by the Rab7 GTPase activity assay. Left panel: Autoradiographs of the GTP hydrolysis products analysed by TLC. Right panel: Quantification of the percentage of GTP hydrolyzed by Rab7 (data are mean n = 2 independent experiments).",
    "molecules": "GTP"
  },
  {
    "caption": "B. ARPI (CSS, ENZA, ODM-201, 14449) decreased, while R1881 increased, SDHA and SDHB subunit protein levels in both LNCaP and LAPC4 cells. PSA is used as a positive control.",
    "molecules": "R1881, 14449, ODM-201, ENZA"
  },
  {
    "caption": "D.  SDH repression during acute phase of ENZA stress increased transcription of different AR-regulated genes without altering AR mRNAs levels in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using two tailed unpaired Student's t-test *, p<0.05 and **, p<0.01 compared between groups.",
    "molecules": "ENZA, enzalutamide"
  },
  {
    "caption": "E-F. Overexpression of SDHA and SDHB subunits decreased AR protein levels (E) and AR transactivation (F) in LNCaP cells. Data information: ENZA: enzalutamide.Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey's test *, p<0.05 and **, p<0.01 compared between groups.",
    "molecules": "ENZA, enzalutamide"
  },
  {
    "caption": "C.  SDH-repressed LNCaP cells were able to maintain higher proliferation rate particularly during ENZA stress with or without Hsp27 silencing in real time cell proliferation assay. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels C (end points) . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups",
    "molecules": "ENZA, enzalutamide"
  },
  {
    "caption": "E. Treatment with p-p38 inhibitors: SB203580 (10 µM) or Genistein (20 µM) for 24 h reduced AR upregulation induced by SDH repression in LNCaP cells (left panel). Bar graph in the right panel shows band intensity normalized to loading control. Data information: ENZA: enzalutamide. Data shown as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA following Tukey's test for panels E . *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups",
    "molecules": "ENZA, enzalutamide, Genistein, SB203580"
  },
  {
    "caption": "F.. Treatment with 5 µM STO-609 for 24 h in LNCaP cells inhibited AR upregulation induced by SDHA/SDHB subunit silencing.",
    "molecules": "STO-609"
  },
  {
    "caption": "G. Thapsigargin, DMM, DMS and ENZA treatment for the indicated time points increased intracellular calcium flux in LNCaP cells as visualized with calcium sensitive Fluo4-AM probe under confocal microscope.",
    "molecules": "calcium, DMS, DMM, ENZA, Fluo4-AM, Thapsigargin"
  },
  {
    "caption": "A-C.  Human patient samples demonstrate relative differences in the immunoexpression of AR, SDHA, p-CaMKK2/p-AMPK, p-p38, and p-Hsp27 in untreated (n=70), neoadjuvant-treated (NHT) (n=130) and CRPC (n=24) samples. Representative  images (A), dot plots (B), compiled bar graph (C) support the AR-SDH loop where inhibition of AR  axis in NHT samples reduced SDHA levels along with upregulation of p-CaMKK2/p-AMPK/p-p38/p-Hsp27 axis. AR-positive CRPC samples restored SDHA as well as the whole axis. Data information: Data shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. **, p<0.01; ***, p<0.001 and ****, p<0.0001. Scale bar: 100µm. All the TMAs were constructed from minimum 2 cores per patient. ",
    "molecules": "neoadjuvant, NHT"
  },
  {
    "caption": "D. IVM reversed the  cell proliferation benefit mediated by SDH repression in LNCaP cells. Data shown as means ± SEM In vitro data represent three independent experiments. Statistical analysis was performed for the last data point *, p<0.05, **, p<0.01 and ***, p<0.001 compared between groups, one-way ANOVA followed by Tukey's test.",
    "molecules": "IVM"
  },
  {
    "caption": "E.  LNCaP xenografts treated with IVM (n=10) (10mg/kg) demonstrated lower serum PSA values (upper panel) and growth of tumours after castration (lower panel) compared to vehicle (n=10). PSA was measured every week. Data shown as means ± SEM",
    "molecules": "IVM"
  },
  {
    "caption": "F. IVM treatment reduced p-Hsp27 levels and therefore AR protein level, with subsequent p-CAMKK2 axis activation in castrated LNCaP xenografts. Bar graph in the right panel shows quantification of data from western blot normalized to loading control. Data shown as means ± SD",
    "molecules": "IVM"
  },
  {
    "caption": "(O-Q"') NR2F1 (red) and SOX2 (green in O-P') or BRN3a (green in Q-Q''') IF on sagittal sections of human GW14 eyes indicating high NR2F1 expression in virtually all NR progenitors (O''',P,P'), differentiating RGCs (Q-Q''') and in the majority of ON astrocytic progenitors (O'). Nuclei counterstaining (blue) was obtained with DAPI. Scale bars 100µm for human sections",
    "molecules": "DAPI"
  },
  {
    "caption": "Q-Q") Cross-sections of DiI-labelled P0 ONs in WT and KO pups",
    "molecules": "DiI"
  },
  {
    "caption": "(S) Quantification of the surface occupied by Tuj1+ or DiI+ fibers in different genotypes and ages, as indicated. data are represented as mean ± SEM; N=4-5 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.",
    "molecules": "DiI"
  },
  {
    "caption": "(V) Histogram quantifying the number of RGC apoptotic cells in WT, HET and KO from E18.5 to P5. Note the significant increase of dying cells at E18.5. Nuclei (blue) were stained with DAPI. data are represented as mean ± SEM; N=3-4 Student t-test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Histograms quantifying the average number of Pax2+ (G,H) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm",
    "molecules": "DAPI"
  },
  {
    "caption": "Histograms quantifying the average number of NF1A+ (I) cells in WT, HET and KO at different stages and axial length of the ON, as indicated. Astrocytic progenitors are significantly increased along the nerve of mutants. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(J,K) NF1A (red, astrocyte precursors) IF on P7 WT and KO ON cross-sections illustrating a high astrocytic density in KO ON (delineated by thin dashed lines). (L) Histogram confirming the increased percentage of NF1A+ astrocytes in HET and KO ONs compared to WT. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(T) Graph quantifying the average number of Sox2+ astrocytes per P28 ON section in KO and WT animals. data are represented as means ± SEM; N=3-5. Statistical significance was obtained by Student t-test (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(D-J) MBP (yellow, fully differentiated oligodendrocytes) IF on cross-sections of WT, HET and KO P7 optic nerves (ONs) at the chiasm (D-F) and between the chiasm and the eye (G-I). Note the dramatic decrease of MBP+ oligodendrocytes at the chiasm in HET and KO, quantified in (J). The ratio of MBP+ cells is calculated over the total DAPI+ cell number per chiasmal/ON section. data are represented as means ± SEM. N=4-5 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(K-N) EM thin sections of P8 WT, HET and KO ONs depicting myelin as a dense, dark staining around axonal fibers illustrate a significantly lower number of myelinated fibers in HET and KO, as quantified in (N). Higher magnification images (insets) showing different degrees of myelination in HET and KO ONs. data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm (4µm)",
    "molecules": "DAPI"
  },
  {
    "caption": "P28 WT and HET confirming persistent hypomyelination in mutants at P28, as quantified in (Q). data are represented as means ± SEM. N=3 Statistical significance was obtained by Student t-test or by two-way ANOVA when comparing 2 or multiple conditions, respectively (*P<0.05; **P<0.01; ***P<0.001). Nuclei (blue) were stained with DAPI. Scale bars: 50µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Experimental design to treat HET pups from P2 to P8 with Miconazole. DMSO (controls) and Miconazole-treated pups were sacrificed at P8 or at P28 to evaluate the ON myelination. (B-E) Sox10 (red, oligodendrocyte precursors) and MBP (green, fully differentiated oligodendrocytes) IF on cross-sections of P8 HET ONs after 6 days of treatment with DMSO (D) or Miconazole (E). Miconazole increased the number of Sox10+ cells compared to HETs treated with DMSO (quantified in B) and rescued MBP staining pixel intensity of HET pups to level comparable to those of WT pups (quantified in C). Nuclei (blue) were stained with DAPI. , the error bars represent the SEM of the means; N=3 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm",
    "molecules": "DAPI, DMSO, Miconazole"
  },
  {
    "caption": "(F-I) Sox10 (oligodendrocyte marker, red) and Phospho-ERK (dually phosphorylated forms of active ERK1 and ERK2, green) IF in WT and HET ONs, treated with DMSO (F) or Miconazole (G) between P2 and P8. As previously reported, Miconazole strongly activate ERK signaling pathway (G). Signal intensity quantification in whole ON area is shown in (H), while specific evaluation of pixel intensity around Sox10+ nuclei is shown in (I). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm",
    "molecules": "DAPI, DMSO, Miconazole"
  },
  {
    "caption": "(J-M) EM thin sections of P28 WT and HET ONs, treated with DMSO (J,K) or Miconazole (L) between P2 and P8. Different degrees of myelin compaction can be appreciated at high magnification, with HET ONs showing non compacted sheaths (white arrows in K) and vacuoles (yellow arrows). Miconazole treatment rescues almost normal myelin g-ratio, as quantified in M. Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm, except (J-L) (500nm).",
    "molecules": "DAPI, DMSO, Miconazole"
  },
  {
    "caption": "(N-P') S100β (green, astrocytes) and Sox2 (red, reactive/proliferative astrocytes) IF in the ONs of WT (N,N') and HET (O-P') animals after 6 days of treatment with DMSO (N-O') or Miconazole (P,P'), then sacrificed at P28. Astrocytes undergo remodeling and express Sox2 in Nr2f1 HET animals (O',P'), even after Miconazole treatment.",
    "molecules": "DMSO, Miconazole"
  },
  {
    "caption": "Histogram showing the average number of S100β+/Sox2+ astrocytes per P28 ON section in WT or HET animals treated with DMSO or Miconazole as indicated, with Sox2 high (magenta columns) and low levels (pink columns). Nuclei (blue) were stained with DAPI. the error bars represent the SEM of the means; ; N=2 Statistical significance was obtained by ANOVA (*P<0.05; **P<0.01; ***P<0.001). Scale bars: 50µm",
    "molecules": "DAPI, DMSO, Miconazole"
  },
  {
    "caption": "Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide (A), cisplatin (B), adriamycin (C), taxol (D), TRAIL (E), bafilomycin A1 (F), thapsigargin (G), and MG132 (H). Means ±S.D. from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO.",
    "molecules": "bafilomycin A1, cisplatin, DMSO, adriamycin, MG132, taxol, temozolomide, thapsigargin"
  },
  {
    "caption": "(I) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs. Cell viability was measured using trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. (*P < 0.05).",
    "molecules": "MG132"
  },
  {
    "caption": "(J) XO8 GSCs were treated with MG132 were cultured in 0.3% soft-agar as described in Materials and Methods. The colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting. Bars, means +S.D. from three independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) XO8 GSCs and DFCs were differentially labeled with PKH67 (green) and PKH26 (red), respectively, and mixed before plated on laminine-coated plates. The cells were treated with 50 nM MG132 and subjected to fluorescent microscopy. Scale bar, 100 μM. (B) Quantitation of (A). Cell numbers from three independent experiments were normalized to those of control cells treated with DMSO and presented as means +S.D. (*p=0.0035, n=3). Student t-test (two-sided, one type) were used for statistical analysis.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(D) XO8 GSCs were treated with various PIs (salinosporamide A, epoxomicin, and PS341), followed by cell viability assays. Bars, means ±S.D. from three independent experiments.",
    "molecules": "PS341, epoxomicin, salinosporamide A"
  },
  {
    "caption": "(E) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay. Bars, means ±S.D. from three independent experiments. (F) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay in serum-containing media. Bars, means ±S.D. from three independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "(J) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy. Scale bar, 100 μM.",
    "molecules": "MG132"
  },
  {
    "caption": "(L) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Real-time PCR analysis of the proteasomal subunits PSMB5, PSMB6, and PSMB7 in XO8 GSCs and XO8 DFCs. Shown are means +S.D. values from the three independent experiments following normalization to those of GSCs. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.053, PSMB6: 0.034, PSMB7: 0.067. (B) Similar to (A) except that the cells were treated with 50 nM MG132 for 17 hrs. Shown are means +S.D. values from the three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.022, PSMB6: 0.08, PSMB7: 0.603.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(C) Immunoblotting analysis of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 and 12. Note that part of Figure 5C is also shown as the left panel of Figure 4A. (D) Same as (C) except that the cells were treated with MG132 for 17 hrs.",
    "molecules": "MG132"
  },
  {
    "caption": "(F) Fractionation analysis of NF-κB in XO8 GSCs and XO8 DFCs treated with 50 nM MG132 for 17 hrs. PARP1 and tubulin were used as markers for the nucleus and cytoplasm, respectively.",
    "molecules": "MG132"
  },
  {
    "caption": "(H) Cell viability assay was performed as in Fig. 2 with cells treated with MG132 alone or in combination with SB203580. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value = 0.724, 0.502, 0.439 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.1 μM MG132, and 0.760, 0.683, 0.498 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.5 μM MG132.",
    "molecules": "DMSO, MG132, SB203580"
  },
  {
    "caption": "(I) Experimental conditions were similar to (H) except that SP600129 was used. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. * P value = 0.009, 0.002, 0.07 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.1 μM MG132, and 0.018, 0.025, 0.09 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.5 μM MG132.",
    "molecules": "SP600129, DMSO, MG132"
  },
  {
    "caption": "(J) Immunoblotting analysis of XO8 GSCs treated with 50 nM MG132 alone or in combination with the JNK inhibitor SP600129.",
    "molecules": "SP600129, MG132"
  },
  {
    "caption": "(A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(C) XO8 GSCs and XO8 DFCs treated with 50 nM MG132 as described in (A) were subjected to immunoblotting.",
    "molecules": "MG132"
  },
  {
    "caption": "(D) XO8 GSCs were treated with MG132, salinosporamide A, epoxomicin, or bortezomib for 24 hrs. Cell lysates were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP, anti-ATF3, anti-ATF3, and anti-BiP antibodies.",
    "molecules": "bortezomib, epoxomicin, MG132, salinosporamide A"
  },
  {
    "caption": "(E) XO8 cells were treated with 50 nM MG132 in the absence or presence of 5μM actinomycin D (ActD) for 6 h. The level of ATF3 mRNA was measured using semi-quantitative real-time PCR in comparison with β-actin.",
    "molecules": "ActD, actinomycin D, MG132"
  },
  {
    "caption": "(A) HEK293 cells were transfected with ATF3 shRNA or vector (pLKO1), treated with 50 nM MG132, and then subjected to immunoblotting.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) Individualized XO8 GSCs were transduced with lentiviral shRNAs against ATF3 or control as described in Materials and Methods. After four days, individualized GSCs were treated with 50 nM MG132, and subjected to immunoblotting.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) XO8 GSCs were transfected with control or CHOP siRNA for 48 hrs and then treated with 50 nM MG132 for 24 hrs. Cell viability was determined using the Trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used.",
    "molecules": "MG132"
  },
  {
    "caption": "(E) Wild-type and CHOP-/- MEF cells were treated with MG132 for 24 hrs. Cell viability was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from three separate experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used.",
    "molecules": "MG132"
  },
  {
    "caption": "(G) The expression of NOXA was assessed with real-time PCR using total RNA extracted from XO8 GSCs treated with 50 nM MG132 alone or in combination with each inhibitor of ER stress-driven apoptosis (salubrinal; 15 µM, STK064652; 20 µM, STK047915; 20 µM). The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: salubrinal, 0.0003; STK064650, 0.0036; STK047915, 0.0019.",
    "molecules": "STK064652, STK047915, DMSO, MG132, salubrinal"
  },
  {
    "caption": "(H) Similar to (G) except that XO8 GSC transduced with lentiviral shRNAs were treated with MG132. The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: #1, 0.026; #5, 0.0033; #6, 0.0482.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(I) XO8 GSCs were treated with 20 nM MG132 alone or in combination with each inhibitor as described in (C), and subjected to immunoblotting. Sal.: salubrinal.",
    "molecules": "MG132, salubrinal"
  },
  {
    "caption": "(A and B) XO8 GSCs were subcutaneously (s.c.) injected in the flank of mice. When the tumor volume reached approximately 180 mm3, GSC-bearing mice were administered intraperitoneally (i.p.) with 1 mg/kg MG132 and 10 mg/kg STK alone or in combination on every day for 2 weeks. Tumors were dissected from the subcutaneous regions of nude mice. Inset picture shows the tumor in respective group. Therapeutic effects were monitored using bioluminescence imaging. (C) The bar diagram represents the tumor volumes. (D) Tumor weights were determined at the end of the study. (E) The tumor sizes in each group were determined by histological analysis at day 21 after tumor inoculation.",
    "molecules": "MG132"
  },
  {
    "caption": "(F) XO10 GSC were orthotopically injected into the brains of BALB/c nude mice. STK047915, MG132, or both were injected intraperitoneally to GSCs-bearing mice at day 14 after XO10 (5×105 cells) inoculation. The PBS was used as a control. Representative photographs of H&E staining from each group show tumor growth. Magnification, ×1.",
    "molecules": "STK047915, MG132"
  },
  {
    "caption": "(E) HUVEC exposed to same conditions as in C, followed by fixation with (upper row) or without (lower row) permeabilization with 0.3% Triton X-100, and staining for VE-PTP (red) and nuclei (blue, Hoechst 33342). (F) VE-PTP on cell surface measured by cell-based ELISA assay of HUVEC after exposure as in C and D. Red and black lines show measurements with anti-VE-PTP antibody and isotype control antibody. Mean ± SEM, n = 6 independent experiments/group. D",
    "molecules": "Hoechst 33342, Triton X-100"
  },
  {
    "caption": "(E) HUVECs cultured on biotinylated gelatin were treated either with control siRNA, VE-PTP siRNA, Tie2 siRNA or VE-cadherin siRNA, then exposed to 15 dyn/cm2 shear stress for 30 minutes followed by 3 min incubation with Streptavidin-Alexa647 (red), washing, fixation and staining for VE-cadherin (green). (F and G) Quantification of Streptavidin leakage area per image. Mean ± SEM, n = 19 pooled in ten independent experiments (F), and n = 6 pooled in three independent experiments (G). D",
    "molecules": "Alexa647, gelatin, Streptavidin"
  },
  {
    "caption": "(B) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 13-18 mice/group.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(E) Fractional area occupied by Oil Red O-stained atheromas in aortic arch, brachiocephalic artery, at intercostal artery ostia, and away from ostia. Each dot is value for one mouse. Mean ± SEM, n = 17 mice/group.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(B) Proteasome inhibitor treatment (10 μM MG132 for 1 to 8 h) promotes accumulation of a ~110KD fragment (asterisk) in HEK293A cells.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) The ~110KD fragment (asterisk) is related to AMOT. Wild-type (WT) and AMOT knockout (KO) HEK293A cells were treated with 10 μM proteasome inhibitor bortezomib (BTZ) for 6 h and subjected to Western blotting analysis using the indicated antibodies.",
    "molecules": "bortezomib, BTZ"
  },
  {
    "caption": "(F and G) Serum and LPA treatments induce AMOT degradation and promote accumulation of a ~110 kDa protein (asterisk) in HEK293A cells. Cells were serum-starved overnight and then treated with 10% FBS or 1 μM LPA for the indicated time. The phosphorylation levels of YAP proteins were detected by Phos-tag gel and pYAP immunoblotting. Protein quantification is shown below. Data represent mean ± SEM from three independent experiments. L.E. indicates long exposure.",
    "molecules": "LPA"
  },
  {
    "caption": "C) Nelfinavir (NFV) and Lopinavir (LPV) block AMOT cleavage. HEK293A cells were pre-treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h. Cell lysates were then subjected to Western blotting.",
    "molecules": "BTZ, Lopinavir, LPV, Nelfinavir, NFV"
  },
  {
    "caption": "(F) Deletion of DDI2 blocks AMOT cleavage. Wild-type (WT) and DDI2 KO cells were serum-starved overnight and then treated with 1 μM LPA for 2 h.",
    "molecules": "LPA"
  },
  {
    "caption": "K) The aspartic catalytic-site mutant DDI2 exhibits a dominant negative effect through inactivation of wild-type DDI2 in HEK293A cells. Wild-type HEK293A cells were transfected with the indicated plasmids and treated with 1 μM LPA (K) before harvest.",
    "molecules": "LPA"
  },
  {
    "caption": "(A) Ubiquitination is required for AMOT cleavage. HEK293A cells were treated with 100 nM E1 inhibitor MLN7243 overnight followed by 10 μM BTZ for 6 h. Cell lysates were then subjected to Western blotting.",
    "molecules": "BTZ, MLN7243"
  },
  {
    "caption": "(B and C) The TNKS1/2-RNF146 axis is crucial for AMOT cleavage. Wild-type (WT), TNKS1/2 double KO (dKO) and RNF146 KO HEK293A cells were treated with the proteasome inhibitor MG132 for 6 h before harvest. Cell lysates were subjected to Western blotting analysis using the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(K) Serum, LPA, and S1P stimulate PARylation and ubiquitination of AMOT. Serum-starved cells were treated with 10% FBS, 1 μM LPA, or 1 μM S1P for 4 h and subjected to an immunoprecipitation (IP) assay.",
    "molecules": "LPA, S1P"
  },
  {
    "caption": "(C) Nelfinavir and Lopinavir block AMOT cleavage in HUVECs. HUVECs were pre-treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h. Cell lysates were subjected to Western blotting. Protein quantification is shown below. (D) Aspartic protease DDI2 is responsible for AMOT cleavage in HUVECs. HUVECs were transfected with two different small interfering RNA (siRNA) and 48 h post-transfection, the cells were treated with 10 μM BTZ for 6 h. Cell lysates were subjected to Western blotting. Protein quantification is shown below.",
    "molecules": "BTZ, Lopinavir, Nelfinavir"
  },
  {
    "caption": "(F RNF146 are indispensable for AMOT cleavage in HUVECs. siRNAs were transfected into HUVECs, the cells were treated with 10 μM BTZ for 6 h before harvest. Protein quantifications are shown below.",
    "molecules": "BTZ"
  },
  {
    "caption": "(H) Representative immunofluorescence images of HUVEC spheroids 18 h after VEGF stimulation. Above: inverted contrast images. Areas within the red dash box indicate region of interest (ROI) and are shown below at higher magnification. Plasma membranes were stained with CellMask (red), actin filaments with Phalloidin (green), and nucleus with DAPI (blue). Scale bar 100 μm. HUVECs infected with empty vector virus were used as control.",
    "molecules": "CellMask, DAPI, Phalloidin"
  },
  {
    "caption": "(G and H) AMOT-CT but not full-length AMOT could rescue impaired angiogenesis in ddi2-deficient embryos. Embryos were injected with ddi2 MO together with mRNA encoding wild-type AMOT (AMOT-WT) or AMOT-CT. Scale bar: 50 μm. (I and J) Nelfinavir (NFV) and XAV939 repress ISV development. Embryos were treated with 5 μM NFV, 10 μM XAV939 at 6 hpf for 24 h. Number of ISV defects was counted at 30 hpf. Scale bar: 50 μm.",
    "molecules": "Nelfinavir, NFV, XAV939"
  },
  {
    "caption": "(F and G) High magnification confocal images and quantification of filopodia extension at the leading edge of the P6 retinas. Tip cells are labeled with IB4. Scale bar: 10 μm. Control (n=10), Nf2iEC-KO (n=10), Nf2iEC-KO; CTiEC-TG (n=10) and Nf2iEC-KO; ΔcutiEC-TG (n=8).",
    "molecules": "IB4"
  },
  {
    "caption": "Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Haematoxylin/eosin staining of paraffin sections of Ronin fl/del and ecto-Ronin teratomas.",
    "molecules": "eosin, Haematoxylin, paraffin"
  },
  {
    "caption": "In vitro differentiation of Ronin fl/del, ecto-Ronin (grown in the presence of Dox) and Ronin del/del ESC.",
    "molecules": "Dox"
  },
  {
    "caption": "Analysis of nascent protein synthesis using OP-puro assay. Ronin fl/del cells treated with protein synthesis inhibitor (cycloheximide) are used as negative control. The nuclei are counterstained with DAPI.   Quantification of the OP-puro signal, normalised to DAPI. Ordinary one-way Anova data represent mean ± SD; n = three independent experiments for each genotype.   ",
    "molecules": "cycloheximide, DAPI, OP-puro"
  },
  {
    "caption": "Blastocyst (E4.5) and diapause embryos stained for Ronin, Sox2 and Sox17. Egg cylinder stage (E5.5) embryo stained for Ronin and Sox17. Nuclei are counterstained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "E4.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E4.5 blastocysts, Mann-Whitney test, data represent mean ± SD; Ronin fl/del n embryos = 26, Ronin del/del n embryos = 27.   Diapause embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in diapause embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 4, Ronin del/del n embryos = 12.   E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E5.5 embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 21, Ronin del/del n embryos = 15.   Reactivated E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in reactivated E5.5 embryos, Mann-Whitney test, data represent mean ± SD, Ronin fl/del n embryos = 17, Ronin del/del n embryos = 12.   ",
    "molecules": "DAPI"
  },
  {
    "caption": "D Immunoblot detection of the relative accumulation of LC3 I and LC3 II in HeLa cells transfected with USP19-specific siRNA or scrambled siRNA with rapamycin (250 nM) treatment for 12 h.",
    "molecules": "rapamycin"
  },
  {
    "caption": "E HeLa cells were transfected with USP19-specific siRNA or scrambled siRNA and treated with indicated concentration of rapamycin for 12 h, p62 levels were detected by immunoblot.",
    "molecules": "rapamycin"
  },
  {
    "caption": "F Human peripheral blood mononuclear cells (PBMCs) transfected with control or USP19-specific siRNA were treated with rapamycin (250 nM) for 18 h, the lysates were analyzed with each antibody.",
    "molecules": "rapamycin"
  },
  {
    "caption": "G Flag-USP19 stably expressing 293T cells were incubated with DMEM or EBSS medium for 3 h in the absence or presence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblot analysis using anti-LC3 antibody.",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "H Flag-USP19 inducible HeLa cells were treated with 200 ng/ml doxycycline (Doxy) for overnight to induce the expression of Flag-USP19. The protein levels of p62 were analyzed after rapamycin treatment for 12 h at indicated concentrations.",
    "molecules": "Doxy, doxycycline, rapamycin"
  },
  {
    "caption": "B HeLa cell line harboring inducible Flag-USP19 was induced with indicated concentration of Doxy for overnight, and the protein was harvested for immunoblot analysis.",
    "molecules": "Doxy"
  },
  {
    "caption": "D Human PBMCs were transfected with USP19-specific or scramble siRNA and treated with rapamycin (250 nM) for 18 h. The extracts were analyzed by immunoblot with indicated antibodies.",
    "molecules": "rapamycin"
  },
  {
    "caption": "H 293T cells were transfected with plasmid encoding Flag-Beclin-1 and treated with CHX (100 μg/ml) alone or with MG132 (10 μM), 3MA (10 mM), CQ (50 μM) as combination for 6 h or transfected with the HA-USP19 plasmid. The cell lysates were analyzed by immunoblot.",
    "molecules": "3MA, CQ, CHX, MG132"
  },
  {
    "caption": "A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 (10 μM) treatment for 3 h. Protein extracts were immunoprecipitated using anti-Beclin-1 antibody or with IgG as a negative control and analyzed by immunoblot using anti-ubiquitin and anti-Beclin-1 antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.",
    "molecules": "MG132"
  },
  {
    "caption": "C HeLa cells were transfected with USP19-specific siRNA and plasmid encoding Flag-Beclin-1, plus K11-linked-Ub. Protein was harvested after MG132 (10 μM) treatment for 3 h and immunoprecipitated with anti-Flag and immunoblotted with anti-HA.",
    "molecules": "MG132"
  },
  {
    "caption": "D 293T cells were transfected with plasmid for Flag-Beclin-1 and HA-K11-Ub, together with the empty vector or expression vector for Myc-USP19 (WT, CS or CS/HA) and treated with MG132 (10 μM). Cell lysates were subjected to immunoprecipitation with anti-Flag and immunoblot analysis with anti-HA.",
    "molecules": "MG132"
  },
  {
    "caption": "C 293T cells were transfected with Flag-Beclin-1 (WT or K437R) and treated with CHX (100 μg/ml) for indicated time points. The expression levels of WT and K437R Flag-Beclin-1 were analyzed by immunoblot.D Quantification of the expression levels of WT and K437R Flag-Beclin-1.",
    "molecules": "CHX"
  },
  {
    "caption": "quantification of the frequency of cell clusters with polarised mCherry-PAR-6B (C) in control and mitomycin  division-blocked mESC  live cells after 24 hours in Matrigel. Data information: Data in (C) are presented as means ± SEM. n = 3 experiments. At least 20 clusters were analysed for each column in every experiment. Two-way ANOVA analysis; P values were listed in the graphs.",
    "molecules": "Matrigel, mitomycin"
  },
  {
    "caption": "C Line-scan profiles of PAR-3   at the cell-cell interface in wild-type control, mitomycin C  treated and Cdh1  KO control, mitomycin C treated 2-cell doublets. D Ratio of  PAR-3  pixel intensity values at central and surrounding regions in >2-cell mESC  clusters. Data information: individual and mean-valued line-scans in (C) individual cell cluster values (small dots), mean experimental values (large dots) and means of 3 experiments (bars) ± SEM in (D) 17-50 cell clusters were analysed for each column in every experiment; 15 doublets for each condition in (C); 17-30 cell clusters were analysed for each condition in every experiment in (D); Two-way ANOVA analysis in (D), P values were listed in the graphs. scale bars: 10 µm.",
    "molecules": "mitomycin C"
  },
  {
    "caption": "A-C Immunofluorescence of PAR-3 and P-cadherin (A), proportions of mESC   clusters with a positive PAR-3  centre (B) and line-scans of PAR-3 at the cell-cell interface (C) in control, P-cadherin  knock-down by RNAi, mitomycin  treated and P-cadherin knock-down mitomycin  treated W4 mESC  doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (B) ; individual and mean-valued line-scans in (C) n = 3 experiments in (B), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (C) Two-way ANOVA analysis in (B) P values were listed in the graphs. scale bars: 5 µm.",
    "molecules": "mitomycin"
  },
  {
    "caption": "D-F Immunofluorescence  of PAR-3  and JAM-A (D), proportions of mESC  clusters with a positive PAR-3 centre (E) and line-scans of PAR-3 at the cell-cell interface (F) in control, JAM-A knock-down by RNAi, mitomycin  treated and JAM-A knock-down mitomycin  treated W4 mESC  doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (E), individual and mean-valued line-scans in (F) n = 3 experiments in (E), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (F), Two-way ANOVA analysis in (E), P values were listed in the graphs. scale bars: 5 µm.",
    "molecules": "mitomycin"
  },
  {
    "caption": "G-I Immunofluorescence  of PAR-3 and Nectin-2 (G), proportions of mESC  clusters with a positive PAR-3 centre (H) and line-scans of PAR-3  at the cell-cell interface (I) in control, Nectin-2 knock-down by RNAi, mitomycin  treated and Nectin-2  knock-down mitomycin  treated W4 mESC  doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (H); individual and mean-valued line-scans in (I). n = 3 experiments in (H), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (I). Two-way ANOVA analysis in (H); P values were listed in the graphs. scale bars: 5 µm.",
    "molecules": "mitomycin"
  },
  {
    "caption": "A, B Immunofluorescence  of PAR-3 (A) and proportions of mESC  doublets with polarised  PAR-3 (B) in wild-type (ES-E14) and E-cadherin knock-out (Cdh1  KO) cells at 30 hours in 0.5% agarose. Data information: Data are presented as means ± SEM in (B); n = 3 experiments in (B) Two-way ANOVA analysis in (B) P values were listed in the graphs. scale bars: 5 µm.",
    "molecules": "agarose"
  },
  {
    "caption": "C Immunofluorescence  of PAR-3  in cell division blocked mESCs  cultured on E-cadherin or Fibronectin  coated glass topped with or without Matrigel  for 24 hours. Data information: scale bars: 5 µm.",
    "molecules": "Fibronectin, Matrigel"
  },
  {
    "caption": "Human  first-trimester  trophoblast  cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with  ZIKV (MOI=2) for 1 h and refreshed with regular media over  time. RNA was collected for measuring viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (A) ZIKV  titers in Sw.71 and HPC trophoblasts. *p < 0.05 by One-way ANOVA.",
    "molecules": "RNA"
  },
  {
    "caption": "(C) Higher  ZIKA titer was shown in ISG20-/- Sw.71 cells. Sw.71 and  ISG20-/- Sw.71 cells were infected with ZIKV  (MOI=2) for 1 h and refreshed with regular media for 48 h, and  RNA  was collected for determining the viral titer and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.",
    "molecules": "RNA"
  },
  {
    "caption": "(D)  IFNβ pre-treatment significantly prevented trophoblast  cells from ZIKV infection, however, this protection was evidently attenuated due to lack of ISG20. Sw.71 and ISG20-/- Sw.71  cells were pre-treated with or without 30 ng/ml IFNβ for 24 h, followed by ZIKV infection (MOI=2) for 1 h and refreshed with regular media for 24 h, and  RNA was collected to determine the viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.",
    "molecules": "RNA"
  },
  {
    "caption": "(D) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in  trophoblast  cells. Supernatants from negative and positive clones were collected and added to ISG20-/- Sw.71 trophoblast  cells together with ZIKA  virus (MOI=2) for 1 h, followed by refreshing with new growth media for 48 h.  RNA was then collected to determine viral titers by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01 by Student's t-test.",
    "molecules": "RNA"
  },
  {
    "caption": "(E) Recombinant ISG20-Fc degrades ZIKA viral RNA  and HSV2 viral DNA. 50ng purified  viral RNA (ZIKV) or DNA  (HSV-2) were incubated with increasing concentrations of rISG20-Fc (5, 50, 500 ng) in the presence of  RNase inhibitor for 90min at 37°C followed by quantification of viral titers by qRT-PCR, and agarose gel was used to evaluate the RNA degradation by  electrophoresis. The representative picture of agarose gel is presented. rISG20-Fc was able to degrade both ZIKV RNA  and HSV-2 DNA  in a dose dependent manner; however, rISG20-Fc was more efficient in degrading viral  RNA than DNA. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01. ***p < 0.001 and ****p < 0.0001 by One-way ANOVA.",
    "molecules": "agarose"
  },
  {
    "caption": "(A) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3  mice  for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test. (B) rISG20-Fc treatment reduces ZIKV  titers in the  fetal brain. Although there was no difference of ZIKV  titer in the placenta, at the fetal  side, there was a significant decrease of viral titer in the  fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test. ( ",
    "molecules": "PBS"
  },
  {
    "caption": "(C) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for  PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and  IFNγ by One-way ANOVA.",
    "molecules": "PBS"
  },
  {
    "caption": "Drp1 knockdown cells were treated with DMSO (control) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax, cytochrome c, and Mic27 or Mic60 (see Appendix Figure S4). Three-color images were taken, whereby Mic27 or Mic60 were recorded in the STED mode. Relying on the Bax and the cytochrome c signals, we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release. The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity, which is a sensitive measure for the distribution of the labelled protein. The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean.",
    "molecules": "actinomycin D, DMSO"
  },
  {
    "caption": "E-H Experimental glucose uptake flux (E), acetate flux (F), and growth rate (G) of E. coli grown on glucose (15 mM) with different concentrations of αMG (0-100 mM), at low (0 mM) and high (30 mM) acetate concentrations. These data were used to quantify the total carbon uptake flux as a function of the αMG concentration (H). Each data point represents an independent biological replicate, and lines represent the best polynomial fits.",
    "molecules": "acetate, carbon, glucose, αMG"
  },
  {
    "caption": "I, J Total carbon uptake (I) and growth rate (J) as a function of the glucose uptake flux, at low (0 mM) and high (30 mM) acetate concentrations. Each data point represents an independent biological replicate, and lines represent the best linear fits. K, L Relationships between the acetate flux and glucose uptake (K), and between the contribution of acetate to carbon uptake and glucose uptake (L). Each data point represents an independent biological replicate, and lines represent the best polynomial fits.",
    "molecules": "acetate, carbon, glucose"
  },
  {
    "caption": "C Experimental acetate flux of E. coli K-12 MG1655 grown on glucose (15 mM) at different acetate concentrations in the presence of αMG (0, 20, 40 or 100 mM). Each data point represents an independent biological replicate, and the lines represent best fits using a logistic function.",
    "molecules": "acetate, glucose, αMG"
  },
  {
    "caption": "D Experimental acetate concentration threshold at which the acetate flux reverses as a function of the αMG concentration, determined from the best fits shown in panel (C).",
    "molecules": "acetate, αMG"
  },
  {
    "caption": "B Experimental growth rate of E. coli K-12 MG1655 grown on glucose (15 mM) as a function of the acetate concentration at high (0 mM αMG, light blue, low (100 mM αMG, dark blue) glycolytic flux. Each data point represents an independent biological replicate and the lines represent best polynomial fits.",
    "molecules": "acetate, glucose, αMG"
  },
  {
    "caption": "C Relative change in the growth rate of E. coli K-12 BW25113 wild-type in the presence of 10 mM acetate at high (0 mM αMG) and low (100 mM αMG) glycolytic flux. Mean values ± standard deviations (error bars) were estimated from n independent biological replicates, as indicated on the figure.",
    "molecules": "acetate, αMG"
  },
  {
    "caption": "D Relative change in the growth rates of E. coli K-12 BW25113 Δpgi and ΔpfkA strains in the presence of 10 mM acetate. Mean values ± standard deviations (error bars) were estimated from three independent biological replicates.",
    "molecules": "acetate"
  },
  {
    "caption": "Relative change in the growth rates of wild-type, Δpta, and Δacs E. coli K-12 BW25113 strains in the presence of 10 mM acetate at low glycolytic flux (100 mM αMG). Mean values and standard deviations (error bars) were estimated from n independent biological replicates, as indicated on the figure.",
    "molecules": "acetate, αMG"
  },
  {
    "caption": "A,B Impact of the presence of 10 mM acetate on the growth rate of wild-type, Δpta, and Δacs E. coli K-12 BW25113 strains grown on (A) 30 mM glycerol and (B) 15 mM galactose. Mean values and standard deviations (error bars) were estimated from three independent biological replicates.",
    "molecules": "acetate, galactose, glycerol"
  },
  {
    "caption": "Immunoblot analysis of proteins biotinylated  and isolated as in (A). Parental HeLa-T-REx cells or cells expressing H3.1-BirA*-HA and treated with biotin for indicated times.",
    "molecules": "biotin"
  },
  {
    "caption": "Immunoblot  analysis of TAP-H3.1 or -CENP-A pulldown of UBR7-LAP (schematic, upper).  293T cells were co-transfected with UBR7-LAP and TAP-tagged constructs, which were purified on IgG-conjugated sepharose beads.",
    "molecules": "sepharose"
  },
  {
    "caption": "Representative images of HeLa  cells transfected with UBR7-FL. DNA is visualized via  DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. The area within yellow dotted box in (E) is magnified 4x in (E'). Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence  intensity in (E). Error bars represent mean +/- standard deviation. One way ANOVA was used to calculate p-values (italics) associated with differences between treatments. Experiment in (E-F) was performed twice.  ",
    "molecules": "DAPI"
  },
  {
    "caption": "Anti-GFP immunoblot analysis of streptavidin pulldown of the indicated biotin-conjugated H3  peptides following incubation with 293T-GFP-UBR7 cell lysate after mutation of residues highlighted in (C).",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in (G). 4.5 h represent newly synthesized histones and 48 h represent pre-existing histones. Wedge represents loading titration: 1x > 0.1x > 0.01x > 0.001x. All other lanes are loaded at 1x concentration. Immunoblot signals were normalized to anti-FLAG signal.   Quantification of anti-FLAG (H3.1-APEX2-3xFLAG) and anti-UBR7 immunoblot represented in (G-H). Lanes 8 and 11 were quantified. Error bar represents mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line).  ",
    "molecules": "streptavidin"
  },
  {
    "caption": "Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that  sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.  ",
    "molecules": "DAPI, Triton X-100"
  },
  {
    "caption": "A,C.  Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide). B,D. Plots represent either normalized  immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.",
    "molecules": "α-amanitin, aphidicolin, hydroxyurea, triptolide"
  },
  {
    "caption": "Immunoblot for LAMP2A in parental 293T cells or cells expressing   UBR7-FL (left) or  sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132.",
    "molecules": "MG-132"
  },
  {
    "caption": "(C) Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody.",
    "molecules": "Biotinylated"
  },
  {
    "caption": "(A) GCI-based kinetic analysis of interactions between immobilized sybodies (indicated above each plot) and wild-type (WT) or mutant RBDs carrying the individual K417N, E484K or N501Y mutations (left panels), the combined triple KEN (Beta) mutations (middle panels), or the double L452R/T478K (Delta) mutations (right panels). Sb#15, Sb#68 and GS4 were immobilized on independent flow-cells via biotinylated Avi-tags, and the RBD variants were sequentially injected at a concentration of 200 nM.",
    "molecules": "biotinylated"
  },
  {
    "caption": "Lentiviral overexpression of miR-200c and miR-203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay. For the changes in EC50 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).",
    "molecules": "gemcitabine"
  },
  {
    "caption": "Overexpression of miR-203 decreases expression of the anti-apoptotic factor survivin and sensitizes to gemcitabine-triggered apoptosis as evaluated by cleaved caspase-3 in Western blot and immunofluorescence. Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine, respectively, for 48 h. Scale bar 20 μm.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR-203 or all miR-200 members. For the changes in EC80 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).",
    "molecules": "gemcitabine"
  },
  {
    "caption": "B, C Histone marks were analyzed using ChIP coupled to qRT-PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT-PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1.",
    "molecules": "mocetinostat"
  },
  {
    "caption": "Relative expression of indicated genes in Panc1 measured by qRT-PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1.",
    "molecules": "mocetinostat"
  },
  {
    "caption": "Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm.",
    "molecules": "mocetinostat"
  },
  {
    "caption": "Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test.",
    "molecules": "mocetinostat"
  },
  {
    "caption": "Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann-Whitney U-test.",
    "molecules": "Mocetinostat, mocetinostat"
  },
  {
    "caption": "Immunoblot and immunofluorescence for cleaved caspase-3 showing strong increase in apoptosis in gemcitabine-resistant Panc1 after combined treatment with mocetinostat (1 μM) and gemcitabine (50 nM). Scale bar 20 μm.",
    "molecules": "gemcitabine, mocetinostat"
  },
  {
    "caption": "MTT assay for Panc1 treated with the indicated concentrations of mocetinostat (left) or SAHA (middle) and gemcitabine (72 h). Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability. In contrast, a combination with SAHA had no effect. For calculation of the CI and synergy between the drugs, see Table2. Comparison of mocetinostat and SAHA alone (right). n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are ***P < 0.001 and ****P < 0.0001; for exact P-values, see Supplementary Table S4).",
    "molecules": "gemcitabine, mocetinostat, SAHA"
  },
  {
    "caption": "Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient-derived pancreatic cancer cells. Note that hPaca1 behaves similar to Panc1, but mocetinostat had no significant effect in hPaca2. Scale bar 20 μm. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).",
    "molecules": "gemcitabine, mocetinostat"
  },
  {
    "caption": "MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU-145 and the docetaxel-resistant subclone DU-145 DR (left). Mocetinostat treatment of DU-145 DR downregulates ZEB1, upregulates E-cadherin, miR-200, and miR-203 expression. For relative miRNA expression, the expression levels in original DU-145 were set to 1 (middle panels, the immunoblot panel derives from the same experiment shown in Fig1A). Mocetinostat sensitizes DU-145 DR to docetaxel (right). For calculation of the CI and synergy between the drugs, see Table2. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05; for exact P-values, see Supplementary Table S4).",
    "molecules": "docetaxel, mocetinostat, Mocetinostat"
  },
  {
    "caption": "Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann-Whitney U-test.",
    "molecules": "gemcitabine, mocetinostat"
  },
  {
    "caption": "Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E-cadherin in tumor tissues of mice treated with mocetinostat. Scale bar 40 μm, inserts for higher magnifications 10 μm. Squares indicate magnified regions.",
    "molecules": "mocetinostat"
  },
  {
    "caption": "Representative pictures of in situ hybridization for miR-203 and control probe showing gain of miR-203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat-treated xenograft tumors. Scale bar 40 μm, inserts for higher magnifications 5 μm. Squares indicate magnified regions.",
    "molecules": "mocetinostat"
  },
  {
    "caption": "Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice. Panc1 cells were pre-treated with mocetinostat (1 μM) and/or gemcitabine (50 nM) for 48 h, followed by a 7-day recovery period before being injected subcutaneously (left). Equal numbers of viable cells were injected in 75 μl volume. At day 9 after injection, tumor growth was detectable in all groups (lower right). To better visualize and compare tumor growth, the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted (upper right). The individual absolute tumor volumes on day 37 (lower left) and the group medians of the absolute tumor volumes over time (lower right) are shown. For cells pre-treated with the combination of mocetinostat and gemcitabine, tumor growth was arrested. n = 4 for each treatment group; nonparametric Mann-Whitney U-test.",
    "molecules": "gemcitabine, mocetinostat"
  },
  {
    "caption": "Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (A) Bacterial growth was measured by optical density at 600 nm. Data are representative of five independent experiments. ",
    "molecules": "glucose"
  },
  {
    "caption": "Cells were grown in MHB-ca medium without or with 20 mM glucose as indicated. (B) PndhC and PndhF YFP reporter constructs were used for following gene expression and data was normalized by growth. Data are expressed as average ± SEM of three independent experiments. *** Denotes p < 0.001 compared to ndhC expression in WT strain grown in MHB-ca medium via two-way ANOVA with Tukey's posttest.",
    "molecules": "glucose"
  },
  {
    "caption": "(A) Oxygen consumption of WT and isogenic mutant strains was measured with Clark oxygen electrode. Cells were grown in MHB-ca medium until exponential phase (OD600 = 0.6) or until late stationary phase (24 h). Cells were washed with PBS and respiration initiated by the addition of 20 mM glycerol. Data are expressed as average ± SD of four independent experiments. *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.",
    "molecules": "glycerol, oxygen"
  },
  {
    "caption": "(B) NADH/NAD+ ratios were measured in cells grown aerobically in MHB-ca medium for 20 h were measured. 100% corresponds to a NADH/NAD+ ratio of 0.003. Data are expressed as average ± SD of four independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "(C) Acetate concentration was monitored in supernatants of WT and isogenic ndh strains at 7 and 24 h of growth in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via two-way ANOVA with Tukey's posttest.",
    "molecules": "Acetate"
  },
  {
    "caption": "(D) MKH2/MK ratios were measured in cells grown aerobically for 20 h in MHB-ca medium. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "MKH2, MK"
  },
  {
    "caption": "(A) The carotenoid pigment of different S. aureus strains was extracted by methanol and quantified by the optical density at 465 nm, and the values were normalized to WT Newman strain, which was set at 100. Data are expressed as the average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "carotenoid, methanol"
  },
  {
    "caption": "(B) Hydrogen peroxide sensitivity was measured by incubating WT and isogenic strains, previously grown for 20 h in MHB-ca, with different concentrations of hydrogen peroxide. Serial dilutions were plated in TSB medium for measuring CFU. Data are expressed as the average ± SD of three independent experiments.",
    "molecules": "Hydrogen peroxide, hydrogen peroxide"
  },
  {
    "caption": "(B) Biofilm formation of Newman WT and ndh deficient mutants after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "glucose, NaCl"
  },
  {
    "caption": "(C) MKH2/MK ratios were measured in cells grown under the same conditions as for biofilm formation. Data are expressed as average ± SD of three independent experiments. * Denotes p < 0.05 compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "MKH2, MK"
  },
  {
    "caption": "(D) NADH/NAD+ ratios were measured in cells grown under the same conditions as for biofilm formation. 100% corresponds to a NADH/NAD+ ratio of 0.09. Data are expressed as average ± SD of four independent experiments performed by duplicates. * Denotes p < 0.05, and *** denotes p < 0.001 compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium without and with 25 mM glucose, 40 mM sodium pyruvate or 60 mM sodium acetate. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of five independent experiments. * Denotes p < 0.05, and *** denotes p < 0.001, ns denotes no significant compared to WT grown in MHB-ca and WT grown in MHB-ca + glucose via two-way ANOVA with Tukey's posttest.",
    "molecules": "glucose, hemoglobin, sodium acetate, sodium pyruvate"
  },
  {
    "caption": "(A) Hemolytic activity of the Newman WT and isogenic mutant strains was measured by incubation of rabbit blood cells with serially diluted supernatants from 20 h cultures grown in MHB-ca medium. Lysis was monitored by the release of hemoglobin. Data are expressed as average ± SD of four independent experiments. *** Denotes p < 0.001, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "hemoglobin"
  },
  {
    "caption": "(B) Biofilm formation of Newman WT and isogenic mutant strains after static incubation at 37 °C for 20 h. The histogram shows the amount of biofilm biomass attached to the microtiter plate when cells were grown in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl. Data are expressed as average ± SD of four independent experiments performed by octuplicates. ** Denotes p < 0.005, ns denotes no significant compared to WT via one-way ANOVA with Dunnett's posttest.",
    "molecules": "glucose, NaCl"
  },
  {
    "caption": "(C) Hemolytic activity of the Newman WT and isogenic mutant strains grown aerobically for 20 h in MHB-ca medium, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of five independent experiments. ** Denotes p < 0.005 and *** denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.",
    "molecules": "oleic acid"
  },
  {
    "caption": "(D) Biofilm formation of Newman WT and isogenic mutant strains after static incubation for 20 h in MHB-ca supplemented with 10% human plasma, 0.5 % glucose and 2% NaCl, in the absence or presence of 10 mg/ml FA-deficient BSA and 0.01% oleic acid. Data are expressed as average ± SD of four independent experiments performed by octuplicates. *** Denotes p < 0.001 compared to each strain without any BSA or oleic acid addition via two-way ANOVA with Tukey's posttest.",
    "molecules": "glucose, oleic acid, NaCl"
  },
  {
    "caption": "a-b Expansions of 1H-15N HSQC spectra are overlaid and Δδ values are plotted vs. the amino acid sequences, along with secondary structures: (a) 10 µM 15N-enriched CXCL12 alone (black peaks) and in the presence of 330 µM Gal-3 CRD (red peaks; assignments reported by Murphy et al.[74]) and (b) 30 µM 15N-enriched Gal-3 CRD alone (black peaks) and in the presence of 500 µM CXCL12 (red peaks; assignments for Gal-3",
    "molecules": "1H, 15N"
  },
  {
    "caption": "C The peritoneal recruitment of classical monocytes after IP injection of PBS (n = 6) or TG in C57BL/6J WT (dark green, n = 10 mice) and Gal-3-/- (black, n = 5 mice) mice was assessed after 18 h. Where indicated, the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment.",
    "molecules": "AMD 3465, PBS, TG"
  },
  {
    "caption": "E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity (MFI) (n = 4 mice).",
    "molecules": "TG"
  },
  {
    "caption": "E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal-3 and Gal-1 alone and with 70 mM lactose, and Gal-3 CRD, was determined by flow cytometry (n = 4).",
    "molecules": "lactose"
  },
  {
    "caption": "Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (F) 1 µM Gal-3, (G) Gal-1, (H) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.",
    "molecules": "cellobiose, DMJ, lactose"
  },
  {
    "caption": "Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (I) 0.1 nM Gal-3 and (J) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.",
    "molecules": "cellobiose, DMJ, lactose"
  },
  {
    "caption": "a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD followed by stimulation with forskolin, an activator of adenylate cyclase. Results are shown as luminescence relative to baseline (RLU, example of n = 3).",
    "molecules": "cAMP, forskolin"
  },
  {
    "caption": "b The effect of 10 nM Gal-3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation, was tested. Control experiments were performed as indicated (n = 3, four technical replicates).",
    "molecules": "forskolin"
  },
  {
    "caption": "F-G Jurkat T cells were incubated with 100 nM of Gal-3 CRD Alexa Fluor 555 alone, with 10 µM AMD 3100, with 100 nM CXCL12 or with CXCL12 and AMD 3100. Experiments were performed (f) without and (g) in the presence of 70 mM lactose (F-G: n = 3, two technical replicates).",
    "molecules": "Alexa Fluor 555, lactose, AMD 3100"
  },
  {
    "caption": "H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal-3 CRD as indicated (n = 4, two technical replicates).",
    "molecules": "FITC"
  },
  {
    "caption": "(A,B)  Net charge distribution (black) of αSyn (A) and Aß (B), plotted together with the binding  distribution of wild-type SERF2  (blue) and the SERF2  charge mutant (red). Blue boxes represent the SERF2 interacting  regions.",
    "molecules": "Aß"
  },
  {
    "caption": "(C) ThT-monitored amyloid kinetics  of 50uM alpha-synuclein in the presence of either wild-type SERF2  or mutant SERF2  in a 1:1 ratio. The average of three biological replicates is represented and error bars indicate mean ± SD.",
    "molecules": "ThT"
  },
  {
    "caption": "(D) ThT-monitored amyloid kinetics  of 1uM amyloid-beta in the presence of either wild-type SERF2  or mutant SERF2  in a 1:4 ratio. The average of four technical replicates is represented and error bars indicate mean ± SD.",
    "molecules": "amyloid-beta, ThT"
  },
  {
    "caption": "(F) ThT-monitored amyloid kinetics  of 2uM human islet amyloid polypeptide in the presence of either wild-type SERF2  or mutant SERF2  in a 1:4 ratio. The average of five technical replicates is represented and error bars indicate mean ± SD.",
    "molecules": "ThT"
  },
  {
    "caption": "(E) Number of aggregates per  retro vesicular ganglion  from three independent cohorts of the indicated  Aβ1-42 strains with moag-4  deletion mutant (del (tm4909)) n=11, moag-4  control mutant (ctrl mutant) n=12, moag-4  charge mutant (charge mutant) n=17, and wild-type n=15. Data are represented as mean ± SD. One-way ANOVA + Bonferroni post-hoc test for significance (* p < 0.05, **** p < 0.0001).",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(E) Oil red O staining show dramatically increased neutral lipids in the head region of lid-1(xd288) and atgl-1(xd314) compared with N2. Scale bar: 10 μm.",
    "molecules": "lipids, Oil red O"
  },
  {
    "caption": "(E) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds supplemented with different fatty acids. AA and EPA but not LA significantly enhance the neurodegeneration triggered by mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. ns: not statistically significant. ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 3, with at least 50 animals per strain analyzed in each experiment.",
    "molecules": "AA, EPA, LA"
  },
  {
    "caption": "(A) Lipidomic data show that the relative content of total C20:3 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (B) Lipidomic data show that the relative content of total C20:4 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (C) Lipidomic data show that the relative content of total C20:5 is slightly increased in atgl-1(xd314); mec-4(d) but is dramatically decreased in fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d), which matches the role of FAT-4 in synthesizing C20:5 (EPA). Data information The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.",
    "molecules": "C20:3, C20:4, C20:5, EPA"
  },
  {
    "caption": "(D) The percentage of C20:3-, C20:4- or C20:5-containing TAGs to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing TAGs to total lipids containing C20:4 is significantly increased in atgl-1 and fat-4 single mutants. The percentage is further increased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.",
    "molecules": "C20:3, C20:4, FA, free fatty acids, C20:5, lipids, phospholipids, PL, PUFA, TAG, TAGs, triacylglycerol"
  },
  {
    "caption": "(E) The percentage of C20:3-, C20:4- or C20:5-containing phospholipids to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing phospholipids to total lipids containing C20:4 is significantly decreased in atgl-1 and fat-4 single mutants. The percentage is further decreased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.",
    "molecules": "C20:3, C20:4, FA, free fatty acids, C20:5, lipids, phospholipids, PL, PUFA, TAG, triacylglycerol"
  },
  {
    "caption": "(G) The percentage of C20:4-containing phospholipids to total lipids that have C20:4 is dramatically decreased in mboa-7(gk399); atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks signify significant differences as compared to mec-4(d). ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.",
    "molecules": "C20:4, lipids, phospholipids"
  },
  {
    "caption": "A) Volcano plot of the fold-change (x-axis) plotted against significance (y-axis) of lipids changing upon HF/HS feeding. Lipids are colored according to fold-change (log2, absolute) > 1 (orange), p-value <0.05 (red) or both (green). Pvalues calculated from differential expression using limma",
    "molecules": "lipids, Lipids"
  },
  {
    "caption": "B) Heatmap of the fold-change (log2) of each lipid in HF/HS compared to chow diet. Only lipid species detected in all mice are shown.",
    "molecules": "lipid"
  },
  {
    "caption": "A) The relative genetic variation of hepatic lipidome composition; all lipids were quantified in proportion to the total lipidome. Each lipid class is shown in a different color where differences can be observed across the strains.",
    "molecules": "lipid, lipids"
  },
  {
    "caption": "B) Heatmap showing correlations between different lipid species (x-axis) and the abundance of gut microbes (y-axis). Microbes were summarized at the levels of order (o_), genus (g_) or family (f_). *=p<0.05, **=p<0.01 Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05).",
    "molecules": "lipid"
  },
  {
    "caption": "A) Heatmap showing correlations among hepatic lipids.",
    "molecules": "lipids"
  },
  {
    "caption": "A) Manhattan plot of genome-wide association for TAG(48:2). Red line shows Bonferroni-corrected threshold of significance calculated based on FaST-LMM pvalues.",
    "molecules": "TAG(48:2)"
  },
  {
    "caption": "(C) Correlations between hepatic expression of Map2k6 and all TAGs identified in the study. Blue represents negative correlations.",
    "molecules": "TAG"
  },
  {
    "caption": "F) Comparison of total hepatic TAG between Map2k6-overexpressing mice (black bar) and control mice (empty bar).",
    "molecules": "TAG"
  },
  {
    "caption": "G) Differences in total phospholipid (PC), total cholesterol (TC) and unesterified cholesterol (UC) between Map2k6-overexpressing mice (black bars) and control mice (empty bars).",
    "molecules": "UC, unesterified cholesterol, cholesterol, PC, phospholipid"
  },
  {
    "caption": "H) Body fat % of both experimental cohorts prior to (week 0) or 8 weeks on a HF/HS diet.",
    "molecules": "fat"
  },
  {
    "caption": "I,J) Plasma concentration of glucose (I) and insulin (J) at the end of the 8 weeks study.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "B-D) Allelic variation plots showing the peak SNP for Ifi203 expression (rs31614030) at the CC or TT allele (x-axis) plotted against expression of Ifi203 (B), levels of hepatic PC(38:3) (C), and plasma insulin levels (D). Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.",
    "molecules": "insulin, PC(38:3)"
  },
  {
    "caption": "E-G) Correlation between the parameters listed above showing significant relationships between hepatic Ifi203 and PC(38:3) (E), hepatic Ifi203 and plasma insulin levels (F) or PC (38:3) and plasma insulin (G). Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)",
    "molecules": "insulin, PC (38:3), PC(38:3)"
  },
  {
    "caption": "Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Ifi203 (I), total PC levels in liver (J) P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).",
    "molecules": "PC"
  },
  {
    "caption": "Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Pemt (K), total liver TAG content (L), or plasma insulin levels (M). P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).",
    "molecules": "insulin, TAG"
  },
  {
    "caption": "(A) ROS burst response in wild-type (WT) and map4k4 lines in response to 1 μM flg22. Values are means ± SEM, n = 16 (biological replicates). Results are representative of three independent experiments. Statistical analysis was performed using a one-way ANOVA with Tukey post-test, p < 0.01. Data information Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.",
    "molecules": "ROS"
  },
  {
    "caption": "(F) qRT-PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour. Values are the means of three biological replicates ± SD, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.",
    "molecules": "H2O"
  },
  {
    "caption": "(C) qRT-PCR analysis of PR1 expression levels in Col-0, map4k4-1 and c867 after mock (MgCl2) or Pst DC3000 inoculation, values are the means ± SD, n = 3 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.",
    "molecules": "MgCl2"
  },
  {
    "caption": "(D) Flg22-protection assay. Col-0, map4k4-1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation. The bacterial population was measured 3 days after infection. Values are the means ± SD, n = 6. **p < 0.01, Results are representative of three independent experiments. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.",
    "molecules": "H2O"
  },
  {
    "caption": "(A) Subcellular localization of MAP4K4-GFP in Arabidopsis mesophyll protoplasts. MAP4K4-GFP (green) partially co-localized with the plasma membrane dye FM4-64 (red). 14 hours after transformation, photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4-64 (< 3 min). Scale bars = 10 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(B) Auto-phosphorylation of recombinant HisMBP-MAP4K4 wild-type, no auto-phosphorylation in the kinase-inactive HisMBP-MAP4K4-D139A protein. The phosphorylation level was determined by Pro-Q Diamond phosphoprotein gel stain (upper panel), protein loading amounts were visualized by Coomassie G-250 staining (lower panel). The predicted molecular weight of HisMBP-MAP4K4 is 118.4 kDa.",
    "molecules": "Pro-Q Diamond phosphoprotein gel, Coomassie G-250, His"
  },
  {
    "caption": "(D) MAP4K4 associates with BIK1 in vivo by Co-IP. BIK1-HA and MAP4K4-GFP were co-expressed in plants by crossing of 35S::BIK1-HA and 35S::MAP4K4-GFP transgenic lines. 14 days old plants were treated (+) with 1 µM flg22 or H2O (-) for 10 min. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of BIK1-HA, MAP4K4-GFP is 49.3 kDa and 103.8 kDa respectively.",
    "molecules": "H2O"
  },
  {
    "caption": "(B) MAP4K4 regulates BIK1 accumulation through the proteasome pathway. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with DMSO (-) or the proteasome inhibitor MG132 (50 µM) for 5h, then the plants were collected for immunoblot analysis.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(C) Flg22-induced BIK1 phosphorylation assay. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with H2O (-) or 1 µM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot.",
    "molecules": "H2O"
  },
  {
    "caption": "(D, E and F) Identification of phosphorylation sites by LC-MS/MS analysis. Five residues in GST-BIK1-K105E were phosphorylated in vitro by HisMBP-MAP4K4. The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions. (G) S233 and T242 were required for BIK1 hyper-phosphorylation. BIK1-HA, BIK1S233-HA and BIK1S242-HA mutants were expressed in Col-0 protoplasts and treated with 100 nM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot. Each experiment was performed at least three times with similar results. ",
    "molecules": "His"
  },
  {
    "caption": "(B) Identification of phosphorylation sites by LC-MS/MS analysis. In vitro phosphorylation site of HisMBP-PP2C38 by HisMBP-MAP4K4. The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions.",
    "molecules": "His"
  },
  {
    "caption": "(E) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity. HisMBP-MAP4K4 was incubated with HisMBP-PP2C38 or HisMBP-PP2C38D88N D289N (phosphatase-inactive) or alone, and the phosphorylation level of MAP4K4 was determined by Pro-Q Diamond phosphoprotein gel stain. All the experiments were performed three times with similar results.",
    "molecules": "Pro-Q Diamond phosphoprotein gel, His"
  },
  {
    "caption": "(A) BIK1 transgene restores flg22-induced ROS burst in map4k4-1. 35S::BIK1-HA WT or 35S::BIK1-HA map4k4-1 were generated by crossing as described before. ROS burst response in the indicated genotypes in response to 1 μM flg22. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.",
    "molecules": "ROS"
  },
  {
    "caption": "(C) ROS burst response in Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.",
    "molecules": "ROS"
  },
  {
    "caption": "(G) ROS burst response in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants. Values are means ± SEM, n = 16 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.",
    "molecules": "ROS"
  },
  {
    "caption": "C Overabundance of MIAA  in  LAM plasma. Top panel shows each control and patient  group (number (n) of samples are indicated); bottom panel shows aggregation of related pulmonary  diseases. The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; top panel, control-LAM P = 0.048, LAM-Langerhans P = 1x10-4, LAM-Sjögren P = 1x10-4, LAM-lupus P = 1x10-3,  LAM-emphysema P = 0.041; bottom panel,  LAM-Langerhans P = 7x10-6 and control-other P = 0.031). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test P = 6x10-4 (top panel) and P = 3x10-5 (bottom panel).",
    "molecules": "MIAA"
  },
  {
    "caption": "E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin-treated (number (n) of samples are indicated). The asterisk indicates significant difference based on two-sided Mann-Whitney test (P = 0.043). Average values are indicated with lilac-colored lines.",
    "molecules": "MIAA, rapamycin"
  },
  {
    "caption": "G Plasma  levels of three other metabolic products derived from monoamine  metabolism and showing significant differences from controls and/or between LAM  and related pulmonary diseases  (individual samples indicated in panel C). The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, and ****P < 0.0001; 4-HPA, control-LAM P = 0.024 and LAM-other P = 0.089; HVA, control-LAM P = 0.008 and LAM-other P = 0.013; and VMA, control-LAM P = 1x10-4 and LAM-other P = 0.10). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test; 4-HPA P = 0.095; HVA P = 0.013; and VMA P = 0.005.",
    "molecules": "4-HPA, HVA, monoamine, VMA"
  },
  {
    "caption": "A Overabundance of  MIAA  in LAM plasma  (UK cohort not treated with rapamycin) relative to healthy  women. The number (n) of individuals in each group is indicated; one of 20 healthy controls did not pass the quality controls for  LC-MS/MS. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.018). Average values are indicated with lilac-colored lines. B Overabundance of MIAA  in plasma of LAM patients with  AMLs. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.042). The number (n) of individuals in each group is indicated. Average values are indicated with lilac-colored lines. ",
    "molecules": "MIAA, rapamycin"
  },
  {
    "caption": "A Representative images of immunohistochemical  positivity of  MAO-A/B in LAM lung  lesions  (top panels, two patients) and lung  tissue from healthy (non-LAM) individuals (bottom panels). In total, seven LAM patients and three healthy controls were analyzed. Brown-stained cells, counter-stained with hematoxylin, are considered positive. Scale bars are shown.",
    "molecules": "hematoxylin"
  },
  {
    "caption": "B Representative images of  immunofluorescence detection of MAO-A/B and αSMA in  LAM lung lesions, nuclei stained with DAPI (merged).",
    "molecules": "DAPI"
  },
  {
    "caption": "D Western blot  results (independent experiments n = 4) from Tsc2-deficient and wild-type  MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10%  FBS. Loading controls are shown. The inferred  protein expression level is indicated by the ratio between the corresponding signal and loading control, standardized to the basal setting",
    "molecules": "DMSO, everolimus"
  },
  {
    "caption": "B  Flow cytometry  results (independent experiments n = 3) showing a higher percentage of MitoSOX red-positive cells in Tsc2-deficient MEFs. C Flow cytometry results (independent experiments n = 3) showing higher intensity (X-axis, single channel intensity FL3 670 nm) of MitoTracker red-positive cells in  Tsc2-deficient MEFs. ",
    "molecules": "MitoSOX red, MitoTracker red"
  },
  {
    "caption": "D Higher  basal cell respiration (as measured by oxygen consumption, Y-axis) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 5x10-4; replicates/condition n = 5, and independent experiments n = 3).",
    "molecules": "oxygen"
  },
  {
    "caption": "F Left panel, western blot results showing overexpression of catalase (CAT) in Tsc2-deficient MEFs, and unaffected by exposure to everolimus (independent experiments n = 2). The CAT expression level is indicated by the ratio of the corresponding signal relative to loading control and basal setting (noted as 1(ref)). Right panel, Cat overexpression (log10-fold change) in  Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 1x10-2; replicates/condition n = 3, and independent experiments n = 2). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).",
    "molecules": "everolimus"
  },
  {
    "caption": "G Representative images of  immunohistochemical  detection of acrolein in  LAM lung  lesions.",
    "molecules": "acrolein"
  },
  {
    "caption": "A Overabundance of histamine in Tsc2 wild-type relative to Tsc2-deficient MEFs, both growth in DMEM 10% FBS. The asterisks indicate significant difference with two-sided t-test (P = 0.017); replicates/condition n = 5, and independent experiments n = 2). Average values are indicated with lilac-colored lines.",
    "molecules": "histamine"
  },
  {
    "caption": "D  Western blot results of HRH1, SLC22A3, and loading control from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% or 0.5% FBS  (independent experiments n = 2). The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal setting",
    "molecules": "DMSO, everolimus"
  },
  {
    "caption": "F Evaluation of cell viability inhibition by  loratadine alone (concentrations shown on X-axis, from 0 to 100 μM) or combined with everolimus (fixed to 20 nM) in Tsc2-deficient and wild-type MEF cultures grown in DMEM 10% FBS for 72 hours. The synergistic combination index (CIx < 1) is shown (replicates/condition n = 3, independent experiments n = 3). Each data point represents the mean and SD.",
    "molecules": "everolimus, loratadine"
  },
  {
    "caption": "H Percentages of viability of Tsc2-dificient and wild-type MEFs exposed to DMEM 10% or 0.5%  FBS with or without L-histidine for 72 hours. The asterisks indicate significant differences with one-sided t-test (10%  FBS, P = 0.017; 0.5%  FBS, P = 5x10-4; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD.",
    "molecules": "L-histidine"
  },
  {
    "caption": "B Inhibition of Tsc2-deficient 105K tumor growth with different monotherapies, as indicated in the inset. Asterisks indicate significant reductions relative to vehicle (two-way ANOVA; clorgyline P = 0.015, loratadine P = 0.018, and rasagiline P = 0.049). Each data point represents the mean and SEM.",
    "molecules": "clorgyline, loratadine, rasagiline"
  },
  {
    "caption": "C Further reduction of Tsc2-deficient 105K tumor growth by  rapamycin  combined with clorgyline or loratadine, relative to rapamycin alone. Asterisks indicate significant reductions relative to  rapamycin alone (two-way ANOVA; clorgyline P = 0.035, loratadine P = 0.045). Each data point represents the mean and SEM.",
    "molecules": "clorgyline, loratadine, rapamycin"
  },
  {
    "caption": "F Inhibition of Tsc2-deficient 105K tumor growth with Hrh1 expression depletion. Asterisk indicates significant reduction relative to control pLKO (two-way ANOVA; P = 0.035) . Each data point represents the mean and SEM. G Inhibition of Tsc2-deficient 105K tumor growth with administration of α-methyl-DL-histidine. Asterisk indicates significant reduction relative to vehicle (two-way ANOVA; P = 0.028). Each data point represents the mean and SEM. ",
    "molecules": "α-methyl-DL-histidine"
  },
  {
    "caption": "A Top panels, western blot results of HRH1 and phospho-Ser616 DRP1  expression in vehicle control and single-drug treated tumors. Bottom panels show quantifications; significant differences correspond to one-sided t-test (*P = 0.011 and **P = 2x10-3; tumors/group n = 4). Average values are indicated with lilac-colored lines. B Western blot  results showing increased HRH1 expression in combinations relative to rapamycin alone, reduced expression of S6 total in the rapamycin-loratadine combination, and raised phospho-Ser616 DRP1 expression in this combination. Bottom panels show quantifications and significant differences with one-sided t-test (HRH1, clorgyline P = 0.011, loratadine P = 0.049, rasagiline P = 0.021; total S6, loratadine P = 4x10-4; phospho-Ser616 DRP1,  loratadine P = 0.037; tumors/group n = 4). Average values are indicated with lilac-colored lines. ",
    "molecules": "clorgyline, loratadine, rasagiline, Ser, rapamycin"
  },
  {
    "caption": "E Representative images of  hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with vehicle or monotherapies. Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis, whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology. Scale bars are shown.",
    "molecules": "eosin, hematoxylin, rapamycin"
  },
  {
    "caption": "F Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with rapamycin alone or rapamycin combinations. With the addition of a second drug to rapamycin, tumors more frequently tended to show glandular differentiation and less atypia. Scale bars are shown.",
    "molecules": "eosin, hematoxylin, rapamycin"
  },
  {
    "caption": "B, LC3-II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS(Ø), FUS-R495x, FUS(Ø)-R495x transfected cells and FUS(Ø)-R495x and altFUS co-transfected cells across 3 independent experiments (n=3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3-II abundance Statistical significance is relative to the mock condition unless otherwise indicated (**** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction).",
    "molecules": "bafilomycin"
  },
  {
    "caption": "C-E, Locomotion assay represented by the percentage of climbing success in control and RU-486-treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS(Ø) (D), and the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x (E) at day 1, 10 and 20 post-induction. Statistical comparisons were made between each population (n=4 - biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU-486-treated population (mean ± SD, n.s. = non-significant, * = p value < 0.05, *** = p value < 0.001, two-way ANOVA with Tukey's multiple comparison correction).",
    "molecules": "RU-486"
  },
  {
    "caption": "C. Pull-down with RNase J1-8xHis tag from stationary phase cells The samples then either were (lane 9) or were not (lane 10) treated with RNase A to detect whether the interaction was via RNA. Lanes 11, 12 - purified proteins were used as markers. The experiment was performed three times (biological replicates) with the same result.",
    "molecules": "RNA"
  },
  {
    "caption": "B. Representative primary data - polyacrylamide gel (the experiment was performed 3x with the same results). Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), 17-18 nt long fragments (RNase J1 stopped by RNAP; indicated with asterisks) and <5 nt fragments (RNA released from TC into buffer - indicative of TC disassembly) are shown; lane 3, the same as lane 1 but included also incubation with RNase R (R); lanes 4 and 5, TCs were denatured by heat prior to RNase addition to demonstrate the activity and cleavage patterns of both enzymes; lanes 6 and 7, the same as lanes 2 and 3 but the buffer was washed off (TCs were retained by streptavidin beads) to demonstrate which RNA fragments were associated with TC; lanes 8, 9 (M1, M2) Mw marker generated by treating the 30 nt RNA with alkali and formamide (M1-4 min treatment, M2-7 min treatment). As reported in (Costanzo et al., 2016) [and references therein], the cleavage by alkali or formamide leaves the phosphate group of the attacked phosphodiester bond bound at 3', initially in the 2',3' cyclic form (upper band in the band couples). This successively opens (lower band in the band couples) yielding a double-banded pattern for short oligoribonucleotides.",
    "molecules": "alkali, formamide, RNA, RNA fragments, streptavidin"
  },
  {
    "caption": "A. Primary data - polyacrylamide gel - a representative result. For description of bands/fragments see next panel legend. Asterisks indicate STUBS (16-19 nt). B. A schematic representation of quantitation of fragments. OUTSIDE (16-30 nt) are RNA fragments that originated by digestion of the full length RNA by RNases that were stopped either before reaching or at the point of reaching RNAP; STUBS (16-19 nt) are RNA fragments that originated by digestion of the full length RNA that were stopped at the point of reaching RNAP; INSIDE (< 16 nt) are RNA fragments (oligonucleotides) that could be only generated after the disassembly of the complex by the torpedo mechanism. The red color indicates parts of RNA that were digested. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments as explained in (B) and indicated in (A). The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments calculated from three biological replicates. ",
    "molecules": "oligonucleotides, RNA, RNA fragments"
  },
  {
    "caption": "A. TCs were assembled on DNA-RNA scaffolds (DNA was biotinylated and attached to streptavidin coated magnetic beads); TCs were then divided into three tubes and challenged with buffer ("-", mock treatment) or RNase J1 of Xrn1. Dissociation of RNAP from DNA was monitored by detecting RNAP with anti-β antibody in two fractions: B - beads (RNAP still bound to DNA), R - released (free in buffer). The gel strip shows representative primary data (Western blot). The graph shows averages (the bars) of two experiments (biological replicates) and the error bars show the range. The combined signal for B+R for each treatment was set as 100 %.",
    "molecules": "DNA, streptavidin"
  },
  {
    "caption": "B. Primary data - polyacrylamide gel. RNAP from E. coli. Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), lane 3, the same as lane 1 but included also incubation with Xrn1. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments",
    "molecules": "RNA"
  },
  {
    "caption": "Representative images of changes in intracellular calcium, as measured in fluorescence ratio images (F340/380) (A) Examples show a TRPM8-expressing DRG neuron (left in A responding to both the TRPM8 agonist menthol (300 μM) and to a cold stimulus from 32°C to 4°C a neuron that responds to cold (right in A but does not express any of TRPM8, TRPA1 or TRPC5, as shown by lack of response to the TRPM8 agonist menthol (300 μM) Calcium increase is due to influx from extracellular solution",
    "molecules": "menthol, calcium, Calcium"
  },
  {
    "caption": "experiments showing an SCG neuron responding to the TRPA1 agonist AITC (left in E and a novel cold-sensitive SCG neuron that responds to cold but does not express any of TRPM8, TRPA1 or TRPC5 (right in E Many glial cells are also visibly activated by AITC but not by cold.",
    "molecules": "AITC"
  },
  {
    "caption": "(C) Mean cold-sensitive increase in calcium-dependent fluorescence ratio F340/380 in SCG neurons is significantly smaller in neurons recorded in voltage-clamp at -60mV (0.56 ±0.08, mean ± SEM, n=21 neurons) than in neurons recorded in current-clamp mode (1.16 ±0.29, n=11 neurons, p=0.0165, unpaired t-test).",
    "molecules": "calcium"
  },
  {
    "caption": "(F) Summary of results of experiments on SCG neurons (mean ±SEM, n=115 neurons, 4 separate cultures). YM58483 caused a significant decrease in cold-response amplitude (p<0.0001, RM one-way ANOVA + Dunnett's test), with only partial recovery. Temperature trace below. (G) In neurons with a small response to cold (F340/380 increase <0.2; n=24 neurons), many responses are potentiated by verapamil (31% of cold-insensitive neurons) and are fully blocked by ORAI channel antagonist YM58483 (p<0.0001, RM one-way ANOVA + Dunnett's test).",
    "molecules": "verapamil, YM58483"
  },
  {
    "caption": "(D) Representative fluorescence images of SCG neurons from neonatal mice (age P5-7) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1. Neurons were cooled from 35°C to 7°C in the presence of 100μM verapamil. Electroporation was indicated by expression of mCherry (# in left-hand panel); in these neurons a cold response was absent (# in right-hand panel) while in neurons not expressing mCherry (*) cold responses were still observed. Scale bar 20μm.",
    "molecules": "verapamil"
  },
  {
    "caption": "(A) Representative immunofluorescence images showing adult mouse stellate ganglion (SG) sections stained for nNOS (red), to label nitrergic neurons, βIII Tubulin (green) to label all neurons, and DAPI to label nuclei. nNOS is widely expressed in neurons of the stellate ganglion, but is absent from glial cells (glial nuclei identifiable in merged image, similar results in n=5 animals). Antibody against nNOS from Abcam; similar results obtained with antibodies from ECM and Millipore. All three antibodies show nNOS widely expressed in SG cell bodies and neurites.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Adult mouse SCG (n=2 animals, upper) and SG (n=5 animals, lower) sections stained for nNOS (red), tyrosine hydroxylase (TH, green) and DAPI (blue) to label nitrergic neurons, noradrenergic neurons and nuclei respectively. Many noradrenergic cells also express nNOS.",
    "molecules": "DAPI, noradrenergic"
  },
  {
    "caption": "Schematic representation of the upstream region of myogenin and amplified regions by RT-PCR (top). RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes (bottom). The presence or absence of reverse transcriptase (RT) is shown by (+) or (-), respectively. The templates (cDNA or genomic DNA) are indicated by C or G, respectively.",
    "molecules": "cDNA, genomic DNA"
  },
  {
    "caption": "The primers used for RT-PCR (top). Strand-specific RT-PCR for the novel transcripts at the upstream region of myogenin in C2C12 myotubes using total RNA (middle and bottom) and poly(A)+ RNA (bottom).",
    "molecules": "poly(A)+ RNA, RNA"
  },
  {
    "caption": "Immunocytochemistry for the detection of myogenin at 24 h (G) or MHC at 72 h (H) in Myoparr-depleted C2C12 cells after differentiation induction. Nuclei were counterstained with DAPI. Bar, 100 μm. The percentage of myogenin-positive cells or fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01. ***p < 0.001 (unpaired two-tailed Student's t-test or unpaired two-tailed Welch's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "Significantly decreased MHC expression in Ddx17-depleted C2C12 cells is shown by immunocytochemistry. Nuclei were counterstained with DAPI. Bar, 100 μm. Fusion index is shown as percent of the control. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Immunocytochemistry for myogenin in Ddx17-depleted C2C12 cells. Bar, 100 μm. Myogenin-positive cells are shown as percent of the control. n = 4, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "Myoparr and Ddx17 are required for C2C12 cell cycle withdrawal. C2C12 cells transfected with each siRNA were cultured in growth medium for 24 h. After differentiation induction, cells were maintained in differentiation medium for 40 h and then treated with EdU for 6 h. EdU-positive cells are shown as percent of the control. Nuclei were counterstained with Hoechst 33342. n = 3, mean ± SD. **p < 0.01 (unpaired two-tailed Student's t-test). Bar, 100 μm.",
    "molecules": "Hoechst 33342, EdU"
  },
  {
    "caption": "C-E. Logistic statistical model based on the (C) single cell sequencing, (D) ImageStream (E) analysis or automated FISH of RPE-1 cells in untreated condition (blue circles) or treated with Auxin for 48 h (red squares). Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated (blue line) or Auxin-treated (red line) condition. Red asterisks (IAA) and Blue (Untreated) indicate significance over the respective mean using a binomial test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.",
    "molecules": "Auxin, IAA"
  },
  {
    "caption": "B-C. (B) Micronuclei frequency formation by live cell imaging in untreated (NT) and Auxin-treated (IAA) condition. (C) Bar graph represents the type of chromosome mis-segregation (independently of the dCas9 signal) observed in the indicated conditions, with (IAA) or without (NT) auxin, in the cell lines expressing dCas9 mScarlet-I and sgRNA targeting chromosome 1 or 3. Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together (n = 45 - 149 cells).",
    "molecules": "Auxin, auxin, IAA"
  },
  {
    "caption": "D. Representative live cell imaging of RPE-1 cells dCas9 mScarlet-I with sgRNA targeting chromosome 1 (red dots) starting from metaphase and showing example of correct (untreated) or mis-aligned chromosome (auxin) leading to the formation of a micronucleus. Yellow arrows mark mis-aligned- and micronucleus-containing chromosome 1. Cells were imaged every five minutes. The numbers indicate the time point at which the presented images were taken. Cells were fixed at the end of the movie to detect Cas9 with an antibody. Scale bar represents 10 µm.",
    "molecules": "auxin"
  },
  {
    "caption": "E. Bar graph representing the proportion of chromosome mis-segregation observed in both cell lines after auxin addition. Only movies in which the dCas9 mScarlet-I dots were clearly visible during the whole division were taken into consideration for the analysis (N = 6 - 10 cells).",
    "molecules": "auxin"
  },
  {
    "caption": "F. Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP-A depletion (28 h auxin). Chromosomes are stained using whole (X) or centromeric (6) FISH probes. Yellow arrows mark lagging chromosomes. Scale bar represents 5 µm. G-H. Bars represent the (G) proportion of chromosome mis-segregation and (H) the type of the different chromosome mis-segregations observed. Error bars represent the SEM of two replicates. n > 66 mis-segregation events. Paired t test lagging chromosomes vs mis-alignment chromosomes, *p = 0.0275. ",
    "molecules": "auxin"
  },
  {
    "caption": "E-F. Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP-C binding following CUT&RUN, sequencing and centromere mapping. Cells were treated for 6 h with IAA to deplete CENP-A. Error bars represent the SEM of three independent experiments and the dashed line represents the mean. Acrocentric chromosomes 13-14, 21 and 22 were marked by a line. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001.",
    "molecules": "IAA"
  },
  {
    "caption": "B. Representative image of centromere 11 (red) and centromere X (green) FISH on RPE-1 CENP-AAID CENP-B KO mutant in control (top) or IAA treated condition (bottom). Scale bar represents 5 µm. Bar graphs show automatic FISH quantification of chromosome mis-segregation in the indicated cell lines with and without IAA addition for 24 h. Error bars represent the SEM of three independent experiments (n> 200 cells per experiment). Dunnett's multiple comparisons test was used to compare conditions. Grey lines separate independent experiments. Fold changes between grey and red bars are also indicated.",
    "molecules": "IAA"
  },
  {
    "caption": "C. Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound. D. Representative image of centromere 17 (red) and centromere X (green) FISH on RPE-1 cells treated with the BAL27862 compound. Scale bar represents 5 µm. ",
    "molecules": "BAL27862"
  },
  {
    "caption": "E. Bars represent the frequency of chromosome mis-segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration. Error bars represent the SEM of three independent experiments, dashed line represents the mean. n > 40 micronuclei were analyzed. Dunnett's multiple comparisons test, *: p<0.05. DMSO was used at the same concentration of BAL27862.",
    "molecules": "BAL27862, DMSO"
  },
  {
    "caption": "B. Representative image of CENP-B box (red) and centromere 3 (green) FISH on RPE-1 interphase nucleus after 48 h IAA treatment. CENP-B box signal at individual homologs are magnified in insets. Scale bar represents 5 µm.",
    "molecules": "IAA"
  },
  {
    "caption": "E. Immunoblot shows expression of the (ΔN)CENP-B-dCas9 after doxycycline induction.",
    "molecules": "doxycycline"
  },
  {
    "caption": "F. Representative images show a micronucleus or the main nucleus containing the Y chromosome (red) detected by FISH in no-doxycycline or doxycycline treated cells respectively. Chromosome X is shown in green. Scale bar represents 5 µm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "G. Bar plot shows the mean (n ≥ 3) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM. n > 200 cells with a micronucleus. Mann-Whitney test. *p=0.0173.",
    "molecules": "doxycycline, IAA"
  },
  {
    "caption": "C. Plot shows the karyotype distribution of DLD-1 and RPE-1 cells in untreated or Auxin-treated condition",
    "molecules": "Auxin"
  },
  {
    "caption": "A  Growth kinetic  measurements of SM exposed to AMSIN or various antibiotics with known cellular targets. The peptide or the compounds used were 4×CL.  VAN,  vancomycin; RIF, rifampicin; KAN, kanamycin. OD600 were calculated as measured values subtracting the background value from broth medium (OD600 = 0.356).",
    "molecules": "KAN, kanamycin, RIF, rifampicin, VAN, vancomycin"
  },
  {
    "caption": "D  Scanning electron microscopic  observation of AMSIN-induced BM  deformation. The doses used were 10×CL for AMSIN and vancomycin.",
    "molecules": "vancomycin"
  },
  {
    "caption": "E Cytoplasmic  accumulation of the soluble cell-wall precursor UMP  in vancomycin-treated (blue line), AMSIN-treated (red dashed line) or not treated (green line) culture of MRSA P1374. Inset: UMP  identified by  UPLC-HRMS with singlely and doubly protonated m/z observed.",
    "molecules": "UMP, vancomycin"
  },
  {
    "caption": "H  ESI-MS detecting the non-covalent complex between AMSIN  and  UMP. The quadruply protonated complex (m/z 1304.8) is observed. Inset: comparison of intensities of the complex between two ratios of  UMP:AMSIN  Here, AMSIN is abbreviated as AMS due to space limitation.",
    "molecules": "UMP"
  },
  {
    "caption": "J The  dose-response unit curve of  UMP binding to AMSIN, which is fitted by the BIAevaluation v2.0.1. software using 1:1 Langmuir binding  model.",
    "molecules": "UMP"
  },
  {
    "caption": "C Comparison of cytotoxic effects of AMSIN and LL-37 on  HL-60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk; *P<0.05, ***P<0.001, ns: no significance",
    "molecules": "LL-37"
  },
  {
    "caption": "D An  inhibition-zone assay  showing the inability of  AMSIN  to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann-Whitney U (ns: no significance",
    "molecules": "DNA"
  },
  {
    "caption": "F Peptide-induced  IL-8 release. A549  cells were treated with AMSIN or  LL-37 for 24 h.  IL-8 levels in culture supernatants from the FBS-containing medium (left) or the serum-free medium (right) were measured with an  ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ***P<0.001, ns: no significance G Peptide-induced MCP-1 release. HL-60 cells were treated with AMSIN or  LL-37 for 24 h. MCP-1 levels in culture supernatants from the  FBS-containing medium (left) or the serum-free medium (right) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student's t-test or Mann-Whitney U test (the latter indicated by a red asterisk or ns; *P<0.05, **P<0.01, ns: no significance",
    "molecules": "LL-37"
  },
  {
    "caption": "Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(d) A cryo-immuno-EM image of a GFP-Rab34 transfected HeLa cell. GFP-Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34 positive membranes. Sites of contact are highlighted by arrows.",
    "molecules": "gold"
  },
  {
    "caption": "(f) Western blot analysis of GST-RILP pull down showing direct interaction between RILP and GTP hydrolysis deficient (Q111L) Rab34 (at 200nM, in the presence of 1mM GTPγS) and FLCN-DENN domain (1μM), but not low nucleotide affinity (T66N) Rab34. FLCN-DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP / Rab34 Q111L binding. Error bars show SEM. ** = p<0.01.",
    "molecules": "GTPγS"
  },
  {
    "caption": "(h) Western blot analysis of GST-RILPpull down experiments with GTPγS loaded (in the presence of 1mM GTPγS) and GDP loaded (in the presence of 1mM GDP) wildtype Rab34, showing FLCN-DENN (500nM) induced interaction with RILP.",
    "molecules": "GDP, GTPγS"
  },
  {
    "caption": "C. The kinetics of EYFP-Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separate compartments of the same dish, enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment (A&O, both 10 µM). The spatial heterogeneity of EYFP-Parkin (coefficient of variation of the intensity of individual pixels) was estimated for individual cells for each time point (n = 39-47 cells; P < 0.0001 difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)",
    "molecules": "antimycin, oligomycin"
  },
  {
    "caption": "D. Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 µM) for 3h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVA",
    "molecules": "DMSO"
  },
  {
    "caption": "A. The pattern of Parkin translocation caused by Miro1 overexpression is different from A&O-induced translocation. The figure illustrates puncta-like EYFP-Parkin on mitochondria in antimycin and oligomycin-treated (both 10 µM for 3h) PC6 cells (middle panels), whereas in Miro1-overexpressing cells, EYFP-Parkin appears along rod-shaped mitochondria visualised with CFP-mito (lower panels). The merged panels present EYFP-Parkin (green) and CFP-mito (red)",
    "molecules": "antimycin, oligomycin"
  },
  {
    "caption": "Miro1 overexpression does not cause mitochondrial depolarization (D) Representative image of Miro1 and EYFP-Parkin-transfected cells stained with TMRE. The merged panels present EYFP-Parkin (green) and TMRE (red)",
    "molecules": "TMRE"
  },
  {
    "caption": "Miro1 overexpression does not cause mitochondrial depolarization (E) Relative TMRE intensity quantified in neurons transfected with Miro1 (detected by CFP-mito co-transfection); the control group is non-transfected cells in the same image. The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs-expressing neurons. Note that FCCP treatment (3 μM for 15 min) almost completely abolished TMRE fluorescence. ****P < 0.0001 vs control group, n = 94-186 cells from 4 dishes per group, Kruskal-Wallis test",
    "molecules": "TMRE, FCCP"
  },
  {
    "caption": "G. Parkin overexpression does not induce degradation of myc-Miro1 in control conditions. Representative Western blot image and analysis of myc-Miro1 expression in HEK cells. Treatment with A&O (both 15 µM) for 3h in the absence of MG132 led to slight degradation of myc-Miro1. Co-expression of EYFP-Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present. However, Parkin co-expression enhanced Miro1 degradation when cells were treated with A&O. *P < 0.05 and ****P < 0.0001, ns: not significant, n = 4 independent experiments, one way ANOVA. Note that only the main band of myc-Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP-Parkin",
    "molecules": "MG132"
  },
  {
    "caption": "H. EYFP-Parkin co-immunoprecipitates with overexpressed myc-Miro1, both in DMSO- or A&O (both 15 µM for 3h) treated HEK cells",
    "molecules": "DMSO"
  },
  {
    "caption": "D. Parkin mutants T240R and C431N interact with Miro1 both in DMSO- or A&O-treated (both 15 µM for 3h) HEK cells. Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin-expressing groups.",
    "molecules": "DMSO"
  },
  {
    "caption": "A. Miro1 K572R mutant interacts with HA-Parkin similarly to Miro1 WT. HEK cells overexpressing myc-Miro1 WT or K572R mutant were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probed for myc-Miro1 and HA-Parkin. Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP, which is missing in the case of the K572R mutant",
    "molecules": "DMSO"
  },
  {
    "caption": "B. Miro1 K572R ubiquitination by Parkin is reduced. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1 K572R and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin-HA and myc-Miro1",
    "molecules": "DMSO, ubiquitin"
  },
  {
    "caption": "A. A Ca2+-insensitive mutant of Miro1 (Miro1-EF) interacts with Parkin. HEK cells overexpressing myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red) and Parkin (green). The high-contrast grayscale image from the green channel of this blot shows that endogenous Parkin co-immunoprecipitated with Miro1 in all conditions. The bar chart shows the quantification of co-immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP-Parkin signal (*P < 0.05 and **P < 0.01, repeated measures ANOVA",
    "molecules": "EYFP, Ca2+, DMSO"
  },
  {
    "caption": "B. Miro1-EF is less susceptible to ubiquitination by Parkin. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin (red), Miro1 (green) and Parkin (green; representative images are from the same experimental material on separate SDS-PAGE-WB membranes). The high-contrast grayscale shows the green channel of this blot, and the bar chart shows quantification of co-immunoprecipitated Miro from 3 different blots (**P < 0.01 and ***P < 0.001, repeated measures ANOVA). Note that EYFP-Parkin co-immunoprecipitated with ubiquitin only after A&O treatment",
    "molecules": "DMSO, ubiquitin"
  },
  {
    "caption": "C. Mutation of Miro1 EF-hands diminishes Miro1 ubiquitination with S65-phosphorylated ubiquitin. HEK cells were transfected with EYFP-Parkin with either myc-Miro1-WT or myc-Miro1-EF with or without PINK1-V5. Cells were treated with either DMSO or A&O (both 10 µM) for 5 hrs with the exception of the PINK1-V5 group. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red), S65-Phosphoubiquitin and PINK1-V5 (green; the weak bands marked with the asterisk are nonspecific).",
    "molecules": "DMSO, S65-phosphorylated ubiquitin, S65-Phosphoubiquitin"
  },
  {
    "caption": "A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)",
    "molecules": "Ca2+, BAPTA-AM"
  },
  {
    "caption": "B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)",
    "molecules": "Bay K 8644, Ca2+"
  },
  {
    "caption": "Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescence",
    "molecules": "Ca2+, glutamate, glycine"
  },
  {
    "caption": "Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test)",
    "molecules": "Ca2+, glutamate, Glutamate, glycine"
  },
  {
    "caption": "E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA)",
    "molecules": "Glutamate, glutamate, glycine"
  },
  {
    "caption": "G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA)",
    "molecules": "Glutamate, glutamate, glycine"
  },
  {
    "caption": "H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test)",
    "molecules": "Glutamate, glutamate, glycine"
  },
  {
    "caption": "I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch\"s A",
    "molecules": "ADP, ATP, Glutamate, glutamate, glycine"
  },
  {
    "caption": "J. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).",
    "molecules": "Glutamate, glutamate, glycine"
  },
  {
    "caption": "A. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.",
    "molecules": "carbohydrates, fat, sucrose"
  },
  {
    "caption": "C. Blood glucose levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "E. Blood glucose levels for insulin tolerance test of control (n=7) and NICDiOE-EC control (n=7) mice. Data represent mean ± SEM, unpaired t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "G. Blood glucose levels for glucose tolerance test of control (n=6) or NICDiOE-EC (n=7) mice. Data represent mean ± SEM, unpaired t-test. H. Quantification of area under the curve for glucose tolerance test in G. Data represent mean ± SEM, unpaired t-test. ",
    "molecules": "glucose"
  },
  {
    "caption": "A. Blood glucose levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "C. Blood glucose levels for insulin tolerance test of control (n=10) and Rbpji∆EC (n=7) mice. Data represent mean ± SEM, unpaired t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "E. Blood glucose levels for glucose tolerance test of control (n=6) or RbpjiΔEC (n=5) mice. Data represent mean ± SEM, unpaired t-test. F. Quantification of area under the curve for glucose tolerance test in E. Data represent mean ± SEM, Welch's t-test. ",
    "molecules": "glucose"
  },
  {
    "caption": "A. Uptake of FITC-insulin and CAV1 expression in primary human umbilical venous ECs (HUVECs) upon Notch induction. Scale bar 20 µm.",
    "molecules": "FITC"
  },
  {
    "caption": "B. Uptake of FITC-insulin and CAV1 expression in (HUVECs) upon Notch blockage. Scale bar 20 µm.",
    "molecules": "FITC"
  },
  {
    "caption": "C. Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. D. Densitometric analysis of western blot in C. n=3, data represent mean ± SEM, unpaired t-test. ",
    "molecules": "insulin"
  },
  {
    "caption": "E. Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. F. Densitometric analysis of western blot in E. n=3, data represent mean ± SEM, unpaired t-test. ",
    "molecules": "insulin"
  },
  {
    "caption": "B. AKT phosphorylation in myogenic C2C12 cells treated with different doses of insulin. n=3, data represent mean ± SEM, unpaired t-test.",
    "molecules": "insulin"
  },
  {
    "caption": "E. Proximity ligation assay showing the association of CAV1 and INSR (insulin receptor) in MCECs upon stimulation with insulin. n=86-120 cells from 8 microscopic high power fields per time point, data represent mean ± SEM, unpaired t-test, *** means p<0.001.",
    "molecules": "insulin"
  },
  {
    "caption": "I. Expression of Cav1, Cav2, Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.",
    "molecules": "carbohydrates, fat, sucrose"
  },
  {
    "caption": "B. Blood glucose levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.",
    "molecules": "2-DG, glucose"
  },
  {
    "caption": "C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.",
    "molecules": "2-DG"
  },
  {
    "caption": "D. 2-DG uptake levels in skeletal muscle of control and NICDiOE-EC mice. n=5, data represent mean ± SEM, unpaired t-test. E. 2-DG uptake levels in visceral white adipose tissue (vWAT) of control (n=4) and NICDiOE-EC (n=5) mice. Data represent mean ± SEM, unpaired t-test. ",
    "molecules": "2-DG"
  },
  {
    "caption": "G. Blood glucose levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.",
    "molecules": "2-DG, glucose"
  },
  {
    "caption": "H. Plasma insulin levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.",
    "molecules": "2-DG"
  },
  {
    "caption": "I. 2-DG uptake levels in skeletal muscle of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test. J. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=5, data represent mean ± SEM, unpaired t-test. ",
    "molecules": "2-DG"
  },
  {
    "caption": "B. Blood glucose levels for glucose tolerance test (GTT) of control (n=4) or RbpjiΔEC (n=5) mice kept on high-fat diet (HFD). Data represent unpaired t-test, mean ± SEM. C. Quantification of area under curve (AUC) for GTT in B. Data represent mean ± SEM, unpaired t-test. ",
    "molecules": "glucose"
  },
  {
    "caption": "D. Blood glucose levels for insulin tolerance test (ITT) of control (n=4) or RbpjiΔEC (n=3) mice kept on HFD. Data represent unpaired t-test, mean ± SEM. E. Quantification of AUC for ITT in D. Data represent mean ± SEM, unpaired t-test. ",
    "molecules": "glucose"
  },
  {
    "caption": "F. HbA1c levels of control (n=4) or RbpjiΔEC (n=6) mice kept on HFD seven weeks after recombination. Data represent mean ± SEM, unpaired t-test.",
    "molecules": "HbA1c"
  },
  {
    "caption": "H. Blood glucose levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.",
    "molecules": "2-DG, glucose"
  },
  {
    "caption": "I. Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.",
    "molecules": "2-DG"
  },
  {
    "caption": "J. 2-DG uptake levels in skeletal muscle of control (n=5) and RbpjiΔEC (n=4) mice on HFD. Data represent mean ± SEM, unpaired t-test. K. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test. ",
    "molecules": "2-DG"
  },
  {
    "caption": "Primary rat cortical neurons transfected with eGFP-GA50 were examined to determine at which time points preceding cell death aggregates are found in neurites. Two days post-transfection, aggregates formed by eGFP-GA50 (green) are detectable in neurites (SMI-32 staining in red). These aggregates remain localized to neurites at 96 hours (4 days) and 288 hours (8 days). Colocalization is indicated by yellow overlay of colors (right panels). Inset below each image shows enlargement of neurite regions containing aggregates, Representative fields from 60x magnification z-stack confocal images, scale bar indicates 20μm. Inset scale bars indicate 5μm.",
    "molecules": "GA50"
  },
  {
    "caption": "Primary rat cortical neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. A) Example of a cortical neuron expressing eGFP-GA100 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.",
    "molecules": "GA, GA100"
  },
  {
    "caption": "Primary rat motor neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. B) Example of a motor neuron expressing eGFP-GA25 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.",
    "molecules": "GA, GA25"
  },
  {
    "caption": "C) Quantification of eGFP-GAn aggregate velocity within cortical neurons.",
    "molecules": "GA"
  },
  {
    "caption": "D) Quantification of eGFP-GAn aggregate velocity within motor neuron neurites.",
    "molecules": "GA"
  },
  {
    "caption": "Primary rat neurons were co-transfected with Td-tomato and eGFP-GAn plasmids or eGFP-alone. The same neurons were imaged at 24-hour intervals to assess viability. A) Representative fields (right) of eGFP and eGFP-GA50 cortical neurons at days 1, 7, and 13 post-transfection follow the survival of individual cells over time (white arrows). 20x magnification, scale bar indicates 25μm. ",
    "molecules": "GA"
  },
  {
    "caption": "Cortical neurons were loaded with FM4-64 dye 48 hours after transfection. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative dye loading showing defined puncta regions along neurites 40x magnification, scale bar indicates 10μm. ",
    "molecules": "FM4-64, KCl"
  },
  {
    "caption": "B) Representative intensities of FM4-64 puncta regions pre- and post- stimulation demonstrate loss of synaptic puncta fluorescence intensity following stimulation in GFP but not GA50 containing neurons. These images visually reflect the process performed in an automated manner through our imaging software to determine puncta fluorescence intensity. An unbiased constant threshold is placed on each image per experiment, then fluorescence intensity is measured over time. The threshold applied isolates puncta from background, giving distinct regions. 8-bit images were thresholded and pseudo-color (red) was applied to enlarged puncta regions of interest, scale bar indicates 10μm.",
    "molecules": "GA50, FM4-64"
  },
  {
    "caption": "C) Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 10 seconds of baseline recording and 10 seconds of the depolarization phase are shown, arrow indicates start of depolarization. Non-linear regression was performed in GraphPad Prism7 using the plateau followed by one phase decay model. The decay constant (k) for GFP was 0.4971 s-1, while for GA50 it was 219.5 s-1. Similarly the tau (τ) values were 2.012 seconds for GFP and 0.00455 seconds for GA50, respectively. Data presented as mean ± SEM for each imaged timepoint. Statistical significance was determined using the Sidak-Bonferroni method, ****p < 0.0001, n=20 puncta regions from 4 biological replicates.",
    "molecules": "GA50"
  },
  {
    "caption": "Primary cortical or motor neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f. After 48 hours, mCherry positive cells were determined. Green fluorescence intensity was then recorded from identified neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative images of a co-transfected cortical neuron expressing mCherry-GA50 (left panel) and GCaMP6f (right two panels). This image shows a representative demonstration of the increase in detectable green fluorescence from the GCaMP6f indicator pre- and post- stimulation (center and right respectively). 40x magnification, scale bar indicates 25μm.",
    "molecules": "GA50, KCl"
  },
  {
    "caption": "B-C) Graphical representation of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The contribution of Ca2+ influx was determined by experiments in which stimulation was performed in ACSF containing high KCl but no Ca2+. Quantification of the peak change in fluorescence in cortical neurons (B) and motor neurons (C).",
    "molecules": "Ca2+, KCl"
  },
  {
    "caption": "eGFP-GAn dipeptides were expressed in primary cortical or motor neurons for 48 hours, then immunostained for neurofilament (gray) and SV2 (red). A-B) Representative z-stack confocal images of neurites from eGFP, eGFP-GA50, and eGFP-GA100 expressing cortical (A) or motor (B) neurons. 60x magnification, scale bar indicates 5μm. ",
    "molecules": "GA"
  },
  {
    "caption": "E) mRNA was Trizol extracted from 3 independent wells of C9-ALS and isogenic i3 cortical neurons. RNA was converted to cDNA using the Superscript First-strand kit, and q-PCR was performed using PowerUp SYBR Green. Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels. Analysis was performed using the ΔΔCT method. 2ΔΔCT ± SE, is presented. Unpaired t-test, **p < 0.01.",
    "molecules": "SYBR Green, Trizol"
  },
  {
    "caption": "A) Cortical neurons were loaded with FM4-64 dye 48 hours after transfection with eGFP-GA50 and transduction with rSV2a-eGFP-pRRL lentivirus. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 60 seconds of baseline recording and 60 seconds of the depolarization phase are shown, arrow indicates start of depolarization. The lack of synaptic release in the case of GA50 expression (red line) was rescued back to levels comparable to eGFP controls (pink line) in the case of coexpression of rSV2a-eGFP-pRRL (orange line). Statistical significance was determined using the Sidak-Bonferroni method, ***p < 0.001, n=10 puncta regions from each of 4 biological replicates.",
    "molecules": "FM4-64, KCl"
  },
  {
    "caption": "B) Cortical neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f, then transduced with rSV2a-eGFP-pRRL. Green fluorescence intensity was recorded from identified mCherry positive neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Quantification of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The significant increase in Ca2+ influx observed following GA50 expression (*p < 0.05) is resolved when rSV2a-eGFP-pRRL is coexpressed. Data presented as mean ± SEM. One-way ANOVA with Sidak's multiple comparisons test, at least 10 cells per condition, pooled from n= 3 independent biological experiments. No significant differences were observed between mCherry or mCherry+SV2 conditions, groups were merged for this analysis.",
    "molecules": "Ca2+, KCl"
  },
  {
    "caption": "(E) Kaplan-Meier survival analysis of motor neurons expressing GA50 or GA50+SV2 followed for 10 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.345, this was decreased to 0.7769 for GA50+SV2. At least 200 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001.",
    "molecules": "GA50"
  },
  {
    "caption": "(Left) Effect of 1,6-Hexanediol treatment on AcGFP-Siwi and AcGFP-Siwi-D670A foci. Z-stacks were taken prior to the addition of 1,6-Hexanediol or medium and after 30 minutes incubation at RT. Z-projections of the middle 6 μm stacks are then normalized and pseudo-colored with Fire LUT to visualize the Siwi foci. Scale bar 10 μm. Scale bars represent pixel intensity in arbitrary units (A.U.) (Right) High granule/cell intensity ratio of Siwi-D670A persists despite 1,6-Hexanediol treatment. Average intensity ratio from each of the independent Z-stacks (n=30 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N = 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.",
    "molecules": "1,6-Hexanediol"
  },
  {
    "caption": "Co-expression of Siwi-D670A increases granule-to-cell intensity ratio of BmSpnE and BmQin foci. (Upper) Representative Z-projections (maximum intensity) of mCherry-BmSpnE and mCherry-BmQin foci. AcGFP signal from FITC channel is not shown. Scale bar 8 μm. (Bottom) Average intensity ratio from each of the independent Z-stacks (n=6 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N ≥ 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.",
    "molecules": "FITC"
  },
  {
    "caption": "SAMHD1 knockdown enhances EV71 replication. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test).",
    "molecules": "RNA"
  },
  {
    "caption": "SAMHD1 knockdown enhances EV71 replication. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, * P < 0.05, ** P < 0.01, paired t-test).",
    "molecules": "RNA"
  },
  {
    "caption": "EV71 viral RNA was detected in cell lysates by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P <0.01, paired t-test).",
    "molecules": "RNA"
  },
  {
    "caption": "RD SAMHD1-HA cell lines were treated with dimethyl sulfoxide (DMSO) or 10 μM MG132 for 12 h prior to harvest and subjected to IB with tubulin as a loading control.",
    "molecules": "dimethyl sulfoxide, DMSO, MG132"
  },
  {
    "caption": "TRIM21 induced the degradation of SAMHD1, and MG132 rescued the TRIM21-mediated degradation of SAMHD1. HEK293T cells were cotransfected with SAMHD1 and VR1012 or TRIM21 expression plasmids with or without MG132 treatment for 12 h prior to harvest, and then subjected to IB with tubulin as a loading control.",
    "molecules": "MG132"
  },
  {
    "caption": "Overexpression of TRIM21 in HEK293T cells increased EV71 replication by degrading SAMHD1. HEK293T cells were transfected with VR1012 or TRIM21-HA for 24 h and then infected with EV71 at a MOI of 0.05. EV71 viral RNA in cell lysates was detected by RT-qPCR with GAPDH as a control (n=3, mean±SD, ** P < 0.01, paired t-test)).",
    "molecules": "RNA"
  },
  {
    "caption": "sh-SAMHD1 and sh-TRIM21 THP-1 cells were treated with PMA for 24 h and infected with the VSV-G pseudotyped reporter HIV-1, then detected 48 h later by flow cytometry analysis (n=3, mean±SD, * P < 0.05, ** P < 0.01, *** P < 0.001, paired t-test).",
    "molecules": "PMA"
  },
  {
    "caption": "TRIM21 interacts with SAMHD1 via PRY and SPRY domains. SAMHD1-flag was cotransfected with VR1012 or TRIM21 WT or the indicated mutant for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.",
    "molecules": "MG132"
  },
  {
    "caption": "SAMHD1-flag 1-547 was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB.",
    "molecules": "MG132"
  },
  {
    "caption": "The microscale thermophoresis curve for the interaction between SAMHD1 and TRIM21. Alexa Fluor®647 labelled SAMHD1 at a concentration of 20 nM were incubated with two-fold dilution series of unlabeled TRIM21 (3 μM to 9.16E-05 μM). The curve represents the signal recorded from three measurements. The normalized fluorescence thermophoretic signals were plotted against the concentration of TRIM21 with mean values ± standard deviation as well as a fitting to a 1:1 binding model (NanoTemper® analysis software, F Norm = F Hot / F Cold). The Kd of this interaction was determined to be 270±92nM. The data was a representive of three independent experiments using different dilution.",
    "molecules": "Alexa Fluor®647"
  },
  {
    "caption": "SAMHD1-flag WT or the G153S or G183R mutant was cotransfected with VR1012 or TRIM21-HA for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB. Tubulin served as a loading control.",
    "molecules": "MG132"
  },
  {
    "caption": "SAMHD1-HA WT and VR1012 or TRIM21 were cotransfected with K48-only and K63-only ubiquitin-flag into HEK293T cells for 24 h, and the cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.",
    "molecules": "MG132"
  },
  {
    "caption": "K48-only ubiquitin-flag and SAMHD1-HA WT or mutant K622R were cotransfected with VR1012 or TRIM21 into HEK293T cells as indicated. The cells were then treated with 10 μM MG132 for 12 h before harvest and subjected to HA IP and IB with tubulin as a loading control.",
    "molecules": "MG132"
  },
  {
    "caption": "(Top) PCR detection of the recombined BrafLox−V600E allele (Lox-V600E) in BVE mouse tissues at 8 weeks of age without tamoxifen induction. Substantial recombination is observed in the lung, while weaker recombination is also detected in the liver. No recombination was detected in hematopoietic tissues (bone marrow and spleen) even after 40 cycles of amplification (right). (Bottom) H&E staining of lung sections from wild-type (WT, 10 weeks of age), BVE (1-10 weeks of age) and Braf+/LSL−V600E mouse 8 weeks after nasal delivery of AdCre is indicated below. V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development. Scale bars, 100 μm.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Intracellular un-esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2+/+ and Npc2+/hypo IMCs. Scale bars, 25 μm. Filipin-stained cells were graded from 0 to 3+ as indicated in the microphotographs (see Materials and Methods for criteria), and % of each grade in IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice is shown in the bar chart. The data were obtained by analysing 176 Npc2+/+ and 242 Npc2+/hypo IMCs from three independent Npc2+/+ and Npc2+/hypo pairs.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Intracellular un-esterified cholesterol distribution as detected by filipin staining in Npc2+/hypo IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 25 μm. Quantitative data in the bar graph were obtained by analysing 265 bNPC2-untreated (-bNPC2) and 252 bNPC2-treated (+bNPC2) cells from three independent cultures using the method described in (A).",
    "molecules": "cholesterol"
  },
  {
    "caption": "Immunoblot analysis of intracellular and secreted (CM) CCL6 in bafilomycin-treated Npc2+/+ IMC culture. IMCs without (left) or with (right) 50 μg/ml bNPC2 pre-loading for 30 min were chased in serum-free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h.",
    "molecules": "bafilomycin, bafilomycin A1"
  },
  {
    "caption": "C. Increased serum levels of the lipid peroxidation marker 4-HNE (a marker of increased oxidative damage) in aged, but not in young VEGF-Ahyper mice. *P-value: 0.0325. N=7 mice/group (2 independent experiments). Graph shows mean ± SEM.",
    "molecules": "4-HNE"
  },
  {
    "caption": "D. VEGF-Ahyper mice have decreased levels of the major antioxidant in the lens, reduced glutathione (μmol/gm lens weight), prior to cataract formation. Lenses with 0 to +1 cataracts from 11-months old mice were used for measurements. ** P-value: 0.0097. N=7/group (2 independent experiments).",
    "molecules": "glutathione"
  },
  {
    "caption": "A-B. Choroidalflat mount staining of a white VEGF-Ahypermouse shows choroidalvessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.",
    "molecules": "fluorescein"
  },
  {
    "caption": "A-B. Choroidalflat mount staining of a white VEGF-A hypermouse shows choroidal vessels (green [fluorescein-conjugated isolectin B4]) from which neovessels (strong red [CD31]) originate and that are covered by a RPEcell layer (white [phalloidin]), which separates the CNV lesion from the photoreceptors. White arrows demarcate CNV lesion. Yellow arrows show round autofluorescent deposits. Scale bars 100 μm.",
    "molecules": "fluorescein"
  },
  {
    "caption": ". Laser-induced CNV experiments demonstrate that treatment with a caspase-1 inhibitor (compared to DMSO treated controls) or a neutralizing TLR2 antibody (compared to isotype matched IgG treated controls) inhibits CNV lesion growth. P-values are shown. N=15 mice/group. Representative CNV lesions in choroidal flat mounts are shown. Scale bars 100 μm.",
    "molecules": "DMSO, caspase-1 inhibitor"
  },
  {
    "caption": "Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2-inhibitor PHOS [42].",
    "molecules": "PHOS"
  },
  {
    "caption": "Graphs showing different parameters of neuronal migration dynamics, tracked via live imaging. Upon ECE2 inhibition, significant decrease in velocity, increase in resting TP and increase in tortuosity are observed. Data shown as z-scores (CTRL = DMSO control; PHOS = Phosphoramidon; Resting TP = Resting timepoints; N=number of individual neurons analysed from 2 batches; Resting TP: *P=0.024; tortuosiy: *P=0.035, ***P < 0.001 in 2-tailed Chi-Sqare test).",
    "molecules": "DMSO, PHOS, Phosphoramidon"
  },
  {
    "caption": "The thickness of the apical F-actin belt is increased in ECE2 KO COs. C, Example images of apical F-actin belt in 60d old CTRL and ECE2 KO COs with F-actin labeled by AlexaFluor594-conjugated Phalloidin (Scalebar=100 µm). D, Quantification of the thickness of apical F-actin belt in Fiji [86] by measuring the area of F-actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs (Box plots: mean=red line, median=black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of analysed ventricles in 2 batches; Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pariwise Multiple Comparison: ***P < 0.001).",
    "molecules": "AlexaFluor594, Phalloidin"
  },
  {
    "caption": "priming.Ammonium chloride (A), were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.",
    "molecules": "Ammonium chloride"
  },
  {
    "caption": "E64d (CatB/L inhibitor) (B) and/or camostat (TMPRSS2 inhibitor) (B) were added to the indicated target cells before transduction with pseudotypes bearing the indicated glycoproteins.",
    "molecules": "E64d, camostat"
  },
  {
    "caption": "(C) 293T cells transiently expressing ACE2 alone or in combination with TMPRSS2 were incubated with CatB/L inhibitor E64d or PBS as control and inoculated with pseudotypes bearing the indicated viral surface proteins. The average of three independent experiments is shown in panels A-C. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest.",
    "molecules": "E64d"
  },
  {
    "caption": "(a) Haematoxylin and eosin (H & E) staining of kidneys from control (Flcnflox/flox), Flcn KO, Flcn/Tfeb DKO, Flcn/Tfe3 DKO, Flcn/Tfe3 DKO; Tfeb-HET and Flcn KO; Tfeb-HET mice at p18 (replicated three times). Scale bars, 3 mm (upper panels). Boxed areas are magnified in the bottom panels. Arrowhead indicates tubular papillary atypical hyperplasia. Scale bars, 100 μm (lower panels).",
    "molecules": "eosin, Haematoxylin"
  },
  {
    "caption": "(b) Blood urea nitrogen (BUN) levels in mice of the indicated genotypes at p18 (mean± SD, n=3). One Way ANOVA and the Tukey's HSD posthoc test (corrected for multiple comparisons) was applied. Significance for each comparison is provided in Methods.",
    "molecules": "nitrogen, urea"
  },
  {
    "caption": "(d) Representative immunohistochemical (IHC) analysis of Tfeb or Tfe3 and Cadherin-16 (Cdh16) on adjacent kidney sections from mice of the indicated genotypes at p2. Tfeb and Tfe3 were stained in DAB, Cdh16 was stained in teal. Nuclei were stained with haematoxylin II (blue). Magnification: 10x, scale bar:100 µm. Magnification: 20x (in inset image), scale bar:25 µm. (e) Quantification of TFEB or TFE3 levels relative to cadherin 16 (indicated as ratio); values represent mean ± S.E.M (n=4 biological replicates for each genotype). A Welch's One Way ANOVA test with the Dunnett's T3 multiple comparisons test was applied. Significance for each comparison is provided in Methods. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "DAB, haematoxylin II"
  },
  {
    "caption": "Western blot of a time course of Mta3 deletion in Mta1∆Mta2∆Mta3Flox/Flox: Cre-ER (Mta12∆3F/F) or Control (Mta12∆3F/F without Cre-ER) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control (αRNAPII). The time course is indicated at the top as Days + tamoxifen.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(B) Southern blots of the IgH/c-Myc translocation assay using genomic DNA extracted from CIT-stimulated CH12F3-2A cells treated with the siRNAs shown at the bottom of each panel. Numbers on the top indicate gel lanes. (C) Frequency of IgH/c-Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample (Boboila et al., 2012b). The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (** p ≤ 0.01, *** p ≤ 0.001). ",
    "molecules": "CIT"
  },
  {
    "caption": "(C) Top: schematic diagram of the position of the ChIP assay PCR products specific to the S region. Bottom: ChIP assay of the indicated DNA repair proteins using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).",
    "molecules": "CIT"
  },
  {
    "caption": "(C) Western blot analysis of the whole cell extract from CIT-stimulated CH12F3-2A cells transfected with the indicated siRNAs. Expression of various proteins including AID, variant H2As and Ku80 were not decresed by Phf5a KD. (ns, non-specific)",
    "molecules": "CIT"
  },
  {
    "caption": "Phloroglucinol staining of wild-type seedlings inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml in (A hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation",
    "molecules": "Phloroglucinol"
  },
  {
    "caption": "B Phloroglucinol staining of wild-type adult leaves inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). The upper images are enlarged ones of the lower boxes at 2 dpi. Scale bars, 100 μm. Data information Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml in (B hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation; IS, infected site; UIS, uninfected site.",
    "molecules": "Phloroglucinol"
  },
  {
    "caption": "C Quantification of lignin content in pathogen-treated seedlings Data are shown as means ± SD (n = 4; 20-30 seedlings each). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation",
    "molecules": "lignin"
  },
  {
    "caption": "D Quantification of lignin content in pathogen-treated wild-type leaves Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation",
    "molecules": "lignin"
  },
  {
    "caption": "E Quantification of lignin content in wild-type leaves treated with different titers of Pst DC3000 (AvrRpm1). Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at the indicated titers in (E). Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. dpi, days post-inoculation",
    "molecules": "lignin"
  },
  {
    "caption": "F Quantification of lignin content in wild-type and rpm1-3 rps2-101c leaves treated with avirulent bacteria. Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation",
    "molecules": "lignin"
  },
  {
    "caption": "B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.",
    "molecules": "CA, coniferyl alcohol, lignin, PA, piperonylic acid"
  },
  {
    "caption": "C Measurements of Pst DC3000 (AvrRpm1) growth. Data are shown as means ± SD (n = 3). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.",
    "molecules": "CA, coniferyl alcohol, PA, piperonylic acid"
  },
  {
    "caption": "D Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). E Quantification of leaves (n ≥ 30) with spreading cell death as in (D). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.",
    "molecules": "CA, coniferyl alcohol, PA, piperonylic acid"
  },
  {
    "caption": "F Quantification of total and free SA in Col-0 and palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 5; 6 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid dpi, days post-inoculation.",
    "molecules": "PA, piperonylic acid, SA"
  },
  {
    "caption": "B Time-lapse images of GFP-Pst DC3000 and Pst DC3000 (AvrRpt2) captured at 0 (magenta), 15, (yellow), and 30 (green) sec Time in seconds in the movies is shown at the top of images. Blue color was used for chlorophyll autofluorescence. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. Scale bars, 10 μm",
    "molecules": "chlorophyll"
  },
  {
    "caption": "C Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in wild-type and palQ plants after PA and CA pretreatments. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm",
    "molecules": "CA, coniferyl alcohol, PA, piperonylic acid"
  },
  {
    "caption": "A CANBD- and CADMAC-incorporated lignin polymerization. CANBD (green) and CADMAC (blue) fluorescence was observed in the lignified infection site at 2 dpi, as revealed by phloroglucinol staining. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site White dash lines indicate the boundary between IS and UIS. Scale bars, 500 μm",
    "molecules": "DMAC, CA, lignin, NBD, phloroglucinol"
  },
  {
    "caption": "B Cross-sections of CANBD- and Pst DC3000 (AvrRpm1)-treated leaves. Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 40 μm",
    "molecules": "CA, NBD"
  },
  {
    "caption": "D CADMAC-incorporated lignin deposition in the GFP-Pst DC3000 (AvrRpt2)-infected site at 2 dpi. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site DC3000, Pst DC3000; AvrRpt2, Pst DC3000 (AvrRpt2); Chl, chlorophyll. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm",
    "molecules": "DMAC, Chl, chlorophyll, CA, lignin"
  },
  {
    "caption": "E Cellular observation of CADMAC-incorporated lignin formation against infecting GFP-Pst DC3000 (AvrRpt2). Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 20 μm",
    "molecules": "DMAC, CA, lignin"
  },
  {
    "caption": "F 3D image of CANBD-polymerized structure. Images were taken at 24 hpi Data information: Bacterial inoculum was at 108 cfu/ml. Scale bars, 20 μm",
    "molecules": "CA, NBD"
  },
  {
    "caption": "Quantification of lignin content in wild-type and caspl mutant leaves after Pst DC3000 (AvrRpm1) inoculation. ata are shown as means ± SD (n = 4; 3-9 leaves each). ata information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); dpi, days post-inoculation.",
    "molecules": "lignin"
  },
  {
    "caption": "A Visualization of CASPL4D1 proteins and lignin by mCherry and CADMAC fluorescence. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars, 20 μm (A)",
    "molecules": "DMAC, Chl, chlorophyll, CA, lignin"
  },
  {
    "caption": "B Magnified views of CASPL4D1 and lignin localization. Images in boxes were enlarged in the 2nd to 4th columns. Arrows indicate lignin deposition between cells. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars 10 μm (B).",
    "molecules": "DMAC, Chl, chlorophyll, CA, lignin"
  },
  {
    "caption": "C Relative fluorescence intensity profiles of CASPL1D1 (mCherry) and lignin (CADMAC) across transects indicated by dotted lines Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1).",
    "molecules": "DMAC, CA, lignin"
  },
  {
    "caption": "D A model showing morphological changes of CASPL- and lignin-deposited cells during the HR response.",
    "molecules": "lignin"
  },
  {
    "caption": "F) Circuit-mediated tuning of BMP4   =using miSFITs library. EV5C.F) Radial marker profiles of each germ-layer marker analyzed from confocal microscopy images. Charts on the left show normalized intensity of marker expression  at a given colony position, where x=0 is the center of the colony and x=1 is the edge of the colony.Representative microscopy  image of a single colony (top) showing composite of Sox2/mCitrine  (yellow), Sox17/mCherry  (red) and TBXT/AF647  (blue). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4   and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm.",
    "molecules": "AF647"
  },
  {
    "caption": "Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 hours. Scale bar: 10µm Morphological parameters quantification within 48 hours after LPS injection",
    "molecules": "LPS"
  },
  {
    "caption": "Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection. Scale bar: 20µm",
    "molecules": "LPS"
  },
  {
    "caption": "Morphological parameters quantification for 5 and 15 months old mice after LPS injection",
    "molecules": "LPS"
  },
  {
    "caption": "Two-photon representative images of wild-type and Nlrp3-/- mice (5 and 15 months old). Scale bar: 20µm. Quantification of morphological parameters for wild-type and Nlpr3-/- mice after LPS injection",
    "molecules": "LPS"
  },
  {
    "caption": "Representative cortical images of MXO4 staining for APP and APP/Nlrp3-/- 15 months old mice. Scale bar: 50µm.",
    "molecules": "MXO4"
  },
  {
    "caption": "Flow cytometry plots from APP and APP/Nlrp3-/- mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation. d) Relative Aβ microglia uptake quantification",
    "molecules": "MXO4"
  },
  {
    "caption": "Iba-1 (green), CD169 (red) and MXO4 staining in plaque-associated areas (cortex and hippocampus) of 5 and 15 months old of APP and APP/Nlrp3-/-. Note the colocalization between Iba-1 and CD169 (white arrows) in APP 15-month-old mice 2 days post LPS injection. Scale bar: 20µm",
    "molecules": "LPS, MXO4"
  },
  {
    "caption": "Iba-1, Ki67 and DAPI staining in the cortex and hippocampus of wild-type and Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows) Scale bar: 20µm. Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01, ***p<0.001).",
    "molecules": "LPS, DAPI"
  },
  {
    "caption": "Iba-1, Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP/Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20µm Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel)",
    "molecules": "LPS, MXO4"
  },
  {
    "caption": "A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi & Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.",
    "molecules": "Moco, molybdenum"
  },
  {
    "caption": "IIA1.6 B lymphoma cells were treated 45 min with indicated inhibitors (CK666 at 25 µM, SMIFH2 at 25µM) or DMSO as control prior to being fixed and stained for α-tubulin (left column) and F-actin (right column). Scale bar: 3 µm.",
    "molecules": "CK666, DMSO, SMIFH2"
  },
  {
    "caption": "Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right, values were normalized with respect to the mean of control condition) and filamentous actin at the centrosome (left, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell). Measurements were pooled from 3 independent experiments; DMSO: n= 91, CK666: n= 82, SMIFH2: n= 74, latrunculinA: n= 96. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test.",
    "molecules": "CK666, DMSO, latrunculinA, SMIFH2"
  },
  {
    "caption": "Percentage differences of centrosomal F-actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of \"0\").",
    "molecules": "DMSO"
  },
  {
    "caption": "The duration of EB3-positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells (left). Error bars correspond to standard deviations. The number of EB3-positive comets exiting the a 2-µm wide centrosomal area was also compared between the two conditions (right). In both cases P values were calculated with Mann-Whitney test.",
    "molecules": "CK666, DMSO"
  },
  {
    "caption": "RPE1 cells stably expressing centrin1-GFP were plated for 3 h on coverslips coated with different ratios (100:0; 50:50 or 1:99) of fibronectin and PLL-PEG prior to fixation and staining for F-actin (top line and magnified views around centrosome below. Scale bars: 10 µm and 2 µm, respectively) and α-tubulin (bottom line. Scale bar: 10 µm).",
    "molecules": "PLL-PEG"
  },
  {
    "caption": "IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm.",
    "molecules": "poly-L-lysine"
  },
  {
    "caption": "B) Binding affinity  of CodB  for cytosine  as measured using the thermostability assay. Cytosine  was titrated into detergent solubilised membranes   from cells overexpressing CodB. The  Kd  was estimated to be 51 ± 9 µM. The measurements are the average of 4 independent titrations with error bars of the s.e.m.",
    "molecules": "cytosine, Cytosine"
  },
  {
    "caption": "Hoxb8-FL cells generated from bone marrow of mice of the indicated genotypes were treated for 24h with graded doses of the CHK1-inhibitors PF-477736 (PF) or CHIR-124 (CHIR). Representative histograms of CHK1i-treated (grey) or DMSO-treated (black) cells, analyzed for cell cycle distribution and cell death using Nicoletti staining and flow cytometry are shown.",
    "molecules": "CHIR, CHIR-124, DMSO, PF, PF-477736"
  },
  {
    "caption": "WT Hoxb8-FL cells were treated with CHIR-124 in the presence or absence of the pan-caspase inhibitor QVD. Cell death was assessed after 24h using flow cytometry as in (A).",
    "molecules": "CHIR-124, QVD"
  },
  {
    "caption": "Western blot of CHIR-treated Hoxb8-FL WT and Bax-/-Bak-/- cells using the indicated antibodies.",
    "molecules": "CHIR"
  },
  {
    "caption": "WT and Bax-/-Bak-/- Hoxb8-FL cells were treated for up to 8h with 500 nM CHIR and p21 mRNA levels were compared to Hprt in response to CHK1-inhibition by real-time RT-qPCR. Bars represent means ± S.D. of independent experiments (n=4-6), performed in duplicates.",
    "molecules": "CHIR"
  },
  {
    "caption": "LSK (Lin- Sca1+ cKit+) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13.5 or the bone marrow of adult mice and treated for 48h with PF-477736 [1.5 μM]. Survival was assessed using AnnexinV-staining and flow cytometry. Bars represent means ± S.E.M. from n=6 wild type and n=3 Vav-BCL2 fetal liver LSK cells (left) and n=3 bone marrow LSK cells (right).",
    "molecules": "PF-477736"
  },
  {
    "caption": "MACS-purified CD34+ human cord-blood derived HSPC were treated with PF-477736 or CHIR-124 ± the caspase-inhibitor QVD (50 μM). Cell death was assessed using AnnexinV/7-AAD staining and flow cytometry.",
    "molecules": "7-AAD, CHIR-124, PF-477736, QVD"
  },
  {
    "caption": "DAPI-negative viable LK- and LSK-cells of the indicated genotypes (littermates) were sorted from E13.5 fetal livers and immediately fixed in 70% EtOH. Fixed cell suspensions were stained for ɣH2A.X and DAPI intracellularly. Shown here are representative dot-plots and quantification of N=4 embroys per genotype; Bars represent means ± S.E.M.",
    "molecules": "DAPI, EtOH"
  },
  {
    "caption": "Total E13.5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK (top panel) from LSK (lower panel) cells. Shown here are representative dot-plots of Ki67 and DAPI staining in LK and LSK cells, quantified in (E). Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre.",
    "molecules": "DAPI"
  },
  {
    "caption": "Analysis of the colony formation potential of E14.5 total fetal liver cells of the indicated genotypes in methylcellulose. N=3 for controls (pooled; 1 Chk1fl/+, 2 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre and N=3 for Chk1fl/- Vav-Cre Vav-BCL2.",
    "molecules": "methylcellulose"
  },
  {
    "caption": "Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Quantification of Chk1-del PCR-product enrichment in DNA from GFP+ vs. Tomato+ bone marrow cells sorted on day 8 after first TAM administration. Transgenic Cre levels were quantified in parallel for reference. Bars represent means ± S.E.M. from n=5 mT/mG Vav-CreERT2 Chk1fl/fl mice.",
    "molecules": "DNA, TAM"
  },
  {
    "caption": "Percentage of living LK or LSK-cells and (F) percentage of GFP+ LK or LSK-cells found in the bone marrow of mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) mice on day 8 or 15 post first TAM administration. Bars in (E)/(F) represent means ± S.E.M. (Day 8 N=8/8, WT/Chk1fl/fl, Day 15 N=7/8).",
    "molecules": "TAM"
  },
  {
    "caption": "GFP+ (CRE) and Tomato+ (CRE-) total bone marrow cells were sorted on day 8 after first TAM-administration and processed for Western Blot using the indicated antibodies. Sorted cells from three animals - mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) - were pooled per lane.",
    "molecules": "TAM"
  },
  {
    "caption": "Thymocytes of the indicated genotypes were sorted based on GFP+ and Tomato+ on day 15 after the first TAM administration for western analysis using the indicated antibodies.",
    "molecules": "TAM"
  },
  {
    "caption": "(A) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 (BafA1) for 24 hrs or cultured in amino acid-deprived medium (Stv) for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.",
    "molecules": "BafA1, Bafilomycin A1"
  },
  {
    "caption": "(A) Immunoblot analysis. Primary hepatocytes were isolated from wild-type (wt), p62f/f; Alb-Cre (p62KO) and Nrf2f/f; Alb-Cre (Nrf2KO) mice. The hepatocytes were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with anti-NBR1 and anti-Actin antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.",
    "molecules": "As (III), sodium arsenite"
  },
  {
    "caption": "(C) Immunofluorescence microscopy. Primary hepatocytes isolated from wild-type mice were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.",
    "molecules": "As (III), sodium arsenite"
  },
  {
    "caption": "A) Immunoblot analysis. Primary hepatocytes were isolated from Nbr1f/f mice and then infected with adenovirus GFP or Cre for 48 hrs. Thereafter, the hepatocytes were divided into 3 groups: 1. Cultured in regular medium (-), 2. Treated with 10 μM As (III) for 12 hr [As (III)] and 3. After treated with 10 μM As (III) for 12 hr, cultured in regular medium for another 12 hr (Wash). Cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.",
    "molecules": "As (III)"
  },
  {
    "caption": "(C) Immunofluorescence microscopy. Nbr1f/f hepatocytes were infected with LacZ or Cre adenovirus for 48 hrs and, when indicated, challenged with 10 μM As (III) for 12 hrs. The cells were then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. **P < 0.01, and ***P < 0.001 as determined by Welch's t-test. (D) Role of NBR1 in the p62-liquid droplets and the p62-mediated Nrf2 activation. Oxidative stress up-regulates the level of NBR1, which enhances the phosphorylation and liquid-droplet formation of p62. The resulting p62-droplets are resistant to autophagic degradation and serve as signaling nodes for the activation of Nrf2. Since p62 is one of the targets of Nrf2, increased level of p62 in cooperation with NBR1 promote the formation of liquid droplets and contributes to the persistent activation of Nrf2 against oxidative stress. ",
    "molecules": "As (III)"
  },
  {
    "caption": "(A) Changes in the initial rates of substrate uptake (gray bars), transport domain dynamics (black bars), and l-Asp dissociation constant (white bars) of the mutant GltPh variants relative to the WT transporter. The transition frequencies were measured under non-equilibrium transport conditions, and frequencies obtained in the presence of d,l-TBOA were subtracted from the data. Error bars represent standard errors for at least three independent measurements.",
    "molecules": "l-Asp"
  },
  {
    "caption": "(A) Dwell-time distributions of the OFS (blue) and the IFS (red) for WT (top) and K290A (bottom) GltPh observed under equilibrium conditions in saturating Na+/l-Asp (left). Lines are fits to three exponentials. Data are averages and standard errors of at least three independent measurements.",
    "molecules": "l-Asp, Na"
  },
  {
    "caption": "(A-H) Transiliac bone sample of the patient at age of 10 years viewed in the light microscope. A-E) Goldner stained sections, F-H): Giemsa stained sections. A) Overview of the entire sample shows scarcity of mineralized bone (stained in green). Black arrowheads point towards the very thin cortices (right side, only a fractured cortex is visible). B) Trabecular features are mostly small and isolated. C) Black arrows point toward three bone resorbing osteoclasts leading to the disconnection of two trabecular features. D) Cortical bone viewed under polarized light: white arrows point towards collagen fibers arranged in a parallel concentric way around osteon next to a region where collagen fibers are randomly arranged, as typical for woven bone (white asterisk). E) Detail from cortical bone showing roundish cells on the periosteal side surrounded by a mainly red, thus, poorly mineralized matrix (white stars). F), G) Details from the cortex in Giemsa stained section show areas with unmineralized cartilage (black arrows) and mineralized woven bone and or mineralized cartilage (dark purple, black asterisks), mineralized bone is stained pink. H) Trabecular feature with mineralized cartilage inclusion stained deep purple (arrowhead), surrounded by bone tissue stained pink. Note many osteoblasts on the trabecular surface (black arrows)",
    "molecules": "Giemsa, Goldner"
  },
  {
    "caption": "C-F) C) COL1A1 and COL1A2 expression in the ER of SEC16B patient and control fibroblasts assessed by western blotting. KDEL, Calnexin, BIP and PDI, all ER-resident proteins were used as ER markers. E) Golgi complex was isolated according to the method described in Current Protocols in Cell Biology, Unit 3.9 from healthy control cells and patient derived fibroblasts. Subsequently, COL1A1 and COL1A2 expression in the Golgi complex of SEC16B patient and control fibroblasts were determined by western blotting. GM130 (Golgi-resident protein) was used as marker for Golgi complex. All cells were treated with ascorbic acid 0.05 mg/ml for 20 hours before the Golgi complex was isolated. D, F) Quantification of the bands that was done with Bio-Rad Image Lab 6.1 software to calculate the expression levels. COL1A1 and COL1A2 band intensities were either normalized to (D) calnexin or (F) GM130. The normalized intensities were reported as fold change patient to control cells. Columns represent the mean of three (D) or two (F) independent experiments (raw values: 0.27 and 0.53 for COL1A1 and 0.29, 0.21 for COL1A2) respectively. Biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two-tailed t test",
    "molecules": "ascorbic acid"
  },
  {
    "caption": "C) Patient and control fibroblasts were electroporated with plasmids encoding mCherry-tagged Mannosidase II (ManII) as part of a RUSH construct. After 24 hours, cells were fixed (T0) or treated with 40 mM biotin for 20 minutes (T20) followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm. D) quantification of 59 to 81 cells from three independent RUSH experiments (biological replicates). Biological replicates were performed. Central band represents the median. Whiskers are minimum and maximum values. Outliers are defined as greater than 1.5 fold of upper quartiles.** highly significant (p < 0.005) differences obtained by unpaired two tailed t-test",
    "molecules": "biotin"
  },
  {
    "caption": "E) 24 hours after plating of patient and control fibroblasts, cells were treated with DMSO, 10mM thapsigargin, or 1 mg/ml tunicamycin for 6 hours. Cells were lysed and immunoblotted for XBP1s as a marker for ER stress and for vinculin to ensure equal loading.",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "Patient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. B) Immunofluorescence labeling of the control and the patient fibroblasts for COL1A1 marker proα1 (I) and rough ER marker KDEL. The staining showed that while COL1A1 is accumulated in the ER of patient cells transfected with empty vector, it is not accumulated in the ER of the patient cells transfected with SEC16B wt. Scale bar is 25 µm",
    "molecules": "puromycin"
  },
  {
    "caption": "Patient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. D) LC3B showed reduced expression in the TF SEC16B mutant cells (similar to expression in healthy control Fig. 5B) when compared to empty vector transfected patient cells. E) Quantification of the bands. All band intensities were normalized to those of Actin and reported as fold change patient to control cells. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); **highly significant (p < 0.001 differences obtained by using unpaired two tailed t-test.",
    "molecules": "puromycin"
  },
  {
    "caption": "D) HepG2 cells were transfected with the indicated siRNA. After 48h, cells were transfected with plasmids encoding signal sequence GFP (ssGFP) as part of a RUSH construct. After 24 hours, cells were treated with 40 mM biotin for 15 minutes followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm.",
    "molecules": "biotin"
  },
  {
    "caption": "D: AS or nocodazole-synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations (IP). Extracts (input) and IP samples were analysed by immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "nocodazole"
  },
  {
    "caption": "E: KI cells synchronised in mitosis with either nocodazole (M Noc.) or STLC (M STLC) were collected by shake-off, and drug-treated cells that remained adherent after shake-off (AS Noc.; AS STLC) were lysed and subjected to GFP TRAP IP. AS cells and free-GFP-expressing 2G-PAI1 U2OS cells were included as controls. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "Noc, nocodazole, STLC"
  },
  {
    "caption": "G, H: AS or nocodazole-synchronised (M) WT U2OS cells were subjected to IP with IgG and either anti-FAM83D-coupled sepharose beads (G), or anti-CK1α-coupled sepharose beads (H). Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "sepharose, nocodazole"
  },
  {
    "caption": "I: KI cells were synchronised in G2 with RO-3306, or arrested in mitosis (M) using STLC. STLC-treated shake-off cells were washed and re-plated, and cells lysed at the indicated time points after STLC wash-out. Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "RO-3306, STLC"
  },
  {
    "caption": "K: AS or STLC-synchronised (M) HeLa, A549 and HaCaT cells were subjected to IP with either IgG- or anti-FAM83D-coupled sepharose beads. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "sepharose, STLC"
  },
  {
    "caption": "A: STLC-synchronised mitotic (M) wild-type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.",
    "molecules": "DAPI, DNA, STLC"
  },
  {
    "caption": "B: STLC-synchronised mitotic (M) FAM83D-/- knockout (KO), FAM83DGFP/GFP knockin (KI) and FAM83DGFP/GFP(F283A) knockin (FA) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µ",
    "molecules": "DAPI, DNA, STLC"
  },
  {
    "caption": "D: The cell lines were STLC-synchronised and mitotic cells (M) isolated by shake off. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to GFP TRAP immunoprecipitation (IP) and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "STLC"
  },
  {
    "caption": "G: The cell lines were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "B: FAM83D-/- (KO), wild-type (WT), and two independent clones from a CRISPR/Cas9-mediated knockin rescue of FAM83D cDNA into FAM83D-/- cells (c. 6 and c. 11), were synchronized in mitosis (M) with STLC. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "STLC"
  },
  {
    "caption": "The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing FAM83D (C) Representative images of mitotic cells are included. Scale bars, 20 µm.",
    "molecules": "STLC"
  },
  {
    "caption": "The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing CK1⍺ (D). Representative images of mitotic cells are included. Scale bars, 20 µm.",
    "molecules": "STLC"
  },
  {
    "caption": "F: The cell lines were STLC-synchronized in mitosis (M) or left AS, lysed and subjected to immunoprecipitation (IP) with anti-FAM83D-coupled sepharose beads, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "sepharose, STLC"
  },
  {
    "caption": "A: Nocodazole-synchronised mitotic FAM83DGFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ-phosphatase. Asynchronous (AS) cells were used as a control. Whole cell extracts (input) and IP samples were subjected to SDS-PAGE and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "Nocodazole"
  },
  {
    "caption": "STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium (B) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "STLC"
  },
  {
    "caption": "STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 (C) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "MG132, STLC"
  },
  {
    "caption": "STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME (D). AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "STLC, ProTAME"
  },
  {
    "caption": "E: Wild-type U2OS cells were either left asynchronous (AS), or arrested in mitosis with STLC and collected by shake-off (M). AS and M cells were incubated in media containing combinations of RO-3306 and MG132 as indicated, prior to lysis. MG132 was applied for 1.5 h, whereas RO-3306 was applied for the last 1 h of incubation prior to lysis. Samples were lysed and extracts were subjected to SDS-PAGE, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "MG132, RO-3306, STLC"
  },
  {
    "caption": "F: STLC-synchronised mitotic wild-type U2OS cells were subjected to qRT-PCR analysis using primers for FAM83D, HMMR, CSNK1A1, and CCNB1. AS cells were used as a control. Error bars, SEM; *, P<0.01; Student's T-test. n=3",
    "molecules": "STLC"
  },
  {
    "caption": "H: STLC-synchronised mitotic wild-type (WT) and HMMR knockout (KO) mouse embryonic fibroblasts (MEFs) were lysed and subjected to IB with the indicated antibodies. AS cells were included as a control. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "STLC"
  },
  {
    "caption": "The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-HMMR antibody (I) DNA is stained with DAPI. Scale bars, 20 µm.",
    "molecules": "DAPI, DNA, STLC"
  },
  {
    "caption": "The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-CK1α antibody (J). DNA is stained with DAPI. Scale bars, 20 µm.",
    "molecules": "DAPI, DNA, STLC"
  },
  {
    "caption": "B: STLC-synchronised mitotic FAM83DGFP/GFP knockin (KI), FAM83DGFP/GFP (F283A) (FA), and FAM83DGFP/GFP(F283A) stably expressing aGFP.16-CK1α (FA + aGFP.16-CK1α) U2OS cells were subjected to GFP TRAP immunoprecipitation (IP), followed by immunoblotting (IB) with the indicated antibodies. Asynchronous (AS) cells were used as controls. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "STLC"
  },
  {
    "caption": "D: Immunofluorescence analysis for the cells described in (B) following synchronisation with STLC. Cells were stained using anti-CK1α antibody, and DNA is stained with DAPI. Scale bars, 20 µm.",
    "molecules": "DAPI, DNA, STLC"
  },
  {
    "caption": "F: FAM83D-/- knockout (KO), KI, and FA cells were synchronised in mitosis (M) using STLC. Lysed extracts were subjected to anti-GFP IPs, followed by ATP [γ-32P] kinase assays, using an optimised CK1 substrate peptide (CK1tide). AS cells were used as controls. Error bars, SEM; P<0.01; ANOVA. n=3. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.",
    "molecules": "ATP, STLC"
  },
  {
    "caption": "A. Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst, taken every 5 min as they progress through division. Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes. Scale bar, 20 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "F. Representative images of mitotic U2OS cells stained with Hoechst and grown on L-shaped micropatterns previously coated with fibronectin. The position of metaphase chromosomes is indicated (yellow line) as is the presence of cortical blebbing (red arrows). Scale bar, 20 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "B. Carfilzomib, Bortezomib and MLN9708 activate  luciferase activity in   M2  macrophages  derived from  Il-1β-luciferase transgenic  mice. BMDMs from  IL-1β-luciferase transgenic mice were treated by  IL-4  (20 ng/mL) for 24 hours to differentiate into mature M2  macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.",
    "molecules": "Bortezomib, Carfilzomib, MLN9708"
  },
  {
    "caption": "F, G. Carfilzomib, Bortezomib  and  MLN9708 promote the mRNA  expression of  Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by  IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from  BMDMs or Raw264.7 cells 6 hours after stimulation and the expression  of Il-1β were quantified through  RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test). H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine  IL-1β  in  M2 macrophages. BMDMs  (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through  ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "Bortezomib, Carfilzomib, DMSO, MLN9708"
  },
  {
    "caption": "A. The most effective one among the candidate compounds,  Carfilzomib, significantly promotes the  expression of M1 macrophage markers, as well as reduces the expression of M2 macrophage  markers. BMDMs were pretreated by IL-4  (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). RNA was extracted from cells 6 hours after stimulation and the  expression of M1 (Il-6/Inos) or  M2 (Cd206/Arg1)  macrophage markers were quantified through  RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test).",
    "molecules": "Carfilzomib, DMSO"
  },
  {
    "caption": "E. Carfilzomib promotes the  phagocytosis  of macrophages. BMDMs  were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by  DMSO or Carfilzomib (500 nM) for 12 hours. After starving for 2 hours and  stained  with Red membrane dye, BMDMs (red) were incubated with  L1210-GFP cells (green) in serum-free medium for another 2 hours. Phagocytosis effect was observed and photographed under fluorescence microscope. Scale bars, 50 μm (up), 20 μm (down). The yellow arrows indicated L1210 that is phagocytosed by macrophages. F. Statistics represent the  number of phagocytosis L1210 in 100  macrophages. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).",
    "molecules": "Carfilzomib, DMSO, Red membrane dye"
  },
  {
    "caption": "M. Carfilzomib  promotes the M1-polarization of Tumor associated macrophages  in vitro. BMDMs  were cultured with tumor culture supernatant (TSN) produced by L1210 cells, followed by activation with DMSO or Carfilzomib  (1 μM). RNA was extracted from cells and the expression of  Il-1β, Il-6, Inos were quantified through  RT-qPCR  6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "Carfilzomib, DMSO"
  },
  {
    "caption": "A, B. Carfilzomib  and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with   IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib  (1 μM), or MG132  (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132  (5 μM)) for 1 hour, then induced with  IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test).",
    "molecules": "Carfilzomib, DMSO, MG132"
  },
  {
    "caption": "E. Carfilzomib, Bortezomib, MLN9708 and MG132 induce ER stress response. BMDMs were pretreated with  IL-4 (20 ng/mL) for 24 hours and then stimulated by  DMSO or  Carfilzomib (1 μM),  Bortezomib  (1 μM), MLN9708  (2 μM), MG132 (5 μM). RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER  stress related genes (Bip, Chop) were detected through  RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "Bortezomib, Carfilzomib, DMSO, MLN9708, MG132"
  },
  {
    "caption": "F. ER  stress agonists, TUN and  TG, promote the  expression of  M1 macrophage  markers, as well as reduce the expression of M2  macrophage markers. BMDMs  were pretreated with IL-4  (20 ng/mL) for 24 hours and then stimulated by DMSO, TUN  (500 nM) or TG (500 nM). RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through  RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "DMSO, TG, TUN"
  },
  {
    "caption": "G. Inhibition of  ER stress impairs the ability of  Carfilzomib, Bortezomib,  MLN9708 and MG132 to reprogram  M2 towards M1-like macrophages. BMDMs were induced by  IL-4 (20 ng/mL) for 24 hours, then pretreated with  4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib  (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β,  Il-6 and  Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "Bortezomib, Carfilzomib, MLN9708, MG132, 4-PBA"
  },
  {
    "caption": "A, B Deficiency of  Ern1 reduces the expression  of inflammatory related genes. Wild-type and Ern1-/- Raw264.7 cells (A) or BMDMs (B) were pretreated by IL-4  (20 ng/mL) for 24 hours, then stimulated by  DMSO or Carfilzomib  (1 μM). The mRNA of Il-1β and Il-6  were quantified through  RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "Carfilzomib, DMSO"
  },
  {
    "caption": "E.  Kira6 inhibits the  expression of ER  stress related genes and inflammatory related genes.  BMDMs  was treated by IL-4 (20 ng/mL) for 24 hours, treated with  Kira6  (100 nM) for 1 hour, and then stimulated by  Carfilzomib (1 μM) for 6 hours. RNA was extracted from cells and the expression  of  Bip, Chop,  Il-1β and  Il-6  were quantified through  RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test). F, G. Carfilzomib has no effect on splicing of XBP1  in M2 macrophages. BMDMs (F) or Raw264.7  cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated with DMSO, Carfilzomib (1 μM) or TUN  (500 nM). The mRNA expression of sXBP1 and unXBP1 were detected through  RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "Carfilzomib, DMSO, Kira6, TUN"
  },
  {
    "caption": "J.  Carfilzomib promotes the association between  IRE1α  and TRAF2  in M2 macrophages. Raw264.7 cells were pretreated by  IL-4 (20ng/mL) for 24 hours, then stimulated by  Carfilzomib (1 μM) for 2 or 4 hours. Coimmunoprecipitation (Co-IP) and IB were performed with indicated antibodies.",
    "molecules": "Carfilzomib"
  },
  {
    "caption": "L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by  IL-4 (20 ng/mL) for 24 hours, then stimulated by  Carfilzomib  (1 μM) for indicated time points. WCL were analyzed by IB  with indicated antibodies.",
    "molecules": "Carfilzomib"
  },
  {
    "caption": "C. Representative images of Hematoxylin and  eosin (H&E) staining  of the  lung tissue obtained from different treatment groups. Expression of Ki-67 in lung tissues was detected by immunohistochemistry  method. D. Statistics of the tumor area  and the number  of Ki67  positive cells in sections of  lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "E. Carfilzomib treatment promotes the infiltration of M1 macrophages. Tumor bearing TD mice  were treated as indicated in (A) and euthanized. Lung tissues were dissected for evaluating M1/M2 populations through analyze the expression  of CD80 and CD206 by flow cytometry. F. Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test). G. Carfilzomib  treatment promotes the infiltration and activation of   CD8+ T cells. Lung tissues of (E) were used for evaluating the infiltration of CD8+ T cells and their expression of CD69 by flow cytometry. H. Statistics about the proportion of CD8+ or CD69+ positive cells in  lung  tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).",
    "molecules": "Carfilzomib"
  },
  {
    "caption": "K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or  LyzM-cre;lsl-dTA to allow tumor xenograft  to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib  for two weeks. The  xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).",
    "molecules": "Carfilzomib"
  },
  {
    "caption": "D. CD4+ and  CD8+ T cells play a role in mediating   cancer-shrinking effect of Carfilzomib. Lung-cancer-bearing TD mice were treated by  saline, Carfilzomib, anti-mouse CD4  antibody (ip.), anti-mouse CD8 antibody (ip.), or Carfilzomib plus anti-mouse CD4/8 antibody. CT scanning was performed after two-week treatment. E. Representative images of H&E staining of the treated lung  tissues.  Expression of  Ki-67 in lung  tissues was detected by immunohistochemistry method. F. Carfilzomib synergized with PD1 antibody in shrinking lung cancers. Lung cancer bearing TD mice  were treated by  saline, anti-PD1 antibody (ip), Carfilzomib, or Carfilzomib plus anti-PD1 (ip). CT scanning  was performed after two-week treatment. G. Representative images of H&E staining of the lung tissues. Expression  of Ki-67 in lung tissue was detected by immunohistochemistry method.",
    "molecules": "Carfilzomib"
  },
  {
    "caption": "A. Carfilzomib, Bortezomib and MLN9708 promote the  expression of M1  macrophage markers (Il-1β,  Il-6,  Tnfα) and reduce the expression  of M2 macrophage  markers (Il-10, Tgfβ) in  macrophages  derived from PBMCs. After induced by  IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages,  PBMCs derived macrophages  were stimulated by Carfilzomib  (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).",
    "molecules": "Bortezomib, Carfilzomib, MLN9708"
  },
  {
    "caption": "C-F. Carfilzomib, Bortezomib  and MLN9708 increase the expression of CD80 (C) and reduce the  expression of CD206  (E) in M2  macrophages derived from  PBMCs. After activated by  IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib  (500 nM) or MLN9708 (500 nM). The representative histogram of CD80  and CD206 expression was analyzed by  flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or  CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test).",
    "molecules": "Bortezomib, Carfilzomib, MLN9708"
  },
  {
    "caption": "(C) Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4-hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to Ethanol treated control cells (See also Appendix figure S1A).",
    "molecules": "4-hydroxy tamoxifen, 4OHT"
  },
  {
    "caption": "(B) Colony formation in ICL and control MEFs. 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days. Representative colony formation images in Ch10 and Ch14 are shown (left) and quantification of colonies formed (right) in the 4 ICL lines (n=3 for each data set and error bars denote SD, p<0.005 for Ch10, Ch12 and Ch14 and ns for Ch9).",
    "molecules": "Methylene blue"
  },
  {
    "caption": "(D) Analysis of metabolites (glucose consumed, lactate produced and glutamine consumed) for the ICL lines. Spent media was analyzed after each ICL & control line was grown in culture for three days. Media in identical culture conditions, but with no plated cells, was used as a baseline for all samples.",
    "molecules": "glucose, glutamine, lactate"
  },
  {
    "caption": "(A) Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post tamoxifen administration.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(E) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO, ionomycin (Iono) or thapsigargin (TG). Total lysates of these cells were subjected to western blotting. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with an arrow.",
    "molecules": "DMSO, Iono, ionomycin, TG, thapsigargin"
  },
  {
    "caption": "(F) LN229 cells treated with DMSO or thapsigargin (TG) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG. The immunoprecipitated products were subjected to western blotting. The number of conjugated ubiquitin (Ub) moieties was marked. Tetra-ubiquitinated Merlin is indicated by an arrow.",
    "molecules": "DMSO, TG, thapsigargin, Ub, ubiquitin"
  },
  {
    "caption": "(G) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were treated with DMSO or thapsigargin (TG). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Both arrowhead and arrow point to the mono-ubiquitinated Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, His, Histidine, nickel, TG, thapsigargin"
  },
  {
    "caption": "(H) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were detached or reseeded as in (C). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Arrowheads and arrows point to the mono- or di-ubiquitinated Merlin (see a longer exposed blotting results in Figure EV1D). The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.",
    "molecules": "His, Histidine, nickel"
  },
  {
    "caption": "(B) LN229 cells treated with DMSO, ionomycin (Iono) or thapsigargin (TG) were lysed and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, Iono, ionomycin, TG, thapsigargin"
  },
  {
    "caption": "(C) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to active RAC1 pulldown assay followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "LN229 cells transduced with vector or EGFP-RAC1(Q61L) (D) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(E) LN229 cells transduced with vector or Myc-PAK1(T423E) (E) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(F) LN229 cells stably transduced with wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG) and analyzed by western blot. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Native Merlin (asterisk); ubiquitinated Merlin (arrow).",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(G) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(B) LN229 cells transduced with a pool of four siRNAs against NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(C) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(E) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA. These cells were detached as in (D), lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.",
    "molecules": "His, Histidine, nickel"
  },
  {
    "caption": "(A) LN229 cells transduced with a pool of four siRNAs against AMOTL1 (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(B) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.",
    "molecules": "His, Histidine, nickel"
  },
  {
    "caption": "(D) Merlin-depleted LN229 cells stably expressing Flag-HA-tagged Merlin were transduced with scrambled shRNA (sh-Co.) or two distinct shRNAs targeting AMOTL1, treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by western blotting.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(F) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(A) LN229 cells stably transduced with Flag-tagged wild-type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS-PAGE. Outlined gel pieces were analyzed by mass spectrometry. (B) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in (A) are shown. ",
    "molecules": "SDS, thapsigargin, Ubiquitin"
  },
  {
    "caption": "(F) Merlin-depleted LN229 cells expressing His-Ubiquitin were stably transduced with the indicated HA-tagged Merlin forms or an empty vector. These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA. Arrows indicate mono-ubiquitinated Merlin. The red asterisk indicates native Merlin and black asterisks indicate non-specific signals. The ratio of mono-ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, His, nickel, thapsigargin"
  },
  {
    "caption": "(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(A) LN229 cells were treated with DMSO or thapsigargin (TG) for the indicated time and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(B) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(C) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(D) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(A) LN229 cells stably transduced withHA-Lats1 (two leftmost panels) or vector only (right panel) were treated with DMSO or thapsigargin (TG) and subjected to PLA using HA and Merlin antibodies. Scale bar = 10 μm. (B) PLA signals (dots) in each cell from the results in (A) were quantified. Mean±s.e.m, two-way ANOVA. *P<0.05, ***P<0.001, ****P<0.0001. Each data point represents an image field containing an average of 10 cells. N=4-9 images for each condition as indicated. All images were collected from one experiment. Two independent experiments were performed and gave similar results. ",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(C) Merlin-depleted LN229 cells were stably transduced with HA-tagged wild-type (WT) Merlin or its mutants. These cells were treated with thapsigargin (TG) for 15 minutes and subjected to PLA using HA and Lats1 antibodies. Scale bar = 20 μm. (D) PLA signals (dots) in each cell from the results in (C) were quantified. Mean±s.e.m, Ordinary one-way ANOVA. ****P<0.0001. Each data point represents an image field containing an average of 10 cells. NVector=8, NWT=16, NK396R=18, NK159R=17 images. All images were collected from one experiment. Two independent experiments were performed and gave similar results. ",
    "molecules": "TG, thapsigargin"
  },
  {
    "caption": "(G) Merlin-depleted LN229 cells were stably transduced by HA-tagged Merlin. These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin (TG). PLA was performed in these cells using HA and Lats1 antibodies, and the PLA signals (dots, showed in Figure EV5E) in each cell were quantified. Mean±s.e.m, two-way ANOVA. ****P<0.0001. Each data point represents an image field containing averagely 10 cells. N=12 images in all conditions, except for Nsi-Control:TG=13 images. All images were collected from one experiment. Two independent experiments were performed and showed similar results.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(H) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to cytosolic (C)/membrane (M) fractionation followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.",
    "molecules": "DMSO, TG, thapsigargin"
  },
  {
    "caption": "(A) Met5-A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (B) The percentage of BrdU positive cells from (A) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.",
    "molecules": "BrdU"
  },
  {
    "caption": "(C) Met5-A cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (D) The percentage of BrdU positive cells from (C) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001. ",
    "molecules": "BrdU"
  },
  {
    "caption": "(E) Meso-33 cells transduced with vector or the indicated Merlin forms were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (F) The percentage of BrdU positive cells from (E) was quantified. Mean±s.e.m, N=3 biological repeats, ordinary one-way ANOVA. *P<0.05, **P<0.01. ",
    "molecules": "BrdU"
  },
  {
    "caption": "(G) Meso-33 cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to the BrdU incorporation assay. The percentage of BrdU positive cells was quantified. Mean±s.e.m, N=3 biological repeats, two-way ANOVA.",
    "molecules": "BrdU"
  },
  {
    "caption": "(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D).",
    "molecules": "DiBAC"
  },
  {
    "caption": "(C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary.",
    "molecules": "DiBAC, FM4-64"
  },
  {
    "caption": "(F, G) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence.",
    "molecules": "DiBAC, ouabain"
  },
  {
    "caption": "(H-H'') Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge.",
    "molecules": "DiBAC"
  },
  {
    "caption": "(I, I') Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).",
    "molecules": "DiBAC"
  },
  {
    "caption": "(B-B'') Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).",
    "molecules": "amiloride, DiBAC"
  },
  {
    "caption": "(C, C') Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary.",
    "molecules": "Amiloride"
  },
  {
    "caption": "(D, E) Expression of rpk RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).",
    "molecules": "DiBAC"
  },
  {
    "caption": "(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.",
    "molecules": "DiBAC"
  },
  {
    "caption": "(G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.",
    "molecules": "DiBAC"
  },
  {
    "caption": "(J-L) Effect of expression and activation of the Cl--selective channelrhodopsin ChloC. An overall reduction in Smo immunostaining was observed following channel activation (K), as compared to control glands kept in the dark (J). (L) Quantification of membrane fluorescence; N=10 glands, data were compared using an unpaired t test, (*** indicates p<0.001), error bars are standard deviations.",
    "molecules": "Cl"
  },
  {
    "caption": "A. Immunostaining  with S9.6 (green) and DAPI  (blue) in U2OS  cells, transfected for 48 hours with siCTRL or siDIS3 and then mock-treated (UNTR) or treated with  RNase H  or RNase III for 1 hour before fixation. Histograms show S9.6 signal intensity per cell, error bars indicate s.e.m. (n=3, biological replicates). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates).). **** = p < 0.0001, *** = p < 0.001, ns = not statistically significant, two-way ANOVA, scale bar is 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Immunostaining  with J2 (green) and DAPI  (blue) in U2OS  cells, as in panel A). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates). For all conditions, differences between siCTRL and siDIS3 were not statistically significant, two-way ANOVA, scale bar is 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Dot blot  analysis of DNA:RNA hybrids formation: serial dilutions (750 ng, 375 ng, 187,5 ng, 93,75 ng, 46,87 ng) of genomic DNA extracted from WT or DIS3  silenced U2OS  cells and pre-treated with RNase H, RNase III or mock treated, were arrayed on a membrane and probed using the S9.6 antibody. The histogram shows the S9.6 signal quantification in 3 biological replicates, error bars indicate s.e.m., normalized on SYBR®-Gold signal (Adj. Vol.), using Image lab software. *** = p < 0.001, ** = p < 0.01, ns= not statistically significant, two-way ANOVA.",
    "molecules": "SYBR®-Gold"
  },
  {
    "caption": "A. Representative confocal images upon irradiation  with the 405nm laser. U2OS  cells were presensitized with BrdU  for 24 hours, micro-irradiated, fixed after 10 minutes, and stained  with antibodies against γH2AX  and DIS3  scale bar is 10 µm. B. Magnification image of the white box in panel A. C. Intensity profiles of γH2AX  (green) and DIS3  signals through the yellow line in panel B. Quantification is performed with ImageJ software scale bar is 10 µm.",
    "molecules": "BrdU"
  },
  {
    "caption": "B. Immunostaining  with S9.6 (green) and DAPI  (blue) in MM.1S, RPMI-8226, PCM6, and OPM2  cells, treated (RNase H) or not (UNTR) with RNase H  for 1 hour. Histograms show S9.6 signal intensity per cell. More than 150 cells were counted, error bars indicate s.e.m. (n=3 biological replicates). **** = p < 0.0001, one-way ANOVA, scale bar is 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": "(C) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(D) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(E) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild-type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": "(C) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells treated with 1, 3 or 10 µM of promethazine and co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.",
    "molecules": "promethazine, TO-PRO-3"
  },
  {
    "caption": "A WT MDFs were treated ± TSI individually or in combination for 3 hrs. Whole cell lysates (WCL) and UBA-pull down (UBA-PD) fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments. Samples of UBA-pull down in following figures were analysed in the same way unless otherwise indicated.",
    "molecules": "UBA"
  },
  {
    "caption": "B WT, Ripk3-/-, Mlkl-/-, Tnfr1-/- and Traf2-/- MDFs were untreated (UT) or treated with TSI for 3 hrs. Nec-1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively.",
    "molecules": "GSK872, Nec-1"
  },
  {
    "caption": "E RIPK3-gyrase were inducibly expressed in Ripk3-/- MDFs by doxycycline (dox) for 5 hrs, and cells were then treated ± combination of TSI, or coumermycin (coum) for 3 hrs.",
    "molecules": "coum, coumermycin, dox, doxycycline"
  },
  {
    "caption": "B UBA-pull down from WT MDFs treated with TSI for 3 hrs were subjected to the DUBs shown in (A). Beads eluates were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.",
    "molecules": "UBA"
  },
  {
    "caption": "C 1.4 mL cleared cell lysate from 4 x 106 TSI-treated WT MDFs was split into three parts of the indicated volume, followed by UBA-pull down and DUB incubation. Bead eluates were analysed by Western blot and probed with the indicated antibodies. Representative of three independent experiments.",
    "molecules": "UBA"
  },
  {
    "caption": "A WT MDFs were treated with TSI for indicated time. Cells were fractionated into cytosol and crude membrane, followed by UBA-pull down. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.",
    "molecules": "UBA"
  },
  {
    "caption": "B WT USP21-CaaX and USP21C221R-CaaX (CR) were inducibly expressed in WT MDFs by doxycycline for 5 hrs. Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were untreated (UT) or treated with TSQ for 6 hrs. Cells were fractionated into cytosol (C) and crude membrane (M). Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. B Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.",
    "molecules": "doxycycline, UBA"
  },
  {
    "caption": "E HT29 cells were stimulated with TSI, ± NSA (500 nM), or left untreated (UT) for 16 hrs, followed by cellular fractionation. Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments. F Cell lysates from (E) were subjected to UBA-pulldown and analysed as described above.",
    "molecules": "NSA, UBA"
  },
  {
    "caption": "A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments. B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments. C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.",
    "molecules": "doxycycline, PI, UBA"
  },
  {
    "caption": "D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments. E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.",
    "molecules": "BAF, Bafilomycin A1, PS, PS341, doxycycline, IDN-6556, UBA"
  },
  {
    "caption": "B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.",
    "molecules": "doxycycline, UBA"
  },
  {
    "caption": "D MLKL-/- HT29 cells stably transfected with constructs encoding human MLKL-USP21 and MLKL-USP21C221R, were treated with doxycycline, NSA (1 μM), TSI or combinations thereof (added simultaneously). Sytox Green positive cells were quantified in real time by live cell imaging. Data are plotted as mean ± SEM of six independent experiments. (A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA). E Human MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in MLKL-/- HT29 cells by doxycycline (10 ng/mL) for 16 hrs, TSI was added for the indicated times but all samples were collected 25 hrs post-induction, followed by UBA-pulldown . Representative of three independent experiments.",
    "molecules": "Sytox Green, doxycycline, NSA, UBA"
  },
  {
    "caption": " C to F Binding of C179 (C), or antibodies present in AAV-HA (D), AAV-cHA (E) or WIV (F) pooled pre-challenge sera (n = 18 mice per group) to Cal/7/9 or PR8 virus after incubation of the virions at pH = 7.2, 5.8, 5.4, 5.0, 4.4 or 4.4+DTT to induce conformational changes in HA or remove the HA1 subdomain. Mean ± SD (n = 3, in technical triplicates). Statistical significance between pH = 7.2 and other conditions was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05, **p<0.01). ",
    "molecules": "DTT"
  },
  {
    "caption": " G and H IHC staining of submucosal glands (SMG) (G) or bronchi (H) with an anti-H1N1 polyclonal antibody (bar: 100 µm; red: influenza antigen (neufuchsin); blue: nuclei (haematoxylin)). ",
    "molecules": "haematoxylin, neufuchsin"
  },
  {
    "caption": "D Respiration of Subject 3 and Subject 1 derived fibroblasts (black and grey, respectively) compared to control fibroblasts (white) using high-resolution respirometry. ROX-corrected analysis of basal respiration, Oligomycin inhibited LEAK respiration and maximum uncoupled respiration (ETS). Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption. Data are mean ± SD from at least 3 experiments and analysed by Student's t-test. **p<0.01, ***p<0.001.",
    "molecules": "oxygen, Oligomycin"
  },
  {
    "caption": "B BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM. Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]).",
    "molecules": "DDM"
  },
  {
    "caption": "C Western blot analysis of whole cell lysates from controls (C1 and C2) and subject cell lines (S1 and S3) that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 100 or 200 ng/ml doxycycline (for 48 and 96 hours respectively) where indicated.",
    "molecules": "doxycycline"
  },
  {
    "caption": "D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls (C1 and C2) and subject cell lines that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 200 ng/ml doxycycline for 72 hours.",
    "molecules": "doxycycline"
  },
  {
    "caption": " Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunostained to examine ciliary Gli2 The white arrowheads (G) point to ciliary tips. Quantification results were from three independent experiments, each using MEFs from different Rabl2-deficient embryos. Gli2 intensities, presented in arbitrary unit, were measured from at least 22 cilia in each experiment and condition and pooled together. ",
    "molecules": "SAG, DMSO"
  },
  {
    "caption": " H. Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunoblotted to examine levels of Gli1 and Gli3 In the samples for immunoblotting (H), GFP-positive cells occupied 82.3% (GFP), 87.3% (GFP-Rabl2), and 81.2% (GFP-Rabl2Q80L), respectively, when 260 cells in each sample were scored. Relative band intensities are shown in red. ",
    "molecules": "SAG, DMSO"
  },
  {
    "caption": " I. Association of endogenous Rabl2-GTP with IFT-B. Mouse testis lysates with or without 1 mM GTPγS were subjected to sucrose gradient ultracentrifugation. Fractions were collected from top (low MW) to bottom (high MW). Note the obvious enrichment of Rabl2 in fraction #16 in the presence of GTPγS. ",
    "molecules": "GTP, GTPγS"
  },
  {
    "caption": " H, I. BBSomes and IFT-B were able to reach the ciliary base through retrograde IFT in the presence of Rabl2Q80L. Ciliary 3NG-Bbs5 (H) or Ift27-GFP (I) in IMCD3 cells co-expressing RFP-Rabl2 or RFP-Rabl2Q80L was live imaged at 4 fps. Snapshots for the ciliary RFP proteins, the first frames from Movies EV2-EV5, corresponding kymographs, and quantification results from the indicated numbers of cilia are presented. For processivity analysis (I), only traceable puncta were scored. ",
    "molecules": "3NG"
  },
  {
    "caption": " E, F. Rabl2D73GQ80L did not repress ciliary translocation of SMO and GPR161. RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were serum-starved for 24 h and treated with DMSO (mock) or SAG for an additional 24 h prior to immunostaining (E). The white arrows (E) point to positions of cilia. In quantification results (F), at least 100 cells were scored in each experiment and condition. ",
    "molecules": "SAG, DMSO"
  },
  {
    "caption": " G. Ciliary Rabl2D73GQ80L did not repress the BBSome export. FLIP assays were performed in RPE1 cells co-expressing 3NG-Bbs5 with RFP-tagged Rabl2Q80L or Rabl2D73GQ80L as depicted in Fig 5F. Photobleaching was executed at t = 0. Quantification results on half-life (t1/2) were from 10 cilia for each condition. ",
    "molecules": "3NG"
  },
  {
    "caption": "e) Relative mRNA expression  of β-catenin target genes in the RPMI8402  cell line (right panel) after knockdown of  β-catenin (left panel) compared to control. Graph represents the mean and sd of 6 independent experiments. f) Relative mRNA expression  of  β-catenin target genes in the RPMI8402  cell line after treatment with the  β-catenin inhibitor ICG-001 (10µM, overnight). Graph represents the mean and sd of 3 independent experiments. g) Relative mRNA expression  of  β-catenin target genes in the RPMI8402  cell lines (right panel) after knockdown of ZBTB33/Kaiso  (left panel) compared to control. Graph represents the mean and sd of 3 independent experiments. Data information: Statistical significance was determined by two-sided Student's t test.",
    "molecules": "ICG-001"
  },
  {
    "caption": "a) Effect of Vincristine  (VCR) treatment on cell viability  of RPMI8402  cells transduced with sh β-catenin  or sh-control. Dose-response curves  represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. *** p ≤ 0.001 of IC50  comparison with respect to sh control Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for a (at day 0)",
    "molecules": "VCR, Vincristine"
  },
  {
    "caption": "Effect of VCR  (b) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves  represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50  comparison with respect VCR-treated cells. Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for , b, (at day 0)",
    "molecules": "ICG-001, VCR"
  },
  {
    "caption": "Methotrexate  (MTX) (c left), L-Asparaginase (L-ASP) (c central) or Cytarabine  (Ara-C) (c right) treatment in the presence or absence of increasing doses of the β-catenin inhibitor  ICG-001.  Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50  Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for c, (at day 0)",
    "molecules": "ICG-001, Ara-C, Cytarabine, L-ASP, L-Asparaginase, Methotrexate, MTX"
  },
  {
    "caption": "c) Percentage of RPMI8402  cells PB, BM, spleen  and liver  at sacrifice. RPMI8402  cells identified as human CD45+HLA-ABC+CD5+ by  flow cytometry and gated on DAPI  negative cells. Control: animals left untreated (n = 2); Chemo   + DMSO: animals receiving chemotherapy   and vehicle (n = 4); Chemo   +  ICG-001: animals receiving chemotherapy   and the  β-catenin inhibitor  ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo   + DMSO  and Chemo   + ICG-001  groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo   + DMSO  and Chemo   + ICG-001  groups was determined by two-sided Student's t test.",
    "molecules": "ICG-001, DAPI, DMSO"
  },
  {
    "caption": "g) H&E staining  of representative images per each mouse  treated as indicated. h) Percentage of liver  area covered by cellular colonies per 10X micrograph. 4 different images were quantified per animal and mean values per animal are represented by dots. Control: animals left untreated (n = 2); Chemo   + DMSO: animals receiving chemotherapy   and vehicle (n = 4); Chemo   + ICG-001: animals receiving chemotherapy   and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo   + DMSO  and Chemo   + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo   + DMSO  and Chemo   +  ICG-001 groups was determined by two-sided Student's t test.",
    "molecules": "ICG-001, DMSO"
  },
  {
    "caption": "Confocal micrographs show CK7 (green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 d. Scale bars: 50 μm.",
    "molecules": "collagen"
  },
  {
    "caption": "Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm.",
    "molecules": "cisplatin, collagen"
  },
  {
    "caption": "EphA2 (total and phosphorylated at S897 or Y588) in OVCAR3, OVCAR4 and OVCAR8 after treatment with 0-20 μM cisplatin for 72 h was assessed by immunoblotting (A) and quantified for pS897/pY588 ratio (B). N = 4.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Cytotoxicity assay results after cell treatment with 0-20 μM cisplatin for 72 h. N = 6.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Quantitative assessment of EphA2 (total and phosphorylated) and pS897/pY588 ratio in early passage patient-derived HGSC cultures treated with 0-20 μM cisplatin for 72 h (see immunoblots in Appendix Fig S2A). N = 6 patients, pooled.",
    "molecules": "cisplatin"
  },
  {
    "caption": "EphA2 (total and phosphorylated) in OVCAR4 and OVCAR8 after treatment with 0-80 μM carboplatin for 72 h (G) along with pS897/pY588 quantification (H). N = 3.",
    "molecules": "carboplatin"
  },
  {
    "caption": "Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors (day 53; after 13 d i.p. treatment). Color scale unit: p/sec/cm2/sr. Charts illustrate the tumor growth (B) and ascites volumes at the end of the follow-up (C). Carboplatin treatment started on day 35. Bioluminescent unit: p/sec/cm2/sr. N = 5 mice/group.",
    "molecules": "carboplatin, Carboplatin"
  },
  {
    "caption": "Representative micrographs of hematoxylin-eosin staining in the xenografts tumors. Scale bars: 100 μm",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Immunoblot images show EphA2 (A; total, pS897 and pY588) and corresponding pS897/pY588 ratios (B) of TYK-nu and TYK-nu.R after treatment with 0-10 μM cisplatin for 72 h . N = 5.",
    "molecules": "cisplatin"
  },
  {
    "caption": "EphA2 (total and phosphorylated) in the cells treated with 0-80 μM carboplatin for 72 h.",
    "molecules": "carboplatin"
  },
  {
    "caption": "EphA2 (total and pS897) in TYK-nu after treatment with inhibitors against MEK (UO126, 10 μM), PKA (H89.2HCl, 10 μM), PI3K (LY294002, 10 μM) or its alpha subunit (BYL-719, 5 μM) and RSK (BI-D1870, 10 μM) in combination with 0-10 μM cisplatin for 72 h (E). Corresponding EphA2-pS897 quantification for BI-D1870 treatment (F) N = 5.",
    "molecules": "BI-D1870, BYL-719, cisplatin, H89.2HCl, LY294002, UO126"
  },
  {
    "caption": "EphA2 (total and pS897) in TYK-nu.R treated with 0-20 μM cisplatin without or with 10 μM BI-D1870 for 72 h.",
    "molecules": "BI-D1870, cisplatin"
  },
  {
    "caption": "EphA2, RSK and ERK1/2 (total and phosphorylated) in TYK-nu (H) and OVCAR4 (I) treated with 0-30 μM carboplatin without or with 50 μM LJH685 for 72 h.",
    "molecules": "carboplatin, LJH685"
  },
  {
    "caption": "Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 10 μM BI-D1870",
    "molecules": "BI-D1870, cisplatin"
  },
  {
    "caption": "Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 50 μM LJH685",
    "molecules": "cisplatin, LJH685"
  },
  {
    "caption": "RSK and EphA2 (total and phosphorylated) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h.",
    "molecules": "cisplatin, LJH685"
  },
  {
    "caption": "Immunoblot images (A) and quantification (B) show GPRC5A 46/41 kDa ratios in OC cells treated with 0-10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h. The ratio in mock cells was set to 1. N = 4.",
    "molecules": "cisplatin, LJH685"
  },
  {
    "caption": "Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm.",
    "molecules": "cisplatin, LJH685"
  },
  {
    "caption": "EphA2 (total and phosphorylated), RSK1/2 and GPRC5A in TYK-nu were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.",
    "molecules": "cisplatin"
  },
  {
    "caption": "EphA2 (total and phosphorylated), RSK1/2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK-nu.R were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Chart illustrates the viability of the RSK1/2 depleted TYK-nu.R after cisplatin treatment. N = 3.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Confocal micrographs show cleaved caspase-3 (clCasp3, green), mRFP (orange) and phalloidin (F-actin, red; only shown for CAF) in 3D OVCAR8-RFP and OCKI_p22 CAF mono- and co-cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 100 μm.",
    "molecules": "cisplatin, LJH685, phalloidin"
  },
  {
    "caption": "Confocal micrographs show cytokeratin 7 (CK7, green) and phalloidin (F-actin, red) in 3D patient OCKI_p13 mono- and co-culture with OCKI_p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 50 μm. Quantitative assessment of clCasp3+ OCKI_p13 cells in 3D co-cultures of patient-derived cells. N = 4.",
    "molecules": "cisplatin, LJH685, phalloidin"
  },
  {
    "caption": "Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.",
    "molecules": "BI-D1870, carboplatin"
  },
  {
    "caption": "Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.",
    "molecules": "BI-D1870, carboplatin"
  },
  {
    "caption": "Charts illustrate EphA2-pS897 inhibition (circles) and EphA2-pY588 increase (squares) by 10 µM BI-D1870 alone (unbroken line) or in combination with 0-10 µM cisplatin (dotted line) in the GPRC5Ahigh HGSC cells. See Appendix S7B for immunoblots.",
    "molecules": "BI-D1870, cisplatin"
  },
  {
    "caption": "Cell viability of the GPRC5Ahigh OCKI_p02 and OCKI_p06 after treatment with a combination of 0-20 µM cisplatin and 10 µM BI-D1870 for 72 h. N = 3.",
    "molecules": "BI-D1870, cisplatin"
  },
  {
    "caption": "Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFs",
    "molecules": "cisplatin, LJH685"
  },
  {
    "caption": "Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 10 µM BI-D1870 in mesothelial cells (G; N = 3).",
    "molecules": "BI-D1870, cisplatin"
  },
  {
    "caption": "Chart illustrates the changes of EphA2-pS897 and EphA2-pY588 by BI-D1870 alone and with cisplatin in mesothelial cells. Upon combination with 10 µM cisplatin for 72 h, 10 µM BI-D1870 decreased EphA2-pS897 and EphA2-pY588. See Appendix Fig S7J for immunoblot.",
    "molecules": "BI-D1870, cisplatin"
  },
  {
    "caption": "Confocal micrographs of EphA2 (red) and GPRC5A (green) in primary OCKI_p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin. Scale bars: 20 μm.",
    "molecules": "cisplatin, collagen"
  },
  {
    "caption": "A, B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR (qPCR). Controls were treated with vehicle for 84 h and set to 100. Data represent the mean of three independent experiments in triplicate, and bars represent SE. P-values ranged between 1.66 × 10−5 and 0.025, and IRE1α expression in R1881 48 h was *P = 0.013 with respect to vehicle-treated cells using unpaired Student's t-test.",
    "molecules": "R1881"
  },
  {
    "caption": "F, G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis. Data presented are representative of three independent experiments.",
    "molecules": "R1881"
  },
  {
    "caption": "After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.A Expression level of AR mRNA upon siRNA treatment for 48 h assessed by qPCR in LNCaP cells. Expression in cells transfected with CTRL siRNA was set to 1. Bars represent SE with *P = 0.001 indicating significant difference between AR siRNA- and control siRNA-transfected cells using paired Student's t-test.",
    "molecules": "R1881"
  },
  {
    "caption": "After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.B-E Same as in (A), but mRNA expression levels of the indicated UPR genes were determined by qPCR at indicated time points after R1881 stimulation. Expression in cells transfected with CTRL siRNA was set to 100. Bars represent SE. P-values are shown indicating significant difference between AR siRNA- and control siRNA-transfected cells using unpaired Student's t-test.",
    "molecules": "R1881"
  },
  {
    "caption": "After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.F Expression of the indicated proteins under conditions indicated on the top label was determined by Western blot analysis. Representative blots for three independent experiments are shown.",
    "molecules": "R1881"
  },
  {
    "caption": "A, B LNCaP (A) or VCaP cells (B) were cultured and treated with vehicle (C) for 48 h or R1881 for 24 and 48 h. The cells were then fixed, and ChIP assay was performed as described in Materials and Methods using AR antibody. The data shown are representative of one experiment in duplicate. Error bars represent SE. *P < 0.01 for LNCaP and *P < 0.04 for VCaP indicate significant difference between C (control) and R1881 using unpaired Student's t-test.",
    "molecules": "R1881"
  },
  {
    "caption": "XBP-1S expression was determined by IHC of a neoadjuvant hormone therapy (NHT) tissue microarray containing samples from hormone naïve (untreated) (n = 25), patients that received NHT for 1-6 months (n = 33), and patients that received NHT for 7-12 months (n = 50), as indicated. Representative images are shown. Scale bars: 100 μm.Quantitative presentation of the data from (C). *P = 0.006 in unpaired Student's t-test.",
    "molecules": "hormone"
  },
  {
    "caption": "B. Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A-Fc and visualized with immunofluorescent staining to detect GFP (green) and phalloidin (red). Average intensity images for all different constructs are shown in grey color. Scale bar 20 µm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "C, D. Defective cilia  formation in ccdc108  morphants. Embryos  at stage 27 were fixed and stained with the acetylated tubulin  (Ac-tub) antibody and/or  Cep164   antibody. mRNA of a membrane-bound form of GFP (mGFP) was co-injected with each morpholino  to indicate targeted cells. Representative  confocal  images (C) and 3D-SIM  images (D) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108   depleted embryos. (C) 20 images of 20 embryos and (D) > 80 MCCs from 6 embryos for each condition. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.",
    "molecules": "morpholino"
  },
  {
    "caption": "G.  In situ hybridization chain reaction (HCR) reveals that ccdc108  mRNA is specially detected in  MCCs. Embryos  at stage 35 were fixed and subjected to in situ HCR (red). Embryos were then incubated with the  Ac-tub   antibody (cyan) and DAPI (gray).",
    "molecules": "DAPI"
  },
  {
    "caption": "C, D. Representative confocal  images and plots show that  ccdc108 morphants displayed  ciliogenesis defects in neuromasts (C) and olfactory placodes (D). Zebrafish embryos were fixed and stained  with the Ac-tub  antibody (green),  phalloidin (purple) and Hoechst  (blue). Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). Mean ± s.e.m values are presented.",
    "molecules": "Hoechst, phalloidin"
  },
  {
    "caption": "C. Representative  3D-SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented.  Apical  area of MCCs in each condition were measured ; 80  MCCs from 6 embryos for each condition. D. Representative confocal  images show a significant reduction in apical actin following depletion of    Ccdc108. Cell boundaries and apical actin   network were marked with mGFP (green) and phalloidin (purple) and phalloidin intensity levels measured and plotted. > 80 MCCs from 6 embryos  for each condition. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.",
    "molecules": "phalloidin"
  },
  {
    "caption": "A-C. The IFT-interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos: (A) impaired  multiciliogenesis, (B) defective apical migration/docking of centrioles, and (C) reduced enrichment of apical F-actin. Embryos at stage 27 were fixed and stained with the acetylated tubulin (A; Ac-tub; green), Cep164 (B; purple) and  Cetn1 (B; green) antibodies or phalloidin (purple). mRNA of a membrane-bound form of GFP was co-injected with each morpholino to indicate targeted cells (A,B). Typical D-SIM images (A,B) and confocal  images (C) are present. Embryos were treated as described in Figure 1C and expressed mGFP or  GFP-Ccdc108 mutants. Plots show results from three independent experiments. (A) > 70 MCCs from 6 embryos, and (C) > 70 MCCs from 6  embryos for each condition. Unpaired two-tailed t-test was performed (*** p<0.001; **p<0.01). Mean ± s.d. values are presented.",
    "molecules": "morpholino, phalloidin"
  },
  {
    "caption": "C. Ift74  depletion affects centriole apical migration in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs show failure of apical trafficking of basal bodies. Basal bodies (Cetn1, green) and distal appendages  (Cep164, purple) were labeled with indicated antibodies. Quantitative plot shows a slight significant reduction in  basal bodies by the depletion of Ift74. >70  MCCs from 6 embryos for each condition. D. Representative 3D-SIM images and plots reveal that apical  expansion of MCCs is slightly affected by Ift74  depletion. >70 MCCs  from 6 embryos  for each condition. Embryos  were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.",
    "molecules": "phalloidin"
  },
  {
    "caption": "E. Representative confocal  images and plot show a significant reduction in  apical F-actin  enrichment upon Ift74 depletion. >70 MCCs  from 6  embryos for each condition.  Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple). Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.",
    "molecules": "phalloidin"
  },
  {
    "caption": "C-E. Ccdc108 depletion affects  centriolar distribution of IFT-B proteins during  centriole migration in MCCs. Embryos were co-injected with GFP-IFTs (C: Ift74; D:  Ift80; E:  Ift88; green), Cetn4-RFP (purple) and each  morpholino, and imaged at stage 18.  HA  tagged Ccdc108  proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to  immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that  Ccdc108-depleted MCCs showed a significant decrease of centriolar  distribution of IFT-B proteins (Ift74, Ift80 and Ift88), and Ccdc108-IFT  interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT-B proteins. >40 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are also presented.",
    "molecules": "morpholino"
  },
  {
    "caption": "A, B. Centriolar distribution of ciliary adhesion complex related proteins  Fak and Cp110  are not reduced following Ccdc108  depletion.  Embryos  were co-injected with GFP-Cp110 (A) GFP-Fak (B),  Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. >30  MCCs from 6 embryos  for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.",
    "molecules": "morpholino"
  },
  {
    "caption": "C, D.  Ccdc108   depletion affects  centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs. Embryos were co-injected with GFP-Drg1 (C) GFP-RBD (D), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18.  HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos  were subjected to immunoblotting to evaluate the protein expression  levels.  α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of  centriolar  distribution of Drg1 and  RBD, and neither of Ccdc108-IFT interaction deficient mutants can rescue the  centriolar phenotypic defects. >30 MCCs  from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.",
    "molecules": "morpholino"
  },
  {
    "caption": "FACS analysis of Lysotracker (B) dye fluorescence in RCS treated with the indicated FGF ligands (50 ng/mL) for 16h. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. Analysis of variance one way (ANOVA) p<0.002 (B) Tukey's post hoc test ***p<0.0005.",
    "molecules": "Lysotracker"
  },
  {
    "caption": "FACS analysis of DQ-BSA (C) dye fluorescence in RCS treated with the indicated FGF ligands (50 ng/mL) for 16h. Concanamycin A was used at 100 nM for 1h to inhibit lysosomal function. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. Analysis of variance one way (ANOVA) =0.005 (C); Tukey's post hoc test ***p<0.0005.",
    "molecules": "DQ-BSA, Concanamycin A"
  },
  {
    "caption": "D. Lamp1 immunofluorescence (red) in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours. Insets show magnification of the boxed area. Nuclei were stained with DAPI (blue). Scale bar 15 μm and 2 μm (higher magnification boxes) . Bar graph shows quantification of Lamp1-positive vesicles/cell. Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. n=45 cells were analyzed. Student paired T-Test *p<0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Representative TEM images of RCS chondrocytes treated with 5% ABS (vehicle) and FGF18 (50 ng/mL) for 16 hours. BafA1 (100 nM; 4 hours) was used to inhibit lysosome activity. Arrows indicate lysosomes. Higher magnification insets showed presence of ER membranes decorated with ribosomes (arrowheads). Scale bar 500nm. Quantification shows average number of ER membranes and mitochondria number/ lysosome vesicle (Lys). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment. ER fragment/vesicle n=60 (vehicle) and n=72 (FGF18) cells were analyzed; Mitochondria number/vesicle: n=40 (vehicle) and n=72 (FGF18) cells were analyzed. Student paired T-Test ***p<0.0005; NS not significant.",
    "molecules": "BafA1"
  },
  {
    "caption": "F. Representative co-immunofluorescence staining of ER (ER tracker BODIPY green) and lysosomes (Lamp1, red) in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 16 hours. Insets show higher magnification of boxed area. Scale bar 15 μm and 2 μm (higher magnification boxes). N=3 biological replicates/treatment.",
    "molecules": "BODIPY"
  },
  {
    "caption": "G. Schematic representation of EATR assay: eGFP fluorescence, but not mCherry, is lost at acidic pH. Chondrocytes with indicated genotypes (ctrl=wild type) were treated with FGF18 (50 ng/mL; 12 hours) and BafA1 (200 nM; 3 hours) where indicated. ER acidification was measured by FACS. Mean +/- standard error of the mean (sem) of N=3 biological replicates. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; **p<0.005; NS not significant.",
    "molecules": "BafA1"
  },
  {
    "caption": "H. FACS analysis of DQ-BSA dye fluorescence in chondrocytes with indicated genotypes (ctrl=wild type) treated with FGF18 (50 ng/mL) for 16 hours. Concanamycin A was used at 100 nM for 1h to inhibit lysosomal degradation. Fluorescence intensities were expressed as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates/treatment/genotype. Analysis of variance (ANOVA) p=0.00279; Tukey's post hoc test **p<0.005; *p<0.05; NS not significant.",
    "molecules": "DQ-BSA dye, Concanamycin A"
  },
  {
    "caption": "J. Representative immunofluorescence staining of ER (CLIMP63, green) and lysosomes (TMEM192-HA, red) in chondrocytes with indicated genotypes (ctrl=wild type) treated with FGF18 (50 ng/mL for 16h). BafA1 was used at 100 nM for 4 hours to inhibit lysosomal degradation. Quantification of relative CLIMP63 fluorescence into TMEM192-HA positive vesicles. N=3 biological replicates; n=21 cells were analyzed. Mean +/- standard error of the mean (sem). Paired Student's T-test NS= not significant. Scale bar 10 μm and 1.3 μm (higher magnification boxes).",
    "molecules": "BafA1"
  },
  {
    "caption": "Representative western blot analysis of IRS1, p-P70S6K (T389), P70S6K, p-AKT (T308), AKT in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/mL) for 12h. MG132 (10 μM for 6h) to inhibit proteasome (C) activity, respectively. β-actin was used as loading control. N=3 independent experiments. Bar graphs showed quantification of indicated proteins normalized to their totals. IRS1 was normalized to β-actin. Mean +/- standard error of the mean (sem). Student's paired T-test **p<0.005; *p<0.05; NS not significant.",
    "molecules": "MG132"
  },
  {
    "caption": "E. Co-immunofluorescence of p-IRS1 S307 mouse (green) and p-S6 S240/S242 (red) ribosomal protein in IRS1-overexpressing RCS chondrocytes treated with FGF18 (50 ng/mL) for 12h. Nuclei (N) were stained with DAPI (blue). Scale bar 15 μm. Quantification analysis of p-S6 fluorescence intensity in IRS1-overexpressing vs non expressing RCS chondrocytes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=35 cells analyzed. Student's paired T-test *p<0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. EATR assay. Chondrocytes were treated with FGF18 (50 ng/mL; 16 hours) and Actinomycin (1μg/mL; last 4 hours) where indicated. ER acidification was measured by FACS. Mean +/- standard error of the mean (sem) of N=17 (veh), N=17 (FGF18), N=5 (actinomycin), N=5 (actinomycin + FGF18) biological replicates. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; NS not significant.",
    "molecules": "Actinomycin, actinomycin"
  },
  {
    "caption": "B. FACS analysis of Lysotracker dye fluorescence in RCS chondrocytes treated with FGF18 (50 ng/mL) for 16 hours. Actinomycin (1μg/mL) was added for the last 4 hours. Fluorescence intensities were calculated as % relative to vehicle (5% ABS). Mean +/- standard error of the mean (sem) of N=3 biological replicates. Analysis of variance one way (ANOVA) p=0.0002; Tukey's post hoc test ***p<0.0005; NS not significant.",
    "molecules": "Actinomycin, Lysotracker"
  },
  {
    "caption": "C. Representative immunofluorescence staining of Lamp1 (red) in RCS chondrocytes treated with vehicle (5% ABS), FGF18 (50 ng/mL; 16 hours) and Actinomycin (1μg/mL; 4 hours). Nuclei were stained with DAPI (blue). Scale bar 10 μm. Representative images of N=3 biological replicates. Scale bar 10 μm.",
    "molecules": "Actinomycin, DAPI"
  },
  {
    "caption": "G. Immunofluorescence staining of CLIMP63 (green) and lysosomes (TMEM192-HA, red) in RCS chondrocytes with indicated genotypes (ctrl=wild type) upon FGF18 treatment (50 ng/mL for 16h). BafA1 (100 nM; 4 hours) was used to inhibit lysosomal degradation; scale bar 20 μm. Insets show magnification of CLIMP63 accumulation into lysosomes; scale bar 2 μm. Quantification of CLIMP63 fluorescence intensity into TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=10 cells/experiment were analyzed. Analysis of variance one way (ANOVA) p=1.55e-7; Tukey's post hoc test ***p<0.0005; NS not significant. H. EATR assay in RCS with indicated genotypes (ctrl=wild type) showing % of cells with acidified ER by FACS analysis. FGF18 was used at 50 ng/mL for 16 hours. Mean +/- standard error of the mean (sem) of N=4 biological replicates/treatment/genotype. Analysis of variance one way (ANOVA) p<0.0001; Tukey's post hoc test ***p<0.0005; NS not significant. ",
    "molecules": "BafA1"
  },
  {
    "caption": "A. TFE3 (green) subcellular localization in RCS with indicated genotypes (control=wild type) treated with FGF18 (50 ng/mL) for 16 hours. Torin1 (1 μM for 2 hours) was used as positive control. Nuclei were stained with DAPI (blue). Bar graph shows quantification (expressed as %) of cells with nuclear TFE3 and TFEB (representative images of TFEB immunofluorescence are shown in EV2E). Mean +/- standard error of the mean (sem) of N=3 biological replicates; n=80 (TFE3) and n=70 (TFEB) cells/experiment were analyzed. Scale bar 10 μm. Analysis of variance one-way (ANOVA) p=3.23e-12 (TFEB), p=2.48e-11 (TFE3); Tukey's post hoc test ***p<0.0005; NS not significant.",
    "molecules": "DAPI, Torin1"
  },
  {
    "caption": "B. Western blot analysis of TFEB, and phospo-TFEB (Serine 142) in RCS chondrocytes stably expressing human TFEB-3XFlag protein. Cells were treated with vehicle (5% ABS) or FGF18 (50 ng/mL) for 16h. β-actin was used as loading control. Representative of three independent experiments.",
    "molecules": "Serine"
  },
  {
    "caption": "C. GFP immuno-EM staining in FGFR3/4KO chondrocytes infected with a constitutive nuclear (and active) mutant TFEB fused to GFP tag (TFEB- S142A:S211A-GFP). GFP positive gold immune-particles showed presence of GFP-puncta into the nucleus (N, stained in green for visualization). Lysosomes were stained in blue for visualization. Insets showed magnification of lysosomes. Quantification of lysosome diameter (nm) in control (wild type) and FGFR3;4KO chondrocytes infected with empty or with TFEB- S142A:S211A-GFP vector. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n=40 (veh) n=78 (FGFR3;4KO) n=57 (FGFR3;4KO +TFEB-S142A:S211A-GFP) cells were analyzed. Scale bar 500 nm. Kruskal Wallis test p=1.43e-13; Nemenyi post hoc test ***p<0.0005; NS not significant.",
    "molecules": "gold"
  },
  {
    "caption": "D. Co-immunofluorescence of p-IRS1 (S307 mouse) and TFEB in IRS1-overexpressing RCS chondrocytes treated with FGF18 (50 ng/mL) for 12h. Nuclei (N) were stained with DAPI (blue) and delimited with dashed line. Scale bar 15 μm. Quantification of TFEB nuclear localization in IRS1-overexpressing vs non-expressing RCS. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n= 124 cells analyzed; Student's un-paired T-test *p<0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Subcellular localization analysis of TFEB (red) and TFE3 (green) in RCS chondrocytes treated with FGF18 (50 ng/mL) for 12h. JNK inhibitor was used at 50 μM for 12h. Nuclei were stained with DAPI (blue). Quantification analysis showed % of cells with nuclear TFEB and TFE3 in RCS chondrocytes with indicated treatments. Scale bar 15 μm. Mean +/- standard error of the mean (sem) of N=3 biological replicates. n= 126 cells (control), 126 cells (FGF18), 95 cells (JNK Inhibitor), 163 cells (JNK Inhibitor + FGF18). Student's paired T-test *p<0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Co-staining of CLIMP63 (green) and TMEM192-HA (lysosomes, red) in control and TFEB;3KO RCS treated with FGF18 (50 ng/mL for 16 hours). BaFA1 was used at 100 nM for 3h. Scale bar 15 μm and 2 μm (higher magnification boxes).",
    "molecules": "BaFA1"
  },
  {
    "caption": "H. ChIP analysis of TFEB binding to Fam134b DNA in RCS cells transfected with TFEB-3XFLAG. Numbers in the CLEAR site (yellow box) refer to the distance [in base pairs] from the transcriptional start site (+1) of Fam134b-2 gene. Immunoprecipitated DNA was normalized to the input and plotted as relative enrichment over a mock control. Bar graph shows fold change enrichment; mean +/- standard error of the mean (sem) of N=3 independent experiments. Student un-paired T-Test **p<0.005.",
    "molecules": "DNA"
  },
  {
    "caption": "C,D. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (TMEM192-HA, red) in control and Sh-FAM134B U2OS cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (C). In (D), Quantification of CLIMP63 fluorescence in TMEM192-HA decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 21 (vehicle ctrl), N=33 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle shFAM134B), N= 30 (TFEB S142A:S211A-GFP shFAM134B) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant.",
    "molecules": "BaFA1"
  },
  {
    "caption": "E,F. Co-immunofluorescence staining of ER (CLIMP-63, green) and lysosomes (Lamp1, red) in WT and Fam134bKO MEF cells over-expressing TFEB S142A:S211A-GFP (purple). BaFA1 was used at 100 nM for 4h. Scale bar 15 μm and 2 μm (higher magnification boxes) (E). In (F), Quantification of CLIMP63 fluorescence in Lamp1 decorated lysosomes. Mean +/- standard error of the mean (sem) of N=3 biological replicates. N= 20 (vehicle ctrl), N=22 (TFEB S142A:S211A-GFP ctrl), N=21 (vehicle Fam134bKO), N= 20 (TFEB S142A:S211A-GFP Fam134bKO) cells were analysed. Student's un-paired T-test ***p<0.0005; NS not significant.",
    "molecules": "BaFA1"
  },
  {
    "caption": "A,B. Representative images of alcian blue (cartilage) and Alizarin red (bone) skeletal staining showing growth retardation in Fgfr3/4 dKO mice compared to age/sex wild type littermate at post-natal day 30. B. Femur, tibia and tail details.",
    "molecules": "Alizarin red, alcian blue"
  },
  {
    "caption": "C. Haematoxylin/Eosin staining of femoral growth plate sections from wild type and Fgfr3/4 dKO mice. Higher magnification insets showed a disorganized hypertrophic chondrocyte layer, in Fgfr3/4 dKO mice. Scale bar 60 μm.",
    "molecules": "Eosin, Haematoxylin"
  },
  {
    "caption": "Alcian blue (cartilage) (I) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) cartilage length (I) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.",
    "molecules": "Alcian blue"
  },
  {
    "caption": "Alizarin Red (bone) (J) staining of scramble and Fam134bmo medaka fish. Graph shows quantification of Ethmoid plate (EP), Palatoquadrate (PQ), Ceratohyal (CH), Paired Prootics (PO), Ceretobranchials 1 to 5 (CB1 to CB5) bone mineralization (J) in Fam134bmo and scramble fish. Values were expressed as % relative to the scramble (100% red dotted line). Mean +/- standard error of the mean (sem) of n=9 fish/genotype. Student unpaired T-Test *p<0.05; **p<0.005; ***p<0.0005. NS not significant.",
    "molecules": "Alizarin Red"
  },
  {
    "caption": " (A) Bcl-xfl/fl;RosaCreERT2+/Ki (n=12) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=16) mice (age 9-12 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Mice were monitored for up to 200 days post-treatment with tamoxifen. Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001. ",
    "molecules": "tamoxifen"
  },
  {
    "caption": " (B) Bcl-xfl/fl;RosaCreERT2+/Ki (n=24) or, as controls, Bcl-xfl/fl (n=9) and RosaCreERT2+/Ki (n=6) mice (males and females, aged 8-14 weeks, numbers also indicated in the figure legend) were lethally γ-irradiated (2 x 5.5 Gy, 3 h apart) and reconstituted with bone marrow from UBC-GFP mice (referred to as GFP-Chimeras). After 8 weeks, reconstituted mice were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses oral gavage) and monitored for up to 200 days (termination of the experiment). Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001. ",
    "molecules": "tamoxifen"
  },
  {
    "caption": " (C-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001. (G) Spleen weights were measured in sick mice (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse and numbers are indicated. Statistical significance was assessed using the Student's t-test; ****p<0.0001. ",
    "molecules": "hemoglobin, HGB, tamoxifen"
  },
  {
    "caption": " (I) Histological analysis of H&E-stained sections of spleens of tamoxifen-treated mice of the indicated genotypes. ",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), or as controls, Bcl-xfl/fl;GFP-Chimeras (n=6) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Total white blood cell counts (WBC) were analyzed by ADVIA in sick mice or at the termination of the experiment (healthy control mice). Data are presented as mean ±SEM. Each data point represents an individual mouse and n numbers are indicated above. Statistical significance was assessed using the Student's t-test. No statistically significant differences were observed. ",
    "molecules": "tamoxifen"
  },
  {
    "caption": " (C) Histological analysis of H&E-stained sections of the sternum of sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras or healthy wild-type and RosaCreERT2+/Ki;GFP-Chimera control mice at the indicated time points post-treatment with tamoxifen (dpt=days post-treatment). ",
    "molecules": "tamoxifen"
  },
  {
    "caption": " (E) ALT (left panel), AST (middle panel) and bilirubin levels (right panel) in the serum were determined in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=18) or in healthy RosaCreERT2+/Ki;GFP-Chimeras (n=4) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; **p<0.01. nd=not detected. ",
    "molecules": "bilirubin"
  },
  {
    "caption": " Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Kidney weights were measured in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; *p<0.05, ***p<0.001. ",
    "molecules": "tamoxifen"
  },
  {
    "caption": " (A) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells. (B) TUNEL+ cells were quantified using a personalized script for the Image J software. Kidney sections were divided into ~40 microscopic images and the total numbers of blue and brown (TUNEL+) nuclei were determined. The percentages of TUNEL+ nuclei were calculated for each picture. The average value for each kidney section was then determined and plotted as a single data point. Error bars represent SEM from at least 3 independent samples for each genotype and treatment. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test (comparing control RosaCreERT2+/Ki (24 h post-tamoxifen n=2, 24 h post-γ-irradiation n=9) with Bcl-xfl/fl;RosaCreERT2+/Ki (24 h post-tamoxifen n=6, 24 h post-γ-irradiation n=5) and control RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=7, End point n=8) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=8, End point n=6) (***p<0.001). ",
    "molecules": "hematoxylin, tamoxifen"
  },
  {
    "caption": " (A, B) Data are presented as % survival and statistical significance was assessed using the Mantel-Cox (Log-rank) test comparing tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=25) with Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=25), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=18), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=15) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=22). *p<0.05, ****p<0.0001. The survival curves for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras ",
    "molecules": "tamoxifen"
  },
  {
    "caption": " (C) Histological analysis of H&E-stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points (dpt = days post-tamoxifen treatment). Pictures are representative of at least 3 mice for each genotype and treatment. ",
    "molecules": "tamoxifen"
  },
  {
    "caption": " (D-F) (D) Total red blood cell (RBC) count, (E) hematocrit (HCT) and (F) hemoglobin (HGB) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test comparing tamoxifen-treated RosaCreERT2+/Ki;GFP-Chimeras (n=6) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=20), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=12), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=15), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=10) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=17) ; ***p<0.001; ****p<0.0001. Data for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras and RosaCreERT2+/Ki;GFP-Chimeras ",
    "molecules": "hemoglobin, HGB, tamoxifen"
  },
  {
    "caption": " (A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimeras and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Mice were monitored for up to 200 days post-treatment (dpt). Data are presented as % survival. ",
    "molecules": "A1331852"
  },
  {
    "caption": " (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Mice were monitored for up to 150 days post-treatment (dpt). Data are presented as % survival. ",
    "molecules": "5-Fluorouracil, 5-FU, A1331852, Cyclophosphamide"
  },
  {
    "caption": " (C) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with the BCL-XL inhibitor A1331852 (n=7, 5 doses by oral gavage, 25 or 100 mg/kg body weight each dose). Total platelet counts were determined by ADVIA at the indicated time points. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (**p<0.01, ***p<0.001, ****p>0.0001). ",
    "molecules": "A1331852"
  },
  {
    "caption": " Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of drug-treated GFP-Chimeras or control mice (n=8 each group) at the termination of the experiment Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001). ",
    "molecules": "hemoglobin, HGB"
  },
  {
    "caption": " Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of in drug-treated C57BL/6-Ly5.1 (wild-type) mice or control mice at the indicated time points (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001). ",
    "molecules": "hemoglobin, HGB"
  },
  {
    "caption": " (A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimera and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated GFP-Chimeras or control mice at the termination of the experiment (n=8 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed. ",
    "molecules": "A1331852"
  },
  {
    "caption": " (B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with the BCL-XL inhibitor A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated mice or control mice at the termination of the experiment (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed. ",
    "molecules": "5-Fluorouracil, 5-FU, A1331852, Cyclophosphamide"
  },
  {
    "caption": "A Freeze-fracture electron micrograph of wild-type yeast treated with DTT for 3 h. The left panel shows two spherical structures, one in a vacuole membrane invagination and one inside the vacuole. The middle panel shows the boxed area of the left panel at higher magnification. Arrows indicate membrane layers that are free of intramembrane particles. The right panel shows the protoplasmic fracture face (P face) of cortical ER with a high density of intramembrane particles. Cy, cytosol; N, nucleus; V, vacuole.",
    "molecules": "DTT"
  },
  {
    "caption": "B Fluorescence images of cells expressing GPD promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. The upper and lower panel show vacuole-associated and perinuclear GFP-Pho8 stretches and an intravacuolar GFP-Pho8 ring, respectively. Scale bars: 2 µm.",
    "molecules": "CMAC"
  },
  {
    "caption": "D Fluorescence images of cells expressing Hmg2-GFP and either additionally expressing Sec63-mCherry or stained with CMAC. The upper and lower panel show cytosolic and vacuolar Hmg2-GFP rings, respectively. Scale bars: 2 µm.",
    "molecules": "CMAC"
  },
  {
    "caption": "A Individual frames from time-lapse imaging of a cell expressing GAL promoter-driven GFP-Pho8 and stained with the vacuole membrane dye FM4-64. Numbers indicate the time in minutes after the start of the image sequence. Scale bar: 2 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "Fluorescence images of ∆atg7 and ∆atg8 cells (A) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.",
    "molecules": "CMAC"
  },
  {
    "caption": "Fluorescence images of ∆snf7 and ∆vps4 cells (B) expressing GAL promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. Scale bar: 2 µm.",
    "molecules": "CMAC"
  },
  {
    "caption": "C Relative activity of the ER-phagy reporter Yop1-Pho8∆60 in tunicamycin-treated cells. The dotted line indicates the activity in ∆atg7 cells. Mean + SEM, n ≥ 3.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "D, E Relative activity of endogenous Pho8 in untreated cells (D) or after tunicamycin treatment (E). Background activity determined in ∆pho8 mutants was subtracted from the activity in all other strains. Mean + SEM, n = 3. WT, wild-type.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "F Western blot of GFP from cells expressing Sec63-GFP and treated with tunicamycin (Tm) where indicated. The two Atg7-independent cleavage products of around 55 kDa were used as readout for micro-ER-phagy. Pgk1 served as a loading control. G Quantification of Sec63-GFP cleavage normalized to the WT from western blots as shown in panel (F). Mean + SEM, n = 4. ",
    "molecules": "Tm, tunicamycin"
  },
  {
    "caption": "A Electron micrograph of wild-type yeast treated with DTT for 3 h. Inset 1 shows an ER whorl and inset 2 shows ER fragments in macroautophagic bodies (white arrows). Cy, cytosol; V, vacuole.",
    "molecules": "DTT"
  },
  {
    "caption": "B Relative activity of cyto-Pho8∆60 after nitrogen starvation, which reports on non-selective macroautophagy of cytosol. Mean + SEM, n ≥ 3. WT, wild-type.",
    "molecules": "nitrogen"
  },
  {
    "caption": "Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after nitrogen starvation (C) Mean + SEM, n ≥ 3.",
    "molecules": "nitrogen"
  },
  {
    "caption": "Relative activity of the ER-phagy reporter Yop1-Pho8∆60 after tunicamycin treatment (D, E). Mean + SEM, n ≥ 3.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "F, G Growth assays of untreated and tunicamycin-treated WT, ∆atg39/40, ∆snf7 and ∆snf7 ∆atg39/40 cells. Numbers indicate growth relative to WT cells based on the areas under the curves. Mean ± SEM, n = 3.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.",
    "molecules": "CMAC"
  },
  {
    "caption": "Fluorescence images of a cell expressing non-fluorescent GFP(Y66F)-Pho8, Snf7-mNeonGreen and the ER marker mCherry-Ubc6, and stained with the vacuole dye CMAC. Numbers indicate the time in minutes after the start of the image sequence. White arrows indicate a Snf7-mNeonGreen punctum adjacent to an ER whorl. Black arrow point to a vacuole membrane invagination. Panel (E) illustrates transient appearance of Snf7 at an ER whorl. Panel (F) illustrates orientation of a Snf7 punctum towards the neck of a vacuole membrane invagination. In this series, mNeonGreen fluorescence was adjusted differently for different time points to compensate signal decay over time. Scale bar: 2 µm.",
    "molecules": "CMAC"
  },
  {
    "caption": "A Electron micrographs of wild-type and ∆snf7 cells treated with DTT for 3 h. Cy, cytosol; V, vacuole. Scale bars: 300 nm.",
    "molecules": "DTT"
  },
  {
    "caption": "C, D Electron micrographs of serial thin sections of a ∆snf7 cell (C) or a wild-type cell (D) treated with DTT for 3 h. Note that the whorl appears separated from the cytosol in some sections (e.g. section 300 nm) but is still in contact with the cytosol (see sections 600 nm and 700 nm). In the wild-type cell (D), the vacuolar whorl is completely separated from the cytosol. Scale bar: 300 nm.",
    "molecules": "DTT"
  },
  {
    "caption": "(J-L) BM12/SJL mice  (recipient, R) were immunized with CD4+ T cells from LN  and OB mice  (donor, D), and then intraperitoneally injected once every day with 2-DG  (200 mg/kg) or PBS  for 3 weeks. Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG  or IgM in serum  from the immunized mice  at the indicated time (J). Immunofluorescent  analysis of anti-nuclear  antibody (ANA) in serum  (top) and IgG  deposition in the kidney  (bottom) (n = 4 or 5, biological replicates). Scale bar: 50 μm. Data are presented as immunofluorescent  images (K) and quantification of bar graphs (L). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.",
    "molecules": "2-DG, dsDNA, histone, PBS, ssDNA"
  },
  {
    "caption": "(L, M) ECAR  measurements of glycolysis of naïve CD4+ T cell  from LN  and OB mice  treated with vehicle or etomoxir  (Eto, 150 μM) that stimulated with α-CD3/CD28  (TCR, 1/1 μg/ml) for 24 h (n = 3, biological replicates). The statistical results are presented as a bar graph (M). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.",
    "molecules": "Eto, etomoxir"
  },
  {
    "caption": "Immunoblot  analysis of cytoplasmic  and nuclear  NF-AT levels (C), that activated by PMA/Ionomycin  (P/I, 10/100 ng/ml) for the indicated time points.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "(A, B) Immunoblot  analysis of cytoplasmic  and nuclear  NF-AT levels in WT and Goliath-KO naïve  CD4+ T cells from lean  (LN) or obese  (OB) mice  that were stimulated by α-CD3/28 (5/5 μg/ml) or PMA/ionomycin  (P/I, 10/100 ng/ml).",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(C-F) ECAR  measurements of glycolysis in WT and Goliath-deficient  naïve  CD4+ T cells treated with DMSO or fenofibrate  (FF, 5 μM) (C, D), or isolated from LN  or OB mice (E, F) that stimulated by α-CD3/28  (1/1 μg/ml) for 24 h (n = 3, biological replicates). The statistical results are presented as bar graphs (D, F). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.",
    "molecules": "DMSO, fenofibrate, FF"
  },
  {
    "caption": "(J-L) BM12/SJL mice  (recipient, R) were adoptively transferred with  CD4+ T cells from WT and Goliath-KO lean  or obese mice  (donors, D) to induce inflammation. The anti-dsDNA, anti-ssDNA  and anti-histone IgG  or IgM  in serum  were examined by ELISA (J). ANA in serum  (top, scale bar: 50 μm) and the kidney IgG  deposition (bottom, scale bar: 50 μm) were visualized using immunofluorescence  (n = 3, biological replicates). Data are presented as Immunofluorescent  images (K) and quantification bar graphs (L). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.",
    "molecules": "dsDNA, histone, ssDNA"
  },
  {
    "caption": "Immunofluorescence  analysis of Goliath protein expression in  CD4+ T cells  from lean  and obese mice  as the indicated treatments. Data are presented as immunofluorescent  images (A, and quantification bar graphs (B, Etomoxir  (Eto) 150 μM for 6 h; Scale bar: 2 μM. Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, **p < 0.01, ***p < 0.001. Error bars represent SD.",
    "molecules": "Eto, Etomoxir"
  },
  {
    "caption": "Immunofluorescence  analysis of Goliath protein expression in CD4+ T cells  from mice  as the indicated treatments. Data are presented as immunofluorescent  images C) Etomoxir  (Eto), 150 μM for 6 h; MG132, 10 μM for the last 4 h. Scale bar: 2 μM.",
    "molecules": "Eto, Etomoxir, MG132"
  },
  {
    "caption": "Immunofluorescence  analysis of Goliath protein expression  in  CD4+ T cells from mice  as the indicated treatments. quantification bar graphs D). Etomoxir  (Eto), 150 μM for 6 h; MG132, 10 μM for the last 4 h. Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, **p < 0.01, ***p < 0.001. Error bars represent SD.",
    "molecules": "Eto, Etomoxir, MG132"
  },
  {
    "caption": "(E, F) Endogenous ubiquitination  of Usp9x (E) and CaN (F) in primary  CD4+ T cells  treated with DMSO  or Gonib32, then stimulated with (+) or without (-) PMA/ionomycin  (P/I, 10/100 ng/ml).",
    "molecules": "Gonib32, DMSO, ionomycin, PMA"
  },
  {
    "caption": "(G, H) Immunoblot  analysis of the interaction  of Usp9x  and CaN, NF-AT and CaN  (G), and NFATc1/2 in nuclear  extract (NE) and cytoplasmic  extract (CE) (H) in primary CD4+ T cells treated with Gonib32, then stimulated with (+) or without (-) P/I (10/100 ng/ml).",
    "molecules": "Gonib32"
  },
  {
    "caption": "ECAR  measurements of glycolysis and proliferation  analysis in lean  and obese  naïve  CD4+ T cells treated with DMSO  or Gonib32  (2 μM) upon α-CD3/CD28  (1/1 µg/ml) stimulation for 24 h (ECAR) (I, J) (n = 3, biological replicates). The statistical results are presented as a bar graph (J",
    "molecules": "Gonib32, DMSO"
  },
  {
    "caption": "(M-O) BM12/SJL mice  (recipient, R) were immunized with CD4+ T cells  from lean  and obese mice  (donors, D), then were injected with Gonib32  (50 mg/kg) or vehicle control (5% DMSO  + 95% saline) once a day for 3 weeks. Anti-dsDNA, anti-ssDNA, and anti-histone IgG  or IgM  in serum  were measured using ELISA (M). Immunofluorescent  analysis of anti-nuclear  antibody in serum  (top) and IgG  deposition in the kidney  (bottom) (n = 4 or 5, biological replicates). Data are presented as immunofluorescent  images (N) and quantification bar graphs (O). All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.",
    "molecules": "Gonib32, DMSO, dsDNA, histone, ssDNA, saline"
  },
  {
    "caption": "Ferrozine-based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points. *P < 0.05 (unpaired Student's t-test versus each time points with FeSO4 100 µM).",
    "molecules": "Ferrozine, iron, FeSO4"
  },
  {
    "caption": "Representative confocal microscope images of L6 cells stained with IP-1 (Iron Probe 1) or transfected with IRE-CFP (Iron Regulatory Element) reporter after iron treatment (FeSO4, 250 µM) for 24 h.",
    "molecules": "IP-1, Iron Probe 1, iron, FeSO4"
  },
  {
    "caption": "Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment (250 µM, 24 h) with iron chelator DPD (500 µM).",
    "molecules": "DPD, PGSK, iron"
  },
  {
    "caption": "Relative gene expressions - ferritin heavy chain (FTH), ferritin light chain (FTL), ferroportin (SLC40A1, transferrin receptor 1 (tfr1, TFRC) - normalized to 18sr RNA expression after iron treatment (FeSO4, 250 µM) for 24 h. *P < 0.05 (unpaired Student's t-test versus Basal).",
    "molecules": "iron, FeSO4"
  },
  {
    "caption": "Representative western blot images and quantification of ferritin (heavy chain) and tfr1 (indicated with arrowhead) over GAPDH after iron treatment (250 µM, 24 h).",
    "molecules": "iron"
  },
  {
    "caption": "Glucose uptake of L6 cells with insulin stimulation (10 nM or 100 nM, 20 min) after iron treatment (250 µM, 1 h or 24 h).",
    "molecules": "insulin, iron"
  },
  {
    "caption": "Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) over GAPDH with insulin stimulation (10 nM or 100 nM, 5 min) after iron treatment (250 µM, 24 h).",
    "molecules": "insulin, iron"
  },
  {
    "caption": "Representative confocal microscope image of L6 cells stably expressing tandem fluorescent-eGFP-mCherry LC3B treated with iron (FeSO4, 250 µM) for the indicated time points (4 h, 8 h, 24 h). Quantification of mean LC3B puncta per cell from (A). Experiments were repeated three times, and one representative experiment is presented here.",
    "molecules": "iron, FeSO4"
  },
  {
    "caption": "Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment (FeSO4, 250 µM) at multiple time points.",
    "molecules": "iron, FeSO4"
  },
  {
    "caption": "Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine (CQ, 30 µM). * Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM.",
    "molecules": "chloroquine, CQ, iron"
  },
  {
    "caption": "Representative confocal microscope z-stack image of mCherry-LC3B L6 cells after iron treatment (250 µM, 24 h).",
    "molecules": "iron"
  },
  {
    "caption": "Quantification of autophagosomes motility from live cell microscopy (Supplementary Movie 1) from mCherry-LC3B L6 cells under basal, iron (250 µM, 24 h) and starvation (amino acid free) condition. Experiments were performed three times, and one representative experiment is presented here. *P < 0.05 (unpaired Student's t-test compared to Iron 24h).",
    "molecules": "amino acid, iron, Iron"
  },
  {
    "caption": "Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment (250 µM, 24 h).",
    "molecules": "iron"
  },
  {
    "caption": "Representative confocal microscope images of eGFP-mCherry-LC3B L6 cells with Lysotracker DeepRed after iron treatment (250 µM, 24 h).",
    "molecules": "Lysotracker DeepRed, iron"
  },
  {
    "caption": "Representative western blot images of phospho-ULK1 (S757), phospho-S6Kp70 (T389), total S6K, GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points.",
    "molecules": "iron, FeSO4"
  },
  {
    "caption": "Representative epi-immunofluorescent images of L6 cells transfected with myc-RHEB Q46L and immuno-stained against LC3B and myc after iron treatment (FeSO4, 250 µM) for 24 h.",
    "molecules": "iron, FeSO4"
  },
  {
    "caption": "Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h.",
    "molecules": "Ferrozine, iron"
  },
  {
    "caption": "Representative confocal images of wt L6 and RHEB-Q64L L6 cells pulsed with Lysotracker Deep Red and immuno-stained against LC3B after iron treatment (250 µM, 24 h).",
    "molecules": "Lysotracker Deep Red, iron"
  },
  {
    "caption": "Representative TEM images of wt L6 and RHEB-Q64L cells after iron treatment (250 µM, 24 h).",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images and quantifications of phospho-IRS1 (Y612, indicated by arrowhead) and AKT (T308, indicated by arrowhead) to GAPDH in wt and RHEB-Q64L L6 cells stimulated with insulin (100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)",
    "molecules": "insulin, iron"
  },
  {
    "caption": "Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment (250 µM, 24 h) followed by 24 h iron withdrawal. Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment (250 µM, 24 h) followed by 24 h withdrawal.",
    "molecules": "PGSK, Ferrozine, iron"
  },
  {
    "caption": "Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in L6 cells iron treatment (250 µM, 24 h) followed by 24 h withdrawal.",
    "molecules": "iron"
  },
  {
    "caption": "Representative epi-immunofluorescent microscope images of L6 cells immuno-stained against LC3B and LAMP1 after iron treatment (250 µM, 24 h) followed by 3 h withdrawal.",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h or 24 h.",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images and quantification of phospho-mTOR S2448, total mTOR and actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h.",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images of phospho-mTOR S2448, total mTOR phsophpo-TSC2 T1462, total TSC2, phospho-AKT S473, total AKT, actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without MK2206 (AKT inhibitor.)",
    "molecules": "MK2206, iron"
  },
  {
    "caption": "Representative western blot images of phospho-UVRAG S550 and total UVRAG in L6 cells transfected with FLAG-UVRAG and FLAG pulldown after iron treatment (250 µM, 4 h and 24 h) followed by withdrawal for 1 h and 3 h.",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images of endogenous phospho-UVRAG S550, total UVRAG expression in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without torin1 (200 nM).",
    "molecules": "iron, torin1"
  },
  {
    "caption": "Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: lysosomal number in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).",
    "molecules": "iron, Torin1"
  },
  {
    "caption": "Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: speed (F) in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).",
    "molecules": "iron, Torin1"
  },
  {
    "caption": "Prussian Perl blue staining in liver and skeletal muscle after iron injections.",
    "molecules": "Prussian Perl blue, iron"
  },
  {
    "caption": "Representative western blot images and quantification of ferritin and tfr1 (indicated by arrowhead) to tubulin in skeletal muscles 24 h after iron injections.",
    "molecules": "iron"
  },
  {
    "caption": "Insulin tolerance test 24 h after iron injections.",
    "molecules": "iron"
  },
  {
    "caption": "Glucose tolerance test (GTT) 24 h after iron injections.",
    "molecules": "iron"
  },
  {
    "caption": "Insulin tolerance test 24 h after iron injections. Quantification of area under curve in ITT (E).",
    "molecules": "iron"
  },
  {
    "caption": "Glucose tolerance test (GTT) 24 h after iron injections. Quantification of area under curve for panel (F).",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in skeletal muscles 24 h after iron injection followed by i.p. insulin injection.",
    "molecules": "insulin, iron"
  },
  {
    "caption": "Representative confocal microscope images of skeletal muscle tissue sections immuno-stained against LC3B (Alexa 647) and LAMP1 (Alexa 555) Quantification of lysosomes (LAMP1 puncta) in skeletal muscles 24 h after iron injections.",
    "molecules": "Alexa 555, Alexa 647, iron"
  },
  {
    "caption": "Representative TEM images of skeletal muscle 24 h after iron injections.",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images of phospho-UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections.",
    "molecules": "iron"
  },
  {
    "caption": "Representative western blot images and quantification of LC3-B and p62 to tubulin in skeletal muscles 24 h after iron injections.",
    "molecules": "iron"
  },
  {
    "caption": "Relative fold repression for let-7a reporter in RAW 264.7 cells during LPS treatment (mean+/- s.e.m., n=3) (B). The schematic representation of the constructs used for luciferase assays are shown.",
    "molecules": "LPS"
  },
  {
    "caption": "Relative levels of pro-inflammatory cytokines TNF-ɑ and IL-6 mRNA, a target of let-7a miRNA, have been plotted against time of LPS stimulation, 18S rRNA was used for normalization (mean+/- s.e.m., n=2 (IL-6), n=3 (TNF-α)) (C).",
    "molecules": "LPS"
  },
  {
    "caption": "Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Ago2 was pulled down using anti-FLAG beads and the levels of Ago-associated let-7a miRNA were measured by qRT-PCR. Quantity of immunoprecipitated Ago2 was detected in western blot analysis with anti-HA antibody and used for normalization of amounts of miRNA detected by qRT-PCR. Levels of cellular let-7a were measured by Northern blot and U6 RNA was used as loading control. Relative levels of cellular and Ago2 bound let-7a were plotted (mean+/- s.e.m., n=3) (D).",
    "molecules": "LPS"
  },
  {
    "caption": "Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Phosphorylated Ago2 (pY-Ago2) level was measured using 4G10 antibody specific for phosphorylated tyrosine. Phosphorylated Ago2 (pY-Ago2) band intensities were normalized against total Ago2 detected with HA specific antibody that was also used for Ago2 pull down (E, upper panel). Relative 4G10 intensities were plotted from three independent experiments (mean+/- s.e.m) (E, lower panel).",
    "molecules": "LPS, tyrosine"
  },
  {
    "caption": "Time dependent Ago2 phosphorylation and its miRNA binding in LPS treated macrophage cells. RAW 264.7 cells were transfected with FH-Ago2 expression plasmid and treated with LPS (1ng/ml) for different time points. Primary cells (PEC) were isolated from BALB/c mice and endogenous Ago2 was immunoprecipitated with anti-Ago2 (eIF2C2) antibody and phospho tyrosine level of Ago2 was measured using 4G10 antibody. Relative intensities of phospho-Ago2 against total amount of immunoprecipitated Ago2 were quantified and mentioned below the lanes (mean+/- s.e.m., n=3) (F, upper panel). Similar experiments were done with FH-Ago1 and Ago3 and levels of Phosphotyrosine in immunoprecipitated FH-Ago1 and FH-Ago3 were measured in RAW 264.7 cells (F, lower panel).",
    "molecules": "LPS, Phosphotyrosine, tyrosine"
  },
  {
    "caption": "Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Knockdown of PP2A, MKP1, MKP3 or SHP1/2 was checked by western blot analysis done with lysates of cells treated with respective siRNAs. β-Actin was used as loading control (G).",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of knockdown of different phosphatases on miRNA-mediated repression in 24hrs LPS treated RAW 264.7 cells. Luciferase based let-7a miRNA reporter assay was done in LPS-treated cells downregulated for specific phosphatases. Levels at 0 hrs time point were taken as units (mean +/- s.e.m., n=4) (H).",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of knockdown of different phosphatases on miRNA-association of Ago2 in 24hrs LPS treated RAW 264.7 cells. Ago2 associated let-7a was estimated by qRT-PCR in LPS treated cells depleted for specific phosphatases. let-7a miRNA level was normalized against respective FH-Ago2 bands (mean+/- s.e.m., n=3) (I).",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Phospho Ago2 levels were measured in siCon and siPP2A transfected cells upon 24hrs of LPS treatment using Phospho Tyrosine specific 4G10 antibody. HA-Ago2 was detected with anti-HA antibody (J). Immunoprecipitated Ago2 content was used for normalization for both miRNA and mRNA levels in above mentioned experiments.",
    "molecules": "LPS, Tyrosine"
  },
  {
    "caption": "Effect of PP2A knock-down on Ago2 phosphorylation and its miRNA association. Ago2 associated IL-1β mRNA level was estimated in siCon and siPP2A treated cells which were stimulated with LPS for 24hrs (mean+/- s.e.m., n=3) (L).",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264.7 cells after LPS stimulation (A).",
    "molecules": "LPS, OA, Okadaic acid"
  },
  {
    "caption": "Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation. The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount (B).",
    "molecules": "LPS, OA, Okadaic acid, Tyr"
  },
  {
    "caption": "Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on miRNA activity. Change in fold repression for a let-7a reporter upon LPS exposure in control and OA treated cells (mean+/- s.e.m., n=3) (C).",
    "molecules": "LPS, OA, Okadaic acid"
  },
  {
    "caption": "Schematic representation of the in vitro phosphatase assay (upper panel). PP2A was immunoprecipitated from 24hrs LPS treated RAW 264.7 cells and were incubated in vitro with wild type or phosphorylation defective FH-Ago2 mutant (Ago2Y529F) isolated from HEK293 cells transfected with respective expression constructs. Phosphorylated Ago2 (pYAgo2) levels were measured by western blot analysis using anti-phospho Tyrosine specific 4G10 antibody. Relative intensities were quantified by densitometric analysis and mentioned below the respective panels. FH-Ago2 was detected by anti-HA antibody (Lower panel).",
    "molecules": "LPS, Tyrosine"
  },
  {
    "caption": "Defective de novo miRNP formation in cells pre-treated with PP2A inhibitor. Ago2 associated (F) and total (G) miR-122 levels were measured by qRT-PCR and normalized against immuneprecipitated FH-Ago2 content and U6 RNA respectively. Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted. All samples were treated for 24 hrs with LPS (mean+/- s.e.m., n=4).",
    "molecules": "DOX, LPS"
  },
  {
    "caption": "PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Expression of PTPA after LPS stimulation in RAW 264.7 cells as well as in PEC isolated from BALB/c mice. β-actin is used as loading control (H).",
    "molecules": "LPS"
  },
  {
    "caption": "PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264.7 cells (I). Phosphorylated Ago2 level was measured by western blot in HA-immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH-Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody (I, left panel). The PTPA was detected with anti PTPA antibody, β-Actin was used as loading control (I, right panel).",
    "molecules": "LPS, phosphotyrosine"
  },
  {
    "caption": "PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264.7 cells treated with LPS for 24 hrs and was incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells and upon the assay reaction, the phospho-Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody (K, right panel).",
    "molecules": "LPS, phosphotyrosine"
  },
  {
    "caption": "Schematic representation of in vitro phosphatase assay (F, upper panel). PP2A, MKP1, MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody (F, lower panel) and measured by densitometry. BC; Bead Control.",
    "molecules": "phosphotyrosine"
  },
  {
    "caption": "Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment (100 nM) were detected. Cells were pre-treated with OA (100 nM) for 2 hrs before infection (J). β-Actin was used as loading control.",
    "molecules": "OA"
  },
  {
    "caption": "Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells and mouse PEC. Phospho-Ago2 level was measured in OA pre-treated; Ld infected RAW 264.7 cells expressing FH-Ago2 and in PEC post 24 hrs of infection with or without OA pre-treatment. Phosphorylated Ago2 levels were also checked (K).",
    "molecules": "OA"
  },
  {
    "caption": "RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. OptiprepR fractions, positive for MVB (fraction number 2,3,4) and ER (fraction number 7,8,9) markers were pooled separately and Ago2 was immunoprecipitated using Anti-FLAG beads. Phosphorylated Ago2 levels were quantified in western blot done with phospho-tyrosine specific 4G10 antibody (M).",
    "molecules": "tyrosine"
  },
  {
    "caption": "Effect of PP2A downregulation or inhibition on pro and anti-inflammatory cytokine levels in Ld infected macrophage. IL-10 levels were measured after Ld infection in PP2A knocked-down RAW 264.7 cells (mean+/- s.e.m., n=3) and in PEC (mean+/- s.e.m., n=3). IL-6 was also measured in RAW cells after OA treatment (mean+/- s.e.m., n=5) and also in PEC pre-treated with OA (mean+/- s.e.m., n=3). Values obtained at 0 hrs (non-infected) or non-OA treated samples were considered as unit in each case. IL-6 protein level (n=5) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock-down RAW 264.7 cells.",
    "molecules": "OA"
  },
  {
    "caption": "Effect of PP2A knock-down on parasite internalization Internalized parasites were imaged in RAW 264.7 cells stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 (red) (B).",
    "molecules": "Alexa 488, Phalloidin"
  },
  {
    "caption": "Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. In images obtained, Ago2 was detected with α-HA (detected at 405nm) and Leishmania was stained for GP63 (detected at 564nm) (E).",
    "molecules": "OA"
  },
  {
    "caption": "Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted. Scale bar 10 µm. RNA was isolated from different experimental sets and TNF-α, IL-1β and IL-10 mRNA levels were estimated (mean +/- s.e.m., n=3) (F).",
    "molecules": "OA"
  },
  {
    "caption": "Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. RAW 264.7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs. RAW 264.7 cells were stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 (red) (G, left panel). Number of internalized parasites per 100 macrophages were calculated and relative values were plotted (G, right panel) (mean+/- s.e.m., n=20). Scale bar 20 µm.",
    "molecules": "Alexa 488, OA, Phalloidin"
  },
  {
    "caption": "LPS induced miRNA export from mammalian macrophage cells. A schematic diagram of the experiments has been described in left panel of (A). Characterization and quantification of exosomes derived from Control and LPS-activated RAW 264.7 cells by Nanoparticle Tracking Analysis (NTA) done for isolated exosomes. Relative size distributions are shown (A, right panel).",
    "molecules": "LPS"
  },
  {
    "caption": "Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264.7 cells. RNA content was normalized against total exosomal proteins (B). β-Actin as loading control for HuR western blot.",
    "molecules": "LPS"
  },
  {
    "caption": "Binding of miRNAs with HuR after LPS exposure was measured. Amount of RNA was normalized against HuR content. Anti GFP-antibody was used for immunoprecipitation control (C). Values are mean+/- s.e.m. and, n=3",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of GW4869, the inhibitor of exosome mediated miRNA export, on exosomal miRNA content released by LPS stimulated cells. The level of miRNAs, let-7a and miR-155 were measured in exosomes (EV) released from LPS activated cell after GW4869 treatment. Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit. Values are mean+/- s.e.m. and, n=3",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "Effect of GW4869 on cellular cytokine levels in LPS treated cells. The effect of GW4869 on cellular expression of pro-inflammatory cytokines IL-1β and TNF-α in LPS-activated macrophages were measured and normalized against 18S rRNA. Values in naive RAW 264.7 cells without GW4869 and LPS treatment were set as unit. Values are mean+/- s.e.m. and, n=3",
    "molecules": "GW4869, LPS"
  },
  {
    "caption": "Effect of siRNA-mediated downregulation of protein HuR on cellular expression of pro-inflammatory cytokines. In the left panel of F, effect of siRNA treatment on cellular cytokine mRNA levels in LPS-stimulated RAW 264.7 cells is shown. Relative levels measured against 18S rRNA are plotted. The effect of HuR depletion on cellular miR-155 content has been measured and relative values normalized against U6 snRNA are plotted (right panel, F).",
    "molecules": "LPS"
  },
  {
    "caption": "The amount of let-7a bound to Ago2 after LPS stimulation in presence and absence of siHuR has been calculated and relative values has been normalized to immunoprecipitated Ago2 (G) .Values are mean+/- s.e.m. and, n=3",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of HuR expression on inflammatory responses in macrophage cells. The effect of HuR expression on production of pro-inflammatory cytokine TNF-α in RAW 264.7 cells was measured. mRNA and protein level of TNF-α was quantified. Values obtained with pCINeo expression and without LPS treatment was taken as unit (I). Transfection was done either with pCIneo (control plasmid) of HA-HuR expression construct and their effect on cellular pro-inflammatory cytokine mRNA levels in RAW64.7 cells was determined. Values in control set were taken as unit. Values are mean+/- s.e.m. and, n=3",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of HA-HuR expression on p38 mediated activation of downstream signalling events. The Levels of p-p38 and p-MSK1 have been monitored by western blotting done with cell extracts prepared from HA-HuR and control plasmid transfected, untreated and LPS treated RAW 264.7 cells. β-Actin blot was used as loading control.",
    "molecules": "LPS"
  },
  {
    "caption": "Downregulation of HuR in Ld-infected macrophages. Effect of proteasomal inhibitor MG132 treatment on HuR protein levels in control and 6 hrs of Ld-infected RAW 264.7 cells. In parallel assays levels of pro-inflammatory cytokine TNF-α was measured in Ld-infected cells either with no treatment or pre-treated with MG132 (L). Values are mean+/- s.e.m. and, n=3.",
    "molecules": "MG132"
  },
  {
    "caption": "Expression profiles of HuR, PP2A and miRNA let-7a are connected to IL-6 expression in RAW 264.7 cells. Data obtained from experiments described in previous figures are summed up to plot the changes in IL-6 mRNA levels against time of LPS treatment along with changes in PP2A, miRNA let-7a and HuR (D).",
    "molecules": "LPS"
  },
  {
    "caption": "Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. HA-HuR was immunostained with anti-HA antibody and detected by secondary antibody tagged with AlexaR564 (A).",
    "molecules": "AlexaR564, CFSE"
  },
  {
    "caption": "Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. In the zoomed part of the merged picture are used to show the internalized parasite. Internalized parasites present in HA-HuR positive cells were counted and compared against untransfected cells without HA-HuR expression (B).",
    "molecules": "CFSE"
  },
  {
    "caption": "Effect of expression of HA-HuR on Ld infection in mice liver. Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA. Values are mean+/- s.e.m. and, n=5 (G).",
    "molecules": "DNA"
  },
  {
    "caption": "Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell. RAW 264.7 cells were transfected with HA-HuR or pCIneo followed by 2 hrs of pretreatment with OA. These cells were then infected with LdR for 24 hrs. Pro-inflammatory cytokines TNF-α (right panel; mean+/- s.e.m., n=3) and IL-1β (left panel; mean+/- s.e.m. and, n=4) were measured at mRNA level and relative values are plotted. Values without OA treatment was set as control.",
    "molecules": "OA"
  },
  {
    "caption": "D HeLa cells were treated with indicated siRNAs, synchronized by double thymidine block, and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor (LBR) to label the NE, and FXR1.",
    "molecules": "thymidine"
  },
  {
    "caption": "E HeLa cells stably expressing GFP-FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414, which labels FG-Nups. The magnified framed regions are shown in the corresponding numbered panels. The arrowheads indicate NE and cytoplasmic localization of GFP-FXR1.",
    "molecules": "FG"
  },
  {
    "caption": "F HeLa cells stably expressing GFP-Nup107 were synchronized by double thymidine block and released for 12 hours, permeabilized with Triton/SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus, respectively, and analysed by immunofluorescence microscopy.",
    "molecules": "digitonin, SDS, thymidine, Triton"
  },
  {
    "caption": "A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.",
    "molecules": "thymidine"
  },
  {
    "caption": "B HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, 1000 cells were analysed for each graph (mean ±SD, **P < 0.01; ***P < 0.001, N = 3).",
    "molecules": "thymidine"
  },
  {
    "caption": "HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C)",
    "molecules": "thymidine"
  },
  {
    "caption": "HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nuclear intensity of FG-Nups labelled by mAb414 (D), RanBP2 (E) and GFP-Nup107 (F) was quantified. 1800 cells were analysed for each graph (mean ±SD, *P < 0.05; **P < 0.01; N = 3).",
    "molecules": "FG, thymidine"
  },
  {
    "caption": "G, H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules (G) was quantified and nuclear intensity of FG-Nups labelled by mAb414 (H) was quantified. 1600 cells were analysed in (G) and 2100 cells were analysed in (H) (mean ±SD, *P < 0.05; *** P<0.001; N = 3).",
    "molecules": "FG"
  },
  {
    "caption": "A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. Arrowheads point to nuclear blebs observed in FXR1-deficient cells.",
    "molecules": "thymidine"
  },
  {
    "caption": "B, HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by Western blot (B)",
    "molecules": "thymidine"
  },
  {
    "caption": "C HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy (C). The percentage of cells with irregular nuclei was quantified, 1000 cells were analysed (mean ±SD, **P < 0.01, ***P < 0.001; N = 3).",
    "molecules": "thymidine"
  },
  {
    "caption": "HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Time from prophase till anaphase (D), from prophase till metaphase (E), from metaphase till anaphase (F) and from anaphase till chromatin decondensation (G) was quantified.",
    "molecules": "histone, thymidine"
  },
  {
    "caption": "HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The selected frames of the movies are depicted and time is shown in minutes (H). Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1-deficient cells.",
    "molecules": "histone, thymidine"
  },
  {
    "caption": "HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Percentage of daughter cells with irregular nuclei was quantified in (I)",
    "molecules": "histone, thymidine"
  },
  {
    "caption": "HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. time from anaphase till nuclear blebs was quantified in (J).",
    "molecules": "histone, thymidine"
  },
  {
    "caption": "A-D HeLa cells stably expressing GFP-Nup107 were treated with indicated siRNAs, synchronized by double thymidine block, released and analysed by live video spinning disk confocal microscopy (A). The selected frames of the movies are depicted and time is shown in minutes. The onset of anaphase is indicated. The magnified framed regions with time indicated in minutes are shown in (B). White arrowheads point to the cytoplasmic GFP-NUP107 granules appearing during nuclear expansion of control and FXR1-deficient cells, yellow arrowheads point to the fussion events of GFP-NUP107 granules with NE in control cells. The percentage of cells with cytoplasmic GFP-Nup107 granules was quantified in (C). Time from anaphase till GFP-Nup107 cytoplasmic granule formation was quantified in (D). 57 cells were analysed (mean ±SD, *P < 0.05; ns = non-significant; N = 3).",
    "molecules": "thymidine"
  },
  {
    "caption": "A HeLa cells stably expressing GFP-Nup133 were synchronized by double thymidine block, released for 12 hours, treated or not with NaAsO2 to induce stress granule formation and with 1,6 Hexanediol, and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.",
    "molecules": "Hexanediol, NaAsO2, thymidine"
  },
  {
    "caption": "B, C HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with or without 1,6 Hexanediol and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (C), 3100 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; ****P < 0.0001; N = 3).",
    "molecules": "Hexanediol, thymidine"
  },
  {
    "caption": "B, C HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Arrowheads indicate blebbed regions of nuclei. The percentage of interphasic cells with Nup granules was quantified (C), 900 cells were analysed (mean ±SD, ***P < 0.001; N = 3).",
    "molecules": "thymidine"
  },
  {
    "caption": "E, F HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414. The magnified framed regions are shown in the corresponding numbered panels in (E). The percentage of cells with cytoplasmic Nup granules was quantified in (F), 2700 cells were analysed (mean ±SD, ***P < 0.001; N = 3).",
    "molecules": "thymidine"
  },
  {
    "caption": "A, B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as indicated and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (B), 5200 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; N = 3).",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "C-E HeLa cells stably expressing GFP-Nup107 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as in (A) and analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted and time is shown in minutes. The magnified framed regions are depicted in the lower rows. White arrowheads point to individual GFP-Nup107-positive granules. Yellow arrowheads point to the granules undergoing fusion events. The percentage of cells with fusion/fission events of GFP-Nup107 granules was quantified in (D), 815 cells were analysed (mean ±SD, **P < 0.01; N = 3). The number of fusion/fission events per cell was quantified in (E), 815 cells were analysed (mean ±SD; N = 3).",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by Western blot (A)",
    "molecules": "Monastrol"
  },
  {
    "caption": "Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy Examples of Nup localization defects are shown in (B)",
    "molecules": "Monastrol"
  },
  {
    "caption": "Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup granules was quantified in (C), 283 cells were analysed (mean ±SD, ***P < 0.001; N = 3).",
    "molecules": "Monastrol"
  },
  {
    "caption": "Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy examples of nuclear architecture defects are shown in (D).",
    "molecules": "Monastrol"
  },
  {
    "caption": "H, I Mouse Embryonic Fibroblasts (MEFs) derived from the Fmr1 knock-out (KO) mice and wild type controls were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy (H). The percentage of cells with cytoplasmic nucleoporin granules was quantified in (I), 2400 cells were analysed (mean ±SD, *P < 0.05; N = 3).",
    "molecules": "Monastrol"
  },
  {
    "caption": "A HeLa cells were transfected with the import/export reporter plasmid XRGG-GFP, treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release. Dexamethasone was added for 3 hours to induce XRGG-GFP nuclear import. Following wash-out the nuclear export of XRGG-GFP was analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted in Figure EV5C. The percentage of cytoplasmic XRGG-GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in (A), 199 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3).",
    "molecules": "Dexamethasone, Monastrol"
  },
  {
    "caption": "B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.",
    "molecules": "thymidine"
  },
  {
    "caption": "Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. The percentage of p-Rb and/or EdU positive cells was quantified in (E), 2100 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3)",
    "molecules": "EdU"
  },
  {
    "caption": "F Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. the percentage of p-Rb and/or cyclin B positive cells was quantified in (F), 3300 cells were analysed (mean ±SD, *P < 0.05; N = 3).",
    "molecules": "EdU"
  },
  {
    "caption": "Mouse YUMM1.7 melanoma  cells were subcutaneously inoculated into C57BL/6  mice and when tumors reached 100 mm3 mice  were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib  and MEKi, Trametinib) or BRAFi/MEKi plus  BIBF  (n=6). (C) Kaplan-Meier survival  curves of  mice treated with the indicated therapies (n = 6). Log rank (Mantel-Cox) statistical test was used for MAPKi vs MAPKi/BIBF1120. ****P≤0.0001.",
    "molecules": "BIBF, BIBF1120, Nintedanib, Trametinib, Vemurafenib"
  },
  {
    "caption": "Mouse YUMM1.7 melanoma  cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice  were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib  and MEKi, Trametinib) or BRAFi/MEKi plus BIBF  (n=6). (F) Quantification of collagen fibers thickness (n = 6 for control, BIBF and  BRAFi/MEKi groups and n = 5 for  BRAFi/MEKi + BIBF group). Two-way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification. **P≤0.01, ***P≤0.001, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated.",
    "molecules": "BIBF, BIBF1120, Nintedanib, Trametinib, Vemurafenib"
  },
  {
    "caption": "Mouse YUMM1.7 melanoma  cells were subcutaneously inoculated into C57BL/6  mice  and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib  and  MEKi,  Trametinib) or  BRAFi/MEKi plus BIBF (n=6). (G) Heatmap showing the differential expression of  ECM  and myofibroblast/CAF  genes in  mice treated with MAPK-targeted therapies with or without  BIBF  compared to control mice (log2 ratio, n = 5).",
    "molecules": "BIBF, BIBF1120, Nintedanib, Trametinib, Vemurafenib"
  },
  {
    "caption": "(H) Heatmap and Western Blot showing the expression of  ECM, myofibroblast/CAF  and phenotype switch markers in human M238R cells compared to the parental M238P  cells. Heatmap represents the mean of  expression  of 3 independent experiments by RT-qPCR. (I) Heatmap showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells treated with BIBF  (2 µM, 72 h) or vehicle alone by RT-qPCR  (n = 3).",
    "molecules": "BIBF"
  },
  {
    "caption": "(B) Relative miRNA  expression levels were quantified in  M238P cells treated for 72 h with BIBF1120 (BIBF, 2 μM), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1µM), or with MAPKi (1 μM) plus BIBF (2 µM) by RT-qPCR  and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. One-way ANOVA was used for statistical analysis. **P≤0.01, ***P≤0.001. Significance was calculated against the control group. Statistical significance of  BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (C) Expression of miR-143-3p and miR-145-5p in control  mice  and mice  treated with the indicated therapies was quantified by RT-qPCR. Data is represented as mean ± SEM from 2 independent experiments performed on 6  mice, with 2 sites of injections. One-way ANOVA was used for statistical analysis. *P≤0.05, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs  BRAFi/MEKi + BIBF was also calculated.",
    "molecules": "BIBF, BIBF1120, Trametinib, Vemurafenib"
  },
  {
    "caption": "(D) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF-β (10 ng/mL) or PDGF-BB (20 ng/mL) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, ***P≤0.001. (E) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib/BIBF1120 (BIBF, 2 μM), the TGF-β receptor kinase inhibitor SB431542 (SB, 10 µM), and the pan-AKT inhibitor GSK690693 (GSK, 10 µM) by RT-qPCR. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, **P≤0.01, ***P≤0.001.",
    "molecules": "GSK, GSK690693, BIBF, BIBF1120, Nintedanib, SB, SB431542"
  },
  {
    "caption": "(B) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143  (LNA-143) or anti-miR-145  (LNA-145) (50 nM) or a combination of the two. ECM markers RT-qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001.",
    "molecules": "LNA, Vemurafenib"
  },
  {
    "caption": "(B) FSCN1 immunofluorescent staining  and quantification of fluorescence intensity in  M238P cells treated or not with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments, Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.",
    "molecules": "LNA, Vemurafenib"
  },
  {
    "caption": "BRAFi-resistant M238R cells overexpressing FSCN1  were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl  lentivirus  were used as control. (H) Crystal violet viability assay  of M238R cells stably overexpressing  FSCN1  during 6 days with the indicated doses of the BRAFi Vemurafenib. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ****P≤0.0001.",
    "molecules": "Crystal violet, Vemurafenib"
  },
  {
    "caption": "(A, M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (B, M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (A-B) Images and quantification of cell area in cells stained for  F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.",
    "molecules": "Trametinib, Vemurafenib"
  },
  {
    "caption": "C) M238P cells were transfected with  miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). D) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (C-D) Images and quantification of focal adhesions  number in cells stained  for pPaxillin (green) and nuclei  (blue). Focal adhesions (FA) number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of  focal adhesions per cell calculated for each field. Paired Student t-test has been used for statistical analysis. *P≤0.01, ***P≤0.001, ****P. ≤0.0001. Scale bar 40 μm.",
    "molecules": "Trametinib, Vemurafenib"
  },
  {
    "caption": " A) Treadmill analysis (n=8/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two-tailed Student's t test ***p=0.005; ***p=0.0007 (T3) and ****p<0.0001 (T4). Only p values for rapamycin-treated vs. untreated Cox15sm/sm mice are shown ",
    "molecules": "rapamycin"
  },
  {
    "caption": " B) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; PAS: periodic acid Schiff reaction; COX: cytochrome c oxidase; SDH: succinate dehydrogenase. White bars correspond to 50µm ",
    "molecules": "eosin, hematoxylin, rapamycin"
  },
  {
    "caption": " C) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: ***p=0.001; ***p=0.006, ****p<0.0001 ",
    "molecules": "rapamycin"
  },
  {
    "caption": " D) Analysis of the number of centralized nuclei in muscle fibers (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0018, **p=0.0052 ",
    "molecules": "rapamycin"
  },
  {
    "caption": " E) Analysis of PAS-reaction intensity in muscle fibers (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0293 (WT vs. Cox15sm/sm) and *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm Rapa) ",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": " F) Analysis of COX-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001; **p=0.0031 ",
    "molecules": "rapamycin"
  },
  {
    "caption": " G) Analysis of SDH-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199, **p=0.0091 ",
    "molecules": "rapamycin"
  },
  {
    "caption": " A) Spectrophotometric activities of the respiratory chain (n=4-5/group). CS: citrate synthase; CIV: complex IV. Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by two way ANOVA with Tukey's correction ****p<0.0001 (CS: WT vs. Cox15sm/sm), ***p=0.0010 (CS: Cox15sm/sm vs. Cox15sm/sm rapamycin), ****p<0.0001 (CIV: WT vs. Cox15sm/sm), **p=0.0060 (CIV/CS: WT vs. Cox15sm/sm) ",
    "molecules": "rapamycin"
  },
  {
    "caption": " B) Western blot immunovisualization of subunits of the respiratory complexes. Note the increased protein levels in Cox15sm/sm vs. WT samples, which are reduced to normal levels upon rapamycin treatment. Additional samples were run on a separate gel (not shown) ",
    "molecules": "rapamycin"
  },
  {
    "caption": " C) BNGE in gel activity for cIV. Sc: supercomplexes. Note that the COX reaction is slightly increased in rapamycin-treated vs. untreated Cox15sm/sm samples ",
    "molecules": "rapamycin"
  },
  {
    "caption": " D) Immunoblot of 1D-BNGE using an anti-cIV antibody (COX1). Note that COX amount is slightly increased in rapamycin treated vs. untreated Cox15sm/sm muscles. SDHB was used as a loading control ",
    "molecules": "rapamycin"
  },
  {
    "caption": " E) State III (succinate-driven) oxygen consumption (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two tail Student's t test **p=0.0016 (WT vs. Cox15sm/sm); *p=0.047 (Cox15sm/sm vs. Cox15sm/sm rapamycin) ",
    "molecules": "oxygen, rapamycin, succinate"
  },
  {
    "caption": " F) Analysis of mtDNA amount (n=9/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey's correction: **p=0.0020; *p=0.0296 ",
    "molecules": "mtDNA"
  },
  {
    "caption": " Note the accumulation of profoundly altered mitochondria in Cox15sm/sm muscles (white arrows), possibly as a result of partially digested organelles within endolysosomes. Black arrows indicate examples of normal mitochondria. In rapamycin-treated Cox15sm/sm animals only a few of these structures are present, which can be detected also in rapamycin-treated WT samples. The bar corresponds to 1136 nm (4400X) ",
    "molecules": "rapamycin"
  },
  {
    "caption": " A) Representative western blot of autophagy markers at steady state in skeletal muscle. GAPDH was used as a loading control. B) Densitometric analysis of immunoblots similar to that shown in A (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0068 (LC3-II WT vs. Cox15sm/sm), **p=0.0063 (P62 WT vs. Cox15sm/sm) *p=0.025 (P62 Cox15sm/sm vs. Cox15sm/sm+rapamycin), **p=0.0098 (S6 WT vs. Cox15sm/sm), *p=0.034 (P-S6 WT vs WT+rapamycin), ***p=0.0002 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), *p=0.014 (P-S6 WT vs. WT+rapamycin) ***p=0.0007 (Cox15sm/sm vs. Cox15sm/sm+rapamycin)",
    "molecules": "rapamycin"
  },
  {
    "caption": " C) Analysis of the autophagic flux (n=3/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rapamycin, or rapamycin plus colchicine. Note that LC3-II was increased in Cox15sm/sm vs. WT samples. Colchicine did not increase LC3-II levels suggesting a block in the autophagic flux; rapamycin plus colchicine treated Cox15sm/sm mice showed higher levels of LC3-II, suggesting that rapamycin increased the autophagic flux in Cox15sm/sm muscles. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0049 (WT vs. WT+colchicine), **p=0.0046 (WT+rapamycin vs. WT+rapamycin+colchicine), *p=0.0225 (WT vs. Cox15sm/sm), **p=0.0057 (Cox15sm/sm colchicine vs. Cox15sm/sm+rapamycin+colchicine), ***p=0.0003 (Cox15sm/sm rapamycin vs. Cox15sm/sm+rapamycin+colchicine)",
    "molecules": "colchicine, Colchicine, rapamycin"
  },
  {
    "caption": " A) Analysis of the autophagic flux (n=4/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rilmenidine, or rilmenidine plus colchicine. Colchicine alone increased LC3-II in WT but not Cox15sm/sm mice; rilmenidine had no effect on WT LC3-II levels, but reduced them in Cox15sm/sm mice. Rilmenidine plus colchicine increased LC3-II levels in both WT and Cox15sm/sm mice. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: **p=0.0093 (WT vs. WT+colchicine), ****p<0.0001 (WT+rilmenidine vs. WT+rilmenidine+colchicine), **p=0.0001 (WT vs. Cox15sm/sm), **p=0.0083 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine), *p=0.0112 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine+colchicine), ****p<0.000",
    "molecules": "colchicine, Colchicine, rilmenidine, Rilmenidine, rapamycin"
  },
  {
    "caption": " B) Treadmill analysis (n=4/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM ",
    "molecules": "rilmenidine"
  },
  {
    "caption": " C) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; GT: Gomori trichrome; PAS: periodic acid Schiff reaction ",
    "molecules": "Gomori trichrome, GT, eosin, hematoxylin, rapamycin"
  },
  {
    "caption": " D) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ***p=0.0002, ****p<0.0001 ",
    "molecules": "rilmenidine"
  },
  {
    "caption": " E) Analysis of the number of centralized nuclei in muscle fibres (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0091 ",
    "molecules": "rilmenidine"
  },
  {
    "caption": " A) anti-TFEB immunofluorescence on rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. CD11C (blue signal) indicates inflammatory cells. Right panel: quantification of n=3 animals/group. The scale bars correspond to 30µm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test B) Quantification of the TFEB-positive nuclei (n=3/group). Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199 (WT vs. WT+rapamycin), *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), ***p=0.0001 ",
    "molecules": "rilmenidine, rapamycin"
  },
  {
    "caption": " A) Real-time PCR analysis of Lamp1 transcript (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test **p=0.008, *p=0.038 ",
    "molecules": "rapamycin"
  },
  {
    "caption": " B) Anti-LAMP1 staining in rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. Rapamycin, but not rilmendine, increases the number of LAMP1-positive vesicles in Cox15sm/sm samples (inset). The scale bars correspond to 30µm ",
    "molecules": "rilmendine, rilmenidine, rapamycin, Rapamycin"
  },
  {
    "caption": "A. Serum  levels of 15-keto-dihydro-PGF2α in  type 2 diabetic patients  without  diabetic retinopathy  (no DR), with non-proliferative diabetic retinopathy (NPDR), and with  proliferative diabetic retinopathy  (PDR) (n=20-24). Data information: n.s. stands for \"not significant.\" Data were analyzed by Kruskal-Wallis test with Dunn's multiple comparisons test (A), Data are represented as mean ± SEM.",
    "molecules": "15-keto-dihydro-PGF2α"
  },
  {
    "caption": "C. Retinal PGF2α  production in OIR  mice  during postnatal days 12-17 (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test (C, Data are represented as mean ± SEM.",
    "molecules": "PGF2α"
  },
  {
    "caption": "D. mRNA expression  of PGF2α  synthases in the retinas  of OIR mice  during postnatal days 12-17 (n= E. mRNA expression  of Ptgfr  in the retinas  of OIR mice  during postnatal days 12-17 (n=6-8). Data information: n.s. stands for \"not significant.\" Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test D, E, Data are represented as mean ± SEM.",
    "molecules": "PGF2α"
  },
  {
    "caption": "F. mRNA expression  of Ptgfr  in each retinal  layer  from normoxic and OIR mice  on postnatal day 16 (n=6). The left-hand image displays representative cross-sections from OIR and normoxic retinas, with  isolectin B4-stained vessels  in green, DAPI-stained nuclei  in blue, and LCM  isolation locations circled by white dashed circles. INL,  inner nuclear layer; ONL, outer nuclear layer; RGC,  retinal ganglion cells. Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test F) Scale bar: 20 μm. Data are represented as mean ± SEM.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Representative images of OIR retinas  from CKO-T and control mice  on postnatal day 17. The green color shows the isolectin B4-stained vessels. The second-row panels are the enlarged images of white boxes from the first-row panels. The third-row images show  neovascular tufts (NV, white) and the fourth-row images show the vaso-obliteration  (VO, white) area. D. Quantitation of oxygen-induced   retinal neovascularization in CKO-T and control mice  (n=12). E. Quantitation of retinal vaso-obliteration in CKO-T and control mice  (n=12). Data information: n.s. stands for \"not significant.\" Data were analyzed by Mann-Whitney test D, E). Scale bars: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.",
    "molecules": "oxygen"
  },
  {
    "caption": "A. Representative images of scratch-induced migration  of PGF2α-treated HRMECs. Yellow solid lines represent scratch locations, dashed lines represent post-migration  locations, and arrows indicate migration  direction. B. Quantitation of the cell migration  distance in A (n=4). C. Effect of PGF2α  treatment on HRMEC proliferation  in a  dose-dependent manner. (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test (B, Kruskal-Wallis test with Dunn's multiple comparisons test (C). Scale bars: 100 μm (A) Data are represented as mean ± SEM.",
    "molecules": "PGF2α"
  },
  {
    "caption": "D. Representative images of staining  for  BrdU incorporation in PGF2α-treated HRMECs. E. Quantification of BrdU+ cells in D (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.",
    "molecules": "BrdU, PGF2α"
  },
  {
    "caption": "F. Representative tube formation images of PGF2α-or vehicle (DMSO)-treated HRMECs. G-J. Quantitation of total tubule  length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test G, H, I, J, Scale bars: 200 μm (F, Data are represented as mean ± SEM.",
    "molecules": "DMSO, PGF2α"
  },
  {
    "caption": "K. Representative images of  endothelial cell sprouting  of PGF2α-treated mouse aortic rings. Yellow lines indicate the EC sprouting  area. L. Quantification of the EC sprouting  area (n=6). Data information: One data point represented the mean value of three technical replicates from one independent biological replicate. Data were analyzed by Mann-Whitney test L) Scale bars: 200 μm K). Data are represented as mean ± SEM.",
    "molecules": "PGF2α"
  },
  {
    "caption": "A. Heat map of upregulated genes in PGF2α-treated HRMECs  (n=3 samples).",
    "molecules": "PGF2α"
  },
  {
    "caption": "C. Effect of PGF2α treatment on ELR+ CXC chemokine expression in HRMECs (n=3). Data information: n.s. stands for "not significant." Data were analyzed by unpaired Student's t-test (C) Data in C.",
    "molecules": "PGF2α"
  },
  {
    "caption": "H. Effect of CXCR2  inhibitor SB265610  (1 μM) on PGF2α-stimulated HRMEC migration. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position after cell migration, and the arrows indicate the migratory direction. I. Quantitation of scratch-induced HRMEC migration  in H (n=4). J. Effect of SB265610  (1 μM) on PGF2α-stimulated HRMEC proliferation  (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (I, J, Data in I. J. are represented as mean ± SEM. Scale bar: 100 μm (H)",
    "molecules": "SB265610, PGF2α"
  },
  {
    "caption": "K. Effect of CXCR2  inhibitor SB265610  (1μM) on tube  formation in PGF2α-treated HRMECs. L. Statistical graph of tube  formation experiments in K, comparing the total tubule  length, number of junctions, number of meshes, and percentage of mesh area (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test L). Data in L are represented as mean ± SEM. Scale bar: 200 μm (K).",
    "molecules": "SB265610, PGF2α"
  },
  {
    "caption": "A. Effect of CXCR2  inhibitor SB265610  (1 μM) on PGF2α-promoted sprouting  of aortic rings. Yellow lines show the sprouting  area. B. Quantitation of the sprouting  areas in A (n=6). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (B, Scale bar: 200 μm (A), image).Data are represented as mean ± SEM.",
    "molecules": "SB265610, PGF2α"
  },
  {
    "caption": "H. Representative images of oxygen-induced retinal angiogenesis   in Cxcl1-expressing lentivirus-injected CKO mice  on postnatal day 17. Green represents the isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show  neovascular tufts (NV, white), and the fourth-row images show the vaso-obliteration (VO, white) area. I. Quantitation of oxygen-induced retinal neovascularization  in H (n=8). J. Quantitation of retinal vaso-obliteration  in H (n=8). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test , I, J). Scale bar: 500 μm (H, unmagnified image), 150 μm (H, magnified image).Data are represented as mean ± SEM.",
    "molecules": "oxygen"
  },
  {
    "caption": "A. Heat map of differentially expressed transcription factors in HRMECs  in response to 500 nM PGF2α  (n=3).",
    "molecules": "PGF2α"
  },
  {
    "caption": "C. Effect of FOS  knockdown on 500 nM PGF2α-induced CXCL8 expression  in HRMECs  (n=4). D. Effect of FOS  knockdown on 500 nM PGF2α-induced HRMEC CXCL8  secretion in the culture medium (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by Mann-Whitney test C, D, Data in C, D, are represented as mean ± SEM. ",
    "molecules": "PGF2α"
  },
  {
    "caption": "E. and F. Effect of RA  (1 μM, AP-1  dimer inhibitor), SN-50  (100 μg/mL, NFκB  inhibitor), and  Inca-6  (2.5 μM, NFAT  inhibitor) on 500 nM PGF2α-induced CXCL8 expression  in HRMECs and HRMEC CXCL8  secretion in the culture medium (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by Mann-Whitney test E, F, Data in E, F, are represented as mean ± SEM. ",
    "molecules": "SN-50, Inca-6, PGF2α, RA"
  },
  {
    "caption": "G. Representative images of scratch-induced migration  of 500 nM PGF2α-treated HRMECs  with or without FOS  knockdown. The yellow solid line indicates the original position of the scratch, the dashed line indicates the position of the cells after migration, and the arrows represent the migratory direction. H. Quantitation of the cell-migration  distance in H (n=4). I. Effect of FOS  knockdown on 500 nM PGF2α-induced HRMEC proliferation  (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (H, I Scale bar: 100 μm (G) Data in H, I, are represented as mean ± SEM. ",
    "molecules": "PGF2α"
  },
  {
    "caption": "J. Representative images of tube  formation by 500 nM PGF2α-treated HRMECs  with or without FOS  knockdown. K. Quantitation of total tube  length, number of junctions, number of meshes, and percentage of mesh area in J (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test K, Scale bar: 200 μm (J, Data in K, are represented as mean ± SEM. ",
    "molecules": "PGF2α"
  },
  {
    "caption": "N. Effect of FOS  inhibitor SR11302  (1 μM) on 500 nM PGF2α-promoted sprouting  of mouse aortic rings. Data information: Scale bar: 200 μm N).",
    "molecules": "PGF2α, SR11302"
  },
  {
    "caption": "P. Effect of FOS  inhibitor SR11302  (1 μM) on 500 nM PGF2α-promoted CXCL1  secretion in mouse aortic ring  culture medium (n=6). Data information: n.s. stands for \"not significant.\" Data were analyzed by Mann-Whitney test P) Data in P are represented as mean ± SEM. ",
    "molecules": "PGF2α, SR11302"
  },
  {
    "caption": "B. Effect of U73122  (10 μM), BAPTA  (50 μM), Y27632  (5 μM), and Ly294002  (15 μM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs  (n=4). C. Effect of KN93  (10 μM) and RO-318220 (250 nM) treatment on 500 nM PGF2α-induced FOS expression  in HRMECs  (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by Mann-Whitney test (B, C Data are represented as mean ± SEM.",
    "molecules": "BAPTA, KN93, Ly294002, PGF2α, RO-318220, U73122, Y27632"
  },
  {
    "caption": "D. Effect of SB203580  (2 μM), PD98059  (20 μM), and SP600125  (60 nM) treatment on 500 nM PGF2α-induced FOS gene expression in HRMECs  (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by Mann-Whitney test Data are represented as mean ± SEM.",
    "molecules": "PD98059, SP600125, PGF2α, SB203580"
  },
  {
    "caption": "E. Effect of U73122  (10 μM), BAPTA  (50 μM), and KN93  (10 μM) treatment on 500 nM PGF2α-induced p38 phosphorylation  in HRMECs. F. Quantitation of the ratio of p38 phosphorylation  to total p38  protein in E (n=3). Data information: n.s. stands for \"not significant.\" Data were analyzed by unpaired student's t-test (F, Data are represented as mean ± SEM.",
    "molecules": "BAPTA, KN93, PGF2α, U73122"
  },
  {
    "caption": "G. Effect of U73122  (10 μM), BAPTA (50 μM), and SB203580  (2 μM) on the phosphorylation  of CAMK2 isoforms in 500 nM PGF2α-treated HRMECs. H. Quantitation of the ratio of CAMK2G phosphorylation  to total CAMK2G  in G (n=3). I. Quantitation of the ratio of phosphorylated CAMK2D  to total CAMK2D  in G (n=3). Data information: n.s. stands for \"not significant.\" Data were analyzed by unpaired student's t-test H, I, Data are represented as mean ± SEM.",
    "molecules": "BAPTA, PGF2α, SB203580, U73122"
  },
  {
    "caption": "J. Effect of CAMK2G  or CAMK2D  knockdown on 500 nM PGF2α-triggered p38 phosphorylation  in HRMECs. K. Quantitation of the ratio of p38 phosphorylation  to total p38  protein in J (n=3). Data information: n.s. stands for \"not significant.\" Data were analyzed by unpaired student's t-test Data are represented as mean ± SEM.",
    "molecules": "PGF2α"
  },
  {
    "caption": "L. Effect of CAMK2G  or CAMK2D  knockdown on 500 nM PGF2α-induced FOS expression  in HRMECs  (n=4). Data information: n.s. stands for \"not significant.\" Data were analyzed by Mann-Whitney test L) Data are represented as mean ± SEM.",
    "molecules": "PGF2α"
  },
  {
    "caption": "M. Effect of U73122  (10 μM), BAPTA  (50 μM), KN93  (10 μM), and SB203580  (2 μM) on ELK-1 phosphorylation  in 500 nM PGF2α-treated HRMECs. N. Quantitation of the ratio of phosphorylated ELK-1 to total ELK-1 in M (n=3). Data information: n.s. stands for \"not significant.\" Data were analyzed by unpaired student's t-test N). Data are represented as mean ± SEM.",
    "molecules": "BAPTA, KN93, PGF2α, SB203580, U73122"
  },
  {
    "caption": "B. Representative images of OIR retinas  in WT and Cxcr2-/- mice  with or without AL8810  treatment. The green color shows the  isolectin B4-stained vessels, the second-row panels display the enlarged images of white boxes in the first-row panels, the third-row images show  neovascular tufts (NV, white), and the fourth-row images show the  vaso-obliteration (VO, white) area. C. Quantitation of oxygen-induced retinal neovascularization  in WT and Cxcr2-/- mice  with or without AL8810  treatment (n=12). D. Quantitation of retinal vaso-obliteration in WT and Cxcr2-/- mice  with or without AL8810  treatment (n=12). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (C, D) Scale bar: 500 μm (unmagnified image), 150 μm (magnified image). Data are represented as mean ± SEM.",
    "molecules": "AL8810, oxygen"
  },
  {
    "caption": "E. Effect of AL8810  on retinal Fos expression  in WT and Cxcr2-/- OIR mice  on postnatal day 16 (n=6). F. Effect of AL8810  on retinal Cxcl1 expression  in WT and Cxcr2-/- OIR mice  on postnatal day 16 (n=6). Data information: n.s. stands for \"not significant.\" Data were analyzed by two-way ANOVA with Mann-Whitney test (E, F). Data are represented as mean ± SEM.",
    "molecules": "AL8810"
  },
  {
    "caption": " A Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist) Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958) ",
    "molecules": "fluprostenol, isoproterenol, lisophosphatidic acid, prostaglandin E1, PGE2, prostaglandin E2, sphingosine-1-phosphate, treprostinil, WIN55,212-2"
  },
  {
    "caption": " B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined Data information: values are mean±s.e.m In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065)",
    "molecules": "PGE2"
  },
  {
    "caption": " D, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist) Data information: values are mean±s.e.m In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1",
    "molecules": "AH6809, L161,982, 798106, L798106, PGE2"
  },
  {
    "caption": " B-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours. Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2 ",
    "molecules": "Gαt, serum, SQ22536, DMSO, PGE2"
  },
  {
    "caption": " A,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments ",
    "molecules": "bisindolylmaleimide, PE, phenylephrine, serum, BPKDi, endothelin-1, ET1, PGE2"
  },
  {
    "caption": " C NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3",
    "molecules": "AIP, serum, BPKDi, DMSO, PGE2"
  },
  {
    "caption": " A NRVMs were serum-starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2. Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot Data information: In (A-C) representative images from at least 3 independent experiments are shown ",
    "molecules": "serum, PGE2"
  },
  {
    "caption": " B NRVMs were infected with recombinant adenoviruses encoding EGFP (AdGFP) or RGS16 24 hours prior to stimulation, then Rac1 activity was measure Data information: In (A-C) representative images from at least 3 independent experiments are shown. In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS16, P=0.0415 (B ), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4",
    "molecules": "PGE2"
  },
  {
    "caption": " C Cells were pretreated with 100 ng/ml pertussis toxin (PTX, Gi/o-inhibitor) for 24 hours or with 50 μM NSC23766 (NSC, Tiam1-Rac1 interaction inhibitor) for 2 hours. Thereafter Rac1 activation was determined by effector pulldown assay Data information: In (A-C) representative images from at least 3 independent experiments are shown In (B-D) values are mean±s.e.m., (B,C) n=4 3; *,P<0.05 (Two-Way ANOVA, RGS1 PTX, P=0.039 (C), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4",
    "molecules": "NSC, NSC23766, PGE2"
  },
  {
    "caption": " D NRVMs were transduced with the 3xMEF2-Luc reporter and were serum-starved for 20 hours. After 2-hour of 50 μM NSC23766 (A) pretreatments, the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours, then MEF2 activity was determined. NSC abolished the MEF2 activation by PGE2 Data information In (B-D) values are mean±s.e.m. (D), n=3; *,P<0.05 (Two-Way ANOVA, RGS1",
    "molecules": "serum, DMSO, NSC, NSC23766, PGE2"
  },
  {
    "caption": " A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC Data information: In (A-C) representative images from 3 independent experiments are shown ",
    "molecules": "NSC, NSC23766, PGE2"
  },
  {
    "caption": " B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC Data information: In (A-C) representative images from 3 independent experiments are shown ",
    "molecules": "NSC, NSC23766, PGE2"
  },
  {
    "caption": " C NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker Data information: In (A-C) representative images from 3 independent experiments are shown ",
    "molecules": "L798106, PGE2"
  },
  {
    "caption": " D MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2 Data information In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5",
    "molecules": "IPA-3, PGE2"
  },
  {
    "caption": " Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6 ",
    "molecules": "BPKDi, IPA-3, PGE2"
  },
  {
    "caption": " MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours A mRNA-levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA-levels, fold increase compared to saline-treated controls, normalized to 18s-content Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values: IL6, P=0.0007 (A); TNFα, P<0.0001 (A)",
    "molecules": "lipopolysaccharide"
  },
  {
    "caption": " MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano-liquid chromatography tandem mass spectrometry Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values PGE2, P=0.0016 (B",
    "molecules": "lipopolysaccharide, PGE2"
  },
  {
    "caption": " MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours C Histologic and macroscopic stainings of β-galactosidase-activity in MEF2-lacZ reporter mice show MEF2-activation (blue cells and precipitates, respectively) in the myocardium upon LPS-treatment. Saline-treated littermates served as control. Scale bar of histological stainings is 100 µm. Quantification of whole-heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity) Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant. In (C) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments. P values blue pixel intensity, P=0.0083 (C)",
    "molecules": "lipopolysaccharide, LPS"
  },
  {
    "caption": " MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours D Induction of PKD, HDAC5 and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, the quantification is in the right panel Data information: values are mean±s.e.m The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant P values ); phospho-PKD-Ser-744/Ser-748, P=0.0049 (D); phospho-PKD-Ser-916, P=0.2684 (D); phospho-HDAC5-Ser-498 (D), P=0.0019; phospho-HDAC5-Ser-259, P=0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P=0.1962 (D); phospho-PAK2-Thr-402, P=0.0446 (D). The exact n and P values can be also found in the Source Data excel file for Figure 7",
    "molecules": "lipopolysaccharide, LPS"
  },
  {
    "caption": "E. Normalized ThT kinetics of 10μM Biot-Aβ1-42 with timepoints of samples that incubated with Aβ1-42 membranes.",
    "molecules": "Aβ1-42, Biot, ThT"
  },
  {
    "caption": "I. ThT kinetics of Biot-Aβ1-42 seeding. 10μM of Biot-Aβ1-42 incubated with 0.5 or 1 μM of Biot-Aβ1-42 seeds.",
    "molecules": "Aβ1-42, Biot, ThT"
  },
  {
    "caption": "D. Binding of Biot-Aβ1-42 to homologue peptides derived from ~520 randomly selected proteins.",
    "molecules": "Aβ1-42, Biot, peptides"
  },
  {
    "caption": "E. Summary of Biot-Aβ1-42 binding throughout 8 membranes, color indicates the mean between membranes and the size of the outline the standard deviation.",
    "molecules": "Aβ1-42, Biot"
  },
  {
    "caption": "A-C. ThT kinetics of 10μM rAβ1-42 alone or in presence (1:1) of 3 homologue peptides (full screen on Appendix Figure S3,S4, n= 2 independent experiments with 4 repeats). Reused images in Appendix figure S3",
    "molecules": "Aβ1-42, ThT"
  },
  {
    "caption": "I. Representative AFM height images of Aβ fibrils alone or in a 1:1 mixture with P3, P5, P8 and P12 peptides are show in the top row. The boxes indicate the magnified regions shown in the second row. Arrows indicate the locations of representative individual fibrils shown in magnified detail, each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data. The scale bar for each row are shown to the left, with both the 3D model and the straightened image data representing 10 nm. The colour scale of the 3D models from blue to yellow indicate the distance (from low to high) between the fibril surface and fibril centre axis to demonstrate their twist patterns. The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single, not fragmented, amyloid fibrils are shown in the bottom row.",
    "molecules": "amyloid fibrils, Aβ fibrils, fibril, fibrils, peptides"
  },
  {
    "caption": "D. Representative images of 3 proteins that can induce aggregation of Aβ1-42 in biosensor cell. Increase aggregation is observed in cells expressing the construct but not GFP alone (left panels). Scale bar: 100μm. FRAP of the resulting aggregates shows no recovery (in detail at Appendix Figure S11). Scale bar: 2.5μm Removal of the homologue regions resulting in reduced aggregation (right panels). (n=3 independent experiments).",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(F) Representative immunofluorescence for GnRH- (red) and BrdU- (green) positive cells in control and mutant E14.5 embryos arising from progenitors born between E9.5 and E10.5. BrdU-positive GnRH neurons (white arrowheads) were more abundant in mutant than in control embryos. Scale bar: 20μm.",
    "molecules": "BrdU"
  },
  {
    "caption": "(G) Number of BrdU-positive GnRH neurons in the nose, olfactory bulb (OB) and ventral forebrain (VFB) at E14.5 and total number of cells in control and mutant embryos. Double-labeled cells were more numerous in the mutant VFB. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 embryos from 2 litters.",
    "molecules": "BrdU"
  },
  {
    "caption": "(I) Number of GnRH neurons in E14.5 embryos injected with neutralizing antibodies to Neuropilin-1 (Nrp1Ab) compared to an isotype goat antibody, or the antiapoptotic compound ZVAD compared to its vehicle, together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age. Note that the data are represented as fold change, as raw values are not comparable with transgenic animals that did not undergo surgery. Unpaired t-test, n=5 to 7 embryos from at least 2 litters.",
    "molecules": "ZVAD"
  },
  {
    "caption": "(G) Plasma LH levels at P45 in control and mutant mice. Mann-Whitney U test, n=5 to 6 mice.",
    "molecules": "LH"
  },
  {
    "caption": "(B) Bacterial survivability  under oxidative stress. Representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were treated with hydrogen peroxide. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (B), were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).",
    "molecules": "hydrogen peroxide"
  },
  {
    "caption": "Wild-type and LGP2-KO HEK293  cells in 24-well plates were transfected with 200 ng/ml of short poly I:C IFN-β  and IP-10  mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). LGP2  KO2 and KO3 are independently isolated LGP2  KO clones. Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "(F) HEK293   cells in 24-well plates were transfected with LGP2    (0.1, 0.3, or 0.6 μg/well) and RIG-I (0.1 μg/well) expression vectors as indicated. 24 h after transfection, cells were stimulated with 200 ng of short poly I:C  for 4 hr. Total RNAs were extracted, and the expression of  IFN-β mRNA was determined by RT-qPCR   and normalized to GAPDH   (n = 3, t-test, *p < 0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "B) A549  cells transfected with HA-tagged ubiquitin  were stimulated with 200 ng/ml of short poly I:C for 6 h. PLA  was performed with anti-HA, anti-RIG-I, and anti-LGP2  antibodies. The number of PLA  signals were counted (t-test, *p < 0.05, n = 20). Each dot represents the number of PLA  signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "D-F) HEK293  cells were transfected with 200 ng/ml of short  poly I:C. Cells were fixed at the indicated  time points, and PLA  was performed with anti-RIG-I, anti-LGP2, and anti-K63-Ub  antibodies (D). The number of PLA  signals were counted (E, F) (t-test, *p < 0.05, n = 30). Each dot represents the number of PLA  signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "(F, G) WT and Riplet  KO HEK293  cells were transfected with 200 ng/ml of short poly I:C for 6 h, and PLA  was performed with anti-LGP2  and anti-K63-Ub  antibodies. The number of PLA  signals were counted (t-test, *p < 0.05, n = 100). Each dot represents the number of PLA  signals in each cell. Scale bars represent 10 μm. Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "(B) HE293  cells stably expressing GFP  (control), LGP2, or LGP2-4KR were transfected with p125luc reporter plasmids and then stimulated with indicated amounts of short poly I:C. WCEs were prepared 24 h after transfection and stimulation, and luciferase activity  was determined. The proteins were subjected to  SDS-PAGE and detected by western blotting with the indicated Abs. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "HEK293  cells stably expressing GFP, LGP2, or LGP2-4KR were transfected with 200 ng/ml of short poly I:C  (C-E), Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5  mRNA was determined by RT-qPCR  and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "(K, L) HEK293  cell clones stably expressing LGP2  or LGP2-4KR at lower levels were transfected with short poly I:C, and the expression of mRNAs were determined by RT-qPCR. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "A) Microarray  analysis was performed as described in materials and methods. A heatmap of the microarray  data of LGP2  and LGP2-4KR stably expressing cells stimulated with short poly I:C  for 12 h.",
    "molecules": "poly I:C"
  },
  {
    "caption": "B, LGP2  and LGP2-4KR stably expressing HEK293  cells were stimulated with 200 ng/ml of short  poly I:C, and the expression of the genes at the indicated  time points were determined by  RT-qPCR and normalized to GAPDH  (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "C) LGP2  and LGP2-4KR stably expressing HEK293  cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR  and normalized to GAPDH  (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "H, I) WT and indicated mutant HEK293  cells were stimulated with 200 ng/ml of short  poly I:C for 8 h. WCEs were prepared and subjected to SDS-PAGE. The proteins were detected with indicated antibodies.",
    "molecules": "poly I:C"
  },
  {
    "caption": "WT, and Riplet   KO HEK293  cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1  mRNA was determined by RT-qPCR  and normalized to GAPDH  (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "B) WT, LGP2  KO, HEK293  cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1  mRNA was determined by  RT-qPCR  and normalized to GAPDH  (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "C, WT and Riplet  KO A549  (C) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100  was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "D) WT and Riplet  KO MEF  (D) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100  was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "H) LGP2  or LGP2-4KR stably expressing WT or Riplet  KO HEK293  cells were stimulated with 200 ng/ml of short poly I:C (H).  IFN-β mRNA levels at indicated time points were determined by  RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Data information: All data are representative at least two independent experiments, and \"n\" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.",
    "molecules": "poly I:C"
  },
  {
    "caption": "(E) Smyhc1 immunohistochemistry stain of 24 hpf (hour post fertilization) zebrafish larvae. Filamentous actin is stained with phalloidin (red). Nuclei are stained with DAPI (blue). Smyhc1 is stained with the F59 antibody (green) (Elworthy et al, 2008). smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae display Smyhc1 in the muscle fibers at 24hpf. smyhc1-/- larvae at 24 do not stain positively for Smyhc1. Scale bar represents 50 µm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(B) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1-/- adults, in contrast to the compression and fusion of vertebrae seen in smyhc1R673H/+ adults. Distal tail regions are highlighted and enlarged.",
    "molecules": "Alizarin red"
  },
  {
    "caption": "(A) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin-rhodamine to detect actin. Puncta of filamentous actin are indicated with white arrowheads, misshapen myofibers are indicated with white arrows and outlined. (B) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin-rhodamine. Puncta of filamentous actin are indicated with white arrowheads, distorted myofibers are indicated with arrows and outlined, and frayed myofibers are indicated with yellow arrowhead. ",
    "molecules": "rhodamine, phalloidin"
  },
  {
    "caption": "(C) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). (D) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). Bundle of actin ringed with α-actinin indicated with white-bordered black arrowhead. ",
    "molecules": "rhodamine, DAPI, phalloidin"
  },
  {
    "caption": "(E) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 54), smyhc1-/- (n = 31), smyhc1R673H/+ (n = 45), and smyhc1R673H/R673H (n = 49). Distance between myosepta was measured perpendicular to rostral-caudal body axis at defined mid-body regions in phalloidin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (F) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 51), smyhc1-/- (n = 32), smyhc1R673H/+ (n = 64), and smyhc1R673H/R673H (n = 38). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. ",
    "molecules": "phalloidin"
  },
  {
    "caption": "(A) Representative morphologies of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 2 dpf after 24 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. All para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis. (B) Representative images of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 3dpf after 48 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. Para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis. ",
    "molecules": "DMSO, para-aminoblebbistatin, Para-aminoblebbistatin"
  },
  {
    "caption": "B. CACNA1A is present as punctae on the Vacuolin-1 enlarged LAMP1 positive lysosomes in primary cerebellar cultures of both CTL and Cacna1atg-la mutants. Scale bars, 20 μm.",
    "molecules": "Vacuolin-1"
  },
  {
    "caption": "D. CACNA1A is concentrated in the lysosomal fractions isolated from mice cerebella with iodixanol gradient.",
    "molecules": "iodixanol"
  },
  {
    "caption": "A. DQ-BSA labeled late endosomes fuse with LAMP1 labeled lysosomes in CTL but not the mutant cells. Scale bar, 20 μm. B. Quantification of DQ-BSA and LAMP1 co-localization in CTL and Cacna1atg-la cells. Data are presented as means ± SD. (ns: not significant; ***: p < 0.001) C. Bepridil but not ω-agatoxin TK (ω-Aga TK) treatment reduces lysosomal fusion in cultured cerebellar cells. DQ-BSA (Green) labeled late endosomes co-localize with LAMP1 (Red) labeled lysosomes with/without ω-Aga TK (1 µM) or Bepridil (10 µM) treatment. Scale bar, 20 μm. D. Quantification of DQ-BSA and LAMP1 co-localization with ω-Aga TK or Bepridil treatment. Data are presented as means ± SD. (ns: not significant; **: p < 0.001).",
    "molecules": "Bepridil"
  },
  {
    "caption": "Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt-villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.",
    "molecules": "β‐NF"
  },
  {
    "caption": "The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1+/+mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5GFP‐positive CBCstem cells.",
    "molecules": "βNF"
  },
  {
    "caption": "A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "VillinCreERT2Snai1fl/florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cell growth in VillinCreERT2Snai1fl/florganoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "A-C Droplet digital PCR analysis of Bmf, Bbc3/Puma and Bcl2l 11/Bim after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 h with tamoxifen. No differences in gene expression were observed (n = 3).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "D qRT-PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 (P = 0.0001) and 72 h (P = 0.006) with tamoxifen. Bars represent mean ± SD. n = 3 mice per group.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "E SW480 cells, which transiently express GFP‐SNAI1, were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1, IL8 and a novel target SERINC3 revealed by microarray analysis. An unrelated DNA region was used as a negative control. Anti‐GFP antibody was used to immunoprecipitate GFP‐tagged SNAI1, while IgG control antibody was used as a negative control. The results are given as relative enrichment compared to input material and are representative of three independent experiments.",
    "molecules": "DNA"
  },
  {
    "caption": "B. Representative immunofluorescence and live-cell images of dynamics MERVL::tdTomato and Gag expression during embryos preimplantation development (left). Quantification of tdTomato and Gag intensity (right). For the live-cell images, average intensity of tdTomato signal intensities relative to 2-cell stage embryos. For the immunofluorescence images, bar graphs showing the relative intensities of Gag/DAPI signal ratio. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 4. **P < 0.01, ***P < 0.001 by two-tailed Student"s\u2028 t-test. Scale bar,",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Representative DAPI staining of blastocysts of tdTomato+ and tdTomato- SCNT embryos after 115 hr. of culture in vitro. Scale bar, 50 μm. D. The tdTomato+ and tdTomato- SCNT blastocyst cell numbers were determined by counting the DAPI-stained cells. Three independent experiment replicates were performed. N, total number of embryos analyzed for each condition. Red bars indicated the mean value. E ",
    "molecules": "DAPI"
  },
  {
    "caption": "F. Dynamic appearance of H3K27me3 during early preimplantation development. Shown are representative images of embryos stained with DNA and H3K27me3. Negative staining of H3K27me3 could be observed in ICSI and SCNT-tdTomato+ embryo at morula stage. Representative images from ≥ 83 embryos analyzed in four independent micromanipulations are shown. Scale bar, 20 μm",
    "molecules": "DNA"
  },
  {
    "caption": "C. The sketch of KDM6A and KDM6B in vitro transcription vector (right), and the integrity of in vitro transcripted mRNA was confirmed by electrophoresis with formaldehyde gels (left). M, marker; T7, in vitro transcription promoter; HA, hemagglutinin epitope tag",
    "molecules": "formaldehyde"
  },
  {
    "caption": "RT-PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (-RT). A fragment of the actin ORF was amplified as control.",
    "molecules": "cDNA, RNA"
  },
  {
    "caption": "HEK293 cells expressing HA-RAMP4-2 alone or in combination with SPPL2c or SPP were treated with 100 µM (Z-LL)2-ketone (ZLL), 5 µM inhibitor X (InX), 5 µM DAPT or DMSO as control. RAMP4-2 band intensity was quantified from blots of three independent experiments and normalised to cells just over-expressing the substrate.",
    "molecules": "(Z-LL)2-ketone, ZLL, DAPT, DMSO, inhibitor X, InX"
  },
  {
    "caption": "Microsomes isolated from testis of wild type and SPPL2c-/- mice were solubilised in 1% digitonin and proteins were separated by blue native PAGE. After transfer to PVDF membrane, SPPL2c and SPP were detected using the polyclonal antisera introduced above.",
    "molecules": "digitonin"
  },
  {
    "caption": "Histochemical visualization of β-galactosidase (β-gal) reporter activity revealed SPPL2c expression within seminiferous tubules. Cryo-sections from PFA-fixed wild type and SPPL2c-/- testis were stained with X-Gal as β-gal substrate.",
    "molecules": "X-Gal, PFA"
  },
  {
    "caption": "SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band.",
    "molecules": "DNA, Hoechst 33342"
  },
  {
    "caption": "Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin-fixed wild type and SPPL2c-/- testis. The SPPL2c antiserum directed against a C-terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed. Nuclei were stained with hematoxylin. Scale bar, 100 µm (upper panel) or 50 µm (lower panel).",
    "molecules": "diaminobenzidine, hematoxylin, peroxidase"
  },
  {
    "caption": "Paraffin sections from Bouin-fixed wild type and SPPL2c-/- testis were stained with hematoxylin & eosin (H&E). Scale bars, 100 µm.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Carbonate-washed total membrane fractions from wild type and SPPL2c-/- testis (n=5) were subjected to a label-free quantitative proteome analysis. For each quantified protein, a log2 intensity ratio between SPPL2c-deficient and wild type samples was calculated. Negative values indicate a depletion and positive values an enrichment in SPPL2c-/- samples. In the depicted volcano plot the negative log10 of the p value (y axis) is plotted versus the log2 intensity ratio of SPPL2c-/- versus wild type samples (x axis). All proteins above the significance level of p<0.01 (unpaired Student's t test) are coloured in red. The hyperbolic curve indicates the threshold for a permutation-based FDR correction for multiple hypotheses with p = 0.05 and s0 = 0.1.",
    "molecules": "Carbonate"
  },
  {
    "caption": "Immunohistochemical detection of PLN in paraformaldehyde (PFA)-fixed testis from wild type and SPPL2c-/- mice. Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate.",
    "molecules": "diaminobenzidine, paraformaldehyde, PFA, peroxidase"
  },
  {
    "caption": "Testis suspensions were stained with 2.5 µM DRAQ5 to determine cellular DNA amount. 1C-co = elongated spermatids, 1C = other haploid cells including round spermatids, 2C = spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells, 4C = primary spermatocytes, G2 spermatogonia.",
    "molecules": "DNA, DRAQ5"
  },
  {
    "caption": "Intracellular Ca2+ concentrations were analysed in testis suspensions from either wild type or SPPL2c-deficient mice using the Ca2+-sensitive probe Fluo4-AM. Individual germ cell populations were identified Bars indicate Median Fluo4-AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples.",
    "molecules": "Ca2+, Fluo4-AM"
  },
  {
    "caption": "B (left) SRM images of paraspeckles in HAP1 NEAT1 ∆3′ mutant cells (∆3′) detected by NEAT1_5′ (green) and  NEAT1_15k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1  3′ ends in the core or in the core and shell (n = 44).",
    "molecules": "MG132"
  },
  {
    "caption": "B SRM images  of the paraspeckles in ∆0-0.8kb and ∆0-1.9kb (∆5′) cells treated with MG132  (5 μM for 6 h) detected by NEAT1_5′, 1k, and 2k (green) and NEAT1_3′ (magenta) FISH  probes. Scale bar, 500 nm.",
    "molecules": "MG132"
  },
  {
    "caption": "D (left)  EM observation of the paraspeckles in ∆5′ cells treated with MG132 (5 μM for 17 h) using NEAT1_5′ probes. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (347 gold particles) within the paraspeckles in ∆5′ cells. (right) Graph showing the proportion of the localization of NEAT1_5′ probes in each paraspeckle in ∆5′ cells (n = 20). The box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).",
    "molecules": "MG132"
  },
  {
    "caption": "B SRM images  of the paraspeckles in MG132-treated (5 μM for 6 h) HAP1 NEAT1  ∆5′/∆3′ cells detected by NEAT1_2k (green) and NEAT1_15k FISH probes (left, magenta) or  NONO immunofluorescence (IF) (right, magenta). Scale bar, 500 nm.",
    "molecules": "MG132"
  },
  {
    "caption": "D (upper)  EM observation of the paraspeckles in MG132-treated (5 μM for 17 h) ∆5′/∆3′ cells using NEAT1_5′ (left) and NEAT1_D2 probes (right). Scale bar, 100 nm. (middle) Graph showing the proportion of the  localization of NEAT1_5′ (left, 310 gold particles) and NEAT1_D2 (right, 294 gold particles) probes within the paraspeckles in HAP1 NEAT1 ∆5′/∆3′ cells. (lower) Graph showing the proportion of the localization of NEAT1_5′ (left, 19 cells) and D2 (right, 22 cells) probes in each paraspeckle in ∆5′/∆3′ cells. Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).",
    "molecules": "MG132"
  },
  {
    "caption": "C Diameters (Sx) of the paraspeckles in WT, ∆3′, ∆5′, and ∆5′/∆3′ cells treated with MG132 (5 μM for 6 h) determined by SRM (WT: n = 129, ∆3′: n = 106, ∆5′: n = 100, ∆5′/∆3′: n = 292). WT (mean size: 387.5 nm), ∆3′ (mean size: 492.4 nm), ∆5′ (mean size: 497.7 nm), and ∆5′/∆3′ mutant (mean size: 753.3 nm). (****: P < 0.0001, compared with WT: Kruskal-Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).",
    "molecules": "MG132"
  },
  {
    "caption": "B Quantitation of NEAT1_2  by RT-qPCR in WT and ∆5′/∆3′/∆PAS cells with or without MG132 treatment (6 h). Data are represented as mean ± SD (n = 3).",
    "molecules": "MG132"
  },
  {
    "caption": " (A) Binding of wild type DUBm-Ubp8 to monoubiquitin. (B) Binding of DUBmUbp8C146A to monoubiquitin. (C) Binding of DUBm-Ubp8C146S to monoubiquitin. (D) Binding of DUBm-Ubp8C146R to monoubiquitin. (E) Binding of DUBm-Ubp8C146A to K48 diubiquitin. (F) Binding of DUBm-Ubp8C146S to K48 diubiquitin. ",
    "molecules": "diubiquitin, monoubiquitin"
  },
  {
    "caption": "Binding was measured by fluorescence polarization using N-terminally TAMRA-labeled monoubiquitin. The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM (Clerici et. al, 2014) and 0.60 ± 0.17 nM, respectively. Error bars are s.d. calculated on five measurements per point.",
    "molecules": "TAMRA, monoubiquitin, ubiquitin"
  },
  {
    "caption": " (A) Equilibrium binding of OTUD1 C320A and C320R to K63-linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63-linked diubiquitin in which the proximal ubiquitin was fluorescently-labeled. Error bars indicate s.d. and are based on three measurements per data point. One representative experiment of two is shown. ",
    "molecules": "diubiquitin, FlAsH, ubiquitin"
  },
  {
    "caption": " Polyubiquitin chains were co-immunoprecipitated with FLAG-PSMD4, an ubiquitin receptor for the 26S proteasome, from cells expressing either USP14 wild-type, C114A, or C114R. One representative experiment of two is shown. ",
    "molecules": "Polyubiquitin, ubiquitin"
  },
  {
    "caption": "(B) HA-ATG9A-BirA* was expressed in HCT-116 ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with WT ATG13  or ATG13  ∆HORMA. Cells were grown in full DMEM media, treated with 50 µM  biotin for 12 hrs, followed by detergent lysis and incubation with streptavidin resin. The graph on right shows quantification of normalized ATG101  infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(D) HA-ATG9A-BirA* was overexpressed in WT and ULK1/2 Double KO MEFs. Cells were subjected to streptavidin pulldown and immunoblotting with indicated antibodies. The graph on right shows quantification of normalized ATG13 infrared signal. Mean ± SEM. n=3 (biological replicates). Significance measured using one-sample t-test compared to hypothetical mean of 1 (right). ns=p>0.05, *=p≤0.05, **= p≤0.01, ***=p≤0.001, ****= p≤0.0001.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(A) Endogenous p62/SQSTM1  level in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13  KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by  immunoblotting  with indicated antibodies. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin  for 24 hrs and whole cell lysates were subjected to  immunoblotting (left). The graph on right shows quantification of normalized p62  infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) Endogenous p62/SQSTM1 level in HEK-293T ATG13 WT,  ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated proteins. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin  for 24 hrs and whole cell lysates were subjected to immunoblotting. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right).",
    "molecules": "Bafilomycin"
  },
  {
    "caption": "(A) Endogenous p62/SQSTM1 level was measured in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO OR ATG13 KO cells reconstituted with ATG13 WT, ATG13 ΔHORMA and ATG13  Δ2AA by immunoblotting  with indicated proteins (left). Cells were grown in full DMEM media and whole cell lysates were subjected to  immunoblotting (left). Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) HCT-116 cells in A were incubated in EBSS for 4 hours then processed and analyzed as in A.",
    "molecules": "EBSS"
  },
  {
    "caption": "(C) Modified pulse chase experiment. HEK293T ATG13 WT and  HEK293T ATG13 KO cells stably expressing HA ATG9A-BirA* were pulse labeled with  biotin for 24 hours, media was replaced with full DMEM and streptavidin pulldown was performed at indicated time points. Quantification of normalized  p62  infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using Student's t test (bottom). ns=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****= p≤0.0001.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(E) Confocal images of EGFP-p62/SQSTM1 colocalization with HA ATG9A and ATG13  in HCT116 ATG9A-HA KI cells stably expressing EGFP-p62/SQSTM1. Cells were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs, fixed, labelled with antibodies for HA  and ATG13 and imaged (Scale bar=10 µm).",
    "molecules": "Wortmannin"
  },
  {
    "caption": "(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested. The ferritin content was determined by ELISA. Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin. The data are presented as the standard deviation from three different experiments.",
    "molecules": "chloroquine, FAC, leupeptin, Ponasterone A"
  },
  {
    "caption": "(B) Cells treated as in (A) were incubated in the presence of 1 μg/ml EGF for 2 h. Cells were fixed and processed for immunofluorescence using mouse anti‐EGF receptor and Alexa 594 conjugated goat anti‐mouse IgG. Chloroquine and leupeptin effectively inhibit degradation of EGF receptor.",
    "molecules": "Chloroquine, leupeptin"
  },
  {
    "caption": "(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 or 10 μM lactacystin in the presence of Ponasterone A for 10 h and harvested. The ferritin content was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.",
    "molecules": "FAC, lactacystin, MG132, Ponasterone A"
  },
  {
    "caption": "(C) Cells were incubated with 1.0 × 10−7 M Tf(59Fe)2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined.",
    "molecules": "59Fe, MG132, Ponasterone A"
  },
  {
    "caption": "(E) Cells were treated as described in (C), ferritin was then immunoprecipitated, eluted using 100 mM glycine, pH 2.5 and measured by ELISA. The amount of ferritin‐associated 59Fe was measured and the specific activity of ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.",
    "molecules": "59Fe, glycine"
  },
  {
    "caption": "(A) FM3A (black) and ts85 (grey) cells were transiently transfected with a plasmid containing CMV‐regulated Fpn‐GFP and incubated in the presence of FAC (10 μM Fe) and 0.5 μM hepcidin for 24 h. Hepcidin was either maintained (+) or removed (−) to allow Fpn‐GFP localization at the plasma membrane. Cells were maintained at the permissive temperature (33°C) or moved to the restrictive temperature (39°C) and incubated for 6 h. Cells were harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.",
    "molecules": "FAC, Fe"
  },
  {
    "caption": "(B) FM3A and ts85 cells were treated as in (A) but FAC was replaced with 1.0 × 10−7 M Tf(59Fe)2. Ferritin was immunoprecipitated and the specific activity of 59Fe‐ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.",
    "molecules": "59Fe"
  },
  {
    "caption": "(D) FM3A and ts85 cells were treated as in (A) but cells that had been incubated at the restrictive temperature (39°C) were then returned to the permissive temperature (33°C) and incubated in the presence of cycloheximide (75 μg/ml) and 10 μM FAC for 1 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.",
    "molecules": "cycloheximide, FAC"
  },
  {
    "caption": "(A) Cells were then incubated at the restrictive temperature (39°C) for 6 h in the presence or absence of 100 μM DFO, with or without 100 μM chloroquine or 10 μM MG132. Cells were then harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.",
    "molecules": "chloroquine, DFO, MG132"
  },
  {
    "caption": "(B) Cells treated as in (A) were incubated in the presence or absence of 5 mM 3‐methyladenine.",
    "molecules": "3‐methyladenine"
  },
  {
    "caption": "(A) Δccc1/pGAL‐H+L cells were transformed with a plasmid containing a methionine regulated CCC1 (pMET3CCC1). Cells were grown in medium with galactose for 20 h, washed and incubated in galactose (•), glucose with 10 × methionine (▵), glucose (○) or glucose without methionine (▾) for 10 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p.",
    "molecules": "galactose, glucose, methionine"
  },
  {
    "caption": "(B) erg6‐2 and erg6‐2 pGAL‐H+L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h. Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h.",
    "molecules": "galactose, glucose, MG132"
  },
  {
    "caption": "(C) Cells were then harvested, ferritin levels determined by ELISA and immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis. Cells with the erg6‐2 allele permit the entry of MG132 ( Lee and Goldberg, 1996).",
    "molecules": "MG132"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (N) Prom1+/+ or Prom1-/- hepatocytes serum-starved for 8 hours were incubated with 2-[3H]deoxyglucose for 10 minutes. Glucose uptake was assessed by calculating the amount of cell-associated radioactivity normalized to the amount of protein (n = 3 mice/group).",
    "molecules": "2-[3H]deoxyglucose, Glucose"
  },
  {
    "caption": "C. Blood glucose levels (mg/dL) of mice after fasted for 24 hours (n = 12 or 14/group).",
    "molecules": "glucose"
  },
  {
    "caption": "D. Serum glucagon level in the fed or fasted mice (n = 3/fed, 7 or 8/fasted).",
    "molecules": "glucagon"
  },
  {
    "caption": "E. Glycogen contents in the liver of mouse in each condition was shown by PAS (periodic acid schiff) staining. Scale bar, 500 μm.",
    "molecules": "Glycogen"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. F. Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation (10 nM) were determined by qRT-PCR. The level of each mRNA was normalized to the level of the 18S rRNA. For these experiments, primary hepatocytes were cultured under 3D conditions using a Matrigel matrix (n = 3 mice/group).",
    "molecules": "glucagon"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. G. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, phospho-Inositol trisphosphate receptor (p-IP3R), IP3R, phospho-Hormone-Sensitive Lipase (p-HSL), HSL, Prom1 and Gapdh 10 minutes after glucagon stimulation (10 nM) were determined by immunoblotting.",
    "molecules": "glucagon"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. H. The nuclear localization of p-CREB 10 minutes after glucagon stimulation (10 nM) was determined by immunofluorescence staining. Scale bar, 20 μm.",
    "molecules": "glucagon"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. I. Relative PKA activities 10 minutes after glucagon stimulation (10 nM) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).",
    "molecules": "IBMX, glucagon"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. J. The cAMP concentration 10 minutes after glucagon stimulation (10 nM) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).",
    "molecules": "cAMP, IBMX, glucagon"
  },
  {
    "caption": "12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (A) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (200 µg/kg body weight, n = 10/group).",
    "molecules": "glucagon, glucose"
  },
  {
    "caption": "12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (B) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting (2 mg/kg body weight).",
    "molecules": "glucagon"
  },
  {
    "caption": "12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (C) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit (2 mg/kg body weight, n = 3/group).",
    "molecules": "glucagon"
  },
  {
    "caption": "12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (D) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit (2 mg/kg body weight, n = 3/group).",
    "molecules": "cAMP, glucagon"
  },
  {
    "caption": "Glucose disposal rates were measured in 12-week-old male mice using glucose, pyruvate (E) tolerance tests (n = 10 mice/group).",
    "molecules": "Glucose, glucose, pyruvate"
  },
  {
    "caption": "Glucose disposal rates were measured in 12-week-old male mice using insulin (F) tolerance tests (n = 10 mice/group).",
    "molecules": "Glucose, insulin"
  },
  {
    "caption": "G. Levels of p-AKT, AKT, p-ERK, ERK, IRβ and GAPDH were determined by immunoblotting after insulin stimulation (10 nM).",
    "molecules": "insulin"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (H) The nuclear localization of p-CREB after isoprenaline stimulation (10 μM) for 10 minutes was determined by immunofluorescence staining. Scale bar, 20 μm.",
    "molecules": "isoprenaline"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (I) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH after isoprenaline stimulation (10 μM) for 0, 10 or 20 minutes were determined by immunoblotting.",
    "molecules": "isoprenaline"
  },
  {
    "caption": "Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group).",
    "molecules": "cAMP, IBMX, isoprenaline"
  },
  {
    "caption": "WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (K) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after epinephrine stimulation",
    "molecules": "epinephrine, glucose"
  },
  {
    "caption": "WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (L) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting.",
    "molecules": "epinephrine"
  },
  {
    "caption": "M-O. WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (M) Blood glucose levels (mg/dL) were measured 0, 60 and 120 minutes after immobilization (n = 10/group).",
    "molecules": "glucose"
  },
  {
    "caption": "WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (O) Serum epinephrine level of mice after a 30 minutes-immobilization was determined (n = 3/control group, 7 or 8/immobilized group).",
    "molecules": "epinephrine"
  },
  {
    "caption": "A. The expression of Prom1, radixin, AKAP7, AKAP8, AKAP8I, AKAP9, AKAP10, AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs. Hepatocytes were then serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. The phosphorylation of PKA substrates was determined by immunoblotting.",
    "molecules": "glucagon"
  },
  {
    "caption": "Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (B) The nuclear localization of p-CREB after glucagon stimulation was determined by immunofluorescence staining. Blue; DAPI, Red; p-CREB. The percentage of cells with nuclear p-CREB staining among more than 300 cells in each group was also statistically analyzed (Figure EV3G). Scale bar, 20 μm.",
    "molecules": "DAPI, glucagon"
  },
  {
    "caption": "Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (C) Levels of p-PKA substrates, p-CREB, CREB, p-IP3R, IP3R, Prom1, radixin and GAPDH after glucagon stimulation (10 minutes) were determined by immunoblotting.",
    "molecules": "glucagon"
  },
  {
    "caption": "12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (E) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon administration (200 µg/kg body weight, n = 10 mice/ group).",
    "molecules": "glucagon, glucose"
  },
  {
    "caption": "12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (F) Levels of p-PKA, p-CREB, CREB, radixin and GAPDH in the liver after a 10-minutes glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting.",
    "molecules": "glucagon"
  },
  {
    "caption": "12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Glucose disposal rates in sh-Con or sh-Rdx (radixin) mice were measured using glucose, insulin and pyruvate tolerance tests (n = 10 mice/group).",
    "molecules": "glucagon, Glucose, glucose, insulin, pyruvate"
  },
  {
    "caption": "G. The cellular localization of radixin and actin in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes was determined by immunofluorescence staining for radixin and phalloidin staining. Scale bar, 20 μm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "I. Detergent-resistant lipid rafts were isolated from WT (Prom1+/+) and KO (Prom1-/-) mouse primary hepatocytes after cholesterol depletion by methyl-β-cyclodextrin (mβCD, 30 mM) for 30 minutes at 4 oC. Levels of the Prom1, radixin, flotillin and transferrin receptor proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting.",
    "molecules": "cholesterol, methyl-β-cyclodextrin, mβCD"
  },
  {
    "caption": "WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. the nuclear localization of p-CREB after glucagon stimulation for 10 minutes (E) was determined by immunofluorescence staining. Scale bar, 20 μm.",
    "molecules": "glucagon"
  },
  {
    "caption": "WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. (F) Ad-FERM-Myc- or Ad-LacZ-expressing WT hepatocytes were serum-starved for 16 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, FERM-Myc and GAPDH were determined by immunoblotting.",
    "molecules": "glucagon"
  },
  {
    "caption": "Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (0.2 mg/kg body weight, n = 10 mice/ group).",
    "molecules": "glucagon, glucose"
  },
  {
    "caption": "Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (H) Levels of p-CREB, CREB, p-PKA substrates, and FERM in the liver 10 minutes after glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting (n = 3 mice/ group).",
    "molecules": "glucagon"
  },
  {
    "caption": "Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (I) Glucose disposal rates in GFP- or FERM-overexpressing mice were measured using glucose, pyruvate and insulin tolerance tests (n = 10 mice/group).",
    "molecules": "glucagon, Glucose, glucose, insulin, pyruvate"
  },
  {
    "caption": "(B) Increasing amount of Ad-RdxR or Ad-LPTDR were infected to WT primary hepatocytes for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, radixin, Myc-radixin and GAPDH were determined by immunoblotting.",
    "molecules": "glucagon"
  },
  {
    "caption": "Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (C) DIO WT (Prom1+/+) and KO (Prom1-/-) mice were intraperitoneally injected with glucagon. Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon (100 µg/kg body weight) stimulation (n = 10 mice/ group).",
    "molecules": "glucagon, glucose"
  },
  {
    "caption": "Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (D) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH were determined by immunoblotting 10 minutes after glucagon (2 mg/kg body weight) stimulation (n = 3 mice/group).",
    "molecules": "glucagon"
  },
  {
    "caption": "Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (E) Glucose disposal rates in DIO WT (Prom1+/+) and KO (Prom1-/-) mice were measured using glucose and insulin tolerance tests (n = 10 mice/group).",
    "molecules": "Glucose, glucose, insulin"
  },
  {
    "caption": "Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (F) DIO KO (Prom1-/-) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours, fasted for 4 hours and intraperitoneally injected with glucagon (100 µg/kg body weight). Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (GST). Glucose disposal rates were measured using the pyruvate tolerance test (PTT) (n = 10 mice/group).",
    "molecules": "glucagon, glucose, Glucose, pyruvate"
  },
  {
    "caption": "(E)  Flow cytometry  analysis of Hela  cells treated with the indicated RNAi for 72h and immune-stained for γH2A.X, DNA staining  with propidium iodide. One of three biological replicates is represented.",
    "molecules": "propidium iodide"
  },
  {
    "caption": "(A) CUL4A  and CUL4B  were analyzed by immunoblotting  of extracts prepared from HeLa Kyoto cells arrested in S-phase (S= thymidine), G2   (R0-3306), or mitosis  (M= nocodazole  or taxol). One of three biological replicates is shown. H3  pS10 (phosphorylated histone H3 at serine 10) was used as a mitotic  marker. Same samples but separate blot  for H3pS10 staining  due to size differences.",
    "molecules": "nocodazole, taxol, R0-3306, thymidine"
  },
  {
    "caption": "(G) In vitro kinase assays  with purified CDK1-cyclinB  and Sf9-purified CUL4B  and mutant proteins in the presence of [γ-32P]ATP. The autoradiogram  (top panel) shows CUL4B phosphorylation, while coomassie staining (lower panel) controls equal loading of CUL4B  and mutants (N=3 technical replicates for WT, P50L; NP1; N=2 for NP1-A49T and NP1-A49T-P50L). Note that the A49T mutation in the NP1 mutant restores phosphorylation of the T49 residue, but only in the presence of the adjacent proline at position 50.",
    "molecules": "ATP, γ-32P"
  },
  {
    "caption": "(A)  Western blot  analysis to monitor CUL4A  or CUL4B  levels in extracts prepared from HeLaFLP  control cells or HeLaFLP  cells deleted for CUL4B by CRISPR/Cas9  (∆CUL4BFLP) but stably expressing no CUL4B  (-), CUL4B  or the indicated NP1 or P50L mutants from the doxycycline-inducible promoter. GAPDH  was included to control equal loading. One of three biological replicates is shown.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(F) In vitro E3  ligase activity  assays using the indicated reconstituted CRL4BCRBN complexes and the neo-substrate CKlα. At time  0, lenalidomide  was added to start the reaction and CKlα ubiquitination  was followed at the times  indicated (in minutes) by immunoblotting  with CKlα  antibody. N≥3 technical replicates for CUL4B, CUL4A, P50L, K895R and L864fs*13; N=1 for NP1. Note that the in vitro E3 ligase activity  of CRL4B  and the P50L patient  mutation is comparable.",
    "molecules": "lenalidomide"
  },
  {
    "caption": "(E) HA-immunoprecipitation  (IP) of extracts (input) prepared from unedited (-) or HA-CUL4B  endogenously expressing HeLa Kyoto  cell lines arrested in S-phase (thymidine) or mitosis  (nocodazole) were probed with antibodies against LIS1, WDR1  and DDB1. The antibody recognizing phospho-H3(S19) controls mitotic  arrest and specificity of the immunoprecipitation.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "(F, G)  HeLa Kyoto  endogenously expressing HA-CUL4B  or the indicated NP1 or P50L mutants were arrested in mitosis with nocodazole. Extracts (input) and HA-immunoprecipitants  were analyzed for the presence of HA-CUL4B  for control and bound DDB1  and LIS1  (F) or DDB1  and WDR1  (G), respectively. One of 3 biological replicates is shown.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(C, D) Western blot of in vitro pulldown  assays of Strep-tagged CUL4B  and FLAG-tagged LIS1  (C) or WDR1  (D) co-expressed in Sf9  cells in the presence (+) or absence (-) of DDB1. One of 3 biological replicates is shown. Note that LIS1  and WDR1  specifically bind CUL4B, but this interaction  is enhanced by the presence of DDB1.",
    "molecules": "Strep"
  },
  {
    "caption": "(G) FP  assay investigating binding  of CRL4B and CRL4A to Alexa-labeled WDR1",
    "molecules": "Alexa"
  },
  {
    "caption": "Fluorescence polarization  assay comparing the binding  of CRL4B and λ-PP-treated CRL4B to Alexa-labeled WDR1. The mean with SD of N=3 technical replicates is depicted. (L) Quantification of in vitro binding assays (Strep-pull down) of Strep-LIS1 with purified CRL4B, or the NP1 phospho-mutant (CRL4BNP1) (N=2 technical replicates), or λ-PP treated CRL4B (CRL4Bλ-PP) (N=1).",
    "molecules": "Alexa, Strep"
  },
  {
    "caption": "(C) Extracts (input) prepared from S-phase (S; thymidine) or mitosis  (M; nocodazole)-arrested  HeLa Kyoto  cells (-) and endogenously expressing HA-tagged WT CUL4B, or the NP1 or P50L mutants were immunoprecipitated  with HA.II-antibodies (IP). Bound ACTR3  or DDB1  was detected using specific antibodies. One of three biological replicates is shown. Note that in contrast to DDB1, ACTR3 binds CUL4B  in a phosphorylation-dependent manner.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "(D) HA-immunoprecipitants   (IP) of extracts (input) prepared from unedited (-) controls or endogenously expressing HA-CUL4B HeLa Kyoto  cells arrested in mitosis  with nocodazole  were probed with antibodies against DDB1, DCTN1  (dynactin) and actin. One of two biological replicates is depicted.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(F) Live cell imaging of HeLa  cells expressing H2B-mcherry   and lifeAct-GFP, treated for 72 h with control oligos (siControl) or RNAi-oligos depleting CUL4B, LIS1  or WDR1. Representative cells are shown in time  intervals of 6 min during metaphase with identical contrast settings. The unanchored spindle  in CUL4B- and LIS1  deleted cells is marked with a white arrow. Scale bar: 10 μm.",
    "molecules": "lifeAct"
  },
  {
    "caption": "A Time course images of the hippocampal CA1 region of Siglechdtr/dtr mice after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). Arrow: live microglia with normal nucleus. Double arrow: apoptotic microglia with pyknotic nucleus. Arrowhead: microglial debris with no nucleus. Scale bar, 50 µm. B Number of apoptotic microglia with pyknotic or fragmented nucleus (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). C Number of live microglia with normal nucleus (n = 5 animals per group, one-way ANOVA with post hoc Tukey's test). D Number of pieces of microglial debris (Iba1+ spheres with a diameter > 2 µm and no nucleus) (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). ",
    "molecules": "DAPI"
  },
  {
    "caption": "E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). A 3D image (rightmost panel) was reconstructed using Imaris software. Arrow: live microglia. Arrowhead: microglial debris. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A Histological analysis of RFP+ cells in Siglechdtr/dtr:Ccr2RFP/+ mice. Lumber spinal cord after EAE induction, and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed. Iba1 immunoreactivity (green) and RFP fluorescence (red) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for all RFP signals (red). Scale bar, 50 µm.",
    "molecules": "PBS"
  },
  {
    "caption": "B Flow cytometric analysis of RFP+Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr/dtr:Ccr2RFP/+ mice 2 days after PBS or DT administration. Representative data and a quantification graph (n = 5 animals per group, two-tailed unpaired Student's t-test) are shown.",
    "molecules": "PBS"
  },
  {
    "caption": "C Immunohistochemical localization of CD206+ perivascular macrophages (arrows) in hippocampal CA1 of Siglechdtr/dtr mice. Sections prepared 2 days after administration of PBS or DT were stained with anti-Iba1 (green), anti-CD206 (red) and anti-CD31 (blue) antibodies. Scale bar, 50 µm. D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals per group, two-tailed unpaired Student's t-test). ",
    "molecules": "PBS"
  },
  {
    "caption": "A qPCR analysis of marker molecules for CNS cell types. The hippocampus of WT and Siglechdtr/dtr mice 2 days after PBS or DT administration was analyzed (n = 3 animals per group, one-way ANOVA with post hoc Tukey's test). Results are normalized to Gapdh, and are shown as ratios to the value of WT mice injected with PBS.",
    "molecules": "PBS"
  },
  {
    "caption": "B Immunohistochemical detection of astrocytes in Siglechdtr/dtr mice. Hippocampal CA1 sections were prepared 2 days after PBS or DT administration, and stained with an anti-GFAP antibody. Images were acquired using the same laser power and sensitivity, and image processing was the same. Scale bar, 30 µm. C Astrocyte number in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals, two-tailed unpaired Student's t-test). ",
    "molecules": "PBS"
  },
  {
    "caption": "A Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1. Sections were prepared from Siglechdtr/dtr mice 2 days after PBS or DT administration, and stained with anti-GFAP (green) and anti-CD11b (red) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Scale bar, 10 µm.",
    "molecules": "PBS"
  },
  {
    "caption": "B CLEM analysis of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. A phagocytic astrocyte, which engulfed degenerated microglial components, was identified by immunohistochemistry using anti-S100β (green) and anti-CD11b (red) antibodies, and DAPI (cyan) (upper and middle panels). Then, a phagocytic cup of the same cell was analyzed by electron microscopy (lower panel). Degenerated microglial cytoplasm/plasma membrane (arrow) and nucleus (double arrow) in a phagocytic cup of the astrocyte, and a piece of microglial debris internalized in astrocytic cytoplasm (arrowhead) are indicated. Scale bar, 50 µm (upper panel), 10 µm (upper panel [inset] and middle panel), and 2 µm (lower panel).",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).",
    "molecules": "chloroquine"
  },
  {
    "caption": "(a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).",
    "molecules": "chloroquine"
  },
  {
    "caption": "(l,m) DHE and DAPI fluorescence at P28 in the DG (l) and SVZ (m) from FIP200GFAP cKO mice with or without NAC treatment, 2cKO mice and Trp53GFAP cKO mice. Dotted lines indicate the boundaries of the granular zone (GZ) (l) and SVZ (m). Arrows mark cells in the SGZ (l) and SVZ (m) and arrowheads mark cells in surrounding regions (the GZ in l and the striatum (ST) in m). n = 5 mice, ≥4 sections per mouse, >20 (h,j,k) or >200 (i) BrdU+ cells counted per mouse. cKO(n), NAC non-responder cKO mice; LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.",
    "molecules": "NAC"
  },
  {
    "caption": "(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ.",
    "molecules": "NAC"
  },
  {
    "caption": "(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28 mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown.",
    "molecules": "NAC"
  },
  {
    "caption": "(d-m) Immunofluorescence of the DG (d-h) and SVZ (i-m) of P28 mice treated by NAC or vehicle control. Representative images are shown in d,g (DG) and k,m (SVZ). Dotted lines indicate boundaries of the granular zone (GZ) (d,g) and SVZ (k,m). Boxed areas in g,k,m are shown in more detail in insets (g,m) and/or panels below (k,m). Lines indicate boundaries of the GZ (d,g) and SVZ (k,m). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,g), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (e,h), and NSCs (i,l), and GFAP+nestin− astrocytes (f,j) per section are shown. (n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28mice treated by NAC or vehicle control. n = 5 mice, ≥4 sections per mouse, >500 cells counted per NAC in n. cKO(n), NAC non-responder cKO mice; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. **P 0.01.",
    "molecules": "NAC"
  },
  {
    "caption": "(a-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28 mice treated with NAC or vehicle (Veh) control. Representative images are shown for PSA-NCAM (a) and NeuN (d). Dotted lines indicate boundaries of the DG (a,d), SVZ (a) and layers of the OB (d). Arrows mark PSA-NCAM+ cells in a. Mean ± s.e.m. of the number of PSA-NCAM+ cells in SVZ (b) and DG (normalized to 1 mm2 area) (c), and the number of NeuN+ cells in DG (e) and IGL of OB (per mm2) (f) per section are shown (n = 5 mice for each; ≥4 sections for each mouse). cKO(n), NAC non-responder cKO mice; GZ, granular zone; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. **P 0.01.",
    "molecules": "NAC"
  },
  {
    "caption": "A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments.",
    "molecules": "PBS, Ca2+, Fluo-4, Fluo-4 dye"
  },
  {
    "caption": "C Average MS1 intensity z-score kinetics for 217 predicted CaMKII-α sites that are shared between both cell lines. For comparison, average (n = 6) calcium abundance kinetics are shown.",
    "molecules": "calcium"
  },
  {
    "caption": "Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed. UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min (5 min for CaMKII). Ten μg protein from RIPA lysates were analysed by Western blot. Kinase marker substrate phosphorylations and corresponding loading controls are shown. For each kinase, two independent replicates were performed and are shown. Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band identity",
    "molecules": "PBS"
  },
  {
    "caption": " The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay. Scratches were made in UACC257 monolayers. Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0.1 μM or 1 μM in triplicates. A Example pictures showing the effect of EDN on migration. Cell free areas are delimited by blue lines. Migration was quantified as the change in cell free area between 0 h and 48 h. B The four inhibitors that were effective at 1 µM are shown in detail, i.e. every point corresponds to one well. C Abundance of E-Cadherin (normalized to GAPDH and Actin) was assessed by LC-SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM. Only the single inhibitor which could reverse EDN induced E-Cadherin repression is shown. * p < 0.05 two-sided unpaired t-test.  ",
    "molecules": "PBS, DMSO"
  },
  {
    "caption": "Solubility of Munc18-1. HEK293T cells transfected as in (C) were solubilized in 0.1% Triton X-100 (TX) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting. TX-soluble Munc18-1 was measured as percent of total Munc18-1 by quantitative immunoblotting. Data are means ± SEM (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Kruskal-Wallis test followed by Dunn's multiple comparison test, or by one-way ANOVA followed by Bonferroni post-hoc test; n = 5-9 independent experiments; exact n and p values are shown in Appendix Table S1).",
    "molecules": "Triton X-100, TX"
  },
  {
    "caption": "Same as in (A, B) except that compounds 9, 10, 11, 13, 14 and 16 were added at 0.25, 1, 5, 20, 100, or 250 µM. Note, that the data for 250 µM were not plotted in (D) because of causing neuronal death. Data are means ± SEM (*p < 0.05, **p < 0.01 by Kruskal-Wallis test with Dunn's multiple comparison test, or one-way ANOVA with Dunnett's post-hoc test; n = 6-15 independent experiments; exact n and p values are shown in Appendix Table S1).",
    "molecules": "compounds 9"
  },
  {
    "caption": "Limited proteolysis. Recombinant purified proteins were incubated with increasing concentrations of trypsin. Remaining protein levels were analyzed by quantitative immunoblotting. Data are means ± SEM (* p < 0.05, **p < 0.01, ****p < 0.0001, by 2-way ANOVA and Dunnett's multiple-comparison test; n = 4 independent experiments; exact p values are shown in Appendix Table S1).",
    "molecules": "trypsin"
  },
  {
    "caption": "Munc18-1 knockout neurons expressing G544D Munc18-1 were subjected to analysis of mean firing rate before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. 16 electrodes per well were analyzed for neuronal firing (A; purple boxes indicate network activity). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test, or Kruskal-Wallis test followed by Dunn's post-hoc test; n = 11-16 independent experiments; exact n and p values are shown in Appendix Table S1).",
    "molecules": "DMSO"
  },
  {
    "caption": "Uptake of synaptotagmin-1 antibody during high K+ stimulation. Neurons expressing cre recombinase and/or WT, R406H or G544D Munc18-1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay. Endocytosed synaptotagmin-1 antibody was quantified by immunostaining (A-C; scale bar = 30 µm; 4-PBA = 4-phenylbutyrate), via counting the number of pixels > intensity of 15 (D). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-10 independent experiments; exact n and p values are shown in Appendix Table S1).",
    "molecules": "4-PBA, 4-phenylbutyrate, K+"
  },
  {
    "caption": "Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 G544D (H) Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible.",
    "molecules": "compound 10"
  },
  {
    "caption": "Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta (N, O). Scale bar in (N) and (O) = 10 µm. 4-PBA = 4-phenylbutyrate.",
    "molecules": "4-PBA, 4-phenylbutyrate"
  },
  {
    "caption": "(B) Representative large image of a brain slice from R6/2 mice after 4-week striatal infusion of fluorescent cholesterol (bodipy-chol, green). In the inlet, infused striatum is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar: 1000 µm.",
    "molecules": "bodipy, chol, cholesterol, Ho, Hoechst"
  },
  {
    "caption": "(C) Cholesterol content measured by mass spectrometry in infused striatum of untreated wt (N=7) and R6/2 (N=4), R6/2 ACSF (N=5), R6/2 chol-low (N=5), R6/2 chol-medium (N=5), and R6/2 chol-high (N=5) mice at 12 weeks of age after 4-week striatal cholesterol infusion. The low, medium, and high doses respectively correspond to 15 µg, 185 µg, and 369 µg of cholesterol infused in the striatum after 4 weeks. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol, Cholesterol, cholesterol"
  },
  {
    "caption": "(D) Latency to fall (seconds) from an accelerating rotarod from 5 weeks (pre-surgery; i.e., before pump implantation) to 10 weeks of age in wt (N=23 at 5 weeks, N=28 at 8 and 10 weeks), R6/2 ACSF (N=35 at 5 weeks, N=31 at 8 weeks and N=30 at 10 weeks), R6/2 chol-low (N=22 at 5 weeks, N=14 at 8 weeks and N=13), R6/2 chol-medium (N=13 at 5 weeks, N=9 at 8 weeks and N=8 at 10 weeks), and R6/2 chol-high (N=19 at 5 weeks, N=17 at 8 and 10 weeks) mice. The graph shows means±standard error for each time point. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol"
  },
  {
    "caption": "Global motor activity (E), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol"
  },
  {
    "caption": "total distance traveled (F), mean velocity (G), stereotyped movements (H), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol"
  },
  {
    "caption": "number of rearings (I) in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol"
  },
  {
    "caption": "(J, K) Representative track plots (J) generated from the open-field test from wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=26 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=11 at 9 weeks, N=7 at 11 weeks), R6/2 chol-medium (N=11 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks) and relative quantification (K) of the time spent in the center and in the periphery (%) of the arena. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol"
  },
  {
    "caption": "(L) Discrimination index (DI; %) in the novel object recognition test at 6 weeks of age (before pump implantation) and 11 weeks of age (4 weeks after cholesterol infusion) of wt (N=16 at 9 weeks, N=21 at 11 weeks), R6/2 ACSF (N=28 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=21 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=10 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks). DI above zero indicates a preference for the novel object; DI below zero indicates a preference for the familiar object. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol, cholesterol"
  },
  {
    "caption": "(B) Spontaneous EPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of -70 mV.",
    "molecules": "chol"
  },
  {
    "caption": "(C-D) Average amplitude (C) and average frequency (D) of EPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol"
  },
  {
    "caption": "(E-G) Number of excitatory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (E) Representative tissue block of striatum (10 µm x 15 µm x 10 µm) used for the 3D analysis. (F) Representative FIB-SEM segmentation and reconstruction of excitatory synapses (yellow) in 200 serial sections of striatum for a total volume of 500 µm3, of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (G) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 animals/group). Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of , the number of synapses counted/µm3 in different blocks of striatal tissue (G) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "chol"
  },
  {
    "caption": "(A) Spontaneous IPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of 0 mV.",
    "molecules": "chol"
  },
  {
    "caption": "(B-C) Average amplitude (B) and average frequency (C) of IPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded (B, C) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).",
    "molecules": "chol"
  },
  {
    "caption": "(D-E) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (D) Representative FIB-SEM segmentation and reconstruction of inhibitory synapses (green) in 200 serial sections of striatum for a total volume of 500 µm3 in wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (E) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 mice/ group). Data information: The data E) are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of synapses counted /µm3 in different blocks of tissue for each group of animals (N=3 animals/group) (E). Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).",
    "molecules": "chol"
  },
  {
    "caption": "(B) 24S-OHC level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2‐ACSF, and R6/2 chol-high mice at 12 weeks of age after a 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.",
    "molecules": "24S-OHC, chol, cholesterol"
  },
  {
    "caption": "(C-E) Lanosterol (C), lathosterol (D), and desmosterol (E) level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2 ACSF, and R6/2 chol-high mice at 12 weeks of age after 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.",
    "molecules": "lathosterol, chol, cholesterol, desmosterol, Lanosterol"
  },
  {
    "caption": "(F-I) Nuclear (white arrows) and perinuclear (yellow arrows) localization of endogenous SREBP2 in the striatum of wt, R6/2 and R6/2-chol mice. Representative confocal image (F and H) and relative quantification (G and I) of SREBP2 (red) in wt and R6/2 mice and in the infused and contralateral striatum of R6/2 chol-high mice (N=4). Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar in F and H: 10 µm. Graphs in (G and I) represents the intensity of SREBP2 normalized on nuclei (%). Data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each image. Statistics: Student's t-test (**p<0.01; ****p<0.0001).",
    "molecules": "chol, Ho, Hoechst"
  },
  {
    "caption": "J, K Representative high-magnification confocal images of immunostaining against SREBP2 (red) and NeuN (J) or GFAP (K) (green) on coronal sections of brains from R6/2 chol-high mice. A cell in the infused striatum, positive for NeuN and GFAP respectively, is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bars: 1 µm.",
    "molecules": "chol, Ho, Hoechst"
  },
  {
    "caption": "Immunolabeling of muHTT aggregates (red) in R6/2 ACSF and R6/2 chol-high mice (N=3/group). Zoom of representative confocal images of immunostaining against muHTT aggregates (red) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum (A) and relative quantification of number (B) and size (C) of aggregates. Hoechst (Ho, blue) was used to counterstain nuclei. 18 images/animal were analyzed from 9 sections throughout the entire striatum. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 10 µm (A) Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).",
    "molecules": "chol, Ho, Hoechst"
  },
  {
    "caption": "Representative confocal images of immunostaining against muHTT (red) and DARPP32 (D) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification (E Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).",
    "molecules": "chol, Ho, Hoechst"
  },
  {
    "caption": "Representative confocal images of immunostaining against muHTT (red) and GFAP (F) (green) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification G). Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).",
    "molecules": "chol, Ho, Hoechst"
  },
  {
    "caption": "(H) TEM images of EM48 pre-embedding immunogold labelling showing muHTT aggregates in the striatal neuron cell bodies of R6/2 ACSF mice and R6/2 chol-high mice (upper panels). In the lower panels, muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images. Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus, and arrowheads indicate single 10-nm gold particles in MSN. Nucleus (N), nucleolus (n), mitochondrion (m), rough endoplasmic reticulum (rer), Golgi apparatus (g), and white arrows indicate the nuclear envelope (N=3 animals/group). Scale bars: 700 nm and 300 nm.",
    "molecules": "chol, gold particles"
  },
  {
    "caption": "Quantification of aggregated and total muHTT in the infused striatum of HD mice after 4-week cholesterol infusion by TR-FRET analysis using different antibody pairs. Preliminary assessment of the sustainability of the assay in wt and R6/2 striata (N=5/group) using 4C9-4C9 and 2B7-MW1 antibodies in combination to detect, respectively, muHTT aggregates (J) and total muHTT (K). Quantification of muHTT aggregates (L) and soluble and other muHTT species (M) in the infused striata of R6/2 ACSF and R6/2 chol-high mice. Data in (J-M) are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from one striatum. Statistics: student's t-test (*p<0.05).",
    "molecules": "chol, cholesterol"
  },
  {
    "caption": "Representative confocal images showing p62 (A, red) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of dots for p62 (B) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in (B are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in (A 10 µm.",
    "molecules": "chol, Ho, Hoechst"
  },
  {
    "caption": "Representative confocal images showing LAMP1 (C, green) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of density for LAMP1 (D) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in D) are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in C): 10 µm.",
    "molecules": "chol, Ho, Hoechst"
  },
  {
    "caption": "(A, B) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles (Dataset EV1). A, all mitochondrial proteins detected. Grey area indicates proteins that changed less than 2-fold in both directions. Subunits of selected mitochondrial machineries are highligted as follows: magenta, pyruate dehydrogenase complex (PDH); blue, mitochondial carriers (Carrier); brown, translocase of the outer membrane (TOM); yellow, translocase of the inner membrane (TIM); green, mitochondrial contact site and cristae organizing system (MICOS); yellow-green, mitochondrial fusion-fission system (Dynamics); red, porin or voltage dependent anion channel (VDAC); B, subunits of OXPHOS complexes (Complex I-V) and mitoribosomes (Ribosome). Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex. For the full gene name, add NDU (complex I), SDH (complex II), UQC (R10), COX (complex IV), ATP (5L, 5J2), MRP (Ribosome) in front of the abbreviated gene names given in the figure.",
    "molecules": "ATP"
  },
  {
    "caption": "(A-F) OXPHOS complexes (A-C) and selected enzymes involved in the energy metabolism of control (143B, solid lines) and ρ0 cells (dashed lines). A, Complex (C) I, III and IV; B, F1, FO domain and inhibitor protein IF1 of complex V; C, subunits SDHA, SDHB-D and assembly factor SDHAF2 of complex II; D, oxoglutarate (OGDH; average of OGDHL, OGDH, DLST) and pyruvate dehydrogenase complex (PDH; average of PDHA1, PDHB, PDHX, DLAT); E, subunits of GDP forming (SUCLG2) and ADP forming (SUCLA2) succinyl-CoA ligase; F, urea cycle enzymes and glutaminase (GLS). Plots and gene names are in corresponding colors.",
    "molecules": "ADP, GDP"
  },
  {
    "caption": "(A-D) Selected metabolite carriers of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, isoforms of the nucleotide carrier (ANT1-3) and phosphate carrier (PiC); B, pyruvate carrier; C, dicarboxylate carriers; D, mitoferrins (MFRN1-2) and glycine transporter (GlyC). Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01 (Student's t-test).",
    "molecules": "dicarboxylate, pyruvate"
  },
  {
    "caption": "C Hematoxylin-eosin staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm) from Cdk4+/+ and Cdk4-/- mice.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "E Hematoxylin-eosin (H&E) staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm (upper panel) and 50 μm (lower panel)) from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "C Hematoxylin-eosin staining in scWAT showing multilocular adipocytes in Cdk4-/- (scale bar 50 μm).",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "E-F Acute cold test (4°C) after treatment with the β-adrenergic antagonist propranolol (Prop) in Cdk4+/+ (n=7) and Cdk4-/- (n=6) animals or with vehicle (NaCl) (Cdk4+/+ (n=7) and Cdk4-/- (n=6)) (E) and corresponding quantification of the area under the curve (AUC).",
    "molecules": "Prop, propranolol, NaCl"
  },
  {
    "caption": "D Hematoxylin-eosin (H&E) staining of iBAT sections (scale bar 100 μm) from Cdk4flox/flox and Cdk4flox/flox Sf1-Cre mice.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "A-D Confocal illustrations of frontal hypothalamic sections from Cdk4+/+ mice (A, C, N=3) and Cdk4-/- mice (B, D, N=3). Low magnification showing nuclei (Hoechst, left pannel) and c-Fos activated-cells (white, right pannel) in the mPOA (A1-2, B1-2) and the ARC, the VMH, the DMH, the LH (C1-2, D1-2). High magnifications corresponding to the frame area in A1-2, B1-2, C2, D2 and showing c-Fos-IR cells (A3-4, B3-4, C3-6, D3-6). Scale bars (A1-2, B1-2, C1-2, D1-2): 200μm, (A3-4, B3-4, C3-4, D3-4): 50μm. E Quantification of c-Fos IR cells in brain sections of Cdk4+/+ and Cdk4-/- mice (n=15-24 slices according hypothalamic nucleus and genotype). ",
    "molecules": "Hoechst"
  },
  {
    "caption": "(B) E. coli and B. subtilis abundances in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG at 6 hr.",
    "molecules": "IPTG"
  },
  {
    "caption": "Time-series measurements of extracellular plasmid or E. coli gDNA concentrations in the co-culture in the presence of (D) 0.02 mM, (E) 0.2 mM or (F) 2 mM IPTG.",
    "molecules": "IPTG"
  },
  {
    "caption": "(H) Abundance of transformants at 6 hr in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG.",
    "molecules": "IPTG"
  },
  {
    "caption": "Time-series measurements of (E) the abundance of E. coli MG1655-rfp, (F) the abundance of engineered B. subtilis Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol were statistically different. Stars (*) indicate p-value of 0.0237 and 0.0283 for 4 hr and 8 hr, respectively.",
    "molecules": "chloramphenicol"
  },
  {
    "caption": "Time-series measurements of (G) extracellular pBB275 plasmid concentration, Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol",
    "molecules": "chloramphenicol"
  },
  {
    "caption": "Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Representative two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively.",
    "molecules": "FAM"
  },
  {
    "caption": "Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM.",
    "molecules": "Tepotinib"
  },
  {
    "caption": "Relative volume of tumours grown in Rag2-/- animals after treatment with Tepotinib or vehicle control. One-way ANOVA, Tukey's multiple comparison. Ns: not significant; ** p<0.024. Mean +/- SEM. A total of 19 Rag2-/- mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls. For tumours with no cMet, 13 Rag2-/- mice were treated with Tepotinib and 9 were used as control.",
    "molecules": "Tepotinib"
  },
  {
    "caption": "Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.",
    "molecules": "Tepotinib"
  },
  {
    "caption": "Quantification of pH3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.",
    "molecules": "Tepotinib"
  },
  {
    "caption": "Quantification of caspase 3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.",
    "molecules": "Tepotinib"
  },
  {
    "caption": "MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells (B-D) Data at 1 h (C), 5 h (D are presented as mean ± SEM; n = 4 (B-D) biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 by paired T-test (C), or one-way ANOVA followed by Dunnett's multiple comparison test (D",
    "molecules": "antimycin A, FCCP, TMRE"
  },
  {
    "caption": "LDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 4 (G) , biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test.",
    "molecules": "decylQ, antimycin A, FCCP, mitoQ"
  },
  {
    "caption": "LDH release was measured after cold stress for 24 h. BAPTA-AM (10 µM) was used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 3, biological replicates, ****p < 0.0001, by unpaired T-test.",
    "molecules": "BAPTA-AM"
  },
  {
    "caption": " (C) HEK293T cells were transfected with pcDNA3.1 encoding FLAG-TIP60WT, FLAG- TIP60S90A or empty vector (vec). The samples were subjected to FLAG affinity-purification, dephosphorylated with shrimp alkaline phosphatase (rSAP) and incubated as indicated with CDK9/cyclinT1 in presence of ATP. Phosphorylation of TIP60 was analysed by a phosphoS90-specific TIP60 antibody, and the blot was further probed with antibodies specific for FLAG and CDK9 ",
    "molecules": "ATP"
  },
  {
    "caption": " (D) p53-/- hRasG12V MEF were treated with DMSO or 1µM SNS-032 (SNS) for 1h. Nuclear lysates were analysed by Western blotting ",
    "molecules": "SNS-032, SNS), DMSO"
  },
  {
    "caption": " (E) p53-/- hRasG12V MEF were treated with DMSO, 1µM SNS-032 (SNS) or 50µM DRB followed by addition of 100nM Calyculin A as indicated. Nuclear lysates were analysed by Western blotting ",
    "molecules": "DRB, SNS, SNS-032, Calyculin A, DMSO"
  },
  {
    "caption": " (E) U2OS cells stably expressing FLAG-tagged TIP60WT, TIP60S90A or empty vector (vec) were treated with 2 µM SNS-032 (SNS) for 3 hours as indicated. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting",
    "molecules": "SNS, SNS-032"
  },
  {
    "caption": " (D) U2OS cells stably expressing FLAG-TIP60WT or empty vector were treated with 2µM SNS-032 (SNS) for 2 hours as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. This material and input lysate were subjected to Western blot analysis ",
    "molecules": "SNS, SNS-032"
  },
  {
    "caption": " (A) U2OS cells stably expressing FLAG-TIP60WT, FLAG-TIP60S90A or empty vector (vec) were treated with 2µM SNS-032 (SNS), 2µM JQ1 (JQ1) or the solvent DMSO (d.) for 2h as indicated. FLAG-immunoprecipitations were performed from nuclear extracts. Immunoprecipitated material and input lysate were subjected to Western blot analysis",
    "molecules": "JQ1, SNS, SNS-032"
  },
  {
    "caption": " (B) U2OS cells stably expressing FLAG-TIP60WT were treated with 2µM JQ1 (JQ1) for 2h. The nucleus was fractionated into nucleoplasm and chromatin. Both fractions were analysed by Western blotting ",
    "molecules": "JQ1"
  },
  {
    "caption": "(E) Characterization of dTAG-induced destruction and recovery. STAT1-EGFP-dTAG expressing cells were induced with IFNγ for 24h, then treated with dTAG13 (100 nM) for DMSO vehicle control (I) or STAT1 depletion (II) for 24h. To determine recovery of STAT1 expression, cells were washed three times with medium and harvested 48h after IFNγ washout (120 hour timepoint, lane 4). In Parallel, (III) cells were treated with dTAG13 (100 nM) for 48h during 24h induction with IFNγ. Immunoblot of STAT1-GFP-dTAG at indicated treatments confirm STAT1 depletion and recovery, respectively. α-Tubulin (Tubulin) was used as a loading control.",
    "molecules": "DMSO, dTAG13"
  },
  {
    "caption": "(A) Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid or nitrogen starvation (‐N) medium supplemented with indicated carbon sources. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐GFP and α‐Cdc11 antibodies. Quantification of ATG8 induction is shown in the lower left panel. Total GFP signals (GFP‐Atg8 and free GFP) were quantified and normalized to Cdc11 signals. Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation. Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals (GFP‐Atg8 and free GFP) after 6 h starvation. The means and s.d. of four (n=4) independent experiments are indicated.",
    "molecules": "amino‐acid, nitrogen"
  },
  {
    "caption": "(B) Fluorescence microscopical analysis. Wild‐type, rho0, and Δatg7 cells expressing prATG8‐GFP‐ATG8 were grown as described in (A) and exposed to amino‐acid (left panel, galactose) or nitrogen starvation (right panel, glucose (‐N)) for 6 h. Vacuoles were visualized by over night FM4‐64 (1 μM) staining (red). Arrowhead indicates a punctate GFP‐Atg8 structure. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Cellular localization of GFP signal was analysed in at least 150 cells (n⩾150) for each strain and condition. Scale bars represent 1.5 μm.",
    "molecules": "amino‐acid, galactose, glucose, nitrogen"
  },
  {
    "caption": "Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation. Wild‐type, rho0, atg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: cbs1), complex IV (CIV: mss51, pet111, pet122), or the F1Fo‐ATP synthase (complex V; CV: atp10) were exposed to amino‐acidstarvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. All strains expressed prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.",
    "molecules": "acetate, amino‐acid, antimycin A, galactose, oligomycin"
  },
  {
    "caption": "(A, B) Wild‐type, rho0, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. ATP and protein from total cells (upper panels) or isolated mitochondria (lower panels) were determined at indicated time points.",
    "molecules": "acetate, amino‐acid, antimycin A, ATP, galactose, oligomycin"
  },
  {
    "caption": "(C) Wild‐type and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate or galactose for 3 h. Wild‐type cells were exposed to antimycin A (AA), oligomycin (O), or CCCP (50 μM) during the amino‐acid starvation period when indicated. Cells were treated with the mitochondrial membrane potential‐dependent dye DiOC6(3) and examined by fluorescence microscopy. Average pixel intensities for at least 10 mitochondrial tubules (n=10) in 5 representative cells were determined for each condition. Values for wild‐type mitochondria in the presence of acetate were defined as 100% and relative values were calculated accordingly. The means and s.d. are shown.",
    "molecules": "acetate, amino‐acid, antimycin A, CCCP, galactose, oligomycin"
  },
  {
    "caption": "(A) Wild‐type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8‐GFP‐ATG8 (upper panel) or prATG8‐GFP (lower panel) were exposed to amino‐acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated.",
    "molecules": "amino‐acid, galactose, rapamycin"
  },
  {
    "caption": "(B) Wild‐type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1‐NPR1‐HA were exposed to amino‐acid starvation medium supplemented with galactose in the absence (upper panels) or presence (lower panels) of rapamycin. The hyperphosphorylated (Npr1‐P) and dephosphorylated (Npr1) forms of Npr1 are indicated. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐HA and α‐Cdc11 antibodies.",
    "molecules": "amino‐acid, galactose, rapamycin"
  },
  {
    "caption": "(C) Wild‐type, rho0, Δnpr2, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with galactose. Additionally, wild‐type cells were treated with rapamycin during starvation (left panel). Phosphorylated (Atg13‐P) and dephosphorylated (Atg13) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α‐Atg13 and α‐Cdc11 antibodies.",
    "molecules": "amino‐acid, galactose, rapamycin"
  },
  {
    "caption": "(A) PKA‐dependent regulation of the autophagic response under amino‐acid starvation. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose. PKA activity in pka was inhibited by addition of 1NM‐PP1 (PP1; 1 μg/ml). Samples were analysed as described in Figure 1A; autophagic flux was determined after 3 and 6 h.",
    "molecules": "1NM‐PP1, PP1, amino‐acid, galactose"
  },
  {
    "caption": "(B)In vivo activity of PKA. Wild‐type, rho0, pka, and ras2G19V‐expressing cells harbouring 6xMYC‐cki12−200(S125/130A) (Cki1) were grown in galactose medium. When indicated, wild‐type cells were grown in galactose medium in the presence of antimycin A (AA) or oligomycin (O) for 6 h. PKA‐dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α‐Myc antibody (upper panels). Ratio of phosphorylated (Cki1‐P) and non‐phosphorylated (Cki1) forms of Cki1 relative to wild‐type cells (wt=1) (lower panels).",
    "molecules": "antimycin A, galactose, oligomycin"
  },
  {
    "caption": "(B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 and prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed to amino‐acid starvation medium supplemented with galactose for 3 h. Wild‐type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP‐Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm.",
    "molecules": "amino‐acid, antimycin A, galactose, oligomycin"
  },
  {
    "caption": "(C) Steady‐state levels of Atg1‐ and Atg13‐mCherry during amino‐acid starvation. Wild‐type, rho0, and Δatg7 cells expressing prATG1‐ATG1‐mCherry (upper panels) or prATG13‐ATG13‐mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α‐dsRed and α‐Cdc11 antibodies.",
    "molecules": "amino‐acid"
  },
  {
    "caption": "B, C Gene expression was measured by Q-RT-PCR after 72 h for MDA-MB-231 (B) and HMLER cells (C) treated daily with DAC (10 µM). PBGD expression level was used as an internal control. Data are expressed as mean ± s.e.m. of at least 3 independent experiments. **** P<0.0001, two-tailed unpaired Student's t-test.",
    "molecules": "DAC"
  },
  {
    "caption": "A, B Cells were treated with DAC (10 μM, 72 h), and/or net1-mAb (10 μg/ml, 48 h) and/or recombinant netrin-1 (5 μg/ml, 48 h). TUNEL assays from 3 independent experiments. *** P<0.0001 one-way ANOVA.",
    "molecules": "DAC"
  },
  {
    "caption": "D Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated tumors, compared with PBS treated tumors. >1700 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.",
    "molecules": "decitabine"
  },
  {
    "caption": "G, H When tumors reached 100 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). (G) Tumor volumes were measured twice a week. The statistical significance of the differences obtained between the control PBS-group and treated (DAC + net1-mAb) group was determined by two-way ANOVA and post-hoc Tukey-test. *** P<0.0001. (H) Tumor weights were measured 3 days after the end of the experiment. The statistical significance of the differences obtained between the groups was determined by one-way ANOVA, *** P<0.0001. Error bars = s.e.m.",
    "molecules": "DAC, decitabine"
  },
  {
    "caption": "A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors, compared with the control PBS-group. The percentage of mean DNA methylation of the 11 DAPK1-CpGs was 94% (550 amplicons analyzed), while that of the 7 NTN1-CpGs was 64% (213 amplicons analyzed). Two-way ANOVA and post-hoc Tukey-test.",
    "molecules": "decitabine"
  },
  {
    "caption": "B Expression of DAPK1, UNC5B, and NTN1 was measured by Q-RT-PCR in PDX tumors after 7 days of in vivo DAC treatment (0.4 mg/kg). The level of PBGD expression was used as an internal control. Data are expressed as mean ± s.e.m. for at least 3 grafts per group. **** P<0.0001,, two-way ANOVA and post-hoc Tukey-test.",
    "molecules": "DAC"
  },
  {
    "caption": "F The combination of net1-mAb and DAC reduces humanbreasttumor growth in immuno-compromised mice. After anesthesia, mice were engrafted in the interscapular area with a ≈ 60 mm3 patient-derived tumor. When the tumors reached 120-150 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg) or with a humanIgG1 control isotype antibody (Ctrl IgG1, 10 mg/kg) from day 1 to day 21. Tumor volumes were measured twice a week; n = 7 mice per group. The statistical significance of the differences obtained between the DAC + Ctrl IgG1-group and the DAC + net1-mAb-group was determined by two-way ANOVA2 and a post-hoc Tukey-test, **** P<0.0001. Error bars = s.e.m.",
    "molecules": "DAC, decitabine"
  },
  {
    "caption": "A, D Stably transfected MDA-MB-231 cells bearing a control (A), DAPK1 (B), UNC5B (C), or NTN1 shRNA (D) were injected into the mammary fat pad of immuno-compromised mice. When tumors reached 100-120 mm3, mice were injected subcutaneously with DAC (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). Tumor volumes were measured twice a week, n = 8 mice per group. The statistical significance of the differences obtained between DAC-group and DAC + net1-mAb-group for shControl, DAC-group and DAC + net1-mAb-group for shDAPK1 and shUNC5B, and PBS-group and DAC-group for shNTN1, respectively, were determined by two-way ANOVA and a post-hoc Tukey-test. **** P<0.0001, , ns = not significant. Error bars = s.e.m.",
    "molecules": "DAC"
  },
  {
    "caption": "A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 24, 48 and 72 hours and imaged after DNA staining with Hoechst. Green arrowheads indicate examples of prometaphase arrested cells, and the red arrowheads indicate examples of dead cells due to Plk1 downregulation. Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments. Scale bar, 20 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "B) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. C) Quantification of nuclear size measurements from Fig 2B. Data derived from 3 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.",
    "molecules": "Hoechst"
  },
  {
    "caption": "D) Solid phase transfection of nontargeting (scrambled), GOLGA2 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Golga2 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. E) Quantification of experiments in Fig 2D. Data derived from 3 independent experiments. P value (scrambled vs GOLGA2) < 2e-16, Mann-Whitney U test. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "F) Solid phase transfection of nontargeting (scrambled), MKI67 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. G) Quantification of experiments in Fig 2F. Data derived from 3 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. Scale bar, 50μm. B) Quantification of experiments in Fig 3A. Data derived from 2 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "C) Solid phase transfection of nontargeting (scrambled), MKI67 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. D) Quantification of experiments in Fig 3C. Data derived from 2 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "(I) Representative image showing changes in  colon morphology and  length following  DSS-induced colitis with concurrent treatment with  MBELNs. (J) Column graph showing changes in colon length. Data are mean ± SEM from seven biological replicates, **P<0.01 using one-way ANOVA. (",
    "molecules": "MBELNs, DSS"
  },
  {
    "caption": "Hematoxylin and eosin  (HE)  staining to show histological  changes in  colon  tissue Scale bar 200 μm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "(A)  Mass spectrometry (MS) analysis of aryl hydrocarbon receptor  (AhR)  pulldown protein from  Mulberry bark derived exosome-like nanoparticles (MBELNs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated MC38 cell lysate. Data from three technical replicates are shown.",
    "molecules": "MBELNs, Mulberry bark derived exosome-like nanoparticles, 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD"
  },
  {
    "caption": "(D) Representative images showing effect of MBELNs  with and without TCDD (1 nm)/ carbobenzoxy-Leu-Leu-leucinal (MG132; 20 µM) on co-expression of AhR and  COPS8 using immunofluorescence. Scale bar 10 μm, images are from three biological replicates.",
    "molecules": "MBELNs, TCDD, carbobenzoxy-Leu-Leu-leucinal, MG132"
  },
  {
    "caption": "(E)  Western blot showing  MBELNs, TCDD  and MG132 effect on co-expression of AhR and COPS8. The data represent as mean ± SEM from three biological replicates **P<0.01, ***P<0.001 using one-way ANOVA.",
    "molecules": "MBELNs, TCDD, MG132"
  },
  {
    "caption": "(F) Heat map showing  MS analysis of AhR pulldown  protein from MC38 cells treated with MBELNs in combination with proteasome inhibitor, MG132. Data are represented from three technical replicates.",
    "molecules": "MBELNs, MG132"
  },
  {
    "caption": "Mice  were treated by 2% dextran sodium sulfate  (DSS) in drinking water for 7 days. (F) Representative  hematoxylin and eosin (HE)  stained sections of colon  and cecum from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 9). Scale bar 100μm, representative data from five biological replicates per genotype. (G) Histopathologically scored sections of distal colon; cecum and colon length were analyzed from COPS8fl/fl and COPS8ΔIEC mice after  DSS-indcued colitis (day 7). Data are represented as mean ± SEM from five biological replicates per genotype. **P<0.01 using Student's t-test. (",
    "molecules": "dextran sodium sulfate, DSS, eosin, hematoxylin"
  },
  {
    "caption": "Mice  were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (H) Cytokine levels (interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)) in the  colon  collected on day 7 in colitis induced COPS8fl/fl and COPS8ΔIEC mice. Data are represented as mean ± SEM from five biological replicates per genotype. *P<0.05, **P<0.01, NS- non-significant using Student's t-test.",
    "molecules": "dextran sodium sulfate, DSS"
  },
  {
    "caption": "Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days. (I) The frequency of CD11b+Ly6C+ and CD11b+Ly6G+ cells in colonic lamina propria  (cLP) of COPS8fl/fl and or COPS8ΔIEC mice  with DSS-induced colitis. The column graph represents percentage of Ly6C+ and Ly6G+ cells, presented as mean ± SEM from three biological replicates per genotype. *P<0.05, NS- non-significant using Student's t-test. (J) Representative  FACS plots and percentage of intracellular staining of  Forkhead box protein P3 (FOXP3), Interferon gamma (IFN-γ) and IL-17A in CD3+CD4+  T cells from  colonic lamina propria  of COPS8fl/fl or COPS8ΔIEC mice with DSS-induced  colitis. The column graph represents percentage of Treg+ and ratio of Th1/Th17 cells, represented as mean ± SEM from three biological replicates per genotype. *P<0.05 using Student's t-test.",
    "molecules": "dextran sodium sulfate, DSS"
  },
  {
    "caption": "(I) Real-time quantitative reverse transcription polymerase chain reaction  (qRT-PCR) analysis of the expression of genes encoding inflammatory cytokines in the ileum. COPS8fl/fl and COPS8ΔIEC mice were either untreated (phosphate buffered saline (PBS)) or treated with the antibiotic cocktail (Abx) for 3 months. Data are mean ± SEM from three biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.",
    "molecules": "Abx, antibiotic, PBS, phosphate buffered saline"
  },
  {
    "caption": "(B) Representative image showing changes in  colon morphology and length following DSS-induced  colitis  with concurrent treatment with MBELNs COPS8fl/fl and COPS8ΔIEC mice. Representative data from seven biological replicates per genotype.",
    "molecules": "MBELNs, DSS"
  },
  {
    "caption": "Hematoxylin and eosin staining (HE) to show  histological  changes Scale bar 200μm",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "(F)  Expression of anti-microbial peptide (AMPs) in the  colon  of COPS8fl/fl and  COPS8ΔIEC mice  while being treated with  MBELNs by oral administration. Data are mean ± SEM from three biological replicates per group. *P<0.05, **P<0.01, NS- non-significant using one-way ANOVA.",
    "molecules": "MBELNs"
  },
  {
    "caption": "(I-J) Transcriptional expression of  nuclear factor kappa B (NF-κB) (I) and inhibitor of nuclear factor kappa-β kinase (IKK-β) (J) mRNA using Real-time quantitative reverse transcription Polymerase chain reaction (qRT-PCR) in colonic epithelial cells upon treatment with MBELNs  in addition to  DSS. Data are mean ± SEM from three biological replicates per group. **P<0.01, NS- non-significant using one-way ANOVA.",
    "molecules": "MBELNs, DSS"
  },
  {
    "caption": "(A)  Western blots showing expression  of AhR in MC38  cells induced by different fractions (protein, lipid and   RNA) derived from Mulberry bark derived exosome-like nanoparticles (MBELNs). Data shown are from three biological replicates.",
    "molecules": "MBELNs, Mulberry bark derived exosome-like nanoparticles, lipid, protein, RNA"
  },
  {
    "caption": "(B) The effect of different size protein fractions of mulberry bark derived exosome-like nanoparticles (MBELNs) on the expression of aryl hydrocarbon receptor  (AhR) demonstrated in  Western blots. Data shown are from three biological replicates.",
    "molecules": "MBELNs, mulberry bark derived exosome-like nanoparticles"
  },
  {
    "caption": "Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA.",
    "molecules": "alphoid DNA"
  },
  {
    "caption": "The relative expression of TERC was measured by RT-qPCR from total RNA of HeLaS3 and AGO2KO cells. HPRT1 was used as internal control (n = 3 experimental replicates). qPCR of 3'-extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers. HPRT1 was used for normalization (n=3 experimental replicates). qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo(dT)10 priming. HPRT1 was used for normalization (n=3 experimental replicates).",
    "molecules": "cDNA, RNA"
  },
  {
    "caption": "HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT-qPCR . 7SK RNA was used for normalization (n=4 experimental replicates).",
    "molecules": "7SK RNA, RNA"
  },
  {
    "caption": "RIP assay was performed from HeLaS3_FH AGO2 and HeLaS3_GFP cell extract using anti-HA antibody or IgG, as negative control, followed by TERC detection by RT-qPCR. 7SK RNA was used for normalization (n=3 experimental replicates).",
    "molecules": "7SK RNA"
  },
  {
    "caption": "HeLaS3 cells were transduced with a lentiviral vector coding for wild-type TERC (HeLaS3_TERCwt), TERC mutated in position 313-340 (HeLaS3_TERC (313-340mut)) or TERC delated in position 12-31 (HeLaS3_TERC (Δ 12-31)). Whole-cell lysates from HeLaS3_TERCwt, HeLaS3_TERC(313-340mut) and HeLaS3_TERC (Δ 12-31) was immunoprecipitated using an anti-AGO2 antibody in order to assessed the impact of TERC mutations on AGO2 binding. For each experiment the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples. Association of AGO2 with TERC(313-340mut and TERC (Δ 12-31) was compared to the association with TERCwt (n=3 experimental replicates).",
    "molecules": "7SK RNA"
  },
  {
    "caption": "C. Biotinylation of the centrosome and microtubules by V5-BirA*-AURKA  in different cell cycle stages. After 18 h biotin incubation, HEK293T::V5-BirA*-AURKA were fixed and stained for the fusion protein with V5 antibody,  biotinylated  proteins with the fluorescent streptavidin and the centrosome with gamma-tubulin  antibody. DNA  was  stained with DAPI. Scale bar, 5 μm.",
    "molecules": "biotin, DAPI, streptavidin"
  },
  {
    "caption": "D. Control and  biotin-treated  HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated  by   streptavidin beads. The initial sample (initial) and captured biotinylated  proteins (pulldown) were run on an SDS gel and  immunoblotted with the fluorescent streptavidin and antibodies against  V5 and vinculin (loading control).",
    "molecules": "biotin, SDS, streptavidin"
  },
  {
    "caption": "C. AURKA interacts  with the centriolar satellite  proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3 and  CP110. HEK293T cells were transiently transfected with  FLAG-BirA* or FLAG-BirA*-AURKA. Following 18 h biotin incubation, cells were lysed, and biotinylated  proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin  and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4 and SSX2IP.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "F. AURKA co-localize with PCM1  at the  peripheral  satellite clusters upon chemical dimerization of satellites with Kif5b motor domain.  U2OS  cells were co-transfected with  GFP-PCM1-FKBP and HA-Kif5b(1-269 a.a.)-FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours. Cells that were not treated with rapamycin  were processed in parallel as a control. Fixed cells were  stained  with antibodies against GFP, AURKA and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 μm. G. Quantification of  pericentrosomal levels of AURKA and gamma tubulin for F. n>25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****p < 0.0001; **p < 0.01; Unpaired Student's t-test.). ",
    "molecules": "DAPI, rapamycin"
  },
  {
    "caption": "D. Cycloheximide chase experiment for quantification of AURKA half-life. Cells were transfected with control or PCM1 siRNA#1 for 48 h, then treated with 200 nM cycloheximide along with serum starvation for indicated time points. AURKA intensities were quantified by immunoblotting for AURKA and vinculin  (loading control) and AURKA levels were normalized to vinculin levels. Data represent mean value from three experiments per condition ± SEM (**p < 0.01, Unpaired Student's t-test)",
    "molecules": "Cycloheximide, cycloheximide"
  },
  {
    "caption": "A. Representative  immunofluorescence images of  cilium assembly experiments in control and PCM1-depleted RPE1  cells treated with DMSO  or MLN8237. Cells were transfected with control or PCM1  siRNA#1 for 48 h and treated with  DMSO (vehicle control) or 0.5 μM MLN8237  in  serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin  antibody. DNA was stained  with DAPI. Scale bar, 10 μm. B. Quantification of  ciliogenesis  efficiency for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; **p < 0.01; ns, non-significant, Unpaired Student's t-test.). C. Quantification of cilium length for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (****p < 0.0001; ns, non-significant, Unpaired Student's t-test.). ",
    "molecules": "MLN8237, DAPI, DMSO"
  },
  {
    "caption": "D. Representative  immunofluorescence images of cilium assembly experiments in control and PCM1-depleted  RPE1  cells treated with DMSO or tubacin. Cells were transfected with control or  PCM1  siRNA#1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h. Cells were fixed and immunostained for the  primary cilium  with Arl13b  and acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with  DAPI. Scale bar, 10 μm E. Quantification of ciliogenesis efficiency for D. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (*p < 0.05; ns, non-significant, Unpaired Student's t-test). ",
    "molecules": "DAPI, DMSO, tubacin"
  },
  {
    "caption": "F. Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium. RPE1 cells were transfected with control or  PCM1 siRNA#1 for 48 h,  serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with antibodies against  IFT88, acetylated tubulin and  gamma tubulin.  DNA was stained with DAPI. Scale bar, 10 μm. G. Quantification of  IFT88 centrosomal intensity and  ciliary concentration for F.  IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections, and average means of the levels in control cells were normalized to 1. Ciliary  concentration was calculated by dividing ciliary fluorescence intensity to the cilium length. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test) ",
    "molecules": "MLN8237, DAPI, DMSO"
  },
  {
    "caption": "H, I. Quantification of cilium  disassembly and centrosomal AURKA levels after  serum stimulation. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with  DMSO or 0.5 μM MLN8237 in  serum stimulation medium for 2 h and 24 h. Cells were fixed and  immunostained with antibodies against   acetylated tubulin  and gamma tubulin, and percentage of ciliated cells were quantified. x-axis indicates the hours after serum stimulation. (H) n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM. (I) n>95 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test).",
    "molecules": "MLN8237, DMSO"
  },
  {
    "caption": "(c) Oil Red O (ORO) staining and fatty acid methyl ester (FAME) analyses of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant L3 larvae show that lysosomal lipases regulate cytosolic fat stores (see Fig. 2d for adult measurements). Means±s.e.m. are presented relative to wild type; significant differences are indicated; n = 3 independent experiments. (d) ORO quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults fasted for 4 h shows that LIPL-1 and LIPL-3 contribute to fat mobilization following fasting. (Mean percentage ± s.e.m. relative to fed wild type). Wild-type fasted worms show 20% less ORO signal than animals fed ad libitum (P≤0.001), whereas lipl-1(tm1954) lipl-3(tm4498) double mutant worms show 7% reduction in ORO signal (a difference that is not significant to well-fed fat levels at P≤0.01 but it is significant at P≤0.05). n = 3 independent experiments.",
    "molecules": "FAME, fatty acid methyl ester"
  },
  {
    "caption": "(e) Wild-type or lipl-1(tm1954) lipl-3(tm4498) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl-3 RNAi plates, incubated for 12 h at 20 °C, and processed for total FAMEs; as a reference, an aliquot of wild-type young adults on control RNAi bacteria was fasted for 6 h. FAME quantification (mean percentage of fed wild type ±s.e.m.) shows that acute mxl-3 inactivation, as fasting, reduces fat stores in a lipl-dependent manner. n = 3 independent experiments. NSD, P-value>0.05.",
    "molecules": "FAME"
  },
  {
    "caption": "(a) Expression analyses of the liver of C57BL/6J 9-week females fasted overnight (ON) for 10 h relative to siblings feeding ad libitum show that LipA (mouse lysosomal acid lipase), Map1lc3a (mammalian lgg-1/lgg-2) and Tfeb (mammalian hlh-30) but not Max (mammalian mxl-3) are transcriptionally linked to nutrients in the mouse liver (median+s.e.m. fold change; ddCt). Levels of expression were normalized to ActB and Cog2 as internal controls; all but Max differences are significant (P0.05), n = 3 independent experiments. Cyp4a14 is a positive control.",
    "molecules": "nutrients"
  },
  {
    "caption": "(b) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2, 4 or 8 h, compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media, show that LAL (human lysosomal acid lipase), MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes, and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent (median dCt+s.e.m.). Levels of expression were normalized to ACTB as internal control; all butMAX differences are significant (P0.05), n = 3 independent experiments. IGFBP is a positive control. EBSS, Earles's balanced salt solution.",
    "molecules": "nutrients"
  },
  {
    "caption": "(b) Live Nile red quantification of wild-type and lipl-1lipl-3 double mutant worms shows that lipl-1 and lipl-3 contribute to the clearance of lipids from the endocytic pathway. Mean+s.e.m. signal intensity is shown as percentage relative to wild type; significant differences are indicated; n = 4 independent experiments.",
    "molecules": "lipids"
  },
  {
    "caption": "Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across (A) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genes",
    "molecules": "tRNA"
  },
  {
    "caption": "(left) Total RNA from the indicated strains were separated on a 2,8% agarose gel. (right) Quantification of overall tRNA levels (mean ± standard deviation from 3 biological replicates).",
    "molecules": "tRNA, agarose, RNA"
  },
  {
    "caption": "Snapshots of ChIP-seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative (A) tRNA gene, (B) 5S rRNA gene and (C) srp7. Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1.",
    "molecules": "tRNA"
  },
  {
    "caption": "Average ChIP-seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1.",
    "molecules": "tRNA"
  },
  {
    "caption": "Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3 Flag epitope tag at the C-terminus of Rpc37.",
    "molecules": "tRNA, adenine"
  },
  {
    "caption": "Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.",
    "molecules": "tRNA"
  },
  {
    "caption": " (A-B) Rates of glucose consumption (A measured in the media 24h after treatment relative to the non-treated (NT) control from three independent experiments (n = 3). Error bars represent the SEM Data information: Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls ",
    "molecules": "glucose"
  },
  {
    "caption": " (B) Radiographic analysis of de novo protein synthesis using 35S-labelling. Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher\"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls ",
    "molecules": "35S"
  },
  {
    "caption": " (B-C) Rates of glucose consumption (B measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD ",
    "molecules": "DMSO"
  },
  {
    "caption": " (B-C) Rates o lactate production (C) measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD ",
    "molecules": "DMSO"
  },
  {
    "caption": " (D) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD ",
    "molecules": "DMSO"
  },
  {
    "caption": " (E) Western blotting of iNOS, tubulin, phospho-Thr172 AMPKα (pAMPK), total AMPKα (AMPK), phospho-Ser79-ACC  (pACC) and total ACC. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα  control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student\"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα  treated controls",
    "molecules": "DMSO"
  },
  {
    "caption": " (F) RT-qPCR analysis of iNOS mRNA levels relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls",
    "molecules": "DMSO"
  },
  {
    "caption": " (G) Nitric oxide (NO) levels in the culture media of cells relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls",
    "molecules": "DMSO, Nitric oxide, NO"
  },
  {
    "caption": " (C) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls ",
    "molecules": "H&E"
  },
  {
    "caption": " (A) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls ",
    "molecules": "H&E"
  },
  {
    "caption": "Nucleosomal array oligomers are globular.A Nucleosomal array oligomers were stained with DAPI and examined using FM (fluorescence microscopy) as described in the Materials and Methods section. Shown are representative images obtained in 4.5 mM and 10mM MgCl2.B Control FM images obtained in 0, 1 and 2.5 mM MgCl2.",
    "molecules": "MgCl2"
  },
  {
    "caption": "C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods. Shown in the left panels are representative images obtained in 4.5 mM, and 10 mM MgCl2. Shown in the right panels are images of the interior of the oligomers (white arrows, left panels) after cropping and re-scaling.",
    "molecules": "MgCl2"
  },
  {
    "caption": "Figure 2Sedimentation velocity analysis of the salt-dependent assembly of nucleosomal array oligomers.A Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl2. The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample (see Materials and Methods). The inset shows the mean second moment sedimentation coefficient the standard error for three replicated experiments.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).",
    "molecules": "MgCl2"
  },
  {
    "caption": "A, B SAXS profiles of the nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).",
    "molecules": "MgCl2"
  },
  {
    "caption": "A 601-nucleosomal arrays were incubated in 0.5, 5 mM MgCl2, 5 mM MgCl2 + MNase, and analyzed by the differential centrifugation assay to determine the fraction oligomeric. The amounts of DNA in the supernatant fraction were measured. Note that for the MNase-digested oligomers the supernatant fraction also includes the digested free linker DNA. Each value is the mean of three measurements and the error bars represent the standard deviation.",
    "molecules": "MNase, DNA, MgCl2"
  },
  {
    "caption": "B Verification of complete MNase digestion. DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase, and then electrophoresed on agarose gel. The position of mono nucleosome is marked with a star symbol.",
    "molecules": "MNase, DNA, MgCl2"
  },
  {
    "caption": "C Nucleosomal array oligomers (left) or with MNase treatment (right) were stained with DAPI and examined using FM. Shown are representative images obtained. Note that the sizes of MNase-treated oligomers are much smaller than those of the control oligomers (left and Fig. 1A).",
    "molecules": "MNase"
  },
  {
    "caption": "A Oligomers assembled from H1-nucleosomal arrays were stained with DAPI and examined using FM. Shown are representative images obtained in 4 mM and 5 mM MgCl2.B Control FM images obtained in 0, 1 and 3 mM MgCl2.",
    "molecules": "MgCl2"
  },
  {
    "caption": "C H1-nucleosomal array oligomers were negatively stained and visualized by TEM. Shown in the left panel is a representative image obtained in 4 mM MgCl2. Shown in the right panel is an image of the interior of the oligomer (white arrow, left panel) after cropping and re-scaling.",
    "molecules": "MgCl2"
  },
  {
    "caption": "Figure 6Sedimentation analysis of salt-dependent H1-oligomer assembly.A Representative experiment showing the second moment sedimentation coefficients of the H1-oligomers as a function of MgCl2. The dashed line indicates the upper limit of measurable sedimentation coefficients (~106 S). The white symbol is intended to show that the sedimentation coefficient of the H1-oligomers in 5 mM MgCl2 is beyond the detectable limit.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).C The g(s*) profiles in 2.5 mM, 3 mM, and 3.5 mM from panel (B) are re-plotted on a smaller scale.",
    "molecules": "MgCl2"
  },
  {
    "caption": "A, B SAXS profiles of the H1-nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).",
    "molecules": "MgCl2"
  },
  {
    "caption": "C, D SAXS profiles of the native chicken chromatin in 0 (TE), 0.5, 1 mM MgCl2 (C) and 2.5, 5 mM MgCl2 (D) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).",
    "molecules": "MgCl2"
  },
  {
    "caption": "A FMimages of chromatin structure in the nuclei with 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right). Insets show the intensity line profiles between the two marked arrow heads in the images.",
    "molecules": "MgCl2"
  },
  {
    "caption": "B SAXS profiles of the isolated HeLa nuclei. SAXS profiles of the nuclei in 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right) are shown in blue. For comparison, the scattering curves of H1-arrays from Figs. 7A and B were overlaid as red lines (left, 0 mM; center and right, 10 mM MgCl2).",
    "molecules": "MgCl2"
  },
  {
    "caption": "A) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D (PP3) and fft2Dfft3D (PP4) mutant strains with new integrations of Tf2-12-neoAI. After 3 days at 32o C.",
    "molecules": "G418"
  },
  {
    "caption": "C) Southern blot probed for neoA fft3D (PP3) and fft2Dfft3D mutant strains (PP4) and derived G418 resistant subclones.",
    "molecules": "G418"
  },
  {
    "caption": "A, B qRT-PCR for EN (A) and αSMA (B) of liver lysates from single high-dose CCl4- or oil-treated wild-type (WT) mice (n = 2 per time point).",
    "molecules": "CCl4, oil"
  },
  {
    "caption": "C Early activated and long-term-activated HSC isolated by differential density gradient centrifugation (8.26% versus 11.5% density layer). qRT-PCR for EN from early activated (8.26%) and long-term-activated HSC (11.5%) from 4-week CCL4-treated mice and vehicle control mice (n = 3-4 per data point).",
    "molecules": "CCL4"
  },
  {
    "caption": "D EN expression in liver lysates from 2-, 4-, and 6-week CCl4-treated mice (n = 5-8) and vehicle control mice (n = 3) determined by qRT-PCR.",
    "molecules": "CCl4"
  },
  {
    "caption": "E-G METAVIR score of 2-, 4-, and 6-week CCl4-treated mice (F-G) Col1a and F4/80 immunofluorescence of 6-week CCl4-treated and vehicle control WT (F) and ENKO (G) mice.H Mean Col1a+ area per high power field (HPF) shown as percentage of the total area (n = 5-8).",
    "molecules": "CCl4"
  },
  {
    "caption": "I Western blot analysis for collagen 1a (Col1a), platelet-derived growth factor receptor beta (PDGFRβ) and β-actin of total liver protein lysates from 6-week CCl4-treated or control vehicle WT and ENKO mice (n = 3-5).",
    "molecules": "CCl4"
  },
  {
    "caption": "J-L Double immunofluorescence of 6-week CCl4-treated and vehicle control WT (J) and ENKO (K) mice for desmin and αSMA (L) shown as percentage of the total area (n = 5-8).",
    "molecules": "CCl4"
  },
  {
    "caption": "M Serum liver enzymes (GPT/ALT and GOT/AST) from 2-, 4-, and 6-week CCl4-treated WT and ENKO mice (n = 5-8).",
    "molecules": "CCl4"
  },
  {
    "caption": "A Representative images of Ki-67 immunohistochemistry after 2, 4, and 6 weeks of CCl4 treatment of WT and ENKO mice (n = 5-8 mice per group and time point). Scale bars: 75 μm.B Quantitation of Ki67+ hepatocytes/HPF after 2, 4, and 6 weeks of CCl4 treatment.",
    "molecules": "CCl4"
  },
  {
    "caption": "Immunoblot analysis of total Aβ secretion and the production of different Aβ species (shown in low and high immunoblot exposures) in conditioned media of cell lines",
    "molecules": "Aβ"
  },
  {
    "caption": "Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of cell lines",
    "molecules": "Aβ"
  },
  {
    "caption": "ELISA analysis of Aβ species in conditioned medium of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Aβ levels are shown relative to DMSO vehicle control (n = 4 biological replicates).",
    "molecules": "RO7019009, Aβ, DMSO"
  },
  {
    "caption": "Dose response curve of RO7019009 in HEK293/sw cells measured using the MSD sandwich-immunoassay (n = 3 biological replicates).",
    "molecules": "RO7019009"
  },
  {
    "caption": "Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines. Comparison of in vitro potencies for Aβ42 inhibition in HEK293/sw cells, IC50 = 14 nM (n = 3) (as in (C)) with those in H4/sw cells, IC50 = 5 nM (n = 7) and N2a/sw cells, IC50 = 2 nM (n = 6) together with that of Notch cleavage inhibition in a HEK293-based Notch1 luciferase reporter cell line, IC50 > 10 µM (n = 2) (all n numbers represent biological replicates).",
    "molecules": "RO7019009, Aβ42"
  },
  {
    "caption": "Left panel: Immunoblot analysis of total Aβ in conditioned media of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Total APPs levels were analyzed to control for normal APP secretion and equal sample loading. Right panel: Quantification of relative Aβ amounts in (E) (n = 2 biological replicates).",
    "molecules": "RO7019009, Aβ, DMSO"
  },
  {
    "caption": "Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293/sw cells after separation by Tris-Bicine urea SDS PAGE; synthetic Aβ peptides were loaded as size markers.",
    "molecules": "RO7019009, Aβ, Bicine, Tris, urea"
  },
  {
    "caption": "ELISA analysis of Aβ species in conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle (DMSO) showing the secretion of Aβ38 (A), Aβ43 (B) and Aβ42 (C) compared to total Aβ (Aβ38 + Aβ40 + Aβ42 + Aβ43) (n = 5 biological replicates). Note that in some experiments Aβ38 generation was below the detection limit for some mutants. Data are presented as mean ± SD.",
    "molecules": "Aβ38, Aβ43, RO7019009, Aβ, Aβ40, Aβ42, DMSO"
  },
  {
    "caption": "Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle (DMSO).",
    "molecules": "RO7019009, Aβ, DMSO"
  },
  {
    "caption": "Immunoblot analysis of C99 V44Bpa crosslinking (CL) to PS1 WT or NTFs (circle) of normally endoproteolysed mutants in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in (A). Immunoblot analysis of C99 V44Bpa crosslinking to full-length (FL) PS1 (triangle) of endoproteolysis-deficient mutants and the corresponding M292D control in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in (C).",
    "molecules": "RO7019009, DMSO"
  },
  {
    "caption": "Analysis of the percentages of dead PC3 DKO cells or PC3 DKO cells with high expression of the remaining OAS, treated with 75 µM H2O2, 40 μM p-Benzoquinone (pBQ) or 2 mM methyl methane sulfonate (MMS). The number of dead cells (Cytotox Green-permeable) was monitored every 3 h using an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.001; *, p<0.05; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.",
    "molecules": "p-Benzoquinone, pBQ, H2O2, methyl methane sulfonate, MMS"
  },
  {
    "caption": "Analysis of the percentages of dead HME DKO cells and HME DKO cells with high expression of the remaining OAS, treated with 500 μM H2O2, and KO1-3 and OAS1-3hi cells treated with 750 μM H2O2. Data were obtained and presented as in (A). Means of triplicates ± SD. ***, p<0.001; **, p<0.01; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.",
    "molecules": "H2O2"
  },
  {
    "caption": "Survival of HME cells, measured by the MTT cell survival assay. Left: control (vec) and KO1-3 cells were treated with 250, 500 or 750 μM of H2O2 and analyzed after 24 h; Right: control, OAS1hi and OAS KO were treated with 500 μM of H2O2 and analyzed after 72 h . The data are presented as mean + SD of triplicates. ***, p<0.001; **, p<0.01; *, p<0.05 in Student's t-test.",
    "molecules": "H2O2"
  },
  {
    "caption": "An autoradiograph showing radiolabeled PAR2-5A bands that were formed in reactions of OAS1 with the activator (pIC) and the acceptor PAR (lanes 11-14). Lanes 1-2 OAS1 only, lanes 3-6 OAS1 with activator pIC; lanes 7-10 OAS1 with substrate PAR; lane 15 negative control without OAS1. 32P-α-ATP was added to all reactions.",
    "molecules": "α-ATP, 2-5A, 32P, pIC, PAR"
  },
  {
    "caption": "Top: specificity of anti-2',5'A, as shown by ELISA. Synthetic 2',5'-p3A3, PAR2-5A #1, PAR2-5A #2 (2 different preparations of PAR2-5A). PAR and 3',5'-pA3 were used as competitors. Bottom: Dot-blot of synthetic PAR and 2-5A-PAR. In vitro synthesized PAR and PAR-2-5-A were applied in 1:2 dilutions (left to right) to nylon membrane and probed with anti-2-5A or anti-PAR.",
    "molecules": "2',5'-p3A3, 3',5'-pA3, 2-5A, 2-5-A, 2',5'A, PAR"
  },
  {
    "caption": "Immunostaining of PAR-2-5A synthesized in vitro. Reaction mixtures were resolved by electrophoresis in a polyacrylamide-8 M urea denaturing gel, transferred to a charged nylon membrane, and probed with mouse anti-PAR 10H (left panel) or rabbit anti-2-5A after stripping (right panel). XC: Xylene Cyanol (apparent size 150 nucleotides), BPB: Bromophenol Blue (apparent size 35 nucleotides). Lane 1, purified PARP1 alone; lane 2, PAR reaction: PARP1, activated DNA, NAD+, and histone; lane 3, 2-5A reaction: OAS1, pIC, ATP; lane 4, 2-5A and PAR reactions: OAS1, pIC, ATP and PARP1, activated DNA, NAD, histone. After incubation at 37oC for 60 min, the proteins were removed by digestion with proteinase K.",
    "molecules": "XC, Xylene Cyanol, ATP, BPB, Bromophenol Blue, histone, NAD, NAD+, 2-5A, pIC, PAR"
  },
  {
    "caption": "Western analysis of PARP1 auto-modified with PAR in vitro in the absence or presence of OAS1. Reaction mixtures were resolved by SDS-PAGE, transferred to a charged nylon membrane, and probed with mouse anti-PARP1. Reactions were set up as in (C), but without proteinase K digestion. Lane 1, PARP1 alone; lane 2, PAR reaction: PARP1, activated DNA and NAD; lane 3, 2-5A reaction: OAS1, pIC, and ATP; lane 4, 2-5A and PAR reactions: OAS1, pIC, and ATP with PARP1, activated DNA, NAD and histone; lane 5, 2-5A and PAR reactions without ATP. The large smeary signals are due to PAR chains attached to PARP1 and the smaller sharp bands are free PARP1. The same blot was probed with anti-PAR, anti-2-5A and anti-OAS1.",
    "molecules": "ATP, histone, NAD, 2-5A, pIC, PAR"
  },
  {
    "caption": "Western analysis of 2-5A precipitated with anti-PAR antibodies from lysates of H2O2-treated HME cells. Cells were treated with 500 M H2O2 for 12 min, and lysates were incubated with control (mouse IgG3) or mouse anti-PAR. Samples were probed with anti-PAR, anti-2-5A or anti-PARP1 antibodies. Flow-thr.: flow-through, WCL: whole cell lysate, IP: immuno-precipitate. Right panels (IP) show exposures for 10 x longer than left panels (WCL, flow-thr). Apparent MW (kDa) is indicated on the right. Experiments were repeated two times with similar results.",
    "molecules": "H2O2, 2-5A, PAR"
  },
  {
    "caption": "Western analysis of 2-5A precipitated with anti-PAR antibodies from lysates of HME control, KO1 and TKO cells, and control cells pre-incubated with olaparib, treated with H2O2 for 12 min. Samples were probed with anti-PAR or anti-2-5A antibodies. Ponceau staining of the membrane is shown as loading control. Experiments were repeated four times with similar results.",
    "molecules": "H2O2, olaparib, 2-5A, PAR"
  },
  {
    "caption": "HME cells were treated with H2O2 for the indicated times and stained with anti-PAR and anti-2-5A. Nuclei were visualized with DAPI. The densities of the 2-5A and PAR fluorescence signals in the nuclei are plotted. Signals in 40+ cells per sample were measured. Data are presented as distribution plot with mean value +/- SD; ***, p<0.001; *, p<0.5; ns, non-significant; as determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.",
    "molecules": "DAPI, H2O2, 2-5A, PAR"
  },
  {
    "caption": "HME control (vector) and TKO cells were treated with H2O2 for 12 min, incubated on ice for 7 min with PBS or detergent-containing Buffer 1, to extract unbound proteins, fixed with methanol/acetone, and stained with anti-2-5A and anti-PAR. Scale bar, 10μm.",
    "molecules": "acetone, H2O2, methanol, 2-5A, PBS, PAR"
  },
  {
    "caption": "HME vector control (vec), DKO, and TKO cells were treated with H2O2 for 12 min. In untreated cells, no PAR staining was detected. The densities of fluorescence signals in the nuclei relative to the untreated vector control are plotted. Signals in 40 or more cells per sample were measured. Data are presented as distribution plot with mean value +/- SD; ***, p<0.001; *, p<0.5; ns, non-significant as determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.",
    "molecules": "H2O2, PAR"
  },
  {
    "caption": "HME control (vector) and KO1 cells were treated with H2O2 for the indicated times and stained with anti-2-5A and anti-PAR. Scale bar, 10μm.",
    "molecules": "H2O2, 2-5A, PAR"
  },
  {
    "caption": "Accumulation of 2-5A and PAR in HME cells pre-incubated with 10 or 100 nM olaparib for 30 min before treatment with H2O2 for 12 min, as visualized by immunostaining with anti-2-5A and anti-PAR. Scale bar, 10μm.",
    "molecules": "H2O2, olaparib, 2-5A, PAR"
  },
  {
    "caption": "Untreated HME (left) or PC3 (right) cells were stained with anti-OAS1-3 and the nuclei were visualized by staining with DAPI. The intensities of OAS1-3 fluorescence signals in the nuclei are plotted. Nuclear and cytoplasmic masks were made based on DAPI and phalloidin staining, respectively. Experiments were repeated at least two times; at least 40 cells per condition were counted. Data are presented as distribution plot with mean value +/- SD; statistical significance was determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "HME control (vec) and KO1 cells were pre-treated with IFNβ for 2 days, followed by a 10-min treatment with H2O2. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method; staining with anti-AIF and anti-HDAC2 staining was used to characterize membrane and nuclear fractions, respectively. Anti-PAR detects all PAR-modified proteins, which appear as smears.",
    "molecules": "H2O2, PAR"
  },
  {
    "caption": "Representative images and analysis of Western blots for PAR, performed with lysates of HME control (vec) OAS single KO cells (KO1-3), treated with H2O2 for 15 or 40 min. β-actin is shown as a loading control. Signal intensity was measured using ImageJ software. Average lane densities, normalized to vector from three independent experiments, are plotted below as mean + SD, * p < 0.05 as assessed by Student's t-test.",
    "molecules": "H2O2, PAR"
  },
  {
    "caption": "HME cells, pre-incubated with IFNβ for 2 days to increase OAS abundance, were treated with H2O2 for 0-60 min at 37oC. Western blots of PAR accumulation (top), shift in the position of PARylated PARP1 (middle), OAS2 induction by IFNβ, and β-actin as a loading control are shown. Experiments were repeated twice.",
    "molecules": "H2O2, PAR"
  },
  {
    "caption": "HME control (vec) and KO1 cells were pretreated with 0-500 IU/ml IFNβ for 2 days, and then treated with 500 μM H2O2 for 10 or 30 min. Western blots of PAR accumulation (top, high and medium exposure; middle, low exposure), OAS1-3 induction by IFNβ, and β-actin as a loading control are shown. The experiment was repeated four times; ns, non-specific band.",
    "molecules": "H2O2, PAR"
  },
  {
    "caption": "HME cells were treated with 750 μM of H2O2, with and without pre-treatment with olaparib. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.01 in two-way ANOVA. The data represent the results of three experiments.",
    "molecules": "H2O2, olaparib"
  },
  {
    "caption": "HME control (vec) and KO1-3 cells were incubated with olaparib for 30 min followed by treatment with 500 μM H2O2 for 15 min. PAR accumulation was assessed by the Western method, with anti-PAR. β-actin is shown as a loading control.",
    "molecules": "H2O2, olaparib, PAR"
  },
  {
    "caption": "HME vec, KO1 and OAS1hi cells were incubated with olaparib for 30 min, then treated with 500 or 1000 μM of H2O2 and allowed to grow for 72 h. Bars represent means of triplicates ± SD from three experiments. * p<0.05; ** p<0.01 (relative to H2O2-treated vec cells) in Student's t-test.",
    "molecules": "H2O2, olaparib"
  },
  {
    "caption": "KO1 cells transfected with vector (KO1-vec), wild-type OAS1 (KO1-OAS1 wt), or mutant OAS1 (D75A/D77A, KO1-OAS1 mut) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method. Loading controls: β-actin and overall protein levels were visualized by stain-free technology. The experiment was repeated three times; a representative image is shown.",
    "molecules": "H2O2"
  },
  {
    "caption": "Quantification of the relative abundance of PAR in (D). Images obtained in three independent experiments were analyzed; the data are plotted as mean + SD *, p<0.05 by Student's t-test.",
    "molecules": "PAR"
  },
  {
    "caption": "KO1 cells transfected with vector, wild-type OAS1 (OAS1 wt), or mutant OAS1 (D75A/D77A, OAS1 mut) were treated with 500 μM of H2O2. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ****, p<0.001 in two-way ANOVA. The data represent the results of three experiments.",
    "molecules": "H2O2"
  },
  {
    "caption": "A, B. Representative images and analysis of AIF accumulation in the nuclei of HME control (vec) and OAS single-knockout cells (KO), and HME control (vec) and OAS double- and triple-knockout cells (DKOs and TKO), 24 h after treatment with 1000 μM H2O2. Data information: Nuclei were visualized by DAPI staining. Arrowheads: AIF translocated to nuclei. Untreated control cells are shown on the left. Right panels, AIF-positive nuclei were counted from pictures taken from three independent fields in two independent experiments. Means ±SD are plotted. HME: * p<0.005; ** p<0.001 compared to H2O2-treated control (vec) cells;",
    "molecules": "DAPI, H2O2"
  },
  {
    "caption": "C. Representative images and analysis of AIF accumulation in the nuclei of PC3 DKO2/3 and PC3 DKO2/3+1 cells, 24 h after treatment with 100, 200 or 400 μM H2O2. Data information: Nuclei were visualized by DAPI staining. Arrowheads: AIF translocated to nuclei. Untreated control cells are shown on the left. Right panels, AIF-positive nuclei were counted from pictures taken from three independent fields in two independent experiments. Means ±SD are plotted. PC3: *** p<0.0001 compared to untreated cells of each genotype as assessed by Student's t-test. Scale bars, 10μm.",
    "molecules": "DAPI, H2O2"
  },
  {
    "caption": "[Ca2+]i changes in individual BMDMs were measured (A) using fura-2 during frustrated phagocytosis (n = 46-113) and (B) by dropping beads onto adherent cells loaded with Calbryte 520 (n = 64-159), in 1.8 mM extracellular Ca2+ (+Ca2+o) or Ca2+-free medium containing 100 μM EGTA (-Ca2+o) with or without prior loading with 25 μM EGTA/AM or 25 μM BAPTA/AM. The maximum fura-2 ratio changes (∆ 350/380) or fluorescence changes normalized to initial fluorescence (∆ F/F0) are plotted.",
    "molecules": "Calbryte 520, BAPTA/AM, Ca2+, EGTA/AM, EGTA, fura-2"
  },
  {
    "caption": "FcγR-mediated phagocytosis was monitored using beads conjugated to Alexa Fluor 488 (in green). External beads were differentially labelled with anti-IgG (in red) and distinguished by their dual labelling (in yellow). Cytochalasin D (10 μM, Cyt D) prevented bead internalization.",
    "molecules": "Alexa Fluor 488, Cyt D, Cytochalasin D"
  },
  {
    "caption": "Experiments conducted in -Ca2+o: (F) 1 μM thapsigarin (Tg) or 10 μM CPA pre-treatment, all but eliminated the 1 μM ionomycin (iono) response; single-cell Ca2+ traces and mean ∆ F/F0, n = 450-603 cells. (G) FcγR activation (as in B) evoked Ca2+ oscillations that were blocked with Tg or CPA treatment, whilst bead phagocytosis was not affected (H), n = 312-428 cells. (I) 1 μM bafilomycin A1 (Baf) for 3h, or Ca2+- binding dextran (Cal520-dx) and non-Ca2+-binding dextrans (FITC-dx and Texas red-dx) trafficked to lysosomes, minimally affected global Ca2+ signals evoked by FcγR activation (J); in parallel, bead uptake was only reduced by bafilomycin A1 or by chelating Ca2+ within the lysosome with Cal520-dx (K), n = 100-162 cells. All cells are WT BMDM and all beads are 3-μm IgG-opsonized.",
    "molecules": "Cal520, Baf, bafilomycin A1, Ca2+, CPA, dextran, dextrans, dx, FITC, iono, ionomycin, Texas red, Tg, thapsigarin"
  },
  {
    "caption": " The  Ca2+ signal elicited by frustrated phagocytosis   was significantly reduced in Ned-19 (10 μM)-treated WT and in all TPC-KO BMDMs; maximum Ca2+  amplitude, expressed as a percentage of WT control (n = 314-641 cells).  Phagocytosis  of opsonized 3-µm (B and C) and 6-μm (D) beads was inhibited by Ned-19 (B) and in BMDMs from Tpcn-/- mice compared to WT (n = 134-280 cells).",
    "molecules": "Ca2+, Ned-19"
  },
  {
    "caption": "Raising cytosolic Ca2+ with ionomycin (1 μM) rescues the Tpcn1-/- and Tpcn2-/- phagocytic defect, whilst UTP (100 μM) and ML-SA1 (50 μM) only weakly rescue (n = 84-192 cells).",
    "molecules": "Ca2+, ionomycin, ML-SA1, UTP"
  },
  {
    "caption": "BMDMs were co-loaded with 2 µM Calbryte590/AM (50 min) and 200 nM Lysotracker Green (LTG, 5 min) and, at room temperature, presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). A three-channel image was recorded every 10 s. The LTG brightness has been enhanced post hoc to visualize the dimmer, peripheral lysosomes. Time Zero is defined as the frame when the first Ca2+ signal was observed. (A) Images represent Ca2+ responses or an overlay of LTG and bead fluorescence. Times of each image are relative to the first Ca2+ spike. (B) Enlargement of the first contact point between lysosomes and bead (red arrow) as depicted by the dashed box, 80 s post-Ca2+. (C) Whole-cell Ca2+ responses from the cell in A. (D) Collation of the post-Ca2+ contact time, displayed as a scatter plot of individual cells and the mean ± S.E.M. (n = 27 cells).",
    "molecules": "Calbryte590/AM, Alexa Fluor 647, Ca2+, LTG, Lysotracker Green"
  },
  {
    "caption": "RAW246.7 cells were transfected with either TPC1-TagRFP-T or TPC2-TagRFP-T (red) and presented with 3-µm opsonized beads labelled with Alexa Fluor 647 (blue). Acute time-series of bead engagement were collected. Contact between TPCs and beads are depicted in the time-stamped micrographs, where time is arbitrary after the start of the image stack (E,F) i.e. 110 s is still prior to engagement. The spatial relationship between TPCs and beads was quantified using a target graticule of 0.5-µm bands centred on the engulfed bead; the mean fluorescence within each band was normalized to the fluorescence of the maximum band and plotted against this radial distance (G,H); n = 35-36 cells.",
    "molecules": "RFP-T, Alexa Fluor 647"
  },
  {
    "caption": "RAW 247.6 macrophages loaded with a cytosolic chemical dye (Cyto dye) to detect global cytosolic Ca2+, and heterologously expressing (B) TPC1-G-GECO1.2 (TPC1-GG) or (C-E) TPC2-G-GECO1.2 (TPC2-GG) to detect peri-endo-lysosomal Ca2+ domains, which were not detected by (F) a TPC2 mutant (D276K)-G-GECO1.2 that blocks ion permeation or (A) unfused cytosolic G-GECO1.2 (Cyto GG). (A-F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of the maximum fluorescence of the indicator, determined by adding 2 μM ionomycin and 10 mM CaCl2 at the end of each experiment (% Fmax) (D) TPC2-G-GECO1.2 activity was inhibited by 10 µM Ned-19, but was unaffected by (E) chelating global Ca2+ with EGTA/AM (100 μM for 2 min prior to bead addition). Summary data of the first spike from cells A-F (subsequent spikes show the same pattern; Appendix Fig. S4B), n = 87-274 cells; ***P<0.001 vs Cyto GG, ### P<0.001 vs TPC2-GG ctrl, ††† P<0.001 vs Cyto GG. Fusion of G-GECO1.2 to TPC2 did not alter the affinity of G-GECO1.2 for Ca2+ determined in permeabilized cells (n = 34-71 cells).",
    "molecules": "Cyto dye, CaCl2, Ca2+, EGTA/AM, ionomycin, Ned-19"
  },
  {
    "caption": "(I) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm beads in -Ca2+o expressed as a percentage of Fmax. (I, J) Pre-emptying the ER stores with CPA or thapsigargin (Tg) abolished the global cytosolic Ca2+ response evoked by beads, but a TPC2-G‑GECO1.2 component remained, n = 17 - 121 cells.",
    "molecules": "Ca2+, CPA, Tg, thapsigargin"
  },
  {
    "caption": "No local Ca2+ signal was detected with TPC2-G-GECO1.2 when Ca2+ spiking was evoked by ER-Ca2+ release with 100 μM UTP (K, L) or 10 μM CPA (M); only a bystander response equivalent to unfused cytosolic G-GECO1.2 was detected (n = 121-182 cells; *P<0.05).",
    "molecules": "Ca2+, CPA, UTP"
  },
  {
    "caption": "Translocation of CB, a non-Ca2+ binding CB mutant (CB mut) or a triple mTagBFP2 (BFP) to the lysosome (LAMP1), as indicated by the increase in the Pearson's correlation coefficient was induced by 250 nM rapalog (30 mins) compared with ethanol (EtOH) control-treated RAW 247.6 cells. n = 11-25 cells; ***P<0.001.",
    "molecules": "Ca2+, ethanol, EtOH, rapalog"
  },
  {
    "caption": "Images of a single RAW 247.6 cell displaying yellow puncta indicative of successful re-positioning of CB (in green) to the lysosome (in red) in the presence of rapalog but not EtOH.",
    "molecules": "EtOH, rapalog"
  },
  {
    "caption": "When CB was brought to the lysosome (+rapalog) uptake of beads was inhibited, whilst the non-Ca2+ binding CB (CB mut; n = 46-187 cells) or triple mTagBFP2 (BFP; n = 38-52 cells) did not block phagocytosis;***P<0.001.",
    "molecules": "Ca2+, rapalog"
  },
  {
    "caption": "Bead uptake in BMDMs with and without loading of 25 µM EGTA/AM and 10 μM Ned-19. EGTA-insensitive phagocytosis of beads was inhibited by Ned-19 (n= 75-102 cells) in WT BMDM; ***P<0.001.",
    "molecules": "EGTA/AM, EGTA, Ned-19"
  },
  {
    "caption": "RAW 247.6 macrophages expressing cytosolic G-GECO1.2 (Cyto GG), TPC2-G-GECO1.2 (TPC2-GG) or TRPML1-G-GECO1.2 (TRPML1-GG). (F) Single-cell Ca2+ traces upon FcγR activation by opsonized 3-μm or 6-μm beads in -Ca2+o expressed as a percentage of the maximum indicator fluorescence (% Fmax = 2 μM ionomycin and 10 mM CaCl2). (G) Summary of first Ca2+ spike; n = 69-121 (3-μm bead), 22-46 (6-μm beads) cells; ***P<0.001, **P<0.01 vs Cyto GG, ### P<0.001 TPC2-GG vs TRPML1-GG.",
    "molecules": "CaCl2, Ca2+, ionomycin"
  },
  {
    "caption": "Dynamin inhibitors (80 μM Dynasore or 20 μM Dyngo-4a) inhibit phagocytosis of opsonized 3-μm beads in WT BMDM; n = 101-149 cells, ***P<0.001.",
    "molecules": "Dynasore, Dyngo-4a"
  },
  {
    "caption": "Rescue of the TPC-KO dynamin defect by the dynamin activator, Ryngo-1-23 (80 μM): mean number of internalised beads per cell as a percentage of the WT control; n = 128-241 cells, **P<0.01, ***P<0.001.",
    "molecules": "Ryngo"
  },
  {
    "caption": "Single WT BMDM expressing HA-tagged dynamin-2 (immunolabelled with anti-HA, in red) undergoing phagocytosis of IgG-3-μm beads (blue). Images were taken 10 or 30 min post-addition of beads; actin (green) labelled with Phalloidin Alexa 488.",
    "molecules": "Alexa 488, Phalloidin"
  },
  {
    "caption": "Calcineurin inhibitors, FK506 (10 μM) or cyclosporin A (CsA, 10 μM) inhibited phagocytosis in WT cells, n = 65-159 cells. Ryngo (80 μM) rescued phagocytosis defects in Tpcn1-/- BMDM even in the presence of FK506 or CsA, n = 103-208 cells.",
    "molecules": "CsA, cyclosporin A, Ryngo, FK506"
  },
  {
    "caption": "Calcineurin activity and cytosolic Ca2+ responses simultaneously monitored in single RAW 247.6 using CaNAR2 and jRGECO1a, respectively, upon addition of 3-μm beads. CaNAR2 signals (FRET Y/C) were inhibited by FK506 (10 μM) and Ned-19 (10 μM); (H) single-cell traces and (I) mean maximum responses. n = 65-169; ***P<0.001 vs control.",
    "molecules": "Ca2+, Ned-19, FK506"
  },
  {
    "caption": "RAW 246.7 macrophages expressing the dynein-binding protein (mTagBFP2-BicD2-FKBP12) for rapalog-induced lysosome repositioning to the MTOC, also co-expressed HsLAMP1-mCherry with or without a C-terminal FRB* (LAMP-FRB* or LAMP, in red). Cells were treated with 0.1% ethanol or 250 nM rapalog for 2 h. Only the tripartite complex of FKBP12-rapalog-FRB induced clustering of lysosomes at the MTOC (A, B). This clustering was selective for lysosomes because ER and mitochondria (KDEL-GFP and MitoTracker Green) were not affected (C). (A, B) Phagocytosis of IgG-3-μm beads was reduced by immobilizing lysosomes at the MTOC, n = 80-564 cells; ***P<0.001.",
    "molecules": "ethanol, MitoTracker, rapalog"
  },
  {
    "caption": "Activation of dynamin-2 with Ryngo 1-23 or expression of constitutively active dynamin-2 (S764A mutant, expression confirmed by the grayscale image) restored phagocytosis of IgG-3-μm beads (n = 34-51 cells), but had no effect on the phagocytosis of IgG-6-μm beads, n = 36-72 cells (E); ***P<0.001 EtOH Ctrl vs rapalog Ctrl, ###P<0.001 rapalog Ctrl vs rapalog Ryngo/Dyn2S764A.",
    "molecules": "EtOH, rapalog, Ryngo, Ryngo 1-23"
  },
  {
    "caption": "D. Top three rows: Parasites expressing Cb EmeraldFP along with myoA-SNAP before and after Ca2+-Ionophore (A23187) or BIPPO treatment. The parasites were also treated with 2 µM of Cytochalasin D or DMSO (as control) for 30 min. After addition of A23187, preferential relocalisation of actin can be observed at the apical tip, while addition of BIPPO caused F-actin accumulation at the basal end. 4th row: myoAKO parasites expressing Cb-EmeraldFP before and after treatment with A23187 and BIPPO. While no apical or basal accumulation of F-actin is observed, some peripheral location of F-actin occurs in presence of A23187 or BIPPO, preferentially in the apical half of the parasite. Bottom row: adf cKD parasites expressing Cb-EmeraldFP before and after treatment with Ca2+ Ionophore or BIPPO. No apparent change in F-actin localisation can be seen upon treatment. E. Skeletonization of movies shown in D. Before addition of A23187 or BIPPO, RH and myoA KO parasites behave similar with no accumulation of F-actin at the apical tip. After treatment with calcium ionophore A23187, preferential accumulation of F-actin at the apical tip can be observed for RH (red arrowhead), while BIPPO presented preferential accumulation in the basal end (red arrowhead). In myoA KO, some relocalisation of actin is observed at the periphery of the parasite (yellow arrowhead). In the case of adf cKD, no relocalisation occurs. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin.",
    "molecules": "A23187, Ca2+, calcium, Cytochalasin D, DMSO, Ionophore, ionophore, BIPPO"
  },
  {
    "caption": "F. Quantification of actin accumulation before and after treatment with Ca2+ Ionophore or BIPPO. The parasites were counted for F-actin accumulation after adding Ca2+ Ionophore or BIPPO. Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip. A minimum of 200 parasites were counted upon 3 biological replicates. Two-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. Error bars represent standard deviation.",
    "molecules": "Ca2+, Ionophore, BIPPO"
  },
  {
    "caption": "A. Wild Type RH parasites mid-invasion. Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes, fixed with 4% PFA before labelling the TJ with anti-Ron2 (yellow) and DAPI (magenta) for nuclear staining. White arrow points at invasion direction.",
    "molecules": "DAPI, PFA"
  },
  {
    "caption": "A. Different invasion stages of P. falciparum merozoites expressing Cb-EmeraldFP. DAPI labels the nucleus (DNA), Cb-EmeraldFP labels actin filaments (CB-EME, green) and anti-RON4 labels the junction (red). Left panel: onset of invasion. Middle panel: A merozoite in the middle of invasion; note the constriction of the nucleus (blue) as it passes through the RON4 junction and F-actin colocalising with it (green). Right panel: A merozoite completing invasion; note the F-actin ring beyond the RON4-junction and actin filaments surrounding the nucleus. Data information: Scale bar represents 1 µm for Plasmodium falciparum",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "B. IFA showing various stages of invasion of erythrocytes by P. knowlesi merozoites. DAPI labels the nucleus (DNA), anti-actin Antibody labels actin filaments (anti-actin, green) and the invasion junction is marked with a C-terminally tagged PkRON2 mCherryHA (red). Left panel: a merozoite beginning invasion; note the formation of an F-actin ring (green) behind the PkRON2 junction (red). Middle panel: A merozoite in the middle of the invasion event; note the constriction of the nucleus (blue) as it passes through the PkRON2 junction and F-actin colocalising with it (green). Right panel: A merozoite completing the process of invasion; note the F-actin ring beyond the PkRON2-junction and actin filaments colocalising with the nucleus. Data information: Scale bar represents 3 µm for Plasmodium knowlesi.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "C. Super-resolution images showing various stages of invasion of erythrocytes by P. falciparum merozoites expressing Cb-EmeraldFP. Left panel: DAPI labels the nucleus (blue), Cb-EmeraldFP labels actin filaments (CB-EME, green) and the invasion junction is marked with an anti-RON4 antibody (red). Brightfield images have been marked with red dots depicting the junction, black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell. Top panel shows an attached merozoite beginning the process of invasion, the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion. Right panel: Schematic depicting merozoite invasion into red blood cells. Letters depict the corresponding event in the model (left) and microscopy pictures (right). White arrows depict actin filaments that colocalise with the nucleus. D. Quantification of Plasmodium falciparum invasion events categorising actin-TJ and actin-nucleus association events. 121 events were counted in total. Data information: Error bars represent standard deviation. Scale bar represents 1 µm for Plasmodium falciparum",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) PI staining of sgControl or sgRnf31 B16-F10 tumor cells treated with combined TNF/IFN-γ (both 10 ng/mL) for 24 hours in the absence and presence of QVD-OPh (10 μM); n.s.: no significance, ***p < 0.001, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.",
    "molecules": "QVD-OPh"
  },
  {
    "caption": "(B) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental B16-F10 tumor cells left untreated or treated with supernatant (Sup) (derived from a previous overnight OT-I/B16-F10 ova co-culture at the indicated E:T ratio for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of Sup); *p < 0.05, **p < 0.01, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.",
    "molecules": "HOIPIN-1"
  },
  {
    "caption": "(C) Representative flow cytometry plots (left) and pooled data (right) showing PI staining of parental A375 tumor cells left untreated or treated with TNF/IFN-γ (both 10 ng/mL) for 48 hours, following a 4 hour pre-treatment with vehicle or HOIPIN-1 (50 μM) (the concentration of vehicle/HOIPIN-1 was halved upon the addition of cytokines); *p < 0.05, **p < 0.01, ****p < 0.0001, Unpaired t-test for n = 3 biological replicates. Pooled data are represented as mean ± SEM.",
    "molecules": "HOIPIN-1"
  },
  {
    "caption": "(b) Sensitivity of RV replication factories to aliphatic alcohols at 4 HPI. Left to right -1,6-hexanediol  (1,6HD); 1,3-propylene diol (1,3PD); 1,2-propylene diol, or  propylene glycol (PG); glycerol (Gly); polyethylene glycol 200 (PEG200). Top panels - before application and bottom panels - 1 min after application of these compounds (4.5% v/v). Scale bar, 30 µm.",
    "molecules": "Gly, glycerol, 1,6-hexanediol, 1,6HD, PEG200, polyethylene glycol 200, 1,2-propylene diol, PG, propylene glycol, 1,3-propylene diol, 1,3PD"
  },
  {
    "caption": "(a) Recombinantly expressed, Atto647-dye labelled  NSP5,  Atto488-dye labelled NSP2 and unlabeled NSP5 protein samples were used for investigating their  phase-separation properties. All labelled protein samples were mixed with the unlabeled NSP5  (1:10 molar ratio) to minimise the effect of labelling on LLPS. Top panel, left to right: NSP5-647/NSP2-488 droplets formed upon mixing of both proteins (10 μM each). Both channels are shown, along with an image of both channels overlaid. 4% (v/v) 1,6-hexanediol (1,6HD) dissolves these condensates. Scale bar, 10 µm. Middle panel, left to right: NSP5-647 (50 μM) +  poly-arginine (polyArg, 5 μΜ);  NSP5-polyArg  condensate + 1,6-HD; NSP5-polyArg condensates + 0.5 M NaCl; NSP5 + R9 peptide (Arg-9, 5 μΜ). Bottom row, left to right: NSP5-647 sample (35 μM) alone; after addition of 10% v/v PEG-20K; NSP2-488 (25 μM) alone; after addition of 10% v/v  PEG-20K.",
    "molecules": "488, Atto488, 647, Atto647, 1,6-HD, 1,6-hexanediol, 1,6HD, PEG-20K, poly-arginine, polyArg, NaCl"
  },
  {
    "caption": "Top panel, left to right:  NSP5-∆CTR labelled with Atto-647 (25 μM) incubated with unlabeled NSP2  (10 μΜ); NSP5-∆CTR in the presence of 10% v/v PEG-20K; and with 5 μΜ of poly-arginine  (polyArg). Bottom panel: Atto-647-labelled NSP5-∆CTR (5 μΜ) incubated with unlabeled NSP5 (25 μΜ) and Atto-488  labelled NSP2 (10 μΜ). NSP5/NSP2  droplets containing labelled NSP2  (green) also contain NSP5-∆CTR-Atto647 (magenta), shown along with an image of both  488/647 channels overlaid. Scale bar, 10 µm.",
    "molecules": "488, Atto-488, 647, Atto-647, Atto647, PEG-20K, poly-arginine, polyArg"
  },
  {
    "caption": "(a) RV-infected   MA-NSP5-EGFP cells at 6 HPI. NSP5-EGFP-tagged viral factories (green) are dissolved in the presence of 4.7% (v/v) propylene glycol (PG, middle). Viral RNA-protein condensates rapidly reform (<10 min) after replacing the PG-containing cell culture medium (PG removed, right). Seg3 (magenta) and Seg4 (cyan) transcripts are detected by smFISH, and colocalising Seg3 and Seg4 RNA signals (white). Scale bars: 50 µm, zoomed-in regions: 10 µm.",
    "molecules": "PG, propylene glycol"
  },
  {
    "caption": "Effects of  PG treatments on the viral production.  PG-containing medium (2% or 4.7% PG), or standard cell culture medium, were applied to RV-infected cells (MOI of 10) 2 HPI (1 hour after virus absorption). (e) Effects of PG treatments on virus production at different infection points. 4.7% (v/v)  PG was applied between 2-7 HPI ('early infection'), and then diluted to 2% between 7-11 HPI, or 4.7% PG was applied between 7-11 HPI ('late infection'), and diluted prior to harvesting the virus. Each group was compared to the control group (10 h treatment with 2% PG). Each group was compared to the control group (10 h treatment with 2% PG). Application of 4.7%  PG for 5 h significantly reduced virus replication (P = 0.0016) between 2-7 HPI compared to the control group.  PG treatment between 7-12 HPI did not significantly reduce the viral titre compared to the control group (P = 0.1141). Infectious titres represent mean±SD values estimated for four independent biological replicates. Statistical analyses were performed using a Kruskal-Wallis test, followed by uncorrected Dunn's multiple comparisons test (**P < 0.002).",
    "molecules": "PG"
  },
  {
    "caption": "(f, g) 4.7% PG  treatment of RV-infected cells results in NSP5 dephosphorylation. (f) Western blot  analysis of  RV-infected cells harvested at 7 and 12 HPI, treated with 2% or 4.7%  PG at different infection time points indicated in the figure. Multiple  phosphorylated forms of NSP5 can be seen as higher MW bands. Treatment with 4.7% PG  reduces  phosphorylation, which is not perturbed in the presence of 2% PG. Treatments administered between 2-7 and 7-12 hpi reduce NSP5 phosphorylation, albeit to a considerably lower degree compared to the 2-12 hpi treatment. (g) Okadaic acid  (5 μM) applied to 4.7% PG-treated infected cells blocks  NSP5  dephosphorylation restoring its  phosphorylation pattern.",
    "molecules": "Okadaic acid, PG"
  },
  {
    "caption": "C57BL/6J mice  were intraarticularly infected with MRSA  (4 × 106 CFU; n = 3-4 per group) and left untreated for 24 hours. K. Paraffin-embedded tissues were sectioned and used to measure inflammation  score and synovial hyperplasia. GLUT1  and MCT4 expression was detected, and the percentage of positively staining  cells were determined (Scale bar: 1,000 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p<0.05 or **p <0.01; N.D. = not detected).",
    "molecules": "Paraffin"
  },
  {
    "caption": "D and E. Infiltrating  immune cell counts within  synovial fluid  and MRSA synovial fluid  bioburden were quantified. MRSA  bioburden in synovial tissue was quantified. F. Paraffin-embedded tissues were sectioned and measured at 7 and 14 days with respect to inflammation  score, synovial hyperplasia, synovial  cellularity, OARSI score, proteoglycan  depletion, osteophyte   formation, bone  erosion, and bone  formation (Scale bar: 1,000 μm). G and H. Lactate  level in synovial fluid was measured at 14 days and expression  of GLUT1, MCT4, cleaved-IL-1β, and NLRP3  in synovial tissue  was measured with GAPDH  as a loading control. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected; N.S. = not significant).",
    "molecules": "Lactate, Paraffin, proteoglycan"
  },
  {
    "caption": "C. Serum  and synovial fluid lactate  levels were measured and the relative luminescence intensity is shown. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).",
    "molecules": "lactate"
  },
  {
    "caption": "F. Blood  was collected, and  complete blood counts (CBC) were measured. G. Paraffin-embedded tissues were sectioned and measured with respect to inflammation  score, synovial hyperplasia, OARSI  score, proteoglycan  depletion, osteophyte  formation, bone  erosion, and bone  formation (Scale bar: 1,000 μm). The expression  of GLUT1  was detected and the percentage of positively staining  cells was determined. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).",
    "molecules": "Paraffin, proteoglycan"
  },
  {
    "caption": "C57BL/6J mice  were intraarticularly infected with MRSA  (4 × 106 CFU; n = 4-5 per group) for 24 hours.  Synovial fluid cells were collected and (5 × 105) seeded on antibiotic-free DMEM medium with or without DMF  (300 µg/ml) for 6 and 24 hours. B. MRSA  expressive of green fluorescent protein (GFP) was detected and measured (Scale bar: 100 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).",
    "molecules": "DMF"
  },
  {
    "caption": "G.  Inhibition  zones in disk fusion assays using vancomycin  (1,000 µg) and DMF  (75 to 300 µg) were measured and presented in a histogram. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).",
    "molecules": "DMF, vancomycin"
  },
  {
    "caption": "J.  RAW264.7 cells were stained  with CellTracker  and infected with MRSA  (4 × 106 CFU) for 2 hours. The cells were then washed to remove extra-cellular MRSA. Medium containing DMF  (700 µg/ml), vancomycin  (100 µg/ml), and DMF  (700 µg/ml) with vancomycin  (100 µg/ml) was added and GFP  intensity was measured at 1-hour intervals for a total of 12 hours. Representative images of differential fluorescence with CellTracker  and GFP  at 4-hour intervals for a total of 12 hours. Intracellular  and extracellular MRSA  were semi-quantitatively measured at 12 hours. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).",
    "molecules": "CellTracker, DMF, vancomycin"
  },
  {
    "caption": "C57BL/6J mice  were subcutaneously treated with vancomycin  (30 mg/kg) and rifampin  (20 mg/kg) for 3 days following MRSA  (8 × 106 CFU) infection, followed by systemic  (14 mg/kg) or local  (70 μg/joint) DMF  treatment at 3 days interval for a total of 3 times (n = 6 per group). D. Paraffin-embedded tissues were sectioned and measured with respect to inflammation  score, synovial hyperplasia, synovial  cellularity, proteoglycan  depletion, osteophyte   formation, bone  erosion, and bone  formation (Scale bar: 2,000 or 500 μm). Bone  resorption by osteoclasts  was measured by  tartrate-resistant acid phosphatase  (TRAP) staining  (Scale bar: 2,000 μm). Lameness score was also measured. E. Illustrated summary of DMF's  effect on MRSA-induced septic arthritis. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected).",
    "molecules": "DMF, DMF's, Paraffin, proteoglycan, rifampin, vancomycin"
  },
  {
    "caption": " [a] Sagittal  sections obtained from mice (p15) of the indicated genotypes were immunostained  for ADP-ribose using the pan-ADP-ribose detection reagent MABE1016. Representative  images showing levels of ADP-ribose in the hippocampal regions CA1, CA3, and dentate gyrus (DG), and in the cerebral cortex. Red dotted boxes highlight the elevated ADP-ribose staining in Xrcc1Nes-Cre cerebellum (left dotted box) and hippocampus (right doted box). Scale bars: 5 mm, 50 μm. WT (n = 4 mice), Xrcc1Nes-Cre (n = 4), Parp1+/- /Xrcc1Nes-Cre (n = 4), Parp1-/-/Xrcc1Nes-Cre (n = 3) and Parp1-/- (n = 3). Summary histograms show mean ± SEM. Pairwise comparisons between WT versus Xrcc1Nes-Cre mice and WT versus Parp1-/-/Xrcc1Nes-Cre mice  were conducted by Kruskal-Wallis ANOVA with Dunn's post-hoc test and statistically significant differences (*p<0.05) are shown. ",
    "molecules": "ADP-ribose"
  },
  {
    "caption": " [b] Protein extracts from wild type (WT), Xrcc1Nes-Cre, and Parp1-/- forebrain tissue, containing hippocampus and cortex, were incubated with 1 mM NAD+ for 45 min in the presence or absence of PARP inhibitor as indicated, and ADP-ribosylation  detected by western blotting using the poly(ADP-ribose)-specific detection reagent MABE1031. Representative images from two or more independent experiments are shown. A western blot showing the level of Parp1 and Xrcc1  in the  forebrain tissue extracts is also shown, Inset. ",
    "molecules": "NAD, poly(ADP-ribose)"
  },
  {
    "caption": " [e] Summary histograms (mean ± SEM) of cumulative seizure-like activity at 5, 10, and 15 min timepoints in cortex, CA1, and CA3  regions of wild type and Xrcc1Nes-Cre brain slices from mice treated or not for 5-8 days ad libitum with PARP1 inhibitor (ABT-888) prior to analysis. WT (n = 8 slices from 4 mice), Xrcc1Nes-Cre (n = 10 from 5 mice), WT +  PARPi (n = 6 from 3 mice), Xrcc1Nes-Cre + PARPi  (n = 9 from 3 mice). Pairwise comparisons at the 15 min time point between WT versus Xrcc1Nes-Cre mice and WT versus Xrcc1Nes-Cre mice  +  PARPi were conducted by Kruskal-Wallis with Dunn's post-hoc tests and statistically significant differences (*p<0.05) are shown. ",
    "molecules": "ABT-888"
  },
  {
    "caption": " [a] Representative  images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes-Cre mouse pups, immunostained for ADP-ribose  (red), NeuN  to identify neurons (green), and counterstained with DAPI  (blue). Cells were pretreated with PARP inhibitor (10 µM) or vehicle for 2 h prior to fixation, with PARG  inhibitor (10 μM) additionally present for the final hour. Scale bar 10 µm. [b] Histogram of mean (± SEM) relative pan-ADP-ribose fluorescence in NeuN-positive hippocampal neurons pretreated with PARP inhibitor (5 µM) or vehicle for 5 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Neurons were cultured from WT (n = 6 mice, > 180 cells per condition), Xrcc1Nes-Cre (n = 6, > 180), Parp1+/-/Xrcc1Nes-Cre (n = 3, > 90), and Parp1-/-/Xrcc1Nes-Cre (n = 3, > 90). * indicates significant differences from WT (Kruskal-Wallis ANOVA, p = 0.0013 and Dunn's post-hoc tests). ",
    "molecules": "ADP-ribose, DAPI"
  },
  {
    "caption": "ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP-PNP and aRF1/aPelota. The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins, forming the post-splitting complex. rps: small subunit ribosomal proteins.",
    "molecules": "AMP-PNP"
  },
  {
    "caption": "Yeast survival of ABCE1 variants (S. solfataricus colored, S. cerevisiae in gray). Most residues connecting to 30S in the post-SC show a growth defect when exchanged for a small one (alanine) or a negative charge (glutamate). ++ no effect, + growth defect, - lethal.",
    "molecules": "alanine, glutamate"
  },
  {
    "caption": "70S splitting efficiency normalized to wildtype. Hinge 2 mutations Y592A/Y593A, R565E and S580E display strongly impaired splitting activity. Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line. ATP turnover per ABCE1 is not affected in all tested mutants.",
    "molecules": "ATP"
  },
  {
    "caption": "C-D: Confocal  images (C) and quantification (D) of immunofluorescence staining  of STAT5b,  actin filaments (phalloidin) and nuclei  (DAPI) in cardiomyocytes  of cryosections from the hearts  of mice  transduced with either control- or ERBB4ECD-AAV. Panel D depicts quantification of colocalization  of STAT5b- and DAPI-specific signals. One dot in the boxplot corresponds to the median value in 20-50 images of stained  sections from one heart  (control AAV: n = 4, ERBB4ECD-AAV: n=5, biological replicates). Two-tailed unpaired T-test was used for statistics. Scale bar 10µm. White arrows point to cardiomyocyte nuclei. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "H-I: Confocal  images (H) and quantification (I) of immunofluorescence staining  of STAT5b  and nuclei  (DAPI) in mouse cardiomyocytes. The cells were treated with either control or Erbb4-targeting shRNAs. Panel I depicts quantification of colocalization  of STAT5b- and DAPI-specific signals (left) and the cell perimeter  (right). One dot in the boxplots corresponds to one cell (Control: n = 24, Erbb4#1: n=26, Erbb4#2: n=35; technical replicates combined from two biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA and Dunn's multicomparison test (colocalization  analysis) and Brown-Forsythe one-way ANOVA and Dunnett's multicomparison test (cardiomyocyte  size analysis) with multiple test correction were used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-B: Proximity ligation assay (PLA) of association of dynamin-2 with ERBB4  in primary mouse cardiomyocytes  treated with either DMSO  or the dynamin  inhibitor dynasore  for 5 hours. Panel A depicts a z projection of a stack of confocal  images. PLA interactions  are shown in red, nuclear stain DAPI  in blue. Panel B depicts quantification of the data. One dot in the boxplot corresponds to the number of PLA  signals in µm2 in one cell (Control: n = 6, DMSO  buffer: n=11, dynasore: n=11 ; technical replicates from one of two biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA and the Dunn's multicomparison test with multiple test correction was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DAPI, DMSO, dynasore"
  },
  {
    "caption": "G: Real-time RT-PCR  analysis of expression of the indicated STAT5b  target genes in primary mouse cardiomyocytes  treated for 1-3 hours with the DMSO  buffer, the ERBB  kinase inhibitor AG1478, or dynasore, and stimulated or not for 30 minutes with  NRG-1. One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation (for each group in  Igf-1 and Cdkn1a  panel: n = 6, for each group expect the dynasore  group in Myc  panel: n=4, for the dynasore  group in Myc  panel: n=3 ; biological replicate experiments). One-way ANOVA and the Dunnett's multicomparison test with multiple test correction was used for statistics. *, P < 0.05; ** P < 0.01; ***, P < 0.001; against DMSO  + NRG-1 treatment (Igf-1 panel: DMSO  without NRG-1 P = 0.0041, AG1478 P = 0.0082, dynasore P = 6.61e-06; Myc  panel: DMSO  without NRG-1 P = 0.0386, AG1478 P = 0.0037, dynasore P = 0.0064; Cdkn1a  panel: DMSO  without NRG-1 P = 0.0123, AG1478 P = 0.0064, dynasore P = 0.0092). Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DMSO, dynasore, AG1478"
  },
  {
    "caption": "H-I: Confocal  images (H) and quantification (I) of immunofluorescence staining   of myosin heavy chain in primary mouse cardiomyocytes. The cells were treated with either DMSO  or dynasore  and stimulated with NRG-1  for two days. The myosin  immunoreactivity was used to quantify cell area. One dot in the boxplot corresponds to one cell (DMSO: n = 22, dynasore: n = 28; technical replicates combined from three biological replicate experiments). Non-parametric Mann-Whitney U-test was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DMSO, dynasore"
  },
  {
    "caption": "Confocal  images (A and quantification (B, of immunofluorescence staining  of  myosin heavy chain (green) and DAPI  (blue) in the hearts  of 4 dpf zebrafish embryos  injected with either the buffer control or NRG-1 into the  pericardial sac at 2 dpf. The myosin  immunoreactivity was used to quantify both the average thickness of the ventricular wall  as well as the cross-sectional area of the ventricles. The number of nuclei  was estimated with the DAPI stain. The amount nuclei  (red) in the ventricle  and the ventricle  area (yellow) was quantified to differentiate between hypertrophic  and  hyperplastic growth. One dot in the boxplots corresponds to one heart  (Control (A-B): n = 10, NRG-1  (A-B): n = 7 biological replicates from two replicate experiments). Unpaired two-tailed T-test was used for statistics. Scale bars 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western  analysis (C) of Stat5, Akt  and Erk phosphorylation  in zebrafish embryos. The embryos  were treated as in A. (DMSO  (Stat5) n = 13, AG1478  (Stat5) n = 7, Lapatinib  (Stat5): n = 4, Gefitinib  (Stat5) n = 6, DMSO  (Akt) n = 9, AG1478  (Akt) n = 3, Lapatinib  (Akt) n = 3, Gefitinib  (Akt) n = 3, DMSO  (Erk) n = 13, AG1478  (Erk) n = 6, Lapatinib  (Erk) n = 4, Gefitinib  (Erk) n = 8 ; biological replicates combined from three replicate experiments).",
    "molecules": "DMSO, Gefitinib, Lapatinib, AG1478"
  },
  {
    "caption": "densitometric quantification (D) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. One dot in the boxplots corresponds to the relative densitometric  value of one pooled sample of 5 zebrafish embryos (DMSO (Stat5) n = 13, AG1478  (Stat5) n = 7, Lapatinib  (Stat5): n = 4, Gefitinib  (Stat5) n = 6, DMSO  (Akt) n = 9, AG1478  (Akt) n = 3, Lapatinib  (Akt) n = 3, Gefitinib  (Akt) n = 3, DMSO  (Erk) n = 13, AG1478  (Erk) n = 6, Lapatinib  (Erk) n = 4, Gefitinib  (Erk) n = 8 ; biological replicates combined from three replicate experiments). One-way ANOVA and the Dunnett's multicomparison test (pStat5, pAkt) or non-parametric Kruskal-Wallis with Dunn's multicomparison test (pErk) was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DMSO, Gefitinib, Lapatinib, AG1478"
  },
  {
    "caption": "E-F: Confocal  images (E) and quantification (F) of immunofluorescence staining  of  myosin heavy chain in hearts  of 4 dpf zebrafish embryos  treated with DMSO  or dynasore  for 2 days. The myosin  immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross-sectional area of the ventricles. One dot in the boxplots corresponds to one heart  (DMSO  (thickness, ejection fraction) n = 11, dynasore  (thickness, ejection fraction) n = 12, DMSO  (area) n = 15, dynasore  (area) n = 14; biological replicates). Unpaired two-tailed T-test was used for statistics. Scale bar 50µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.",
    "molecules": "DMSO, dynasore"
  },
  {
    "caption": "At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA (10 nM each), the intracellular localization of TfnR in HeLa cells was examined (shown as green). Nuclear DNA was stained with Hoechst 33342 (shown as blue). Control knockdown (left panel), Rab8a knockdown (center panel), and BAG6 knockdown (right panel).",
    "molecules": "Hoechst 33342, DNA"
  },
  {
    "caption": "Intracellular localization of Ptc1 (green) in HeLa cells. Nuclei were stained with Hoechst 33342 (shown as blue).",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "BAG6 protein co-precipitated Rab8a, while neither Rab7 nor Rab11a were co-precipitated with BAG6. Flag-tagged Rab8a, Rab7, Rab11a, and luciferase-CL1 (Lc-CL1; a positive control) were expressed in HeLa cells and the cells were treated with 10 μM MG-132 for 4 h. Flag-immunoprecipitates were blotted with anti-BAG6 and anti-Flag antibodies, respectively. Note that all cells used were treated with 10 μM MG-132 for 4 h.",
    "molecules": "MG-132"
  },
  {
    "caption": "S-tagged BAG6 pull-down efficiently co-precipitated Rab8a (T22N), a GDP-bound mutant, while BAG6 scarcely co-precipitated Rab8a (Q67L), a constitutively active mutant. Co-precipitation of Rab8a WT with BAG6 was used as a standard. MG-132 (10 μM) was included in the cell culture for 4 h, as indicated. Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L, and that this was partly due to increased degradation, as will be shown later. S-BAG6 stands for N-terminally S-tagged BAG6 protein",
    "molecules": "GDP, MG-132"
  },
  {
    "caption": "Anti-Flag signals in (C) were quantified, and relative signal intensities are presented. The value of the WT Rab8a signal with MG-132 was defined as 1.0. Note that all signal intensities of Flag-tag were normalized by that of the Rab8a input signals.",
    "molecules": "MG-132"
  },
  {
    "caption": "A series of Flag-Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag-Rab8a. Note that all cells used were treated with 10 μM MG-132 for 4 h.",
    "molecules": "MG-132"
  },
  {
    "caption": "Deficiency of Rabin8, a GEF for Rab8a, enhanced the physical interaction between BAG6 and WT Rab8a proteins. Flag-tagged WT Rab8a was expressed in Rabin8 siRNA-treated cells, and Flag immunoprecipitates were probed with an anti-BAG6 antibody. Note that all cells used were treated with 10 μM MG-132 for 4 h.",
    "molecules": "MG-132"
  },
  {
    "caption": "The N-terminal GTPase domain of Rab8a was essential for BAG6 recognition. A series of Flag-tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S-tagged BAG6 and treated with (+) or without (-) protease inhibitors for 4 h. Flag-Rab8a substrates were immunoprecipitated and probed with an anti-BAG6 antibody.",
    "molecules": "protease inhibitors"
  },
  {
    "caption": "CHX chase experiments show that GDP-bound Rab8a (T22N) was a highly labile protein, while the GTP-bound active mutant (Q67L) was a stable protein (E). Instability of the T22N mutant was not perturbed by the C204S mutation (F). Actin was used as a loading control.",
    "molecules": "CHX, GDP, GTP"
  },
  {
    "caption": "N-terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor, while the ∆N100 fragment was not.",
    "molecules": "proteasome inhibitor"
  },
  {
    "caption": "Rab8a (T22N) was polyubiquitinated in the presence of the proteasome inhibitor. Asterisks indicate non-specific bands.",
    "molecules": "proteasome inhibitor"
  },
  {
    "caption": "Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins.",
    "molecules": "amino acid"
  },
  {
    "caption": "Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control.",
    "molecules": "CHX"
  },
  {
    "caption": "Anti-Flag blot signals in the control or BAG6 siRNA-treated cells were quantified, and relative signal intensities after CHX addition were calculated. The value of the Flag signal at 0 h was defined as 1.0. Note that all signal intensities of the Flag-tag were normalized by that of actin, a loading control, in each sample.",
    "molecules": "CHX"
  },
  {
    "caption": "Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a. Right panel shows a merged image of TfnR staining (shown as green), Rab8a (T22N) staining (magenta), and Hoechst 33342 nuclear staining (blue).",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans-Golgi membrane protein Stx6 (green) in BAG6-suppressed CHO cells with a Chinese hamster-specific siRNA (cBAG6 siRNA#2). Scale bar: 10 μm (A). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Images of the cis-Golgi membrane protein GS28 and the cis-Golgi matrix protein GM130 in BAG6-suppressed CHO cells with another Chinese hamster-specific siRNA (cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S.D. calculated from 10 independent biological replicates (E).",
    "molecules": "glycoproteins"
  },
  {
    "caption": "Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Lectin GS-II-derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment t-test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down-regulated the cell surface expression of glycoproteins (F).",
    "molecules": "glycoproteins"
  },
  {
    "caption": " A. HeLa cells released from double thymidine synchronization in early S phase were treated with 5-azadC for 30 min, washed and collected at the indicated times. Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies. ",
    "molecules": "5-azadC, thymidine"
  },
  {
    "caption": " H. Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry-RNF4 expression plasmid. Cells were treated with 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and SUMO2/3 antibodies. Scale bar, 5 μm. ",
    "molecules": "5-azadC"
  },
  {
    "caption": " J. GFP-tagged DNMT1 from extracts of HeLa/GFP-DNMT1 cells treated or not with 5-azadC was immobilized on GFP-Trap agarose, subjected to stringent washing to remove proteins non-covalently bound to GFP-DNMT1 and incubated with recombinant HA-ubiquitin, E1 and E2 (UbcH5a) enzymes and RNF4 proteins (STUbL reaction) at 37 °C for 1 h. Samples were then subjected to immunoblotting to assay for RNF4-dependent STUbL activity towards GFP-DNMT1. ",
    "molecules": "5-azadC, ubiquitin"
  },
  {
    "caption": " I. Representative images of U2OS cells transfected with Myc-PIAS4 expression plasmid that were left untreated or exposed to 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and Myc antibodies. Scale bar, 5 μm. ",
    "molecules": "5-azadC"
  },
  {
    "caption": " J. HeLa or HeLa/GFP-DNMT1 cells left untreated or exposed to 5-azadC for 30 min were lysed and subjected to GFP IP under stringent conditions. After extensive washing, individual IPs were incubated with an equal amount (800 μg) of whole cell lysate of HeLa cells transfected with Myc-PIAS4 expression construct (Fig. EV2J), washed and immunoblotted with antibodies to Myc, SUMO2/3 and GFP. *, cross-reactive band. ",
    "molecules": "5-azadC"
  },
  {
    "caption": " K. HeLa/GFP-DNMT1 cells transfected with previously validated siRNAs targeting established SUMO E3 ligases (Fig. EV2L and Methods section) were treated with 5-azadC for 30 min, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with antibodies to SUMO1, SUMO2/3, ubiquitin and GFP. ",
    "molecules": "5-azadC, ubiquitin"
  },
  {
    "caption": " B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair. C. Samples in (B) were recovered by stringent DPC pull-down and immunoblotted for crosslinked M.HpaII. ",
    "molecules": "ATP, 32P"
  },
  {
    "caption": " D. HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse-labeled with 5-azadC in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated. ",
    "molecules": "5-azadC, DNA, thymidine"
  },
  {
    "caption": " E. HeLa cells transfected with indicated siRNAs were treated with 5-azadC and/or SUMOi in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated. ",
    "molecules": "5-azadC, DNA"
  },
  {
    "caption": " F. HeLa cells transfected with indicated siRNAs were pulse-labeled or not with 5-azadC for 30 min in late S phase according to the experimental setup in (A). Following 5-azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies. ",
    "molecules": "5-azadC, nocodazole"
  },
  {
    "caption": " E. HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six h after release from the block, cells were pulse-labeled with 5-azadC for 30 min. Mitotic cells were isolated by shake-off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies. ",
    "molecules": "5-azadC, thymidine"
  },
  {
    "caption": "D Left: Representative images of EpSC-like (n=5) and Keratinocyte-like (n=5) cSCC  tumors stained  for the EMT-marker ZEB2  (green) and the keratinocyte  marker TP63  (red). Nuclei  were counterstained  with DAPI. Images are shown at 40x original magnification. Scale bar, 50 µm. Right: Quantification of ZEB2-positive tumor cells (TP63-positive) in three independent regions per sample. Each dot represents a region with at least 500 tumor cells (TP63-positive) counted. Data information: Statistical analysis in D was performed using an unpaired two-sided t-test (EpSC-like: n= 5 samples, 15 technical replicates; keratinocyte-like: n= 5 samples, 15 technical replicates). Central bar represents the mean and error bars represent the standard deviation. *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Western blot showing that LC3-II levels were similar in control and Vps24-depleted cells treated with Bafilomycin A.",
    "molecules": "Bafilomycin A"
  },
  {
    "caption": "(F) Degradation of [14C]-valine- labeled proteins in cells incubated in complete medium (control), serum- and amino acid-deficient medium (starve), or starvation media in the presence of 3-methyl adenine (3-MA) to inhibit autophagic degradation. Average degradation per hour from four experiments done in duplicates ±1 SEM are shown.",
    "molecules": "14C, 3-methyl adenine, amino acid, proteins"
  },
  {
    "caption": "Autophagosomes and amphisomes do form in ESCRT-depleted cells. HeLa cells stably expressing GFP-LC3 were transfected with control (A), Tsg101 (B, D, and E), or Vps24 (C) siRNA and processed for immuno-EM analysis. Cryosections were incubated with antibodies against GFP (15-nm gold) and LBPA (10-nm gold). Amphisomes (Am), characterized by their content of electron-dense LC3-positive areas and intraluminal vesicles, were observed in both control (A) and ESCRT-depleted cells (B and C). (D) In the Tsg101-depleted cells we found large clusters of more typical autophagosomes (Au) and (E) clusters of double-membrane structures, consisting of autophagosomes and tubular structures which might represent phagophores, all labeling strongly for GFP-LC3. We never observed similar clusters in control cells and very rarely in Vps24-depleted cells. Bars, 200 nm. (F) Quantification of amphisomes in control cells and cells depleted for Tsg101 or Vps24. Approximately 20 cells were included per siRNA depletion per experiment (n = 3). Error bars = SEM.",
    "molecules": "gold, LBPA"
  },
  {
    "caption": "Immuno-EM of p62-positive structures in Vps24- and Tsg101-depleted cells. Cryosections of HeLa cells transfected with Vps24 (A, C, and E) or Tsg101 (B, D, and F) siRNA were incubated with antibodies against p62 (15-nm protein A gold, 10-nm in D). Membrane-free p62-positive aggregates (C and D) or p62-positive structures contained within dense clusters of vesicular-tubular elements (A and B) were detected. Vesicles with early endosomal morphology (EE) or multivesicular appearance (MVB) associated with these clusters. We also observed p62 labeling in electron-dense structures sequestered within amphisomes in cell depleted of Vps24 (E) or Tsg101 (F). The amphisomes typically consist of an electron-dense p62 positive body (E and F, arrowhead), and a multivesicular endosomal structure (E and F, arrow). Bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "Vps24 is required for efficient clearance of Htt inclusions. HeLa cells expressing HttQ65-mCFP (A) or HttQ103-mCFP (B) and N2a cells expressing HttQ103-mCFP (C) were transfected with control or Vps24 siRNA for 2 d and then incubated in the absence or presence of doxycycline for 3 d to turn off expression of HttQ65/Q103-mCFP. Vps24 depletion was determined by Western blot analysis. (A and C) Clearance of SDS-insoluble Htt inclusions was analyzed by filter-trap assays. (B) Alternatively, aggregate clearance was analyzed by confocal quantification of the number of HeLa HttQ103 cells having visible inclusions after 3 d in the absence or presence of dox. 300 cells from three independent experiments were counted for each condition. Error bars = SD.",
    "molecules": "SDS, doxycycline"
  },
  {
    "caption": "(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398). Drosophila S2 cells untreated (lane 2) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "(b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells untreated (lanes 1-3) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC, negative control RNAi.",
    "molecules": "amino acids"
  },
  {
    "caption": "(a) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng) into HEK293 cells. Cells were starved of amino acids for 1 h before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.",
    "molecules": "amino acids"
  },
  {
    "caption": "(b) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid (AA) stimulation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K into HEK293 cells. Cells were starved of amino acids for 1 h (-AA) and either remained in the starvation medium or were stimulated with amino-acid-containing-medium (+AA) for 30 min before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.",
    "molecules": "amino acid, amino acids, amino-acid"
  },
  {
    "caption": "(c) RagAT21N and RagCQ120L suppress insulin-induced stimulation of S6K phosphorylation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K (20 ng) or GST-Akt (100 ng) into HeLa cells. Cells were serum-starved overnight and stimulated with insulin (400 nM) for 30 min. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.",
    "molecules": "insulin"
  },
  {
    "caption": "(c, d) dRag GTPases positively regulate larval fat body cell size. (c) Cell area of clonally-induced dRagA-expressing cells or dRagC homozygous mutant cells relative to neighbouring wild-type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 h starved larvae. Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions. Cells expressing dRagAT61N and dRagC loss-of-function cells were significantly smaller than control cells only under nutrient replete conditions. Data are means ± s.d., *P = 2.04 × 10−3 (n = 5), **P = 2.94 × 10−6 (n = 14), ***P = 3.79 × 10−7 (n = 14), ****P = 1.36 × 10−5 (n = 30), Student's two-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(a) Rag acts through TORC1 to regulate S6K phosphorylation. HEK293 cells were transfected with constructs as indicated. Co-expression of of mTORKD construct (600 ng) or rapamycin treatment (rapa, 20 nM, 30 min) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation. The protein level of mTORKD was determined by immunoblotting with anti-mTOR antibody.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or ′ constructs as indicated. Amino acid starvation for 1 h (-AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.",
    "molecules": "Amino acid"
  },
  {
    "caption": "(e) RagA regulates LC3 conversion in mammalian cells. Myc-LC3 was co-transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated. One day after transfection, cells were cultured in amino-acid-sufficienct medium (+AA) or amino-acid-depleted medium (−AA) for 4 h before collection. Western blotting for Myc-LC3 and HA-Rag were performed. Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant-negative RagA. Full scans of blots are provided in Supplementary Information, Fig. S6.",
    "molecules": "amino-acid"
  },
  {
    "caption": "Localization of CASPR in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelled with antibodies against CASPR1 and RIBEYE (A-B OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm",
    "molecules": "CFA"
  },
  {
    "caption": "Localization o visual-arrestin (v-arrestin) in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelle with antibodies against v-arrestin and RIBEYE (C-D) V-arrestin, visual arrestin; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm",
    "molecules": "CFA"
  },
  {
    "caption": "Localization of CNTN1 in semi-thin (0.5μm-thin) sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). (A,B) Sections were double-immunolabelled with antibodies against CNTN1 and RIBEYE proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer",
    "molecules": "CFA"
  },
  {
    "caption": "Quantification of the immunosignals of the indicated proteins in the OPL and IPL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer; n.s., non-significant; ***, p<0.001 (Mann-Whitney U test, actual p-values are given in C) (N=6 independent set of experiments; n=295 images (CFA); n=274 images (MOG/CFA) for quantification of RIBEYE immunosignals; n=95 (CFA); n=92 (MOG/CFA) for quantification of CASPR1 immunosignals; n=91 (CFA), n=82 (MOG/CFA) for CNTN1 quantification; n=109 (CFA); n=100 (MOG/CFA) for quantification of v-arrestin. Scale bars: 20μm",
    "molecules": "CFA"
  },
  {
    "caption": "MOG/CFA- but not CFA-injected mice contain reactive antibodies for retinal proteins in blood and show altered spatial visual behavior. (A) 7-9 days after injection, blood from MOG/CFA-injected (red) but not from CFA-injected mice (black), elicited strong antibody responses in ELISA. Antibodies for RIBEYE and SV2 were used as positive controls (green), secondary antibody only as a negative control (white). Normalized data are given as mean ± S.E.M. (N=4 (CFA); N=5 (MOG/CFA). ** p<0.01 (significance was assessed by Mann-Whitney test, precise p-values given in the figure)",
    "molecules": "CFA"
  },
  {
    "caption": "(C,D) Western blot of protein lysates as in B) probed by blood samples from mice before and 7 to 9 days after MOG/CFA injection. Only blood samples obtained from MOG/CFA-injected mice revealed immunoreactivity against CASPR1 (lanes 15,16; top panels) whereas samples obtained before immunization (lanes 7,8; top panels) did not. The same filters were re-probed with anti-actin antibodies as loading control",
    "molecules": "CFA"
  },
  {
    "caption": "(C,D) Western blot of protein lysates as in B) probed by blood samples from mice before and 7 to 9 days after MOG/CFA injection. Only blood samples obtained from MOG/CFA-injected mice revealed immunoreactivity against CASPR1 (lanes 15,16; top panels) whereas samples obtained before immunization (lanes 7,8; top panels) did not. The same filters were re-probed with anti-actin antibodies as loading control. (E) Summary of data from (C,D) shown as mean ± S.E.M. (N=4, before injection; N=5, MOG/CFA-injected). ***, p<0.001; n.s., non-significant (unpaired two-tailed Student's t-test, precise p-values given in the figure)",
    "molecules": "CFA"
  },
  {
    "caption": "Spatially guided visual behavior, measured as frequency threshold with a virtual optokinetic system 9 days after immunization, is significantly decreased in MOG/CFA-injected mice compared to CFA-injected control litters. Data are shown as median; boxes and whiskers illustrate 25-75 and 5-95 percentiles of values, respectively. Points below and above whiskers are drawn as individual points. At each trial, mice were measured three times. Home-made and commercial MOG/CFA suspensions were compared as well as PLP-injected mice with control-injected mice. Statistical analysis was done by ANOVA (0,05 significance level), mean comparison probabilities were Bonferroni corrected. N=32, n=96 (MOG/CFA); N=24, n=72 (CFA). ***, p<0.001 (Mann-Whitney test). Results from mice injected with home-made and commercial MOG/CFA suspensions as well as results obtained from PLP/CFA-injected mice (and respective control-injected mice) are displayed",
    "molecules": "CFA"
  },
  {
    "caption": "Optic nerve parameters are unaltered in early EAE (9 days after injection). (A,B) Cryostat sections of the optic nerve from MOG/CFA-injected mice (own lab-made suspension; for comparison with commercial MOG/CFA suspensions, see Appendix Fig. S12) and CFA-injected control mice were stained with anti-Nav antibody (1:50 dilution) to label the node of Ranvier and with anti-CASPR1 antibody (1:500 dilution) to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in C) D)",
    "molecules": "CFA"
  },
  {
    "caption": "(E1, E2) Richardson-Blue stained semi-thin sections from the indicated optic nerve samples. (E2, E3, F2, F3) Transmission electron micrographs of the optic nerves from MOG/CFA- and CFA-injected animals. Abbreviations: ax, axon; my, myelin sheath. Axon number and myelin thickness were quantified in G) and H). Scale bars: 1μm (A,B,E,F)",
    "molecules": "CFA"
  },
  {
    "caption": "Retinal slices from MOG/CFA-injected and CFA-injected SypHy mice were stimulated by applying a 25mM K+-containing depolarization solution for 1min (Fig. 6A). In response to depolarization, we observed a strong increase in fluorescence (Fig. 6A) that can be best fitted by a double-exponential curve (B). Amplitudes of fast and slow release (together with the respective time constants) of the first depolarization response are plotted in C)",
    "molecules": "CFA, K+"
  },
  {
    "caption": "The amount of CASPR1 protein in the synapse is also altered in the PLP mouse model of multiple sclerosis. Semi-thin sections of PLP/CFA and CFA-injected mice were probed with the indicated antibodies (A, B; see also Appendix Fig. S13)",
    "molecules": "CFA"
  },
  {
    "caption": "The synaptic amount of CASPR1 is strongly reduced both in the OPL and IPL. RIBEYE is only reduced in the OPL but not significantly in the IPL. CNTN1 is strongly reduced in both OPL and IPL (C, see also Appendix Fig. S13). N=4 PLP/CFA-injected mice; N=4 CFA-injected mice; n=140 slices (CFA, RIBEYE); n=143 slices (PLP/CFA, RIBEYE); n=47 slices (CFA, CASPR1); n=53 slices (PLP/CFA, CASPR1); n=49 slices (CFA, CNTN1); n=46 slices (PLP/CFA, CNTN1); n=44 slices (CFA, v-arrestin); n=44 slices (PLP/CFA, v-arrestin)",
    "molecules": "CFA"
  },
  {
    "caption": "(D,E) Cryostat sections of the optic nerve from PLP and control mice were stained with anti-Nav-channel antibody to label the node of Ranvier and with anti-CASPR1 antibody to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in G) H). (F) transmission electron micrographs of the optic nerves from PLP/CFA- and CFA-injected animals (F1-F4). Axon numbers and myelin thickness were quantified in I) J)",
    "molecules": "CFA"
  },
  {
    "caption": "D, Affinity  capture of proteins by OC43p  and  OC43-SCR bound   magnetic beads. Specific reactivity to Atp1a1  and Atp1a2  was detected by protein immunoblotting  using specific primary antibodies.  OC43p-SCR-coated and uncoated beads (Beads) were used to control non-specific binding.  Spinal cords  were isolated from male  (M) and female  (F) rats  (n=2/group). Input protein lysates (right side, in duplicates) served as loading controls. Approximate molecular weights (in kDa) are indicated by arrows.",
    "molecules": "OC43-SCR, OC43p, OC43p-SCR"
  },
  {
    "caption": "d, e, LDH release and IL-1β secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS.",
    "molecules": "alum, LPS, nigericin, poly(dA:dT), monosodium urate"
  },
  {
    "caption": "b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).",
    "molecules": "3-methyladenine, LPS"
  },
  {
    "caption": "b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).",
    "molecules": "3-MA, 3-methyladenine, LPS"
  },
  {
    "caption": "b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).",
    "molecules": "3-MA, 3-methyladenine, LPS"
  },
  {
    "caption": "b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).",
    "molecules": "3-MA, 3-methyladenine, LPS"
  },
  {
    "caption": "h, i, LDH release and IL-1b secretion from BMDMs infected for 16 h or transfected with LPS. Graphs show mean and s.d. of quadruplicate wells and data are representative of two (e, i) and three (a-d, f-h) independent experiments. *P  0.05, **P  0.01; NS, not significant (two-tailed t-test).",
    "molecules": "LPS"
  },
  {
    "caption": "HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control. (n=5 biological replicates).",
    "molecules": "3-NP"
  },
  {
    "caption": "Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO= Hoechst (blue). The scale bar represents 100 µm.",
    "molecules": "HO, Hoechst"
  },
  {
    "caption": "Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green) and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar presents 2 mm, and the enlarged image scale bar is 40 µm. (n=6 mice/group).",
    "molecules": "DAPI"
  },
  {
    "caption": "The mtDNA content was examined with primers of D-loop, mtCO1, mtCO2 and mtND2 by qPCR in control or Flag-mtHSF1-expressing striatal cells. Scatterplot with bar showed the relative percentage of mtDNA content. (n=3 biological replicates).",
    "molecules": "mtDNA"
  },
  {
    "caption": "Cells were treated with EtBr for six days to delete mtDNA. mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr. (n=3 biological replicates).",
    "molecules": "mtDNA, EtBr"
  },
  {
    "caption": "qPCR analysis of the mtDNA content of AAV-Con-injected mice or AAV-mtHSF1-injected mice. Scatterplot with bar showed the relative percentage of mtDNA content (n=3 mice/group).",
    "molecules": "mtDNA"
  },
  {
    "caption": "Representative images and scatterplot showing the number of mtDNA (purple) colocalized with mitochondria (Tom20, green) in striatal organoids injected with AAV-Con or AAV-mtHSF1. The data were obtained from 3 independent biological experiments. Con: n=65 cells from 20 organoids; Flag-mtHSF1: n=70 cells from 20 organoids. Two-tailed unpaired t-test was used. Images were captured by Structured Illumination Microscopy (SIM).",
    "molecules": "mtDNA"
  },
  {
    "caption": "HdhQ7 or HdhQ111 cell extracts were incubated with DSS (1 mM) for 30 min at RT. SSBP1 oligomers were detected by immunoblotting with an anti-SSBP1 antibody. (n=3 biological replicates).",
    "molecules": "DSS"
  },
  {
    "caption": "Cells were transfected with an empty vector or Flag-mtHSF1 for 48 h. The total cell lysates were harvested and crosslinked with DSS. SSBP1 oligomers were tested by immunoblotting. (n=4 biological replicates).",
    "molecules": "DSS"
  },
  {
    "caption": "Flag-mtHSF1 or sh-SSBP1 was delivered into cells via lentiviral infection. The expression of SSBP1 and mtHSF1 was detected by WB analysis. The mtDNA content was examined by qPCR analysis. (n=3 biological replicates).",
    "molecules": "mtDNA"
  },
  {
    "caption": "GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1. (n=3 biological replicates).",
    "molecules": "DH1"
  },
  {
    "caption": "HdhQ7 or HdhQ111 cells (1 μM/day, 3 days), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "H. YAC128 or age-matched littermates (3 mg/kg/day, 3 months), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "I. striatal organoids differentiated from patient iPSCs or control iPSCs (1 μM/day, 7 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "J. patient fibroblasts (1 μM/day, 5 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "HdhQ7 or HdhQ111 were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "L. patient fibroblasts were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "Immunostaining for mitochondria (Tom20, green) and mtDNA (anti-DNA, red) in striatal organoids treated with DH1 or TAT at D60. Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria. The data were obtained from 3 independent biological experiments. Left, n=222-311 mitochondria per group; Right, n=14-20 organoids per group. Images were taken using Structured Illumination Microscopy (SIM). The scale bar represents 10 μm.",
    "molecules": "DH1, mtDNA, TAT"
  },
  {
    "caption": "Immunostaining for mitochondria (Tom20, red) and p-Drp1 S616 (green) in striatal organoids treated with DH1 or TAT at D60. Scatterplot with bar showed the number of p-Drp1 S616 per cell in organoids. The data were obtained from 3 independent biological experiments. n=29-34 cells from at least 10 organoids. The scale bar represents 5 μm.",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "Scatterplot with bar showed the ratio of MSNs (DARPP32, green) in striatal organoids in the presence or absence of DH1. The data were obtained from 3 independent biological experiments. n=30 organoids per group.",
    "molecules": "DH1"
  },
  {
    "caption": "Representative tracing images of DARPP32+ neurons treated with DH1 or TAT at D45. Scatterplot with bar showed the results of longest neurites length. The data were obtained from 3 independent biological experiments. n=45-92 neurons per group.",
    "molecules": "DH1, TAT"
  },
  {
    "caption": "Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1. The arrows (white) mark fragmented nuclei. The data were obtained from 3 independent biological experiments. n=7-10 organoids per group. The scale bar represents 20 μm.",
    "molecules": "DH1"
  },
  {
    "caption": "Mouse behavior was tested after DH1 treatment. (n=9-12 mice/group).",
    "molecules": "DH1"
  },
  {
    "caption": "Genomic distribution of (C) identified ZF-D3A binding sites, and (D) differentially methyl- ated regions (DMRs) between MCF7-control and ZF-D3A +dox.",
    "molecules": "dox"
  },
  {
    "caption": "(E) Shown for all DMRs identified between ZF-D3A +dox and MCF7-control is: ∆mCG (difference in the methylation level [ratio of C base calls to total base calls for all CG dinucleotides in region, mCG/CG] between ZF-D3A +dox and MCF7-control), ZF-D3A ChIP-seq signal (normalized ChIP-seq read density, ±1kb flanking DMR center), CpG dinucleotide density (CpG/100bp, ±1kb flanking DMR center), and classification of each DMR as promoter or non-promoter located.",
    "molecules": "dox"
  },
  {
    "caption": "(F) DNA methylation level in MCF7-control and ZF-D3A +dox cells flanking (±1 kb, 100 bp bins) the center of ZF-D3A ChIP-seq peaks identified in ZF-D3A +dox.",
    "molecules": "DNA, dox"
  },
  {
    "caption": "(A) Line histogram of DMR methylation levels in each sample. Kernel density estimate smoothed scatterplot comparison of DMR methylation levels in MCF7-control and (B) ZF-D3A +dox, or (C) ZF-D3A dox-wd.",
    "molecules": "dox"
  },
  {
    "caption": "(D) Line histogram of UMR methylation levels in each sample. Scatterplot compari- son of UMR methylation levels in MCF7-control and (E) ZF-D3A +dox, or (F) ZF-D3A dox-wd.",
    "molecules": "dox"
  },
  {
    "caption": "(B) H3K4me3 ChIP-seq normalized read counts in DMRs with ∆mCG >0.2, from H3K4me3 ChIP-seq in ZF-D3A no-dox and ZF-D3A +dox cells. Black line represents equal normalized read counts in ZF-D3A no-dox and ZF-D3A +dox cells; blue line represents a linear regression of the data.",
    "molecules": "dox"
  },
  {
    "caption": "(C) Comparison of UMR ∆mCG and difference in H3K4me3 ChIP-seq normalized read counts between ZF-D3A no-dox and ZF-D3A +dox. Blue line represents a linear regression of the data.",
    "molecules": "dox"
  },
  {
    "caption": "(D) DNA methylation levels of UMRs that intersect with H3K4me3 ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-H3K4me3 antibody.",
    "molecules": "dox"
  },
  {
    "caption": "(E) DNA methylation levels of UMRs that intersect with phos- pho-ser5 RNA polymerase II ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-phospho-ser5 RNA polymerase II antibody.",
    "molecules": "dox"
  },
  {
    "caption": "(A) Western blot measurement of ZF-D3A protein abundance upon doxycycline induction and withdrawal.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) Box and whisker plots of the DNA methylation levels of the 121 CpG sites in the DACH1 promoter region through- out doxycycline induction and withdrawal. Whiskers indicate 1.5 times the interquartile range or the most extreme data point, whichever is lower.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C) Average DNA methylation level at the 121 CpG sites in the DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal and cell cycle inhibition by growth in doxycycline-free media containing different cell cycle inhibitors (Lovastatin, RO-3306, thymidine).",
    "molecules": "RO-3306, doxycycline, Lovastatin, thymidine"
  },
  {
    "caption": "(D) DMR mCG and hmCG levels in MCF7-control and ZF-D3A +dox cells, ordered by the difference in DMR hmCG level (hmCG/CG) between MCF7-control and ZF-D3A +dox cells, and heatmap representation of H3K4me3 levels in ZF-D3A +dox cells flanking (±2.5 kb, 50 bp bins) the centre of the DMRs.",
    "molecules": "dox"
  },
  {
    "caption": "(A) Scatter plot of the difference in UMR DNA methylation levels, versus the fold change in mRNA abun- dance of UMR-associated expressed genes, between MCF7-control and ZF-D3A +dox. Point color indicates the gene differential expression significance: red indicates FDR <0.01, black indicates FDR >0.01. Trend line (green) was fitted using a generalized additive model, and contour lines represent the relative density of UMRs.",
    "molecules": "dox"
  },
  {
    "caption": "(B) Distribution of the ratios of mRNA levels between ZF-D3A +dox and MCF7-control, for expressed genes that have an associated UMR with a ∆mCG >0.3. Column color indicates the class of mRNA abundance ratios between ZF-D3A +dox and MCF7-control: blue = no decrease; green = small decrease; red = decrease.",
    "molecules": "dox"
  },
  {
    "caption": "(C) Representative genes with high induction of promoter DNA methylation in ZF-D3A +dox and no reduction in mRNA abundance.",
    "molecules": "dox"
  },
  {
    "caption": "(H) Distribution of per-read methylation levels (blue line) for selected ZF-D3A +dox WGBS reads (≥10 cytosines in the CpG context) that align within robustly methylated DMRs (DMR mean methylation level [mCG/CG] of 0.4-0.6 in ZF-D3A +dox and <0.1 in MCF7-control), and average CG methylation levels from ZF-D3A +dox WGBS data of the DMR genomic intervals covered by the selected reads (red line).",
    "molecules": "dox"
  },
  {
    "caption": "HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (A) Concentrations of IL-6 were determined (CTRL, n=9; Bax/Bak, n=6; Bcl-XL, n=3; STING, n=3).",
    "molecules": "ABT-737"
  },
  {
    "caption": "HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (B) Concentrations of IL-8 were determined (CTRL, n=7; Bax/Bak, n=4).",
    "molecules": "ABT-737"
  },
  {
    "caption": "HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (C) Concentrations of CXCL1 were determined (CTRL, n=8; Bax/Bak, n=5).",
    "molecules": "ABT-737"
  },
  {
    "caption": "HeLa cell lines were treated with the small-molecule Bcl-2/Bcl-XL-inhibitor ABT-737 for 72 h. Cytokines were measured by ELISA. (D) Concentrations of FGF-2 were determined (CTRL, n=7; Bax/Bak, n=4).",
    "molecules": "ABT-737"
  },
  {
    "caption": "HeLa cell lines were treated with the Mcl-1-inhibitor S63845 for 72 h. Cytokines were measured by ELISA. (E) HeLa CTRL cells and Bax/Bak-deficient cells were treated with the Mcl-1-inhibitor S63845. Concentrations of IL-6 in supernatants are shown (CTRL, n=8; Bax/Bak, n=7).",
    "molecules": "S63845"
  },
  {
    "caption": "F 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing Bcl-XL were treated with ABT-737. Concentrations of IL-6 in the supernatants were measured after 48 h (n=5).",
    "molecules": "ABT-737"
  },
  {
    "caption": "G 1205Lu human melanoma cells or 1205Lu melanoma cells over-expressing mouse Bcl-XL were treated with various concentrations of ABT-737. To some aliquots the caspase-inhibitor zVAD-fmk (50 μM) was added. Relative numbers of apoptotic cells were determined as in EV 1B after 48 h. Data are means/SEM of at least three independent experiments. (1205Lu, n=4; 1205Lu 5 µM ABT-737, n=3; 1205Lu/Bcl-XL and 1205Lu + zVAD, n=3).",
    "molecules": "ABT-737, zVAD, zVAD-fmk"
  },
  {
    "caption": "H HeLa-UL12.5 cells untreated or treated with tamoxifen (100 nM, 24 h) were stained with Picogreen (3 µl/ml) to detect mitochondrial DNA. Scale bare, 10 µm",
    "molecules": "DNA, Picogreen, tamoxifen"
  },
  {
    "caption": "I HeLa CTRL cells and HeLa-UL12.5 cells were treated with ABT-737 (10µM). Concentration of IL-6 in the supernatant were measured after 72h (n=3).",
    "molecules": "ABT-737"
  },
  {
    "caption": "A, The caspase-3-reporter cell line (labelled DEVD) and the control cell line (DEVG; see methods) were infected and analysed at various time points post-infection by flow cytometry (Chlamydia trachomatis (Ctr; MOI=1, 30 h); Salmonella Typhimurium (STy; MOI=50, 24h); Toxoplasma gondii (MOI=5, 16 h); MVA (MOI=10, 16h); Influenza A virus (IAV; MOI=1, 16h); Herpes simplex virus 1 (HSV-1; MOI=1, 16h). Staurosporine treated cells (1 μM, 3h) were used as a positive control. Percentages of cells positive for caspase-3-activation (FRET-loss) were determined. Data are means/SEM of three independent experiments. (n=3)",
    "molecules": "Staurosporine"
  },
  {
    "caption": "B, C Cells were infected with modified vaccinia virus Ankara (MVA) (MOI=10). The caspase-inhibitor zVAD-fmk was used at 100µM; cells were analysed 16 h post-infection.",
    "molecules": "zVAD-fmk"
  },
  {
    "caption": "C Chlamydial genomes per cell culture (expressed as chlamydial DNA in 50 ng of total DNA, determined by quantitative PCR). *, p<0.05 between control and mutant cells. (n=3)",
    "molecules": "DNA"
  },
  {
    "caption": "Setd1afl/fl;CreER cells were transfected with SETD1A  fragment and endogenous Setd1a  was deleted by tamoxifen, then assayed for colony-forming potential. Representative data from 1 out of 3 independent experiments with 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "qRT-PCR  was performed at 3 days post-tamoxifen  to analyze Fancd2  expression in human SETD1A-expressing Setd1afl/fl;CreER MLL-AF9 leukemia   cells. qRT-PCR  with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "293T  cells were transfected with SETD1A-GFP  fusion constructs. Cell nucleus  was visualized with DAPI. The percentage of GFP nuclear localization  is indicated in each image. Asterisks in images indicate the cells with nuclear  specific SETD1A-GFP  signals. Scale bars: 10 µm. G−H. Mouse Setd1afl/fl;CreER MLL-AF9 leukemia  cells were transfected with FO3-GFP  fusion constructs. (G) Subcellular localization  of GFP  fusion constructs was visualized by immunofluorescence  and nucleus  was stained  with DAPI. The percentage of GFP nuclear localization  is indicated in each image. Scale bars: 10 µm. (H) FO3-GFP  fusion expressing cells were treated with tamoxifen  and colony assay with 3 biological replicates was performed. Data information: data are presented as mean ± SD. **P ≤ 0.01, *P ≤ 0.05 (Student's t-test).",
    "molecules": "DAPI, tamoxifen"
  },
  {
    "caption": "Survival  of recipient mice  harboring dox-inducible shRNA-expressing leukemia  cells was plotted (n = 8-10 / group). Data information: **P ≤ 0.01  Kaplan-Meier  for (H)).",
    "molecules": "dox"
  },
  {
    "caption": "293T  cells were transfected with Myc-DDK-tagged wild-type BuGZ  and the cells were treated with 1,6-hexanediol for 3 h. The protein extracts were used for the Co-IP experiments.",
    "molecules": "1,6-hexanediol"
  },
  {
    "caption": "SETD1A-GFP  fusion-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, then assayed for  colony-forming potential. The FUCCI reporter (mCherry-Cdt1(30-120)/Citrine-Geminin(1-110))-expressing cells were used as control. Data from 3 biological replicates are shown. Data are presented as mean ± SD.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "SGC-expressing Setd1afl/fl;CreER MLL-AF9 leukemia  cells were treated with tamoxifen, and RNA-seq  analysis was performed with sorted cells. Data are shown as log2 fold change over tamoxifen  non-treated GFP-expressing control.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(B) Changes in plasma level of each cocktail substances in both supplementation and control studies (NR is detected in control study) compared to time baseline based on untargeted metabolomics measurement. The grey shaded area represents the 95% confidence level interval. For boxplots limits, the middle line represents the median. The upper and lower box limits represent the 25% quantiles. The upper and lower error bars correspond to 75% quantiles. The p-values are derived from one-way ANOVA (FDR< 0.05).",
    "molecules": "NR"
  },
  {
    "caption": "(B) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray.",
    "molecules": "serine"
  },
  {
    "caption": "(D) Plasma L-carnitine interpolations for each of the nine subjects.",
    "molecules": "L-carnitine"
  },
  {
    "caption": "(E) Model fit to the target concentration curve (mean interpolation of plasma L-carnitine over 24 hours) with standard deviation indicated in gray.",
    "molecules": "L-carnitine"
  },
  {
    "caption": "(G) Plasma NAC interpolations for each of the nine subjects.",
    "molecules": "NAC"
  },
  {
    "caption": "(H) Model fit to the target concentration curve (mean interpolation of plasma serine over 24 hours) with standard deviation indicated in gray.",
    "molecules": "serine"
  },
  {
    "caption": " The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating (n = 192 plants), expanding (n = 16 plants) and mature (n = 16) leaf developmental stage. Expression levels in wild-type plants transferred to mannitol-induced stress were compared to those transferred to control conditions at the same developmental stage. Data information: Data are presented as mean ± SEM, n = 4 independent experiments. FC = Fold change. * = FDR < 0.05, Unpaired two-sided Student's t-test. ",
    "molecules": "mannitol"
  },
  {
    "caption": "A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. The first group contains TFs that reached the log2(FC) threshold 40 min after mannitol treatment (A) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1. ",
    "molecules": "mannitol"
  },
  {
    "caption": "A The expression level of two GA-degradation and two GA-biosynthesis enzymes in expanding leaf tissue (third leaf at 15 DAS) of wild-type plants 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h after mannitol treatment. The fold changes (FC) were calculated relative to control conditions.Data information: In A, data are presented as mean ± SEM, n = 4 independent experiments, *= FDR<0.05 (unpaired two-sided Student's t-test)",
    "molecules": "mannitol"
  },
  {
    "caption": "Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I).",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells were immunoblotted for Beclin1, Atg7, capsid and actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. CHIKV, Chikungunya virus; CTRL, control; GFP, green fluorescent protein; DMSO, dimethylsulphoxide; siRNA, short interfering RNA.",
    "molecules": "DMSO"
  },
  {
    "caption": "Cells were infected for 9, 15 or 24 h and treated with DMSO (CTRL) or bafilomycin 3 h before the end of experiment. Whole‐cell lysates were immunoblotted for capsid or actin (I).",
    "molecules": "bafilomycin, DMSO"
  },
  {
    "caption": "Cells were treated with control (CTRL) (left) or NDP52 (right) siRNA, infected for 15 h, then labeled using antibodies to NDP52, nsP2 and puromycin. Quantitative analysis was performed by counting the percentage of infected cells with TGN‐associated RCs (n=30 cells per experiment) (D).",
    "molecules": "puromycin"
  },
  {
    "caption": "Cells were treated with control (CTRL) or NDP52 siRNA, infected for 15 and 24 h and treated with puromycin. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin. Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin (G).",
    "molecules": "Puromycin, puromycin"
  },
  {
    "caption": "A, B Intracellular NAD levels and NAD/NADH ratio in DC and age-matched healthy control fibroblasts. All values are presented as mean ± SD of four and eight replicates in A and B, respectively. Student's t test was performed on DC cells vs controls.",
    "molecules": "NAD, NADH"
  },
  {
    "caption": "Immunoblots of the expression of NAD synthesis and consuming proteins, and their activities in DC and control cells. Protein levels are normalized to GAPDH.",
    "molecules": "NAD"
  },
  {
    "caption": "E Immunoblots of the expression of NAD synthesis and consuming proteins Quantification values are presented as mean ± SD of four controls versus five DC samples as shown in D. Student's t test was performed on DC cells vs controls. Con: control fibroblasts.",
    "molecules": "NAD"
  },
  {
    "caption": "F Effects of NR and NAM supplementation (24 hours) on the NAD levels in DC fibroblasts. All values are presented as mean ± SD of four replicates. One-Way ANOVA was performed on DC cells in indicated conditions.",
    "molecules": "NR, NAD, NAM"
  },
  {
    "caption": "Immunoblots and quantification of indicated proteins in DC fibroblasts treated with DMSO and with 2 μM ATM inhibitor, KU-55933 for 48 hours. Mean (± SD) quantification values of three DC lines with and without ATMi treatment are shown. P values on the basis of Student's t test.",
    "molecules": "DMSO, KU-55933"
  },
  {
    "caption": "Intracellular NAD levels in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.",
    "molecules": "EX 527, NAD, Olaparib"
  },
  {
    "caption": "immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.",
    "molecules": "EX 527, Olaparib"
  },
  {
    "caption": "Intracellular NAD levels in brain tissues from 6-month- old G1 and G3 Tert-/- female mice.",
    "molecules": "NAD"
  },
  {
    "caption": "immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues.",
    "molecules": "NAD"
  },
  {
    "caption": "D NADase activity in G1 and G3 Tert-/- mouse brain lysate was determined at various time points during the reaction. NADase activity was not detectable in the extracts treated with the CD38 inhibitor, 78c, at a concentration of 50nM.",
    "molecules": "78c"
  },
  {
    "caption": "A Representative immunoblots of the expression of PGC1-1α in DC cells treated with vehicle or 3 mM NR. Quantification of the indicated proteins is from three immunoblots.",
    "molecules": "NR"
  },
  {
    "caption": "B-D Cellular and mitochondrial ROS in DC and age-matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates.",
    "molecules": "NR, ROS"
  },
  {
    "caption": "Cellular and mitochondrial ROS in DC and age-matched healthy control cells in the scramble and CD38 knockdown DC cells (D) were measured by flow cytometry from three replicates.",
    "molecules": "ROS"
  },
  {
    "caption": "E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR. White arrows: mitochondria. Yellow arrow: autophagosome-like structures with engulfed mitochondria. Enlarged image within white frame is shown. F-H Quantifications of percentage of damaged mitochondria per cell (F), mitochondrial length (G), and mitochondrial diameter (H). A minimum of 200 mitochondria counted per group.",
    "molecules": "NR"
  },
  {
    "caption": "Representative images of mitophagy showing co-localization between the mitophagy dye (red) and the lysosome dye (green) in DC and control fibroblasts treated with vehicle or 3 mM NR. White arrows: mitophagy. B The mean value of fluorescence intensity/cells in each image was scored. At least 15 images (~ 200 cells) were counted per group. The relative values in each group was normalized to Con1. ",
    "molecules": "NR"
  },
  {
    "caption": "C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR. Quantification of immunoblots from the indicated proteins is from three replicates.",
    "molecules": "NR"
  },
  {
    "caption": "A PCR amplification efficiency at the telomere in the mock- and FPG-treated DC and age-matched healthy control fibroblasts supplemented with vehicle or 3 mM NR. All values are presented as mean ± SD of three replicates. The relative telomere PCR amplification in each sample was normalized to the 36B4 reference gene.",
    "molecules": "NR"
  },
  {
    "caption": "B Telomere restriction fragment analysis in DC and control fibroblasts treated with vehicle or 3 mM NR. Genomic DNA was isolated from the indicated cell lines at 0 d or 14 d, with or without 3 mM NR treatment. At left, DNA molecular mass markers, in kilobase pairs (Kb).",
    "molecules": "NR"
  },
  {
    "caption": "C, D Representative images of γH2AX (green), telomere immuno-FISH (red) in DC cells treated with vehicle or 3 mM NR. White arrows: co-localization of γH2AX foci and telomeric DNA (or TIF). Enlarged views of co-localizing foci are shown at the right panel and in Figure EV1. Percentage of DC and control cells with indicated TIFs per nuclei. ~ 100 cells per condition were scored.",
    "molecules": "DNA, NR"
  },
  {
    "caption": "A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR. Each data point is represented as mean ± SD of three replicates.",
    "molecules": "NR"
  },
  {
    "caption": "B, C Representative images of BrdU (red) and DAPI (blue) staining of DC and age-matched healthy control fibroblasts treated with vehicle or NR. Percentage of BrdU -positive cells per condition. Each dot represents the percentage of cells with BrdU staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.",
    "molecules": "BrdU, DAPI, NR"
  },
  {
    "caption": "D, E Representative images of SPiDER-β-gal (green) and DAPI (blue) staining of DC and age-matched healthy control cells treated with vehicle or 3 mM NR. Percentage of SA-β-gal-positive cells per condition. Each dot represents the percentage of cells with SA-β-gal staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.",
    "molecules": "DAPI, NR"
  },
  {
    "caption": "F The levels of IL-6, IL-8, and MCP-1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR. All values are represented as mean ± SD of three replicates.",
    "molecules": "NR"
  },
  {
    "caption": "G, H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR. Quantification of the indicated proteins are presented as mean ± SD from three immunoblots.",
    "molecules": "NR"
  },
  {
    "caption": "(C) Mean percentages (± S.E.M.) of CD31+ area in E0771 shRenilla (n=3) or shApln (n=3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. (D) Mean percentages (± S.E.M.) of extravasated Dextran in E0771 shRenilla (n=9) or shApln (n=12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm",
    "molecules": "DAPI, Dextran"
  },
  {
    "caption": "(E) Mean counts (± S.E.M.) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n=6) or shApln (n=4) into C57BL/6J wild-type mice (5x105 cells/mouse). *P<0.05; t test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; Scale bars = 200 μm (upper panels) and 50 μm (lower panels).",
    "molecules": "pimonidazole"
  },
  {
    "caption": "(C) Differentially expressed genes using RNAseq transcriptome analysis of CD31+ endothelial cells (ECs) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF (30ng/mL) and DMSO (-Apln) and repaired Apelin GO sister cells stimulated with VEGF and Apelin (1000nM) (+Apln). VEGF target genes and angiogenesis-related genes, predicted by Ingenuity Pathway Analysis (IPA) software, are indicated by bars on the upper axis of the heatmap. GO terms were analyzed by DAVID on-line software.",
    "molecules": "DMSO, VEGF"
  },
  {
    "caption": "(A) Experimental set up and (right) Kaplan Meier survival plot of NeuT;Apln+/+ and NeuT;Apln-/- mice with mammary cancer, left untreated (control) or treated with the indicate dose of the broad VEGFR blocker sunitinib. NeuT;Apln+/+ Control (n=8), NeuT;Apln-/- Control (n=11), NeuT;Apln+/+ Sunitinib (n=11), NeuT;Apln-/- Sunitinib (n=12); *P<0.05; Log rank test. Mice were sacrificed when the tumor size reached 1cm3, following ethical guidelines. The dotted line indicates 50% of survival.",
    "molecules": "sunitinib, Sunitinib"
  },
  {
    "caption": "(B) Percentages (mean ± S.E.M.) of tumor burden in mammary glands of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 4 weeks after tumor onset. NeuT;Apln+/+ Control (n=6), NeuT;Apln-/- Control (n=8), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); *P<0.05; ***P<0.001; one-way ANOVA. Representative H&E images are shown for each genotype in the right panels. Scale bars = 1000 μm (large panels) and 50 μm (insets).",
    "molecules": "sunitinib, Sunitinib"
  },
  {
    "caption": "(C) Tumor volumes of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mammary tumors, size-matched at 20-70mm3 and followed over time by MRI analysis; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=6) mice per group; Lines represent nonlinear fit of tumor growth. Box and arrow indicate the time point used for analysis in panel D. (D) Mitotic counts (mean ± S.E.M.) and representative H&E images of mammary tumors in untreated (control) ­and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after tumor onset.; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=6), NeuT;Apln+/+ Sunitinib (n=9), NeuT;Apln-/- Sunitinib (n=4) tumors per group; *P<0.05; one-way ANOVA to sunitinib-treated NeuT;Apln-/-. White arrows indicate mitotic figures. Scale bars = 50 μm",
    "molecules": "sunitinib, Sunitinib"
  },
  {
    "caption": "(E) Tumor volumes, followed over the indicated time, of orthotopically injected E0771 shRenilla cells left untreated (control) or treated three times a week from day 8 after tumor injection with an Apelin antagonist alone (MM54, 0.4μg/g), sunitinib alone (60mg/kg) or a combination of both. Tumor volumes were measured using calipers and are shown as mean tumor volumes ± S.E.M. E0771 shRenilla Control (n=6), MM54 (n=5), Sunitinib (n=5), Sunitinib+MM54 (n=5); *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA.",
    "molecules": "MM54, sunitinib, Sunitinib"
  },
  {
    "caption": "(B) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of relative tumor blood volume (rTBV) over time after NeuT+ mammary tumors were size-matched at 20-70mm3 (0 weeks). Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); **P<0.01, and ***P<0.001 compared to untreated NeuT;Apln+/+ mice and #P<0.05 compared to untreated control NeuT;Apln-/- mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analyzed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus, we did not perform any statistical analysis on the 6 weeks timepoints.",
    "molecules": "Sunitinib"
  },
  {
    "caption": "(D) Analysis (mean values ± S.E.M.) of CD31+ area (x104 μm2)/field, number of dilated tumor vessels and percentage of alphaSMA+ area per CD31+ blood vessels in mammary tumors of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=4); *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA and Kruskal-Wallis test. (E) Representative immunofluorescence and immunohistochemistry images from Fig. 5D quantification. Dilated blood vessels are marked by a red asterisk. DAPI (blue) is shown as a counterstain to visualize nuclei. Scale bars = 100 μm (upper panels), 50 μm (middel panels) and 20 μm (lower panels).",
    "molecules": "DAPI, sunitinib, Sunitinib"
  },
  {
    "caption": "(A) Percentages of CA9+ cells adjacent to CD31+ tumor blood vessels in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=4); 100-200 peri-vascular intra-tumoral regions per group were counted. ***P<0.001; one-way ANOVA. Right panels show representative immunofluorescent images. Areas limited by dotted white lines indicate CA9+ areas. Scale bars = 50 μm.",
    "molecules": "sunitinib, Sunitinib"
  },
  {
    "caption": "(B) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of vessel permeability (K2) in NeuT+ mammary tumors followed over time. Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=5); ***P<0.001, compared to untreated control NeuT;Apln+/+ mice and §P<0.05 compared to sunitinib-treated NeuT;Apln+/+ mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analysed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus we did not perform any statistical analysis on the 6 weeks timepoints.",
    "molecules": "Sunitinib, sunitinib"
  },
  {
    "caption": "(A) Number of metastatic lung foci in untreated (control) and sunitinib-treated (60mg/kg, three times a week from tumor initiation) NeuT;Apln+/+ or NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. Data of individual lung sections and means (black bars) are shown. Right panels show representative H&E images, where black arrows and insets indicate metastatic foci. Scale bars = 1000 μm (large panels) and 50 μm (insets). *P<0.05, **P<0.01; ***P<0.001; Kruskal-Wallis test; n=3 mice per cohort and three sections per lung were analysed.",
    "molecules": "sunitinib"
  },
  {
    "caption": "(C) Kaplan-Meier plot for progression-free survival stratifying RCC patients with high and low APELIN serum levels 3-5 months after the start date of sunitinib treatment. *P=0.0367; Log rank test.",
    "molecules": "sunitinib"
  },
  {
    "caption": "(D) Kaplan Meier plots for progression-free survival in RCC patients stratified into groups of high or low levels of APELIN and VEGF. Cut-off levels were set by the median. Serum was analysed 3-5 months after the start date of sunitinib treatment. *P<0.05, **P<0.01; Log rank test.",
    "molecules": "sunitinib"
  },
  {
    "caption": "A AP was induced by 5 injections (IP) of cerulein at a dosage of 50 μg/kg. SAP was induced by AP and one injection of lipopolysaccharide (LPS, IP) at a dosage of 20 mg/kg. AP+ANGPTL4 (2 mg/kg) was induced by AP and one injection of ANGPTL4 (IP) at a dosage of 2 mg/kg. ANGPTL4 (4 mg/kg) was induced by two injections of ANGPTL4 (IP) at a dosage of 2 mg/kg. Mice were sacrificed 6 h after pancreatitis induction. (H&E and immunofluorescent staining for IL-1β (red) and amylase (green) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues (n = 10, each group). Scale bar represents 50 µm",
    "molecules": "cerulein, lipopolysaccharide, LPS"
  },
  {
    "caption": "A For macrophage depletion models, mice (six weeks) were injected with GdCl3 (15 mg/kg). After the induction of macrophage depletion in the mice, pancreatitis was induced by cerulein, LPS, and ANGPTL4 for 24 h (n = 10, each group). H&E staining of pancreatic tissue, and levels of amylase and lipase in the serum. Scale bar represents 100 µm",
    "molecules": "cerulein, GdCl3, LPS"
  },
  {
    "caption": "A Cell morphology and the mRNA levels of inflammatory cytokines, including and TNF-α, IL-6, and IL-1β in mouse primary macrophages treated with LPS and ANGPTL4 (100 ng/ml) using qPCR and RT-PCR, and levels of TGF-β and NO in the LPS- and ANGPTL4-treated primary macrophages (n = 5). Scale bar represents 100 µm",
    "molecules": "LPS, NO"
  },
  {
    "caption": "C Mouse primary acinar cells and macrophages were labelled with fluorescence dyes (macrophages, red and acinar cells, green). Co-cultured cells treated with 100 ng/ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h. Scale bars represent 80 µm (red) and 40 µm (white).",
    "molecules": "LPS"
  },
  {
    "caption": "D To explore whether ANGPTL4 affects JNK, AMPK, and PI3K/AKT signaling, each inhibitor (SP600125, compound C, and HS-173, 1 µM) was treated to in THP1-derived macrophages for 2 h after ANGPTL4 treatment (100 ng/ml) for 6 h.",
    "molecules": "SP600125, compound C, HS-173"
  },
  {
    "caption": "E C5a, C5aR, TNF-α, IL-6, and IL-1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS (100 ng/ml). In the transwell experiment, mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 (100 ng/ml). Expression of ANGPTL4, C5aR, and C5a was determined in siANGPTL4-treated THP1-derived macrophage after LPS activation.",
    "molecules": "LPS"
  },
  {
    "caption": "F In the transwell experiment, TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist (W-54011, 10 nM) pretreatment for 12 h prior to treatment with ANGPTL4, LPS, and C5a (100 ng/ml) for 24 h to macrophage in transwell. Scale bar represents 20 µm",
    "molecules": "LPS, W-54011"
  },
  {
    "caption": "C After macrophages were activated with LPS (100 ng/ml), the cells were treated with either neutralizing ANGPTL4 or C5a antibody (100 ng/ml) or an isotype control (IgG). ANGPTL4, C5a, TNF-α, and IL-6 levels were measured in culture media (n = 5).",
    "molecules": "LPS"
  },
  {
    "caption": "D. Expression data of genes associated with inflammatory monocytes, activated neutrophils and pro-inflammatory cytokines were compared between acute asymptomatic (n=30) and asymptomatic (n=26) COVID-19 patients and were represented by ratio with respect to expression in asymptomatic patients. Bar graphs show DEGs with FDR adjusted P value < 0.05 and fold change values are represented in the log2 scale.",
    "molecules": "cytokines"
  },
  {
    "caption": "f, cytokines and chemokines transcripts in the nasal turbinates at 4dpi in male and female SARS-CoV-2 infected hamsters, treated with saline or with 400 µg/kg ivermectin. Data information: Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.",
    "molecules": "ivermectin, saline"
  },
  {
    "caption": "a, representative  histopathology photomicrographies of lungs according to the different groups: mock_saline, CoV_saline and CoV_ivermectin. Top panels: whole lung  sections. Bottom panels: high magnification. CoV_saline section exhibits important congestion (*), edema associated with few mononuclear cells (white arrowheads). Note the thickening of the  alveolar walls. CoV_ivermectin section exhibits important amounts of mononuclear cells (black arrowheads) and less marked signs of congestion  or edema. Hematoxylin and Eosin. Scale bars = 1 mm (top panels) and 20 μm (bottom panels).",
    "molecules": "Eosin, Hematoxylin, ivermectin, saline"
  },
  {
    "caption": "c, quantification of  Iba1+ cells, Arg1+ cells and Iba1+Arg1+ cells in the lungs. mock_saline n=3 (males), mock_ivermectin n=4 (males), CoV_saline n=9 (6 males and 3  females),  CoV_ivermectin n=6 (4 males and 2 females). Data information: M: male hamsters; F: female hamsters. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test",
    "molecules": "ivermectin, saline"
  },
  {
    "caption": "C, D Growth rates as measured by the change in optical density (OD595) over time for wild type (C) and Torin1-resistant (D) cells upon 5 µM of Torin1 treatment (at T = 0 min). Respective strains were treated with the DMSO vehicle alone to control for potential effects of the solvent on cell growth. Logarithmic values of the OD595 increase are plotted against time in minutes. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD). Linear regressions were fitted using the curve fitting function on Prism9. Ratios in the rate of mass doubling (as calculated from gradient of regression lines detailed in Methods) of Torin1 treated cells to DMSO control were 0.34 in wild type (C) and 0.97 for Torin1-resistant (D) cells.",
    "molecules": "DMSO, Torin1"
  },
  {
    "caption": "E Rate of protein synthesis as measured by the amount of HPG incorporation after 20 mins of differing time intervals (0 - 60 mins) of 5 µM Torin1 for wild type (green) and tor1∆ (yellow). Values are normalised to T = 0 minutes. Exponential one-phase decay non-linear regressions were fitted using the curve fitting function on Prism9. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD).",
    "molecules": "HPG, Torin1"
  },
  {
    "caption": "C - F Phosphorylation changes of the conserved phosphosites, S235 and S236, on Rps601(C) and Rps602 (D - F), over 85 minutes of Torin1 (5 µM) treatment wild type (green) and Torin1-resistant (brown) cells. M-values (in brackets) represent the different multiplicities of the same phosphosites. Lines were fitted with the curve fitting function on Prism9 using the the non-linear regressions exponential one-phase decay (C, D) or plateau followed by one-phase decay (E, F) functions respectively. Grey dashed lines indicate 2-fold threshold.",
    "molecules": "Torin1"
  },
  {
    "caption": "A Relative protein expression levels of the 3 S6 kinase homologues, Psk1, Sck1 and Sck2, plotted against time of Torin1 addition. Respective proteins are represented by the different coloured lines as detailed in the legend.",
    "molecules": "Torin1"
  },
  {
    "caption": "A, B Phosphosites identified on (A) Tif212 and (B) Erf1 respectively showing 2-fold phosphorylation change within 20 minutes of Torin1 (5 µM) addition. Both wild type (green) and tor1∆ (yellow) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells (T=0 minutes). Grey dashed lines indicate 2-fold threshold.",
    "molecules": "Torin1"
  },
  {
    "caption": "E, F Changes in rates of protein synthesis of the tif471_18A mutant compared to the wild type control (tif471+) upon Torin1 (5 µM) treatment. Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17% for the tif471_18A phosphomutant and 4.6% for the tif471+ control relative to starting levels in the respective strains. The two graphs represent the untransformed (E) and log2 transformed (F) rates respectively. Error bars represent the standard deviation (SD) of data aggregated from 3 independent experiments.",
    "molecules": "Torin1"
  },
  {
    "caption": "C The fluorescence signals of PIWI proteins were determined in  locust brains.  Hochest33342  was used to label cell nuclei in locust brains. The scale bars represent 20 µm.",
    "molecules": "Hochest33342"
  },
  {
    "caption": "C Representative images of Nile red staining  of lipid droplets in the fat bodies  of dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). The scale bars represent 50 µm. D Quantification of  lipid droplet staining   in dsPiwi1-treated locusts  and dsGFP-treated control  locusts (n=5 biological replicates). E Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.",
    "molecules": "Nile red"
  },
  {
    "caption": "E, F Measurements of  glycogen (n=5 biological replicates) and TAGs (n=10 biological replicates) in fat bodies. Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old  fourth-instar locusts.",
    "molecules": "glycogen, TAGs"
  },
  {
    "caption": "D, E Representative images (D) and quantification (E) of Nile red staining  of fat body lipid droplets in dsNPF1-treated locusts  and dsGFP-treated controls (n=5 biological replicates). The scale bars represent 50 µm, the data are shown as the mean ± SEM (Student's t-test, **P<0.01).",
    "molecules": "Nile red"
  },
  {
    "caption": "A U6 RNA was used as a nuclear RNA control (relative value set to 1 in the nuclear  fraction), and glycine tRNA was used as the cytoplasmic  RNA control (relative value set to 1 in the  cytoplasmic fraction) (n=4 biological replicates), U6 and   tRNA-Gly  was used as an endogenous control.",
    "molecules": "glycine tRNA, tRNA-Gly"
  },
  {
    "caption": "Oligo-FISH images for the representative piRNA target region described in Fig 4A, upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin immunofluorescence staining. Scale bar, 5μm. Ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.",
    "molecules": "DAPI"
  },
  {
    "caption": "Oligo-FISH images for changes in the intra-TAD interaction at the TAD described in (C), upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, green and magenta indicates FISH signal at the different positions of TADs (TAD-1 and TAD-2). Details of TAD probe design is described in Fig EV2C. Scale bar, 5μm. Distance between two FISH signals is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.",
    "molecules": "DAPI"
  },
  {
    "caption": "Boxplots showing fold changes in the histone mark association of piRNA target or non-target TEs based on ChIP-seq upon Piwi-KD (Definition of piRNA target and non-target TEs are described in Fig EV1E). Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated by Wilcoxon rank-sum test.",
    "molecules": "histone"
  },
  {
    "caption": "Oligo-FISH images for the reporter of the tethering system, at 0h (control), 48h hours, 96 hours after transfection of λN-Nxf2 expression vector. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin staining. Scale bar, 5μm. The ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.",
    "molecules": "DAPI"
  },
  {
    "caption": "D) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Inset: Example of surface synaptic AMPARs cluster (GluA&Bsn). Scale bar: 5 µm. E) Quantification of the surface synaptic AMPARs (GluA&Bsn) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure (Ctr=1±0.051, TTX=1.512±0.080, PTX3=1.294±0.081, PTX3 heat-inactivated=0.959±0.044; number of fields examined: 27, 36, 37, 22, 19 respectively; one-way ANOVA analysis of variance, p<0.0001, followed by post hoc Tukey test for multiple comparison as indicated in figure; 3 independent experiments, data are presented as normalized mean values±SEM). F) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content (Ctr: 1±0.065, PTX3: 1.023±0.068; number of fields: Ctr=26, PTX3=26; unpaired t-test, 3 independent experiments, data are presented as normalized mean values±SEM)",
    "molecules": "TTX"
  },
  {
    "caption": "J) Representative images of 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. K) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase upon N-terminal peptide application, but not upon C-terminal peptide exposure. TTX is used as positive control (Ctr=1.000±0.066. TTX=1.395±0.113, C-term=0.717±0.047, N-term=1.349±0.069. Number of fields examined: 55, 31, 18, 54, respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM). ",
    "molecules": "TTX"
  },
  {
    "caption": "E) Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan (red), DAPI (cyan) and βIII-tubulin (green) and confocal analysis. Scale bar: 20µm",
    "molecules": "DAPI, hyaluronidase"
  },
  {
    "caption": "G) HAse treatment blocks PTX3-induced synaptic surface AMPA receptors clustering (Ctr=1.000±0.075, PTX3=1.425±0.088, PTX3+HAse=0.961±0.053, HAse=1.080±0.087. Number of fields examined: 37, 23, 53, 28 respectively; Kruskall-Wallis test, p=0.0004 followed by post hoc Tukey test as indicated in figure; 3 independent experiments, data are presented as normalized mean value±SEM)",
    "molecules": "HAse"
  },
  {
    "caption": "I) Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures. On the contrary WT cultures (from littermates) display increased surface GluA&Bsn/Bsn upon PTX3 treatment. A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures (WT=1±0.06; WT+PTX3=1.389±0.113; WT+TTX=1.698±0.109; Number of fields examined: 40, 39, 32 respectively; Kruskall-Wallis test p<0.0001 followed by post hoc Dunn"s test. TSG6 KO=1.000±0.047; TSG6 KO+PTX3=0.979±0.048; TSG6 KO+TTX=1.363±0.077. Number of fields examined: 54, 57, 29 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. n=3 independent experiments, data are presented as normalized mean values",
    "molecules": "TTX"
  },
  {
    "caption": "D) Representative mEPSC traces recorded from the indicated experimental conditions. E) mEPSC frequency quantitation showing that pre-incubation with PD98059 (30μM) completely prevents the PTX3-dependent increase of mEPSC frequency (Hz, Ctr=0.911±0.104; PTX3=1.748±0.273; PTX3+PD=0.518±0.055; PD=0.491±0.104. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. One-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. Data are presented as a distribution plus mean±SEM). F) inset: average mEPSC amplitude quantitation (pA), Ctr=14.19±0.70; PTX3=19.09±1.71; PTX3+PD=14.31±0.70, PD=16.51±1.17. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. Kruskall-Wallis test, p=0.015 followed by Dunn"s test as indicated in figure, data are presented as a distribution plus mean±SEM. Cumulative probability plot of mEPSC amplitudes are analyzed with Kolmogorov-Smirnov test: Ctr vs PTX3: p<0.0001, D=0.183, PTX3 vs PTX3+PD: p<0.0001, D=0.251, PTX3 vs PD: p<0.0001, D=0",
    "molecules": "PD, PD98059"
  },
  {
    "caption": "H) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn [(GluA&Bsn)/Bsn] shows a statistically significant increase after PTX3 exposure, while pre-incubation with PD98059 prevents the PTX3-dependent increase (Ctr=1±0.042, PTX3=1.499±0.121, PTX3+PD=1.093±0.060, PD=1.017±0.117; number of fields examined: 49, 15, 15, 20 respectively; one-way ANOVA analysis of variance, p=0.0002 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM)",
    "molecules": "PD, PD98059"
  },
  {
    "caption": "I) Quantification of surface synaptic GluA upon PTX3 application and simultaneous inhibition of β1 integrin and ERK1/2 signaling pathways reveals no additive effects with respect to single αCD29 or PD applications (Ctr=1±0.061; PTX3=1.828±0.151; αCD29+PD+PTX3=0.766±0.070; PTX3+αCD29=1.043±0.073; PTX3+PD=1.016±0.061; PD=0.969±0.090; number of fields examined: 29, 31, 30, 23, 14, 33 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Dunn's test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).",
    "molecules": "PD"
  },
  {
    "caption": "Cell viability analysis using CellTiter-Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs (n = 4 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). SM represents SM-164. Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.",
    "molecules": "DMSO, Mel, SM, SM-164"
  },
  {
    "caption": "TNF-induced complex-II immuno-precipitation. BN175 cells were treated with the indicated agents for 8 hrs. FADD immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).",
    "molecules": "DMSO, Mel, SM, zVAD"
  },
  {
    "caption": "Proximity ligation assay between Ripk1 and Casp-8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs (n = 3 biological replicates, shown are representative images). Scale bar represents 10 μm. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).",
    "molecules": "DMSO, Mel, SM, zVAD"
  },
  {
    "caption": "DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.",
    "molecules": "DMSO, Mel, SM"
  },
  {
    "caption": "Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 knockouts (KO) cells treated with the indicated agents for 18 and 48 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.",
    "molecules": "SM, SM-164"
  },
  {
    "caption": "TNF-induced complex-II immuno-precipitation. HT1080 cells were treated with the indicated agents for 8 hrs. CASP-8 immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml).",
    "molecules": "DMSO, SM, zVAD"
  },
  {
    "caption": "Cell death analysis measured by Celigo of PI-positive HT1080 cells, cells were treated with the indicated agents for 24 hrs. (n = 3 biological replicates). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and an unpaired t-test was performed, **** P ≤ 0.0001.",
    "molecules": "DMSO, PI, SM, SM-164, zVAD"
  },
  {
    "caption": "Cell death analysis by Celigo of PI-positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed, **** P ≤ 0.0001.",
    "molecules": "DMSO, PI, SM, SM-164"
  },
  {
    "caption": "TNF-induced complex-II immuno-precipitation. A375 cells were treated with the indicated agents for 6 hrs. Caspase-8 immuno-precipitation was performed followed by western blot analysis. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) (n = 3 biological replicates, shown is a representative experiment).",
    "molecules": "DMSO, SM, zVAD"
  },
  {
    "caption": "Cell death analysis by Celigo of PI-positive A375 treated with the indicated agents for 12 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.",
    "molecules": "DMSO, PI, SM, SM-164, zVAD"
  },
  {
    "caption": "Cell viability analysis using CellTiter-Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL, **** P ≤ 0.0001.",
    "molecules": "DMSO, SM, SM-164"
  },
  {
    "caption": "Cell death analysis by Celigo of PI-positive MeWo (G), DO4 (H) or SW-872 (I) cells. Cells were treated with the indicated agents for 24 hrs (MeWO and SW-872) or 12 hrs (DO4). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.",
    "molecules": "DMSO, PI, SM, SM-164, zVAD"
  },
  {
    "caption": "Cell death analysis by Celigo of PI-positive HS-ES-2R Cells were treated with the indicated agents for 48 hrs (HS-ES-2R) DMSO (Unt), zVAD (10 µM), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.",
    "molecules": "DMSO, PI, SM, SM-164, zVAD"
  },
  {
    "caption": "Cell death analysis by Celigo of PI-positive HS-SY-II cells. Cells were treated with the indicated agents for 24 hrs (HS-SY-II). DMSO (Unt), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.",
    "molecules": "DMSO, PI, SM, SM-164"
  },
  {
    "caption": "Cell Death analysis by IncuCyte Zoom of CellTox Green-positive MET1 (L), MET2 (M) and IC8 (N) cells. Cells were treated with the indicated agents for 40 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=6, and statistical analysis was performed with a one-way Anova, *** P ≤ 0.001, **** P ≤ 0.0001.",
    "molecules": "CellTox Green, DMSO, SM, SM-164, zVAD"
  },
  {
    "caption": "Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, * P ≤ 0.05, *** P ≤ 0.001.",
    "molecules": "Birinapant, SM"
  },
  {
    "caption": "The addition of SM to the standard-of-care treatment ILP-TNF/Mel significantly prolonged survival compared to either modality alone (n = 6 rats per cohort). Statistical analysis was performed with a log-rank test, ** P ≤ 0.01.",
    "molecules": "SM, Mel"
  },
  {
    "caption": "Median TNF/Mel is 0.4472 and TNF/Mel/SM is 1.154. TNF/Mel 25th percentile limit 0.02887 and 75th percentile limit 0.7627. TNF/Mel/SM 25th percentile limit 1.136 and 75th percentile limit 1.67. TNF/Mel Min error bar -0.2389 and Max 0.7687. TNF/SM/Mel Min error bar 1.12 and Max 1.813. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ** P ≤ 0.01.",
    "molecules": "SM, Mel"
  },
  {
    "caption": "DEVDase activity assay of parental BN175 cells and drug-tolerant tumour cell lines isolated from ILP-TNF/Mel/SM treated animals (124, 133, 136), treated with the indicated drugs for 6 hrs (n = 4 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml).",
    "molecules": "SM, DMSO, Mel"
  },
  {
    "caption": "Cell viability analysis using CellTiter-Glo of BN175 parental and 124, 133 and 136 cells treated with the indicated drugs for 24 hrs (n = 3 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Statistical analysis was performed with a one-way Anova, ns=not significant.",
    "molecules": "DMSO, Mel, SM, SM-164"
  },
  {
    "caption": "Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 KO cells treated with the indicated agents for 24 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), SM (100 ng/ml) and Riboxxol (1 µg/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.",
    "molecules": "DMSO, Riboxxol, SM, SM-164"
  },
  {
    "caption": "Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP with TNF/Mel/SM significantly increased infiltration by CD3+ lymphocytes (B) and CD3-CD161+ NK cells (C).",
    "molecules": "Mel, SM"
  },
  {
    "caption": "Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). While ILP treatment with TNF/Mel significantly reduced Granzyme B positive NK cells (D), the numbers of Granzyme B positive NK cells were restored to normal levels upon co-treatment with SM (TNF/Mel/SM).",
    "molecules": "Mel, SM"
  },
  {
    "caption": "Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). Importantly, ILP-mediated treatment with TNF/Mel/SM significantly increased the numbers of CD3+CD8+ cytotoxic T cells (E).",
    "molecules": "Mel, SM"
  },
  {
    "caption": "Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP-TNF/Mel/SM significantly upregulated Granzyme B in CD3+CD8+CD161- cytoxtoxic T cells",
    "molecules": "Mel, SM"
  },
  {
    "caption": "ILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H).",
    "molecules": "Mel, SM"
  },
  {
    "caption": "Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD.",
    "molecules": "Birinapant, SM"
  },
  {
    "caption": "The addition of either anti-CTLA-4 or anti-PD-1 antibodies prolonged survival in comparison to ILP-TNF/Mel/SM alone (n = 6 animals per cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.",
    "molecules": "Birinapant, SM, Mel"
  },
  {
    "caption": "Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-CTLA-4 antibodies led to a significant increase in CD4+ Foxp3- helper T cells (C) accompanied by a depletion of CD4+ Foxp3+ regulatory T cells (D). SM represents Birinapant.",
    "molecules": "Birinapant, SM"
  },
  {
    "caption": "Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-PD-1 antibodies led to an increase in Granzyme B (GzmB) expression on NK cells (E). SM represents Birinapant.",
    "molecules": "Birinapant, SM"
  },
  {
    "caption": "Tumour volumes at the time of surgery following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ****, P ≤ 0.0001.",
    "molecules": "Birinapant, SM, Mel"
  },
  {
    "caption": "Individual growth curves following surgery indicated improved control following ILP-TNF/Mel/SM (n=6 animals per cohort).",
    "molecules": "SM, Mel"
  },
  {
    "caption": "Tumour volume on re-challenge with BN175 sarcoma on contralateral limb of long-term survivors following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). Unless otherwise stated, SM represents Birinapant. Error bars represent S.D.",
    "molecules": "Birinapant, SM, Mel"
  },
  {
    "caption": "ILP-TNF/Mel/SM significantly delayed growth leading to prolonged survival on re-challenge (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.",
    "molecules": "Birinapant, SM, Mel"
  },
  {
    "caption": "A Confocal images showing the basal regions of cochlear explants treated with either culture medium alone or medium containing 5 µM, 10 µM, or 20 µM CDDP for 3 days. The explants were labeled with Myosin 7A (red) to identify hair cells and NF200 (green) to highlight auditory nervefibers. Scale bar=20 µm. nf: nerve fibers, OHCs: outer hair cells, IHCs: inner hair cells.",
    "molecules": "CDDP"
  },
  {
    "caption": "B Dose-response curves of CDDP-induced loss of OHCs (blue line) and IHCs (red line) in basal cochlear regions. Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).",
    "molecules": "CDDP"
  },
  {
    "caption": "C Effect over time on OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively). Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).",
    "molecules": "CDDP"
  },
  {
    "caption": "D Representative Western blot analysis using antibodies against β-actin and cleaved caspase 3 in whole cochlear extracts.E Histogram representing the change in cleaved caspase 3 expression levels over time in control and CDDP group (n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P ≤ 0.028, **P=0.0007, cochleae at the different times after CDDP exposure vs. control 12h).",
    "molecules": "CDDP"
  },
  {
    "caption": "F, G Confocal images showing the basal region of cochlear explants treated with either culture medium alone or containing 10 µM CDDP for 1 (F) or 2 (G) days. Hair cells were identified using Myosin 7A (red), phosphatidylserine sites on the cell membrane surface were detected using fluorochrome-labeled Annexin V (green in F), and apoptotic DNA fragmentation was identified using a TUNEL apoptosis kit (green in G). The white arrow heads indicate cell surface Annexin V-positive labeling (lower left) and TUNEL-positive nuclei (lower right). Scale bar=15 µm.",
    "molecules": "phosphatidylserine, CDDP, DNA"
  },
  {
    "caption": "A, F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and γH2AX (green in A), or Myosin 7A (red) and 53BP1 (green in F). The white arrow heads indicate CDDP-induced increase of γH2AX foci in both OHCs and IHCs. Scale bars: A and F=24 µm.B, G Higher magnification images of representative OHC and IHCnuclei from all conditions tested. Scale bar=5 µm.C, H Histograms displaying green fluorescent signal intensity of x-projections and 3D reconstruction images from OHC and IHCnuclei presented in B and G, respectively. F0 corresponds to background noise, gray dashed lines represent the threshold used to detect specific foci labeling. 3D images were reconstructed according to the threshold defined in the histograms of all conditions tested. Scale bar=5 µm.D, E, I Quantification analysis of γH2AX foci number (D); total volume of foci per nucleus (E); 53BP1 foci number (I) from OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (D: **P=0.007; E and I: **P ≤ 0.009. CDDP-exposed OHCsvs. CDDP-exposed IHCs).J Higher magnification images showing representative OHCnuclei from the basal region of the organ of Corti treated with 10 µM CDDP for 1 day and immunolabeled for 53BP1 (red), γH2AX (green) and counterstained with Hoechst (blue). The 3D image shows co-localization of smaller sized 53BP1 foci within the γH2AX foci. Scale bar=5 µm.",
    "molecules": "CDDP"
  },
  {
    "caption": "A Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-ATM (green). Scale bar=15 µm.B Higher magnification images showing representative OHC and IHCnuclei from control and CDDP-exposed cells. Scale bar=5 µm.C Histograms displaying green fluorescent signal intensity of x-projections of OHC and IHCnuclei from control and CDDP-exposed cells.D Quantification analysis of p-ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone (light blue and red lines for OHCs and IHCs, respectively) or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (**P<0.008, ***P=0.00005, CDDP-exposed OHCsvs. CDDP-exposed IHCs).E Higher magnification confocal and 3D images showing the co-localization of p-ATM foci (green) with 53BP1 foci (red) in an OHCnucleus from the basal region of the organ of Corti culture treated with 10 µM CDDP for 1 day. Scale bar=5 µm.",
    "molecules": "CDDP"
  },
  {
    "caption": "F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-Chk2 (green). Scale bar=15 µm.G Histograms of green fluorescent signal intensity from OHC and IHCnuclei of a CDDP-treated organ of Corti explant.H Quantification of Chk2-positive nuclei in both IHCs (red bars) and OHCs (blue bars) from control and CDDP-treated organ of Corti cultures (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (*P=0.045, ***P=0.0002, CDDP-exposed IHCsvs. control IHCs; CDDP-exposed OHCsvs. control OHCs).",
    "molecules": "CDDP"
  },
  {
    "caption": "I Representative western blots using antibodies against p-p53 (serine 15), p53 and β-actin in control and 10 µM CDDP-exposed whole cochlear extracts.J-K Histograms representing the levels of total p53 protein (J) and p53 phosphorylation (K) in control (gray bars) and CDDP-exposed cochleae (green bars, n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (J: *P ≤ 0.023; K: *P=0.032, **P=0.007, ***P ≤ 0.0008, cochleae at the different times after CDDP exposure vs. control 12h).",
    "molecules": "CDDP"
  },
  {
    "caption": "A 3D images of OHCnuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.B Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0004, CDDPvs. control, or CDDPvs. CDDP+KU55933).",
    "molecules": "KU55933, CDDP"
  },
  {
    "caption": "C Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days and immunolabeled for Myosin 7A (red). Scale bar=24 µm.D Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days (n=5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P=0.03, **P =0.004, ***P ≤ 0.0008, CDDPvs. control, or CDDPvs. CDDP+KU55933).",
    "molecules": "KU55933, CDDP"
  },
  {
    "caption": "E 3D images of OHCnuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 µM PFT-α, 10 µM CDDP or 10 µM CDDP in combination with 100 µM PFT-α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.F Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0007, CDDPvs. control).",
    "molecules": "CDDP, PFT-α"
  },
  {
    "caption": "A Representative auditory brainstem response (ABR) waveforms evoked by 16 kHz tone-bursts in p53wt mice treated with saline (black plot) or CDDP (green plot), and p53-/- mice treated with saline (light blue plot) or CDDP (dark blue plot) for 5 days.B ABR thresholds recorded in p53wt mice before (gray plot) and after 5 days of saline (black plot) or CDDP treatments (green plot), and ABR thresholds recorded in p53-/- mice before (light gray plot), and after 5 days of saline (light blue plot) or CDDP treatment (dark blue plot).C Mean ABR threshold from 4 kHz to 32 kHz derived from B. Data are expressed as mean ± SEM (saline treated-group: n=7, CDDP treated-group: n=12). One-way ANOVA test followed by post hoc Tukey's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt, before or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).",
    "molecules": "CDDP"
  },
  {
    "caption": "D Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP-treated p53wt and p53-/- mice after 5 days. Scale bar=15µm.",
    "molecules": "CDDP"
  },
  {
    "caption": "E Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apex provided from saline-treated p53wt mice (black bars), CDDP-treated p53wt mice (green bars) or p53-/- mice (blue bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt+saline, d5 or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).",
    "molecules": "CDDP"
  },
  {
    "caption": "F ABR thresholds from p53wt mice recorded prior to (gray plot) or after 5 days systemic treatment with: DMSO (yellow plot), CDDP+DMSO (green plot), CDDP+PFT-α (pink plot).G Mean ABR threshold from 4 kHz to 32 kHz derived from F. Data are expressed as mean ± SEM (DMSO treated-group: n=7; CDDP+DMSO treated-group: n=12; CDDP+ PFT-α-treated group: n=12). One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0005, CDDP+DMSO, d5 vs. before or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "H Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from p53wt mice treated with: DMSO (yellow bars), CDDP+DMSO (green bars), CDDP+PFT-α (pink bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP+DMSO, d5 vs. DMSO, d5 or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "I ABR thresholds from p53wt mice recorded prior to (gray plot and black plot for left and right ear, respectively) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear (green plot), and of PFT-α into the right ear (pink plot).J Mean ABR threshold from 4 kHz to 32 kHz derived from I. Data are expressed as mean ± SEM (n=14). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003 , CDDP+left ear, DMSOvs. before, left ear or CDDP + left ear, DMSOvs. CDDP + right ear, PFT-α.",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "K Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from the DMSO-treated left ear (green bars) and PFT-α treated right ear (pink bars) of the same CDDP-treated p53wt mice after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP + right ear, PFT-α vs. CDDP+left ear, DMSO).",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "A ABR thresholds recorded prior to (gray plot) or at day 28 in HBCx-90 (TP53 wt) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).B Mean ABR threshold from 4 kHz to 32 kHz derived from A. Data are expressed as mean ± SEM (before: n=20; DMSO: n=5; PFT-α: n=5; CDDP: n=5; CDDP+PFT-α: n=5). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0002, CDDP vs. before; CDDP vs. CDDP+PFT-α.E ABR thresholds recorded prior to (gray plot) or at day 35 in HBCx-14 (TP53 mutant) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).F Mean ABR threshold from 4 kHz to 32 kHz derived from E. Data are expressed as mean ± SEM (before: n=30; DMSO: n=5; PFT-α: n=5; CDDP: n=10; CDDP+PFT-α: n=10). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003, CDDPvs. before or CDDPvs. CDDP+PFT-α.",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "C Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 28 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.007, CDDP, d28 vs. DMSO, d28 or CDDP, d28 vs. CDDP+PFT-α, d28).G Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 35 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP, d35 vs. DMSO, d35 or CDDP, d35 vs. CDDP+PFT-α, d35).",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "D Tumor growth curves alongside images of dissected tumors collected on day 28. Note the partial inhibition of growth in CDDP and CDDP+PFT- α-treated mice.H Tumor growth curves alongside images of dissected tumors collected on day 35. Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP+PFT- α-treated mice.",
    "molecules": "CDDP, PFT- α"
  },
  {
    "caption": "A Representative confocal images of microvessels in transversal tumor sections from HBCx-14 (TP53mutant) tumors treated with either DMSO, CDDP or a combination of CDDP and PFT-α. The sections were immunolabeled for CD31 (red) and viewed with a 20x objective. The basal-like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8. Upper right panel is a 2D projection from the white boxed area in upper left panel. The red area corresponds to CD31 labeled endothelial area and the white area represents the lumen area. The white line shows the vessel perimeter. Scale bars: left panel=50 µm, right panels=35 µm. The tumor samples were collected at day 21.B Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula: vessel/vascular area = area of CD31-positive objects + lumen area per field area × 100 %. The both HBCx-14 (TP53mutant) and HBCx-90 (TP53wt) tumors from the different treated groups were collected at day 21 (n=4 sections/tumor and 3-4 tumors/group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (***P ≤0.0004, CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "D Representative western blot analysis using antibodies against β-actin, p53 and p21 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO or CDDP and collected at day 18. Note the higher CDDP-induced increase of p53 and p21 expression in TP53wt HBCx-90tumors.",
    "molecules": "CDDP, DMSO"
  },
  {
    "caption": "E, F Representative western blot analysis using antibodies against β-actin, p-Chk1, Beclin 1, LC3-I/II and Rab7 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO, PFT-α, CDDP or a combination of CDDP and PFT-α. The tumor samples were collected at day 18.G, H, I Histograms representing the levels of Beclin 1, LC3-II and Rab7 in HBCx-14 and HBCx-90tumors treated with the different regimens (n=3-4 tumors per group, all experiments were performed in triplicate). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (G: *P = 0.03, **P =0.006 , ***P ≤ 0.0004 ; H: *P =0.02, ***P = 0.0005 ; I: *P =0.03, ***P ≤ 0.0006 , CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).",
    "molecules": "CDDP, DMSO, PFT-α"
  },
  {
    "caption": "(B) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).",
    "molecules": "DAPI, PI"
  },
  {
    "caption": "(B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting",
    "molecules": "z-VAD"
  },
  {
    "caption": "C) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay",
    "molecules": "z-VAD"
  },
  {
    "caption": "(E HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting",
    "molecules": "cysteine, serine"
  },
  {
    "caption": "F) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assays",
    "molecules": "cysteine, serine"
  },
  {
    "caption": "(D) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.",
    "molecules": "DAPI, rhodamine-phalloidin"
  },
  {
    "caption": "(D HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting",
    "molecules": "DMSO"
  },
  {
    "caption": "E) HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay",
    "molecules": "DMSO"
  },
  {
    "caption": "scRNA-Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only (GPI) in primary CD4+ T-cells infected in vitro (D,E) or CD4+ T-cells of PLWH (F). In panels (D, E), cells were infected with VSVG-HIV-1-GFP and sorted for viral expression as detailed in (Golumbeanu et al, 2018). Following latency establishment, cells were left untreated or HIV-1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α-CD3-CD28 engagement. Clusters 1 and 2 were identified by principal component analysis as described in (Golumbeanu et al, 2018). In panel (F), CD4+ T-cells were isolated from total blood of PLWH under ART as described in (Cohn et al, 2018). Viral expression was reactivated by treatment with phytohemagglutinin (PHA) and cells were sorted using antibodies against Env and Gag. Sorted cells were then subjected to scRNA-Seq analysis. The expression level of the HUMAN-GLYCOLYSIS pathway in (D) was calculated as the average expression of genes comprising the gene list; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test. For panels (E, F), significance of GPI differential expression level between clusters (E) or between control and Env+ Gag+ conditions (F) was assessed by the Wilcoxon rank sum test encoded in FindMarkers Seurat R function. ** p< 0.01, *** p< 0.001; *** p< 0.0001. For panels (D,E) n = 1 donor, 43 cells (untreated), 90 cells (SAHA ), 91 cells (TCR), for panel (F) n = 3 donors, 109 cells (control) and 85 cells (Gag+ Env+).",
    "molecules": "PHA, phytohemagglutinin, SAHA, suberoyl anilide hydroxamic acid"
  },
  {
    "caption": "Relative ratios of NADH/NAD+ (D) and ATP/ADP (E) in latently infected and reactivated cells. Data were normalized using the matching uninfected control.",
    "molecules": "ADP, ATP, NAD+, NADH"
  },
  {
    "caption": "relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e. G6PD (B) in HIV-1 infected as compared to mock infected cells.",
    "molecules": "pentose phosphate"
  },
  {
    "caption": "relative expression of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e. TrxR1 (C) and GCLC (D), in HIV-1 infected as compared to mock infected cells.",
    "molecules": "glutathione, thioredoxin"
  },
  {
    "caption": "Reactivation from HIV-1 latency (A) and relative cell viability (B) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin (AF; 500 nM), the GSH inhibitor buthionine sulfoximine (BSO; 250 μM), or a combination of the two. GFP-HIV-1 expression was determined by FACS. Data are expressed as mean ± SD of three replicates and were analyzed by One-Way ANOVA followed by Tukey's post-test (A) or Two-Way ANOVA followed by Sidak´s post-test (B). Solid lines represent the means.",
    "molecules": "AF, auranofin, GSH, BSO, buthionine sulfoximine"
  },
  {
    "caption": "Levels of integrated HIV-1 DNA following treatment for 48 h with AF and/or BSO in CD4+ T-cells derived from PLWH under suppressive antiretroviral therapy. Live cells were sorted after treatment, and integrated DNA was measured by Alu-PCR. The latency reactivating agent SAHA was used as a reference compound (Archin et al, 2012). Data were analyzed by non-parametric Friedman´s test followed by Dunn's post-test.",
    "molecules": "AF, BSO, SAHA"
  },
  {
    "caption": "D.  Yeast two-hybrid interaction  analysis  between Sae2C and Sir3SaID domains in WT or sir4Δ cells. Growth on -His + 3AT  and blue coloration on X-gal indicate an  interaction.",
    "molecules": "X-gal, 3AT, His"
  },
  {
    "caption": "F. Representative  silver-stained gels of in vitro GST-pulldown of  GST or GST-Sir3SaID, GST-sir3-T557ISaID and Sae2C purified peptides. Control: Sae2C (300 ng, lane 6).",
    "molecules": "silver"
  },
  {
    "caption": "C, D Proinflammatory cytokine production in tumor-bearing mice with and without Treg-specific TRAF6 expression. Cell suspensions of the tumor-draining lymph node and tumor-infiltrating leukocytes (TILs) were recovered after 21 days of tumor growth. Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL-17 and flow cytometry analysis.",
    "molecules": "ionomycin, Golgistop, PMA"
  },
  {
    "caption": "A Assessment of K63-type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants. 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6, the enzymatically deficient L74H or C70A TRAF6 mutants, HA-FOXP3, and FLAG-labeled ubiquitin molecules possessing a single lysine residue (K63) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63-type only. Cells were lysed and proteins modified by K63-ubiquitin chains were recovered from lysates by pulling down using a bead-immobilized K63-specific TUBE reagent. The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre-IP whole cell lysate (WCL).",
    "molecules": "lysine, ubiquitin"
  },
  {
    "caption": "C　293T cells were transfected with the indicated combinations of expression constructs encoding HA-FOXP3, Myc-TRAF6, and FLAG-tagged ubiquitin molecules - either wild type (FLAG-Ub), or a variant with a K-to-R mutation at either lysine residue 48 (K48R) or 63 (K63R) responsible for preventing K48- and K63-type polyubiquitination, respectively. .",
    "molecules": "lysine, Ub"
  },
  {
    "caption": "E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl/flFoxp3Cre+ and Traf6wt/wtFoxp3Cre+mice. CD4+/YFP+ cells from Traf6fl/flFoxp3Cre+ ("Traf6-/-") or wild type Traf6wt/wtFoxp3Cre+mice ("Traf6+/+") were isolated by FACS, lysed and either total FOXP3 (right) or K63-ubiquitin-modified proteins (left) were immunoprecipitated. Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti-K63-ubiquitin and FOXP3, respectively, and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG.",
    "molecules": "arginines, lysine"
  },
  {
    "caption": "A Impact of K63-ubiquitination loss on FOXP3's gene silencing capacity. Jurkat T cells transfected with a wild type Foxp3 expression vector, one encoding the K262R mutant, or an empty (control) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter. After activation with PMA and ionomycin for 8 hours, luciferase activity was assayed.",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "C Cellular distribution of wild type and K262R FOXP3. Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant. The relative overlap of FOXP3 protein signal and DAPI-stained nuclei was observed by fluorescence microscopy. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).",
    "molecules": "DAPI"
  },
  {
    "caption": "D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).",
    "molecules": "DAPI"
  },
  {
    "caption": "The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.",
    "molecules": "CFSE, lysine"
  },
  {
    "caption": "The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered \"Tregs\" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.",
    "molecules": "CFSE, lysine"
  },
  {
    "caption": "D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).",
    "molecules": "formalin"
  },
  {
    "caption": "Localization of SPATC1L in germ cells during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and γ-tubulin (green). γ-Tubulin was used as a centrosome marker, and cells were counterstained with the nuclear dye Hoechst 33342 (blue). Fluorescence images of individual staining are shown at the top and merged images are shown in the middle. Insets are magnified iamges. Sc, spermatocyte; RS, round spermatid; ES, elongating spermatid; TS, testicular sperm; MS, epididymal mature sperm; BF, overlay with bright field image.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "Immunoprecipitation of SPATC1L in the testis using an anti-SPATC1L antibody. IgG was used as a control. In, input; IgG, normal rabbit serum.",
    "molecules": "IgG"
  },
  {
    "caption": "Interaction between SPATC1L and PKA subunits. Samples precipitated with an anti-SPATC1L antibody were analyzed by immunoblotting with anti-RIα and anti-Cα antibodies. Immunoprecipitation of SPATC1L was confirmed by immunoblotting with anti-SPATC1L antibody. IgG was used as a control. IP, immunoprecipitation; In, input; IgG, immunoglobulin G from normal rabbit serum.",
    "molecules": "IgG, immunoglobulin G"
  },
  {
    "caption": "Localization of SPATC1L and RIα during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and RIα (green), and counterstained with the nuclear dye Hoechst 33342 (blue). Overlay with bright field image is shown at right in each cell type. TC, testicular cell (spermatocyte); RS, round spermatid; ES, elongating spermatid; TS, testicular sperm. Scale bar = 5 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "PKA activity after overexpression of SPATC1L or SPATC1LΔC. Graphs show the average PKA activity of Spatc1l-transfected cells (white bar) and Spatc1lΔC-transfected cells (black bar) in the presence or absence of cAMP, expressed relative to control cells (mock) as a percentage. Representative signals observed in the assay are shown above. The overexpression of SPATC1L was confirmed in every experiment by immunoblotting (data not shown).",
    "molecules": "cAMP"
  },
  {
    "caption": "The amount of Cα associated with RIα in the presence or absence of SPATC1LΔC. After IP of RIα in cells overexpressing SPATC1LΔC or control, immunoblot analysis was performed using anti-Cα antibody. Graph shows densitometric analysis of Cα in relation to immunoprecipitated RIα in immunoblot analysis . In, input; IgG, immunoglobulin G from normal rabbit serum.",
    "molecules": "IgG, immunoglobulin G"
  },
  {
    "caption": "Histological analysis of hematoxylin and eosin-stained sections of seminiferous tubules prepared from testes of WT and KO mice. Scale bar = 100 μm (upper) and 50 μm (lower).",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Immunostaining analysis of sperm heads and tails in WT and KO mice. Sperm tails were observed by immunostaining for α-tubulin antibody (red), and counterstaining with the nuclear dye Hoechst 33342 (blue). Sperm were also observed by differential interference contrast (DIC) microscopy. Scale bars = 20 μm (upper) and 5 μm (lower).",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "Representative ProQ Diamond-stained 2D gel images of isolated proteins from testes in WT (left) and KO (right) mice. Testes lysates were separated by 2DE, and phosphoproteins were stained with ProQ diamond. Images in the boxes are enlarged in insets shown below. Circles with numbers indicate spots with significantly changed volume relative to those on WT gels.",
    "molecules": "Diamond, diamond"
  },
  {
    "caption": "Phosphorylation of CPAZB3 by cAMP-dependent protein kinase (PKA). CAPZB3 was incubated with PKA and 32P, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower).",
    "molecules": "cAMP, Coomassie Brilliant Blue, 32P"
  },
  {
    "caption": "Phosphorylation of CPAZB3 in the presence or absence of SPATC1L. Phosphorylation of purified CAPZB3 was labeled with 32P via PKA in the presence or absence of SPATC1L, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower). Graph shows densitometric analysis of phopho-CAPZB3 (p-CAPZB3) in reactions without (control; white bar) or with SPATC1L (black bar), expressed relative to control as a percentage.",
    "molecules": "Coomassie Brilliant Blue, 32P"
  },
  {
    "caption": "Observation of filamentous actin (F-actin) in the presence (lower) or absence (upper) of SPATC1L. F-actin in pEGFP vector-transfected control cells (mock; upper, green) and cells overexpressing SPATC1LΔC (lower, green) was visualized by staining using Phalloidin (red). White bar shows the length of transfected cell. Scale bar = 50 μm.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "Cross-presentation of OVA by wild type and TAP1-/- BMDC expressing hβ2m were compared to control BMDC, by measuring IL-2 production by a Kb-SIINFEKL specific T-cell hybridoma (B3Z) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA-coated latex beads for 6hr, prior to fixation.",
    "molecules": "paraformaldehyde"
  },
  {
    "caption": "TAP1-/- BMDC transduced with vector control (A) or hβ2m (C) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb-binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry. Representative plots are shown (A, C). The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin, normalized to that on cell incubated without Epoxomicin (n=3) (B, D).",
    "molecules": "Epoxomicin"
  },
  {
    "caption": "The effect of Epoxomicin on cross-presentation of OVA-coated beads by TAP1-/- BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured",
    "molecules": "Epoxomicin"
  },
  {
    "caption": "293T-FcR-Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.",
    "molecules": "Epoxomicin"
  },
  {
    "caption": "293T-FcR-Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.",
    "molecules": "Epoxomicin"
  },
  {
    "caption": "293T-FcR-Kb cells expressing Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.",
    "molecules": "Epoxomicin"
  },
  {
    "caption": "EM micrographs of double immuno-gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC, WT MEF and LMP2 KO MEF. Large gold particles (15 nm, black arrow head) labels LMP2 and small gold particles labels LAMP1 (5 nm, white arrow head) (Scale bars = 500nm). The insets are the magnification of region of interest containing LAMP1 positive vacuole (Scale bars = 100nm), marked by the black rectangle.",
    "molecules": "gold"
  },
  {
    "caption": "The distribution of LMP2 positive signals (n = 22 images) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles.",
    "molecules": "gold"
  },
  {
    "caption": "Immuno-electron microscopy images (n = 23) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments (D). These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle (E).",
    "molecules": "gold"
  },
  {
    "caption": "BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647-OVA coated bead uptake. A single optical section of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)",
    "molecules": "Alexa 647, Alexa-647, doxycycline"
  },
  {
    "caption": "BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D-rendering of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)",
    "molecules": "Alexa-647"
  },
  {
    "caption": "The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647-OVA enclosed within a limiting membrane positive for LAMP1. The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647-OVA was calculated and data compiled from independent experiments (n=3) are plotted (H).For each transduced cell type, images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point.",
    "molecules": "Alexa 647, Alexa647"
  },
  {
    "caption": "BMDC co-expressing LMP2-RFP and GFP-Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively.",
    "molecules": "Alexa647, doxycycline"
  },
  {
    "caption": "BMDC co-expressing LMP2-RFP and GFP-Rab22ACA (J, K) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively. The boxed region in panel (J) marks a phagosome that has been enlarged in panel (K) (Bar 1μm).",
    "molecules": "Alexa647, doxycycline"
  },
  {
    "caption": "Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent-treated phagosomes treated or untreated with Epoxomicin. The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of Epoxomicin",
    "molecules": "Epoxomicin"
  },
  {
    "caption": "Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647-OVA. Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP, lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs. Post incubation latex beads were treated with detergent and analyzed by flow cytometry (B). The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted (n=4) (C).",
    "molecules": "Alexa647, ATP, Epoxomicin"
  },
  {
    "caption": "b) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti-pTyr-416-Src, anti-Src, anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(c) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti-pTyr-416-Src, anti-Src, anti-p53 and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.",
    "molecules": "epoxomicin"
  },
  {
    "caption": "(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.",
    "molecules": "chloroquine, dasatinib"
  },
  {
    "caption": "(e) Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. Uncropped images of blots are shown in Supplementary Fig. S9.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(a) Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies.",
    "molecules": "agarose"
  },
  {
    "caption": "(b) FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm.",
    "molecules": "PBS"
  },
  {
    "caption": "(a) Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h). Cells were then left at 37 °C for 1 week to allow colony formation, fixed and then stained with crystal violet. Bottom, the number of colonies formed was then quantified and normalized against untreated controls. Data are presented as mean±s.d. and significance is P0.001 for 3-MA, P0.01 for chloroquine and P>0.5 for dasatinib (n=3).",
    "molecules": "3-MA, chloroquine, dasatinib"
  },
  {
    "caption": "(b) Top, wild-type FAK and FAK−/− cells were transiently transfected with two individual Atg5 shRNAs (1 and 2), selected in puromycin and then the clonogenic assay was carried out as described above. Bottom, quantification; data are presented as mean±s.d. and significance is P>0.1 for Atg5 A and B in wild-type FAK cells and P0.01 for Atg5 A and P0.05 for Atg5 B in FAK−/− cells (n=3).",
    "molecules": "puromycin"
  },
  {
    "caption": "(c) Cells were also stained with TUNEL. The positive control was DNase1-treated cells and the negative control lacked TUNEL reaction mix. Quantification is shown and data are presented as mean±s.d. and significance is P0.005 for 3-MA and P0.001 for Atg5 siRNA (n=3).",
    "molecules": "3-MA"
  },
  {
    "caption": "(d) FAK−/− cells were untreated or treated with dasatinib, 3-MA, chloroquine or bafilomycin A (BFA) for 24 h, or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti-PARP antibody. Uncropped images of blots are shown in Supplementary Fig. S9.",
    "molecules": "3-MA, bafilomycin A, BFA, chloroquine, dasatinib"
  },
  {
    "caption": "(a) Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate co-localization in autophagosomes. Scale bars, 20 μm.",
    "molecules": "chloroquine"
  },
  {
    "caption": "A. Slices (DIV15) from control mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images are presented at baseline and after application of high potassium. Note the increase in intracellular Ca++ ([Ca++]i) after KCl application.B. Slices (DIV15) from hTauAT mice were loaded with the Ca++ sensitive dye Fura-2AM. Ratiometric images at baseline and after application of high potassium in area CA3 are depicted. Note the increase in [Ca++]i (false color legend) under both conditions in hTauAT slices compared to controls.",
    "molecules": "Ca, KCl, potassium"
  },
  {
    "caption": "C. Electrophysiological example traces (control = black; hTauAT = red) of excitatory postsynaptic field potentials (fEPSP) depicts examples of depolarizations that are induced by a single high potassium chloride application in stratum pyramidale (s.p.) of area CA3 in slices in which calcium imaging experiments were conducted. The example depolarization evokes averaged calcium influxes into neurons depicted in the calcium imaging graph. The graph shows the mean of [Ca++]i changes in response to high KCl in stratum radiatum and stratum pyramidale of area CA3 in Fura2-AM loaded hippocampal slices. After depolarization [Ca++]i rises up to ~600 nM in hTauAT slices (red trace, n=6 slices, prepared from at least three animals), compared to ~300 nM in control slices (black trace, n=6 slices; prepared from at least three animals). Note that even under resting conditions [Ca++]i is elevated to ~120nM due to hTauAT expression.",
    "molecules": "Ca, calcium, KCl, potassium chloride"
  },
  {
    "caption": "D. Quantification of the maximum [Ca++]i from ratiometric images as depicted in (A) and (B) after background subtraction and selection of regions of interest (10 circles of fixed diameter) in stratum radiatum border to stratum pyramidale of area CA3. Under resting conditions [Ca++]i was elevated in transgenic slice cultures (~120 nM, n=11) compared to controls (~80 nM, n=8). Slices were incubated with one of the following channel blockers: Nifedipine (NIF, VGCC; Ctrl n=8; A152T: n=7), APV (NMDAR; Ctrl n=7; A152T n=8), ifenprodil (IFEN, NMDAR; Ctrl n=8; A152T n=7), memantine (MEM, NMDAR; Ctrl/A152T n=6), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPAR; Ctrl n=8; A152T n=7)), tetrodotoxin (TTX, VGNaC; Ctrl n=4; A152T n=5), tetanus neurotoxin (TeNT, neurotransmitter release; A152T n=6) or in Ca++ free buffer (Ctrl n=10; A152T n=7). For each experiment slice preparations from at least three different mice were used. One-way ANOVA followed by Tukey's post-hoc test; Ctrl: F(7/51) .3,533; p=0.0036; A152T: F(8/55)=5,230; p<0.0001.For an overview of drug concentrations see EV Table 1.E. Quantification of the maximum peak increase in intracellular calcium in the same slices than in (D) after high KCl stimulation. One way ANOVA followed by Tukey's post-hoc test Ctrl: F(7/45)=4,080; p=0.0015; A152T: F(8/53)=7,580; p<0.0001).Error bars represent SEM; One-Way ANOVA *p<0.05; **p<0.01 and ***p<0.001.",
    "molecules": "APV, 6-cyano-7-nitroquinoxaline-2,3-dione, CNQX, Ca, calcium, IFEN, ifenprodil, MEM, memantine, NIF, Nifedipine, KCl, tetrodotoxin, TTX"
  },
  {
    "caption": "A. Glutamate content in the culture medium ([Glu]e) from either hTauAT (n=10 culture inserts containing 6 slices each) or control slices (n=5-12 culture inserts) analyzed at DIV 5, 10, 20 and 25. Glutamate content from medium cultured with hTauAT slices is shown in percent of control at each time point. Increased levels were observed between DIV 5 and DIV 20. At DIV 25 glutamate levels already declined to control levels. One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01 ***p<0.001.B. Lactate dehydrogenase (LDH) release (a measure of cytotoxicity) analyzed in hTauAT (n=17-19 culture inserts containing 6 slices each) or control slices (n=15-16 culture inserts) at DIV 5, 10, 20 and 25. LDH release of hTauAT slices is shown in percent of control at each time point. A pronounced increase (~25%) in LDH release was observed due to hTauAT expression starting at DIV 10 but not at DIV 5 One-way ANOVA: *p<0.05; **p<0.01 and ***p<0.001.",
    "molecules": "Glutamate, glutamate"
  },
  {
    "caption": "C. Slice cultures from control (left), hTauAT (middle) or hTauAT treated with ceftriaxone (CEF; 100 µM; right) stained against neuronal nuclear protein (NeuN) at DIV 30. Representative neuronal cell layers are shown. hTauAT slices show neuronal loss in the granular cell layer of the dentate gyrus and the pyramidal cell layer in the region of the hilus in area CA3 (middle panel, red arrows). Treatment with ceftriaxone for 30 days prevents neuronal loss in DG and area CA3.D. NeuN positive cell bodies were counted in the DG and within the pyramidal cell layer in area CA3 and CA1 at DIV 30 in defined regions of interest (ROI). The number of neurons was reduced in hTauAT slices, both within area DG and CA3, whereas the number of neurons in the CA1 was only slightly affected. Neuronal loss was largely prevented in cultures treated with ceftriaxone (CEF) in dentate gyrus and area CA3 (n=14-19 slices per group and area; prepared from at least 6 animals). One-way ANOVA followed by Tukey's post-hoc test *p<0.05; **p<0.01",
    "molecules": "CEF, ceftriaxone"
  },
  {
    "caption": "A. Example of electrophysiological fEPSP recording in stratum pyramidale of area CA3 in a control slice (DIV30) during wash-in of 50 µm picrotoxin. This leads to blockage of GABAergic transmission followed by epileptiform discharges (burst) occurring approximately after 3 min. The zoom-in reveals that such an epileptiform discharge (burst) consists of repetitive firings.B. Similar example trace as in (A) recorded in a hTauAT expressing slice (red) demonstrating an increase in burst frequency as well as in firings per burst due to hTauAT expression.C. Quantification of bursts per minute in DIV 10 slices from control littermates and hTauAT slices demonstrating a ~2-fold increase in hTauAT slices (n=8 slices per group).D. Firings per burst are increased ~1.5-fold already at the early time point DIV10 due to hTauAT expression.E. Analysis of burst frequency in slices at DIV 30 demonstrates the progression of the pathological effect beyond that observed at DIV 10. Burst frequency is enhanced in hTauAT expressing slices (n = 10 slices). If Ceftriaxone (CEF) is applied to hTauAT slices the pathological burst frequency enhancement is partly prevented (n = 5 slices).F. Firings per burst are strongly enhanced due to hTauAT expression in slices at DIV 30. Ceftriaxone prevents this pathological phenotype if applied during the entire period of cultivation (n = 6 slices).",
    "molecules": "CEF, Ceftriaxone, picrotoxin"
  },
  {
    "caption": "A. Quantification of glutamate concentrations in the medium of control and hTauAT cultures in the presence of either 50 nM tetanus neurotoxin (TeNT) or 1 µM tetrodotoxin (TTX) at DIV5. Under both conditions, glutamate levels in the culture medium of hTauAT slices decreased to control levels (n=6 culture inserts containing 6 slices each / group and condition).B. Quantification of extracellular glutamate concentrations in culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV1-DIV20 demonstrating a possible contribution of astrocytic glutamate dysregulation to increased extracellular glutamate in hTauAT slices (n=8-9 culture inserts containing 6 slices each / group and condition). One-Way ANOVA F(3,30)=14,89; p<0.001",
    "molecules": "benzyloxy-aspartic acid, TBOA, glutamate, tetrodotoxin, TTX"
  },
  {
    "caption": "C. Quantification of LDH release into the culture medium of either control or hTauAT slices at DIV 20 in the presence or absence of 50 µM benzyloxy-aspartic acid (TBOA) from DIV 1 to DIV 20 (n=6-12 culture inserts containing 6 slices each / group and condition). One-Way ANOVA followed by Tukey's post-hoc test.",
    "molecules": "benzyloxy-aspartic acid, TBOA"
  },
  {
    "caption": "D. Quantification of extracellular glutamate concentrations in either control or hTauAT cultures treated (n=7-8 culture inserts containing 6 slices each / group and condition) with ceftriaxone from DIV 1 to DIV 25 demonstrating the preventative effect of CEF on the increase in extracellular glutamate in hTauAT slices. One-Way ANOVA F(3,25)=9.075; p=0.0003).",
    "molecules": "CEF, ceftriaxone, glutamate"
  },
  {
    "caption": "E. LDH release at DIV 25 is strongly reduced due to long term treatment with ceftriaxone in hTauAT slice cultures (n=8-10 culture inserts containing 6 slices each / group and condition). One Way ANOVA F(3,32)=7.685; p=0.0005).",
    "molecules": "ceftriaxone"
  },
  {
    "caption": "F. Intracellular calcium rise after KCl stimulation is not distinguishable from control slices in hTauAT slices treated with ceftriaxone for 15 days (n=5-9 slices; prepared from at least three animals). One way ANOVA F((3,25)=7.317; p=0.0011).",
    "molecules": "calcium, ceftriaxone, KCl"
  },
  {
    "caption": "RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Scatter plot showing log fold change (LFC) of all GW7647-upregulated genes (LFC > 0.8 and P < 0.05) in sham versus their LFC 10h after CLP. The red line represents the diagonal, the black line represents the real slope (0.3115) of the data.",
    "molecules": "GW7647"
  },
  {
    "caption": "RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Heatmap of differentially expressed genes in sham mice after GW7647 treatment, involved in β-oxidation of fatty acids (unit scale bar = log2 of the normalized counts).",
    "molecules": "GW7647"
  },
  {
    "caption": "Oxygen consumption rates (OCR) of liver tissue explants 24h post sham or CLP. Liver tissue was isolated 24h post-surgery and OCR was measured via Seahorse with BSA or palmitic acid (PA) as a substrate for 42 min. n = 4/group.",
    "molecules": "PA, palmitic acid"
  },
  {
    "caption": "Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Plasma was isolated and (F) FFA concentration and (G) glycerol concentration was determined as described in the method section. P-values were calculated with 2-way ANOVA tests. Combined data of 2 independent experiments.",
    "molecules": "FFA, glycerol"
  },
  {
    "caption": "Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Normalized abundances of (H) palmitic acid and (I) palmitoyl-carnitine determined via liquid chromotography-mass spectrometric lipidomics. Values were normalized to IQ values , P-values were calculated with 2-way ANOVA tests. n = 8/group.",
    "molecules": "palmitic acid, palmitoyl-carnitine"
  },
  {
    "caption": "Immunofluorescent images of liver and kidney 24h after sham (with or without starvation) or CLP (n= 6-7/group, combined data of 2 independent experiments). White scale bar = 20µm. Cryosections were stained with Acti-stain (green), Hoechst (blue), and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Quantification of lipid peroxidation by determination of (C) MDA and (D) 4-HNE concentrations in liver homogenates 24h post-surgery in sham and CLP mice as described in methods (n = 6-7/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.",
    "molecules": "4-HNE, MDA"
  },
  {
    "caption": "Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured by TUNEL staining, and presented as % PI positive cells/µm² tissue area. (n= 6/7 mice/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.",
    "molecules": "PI"
  },
  {
    "caption": "Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 3 experiments, n = 20/group.",
    "molecules": "Pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Liver samples were isolated 24h after CLP (n= 5-7/group, combined data of 2 independent experiments), mRNA was prepared and gene expression levels of Ppara was analyzed via qPCR. Gene expression values are shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated via 2-way ANOVA test.",
    "molecules": "Pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was isolated 24h after sepsis and FFA concentration and (D) glycerol concentration were determined as described in the method section. P-values were calculated with 2-way ANOVA tests. n = 5-7/group, combined data of 2 independent experiments. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.",
    "molecules": "FFA, glycerol, Pemafibrate, pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Immunofluorescent images of cryosections of liver 24h post-surgery that were stained with Acti-stain (green), hoechst (blue) and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section. White scale bar = 20µm.",
    "molecules": "hoechst, Pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in liver homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.",
    "molecules": "MDA, Pemafibrate, pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in kidney homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.",
    "molecules": "MDA, Pemafibrate, pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Oxygen consumption rates (OCR) of liver tissue explants 24h post CLP (vehicle or pemafibrate treated) after supplementation of palmitic acid (PA), measured via Seahorse. Visualization of T42(min) OCR. P-values were calculates using 2-way ANOVA analysis. One experiment, n = 3/group.",
    "molecules": "PA, palmitic acid, Pemafibrate, pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and (A) Alanine-amino transferase (ALT), were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.",
    "molecules": "pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and creatinine concentrations, were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.",
    "molecules": "creatinine, pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and IL-6 levels were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.",
    "molecules": "pemafibrate, NaCl"
  },
  {
    "caption": "Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.",
    "molecules": "pemafibrate, PI, NaCl"
  },
  {
    "caption": "Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in kidney paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.",
    "molecules": "pemafibrate, PI, NaCl"
  },
  {
    "caption": "Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Systemic bacterial load (CFU/ml blood) 24h post-sepsis in vehicle or pemafibrate treated mice. P-values were calculated with 2-way Student's t-test.",
    "molecules": "pemafibrate, NaCl"
  },
  {
    "caption": "Pemafibrate (1 mg/kg) or vehicle (0,9% NaCl) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via Log rank tests",
    "molecules": "Pemafibrate, NaCl"
  },
  {
    "caption": "Mice were injected with the PPARα antagonist GW6471 (10µg/g) or vehicle (DMSO) 3h pre- and 3h post-CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 2 independent experiments, n = 16/group.",
    "molecules": "DMSO, GW6471"
  },
  {
    "caption": "Blood samples were collected from healthy volunteers and septic patients (n= 13 septic patients, = 15 healthy controls). Plasma was prepared and (A) FFA and (B) glycerol concentrations were determined as described in the methods. P-values were calculated with two-way Student t-tests.",
    "molecules": "FFA, glycerol"
  },
  {
    "caption": "BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.",
    "molecules": "BrdU"
  },
  {
    "caption": "BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.",
    "molecules": "BrdU"
  },
  {
    "caption": "B Representative images showing co-expression of the stage-specific markers Nestin, Tbr2, Doublecortin (DCX), and Calbindin (all in green), with the β-Galactosidase (β-Gal, red) reporter in 8-week old BATGAL and Axin2LacZ/+ mice. Nuclei are counterstained with DAPI (in blue). Scale bar= 10µm. Insets show a 1.5x magnification of selected cells (position indicated by dashed box). Scale bar= 5µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "F Quantification of BrdU+ cells expressing β-Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [0 vs. 7dpi p=0.0036, 7 vs. 28dpi p=0.0115, 7 vs. 42dpi p=0.0007] (0dpi time-point; n= 4 animals), 3dpi (n= 3 animals), 7dpi (n= 3 animals), 14dpi (n= 4 animals), 28dpi (n= 6 animals) and 42dpi (n= 6 animals).",
    "molecules": "BrdU"
  },
  {
    "caption": "G Total cell fluorescence of β-Gal in BrdU+ cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [0 vs. 3dpi p<0.0001, 7 vs. 14dpi p<0.0001, 14 vs. 28dpi p<0.0001, 28 vs. 42dpi p=0.0333] (0dpi n= 194 cells, 3dpi n= 245 cells, 7dpi n= 224 cells, 14dpi n= 72 cells, 28dpi n= 103 cells and 42dpi n= 114 cells)",
    "molecules": "BrdU"
  },
  {
    "caption": "B Expression of DCX and Calbindin in BrdU+ neurons was comparable between experimental groups at 3dpT and 13dpT (control: n= 5 animals, β-catex3 iDCX: n= 5 animals).",
    "molecules": "BrdU"
  },
  {
    "caption": "B Representative images of BrdU+ cells (red) expressing DCX (grey) or β-Galactosidase (green) at 14 days post BrdU injection (dpBrdU), 28dpBrdU and 42dpBrdu in 8-week old and 24-week old BATGAL mice. Scale bar= 10µm. C Percentage of BrdU+ cells expressing DCX in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the prolonged expression of DCX in 24-week old mice [28dpBrdU p=0.0155]. D Percentage of BrdU+ cells expressing the β-Galactosidase reporter in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the reduced reporter expression in 24-week old mice [28dpBrdU p=0.0117, 42dpBrdU p=0.0034]. ",
    "molecules": "Brdu, BrdU"
  },
  {
    "caption": "B Representative images of recombined (GFP+, green) BrdU+ (grey) neurons in 24-week old control and β-catex3 iDCX animals at 13dpT expressing the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm. C Quantification of DCX and Calbindin expression in BrdU+ recombined cells. At 13dpT, labeled neurons in β-catex3 iDCX animals show a shift towards a more mature marker profile [DCX p=0.0238, Calbindin p=0.0714] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 8 animals; 13dpT control: n= 5 animals, β-catex3 iDCX: n= 5 animals). ",
    "molecules": "BrdU"
  },
  {
    "caption": "B) U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP-464, 1 μM), or DNA-PK (NU7441, 1 μM) inhibitors 30 minutes prior to exposure to IR (15 Gy, 2h recovery). Immunoblots were performed with indicated antibodies.",
    "molecules": "KU 55933, NU7441, ETP-464"
  },
  {
    "caption": "C) U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 hour time point was treated with phosphatase (Ppase). Immunoblots were performed with indicated antibodies.",
    "molecules": "HU"
  },
  {
    "caption": "D) Untreated U2OS cells or cells exposed to either HU (2 mM, 24h) or IR (15 Gy, 2h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP-464, 1 μM; ATR#2: AZ-20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 minutes prior to HU or IR treatment. Immunoblots were performed with indicated antibodies.",
    "molecules": "AZ-20, KU55933, ETP-464, HU"
  },
  {
    "caption": "E) ATR kinase assay was performed with N-terminal PALB2 (aa 1-560; GST-PALB2-N) as a substrate and HA-tagged ATR as kinase and visualized by Fujifilm Phosphoimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ-20, 30 μM) are controls. HA-ATR and GST-PALB2-N was controlled by immunoblotting with HA and GST antibodies.",
    "molecules": "GST, AZ-20"
  },
  {
    "caption": "B) Characterization of the Doxycycline-inducible cell lines expressing siRNA resistant wild type (WT) or mutant (TMA or TMD) FLAG/HA-tagged PALB2. The cell lines were transfected with UNC (Negative Control) or PALB2 siRNA for 24 hours prior to induction with Doxycycline. Immunoblots were performed with indicated antibodies.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "E) Doxycycline-inducible stable U2OS cell lines were left untreated or treated with Doxycycline and/or IR (15 Gy, 2h recovery). Following protein extraction the lysates were immunoprecipitated (IP) with IgG or antibody recognizing phosphorylated S/Q sites (pS/Q) and analyzed by SDS-PAGE. Immunoblots were performed with indicated antibodies.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "B) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU. 24 hours later the cells were fixed and processed for IF as in Figure 3A. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar=5μm.",
    "molecules": "HU"
  },
  {
    "caption": "D) Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 hours then induced for expression of PALB2 (WT and TMA). 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji (* p<0.0001, unpaired Student's t-test). Representative images of displaying RAD51 (green) and HA-PALB2 (red) localization in HU treated EdU positive (not shown) cells. Scale bar=5μm.",
    "molecules": "HU"
  },
  {
    "caption": "A) U2OS and PALB2 cell lines were transfected with UNC (Negative Control) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines. Left panel, cells were treated the following day with IR (5 Gy) and 2 hours later Nocodazole was added for 6 hours, or treated with only Nocodazole for 6 hours. Right panel, cells were treated with IR (5 Gy) and left to recover for 16 hours. Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry. The percentages of mitotic cells in the IR+Nocodazole samples were normalized to the Nocodazole samples (left panel, n=3, error bars=SEM). For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT-PALB2 expressing cells was set to one (right panel). (n=3, **p<0.005, *p<0.05, unpaired Student's t-test, error bars=SEM).",
    "molecules": "Nocodazole"
  },
  {
    "caption": "C) Number of 53BP1 nuclear in TMA-PALB2 expressing G1 daughter cells. Cell lines were prepared as above and left untreated (NT) or exposed to IR (3 Gy), and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis. The irradiated bi-nucleated cells were scored for 53BP1 nuclear bodies. Scale bar = 10μm.",
    "molecules": "Cytochalasin B"
  },
  {
    "caption": "(E) Analysis of Cxcl12  (qRT-PCR) in tumor cells upon pretreatment with the indicated inhibitors for 45 minutes (GW, BOP, DMSO and PBS as control respectively) or 60 minutes (Sunitinib) and 6 hours (Cli95) (6 hours DMSO as control) before incubation with TNC for 24 hours. N = 6 independent experiments, ** p = 0.0022, Mann Whitney test. Mean ± SEM.",
    "molecules": "GW, DMSO, BOP, PBS, Sunitinib, Cli95"
  },
  {
    "caption": "CXCL12 binding to TNC as determined by negative  EM imaging (G), followed by quantification (H). (G) scale bars, 100 nm (left), 50 nm (right), the arrowheads point at gold labeled CXCL12 and the asterisk points at the fibrinogen globe of the murine TNC monomer. (H) Representation of TNC monomer, oligomerization domain (triangle) to form hexamers as seen in (G), FNIII repeats (grey boxes, contant domains, white boxes, alternative domains), fibrinogen like domain (circle). Representative result (3 independent experiments).",
    "molecules": "fibrinogen"
  },
  {
    "caption": "Attraction and retention were determined towards coatings with collagen I (COL), fibronectin (FN) or TNC in the presence of CXCL10, CCL21, CXCL12 (B, C) 3 independent experiments in duplicates, Mann-Whitney test, ns > 0.05; * p <0.05; **p < 0.01; ***p < 0.005. Mean ± SEM.",
    "molecules": "COL, collagen I, fibronectin, FN"
  },
  {
    "caption": "(B, C)  Imaging and signal quantification upon staining for cl. casp3 and DAPI in PBS and  AMD treated tumors, N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50µm, arrows point at cl. Casp3.",
    "molecules": "DAPI, PBS, AMD"
  },
  {
    "caption": "(D, E) CD8 TIL abundance and  localization in PBS and AMD  treated tumors, arrow points at CD8 TIL, scale bar, 50µm (D). (E) Quantification of CD8 T cells per randomly chosen fields. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. (F) Gzmb levels in  PBS and AMD treated tumors. N = 10 tumors, **** p < 0.0001, unpaired t-test. Mean ± SEM. ( ",
    "molecules": "PBS, AMD"
  },
  {
    "caption": "(I, J)  Abundance and quantification of F4/80+ macrophages in PBS  and  AMD treated tumors, (I) staining for the indicated molecules, (arrow, macrophages in the stroma; asterisk, macrophages  in the tumor  nest), (J) quantification of F4/80+ cells inside the tumor nest. N = 5 tumors per condition, 2 sections per tumor, 5 random fields, ** p = 0.0079, Mann-Whitney test. Mean ± SEM. Scale bar, 50 µm.",
    "molecules": "PBS, AMD"
  },
  {
    "caption": "(I-L) qRT-PCR assessment of Neu (I), Cd8a (J), Ifnγ (K) and Gzmb (L) by qRT-PCR. PBS (N = 16) and AMD (N = 19) treated mice, *, p = 0.0364, Mann whitney test (I), **, p = 0.0086 unpaired t-test (J), **, p = 0.0055, unpaired t-test (K), ***, p = 0.0003 unpaired t-test (L), respectively. The central band represents the median, the ends of the box the first and the third quartiles, and the whiskers reach from each quartile to the minimum or maximum.",
    "molecules": "PBS, AMD"
  },
  {
    "caption": "B Representative confocal microscopy of human PBMCs stained with DAPI (white arrows) co-cultured with previously labeled UC-MSC with MitoTracker-Green (top image) and specific antibody for human CD90 (bottom image).",
    "molecules": "DAPI, MitoTracker-Green"
  },
  {
    "caption": "C Percentage of mitochondrial transfer (MitoT) on total human CD45+ cells after 24 hrs co-culture with MitoTracker-Green labeled bone marrow (BM), menstrual (Mens) or umbilical cord (UC)-MSCs (MSC:PBMC ratio 1:25) (n=3 biological replicates, with 3 different donors of BM, Mens and UC-MSCs).",
    "molecules": "MitoTracker-Green"
  },
  {
    "caption": "D FACS analysis of MitoT on T CD3+ (n=4), B CD19+ (n=4) and natural killer (NK) CD56+ (n=3) cells after 24 hrs co-culture with MitoGreen labeled UC-MSCs (ratio 1:25) (Biological replicates, with different donors of UC-MSCs).",
    "molecules": "MitoGreen"
  },
  {
    "caption": "I Mean fluorescence intensity (MFI) of MitoTracker-Green+ UC-MSCs at baseline or after 72 hours PBMC co-culture. Representative FACS plot (left panel) and average of MFI (n=4 biological replicates, with 4 different UC-MSCs) (right panel).",
    "molecules": "MitoTracker-Green"
  },
  {
    "caption": "J Confocal microscopy of human PBMCs after co-culture with MitoTracker-Green stained UC-MSCs. Images showed MSC-mitochondria (green) inside the CD3+ T cells (red).",
    "molecules": "MitoTracker-Green"
  },
  {
    "caption": "M FACS analysis of in vivo MitoT from UC-MSCs to Balb/c mouse mesenteric lymph node and spleen cells, 24 hours after intraperitoneal transplantation of 5*106 MitoTracker-labeled MSCs or without cells (control) (n=1).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "B Confocal microscopy images of human CD3+ T cells with endogenous MT-labeled with MitoTracker Red CMXRos (white arrows, left panel) or after mitoception with MitoTracker Green labeled-MT from UC-MSCs (white asterisk, right panel).",
    "molecules": "MitoTracker Green, MitoTracker Red CMXRos"
  },
  {
    "caption": "Representative proliferation FACS histogram of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells (ratio 1:1) after 5-7 days of CD4+ naïve cell activation with Treg induction/differentiation media (anti-CD3, IL-2 and TGF-b1). PBMC alone represents positive control of proliferation and PBMC co-culture with MitoTneg correspond to the inhibition control with induced Treg cells.",
    "molecules": "CTV"
  },
  {
    "caption": "C Percentage of immunosuppression of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells after 5-7 days of CD4+ naïve cell activation with differentiation media (ratio MitoT cells:PBMC) (n=3 biological replicates, with different donors of PBMCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.",
    "molecules": "CTV"
  },
  {
    "caption": "B Representative FACS plots of mitoception (% of MitoTracker+) in CD4+ or CD8+T cells from mitocepted PBMC compared to control PBMC, before intravenous injection.",
    "molecules": "MitoTracker"
  },
  {
    "caption": "(G) Representative immunofluorescence staining  of ∆FosB  (green) in cultured MSNs  exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297  (SKF) or their combination as indicated. Counterstaining  with MAP2  and DAPI  is shown in the lower row. Scale bar, 25μM. (H) Quantification of ∆FosB+ nuclei  in cultured MSNs  after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB  in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "Act, activin A, DAPI, SKF, SKF81297"
  },
  {
    "caption": "(I) Representative immunofluorescence staining  of  ∆FosB (green) in cultured MSNs  exposed to consecutive stimulation, with different 1st and 2nd treatments with activin A  (Act), SKF81297  (SKF) or their combination as indicated. Counterstaining  with MAP2 and DAPI  is shown in the right column. Scale bar, 25μM. (J) Quantification of  ∆FosB+ nuclei  in cultured MSNs  after different 1st and 2nd treatments with activin A  (Act), SKF81297  (SKF) or their combination as indicated. Results are expressed as average ± SEM of % of  MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "Act, activin A, DAPI, SKF, SKF81297"
  },
  {
    "caption": "(K) Representative immunofluorescence staining  of ∆FosB (green) in cultured MSNs  extracted from mutant mice  lacking ALK4  in GABAergic neurons  (Gad67Cre;Alk4fl/fl) and control mice  (Alk4fl/fl) after exposure to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297  (SKF) or their combination as indicated. Counterstaining  with MAP2  and DAPI  is shown in the second and fourth rows. Scale bar, 25μM. (L) Quantification of  ∆FosB+ nuclei  in cultured MSNs  from mutant and control mice  after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying  ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "Act, activin A, DAPI, SKF, SKF81297"
  },
  {
    "caption": "(A)  Western blot analysis of Protein Kinase A phospho-substrates (PKA  Ⓟ-subs) in PCBP1 immunoprecipitates  from HeLa  cells exposed to sequential stimulation (same treatment in both treatment days) with forskolin  (Fsk) in the absence or presence of the PKA  inhibitor PKI14-22  (PKI). Reprobing with anti-PCBP1 antibodies is shown in the middle blot. Lower blot  shows the total amount of PCBP1  in the cell lysates. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1  levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "PKI, PKI14-22, forskolin, Fsk"
  },
  {
    "caption": "(B) Nuclear localization of PCBP1  analyzed by Western blotting  of extracts from HeLa  cells exposed to sequential stimulation (same treatment in both treatment days) with activin A (Act), forskolin  (Fsk), PKA  inhibitor (PKI) and the ALK4  inhibitor SB431542  (SB) as indicated. Blots  of nuclear  and cytosolic  fractions were reprobed with anti-PCNA  and anti-αTubulin (αTub) antibodies, respectively. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle (first column) normalized to PCBP1  levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "PKI, Act, activin A, forskolin, Fsk, SB, SB431542"
  },
  {
    "caption": "(C) Western blot  analysis of Smad3  in PCBP1 immunoprecipitates  from HeLa  cells exposed to consecutive stimulation (same treatment in both treatment days) with activin A  (Act), forskolin  (Fsk) or their combination as indicated. The Act+Fsk extracts were also immuoprecipitated  using control IgG antibodies as additional control. Reprobing with anti-PCBP1  antibodies is shown in the middle blot. The lower blot  shows unaltered Smad3  levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of Smad3/PCBP1 co-immunoprecipitation (coIP) over vehicle (first column) normalized to PCBP1  levels. (D)  Western blot  analysis of PCBP1  in Smad3 immunoprecipitates  from HeLa  cells using conditions identical to panel (C). The lower blot  shows unaltered PCBP1  levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of PCBP1/Smad3 co-immunoprecipitation  (coIP) over vehicle (first column) normalized to Smad3  levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "Act, activin A, forskolin, Fsk"
  },
  {
    "caption": "(E) Representative immunofluorescence staining  of Smad3  (green) and PCBP1  (red) in cultured MSNs  exposed to consecutive stimulation (same treatment in both treatment days) with activin A  (Act), SKF81297  (SKF) or their combination as indicated. Counterstaining  with MAP2  and DAPI  is shown in the lower row. Scale bar, 25μM.",
    "molecules": "Act, activin A, DAPI, SKF, SKF81297"
  },
  {
    "caption": "(J) Representative images of PLA  (red) between Smad3  and PCBP1  (S3/PCBP1) in cultured MSNs  exposed to consecutive stimulation (same treatment in both treatment days) with  activin A (Act), SKF81297  (SKF), PKA  inhibitor (PKI) or SB431542  (SB) as indicated. Counterstaining  with MAP2  and DAPI  is shown in the lower row. Scale bar, 25μM. (K) Quantification of PLA between Smad3  and PCBP1  in cultured MSNs  after the indicated treatments. Results are expressed as average ± SEM of number of PLA  dots per cell. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "PKI, Act, activin A, DAPI, SB, SB431542, SKF, SKF81297"
  },
  {
    "caption": "(D) Representative images of RNA-PLA  (green) between PCBP1  (left) or Smad3  (right) and sequences in exon 4 (pE4) of FosB  mRNA, respectively, in HeLa  cells exposed to consecutive stimulation (same treatment in both treatment days) as indicated. Counterstaining  with phalloidin and DAPI  is shown in the second and fourth columns. Scale bars, 20μM. (E, F) Quantification of RNA-PLA between PCBP1  (E) or Smad3  (F) and pE4 in HeLa  cells after the indicated treatments. Results are expressed as average ± SEM of number of  RNA-PLA  dots per cell. Only nuclear  dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(G) Representative images of RNA-PLA  (green) between PCBP1  (left) or Smad3  (right) and sequences in exon 4 (pE4) of FosB  mRNA, respectively, in cultured MSNs  exposed to consecutive stimulation as indicated. Counterstaining  with phalloidin  and DAPI  is shown in the second and fourth columns. Scale bars, 20μM. (H, I) Quantification of RNA-PLA   between PCBP1  (E) or Smad3  (F) and pE4 in cultured MSNs  after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA  dots per cell. Only nuclear  dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(D) Representative immunofluorescence staining  of ∆FosB (red) in cultured MSNs  treated with scrambled and shPCBP1 lentiviruses  and exposed to consecutive stimulation (same treatment in both treatment days) with activin A  (Act), SKF81297  (SKF) or their combination as indicated. Counterstaining  with green fluorescent protein (GFP, expressed by the lentiviruses) and DAPI  is shown in the second and fourth rows. Scale bar, 25μM. (E) Quantification of ∆FosB+ nuclei  in cultured MSNs  treated with scrambled and shPCBP1 lentiviruses  followed by the indicated treatments. Results are expressed as average ± SEM of % of GFP+ cells displaying ∆FosB in the nucleus. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; two-way ANOVA followed with Tukey's multiple comparisons test in E). N denotes biological replicates.",
    "molecules": "Act, activin A, DAPI, SKF, SKF81297"
  },
  {
    "caption": "(A-F) RT-qPCR analysis of FosB  (A, D), c-Fos (B, E) and  ∆FosB  (C, F) mRNAs in NAc  of Gad67Cre;Alk4fl/fl (A-C) mutant mice  1 hour (A-C) or 24 hour (D-F) after the second saline  (Sal) or cocaine  (Coc) injection of TIPS. Results are expressed as average ± SEM of fold increase over the saline  group (Sal/Sal) of  Gad67Cre mice. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.",
    "molecules": "Coc, cocaine, saline"
  },
  {
    "caption": "(C) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with 2, 4, 6, 8 or 10% 1,6 hexanediol showing dissolution of NONO puncta with increasing concentration. (D) Dot plot of NONO foci area (µm2) per nucleus at different concentrations of 1,6 hexanediol as in (C). Bars are SD. The numbers of biological replicates are indicated in (C).",
    "molecules": "1,6 hexanediol"
  },
  {
    "caption": "(F) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with PBS, RNase A or DNase I, as indicated. (G) Dot plot of summed green fluorescence per nucleus in (F). Bars are SD. The numbers of biological replicates are indicated in (F).",
    "molecules": "PBS"
  },
  {
    "caption": "(A) Fluorescence micrograph images of representative cells stained for NONO and NEAT1_2 after transfection with plasmids expressing YFP fused NONO_WT and NONO_∆RRM1 protein. DAPI (blue) stain indicates cell nuclei, YFP fused NONO (green) and NEAT1_2 RNA FISH (red). (B) The enrichment of mean NONO fluorescence detected within RNA FISH foci is quantitatively determined as a ratio relative to mean nuclear NONO fluorescence in (A). Bars are SD. The numbers of biological replicates are indicated in (A).",
    "molecules": "DAPI"
  },
  {
    "caption": "Confirmation of peptide entry into neurons. Cultures were incubated with Bio-TMyc (25 µM, 1 h) (panel b) or left untreated (panel a). Arrowheads highlight peptide permeability, detected by fluorescein Avidin D (green), into neurons labeled with neuronal-specific antibody NeuN (red). Peptide is not detected in some neurons (arrow) and non-neuronal cells (asterisk). Confocal microscopy images correspond to single sections and are representative of five independent experiments. Scale bar, 10 μm.",
    "molecules": "Bio, fluorescein"
  },
  {
    "caption": "Effect of TMyc on neuronal survival. Primary cultures were incubated with TMyc (5, 15 or 25 μM, 30 min) and subjected to treatment with NMDA (100 µM) and glycine (10 µM) for 4 h. Specific neuronal viability was established and expressed relative to values in cultures with no treatment. Means ± SEM are represented (n = 8) and statistical analysis was performed by ANOVA test followed by post-hoc Tukey HSD test.",
    "molecules": "glycine, NMDA"
  },
  {
    "caption": "Effect of TFL457, TFL482 and TFL541 on TrkB-FL levels. Cultures preincubated with TrkB-FL peptides or TMyc (25 µM, 30 min), followed by chronic NMDA treatment (2 h), were compared to cells without peptide. Levels of full-length (FL) TrkB were established with panTrkB, an antibody for the extracellular domain that also detects the truncated forms (tTrkB). Quantitation of peptide interference of TrkB-FL downregulation. Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA. Means ± SEM are represented and analysis was performed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.02; n = 4).",
    "molecules": "NMDA"
  },
  {
    "caption": "Effect of TFL457, TFL482 and TFL541 on neuronal viability after chronic excitotoxicity. Cultures were preincubated with peptides and treated with NMDA for 0-6 h as before. Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA. Mean ± SEM of eleven (TFL457), three (TFL482) and seven (TFL541) independent experiments is given.",
    "molecules": "NMDA"
  },
  {
    "caption": "Effect of TFL457 on neuronal viability after acute excitotoxicity. Cells were induced with NMDA (50 μM) and glycine (10 μM) for 1 h and, after agonists removal, culture proceeded for 20 h with DL-AP5 (200 μM) and TMyc or TFL457 (15 μM). Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented.",
    "molecules": "DL-AP5, glycine, NMDA"
  },
  {
    "caption": "Effect of TFL457 on Y515 and Y816 phosphorylation. Cultures preincubated with TMyc or TFL457 (25 μM, 30 min) were treated with NMDA (0-6 h) and analyzed with panTrkB or phosphospecific antibodies. Quantitation of pY515 and pY816. Mean ± SEM of normalized pY515 (n = 7) or pY816 (n = 4) levels obtained after NMDA-treatment (2 h) are represented relative to those found in cells with the same peptide but no NMDA. Statistical analysis was performed by unpaired Student t test (*p = 0.046; n.s. = non-significant).",
    "molecules": "NMDA"
  },
  {
    "caption": "Analysis by immunoprecipitation of TFL457 effect on pY816 levels. Cultures preincubated and treated as before with NMDA (2 h), were immunoprecipitated with antibodies specific for phosphorylated tyrosine (pY). pY816 was analyzed by WB in immunoprecipates.",
    "molecules": "NMDA, tyrosine"
  },
  {
    "caption": "TFL457 effects on neuronal viability after inhibition of TrkB-FL signaling. Cultures were preincubated (30 min) with inhibitors specific for PI3K (Wortmannin, 100 nM), MAPK/ERK (UO126, 300 nM) or PLCγ (U-73122, 5 μM) before incubation with TMyc or TFL457 (25 μM, 30 min). Viability was established 4 h after NMDA treatment. Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post-hoc Tukey HSD test (***p = 0.0001, **p = 0.009, *p = 0.02, n.s. = non-significant, respectively for TMyc vs TFL457 in untreated or Wortmannin, UO126 or U-73122-treated cells; **p = 0.008 for untreated vs U-73122-treated cells preincubated with TFL457; n = 5).",
    "molecules": "NMDA, U-73122, UO126, Wortmannin"
  },
  {
    "caption": "Dose-dependent TFL457 effects. Cultures incubated with TMyc o TFL457 (5, 15 o 25 μM, 30 min) were treated with NMDA (2 h) and compared to cells without peptide or NMDA.",
    "molecules": "NMDA"
  },
  {
    "caption": "Time-course TFL457 effects. Cells preincubated with peptides (25 μM, 30 min) were treated with NMDA (0-6 h).",
    "molecules": "NMDA"
  },
  {
    "caption": "Quantitation of peptide effects. Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA. Means ± SEM are given. Results were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test, comparing TMyc or TFL457-treated cells for each time-point (TrkB-FL, ***p = 0.0009 and *p = 0.02; CREB, *p = 0.0149 and *p = 0.0148; pCREB, ***p = 0.0007 and **p = 0.0012; MEF2D, **p = 0.0023 and **p = 0.0047. All values are respectively for 2 or 4 h; n = 6).",
    "molecules": "NMDA"
  },
  {
    "caption": "Effect of TFL457 on MEF2-promoter activity. Cultures transfected with pMEF2 (two minimal wild type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 µM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented.",
    "molecules": "NMDA"
  },
  {
    "caption": "Effect on CRE-promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG-501 (10 µM). Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG-501.",
    "molecules": "KG-501, NMDA"
  },
  {
    "caption": "Effects on BDNF promoter III or TrkB promoter. Cells transfected with pIIIBDNF (n = 9), pTrkB (n = 7) or pCRE (n = 5), as a control, were processed as above but using 20 µM NMDA for 4 h. Data are presented and analyzed as in panel A (pCRE, **p = 0.0098; pIIIBDNF, *p = 0.029; pTrkB, *p = 0.033).",
    "molecules": "NMDA"
  },
  {
    "caption": "Effects of TFL457 on mRNA levels of CREB/MEF2-regulated genes. Total RNA was extracted from cultures preincubated with CPPs (25 μM, 30 min) and treated or not with NMDA (100 µM, 4 h). Levels of mRNA were normalized to NSE (genes expressed in neurons, left panel) or GAPDH (neuronal and glial expression, right panel). Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented. Differences found in excitotoxicity were analyzed by ANOVA test followed by post-hoc Tukey HSD test (GluN1, ***p = 0.00096; GluN2A, p = 0.98; TrkB-FL, ***p = 0.00037; BDNF, *p = 0.045; n = 8).",
    "molecules": "NMDA"
  },
  {
    "caption": "TFL457 interference of RIP and calpain-processing in excitotoxicity. Cultures were preincubated with peptides (25 µM, 30 min) or left untreated before NMDA addition (2 h). Different exposures are shown to facilitate visualization of FL, f42/39 or f32. Quantitation of normalized f32 and f42/39 levels. Means ± SEM are shown relative to cultures treated with NMDA and no peptide.",
    "molecules": "NMDA"
  },
  {
    "caption": "Effect of TFL457 on neuronal viability after calpain inhibition. After 30 min preincubation with calpeptin (Calp, 10 µM) and calpain inhibitor III (CiIII, 10 µM), cultures were treated with CPPs and NMDA (4 h) as before. Means ± SEM relative to those obtained in untreated cultures are given.",
    "molecules": "Calp, calpeptin, NMDA, calpain inhibitor III, CiIII"
  },
  {
    "caption": "Effect of TFL457 on neuronal viability after metalloproteinase inhibition. Cultures preincubated with GM6001 (10 µM, 30 min) were treated with CPPs and NMDA as before. Analysis was performed as above (without GM6001, ***p = 0.0007; with GM6001, ***p = 0.0006; n = 4).",
    "molecules": "metalloproteinase, GM6001, NMDA"
  },
  {
    "caption": "Effect of NMDA on total and cell-surface TrkB-FL levels. Cultures were incubated with NMDA (100 µM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB-FL levels are expressed relative to untreated cells.",
    "molecules": "NMDA"
  },
  {
    "caption": "Effect of TFL457 on the decrease of TrkB-FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA-treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA.",
    "molecules": "NMDA"
  },
  {
    "caption": "Effect of endocytosis inhibition on TrkB-FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB-FL and f32. Quantitation of TrkB-FL and TrkB-T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region.",
    "molecules": "dynasore"
  },
  {
    "caption": "Confirmation of Bio-TFL457 delivery to mice cortex. Biotinylated TFL457 (Bio- TFL457, 4 nMole/g, retro-orbitally injected) was detected in coronal sections stained with NeuN and DAPI. Representative confocal microscopy images of cortical areas correspond to single sections. Scale bar, 10 µm.",
    "molecules": "Bio, DAPI"
  },
  {
    "caption": "Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457.",
    "molecules": "TTC"
  },
  {
    "caption": "(A) HEK GFP-LC3 cells transfected with control (siCtrl) or two different SNX18 siRNA oligos (siSNX18-3 and siSNX18-5) were starved or not starved for 2 h in the presence or absence of BafA1. GFP-LC3 lipidation and SNX18 protein knockdown were monitored by immunoblotting. The graph shows the average GFP-LC3-II relative to actin normalized to siCtrl-1 fed ± SEM (error bars), n = 4.",
    "molecules": "BafA1"
  },
  {
    "caption": "(B) The degradation of long-lived proteins in HeLa cells transfected with control or SNX18 siRNA was quantified after 4 h of starvation in the absence or presence of 3-MA and normalized to the degradation in fed cells (mean ± SEM [error bars], n = 3).",
    "molecules": "3-MA"
  },
  {
    "caption": "(G and H) HEK GFP-LC3 cells transfected with myc-SNX18 or a myc control plasmid alone (G) or together with CFP-FRB and mRFP-FKBP or mRFP-FKBP 5pase (H) for 6 h were treated or not treated with 5 µM ionomycin (G) or 2.5 µM A/C heterodimerizer (rapalog; H) for the last 3 h before analysis of the total intensity of GFP-LC3 spots per transfected cell (graph shows mean ± SEM [error bars], n = 3).",
    "molecules": "rapalog, ionomycin"
  },
  {
    "caption": "(B) Lysates from fed or starved MDBK cells were incubated with the indicated glutathione-Sepharose-bound GST-tagged proteins. SNX18 was detected by immunoblotting. Ponceau staining shows the GST proteins.",
    "molecules": "GST, Sepharose, glutathione"
  },
  {
    "caption": "(C) In vitro translated GFP-p62 or GFP-SNX18 were incubated with the indicated GST-tagged Atg8 family proteins. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.",
    "molecules": "GST"
  },
  {
    "caption": "(A) GFP-p62 or -SNX18 full-length, SH3-LC, or PX-BAR regions were in vitro translated and incubated with GST-LC3B or -GABARAP. The resulting pulldowns were separated by SDS-PAGE. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.",
    "molecules": "GST"
  },
  {
    "caption": "(B) 18-mer peptides covering the entire sequence of the SNX18 SH3-LC region were spotted on a membrane that was incubated with GST or GST-GABARAP, which were detected by immunoblotting against GST. The peptide sequences that specifically bound GST-GABARAP are shown with the common WDDEW motif in bold.",
    "molecules": "GST"
  },
  {
    "caption": "(C) Peptides with the sequence YGGYQASQGSDDDWDDEWDDSSTVADEPGAL (SNX18 WT) or with the first (SNX18 W154S), the second (SNX18 W158S), or both (SNX18 W154S/W158S) W mutated to S were spotted on membranes that were incubated with GST, GST-LC3B, or GST-GABARAP. Binding was analyzed as in B.",
    "molecules": "GST"
  },
  {
    "caption": "(D) GFP-SNX18 WT and the indicated mutants were in vitro translated and incubated with GST-LC3B or -GABARAP, and their binding was analyzed as in A.",
    "molecules": "GST"
  },
  {
    "caption": "(B and C) RNAi against SH3PX1 (GFP-positive clones) decreases formation of LTR punctae in response to 4 h of starvation in 20% sucrose (B) and inhibits formation of mCherry-Atg8a-positive autophagosomes in response to 2 h starvation (C). Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;",
    "molecules": "sucrose"
  },
  {
    "caption": "(G) Decreased formation of mCherry-Atg8a-positive autophagosomes in SH3PX1 mutant clones (GFP negative, outlined) in fat bodies from larvae starved in 20% sucrose for 3 h. Bars, 20 µm. (H) The graph shows quantification of total mCherry-Atg8a spot intensity per cell for WT and SH3PX1 mutant cells. The graph shows mean ± SEM (error bars); ***, P < 0.001. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B",
    "molecules": "sucrose"
  },
  {
    "caption": "A, B Representative size-exclusion chromatography profiles of the MICU1-MICU2 complex and chimaera in 2 mM EGTA (A) and 2 mM CaCl2 (B) conditions.",
    "molecules": "CaCl2, EGTA"
  },
  {
    "caption": "A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA. B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+. ",
    "molecules": "Ca2+, EGTA"
  },
  {
    "caption": "E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3.",
    "molecules": "calcium, Carbachol, CCh"
  },
  {
    "caption": "F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible.",
    "molecules": "Ca2+"
  },
  {
    "caption": "G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible.",
    "molecules": "Ca2+, CCh"
  },
  {
    "caption": "H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide.",
    "molecules": "Ca2+, PBS"
  },
  {
    "caption": "I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4. Data information: Error bars for some points are too small to be visible.",
    "molecules": "Ca2+, histamine"
  },
  {
    "caption": "(A) Drosophila egg chambers at stages 9/10 of oogenesis expressing a Hpo kinase dimerization sensor that forms small punctae at the apical domain of the follicle cell epithelium that surrounds the oocyte. (B) Treatment of egg chambers with 1,6-hexanediol (10%) for 5 min abolishes formation of Hpo sensor punctae. (C) Treatment of egg chambers with 1M Sorbitol to osmotically withdraw water, and thereby induce macromolecular crowding, is sufficient to induce formation of large micron-scale Hpo sensor punctae. (D) Nutrient restriction (starvation) of adult females for 24h induces large micron-scale Hpo sensor punctae that are similar in size to those in (C). (E) Treatment of egg chambers from adult females starved for 24h with 1,6-hexanediol (10%) for 5 min abolishes formation of Hpo sensor punctae.",
    "molecules": "Sorbitol, 1,6-hexanediol"
  },
  {
    "caption": "(A) Apical cross-sections of Drosophila ovarian follicle cell epithelia at high magnification, showing accumulation of Hpo-YFP punctae at the apical domain, which is strongly enhanced upon 24h starvation to reduce Insulin/IGF-1 signalling. Hpo puncta are lost following a short treatment with 1,6-hexanediol. Samples were fixed before imaging. (B) Quantification of Hpo-YFP puncta number and size in (A). Biological replicates are plotted as individual data points (n>13 for all samples), error bars represent one standard deviation from the mean, statistical significance was determined using a t-test ****P<0.0001 ***P<0.001 **P<0.01 *P<0.05.",
    "molecules": "1,6-hexanediol"
  },
  {
    "caption": "(G) Moderate overexpression of untagged Kib is sufficient to induce formation of large micron-scale HpoKD-Venus condensates that primarily localise apically. DAPI marks nuclei (blue). (H) Overexpression of Kib-GFP with the Gal4/UAS system is sufficient to induce formation of large micron-scale cytoplasmic condensates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Expression of KIB-GFP, KIB∆C-GFP or KIB∆M+C-GFP in human HEK293T cells and co-staining for pLATS. DAPI marks nuclei in blue.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Human Caco2 intestinal epithelial cells are relatively flat when cultured as cell lines in 2D (Caco-2 cells, top, middle) and cuboidal when cultured in 3D (spheroid, bottom). Punctae of pLATS (green) are strongly enriched at the apical domain of cuboidal cells grown in 3D culture but not in flattened cells grown in 2D culture. Note the increased apical enrichment in medium-density culture in 2D, where cells become more cuboidal than at low-density. Phalloidin staining marks the actin cytoskeleton in red. DAPI marks nuclei in blue.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "Acute hippocampal slices of cDKO were pre-incubated for 1 hour either with 10 nM CTα16 or CTα16scr before inducing LTP by TBS (arrowhead). Averaged potentiation levels at LTP induction (t20-25) revealed a significantly enhancement by CTα16 as compared to CTα16scr present over the whole recoding time. Potentiation levels at t75-80 are significantly increased in the presence of CTα16 (**p<0.01). The LTP induction rate is shown as percentage % of mean baseline slope. Data points were averaged over 6 time points. N CTα16 significantly enhances short-term synaptic plasticity at inter-stimulus intervals of 40 (**p<0.01), 80 (*p<0.05), and 160 (**p<0.01) ms compared to CTα16scr.",
    "molecules": "CTα16, CTα16scr"
  },
  {
    "caption": "Acute application of CTα16 and recAPPsα elevates LTP in cDKO slices to the same extent. O, P After 1 hour pre-incubation with either 10 nM CTα16 or 10 nM recAPPsα fEPSPs were recorded. The TBS induced LTP curve was closely overlapping for both conditions and resulted in similar, statistically not significantly different LTP values Q CTα16 and recAPPsα modulate presynaptic function of cDKO mice in the same manner. Age: 5-6 months. n=number of recorded slices. N=number of animals. Data represent mean ± SEM. Data were analyzed by unpaired two-tailed Students t-test.",
    "molecules": "CTα16"
  },
  {
    "caption": "Treatment with 10 nM α-BTX (30 min prior to TBS, black circles) significantly reduces LTP induction (t20-25, phase of post tetanic potentiation) and maintenance (t75-80) in LM controls expressing endogenous APP and APPsα. The LTP induction rate is shown as percentage % of mean baseline slope. Data points were averaged over 6 time points.",
    "molecules": "α-BTX"
  },
  {
    "caption": "Co-application of BTX and recAPPsα significantly inhibits synaptic plasticity in cDKO mice. After 1 hour pre-incubation of acute slices with 10 nM recAPPsα or 10 nM recAPPsα and 10 nM BTX fEPSPs were recorded. Hippocampal acute slices treated with 10 nM BTX and recAPPsα revealed significantly impaired induction (t20-25) and maintenance (t75-80) of LTP in comparison to slices of cDKO mice recorded in the presence of recAPPsα alone.",
    "molecules": "BTX"
  },
  {
    "caption": "Co-application of BTX and CTα16, the C-terminal domain of APPsα, significantly inhibits synaptic plasticity in cDKO mice. After 1 hour pre-incubation of acute slices with 10 nM CTα16, or 10 nM CTα16 and 10 nM BTX fEPSPs were recorded. Hippocampal acute slices treated with 10 nM BTX and CTα16 revealed similar to recAPPsα, a significantly impaired induction (t20-25) and maintenance (t75-80) of LTP in comparison to slices of cDKO mice recorded in the presence of CTα16 alone. Age: 5-6 months of age. n=number of recorded slices. N=number of animals. Data represent mean ± SEM. Data were analysed by unpaired two-tailed Students t-test. *p<0.05, **p<0.01.",
    "molecules": "CTα16, BTX"
  },
  {
    "caption": "Nicotine- and acetylcholine-induced whole-cell current traces of α7-nAChRs expressed in Xenopus oocytes recorded in the absence or presence of CTα16 or CTα16scr, respectively. Note specific robust potentiation of nicotine- and acetylcholine-induced currents by CTα16, but no direct activation of α7-nAChRs by either peptide in the absence of agonist (not shown). Bars indicate application of EC50 agonist concentrations and of the peptide indicated (green: CTα16: grey: CTα16scr). Quantification of potentiation shows that CTα16 enhances nicotine- and acetylcholine-induced currents to a similar extend whereas CTα16scr had no effect.",
    "molecules": "CTα16, CTα16scr, acetylcholine, Nicotine, nicotine"
  },
  {
    "caption": "CTα16 dose-response curve obtained with submaximal nicotine concentrations (100 µM) with a corresponding EC50 value of 10.7 ± 4.6 nM and a hill-coefficient of 2.1 ± 0.3 (n=4).",
    "molecules": "CTα16, nicotine"
  },
  {
    "caption": "Nicotine-induced whole-cell current traces of α7-nAChRs in the presence of 20 nM recAPPsα (green bar) or 20 nM recAPPsβ (blue bar). Note that only recAPPsα efficiently potentiates α7-nAChRs, which is blocked by BTX (orange bar). Quantification of potentiation shows that recAPPsα enhances nicotine-induced currents to a similar extend as CTα16 (see for comparison Figure 7B) while recAPPsβ had no effect.",
    "molecules": "CTα16, Nicotine, nicotine, BTX"
  },
  {
    "caption": "Nicotine dose-response curve obtained either in the presence of 20 nM recAPPsα (green circles, left) or without treatment (black circles, right). Note that recAPPsα treatment shifts the curve to the left, significantly reducing the apparent agonist affinity by about 2-fold (EC50 of 40.3 ± 6.0 µM, green curve vs. EC50 of 80.5 ± 12.7 µM, black curve; n=5; *p<0.05). For statistical evaluation of potentiation we performed a paired two-tailed Students t-test of the agonist-induced currents in the absence and presence of the indicated peptide. n=number of oocytes. Data represent mean ± SEM. nsp>0.05, *p<0.05.",
    "molecules": "Nicotine"
  },
  {
    "caption": "A. Pull-down experiments using as a bait the biotinylated peptides mimicking the N-terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot (LxxR motif in red, biotin at the C-terminus). Immunoblotting shows affinity-captured GOLPH3 (endogenous and recombinant).",
    "molecules": "biotin"
  },
  {
    "caption": "C. Pull-down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail of LCS WT and LCS mutants (mut. 1, 2 and 3) is shown on the top of the blot (mutations are in blue, LxxR motif in red, biotin is at the C-terminus).",
    "molecules": "biotin"
  },
  {
    "caption": "D. HeLa cells were transfected with LCS-GR, subjected to the synchronisation protocol (see Materials and Methods), fixed and processed for cryoimmunolabeling with an anti-GFP antibody (15 nm gold particles) and anti-GM130 antibody (10 nm gold particles, black arrowheads) peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. Scale bar, 150 nm.",
    "molecules": "gold"
  },
  {
    "caption": "E, F. Quantification of the distribution of LCS-GR as measured by Frequency Distribution Analysis (E) and linear density (LD, in F) (data are means± SEM, number of Golgi stacks is n=15 at 0 min, n=14 at 10 min, n=17 at 20 min. (E) The red arrows indicate the fraction of LCS-GR gold particles (%) in vesicles at each indicated time-point.",
    "molecules": "gold"
  },
  {
    "caption": "A. PHFs GOLPH3 OE (left), or KD (right), were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), TGN46 or DAPI (blue). Dashed lines indicate cells overexpressing or interfered for GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi; data are means ± SEM derived from two biological replicates, n=42 in CTRL, n=49 in GOLPH3 KD and n=27 in GOLPH3 OE; ***p <0.001 [Student's t-test]).",
    "molecules": "DAPI"
  },
  {
    "caption": "G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm.",
    "molecules": "BafA1, bafilomycin A1, DAPI"
  },
  {
    "caption": "A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).",
    "molecules": "glycosphingolipid, lipid, sphingosine, 3H"
  },
  {
    "caption": "B. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.",
    "molecules": "sphingolipid"
  },
  {
    "caption": "C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674.",
    "molecules": "Gb3, lipid"
  },
  {
    "caption": "E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.",
    "molecules": "C16:0 Cer, ceramide"
  },
  {
    "caption": "B. Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate (DSS) and analyzed by mass spectrometry. Schematic representation of the major crosslinking connectivity of Elp123 is shown.",
    "molecules": "disuccinimidyl suberate, DSS"
  },
  {
    "caption": "B. Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto -Ura media containing caffeine. The pRS416 vector is used for these experiments.",
    "molecules": "caffeine, Ura"
  },
  {
    "caption": "C. Growth curve analysis of loop mutants in -Ura media with and without K. lactis toxin zymocin. Shaded regions correspond to the SEM at each time point. N=3.",
    "molecules": "Ura"
  },
  {
    "caption": "C. Size comparison of a representative tRNA molecule (1VTQ) and the cavity observed through the middle of the Elp456 hexamer.",
    "molecules": "tRNA"
  },
  {
    "caption": "A. Silver-stained SDS-PAGE of Elongator glycerol gradient fractions purified using low salt (75 mM NaCl) buffer. The fraction containing the most Elp4, Elp5, and Elp6 is designated by an asterisk (*) and the corresponding fraction from a glutaraldehyde-containing glycerol gradient is used for EM analysis.",
    "molecules": "NaCl"
  },
  {
    "caption": "B. Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations (150 mM and 75 mM NaCl, respectively). Densities resembling a second copy of Elp456 is indicated by white triangles.",
    "molecules": "NaCl"
  },
  {
    "caption": "(D-E)  Western blot analysis of RIPA soluble-fraction and RIPA-insoluble, urea-soluble fraction of proteins prepared from HEK293T  cells expressing empty plasmid (control), FLAG-KIF5A  WT, FLAG-ΔExon27. Insoluble/soluble KIF5A  fractions were detected with anti-FLAG  and actin  was used as a loading control, three biological replicates. Statistical analysis was performed using student t-test (*p < 0.05, **p<0.01, ***p<0.001). Data represent the mean ± s.d.",
    "molecules": "urea"
  },
  {
    "caption": "C) Results of a competitive uptake assay showing inhibition of 0.2 µM L-[3H]Trp or L-[3H]Leu uptake in MhsT-WT-expressing MQ614 cells, measured for 30 seconds in the absence (-) or presence of 12 µM of the natural amino acids (the one-letter code is used) or 4-fluoro-phenylalanine (4FP). Data are the mean ± S.E.M. of three independent experiments each performed as technical triplicates.",
    "molecules": "4-fluoro-phenylalanine, 4FP, Leu, 3H, Trp"
  },
  {
    "caption": "A) Uptake of 0.1 μM L-[3H]Leu was measured for 10-second periods in the presence or absence of the indicated natural amino acids and 4-fluorophenylalanine (4FP, all compounds tested at 100 μM final concentration) in MQ614 expressing MhsT-WT or -M236F.",
    "molecules": "4-fluorophenylalanine, 4FP, Leu, 3H"
  },
  {
    "caption": "B, C) Time course of 0.1 µM L-[3H]Leu (B) or 0.1 µM L-[3H]Trp (C) by MQ614 expressing MhsT-WT () or -M236F (), or MQ614 transformed with the control plasmid, pQE60 ().",
    "molecules": "Leu, 3H, Trp"
  },
  {
    "caption": "D, E) Kinetics of L-[3H]Leu (D) or L-[3H]Trp (E) uptake.",
    "molecules": "Leu, 3H, Trp"
  },
  {
    "caption": "(E)  Localization and expression  of CRAMP (red), E-cadherin  (green), F4/80 (green) and  Ly6G (green) in duodenum by immunofluorescent staining. Representative photomicrographs of individual and merged  staining were shown. Nuclei  were stained with DAPI (blue). Scale bar: 50 μm. Data information: Data were representative from three independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.",
    "molecules": "gliadin"
  },
  {
    "caption": " EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section. ",
    "molecules": "Biotin, biotin"
  },
  {
    "caption": " ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3) ",
    "molecules": "CX5461, DMSO"
  },
  {
    "caption": " Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm. ",
    "molecules": "CX5461"
  },
  {
    "caption": " Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3) ",
    "molecules": "DMSO"
  },
  {
    "caption": " Confocal images to show the subcellular localization of NFATc2 in CD3+ T cells treated with P/I and FK506 or CX5461. Scale bar, 10 μm. ",
    "molecules": "CX5461, FK506"
  },
  {
    "caption": " Flow cytometry analysis of CD69 expression in P/I-stimulated CD3+ T cells treated with or without FK506 or CX5461. ",
    "molecules": "CX5461, FK506"
  },
  {
    "caption": " Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d. ",
    "molecules": "CFSE, CX5461, DMSO, FK506"
  },
  {
    "caption": " Skin graft survival curve in mice treated with DMSO, 2.0 mg/kg FK506 or 0.5 -2.0 mg/kg CX5461. (n = 5) ",
    "molecules": "CX5461, DMSO, FK506"
  },
  {
    "caption": " The survival time (days) of cardiac graft in mice treated with DMSO, 2.0 mg/kg FK506 or 2.0 mg/kg CX5461. (n=5) ",
    "molecules": "CX5461, DMSO, FK506"
  },
  {
    "caption": "Luciferase assay with T-binding elements (TBEs) around the indicated loci as shown in (A) and Fig EV3E. Relative luciferase activities of sorted cell fractions are shown with SDs. (three independent experiments with two technical replicates).",
    "molecules": "luciferase"
  },
  {
    "caption": "Luciferase assay with the specific sequence of the locus bound by T around L#1 and L#3 as shown in (A). Relative luciferase activities of low T-inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs. (three independent experiments with two technical replicates).",
    "molecules": "luciferase"
  },
  {
    "caption": "(C) A549 cells expressing control or p53 shRNA were treated with doxorubicin (Dox, 1μg/ml) for the indicated periods of time. WDR63 mRNA and protein levels were then examined. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.",
    "molecules": "Dox, doxorubicin"
  },
  {
    "caption": "(H) A549 cells expressing control or p53 shRNA were transfected with pGL3-BS1 and Renilla luciferase plasmids. Twenty-four hours later, cells were treated with or without Nutlin (10 μM) for another 12 h. Reporter activity was then measured. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; two-tailed Student's t-test.",
    "molecules": "Nutlin"
  },
  {
    "caption": "(E) Purified GST or GST-Arp2 proteins immobilized on glutathione beads were incubated with purified Flag-WDR63. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.",
    "molecules": "glutathione"
  },
  {
    "caption": "(F) Purified GST or GST-WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.",
    "molecules": "glutathione"
  },
  {
    "caption": "(B and C) Effects of full-length WDR63 (B) and WDR63 (1-200) (C) on Arp2/3-mediated actin polymerization were examined by pyrene actin polymerization assays. Various concentrations of WDR63 (B) or WDR63 (1-200) (C) were incubated with 2 μM actin (50% pyrene-labeled), 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Data shown represent three independent experiments.",
    "molecules": "pyrene"
  },
  {
    "caption": "(D) Effect of WDR63 on Arp2/3-mediated branched actin polymerization was analyzed by electron microscopy. 0.6 μM WDR63 was incubated with 2 μM actin, 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Actin structures were then stabilized with addition of 2μM phalloidin, followed by electron microscopy analysis. The arrows indicate actin branch junctions. The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments. For each condition, over 150 actin filaments were counted. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test. Scale bar: 200 nm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "Representative images of wild-type (WT) and Ctns−/− mouse fibroblasts transfected with the ptfLC3 vector under resting conditions (fed), or after serum starvation (Serum Starv.) in the presence or absence of the alkalinizing drug chloroquine (CQ). GFP and RFP staining was analyzed by confocal microscopy. Examples of red-only puncta (mature autophagosomes) are indicated with arrows. Scale bar: 5 μm.The percentage of mature autophagosomes (red-only vesicles) was calculated based on the ratio between the number of red-only puncta and the total number of autophagosomes (number of green and red + red-only puncta). The graph is representative of three different experiments with similar results. Results are mean ± SEM (n = 7 cells per condition). In addition to the experimental conditions shown in (A), the Aa/serum starvation condition is included.",
    "molecules": "Aa, chloroquine, CQ"
  },
  {
    "caption": "WT and Ctns−/− fibroblasts under resting conditions were treated with 100 nM bafilomycin A (BafA) for 2 h and LC3B-II levels were analyzed by Western blot (WB).",
    "molecules": "BafA, bafilomycin"
  },
  {
    "caption": "Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B-II levels in WT and Ctns−/− fibroblasts were measured by WB under resting conditions (−), withdrawal of both amino acids and serum (Aa/Ser. Starv.) and subsequent recovery by replacement of starvation medium with normal cell growth medium (Rec), in the presence or absence of 100 nM BafA for the indicated time.",
    "molecules": "Aa, amino acids, BafA"
  },
  {
    "caption": "WT,Ctns−/− or Ctns−/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns−/− cells. Scale bars: 5 μm. Inset scale bars: 2 μm.",
    "molecules": "BafA, bafilomycin A, chloroquine, CQ, Leu, leupeptin"
  },
  {
    "caption": "Lysosomes were isolated from livers of starved wild-type (WT) and Ctns−/− mice as described in Supplementary Materials and Methods and incubated at 37°C for 30 min with the CMA substrate GAPDH, in the presence or absence of ATP (necessary for CMA) and protease inhibitors (Prot. Inhib.). A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95°C for 5 min, followed by SDS-PAGE and GAPDH and LAMP1 immunoblotting.Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns−/− mice. Results are mean ± SEM. ***P = 0.0005; **P = 0.0041; N.S., not significant (unpaired t-test).",
    "molecules": "ATP, Prot. Inhib, protease inhibitors"
  },
  {
    "caption": "The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X-100-treated WT and Ctns−/− lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input (reaction with no lysosomes). Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns−/− mice shows no significant difference in protease activity. Results are mean ± SEM.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "WT and Ctns−/− mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat (PQ) for 4 h and stained with (A) FITC-annexin V or (B) propidium iodide (PI) and analyzed by FACS. Results from 3 different experiments are shown as mean ± SEM. In (A), *P = 0.05; ***P = 0.0002. In (B), *P = 0.046; **P = 0.0065. Unpaired t-test.",
    "molecules": "H2O2, paraquat, PQ"
  },
  {
    "caption": "Cystine content in WT,Ctns−/−, cysteamine-treated Ctns−/− fibroblasts or Ctns−/− fibroblasts expressing GFP-CTNS was assessed by mass spectrometry.",
    "molecules": "cysteamine, Cystine"
  },
  {
    "caption": "WT, Ctns−/−, Ctns−/− fibroblasts treated with 1 mM cysteamine (48 h), or Ctns−/− fibroblasts expressing GFP-CTNS were fixed and stained with anti-LAMP1 and LAMP2A antibodies as indicated. For better visualization of LAMP1/LAMP2A colocalization, GFP-CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green (lower panels). Some lysosomes are indicated with arrows. Arrowheads indicate LAMP2A distribution to structures different from lysosomes (only observed in Ctns−/− cells and in cysteamine-treated Ctns−/− cells). Rescue of the LAMP2A localization to lysosomes was observed in Ctns−/− cells expressing GFP-CTNS. Scale bar: 2 μm.Quantification of the colocalization analysis described in (B). Calculation of the Pearson's colocalization coefficient was done by analyzing 113 WT, 251 Ctns−/−, 88 cysteamine-treated and 164 GFP-CTNS-expressing Ctns−/− cells, by using the ZEN 2010 software. Results are mean ± SEM. ***P < 0.001; NS, not significant (one-way ANOVA, Bonferroni's multiple comparisons test).",
    "molecules": "cysteamine"
  },
  {
    "caption": "Cystine content in WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers was assessed by mass spectrometry. Results are mean ± SEM (n = 4). ***P < 0.001; **P = 0.0026 (one-way ANOVA, Bonferroni's multiple comparisons test).",
    "molecules": "cysteamine, Cystine"
  },
  {
    "caption": "Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers.",
    "molecules": "cysteamine"
  },
  {
    "caption": "Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.",
    "molecules": "cysteamine, protease inhibitors"
  },
  {
    "caption": "C. Top views of brains of P7 Mpdz+/+ and Mpdz-/- mice that show the sites of Evans blue injection, and the midline planes (dashed line) sectioned to produce the sagittal images below. They show the extent of Evans blue spread in the ventricles and surrounding tissue (one out of three experiments). In, injection; LV, lateral ventricle; SA, Sylvian Aqueduct; V3, third ventricle; V4, fourth ventricle. Scale bars, 1 mm; insets, 0.25 mm.",
    "molecules": "Evans blue"
  },
  {
    "caption": "A. TEM images of CPEC sections from lateral ventricle villi of two Mpdz+/+ and two Mpdz-/- P14-P16 mice injected with HRP. The CPs were reacted with hydrogen peroxide and DAB ex-vivo. The dark particles are DAB deposits internalized by micropinocytosis. The magnified fields to the right show individual particles engulfed in macropinosomes. Note the layered structure of the particles, and the macropinosome that is open to the ventricular space in the Mpdz-/- section. Scale bars, 1 μm; insets, 100 nm. B. Mean number of engulfed DAB particles per cell in Mpdz+/+ and Mpdz-/- mice (mean ± SD, n=22).",
    "molecules": "DAB, hydrogen peroxide"
  },
  {
    "caption": "C. A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field. Scale bars in panels C and D, 1 μm; insets, 200 nm. D. A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell, and a magnified field that contains several macropinosomes. E. Mean numbers of DAB-containing macropinosomes close to the apical or basal sides of CPECs from Mpdz+/+ or Mpdz-/- mice (mean ± SD, n=20).",
    "molecules": "DAB"
  },
  {
    "caption": "Chemotactic response of N2 worms to varying dilutions of 1-undecene. n ≥ 3 assays. Error bars indicate SEM.",
    "molecules": "1-undecene"
  },
  {
    "caption": "(i) Schematic of setup used to record the movement of 5 worms each under control and 1-undecene odor exposure. (ii) Movement trajectories of N2 worms on E. coli OP50 lawn exposed to 1-undecene odor for 30 min and 60 min, each color represents the trajectory of single worm. Scale bar = 10 mm.",
    "molecules": "1-undecene"
  },
  {
    "caption": "Average calcium responses of AWB::GCaMP3 worms exposed to 1-undecene at indicated concentrations. Each worm was recorded for 180 s. Worms were under stimulus between 11 s-130 s, window shown in grey, and stimulus withdrawal beyond 130 s. Shaded regions around the curves represent SEM. n = 3. Two dashed box represents 10 s time window after stimulus addition and stimulus removal respectively.",
    "molecules": "1-undecene, calcium"
  },
  {
    "caption": "Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, odr-3(n2150) and odr-3(n2046) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM. Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, lim-4(ky403) and lim-4(yz12) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 as determined by two-tailed unpaired t-test. Error bars indicate SEM.",
    "molecules": "1-undecene"
  },
  {
    "caption": "Kaplan Meier survival curve on the full lawn of P. aeruginosa for N2 (naive) worms and N2 worms pre exposed to 1-undecene odor. Survival assay was performed at 20°C.",
    "molecules": "1-undecene"
  },
  {
    "caption": "(a) Microscopy of hemocytes from third instar larvae cultured ex vivo and infected with wild-type L. monocytogenes. Hemocyte nuclei (filled arrowhead) and DNA of L. monocytogenes (open arrowhead) are visualized by DAPI staining (blue or white); the actin cytoskeleton is stained with rhodamine-labeled phalloidin (red). hml-Gal4>Atg5IR, expression of an inverted repeat sequence of Atg5 to induce RNAi against Atg5. Scale bars, 10 μm.",
    "molecules": "rhodamine, DNA"
  },
  {
    "caption": "(b) Intracellular wild-type L. monocytogenes (WT Lm) or Δhly L. monocytogenes (Δhly Lm) counted manually in DAPI-stained infected hemocytes. LE112,hml>LE, hml-Gal4-driven expression of PGRP-LE in PGRP-LE112 hemocytes; imd1, imd; RelE20, RelishE20; hml>Atg5IR, inverted repeat sequence described in a; w, hml>Atg1, hml-Gal4-driven expression of Atg1 in w hemocytes; LE112,hml>Atg1, hml-Gal4-driven expression of Atg1 in PGRP-LE112 hemocytes; PGRP-LE112+rap, PGRP-LE112 hemocytes plus rapamycin. *, P 0.001, compared with wild type (t-test). Data are representative of four experiments (a) or at least three independent experiments (b; error bars, s.d. of triplicate measurements).",
    "molecules": "rap, rapamycin"
  },
  {
    "caption": "(a) Growth of L. monocytogenes in S2 cells (S2) or S2 cells expressing PGRP-LE (S2-LE) transfected with double-stranded RNA (RNAi) specific for Atg5, Rel or Dif and dl (Dif-dl) and infected for 1.5 h with wild-type or Δhly L. monocytogenes, followed by 6 h of incubation in medium containing CuSO4 and gentamicin, quantified by plate assay of colony-forming units. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).",
    "molecules": "CuSO4, gentamicin"
  },
  {
    "caption": "(b,c) Fluorescence confocal microscopy of the localization of PGRP-LE together with wild-type L. monocytogenes in S2 cells engineered to express YFP-PGRP-LE (YFP-LE) and infected for 0.5 h with wild-type L. monocytogenes (b) or Δhly L. monocytogenes (c), followed by 1 h of incubation in gentamicin-containing medium, then DAPI staining. Merge, YFP-PGRP-LE (green) and DAPI (magenta). Filled arrowheads indicate L. monocytogenes; open arrowhead indicates accumulation of YFP-PGRP-LE around the bacteria. Scale bars, 5 μm. Data are representative of three experiments.",
    "molecules": "gentamicin"
  },
  {
    "caption": "(a) Dot- or ring-shaped GFP-LC3 (LC3 dot) signals in S2 cells expressing both PGRP-LE and GFP-LC3 under the control of an actin promoter (PGRP-LE GFP-LC3) or GFP-LC3 alone (GFP-LC3) after infection with wild-type or Δhly L. monocytogenes or after 1.5 h of incubation with 5 μM rapamycin (rap). -, no infection.",
    "molecules": "rap, rapamycin"
  },
  {
    "caption": "(d) Immunoblot analysis of lysates of S2 cells left untreated (-) or infected with wild-type L. monocytogenes or treated with 5 μM rapamycin.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(l) Immunoblot analysis of lysates of hemocytes from third instar larvae cultured ex vivo and infected with L. monocytogenes or treated with rapamycin. Arrowhead indicates processed form of GFP-LC3.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(m) GFP-LC3 dots in S2 cells expressing PGRP-LE and GFP-LC3 and treated with RNAi (below graph) and infected for 0.5 h with L. monocytogenes then incubated for 1 h in gentamicin-containing medium; dots quantified by confocal microscopy. Scale bars, 5 μm (c,j), 1 μm (e,f) or 500 nm (g). *, P 0.001 (t-test). Data are representative of three (a,k,m), two (b,e-i), six (c), four (d) or five (j,l) experiments (error bars, s.d. of triplicate measurements (a,b,l) or at least triplicate measurements (m)).",
    "molecules": "gentamicin"
  },
  {
    "caption": "(a) GFP-LC3 dots in S2 cells expressing GFP-LC3 alone or PGRP-LE and GFP-LC3, treated for 2 h with TCT (100 nM), highly purified DAP-type PGN from L. plantarum (DAP; 100 μg/ml) or lysine-type PGN from S. epidermidis (Lys).",
    "molecules": "TCT"
  },
  {
    "caption": "(b) Dot- or ring-shaped GFP-LC3 signals in hemocytes after infection with wild-type or Δhly L. monocytogenes or treatment with rapamycin (5 μM) or with TCT, DAP-type PGN or lysine-type PGN. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).",
    "molecules": "TCT, rapamycin"
  },
  {
    "caption": "(C, D) 100 μg of purified HeLa S3 mitochondria were subjected to limited protease K (at the indicated amount) digestion in a final 50 μl reaction volume at 37C for 30 minutes (C) or sodium carbonate (100 mM) verse sodium chloride (133 mM) extraction followed by western blot (D).",
    "molecules": "sodium carbonate, sodium chloride"
  },
  {
    "caption": "(A, B) HCT116, DNA-PKcs-/-, and Ligase 4-/- cells were subjected to ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye. The exchange was stimulated by 5mM ADP and halted by ANTs inhibitor carboxyatractyloside (cATR). Normalized MgGreen 5K+ fluorescence was first measured to indicate changes in free magnesium concentration (A). The decreased free magnesium concentration was converted into efflux of ATP ([ATP]e, mM) after the addition of ADP (B). Bar graph on the right shows the normalized MgGreen 5K+ fluorescence and efflux of ATP concentration after ADP until the reaction was stopped by cATR treatment.",
    "molecules": "MgGreen 5K+, ADP, ATP, carboxyatractyloside, cATR, magnesium"
  },
  {
    "caption": "(F) shGFP and shATM pair of HeLa (Right) or MCF7 (Left) cells were subjected to an ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye.",
    "molecules": "MgGreen 5K+"
  },
  {
    "caption": "(L) Treatment with VDAC2 siRNA in Hela cells attenuated ADP-ATP exchange. Bar graph on the right shows the efflux of ATP concentration after adding ADP.",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "(B) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 was disrupted by 10 Gy irradiation, but prevented by pre-treatment with 5 mM NAC. Quantification shown at the right. Data information: The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).",
    "molecules": "NAC"
  },
  {
    "caption": "(A, B) Paired shGFP and shATM HeLa (A) and MCF7 cells (B) were subjected to ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye. The decreased free magnesium concentration was converted into efflux of ATP ([ATP]e, mM) after the addition of ADP. The bar graphs were generated from three independent biological replicates analyses;",
    "molecules": "MgGreen 5K+, ADP, ATP, magnesium"
  },
  {
    "caption": "(E) HeLa cells expressing Flag-tagged VDAC2 and V5-tagged ANT2 were subjected to Co-IP using anti-Flag antibody and analyzed by western blot as indicated. ANT2, but not VDAC2, temporarily dissociated from DNA-PKcs after treatment of 10Gy IR. ATM knockdown and inhibition (AZD1390, 10 nM) disrupted the dissociation.",
    "molecules": "AZD1390"
  },
  {
    "caption": "(A-G) Non-transformed cell lines including (A, B) human retinal pigment epithelial-1 (RPE-1), (C, D) primary human skin fibroblasts (HSF), (E) hTERT immortalized human skin fibroblasts 48BR, (F) primary mouse embryonic fibroblasts (MEF) RPE-1, and (G) A-T patient derived skin fibroblasts GM02052 were subjected to ADP-ATP exchange assay at various time points (up to 16 hrs) in response to 100 μM of H2O2 or 10 Gy IR as indicated. ATM inhibitor (AZD1390, 10 nM) was included in parallel analyses. The grafts display sham (white), bottom (red), and recovery (blue) of ADP-ATP exchange activities after treatments. The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD.",
    "molecules": "AZD1390, H2O2"
  },
  {
    "caption": "(A) Western blot results show knockdown of ATM attenuated 100 μM H2O2 induced T2609 and T2647 phosphorylation of DNA-PKcs in HeLa cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "T-WGBS data of primary fibroblasts from no COPD and COPD (II-IV) patients was analyzed DMRs E Detailed view of a representative hypo- (top) and hypermethylated (bottom) DMR (grey box). CpG methylation levels of each individual donor (dots) and the group average (lines) methylation profile of three no COPD (blue) and five COPD (II-IV) (dark green) donors are displayed. RefSeq annotated genes and CpG islands are indicated.",
    "molecules": "CpG"
  },
  {
    "caption": "D Genome browser view of an example DMR at a putative enhancer region. Group median CpG methylation is shown for no COPD (blue), COPD (I) (light green) and COPD (II-IV) (dark green). At the bottom the level of enhancer marks is depicted as fold-change over control: H3K4me1 (ENCODE accession: ENCFF102BGI) and H3K27ac (ENCODE accession: ENCFF386FDQ).",
    "molecules": "CpG"
  },
  {
    "caption": "A-H Live confocal images of ddaC neurons at WP stage and 16 h APF. Treatment with colchicine significantly suppressed the pruning defects of efa6 RNAi neurons (A, B), while taxol treatment enhanced the pruning defects (C, D). Removal of one copy of γTub23C (F) or expression of γTub23C RNAi (H) almost fully rescued the dendrite pruning defects in efa6 mutant (E) or RNAi neurons (G), respectively. Red arrowheads point to the ddaC somas.",
    "molecules": "colchicine, taxol"
  },
  {
    "caption": "A, Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue, cysteine 101. Recombinant PRL2 was treated with 3-O-methylfluorescein phosphate (OMFP) and analyzed by SDS-PAGE with the Phos-tag reagent. Phosphocysteine is hydrolyzed by boiling.",
    "molecules": "3-O-methylfluorescein phosphate, OMFP"
  },
  {
    "caption": "B, Detection of phosphorylated by electrospray mass spectrometry. OMFP-treatment increased the mass of the intact protein by 79.99 amu but had no effect on the C101A mutant.",
    "molecules": "OMFP"
  },
  {
    "caption": "D, Cysteine phosphorylated PRL2 does not bind to CNNM3. Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull-down by the CBS-pair domain of CNNM3 as a GST fusion. Only unphosphorylated PRL2 was observed in the pull-down lanes while phosphorylated PRL2 was in the flow-through lanes.",
    "molecules": "OMFP"
  },
  {
    "caption": "E, ITC measurement of the affinity of PRL2 for the CNNM3 GST-CBS-pair domain. Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200-fold.",
    "molecules": "OMFP"
  },
  {
    "caption": "B & C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.",
    "molecules": "Mg2+"
  },
  {
    "caption": "B & C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.",
    "molecules": "Mg2+"
  },
  {
    "caption": "(C) Sulfo-NHS-biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood-brain barrier permeability, i.e., extravasation is indicative of leakage from the intravascular lumen. Sulfo-NHS-Biotin was confined to the capillaries of the meninges and cortex (as shown with arrows), and there was no evidence of extravasation. Scale bar = 100 μm.",
    "molecules": "Sulfo-NHS-biotin, Sulfo-NHS-Biotin"
  },
  {
    "caption": "(D) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor (gefitinib).",
    "molecules": "gefitinib"
  },
  {
    "caption": "(E) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control (n = 5) or gefitinib (10 mg/kg) (n = 6), infected with strain K79 for 1 h.",
    "molecules": "gefitinib"
  },
  {
    "caption": "(F) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection.",
    "molecules": "Tyrosine"
  },
  {
    "caption": "(A) Tyrosine phosphorylation of EGFR in HBMEC pre-treated with 20 μM S1P2 antagonist (JTE-013) or vehicle control and infected with GBS strain K79 infection for 60 min. EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79.",
    "molecules": "JTE-013, Tyrosine"
  },
  {
    "caption": "(B) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors. All the inhibitors were used at 10 μM. From left to right, p value of DJB-V-39 is 0.0047, p value of SKI is 0.0062, p value of FTY720 is 0.0022, and p value of HB56 is 0.0045.",
    "molecules": "DJB-V-39, HB56, SKI, FTY720"
  },
  {
    "caption": "(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC.",
    "molecules": "gefitinib, Serine"
  },
  {
    "caption": "(B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC.",
    "molecules": "Serine"
  },
  {
    "caption": "(H Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel)",
    "molecules": "montelukast"
  },
  {
    "caption": "(A) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79. Data are presented as a Kaplan-Maier plot with a log rank test used to compare percentage of survival between the groups, CFX group n = 18, and CFX + MONT group n = 10*p = 0.015. (CFX: ceftriaxone; MONT: montelukast).",
    "molecules": "ceftriaxone, CFX, MONT, montelukast"
  },
  {
    "caption": "(B) Nissl staining of the hippocampus CA1, CA2 & CA3 regions (low magnification) and cortex region (higher magnification) (left) and histograms showing the number of Nissl-stained bodies (right) Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value between control group and Ceftriaxone group is 0.00021, p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0.013.",
    "molecules": "Ceftriaxone, Montelukast"
  },
  {
    "caption": "(C-D) Representative immunofluorescence images (left) and quantification (right) of TUNEL bodies, astrocyte (GFAP), microglia (Iba1) in cortex of wild type mice after 7 days of therapy. Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value of TUNEL bodies between CFX group and MONT + CFX group is 0.0012, p value of astrocyte between CFX group and MONT + CFX group is 0.0068, and p value of microglia cells between CFX group and MONT + CFX group is 0.0029. (E) Y-maze test for spatial learning and memory of different group of wild type mice. The total duration of time spent at novel arm (left) and the percentage of entries to novel arm of Y-maze (right). Data represent the means ± SEM was statistically analyzed by One-way ANOVA, p value of total duration of time spent at novel arm between control group (n = 6) and CFX group (n = 5) is 0.00012, p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.00017; p value of total duration of % of entry to novel arm between control group (n = 6) and CFX group (n = 5) is 0.0102, and p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.0216.",
    "molecules": "CFX, MONT"
  },
  {
    "caption": "A. GAPDH smFISH and IF analysis using anti-G3BP antibody (G3BP) and anti-Dcp1b antibody (DCP1) in A549 cells (RL-WT) or A549 RL-KO cells either mock transfected (mock) or transfected with poly(I:C) (PIC) for 5 hours. Scale bar 10 microns. B. Graph of the fraction of G3BP foci with different volumes in PIC treated RL-WT and RL-KO cells. C. Number of DCP1 foci per cell in PIC treated RL-WT and RL-KO cells relative to the number in mock treated cells. D. Average volume of individual Dcp1 foci in mock and PIC treated RL-WT and RL-KO cells.",
    "molecules": "PIC, poly(I:C)"
  },
  {
    "caption": "C. FISH with probes to GAPDH mRNA or oligo(dT) to detect poly(A)+ RNA in A549 cells without or with either NLS-RL-WT or NLS-RL-CM either mock transfected or transfected with poly(I:C) for 4 hours. Scale bar 5 micron.",
    "molecules": "poly(I:C), poly(A)+ RNA"
  },
  {
    "caption": "D. Graph depicting median and the value of the intensity of oligo(dT) fluorescence in individual nuclei divided by nuclear volume in the indicated cell types either mock transfected or transfected with poly(I:C) for 4 hours. At least 43 nuclei analyzed per condition.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. A. FISH analysis to detect poly(A)+ RNA and nucleolar-localized snoRD3A RNA. IF analysis to detect nucleolar granular component protein nucleophosmin (NPM1). B. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 17 nuclei mock treated cells, 31 nuclei PIC treated cells. C. Fraction of nuclei with NPM1 protein enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm. 17 nuclei mock treated cells, 31 nuclei PIC treated cells.",
    "molecules": "PIC, poly(I:C), poly(A)+ RNA"
  },
  {
    "caption": "Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. D. IF analysis to detect dense fibrillar component protein, ribosome processing factor 1 (RPF1). E. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 43 nuclei mock treated cells, 69 nuclei PIC treated cells. F. Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells.",
    "molecules": "PIC, poly(I:C)"
  },
  {
    "caption": "Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. G. FISH analysis to detect poly(A)+ RNA and nuclear speckle-localized MALAT1 RNA and IF analysis with anti-sc35 antibody to detect nuclear speckle protein SRRM2. H. Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei. 51 nuclei mock treated cells, 56 nuclei PIC treated cells. I. Graph of the median and value of the volume of individual SRRM2 foci in nuclei. Mock 1526 SRRM2 foci in 51 nuclei. PIC 136 SRRM2 foci in 20 nuclei that contain <10 SRRM2 foci.",
    "molecules": "PIC, poly(I:C), poly(A)+ RNA"
  },
  {
    "caption": "Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. J. FISH analysis to detect poly(A)+ RNA and IF analysis to detect Cajal body protein coilin. K. Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo(dT) with different distributions of coilin protein. 90 nuclei in mock treated cells, 192 nuclei in PIC treated cells.",
    "molecules": "PIC, poly(I:C), poly(A)+ RNA"
  },
  {
    "caption": "Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. A. FISH analysis to detect poly(A)+ RNA and IF analysis against FUS protein. Images are a single 0.2 micron Z section.",
    "molecules": "PIC, poly(I:C), poly(A)+ RNA"
  },
  {
    "caption": "Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. B. Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo(dT) signal. All the nuclei (total) in which nuclear RNA was degraded (N=64) or the same nuclei classified as to whether or not they contained residual poly(A) foci. Nuclei with poly(A) foci (N=14). Nuclei without poly(A) foci (N=50).",
    "molecules": "poly(A), PIC, poly(I:C)"
  },
  {
    "caption": "Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. C. FISH analysis to detect poly(A)+ RNA and IF analysis against mediator complex protein, MED1, to detect super-enhancer condensates. D. Number of MED1 foci in mock and PIC treated NLS-RL-WT cells. E. Average volume of individual MED1 foci in mock and PIC treated NLS-RL-WT cells.",
    "molecules": "PIC, poly(I:C), poly(A)+ RNA"
  },
  {
    "caption": "Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. F. FISH analysis to detect poly(A)+ RNA and IF analysis against BRD4, to detect super-enhancer condensates. G. Number of BRD4 foci in mock and PIC treated NLS-RL-WT cells. H. Average volume of individual BRD4 foci in mock and PIC treated NLS-RL-WT cells.",
    "molecules": "PIC, poly(I:C), poly(A)+ RNA"
  },
  {
    "caption": "A LC-ESI-MS/MS spectrum of Dnmt3a pulled down by Biotin-As in NIH3T3 cells. NIH3T3 cells were treated with 10 μg/mL Biotin-As for 2 hours, followed by pull-down assay and determination using LC-ESI-MS/MS.",
    "molecules": "As, Biotin"
  },
  {
    "caption": "B-D Validation of the binding between DNMT3A and Biotin-As at 5 μg/mL in NIH3T3 (B), MEFs (C) and CCRF-CEM (D) cells in the Biotin-As pull-down assay.",
    "molecules": "As, Biotin"
  },
  {
    "caption": "F Measurement of DNMT3A-ADD thermodynamic stability upon ATO treatment. Purified DNMT3A-ADD was mixed with ATO at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Melting curves were measured by DSF (left panel), and the calculated ΔTm values are shown (right panel).",
    "molecules": "ATO, HEPES"
  },
  {
    "caption": "G LC-MS spectra of purified DNMT3A-ADD before and after ATO incubation at 1:5 molar ratio for 16 hours.",
    "molecules": "ATO"
  },
  {
    "caption": "H, I Biotin-As pull-down assays for the transfected DNMTs (H) and DNMT3A-C520A/C524A mutation (I) in 293T cells treated with 5 μg/mL Biotin-As.",
    "molecules": "As, Biotin"
  },
  {
    "caption": "A Immunoblotting of DNMT3A protein levels in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO in the indicated conditions.",
    "molecules": "ATO"
  },
  {
    "caption": "B MEFs (upper panel) and CCRF-CEM cells (lower panel) were pretreated with 20 μM DMSO, 1 μM MG132, 0.02 μM Concanamycin A (Conc A), 20 μM Calpeptin (Calp), or 20 μM Z-VAD-FMK (Z-VAD) for 2 hours, followed by 16-hour ATO co-treatment at 1 μg/mL for MEFs or 0.3 μg/mL for CCRF-CEM cells. Immunoblotting of DNMT3A in whole cell lysate is shown.",
    "molecules": "Calp, Calpeptin, Conc A, Concanamycin A, ATO, DMSO, MG132, Z-VAD, Z-VAD-FMK"
  },
  {
    "caption": "C HCT116 cells were pretreated with 3 μM MG132 for 2 hours, followed by ATO co-treatment for 16 hours at the indicated concentrations. Immunoblotting of DNMT3A in whole cell lysate is shown.",
    "molecules": "ATO, MG132"
  },
  {
    "caption": "D In vivo ubiquitination assay. 293T cells were transiently transfected with HA-ubiquitin and Myc-DNMT3A. After 24 hours, cells were treated with or without MG132 for 2 hours at the indicated concentrations, followed by ATO co-treatment for 6 hours at the indicated concentrations. Myc-DNMT3A was immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) as indicated.",
    "molecules": "ATO, MG132, ubiquitin"
  },
  {
    "caption": "E Immunoblotting of the transfected wild-type DNMT3A, C520A/C524A and C541A mutants in 293T cells upon 1 μg/mL ATO treatment for the indicated durations.",
    "molecules": "ATO"
  },
  {
    "caption": "F Measurement of DNMT3A-ADD thermodynamic stability upon ATO or AsPh3 treatment. Purified DNMT3A-ADD was mixed with ATO or AsPh3 at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Chemical structure of AsPh3 is shown. Melting curves were measured by DSF (upper panel), and the calculated ΔTm values are shown (lower panel).",
    "molecules": "ATO, HEPES, AsPh3"
  },
  {
    "caption": "G Immunoblotting of DNMT3A in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO or AsPh3 in the indicated conditions.",
    "molecules": "ATO, AsPh3"
  },
  {
    "caption": "H, I Immunoblotting of endogenous DNMT3A, DNMT3B and DNMT1 protein levels in CCRF-CEM (H) and THP-1 (I) cells treated with DAC or ATO in the indicated conditions. DAC: +, 0.2 μM. ATO: +, 0.06 μg/mL; ++, 0.2 μg/mL; +++, 0.6 μg/mL. Red asterisk indicates the specific band.",
    "molecules": "DAC, ATO"
  },
  {
    "caption": "A Dot blot analysis of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ. The full blotting image of Fig 4A is shown in Fig EV4D, the treatment is the same.",
    "molecules": "DAC, 5meC, ATO"
  },
  {
    "caption": "B COBRA-IAP of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The signal density of unmethylated and methylated DNA were quantified by ImageJ.",
    "molecules": "DAC, ATO"
  },
  {
    "caption": "C 5meC enrichment analysis of the IAP, Xist, H19 loci in MEFs upon ATO treatment at 0.3 μg/mL for 4 or 7 days as measured by MeDIP-qPCR.",
    "molecules": "5meC, ATO"
  },
  {
    "caption": "D Dot blot analysis of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ.",
    "molecules": "DAC, 5meC, ATO"
  },
  {
    "caption": "E COBRA-LINE-1 of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment in the indicated conditions. The signal density of unmethylated and methylated DNA were quantified by ImageJ.",
    "molecules": "DAC, ATO"
  },
  {
    "caption": "F 5meC enrichment analysis of the XIST, H19 loci in CCRF-CEM cells upon ATO treatment at the indicated concentrations for 63 days as measured by MeDIP-qPCR.",
    "molecules": "5meC, ATO"
  },
  {
    "caption": "Ca2+-current-voltage relationships evoked by incremental 15 ms step depolarizations from -87 mV to +58 mV revealed a ~25% reduction of ICa in 1/3 DKO and TKO mice. (A') Due to the perinatal lethality of TKO mice, detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically-cultured organs of Corti after 7 days in vitro (DIV). C57BL/6J (Wt) and 1/3-DKO served as controls. Please note that all recordings from cultured IHCs were performed at [Ca2+]e of 10 mM to maximize IHC exocytic performance. (A") Quantification and statistical analysis of individual maximum ICa amplitudes (Imax) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Ca2+-current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1/3-DKO cells. (B') Quantification and statistical analysis of the residual current at the end of the test pulse. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Representative ICa (upper panel) and Cm (lower panel) in response to a 50 ms depolarizing step to the potential eliciting Imax. (C') Exocytic ∆Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2-100 ms for all respective genotypes and at [Ca2+]e = 6 mM for a second set of recordings from wild-type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants. (C") Magnification of the initial, short depolarizing steps (2-20 ms) for clarity. Exocytic ∆Cm of cultured endophilin-deficient IHCs appears strongly reduced. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis). The reduced Ca2+-efficiency of exocytosis (∆Cm/QCa) in endophilin-deficient IHCs indicates that diminished Ca2+-influx cannot fully account for the reduction of exocytosis. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Table S4 for detailed statistical analysis).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Representative Ca2+-currents (upper panel) and exocytic membrane capacitance increments (∆Cm; lower panel) in response to a 200 ms depolarizing step to -14 mV.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Exocytic ∆Cm (top) and corresponding Ca2+-current integrals QCa (bottom) of Wt and endophilin-deficient IHCs in response to voltage steps from -84 to -14 mV of variable stimulus duration (5 to 200 ms). In 1/3-DKO IHCs, our data indicate reduced sustained exocytosis to a strong depolarization (200 ms; *p = 0.0470, K-W with Dunn's post-hoc test; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis). Inset: Initial ∆Cm showed comparable RRP exocytosis in endophilin-deficient IHCs.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Ca2+-efficiency to drive exocytosis was reduced for strong depolarizations (200 ms) in 1/3-DKO IHCs; *p = 0.0323, K-W with Dunn's post-hoc test; please also refer to Appendix Table S7 for detailed statistical analysis.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Representative electron micrographs of Wt and 1/2-DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity (radius r = 1 µm from the center of the ribbon) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K+. Scale bars: 200 nm.",
    "molecules": "K+"
  },
  {
    "caption": "(a) Left: starvation increases the level of RFP-LC3 puncta (arrows) in rat cortical neurons. Middle: quantification of the percentage of cells with RFP-LC3 puncta. Right: pretreatment with 10 mM 3-MA attenuates the starvation-induced increase in LC3-II level in neurons. Data are means ± s.e.m.; n=3. Samples were run on the same gel and spliced together to create the image shown.",
    "molecules": "3-MA"
  },
  {
    "caption": "(d,e) Pretreatment with Ros (d) or knockdown of Cdk5 (e) attenuates starvation-induced EndoB1 phosphorylation at Thr 145 and autophagy induction in neurons. Data are means ± s.e.m.; n=3 for d and n=8 for e. DMSO, dimethylsulphoxide.",
    "molecules": "Ros"
  },
  {
    "caption": "(a) Left: lipid binding of EndoB1 is inhibited by mutating the five lysine/arginine residues between amino acids 176 and 183 of EndoB1 to glutamic acid (5E mutant). Right: purified GST protein was included as a negative control. S, supernatant fraction; P, pellet fraction that contains the lipid-bound proteins.",
    "molecules": "lipid"
  },
  {
    "caption": "(e) Mutation of Thr 145 to alanine (T145A) or glutamic acid (T145E) has negligible effect on the lipid-binding property of EndoB1. Conversely, the 5E mutant is less able to bind lipid. The right panel shows quantified data as means ± s.e.m.; n=3.",
    "molecules": "lipid"
  },
  {
    "caption": "(a) MPTP administration decreases the numbers of tyrosine hydroxylase (TH)- and cresyl violet (CV)-positive neurons in the substantia nigra. Representative images of tyrosine hydroxylase-stained brain sections are shown. Right panels show quantified data as means ± s.e.m.; n=9 for tyrosine hydroxylase and n=3 for cresyl violet.",
    "molecules": "MPTP"
  },
  {
    "caption": "(b) MPTP administration upregulates the levels of LC3-II and Thr 145-phosphorylated EndoB1 in the midbrain. Lower panels show quantified data as means ± s.e.m.; n=3.",
    "molecules": "MPTP"
  },
  {
    "caption": "(c) Knockdown of EndoB1 or Cdk5 inhibits MPP+-induced autophagy in cultured neurons. Data are means ± s.e.m.; n=5.",
    "molecules": "MPP"
  },
  {
    "caption": "(d) Cdk5 mediates the MPP+-induced EndoB1 phosphorylation at Thr 145 and the MPP+-induced increase in the LC3-II level in neurons.",
    "molecules": "MPP"
  },
  {
    "caption": "(e) Overexpression of EndoB1T145A attenuates MPP+-induced autophagy in neurons. Data are means ± s.e.m.; n=7.",
    "molecules": "MPP"
  },
  {
    "caption": "(f) MPTP fails to increase LC3-II and Thr 145-phosphorylated EndoB1 levels in the midbrain of p35−/− mice. The concomitant reduction in tyrosine hydroxylase level triggered by MPTP injection is also markedly attenuated. Right panels show quantified data as means ± s.e.m.; n=3. Uncropped images of blots are shown in Supplementary Fig. S8.",
    "molecules": "MPTP"
  },
  {
    "caption": "(a) Pretreatment with 3-MA, but not z-VAD-fmk, significantly reduces MPP+-induced neuronal death. Data are means ± s.e.m.; n=6.",
    "molecules": "3-MA, MPP, z-VAD-fmk"
  },
  {
    "caption": "(b) Knockdown of Atg5 expression abrogates MPP+-induced neuronal loss, whereas it reduces neuronal survival in untreated cells. Data are means ± s.e.m.; n=3.",
    "molecules": "MPP"
  },
  {
    "caption": "(c) Knockdown of EndoB1 or Cdk5 expression abolishes MPP+-induced neuronal death. Data are means ± s.e.m.; n=8.",
    "molecules": "MPP"
  },
  {
    "caption": "(d) Overexpression of EndoB1T145A inhibits MPP+-induced neuronal death. Data are means ± s.e.m.; n=5.",
    "molecules": "MPP"
  },
  {
    "caption": "(e) Pretreatment with 3-MA, but not z-VAD-fmk, reduces α-synucleinA53T mutant-induced neuronal death. Data are means ± s.e.m.; n=6.",
    "molecules": "3-MA, z-VAD-fmk"
  },
  {
    "caption": "PCR fragments amplified with specific primers for Vκ and Jκ5 from genomic DNA of purified splenic B cells from Ptenf/f x 3-83ki and Ptenf/f x mb1-cre x 3-83ki mice on the respective backgrounds. Genomic tail DNA from a 3-83ki mouse and DNA from purified splenic B cells of a control (ctrl) mouse were used as controls. PCR for the splicing factor Srp20 served as a loading control. Kb and Kd indicate the respective background of the mice, (H2-Kb: +Ag).",
    "molecules": "genomic DNA, Genomic tail DNA"
  },
  {
    "caption": "B | Intracellular Ca2+ influx was measured in CD90.2/Thy1.2- splenocytes derived from mice of the indicated genotypes following stimulation with 10 μg/ml α-κLC antibody. Figures are representative of at least 3 individual mice per genotype.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D | Representative intracellular Ca2+ influx in HH10-mu IgM- and HH10-mu IgD-expressing cells upon stimulation with multivalent HEL (complex cHEL), monovalent (soluble sHEL) (both at a concentration of 1 μg/ml), or 10 μg/ml α-mouse κLC antibody, respectively.",
    "molecules": "Ca2+"
  },
  {
    "caption": "E | Representative intracellular Ca2+ influx of HH10-mu IgM- (top) and HH10-mu IgD- (bottom) expressing cells upon stimulation with indicated ratios of 1 μg/ml cHEL and sHEL, and a 1 : 25 mixture of cHEL with bovine serum albumin (BSA), where 1 = 1 μg/ml.",
    "molecules": "Ca2+"
  },
  {
    "caption": "| Representative intracellular Ca2+ influx measured in Thy1.2- splenocytes, derived from mice of the indicated genotypes, upon stimulation with cHEL, monovalent sHEL (both at a concentration of 1 μg/ml) or 10 μg/ml α-mouse κLC antibody, respectively. Line diagrams below the dot plots show the overlayed MFI of the Ca2+ influx kinetics displayed in the plots above.",
    "molecules": "Ca2+"
  },
  {
    "caption": "C | Quantification of intracellular Ca2+ influx The median area under the curve of the Ca2+ influx kinetics was calculated, single dots represent data from individual mice, mean ±SD. Statistical significance was calculated by applying the Mann-Whitney-U test n. s. = not significant, ** p ≤ 0.01.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D | Analysis of GC in spleen sections from WT and IgD-/- animals after 4, 7 and 10 days post immunization. Representative images of 2 x 2 mm sections stained with DAPI (nuclei) and peanut agglutinin (PNA) are shown for the indicated time points. Scale bar represents 500 μm. Dashed white or yellow lines mark distinct GC foci in the merged images. E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs. As no distinct GC foci were observed in IgD-/- samples from days 4 and 7, an identical 800 x 800 μm region is shown in each case. F | Quantification of number of GCs per mm2 area of spleen sections from WT (black) and IgD-/- (green) animals after 4, 7 and 10 days post immunization. Data represent mean ±SD of two sections (from 3 animals per group and time point) and were analyzed by two-tailed unpaired t-test. ",
    "molecules": "DAPI"
  },
  {
    "caption": "G | Total serum α-TNP IgG. Sera from PBS- or TNP-Ova-immunized WT (left) and IgD-/- (middle) mice, collected at day 7 following immunization, were adjusted to an IgG concentration of 1 μg/ml and applied in duplicates and dilution steps of 1 : 3 to TNP-coated plates, respectively, mean ±SEM. Right: Overlay of α-TNP IgG titers from TNP-Ova-immunized WT and IgD-/- mice. Statistical significance was calculated by using the Mann-Whitney-U test (see also Table S7), n. s. = not significant, * p ≤ 0.05, ** p ≤ 0.01.",
    "molecules": "TNP"
  },
  {
    "caption": " Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Developmental loss of VE-cadherin leads to severe edema. Fetuses of the genotypes listed in the top row were analyzed at the developmental stages in the first column. Numbers in brackets denote embryos with signs of edema. Last column lists the percentage of viable fetuses in the VE-cadherin deleted cohort Fetuses were explanted at day E14.5. Arrowheads indicate prominent edema along the back. The images are representative for 42 (Cdh5lox/lox) and 25 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals",
    "molecules": "4-OHT"
  },
  {
    "caption": " Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Skin wholemount preparations stained for PROX1, PECAM1 and VEGFR-3. Maximum intensity projections (MIPs) of tiled confocal stacks (Cdh5lox/lox 2075µm x 2075µm, z=50µm; Cdh5lox/lox; Prox1-CreERT2 3765µm x 3765 µm, z=61µm). Scale bars = 100 μm. The data are representative for 6 (Cdh5lox/lox) and 6 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 3 litters",
    "molecules": "4-OHT"
  },
  {
    "caption": " Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Enumeration of the PROX1-expressing (PROX1+) nuclei in three different VE-cadherin deleted wholemount preparations analogous to Fig. 1B. Nuclei were counted in a MIP corresponding to an area of 1.5 mm2 and normalized to the number of nuclei in Cdh5lox/lox control preparations The area covered by lymphatic vessels was determined in the samples evaluated in (D) and is depicted as relative area compared to Cdh5lox/lox controls. The calculations and measurements were obtained from three animals and three confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji",
    "molecules": "4-OHT"
  },
  {
    "caption": " Deletion of VE-cadherin in lymphatic endothelial cells of newborn pups was induced by two optimal doses of 4-OHT at days P2 and P4. For analysis 6 weeks later ear punches were subjected to wholemount immunostaining of the dermal vasculature. A-H (A, B, E, F) Overview MIPs of ear skin wholemount preparations immunostained for the indicated antigens. VEC, VE-cadherin; PRX, PROX1. (C, D, G, H) Magnified views of the areas in the red boxes in (A, E). Areas delimited by the stippled yellow boxes in (C) and (G) are enlarged in the insets (yellow solid boxes). Yellow arrows indicate widened lumen, yellow arrowheads denote oak leaf-shaped LECs with discontinuous junctions. Scale bars in (A and E) correspond to 100 μm and in (C and G) to 50 µm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters ",
    "molecules": "4-OHT"
  },
  {
    "caption": " A-L VE-cadherin deletion was initiated either in newborn pups (A, B, E, F) by two applications of 4-OHT at days P2 and P4 or in mice at 11 weeks (C, G) and 42 weeks (D, H) of age (three applications of Tamoxifen at 2 day intervals via oral gavage). Ear skins were prepared 6 weeks (A, B, E, F) or 5 weeks (C, D, G, H) after induction and immunostained for the indicated proteins. Shown are MIPs of confocal tile scans (approx. 750 µm x 750 µm) covering 40 µm in depth. White arrows in (F) highlight distorted and partially fragmented lymphatic vessels and white arrowheads denote aberrantly pointed vessels (F). (I-L) Lymphatic valves in the dermis of the ear were maintained for 5 weeks despite deletion of VE-cadherin at 11 weeks of age (yellow arrows in K, L). PEC1, PECAM1; PRX1, PROX1;VR3, VEGFR-3. Scale bars correspond to 100 μm. The data represent wholemount stainings from 6 (A, E), 5 (B, F), 6 (C, G, K, L), 3 (D, H) and 6 (I, J) analyzed animals ",
    "molecules": "4-OHT, Tamoxifen"
  },
  {
    "caption": "  Fig. 4: VE-cadherin is indispensable for the maintenance of mesenteric lymphatic vessels at all ages. A-F Deletion of VE-cadherin was induced in mice at various age (4 weeks (A), neonate (B), 6 month (C, D, E) or 1 year (F)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages). The mesentery was prepared as indicated 4, 6, 8 or 11 weeks later and the wholemount preparations were immunostained for the proteins specified in color (left). (B) Inset in top panel shows a magnification of the area outlined by the white dashed line (for further magnification see expanded view Fig. 1A and B). Arrows indicate the previous position of lymphatic valves; white arrowheads indicate PECAM1+ capillaries supplying the intestinal fat tissue lining the vessels (magnified in expanded view Fig. 1C); yellow arrowheads denote island of LECs that display intracellular VEGFR-3 staining. (C) Arrows denote LECs that have converted from growth as tubes to sheets; white arrowheads, thinning of lymphatic vessels; yellow arrowheads, aberrant sprouting of LECs. The area outlined by a white box is depicted magnified in (D) and provides details on LECs growing as a sheet of cells. (E) Formation of ectopic sprouts from the valve area of degenerating lymphatic vessels. (F) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet-like growth. PEC1, PECAM1; PRX1, PROX1; VR3, VEGFR-3. Scale bars = 100 μm. The data represent n=5 (A), n=5 (B), n=3 (C-E) and n=6 (F) animals.  ",
    "molecules": "4-OHT, Tamoxifen"
  },
  {
    "caption": "  Fig. 5: Deletion of VE-cadherin results in fragmentation and distension of the intestinal lacteals. A-E Deletion of VE-cadherin was induced in mice at various ages (neonatal (C), 16 weeks (D) or 1 year (E)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages).Control mice of the same age were subjected to an identical regimen (A, B). Intestinal wholemount preparations of the jejunum and ileum were generated 5 - 8 weeks after Tamoxifen induction and immunostained for the indicated proteins, scale bars = 100 μm. The data represent wholemount stainings from 3 (D), 5 (B,E) and 6 (A, C) animals analyzed for each genotype.  ",
    "molecules": "4-OHT, Tamoxifen"
  },
  {
    "caption": "Deletion of VE-cadherin was induced in adult mice (13 weeks) and EdU incorporation into PROX1-positive cell nuclei was determined 6 weeks after Tamoxifen administration. Shown are mesenteric wholemount stainings prepared from control (A, B; n=3) or VE-cadherin-deleted mice (C, D; n=3). (B) and (D) show a magnification of the area outlined by the white dashed line in (A) and (C). Antigens shown in addition to the EdU incorporation are depicted in colour on the left side of the first panel. PEC, PECAM1; PRX1, PROX1. Scale bars = 100 µm Enumeration of EdU-positive LECs. The measurements were obtained from three animals and nine confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji. Co-localization of PROX1 and EdU was determined manually in each confocal plane, using the cell counting tool of Fiji. The data represent mean ± SD. **** p ≤ 0.0001. Two-tailed unpaired Student's t test",
    "molecules": "EdU, Tamoxifen"
  },
  {
    "caption": "Aberrantly enhanced VEGFR-3 phosphorylation in VE-cadherin-deficient mesenteric LECs of six weeks old mice that had received 4-OHT injections at P2 and P4. Shown are wholemount immunostainings for the antigens shown on the left in color. pVR3, pVEGFR-3, PRX1, PROX1; PEC1, PECAM1. (G) and (I) show a magnification of the area outlined by the white dashed box in (F) and (H). Scale bars = 50 μm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters",
    "molecules": "4-OHT"
  },
  {
    "caption": "Expression of the transcriptional co-activators YAP and TAZ was analyzed in mesenteric wholemount preparations 8 weeks after induction of VE-Cadherin deletion at 1 year of age by three doses of Tamoxifen. Respective antigens depicted are indicated on the top of the panels in colour. The highly related molecules YAP and TAZ were both recognized by the antibody used and are therefore collectively abbreviated Y/T. Scale bars = 100 μm",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "  Fig. 7: Loss of VE-cadherin from lymphatic endothelial cells results in increased circumferential actin and a reduced number of focal adhesions. A, B Primary dermal LECs were isolated as described in appendix Fig. S8 A. PdLECs were used for up to four passages and control and VE-cadherin-deleted pdLECs had been cultured for the same number of passages. Cells were seeded at a density of 2.5 x 104 cells on gelatine-coated 15mm glass coverslips and co-stained after 3 days of culture for the actin cytoskeleton (Phalloidin) and either Vinculin (A) or FAK (B) for the visualization of focal adhesions. PROX1 staining in (A) identifies LECs, nuclei in (B) were counterstained with Hoechst. Scale bars correspond to 50 µm. (C, D) Bar diagrams show the number of focal adhesions/cell, which were enumerated using Image J from the Vinculin and FAK stainings shown in (A and B). Data are mean values + S.E.M. derived from three cell lines and 9 viewfields of 256 µm x 256 µm per biological group. *** p ≤ 0.001. Two-tailed unpaired Student's t test.  ",
    "molecules": "Hoechst, gelatine, Phalloidin"
  },
  {
    "caption": "Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured at low/high density Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without serum Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without Aphidicolin (Aphi) (C). Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.",
    "molecules": "Aphi, Aphidicolin, DAPI, phalloidin"
  },
  {
    "caption": "Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on HR in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.",
    "molecules": "cGAMP"
  },
  {
    "caption": "Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on NHEJ in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.",
    "molecules": "cGAMP"
  },
  {
    "caption": "cGAS is not recruited to DSB sites: Confocal microscopic images of GFP-NLS- or GFP-hcGAS-expressing U2OS-DSB reporter cells incubated (or not) with Shield-1 and 4-OHT to induce the expression and translocation of mCherry-LacI-FokI (red) to specific DSB sites. Scale bar: 10μm. The arrowheads indicate DSB sites.",
    "molecules": "4-OHT, Shield-1"
  },
  {
    "caption": "Pre-incubation of dsDNA with mcGAS-cat prevents D-loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D-loop formed in each reaction (left) was graphed as the average of triplicates ± SD.",
    "molecules": "dsDNA"
  },
  {
    "caption": "Pre-incubation of template dsDNA with hcGAScat blocks subsequent D-loop formation. The percentage of D-loop formation (below) was graphed as the average of triplicates±SD.",
    "molecules": "dsDNA"
  },
  {
    "caption": "Negative-stain electron micrographs of cGAS-dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm.",
    "molecules": "dsDNA"
  },
  {
    "caption": "Effect of indicated hcGAS variants on D-loop formation when pre-incubated with dsDNA. Percentage of D-loop formed in each reaction (left) graphed as the average of triplicates± SD.",
    "molecules": "dsDNA"
  },
  {
    "caption": "hcGASΔDNA-Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24.",
    "molecules": "cGAMP, dsDNA"
  },
  {
    "caption": "hcGASΔDNA-Y215E and hcGASΔcGAMP are defective in synthase activity.",
    "molecules": "cGAMP"
  },
  {
    "caption": "Negative-stain electron micrographs showing that hcGAScat-ΔDNA-Y215E is defective in inducing cGAS-dsDNA complexes. Scale bar: 100 nm.",
    "molecules": "dsDNA"
  },
  {
    "caption": "L. Representative images of HeLa  cells expressing the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK  constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS), and stained  with markers of the  endoplasmic reticulum (KDEL), plasma membrane  (CellMask) and mitochondria  (cytochrome C). Data information: Scale bar represents 5 μm in (L)",
    "molecules": "CellMask, cytochrome C"
  },
  {
    "caption": "J-L. Representative stills (J), kymographs  (K) and quantification of the number of DLK-GFP  positive particles (L) from time-lapse recordings of DIV6 DRG neurons  overexpressing GFP-tagged DLK   and treated with control (DMSO) or DLK  inhibitor GNE-3511 (DLKi) for 3h (n = 22-46 neurons/condition; Unpaired t test). Data information: Arrowheads point to individual DLK-GFP  positive structures in J. All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001. Scale bar represents 10 μm in (J), 5 μm in (K)",
    "molecules": "DMSO, GNE-3511"
  },
  {
    "caption": "E-F. Lipidomic analysis of the immunoprecipitates  obtained from the HA-tagged protein immunoprecipitations  from extracts of HeLa  cells overexpressing HA  alone, DLK-WT-HA  and DLK-CS-HA. Quantification of lipid  classes (E) and sphingomyelin  fatty acid species (F) measured in the immunoprecipitates  (n = 3-4 independent experiments; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. CE, cholesteryl ester; CER, ceramide; DAG, diacylglycerol; DCER, dihydroceramides; HCER, hexosylceramides, LCER, lactosylceramides; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; TAG, triacylglycerol.",
    "molecules": "HCER, hexosylceramides, lactosylceramides, LCER, CER, ceramide, CE, cholesteryl ester, DAG, diacylglycerol, DCER, dihydroceramides, lipid, LPC, lysophosphatidylcholine, LPE, lysophosphatidylethanolamine, PC, phosphatidylcholine, PE, phosphatidylethanolamine, SM, sphingomyelin, TAG, triacylglycerol"
  },
  {
    "caption": "G-I. Representative Western blots  of DLK, p-cJun  and GAPDH (G) and quantification of relative c-Jun phosphorylation  (H) and DLK  levels (I) from 3DIV cultured embryonic DRG neurons  treated with D609  for 3h in the presence or absence of NGF  (n = 8 biological replicates; series of unpaired t tests with control NGF  withdrawal condition followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "D609"
  },
  {
    "caption": "Representative stills (J) from  time-lapse recordings from 6DIV cultured embryonic DRG neurons  overexpressing GFP-tagged DLK  and treated with D609  for 3h (n = 29-46 neurons  Arrowheads in J point to individual DLK-GFP  positive structures. Data information: Scale bar represents 10 μm in (J)",
    "molecules": "D609"
  },
  {
    "caption": "kymographs  and quantification of the number of DLK-GFP  positive particles (K) from  time-lapse recordings from 6DIV cultured embryonic DRG neurons  overexpressing GFP-tagged DLK  and treated with D609  for 3h (n = 29-46 neurons; Mann-Whitney U test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents , 5 μm in (K)",
    "molecules": "D609"
  },
  {
    "caption": "L. Representative Western blots of DLK, α-tubulin and Na+/K+-ATPase from cytosolic, membrane  and insoluble fractions of 3DIV cultured embryonic DRG neurons  treated with D609  for 3h.",
    "molecules": "D609"
  },
  {
    "caption": "A.  Western blot  analysis of endogenous DLK, p-cJun, Rab6  and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons  treated with dynasore  (DYN) or brefeldin A  (BFA) for 3h in the presence or absence of NGF (n = 6-12 biological replicates; series of unpaired t tests /Mann-Whitney U tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "BFA, brefeldin A, DYN, dynasore"
  },
  {
    "caption": "B. Representative Western blots of p-MKK4, MKK4  and GAPDH, and quantifications of the relative MKK4 phosphorylation levels in 3DIV cultured embryonic DRG neurons  treated with dynasore  (DYN) for 3h in the presence or absence of NGF  (n = 6 biological replicates, series of unpaired t tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "DYN, dynasore"
  },
  {
    "caption": "Representative stills (C), kymographs (D) and quantification of the orientation (E) of DLK-GFP  positive particles from time-lapse recordings of DIV6 DRG neurons  overexpressing GFP-tagged DLK  and treated with dynasore  (DYN) or brefeldin A (BFA) for 3h (n = 34-101 neurons, Chi-square test in E Arrowheads in C point to individual DLK-GFP  positive structures. Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (C) and , 5 μm in (D)",
    "molecules": "BFA, brefeldin A, DYN, dynasore"
  },
  {
    "caption": "quantification of number of DLK-GFP  positive particles (F) from  time-lapse recordings of DIV6 DRG neurons  overexpressing GFP-tagged DLK  and treated with dynasore  (DYN) or  brefeldin A (BFA) for 3h (n = 34-101 neurons, series of Mann-Whitney tests with control followed by a Holm-Sidak correction in F). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "BFA, brefeldin A, DYN, dynasore"
  },
  {
    "caption": "G-H. Representative Western blots of DLK, α-tubulin and  Na+/K+-ATPase (G) and quantifications (H) from cytosolic  and membrane  fractions of 3DIV cultured embryonic DRG neurons  with dynasore  (DYN) for 3h in the absence of NGF  (n = 5-11 biological replicates, series of unpaired t tests /Mann-Whitney U tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "DYN, dynasore"
  },
  {
    "caption": "I-K. Representative stills (I), kymographs  (J) and quantification of the number of DLK-GFP  positive particles (K) from cultured embryonic DRG neurons  treated with chlorpromazine  (CHP), dansylcadaverine  (DCV) or methyl-β-cyclodextrin (MCD) for 3h (n = 12 neurons, series of Mann-Whitney U tests with control situation followed by a Holm-Sidak correction). Arrowheads in I point to individual DLK-GFP  positive structures. Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (I), 5 μm in (J).",
    "molecules": "chlorpromazine, CHP, MCD, methyl-β-cyclodextrin, dansylcadaverine, DCV"
  },
  {
    "caption": "L. Representative  Western blots  of p-cJun  and GAPDH, and quantification of relative  c-Jun phosphorylation  levels in 3DIV cultured embryonic DRG neurons  treated with chlorpromazine  (CHP), dansylcadaverine  (DCV) or methyl-β-cyclodextrin (MCD) for 3h in the absence of NGF  (n = 6-24 biological replicates, series of Mann-Whitney U tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "chlorpromazine, CHP, MCD, methyl-β-cyclodextrin, dansylcadaverine, DCV"
  },
  {
    "caption": "M. Representative Western blots  of p-cJun  and GAPDH, and quantification of relative c-Jun phosphorylation  levels in 3DIV cultured embryonic DRG neurons  treated with vincristine  (VNC) and dynasore  (DYN) for 3h (n = 7 biological replicates, series of Mann-Whitney U tests with vincristine  condition followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "DYN, dynasore, vincristine, VNC"
  },
  {
    "caption": "A. Western blot  analysis of cleaved  caspase-3 (cCasp3) and GAPDH, and quantifications of cCasp3 levels in 3DIV cultured embryonic DRG neurons  treated with dynasore  (DYN) for 8h in the absence of NGF  (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "DYN, dynasore"
  },
  {
    "caption": "B. Quantifications of cCasp3 levels from 3DIV DRG neurons  treated with chlorpromazine  (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 8h in absence of NGF  (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.",
    "molecules": "chlorpromazine, CHP, MCD, methyl-β-cyclodextrin, dansylcadaverine, DCV"
  },
  {
    "caption": "D. Representative images of axons  from E12.5 DRG  explants cultured for 2 days, subsequently deprived of NGF and treated with different concentrations of dynasore  (DYN) for 24 h, and visualized by immunostaining  for βIII-tubulin. Quantification of the axonal degeneration index  as the ratio of fragmented axon  area over total axon  area (n = 13-15 explants, series of unpaired t-test/Mann-Whitney U tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 20 μm in (D) and 10 μm in zooms in (D).",
    "molecules": "DYN, dynasore"
  },
  {
    "caption": "E. Representative images of axons  from cultured embryonic DRG neurons  cultured for 2 days, subsequently deprived of NGF  and treated with different concentrations of dynasore  (DYN) for 24 h, and visualized by imaging SiR-tubulin  probe. On the right, quantification of fragmented axons  using the degeneration index  (n = 15 images from 3 independent cultures, series of unpaired t-test/Mann-Whitney U tests with control NGF  withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 100 μm in (E)",
    "molecules": "SiR, DYN, dynasore"
  },
  {
    "caption": "(F) qRT-PCR analysis showing the dynamic changes of Islr in colonic tissues from mice upon DSS treatment and after DSS removal. n = 4 at each time points.",
    "molecules": "DSS"
  },
  {
    "caption": "(G) In situ hybridization for Islr with RNAscope probe in mouse colons without or with 3-day or 5-day DSS treatments. Scale bar: 50 μm.",
    "molecules": "DSS"
  },
  {
    "caption": "L) In situ hybridization for ISLR/Islr with RNAscope probes in mouse colon tumors from AOM-DSS model (L). t indicating tumor; a indicating adjacent tissues of tumor. Scale bar: 50 μm.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "(D) qRT-PCR for Ets1 in colons with or without 5-day DSS treatment. n = 4.",
    "molecules": "DSS"
  },
  {
    "caption": "(E) Double immunofluorescence for Ets1 and β-catenin in normal mouse colons without DSS treatment, mouse colons with 5-day DSS treatments, and AOM-DSS mouse colon tumors. Scale bar: 50 μm.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "(J) Chromatin immunoprecipitation assay was carried out on primary intestinal stromal cells isolated from WT mice without DSS treatment or WT mice after 5-day DSS treatment using antibodies against ETS1 and Histone H3. The antibody against Histone H3 was used as a positive control. IgG was used as a negative control. The enrichment of Ets1 binding to Islr promoter was quantified using qPCR. n = 3 technical replicates.",
    "molecules": "DSS"
  },
  {
    "caption": "(A) Quantification of body weight change in control (n=12) and cKO (n=13) mice after DSS treatment and DSS removal.",
    "molecules": "DSS"
  },
  {
    "caption": "(C) Histological images of colonic tissues from control (n=10) and cKO (n=10) mice at indicated timepoints after DSS treatment. The Panels with non DSS are identical to Panels in Fig EV1F. Scale bar: 100 μm.",
    "molecules": "DSS"
  },
  {
    "caption": "(E) Gross images of colons and quantification of colon length from control (n=6) and cKO (n=6) mice two days after 5-day DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "(F) Double immunofluorescence for Ki67 and β-catenin in colons from control (n=4) and cKO (n=4) mice two days after 5-day DSS treatment. Quantification of percentage of Ki67+ epithelial cells. Scale bar: 100 m.",
    "molecules": "DSS"
  },
  {
    "caption": "(C) Gross images of AOM-DSS tumors in control (n=9) and cKO (n=10) mice.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "(A) Heatmap of the altered Hippo related genes in colons from control and cKO mice one day after 5-day DSS treatment. The parameter of the color key indicated the fold changes converted to log2 of signal value normalized.",
    "molecules": "DSS"
  },
  {
    "caption": "(C) Western blotting for pMst1/2, Mst1, pMob1 and Mob1 in colon epithelium from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The quantification of pMst1/2 versus Mst1 and pMob1 versus Mob1 were shown under the corresponding protein bands.",
    "molecules": "DSS"
  },
  {
    "caption": "(D) Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice two days after 5-day DSS treatment. Histone H3, a positive control for nuclear proteins. α-Tubulin, a positive control for cytoplasmic proteins. The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band.",
    "molecules": "DSS"
  },
  {
    "caption": "(E and F) Double immunofluorescence for Yap1 and β-catenin in the colons from control (n=4) and cKO (n=4) mice two days after DSS removal (E), and in AOM/DSS tumors from control (n=4) and cKO (n=4) mice (F). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "(G) Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice two days after 5-day DSS treatment. β-actin was used as a loading control. The ratio of Yap1/pYap1 were quantified. n=3.",
    "molecules": "DSS"
  },
  {
    "caption": "(E) To analyze changes in oxidative stress in PV+ interneurons, the somatosensory and motor cortices of WT and Cox6a2-/- mice were stained with an antibody against 8-OHdG, a marker of oxidative DNA damage. (F) 8-OHdG immunoreactivity was significantly higher in PV+ null in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests. ",
    "molecules": "8-OHdG"
  },
  {
    "caption": "(C) Changes in expression of oxidative stress-related genes (green), genes coding for AMPA receptors (orange), sodium channels (red), potassium channels (magenta), and internal calcium signaling proteins (blue). Color scale bars show normalized read counts.",
    "molecules": "calcium"
  },
  {
    "caption": "(E) PV+ interneurons from PV-CRE;Cox6a2fl/fl mice exhibited significantly lower ATP-to-ADP ratios than PV+ interneurons from PV-CRE mice throughout the entire duration of the recordings (23 cells from 4 PV-CRE mice; 15 cells from 6 PV-CRE;Cox6a2-/- mice).",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "(H) Recorded PV+ interneurons were filled with biocytin and their dendritic morphology was reconstructed using Imaris software. Scale bars: 30 μm.",
    "molecules": "biocytin"
  },
  {
    "caption": "A, B. Sum projections of the skeletonized ridge network and microvilli (A) and segmented IgM-BCR clusters (B) in an untreated and 100 µM CK-666-treated cell for 15 consecutive time frames amounting to 37.5 s.",
    "molecules": "CK-666"
  },
  {
    "caption": "G. Continuous analysis of IgM-BCR cluster density in 15-30 min 100 µM CK-666-treated cells using geodesic distance maps initiating from distinct surface features (centerline of ridges, nodes and microvilli tips) (to compare to untreated in Fig 3F). Data points and the dotted lines represent mean ± SD. H. Time-course images of EGFP-CaaX and IgM-BCR intensity in an elongating microvillus (purple, MV mask).",
    "molecules": "CK-666"
  },
  {
    "caption": "A. IgM-BCR cluster distribution upon Ag stimulation. Ramos B cells in the Matrigel were stimulated with 50 ng/ml NIP-15-BSA Ag for at least 15 minutes. Ag-MC on the B cell surface were segmented and overlaid with the ridge network (including microvilli) with a 1.25x and 3.0x local intensity cut-off for cluster detection.",
    "molecules": "NIP-15"
  },
  {
    "caption": "A. Phalloidin-Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP-CaaX and IgM-BCR with or without 30 min 2 µM LatA treatment.",
    "molecules": "Alexa555, LatA, Phalloidin"
  },
  {
    "caption": "D. Surface-masked EGFP-CaaX and IgM-BCR signal and overlay of segmented elevations (yellow) with the surface-masked IgM-BCR signal in cells treated with LatA and LatA+Ag (50 ng/ml NIP-15-BSA). E. Time frames from a LatA+Ag cell showing directed movement of Ag-MC towards the elevations (yellow). Each Ag-MC shown with a different colored arrow.",
    "molecules": "NIP-15, LatA"
  },
  {
    "caption": "F. IgM-BCR intensity enrichment within all the segmented clusters in LatA and Lat+Ag cells. G. Cluster density at surface features. Quantification of the IgM-BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM-BCR clusters (left panel, local contrast cutoff = 1x) and for Ag-MC (right panel, local contrast cutoff = 3x). H",
    "molecules": "Lat, LatA"
  },
  {
    "caption": "I. Surface-masked EGFP-CaaX and IgM-BCR signal for a LatA and LatA+Ag cell and the sum projections of their segmented IgM-BCR clusters (duration: 37.5 s). IgM-BCR clusters are color-coded (green and yellow) according to their assigned surface region.",
    "molecules": "LatA"
  },
  {
    "caption": "a, Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 ()), with and without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD, vacuoles were stained with FM4-64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30 and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4-64. Gray: DIC. Scale bar 5 µm.",
    "molecules": "FM4-64, NaCl"
  },
  {
    "caption": "atg18 cells expressing plasmids carrying Atg18-GFP or Atg18FGGG-GFP, a mutant in the lipid binding sites, were stained with FM4-64. a, Confocal z-stacks were taken before and 2, 6 and 10 min after addition of 0.4 M NaCl. Stacks were processed into maximum projections using ImageJ. Scale bar: 5 µm.",
    "molecules": "FM4-64, NaCl"
  },
  {
    "caption": "d, Localisation of Atg18-GFP 5 min after salt shock. Experiment as in a. Scale bar: 1 µm.",
    "molecules": "salt"
  },
  {
    "caption": "b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for b, the wildtype peptide",
    "molecules": "hexafluoro-2-propanol, HFIP"
  },
  {
    "caption": "b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for c, the SLoop peptide.",
    "molecules": "phospholipid"
  },
  {
    "caption": "Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for a, Atg18wt",
    "molecules": "Cholesterol"
  },
  {
    "caption": "Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for b, Atg18FGGG;",
    "molecules": "Cholesterol"
  },
  {
    "caption": "Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for c, Atg18DLoop;",
    "molecules": "Cholesterol"
  },
  {
    "caption": "Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for d, Atg18SLoop.",
    "molecules": "Cholesterol"
  },
  {
    "caption": "a, Time lapse analysis of Atg18 added to liposomes. 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5% PI3P and 1% PI(3,5)P2. The sample was imaged on a spinning disc confocal microscope. Most of the tubules underwent scission into small vesicles (See movie EV6). Scale bar 5 µm. Areas in boxes 1 and 2 of c are shown at higher magnification in b and c. Scale bars: 2.5 µm.",
    "molecules": "PI(3,5)P2, PI3P"
  },
  {
    "caption": "a, 1µM Atg18 was added to wells containing sedimented GUVs with 10% PI3P or PI(3,5)P2 while imaging a single plane by spinning disc microscopy. Membrane tension exerted by Atg18 binding suffices to induce liposome collapse, which allows to better judge the mobility of the tubular or clustered membranes that remain. Images are extracted from movies EV2 to EV5. Scale bar 5 µm.",
    "molecules": "PI(3,5)P2, PI3P"
  },
  {
    "caption": "b, Atg18 was covalently labeled with the green fluorescent dye Alexa488 TFP (ThermoFisher) and used in experiments as in a, with rhodamine-phosphatidylethanolamine-labeled (Rh-PE) GUVs containing the indicated phosphoinositides. Scale bar 5 µm",
    "molecules": "TFP, Alexa488, phosphatidylethanolamine, rhodamine"
  },
  {
    "caption": "a, Cross-linking of Atg18. Purified recombinant Atg18wt or Atg18FGGG (1.5 µM) was incubated with liposomes containing 5% PI3P, PI(3,5)P2, PI(4,5)P2 or 15% PS as a control for negative charge. After addition of a cleavable cross-linker or buffer only, liposomes were pelleted, dissolved in sample buffer, incubated in the presence or absence of 100 mM dithiothreitol (DTT) for 5 min at room temperature and analysed by SDS-PAGE and Coomassie staining. Cross-link products migrate at the upper limit of the separating gel.",
    "molecules": "PI(3,5)P2, dithiothreitol, DTT, PI(4,5)P2, PS, PI3P"
  },
  {
    "caption": "b, Cross-linking experiment with recombinant Atg18wt as in a, performed in the presence of increasing concentrations of liposomes containing 5% PI(3,5)P2.",
    "molecules": "PI(3,5)P2"
  },
  {
    "caption": "(b) Rates of degradation of long-lived proteins in control and RARα(-) cells maintained in the presence or absence of serum for 12 h. Values are expressed as percentage of proteolysis; n = 3.",
    "molecules": "proteins"
  },
  {
    "caption": "(c,d) Percentage of degradation due to lysosomes (c) and macroautophagy (d) in cells assayed as in b but treated with inhibitors of lysosomal proteolysis (c) or with 3-methyladenine (3MA) to block macroautophagy (d). Values are expressed as percentage of total protein degradation sensitive to the lysosomal inhibitors; n = 3. In all panels, all values are mean ± s.e.m., and differences with control are significant for *P 0.05.",
    "molecules": "3-methyladenine, 3MA, protein"
  },
  {
    "caption": "(a) Rates of degradation of long-lived proteins in mouse fibroblasts untreated (None) or treated with 40 μM ATRA and maintained in the presence or absence of serum. Values are expressed as a percentage of proteolysis; n = 3.",
    "molecules": "ATRA, proteins"
  },
  {
    "caption": "(d,e) Autophagic flux in untreated and ATRA-treated cells expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged-channel images. Arrows indicate autolysosomes (red). Scale bar, 2 μm. Shown in e are the number of autophagosomes (left) and percentage of autolysosomes (right) after quantification of >50 cells. AV, autophagic vacuoles.",
    "molecules": "ATRA"
  },
  {
    "caption": "(d,e) Autophagic flux in untreated and ATRA-treated cells expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged-channel images. Arrows indicate autolysosomes (red). Scale bar, 2 μm. Shown in e are the number of autophagosomes (left) and percentage of autolysosomes (right) after quantification of >50 cells. AV, autophagic vacuoles.",
    "molecules": "ATRA"
  },
  {
    "caption": "(f) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter with or without ATRA and after photoactivation maintained in the presence or absence of serum. Left, representative images. Nuclei are labeled with DAPI. Scale bars, 10 μm. Graph shows quantification of the number of puncta per cell in >50 cells. All values in a-c, e and f are mean ± s.e.m., and differences with untreated (marked with asterisk) or with serum-supplemented cells (marked with §) are significant for P 0.01. Full-field fluorescence images and full-length blots are shown in Supplementary Figures 3 and 21, respectively.",
    "molecules": "ATRA"
  },
  {
    "caption": "(a) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter without (None) or with 20 μM of the indicated compounds imaged 16 h after photoactivation. Insets show higher-magnification images. Nuclei are labeled with DAPI. Scale bars, 10 μm. (b) Quantification of the effect of increasing concentrations of GR1 on the same cells. Untreated cells and cells treated with 40 μM ATRA are also shown. Representative images are shown in Supplementary Figure 7. Graph shows the average number of fluorescent puncta per cell, quantified in >50 cells. All values are mean ± s.e.m.",
    "molecules": "ATRA, GR1"
  },
  {
    "caption": "(c) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units (RLU) detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12h .",
    "molecules": "ATRA"
  },
  {
    "caption": "(d) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection.Cells transfected as in c were treated with 100 nM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU.",
    "molecules": "ATRA, BMS614"
  },
  {
    "caption": "(e) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h.",
    "molecules": "ATRA"
  },
  {
    "caption": "(f) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h. Cells transfected as in e were treated with 10 μM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU. Values in c-f are mean ± s.e.m.; n = 4-6.",
    "molecules": "ATRA, BMS614"
  },
  {
    "caption": "(a) Rates of degradation of long-lived proteins in control mouse fibroblasts or RARα(−) or LAMP-2A(−) (L-2A(−)) cells left untreated (None) or treated with 20 μM of the indicated compounds. Values are expressed as the fold change in proteolytic rate relative to the rate in untreated cells for each group; n = 3.",
    "molecules": "proteins"
  },
  {
    "caption": "(b-d) Control mouse fibroblasts or RARα(−), LAMP-2A(−) or LAMP-2B(−) (L-2B(−)) cells were transfected with the KFERQ-mCherry1 photoactivatable reporter with or without the indicated compounds (20 μM). In b are shown the representative fields and high-magnification images (insets) for GR1. Nuclei are labeled with DAPI. Representative fields for GR2 and AR7 are shown in Supplementary Figure 13. Scale bars, 10 μm. (c,d) Average number of fluorescent puncta per cell quantified in >50 cells in at least four different fields in control cells and RAR(−) (c) or LAMP-2B(−) (d) cells. No puncta were detected in LAMP-2A(−) cells.",
    "molecules": "AR7, GR1, GR2"
  },
  {
    "caption": "(e) Mouse fibroblasts transfected with the KFERQ-mCherry1 photoactivatable reporter with the indicated concentrations of AR7 or GR1 or with both compounds to reach the same final concentration, as indicated. Top, representative fields and high-magnification images (insets). Nuclei are labeled with DAPI. Bottom, quantification of the number of fluorescent puncta per field in each condition. Scale bars, 10 μm. In a, c, d and e, values are mean ± s.e.m.; n > 50 cells. Differences with untreated samples (marked with asterisk) or between single and combined treatments (§) are significant for P 0.01.",
    "molecules": "AR7, GR1"
  },
  {
    "caption": "(b) mRNA levels of LAMP-2A in control mouse fibroblasts (left) or RARα(−) cells (right) treated with AR7 or paraquat (PQ) as in a; n = 4-5. Values are presented as fold change in mRNA relative to untreated cells.",
    "molecules": "paraquat, AR7"
  },
  {
    "caption": "(c) Cellular viability of control (left) or LAMP-2A(−) (right) fibroblasts exposed to 2 mM or 0.5 mM paraquat, respectively, and treated with the indicated compounds for 12 h before or after the paraquat treatment; n = 3.",
    "molecules": "paraquat"
  },
  {
    "caption": "(d) Viability of mouse fibroblasts transfected with the indicated concentrations of a plasmid encoding α-synuclein and left untreated (None) or treated with 1 mM paraquat alone or in the presence of 20 μM AR7; n = 3. In b-d, all values are mean ± s.e.m. Differences with cells that are untreated (marked with asterisk) or those treated only with paraquat (§) were significant for P 0.001. Full-length blots are shown in Supplementary Figure 21.",
    "molecules": "paraquat, AR7"
  },
  {
    "caption": "B. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test). D ",
    "molecules": "TRITC"
  },
  {
    "caption": "H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.",
    "molecules": "TRITC"
  },
  {
    "caption": "F, G Lysates from T47D or BT20 treated with DMSO, PD173074, AG1478, or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα (F); T47D or BT20 treated with DMSO, or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα (G) were immunoblotted with the indicated antibodies.",
    "molecules": "AG1478, MEK162, BMS582949, DMSO, PD173074, U0126"
  },
  {
    "caption": "Lysates from T47D stimulated or not with FGF10, EGF for different time periods or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies.",
    "molecules": "AG1478, MEK162, PD173074"
  },
  {
    "caption": "G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).",
    "molecules": "AG1478, MEK162, PD173074"
  },
  {
    "caption": "(D) Immunofluorescence  of neurons  expressing BioID2-tau   or BioID2  (myc) or non-transduced control cells. Cells were incubated in biotin  medium (50 μM) or vehicle medium for 30 h prior to fixation and probed for biotin  (streptavidin, SA) and cell nuclei  (DAPI) (n = 3 biological replicates). BioID2-tau   and incorporated biotin co-localise in axons  and somata. Scale bar, 25 μm.",
    "molecules": "biotin, DAPI, SA, streptavidin"
  },
  {
    "caption": "(C) Immunofluorescence  of P35 mouse brain  expressing BioID2-tau. Hippocampal Cornu ammonis 1 (CA1) region was probed for BioID2-tau  (myc), for incorporated biotin  (streptavidin, SA) and for cell nuclei  (DAPI) (n = 3-5 biological replicates). Scale bar, 50 μm.",
    "molecules": "biotin, DAPI, SA, streptavidin"
  },
  {
    "caption": "(F) Microscale thermophoretic (MST) measurement of direct tau   and NSF interaction. MST binding  curve of recombinant hexameric  NT-647-NHS-labelled NSF  (27 pM) and recombinant tau   (18 mM - 0.275 nM). Dissociation constant: KD  = 146 ± 70 nM (sigmoidal curve fit, R2 = 0.844). (n = 5 technical replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.",
    "molecules": "NT-647-NHS"
  },
  {
    "caption": "(H) Rate constants derived from kinetic  data in i. (n = 3 technical replicates) ATP  hydrolysis is similar in NSF   and NSF  incubated with control peptide TAT-Scr, albumin, or denatured tau, yet lower with increasing concentrations of tau   or after addition of NSF-inhibiting peptide TAT-NSF. Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Impaired AMPA-type glutamate receptor  surface expression  in cultured tau-/- neurons. Cortical tau+/+ or tau-/- neurons  were surface biotinylated after incubation with non-competitive NMDAR  antagonist MK-801  (50 μM, 3 min) and/or GABAAR antagonist bicuculline (30 μM, 10 min) at 37°C. Surface GluA1  levels were detected by immunoblot  after with streptavidin  (SA) enrichment. Input was probed for total cellular GluA1, NSF, tau   and  βIII-tubulin. (n = 3 biological replicates) Input and SA-enrichment from non-surface biotinylated neurons  served as negative controls. (B) Reduced neuronal  surface GluA1  in tau-/- neurons. Quantification of tau+/+ and tau-/- surface GluA1  upon treatment and SA-enrichment shown in (A). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test. ",
    "molecules": "bicuculline, MK-801, SA, streptavidin"
  },
  {
    "caption": "(C) Cell surface GluA1  and total NSF immunostaining  in primary mouse hippocampal neurons (DIV15). Tau+/+, tau-/- neurons  and tau-/- expressing human tau  (tau-/-.AAVtau) were treated with either vehicle (Veh; DMSO) or MK-801 (50 μM) for 3 min. Representative confocal  images of whole cell (left) and neurites  (right) are shown (n = 3 biological replicates). NSF, surface GluA1  and cell nuclei  (DAPI) were visualized. Scale bar, 25 μm. (D-E) Quantification of neurite  surface GluA1  and neurite NSF  relative to tau+/+ Veh control. Reduced surface GluA1  (D) and total NSF  (n = 10-12 neurites  from 3 biological replicates) (E) localized  to neurites  in tau-/- neurons  treated with MK801  (n = 21-23 neurites  from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test. ",
    "molecules": "DAPI, DMSO, MK-801, MK801"
  },
  {
    "caption": "(C) Cued fear conditioning memory in tau+/+, tau-/- and tau-/-.AAVtau mice  upon bilateral infusion of TAT-NSF   or TAT-Scr. Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included. Note that infusion of  TAT-NSF lowers memory performance to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. (n = 2-5 biological replicates) Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.",
    "molecules": "isoflurane"
  },
  {
    "caption": "(D) Novel object preference  (fold preference over familiar object) in tau+/+, tau-/- and tau-/-.AAVtau mice  upon bilateral infusion of TAT-NSF  or TAT-Scr (n = 2-5 biological replicates). Due to infusion of Tat-fusion peptides occurring under brief isoflurane  anaesthesia, a general anaesthesia (GA) control is included (n = 2 for GA). Note that infusion of TAT-NSF  lowers object discrimination to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.",
    "molecules": "isoflurane"
  },
  {
    "caption": "F. FACS  analysis to evaluate cell cycle progression  by  Propidium Iodide (PI) staining  in BJ  cells treated as indicated in E.",
    "molecules": "Propidium Iodide"
  },
  {
    "caption": "Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2-/-) (red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2-/- sperm",
    "molecules": "NH4Cl, Ca2+, ionomycin, progesterone, PGE1"
  },
  {
    "caption": "Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2-/- sperm form DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labelled with DAPI (blue). Scale bars represent 10 µm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "Representative distribution of rotation frequencies of freely swimming non-capacitated (0 mM bicarbonate; black; n = 218) and capacitated sperm (25 mM bicarbonate; red; n = 232) determined by dark-field imaging.",
    "molecules": "bicarbonate"
  },
  {
    "caption": "Rolling frequency (mean ± SD) of non-capacitated (0 mM bicarbonate, n = 1,455) and capacitated sperm (25 mM bicarbonate, n = 1,097).",
    "molecules": "bicarbonate"
  },
  {
    "caption": "Rolling frequency (mean ± SD) of freely swimming sperm in 0 (n = 1,175), 0.2 (n = 832), and 1 % (w/v) (n = 599) methyl cellulose. .",
    "molecules": "methyl cellulose"
  },
  {
    "caption": "Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 73) and capacitating conditions (25 mM bicarbonate; red; n = 272).",
    "molecules": "bicarbonate"
  },
  {
    "caption": "Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm incubated under non-capacitating (0 mM bicarbonate, n = 1009) and capacitating (25 mM bicarbonate, n = 946) conditions.",
    "molecules": "bicarbonate"
  },
  {
    "caption": "Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm in 0 (n = 457), 0.2 (n = 389), and 1 % (w/v) (n = 187) methyl cellulose.",
    "molecules": "methyl cellulose"
  },
  {
    "caption": "Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream. The green trace indicates the fluorescence of fluorescein included in the barrier stream. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream.",
    "molecules": "fluorescein"
  },
  {
    "caption": "Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate.",
    "molecules": "bicarbonate"
  },
  {
    "caption": "Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2+. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies in the presence and absence of extracellular Ca2+.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Fraction of motile sperm (mean ± SD) in a sperm population incubated in the presence (black) and absence (at t = 0) of extracellular Ca2+ (red) (n ≥ 5).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Distribution of rotation frequencies in populations of freely swimming sperm in the presence (black, n = 335) and absence (red, n = 224) of extracellular Ca2+ (red).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Graphs show changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of GEM, and WST assays were performed 48 hr after treatment (n=3, biological replicates).",
    "molecules": "GEM"
  },
  {
    "caption": "Levels of endpoint metabolites of the purine synthesis pathway in GR cells relative to those in WT cells as determined by CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. n.s.= non-significant   Levels of endpoint metabolites of the pyrimidine synthesis pathway in GR cells relative to those in WT cells as determined by CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01.  ",
    "molecules": "purine, pyrimidine"
  },
  {
    "caption": "Levels of intermediate metabolites of the tricarboxylic acid cycle (TCA) cycle in GR cells relative to those in WT cells based on targeted CE-MS metabolomics. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01.",
    "molecules": "TCA, tricarboxylic acid"
  },
  {
    "caption": "Relative 2-hydroxyglutarate (2-HG) production level based on targeted CE-MS metabolomics comparing among WT and GR cells (left panel: T24, right panel: UMUC3). The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. *p<0.05, **p<0.01. Relative mRNA expression levels of IDH2 and EGLN1 genes compared between WT and GR cells (left panel: T24, right panel: UMUC3). The expression levels were analyzed by Student's t test and plotted relative to expression levels in WT cells. The data are shown as the means ± SEs (n=3, biological replicates. *p<0.05, ***p<0.001.",
    "molecules": "2-HG, 2-hydroxyglutarate"
  },
  {
    "caption": "Graph showing the viability of GR cells exposed to various concentrations of GEM for 48 hr after transfection with NTC and IDH2-targeting (siIDH2#1 and #2) siRNA (left: T24GR, right: UMUC3GR) The data are shown as the means ± SDs (n=3, biological replicates).",
    "molecules": "GEM"
  },
  {
    "caption": "Graph showing the viability of J82WT and J82-IDH2ox cells exposed to various concentrations of GEM for 48 hr. The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. ***p<0.001.",
    "molecules": "GEM"
  },
  {
    "caption": "Changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of CDDP for 48 hr. The data are shown as the means ± SDs (n=3, biological replicates).",
    "molecules": "CDDP"
  },
  {
    "caption": "Immunohistochemistry (IHC) staining of Ki-67 in UMUC3WT and GR cells exposed to CDDP/GEM. Ki-67 staining quantitation of UMUC3WT and UMUC3GR cells exposed to CDDP/GEM are shown as bar graph The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by one-way ANOVA with the Bonferroni test. ** p<0.05, ***p<0.001. n.s.= non-significant",
    "molecules": "CDDP, GEM"
  },
  {
    "caption": "NADPH/NADP ratios of GR cells transfected with NTC, IDH2 siRNA#1, and siRNA#2 (left: T24GR, right: UMUC3GR). The data are shown as the means ± SDs (n=3, biological replicates) and were analyzed by Student's t test. ** p<0.01.",
    "molecules": "NADP, NADPH"
  },
  {
    "caption": "Graph showing the viability of GR cells exposed to various concentrations of GEM for 48 hr in combination with DMSO or 10 µM AGI6780 (upper: T24GR, lower: UMUC3GR). The data are shown as the means ± SDs (n=3, biological replicates).",
    "molecules": "AGI6780, DMSO, GEM"
  },
  {
    "caption": "IHC staining for IDH2, Ki-67, CAIX, TIGAR, TKT, and CTPS1 in groups with GEM alone, AGI6780 alone, and the combination of GEM and AGI6780.",
    "molecules": "AGI6780, GEM"
  },
  {
    "caption": "A: cell growth in glucose (GLU) and galactose (GAL). C29V, C47V, TpLV: immortalized fibroblast cell lines from control individuals; PITRM1V: immortalized fibroblasts from subject II-2, carrying the PITRM1R183Q mutation. pLKO.1: empty vector; sh38: shRNA38; sh41: shRNA41. Each cell line was measured six times. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA post-hoc Bonferroni test ***p<0.001.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A: oxidative growth. W303-1Bcym1Δ strains harbouring the wild-type CYM1 allele (CYM1wt), the cym1R163Q mutant allele or the empty vector were serially diluted from 107 to 104 cells/ml. Five microliters of each dilution were spotted on SC agar plates without uracil, supplemented with 2% glucose, 2% glycerol or 2% ethanol. Plates were incubated at 37°C for 3-7 days.",
    "molecules": "ethanol, glucose, glycerol"
  },
  {
    "caption": "B: oxygen consumption rate (OCR). Cells grown at 37°C SC medium without uracil were supplemented with 0.5% glucose. Values were normalized to the OCR of the CYM1wt strain (49 nmol O2 min−1 mg−1) and represented as the mean of at least three values ± SD. Statistical analysis was by unpaired, two-tail Student's t test: ***p<0.001.",
    "molecules": "glucose, O2"
  },
  {
    "caption": "G: Western blot of Aβ1-42myc monomer and dimer incubated with purified mitochondrial extracts from cells grown at 37°C in SC medium supplemented with 0.15% glucose and 2% galactose. VDAC is used as a loading control. Each experiment was performed in triplicate.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A APB Northern blot using a tRNAHis probe. m5C38 levels of tRNAAspGUC were measured by 454 bisulfite sequencing at the indicated time points. Both queuosinylation and methylation levels could be restored by the addition of queuine. The slower migration of tRNAHis is eliminated by oxidizing the ribose with periodate, producing a single faster-migrating band (ox). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).",
    "molecules": "periodate, ribose, APB, Asp, guanine, His, queuine, queuosine, tRNA"
  },
  {
    "caption": "B The addition of queuine to HCT116 cells cultured in serum free medium resulted in an increase of both queuosinylation and m5C38 levels. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).",
    "molecules": "guanine, queuine, queuosine"
  },
  {
    "caption": "C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa, whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).",
    "molecules": "guanine, His, queuine, queuosine, tRNA"
  },
  {
    "caption": "B Corresponding queuosinylation and methylation levels, as determined by Northern blot using a tRNAHisGUG probe and bisulfite sequencing of tRNAAspGUC. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).",
    "molecules": "Asp, guanine, His, queuine, queuosine, tRNA"
  },
  {
    "caption": "D Representative metagene plot of 27-30 mer ribosome footprints in S and SF culture conditions for two biological replicates (rep1 and rep2). Data information: SF (serum free medium), q (queuine)",
    "molecules": "queuine"
  },
  {
    "caption": "A Heatmap showing codon occupancy according to the color scale. Green bars indicate Q-decoded codons. The Asp-GAC codon translated by Q- and m5C38-tRNAAspGUC is shown in yellow and Lys and Glu codons are in orange. Lack of Q reduces ribosome translation speed at all Q dependent codons and at near-cognate decoded codons. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).",
    "molecules": "Asp, Glu, guanine, Lys, queuine, queuosine, tRNA"
  },
  {
    "caption": "B Translational speed of C ending codons relative to U ending codons. Error bars: ±SE of the permutated ratios of C-ending codon occupancy relative to U for the indicated conditions; (n=4). Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).",
    "molecules": "guanine, queuine, queuosine"
  },
  {
    "caption": "D Codons translated by Q-tRNA are depleted in down-regulated proteins and increased in up-regulated proteins in both forward and reverse SILAC.",
    "molecules": "tRNA"
  },
  {
    "caption": "A The volcano plot shows differentially translated transcripts against adjusted p-values for SF compared to SF+q. Red dots indicate padj< 0.1 in multiple tests. Data information: S (standard medium), SF (serum free medium), q (queuine)",
    "molecules": "queuine"
  },
  {
    "caption": "C Representative polysome profiles of HeLa cells grown in S and SF medium in the absence or presence of q. As a measurement for the global translation rate the fraction of polysomal ribosomes was quantified; *p <0.05 (t-test); n=5. Data information: S (standard medium), SF (serum free medium), q (queuine), RO (ribosome occupancy).",
    "molecules": "queuine"
  },
  {
    "caption": "D Proliferation analysis of HeLa cells grown under the indicated culture conditions. Population doubling levels of three biological replicates were calculated for each time point. Error bars: ±SD; n = 3. Data information: S (standard medium), SF (serum free medium), q (queuine)",
    "molecules": "queuine"
  },
  {
    "caption": "A Representative fluorescence confocal images of live HeLa cells expressing Fluc-EGFP and FlucR188Q-EGFP maintained in SF compared to SF+q. Scale bars, 10 μm. Data information: SF (serum free medium), q (queuine)",
    "molecules": "queuine"
  },
  {
    "caption": "B Fluorescence intensity of aggregates was quantified as a fraction of fluorescence of the entire cell. The number of cells showing at least one aggregate is indicated. 10 cells were imaged for Fluc-EGFP and 20 cells for FlucR188Q-EGFP from 3 independent transfections; *p < 0.05 (t-test). Data information: S (standard medium), SF (serum free medium), q (queuine), AU (arbitrary units).",
    "molecules": "queuine"
  },
  {
    "caption": "C Specific luminescence activity of EGFP-tagged sensor proteins maintained in SF compared to SF+q. Luci­ferase activity was normalized to the luciferase protein content in the sample. Error bars: ±SD; *p < 0.05 (t-test); n = 8. Data information: SF (serum free medium), q (queuine)",
    "molecules": "queuine"
  },
  {
    "caption": "A Transmission electron microscopy images showing the increase of cystic ER vacuolae upon q depletion. Arrowheads indicate rough endoplasmic reticulum while expansions are indicated by asterisks. Scale bar 250 nm. Data information: S (standard medium), SF (serum free medium), q (queuine).",
    "molecules": "queuine"
  },
  {
    "caption": "B Representative images showing immunofluorescence of HeLa cells grown in S and SF medium in the absence or presence of Q. Anti-KDEL (ER stress marker); anti-eIF2-pS51 (ER stress-induced inhibitor of translation); anti-ATF4 (transcription factor that activates ER stress-responsive genes); anti-Hps70 (chaperone). Nuclei were stained with Hoechst. Scale bar 10 µm. Data information: S (standard medium), SF (serum free medium), q (queuine).",
    "molecules": "Hoechst, queuine"
  },
  {
    "caption": "A APB Northern blot using a tRNAHis probe. m5C38 levels were determined by 454 bisulfite sequencing of RNA from the same liver tissue. Both queuosinylation and methylation levels could be restored by the addition of q to the feed. The results are shown for three biological replicates. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.",
    "molecules": "APB, His, queuine, RNA, tRNA"
  },
  {
    "caption": "B Representative polysome profiles showing a reduction of the protein translation rate in the liver of axenic mice fed with a q-free synthetic diet. Addition of q to the q-free diet significantly rescued the translation rate. n=3. Asterisks indicate statistically significant (p<0.05, t-test) differences. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.",
    "molecules": "queuine"
  },
  {
    "caption": "C Box plot showing the body weight of mice fed a q-free synthetic diet for 60 days, center lines show the medians; box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range; n=3. Addition of q to the q-free diet significantly rescued body weights. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.",
    "molecules": "queuine"
  },
  {
    "caption": "D Dimethyl-labeling analysis of -q versus +q liver tissue. Top 10% of deregulated proteins are indicated in red and blue. UPR effectors: HSPA5/BiP, HSP90b1 and CALR are in orange. Data information: -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.",
    "molecules": "queuine"
  },
  {
    "caption": "F Liver sections from indicated mice were stained with anti-KDEL antibody. Arrowheads indicate ER stressed hepatocytes. Scale bar 25 µm. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.",
    "molecules": "queuine"
  },
  {
    "caption": "(A) Competition between Beclin‐1 BH3 and ABT737 for Bcl‐XL binding. A fluorescent 25‐mer peptide containing the BH3‐like domain of Beclin‐1 ( Figure 1D) was docked to recombinant Bcl‐XL ΔTM protein in the absence or presence of ABT737. The IC50 of ABT737, as measured in the presence of 15 nM of peptide and 100 nM of Bcl‐XL ΔTM, was 1.7 μM.",
    "molecules": "ABT737"
  },
  {
    "caption": "(B, C) Abolition of the interaction between Bcl‐XL and Beclin‐1 by ABT737 in intact cells. Co‐immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E. Sixteen hours before the immunoprecipitation, cells were exposed to ABT737. Similar results were obtained for co‐transfected Bcl‐XL and Beclin‐1 (B) and for endogenous Beclin‐1 interacting with Flag‐tagged Bcl‐XL (C).",
    "molecules": "ABT737"
  },
  {
    "caption": "(D, E) Differential effect of ABT737 on the interaction between Bcl‐2 (D) or Mcl‐1 (E) and Beclin‐1. This experiment was designed as (B). All experiments have been performed at least three times, with similar results.",
    "molecules": "ABT737"
  },
  {
    "caption": "(A, B) Detection of autophagic vacuoles by LC3‐GFP and their modulation by ABT737 and by Beclin‐1‐specific siRNAs. HeLa cells were transfected with control or Beclin‐1‐specific siRNAs, 24 h later re‐transfected with LC3‐GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient‐free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3‐GFP‐transfected cells bearing LC3‐GFP aggregates in the cytoplasm (LC3‐GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin‐1‐specific siRNAs, as quantified by immunoblot.",
    "molecules": "ABT737, nutrient"
  },
  {
    "caption": "(E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown.",
    "molecules": "ABT737"
  },
  {
    "caption": "Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin‐1 at 0 h) were re‐transfected with LC3‐GFP finally cultured in nutrient‐free (NF) conditions (60-72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A).",
    "molecules": "ABT737, nutrient"
  },
  {
    "caption": "(A-D) Modulation of Beclin‐1‐induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP‐LC3 together with an empty control vector or Beclin‐1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3‐GFP‐positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3‐GFP method.",
    "molecules": "ABT737"
  },
  {
    "caption": "(E) BH3‐dependent modulation of Beclin‐1‐induced autophagy by anti‐apoptotic Bcl‐2 proteins. Cells were transfected simultaneously with Beclin‐1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl‐XL, Bcl‐XL G138A, Bcl‐2 or Mcl‐1 (or control vector), as well as GFP‐LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h‐long experiment. The percentage of cells exhibiting the accumulation of LC3‐GFP in vacuoles (LC3‐GFPvac) is quantified as means±s.d. (n=3 separate experiments).",
    "molecules": "ABT737"
  },
  {
    "caption": "(A) Immunoprecipitation of endogenous Bcl‐2 and Beclin‐1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient‐free EBSS or with ABT737 (1 μM).",
    "molecules": "ABT737, nutrient"
  },
  {
    "caption": "(B) Immunoprecipitation of wild‐type and ER‐targeted Bcl‐2 with Beclin‐1. Cells stably transfected with wild type, ER‐ and mitochondrion‐targeted Bcl‐2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1.",
    "molecules": "ABT737"
  },
  {
    "caption": "(A, B) Interactions between Bcl‐XL, Beclin‐1 and Bad in conditions of autophagy induction. Cells were either treated by nutrient depletion (A) or addition of 1 μM rapamycin, 1 mM lithium chloride, 100 μM L‐690,330 or 50 μM carbamazepine (B), followed by immunprecipitation of Bcl‐XL (as in Figure 2C) and revealing the immunoblots by antibodies specific for Bcl‐XL, Beclin‐1 or Bad.",
    "molecules": "L‐690,330, carbamazepine, lithium chloride, nutrient, rapamycin"
  },
  {
    "caption": "(C) Interaction between endogenous Bad and Bcl‐2. HeLa cells were treated with ABT737 (1 μM) or nutrient‐depleted, followed by immunoprecipitation of Bcl‐2 and immunodetection of Bad.",
    "molecules": "ABT737, nutrient"
  },
  {
    "caption": "(D, E) Impact of Bad depletion on autophagy. Cells were transfected with LC3‐GFP together with siRNAs specific for Bad, siRNAs specific for emerin or the Beclin‐1‐associated PI3 kinase Vps34. Forty‐eight hours later, when the siRNAs had down‐regulated the proteins of interest (D), cells were subjected by nutrient depletion (NF) or addition of rapamycin (E) and the frequency of cells exhibiting LC3‐GFP aggregation as a marker of autophagy was assessed after 18 h (means±s.d., *P0.05, n=3 separate experiments).",
    "molecules": "nutrient, rapamycin"
  },
  {
    "caption": "(F) Effect of the Bad knock‐out on autophagy. Wild Type (WT) or Bad−/− MEF were transfected with LC3‐GFP and then subjected to nutrient depletion (NF) and/or treatment with ABT737 (18h; means±s.d., n=3). The asterisks denotes a significant (P0.05) effect of Bad deficiency.",
    "molecules": "ABT737, nutrient"
  },
  {
    "caption": "(H) Overexpression of Bad triggers autophagy. Cells were transfected with LC3‐GFP alone (Control), together with vector‐only or with a vector encoding Bad, and subjected 48 h later to nutrient depletion (NF). Cells were cultured in the continuous presence or absence of the pan‐caspase inhibitor Z‐VAD‐fmk (50 μM) for 16 h, followed by assessment of autophagy as in (E) (means±s.d., n=3 separate experiments, *P0.01). Immunoblots are representative of at least three independent experiments.",
    "molecules": "nutrient, Z‐VAD‐fmk"
  },
  {
    "caption": "D Representative confocal  images of HEK293  cells expressing various GFP-fused proteins (green) and supplemented with TopFluor® TMR-PS (red); scale bar = 10 µm. Yellow arrows indicate high intensity PS  fluorescent regions. E %PM  with PS  clusters = area of high intensity fluorescent PS  clusters over total  plasma membrane area from images in panel D. Black bars are control proteins and blue bars are eVP40    proteins. Values are reported as mean ± s.d.; N>18 cells per biological replicate, n=3 for 3 biological replicates; A one-way ANOVA was performed with multiple comparisons compared to the control GFP  %PS  clustering (***p=0.0007, **p=0.004). Data information: PS: phosphatidylserine; PM: plasma membrane.",
    "molecules": "TopFluor® TMR-PS, phosphatidylserine, PS"
  },
  {
    "caption": "D, E PS  concentration in LUVs  enhances the ability of His6-eVP40  to oligomerize on membranes. D Representative western blot of chemical crosslinking performed on His6-WT-eVP40   following incubation with LUVs  of varying PS  content (detected by Mouse α-His  antibody & HRP-Sheep α-Mouse). E Oligomerization  capacity was determined from the western blot  band density ratio of oligomers/(monomer + dimer) from chemical crosslinking experiments. A one-way ANOVA was performed with multiple comparisons compared to the control 0% PS LUVs  control (30% PS  *p= 0.02; 60% PS  *p=0.017). n=3 biological replicates. Values are reported as mean ± s.d.; Data information: LUVs:  large unilamellar vesicles; PS: phosphatidylserine; HRP: horseradish peroxidase.",
    "molecules": "phosphatidylserine, PS"
  },
  {
    "caption": "D, E Analysis of PS  clustering in HEK293  cells in response to fendiline  treatment through N&B analysis. D Left panel: Representative images from  time-lapse  (30 frames) imaging of HEK293  expressing GFP-LactC2  and treated with fendiline  for 48 hours; scale bar= 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization  state (b value) (red: monomer-5mer, green: 5mer-10mer, blue: >10mer). E Average % pixels quantification from panel (d)= Percentage of GFP-LactC2  with brightness values corresponding to monomer-5mer (~1.-1.5), 5mer-10mer (~1.5-1.9) and >10mer (>1.9) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons comparted to the control DMSO  % average pixels (****p<0.0001, **p=0.0043). Data information: GFP-LactC2: phosphatidylserine  sensor; N&B:  Number & Brightness analysis; PS: phosphatidylserine.",
    "molecules": "fendiline, DMSO, phosphatidylserine, PS"
  },
  {
    "caption": "Effect of fendiline  on EBOV infection. A Representative confocal  images of Vero E6  cells infected with EBOV  (Kikwit) at the indicated MOI and treated with the indicated concentration of fendiline. Cells were pretreated 24 hours prior to infection  with the indicated concentration of fendiline. Post infection, cells were treated 1 hour later (d -1/0), treated every day (e.d), or treated every other day (e.o.d) and fixed at either 48 hours, 72 hours or 96 hours post infection. (green=EBOV; blue= nuclei). White numbering in top right corner indicates %infection. Scale bar = 100 μm. Data information: EBOV: Ebola virus; MOI: multiplicity of infection",
    "molecules": "fendiline"
  },
  {
    "caption": "Effect of fendiline  on EBOV  infection. B-D Quantification of % inhibition  of EBOV  by fendiline. B 48 hours (MOI 1.0) C 72 hours (MOI 0.1) D 96 hours (MOI 0.1). Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons was performed. n=3 biological replicates. Data information: EBOV: Ebola virus; MOI: multiplicity of infection; d. -1/0: treatment 1 hour after infection; e.d.: treatment every day; e.o.d.: treatment every other day.",
    "molecules": "fendiline"
  },
  {
    "caption": "A-C Effect of fendiline  on eVP40  and mVP40 PM localization  in HEK293  cells after 48 hours of treatment. A Representative confocal  images from live cell imaging  experiments of HEK293  cells expressing EGFP-WT-eVP40 (top panel) and EGFP-WT-mVP40   (bottom panel) after 48 hours of fendiline  treatment. scale bars= 10 µm. Effect of fendiline  on mVP40 (B) and eVP40 (C) PM localization  was quantified by the ratio of EGFP  fluorescence intensity at the PM / total EGFP  fluorescence intensity (and normalized to DMSO  control). N>15 cell per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed compared to the DMSO  control. Data information: PM: plasma membrane",
    "molecules": "fendiline, DMSO"
  },
  {
    "caption": "D, E Analysis of eVP40 oligomerization  in HEK293  cells in response to 48 hour fendiline  treatment using N&B analysis. D Left panel: Representative images from  time-lapse  (30 frames) of HEK293  expressing EGFP-WT-eVP40 and treated with fendiline  for 48 hours. scale bar = 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization  state (b value) (red: monomer-hexamer, green: hexamer-12mer, blue: 12mer-24mer, pink: >24mer). E Average % pixel quantification from panel (d)= % of GFP-WT-eVP40 with brightness values corresponding to monomer-hexamer (~1.-1.6), hexamer-12mer (~1.6-2.0), 12mer-24mer (2.0-3.2) and >24mer (>3.2) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9 cell per biological replicate, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons compared to the control DMSO  % average pixels (**p=0.0035). Data information: N&B: Number & Brightness analysis.",
    "molecules": "fendiline, DMSO"
  },
  {
    "caption": "A-D Functional budding assays  assessed at 24 hours (A-B) and 48 hours (C-D) post treatment. A Representative western blot  of budding assays  performed at 24 hours post transfection with vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline  treatment. VLP  samples (top panel) and cell lysate  samples (bottom panel) collected from HEK293  cells and immunoblotted  for eVP40 expression; GAPDH  served as a loading control. eVP40  detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH  detected by mouse α-GAPDH  and HRP-Sheep α-Mouse) B Quantification of relative budding  index at 24 hours post vehicle or fendiline  treatment. Relative budding index was determined by the western blot  band density of eVP40  in the VLP  fraction/(total eVP40 cell lysate + eVP40 VLP  band density) and was normalized to the DMSO  control. Cell lysate eVP40  band density was normalized to GAPDH  band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO  control. C Representative  western blot of budding assays  performed at 48 hours post vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline  treatment. VLP  samples (top panel) and cell lysate samples (bottom panel) collected from HEK293  cells and immunoblotted  for eVP40  expression; GAPDH served as a loading control. eVP40  detected by (Rabbit α-eVP40    and HRP-Goat α-Rabbit); GAPDH  detected by (Mouse α-GAPDH  and HRP-Sheep α-Mouse) D Quantification of relative budding index at 48 hours post fendiline  treatment. Relative budding index was determined by the  western blot band density of eVP40   in the VLP  fraction/(total eVP40 cell lysate + eVP40 VLP  band density) and was normalized to the DMSO  control. Cell lysate eVP40  band density was normalized to GAPDH  band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO  control. (*p=0.0260) Data information: VLPs: virus-like particles; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HRP: horseradish peroxidase.",
    "molecules": "fendiline, DMSO"
  },
  {
    "caption": "A-C TEM  analysis of eVLP morphology. A Representative transmission electron micrographs  of eVLPs  purified from HEK293  cells expressing FLAG-eVP40 and eGP following 48 hours of DMSO  (left panel) or 5 µM fendiline  treatment (right panel). B Quantification of eVLP  length (µm) of DMSO-derived eVLPs  (black) and fendiline-derived eVLPs  (blue). N>50 VLPs   per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (**p=0.0139). C Quantification of eVLP   diameter (nm) of DMSO-derived eVLPs  (black) and fendiline-derived eVLPs  (blue). N>50 VLPs   per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (*p=0.0430). Data information: TEM: transmission electron microscopy; eVLPs: entry-competent VLPs; eGP: Ebola virus glycoprotein",
    "molecules": "fendiline, DMSO"
  },
  {
    "caption": "D, E Fluorescence based DiI TIM-1    dependent entry assay. D Representative confocal  images from the DiI-entry assay comparing entry of eVLPs  produced from DMSO  (top panel) and fendiline-treated HEK293  cells (bottom panel) into target cells (HEK293  cells transiently expressing increasing amounts of TIM-1; 0.0 µg, 0.5 µg, 1.0 µg). A stack of 10 frames was acquired for each image. DiI  (initially red) was recolored to yellow for easier observation in print; blue (Hoechst 3342 stain); scale bar = 10 µm. E Quantification of eVLP  entry was performed by calculating the total number of DiI  punctate / the total number of DiI-positive cells. Three images from each z-stack were quantified. N=9 cell per replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons against the 0.0 µg TIM-1  condition for both DMSO- and fendiline  derived eVLPs.(****p<0.0001; **p=0.0093). Data information:  TIM-1:  t-cell immunoglobulin receptor-1; eVLPs: entry-competent VLPs; DiI: 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate.",
    "molecules": "Hoechst 3342, fendiline, 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate, DiI, DMSO"
  },
  {
    "caption": "(A) Waterfall plot showing the percentage of tumor volume change in olaparib-treated tumors compared to the tumor volume on day 1. +20% and -30% are marked by doted lines to indicate the range of PR, SD and PD. The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation. TNBC, Triple Negative Breast Cancer; ER+BC, Estrogen Receptor positive Breast Cancer; P, primary; M, metastasis. Error bars indicate SEM from independent tumors (n ≥ 3).",
    "molecules": "olaparib"
  },
  {
    "caption": "(B) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort-1. Olaparib-sensitive models are represented with discontinuous lines. Acquisition of PARPi-resistance in PDX302, STG201 and PDX093 after prolonged exposure to olaparib is shown.",
    "molecules": "olaparib, Olaparib"
  },
  {
    "caption": "(C) YFP-PALB2 recruitment to laser-induced DSBs is impaired in HeLa cells expressing PALB2 p.M296Nfs (n=4, unpaired t-test at 16min).",
    "molecules": "DSBs"
  },
  {
    "caption": "(C) Quantification of geminin-positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib. Paired t test in PARPi-sensitive (CR/PR) vs. PARPi-resistant (PD) PDXs.",
    "molecules": "olaparib"
  },
  {
    "caption": "(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells in FFPE samples from untreated PDX tumors. Color bars indicate the presence of pathogenic variants in the indicated genes. Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: black box: PR/CR; white box: PD. Box colors indicate the PARP inhibitor treatment. Boxes with two colors indicate the same response to both treatments. Olaparib 100mg/kg & 50mg/kg indicates that both doses were tested and resulted in the same response categorization.",
    "molecules": "Olaparib"
  },
  {
    "caption": "A Immunoblot analysis with the indicated antibodies of immunoprecipitates (IP) prepared from the SSU fraction of eIF3D-V5/eIF4E-FLAG dKI HEK293T cells (eIF3D/4E dKI) or wild-type (WT) cells with antibodies to the epitope tags. The SSU fraction (input) was also analyzed directly. Cells were fixed with formalin, cell lysates were treated with RNase, and the SSU and RS fractions were separated by sucrose density gradient centrifugation before immunopurification. Molecular sizes are indicated in kilodaltons.",
    "molecules": "formalin"
  },
  {
    "caption": "C Similar to CSR-1, the majority of small RNAs interacting with CSR-2 in vivo have 5′PPP ends. The RNAs in the immunopurified Argonaute complexes were treated with or without Vaccinia virus capping enzyme followed by an alkaline phosphatase, and finally labeled with [γ-32P]ATP and a polynucleotide kinase. RNA products were resolved by 15% sequencing gels.",
    "molecules": "[γ-32P]ATP"
  },
  {
    "caption": "E Immunofluorescence images of H3K9me2 and SYP-1 in male nuclei of the WT and the Argonaute mutants. High-resolution fluorescence images, 3D reconstruction images (3D CG), and maximum intensity projection (MIP) images of segmented H3K9me2 signals are presented. In the male nuclei of the csr-1 mutant and the csr-2; csr-1 double mutant, some H3K9me2 segments showed abnormally large volumes (narrow arrow) and/or overlapped partially with SYP-1 signals (arrowhead). In the double mutant, DAPI signals were often detected in an aggregated state (dashed line).",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) The survival plot of Albumin-Cre/+; Wdr6f/f mice with DEN-induced HCC.",
    "molecules": "DEN"
  },
  {
    "caption": "(A) The effects of NF‐κB inhibitor IMD0354 or IκBα‐SR on TNFα expression in MHCC-97L cells with or without WDR6 overexpression.",
    "molecules": "IMD0354"
  },
  {
    "caption": "(Di-ii) The effects of autophagic deficiency using 3-MA on the levels of p65 protein and TNFα secretion in HCC cells with WDR6 knockdown.",
    "molecules": "3-MA"
  },
  {
    "caption": "(Ei) WDR6 mRNA levels in Hep3B and MHCC-97L cells treated with or without TNFα in culture media. (Eii) Schematic diagram showing three candidate NF‐κB-binding sites in WDR6 promoter. (Eiii) The impact of IMD0354 on the luciferase reporter of WDR6 proximal promoter in HCC cells. (Eiv-v) ChIP assays using HCC cells or HCC tissues from patients (P#36 and P#54). Da",
    "molecules": "IMD0354"
  },
  {
    "caption": "(B) CHX assays of UVRAG stability in HCC-LM3 cells transfected with WDR6 shRNA.",
    "molecules": "CHX"
  },
  {
    "caption": "(C) The effects of WT-WDR6 or WDR6-R123A mutant on UVRAG levels in MHCC-97L cells in the presence of MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) HEK293T cells with indicated constructs were treated with 5 μM Pep2-WDxR or Pep2-con or Pep2-WDxA mutant for 16 hours, respectively. Then co-IP assay was used to assess the impacts of these peptides on the WDR6-UVRAG binding.",
    "molecules": "Pep2"
  },
  {
    "caption": "(C) BALB/c mice introduced with H22-WDR6 overexpression cells were administered with Pep2-WDxR or Pep2-con with or without anti-PD-L1 antibody. The images and sizes of HCC (Ci), hematoxylin-eosin (H&E) staining and metastasis nodules of lung were presented (Cii). The survival analysis of the mouse after treatment was performed (n = 9/group) (Ciii). In the BALB/c mice from (C), Flow cytometry was performed to analyze PMN-MDSC, M-MDSC, and IFN-γ+ CD8+ T cells in tumors (Civ).",
    "molecules": "eosin, hematoxylin, Pep2"
  },
  {
    "caption": "(D) The survival test of DEN- and CCl4-induced HCC using WDR6-knockout and WT mice. At twenty-six weeks, mice were administered with or without anti-PD-L1 antibody (n = 6/group).",
    "molecules": "DEN, CCl4"
  },
  {
    "caption": "C,D. Immunolabeling of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody (in green in C, in brown in D, A375P:GFP cells), an anti-HNK1 antibody (in white, migrating and early post-migrating NCCs), an anti-Ki67 antibody (in red in C, cycling cells), an anti-SOX10 antibody (in red in D, also stains chick endogenous NCCs) and an anti-MITF antibody (in yellow in D). Nuclei were stained with Hoechst (in blue). In C, the upper left photo shows a grafted embryo prior to cryosection. In D, right panels are enlargements of the lower left panel. Scale bar: 200 µm.",
    "molecules": "Hoechst, HNK1"
  },
  {
    "caption": "G. Immunofluorescent labeling of HH25 chick embryo sections 50 hours post-engraftment of OF-MEL-020 patient sample, labeled with CFSE (in green) prior to the graft. An anti-SOX10 antibody (in red) and an anti-MITF antibody (in yellow) were used. Nuclei were stained with Hoechst (in blue). Right panels are enlargements of the dotted area in the left panel.",
    "molecules": "Hoechst, CFSE"
  },
  {
    "caption": "A,B. Immunofluorescent labeling (A) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody, an anti-MITF antibody (pink) and an anti-SOX10 (white) antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in left panel. In B, the mean fraction of MITF-negative (MITFneg), -low (MITFlow) and -positive (MITFpos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 16 slices from 3 embryos). Error bars show SEM. Mann-Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed, p-values are indicated on the graph.",
    "molecules": "Hoechst"
  },
  {
    "caption": "C,D. Immunofluorescent labeling (C) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody and an anti-Ki67 antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in the left panel. In D, the mean fraction of Ki67-positive (Ki67pos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 25 slices from 3 embryos). Arrows point at cells having a mesenchymal morphology. Error bars show SEM. Student t-test, **p = 0.0070.",
    "molecules": "Hoechst"
  },
  {
    "caption": "H-K. 3D views (H, J) and quantification of tumor volumes (I, K) of HH25 chick embryos engrafted with GLO (H, I) or GLO-R (J, K) cells and treated with a combination of Vemurafenib and Cobimetinib, or Vorinostat alone, or a combination of the three molecules. The number of embryos analyzed for each experimental condition are indicated on the graphs. Bars and error bars indicate mean + SEM. Student t-tests, **: p < 0.01, ns: not significant. Exact p-values are indicated on the graphs.",
    "molecules": "Cobimetinib, Vemurafenib, Vorinostat"
  },
  {
    "caption": "A,B. 3D views (A) and quantification of variations in the mean tumor volume (B) of HH25 chick embryos engrafted with BRAFwt (OF-MEL-028) or BRAFV600E (OF-MEL-020, OF-MEL-027) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib. The number of embryos analyzed for each patient sample is indicated on the graphs. Student t-test for OF-MEL-020; Mann-Whintey test for OF-MEL-028, *: p < 0.05, ns: not significant. Exact p-values are indicated on the graphs.",
    "molecules": "Cobimetinib, Vemurafenib"
  },
  {
    "caption": "C,D. Survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of Dabrafenib (C) and Trametinib (D). The maximum tolerated dose (MTD, in red on the X-axis) was defined as the higher dose of drug associated with a survival rate higher than 75% and a mean BSA similar (i.e., non-statistically different) from embryos treated with excipient (17.1 and 0.0228 mg/kg respectively). N=10 replicates per experimental group. Error bars indicate SEM. ns, non-significant using Student's t-test compared with excipient.",
    "molecules": "Dabrafenib, Trametinib"
  },
  {
    "caption": "(F-H) of HH25 chick embryos engrafted with 3 different patient samples (OF-MEL-033 (F), OF-MEL-034 (G), OF-MEL-035 (H)) and treated with excipient or a combination of Dabrafenib and Trametinib. The clinical response of each patient after a 3 months treatment with Dabrafenib/Trametinib is indicated above the graphs. Error bars indicate SEM. Mann-Whitney tests, exact p-values are indicated on the graphs.",
    "molecules": "Dabrafenib, Trametinib"
  },
  {
    "caption": "NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells. Recombinant HALO-NEDP1 C163A (CA) conjugated to HALO-Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells. Enriched proteins were resolved by SDS-PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies. HALO-NEDP1 CA specifically enriches for NEDD8-reactive proteins in both WT and NEDP1 KO cells, but does not enrich for Ubiquitin-modified proteins in either cell line.",
    "molecules": "Ubiquitin"
  },
  {
    "caption": "Poly-NEDD8 chains can be generated by in vitro reactions (Rxn). NAE (0.15 μM), UBE2M, and NEDD8 (20 μM) were incubated on ice or incubated at 30°C for 3 hours and reactions were stopped by addition of LDS loading buffer. Reactions were resolved by SDS-PAGE and stained with colloidal Coomaisse. Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis. The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2-N4. Unconjugated NEDD8 is indicated by N1. UBE2M modified by NEDD8 is indicated with an asterisk.",
    "molecules": "Coomaisse, trypsin"
  },
  {
    "caption": "Neddylated species are NEDD8 E1 dependent. WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time. Lysed cells were then processed for Western blot analysis. NEDD8 E1 inhibition results in a time-dependent decrease in the amount of Cullin and non-Cullin NEDD8 reactive bands.",
    "molecules": "MLN4924"
  },
  {
    "caption": "Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow). WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis.",
    "molecules": "H2O2"
  },
  {
    "caption": "(Left) The 25-kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells. U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Right) The same 25 kDa NEDD8 species, which is strongly induced in WT cells, is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 (as indicated by the arrow).",
    "molecules": "H2O2"
  },
  {
    "caption": "U2OS NEDP1 KO cells are resistant to the PARP-1 inducer, H2O2. WT and NEDP1 U2OS cells were plated in 96-well plates and after 24 hours were treated with the indicated concentration of H2O2. They were assessed for viability 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "NEDP1 KO cells are re-sensitized to H2O2, following re-expression of NEDP1 by transient transfection. U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector. 48 hours after plating, cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2. WT, NEDP1 KO, and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2. 24 hours after treatment, cell viability was measured using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "PARP-1 activity is reduced in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time. PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "PARP-1 activity is rescued by re-expression of NEDP1 in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs. 72 hours after transfection, cells were treated with the indicated amount of H2O2 and processed for Western blot analysis. PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re-expression of NEDP1.",
    "molecules": "H2O2"
  },
  {
    "caption": "PARP-1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO. U2OS cells were plated in 96 well plates and 24 hours later pre-treated with Olaparib (10 μM) for 1 hour before treatment with the indicated concentration of H2O2. 24 hours later, cell viability was measured using the CellTiterGlo assay. NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP-1 inhibition in WT cells. Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment.",
    "molecules": "H2O2, Olaparib"
  },
  {
    "caption": "AIF translocation to the nucleus is impaired in NEDP1 KO cells. WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 (600 μM) for the indicated amount of time and then processed for immunofluorescence analysis using α-AIF antibodies and DAPI staining. PARP-1 dependent cell death is induced in WT U2OS cells, as indicated by translocation of AIF from the mitochondria to the nucleus (Scale bar 10 μm).",
    "molecules": "DAPI, H2O2"
  },
  {
    "caption": "Total nuclear AIF intensity from (D) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar. AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "NEDP1 KO U2OS cells are not resistant to induction of apoptosis from combined treatment with TNF-α and cycloheximide. U2OS cells were plated in 96 well plates and incubated with cycloheximide (10 μg/mL) before addition of the indicated amount of TNF-α. Cell survival was determined 24 hours later using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "NEDP1 KO U2OS cells are not resistant to treatment with camptothecin (CPT). WT U2OS and NEDP1 KO cells were plated in 96-well plates and treated with the indicated amount of CPT. 48 hours after exposure cell survival was measured using the CellTiterGlo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.",
    "molecules": "camptothecin, CPT"
  },
  {
    "caption": "NEDD8 trimers form in WT U2OS cells after treatment with H2O2 and co-immunoprecipitate with endogenous PARP-1 but not with immunoglobulin control (IGG). WT U2OS cells were left untreated of treated with H2O2 (600 μM) for 30 min and were harvested for immunoprecipitation and Western blot analysis",
    "molecules": "H2O2"
  },
  {
    "caption": "NEDD8 trimers bind to GFP-PARP-1. WT or NEDP1 U2OS KO cells were transfected with GFP or with full-length GFP-PARP-1 and 24 hours later treated as indicated with H2O2 (600 μM) for 30 min before cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from WT cells only after H2O2 treatment or from both treated or untreated NEDP1 KO cells.",
    "molecules": "H2O2"
  },
  {
    "caption": "HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.",
    "molecules": "NaB, sodium butyrate"
  },
  {
    "caption": "HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.",
    "molecules": "NaB, sodium butyrate"
  },
  {
    "caption": "Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.",
    "molecules": "H2O2"
  },
  {
    "caption": "In vitro de-neddylation by NEDP1 is sensitive to oxidation. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST-NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H2O2). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.",
    "molecules": "DTT, H2O2, MLN4924"
  },
  {
    "caption": "In vitro neddylation reactions with UBE2M are insensitive to H2O2. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. Before reactions were started, increasing concentrations of H2O2 (as indicated) were added to the NEDD8 reactions. Subsequently the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.",
    "molecules": "H2O2"
  },
  {
    "caption": "(G) Western blot validating the degradation effect of PABPN1   under the treatment of dTAG13  for 0, 3, and 6 hours.",
    "molecules": "dTAG13"
  },
  {
    "caption": "(G) Splicing  analysis of the C3, C4, and polyA  reporters in Figure 3F for endogenous genes LARP7, COPS5, and NSUN5  by RT-PCR upon PABPN1  knockdown (top) and the quantified intron retention levels (IR score) of these reporters (bottom). Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).",
    "molecules": "polyA"
  },
  {
    "caption": "(D) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 3 month-old ASMko and wt mice. DAPI staining shows cell nuclei. Scale bars, 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Immunofluorescence staining against Iba-1 in Cb of ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 100 μm. (B) Magnified images (from A) showing ramified (left) versus amoeboid (right) morphology in wt and ASMko microglia treated or not with PLX. Scale bars, 30 μm. (C) Mean ± SEM number of Iba-1 positive cells in the Cb of the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).",
    "molecules": "DAPI, PLX"
  },
  {
    "caption": "(D, E) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials (D) or the mean ± SEM time of the four trials (E) (n = 7 mice per group, Two-way Anova, Games-Howell (D) and Bonferroni (E) post hoc).",
    "molecules": "PLX"
  },
  {
    "caption": "(F) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long-term treatment (n= 7 mice per group, Two-way Anova, Bonferroni post hoc).",
    "molecules": "PLX"
  },
  {
    "caption": "(G) Survival curve of ASMko and wt mice with or without long-term PLX treatment (n = 7 mice per group).",
    "molecules": "PLX"
  },
  {
    "caption": "(H) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 500 μm. (I) Mean ± SEM number of Calbindin positive cells in the anterior (lobules I-V), middle (lobules VI-VIII) and posterior zone (lobules IX-X) of the Cb from the different mouse groups (n = 7 mouse per group, Two-way Anova, Bonferroni post hoc).",
    "molecules": "DAPI, PLX"
  },
  {
    "caption": "(E) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone (n = 8 mice per group).",
    "molecules": "dexamethasone, LPS"
  },
  {
    "caption": "(F) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months. Scale bar, 50 μm.",
    "molecules": "PLX"
  },
  {
    "caption": "(H) High magnification of immunofluorescence staining against MBP in ASMko mice treated or not with PLX for 2 months. White arrows show MBP aggregates indicative of myelin debris. Scale bar, 50 μm. (I) Mean ± SEM number of MBP aggregates per cell area in the different mouse groups (n = 7 mice per group, Student´s t-test). ",
    "molecules": "PLX"
  },
  {
    "caption": "(K) Amplified image and its 3D rendered replicate of amoeboid microglia containing myelin debris (arrowheads) in the ASMko cerebellum. The orthogonal projection of this cell in the lower panels confirms the presence of the myelin debris inside the cell and underneath the plasma membrane (arrowhead). DAPI staining shows cell nuclei. Scale bars, 10 μm. (L) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and ASMko mice (n = 7 mice per group). ",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without SM (B) Scale bar, 10 μm.",
    "molecules": "SM"
  },
  {
    "caption": "Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without Siramesine (C). Scale bar, 10 μm.",
    "molecules": "Siramesine"
  },
  {
    "caption": "(E) Lysotracker-Red staining in BMDMs from wt and ASMko mice. Scale bar, 10 μm.",
    "molecules": "Lysotracker-Red"
  },
  {
    "caption": "(F) Western blot of CathB levels and the cytosolic protein GAPDH in the cellular and cytosolic fractions of wt and ASMko BMDMs extracted with digitonin (left). Mean ± SEM pro-CathB and CathB levels in the cytosolic fractions (right) (n=2 cultures per genotype, Student´s t-test).",
    "molecules": "digitonin"
  },
  {
    "caption": "(G) Immunofluorescence staining against Iba-1 (green) and CathB (red) in Cb of ASMko and wt mice. Arrowheads show double positive cells. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (H) Immunofluorescence staining against CathB (green), Arg-1 (red, anti-inflammatory microglia) and F4/80 (yellow, all microglia) in Cb of ASMko mice. DAPI staining shows cell nuclei. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (I) Table showing mean ± SEM percentage of microglia presenting CathB staining (CathB +/Iba-1+), and the number of CathB positive microglia co-expressing Arg-1 (CathB+/Arg-1+) in Cb of ASMko and wt mice (n = 5 mice, >10 cells per mouse). ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt BMDMs. Staining of Ponceau-S is shown as loading control. (B) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs (n = 5 independent cultures, Student´s t-test). ",
    "molecules": "Ponceau-S"
  },
  {
    "caption": "(I) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 (n = 4 independent cultures Two-way Anova, Bonferroni post hoc).",
    "molecules": "Ca074, MTT"
  },
  {
    "caption": "(L) Western blot of Cath B and β-Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX. (M) Mean ± SEM of CathB levels normalized by β-Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX (n= 6 mice Two-way Anova, Bonferroni post hoc). ",
    "molecules": "PLX"
  },
  {
    "caption": "(O) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle (veh) or PLX (PLX). Below, Western blots of organotypic culture lysates against Iba-1, to confirm microglia depletion, and against GAPDH as loading control.",
    "molecules": "PLX"
  },
  {
    "caption": "(P) Mean ± SEM increase in pro-CathB (left) and CathB (right) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX (n = 3 mice per group Two-way Anova, Bonferroni post hoc).",
    "molecules": "PLX"
  },
  {
    "caption": "(A) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment (n = 7 mice per group).",
    "molecules": "Ca074Me"
  },
  {
    "caption": "Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials (B)",
    "molecules": "Ca074Me"
  },
  {
    "caption": "Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time of the four trials (C)",
    "molecules": "Ca074Me"
  },
  {
    "caption": "(D) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month. DAPI staining shows cell nuclei. Scale bar, 200 μm.",
    "molecules": "Ca074Me, DAPI"
  },
  {
    "caption": "(A) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 2 month-old Npc1nmf164 and wt mice. DAPI staining shows cell nuclei. Scale bars, 30 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) High magnification images and their 3D rendered replicates from cerebellar Npc1nmf164 microglia stained against Iba-1 and MBP containing myelin debris (arrowheads). Scale bars, 10 μm (up), 2 μm (down). The orthogonal projection of the cells in the right panels confirms the presence of myelin debris inside the cell and underneath the plasma membrane. DAPI staining shows cell nuclei. Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(I) Immunofluorescence staining against Iba-1 and CathB in Cb of a NPC patient. DAPI shows cell nuclei. Scale bar, 50 μm. A merged magnified image of the selected area is shown in the right down panel, scale bar 10 μm. Table shows mean ± SEM percentage of microglia presenting CathB staining in NPC and control children.",
    "molecules": "DAPI"
  },
  {
    "caption": "E EdU staining marks cells that were in S-phase during the 1h incubation time prior to microscopic imaging. Scale bar = 50 µm, n= 12. F DAPI staining was used to identify cells currently in mitosis (examples are highlighted by yellow asterisks).",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "A Root growth assay of seedlings grown for 7 days in the presence of increasing concentrations of auxin biosynthesis inhibitors kynurenine and yucasin (n = 14-26, both inhibitors always in equal concentrations) at the indicated temperatures.",
    "molecules": "yucasin, auxin, kynurenine"
  },
  {
    "caption": "D Temperature induced root growth is unaffected in 8 days-old seedlings if 0.5 mM NPA is applied to the root shoot junction on day 5 (n = 13-16) at the indicated temperatures.",
    "molecules": "NPA"
  },
  {
    "caption": "G (Co-)incubation of 5 days-old seedlings for 3h with 10 μM EdU only or in combination with either 100 nM NAA or 50 μM PEO-IAA (n = 11-24),",
    "molecules": "NAA, PEO, EdU, IAA"
  },
  {
    "caption": "C EdU staining of 5 days-old seedlings at indicated temperatures (n = 11-14).",
    "molecules": "EdU"
  },
  {
    "caption": "B: Hippocampal neurons transduced with GFP-TDP-25 constructs (DIV5+8) or GFP-TDP-43 (DIV5+4 due to higher toxicity) were sequentially extracted with RIPA buffer followed by 2% SDS buffer and analyzed via immuoblotting. Left panel shows representative immunoblot for GFP and loading control Calnexin. Right panel shows quantification of the ratio of the respective GFP densiometric signal in SDS extract to RIPA extract. Barplots showing mean ± SD from n=3 independent experiments. TDP-43 (0.039 ± 0.029, mean ± CI) vs TDP-25 wt (1.22 ± 0.296) vs TDP-25 mut (0.928 ± 0.564): F(2,6)=51.4, ***p=0.000168, η²=0.94. TDP-43 vs TDP-25 wt: ***p=1.67*10-4, TDP-43 vs TDP-25 mut: p=7.983*10-4, TDP-25 wt vs TDP-25 mut: p=0.1182,",
    "molecules": "SDS"
  },
  {
    "caption": "E: Immunofluorescence of hippocampal neurons transduced with GFP, GFP-TDP-25 wild-type or mutant and treated with MG132 (10 µM, 14h, DIV5+8). Representative images of GFP-TDP-25 inclusions and diffuse cytoplasmic staining. Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei (omitted here for clarity) in MG132 treated cells compared to DMSO control. Counterstain to label the neuronal cytoskeleton (MAP2). Scale bar = 10 µm. Box plot as in Fig. 3C. GFP (1.23 ± 0.393, n=5 independent experiments) vs GFP-TDP-25 wt (2.11 ± 0.804, n=5) vs GFP-TDP-25 mut (2.36 ± 1.16, n=5): H(1)=7.34, df=2, p=0.02548, η²[H]=0.445, Kruskal-Wallis test. GFP vs GFP-TDP-25 wt: *p=0.048, GFP vs GFP-TDP-25 mut: *p=0.048, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.690, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.",
    "molecules": "DAPI, DMSO, MG132"
  },
  {
    "caption": "Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants. Scale bar, 10 µm.",
    "molecules": "propidium iodide"
  },
  {
    "caption": "Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point.",
    "molecules": "Starch"
  },
  {
    "caption": "Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.",
    "molecules": "CO2"
  },
  {
    "caption": "Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.",
    "molecules": "CO2"
  },
  {
    "caption": "Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.   ",
    "molecules": "Fru, fructose, Glc, glucose, Suc, sucrose"
  },
  {
    "caption": "Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point.",
    "molecules": "starch"
  },
  {
    "caption": "Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype.",
    "molecules": "CO2"
  },
  {
    "caption": "Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.",
    "molecules": "Glc, glucose"
  },
  {
    "caption": "Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   ",
    "molecules": "Fru, fructose, starch, Suc, sucrose"
  },
  {
    "caption": "Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2.",
    "molecules": "CO2"
  },
  {
    "caption": "Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm.",
    "molecules": "CO2, propidium iodide"
  },
  {
    "caption": "Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.",
    "molecules": "CO2"
  },
  {
    "caption": "Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. plasma glucose (D) was measured every day. Plasma insulin (C) was analyzed at day 7 using insulin ELISA kit. Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.",
    "molecules": "glucose"
  },
  {
    "caption": "(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT and fzo-1 mutants animals. Dashed lines outline regions where fluorescence intensity was quantified.",
    "molecules": "TMRE"
  },
  {
    "caption": "(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT animals with and without needle wounding. Dashed lines outline regions where fluorescence intensity was quantified.",
    "molecules": "TMRE"
  },
  {
    "caption": "(A) Representative confocal images show the TMRE and mito::GFP signals in WT, miro-1(tm1966), miro-1(ΔEF) (EF-hand domain mutation), and miro-1(OE, overexpression) animals. (B) Quantitation of TMRE and mito::GFP signal (normalized ratio of TMRE fluorescence to mito::GFP fluorescence). n > 200 mitochondria.",
    "molecules": "TMRE"
  },
  {
    "caption": "(D) ATP content of crude isolated mitochondria from WT and miro-1(tm1966) mutants as measured with a commercial luminescence kit. n = 3, biological replicates.",
    "molecules": "ATP"
  },
  {
    "caption": "(D) Western blot analysis of the interaction between MIRO-1 and VDAC-1 by coimmunoprecipitating with GFP nanobody-conjugated beads in GFP::MIRO-1 animals and detected by GFP and VDAC-1 antibodies.",
    "molecules": "nanobody"
  },
  {
    "caption": "(A) Schematic of the vdac-1 transgene by CRISPR-Cas9 method. The images below show the young adult stage worms of WT and vdac-1(zju247) mutants. (B) Representative confocal images showing TMRE staining and mito::GFP in L4440, vdac-1 RNAi, vdac-1(zju247), and vdac-1 OE animals.",
    "molecules": "TMRE"
  },
  {
    "caption": "(E) ATP content in crude isolated mitochondria of WT animals and vdac-1(zju247) mutants. Quantification is conducted with a commercial luminescence kit. n = 3, biological replicates.",
    "molecules": "ATP"
  },
  {
    "caption": "(B) Immunoprecipitation of GFP::MIRO-1 and GFP::MIRO-1(E473G) with GFP nanobody-conjugated beads.",
    "molecules": "nanobody"
  },
  {
    "caption": "(F) Immunoprecipitation of GFP::MIRO-1 in fzo-1 mutant with GFP nanobody-conjugated beads. The interaction between GFP::MIRO-1 and VDAC-1 is increased in fzo-1 mutants.",
    "molecules": "nanobody"
  },
  {
    "caption": "(G) BN-PAGE and immunoblot analysis of VDAC-1 oligomerization crosslinked with BMH in WT and miro-1(tm1966) mutants using VDAC-1 antibody.",
    "molecules": "BMH"
  },
  {
    "caption": "F  HT-29 cells were transfected with NC, siMLKL,  miR-324-5p, or anti-miR-324-5p. After 48 h, cells were treated with DMSO  or T+S+Z for an additional 24 h. Cell survival  was determined by measuring ATP  levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP, DMSO"
  },
  {
    "caption": "F, G HeLa-endogenous MLKL  cells (F) and HeLa-exogenous MLKL  (G) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with T+S+Z for 24 h. Cell survival  was determined by measuring ATP  levels. Data information: The number of surviving  cells was normalized to the number of surviving  control cells, which were treated with DMSO. data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP, DMSO"
  },
  {
    "caption": "H HeLa  cells expressing MLKL(1-190 aa) fused to DmrB  (HeLa-MLKL(1-190)-Dmir  cells) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with DMSO  or the dimerization agent AP20187  (60 nM) for 24 h. Cell survival   was determined by measuring ATP  levels. Data information: The number of surviving  cells was normalized to the number of surviving  control cells, which were treated with DMSO. data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "AP20187, ATP, DMSO"
  },
  {
    "caption": "E MEFs  were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with DMSO  or T+S+Z for 24 h. Cell viability was determined by measuring ATP  levels. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP, DMSO"
  },
  {
    "caption": "A U937  cells were treated with 100 ng/ml IFN-α, 100 ng/ml  IFN-β, 100 ng/ml  IFN-γ, 100 ng/ml LPS, or 25 μg/ml poly(I:C) for 24 h. Identical concentrations were used in later experiments unless otherwise stated. qPCR  analysis for the expression of MLKL. C U937  cells were treated with PBS, IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C)  for 24 h. qPCR  analysis for the expression of miR-324-5p. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "LPS, PBS, poly(I:C)"
  },
  {
    "caption": "B U937 cells were treated with IFN-α,  IFN-β,  IFN-γ, LPS, or poly(I:C)  for 24 h. and western blotting analysis of MLKL  and β-actin (left). Quantification of MLKL  normalized to  β-actin  levels (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "LPS, poly(I:C)"
  },
  {
    "caption": "F, G U937  cells were incubated with 300 nM ruxolitinib  for 2 h prior to  IFN-β (F) or   IFN-γ (G) treatment. After 24 h, qPCR  analysis for the expression of miR-324-5p. After 48 h, western blotting  analysis of p-STAT1, STAT1, MLKL  and β-actin. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "A U937  cells were transfected with NC, siMLKL-3'UTR, or  miR-324-5p. After 48 h, cells were exposed to 100 ng/ml IFN-γ plus 20 μM  z-VAD for 24 h. Identical concentrations were used in later experiments unless otherwise stated. Cell viability was determined by measuring ATP  levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP, z-VAD"
  },
  {
    "caption": "B qPCR  analysis for the expression of MLKL  (left) and miR-324-5p (middle) in PBMC-derived macrophages  that were treated with PBS  or IFN-γ for 24 h. The corresponding  western blotting analysis of MLKL  and β-actin (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "PBS"
  },
  {
    "caption": "C, D PBMC-derived macrophages  (C) and HT-29 cells (D) were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were exposed to S+Z or  IFN-γ+S+Z for 24 h. Cell viability  was determined by measuring ATP  levels (left). The corresponding western blotting analysis of MLKL  and β-actin (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP"
  },
  {
    "caption": "F MiR-324-5p+/+ HT-29, miR-324-5p-/- -19# , and miR-324-5p-/- -36# cells were treated with T+S+Z for the indicated times. Cell viability  was determined by measuring ATP  levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP"
  },
  {
    "caption": "G, H MiR-324-5p+/+ HT-29, and miR-324-5p-/- HT-29 cells were treated with IFN-β  (G) or IFN-γ (H) plus 20 μM z-VAD for 24 h. Cell viability was determined by measuring ATP  levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP, z-VAD"
  },
  {
    "caption": "A qPCR  analysis for the expression of IFN-β  (left) and IFN-γ (right) in PBMC-derived macrophages  that were infected with IAV  (H1N1 strain PR8) at an multiplicity of infection (MOI) of 0.2 for 24 h. B qPCR  analysis for the expression of MLKL  (left) and miR-324-5p (right) in PBMC-derived macrophages  that were treated with the 300nM ruxolitinib  for 2 h prior to the infection with IAV  (MOI=0.2) for 24 h. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "C PBMC-derived macrophages  were transfected with NC, siMLKL, or miR-324-5p. After 48h, cells were treated with S+Z in the presence or absence of IAV  (MOI=0.2) for 24 h. Cell viability was determined by measuring ATP  levels. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.",
    "molecules": "ATP"
  },
  {
    "caption": "H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.",
    "molecules": "doxycycline"
  },
  {
    "caption": " I Electron micrograph of negatively-stained fish egg coat filaments. On the right are representative 2D classes, indicating a separation of ~65 Å between repeating units within the polymers (white arrows). Scale bars: 100 nm (left panel) and 10 nm (bottom right panel). ",
    "molecules": "polymers"
  },
  {
    "caption": " A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl (top). Analysis of this material by correlation averaging reveals a periodic pattern (bottom) with a two-fold symmetry axis perpendicular to the plane of the sheet (white symbol). Scale bars: 250 Å (top) and 125 Å (bottom). ",
    "molecules": "NaCl"
  },
  {
    "caption": "(D) qRT-PCR analysis of Mapk12 (p38gamma) and Mapk13 (p38delta) mRNA expression in liver extracts prepared from wild-type mice (WT) fed a diet deficient in methionine and choline (MCD) or control diet (ND) for 3 weeks; mRNA expression was normalized to the amount of Gapdh mRNA. (n=5-10)",
    "molecules": "choline, methionine"
  },
  {
    "caption": "(B) Liver triglycerides and (C) plasma transaminase activity (ALT) measured in WT and p38γ/δ-/- mice after 3 weeks of MCD diet.",
    "molecules": "triglycerides"
  },
  {
    "caption": "(D) TBARS and hydrogen peroxide detected in liver samples from mice fasted overnight after the 3-week MCD diet.",
    "molecules": "hydrogen peroxide, TBARS"
  },
  {
    "caption": "Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a MCD diet for 3 weeks. (B) Liver triglycerides (C) and plasma ALT at the end of the diet period.",
    "molecules": "triglycerides"
  },
  {
    "caption": "Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (B) liver triglycerides (n=5-10).",
    "molecules": "triglycerides"
  },
  {
    "caption": "Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (D) Glucose tolerance measured at the end of the diet period. Blood glucose concentration was measured in mice given an intraperitoneal glucose injection (1g/kg) after overnight fasting (n=5-10).",
    "molecules": "Glucose, glucose"
  },
  {
    "caption": "Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (E) Basal blood glucose in overnight-fasted ND and HFD-fed Lyzs-Cre and p38γ/δLyzs-KO mice (n=5-10). Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 refers to p38γ/δLyzs-KO versus Lyzs-Cre; ##P<0.01; ###P<0.001 refers to ND versus HFD (2-way ANOVA coupled to Bonferroni's post tests or Newman-Keuls post test for liver triglycerides).",
    "molecules": "glucose"
  },
  {
    "caption": "Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (C) Livertriglyceride content.",
    "molecules": "triglyceride"
  },
  {
    "caption": "Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BMLyzs-Cre) or p38γ/δLyzs-KOmice (Cx BMp38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (E) Fasted glucose, detected at the end of the diet period in overnight-fasted mice. Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni's post tests).",
    "molecules": "glucose"
  },
  {
    "caption": "(D and E) WT mice fed the MCD diet were i.v. injected with a 1:1 mix of DiO-labeled Lyzs-Cre neutrophil and DiD-labeled p38γ/δLyzs-KO neutrophils (6x106 cells in total; n=10). One hour after injection, liver-infiltrating neutrophils were assessed by flow cytometry (D) and fluorescence micrography on liver sections (E).",
    "molecules": "DiD, DiO"
  },
  {
    "caption": "Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (C) Liver triglyceride and (D) plasma transaminase activity (ALT) at the end of the diet period.",
    "molecules": "triglyceride"
  },
  {
    "caption": "(A, B and C) Plasma levels of 3-hydroxybutyrate in WT and p38γ/δ-/- (A), Lyzs-Cre and p38γ/δLyzs-KO mice (B) and Lyzs-Cre Ly6G Ab treated after MCD diet (C).",
    "molecules": "3-hydroxybutyrate"
  },
  {
    "caption": "(A) Respiratory chain super-complex levels are slightly restored by over-expression of Mcp3. Mitochondria isolated from the indicated strains were lysed in 1% digitonin and subjected to a 4-8% BN-PAGE. Proteins were analysed by immunodecoration with antibodies against either Cor1 of complex III or Cox2 of complex IV.",
    "molecules": "digitonin"
  },
  {
    "caption": "(B) Assembly defects in TOM and TOB complexes in mdm10 cells are partially restored by Mcp3 over-expression. Mitochondria of the indicated cells were lysed in 1% digitonin and subjected to a 6-13% BN-PAGE and immunoblotting with antibodies against Tom40 (left) or Tob55 (right). The assembled TOM complex and an unassembled Tom40 species at ca. 100 kDa (arrowhead) are indicated as well as the TOB complex and an additional higher molecular weight species of the TOB complex (asterisk).",
    "molecules": "digitonin"
  },
  {
    "caption": "(C) Over-expression of Mcp3 partially rescues the alterations in lipid composition of cells lacking Mdm10. Lipids were extracted from highly pure mitochondria isolated from the indicated cells and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from other cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD; *, p < 0.05) is given.",
    "molecules": "ERG, phospholipid, PL"
  },
  {
    "caption": "(D) Mcp3 rescues loss of Mdm12. Wild-type (WT) or mdm12Δ cells transformed with the empty plasmid pYX142 () or mdm12Δ cells over-expressing either Mcp3 or Mdm12 (as a control) were analysed at 30°C or 37°C by drop dilution assay on rich medium containing glycerol (YPG).",
    "molecules": "glycerol"
  },
  {
    "caption": "(C, D) Loss of Mcp3 has no effect on the phospholipid composition of mitochondria. Highly-pure mitochondria were isolated from the indicated yeast cells. Lipids were extracted and then analysed by mass spectrometry analysis. The level of each phospholipid species in wild-type mitochondria was set to 100% and the ERG/PL ratio to 1. Relative changes in mitochondria from mcp3 cells are presented. The mean with standard deviations of three biological repeats with two technical repeats for each (n=6; SD) is given.",
    "molecules": "ERG, phospholipid, PL"
  },
  {
    "caption": "(H) Mcp3 is a MOM protein. Mitochondria isolated from cells expressing HA-Mcp3 were treated with proteinase K (PK, 100 µg/ml) in the absence or presence of the detergent Triton X-100 (TX). Samples were precipitated with TCA and analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag or the indicated mitochondrial proteins. Tom20, a MOM protein exposed to the cytosol; Dld1, a protein located in the IMS.",
    "molecules": "TCA, Triton X-100"
  },
  {
    "caption": "(A) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors. Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70, Tom20 or Tom22 prebound to glutathione beads or with unloaded beads. After washing bound material was eluted with sample buffer. Proteins were analysed by SDS-PAGE, blotting onto a membrane and autoradiography. To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used.",
    "molecules": "glutathione"
  },
  {
    "caption": "(B) Mcp3 precursor binds to Tom22 in organello. Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His-Tom22 containing cells. Mitochondria were pre-incubated with CCCP (40 µM) to halt import. After lysis with β-dodecyl maltoside samples were incubated with Ni-NTA beads. After washing bound proteins were eluted with sample buffer and analysed by SDS-PAGE and autoradiography. I, 5% of 35S-Mcp3 used; pel., 2% insoluble material after clarifying spin; sup., 2% of material incubated with Ni-NTA; n.b. 2% of non-bound material after binding to Ni-NTA; eluate, 100% of bound material.",
    "molecules": "35S, CCCP, β-dodecyl maltoside"
  },
  {
    "caption": "(C) Radiolabelled Mcp3 was incubated with organelles described in (B). After import mitochondria were solubilized in digitonin, the lysate was cleared by centrifugation and incubated with or without an antibody against the His-tag (α-His). Samples were analysed by BN-PAGE, autoradiography and afterwards immunodecoration with an antibody against Tom40. Bands shifted by the anti-His antibody are indicated by arrowhead.",
    "molecules": "digitonin"
  },
  {
    "caption": "(C) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential. Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9-DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o-phenantroline and EDTA (o-Phen./EDTA). After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. L+MPP: radiolabelled proteins were incubated with recombinant MPP.",
    "molecules": "EDTA, CCCP, o-phenantroline"
  },
  {
    "caption": "(H) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria. Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP. After import mitochondria were incubated with PK as in (G) and analysed by SDS-PAGE and autoradiography. I, 20% of radiolabelled precursor protein used in each import reaction; The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3.",
    "molecules": "CCCP"
  },
  {
    "caption": "Quantification by western-blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation. UT: untransformed, PN: pre-neoplastic, TR: transformed. Histone H3 is used as a loading control. The graph on the right displays the H3 normalized densitometric values of western-blot band from untreated and DMSO treated cells. Values represent mean ± SEM from three biological replicates. One asterisk indicates p-value < 0.05 (one-tailed unpaired Student's t-test). ns: non-significant.",
    "molecules": "DMSO, H3K27me3"
  },
  {
    "caption": "Western-blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor (EZH2i) or DMSO as control for 12 days. Histone H3 is used as a loading control. Values represent H3-normalized densitometric values of the H3K27me3 bands, and are expressed relative to the relevant DMSO control. UT: untransformed, PN: pre-neoplastic, TR: transformed.",
    "molecules": "DMSO, H3K27me3"
  },
  {
    "caption": "ChIP-seq signal for EZH2 and H3K27me3 (left) at EMX2 and HOXB9 loci. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. mRNA expression of EMX2 and HOXB9 (right) as detected by RNA-seq. Expression values represent mean ± SEM from three biological replicates. UT: untransformed, PN: pre-neoplastic, TR: transformed. TPM: transcripts per million.",
    "molecules": "H3K27me3"
  },
  {
    "caption": "qRT-PCR showing the expression levels of EMX2 and HOXB9 in the indicated cells treated with EZH2i or a DMSO control for 12 days. Values represent mean ± SEM from three biological replicates. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). UT: untransformed, TR: transformed.",
    "molecules": "DMSO"
  },
  {
    "caption": "Quantification of EMX2 levels by qRT-PCR in five different GBM cell lines upon treatment with 1μM, 3μM or 10μM of EZH2i for eight days, or a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Two asterisks indicate p-value <0.01 comparing EZH2i- and DMSO-treated cells (Two-way ANOVA).",
    "molecules": "DMSO"
  },
  {
    "caption": "Relationship between the extent of EMX2 upregulation induced by EZH2i and the DNA methylation level at the EMX2 promoter in GBM cell lines. Values for EMX2 upregulation are the fold-change in mRNA expression induced upon treatment with 10μM EZH2i (see: panel G), and are expressed relative to a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Methylation values are averages of DNA methylation at CpGs across the promoter of EMX2 in GBM cell lines. All data sourced from the CCLE. The significance of the anti-correlation between mRNA levels and DNA methylation levels across cell lines is indicated (Spearman rank correlation).",
    "molecules": "DMSO"
  },
  {
    "caption": "Hematoxylin and eosin (H&E) staining of a human GBM tumour. The black square represents the approximate location of the field shown in G (left image). Serial sections were used for H&E and RNA-FISH. Scale bar: 300 µm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "Visualisation of EZH2 (green) and EMX2 mRNA (red) in a human GBM tumour by RNA FISH. Nuclei were counterstained with DAPI. Scale bar: 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45-negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 µm in diameter, 1, 2) and large size (larger than 10µm in diameter, 2,3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of 4-8 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (4, 7, 8).",
    "molecules": "DAPI"
  },
  {
    "caption": "B ICC staining of CTC-ITB-01 for pan-keratin (orange), ER (green), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01 (1) and (2). MCF-7 cells are depicted as reference cell line.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR™ assay. CTC-ITB-01 is compared to A549 as recommended control cell line. An internal (DEAB) control is also depicted. 77.92% of the gated CTC-ITB-01 population are ALDH+.",
    "molecules": "DEAB"
  },
  {
    "caption": "A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.",
    "molecules": "DAPI"
  },
  {
    "caption": "B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "A Equal numbers of CTC-ITB-01 cells were seeded on 6-well culture plates and grown in the presence or absence of different 17-β-estradiol concentrations as indicated. After 10 days cells were fixed and stained with Coomassie blue staining (right panel). Macroscopic photos are shown of the fixed cell colonies from cells grown in the absence or presence of the indicated E2 concentrations (right panel). Scale bar 200 µm.",
    "molecules": "17-β-estradiol, E2"
  },
  {
    "caption": "C CTC-ITB-01 and MCF-7 cells were grown under the same culture conditions in the presence or absence of E2 as indicated. 24h after E2 deprivation or E2-treatment cells were harvested. Western blots of the protein lysates were probed with antibodies targeting ER-alpha, FOXM1, Bcl-2, PR-A, PR-B and ID1, actin was used as loading control, visualized at long exposure (l.e.) and/or short exposure (s.e.).",
    "molecules": "E2"
  },
  {
    "caption": "(D) Growth curves of CTC-ITB-01 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.",
    "molecules": "Palbociclib"
  },
  {
    "caption": "E) Growth curves of MCF-7 cells under varying concentrations of Palbociclib treatment were measured using an IncuCyte Zoom live cell imaging system. The mean values from three technical triplicates (one experiment) with standard deviation are shown.",
    "molecules": "Palbociclib"
  },
  {
    "caption": "(F) Influence of Palbociclib on CTC-ITB-01 growth after 300 hours Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.",
    "molecules": "dimethyl sulfoxide, DMSO, Palbociclib"
  },
  {
    "caption": "G) Influence of Palbociclib on MCF-7 after 80 hours. Data was chosen from one representative time point during the exponential growth phase. Statistical significance was analyzed with a one-way ANOVA with Dunnett's multiple comparisons test and compared to the vehicle substance dimethyl sulfoxide (DMSO)-. The mean values from three technical triplicates (one experiment) with standard deviation are shown.",
    "molecules": "dimethyl sulfoxide, DMSO, Palbociclib"
  },
  {
    "caption": "(H) Effect of Palbociclib on the growth of CTC-ITB-01 cells Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.",
    "molecules": "Palbociclib"
  },
  {
    "caption": "I) Effect of Palbociclib on the growth of MCF-7 cells. Concentrations were transformed to common logarithm. Three-parameter non-linear logistic regression was used to determine the IC50. The mean values from three technical triplicates (one experiment) with standard deviation are shown. Error bars for standard deviation smaller than the symbols are not displayed.",
    "molecules": "Palbociclib"
  },
  {
    "caption": "F, G Western Blotting analysis of phosphorylated IκB  (F) and quantification of gray value (G) in fibroblasts  (L929) that pre-treated with LPS  and then cultured with CMs  from TAMEMs  and other controls for 24 h (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the LPS-treated group; n=3, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G, in Graph Pad Prism 8.",
    "molecules": "LPS"
  },
  {
    "caption": "G H&E (day 7 and 14) and Masson's  (day 14) staining  for full-thickness skin  samples containing the entire wound  area (scale bar: 500 μm; n=8), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates), (gt: granulation tissue, he:  hyperproliferative epithelium, black arrow: wound  edges).",
    "molecules": "Masson's"
  },
  {
    "caption": "H- J Quantitative analysis of  regenerated granulation  (H) on day 7 and Re-epithelialization of wound  bed (I) and Masson's trichrome-stained  tissue (J) on day 14 (blue for collagen) (***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (H, I, J, in Graph Pad Prism 8.",
    "molecules": "Masson's trichrome, collagen"
  },
  {
    "caption": "(a) Primary neurons were labeled with 35S-labeled methionine, and TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or nonspecific IgG antibodies (IP-IgG). (b) Log-normal plot of normalized TDP43 levels. TDP43 half-life was determined by fitting a first-order exponential curve to the data (R2 = 0.8984). Values were pooled from five independent experiments.",
    "molecules": "35S-labeled methionine"
  },
  {
    "caption": "(c) Potential confounders in half-life analyses. Top, total protein ideally decreases over time in a predictable manner (top right panel). Middle, toxicity reduces the amount of measured protein, potentially shortening the calculated half-life (middle right panel). Bottom, protein aggregation might also shorten half-life estimations if the protein cannot be immunoprecipitated (black arrows, bottom right panel) or lengthen it if the protein is more stable and successfully immunoprecipitated (gray arrows, bottom right panel).",
    "molecules": "protein"
  },
  {
    "caption": "(c,d) Probability density plots of single-cell half-life measurements for TDP43(WT)-Dendra2 (c) and TDP43A315T-Dendra2 (d). At 0.5 μM, all three of the compounds significantly reduced TDP43(WT)-Dendra2 half-life compared to vehicle control (DMSO, n = 110; FPZ, n = 96, f* P = 1 × 10−4; MTM, n = 129, m* P = 2 × 10−7; NCP, n = 111, n* P = 2 × 10−5, two-sided Kolmogorov-Smirnov test). The compounds also reduced TDP43A315T-Dendra2 half-life, in comparison to vehicle control (DMSO, n = 120; FPZ, n = 155, f* P = 2 × 10−9; MTM, n = 152, m* P = 0.004; NCP, n = 111, n* P = 0.005, two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons in b-d. Values were obtained from eight wells per condition, with experiments performed in triplicate.",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": "(e) The change in red fluorescence intensity over time was used to calculate LC3-Dendra2 half-life, as in Figure 2 (DMSO, R2 = 0.7333; Beclin, R2 = 0.6968, FPZ, R2 = 0.8752; MTM, R2 = 0.8447; NCP, R2 = 0.8357; *P 0.0001 for global trend, F = 12.6, extra sum-of-squares F-test). Error bars represent s.e.m. (",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": ". (f) The median single-cell half-life of LC3-Dendra2 was significantly reduced by beclin expression or treatment with 0.5 μM NCP, MTM or FPZ (*P 0.01, **P 0.001, Kruskal Wallis statistic = 52.56, Kruskal-Wallis with Dunn's test).",
    "molecules": "NCP, FPZ, MTM"
  },
  {
    "caption": "(g) Probability density plot of LC3-Dendra2 half-life measurements from individual neurons treated with vehicle control (DMSO, n = 140), NCP (n = 128), MTM (n = 91), FPZ (n = 77) or those expressing beclin (n = 101). n*, P = 6 × 10−4; m*, P = 1 × 10−10; f*, P = 5 × 10−9; b*, P = 2 × 10−3; two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons. Values were pooled from eight wells per condition, with experiments performed in triplicate.",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": "(a) FPZ (n = 490) and MTM (n = 580) but not NCP (n = 523) lowered total TDP43(WT)-EGFP levels in comparison to vehicle (DMSO, n = 1,007).",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": "(c) Each compound also reduced TDP43A315T-EGFP levels (DMSO, n = 983; FPZ, n = 550; MTM, n = 542; NCP, n = 493).",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": "(d) Histogram of single-cell TDP43(A315T)-EGFP levels. Asterisks in b and d denote median values for cells exposed to vehicle (gray), FPZ (green), MTM (blue) or NCP (purple). For FPZ, MTM and NCP versus DMSO in a and d, P 1 × 10−3 by two-sided Kolmogorov-Smirnov test.",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": "e) No compound reduced TDP43(WT)-EGFP inclusions 48 h after transfection (DMSO, n = 360; FPZ, n = 194; MTM, n = 234; NCP, n = 203).",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": "(f) FPZ and MTM but not NCP reduced TDP43A315T-EGFP inclusions at 48 h (DMSO, n = 327; FPZ, n = 226; MTM, n = 262; NCP, n = 253). (g) No compound affected TDP43(WT)-EGFP localization 24 h after transfection (DMSO, n = 256; FPZ, n = 212; MTM, n = 250; NCP, n = 241). (h,i) Each compound (h) prevented TDP43A315T-EGFP cytoplasmic mislocalization (DMSO, n = 233; FPZ, n = 268; MTM, n = 263; NCP, n = 270) and reduced cytoplasmic TDP43(A315T)-EGFP levels (DMSO, n = 125; FPZ, n = 102; MTM, n = 121; NCP, n = 144. *P 0.05; NS, P > 0.05) (i). For a, c and e-i, *P 0.05; nonsignificant (NS), P > 0.05, one-way ANOVA with Dunnett's test. Error bars represent s.e.m. Values in a-d were pooled from 8-12 wells per condition from six experiments, values in e-h were pooled from eight wells per condition from three experiments, and values in i were pooled from five wells per condition from two experiments.",
    "molecules": "NCP, DMSO, FPZ, MTM"
  },
  {
    "caption": "(a,b) Primary neurons transfected with TDP43(WT)-EGFP (a) or TDP43A315T-EGFP (b) were treated with autophagy inducers and survival determined by AFM. Data were obtained from eight wells per condition, with experiments performed in triplicate. Supplementary Table 2 lists the number of neurons per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis. (c) Autophagic induction improved survival in TDP43M337V HB9-positive human iPSC-derived MNs. (d) FPZ and MTM also reduced the risk of death in MAP2-positive human iPSC-derived MNs Values in c and d were pooled from 18 wells per condition, with experiments performed in triplicate. (e) Astrocytes differentiated from human iPSCs exhibit mutant TDP43-related toxicity that is mitigated by FPZ and MTM. Values were pooled from 18 wells per condition, performed in triplicate. Supplementary Table 3 lists the number of human iPSC-derived neurons and astrocytes per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis.",
    "molecules": "FPZ, MTM"
  },
  {
    "caption": "U2OS WT or U2OS GFP-ATG2A knock-in (KI) cells were grown in CM or starvation media for 2 h, lysed and incubated with anti-GFP nanobody beads coupled to agarose to immunoprecipitate (IP) GFP-ATG2A. IP samples and 2% input lysates were run on 4-12% gradient gel and processed for western blotting. Anti-ATG2A, anti-WIPI4 and anti-LC3B, anti-GABARAP (pan) were used to probe for the presence/absence of autophagy proteins in the immunoprecipitated samples. p-p70S6K (T389) was used as a marker for starved cells and total p70S6K as loading control.",
    "molecules": "agarose"
  },
  {
    "caption": "Myc-tagged ATG2A-wild type (WT) and ATG2A-mLIR (orange) were co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP in HEK293T cells, lysed and anti-GFP nanobodies coupled to agarose were used to immunoprecipitate GFP-tagged proteins. Samples were subjected to western blotting and probed for the presence of Myc-ATG2A in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   As in (B) but using Myc-tagged ATG2B-WT or ATG2B-mLIR (orange) co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP. Samples were subjected to western blotting and probed for the presence of Myc-ATG2B in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   ",
    "molecules": "agarose"
  },
  {
    "caption": "U2OS ATG2A/B double knock-out (DKO) CRISPR/Cas9 cells stably expressing Tandem tagged LC3B (mCherry-GFP-LC3B) were retrovirally transduced to express vector, or HA-tagged ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA). Cells were grown in complete medium (CM) or starved for 2 h (EBSS) or treated with CM plus BafilomycinA1 (200nM, 4 h), fixed and analysed by confocal microscopy. Merged images of GFP (green) and mCherry (red) channels show the presence of autophagosomes/phagophores (GFP and mCherry positive, yellow puncta) or autolysosomes (mCherry only, red puncta) Scale bar 10µm. Images are representative of n=3 independent experiments.",
    "molecules": "BafilomycinA1"
  },
  {
    "caption": "U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).",
    "molecules": "BafA1, BafilomycinA1"
  },
  {
    "caption": "U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments.",
    "molecules": "BafA1, BafilomycinA1, digitonin, TritonX-100"
  },
  {
    "caption": "U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.   Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.   ",
    "molecules": "BafA1, BafilomycinA1, DAPI, DNA"
  },
  {
    "caption": "B) Lysates of HEK293A cells expressing either GFP or GFP-TBC1D14 were subjected to immunoprecipitation (IP) with GFP-Trap, and then immunoblotted for GFP, TRAPPC12 and TRAPPC4.",
    "molecules": "Trap"
  },
  {
    "caption": "C) Lysates of cells expressing GFP or GFP-TRAPPC3 were subjected to IP with GFP-Trap, and then immunoblotted for TRAPPC4, GFP and TBC1D14.",
    "molecules": "Trap"
  },
  {
    "caption": "D) HeLa C1 cells transfected with CFP or CFP-TBR were treated with D/D solubiliser (Clontech) or D/D solubiliser plus 2.5 μg/ml Brefeldin A (BFA) for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. Results are expressed as a percentage of GFP levels at t=0, and the mean of 3 independent experiments ± s.e.m.",
    "molecules": "D/D solubiliser, BFA, Brefeldin A"
  },
  {
    "caption": "A) HEK293A cells expressing GFP, GFP-TBC1D14 224-669 or GFP-TBR were treated in duplicate with EBSS, EBSS plus 100 nM BafA1 or not for two hours, lysed and subjected to immunoblotting for LC3B, tubulin and GFP. The amount of LC3B/tubulin for each condition from three independent experiments is shown on the bar graph, ±s.e.m. * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.",
    "molecules": "BafA1"
  },
  {
    "caption": "B) Samples prepared according to (A) (8 × 107 HEK293A cells per transfection) were lysed in TNTE, split equally and denatured with 1% SDS (D) or not (ND) and subjected to 1 hour streptavidin resin pulldown at room temperature. The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins.",
    "molecules": "Biotin, SDS"
  },
  {
    "caption": "D) HEK293T cells stably depleted of TRAPPC8 (shC8) or not (shCtrl) were transfected with constructs encoding GFP or GFP-TRAPPC3 and subjected to GFP Trap IP. Precipitated proteins were immunoblotted for GFP, TRAPPC4 and TRAPPC8. Bar chart: the amount of TRAPPC4 isolated was normalised to the amount of GFP-TRAPPC3 precipitated from three independent experiments and plotted ± s.e.m., ns = non-significant, unpaired T-test.",
    "molecules": "Trap"
  },
  {
    "caption": "C) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B (knockdown confirmed by immunoblot analysis), were treated with D/D solubiliser for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. The line graph shows the GFP fluorescence as a percentage of t=0, ± s.e.m. from three independent experiments.",
    "molecules": "D/D solubiliser"
  },
  {
    "caption": "(B) Representative immunoblot of Nrxn1β-Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against the Fc-tag and the 3G10 epitope (to detect the HS stub which reveal HS after heparinase digestion). (C) Quantification of 3G10 (reflecting HS) signal normalized to that of Fc (Nrxn1). Bar graph shows means ±SEM of 3 independent experiments. ",
    "molecules": "3G10, HS"
  },
  {
    "caption": "HEK293 cells: (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5.",
    "molecules": "3G10, HS"
  },
  {
    "caption": "(F-H) Primary cortical neurons: Cortical neurons were infected with control lentivirus (empty vector) or lentivirus expressing FLAG-tagged CA10, and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment. (F) Samples were analyzed by immunoblotting using antibodies against neurexins, 3G10 (for the HS stub) and FLAG (for CA10). Stars indicate non-neurexin bands (see results). (G) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (H) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests (n=3).",
    "molecules": "3G10, HS"
  },
  {
    "caption": "(I-K) Mouse brains: Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment. (I) Samples were analyzed by immunoblotting using antibodies against neurexins, the 3G10 epitope (for the HS stub) and CA10. Stars indicate non-neurexin bands (see results). (J) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (K) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of 3 biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests.",
    "molecules": "3G10, HS"
  },
  {
    "caption": "(B) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against Fc (for Nrxn1β), the 3G10 epitope (for the HS stub) and V5 (for CA10). Input samples were analyzed separately for CA10 to ensure equal expression (lower panel). (C) Quantification of the 3G10 epitope signal, normalized to Fc (Nrxn1β). Data shown are mean ±SEM of 3 independent replicates. *, p< 0.05; by Mann-Whitney tests comparing each Nrxn1 mutant with and without CA10 (n=3). No significance difference was observed for S&T>G (p=0.1143), GIGG (p=0.9714) or DIRR (p=0.4857) variants. ",
    "molecules": "3G10, HS"
  },
  {
    "caption": "(E) representative immunoblots analyzed using antibodies against the Fc-tag (for Nrxn1β-Fc) the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. (F, Quantification of the 3G10 epitope signal normalized to Fc (for Nrxn1β-Fc) signals, Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.",
    "molecules": "3G10, HS"
  },
  {
    "caption": "(G) show representative immunoblots analyzed using antibodies against the HA tag (Nrxn1α-HA), the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. H) Quantification of the 3G10 epitope signal normalized to HA (for Nrxn1α-HA) signals, respectively. Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.",
    "molecules": "3G10, HS"
  },
  {
    "caption": "(D) Comparison of 1H-13C HSQC spectra of Nrxn1 stalk (red) and Nrxn1 stalk/CA10 (black) at a 2:1 molar ratio (50 mM phosphate buffer, pH 7.4, 0.5 mM d10-DTT, 10% D2O). Nrxn1 residues with stronger chemical shift changes are indicated blue boxes. Asterisk (*) indicates an impurity from glycerol.",
    "molecules": "DTT, 13C, D2O, glycerol, 1H, phosphate"
  },
  {
    "caption": "(C) α-Neurexin immunoprecipitated from forebrain (Fb) and cerebellum (Cb) using an anti-pan-neurexin antibody was heparinase-treated ('Heps') and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal (to detect the HS stub). Stars indicate non-neurexin bands (see results). (D) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ±SEMof 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum. (E) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Bar graphs show mean ±SEM of 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum. ",
    "molecules": "3G10, HS"
  },
  {
    "caption": " C Wild-type and tel1∆ strains expressing a fully functional Top1-HA tagged protein were arrested in G1 with α-factor (αf) and released into YEPD supplemented with CPT (50 μM). Western blot with anti-HA antibodies (top) and Coomassie staining (bottom) of protein extracts prepared at the indicated time points ",
    "molecules": "α-factor, αf, CPT"
  },
  {
    "caption": " A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti-Rad53 antibodies (C)",
    "molecules": "α-factor, αf, CPT"
  },
  {
    "caption": " E, F G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM) (E) or in YEPD supplemented with CPT (10 μM) with (+noc) or without (-noc) nocodazole (F). Western blot analysis with anti-Rad53 antibodies ",
    "molecules": "αf, CPT, noc, nocodazole"
  },
  {
    "caption": " A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM). (A) Western blot analysis with anti-Rad53 antibodies ",
    "molecules": "α-factor, αf, CPT"
  },
  {
    "caption": " (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodie ",
    "molecules": "αf, CPT"
  },
  {
    "caption": " (B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodies (B, D ",
    "molecules": "αf, CPT"
  },
  {
    "caption": " F, G G1-arrested cell cultures (αf) were released into YEPD supplemented with CPT (50 μM). Western blot analysis with anti-Rad53 antibodies ",
    "molecules": "αf, CPT"
  },
  {
    "caption": "(A) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into 10 mM Tris buffer of the indicated pH (left panel) or into 10 mM Tris pH 7.4 buffer supplemented with increasing amounts of NaCl (1 - 250 mM; right panel), and reduction sensitivity of DNGR-1 was assessed by reducing SDS-PAGE and Western blot. (B) 0.25 μg of purified DNGR-1 long mouse ECD was diluted into buffers of different pH and ionic strength, and its reduction sensitivity was assessed by reducing SDS-PAGE and Western blot. The intensity of bands corresponding to dimer and monomer was determined densitometrically, and the ratio was plotted as a function of buffer ionic strength and pH.",
    "molecules": "NaCl"
  },
  {
    "caption": "(C) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffer, and its reduction sensitivity was tested under mildly (Laemmli buffer) or strongly denaturing (8 M urea) conditions by reducing SDS-PAGE and Western blot.",
    "molecules": "MES, urea"
  },
  {
    "caption": "(D) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffers, and its reduction sensitivity after different lengths of heat-denaturation in Laemmli buffer was tested by reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.",
    "molecules": "MES"
  },
  {
    "caption": "(A) 1 μg of purified long mouse DNGR-1 ECD was transferred into 10mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS-PAGE and Western blot.",
    "molecules": "MES"
  },
  {
    "caption": "(C) Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS-PAGE and Western blot under reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction sensitive protein with a +.",
    "molecules": "MES"
  },
  {
    "caption": "(A) Long mouse and human DNGR-1 ECD proteins were diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent far-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition)",
    "molecules": "MES"
  },
  {
    "caption": "(B) Long mouse DNGR-1 ECD was diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent near-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition and the blue line shows the difference between the two).",
    "molecules": "MES"
  },
  {
    "caption": "(D) Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10mM MES pH 6.1 buffers and the lysates were analyzed by SDS-PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.",
    "molecules": "MES, SDS"
  },
  {
    "caption": "(A) Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and quantitation of four independent experiments is shown.",
    "molecules": "MES"
  },
  {
    "caption": "(d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4 day culture period.",
    "molecules": "3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester, Brusatol, N-acetyl-L-cysteine, ROS"
  },
  {
    "caption": "(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (b) representative histological lung sections (haematoxylin and eosin (HE) staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 (intraperitoneally; i.p. 10 mg kg−1 daily) to inhibit CD39 activity. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.",
    "molecules": "POM1"
  },
  {
    "caption": "(c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (d) representative histological lung sections (HE staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 (i.p. 10 mg kg−1 daily). n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.",
    "molecules": "PSB1115"
  },
  {
    "caption": "(e,f) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates treated with vehicle control or the CD39 blocker polyoxometalate-1 (POM1) and treated with vehicle or the adenosine receptor inhibitor PSB1115 (4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulphonic acid potassium salt). Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least four mice per genotype were analysed. Scale bars, 50 μm. **P0.01. NS, not significant; (χ2-test assessing the genotype effect in a generalized linear model with logit link).",
    "molecules": "4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulphonic acid potassium salt, PSB1115, polyoxometalate-1, POM1"
  },
  {
    "caption": "The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or a siRNA targeting AGR2 (siAGR2) for 24 h (C) Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2, anti-ERK1 antibodies (for loading control).",
    "molecules": "DSP"
  },
  {
    "caption": "The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or treated or not with tunicamycin (Tun) prior to cross-linking (D). Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2 anti-calnexin (CANX) antibodies (for loading control).",
    "molecules": "DSP, Tun, tunicamycin"
  },
  {
    "caption": "The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. G) Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT. The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors (n = 4).",
    "molecules": "DTT"
  },
  {
    "caption": "A) Co-immunoprecipitation of AGR2 with TMED2 under basal and tunicamycin induced ER stress in intestinal epithelial cells treated in situ. *indicates Immunoglobulin heavy and light chains.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "C) Changes in AGR2 dimer and monomer ratio in TMED2 overexpressing cells. DSP-stabilized AGR2 was analyzed under non-reducing (top blot) or reducing conditions (bottom blot). D=dimeric, M=monomeric AGR2.",
    "molecules": "DSP"
  },
  {
    "caption": "A) Formation of GFP-LC3 autophagic puncta in TMED2 overexpressing HEK239T cells as monitored using confocal microscopy (right panel). DAPI was used for nuclear staining visualization. Percentage of GFP-LC3 puncta in control (CTL) and TMED2 overexpressing HEK293T cells as quantified from three independent experiments by counting 240 GFP-LC3 positive cells for each condition (left panel). (**): p=0.0032.",
    "molecules": "DAPI"
  },
  {
    "caption": "B) Western blot detection (left panel) and quantification (right panel) of LC3 level in control and TMED2 overexpressing HEK293T cells treated or not with 50 µM chloroquine for 2 hours. Actin (ACT) served as a loading control. (*): p=0.0498.",
    "molecules": "chloroquine"
  },
  {
    "caption": "C) Western blot analysis of AGR2 expression in control and TMED2 overexpressing HEK293T cells upon autophagy inhibition with 20 µM and 10 µM chloroquine treatment. Actin (ACT) served as a loading control.",
    "molecules": "chloroquine"
  },
  {
    "caption": "D) HEK293T cells were transfected with control (ev) or TMED2 plasmid and then used in the ERMIT assay with AGR2 WT the presence of gradual amounts of chloroquine.",
    "molecules": "chloroquine"
  },
  {
    "caption": "A. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole cell extracts were then analysed by western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Representative of two independent experiments.",
    "molecules": "HU, Ser, XL413"
  },
  {
    "caption": "B. U2OS cells were either mock treated or treated with 10 μM XL413, 4 mM HU or both for 24 hours before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μM. In the dot plots ~800 comets per each condition were analysed, means are indicated with a red line and their value shown above the plot. Data are from two independent experiments.",
    "molecules": "HU, XL413"
  },
  {
    "caption": "C. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and, where indicated, after 30 minutes 4 mM HU was added for a further 2 hours. Whole cell extracts were analysed by western blotting with the indicated antibodies. Total protein stain (TPS) as a loading control. Representative of two independent experiments.",
    "molecules": "AZD6738, HU, XL413"
  },
  {
    "caption": "D. U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior (pre-treatment) or at the same time (co-treatment) to the addition of HU. Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain (TPS) is displayed as a loading control. Representative of two independent experiments.",
    "molecules": "TPS, HU, XL413"
  },
  {
    "caption": "C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments.",
    "molecules": "Histone"
  },
  {
    "caption": "A. U2OS cells were labelled with EdU for 30 minutes, then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine. At the indicated time points cells were fixed and proteins binding to EdU-labelled DNA captured by DNA mediated chromatin Pull down technique (DmChP). Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material.",
    "molecules": "EdU, thymidine"
  },
  {
    "caption": "B. U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by western blot as above.",
    "molecules": "EdU, HU, XL413"
  },
  {
    "caption": "C. As in panel B but incubation with HU was extended to 24 hours. Black arrow indicates MRE11 electrophoretic-mobility shift.",
    "molecules": "HU"
  },
  {
    "caption": "D. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells incubated for further 24 hours. Extracts prepared from HU treated cells were then incubated in presence or absence of λ-phosphatase. Proteins were analysed by western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic-mobility shift.",
    "molecules": "TPS, HU, XL413"
  },
  {
    "caption": "E. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Inset I-II represent enlargements of selected region of the merged images.",
    "molecules": "HU, XL413"
  },
  {
    "caption": "A. MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test (** p˂0.01",
    "molecules": "CldU, IdU, HU, XL413"
  },
  {
    "caption": "B. U2OS cells were either mock or treated with 10 μM XL413, 50 μM Mirin or both, and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413 and/or Mirin. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test (*p<0.05, ** p˂0.01",
    "molecules": "CldU, IdU, HU, Mirin, XL413"
  },
  {
    "caption": "C. U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413, 50 μM Mirin or both, and labelled with CldU (red). Then 50 nM CPT was added and cells labelled with of IdU (green) for further 30 minutes. A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.",
    "molecules": "CIdU, CldU, IdU, CPT, Mirin, XL413"
  },
  {
    "caption": "D. U2OS cells were transfected with control (siLuc) or MRE11 targeting siRNA (siMRE11). After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU (red) for 30 minutes. Where indicated 50 nM CPT was added and then cells were labelled with IdU (green). A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentileswith data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.",
    "molecules": "CIdU, CldU, IdU, CPT, XL413"
  },
  {
    "caption": "B. Representative western blot of indicated proteins obtained from whole cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413.",
    "molecules": "XL413"
  },
  {
    "caption": "C. Ratios of IdU/CldU tracts length is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analyzed for each condition.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "D. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: student t-test; ns, not significant; **P<0.01, ***P < 0.001",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "F. Frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413. The number of replication intermediates analyzed is indicated in parentheses. Similar results were obtained in two independent experiments (Appendix Table",
    "molecules": "HU, XL413"
  },
  {
    "caption": "G. Amount of ssDNA length at the junction (Red arrows in Figure 4D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork and the value is displayed above the plot",
    "molecules": "ssDNA, XL413"
  },
  {
    "caption": "A. Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU. Scale bar = 5 μM. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*",
    "molecules": "HU"
  },
  {
    "caption": "D-E. U2OS and AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs. 48 hours post transfection cells were either mock-treated or treated with 10 μM XL413 or 10 μM 3MB-PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (*p<0.05, *** p<0.001",
    "molecules": "3MB-PP1, Nocodazole, XL413"
  },
  {
    "caption": "a.  Immunofluorescent images  of skin sections from  PDGFRα-eGFP reporter mice with normal (WT, n=4), inflamed (InvEE, n=4), wounded (d14 post-wounding;  wound, n=4), or tumour-associated (Papilloma, Pap d17 and d30 post-wounding, n=4 per condition) skin. Nuclei  were  stained  with Dapi  (4', 6-diamidino- 2- phenylindole; blue). Dotted line represents  epidermal-dermal   boundary. Scale bars: 50 μm. b. Quantification of the  number of fibroblasts present in different skin conditions (n=4 mice per condition; ** p<0.01; *** p<0.001; **** p<0.0001; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M. ",
    "molecules": "4', 6-diamidino- 2- phenylindole, Dapi"
  },
  {
    "caption": "c.   Herovici staining  of sections of normal (WT), inflamed (InvEE), wound, or tumour-associated (Papilloma) skin. Scale bars: 100 μm.",
    "molecules": "Herovici"
  },
  {
    "caption": "c, d. Incidence of  papilloma  formation (ns: non-significant; Gehan-Breslow-Wilcoxon test) (c) and number of tumours per mouse (d) in wild-type (WT, n=17) and PRSS35-/- (n=22) mice treated with DMBA-TPA (*p=0.0171; Wilcoxon matched-paired rank test). Data represent means ± S.E.M.",
    "molecules": "DMBA, TPA"
  },
  {
    "caption": "d.  Picrosirius red staining of normal  skin, day 14 post-wounding (d14pw) skin or wound-induced papillomas from WT and PRSS35 KO mice. Red colouring indicates thick  fibres, green colouring indicates thin fibres. Scale bars: 200 µm. e. Quantification of picrosirius red staining as total collagen  density (upper panel) and ratio of red versus green picrosirius staining (lower panel). Using QuPath Bioimage analysis software pixels colouring red or green were determined per area. (n≥4 technical replicates per condition) (* p<0.05, ** p<0.01, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M. ",
    "molecules": "picrosirius, Picrosirius red, picrosirius red"
  },
  {
    "caption": "a. Human skin  sections were stained with a PRSS35-specific antibody (red) and counterstained with  Dapi (n=5 per condition). Boxed areas are magnifications as shown in the lower panel and dotted line represents the  epidermal-dermal boundary. Scale bars: 100 μm. b. Quantification of PRSS35 positive signal by  Volocity analysis (n≥4 per condition; ** p<0.01; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M. ",
    "molecules": "Dapi"
  },
  {
    "caption": "(d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm.",
    "molecules": "LPS"
  },
  {
    "caption": "(e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared RBCL (e).",
    "molecules": "LPS"
  },
  {
    "caption": "Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO, Hypoxanthine (Hyx) were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).",
    "molecules": "Hypoxanthine, Hyx"
  },
  {
    "caption": "Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. XO added to macrophages for 24 h. Febuxostat (Feb) was pre-incubated for 30 minutes with XO before addition to macrophages. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).",
    "molecules": "Feb, Febuxostat"
  },
  {
    "caption": "Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO the anti-oxidants N-acetyl-L-cysteine (NAC) and 1-Thioglycerol (1-TG), and heat inactivated (HI) XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).",
    "molecules": "1-TG, 1-Thioglycerol, N-acetyl-L-cysteine, NAC"
  },
  {
    "caption": "(a) Plasma levels of TNF and IL8 from patients with uncomplicated (black circles, n=14) or cerebral (red circles, n=9) malaria correlate with levels of XO-produced ROS detected in each sample. ROS is expressed as fold change over plasma from healthy controls. Uncomplicated malaria, gray circles, cerebral malaria, red circles. Linear regression (a) were performed to determine statistical significance (*p<0.05, **p<0.01).",
    "molecules": "ROS"
  },
  {
    "caption": "(c) Plasma from malaria patients induce macrophages to secrete cytokines, which are inhibited by a XO specific inhibitor. Plasma from a healthy control (HP) and from three patients: P1 (with cerebral malaria), P2 and P3 (both with uncomplicated malaria) was pre-incubated for 30 min with febuxostat (Feb) or alone at 37ºC before addition to macrophages at 1:2 dilution in media for 30 min. Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation. unpaired t-tests (c) were performed to determine statistical significance (*p<0.05, **p<0.01).",
    "molecules": "Feb, febuxostat"
  },
  {
    "caption": "A, B. Western blot (A) and quantitative (B) analyses of MiD49  and Mcl1  degradation in WT and ∆MARCH5 HeLa  cells. Cycloheximide  (CHX: 200 μg/ml; protein synthesis inhibitor); MG132: 20 μM, proteasome  inhibitor. Data information: Data are shown as mean ± SE from three biological repeats (B, Statistics: two-tailed unpaired Student's t-test (B, ; *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.",
    "molecules": "CHX, Cycloheximide, MG132"
  },
  {
    "caption": "Cell survival   analysis of the indicated HeLa  cells treated with Actinomycin D (ActD, 1 μM) or Doxorubicin  (Doxo, 10 μM). Representative images are shown. Cell viability was measured by Cell Titer-Glo. Scale bar, 50 μm. Data information: Data are shown as mean ± SE from three biological repeats D, Statistics: two-tailed unpaired Student's t-test D, ; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "ActD, Actinomycin D, Doxo, Doxorubicin"
  },
  {
    "caption": "Cell survival  analysis of the indicated HeLa  cells. 7 × 105 cells in one well of 6-well plate were treated with ActD  (200 nM) for 4 hours or Doxo  (50 nM) for 2 hours and subsequently cultured in normal medium for 21 days. Surviving colonies were stained  by 0.1% (W/V) methylene blue. Representative images and quantifications of the surviving colony numbers are shown. Data information: Data are shown as mean ± SE from three biological repeats G). Statistics: one-way ANOVA (G); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "ActD, Doxo, methylene blue"
  },
  {
    "caption": "Western blot analysis of BH3-domain containing proteins in the indicated HeLa  cells treated with Doxo  (10 μM). Three proteins with enhanced level in ∆UBXD8 cells (Noxa, Bik, and Bnip3) are highlighted in red.",
    "molecules": "Doxo"
  },
  {
    "caption": "C. Mitochondrial fraction from 293T cells ectopically expressing GASZ, MitoPLD, AIFM1, or SIRT3 were treated in the presence or absence of Triton X-100 and/or Proteinase K, and examined by Western blots.",
    "molecules": "Proteinase K, Triton X-100"
  },
  {
    "caption": "A. Mutant ALX1165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L/165L NCC (black). Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24-hour period. For fluorescent pictures, cells were stained in serum free media containing 3.6µM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 hours using a Keyence BZ-X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200µm. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.*: Significantly different from control NCC (p<0.0001).",
    "molecules": "CellTracker Green, CMFDA"
  },
  {
    "caption": "C. Addition of soluble BMP2 or CV2, a BMP9 antagonist, to the culture medium could partially rescue the migration defect of ALX1165F/165F NCC. At the beginning of the assay, 10, 50, or 100 ng/ml of soluble BMP2, CV2, or a combination of the two at 100 ng/ml each were added to the culture medium, and the cells were monitored over the next 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar: 400µm.",
    "molecules": "CV2"
  },
  {
    "caption": "A. Dissected flatmount wild type and alx1-/- zebrafish larvae craniofacial cartilages after Alcian blue staining, the anterior points to the left of the page in all images. The ventral cartilages appear normal, but the alx1-/- anterior neurocranium (ANC) appears narrow, with the midline element that is derived from the frontonasal NCC being absent. The Meckel's cartilage (arrow, MC) is also diminutive. Scale bar: 200 µm.",
    "molecules": "Alcian blue"
  },
  {
    "caption": "C. Dissected flatmount of zebrafish embryos injected with Alx1DN, after Alcian blue staining. The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium (ANC) and a profound hypoplasia of the Meckel's and ventral cartilages. In the most severely affected zebrafish, a nearly abrogated ANC was observed. Scale bar: 200 µm.",
    "molecules": "Alcian blue"
  },
  {
    "caption": " (B) Bar diagram representation of the relative SUMO conjugation activity of Nse2, full-length Smc5-Nse2, ΔHinge/Smc5-Nse2, ΔHead/Smc5-Nse2 and Arm/Smc5-Nse2 truncation constructs (schematic representation above). Orange bars indicate the presence of ssDNA (virion ϕx174) and red bars absence of ssDNA. Reaction rates were performed at least in three different independent experiments (see Fig EV2). Data values are mean ± s.e.m.; and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. **P<0.01 ",
    "molecules": "ssDNA"
  },
  {
    "caption": " (C) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHead/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer ",
    "molecules": "SYPRO, ssDNA"
  },
  {
    "caption": " (D) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer ",
    "molecules": "SYPRO, ssDNA"
  },
  {
    "caption": " (E) Western blot of the time-course SUMO conjugation reaction in the presence of ssDNA (virion ϕx174) using either Nse2 (left) or Arm/Smc5-Nse2 complex (right). The reactions were run in the presence or absence of cNse4 external substrate. Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2 and pS2, poly-SUMO2) ",
    "molecules": "ssDNA"
  },
  {
    "caption": " (D) Bar diagram representation of the SUMO conjugation rates of activity assays of Arm/Smc5-Nse2 KE mutants in the presence (orange bars) or absence (red bars) of ssDNA (virion ϕx174), relative to wild-type (set to 1). Reactions rates were performed at least in three different independent experiments. Data values are mean ± s.e.m. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. *P<0.05, **P<0.01, ***P<0.001 ",
    "molecules": "ssDNA"
  },
  {
    "caption": " (C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001 ",
    "molecules": "60b linear ssDNA"
  },
  {
    "caption": "E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) and B (F) after 6 d C-deprivation. data are means ± standard deviation (s.d.) of 3 biological replicates.",
    "molecules": "Chlorophyll, protein"
  },
  {
    "caption": "A, Interaction  between ARF2  and PIF5  in the yeast two-hybrid assay. Growth of yeast  cells co-transformed with indicated BD and AD vectors was monitored on quadruple dropout medium (QDO, SD-Ade/-His/-Leu/-Trp) and QDO medium containing X-α-gal.",
    "molecules": "X-α-gal"
  },
  {
    "caption": "C, Chlorophyll  and total cellular protein  content reduction in genotypes shown in A after 6 d C-deprivation. Color legends in C are the same as in B. data are means ± s.d. of 3 biological replicates.",
    "molecules": "Chlorophyll, protein"
  },
  {
    "caption": "F, Chlorophyll  and total cellular protein  content reduction in genotypes shown in D after 6 d C-deprivation. Color legends in F are the same as in E. data are means ± s.d. of 3 biological replicates.",
    "molecules": "Chlorophyll, protein"
  },
  {
    "caption": "H, Chlorophyll  and total cellular protein  content reduction in genotypes shown in G after 12 d C-deprivation. data are means ± s.d. of 3 biological replicates.",
    "molecules": "Chlorophyll, protein"
  },
  {
    "caption": "J, Chlorophyll  and total cellular protein  content reduction in genotypes shown in I after 8 d C-deprivation. data are means ± s.d. of 3 biological replicates.",
    "molecules": "Chlorophyll, protein"
  },
  {
    "caption": "E and F, Chlorophyll  and total cellular protein  content reduction in genotypes shown in A (E) after 6 d and 9 d C-deprivation and in genotypes shown in B (F) after 6 d and 11 d C-deprivation. Color legends in E are the same as in A. Color legends in F are the same as in B. data are means ± s.d. of 3 biological replicates.",
    "molecules": "Chlorophyll, protein"
  },
  {
    "caption": "B In-vitro colony forming assays comparing RAD51-overexpressing and control HGSOC cell line, Caov-3, after treatment with increasing doses of carboplatin. Mean and SD of four biological replicates (left) a representative experiment (right). P-value for a comparison between cell lines for each drug treatment condition is indicated above the bars. T-test.",
    "molecules": "carboplatin"
  },
  {
    "caption": "Whole cell extracts of High Five insect cells individually expressing FLAG-SHLD3, Strep-RIF1, and 6xHis-REV7 through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for REV7 or the Strep or FLAG epitopes. Results are representative of three biologically independent experiments. IB: immunoblot, FL: full-length. For SHLD3, N and C correspond to residues 1-125 and 126-250, respectively.",
    "molecules": "Strep, streptactin"
  },
  {
    "caption": "Representative micrographs of the LacR-FokI assay to evaluate DNA damage recruitment of eGFP-tagged SHLD3 variants after induction of LacR-FokI expression.",
    "molecules": "DNA"
  },
  {
    "caption": "Whole cell extracts of High Five insect cells individually expressing the wild-type (WT) or indicated alanine substitution FLAG-SHLD3C (residues 126-250) variants and Strep-RIF1N (residues 1-980) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.",
    "molecules": "streptactin, alanine"
  },
  {
    "caption": "Quantification of LacR/LacO assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to LacO arrays in U2OS 2-6-3 cells by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 134, 125, 120, 130, 104, 129, 117 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). EV: empty vector. Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ***: P = 0.0004. **: P = 0.002. *: P = 0.02. ns: P > 0.05.",
    "molecules": "alanine"
  },
  {
    "caption": "Quantification of LacR-FokI assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to sites of DNA double-strand breaks induced by mCherry-LacR-FokI in U2OS 2-6-3 cells. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent means (n = 103, 136, 159, 151, 139, 145, 140 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ns: P > 0.05.",
    "molecules": "alanine"
  },
  {
    "caption": "Representative micrographs of immunofluorescence experiments (from 3 biologically independent experiments) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3-KO cells. 3xFLAG-SHLD3-complemented RPE SHLD3-KO cells were treated with 200 nM aphidicolin (Aphi) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells. Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler. Pearson's correlation coefficients (PCC) were calculated for pixels within each cyclin A-negative, 53BP1 body-positive nucleus between the 53BP1 and FLAG channels. Each point represents the PCC value of an individual nucleus. Bars represent means. (n = 120, 130 for EV, SHLD3 from three biologically independent experiments). Analysis was performed using two-tailed Mann-Whitney test. ****: P < 0.0001.",
    "molecules": "Aphi, aphidicolin"
  },
  {
    "caption": "Representative micrographs of immunofluorescence experiments analyzing localization of FLAG-SHLD3 alanine substitution variants stably expressed in RPE SHLD3-KO cells by lentivirus transduction. Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells.",
    "molecules": "alanine, Aphi"
  },
  {
    "caption": "Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3-KO cells with or without 24-hour 200 nM Aphi treatment. Bars represent means ± s.d. (n = 3 biologically independent experiments with ≥ 30 nuclei imaged each).",
    "molecules": "Aphi"
  },
  {
    "caption": "Whole cell extracts of High Five insect cells individually expressing the FLAG-SHLD3C (residues 126-250) and the indicated alanine substitution Strep-RIF1 (residues 1-980) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.",
    "molecules": "Strep, streptactin, alanine"
  },
  {
    "caption": "Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. DNA damage was induced in U2OS 2-6-3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies. Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1-labeled irradiation stripe and the average nuclear intensity. Only nuclei containing RIF1 stripes are quantified. Bars represent means (n = 206, 215 for RIF1WT, RIF1D28N from 2 biologically independent experiments). Analysis was performed using Welch's t-test. ****: P < 0.0001.",
    "molecules": "DNA"
  },
  {
    "caption": "C The indicated 35S−labelled in vitro−translated GFP−tagged constructs were incubated with GST, GST−LC3B or GST−GABARAP conjugated to glutathione Sepharose, and their binding was evaluated by autoradiography (ARG). 5% of the in vitro−translated protein (arrow head) used was loaded. Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments.",
    "molecules": "35S"
  },
  {
    "caption": "B 35S−labelled in vitro−translated GFP−ALFY (aa 2981-3526) wild−type and different LIR mutants were incubated with GST−GABARAP or −LC3C and binding evaluated by ARG. 10 and 2% of the in vitro−translated proteins used were loaded to illustrate binding affinity. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.",
    "molecules": "35S"
  },
  {
    "caption": "E 35S−labelled in vitro−translated GFP or GFP−Bchs (aa 3326-3341) were incubated with GST or GST−dAtg8a and binding evaluated by ARG. 5% of the in vitro−translated protein used was loaded. Ponceau staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.",
    "molecules": "35S"
  },
  {
    "caption": "G 35S‐labelled in vitro‐translated GFP‐ALFY (aa 2981-3526) wild‐type and K3343A/D3344A/Y3351A mutant were incubated with GST‐LC3B and GABARAP and binding evaluated by ARG. 10% of the in vitro‐translated proteins used was loaded. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.",
    "molecules": "35S"
  },
  {
    "caption": "A HeLa FlpIn GFP−GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti−ALFY antibodies. Scale bar, 10 μm.",
    "molecules": "amino acid, MG132"
  },
  {
    "caption": "D HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX−100−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.",
    "molecules": "bafilomycin A1, puromycin, TX−100"
  },
  {
    "caption": "E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X−100 (TX−100)−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.",
    "molecules": "bafilomycin A1, puromycin, Triton X−100, TX−100"
  },
  {
    "caption": "B. Flow cytometry for Bu-1a expression in wild type cells with (GAA)n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the allele Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1)",
    "molecules": "GAA"
  },
  {
    "caption": "C, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.",
    "molecules": "GAA"
  },
  {
    "caption": "Human PrimPol, or mutants, tagged with YFP were expressed in primpol cells harbouring (GAA)10 sequence in the BU-1A locus. Bu-1a and YFP double positive cells were sorted and expanded for 2 weeks, and then analysed for Bu-1a expression variants. For each complementation, at least two independently derived clones were subjected to fluctuation analysis. As previously observe expression of hPrimPol[AxA] and hPrimPol[ZfKO] is deleterious and unstable. Cells expressing these mutations and remaining YFP-positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis. Pooled results from at least three independent fluctuation analyses are represented with mean ±SD indicated with red bar and whiskers. Statistical significance: **** p < 0.0001, *** p<0.001, * p ≤ 0.05, ns = not significant; Kruskal-Wallis test",
    "molecules": "GAA"
  },
  {
    "caption": "A. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA)10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significant",
    "molecules": "GAA"
  },
  {
    "caption": "B. DNA:RNA hybrids in primpol BU-1A(GAA)10:GgRNase H and primpol BU-1A(GAA)10:hPrimPol. An unpaired t-test on three biological replicates was used to compare differences to primpol BU-1A(GAA)10 for each matched amplicon. The bar represent the mean and whiskers the SD. *** p ≤ 0.001, ** p ≤ 0.01, ns = not significant",
    "molecules": "GAA"
  },
  {
    "caption": "C. Overexpression of chicken RNase H1 prevents (GAA)10 induced BU-1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU-1A (GAA)10 clones. One-way ANOVA was used to calculate the significance of differences in BU-1 instability between primpol BU-1A ΔG4 and other cell lines. **** p ≤ 0.0001, ns = not significant",
    "molecules": "GAA"
  },
  {
    "caption": "A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication",
    "molecules": "Hoechst 33342, DNA"
  },
  {
    "caption": "B. Bu-1a fluctuation analysis of two independently derived primpol BU-1A(GAA)10: Gg RNase H1-YFP-geminin degron clones. Since the expression of the RNase H1-YFP-geminin degron construct is not stable (unlike the RNase H1-YFP construct without the degron), Bu-1a expression was assessed separately in the YFP +ve and YFP -ve cells within each clone. Statistical differences calculated the Kruskal-Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant",
    "molecules": "GAA"
  },
  {
    "caption": "C. DRIP-qPCR for R-loops around the engineered +3.5 (GAA)10 repeat in BU-1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU-1 DRIP signal was normalised to -0.5 kb amplicon in G1-arrested cells (t = 0 h) Black: wild type; red: primpol. Error bars = SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05",
    "molecules": "GAA"
  },
  {
    "caption": "E. Validation of analysis of nascent DNA:RNA hybrid formation in BU-1 locus. Enrichment of 4SU labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** p ≤ 0.01, * p ≤ 0.05, ns = not significant; unpaired t-test",
    "molecules": "4SU, DNA, RNA"
  },
  {
    "caption": "Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates.",
    "molecules": "Docetaxel, Gemcitabine"
  },
  {
    "caption": "Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicates",
    "molecules": "DMSO, Docetaxel"
  },
  {
    "caption": "Representative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicates",
    "molecules": "DMSO, Docetaxel, Gemcitabine"
  },
  {
    "caption": "Quantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.",
    "molecules": "ATP"
  },
  {
    "caption": "1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human b-Raf kinase (WT or V600E) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.",
    "molecules": "ATP"
  },
  {
    "caption": "1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.",
    "molecules": "ATP"
  },
  {
    "caption": "2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib.",
    "molecules": "dabrafenib, vemurafenib"
  },
  {
    "caption": "1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794",
    "molecules": "Ku0063794, rapamycin"
  },
  {
    "caption": "Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2",
    "molecules": "Phosphopeptide"
  },
  {
    "caption": "Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600E",
    "molecules": "Phosphopeptide"
  },
  {
    "caption": "Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTOR",
    "molecules": "Phosphopeptide"
  },
  {
    "caption": "Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of n=3 (RTKi in G (DMSO in G",
    "molecules": "DMSO"
  },
  {
    "caption": "Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in and ERKi in H)",
    "molecules": "DMSO"
  },
  {
    "caption": "Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki in",
    "molecules": "DMSO"
  },
  {
    "caption": "Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki in",
    "molecules": "DMSO"
  },
  {
    "caption": "Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO",
    "molecules": "DMSO"
  },
  {
    "caption": "Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of Docetaxel (DMSO",
    "molecules": "DMSO, Docetaxel"
  },
  {
    "caption": "Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in C: Vehicle n=8, PI3Ki n=16, Docetaxel n=16, Combination n=8 Pvalues represent the mean of all the time points",
    "molecules": "Docetaxel"
  },
  {
    "caption": "Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in D: Vehicle n=16, PI3K n=16, Gemcitabine n=4, Combination n=4. Pvalues represent the mean of all the time points",
    "molecules": "Gemcitabine"
  },
  {
    "caption": "Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in G: Vehicle n=15, PI3Ki n=10, Docetaxel n=8, Combination n=4",
    "molecules": "Docetaxel"
  },
  {
    "caption": "Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in H: Vehicle n=15, PI3K n=10, Gemcitabine n=8, Combination n=6.",
    "molecules": "Gemcitabine"
  },
  {
    "caption": "Cell counting experiments analyzing the response to trastuzumab (50 nM, 5 days) of naïve BT474 as well as the resistant BTRH pool and the BTRH#10 clone.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "Western blot analyses of the expression of HER2, pHER2, HER3 and pHER3 in BT474, BTRH and BTRH#10 cells. HER2 and HER3 were immunoprecipitated from 100 or 500 μg of cell extracts, respectively, and blots probed with anti-HER2, anti-HER3 or anti-phosphotyrosine. Calnexin was used as a loading control. Numbers below the blots show the quantitation of the signal of each band, referred to BT474 cells (intensity of band in BT474 cells=1).",
    "molecules": "phosphotyrosine"
  },
  {
    "caption": "Response of BT474, BTRH and BTRH#10 to EV20/MMAF. Cells treated with the indicated doses of EV20/MMAF for 5 days were counted.",
    "molecules": "MMAF"
  },
  {
    "caption": "Action of the nude EV20 antibody or EV20/MMAF (10 nM each, 5 days) on BT474, BTRH and BTRH#10 cells.",
    "molecules": "MMAF"
  },
  {
    "caption": "Comparison of the effect of MMAF and EV20/MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs.",
    "molecules": "MMAF"
  },
  {
    "caption": "Response to trastuzumab (50 nM, 5 days) of BT474, BTRH, patient derived cells (PDX118) and their trastuzumab-resistant derivatives TR1 and TR2.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "Effect of EV20/MMAF on BT474, BTRH, PDX118, TR1 and TR2. Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.",
    "molecules": "MMAF"
  },
  {
    "caption": "Effect of trastuzumab (50 nM, 5 days) in HER2 positive breast cancer cell lines (SKBR3, BT474, BTRH, MDA-MB-361, HCC1419, HCC1569 and HCC1954). The blue line indicates the threshold (established from BTRH) used to consider a cell line resistant to trastuzumab.",
    "molecules": "trastuzumab"
  },
  {
    "caption": "Effect of EV20/MMAF on the cell lines used in (D). Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.",
    "molecules": "MMAF"
  },
  {
    "caption": "Effect of HER3 knockdown on the antiproliferative action of EV20/MMAF analyzed by cell counting after 5 days of treatment with the ADC.",
    "molecules": "MMAF"
  },
  {
    "caption": "Internalization of EV20/MMAF (10 nM) at indicated times in BT474 and BTRH cells. Scale bar: 20 µm. HER3 staining: red; DAPI: blue.",
    "molecules": "DAPI, MMAF"
  },
  {
    "caption": "Internalization of biotin-S-S-EV20/MMAF in BT474 and BTRH cells. These cell lines were pulsed with 10 nM of biotin-S-S-EV20/MMAF for 1 hour at 4°C and chased at 37°C at the indicated times. Subsequently, surface accessible biotin was cleaved, cells were lysed, and extracts precipitated with streptavidin-sepharose. EV20/MMAF was analyzed by Western blotting using anti-human-HRP. Quantitative plot of the Western studies shown in (B). Data are presented as the mean + SD of two independent experiments.",
    "molecules": "sepharose, biotin, MMAF, streptavidin"
  },
  {
    "caption": "Co-localization (yellow) between EV20/MMAF and LAMP1 at the indicated incubation times with EV20/MMAF in BT474 and BTRH cells. Scale bar: 5 µm. HER3 staining: red; LAMP1: green, DAPI: blue.",
    "molecules": "DAPI, MMAF"
  },
  {
    "caption": "Western blot analyses of the effect of EV20/MMAF (10 nM) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC. The same Western was probed for HER2 or HER3 expression using two differently labelled species-specific secondary fluorescent antibodies. Quantitative representation of the Western studies shown in (E).",
    "molecules": "MMAF"
  },
  {
    "caption": "Cell cycle analysis by flow cytometry of BT474 and BTRH cells (50,000 events) treated with EV20/MMAF (10 nM) for the indicated times.",
    "molecules": "MMAF"
  },
  {
    "caption": "BT474 and BTRH cells were treated for the indicated days with EV20/MMAF (10 nM), lysed and the amount of different proteins analyzed by Western. Numbers below the blots show the quantitation of the signal of each band, referred to time 0.",
    "molecules": "MMAF"
  },
  {
    "caption": "Action of EV20/MMAF on mitotic spindle formation. BT474 and BTRH cells were treated (10 nM, 48 hours), fixed, stained for β-tubulin (green) and DAPI (blue). Scale bar: 7.5 μm.",
    "molecules": "DAPI, MMAF"
  },
  {
    "caption": "Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20/MMAF (10 nM, 48 hours) in BT474 and BTRH cells. Bars represent the mean + SD of two independent experiments, calculated as follows: (number of mitotic cells with abnormal spindles/total number of mitotic cells) ×100 (%).",
    "molecules": "MMAF"
  },
  {
    "caption": "Cell death induction by EV20/MMAF (10 nM) analyzed by double annexin V and PI staining in BT474 and BTRH cells (50,000 events).",
    "molecules": "MMAF, PI"
  },
  {
    "caption": "Time-course analysis of the effect of EV20/MMAF (10 nM) on cleaved-caspase-3, analyzed by Western blotting in BT474 and BTRH cells. GAPDH was used as a loading control. Samples with intensity values of 1 were used as reference.",
    "molecules": "MMAF"
  },
  {
    "caption": "Fluorimetric analyses of caspase-3 activity in BT474 and BTRH cells treated with 10 nM EV20/MMAF. Data show the mean + SD of triplicates of an experiment that was repeated twice.",
    "molecules": "MMAF"
  },
  {
    "caption": "In vivo effect of EV20 (10 mg/kg, n=4), untreated control (PBS, n=4) and EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4) on tumor growth in mice injected with BTRH cells. Mice were treated when tumors reached a volume of 500 mm3. Data are represented as mean + SEM.",
    "molecules": "MMAF"
  },
  {
    "caption": "In vivo effect of single-dose EV20 (10 mg/kg, n=3) untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with BTRH#10 cells. Data are represented as mean + SEM. In vivo effect of single-dose EV20 (10 mg/kg, n=4), untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with HCC1954 cells. Data are represented as mean + SEM.",
    "molecules": "MMAF, trastuzumab"
  },
  {
    "caption": "Analysis of pHistone H3, HER3, pHER3 and cleaved caspase-3 in tumors of mice injected with BTRH and treated (T) or not (C) with EV20/MMAF (3.3 mg/kg for 24 hours or two weeks). Numbers below the blots show the quantitation of the signal of each band, referred to that of a control untreated tumor.",
    "molecules": "MMAF"
  },
  {
    "caption": "Long-term monitoring of mice injected with BTRH cells and treated with EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4). Where indicated, mice were treated with EV20/MMAF. Data were represented as mean + SD of the different mice groups.",
    "molecules": "MMAF"
  },
  {
    "caption": "Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to neratinib (BTRN#5 and BTRN#24)",
    "molecules": "MMAF, neratinib"
  },
  {
    "caption": "Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of (B) cells resistant to neratinib",
    "molecules": "neratinib"
  },
  {
    "caption": "Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to lapatinib (BTRL, BTRL#109 and BTRL#3)",
    "molecules": "lapatinib, MMAF"
  },
  {
    "caption": "Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to lapatinib",
    "molecules": "lapatinib"
  },
  {
    "caption": "Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to T-DM1 (BT-TDM1R#1 and BT-TDM1R#6).",
    "molecules": "MMAF, T-DM1"
  },
  {
    "caption": "Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to TDM1.",
    "molecules": "TDM1"
  },
  {
    "caption": "A. A549 cells were transfected with 2 μg of poly(I:C) for 6 hrs and then infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment) or infected with ZIKV first for 6 hrs and then transfected with poly(I:C) for 12 hrs (Post-treatment). Levels of ZIKVgenomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. ***P<0.001 (Student's t-test), N=3",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "B. A549 cells infected with ZIKV (MOI=3) for 6 hrs were transfected with 2 μg poly(I:C) for 12 hrs after which levels of ifn-",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "C. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "D. HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins. Sixteen hours later they were transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test), N=4",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "E. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "F. HEK293T cells were transfected with plasmids encoding individual ZIKV proteins, the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 0.4 μg of poly(I:C). Twenty-four hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "B. A549 cells were infected with ZIKV (MOI=5) for 24 hrs and then treated with DMSO, MG132 (20 µM) or epoxomicin (Epox) (400 nM) for 12 hrs. Cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.",
    "molecules": "DMSO, epoxomicin, MG132"
  },
  {
    "caption": "C. A549 cells were transfected with plasmids encoding FLAG-tagged ZIKVNS5 or FLAG alone for 24 hrs and then treated with epoxomicin (400 nM) for 24 hrs. Cells were harvested and process for immunoprecipitation (IP) using a mouse anti-FLAG or anti-myc antibody followed by SDS-PAGE and Immunoblotting. Representative panels are shown. The experiment was repeated three times.",
    "molecules": "epoxomicin"
  },
  {
    "caption": "Changes in fibroblast proliferation and collagen deposition during dermal development in mice. Immunofluorescence staining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin at indicated developmental time points (upper panel). Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points (lower panel).",
    "molecules": "collagen, Picrosirius red"
  },
  {
    "caption": "Quantification of fibroblast proliferation (left; n= 4 E16.5, P10; n= 3 E10.5, E18.5, P2, P21, P44, P50; n= 2 E17.5, P0 biological replicates) and collagen density (right; n= 4 E16.5, P2, P6, P10, P14, P16, P21, P50; n= 3 E10.5, E18.5, P2, P21, P44, P50 biological replicates) with age.",
    "molecules": "collagen"
  },
  {
    "caption": "Changes in dermal cell proliferation and collagen deposition in human skin. (G) Immunofluorescence staining for Vimentin (red) and Ki67 (green) of back skin at indicated time points (upper panel). Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points (lower panel). (H) Quantification of proliferating cells in the upper and lower dermis (n=3 biological replicates per time point). (I) Quantification of dermal cell proliferation (Ki67+) (left) and collagen density (right) with age (n= 3 biological replicates per time point). Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent, non-proliferative state before collagen was efficiently deposited. Data shown are means ±s.d. Nuclei were labelled with DAPI (blue in B,E,G). Scale bars, 100µm. wk, week.",
    "molecules": "collagen, DAPI, Picrosirius red"
  },
  {
    "caption": "Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in (triangle) and outside (circle) collagen gels for up to 5 days. Cell number was measured by CellTiterGlo assay at indicated time points (n= 3 technical replicates of one representative experiment repeated 3 times).",
    "molecules": "collagen"
  },
  {
    "caption": "Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days (triangle). Controls were kept on TCP for 3 days before plating (circle). Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown (n= 3 technical replicates).",
    "molecules": "collagen"
  },
  {
    "caption": "ECM degradation in DED organotypic cultures by collagenase treatment. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures at indicated time points after collagenase treatment. Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment (n= 8 section per condition of one representative experiment repeated two times). Note that fibroblasts around the injection sides start to proliferate after 48 hours. Nuclei were labelled with DAPI (blue) (D,F). Scale bars, 100µm (D,F). Data shown are means ± s.d. Fb, fibroblasts; PD, post digestion; TCP, tissue culture plastic; h, hours.",
    "molecules": "DAPI"
  },
  {
    "caption": "Live imaging of adult PDGFRαH2BEGFP mouse back skin. Representative time-lapse images of adult PDGFRαH2BEGFP (green) dermis with collagen shown as second harmonic generation (SHG) in purple at indicated imaging time points. Scale bar, 100 µm.",
    "molecules": "collagen"
  },
  {
    "caption": "Fibroblast activation during wound repair. Immunofluorescence staining for α−sma (red) of PDGFRαH2BEGFP (green) wound sites at indicated post wounding (PW) times Nuclei were labelled with DAPI (blue)",
    "molecules": "DAPI"
  },
  {
    "caption": "Changes in fibroblast density, proliferation and collagen deposition during wound repair. Immunostaining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin sections at indicated wound healing time points. Nuclei were labelled with DAPI",
    "molecules": "collagen, DAPI"
  },
  {
    "caption": "Changes in fibroblast density, proliferation and collagen deposition during wound repair. Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).",
    "molecules": "collagen"
  },
  {
    "caption": "Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding.",
    "molecules": "Picrosirius red"
  },
  {
    "caption": "Quantification of fibroblast proliferation (left; n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates) and collagen density (right; n= 4 PW0, PW2; n= 3 PW4, PW7, PW10, PW14; n= 2 PW21 biological replicates) inside (A) and outside (B) the wound bed over time.",
    "molecules": "collagen"
  },
  {
    "caption": "Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. Representative wound bed Z-stack stitched maximum projections of the entire wound bed (upper panel) showing fibroblasts (green) and collagen with second harmonic generation (SHG) in purple at indicated time points after wounding and before live imaging. Dotted line indicates wound edge and boxed area indicates the position of the magnified area below.",
    "molecules": "collagen"
  },
  {
    "caption": "(B-C) Western blot analysis (B) and densitometric quantification (C) of OTUD1 in aortas of control and Ang II-induced mice. β-actin was used as the loading control (n=5 biological replicates). (D) mRNA levels of OTUD1 in aortas of control and Ang II-induced mice were determined by RT-qPCR. The values were normalized to Rn18s (n=5 biological replicates).",
    "molecules": "Ang II"
  },
  {
    "caption": "(G-H) Time-course of OTUD1 induction in response to Ang II in HUVECs. HUVECs were exposed to 1 μM Ang II for indicated time. Western blot analysis (G) and densitometric quantification (H) of OTUD1 were shown (n = 3 biological replicates). GAPDH was used as the loading control.",
    "molecules": "Ang II"
  },
  {
    "caption": "(A) Representative images of H&E staining of aortic sections of the mice from four groups (scale bar=25 μm). E, endothelium; M, media; A, adventitia. (B) The quantification of arterial wall thickness in panel A; n=5 biological replicates. (C) Representative images of Masson's Trichrome staining of aortic sections of the mice (scale bar=50 μm). (D) The quantification of collagen deposition in panel C; n=5 biological replicates.",
    "molecules": "Masson's Trichrome"
  },
  {
    "caption": "(B) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.",
    "molecules": "Ang II"
  },
  {
    "caption": "(E) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.",
    "molecules": "Ang II"
  },
  {
    "caption": "(G) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (H) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and Flag-OTUD1 or Flag-OTUD1(C320S), and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (I) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, Flag-OTUD1, and HA-Ub, HA-Ub-K48 (K48 only), or HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "(G) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "(H) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3 and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. IgG, immunoglobulin G.",
    "molecules": "MG132"
  },
  {
    "caption": "(A-B) Representative images (A) of H&E staining of aortic sections (scale bar=25 μm) and quantification of arterial wall thickness (B); n=5 biological replicates. E, endothelium; M, media; A, adventitia. (C-D) Representative images (C) of Masson's Trichrome staining of aortic sections (scale bar=25 μm) and quantification of collagen deposition (D); n=5 biological replicates.",
    "molecules": "Masson's Trichrome"
  },
  {
    "caption": "A. Representative pseudo colour images of sypHy    fluorescence in WT and BsnGT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20Hz (in the presence of bafilomycin A1) and upon application of NH4Cl to visualise SVs  that were refractory to electrical stimulation. B. Average traces of the normalised fluorescence change (ΔF/F0) of sypHy    in WT (black) and  BsnGT (red) neurons  as described in A. Intensities were normalized to the peak of NH4Cl response. C. Quantifications of mean RRP   fraction in WT and BsnGT. D. Frequency distribution histograms of the response amplitudes of 1050 individual synaptic  puncta (from 6 independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in BsnGT. E. Quantifications of mean TRP   fraction in WT and BsnGT. F. Frequency distribution histograms of synaptic  response amplitudes in BsnGT and WT neurons  stimulated with 900 AP. Note the shift in distribution between genotypes. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) In D and F, the data of 1050 synapses per genotype were processed. Data is obtained from 6 (A-F) independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 2 µm in A",
    "molecules": "NH4Cl, bafilomycin A1"
  },
  {
    "caption": "J. Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining  were applied as in G. K. Quantification of normalized IF  of Syt1Ab uptake on experiments from J. L. Quantification of fraction of active excitatory  and inhibitory synapses in J. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in K depict IF  in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images J). Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in J.",
    "molecules": "KCl"
  },
  {
    "caption": "A, B. Representative pseudo colour images (A) and average traces (B) of sypHy  fluorescence plotted for WT and BsnGT neurons  treated with a CDK5  inhibitor roscovitine  (rosc, 100µM, gray and pink trace) or vehicle (black and red trace) for 30 minutes before stimulation with 40 and 900 APs in the presence of  bafilomycin A. C, D. Plots show mean values of RRP  (C) and TRP  (D) fraction for both genotypes before and after treatment. E. Roscovitine  treatment has a significantly higher effect on RRP  and TRP  in BsnGT neurons compared to WT. Data information: n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with Tukey´s multiple comparison test (C, D) and by Student's t-test (E) *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.",
    "molecules": "bafilomycin A, rosc, roscovitine, Roscovitine"
  },
  {
    "caption": "A. Representative immunoblot  with antibody against CDK5-dependent pSer551 of Syn1  (pSer551Syn1) and total Syn1  and total protein stain  used as loading control for quantification of Syn1  on P2 fractions prepared from hippocampal  tissue lysates of WT and BsnGT mice. B, C. Quantification of pSer551Syn1 / Syn1  ratio (in B) and total Syn1  (in C) abundance on blots  from A. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in B and C For statistics, student's t-test in B and C were used, *p≤0.05, **p < 0.01, ***p< 0.001.",
    "molecules": "Ser"
  },
  {
    "caption": "E. Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained  for pSer551Syn1  and total Syn1. F. Quantification of IF  intensity of pSer551Syn1 in synapses  from WT,  BsnGT, and WT cells treated with roscovitine. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images (E, quantified from two (F) independent cultures per condition. For statistics, one-way ANOVA with Sidak´s multiple comparisons test in F were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in",
    "molecules": "roscovitine, Ser"
  },
  {
    "caption": "G. Representative images of 21 DIV hippocampal WT and BsnGT neurons  at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h) H. Quantification of experiment in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images , G) quantified from four (H) independent cultures per condition. For statistics two-way ANOVA with Tukey´s multiple comparisons test in H were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in G.",
    "molecules": "BIC, TTX"
  },
  {
    "caption": "A, B. Representative pseudo colour images (A) and average traces (B) of sypHy  fluorescence plotted from WT and  BsnGT hippocampal neurons treated with calcineurin  inhibitor FK506. Data information: Scale bar is 2 µm.",
    "molecules": "FK506"
  },
  {
    "caption": "E. Representative Western blot  on P2 fractions prepared from hippocampal  tissue of WT and BsnGT mice  probed with antibodies against SNAP25 phosphorylated  onThr138 and total SNAP25. F. Quantification of blot  in E shows lower pThr138SNAP25 to total SNAP25 IF  ratio in BsnGT compared to WT. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB  in F Statistical significance was assessed using Student's t-test in F, significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.",
    "molecules": "Thr"
  },
  {
    "caption": "A, B. Representative pseudo colour images (A) and average traces (B) of sypHy  fluorescence plotted for WT and  BsnGT neurons  without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin  (gray and pink) or PKA  inhibitor H89  (light grey and orange). Data information: The scale bar is 2 µm in A",
    "molecules": "forskolin, H89"
  },
  {
    "caption": "E. Representative immunoblot  with antibody against pSer9Syn1 and total Syn1  and total protein stain  of hippocampal  P2 fraction from WT and BsnGT mice  prepared in the presence of KN 93 to isolate PKA-dependent regulation of pSer9Syn1. F, G. Quantification of pSer9Syn1/Syn1  ratio (in F) and Syn1  (in G) on blots  from E. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G, The statistical significance was assessed in F and G using Student's t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001.",
    "molecules": "KN 93, Ser"
  },
  {
    "caption": "H. Representative images of  hippocampal neurons from WT and  BsnGT mice  labelled with antibodies against pSer9Syn1 and total Syn1. Neurons  were pre-treated (4 µM, 1hr) with KN93 to isolate PKA-dependent phosphorylation  and then treated in addition with forskolin  or H89  before fixation. I. Quantification of staining  in H. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I. The statistical significance was assessed in I using two-way ANOVA with Tukey´s multiple comparison test as *p≤0.05, **p < 0.01, ***p< 0.001. The scale bar is 5 µm in H.",
    "molecules": "forskolin, KN93, H89, Ser"
  },
  {
    "caption": "A, B. Representative pseudo colour images (A) and average traces (B) of sypHy  fluorescence plotted for untreated WT and BsnGT neurons  (black and red) and cultures treated with of 6-Bnz-cAMPS (50 µM, 1 hour; gray and pink) and rolipram  (1 µM, 30 min, light grey and orange). Data information: Scale bar is 2µm.",
    "molecules": "6-Bnz-cAMPS, rolipram"
  },
  {
    "caption": "H. Quantification of pSer145PDE4B to the total PDE4B  ratio from experiment in I. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student's t-test in H and is given as: *p≤0.05, **p < 0.01, ***p< 0.001.",
    "molecules": "Ser"
  },
  {
    "caption": "I. Representative immunoblot  with antibody against CDK5-dependent pSer145PDE4B and total PDE4B  on hippocampal  tissue from WT and BsnGT animals.",
    "molecules": "Ser"
  },
  {
    "caption": "A-B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio.",
    "molecules": "bafilomycin A1, rapamycin"
  },
  {
    "caption": "C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.",
    "molecules": "rapamycin"
  },
  {
    "caption": "A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated.",
    "molecules": "Ca2+, ATP, EGTA, ionomycin, rapamycin, thapsigargin"
  },
  {
    "caption": "C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA.",
    "molecules": "Ca2+, DMSO, rapamycin"
  },
  {
    "caption": "D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3.",
    "molecules": "Ca2+, IP3, rapamycin"
  },
  {
    "caption": "E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.",
    "molecules": "Ca2+, IP3, rapamycin"
  },
  {
    "caption": "A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4).",
    "molecules": "Ca2+, rapamycin"
  },
  {
    "caption": "C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4).",
    "molecules": "rapamycin"
  },
  {
    "caption": "D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time.",
    "molecules": "Ca2+, A23187, rapamycin"
  },
  {
    "caption": "E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).",
    "molecules": "Ca2+"
  },
  {
    "caption": "F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.",
    "molecules": "Ca2+"
  },
  {
    "caption": "A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3).",
    "molecules": "DMSO, Dox, doxycycline, rapamycin"
  },
  {
    "caption": "B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.",
    "molecules": "Ca2+, ATP, doxycycline, EGTA, rapamycin"
  },
  {
    "caption": "B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.",
    "molecules": "Ca2+, doxycycline, EGTA, Iono, ionomycin, rapamycin"
  },
  {
    "caption": "D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.",
    "molecules": "ATP, Dox, Iono"
  },
  {
    "caption": "2+-dependent. Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO, 1 µM rapamycin (Rapa), 10 µM BAPTA-AM or both. One hour before harvesting, 100 nM bafilomycin A1 was added. Left: representative Western blots; right: quantification of the LC3-II/GAPDH ratio (n = 6). * p<0.05, repeated measurements ANOVA.",
    "molecules": "bafilomycin A1, BAPTA-AM, rapamycin"
  },
  {
    "caption": "Immunoblotting of c-MYC in melanoma cells treated for 72 hr with siASNS, L-Asn, or both (G)",
    "molecules": "L-Asn"
  },
  {
    "caption": "A. Immunoblotting of c-MYC in A375 cells treated with NT-siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints (left). Plot showing relative protein levels of c-MYC (y-axis) as a function of time lapsed (x-axis) in the left panel (right).",
    "molecules": "cycloheximide"
  },
  {
    "caption": "Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, PLX-4032, or both (D).",
    "molecules": "PLX-4032"
  },
  {
    "caption": "E. Immunoblotting of indicated proteins in A375 cells cultured in L-Asn and treated with L-A'ase, PLX-4032, or both for 72 hr.",
    "molecules": "L-Asn, PLX-4032"
  },
  {
    "caption": "F. A375 cells were treated with siASNS, SCH772984, or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3-β.",
    "molecules": "SCH772984"
  },
  {
    "caption": "C. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, sicMYC, PLX-4032, or a combination of siASNS and sicMYC, or siASNS and PLX-4032.",
    "molecules": "PLX-4032"
  },
  {
    "caption": "Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, BCH, or both (H)",
    "molecules": "BCH"
  },
  {
    "caption": "D: Flow cytometric analysis of Esrrb and Nanog fluorescent reporter expression in SSEA1+ TNG E-tdT ESCs cultured for three days in GMEMβ/FCS/LIF with or without puromycin.",
    "molecules": "puromycin"
  },
  {
    "caption": "E: Immunofluorescent detection and quantification of total Nanog and Esrrb protein in E14Tg2a ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental E14Tg2as are shown as a reference. The red lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid.",
    "molecules": "retinoic acid"
  },
  {
    "caption": "B,C: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured in N2B27/LIF/2i and released in GMEMβ/FCS/LIF for 1, 2 or 3 days. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. (B) The red vertical and horizontal lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid. The red trendline represents the moving average of the data distribution at day 3. Representative of two independent experiments each including at least 1000 nuclei. (C) Histogram plots of the data presented in B.",
    "molecules": "retinoic acid"
  },
  {
    "caption": "A: Percentage of methylated CpG dinucleotides profiled across the Esrrb and Nanog enhancer and the Nanog promoter in sorted SSEA-1+ / EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs and TNG E-2a-tdT EpiSC. CpG methylation was assessed by measuring protection from digestion of the HpaII, AciI, Hin6I or TaqI restriction sites (indicated by vertical lines in the gene structure maps derived from the mouse reference genome (mm9) and expressed relative to the TSS in ESCs Values represent total CpG methylation levels (5mC + 5hmC). Error bars: standard deviation of the measures in four independent experiments.",
    "molecules": "CpG, CpG dinucleotides"
  },
  {
    "caption": "B: Methylated CpG dinucleotides across the Esrrb enhancer were assessed SSEA-1+ / EsrrbHi E-GFPd1 ESCs were sorted and placed back in culture overnight before resorting and methylation analysis relative to Esrrb- E-GFPd1 ESCs. Error bars: standard deviation of the measures in three independent experiments.",
    "molecules": "CpG dinucleotides"
  },
  {
    "caption": "D: Quantitative ChIP-PCR analysis of OCT4, NANOG and ESRRB binding and histone modifications at the Esrrb and Nanog enhancers and the Nanog promoter in the sorted populations Error bars: standard deviation of the measures in two (NANOG, ESRRB, H3K27ac) or three (OCT4, H3K4me3, H3K4me1) independent ChIP experiments, each performed on pooled chromatin from at least three independently sorted samples. The diagrams at the bottom show the approximate position of the regions analysed for transcription factor binding or histone modifications (in color or black), along with the relative control genomic locations (grey).",
    "molecules": "H3K4me1, H3K4me3"
  },
  {
    "caption": "(D) Representative smFISH images in DRG sections and quantitative results showed that 2310002F09Rik and Gm14273 were more enriched in the nucleus, whereas CLAP and Gm11549 were mainly distributed in the cytoplasm. DAPI served as a nuclear marker. Scale bar, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Representative bright-field and fluorescent images showing the Sst-cre; Ai9 neurons for calcium imaging following siCLAP transfection. Scale bar, 10 μm.",
    "molecules": "calcium"
  },
  {
    "caption": "The percentage of Sst-Cre; Ai9 neurons evoked by histamine (I) was decreased by knockdown of CLAP, whereas the percentage of these neurons affected by 5-HT (J), chloroquine (K), capsaicin (L) were not changed. The results are presented as mean ± SEM (n = 4). ** P < 0.01 versus siNC.",
    "molecules": "capsaicin, chloroquine, histamine, 5-HT"
  },
  {
    "caption": "(C-E) Knockdown of CLAP reduced itch scratching evoked by histamine (C), but not 5-HT (D) and chloroquine (E). The results are presented as mean ± SEM (For histamine and 5-HT, siNC = 12，siCLAP = 15; for chloroquine, siNC = 5，siCLAP = 8 ). ** P < 0.01 versus siNC.",
    "molecules": "chloroquine, histamine, 5-HT"
  },
  {
    "caption": "A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).",
    "molecules": "cobalt chloride, CoCl2"
  },
  {
    "caption": "B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.",
    "molecules": "oxygen"
  },
  {
    "caption": "C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen.",
    "molecules": "oxygen"
  },
  {
    "caption": "D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.",
    "molecules": "oxygen"
  },
  {
    "caption": "B. WT, rbd2Δ and sre2Δ cells were treated with bortezomib (Bz) for 3 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum.",
    "molecules": "bortezomib, Bz"
  },
  {
    "caption": "C. WT, dsc1Δ and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag-Sre2MS (MS) were treated with bortezomib (Bz) for 3 hr. Whole cell lysates were analyzed by western blot with anti-Flag antibody.",
    "molecules": "bortezomib, Bz"
  },
  {
    "caption": "C. Biotin-phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2-Flag-APEX2 (rbd2-F-AX) or rbd2-G246R-Flag-APEX2 (G246R-F-AX) plasmid were lysed after biotin-labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1%SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Cdc48 serum, or anti-FlagIgG.",
    "molecules": "Biotin, biotin, H2O2, SDS"
  },
  {
    "caption": "A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).",
    "molecules": "cobalt chloride, CoCl2"
  },
  {
    "caption": "B. Western blot of phosphatase-treated, whole cell lysates from strains in (A) grown for 3 hr in the presence or absence of oxygen was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.",
    "molecules": "oxygen"
  },
  {
    "caption": "H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.",
    "molecules": "bortezomib, Bz"
  },
  {
    "caption": "B. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).",
    "molecules": "cobalt chloride, CoCl2"
  },
  {
    "caption": "D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.",
    "molecules": "biotin, Bz"
  },
  {
    "caption": "(A) Representative images showing the myogenic differentiation, as assessed by immunostaining for MyHC (green), of human MuSCs (hMuSCs) isolated from muscle biopsies of DMD patients, either before the beginning of the clinical trial (hMuSCs (-)) or after 1 year of treatment with Givinostat (hMuSCs Giv (-)). Cells were cultured alone or (only for hMuSCs -) in transwell co-culture with FAPs isolated from the same patients and exposed in vitro to Giv (+ehFAPs Giv Vitro) or control vehicle (+ehFAPs). The upper panels represent myogenic differentiation of DMD-1 patient; bottom panels represent myogenic differentiation of DMD-2 patient. Dose and timing of Giv treatment as described in Bettica et al., 2016 (23). Scale bar = 50 μm. (B) Graph showing the differentiation index of hMuSCs described in (A). Two technical replicates are shown. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.",
    "molecules": "DAPI, Givinostat, Giv"
  },
  {
    "caption": "(C) Representative images showing the myogenic differentiation of MuSCs isolated from 1.5 month old mdx mice, as assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with mdx FAPs, which were pretreated with DMSO or GW4869 (GW 10mM added to FAPs 30 minutes before starting the co-culture with MuSCs). Scale bar = 50 μm. (D) Graph showing the fusion index of MuSCs in the conditions described in (C), (n=5, biological replicates). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), ** p < 0.01; Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-cultured with FAPs not treated (DMSO), # p<0.05; § represents statistical analysis by 2Way Anova test; §§§ p < 0,001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.",
    "molecules": "DAPI, DMSO, GW, GW4869"
  },
  {
    "caption": "(E) Representative images of PKH-67 (green) and DAPI (blue) staining in a transwell co-culture between MuSCs and FAPs isolated from 1.5 month old mdx mice. FAPs exposed to PKH-67 prior to the co-culture with MuSCs. Scale bar = 50 μm. (F) Graph showing the percentage of PKH-67 positive MuSCs after the co-culture with FAPs, (n=3, biological replicates). Star (*) indicates statistical analysis by t-test;**** p < 0.0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.",
    "molecules": "PKH-67, DAPI"
  },
  {
    "caption": "(G) Representative images of myogenic differentiation of mdx MuSCs, as assessed by immunostaining for MyHC (green). MuSCs were cultured either alone (-) or incubated with EVs isolated from 1.5 month old mdx mice (EVs FAPs) treated with vehicle (CTR) or exposed to TSA in vivo (TSA) or in vitro (TSA vitro). Scale bar = 50 μm. (H) Graph showing the fusion index of MuSCs in the condition described in (G), (n=3, biological replicates). Star (*) indicates statistical analysis by Tukey test on the % of n>5 (% of nuclei that were MyHC+ inside myotubes containing more than 5 nuclei) relative to MuSCs cultured alone (-), **p <0.01, ****p<0,0001; Hash (#) indicates statistical analysis by Tukey test relative to EVs FAPs CTR, ##p <0.01, ###p<0,001; ns= not significant; § represents statistical analysis by 2way Anova test, §§§§p < 0,0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.",
    "molecules": "DAPI, TSA"
  },
  {
    "caption": "(A) Graph showing the relative expression of Drosha in FAPs after Drosha down-regulation by siRNA (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by t-test relative to FAPs TSA treated and transfected with scramble (Scr); **p<0.01.",
    "molecules": "TSA"
  },
  {
    "caption": "(B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm. (C) Graph showing the fusion index of MuSCs in the conditions described in (B) (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by Tukey test relative to MuSCs alone (-); *p<0.05, ****p<0.0001. Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-culture with FAPs CTR transfected with scramble (Scr); ##p < 0.01. Cross (†) means Tukey analysis compared to MuSCs in co-culture with FAPs TSA transfected with scramble (Scr): ††††p < 0,0001. § represents statistical analysis by 2way Anova test. §§§p < 0,001.",
    "molecules": "TSA"
  },
  {
    "caption": "(A) Representative images of Sca-1 (green), Laminin (white), DAPI (blue) immunofluorescence, and miR-206-3p (violet) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior (TA) of young mdx mice (CTR) and after TSA treatment (TSA) (0.6 mg/kg/day for 15 days by i.p.) (n=6). Scale bar = 25 μm. (B) Graph showing the quantifications of miR-206-3p violet area relative to conditions indicated in (A). Star (*) indicates t-test analysis **p<0.01. Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI, TSA"
  },
  {
    "caption": "(C) Graph representing the miR-206-3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice (CTR), mdx young and old mice (control -CTR- and TSA treated) (n=4). Star (*) indicates Tukey analysis compared to wild type mice (CTR), ****p < 0.0001; hash (#) indicates Tukey analysis compared to mdx young CTR, #### p<0.0001. § represents statistical analysis by Anova test. §§§§ p<0,0001.",
    "molecules": "TSA"
  },
  {
    "caption": "(D) Representative images of CD90 (green), Dapi (blue) and miR-206-3p (violet) immunohistochemistry in brachial biceps bioptic samples of DMD patient before (CTR) and after Givinostat treatment (GIV). Scale bar = 50 μm. (E) Graph showing the quantifications of miR-206-3p violet area measured as pixel^2/field relative to the experimental points indicated in (D) (n=18, biological replicates). Star (*) indicates t-test analysis. **p<0.01. Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI, Dapi, Givinostat, GIV"
  },
  {
    "caption": "(F) Graph showing the relative expression of miR-206-3p in EVs isolated from a human population of muscle resident cells enriched in FAPs (ehFAPs) isolated from biopsies of two DMD patients (DMD-1 and DMD-2) before (CTR) and after (GIV) Givinostat treatment, Two technical replicates are shown.",
    "molecules": "Givinostat, GIV"
  },
  {
    "caption": "(A) Flow cytometry analysis of mdx muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA. The left panel shows the percentage of PKH-67 positive cells in whole muscle. The right panel shows the percentage of the PKH-67 positive MuSCs.",
    "molecules": "PKH-67, PKH67, TSA"
  },
  {
    "caption": "(B) Representative images of PKH-67 (green) and DAPI (blue) staining in MuSCs freshly isolated from muscles previously injected with PKH67-labeled EVs. Scale bar = 25 μm. Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "PKH-67, PKH67, DAPI"
  },
  {
    "caption": "(C) Graph showing the relative expression of miR-206-3p in MuSCs isolated as described in (A and B) and in MuSCs from muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA, and then transfected or not with Drosha siRNA (siRNA Drosha) (n=4). Star (*) means significance relative to MuSCs that not uptake PKH67-labled EVs, **p < 0.01. Hash (#) means significance compared to MuSCs that uptake EVs-FAPs TSA, ##p < 0.01. § indicates Anova analysis, §§p<0,01.",
    "molecules": "PKH67, TSA"
  },
  {
    "caption": "(D) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05, **p<0.01",
    "molecules": "TSA"
  },
  {
    "caption": "(E) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs-derived EVs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05.",
    "molecules": "TSA"
  },
  {
    "caption": "Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (F) Representative images of immunofluorescence for embryonic myosin heavy chain (eMyHC-red) and laminin (Lam-cyan) stainings. Scale bar = 50 μm. (G) Graph showing the quantification of cross-sectional area (CSA). (H) Graph showing the quantification of muscle regeneration (eMyHC). Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI, TSA"
  },
  {
    "caption": "Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (I) Representative images of Masson's Trichrome staining. Scale bar = 50 μm. (J) Graph showing the quantifications of muscle fiber area fraction (MFAF). (K) Graph showing the quantifications of fibrotic area. Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI, TSA"
  },
  {
    "caption": "Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (L) Representative images of myeloperoxidase staining (MPO-red). Scale bar = 50 μm. (M) Graph showing the quantifications of inflammation (MPO). Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI, TSA"
  },
  {
    "caption": "A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm. (B) Graph showing the fusion index of MuSCs in the condition described in (A). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), *p < 0.05, ****p>0,0001; hash (#) means significance compared to EVs-FAPs CTR -, ###p<0,001; and (†) means significance to EV-FAPs TSA -, †††† p<0,0001. § indicates significance by Anova test; §§§§ p>0,0001.",
    "molecules": "DAPI, TSA"
  },
  {
    "caption": "(C) Graph relative to Notch3 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) (n=3). Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR, ##p<0,01. § indicates significance by Anova test; §§ p>0,01.",
    "molecules": "TSA"
  },
  {
    "caption": "(D) Graph relative to Notch1 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) n=3. Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR; *p < 0.05. § indicates significance by Anova test; §§ p>0,01.",
    "molecules": "TSA"
  },
  {
    "caption": "(E) Representative images showing myofibers cultured alone (Ctr) or with extracellular vesiclesisolated from FAPs (EVs-FAPs) and stained with PKH-67 (green) and MyoD (red). Nuclei were counterstained with DAPI (blue). Arrows represent nuclei positive for both PKH-67 and MyoD. Scale bar = 25 μm.",
    "molecules": "PKH-67, DAPI"
  },
  {
    "caption": "(F) From left to right: representative images of myofibers cultured for 24 hours alone (-) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 (MEDIA-FAPs) or extracellular vesicles (EVs-FAPs) collected from FAPs exposed or not to TSA in vivo (CTR and TSA) and transfected with antagomiR-206 (TSA A-206). Myofibers have been stained for EdU(Cyan), immunofluorescence with Pax7 (green), MyoD (red). Nuclei were counterstained with DAPI (blue). (n=3). Scale bar = 25 μm. (G) Graph showing the average of nuclei positive for Pax7 (green), MyoD (red) in the conditions indicated in (F). Star (*) indicates statistical analysis by Tukey test relative to myofibers cultured alone (-), (n=3, biological replicates). *p<0,05, **p< 0.01; ***p<0,001; ***p<0,0001; hash (#) means significance compared to MEDIA FAPs CTR, #p<0,05; (†) means significance to EV-FAPs CTR, † p<0,05; while ¶ means significance compare to EVs-FAPs TSA, ¶¶¶¶ p<0,0001. § means significance by Anova test; §§ p < 0.01.",
    "molecules": "EdU, DAPI, GW4869, TSA"
  },
  {
    "caption": "WT mixed glial cultures were co-treated with MPSIIIA GAG (4 μg/ml) and/or the TLR4 inhibitor CLI-095 (1 μg/ml) for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus MPSIIIA GAG + vehicle.",
    "molecules": "CLI-095, MPSIIIA GAG"
  },
  {
    "caption": "Multi-heparinase (HepM) and/or chondroitinase ABC (cABC) digests were performed on MPSIIIA GAG, and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours alongside heparin, bovine HS (bHS), porcine DS (pDS) and WT GAG. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001. Symbols above bars are versus MPSIIIA GAG alone.",
    "molecules": "DS, GAG, HS, heparin, oligosaccharides, MPSIIIA GAG"
  },
  {
    "caption": "MPSIIIA GAG was 2-O-desulphated (hashed bars) utilising 120 mM NaOH, and compositional HS disaccharide analysis performed via RP-HPLC against untreated MPSIIIA GAG (black bars) to validate complete 2-O-desulphation. Percentage contribution of each modification were determined (n=3 GAG samples). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; 2S MPSIIIA GAG vs. 2S 2-ODS MPSIIIA GAG P<0.0001. NS, N-sulphated glucosamine; 2S, 2-O-sulphate group; 6S, 6-O sulphate group.",
    "molecules": "2-O-sulphate group, 2-ODS MPSIIIA GAG, 2S, 6-O sulphate group, 6S, HS disaccharide, GAG, N-sulphated glucosamine, NS, NaOH, MPSIIIA GAG"
  },
  {
    "caption": "MPSIIIA GAG was 2-O-desulphated and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; MPSIIIA GAG vs. 2ODS MPSIIIA GAG P<0.0001. 2ODS, 2-O-desulphated.",
    "molecules": "2ODS MPSIIIA GAG, oligosaccharides, MPSIIIA GAG"
  },
  {
    "caption": "Representative sections from 9 month old WT and MPSIIIA brains showing secondary storage of GM2 ganglioside, cholesterol, protein aggregates and amyloid beta peptide 1-40 and 1-42 within cortical layers II to V/VI, x20, scale bar: 50 μm. Inserts, 100x, scale bar: 10μm. (n=4 mice per group)",
    "molecules": "amyloid beta peptide 1-40, cholesterol, GM2 ganglioside"
  },
  {
    "caption": "ATP levels in whole brain homogenate from 9 month old WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.02",
    "molecules": "ATP"
  },
  {
    "caption": "WT (E-G) and Nlrp3-/- (H-J) mixed glia were primed with MPSIIIA GAG or LPS. ATP (5 mM), cholesterol crystals (1 mg/ml) or amyloid-β fibrils (Aβ1-40 4.3 μM and Aβ1-42 5 μM) were added 4 hours post-priming stimulus, and the secretion of IL-1β into the media measured via ELISA after a further 20 hours (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001",
    "molecules": "Aβ1-40, Aβ1-42, ATP, cholesterol, LPS, MPSIIIA GAG"
  },
  {
    "caption": "Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with anti-mouse glial fibrillary acidic protein (GFAP; red)/DAPI (nuclear, blue) to identify activated astrocytes, 20x, scale bar: 50μm Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with isolectin B4 (ILB4) to identify activated microglia, 20x, scale bar: 50μ",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Western blot  analysis of PMA-differentiated THP1 monocytes  stably transduced with either V5-tagged VZV ORF9  or GFP  using the indicated antibodies. Data information: Panels (D) are representing two independent biological experiments. In (E) each data point represents a technical replicate.",
    "molecules": "PMA"
  },
  {
    "caption": "(E) THP1 monocytes  stably transduced with either VZV ORF9  or GFP were PMA-differentiated and transfected with indicated doses of dsDNA. Expression of IFNB1  and IFNL1  was assessed by RT-qPCR. Graphs show expression relative to GAPDH. Data information: Panels (E) are representing two independent biological experiments. In (E) each data point represents a technical replicate.",
    "molecules": "dsDNA, PMA"
  },
  {
    "caption": "(F) THP1 monocytes  stably transduced with either VZV ORF9  or GFP  were treated with the STING  agonist diABZi  (0.005 μM). Expression of IFNB1, IFIT1  and IFI44  was assessed by RT-qPCR. Graphs show expression relative to GAPDH. Data information: Panel (F) shows pooled data from three and two repeats, respectively, where each data point represents an independent biological experiment (horizontal bars show means). Statistical analysis in (F) were paired t-tests.",
    "molecules": "diABZi"
  },
  {
    "caption": "(I) PMA-differentiated THP1  cells were co-cultured with VZV-ORF9ecDHFR-infected MeWo  cells for 1h in the presence of TMP, washed, and incubated for another 24h in the presence of TMP. Cells were then washed extensively and fresh medium was added that was supplemented or not with TMP. Expression levels of IFNB1, IFIT1, and IFI44  were measured by  RT-qPCR after a further 24h incubation. Graphs show expression relative to GAPDH. Mean fold changes are indicated. Data information: Panel (I) shows pooled data from three repeats, where each data point represents an independent biological experiment. Statistical analysis in (I) were paired t-tests.",
    "molecules": "PMA, TMP"
  },
  {
    "caption": "(B) THP1 monocytes  stably transduced with either VZV FLAG-ORF9  or FLAG-GFP  were PMA-differentiated overnight. The next day, cells were lysed and ectopically expressed proteins were immunoprecipitated  using α-FLAG  antibody. Input, unbound and IP  fractions were subjected to immunoblotting  using the indicated antibodies. Data information: Panels (B), are representative of three independent experiments.",
    "molecules": "PMA"
  },
  {
    "caption": "(C) HaCaT  cells and PMA-differentiated THP1  cells were infected with WT VZV  or VZVORF9-V5 through co-culture with infected MeWo  cells for 48 hours. Cells were lysed and ORF9  was immunoprecipitated  using α-V5 antibody. Input and IP  fractions were subjected to immunoblotting  using the indicated antibodies. Data information: Panel (C) is representative of two (HaCaT) and three (THP1) independent experiments.",
    "molecules": "PMA"
  },
  {
    "caption": "(D) HEK293T  cells were seeded onto glass coverslips and were transfected with human cGAS-V5, FLAG-ORF9, or both together. The next day, cells were fixed, permeabilised and stained  using α-V5-FITC, rabbit-α-FLAG, and goat-α-rabbit-AF647  antibodies, and DAPI. Mounted coverslips were imaged using  confocal microscopy. Scale bars: 15 µm. Data information: Panels (D), are representative of three independent experiments.",
    "molecules": "AF647, DAPI, FITC"
  },
  {
    "caption": "(D) Recombinant cGAS, FLAG-ORF9, FLAG-GFP, and biotinylated VACV70mer dsDNA  were incubated in the indicated combinations. Recombinant proteins were precipitated using streptavidin  beads. Fractions were analysed by immunoblotting. Data information: Results shown are representative of two independent experiments.",
    "molecules": "dsDNA, streptavidin"
  },
  {
    "caption": "(E) HEK293T  cells were transfected with expression plasmids for FLAG-GFP  or FLAG-ORF9. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA  was precipitated using streptavidin  beads and fractions were subjected to immunoblotting  using the indicated antibodies. Data information: Results shown are representative of two independent experiments.",
    "molecules": "DNA, dsDNA, streptavidin"
  },
  {
    "caption": "(F) VACV70mer dsDNA  was incubated with indicated proteins and analysed by agarose  gel EMSA. Triangles indicate concentrations of ORF9  (0.7, 1.4, 2.9, 5.7, 8.6 µM), cGAS  (0.6, 1.1, 2.2, 4.5, 6.7 µM), and ORF9  (as before) in the presence of 4.5 µM cGAS. Data information: Results shown are representative of two independent experiments.",
    "molecules": "agarose, dsDNA"
  },
  {
    "caption": "(C) HEK293T  cells were transfected with expression plasmids for FLAG-GFP  or FLAG-ORF9  as indicated. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA  was precipitated using streptavidin  beads and proteins were analysed by immunoblotting  using the indicated antibodies. Data information: Data shown in panels (C) are representative of two independent experiments.",
    "molecules": "DNA, dsDNA, streptavidin"
  },
  {
    "caption": "(E) THP1  cells stably transduced with GFP, ORF9, or ORF9-DM were transfected with the indicated amounts of E.coli dsDNA. The next day, IFNB1  expression was assessed by RT-qPCR. Data information: Panel (E) shows pooled data from two repeats, where each data point represents an independent biological experiment (n=2 +/- range).",
    "molecules": "dsDNA"
  },
  {
    "caption": "(F) In vitro cGAS  activity assay. Recombinant hcGAS  was incubated with ATP, GTP, and radioactive α32P-ATP  at 37°C with addition of dsDNA  and other proteins as indicated (triangles represent concentrations of 0.5, 1, 2, 4, 6 µM). ORF9-DM was tested only at 0.5, 1, 2 µM. Reactions were treated with calf-intestinal phosphatase  and products were analysed by thin-layer chromatography and phosphorimaging. (G) Signal intensities from (F) were determined by densitometry  analysis and normalized to the average BSA  signal. Data information: Panel (F) is representative of three independent experiments. Panel (G) shows pooled data from three repeats, where each data point represents an independent biological experiment (n=3 +/- SD).",
    "molecules": "ATP, dsDNA, GTP, α32P"
  },
  {
    "caption": "(B) Oxygen consumption (VO2), (C) carbon dioxide production (VCO2) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4).",
    "molecules": "CO2, O2"
  },
  {
    "caption": "(E) VO2, (F) VCO2 and (G) energy expenditure in 10‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=3-5).",
    "molecules": "CO2, O2"
  },
  {
    "caption": "(L) Serum‐free fatty acid (FFA) and glycerol (Gly) from 4‐ to 6‐mo‐old Con and KO mice untreated (basal) or treated with isoproterenol (Iso) i.p. for 20 min (n=4-6), and (M) from 4‐mo‐old RD‐fed cold‐challenged (75 min) mice (n=3-4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.",
    "molecules": "free fatty acid, glycerol, isoproterenol"
  },
  {
    "caption": "(A) Blood glucose in 6‐ to 10‐month (mo)‐old mice (n=12-15), and (B) serum insulin levels in 10‐mo‐old fed control (Con) and KO mice (n=6).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(C) Glucose tolerance tests in 10‐mo‐old chow diet (RD)‐fed (n=5), and (D) in 10‐ to 12‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=4-9).",
    "molecules": "Glucose"
  },
  {
    "caption": "Representative images of mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 days following 10Gy X-irradiation. Left panels represent troponin C-positive embryonic cardiomyocytes (troponin C - magenta; DAPI - light blue). Middle panels display γH2AX foci (green) and telomeres (red) in Z projections of 0.1µm slices, with white arrows indicating co-localisation. Co-localising foci are amplified in the right-hand panels (amplified images represent a single Z-plane where co-localisation was observed). Scale bars represent 10μm. Scale bars in single plane images 500nm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Mean number of both TAF and non-TAF in neonatal rat cardiomyocytes at days 0, 3, 5, 10 days following treatment for 24h with H2O2. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition.",
    "molecules": "H2O2"
  },
  {
    "caption": "Representative images of immunofluorescent staining against α-SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs.",
    "molecules": "EdU"
  },
  {
    "caption": "Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins: Tgfb2, Edn3, and Gdf15 for 48h and immunostained against α-SMA and EdU (fibroblasts) and α-actinin (CMs).",
    "molecules": "EdU"
  },
  {
    "caption": "EdU positive neonatal fibroblasts (H) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.",
    "molecules": "EdU"
  },
  {
    "caption": "Mean % of 4-HNE- (top) or 8oxodG- (bottom) positive CMs from 3 month (young) or 30 month (old) aged mice. Data are mean ± SEM of n=4 per age group. 100 CMs were quantified per age group.",
    "molecules": "4-HNE, 8oxodG"
  },
  {
    "caption": "Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in wild-type (control) compared to MAO-A transgenic mice with or without drinking water supplemented with 1.5g/kg/day NAC from the age of 4 to 24 weeks Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.",
    "molecules": "NAC"
  },
  {
    "caption": "Schematic depicting isolated mouse adult CMs isolated from 4 animals were treated with or without 100nM rotenone either in the presence of 5mM NAC or vehicle control (pre-treated for 30 minutes before rotenone treatment), for 24 h before fixation. Mean number of TAF (top graph) and mean % of TAF-positive nuclei (bottom graph). Data are mean ± SEM from 4 separate CM cultures isolated from 3 month-old mice. 50 CMs were quantified per condition.",
    "molecules": "NAC, rotenone"
  },
  {
    "caption": "Comparison between the % of p16- or eGFP-positive CMs by RNA-in situ hybridization per plane in INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n=5 per age group. 100 CMs were analysed per mouse.",
    "molecules": "AP20187"
  },
  {
    "caption": "% of fibrotic area evaluated by Sirius Red staining.",
    "molecules": "Sirius Red"
  },
  {
    "caption": "Quantification of mean number of TAF and % of TAF-positive CMs in 24 months old wild-type mice treated with vehicle or navitoclax (50mg/kg/day). Data are mean± SEM of n=5-8 mice per group. More than 100 CMs were quantified per animal.",
    "molecules": "navitoclax"
  },
  {
    "caption": "CM cross-sectional area in 24 months old wild-type mice treated with vehicle or navitoclax.",
    "molecules": "navitoclax"
  },
  {
    "caption": "(Left) Representative images of Sirius red staining and (right) % of fibrosis area in 24 month-old mice treated or not with navitoclax.",
    "molecules": "navitoclax, Sirius red"
  },
  {
    "caption": "MRI analysis of ejection fraction (EF%) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.",
    "molecules": "navitoclax"
  },
  {
    "caption": "left ventricle mass (LVmass) index in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.",
    "molecules": "navitoclax"
  },
  {
    "caption": "% of ventricle wall thickness (%WTC) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.",
    "molecules": "navitoclax"
  },
  {
    "caption": "Examples of confocal microscopy images of CMs positive for CM marker troponin C (TropC), EdU, Ki67 and Aurora B from navitoclax treated animals. (Upper Right panel) white arrows identify two nuclei in the same CM that have incorporated EdU. (Lower Right panel) white arrow identify EdU expressing Aurora B symmetrically between two nuclei. Scale bars represent 20µm.",
    "molecules": "EdU, navitoclax"
  },
  {
    "caption": "Quantification of EdU positive CMs (mono- or multi-nucleated) in vehicle and navitoclax treated animals. Data are mean±S.E.M of n=5-6 mice per group.",
    "molecules": "EdU, navitoclax"
  },
  {
    "caption": "% of Ki67 positive CMs in 29 month old INK-ATTAC mice treated with vehicle or AP20187. Data are mean±S.E.M of n=4-6 mice per group.",
    "molecules": "AP20187"
  },
  {
    "caption": "BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4-PHA lectin (lower) or anti-BACE1 or APP antibodies (upper). hAPP indicates the APP23 transgenic mouse model for AD.",
    "molecules": "E4-PHA lectin"
  },
  {
    "caption": "LC-MS base peak chromatogram of desialo-alditol N-glycans derived from mouse brain BACE1. To simplify the results, N-glycans were chemically desialylated before LC-MS analysis. BACE1-specific glycans, judged by comparison with N-glycan structures from anti-BACE1 IgG (shown in Supplementary Fig S2C), are highlighted by red squares. Asterisks indicate glycans demonstrated by MS/MS analysis to contain a bisecting GlcNAc structure. Numbers in parentheses indicate the charge state.",
    "molecules": "N-glycans"
  },
  {
    "caption": "BACE1 from temporal lobe membrane fractions of NC, eAD, or AD patients was immunoprecipitated and blotted with E4-PHA (upper) or anti-BACE1 (lower). The signal intensity of E4-PHA relative to that of BACE1 was calculated (n = 10). The graph shows means ± SEM (*P < 0.05; one-way ANOVA with post hoc Tukey-Kramer test. P = 0.014 for NC versus eAD, P = 0.028 for NC versus AD).",
    "molecules": "E4-PHA"
  },
  {
    "caption": "Immunoblot of BACE1 or GAPDH (loading control) from immortalized MEFs treated with a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine; CQ).",
    "molecules": "chloroquine, CQ, MG132"
  },
  {
    "caption": "L, M, N, O The blood glucose level of mice after intraperitoneal injection of glucose or insulin for glucose (GTT) (L) and insulin tolerance tests (ITT) (N), and AUC, area under the curve (M, O).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "A ORO staining of SVF cells isolated from iWAT of male mice. Differentiation was induced with curcumin up to day 8. C ORO staining of 3T3-L1 cells. Differentiation was induced with curcumin up to day 8.",
    "molecules": "curcumin, ORO"
  },
  {
    "caption": "B mRNA expression of adipogenesis-related factors of SVF cells isolated from iWAT of male mice. D mRNA expression of PPARγ, C/EBPα and FABP4 expression of 3T3-L1 cells treated with curcumin.",
    "molecules": "curcumin"
  },
  {
    "caption": "protein expression levels of m6A regulator in iWAT from mice in HFD and HFD+CUR groups. protein expression levels of m6A regulator in 3T3-L1 cells were analyzed",
    "molecules": "CUR, m6A"
  },
  {
    "caption": "K Oil Red O staining of Vector or OE-ALKBH5 transfected 3T3-L1 cells. Differentiation was induced with curcumin up to day 8.",
    "molecules": "curcumin, Oil Red O"
  },
  {
    "caption": "E Oil Red O staining of control, ALKBH5-depleted and ALKBH5+TRAF4 depleted cells after induced for 8 days.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "L Oil Red O staining of control, ALKBH5-depleted and ALKBH5+YTHDF1 depleted cells on day 8 of differentiation.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "C, D Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells in the absence or presence of MG132 or 3MA and quantification of protein levels normalized to β-actin expression.",
    "molecules": "3MA, MG132"
  },
  {
    "caption": "E Ubiquitination of endogenous PPARγ in control and TRAF4 overexpressing cells were differentiated by treatment with DMI. Cells were treated with MG132 for 6 h.",
    "molecules": "DMI, MG132"
  },
  {
    "caption": "L CM-H2DCFDA (green) indicating cellular ROS  levels of WT or TMEM175  KO HEK293T  cells pre-treatment with vehicle or 10 μM HA14-1 for 20 min. Scale bars = 10 μm. M Statistics of CM-H2DCFDA  fluorescence intensity of (L). The n values represent the cell numbers counted within the same experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests M) and is indicated with NS for not significant (P > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. In panel M, n value means the number of technical replicates.",
    "molecules": "HA14-1, CM-H2DCFDA, ROS"
  },
  {
    "caption": "H Co-localization  of mitochondrial  indicator TOM20-mCherry  and lysosomal  indicator  LysoTracker in WT (upper row) or TMEM175  KO (lower row) HEK293T  cells. Co-localization  of mitochondrion  and lysosome  is observed as a yellow colour in the merged image (marked by white arrows). Scale bars = 10 μm. I The number of merged puncta per cell in (H). We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175  KO group from 42 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels I, n value means the number of technical replicates.",
    "molecules": "LysoTracker"
  },
  {
    "caption": "D TMEM175  mRNA levels in the midbrains  of WT or TMEM175  KO mice  were detected by RT-qPCR using SYBR  Green PCR Master Mix. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.",
    "molecules": "SYBR  Green"
  },
  {
    "caption": "H, Quantification of rears in the cylinder (H) pre- and post-MPTP   treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.",
    "molecules": "MPTP"
  },
  {
    "caption": "I Quantification of the latency to fall  from a  wire hang  apparatus (I) pre- and post-MPTP  treatment. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.",
    "molecules": "MPTP"
  },
  {
    "caption": "J Swing Duration CV (CV%) (Left panel) and Ataxia  Coefficient in  Gait tests 7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.",
    "molecules": "MPTP"
  },
  {
    "caption": "K Latency to fall in rotarod tests  7 days post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.",
    "molecules": "MPTP"
  },
  {
    "caption": "L Immunoblots of the TH  levels post-MPTP  treatment in WT and TMEM175  KO mouse midbrains. M Quantification of the relative TH  levels normalized to GAPDH  post-MPTP. Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.",
    "molecules": "MPTP"
  },
  {
    "caption": "N TH immunofluorescent labeling on substantia nigra slices of WT or TMEM175-KO mice  post-MPTP  treatment. Scale bars = 100 μm. O Statistics of the TH-positive neurons  in (N). Data information: The data are presented as the mean ± SEM. Statistical significance was analyzed with two-sided student's t-tests, and is indicated with NS for not significant (p > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.",
    "molecules": "MPTP"
  },
  {
    "caption": "A. Representative immunoblots and quantifications (below) of the indicated proteins in lysates of ARPE-19 cells treated with 1 mM DFP for the indicated time (h, hours).",
    "molecules": "DFP"
  },
  {
    "caption": "B-C. Representative confocal images of ARPE-19 cells stably expressing the mito-QC reporter (B), or the pexo-QC reporter (C) treated with 1 mM DFP for the indicated time (h, hours). Enlarged images of the area outlined in white are shown in the lower corners. The 'red-only' mask is generated during images analysis and highlights red-only puncta (autolysosomes) based on the mCherry/GFP ratio and thresholding criteria. Nuclei were stained in blue (Hoechst). Scale bar: 10μm. At right, quantification of total red-only punctate per cell (mitolysosomes or pexolysosomes).",
    "molecules": "Hoechst, DFP"
  },
  {
    "caption": "D. ARPE-19 cells were transfected with 50 pmol of non-targeting siRNA (siNT) or 50 pmol of siRNA targeting ATG13 or ULK1. 48 hours post knockdown, cells were treated with 1 mM DFP for an additional 48 hours. Cell lysates were subject to immunoblotting with the indicated antibodies. Quantification is shown on the right.",
    "molecules": "DFP"
  },
  {
    "caption": "A. Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells, treated with 1mM DFP for 48 hours.",
    "molecules": "DFP"
  },
  {
    "caption": "B. Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo-QC reporter and treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).",
    "molecules": "DFP"
  },
  {
    "caption": "C. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins in lysates of WT or two NIX KO ARPE-19 clones (Cl), treated with 1 mM DFP for the indicated time (h, hours).",
    "molecules": "DFP"
  },
  {
    "caption": "D-E. Representative confocal images of WT or NIX KO ARPE-19 cells stably expressing the mito-QC reporter (D) or the pexo-QC reporter (E) treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).",
    "molecules": "DFP"
  },
  {
    "caption": "F. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins. NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe empty vector (∅) or a pBabe NIX vector, were treated with 1 mM DFP for 48 hours prior lysis. Two biological replicates are shown in the immunoblot.",
    "molecules": "DFP"
  },
  {
    "caption": "A-B. Representative confocal images of untreated (CTRL) and DFP-treated ARPE-19 cells (1mM, 48 hours), stably expressing GFP-WT NIX (green) and stained with anti-catalase antibody (A, red) or anti-PMP70 antibody (B, red). At right, quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70, with or without DFP treatment for 48 hours. Peroxisomes enriched for GFP-NIX or having minimal signal for GFP-NIX are depicted with white arrowheads or white arrows, respectively.",
    "molecules": "DFP"
  },
  {
    "caption": "C. Representative confocal images of ARPE-19 cells stably expressing GFP-WT NIX, GFP-NIXΔTM or GFP-NIX TM only (green), treated with DFP (1mM) for 48 hours and immunostained with anti-catalase antibody (red). At right, quantification of Pearson correlation coefficients between the green and the red signal.",
    "molecules": "DFP"
  },
  {
    "caption": "B. Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe Flag vector (∅), a pBabe Flag-NIX vector (NIX), a pBabe Flag-NIX W36A\L39A vector (W36A\L39A) or a pBabe Flag-NIX S34E\S35E vector (S34E\S35E), and treated with 1 mM DFP for 48 hours prior lysis.",
    "molecules": "DFP"
  },
  {
    "caption": "C-D. Representative confocal images of cells as in B, stably expressing the mito-QC reporter (C) or the pexo-QC reporter (D) and treated with 1 mM DFP for 24 hours (C) or 48 hours (D) (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel). Enlarged images of the area outlined in white are shown in the lower corners. Nuclei were stained in blue (Hoechst)",
    "molecules": "Hoechst, DFP"
  },
  {
    "caption": "A. Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers (PMP70 and catalase) or a lysosomal marker LAMP1. Nuclei are coloured in white (DAPI). RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL; inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells. B. Quantification of signal area in the ONL and INL from images as A. Each data point represents a single mouse (n=3-4 per group).",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.",
    "molecules": "HU"
  },
  {
    "caption": "Analysis of replication fork progression at the single-molecule level by DNA combing. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477).",
    "molecules": "BrdU, DNA fibers, ssDNA"
  },
  {
    "caption": "(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent.",
    "molecules": "HU, tRNA"
  },
  {
    "caption": "(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin.",
    "molecules": "HU"
  },
  {
    "caption": "(E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.",
    "molecules": "HU"
  },
  {
    "caption": "(F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.",
    "molecules": "HU"
  },
  {
    "caption": "(B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted.",
    "molecules": "HU"
  },
  {
    "caption": "(E) Position relative to the nuclear envelope (Zone 1 of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition.",
    "molecules": "galactose, glucose, HU"
  },
  {
    "caption": "A S. cerevisiae wt or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐luciferase‐DM‐NLS (green) were grown at 30°C and shifted to 37°C for 90 min. MG132 was added prior to temperature upshift. Changes in protein localizations were recorded. DNA was stained by DAPI (blue).",
    "molecules": "DNA, MG132"
  },
  {
    "caption": "A-D Cryo‐sections of (A) S. cerevisiae wt cells expressing GFP‐luciferase‐DM‐NLS, (B) hsp42Δ cells expressing GFP‐VHL. Sections were immunogold‐labeled with GFP‐specific antibodies. Gold particles are marked (black arrows). Electron‐dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.E, F Average numbers of gold particles associated with nuclear and cytosolic protein aggregates or distributed throughout the cytosol or nucleus were determined.",
    "molecules": "Gold, gold"
  },
  {
    "caption": "A-D Cryo‐sections of S. cerevisiae (C, D) wt or hsp42Δ cells expressing Hsp104‐GFP. Sections were immunogold‐labeled with GFP‐specific antibodies. Gold particles are marked (black arrows). Electron‐dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.E, F Average numbers of gold particles associated with nuclear and cytosolicprotein aggregates or distributed throughout the cytosol or nucleus were determined.",
    "molecules": "Gold, gold"
  },
  {
    "caption": "A S. cerevisiae wt cells expressing GFP‐VHL (green) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132. Ubiquitin (red) was stained by immunofluorescence microscopy using specific antibodies. DNA was stained by DAPI (blue). Changes in protein localizations were recorded. Scale bars, 2 μm.",
    "molecules": "DNA, MG132"
  },
  {
    "caption": "B tGnd1‐GFP is stabilized in ubr1Δ san1Δ cells. S. cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1‐GFP were grown at 30°C. Cycloheximide was added, and protein levels were determined at the indicated time points by Western blot using GFP‐specific antibodies. Zwf1 levels are given as a loading control.",
    "molecules": "Cycloheximide"
  },
  {
    "caption": "A, B S. cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry‐VHL (red) were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. Scale bars, 2 μm. The total number of mCherry‐VHL foci per cell was determined.",
    "molecules": "MG132"
  },
  {
    "caption": "S. cerevisiae hsp42Δ and hsp42Δ tet‐off sis1 cells expressing GFP‐VHL were grown for 20 h in the absence (−Dox) or presence (+Dox) of doxycycline at 30°C and shifted for 90 min to 37°C in the presence of MG132. Changes in protein localization were monitored. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.",
    "molecules": "Dox, doxycycline, DNA, MG132"
  },
  {
    "caption": "B S. cerevisiae wt, hsp42Δ, sti1Δ, and hsp42Δ sti1Δ cells expressing GFP‐VHL were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.",
    "molecules": "MG132"
  },
  {
    "caption": "A, B S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing GFP‐VHL (green, A) or GFP‐luciferase‐DM‐NLS (green, B) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132, and protein localizations were recorded. DNA was stained by DAPI (blue), and the nuclear envelope was stained by Nsp1immunofluorescence microscopy (red). Solubilities of GFP‐VHL and GFP‐luciferase‐DM‐NLS were determined after stress application by Western blot using GFP‐specific antibodies. Solubility of actin was determined as a control. T, total fraction; S, soluble fraction; P, pellet fraction. Scale bars, 2 μm.",
    "molecules": "DNA, MG132"
  },
  {
    "caption": "C S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing mCherry‐VHL were heat‐shocked to 38°C. Upon return to 30°C, protein synthesis was stopped by cycloheximide (CHX) addition and degradation of mCherry‐VHL was monitored by Western blot analysis. The stability of GFP‐luciferase‐DM‐NLS expressed in S. cerevisiae wt and btn2Δ cells was determined accordingly. Ccs1 or Zwf1 levels are given as loading controls.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "A, B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence microscopy. DNA was stained by either Htb1‐mCherry or DAPI. Scale bars, 2 μm. In case of Hsp42 immunofluorescence, a line intensity plot of a deconvoluted widefield image is given.",
    "molecules": "DNA"
  },
  {
    "caption": "C S. cerevisiae btn2Δ hsp42Δ Hsp42‐NLS cells expressing GFP‐VHL (green) were grown at 30°C and heat‐shocked to 37°C for 30 min in the presence of MG132. Changes in protein localizations were recorded. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.",
    "molecules": "DNA, MG132"
  },
  {
    "caption": "D S. cerevisiaebtn2Δ hsp42Δ Hsp42‐NLS cells expressing mCherry‐VHL (red) were treated as described in (C). The nuclear membrane was visualized by Nsp1 immunofluorescence (green) and colocalization of mCherry‐VHL foci with Hsp42 was probed by Hsp42 (green) immunofluorescence microscopy. DNA was stained with DAPI (blue). Scale bars, 2 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "A S. cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points. Zwf1 levels are given as a loading control.",
    "molecules": "MMS"
  },
  {
    "caption": "B, C S. cerevisiae wt and btn2Δ cells expressing Hos2‐GFP (green, B) or GFP‐VHL (green, C) were grown at 30°C and treated with MMS. Changes in protein localizations were recorded at the indicated time points. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined.",
    "molecules": "DNA, MMS"
  },
  {
    "caption": "B. Parental HEK293T,  HET and HOM1 cells were treated with either  Activin A or BMP4/7 (both at 20 ng/mL) for 1 h, in the presence or absence of either 1 µM LDN-193189  or 10 µM SB-431542. Quantifications are the levels of pSMAD1/5 normalized to the loading control (Actin or Tubulin), expressed as fold change relative to untreated for each clone. Quantifications are the average normalized intensities ± SEM of 3-5 independent experiments. Note that the  Activin A quantifications also includes data generated with high dose Activin A  in Fig. 3A as they are independent replicates. Data information: In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. hi, high; Act, Activin A; SB, SB-431542; LDN,  LDN-193189. The p-values (for A, B and D) are from a t-test with Holm-Sidak correction, for comparison between indicated pairs. ns, not significant; *, p< 0.05; **, p<0.01, ***, p<0.001; ****, p<0.0001. The p-values in C are from two-way ANOVA with Tukey's post hoc correction.",
    "molecules": "LDN, LDN-193189, SB, SB-431542"
  },
  {
    "caption": "C. NIH-3T3 cells were either  untransfected or  transfected  with either FLAG-SMAD1 or  GFP-SMAD3 together with Opto-ACVR1B*. Post  transfection, cells were either kept in the  dark or exposed to blue light for 1 h, in the presence or absence of 50 µM SB-505124 or 1 µM LDN-193189. Data information: In all panels, Western blots of whole cell lysates were probed with antibodies against pSMAD1/5 (that detected both endogenous and pFLAG-SMAD1), SMAD1 (which detects both endogenous and FLAG-SMAD1), HA (which detects the Opto-receptors), pSMAD3 (which detects pGFP-SMAD3), SMAD3 (which detects GFP-SMAD3) and either Tubulin  or Actin as the loading controls. Lane numbers are given below each blot for clarity. ",
    "molecules": "LDN-193189, SB-505124"
  },
  {
    "caption": "H. Average brightness of  His-Activin A-Atto647N in conditions (A-G). Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-3 biological replicates. Data information: In H and I the p values are from one-way ANOVA with Tukey's post hoc correction. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.",
    "molecules": "Atto647N"
  },
  {
    "caption": "I. Average brightness of His-Activin A-Atto647N in  ICR-B169 and  HSJD-DIPG-007 cells alone or with pre-incubation with 1.5 µg/mL of rhACVR2A Fc and 15 µg/mL of rhACVR2B Fc. Source data is in Fig. EV4B-F. Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-4 biological replicates. Data information: In H and I the p values are from one-way ANOVA with Tukey's post hoc correction. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.",
    "molecules": "Atto647N"
  },
  {
    "caption": "A, B.  HEK293T HOM1 cells were plated on a planar lipid bilayer containing (NIP)1-H12-Hylight647 (A) or  His-Activin A-Atto647N (B) and an automated time-lapse was started, where 2-4 positions within a well were  imaged every 40 s during a 27-min time period; 3 time points from one position are shown. TIRF, upper panels, brightfield, lower panels. The cluster intensities from all positions were quantified at 6 time points indicated (right panel) and plotted as individual cluster values. The horizontal gray bar denotes the mean. Cells + bilayer denotes bilayer areas under the cells, as identified from the brightfield images, and bilayer denotes areas where no cells were present. A representative experiment from 2 biological replicates quantified is shown. Scale bar 10 μm. In B, the region under cell 2 is shown enlarged in the top right-hand corner.",
    "molecules": "Atto647N, H12, Hylight647, (NIP)1"
  },
  {
    "caption": "PA binding of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a PA precipitation assay and a liposome flotation assay. The POPC:POPE:POPA ratios in each liposome were 50:50:0, 50:40:10, 50:30:20, and 50:20:30. Diagrams of the liposome precipitation and flotation assays are illustrated separately.",
    "molecules": "POPE, POPA, POPC, PA"
  },
  {
    "caption": "Results of fluorescence polarization (FP) experiments to monitor the interaction between the FFAT-like motif-containing peptide of PTPIP51 and VAPB_ΔTM. The Kd value of the interaction determined in the FP experiment was ~53 µM. Peptides with mutation of conserved phenylalanine and tyrosine residues in the FFAT motif were also tested. The data are presented as the mean ± SD of technical quadruplicate experiments.",
    "molecules": "phenylalanine, tyrosine"
  },
  {
    "caption": "CL levels after PTPIP51 depletion and reconstitution were monitored. A doxycycline-induced shRNA expression system was used to deplete the PTPIP51 protein in HeLa cells. Left, western blot analysis of HeLa cells in which PTPIP51 was depleted and restored. β-ACTIN was used as the loading control. Right, the mitochondrial CL level (nmol/mg) according to PTPIP51 expression. To reconstitute PTPIP51, the full-length PTPIP51 gene was transiently expressed. The data are presented as the mean ± SD of technical triplicate experiments from one representative experiment (n = 3), and p-values calculated using Student's t-test are shown.",
    "molecules": "CL, doxycycline"
  },
  {
    "caption": "Quantification of CL in mitochondria using lipidomic analysis after PTPIP51 depletion and reconstitution. Color codes for each sample were presented in the lower right subpanel. Experiments were technically repeated three times, and the results are represented as the means ± SD of technical replicates (n = 3). *p < 0.05; **p < 0.01 (Student's t-test). Quality control (QC) samples, which were pooled identical aliquots of the mitochondria samples, were measured four times throughout the run for data reproducibility. Upper right, western blot analysis of HeLa cells in which PTPIP51 WT or ∆FFAT mutant were depleted and restored. β-Actin was used as a loading control.",
    "molecules": "CL"
  },
  {
    "caption": "Western blot analysis of nephrin expression in immortalized podocytes cultured in normal (5 mM) or high glucose (HG; 30 mM) or high mannitol (Mann; 5 mM glucose plus 25 mM mannitol) for 24, 48 and 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).",
    "molecules": "glucose, Mann, mannitol"
  },
  {
    "caption": "B Analysis of the methylation status at nephrin gene promoter in podocytes cultured in normal, high glucose or high mannitol at the indicated time points. U, unmethylated-specific primers; M, methylated-specific primers.",
    "molecules": "glucose, mannitol"
  },
  {
    "caption": "C Effect of pargyline hydrochloride, a pan-KDM inhibitor, on high glucose-mediated nephrin expression. Immortalized mouse podocytes were cultured in the indicated cultured media in the presence or absence of pargyline hydrochloride (1 μM) for 48 hours. Protein lysates from the treated podocytes were analyzed for nephrin expression by immunoblotting. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Wilcoxon two-sample test; n = 3).",
    "molecules": "glucose, pargyline hydrochloride"
  },
  {
    "caption": "D Effect of pargyline hydrochloride on high glucose-mediated DNA methylation at nephrin gene promoter. Genomic DNA samples from the treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation-specific PCR.",
    "molecules": "glucose, pargyline hydrochloride"
  },
  {
    "caption": "E Quantitative RT-PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).",
    "molecules": "glucose, mannitol"
  },
  {
    "caption": "F Western blot analysis of KDM6A expression in podocytes cultured in normal, high glucose or high mannitol conditions for 24, 48 ad 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).",
    "molecules": "glucose, mannitol"
  },
  {
    "caption": "G Western blot analysis of H3K27me1, H3K27me2, H3K27me3 and pan-methyl H3K9 in podocytes cultured in normal, high glucose or high mannitol conditions for 9 and 24 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).",
    "molecules": "H3K27me2, methyl H3K9, glucose, H3K27me3, mannitol"
  },
  {
    "caption": "A Effect of KDM6A knockdown on the expression of nephrin and WT-1 in immortalized mouse podocytes cultured under high glucose conditions. Western blot analyses were performed using protein lysates of podocytes that were transfected with scrambled siRNAs or KDM6A-specific siRNAs, and cultured in high glucose (HG) or high mannitol (Mann) for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "glucose, Mann, mannitol"
  },
  {
    "caption": "C Changes in H3K27me3 levels in podocytes after treatment with high glucose, the combination of high glucose and KDM6A knockdown, or KDM6A overexpression for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "glucose, H3K27me3"
  },
  {
    "caption": "D Changes in DNA methylation at nephrin gene promoter by high glucose, by the combination of high glucose plus KDM6A knockdown, or by KDM6A overexpression in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers. Experiments were repeated three times and a representative gel from one experiment is shown.",
    "molecules": "glucose"
  },
  {
    "caption": "E Effect of GSK-J4 (40 μM), a specific KDM6A inhibitor, on high glucose-mediated changes in levels of nephrin, WT-1 and H3K27me3. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "glucose, GSK-J4, H3K27me3"
  },
  {
    "caption": "F Changes in DNA methylation at nephrin gene promoter by GSK-J4 (40 μM) in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers.",
    "molecules": "GSK-J4"
  },
  {
    "caption": "C Changes in body weights and levels of blood glucose in normal, diabetic and GSK-J4-treated diabetic mice. As noted, GSK-J4 treatment did not significantly affect body weights or blood glucose levels in diabetic mice during the experimental period of 12 weeks (Wilcoxon two-sample test; n = 8).",
    "molecules": "glucose, GSK-J4"
  },
  {
    "caption": "D Levels of urinary protein excretion, weights of kidney, and levels of HbA1c in normal, diabetic and GSK-J4-treated diabetic mice. Urinary total protein excretion, kidney weight and levels of HbA1c were measured at 12 weeks after diabetic induction. The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).",
    "molecules": "GSK-J4"
  },
  {
    "caption": "E Western blot analysis of KDM6A, nephrin, WT-1 and H3K27me3 expressed in kidney glomeruli of normal, diabetic and GSK-J4-treated diabetic mice at 12 weeks after diabetic induction. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "GSK-J4"
  },
  {
    "caption": "F Immunofluorescence analysis of KDM6A, nephrin and WT-1 in kidney sections from normal, diabetic, and GSK-J4-treated diabetic mice. Green: nephrin or WT-1; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "DAPI, GSK-J4"
  },
  {
    "caption": "G Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from normal, diabetic, and GST-J4-treated diabetic mice. Green: F-actin; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. Presented experiments were performed at least three times independently.",
    "molecules": "DAPI, GST-J4"
  },
  {
    "caption": "C Changes in body weights and levels of blood glucose in wild-type or KDM6A-KO mice with or without STZ treatment. No significant differences in body weights or blood glucose levels between wild-type and KDM6A-KO mice with diabetes were observed during the 8-week experimental period (Wilcoxon two-sample test; n = 8).",
    "molecules": "glucose, STZ"
  },
  {
    "caption": "D Levels of urinary protein excretion, weights of kidney and levels of HbA1c in wild-type and KDM6A-KO mice with or without STZ treatment (at 8 weeks after the onset of diabetes). The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).",
    "molecules": "STZ"
  },
  {
    "caption": "E Immunofluorescence images of kidney sections stained with KDM6A and nephrin in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "F Immunofluorescence images of kidney sections stained with KDM6A and H3K27me3 in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "H Western blot analysis of nephrin, KDM6A and WT-1 expressed in primary podocytes isolated from the above treated mice. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "I Effect of high glucose on the expression of podocyte-related markers in primary cultured podocytes isolated from wild-type or KDM6A-KO mice. The primary podocytes isolated from wild-type or KDM6A-KO mice were cultured in normal or high glucose (30 mM) for 48 hours. Protein lysates from the cultured podocytes were subjected to Western blot analysis with the indicated antibodies. *P < 0.05 versus wild-type podocytes in normal glucose, #P < 0.05 versus wild-type podocytes in high glucose (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "glucose"
  },
  {
    "caption": "C Increased expression of KDM6A and KLF10 in primary podocytes cultured in high glucose for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).",
    "molecules": "glucose"
  },
  {
    "caption": "D Effect of KLF10 knockdown on high glucose-mediated reduction of nephrin in primary podocytes. As noted, knockdown of KLF10 prevented downregulation of nephrin and upregulation of KDM6A in high glucose-treated podocytes. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "glucose"
  },
  {
    "caption": "E Immunofluorescence analysis of KLF10 and nephrin in renal sections of normal, diabetic, and GSK-J4-treated diabetic mice. Scale bars, 20 μm. *P < 0.05 versus the normal control group, #P < 0.05 versus the untreated diabetic group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "GSK-J4"
  },
  {
    "caption": "F Immunofluorescence images of KLF10 and nephrin in renal sections of wild-type or KDM6A-KO mice with or without STZ treatment. Scale bars, 20 μm. *P < 0.05 versus the untreated wild-type group, #P < 0.05 versus the STZ-treated wild-type group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "G Western blot analysis of KDM6A, KFL10 and nephrin expression in primary podocytes isolated from normal, diabetic, and GSK-J4-treated diabetic mice. *P < 0.05 versus normal controls, #P < 0.05 versus the untreated diabetic group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "GSK-J4"
  },
  {
    "caption": "H Western blot analysis of KDM6A, KLF10 and nephrin expression in primary podocytes isolated from wild-type or KDM6A-KO mice with or without STZ treatment. *P < 0.05 versus the untreated wild-type group, #P < 0.05 versus the STZ-treated wild-type group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "ChIP analysis of KLF10, acetyl-Histone H4 (H4-Ac), Dnmt1 and Dnmt3 binding to nephrin gene promoter. ChIP assays were carried out using cross-linked chromatin from primary podocytes that were cultured in normal or high glucose conditions. *P < 0.05, significant difference versus the normal control group (Wilcoxon two-sample test; n = 3).",
    "molecules": "glucose"
  },
  {
    "caption": "B Western blot analysis of KLF10 expression in kidney samples of wild-type or KLF10-KO mice treated with or without STZ at 8 weeks after the onset of diabetes.",
    "molecules": "STZ"
  },
  {
    "caption": "C Changes in body weight and blood glucose levels in wild-type or KLF10-KO mice with or without STZ treatment. No significant differences in body weights or blood glucose levels between diabetic wild-type mice and diabetic KLF10-KO mice were detected (Wilcoxon two-sample test; n = 8).",
    "molecules": "glucose, STZ"
  },
  {
    "caption": "D Levels of urinary protein excretion, weights of kidney and levels of HbA1c in wild-type or KLF10-KO mice with or without STZ treatment. The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).",
    "molecules": "STZ"
  },
  {
    "caption": "E Immunofluorescence images of kidney sections stained with KLF10 and nephrin in wild-type or KLF10-KO mice untreated or treated with STZ. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "F Immunofluorescence images of kidney sections stained with KLF10 and KDM6A in wild-type or KLF10-KO mice untreated or treated with STZ. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "G Immunofluorescence staining of F-actin and KLF10 in primary podocytes isolated from the wild-type or KLF10-KO mice untreated or treated with STZ. Presented experiments were performed at least three times independently. Scale bars, 20 μm.",
    "molecules": "STZ"
  },
  {
    "caption": "H Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from the wild-type or KLF10-KO mice untreated or treated with STZ. Presented experiments were performed at least three times independently. Scale bars, 20 μm.",
    "molecules": "STZ"
  },
  {
    "caption": "I Western blot analysis of KLF10, KDM6A, nephrin and WT-1 expression in primary podocytes isolated from the wild-type or KLF10-KO mice that were untreated or treated with STZ. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).",
    "molecules": "STZ"
  },
  {
    "caption": "B) Single point activity assays of USP1WT and deletion mutants on Ub-Rho show signifi­cantly increased activity in mutants where insert L1 and L3 are both deleted.",
    "molecules": "Rho, Ub"
  },
  {
    "caption": "C) Single point activity assays of USP1WT and deletion mutants (± UAF1) on Ub-Rho show the loss in hyper-activation of USP1ΔL1L3 upon addition of UAF1.",
    "molecules": "Rho, Ub"
  },
  {
    "caption": "D) Michaelis-Menten analysis of USP1 deletion mutants against ubiquitin-rhodamine (Ub-Rho) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants (n=2, biological replicates).",
    "molecules": "Rho, rhodamine, Ub, ubiquitin"
  },
  {
    "caption": "E) Michaelis-Menten analysis of USP1 deletion mutants (+UAF1) against Ub-Rho shows that all deletions mutants have similar catalytic activity (n=2, biological replicates).",
    "molecules": "Rho, Ub"
  },
  {
    "caption": "F) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i.e. LRGG-AMC (100µM). USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT (n=2, technical replicates).",
    "molecules": "AMC, LRGG"
  },
  {
    "caption": "G) Comparing activity of USP1WT and USP1ΔL1L3 (+UAF1) on a peptide substrate i.e. LRGG-AMC (100µM). Addition of UAF1 allows cleavage of LRGG-AMC by both USP1WT and USP1ΔL1L3 (n=2, technical replicates).",
    "molecules": "AMC, LRGG"
  },
  {
    "caption": "A) Quantification of gel-based activity assays where the activity of USP1WT and deletion mutants (+UAF1) on PCNA-UbTAMRA is compared. The USP1ΔL1 mutant has reduced activity on PCNA-UbTAMRA compared to the other USP1 variants tested (n=2, biological replicates).",
    "molecules": "TAMRA, Ub"
  },
  {
    "caption": "C) Quantification of gel-based activity assays where the activity of USP1 PIP mutants (+UAF1) on PCNA-UbTAMRA is compared. Both the PIP mutants have reduced activity on PCNA-Ub compared to USP1WT (n=2, biological replicates).",
    "molecules": "TAMRA, Ub"
  },
  {
    "caption": "D) FP based binding assays of USP1 mutants (+ UAF1) with PCNA-UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT (n=2, technical replicates).",
    "molecules": "TAMRA, Ub"
  },
  {
    "caption": "E) Quantification of gel-based activity assays where the activity of USP1WT+UAF1 on PCNA-UbWT is compared with PCNA-UbPIM1 shows that PCNA-UbWT is cleaved much faster compared to PCNA-UbPIM1 (n=2, technical replicates (example shown here from 1 of 2 biological replicates))",
    "molecules": "Ub"
  },
  {
    "caption": "F) Comparing activity of USP1WT and USP1PIP1 (+UAF1) on PCNA-UbWT and PCNA-UbPIM1 in gel-based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA, whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).",
    "molecules": "Ub"
  },
  {
    "caption": "A) Schematic representation of loading and purification of PCNA-Ub on nicked circular DNA. The DNA-loaded PCNA-Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA-loaded PCNA-Ub.",
    "molecules": "Ub"
  },
  {
    "caption": "B) Michaelis-Menten analysis of USP1+UAF1 with and without DNA (65bp dsDNA) shows that DNA binding alone has no effect on USP1 activity (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).",
    "molecules": "DNA, dsDNA"
  },
  {
    "caption": "C) Coomassie stained gel of in vitro activity assay showing increased activity of USP1-UAF1 on DNA-loaded PCNA-Ub compared to PCNA-Ub (-DNA) and PCNA-Ub (+DNA, +RFC and not ATP).",
    "molecules": "ATP, DNA, Ub"
  },
  {
    "caption": "D) Quantification of gel-based activity assays showing enhanced activity of USP1+UAF1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).",
    "molecules": "DNA, Ub"
  },
  {
    "caption": "E) Quantification of gel-based activity assays showing enhanced activity of USP1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).",
    "molecules": "DNA, Ub"
  },
  {
    "caption": "A) Gel based quantification of USP1-UAF1 activity on PCNA-Ub (DNA loaded and free) for three different concentration of enzyme and substrate.",
    "molecules": "DNA, Ub"
  },
  {
    "caption": "B) FP based activity assays on free PCNA-Ub (three concentrations of USP1 and PCNA-Ub) and activity of USP1 on increasing concentrations of Ub-Rho.",
    "molecules": "Rho, Ub"
  },
  {
    "caption": "A) Comparison of USP1WT and deletion mutants (+UAF1) for activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows no increase in USP1ΔL1 activity on the loaded substrate. Left panel: Quantification of gel-based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, biological replicates), Right panel: Quantification of the activation fold observed in USP1WT and mutants on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, biological replicates).",
    "molecules": "DNA, Ub"
  },
  {
    "caption": "B) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species. Three groups of positively charged residues (***) are mutated separately and tested for binding to double stranded DNA (65bp) using SPR which shows the importance of this region for DNA binding of USP1.",
    "molecules": "DNA"
  },
  {
    "caption": "C) Comparison of USP1DM1, USP1PIP1 and USP1PIP1+DM1 (+UAF1) activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows reduced increase in activity of these mutants compared to USP1WT. Left panel: Quantification of gel based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, technical replicates), Right panel: Quantification of the activation fold observed in USP1WT, USP1PIP1, USP1DM1 and USP1PIP1+DM1 on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, technical replicates).",
    "molecules": "DNA, Ub"
  },
  {
    "caption": "D) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA.",
    "molecules": "DNA"
  },
  {
    "caption": "BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP- expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.",
    "molecules": "cytokine"
  },
  {
    "caption": "BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low-cells expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.",
    "molecules": "cytokine"
  },
  {
    "caption": "B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs, and after three days cytokine release (B) were measured using a 3H-thymidine uptake- and a Legendplex assay, respectively. Mean values and standard deviations from triplicate samples.",
    "molecules": "cytokine"
  },
  {
    "caption": "C-F. 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors (5 animals per group), and uptake at the indicated locations was determined by PET quantification over time (C, D) and at termination of the experiment after 48 hours (E, F) as described in the methods section. Error bars represent standard deviation of the mean value.",
    "molecules": "Cu"
  },
  {
    "caption": "(B) MPL-MIL HCM assay in HeLaWT and HeLaHexaKO expressing HT-LC3B, starved in EBSS for 90min ± 100 nM BafA1. Cells were sequentially incubated with HT ligands MIL and MPL, and HCM quantification carried out. Plasma membrane permeabilization was carried out with 4 nM Agilent XF Plasma Membrane Permeabilizer/PMP. PMP was used during the MIL staining and prior to fixation in 4% paraformaldehyde. MPL staining followed the fixation. (B i) MPL-accessible membrane-bound HT-LC3B (remaining available to MPL after MIL saturation). (B ii) MIL-accessible membrane-bound HT-LC3B; (B iii) MIL/MPL puncta ratio. Circles, control samples (full medium); rectangles (EBSS); triangles (EBSS+BafA1); green, MIL; red, MPL; gray, MIL/MPL ratios. Images and statistics, HCM images (masks: white, primary objects/cells; red, MPL profiles; green, MIL profiles), one of 60 fields/well, 1,000 primary objects (cells) per well; 6 wells per plate (sampling error), triplicate plates (independent biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent replicates.",
    "molecules": "MIL, MPL, BafA1, paraformaldehyde"
  },
  {
    "caption": "(C-E) Selected electron microscopy (EM) images of HeLaWT and HeLaHexaKO EBSS-induced for autophagy for 90 min and their quantification (graph in F). AP, autophagosomes (phagophores or double membrane autophagosomes with content of similar electron-density to surrounding cytosol); Amph, amphisomes; AL, autolysosomes; MVB, multivesicular bodies; LELy, late endosomes or lysosomes. Statistics, unpaired t-test, two groups: HeLaWT and HeLaHexaKO (93 images each; sample mean, SE). Blue single asterisk, cytoplasmic cargo inside amphisome or autolysosome; double asterisk, phagophore structures; arrows, phagophores or autophagosomes. (F) Super-resolution (dSTORM) of HeLaHexaKO HT-LC3B stained with MIL (note staining of the interiors of the globular structures).",
    "molecules": "MIL"
  },
  {
    "caption": "(B) Representative images of SolVit assay with HeLaWT and HeLaHexaKO as donors, and HeLaHexaKO HT-LC3B as acceptor. SolVit HCM images (example). MPL+ (red), sealed LC3B+ membranes MIL+ (green), unsealed LCB+ membranes. (C) Quantification of MPL+ and MIL+ profiles (60 fields/well acquired): (C i) MPL+ (red) puncta per field. (C ii) MIL+ (green) puncta per field. (C iii) MIL+/MPL+ profile ratios (gray). Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent replicates, each HCM experiment: 1,000 valid primary objects/cells per well (max fields per well, 60), 5 wells/sample.",
    "molecules": "HT, MIL, MPL"
  },
  {
    "caption": "(B) Cells were starved in EBSS for 90 min ±100 nM BafA1, sequentially incubated with MIL and MPL. MPL (B i; red), marker of sealed membranes; MIL (B ii; green), marker of unsealed membranes; profiles quantified by HCM. (B iii) MIL+/MPL+ puncta ratio (gray),",
    "molecules": "MIL, MPL, BafA1"
  },
  {
    "caption": "(C) HelaWT, HeLaHexaKO, HeLaLC3TKO, HeLaGABATKO stably expressing HT-LC3B were starved with EBSS for 90min or 6h. Cell were incubated with ± HaloTag ligand fluorescently labeled with TMR. In-gel fluoresce (TMR) and Western blots (anti-HaloTag antibody) of freed HaloTagTMR were imaged and band intensity quantified. (D) Quantification of in-gel fluorescence band intensity and Western blot with anti-Halo antibody.",
    "molecules": "TMR"
  },
  {
    "caption": "(C) SolVit complementation/rescue analysis (quantification) of acceptor HeLaLC3TKO-HT-LC3B PNS with donor PNS from HeLaWT, HeLaLC3TKO or HelaGABATKO cells ±ATP, 1 h, 37˚C. (D) SolVit complementation/rescue analysis (quantification) of acceptor HeLaGABATKO-HT-LC3B PNS with donor PNS from HeLaWT, HeLaLC3TKO and HelaGABATKO cells ±ATP, 1 h, 37˚C.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) GST pulldown analysis of in vitro translated and radiolabeled [35S] Myc-VPS37A with GST or GST tagged LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2. 2% of input was loaded.",
    "molecules": "35S"
  },
  {
    "caption": "(I) Complementation of VPS37A KO with VPS37A constructs that have or lack mATG8-binding region. Quantification of MIL+ puncta in Huh7WT HT-LC3B (circles) and Huh7VPS37AKO HT-LC3B cells (squares), transfected with empty FLAG, full length VPS37A (FLAG-VPS37AFL) or mutant VPS37A (FLAG-VPS37AdeltaN(1-20)). Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are means ± SD, n = 3 biologically independent replicates, each HCM experiment: 1,000 valid primary objects/well, 6 wells/sample. (J) HCM images, examples corresponding to Fig 5I, scale bar, 10 μm.",
    "molecules": "MIL"
  },
  {
    "caption": "(B) MPL-MIL HCM assay in Huh7WT and Huh7VPS37AKO stably expressing HT-LC3B. Quantification: (B i) membrane-permeant HT ligand (MPL) staining of LC3B-II sequestered within sealed membranes. (B ii) MIL staining of membrane-bound HT-LC3B-II accessible to the cytosol and (B iii) MIL/MPL puncta ratio.",
    "molecules": "membrane-permeant HT ligand, MIL, MPL"
  },
  {
    "caption": "(F-H) MPL-MIL HCM assay in Huh7WT (control or CHMP2A siRNA-treated cells; immunoblot in F) stably expressing HT-LC3B during CCCP-induced mitophagy in ± BafA1 (100 nM). (G) Quantification: (G i) MPL+ sealed membranes; (G ii) MIL+ unsealed membranes; (G iii) MIL/MPL puncta ratio. (H) HCM images corresponding to panel G.",
    "molecules": "MIL, MPL, BafA1, CCCP"
  },
  {
    "caption": "(B) Quantification of puncta: (B-i) MPL+ puncta (red), sealed LCB+ membranes; (B-ii) MIL+ (green) puncta, unsealed/ligand-accessible LCB+ membranes; (B-iii) MIL/MPL ratios (gray).",
    "molecules": "MIL, MPL"
  },
  {
    "caption": "(C) Quantification of MIL+ puncta (unsealed/ligand-accessible LC3B+ membranes) in Huh7WT (circles) or Huh7VPS37AKO (squares) cells stably expressing HT-LC3B. Cells were transfected with empty FLAG vector, FLAG-VPS37A full length (VPS37AFL), FLAG-VPS37AdeltaN(1-90), or FLAG-VPS37AdeltaN(1-20) constructs. CCCP, 6 h treatment. (D) HCM images of Huh7VPS37AKO HT-LC3B with VPS37A constructs (VPS37AFull, FLAG-VPS37AdeltaN(1-90), FLAG-VPS37AdeltaN(1-20) and FLAG-VPS37ANter-GFP). FLAG-Tag VPS37A constructs, expression detected as red; and MIL+ puncta, pseudo color green. Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent experiments, each HCM experiment: 1,000 valid primary objects/cells per well, 6 wells/sample, Scale bar, 10μm. ",
    "molecules": "MIL, CCCP"
  },
  {
    "caption": "Gene expression analyzed by  quantitative PCR in brown adipocytes that were fed with or without 1mM  asparagine as in (A). n=3 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.",
    "molecules": "asparagine"
  },
  {
    "caption": "Preadipocytes  bearing the shRNA targeting  Pgc1α were induced differentiation with or without 1mM  asparagine. Protein expression was analyzed on day 6. n=3 biological replicates.",
    "molecules": "asparagine"
  },
  {
    "caption": "Brown preadipocytes were infected by lentiviral shRNAs against  Asns. Cells were differentiated in culture media with or without 1mM asparagine. Western blot  was performed on day 6.",
    "molecules": "asparagine"
  },
  {
    "caption": "I,J.  Mice  supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal (I) and CL316,243-stimulated (J) states. n=5  mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. ",
    "molecules": "asparagine, CL316,243"
  },
  {
    "caption": "Hematoxylin  and  eosin staining of BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) of  mice in (A).",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "Glucose tolerance test was performed after a high-fat diet  feeding for 17 weeks in  mice of (A). n=5 mice each group.   Serum insulin  levels in mice of (A). n=5 mice each group.   Serum triglyceride levels in mice  of (A). n=5 mice each group.   Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.",
    "molecules": "insulin, triglyceride"
  },
  {
    "caption": "Heat map of metabolite relative levels  in brown fat cells treated with or without 1mM asparagine.",
    "molecules": "asparagine"
  },
  {
    "caption": "Immunoblot detection of the  glucose  transporters, glycolysis and TCA key enzymes in primary brown fat cell  line Asparagine was supplemented to cells during differentiation.",
    "molecules": "Asparagine, glucose"
  },
  {
    "caption": "Immunoblot detection of the glucose  transporters, glycolysis and TCA key enzymes in immortalized brown fat cell line (E), BAT from  mice fed asparagine (F) and injected asparaginase (G). Asparagine  was supplemented to cells during differentiation.",
    "molecules": "asparagine, Asparagine, glucose"
  },
  {
    "caption": "Intracellular abundance of 13C-isotopologue metabolites downstream of glycolysis in brown adipocytes. n=5 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.",
    "molecules": "13C"
  },
  {
    "caption": "C,D. Quantification of glucose (C) and lactate (D) concentration from media. The media was collected after incubated with mature brown adipocytes for 48 h. n=3 independent experiments in (C), n=6 independent experiments in (D). Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test. ",
    "molecules": "glucose, lactate"
  },
  {
    "caption": "Quantification of intracellular amino acid levels in brown fat cells (day 6) treated with or without 1mM asparagine. n=4 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.",
    "molecules": "amino acid, asparagine"
  },
  {
    "caption": "B-D.  mTORC1  pathway activity was analyzed by immunoblotting for the phosphorylation of 4E-BP1 and S6K in brown adipocytes (B), BAT from mice fed asparagine (C) or asparaginase  (D). (B) Asparagine was added to cultured media from day 4 to day 6.",
    "molecules": "asparagine, Asparagine"
  },
  {
    "caption": "Brown preadipocytes were induced to differentiation with or without 1mM  asparagine. Rapamycin (75nM) was added to cells for 48h. Then western blot detected protein expression on day 6.",
    "molecules": "asparagine, Rapamycin"
  },
  {
    "caption": "Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation. Protein expression analysis was performed on day 6.",
    "molecules": "asparagine"
  },
  {
    "caption": "(A) A431 WT and SKO cells -/+ GFP-fused WT or A212P-seipin were delipidated, treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (B) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/cell in indicated size category, mean ± SEM, n=57-61 cells/genotype, 2 experiments, p**< 0.005 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(E) SKO + WT-seipin-GFP cells were delipidated and treated with OA for 1h, fixed and stained with LipidTox Red. Single focal plane of a 3D-SIM z-stack. (F) Analysis of (E), bars: % of LDs/cell with the indicated number of WT-seipin-GFP punctae associated, mean ± SEM, n=12 cells (693 LDs), 2 experiments, p**< 0.005 (Tukey multiple comparisons test).",
    "molecules": "OA"
  },
  {
    "caption": "(G) SKO + WT- or A212P-seipin-GFP cells were delipidated, incubated with OA for 30 min, and immunolabelled for TEM with anti-GFP antibodies. Black arrowheads: immuno-gold, orange arrowheads: ER-LD junctions. See also Appendix Fig S3. (H) Analysis of (G), bars: relative labeling index in different cellular compartments, n=3456-4512 immuno-gold clusters. Distribution of both WT- and A212P-GFP-seipin immunolabel is non-random (chi square test, **p<0.005), but there is significant enrichment of WT- but not A212P-seipin-GFP at ER-LD contacts relative to the ER (binomial test, **p<0.005). See also Appendix Table S1 and S2.",
    "molecules": "OA"
  },
  {
    "caption": "(I) A431 WT cells with seipin tagged with sf-GFP at its chromosomal locus and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 1 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.",
    "molecules": "OA"
  },
  {
    "caption": "(A) SKO cells stably expressing WT-seipin-GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30-75 min. Airyscan video snapshots, upper panel: an example of 2 seipin-GPF punctae, one stably associated with a LD (blue arrowhead) and another not LD associated (orange arrowhead). Lower panel: an example of a mobile LD, with a single seipin puncta moving together with it (blue arrowhead). (B) Analysis of (A), bars: mean velocity of tracked seipin puncta ± SEM, n=13 ROIs from 13 cells, 90 puncta, 2 experiments, **p<0.005 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(C) Analysis of LD mobility in WT and SKO cells. Cells were incubated with OA for 45-75 min, LDs were visualized with LD540 and cells imaged by widefield microscopy. Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n= 149-151 cells, 2 experiments, **p<0.005 (Mann Whitney test). (D) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD. (E) Analysis of the LD mobility from (C) and (D), individual LDs plotted for volume and mean velocity.",
    "molecules": "OA"
  },
  {
    "caption": "(F) WT and SKO cells were transfected with ER marker plasmid (sfGFP-ER-3, green), delipidated overnight and incubated with OA and LipidTox Deep Red (magenta) for 15-75 min. Airyscan video snapshots exemplifying different modes of LDER-associated movement, note the different time scales. Panel I and II: WT cell, panel III: SKO cell. (G) Analysis of LDER-associated movement from live airyscan microscopy as in (F). Bars: % of LDs/ROI displaying indicated mode of movement, ± SEM, n= 43-55 ROIs (201-467 LDs) from 4 experiments, **p<0.005 (Mann Whitney test). (H) Analysis of LD velocity. Bars: mean velocity of tracked LDs, ± SEM, n=29-42 LDs/group, 2 experiments, **p<0.005 (Kruskal-Wallis test, followed by Dunn test).",
    "molecules": "OA"
  },
  {
    "caption": "(I) WT and SKO cells stably expressing HSP47-APEX were delipidated, treated with OA for 1 h and processed for SB-EM. 3D models of LDs and surrounding ER and mitochondria profiles shown on top of a block face image from SB-EM dataset. Insets are representative cross sections from modeled area highlighting the ER-LD association. Red= LD, Yellow= ER, Green=mitochondria. (J) Analysis of LDs from (I). Black lines indicate median, boxes third quartiles, n= 98-227 LDs. (K) Analysis of LDs displaying no ER contacts from (I). Bars: mean % of LDs in ROIs not in proximity to the ER, ± SEM, n= 11-16 ROIs/genotype, **p<0.005 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(A) WT and SKO cells were treated with OA for 1 h and processed for ET. Representative single tomographic slices of ER-LD contacts. Orange arrowheads indicate narrow ER-LD contacts, blue arrowheads indicate widened ER profiles seen in SKOcells. (B) 3D-models of LDs and ER-LD contacts based on tomograms in (A). Yellow: ER, brown: LD, red: ER-LD contacts.",
    "molecules": "OA"
  },
  {
    "caption": "(C) Exemplary TEM image of a WT cell incubated with OA for 1h. A grid is randomly placed on top of the image and the width of perpendicularly oriented ER profiles passing through grid lines are measured. Yellow lines indicate measured sites. (D) ER profile analysis of (C). Bars: average profile width/cell, ± SEM, n= 11-9 cells (149-224 profiles), *p<0.05 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(A) WT and SKO cells stably expressing ACSL3-Cherry were transfected with ADRP-GFP for 24 h, incubated with OA for 1 h, fixed and stained with LD540. Single confocal planes of deconvolved confocal z-stacks. (B) Colocalization analysis of (A), from single confocal sections. Bars: correlation coefficient m1 ± SEM, n=34-35 cells/genotype, 2 experiments, **p<0.005 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(C) WT and SKO cells were transfected with ADRP-GFP for 24 h, incubated with OA for 2 h and co-plated with delipidated WT or SKO cells stably expressing ACSL3-Cherry. After co-plating, cells were incubated for 2.5 h in normal growth medium, then fused together using PEG. Cells were fixed and imaged 1 h post-fusion. Single confocal planes.",
    "molecules": "OA, PEG"
  },
  {
    "caption": "(E) WT and SKO cells were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, single confocal planes of deconvolved z-stacks. (F) Analysis of (E), insets illustrate assigned categories of ACSL3LD association. Bars: % of LDs/ROI in the indicated category, mean ± SEM, n= 9-10 peripheral ROIs from 9-10 cells/treatment (616-968 LDs), 3 experiments, **p<0.005 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(G) TEM images of anti-ACSL3-labeled WT and SKO cells treated with OA for 1 h. (H) Analysis of (G), n= 66-119 LDs from 8 cells/genotype.",
    "molecules": "OA"
  },
  {
    "caption": "(A) WT and SKO cells stably expressing HPos-Cherry were incubated with OA for 4 h and LDs were labelled with LipidTox Green. The HPos-Cherry signal was bleached and signal recovery to LDs monitored using airyscan imaging. Representative images of 10 acquisitions/genotype, 2 experiments. Circle denotes bleached region, arrowheads indicate HPos signal accumulating around the LD. (B) Analysis of HPos-Cherry recovery to LDs after photobleaching, performed by confocal microscopy after 2-4 h OA loading. Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=22-25 bleached LDs/genotype, 2 experiments.",
    "molecules": "OA"
  },
  {
    "caption": "(C) WT and SKO cells were delipidated for 72 h, with the last 24 h in the presence of 1 µM BPY-C12 to label cellular lipids. LD biogenesis was induced by incubating cells in the presence of 0.1 mM OA and LipidTox Green for 1-2 h. BPY-C12 on LDs was bleached and recovery to LDs monitored using airyscan imaging or conventional confocal imaging. (D) Airyscan video snapshots of BPY-C12 recovery to LDs as described in (C). Circle denotes bleached ROI. For the last images of both panels brightness of BPY channel has been increased. (E) Analysis of BPY-C12 recovery to LDs from photobleaching experiments described in (C), data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=22 bleached LDs/genotype from 3 experiments.",
    "molecules": "lipids, OA"
  },
  {
    "caption": "(F) Delipidated WT and SKO cells were incubated with OA and LipidTox green for 1 h to induce LD formation, followed by 10 min labeling with BPY-C12. Live cellconfocal images were captured at indicated chase time points in the presence of OA and LipidTox green. (G) Colocalization of BPY-C12 and LDs (Pearson's colocalization correlation coefficient) ± SEM, n=8-26 cells/time point, 2 experiments, *p<0.05, **p<0.005 (unpaired T-test, between WT and SKO at the same time point).",
    "molecules": "OA"
  },
  {
    "caption": "(H) Delipidated WT and SKO cells were pulsed for 10 min with 0.1 mM alkyne-OA either directly (1-2) or after pre-incubation with 0.1 mM OA for 1 h (3-4). After alkyne-OA incubation cells were collected (1 and 3) or further incubated for 20 min in 10% FBS medium (2) or 0.1 mM OA medium (4). Bars: % of alkyne-OA in neutral lipids (triacylglycerol and cholesterol esters), normalized to WT pulse (1), ± SEM, n=6-9 samples, 2-3 experiments, *p<0.05, **p<0.005 (unpaired T-test).",
    "molecules": "alkyne-OA, cholesterol esters, OA, triacylglycerol"
  },
  {
    "caption": "(A) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 20 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.",
    "molecules": "OA"
  },
  {
    "caption": "(B) WT and SKO cells were treated with OA for 20 h, fixed and stained with LD540. Maximum projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (C) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/field of view in indicated size category, mean ± SEM, n=25-30 fields of view (100-120 ROIs of not clustered LD areas from 100-120 cells), 2 experiments, p**< 0.005 (unpaired T-test). See also Appendix Figure S5D.",
    "molecules": "OA"
  },
  {
    "caption": "(D) TEM micrographs of WT and SKO cells treated with OA for 20 h.",
    "molecules": "OA"
  },
  {
    "caption": "(G) WT and SKO cells were treated with OA for 20 h, fixed and stained with anti-ACSL3 antibodies and LD540. Maximum intensity projections of deconvolved confocal z-stacks. Insets show small and large LDs. (H) Analysis of (G). Bars: % of small (diameter <0.5 µm) or large (diameter >0.5 µm) LDs/cell in the indicated category, mean ± SEM, n=10 ROIs from 10 cells/treatment (305-581 LDs), **p<0.005 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(I) WT and SKO cells stably expressing HPos-Cherry were incubated with OA for 20 h, and LDs were labelled with LipidTox Green prior to imaging. The HPos-Cherry signal was bleached and signal recovery to LDs was monitored. (J) Analysis of (I). Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=51-61 bleached LD areas/genotype, 3 experiments.",
    "molecules": "OA"
  },
  {
    "caption": "(A) Two control and BSCL2fibroblast cell lines were delipidated and treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks.",
    "molecules": "OA"
  },
  {
    "caption": "(B) Control and BSCL2fibroblasts were incubated with OA for 75-90 min, LDs were stained with LD540 and cells imaged live with widefield microscopy. Insets show overlay of two subsequent frames (400 ms apart). (C) Analysis of (B). Bars = 1 - [Pearson's colocalization coefficient between subsequent frames/ROI], ± SEM, n=120-140 ROIs (60-70 cells), 2 experiments, **p<0.005 (Mann Whitney test).",
    "molecules": "OA"
  },
  {
    "caption": "(D) Control and BSCL2fibroblasts were transfected with ER marker plasmid (sfGFP-ER-3) for 24 h and treated with OA for 10-60 min. LipidTox Deep Red or LD540 was used to label LDs and cells were imaged live by airyscan microscopy. Examples of LDs with high mobility are shown, blue arrowheads indicate a LD moving along the ER, orange arrowheads indicate LDs moving independent of the ER. (E) Analysis of ER associated movement of LDs from live airyscan microscopy as in (D). Bars: % of LDs/ROI displaying indicated mode of movement, mean ± SEM, n= 50-42 ROIs (218-374 LDs), 2 experiments, **p<0.005 (Mann Whitney test).",
    "molecules": "OA"
  },
  {
    "caption": "(F) Control and BSCL2fibroblasts were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, maximum intensity projections of deconvolved confocal z-stacks. (G) Analysis of (F), with insets illustrating assigned categories of ACSL3 LD association. Insets are the same as in Fig 4F. Bars: % of LDs/ROI showing the category indicated, mean ± SEM, n= 11-10 peripheral ROIs from 11-10 cells/group (1200-2019 LDs), 2 experiments, **p<0.005 (unpaired T-test).",
    "molecules": "OA"
  },
  {
    "caption": "(H) Delipidated fibroblasts were pulsed for 20 min with 0.1 mM alkyne-OA either directly (1) or after pre-incubation with 0.1 mM OA for 1 h (2), and chased in the presence of 0.1 mM OA for 40 min. Bars: % of alkyne-OA in neutral lipids (triacylglycerol and cholesterol esters), normalized to control cells in (1), mean ± SEM, n=11-12 samples, 2 experiments, *p<0.05 (unpaired T-test). For C,E,G and H the results are pooled of 2 controls and 2 patients.",
    "molecules": "alkyne-OA, cholesterol esters, OA, triacylglycerol"
  },
  {
    "caption": "B Equilibrium titration of Hsf1 binding to Alexa Fluor® 488-labeled HSE-DNA. Fluorescence polarization plotted versus the Hsf1 trimer concentration. KD = 4.7 ± 1.9 nM (n = 4).",
    "molecules": "Alexa Fluor® 488, DNA"
  },
  {
    "caption": "C Hsf1 rapidly switches between different HSE-containing double stranded DNA oligonucleotides. Trimeric Hsf1 was pre-incubated with Alexa Fluor® 488-labeled HSE-DNA in MgCl2-free buffer. At timepoint 0 (arrow) buffer, 4 mM MgCl2, 2 mM ATP+4 mM MgCl2, control DNA (DNACtrl at 10-fold molar excess over HSE-DNA), and HSE-containing DNA was added as indicated and fluorescence polarization followed over time.",
    "molecules": "Alexa Fluor® 488, ATP, DNA, double stranded DNA, MgCl2"
  },
  {
    "caption": "D Trimeric Hsf1 were bound to HSE-DNA and the indicated components added at timepoint 0.",
    "molecules": "DNA"
  },
  {
    "caption": "E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70. Rates were determined",
    "molecules": "DNA"
  },
  {
    "caption": "The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on temperature (for 25°C n = 6, for 37°C n = 5, T-test, p = 0.011) (F)",
    "molecules": "DNA"
  },
  {
    "caption": "The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of Hsc70 and DnaJB1 (n = 3) (G)",
    "molecules": "DNA"
  },
  {
    "caption": "The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of ATP (n = 3) (H)",
    "molecules": "ATP, DNA"
  },
  {
    "caption": "The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of the nucleotide exchange factor Apg2 (HSPA4) (n = 3, ANOVA, **, p < 0.01; ****, p < 0.0001) (I). Shown are mean ± SD.",
    "molecules": "DNA"
  },
  {
    "caption": "A,B Standard Hsc70/DnaJB1-mediated dissociation reaction of Hsf1 from Alexa Fluor® 488-labeled HSE-DNA monitored by fluorescence polarization (A). The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x-axis, the reaction stopped by addition of blue-native loading buffer and stored on ice until separation by blue-native PAGE, blotted onto a PVDF membrane and detected with an Hsf1-specific antiserum (B). Lanes 1, purified Hsf1 trimer (T); 2, purified Hsf1 monomer (M); 3-11, samples from the Hsc70/DnaJB1-mediated Hsf1 dissociation reaction (0 to 80 min); 12-15, Dissociation reaction incubated for 80 min missing individual components as indicated. HO, higher order oligomers; T, trimer; D, dimer; M, mono­mer. C Quantification of the amounts of Hsf1 species of the blot shown in (B) and five similar blots. For each lane the intensities of the two areas indicated by the brackets to the right were integrated; upper bracket, DNA bound timers and higher order oligomers (Hsf1b); lower bracket, monomers and Hsf1-species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA (Hsf1d). Shown are means ± SD (n = 3). Inset, deoligomerization rate as determined by fitting an exponential decay function to the data. Shown are means ± SD (n = 3) ",
    "molecules": "Alexa Fluor® 488, DNA"
  },
  {
    "caption": "E Relative deuteron incorporation of the low (solid bars) and high (open bars) exchanging subpopulations.",
    "molecules": "deuteron"
  },
  {
    "caption": "C Hsf1(1-408) with a deleted TAD is dissociated from HSE-DNA by Hsc70, DnaJB1 and ATP with similar rates as Hsf1wt. Fluorescence polarization assays containing Alexa Fluor® 488-HSE-DNA bound Hsf1(1-408) trimers and the indicated components.",
    "molecules": "Alexa Fluor® 488, ATP, DNA"
  },
  {
    "caption": "D The HR-B proximal Hsc70 binding site is essential for Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1.",
    "molecules": "DNA"
  },
  {
    "caption": "A The rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1 depends on the number of HR-B proximal Hsc70 binding sites in the Hsf1 trimer. Hsf1wt (wt) and Hsf1∆(202-213) (∆) monomers were mixed in the indicated ratios and heat shocked at 42°C for 10 min to form mixtures of different homo- and heterotrimers as indicated with the cartoons (the red line and the arrow head indicate the Hsc70 binding site). Numbers indicate the relative fraction of the different species.",
    "molecules": "DNA"
  },
  {
    "caption": "B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA-bound Hsf1. Hsf1_t10/20/30, region 202-213 moved by 10, 20, or 30 residues towards the C-terminus.",
    "molecules": "DNA"
  },
  {
    "caption": "C Anti-FLAG antibodies are not able to dissociate HSE-DNA-bound Hsf1. Red lines in the cartoon indicate the Hsc70 binding site; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK. Hsf1_i201/211/221, FLAG-epitope inserted after residue 201, 211 or 221. Anti-FLAG antibodies were split in halfmers by incubation with 2 mM DTT",
    "molecules": "DTT, DNA"
  },
  {
    "caption": "Anti-FLAG antibody halfmers bound to FLAG-epitope containing Hsf1 variants. Hsf1wt and FLAG-insertion variants were analyzed by blue-native gel electrophoresis in the absence or presence of DTT-treated anti-FLAG antibodies as indicated and Hsf1 detected by immunoblotting; t-bound, FLAG antibody-bound Hsf1 trimers; m-bound, FLAG antibody bound Hsf1 monomer.",
    "molecules": "DTT"
  },
  {
    "caption": "G Rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1wt and Hsf1-I190S,I194S",
    "molecules": "DNA"
  },
  {
    "caption": "D. Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. The dividing lines correspond to gels sections visible in the raw data file.",
    "molecules": "digitonin"
  },
  {
    "caption": "Figure 3. Decrease of nucleoid number without mtDNA depletion in patient fibroblasts. A. In control and patient fibroblasts, nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p<0.001). Nucleoid number (B): patient 1 versus control ***: p=0.0001, patient 2 versus control ***: p=0.001. Nucleoid size (C): patient 1 versus control ***: p=0.0001. D. Mitochondrial DNA content in control and patientfibroblasts was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nuclear DNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).",
    "molecules": "DNA"
  },
  {
    "caption": "Figure 4. Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles. A. Transfections were performed with empty vector (EV) or vectors encoding wild-type CHCHD10-FLAG (WT), CHCHD10-FLAG (S59L) or CHCHD10-FLAG (P34S) mutants. Mitochondria were stained with Mitotracker. Cells overexpressing wild-type and mutant CHCHD10 were labeled with FLAG antibodies. Nucleoids were visualized with an antiserum against DNA. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): very significant (**: p=0.0280) or extremely significant (***: p=0.0001). D. Mitochondrial DNA content was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nDNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).",
    "molecules": "DNA, nDNA"
  },
  {
    "caption": "Figure 5. Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress. A. Total extracts from control (C) fibroblasts and intact isolated mitochondria from control and patient fibroblasts (P1, P2) were analyzed by immunoblotting using antibodies against PCNA (nuclear protein), GAPDH (cytosolic protein) or SMAC (mitochondrial intermembrane space protein) (upper panel). Mitochondria from patient and control fibroblasts were incubated with NP-40 and separated into pellets (P) and supernatants (S). The fractions of each extraction were subjected to western blot analysis. VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins, respectively (middle panel). Ratio of mtDNA amplified from supernatant/mtDNA amplified from pellet, by qPCR, was quantified in control and patient fibroblasts (lower panel).",
    "molecules": "NP-40"
  },
  {
    "caption": "B. Determination of the mtDNA copy number after H2O2 treatment. mtDNA/nDNA values in control, patient 1 and patient 2 fibroblasts. mtDNA : mitochondrial DNA, nDNA: nuclear DNA. C. mtDNA repair after H2O2 treatment. A long-range PCR was used to evaluate the oxidative damage, induced by H2O2 treatment, in mtDNA. The relative PCR amplification of a 15.6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment. mtDNA repair activity in control, patient 1 and patient 2 relative PCR. B-C. Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times. Untreated control cultures were incubated in serum-free medium alone. Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed. Values were normalized to untreated cells and differences were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01), very significant (**:0.01>p>0.001). Patient 1 versus control: *: p=0.015 (recovery 1h), *: p=0.022 (recovery 2h), **: p=0.003 (recovery 4h). Patient 2 versus control: *: p=0.0230 (recovery 2h), *: p=0.041 (recovery 4h). NT: cells not treated with H2O2",
    "molecules": "serum, nDNA, nuclear DNA, H2O2"
  },
  {
    "caption": "Figure 6. No autophagy inhibition (A-C) and no mitophagy defect (D-E) in patient fibroblasts. A. Immunoblot of LC3B-I/-II expression in fibroblasts of patient 2 (1,2), patient 1 (3,4) and control individual (5,6) in absence (-) or presence (+) of chloroquine (CLQ). B. Quantitative analysis of LC3B-II/β-tubulin ratio from immunoblots (n=3). C. Quantitative analysis of LC3B-II + CLQ/ LC3B-II - CLQ ratio from immunoblots (n=3).",
    "molecules": "chloroquine, CLQ"
  },
  {
    "caption": "Figure 7. CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts. A. Control (C1, C2) and patient (P1, P2) fibroblasts were treated with 1 μM of staurosporine (STS) for 16 h. Cell death was determined by flow cytometry using Annexin V/DAPI staining. Three independent experiments were performed per condition with two points analyzed per experiment. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*: p=0.0116) or extremely significant (***: p=0.0001).",
    "molecules": "staurosporine, STS"
  },
  {
    "caption": "B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control)",
    "molecules": "STS"
  },
  {
    "caption": "B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control) or by DEVDase activity measurement (C). Two (B) or three (C) independent experiments were performed per condition. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001).",
    "molecules": "STS"
  },
  {
    "caption": "D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).",
    "molecules": "STS"
  },
  {
    "caption": "D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).",
    "molecules": "STS"
  },
  {
    "caption": "B-D. HeLa cells transiently expressing the WT or mutated forms of CHCHD10-FLAG (P34S or S59L) were treated with 1 μM Actinomycine D (ActD) for 4, 6 or 8 h with measurement of Annexin V/DAPI staining (B), DEVD-ase activity (C) from three independent experiments. Differences between the mutated and non mutated alleles were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01) or very significant (**:0.01>p>0.001). P34S versus WT: **: p=0.0055 (4h), *: p=0.0103 (8h). S59L versus WT: *: p=0.0304 (4h), *: p=0.0129 (8h).",
    "molecules": "ActD, Actinomycine D"
  },
  {
    "caption": " (B) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay. ",
    "molecules": "Amylose"
  },
  {
    "caption": " (C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads. ",
    "molecules": "amylose"
  },
  {
    "caption": "Figure 1. Identification of non-canonical p53 binding sites in human subtelomeres. (A and B) ChIP-seq tracks for p53 in camptothecin (CPT) treated or untreated HCT116 cells were aligned with RNAPII, CTCF, RAD21, and SMC1 for human chromosome18q (A) or 13q (B) subtelomeres. Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top. Subtelomeric DNA paralogy segment tracks, internal telomere-like sequence tracks (red CCCTAA), and annotated RefSeq gene tracks are indicated above the ChIP-seq tracks. (C) Alignment of human subtelomeric elements containing non-canonical p53 recognition sequences. The position of the RepeatMasker-defined LTR10c element is shown; also note the (TG)n microsatellite repeat adjacent to the p53 binding site. (D) Schematic organization of non-canonical p53 binding sites found in human subtelomeres.",
    "molecules": "camptothecin, DNA"
  },
  {
    "caption": "Stress-induced p53 binding at human subtelomeres. (A) Western blot of HCT116 p53-/- and p53+/+ cells treated with DMSO (D) or 50 µM etoposide (E) for 24 hrs. Cell lysates were assayed for total p53, phospho-Ser15 p53 (p-p53), γH2AX, or GAPDH.",
    "molecules": "DMSO, etoposide"
  },
  {
    "caption": "(B) ChIP-qPCR was assayed in HCT116 p53-/- or p53 +/+ cells treated with DMSO or etoposide as in panel A, and assayed by ChIP with antibodies to p53 (Ab6) or control IgG, followed by qPCR for p53 binding site in subtelomere 18q (top panel) or 13q (lower panel).",
    "molecules": "DMSO, etoposide"
  },
  {
    "caption": "(C) HCT116 p53-/- and p53+/+ cells were cultured in DMEM with 10% FBS or Hank's buffered saline solution (HBSS) lacking FBS and then assayed by ChIP with either IgG control (grey) or two different p53-specific antibodies Ab6 (red) or FL(black). ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q, 13q, or p21 promoter.",
    "molecules": "HBSS"
  },
  {
    "caption": ". (D) Doxycyclin (Dox)-inducible p53 H1299 cells were treated without (-) or with (+) Dox in media containing 10% serum or in serum free HBSS, and then subject to ChIP assay with control IgG or p53-specific Ab6 antibody. ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 (top panel), p53-negative control IGX1A (middle panel), or p21 positive control (lower panel). Error bars represent SD, and * indicates p value < .05. p-values were determined by Student t-test.",
    "molecules": "HBSS, serum, DNA, Dox, Doxycyclin"
  },
  {
    "caption": "(B) RT-qPCR measure of p21 (top panel) or PARD6G (lower panel) in HCT116 p53-/- or p53+/+ cells after treatment with DMSO or 50 µM etoposide.",
    "molecules": "DMSO, etoposide"
  },
  {
    "caption": "(D) RT-qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 (top panel) or pr2 (lower panel) from HCT116 p53+/+ cells cultured with serum (grey bars) or in HBSS lacking serum (black bars). Error bars represent SD and * indicates p value <.05 using student t-test comparing treated and untreated samples.",
    "molecules": "HBSS, serum"
  },
  {
    "caption": ". p53-dependent cell viability and telomere protection in response to DNA damage. (A) Western blot of total p53, γH2AX, and GAPDH in HCT116 p53-/- or p53+/+ cells treated with 1 µM or 50 µM etoposide for 24 hrs.",
    "molecules": "etoposide"
  },
  {
    "caption": "s. (B) Cell cycle analysis of HCT116 p53-/- or p53+/+ cells untreated or treated with 50 µM etoposide for 24 hrs. Cell cycle was determined by FACS analysis of propidium iodide stained cells.",
    "molecules": "etoposide"
  },
  {
    "caption": "(C) Cell clonal survival assay for HCT116 p53-/- or p53+/+ cells treated with DMSO or 1, 1.5 or 2 µM etoposide for 24 hr prior to replating.",
    "molecules": "DMSO, etoposide"
  },
  {
    "caption": ". (D) Quantification of clonal survival assay relative total cell numbers (top panel) or normalized to DMSO controls (lower panel).",
    "molecules": "DMSO"
  },
  {
    "caption": "E) TIF assay for HCT116 p53+/+ or p53-/- cells treated with 1 µM etoposide for 3 hrs. Telomere DNA FISH with TelC probe (red), γH2AX (green), Dapi (blue).",
    "molecules": "DNA, etoposide"
  },
  {
    "caption": "G) Telomere length assay showing p53-/- or p53+/+ cells treated with either DMS0, 1 or 50 µM etoposide. Ethidium bromide (EtBr) stained agarose gels (left panel), Southern blot hybridized with Tel C probe (middle panel) or with α Satellite DNA promote (right panel). Quantification of telomereDNA signal relative to invariant bands in α Sat. Error bars represent SD and * indicates p values <.05 determined by student t-test (TLA), or chi-square (TIF assay).",
    "molecules": "DNA, etoposide"
  },
  {
    "caption": "(H) Two-dimensional agarose gel analysis of telomeric DNA from HCT p53+/+ or or p53-/- treated with 50 µM etoposide for 24 hrs. Ethidium gel (top) and Southern blot probed with TelG probe (lower panel). Single stranded telomere DNA (red arrow) and recombination/ replication-associated structures (blue arrow) are indicated.",
    "molecules": "DNA, etoposide"
  },
  {
    "caption": "Figure 5. p53-dependent histone modification changes in response to DNA damage stress. HCT116p53-/- (left panels) or p53 +/+ (right panels) were treated with DMSO (black) or 50 µM etoposide (red) for 24 hrs and assayed by ChIP-qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere (panel A) or 13qsubtelomere (panel B) or for control regions (p21 promoter, GKN1/2, PARKIN) as indicated below. ChIP-qPCR for p53, H3K9Ac, H3K27Ac, γH2AX, or IgG control were quantified as % input. Error bars indicates SD and * indicates p value<.05 using student t-test comparing ChIP values in p53+/+ relative to p53-/- cells for specific primer pairs.",
    "molecules": "DMSO, etoposide"
  },
  {
    "caption": "(C) Dot blot analysis of telomere repeat or Alu repeat DNA from p53+/+ or p53-/- HCT116 cells treated with DMSO or etoposide (ETP) (as described in panel A), that were subject to ChIP with antibodies to IgG, p53, γH2AX, TRF2, or TRF1. Quantitation of three independent ChIP-assays for TRF1 and TRF2 is shown in the panel to the right. (",
    "molecules": "DMSO, etoposide, ETP"
  },
  {
    "caption": "(A) HCT116 p53+/+ or p53-/- cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53, γH2AX, H3K9Ac, panH3, and GAPDH at times 0, 8, 16, or 24 hr post-treatment.",
    "molecules": "etoposide"
  },
  {
    "caption": "C-D) RT-qPCR analysis for cells treated as shown in panel A. (C) RT-PCR for the p21 (top), or PARD6G mRNA (lower). (D) RT-PCR for primer pairs spanning subtelomere18q as indicated below. Lower panel shows the No-RT control for 18qsubtelomere primers. RT-qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value <.05 using student t-test comparing etoposide (24 hr) relative to untreated for specific primer pairs.",
    "molecules": "etoposide"
  },
  {
    "caption": "(E) Meta-data analysis of published GRO-Seq and ChIP Seq data sets. Gro-Seq Minus (red/maroon) or Plus (purple/pink) RNA strand from Control or Nutlin -3a treated MCF7 cells was mapped to human 18q subtelomere. Histone H3K27ac, H3K4me1, and H3K4me3ChIP-Seq data sets from untreated MCF7 are also shown for these subtelomeric regions. Right panel shows a zoom in of approximately 1000bp (light blue on left panel) surrounding the p53 response element within the subtelomere.",
    "molecules": "Nutlin -3a, RNA"
  },
  {
    "caption": "(B) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO (D) or 50 µM etoposide (E) for 24 hr. Blots were probed with pan-p53, phospho-p53, γH2AX, or GAPDH (as loading control)",
    "molecules": "DMSO, etoposide"
  },
  {
    "caption": "(C-E) RT-qPCR for p21 (C), PARDG6 (D), or 18q subtelomeric RNA (E) in 18q WT or18q Δp53 treated with DMSO (black) or 50 μM etoposide (red) for 24 hr. (F) ChIP-qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B-E.",
    "molecules": "DMSO, etoposide"
  },
  {
    "caption": "F Examples of electrophysiological properties of humanneurons: control individual (top); PKAN patient (bottom). Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current-clamp mode. Middle traces show Na+ and K+ currents (down- and upward-deflecting from baseline, respectively) in response to a 60 mV step from a holding voltage of -70 mV in voltage-clamp mode. Insets on the right display enlarged portions of the traces to magnify fast Na+ currents. G Histogram with percentages of recorded cells showing repetitively firing (z-test).",
    "molecules": "K+, Na+"
  },
  {
    "caption": "A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM, the neuronal specific anti-NCAM antibody and the nuclear staining Hoechst. Left panel, basal conditions. Right panel, after addition of the mitochondrial uncoupler FCCP. Scale bar 20 μm. Plot showing the quantification of TMRM fluorescence signal from NCAM+neurons. Data presented as means + SEM of three independent experiments (unpaired, two-tailed t-test).",
    "molecules": "FCCP"
  },
  {
    "caption": "C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls, PKAN patients and PKAN patients overexpressing PANK2. The plots show OCR normalization to cell number. OCR was measured in basal conditions, and after oligomycin and FCCP addition. Bars indicate means + SEM of three independent experiments (Two-way ANOVA).",
    "molecules": "FCCP"
  },
  {
    "caption": "A Representative images of neurons stained with the neuron specific anti-NCAM antibody, ROS sensible fluorescent probe DCF, and the nuclei dye Hoechst. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM positive controls- and PKAN humanneurons, infected or not with Ngn2-PANK2-LV. Data presented as means + SEM of at least three independent experiments (One-way ANOVA).",
    "molecules": "ROS"
  },
  {
    "caption": "C Hemequantification by absorbance at 400 nm of the soluble cell lysates.",
    "molecules": "Heme"
  },
  {
    "caption": "B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days. Repetitive firing activity (left) and relatively large Na+ and K+ currents (right), were restored by CoA. The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs. CoA-treated neurons from control and PKAN patients (z-test).",
    "molecules": "K+, Na+"
  },
  {
    "caption": "C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst. Anti-NCAM antibody was used to detect neurons. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM+ controls- and PKAN humanneurons differentiated or not in the presence of CoA (25 μM) in the medium for 3 weeks. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).",
    "molecules": "ROS"
  },
  {
    "caption": "D Oxygen consumption rate (OCR) with and without CoA on controls and PKAN patients. Basal and uncoupled (FCCP) respiration increased after CoA supplementation. Data presented as means + SEM of 24 independent replicates for each condition (Two-way ANOVA).",
    "molecules": "FCCP"
  },
  {
    "caption": "E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).",
    "molecules": "Heme"
  },
  {
    "caption": "Lysates of FHM   cells overexpressing CiTLR5a   were respectively incubated with FlaA, FlaB, FlaC, at pH  5.0 or pH  7.4 for 1 h at 4 °C, mixed with HA  Ab-coupled beads or streptavidin agarose  beads for 2 h at 4 °C, then analyzed by immunoblotting  (IB) with anti-HA  or anti-His  antibodies.",
    "molecules": "agarose, streptavidin"
  },
  {
    "caption": "Lysates of FHM  cells overexpressing CiTLR5b  were respectively incubated with FlaA, FlaB, FlaC, at pH  5.0 or pH  7.4 for 1 h at 4 °C, mixed with HA  Ab-coupled beads or streptavidin agarose  beads for 2 h at 4 °C, then analyzed by immunoblotting  (IB) with anti-HA  or anti-His  antibodies.",
    "molecules": "agarose, streptavidin"
  },
  {
    "caption": "Lysates of FHM  cells overexpressing CiTLR5a and CiTLR5b were respectively incubated with biotin-poly(I:C), biotin-ssRNA  (1 μg/mL) at pH  5.0 or pH  7.4 for 1 h at 4 °C, mixed with HA  Ab-coupled beads for 2 h at 4 °C, then analyzed by immunoblotting  (IB) with anti-HA  antibodies.",
    "molecules": "biotin, poly(I:C), ssRNA"
  },
  {
    "caption": "Lysates of FHM  cells overexpressing  CiTLR5a  and CiTLR5b   (including region deletants) were incubated with biotin-poly(I:C). Vector was used as a control. Pull-down  and immunoblotting  (IB) were performed.",
    "molecules": "biotin, poly(I:C)"
  },
  {
    "caption": "H-K Lysates of FHM  cells overexpressing  CiTLR5a  and CiTLR5b  site mutants were incubated with biotin-poly(I:C). Vector and wild type were used as controls. Pull-down and immunoblotting  were carried out (H, J). (I, K) Relative band intensity ratios of (H) and (J) were analyzed by ImageJ, respectively (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (I), and (K). n represents biological replicates. *P < 0.05, **P < 0.01.",
    "molecules": "biotin, poly(I:C)"
  },
  {
    "caption": "Lysates of FHM  cells overexpressing CiTLR5a or CiTLR5b  mutants were respectively incubated with biotin-poly(I:C) (I) Vector and wild type were used as controls. Pull-down and immunoblotting  assays were conducted (I, (J, Relative band intensity ratios of (I) were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (J), n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "biotin, poly(I:C)"
  },
  {
    "caption": "Lysates of FHM  cells overexpressing LoTLR5 (F), DrTLR5a (G),  DrTLR5b (G),  IpTLR5-1 (H),  IpTLR5-2 (H), IpTLR5S (H), were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH  5.0 or pH 7.4. Complexes were pulled down by streptavidin  beads and analyzed via immunoblotting with anti-GFP  antibody.",
    "molecules": "biotin, poly(I:C), streptavidin"
  },
  {
    "caption": "Lysates of FHM cells overexpressing OmTLR5M  (I), OmTLR5S (I), and HsTLR5 (J) were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH  5.0 or pH  7.4. Complexes were pulled down by streptavidin  beads and analyzed via immunoblotting  with anti-GFP  antibody.",
    "molecules": "biotin, poly(I:C), streptavidin"
  },
  {
    "caption": "Lysates of FHM  cells overexpressing  DrTLR5a and DrTLR5b mutants were incubated with biotin-poly(I:C) and analyzed by immunoblotting  (N), and relative band intensity ratios were analyzed by ImageJ (O) (n = 3). Vector and wild type (WT) were used as controls. Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (O). n represents biological replicates. *P < 0.05, **P < 0.01.",
    "molecules": "biotin, poly(I:C)"
  },
  {
    "caption": "Bisulfite sequencing maps of 454 reads showing the average methylation status of individual cytosines (boxes) within tRNAs from bone marrow (BM), mesenchymal stromal cells (MSC), and in vitro differentiated adipocytes and osteoblasts. Methylation levels are indicated on a color scale from green (unmethylated) to red (completely methylated).",
    "molecules": "tRNA, cytosines"
  },
  {
    "caption": "Representative Northern blot showing tRNA(Gly) and tRNA(Asp) fragmentation in bone marrow from 8-day-old and adult mice. tRNA(Ser) was included as a Dnmt2 non-substrate tRNA, and 5S rRNA was included as a loading control.",
    "molecules": "tRNA, Asp, Gly, rRNA, Ser"
  },
  {
    "caption": "tRNA fragment coverage for the Dnmt2 substrates tRNA(Gly) and tRNA(Asp), and the non-substrate tRNA(Ser) in bone marrow, as determined by deep sequencing. Wild-type coverage is represented in red, and Dnmt2−/− coverage is indicated in blue. The plots illustrate the specific increase of 5′-halves from tRNA-Gly and 3′-halves from tRNA-Asp fragments in Dnmt2 mutants.",
    "molecules": "tRNA, Asp, Gly, Ser"
  },
  {
    "caption": "Analysis of protein synthesis by dynamic SILAC. The top 10% of proteins that are translated slower in Dnmt2−/− versus wild-type cells are indicated in orange. The top 10% of proteins that are translated faster in Dnmt2−/− versus wild-type cells are shown in blue. A change of >  twofold is indicated by the black lines.",
    "molecules": "protein, proteins"
  },
  {
    "caption": "Dimethyl-labeling analysis of 8-day-old mouse bone marrow tissue. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. Nestin and periostin are marked with arrows.",
    "molecules": "Proteins, proteins"
  },
  {
    "caption": "Correlation between protein and mRNA expression for differentially expressed proteins. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. The Pearson correlation coefficient is −0.03.",
    "molecules": "mRNA, protein, proteins, Proteins"
  },
  {
    "caption": "Frequency of amino acid substitutions in the dSILAC dataset. Differences between Asp > Glu and Glu > Asp transitions versus Asn > Lys and His > Gln were found to be highly significant (P < 0.01, Fisher's exact test).",
    "molecules": "amino acid, Asn, Asp, Gln, Glu, His, Lys"
  },
  {
    "caption": "(C-D) ITC measurements of purified CEP19 titrated with CrRabL2 in the presence of GTP (C) or GDP (D).",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "(G) SEC profile (top) and Coomassie stained SDS gel (bottom) of the complex between RabL2 and a minimal binding-region of CEP19 (residues 107-195) demonstrating a direct interaction.",
    "molecules": "SDS"
  },
  {
    "caption": "(A) SEC profile of CrRabL2Q83L co-purification with CrIFT81/74/27/25/22 (top) in the presence of non-hydrolysable GTP homologue, GTPγS. Coomassie stained SDS gel of SEC fraction highlighted by dashed lines (bottom). (B) SEC profile for the purification of the HsIFT81/74/27/25/22/RabL2 complex in the presence of GTPγS (top) and the corresponding Coomassie stained SDS gel (bottom).",
    "molecules": "GTP homologue, GTPγS, SDS"
  },
  {
    "caption": "(E-H) GTPases assays with the indicated proteins following the release of inorganic phosphate upon GTP hydrolysis as a function of time. Each experiment was carried out in three technical replicates. Concentrations for experiments in (E) were 55µM CrIFT-B1 pentamer/CrRabL2, 60µM CrIFT-B1 pentamer and 70µM CrIFT-B1 tetramer and 1mM GTP. Concentrations for experiments in (G) were 60µM protein and 30µM GTP. The curves represent averages and error bars indicate standard deviation of measurements for every 100s for panel (E) and every 200s for panel (G). (F, H) Quantification of the reaction rates (arbitrary units of Absorbance (Abs) per second(s)) for each experiment shown in panels (E) calculated using linear regression of the first 500s and (H) using single exponential fit, and agreement with the fit to the curves in (E, G) are indicated by the error bars and R2-value.",
    "molecules": "GTP, inorganic phosphate"
  },
  {
    "caption": "(A) SEC profile showing that a minimal CrIFT81460-533/CrIFT74460-532 complex co-purifies with CrRabL2Q83L in the presence of GTPγS (left). The right panel displays the Coomassie stained SDS gel of SEC fractions (horizontal top dashed line).",
    "molecules": "GTPγS, SDS"
  },
  {
    "caption": "(B) An N-terminal hexa-histidine tagged CrIFT81460-533/74460-532 complex interacts with untagged CrRabL2Q83L in a GTPγS-dependent manner in pulldown assays.",
    "molecules": "GTPγS"
  },
  {
    "caption": "(C) SEC profile showing that human RabL2B co-purifies with Chlamydomonas IFT-B1 pentamer (left). Coomassie stained SDS gel of SEC fractions highlighted with dashed lines on the SEC profile (right).",
    "molecules": "SDS"
  },
  {
    "caption": "(E) RT-qPCR quantification of relative miR-21-3p level in the epidermis of Ppard +/+ and -/-mice, acutely irradiated (Ac-UV; +) or non-irradiated (-), treated with the PPAR/ antagonist GSK0660 (+) or vehicle (-), as indicated. miR-21-3p: Ppard +/+ no UV vs Ppard +/+ Ac-UV P = 0.005, Ppard +/+ Ac-UV vs Ppard +/+ Ac-UV/GSK0660 P = 0.04, N=5 (Ppard +/+) to 3 (Ppard -/-) animals per group. One representative experiment is shown out of two independent replicates",
    "molecules": "GSK0660"
  },
  {
    "caption": "(F) RT-qPCR quantification of relative miR-21-3p levels in HaCat cells treated with the PPAR/ agonists GW501516, GW0742 (+) or vehicle (-) as indicated. miR-21-3p: Veh vs GW501516 P = 4E-04, Veh vs GW0742 P = 2E-04. N=2-3 biological replicates, one representative experiment is shown out of two independent replicates.",
    "molecules": "GW0742, GW501516"
  },
  {
    "caption": "(A) RT-qPCR quantification of relative pri-miR-21 level in HaCaT human keratinocytes treated with the PPAR/ agonist GW0742 (+) or vehicle (-), with (+) or without (-) cycloheximide (Cyclo) as indicated. Pri-miR-21: Veh vs GW0742 P = 0.009. N=3 biological replicates, one representative experiment is shown out of two independent replicates.",
    "molecules": "Cyclo, cycloheximide, GW0742"
  },
  {
    "caption": "(C) RT-qPCR quantification of pri-miR-21, pre-miR-21, miR-21-5p and miR-21-3p levels in HaCat cells treated for 24h with the PPAR/ agonist GW0742 (+), TGFβ receptor inhibitor SB431542 (+), or vehicle (-) as indicated. Pri-miR-21: GW0742 vs SB431542 P = 0.004, GW0742 vs GW0742/SB431542 P = 0.015; Pre-miR-21: Veh vs GW0742 P = 0.022, GW0742 vs SB431542 P = 0.036, GW0742 vs GW0742/SB431542 P = 0.024; miR-21-5p: GW0742 vs SB431542 P = 0.034, GW0742 vs GW0742/SB431542 P = 0.024; miR-21-3p: Veh vs GW0742 P = 0.036, GW0742 vs SB431542 P = 0.011, GW0742 vs GW0742/SB431542 P = 0.008. N=3 biological replicates, one representative experiment is shown out of two independent replicates.",
    "molecules": "GW0742, SB431542"
  },
  {
    "caption": "(E) RT-qPCR quantification of relative miR-21-5p and miR-21-3p levels in the skin of Ppard +/+ mice treated with the TGFβ receptor inhibitor SB431542 (+) or vehicle (-), with (+) or without (-) acute UV exposure (Ac-UV). miR-21-5p: no UV vs Ac-UV P = 0.038, Ac UV vs Ac UV/SB431542 P = 0.028; miR-21-3p: no UV vs Ac-UV P = 0.017; Ac-UV vs Ac-UV/SB431542 P = 0.035. N=3 animals per group, one representative experiment is shown out of three independent replicates.",
    "molecules": "SB431542"
  },
  {
    "caption": "Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10µM for 6h) under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (C) (*P<0.05, ****P<0.0001; unpaired t-test, n=3, mean +SEM).   ",
    "molecules": "CB5083"
  },
  {
    "caption": "Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline-induced mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (E) (**P<0.01, ****P<0.0001; unpaired t-test, n=4, mean +SEM).   ",
    "molecules": "doxycycline"
  },
  {
    "caption": "Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA-mediated p97 depletion.   Graph represents the quantifications of (H) (***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM) and Western blot for efficacy of siRNA depletion of p97 (right).   ",
    "molecules": "CHX"
  },
  {
    "caption": "Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).   Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   ",
    "molecules": "CHX, MG132"
  },
  {
    "caption": "Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10µM for 6h).",
    "molecules": "MG132"
  },
  {
    "caption": "Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.   Graph represents the quantifications of (E) (ns P>0.05, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).   ",
    "molecules": "CHX"
  },
  {
    "caption": "Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ∆ATX3 cell extract. Arrow represents the main RNF8 band and asterisks represents unspecific bands.   Graphs represent the quantifications of (A). RNF8 level at starting point (0h) was shown without equalisation (left). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate (right). (**P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   ",
    "molecules": "CHX"
  },
  {
    "caption": "Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ATX3 knockdown HeLa cells. The degradation was completely blocked after simultaneous inhibition of proteasome (MG132, 10µM).",
    "molecules": "CHX, MG132"
  },
  {
    "caption": "Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm+nucleoplasm) of HeLa cells, expressing DOX-inducible GFP-ATX3-C14A variant as compared to GFP-ATX3-WT. Endogenous ATX3 was depleted with siRNA targeting 3'UTR region of ATXN3.   Graph represents the quantifications of (A). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (*P<0.05, **P<0.01; two-way ANOVA, n=2, mean +SEM).   ",
    "molecules": "CHX, DOX"
  },
  {
    "caption": "Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm + nucleoplasm) of HeLa cells, expressing DOX-inducible ATX3-UIM* variant as compared to ATX3-WT. Endogenous ATX3 was depleted with siRNA ATX3_3'UTR.   Graph represents the quantifications of (C). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (ns P>0.05, *P<0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   ",
    "molecules": "CHX, DOX"
  },
  {
    "caption": "Western blot analysis showing endogenous RNF8 protein level in soluble fraction (Cytosol and Nucleosol) of HeLa cells under indicated conditions. RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX-inducible expression of GFP-ATX3-WT but not with GFP-ATX3-VBM.   Graphs represent the quantifications of (E). (ns P>0.05, * P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).   ",
    "molecules": "DOX"
  },
  {
    "caption": "CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-C14A or ATX3-UIM* variant. (ns P>0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-VBM variant. (ns P>0.05, *P<0.05, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   ",
    "molecules": "DOX"
  },
  {
    "caption": "Representative IF images and quantification showing endogenous K63-Ub and γ-H2AX at UV-A micro-laser-induced DNA damage tracks in U2OS cells under indicated siRNA-depleted conditions. (Scale bar; 10µm).   Quantification of (I). Graph represents the average intensity of the K63-Ub (ns P>0.05, **P<0.01, ***P<0.001; unpaired t-test, n=2, mean +SEM, more than 50 nuclei were analysed).   ",
    "molecules": "Ub"
  },
  {
    "caption": "Representative IF-images showing Rad51 foci in S-phase (Edu positive) U2OS nucleus after 5h of IR (2Gy) exposure. (Scale bar; 10µm).   Graph represents the quantification of (E) showing average number of Rad51 foci per S-phase (Edu positive) nucleus. (ns P>0.05, ****P<0.0001; unpaired t-test, n=2, mean +SEM, at least 50 nuclei per condition and experiment).   ",
    "molecules": "Edu"
  },
  {
    "caption": "b. In vitro activity. Left y-axis: 13C2-oxalate   in mM, right y-axis: 13C-formate  in mM, x-axis: time  in minutes. SYNB8802       (blue, circles, solid line: oxalate, dotted line: formate) The control EcN   (pink, triangle, solid line: oxalate, dotted line: formate). Three biological replicates were run and plotted separately.",
    "molecules": "13C, 13C2, formate, oxalate"
  },
  {
    "caption": "c. In vitro simulation  (IVS). Left y-axis: Rate of oxalate degradation  in μmol/h/109 cells. X-axis = time  in hours. Left X-axis 0 - 4 hours, Right X-axis 6 - 48 h. Three biological replicates were run and plotted separately. Data points in the orange box represent incubation in simulated gastric fluid (SGF). Data points in the light blue box represent incubation in simulated intestinal fluid  (SIF). Data points in the pink box represent incubation in simulated colonic fluid (SCF).",
    "molecules": "oxalate"
  },
  {
    "caption": "b. Acute mouse  study of UOx lowering with SYNB8802. The y-axis shows the urinary 13 C2-oxalate  normalized to creatine. The x-axis shows the two groups, EcN (control, pink) vs  SYNB8802AbxR (light blue; n = 15 mice  per group). Individual dots represent each metabolic cage (n = 3 mice  per metabolic cage). Error bars are calculated as SEM. (unpaired t-test with Welch's correction, **** p < 0.0001)",
    "molecules": "13 C2, creatine, Ox, oxalate"
  },
  {
    "caption": "d. Nonhuman primate  model development study. The y-axis shows urinary oxalate  normalized to creatine. X-axis shows baseline urinary oxalate  levels after overnight fast, and urinary oxalate  measured 6 hours post administration of either a spinach   preparation (triangle, pink) or water (circle, blue) (n=6 for each group). Individual dots represent individual animals. Error bars are calculated as SEM. (Two-way ANOVA followed by Sidak's multiple comparison analysis, **** p < 0.0001).",
    "molecules": "creatine, oxalate"
  },
  {
    "caption": "f. Urinary recovery of oxalate in nonhuman primates. The y-axis shows change in urinary oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatment groups). Individual dots represent normalized urinary oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, * p < 0.05)",
    "molecules": "oxalate"
  },
  {
    "caption": "g. Urinary recovery of 13C2 oxalate in nonhuman primates. The y-axis shows change in urinary 13C2-oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatmentgroups). Individual dots represent normalized urinary 13C2-oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, ** p < 0.01)",
    "molecules": "13C2, oxalate"
  },
  {
    "caption": "A, B. A fraction of OTULIN is localized at mitochondria where it suppresses M1-linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1-linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "E. Mitochondria and M1-linked ubiquitin co-localize after TNF treatment. SH-SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1-ubiquitin (1E3, red) and analyzed by SR-SIM.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "G. TNF induces a fast and transient increase in M1- and K63-specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1-, K63-, and K48-specific ubiquitin antibodies.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "A, B. PINK1 is stabilized by TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) before harvesting or incubated with OTULIN-specific siRNA for 48 h (A). Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies. Quantification of PINK1-specific signals normalized to TIM23 is shown in the right panel. Data represent the mean values with standard deviations of 4 independent experiments. *p < 0.05. A two-tailed non-parametric Mann-Whitney U-test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1.",
    "molecules": "CCCP"
  },
  {
    "caption": "C. TNF-induced PINK1 stabilization does not induce mitophagy. HeLa cells expressing mt-mKeima were treated with TNF (25 ng/ml) for 30 min or 16 h. As a control, mt-mKeima -expressing Hela cells were treated with antimycin A and oligomycin (A/O, 10 µM each) for 1 h. The analysis was done by flow cytometry gating of lysosomal and neutral mt-mKeima.",
    "molecules": "antimycin A, oligomycin"
  },
  {
    "caption": "D. Catalytically active HOIP ubiquitinates overexpressed PINK1. HEK293T cells were transfected with the plasmids indicated. After 24 h, the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "E. Recombinant HOIP ubiquitinates PINK1 in vitro. V5-tagged-PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1, UBE2L3, C-terminal HOIP and ubiquitin for in vitro ubiquitination. The samples were then analyzed by immunoblotting using V5 antibodies.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "F. Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min), then endogenous PINK1 was immunoprecipitated from mitochondrial fractions. Cells mildly overexpressing PINK1-V5 were also included (lanes 4, 5, 8), to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1. As a control for the presence of M1-linked ubiquitin chains, immunoprecipitated PINK1 was treated with recombinant OTULIN. As controls for the specificity of the immunoprecipitation, beads only (lanes 6 - 8) and beads plus IgG (lane 9) were included. The samples were analyzed by immunoblotting using antibodies against PINK1, M1-ubiquitin, and phosphorylated ubiquitin. For immunoprecipitation of overexpressed PINK1, only 50% of cells were used in comparison to the immunoprecipitation of endogenous PINK1.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "A. PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL-1L, were transfected with PINK1, WT OTULIN or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep-tagged UBAN domain of NEMO to enrich proteins modified with M1-linked ubiquitin. Proteins affinity-purified by Strep-Tactin beads were immunoblotted against ubiquitin.",
    "molecules": "Strep, ubiquitin"
  },
  {
    "caption": "B. Catalytically active PINK1 increases p-S65-ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase-dead (K/D) PINK1. 48 h after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p-S65-ubiquitin, PINK1, and VDAC (loading control).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "C. The TNF-induced increase in p-S65-ubiquitin is abolished in PINK1-deficient cells. HEK293T cells were transfected with control or PINK1-specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies.",
    "molecules": "CCCP, ubiquitin"
  },
  {
    "caption": "D. TNF-induced M1 ubiquitination is reduced in PINK1-deficient cells. WT and PINK1-KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep-tagged UBAN domain, as described in A, followed by immunoblotting using M1-ubiquitin-specifc antibodies, Strep-Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1-ubiquitin-positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of 3 independent experiments; n.s. not significant, ***p < 0.001. A two-way ANOVA test was used to analyze statistical significance.",
    "molecules": "Strep, ubiquitin"
  },
  {
    "caption": "A. STS-induced mitochondrial Bax recruitment is reduced by TNF. HeLa cells were treated with STS (1 μM, 1 h) with or without a 15 min pretreatment with TNF (25 ng/ml) and then harvested. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1-ubiquitin. The input was immunoblotted for TIM23 (upper panel). Bax-specific signal intensities were quantified and normalized to TIM23-specific signals (lower panel).",
    "molecules": "STS, ubiquitin"
  },
  {
    "caption": "B. STS-induced cytochrome C release is decreased by TNF. HeLa cells were treated as described in A. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. GAPDH was used as a reference. Quantification of 5 biological replicates is shown in the lower panel. Signal intensities were quantified and normalized to that of GAPDH. *p < 0.05. Kolmogorow-Smirnov normality test, paired t-test, two-tailed, n=5.",
    "molecules": "STS"
  },
  {
    "caption": "E. The fast anti-apoptotic effect of TNF is not affected by the NF-κΒ inhibitor IκΒα. SH-SY5Y cells were transiently transfected with the NF-κΒ super-repressor IᴋBα-2S or luciferase as a control. 24 h later, the cells were treated with STS (5 µM, 2 h) with or without a 15 min pretreatment with TNF (25 ng/ml). Cells were fixed and stained by antibodies against active caspase-3. Signal intensities were quantified by immunocytochemistry and fluorescence microscopy. Expression of IᴋBα-2S was tested by immunoblotting using antibodies against the HA tag. Data represent the standard deviation of three independent experiments; at least 300 cells were counted per experiment. *p < 0.05, **p < 0.01. Student's t-test, two-tailed, n=3.",
    "molecules": "STS"
  },
  {
    "caption": "F. The protective effect of TNF is dependent on HOIP expression. HeLa cells were transiently transfected with control or HOIP siRNA. 48 h after transfection, cells were treated with STS (1 μM, 1 h) with or without a 15 min TNF pretreatment (25 ng/ml) and then harvested. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP. GAPDH and actin were immunoblotted as input controls.",
    "molecules": "STS"
  },
  {
    "caption": "B. Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment. Representative immunofluorescence images of SH-SY5Y cells using the proximity ligation assay (PLA) between phospho-S536-p65 (rabbit) and TOM20 (mouse) couple antibodies. One set of cells were treated with TNF (25 ng/ml, 15 min) before fixation. Nuclei were stained with DAPI, mitochondria were stained with MitoTrackerTM Red CMXRos (red), and the PLA amplification reaction was visualized by green foci. As a positive control, fixed cells were incubated with primary TOM20 (mouse) and TOM70 (rabbit) antibodies and subjected to the PLA assay. As negative controls, fixed cells were incubated with either TOM20 or p-p65 antibodies prior to the PLA assay.",
    "molecules": "DAPI, MitoTrackerTM Red"
  },
  {
    "caption": "B. PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co-transfected with V5-tagged wildtype PINK1 or kinase-dead PINK1-K/D and HA-tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p-S65-ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p-S65-ubiquitin, and GAPDH.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "C. Linear ubiquitination of NEMO is reduced in PINK1-deficient cells. HEK293T cells were co-transfected with HA-NEMO and control or PINK1-specific siRNAs. 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using M1-ubiquitin antibodies. The input was immunoblotted for NEMO and actin. PINK1 silencing efficiency was determined by real-time RT-PCR. Bars represent mean± SD with three technical replicates.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "A. TNF induces the movement of peripheral mitochondria towards the nucleus. Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF (25 ng/ml). The motility of mitochondria was analyzed using Imaris 9.8 spot function. The graphical representation of the top 25% peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment. Exemplified dataset shows the median distance with the upper and lower quartile (shaded area) of n=105-187 mitochondrial spots per time point.",
    "molecules": "Hoechst 33342, MitotrackerTM Green"
  },
  {
    "caption": "B. Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1/2 expression. HeLa cells were transfected with control or Miro1 and Miro2 siRNAs. 48 h later, cells were treated with TNF (25 ng/ml, 15 min), fixed and stained using antibodies against p65 and tubulin and DAPI. Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4.2.1. Quantification is based on 3 biological replicates. Kruskal-Wallis test followed by Dunn's multiple comparison test, n=79-157, *p < 0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "E, F, G. TNF increases mitochondria-nucleus contact sites. Representative immunofluorescence images (E, F) and quantification (G) of mitochondria-nucleus contact sites in HeLa cells transfected with the SPLICSNU-MT and treated with 25 ng/ml TNF or PBS for 15 min. TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation. Nuclei were stained with Hoechst33342 (ThermoFisher, 1 μg/ml). Unpaired two-tailed t-test, n=43 (control) or 51 (TNF),",
    "molecules": "Hoechst33342, PBS"
  },
  {
    "caption": "H, I, J. TNF increases mitochondria-nucleus contact sites in a HOIP-dependent manner. Representative immunofluorescence images (H, I) and quantification (J) of mitochondria-nucleus contact sites in control or HOIP-silenced (HOIP siRNA) HeLa cells transfected with the SPLICSNU-MT probe treated with 25 ng/ml TNF or PBS for 15 minutes. Mitochondria and nuclei were stained as described in E and F.",
    "molecules": "PBS"
  },
  {
    "caption": "(A) Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a 3-fold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively",
    "molecules": "DRB"
  },
  {
    "caption": " (B) Hek293 cells were thymidine-arrested for 20h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI and then subjected to ChIP-multiplex PCR ",
    "molecules": "OHT, thymidine"
  },
  {
    "caption": "Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed by IP-Western",
    "molecules": "OHT, Dox"
  },
  {
    "caption": "Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed b IFM for γH2AX- and Myc-separase-positive foc The quantification of the IFM in (E) shows averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each",
    "molecules": "OHT, Dox"
  },
  {
    "caption": "Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (A",
    "molecules": "OHT, Ethanol"
  },
  {
    "caption": "Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected t flow cytometry to quantify the percentage of GFP positive cells (B The GFP quantification in (B) displays averages (bars) of 3 independent experiments (dots)",
    "molecules": "OHT, Ethanol"
  },
  {
    "caption": "Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls Two days later PI-stained cellular DNA (C)",
    "molecules": "DNA, OHT, Ethanol"
  },
  {
    "caption": "(A) Transgenic Hek293 cells constitutively expressing a separase sensor (cartoon below) and inducibly expressing Myc-separase were transiently transfected to express Flag-AsiSI-ER, Dox- and/or OHT-treated in G2-phase and analysed by (IP-)Western",
    "molecules": "OHT, Dox"
  },
  {
    "caption": " (C) Sensor expressing cells were treated in G2-phase with DRB and nocodazole for 6 and 2 h, respectively, prior to chromosome spreading and IFM using Hec1- and γH2AX-antibodies. The separase sensor was detected based on autofluorescence of eGFP and mCherry, while Hec1- and γH2AX-antibodies were detected with corresponding Cy5- and marina blue-labeled secondary antibodies, respectively. Note that sizes of spread chromosomes vary greatly with buffer conditions, which is why no scale bar is shown ",
    "molecules": "marina blue, DRB, nocodazole"
  },
  {
    "caption": "Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst others, a pan-specific antibody against phosphorylated serine (A, pan-pS",
    "molecules": "DRB, RO-3306, thymidine"
  },
  {
    "caption": "Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst other a separase antibody specific for phosphorylated Ser1660 (B, pS1660)",
    "molecules": "DRB, RO-3306, thymidine"
  },
  {
    "caption": "Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated (C) DNA damage-induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition. G2-enriched Hek293 cells were treated with KU-55933 (0.3 μM) and/or DRB and analyzed by (IP-)Western 12 h thereafter using the indicated antibodies",
    "molecules": "DRB, KU-55933, RO-3306, thymidine"
  },
  {
    "caption": " (D) Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs. HeLaK cells expressing N-terminally NLS-eGFP-tagged separase variants were treated with DRB or carrier solvent (− DRB) for 4 h and then subjected to IFM using anti-γH2AX and anti-Nup153 to visualize sites of DNA damage and nuclear pore complexes, respectively. Transgenic separase was detected based on the eGFP autofluorescence. Note that due to their relatively high nuclear concentration co-localization of separase-WT and -S1660D with γH2AX-foci is not discernable. Scale bar = 5 μm ",
    "molecules": "DRB"
  },
  {
    "caption": " (E) In vitro phosphorylation of separase on Ser1660 by ATM kinase. Incubation of GST-p53 (amino acids 9-22), GST, separase-WT, or separase-S1660 with recombinant ATM-WT, ATM-KD (kinase dead), and/or KU-55933 in presence of [γ-33P]-ATP was followed by immunoblotting and autoradiography",
    "molecules": "ATP, KU-55933"
  },
  {
    "caption": " (F) Arg-methylation of RG-repeats mediates recruitment of separase to DSB-containing chromatin. Myc-separase-WT or -KG expressing cells were treated with DRB as indicated and analysed by (IP-)Western and Coomassie staining ",
    "molecules": "Arg, DRB"
  },
  {
    "caption": " (G) In vitro Arg-methylation of separase's RG-repeats by PRMT1. Incubation of histone H4, separase-WT, or separase-KG with recombinant PRMT1 or reference buffer in presence of S-adenosyl-L-[methyl-3H]-methionine was followed by Coomassie staining and autoradiography ",
    "molecules": "S-adenosyl-L-[methyl-3H]-methionine, Arg"
  },
  {
    "caption": "(A) Persistence of γH2AX- and MDC1-positive foci in absence of NES-phosphorylation or RG-methylation of separase. Transfected Hek293T cells were siRNA- and Dox-treated to deplete endogenous separase and induce expression of the indicated, siRNA resistant Myc-tagged separase variants, respectively. Then, they were constitutively (− washout) or transiently exposed to DRB (+ washout) and finally quantitatively assessed by IFM for γH2AX- and MDC1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm",
    "molecules": "DRB, Dox"
  },
  {
    "caption": " (D) Interaction of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG with γH2AX. Cells from (A) were subjected to (IP-)Western analysis as indicated. Lanes that illustrate cellular levels of cyclin A2 and γH2AX after DRB-washout and those that analyse interaction of Myc-separase variants with γH2AX in presence of DRB are labeled by red arrows and blue arrow heads, respectively",
    "molecules": "DRB"
  },
  {
    "caption": "Separase is sumoylated in response to DSBs. Hek293T cells transfected to express His6-separase (A were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodies",
    "molecules": "DRB"
  },
  {
    "caption": "Separase is sumoylated in response to DSBs. Hek293T cells transfected to expres His6-Sumo2 (B) were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodies",
    "molecules": "DRB"
  },
  {
    "caption": "Lys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressing His6-Sumo2 (C or -K1034R (D) were DRB- or mock-treated and subjected to denaturing IMA Input samples and eluates were immunoblotted using the indicated antibodies",
    "molecules": "DRB"
  },
  {
    "caption": "Lys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressin Myc-separase-WT or -K1034R (D) were DRB- or mock-treated and subjected t Myc-I Input samples and eluates were immunoblotted using the indicated antibodies",
    "molecules": "DRB"
  },
  {
    "caption": " (E) Mms21-dependent in vitro sumoylation of separase at Lys-1034. Myc beads were loaded with separase-WT or -K1034R or left empty (last lane), combined with recombinant Ubc9 and/or Mms21, as indicated, and incubated in presence of His6-Sumo2, Sae1-Sae2 and ATP. Supernatant and washed beads were analysed by Coomassie staining (lower panels) and immunoblotting (upper panels), respectively ",
    "molecules": "ATP"
  },
  {
    "caption": " (F) Cells expressing the indicated separase variants were DRB- or mock-treated and then lysed. Lysates and chromatin pelleted therefrom were assessed by immunoblotting and Coomassie staining, as indicated. Note that the lanes shown in the upper panels, although not directly juxtaposed, nevertheless originate from the same gel ",
    "molecules": "DRB"
  },
  {
    "caption": "(A, B) SEPARASE+/+ and SEPARASE+/- MEFs were constitutively (− washout) or transiently exposed to DRB (+ washout) and then analysed by IF for cells with γH2AX- and 53BP1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm. (C) Cell lysates from one experiment in (A) were analysed by immunoblotting using the indicated antibodies ",
    "molecules": "DRB"
  },
  {
    "caption": "SEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed by immunoblotting of total cell lysates (D",
    "molecules": "DRB"
  },
  {
    "caption": "SEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed b IPs using anti-Rad21 or unspecific IgG (E). The lower right two lanes show in vitro-expressed mouse Rad21 treated with hyperactive (SA) separase or a protease-dead (PD) variant",
    "molecules": "DRB"
  },
  {
    "caption": " (B) Two-stage DMBA (initiation) plus TPA (promotion) skin carcinogenesis. The number and size of skin papillomas are plotted against time for each genotype: SEPARASE+/+ (n=8) versus SEPARASE+/- (n= 11) ",
    "molecules": "DMBA, TPA"
  },
  {
    "caption": " (C) Representative hematoxylin- and eosin-stained sections from SEPARASE+/- biopsies. (i) glandular hyperplasia; (ii) benign papilloma; (iii) squamous cell carcinoma. Scale bars are 200 µm in (i) and 500 µm in (ii) and (iii). (D) Quantitative histological assessment of the DMBA-TPA skin cancerogenesis assay. Given are numbers of total, and corresponding percentage in brackets ",
    "molecules": "eosin, hematoxylin, DMBA, TPA"
  },
  {
    "caption": "A) Murine small intestines immunostained for the proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from 4 control (Yapfl/fl Tazfl/fl) and 4 YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a moderate increase in apoptosis (Cas3+) in crypt base stem cells, quantified on the right, but no overall effect on the morphology of the small intestine. Arrows point to apoptotic cells. B) Murine large intestines immunostained for proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from control (Yapfl/fl Tazfl/fl) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to an increased number of apoptotic (Cas3+) cells within the transverse folds of the ascending colon.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "C) Murine small intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a mild increase in apoptosis (Cas3+) in crypt base stem cells, but no overall effect on the morphology of the small intestine. Arrows point to stem cells (YAP nuclear staining), while arrows point to apoptotic cells (Cas3 staining). N > 10 animals per genotype. D) Murine large intestines immunostained for YAP or cleaved caspase 3 (Cas3 ) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to increased apopotosis (Cas3+) and severe morphological defects in the transverse folds of the ascending colon, but does not affect morphology of the remaining colon regions. Quantitatively, 75% of n = 4 animals showed this severe phenotype.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "A) Murine small (top) and large (bottom) intestines isolated from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt-villus axis in controls, with nuclear YAP and Ki67 positive cells restricted to the crypt base (representative images from n=5 mice). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells. Note the gradient of YAP expression levels is maintained along the crypt-villus axis (representative images from n=5 mice for each genotype). (Right) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base. (Bottom right) Q-PCR analysis of Wnt pathway target genes reveals that Lats1/2 dKO causes a mild reduction in Lgr5 expression, with complete loss of Olfm4.",
    "molecules": "tamoxifen, Tamoxifen"
  },
  {
    "caption": "B) Axin2 mRNA expression was measured by RNAscope (red) and found to be increased in both intensity and uniformity of staining along the crypt-villus axis in Lats1/2 dKO after 3 days post i.p. injection (dpi) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2. At 7dpi, Axin2 mRNA levels remain uniform along the crypt-villus axis in Lats1/2 dKO animals, although their total level has declined compared to 3dpi.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "B) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype)",
    "molecules": "tamoxifen, Tamoxifen"
  },
  {
    "caption": "C) Q-PCR analysis reveals a progressive increase in the YAP-TEAD target gene Ctgf in Lats1/2 dKO intestines at 3-days and 7-days post i.p. injection with tamoxifen, as well as similarly increased TEAD1 and TEAD4 expression, confirming that TEAD1 and TEAD4 are YAP-responsive genes. Notably, Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1/2 deletion, consistent with Axin2 being a known Wnt-specific target gene, and with Wnt signalling remaining active in Lats1/2 dKO intestines.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "B) YAP immunostaining of 3-day old wild type organoids treated with DMSO (Control) or Porcupine inhibitor LGK974 at 5µM. Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation. Quantification of YAP protein levels is shown below, with statistical significance in an unpaired 2-tailed t-test.",
    "molecules": "DMSO, LGK974"
  },
  {
    "caption": "A) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d. n > 10 organoids in each experiment. B) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** p <0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired student's t-test. C ",
    "molecules": "Dasatinib"
  },
  {
    "caption": "C) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. Q-PCR analysis of the YAP-TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired student's t-test.",
    "molecules": "Dasatinib, eCF506"
  },
  {
    "caption": "A) Mouse intestinal organoids immunostained for YAP (green). Note the mostly cytoplasmic localisation of YAP at 48h in the organoid. Irradiation with 4Gy of X-rays drives strong nuclear localisation of YAP in many cells, which is reversible by treatment with the Src family kinase inhibitor Dasatinib. The Dasatinib treated organoids exhibit increased cell death after irradiation, as indicated by pyknotic nuclei (white arrow). n > 10 organoids in each experiment. This experiment employs the same control batch of 2-3d organoids also shown in Figure 6A.",
    "molecules": "Dasatinib"
  },
  {
    "caption": "C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition).",
    "molecules": "LGK974"
  },
  {
    "caption": "D) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.",
    "molecules": "LGK974"
  },
  {
    "caption": "(b) Quantification of CTM-GFP and mCTM-GFP relative to WT-GFP in HEK cells 24 h after individual transfections with WT-GFP, mCTM-GFP (n = 6) or CTM-GFP. Cells transfected with WT-GFP or CTM-GFP were treated without (no treatment; n = 13) or with serum deprivation (+ SD; n = 5; to enhance CMA activity) or with macroautophagy inhibitor 3-methyladenine (+ 3-MA; 10 mM; n = 5), proteasome inhibitor (+ MG132; 5 μM; n = 6), lysosome inhibitor (+ NH4Cl; 20 mM; n = 6) or pepstatin A (+ Pep A; 10 μM; n = 5). Top, representative immunoblots of cell lysates for GFP. Membranes reprobed for β-actin were used as a loading control. Bottom, means ± s.e.m. of relative protein levels, normalized to WT-GFP (arbitrarily set as 1; white bar); n represents the number of independent experiments from at least 4 separate cultures. One-way ANOVA with Fisher least significant difference test, F(12,73) = 20.939. ***P 0.001 versus WT-GFP (white bar); ΔΔΔP 0.001 relative to untreated CTM-GFP levels (gray bar). Full-length blots are presented in Supplementary Figure 9.",
    "molecules": "3-MA, 3-methyladenine, NH4Cl, MG132, Pep A, pepstatin A"
  },
  {
    "caption": "(c) Representative confocal images of colocalization of GFP with the lysosome marker LAMP-1 in COS-7 cells transfected with either WT-GFP (n = 17) or CTM-GFP (n = 16). Scale bar, 20 μm.",
    "molecules": "CTM"
  },
  {
    "caption": "b) Reciprocal coimmunoprecipitation (IP) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1. Flag-tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA-GluN2Bct-CTM or HA-GluN2Bct-CTMm at various ratios in HEK cells, and coimmunoprecipitation and/or immunoblotting were performed 24 h thereafter. Anti-HA was used to detect HA-GluN2Bct-CTM and HA-GluN2Bct-CTMm, anti-FLAG to detect wtDAPK1 and cDAPK1.",
    "molecules": "CTM"
  },
  {
    "caption": "(c) HA-GluN2Bct-CTM specifically and dose-dependently decreased the level of cDAPK1 (n = 4 independent experiments from 4 separate cell cultures and transfections; P 0.001; F(5,18) = 18.27) but not wtDAPK1",
    "molecules": "CTM"
  },
  {
    "caption": "(d; n = 3 independent experiments from 3 separate cell cultures and transfections; P = 0.933; F(5,12) = 0.249). HA-GluN2Bct-CTM-mediated cDAPK1 knockdown was significantly reduced by NH4Cl (c; 20 mM; n = 4; ΔΔΔP 0.001 compared to HA-GluN2Bct-CTM:cDAPK1 8:1 group) and by mutational inactivation of CTM",
    "molecules": "NH4Cl, CTM"
  },
  {
    "caption": "(e; HA-GluN2Bct-CTMm; P = 0.785 compared to control (white bar); F(4,35) = 0.432, 8 independent experiments from 8 separate cell cultures and transfections). Levels of cDAPK1 or wtDAPK1 cotransfected with pcDNA3.0 vector (0:1, white bar) represent the control values, arbitrarily set as 1. *P 0.05, **P 0.01, ***P 0.001, compared with the control; n.s., not significant. Bars represent relative means ± s.e.m. Full-length blots are presented in Supplementary Figure 9. Membranes reprobed for β-actin were used as loading controls. One-way ANOVA with Fisher least significant difference test.",
    "molecules": "CTMm"
  },
  {
    "caption": "(a) Left, NMDA (50 μM; 30 min) activated DAPK1, resulting in a time-dependent decrease in its phosphorylation levels (pDAPK1, n = 4). Right, coimmunoprecipitation (IP) with anti-GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA-induced association between DAPK1 and GluN2B (n = 3).",
    "molecules": "NMDA"
  },
  {
    "caption": "(b) Design and production of TAT-GluN2Bct-CTM and TAT-GluN2Bct peptides (left) using an E. coli expression system. Coomassie blue staining after SDS-PAGE assessed their purity (right); left lane, size marker.",
    "molecules": "CTM"
  },
  {
    "caption": "(c) Bath application of TAT-GluN2Bct-CTM (200 μM; n = 9), but not TAT-GluN2Bct (200 μM; n = 6), knocked down activated DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; one-way ANOVA; P 0.001, F(5,36) = 10.891) and dose- (d, doses in μM; n = 4; P 0.001; F(6,21) = 18.14) and time-dependent (e; P 0.001; F(8,44) = 12.074). (f) A single pretreatment of TAT-GluN2Bct-CTM (sing; 200 μM, 60 min before and during the 30-min NMDA stimulation) produced a transient reduction of DAPK1, returning to baseline within 7 h (n = 4; P = 0.888), and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h (mult; n = 4; ΔΔP = 0.002). One-way ANOVA; P 0.001, F(4,15) = 10.389.",
    "molecules": "NH4Cl, NMDA, CTM"
  },
  {
    "caption": "(g) Synthetic peptides TAT-GluN2B and TAT-GluN2BCTM.",
    "molecules": "CTM"
  },
  {
    "caption": "(h) TAT-GluN2BCTM (25 μM; n = 5; P = 0.001), but not control TAT-GluN2B (25 μM; n = 4; P = 0.223), decreased native DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; P = 0.302). One-way ANOVA, P 0.001, F(5,24) = 13.591. Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control (white bar; *P 0.05, **P 0.01 and ***P 0.001) or NMDA-treated group (gray bar; ΔP 0.05, ΔΔP 0.01 and ΔΔΔP 0.001). Membranes reprobed for β-actin were used as a loading control. Bars represent relative means ± s.e.m. Sample sizes represent number of individual experiments. Full-length blots are presented in Supplementary Figure 9.",
    "molecules": "saline, NH4Cl, NMDA, CTM"
  },
  {
    "caption": "(a) Top, the synthetic cell-penetrating α-synuclein targeting peptide TAT-βsynCTM and its control TAT-βsyn. Middle, immunoblots demonstrate that TAT-βsynCTM (25 μM; n = 5), but not the CTM-lacking control peptide TAT-βsyn (25 μM; n = 5), specifically decreased the targeted endogenous α-synuclein (one-way ANOVA, Tukey post hoc, P 0.001, F(3,16) = 12.435), without affecting the level of unrelated control protein PSD-95 at 4 h (bottom), and this reduction was prevented in the presence of NH4Cl (20 mM; n = 5). Sample sizes represent individual experiments from at least 3 separate primary cultures. (",
    "molecules": "NH4Cl, CTM"
  },
  {
    "caption": "(b) Top, PSD-95 targeting peptide TAT-GluN2B9cCTM and control TAT-GluN2B9c. Middle, TAT-GluN2B9cCTM (25 μM; n = 4), but not Tat-GluN2B9c (25 μM; n = 4), effectively degraded endogenous PSD-95 (one-way ANOVA, Tukey post hoc, P 0.001, F(3,12) = 18.154) without perturbing untargeted protein α-synuclein (bottom). NH4Cl rescued PSD-95 degradation. Sample sizes represent individual experiments from at least 2 separate primary cultures. Membrane reprobing for β-actin was used as an additional specificity and loading control. ΔP 0.05, **,ΔΔP 0.01 and ***P 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (arbitrarily set as 1). Full-length blots are presented in Supplementary Figure 9.",
    "molecules": "saline, NH4Cl, CTM"
  },
  {
    "caption": "(a) Immunoblotting for phosphorylated DAPK1 (pDAPK1) revealed a time-dependent activation (dephosphorylation) of DAPK1 by H2O2 treatment (300 μM; 30 min; n = 4).",
    "molecules": "H2O2"
  },
  {
    "caption": "(b) Bath application of 100 μM TAT-GluN2Bct-CTM (36.54 ± 7.1% of control; n = 8; P = 0.001), but not TAT-GluN2Bct (89.13 ± 10.78%; n = 7; P = 0.311), 60 min before and during H2O2 treatment (300 μM; 30 min) knocked down DAPK1 at 2 h after washout, and this effect was rescued by NH4Cl (20 mM; n = 8; **P = 0.003 to control; P = 0.106 to H2O2-treated). One-way ANOVA, F(4,34) = 11.628, P 0.001. Bars represent DAPK1 levels relative to saline control group. β-actin was used as a loading control.",
    "molecules": "saline, NH4Cl, H2O2, CTM"
  },
  {
    "caption": "(c) LDH assay revealed that H2O2 treatment (300 μM; 30 min) resulted in a significant increase in neuronal death 12 h after treatment (n = 8; 2.50 ± 0.12; P 0.001 to control), which was rescued by breaking down H2O2 with catalase (100 U in 10 μl phosphate-buffered saline; n = 4; 1.17 ± 0.02; P = 0.001 to H2O2 group). H2O2-induced neurotoxicity was significantly reduced by TAT-GluN2Bct-CTM (50 μM; applied 60 min before and maintained throughout the experiments; n = 9; 1.56 ± 0.08; P = 0.001 to H2O2 group), but not by TAT-GluN2Bct (50 μM; n = 9; 2.63 ± 0.10; P = 0.105 to H2O2 group) or the NMDAR antagonist APV (1 μM; n = 4; 2.16 ± 0.14; P = 0.169 to H2O2 group). NH4Cl abolished the neuroprotective effect of TAT-GluN2Bct-CTM (n = 4; 2.39 ± 0.27; P = 0.538 compared to H2O2 group). One-way ANOVA, P 0.001, F(6,41) = 26.842. ***,ΔΔΔP 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (white bar, arbitrarily set as 1). n represents individual experiments from at least 3 separate primary cultures. Full-length blots are presented in Supplementary Figure 9.",
    "molecules": "saline, APV, NH4Cl, catalase, H2O2, CTM"
  },
  {
    "caption": "(d) Immunoblots demonstrated specific DAPK1 knockdown in the infarct (Ipsi) but not contralateral (Contra) side following application of TAT-GluN2BCTM (10 mg per kilogram, i.v.; n = 3; t(4) = 14.459, P 0.001) but not TAT-GluN2B (10 mg per kilogram, i.v.; n = 3; t(4) = 0.739, P = 0.501). β-actin was used as a loading control; two-tailed Student's t-test ***P 0.001; n.s., not significant.",
    "molecules": "CTM"
  },
  {
    "caption": "(e) HE (left) and immunohistochemical DAPK1 (right) staining of adjacent brain sections. Compared with those in saline (top) and TAT-GluN2B-treated (middle) controls, TAT-GluN2BCTM treatment (bottom) selectively reduced infarct area (left) and DAPK1 levels (right) ipsilaterally. Black outlining delineates infarct areas as visualized with HE staining.",
    "molecules": "saline, CTM"
  },
  {
    "caption": "(f) Left, brain sections stained with Fluorojade B in rats injected with saline (n = 6), TAT-GluN2B (n = 5) or TAT-GluN2BCTM (n = 5) after treatment as shown in a. Right, quantification of cellular damage by counting the number of Fluorojade B-positive cells in each 640 × 640 pixel image at 10× magnification. TAT-GluN2BCTM (10 mg per kilogram) displayed more prominent neuroprotection in the cortex (P 0.001) and striatum (P 0.001) as compared to TAT-GluN2B (10 mg per kilogram). Cortex: H(2) = 41.235; P 0.001; striatum: H(2) = 38.808; P 0.001. Kruskal-Wallis ANOVA on ranks with Dunn's post hoc; bars represent relative means ± s.e.m., ***,ΔΔΔP 0.001. n values represent tissue from 3 animals collected from at least 2 litters. Scale bars 1 mm in b,c,e, 20 μm in f. Full-length blots are presented in Supplementary Figure 9.",
    "molecules": "saline, CTM"
  },
  {
    "caption": "Histochemical  (H&E) and immunostaining  (red) staining  of T cells (CD3+), neutrophils  (Ly6b+) and phosphorylated Stat3  (p-Stat3) (red nuclear staining, black arrows) in skin  thin sections prepared at 14 days post-IR  from mice  treated by daily topical application of acquosum   (control), ruxolitinib  or tofacitinib  following irradiation  (15 Gy). Scale bar, 100 μm. (CD3  and Ly6b immunostaining  images from Tofacitinib  treated mice  represent seperate analyses performed on serial thin sections.) Quantification of epidermal  thickness,  T cells (CD3+) and neutrophils  (Ly6b+), and p-STAT3+ cells in (B), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.",
    "molecules": "ruxolitinib, tofacitinib, Tofacitinib"
  },
  {
    "caption": "Photographic images showing hair  loss in mice  at 14 days post-IR   (15 Gy) following treatment by daily topical application of acquosum  (carrier control) or JAK  inhibitors, ruxolitinib  or tofacitinib. Chest  and neck hairs  of mice  in all groups were clipped prior to irradiation   to enhance drug absorption. Quantification of the area of  ventral hair  loss (in pixels) in mice  in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.",
    "molecules": "ruxolitinib, tofacitinib"
  },
  {
    "caption": "Photographic images showing hair  loss in control (PBS) and cyclosporine  (CsA) treated WT mice  at 14 days post-IR  (15 Gy). Quantification of the area of ventral hair loss in mice  in (A), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.",
    "molecules": "CsA, cyclosporine, PBS"
  },
  {
    "caption": "Histochemical  (H&E) and immunostaining  (red) show  T cell (CD3+) and neutrophil  (Ly6b+) infiltration in the skin  of mice  in (A). Scale bar, 100 μm. (Images of CD3  and Ly6b immunostaining  from CsA  treated mice  represent seperate analyses performed on serial thin sections.) Quantification of epidermal  thickness, and T cell (CD3+) and neutrophil  (Ly6b+) infiltration (red  staining) in skin  thin sections from (C), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.",
    "molecules": "CsA"
  },
  {
    "caption": "Quantification by  real-time qPCR analysis of Il6, Il17a, II22, Ccl3, Ccr6, and Ccl20  mRNAs in whole skin  of naïve and irradiated  (15Gy) control (PBS) and CSA  treated WT mice  at 14 days post-IR, (n = 5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.",
    "molecules": "CSA, PBS"
  },
  {
    "caption": "Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. RKO (WT and cTAZ-/-) cells treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF using STAT1 antibody (green). DNA was labeled by DAPI (blue). Scale bar, 50 μm. Quantification was performed using ImageJ software (right). N>C: nuclear; N=C: nuclear plus cytoplasmic; N<C: cytoplasmic localization.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "Depletion of cTAZ repressed viral replication. Cells were treated the viral genomic RNA was quantified by qPCR (normalized to human GAPDH). Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.",
    "molecules": "genomic RNA"
  },
  {
    "caption": "Overexpression of cTAZ promotes virus infection. DOX-inducible cTAZ-overexpressing RKO cell lines were pre-treated with DOX for 48 hours (1 μg/ml). gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.",
    "molecules": "DOX"
  },
  {
    "caption": "(B) Representative immunofluorescence images showed the expression of multiple RPE markers Pax6, MITF and Bestrophin in the chimeric fetuses. Blue, Hochst3342. WT, Bama WT fetus as positive control. NW-2, the chimeric fetus. MITFL247S/L247S fetus as negative control. Scale bars, 50 μm",
    "molecules": "Hochst3342"
  },
  {
    "caption": "(A) WASH deficiency enhances autophagy induction. WASH+/+ or WASH−/− MEFs were treated with EBSS for the indicated times in the presence or absence of 20 nM bafilomycin A1 (BafA1), and harvested for immunoblotting. Ratios of LC3‐II/β‐actin were calculated and shown at the right panel.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(C) Poly‐ubiquitinated proteins are reduced in WASH−/− MEFs. WASH+/+ and WASH−/− MEFs were starved in EBSS for 2 h treated with or without 20 nM BafA1 or 10 μM MG132. Cells were harvested for immunoblotting with anti‐poly‐ubiquitin antibody (Enzo, clone FK1) that only recognizes poly‐ubiquitinated ubiquitin chains.",
    "molecules": "BafA1, MG132"
  },
  {
    "caption": "(D) WASH does not localize in autolysosomes. HeLa cells were treated with CM or 1 h in the presence or absence of 20 nM BafA1 at 37°C for 1 h. WASH and LAMP1 were visualized by staining with anti‐WASH and anti‐LAMP1 antibodies.",
    "molecules": "BafA1"
  },
  {
    "caption": "(I) K437R‐Beclin 1 mutant suppresses autophagosome formation. Beclin 1‐knockdown HeLa cells expressing WT‐Beclin 1 or K437R‐Beclin 1 were infected with lentivirus encoding GFP‐LC3, and stimulated with EBSS for 1 h in the presence or absence of 20 nM BafA1. GFP‐LC3 dots were visualized by confocal microscopy. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.",
    "molecules": "BafA1"
  },
  {
    "caption": "(A) WASH overexpression suppresses the ubiquitination of Beclin 1. HEK293T cells were transfected with the indicated vectors for 24 h followed by an Ni‐NTA‐based pull‐down assay and immunoblotted with anti‐poly‐ubiquitin (anti‐poly‐Ub) (left panel), and anti‐K63‐specific ubiquitin (anti‐K63‐Ub) (middle panel) antibodies. The same blot was stripped and probed with anti‐K48‐specific ubiquitin (anti‐K48‐Ub) antibody (right panel).",
    "molecules": "Ni, NTA"
  },
  {
    "caption": "(B) WASH overexpression hinders K63‐linked ubiquitination of Beclin 1. HeLa cells stably expressing vector or Flag-WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibodies against endogenous Beclin 1 and K63‐linked polyubiquitin (K63‐Ub). The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and K63‐Ub was calculated and shown in the right panel.",
    "molecules": "BafA1"
  },
  {
    "caption": "(C) WASH overexpression inhibits LC3 lipidation. HeLa cells stably expressing vector or Flag-WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibody against endogenous LC3. LC3 dots were calculated and shown in the right panel.",
    "molecules": "BafA1"
  },
  {
    "caption": "A. Ribbon diagram showing the 2-fold dimerization interface of Mdm12. Oxygen and nitrogen atoms are shown in red and blue, respectively. The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12. The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N-terminus β1-strand. Ten orthologs are aligned from residues 1 to 11. Absolute and highly conserved residues are indicated in red and orange, respectively.",
    "molecules": "nitrogen, Oxygen"
  },
  {
    "caption": "D. Mdm12 binds NBD-PE. Wild-type and monomeric (I5P mutant) Mdm12 were incubated with NBD-PE and separated from free NBD-PE in native PAGE. Coomassie staining (left) and fluorescent (right) detection indicates that Mdm12 directly interacts with NBD-PEin vitro.E. Quantitative data showing binding affinities for NBD-PE by Mdm12. The binding affinities of Mdm12 (monomer/dimer shown in Native PAGE and I5P mutant) for NBD-PE was measured with a NBD-PE concentration-dependent manner. All experiments were carried out three times and the means ± SD are given.",
    "molecules": "PE"
  },
  {
    "caption": "F. Mdm12 mutants (L256W, I262W, and L256W/I262W double mutant) were incubated with NBD-PE, and subjected to native PAGE. Because wild-type Mdm12 separates as both monomer and dimer on native PAGE, the purely monomeric form (I5P) of Mdm12 was used as the wild type for clarity. The graph in the right indicates the quantities measured in the experiments. The bar shows the relative amounts of the band ratio (fluorescence/Coomassie). Values represent the means and SD from three independent experiments.",
    "molecules": "PE"
  },
  {
    "caption": "A. Structural views suggesting that Mdm12 might have a preference for certain phospholipids. The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution (left) and the sequence conservation (right) in the same orientation. Surface electrostatics and sequence conservation were calculated using an APBS program [31] with the nonlinear Poisson-Boltzmann equation and contoured at ±3kT/e, and ConSurf website (consurf.tau.ac.il) [32] with 34 different yeast orthologs, respectively. The ribbon diagram shown in the middle indicates the overall orientation of Mdm12.",
    "molecules": "PE"
  },
  {
    "caption": "B. Lipid displacement experiments to identity the natural ligands of Mdm12in vitro (see material and methods section for details). NBD-PE-preloaded Mdm12-GST was mixed with a series of glycerophospholipids at different concentrations, and displacement of NBD-PE by non-labeled ligand was estimated from the decrease in fluorescence. Means ± SD are shown (n=3 independently performed experiments).",
    "molecules": "PE"
  },
  {
    "caption": "(C) TAK1-/-MEF were infected to express a CRISPR/Cas9 system targeting luciferase or the spata2 gene, from which single clones were generated. The cells were treated with mTNF (10 ng/ml) or mTNF along with Necrostatin-1 (Nec-1) for 1-3 h as indicated. The blot was sequentially probed with antibodies recognizing PARP, cleaved caspase-3 and tubulin.",
    "molecules": "Nec-1, Necrostatin-1"
  },
  {
    "caption": "(D). The same cells as used in (C) were treated as before with TNF (10 ng/ml) or TNF along with Necrostatin-1 (Nec-1) for one and 2 h as indicated. Caspase activity was determined using the fluorogenic substrate DEVD-AMC.",
    "molecules": "Nec-1, Necrostatin-1"
  },
  {
    "caption": "Representative images of GINs cultures showing GABA and TUJ1 expression. Scale bar = 100 μm.",
    "molecules": "GABA"
  },
  {
    "caption": "The proportion of GABA+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at day 35. (CTRL, n=120 fields were counted from 5 cell lines; sMDD, n=134 fields were counted from 6 cell lines).",
    "molecules": "GABA"
  },
  {
    "caption": "G. Traces of Na+/K+ currents recorded from GINs in sMDD and CTRL groups. Quantification showed average of peak values of Na+ currents (CTRL, n=46 neurons from 3 lines; sMDD, n=38 neurons from 3 lines). Nested t-test, **p＜0.01. H. Average fast potassium currents of GINs in sMDD and CTRL groups (CTRL, n=43 neurons from 3 lines; sMDD, n=36 neurons from 3 lines).",
    "molecules": "potassium, Na+"
  },
  {
    "caption": "Representative images of calcium imaging in CTRL and sMDD GINs at each timepoint. Scale bar = 20 μm.",
    "molecules": "calcium"
  },
  {
    "caption": "The representative trajectory of average intensity changes over time from CTRL (red line) and sMDD (blue line) neurons in response to 67 mM KCL. CTRL, n=6 neurons; sMDD, n=6 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=155 neurons derived from 5 CTRL cell lines, n=198 neurons derived from 6 sMDD cell lines). Nested t-test, **p=0.0022 for CTRL versus sMDD.",
    "molecules": "Ca2+, KCL"
  },
  {
    "caption": "Immunostaining for the GABA interneuron marker GAD67, neural progenitor marker SOX2 and ventral prosencephalic progenitor marker NKX2.1 at day 30 in CTRL and sMDD ventral forebrain organoids.",
    "molecules": "GABA"
  },
  {
    "caption": "Representative images of calcium imaging in CTRL and sMDD ventral forebrain organoids at different states.",
    "molecules": "calcium"
  },
  {
    "caption": "Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per cell line (n=156 neurons derived from 4 CTRL cell lines, n=88 neurons derived from 4 sMDD cell lines). Nested t-test, ***p=0.0005 for CTRL versus sMDD.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Calcium imaging was performed on GFP+ neurons in the knockdown group. The representative image shows the rest state of GFP+ cells and the state of cells after KCl stimulation. The pseudo-color showed Rhod-4 calcium fluorescent dye, and the selected cells are GFP+ neurons.",
    "molecules": "Rhod-4, Calcium, calcium, KCl"
  },
  {
    "caption": "The representative trajectory of average intensity changes over time in Vehicle virus and KD virus groups. CTRL, n=10 neurons; sMDD, n=10 neurons. Traces are from a representative experiment (the whole quantification result is shown in I). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per virus group. n=89 for Vehicle virus, n=118 for KD virus, respectively from 4 batches. T-test, ****p<0.0001.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Calcium imaging was performed on GFP+ neurons in the overexpression group. The representative image shows the rest state of GFP+ cells and the state of cells after KCl stimulation. The pseudo-color showed Rhod-4 calcium fluorescent dye, and the selected cells are GFP+ neurons.",
    "molecules": "Rhod-4, Calcium, calcium, KCl"
  },
  {
    "caption": "The representative trajectory of average intensity changes over time in Vehicle virus and OE virus groups. CTRL, n=10 neurons; sMDD, n=10 neurons. Traces are from a representative experiment (the whole quantification result is shown in D). Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per virus group. n=166 for Vehicle virus, n=81 for OE virus, respectively from 3 batches.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Representative images of calcium imaging in CTRL (from RC01001-C) and sMDD (from SA004) GINs at resting and stimulus state with different treatments.",
    "molecules": "calcium"
  },
  {
    "caption": "Quantification of peak [Ca2+] (Fmax-F0)/F0 shown with different treatments including BDNF, Trzd, PNU, Ro at concentrations of 1 μM and 10 μM. T-test, **p=0.0042 for sMDD+BDNF versus sMDD; one-way ANOVA, ****p＜0.0001 for sMDD+10 μM Trzd versus sMDD. n≥7 neurons from each group.",
    "molecules": "Ro, BDNF, Ca2+, PNU, Trzd"
  },
  {
    "caption": "Dot plots showing the expression level and percentage of genes selected from functional gene sets across CTRL, sMDD and sMDD with Trzd GINs clusters.",
    "molecules": "Trzd"
  },
  {
    "caption": "Top: Violin plots showing the expression level of CAMK2N1 and CAMK2N2 in three groups. Bottom: Scatter plot showing the correlations of foldchanges according to the expression of genes encoding CaMK family proteins between CTRL GINs and sMDD GINs and between sMDD with Trzd GINs and sMDD GINs. A linear regression line is added to the plot based on these two variables. sMDD+Trzd vs. sMDD, n=14 genes; CTRL vs. sMDD, n=14 genes.",
    "molecules": "Trzd"
  },
  {
    "caption": "Representative images of single neuron from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs.",
    "molecules": "Trzd"
  },
  {
    "caption": "Representative line chart of Sholl intersection number over distance from soma in CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs at day 45. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines. Quantification of maximum intersections from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs via Sholl analysis. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines.",
    "molecules": "Trzd"
  },
  {
    "caption": "Quantification of sum of intersections from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs via Sholl analysis. CTRL, n=81 neurons from 5 cell lines; CTRL+Trzd, n=81 neurons from 5 cell lines; sMDD, n=73 neurons from 6 cell lines; sMDD+Trzd, n=71 neurons from 6 cell lines. One-way ANONA, ****p＜0.0001 for CTRL versus sMDD; ***p=0.0002 for sMDD versus sMDD+Trzd. The representative trajectory of average intensity changes over time from CTRL, CTRL+Trzd, sMDD and sMDD+Trzd GINs in response to 67 mM KCL. CTRL, n=20 neurons; CTRL+Trzd, n=20 neurons; sMDD, n=20 neurons; sMDD+Trzd, n=21 neurons. Tracs are from a representative experiment (the whole quantification result is shown in Fig EV5E).",
    "molecules": "KCL, Trzd"
  },
  {
    "caption": "Quantification of peak [Ca2+] (Fmax-F0)/F0 shown per group, CTRL, CTRL+Trzd, sMDD and sMDD+Trzd all from 3 cell lines. T-test, *p=0.0495 for CTRL versus sMDD, **p=0.0075 for sMDD versus sMDD+Trzd.",
    "molecules": "Ca2+, Trzd"
  },
  {
    "caption": "Representative images of calcium imaging in sMDD and sMDD+Trzd ventral forebrain organoids at different states.",
    "molecules": "calcium, Trzd"
  },
  {
    "caption": "Quantification of the effect of Trzd on peak [Ca2+] (Fmax-F0)/F0 in ventral forebrain organoids (CTRL from IMR90-4, n=40 cells; CTRL+Trzd from IMR90-4, n=58 cells; sMDD from SA007, n=49 cells; sMDD+Trzd from SA007, n=64 cells). Two-tailed test, ****p＜0.0001 for CTRL versus sMDD; ***p=0.0007 for sMDD versus sMDD+Trzd.",
    "molecules": "Ca2+, Trzd"
  },
  {
    "caption": "Representative electrophysiological traces of AP at a holding potential of -70 mV from GINs in sMDD and sMDD+Trzd groups. Average number of APs evoked during 500ms stepwise depolarization (CTRL, n=35 neurons from 3 lines; CTRL+Trzd, n=30 neurons from 3 lines; sMDD, n=55 neurons from 3 lines; sMDD+Trzd, n=44 neurons from 3 lines).",
    "molecules": "Trzd"
  },
  {
    "caption": "Traces of Na+/K+ currents were recorded from GINs in sMDD and sMDD+Trzd groups. Average peak values of Na+ currents (CTRL, n=46 neurons from 3 lines; CTRL+Trzd, n=32 neurons; sMDD, n=38 neurons from 3 lines; sMDD+Trzd, n=36 neurons from 3 lines).",
    "molecules": "Na+, Trzd"
  },
  {
    "caption": "(D) Serum fatty acids and (E) glycerol from fed or 6‐h‐fasted Con and KO mice on RD (n=7-9).",
    "molecules": "fatty acids, glycerol"
  },
  {
    "caption": "(F) Serum fatty acids and (G) glycerol after intraperitoneal isoproterenol (Iso) in 6‐h‐fasted Con and KO mice on RD (n=5-6).",
    "molecules": "fatty acids, glycerol, Iso, isoproterenol"
  },
  {
    "caption": "(A) Blood glucose levels in 6 mo (n=7-9) and (B) 12 mo Con and KO mice on RD (n=6), and in (C) 12‐mo‐old Con and KO mice on HFD (n=4).",
    "molecules": "glucose"
  },
  {
    "caption": "(D) Serum insulin levels in 6 mo, (n=7-9) and (E) 12‐mo‐old RD‐fed Con and KO mice that were fed or 6 h fasted (n=6).",
    "molecules": "insulin"
  },
  {
    "caption": "(L) Insulin tolerance tests and (M) body weights of 1‐mo‐old Con and KO mice on RD (n=3-5). Values are mean±s.e.m. P values are as compared with diet‐ and age‐matched controls. *P0.05, **P0.01, ***P0.001. Con, control; HDF, high‐fat diet; HOMA, homeostasis model of insulin resistance; KO, knockout; mo, month; POMC, proopiomelanocortin; RD, regular chow; Stv, fasted.",
    "molecules": "insulin"
  },
  {
    "caption": "(J) Serum fatty acids and (K) glycerol from fed or 6‐h‐fasted (Stv) 3 mo and 22 mo mice (n=7-9), and (L,M) after intraperitoneal Iso in 6‐h‐fasted 3 mo and 22 mo mice (n=5-6).",
    "molecules": "fatty acids, glycerol, Iso"
  },
  {
    "caption": "(D) qPCR  analysis of TAPT1  expression in 3 WT (WT1, WT2, WT3) and 3 affected (V.1 (F1), V.5 (F1), IV.1 (F2)) primary fibroblasts  treated with cycloheximide  (CHX). CHX  was used to block nonsense mediated decay (NMD). Our results showed a time dependent increase in the level of TAPT1  transcripts in all 3 patient cells while TAPT1  RNA level remained constant in the WT cells. For each graph, fold change relative to non-treated condition is plotted as mean ± SD. Asterisks indicate conventional statistical significance (Student t-test; n.s. p-value > 0.05, **** p-value < 0.0001).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "d: Quantification of the number of ZIKV E proteins on the EV surface. The percentage of 10nm gold sphere-labeled IC-EVs was counted based on the immuno-EM images of IC-EVs. N indicates the total number of counted IC-EVs.",
    "molecules": "gold"
  },
  {
    "caption": "(c) MN-1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux. A representative LC3 western blot is shown. All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (d) The ratio of LC3-II:actin for c determined by densitometry analysis using ImageJ. n = 3 independent experiments, F = 0.4501, one-way ANOVA with post hoc Tukey test. *P 0.05.",
    "molecules": "ammonium chloride"
  },
  {
    "caption": "(e) MN-1 cells were treated with ammonium chloride to block lysosomal activity, and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6-h recovery period. We performed p62 western blot analysis on MN-1 cells at baseline (-), after ammonium chloride treatment (+) and after the recovery period (Rec). All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (f) On the basis of the p62 values obtained in e, we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline/recovery values to baseline/recovery for MN-1 WT cells. n = 3 independent experiments, F = 5.45, one-way ANOVA with post hoc Tukey test. *P 0.05, ** P 0.01. Data are presented as mean ± s.e.m. Scale bar, 20 μm. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.",
    "molecules": "ammonium chloride"
  },
  {
    "caption": "(b) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation. Untreated: n = 3 independent experiments, F = 3.48, one-way ANOVA with post hoc Tukey test. **P 0.01. Sucrose treatment: n = 3 independent experiments, **P 0.01, t-test, WT t(10) = 8.133; ***P 0.001, t-test, AR 24Q t(10) = 6.11; P not significant (n.s.), AR 65Q t(10) = 0.644. Sucrose-treated MN-1 AR24Q cells displayed significantly higher 4X-CLEAR luciferase activity: n = 3 independent experiments, F = 2.49, one-way ANOVA with post hoc Tukey test. ***P 0.001. All ratios were normalized to untreated MN-1 WT cells, whose value was set to 1.",
    "molecules": "sucrose, Sucrose"
  },
  {
    "caption": "(c) We exposed MN-1 cells to ammonium chloride, an inhibitor of lysosomal activity, to determine the capacity for TFEB induction. RT-PCR analysis revealed lack of TFEB target gene induction in MN-1 AR65Q cells for four gene targets: Lamp1 (lysosomal-associated membrane protein 1), F = 37.51; Atp6v1h (vesicular ATPase V1 subunit H), F = 57.26; Mcoln1 (mucolipin 1), F = 251.0; and Gla (galactosidase-α), F = 75.51; n = 3 independent experiments, *P 0.05, **P 0.01, ANOVA with post hoc Tukey test. For Lamp1 and Mcoln1, MN-1 AR24Q cells yielded significantly higher expression. Differences in the expression of TFEB target genes at baseline were negligible.",
    "molecules": "ammonium chloride"
  },
  {
    "caption": "(b) We transfected MN-1 AR65Q cells with the 4X-CLEAR luciferase reporter and with BFP-TFEB or BFP empty vector and subjected the MN-1 AR65Q cells to conditions that promote TFEB activation, as shown. MN-1 WT cells were also transfected with the 4X-CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes. Results are shown normalized to untreated MN-1 WT cells expressing BFP-empty. Untreated, F = 182.7; starvation, F = 63.64; NH4Cl, F = 291.9; Rapamycin, F = 3128; ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001.",
    "molecules": "NH4Cl, Rapamycin"
  },
  {
    "caption": "(d) MN-1 WT, MN-1 AR24Q and MN-1 AR65Q cells were cultured in standard medium (Ctl) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB. Cells were scored for nuclear or cytosolic localization of TFEB. Scale bars, 20 μm. (e) Quantification of d. Untreated, F = 0.56; R1881, F = 5.51; ANOVA with post hoc Tukey test. **P 0.01.",
    "molecules": "R1881"
  },
  {
    "caption": "(f) We transfected MN-1 WT cells with the 4X-CLEAR luciferase reporter and either BFP-empty or AR25Q-BFP, under standard culture conditions (control), ammonium chloride treatment or rapamycin treatment. Control, t(3) = 15.73; NH4Cl, t(4) = 4.108; rapamycin. t(4) = 4.58; t-test. **P 0.01.",
    "molecules": "ammonium chloride, rapamycin"
  },
  {
    "caption": "(g) We transfected MN-1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X-CLEAR luciferase reporter activity under baseline conditions (control) or after rapamycin. Control, t(4) = 0.51; rapamycin, t(4) = 7.15; t-test. **P 0.01.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP). Immunoblotting (IB) for AR confirmed expression of AR in control (Ctrl) and SBMA NPCs, and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs.",
    "molecules": "DHT, dihydrotestosterone"
  },
  {
    "caption": "Flow cytometric analysis of γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse and line the median. *p < 0.05, **p < 0.01, ***p < 0.001 using Mann-Whitney test. Numbers of γδT17 cells in the LN (A) and skin (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments.",
    "molecules": "IMQ"
  },
  {
    "caption": "Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.",
    "molecules": "IMQ"
  },
  {
    "caption": "Flow cytometric analysis of γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). In graphs, each symbol represents a mouse and line the median. Numbers of γδT17 cells in the LN and skin (staining as in Fig EV1) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 11; 4 experiments.",
    "molecules": "IMQ"
  },
  {
    "caption": "Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 6; 3 experiments, IMQ: n = 8; 3 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.",
    "molecules": "IMQ"
  },
  {
    "caption": "Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-17A (A) or IL-17F (D) and IFNγ production in γδ T cells before (steady-state) and after IMQ-induced psoriasis. Numbers in gate indicate % positive cells; numbers outside the gate indicate mean fluorescence intensity of IL-17A or IL-17F. (B, E) Frequency of IL-17A+ (B) and IL-17F+ (E) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C, F) Quantification of mean fluorescence intensity (MFI) of IL-17A (C) and IL-17F (F) staining in γδ T cells after IMQ-induced psoriasis (each color represents a different experiment).",
    "molecules": "IMQ"
  },
  {
    "caption": "Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (A) and frequency of IL-22+ γδ T cells (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 8-9; 3 experiments.",
    "molecules": "IMQ"
  },
  {
    "caption": "Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). (A, ) Frequency of IL-17A+ (A) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (B) Frequency of IL-17F+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C) Quantification of mean fluorescence intensity (MFI) of IL-17A staining in γδ T cells after IMQ-induced psoriasis (D) Quantification of mean fluorescence intensity (MFI) of IL-17F staining in γδ T cells after IMQ-induced psoriasis.",
    "molecules": "IMQ"
  },
  {
    "caption": "Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis.",
    "molecules": "IMQ"
  },
  {
    "caption": "(A) Representative traces showing SQR-driven oxygen consumption in 3x106 control (C) and five patientsfibroblasts (P1-5) in the absence (solid traces) or presence (dashed traces) of CoQ10 in the culture medium. See Materials and Methods for the role and concentration of the compounds used. (B) Relative rates of SQR-driven oxygen consumption are normalized to CIV respiration rates.",
    "molecules": "oxygen"
  },
  {
    "caption": "(A) Representative western blots showing SQR, TST, ETHE1 and SUOX of two control fibroblasts treated with DMSO (-) or with DMSO+ 4-NB (+). (B) Quantification of proteins levels in fibroblasts treated with 4-NB relative to control.",
    "molecules": "4-NB, DMSO"
  },
  {
    "caption": "(A) Total GSH amount normalized to protein from kidney and (B) brain of wild-type (WT) and mutant (Mut) mice. Error bars represent SDs of at least 3 animals per group.",
    "molecules": "GSH"
  },
  {
    "caption": "(C) Thiosulfate levels in urine and (D) plasma normalized to protein amount. Error bars represent SDs of 9 animals per group (urine) and 6 animals per group (plasma).",
    "molecules": "Thiosulfate"
  },
  {
    "caption": "(E) H2S amount normalized to tissue from kidney and (F) brain. Error bars represent SDs of 7/8 animals per group.",
    "molecules": "H2S"
  },
  {
    "caption": "(G) Acylcarnitine C4, C5 and C6 levels from plasma, normalized to volume. Error bars represent SDs of 5 wild-type and 3 mutants.",
    "molecules": "Acylcarnitine C4"
  },
  {
    "caption": "(A Total PD-L1, PARP from colon (Colo-205), lung (A549), pancreatic (PANK1) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2 GAPDH is loading control",
    "molecules": "KMTR2, lexatumumab, Lexa"
  },
  {
    "caption": "Total PD-L1, PARP from triple negative breast cancer cell (MDA-MB-436) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2, BaCa, GAPDH is loading control",
    "molecules": "BaCa, KMTR2, lexatumumab, Lexa"
  },
  {
    "caption": "C) Total PD-L1, PARP, cleaved caspase-3 from ovarian cell (Cavo-3) lysates treated with indicated DR5 agonist antibodies named tigatuzumab. GAPDH is loading control",
    "molecules": "tigatuzumab"
  },
  {
    "caption": "(D) Total PD-L1, CD47, Calreticulin, PARP from MDA-MB-436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z-VAD. GAPDH is loading control.",
    "molecules": "Z-VAD"
  },
  {
    "caption": "(E) Total PD-L1 western blotting signal from the lysates of OVCAR-3 (n=3) and MDA-MB-436 (n=3) cells after 6 hrs treatment of 100nM lexa (DR5 agonist antibody) was normalized to GAPDH.",
    "molecules": "lexa"
  },
  {
    "caption": "(F) Surface biotinylation of PD-L1 from indicated tumor cells after indicated DR5 agonist treatments. Cleaved caspase-3 and PARP indicates activation of DR5 signaling. In lane 3 and 4, KMTR2 was pre-neutralized either with recombinant DR5 (rDR5) or recombinant FOLR1 (rFOLR1).",
    "molecules": "KMTR2"
  },
  {
    "caption": "(K) ER (-), PR (-) and HER2 (-) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, following by treatment with IgG1 or KMTR2 (50μg, 4 doses). Harvested tumors were analyzed for PD-L1, CD47 and PARP in lysates.",
    "molecules": "KMTR2"
  },
  {
    "caption": "(L) KMTR2 treated (50μg, 4 doses) UCD52 TNBC PDX tumors were stained for PD-L1 using immunohistochemistry (IHC).",
    "molecules": "KMTR2"
  },
  {
    "caption": "(C) Luciferase activity of reporter lines from tumor cell-Jurkat cell co-culture assay using MDA-MB-436 and OVCAR3 cells after treatment (50nM) with indicated DR5 agonists (tiga: tigatuzumab, AMG: AMG655, KMTR1, Lexa: Lexatumumab). The background luciferase signal from untreated cells (due to basal surface PD-L1) was subtracted. Various controls treatments (IgG1, anti-PD-L1, anti-EGFR) are also shown (n=3). (D) Same as (C) except tumor cells were pre-treated with indicated inhibitors for AKT, ERK, mTOR, MEK, STAT3, PARP-i and NF-Kβ along with DR5 agonist KMTR2 antibody prior to co-culture. ",
    "molecules": "KMTR1, KMTR2, Lexatumumab, tigatuzumab, AMG655, AMG, Lexa, tiga"
  },
  {
    "caption": "(H) Flow cytometry analysis of MDA-MB-436 cells treated with TNFα ± MG132 (top) and indicated DR5 agonist ± MG132 (bottom).",
    "molecules": "MG132"
  },
  {
    "caption": "(I) Total PD-L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "(J) Flow cytometry surface PD-L1 analysis of OVCAR-3 cells treated with indicated DR5 agonist ± MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "(K) MDA-MB-436 cells were treated with MG132 for indicated times and total PD-L1 from lysates was analyzed. GAPDH is loading control.",
    "molecules": "MG132"
  },
  {
    "caption": "(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.",
    "molecules": "KMTR2, ubiquitin"
  },
  {
    "caption": "ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots.",
    "molecules": "KMTR2"
  },
  {
    "caption": "ApoEVs isolated after IgG1 and tigatuzumab treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.",
    "molecules": "tigatuzumab"
  },
  {
    "caption": "ApoEVs isolated after IgG1 and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.",
    "molecules": "lexatumumab"
  },
  {
    "caption": "(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.",
    "molecules": "HPG, Met"
  },
  {
    "caption": "(G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVs",
    "molecules": "KMTR2"
  },
  {
    "caption": "after addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2).",
    "molecules": "KMTR2, lexatumumab"
  },
  {
    "caption": "(I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment.",
    "molecules": "KMTR2, Lexa"
  },
  {
    "caption": "(K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band",
    "molecules": "KMTR2, ZDEVD"
  },
  {
    "caption": "(L) Relative caspase-8 and caspase-3 activity assays caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3).",
    "molecules": "ZDEVD"
  },
  {
    "caption": "(M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) , cells were allowed to grow 24 hrs, followed by cell viability analysis.",
    "molecules": "KMTR2, ZDEVD"
  },
  {
    "caption": "(A KMTR2, KMTR2+Z-VAD treated MDA-MB-436 lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1 PARP as indicated.",
    "molecules": "KMTR2, Z-VAD"
  },
  {
    "caption": "B) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.",
    "molecules": "lexatumumab"
  },
  {
    "caption": "(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.",
    "molecules": "ionophore A23187, lexatumumab"
  },
  {
    "caption": "(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)",
    "molecules": "KMTR2, GSK269, GSK269962A, GSK429, GSK429286A"
  },
  {
    "caption": "(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)",
    "molecules": "KMTR2, GSK269, GSK269962A, GSK429, GSK429286A"
  },
  {
    "caption": "Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads. (G) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.",
    "molecules": "KMTR2, avelumab, GSK269962"
  },
  {
    "caption": "(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.",
    "molecules": "avelumab"
  },
  {
    "caption": "(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.",
    "molecules": "MD5-1, GSK269962A"
  },
  {
    "caption": "(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).",
    "molecules": "KMTR2, lexatumumab"
  },
  {
    "caption": "(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry",
    "molecules": "avelumab, lexatumumab"
  },
  {
    "caption": "(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).",
    "molecules": "GSK269962A"
  },
  {
    "caption": "(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). average tumor weight is shown (n=4).",
    "molecules": "GSK269962A"
  },
  {
    "caption": "(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.",
    "molecules": "avelumab"
  },
  {
    "caption": "(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size.",
    "molecules": "GSK269962A, lexatumumab, Lexatumumab"
  },
  {
    "caption": "(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6).",
    "molecules": "avelumab, lexatumumab"
  },
  {
    "caption": "Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). (O) KMTR2 (8 doses) antibody used as the DR5 agonist for the experiment. (P) Average of tumor weights of data shown in (O). ",
    "molecules": "KMTR2, GSK269962A"
  },
  {
    "caption": "(Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.",
    "molecules": "KMTR2, avelumab, GSK269962A, PBS"
  },
  {
    "caption": "(A) Chi-G4S-DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab +ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2mg/kg dose (6 total). Various treatments were started when tumors were ~400mm3 size. Harvested tumors were grouped together and sized matched (3 independent sets: n=2-6 tumors in each set) followed by TIL isolation CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry.",
    "molecules": "avelumab, lexatumumab, PBS"
  },
  {
    "caption": "Chi-G4S-DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab +ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.",
    "molecules": "avelumab, lexatumumab"
  },
  {
    "caption": "(H) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5-1 and bispecific avelu-MD5 antibody.",
    "molecules": "MD5, MD5-1, avelu"
  },
  {
    "caption": "(K) ~150-200mm3 size MC38 tumor bearing C57LB/6 mice were treated with indicated MD5-1, avelumab and bispecific antibodies along with control IgG1, 6 total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.",
    "molecules": "MD5-1, avelumab"
  },
  {
    "caption": "(L) 6-8 weeks old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150-200mm3, animals were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day. On day 18, tumors were harvested, sized matched and pooled by treatment group, exposed to collagenase/DNase and were single cell suspensions enriched for CD8+ cells. Enriched CD8+ T-cells from various treatments were restimulated with anti-CD3 (OKT3) antibody for 4 additional hours. CD8 gated cells were next analyzed for IFN-γ intracellular expression using flow cytometry.",
    "molecules": "OKT3"
  },
  {
    "caption": "a, b. The amount of FDP-lysine, which was major protein-bound acrolein, in the sera of healthy controls and PD patients was determined by an ELISA (a. all patients, (control n= 22; PD n=94) b. staged by the H&Y scale, a five-steps index of PD severity. Larger number indicates more severe condition. n=94; H&Y stage I (n=18), stage II (n=42) stage III (n=23), stage IV (n=7), and stage V (n=4).",
    "molecules": "acrolein, FDP-lysine"
  },
  {
    "caption": "SH-SY5Y cells were treated with the indicated chemicals at 50 μM for 4 h (a) Cells were fixed and stained with an anti-LAMP2 (green) antibody as a lysosomal marker. Nuclei were stained with DAPI (blue). Cell membrane is indicated by the white line. Clustered relative to whole lysosomes were quantified as lysosomal clustering value (a, right).",
    "molecules": "DAPI"
  },
  {
    "caption": "e. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.",
    "molecules": "acrolein"
  },
  {
    "caption": "h. SH-SY5Y cells were treated with acrolein for 2 h. Cell lysates were immunoblotted with the indicated antibodies.",
    "molecules": "acrolein"
  },
  {
    "caption": "d. SH-SY5Y cells were transfected with the indicated siRNAs for 48 h and then treated with 40 µM acrolein for 2 h. (upper) Cells were fixed and stained with an anti-LAMP2 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.",
    "molecules": "acrolein, DAPI"
  },
  {
    "caption": "a. Cells were transfected with the TMEM55B-myc-DDK plasmid for 24 h and then treated with 40 μM acrolein for 2 h. Cell lysates were immunoprecipitated with flag magnetic beads and immunoblotted with the indicated antibodies.",
    "molecules": "acrolein"
  },
  {
    "caption": "b. SH-SY5Y cells were treated with 40 μM acrolein for 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).",
    "molecules": "acrolein, DAPI"
  },
  {
    "caption": "c. SH-SY5Y cells treated with the indicated concentration of acrolein for 2 h were lysed, subjected to Phos-tag PAGE, and immunoblotted with an anti-JIP4 antibody. d. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI (Jak3 i VI) for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells lysates were subjected to Phos-tag PAGE and immunoblotted with an anti-JIP4 antibody.",
    "molecules": "acrolein"
  },
  {
    "caption": "e. SH-SY5Y cells were pre-treated with 10 µM Jak3 inhibitor VI for 1 h and then treated with 40 µM acrolein for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue).",
    "molecules": "acrolein, DAPI"
  },
  {
    "caption": "SH-SY5Y cells transfected with indicated siRNAs for 48 h were treated with 40 μM acrolein for 2 h. Cell lysates were subjected to Phos-tag PAGE and immunoblotted with anti-JIP4 antibody (f, left). CaMK2G knockdown efficiency was confirmed by qRT-PCR.; ***p <0.001, n=3 technical replicates. Values are mean ± SD. At least three experiments were replicated (f, right).",
    "molecules": "acrolein"
  },
  {
    "caption": "j. His6-tagged recombinant truncated JIP4 (100-300 aa) was purified from E. coli and incubated with recombinant CaMK2G with/without Ca2+ and calmodulin at 30°C　for 1 h. The reactant was subjected to Phos-tag PAGE and immunoblotted with anti-His antibody. Jak3 inhibitor VI was pre-reacted with CaMK2G for 30 min.",
    "molecules": "Ca2+, calmodulin"
  },
  {
    "caption": "c. GFP or GFP-ALG2 transfected TRPML1-mCherry stably expressing cells were treated with acrolein for 2 h. Cell lysates were immunoprecipitated with anti-RFP magnetic beads and immunoblotted with the indicated antibodies.",
    "molecules": "acrolein"
  },
  {
    "caption": "f. SH-SY5Y cells transfected with GFP or GFP-ALG2 for 24 h were treated with acrolein for 2 h and then spatial proximity of JIP4 and GFP was detected by a PLA using JIP4 and GFP antibodies.",
    "molecules": "acrolein"
  },
  {
    "caption": "Parental and JIP4 KO SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 for 2 h (a). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.",
    "molecules": "acrolein, bafilomycin A1"
  },
  {
    "caption": "SH-SY5Y cells were treated with 40 µM acrolein with or without 100 nM bafilomycin A1 in the presence of 10 µM Jak3 inhibitor VI (Jak3 i VI) for 2 h (b). Cell lysates were immunoblotted with the indicated antibodies. Autophagic flux was determined as the LC3-II production rate calculated as the level of LC3-II with bafilomycin A1 treatment.",
    "molecules": "acrolein, bafilomycin A1"
  },
  {
    "caption": "d. (upper) SH-SY5Y cells were pretreated with 10 µM BAPTA-AM or 10 µM Jak3 inhibitor VI for 1 h and then treated with 500 µM H2O2 for an additional 2 h or cultured in starvation medium for an additional 2 h. Cells were fixed and stained with anti-JIP4 (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (lower) Clustered relative to whole lysosomes were quantified as lysosomal clustering value.",
    "molecules": "BAPTA-AM, DAPI, H2O2"
  },
  {
    "caption": "e. SH-SY5Y cells were treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were lysed, subjected to Phos-tag PAGE, and immunoblotted with anti-JIP4 antibody.",
    "molecules": "acrolein, H2O2"
  },
  {
    "caption": "f. (left) SH-SY5Y cells were transfected with TMEM55B siRNAs for 48 h and then treated with 40 µM acrolein and 500 µM H2O2 or cultured in starvation medium for 2 h. Cells were fixed and stained with anti-γ-tubulin (green) and anti-LAMP2 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (right) Clustered relative to whole lysosomes were quantified as lysosomal clustering value. ***",
    "molecules": "acrolein, DAPI, H2O2"
  },
  {
    "caption": "b. Parental and JIP4 KO SH-SY5Y cells were treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay. c. SH-SY5Y cells were transfected with TRPML1 siRNAs for 48 h and then treated with various concentrations of acrolein for 24 h. Cytotoxicity was measured by an LDH assay.",
    "molecules": "acrolein"
  },
  {
    "caption": "E-F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood.Cells (E-F) were restimulated with PMA/Ionomycin for 4 h. Data consist of PBMC isolated from n=4 healthy individuals.Data (E) are FACS plots of cells gated on CD14-CD3+CD4+cells with mean frequencies per group and quantifications shown as bar diagram (mean +SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated cell populations with mean fluorescent intensity (MFI) ±SEM or mean frequencies per group. *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "A. Analysis of CD4T cells isolated from the dLN and restimulated with PMA/Ionomycin for 4 h. Data are representative FACS plots, gated on VD-CD4+cells with mean frequencies per group.Cells (A-D) were isolated at day 9 after MOG/CFA/PTx immunization. Data consist of n=5 mice of each genotype. *p < 0.05, **p < 0.01, N.S. - not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "I-K. Analysis of Treg cells isolated from mice shown in (D). (I) Total Treg cell numbers (mean +SEM) isolated from CNS and dLN. (J) Ratio of Treg and Th17cells isolated from the CNS. Data are representative FACS plots, gated on VD-CD90+CD4+cells with mean frequencies per group. (K) Quantification of Treg/Th17 cell ratio (mean +SEM) within CNS and dLN.Cells were restimulated with MOG for 6 h and (I-N) in the presence of PMA/Ionomycin for 4 h.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "B Immunostaining analysis of protein localization. U2OS cells were transfected with construct encoding ORF3a. Cells were fixed and stained with the indicated antibodies. The green signal is CLCC1 or lysosome marker LAMP1, the red signal is flag (for SFB-ORF3a), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C,D Effect of SARS-CoV-2 N protein on stress granule formation. A549 (C) and MCF10A (D) cells were transfected with pinducer20-N protein of SARS-CoV-2. All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression. Cells were fixed and stained with the indicated antibodies. The green signal is G3BP1, the red signal is HA (for N protein), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.",
    "molecules": "DAPI, Dox, sodium arsenite"
  },
  {
    "caption": " Co-culture model of HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and an UbG76V-GFP proteostasis reporter in donor and receiver compartment (48 h). (C) Separate analysis of both compartments by immunoblot and (D) immunoblot quantification. For quantitative analysis of immunoblots UbG76V-GFP was normalized to calnexin. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Red dashed line indicates the control's expression level. * denotes p<0.05, *** denotes p<0.001. ",
    "molecules": "Ub"
  },
  {
    "caption": " (F,G) Flow cytometry analysis of a UbG76V-GFP reporter cell line incubated 48h with conditioned media from RFP or GA175-RFP transfected cells upon immunodepletion of poly-GA or control depletion using unspecific IgG. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05. (G) Comparisons of the corresponding histograms for compensated RFP and UbG76V-GFP fluorescence from one representative experiment that shows specific transmission of GA175-RFP associated with accumulation of UbG76V-GFP in cells incubated with GA175-RFP conditioned media. ",
    "molecules": "Ub"
  },
  {
    "caption": " Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP-43 levels in neurons with poly-GA aggregates or treated with MG132. Arrows mark punctate TDP-43 staining. Rolipram treatment reduced cytoplasmic TDP-43 in GA175-GFP neurons. Scale bar denotes 20 µm. (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced neurons. n=4 biological replicates. In total 462 GFP and 371 GA175-GFP cells treated with vehicle, and 386 GFP and 529 GA175-GFP cells treated with MG132, 513 GFP and 434 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "DMSO, MG132, rolipram, Rolipram"
  },
  {
    "caption": " Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (C) Immunoblot to show effects of MG132 and rolipram on GA175-GFP and GFP expression. ",
    "molecules": "DMSO, MG132, rolipram"
  },
  {
    "caption": " Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. Filter trap assay with quantification of SDS-insoluble aggregated GA175-GFP. n=5 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "DMSO, MG132, rolipram"
  },
  {
    "caption": " (F,G) HeLa cells were co-transfected with RFP-TDP-CTF and GFP or GA175-GFP for 2 days. For the final 16h, cells were treated with rolipram (30 µM) or MG132 (10 µM). Filter trap assay of SDS-insoluble TDP-CTF aggregates quantified by densitometry. n=4 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "MG132, rolipram"
  },
  {
    "caption": " (H,I) HeLa cells were co-transfected with TDP-43ΔNLS-GFP and iRFP or GA175-iRFP for 2 days. For the final 16h, cells were treated with either vehicle or rolipram (30 µM). Filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates quantified by densitometry. n=3 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "rolipram"
  },
  {
    "caption": " Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP-reporter. White arrows indicate cells with cytoplasmic TDP-43. (B) Automated quantification of cells with cytoplasmic TDP-NLS reporter in GFP and GA175-GFP positive cells. n=4 biological replicates. In total 345 GFP and 386 GA175-GFP cells treated with vehicle, 371 GFP and 404 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "rolipram"
  },
  {
    "caption": " Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. (C) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "rolipram"
  },
  {
    "caption": " Immunofluorescence of Hela cells transfected with GFP or GA175-GFP showing reduced poly-GA aggregation upon rolipram treatment (30 µM, 16 h). (H) Automated quantification of poly-GA aggregate number per cell. n=3 biological replicates. In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. ",
    "molecules": "rolipram"
  },
  {
    "caption": " (C HeLa cells were co-transfected with the indicated TDP-NLS reporters as well as GFP or GA175-GFP, and treated with MG132 (10µM) or vehicle for 16h. ",
    "molecules": "MG132"
  },
  {
    "caption": " HeLa cells were co-transfected with wildtype or K95A GFP-TDP NLS, HA-ubiquitin and iRFP or GA175-iRFP. 24h after transfection, cells were treated with rolipram (30 µM), MG132 (10 µM) or DMSO (vehicle) for 16h. Lysates were immunoprecipitated with Protein G beads coupled with anti-GFP antibody. (A) Immunoblotting of input (left panels) and anti-GFP immunoprecipitates (right panels) to show GFP-reporter levels and poly-ubiquitination. (B) Quantification of HA-ubiquitin levels normalized to total GFP- NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05, and *** denotes p<0.001. Red dashed line indicates the control's expression level. ",
    "molecules": "DMSO, MG132, rolipram, ubiquitin"
  },
  {
    "caption": "D-I eIF2α phosphorylation  in WT and Perk  KO astrocytes  (N=3) and neurons  (N=6) ± 20-24 hours of TM-induced ER  stress. Representative WB  (D, G), quantification of absolute phosphorylated eIF2α  level (p-eIF2α/eIF2α) (E, H) and the ER  stress-induced p-eIf2α  response (F, I). Data information: (E, H, Data are normalised to untreated WT. (F, I Data are related to untreated cells of the same genotype. Baseline level (without ER  stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, H, Students t-test (F, I)",
    "molecules": "TM"
  },
  {
    "caption": "J-O Protein synthesis in WT and Perk  KO astrocytes  (N=5) and neurons  (N=5) ± 20-24 hours of TM-induced ER  stress. Representative images obtained by high-content microscopy  showing puromycinilated proteins detected by immunofluorescence  (J, M), quantification of puromycin  intensity as measure for de novo protein synthesis (K, N) and the ER  stress-induced protein synthesis response (L, O). Astrocytic soma  (GFAP, white), dendrites  (MAP2, white), nuclei  ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green). Scale bar: 25 μm. Data information: K, N) Data are normalised to untreated WT. , L, O) Data are related to untreated cells of the same genotype. Baseline level (without ER  stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test K , N); Nested t-test (L, O).",
    "molecules": "puromycin, Puromycin, TM"
  },
  {
    "caption": "A-F ATF4 expression   in WT and Perk  KO astrocytes  (N=3) and neurons  (N=9) ± 20-24 hours of TM-induced ER  stress. (A, D) Representative confocal  images of ATF4 immunofluorescence. Astrocytic soma  (GFAP, cyan), dendrites  (MAP2, cyan), nuclei  ((Δ)Cre, green), ATF4  (magenta). Scale bar: 10 μm. (B, E) Quantification of absolute nuclear ATF4  intensity. Data are normalised to untreated WT. (C, F) Quantification of the ER  stress-induced ATF4 response. Data are related to untreated cells of the same genotype. Baseline level (without ER  stress) is depicted by a dashed line. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F).",
    "molecules": "TM"
  },
  {
    "caption": "G-J (G, I) Venn diagram showing the number of significantly upregulated ATF4  target genes in WT (grey and blue) and Perk  KO (green and blue) astrocytes  (G, N=3) and neurons  (I, N=3) upon 20-24 hours of TM-induced ER  stress determined by mRNA-seq. Percentage: fraction of upregulated ATF4  target genes in WT cells that are also upregulated in Perk  KO cells. (H, N=3; J, N=3) Log2 fold change of ATF4  target genes that are significantly upregulated in both WT and Perk  KO cells (blue in Venn diagram). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant.",
    "molecules": "TM"
  },
  {
    "caption": "Protein synthesis and ATF4 expression  in mouse  (A-F) astrocytes  (A-C and neurons  (D-F ± 20-24 hours of TM-induced ER  stress ± PERK-i (Pi). (A, D, Representative images obtained by  high-content microscopy. Astrocytic soma  (GFAP dendrites  (MAP2, white), nuclei  (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4  (magenta). Scale bar: 25 μm. (B, N=5; E, N=5; Quantification of puromycin  intensity as measure for de novo protein synthesis. (C, N=5; F, N=7; Quantification of absolute nuclear ATF4  intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.",
    "molecules": "Puromycin, puromycin, TM"
  },
  {
    "caption": "Protein synthesis and ATF4 expression  in human  (G-L) astrocytes  G-I, ) and neurons  J-L) ± 20-24 hours of TM-induced ER  stress ± PERK-i (Pi). G, J) Representative images obtained by high-content microscopy. Astrocytic soma Phalloidin, white), dendrites  (MAP2, white), nuclei  (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4  (magenta). Scale bar: 25 μm. ; H, N=3; K, N=3) Quantification of puromycin  intensity as measure for de novo protein synthesis. I, N=4; L, N=3) Quantification of absolute nuclear ATF4  intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.",
    "molecules": "Phalloidin, Puromycin, puromycin, TM"
  },
  {
    "caption": "D-G ROS  accumulation in WT and Perk KO neurons (N=3) and astrocytes  (N=3) ± 20-24 hours of TM-induced ER  stress. (D, F) Representative images obtained by high-content microscopy. Dendrites   (MAP2, white), astrocytic soma  (Phalloidin, white), nuclei ((Δ)Cre, red), ROS  (CellROX  green, green). Scale bar: 25 μm. (E, G) Quantification of CellROX  green intensity. Data information: (E, G, Data are normalised to untreated WT. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, G,",
    "molecules": "CellROX, Phalloidin, ROS, TM"
  },
  {
    "caption": "I Quantification of the increase in nuclear ATF4  intensity in WT neurons  ± HRI  KD ± 20 hours of sodium arsenite  (Ars) treatment (N=3). J The remaining ATF4  response (%) upon HRI  KD, related to ATF4  response of WT transduced with shRNA control (100%, represented by a dashed line) (N=3). Calculated from data in (I): [[(KD_Ars+-KD_Ars-)/KD_Ars-]/[(WT_Ars+-WT_Ars-)/WT_Ars-]]*100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test I, One sample t-test with a hypothetical value of 100 J)",
    "molecules": "Ars, sodium arsenite"
  },
  {
    "caption": "K-M eIF2α phosphorylation in HRI  KD WT and Perk  KO neurons (N=3) ± 20-24 hours of TM-induced ER  stress. Representative WB  (K), quantification of the ER  stress-induced p-eIf2α  response (L) and absolute phosphorylated eIF2α  level (p-eIF2α/eIF2α) (M). Data information: M, Data are normalised to untreated WT. (L, Data are related to untreated cells of the same genotype and normalised to WT without HRI  KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test L, Students t-test (M)",
    "molecules": "TM"
  },
  {
    "caption": "N-S Protein synthesis (N=3) and ATF4 expression  (N=4) in HRI  KD WT and Perk  KO neurons  ± 20-24 hours of TM-induced ER  stress. Representative images obtained by (N) confocal  and (Q)  high-content microscopy. Dendrites  (MAP2, white/cyan), nuclei  ((Δ)Cre, green/red), de novo synthesized proteins (Puromycin, green), ATF4  (magenta). Scale bar: 25 μm. Quantification of the ER  stress-induced protein synthesis  response (O) and puromycin intensity as measure for de novo protein synthesis (P). Quantification of the ER  stress-induced ATF4  response (R) and absolute nuclear ATF4  intensity (S). Data information: P, S) Data are normalised to untreated WT. O, R) Data are related to untreated cells of the same genotype and normalised to WT without HRI  KD. Baseline level (without ER  stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, R); Nested t-test (P, S).",
    "molecules": "Puromycin, puromycin, TM"
  },
  {
    "caption": "A-F Protein synthesis (A-C, N=3) and ATF4 expression  (D-F, N=5) in WT and Perk  KO neurons  overexpressing eIF2αWT or phospho-deficient eIF2αS51A ± 20-24 hours of TM-induced ER  stress. Representative images obtained by (A) confocal  and (D)  high-content microscopy. Dendrites  (MAP2, white/cyan), nuclei  ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4  (magenta). Scale bar: 25 μm. Quantification of the ER  stress-induced protein synthesis (B) and ATF4  response (E). Data are normalised to WT eIF2αWT. Quantification showing the percentage change in the ER  stress-induced protein synthesis (C) and ATF4  (F) response induced by eIF2αS51A compared to eIF2αWT. (C) Calculated from data in (B): [(eIF2αS51A-eIF2αWT)/eIF2αWT] *100. (F) Calculated from data in (E): [(eIF2αS51A-eIF2αWT)/eIF2αWT]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E, Nested t-test (C, F).",
    "molecules": "Puromycin, TM"
  },
  {
    "caption": "Expression   of p-eIF2α  sensitive (5'ATF4-uORFWT) and p-eIf2α   insensitive (5'ATF4-uORFMut) EYFP translational reporters in WT and Perk  KO neurons  ± 20-24 hours of TM-induced ER  stress (N=3). (I, K) Representative confocal  images. Dendrites  (MAP2, cyan), nuclei  ((Δ)Cre, red), endogenous ATF4  (magenta), ATF4  reporter (green). Scale bar: 10 μm. (J, L) Quantification of EYFP  intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test J, L)",
    "molecules": "TM"
  },
  {
    "caption": "A-F  Protein synthesis and ATF4 expression  in ISRIB-treated WT and Perk  KO neurons  ± 20-24 hours of TM-induced ER  stress (N=4). (A, D) Representative images obtained by high-content microscopy. Dendrites  (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4  (magenta). Scale bar: 25 μm. Quantification of the ER  stress-induced protein synthesis (B) and ATF4  (E) response. Data are normalised to WT without ISRIB. Baseline level (without ER  stress) is depicted by dashed line. Quantification showing the percentage change in the ER  stress-induced protein synthesis (C) and ATF4  (F) response induced by ISRIB. (C) Calculated from data in (B): [(ISRIB+-ISRIB-)/ISRIB-]*100. (F) Calculated from data in (E): [(ISRIB+-ISRIB-)/ISRIB-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F)",
    "molecules": "ISRIB, Puromycin, TM"
  },
  {
    "caption": "G-M Protein synthesis and ATF4 expression  in 2b5he and 2b5ho astrocytes  and neurons  ± 20-24 hours of TM-induced ER  stress in the presence or absence of PERK-i (Pi) (N=3). (G, I, K, M) Representative images obtained by high-content microscopy. Astrocytic soma  (Phalloidin, white), dendrites  (MAP2, white), nuclei  (DAPI, red), de novo synthesized proteins (Puromycin, green), ATF4  (magenta). Scale bar: 25 μm. (H, L) Quantification of puromycin  intensity as measure for de novo protein synthesis and (J, N) absolute nuclear ATF4  intensity. Data are normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Three-way ANOVA with Tukey's post hoc test (H, J, L, N).",
    "molecules": "DAPI, Phalloidin, Puromycin, puromycin, TM"
  },
  {
    "caption": "A Differentially expressed genes (DEGs) involved in oxidative stress related processes comparing TM-treated Perk  KO and WT neurons. Blue arrow: Ang.",
    "molecules": "TM"
  },
  {
    "caption": "B, C Ang  mRNA level in WT and Perk  KO neurons  and astrocytes  ± 20-24 hours of TM-induced ER  stress, determined by mRNA-seq (N=3). FPMK: Fragments Per Kilobase of transcript per Million. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, C,",
    "molecules": "TM"
  },
  {
    "caption": "F, G tRNA  unfolding in WT and Perk  KO neurons  ± 20-24 hours of TM-induced ER  stress (N=4). (F) Representative images obtained by  high-content microscopy   of 1-methyladenosine (m1A) immunofluorescence  to determine tRNA  unfolding. Dendrites  (MAP2, white), nuclei  ((Δ)Cre, red), unfolded tRNA  (m1A, green). Scale bar: 25 μm. (G) Quantification of m1A  intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test G,",
    "molecules": "1-methyladenosine, m1A, tRNA, TM"
  },
  {
    "caption": "H-M Abundance of small RNAs in WT and Perk  KO astrocytes  (N=3) and neurons  (N=6) ± 20-24 hours of TM-induced ER  stress, determined by high-resolution automated electrophoresis  (H, K) Representative gel image. Electropherogram  (I, L) and zoom (J, M) showing quantified fluorescence units (FU) of  small RNAs, size ranges of  tRNAs and tsncRNAs  are indicated. Shown is the mean of the biological replicates.",
    "molecules": "small RNAs, tsncRNAs, tRNAs, TM"
  },
  {
    "caption": "N-R Protein synthesis  and ATF4 expression  in WT and Perk  KO neurons  ± 20-24 hours of TM-induced ER  stress in the presence or absence of ANG-I (N=3). (N) Representative images obtained by  high-content microscopy. Dendrites  (MAP2, white), nuclei  ((Δ)Cre, red), de novo synthesis of proteins (Puromycin, green). Scale bar: 25 μm. Quantification of the ER  stress-induced protein synthesis (O) and ATF4  response (Q). Data are normalised to WT without ANG-i. Baseline level (without ER  stress) is depicted by dashed line. Quantification showing the percentage change in the ER  stress-induced protein synthesis (P) and ATF4  response (R) induced by ANG-i. (P) Calculated from data in (O): [(ANG-i+-ANG-i-)/ANG-i-]*100. (R) Calculated from data in (Q): [(ANG-i+-ANG-i-)/ANG-i-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, Q); Nested t-test (P, R).",
    "molecules": "Puromycin, TM"
  },
  {
    "caption": "A  PHIP-1 interaction  with GPB-1 by  yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4  DBD-PHIP-1(H45A) and GAL4  AD-GPB-1, as indicated. Yeast  strains carrying the indicated plasmids were cultured on a selective plate lacking histidine  and containing 5 mM 5-aminotriazole for 4 days.",
    "molecules": "5-aminotriazole, histidine"
  },
  {
    "caption": "A  PHIP-1 interaction  with   UNC-51  by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4  DBD-PHIP-1(H45A) and GAL4  AD-UNC-51(274-856), as indicated. Yeast  strains carrying the indicated plasmids were cultured on a selective plate lacking histidine  and containing 5 mM 5-aminotriazole for 4 days.",
    "molecules": "5-aminotriazole, histidine"
  },
  {
    "caption": "B) Tumour growth curves for successive four-generation xenografts treated with 5-Fu or PBS. Twelve mice per group.",
    "molecules": "5-Fu, PBS"
  },
  {
    "caption": "A) Heat map analysis of the expression of circRNAs (both human circRNAs and ebv-circRNAs) in the fourth passage xenografts treated with 5-Fu or PBS. Red colour represents upregulated circRNAs, and green colour represents downregulated circRNAs.",
    "molecules": "5-Fu, PBS, circRNAs"
  },
  {
    "caption": "(B) Volcano plot representing the expression fold changes in circRNAs. The red dots represent upregulated circRNAs, and green dots represent downregulated circRNAs. (C) The back-spliced read distribution of ebv-circRNAs (n=261). ",
    "molecules": "circRNAs"
  },
  {
    "caption": "(D) The length distribution of ebv-circRNAs.",
    "molecules": "circRNAs"
  },
  {
    "caption": "(E) Genomic origin of ebv-circRNAs.",
    "molecules": "circRNAs"
  },
  {
    "caption": "(F) Number of ebv-circRNAs produced from one gene.",
    "molecules": "circRNAs"
  },
  {
    "caption": "(D) Real-time PCR analysis of circLMP2A and LMP2A expression after treatment with actinomycin D at the indicated time points in SNU-4th cells.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "(I) RNA ISH showing the co-localization between circLMP2A and miR-3908 in SNU-4th cells. circLMP2A probes were labelled with Cy3, and miR-3908 probes were labelled with FITC. Nuclei were stained with DAPI.",
    "molecules": "Cy3, DAPI, FITC"
  },
  {
    "caption": "N. Immunofluorescence for GATA4 (green) with DAPI staining (blue) of 10 µm testis cryo-sections from adult CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 100 µm. Dashed lines highlight the STs. O. Quantification of (N), CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 50 STs were analyzed for each mouse. See Appendix Fig S2 for ST number per section and ST diameter. Data information: In O), data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Immunofluorescence for MVH (green) with DAPI staining (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-. Scale bars: 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "G. C-CASP3 (green) and MVH (magenta) staining with DAPI (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-, scale bars: 20 µm. H. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total C-CASP3 positive cells per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. I. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total MVH positive cells (germ cells) per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. Data information In H, I), data are presented as mean ± SEM. n.s., not significant, ***P < 0.005 , ****P < 0.0001 (the Mann-Whitney test).",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Immunofluorescence staining for PLZF (red) with DAPI staining (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P5, P10 and P14. Scale bars: 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Immunofluorescence staining for SCP1 (green), PLZF (red) and DAPI (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P2, P3, P5 and P7. Arrowheads highlight PLZF- SCP1+ (green) cells, which represent differentiated spermatogonia. Scale bars: 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Representative images of mouse seminiferous tubule (ST) sections stained for PLZF (red), SCP1 (green) and DAPI (blue) at P4, P6 and P7. Scale bars: 20 μm. Arrowheads highlight PLZF- SCP1+ (green) cells localize to the middle of the ST lumen, which represent differentiated spermatogonia that translocated to the middle of the ST lumen. C. Quantification of (B). Shown is the number of PLZF- SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per ST section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. D. Quantification of (B). Shown is the total number of SCP1+ cells which translocated inwards the seminiferous tubule lumen per ST section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. E. Quantification of (B). Shown is the number of PLZF+ SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. Data information: In (C-E), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the Mann-Whitney test).",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Representative images of P4 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. B. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. (C Quantitation of spindle angle of anaphase GSCs in P4 ST sections (from A). Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (C), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. (D Quantitation of spindle angle of anaphase GSCs in P5 ST sections (from B). for P5. In (D), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and DAPI (blue) showing GSCs in interphase. Scale bars: 5 μm. C. Quantification of (B). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 100 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In (C data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates. (B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. ",
    "molecules": "AT-406, ATP, GSK872, Nec-1, zVAD"
  },
  {
    "caption": "(D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2). ",
    "molecules": "BMS-345541, ATP, GSK872, Nec-1, zVAD"
  },
  {
    "caption": "(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD.",
    "molecules": "AT406, zVAD"
  },
  {
    "caption": "(D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates. (E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates. ",
    "molecules": "AT-406, zVAD"
  },
  {
    "caption": "(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed. (B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed. ",
    "molecules": "AT-406, AT406, zVAD"
  },
  {
    "caption": "(A) Pml+/+ and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated timeframes. The contents of p-IKKα/β, IKKα/β, IκBα, p-MK2 and MK2 was analysed by immunoblotting. (B) Pml+/+and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time points were then determined. ",
    "molecules": "zVAD"
  },
  {
    "caption": "(C, D, E, F) WT (Pml+/+) and Pml-/- mice were treated with DMSO or Nec-1s (6 μg/g) or MK2 inhibitor PF-3644022 (75 μg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 μg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± S.E.M is shown (C, E).",
    "molecules": "DMSO, Nec-1s, PF-3644022"
  },
  {
    "caption": "(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points.",
    "molecules": "AT-406, zVAD"
  },
  {
    "caption": "(C) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. ",
    "molecules": "AT-406, ATP, GSK872, Nec-1, zVAD"
  },
  {
    "caption": "(D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed.",
    "molecules": "zVAD"
  },
  {
    "caption": "(E) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting.",
    "molecules": "His"
  },
  {
    "caption": "(F) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.",
    "molecules": "DTT, ATP, His, MgCl2"
  },
  {
    "caption": "(C) Immunoelectron micrograph of S-GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads).",
    "molecules": "gold"
  },
  {
    "caption": "(D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(A) Immuno-EM of GFP-p62. S-GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A-gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo).",
    "molecules": "gold"
  },
  {
    "caption": "(B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1.",
    "molecules": "PSI"
  },
  {
    "caption": "(C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.",
    "molecules": "gold"
  },
  {
    "caption": "(A) Comparison of p62 degradation using pulse-chase labeling with 35S-methionine and immunoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed with 35S-methionine and incubated for the indicated times in nonradioactive medium. After immunopurification, the amount of radioactive p62 was determined by autoradiography, and the total amount of p62 was determined by an immunoblot of the same membrane. The p62 level in total cellular lysates after different times of 10 μg/ml cycloheximide treatment determined by immunoblotting (bottom).",
    "molecules": "35S, CHX, cycloheximide, methionine"
  },
  {
    "caption": "(B) The levels of p62 and LC3-II change with autophagic activity. HeLa or S-GFP-p62 cells were either left untreated or rapamycin (10 μg/ml) or bafilomycin A1 (Baf. A1; 10 μg/ml) was added for 18 h. Immunoblots were sequentially probed using LC3, p62, and actin antibodies.",
    "molecules": "Baf. A1, bafilomycin A1, rapamycin"
  },
  {
    "caption": "(C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S-GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3-positive cells with punctuate localization are indicated to the right.",
    "molecules": "amino acids"
  },
  {
    "caption": "(B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.",
    "molecules": "amino acids"
  },
  {
    "caption": "(B) The active fragment of mTOR or active Akt2 was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC-0068.",
    "molecules": "GDC-0068, Torin"
  },
  {
    "caption": "(C) HEK-293E Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.",
    "molecules": "insulin"
  },
  {
    "caption": "(D) HEK-293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.",
    "molecules": "amino acids"
  },
  {
    "caption": "(E) Control or iRapKO MEFs were treated with or without 2 μM 4-Hydroxytamoxifen (4-OHT) for 4 days to induce Raptor knockout. Treated cells were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.",
    "molecules": "4-Hydroxytamoxifen, 4-OHT, insulin"
  },
  {
    "caption": "(F) SIN1 WT was expressed in SIN1 KO MEFs, and cells were selected by FACS. Cell lines were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.",
    "molecules": "insulin"
  },
  {
    "caption": "The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Mass Spectrum for pRagC T394",
    "molecules": "Rapamycin, Torin"
  },
  {
    "caption": "The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Western blot for pRagC S21",
    "molecules": "Rapamycin, Torin"
  },
  {
    "caption": " (A) The HA-RagC HeLa Flp-In T-Rex stable cell lines were created (See methods for details) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight. Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3). (B) Expression of RagC WT, 3A or 3E was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3) ",
    "molecules": "insulin, tetracycline"
  },
  {
    "caption": " (C) Expression of RagC WT and 3A was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for indicated time. Left: Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Right: Graph shows mean ± S.E.M. of quantitative analyses of the Western blots after normalization for total protein, n=3 ",
    "molecules": "insulin, tetracycline"
  },
  {
    "caption": " (D) HEK-293E cells stably expressing RagC WT, 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, ** p<0.01, two tailed Student's t test, n=3) ",
    "molecules": "amino acids"
  },
  {
    "caption": " (A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4) ",
    "molecules": "tetracycline"
  },
  {
    "caption": " (E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4 ",
    "molecules": "Chloroquine"
  },
  {
    "caption": " (F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm ",
    "molecules": "Chloroquine"
  },
  {
    "caption": " (D) Expression of HA-RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells. Total cell lysates (TCL) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (*** p<0.001, Student's t test, n=3)",
    "molecules": "tetracycline"
  },
  {
    "caption": "B, C  Cell death measured by lactase dehydrogenase (LDH) (B) and cell viability  measured by 3-(4,5- dimethyl-thiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (C) in T98 cells 42 hrs post-transfection of pure and interrupted   SCA8  repeat tracts; LDH n=8, MTT n=12, n=independent experiments, * p < 0.05, NT not transfected, EV: empty vector; unpaired t test; mean ± SEM.",
    "molecules": "3-(4,5- dimethyl-thiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide, MTT"
  },
  {
    "caption": "B, C Cell death (B; LDH, n=8) and viability (C; MTT, n=6) assays in T98 cells 42-hrs post-transfection. NT: not transfected; EV: empty vector; n=independent experiments; * p < 0.05; unpaired t test; mean ± SEM.",
    "molecules": "MTT"
  },
  {
    "caption": "Protein  blots of polyGln  proteins expressed in transfected HEK293T cells with interrupted or pure    polyGln  repeats; EV: empty vector; n=3 transfections",
    "molecules": "polyGln"
  },
  {
    "caption": "Immunofluorescence  of  intranuclear inclusions for     polyGln   expressed from Alt.  polyGln  and Alt. Int.  polyGln constructs in  HEK293T cells; scale bar: 50μm; green arrow heads indicate cells positive for Alt. Int.   polyGln intranuclear inclusions; dashed white box in merge panels indicates the region show in the orthogonal (Ortho) zoomed images which show cross section and localization of diffuse   polyGln staining and nuclear polyGln(Arg)  inclusions  with alpha tubulin as a cytoplasmic marker; n=4 experiments with n>55 cells per experiment",
    "molecules": "Arg, polyGln"
  },
  {
    "caption": "K PolyGln(Arg) proteins but not pure polyGln  proteins colocalize with  nucleophosmin in  HEK293T cells; scale bars: 20µm; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co-localization of polyGln inclusions and nucleophosmin in three dimensions.",
    "molecules": "Arg, PolyGln, polyGln"
  },
  {
    "caption": "A-C Protein blotting (A) and quantification (B, C) of polySer RAN proteins in HEK293T cells from interrupted (Int.95; Int.102) or pure (Pure 96; Pure 104) CAG repeats. EV: empty vector, n=3 transfections, ** p<0.01, *p<0.05, unpaired t test, mean ± SEM.",
    "molecules": "polySer"
  },
  {
    "caption": "D, Immunofluorescence of  RAN polySer protein aggregates from CGG interrupted and pure CAG  repeat tracts in HEK293T  cells; scale bar: 10μm. n>20 cells per construct for each experiment (Exp.)",
    "molecules": "polySer"
  },
  {
    "caption": "F, Protein blot  of polyAla RAN  proteins expressed from pure or interrupted constructs; n=3 transfections",
    "molecules": "polyAla"
  },
  {
    "caption": " (C) Flow cytometric analysis of living Zombie- tumour cells with B220+CD11c+NK1.1+ cells that were primed by NoCM or LuCM. NoCM: N=3, LuCM: N=4. Shown are averages with SEM. Welch"s t-test. Rhodamin+ tumour cells were counted as viable cells ",
    "molecules": "Zombie, Rhodamin"
  },
  {
    "caption": " (D) Representative immunohistochemical stainings of rhodamin+ (red) Zombie+ (green) tumour cells (scale bar, 10 μm) ",
    "molecules": "Zombie, rhodamin"
  },
  {
    "caption": " (A) Representative immunostaining of fibrinogen depositions in tumour-bearing mouse lungs received an injection of rhodamine-labelled B220+CD11c+NK1.1+HepELs derived from tumour-bearing- FX+/− or FX−/− mice (scale bar, 20 μm) ",
    "molecules": "rhodamine"
  },
  {
    "caption": " (B) Tumour cell homing in the lungs pre-treated with LiCM-primed B220+CD11c+NK1.1+cells from tumour-bearing WT or FX−/− mouse liver. Representative photos of homing of rhodamine-labelled tumour cells (upper, scale bar, 20 μm). Number of homing tumour cells are shown (lower). Shown are averages (N = 60 sections, 5/group, all field count/section, 12 sections/sample) with SEM and Welch"s t-test ",
    "molecules": "rhodamine"
  },
  {
    "caption": "G.  Western blot  of TREM2 in lysates  and media of cultured  human macrophages isolated from GRN-FTLD #1 and HC #1 upon treatment with Ab1  and Ab2. An isotype antibody was used as a negative control. ADAM protease inhibition (GM) does not further increase mTREM2 levels in GRN-FTLD patients. Equal amounts of protein were loaded. GAPDH was used as loading control.",
    "molecules": "GM"
  },
  {
    "caption": "F. Immunohistochemical analysis of  lipofuscin (green) in coronal  brain  sections. Representative images of thalamus  are shown. Scalebars = 50 μm. G. Quantification of lipofuscin autofluorescence. Five images per mouse were taken, means were normalized to WT samples (n=3 per genotype, female). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.",
    "molecules": "lipofuscin"
  },
  {
    "caption": "E-G. Abundance of BMP species and  glucosylsphingosine (GlcSph) in total brain of 6-month-old  Grn-/-, Trem2-/-, Double-/- and WT mice  (n=4-5 per genotype). H. Glucocerebrosidase (GCase) activity in whole  brain lysates from 6-month-old male Grn-/-, Trem2-/-, Double-/- and WT mice. The linear increase of fluorescence signal was measured and then normalized to WT mice (n=3-6 per genotype). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey`s post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at ***, p < 0.001; ****, p < 0.0001, and ns, not significant.",
    "molecules": "GlcSph, glucosylsphingosine"
  },
  {
    "caption": "(G) FACS  analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) Immunofluorescence  images of SYCP3  (red), DAZL  (yellow) and DAPI  at agg11 of IVDi tissue maturation from XGFP- PGCLCs. IVDi = in vitro differentiation. White squares indicate the positions of the magnified section shown below. Top panel scale bar = 100 μm. Middle panel scale bar = 10 μm. Bottom panel scale bar = 50 μm. (I) Quantification of SYCP3+ cells (oocytes  in cyst and  primary follicles) in IVDi tissues at agg11. Each dot represents one IVDi tissue performed in 3 biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic  capacity of XGFP- and XGFP+ PGCLCs. Meiosis  is induced via addition of Retinoic Acid (RA) and Bone Morphogenetic Protein 2  (BMP2). c0 = starting day of culture, c9 = culture day 9 and last day of culture. (B) Representative images for the expression  of XGFP  (green) and SYCP3  (red) in germ cells at c5, c7 and c9 from XGFP+ and XGFP- PGCLCs. All cells with SYCP3  signal irrespective of localization  pattern or intensity were scored as SYCP3+ cells. Cells were counterstained  with DAPI  (grey). Scale bars = 10 μm. (C) Number of SYCP3+ cells per m220 culture day originating from XGFP+ and XGFP- PGCLCs. Each white dot represents a biological replicate (n = 3). P-values shown are from a two-sided Mann-Whitney U test. n.s. = not significant. (D) Percentage of XGFP+ cells among SYCP3+ cells at the indicated m220 culture day, originating from XGFP+ or XGFP- PGCLCs. Green bars show constitutive GFP expression in XGFP+ PGCLC-derived cells, while striped bars signify XGFP reactivation  in XGFP- cells. Each white dot represents a biological replicate (n = 3).",
    "molecules": "DAPI, RA, Retinoic Acid"
  },
  {
    "caption": "(G) Representative tile scan merged images of XGFP  (green) and Xtomato  (red) in  germ cells at c7 from XGFP+ and XGFP- PGCLCs. Cells were counterstained  with DAPI  (grey). Scale Bar = 1 mm. The white squares contain a magnified image of the region depicted by the white dotted squares (Scale Bar = 1 µm). (H) Density ridge plot showing size of cellular aggregates in (G) measured using the XTomato  signal. X-Axis shows area in µm2 scaled as log2. Dashed line represents the mean. XFGP- mean = 1320 µm2, XGFP+ mean = 5514 µm2. XGFP- n = 297 aggregates, XGFP+ n = 373 aggregates. P-value from two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) ArfGAP1 was stably knocked down in HEK293A cells using shRNAs against ArfGAP1 (shArfGAP1) or GFP (shGFP) control. Cells were cultured under normal condition (NC; lanes 1 and 7), or starved of amino acids for 1 hour (-AA 1h; lanes 2 and 8), followed by either maintaining in amino acid starvation media (-AA; lanes 3 and 9), or addition of 1 mM of Leu (+Leu; lanes 4 and 10), Gln (+Gln; lanes 5 and 11), or Asn (+Asn; lanes 6 and 12) for 2 hours. mTORC1 activity was analyzed similar to (B). Protein levels of ArfGAP1 were confirmed by immunoblotting. S6K1 and Actin were probed as controls.",
    "molecules": "AA, amino acid, amino acids, Asn, Gln, Leu"
  },
  {
    "caption": "(E) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) with Myc-tagged S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4mM of Gln or Asn were added for 2 hours. mTORC1 was analyzed similar to (B). IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at Thr 389. Actin serve as a loading control.",
    "molecules": "AA, amino acids, Asn, Gln, Thr"
  },
  {
    "caption": "(B) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control.",
    "molecules": "AA, amino acids"
  },
  {
    "caption": "(D) MIA Paca-2 cells stably expressing FLAG-tagged or HA-tagged TMEM192 cells were cultured under normal condition (NC) or starved of amino acids (-AA), and then stimulated with 2 mM glutamine and asparagine (+Gln/Asn) for 2 hours. HA-tagged TMEM192 was immunoprecipitated (IP) with HA beads and IP and whole cell lysates (WCL) were probed for ArfGAP1, HA, RagA, or p18 (LAMTOR1). Flag, pS6K1 (mTORC1 activity), S6K1, and Vinculin were probed for as controls.",
    "molecules": "AA, amino acids, Asn, asparagine, Gln, glutamine"
  },
  {
    "caption": "(D) MIA Paca-2 cells stably expressing short hairpin RNAs (shRNAs) against ArfGAP1 or GFP (control) were plated and treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 48 hours prior to cell counting. Columns represent Mean ± SEM (N = 6). Student's t-test shGFP Vehicle vs. shGFP Torin 1 (P = 2.83 E-07), shGFP Vehicle vs. shArfGAP1 Vehicle (*P = 0.025), shGFP Torin 1 vs, shArfGAP1 Torin 1 (P = 0.139), and shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 1.22 E-07). "ns" stands for "not significant".",
    "molecules": "Torin 1"
  },
  {
    "caption": "Representative images of euploid control or ArCK  cells interacting with NK cells. The  NK cell-mediated killing was measured at a 2.5:1 (NK cells: target cells) ratio and was recorded by  live cell imaging for 36 hours at a 30-min interval. TO-PRO-3 (1 μM) was added to the medium at the same time  of NK cell  addition to measure  cell membrane integrity.  Phase contrast (top) and  TO-PRO-3 signal (bottom) from the same field were presented. Arrowheads indicate ArCK cell death. All images were acquired at the same exposure time and light  intensity. Scale bar 20 μM.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": "NK cell-mediated killing for doxorubicin (Doxo), palbociclib (Palbo), and nutlin3 (Nutlin) treated samples (NK cell: target cell =2.5:1). n=3 biological replicates; mean± SEM. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p< 0.0001; Ctrl vs. Nutlin, p< 0.0001; KS test.",
    "molecules": "Doxo, doxorubicin, Nutlin, nutlin3, Palbo, palbociclib"
  },
  {
    "caption": "NK cell-mediated cytotoxicity towards  torin1-treated cells. Torin1 treated (Torin) cells were generated as described in (F) and the NK cell killing assay was performed as described in Figure 1. n=3 biological replicates; mean± SEM. Ctrl vs. Torin, p= 0.79, not significant (n.s.); KS test.",
    "molecules": "Torin, torin1, Torin1"
  },
  {
    "caption": "   γ-H2AX foci were analyzed in ArCK  and G1 arrested cells (generated as described in Figure 2). At least 50 cells were analyzed per condition per replicate. n=2 biological replicates; individual values and mean are shown. The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov-Smirnov test. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p= 0.07, n.s.; Ctrl vs.  Nutlin, p= 0.11, n.s.; Ctrl vs. ArCK, p= 0.0007.   ",
    "molecules": "Doxo, Nutlin, Palbo"
  },
  {
    "caption": "ArCK or euploid proliferating control cells were treated with either DMSO or the NF-κB inhibitor BMS-345541 (5 μM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK  cell co-culture assay. n=3 biological replicates; mean± SEM. ArCK vs  ArCK NF-κB inhibitor, p< 0.0001; KS test. (H, I) The effect of inactivating both  RELA and  RELB on  NK cell-mediated cytotoxicity  in 7-day doxorubicin (H) and nutlin3-treated (I) cells. n2 biological replicates; Doxo-c1 vs.  Doxo RELA RELB-c1, p = 0.02. Nutlin-c1 vs.  Nutlin RELA RELB-c1, p = 0.44, n.s.; KS test. ",
    "molecules": "BMS-345541, DMSO, Doxo, doxorubicin, Nutlin, nutlin3"
  },
  {
    "caption": "HCT116 (left) or DLD1 cells (right) were treated with either DMSO or the Mps1 inhibitor reversine (500 nM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the  NK cell co-culture assay. n=3 biological replicates; mean±SEM. HCT116 vs. HCT116 Mps1 inhibitor, p= 0.44, n.s.; DLD1 vs. DLD1 Mps1 inhibitor, p= 0.44, n.s.; KS test.",
    "molecules": "DMSO, reversine"
  },
  {
    "caption": "(b) Levels of spermidine in the heads of wild-type flies fed with food supplemented by 1 or 5 mM spermidine (Spd1mM+ or Spd5mM+, respectively) or food with no spermidine (Spd−). Data are shown normalized to spermidine levels of 1-d-old (1 d) Spd− flies (n = 4 independent experiments; F = 14.08 for Spd− flies, F = 5.30 for Spd1mM+ flies, F = 2.62 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). 15 d, 15 d old; 30 d, 30 d old.",
    "molecules": "food, Spd, spermidine"
  },
  {
    "caption": "(c) Levels of putrescine in the heads of aged Spd1mM+ or Spd5mM+ flies compared with Spd− flies. Data are shown normalized to putrescine levels of 1-d-old Spd− flies (n = 4 independent experiments; F = 24.31 for Spd- flies, F = 0.22 for Spd1mM+ flies, F = 6.67 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). *P 0.05; **P 0.01; ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.",
    "molecules": "putrescine, Spd"
  },
  {
    "caption": "(a) Aversive associative memory performance at 3 min after training (STM) of 3-d-old (3 d) Spd1mM+ and Spd5mM+ flies in comparison to Spd− flies (n = 8-9 independent experiments, F = 2.28, one-way-ANOVA with Bonferroni correction).",
    "molecules": "Spd"
  },
  {
    "caption": "(b) Aversive associative memory performance at 3 h after training (ITM), ARM and ASM of 3-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 8-9 independent experiments; F = 2.05 for ITM, F = 0.02 for ARM, F = 1.74 for ASM; one-way ANOVA with Bonferroni correction).",
    "molecules": "Spd"
  },
  {
    "caption": "(c) STM of 30-d-old (30 d) Spd1mM+ and Spd5mM+ and Spd− flies (n = 8-9 independent experiments, F = 14.48, one-way ANOVA with Bonferroni correction).",
    "molecules": "Spd"
  },
  {
    "caption": "(d) ITM, ARM and ASM of 30-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 7-9 independent experiments; F = 43.26 for ITM, F = 1.74 for ARM, F = 26.12 for ASM; one-way ANOVA with Bonferroni correction).",
    "molecules": "Spd"
  },
  {
    "caption": "(e) Spermidine feeding for 10 d before measuring memory was sufficient to suppress AMI in aged (30 d old) flies (n = 9-11 independent experiments, F = 12.32, one-way ANOVA with Bonferroni correction).",
    "molecules": "Spermidine"
  },
  {
    "caption": "(f,g) Spermidine feeding on days 21-30 resulted in a significant increase in the levels of spermidine and putrescine in 30-d-old flies (n = 3 independent experiments, F = 32.96 (f) and 64.77 (g), one-way ANOVA with Bonferroni correction). The feeding regime of the flies is presented beneath the graphs, with + and − indicating food with or without spermidine. *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.",
    "molecules": "food, putrescine, Spermidine, spermidine"
  },
  {
    "caption": "(a) Expression of GCaMP3.0 in the mushroom body of an individual fly, focused on the horizontal lobes. The red line indicates a region of interest used to determine changes in fluorescence emission. Scale bar represents 50 μm. (b) False color-coded image of Ca2+ activity in the horizontal mushroom body lobes shown in a. Warm colors indicate high levels, cold colors indicate low levels or no Ca2+ activity. The numbers indicate changes in fluorescence ΔF (%).",
    "molecules": "Ca2+"
  },
  {
    "caption": "(c,d) Time course of Ca2+ increase in horizontal mushroom body lobes of 3-d-old and 30-d-old Spd− flies evoked by the odors 4-methyl-cyclohexanol (MCH) or 3-octanol (OCT) in comparison with the diluent, mineral oil (n = 5 flies, non-parametric Mann-Whitney U test found no substantial difference between the Ca2+ increase of 3-d-old and 30-d-old Spd− flies evoked by the odors). The gray bars indicate the duration of the odor stimuli. Data are presented as mean ± s.e.m.",
    "molecules": "mineral oil, 3-octanol, Ca2+, 4-methyl-cyclohexanol, Spd"
  },
  {
    "caption": "(b) Western blot analysis of poly-ubiquitinated proteins (SDS-soluble protein fraction) from head extracts from Spd− (3 and 30 d old) flies compared with Spd5mM+ (3 and 30 d old) flies. Right, quantification of poly-ubiquitinated proteins levels normalized to α-tubulin (n = 10 or 5 technical replicates from two independent biological aging replicates for all data, F = 6.32, one-way ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9b.",
    "molecules": "SDS, Spd"
  },
  {
    "caption": "(c-f) Adult brains of 3-d-old and 30-d-oldSpd− flies, as well as 3-d-old and 30-d-oldSpd5mM+ fliesimmunostained for ref(2)P. Scale bar represents 25 μm. (g) Quantification of ref(2)P intensity in the central brain region normalized to 3-d-oldSpd− (n = 12-13 independent brains for all data, F = 26.58, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; P > 0.05. Data are presented as mean ± s.e.m.",
    "molecules": "Spd"
  },
  {
    "caption": "(d) Similarly, STM was markedly decreased in 20-d-old Atg8a−/− (Atg8aEP362/Atg8aEP362) mutant female flies compared with control flies (Atg8a+/+) without any effect by spermidine feeding (n = 8-9 independent experiments, F = 68.43, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m. The aversive olfactory avoidance and shock reactivity of different mutant are shown in Supplementary Table 5.",
    "molecules": "spermidine"
  },
  {
    "caption": "(b) Cohesin loading assay. Scc1wt cohesin tetramer was incubated with nicked circular (CN) or 1x, 2x or 4x concentration of linearised (L) plasmid DNA, immunoprecipitated with anti-Scc1 antibodies, washed with high salt buffer and then eluted with Scc1 peptide. Recovered DNA was separated by agarose gel electrophoresis and stained with GelRedDNA stain. Input DNA = 7 %.",
    "molecules": "DNA"
  },
  {
    "caption": "(d) Non-cleaved and cleaved Scc1Halo-TEVcohesin tetramer was incubated with nicked circular plasmid DNA and processed as in (b). Input DNA = 4 %.",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Kymograph of Scc1GFP-TEVcohesin bound to doubly tethered λ-DNA in cohesin binding buffer and washed with 750 mM NaCl buffer + Sytox Orange. XhoI flow in induced DNA cleavage and cohesin release. Flow in from top and scale bar = 5 µm in this and all subsequent kymographs.",
    "molecules": "DNA, NaCl"
  },
  {
    "caption": "(f) Kymographs of Scc1GFP-TEVcohesin and Scc1GFPcohesin bound to doubly tethered λ-DNA and washed with 750 mM NaCl buffer. TEV protease flow in released Scc1GFP-TEVbut not Scc1GFP from DNA.",
    "molecules": "DNA, NaCl"
  },
  {
    "caption": "(a) Kymograph of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex after 750 mM NaCl buffer wash. (b) Distribution of background subtracted EMCCD counts of Scc1Halo-TMR-TEVcohesin complexes immobilized on the coverslip. Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively. n = 290 regions with fluorescent molecules, 228 regions with background. (c) Photobleaching kinetics of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex. (d) Diffusion coefficients of Smc1/3 wild type or K38A ATP binding deficient 'KA' forms of Scc1Halo-TMR-TEV-cohesin in the presence or absence of ATP. Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period.",
    "molecules": "TMR, ATP, DNA, NaCl"
  },
  {
    "caption": "(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (a) TetO-DNA-bound TetRHalo-TMR.",
    "molecules": "TMR"
  },
  {
    "caption": "(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (b) DNA-bound Halo-TMREcoRIE111Q.",
    "molecules": "TMR"
  },
  {
    "caption": "(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (c) a DNA-bound TMR-labelled nucleosome.",
    "molecules": "TMR"
  },
  {
    "caption": "(d) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound QDotEcoRIE111Q. DNA was stained with Sytox Orange.",
    "molecules": "DNA"
  },
  {
    "caption": "(a) Kymograph of two Halo-TMRT7 RNAP transcription elongation events. (b, c) Kymographs showing (b) Halo-TMRT7 RNAP transcription stalling following removal of NTPs and (c) subsequent resumption after NTP flow in.",
    "molecules": "TMR, NTPs"
  },
  {
    "caption": "(d) Kymograph of Halo-TMRT7 RNAP (diameter ~ 8 nm excluding HaloTag) displacing Scc1GFP-TEV-cohesin following resumption of transcription in T7 reaction buffer + NTPs.",
    "molecules": "TMR, NTPs"
  },
  {
    "caption": "(e) Kymograph of Halo-TMRT7 RNAP constraining translocation of salt-resistant Scc1GFP-TEV-cohesin.",
    "molecules": "TMR"
  },
  {
    "caption": "(a) Representative field of view showing Halo-TMRCTCF bound to pPlat-4xCTCF following 75 mM NaCl, 75 mM KCl buffer wash. Arrows denote CTCF bound at predicted site of 4xCTCF array.",
    "molecules": "TMR, KCl, NaCl"
  },
  {
    "caption": "(b) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound Halo-TMRCTCF. DNA was post-stained with Sytox Green.",
    "molecules": "TMR, DNA"
  },
  {
    "caption": "B - D GST pull-down experiments were conducted in duplicates using the indicated GST fusion proteins. Eluates (30%) were subjected to immunoblotting together with an aliquot (1%) of the input. Blots were stained with Ponceau S and MAD2 was detected by immunoblotting. Densitometric signals were quantified using ImageJ software. Results were compared using unpaired two-tailed t-tests; ****p < 0.0001, ns - not significant; exact p-values are indicated; error bars represent SD of 8 independent biological replicates (n = 8).",
    "molecules": "Ponceau S"
  },
  {
    "caption": "C Co-immunoprecipitation experiments using lysates from HEK-293 cells stably expressing the indicated YFP-tagged transporters. Eluates (20%) as well as 0.1% total cell lysate were analyzed by immunoblotting. The immunoblot for mouse anti-β2-adaptin was subsequently used for incubation with rabbit anti-GFP primary and IRDye 800CW anti-rabbit secondary antibody. IRDye signals were detected using the LI-COR Odyssey CLx imaging system. Densitometric signals were quantified using ImageJ software.",
    "molecules": "IRDye, IRDye 800CW"
  },
  {
    "caption": "B Cell surface biotinylation using HEK-293 cells stably expressing YFP-hSERT was performed Whole cell lysates and streptavidin-conjugated fractions (i.e., surface protein) were subjected to immunoblotting.",
    "molecules": "streptavidin"
  },
  {
    "caption": "E Primary rat dorsal raphe neurons were cultured After 14 days, cells were infected with lentiviral particles encoding either scramble-shRNA or shRNA directed against MAD2. Five days after infection, cells were fixed in acetone/methanol (1:1) and subjected to immunofluorescence with the indicated primary and secondary antibodies. Confocal images were captured on a Nikon A1 laser scanning confocal microscope at either 20× (Fig. 5 E, left panel) or 60× (Fig. 5 E, right panel) magnification, representative images are shown. Due to imaging conditions the optical section thickness is increased at 20× compared to 60× magnification. Calibration bars in images showing rMAD2 represent arbitrary fluorescence units. Scale bars represent 50 and 10 µm for 20× and 60× magnification, respectively.",
    "molecules": "acetone, methanol"
  },
  {
    "caption": "The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (C) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.",
    "molecules": "H2O2, Leu"
  },
  {
    "caption": "The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (D) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.",
    "molecules": "H2O2, Leu"
  },
  {
    "caption": "The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (E) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.",
    "molecules": "antimycin A, Leu"
  },
  {
    "caption": "The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (F) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.",
    "molecules": "antimycin A, Leu"
  },
  {
    "caption": "(G) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes expressed in BY4742 wild-type and Δtsa1Δtsa2 cells, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls, showing the probe response upon the addition of water.",
    "molecules": "H2O2, Leu, water"
  },
  {
    "caption": "(H) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes, expressed in wild-type and Δtsa1Δtsa2 cells, to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls showing the probe responses upon addition of 0.1% (v/v) ethanol.",
    "molecules": "antimycin A, ethanol, Leu"
  },
  {
    "caption": "(I) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells, to boli at exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of water.",
    "molecules": "H2O2, Leu, water"
  },
  {
    "caption": "(J) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of 0.1% (v/v) ethanol.",
    "molecules": "antimycin A, ethanol, Leu"
  },
  {
    "caption": "(A) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δprx1, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Lighter colored curves are controls showing the probe response to the addition of water.",
    "molecules": "H2O2, Leu, water"
  },
  {
    "caption": "(B,C) The response of a cytosolic roGFP2-Tsa2ΔCR probe, expressed in wild-type, Δtrx3 and Δprx1 cells, to the addition of 0.1 mM (B) or 1 mM (C) exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.",
    "molecules": "H2O2, Leu"
  },
  {
    "caption": "(D) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid, to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase. The light grey curve in both panels represents the wild-type control, treated with the same concentration of exogenous H2O2.",
    "molecules": "H2O2"
  },
  {
    "caption": "(E) The profile of mRNA expression of the yeast redox or redox-related enzymes in Δprx1+empty vector cells, compared to Δprx1+Prx1-WT cells, both grown to an early exponential phase in SGal (-Ura) medium (n = 3 biological replicates, with cells obtained from 3 independent cultures).",
    "molecules": "Ura"
  },
  {
    "caption": "(F) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1-WT plasmid to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential phase",
    "molecules": "H2O2"
  },
  {
    "caption": "(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtsa1Δtsa2, Δpor1, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.",
    "molecules": "H2O2, Leu"
  },
  {
    "caption": "(B) Left panel: The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of 2.5 mM H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: Scheme depicting Prx1 and its peroxidatic cysteine at position 91.",
    "molecules": "cysteine, H2O2, Leu"
  },
  {
    "caption": "(C) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe to exogenous 2.5 mM H2O2. The probe was expressed in wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a vector encoding wild-type Prx1 or a vector encoding Prx1-C91A, grown in SGal (-Leu, -Ura). (D) The response to of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in either wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a Prx1-WT or a Prx1-C91A plasmid to exogenous H2O2 at a concentration of 2.5 mM. Cells were grown to SGal (-Leu, -Ura) medium and harvested at early exponential phase.",
    "molecules": "H2O2, Leu, Ura"
  },
  {
    "caption": "(B) 'Acute stress-washout' assay. Δprx1 cells containing a Prx1-WT plasmid and expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix-localized Grx1-roGFP2 probe to the addition of 1 mM H2O2 was measured. The middle panel is an enlargement of the left panel. The graph in the right panel shows the steady state Grx1-roGFP2 oxidation following H2O2 pre-treatment as well as the maximum Grx1-roGFP2 oxidation in response to the subsequent second H2O2 treatment.",
    "molecules": "H2O2, Leu, Ura"
  },
  {
    "caption": "'Acute stress-washout' assay. Δprx1 cells expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. (C) The same experiment as in (B) performed with Δprx1 cells transformed with a Prx1-C91A plasmid.",
    "molecules": "H2O2, Leu, Ura"
  },
  {
    "caption": "(D) ∆prx1 cells transformed with a vector encoding wild-type Prx1, a vector encoding the matrix-targeted D-aminooxidase (su9-DAO) and expressing a mitochondrial matrix-localized Grx1-roGFP2 were grown to exponential phase in SGal (-Leu, -Ura, -His) and then pre-treated with 0.15 M D-alanine for 0, 0.5, 1.5 hours. Afterwards, the cells were washed and the response of Grx1-roGFP2 to the addition of 1 mM H2O2 was measured.",
    "molecules": "alanine, His, H2O2, Leu, Ura"
  },
  {
    "caption": "(E) Left panel: the response of mitochondrial matrix-localized roGFP2-Prx1 and roGFP2 probes expressed in wild-type cells to exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2-Prx1 sensor leads to a roGFP2-like behavior.",
    "molecules": "H2O2, Leu"
  },
  {
    "caption": "(H) Redox shift assays of Prx1. Δprx1 cells transformed with a plasmid encoding wild-type Prx1 were grown to early exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins. Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP.",
    "molecules": "cysteine, H2O2, TCEP, Ura"
  },
  {
    "caption": "(J) The response of a mitochondrial matrix-localized Grx1-roGFP2, expressed in wild-type cells grown to the addition of exogenous 1 mM H2O2. Cells were grown in SGal (-Leu) and harvested at early exponential phase. Left panel, the pre-treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH. Right panel, newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix.",
    "molecules": "H2O2, Leu"
  },
  {
    "caption": "(B) The response of a mitochondrial matrix-localized roGFP2-Prx1-P233stop probe expressed in wild-type cells to the indicated concentrations of exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).",
    "molecules": "H2O2, Leu"
  },
  {
    "caption": "(C) Maleimide-based gel-shift assay to assess Prx1 cysteine redox state. Wild-type cells transformed with an empty plasmid or ∆prx1 cells with either an empty plasmid or a plasmid encoding wild-type Prx1, the hyperoxidation-resistant Prx1-P233stop or the inactive Prx1-C91A variants, were grown to exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24.",
    "molecules": "cysteine, H2O2, Maleimide, mm, PEG, Ura"
  },
  {
    "caption": "(D) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe, expressed in Δprx1 cells containing a plasmid encoding either wild-type Prx1, Prx1-P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2. Cells had been pre-treated with either 10 mM H2O2 or with water as a control. For these experiments, cells were grown in SGal (-Leu, -Ura) medium and harvested at early exponential phase. OxD refers to the degree of sensor oxidation. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).",
    "molecules": "H2O2, Leu, Ura, water"
  },
  {
    "caption": "(E) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with empty vector or with a vector encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SD (-Ura) and treated with the indicated concentrations of H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 5-8 biological replicates, with cells taken from independent cultures for each individual biological replicate).",
    "molecules": "H2O2, Hydrogen peroxide, Ura"
  },
  {
    "caption": "(A) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with an empty plasmid or a plasmid encoding Prx1, Tsa1, PRDX3, PRDX5, PRDX6, PfAOP or PfAOP-L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre-treated with 0, 1, 5, 10, 25 mM H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume was plated on YPD plates. The number of viable colonies was counted after 2 days of growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3-8 biological replicates, with cells taken from independent cultures for each individual biological replicate). Significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001.",
    "molecules": "H2O2, Hydrogen peroxide"
  },
  {
    "caption": "(B) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress. Data from viability and matrix Grx1-roGFP2 responses during acute H2O2 stress were correlated.",
    "molecules": "H2O2"
  },
  {
    "caption": "(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe to 1 mM H2O2. in BY4742 wild-type cells with an empty vector, in Δglr1 cells transformed either with an empty vector or a vector encoding wild-type Glr1 or the cytosolic form of Glr1, where the MTS-encoding region was removed, or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1. Cells were grown to exponential phase in SGal (-Leu, -Ura) medium. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).",
    "molecules": "H2O2, Leu, Ura"
  },
  {
    "caption": "(C) H2O2 'acute stress' assay. Wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells pre-grown in SGal medium complemented with all amino acids to early exponential phase. Cells were treated with H2O2 at the indicated concentrations for 30 mins. Subsequently cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate). An inset with a different scale on the y-axis is presented for the 1 and 5 mM concentration to allow better interpretation.",
    "molecules": "H2O2"
  },
  {
    "caption": "(D) H2O2 'acute stress' assay. Δprx1Δglr1 cells co-transformed with an empty plasmid or with a plasmid encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SGal (-Ura) medium to early exponential phase. Cells were treated with the indicated amounts of H2O2 for 30 mins. Subsequently, the cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate).",
    "molecules": "H2O2, Ura"
  },
  {
    "caption": "(A) Representative kymographs showing tetramethylrhodamine (TMR)-labeled DDB movement on each type of recombinant MT lattice, as indicated. Scale bars, 5 μm and 25 sec. Pink arrowhead marks a diffusive DDB complex. See also Movie EV1. (B) Quantification of DDB velocity on MT lattices lacking each of the tubulin-CTTs (n ≥ 100 DDB complexes per condition from at least three independent experiments). No statistical difference was observed between WT and mutant MTs (P ≥ 0.1, unpaired t test) (C-D) Quantification of the number of processive (C), or diffusive (D) DDB complexes relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Processive (unidirectional motion exceeding 0.5 μm) and diffusive (back-and-forth motion) events were scored in a field of view and then divided by the lengths of MTs and time, thus normalizing to a value of number of motility events per μm MT per sec. Error bars SD.",
    "molecules": "tetramethylrhodamine, TMR"
  },
  {
    "caption": "(E-F) TIRF images of 1 nM TMR-labeled GST-hDyn (2 mM ATP) (E), or 0.3 nM TMR-labeled p150 (F) molecules (green) bound to either Δα-CTT (top), or Δβ-CTT MTs (bottom). Pink arrows show mutant MTs in both the raw, and SD map images, highlighting differences in binding between WT and mutant MTs that are apparent in the SD map. Right, standard deviation maps from an entire time sequence reveal ensemble binding and dissociation events that lead to variations in pixel intensities (see Methods). Scale bars are 5 μm. (G) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. Image shows first frame from time series. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD. P value (unpaired t test) of Δα-CTT vs. Δβ-CTT MTs = 0.5208 for GST-hDyn, and ≤ 0.0001 for p150.",
    "molecules": "TMR, ATP"
  },
  {
    "caption": "(A) TIRF image of TMR-labeled DDB molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. A standard deviation map of the whole time sequence is shown, revealing the preferential association of DDB with tyrosinated MTs versus detyrosinated MTs (pink arrow). Representative kymographs of DDB movement are shown from each type of MT present in the same chamber. Scale bars are 5 μm and 25 sec. See also Movie EV2. (B-C) Quantification of the number of processive (B) or diffusive (C) DDB complexes per μm MT per sec on detyrosinated MTs relative to tyrosinated MTs in the same chamber (n ≥ 20 MTs quantified for each condition from three independent experiments). Error bars represent SD. Data from Δα-CTT MTs (Fig. 1) are re-plotted for comparison. (D) Quantification of the frequency of processive DDB molecules as a function of the amount of tyrosinated tubulin incorporated into the lattice normalized to fully tyrosinated MTs. Data points from two independent experiments are shown.",
    "molecules": "TMR"
  },
  {
    "caption": "(E) TIRF images of 0.3 nM TMR-labeled p150 (top) or 1 nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either tyrosinated (red) or detyrosinated (blue) MTs. Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either tyrosinated or detyrosinated MTs are shown. Scale bars are 5 μm and 25 sec. (F) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2 mM ATP) or p150 bound to the indicated mutant MT relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT. (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.",
    "molecules": "TMR, ATP"
  },
  {
    "caption": "(B) TIRF image of TMR-labeled DDB molecules (green) on either WT (red) or CPA-treated (blue) pig brain MTs. Standard deviation map of the entire time sequence shows DDB preference for WT MTs and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec. (C) Quantification of the number of processive DDB complexes per μm MT per sec on the CPA-treated MTs, relative to WT MTs in the same chamber (n ≥ 7 MTs of each type quantified from at least three independent experiments). Error bars represent SD.",
    "molecules": "TMR"
  },
  {
    "caption": "(D) TIRF images of 0.3nM TMR-labeled p150 (top) or 1nM TMR-labeled GST-hDyn (bottom) molecules (green) bound to either WT (red), or CPA-treated MTs (blue). Right, standard deviation maps of protein binding over an entire time sequence and kymographs from either WT or CPA-treated are shown. Scale bars are 5 μm and 25 sec for p150, and 5 μm and 12 sec for GST-hDyn (E) Quantification of mean fluorescence intensity (arbitrary units) per μm MT for TMR-labeled GST-hDyn (2mM ATP) or p150 bound to CPA-treated MTs relative to WT MTs in the same chamber. The intensity of GST-hDyn was quantified from maximum intensity projections of the entire time sequence due to transient binding of GST-hDyn to the MT (n ≥ 10 MTs quantified for each condition from at least three independent experiments). Error bars represent SD.",
    "molecules": "TMR, ATP"
  },
  {
    "caption": "(B) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated (red) and detyrosinated (blue) sections. Most TMR-labeled DDB molecules (green) move processively through the annealed junction between the two types of MT lattice (65%, n=836 processive complexes, 65 MT-MT junctions, three independent experiments). Diffusive DDB molecules (white arrow) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary. Right, video frames showing a processive DDB complex (pink arrowhead) moving across the junction (yellow arrowhead) of tyrosinated and detryosinated MT. The (+) and (-) signs denote MT polarity inferred from the directionality of DDB movement. Scale bars are 5 μm and 25 sec. See also Movie EV3.",
    "molecules": "TMR"
  },
  {
    "caption": "(C) TIRF images of low (0.3 nM) and high (10 nM) concentrations of TMR-labeled p150 (green) bound to chimeric yeast MTs containing tyrosinated (red) and detyrosinated (blue) sections of MT. Representative kymographs below show diffusive behavior of p150 molecules on the MT. Note that p150 rarely diffuses into the detyrosinated section of MT, even at high concentrations. Scale bars are 5 μm and 10 sec.",
    "molecules": "TMR"
  },
  {
    "caption": "(D-E) TIRFimages and kymographs of annealed WT and CPA-treated porcineMTs(D), or annealed WT MTs(E). The majority of DDB complexes (78%, n=960 processive complexes, 45 MT-MT junctions, three independent experiments) move processively across the boundary between WT and CPA-MTs(D). Note TMR-labeled DDB complexes (green) in (E) traverse the annealed boundary without pausing (93%, n=677 processive complexes, 45 MT-MT junctions, three independent experiments), suggesting that the annealed junction does not affect DDB motility.",
    "molecules": "TMR"
  },
  {
    "caption": " D. CBP-Nmd4 was mixed with hUPF1-HD or yUPF1-HD (all the proteins overexpressed in E. coli and purified). Protein mixtures before (input, 20% of total) or after purification on calmodulin affinity beads were separated on 10% SDS-PAGE (w/v) acrylamide gels ",
    "molecules": "acrylamide, calmodulin"
  },
  {
    "caption": " E. Upf1 interaction with a 30-mer 3' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution ",
    "molecules": "RNA, NaCl, streptavidin"
  },
  {
    "caption": "A. Infection by a L. pneumophila strain overexpressing MvcA but not the inactive mutant MvcAC83A interferes with UBE2N modification. Raw264.7 cells were infected with the indicated bacterial strains at an MOI of 5 for 2 h. Saponin soluble proteins resolved by SDS-PAGE were probed with a UBE2N-specific antibody (top panel). The delivery of MavC and MvcA into infected cells was probed with antibodies specific to each of these two proteins. Note that MavC but not MvcA is detectable in cells infected with wild type bacteria (middle panels labeled as "translocation"). Tubulin was probed as a loading control. Protein levels of MavC and MvcA associated with the bacteria were similarly probed in the saponin insoluble fraction (lower panels labeled as "expression"). The bacterial metabolism enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. Note that endogenous MvcA was not detectable in bacteria grown in bacteriological medium (the middle panel of the lower portion).",
    "molecules": "Saponin, saponin"
  },
  {
    "caption": "C. MvcA interferes with MavC-induced UBE2N ubiquitination. Reactions containing the indicated combinations of proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by Coomassie Brilliant blue (CBB) staining. Note that MvcA (inclusion of MvcA with MavC, 7th lane. Compare to MavC alone in 3rd lane) but not the MvcAC83A mutant (8th lane) abolished MavC-induced UBE2N modification.",
    "molecules": "Coomassie Brilliant blue"
  },
  {
    "caption": "A. The hydrolytic cleavage of UBE2N-Ub by MvcA into UBE2N and ubiquitin (Q40E variant). Reactions containing the indicated proteins were allowed to proceed for 30 min and samples resolved by SDS-PAGE were detected by CBB staining (left). Mass spectrometric analysis of the protein band corresponding to ubiquitin produced by MvcA from UBE2N-Ub (right). The ubiquitin product is the Glu40 variant of ubiquitin.",
    "molecules": "Glu, Ub, ubiquitin"
  },
  {
    "caption": "C. Dose-dependent cleavage of UBE2N-Ub by MvcA. A series of reactions containing UBE2N-Ub and MvcA at the indicated molar ratios were established and the reactions were allowed to proceed for 30 min before SDS-PAGE and CBB staining. Note that the activity was detectable in a reaction starting at the molar ratio of between UBE2N-Ub and MvcA is 2048:1.",
    "molecules": "Ub"
  },
  {
    "caption": "D. Time-dependent cleavage of UBE2N-Ub by MvcA. UBE2N-Ub was incubated with MvcA at the molar ratio of 128. Samples taken at the indicated time points were resolved by SDS-PAGE and detected by CBB staining.",
    "molecules": "Ub"
  },
  {
    "caption": "E. UBE2N produced from UBE2N-Ub via MvcA catalyzed deubiquitination is active in catalyzing the formation of polyubiquitin chains. UBE2N-Ub was preincubated with MvcA or its inactive mutant for 30 min. Then a cocktail containing E1, UBE2V2, 3xHA-ubiquitin, the E3 enzyme TRAF6 and ATP was added into reactions. Ubiquitination was allowed to proceed for 30 min. Proteins in samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes and blotted with the HA specific antibody. Note the robust formation of polyubiquitin chains in the reaction receiving MvcA (middle lane).",
    "molecules": "polyubiquitin chains, ATP, Ub, ubiquitin"
  },
  {
    "caption": "H. Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis. For deubiquitination reactions, the indicated amounts of MvcA or its mutants were incubated with 2.0 μg UBE2N for 5 min in 25 μl reactions. For ubiquitin deamidase activity, the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "I-J. The roles of ubiquitin contact residues in the activity of MvcA. I. The indicated amounts of MvcA or its mutants were incubated with 2 μg UBE2N-Ub for 5 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM). J. The activity of the MvcA mutant harboring three mutations. 0.05 μg, 0.25 μg and 1.25 μg of MvcA or the mutant was incubated with 2 μg UBE2N-Ub in 25 μl reactions. After 30 min incubation, the products resolved by SDS-PAGE were detected by CBB staining. Note that the mutant did not detectable cleave UBE2N-Ub even in the reaction received the highest amount of protein.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "D. MvcA is required for reversal of UBE2N modification during L. pneumophila infection. U937 cells infected with wild type or the ∆mvcA mutant were withdrawn at the indicated time points. Samples resolved by SDS-PAGE were probed for UBE2N, translocated MavC and MvcA. Tubulin was probed as a loading control. Note that in samples infected with the ∆mvcA strain, the level of UBE2N-Ub decreased at slower rates.",
    "molecules": "Ub"
  },
  {
    "caption": "F. MvcA restores the nuclear translocation of p65 inhibited by MavC during L. pneumophila infection. Raw264.7 cells were infected with the indicated L. pneumophila strains for 2 h at an MOI of 1. Fixed cells were immunostained with antibodies specific for p65 and the bacterium, respectively. Host nuclei were labeled with Hoechst. Representative images of cells in each sample category were shown (upper panels); nuclear localization of p65 was determined by scoring at least 300 infected cells (lower panel). Results shown were from three independent experiments done in triplicate and the values shown were mean +/- SE; t-test.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.65 μM each) with recombinant SIRT1 in the presence or absence of NAD+.",
    "molecules": "NAD"
  },
  {
    "caption": "(f) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells. Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM (10 mM) for 5h and subsequent enrichment for acetylated proteins via acetyl-lysine immunoprecipitation. As a control cells were either treated with control siRNA or siSUMO2/3. The samples were analyzed by immunoblotting with α-SUMO2/3 and α-SUMO2K11Ac antibodies. The α-SUMO2/3Ac and α-SUMO2K11Ac reactive species migrating at ~200 kDa are marked with an asterisk.",
    "molecules": "acetyl-lysine, NAM"
  },
  {
    "caption": "(b) Western blot analysis with α-RGS-His and α-HA was performed to monitor poly-SUMOylation of HA-PML. The remaining samples were measured by LC-MS/MS and final data analysis was done with MaxQuant. A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49.3. Intensities for this SUMO2 peptide for the control (gray) and arsenic (purple) treated samples are blotted as a bar graph. The diagram shows a representative dataset derived from two replicates. The corresponding peptide is displayed below the graph. The SUMOylation site is marked with SUMO2 (green) and peptide fragment ions are indicated by y- (red) and b- (blue).",
    "molecules": "arsenic"
  },
  {
    "caption": "(c) The degradation of the PML reactive band (~ 120 kDa) was determined by immunoblotting. HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1/2/3. The expression of RGS-His-tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h. Prior to lysis, cells were treated with 1 μM As2O3 for 6 h. Western blot analysis for wild-type SUMO2 and SUMO2K11Q were blotted with α-RGS-His. Immunoblotting with α-Tubulin served as a loading control. Immunoblots for knockdown controls (SUMO1 and SUMO2/3) can be found",
    "molecules": "As2O3, DOX"
  },
  {
    "caption": "Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type (WT) or Nlrc3-/- mice and treated with LPS (100 ng/ml) for 48 hours.",
    "molecules": "LPS"
  },
  {
    "caption": "The intracellular production of IFN-γ and IL-17 by CD4+ T cells among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated",
    "molecules": "MOG"
  },
  {
    "caption": "cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated",
    "molecules": "MOG"
  },
  {
    "caption": "thymidine incorporation proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated",
    "molecules": "MOG"
  },
  {
    "caption": "CFSE proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated",
    "molecules": "MOG"
  },
  {
    "caption": "WT and Nlrc3-/- mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized WT (n= 10) and Nlrc3-/- mice (n= 10).",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells from the spinal cord and brain of WT or Nlrc3-/- mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.",
    "molecules": "MOG"
  },
  {
    "caption": "DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide.",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Recall response to MOG(35-55) by splenocytes isolated from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6, Il23 and Il27 mRNA in DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. CFSE proliferation assay of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.",
    "molecules": "CFA, CFSE, MOG, pertussis toxin"
  },
  {
    "caption": "DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. cytokine secretion of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated with LPS (100 ng/ml) for specified time. DC lysates were probed for phosphorylated p65 (p-p65), total p65, p-AKT, AKT, p-p38, p38, p-ERK, ERK, p-JNK, JNK and GAPDH.",
    "molecules": "LPS"
  },
  {
    "caption": "DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Concentrations of IL-12, IL-6, IL-23 and IL-27 in supernatants were detected by ELISA.",
    "molecules": "LPS, SB203580"
  },
  {
    "caption": "DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.",
    "molecules": "LPS, MOG, SB203580"
  },
  {
    "caption": "DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.",
    "molecules": "CFSE, LPS, MOG, SB203580"
  },
  {
    "caption": "DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Frequencies of CD4+ T cells that express IFN-γ and IL-17A in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6 and Il23 mRNA in DCs sorted from spleens of DC(WT), DC(NLRC3-KO), DC(p38-KO) and DC(p38+NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.",
    "molecules": "CFA, MOG, pertussis toxin"
  },
  {
    "caption": "DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. DC lysates were probed for p-p38, p38, NLRC3 and GAPDH. Densitometry quantification of band intensity were presented in the right panel.",
    "molecules": "LPS"
  },
  {
    "caption": "DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Enzyme-linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours.",
    "molecules": "LPS"
  },
  {
    "caption": "DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.",
    "molecules": "LPS, MOG"
  },
  {
    "caption": "DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.",
    "molecules": "CFSE, LPS, MOG"
  },
  {
    "caption": "EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Mean clinical scores of EAE (n= 5 mice per group ). Arrows indicate DC vaccine administration.",
    "molecules": "MOG"
  },
  {
    "caption": "EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Recall proliferative to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.",
    "molecules": "MOG"
  },
  {
    "caption": "EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. cytokine response (D) to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.",
    "molecules": "MOG"
  },
  {
    "caption": "(A) Wild‐type (YW5‐1B), apg10‐1 mutant (MT91‐4‐2) and Δapg10 (TFD10‐L1) cells were incubated in SD(−N) medium containing 1 mM PMSF at 30°C. After incubation for 6 h, cells were observed under light microscopy (Nomarski images).",
    "molecules": "PMSF"
  },
  {
    "caption": "(A) Two‐hybrid assay for interactions of Apg10p with Apg5p, Apg7p and Apg12p. Yeast cells (PJ69‐4A) harboring the indicated plasmids were streaked out on plates with medium lacking adenine to assay for interaction‐dependent activation of the ADE2 gene.",
    "molecules": "adenine"
  },
  {
    "caption": "Effects of the deletion of APG5 or APG7 on Apg12p-Apg10p thioester formation. Coimmunoprecipitation was performed with wild‐type (KA311B), Δapg5 (YNM122) or Δapg7 (YTS12) cells harboring HAAPG10 and MycAPG12 on 2μ plasmids. The resulting precipitates were analyzed by Western blotting. The DTT‐resistant Apg12p-Apg5p conjugate is indicated by ●.",
    "molecules": "DTT"
  },
  {
    "caption": "(A) Autophagic activities were measured by an ALP assay ( Noda and Ohsumi, 1998). YTS3 cells (Δapg10PHO8::pho8Δ60) harboring each mutant APG10 on CEN plasmids were grown to 1 OD600/ml in SC medium lacking tryptophan and then transferred to SD(−N) medium. Lysates from the cells after incubation for 0 and 4 h were used for assay. Error bars indicate the SD of three independent experiments.",
    "molecules": "tryptophan"
  },
  {
    "caption": "E. Immunoblot using anti-His antisera to detect dimer and oligomer formation by purified polyhistidine (6xHis)-tagged WT or dominant-negative LukED toxins in the absence or presence of 2-methyl-2,4-pentanediol (MPD, 40% v/v). Emut1 = LukEmut1, Dmut = LukDmut. A representative of two independent experiments is shown.",
    "molecules": "MPD"
  },
  {
    "caption": "A. Survival of Swiss-Webster mice following intravenous administration of purified recombinant toxins: LukED (10 μg per subunit), HlgAB (5 μg per subunit), HlgCB (35 μg per subunit) and LukSF-PV/PVL (35 μg per subunit). Buffer: n = 2 mice; LukED: n = 3 mice; HlgAB: n = 3 mice; HlgCB: n = 2 mice; PVL: n = 3. A representative of two independent experiments is shown.",
    "molecules": "mice"
  },
  {
    "caption": "A. Lysates from 293T cells transfected with the indicated DNA constructs were incubated with 1 mM Mg++ plus 0.1 mM GTPγS, GDP or 2 mM EDTA and immunoprecipitated with anti-Myc antibody. Rab18-HA co-immunoprecipitated with Myc-TRAPPC9 when it was nucleotide free (+EDTA).",
    "molecules": "EDTA, Mg++, GDP, GTPγS"
  },
  {
    "caption": "D. GST-Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay. GST-Rab18 was pre-loaded with GTPγS or GDP, or stripped off nucleotide by EDTA. Lysates from 293T cells were subjected to GST-Rab18 pull-down. The presence of various TRAPP subunits was detected by immunoblotting. TRAPPII, but not TRAPPIII complex was co-isolated with GST-Rab18, most strongly with Rab18 in nucleotide-free state.",
    "molecules": "EDTA, GDP, GTPγS"
  },
  {
    "caption": "B-D. TRAPP stimulated guanine nucleotide exchange on purified Rab18, Rab1 and Rab2 GST fusion proteins measured by release of 3H-GDP that was pre-loaded onto the Rab proteins.",
    "molecules": "GDP"
  },
  {
    "caption": "E-G. Nucleotide exchange reactions measured by time-dependent binding of [35S]GTPγS. Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested. In the 3H-GDP release experiments, the reactions were incubated for 30 minutes at 37°C before the Rab proteins were subjected to nitrocellulose filter binding. In the [35S]GTPγS binding experiments, the reactions were incubated for the indicated amount of time before filter binding.",
    "molecules": "GDP, GTPγS"
  },
  {
    "caption": "H-I. Anti-Myc antibody immunoprecipitates from lysates of HEK293T cells transfected Myc-TRAPPC9 were used as the GEF for GST-Rab1 or GST-Rab18 in 30 min incubations at 37°C with [35S]GTPγS.",
    "molecules": "GTPγS"
  },
  {
    "caption": "A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. Representative fluorescence images show the status of lipid droplets in these siRNA treated cells after oleic acid incubation. Bar =10 μm.",
    "molecules": "oleic acid"
  },
  {
    "caption": "B. After loading with oleic acid, human skin fibroblasts containing a R475* mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a TRAPPC9 wildtype human individual. Bar = 10 μm.",
    "molecules": "oleic acid"
  },
  {
    "caption": "A. HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with bodipy 493/503. Bar = 10 μm.",
    "molecules": "oleic acid"
  },
  {
    "caption": "B. Wildtype or TRAPPII deleted cells were loaded with oleic acid and stained with bodipy 493/503 (green) and endogenous Rab18 by immunofluorescence (red). Bar = 10 μm.",
    "molecules": "oleic acid"
  },
  {
    "caption": "C. DsRed-Rab18 was transfected into wildtype (top panels and insets) or TRAPPII deleted HEK293T cells (bottom panels and insets) that were loaded with oleic acid and stained with bodipy 493/503 (green). Bar = 10 μm.",
    "molecules": "oleic acid"
  },
  {
    "caption": "B. Huh-7 cells were treated with or without BFA for 6 hours before staining for Rab18 (red) and LD (green). Bar = 10 μm.",
    "molecules": "BFA"
  },
  {
    "caption": "C. TRAPPC9 were detected on Golgi but not LD surface. Huh-7 cells were permeabilized with digitonin before fixation and staining. LDs were labeled with bodipy 493/503 and pseudocolored in white. TRAPPC9 (red) and LD surface marker ADRP (green) were detected by their respective antibodies. Bar = 10 μm.",
    "molecules": "digitonin"
  },
  {
    "caption": "Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.A. In the merged images, bodipy 493/503 was pseudocolored in green, TRAPPC9 in red, and ADRP in blue. In the colocalization between TRAPPC9 and ADRP on the right side, the ADRP signal was re-colored to green for easy visualization. Bar = 10μm.",
    "molecules": "oleic acid"
  },
  {
    "caption": "Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.B. Bodipy 493/503 was pseudocolored in green, Rab18 in red, and γ-COP in blue. In the colocalization between Rab18 and γ-COP on the right side, the γ-COP signal was re-colored to green for easy visualization. Bar = 10μm.",
    "molecules": "oleic acid"
  },
  {
    "caption": "Huh-7 cells were first starved with serum free medium and then incubated with oleic acids for 12 hours. Condition 1 (control), the cells were not treated with BFA at any time point (left column). Condition 2, the cells were incubated with both 5 μg/ml of BFA and oleic acid incubation for 6 h and then in oleic acid alone for 10 h (middle column). Condition 3, the cells were treated with 5 μg/ml of BFA starting at 12 hours after oleic acid incubation (right column). Bar = 10μm.",
    "molecules": "BFA, oleic acid, oleic acids"
  },
  {
    "caption": "C, Diarylquinoline and SQ31f inhibition of ATP hydrolysis by purified ATP synthase bearing truncated α subunits. Mean relative activity (filled symbols) ± s.d from n=3 separate assays (empty symbols) for each of four different inhibitors (triangles, circles, diamonds, and squares).",
    "molecules": "SQ31f"
  },
  {
    "caption": "D, Inhibition of ATP hydrolysis activity in M. smegmatis IMVs containing ATP synthase with truncated α subunits. Initial ATP hydrolysis activity of the ATP synthase is defined as the activity that is sensitive to 100 μM DCCD. Mean relative activity from n=3 separate assays is shown.",
    "molecules": "DCCD"
  },
  {
    "caption": "E, Uncoupling activity of diarylquinoline analogs in M. smegmatis IMVs monitored with ACMA fluorescence. Each compound was assayed separately, and the initial fluorescence was set to 1.0. RFU: relative fluorescence units.",
    "molecules": "ACMA"
  },
  {
    "caption": "A, Uncoupling activity of SQ31f in M. smegmatis IMVs monitored with acridine orange fluorescence.",
    "molecules": "acridine orange, SQ31f"
  },
  {
    "caption": "B, SQ31f inhibition of ATP hydrolysis by purified ATP synthase with truncated α subunits (blue curve) occurs at higher concentrations than inhibition of ATP synthesis by M. smegmatis IMVs (purple curve). Mean relative activity (filled symbols) ± s.d from n=3 separate assays (triangle and circles).",
    "molecules": "SQ31f"
  },
  {
    "caption": "(D) SCFA faecal  quantification of 3m C57Bl (n=3) and mdx  (n=4-5) mice. Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).",
    "molecules": "SCFA"
  },
  {
    "caption": "(E) Colon  images captured with the iMScope TRIO described altered pattern of expression of different Phosphatidylcholines  (PC) and Lysophosphatidylcholines  (LysoPC) (as indicated by m/z values) in 3m mdx mice  (n=3). Scale bar: 50 μm. For each lipid, the mean intensities measured at 12 positions throughout colon  images are shown on the right side where bars are mean ± SEM (n = 3). Data information: Data are presented as mean ± SD (*p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; Student t-test).",
    "molecules": "LysoPC, Lysophosphatidylcholines, PC, Phosphatidylcholines"
  },
  {
    "caption": "(E) Faecal  content quantification of SCFAs  in 3m mdx  and 3m mdx+ABX mice  (n=5 per group). Data equal to 0. Data are presented as mean ± SD (**p< 0.01, ***p< 0.001; Student t-test).",
    "molecules": "SCFAs"
  },
  {
    "caption": "(H) Representative images of TA muscles  from 3m mdx  and GFmdx mice stained  for CD45  (in green), isolectin  (in red) and phalloidin  (in purple). Nuclei  were counterstained  with DAPI  (in blue). Scale bar: 10 μm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(I) Absolute number of CD3+  inflammatory cells  (white arrows) were quantified in n=12 images of TA  of 3m C57Bl, 3m mdx  and 3m GFmdx mice  (n=6 each). CD3 staining  is shown in green and DAPI  in blue. Scale bars: 50 μm. Data information: Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 The comparisons among the averages of CD3+ cells were evaluated using unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "A Co-localisation of YFP-ABCB15 with Propidium Iodide (PI) in root epidermal cells of 3-day-old seedlings. A plot of the fluorescence intensity of YFP and PI along the dashed lines shows the colocalization of the YFP and PI intensity peaks.",
    "molecules": "PI, Propidium Iodide"
  },
  {
    "caption": "C IAA export assay. Export of [3H]-IAA, assayed in parallel from tobacco mesophyll protoplasts expressing ABCB1, ABCB15-22 and ABCB17P980G against vector control. mean ± SE; n = 26 (vector control), 44 (ABCB1), 5 (ABCB15), 4 (ABCB16), 9 (ABCB17), 5 (ABCB18), 9 (ABCB22) and 8 (ABCB17P980G),transport experiments generated from independent tobacco transfections.",
    "molecules": "IAA, [3H]"
  },
  {
    "caption": "B Analysis of DR5::VENUS expression in the root elongation zone of 4-day-old pWOX5:XVE>>YUC1-2A-TAA1, in Control and amiR-2572 treated with β-estradiol (5 µM) for 0, 7.5 and 9h. Images are composed of several tiles generated in a single snap with automatic assembly, PI in gray. The zoomed images of the yellow squares are presented below each root showing the accumulation of the DR5::VENUS signal in the elongation zone.",
    "molecules": "β-estradiol, PI"
  },
  {
    "caption": "A, B Expression pattern of proABCB15:NLS-GFP-GUS, proABCB16:NLS-GFP-GUS, proABCB17:NLS-GFP-GUS, proABCB18:NLS-GFP-GUS, and proABCB22:NLS-GFP-GUS expression in roots of 3-day-old seedlings, using confocal microscopy showing longitudinal overview pictures (Propidium iodide in magenta), and images zoomed on the epidermis and LRC corresponding to yellow squares (A); cytological sections of GUS stained roots of 3-day-old seedlings, counterstained with ruthenium red (B). Scale bars represent 20 µm for both graphs.",
    "molecules": "Propidium iodide, ruthenium red"
  },
  {
    "caption": "A Fluorescence of YFP-ABCB17 in the root meristem and leaf in 3-day-old F1 crosses with WT, syn-tasi-1522A#1 and amiR-2572, propidium iodide (PI) in magenta. Red arrows highlight the position of the epidermis . Scale bars = 50 µm. B Schematic representation of root geometry with the indication of regions of interest for YFP-ABCB17 signal quantification. Red (Stele), and black (epidermis/LRC). C, D Quantification of YFP-ABCB17 fluorescence intensity in the stele (C) and epidermis/LRC (D) of A, measured at regions of interest corresponding t",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "B. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).",
    "molecules": "Ca2+, Na+"
  },
  {
    "caption": "C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+.",
    "molecules": "Na+"
  },
  {
    "caption": "D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.",
    "molecules": "Ca2+"
  },
  {
    "caption": "E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.",
    "molecules": "Ca2+, MONNA, NFA, niflumic acid"
  },
  {
    "caption": "A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test).",
    "molecules": "Ca2+, Na+"
  },
  {
    "caption": "C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA.",
    "molecules": "Ca2+, MONNA, Na+"
  },
  {
    "caption": "D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).",
    "molecules": "Ca2+"
  },
  {
    "caption": "E. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA.",
    "molecules": "Ca2+, MONNA"
  },
  {
    "caption": "F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used.",
    "molecules": "Ca2+, MONNA, NMDG"
  },
  {
    "caption": "G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).",
    "molecules": "45Ca, water"
  },
  {
    "caption": "Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.",
    "molecules": "Na+"
  },
  {
    "caption": "Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (C) Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.",
    "molecules": "Ca2+, Na+"
  },
  {
    "caption": "Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 70 mM Ca2+ (D). Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.",
    "molecules": "Ca2+, Na+"
  },
  {
    "caption": "Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ (G), , showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.",
    "molecules": "Na+"
  },
  {
    "caption": "Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2+ (H), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.",
    "molecules": "Na+"
  },
  {
    "caption": "Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.",
    "molecules": "NMDG, NMDG+"
  },
  {
    "caption": "Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.",
    "molecules": "Na+"
  },
  {
    "caption": "Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.",
    "molecules": "NMDG, NMDG+"
  },
  {
    "caption": "Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).",
    "molecules": "Ca2+, Na+"
  },
  {
    "caption": "Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).",
    "molecules": "Ca2+, Na+"
  },
  {
    "caption": "RNAscope-based whole-mount in situ hybridization (C) and quantification of Efnb2 transcript levels (D) in P7 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).",
    "molecules": "RNA"
  },
  {
    "caption": "First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017.",
    "molecules": "nucleotides"
  },
  {
    "caption": "Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.).",
    "molecules": "polyacrylamide"
  },
  {
    "caption": "AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.",
    "molecules": "nucleotides"
  },
  {
    "caption": "AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001.",
    "molecules": "nucleotides"
  },
  {
    "caption": "Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test).",
    "molecules": "ponceau S, Puromycin"
  },
  {
    "caption": "[35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).",
    "molecules": "[35S]-methionine, Coomassie"
  },
  {
    "caption": "D After whole-brain electrophoretic staining with polythiophenes, sagittal 500 µm slices were cut and plaques were counted. By passively re-staining the same slice, no additional plaques were detected (\"FEC + passive\"). Focal shifts after slice reprocessing account for the slight differences between the images. One biological replicate. Scale bars: 100 µm",
    "molecules": "polythiophenes"
  },
  {
    "caption": "A, B The distinct significant effect of each of the strongest treatments on plaque size cluster, NB360 5-month and LIN5044 14-month, as well as their overlap, using either (A) neuroanatomical boundaries, or (B) SAVs.",
    "molecules": "LIN5044, NB360"
  },
  {
    "caption": "(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference.",
    "molecules": "ammonia, NH4Cl"
  },
  {
    "caption": "(B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA).",
    "molecules": "TB-1, orotic acid"
  },
  {
    "caption": "(C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test).",
    "molecules": "TB-1"
  },
  {
    "caption": "(E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.",
    "molecules": "TB-1, L-Arg, L-Arginine, L-arginine, Na-benzoate"
  },
  {
    "caption": "(F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.",
    "molecules": "TB-1, ammonia, L-Arg, L-arginine, Na-benzoate"
  },
  {
    "caption": "(A) Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 hours starting at day 10 of age) or vehicle. WT + Vehicle n=8; WT + TB-1 n=8; AslNeo/Neo + Vehicle n=20; AslNeo/Neo + TB-1 n=16. *p<0.05 (Log-rank Mantel-Cox test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.",
    "molecules": "TB-1"
  },
  {
    "caption": "(B) Isotopic enrichment of 15N-labeled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n=15) or vehicle (n=9). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.",
    "molecules": "TB-1, 15NH4Cl, 15N, urea"
  },
  {
    "caption": "(C) Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls. (D) Densitometric quantifications. AslNeo/Neo + Vehicle n=5; AslNeo/Neo + TB-1 n=9. **p<0.01, *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.",
    "molecules": "TB-1"
  },
  {
    "caption": "(E) Argininosuccinate in dried blood spots (DBS) of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n=4-14 mice/group). ***p<0.001, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.",
    "molecules": "TB-1, Argininosuccinate"
  },
  {
    "caption": "(F) Isotopic enrichment of 15N-labeled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n=11) or vehicle (n=5). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.",
    "molecules": "TB-1, argininosuccinate, 15N"
  },
  {
    "caption": "(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.",
    "molecules": "TB-1, eosin, Hematoxylin, periodic acid, Schiff"
  },
  {
    "caption": "(C) Quantification of hepatic glycogen in vehicle (n=6)- and TB-1-treated AslNeo/Neo mice (n=12) compared to WT (n=5) controls. **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.",
    "molecules": "TB-1, glycogen"
  },
  {
    "caption": "(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control. Data information: All values are shown as averages ± S.E.M.",
    "molecules": "TB-1"
  },
  {
    "caption": "(E) Representative electron microscopy images of liver samples harvested from WT and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bar: 900 nm.",
    "molecules": "TB-1"
  },
  {
    "caption": "(A) Representative electron microscopy images of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. False color on the images indicates glycogen within the nuclei. Scale bar: 1 μm. (B) Quantification of nuclei containing glycogen (approx. 180 nuclei were analyzed in total per condition, n=3 mice/group). ",
    "molecules": "TB-1, glycogen"
  },
  {
    "caption": "Representative still images of control, Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus-Rod treated with nocodazole and time in minutes indicated. Scale bar, 5 μm.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Time from NEBD to mitotic exit in control depleted, Rod depleted, Bub1 depleted and Rod and Bub1 co-depleted HeLa, U2OS and RPE1 cells. Cells were treated with nocodazole.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells. Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.",
    "molecules": "nocodazole, RO3306"
  },
  {
    "caption": "Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Time from NEBD to exit of indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.",
    "molecules": "taxol"
  },
  {
    "caption": "Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Time from NEBD to exit in indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.",
    "molecules": "taxol"
  },
  {
    "caption": "Localization of Venus-tagged Mad1 in parental HeLa cells, Bub1 CR cells and Rod CR cells in the presence of nocodazole. Scale bar, 5 μm.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Venus-tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells (left) or in cells arrested with nocodazole (right). Data combined from 3 independent experiments and standard deviation indicated.",
    "molecules": "nocodazole"
  },
  {
    "caption": "A HeLa cell line stably expressing Mad1-BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole. Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies. Representative of 2 independent experiments.",
    "molecules": "Biotin, nocodazole, thymidine"
  },
  {
    "caption": "Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(A) Upper panel: Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX-011 (n=8) or SCR ASO control (n=6). Vinculin was used as loading control. Lower panel: Quantification of the protein levels, relative to the loading control, reported as dot plots. *p<0.05 by t-test followed by Welch's correction (CLU p= 0.049, Cdc25C p= 0.024).",
    "molecules": "OGX-011"
  },
  {
    "caption": "(C) Immunoistochemistry staining for CLU and Cdc25C of radical prostatectomy samples from historical control specimens with no neoadjuvant therapy (n=5) or neoadjuvant hormone therapy, (NHT) (n=5) or the combination of NHT and OGX-011 (n=9) for 3 months prior surgery. Average score staining from the 3 groups (left) and representative sections for CLU and Cdc25C (right). Error bars represent mean ± SEM. CLU: *p<0.05; untreated versus 3mNHT (p=0.0213); 3mNHT versus 3mNHT +OGX-011 (p=0.012) by ANOVA followed by Bonferroni post hoc analysis. All cases were normalized by clinical stage, Gleason score and serum PSA. For each patient, the pathologist selected the area with the highest Gleason score. Scale bar represents 100 µm",
    "molecules": "OGX-011, hormone"
  },
  {
    "caption": "(B) Dual immunofluorescence staining with Cdc25C (green) and CLU (red) antibodies in PC3 cells. Confocal microscopy was used to identify the interaction (white arrows). DNA was counterstained with (DAPI) (blue). Scale bar represent 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells. Confocal microscopy was used to detect the interaction (red dots). DNA was counterstained with DAPI (blue). PC3 cells transfected with siCLU were used as a negative control. Scale bar represent 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Left panel: Western blot for CLU, Cdc25C, Cdc25C-T48, Cdc25C S216 and Cdc25C S198 in PC-3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Vinculin was used as loading control. Right panel: Immunofluorescence microscopy for Cdc25C-T48 in PC3 cells after siSCR or siCLU transfection. DNA was counterstained with (DAPI) (blue). Scale bar represents 10 µm.",
    "molecules": "DNA, nocodazole"
  },
  {
    "caption": "(B) Western blot for CLU, Cdc25C-T48, Cdk1 and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid (OA) (100 nM) for 12 hours. Proteins were extracted 15 min after nocodazole release. Vinculin was used as loading control.",
    "molecules": "nocodazole, okadaic acid"
  },
  {
    "caption": "(C) Western blot for PP2A, CLU, Cdc25C-T48, Cdc25C, Wee1 and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocodazole released at indicated time points. Vinculin was used as loading control.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(D) PP2A phosphatase activity measurement in PC3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Error bar represent mean ± SEM, n=3, ** p<0.01 (p=0.0025) by ANOVA followed by Bonferroni's post hoc comparisons. Cells treated with no peptide were used as a negative control.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(A) Western blot for CLU, Cdk1 Y15, Cdk1 and Wee1 in PC-3 cells after siSCR or siCLU transfection followed by synchronization and nocodazole release at indicated time points. Vinculin was used as loading control",
    "molecules": "nocodazole"
  },
  {
    "caption": "(C) FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after transfection with siSCR or siCLU in presence and absence of Wee1 inhibitor, MK-1775. Error bar represent mean ± SEM, n=3, ****p<0.0001 by Mann-Whitney",
    "molecules": "MK-1775"
  },
  {
    "caption": "(D) Western blot for CLU, Wee1, Cdk1 Y15, Cdk1 and Cyclin B1 in PC3 cells after transfection with siSCR or siCLU followed by synchronization and treatment with or without MK-1775, a specific Wee1 inhibitor, for 12 hours. Cells were harvested at different time points after nocodazole release. Vinculin was used as loading control.",
    "molecules": "MK-1775, nocodazole"
  },
  {
    "caption": "(A) Cabazitaxel IC50 calculation using WST1 assay in PC3 cells after transfection with siSCR or siCLU (left), and SCR or OGX-011 (right) followed by 72h of cabazitaxel treatment. Percentage of surviving cells was calculated relative to control. Error bar represent mean ± SEM, n=3. Inset: Western blot was performed to verify CLU knockdown in cells.",
    "molecules": "WST1, Cabazitaxel, cabazitaxel, OGX-011"
  },
  {
    "caption": "(B) Mitotic cells and cells in mitotic catastrophe were counted using DAPI in PC-3 cells after SCR or OGX-011 transfection followed by different concentration of cabazitaxel as indicated (>1000 counts) Error bar represent mean ± SEM.,*p<0.05, ***p<0.001, ****p<0.0001 by Student's t-test.( caba 1.5 nM p=0.00115 ; 3 nM p=0.0105 ; 4.5 nM p=1.93x10-7, ; 6.25 nM p=8.9x10-9; 12.5 nM p=6.8x10-9; 25 nM p=1.2x10-9).",
    "molecules": "cabazitaxel, OGX-011"
  },
  {
    "caption": "(C) Clonogenic assay in PC-3 cells after SCR or OGX-011 transfection followed by treatment with different concentrations of cabazitaxel as indicated. The number of survival clones was assessed after 15 days using crystal violet assay. Error bars represent mean ± SEM n=3,*p<0.05, **p<0.01 by Mann-Whitney (0 nM caba p=0.039; 0.5 nM p=0.0079; 2 nM p=0.0079)",
    "molecules": "cabazitaxel, OGX-011"
  },
  {
    "caption": "(A) Cell cycle distribution of syncronized PC3 and PC3-R-caba cells 60 min after nocodazole release at indicated time points.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(B) Western blot for CLU, Cdc25C, Cdc25C-T48, Cdk1, Cdk1-Y15 and Wee1 in IGRCaP-1 and PC3 parental (WT) or cabazitaxel-resistant (R-caba) cells as well as for PC3-R-caba cells transfected with siSCR or siCLU. Vinculin was used as loading control.",
    "molecules": "cabazitaxel"
  },
  {
    "caption": "(C) Cell survival measured using WST1 in PC3-R-caba cells after SCR or OGX-011 transfection and treatment with 10 nM cabazitaxel for 48h. Error bar represent mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni's post hoc correction.",
    "molecules": "WST1, cabazitaxel, OGX-011"
  },
  {
    "caption": "(D) Cell survival measured using WST1 in PC3 resistant cells after transfection with siSCR or siCLU followed by treatment with cabazitaxel (10 nM; C10), MK-1775 (10 nM; MK10) or both for 72 hours. .Error bar represent the mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni's post hoc correction.",
    "molecules": "WST1, cabazitaxel, MK-1775"
  },
  {
    "caption": "(F) Left panel: Quantification of tumour volume over 52 days in nude male mice xenotransplanted with PC3-docetaxel resistant cells and treated with either MK-1775 (M, black line), MK-1775 and micellar taxane (MT, blue line), micellar taxane and OGX-011 (TO, red line) or MK-1775, micellar taxane and OGX-011 (MTO, green line). The error bars indicate mean ± SEM, n=8. Right panel: Slopes of tumor growth between groups, were compared by ANOVA followed by Dunnett's post hoc analysis..*p<0.05, ***p<0.001 .p=0.0488 (M to TO); p=0.0007 (M to MTO).",
    "molecules": "OGX-011, docetaxel, MK-1775, micellar taxane"
  },
  {
    "caption": "Figure 2. Rpp30 is required for pre-tRNA processing.Whole fly RNA extracts were used to study tRNA processing by Northern Blot. Top box : the three different probes used are depicted : 5' and 3' probes (red and green, respectively) can detect full pre-tRNAs and tRNAs intermediates, but will not detect mature tRNAs. An Internal Probe (IP, blue) corresponding to the anti-codon region was used to detect all non-mature and mature tRNAs. The genotypes used in this experiment are numbered at the bottom (1-6). Northern Blot panels: tRNA-his 5' probe (top), tRNA-his 3' probe (middle) and tRNA-his IP (bottom). U6 probe was used as loading control.",
    "molecules": "his, tRNA"
  },
  {
    "caption": "  ASPC-1, MiaPaCa-2, Colo-357, Panc-1, Panc-89 and PancTu-1 cells were grown in the absence or presence of the indicated concentrations of omeprazole, 5-fluorouracile, pantoprazole or gemcitabine, respectively, for 4 days to determine the IC50 values. The IC50 ies and the sigmoidicities are gradually differing between the cell lines. In lower concentrations a slight growth-stimulatory effect (hormesis) was observed.  ",
    "molecules": "5-fluorouracile, gemcitabine, omeprazole, pantoprazole"
  },
  {
    "caption": "  The cell lines ASPC-1, MiaPaCa-2 , Colo357, Panc-1, Panc89 and PancTu1 were untreated or treated with 5-FU alone or in combination with the indicated concentrations of OMP for 4 days. At lower doses of 5-FU alone (black lines, 0 µg/ml OMP) a growth-stimulatory effect (hormesis) was observed in the cell lines ASPC-1, Panc-1 and PancTu-1. The data points show the means of 8 measurements. The curves are fitted in these cell lines using the Brain-Cousens model (see sub-section 4.11). In MiaPaCa-2 and Panc-89 cells no hormesis occured, these curves were fitted using a three parameter logistic model. In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations (data points shown). The red, green and blue lines indicate the dose-effect curves of 5-FU when various concentrations of OMP were added (10, 20 and 40 µg/ml, respectively). In ASPC-1 and Panc-1 cells the hormesis of 5-FU was reversed and in PancTu-1 cell it was mitigated by OMP depending on the concentration of 5-FU. In MiaPaCa-2 cells we found an additive interaction of 5-FU with OMP. In Panc-89 cells the interaction was antagonistic. ",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "  Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU , OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours. ",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "(A) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present.",
    "molecules": "omeprazole, OMP"
  },
  {
    "caption": "(E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "  The ASPC-1 cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. The most obvious changes is that the PC/Cho ratio is significantly lower in the OMP group compared to the control group. Upon 5-FU, the Cho/Acetate ratio decrease is the only significant change. In the 5-FU+OMP group, there are several significant changes, i.e.the FACH2/CH = CH ratio is significantly higher. Moreover, the Cho/Acetate ratio changed upon 5-FU+OMP as in the 5-FU group, but also the citrate/GSH ratio. The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline, Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.  ",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "  The cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. Upon OMP, the PC/Cho ratios are significantly lower compared to control. Furthermore the acetate/FACH2 ratio is significantly decreased in the OMP group. The latter also showed a higher CH = CH level, the ratio to FACH2 is, however, decreased. Upon 5-FU and 5-FU+OMP, similar changes could be observed. Furthermore, in contrast to ASPC-1 cells, the Ala/Gln/AMP pathway is also involved. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline. Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids. ",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "(B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one.",
    "molecules": "OMP"
  },
  {
    "caption": "  The experiment was repeated three times with nearly identical results. The LC3-I fractions can be clearly distinguished above the LC3-II signals. Bafilomycin A1 elevated slightly the LC3-II signal intensity compared to control in both cell lines. There was a marked and dose-dependent increase of the signal strength of both fractions when OMP was used. 5-FU alone did not relevantly influence the LC3-level. The corresponding β-Actin level is shown below the LC3-WB to confirm the correctness of this semiquantitative evaluation. ",
    "molecules": "5-FU, Bafilomycin A1, OMP"
  },
  {
    "caption": "  Quantification of mRNAs in ASPC-1 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. The stars indicate significant differences after 24 hours compared to control. The pro-apoptotic bad-mRNA was upregulated after 5-FU- and 5-FU+OMP-treatment. The antiapoptotic bcl-2 mRNA was downregulated by 5-FU and bcl-XL by 5-FU+OMP. OMP led to downregulation of bad and survivin and to upregulation of the mdr-1mRNA.  ",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "  mRNA quantification in MiaPaCa-2 cells untreated or treated with OMP 80 µg/ml, 5-FU 5 µg/ml or the combination of both was performed at different time points throughout 24 hours, and the means of three replicates are shown for every time point. While the mdr-1 mRNA is not signicantly changed, the vATPase mRNA is upregulated in the 5-FU+OMP group after 24 hours compared to control.  ",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "  The Bad , Puma and Atg12 proteins were detected in MiaPaCa-2 and ASPC-1 cells. The Bad expression is slightly increased by 5-FU and suppressed by OMP and 5-FU+OMP in ASPC-1, but not in MiaPaCa-2 cells. Puma is induced by OMP in both cell lines indicating involvement of the BH3-only pathway. Atg12 is strongly enhanced by OMP in both cell lines. Abbreviations: K - control, F - 5-FU, O - OMP, FO - 5-FU+OMP. ",
    "molecules": "5-FU, OMP"
  },
  {
    "caption": "B Composition and levels of bioactive lipid mediators (LMs), including prostaglandins, leukotrienes, lipoxins, thromboxanes, and their precursors in culture with CVF, CLys and CSN quantified by LC-MS/MS-based profiling. Heat maps show relative amounts of LMs detected by LC-MS/MS in supernatants. Asterisks show statistical differences analyzed using two-way ANOVA followed by Turkey's multiple comparison test.",
    "molecules": "leukotrienes, lipoxins, prostaglandins, thromboxanes"
  },
  {
    "caption": "C Dynamic of PGE2 release correlated with Treg induction by CLys (upper panel) and CSN (lower panel) using a Pearson correlation.",
    "molecules": "PGE2"
  },
  {
    "caption": "D Treg induction by CLys or CSN following PBMC pre-incubation with 10µM mPGES1 inhibitor 934 (or DMSO as a control).",
    "molecules": "934, DMSO"
  },
  {
    "caption": "E Induction of Tregs with PGE2 analog (DINP) (5µM) and/or recombinant human IL-10 (rhIL-10) (10ng/ml) as compared to CLys.",
    "molecules": "DINP, PGE2"
  },
  {
    "caption": "B Non-monocyte fractions were pre-incubated with IL-10R inhibitor and PGE2 receptor (EP2 and EP4) antagonists (or DMSO as a control) and incubated with CLys (left upper panel) or CSN (10µg/ml) (left lower panel) in presence or absence of monocytes and Treg induction was analysed after 72h. On the right panel, PGE2 receptors were first blocked on monocytes (2) or non-monocytes (3) or both (4) before addition of non-monocytes (2) or monocytes (3) and incubation with CSN for 72h and analysis of Treg induction compared to control condition (1).",
    "molecules": "DMSO"
  },
  {
    "caption": "E Levels of LMs and PGE2 released by macrophages in presence of CVF, CLys and CSN Additionally, macrophages were lysed and gene expression of PTGS2, PTGES, ALOX5, ALOX15 and MaR1 quantified by RT-PCR.",
    "molecules": "PGE2"
  },
  {
    "caption": "C PBMCs were treated with GDH inhibitor bithionol (BT) (20µM) or IDH inhibitor IDH-305 (50nM) alone or in combination before incubation with CLys or CSN. Percentage of Treg was determined and PGE2 and IL-10 levels were evaluated.",
    "molecules": "bithionol, BT, IDH-305"
  },
  {
    "caption": "D Treg induction from PBMC by CLys, CSN and TsGDH (5µg/ml) and levels of PGE2 and IL-10 in culture supernatants.",
    "molecules": "GDH"
  },
  {
    "caption": "E Treg induction from monocyte-naïve CD4+-TsGDH culture and levels of PGE2 and IL-10 in monocyte-CD4+-TsGDH and monocyte-TsGDH culture supernatants.",
    "molecules": "GDH"
  },
  {
    "caption": "(J-K) Representative immunofluorescence image of control (J) and 15 Gy IR treated (K) MC38 and B16F10 tumors stained for proliferation (EdU), EC nuclei (anti-ERG) and tumor vessels (anti-CD31). (L-M) Quantification of proliferating cells (L) and proliferating TECs (M) in MC38 tumors (n= 8 biological replicates per group). (N-O) Quantification of proliferating cells (N) and proliferating TECs (O) in B16F10 tumors (n= 4 to 9 biological replicates per group). (P-Q) Quantification of proliferating cells (P) and TECs (Q) in MC38 tumors after fractionated IR (n=5-7 biological replicates per group). (R-S) Quantification of proliferating cells (R) and TECs (S) in B16F10 tumors after fractionated IR (n= 5-7 biological replicates per group). (T-U) Measurement of proliferating TECs in (T) MC38 tumors (n= 9 biological replicates) and (U) B16F10 tumors (n= 5 biological replicates) in small (area <250 µm2) and large (area <250 µm2) tumor blood vessels.",
    "molecules": "EdU"
  },
  {
    "caption": "(B) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and cell density recorded (x105/mL). 3 independent experiments are shown with mean and SEM. The dotted line shows seeding density (10,000 cells/100µL). Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed",
    "molecules": "arginine"
  },
  {
    "caption": "(C) Indicated cell lines grown for 72 hours in complete medium or medium deficient in arginine and viability measured. 3 independent experiments are shown with mean and SEM. The live cell fractions (Annexin V-, 7-AAD-) were used for statistical analysis. Data information: For statistical analysis, an ordinary one-way ANOVA with Dunnett's correction for multiple comparisons was performed",
    "molecules": "7-AAD, arginine"
  },
  {
    "caption": "(D) Western blotting was used to visualise ASS1 protein levels in untreated or imatinib treated (2µM, 48 hours) CML CD34+ samples, normal CD34+ samples and indicated cell lines following 16 hours arginine starvation.",
    "molecules": "arginine, imatinib"
  },
  {
    "caption": "(A) CTV analysis of CML CD34+ patient samples after 72 hours arginine starvation (n=3). Data are shown with mean and SEM. Data information: For statistical analysis, multiple unpaired tests with Benjamini, Krieger, and Yekutieli Benjamini two-stage step up correction was performed out on log-transformed data in A",
    "molecules": "arginine"
  },
  {
    "caption": "(D) CFCs from CML CD34+ patient samples generated after 72 hours starvation of indicated amino acid. The upper panel shows representative image of colonies, counts are plotted in the lower panel. Data are shown are from 4 (-arginine) or two (-ornithine or -citrulline) biological replicates. Mean and SEM are shown. (E) CFCs from Normal CD34+ samples generated after 72 hours starvation of indicated amino acid. Upper panel shows representative image of colonies, counts are plotted in lower panel. Data are shown are from 3 biological replicates. Mean and SEM are shown. Data information: Mann-Whitney tests were performed.",
    "molecules": "arginine, citrulline, ornithine"
  },
  {
    "caption": "(B) Indicated cell lines (Control or knock-down (sg-2 or sg-3)) grown for 24 hours in absence or presence of BCT-100 (1000ng/mL) and ASS1 protein levels measured. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed",
    "molecules": "BCT-100"
  },
  {
    "caption": "(C) Indicated cell lines (Control or knock-down (sg-2 or sg-3)) grown for 72 hours in absence or presence of BCT-100 (1000ng/mL) and viability measured. 3 independent experiments are shown with mean and SD. Live cells (Annexin V-, 7-AAD-) were analysed. Data information: For statistical analysis, ordinary one-way ANOVA with Tukey's correction for multiple comparisons was performed",
    "molecules": "BCT-100, 7-AAD"
  },
  {
    "caption": "(B) Volcano plot showing differentially expressed (DE) genes between CML CD34+ vehicle and BCT-100 treated cells, red denotes q-value <0.1. Data information: , DESEQ2 and GSEA were used as described in methods.",
    "molecules": "BCT-100"
  },
  {
    "caption": "(G) The percentage of CD34+CD38- cells (from CD34+) is shown. Biological replicate data from all mice, average and SD are plotted. Vehicle: n=8 mice, Imatinib: n=8 mice, BCT-100: n=6 mice, Combo: n=7 mice. Data information A Kruskal-Wallis test was used to analyse data in G",
    "molecules": "BCT-100, Imatinib"
  },
  {
    "caption": "(H) The absolute number of CD34+CD38- cells is shown. Biological replicate data from all mice, average and SD are plotted. Vehicle: n=8 mice, Imatinib: n=8 mice, BCT-100: n=6 mice, Combo: n=7 mice. Data information A Kruskal-Wallis test was used to analyse data in H.",
    "molecules": "BCT-100, Imatinib"
  },
  {
    "caption": "(C, D and E) HeLa  cells stable expressing ORF6-Flag  were treated with or without 200 μM OA  for 12 h, then fixed and stained  with anti-Flag  (red). Initial LDs  were labeled with GFP-GPAT4152-208 (green), mature LDs  were labeled with BODIPY-493/503 (green) or GFP-Plin2  (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.",
    "molecules": "BODIPY-493, OA"
  },
  {
    "caption": "(B) Analysis of the homodimerization  of ORF6  by cross-linking with DSS. HEK293T  cells expressing ORF6-Flag  were treated with 0, 0.1, 0.2 and 0.3 mM DSS  for 30 min. Cell lysates were analyzed via WB.",
    "molecules": "DSS"
  },
  {
    "caption": "(F) HEK293T  cells were transfected with ORF6-Flag  or ΔNTD-Flag  or ΔAH1-Flag  or ΔAH2-Flag  and were treated with 0.1 mM DSS  for 30 min. Cell lysates were analyzed via WB.",
    "molecules": "DSS"
  },
  {
    "caption": "(G) HeLa  cells were transfected with ORF6-Flag  or indicated mutants and were treated with 200 μM OA  for 12 h, and then fixed and stained  with anti-Flag  (red). LDs  were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.",
    "molecules": "BODIPY-493, OA"
  },
  {
    "caption": "(A) HeLa  cells were transfected with vector or ORF6-Flag  for 24 h, and then treated with 200 μM OA  for indicated times. Cells were fixed and stained  with anti-Flag  (green). LDs  were labeled with LipidTox Deep Red  (red). The nuclei  were stained  with DAPI. Cells were imaged by  confocal microscopy. Scale bar represents 10 μm. White ROIs indicate cells expressing ORF6  and yellow ROIs indicate the cells without ORF6 expression.",
    "molecules": "LipidTox Deep Red, DAPI, OA"
  },
  {
    "caption": "(D) HeLa  cells stable expressing vector or ORF6-Flag  were treated or untreated with 50 μM ATGL  inhibitor (ATGLi) for 24 h, and then were fixed and stained  with anti-Flag  (red). LDs  were labeled with  BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (E) Mean number of LDs  in each cell in (D) was counted from 25 cells of three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ****p < 0.0001. Error bars represent the mean ± SD.",
    "molecules": "BODIPY-493"
  },
  {
    "caption": "(H) HeLa  cells stable expressing vector or ORF6-Flag  were treated or untreated with 50 μM ATGL  inhibitor, along with DMSO  or 1 μM DGAT1  inhibitor (DGAT1i) or/and 2 μM DGAT2  inhibitor (DGAT2i) for 24 h. LDs  were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (I) Mean number of LDs in each cell in (H) was counted from 35 cells of three independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ****p < 0.0001, ns means no significance. Error bars represent the mean ± SD.",
    "molecules": "BODIPY-493, DMSO"
  },
  {
    "caption": "(E) ORF6-Flag  or the mutants were co-expressed with mCheey-RAMP4. Cells were treated with 200 μM OA  for 12 h, and then fixed and stained  with anti-Flag  (red). The ER  was visualized with mCheey-RAMP4  (red). LDs  were labeled with LipidTOX Deep Red (white). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.",
    "molecules": "LipidTOX Deep Red, OA"
  },
  {
    "caption": "(J) HeLa  cells stable expressing ORF6-Flag  were transfected with si-BAP31  and/or si-USE1  for 24 h, and were further treated with 50 μM ATGL  inhibitor for 24h. Cells were fixed and stained  with anti-Flag  (red). LDs  were labeled with BODIPY-493/503 (green). The nuclei  were stained  with DAPI. Cells were imaged by  confocal microscopy. Scale bar represents 10 μm. (K) Mean number of LDs  in each cell in (J) was counted from 25 cells of three independent experiments. Two-tailed Unpaired Student's t-test, *p < 0.05, **p < 0.01. Error bars represent the mean ± SD.",
    "molecules": "BODIPY-493, DAPI"
  },
  {
    "caption": "(A) HeLa  cells were transfected with vector or ORF6-Flag  for 12 h and treated with 200 μM OA  for another 12 h, the medium was removed and replaced with fresh complete culture containing 1 μM DGAT1  inhibitor and 2 μM DGAT2  inhibitor for 24 h. Meanwhile, 50 μM ATGL  inhibitor or 100 μM Chloroquine  (CQ) was added and allowed to incubate for 24 h or 4 h to block lipolysis  or lipophagy, respectively. Cells were fixed and stained  with anti-Flag  (red). LDs  were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. White ROIs indicate cells expressing ORF6  and yellow ROIs indicate the cells without ORF6 expression.",
    "molecules": "BODIPY-493, Chloroquine, CQ, OA"
  },
  {
    "caption": "(C) Representative transmission electron micrograph  of ORF6-Flag  stable expressing or vector HeLa  cells upon OA  treatment. Red arrows mark the contact sites between LDs  and mitochondria. M, mitochondria. LD,  lipid droplets. Scale bar represents 1 μm. (D) The number of mitochondria-LD  contacts per cell was counted from 20 cells. The number of mitochondrial  engaged in mitochondrial-LD  contact per LD  in (C) was counted from 20 LDs. Two independent experiments. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.",
    "molecules": "OA"
  },
  {
    "caption": "(F and G) SARS-CoV-2-infected Vero-E6 cells were fixed and stained  with anti-Tom20  (cyan). Tom20  represents mitochondria  marker. LDs  were labeled with LipidTOX Deep Red (red). Cells were imaged by  confocal microscopy. Scale bar represents 10 μm. Colocalization  of LDs  and mitochondria  (Pearson's Coefficient), n=20 cells, two independent experiments. Two-tailed Unpaired Student's t-test, **p < 0.01. Error bars represent the mean ± SD.",
    "molecules": "LipidTOX Deep Red"
  },
  {
    "caption": "(H) Cos7  cells expressing ORF6-Flag  and vector were treated with 200 μM OA  for 12 h, and then were fixed and stained  with anti-Flag  (white) and anti-Tom20  (red). Tom20  represents mitochondria  marker. LDs  were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm.",
    "molecules": "BODIPY-493, OA"
  },
  {
    "caption": "(L) Seahorse FAO assays  to examine the oxidation of endogenous FAs  in vector, or ORF6-Flag  or ORF64Q-Flag  over-expressed HeLa  cells. Three biological replicates were performed with similar results. Error bars represent the mean ± SD.",
    "molecules": "FAs"
  },
  {
    "caption": "(M) The concentration of ATP  in cells was analyzed according to manufacturer's instructions. Error bars, mean ± SD of four independent experiments. Two-tailed Unpaired Student's t-test, ***p < 0.001, ns means no significance.",
    "molecules": "ATP"
  },
  {
    "caption": "(F) Cos7  cells expressing ORF6-Flag  were transfected with negative or si-MTX1/si-MTX2/si-SAMM50  (Triple KD) for 36 h and treated with 200 μM OA  for another 12 h. Cells were fixed and stained  with anti-Flag  (white) and anti-Tom20  (red). Tom20  represents mitochondria  marker. LDs  were labeled with BODIPY-493/503 (green). Cells were imaged by confocal microscopy. Scale bar represents 10 μm. (G) Quantification of average number of LD-mitochondria  contacts per LD  from (F). 25 cells (Negative), 50 cells (Triple KD) from three independent experiments were calculated. Two-tailed Unpaired Student's t-test, ****p < 0.0001. Error bars represent the mean ± SD.",
    "molecules": "BODIPY-493, OA"
  },
  {
    "caption": "Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm",
    "molecules": "GCaMP6m"
  },
  {
    "caption": "Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells)",
    "molecules": "GCaMP6m, calcium"
  },
  {
    "caption": "Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n=3 independent experiments)",
    "molecules": "2,5-di-tert-butylhydroquinone, TBHQ, Ca2+"
  },
  {
    "caption": "Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)",
    "molecules": "Ca2+, CPA, cyclopiazonic acid, EGTA"
  },
  {
    "caption": "Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1-KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n=3 independent experiments; 30-56 cells)",
    "molecules": "GCaMP6m, calcium"
  },
  {
    "caption": "Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n=3 independent experiments.)",
    "molecules": "TBHQ, Ca2+"
  },
  {
    "caption": "Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA (n=3 independent experiments).",
    "molecules": "Ca2+, CPA, EGTA"
  },
  {
    "caption": "HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm",
    "molecules": "rapamycin"
  },
  {
    "caption": "Hek293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into Cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": "Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": "Mitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localizations. Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture. OMM: outer mitochondrial membrane; IMS: intermembrane space",
    "molecules": "sucrose"
  },
  {
    "caption": "Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of vehicle- or rapamycin (Rapa., 1 μM for 4 h)-treated Hek293T cell mitochondria following carbonate extraction at pH 11.5. Akt, phosphorylated (S473) Akt (p-Akt), MICU1, the established integral membrane proteins TIM23 and MCU, and the soluble protein cytochrome c (Cyt. c) was analyzed",
    "molecules": "Rapa, rapamycin, carbonate"
  },
  {
    "caption": "Hek293T cells were transfected with the GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). GFP-MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphorylated (S473) Akt (p-Akt) antibodies by western blotting",
    "molecules": "Rapa, rapamycin, triciribine"
  },
  {
    "caption": "Resting mitochondrial calcium levels in the control (ctrl) and Akt D/D-expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 44-49 cells)",
    "molecules": "GCaMP6m, calcium"
  },
  {
    "caption": "Resting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells)",
    "molecules": "GCaMP6m, calcium"
  },
  {
    "caption": "Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m (n=3 independent experiments; 38-50 cells)",
    "molecules": "GCaMP6m, calcium"
  },
  {
    "caption": "Representative kinetics (H) and analysis (I) of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [Ca2+] (n=3 independent experiments).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. Where indicated, the cells were treated with 5 μM MG132 for 4 h. pre: precursor; int: intermediate; m: mature form",
    "molecules": "MG132"
  },
  {
    "caption": "Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs, following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. The cells were treated with 5 μM MG132 for 4 h before fractionation",
    "molecules": "MG132, carbonate"
  },
  {
    "caption": "Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionation",
    "molecules": "MG132, carbonate"
  },
  {
    "caption": "ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1), both WT and mutants, is represented relative to the amount at time 0. (n=3 independent experiments)",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were harvested, and total protein was extracted and subjected to western blotting analysis with the indicated antibodies. Immunoblotting was performed under reducing (with the presence of DTT) or non-reducing (non-red) conditions (without DTT). Cells were treated with 5 μM MG132 for 4 h before lysis",
    "molecules": "DTT, MG132"
  },
  {
    "caption": "Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells).",
    "molecules": "GCaMP6m, calcium"
  },
  {
    "caption": "Western blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis",
    "molecules": "MG132"
  },
  {
    "caption": "Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers",
    "molecules": "carbonate"
  },
  {
    "caption": "Western blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria transfected with HA-tagged MICU1 WT following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. pre: precursor; int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysis",
    "molecules": "MG132, carbonate"
  },
  {
    "caption": "Control or IMMP1L-silenced HeLa cells were transfected with HA-tagged MICU1 WT. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1) is represented relative to the amount at time 0. (n=3 independent experiments)",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Representative kinetics (F) and analysis (G) of aequorin-based [Ca2+]m measurements in control or IMMP1L-silenced HeLa cells challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)",
    "molecules": "Ca2+, CPA, cyclopiazonic acid, EGTA"
  },
  {
    "caption": "Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells).",
    "molecules": "GCaMP6m, calcium"
  },
  {
    "caption": "Representative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unit",
    "molecules": "MitoSOX, ROS"
  },
  {
    "caption": "ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure.",
    "molecules": "H2O2, ROS, Tempol"
  },
  {
    "caption": "(j)  Live cell imaging of  HE and HSC-like cells stained with TMRE (red) at day 3 of subculture. Representative merged brightfield/TMRE  and TMRE images are shown. Scale bars, 100 µm. Bar graphs show means of TMRE staining intensity from all replicate wells across all experiments (HE spindle  and HE round, n=3 biological replicates with 3 technical replicates each; HSC-like, n=2 biological replicates with 3 technical replicates each, Kruskal-Wallis test with multiple comparisons). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001",
    "molecules": "TMRE"
  },
  {
    "caption": "(b-e)  FACS-sorted HE  cells were subcultured with or without  UK5099  (10 µM) or DCA (3 mM). Subculture day 3 representative GPA/CD43 plots (b, d) and subculture day 6 representative CD45/CD43 plots (c, e) are shown Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "UK5099, DCA"
  },
  {
    "caption": "(g) Fold change in the  expression  of HBE1 or HBG1-2  transcripts ± SEM normalized to  KLF1 in CFUs obtained from HE  cells treated with UK5099 (10 µM) or  DCA (3 mM) relative to non-treated cells (n=3 biological replicates, paired t-test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "UK5099, DCA"
  },
  {
    "caption": "(i-k) Pregnant mice  were injected with UK5099 or DCA  at E9.5 and  fetal livers  were analyzed at E14.5 by flow cytometry. FL, fetal liver. Levels of T and B cells (i) and LT-HSCs (j) as percentages in  fetal liver  are shown for control (n=10 biological replicates), UK5099-treated (n=14 biological replicates) and DCA-treated (n=16 biological replicates) conditions (one-way ANOVA test). (k) The ratio of BFU-E to  CFU-GM colonies obtained from sorted  LT-HSCs are shown CFU, colony forming unit; BFU, burst forming unit; E, erythroid; M, macrophage; G, granulocyte. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "UK5099, DCA"
  },
  {
    "caption": "(l-p) HE cells co-cultured with   OP9-DL1 stroma   were treated with DCA  for 3 days and transplanted into irradiated NSG  mice. Bone marrow  (BM) and thymi were harvested on week 12. (l) The percentages ± SEM of  human CD4+CD8+ double positive  thymocytes in huCD45+ cells from the  thymus are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). The percentages ± SEM of human B cells (m), CLPs (n) from the BM  at week 12, as well as myeloid cells from PB at week 8 (o) and myeloid cells from BM at week 12 (p) in huCD45+ cells are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). PB, peripheral blood. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001",
    "molecules": "DCA"
  },
  {
    "caption": "(d) Dot plots show  expression levels of the indicated hematopoietic  transcription factors in clusters 6 and 7 for HE ctrl, HE + UK5099 and HE  + DCA  conditions, detected by scRNAseq and based on percent expressed (size of the dots) and average level of  expression  (color intensity).",
    "molecules": "UK5099, DCA"
  },
  {
    "caption": "(d)  HE  cells were transduced with shScrambled (shScr) or shLSD1 with or without UK5099 (10 µM) the day after the sort  and day 3  CD43+/GPA+ cell frequencies ± SEM relative to shScr are presented (n=3 biological replicates 1-2 technical replicates each, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001",
    "molecules": "UK5099"
  },
  {
    "caption": "(f-g) FACS-sorted  HE  cells were subcultured with  ACSS2i (5 µM), DCA  (3 mM), or both (n=5 biological replicates, one-way ANOVA test) (f) or with C646  (10 µM), DCA (3 mM), or both (n=3 biological replicates, one-way ANOVA test) (g) and day 6  CD43+CD45+ cell frequencies ± SEM are shown. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001",
    "molecules": "C646, DCA"
  },
  {
    "caption": "(j) FACS-sorted  HE  cells were subcultured with CP (5 µM), DCA (3 mM) or both and day 6 CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=4 biological replicates, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001",
    "molecules": "CP, DCA"
  },
  {
    "caption": "(k) Cholesterol content ± SEM in HE cells was measured by confocal microscopy at day 2 of DCA (3 mM) treatment by  filipin III staining  (n=3 biological replicates, paired t test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001",
    "molecules": "Cholesterol, DCA, filipin III"
  },
  {
    "caption": "(m)  FACS-sorted HE  cells were subcultured with  Ato (0.5 µM), DCA (3 mM) or both and day 3  CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=3 biological replicates for control/DCA, n=2 biological replicates with 2 technical replicates for Ato/Ato+DCA, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001",
    "molecules": "Ato, DCA"
  },
  {
    "caption": "B. Representative 2D projection of confocal  images showing caspase  activation at the dissection  time point  (red channel) and in the recent past (green channel, arrows) within putative escort somatic cells via DBS-S-QF  sensor; TUNEL staining  indicates apoptosis  (grey, arrowhead); DAPI  labels the nuclei  in the entire Figure. Experimental flies  were kept after eclosion from the pupae  for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; ;QUAS-Gal4  /+ (BL83123). C. Representative 2D projection of confocal  images showing escort  and follicular somatic cells permanently labelled with DBS-S-QF sensor (green channel, arrows); the arrowhead indicates the presence of  germline cells  positive for TUNEL staining  (red, arrowhead). Notice the lack of TUNEL  signal and therefore apoptosis  in somatic cells  permanently labelled with  DBS-S-QF sensor (green). Experimental flies  were kept after eclosion from the pupae  for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; QUAS-flippase  (BL30126)/+; Actin5C  FRT-stop-FRT lacZ-nls/+ (BL6355) Data information: Scale bars represents 10 µm in the entire Figure.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-B. Representative 2D projection of confocal  images showing the expression  of the cell identity Castor  in follicular cells  control with Dronc expression  (A: 109-30-Gal4  (BL7023)/+; DroncKO UAS-Histone-RFP  (BL56555) Tub-G80ts (BL7019)/+) and without Dronc  (B: 109-30-Gal4  (BL7023)/+; DroncKO UAS-Histone-RFP  (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF). Notice the reduction in the number of Castor-expressing cells in the FCD  and the reduction in size of this region in B. Nuclei  are labelled with DAPI  (blue); Castor  (green) and FasIII  (grey) label the follicular cells; UAS-Histone-RFP  labels the nuclei  of Gal4-expressing cells (red). Data information: Scale bars represents 10 µm. Experimental flies  were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).",
    "molecules": "DAPI"
  },
  {
    "caption": "J-K. Representative 2D projection of confocal  images of the genotypes quantified in I. Nuclei  are labelled with DAPI  (blue). Red and green nuclei  show the expression  of different Fucci  markers. FasIII  (grey) was used to locate  the FCD. Notice the reduction of follicular cells  and limited overlap between the green and red fluorescent signals (white arrowheads) in K. Data information: Scale bars represents 10 µm. Experimental flies  were kept after eclosion from the pupae  for 14 days at 29°C prior to dissection (applicable to all panels).",
    "molecules": "Fucci, DAPI"
  },
  {
    "caption": "H. Western blot showing the expression levels of the autophagy marker p62  (upper lane), Caspase-9 (middle lane) and Actin  (bottom lane, loading control) in either scrambled or Caspase-9  deficient  OVCAR-3 cells; the protein levels of the different read outs were measured at 72h after siRNA treatment in cells grown during the last 4 h before sample processing in our standard cell culture conditions, in cell culture media containing EtOH  (0.2%), and in cell culture media containing EtOH  (0.2%) +  bafilomycin A1  (400nM).",
    "molecules": "bafilomycin A1, EtOH"
  },
  {
    "caption": "A. Cartoon representation of the Dln1 dimer (PDB 4ZNO). Subunit A is colored in gray. The lectin module of subunit B is colored in yellow, whereas the middle and C-terminal moieties of the aerolysin module are colored in hot pink and green, respectively. The pre-stem hairpin is colored in blue. The bound sucrose molecules are shown as light pink sticks.B. The dimeric interface. Residues involved in dimeric interactions are shown as sticks.",
    "molecules": "sucrose"
  },
  {
    "caption": "A. A close-up view of the sucrose-binding pocket. Residues hydrogen-bonding with sucrose are shown as yellow sticks, and the sucrose molecule is shown as light pink stick.",
    "molecules": "sucrose"
  },
  {
    "caption": "B. The glycan binding profile of Dln1. This profile represents FITC-labeled Dln1 at 200 μg/ml screened against 609 printed glycans on Mammalian Printed Array version 5.2 in replicates of six. A list of top 8 hits reacted with a concentration of 200 μg/ml is shown in Appendix Table S1. The two kinds of Dln1-binding glycan structures (No. 214 and 316) are illustrated in schematic.",
    "molecules": "glycans"
  },
  {
    "caption": "C&D. Close-up views of Manα1-2Man and Manα1-3Man binding sites. The mannobiose molecules are shown as cyan and gray sticks, respectively. The Fo-Fc electron density maps of disaccharides and the disaccharide-binding sites are shown as gray mesh and countered at 3.0 . The hydrogen-bonding residues are shown as yellow sticks. Polar interactions with the mannobiose are shown as dashed lines.",
    "molecules": "Manα1-2Man, Manα1-3Man, mannobiose"
  },
  {
    "caption": "A&B. ELISA of Dln1 towards glycosylated gp120 or yeast mannan. The curve represents the average of triplicate detections, using BSA as the control. Points are averages (±standard error of the mean) of triplicate determinations.",
    "molecules": "mannan"
  },
  {
    "caption": "A. Oligomerization of Dln1 in the presence of S. cerevisiae mannan or liposomes at different pH values. Histograms are averages (±standard error of the mean) of triplicate determinations",
    "molecules": "liposomes, mannan"
  },
  {
    "caption": "B&C. Dye release velocity from the liposomes in the presence of Dln1 at different concentrations, and various pH values. Liposomes were incubated with Dln1 of different concentrations at pH 5.5, and 1 μM Dln1 at various pH values, respectively.",
    "molecules": "liposomes, Liposomes"
  },
  {
    "caption": "D. Oligomerization of Dln1M5 with or without liposomes at different pH values. All samples for electrophoresis were prepared with boiling at 95°C for 5 min, and the comparative gels were processed in parallel. The oligomer band was quantified by Image J software with the average of triplicate detections, using the marker band as a sample processing control. The SDS-PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4. Histograms are averages (±standard error of the mean) of triplicate determinations.",
    "molecules": "liposomes"
  },
  {
    "caption": "E. Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5.5.",
    "molecules": "Liposomes"
  },
  {
    "caption": "F. The inhibition of dye release in presence of 0.5 M Dln1M5 and 25 mM polyethylene glycols (PEG) of different molecular weights at a fixed pH value of 5.5. Dye efflux was monitored in a period of 1400 sec. Triton X-100 at 0.1% was added to completely disrupt liposomes. Dye efflux was measured as a percentage to the maximal release upon addition of Triton X-100. Carboxyfluorescein was loaded in the liposomes for the detection of dye efflux.",
    "molecules": "Carboxyfluorescein, liposomes, PEG, polyethylene glycols, Triton X-100"
  },
  {
    "caption": "A. Negative-stain electron microscopy image of Dln1M5 in presence of liposomes.B. Side views of Dln1M5 oligomers attached to the membrane of liposomes. The inset represents the respective class averages.",
    "molecules": "liposomes"
  },
  {
    "caption": "C&D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings.",
    "molecules": "lipid monolayer"
  },
  {
    "caption": "C&D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings.",
    "molecules": "lipid monolayer"
  },
  {
    "caption": "E. EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer. The inset represents the computed diffraction pattern Fourier transform of the image.F. Projection map with a P4 symmetry.",
    "molecules": "lipid monolayer"
  },
  {
    "caption": "(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the muscle cytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the muscles than either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.",
    "molecules": "chloroquine, CQ, Rap, rapamycin"
  },
  {
    "caption": "(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the musclecytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the musclesthan either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.",
    "molecules": "chloroquine, CQ, Rap, rapamycin"
  },
  {
    "caption": "(E) Control white RNAi muscles starved and treated with CQ accumulate GFP-Atg8a punctae, but very few colocalize with ubiquitin (Ubi). (F) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP-Atg8a punctae. The remainder colocalize with ubiquitin. (G) Quantification of the mean GFP-Atg8a punctae area and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in E and F. Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals, with p-values for Rpn1 RNAi relative to white RNAi control (***p<.001).",
    "molecules": "CQ"
  },
  {
    "caption": "(A-D) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant (DTS). Dmef2-Gal4, UAS-GFP-Atg8a control and UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection. 18 hrs post-shift (PS), both control (A) and DTS muscles (B) accumulate GFP-Atg8a punctae, but the latter also accumulate ubiquitin (Ubi) aggregates. 48 hrs PS, control muscles (C) maintain strong autophagy induction, but DTS mutant muscles (D) have reduced GFP-Atg8a punctae accumulation, similar to the Rpn1 knockdown phenotype. (E) Quantification of the mean GFP-Atg8a punctae and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in A-D (SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p-values of DTS relative to control). Dark green = Dmef2-Gal4, UAS-GFP-Atg8a; Light green = UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a.",
    "molecules": "CQ"
  },
  {
    "caption": "(D-E) tyramine (Tyr) treatment reduces GFP-Atg8a punctae in larval muscle. UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals were placed on 100mg/ml Tyr (D) or control food (E), shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection.",
    "molecules": "Tyr, tyramine"
  },
  {
    "caption": "(F-H) Effect of Tyr treatment on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and Dmef2-Gal4, UAS-GFP-Atg8a animals, shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. Aggregate (F) and GFP-Atg8a punctae (G) numbers in muscles are significantly reduced with 100 mg/ml Tyr treatment in DTS but not control animals 18 hrs post-shift (PS). (H) 100 mg/ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS. n = 12 and p-values shown relative to non-Tyr treated animals. For all measurements, SEM is shown with *p<.05; **p<.01; ***p<.001.",
    "molecules": "Tyr"
  },
  {
    "caption": "(I) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-G4 control with or without 100 mg/ml Tyr. After 6hrs starvation, Atg8a-II levels are reduced in DTS + 100 mg/ml Tyr relative to untreated DTS.",
    "molecules": "Tyr"
  },
  {
    "caption": "(D-H)Dmef2-Gal4 drives expression of UAS-GFP-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. RNAi knockdown of CG41099 (E), CG7112 (F), CG31935 (G), and Rab3-GAP (H), blocks the formation of GFP-Atg8a labeled autophagosomes compared to knockdown of white (D).",
    "molecules": "CQ, nutrient"
  },
  {
    "caption": "(I-M) Dmef2-Gal4 drives expression of UAS-myc-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. The effect of the RAB3GAPs is not via Rab3 or Rab5 function, as a Rab3 null mutation (J), expression of a constitutive active form of Rab3 (Q80L) (K) or Rab5 (Q88L) (L) has no effect on muscle autophagy compared to control (I). Expression of a constitutive active form of Rab39 (Q69L) (M) reduces myc-Atg8a labeled vesicles. (N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.",
    "molecules": "CQ, nutrient"
  },
  {
    "caption": "(O) Atg8a and Rab3-GAP colocalize to autophagosomes. Dmef2-Gal4 drives expression of UAS-myc-Atg8a and UAS-CFP-Rab3-GAP in animals starved and treated with CQ for 6 hrs. In all panels green = GFP-Atg8a and red = myc-Atg8a, and blue = CFP-Rab3Gap2.",
    "molecules": "CQ"
  },
  {
    "caption": "(A) Primary cultured muscles with control lacZ RNAi, treated for 12 hrs with Rap+CQ. Note the formation of large GFP-Atg8a vesicles throughout the muscles. (B) GFP-Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi, treated for 12 hrs with Rap+CQ. Knockdown of Pfk (C) or CG9485 (D) causes increased accumulation of GFP-Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap+CQ. (E) Quantification for A-D of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). SEM is indicated with *p<.05; **p<.01.",
    "molecules": "CQ, Rap"
  },
  {
    "caption": "(F) Control white RNAi muscles accumulate large autophagosomes at 1 mg/ml CQ. (G) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle. (H) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle. (I) Quantification of the GFP-Atg8a punctae area in muscles from white, CG9485, and AGBE knockdowns.n = 6 larval muscles with SEM indicated and *p<.05.",
    "molecules": "CQ"
  },
  {
    "caption": "RFP+ (G1 phase), GFP+ (G2 phase), and GFP+/RFP+ (S phase)   RPE-FUCCI cells were sorted by flow cytometry, RNA was extracted and analyzed using qRT-PCR (n=3). Expression is normalized to GAPDH  mRNA. Bars show mean ± SD.",
    "molecules": "FUCCI"
  },
  {
    "caption": "RPE cells were released for indicated  times from a thymidine block and collected cells were analyzed by  immunoblotting and FACS. Nocodazole was added after 4 h to arrest cells in mitosis. Alternatively,  RO3306 inhibitor was added to prevent mitotic entry. Where indicated sample was collected by mitotic shake-off (MS) to obtain pure mitotic population. Samples were analyzed by immunoblotting (upper panel). Staining for  pS10-H3 was used as a marker of mitosis, TFIIH as a loading control. In parallel, cell cycle progression was followed by flow cytometry using DAPI  and mpm2 staining. Representative plot is shown, M indicates percentage of mitotic cells (middle panel). Signal of the  rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the  thymidine arrested sample (lower panel). Bars show median ± SD.",
    "molecules": "RO3306, DAPI, Nocodazole, thymidine"
  },
  {
    "caption": "RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA, probed with  rabbit polyclonal antibody against  FAM110A and mouse monoclonal to  γ-tubulin, and were analyzed by confocal microscopy. Shown is maximal projection of representative mitotic (upper panel) and interphase (lower panel) cells. Scale bars indicate 10 μm.",
    "molecules": "PFA"
  },
  {
    "caption": "RPE  cells transfected with control or FAM110A  siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase. Impaired  chromosomal alignment and multipolar spindles were scored and representative  images are shown. Scale bars indicate 20 μm, arrowheads point to misaligned chromosomes. More than 30  metaphases were evaluated per condition (n=3). Bars indicate median ±SD. Statistical significance was determined by two-tailed t-test, (*** p<0.001, * p<0.05).   Quantification of lagging chromosomes  in anaphase  cells from (E). More than 30 anaphases were evaluated per condition (n=3). Representative images of abnormal anaphase are shown, scale bars indicate 20 μm, arrowheads point to incorrectly segregating chromosomes. Statistical significance was determined by two-tailed t-test. Bars indicate median ±SD (*** p<0.001).   ",
    "molecules": "MG132"
  },
  {
    "caption": "RPE cells arrested in mitosis by nocodazole (NZ) were treated with  DMSO, PLK1 inhibitor BI2536 (100 nM), Aurora-A inhibitor MLN8054 (0.25 μM), CDK1 inhibitor  RO3306 (20 nM) or CK1 inhibitor PF-670462 (1 μM) for 60 min, collected and analyzed by immunoblotting with indicated antibodies. Asynchronously growing RPE cells are shown for comparison (left line). Staining for H3-pS10 served as a marker of mitosis (n=3).",
    "molecules": "MLN8054, RO3306, BI2536, DMSO, nocodazole, NZ, PF-670462"
  },
  {
    "caption": "U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 (100 nM) and collected by mitotic shake off. Part of the nocodazole arrested cells was further treated with  CK1 inhibitor PF-670462 (1 μM) for 60 min. Whole cell lysates were probed with indicated antibodies.  Staining for pS10-H3  served as a marker of mitosis.",
    "molecules": "BI2536, nocodazole, PF-670462"
  },
  {
    "caption": "RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC), FAM110A  or CSNK1D siRNA and grown for 48 h. Alternatively, cells were incubated with PF670462  for the last 12 h. Cells were treated with MG132 for 30 min prior fixation. Impaired chromosomal alignment was scored in >30 metaphase cells per condition. Error bars indicate median ±SD. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001, *** p<0.001).",
    "molecules": "MG132, PF670462"
  },
  {
    "caption": "RPE1 were transfected with control siRNA or CSNK1D  siRNA or were treated with PF670462  prior fixation. Cells were probed with rabbit polyclonal to FAM110A  and mouse monoclonal to γ-Tubulin. Representative  images are shown, scale bars indicate 10 μm.   Quantification of (A). Plotted is median FAM110A intensity at  spindle poles positive for  γ-tubulin ± SD. Each dot represents a single  spindle pole. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001).   ",
    "molecules": "PF670462"
  },
  {
    "caption": "Cells expressing the wild-type or mutant EGFP-FAM110A were fixed and stained for γ-tubulin and α-tubulin and analyzed by  confocal microscopy (z-stack 0.16 µm). Representative images of maximal projections of the metaphase cells are shown, scale bars indicate 10 μm. Merged images show signal of DAPI,  γ-tubulin and  EGFP. Insets show a single Z plane at the spindle pole.",
    "molecules": "DAPI"
  },
  {
    "caption": "J. Whole mount immunofluorescence    of the organ of Corti   from 2-month old control and Cgn-cKO mice. Phalloidin  (F-actin, magenta); Myosin7a  (hair cells, green). White arrows label the lost OHCs.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "J. Whole mount immunofluorescence  of the organ of Corti  from 7-month old CgndelG mice. White arrows indicate lost OHCs  by F-actin  labeling (Phalloidin).",
    "molecules": "Phalloidin"
  },
  {
    "caption": "(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei  of the indicated cell types stained  with DAPI. Scale bars, 3 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Areas of DAPI-dense regions (top), distance of DAPI-dense regions from the  nuclear periphery (middle), and variance of DAPI  signals (bottom). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI  dense regions = 950/1450/839/1535/736 and number of slices = 90/115/95/135/110 for mESC/EpiLC/d2/d4c7 mPGCLC/GSC. Significances are computed using Wilcoxon rank-sum tests, p-values from top to bottom 4.37e-3, 1.62e-3, 2.99e-2, 2.03e-10, 4.03e-1, <2.2e-16, 1.31e-3, 8.94e-3, 1.06e-4, 5.62e-2, 4.63e-5, 7.65e-13. P-value symbol brackets: ∗∗∗∗ = [0, 0.0001); ∗∗∗ = [0.0001, 0.001]; ∗∗ = [0.001, 0.01); ∗ = [0.01, 0.05); ns = [0.05,",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) (left) Fluorescence in situ hybridization  (FISH) against chromosome 16 (red) with DAPI staining  (grey). Z-stacked representative images are paired with magnified views. (right) Distributions of surface volumes for  chr16. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 51/68/53 for mESC/EpiLC/GSC. Scale bars, 5 μm. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 4.16e-2, 4.33e-6, 8.68e-9.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei  of GSCs  and GSCLCs stained  with DAPI. Scale bars, 3 μm. (C) Areas of DAPI-dense regions (left), distance of DAPI-dense regions from the nuclear periphery  (middle), and variance of DAPI  signals (right). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI  dense regions = 1535/736/1227 and number of slices = 135/110/120 for d4c7 mPGCLC/GSC/GSCLC. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 2.03e-10, 1.69e-9, 0.123, 0.00894, 8.02e-8, 0.0707, 7.65e-13, 0.417, 2.07e-12.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F-G)  IF analysis of Cep164 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164  antibodies. (F) Representative images. Scale bar, 1 μm. (G) Quantification of Cep164 intensity at the  mother   centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=147 cells; OFD1 KD, n=146 cells; MyoVI  KD, n=150 cells;  MyoVI + OFD1 KD, n=151 cells, from four independent experiments. ** P<0,005; **** P<0,0001 by Kruskal-Wallis test.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(I) Quantification of OFD1 intensity at the mother or daughter centrioles. hTERT-RPE1  cells treated or not with Nutlin-3, were incubated with nocodazole  for 1 hour (6 μg/ml) and  immunostained  with anti-OFD1, anti-centrin1  and anti-Cep164 antibodies. Mother  centrioles were identified by the coincident staining of centrin1  and  Cep164, while daughter  centrioles were  centrin1-only stained. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mother centrioles: Mock, n=148 cells; Nutlin-3, n=169 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; Nutlin-3, n=122 cells, from three independent experiments. ns, not significant by Mann-Whitney test.",
    "molecules": "nocodazole, Nutlin-3"
  },
  {
    "caption": "(J) Quantification of Cep164 intensity at the mother  centrioles in hTERT-RPE1 cells treated as in (I). Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=195 cells; Nutlin-3, n=196 cells, from four independent experiments. ns, not significant by Mann-Whitney test.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "FRAP  analysis of centriole-associated  GFP-OFD1.  hTERT-RPE  cells stably expressing GFP-OFD1 and centrin1-dTomato were transfected with siRNA against  myosin VI. After four days, cells were treated with nocodazole (1 hour, 6 μg/ml) and subjected to live-cell imaging. (E) A representative graph of one out of three experiments. For each  time point, the fraction of recovery of GFP-OFD1 is shown. Results are expressed as means with 95% confidence interval. n=12 cells (Mock), n=13 cells (MyoVI KD). (F) Quantification of the half-time  of fluorescence recovery (t1/2,) and of the mobile fraction of GFP-OFD1. Results are expressed as mean ±SD. n=41 cells (Mock), n=31 cells (MyoVI  KD), from three independent experiments. ns, not significant; *** P<0,0005 by Unpaired T-test.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(B)  IF analysis of PCM1 signal. hTERT-RPE1  cells were transfected with siRNA against  myosin VI  and immunostained  with anti-PCM1 and anti-Cep135 antibodies. Upper panel, representative images, scale bar, 2 μm. Lower panel, quantification of PCM1  intensity. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=96 cell; MyoVI KD, n=98cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (C)  IF analysis of  PCM1 signal. hTERT-RPE1 cells were treated with Nutlin-3 for 24 hours and immunostained  with anti-PCM1 and anti-Cep135 antibodies. Panels as in B. Mock, n=96 cells; Nutlin-3, n=100 cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test. (D)  IF  analysis of PCM1 signal. hTERT-RPE1 p53 KO cells were transfected with siRNA against  myosin VI and immunostained with anti-PCM1 and anti-Cep164  antibodies. Panels as in B. Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole, identified with anti-Cep164 staining. Mock, n=128 cells; MyoVI KD, n=114 cells from three independent experiments. ns, not significant by Mann-Whitney test.",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "(D) IF analysis of primary cilium  upon  serum starvation. hTERT-RPE1 p53  KO cells were transfected with siRNA against  myosin VI. After four days, cells were fixed (growing) or serum starved    for 24 hours (SS). Cells were immunostained with anti-acetylated tubulin (to identify the  cilia), anti-Cep135 (to identify the centrioles) and  DAPI. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. n=3 independent experiments. 100-200 cells/condition were counted for each experiment. ***P < 0.001 by two-way ANOVA test.",
    "molecules": "DAPI"
  },
  {
    "caption": "F, G MCF-7  cells expressing GFP-INPP4B  or GFP-vector were cultured in growth media or EBSS  for 4 hours, then fixed and immunostained  with p62  antibodies, and co-stained  with DAPI  and phalloidin  (F). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (G). Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F p values determined by one-way ANOVA with Tukey post hoc test in G,",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "H, I MCF-7 cells expressing GFP-INPP4B  or GFP-vector were treated with 100 nM bafilomycin A1  or DMSO  as a vehicle control for 4 hours, then lysed and immunblotted  with LC3B  antibodies and GAPDH  antibodies as a loading control (H). Data represent the relative  LC3B-II  levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (I) (n=3 experiments). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in , I,",
    "molecules": "bafilomycin A1, DMSO"
  },
  {
    "caption": "J, K MCF-7 cells expressing GFP-INPP4B  or GFP-vector were treated with 100 nM bafilomycin A1 or DMSO  as a vehicle control for 4 hours, then fixed and immunostained  with p62  antibodies, and co-stained with DAPI  and phalloidin  (J). Data represent the number of p62+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (K). Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in J. p values determined by one-way ANOVA with Tukey post hoc test in K,",
    "molecules": "bafilomycin A1, DAPI, DMSO, phalloidin"
  },
  {
    "caption": "L, M MCF-7 cells expressing GFP-INPP4B  or GFP-vector were treated with 2 µM BYL719  or DMSO  as a vehicle control for the indicated times. Cells were lysed and immunblotted  with LC3B, pAKTS473, AKT(pan), pS6KT389 or S6K  antibodies and GAPDH  antibodies as a loading control (L). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (M). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in M,",
    "molecules": "BYL719, DMSO"
  },
  {
    "caption": "N, O MCF-7 cells expressing GFP-INPP4B  or GFP-vector were treated with 1 µM or 10 µM SAR405  or DMSO  as a vehicle control for 4 hours. Cells were lysed and immunblotted  with LC3B  antibodies and GAPDH antibodies as a loading control (N). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (O). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA with Tukey post hoc test in O,",
    "molecules": "DMSO, SAR405"
  },
  {
    "caption": "A, B MCF-7 cells expressing NT, INPP4B  #1 or INPP4B  #2 shRNA were fixed and immunostained  with recombinant GST-2xFYVE and LAMP2  antibodies, and  co-stained  with DAPI  (A). Data represent the proportion of 2xFYVE+ lysosomes  (n=3 experiments, >40 cells/experiment) (B). Arrows indicate  co-localization  between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by one-way ANOVA with Tukey post hoc test in B",
    "molecules": "DAPI"
  },
  {
    "caption": "C MCF-7 cells were treated with 0.02% (w/v) saponin  for 30 seconds, then fixed and immunostained  with INPP4B  and either LAMP1  or CD63  antibodies, and co-stained with DAPI. Arrows indicate co-localization  between INPP4B  and CD63. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C,",
    "molecules": "DAPI, saponin"
  },
  {
    "caption": "D, E MCF-7  cells co-expressing GFP-INPP4B  or GFP-vector, and Hrs  or NT shRNA, were fixed and immunostained  with recombinant GST-2xFYVE and LAMP2  antibodies, and co-stained  with DAPI  (D). Data represent the proportion of 2xFYVE+ lysosomes  (n=3 experiments, >30 cells/experiment) (E). Arrows indicate  co-localization  between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D, p values determined by one-way ANOVA with Tukey post hoc test in",
    "molecules": "DAPI"
  },
  {
    "caption": "F, G  MCF-7 cells co-expressing GFP-INPP4B  or GFP-vector, and Hrs  or NT shRNA, were fixed and immunostained  with recombinant GST-2xFYVE   and CD63  antibodies, and co-stained  with DAPI  (F). Data represent the proportion of 2xFYVE+ late endosomes (n=3 experiments, >30 cells/experiment) (G). Arrows indicate co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in F. p values determined by one-way ANOVA with Tukey post hoc test in G,",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B MCF-7  cells expressing GFP-INPP4B  or GFP-vector were fixed and immunostained  with LAMP1  or LAMP2  antibodies, and co-stained with DAPI  and phalloidin  (A). Data represent the number of LAMP1+ or LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (B). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in A, p values determined by two-tailed unpaired t test in B,",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "C, D Snapshots of  Magic Red cathepsin B   substrate and Hoechst 33342  captured in live MCF-7 cells expressing GFP-INPP4B  or GFP-vector (C). Data represent the number of  Magic Red cathepsin B+ puncta per cell (n=3 experiments, >50 cells/experiment) (D). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in C, p values determined by two-tailed unpaired t test in D,",
    "molecules": "Magic Red, Hoechst 33342"
  },
  {
    "caption": "E, F MCF-7 cells expressing NT or INPP4B  #1 shRNA were cultured in growth media  or EBSS  for the indicated times, then fixed and immunostained  with LAMP1  antibodies and co-stained  with DAPI  and phalloidin  (E). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >30 cells per experiment) (F). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in p values determined by one-way ANOVA with Tukey post hoc test in F",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "G, H  MCF-7  cells expressing GFP-INPP4B  or GFP-vector were transfected with NT, PIK3CA  #1 or PIK3CA  #2 siRNA. After 24 hours, cells were fixed and immunostained  with LAMP1  antibodies, and co-stained with DAPI  and phalloidin  (G). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (H). Data information: Data is presented as mean ± SD. Scale bar is 10 µm in G. p values determined by one-way ANOVA in H.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "A, B Snapshots of LAMP1-GFP, Magic Red cathepsin B  substrate and  Hoechst 33342 captured in live HeLa  cells transfected with INPP4B  or NT siRNA (A). Data represent the number of LAMP1+/Magic Red Cathepsin B+ and LAMP1+/Magic Red Cathepsin B− lysosomes   (terminal storage lysosomes) per cell (n=3 experiments, >50 cells/experiment) (B).Yellow arrows indicate LAMP1+/MR Cathepsin B+ lysosomes, and white arrows indicate terminal storage lysosomes. Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by two-way ANOVA with Holm-Sidak post-hoc test in B",
    "molecules": "Magic Red, Hoechst 33342"
  },
  {
    "caption": "A, B  MCF-7 cells expressing GFP-INPP4B  or GFP-vector were treated with 5 µM YM201636  for 2 hours. Cells were fixed and immunostained  with recombinant GST-2xFYVE and LAMP2  antibodies, and co-stained with DAPI  (A). Data represent the proportion of 2xFYVE+ lysosomes  (n=3 experiments, >30 cells/experiment) (B). Arrows show co-localization between 2xFYVE and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A p values determined by one-way ANOVA in B",
    "molecules": "YM201636, DAPI"
  },
  {
    "caption": "D-F  MCF-7  cells expressing GFP-INPP4B  or GFP-vector were treated with 5 µM YM201636  or DMSO  as a vehicle control for 2 hours, then YM201636  was washed out for 1, 2 or 4 hours. Cells were fixed and immunostained  with LAMP1  antibodies, and co-stained  with DAPI  and phalloidin  (D). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (E), and the lysosome  fold change relative to the YM201636-treated condition for each cell line (F) (n=3 experiments, >30 cells per experiment). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D. p values determined by one-way ANOVA in E, or by two-tailed unpaired t test of the area under the curve in F.",
    "molecules": "YM201636, DAPI, DMSO, phalloidin"
  },
  {
    "caption": "G, H MCF-7 cells expressing GFP-INPP4B  or GFP-vector were treated with 1, 2 or 5 µM YM201636  or DMSO  as a vehicle control for 4 hours. Cells were lysed and immunoblotted  with LC3B  antibodies, and GAPDH  antibodies as a loading control (G). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (H). Data information: Data is presented as mean ± SD. p values determined by one-way ANOVA in H,",
    "molecules": "YM201636, DMSO"
  },
  {
    "caption": "A, B MCF-7  cells expressing GFP-INPP4B  or GFP-vector were transfected with NT, SNX1  or SNX2  siRNA. After 24 hours, cells were fixed and immunostained  with LAMP2  antibodies, and  co-stained with DAPI  and phalloidin  (A). Data represent the number of LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (B). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, p values determined by one-way ANOVA in with Tukey post hoc test in B",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "D, E MCF-7  cells expressing GFP-INPP4B  or GFP-vector were treated with 5 µM YM201636  for 2 hours, then YM201636  was washed out for 1 hour. Cells were fixed and immunostained  with SNX2  and LAMP1  antibodies, and co-stained with DAPI  (D). Data represent the proportion of SNX2+ LAMP2  puncta (n=3 experiments, >40 cells/experiment) (E). Arrows show  co-localization between SNX2  and LAMP2. Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in D, p values determined by one-way ANOVA in with Tukey post hoc test in E,",
    "molecules": "YM201636, DAPI"
  },
  {
    "caption": "F HeLa  cells were transfected with GFP-SNX2. After 24 hours, cells were treated with 5 µM YM201636  for 2 hours, then YM201636  was washed out for 1 hour. Cells were fixed and subjected to  immuno-electron microscopy  analysis using GFP  (10 nm) antibodies. Arrows show GFP-SNX2 localization  to lysosome. Data information: Scale bar is 100 nm in F.",
    "molecules": "YM201636"
  },
  {
    "caption": "G HeLa  cells were transfected with GFP-PIKfyve. After 24 hours, cells were were treated with 5 µM YM201636  for 2 hours, then YM201636  was washed out for 1 hour. Cells were fixed and immunostained  with SNX2, GFP  and LAMP2  antibodies, and imaged using super resolution microscopy. Arrows show  co-localization  between GFP-PIKfyve, SNX2  and LAMP2. Data information: The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in",
    "molecules": "YM201636"
  },
  {
    "caption": "A, B HeLa   cells transfected with INPP4B  or NT siRNA were treated for 1 hour with 5 µg/mL puromycin. Cells were fixed and immunostained  with ubiquitin  antibodies, and co-stained  with DAPI  and phalloidin (A). Data represent the percentage of cells with ubiquitinated  protein aggregates (n=3 experiments, >200 cells/experiment) (B). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A p values determined by one-way ANOVA in with Tukey post hoc test in B",
    "molecules": "DAPI, phalloidin, puromycin, ubiquitin"
  },
  {
    "caption": "C, D HeLa  cells were treated with 5 µM YM201636  or DMSO  as a vehicle control for 2 hours, then 5 µg/mL puromycin  for 1 hour. Cells were fixed and immunostained  with ubiquitin  antibodies, and co-stained  with DAPI  and phalloidin  (C). Data represent the percentage of cells with ubiquitinated  protein aggregates (n=3 experiments, >200 cells/experiment) (D). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in C, p values determined by one-way ANOVA in with Tukey post hoc test in D,",
    "molecules": "YM201636, DAPI, DMSO, phalloidin, puromycin, ubiquitin"
  },
  {
    "caption": "E, F MCF-7  cells expressing GFP-INPP4B  or GFP-vector were treated for 4 hours with 10 µg/mL puromycin, 5 µm MG132  and either 5 µM YM201636  or DMSO  as a vehicle control. Cells were fixed and immunostained  with ubiquitin  antibodies, and co-stained with DAPI  and phalloidin  (E). Data represent the percentage of cells with ubiquitinated  protein aggregates (n=3 experiments, >200 cells/experiment) (F). Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in E. p values determined by one-way ANOVA in with Tukey post hoc test in",
    "molecules": "YM201636, DAPI, DMSO, MG132, phalloidin, puromycin, ubiquitin"
  },
  {
    "caption": "G HeLa  cells transfected with INPP4B  or NT siRNA were treated with 10 µg/mL puromycin  for 2-6 hours, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative  cell viability normalized to untreated NT siRNA cells (n=3 experiments). Data information: p values determined by two-tailed unpaired t test of the area under the curve in G",
    "molecules": "puromycin"
  },
  {
    "caption": "H HeLa  cells were treated 10 µg/mL puromycin  ± 5 µM YM201636  or DMSO  as a vehicle control for 2-6 hours, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to DMSO-treated cells (n=3 experiments). Data information: p values determined by one-way ANOVA with Tukey post hoc test of the area under the curve in H.",
    "molecules": "YM201636, DMSO, puromycin"
  },
  {
    "caption": "f, Confocal microscopy images of humanlung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "A, B. Aggregate number of detected and undetected cysteines (A) and lysines  (B) in 3,840 CpDAA-containing proteins.",
    "molecules": "CpDAA, cysteines, lysines"
  },
  {
    "caption": "E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.",
    "molecules": "IETD-AFC"
  },
  {
    "caption": "E Quantification of Q35::YFP foci in WT and hsp-110 hairpin background. Shown is the mean ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01.",
    "molecules": "Q35"
  },
  {
    "caption": "A Maximum intensity projections of confocal z-stacks of 4-day-old animals expressing the indicated transgenes or harboring the indicated temperature-sensitive (ts) mutations. Muscle cells were stained with anti-myosin (green) and Alexa Flour 647-phalloidin (purple). Scale bar: 5 µm. Myosin misfolding reflects the exposure of the ts mutant phenotype of myosin(ts) [unc-54(e1301)] at the permissive temperate of 15°C by co-expression of the hsp-110 hairpins.",
    "molecules": "Alexa Flour 647"
  },
  {
    "caption": "A Representative electron micrographs of HeLa cells treated or not with 250 µM LLOMe for 5 min or 10 min before chemical fixation. Cells were incubated for 4 h following washing steps and overnight incubation with 15 nm gold nanoparticles conjugated to bovine serum albumin to visualize lysosomes (black spots inside lysosomes are internalized gold nanoparticles) before treatment with LLOMe and processing of samples for electron microscopy. Arrows indicate areas with increased lysosome-ER contacts. B Quantification of electron micrographs showing an increase in membrane contact sites between ER and lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes. Error bars denote +/- SD from n=3 independent experiments, >48 lysosomes were quantified per experiment for each condition.",
    "molecules": "gold, LLOMe, nanoparticles"
  },
  {
    "caption": "C Representative electron micrographs of VAP double knockout HeLa cells that were either left untreated or incubated with 250 µM LLOMe for 5 min or 10 min before chemical fixation. Cells were incubated with 15 nm gold nanoparticles conjugated to bovine serum albumin for 4 h to visualize lysosomes. After 4 h, gold nanoparticles were washed off and cells were incubated overnight before treatment with LLOMe and processing samples for electron microscopy (same as in A). Quantification of electron micrographs showing no increase in membrane contact sites between ER and lysosomes following 250 µM LLOMe treatment for 5 or 10 minutes in VAP double knockout cells. Error bars denote +/- SD from n=3 independent experiments, >40 lysosomes were quantified per experiment for each condition.",
    "molecules": "gold, LLOMe, nanoparticles"
  },
  {
    "caption": "D Representative movie stills of a live-cell imaging experiment illustrating the recruitment of transiently expressed eGFP-ORP1L to lysosomes following 250 µM LLOMe treatment. Cells were pre-treated for 30 min with 75 nM Lysotracker Deep Red to monitor lysosome damage (judged by the decreased number of Lysotracker spots after LLOMe treatment). The graph represents quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment.",
    "molecules": "Lysotracker, Lysotracker Deep Red, LLOMe"
  },
  {
    "caption": "A Movie stills from a live-cell imaging experiment showing rapid 2xSidM-eGFP recruitment to damaged lysosomes upon 250 µM LLOMe treatment (indicated in green). Lysosomes were visualized using transiently overexpressed LAMP1-mCherry (indicated in red). Yellow spots show colocalized PtdIns4P probe (2xSidM-eGFP) and LAMP1-mCherry after 3 and 10 min of LLOMe treatment. The graph shows the quantification of 2xSidM-eGFP foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >20 cells were analyzed per experiment.",
    "molecules": "LLOMe, PtdIns4P"
  },
  {
    "caption": "B HeLa cells were transfected with control siRNA or siRNA targeting PI4K2A or PI4K2B for 48 h and transiently transfected with the 2xSidM-eGFP probe to monitor PtdIns4P recruitment after LLOMe-treatment by live-cell imaging. Representative movie stills show the dynamics of 2xSidM-eGFP recruitment in control, PI4K2A and PI4K2B depleted cells. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SEM from n=3 independent live-cell imaging experiments, >25 cells were analyzed per experiment for each condition.",
    "molecules": "LLOMe, PtdIns4P"
  },
  {
    "caption": "C CRISPR-Cas9 mediated knockout of PI4K2A abolishes recruitment of the 2xSidM-eGFP probe (PtdIns4P) to damaged lysosomes. Two different clones (clone 2 and clone 3) were tested. The graph represents the quantification of 2xSidM-eGFP foci per cell after LLOMe-treatment. Error bars denote +/- SD from n=2 independent live-cell imaging experiments, >20 cells were analyzed per experiment for each condition.",
    "molecules": "LLOMe, PtdIns4P"
  },
  {
    "caption": "D Representative movie stills from live fluorescence microscopy showing reduced eGFP-ORP1L recruitment in PI4K2A knockout cells compared to wild type HeLa cells upon treatment with 250 µM LLOMe. The graph represents the quantification of eGFP-ORP1L foci per cell after LLOMe treatment. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >80 cells were analyzed per experiment for each condition.",
    "molecules": "LLOMe"
  },
  {
    "caption": "Representative movie stills from live-cell imaging experiments monitoring Lysotracker recovery after induced lysosomal damage using 250 µM LLOMe. Cells were pre-treated with 75 nM Lysotracker Deep Red for 30 min before addition of LLOMe. The graph represents the quantification of bright Lysotracker positive foci per cell. While the decrease in the number of Lysotracker spots is quickly recovering in HeLa cells, there is a severe impairment in lysosomal repair in HeLa-PI4K2A-KO cells.",
    "molecules": "Lysotracker, Lysotracker Deep Red, LLOMe"
  },
  {
    "caption": "A HeLa cells were co-transfected with siRNAs targeting ESCRT proteins TSG101 and ALIX (siTSG101+siALIX) or with non-targeting siRNA control (siControl), and incubated for 24 h before transfection with the PtdIns4P binding probe 2xSidM-eGFP. 24 h post-transfection lysosomes were damaged with 250 µM LLOMe and PtdIns4P recruitment to damaged lysosomes was monitored using the 2xSidM-eGFP probe in live-cell imaging experiments. The graph shows quantification of 2xSidM-eGFP foci per cell in cells co-depleted of TSG101+ALIX and control (siControl) cells.",
    "molecules": "LLOMe, PtdIns4P"
  },
  {
    "caption": "A Representative movie stills of a live-cell imaging experiment illustrate faster recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes when compared to cholesterol as indicated by the mCherry-D4 probe. The graph shows quantification of 2xSidM-eGFP and mCherry-D4 foci per cell. Error bars denote +/- SD from n=2 independent live-cell imaging experiments",
    "molecules": "cholesterol, PtdIns4P"
  },
  {
    "caption": "D HeLa cells expressing an eGFP-tagged ORP1L mutant incapable of binding to VAP (mFFAT) show no accumulation of the cholesterol reporter mCherry-D4 upon lysosomal damage induced with 250 µM LLOMe when compared to wildtype (wt) eGFP-ORP1L. The quantification graph shows D4-mCherry foci per cell. Error bars denote +/- SEM from n=4 independent live-cell imaging experiments, >50 cells were analyzed per experiment for each condition.",
    "molecules": "cholesterol, LLOMe"
  },
  {
    "caption": "A Representative movie stills from live-cell imaging experiments indicate recruitment of CHMP4B-eGFP to damaged lysosomes in control and U18666A treated cells. HeLa cells stably expressing CHMP4B-eGFP were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of CHMP4B-eGFP foci per cell. Error bars denote +/- SEM from n=4, >30 cells per experiment for each condition.",
    "molecules": "U18666A, LLOMe"
  },
  {
    "caption": "B Representative immunofluorescence images illustrate Galectin-3 recruitment kinetics on the damaged lysosomes upon treatment with 2 µM U18666A for 17 h. HeLa cells were treated or not (Control) with U18666A and +/- LLOMe for the indicated time points, fixed and processed for immunofluorescence microscopy. Endogenous levels of Galectin-3 were visualized.",
    "molecules": "U18666A, LLOMe"
  },
  {
    "caption": "D Representative movie stills from live-cell imaging experiments indicate recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes in control but not in the U18666A treated cells. HeLa cells were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of 2xSidM-eGFP foci per cell.",
    "molecules": "U18666A, LLOMe, PtdIns4P"
  },
  {
    "caption": "A Representative movie stills of a live-cell imaging experiment show recruitment dynamics of transiently expressed eGFP-OSBP in HeLa cells treated with 250 µM LLOMe. The graph shows quantifications of OSBP foci per cell. Error bars denote +/- SEM from n=5 independent live-cell imaging experiments, >22 cells per experiment.",
    "molecules": "LLOMe"
  },
  {
    "caption": "Subcellular localizations of zRbm14a. The representative confocal micrographs (D) were acquired from the animal pole of zebrafish embryos. Exposures were kept the same during the imaging for comparison. DAPI labels nuclear DNA. Framed regions were magnified to show details. Arrows denote representative cytoplasmic puncta, whereas arrowheads point to perinuclear condensates.",
    "molecules": "DAPI"
  },
  {
    "caption": "Phenotypes of zRbm14 morphants. Totally 8 ng of ctrl-MO, 14-tMOs, or 14-MOs were co-injected with 100 pg of in-vitro transcribed GFP mRNA (as a tracer) into 1-cell embryos to generate control, maternal zRbm14, and zygotic zRbm14 morphants, respectively. Representative embryos, imaged under a dissecting microscope, are shown in (A)",
    "molecules": "MO, MOs"
  },
  {
    "caption": "Phenotypes of zRbm14 morphants. Totally 8 ng of ctrl-MO, 14-tMOs, or 14-MOs were co-injected with 100 pg of in-vitro transcribed GFP mRNA (as a tracer) into 1-cell embryos to generate control, maternal zRbm14, and zygotic zRbm14 morphants, respectively. phenotyping results in (B). Total numbers of embryos analyzed are listed over histograms. Sample dots are included for quantification results with no error bars.",
    "molecules": "MO, MOs"
  },
  {
    "caption": "Maternal zRbm14 morphants displayed reduced cell proliferation in MZT. Embryos stained with Hoechst 33342 and Pyronin Y to mark the nucleus and the cytoplasm, respectively, were imaged from the animal pole to cover a 10-μm z-depth with a confocal microscope (C). Maximum intensity-projected images (C)",
    "molecules": "Hoechst 33342, Pyronin Y"
  },
  {
    "caption": "D. Hexanediol (Hex) treatment from 2.5 hpf blocked the blastula development. Representative images are presented for each group of embryos, with phenotypes indicated by insets of colored boxes. Total embryo numbers analyzed are listed over histograms.",
    "molecules": "Hex, Hexanediol"
  },
  {
    "caption": "D. Expression patterns of four clusters of representative transcripts, categorized from RNA-seq results of ctrl-MO embryos. The transcripts in the maternal-early and the zygotic-early clusters respectively displayed continual degradation or accumulation from 2.5 hpf, whereas those in the maternal-late and zygotic-late clusters respectively started degradation or accumulation from 4 hpf.",
    "molecules": "MO"
  },
  {
    "caption": "EU incorporated into nascent transcripts were visualized through a click reaction. Hoechst 33342 was used to counterstain the nucleus. A representative maximum intensity-projected confocal micrograph, taken from the animal pole, is shown for each group of 10 embryos (I).",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "Depletion of zRbm14 resulted in accumulation of poly(A)-containing maternal mRNAs. Total RNAs purified from the indicated zebrafish embryos were subjected to poly(A) length assays. One set of representative PCR results for org and trip10a (B) As the PCR products of the poly(A)-transcripts mainly emerged as a single band, only intensities of this major band were measured.",
    "molecules": "poly(A)"
  },
  {
    "caption": "Depletion of zRbm14 resulted in accumulation of poly(A)-containing maternal mRNAs. Total RNAs purified from the indicated zebrafish embryos were subjected to poly(A) length assays. quantification results from three independent experiments (C) are presented. As the PCR products of the poly(A)-transcripts mainly emerged as a single band, only intensities of this major band were measured.",
    "molecules": "poly(A)"
  },
  {
    "caption": "G, H. zParn was highly expressed from 4-cell to 32-cell stages and enriched in zRbm14 condensates. Immunoblotting (G) and immunostaining (H) were performed using a rabbit anti-zebrafish Parn antibody. Lysates from 5 embryos were loaded in each lane in (G). α-tubulin served as internal controls. Representative single optical sections are presented in (H). Chromatin DNA was stained with DAPI. Arrows point to typical regions abundant in condensates.",
    "molecules": "DAPI"
  },
  {
    "caption": "J. zRbm14b and zParn efficiently co-phase separated in deadenylation experiments. The addition of SDS disrupted the condensates to terminate the deadenylation reaction.",
    "molecules": "SDS"
  },
  {
    "caption": "Co-phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn. RNAs extracted from the indicated samples were subjected to immuno-northern blotting using an antibody against m6A (K). were quantified from five sets of independent results.",
    "molecules": "m6A"
  },
  {
    "caption": "Co-phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn. Relatively proportions of Cap-10m6A-A0 in total RNAs (L) were quantified from five sets of independent results.",
    "molecules": "m6A"
  },
  {
    "caption": "Severe down-regulation of Gata6 and Nanog in Rbm14-depleted mouse blastocysts. Representative immunofluorescent images (H) were from single option sections. DAPI stained nuclear DNA. As Gata6, Nanog, and Rbm14 localize to the nucleus in blastocysts",
    "molecules": "DAPI"
  },
  {
    "caption": "Scatter plots representing the association of CSF PGRN with temporo-parietal FDG-PET uptake within the subjects of the Alzheimer's continuum group (n = 474). Abbreviations: FDG-PET: Fludeoxyglucose positron emission tomography",
    "molecules": "FDG, Fludeoxyglucose"
  },
  {
    "caption": "Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in non-carriers (NC, blue; A, C, E and G) and in mutation carriers (MC, red; B, D, F and H). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181.",
    "molecules": "Threonine"
  },
  {
    "caption": "Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in healthy controls (blue; A, D, G and J), Alzheimer's continuum (red; B, E, H and K) and 'suspected non-Alzheimer's pathophysiology (SNAP)' groups (green; C, F, I and L). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD, the analysis including these outliers yielded similar results (Appendix Table S8). The Aβ1-42 values used for the associations test are those based on an extrapolation curve since the upper technical limit is 1700pg/ml. We also tested the associations with Aβ1-42 values truncated at the upper technical limit and the result was similar. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181; SNAP: suspected non-Alzheimer's pathophysiology.",
    "molecules": "Threonine"
  },
  {
    "caption": "Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. Proliferation of ECs were measured by flow cytometry Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups",
    "molecules": "O2"
  },
  {
    "caption": "Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. migration of ECs were measured by wound healing assay Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups",
    "molecules": "O2"
  },
  {
    "caption": "Tg and WT rats were injected with saline or MCT. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Data information: Values are expressed as mean ± SEM.",
    "molecules": "MCT"
  },
  {
    "caption": "Tg and WT rats were injected with saline or MCT. Levels of miR-483-3p/-5p in serum and lung tissues were measured by qPCR (F). Data information: Values are expressed as mean ± SEM.",
    "molecules": "MCT"
  },
  {
    "caption": "Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated miRNAs (G) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. ",
    "molecules": "MCT"
  },
  {
    "caption": "Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. ",
    "molecules": "MCT"
  },
  {
    "caption": "EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of mPAP for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).",
    "molecules": "MCT"
  },
  {
    "caption": "EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).",
    "molecules": "MCT"
  },
  {
    "caption": "Vascular integrity evaluated by intravenous injection of Evans blue dye (D). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 0.5 cm (D).",
    "molecules": "Evans blue dye"
  },
  {
    "caption": "EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).",
    "molecules": "DMSO, SU5416"
  },
  {
    "caption": "EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of RV hypertrophy [RV/(LV+S)] (F) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).",
    "molecules": "DMSO, SU5416"
  },
  {
    "caption": "qPCR analysis of miR-483-3p/-5p in serum from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups",
    "molecules": "MCT"
  },
  {
    "caption": "qPCR analysis of miR-483-3p/-5p in lung tissue from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups",
    "molecules": "MCT"
  },
  {
    "caption": "(G)  U2OS (left panel, n=6 biological replicates) or HeLa cells (right panel, n = 3 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Flow cytometry analysis of  phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal. Data are represented as a bar graph showing the mean ± SEM, each replicate being represented by a round symbol. Significance was determined by two-way ANOVA followed by a Holm-Sidak's test. *P<0.05, **P<0.01, ***P<0.0005.",
    "molecules": "phleomycin"
  },
  {
    "caption": "(H) U2OS (n=8 biological replicates) were transfected with the indicated siRNA; 48 hours post-transfection, they were treated with the radiomimetic antibiotic, phleomycin (50 μg/mL), and collected at the indicated time points. Data are represented as a bar graph showing the mean ± SEM, each replicate being representing as a round symbol. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05.",
    "molecules": "phleomycin"
  },
  {
    "caption": "(A) U2OS  (left panel), RPE1-hTERT (middle panel), and  HeLa  cells (right panel) were monitored for their sensitivity to the radiomimetic drug NCS using the SRB assay. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ  (siPOGZ-1 or -2); RPE1-hTERT cells were transduced a control shRNA (shCtrl) or a shRNA directed against  human POGZ (shPOGZ-1 or -2); HeLa cells expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were pulsed with NCS at the indicated concentrations for 1 hour, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round symbol (3 biological replicates). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.01.",
    "molecules": "NCS"
  },
  {
    "caption": "(F) Quantification of γ-H2AX  foci in  HeLa  cells where POGZ has been targeted by  CRISPR  technology (POGZ∆-1 or -2) and in control HeLa  cells (sgCtrl). Cells were exposed to 1 Gy before being pulsed with  Edu for 1hr and were recovered at the indicated time points. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each  time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0005. (G) Representative images used for quantification in (F). Scale bar = 5 μm. ",
    "molecules": "Edu, EdU"
  },
  {
    "caption": "(A) U2OS stably expressing mCherry-LacR-FokI were transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield1 and 4-OHT. Immunofluorescence  against the indicated DNA repair proteins was subsequently performed to monitor their accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear  background. At least 100 cells per condition were counted (n=3 biological replicates). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.005, **P≤0.0005.",
    "molecules": "4-OHT, Shield1"
  },
  {
    "caption": "(B)  U2OS  (left panel), RPE1-hTERT (middle panel), and HeLa cells (right panel) were monitored for their capacity to form IR-induced BRCA1 foci. For each cell line, the following conditions were used: U2OS cells were transfected with a nontargeting siRNA (siCtrl) or an siRNA targeting human POGZ (siPOGZ-1 or -2); RPE1-hTERT  cells were transduced a control shRNA (shCtrl) or a shRNA directed against  human POGZ (shPOGZ-1 or -2); HeLa cells were expressing a non-targeting sgRNA (sgCtrl) or a sgRNA targeting human POGZ and sub-cloned (POGZ∆-1 or -2). Cells were exposed to 1 Gy before being pulsed with  Edu for 1hr and were recovered 1h post-exposure to IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.001.",
    "molecules": "Edu, EdU"
  },
  {
    "caption": "(E) U2OS stably expressing  mCherry-LacR-FokI were transduced with the indicated shRNA and subsequently transfected with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1 and  4-OHT. Immunofluorescence against BRCA1 was subsequently performed to monitor its accumulation at sites of DNA damage by confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear  background. At least 50 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.0001.",
    "molecules": "4-OHT, Shield-1"
  },
  {
    "caption": "(G)  U2OS stably expressing mCherry-LacR-FokI were transduced with the indicated siRNA. 24h post-transfection, DNA damage was induced using Shield-1  and 4-OHT. Immunofluorescence  against the indicated  HP1 isoform was subsequently performed to monitor its respective accumulation at sites of DNA damage by  confocal microscopy. Data are represented as a box-and-whisker (10-90 percentile) where the fluorescent signal at the mCherry focus was normalized to nuclear  background. At least 75 cells per condition were counted. Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.005, **P<0.0001.",
    "molecules": "4-OHT, Shield-1"
  },
  {
    "caption": "(C)  HeLa cells transfected with the indicated construct were monitored for their capacity to form  IR-induced BRCA1 foci. HeLa cells where POGZ has been targeted by  CRISPR technology (POGZ∆-1) and in control HeLa cells (sgCtrl) were transfected by an empty Flag vector (EV) or a sgRNA-resistant FLAG-tagged POGZ  cDNA construct corresponding to indicated rescue mutant (full-length, FL; POGZ801-848, HPZ). Cells were exposed to 1 Gy before being pulsed with  Edu for 1hr and were recovered 1h post-exposure to  IR. Cells were fixed, stained, and imaged via confocal microscopy. Data are the total number of ΒRCA1 foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05, **P<0.0001. (D) Representative images used for quantification in (C). Scale bar = 5 μm. ",
    "molecules": "Edu, EdU"
  },
  {
    "caption": "(E) Similar as in (C) except that  γ-H2AX foci were monitored at the indicated  time points  by confocal microscopy. Data are the total number of γ-H2AX foci in EdU+ cells and represented as a bar graph showing the mean ± SD (n = 3 biological replicates, with at least 100 cells analyzed for each  time point). Significance was determined by two-way ANOVA followed by a Dunnett's test. *P<0.05. (F) Representative images used for quantification in (E). Scale bar = 5 μm. ",
    "molecules": "EdU"
  },
  {
    "caption": "(D) MEFs  were monitored for their sensitivity to the radiomimetic drug phleomycin using the SRB assay. Immortalized MEFS obtained from the indicated genotype were treated with increasing concentrations of phleomycin for 1hr, replenished with fresh medium and incubated for 4 days before being processed for SRB assays. Data are represented as a bar graph showing the relative mean ± SEM, each replicate being representing as a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005, **P<0.0005.",
    "molecules": "phleomycin"
  },
  {
    "caption": "TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). (C) DNA incorporation of EdU was detected by flow cytometry. The percentage of proliferating cells is indicated (n=4, mean±SEM; ***p<0.0001 versus scr-shRNA ECs by Student's t-test).",
    "molecules": "EdU"
  },
  {
    "caption": "TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). DNA content was determined by propidium iodide staining and assessed by fluorescences-activated cell sorter analysis (representative experiment out of 4 with similar results).",
    "molecules": "DNA"
  },
  {
    "caption": "TFEB silencing reduced human EC morphogenesis. Representative images of tube-like-structure of scr-shRNA and sh-TFEB human ECs stained with phalloidin-555. Bar graph indicates the percentage of phalloidin+ area in sh-TFEB and scr-shRNA ECs (scale bars: 0.25 mm; n=6, mean±SEM; ***p<0.0001 versus scr-shRNA by Student's t-test).",
    "molecules": "phalloidin, phalloidin-555"
  },
  {
    "caption": "Silenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction. Representative immunoblot of PM biotinylated portion and total cell lysates of scr-shRNA and sh-TFEB ECs after VEGF-A stimulation (30 ng/ml). Blots of total or PM cell lysates were probed with anti-VEGFR2. Blots of total cell lysates were probed with anti-p-Y1175-VEGFR2, anti-PLCγ, p-PLCγ, anti-ERK-1/2, anti-pERK1/2, anti-p-Src, anti-Src, anti CD31 and α-tubulin Abs. The bar graphs (i,ii) show densitometric analysis of stimulated versus unstimulated scr-shRNA and sh-TFEB ECs expressed as: (i) % of VEGFR2 on PM fraction (n=3, mean±SEM; ANOVA p<0.02; **p<0.001 versus scr-shRNA by Bonferroni post-test), (ii) % of VEGFR2 total",
    "molecules": "biotinylated"
  },
  {
    "caption": "MYO1C silencing reverses the effect of TFEB silencing on the up-regulation of PM VEGFR2. Analyses were performed on human ECs carrying appropriate scr-shRNA or sh-TFEB in the presence or absence of sh-MYO1C. Representative western blot of MYOC1, total and PM biotinylated VEGFR2 (representative experiment out of 4 with similar results).",
    "molecules": "biotinylated"
  },
  {
    "caption": "B-D. Mice were analyzed by Kaplan-Meier survival plot (B), the percentage of body weight (C), and the clinical scores (D) of WT and GPx8-/- mice with colitis induced by 4% DSS for 6 days (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS"
  },
  {
    "caption": "Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (E) Images and statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS"
  },
  {
    "caption": "Colons treated with 4% DSS for 5 days were collected on day 14. , 5-6 mice were used for each experiment. (F) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections. The infiltrated immune cells at the transmural extensions were indicated by arrowhead. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS, eosin, hematoxylin"
  },
  {
    "caption": "Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (G) Colon sections were stained with markers for macrophages (F4/80) and anti-GPx8. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS"
  },
  {
    "caption": "Colons treated with 4% DSS for 5 days were collected on day 14. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS"
  },
  {
    "caption": "B. Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages (F4/80).",
    "molecules": "liposomes, clodronate"
  },
  {
    "caption": "After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). (C) The percentage of body weight. (D) The clinical scores of mice. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).",
    "molecules": "DSS"
  },
  {
    "caption": "After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (E) Statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).",
    "molecules": "DSS"
  },
  {
    "caption": "After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (F) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).",
    "molecules": "DSS, eosin, hematoxylin"
  },
  {
    "caption": "After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (G) Colon sections stained with anti-F4/80. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).",
    "molecules": "DSS"
  },
  {
    "caption": "After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). D) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test). ",
    "molecules": "DSS"
  },
  {
    "caption": "A. Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency. BMDMs were primed for 6 h with 0.5 μg/ml LPS and then transfected with LPS (TF LPS) for 8 h, stimulated with ATP (5 mM) for 3 h, MSU (150 μg/mL) for 8 h, or nigericin (5 μM) for 3 h. Activation of inflammasome was demonstrated by secreted IL-1β. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "ATP, LPS, MSU, nigericin"
  },
  {
    "caption": "B. GPx8 deficiency enhanced noncanonical inflammasome activation. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "LPS"
  },
  {
    "caption": "C. Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs. BMDMs were primed for 6 h with 1 μg/ml poly(I:C) and then transfected with LPS for 8 h. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "LPS, poly(I:C)"
  },
  {
    "caption": "G. RNA expression of caspase-11 was not altered by GPx8 deficiency. Caspase-11 RNA expression of BMDMs isolated from GPx8-/- mice was measured after LPS priming. Data information: ata are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.",
    "molecules": "LPS"
  },
  {
    "caption": "H. GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo. Colon samples from 5 WT or GPx8-/- mice treated with DSS for 5 days were collected on day 7. Levels of full-length and processed caspase-1, -11 and gasdermin D (GSDMD) in tissue lysates were individually analyzed by immunoblots.",
    "molecules": "DSS"
  },
  {
    "caption": "GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (I) Survival of mice primed with ultra pure LPS (E. coli O55:B5, 400 μg/kg) and re-challenged 6 h later with LPS (40 mg/kg) (n = 8 per group).",
    "molecules": "LPS"
  },
  {
    "caption": "GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (J) Survival of mice primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (10 mg/kg) (n = 9 per group).",
    "molecules": "LPS, poly(I:C)"
  },
  {
    "caption": "GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (K) IL-1β production levels in mice sera were measured 2 h after being primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (1 mg/kg) (n = 8-9 per group). In (K), data are presented as the mean ± SEM. NS, no significance. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "LPS, poly(I:C)"
  },
  {
    "caption": "C. Interaction between GPx8 and caspase-4 in THP-1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay (PLA) (red dots, labeled by arrowheads). GPx8-Myc stably expressing THP-1 were used to detect the interaction between GPx8-Myc and endogenous caspase-4. Untreated cells (Ctrl) or cells primed with LPS (primed LPS) were incubated with anti-Myc and anti-caspase-4 Abs, according to the manufacturer's instructions, and the nucleus was stained with DAPI (blue). Results were quantified by counting at least 5 different fields with an average of 300 cells. Reactions without primary Abs were used as the control for the PLA assay (PLA Ctrl). Data information: All data are representative of at least 3 independent experiments. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DAPI, LPS"
  },
  {
    "caption": "293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (D) Complexes of GPx8 and caspase-4 were demonstrated by co-IP assays. Cysteine-dependent covalent interactions of GPx8 and caspase-4 (C258S) were analyzed under non-reducing and reducing conditions. Disulfide-linked complexes of Gpx8 and caspase-4 are indicated.",
    "molecules": "Cysteine"
  },
  {
    "caption": "293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (E) Interactions of GPx8 and caspase-4 in response to H2O2 treatment were demonstrated by co-IP assays of lysates prepared from 293T cells treated with H2O2 (200 μM).",
    "molecules": "H2O2"
  },
  {
    "caption": "C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. (H and I) Cells were treated with 12-O-tetradecanoylphorbol 13-actate (PMA) (0.1 μM) and differentiated for 3 days then primed and transfected with LPS. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (H) and LDH release (I). Data information: All data are representative of at least 3 independent experiments. In H and I, data are presented as the mean ± SD, n=4, technical repeats. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "LPS, 12-O-tetradecanoylphorbol 13-actate, PMA"
  },
  {
    "caption": "J. E. coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase-4 (C258S) to form a disulfide-linked complex (oxidized GPx8: oGPx8; reduced GPx8: rGPx8). Lanes 2 and 3, GPx8 pretreated with 1 and 5 mM H2O2, respectively. Lanes 4 and 5, Gpx8 pretreated with 50 and 200 mM DTT, respectively. Disulfide-linked complexes of recombinant Gpx8 and caspase-4 (C258S) are indicated.",
    "molecules": "DTT, H2O2"
  },
  {
    "caption": "K. GPx8 inhibited the activation of caspase-4 by decreasing the oligomerization of caspase-4. Oxidized GPx8 and caspase-4 (C258S) were co-incubated for 30 min. Different amounts of LPS were added to the complex and incubated for another 30 min. Oligomerized caspase-4 was analyzed by native blue PAGE according to the manufacturer's instructions. Mock: without GPx8; oxidized GPx8: oGPx8; oxidized GPx8C79S: oC79S. Data information: All data are representative of at least 3 independent experiments. In K, quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers/total proteins. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "LPS, oligomers"
  },
  {
    "caption": "A. Casp4C118S mutant partially abolished the covalent disulfide-link with GPx8. Interacting GPx8 were co-immunoprecipitated with FLAG-tagged caspase-4 (WT, C118S, C329S mutants) and analyzed under reducing (+DTT) or non-reducing (-DTT) conditions. Disulfide-linked complexes of GPx8 and caspase-4 are indicated. Fold change of complexes was normalized with internal controls and indicated below blots.",
    "molecules": "DTT"
  },
  {
    "caption": "C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. (C-D) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS. THP-1 cells were differentiated, primed, and transfected with LPS. Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL-1β (C) and LDH (D). In C and D, data are presented as the mean ± SD, n=4, technical repeats. ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "LPS"
  },
  {
    "caption": "A. The caspase-4/11 and -1 inhibitor, VX-765, inhibited noncanonical inflammasome activation. BMDMs isolated from mice were primed with LPS for 5-6 h, treated with 20 μM VX-765, and transfected with LPS for 12 h. Data information data are presented as the mean ± SD, n=5, technical repeats. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "LPS, VX-765"
  },
  {
    "caption": "Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and analyzed by a Kaplan-Meier survival plot (B) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). Data information: , data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS, VX-765"
  },
  {
    "caption": "Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and the percentage of body weight (C) and clinical scores (D) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS, VX-765"
  },
  {
    "caption": "Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Full-length and cleavage forms of caspase-11, caspase-1, and GSDMD (E) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS, VX-765"
  },
  {
    "caption": "Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Production of IL-1β and IL-6 (F) in colon tissue lysates was assessed on day 14 after treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).",
    "molecules": "DSS, VX-765"
  },
  {
    "caption": "(b) PC12 cells transfected with either 60Q or 60Q-HSC70bm. The presence of two HSC70bm motifs in 60Q-HSC70bm reduced the number of inclusions as compared to 60Q (scale bar, 20 μm). (c) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q-HSC70bm. 10 mM 3-methyladenine (3-MA), 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy, the proteases, cathepsins D and E, and cathepsin A, respectively. Bars in c represent relative mean values ± s.e.m. from four independent experiments, with levels of aggregation observed for 60Q normalized to a value of 1. Quantifications were performed by ArrayScan.",
    "molecules": "3-methyladenine, leupeptin, pepstatin A"
  },
  {
    "caption": "(a) Western blot analysis of the chase experiment in 60Q Neuro2a cells. Full-length western blots are presented in Supplementary Figure 13. (b) RHQ enhanced the tNHTT-60Q-EGFP degradation during the chase period. 5 μM MG132, 10 mM 3-methyladenine and 15 mM ammonium chloride (NH4Cl) were used to inhibit proteasome, macroautophagy and lysosomal degradation, respectively. NH4Cl had no effect on proteasome activity and did not affect cell viability (Supplementary Fig. 4a,b). Bars in b represent relative mean values ± s.e.m. from three independent experiments. Protein levels at time 0 represent the control condition (arbitrarily set as 1).",
    "molecules": "3-methyladenine, ammonium chloride, NH4Cl, MG132"
  },
  {
    "caption": "(B) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and LAMP1-mCherry and treated with 1 mM LLOMe for the indicated time. Scale bar = 10 µM.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(B) Representative live-cell fluorescent images of HeLa cells co-transfected with the indicated EGFP-TECPR1 deletion mutant and LAMP1-mCherry before and after treatment with 1 mM LLOMe. Scale bars = 10 µM. To the right is the corresponding quantification of the fold change in EGFP-TECPR1 lysosomal fluorescence intensity after a 15-minute treatment with vehicle or 1 mM LLOMe. Grey points represent individual cells from three independent experiments.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(A,B) Representative live-cell fluorescent images of HeLa cells co-transfected with EGFP-TECPR1 and mCherry-Gal3 and treated with 1 mM LLOMe for the indicated time. Arrowheads in B indicate the appearance of TECPR1 (green arrow) and Gal3 (magenta arrow). Scale bars = 10 µM for A and 1 µM for B. (C) Quantification of TECPR1 and Gal3 recruitment to damaged lysosomes. Data are presented as mean ± SD from 5 independent experiments (each experiment represents a single cell with at least 3 individual lysosomes quantified).",
    "molecules": "LLOMe"
  },
  {
    "caption": "(D) Representative confocal image of a HeLa cell transfected with EGFP-TECPR1, treated with LLOMe for 5 minutes, and immunostained for CHMP2A and LAMP1. Scale bars = 10 µm for whole image and 1 µm for insets. Fluorescence intensity profiles of the indicated channels across the dotted lines are shown in the lower subpanel. (E) Representative confocal images of a HeLa cells transfected with EGFP-TECPR1 and TagBFP-TMEM192 (a lysosomal/late endosomal protein), treated with 1 mM LLOMe for the indicated time, and immunostained for ALIX and Gal3. Scale bars = 10 µm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(A) Western blot analysis of LC3 lipidation status in wild type (WT), FIP200 KO and ATG16L1 KO HeLa cells treated with the indicated concentrations of LLOMe for 30 minutes.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(B) Quantification of LC3-II levels in HEK, HeLa and MEF WT and ATG16L1-deficient cell lines treated with and without 1 mM LLOMe for 30 minutes. Bars show mean ± SD from three biologically independent experiments represented as data points. Significance was determined from biological replicates using a Student's t tests. *p = 0.0177, ** p = 0.0082, *** p = 0.0001.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(C) Western blot analysis of LC3 lipidation status in WT, ATG7 KO and ATG16L1 KO HeLa cells (top) and WT, ATG5-/- and ATG16L1Δ/Δ MEFs (bottom) treated with the indicated concentration of LLOMe for 30 minutes.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(F) Western blot analysis of lyso-IP samples collected from HeLa ATG16KO cells treated with or without 1 mM LLOMe for 30 minutes. Bars show mean ± SD from three biologically independent experiments represented as data points.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(G) Representative live-cell fluorescent images of HeLa cells co-transfected with TagBFP-TMEM192, EGFP-ATG5 and mCherry-TECPR1, before and after a 15-minute treatment with 1 mM LLOMe. Scale bars = 10 µm for whole images and 2 µm for zoom. To the right is the corresponding quantification of the fold change in ATG5/TECPR1 lysosomal fluorescence intensity after a 15-minute treatment with vehicle or 1 mM LLOMe. Grey points represent individual cells from three independent experiments. Green/magenta points represent the means of individual experiments (n > 25 cells per experiment).",
    "molecules": "LLOMe"
  },
  {
    "caption": "(H) Western blot analysis of LC3 lipidation status in HEK TECPR1/ATG16L1 KO cells treated with 1 mMLLOMe for 30 minutes. To the right is the corresponding quantification of LC3-II protein levels. Bars show mean ± SD from three or four biologically independent experiments which are represented as data points.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(A) Representative live-cell images of HEK cells treated as indicated and stained with LysoTracker Red. Nuclei were stained with Hoechst 33342. Scale bars = 20 µm. (B) Quantification of LysoTracker Red puncta from A. Grey points represent individual cells from three independent experiments. Red points represent the means of individual experiments (n > 100 cells per experiment).",
    "molecules": "Hoechst 33342, LysoTracker Red"
  },
  {
    "caption": "(C) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point.",
    "molecules": "CNP520"
  },
  {
    "caption": "(A) Subjective assessment of hair depigmentation (score 0-5, with 0 = completely black and 5 = completely white) at 2 doses of either CNP520 or the BACE-1/-2 inhibitor NB-360 (mean ± SEM, n=8/group), statistical comparisons were made for every scoring day, versus vehicle using Kruskal-Wallis with Dunn's post-hoc test.",
    "molecules": "CNP520, NB-360"
  },
  {
    "caption": "(B) Summary of doses, steady-state effects on Aβ40 in brain (% compared to vehicle-treated mice) and the ratios between total skin concentrations of CNP520 and NB-360 and their respective IC50 values for inhibition of BACE-2 in vitro.",
    "molecules": "CNP520, NB-360"
  },
  {
    "caption": "(B) Time-dependent reduction of Aβ40 in rat brain and CSF after a single 15.4 mg/kg (30 µmol/kg) oral dose of CNP520.",
    "molecules": "CNP520"
  },
  {
    "caption": "(C) CNP520 levels in dog blood and CSF, following a 3.1 mg/kg (6 µmol/kg) oral dose (mean ± SD = 4).",
    "molecules": "CNP520"
  },
  {
    "caption": "(D) Time-dependent reduction of Aβ40 and 42 in dog CSF, after a 3.1 mg/kg oral CNP520 dose (mean ± s.d.).",
    "molecules": "CNP520"
  },
  {
    "caption": "Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (A) CNP520 levels in blood and cerebellum.",
    "molecules": "CNP520"
  },
  {
    "caption": "Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (B) Total cortical plaque area (normalized to total sample area (*10−2), stained with antibody NT-12.",
    "molecules": "CNP520"
  },
  {
    "caption": "Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (C) Formic acid-soluble Aβ40 in forebrain. (D) Formic acid-soluble Aβ42 in forebrain.",
    "molecules": "CNP520, Formic acid"
  },
  {
    "caption": "Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (E) Double-immunofluorescence stained brain sections with NT12 (against amyloid-β, green) and Iba1 (against microglia, red) or GFAP (astrocytes, red) for the different treatment groups as indicated. Scale bar: 500 µm.",
    "molecules": "CNP520"
  },
  {
    "caption": "Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (F) Quantification of total Iba1 positive microglia (normalized to total area (*10−2), only a subset of samples analyzed.",
    "molecules": "CNP520"
  },
  {
    "caption": "Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (G) Quantification of total GFAP positive astrocyte area (normalized to total area (*10−2). Data were analyzed with Dunnett's multiple comparison test.",
    "molecules": "CNP520"
  },
  {
    "caption": "(C) Time vs CSF concentration profiles after single dose of CNP520 (Mean ± SEM, n = 6-8).",
    "molecules": "CNP520"
  },
  {
    "caption": "(A) Changes in Aβ40 relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).",
    "molecules": "CNP520"
  },
  {
    "caption": "(B) Changes in sAPPβ relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).",
    "molecules": "CNP520"
  },
  {
    "caption": "(C) Changes in sAPPα relative to baseline values. CSF was sampled via a lumbar catheter at time points from 2 hours pre- to 34 hours post-administration of a single dose of CNP520 (mean ± SEM, n = 6-8).",
    "molecules": "CNP520"
  },
  {
    "caption": "(D) Dose response of Aβ40 (mean ± SD) to CNP520 relative to baseline Aβ40 levels after 2 weeks of dosing. (D) CNP520 was administered daily for 14 days; CSF was sampled via a lumbar catheter 2 hours before dosing, and 24 hours after the last dose (mean ± SD, n = 8 in treatment groups, n = 16 in placebo group).",
    "molecules": "CNP520"
  },
  {
    "caption": "(A) Mouse forebrain Aβ40 levels in homozygous male and female APOE4-TR mice with and without CNP520 treatment, 4 hours after dosing, shown are mean ± SEM, n = 6/group.",
    "molecules": "CNP520"
  },
  {
    "caption": "Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group. Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations.",
    "molecules": "Calcein"
  },
  {
    "caption": "Three-dimensional CT analyses of BMD, BV/TV, Tb.N, and Tb.Sp in trabecular bone from Vehicle (n=5 independent biological replicates) and ROS (n=6 independent biological replicates) model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice. Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.  ",
    "molecules": "ROS"
  },
  {
    "caption": "TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (D).",
    "molecules": "ROS"
  },
  {
    "caption": "OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (F).",
    "molecules": "ROS"
  },
  {
    "caption": "B. Limited proteolysis of a purified preparation of the Nse5/Nse6 complex by trypsin. Samples taken at different time points were analyzed by  SDS-Page and  Coomassie staining. Selected stable fragments identified by mass spectrometry are indicated.",
    "molecules": "trypsin"
  },
  {
    "caption": "B. Salt stability of  Smc5/Smc6-Twin-Strep interactions with Nse5/6 and Nse6(1-179). Immobilized Smc5/Smc6-Twin-Strep was mixed with  Nse5/6 or Nse6(1-179)-CPD-His ('in'). Beads were washed with buffers containing the indicated salt concentrations. Bead fractions were analysed by SDS-Page and Coomassie staining. Pulldown efficiencies were estimated from the intensity of Coomassie gel bands.",
    "molecules": "Coomassie"
  },
  {
    "caption": "D. Analysis of spore viability by yeast tetrad dissection. Diploid strains heterozygous for alleles of Nse6 (wt, 86-C, 177-C and 179-C) were sporulated. Isolated  spores were grown on YPD plates. Viable clones were tested for the marker cassette conferring resistance to G418 (marked by circles in green colours). Dead spores were marked by circles in red colours.",
    "molecules": "G418"
  },
  {
    "caption": "A. ATP  hydrolysis rates (given per Smc5/6  complex) were measured by an  enzyme-coupled assay in the absence and presence of plasmidDNA (conc. 1.25 nM) for the Smc5/6 hexamer (conc. 150 nM) and the octamer (conc. 150 nM) with increasing concentrations of ATP. The curves were fitted to Michaelis Menten equation and Km and kcat values were determined. Please note that the hexamer  without plasmidDNA shows cooperative behaviour, thus the Michalis-Menten kinetics is not formally applicable (marked by asterisk). Assays were performed in biological triplicates, mean values are shown with error bars indicating standard deviations. For bar graphs individual data points are also displayed. B. Same as in (A) using 40bpDNA (annealed 40 bp oligo DNA) (conc. 1 µM) instead of plasmidDNA. Note that the data points and curves for samples without DNA (hexamer -DNA; octamer -DNA) are identical to (A). ",
    "molecules": "ATP, DNA"
  },
  {
    "caption": "D. Pulldown experiments with Smc5/6 complexes and circular plasmid (2.8 kbp). DNA is retained after high salt washes (1 M NaCl) only in the presence of both Nse5/6 and ATP. The graph on the right shows a quantification (mean values and standard deviations) of the amount of co-purified  DNA from technical triplicates. Individual data points are also displayed.",
    "molecules": "ATP, DNA, NaCl"
  },
  {
    "caption": "F. Salt-stable DNA association requires ATP binding  and Smc5/6 head-engagement, but not  ATP hydrolysis. Same experimental setup as in (D) using mutant versions of the Smc5/6 hexamer as well as the non-hydrolyzable analogue of ATP, ATPγS, as indicated.",
    "molecules": "ATPγS, ATP, DNA"
  },
  {
    "caption": "B. Cross-linking of engineered variants of the Smc5/6 hexamer. As in (a) using  Smc5 and Smc6 cysteine mutants. Schemes indicate the location of engineered cysteines and their expected ability to cross-link in a rod-like and a ring-like conformation. High-molecular weight species were analysed by SDS-Page and Coomassie staining. Wild-type hexamer ('wt') is included as cross-linking control. Species occurring only in the presence of engineered cysteines are labelled by coloured arrowheads. Cross-linking efficiencies were calculated from the  intensity of Coomassie-stained bands by comparing the band of the corresponding cross-linked species to the bands of unmodified Smc5 and Smc6. Numbers below the gel quantify the percentage of cross-linked protein species in the displayed gel. Comparable numbers were obtained in at least one additional independent experiment.",
    "molecules": "Coomassie, cysteine, cysteines"
  },
  {
    "caption": "E. Effects of 40bpDNA and plasmidDNA on head engagement (as judged by E-Cys cross-linking) of the Smc5/6 complex with wildtype (wt) or hydrolysis-deficient (EQ) heads, in both the presence or absence of the Nse5/6 complex. Cross-linking efficiencies were calculated from the intensity of Coomassie-stained bands by comparing the band of the corresponding cross-linked species to the bands of unmodified Smc5  and Smc6. Numbers below the gel quantify the percentage of cross-linked protein species in the displayed gel.",
    "molecules": "Coomassie, Cys, DNA"
  },
  {
    "caption": "F-G. Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron (n=10-15; **p<0.05; ****p<0.0001, one-way ANOVA).",
    "molecules": "U0126"
  },
  {
    "caption": "A-B. GABAergic neuron  defects in wild-type  fish treated with ER/Golgi traffic inhibitor BFA (n=9; ****p<0.0001; ns, not significant; One-way ANOVA).",
    "molecules": "BFA"
  },
  {
    "caption": "O. Representative images  of GABAergic neurons and total number of GABAergic neurons (GFP+ cells) in the optic tectum  (OT) and cerebellum (CB) regions of wild-type, chd7 mutant and ephedrine-treated fish (n=9; ****p<0.0001; One-way ANOVA). Treatment with ephedrine ameliorated the number of GABAergic neurons.",
    "molecules": "ephedrine"
  },
  {
    "caption": "(A) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide (CHX), with or without SIRT7 overexpression. (B) Quantification of NFATc1 levels in (A) from 3 independent experiments. ",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(G-H) Immunoblots (G) and quantitative data (H, n=3) showing NFATc1 levels in HEK293T cells transfected with NFATc1-CA, with or without ectopic SIRT7, treated with MG132.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7+/+ and Sirt7-/- mice with or without Cyclosporin A (CsA) treatment. The black dashed line indicates bulge (Bu) of hair follicle. (B) Quantitative analysis of Nfatc1 levels in (A, n=10 hair follicles) using ImageJ plus 6.0. ",
    "molecules": "CsA, Cyclosporin A"
  },
  {
    "caption": "(C) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7+/+ and Sirt7-/- HFs with or without CsA treatment.",
    "molecules": "CsA"
  },
  {
    "caption": "C. EdU incorporation assay of GSCs transduced with shNT or shSATB2. Scale bar: 50 μm. D. Quantification of (C) showing the percentage of EdU+ cells (n=5). ",
    "molecules": "EdU"
  },
  {
    "caption": " F. qPCR analysis of ChIP assays with the indicated antibody (AcH3K18, AcH3K27, AcH4) using T3359 GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced acetylation of H3K18, H3K27 and H4 levels on the MAR of FOXM1 locus. ",
    "molecules": "AcH3K18, AcH4, AcH3K27"
  },
  {
    "caption": " A. Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control (n=5). ",
    "molecules": "C646"
  },
  {
    "caption": " B. Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days. Scale bar: 100 μm. ",
    "molecules": "C646"
  },
  {
    "caption": " C. qPCR analysis of SATB2, FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours (n=3). ",
    "molecules": "C646"
  },
  {
    "caption": "E. Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase-labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation (n=5 mice per group). F. Quantification of tumor growth from (E) (n=5 mice per group).",
    "molecules": "C646"
  },
  {
    "caption": " G. Kaplan-Meier survival curves of mice bearing T3359 GSC-derived xenografts treated with C646 or the vehicle control (Vehicle control: n=9 mice; C646: n= 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days. ",
    "molecules": "C646"
  },
  {
    "caption": " H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm. I. Quantification of Ki67 positive cells in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). ",
    "molecules": "C646"
  },
  {
    "caption": "Labeling of siRNA-transfected HUVECs with phalloidin (white) 30 min after Sema3E stimulation. Scale bar, 50 µm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "Trajectory plots (left) and rose plots (right) of HUVECs pre-treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses. P values for the Rayleigh test represent non-random distributions of cell endpoints. The graphs show FMI along the y- and x-axes during cell migration. n = 90 cells per group.",
    "molecules": "SB203580"
  },
  {
    "caption": "Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µ",
    "molecules": "SYTO dye"
  },
  {
    "caption": "Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.   Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.   ",
    "molecules": "4-hydroxytamoxifen, 4OHT"
  },
  {
    "caption": "Labeling for EdU (green), ERG (red), and CD31 (gray) in retinas of P4 RhojWT/WT and RhojGFP/GFP mice 2 h after i.p. EdU injections. Scale bar, 20 µm. The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts. n = 12 per group.",
    "molecules": "EdU"
  },
  {
    "caption": "Labeling for CD31 in retinas of P4 Rhojflox/flox and Pdgfb-iCreERT2:Rhojflox/flox (RhojiΔEC) mice after single i.p. injection of 100 µg of 4OHT at P1. Scale bar, 500 µm.",
    "molecules": "4OHT"
  },
  {
    "caption": "Labeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm. Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.",
    "molecules": "4OHT"
  },
  {
    "caption": "(D) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca). IPs were blotted with different antibodies as indicated. Star denotes KDF1.",
    "molecules": "Ca, calcium"
  },
  {
    "caption": "(C) Immunoblots of WCL collected at 0, 1 and 2 days after calcium shift with different antibodies as indicated.",
    "molecules": "calcium"
  },
  {
    "caption": "B) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated. Expression f exogenous KDF1 was induced by doxycycline (DOX) at different concentration.",
    "molecules": "doxycycline, DOX"
  },
  {
    "caption": "C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05",
    "molecules": "calcium"
  },
  {
    "caption": "(C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05",
    "molecules": "calcium"
  },
  {
    "caption": "(A) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated. Lo: low calcium; Hi: high calcium.",
    "molecules": "calcium"
  },
  {
    "caption": "(B-C) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide (CHX). WCL was collected at 0, 30, 60, 90, 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody (B). Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified (C). Statistical analysis is conducted using 2-way ANOVA. N=3 (biological replicates). *, p<0.05. Error bar represents S.D. (standard deviation).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(D-E) WT and KDF1 KO keratinocyte (right panels) or cells transfected with plasmid encoding exogenous IKKα (left panels) were treated with MG132 at 10µM for 6 hours, then were subjected to immunoprecipitation using anti-ubiquitin (Ub) antibody. IP and WCL were analyzed by immunoblots with α-IKKα antibody (D). Overall intensity of all ubiquitinated IKKα bands was determined by densitometry. Ratio of ubiquitinated IKKα was quantified and presented as bar graphs (E). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). *, p<0.05. Kd: kilodalton for molecular weight markers.",
    "molecules": "MG132, Ub, ubiquitin"
  },
  {
    "caption": "(F) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant, under both low and high calcium conditions, was examined and quantified by western blotting",
    "molecules": "calcium"
  },
  {
    "caption": "B) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca), same as in Fig. 1D. IPs were blotted with different antibodies as indicated. The star denotes KDF1.",
    "molecules": "Ca, calcium"
  },
  {
    "caption": "D) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift.",
    "molecules": "calcium"
  },
  {
    "caption": "E) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours, then subjected to immunoprecipitation using anti-ubiquitin antibody. Precipitated product was analyzed by immunoblotting with IKKα antibody. Band intensity was determined by densitometry and shown as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). **, p<0.01. Kd: kilodalton for molecular weight markers.",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "F-G) Immunoblot of Krt10 and Loricrin (Lor) with WT and USP7 KO cells before and after calcium shift (F). Star denotes an unspecific band in α-Loricrin blots. Band intensity was determined by densitometry and shown as bar graphs",
    "molecules": "calcium"
  },
  {
    "caption": "(D) Lysosomal inhibition increases HA-LC3II levels promoted by CD16:7-263-281. 293 cells were transfected with the indicated chimera and HA-LC3A, and subjected to anti−CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules.",
    "molecules": "bafilomycin"
  },
  {
    "caption": "(B-D) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione (IP, immunoprecipitation; TL, total lysate). Shown are WBs against the indicated molecules. (B) AU-ATG16L1 co‐precipitates with wild‐type (WT) TMEM59-GST but not with a mutated version where the four essential amino acids were mutated to alanine (TMEM59-4M-GST). (C) Full‐length TMEM59 and TMEM59-Δ282 co‐precipitate with GST-ATG16L1, whereas the respective 4M versions do not. (D) AU-ATG16L1 co‐precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 (GST-263-281), but not with a 4M version (GST-263-281-4M) or a GST fusion protein with the inactive portion of TMEM59-ID (GST-282-323).",
    "molecules": "glutathione"
  },
  {
    "caption": "(E) HA-ATG16L1 expressed in bacteria co−precipitates with a GST-263-281 recombinant protein purified from bacterial cultures, but not with a 4M version of the same construct or GST-282-323. The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione. The loaded beads were then used for HA-ATG16L1 pull−down from crude bacterial lysates. Shown are WBs against the indicated molecules (PD, pull−down). A Coomasie staining of a protein gel with the purified GST fusion proteins is shown. The right panel compares the amount of HA-ATG16L1 pulled down by GST-263-281 with the signal provided by direct anti−HA immunoprecipitation. This result shows that about 20-25% of the available HA-ATG16L1 protein is precipitated by GST-263-281. Asterisks indicate irrelevant bands in B and D.Source data for this figure is available on the online supplementary information page.",
    "molecules": "glutathione"
  },
  {
    "caption": "(A-F) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (A) A deleted version of ATG16L1 lacking the WD domain (HA-ATG16L1-ΔWD) does not co‐precipitate with TMEM59-GST. (B) TMEM59 does not co‐precipitate with ATG16L1-ΔWD fused to GST. (C) The WD domain of ATG16L1 (HA-ATG16L1-WD) suffices to co‐precipitate with TMEM59-GST. (D) TMEM59 co‐precipitates with ATG16L1-WD fused to GST. (E) HA-ATG16L1-WD does not bind a 4M version of TMEM59-GST. (F) TMEM59-4M does not co‐precipitate with the ATG16L1-WD fused to GST.Source data for this figure is available on the online supplementary information page.",
    "molecules": "glutathione"
  },
  {
    "caption": "(C, D, E, G, H) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (C) The N‐terminal CARD of NOD2 (NOD2-CARD1-HA), but not the CARD of NOD1 (NOD1-CARD-HA), co‐precipitates with GST-ATG16L1. (D) The ID of TLR2 (HA-TLR2-ID) co‐precipitates with GST-ATG16L1. (E) Mutated versions (MUT) of NOD2-CARD1-HA and HA-TLR2-ID (as shown) do not co‐precipitate with GST-ATG16L1. (F) Amino‐acid sequences of T3JAM and DEDD2 including the motif. Residues identified by the Prosite algorithm are highlighted. (G) Wild‐type (WT) versions of HA-T3JAM and HA-DEDD2 (as indicated) co‐precipitate with GST-ATG16L1. (H) Mutated versions (MUT) of HA-T3JAM and HA-DEDD2 do not co‐precipitate with GST-ATG16L1. Asterisks indicate irrelevant bands in C, D and G.Source data for this figure is available on the online supplementary information page.",
    "molecules": "glutathione"
  },
  {
    "caption": "G, H The period of DD locomotor rhythms of flies treated with RU486 to activate the pdfG4-geneswitch (pdf-GS) driver (G) or vehicle control (H). Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.",
    "molecules": "RU486"
  },
  {
    "caption": "B ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim, Pdp1ε, per and clk in Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) (n≥3). Data information: Student's t-test, *P < 0.05. Two-way ANOVA, significant effect between ub-H2B and IgG were found for all genomic regions tested (P<0.001). Significant effect of genotypes were found for tim E-box (P<0.05), Pdp1ε E-box (P<0.01), tim TSS (P<0.01) and Pdp1ε TSS (P<0.001).",
    "molecules": "ub"
  },
  {
    "caption": "D ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim and Pdp1ε in not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) (n=3). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical difference is measured using two-way ANOVA, P<0.001, significant effect of genotypes were found for tim E-box (P<0.01), Pdp1ε E-box (P<0.05), tim TSS (P<0.05) and Pdp1ε TSS (P<0.001) and tim gene body (P<0.01). G4, GAL4; U, UAS.",
    "molecules": "ub"
  },
  {
    "caption": " D Representative picture of 14-day-old plants grown 9 days in liquid 1/2MS supplemented with 1 µM flg22. The scale bar representing 0.6cm is included in the picture. E Lateral root density of WT and two independent RNAi-ASCO lines 9 days after transfer in 1/2MS supplemented with 0.1 µM or 1 µM flg22. ",
    "molecules": "flg22"
  },
  {
    "caption": " F Representative picture of root apical meristems after cell wall staining, in response to flg22. TZ: Transition Zone; QC: Quiescent Center. G Root apical meristem size of WT and RNAi-ASCO1 (eg distance from QC to TZ in µm). ",
    "molecules": "flg22"
  },
  {
    "caption": "B. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO, thapsigargin (TH), ISRIB, or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented at each lane.",
    "molecules": "DMSO, ISRIB, TH, thapsigargin"
  },
  {
    "caption": "D, E. MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, GADD34, and Endouc-Flag using Western blot. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane.",
    "molecules": "DMSO, TH"
  },
  {
    "caption": "A. MEF wild-type (WT) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin (TH) for 4 hr followed by analysis of the protein level of p-PKR, total PKR, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.",
    "molecules": "DMSO, TH, Thapsigargin"
  },
  {
    "caption": "B. MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p-eIF2α, total eIF2α, CHOP, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.",
    "molecules": "DMSO, TH"
  },
  {
    "caption": "D, E. The cleavage activity of Endouc was studied using materials presented at each lane. EndoucK242A: Endouc mutant; Extract: incubated in cell extracts; and Buf: reaction buffer. RNA probe without adding reaction buffer served as a negative control. The predicted cleavage sites (U) were mutated to generate mutant huORFchop transcript (unlabelled huORF-mt) and labelled by biotin (biotin-huORF-mt). Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe.",
    "molecules": "biotin"
  },
  {
    "caption": "A. HEK 293T cells were transfected with pCS2 vector (black), pCS2-Endouc (red), and pCS2-EndoucK242A (green) for 24 hr and subjected to either DMSO (control) or thapsigargin (TH; stress) treatment for 1 hr. The lysates of transfected cells were applied to polysome profile analysis (PPA). The sucrose gradient from top to bottom was shown from left to right, respectively. The sedimentation of 40S, 60S, 80S, and polysomes was indicated. Lysate from control, Endouc-expressing, and EndoucK242A-expressing cells were applied to PPA. The resulting fractions were TCA-precipitated, analysed by SDS-PAGE, and subjected to Western blot using antibodies against Flag and S6.",
    "molecules": "DMSO, TH, thapsigargin, TCA"
  },
  {
    "caption": "B. The luc activity of Fluc (solid column) versus Rluc (blank column) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures A was presented as an increase in fold over that obtained from control group of RF which was normalized as 1. These data suggested that the change of Fluc/Rluc ratio shown in Figures A was entirely dependent on the change of Fluc activity. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (**: P<0.005).",
    "molecules": "RF"
  },
  {
    "caption": "A-F. Schematic representation of capped (m7G-capped) and A-capped (non-functional capped) monocistronic mRNAs with poly(A)-tail-encoded firefly luciferase (Fluc) and fused upstream with (A) c-myc IRES, (B) no insertion, (C) non-specific 105-nt luc RNA (Non105), (D) huORFchop-1-105-nt, (E) huORFchop-69-105-nt, and (F) huORFchop-81-105-nt as shown above each bar graph. The RNA transfection assay of each transcript equipped with m7G-capped or A-capped monocistronic mRNA in HEK 293T cells was compared. To normalize the luc activities of capped and A-capped Fluc transcripts, the m7G-capped Renilla luc (Rluc) mRNA was used as an internal control. The luc activity of cells transfected with Cap-monocistronic mRNA in each graph was set as 100%.",
    "molecules": "poly(A)-tail-"
  },
  {
    "caption": "GFP expression under Pdgfrb-promoter in mice is present in mesangial cells of glomeruli and interstitial cells of cortex and medulla, but not in tubular cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µm",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative Ki67 immunofluorescence staining (green) in wt and Foxd1Cre::Pdgfrb+/J mice, showing increased proliferation in the transgenic mice. Glomeruli are outlined with circles, arrowheads point to Ki67 positive interstitial cells. Nuclei are stained with DAPI (blue). Scale bar = 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Isolated primary fibroblasts and mesangial cells from Foxd1Cre::Pdgfrb+/J mice have higher proliferation rates assessed by bromodeoxyuridine (BrdU) incorporation assay compared to cells from wt mice. Bar graphs show means ± SD, n = 4 per group, *: p≤0.05 compared to wt of same time point.",
    "molecules": "bromodeoxyuridine"
  },
  {
    "caption": "PAS staining of glomeruli of 25 weeks old wt and of 6 and 35 weeks old Foxd1Cre::Pdgfrb+/J mice shows pathological changes of the glomeruli in Foxd1Cre::Pdgfrb+/J mice. Glomerular tuft size of Foxd1Cre::Pdgfrb+/J mice was significantly increased compared to wt mice during the time course.",
    "molecules": "PAS"
  },
  {
    "caption": "Creatinine clearance was measured in mice of an age of 10, 14, 25 and 35 weeks and decreased continuously in Foxd1Cre::Pdgfrb+/J mice, with a predicted complete loss of renal function 62 weeks in transgenic mice using linear regression analyses as described previously (Steiger et al, 2018). 10 weeks n = 5 wt and n = 4 Foxd1Cre::Pdgfrb+/J, 14 weeks n = 2 per group, 25 weeks n = 8 wt and n = 8 Foxd1Cre::Pdgfrb+/J, 35 weeks n = 3 per group ",
    "molecules": "Creatinine"
  },
  {
    "caption": "The blood urea nitrogen (BUN) concentrations were higher in Foxd1Cre::Pdgfrb+/J mice compared to wt mice, apart from 25 weeks old group. Bar graphs show means ± SD, 10 weeks n = 7 wt and n = 5 Foxd1Cre::Pdgfrb+/J, 14 weeks n = 3 per group, 25 weeks n = 11 wt and n = 12 Foxd1Cre::Pdgfrb+/J, 35 weeks n = 3 per group",
    "molecules": "nitrogen, urea"
  },
  {
    "caption": "hemoglobin content (C) decrease in Foxd1Cre::Pdgfrb+/J mice continuously. Horizontal lines are means ± SD of n = 3 per group. *: p≤0.05 compared to same wt of same time point.",
    "molecules": "hemoglobin"
  },
  {
    "caption": "Angiotensin II infusion via an osmotic pump induced hypertension similarly in wt and Foxd1Cre::Pdgfrb+/J mice (C).",
    "molecules": "Angiotensin II"
  },
  {
    "caption": "In this model of hypertensive injury, the kidney function and even the histopathology was largely unaffected in wt mice, whereas the Foxd1Cre::Pdgfrb+/J mice showed a significantly increased BUN values and prominent glomerulopathy after 28 days compared to the starting values (day 0), to wt animals after 28 days and to age-matched Foxd1Cre::Pdgfrb+/J mice without Angiotensin II infusion (D).",
    "molecules": "Angiotensin II"
  },
  {
    "caption": "Consistently the creatinine clearance per g bodyweight decreased in the hypertensive Foxd1Cre::Pdgfrb+/J mice (E).",
    "molecules": "creatinine"
  },
  {
    "caption": "PAS staining showed more prominent mesangial expansion and intratubular protein casts indicative of proteinuria only in hypertensive Foxd1Cre::Pdgfrb+/J mice.",
    "molecules": "PAS"
  },
  {
    "caption": "22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Histomorphometric quantifications showed a significant reduction of α-SMA abundance in the glomerular tufts of imatinib treated Foxd1Cre::Pdgfrb+/J mice.",
    "molecules": "imatinib"
  },
  {
    "caption": "22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Glomerular cellularity evaluated by counting the total number of cells per glomerular tuft, normalized to the area, were significantly reduced in imatinib treated Foxd1Cre::Pdgfrb+/J mice compared to control mice.",
    "molecules": "imatinib"
  },
  {
    "caption": "22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Histomorphometric quantifications of collagen I showed no effect of imatinib treatment on mesangial sclerosis in the glomeruli (K), whereas reduced interstitial fibrosis in imatinib treated Foxd1Cre::Pdgfrb+/J mice compared to controls was found (L).",
    "molecules": "imatinib"
  },
  {
    "caption": "22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Hemoglobin serum levels were significantly improved in imatinib treated mice.",
    "molecules": "Hemoglobin, imatinib"
  },
  {
    "caption": "22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Representative pictures of the histological stainings of α-SMA (left) and collagen I (middle: glomeruli, right: cortical interstitium). Scale bar = 50 µm",
    "molecules": "imatinib"
  },
  {
    "caption": "22 weeks old Foxd1Cre::Pdgfrb+/J mice were treated with imatinib (daily gavage, 50mg/kg bodyweight for 21 days; Foxd1Cre::Pdgfrb+/J + imatinib) and were compared to control Foxd1Cre::Pdgfrb+/J mice receiving vehicle (water; Foxd1Cre::Pdgfrb+/J + water). Aged-matched wt mice are indicated as dashed lines. Total PDGFR-β protein levels were decreased in Foxd1Cre::Pdgfrb+/J mice after imatinib treatment, as shown using western blot (O) and its densitometric evaluation (P).",
    "molecules": "imatinib"
  },
  {
    "caption": "(e) Immunoblots of Triton X-100 soluble ('TX100') and insoluble ('1% SDS') fractions of cerebella obtained from control and Miz1ΔPOZNes mice of the indicated ages. Blots were probed with antibodies recognizing either mono- or polyubiquitinated proteins. Staining of the membrane with amido black was used as loading control.",
    "molecules": "SDS, Triton X-100, TX100"
  },
  {
    "caption": "(a) Genomic PCR documenting recombination of the floxed Miz1 alleles in Cre-ER-positive and -negative MEFs. 4-OHT was added to the media for 4 days.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(c) Immunoblots using an α-Lc3 antibody detecting Lc3-I and conjugated Lc3-II. 4-OHT-treated and control-treated MEFs of the indicated genotype were incubated with leupeptin and ammonium chloride (lys PI) for 2 h in DMEM (+amino acids) or in EBSS (−amino acids) medium. The expression of α-tubulin (upper panels) or α-vinculin (lower panels) was used as loading control.",
    "molecules": "4-OHT, amino acids, ammonium chloride, leupeptin"
  },
  {
    "caption": "(f) Recombination of Miz1flox/floxCre-ER+ neurospheres was tested by genomic PCR. 4-OHT was added to the media for 3 days.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(g) Immunoblots using an α-Lc3 antibody detecting Lc3-I and Lc3-II. 4-OHT-treated and non-treated Miz1flox/floxCre-ER+ neurospheres were incubated with leupeptin and ammonium chloride (lys PI) for 30 min. The expression of α-tubulin was used as loading control.",
    "molecules": "4-OHT, ammonium chloride, leupeptin"
  },
  {
    "caption": "Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).",
    "molecules": "Pentixafor, 68Ga"
  },
  {
    "caption": "Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).",
    "molecules": "Pentixafor, FDG, 18F, 68Ga"
  },
  {
    "caption": "Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.",
    "molecules": "Pentixafor, 68Ga"
  },
  {
    "caption": "Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.",
    "molecules": "Pentixafor, 68Ga"
  },
  {
    "caption": "Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.",
    "molecules": "Pentixafor, AMD3100"
  },
  {
    "caption": "[68Ga]PentixaforPET/CT and [18F]FDGPET/CTA-D Maximum intensity projections (MIP) of [68Ga]Pentixafor (A) and [18F]FDGPET/CT (B) of a 68-year-old male with histologically proven multiple myeloma indicating the better lesion-to-background contrast for [68Ga]Pentixafor in the corresponding myeloma manifestations. Trans-axial views of the upper thorax (C) and the pelvis (D) underline the higher uptake values of the bone manifestations (yellow arrows) of [68Ga]Pentixafor compared to [18F]FDG.",
    "molecules": "Pentixafor, FDG, 18F, 68Ga"
  },
  {
    "caption": "Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).",
    "molecules": "Pentixafor, FDG, 18F, 68Ga"
  },
  {
    "caption": "[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.",
    "molecules": "Pentixafor, 68Ga"
  },
  {
    "caption": "c, d, Atg5-/- and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 µg ml-1) and cyclohexamide (CHX, 5 µg ml-1) for 0, 4, 8 and 12 h. Cell lysates were analysed by western blot for loss of P62 expression (c). P62 levels were quantified by densitometry normalized to actin (n = 3; d). *P 0.04; **P 0.008; ***P = 0.0003",
    "molecules": "cyclohexamide, rapamycin"
  },
  {
    "caption": "e, f, Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin (50 µg ml-1) or dimethylsulphoxide (DMSO) control for 4 h (e). LC3-II expression in the presence of rapamycin was quantified and normalized to actin (n = 6; f).",
    "molecules": "dimethylsulphoxide, rapamycin"
  },
  {
    "caption": "c-f, Immunofluorescence images of Paneth cells from control patients (c, e) and patients with the risk allele (d, f) stained for lysozyme (red; c, d) and double-labelled additionally for leptin (green; e, f; yellow dashed line denotes the crypt unit).",
    "molecules": "leptin"
  },
  {
    "caption": "g, Aberrant lysozyme expression was quantified using the same criteria as that used for mouse sections in Fig. 3 (n = 6,829 cells for at risk patients and 8,182 for control, n = 5 patients for each genotype). Leptin-positive D3 cells were quantified in patient samples (n = 4 patients per genotype) homozygous for the risk allele (76 out of 322 D3 cells were positive from a total of 580 crypts examined) and homozygous for the safe allele (11 out of 93 D3 cells were positive from a total 749 crypts examined). Scale bars: a-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.",
    "molecules": "Leptin"
  },
  {
    "caption": "(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in large DRG neurons in response to repetitive mechanical indentations with a blunt glass probe; experiments were performed as described in the methods section. The applications of 25 μM baclofen (B) or vehicle (A) are indicated by the red horizontal lines. The downwards spikes represent individual MA currents induced by repetitive mechanical stimuli every 30 s for 200 ms stimulus duration.",
    "molecules": "baclofen"
  },
  {
    "caption": "(C) Quantification of normalized MA current amplitudes from DRG neurons, vehicle (white, n=7), neurons responding to baclofen (R, red) (n=6, **P<0.01, Paired t-test and non-responding DRG neurons (NR, blue, n=6). Striped column shows the average normalized current amplitudes 3-10 minutes after removal of baclofen in responding neurons (Washout) (n=6). Data are shown as mean ± SEM and scatter plots.",
    "molecules": "baclofen"
  },
  {
    "caption": "(D-E) Representative MA currents recorded at -60 mV in large DRG neurons in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-6 µm in 0.4 µm increments every 15 s (step protocol) before (black) and after (red) exposure to 25 µM Baclofen. Not every trace is shown for clarity. Inset above the current traces show the mechanical step protocol indicating the displacement of the mechanical probe.",
    "molecules": "Baclofen"
  },
  {
    "caption": "(F) Quantification of normalized MA currents of responding large DRG neurons [n=13, *P<0.05, **P<0.01, ***P<0.001, before (black) and after (red) baclofen treatment. Dashed grey line shows average MA currents 3-15 minutes after washout of baclofen. Repeated measures ANOVA with student's t-test no corrections was used to compare baseline and baclofen treatment. Data are shown as mean ± SEM.",
    "molecules": "baclofen"
  },
  {
    "caption": "(H-I) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=13, **P<0.01, Paired t-test) and non-responding large (NR) DRG neurons (n=9, NS, not significant) before (gray) and after (red) baclofen application. Data are shown as mean ± SEM and scatter plots.",
    "molecules": "baclofen"
  },
  {
    "caption": "(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in HEK293 cells transiently transfected with Piezo2 +GABAB1+ GABAB2 + GFP (for visualization) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-5.6 µm in 0.4 μm increments every 15 s before (black) and after (red) exposure to 25 µM baclofen. Not every trace is shown for clarity.",
    "molecules": "baclofen"
  },
  {
    "caption": "(C) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP [C, n=13, *P<0.05, **P<0.01, before (black) and after (red) baclofen treatment, Repeated measures ANOVA with student's t-test no corrections]. Data are shown as mean ± SEM.",
    "molecules": "baclofen"
  },
  {
    "caption": "(D) Quantification of MA current inactivation rate (Tau) in HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP before (gray) and after (red) baclofen application (n=13, NS, not significant; Paired t-test). Data are shown as mean ± SEM and scatter plots.",
    "molecules": "baclofen"
  },
  {
    "caption": "Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP (E, n=7) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.",
    "molecules": "baclofen"
  },
  {
    "caption": "Quantification of normalized MA currents of HEK293 cells transfected Piezo2+GABAB1+ GABAB2 + βARKct-mCherry (F, n=8) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.",
    "molecules": "baclofen"
  },
  {
    "caption": "Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+D2RS-YFP (A, n=13) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.",
    "molecules": "quinpirole"
  },
  {
    "caption": "Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP (B, n=5) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.",
    "molecules": "quinpirole"
  },
  {
    "caption": "(D,E) Quantification of normalized MA currents in responding large DRG neurons (D, n=5) versus non-responding DRG neurons (E, n=3) before (black) and after (red) application of sumatriptan 10 µM to activate the Gi-coupled Htr1d receptors. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.",
    "molecules": "sumatriptan"
  },
  {
    "caption": "(G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.",
    "molecules": "Lubrol"
  },
  {
    "caption": "Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the PI3K inhibitor Wortmannin 50 nM (B, n=10) Cells were treated with wortmannin 30 min before the experiments, and were present throughout the measurements.",
    "molecules": "Wortmannin, wortmannin"
  },
  {
    "caption": "Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the MAPK inhibitor U0126 10 µM (C, n=7). Cells were treated with U0126 for 30 min before the experiments, and were present throughout the measurements.",
    "molecules": "U0126"
  },
  {
    "caption": "Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (A-C) Percentage of withdrawal response for female mice using von Frey filaments at: 0.07 g (A), 0.16 g (B) and 0.4 g (C). (n= 11-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots. ",
    "molecules": "sumatriptan, U0126, wortmannin"
  },
  {
    "caption": "Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (D-F) Percentage of withdrawal response for male mice using von Frey filaments at: 0.07 g (D), 0.16 g (E) and 0.4 g (F). (n= 12-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots.",
    "molecules": "sumatriptan, U0126, wortmannin"
  },
  {
    "caption": "A Western blots of acid-extracted histones from activated CD4+ T cells showing global histone lactylation and H3K18 lactylation levels in the presence and absence of glucose at 24 hours after stimulation of cells. One of three similar experiments is shown.",
    "molecules": "glucose"
  },
  {
    "caption": "C, D Western blots of acid-extracted histones from activated CD4+ T cells in the presence of glycolysis inhibitor 2-DG and PDK inhibitor DCA, respectively. Cells were analysed for global lactylation and H3K18 lactylation 24 hours after activation of TCR with anti-CD3 (5 µg/ml).",
    "molecules": "2-DG, DCA"
  },
  {
    "caption": "E Immunoblotting of acid-extracted lactylated histones from anti-CD3-activated CD4+ T cells in the presence of extracellular lactate (25 mM) at 24 hours of cell culture. One representative out of three experiments is shown. For all immunoblotting experiments, ex vivo purified, non-activated CD4+ T cells were used as control lymphocytes.",
    "molecules": "lactate"
  },
  {
    "caption": "F ChIP-sequncing showing H3K18 lactylation signals in control Th17 cells and lactate-treated Th17 lymphocytes (25 mM). Biological duplicates are shown.",
    "molecules": "lactate"
  },
  {
    "caption": "CD4 and CD8 T cells were stimulated with cGAMP  (20 μg/ml) and αCD3/CD28  beads as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. Blots  depict one representative donor out of three.",
    "molecules": "cGAMP"
  },
  {
    "caption": "CD4 T cells  were stimulated with cGAMP  (20 μg/ml) and αCD3/CD28; and human IFNβ   (top) and IP10  levels (bottom) in the supernatant were analyzed by ELISA  at the indicated  time points. Data panels depict mean ± SD of biological duplicates from three independent donors.",
    "molecules": "cGAMP"
  },
  {
    "caption": "CD4 T cells were pre-treated with cycloheximide  (2 μg/ml) for 2 h, then treated with cGAMP  (20 μg/ml) and αCD3/CD28 (3/28) for 4 h, then analyzed by  quantitative PCR for IFNB1  and OASL  expression. Data are depicted as mean + SEM of 3 independent donors (for cGAMP  and αCD3/CD28 treated cells without CHX  only 2 data points were obtained). Statistics were determined by a two-way ANOVA on log-transformed data using Šidák correction for multiple testing: ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant.",
    "molecules": "cGAMP, CHX, cycloheximide"
  },
  {
    "caption": "CD4 T cells were pre-treated with Dasatinib  (100, 10, 1 and 0.1 nM), Tacrolimus  (1000, 100, 10 and 1 nM), TPCA-1 (4, 2, 1 and 0.5 μM) and Torin1 (50, 5, 0.5 and 0.05 nM) for 30 min, then treated with cGAMP  (20 μg/ml) and αCD3/CD28 for 16 h. Ηuman IFNβ levels in the supernatant were analyzed by ELISA. Data panels depict mean of biological duplicates from three independent donors.",
    "molecules": "cGAMP, Dasatinib, Tacrolimus, Torin1, TPCA-1"
  },
  {
    "caption": "CD4 T cells were treated with cGAMP  and indicated stimuli together with increasing concentrations of TPCA-1 for 4 h. Cell lysates were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells were stimulated with cGAMP  (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown. CD4 T cells  were stimulated with cGAMP  (20 μg/ml) and additional stimuli as indicated. Cell lysates after 4 h of stimulation were analyzed by immunoblotting. One representative donor out of two is shown.",
    "molecules": "cGAMP, TPCA-1"
  },
  {
    "caption": "Freshly isolated CD4 T cells  were treated with cGAMP, αCD3/CD28  beads and ABT737/S63845  (A/S) for 48 h and analyzed by flow cytometry. Treatment induced cell death  was assessed by  Annexin V  and propidium iodide staining. Data are depicted as mean + SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Dunnett correction for multiple testing: ***p < 0.001; *p < 0.05; ns, not significant.",
    "molecules": "ABT737, cGAMP, S63845"
  },
  {
    "caption": "CD4 T cells were treated with indicated concentrations of cGAMP  for 18 h, and ABT737/S63845  for 2 h. Lysates  and supernatant  precipitations were analyzed by immunoblotting. One representative donor out of two is shown.",
    "molecules": "ABT737, cGAMP, S63845"
  },
  {
    "caption": "CRISPR/Cas9  targeted CD4 T cells for indicated genes were treated with the indicated stimuli for 48 h and analyzed by flow cytometry. Each panel shows propidium iodide  and  Annexin V  intensity for one representative donor out of three. Digits indicate the percentage of the parental population (single events without subcellular debris).",
    "molecules": "propidium iodide"
  },
  {
    "caption": "CellTrace Violet (CTV) stained, freshly isolated CD4 and CD8 T cells   were activated with αCD3/CD28 beads, stimulated with cGAMP  for 96 h and analyzed by  flow cytometry. Each panel shows profiles from activated (black), cGAMP-treated (green) and resting (light gray) single and live cells from one representative donor.",
    "molecules": "CellTrace Violet, CTV, cGAMP"
  },
  {
    "caption": "Freshly isolated CD4 T cells were treated with cGAMP (40 μg/ml) and αCD3/CD28  (3/28) for 48 h and analyzed by Seahorse XF mito  stress test. Data are depicted as mean ± SEM of 3 independent donors. Statistics indicate significance by two-way ANOVA with Šidák correction for multiple testing: **p < 0.01; *p < 0.05; ns, not significant.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(B) WT and MPS-IIIA hippocampal neurons (DIV14) were transfected with v-Glut1-pHluorin-mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80-100 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensity was quantified at each bouton during KCl perfusion, normalized to the fluorescence value obtained upon rapid alkalization (NH4Cl perfusion) and expressed as ∆fluorescence (normalized to baseline; ΔFKCl/ΔF NH4Cl). Values were displayed both as fluorescence traces (means ± s.e.m. at each time frame) and maximum fluorescence after KCl perfusion (means ± s.e.m.). Representative panels on the right show the fluorescence intensity change in control neurons (WT). Pseudo-colour was applied to better reveal fluorescence changes.Note that NH4Cl alkalinizes all vesicles revealing the total (recycling + resting) pool in neuronal cells analyzed.",
    "molecules": "NH4Cl, KCl"
  },
  {
    "caption": "(C) VAMP2 and SNAP-25 protein levels were quantified by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.",
    "molecules": "cycloexamide"
  },
  {
    "caption": "(D) SDS-resistant complex levels were evaluated in WT and MPS-IIIA total brain samples at indicated ages by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.",
    "molecules": "SDS"
  },
  {
    "caption": "(B) α-Synuclein was immunoblotted in WT and MPS-IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.) and highly insoluble (H. Insol.) forms correspond to the protein solubilized respectively in Triton-X100, SDS 10% and 8M UREA.",
    "molecules": "SDS, Triton-X100, UREA"
  },
  {
    "caption": "(G) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(H) Palmitoylation-dependent shift in the molecular weight of CSPα was evaluated in WT and MPS-IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents (beta-mercaptoethanol or dithiothreitol).",
    "molecules": "beta-mercaptoethanol, dithiothreitol"
  },
  {
    "caption": "(G) SDS-resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non-boiled samples with VAMP2 or SNAP-25 antibodies. The amounts of SNARE complexes were quantified.",
    "molecules": "SDS"
  },
  {
    "caption": "(B) SDS-resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.",
    "molecules": "SDS"
  },
  {
    "caption": "(F) P0 lungs were stained with Hematoxylin and Eosin (H&E) and representative images are shown. Scale bar = 100 µm. (G) Representative images of diaphragms (Dps) isolated from P0 Ctrl or YY1cKO littermates.",
    "molecules": "Eosin, Hematoxylin"
  },
  {
    "caption": "(B) SCs were FACS sorted 3 days after the last TM injection and cultured for 1.5 days; RT-qPCR detection of YY1 mRNA shows the ablation in YY1iKO cells.",
    "molecules": "TM"
  },
  {
    "caption": "(F) Representative FACS plots. About 100,000 cells from 2-month-old Ctrl and YY1iKO mice were sorted by FACS 3 weeks post-TM injection. The percentage of SCs was shown. (n=3 mice, each).",
    "molecules": "TM"
  },
  {
    "caption": "(G) H&E staining was performed on the injured TA muscles collected from the designated times post-CTX injection to visualize the degree of regeneration. Scale bar = 100 µm.",
    "molecules": "CTX"
  },
  {
    "caption": "(H) IF staining for eMyHC on the TA muscles 5 days post-CTX injury. Quantifications of the number of eMyHC+ fibers are shown on the right (n=3, each). Scale bar = 50 µm.",
    "molecules": "CTX"
  },
  {
    "caption": "(I) Left: representative images of TA muscles isolated from Ctrl or YY1iKO mice 60 days post-CTX injury. Right: Masson's Trichrome staining of the above muscles to visualize the degree of fibrosis. Scale bar = 100 µm.",
    "molecules": "CTX"
  },
  {
    "caption": "(K) Representative FACS plots showing the percentage of SCs sorted from TA muscles 3 days after CTX injury of Ctrl and YY1iKO mice.",
    "molecules": "CTX"
  },
  {
    "caption": "Immunostaining for Pax7 together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of Pax7+ SCs per area are shown on the right (n=3 mice, each).",
    "molecules": "CTX"
  },
  {
    "caption": "Immunostaining for MyoD together with Laminin was performed on the TA muscles 3 days post-CTX injury. Scale bar = 100 µm. Quantifications of the numbers of MyoD+ SCs per area are shown on the right (n=3 mice, each).",
    "molecules": "CTX"
  },
  {
    "caption": "(N) IF staining for Pax7 and Laminin on TA muscles 4 weeks after CTX injury of Ctrl or YY1iKO mice. Quantifications of the numbers of Pax7+ SCs per 100 fibers are shown on the right (n=3 mice, each). Scale bar = 100 μm.",
    "molecules": "CTX"
  },
  {
    "caption": "(P) H&E staining was performed on TA muscles of Ctrl or YY1iKO mice 7 day after second CTX injury. Scale bar = 400 μm (left) or 100 μm (right).",
    "molecules": "CTX"
  },
  {
    "caption": "(S) IF staining for eMyHC and Laminin on TA muscles 7 days after the second CTX injury of Ctrl or YY1iKO mice. Quantifications of the number of eMyHC+ fibers per area are shown on the right (n=3 mice, each). Scale bar = 100 μm.",
    "molecules": "CTX"
  },
  {
    "caption": "(B) SCs were FACS sorted 4 months after the last TM injection; RT-qPCR detection of YY1 mRNA shows the ablation in YY1dKO cells.",
    "molecules": "TM"
  },
  {
    "caption": "(C) Representative images of Ctrl or YY1dKO mice 4 months after the TM administration.",
    "molecules": "TM"
  },
  {
    "caption": "(F) Representative images of Dp muscles isolated from Ctrl or YY1dKO mice 4 months after the TM injection.",
    "molecules": "TM"
  },
  {
    "caption": "(C) An equal number of FACS-isolated SCs from Ctrl or YY1iKO mice were cultured for 48 hrs and EdU labelled for 8 hrs, followed by immunostaining for Pax7 (green) and EdU (red). Quantifications of the percentage of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.",
    "molecules": "EdU"
  },
  {
    "caption": "(G) SCs expansion in vivo was determined by EdU labeling for 12 hrs 2.5 days after CTX injury. Quantifications of the numbers of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.",
    "molecules": "EdU, CTX"
  },
  {
    "caption": "(H) An equal number of FACS-isolated SCs from Ctrl or YY1iKO mice were cultured for 24hrs and EdU labeled for 12 hrs, followed by immunostaining for Pax7 (green) and EdU (red) was performed. Quantifications of the percentage of EdU+ cells are shown on the right (n=3 mice, each). Scale bar = 100 µm.",
    "molecules": "EdU"
  },
  {
    "caption": "ChIP-qPCR was performed to show the enrichment of H3K27 me3 (R) binding on selected mitochondrial genes.",
    "molecules": "H3K27 me3"
  },
  {
    "caption": "(A) An equal number of FACS-isolated SCs from C57 mice were cultured for 24 hrs and treated with 0, 1, 10, or 20mM 2-DG (glycolytic inhibitor) or NaN3 (respiration inhibitor) for 3 hrs before measurement of ATP production. The relative ATP levels normalized to the 0 mM values are plotted. N=3 mice.",
    "molecules": "2-DG, ATP, NaN3"
  },
  {
    "caption": "(B) SCs from C57 mice were treated with 10mM 2-DG or 10mM NaN3 for 36 hrs before measurement of proliferation rate by MTS assay (n=3 mice, each).",
    "molecules": "2-DG, MTS, NaN3"
  },
  {
    "caption": "(C) SCs from C57 mice were treated with 10mM 2-DG for 24 hrs and subject to EdU labeling for 6 hrs. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified (n=3 mice, each).",
    "molecules": "2-DG, EdU"
  },
  {
    "caption": "(D) Ctrl and YY1iKO ASCs were stained with 60 ug/mL 2-NBDG for 45 mins and the fluorescence intensity (MFI) of 2-NBDG was measured by flow cytometry (n=3 mice, each).",
    "molecules": "2-NBDG"
  },
  {
    "caption": "An equal number of Ctrl and YY1iKO cells were cultured for 36h Lactate concentration (F) were measured and normalized with cell numbers. N=3 mice. by",
    "molecules": "Lactate"
  },
  {
    "caption": "An equal number of Ctrl and YY1iKO cells were cultured for 36h ATP production (G) were measured and normalized with cell numbers. N=3 mice.",
    "molecules": "ATP"
  },
  {
    "caption": "(K) FACS-isolated SCs from Pax7-nGFP mice were transfected with Hif1α or control siRNAs and EdU labeled for 5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.",
    "molecules": "EdU"
  },
  {
    "caption": "(A-B) Ctrl or YY1iKO ASCs were treated with CHX (Cycloheximide) for 90 mins and Hif1α protein was measured by flow cytometry. The numbers indicate the degradation rate after CHX treatment. N=3 mice. (C) The relative degradation rate was calculated by the ratio of degradation rate in YY1iKO (11.14% ± 1.71%) vs Ctrl (5.08% ± 0.796%). N=3 mice.",
    "molecules": "CHX, Cycloheximide"
  },
  {
    "caption": "(G) SCs were isolated from Ctrl or YY1iKO mice and infected with the above viruses. EdU labeling was then performed for 5.5 hrs. The percentage of MyoD+EdU+ cells over the total number of MyoD+ cells was quantified. Scale bar = 100 µm. N=3 mice.",
    "molecules": "EdU"
  },
  {
    "caption": "(J) YY1(WT) or YY1(S365D) mutant was expressed in YY1iKO ASCs and EdU labeled for 6 hrs. (K) Similarly, YY1 (WT) or YY1 (1-394) mutant was expressed and EdU labeled for 6hrs. A GFP expressing plasmid was used the negative control. The percentage of Pax7+/EdU+ cells over the total number of Pax7+ cells was quantified. Error bars represent s.d.'s of the mean. Student's t-test (two-sided): N.S = non-significant, *P<0.05, **P<0.01, ***P<0.001.",
    "molecules": "EdU"
  },
  {
    "caption": "A, B Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in PBMCs (A) and BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at the indicated time points.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "E A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right).",
    "molecules": "Baf A1, PS-341, PR-171, CQ, poly (I:C)"
  },
  {
    "caption": "H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR171 (10 μM).",
    "molecules": "PR171, DMSO, ubiquitin"
  },
  {
    "caption": "C qRT-PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP-/- 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP.",
    "molecules": "poly(dA:dT), poly(I:C)"
  },
  {
    "caption": "I, J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies.",
    "molecules": "poly(dA:dT), poly(I:C)"
  },
  {
    "caption": "A, B Immunoblot analyses of total and phosphorylated (p-) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) (A) or poly (dA:dT) (B) at different time points.",
    "molecules": "poly (dA:dT), poly (I:C)"
  },
  {
    "caption": "F, G Abundance of IRF3 (F) and IFNAR1 (G) transcripts (detection site-1 and -2) among mRNA immunoprecipitated with anti-m6A antibody from shCtrl and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations.",
    "molecules": "m6A"
  },
  {
    "caption": "A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly (I:C) or poly (dA:dT) at the indicated time points. B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV-eGFP at the indicated time points.",
    "molecules": "poly (dA:dT), poly (I:C)"
  },
  {
    "caption": "H, Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. Representative images from 3 independent biological experiments are shown (H),",
    "molecules": "PR-171, DMSO, poly (I:C)"
  },
  {
    "caption": "protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. the relative levels of IFNAR1 (left) and IRF3 (right) are presented as the mean ± SD (I).  ",
    "molecules": "PR-171, DMSO, poly (I:C)"
  },
  {
    "caption": "(H) Representative immunofluorescence images of Iba1 (green) and APOE (red) on cortical sections from SPF and GF mice. Nuclei  were stained with DAPI  (blue). Scale bar: 50 μm. White arrowheads indicate APOE+/Iba1+ microglia and arrows show APOE-/Iba1+ microglia. Quantification of APOE+/Iba1+ microglia. Each symbol represents one  mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by unpaired t-test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) Representative  Iba1+ immunohistochemistry  in the choroid plexus of SPF, GF,  ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient  mice. Scale bar = 50 µm. (I-O) Quantification of stromal CPMΦ. Each symbol represents one mouse. Three to four sections per  mouse  were examined. Means ± s.e.m. are indicated. Significant differences were evaluated by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of at least two independent experiments. ",
    "molecules": "ABX, SCFA"
  },
  {
    "caption": "(H) Representative  Iba1+ immunohistochemistry in the perivascular space of SPF,  GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ perivascular macrophages. Scale bar = 50 µm. (I-O) Quantification thereof. Each symbol represents one  mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments. ",
    "molecules": "ABX, SCFA"
  },
  {
    "caption": "(H) Representative Iba1+ immunohistochemistry in the leptomeninges of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Arrows indicate Iba1+ meningeal macrophages. Scale bar = 50 μm. (I-O) Quantification thereof. Each symbol represents one  mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. No significant differences were determined by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test. Data are representative of at least two independent experiments. ",
    "molecules": "ABX, SCFA"
  },
  {
    "caption": "(C, D) Representative immunofluorescence images (left) of (C) Iba1  (green) and KI67 (red) or (D) CD74 (red) on cortical  sections from SPF and GF mice. Nuclei  were stained  with  DAPI  (blue). Scale bars: 25 μm. White arrows indicate KI67+/Iba1+ or CD74+/Iba1+ macrophages and arrowheads show KI67-/Iba1+ or CD74-/Iba1+ macrophages. Quantification thereof (right). Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were evaluated by two-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of two independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-C) Representative immunofluorescence images visualizing (A) cortical  mMΦ, (B) cpMΦ located in the lateral ventricle and (C) hippocampal pvMΦ from SPF (left), GF  (middle) and ABX-treated 5xFAD  mice (right). Iba1  (green) and Aβ  (6E10, red). Nuclei  were stained  with DAPI (blue). White arrowheads indicate  Iba1+Aβ+ MΦ and white arrows show Iba1+Aβ- MΦ. Scale bar: 50 μm.",
    "molecules": "ABX, DAPI"
  },
  {
    "caption": "(H-J) Percentage of Iba1+Aβ+ (H) cortical mMΦ, (I)  cpMΦ and (J) hippocampal pvMΦ of SPF, GF  and ABX-treated 5xFAD mice. Means ± s.e.m. are indicated. Significant differences were evaluated by one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.",
    "molecules": "ABX"
  },
  {
    "caption": "B. A representative example of a 7 d intestine reared at 29 Co in experimental conditions that protect the epithelial integrity and labelled with ReDDM cell-lineage tool. esg (green cytoplasmic signal) labels intestinal progenitor cells, whilst Histone-RFP (red nuclear signal) acts as a semi-permanent marker that allows the visualization of differentiated cells. Note the extensive overlap between the two markers and the absence of differentiated cells only showing the Histone-RFP labelling, as an indication of negligible epithelial turnover. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining (not shown) as a reference. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019 C. Quantification of intestinal cell subpopulations labelled with ReDDM system at high magnification (GFP and Histone-RFP) and different time points post ReDDM activation (3- and 7-days) in experimental conditions that protect the epithelial integrity; note that none of the cell populations in the gut (GFP (n.s. no significant , P = 0.5267) or Histone-RFP (n.s. , P =0.2752) significantly increase in number overtime (Quantifications were made using N ≥ 2 biological replicates, total number of guts analysed at 3 days n =34 and at 7 days n=45; Mann-Whitney, C). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019)",
    "molecules": "DAPI"
  },
  {
    "caption": " D. A representative example of an adult female posterior midgut at low (A) and high magnification (B, Region 4-Region 5) showing the initiator caspase reporter DBS-S-QF (Red, immunostaining anti-HA) after 16 hours of paraquat treatment in Oxford food at 25 Co; note the expansion of the labelling with DBS-S-QF to large intestinal cells (ECs) (compare D with A). The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining as a reference. Genotype: y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF (BL83131) ",
    "molecules": "DAPI, paraquat"
  },
  {
    "caption": " E. ReDDM lineage-tracing in an adult intestine reared in Oxford Medium and paraquat (20 mM) during 16 hours at 29 Co; note the abundance of Histone-RFP cells without GFP signal, as an indication of epithelial damage and subsequent differentiation of progenitor cells. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019) F. Quantification of ReDDM labelling at high magnification after paraquat treatment; note the statistically significant increase (**; P = 0.0099) of Histone-RFP expressing cells without GFP signal (Quantifications were made using N ≥ 2 biological replicates; Unpaired two-tailed t test, 3d n = 9, 7d n =7). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019) ",
    "molecules": "paraquat"
  },
  {
    "caption": " H. Representative example of an intestine 7d old expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-gal4 (green immunostaining with antibody against P35) at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-P35 (BL5072); UAS-P35 (BL5073)/+ I. ReDDM lineage-tracing system in a Drosophila intestine expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-Gal4 and experimental conditions that protect the epithelial integrity at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073) J. ReDDM quantification corresponding to the intestines described in (I); no significant increase in either esg (n.s. , P = 0.1352) or Histone-RFP (n.s. , P = 0.9801) cell number is observed (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, 3d n = 12, 7d n = 11). Error bars represent Standard Error of the Mean. DAPI (blue) labels the nuclei in panels A, D and H. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073) ",
    "molecules": "DAPI"
  },
  {
    "caption": "A. ReDDM activation in Dronc heterozygous (+/-) intestine 7 days after temperature shift at 29 Co; esg expression (green) labels the intestinal progenitor cells, Histone-RFP (red) is a semi-permanent marker retained in differentiated cells and Pdm-1 (grey) labels differentiated ECs. The red arrows indicates the enlarged areas depicted in the insets in the entire figure. DAPI labels the DNA In the entire Figure (blue). All of the experiments in the entire figure have been made under experimental conditions that protect the epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Relative number of esg-expressing cells normalised to DAPI; notice that the relative percentage of esg-labelled cells is significantly higher in Dronc fully mutant conditions (-/-) at 3 days (***; P = 0.0007) and 7d (****; p <0.0001) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t-test, +/- n=32, -/- n=25).",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Relative number of esg-negative cells expressing Histone-RFP normalised to DAPI; notice that the number of Histone-RFP cells without esg expression is significantly higher in Dronc fully mutant (-/-) conditions at 7d (*; P = 0.0046) (Quantifications were made using N biological replicates ≥ 2; Mann-Whitney test, +/- n = 24, -/- n = 20.",
    "molecules": "DAPI"
  },
  {
    "caption": " . A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Dl-Gal4. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity (flies transferred every two days into vials with fresh food). DAPI (blue) labels the DNA in the entire Figure. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO/+ (recombinant chromosome made for this study; the parental line delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP was obtained from Maria Dominguez) B. A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Dl-Gal4. Dl-expressing cells are labelled with UASGFP (green); there are no morphological differences between A and B. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Relative number of GFP+ Delta-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (ns; P = 0.9231) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 22, -/- n = 13). Error bars represent Standard Deviation of the Mean. Genotypes: +/- :w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry -/- : w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry D. Average Delta cell size (μm²); notice that the cell size does not change between heterozygous and homozygous Dronc mutant ISCs (ns; P = 0.9694) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 42, -/- n = 21). Error bars represent Standard Deviation of the mean. Genotypes:",
    "molecules": "DAPI"
  },
  {
    "caption": ". A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Su(H)-Gal4. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ A 7d intestine in which the Enteroblasts have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Su(H)-Gal4. Su(H)-Gal4-expressing cells are labelled with UASGFP (green) and UAS-Histone-RFP; note the increase in cell size when compared to E and the absence of Histone-RFP cells without GFP signal. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry Relative number of Su(H)-Gal4-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (n.s. ; P = 0.5099) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 30, -/- n = 29). Error bars represent Standard Deviation of the mean. Genotypes: +/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ -/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry H. Average Su(H) cell size (μm²), there is a significant increase in Su(H) Dronc null cells (-/-) when compared to the heterozygous control condition (**; P = 0.0064) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 49, -/- n = 33). Error bars represent Standard Deviation of the mean. Genotypes: ",
    "molecules": "DAPI"
  },
  {
    "caption": "F. There is extensive overlap between the expression of the EB marker Su(H) (green, immunostaining against Beta-galactosidase; white arrow) and the apical caspase reporter DBS-S-QF (red, immunostaining against HA), but it is also present in progenitor cells without Su(H) expression (yellow arrowheads). DAPI (Blue) labels cell nuclei in all the panels.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Representative ReDDM labeling of a Drosophila Dronc heterozygous (+/-) intestine overexpressing an RNAi against Notch for 3 days; note the lack of fully differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP). DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity. Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + B. Representative ReDDM labelling of a Drosophila Dronc mutant homozygous (-/-) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC; note the lack of differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP), as well as the increase of GFP-positive cells (compare A to B). Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Quantification of the number of Histone-RFP cells normalised to DAPI (proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels); note the statically significant increase in Histone-RFP positive cells in Dronc homozygous mutant intestines compared to controls (**; P =0.0080) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 8, -/- n = 15). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118 UAS-Notch-RNAi; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry ",
    "molecules": "DAPI"
  },
  {
    "caption": " D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / + E. Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation (compare D and E). The white arrows indicate the position of insets 500μm from the posterior region. Note the complete loss of esg labelled cells in this region. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry F. Relative number of esg-positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch-Intra; note the significant reduction of esg-expressing cells (****; P < 0.0001) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 17, -/- n = 17). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118; esg-Gal4 UAS-CD8 -GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry",
    "molecules": "DAPI"
  },
  {
    "caption": "I. Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat. Note the absence of large Su(H) positive, GFP (-) cells compared to (H). The white arrows indicate the enlarged area depicted in the insets. Genotypes: +/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118, Su(H)GBE-LacZ ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry",
    "molecules": "paraquat"
  },
  {
    "caption": "B. Growth comparisons of selected kinases with the kinase‑dead mutant match. 24h growth curves (OD595) from liquid culture of yeast strains (W303) expressing human kinase in the presence or absence of 3.2% (v/v) DMSO. Upper panel: BUB1: BUB1 wild type, BUB1-KD: BUB1(K821M); upper middle panel: NEK6: NEK6 wild type, NEK6-KD: NEK6(K74M&K75M); lower middle panel: PKCα-ac: PKCα(A25E), PKCα-KD: PKCα(K368R); lower panel: Control with empty plasmid. Error bars represent SD from three biological replicates.",
    "molecules": "DMSO"
  },
  {
    "caption": "C. Human protein kinases phosphorylate large sets of yeast proteins. Upper panel: In total, 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho-motif antibodies on yeast proteins in comparison to other human kinases, kinase‑dead mutant versions or vector control samples. Commercially available pS/T-antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases. Lower panel: Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded. When developed with the indicated phospho-motif recognizing antibodies, immunoreactive bands indicated phosphorylation of yeast proteins.",
    "molecules": "phospho"
  },
  {
    "caption": "C. Y2H interaction experiment with human AAR2 (FL) as bait and PRP8 (aa1755-2335) as prey. Growth on selective media is abolished when AAR2 carries a S284E phospho-mimicry amino acid substitution. Mutation of second candidate phospho-site, AAR2(T356), does not influence the AAR2-PRP8 protein interaction.",
    "molecules": "phospho"
  },
  {
    "caption": "C. Phosphorylation of ERα in mammalian cells. ERα was co-expressed with the indicated wildtype kinases or kinase‑dead versions in HEK293 cells and immunoprecipitated. Phosphorylation status of ERα was analyzed with an in-gel phosphostain (Pro-Q® Diamond phosphoprotein gel stain, Invitrogen), relative to the whole protein stain (SYPRO®Ruby protein gel stain, Invitrogen).",
    "molecules": "Pro-Q® Diamond, SYPRO®Ruby"
  },
  {
    "caption": "E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.",
    "molecules": "17β-estradiol, E2"
  },
  {
    "caption": "B-J: Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs. Antibodies against Tuj1, Gfap and Olig2 were used to recognize neurons, astrocytes and oligodendrocytes. In D, G and J the percentage of cells positive (mean ± SEM) for each marker was counted over the total of DAPI positive cells (100%). No difference between genotypes was highlighted (Two-way ANOVA); the variation through time resulted significant for the levels of Tuj1 and Gfap (Two-way ANOVA: F(1,16)=9.542, p-value<0.05 and F(1,10)=68.95, p-value<0.0001 respectively). Sample size: n ≥3 wells obtained from two independent preparations; for each well 10 random fields were acquired. Scale bars: 30 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "H-K: Raster plots (H and I) and their quantitation under basal conditions and upon 100µM 4-AP exposure. The magnitude of the response after stimulation is expressed either as fold change (J) or as the averaged maximal values reached after 4-AP exposure (K). Dotted line in J represents untreated controls (set to 1). Values are represented as mean ± SEM. Student's t-test, *: p-value<0.05. Sample size n=8 wells deriving from three independent preparations.",
    "molecules": "4-AP"
  },
  {
    "caption": "L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells. ",
    "molecules": "DAPI"
  },
  {
    "caption": "Bar graphs represent the expression values (mean ± SEM) of 27 genes, selected for their downregulation in null samples at DIV 14 (p-value <0,07; see Appendix Figure S2), after CX546 administration in the two different time windows (early in G and late in H). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls (set to 1, red dotted line). *: p-value<0.05; **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001. Sample size: n≥8 wells deriving from two independent preparations. Each point is representative of a single well.",
    "molecules": "CX546"
  },
  {
    "caption": "F,G: Violin plots represent the average values of MQAE intensity for early (F) or late (G) treated null neurons and their relative controls. One-way ANOVA followed by Tukey's multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons. ***: p<0.001; ****: p <0.0001. The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles. Sample size: n≥300 cells from five different embryos per genotype.",
    "molecules": "MQAE"
  },
  {
    "caption": "I,J: In I One-way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons (set to 100), null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4. * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001); # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine (#: p-value<0.05; ##: p-value<0.01). Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 (expressed as 2-∆ct). Box plot shows the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 3 wells obtained from four different embryos. One-way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones. **: p-value<0.01. Sample size: n≥3 wells from four different embryos.",
    "molecules": "CX546, Nifedipine"
  },
  {
    "caption": "B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01.",
    "molecules": "CX546"
  },
  {
    "caption": "F,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal.",
    "molecules": "Ampakine, CX546"
  },
  {
    "caption": "D Immunocytochemistry analysis of sperm stained with anti‐ApCatSper 3, anti‐GC, or anti‐CNGK antibodies; superposition of images obtained by fluorescence microscopy and bright‐field light microscopy; scale bar = 10 μm. The DNA was stained with DAPI (blue).",
    "molecules": "DNA"
  },
  {
    "caption": "A Alkaline‐evoked Ca2+ signals in sperm mixed with NH4Cl; sperm were loaded with the Ca2+ indicator Fluo‐4. ΔF/F (%) indicates the change in Fluo‐4 fluorescence (ΔF) with respect to the basal fluorescence (F, mean of the first 3-5 data points).B Ca2+ signals evoked by mixing of sperm simultaneously with NH4Cl (30 mM) and the CatSper inhibitor MDL12330A.C Dose-response relation of inhibition of the Ca2+ signals shown in panel (B), Ki = 20 μM, amplitudes were determined at t = 4 s.",
    "molecules": "Ca2+, NH4Cl, MDL12330A"
  },
  {
    "caption": "A Changes in pHi evoked by mixing with pHi‐clamp solutions (see explanation in the text); sperm were loaded with the pHi indicator BCECF. ΔR/R (%) indicates the change in the BCECF fluorescence emission ratio (ΔR = F494/F540) with respect to the basal ratio (R, mean of the first 3-5 data points).B Steady‐state change (at t = 14 s) of BCECF fluorescence for the pHi signals shown in (A). The intercept of the fitted straight line with the x‐axis yields the resting pHi; the slope of the straight line yields the ΔR/R (%) × ΔpH−1. Inset: calibrated changes in pHi evoked by various pHi‐clamp solutions.",
    "molecules": "BCECF"
  },
  {
    "caption": "C Ca2+ signals evoked by mixing of sperm with pHi‐clamp solutions.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D Dose-response relation for the Ca2+ signals shown in (C).",
    "molecules": "Ca2+"
  },
  {
    "caption": "E Calibrated pHi increase (red) and respective Ca2+ response (black) evoked by mixing of sperm with a pHi 7.6‐clamp solution; depicted are, on an extended time scale, the first 500 ms of the respective pHi increase and Ca2+ signal shown in (B, inset) and (C), respectively. The threshold pHi for CatSper activation was deduced from the latency of the Ca2+ signal.F Threshold pHi and latency of Ca2+ signals evoked by various pHi‐clamp solutions (mean ± SD; n ≥ 3).",
    "molecules": "Ca2+"
  },
  {
    "caption": "A Depolarization‐evoked Ca2+ signals in sperm mixed with ASW containing high KCl concentrations.",
    "molecules": "Ca2+, KCl"
  },
  {
    "caption": "B Ca2+ signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A.",
    "molecules": "Ca2+, MDL12330A, KCl"
  },
  {
    "caption": "C Ca2+ signals evoked by mixing of sperm with 160 mM KCl and MDL12330A.",
    "molecules": "Ca2+, MDL12330A, KCl"
  },
  {
    "caption": "D Dose-response relation for the Ca2+ signals shown in (B, C) at t = 1-2 s.",
    "molecules": "Ca2+"
  },
  {
    "caption": "E Threshold pHi for Ca2+ signals evoked by pHi‐clamp solutions in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (mean ± SD; n ≥ 3); data for 9 mM KCl are from Fig F.F Resting pHi and resting Vm in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (black) (mean ± SD; n ≥ 3). Mean threshold pHi for CatSper activation at different membrane potentials (red); mean threshold pHi was derived from data shown in (E).",
    "molecules": "Ca2+, KCl"
  },
  {
    "caption": "A Ca2+ signals in sperm evoked by photorelease (at t = 0) of resact from caged resact in the presence of the CatSper inhibitor MDL12330A.",
    "molecules": "Ca2+, MDL12330A, resact"
  },
  {
    "caption": "B Ca2+ signals evoked by intracellular photorelease (at t = 0) of cGMP in sperm loaded with caged cGMP in the presence of the CatSper inhibitor MDL12330A.",
    "molecules": "Ca2+, cGMP, MDL12330A"
  },
  {
    "caption": "C Normalized dose-response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in (A, B) (Ki = 6.2 and 4.3 μM, respectively).",
    "molecules": "Ca2+, cGMP, resact"
  },
  {
    "caption": "D Normalized dose‐response relation for inhibition of the resact‐ and cGMP‐induced Ca2+ signals shown in Supplementary Fig S4 by the CatSper inhibitor mibefradil (Ki = 7.7 and 20.9 μM, respectively).",
    "molecules": "Ca2+, cGMP, mibefradil, resact"
  },
  {
    "caption": "A Calibrated Vm (left) and pHi (right) changes and Ca2+ signals (middle) evoked by resact.",
    "molecules": "Ca2+, resact"
  },
  {
    "caption": "B Normalized Vm, pHi, and Ca2+ signals evoked by 10 pM resact; the first 600 ms after mixing are shown. The hyperpolarization precedes the pHi increase, whereas the pHi increase precedes the Ca2+ increase.",
    "molecules": "Ca2+, resact"
  },
  {
    "caption": "C Latency of the Vm, the pHi, and the Ca2+ signals evoked by various resact concentrations (mean ± SD; n = 3).",
    "molecules": "Ca2+, resact"
  },
  {
    "caption": "D Normalized pHi and Ca2+ signals evoked by photorelease of cGMP in sperm loaded with caged cGMP.",
    "molecules": "Ca2+, cGMP"
  },
  {
    "caption": "E Calibrated Vm and Ca2+ signals evoked by 1 nM resact; the threshold voltage (Vthr) for the Ca2+ influx was deduced from the latency of the Ca2+ signal.",
    "molecules": "Ca2+, resact"
  },
  {
    "caption": "F Calibrated pHi and Ca2+ signals evoked by 1 nM resact; the threshold pHi (pHthr) at which the Ca2+ influx commences was deduced from the latency of the Ca2+ signal.",
    "molecules": "Ca2+, resact"
  },
  {
    "caption": "G Linear relationship between pHthr and Vthr for activation of Ca2+ influx by various resact concentrations (data derived from Supplementary Fig S6; mean ± SD; n ≥ 3).",
    "molecules": "Ca2+, resact"
  },
  {
    "caption": "A Dark‐field light microscopy images of a sperm suspension before (top) and after (bottom) photorelease of a resact gradient (middle) in the absence (control; left) or presence of the CatSper inhibitor MDL12330A (10 μM; right). MDL12330A abolishes resact‐induced sperm accumulation.B Relative change of the sperm dispersion in the field of view evoked by photorelease of resact (t = 0, flash) in the absence (control; blue) or presence of MDL12330A (red); a decrease in dispersion indicates sperm accumulation in the irradiated area (mean ± SD; n = 4).",
    "molecules": "MDL12330A, resact"
  },
  {
    "caption": "C Changes in pHi evoked by repetitive release (arrows) of cGMP from caged cGMP. The first UV flash was delivered at t = 0.",
    "molecules": "cGMP"
  },
  {
    "caption": "(A, B) qRT-PCR analysis of IL6 expression upon indicated stimulations in presence or absence of NOD1 (A) or NOD2 (B). HeLa inducible NOD1 cells (A) or NOD2 cells (B) were induced in absence or presence of doxycycline overnight and afterwards stimulated with various stimuli for 4 h.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(D) Western blot analysis of MAPKs and NF-κB activation upon S1P stimulation. HeLa NOD1 or NOD2 cells were treated with cytosolic S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) for indicated times.",
    "molecules": "iE-DAP, MDP, S1P"
  },
  {
    "caption": "Multiplex analysis of CXCL2 (I), CCL5 (J) and IL16 (K) production in supernatants of WT, Nod1/2 and Rip2 KO BMDMs upon S1P (10 µM) or MDP (10 µM) stimulations together with digitonin (2.5 µg/ml) for 20 h.",
    "molecules": "digitonin, MDP, S1P"
  },
  {
    "caption": "(A, B) Immunoprecipitation with various lipid-coated beads using HeLa NOD1-GFP (A) and NOD2-GFP cells (B). Cell lysates of HeLa NOD1 or HeLa NOD2 cells were incubated with lipid-coated beads at room temperature for 2 h.",
    "molecules": "lipid"
  },
  {
    "caption": "(E, F) Single particle tracking of NOD1-GFP (E) or NOD2-GFP (F) with S1P-TAMRA. After doxycycline induction overnight, HeLa NOD1 or NOD2 cells were stimulated with S1P-TAMRA (20 µM) for 1 h together with digitonin. Scale bar: 5 µm.",
    "molecules": "digitonin, doxycycline, S1P"
  },
  {
    "caption": "(D) MST analysis of direct binding of NOD1 to S1P and iE-DAP. (E) MST analysis of direct binding of NOD2 with S1P and MDP.",
    "molecules": "iE-DAP, MDP, S1P"
  },
  {
    "caption": "(F) ELISA analysis of IL8 in supernatants of HEK293T NOD1/2 KO cells transfected with indicated NOD1 or NOD2 mutants. After 24 h transfection, cells were stimulated with S1P (20 µM), iE-DAP (20 µM) or MDP (20 µM) together with digitonin for 20 h.",
    "molecules": "digitonin, iE-DAP, MDP, S1P"
  },
  {
    "caption": "(A, B) MST analysis of direct binding of NOD1 (A) or NOD2 (B) with ADP in the presence of S1P (2 µM).",
    "molecules": "ADP, S1P"
  },
  {
    "caption": "A, Bar graph depicting increased ratio of S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) in SUM159PT KOd and KOs compared to WT cells. n = 3 technical replicates. *P < 0.05; **P < 0.01; Mann-Whitney U-test. All data are means ± SD.",
    "molecules": "S-adenosylhomocysteine, SAH, S-adenosylmethionine, SAM"
  },
  {
    "caption": "A, Representative images and bar graph quantification depicting 5-methylcytosine (5-mC) signal by immunofluorescence in SUM159PT WT, KOs, and KOd cells. n = 2 experimental replicates with 5 to 6 technical replicates each. ***P < 0.001; Kruskal-Wallis test. Data are means ± SD. Scale bar, 50 µm.",
    "molecules": "5-mC, 5-methylcytosine"
  },
  {
    "caption": "D, Bar graphs depicting  5-mC abundance at promoters of the indicated genes shown as fold enrichment of methylated DNA immunoprecipitate (MedIP) over IgG control. n = 3 experimental replicates with 2 to 3 technical replicates each. *P < 0.05, ** P < 0.01, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.",
    "molecules": "5-mC"
  },
  {
    "caption": "E, Bar graphs representing average PRDM5  and collagen gene mRNA expression  upon 5- aza  treatment in SUM159PT  WT, Kos, and KOd cells. n = 3 experimental replicates with 2 technical replicates each. *P < 0.05, n.s., not significant; Kruskal-Wallis test. Data are means ± SEM.",
    "molecules": "5- aza"
  },
  {
    "caption": "A, Bar graph quantification (left panel) and representative bioluminescence images (right panel) of metastases  at day 27 after injection of  MDA-MB-231 cells into the  tail veins  of mice  bearing short hairpin sh NT (non-targeting), sh1 or sh2 targeting PRDM5. Mice  were fed with doxycycline  food to sustain shRNA expression during the whole experiment. n = 7 to 8 animals per group. **P < 0.01; One-way ANOVA. All data are means ± SD.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Domain architecture and structure of Miro. (A) Bar diagram of Miro domain structure. Numbering corresponds to dMiro isoform D, domain names are described in the text (B) Ribbon diagram of the MiroS structure in the 'side view'. Note three distinct domains: ELM1 (blue), ELM2 (green), cGTPase (grey/yellow), joined by two linkers: Lnk1 (orange), and Lnk2 (red). The structure shown is MgGDP‐MiroS, with the Ca2+ ion from the Ca‐MiroS structure overlaid for orientation ( Ca2+/Mg2+/GDP as yellow/purple/multicoloured spheres). cGTPase, C‐terminal GTPase; dMiro, Drosophila Miro; ELM, EF hand pair with ligand mimic.",
    "molecules": "Ca, Ca2+, GDP, Mg"
  },
  {
    "caption": "Details of the EF hand loops. All loops are shown in approximately the same orientation, with entering and exiting helices ghosted for clarity. Residues of note are coloured in orange. cEF1 is shown in its apo state. cEF2 is shown in its apo, Ca2+‐bound and Mg2+‐bound states. In the cEF2 apo state, E354 is stabilized by nearby loop residue R344. In the Ca2+‐bound state, cEF2 loop residues coordinate Ca2+ in a pentagonal, bipyramidal arrangement, with E354 as a bidentate ligand. An ordered water molecule at the apex of one of the pyramids is represented as a small red sphere. In the Mg2+‐bound state, the coordination is octahedral with E354 as a monodentate ligand. The hEF1 and hEF2 loops adopt a conformation quite different from the cEF hands, and also lack the acidic residues that typically coordinate cations. Note the conserved hEF1 272VTL274 and hEF2 386VTL388 residues, which overlay identically in Fig 2A. cEF, canonical EF hand; hEF, hidden EF hand.",
    "molecules": "Ca, Mg"
  },
  {
    "caption": "(A) The ELM2/cGTPase interface can be divided into three regions (insets 1-3). Select residues are shown as sticks, polar contacts as dashed lines, and Mg2+ and MgGDP as spheres. Region 1: αC2 and the hEF2 loop form both backbone and side‐chain contacts with αG1 and Switch I in the cGTPase, including the Switch I threonine T481. Region 2: αD2 in hEF2 packs against the twisting cGTPase β‐strands Gβ1‐3. Region 3: the candidate Pink1 phosphorylation site residue S324 in Lnk1 hydrogen bonds with R444 in Lnk2, which also forms a salt bridge with D521, at the end of the Switch II loop.",
    "molecules": "GDP, Mg"
  },
  {
    "caption": "(B) cGTPase rearrangements in MgGDP binding. Displayed is the MiroS‐MgGDP structure, with select residues from the aligned apo‐MiroS structure shown in orange. GDP is shown as sticks (2Fo‐Fc map, 1.2σ), and Mg2+ as a purple sphere. In the apo state, D505 in the Switch II 503DIDV506 motif is far from the nucleotide‐binding pocket and is hydrogen bonded to R507, which is also stabilized by interactions with P512. In the MgGDP‐bound state, the D505 side‐chain rotates ∼7 Å to interact with the β‐phosphate (arrow), locking the Switch II loop in a β‐strand conformation and extending Gβ3. V506 rotates away from the nucleotide‐binding pocket, and both R507 and Y537 become solvent exposed. cGTPase, C‐terminal GTPase; Sw I/II, Switch I/II; P loop, phosphate‐binding loop.",
    "molecules": "GDP, Mg"
  },
  {
    "caption": "A-B Time dependent effects of ingesting 0, 0.1, 1, 5, 10, 30, and 50 mM vitamin C without any further nutrition on the survival of wild-type control flies (w1118) (A) males (B) females, n = 6. Kaplan-Meier survival analyses were performed to compare the survival rates of agar only and fed conditions at different concentrations of vitamin C",
    "molecules": "agar, vitamin C"
  },
  {
    "caption": "F Electrophysiology mapping of 31 sensilla with control to 50 mM vitamin C, n = 10-18.",
    "molecules": "vitamin C"
  },
  {
    "caption": "G Tip recordings from the L4 and L6 sensilla of specific GRN-ablated flies with 50 mM vitamin C. +/- indicates the presence or absence of the transgene, respectively, n = 10-13.",
    "molecules": "vitamin C"
  },
  {
    "caption": "D Behavioral rescue of Gr5a∆5, Gr61a1, Gr64bLEXA, Gr64cLEXA, and Gr64eLEXA deficits in the vitamin C attraction via the expression of the respective UAS transgenes under the control of their specific GAL4s or Gr64f-GAL4, n = 6-8.",
    "molecules": "vitamin C"
  },
  {
    "caption": "(B) Knock-out of several genes with different functionalities in the cell envelope increase death rates. Growth in glycerol minimal medium, starvation induced by glycerol depletion.",
    "molecules": "glycerol"
  },
  {
    "caption": "(C) 8 µg/ml Polymyxin B and 4 µg/ml Colistin, added daily to cultures previously grown on glucose minimal medium (reference condition) with the first dose at 24h. Antibiotic concentration was 10x minimum inhibition concentration during growth (MIC). Each data point is the average of two biological repeats. Error bars are not shown because the variation is smaller than symbol size. Viabilities indicated at <101 mean no colonies were detected from 100 µl culture.",
    "molecules": "Colistin, Polymyxin B"
  },
  {
    "caption": "(D) Death rates for different Mg2+ concentrations. Reference condition (black circle in Fig. 1A) has 0.41 mM Mg2+. DNP+ cultures were supplemented with 1 mM 2,4-dinitrophenol during starvation in reference medium. Each data point is from a single experiment. All cultures were grown in glucose minimal medium. P-values (Student's t-test, two-tailed) are indicated in comparison to the reference condition.",
    "molecules": "2,4-dinitrophenol, DNP, Mg2+"
  },
  {
    "caption": "Death rate plotted versus growth rate on a semi-log scale for experiments in 'osmo-balanced' medium. Colors indicate carbon limitation (C, 'blue', acetate and glycerol), anabolic limitation (A, 'green', 3µM IPTG), ribosome limitation (R, 'yellow', 4µM Chloramphenicol), rich medium (L, 'red', LB), LacZ overexpression (O, 'grey', 10ng/ml cTc), stationary phase (S, 'white') and the glucose reference condition ('black'), as defined in Fig. 1A. The data shows weak anti-correlation with growth rate; solid black line is an exponential fit to the data (R2 = 0.34). Dashed lines are the exponential fits to the CARLOS data in Fig. 1A.",
    "molecules": "acetate, carbon, Chloramphenicol, cTc, glycerol, IPTG"
  },
  {
    "caption": "H NF2 and WWC2 interact with LATS1 as indicated by BioID labeling. HEK293A cells stably expressing BirA-NF2, WWC2, MOB1A, or SAV1 were labeled with biotin overnight, and endogenous kinases were immunoprecipitated and subjected to immunoblotting using Streptavidin-HRP. No biotinylation of MAP4K4 was detected.",
    "molecules": "biotin, Streptavidin"
  },
  {
    "caption": "C Both HPO1 and HPO2 are required for LATS1/2 activation by actin disrupting agent LatB. Control or KO HEK293A cells were treated with 0.2 μg/ml LatB for 30 min, and cell lysates were subjected to immunoblotting to analyze phosphorylation of LATS1, YAP, NF2, and MOB1. Asterisk indicates S13 phosphorylated NF2.",
    "molecules": "LatB"
  },
  {
    "caption": "A Measurement of urinary albumin/creatinine by ELISA (albumin-to-creatinine ratio: day 14: Phb2fl/fl 0.09 ± 0.03 mg/mg, n = 4, versus Phb2pko 0.37 ± 0.21 mg/mg, n = 4, P = 0.2350; day 21: Phb2fl/fl 0.65 ± 0.16 mg/mg, n = 4, versus Phb2pko 193.10 ± 26.81 mg/mg, n = 4, ***P = 0.003; day 28: Phb2fl/fl 0.57 ± 0.07 mg/mg, n = 4, versus Phb2pko 470.40 ± 131.30 mg/mg, n = 4, *P = 0.0117).",
    "molecules": "creatinine"
  },
  {
    "caption": "D Measurement of serum creatinine levels of mice in their fifth week of life (Phb2fl/fl/het 14.74 ± 3.75 μmol/l versus Phb2pko 74.73 ± 2.20 μmol/l, n = 3 for both groups; ***P = 0.0002).",
    "molecules": "creatinine"
  },
  {
    "caption": "E Measurement of serum urea levels of mice in their fifth week of life (Phb2fl/fl/het 44.00 ± 7.02 mg/dl versus Phb2pko 493.70 ± 45.34 mg/dl, n = 3 for both groups; ***P = 0.006).",
    "molecules": "urea"
  },
  {
    "caption": "A PAS staining of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).B Immunohistochemistry for podocin on kidney sections of tPod-Phb2pko and control mice 2.5 weeks after the end of tamoxifen treatment (scale bar: 20 μm).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "A Serum creatinine and urea levels in the fifth week of life (left graph: Phb2pko 74.73 ± 2.20 μmol/l versus Phb2pko/Insrpko/Igf1rpko 37.36 ± 8.83 μmol/l, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0169; right graph: Phb2pko 493.70 ± 45.34 mg/dl versus Phb2pko/Insrpko/Igf1rpko 272.80 ± 49.29 mg/dl, n = 3 for Phb2pko and n = 4 for Phb2pko/Insrpko/Igf1rpko, *P = 0.0247).",
    "molecules": "creatinine, urea"
  },
  {
    "caption": "F Measurements of mitochondrial respiration with complex II substrates (succinate and glycerol-3-phosphate) did not show significant differences between Phb2-deficient and control podocytes (n = 6).",
    "molecules": "glycerol-3-phosphate, succinate"
  },
  {
    "caption": "H FACS analysis of MitoTracker- and MitoSOX-stained Phb2-deficient podocytes and control podocytes.I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2-deficient podocytes compared to control podocytes (n = 3, P = 0.7782 for MitoTracker, P = 0.1088 for MitoSOX).",
    "molecules": "ROS"
  },
  {
    "caption": "A Phb2-deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t-test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein.",
    "molecules": "rapamycin"
  },
  {
    "caption": "C Kaplan-Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle-treated Phb2pko mice (n = 11 for Phb2fl/fl + rapamycin, n = 15 for Phb2pko + vehicle, n = 16 for Phb2pko + rapamycin).D Statistical analysis comparing rapamycin-treated Phb2pko mice with vehicle-treated Phb2pko mice.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(D) Livers from WT, Clec4F-DTR+ or CD169-DTR+ mice treated i.p. with DT, or WT mice treated i.v. with PLL or CLL were analyzed by IHC to visualize F4/80+ macrophages (brown). Two representative sections for each group are shown.",
    "molecules": "CLL, PLL"
  },
  {
    "caption": "(B) WT C57BL/6 mice were treated i.v. with PLL or CLL 42 h prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was quantified by FFA. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; ***P < 0.001.",
    "molecules": "CLL, PLL"
  },
  {
    "caption": "(A) CD169-DTR+ mice were treated i.p. with PBS or DT prior to collection of the popliteal LN. Frozen LN sections were stained for CD169+ macrophages (green), B220+ B cells (blue) or ERTR-7+ stromal cells (red). Image shown is representative of three biological replicates. Scale bars: 200 μm.",
    "molecules": "PBS"
  },
  {
    "caption": "1D 1H-NMR spectra of a metabolite mix and the individual metabolites contained therein. Identification of single compound peaks from 1D 1H-NMR spectra of a metabolite mix. Compound detection is exemplified by showing sections of the 1D 1H-NMR spectra of ATP, IMP, proline (PRO), malate (MAL), shikimate (SKM) and methionine (MET).",
    "molecules": "ATP, IMP, MAL, malate, MET, methionine, PRO, proline, shikimate, SKM"
  },
  {
    "caption": "Relative activity of NADP+-dependent glucose-6-phosphate dehydrogenase in the presence of potential regulators, error bars represent the s.e.m.. All regulators were tested at two concentrations in four distinct replicates (n=4, ATP: 5mM, 18 mM; GTP: 2 mM, 10 mM; IMP and AMP: 1 mM, 5 mM), the asterisk denotes significant inhibition (18 mM ATP: p-value = 0.029, 10 mM GTP: p-value = 0.025, one-tailed t-test).",
    "molecules": "AMP, ATP, GTP, IMP, NADP"
  },
  {
    "caption": "Relative activity of phosphate acetyltransferase in the presence of phenylpyruvate and tryptophan, error bars represent the s.e.m.. All regulators were tested at three concentrations in three distinct replicates (n=3, phenylpyruvate: 0.1, 1, 5 mM, L-tryptophan: 0.1, 1, 4.4 mM).",
    "molecules": "L-tryptophan, tryptophan, phenylpyruvate"
  },
  {
    "caption": "Relative activity of fructose-bisphosphate aldolase class II in the presence of 5 mM of potential regulators, except hypoxanthine, which was tested at 3.22 mM. All regulators were tested in three distinct replicates (n=3), error bars represent the s.e.m.. The asterisk denotes significant activation (5 mM 3PG: p-value = 0.025, 5 mM ATP: p-value = 0.024, 5 mM PEP: p-value = 0.046, one-tailed t-test).",
    "molecules": "3PG, ATP, hypoxanthine, PEP"
  },
  {
    "caption": "B, C Kinetic change of mRNA expression  of Top1, Btbd1, Tdp1, and Srsf1  in microglia  following LPS/IFNγ  stimulation (n = 6, technical replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using two-way ANOVA with Dunnett's Multiple Comparison test for (B and C),",
    "molecules": "LPS"
  },
  {
    "caption": "F-H Immunostaining  of TOP1  (red) in stimulated microglia  (4 h after LPS/IFNγ  stimulation) or resting microglia, co-stained with Hoechst  (blue) (bar = 20 μm); for the lower-left panel in (H), n = 3 technical replicates per group (2 to 5 random fields per well were analyzed); for the lower right panel, 201 cells from unstimulated microglia  and 256 cells from stimulated microglia  were analyzed. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using the student's unpaired two-tailed t-test.",
    "molecules": "Hoechst, LPS"
  },
  {
    "caption": "G Immunofluorescent staining  of spinal cord sections from mice  at EAE  peak and control mice  with anti-F4/80 (green), TOP1  (magenta), and nuclei  with Hoechst  (blue). For the left panels, bar = 400 μm; for the magnified panels, bar = 100 μm. The yellow arrowhead indicates a representative F4/80+ cell with high TOP1 expression.",
    "molecules": "Hoechst"
  },
  {
    "caption": "M,  Western blotting of TOP1 expression  from brain  homogenates of mice  receiving intracisternal LPS  injection or PBS  after 8 h (n = 4 or 5 biological samples).",
    "molecules": "LPS, PBS"
  },
  {
    "caption": "TOP1 expression  from brain  homogenates of mice  receiving intracisternal LPS injection or PBS  after 8 h (n = 4 or 5 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.",
    "molecules": "LPS, PBS"
  },
  {
    "caption": "O Microglia  were sorted from LPS  challenged mice  (i.p) after 4 h or naive mice, and the mRNA expression  of TOP1  was graphed (n = 5 or 6 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.",
    "molecules": "LPS"
  },
  {
    "caption": "P Brain  sections from healthy control and MS brain  tissues, including the active MS  lesion, chronic active lesion, and the normal-appearing white matter (NAWM), were stained  with anti-HLA-DR, anti-TOP1, and co-stained  with Hoechst  (bar = 50 μm).",
    "molecules": "Hoechst"
  },
  {
    "caption": "C, D Immunostaining of iNOS  (red) and Phalloidin  (green) in microglia  (n = 3 technical replicates with more than 5 random fields per well) (bar = 50 μm). Data information: Data shown are mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; n.s. = no statistical significance. Statistical analyses were performed using one-way ANOVA with Dunnett's Multiple Comparison Test for (D)",
    "molecules": "Phalloidin"
  },
  {
    "caption": "C, D Fluorescent intensity of Iba1  in hippocampus  and thalamus  in (B); n = 2, 4, 5 mice  for naive, LPS, and LPS  + TPT  group, respectively (2-6 sections per mouse  were analyzed). Data information: Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses comparing the  LPS_PBS group with the LPS_TPT group were performed using the Student's unpaired two-tailed t-test",
    "molecules": "LPS, PBS, TPT"
  },
  {
    "caption": "L, M Cytokine array  on brain  homogenates after 24 h of LPS  challenge (n = 2 mice  for the naïve group; n = 3 mice  for the LPS  and LPS  + TPT  groups).",
    "molecules": "LPS, TPT"
  },
  {
    "caption": "K Spinal cord  sections dissected from Day 30 from the EAE  batch in (H) were stained  with CD11b  (red), Fluoromyelin   (green), and Hoechst  (blue) (bar = 200 μm). L The CD11b immunofluorescent  intensity in (K) was graphed (n = 2 mice  for the control group; n = 4 mice  for the EAE-PBS  and EAE-TPT  groups). M The percentage of demyelinated area in the white matter of the spinal cord  was graphed (n = 2 mice  for the control group, n = 4 mice  for the EAE-PBS  and EAE-TPT  groups). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; the Student's unpaired two-tailed t-test was used for comparing the EAE-PBS  group with the EAE-TPT  group in L, and M.",
    "molecules": "Fluoromyelin, Hoechst, PBS, TPT"
  },
  {
    "caption": "E) Western blots of spinal cord mitochondria at disease end stage, probed for lysine 48 (left panel) and lysine 63 (right panel) ubiquitin chains. Citrate synthase is used as loading control. Asterisks indicate ubiquitinated proteins with different abundance in SOD1-G93A samples.",
    "molecules": "lysine, ubiquitin"
  },
  {
    "caption": "GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates.",
    "molecules": "Gly"
  },
  {
    "caption": "(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.",
    "molecules": "Gly"
  },
  {
    "caption": "(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.",
    "molecules": "diGly, MitoTracker"
  },
  {
    "caption": "Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized. Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).",
    "molecules": "Gly, Leu"
  },
  {
    "caption": "Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.",
    "molecules": "IMP-1088"
  },
  {
    "caption": "Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.",
    "molecules": "IMP-1088"
  },
  {
    "caption": "Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.",
    "molecules": "Ala"
  },
  {
    "caption": "B. TBK1/IKKε phosphorylate mTOR S2159 directly in vitro: In vitro kinase (IVK) assays with recombinant (re) active TBK1 or IKKε [50 ng] (Invitrogen) and recombinant GST-mTOR substrate [200 ng] for 30 min at 30oC. Reactions were pre-incubated on ice 30 min with amlexanox [500, 250 or 50 μM], BX-795 [10 μM] or MRT-67307 [10 μM] and immunoblotted (IB) as indicated.",
    "molecules": "amlexanox, MRT-67307"
  },
  {
    "caption": "H. The TBK1- and IKKε-activating agonists poly(I:C) and LPS increase mTOR P-S2159 in a BX-795 sensitive manner in cultured macrophages: RAW264.7 macrophages were pre-treated with BX-795 [10 μM] (2 hr.) and stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).",
    "molecules": "LPS, BX-795, poly (I:C), poly(I:C)"
  },
  {
    "caption": "I. Poly(I:C) and LPS increase mTOR P-S2159 in primary bone marrow-derived macrophages (BMDMs): BMDMs were stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).",
    "molecules": "LPS, poly (I:C), Poly(I:C)"
  },
  {
    "caption": "J. LPS increases mTOR P-S2159 in vivo: Mice (C57BL6, 6 wks old) were injected intraperitoneally with PBS or LPS [1 mg/kg-BW] (2 hr.). mTOR was immunoprecipitated from spleen tissue, and IPs and WCL were immunoblotted as indicated. The graph depicts levels of mTOR P-S2159 relative to total mTOR in spleen tissue -/+ LPS. n=8 from 3 independent experiments +/- SD. *p=.004 relative to PBS treated control mice by paired t-test (two-tailed).",
    "molecules": "LPS"
  },
  {
    "caption": "A. EGF increases mTORC1 signaling in a TBK1-dependent manner: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min), and stimulated -/+ EGF [25 ng/mL] (0-60 min). Whole cell lysate (WCL) were immunoblotted (IB) as indicated.",
    "molecules": "Ku-0063794"
  },
  {
    "caption": "E. TBK1 is not required for insulin-stimulated mTORC1 signaling: TBK1+/+ and TBK1-/- MEFs were stimulated with insulin as in C.",
    "molecules": "insulin"
  },
  {
    "caption": "F. Side-by-side comparison of EGF- vs. insulin-stimulated mTORC1 and mTORC2 signaling in TBK1+/+ vs. TBK1-/- MEFs: MEFs were treated as in C.",
    "molecules": "insulin"
  },
  {
    "caption": "G. Pharmacologic TBK1 inhibition reduces EGF-induced mTORC1 signaling: HEK293 cells were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min) or amlexanox [50 μM] (2 hr.), and stimulated -/+EGF [25 ng/mL] (30 min) or insulin (INS) [100 nM] (30 min).",
    "molecules": "amlexanox, insulin, Ku-0063794"
  },
  {
    "caption": "H. EGF but not insulin increases mTOR P-S2159: TBK1+/+ vs. TBK1-/- MEFs: MEFs were treated as in C.",
    "molecules": "insulin"
  },
  {
    "caption": "A. mTORC1 signaling remains intact in MEFs lacking mTORC2 function: Rictor-/- MEFs stably expressing vector control (V) or rescued stably with HA-rictor were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min), and stimulated -/+epidermal growth factor (EGF) [25 ng/mL] (30 min). Whole cell lysate (WCL) were immunoblotted as indicated.",
    "molecules": "Ku-0063794"
  },
  {
    "caption": "B. TBK1 is required for mTORC1 signaling in MEFs with reduced Akt S473 phosphorylation: TBK1 expression was reduced using lentivirally-delivered TBK1 shRNA in Rictor-/- MEFs stably expressing vector control (V). Cells were EGF stimulated as above, except Torin1 [100 nM] was used to inhibit mTOR.",
    "molecules": "Torin1"
  },
  {
    "caption": "A. mTOR S2159 phosphorylation is required for EGF stimulated mTORC1 signaling: HEK293 cells were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were serum starved (20 hr.), treated -/+ rapamycin (30 min) to ablate endogenous mTORC1 function, and stimulated -/+EGF [25 ng/mL] (30 min). HA-S6K1 was immunoprecipitated, and immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) as indicated.",
    "molecules": "rapamycin"
  },
  {
    "caption": "D. TBK1 is required for EGF stimulated mTOR auto-phosphorylation within mTORC1: TBK1+/+ and TBK1-/- MEFS were serum starved, pre-treated with Ku-0063794 [1 μM], and EGF stimulated as in A. Raptor was immunoprecipitated, and IPs and WCL were analyzed.",
    "molecules": "Ku-0063794"
  },
  {
    "caption": "E. mTOR S2159 phosphorylation is required for EGF stimulated mTOR auto-phosphorylation: HEK293 cells were transfected with Myc-mTOR wild type (WT), S2159A, and kinase dead (KD). Cells were then serum starved, pre-treated with Ku-0063794 (30 min), and stimulated -/+ EGF as in A.",
    "molecules": "Ku-0063794"
  },
  {
    "caption": "A. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in cultured macrophages: RAW264.7 cells cultured in full serum were pre-treated with amlexanox ([50 μM or 100 μM]) (2 hr.), rapamycin [20 ng/mL] (30 min), or Ku-0063794 [1 μM] (30 min) and stimulated -/+poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Whole cell lysate (WCL) was immunoblotted as indicated.",
    "molecules": "LPS, amlexanox, Ku-0063794, poly (I:C), rapamycin"
  },
  {
    "caption": "B. Knockdown of TBK1 with shRNA reduces TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-infected with shRNA-containing lentiviruses targeting TBK1 and IKKε shRNA or infected with scrambled control (Scr), selected in puromycin, and treated as in A.",
    "molecules": "puromycin"
  },
  {
    "caption": "C. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in primary macrophages: Bone marrow derived macrophages-monocytes (BMDMs) were pre-treated with amlexanox [100 μM] (2 hr.) or Ku-0063794 [1 μM] (30 min) and stimulated -/+ poly(I:C) or LPS as in A.",
    "molecules": "LPS, amlexanox, Ku-0063794, poly(I:C)"
  },
  {
    "caption": " D. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 in HEK293-TLR3 cells. Cells were serum starved (20 hr.), pre-treated with amlexanox or Ku-0063794 as in C above, and stimulated -/+ poly(I:C) [50 ug/mL] (60 min).",
    "molecules": "amlexanox, Ku-0063794, poly(I:C)"
  },
  {
    "caption": "E. TLR4/LPS-stimulated mTORC1 signaling requires TBK1: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.), pre-treated with amlexanox [50 μM] (2 hr.) or Torin1 [100 nM] (30 min), and stimulated -/+ LPS as in A.",
    "molecules": "Torin1, LPS, amlexanox"
  },
  {
    "caption": "A. TBK1 activity is required for TLR3- and TLR4-stimulated mTORC1 catalytic activity: RAW264.7 macrophages were pre-treated with amlexanox ([50 μM] or 100 μM]) (2 hr.), rapamycin [20 ng/mL] (30 min), or Ku-0063794 [1 μM] (30 min) and treated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Raptor was immunoprecipitated (IP), and IPs and whole cell lysate (WCL) were immunoblotted (IB) as indicated.",
    "molecules": "poly (I:C), rapamycin"
  },
  {
    "caption": "B. and C. mTOR S2159 phosphorylation is required for TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were treated with rapamycin (+) to ablate endogenous mTORC1 function and stimulated with poly(I:C) (A) or LPS (B) as in A. HA-S6K1 was immunoprecipitated, and IPs and WCL was immunoblotted as indicated.",
    "molecules": "LPS, rapamycin"
  },
  {
    "caption": "B. and C. mTOR S2159 phosphorylation is required for TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were treated with rapamycin (+) to ablate endogenous mTORC1 function and stimulated with poly(I:C) (A) or LPS (B) as in A. HA-S6K1 was immunoprecipitated, and IPs and WCL was immunoblotted as indicated.",
    "molecules": "LPS, poly(I:C), rapamycin"
  },
  {
    "caption": "D. Pharmacologic inhibition of PI3Kα but not Akt reduces TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were pre-treated with the PI3Kα class I inhibitor BYL-719 [10 μM], the Akt inhibitor MK-2206 [10 μM], amlexanox [100 uM], or Ku-0063794 [1 μM] (30 min) and stimulated -/+poly (I:C) or LPS as in A.",
    "molecules": "LPS, amlexanox, BYL-719, Ku-0063794, MK-2206, poly (I:C)"
  },
  {
    "caption": "A. Rapamycin suppresses TLR3-stimulated IFNβ protein production in cultured macrophages: RAW264.7 macrophages were pre-treated with rapamycin [20 ng/mL] (30 min), Ku-0063794 [1 μM] (30 min), or amlexanox [50 μM] (2 hr.) and stimulated -/+poly (I:C) [30 μg/ml] for 6 hr. The secretion of IFNβ was measured by ELISA. Results represent the mean +/- SD of quadruplicate samples from one experiment. *p=.0009 relative to +Poly(I:C) by paired t-test (two-tailed).",
    "molecules": "amlexanox, Ku-0063794, poly (I:C), Poly(I:C), Rapamycin, rapamycin"
  },
  {
    "caption": "B. Rapamycin suppresses TLR3-stimulated IFNβ mRNA production in cultured macrophages: RAW264.7 macrophages were treated as above but stimulated -/+poly (I:C) [30 μg/ml] for 3 hr. q-RT-PCR measured IFNβ gene expression. Results represent the mean +/- SD of triplicate samples from one experiment. *p=.03 relative to no Poly(I:C) by paired t-test (one-tailed); **p=.04 relative to +Poly (I:C) by paired t-test (one-tailed).",
    "molecules": "poly (I:C), Poly (I:C), Poly(I:C), Rapamycin"
  },
  {
    "caption": "C. Rapamycin suppresses TLR3-stimulated IFNβ protein production in primary macrophages: BMDMs were pre-treated with rapamycin or amlexanox and analyzed as in A. Results represent the mean +/- SD of triplicate samples from one experiment. *p=.002 relative to +Poly (I:C) by paired t-test (two-tailed).",
    "molecules": "amlexanox, Poly (I:C), Rapamycin, rapamycin"
  },
  {
    "caption": "D. Rapamycin suppresses TLR3-stimulated IRF3 nuclear translocation in RAW264.7 macrophages: Cells were pre-treated with rapamycin and stimulated -/+ poly (I:C) for 3 hr. as in A, fixed, and processed for confocal immunofluorescence microscopy using an anti-IRF3-Alexa 488 antibody and Dapi staining. The graph represents the mean +/- SD of at least 400 cells total from three independent experiments. *p=.002 relative to no Poly(I:C) by paired t-test (two-tailed); **p.003 relative to +Poly (I:C) by paired t-test (two-tailed). Scale bar = 10 μM.",
    "molecules": "Alexa 488, Dapi, poly (I:C), Poly (I:C), Poly(I:C), Rapamycin, rapamycin"
  },
  {
    "caption": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"> <?xml encoding=\"UTF-8\">",
    "molecules": "Alexa 488"
  },
  {
    "caption": "A. Reduced TLR3-induced mTORC1 signaling in mTOR S2159A knock-in primary macrophages (mTORA/A) relative to wild type (mTOR+/+): BMDMs were pre-treated with Torin 1 [100 nM] (30 min) and stimulated -/+ poly (I:C) [30 μg/ml] for 30 min. Whole cells lysate (WCL) was immunoblotted as indicated. The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with poly (I:C) was set at 100%. *The confidence interval at 95% (52.7-59.1) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs.",
    "molecules": "Torin 1, poly (I:C)"
  },
  {
    "caption": "B. Reduced TLR4-induced mTORC1 signaling in mTORA/A primary macrophages: BMDMs were treated as in A except stimulated -/+ LPS [100 ng/mL] (30 min). The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with LPS was set at 100%. *The confidence interval at 95% (58.6-71.2%) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs.",
    "molecules": "LPS"
  },
  {
    "caption": "C. Reduced TLR3-stimulated IFNβ protein production in mTORA/A primary macrophages: BMDMs were serum starved for 6 hr. and stimulated -/+ poly (I:C) [30 μg/ml] for 6 hr. The secretion of IFNβ was measured by ELISA. Results represent the mean +/- SEM from three independent experiments, n=8 samples total. *p=.0002 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.0001 relative to mTOR+/+ + Poly(I:C) by paired t-test (two-tailed).",
    "molecules": "poly (I:C), Poly(I:C)"
  },
  {
    "caption": "D. Reduced TLR3-stimulated IRF3 nuclear translocation in mTORA/A primary macrophages: BMDMs were treated -/+ poly (I:C) for 3 hr as in A, fixed, and processed for confocal immunofluorescence microscopy using an anti-IRF3-Alexa 488 antibody and Dapi stain. The graph represents the mean +/- SEM of at least 270 cells total from four independent experiments. *p=.001 relative to mTOR+/+ no Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.02 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). Scale bar = 10 μM.",
    "molecules": "Alexa 488, Dapi, poly (I:C), Poly(I:C)"
  },
  {
    "caption": "Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).",
    "molecules": "3-methyladenosine, MG132, PLX3397, rapamycin"
  },
  {
    "caption": "Immunocytochemistry of microglia isolated from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ P0 mouse pup brains. Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker(red). Arrowheads (white) indicate bead+ cells. Quantification of mouse microglial phagocytic activity expressed as percentage bead+ cells (n = 4 assays, error bars indicate SEM).",
    "molecules": "latex beads, LPS, MitoTracker"
  },
  {
    "caption": "L) Immunocytochemistry of macrophages differentiated in vitro from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mouse bone marrow. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells. M) Quantification of mouse macrophage phagocytic activity expressed as percentage bead+ cells. (* p = 0.024, Student's t-test with Welch's correction, n = 4 assays, error bars indicate SEM).",
    "molecules": "latex beads, DAPI, LPS, MitoTracker"
  },
  {
    "caption": "A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(H, I) RNA EMSA evaluation of EZh2 binding of p53 mRNA. GST-EZH2 recombinant proteins (EZ1 to EZ4) were incubated with biotin-labeled in vitro transcribed p53 5'UTR (biotin-labeled probe) in the presence or absence of 100-fold unlabeled p53 5'UTR (unlabeled probe), followed by PAGE and immune blotting with HPR-conjugated streptavidin.",
    "molecules": "biotin, HPR, streptavidin"
  },
  {
    "caption": "(A) Biotin pull down assay by incubating biotin-labelled different fragments of p53 mRNA and HOTAIR (positive control) with C4-2 cell lysate followed by western blot with EZH2 antibody.",
    "molecules": "Biotin, biotin"
  },
  {
    "caption": "(B) Biotin pull down assay as in (A) using unmutated and various internally deleted mutants of 5'UTR of p53 mRNA. Top, diagram of different p53 5'UTR deletion mutants.",
    "molecules": "Biotin"
  },
  {
    "caption": "(F) U2OS cells were transfected with control (siC) or EZH2-specific siRNAs and treated with 200 nM of CPT followed by western blot analysis for indicated proteins.",
    "molecules": "CPT"
  },
  {
    "caption": "(C) Co-IP of endogenous EZH2 with eIF4G2 or PABP1 from C4-2 cell lysate pre-treated with RNaseA in 37oC for 30 min. The effectiveness of RNaseA treatment was monitored by RT-PCR analysis of the presence of GAPDH mRNA.",
    "molecules": "RNaseA"
  },
  {
    "caption": "(A, B) VCaP cells stably expressing control (sh-control) or p53-specific shRNA were infected with lentivirus for empty vector (EV) or deletion mutants of EZH2. Cell growth in 2D (A) and 3D (B) conditions were determined by MTS assay and measurement of clone size, respectively. Statistical significance was determined by two-tailed Student's t-test. * P < 0.05, *** P < 0.001.",
    "molecules": "MTS"
  },
  {
    "caption": "(E-G) Luciferase-expressing MIA PaCa-2 cells (2×106) infected with lentivirus were injected via tail vein into NSG mice (n = 6/group). At 12 weeks after injection, mice were subjected to bioluminescent imaging, and images were recorded (E) and bioluminescent signals were quantified (F). Bioluminescent flux (photons s-1 sr-1 cm-2) was determined for lesions in lung, the ends of the box are the upper and lower quartiles and the box spans the interquartile range; the median is marked by a vertical line inside the box; the whiskers are the two lines outside the box that extend to the highest and lowest observations. Lungs were isolated from mice, stained with Bouin's solution, and photographed (G). The white spots on lungs (stained in yellow) are metastatic tumors. * P < 0.05; NS, no significance.",
    "molecules": "Bouin's solution"
  },
  {
    "caption": "VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by western blots with indicated antibodies (C) Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.",
    "molecules": "DZNep, GSK-126"
  },
  {
    "caption": "VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by MTT assay (D). Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.",
    "molecules": "MTT, DZNep, GSK-126"
  },
  {
    "caption": "(C) Representative images of Alizarin Red staining at 21 days of osteogenic induction (left panel) and quantification of calcification (right panel) of BMSCs isolated from 2-month-old and 24-month-old mice by detecting the amount of Alizarin Red extracted from the matrix.",
    "molecules": "Alizarin Red"
  },
  {
    "caption": "(F and G) Representative images of Alizarin Red staining (F) and quantification of calcification (G) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with adenovirus driven control and Ybx1 shRNA at 21 days of osteogenic induction.",
    "molecules": "Alizarin Red"
  },
  {
    "caption": "(H and I) Representative images of Oil Red O staining (H) and quantification of lipid formation by detecting the amount of Oil Red O extracted from the matrix (I) in BMSCs after 10 days of adipogenic induction.",
    "molecules": "lipid, Oil Red O"
  },
  {
    "caption": "(G) Representative images of H&E staining (Hematoxylin-Eosin Staining) in distal femora. Scale bar: 100μm. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin (OCN) immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. Scale bar: 50 μ (J) Quantification of osteocalcin positive cells on the bone surface. Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. Scale bar: 100 μm. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm).",
    "molecules": "TRAP, Trap, Eosin, Hematoxylin"
  },
  {
    "caption": "(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50 μm. (N) Quantification of the bone formation rate (BFR) based on calcein double labeling. (O) Quantification of the mineral apposition rate (MAR) based on calcein double labeling.",
    "molecules": "calcein"
  },
  {
    "caption": "(G) Representative images of H&E staining in distal femora. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. (J) Quantification of osteocalcin positive cells in bone surface . Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). (M) Representative images of calcein double labeling of trabecular bone. (N) Quantification of the bone formation rate (BFR) and mineral apposition rate (MAR) based on calcein double labeling.",
    "molecules": "TRAP, Trap, calcein"
  },
  {
    "caption": "(D-G) Representative images of Alizarin Red staining (D and F) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (E and G) in BMSCs transfected with different isoforms of Sp7 or different isoforms of Spp1.",
    "molecules": "Alizarin Red"
  },
  {
    "caption": "(H-I) Representative images (H) and quantification of Oil Red O staining (I) in BMSCs transfected with different isoforms of Spp1 with 10 days of adipogenic induction.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(L-T) BMSCs were isolated from Ybx1Prx1-CKO mice and Ybx1flox/flox mice, among them, the BMSCs from Ybx1flox/flox mice were transfected with the blank control, BMSCs from Ybx1Prx1-CKO mice were transfected with the blank control or different isoforms of the target genes. (L-P) Representative images of Alizarin Red staining (L and O) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (M and P). (Q-R) Representative images (Q) and quantification of Oil Red O staining (R) in BMSCs after 10 days of adipogenic induction. (S-T) Representative images of β-Gal staining (S) and quantification (T) of β-Gal positive cells among BMSCs.",
    "molecules": "Alizarin Red, Oil Red O"
  },
  {
    "caption": "(D) Representative images of Alizarin Red staining (up panel), Oil Red O staining (middle panel, scale bar: 100 μm) and β-Gal staining (bottom panel, scale bar: 100 μm) in BMSCs treated with different compounds. (E-G) Quantification of calcium mineralization based on Alizarin Red staining (E), quantification of Oil Red O based on Oil Red O staining (F), and quantification of β-Gal positive cells based on β‑Gal staining in BMSCs treated with different compounds (G).",
    "molecules": "Alizarin Red, calcium, Oil Red O"
  },
  {
    "caption": "(M) Western blotting analysis of FBXO33 and YBX1 levels in BMSCs treated with different concentrations of sciadopitysin.",
    "molecules": "sciadopitysin"
  },
  {
    "caption": "(N) Semi-quantitative PCR showed the isoforms of Fn1, Nrp2 and Sirt2 in cultured BMSCs isolated from 2-month-old or 24-month-old mice then treated with or without sciadopitysin.",
    "molecules": "sciadopitysin"
  },
  {
    "caption": "(B-F) Representative μCT images, Scale bar: 1mm. (B) and quantitative analysis of trabecular bone microarchitecture (C-F) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).",
    "molecules": "sciadopitysin"
  },
  {
    "caption": "(G, H) Representative images (G) and quantification (H) of osteocalcin (OCN) positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of Ocn+ cells per bone perimeter (N. Ocn+/B.Pm). Arrows point to osteocalcin positive cells. Scale bar: 50 μ",
    "molecules": "sciadopitysin"
  },
  {
    "caption": "(I, J) Representative images (I) of H&E staining in distal femora and quantification (J) of the number of adipocytes related to the tissue area (right panel, N. adipocytes/T.Ar) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Scale bar: 100 μm.",
    "molecules": "sciadopitysin"
  },
  {
    "caption": "(K, L) Representative images (K) and quantification (L) of TRAP positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). Scale bar: 50 μm.",
    "molecules": "TRAP, Trap, sciadopitysin"
  },
  {
    "caption": "(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50μm. (N, O) Quantification of the bone formation rate (BFR, N) and mineral apposition rate (MAR, O) based on calcein double labeling.",
    "molecules": "calcein"
  },
  {
    "caption": "C Thionine-stained motor neurons  of Atf3:BAC2 Tg and Rpt3  CKO mice at 7, 14 and 17 days after hypoglossal nerve axotomy. Arrows indicate the loss of  motor neurons. D The graph showing the percentage of surviving motor neurons  on the injured  side compared with that on the control side. Data are shown as the mean ± s.e.m. (** P = 0.0002, *** P < 0.0001, determined by unpaired t-test, n = 5 mice  per group at each  time point). Data information: Dashed lines outline hypoglossal nucleus in XII, hypoglossal nucleus; cc, central canal. Scale bars, 150 μm in C)",
    "molecules": "Thionine"
  },
  {
    "caption": "B  Ubiquitination assay of AnkG  protein. The lysates of COS-7  cells expressing the indicated truncated AnkG tagged with GST and HA-Ubiquitin  were  pulled down with glutathione Sepharose  beads, followed by western blotting with anti-HA  and anti-GST  antibodies.",
    "molecules": "Sepharose, glutathione"
  },
  {
    "caption": "L Thionine-stained hypoglossal motor neurons of Rpt3  CKO mice  at 10 days after injury. Rpt3  CKO mice  are infected by AnkG  shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc,  central canal. M The percentage of surviving motor neurons on the injured  side compared with that on the control side in Rpt3  CKO mice  after infection of AnkG  shRNA or scramble shRNA AAVs  (n= 4 mice  per group). Data information: For M), data are shown as the mean ± s.e.m. , * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 100 μm in (L).",
    "molecules": "Thionine"
  },
  {
    "caption": "Quantification of dissected distal germline from wild-type animal stained eithe 5-ethynyl-2′-deoxyuridine (EdU -green) (E) relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons",
    "molecules": "5-ethynyl-2′-deoxyuridine, EdU"
  },
  {
    "caption": "Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit 100µM cisplatin Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey\"s multiple comparisons",
    "molecules": "cisplatin"
  },
  {
    "caption": "Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue) and either anti-PARP-1 (red) (A Scale bar: 20µ (A Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue and eithe anti-RPA-1 (green) and anti-RAD-51 (red) antibodies and treated with gamma radiation (125 Gy) (B) (untreated animals displayed no staining of RPA-1 and RAD-51, not shown) 15µm (B Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition",
    "molecules": "DAPI"
  },
  {
    "caption": "Fertilit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple comparisons",
    "molecules": "Hydroxyure"
  },
  {
    "caption": "fecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, an left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presente as mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compa",
    "molecules": "Hydroxyure"
  },
  {
    "caption": "C. Top: schemas illustrating the behavioral tasks examined in WT mice. Bottom: the plots represent the discrimination index for the novel object recognition task (Vehicle N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12), the sociability index (Vehicle: N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12) and the discrimination index for the social discrimination task (N=8 for each group), measured in each condition. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line to a discrimination index (exploration of novel object - exploration of familiar object/total object exploration) equal to zero.",
    "molecules": "THC, SB, Rapa"
  },
  {
    "caption": "E. Top: schemas illustrating the behavioral tasks in 5-HT6-/- mice. Bottom: the plots represent the discrimination index for the novel object recognition test (discrimination index: 0.30 ± 0.05, N=10 and 0.31 ± 0.03, N=11, for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), the 3-chamber social preference test (sociability index: 0.49 ± 0.04, N=10 and 0.48 ± 0.07, N=11 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05) and the social discrimination test (discrimination index: 0.15 ± 0.06, N=8 and 0.21 ± 0.06, N=8 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), measured in each condition. One-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line corresponds to a discrimination index equal to zero).",
    "molecules": "THC"
  },
  {
    "caption": "C. The plots represent the discrimination index measured in each condition. *** p < 0.001, one-way ANOVA followed by Bonferroni test. The discrimination index for the novel object recognition task is 0.34 ± 0.06 (N=14) and -0.08 ± 0.07 (N= 20), for mice injected with Vehicle and THC, respectively, p < 0.001 and -0.09 ± 0.08 (N=21) and 0.00 ± 0.06 (N=18), for mice treated with THC+SB258285 and THC+Rapa, respectively, p > 0.05 vs. THC-injected mice (error bars correspond to the mean ± SEM, the dotted line to a discrimination index equal to zero).",
    "molecules": "THC, SB258285, Rapa"
  },
  {
    "caption": "B. Left: representative traces of GABA mIPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of GABA mIPSC frequency and amplitude measured during the last minute of recording. The mIPSC frequency is: 3.2 ± 0.4 Hz and 1.3 ± 0.2 Hz for Vehicle (n=9 from N= 4) and THC (n=7 from N= 4) conditions, respectively, p < 0.001 and 2.5 ± 0.2 Hz and 4.1 ± 0.4 Hz for THC+SB258585 (n=8 from N= 4) and THC+Rapa (n=8 from N= 4) conditions, respectively, p < 0.01 and p < 0.001 vs. THC-injected mice.",
    "molecules": "THC, GABA, SB258585, Rapa"
  },
  {
    "caption": "C. Left: representative traces of AMPA mEPSCs recorded in layer V pyramidal neurons are illustrated. Right: the histograms represent means ± S.E.M. of AMPA mEPSC frequency and amplitude measured during the last minute of recording, n = neurons from 3 to 5 mice per group. The mEPSC frequency is: 4.7 ± 0.4 Hz for Vehicle (n=9 from N= 7) and 6.8 ± 0.4 Hz for THC (n=9 from N= 5) conditions, respectively, p < 0.01and 5.3 ± 0.5 Hz and 4.6 ± 0,3 Hz for THC+SB258585 (n=10 from N= 6) and THC+Rapa (n=9 from N= 5) conditions, respectively, p < 0.05 and p < 0.01 vs. THC-injected mice. In B and C, * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Newman-Keuls test.",
    "molecules": "AMPA, THC, SB258585, Rapa"
  },
  {
    "caption": "A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. A. The resting membrane potential (RMP) was determined immediately after whole-cell formation: -71.8 ± 1.2 mV and -60.1 ± 1.5 mV for Vehicle and THC conditions, respectively, p < 0.001.",
    "molecules": "THC, SB258585, Rapa, rapamycin"
  },
  {
    "caption": "A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. B. Action potentials were evoked by a ramp current injection with a 10 pA-step for 2 ms and only the first action potential (AP) were used to estimate the AP threshold: -34.1 ± 0.7 mV and -39.7 ± 0.8 mV for Vehicle- and THC-injected mice, respectively, p < 0.001.",
    "molecules": "THC, SB258585, Rapa, rapamycin"
  },
  {
    "caption": "A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. C. The rheobase represents the minimal current required to induce neuronal firing, it was lowered in THC condition: 670 ± 43 pA and 376 ± 43 pA for Vehicle and THC conditions, respectively, p < 0.001.",
    "molecules": "THC, SB258585, Rapa, rapamycin"
  },
  {
    "caption": "A-D. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) were administered concomitantly with THC (n=20 from N=5) or vehicle (n=18 from N=6). Electrophysiological recordings were performed from PND 60. D. The firing rate induced by a 150 pA current injection during 250 ms is expressed as the number of APs.",
    "molecules": "THC, SB258585, Rapa, rapamycin"
  },
  {
    "caption": "E. top: Voltage sag in response to hyperpolarizing current injection (50 pA-increments, from -400 to 0 pA) in PFC pyramidal neurons from mice injected with vehicle (Veh, n= 10 from N=5), THC (n = 11 from N=5), THC+SB258585 (THC/SB, n=7 from N=4) or THC+Rapa (THC/Rapa, n=7 from N=4) during adolescence. *** p < 0.001, one-way ANOVA followed by Bonferroni test, vs. THC-injected mice. Errors bars correspond to the mean ± SEM. Voltage sag is indicated by arrowheads.",
    "molecules": "THC, SB, SB258585, Rapa"
  },
  {
    "caption": "F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (F) resting potential membrane: THC + ZD: -74.14 ± 1.70 mV, p < 0.001 vs. THC condition, THC + DDOA: -70.47 ± 1.19 mV, p < 0.001 vs. THC condition, THC + ODQ: -63.10 ± 0.81 mV, p < 0.001 vs. THC condition,",
    "molecules": "DDOA, ODQ, THC, ZD7288"
  },
  {
    "caption": "F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (G) AP threshold: THC + ZD: -33.59 ± 2.96 mV, p < 0.01 vs. THC condition, THC + DDOA: -34.28 ± 0.98mV, p < 0.001 vs. THC condition, THC + ODQ: -36.84 ± 0.79 mV, p < 0.001 vs. THC condition",
    "molecules": "DDOA, ODQ, THC, ZD7288, ZD"
  },
  {
    "caption": "F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (H) rheobase: THC + ZD: 806 ± 63 pA, p < 0.001 vs. THC condition, THC + DDOA: 607 ± 43 pA, p < 0.001 vs. THC condition, THC + ODQ: 422 ± 34 pA, p < 0.001 vs. THC condition",
    "molecules": "DDOA, ODQ, THC, ZD7288, ZD"
  },
  {
    "caption": "F-I. Effect of ZD7288 (ZD, 10 μM, n = 10 from N=3), DDOA (15 μM, n=9 from N=4) or ODQ (10 μM, n=12 from N=4) on intrinsic properties of PFC pyramidal neurons. THC and vehicle conditions are similar to those on panel A-D. (I) firing rate. Note that Vehicle- and THC-injected mice are the same as those used in experiments illustrated on Figure 5A-D. *** p < 0.001, ** p < 0.01, * p < 0.05, n.s. p > 0.05 one-way ANOVA followed by Bonferroni test.",
    "molecules": "DDOA, ODQ, THC, ZD7288, ZD"
  },
  {
    "caption": "A. In left, normalized peak amplitudes of isolated AMPA EPSCs recorded at -60 mV, before and after pairing protocol, are illustrated. Representative traces of AMPA EPSCs before (1) or after (2) the pairing protocol are also illustrated (right panel) for each experimental condition: Vehicle-injected mice (white circles, n=3 from N=3), THC-injected mice (green circles, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black circles, n=3 from N=3). Errors bars correspond to mean ± SEM.",
    "molecules": "AMPA, THC, SB258585"
  },
  {
    "caption": "B. The histogram represents the means ± SEM of AMPA EPSCs in % of baseline, measured during the last 5 min of baseline or the last 5 min of recording, for each experimental condition: vehicle-injected mice (white bar, n=3 from N=3), THC-injected mice (green bar, n=3 from N= 3) and THC-injected mice treated with SB258585 during adolescence (black bar, n=3 from N=3), n.s. p > 0.05, * p< 0.05, one-way ANOVA followed by Tukey test.",
    "molecules": "AMPA, THC, SB258585"
  },
  {
    "caption": "(D) Cross sections from OIR S1pr1f/stop/f, S1pr1 ECTG and S1pr1 ECKO P17 pups. Blood vessels are stained with isolectin (red) and nuclei with Hoechst (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) Flat-mounted retinas from SAR247799-treated, OIR WT pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 10 pups /group)",
    "molecules": "SAR247799"
  },
  {
    "caption": "(D) Flat-mounted retinas from ApoM-Fc-S1P-treated, OIR S1pr1f/stop/f or S1pr1 ECTG pups at P17, stained with isolectin. Avascular area, total and average neovascular tuft areas are quantified. (n= 6 pups /group)",
    "molecules": "S1P"
  },
  {
    "caption": "(b, c) Immunoblots of recruitment reactions performed as described in (a), with the indicated proteins omitted. In (c), a mid reaction wash in high salt (0.5 M NaCl) buffer (HSW) was included following DDK phosphorylation as indicated.",
    "molecules": "NaCl"
  },
  {
    "caption": "(h) Reaction performed as in (g). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Reaction performed as in (a), except Sld3/7 was incubated with Rad53 before addition to reactions. ATP was omitted from this pre-incubation as indicated.",
    "molecules": "ATP"
  },
  {
    "caption": "(c) Recruitment reaction conducted as in Fig. 3a, with washes performed at 0.3 M KCl.",
    "molecules": "KCl"
  },
  {
    "caption": "A. Sorbitol stimulates YAP phosphorylation. HEK293A cells were cultured in the presence or absence of serum, and treated with 0.2 M sorbitol for the indicated time points. Phos-tag gel was used to assess total YAP phosphorylation based on mobility shift. Note that YAP and TAZ are recognized by the same antibody. l.e. denotes long exposure of YAP and TAZ.",
    "molecules": "sorbitol"
  },
  {
    "caption": "B. Osmotic stress induces LATS phosphorylation. HEK293A cells were treated with 0.2 M sorbitol for 30 or 60 minutes in the presence or absence of serum. LATS phosphorylation at the hydrophobic motif (HM) was detected with the phospho-specific pLATS antibody.",
    "molecules": "sorbitol"
  },
  {
    "caption": "C. Osmotic stress increases LATS kinase activity. HEK293A cells were treated with 0.2 M sorbitol for 30 minutes. Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP was determined by immunoblotting with phospho-YAP (S127) antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "sorbitol"
  },
  {
    "caption": "D. Osmotic stress does not lead to YAP cytoplasmic localization. HEK293A cells were treated with 0.2 M sorbitol for 1 hr in the presence of serum. YAP subcellular localization was determined by immunofluorescence staining (red). DAPI (blue) was used to stain for DNA. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields, each with approximately 100 cells.",
    "molecules": "sorbitol, DNA"
  },
  {
    "caption": "E. Osmotic stress does not reduce YAP target gene expression. HEK293A cells were treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. n.s means p > 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "sorbitol"
  },
  {
    "caption": "F. Osmotic stress does not disrupt the interaction between YAP and TEAD1. HEK293A cells were treated with 0.2 M sorbitol for 1 hr. Endogenous YAP was immunoprecipitated with YAP antibody, and TEAD1 and YAP were detected by Western blot. Cells were serum starved for 1 hr as indicated (- serum).",
    "molecules": "sorbitol"
  },
  {
    "caption": "G. Osmotic stress does not increase the interaction between YAP and 14-3-3. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the co-precipitated Flag-YAP was detected.",
    "molecules": "sorbitol"
  },
  {
    "caption": "A. Osmotic stress induces YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. YAP/TAZ (red, upper panels) or YAP (red, lower panels) were stained with two specific antibodies. DAPI (blue) was used to visualize cell nuclei. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields.",
    "molecules": "sorbitol"
  },
  {
    "caption": "B. Osmotic stress induces TAZ nuclear translocation. YAP knockout (KO) HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. TAZ localization was determined by immunofluorescence staining with the YAP/TAZ antibody (red). Scale bar: 20 μm.",
    "molecules": "sorbitol"
  },
  {
    "caption": "C. Osmotic stress induces a transient YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for the indicated time points.",
    "molecules": "sorbitol"
  },
  {
    "caption": "D. Osmotic stress induces YAP target gene expression. Wild type (WT) or YAP/TAZ double knockout (Y/T KO) HEK293A cells were serum starved for 1 hr and treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "sorbitol"
  },
  {
    "caption": "E. Osmotic stress decreases YAP and 14-3-3 interaction. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, and were serum starved for 16 hr. Cells were then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the associated Flag-YAP was detected by Western. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "sorbitol"
  },
  {
    "caption": "A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Quantification of YAP/TAZ subcellular staining is shown. Scale bar: 20 μm.",
    "molecules": "sorbitol, SP600125, SB203580"
  },
  {
    "caption": "A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK-2 and JNK substrate c-Jun.",
    "molecules": "sorbitol, SP600125, SB203580"
  },
  {
    "caption": "B. NLK knockout blocks osmotic stress-induced YAP nuclear localization. HEK293A cells were transiently transfected with CRISPR/Cas9 to knockout NLK. Wild type (WT) cells and the NLK KO cell pool were treated with 0.2 M sorbitol for 1 hr in the absence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining. Scale bar: 20 μm.",
    "molecules": "sorbitol"
  },
  {
    "caption": "E. NLK phosphorylates YAP in vitro. NLK-WT and NLK-KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate in the presence of ATP-γ-S. Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody, which identifies the alkylated thiophosphorylation on YAP.",
    "molecules": "ATP-γ-S"
  },
  {
    "caption": "C. Osmotic stress induces YAP phosphorylation at Ser 128. In the left panel, HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated. In the right panel, Flag-YAP was transfected into HEK293A cells. Flag-YAP was immunoprecipitated, YAP Ser 128 phosphorylation was detected by a phosphospecific antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "sorbitol"
  },
  {
    "caption": "D. NLK deficiency reduces YAP S128 phosphorylation. Two CRISPR/CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A. WT cells and two pools of NLK CRISPR/CAS9 transfected cells were treated with 0.2 M sorbitol for the indicated time points. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 4).",
    "molecules": "sorbitol"
  },
  {
    "caption": "F. YAP Ser 128 phosphorylation is required for disruption of YAP-14-3-3 interaction by osmotic stress. Flag-YAP WT or Flag-YAP S128A constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs, and treated with 0.2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr. Refreshing medium served as a control to disrupt YAP and 14-3-3 binding.14-3-3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "sorbitol"
  },
  {
    "caption": "B. YAP Ser 128 phosphorylation is required for osmotic stress-induced YAP nuclear localization. HEK293A YAP KO cells reconstituted with Flag-YAP WT, YAP S128D, or YAP S128A mutant were treated with 0.2 M sorbitol for 1 hr. YAP subcellular localization was determined by Flag immunofluorescence staining (green). Scale bar: 20 μm.",
    "molecules": "sorbitol"
  },
  {
    "caption": "A. YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions. YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D were cultured in the absence (left panel) or presence of 0.2M sorbitol (right panel) for the indicated amount of time. Cell numbers were counted and normalized to day 0. Data are presented as mean ± SEM, n = 3.",
    "molecules": "sorbitol"
  },
  {
    "caption": "B. YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment. Cell cycle analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. Propidium iodine (PI) was used for DNA staining (Fig EV5). Quantification of sub-G1 cells is shown. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "sorbitol, DNA"
  },
  {
    "caption": "C. YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions. Annexin V analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. PE-Annexin V and 7-AAD stained for phospholipid phosphatidylserine (PS) and DNA, respectively. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).",
    "molecules": "phosphatidylserine, sorbitol, DNA"
  },
  {
    "caption": "A Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF  cells, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.",
    "molecules": "Ser, Thr"
  },
  {
    "caption": "D NHF and Lowe 1676 cells were grown in  complete media, amino-acid (aa)/FBS starved for 5 h, or starved  and then recovered in amino-acid/FBS-containing medium, then  immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.",
    "molecules": "aa, amino-acid, Ser, Thr"
  },
  {
    "caption": "C WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs derived from the corresponding mice were  costained with anti-α-tubulin and anti- γ-tubulin antibodies.  Nuclei were stained with DAPI (blue). Scale bar, 10 μm. D Quantitative analyses of focused microtubule arrays  numbers at  γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in C. n = 3 per group in which 30 cells were counted per condition per experiment. E Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.",
    "molecules": "DAPI"
  },
  {
    "caption": "E Microtubule regrowth assay was performed on NHF, Lowe 1676 and  Lowe 3265 cells with 20 μM  nocodazole for 2 h on ice, followed by rinse with PBS, and addition of pre-warmed medium to induce microtubule regrowth. Cells were then  immunostained with anti-α-tubulin and anti-γ-tubulin antibodies at the indicated  time point after addition of pre-warmed medium. The magnified view shows the cytoplasmic microtubules organization center. Scale bar, 10 μm. F Quantitative analyses of focused microtubule arrays  numbers at γ-tubulin (microtubule organizing center, MTOC) measured in the experiments shown in E. n = 3 per group in which 30 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.",
    "molecules": "nocodazole"
  },
  {
    "caption": "A RPE-1 cells transfected with nontargeting control siRNA or OCRL siRNA, were left for 48 h, then subjected to IF microscopy to visualize PI(4,5)P2 (green), γ-tubulin (red) and nuclei  (blue, DAPI staining). Scale bar, 10 μm. B Quantitative analyses of centrosomal PI(4,5)P2 fluorescence intensity in the experiments shown in A. n = 3 per group in which 50 cells were counted per condition per experiment. C Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.",
    "molecules": "PI(4,5)P2, DAPI"
  },
  {
    "caption": "C NHF, Lowe 1676 and Lowe 3265 cells were subjected to IF microscopy to visualize PI(4,5)P2 (green), gamma-tubulin (red) and nuclei (blue, DAPI staining). Scale bar, 10 μm. D The fluorescence intensity of PI (4,5)P2 on centrosomes in C was measured. n = 3 per group in which 50 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. n.s., not significant, calculated by calculated by two-tailed Student's t-test.",
    "molecules": "PI (4,5)P2, PI(4,5)P2, DAPI"
  },
  {
    "caption": "D NHF, Lowe 1676 and  Lowe 3265 cells were transfected with wildtype GFP-SSX2IP  or GFP-PACT-SSX2IP for 48 h, then immunostained with anti-β-tubulin (red) antibody. Nuclei (blue) were  stained with DAPI. The centrosomal microtubule nucleation in  GFP-PACT-SSX2IP transfected cells was indicated by white arrowhead. Scale bar, 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(c) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4 h. Full fields are shown in Supplementary Fig. 1d.",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "(h) Immunoblot for the indicated proteins of immunoprecipitates (IP) of endogenous Cx43 from untreated WT MEFs, on incubation with 3-methyladenine (3MA) or in MEFs from Atg5-null mice (Atg5−/−).",
    "molecules": "3-methyladenine, 3MA"
  },
  {
    "caption": "(a) Immunoblot for LC3 in MOBs from WT or Cx43-null mice (Cx43−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA). Glyceraldehyde-3-dehydrogenae (GPD) is shown as the loading control. Levels of LC3-II (left) and LC3-II flux (right; n = 3 independent experiments).",
    "molecules": "18α GA, 18α glycyrrhetinic acid"
  },
  {
    "caption": "(b) Immunofluorescence for LC3 in MOBs treated with vehicle or 18α glycyrrhetinic acid. Where indicated, chloroquine (CQ) was added to block lysosomal degradation. Bottom: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment).",
    "molecules": "18α glycyrrhetinic acid, chloroquine, CQ"
  },
  {
    "caption": "(a) Immunoblots for the indicated proteins in MEFs treated with tamoxifen (Tx) or lindane (Lind) in the absence or presence of lysosomal protease inhibitors (PI). Right: Changes in LC3-II levels relative to those detected in untreated cells (n = 4 independent experiments).",
    "molecules": "lindane, tamoxifen"
  },
  {
    "caption": "(b) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine (CQ) for 3 h. Right: Quantification of the average number of LC3-positive puncta per cell (n = 3 wells, 3 independent experiments, >30 cells per experiment). (c) Time course of changes in the average LC3-positive puncta per cell after addition of lindane to control NRK cells.",
    "molecules": "lindane, chloroquine, CQ"
  },
  {
    "caption": "(d) Fluorescence images of WT or Cx43−/− MOBs transfected with mCherry-GFP-LC3 and incubated or not with tamoxifen or lindane. Bottom: Quantification of autophagolysosomes (n = 3 wells, 3 independent experiments, >50 cells per experiment).",
    "molecules": "lindane, tamoxifen"
  },
  {
    "caption": "(f) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 treated with lindane and untreated. Single channel images in inverse black and white and merged colour images are shown. Outlined areas are shown at higher magnification. Arrows: double (yellow) and triple (white) co-localization.",
    "molecules": "lindane"
  },
  {
    "caption": "(i) Immunoblot for Atg16 and Atg14 in immunoprecipitates (IP) of endogenous Cx43 from WT MEFs maintained in the absence of serum (4 h) or incubated in the presence of tamoxifen or lindane. I: input; FT: flow through. All values are mean + s.e.m and differences from untreated cells are significant for *P 0.01. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.",
    "molecules": "lindane, tamoxifen"
  },
  {
    "caption": "(a-c) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP-Cx43 maintained in the presence of serum (a), absence of serum (b) or treated with lindane (c). Single black and white inverted channels, merged channels and higher magnification insets are shown.",
    "molecules": "lindane"
  },
  {
    "caption": "(f-h) Fluorescence images of NRK cells expressing GFP-Cx43 maintained in the presence of serum (f), absence of serum (g) or treated with lindane (h) and incubated with Alexa594-transferrin for 15 min before fixation. Top: single and merged channels and outlined area at higher magnification. Bottom: 3D reconstructions and higher magnification details of the outlined areas. Nuclei are highlighted with DAPI. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.",
    "molecules": "lindane"
  },
  {
    "caption": "(b,c) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum (b) or treated or not with 3-methyladenine (c). Atg5 is shown as the negative control for immunoprecipitation.",
    "molecules": "3-methyladenine"
  },
  {
    "caption": "(h) Immunofluorescence of endogenous Cx43 in control NRK cells or those knocked down for Atg14, treated (or not) with tamoxifen or lindane (left). E-cadherin staining (red) is shown to highlight PM in g and h. Full fields are shown in Supplementary Fig. 6h. Quantification of intracytoplasmic Cx43-positives vesicles (right; n = 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean + s.e.m and significant for *P 0.01 and **P 0.001 using analysis of variance + Bonferroni test. Scale bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.",
    "molecules": "lindane, tamoxifen"
  },
  {
    "caption": "D. HAT1-catalyzed   histone H3 succinylation was analyzed by mixing purified HAT1,  histone H3, and succinyl-CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies.",
    "molecules": "CoA, succinyl-CoA"
  },
  {
    "caption": "E. HAT1-mediated  histone H3 succinylation  was assessed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without acetyl-CoA (2 μM). Western blot analysis  was performed with the indicated antibodies.",
    "molecules": "acetyl-CoA, succinyl-CoA"
  },
  {
    "caption": "A. The levels of histone H3K122 succinylation, histone H3K122 acetylation, histone H3K27 acetylation, histone H3, HAT1, and β-actin were examined by Western blot analysis in HepG2, HAT1 knockout (KO) Clone 1 HepG2 cells, and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 (T188A).",
    "molecules": "H3K122, H3K27"
  },
  {
    "caption": "D. The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti-H3K122 succinylation antibody and anti-H3K122 acetylation antibody, and followed by quantitative PCR with primers of the promoter regions of CREBBP, BPTF and RPTOR  in WT HepG2 cells, HAT1  KO HepG2  cells and HAT1 KO HepG2  cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data were presented as mean ± SD. Student's t-test, ***P < 0.001; ns, no significance.",
    "molecules": "H3K122"
  },
  {
    "caption": "D.  HAT1-catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1, PGAM1, and succinyl-CoA/acetyl-CoA in the in vitro succinylation assays. Western blot analysis was performed with indicated antibodies.",
    "molecules": "acetyl-CoA, succinyl-CoA"
  },
  {
    "caption": "F. The decrease of glucose  and increase of lactate in the culture medium and the relative amounts of  2-PG and 3-PG were analyzed by ELISA  assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1  mutant (K99R). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.",
    "molecules": "2-PG, 3-PG, glucose, lactate"
  },
  {
    "caption": "Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi-defined CSY media with or without sucrose, 4% and 1% respectively. (+), inoculated wells; (-), non-inoculated control. The picture was taken after a 24-hour incubation period.",
    "molecules": "sucrose"
  },
  {
    "caption": "The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L).",
    "molecules": "sucrose"
  },
  {
    "caption": "Sucrose specific uptake rate (e) and combined lactate/acetate specific secretion rates (f) at varying initial sucrose concentrations in CSY medium. Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M. florum cultures. Whiskers indicate minimal and maximal values. Dotted lines indicate the selected uptake (-5.26 mmol∙gDW-1∙hr-1) and secretion rate (8.69 mmol∙gDW-1∙hr-1) values used for modeling. Sucrose quantifications were performed in technical duplicate, whereas lactate and acetate quantifications were performed in single replicates. See Appendix Figure S5 for further details.",
    "molecules": "acetate, lactate, Sucrose, sucrose"
  },
  {
    "caption": "A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIA1. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.",
    "molecules": "DTT"
  },
  {
    "caption": "B Analysis of the PDIA1 structure to model the effects of the R300H mutation. The close association between Arg300 located to the b' domain of PDIA1 with Trp396 located to the a' domain adjacent to the active site motif CGHC (designated as AS in yellow) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues. A potential stabilization of the interaction between the b' and a' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396.",
    "molecules": "His, imidazole, Arg, Trp"
  },
  {
    "caption": "E Representative electrophoresis analysis of Proteinase K-treated recombinant PDIA1 variants. Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples (arrows indicate band 1 and band 2) indicated differences in the removal of the x-linker region. Bottom panel: Ratios of band 1 to band 2 were quantified in 4 independent experiments.",
    "molecules": "trypsin"
  },
  {
    "caption": "G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis. The percentages of different BPTI species was calculated (6H, fully reduced; 1S, one disulfide bond; 2S, two disulfide bonds; 3S, three disulfide bonds) at time point 2.5 min in four independent experiments.",
    "molecules": "BPTI"
  },
  {
    "caption": "H Measurement of H2O2 levels at the ER lumen of living NSC34. Left panel: Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen (O2), generating hydrogen peroxide (H2O2) as a product of PDIs activity. Right panel: NSC34 cells were transiently co-transfected with ER luminal HyPer sensor and indicated PDIs. After 48 h the 490/420 nm fluorescence ratio was recorded for 2 min under basal conditions. Means and SEM derived from all cells per condition (n=55-74) monitored in four independent experiments are shown.",
    "molecules": "H2O2, hydrogen peroxide, oxygen"
  },
  {
    "caption": "A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant ERp57. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.",
    "molecules": "DTT"
  },
  {
    "caption": "G The gain of an N-glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector and treated with 1 µg/ml tunicamycin (Tm) for 20 h to inhibit N-glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N-glycosylation was analyzed by Western blot using anti-V5 antibody.",
    "molecules": "PNGase F, Asn, Asp, tunicamycin"
  },
  {
    "caption": "C Whole-mounted diaphragms from ERp57WT and ERp57Nes-/- mice were co-stained with anti-neurofilament (red) and α-BTX to reveal the postsynaptic densities (green). 3D reconstructions (lower panel) of higher magnification. ERp57WT NMJs are fully innervated pretzel-like, whereas ERp57Nes-/- NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Movies EV4-7).",
    "molecules": "α-BTX"
  },
  {
    "caption": "B, C Inhibitory effect of mycolactone on cell expression  of CD38 (Type II) and CD138, IL-6R, and CD40  (Type I) transmembrane proteins (B). Stimulatory effect of mycolactone  on cell expression  of BCMA, a Type III transmembrane protein (C). Mean Fluorescence Intensities (MFIs) were measured in live cells 48 h after addition of mycolactone. Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments, relative to vehicle-treated controls (Ctrl).",
    "molecules": "mycolactone"
  },
  {
    "caption": "C Synergy between mycolactone and BZ in the MM.1S, JIM3 and KMS-11 cell lines, when treated as in (B). Data are Mean Loewe scores ± SD shown as heatmaps. N = 6 (cumulative data of 3 independent experiments with technical duplicates) for MM.1S and KMS-11, and N = 4 (cumulative data of 2 independent experiments with technical duplicates) for JIM3. Statistical significance was assessed by Student's t-test: *p<0.05; **p<0.01; ***p<0.001.",
    "molecules": "BZ, mycolactone"
  },
  {
    "caption": "D,E MM.1S  cell were treated as in (B) but using carfilzomib instead of BZ  (D). Synergy scores (E) were calculated as in (C). Data are Mean Loewe scores of technical duplicates, shown as heatmaps.",
    "molecules": "BZ, carfilzomib"
  },
  {
    "caption": "In  MM.1S  cells treated with mycolactone  and/or BZ  at the indicated concentrations, or vehicle as control for 6 h: (D) CHOP  and GADD34 mRNA levels were quantified by  qPCR Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated.  Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) were used as positive controls.",
    "molecules": "BZ, mycolactone, Tg, Thapsigargin, Tu, Tunicamycin"
  },
  {
    "caption": "In  MM.1S  cells treated with  mycolactone and/or  BZ  at the indicated concentrations, or vehicle as control for 6 h: (E) ATF6  and GAPDH  protein levels in cell lysates were assessed by Western blot with molecular weight markers (kDa) indicated on the right; Bands corresponding to glycosylated (G-ATF6), non-glycosylated  (NG-ATF6) and cleaved  ATF6 are indicated by arrows; Data information: DTT (4 mM, 2 h) were used as positive controls. shown data are representative of 2 independent experiments with similar results.",
    "molecules": "DTT, BZ, mycolactone"
  },
  {
    "caption": "In MM.1S  cells treated with mycolactone and/or BZ  at the indicated concentrations, or vehicle as control for 6 h: (F) Total RNA was isolated and used as a template for RT-PCR of XBP-1 (upper panel) which was then digested with  Pst1  and separated on a 2% agarose gel (lower panel) with molecular weight markers (bp) indicated on the left; spliced XBP1 (sXBP1) mRNA levels were also quantified by qPCR  and normalized to total (spliced + unspliced) XBP1 mRNA levels, (G) BiP  mRNA level were quantified by  qPCR. Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) were used as positive controls. shown data are representative of 2 independent experiments with similar results.",
    "molecules": "agarose, BZ, mycolactone, Tg, Thapsigargin, Tu, Tunicamycin"
  },
  {
    "caption": "C MM.1S BzR cells were treated for 24 h with  mycolactone and/or BZ at the indicated concentrations, or vehicle as control. Data are Mean % ± SD of live, apoptotic and dead cells relative to total cells. N = 6 (cumulative data of 3 independent experiments with technical duplicates).",
    "molecules": "BZ, Bz, mycolactone"
  },
  {
    "caption": "E ATF4, CHOP, sXBP1 and BiP  mRNA levels were quantified by qPCR in MM.1s BzR cells treated as in (C). sXBP1 mRNA levels were normalized to total (spliced + unspliced) XBP1 mRNA level. Data are Mean RNA fold changes (2-∆∆CT) ± SD, relative to untreated controls. N = 5 (cumulative data of 2 independent experiments with technical duplicates and triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.",
    "molecules": "Bz, Tg, Thapsigargin"
  },
  {
    "caption": "C  v-abl pro-B cells were treated with  mycolactone and/or BZ  at the indicated concentrations for 6 h. CHOP mRNA levels were quantified by  qPCR. Data are Mean RNA fold changes (2-∆∆CT) ± SD relative to untreated controls. N = 6 (cumulative data of 3 independent experiments with technical duplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Data information: Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.",
    "molecules": "BZ, mycolactone, Tg, Thapsigargin"
  },
  {
    "caption": "D v-abl pro-B cells were treated for 6 h with mycolactone and/or  BZ  at the indicated concentrations. ATF4 protein levels were assessed in cell lysates by Western blot (top panel) and quantified relative to GADPH levels (lower panel). Data information: Data shown are representative of experiments performed with two independent v-abl cell clones, with similar results. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.",
    "molecules": "BZ, mycolactone, Tg, Thapsigargin"
  },
  {
    "caption": "Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients  were treated with mycolactone and/or  BZ  at the indicated concentrations, or vehicle as control, for 18 h. Then, MM  cells were identified by  staining  with anti-CD38  and anti-CD138 antibodies A Following treatment, induction of apoptosis/necrosis was measured by exposure of annexin V  and PI incorporation. Data are Mean % of live cells from technical duplicates, relative to controls.",
    "molecules": "BZ, mycolactone, PI"
  },
  {
    "caption": "Mononuclear cells from  bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or  BZ at the indicated concentrations, or vehicle as control, for 18 h. B Synergy between  mycolactone and BZ in treated tumors. Data are Mean Loewe scores from technical duplicates, shown as heatmaps.",
    "molecules": "BZ, mycolactone"
  },
  {
    "caption": "A C57BL/6  mice (N=2/group) were injected intraperitoneally with BZ (0.5 mg.Kg-1), BZ (0.5 mg.Kg-1) + mycolactone  (0.3 mg.Kg-1), DMSO as vehicle controls every 3.5 days, or left untreated.  Blood samples were analyzed 5, 15, 20 and 25 days after the first injection and percentages of  CD3+ and CD19+ cells determined by  flow cytometry. Results are expressed as % of positive cells among total mononuclear cells, each dot representing the Mean % from 2 mice.",
    "molecules": "BZ, DMSO, mycolactone"
  },
  {
    "caption": "B NSG mice (N=9/group) were injected subcutaneously with 3.106 MM.1S cells at day 0. Seven days later, mice were injected with DMSO, BZ (0.5 mg.Kg-1) and/or mycolactone (0.3 mg.Kg-1) every 3.5 days intraperitoneally and tumor growth was followed by daily measurement of the tumor diameter. Data are Mean tumor diameters ± SD and represent cumulative data from 3 independent experiments with 3  mice/group. Difference between groups were analyzed with Tukey's multiple comparison test using Two-way Anova, with mixed effect model for each time points: *p<0.05; **p<0.01; ***p<0.001 (exact p-values indicated). Only significant differences are shown.",
    "molecules": "BZ, DMSO, mycolactone"
  },
  {
    "caption": "E, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 0.1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. Representative image of filter after Crystal violet staining is shown. Scale=200µm. n = 3 in each group. F, Murine pancreatic tumour cells R211 were treated with vehicle, BYL-719 1 µM, AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 4 in each group. G ",
    "molecules": "BYL-719, AZD4547"
  },
  {
    "caption": "A-B A, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle, α-specific A66 or pan-PI3K inhibitor BKM120 at 0.01, 0.05, 0.1, 0.5, 1 or 5 µM in the presence of 10% FBS and protein levels of P-Akt (Ser473), total Akt and β Actin were observed by western blot. B, P-Akt on Ser473 was quantified and normalised with β Actin. n = 3 in each group.",
    "molecules": "A66, BKM120, Ser"
  },
  {
    "caption": "R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM D, migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle (DMSO). n = 3 in each group.",
    "molecules": "A66, BKM120, DMSO"
  },
  {
    "caption": "E, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle (0.01% DMSO) or α-specific A66 or pan-PI3K inhibitor BKM120 at 1µM. Phospholipids were extracted, total PIP, PIP2 and PIP3 were quantified and compared to the vehicle (DMSO). n = 3 in each group.",
    "molecules": "PIP3, PIP2, PIP, A66, BKM120, DMSO"
  },
  {
    "caption": "G, Survival curve of KPC mice treated with the vehicle or BYL-719. Log rank test, * p<0.05.",
    "molecules": "BYL-719"
  },
  {
    "caption": "H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).",
    "molecules": "BYL-719, methyl cellulose, Tween-80"
  },
  {
    "caption": "S, Murine pancreatic tumour cells R211 were treated with vehicle, TNF-α 20 ng/mL, BYL-719 1 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n= 4 independent values.",
    "molecules": "BYL-719"
  },
  {
    "caption": "B: Immunofluorescence staining of stem cell markers POU5F1 (green) and SSEA4 (red): upper panel - Detroit 551 control iPSCs, middle panel - WS5A iPSCs and lower panel - CP2A iPSCs (Scale bar, 100 µm). Nuclei are stained with DAPI (blue).",
    "molecules": "SSEA4, DAPI"
  },
  {
    "caption": "D, E: Flow cytometric quantification of expression level of SSEA4 (D, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) and POU5F1 (E, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control, WS5A and CP2A fibroblasts.",
    "molecules": "SSEA4"
  },
  {
    "caption": "A: Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN (red) and HNF4A (green) (a) (scale bar, 100 µm), mesodermal derived cardiomyocytes with positive expression of TNNT2 (red) (b) (scale bar, 100 µm) and ectodermal derived dopaminergic neurons with positive expression of TH (green) and MAP2 (red) (c) (scale bar, 10 µm). Nuclei are stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "B: Confocal images of mitochondrial morphology for iPSC lines with co-staining of MTG (upper panel) and TMRE (lower panel) (scale bars, 25 µm). Nuclei are stained with DAPI (blue).",
    "molecules": "DAPI, MTG, TMRE"
  },
  {
    "caption": "Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for mitochondrial volume (MTG) (C, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A)",
    "molecules": "MTG"
  },
  {
    "caption": "Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for total MMP (TMRE) (D, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) and specific MMP (E, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "F: Intracellular ATP production in iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts (n=3, technical replicates per clone for control and WS5A; n=4, technical replicates per clone for CP2A).",
    "molecules": "ATP"
  },
  {
    "caption": "B: Representative phase contrast images (upper panel) and immunostaining for specific stages during neural induction from iPSCs to NSCs. Upper panel displays the morphology in culture of different cell types during neural induction to NSCs from iPSCs including iPSCs (a); neuroepithelium with rosette-like structures (b); neurospheres with defined round shapes in suspension culture (c) and NSCs in monolayers (d) (scale bars, 50 µm). Lower panel demonstrates the representative phase contrast images for the immunostaining corresponding to the specific stages in the upper panel: iPSCs with positive staining of SSEA4 (red) and POU5F1 (green) (e) (scale bar, 50 µm); neuroepithelium with rosette-like structures with positive staining of PAX6 (green) and NESTIN (red) (f) (scale bar, 50 µm); neurospheres with positive staining of NESTIN (red) (g) (scale bar, 100 µm); NSCs with positive staining of PAX6 (green) (h) (scale bar, 50 µm).",
    "molecules": "SSEA4"
  },
  {
    "caption": "D: Representative images of the immunofluorescent labeling for NSC markers SOX2 (green) and NESTIN (red) (scale bar, 25 µm) from Detroit 551 control iPSC-derived NSCs. Nuclei are stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "E: Representative confocal images showing glial and neuronal lineages derived from NSCs. a, Immunostaining of NSC-derived astrocytes with GFAP (red) staining (scale bar, 50 µm). b, Immunostaining of oligodendrocytes showing GALC (red) positive labeling (scale bar, 50 µm). c, Dopaminergic neurons showing TH (green) and TUJ1 (red) positive staining (scale bar, 25 µm). Nuclei are stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "Flow cytometric analysis of mitochondrial volume (MTG) (B, n=5, technical replicates per clone), total MMP (TMRE) (C, n=5, technical replicates per clone) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG in NSCs generated from control lines, WS5A and CP2A iPSCs.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "E: Intracellular ATP production in NSCs (n=3, technical replicates per clone).",
    "molecules": "ATP"
  },
  {
    "caption": "Flow cytometric analysis of mitochondrial volume (MTG) (F, n=6, technical replicates for control; n=5, technical replicates for WS5A and CP2A), total MMP (TMRE) (G, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "Flow cytometric analysis of specific MMP (TMRE/MTG) (H, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.",
    "molecules": "MTG, TMRE"
  },
  {
    "caption": "I: Intracellular ATP production in Detroit 551, WS5A and CP2A fibroblasts (n=6, technical replicates for control; n=3, technical replicates for WS5A and CP2A).",
    "molecules": "ATP"
  },
  {
    "caption": "H: Representative images of confocal microscopy with immunofluorescence labeling of DA neuron-specific marker TH (green) and neuron-specific marker MAP2 (red) for iPSC-derived DA neurons (scale bars, 25 µm). Nuclei are stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "B: Representative confocal images of immunostaining for mitocondrial complex I subunit NDUFB10 (green) and TOMM20 (red) in control, WS5A and CP2A NSCs (scale bars, 50 µm). Nuclei are stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "A-C: LCMS-based metabolomics for quantitative measurements of NAD+/NADH ratio (A, n=3, technical replicates per clone), NAD+ (B, n=3, technical replicates per clone) and NADH (C, n=3, technical replicates per clone) level in NSCs.",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "D-F: LCMS-based metabolomics for quantitative measurement of NAD+/NADH ratio (D, n=3, technical replicates per clone), NAD+ (E, n=3, technical replicates per clone) and NADH (F, n=3, technical replicates per clone) level in iPSCs.",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "G: Intracellular ROS production measurements of the specific ROS level calculated by total ROS/MTDR in control, WS5A and CP2A iPSC-derived NSCs using DCFDA and MTDR (n=6, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=3, technical replicates per clone for CP2A).",
    "molecules": "DCFDA, MTDR, ROS"
  },
  {
    "caption": "H: Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551, WS5A and CP2A iPSC-derived NSCs calculated by total ROS (MitoSox Red)/MTG (n=4, technical replicates per clone for control and CP2A; n=3, technical replicates per clone for WS5A).",
    "molecules": "MitoSox Red, MTG, ROS"
  },
  {
    "caption": "K-L: Representative images (K) and quantification (L) for western blotting with Phospho-SirT1 (Ser47), LC3B, UCP2, PINK1, Parkin, BNIP3 and β-ACTIN. Three independent experiments are included.",
    "molecules": "Ser"
  },
  {
    "caption": "B. Normalized number of filaments nucleated over time, from SPIN90-Arp2/3 exposed to 2 μΜ G-actin (15% labeled with Alexa488) and 1 μΜ profilin, with 0 or 0.5 µM of GST-VCA from different NPFs. Solid lines are exponential fits, yielding nucleation rates knuc=(1.06±0.03)х10-3 s-1 without VCA, and knuc=(3.23±0.08)х10-3, (2.58±0.05)х10-3, and (2.28±0.03)х10-3 s-1 with VCA from N-WASP, WASP and WASH, respectively.",
    "molecules": "Alexa488"
  },
  {
    "caption": "GMF and VCA interfere with the SPIN90-Arp2/3 interaction, in the absence of filament. (G) Immuno-blots from pull-down assays where GST beads, decorated with GST or GST-SPIN90-Cter (the functional domain of SPIN90), were incubated with 0.4 μΜ Arp2/3 and gradient concentrations of GMF or VCA for 1 hour at room temperature. After the unbound protein was washed out, the amount of Arp2/3 attached to the beads was detected by Anti-ArpC2 antibody. Ponceau red straining of the membrane verified that the beads were loaded with equal amounts of GST or GST-SPIN90 (Appendix Figure S2).",
    "molecules": "Ponceau"
  },
  {
    "caption": "(E) Representative confocal micrographs of p-tyrosine (p-Tyr) polarization at B-T cell contacts after 1-2h of co-culture. Cell conjugates were stained with anti-pTyr antibody. Scale bar, 5µm. The top panels display only the p-Tyr signal in grey, the bottom panel depicts p-Tyr (red), BHEL cells (blue) and CD4OT-II cells (green). (F) Quantification of p-Tyr accumulation at the B-T interface as shown in E. Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment. ",
    "molecules": "Tyr, tyrosine"
  },
  {
    "caption": "(G) NEF prevents induction of F-actin remodeling in BHEL during IS. BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated slides and subjected to live-cell time-lapse imaging. Shown are representative still images from the indicated time points post contact formation (see Movies EV1 and EV2). Red signals indicate actin polymerization specifically in BHEL cells. Scale bars, 5µm. Filled and empty arrowheads indicate examples of accumulation of polarized actin or p-Tyr signal at B-T interface during IS formation or lack thereof, respectively. Asterisks mark pairs of interacting T and B cell. (H) Quantification of B cell actin polymerization as shown in G with the frequency of actin polymerization in B cells co-cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 50 conjugates counted per experiment. ",
    "molecules": "Tyr"
  },
  {
    "caption": "(L) Relative B cell proliferation (quantified by gating on the last 3-4 generations) is plotted as mean values with SD from four independent experiments. Control T cells +Ag were arbitrarily set to 1.",
    "molecules": "Ag"
  },
  {
    "caption": "(A) Representative cross-sections of draining LNs (dLNs) from mice 7d post immunization with HEL-OVA/CFA following adoptive transfer of BHEL and transduced CD4OT-II cells. dLN were cryosectioned and stained with fluorophore-conjugated antibodies against GL-7 to reveal GCs (green), CD21/35 to stain for follicular dendritic cells (FDC, red) and Hoechst to stain for cell nuclei (blue). Scale bar, 250µm. (B, C) Quantification of the number of GCs relative to B cell follicles (B) and size of GC (C) on confocal micrographs as shown in A. Shown are mean values with SD from dLNs from three mice in two independent experiment. Each dot represents one LN (3 LNs/mouse) in B and one GC in C. ",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Still images of the Movies EV3 (Control, upper panel) and EV4 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 24hrs post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel), respectively. High endothelial venules (HEVs) (white) were labeled by intravenous injection of Alexa660-labeled Meca-79 antibody and collagen (blue) is visualized by second harmonic generation (SHG). Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (B) Quantification of the duration of interactions as indicated by the arrows in A. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Data is derived from 5 to 6 movies, acquired from 4 mice in 2 independent experiments. Percentages on top indicate frequency of interactions that lasted 20min or longer. ",
    "molecules": "Alexa660, collagen"
  },
  {
    "caption": "(C) Still images of the Movies EV5 (Control, upper panel) and EV6 (NEF, lower panel), obtained by intravital time lapse 2PM imaging of draining popliteal LNs 8d post immunization with HEL-OVA/CFA, following adoptive transfer of GFP BHEL cells (green) and mTomato CD4OT-II cells (red) expressing empty vector (Control; upper panel) or NEF WT (lower panel). Follicular dendritic cells (FDCs) (white) were labeled by subcutaneous injection of Alexa647-labeled anti-CD21/CD35 antibody and collagen (blue) is visualized by SHG. Yellow and orange arrows indicate stable and transient ISs, respectively. Numbering of ISs corresponds to that in the respective Supplemental movies. Scale bar 20µm. (D) Quantification of the duration of interactions as indicated by the arrows in C. Touching T and B cells were manually quantification using virtual reality (InViewR, Arivis). Each dot represents one T-B interaction pair. Red bar shows median. Shown is one representative dataset out of two, derived from 5 to 8 movies, acquired from 4 mice. Percentages on top indicate frequency of interactions that lasted 20min or longer. ",
    "molecules": "Alexa647, collagen"
  },
  {
    "caption": "(A) Left panel: HeLa cells were starved for 2 h in the presence or absence of either 100 nM wortmannin or 10 mM 3MA, after which they were treated with DCFDA and analyzed by a fluorimeter as described above. Right panel: the rate of degradation of long‐lived proteins was measured in cells incubated in either α‐MEM medium or EBSS medium in the absence or presence of 100 nM wortmannin or 10 mM 3MA. Values are represented as the means±s.d. of three separate determinations.",
    "molecules": "3MA, wortmannin"
  },
  {
    "caption": "(A) Upper panel: CHO cells stably transfected with GFP‐GATE‐16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ/ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs, or grown in a control medium containing the drugs for 2 h. The cells were then fixed, permeabilized and stained with anti‐GFP monoclonal antibodies. Representative images are shown. Lower panel: HEK 293 cells were transfected with GFP‐GATE‐16. At 24 h post‐transfection, the cells were treated with NAC or catalase and starved as explained above, lysed in Ripa buffer and 100 ‐g of each lysate was separated on 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16 and anti‐tubulin antibodies as control. The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE‐16. (*) indicates non‐lipidated and (**) indicates lipidated GFP‐GATE‐16.",
    "molecules": "catalase, NAC"
  },
  {
    "caption": "(B) CHO cells treated with NAC or catalase and starved as detailed in (A) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1.",
    "molecules": "catalase, NAC"
  },
  {
    "caption": "(C) The rate of degradation of long‐lived proteins was measured in CHO cells incubated in either α‐MEM medium or EBSS medium, or following pretreatment with 10 mM NAC for 10 min or with 1000 μ/ml catalase overnight. A representative experiment is shown.",
    "molecules": "catalase, NAC"
  },
  {
    "caption": "(B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS-PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS-PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown.",
    "molecules": "DTT, H2O2"
  },
  {
    "caption": "(D) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS-PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS-PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.",
    "molecules": "[35S]methionine"
  },
  {
    "caption": "(A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.",
    "molecules": "DTT"
  },
  {
    "caption": "(B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A).",
    "molecules": "DTT, H2O2"
  },
  {
    "caption": "(C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.",
    "molecules": "DTT, H2O2"
  },
  {
    "caption": "(A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.",
    "molecules": "DTT"
  },
  {
    "caption": "(A) HeLa cells were transiently transfected with GFP-HsAtg4BWT or GFP‐HsAtg4BC78S. At 24 h post‐transfection, cells were starved for 2.5 h in the presence of 4 mM H2O2 after which they were fixed, permeabilized and incubated with anti‐LC3 polyclonal antibodies. Cells were visualized (upper panel) and quantified (lower panel) as explained in Figure 8. Representative autophagosomes are indicated in the upper panel by arrows. The results presented in the lower panel are the means±s.d. of a total of 100 cells from three separate experiments. (*) indicates significance at P0.001.",
    "molecules": "H2O2"
  },
  {
    "caption": "(a-d) Rotenone (Rot) increased the number of GFP-LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a-c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle.",
    "molecules": "Rot, Rotenone"
  },
  {
    "caption": "(e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown.",
    "molecules": "Rotenone"
  },
  {
    "caption": "(g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control; †P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.",
    "molecules": "Baf, bafilomycin, Rot, Rotenone"
  },
  {
    "caption": "(a,b) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC-MS analysis. (a) MS spectra demonstrated increased cardiolipin (CL) content of the OMM after rotenone (Rot) treatment, with diversification of the cluster distribution to the 7 clusters exhibited by the IMM. Veh, vehicle. (b) Pie charts showing the CL distribution between IMM and OMM fractions from toxin-treated neurons, normalized to mitochondrial lipid phosphorus.",
    "molecules": "cardiolipin, rotenone"
  },
  {
    "caption": "(c,d) Treatment with rotenone caused significant increases in PLA2-hydrolysable (surface accessible) CL assessed by LC-MS in primary cortical neurons (c; 250 nM) and SH-SY5Y cells (d; 1 μM).",
    "molecules": "CL, rotenone"
  },
  {
    "caption": "(e,f) Treatment with 6-OHDA and CCCP increased surface exposure of CL probed with annexin V in SH-SY5Y (e, 120 μM) and parkin-expressing HeLa cells (f, 20 μM), respectively. Mean ± s.d. of n = 3 independent experiments for c-f (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control.",
    "molecules": "CL, CCCP, 6-OHDA"
  },
  {
    "caption": "(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647-annexin V binding.",
    "molecules": "Anionic phospholipids, rotenone"
  },
  {
    "caption": "(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (b-d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM).",
    "molecules": "6-OHDA, Rot, rotenone"
  },
  {
    "caption": "(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression.",
    "molecules": "rotenone"
  },
  {
    "caption": "(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f)",
    "molecules": "rotenone"
  },
  {
    "caption": "(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of n = 4 independent experiments for b,c, and n = 3 independent experiments for a,d-f,h-i (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle; †P0.05 versus toxin-treated control siRNA; **P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.",
    "molecules": "Rot, rotenone"
  },
  {
    "caption": "(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain.",
    "molecules": "rotenone, STS"
  },
  {
    "caption": "(b,c) Likewise, SH-SY5Y cells did not show beclin 1 cleavage on exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin.",
    "molecules": "6-OHDA, rotenone"
  },
  {
    "caption": "(d) Cell lysates from stable PINK1-Flag-expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), rotenone (1 μM) or FCCP (2 μM), and analysed by Flag immunoblot for PINK1-Flag levels.",
    "molecules": "FCCP, 6-OHDA, rotenone"
  },
  {
    "caption": "(e) SH-SY5Y cells transfected with HA-parkin were treated with the indicated toxins, immunolabelled for HA (green) and mitochondrial p60 antigen (red) and analysed for HA-parkin redistribution to mitochondria. FCCP elicited translocation of parkin to mitochondria (arrows), which was not seen with the other mitophagy stimuli.",
    "molecules": "FCCP"
  },
  {
    "caption": "(f) Cytosolic and mitochondrial pellets were prepared from SH-SY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or rotenone (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers.",
    "molecules": "6-OHDA, rotenone"
  },
  {
    "caption": "(g,h) Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or rotenone-treated cells. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.",
    "molecules": "FCCP, rotenone"
  },
  {
    "caption": "(a) Recombinant LC3 incubated with tetraoleoyl-CL (TOCL) or dioleoylphosphatidylcholine (DOPC) liposomes at the indicated molar ratios was analysed by blue native PAGE; liposome binding impaired the gel entry of proteins.",
    "molecules": "dioleoylphosphatidylcholine, DOPC, tetraoleoyl-CL, TOCL"
  },
  {
    "caption": "(b) Titration of phospholipids (for example, TOCL) to LC3 was performed to evaluate the ratio that prevents 50% of the LC3 from entering the gels (IC50) as an index of relative affinity. Bottom left inset: representative gel for TOCL/LC3 = 12; top right inset: comparison of IC50 values for TOCL versus dioleoyl-phosphatidic acid (DOPA) and tetralinoleoyl-CL (TLCL) versus monolyso-trilinoleoyl-CL (lyso-CL). The IC50 for DOPG was >15. *P0.05 versus TOCL; †P0.05 versus TLCL.",
    "molecules": "dioleoyl-phosphatidic acid, DOPA, DOPG, phospholipids, tetralinoleoyl-CL, TLCL, TOCL, monolyso-trilinoleoyl-CL"
  },
  {
    "caption": "(f-j) The arrowhead in e shows the position of the N-terminal truncation used to create GFP-LC3 deletion mutants, which were analysed for GFP-LC3 puncta formation (f,g,i) and participation in mitophagy (h,j) in response to the indicated stimuli. Rot, rotenone; Veh, vehicle.",
    "molecules": "rotenone"
  },
  {
    "caption": "(e,k) Dual point mutations were prepared on the basis of predicted CL contact residues (e), and analysed for participation in mitophagy elicited by Rot or 6-OHDA (k). Inset: expression of GFP-LC3 plasmids (uncropped blots in Supplementary Fig. S7). UT, untransfected. Mean ± s.d. of n = 3 independent experiments for f-h, and n = 4 independent experiments for i,j (see Supplementary Table S4 for statistics source data); *P0.05 versus respective vehicle. †P0.05 versus toxin-treated wild type (WT).",
    "molecules": "CL, 6-OHDA, Rot"
  },
  {
    "caption": "C. SPATA2 is involved in TNF-α-induced necroptosis. SPATA2 was knocked down in L929 cells using RNAi, and cells were pretreated with DMSO, Z-VAD, or Z-VAD+NEC1 for 1 hour followed by treatment with TNF-α for 2.5 hours. Cell viability was assessed with propidium iodide staining and flow cytometry. Knockdown of SPATA2 mRNA was verified using real time PCR. p** < 0.05 (two-tailed, unpaired Student´s t-test).",
    "molecules": "NEC1, DMSO, Z-VAD"
  },
  {
    "caption": "(G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction.",
    "molecules": "ATP"
  },
  {
    "caption": "(H) Left panel: Glucose quantification in wt, gas-1 and age-1; gas-1 nematode lysates with NMR, n=7, ns= non-significant, Mann Whitney U test. Right panel: Quantification of total carbohydrates in the indicated strains with phenol-sulfuric assay. Error bars: mean ± SEM, 2 biological replicates (5 technical replicates each), normalized to wt and number of animals used. **p value < 0.01, ns= non-significant, Mann-Whitney U test.",
    "molecules": "carbohydrates, Glucose"
  },
  {
    "caption": "ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes;",
    "molecules": "ATP"
  },
  {
    "caption": "ATP measurements in: (I) age-1; gas-1 (EV) (a; g (EV)) and age-1; gas-1; kin-1 (RNAi) (a; g; kin-1(RNAi)). Error bars: Mean ± SEM, n=4, *p value <0.05, Mann Whitney U test.",
    "molecules": "ATP"
  },
  {
    "caption": "A) Volcano plot of lipidomic profiling in gas-1 versus age-1; gas-1 mutants. Red stars: significantly altered lipids in age-1; gas-1 versus gas-1 (5% FDR; n=5).",
    "molecules": "lipids"
  },
  {
    "caption": "F) Immunoblot analysis of phospho-Akt (S473), pan Akt, phospho-PRAS40 (T246) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0.2% DMSO (-) or AMP (2 μM), ENPF (2 μM) and PTX (36 μM) (+) for 24 h (actin loading control).",
    "molecules": "AMP, DMSO, ENPF, PTX"
  },
  {
    "caption": "(B) Locomotory activity of 10-day-old gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines. Values represent arbitrary units from 3 biological replicates with 4 technical replicates each, 35 worms per technical replicate. Error bars: mean ± SEM, **p value< 0.01, ***p value< 0.001, one-way ANOVA, Dunnett's post-hoc correction.",
    "molecules": "DMSO"
  },
  {
    "caption": "(E-F) Immunoblot analysis of (E) KIN-1 phospho-substrates in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).",
    "molecules": "DMSO"
  },
  {
    "caption": "(E-F) Immunoblot analysis of (F) phospho-AAK-2 in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).",
    "molecules": "DMSO"
  },
  {
    "caption": "(G) Left panel: FRAP in vehicle or PTX-treated gas-1 animals. Each point: mean recovery of 14 bleached regions (2 regions per animal, 7 animals per condition) ± SEM. Right panel: maximum (total) recovery per condition ± SEM. **p value< 0.01 unpaired t test. For all immunoblot panels: numbers correspond to band densitometries across biological replicates and for all lifespan curves: the legend shows Mean median lifespan ± SEM across all biological replicates.",
    "molecules": "PTX"
  },
  {
    "caption": "(B) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Lysosomes were inhibited by treating mice with 50 mg/kg of chloroquine for 7 days. Chloroquine treatment started at the day of denervation and lasted for 7 days, whereas for fasting experiments at the 6th day of chloroquine injection food was removed for 24 h. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.",
    "molecules": "chloroquine, Chloroquine"
  },
  {
    "caption": "(B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (*P0.001).",
    "molecules": "FCCP, Oligomycin"
  },
  {
    "caption": "(B) Immunoblotting analysis shows that AICAR treatment causes phosphorylation of AMPK and the downstream target ACC but does not affect phosphorylation of AKT and FoxO3.",
    "molecules": "AICAR"
  },
  {
    "caption": "(C) FoxO3 binding to atrogin‐1 and MuRF‐1 promoters, as determined by ChIP, is induced by AICAR treatment.",
    "molecules": "AICAR"
  },
  {
    "caption": "A-E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs (A-C) and dermal HMVECs (D-E) were grown to 100% confluence over 8-μm-pore insert membranes. ECs were first treated with Vasculotide (VT), PBS (vehicle/negative control), Ang1 (positive control), PEG-Cys (polyethylene glycol backbone), T7c (non-PEGylated CHHHRHSF peptides), or VT in the presence of 100-fold molar excess of T7c. The concentrations used, 10-20 ng/mL VT and 200-400 ng/mL Ang1, are estimated molar equivalents (0.71-1.43 nM). Thirty minutes later, ECs were stimulated with thrombin, 0.1% BSA (vehicle/negative control), or EDTA (positive control). Another 30mins later, the amount of FITC-dextran diffusion into the lower chambers (A, D) provides a measure of endothelial permeability. CMTPX-labeled tumor cells (TCs) were then dispensed into inserts, and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h (B-C) or 28 h (E) reflects the efficiency of trans-endothelial TC migration. Representative fluorescent images (10×) of membrane undersides are shown in (C), where DAPI-stained nuclei of ECs/TCs are shown in blue, 'extravasated' CMTPX-labeled TCs are shown in red, and the # of CMTPX+ pixels are shown numerically in red. Means ± SEM are shown (A,B,D,E). Three experiments (twice with HMVEC-LBl and once with HMVEC-DBl) were run with n = 3-5 inserts (independent biological replicates) per group and analyzed by two-sampled unpaired t-tests.",
    "molecules": "dextran, PEG-Cys, T7c, Vasculotide, VT, EDTA"
  },
  {
    "caption": "A-H LM2-4luctumor cells were implanted intravenously into SCIDmice, generating predominantly lung-specific metastases in one experiment (n = 5-6; A-D) and predominantly lymphatic-specific metastases in another experiment (n = 12; E-H). (C and G) Photographs of typical metastases in the lungs and caudal lymphatics, respectively. Kaplan-Meier survival analysis showed that Vasculotide (VT) significantly delayed mortality due to lungmetastases (A) but not lymphaticmetastases (E). Quantitative analysis of in vivo bioluminescent images taken at 24DPI (B) showed that concurrent VT treatment effectively reversed the sunitinib (SU)-induced acceleration of lungmetastases (D). With regard to lymphaticmetastases (F, H), SU treatment did not accelerate their progression. Geometric means ± 95%CI are depicted in (D and H); P values were derived by one-way ANOVA (D) and Kruskal-Wallis test (H). The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5.",
    "molecules": "Vasculotide, VT, SU, sunitinib"
  },
  {
    "caption": "A, B In modified Boyden chamber experiments, confluent dermal HMVECs were stimulated with tumor cell-conditioned media (TC-CM)-that is, media conditioned by either LM2-4luc, SN12luc, or HT29luc tumor cells-and treated with 20 ng/mL Vasculotide vs. 400 ng/mL Ang1 (positive control) vs. PBS (negative control). Meta-analyses of treatment effects on dextran permeability (A) and tumor cell migration (B), where each datapoint represents the mean of an independent experiment, averaged over 2-3 biological replicates (inserts) per treatment group and normalized internally within that same experiment. Statistical significance of overall Ang1 or VT treatment effects was determined by paired t-tests, n = 11 (A) or 6 (B) experimental replicates.C Representative fluorescent images (10×) of 'extravasated' CMTPX-labeled TCs (red) fixed on membrane undersides.",
    "molecules": "dextran, Vasculotide, VT"
  },
  {
    "caption": "A-C Vasculotide (VT) did not alter the growth kinetics of orthotopic LM2-4luc tumors; n = 8 mice per group. Sunitinib (SU) treatment significantly lowered primary tumor burden as measured by volume (B: one-way ANOVA, P < 0.01) or bioluminescent activity (C: one-way ANOVA, P < 0.001).",
    "molecules": "Vasculotide, VT, SU, Sunitinib"
  },
  {
    "caption": "D VT treatment did not alter the mean thickness of the viable peripheral regions of primary tumors, as quantified by H&E histological staining.",
    "molecules": "VT"
  },
  {
    "caption": "E VT treatment did not alter the mean thickness of the CAIX+ hypoxic edge between the viable periphery and necrotic core of a primary tumor; IHC control and sample images taken at 5× are shown.",
    "molecules": "VT"
  },
  {
    "caption": "F Ex vivo bioluminescent imaging suggests that VT treatment lowered the proportion of mice with metastatic involvement in the lungs, without the corresponding effect on lymphatic metastases. Representative bioluminescent vs. photographic images of dissected lungs in a 24-well plate are shown.",
    "molecules": "VT"
  },
  {
    "caption": "G Human LM2-4luc cells can be identified on histological sections of mouse lung tissue by IHC staining of human vimentin, a cytoskeletal protein. Size distribution analysis show VT monotherapy reducing the occurence of bigger metastatic nodules.",
    "molecules": "VT"
  },
  {
    "caption": "B Bioluminescent images of tumor burden.C-D Total bioluminescent fluxes, at 35 days post-implantation (while the 'SU_3 wk' group was still receiving 60 mg/kg/day sunitinib treatment), were separately quantified from the thoracic (C) and abdominal (D) regions. Geometric means and P values from Mann-Whitney tests are depicted in (C and D). Percentages listed below the x-axes in panels (C and D) refer to treatment-associated changes in the geometric mean bioluminescence, relative to the vehicle group.",
    "molecules": "SU, sunitinib"
  },
  {
    "caption": "A-F Primary human umbilical vein endothelial cells (HUVECs) or primary human dermal microvascular endothelial cells (HMVEC-DBls) were stimulated at 80% confluency with Vasculotide (treatment), Ang1 (positive control; ligand of Tie2), PBS (vehicle control), or VEGF (negative control; non-ligand of Tie2). Tie2 was immunoprecipitated from cell lysates and immunoblotted for total Tie2 or phosphotyrosine. Vasculotide treatment did not lead to increases in the pTie2/Tie2 ratio in HUVECs after 45-min (A-D) or 15-min (E) treatments, with (E) or without (A-D) serum/supplement starvation. Vasculotide also did not activate Tie2 receptors in HMVEC-DBls, whether in the presence or absence of serum, growth factor, and cytokine supplements (F).",
    "molecules": "Vasculotide"
  },
  {
    "caption": "Healthy non-tumor-bearing mice (n = 8-9 per group) were treated for 1 week with vehicle (Veh); Vasculotide 250 ng/2 days IP (VT); sunitinib 60 mg/kg/day PO (SU); or VT + SU. Tie2 immunoprecipitates from lung, kidney, or liver homogenates were pooled from two mice per treatment group and immunoblotted with an anti-Tie2 antibody ('Tie2') or an anti-phosphotyrosine antibody ('p-Tie2').Densitometry analysis of immunoblots from (A). pTie2/Tie2 ratios were internally normalized to the vehicle group within each blot. Means ± SEM and P values (unpaired t-tests) are depicted.",
    "molecules": "Vasculotide, VT, SU, sunitinib"
  },
  {
    "caption": "A-B Pull-down assay. Input ligands-rhAng1 (A) and VT (B)-were incubated with either protein A sepharose beads only, bead-conjugated mouse Tie2-Fc (mTie2), or bead-conjugated human Tie2-Fc (hTie2). The 'bound' fraction shows the total amount pulled down by beads. The 'unbound' fraction is 7% of residual/free ligands remaining in the supernatant.",
    "molecules": "VT"
  },
  {
    "caption": "C Quantitative kinetics analysis by SPR. All analytes were run at increasing concentrations of 0.625, 1.25, 2.5, 5, and 10 nM, at 37°C and pH 7.4. The positive controls-rhAng1, BowAng1, COMP-Ang1-all bound strongly to immobilized hTie2-Fc. There was no detectable binding of VT, or the negative control, rhVEGF, to immobilized hTie2-Fc.",
    "molecules": "VT"
  },
  {
    "caption": "The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.A, B. A-site bound substrates ACC-Puromycin (A) and ACCA-Pro (B).",
    "molecules": "peptidyl-tRNA, Pro, Puromycin"
  },
  {
    "caption": "The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.C, D. P-site bound substrates ACCA-Leu-Phe (C) and ACCA-Pro-Pro (D).",
    "molecules": "Leu-Phe, peptidyl-tRNA, Pro-Pro"
  },
  {
    "caption": "This figure compares conformation of aminoacyl residues of the two A-site substrates:A. ACC-PuromycinB. ACCA-Pro.To show position of the A-site substrates relative to the P-site, the model P-site substrate tRNA-Phe is shown as in the pre-attack complex of the ribosome (pdb id 1vy4). In both illustrations, the A site is viewed as through the peptide exit tunnel. In addition to S. cerevisiae residues numbering, E. coli numbering is shown in parenthesis. Comparison of the two structures shows that the side chain of the methyl-tyrosine is bound to the conserved hydrophobic A-site cleft, whereas the side chain of the proline residue penetrates the actual site of peptide bond formation where it may interfere with alignment of the reacting groups and proper positioning of the attacking electron pair of the α-amine.",
    "molecules": "Pro, Puromycin, tRNA-Phe"
  },
  {
    "caption": "This figure compares conformation of two dipeptides in the ribosome nascent peptide tunnel:A. Phe-Leu peptideB. Pro-Pro peptide.",
    "molecules": "Phe-Leu, Pro-Pro"
  },
  {
    "caption": "(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n =131) groups. Aβ positivity was defined as CSF Aβ42/40 ratio < 0.071.",
    "molecules": "Aβ42"
  },
  {
    "caption": "(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of CSF Aβ42/40. The horizontal-axes directions were inverted; lower CSF Aβ42/40 ratio reflects higher Aβ pathology. For each Aβ status group, we computed the standardized regression coefficients (β) and the P-values, adjusted for age and sex. The solid lines indicate the regression line and the 95% confidence intervals for each of the Aβ status groups. The dashed green lines indicate the CSF Aβ42/40 cutoff. Participants were also colour-coded based on the Aβ PET CL scale (≤12CL, black; >12CL, turquoise; Aβ PET non-available, grey).",
    "molecules": "Aβ42"
  },
  {
    "caption": "(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of Aβ PET Centiloids (CL). The standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for age and sex. The solid lines indicate the regression line and the 95% confidence intervals. The dashed green lines indicate the CL12 and CL30 cutoffs. Participants were also colour-coded based on the CSF Aβ42/40 ratio (A-, black; A+, turquoise).",
    "molecules": "Aβ42"
  },
  {
    "caption": "A-C ROC analysis was performed to test the accuracy to discriminate between Aβ-positive (A+) from Aβ-negative (A-) individuals. Aβ positivity was defined as CSF Aβ42/40 < 0.071 (A), Aβ PET positive visual read (B) or Aβ PET Centiloid (CL) > 12 (C). Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.",
    "molecules": "Aβ42"
  },
  {
    "caption": "A-D Dot and box-plot showing the levels of each p-tau biomarker in each of the AT groups. Aβ-positive (A+) was defined by a CSF Aβ42/40 < 0.071 and Tau-positive (T+) by an Elecsys CSF Mid-p-tau181 > 24 pg/ml. The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers). The horizontal dashed line indicates the median of the p-tau biomarker in the A-T- group. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.",
    "molecules": "Aβ42"
  },
  {
    "caption": "The graphs represent the z-scores changes of each CSF biomarker as a function of CSF Aβ42/40 ratio (as proxy of disease progression) using a robust local weighted regression method. The z-scores were calculated using the mean and the SD of each CSF biomarker in the A-T- group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed line indicates the CSF Aβ42/40 cutoff for A+. Note that the CSF p-tau biomarkers reach the 2 z-scores (depicted with an horizontal dashed line) with the following sequence: Mid-p-tau231, N-p-tau181, N-p-tau217, Mid-p-tau181 and t-tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, Neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.",
    "molecules": "Aβ42"
  },
  {
    "caption": "B. Binding assay using MBP-fused Apc1-loop500 fragments. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, 2A (T532A/T539A) and S558A) was incubated with anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by immunoblotting with Plx1 antibody and Ponceau S staining.",
    "molecules": "amylose, Ponceau S"
  },
  {
    "caption": "D. Specific binding of Plx1-PBD to Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives 2A (T532A/T539A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated in the presence of 10 µg of WT Plx1-PBD for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by Coomassie",
    "molecules": "amylose, Coomassie"
  },
  {
    "caption": "E. In vitro phosphorylation by Plx1. Apc3-loop fragment, α-Casein, N-terminus of Cdc20 (N159) or MBP protein was incubated with purified Plx1 in the presence of [γ32P]-ATP at 23˚C for indicated times, separated by SDS-PAGE and detected by autoradiography and Coomassie brilliant blue (CBB) staining.",
    "molecules": "ATP, CBB, Coomassie brilliant blue, γ32P"
  },
  {
    "caption": "E. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. MBP-fused Apc1-loop500 WT or its derivatives (T532A, T539A, T532A/T539A (2A) and S558A) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. The recovery of WT (x1/2 and x1) was run as a standard. F. Quantification of (E). The bar graph is quantification of bound B56γ. The intensities of WT were arbitrarily set to 1.0. Error bars, SEM from three independent experiments. ",
    "molecules": "amylose, CBB, Coomassie brilliant blue, 35S"
  },
  {
    "caption": "A. Plx1 and PP2A-B56 competes for Apc1-loop500. MBP-fused Apc1-loop500 WT or its derivatives (∆11, deletion of 11 residues including the B56-binding motif) was incubated with the 35S-labelled Flag-B56γ in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with indicated amounts of WT Plx1-PBD or Plx1-PBD Pincer mutant (Pin) for 15 min. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining.",
    "molecules": "amylose, CBB, Coomassie brilliant blue, 35S"
  },
  {
    "caption": "C. Dephosphorylation of Apc1-loop500 by PP2A-B56 in the presence of Plx1-PBD. MBP-fused Apc1-loop500 WT or its derivatives (L557A/V560A or E562A) was incubated in anaphase extracts supplemented with non-degradable cyclin B at 23˚C for 1 h, and further incubated with 10 µg of WT Plx1-PBD for 15 min. The complexes were isolated by amylose beads and incubated in the presence (78 nM) or absence of purified PP2A- B56γ at 23˚C for 20 min. The proteins were recovered by amylose beads, and analysed as described in (B).",
    "molecules": "amylose"
  },
  {
    "caption": "(B) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB).",
    "molecules": "streptavidin"
  },
  {
    "caption": "(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining.",
    "molecules": "Glutathione"
  },
  {
    "caption": "(D) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(G) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(I) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.",
    "molecules": "NTA"
  },
  {
    "caption": "(B) SFB-PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads. Interaction with α-catenin was determined by western blotting.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(C) In vitro phosphorylated MBP-tagged α-Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant (D496A) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represents mean absorbance from three independent experiments. Error bars indicate SD, **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).",
    "molecules": "malachite green, phosphate"
  },
  {
    "caption": "(L) SFB-PPM1G and SFB-PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells. Lysates were pulled down using streptavidin beads and interaction with α-catenin was detected by western blotting using α-catenin antibody.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(C) Control, PPM1G and B56δ shRNA transduced cells were transfected with SFB-α-catenin. Cell lysates were pulled down using streptavidin beads and interaction with β-catenin was determined by western blotting.",
    "molecules": "streptavidin"
  },
  {
    "caption": "(D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm.",
    "molecules": "Phalloidin, rhodamine"
  },
  {
    "caption": "E Phalloidin staining of F-actin organization. Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed. Micrographs of Sc and Sc_NI cells are reproduced from Fig 2E. Scale bar: 3 µm.",
    "molecules": "Phalloidin, phalloidin"
  },
  {
    "caption": "I Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm J Quantification of actin patch resistance to CK-666 treatment. Bar graphs represent the percentage of cells with a given number of visible actin patches after CK-666 treatment. (n = 41 for ScNI, 55 for N2, 31 for Ca, 41 for Op, 55 for Hs).",
    "molecules": "CK-666, phalloidin"
  },
  {
    "caption": "B (Left) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa-488-labeled ADF/cofilin (replacement of unlabeled ADF/cofilin). (Right) Quantification of ADF/cofilin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 34 for Act_Sc branched, n = 28 for Act_N2 branched, n = 43 for Act_Ca branched, n = 24 for Act_Sc linear, n = 15 for Act_N2 linear and n = 27 for Act_Ca linear). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).",
    "molecules": "Alexa-488"
  },
  {
    "caption": "C (Left) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488-tropomyosin. (Right) Quantification of tropomyosin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 45 for Act_Sc branched, n = 63 for Act_N2 branched, n = 57 for Act_Ca branched, n = 22 for Act_Sc linear, n = 32 for Act_N2 linear and n = 27 for Act_Ca linear). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).",
    "molecules": "Alexa 488"
  },
  {
    "caption": "C Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. D In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). E Polarity Indexes. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).",
    "molecules": "Phalloidin"
  },
  {
    "caption": "F Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. G In vivo actin network deviation indexes of cells treated with DMSO or CK-666. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).",
    "molecules": "CK-666, DMSO, phalloidin"
  },
  {
    "caption": "GST-Mif2_1-41 was immobilized on GSH Sepharose beads and incubated with Mtw1-Nnf1 complex (MN) in the presence of increasing concentrations of Ame1_1-20 peptide (lanes 4-9). GST-Ame1_1-30 served as control (lane 2 and 3). After washing, bound complexes were eluted by adding SDS sample buffer. Protein bands in Coomassie stained gel are highlighted in the indicated color. Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST-Mif2 amounts in the same lane.",
    "molecules": "GSH, Sepharose, SDS"
  },
  {
    "caption": "Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex. Mif2 and Ame1 wild-type N-termini serve as a control, protein bands are highlighted in the indicated color.",
    "molecules": "Alanine"
  },
  {
    "caption": "Analysis of these Mtw1 mutants in vivo using the anchor-away technique. Mtw1-FRB strains containing RPL13-FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "Analysis of Ame1 N-terminal deletion and swap mutant using the anchor-away technique. Ame1-FRB strains containing RPL13-FKBP12 for ribosome anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "Phenotypic analysis of different Ame1 rescue constructs using fluorescence microscopy. Images of representative large budded cells immediately and 4 h after Rapamycin addition are shown, indicated as t = 0 h and t = 4 h. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. One kinetochore cluster is enlarged in the white box. Scale bar represents 5 µm.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "Co-purification of Strep-Mtw1c through Flag-Mif2 after co-expression in insect cells. Proteins were detected by western blotting using their respective tags.",
    "molecules": "Strep"
  },
  {
    "caption": "Cross-link (XL) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross-linker Bis[sulfosuccinimidyl] glutarate. Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration. The corresponding Coomassie-stained SDS-PAGE gels of Mif2-wt alone or in combination with Cse4n are shown. Fractions used for cross-linking are highlighted. Topological map of Mif2 based on the identified intra-molecular cross-links. Common cross links that are found in both samples are shown in red, whereas unique crosslinks that can be only found in one of the samples are highlighted in blue. Conserved regions and their described functions are indicated. A list of cross-links that link N-terminal residues to C-terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2.",
    "molecules": "Bis[sulfosuccinimidyl] glutarate"
  },
  {
    "caption": "Mif2Flag-wt or -∆sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose, washed and incubated with Mtw1-Nnf1 complex (MN). After washing, bound complexes were eluted by adding 3xFlag peptide. Western blotting against the His-tag on Nnf1 confirmed increased binding of MN to Mif2-∆sigAT, already visible in the Coomassie stained SDS-PAGE gel.",
    "molecules": "Agarose"
  },
  {
    "caption": "Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system. Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.",
    "molecules": "galactose, Galactose, Glucose, Raffinose"
  },
  {
    "caption": "Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter. Strains were plated on YEP-Glucose and YEP-Raffinose/Galactose plates.",
    "molecules": "Galactose, Glucose, Raffinose"
  },
  {
    "caption": "Analysis of chromosome segregation in strains from (B). Distribution of fluorescently labeled Chromosome III was analyzed in large-budded cells 16 hours after switching the medium to Galactose. Scale bar represents 2 μm.",
    "molecules": "Galactose"
  },
  {
    "caption": "Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1-GFP and Spc42-mCherry. Images of representative large budded cells after 16h in Galactose are shown. One kinetochore cluster is enlarged in the white box for each strain. Scale bar represents 5 µm. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. Quantification of (D) in which large budded cells were classified according to their Mtw1-GFP signal into the four indicated groups. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 2 independent experiments.",
    "molecules": "Galactose"
  },
  {
    "caption": "Phenotypic analysis of Mif2-∆sigAT mutant using the same galactose inducible overexpression system as in (A). Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.",
    "molecules": "galactose, Galactose, Glucose, Raffinose"
  },
  {
    "caption": "(A) DNA damage induces H2AX ADP-ribosylation. 293T cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes). ADP-ribosylated proteins were IPed with anti-ADP-ribose (anti-ADPR) antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.",
    "molecules": "ADP-ribose, ADPR, H2O2, MMS, PBS"
  },
  {
    "caption": "(B) H2AX is ADP-ribosylated at glutamate 141 (E141). shPARG HCT116 cells were treated with H2O2 (2 mM in PBS, 5 minutes) and ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. Glutamate 141 (E141) is indicated by an asterisk.",
    "molecules": "glutamate, Glutamate, H2O2, hydroxamic acid, NH2OH, PBS"
  },
  {
    "caption": "(C) The E141A mutation abolishes the ADP-ribosylation of H2AX. Empty vector (EV), wild type (WT) or the E141A mutant (E141A) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2. ADP-ribosylated proteins were IPed with anti-ADPR antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.",
    "molecules": "ADPR, H2O2"
  },
  {
    "caption": "(D) ADP-ribosylation on H2AX is suppressed by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. The samples were examined by IP and Western blotting.",
    "molecules": "DMSO, olaparib"
  },
  {
    "caption": "(E) PARP1, but not PARP2, mediates the ADP-ribosylation on H2AX. Wild type (WT) cells, PARP1-null cells or PARP2-null cells were treated with H2O2.",
    "molecules": "H2O2"
  },
  {
    "caption": "(A) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 hours using MTT assay. Values are mean ± SD of three assays.",
    "molecules": "H2O2, MMS"
  },
  {
    "caption": "(B) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes), subsequently; the cells were cultured in fresh DMEM medium for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays.",
    "molecules": "H2O2, MMS, PBS"
  },
  {
    "caption": "(C) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes) and harvested at the indicated recovery time points. Then FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays.",
    "molecules": "H2O2, MMS, PBS"
  },
  {
    "caption": "(D) DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes), followed by BrdU incorporation (30 μM in medium, 30 minutes). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample, Values are mean ± SD of three assays.",
    "molecules": "BrdU, DAPI, DNA, H2O2, MMS, PBS"
  },
  {
    "caption": "(E) Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays.",
    "molecules": "H2O2, MMS"
  },
  {
    "caption": "(A) ADP-ribosylation mediates the recruitment of Neil3. GFP-Neil3 was expressed in U2OS cells, and cells were treated with olaparib (1 µM, 1 hour) or mock (0.1 % DMSO in medium, 1 hour). Following laser microirradiation treatment, the recruitment of Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.",
    "molecules": "DMSO, olaparib"
  },
  {
    "caption": "(C) PARP inhibitor treatment (olaparib: 1 µM, 1 hour) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions. The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.",
    "molecules": "olaparib"
  },
  {
    "caption": "(D) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. Oxidative damage was induced by KillerRed (KR) system with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 minutes. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of Neil3 foci co-localized with KR was quantified.",
    "molecules": "DMSO, olaparib"
  },
  {
    "caption": "(A) The GRFs of Neil3 recognizes PAR. The recombinant GST fusion proteins were incubated with PAR and the protein-PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted three-fold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full length Neil3, N-ter represents N-terminal Neil3 (1-300 aa), Mid represents middle Neil3 (300-404 aa), and GRFs represents C-terminal Neil3 GRFs domain (480-606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (middle panel). The relative PAR-binding in the pull down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays.",
    "molecules": "agarose, glutathione, nitrocellulose"
  },
  {
    "caption": "(A) PARP inhibition facilitates γH2AX following oxidative stress. The cells were pre-treated with olaparib (1 µM, 1 hour) prior to H2O2 stimulation. 0.1 % DMSO in medium for 1 hour as mock. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.",
    "molecules": "DMSO, H2O2, olaparib"
  },
  {
    "caption": "(G) DNA replication of U2OS cells expression with individual wild type (WT), the H2AX KO + WT, the E141A mutant (E141A) or the Neil3/MDC1 Chimera (Chimera) were analyzed by BrdU incorporation following treatment with H2O2, MMS or mock (PBS). Represented images are shown in the left panel. The graph shows percentages of BrdU-positive cells expressing WT, E141A or Chimera (right panel).",
    "molecules": "BrdU, DNA, H2O2, MMS, PBS"
  },
  {
    "caption": "A, B. The recruitment of the mCherry fused p62 derived sensor AS3_p62 to the autophagosomes positive for overexpressed EGFP-LC3B (A) or endogenous LC3B (B) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment.",
    "molecules": "KU, Baf"
  },
  {
    "caption": "A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored in basal conditions (A) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored upon starvation (EBSS) (B) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored after autophagy induction by Torin I (C) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.",
    "molecules": "doxycycline, Torin I"
  },
  {
    "caption": "D. FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA-Parkin, mt-mKeima, and a GFP fusion of the indicated sensor. Cells were treated with OAQ (oligomycin, antimycin, Q-VD) for indicated times and analyzed by FACS for lysosomal-positive mt-mKeima (561nm). The average of three independent experiments is presented. Representative FACS data from experiments (GFP control and AS3_67) are shown in the lower panels. Error bars indicate standard deviation. Significance calculation: 2-way anova of one-way anova. n.s.= not significant. *** indicates p-value of 0.0001.",
    "molecules": "Q-VD, antimycin, oligomycin"
  },
  {
    "caption": "A. LC3C specific sensor mCh-AS3_67 is recruited to Salmonella (SFH2) escaping SCV. Salmonella SFH2 strain is carrying a GFP-reporter gene activated by availability of glucose-6-phosphate in the host's cytosol.",
    "molecules": "glucose-6-phosphate"
  },
  {
    "caption": "A,B. LC3C specific sensor mCh-PB1-AS3_67 is recruited to a subset of mitochondria undergoing mitophagy. Mitochondria were labeled with MitoTracker Deep Red (ThermoFisher). U2OS cells were transiently co-transfected for indicated sensor and mATG8 proteins. Mitophagy was induced by CCCP treatment over three hours. Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 (Baf).",
    "molecules": "Baf, BafilomycinA1, CCCP"
  },
  {
    "caption": "Nucleus height was measured by immunofluorescence using Hoechst staining",
    "molecules": "Hoechst"
  },
  {
    "caption": "Nucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Representative HeLa-FUCCI cells in S-G2-M (green) and G1 phase (red) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 31 µm.",
    "molecules": "AP, Blebb"
  },
  {
    "caption": "Representative flow cytometry histograms for EdU positive cells in Ctrl, Blebb and Blebb + AP conditions. Corresponding percentage of EdU positive cells.",
    "molecules": "EdU, AP, Blebb"
  },
  {
    "caption": "Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb).",
    "molecules": "AP, Blebb, blebbistation"
  },
  {
    "caption": "TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5).",
    "molecules": "AP, blebbistatin"
  },
  {
    "caption": "Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. (b) Corresponding quantifications of p-Jun Ser63 nuclear intensity.",
    "molecules": "AP, Blebb, DNA, Ser, Hoechst"
  },
  {
    "caption": "Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (ratio nucleus/cytosol).",
    "molecules": "AP, Blebb, DNA, Hoechst"
  },
  {
    "caption": "Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP.",
    "molecules": "AP, Blebb, Ser, Y27632"
  },
  {
    "caption": "Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition.",
    "molecules": "AP, Cytochalasin D, CytoD, Ser"
  },
  {
    "caption": "Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2.",
    "molecules": "Ser"
  },
  {
    "caption": "Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity.",
    "molecules": "agarose, DNA, Ser, Hoechst"
  },
  {
    "caption": "Quantifications of YAP activity in Ctrl, Blebb and Blebb + AP conditions, treated or not with importazol (nucleo-cytoplasmic ratio).",
    "molecules": "AP, Blebb, importazol"
  },
  {
    "caption": "Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.",
    "molecules": "AP, Blebb, ser, Thr"
  },
  {
    "caption": "Corresponding quantification of p-YAP (Ser125) relative to Ctrl and normalized to GAPDH. Corresponding quantification of p-LATS (thr1079) relative to Ctrl and normalized to GAPDH.",
    "molecules": "Ser, thr"
  },
  {
    "caption": "Quantifications of p-Jun Ser63 nuclear intensity in Ctrl, Blebb, and Blebb + AP conditions for cells treated with DMSO, SB203580, a MAP Kinase inhibitor and SP600125, a JNK inhibitor.",
    "molecules": "SP600125, AP, Blebb, DMSO, Ser, SB203580"
  },
  {
    "caption": "Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions for cells depleted or not for lamin-A/C. Scale bar = 10 µm. Corresponding quantification of p-Jun Ser63 nuclear intensity.",
    "molecules": "AP, Blebb, Ser"
  },
  {
    "caption": "WT and CKO mice  aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (n=10，there were 10  mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). L, M Representative images of Ocn+ (red) and DAPI (blue)  immunostaining (L) and quantification of Ocn+ cells on  trabecular (M), scale bar, 50µm (n=6). N, O Representative images of TRAP (red) and DAPI (blue) immunostaining (N) and quantification of TRAP cells on trabecular (O), scale bar, 50µm (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).",
    "molecules": "DAPI"
  },
  {
    "caption": "BMT  was conducted on 12-month-old CKO and WT  mice with normal diet, and then the recipients from each group were performed for analysis 3 months after interventions (n=10，there were 10  mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). H Representative images of H&E staining (upper panel), TRAP staining (middle panel) and calcein double-labeling (lower panel) in distal femora, scale bar, 100 µm for H&E staining. 50 µm for TRAP staining, 50μm for calcein  double-labeling. I, J Quantification of number for osteoblasts (I) and number of osteoclasts (J) in distal femora (n=6). K Quantification of bone  formation rate per bone surface  (BFR/BS) (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).",
    "molecules": "calcein"
  },
  {
    "caption": "in situ myeloid  specific MYDGF  overexpression in bone marrow was performed on CKO mice  aged 9 months. Micro-CT  analysis and histomorphometric analysis of distal  femora  were performed after 3 months intervention. H Representative images of H&E in distal femora, scale bar, 100 µm. I Quantification of number for  osteoblasts  in distal femora  (n=6). J Representative images of TRAP staining  in distal  femora, scale bar, 50µm K Quantification of number for osteoclasts in distal femora (n=6). L Representative images of calcein double-labeling, scale bar, 50μm. M Quantification of bone formation rate per  bone surface (BFR/BS) (n=6). Data information: Results are shown as mean ±SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t test).",
    "molecules": "calcein"
  },
  {
    "caption": "N-P  BMMs were co-cultured with  osteoclasts in presence of 1,25(OH)2D3 and PGE2  with conditioned medium of BMCs from WT or CKO mice for 5 days. Representative images of osteoclasts co-cultured with osteoblasts, scale bar, 200μm (N). Quantification of osteoclast number (O) and percentage of osteoclast area (P). Results are shown as mean ± SEM, *P < 0.05 (Student's t test). Data information: Each experiment was repeated 5 times.",
    "molecules": "1,25(OH)2D3, PGE2"
  },
  {
    "caption": "G-I  Primary  calvarial osteoblasts   cultured in osteogenesis induction medium with treatment of conditioned medium of BMCs from WT or CKO mice for ALP staining assay at 7 days or ARS staining assay at 14 days. Representative images of ALP staining and ARS staining  of osteoblasts, scale bar, 100μm (G). Quantitative analysis of ALP activity (H) and calcium mineralization (I). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each experiment was repeated 5 times.",
    "molecules": "ARS, calcium"
  },
  {
    "caption": "J-L Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of 0ng/ml (vehicle) or 100ng/ml rMYDGF. Representative images of ALP staining and ARS staining of osteoblasts, scale bar, 100μm (J). Quantitative analysis of ALP activity (K) and calcium mineralization (L). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each experiment was repeated 5 times.",
    "molecules": "ARS, calcium"
  },
  {
    "caption": "M BMMs were treated with control, RANKL or MG132 for osteoclastogenesis, then with or without rMYDGF for 24 hours. IgG, P65 and histone antibodies were used to  ChIP and RT-PCR was performed to determine Nfatc1 promoters. Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).",
    "molecules": "MG132"
  },
  {
    "caption": "H The mRNA levels of Runx2 in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated interventions. I, J Quantitative analysis of ALP  activity (I) and calcium mineralization (J) in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated intervention. Data information: Results are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001(Student's t test).",
    "molecules": "calcium"
  },
  {
    "caption": "(C) Lysates from control or myc-tagged VCP-WT, ATPase-inactive VCP-EQ, or one of two IBMPFD mutants, VCP-RH or -AE, expressed for 16 h from stably transfected tetracycline-inducible U20S cells and immunoblotted with antibodies to myc, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. Note the increase in LC3II and p62 in VCP siRNA KD and mutant-expressing cells.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A-C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm.",
    "molecules": "Baf, DMSO, tetracycline"
  },
  {
    "caption": "(A) Epifluorescent images for GFP and mRFP in U20S or VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD- or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.",
    "molecules": "Baf, rapamycin"
  },
  {
    "caption": "(D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)-expressing cells. Bars, 15 µm.",
    "molecules": "Baf, tetracycline"
  },
  {
    "caption": "(A) Lysates from siRNA-treated U20S cells (scramble control [Scr] or VCP siRNA KD) after autophagic induction via nutrient deprivation for 0, 2, or 4 h and immunoblotting for LC3 and α-tubulin. LC3II degrades over time in control but not in KD cells. One of two experiments is shown.",
    "molecules": "nutrient"
  },
  {
    "caption": "(B) Lysates from U20S or tetracycline-inducible VCP-WT, -EQ, -RH, or -AE cells after autophagic induction via nutrient deprivation for 0, 2, 4, or 6 h and immunoblotting for LC3 and α-tubulin. LC3II degrades in control and VCP-WT but degrades less efficiently in mutant (EQ, RH, or AE)-expressing cells. (C) Graphical representation of densitometric evaluation of LC3II and α-tubulin at each time point from three independent experiments.",
    "molecules": "nutrient, tetracycline"
  },
  {
    "caption": "(A) Epifluorescent images for GFP-LC3 (LC3) and LTR of VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Open arrows highlight autolysosomes (GFP and LTR colocalized), and closed arrows show autophagosomes (GFP only). (B) Pearson's coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(C) Epifluorescent images for GFP-LC3 and endogenous Lamp1 immunohistochemistry of U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells or U20S cells cotreated with Baf transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Arrows highlight autolysosomes (GFP and Lamp1 colocalized). (A and C) The boxed regions in the merge field are enlarged in the adjacent panels. (D) Pearson's coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments. (B and D) Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.",
    "molecules": "Baf, rapamycin"
  },
  {
    "caption": "(E) Electron microscopy of tetracycline-inducible control or VCP-WT-, VCP-RH-, or VCP-AE-expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h. Note the accumulation of autophagic structures in the mutant-expressing cell lines. Arrows denote autophagic structures.",
    "molecules": "rapamycin, tetracycline"
  },
  {
    "caption": "(A) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH-expressing mice (RH12) 2 d after electroporation (baseline) or after 24 h of nutrient deprivation (starvation) of polyQ80 (Q80)- or polyQ19-luciferase (Q19) in the right and left tibialis anterior, respectively. The ratio of polyQ80-luciferase/polyQ19-luciferase is indicated below each image set. Values are in ×104 photons per second per square centimeter per steradian. (B) Box and whisker plot of the change (Δ) in the ratio of polyQ80-luciferase/polyQ19-luciferase activity in the left and right tibialis anterior muscle of control, VCP-WT, or one of two VCP-RH (RH12 or RH9) transgenic mouse lines after 24 h of starvation. The graph is representative of three animals per group. The p-value for RH12 was 0.05 and 0.06 when compared with control animals or VCP-WT transgenic. The p-value for RH9 was 0.03 when compared with either control or VCP-WT transgenic. The p-value was 0.01 and 0.02 when combined RH12 and RH9 animals were compared with control or VCP-WT groups, respectively. There was no statistical difference between control or VCP-WT groups.",
    "molecules": "nutrient"
  },
  {
    "caption": "(A) Quantitation of mCherry-TDP-43 distribution (nuclear only or cytoplasmic) in control U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells, or control U20S cells treated with 10 µM Baf or 50 µM chloroquine (Chlq) for 4 h from 10 different fields from two independent experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.02 when compared with VCP-WT-expressing cells. (B) Epifluorescent images for mCherry-TDP-43 (red) and DAPI (blue) in control U20S, VCP-WT-, VCP-RH-, or VCP-AE-expressing cells, or control U20S cells treated with Baf or chloroquine for 4 h. Arrows denote cytosolic TDP-43 and perinuclear TDP-43 inclusions.",
    "molecules": "Baf, chloroquine, Chlq"
  },
  {
    "caption": "(C, top) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of mCherry-TDP-43-transfected VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells. Note the increase in cytosolic TDP-43 from IBMPFD mutant- and EQ-expressing cells. (bottom) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry-TDP-43 or similarly transfected cells treated with 30 µg/ml (cq1) or 120 µg/ml (cq2) chloroquine diphosphate or 200 ng/ml Baf. Data are representative of three independent experiments. Bar, 15 µm.",
    "molecules": "Baf, chloroquine diphosphate"
  },
  {
    "caption": "(B) TDP-43 immunostaining of quadriceps skeletal muscle from 3-mo-old mice treated with saline or 50 mg/kg/d intraperitoneal chloroquine (Chlq) for 4 wk. (A and B) Insets show one myonuclei from each field. Bars, 30 µm.",
    "molecules": "chloroquine, Chlq"
  },
  {
    "caption": "B. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207 and KY594-1.",
    "molecules": "NaCl"
  },
  {
    "caption": "C. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strain KY594-1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 [Hog1-P (%)] were calculated as explained in Materials and Methods, and are shown beneath the panel. Data information: Representative results from three independent experiments.",
    "molecules": "NaCl"
  },
  {
    "caption": "D. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single-copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild-type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (-) 1 M NaCl for 5 min.",
    "molecules": "NaCl"
  },
  {
    "caption": "E-H. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strains (E) KY603-3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min. Data information: Representative results from three independent experiments.",
    "molecules": "NaCl"
  },
  {
    "caption": "I. Comparison of the NaCl dose-responses of Hog1 activation by various strains. Phos-tag band-shift assays were independently repeated three times, and average values were plotted. Data information: (I) Error bars are SEM (n=3).",
    "molecules": "NaCl"
  },
  {
    "caption": "A-C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel, and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type.",
    "molecules": "NaCl"
  },
  {
    "caption": "D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min.",
    "molecules": "NaCl"
  },
  {
    "caption": "E. Comparison of the NaCl dose-responses of Hog1 activation by constitutively-active Ste11-Q301P and constitutively-active Pbs2-DD. Phos-tag band-shift assays were independently repeated three times, and the average values were plotted. Data information: (E) Error bars are SEM (n=3).",
    "molecules": "NaCl"
  },
  {
    "caption": "D-G. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strain (D-E) KT235 or (F-G) KT290 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was transformed with either pRS416-Hog1 (WT) or its indicated mutant derivatives, and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarizes the averages of three independent experiments. Data information: (E, G Error bars are SEM (n=3).",
    "molecules": "NaCl"
  },
  {
    "caption": "H. Phos-tag band-shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single-copy expression plasmid pRS416-Hog1 (WT) or pRS416-Hog1-ΔL16, and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown. Data information: Error bars are SEM (n=3).",
    "molecules": "NaCl"
  },
  {
    "caption": "A. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. The yeast strain FP4 (hog1Δ) was transformed with the single-copy expression plasmid pRS416-Hog1-ΔL16, and was stimulated with the indicated concentrations of NaCl for 5 min. B. Averages of three independent experiments from A were plotted. Results for Hog1-WT (from Fig 1I) are included for comparison. Data information: Representative results from three independent experiments. Error bars are SEM (n=3).",
    "molecules": "NaCl"
  },
  {
    "caption": "Detection of Pbs2 phosphorylation at S514 The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (A) Phos-tag band-shift assay In (A), the positions of Pbs2-HA phosphorylated (S514-P) and unphosphorylated (S514-OH) at S514 are indicated.",
    "molecules": "NaCl"
  },
  {
    "caption": "Detection of Pbs2 phosphorylation at T518. The yeast strains KT003 (pbs2Δ), KT005 (ste11Δ pbs2Δ), TM280 (ssk2/22Δ pbs2Δ) and KT043 (ste11Δ ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extract, and phosphorylated Pbs2 was analyzed by (B) anti-phospho-T518 immunoblotting.",
    "molecules": "NaCl"
  },
  {
    "caption": "C-F. NaCl dose-response analyses of Pbs2 phosphorylation. (C-D) KT005 (ste11Δ pbs2Δ) and (E-F) TM280 (ssk2/22Δ pbs2Δ) were transformed with YCplac22I'-Pbs2-HA, and were treated with the indicated concentrations of NaCl for 5 min. (C and E) S514 phosphorylation was analyzed using the Phos-tag band-shift assay. PC; Positive Control. (D and F) T518 phosphorylation was analyzed using anti-phospho-T518 immunoblotting. G-H. Average values of three independent experiments from (C-D) and (E-F), respectively, were plotted. AU, arbitrary unit. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).",
    "molecules": "NaCl"
  },
  {
    "caption": "I. Detection of di-phosphorylated Pbs2. KT005 (ste11Δ pbs2Δ) transformed with YCplac22I'-Pbs2-HA was treated with the indicated concentrations of NaCl for 5 min. Pbs2-HA was immunoprecipitated from cell extracts, and subjected to Phos-tag SDS-PAGE. Blots of these gels were probed with (upper panel) anti-phospho-T518 or (lower panel) anti-HA. J. Same as in (I), except that the yeast strain KT003 (pbs2Δ) was used. ",
    "molecules": "NaCl"
  },
  {
    "caption": "B. Phos-tag band-shift analyses of osmostress-induced Hog1 phosphorylation. KT005 (ste11Δ pbs2Δ) was transformed with YCplac22I'-Pbs2 -HA (WT) or its indicated mutant derivatives. Cells were stimulated with (+) or without (-) 0.6 M NaCl for 5 min.",
    "molecules": "NaCl"
  },
  {
    "caption": "C-F. Phos-tag band-shift analyses of Hog1 phosphorylation. KT291 (ste11Δ pbs2Δ hog1Δ) was transformed with (C and D) pRS416-Hog1 or (E and F) pRS416-Hog1-ΔL16, together with (C and E) YCplac22I'-Pbs2-S514A-HA or (D and F) YCplac22I'-Pbs2-T518A-HA. Cells were stimulated with the indicated concentrations of NaCl for 5 min. G-H. Average values of three independent experiments from (C and E) and (D and F), respectively, are plotted. Data information: (C-F) Representative results from three independent experiments. (G-H) Error bars are SEM (n=3).",
    "molecules": "NaCl"
  },
  {
    "caption": "A. KT299 (MATa hkr1Δ msb2Δ ssk2/22Δ) was exposed to the indicated concentrations of α-factor for 15 min in the absence of osmostress. Phosphorylation of Fus3 and Kss1 was detected by immunoblotting.",
    "molecules": "α-factor"
  },
  {
    "caption": "B. KT299 (MATa ssk2/22Δ hkr1Δ msb2Δ) was exposed to the indicated concentrations of α-factor for 15 min in the presence or absence of 0.8 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Data information: (B) Representative results from three independent experiments.",
    "molecules": "NaCl, α-factor"
  },
  {
    "caption": "D. HM06-1 (MATa ΔS/O/H/M ssk2/22Δ) was exposed to the indicated concentrations (log scale) of α-factor for 15 min in the presence or absence of 1.0 M NaCl, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average values of three or more independent experiments were plotted. Data information: Error bars are SEM: (D), n=3 or more",
    "molecules": "NaCl, α-factor"
  },
  {
    "caption": "E. KT306 (MATa hkr1Δ msb2Δ ssk2/22Δ hog1Δ) was transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N/H D/G), and was exposed to 10 μM α-factor for the indicated time in the absence of osmostress, and Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Average of three independent experiments are plotted. Data information: Error bars are SEM (E) n=3.",
    "molecules": "α-factor"
  },
  {
    "caption": "A. An example of the time-course experiments for the osmostress-induced Hog1 phosphorylation. The yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was stimulated with 1.0 M NaCl for the indicated times, and the percentage of phosphorylated Hog1 (Hog1-P) was determined using a Phos-tag band-shift assay.",
    "molecules": "NaCl"
  },
  {
    "caption": "Compilations of the time-courses of osmostress-induced Hog1 activation in (B) KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P), For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. Data information: For each data point, n=1 or more.",
    "molecules": "NaCl"
  },
  {
    "caption": "Compilations of the time-courses of osmostress-induced Hog1 activation in (C) TM257 (ssk2/22Δ), and (D) TM142 (wild-type). For clarity, time-course curves are shown in two panels for lower and higher ranges of NaCl concentrations. The color chart below (D) indicates the concentrations of NaCl used. Data information: For each data point, n=1 or more.",
    "molecules": "NaCl"
  },
  {
    "caption": "E. Effects of Hog1 kinase activity and osmostress-induced glycerol accumulation on the time-courses of Hog1 activation. Cells were stimulated with 0.4 M NaCl (left panel) or 1.6 M NaCl (right panel) for the indicated times, and the percentage of phosphorylated Hog1 was determined using Phos-tag band-shift assay. Strains used were: TM142 (WT), KT254 (gpd1Δ gpd2Δ stl1Δ), and TM232 (hog1Δ) carrying pRS416-HOG1-K52S K53N. K/S K/N, K52S K53N; gpd1/2, gpd1Δ gpd2Δ. Data information: (E) Error bars are SEM (n=3 or more).",
    "molecules": "glycerol, NaCl"
  },
  {
    "caption": "C. HME cells expressing wild-type STAT2 were seeded in complete medium. On the second day, the cells were re-fed with medium lacking supplements. On the third day, the cells were treated with each supplement for 4 h, and total lysates were analyzed by the Western method. 1. EGF, 2. Insulin, 3. Hydrocortisone, 4. GA-1000, 5. BPE.",
    "molecules": "Hydrocortisone"
  },
  {
    "caption": "D. HME cells were pretreated with hydrocortisone (0.04mg/ml and 0.4mg/ml) for 3 h and then treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.",
    "molecules": "hydrocortisone"
  },
  {
    "caption": "F. HCC827 and LnCAP cells expressing wild-type STAT2 were treated with CDK inhibitors (1 µM) for 4 h and whole-cell lysates were analyzed by the Western method. 1. Flavopiridol, 2. AT7519, 3. Dinaciclib.",
    "molecules": "Dinaciclib, AT7519, Flavopiridol"
  },
  {
    "caption": "A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 30 min or were untreated. The cells were washed with PBS and the media was replaced with fresh media containing staurosporine (500 nM). Whole-cell lysates were harvested and analyzed by the Western method;",
    "molecules": "staurosporine"
  },
  {
    "caption": "(B) Upper panel. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002. PTEN, PI3KCA, GSK3 and phospho-AKT Ser-473 protein levels were analyzed by Western blot. GAPDH was used as a loading control. AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p-AKT-Ser-473 antibody and using chimeric GSK-3 as a substrate. A two-tailed Student's t test was used to compare the two groups (p<0.05). SEM, standard error of the mean. Lower panel. SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN, miR-10b or both and phospho-AKT, AKT or tubulin levels analyzed by Western Blot assays. Control is empty vector-transfected cells.",
    "molecules": "insulin, LY294002, Wortmannin"
  },
  {
    "caption": "(C) Clonogenic ability of miR-10b-depleted CD44+ MCF-7 cells after PI3K pathway inhibition. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002. Cells transfected with anti-miR-10b or a scrambled control (anti-miR-ctl) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.",
    "molecules": "insulin, LY294002, wortmannin"
  },
  {
    "caption": "(D) miR-10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation. MCF-7 stable miR-Scr-OE or miR-10b-OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.",
    "molecules": "insulin"
  },
  {
    "caption": "HIF-1α ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated",
    "molecules": "oxygen"
  },
  {
    "caption": "(C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated",
    "molecules": "oxygen"
  },
  {
    "caption": "Frequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines)",
    "molecules": "oxygen"
  },
  {
    "caption": "The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations",
    "molecules": "oxygen"
  },
  {
    "caption": "Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell lines",
    "molecules": "oxygen"
  },
  {
    "caption": "Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygen",
    "molecules": "oxygen"
  },
  {
    "caption": "Sites that bound (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments",
    "molecules": "oxygen"
  },
  {
    "caption": "Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia",
    "molecules": "oxygen"
  },
  {
    "caption": "Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536, GSK461364, BRD-K70511574, and GW-843682X), based on data from the Cancer Therapeutics Response Portal v2 database and protein expression data derived from the MD Anderson Cell Line Project database (Li et al, 2017). Proteins with expression levels that correlate with area under the curve (AUC) values for at least two drugs are included in the graph. The color and size of the data point represent the direction and significance (p value) of the correlation. Red indicates a positive correlation with drug resistance and green a positive correlation with drug sensitivity (i.e., proteins in red are more highly expressed in resistant cell lines).",
    "molecules": "BI2536, BRD-K70511574, GSK461364, GW-843682X"
  },
  {
    "caption": "Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536 GSK461364 AUC values for two Plk1 inhibitors are compared with expression of selected proteins (cMet, E-cadherin, β-catenin, and Snail); each data point represents an individual non-small cell lung cancer cell line. E-cadherin, β catenin, and cMet protein expression correlated with drug resistance.",
    "molecules": "BI2536, GSK461364"
  },
  {
    "caption": "Parental and TGF-β isogenic cell lines were treated with 50nM volasertib for 72 hours. Cells were then harvested, and lysates were immunoblotted for cleaved PARP and γH2AX proteins were subsequently quantitated and normalized with β-actin (B).",
    "molecules": "volasertib"
  },
  {
    "caption": "Thirty-three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib, including those involved in the cMet/FAK/Src signaling axis (blue text) and those involved in the PI3K/Akt signaling axis (orange text).",
    "molecules": "volasertib"
  },
  {
    "caption": "Cell viability was measured using CellTiter-Glo in NSCLC cell lines treated with volasertib, tepotinib, or a 1:2 ratio of both for 72 hours. Left: representative cell viability graph of cells treated with the indicated drugs. Right: heatmap depicting the calculated combination indices at Fa = 0.25 and Fa = 0.5.",
    "molecules": "tepotinib, volasertib"
  },
  {
    "caption": "Mice bearing epithelial (red text) and mesenchymal (blue text) non-small cell lung cancer tumors were treated with vehicle control, volasertib (30 mg/kg per week intravenously), tepotinib (25 mg/kg per day orally), or the combination for 5 weeks to generate tumor growth curves of patient-derived xenografts (PDX; A) and cell line xenografts The percent change in tumor volume at the end of treatment (normalized to day zero) was calculated with each data point representing an individual mouse, and the bar is the mean value ± standard error of 10 mice for each group",
    "molecules": "tepotinib, volasertib"
  },
  {
    "caption": "Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies. *p < 0.05. **p < 0.01.",
    "molecules": "volasertib"
  },
  {
    "caption": "Cell viability measured by CellTiter-Glo in Calu6 parental and Calu6-VAR cells treated with different concentrations of volasertib for 72 hours.",
    "molecules": "volasertib"
  },
  {
    "caption": "Epithelial and mesenchymal non-small cell lung cancer (NSCLC) cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.",
    "molecules": "volasertib"
  },
  {
    "caption": "Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoprecipitated (IP) using β1-integrin, vimentin, and Plk1 antibodies, then immunoblotted for the indicated proteins.",
    "molecules": "volasertib"
  },
  {
    "caption": "D-E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb-/- whole brains. OCR measurements under Complex 1 substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N=3-4 mice per group; bar graphs show mean + SE.",
    "molecules": "ADP, antimycin A, FCCP, malate, oligomycin, pyruvate, rotenone"
  },
  {
    "caption": "K. Immunofluorescence of 8OHdG in CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 microns. L. Quantification of 8OHdG by ELISA in brain tissues in young (aged 1.5-3 months) and old (aged 6-12 months) Ifnb+/+ and Ifnb-/- mice. ",
    "molecules": "Nissl, 8OHdG"
  },
  {
    "caption": "H. Projections of 3D images from immunostaining of Drp1 (blue), Hsp60 (mitochondria, green), INF2 (yellow), and calnexin (ER, magenta) in Ifnb-/- MEFs with or without rIFN-β treatment for 6 h. Scale bar equal 2 microns. Arrows indicate fission foci. I. Quantifications of triple-positive MT-DR+Drp1+Cnx+ in MEFs. Images are shown in Fig EV3. ",
    "molecules": "MT-DR"
  },
  {
    "caption": "E-K. Immunofluorescence in the striatal region in mice treated with 6OH-DA and rIFN-β for optineurin (E, quantified in F) and Tom20 (E), with quantification of individual mitochondrial size by mitochondrial section area (G). 8OHdG- (H-I), OxDJ1- (J-K), and DAPI-labeled nuclei. Scale bars equal 10 microns.",
    "molecules": "8OHdG, DAPI, 6OH-DA"
  },
  {
    "caption": "F) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80Alexa488 complexes on taxol-stabilized microtubules that were attached to a passivated glass surface. Kymographs show Ndc80 complexes at a concentration of 0.2 nM with (FL, blue) or without (ΔL, orange) the loop. Scale bars: vertical (5 µm), horizontal (5 s). G) Quantification of Ndc80 residence times for data as in panel F. Solid line represents a single exponential fit.",
    "molecules": "Alexa488, taxol"
  },
  {
    "caption": "J) Typical fields of view showing decoration of taxol-stabilized microtubules (cyan) incubated with full-length or loopless Ndc80 (yellow) at the indicated concentration. Images show an average projection of 200 frames. The contrast between individual fluorescent channels (inverted grayscale) was fixed. Auto-contrast was used for the composite images to highlight the differences in the uniformity of Ndc80 decoration.",
    "molecules": "taxol"
  },
  {
    "caption": "TMR-labelled Ndc80 trimers with (FL, blue) or without (ΔL, orange) the loop were added to taxol-stabilized microtubules to measure their residence time (panel C) and one-dimensional diffusion (panel D). Ndc80 trimers with and without the loop are shown. Scale bars: vertical (1000 s), horizontal (5 µm).",
    "molecules": "TMR, taxol"
  },
  {
    "caption": "C) Quantification of the time that cells spent in mitosis following various treatments. Each dot represents a cell and the red lines indicate median values. Nocodazole was added 17 hours after electroporation and 1 hour before microscopy. A minimum of 30 cells were analysed for each condition.",
    "molecules": "Nocodazole"
  },
  {
    "caption": "B) Total Internal Reflection Fluorescence (TIRF) microscopy was used to investigate Ndc80Alexa488 complexes (0.6 nM) added to trimeric Ndc80TMR (10 pM) on fluorescent taxol-stabilized microtubules that were attached to a passivated glass surface. Typical kymographs showing virtually motionless Ndc80 trimers (magenta) and transiently binding Ndc80 monomers (yellow). Wild-type (wt) monomers associate with wt trimers (left), but not with M5 trimers (right). Scale bars: vertical (100 s), horizontal (5 µm).",
    "molecules": "TMR, Alexa488, taxol"
  },
  {
    "caption": "C) Quantification of the intensity of the monomeric Ndc80 associating with microtubule-bound trimeric Ndc80. A threshold for binding was set at an intensity equivalent to one Alexa488 copy. Intensities well above 1 (yellow) could thus reflects multiple monomers binding simultaneously.",
    "molecules": "Alexa488"
  },
  {
    "caption": "B) Diffusion of full-length and loopless Ndc80 trimers in absence and presence of AB-849. The primary rabbit polyclonal AB-849 was detected using a Alexa650-labelled anti-rabbit secondary IgG antibody. Scale bars: vertical (100 s), horizontal (5 µm).",
    "molecules": "Alexa650"
  },
  {
    "caption": "C. Soft-agar assay of NIH-3T3 ROS1D2113N compared with NIH-3T3 SLC-ROS1 fusion cells (positive control) or ROS1K1980E (kinase-dead, negative control). +/- lorlatinib treated samples were compared for significance using two-way RM ANOVA (matching across rows, N = 3, biological replicates) with Šídák's multiple comparisons test (P values are indicated in figure)",
    "molecules": "Soft-agar, lorlatinib"
  },
  {
    "caption": "Cell proliferation data showing effects of treatment with DMSO (Vehicle), crizotinib (250nM), lorlatinib (250 nM), and NVL-520 (250 nM) in MCF10A ROS1 wildtype (WT) (G), D2113N (H), D2113G (I) and SLC34A2-ROS1 (SLC-ROS1) (J) as indicated. Ordinary One-way ANOVA with Dunnett's multiple comparisons test was used to determine significant differences. N = 6 wells, biological replicates (H-I). Error bars in figure represent mean ± SEM.",
    "molecules": "crizotinib, DMSO, lorlatinib, NVL-520"
  },
  {
    "caption": "C. Immunoblotting was performed on cell lysates generated from stable HEK293A ROS1D2113N, HEK293A CD74-ROS1 and NIH ROS1D2113N and NIH3T3 CD74-ROS1 after treatment with ROS1 TKI. HEK293A stable cell lines were treated with DMSO, 100 nM entrectinib and 100 nM lorlatinib, and NIH3T3 stable cell lines were treated with 25 nM and 100 nM lorlatinib. The targets interrogated are indicated in the labels on the left side of the image panels in HEK293 images and on the right side of image panels in NIH3T3 images. D. Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1, SHP2, ERK1/2 and STAT3 and AKT1 phosphorylation from two independent replicate experiments. Pixel density of phosphorylated protein signal was divded by total protein signal, and this ratio was internally normalized to DMSO (Vehicle) treated cell ratio. Note that only the 100 nM lorlatinib condition that was common to the HEK293A and NIH3T3 experiments is depicted in graphs.",
    "molecules": "DMSO, entrectinib, lorlatinib"
  },
  {
    "caption": "A-C. Cell proliferation effects after treatment with SHP2 inhibitors (TNO155, RMC4550), MEK1/2 inhibitors (selumetinib, trametinib), and JNK1/2/3 inhibitor, SP600125 as indicated (N = 6, biological replicates).",
    "molecules": "SP600125, RMC4550, selumetinib, TNO155, trametinib"
  },
  {
    "caption": "D-F. Cell proliferation effects after treatment with AKT1/2/3 inhibitor afuresertib, and MTOR1 inhibitor, everolimus, as indicated (N = 6, biological replicates).",
    "molecules": "afuresertib, everolimus"
  },
  {
    "caption": "G-I. Cell proliferation effects after treatment with JAK1/2 inhibitor ruxolitinib, and TGFBR1 inhibitor galunisertib (N = 6, biological replicates). All inhibitors were used at 250 nM final concentration, with the exception of galunisertib, which was used at 500 nM final concentration. Note that the DMSO data are the same in for each cell line when compared to different class of inhibitors. Error bars in figure represent mean ± SEM.",
    "molecules": "DMSO, galunisertib, ruxolitinib"
  },
  {
    "caption": "G. Immunoblot analysis of the phosphorylated (p) and total (t) proteins from NIH-3T3 ROS1D2113N P1 cell lysates prepared from cells treated with vehicle (DMSO) or 10, 100, or 500 nM of crizotinib, entrectinib, or lorlatinib for 4 hours.",
    "molecules": "crizotinib, DMSO, entrectinib, lorlatinib"
  },
  {
    "caption": "B) Histology  of Dot1l-KO and CTL testes  (periodic acid-Schiff staining). Scale bars indicate 200µm.",
    "molecules": "periodic acid-Schiff"
  },
  {
    "caption": "G) Coomassie-stained  protamine extracts from CTL and Dot1l-KO spermatozoa  following acid urea gel electrophoresis  (same gel, two different intensities). The same quantity of material has been loaded in each well (i.e. extracts from 1.4 million spermatozoa). Protamine 1  and 2 bands (PRM1  and PRM2, respectively) are detected at the bottom of the gel. The rectangle highlights the bands that are likely immature forms of Protamine 2 (i.e. non-cleaved precursor forms). The right panel shows a western blot detection using anti-PRM2  antibody which confirms that one of the high molecular weight band only observed in Dot1l-KO spermatozoa  is an immature form of  Protamine 2 (Pre-PRM2). The corresponding Coomassie-stained gel is also shown.",
    "molecules": "urea"
  },
  {
    "caption": "I Representative Sirius Red staining (left) with quantitative results (right) on the kidney sections of WT (n = 7) and Ehmt2Ksp (n = 4) mice at day seven after renal I/R injury (I/R 7D).",
    "molecules": "Sirius Red"
  },
  {
    "caption": "B Representative immunoblots (left) with quantitative results (right) of G9a and H3K9me1/2 in cisplatin-injured mouse kidneys.",
    "molecules": "cisplatin"
  },
  {
    "caption": "C mRNA levels of Kim1 and Ngal of PBS+NI (n = 6), A366+NI (n = 5), PBS+I/R 3D (n = 6) and A366+I/R 3D (n = 5) groups.",
    "molecules": "A366, PBS"
  },
  {
    "caption": "Representative renal TG (triglyceride) and TC (cholesterol) levels (E) of PBS+I/R 3D (n = 12) and A366+I/R 3D (n = 12) groups.",
    "molecules": "A366, cholesterol, TC, PBS, TG, triglyceride"
  },
  {
    "caption": "F mRNA levels of fatty acid oxidation related genes of PBS+I/R 3D (n = 6) and A366+I/R 3D (n = 5) groups.",
    "molecules": "A366, PBS"
  },
  {
    "caption": "K Representative immunostaining for CD3, Ly6G, and F4/80 and quantitative results of PBS+I/R 3D (n = 6) and A366+I/R 3D (n = 5) groups.",
    "molecules": "A366, PBS"
  },
  {
    "caption": "Representative immunoblots (left) with quantitative results (right) of p-Rip3 for PBS+I/R 3D (n = 3) and A366+I/R 3D (n = 3) groups.",
    "molecules": "A366, PBS"
  },
  {
    "caption": "Representative images (M) with quantitative results of lipid droplet areas (N) and percentage of different lipid droplet size (O), in labeled-PA treated mPTECs isolated from WT and Ehmt2Ksp mice at one hour after treatment. The results were obtained from three biological replicates; a total of 20 cells were imaged in each group for quantification of lipid droplet area (N, O)",
    "molecules": "PA"
  },
  {
    "caption": "Q-S Seahorse studies (Q) with quantitative results of basal (R) and maximal (S) respiration in PA-treated mPTECs isolated from WT and Ehmt2Ksp mice. OCR, oxygen consumption rate; n = 4 cultures.",
    "molecules": "PA"
  },
  {
    "caption": "E Representative Oil Red O staining with quantification of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.",
    "molecules": "Oil Red O, WWL"
  },
  {
    "caption": "F mRNA levels of fatty acid oxidation related genes of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.",
    "molecules": "WWL"
  },
  {
    "caption": "G Representative Cpt1a immunostaining with quantitative analysis in the kidneys of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups. Scale bar = 50 μm. IOD, integral optical density.",
    "molecules": "WWL"
  },
  {
    "caption": "Representative immunostaining for CD3 and F4/80 (K) of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.",
    "molecules": "WWL"
  },
  {
    "caption": "Representative CD3 and F4/80 quantitative results (L) of WT+Veh (n = 5), WT+WWL (n = 4), Ehmt2Ksp+Veh (n = 4) and Ehmt2Ksp+WWL (n = 4) groups.",
    "molecules": "WWL"
  },
  {
    "caption": "M Representative immunoblots (left) with quantitative results (right) of p-Mlkl for WT+Veh (n = 3), WT+WWL (n = 3), Ehmt2Ksp+Veh (n = 3) and Ehmt2Ksp+WWL (n = 3) groups.",
    "molecules": "WWL"
  },
  {
    "caption": "Representative Oil Red O staining with quantification (C of indicated groups.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "A Representative H&E staining (left) and pathological scores (right) of injured kidneys from mice received intervention treatment of saline (n = 4) or atorvastatin (20 mg/kg, i.g., first dose at 6-hours after ischemia, n = 5, or 14-hours after ischemia, n = 5).",
    "molecules": "atorvastatin, saline"
  },
  {
    "caption": "C, D Representative Oil Red O staining with quantification (C, scale bar = 100 μm), renal triglyceride and cholesterol levels (D) of saline (n = 4), 6 h-ATO (n = 5) and 14 h-ATO (n = 5) groups.",
    "molecules": "ATO, cholesterol, Oil Red O, saline, triglyceride"
  },
  {
    "caption": "E mRNA levels of fatty acid oxidation related genes of saline (n = 4) and 6 h-ATO (n = 5).",
    "molecules": "saline"
  },
  {
    "caption": "representative immunoblots (H, left) with quantitative results (H, right) of Ces1 and G9a of saline (n = 4) and 6 h-ATO (n = 5).",
    "molecules": "ATO, saline"
  },
  {
    "caption": "L Representative immunoblots (left) with quantitative results (right) of p-Rip3 and p-Mlkl for saline (n = 4) and 6 h-ATO (n = 5).",
    "molecules": "ATO, saline"
  },
  {
    "caption": "Growth phenotypes of the wild-type (WT, Col-0), bri1-null mutant, and two independent ubp12i/ubp13 double mutant lines grown on 1/2MS medium supplemented with 10 μM DEX (DEX medium) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution (0.1% [v/v] EtOH) for additionally 8 days. Scale bars: 10 mm.",
    "molecules": "BL, DEX, EtOH"
  },
  {
    "caption": "Phenotype of the WT and ubp12i/ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days. Scale bars: 10 mm.   Hypocotyl length relative to the mock control of seedlings (B). Experiments were done in triplicate (n >15 seedlings for each line).  ",
    "molecules": "BL, DEX"
  },
  {
    "caption": "Western blot (WB) analysis of BES1 dephosphorylation after BL treatment with the α-BES1 antibody. Total proteins were isolated from 6-day-old seedlings grown on DEX medium in the light, either in the presence of 100 nM BL or mock. PEPC was used as a loading control.   Percentage of dephosphorylated BES1 relative to the total BES1 from (D) (n = 6 biological replicates).  ",
    "molecules": "BL, DEX"
  },
  {
    "caption": "Western blot analysis of BRI1 protein amount in ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from 18-day-old plants grown on 1/2MS medium supplemented with 10 μM DEX (+) or not (-).   Western blot analysis of BRI1 protein level in concanamycin A (ConcA)-treated plants. Eighteen-day-old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation.  ",
    "molecules": "ConcA, concanamycin A, DEX"
  },
  {
    "caption": "Deubiquitinating activity of UBP13 against K48- and K63-linked ubiquitination. Diubiquitins linked through K48 or K63 (K48 Di-Ub or K63 Di-Ub) were incubated alone or with GST-UBP13 or GST-UBP13C207S for 18 h. Deubiquitination of the diubiquitins was analyzed by western blot with the α-ubiquitin (α-Ub, P4D1) antibody. The presence of GST-UBP13/UBP13C207S recombinant proteins was confirmed by the α-GST antibody.",
    "molecules": "diubiquitins, Diubiquitins, K48 Di-Ub, K63 Di-Ub, Ub, ubiquitin"
  },
  {
    "caption": "UBP13 deubiquitination of K63-ubiquitinated BRI1 in vitro. Polyubiquitinated MBP-BRI1CD was generated by incubation with GST-PUB13 (Ub reaction) and then with GST-UBP13 or GST-UBP13C207S (DUB reaction-MBP pull-down, input). After in vitro deubiquitination, MBP-BRI1CD was purified by an MBP pull-down to analyze the BRI1CD ubiquitination (DUB reaction-MBP pull-down, SE). The ubiquitinated proteins were detected by western blot with an α-ubiquitin antibody (FK2). Ubiquitin chain specificity was detected with an α-K63Ub antibody (Apu3). The presence of recombinant proteins was confirmed by the α-GST and α-MBP antibodies.",
    "molecules": "Ub, ubiquitin, Ubiquitin"
  },
  {
    "caption": "Growth phenotype comparison of BRI1-mCit/bri1/ubp12i/ubp13 and BRI125KR-mCit/bri1/ubp12i/ubp13. The indicated transgenic lines were grown on DEX medium for 18 days. Scale bars: 10 mm.",
    "molecules": "DEX"
  },
  {
    "caption": "A-D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 (A) or BRI125KR (C) tagged with mCitrine (mCit) in either bri1/UBP12/UBP13 or bri1/ubp12i/ubp13 background. Six-day-old seedlings were grown on DEX medium, and kept in the dark for 2 h in the presence of 50 μM CHX before imaging. Confocal images of epidermal cells from root meristem are shown. Scale bars: 10 μm. Measurements of the relative PM/intracellular BRI1-mCit fluorescence intensity. For each line, at least 55 and 25 cells from over 25 and 15 seedlings were measured in (B) and (D), respectively.",
    "molecules": "CHX, DEX"
  },
  {
    "caption": "E. Western blot  analysis of an immunoprecipitation  by anti-mCherry  antibody (PDL1) from indicated DG-75 cell lysates, with and without the BS3  crosslinker, and probed for mCherry  (PD-L1) or GFP  (CD80).",
    "molecules": "BS3  crosslinker"
  },
  {
    "caption": "A-B. APC-labelled PD-1 Ig (0.75µg/ml) was used to detect PD-L1 in DG-75 cells co-expressing CD80GFP  (A) or CD86GFP (B). Staining  was carried out following transendocytosis  using Jurkat  cells expressing no CTLA-4, CTLA-4 WT or CTLA-4  Del36 (at a ratio of 1:1) for the indicated durations. (A) shows representative FACS  plots of CD80GFP  vs.  PD-1 Ig at the time points indicated, with full kinetic  data plotted below (mean ± SEM, 3 independent experiments, ***P≤0.001, ****P≤0.0001: two-way ANOVA with Tukey's multiple comparisons test). (B) As for (A) except using CD86-PD-L1  expressing cells.",
    "molecules": "APC"
  },
  {
    "caption": "A. Staining  of free PD-L1  with APC-conjugated PD-1 Ig in DG-75 cells expressing only PD-L1mCherry, using cell lines with increasing ratios of CD80GFP:  PD-L1mCherry compared to CD80GFP only. FACS  plots are representative of 4 independent experiments.",
    "molecules": "APC"
  },
  {
    "caption": "A. DG-75 B cells expressing CD80GFP  or  CD86GFP (green) and PD-L1mCherry (red) were incubated with Jurkat  cells (nuclei  shown in grey) expressing CTLA-4  WT or mutant  CTLA-4 Del36 for the indicated durations and analysed by  confocal microscopy. 2 hours prior to assay endpoint, APC-labelled  PD-1  Ig (0.75µg/ml) was added. CD80/CD86  and PD-L1 co-localisation  at the immune synapse is shown in yellow with PD-1 Ig binding  shown in purple. Scale bar, 10µm.",
    "molecules": "APC"
  },
  {
    "caption": "B.  Flow cytometry plots of CD80GFP levels on DG-75 B-cells (gated on CTFR+ cells) at 24h with graphical representation of the MFI in each condition (RH graph). The Jreg  cells used, and the anti-PD-1 status is indicated above the plot. C. Flow cytometry  plots of CD28  levels indicating contact between the CD80+ APC  and the PD-1+ CD28+ responder Jurkat T-cells, following CD80 transendocytosis. Graphical representation of the CD28  MFI in each condition is shown on the right. D.  Flow cytometry of CD69  levels on responder Jurkat T-cells and graphical representation of CD69  MFI in each condition. E. Flow cytometry  of CD69  levels on  CD28KO (PD-1+) responder Jurkat T-cells and graphical representation of the CD69+ cells MFI in each condition. Cartoons on the left of B-E illustrate the cell type analysed. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test.",
    "molecules": "APC"
  },
  {
    "caption": "B. Titration  of Abatacept staining  (left hand panel) and EC50  values of Abatacept  (right hand panel) for CD80  only or PD-L1/CD80  co-expressing  DG-75. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: paired t-test (B)",
    "molecules": "Abatacept"
  },
  {
    "caption": "D. Comparison of PD-L1  detection using  PD-1-Ig or anti-PD-L1  antibodies (MIH3 or 29E.2A3 clone) with and without prior incubation with 10µg/ml of abatacept, based on data from (C). E. Graphs showing PD-L1 detection under conditions used in (C) following different Abatacept  incubation periods (30mins, 3h or 6h). Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: RM one-way ANOVA (D).",
    "molecules": "abatacept, Abatacept"
  },
  {
    "caption": "A. Total lipids were extracted from PfCDPK7-KO or 3D7 parasites and separated by HPTLC. After quantification by GC-MS content relative to total fatty acids was determined. Relative abundance of individual  phospholipids  was quantified. Disruption of  PfCDPK7  caused a significant decrease in  PC (mean± SEM, n=3; biological replicates, ***p<0.001, t-test).",
    "molecules": "fatty acids, lipids, PC, phospholipids"
  },
  {
    "caption": "E. 3D7 or PfCDPK7-KO parasites were cultured in the presence or absence of 200 μM choline in culture medium. After 3 days,  parasitemia was determined by counting parasites on thin blood  smears (mean± SEM, n=6; biological replicates, *p< 0.05, ***p<0.001, ANOVA).",
    "molecules": "choline"
  },
  {
    "caption": "A. 3D7  parasites were treated with DMSO or 50nM MMV048 for 24h. Subsequently, parasites were fixed and IFA  was performed to detect ER marker BiP  and  PfCDPK7. While PfCDPK7  was present in puncta proximal to ER  (left panel), inhibitor treatment caused a loss in this  vesicular localization and resulted in a more diffuse distribution of the enzyme (right panel) (Scale bar-1 μm).",
    "molecules": "DMSO, MMV048"
  },
  {
    "caption": "C. 3D7  parasites were treated with  DMSO or 50nM MMV048 for 20-24h. Subsequently, IFA  was performed ~24h post-invasion to detect RAP1 and MSP1 (left panel) Right panel, Quantitation of parasites exhibiting abnormal  staining for  RAP1 outside the  PV was observed (mean± SEM, n=3; biological replicates, **p<0.01, t-test; Scale bar-1 μm).",
    "molecules": "DMSO, MMV048"
  },
  {
    "caption": "A-B.  3D7  parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10-12h. Subsequently, parasites were incubated with 14C-Cho (A) or 14C-Eth (B) and were harvested after 12h. Total lipids were extracted and separated by TLC. The  radiolabeled lipids were detected by phosphorimaging  and quantitated by densitometry  (upper panel). Bottom Panel (A and B), fold change in PC and PE formation in inhibitor treated parasites with respect to  DMSO treatment was determined (mean±SEM, n=4; biological replicates, **p<0.01, ***p<0.001, ANOVA; ns, not significant).",
    "molecules": "14C, Cho, DMSO, Eth, lipids, MMV048, PC, PE, UCT943"
  },
  {
    "caption": "C-D. UCT943  and MMV048 treated 3D7 (C) or EK-GFP overexpressing parasites (D) were subjected to Western blotting using anti-PMT (C) and anti-GFP (D) antibodies (left panels). Right Panels:  densitometry  was performed for bands corresponding to  PMT (C) or EK-GFP (D), which were normalized with respect to Actin. Fold change in PMT or  EK was determined with respect to DMSO treated parasites (Mean± SEM, n=4; biological replicates, *p<0.05, ***p<0.001, ANOVA).",
    "molecules": "DMSO, MMV048, UCT943"
  },
  {
    "caption": "B. Ring-infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4-64 and DAPI  to assess lipid uptake. Subsequently, FM4-64  associated fluorescence was detected by fluorescence microscopy (upper panel) or quantitated by  FACS (lower panel) and % change in  FM4-64 uptake in inhibitor-treated parasites was determined. (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).",
    "molecules": "DAPI, FM4-64, lipid, MMV048"
  },
  {
    "caption": "D. Trophozoite stage infected erythrocytes were incubated with FM4-64 and DAPI  to assess lipid uptake by 3D7  or PfCDPK7-KO parasites. Subsequently, FM4-64  associated fluorescence was visualized by live microscopy (top panel) and also analyzed by FACS  and % change in FM4-64 uptake in KO parasites was determined (bottom panel) (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).",
    "molecules": "DAPI, FM4-64, lipid"
  },
  {
    "caption": "d, GFP-LC3 loss in cultured Gfp-Lc3 HSCs (n = 3). Results are expressed as percentage of GFP-LC3 MFI in -BafA compared to +BafA conditions.",
    "molecules": "BafA"
  },
  {
    "caption": "Autophagy flux in cultured FoxO3a−/−::Gfp-Lc3 HSCs (n = 3). Results are expressed as percent GFP-LC3 MFI in -BafA vs. +BafA conditions, and normalized to FoxO3a+/+−::Gfp-Lc3 HSCs. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.",
    "molecules": "BafA"
  },
  {
    "caption": "2-NBD glucose uptake in cultured young and old HSCs (n = 3).",
    "molecules": "2-NBD glucose"
  },
  {
    "caption": "Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate (M-Pyr) (n = 3). Colonies were counted at day 10 and normalized to -BafA conditions. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.",
    "molecules": "BafA, methylpyruvate"
  },
  {
    "caption": "B Expression (cells) and secretion  (media) of VgrG1 from  V. parahaemolyticus RIMD 2210633 derivative POR1  (Vpara; T6SS1+) and its T6SS1- mutant (Vpara/∆hcp1), and from  V. natriegens  (Vnat) carrying the indicated plasmids. Samples were treated (+) or not (-) with 20 µM phenamil  to activate surface sensing in media containing 3% NaCl at 30 °C for 5 h. Loading control (LC) is shown for total protein lysates. Arrows denote bands corresponding to VgrG1. Asterisks denote non-specific bands detected in  Vnat samples.",
    "molecules": "phenamil, NaCl"
  },
  {
    "caption": "C  Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with the indicated attackers, as described in B, on media containing 3% NaCl  at 30 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit.",
    "molecules": "NaCl"
  },
  {
    "caption": "B Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with wild type V. natriegens  attackers harboring the indicated plasmids, on media containing 3%  NaCl at 30 °C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h  timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.",
    "molecules": "NaCl"
  },
  {
    "caption": "C  Expression (cells) and secretion  (media) of  VgrG1  from V. natriegens containing an arabinose-inducible vp1407 in the  chromosomal dns  locus (VnatReg) and harboring an empty plasmid (pEmpty) or plasmids containing VpT6SS1 that lacks vp1407 (pT6SS1Ind) or both vp1407  and hcp1 (pT6SS1Ind/∆hcp1;  T6SS-). Samples were grown in media containing 3% NaCl and either supplemented (+) or not (-) with 0.1%  arabinose  (Ara) at 30 °C. Loading control (LC) is shown for total protein lysates.",
    "molecules": "Ara, arabinose, NaCl"
  },
  {
    "caption": "A, B  Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3%  NaCl  and 0.1%  arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.",
    "molecules": "arabinose, NaCl"
  },
  {
    "caption": "C-F Viability  counts of the indicated prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose  at 28 °C. Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h  timepoints  by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05. ",
    "molecules": "arabinose, NaCl"
  },
  {
    "caption": "Viability counts of  V. natriegens  (cyan), V. vulnificus (purple), A. jandaei ∆tssB (brown),  E. coli  (green), and S. enterica (gray) prey before (0 h) and after (4 h) co-incubation with VnatReg attackers (yellow) harboring the indicated plasmids, on media containing 3% NaCl  at 28 °C. The left panels show prey survival when competing alone (single prey) against the attacker at a 4:1 (attacker:prey) ratio; the right panels show the results of a competition experiment in which all prey were mixed together with the attacker at a 10:1:1:1:1:1 (attacker:prey) ratio. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h  timepoint  by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.",
    "molecules": "NaCl"
  },
  {
    "caption": "A-D (A, B, D) Viability counts of  V. parahaemolyticus RIMD 2210633 ∆hcp1 (A),  V. alginolyticus 12G01 ∆hcp1/∆hcp2 (B), and V. natriegens  (D) prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h  timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. ( C) Illustration summarizing the competitions shown in (A, B). Effector and immunity modules expressed by VnatReg carrying pT6SSEffectorless (yellow) are colored to match the bacterium from which they were derived.",
    "molecules": "arabinose, NaCl"
  },
  {
    "caption": "Semi-thin cross sections of   femoral quadriceps nerves stained with  alkaline methylene blue. Note the smaller size of Ccp1-/- and Ccp1-/-Ttll7-/- nerves (A).",
    "molecules": "alkaline methylene blue"
  },
  {
    "caption": "Semi-thin cross sections of  femoral quadriceps nerves stained with  alkaline methylene blue. These sections were quantified for (B) number of myelinated axons, (C) nerve  area, (D) nerve perimeter (Fig. EV2) of  femoral quadriceps nerves from wild-type, Ccp1-/-, Ttll7-/-, Ccp1-/-Ttll7-/-, Ttll1-/- and Ccp1-/-Ttll1-/- mice. Scatter dot plots in which each point represents one nerve of an individual mouse are shown. The black lines indicate means ±SEM. Significance was tested with one-way ANOVA and Holm-Šídák's multiple comparisons test. Note that all three parameters are decreased for Ccp1-/- and Ccp1-/-Ttll7‑/- mice, while Ccp1-/-Ttll1-/- mice showed no difference to the Ttll1-/- control.",
    "molecules": "alkaline methylene blue"
  },
  {
    "caption": "A) Representative  images of DIV4 primary  hippocampal neurons from wild type, Ttll1-/- and  Ttll7-/- mice Cells were incubated with MitoTracker® (black dots) and mitochondria movements were recorded in the longest neurite  (orange lines).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "A Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.",
    "molecules": "tail DNA"
  },
  {
    "caption": "I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.",
    "molecules": "EdU, DNA, propidium iodide"
  },
  {
    "caption": "Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E. ",
    "molecules": "7-AAD, 7AAD"
  },
  {
    "caption": "G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments. ",
    "molecules": "LPS"
  },
  {
    "caption": "I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.",
    "molecules": "TNP"
  },
  {
    "caption": "A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar, 500 µm. 4× magnification. D Forward scatter (FSC) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison.",
    "molecules": "LPS"
  },
  {
    "caption": "Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKO",
    "molecules": "EdU, DNA, propidium iodide"
  },
  {
    "caption": "Representation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated for 48 h. Scale bar, 10 µm.",
    "molecules": "IdU, CldU, nucleotide"
  },
  {
    "caption": "I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.",
    "molecules": "LPS"
  },
  {
    "caption": "C qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.",
    "molecules": "RNA"
  },
  {
    "caption": "Immunoblots of acyl-biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation-deficient mutant form of the protein with cysteines replaced with alanine residues. The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull-down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein.",
    "molecules": "alanine, cysteines, HA, hydroxylamine"
  },
  {
    "caption": "A significant reduction in the Golga7b signal is observed in the + HA condition of the pull-down when DHHC5 is depleted with siRNA. The graph displays mean +/- s.e.m. N=4, *=p<0.05, paired t-test.",
    "molecules": "HA"
  },
  {
    "caption": "Immunoblots of WT and non-palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants (DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine, C-terminal mutant = mutant where the 3 C-terminal palmitoylation sites of DHHC5 have been mutated to alanine residues.). WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C-terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C-terminus of DHHC5 is essential for the stability of this interaction.",
    "molecules": "alanine, cysteine, serine"
  },
  {
    "caption": "30 minute DMSO treatment of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged). 30 minute Dynasore treatment (25µM) of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged).",
    "molecules": "DMSO, Dynasore"
  },
  {
    "caption": "Quantification of plasma membrane signal of DHHC5 in (A and B). ****=p<0.001, unpaired t-test. N=37, +Dynasore. N=28, +DMSO.",
    "molecules": "DMSO, Dynasore"
  },
  {
    "caption": "Biotin endocytosis assays of HeLa cells with endogenous DHHC5, over-expression of DHHC5 alone, co-expression of DHHC5 and WT Golga7b, co-expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C-terminal mutant. Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5. The lack of signal in the pull-down of the DHHC5 C-terminal mutant or when DHHC5 and WT Golga7b are co-expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions. The robust signal in the pull-down of endocytosed proteins from cells co-expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition. N=3, *=p<0.05, paired t test\"",
    "molecules": "Biotinylated, streptavidin"
  },
  {
    "caption": "ABE purifications from HeLa cells treated with 10nM non-targeting siRNA (nt siRNA) or DHHC5 siRNA were immunoblotted for Dsg2. Loss of the band in the +HA pull-down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5.",
    "molecules": "HA"
  },
  {
    "caption": "Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments.",
    "molecules": "calcium"
  },
  {
    "caption": "Confocal images of DHHC5, Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. See Appendix Figure S6 for quantification of data in panels B-D.",
    "molecules": "calcium"
  },
  {
    "caption": "Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium (5µM calcium) conditions, with quantification of the intensity of the DSG2 signal in the pulldown normalised to input. There is a significant reduction in the Dsg2 signal in the +HA pull-down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition. *=p<0.05, paired t-test, N=4, error bars represent s.e.m. All scale bars 10μm.",
    "molecules": "calcium, HA"
  },
  {
    "caption": "E. Phospholipids identified in autophagic vesicles isolated upon different conditions. Relative amounts were calculated based on total levels of detected phospholipids. Abbreviations are depicted in A.",
    "molecules": "Phospholipids, phospholipids"
  },
  {
    "caption": "A. Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd). B. Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd).",
    "molecules": "MG132"
  },
  {
    "caption": "(b) When the H3R antagonist thioperamide (THIO, 10−6 mol l−1 at reperfusion) and the agonist immepip (IMME, 10−6 mol l−1 at reperfusion) were administered, viability was tested in cultured neurons by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after OGD/R (n=7 per condition; ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).",
    "molecules": "IMME, immepip, THIO, thioperamide"
  },
  {
    "caption": "(c) 2,3,5-triphenyltetrazolium chloride-stained brain sections showing the infarct area in WT and H3R−/− mice receiving saline, the H3R antagonists A331440, clobenpropit (CLOB) and thioperamide, (THIO; 5 mg kg−1, i.p., immediately at reperfusion and 6 h later), and the H3R agonist immepip (IMME, 1 μg, i.c.v., at reperfusion) after I/R (1 h MCAO followed by 24 h reperfusion; scale bar, 5 mm).",
    "molecules": "A331440, CLOB, clobenpropit, IMME, immepip, saline, THIO, thioperamide"
  },
  {
    "caption": "The bar graphs show infarct volume (d) and neurological score (e) (n=6-8 mice per condition, *P0.05, **P0.01, ***P0.001 versus WT saline group with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.",
    "molecules": "saline"
  },
  {
    "caption": "(a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).",
    "molecules": "thioperamide"
  },
  {
    "caption": "(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6-7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test)",
    "molecules": "thioperamide"
  },
  {
    "caption": "(c) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).",
    "molecules": "thioperamide"
  },
  {
    "caption": "(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.",
    "molecules": "thioperamide"
  },
  {
    "caption": "(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm).",
    "molecules": "α-FMH, CIME, cimetidine, PYRI, pyrilamine, saline, THIO, thioperamide"
  },
  {
    "caption": "(a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.",
    "molecules": "LiCl, thioperamide"
  },
  {
    "caption": "(c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm).",
    "molecules": "LiCl, thioperamide"
  },
  {
    "caption": "(d) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).",
    "molecules": "LiCl, thioperamide"
  },
  {
    "caption": "(e) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.",
    "molecules": "IMME, immepip, THIO, thioperamide"
  },
  {
    "caption": "(b,c) When thioperamide was administered in vivo, LC3 expression was assessed by western blot after I/R in WT mice (b) (*P0.05, ***P0.001; n=6 mice per condition, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test), and in H3R−/− mice (c) (***P0.001 versus H3R−/− sham group; n=5-6 mice per condition, with ANOVAs followed by the Bonferroni/Dunn post hoc test.) Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.",
    "molecules": "thioperamide"
  },
  {
    "caption": "(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13.",
    "molecules": "3-MA"
  },
  {
    "caption": "(b) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6-8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).",
    "molecules": "3-MA"
  },
  {
    "caption": "(d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4-6 mice per condition).",
    "molecules": "thioperamide"
  },
  {
    "caption": "(e) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).",
    "molecules": "3-MA, thioperamide"
  },
  {
    "caption": "(g) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.",
    "molecules": "thioperamide"
  },
  {
    "caption": "(a) Representative co-immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD/R. Full-size blots are shown in Supplementary Fig. 14.",
    "molecules": "thioperamide"
  },
  {
    "caption": "A, Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA, or 5 nM Actinomycin D (ActD). GAPDH served as a loading control.",
    "molecules": "ActD, Actinomycin D, MPA"
  },
  {
    "caption": "B, HCT116 cells were treated for 24 hrs with vehicle alone (-), 1 µM MPA, 10 µM MPA, or the combination of 10 µM MPA with 400 µM guanosine. Guanylate nucleotides levels were measured by LC-MS, and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate. Data information data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.",
    "molecules": "guanosine, MPA"
  },
  {
    "caption": "Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.",
    "molecules": "dox, doxycycline, tetracycline"
  },
  {
    "caption": "Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). Whole cell extracts were analyzed on Western blots with the indicated antibodies. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 is shown. Mean ± SD is representative of four independent experiments. (right panel) (D). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.",
    "molecules": "dox, doxycycline, tetracycline"
  },
  {
    "caption": "E, Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA, in presence of dimethyl sulfoxide (DMSO) or 400 µM guanosine. GAPDH was used as a loading control.",
    "molecules": "dimethyl sulfoxide, DMSO, guanosine, MPA"
  },
  {
    "caption": "A, HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone (-) or the indicated concentration of MPA for 24 hrs. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 of four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.",
    "molecules": "MPA"
  },
  {
    "caption": "B, HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.",
    "molecules": "MPA"
  },
  {
    "caption": "C, HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence (-) or the presence (+) of the combination of 10 µM of the ATR inhibitor VE-821 (ATRi) and 10 µM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone (-) or MPA 10 µM for additional 24 hrs. The levels of p53 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least two independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.",
    "molecules": "KU-55933, MPA, VE-821"
  },
  {
    "caption": "A, HCT116 cells were treated with either vehicle alone (-), 1 µM MPA, 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA. Data information: , data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.",
    "molecules": "ActD, MPA"
  },
  {
    "caption": "B, Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section.",
    "molecules": "Ethidium Bromide"
  },
  {
    "caption": "C, Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysates",
    "molecules": "tRNAs, urea"
  },
  {
    "caption": "F, HCT116 cells were pretreated with vehicle alone (-), 1 µM, or 10 µM MPA for 24 hrs, and the levels of p53 and p21 were followed on Western blots in presence of 100 µg/mL cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment.",
    "molecules": "CHX, cycloheximide, MPA"
  },
  {
    "caption": "G, HCT116 cells were pretreated as in (F) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel. Data information: In panels data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.",
    "molecules": "MG132"
  },
  {
    "caption": "B, Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 µM MPA (dark) or 10 µM MPA (clear) over vehicle treated cells.",
    "molecules": "MPA"
  },
  {
    "caption": "C, IdU track length distribution, and distribution of long fork / short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see methods). ***p<0.001, ****p<0.0001 by Kruskal-Wallis test of the means. D, Representative images from (C). ",
    "molecules": "IdU"
  },
  {
    "caption": "E, HCT116 cells, pre-incubated with 10 µM BrdU for 48 hrs were treated with Vehicle (-), 10µM MPA for 6 or 24 hrs, or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation (see Method). Cells were immunostained for native BrDU (green), or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 μm.",
    "molecules": "BrdU, BrDU, DAPI, DNA, Hydroxyurea, MPA"
  },
  {
    "caption": "F, HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins. α-tubulin was used as a loading control.",
    "molecules": "MPA"
  },
  {
    "caption": "A, HCT116 cells were treated with either the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs, before p21 immuno-labeling, propidium iodide staining and FACS analysis. Cell cycle profiles of the total cell population (upper panel, left), the total intensity of p21 and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels.",
    "molecules": "MPA, propidium iodide"
  },
  {
    "caption": "B, HCT116 p21-/- isogenic cell lines were transfected for 24 hrs with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), then cells were serum-deprived for 16 hrs before the re-addition of serum for additional 24 hrs in absence or presence of 10 µM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining and FACS analysis. The results are representative of two independent experiments.",
    "molecules": "MPA"
  },
  {
    "caption": "A, HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in (Fig 5B). Here are shown the cell cycle profiles after 16 hrs of serum deprivation (top panels), or after an additional 24 hrs of serum re-addition in absence (middle panels), or presence of 10 µM MPA (bottom panels). The results are representative of at least three independent experiments.",
    "molecules": "MPA"
  },
  {
    "caption": "B, HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs. The levels of γ-H2AX were analyzed on Western blots. GAPDH was used as a loading control.",
    "molecules": "MPA"
  },
  {
    "caption": "E, Representative images cells labeled with γ-H2AX (green), 53BP1 (red) and DAPI (blue). Scale bars correspond to 10 μm",
    "molecules": "DAPI"
  },
  {
    "caption": "F, HCT116 cells treated were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****p<0.0001 by Kruskal-Wallis test",
    "molecules": "tail DNA, DNA"
  },
  {
    "caption": "A stable inducible HEK-293T cell line expressing myc-PP2AC  (panel A were treated with DMSO, 20 μM  DT-061 or 2 μM iHAP1  for 2 hours and myc-PP2AC affinity purified, respectively. The binding  of the indicated proteins was analysed by  Western blotting A) representative of 3 independent experiments",
    "molecules": "DMSO, DT-061, iHAP1"
  },
  {
    "caption": "A stable inducible  HEK-293T cell line expressing myc-PP2AC  (panel B) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1  for 2 hours and myc-PP2AC  by quantitative mass spectrometry (B). B) analysis of a biological triplicate.",
    "molecules": "DMSO, DT-061, iHAP1"
  },
  {
    "caption": "a stable inducible HeLa  cell line expressing myc- PP2R1A  (C) were treated with DMSO, 20 μM  DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2R1A affinity purified, respectively. The binding  of the indicated proteins was analysed by Western blotting C) representative of 2 technical repeats",
    "molecules": "DMSO, DT-061, iHAP1"
  },
  {
    "caption": "D) HEK-293T cells were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1  for 2 hours and protein lysates were incubated with a microcystin  affinity column to capture PPP complexes. Bound complexes were analysed by mass spectrometry to identify PP2A components. Analysis conducted on a biological triplicate.",
    "molecules": "DMSO, DT-061, microcystin, iHAP1"
  },
  {
    "caption": "E) Doxycyclin-inducible CRISPR-Cas9 HeLa  cell lines allow for depletion of specific B56 subunits. Growth assays in the presence of the indicated concentrations of  DT-061  or iHAP1  measured after 12 days of treatment. Mean and SD of three independent experiments are shown.",
    "molecules": "Doxycyclin, DT-061, iHAP1"
  },
  {
    "caption": "B) Validation of CRISPR  screen results using RNAi depletion followed by SRB growth assay. Cells were treated with DMSO  (Ctr) or DT-061  5.5 µM or iHAP1  0.5 µM. C) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO  or 5.5 μM  DT-061  or 0.5 μM iHAP1  treatment. Data information: B-C) Mean and SD of three independent experiments is shown.",
    "molecules": "DMSO, DT-061, iHAP1"
  },
  {
    "caption": "A) In vitro tubulin polymerization  assay showing concentration dependent effect of iHAP1  on polymerization  dynamics. Taxol  and Nocodazole served as controls for the experimental setup. The assay was performed in the presence of the indicated drug concentrations. Mean and SEM are shown from three to six independent experiments.",
    "molecules": "Nocodazole, Taxol, iHAP1"
  },
  {
    "caption": "D) Representative temporal projection of mitotic spindles  of U2OS EB1-GFP cells following specified treatment. DNA counterstained  with SiR-DNA  shown in cyan in merged images. Scale bar 10µm.",
    "molecules": "SiR"
  },
  {
    "caption": "E) Analysis of number of EB1-GFP  comets per  mitotic spindle. The mean and SD are plotted from three independent experiments (Total number of cells = 26 (DMSO), 20 (30µM DT-061), 27 (DMSO+APC/C in.) and, 26 (2µM iHAP1+APC/C  in). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.",
    "molecules": "DMSO, DT-061, iHAP1"
  },
  {
    "caption": "A) Measurement of time spent in mitosis  by  time-lapse imaging  taking into consideration whether cells exited mitosis  or underwent cell death during mitosis. n indicates the number of cells analyzed from at least 2 independent experiments. Schematic of experimental protocol on the top. Cells were treated with iHAP1  when indicated at the concentration stated in the figure. B) Representative stills from  time-lapse imaging  experiments depicting the two phenotypes. Scale bar represents 20 µm.",
    "molecules": "iHAP1"
  },
  {
    "caption": "A)  Time-lapse imaging  of HeLa  cells expressing a marker staining  the  Golgi apparatus  (green) (n= 87 of cells from 2 experiments analysed all showing dispersal of marker). At time  0, DT-061 was added. On the right-hand side, the co-localisation  of  DT-061  (red) and the  Golgi  marker is shown. B) As in A) but with a marker for ER and either with the addition of DT-061  alone or DT-061 + okadaic acid (50 nM) Data information: Experiments were repeated at least twice and stills show a representative cell for each treatment condition. Cells were treated with DT-061  at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.",
    "molecules": "DT-061, okadaic acid"
  },
  {
    "caption": "A-B) Time-lapse imaging  of H358  cells expressing a marker for the Golgi  (A) or the ER  (B). Cells were treated with DT-061 at time 0. (B, low panel). For A) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi  marker. Arrow indicates addition of compound. Data information: Experiments were repeated at least twice except filming of the Golgi  marker which was done once. Stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.",
    "molecules": "DT-061"
  },
  {
    "caption": "B) Increase in the molar quantity of sphingolipid  classes assessed in MCF7  cells after feeding with the precursor sphingosine. MCF7  cells were co-treated with 15 μM DT-061, 1.5 μM iHAP1, 3 µg/mL Brefeldin A, or vehicle (DMSO). The determined molar quantities of sphingolipid classes were normalized to that of PC. The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t-test. *p<0.05, **p<0.01, ***p<0.001. Cer: ceramide, HexCer: hexosylceramide, PC: phosphatidylcholine, and SM: sphingomyelin.",
    "molecules": "Brefeldin A, Cer, ceramide, DMSO, DT-061, HexCer, hexosylceramide, PC, phosphatidylcholine, sphingolipid, SM, sphingomyelin, sphingosine, iHAP1"
  },
  {
    "caption": "Key marker genes for the four mesenchymal clusters (RBP1, COL1A1, RGS5, and MYH11). Light gray dots denote cells originating from non-tumor samples. Dark gray dots denote cells originating from the tumor samples. Left - Representative smFISH image of patient p1 stained for RGS5 and COL1A1 showing distinct spatial localization of CAFs and Pericytes. Scale bar 10µm. Dashed lines mark the shortest distance of cells (2a) and (2b) from the cell (1). Middle - zoom-in of (1) from left panel, showing a blood vessel like structure formed by endothelial cells marked by PDGFB (magenta) wrapped by pericytes marked by RGS5 (green). Dashed lines are two consecutive cell layers of endothelial cells and pericytes. Scale bar 2.5µm. Right - zoom-in of (2a and b) from the left panel, showing two distant CAFs expressing high COL1A1 signal but not RGS5. DAPI used for nuclei staining. Scale bar 2.5µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-G Hematoxylin and eosin (H&E) staining of the frontal sections of miniature pig mandible slices showing morphological changes during the permanent canine (PC) initiation stage from embryonic day 50 (E50) to postnatal day 10 (PN10); (A'-F') are magnifications of boxed regions in their corresponding figure panel. DC, deciduous canine; PC, permanent canine; SDL, successional dental lamina; n = 3. Scale bars = 100 µm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126). X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W). Data information: Data represent the means ± SEM. n = 3. Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for X, *P < .05, **P < .01, ***P < .001; NS, not significant.",
    "molecules": "U0126"
  },
  {
    "caption": "J Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs treated with IWR-1-endo. Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR-1-endo treatment.",
    "molecules": "IWR-1-endo"
  },
  {
    "caption": "C) Discharging assay of Ub-charged UBE2D1 was carried out in the presence of CHN-1 or CHN-1-UFD-2P951A. The experimental sample was run together with the control with and without a reducing agent (50 mM DTT). The reaction was stopped after 30 min by boiling the samples in 1x SDS sample buffer. Proteins were resolved via SDS-PAGE and immunoblotted with anti-UBE2D1 antibodies. Below, quantification of uncharged UBE2D1 plotted as the uncharged UBE2D1 fraction vs. UBE2D1-Ub (µM). The graph representing the UBE2D1 fraction vs. UBE2D1-Ub (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan) and Plotted data are the mean of three technical replicates. Significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [UBE2D1 vs UBE2D1-Ub(µM) in the presene of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1-Ub(µM)].",
    "molecules": "DTT"
  },
  {
    "caption": "B) CHN-1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor (MG-132, 10µM) and DUB inhibitor (NEM, 100mM). Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels plotted as control (black), or MG-132 treated (magenta) in N2 (wild-type) and ufd-2(tm1380). Plotted data are the mean of three biological replicates.",
    "molecules": "MG-132, NEM"
  },
  {
    "caption": "C) Protein level of endogenous AHCY-1 in N2 (wild-type), chn-1(by155), CHN-1 OE, and ufd-2(tm1380) young adult worms treated with the proteasome inhibitor (MG-132, 10 µM) and DUB inhibitor (NEM, 100 mM). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Tubulin served as a loading control. Right, quantification of the modified AHCY-1 signals plotted as Ub-modified AHCY-1 species normalized to unmodified endogenous AHCY-1 signal and plotted for N2 (wild-type; black), chn-1(by155) (magenta), CHN-1 OE (yellow), and ufd-2(tm1380) (cyan). Plotted data are the mean of three biological replicates.",
    "molecules": "MG-132, NEM"
  },
  {
    "caption": "C G-MDSCs purified from spleen of tumor-bearing WT or Tet2-/- mice were co-cultured with CD8+ T cells at the indicated cellular ratios for T cell activation assays, quantified by CFSE-labeling and dye dilution for 3 days in vitro. Representative histograms show CFSE signals in the in vitro T cell activation assays. D Quantification of CD8+ T cell proliferation in panel C. Each point represents a co-culture experiment (n=3). ns, not significant. Graphs show mean ± SEM and statistical analysis by Student's t-test. ",
    "molecules": "CFSE"
  },
  {
    "caption": "A. IFT22 nucleotide-binding experiments. Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22/74342-401/81397-450 core complex incubated with mant-labeled GDP (mant-GDP) or non-hydrolysable GTP / ATP analogs (mant-GMPPNP / mant-AMPPNP). The fluorescence intensity is plotted as a function of protein concentration. Data were fitted to a single-site binding equation for determination of the dissociation constant (Kd). Kd values and standard deviations are calculated from three independent experiments.",
    "molecules": "AMPPNP, ATP, GDP, GTP, GMPPNP, mant"
  },
  {
    "caption": "D. Top: Cartoon representation of IFT22 (grey) with positions of two nucleotide-binding mutants highlighted. GTP is shown in stick representation and Mg2+ as a ball. D175 (blue) is the unusual residue binding the guanine base while S19 (pink) is a conserved residue required for coordination of the Mg2+ cation and is commonly mutated to an asparagine to prevent nucleotide binding. Bottom: Nucleotide-binding experiments of IFT22 nucleotide-binding mutants D175E, D175A and S19N with fluorescently labeled nucleotides. Only the S19N mutation (light pink) abolished IFT22 nucleotide-binding ability completely.",
    "molecules": "asparagine, GTP, guanine, Mg2+"
  },
  {
    "caption": "C. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged TbIFT22 (WT and mutants) and untagged CrIFT25/27/74/81. WT TbIFT22 is able to pull down the Cr tetrameric complex, thus forming a chimeric IFT-B1 pentamer, while both the A86R and S19N mutant fail to bind the complex.",
    "molecules": "Ni2+-NTA"
  },
  {
    "caption": "A. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged IFT74342-401/81397-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT74342-401/81397-450 complex in the pull-down experiment.",
    "molecules": "Ni2+-NTA"
  },
  {
    "caption": "B. SDS-PAGE gel of a Ni2+-NTA pulldown using the His-tagged IFT7479-401/811-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT7479-401/811-450 complex in the pull-down experiment.",
    "molecules": "Ni2+-NTA"
  },
  {
    "caption": "C. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and S19N mutant). The nucleotide-binding deficient mutant IFT22S19N interacts with IFT25/27/74/81 in the pull-down experiment.",
    "molecules": "Ni2+-NTA"
  },
  {
    "caption": "D. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and A86R mutant). The IFT74/81-binding mutant IFT22A86R fails to pull down the full-length tetrameric complex.",
    "molecules": "Ni2+-NTA"
  },
  {
    "caption": "B. IFA of the indicated trypanosome cell lines using the mAb25 (marker for the axoneme, left panels) and an anti-IFT172 antibody (marker for IFT, right panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. Scale bars correspond to 5 µm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "B. IFA with the indicated cell lines using the mAb25 (marker for the axoneme, central panels) and an anti-IFT172 antibody (marker for IFT, bottom panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells. Scale bars correspond to 5 µm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Representative images of unseparated (< 1 μm apart), initial separation (1-5 μm apart), intermediate separation (5-11 μm apart), and pole separation (>11 μm apart) of centrosomes. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT (purple), CETN3 (green), and SYCP3 (red), and stained with DAPI (blue). Zoomed images of centrosomes below each panel. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "I. Representative images of spermatocytes containing monopolar, bipolar, and multipolar spindles immunolabeled against alpha-tubulin (red), centromere (green), and stained with DAPI. J. Spindle pole number per primary spermatocyte was quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO. n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Spermatocytes were immunolabeled against CETN3 (green), CEP164 (red), SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Spermatocytes were immunolabeled against gamma-tubulin (green), alpha-tubulin (red), and stained with DAPI (blue). Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blots of gamma-tubulin from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (E). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.",
    "molecules": "2,2,2-Trichloroethanol, TCE"
  },
  {
    "caption": "Western blots of gamma-tubulin from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (F). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.",
    "molecules": "2,2,2-Trichloroethanol, TCE"
  },
  {
    "caption": "A. Spermatocytes were immunolabeled against TACC3 (green), SYCP3 (red), CETN3 (magenta), and stained with DAPI (blue). Zoom images of the centrosomes are presented to the right. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (D). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.",
    "molecules": "2,2,2-Trichloroethanol, TCE"
  },
  {
    "caption": "Western blots of TACC3 from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (C). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.",
    "molecules": "2,2,2-Trichloroethanol, TCE"
  },
  {
    "caption": "E, F. Spermatocytes were immunolabeled against beta-catenin (green), SYCP3 (red), PCNT (magenta), and stained with DAPI (blue). Middle columns emphasize beta-catenin signal in diplonema and diakinesis spermatocytes. Scale bars = 5 μm. G. Quantification of beta-catenin positive centrosomes per spermatocyte at diakinesis. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 89, 106, 122, and 93, respectively. ",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C BreastMark microarray platform; correlation of ESRP1 expression with overall survival in tamoxifen-treated patients. A log rank test was used to calculate P=1.218, e-06 (n= 210, number of events= 49).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "C Relative cell density was determined by crystal violet assay. Cells were treated with TAM (10-6 M) or ICI (10-9 M) for 6 days in the presence and absence of E2 (β-estradiol; 10-10 M). Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates. ",
    "molecules": "E2, β-estradiol, ICI, TAM"
  },
  {
    "caption": "D Cell viability was determined using Cell Biolabs CytoSelect™ Cell Transformation Assay using 2-control, 2C3, 9-control and 9C2 cells in CSM-charcoal stripped media, E2 (β-estradiol; 10-10 M) and 10% fetal bovine serum. Data (mean + SD) were calculated using two-way ANOVA based on three independent biological replicates.",
    "molecules": "charcoal, E2, β-estradiol"
  },
  {
    "caption": "E Impact of ESRP1 knockdown on in vivo tumor growth. Five million cells (2-control, 2C3, 9-control and 9C2) were implanted into mammary fat pads of athymic mice (five mice per group) in the presence of supplemental estrogen. Tumors were measured weekly using calipers for external measurements. Tumor volume was calculated as L x W2/2, where L is length and W is width (note the different scale for tumor volumes). Data (mean ± SD) were calculated using two-way ANOVA (n= 5 mice per group).",
    "molecules": "estrogen"
  },
  {
    "caption": "A Representative images of E18.5 coronal cortical sections from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmids encoding EGFP, EGFP+Cre (Cre) or EGFP+Cre+KCC2FL (Cre+KCC2FL). DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. B Quantification of the number of EGFP+ neurons/ROI from embryos electroporated with constructs in (A). Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01. Data are presented as mean ± S.E.M., n (EGFP) = 8 embryos; n (Cre) = 8 embryos; n (Cre+KCC2FL) = 13 embryos. ",
    "molecules": "DAPI"
  },
  {
    "caption": "C Representative image of cleaved Caspase 3 (Cas-3, upper panel) and TUNEL (lower panel) staining in coronal sections from Kcc2lox/lox cortex at E16.5 electroporated with EGFP or EGFP+Cre are shown. Arrowheads point to neurons expressing Cas-3 (upper panel) and TUNEL (lower panel). DAPI staining (blue) marks cell nuclei. CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone; Sp, subplate; IZ, intermediate zone. Large scale bar: 50 µm, small scale bar: 20 µm. D Quantification of the percentage of EGFP+ neurons expressing apoptotic markers at E16.5 from embryos electroporated with EGFP ± Cre. Statistical significance was determined using Mann-Whitney U test (Cas-3); and two-tailed t test (TUNEL), ***P < 0.001. Data are presented as mean ± S.E.M., n (-Cre) = 6 embryos; n (+Cre) = 6 embryos. E The number of EGFP+Cre neurons as a percentage of neurons expressing EGFP alone. Statistical significance was determined using a two-tailed Student's t test. Data are presented as mean ± S.E.M., n = 6 embryos. ",
    "molecules": "DAPI"
  },
  {
    "caption": "D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm. E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(F) Western blot analysis of ubiquitinated proteins using an anti-multi-ubiquitin antibody in the 6 months old WT and p32cKO heart (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(B) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart. Tissues were excited at a wavelength of 540 (upper panel) or 470 (lower panel) and emission spectra were collected with a confocal microscope at wavelengths (band path) of 580-630 nm (upper panel) or 510-560 nm (lower panel). Enlarged view of the white squares is shown right. Scale bars, 20 µm. The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM (three months old mice, n = 11; 6 months old mice, n = 9-14, 9 months old mice, n = 9-15, 12 months old mice, n = 11-21). Statistical significance was assessed by Student's t-test, ***p < 0.005.",
    "molecules": "DAPI, lipofuscin"
  },
  {
    "caption": "(A) LC-MS/MS metabolomic analysis of NAD+, NADH, NADP+, nicotinamide and NAAD in the 6 months old WT and p32cKO hearts. NAD+ and NADP+ showed significantly different levels between WT and p32cKO hearts (n = 12). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.001, ***p < 0.0002.",
    "molecules": "NAD+, NADH, NADP+, nicotinamide, NAAD"
  },
  {
    "caption": "(B) Real-time PCR analysis of RNA expression of NAD-synthesizing enzymes in the 6 months old WT and p32cKO hearts. Error bars are presented as mean ± SD. Student's t-test was performed on WT mice vs p32cKO mice (n = 6), ***p < 0.005, *p < 0.05. The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows. NAM: nicotinamide, NA: nicotinic acid, QA: quinolic acid, NMN: nicotinamide mononucleotide.",
    "molecules": "QA, quinolic acid, NAD, NAM, nicotinamide, nicotinamide mononucleotide, NMN, nicotinic acid"
  },
  {
    "caption": "(G) Nmnat3 mRNA expression in 3T3-L1 cells after 1 mM CAP treatment for 72 h. The HIF1α inhibitor (20 or 50 µM) was added 2 h before the CAP treatment. Error bars are presented as mean ± SD of three independent experiments. Student's t-test was performed on WT cells vs WT cells treated CAP and (or) HIF1α inhibitor,**p < 0.01, *p < 0.05.",
    "molecules": "CAP"
  },
  {
    "caption": "(I) Nmnat3 mRNA expression in 3T3-L1 cells after 150 µM CoCl2 treatment for 72 h (n = 3). Error bars are presented as mean ± SD of three independent experiments. Statistical significance was assessed by Student's t-test, **p < 0.01.",
    "molecules": "CoCl2"
  },
  {
    "caption": "(A) Lysosomal acidification is impaired in p32KO MEFs. Representative images of WT and p32KO MEFs, stained with dextran-Oregon Green (488) and dextran-TMRM. Scale bar, 5 µm. Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification, and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification. Dextran-Oregon Green is quenched under acidic pH, and thus an increased green:red ratio denotes impaired lysosomal acidification. The average ± SEM of the green:red ratio in at least 30 cells in two independent experiments is presented in the plot (normalized to WT green/red = 1). Statistical significance was assessed by Student's t-test, *p < 0.05, ***p < 0.001.",
    "molecules": "CAP, dextran, Dextran, NMN, Oregon Green, Oregon Green (488), TMRM"
  },
  {
    "caption": "(E) Nmnat3(v1) overexpression in p32KO MEFs rescued lysosomal function. LysoTracker Red-stained p32KO MEFs were quantified by flow cytometry. Error bars are presented as mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.",
    "molecules": "LysoTracker Red"
  },
  {
    "caption": "(A) WT MEFs were stained with dextran-Oregon Green and dextran-TMRM to examine lysosomal acidification after 10 nM FK866 treatment. FK866 decreased lysosomal acidification, which was rescued by 1 mM NMN pretreatment. The stained MEFs were quantified using a microscope (BZ-X800, KEYENCE). Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.",
    "molecules": "dextran, FK866, NMN, Oregon Green, TMRM"
  },
  {
    "caption": "(F) To examine autophagosome and autolysosome formation, we used DAPRed, which indicates autophagosomes, and DALGreen, which indicates autolysosomes. After starvation, co-localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells. Scale bars, 10 µm. Quantification is shown on the right. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.",
    "molecules": "DALGreen, DAPRed, bafilomycin A, FK866"
  },
  {
    "caption": "(D) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs, the membrane pellet (bound) and supernatant (free) were subjected to immunoblotting with GAPDH and PGK1 antibodies.",
    "molecules": "NaCl"
  },
  {
    "caption": "(I) After addition of GAP, NAD and Pi, ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin/luciferase kit. Iodoacetate (IA; 4 µM) was used to inhibit GAPDH activity. Three independent experiments were performed. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, **p < 0.002.",
    "molecules": "ATP, GAP, Pi, IA, Iodoacetate, luciferin, NAD"
  },
  {
    "caption": "B Co-immunofluorescence staining  of pre- versus post-menopausal tissue for E-cadherin  (cyan), PDGFRβ (yellow) and F-actin  (pink). DAPI  is shown in grey. The arrowheads in (v) depict fibroblasts  in direct contact with the myoepithelial  layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10  postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).",
    "molecules": "DAPI"
  },
  {
    "caption": "G Co-immunofluorescence of tissue stained  for the epithelial  marker (cytokeratin 19; yellow) and the  tumor-associated macrophage marker CX3CR1 (magenta). DAPI is shown in white (n=2 preneoplastic samples; n=2 tumors). Scale bar, 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C-F Representative confocal  images of ER+ (ER-0032) and  triple negative tumors  ( TN-0066) immunolabeled  for K8/18 (yellow), CD68  (green) and Ki67  (red) (C, E) or K8/18 (yellow), CD8 (green) and  Ki67 (red) (D, F). DAPI  is shown in blue. Arrows depict proliferative T cells  (CD8+Ki67+) or macrophages  (CD68+Ki67+). Enlargements on shown in the right-hand panels. Scale bars, 200 µm for large tilescans; 50 µm for enlargements and smaller tilescans.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunoblots showing expression levels of MYC, TSC1, TSC2 or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (-Tet).",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "Relative TSC1 and TSC2 mRNA expression levels determined by qRT-PCR for high MYC (-Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± st.dev., n=3 technical replicates) *p< 0.05; **p< 0.01; statistical relevance was determined by unpaired t-test (two-tailed).",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "qRT-PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h-72h (+Tet). Immunoblots for 24h and 48h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet) the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control.",
    "molecules": "Tet"
  },
  {
    "caption": "Immunoblots of indicated proteins in P493-6 cells with high MYC (-Tet, 72 hours) or low MYC (+Tet, 72 hours) levels either treated with rapamycin or solvent.",
    "molecules": "rapamycin, Tet"
  },
  {
    "caption": "Elevated TSC1 and MYC expression in BL (cohort 1). Example of immune staining of TSC1, MYC, the B-cell marker CD20, and DAPI nuclear DNA-staining in germinal centers of control lymph nodes (upper rows) and samples from BL patients (lower rows). Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples (see materials and methods) (the horizontal line shows the median, whiskers show maximum and minimum data points and the box represents the first to the third quartiles, n=56 fields for tumor samples and n=21 fields for control germinal centers; scale bar = 100 μm).",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates).",
    "molecules": "7AAD, Tet"
  },
  {
    "caption": "Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).",
    "molecules": "Rapamycin, rapamycin, Tet"
  },
  {
    "caption": "TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates).",
    "molecules": "7AAD, hydroxytamoxifen, rapamycin"
  },
  {
    "caption": "Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection).",
    "molecules": "Puromycin, Rapamycin"
  },
  {
    "caption": "TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicated",
    "molecules": "rapamycin, Tet, tetracycline"
  },
  {
    "caption": "TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates).",
    "molecules": "DNP, oligomycin"
  },
  {
    "caption": "Ratio of mitochondrial to genomic DNA determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).",
    "molecules": "DNA, rapamycin, Tet"
  },
  {
    "caption": "expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).",
    "molecules": "rapamycin, Tet"
  },
  {
    "caption": "TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates).",
    "molecules": "DCF-DA, ROS, rapamycin, Tet, tetracycline"
  },
  {
    "caption": "TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control.",
    "molecules": "Thr, Tyr, Tet"
  },
  {
    "caption": "Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).",
    "molecules": "BHA, Tet"
  },
  {
    "caption": "Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates).",
    "molecules": "EU, Tet"
  },
  {
    "caption": "TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates).",
    "molecules": "tetracycline"
  },
  {
    "caption": "miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates).",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).",
    "molecules": "DNP, oligomycin, Tet"
  },
  {
    "caption": "(J-O) Impaired granulation tissue formation and angiogenesis in αSMA-TK mice. (J) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines, triangles (open: incomplete closure, filled: closed wounds) denote the epithelial tongues. Epi, epidermis; derm, dermis regions denoted. Scale bar: 200 μm. (K) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control (set to 100%). NA (not assessed) reflects the lack of granulation in αSMA-TK mice. WT, N=5; TK, N=4 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (M) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=3; TK, N=5 biological replicates. (N) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=5; TK, N=6 biological replicates.",
    "molecules": "hypoxyprobe"
  },
  {
    "caption": "(H-J) Multispectral imaging of changes in fibroblast subsets during wound repair. (H) Representative images (20x magnification) of orthogonal wound tissue sections at day 6 post-wounding displaying αSMA, FAP, and FSP1 overlaid with DAPI in the wound bed. Subcutaneous (subq), dermis (derm), epidermis (epi), and scar regions are denoted. Individual αSMA, FAP, and FSP1 channel images overlaid with DAPI. Scale bar, 50 µm. (I) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points. Data are presented as the fractional area of each image positive for the indicated markers and negative for CD31. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates. One-way ANOVA with Dunnett's multiple comparison test performed. (J) Percent overlap of αSMA with FAP and FSP1 throughout wound healing. αSMA+ denotes cells positive for αSMA but not FAP or FSP1. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(L) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines. Scale bar, 200 μm. (M) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control. WT, N=3; FAP-TK, N=3; WT, N=17, FSP1-TK, N=9 biological replicates. (N) Immunofluorescent staining for CD31 (red) and nuclei on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=4; FAP-TK, N=4; WT, N=3; FSP1-TK, N=3 biological replicates. (P) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (Q) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=3; FAP-TK, N=3; WT, N=3; FSP1-TK, N=4 biological replicates. (R) W",
    "molecules": "hypoxyprobe"
  },
  {
    "caption": "(A) IHC for collagen I on wound tissue sections at day 17 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 50 μm. (B) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=5; TK, N=5 biological replicates. (C) Polarized light images of picrosirius red stained wound tissues at day 17 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (D) Quantification of collagen fiber (picrosirius red) percent area normalized to the average area in WT controls. WT, N=3; TK, N=3 biological replicates. (E-",
    "molecules": "picrosirius red"
  },
  {
    "caption": "(E-H) Reduced collagen deposition in Col1α1cKO wounds. (E) IHC for collagen I on wound tissue sections at day 14 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 25 μm. (F) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=7; Col1α1cKO, N=4 biological replicates. (G) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (H) Quantification of collagen fiber percent area and fiber width normalized to the average area and width, respectively, in WT controls. WT, N=4; Col1α1cKO, N=5 biological replicates.",
    "molecules": "picrosirius red"
  },
  {
    "caption": "(F-K) Reepithelization, granulation tissue formation, and vascularization in WT and Itgb1cKO mice. (F) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (G) Quantification of epidermal thickness. WT, N=4; Itgb1cKO, N=6 biological replicates. (H) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (I) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Itgb1cKO, N=6 biological replicates. (J) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in right panels. Scale bar, 50 µm. (K) Quantification of collagen fiber percent area normalized to the average area in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. (L) Quantification of collagen fiber width normalized to the average width in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. D",
    "molecules": "picrosirius red"
  },
  {
    "caption": "(A) Dose-response curves  (normalized  growth  rate  as a function of drug concentration) for different antibiotics. Growth rate was measured via optical density measurements over time  Antibiotics used: Trimethoprim  (TMP), tetracycline  (TET), chloramphenicol  (CHL), ciprofloxacin  (CPR), lincomycin  (LIN), nitrofurantoin  (NIT), and mecillinam  (MEC). The TMP dose-response curve  (dark blue) is by far the shallowest. Lines are fits of the Hill function $\frac{g\left( c \right)}{g(0)} = \frac{1}{1 + \left( \frac{c}{IC_{50}} \right)^{n}}\ $to the data. Drug concentrations were arbitrarily rescaled to better visualize  dose-response curve steepness; Error bars show standard deviation of 12 biological replicates.",
    "molecules": "CHL, chloramphenicol, ciprofloxacin, CPR, LIN, lincomycin, MEC, mecillinam, NIT, nitrofurantoin, TET, tetracycline, TMP, Trimethoprim"
  },
  {
    "caption": "(C) Density scatterplot showing growth  response to TMP  versus normalized drug-free growth rate  for 3,913 gene deletion strains these are essentially all viable gene deletion strains in E. coli, no selection of strains was made. These gene deletion strains exhibit diverse  growth rates, offering an unbiased way to test the relation between the drug-free growth rate and the response to antibiotics. Response is defined as growth rate in the presence of TMP  normalized to the drug-free growth rate of the respective deletion strain. TMP was used at a fixed concentration that inhibits wild type growth  by about 30% Spearman correlation coefficient ρs is shown.",
    "molecules": "TMP"
  },
  {
    "caption": "(D) Bar chart showing negative Spearman correlation coefficients  − ρs compared across antibiotics Error bars show bootstrap standard error of ρs. TMP  (blue) exhibits by far the strongest negative correlation, indicating the existence of a particularly strong growth-mediated negative feedback loop for this antibiotic.",
    "molecules": "TMP"
  },
  {
    "caption": "(A) Growth rate  under glucose  limitation achieved by adding the non-metabolizable structural glucose  analog α-methyl glucoside (αMG) at different ratios to glucose  in a minimal medium Data information: Error bars in A, show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate measurements is high",
    "molecules": "glucose, α-methyl glucoside, αMG"
  },
  {
    "caption": "(B) Normalized  growth rate (gray scale) from a checkerboard assay in a two-dimensional concentration gradient of TMP  and αMG. Dashed black line shows contour line of 90%  growth inhibition (IC90  line). Red arrow shows increase in IC90  as growth  is lowered. Inset: Normalized  growth rate  as a function of TMP  concentration along the column marked in blue.",
    "molecules": "αMG, TMP"
  },
  {
    "caption": "(C) Fold-change in IC90  at αMG/glucose  ratio 2.5 in assays as in B for different antibiotics Lowering growth rate increases IC90  for TMP  but not for other antibiotics. Data information: Error bars in C show standard deviation from three neighboring αMG/glucose  ratios",
    "molecules": "glucose, αMG, TMP"
  },
  {
    "caption": "(D) Growth rate  in rich medium (LB) under different levels of overexpression of a gratuitous  protein from a T5-lac   promoter; overexpression burden is controlled by IPTG  concentration Data information: Error bars in D, show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate  measurements is high",
    "molecules": "IPTG"
  },
  {
    "caption": "(E) As B but for  growth rate  reduction by protein overexpression in a two-dimensional concentration gradient of TMP  and IPTG.",
    "molecules": "IPTG, TMP"
  },
  {
    "caption": "(F) Fold-change in IC90  at 1.25 mM IPTG  in assays as in E for different antibiotics Overexpression of unnecessary protein increases IC90  for TMP  by almost five-fold; no comparable increase occurs for other antibiotics. Data information: Error bars in F show standard deviation from IPTG  concentrations centered at 2.5 and 1.25 mM, respectively. CHL  was not used in the protein overexpression assay in F since the plasmid used for overexpression has a CHL-resistance marker",
    "molecules": "CHL, IPTG, TMP"
  },
  {
    "caption": "(G) Growth rate  in minimal medium containing different carbon sources Glucose  (GLU), fructose  (FRU), mannose  (MAN), galactose  (GAL), and glycerol  (GLY). Data information: Error bars in show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of  growth rate measurements is high",
    "molecules": "carbon, FRU, fructose, GAL, galactose, GLU, Glucose, GLY, glycerol, MAN, mannose"
  },
  {
    "caption": "(H) Normalized growth rates (gray scale) on different carbon  sources (x-axis) at different TMP  concentrations (y-axis).",
    "molecules": "carbon, TMP"
  },
  {
    "caption": "(A) TMP dose-response curve in minimal medium with glucose  as carbon  source and at lower drug-free growth rate due to glucose  limitation, achieved by increasing the αMG  /glucose  ratio from 0 (black) to 5 (gray). Glucose  limitation results in a steeper dose-response curve. Lines show Hill function fits (B) Steepness of TMP dose-response curves (dose-sensitivity n) versus drug-free  growth rate  at different αMG  concentrations Numbers next to data points show αMG/glucose  ratio. Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in B show standard deviation of parameter estimates from Hill function fit.",
    "molecules": "carbon, glucose, Glucose, αMG, TMP"
  },
  {
    "caption": "(C,D) As A,B but for growth  limitation by gratuitous protein overexpression in rich growth  medium Inducing overexpression with IPTG  at 10 mM (light gray) steepens the dose-response curve compared to no induction (black). Numbers next to data points in D show IPTG  concentration in mM. Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in D, show standard deviation of parameter estimates from Hill function fit.",
    "molecules": "IPTG"
  },
  {
    "caption": "(E,F) As A,B but for growth  limitation by varying the carbon  source in a minimal medium Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in F show standard deviation of parameter estimates from Hill function fit.",
    "molecules": "carbon"
  },
  {
    "caption": "(A) Dependence of growth rate (black) and folA expression  (green) on TMP  concentration. Schematic: FolA expression  was measured using a promoter-GFP   reporter inserted at a neutral site in the genome Data information: Black line in A shows Hill function fit other lines show polynomial fits of second order (A to guide the eye. Horizontal dotted green line shows folA expression  level in the absence of TMP. Error bars show standard deviation of three biological replicates.",
    "molecules": "TMP"
  },
  {
    "caption": "(B) Growth rate  (black) and folA expression  (green) in the absence of TMP  at different  growth rates  achieved by different ratios of αMG/glucose. Data information: other lines show polynomial fits of second order B) to guide the eye. Horizontal dotted green line shows folA expression  level in the absence of TMP. Error bars show standard deviation of three biological replicates.",
    "molecules": "glucose, αMG, TMP"
  },
  {
    "caption": "(C) Scatterplot of folA expression  level with growth rate  across all combinations of TMP  concentrations and αMG/glucose  ratios Pearson's correlation coefficient ρ and p-value from two-sided t-test are shown. FolA expression  is largely determined by growth rate, suggesting that this regulation is  growth rate-dependent and not mediated by a specific molecular mechanism. Data information: ; other lines show polynomial fits of first (C) order to guide the eye. Horizontal dotted green line shows folA expression  level in the absence of TMP. Error bars show standard deviation of three biological replicates.",
    "molecules": "glucose, αMG, TMP"
  },
  {
    "caption": "(D) Dependence of folA expression  on TMP  concentration at four different αMG/glucose  ratios (0, 2.5, 5, and 10 as shown). Darker green indicates greater αMG/glucose  ratio. FolA expression  converges approximately to the same level at high TMP  concentrations. Data information: Horizontal dotted green line shows folA expression  level in the absence of TMP. Error bars show standard deviation of three biological replicates.",
    "molecules": "glucose, αMG, TMP"
  },
  {
    "caption": "(B) Growth rate as a function of TMP  concentration for different paths through IPTG-TMP  concentration space Constant FolA   is shown in black and inverted FolA   regulation in red. Wild-type  dose-response curve  (blue line; is shown for comparison. (C) Steepness of the  dose response curve  (quantified as dose-sensitivity n) for the three cases in B. Inset: Normalized FolA expression  level as a function of TMP  concentration for the three cases in B; WT (blue) colors as in the bar chart and in B. Data information: Error bars in B show standard deviation of the measured growth rates  used for interpolating the values shown; the entire experiment was replicated once Error bars in C show standard deviation of parameter estimates from Hill function fit.",
    "molecules": "IPTG, TMP"
  },
  {
    "caption": "(D) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells 56 h after transfection with the indicated siRNAs. When indicated, cells were exposed to 100 nM rapamycin for the last 3 h. RPTOR (raptor) and BECN1 (beclin1) siRNAs served as positive and negative controls, respectively.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(E) Autophagic flux was measured as the ratio between luciferase activities in MCF7-RLuc-LC3WT and MCF7-RLuc-LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h. Error bars are SDs for a representative (n = 5) triplicate experiment with a minimum of 4 × 10 randomly chosen areas/sample analyzed (D) or three independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with similarly treated control siRNA-transfected samples.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Representative immunoblots of indicated proteins from whole-cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0-40 min of amino acid starvation [−AA]).",
    "molecules": "amino acid"
  },
  {
    "caption": "(B) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar (siRNA-resistant CIP2A)-encoding lentivirus, transfected with the indicated siRNAs for 53 h, and treated with 100 nM rapamycin (Rapa) for 0-20 min.",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": "(C) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin (Rapa) for 72 h. The viable cells were either visualized after fixation and crystal violet staining (left; representative figure, n = 4) or trypsinized and counted with NucleoCounter NC-3000 equipped with Via1-Cassettes (right).",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": "(D) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA, 2 µM MG132, 5 µM siramesine (Sira), or 100 µM etoposide (Etop) for 48 h, and the metabolic activity (cell density) was measured by the MTT assay (left), and cell death was measured by the LDH assay (right).",
    "molecules": "Etop, etoposide, MG132, Sira, siramesine"
  },
  {
    "caption": "(F) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF10A (left) and HEK-293 (right) cells infected as in E and treated with 100 nM rapamycin for the indicated times. Black lines indicate that intervening lanes have been spliced out.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(D) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control (Ctr) or CIP2A shRNA lentiviruses (Fig. 3 A) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.",
    "molecules": "amino acids"
  },
  {
    "caption": "(F) HA-S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses (Fig. 3 A), transiently transfected with HA-RPS6K1 cDNA and starved for amino acids for 20 min when indicated, were analyzed by immunoblotting. Immunoblots of corresponding cell lysates are shown on the right.",
    "molecules": "amino acids"
  },
  {
    "caption": "(H) Representative confocal microscopy images of untreated and amino acid-starved (4 h) MCF7 cells stained for endogenous CIP2A and raptor. Black lines indicate that intervening lanes have been spliced out. −AA, amino acid starvation; IP, immunoprecipitation; a.u., arbitrary unit. Bar, 10 µm.",
    "molecules": "amino acid"
  },
  {
    "caption": "(A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0-24 h.",
    "molecules": "amino acid, rapamycin"
  },
  {
    "caption": "(B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments.",
    "molecules": "amino acid"
  },
  {
    "caption": "(C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control.",
    "molecules": "amino acid"
  },
  {
    "caption": "(D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0-5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA.",
    "molecules": "CHX, cycloheximide, rapamycin"
  },
  {
    "caption": "(E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control.",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": "(G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (<100 kD), medium (>150 kD), and long (100-130 kD) exposures are shown.",
    "molecules": "MG132, rapamycin"
  },
  {
    "caption": "(J) Lysates from MCF7 cells transfected with the CIP2A-FLAG construct (Junttila et al., 2007) and the indicated ubiquitin-HA constructs were subjected to anti-FLAG immunopurification in denaturing conditions with 0.1% SDS followed by immunoblotting with the indicated antibodies. Black lines indicate that intervening lanes have been spliced out. Ub, ubiquitin; WT, wild type.",
    "molecules": "SDS"
  },
  {
    "caption": "Starvation triggers accumulation of nucleosides, bases and S7P. Wild‐type S. cerevisiae cells growing in minimal media were switched to minimal media containing no carbon, no nitrogen or no phosphate. After the indicated duration of starvation, the metabolome was quantified by LC‐MS. For media composition, see Supplementary Table S2. For the list of absolute concentration of metabolties, see Supplementary Table S3. Data are shown in heat map format, with each line reflecting the dynamics of a particular compound in a particular culture condition. Metabolite levels of biological duplicates were averaged, normalized to cells growing steadily in glucose (time zero), and the resulting fold changes log2 transformed.",
    "molecules": "carbon, glucose, nitrogen, bases, nucleosides, phosphate, S7P"
  },
  {
    "caption": "(B) Fraction of unlabeled nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild‐type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates).",
    "molecules": "carbon, bases, nucleosides"
  },
  {
    "caption": "(C) Ratio of metabolite levels in atg7 strain versus wild‐type strain in carbon starvation.",
    "molecules": "carbon"
  },
  {
    "caption": "(D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild‐type strain in carbon starvation.",
    "molecules": "carbon"
  },
  {
    "caption": "(E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild‐type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.",
    "molecules": "nitrogen, rapamycin"
  },
  {
    "caption": "(A) Ratio of metabolite levels in sdt1 and phm8 strains versus wild‐type strain in carbon starvation. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.",
    "molecules": "carbon"
  },
  {
    "caption": "(C) Screening of Phm8's phosphatase activity against 90 phosphorylated compounds. Phosphatase activity was measured in the presence of 0.5 mM substrate and 5 mM Mg2+ (pH=7.0, 30°C). Compounds with specific activity higher than 0.1 μmol/mg/min are shown. The x axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).",
    "molecules": "phosphate"
  },
  {
    "caption": "(D) Top table: absolute intracellular concentration of nucleotide monophosphates in carbon starvation. Bottom plots: Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration. The x axis represents CMP concentration and the y axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).",
    "molecules": "carbon, CMP, nucleotide monophosphates, phosphate"
  },
  {
    "caption": "Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase, Urh1 is the pyrimidine hydrolase, and Prm15 (Pgm3) is the phosphoribomutase. Ratio of metabolite levels in pnp1/urh1, pnp1, urh1 and prm15 (pgm3) strains versus wild‐type strain during carbon starvation. Data are shown in heat map format, with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild‐type strain. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.",
    "molecules": "carbon"
  },
  {
    "caption": "(B‐E) DHAP, SBP and S7P levels in wild type, tkl1/tkl2 (B), tal1/nqm1 (C) and pfk1 (E) strains upon carbon starvation, and wild‐type strain upon abruptly switching from glucose to no carbon (WT) versus to dihydroxyacetone (WT+DHA) as the sole carbon source (D). The x axis represents hours after carbon starvation, and the logarithmic y axis represents absolute intracellular concentration (mean±range of N=2 biological replicates).",
    "molecules": "DHA, carbon, glucose, DHAP, SBP, S7P"
  },
  {
    "caption": "(A) Level of hallmark metabolites and energy charge in wild type, phm8 and pnp1/urh1 strains in starvation. The x axis represents hours after starvation, and the logarithmic y axis represents energy charge ([ATP]+0.5[ADP]/([ATP]+[ADP]+[AMP]) or absolute intracellular concentration (mean±range of N=2 biological replicates).",
    "molecules": "ADP, AMP, ATP"
  },
  {
    "caption": "(D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).",
    "molecules": "ethanol, glucose, glycerol"
  },
  {
    "caption": "(C and D) DHAP, S7P, G6P, and glutathione levels and NADPH/NADP+ ratio in wild type, phm8 and pnp1/urh1 strains in the experiment shown in (A) The x axis represents minutes after oxidative stress, and the y axis represents absolute intracellular concentration or ratio of intracellular concentration (mean±range of N=2 biological replicates).",
    "molecules": "G6P, glutathione, DHAP, NADP, NADPH, S7P"
  },
  {
    "caption": "(E) Viability of wild type, phm8 and pnp1/urh1 strains in oxidative stress. The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment (mean±range of N=2 biological replicates).",
    "molecules": "H2O2"
  },
  {
    "caption": "Chromatogram of McjD C547 (black) labelled with Alexa 555 (blue) and Alexa 647 (red) maleimide fluorophore using a Superdex 200 size-exclusion column. The fraction containing the highest degree of labeling is marked by a square",
    "molecules": "Alexa 555, Alexa 647, maleimide"
  },
  {
    "caption": "The ATPase activity of all labelled variants C547, Y64C, L67C was determined in liposomes and compared to wild-type, wt. All labelled McjD variants show basal- and ligand-induced ATPase activities in liposomes comparable to that of wild type protein, indicating that labelling does not interfere with their activity. Error bars were calculated from two replicates from two independent reconstitutions (mean ± standard deviation, n=2). The ruler character of FRET using the fluorophore pair Alexa555/Cy5 was supported by data of dsDNA in Figure EV1B, where different base-pair distances between both dyes correlate directly with the apparent FRET efficiency",
    "molecules": "Alexa555, Cy5"
  },
  {
    "caption": "Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicated",
    "molecules": "ALEX"
  },
  {
    "caption": "Apparent ligand binding affinity values of McjD for AMPPNP (top), ATP (middle), and ADP-vanadate (bottom). Data points were obtained from the areas of fits from the E* histograms considering the ratio ligand-free/(ligand-free+ligand-bound) at the indicated substrate concentrations. Extraction of Kd values from a fit to the data is described in the Material and Methods section. Data for detergent-solubilized McjD for additional biochemical conditions is shown in Figure EV2",
    "molecules": "vanadate, ADP, AMPPNP, ATP"
  },
  {
    "caption": "Representative fluorescence time traces (blue, donor signal; red, acceptor signal; black, FRET signal; yellow, fit) of McjD labeled with Alexa555 and Alexa647-maleimide with indicated concentrations of corresponding substrate: (B) McjD L67C with 10 mM ATP and 200 µM MccJ25, McjD Y64C with 10 mM ATP and 200 µM MccJ25, and McjD C547 with 10 mM ATP. The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant. We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted. Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to detergent",
    "molecules": "Alexa555, Alexa647, ATP, maleimide"
  },
  {
    "caption": "Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide under apo conditions at 10 ms time resolution. Approximately ~85% of all apo-McjD traces show no significant fluctuations beyond shot-noise. Apo-McjD is predominantly in the low FRET and thus open state with E* values <0.3 Within the complete data set of apo-McjD (N = 80 traces) comprising donor and acceptor, ~15%, show infrequent fluctuations to a higher FRET efficiency state with a lifetime of (C) 82 ± 25 ms where E* values > 0.5 are observed during short dwells. The data set represents a total recording time of 4.6 mins with a temporal resolution of 10 ms. To exclude unwanted influence on McjD due to surface-immobilization, experimental verification is provided that ATP-induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure EV4",
    "molecules": "Alexa 555, Alexa 647, ATP, maleimide"
  },
  {
    "caption": "Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide in apo conditions at 10 ms time resolution. This panel shows stable ATP-loaded (left) and ATP-free (right) McjD Representative FRET time traces that show switching between ATP free McjD (E* ~ 0.3) and ATP-loaded (high FRET >0.5) at ~4s s (left) and ~1.4 s (right). The traces show photobleaching events of the acceptor after ~10 s (left) and ~3.3 s (right) Representative FRET time traces that show switching between ATP-loaded (high FRET >0.5) and ATP free McjD (E* ~ 0.3) at 2 s (left) and 5 s (right). The traces show photobleaching events after ca. 40 s (left, donor-bleaching) and 70 s (right, acceptor-bleaching)",
    "molecules": "Alexa 555, Alexa 647, ATP, maleimide"
  },
  {
    "caption": "K-L: Percentage of pDCs grouped by COVID-19  severity, as determined by  oxygen  supplementation (no oxygen n=43, nasal oxygen n=65 and high flow oxygen n=5). Percentage of pDCs (K) was correlated to COVID-19 disease severity (L) using simple linear regression. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal-Wallis test and simple linear regression *<p0.05, **<p0.01 ***<p0.001, ns= not significant.",
    "molecules": "oxygen"
  },
  {
    "caption": "C- J: The FR2020 strain was used in subsequent experiments where  pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL  R837, blue) or TLR3  agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6  (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA. Data information: Bars represent mean values with standard error of the mean and symbols represent individual  pDC donors (n=3-4). Equal symbols represent equal donors Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition *<p0.05, **<p0.01 ***<p0.001.",
    "molecules": "R837, poly(I:C)"
  },
  {
    "caption": "F,  DUB activity assay  for OTUD6B  in asynchronous, G1/S- or mitotically synchronized   and released A549  cells using HA-ubiquitin vinyl-sulfone to isolate active forms of DUBs. Analysis was performed by immunoblotting  using the indicated antibodies.",
    "molecules": "vinyl-sulfone"
  },
  {
    "caption": "B, In-vivo ubiquitylation  analyses of LIN28B  in HEK293T  cells in which OTUD6B  was silenced by siRNA. Cells were transfected with the indicated siRNAs and overexpression constructs, then treated with MG132  for 3 hrs. Lysis and IP  was done under denaturing conditions followed by WB  analysis. Data information: For B, exemplary blot  of two independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "C, In-vivo ubiquitylation  assay of LIN28B  in OTUD6B   and a catalytically inactive variant of OTUD6B  (OTUD6B-C158A) overexpressing cells. HEK293T  cells were transfected with indicated combinations of FLAG-LIN28B, HA-Ubiquitin, OTUD6B, OTUD6B-C158A and EV control and treated with MG132  for 3 hrs 24 hrs later. Denatured WCE were subjected to FLAG-IP. WCE and IP  were analyzed by immunoblotting  including a K48-specific ubiquitin  antibody. Exemplary blots  from three independent experiments are shown.",
    "molecules": "MG132"
  },
  {
    "caption": "B, Immunoblot  analysis of LIN28B  and OTUD6B  protein levels throughout the cell cycle. RPMI  (B) cells were synchronized  at the G1/S  transition by a double thymidine  block or in mitosis  by a sequential thymidine/nocodazole  block. Synchronized  cells were released, harvested at the indicated  time points and analyzed by immunoblot  using the indicated antibodies. Data information: All blots  are representations of three independent experiments.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "C, Immunoblot  analysis of LIN28B  and OTUD6B  protein levels throughout the cell cycle. A549  (C) cells were synchronized  at the G1/S transition by a double thymidine  block or in mitosis  by a sequential thymidine/nocodazole  block. Synchronized  cells were released, harvested at the indicated  time points and analyzed by immunoblot  using the indicated antibodies. Data information: All blots  are representations of three independent experiments.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "D, Immunoblot  analysis of LIN28B  protein  half-life in  G1/S-synchronized (upper panel) and asynchronous (lower panel) A549  cells upon OTUD6B  depletion. Cells transfected with the respective siRNAs were synchronized  in G1/S-phase or not and treated with cycloheximide  (CHX) as indicated. Data information: All blots  are representations of three independent experiments.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Male C57BL/6 Nr4a2  cKO or wildtype littermate C57BL/6 mice  were immunised twice with NP-KLH   emulsified in alum. After 2 weeks, 7 μm spleen  sections were stained  with Red:Alexa-Fluor-594-CD4  and Green: FITC-B220   (D) or FITC-GL7    (E). Scale bar represent 100 nm.",
    "molecules": "NP, Alexa-Fluor-594, alum, FITC"
  },
  {
    "caption": "Male C57BL/6 Nr4a2  cKO or wildtype littermate C57BL/6 mice  were immunised twice with NP-KLH emulsified in alum. Serum  was assessed for level of IgG  antibodies binding NP-31 and NP-4 (F).",
    "molecules": "NP, alum"
  },
  {
    "caption": "(B) BAX/BAK-deficient HeLa cells reconstituted with BAX and where indicated HA-Parkin were treated with antimycin A and oligomycin (AO) for 2 h and then 1 µM of each of the BH3 mimetics ABT-737 and S63485 for 6 h. Cell death was assessed at the indicated times by LDH assay. Data is mean +/- SD of four independent experiments.",
    "molecules": "ABT-737, antimycin A, oligomycin, S63485"
  },
  {
    "caption": "(C) BAX/BAK-deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin (AO, 3 h), MG132 (20 µM, 3.5 h) and BH3 mimetics (1 µM of each of ABT-737 and S63485, 1 h). Cytosol and membrane fractions were immunoblotted as indicated. Graph shows densitometric analysis of non-ubiquitinated membrane proteins (top, relative to untreated control) or cytochrome c release (bottom; cytosol/cytosol+membrane, relative to untreated controls) from three independent experiments. Error bars represent mean +/- SD.",
    "molecules": "ABT-737, antimycin A, MG132, oligomycin, S63485, ubiquitinated"
  },
  {
    "caption": "(E) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment of BAX/BAK-deficient HeLa cells ectopically expressing HA-Parkin and BAX for 3 h. Representative immunoblot from two independent experiments.",
    "molecules": "antimycin A, oligomycin, ubiquitinated"
  },
  {
    "caption": "(A) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA-Parkin following 2 h of antimycin A and oligomycin (AO) treatment.",
    "molecules": "antimycin A, oligomycin"
  },
  {
    "caption": "(B) Immunoblotting of whole cell lysates immunoblotted following treatment with antimycin A and oligomycin (AO) for 3 h with MG132 (20 µM). Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/- SD.",
    "molecules": "antimycin A, MG132, oligomycin, ubiquitinated"
  },
  {
    "caption": "(C) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment for 3 h and treatment with the non-specific DUB USP2 for 30 min at 37°C, representative immunoblot from two independent experiments.",
    "molecules": "antimycin A, oligomycin, ubiquitinated"
  },
  {
    "caption": "(E) Whole cell lysates of HA-Parkin and BAK variant-expressing HeLa cells following 3 h antimycin A and oligomycin (AO) treatment with 30 min pre-treatment with MG132 (20 µM), representative immunoblots from three independent experiments.",
    "molecules": "antimycin A, MG132, oligomycin"
  },
  {
    "caption": "(A) HeLaParkin cells were treated with CCCP (10 μM), antimycin A and oligomycin (AO) for 2h or GTPP (10 μM) for 6h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/-",
    "molecules": "antimycin A, CCCP, GTPP, oligomycin, ubiquitinated"
  },
  {
    "caption": "SH-SY5Y cells generated in (B) were treated with antimycin A and oligomycin (AO) for 3 h prior to cell lysis and immunoblotting. Mono and di-ubiquitinated BAK in Parkin-expressing cells following AO are indicated (asterisk). Data is representative of three independent experiments.",
    "molecules": "antimycin A, oligomycin, ubiquitinated"
  },
  {
    "caption": "(D) Domain architecture of human Parkin with positions of selected PD-associated mutations. Ubl, ubiquitin-like; IBR, in-between-RING HeLa cells expressing wild-type Parkin or the indicated PD-associated Parkin mutants were treated with antimycin A and oligomycin (AO) for 2h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins in AO-treated samples relative to untreated control from three independent experiments. Error bars represent mean +/- SD. All Parkin constructs were N-terminally FLAG-tagged except R104W.",
    "molecules": "antimycin A, oligomycin, Ub, ubiquitin, ubiquitinated"
  },
  {
    "caption": "(A) BN-PAGE and SDS-PAGE of HeLa+HA-Parkin cells treated with 1 µM of each of ABT-737 and S63485 for 3 h (BH3), antimycin A and oligomycin (AO) for 2 h or a combination of the two. Experiment was performed in the presence of 10 µM QVD.oph and immunoblots are representative of three independent experiments.",
    "molecules": "ABT-737, antimycin A, oligomycin, QVD.oph, S63485"
  },
  {
    "caption": "(B) UBA enrichment of ubiquitinated proteins from HeLa+HA-Parkin cells, BAX-/- BAK-/- (DKO) HeLa cells or wild-type (WT) HeLa cells expressing HA-Parkin in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of QVD.oph (10 μM). Representative of three independent experiments.",
    "molecules": "ABT-737, QVD.oph, S63485, ubiquitinated"
  },
  {
    "caption": "(D) Parkin activity induced by apoptotic mitochondrial damage is PINK1-dependent. UBA enrichment of ubiquitinated proteins from HeLa cells generated in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of Q-VD.oph (10 μM). *, ubiquitinated protein. Representative of two independent experiments.",
    "molecules": "ABT-737, Q-VD.oph, S63485, ubiquitinated"
  },
  {
    "caption": "(B) Non-reducing SDS-PAGE and immunoblot of recombinant BAK and BAK-Ub following treatment with 25 mM TCEP",
    "molecules": "TCEP, Ub"
  },
  {
    "caption": "(C) Timecourse monitoring fluorescence increase on liposomes following addition of 4 µM BID-BH3 peptide to 50 nM BAK or BAK-Ub in the presence or absence of 25 mM TCEP. Data is mean +/- SD of three independent experiments.",
    "molecules": "TCEP, Ub"
  },
  {
    "caption": "(D) BN-PAGE of BAK and BAK-Ub on liposomes following incubation with 4 µM BID-BH3 peptide in the presence or absence of 25 mM TCEP, immunoblotted for BAK. Data representative of three independent experiments. Graphs shows densitometric analysis of the BAK tetramer (4x) from three or two independent experiments. Error bars represent mean +/- SD for n=3 or mean and range for n=2.",
    "molecules": "TCEP, Ub"
  },
  {
    "caption": "(A) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK.",
    "molecules": "Cys"
  },
  {
    "caption": "(B) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function. Mitochondrially-enriched membranes from MEFs expressing BAK or BAK K113C were cross-linked (CuPhe) with UbG76C and treated with cBID for 30 min at 30°C. Supernatant (S) and pellet (P) fractions were then immunoblotted for BAK under non-reducing conditions, or cytochrome c or ubiquitin under reducing conditions. Representative of three independent experiments. Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS-PAGE.",
    "molecules": "CuPhe, Ub, ubiquitin"
  },
  {
    "caption": "(C) Parkin-mediated ubiquitination limits BAK activation. HeLa+HA-Parkin cells were treated with antimycin A and oligomycin (AO) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT-737 and S63485 (BH3 mim) for 1 h. Intracellular flow cytometry was performed using the conformation-specific anti-BAK antibody G317-2. Representative histograms are shown and the fold increase in mean fluorescence intensity (MFI) is plotted from three independent experiments with error bars showing SD. Data are normalised to untreated control.",
    "molecules": "ABT-737, antimycin A, oligomycin, S63485, ubiquitination"
  },
  {
    "caption": "(D) Parkin-mediated ubiquitination limits BAK apoptotic activity. BAK-/-BAX-/- HeLa cells expressing Parkin and BAK were treated with AO (2 h) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction. Error bars represent mean +/- SD.",
    "molecules": "ubiquitination"
  },
  {
    "caption": "(E) Parkin activity limits cytochrome c release. HeLa cells or HeLa cells expressing Parkin were treated and analysed Immunoblots are representative of three independent experiments. Ubiquitinated BAK indicated (arrow). Graph shows densitometric analysis of three independent experiments showing cytochrome c release (supernatant/supernatant+membrane) relative to 100 nM cBID as 100% release. Error bars represent mean +/- SD.",
    "molecules": "Ubiquitinated"
  },
  {
    "caption": "(G) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv  3.5 h after elicitation with 500 nM  flg22  or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22  set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01).",
    "molecules": "Ethylene"
  },
  {
    "caption": "(H) Mean cfu of  Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).",
    "molecules": "COR"
  },
  {
    "caption": "(B) ROS burst in WT after elicitation with 10 nM   flg22, 1 μM GLV2, co-treatment or pre-treatment with 1 μM GLV2 for 5 h prior to elicitation with   flg22. Shown is the mean of total ROS production (Total RLU) over 40 minutes normalized to the  flg22  response set as 100% -/+ SD, n=96-120 biological replicates from 15 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b/c/d, p<0.001; b-c, p<0.05; b-d, p<0.001",
    "molecules": "ROS"
  },
  {
    "caption": "(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM  flg22. Ethylene accumulation was normalized to the average of WT responses to   flg22  set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).",
    "molecules": "Ethylene"
  },
  {
    "caption": "(E)  Ethylene  production was measured in WT and rgi5x and  rgi5x pRGI3::RGI3  #2 and #3 3.5 h after elicitation with 500 nM flg22. Ethylene  production was normalized to the average of WT responses to  flg22  set as 1. Shown is the mean of n=8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05).",
    "molecules": "Ethylene"
  },
  {
    "caption": "(F) 5-day-old seedlings  of the indicated genotypes were treated with 100 nM  elf18 for 7 days before measuring fresh weight. Shown is the mean of relative fresh  weight  compared to the  MS medium control -/+ SD, n=36 biological replicates from 3 pooled experiments (Students T-test, ***p<0.001).",
    "molecules": "elf18"
  },
  {
    "caption": "(E) Co-immunoprecipitation   experiment upon co-expression of BAK1-HA and RGI3-GFP in  N. benthamiana.  IP was performed using  GFP-TRAP agarose beads.  Western blots for protein detection were probed with α-GFP or α-HA antibodies. Similar results were obtained in 3 independent experiments.",
    "molecules": "agarose"
  },
  {
    "caption": "(C)  Co-immunoprecipitation  experiment upon co-expression of FLS2-HA, BAK1-HA  and RGI3-GFP in  N. benthamiana. Protein extraction was performed 30 min after mock, 1 μM  flg22 or 1 μM GLV2 treatment. IP  was performed using GFP-TRAP  agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Data information: Similar results were obtained in 3 independent experiments.",
    "molecules": "agarose"
  },
  {
    "caption": "(C) Protein extraction was performed from 12-day-old  seedlings of the indicated genotypes. Western blot was probed with α-FLS2 antibodies. (D) Protein extraction was performed from 12-day-old  seedlings after 1 hour treatment with mock or 50 μM MG132. Western blots were probed with α-FLS2  antibodies. Data information: Similar results were obtained in 3 independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "B Yeast two-hybrid analysis for interaction between the above-shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257-444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica-plated on medium with His or without His, but containing 3-amino-1,2,4-triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination.",
    "molecules": "3-amino-1,2,4-triazole, 3AT, leucine, Trp"
  },
  {
    "caption": "To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared to untreated cells (-CQ, A'",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin3 (mCh-Gal3), mRuby3-galectin8 (mRuby-Gal8), mCherry-FIP200, or -ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin3 or mRuby3-galectin8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin3 (Gal3) or mRuby3-galectin8 (Gal8) were quantified (C'; mean ± S.D.",
    "molecules": "chloroquine, Ub, ubiquitin"
  },
  {
    "caption": "J, K HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15 and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± S.D. of three independent experiments;",
    "molecules": "chloroquine"
  },
  {
    "caption": "A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (-) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. B WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: *P<0.05, **P<0.01). C",
    "molecules": "chloroquine, CQ, Torin1"
  },
  {
    "caption": "C HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. D WIPI2 or LC3B puncta per cell were quantified (mean ± S.D. of three independent experiments).",
    "molecules": "LLOMe"
  },
  {
    "caption": "A HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ATG13 (siATG13) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ATG13 knockdown was assayed by immunoblotting below on the right using GAPDH as loading controls. B WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments; ANOVA: **P<0.01). C",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing ULK1 (siULK1), ULK2 (siULK2), or both (siULK1+2) for 72 h, treated without (-) or with 60 µM chloroquine (+CQ) for 150 minutes, and fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. Efficiency of ULK1 knockdown was assayed by immunoblotting The efficiency of ULK2 knockdown was assayed by quantitative RT-PCR and the result is shown in panel C D WIPI2 of LC3B puncta per cell were quantified for each condition (mean ± S.D. of three independent experiments",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "E HEK293A cells were incubated without (-), with 60 µM chloroquine (+CQ), or with chloroquine and 5 µM MRT68921 (+CQ +MRT) for 150 min and fixed and stained for WIPI2 and LC3B. F WIPI2 of LC3B puncta per cell were quantified for each condition as in panel B (mean ± S.D. of three independent experiments).",
    "molecules": "chloroquine, CQ, MRT, MRT68921"
  },
  {
    "caption": "B Co-immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP-KO). Anti-HA antibodies were used as a control (IP HA). On the right, HEK293A and FIP200 knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry-ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatment",
    "molecules": "chloroquine"
  },
  {
    "caption": "A By immunoblot analysis, the levels of Rabaptin5 and as a loading control of tubulin (Tub) were assessed in wild-type HEK293A cells, HEK+Rbpt5 cells stably overexpressing Rabaptin5, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA). B The same stable HEK293A-derived cell lines were treated without (-CQ) or with 60 µM chloroquine for 150 min (+CQ) and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. Scale bar, 10 µm. C",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "D, E The HEK293A-derived cell lines were treated with 60 µM chloroquine for 30min and non-lipidated and lipidated LC3B (I and II, resp.) were assayed by immunoblot analysis (D). The increase of the LC3B-II fraction of total LC3B upon chloroquine treatment was quantified (mean ± S.D. of three independent experiments;",
    "molecules": "chloroquine"
  },
  {
    "caption": "F, G HEK+Rbpt5 cells and Rabaptin5-knockout cells stably re-expressing wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were transfected with mCherry-ATG16L1, treated with 60 µM chloroquine (CQ) for 0, 15 and 30 min, and analyzed by immunofluorescence microscopy for Rabaptin5, mCherry-ATG16L1, and WIPI2. Manders' colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (F) or WIPI2 (G) and M2 showing the inverse (mean ± S.D. of three independent experiments",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "H Wild-type HEK293A cells, HEK+Rbpt5 cells, Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA-mutant Rabaptin5 (Rbpt5-KO+AAA) were stained with lysotracker and with DAPI for nuclei. Scale bar, 10 µm. I The number of lysosomes (lysotracker-positive structures) per cell was quantified from cells as in panel C (mean ± S.D. of three independent experiments). Images for at least 15 cells per sample were quantified. The value for HEK+Rbpt5 cells is an underestimate, because the density of puncta makes them difficult to distinguish as separate structures.",
    "molecules": "DAPI, lysotracker"
  },
  {
    "caption": "A HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500×g for 5min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± S.D. of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± S.D. of three independent experiments; ANOVA: *P<0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti-transferrin receptor and anti-LC3B as cellular markers. Z-stacks for >5'000cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± S.D. of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively).",
    "molecules": "gentamicin"
  },
  {
    "caption": "C Wild-type HEK293A, HEK+Rbpt5, Rbpt5-KO, Rbpt5-KO+AAA, and FIP200-KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR- and/or LC3B-positive compartment - as illustrated on the top left (scale bar, 2 µm) - during the first hour after infection. In the absence of Rabaptin5, LC3-positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (Mean ± S.D. of three independent experiments, analyzing >50 bacteria for each time point.)",
    "molecules": "gentamicin, methanol"
  },
  {
    "caption": "(D-F) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) in involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20 µM IPZ. Data for each panel are from at least 2 separate experiments. Means ± SDs, t-test, oocyte numbers indicated in brackets.",
    "molecules": "IPZ"
  },
  {
    "caption": "(D-F) Quantification of the RBP-3 gradients (top panels) and average cellular RBP-3 E (bottom) in separate experiments involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20µM IPZ. Means ± SDs, t-test, oocyte numbers indicated in brackets.",
    "molecules": "IPZ"
  },
  {
    "caption": "(I) RCC1-mCherry half-time fluorescence recovery (right, means ± SDs, t-test.) and an immobile fraction (left, means ± SDs, Mann-Whitney-test) in control and IPZ-treated cells, oocyte numbers indicated in brackets.",
    "molecules": "IPZ"
  },
  {
    "caption": "(A) Dose-dependent effects of IPZ on MI spindle assembly are shown by maximum z-projection images from confocal time-lapse imaging of oocytes obtained from transgenic histone H2B-EGFP mice stained with SiR-tubulin. Dashed circles indicate oocyte edges; scale bars, 10 µm.",
    "molecules": "IPZ"
  },
  {
    "caption": "(B-C) Live-cell imaging of oocytes was used to quantify the effects of IPZ on GVBD (B) and anaphase I entry (C). Anaphase I was defined as the first time point when chromosome segregation was visible. Fisher's exact test, oocyte numbers are indicated in brackets.",
    "molecules": "IPZ"
  },
  {
    "caption": "(D) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars.",
    "molecules": "IPZ"
  },
  {
    "caption": "(E) MI spindle formation in oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin matured in the presence of 15 µM IPZ. Representative still images of maximum z-projections are shown. Time in hh:mm is relative to GVBD. Scale bar, 10 µm.",
    "molecules": "IPZ"
  },
  {
    "caption": "(F) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group (means ± SDs, Mann-Whitney-test). Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ-treated oocytes were analyzed. (G) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group (means ± SDs). Oocyte numbers are indicated in brackets. ",
    "molecules": "IPZ"
  },
  {
    "caption": "(H) Oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin were treated with 15 µM IPZ after being cultured for 5h in control media. Maximum intensity z-projections from confocal live-cell imaging are shown. Time in hh:mm is relative to GVBD, DMSO or 15 uM IPZ was added at time 5:00. Scale bars, 10 µm.",
    "molecules": "DMSO, IPZ"
  },
  {
    "caption": "(I). Spindle volume at 1h and metaphase I after IPZ addition. The metaphase time was defined as the last time point before anaphase or, in the IPZ-treated oocytes that failed anaphase entry, as the average control metaphase time. Means ± SDs, Mann-Whitney test, oocyte numbers are indicated in brackets.",
    "molecules": "IPZ"
  },
  {
    "caption": "(J) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media. Oocyte numbers are indicated in brackets.",
    "molecules": "IPZ"
  },
  {
    "caption": "(A) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B-mCherry from microinjected mRNA, co-stained with SiR-tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2. Maximum intensity z-stack projections from confocal live-cell movies 30 min after GVBD, at metaphase I and anaphase I. Time in hh: mm is relative to GVBD. Scale bars, 10 µm.",
    "molecules": "IPZ"
  },
  {
    "caption": "(A). Maximum intensity z-stack projections of confocal IF images (DNA stain, indirect IF for tubulin and TPX2) in untreated oocytes (control), oocytes expressing RanT24N or injected with importin β(71-876). The representative of at least 5 experiments is shown.",
    "molecules": "DNA"
  },
  {
    "caption": "(G) Maximum intensity z-stack projections of confocal IF images stained for DNA, pericentrin, and tubulin (directly labeled primary antibody). A representative example of at least 2 experiments is shown. Scale bar, 20 µm. (H-I) The number and size of pericentrin foci were quantified in maximum-intensity z-stack projections of IF images stained as in (G) in at least 2 separate experiments. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests. ",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Western blot analysis with representative blot including lamin A/C, NLRP3, caspase 1, and actin levels in skin fibroblats from patients with HGPS, n = 2 controls and 2 patients. Positive control correspond to THP1 cells stimulated with LPS+ATP.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(C) Protein expression of NLRP3 and caspase 1 in lymphoblasts from control and one patient after stimulation with uric acid and cholesterol crystal.",
    "molecules": "cholesterol, uric acid"
  },
  {
    "caption": "(D) IL-1β and IL-18 medium release from lymphoblasts which were assessed after a 24 hr incubation with uric acid and cholesterol. ***P < 0.001, **P < 0.005, * treatment vs no treatment; aaaP < 0.001; aaP < 0.01 control cells vs patient cells.",
    "molecules": "cholesterol, uric acid"
  },
  {
    "caption": "(A) Cell growthn (lower subpanel left patient 1 and right patient 2) and morphological aspect (upper subpanel) with MCC950 determined in healthy and representative HGPS fibroblasts. **P < 0.01, *P < 0.05 patient cells no treatment vs treatment. Results are presented as the mean ± SD of three independent experiments.",
    "molecules": "MCC950"
  },
  {
    "caption": "(B) Western blot analysis with representative blots including lamin A/C, NLRP3, IL-1β and actin levels in skin fibroblats from control and HGPS patient after 48 hr of vehicle and MCC950 treatment.",
    "molecules": "MCC950"
  },
  {
    "caption": "(C-E) Representative fluorescence images of HGPS and control fibroblasts to evaluate the effect of the MCC950 in the nuclear morphology (D) and NLRP3 expression (E). Scale bar: 30µm. Results are presented as the mean ± SD of three independent experiments. ***P < 0.01 patient vs control cells, aaaP < 0.01 patient cells no treatment vs treatment. One-way ANOVA test was used for statistical analysis.",
    "molecules": "MCC950"
  },
  {
    "caption": "(H) Analysis of serum concentrations of IL-1β measured by ELISA. N=6 per group. Data are shown as means ± SD. ***P < 0.001, wild-type vs Zmpste24-/- mice; aaaP < 0.001; vehicle vs MCC950.",
    "molecules": "MCC950"
  },
  {
    "caption": "Immunofluorescence  labeling of QSOX1  and a Golgi  marker (GM130) in epithelial cells isolated from murine colon. QSOX1  is green, GM130  is red, and DAPI staining  of nuclei  is blue in the merged image. Scale bar is 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Top, average colon  length of WT and QSOX1  KO mice  following indicated treatments. For untreated mice, colon  lengths were calculated for 3 WT and 3 KO. For treated mice, lengths were calculated from 5 mice  except for the DSS-treated WT, for which 3 mice  were analyzed. Error bars are standard deviation. Statistical analysis was performed using Tukey multiple comparison of means (** p<0.01). Administration of QSOX1  inhibitory antibody MAb316.1 (αQSOX1), did not enhance the sensitivity of WT colons  to DSS  (p>0.4 for MAb316.1-treated WT vs. WT control antibody (IgG)-treated; p>0.5 for MAb316.1-treated WT vs. WT without antibody treatment). Bottom, representative images of colons following DSS  treatment. Scale bar is 1 cm.",
    "molecules": "DSS"
  },
  {
    "caption": "Alcian blue and PAS staining  of reduced guanidine-insoluble mucins  from WT and QSOX1  KO mice, separated on a 6% polyacrylamide  gel. Data information: each lane corresponds to a sample from one mouse.",
    "molecules": "Alcian blue, PAS, guanidine, polyacrylamide"
  },
  {
    "caption": "Muc5b  fluorescent immunoblot  of WT and QSOX1  KO lung  lavage samples separated on agarose  gels. Data information: each lane corresponds to a sample from one mouse.",
    "molecules": "agarose"
  },
  {
    "caption": "Western blot  analysis of blood Vwf  from WT and QSOX1  KO mice  separated on agarose  gels. Data information: each lane corresponds to a sample from one mouse.",
    "molecules": "agarose"
  },
  {
    "caption": "QSOX1 immunofluorescence  (green) in control (siControl) and QSOX1  knockdown (siQSOX1) MDA-MB-231  cells. Blue is DAPI staining  of nuclei. Scale bar is 20 µm. Data information: The results described in are representative of 3 experiments that were performed.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blot analysis of colon epithelial cell  lysates from WT and QSOX1  KO mice. Lysates were treated with   PEG-mal  2  kDa or NEM  as indicated above each blot  and probed using antibodies to St6gal1. As for panel A but using antibodies to St3gal1. As for panel A but using antibodies to B3galt5. Data information: For the experiments in panels A-C, equal amounts of total protein were applied to each gel lane, and no reducing agent was added. Experiments were repeated 3 times with fresh new lysates, and representative blots  are shown.",
    "molecules": "PEG-mal, NEM"
  },
  {
    "caption": "QSOX1  oxidizes St6gal1 in vitro. MAb492.1 is a monoclonal antibody that inhibits human QSOX1 St6gal1  with one pair of free cysteines is modified by two  PEG-mal  additions (2PEG), whereas protein with two pairs of free cysteines acquires four PEG-mal  additions (4PEG). The change in migration per two PEG-mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein QSOX1 oxidation  of St6gal1  mutants. The C350-C361 disulfide (the redox-active disulfide present in the C139A-C403A mutant) is oxidized rapidly, while the C142-C406 disulfide (present in the C350A-C361A mutant) is oxidized slowly. Data information: Experiments were repeated 3 times with fresh new lysates, and representative blots  are shown.",
    "molecules": "PEG-mal, SDS"
  },
  {
    "caption": "B Analysis of APPL1, EEA1, LAMP1 and Rab11a staining in sh-Control and sh-PI3KC2β hCMEC/D3 cells. White dotted lines delineate cells; Scale bar, 10µm; nuclei are shown in blue (DAPI).",
    "molecules": "DAPI"
  },
  {
    "caption": "Quantification was performed to determine number of positive vesicles  per cell (ImageJ  software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Representative VE-cadherin staining and graph quantification of fluorescence intensity normalized to total number of cells per field (DAPI  labelling) in sh-Control and sh-PI3KC2β hCMEC/D3 in resting condition and after 24 hours activation with 25 ng/ml of TNFα. Control normalized to 100%. Scale bar, 15 µm. Data represent mean ± SEM of cell fields randomly selected (dots n=14-19). Data are collected from 4-5 independent experiments; ***p<0.001, unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Graph quantification of fluorescence intensity  of VE-Cadherin normalized to total cell number per field (DAPI  labelling) in control condition and after 5 hours in oxygen-glucose deprivation (OGD) condition. Control normalized to 100%. Data represent mean ± SEM of cell fields randomly selected (dots n=16). Data were collected from 5 independent experiments);***p<0.001; **p<0.01, Mann-Whitney test.",
    "molecules": "DAPI"
  },
  {
    "caption": "A Representative  images of PI3P and PI(3,4)P2 labeling and quantification using mCherry-FYVEHRS and GFP-PH(TAPP1) probes respectively in sh-Control or sh-PI3KC2β hCMEC/D3. Scale bar, 10 µm. Nuclei are shown in blue (DAPI). Staining intensity was quantified by ImageJ  software and normalized to total  number of cells (DAPI labelling) per field. Data represent mean ± SEM of cell fields randomly selected for PI3P (dots n=21) and for PI(3,4)P2 (dots n=14-16). Data were collected from at least 5 independent experiments; ***p<0.001, unpaired t-test with Welch's correction.",
    "molecules": "PI(3,4)P2, DAPI, PI3P"
  },
  {
    "caption": "(D-H) C57BL/6 mice (n=4 per group), which received 2x106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (D) CFSE dilution of CD8+ T cells, (E) CFSE dilution of antigen experienced Kb-SIINFEKL+ CD11ahi CD8+ T cells and stained ex vivo (F-H) for effector CD8+ T cell surface markers. Shown are flow cytometry plots of Kb-SIINFEKL co-staining with markers of effector phenotypes: (F) CD11ahi, (G) CD62Llo and (H) CD49dhi.",
    "molecules": "CFSE"
  },
  {
    "caption": "(G) Control and Rab12‐knockdown MEFs were cultured under N or S conditions in the absence or presence of 100 μM bafilomycin A1 for 1 h. Cell lysates were analysed by immunoblotting with the antibodies indicated, and the intensity of the LC3‐II band was quantified. The normalized amount (arbitrary units) of LC3‐II in lanes 1, 2, 3, 4, 5 and 6 is 1.0, 2.3, 4.3, 0.9, 1.6 and 3.8, respectively.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(A) Control and Rab12‐knockdown MEFs were cultured under nutrient‐rich or starved conditions, fixed and then immunostained with the antibodies indicated. Scale bar, 20 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "B. ERK71 mutants have elevated triacylglycerol (TAG) levels (N=4 replicates of ≥10 larvae/ replicate for each genotype). C. Fat body specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels (N=4 replicates of 10 larvae/replicate for each genotype). ",
    "molecules": "TAG, triacylglycerol"
  },
  {
    "caption": "D. Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK71 mutant fat bodies of third instar larvae. Scale bar: 50 µm. E. ERK71 mutant fat body cells contain more lipid droplets than control cells (N=30 cells for each genotype). ",
    "molecules": "DAPI"
  },
  {
    "caption": "F. After fed [13C]glucose, the ERK71 mutant fat bodies display elevated [13C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry-based lipidomics, N=3 replicates of 15 fat bodies/replicate, TAG=triacylglycerol, PE=phosphatidylethanolamine)",
    "molecules": "13C, glucose, PE, phosphatidylethanolamine, TAG, triacylglycerol"
  },
  {
    "caption": "G. ERK7 overexpression in the fat body results in reduced TAG levels, while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage (N=4 replicates of ≥10 larvae/replicate for each genotype).",
    "molecules": "TAG"
  },
  {
    "caption": "A. Fatty acid composition of total lipids in CG>control and CG>ERK7 larvae, measured by gas chromatography (N=3 replicates of 15 larvae/replicate for each genotype).",
    "molecules": "lipids"
  },
  {
    "caption": "C. Amount of glucose-derived [13C]-labeled carbons incorporated into the TAG pool of control and CG>ERK7 fat bodies (N=3 replicates of 15 fat bodies/replicate for each genotype and diet).",
    "molecules": "13C, carbons, glucose, TAG"
  },
  {
    "caption": "D. Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels (N=3 replicates of 15 larvae/replicate for each genotype). E. Circulating glucose levels are decreased in ERK71 mutants (N=4 replicates of 15 larvae/replicate for each genotype). ",
    "molecules": "glucose"
  },
  {
    "caption": "D. Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism. Color key displays scaled log2 gene expression.",
    "molecules": "lipid"
  },
  {
    "caption": "A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies.",
    "molecules": "DAPI"
  },
  {
    "caption": "D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10).",
    "molecules": "DAPI"
  },
  {
    "caption": "A. ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos-tag Western blot (marked with red arrow). Insulin-inducible bands (dark blue) remain unresponsive to ERK7.",
    "molecules": "Insulin"
  },
  {
    "caption": "F. Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels (N=4 replicates of ≥10 larvae/replicate for each genotype).",
    "molecules": "TAG"
  },
  {
    "caption": "G. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG-GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. H, I. Fat body specific knockdown of PWP1 suppresses increased lipid droplet number (H; N>30 cells per genotype) and increased nuclear area (I; N>45 cells per genotype) caused by ERK7 knockdown (BDSC 56939). ",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants (sug17Δ/Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. E. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK71 mutants (N>20). ",
    "molecules": "DAPI"
  },
  {
    "caption": "F. Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae (N=4 replicates of ≥10 larvae/replicate for each genotype).",
    "molecules": "TAG"
  },
  {
    "caption": "J. Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet (p<0.0001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype and diet).",
    "molecules": "sucrose"
  },
  {
    "caption": "D. Table showing the type of movement found for wild-type, mutant 2, mutant 5, and mutant 13 in a modified single-molecule assay with and without ATP. Classification of type of movement is based on two repetitions of different dynein preparations.",
    "molecules": "ATP"
  },
  {
    "caption": "A-D Microtubule affinity measured by a cosedimentation assay in the apo state (full line) and in the presence of ATP (dashed line), and AMPPNP (dotted line) for wild-type (A), mutant 2 (B), mutant 5 (C), and mutant 13 (D).",
    "molecules": "AMPPNP, ATP"
  },
  {
    "caption": "WT and Batf  KO mice were orally infected by gavage with 2x109 cfu/ml of Citrobacter rodentium  (Cr) expressing GFP Cr  infected WT and Batf  KO were sacrificed on day 4 and day 10. Cells were isolated from the colon, re-stimulated with PMA  and ionomycin, and stained  intracellularly for  flow cytometric analysis of cytokine production  and Foxp3 expression  presented as density plots (P). Data are gated on viable CD4+TCRβ+. Data information: Data are representative of two independent experiments",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "Naïve WT and Batf  KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5  inhibitor. On day 5, cells were harvested and used for RNA extraction to assess  gene expression (A; data were normalized to WT DMSO; Foxp3, unstimulated; Il17a  and Ifng, 6 h 2 ug/ml anti-CD3  restimulation) Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.",
    "molecules": "DMSO"
  },
  {
    "caption": "Nuclear  extracts from WT Th17  cells were incubated with biotinylated oligonucleotides  containing Ets1-Runx1-Stat5-specific binding  sites at the indicated genomic regions (wild type or mutant (∆) as indicated). Immunoblots  of precipitated proteins (D)",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "E, Nuclear  extracts from WT Th17  cells were incubated with biotinylated oligonucleotides  containing Ets1-Runx1-Stat5-specific binding  sites at the indicated genomic regions (wild type or mutant (∆) as indicated). densitometry  measurements (E). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "(b) N2a cells coexpressing YFP-PrP and HLA-A2-CFP were subjected to 6 mM trypsin for 100 s. Images were taken before and after trypsin treatment as indicated. Note that the membrane signal of YFP-PrP was destroyed by the protease, whereas HLA-A2-CFP's membrane signal remained unaffected.",
    "molecules": "trypsin"
  },
  {
    "caption": "(d) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP. N2a cells expressing GFP were treated with 20 μM digitonin. Images were taken before and after digitonin application at the indicated time points. Scale bars, 10 μm.",
    "molecules": "Digitonin, digitonin"
  },
  {
    "caption": "(b) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ-CFP. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.",
    "molecules": "digitonin, trypsin"
  },
  {
    "caption": "(b) NRK cells expressing CD3δ-CFP and YFP-CD3δ were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.",
    "molecules": "digitonin, trypsin"
  },
  {
    "caption": "c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.",
    "molecules": "Triton X-100, trypsin"
  },
  {
    "caption": "(a) Cartoon of the FPP assay illustrating the cellular localization of RFP-SKL (red) and Pex3p-GFP (green) and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing RFP-SKL and Pex3p-GFP were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin as indicated. Scale bar, 10 μm.",
    "molecules": "digitonin, trypsin"
  },
  {
    "caption": "(a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane. GAPDH-RFP is shown in red and CFP-LC3 is shown in green. (b) NRK cells stably expressing CFP-LC3 were transiently transfected to express GAPDH-RFP and subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin. The inset shows the overlap of the GAPDH-RFP and CFP-LC3 signals (4 × magnification of the central area (outlined by a box) of the micrographs. Scale bar, 10 μm.",
    "molecules": "digitonin, trypsin"
  },
  {
    "caption": "B. MASTL  was ablated in MDA-MB-231  cells using the isgMASTL system. 72 h after Dox, cells were starved  of glucose  in the media in presence of 10% dFBS for 1 h (-) and then  re-stimulated with 25 mM of glucose  for 10 min before recovery (+). Whole-cell lysates were blotted  with the indicated antibodies. * in indicates unspecific band. β-Actin  was used as a loading control.",
    "molecules": "Dox, glucose"
  },
  {
    "caption": "C. MASTL  was depleted in MDA-MB-231 cells using shRNAs against MASTL  (+) or scrambled shRNAs (−) as control. Cells were starved  from glucose  for 2 h and re-stimulated  with 5 mM glucose  for 15 min and 1 h. Total extracts were recovered and tested for the indicated antibodies. β-Actin was used as a loading control. D. Quantification of phospho-AKT  T308 levels in glucose deprivation  or glucose stimulation  (15 min) conditions in MDA-MB-231 control cells and MASTL-depleted cells either using shRNA or the inducible sgRNA. Bars displays mean data + SEM from three independent experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).",
    "molecules": "glucose"
  },
  {
    "caption": "E. Immunoblot  analysis with the indicated antibodies in BT-549  cells upon glucose deprivation  and 15 min glucose stimulation. MASTL  knockdown was performed by infection with a shRNA for MASTL  (+) or a Scramble shRNA (−) as a control.  β-Actin was used as a loading control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant). F. Immunoblot  analysis with the indicated antibodies in MCF-7 cells upon glucose deprivation  and glucose stimulation  for 15 min. MASTL  knockdown was performed by infection with a shRNA for MASTL  (+) or a Scramble shRNA (−) as a control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).",
    "molecules": "glucose"
  },
  {
    "caption": "A. Time course  experiment at the indicated  time points  after 100 nM insulin  stimulation in 6 h serum-starved  control and MASTL  knockout MDA-MB-231 cells using the isgMASTL system. The plot shows the quantification of phospho-AKT  T308 after 30 min of insulin stimulation. Plots displays mean data + SEM from 5 independent experiments. *P<0.05, Student's t-test.",
    "molecules": "insulin"
  },
  {
    "caption": "B. TSC2  was knocked down in control or MASTL-null MDA-MB-231 cells using specific (+) or Scrambled (−) shRNAs. Cells were starved for glucose  for 1 h. The plot shows the quantification of phospho-AKT  T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.",
    "molecules": "glucose"
  },
  {
    "caption": "C. MDA-MB-231  control (−) or MASTL  null (+) cells were treated with 100 nM rapamycin  or vehicle as control. Cells were starved  for glucose  for 1 h and  re-stimulated  with glucose  for 15 min; rapamycin  was added 15 min before glucose stimulation. The plot shows the quantification of phospho-AKT  T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.",
    "molecules": "glucose, rapamycin"
  },
  {
    "caption": "A. Immunofluorescence  for GLUT4  (magenta) in  MDA-MB-231 cells in conditions of serum starvation  and insulin stimulation. DAPI  (DNA) is in cyan and a  cell mask is in yellow. Arrowheads indicate positive cells for GLUT4  enrichment at the plasma membrane (PM). Representative magnifications are also shown. Scale bars, 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation  at the PM. Data are mean fold increase vs serum  deprivation in scramble condition + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA. Control inmunoblotting  in conditions of insulin stimulation  is also shown for the indicated antibodies.",
    "molecules": "cell mask, DAPI, insulin"
  },
  {
    "caption": "E. GLUT4 staining  (magenta) in skeletal muscle sections. Vinculin  (membrane) and DAPI  (DNA) staining  is shown in yellow and cyan, respectively. Scale bars, 50 µm. Mice  were fasted  overnight for 16 h, and  re-fed  for 2 h before sample collection. Quantification of the mean intensity [relative units x103; n=3 Mastl(+/+) and 3 Mastl(∆/∆)] is shown. Data points reflect the mean + SEM; *P<0.05; ***P <0.001; unpaired Student's t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "G. Immunoblot  with the indicated antibodies in liver  tissues from Mastl(+/+) (n=6) and Mastl(Δ/Δ) (n=7) mice. Mice  were fasted overnight for 16 h, injected intraperitoneally with glucose  (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of phospho-AKT  T308 (normalized to total AKT  level). n=6 Mastl(+/+) and 7 Mastl(Δ/Δ) mice. Charts reflect the mean + SEM; *P<0.05; unpaired Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "A. Co-depletion of ENSA and ARPP19  proteins in the MDA-MB-231 cell line using specific siRNAs or control siRNAs against luciferase  (Luc). 72 h after transfection cells were starved  for glucose  for 1 h and re-stimulated  with glucose  for 15 min. Total cell extracts were collected and analyzed for the indicated antibodies.",
    "molecules": "glucose"
  },
  {
    "caption": "B. isgMASTL  cells were previously infected with lentiviral  supernatants expressing FLAG- and HA-tagged versions of ENSA  and ARPP19  phospho-mimetic mutants (S67D and S62D) or GFP  as a control. Cells were treated with Dox  to induce MASTL CRISPR/Cas9-dependent knock-out, and 72 h later cells were treated as specified in A. The plot displays mean + SEM from 3 independent experiments. Significance determined by Student's t-test comparing ENSA/ARPP19  overexpression versus control cells (ns, not significant; *P<0.05).",
    "molecules": "Dox"
  },
  {
    "caption": "C. Control (Dox  −) and MASTL  knockout (Dox  +) cells were treated with 5 µM fostriecin, 50 nM  okadaic acid (OA) or vehicle (Veh) 10 min before insulin  stimulation for 30 min. Total lysates were analyzed for the indicated antibodies and P-T308-AKT  signal was quantified. β-Actin was used as a loading control. The plot represents mean + SEM from 3 independent experiments, except for fostriecin  treatment in which only data from two experiments were obtained and no error bars are shown. *P<0.05; Student's t-test.",
    "molecules": "Dox, fostriecin, insulin, OA, okadaic acid"
  },
  {
    "caption": "B. Immunodetection  of the indicated antigens in control (−) and MASTL  knockout (+) cells starved  from glucose  and re-stimulated with glucose  for 15 min.",
    "molecules": "glucose"
  },
  {
    "caption": "C. siRNA-mediated genetic depletion of B55alpha  and delta subunits (B55αδ) in control (Dox  −) and MASTL-null (Dox  +) cells. The phosphorylation  status of the different proteins was scored in conditions of glucose  re-addition. n=3 independent experiments. The plot shows mean + SEM from 3 independent experiments. *P<0.05, 1-way ANOVA.",
    "molecules": "Dox, glucose"
  },
  {
    "caption": "D. Immunoblot  with the indicated antibodies in  skeletal muscle (gastrocnemius) extracts. Mice  were fasted overnight for 16 h, injected intraperitoneally with glucose  (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of total IRS1, and phospho-S6K1  T389 (normalized to total S6K1  level; n=6 Mastl(+/+) and 7 Mastl(∆/∆)). vinculin  were used as a loading control. Data are mean + SEM; *P<0.05, unpaired Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "A.  MDA-MB-231  cells were starved  for 1 h and re-stimulated with glucose  for 15 min and T194 phosphorylation  was tested with a specific antibody. The plot shows the quantification of phospho-MASTL  T194 (n=2 independent experiments). Data are mean + SEM from 3 independent experiments. ns, not significant; Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "B. Cas9-expressing control (−) cells or cells with CRIPSR/Cas9-mediated TSC2  knockout (sgTSC2) were treated with rapamycin  (Rap), Torin1  (Tor), or vehicle as control (V). MASTL-knockdown cells (shMASTL) were used as an additional control of the specificity of the antibody. Inhibitors were added 15 min before glucose  re-stimulation for additional 15 min and total cell lysates were harvested for immunoblot. The plot shows the quantification of phospho-MASTL  T194 as mean from 2 independent experiments.",
    "molecules": "glucose, Rap, rapamycin, Tor, Torin1"
  },
  {
    "caption": "C. In vitro  kinase assay using purified GST-human MASTL  as a substrate, and recombinant mTORC1  and S6K1   kinases. Red arrowheads show the phosphorylation  of MASTL  in the presence of mTORC1  or S6K1. Note the appearance of other phosporylation  signals in mTORC1  and S6K1  reactions, independently of the presence of MASTL, which correspond to autophosphorylation  of these kinases. Imperial staining    shows the amount of GST-MASTL  protein used as a substrate in each reaction.",
    "molecules": "Imperial"
  },
  {
    "caption": "G. isgMASTL MDA-MB-231 cells were transduced with the indicated Mastl  mutants or GFP  alone as a control, and treated with doxycycline  (Dox) to induce Mastl  deletion. Cells were collected after glucose  stimulation, and total lysates blotted  for the indicated antibodies.",
    "molecules": "Dox, doxycycline, glucose"
  },
  {
    "caption": "(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm.",
    "molecules": "NEFA, Oil Red O, TG"
  },
  {
    "caption": "(A) HepG2 cells were stably transfected with vector or FLAG-JFK. Cellular extracts were immunopurified with anti-FLAG immunoaffinity resin and eluted with FLAG peptides. The eluates were resolved on SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry.",
    "molecules": "silver"
  },
  {
    "caption": "(E) HepG2 cells were transfected with FLAG-JFK and treated with 50 μg/ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING5/β-actin.",
    "molecules": "CHX, MG132"
  },
  {
    "caption": "(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).",
    "molecules": "fatty acid, ferricyanide"
  },
  {
    "caption": "Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test).",
    "molecules": "NEFA, TG"
  },
  {
    "caption": "C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors.",
    "molecules": "erlotinib"
  },
  {
    "caption": "D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining).",
    "molecules": "erlotinib"
  },
  {
    "caption": "A. Representative H&E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6).",
    "molecules": "erlotinib"
  },
  {
    "caption": "C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice).",
    "molecules": "erlotinib"
  },
  {
    "caption": "E. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).",
    "molecules": "erlotinib"
  },
  {
    "caption": "HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.",
    "molecules": "erlotinib"
  },
  {
    "caption": "HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.",
    "molecules": "erlotinib"
  },
  {
    "caption": "HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.",
    "molecules": "erlotinib"
  },
  {
    "caption": "HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.",
    "molecules": "erlotinib"
  },
  {
    "caption": "G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed.",
    "molecules": "erlotinib"
  },
  {
    "caption": "A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting.",
    "molecules": "erlotinib"
  },
  {
    "caption": "B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm.",
    "molecules": "erlotinib"
  },
  {
    "caption": "C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay. Data are representative of at least three independent experiments.",
    "molecules": "erlotinib"
  },
  {
    "caption": "C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.",
    "molecules": "erlotinib"
  },
  {
    "caption": "A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting.",
    "molecules": "G594, erlotinib"
  },
  {
    "caption": "B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay.",
    "molecules": "G594, erlotinib"
  },
  {
    "caption": "C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs.",
    "molecules": "G594, erlotinib"
  },
  {
    "caption": "HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by quantification of the subG1 cell population (D).",
    "molecules": "G594, erlotinib"
  },
  {
    "caption": "HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by detection of cleaved PARP and caspase-3 (E).",
    "molecules": "G594, erlotinib"
  },
  {
    "caption": "A. Representative H&E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both.",
    "molecules": "G594, erlotinib"
  },
  {
    "caption": "C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D).",
    "molecules": "G594, erlotinib"
  },
  {
    "caption": "The fractions of vesicles that fall into each density category for each RyR1 sample (EDTA: n=64; 1 mM Ca2+: n = 32; 5 mM Ca2+: n=21, technical replicates). Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density, orange to segregated density, and grey to empty vesicles.",
    "molecules": "Ca2+, EDTA"
  },
  {
    "caption": "Structures of RyR1 determined in presence of increasing Ca2+ concentration (1 mM and 5 mM), all with intra-SR extensions. Scale bars: 10 nm in (A) and (E), 20 nm in (C).",
    "molecules": "Ca2+"
  },
  {
    "caption": "(A) in vitro kinase assays were performed using purified recombinant kinases (ULK1 and ULK2) and substrate (ATG16L1) in the presence of radiolabelled ATP. ULK and ATG16L1 inputs were examined by western blot (WB) and substrate phosphorylation was analyzed by autoradiography (AR). Data information: Unless otherwise indicated experiments were performed three times.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.",
    "molecules": "amino acid"
  },
  {
    "caption": "(B) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.",
    "molecules": "gentamycin"
  },
  {
    "caption": "(C) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 µM) for 4 hours. Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.",
    "molecules": "Z-VAD-FMK"
  },
  {
    "caption": "(A) Wild-type MEF cells were infected with Salmonella for 25 minutes. Phospho-ATG16L1, total ATG16L1, and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 μm and 1 μm). Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Wild-type and ULK1/2 dKO were infected with Salmonella for 25 minutes. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 μm and 2 μm). Quantification of ATG16L1-positive bacteria from 7 fields of view from a representative experiment is shown in the right panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5μm and 1 μm). Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.",
    "molecules": "LPS"
  },
  {
    "caption": "(D) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5μm and 1 μm). Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Autophagic flux was analyzed in gba+/+ and gba−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. *p < 0.05.",
    "molecules": "bafilomycin A1, rapamycin"
  },
  {
    "caption": "(E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba+/+ and gba−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control.",
    "molecules": "SDS, TX-100, Urea"
  },
  {
    "caption": "(B) Neurons from gba+/+ and gba−/− bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 1 min, 1 μM oligomycin was added followed by 1 μM FCCP (n = 5, three cells analyzed/experiment).",
    "molecules": "FCCP, oligomycin"
  },
  {
    "caption": "(C) Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).",
    "molecules": "FCCP, methyl pyruvate, oligomycin"
  },
  {
    "caption": "(D) As in (C), except cells were bathed in 10 mM methyl succinate, (n = 3, four cells/experiment). All data in this figure represent the mean ± SEM, *p < 0.05. Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).",
    "molecules": "FCCP, methyl succinate, oligomycin"
  },
  {
    "caption": "(A) Oxygen consumption rates of gba+/+, gba+/−, and gba−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment).",
    "molecules": "antimycin A, FCCP, oxygen"
  },
  {
    "caption": "(E) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment).",
    "molecules": "decylTPP, MitoQ10"
  },
  {
    "caption": "(F) gba+/+and gba−/− cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, *p < 0.05.",
    "molecules": "decylTPP, MitoQ10"
  },
  {
    "caption": "(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(B) Midbrain gba+/+ and gba−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.(C) TMRM fluorescence intensity of cells from (B) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment).",
    "molecules": "FCCP"
  },
  {
    "caption": "(D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba+/+ and gba−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.",
    "molecules": "FCCP"
  },
  {
    "caption": "(J) Left panel: Electron micrographs of the  posterior pituitary gland  from Shank2+/+ and Shank2-/- mice in lower (left panel) and higher magnification. Scale bar (500 nm). Right diagram: No significant difference was detected in the number of vesicles in the  posterior pituitary gland between Shank2+/+ and Shank2-/- mice, Mann-Whitney test, P=0.507, Shank2+/+ n = 3, Shank2-/- n = 3. (K-M) Additionally, Shank2-/- mice display no significant difference in: (K) Estradiol-, Mann-Whitney test, P=0.475, Shank2+/+ n = 6, Shank2-/- n = 7, (L) Progesterone, unpaired, two-tailed Student's t-test, P=0.570, Shank2+/+ n = 5, Shank2-/- n = 5 or (M) Prolactin- plasma concentrations, unpaired, two-tailed Student's t-test, P=0.980; Shank2+/+ n = 3, Shank2-/- n = 3. Data information: All data are presented as mean ± SEM, NS: not significant. E, endothelium; CL, capillary lumen; M,  mitochondria; NG, neurosecretory granule; AE, axonal endings, HYP, Hypothalamus; PIT, Pituitary gland.",
    "molecules": "Estradiol, Progesterone, Prolactin"
  },
  {
    "caption": "(D-G) DREADD activation by CNO injection selectively rescues major components of social attachment behavior in Shank2-/- mice: (D) grooming, two-way ANOVA, effect of genotype: *P=0.018, effect of treatment: **P=0.002, genotype x treatment interaction: ***P=0.001. (E) Crouching, two-way ANOVA, effect of genotype: P=0.091, effect of treatment: P=0.869, genotype x treatment interaction: P=0.663, (F) nest building, two-way ANOVA, effect of genotype: P=0.415, effect of treatment: P=0.341 genotype x treatment interaction: P=0.093, (G) Maternal interaction, two-way ANOVA, effect of genotype: *P=0.034, effect of treatment: P=0.149, genotype x treatment interaction: *P=0.045. Shank2+/+ vehicle injected n = 6, Shank2-/- vehicle injected n = 6, Shank2+/+ CNO injected n = 6, Shank2-/- CNO injected n = 7. Data information: All data are presented as mean ± SEM, NS: not significant. ",
    "molecules": "CNO"
  },
  {
    "caption": "b, MALDI mass spectrum of His6-Apg8. Purified His6-Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4(Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6-Apg8FG.",
    "molecules": "His6, β-octylglycoside"
  },
  {
    "caption": "c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.",
    "molecules": "immonium ions"
  },
  {
    "caption": "d, Thioester formation between Apg8 and Apg7. Cell extracts of Δapg7Δapg8 cells expressing Apg7-Myc and/or HA-Apg8 from 2µ plasmids were immunoprecipitated with anti-HA antibody, and immunoblotted (IB) with anti-Myc and anti-HA antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg7 conjugate.",
    "molecules": "DTT, Thioester"
  },
  {
    "caption": "d, Thioester formation between Apg8 and Apg3. Δapg3, Δapg7 and Δapg10 cells were transformed with both Myc-APG8 and Apg3, or only Apg3, on 2µ plasmids. Cell extracts were immunoprecipitated with anti-Myc antibody, and subjected to immunoblot (IB) using anti-Myc and anti-Apg3 antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg3 conjugate; diamond, possible Myc-Apg8 dimer.",
    "molecules": "DTT, Thioester"
  },
  {
    "caption": "a, Apg7 and Apg3 are necessary for attachment of Apg8 to PE. Δapg cell lysates were subjected to SDS-PAGE containing 6 M urea, and immunoblotted with anti-Apg8 antibody.",
    "molecules": "PE, urea"
  },
  {
    "caption": "b, Autophagic activity in apg3 mutants. Wild-type, Δapg3 and apg3C243A cells were grown in rich medium (open bar) and subjected to nitrogen starvation for 4 h (filled bar). The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity. The apg3C243A mutant showed defective autophagy.",
    "molecules": "nitrogen"
  },
  {
    "caption": "B) vam3∆ or   ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown.",
    "molecules": "Triton X-100, TX100"
  },
  {
    "caption": "C) Trichloroacetic acid extracts were prepared from  ykt6ts cells containing an empty plasmid, 3HA-Ykt6  wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.",
    "molecules": "Trichloroacetic acid"
  },
  {
    "caption": "D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid  extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.",
    "molecules": "Trichloroacetic acid, urea"
  },
  {
    "caption": "(c,d) Impaired mTOR-dependent TOP translation in cells expressing mutant huntingtin. (c) PC12 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin were transfected with luciferase constructs containing 5′ untranslated sequence with (+) or without (−) a TOP sequence along with β-gal. Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin. Data shown are from one representative experiment (of three) done in sextuplicate (error bars represent s.e.; raw data are given in Supplementary Fig. 4 online). (d) COS-7 cells were cotransfected with wild-type or mutant huntingtin, a nontypical TOP or control luciferase vector and β-gal. The experiment was done and analyzed as in c (raw data are given in Supplementary Fig. 4 online) (e-g) Relevance of mTOR sequestration to other polyglutamine expansion diseases",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b) Immunofluorescence analysis of COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates (arrow). COS-7 cells without the primary antibody (negative) and COS-7 cells without any treatment (untreated) stained for LC3 are also shown. The numbers of EGFP-positive cells expressing wild-type or mutant huntingtin with (+agg) and without (−agg) aggregates that had higher numbers of autophagic vacuoles (>15-20 vesicles per cell) is shown in the graph. COS-7 cells grown in starvation medium (starvation) or treated with rapamycin (Rap) for 4 h to induce autophagy had more autophagic vacuoles (arrows).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b,c) Treatment with rapamycin rescues huntingtin-induced degeneration in flies 2 d after eclosion. Photographs of ommatidia of flies expressing mutant (Gln120; Q120) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood. More rhabdomeres are visible in the ommatidia of flies treated with rapamycin. This effect is significant (P 0.0001; Mann-Whitney U test). At 2 d after eclosion, there is no reduction in the number of visible rhabdomeres in wild-type untreated flies (data not shown).",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "Open bars, mice treated with a placebo; filled bars, mice treated with CCI-779. (a-c) Behavioral tests in mice expressing mutant huntingtin after treatment at 16 weeks (CCI-779, n = 14; placebo, n = 14), 18 weeks (CCI-779, n = 10; placebo, n = 9), 20 weeks (CCI-779, n = 9; placebo, n = 7) and 22 weeks (CCI-779, n = 5; placebo, n = 6) of age. Scores are explained in Methods. Data for all time points are given in Supplementary Figure 13 online. (a) Tremors. Overall effect from all treated time points: P = 0.0006, odds ratio (OR) = 0.21, 95% confidence interval (95% c.i.) = 0.098-0.45. 16 weeks, P = 0.01; 18 weeks, P = 0.08; 20 weeks, P = 0.14; 22 weeks, P = 0.07. (b) Wire maneuver. Overall effect from all treated time points: P = 0.02, OR = 0.51, 95% c.i. = 0.29-0.92. 16 weeks, P = 0.29; 18 weeks, P = 0.40; 20 weeks, P = 0.003; 22 weeks, P = 0.008. (c) Grip strength. Overall effect from all treated time points: P 0.0001 OR = 12.73, 95% c.i. = 4.35-37.2. 16 weeks, P = 0.1827; 18 weeks, P = 0.0412; 20 weeks, P = 0.0021; 22 weeks, P = 0.0285.",
    "molecules": "CCI-779"
  },
  {
    "caption": "(d) Accelerating rotarod tests in mice expressing mutant huntingtin after treatment at 4 weeks, (CCI-779, n = 16; placebo, n = 17), 14 weeks (CCI-779, n = 11; placebo, n = 10), 16 weeks (CCI-779, n = 14; placebo, n = 14) and 18 weeks (CCI-779, n = 10; placebo, n = 9) of age. Overall effect from all treated time points: P = 0.035. 4 weeks, P = 0.227; 14 weeks, P = 0.012; 16 weeks, P = 0.0025; 18 weeks, P = 0.057. CCI-779 had no discernable effects on the performance of nontransgenic mice on any of these behavioral tests (data not shown). There was no difference in the male:female ratios of the groups treated with placebo or with CCI-779.",
    "molecules": "CCI-779"
  },
  {
    "caption": "(a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo. (b) Brain weights of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.023), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.90) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.22) of age. (c) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.06), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.38) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.56) of age.",
    "molecules": "CCI-779"
  },
  {
    "caption": "(a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin. Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age. Mice treated with CCI-779 had fewer aggregates per unit area in the striatum than mice treated with a placebo. Overall cell staining in the tissues of mice treated with CCI-779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates (data not shown). Aggregates, when visible, were smaller in mice treated with CCI-779 than in those treated with a placebo, as apparent in the higher magnification images of single cells with aggregate (bottom panels). Six coronal sections per mouse were analyzed; these corresponded to the bregma ± 1 mm. We counted aggregates in five different high-power fields (60× objective) per slide on each side of the brain, sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule. The striata of mice treated with CCI-779 had 70% fewer aggregates than the striata of mice treated with a placebo (P = 0.0001, two-tailed t-test).",
    "molecules": "CCI-779"
  },
  {
    "caption": "(b) CCI-779 blocks mTOR signaling in vivo. Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6, in the cortical area and striatum. Levels of phosphorylated S6 were lower in mice treated with CCI-779 than in those treated with a placebo.",
    "molecules": "CCI-779"
  },
  {
    "caption": "E: Dissociation constants between peptides corresponding to different kind of FFAT motifs and the MSP domains of VAP-A and VAP-B. The kinetic experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of n independent experiments: n=2 for pS209 and pS209+pS213; 3 for pS213; and 4 for the non-phosphorylated FFAT. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.",
    "molecules": "HCl, P20, surfactant polysorbate 20, NaCl, Tris"
  },
  {
    "caption": "B: SDS-PAGE gel and Western blot analysis of purified cSTD3 and pS209 cSTD3 proteins. Top: the gel was stained with Sypro Orange to visualize proteins and molecular weight markers. Bottom: two similar gels were blotted onto nitrocellulose and analysed for the presence of total and STARD3-pS209 using specific antibodies.",
    "molecules": "Sypro Orange"
  },
  {
    "caption": "C: Aggregation assays. LA liposomes (50 µM total lipids) were incubated for 5 minutes with cSTD3 or pS209 cSTD3 (380 nM). Then, LB liposomes (50 µM total lipids) and VAP-AHIS6 or VAP-A KD/MDHIS6 (700 nM) were successively added. Left panels: mean radius (black dots) and polydispersity (shaded area) over time. Right panels: size distribution before (grey bars) and after the reaction (black bars).",
    "molecules": "liposomes, lipids"
  },
  {
    "caption": "D: DHE transport assay. DOPC (1.2-dioleoyl-sn-glycero-3-phosphocholine) liposomes (62.5 µM total lipids, LA) containing 3 mol% MPB-PE were mixed with cSTD3 or pS209 cSTD3 (475 nM). After 5 minutes, liposomes (DOPC/DOGS-NTA-Ni2+/DNS-PE/DHE liposomes 77.5/10/2.5/10 mol/mol, 62.5 µM total lipids, LB), covered or not with 1 µM of VAPHIS6 or VAP-A KD/MDHIS6, were added. FRET between DHE and DNS-PE in the LB liposomes diminishes as DHE is transported towards LA liposomes. The signal was converted into amount of DHE present in LB liposomes (in mol%).",
    "molecules": "liposomes, 1.2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, DNS-PE, DOGS-NTA-Ni2+, MPB-PE, DHE, lipids"
  },
  {
    "caption": "E: Initial DHE transport rate measured with cSTD3 or pS209 cSTD3 in the presence or absence of VAP-A HIS6 or VAP-A KD/MDHIS6. Data are represented as mean ± S.E.M. (n = 3 for cSTD3-VAP and n=4 for all other data). Mann-Whitney test (*P < 0.05).",
    "molecules": "DHE"
  },
  {
    "caption": "A-E: HeLa/Ctrl (A), HeLa/STARD3 (B), HeLa/STARD3 S209A (C), HeLa/STARD3 S209D/P210A (D) and HeLa/STARD3 Conv-FFAT (E) cells were labeled with anti-STARD3 antibodies (magenta), anti-Lamp1 antibodies (green) and with the fluorescent cholesterol probe filipin (Cyan Hot). Nuclei are shown in gray (TO-PRO-3). Higher magnifications images (2x) of the area outlined in white are shown on the right. The filipin, STARD3 and Lamp1 merged image is labeled Overlay. Scale bars: 10 µm. Inset scale bars: 5 µm.",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "G: Relative fluorescence intensity of intracellular filipin signals in endosomes of HeLa/Ctrl, HeLa/STARD3, HeLa/STARD3 S209A, HeLa/STARD3 S209D/P210A and HeLa/STARD3 Conv-FFAT cells. Data are displayed as a Superplot (Lord et al, 2020) showing the relative filipin fluorescence intensity in endosomes of individual cells (small circles) from 6 independent experiments (mean of each experiment shown as a large circle). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. Kruskal-Wallis with Dunn's multiple comparison test (*P < 0.05; **P < 0.01; n=6 independent experiments).",
    "molecules": "filipin"
  },
  {
    "caption": "SPR analysis of the MSP domain of MOSPD2 binding onto immobilized pS209 (D) STARD3 FFAT. Representative sensorgrams resulting from the interaction between the MSP domain of MOSPD2 injected at different concentrations and the different FFAT peptides are shown in (D left). Binding curves display the SPR signal (RU) as a function of time. Concentrations printed in bold indicate samples measured twice. Signal obtained for the negative control peptide immobilized on another flowcell is systematically subtracted, as well as the bulk effect recorded with buffer only. (D right) steady-state analysis of the interaction between pS209 STARD3 FFAT peptide and the MSP domain of MOSPD2. Equilibrium responses (Req) extracted from the left panel were plotted as a function of the monomeric concentration of the MSP domain of MOSPD2, and fitted with a 1:1 binding model. The experiments were performed at 25 °C in Tris-HCl 50 mM, pH 7.5, NaCl 75 mM buffer supplemented with 0.005% (v/v) surfactant polysorbate 20 (P20, GE Healthcare). Mean of 2 independent experiments. Uncertainties are obtained from the standard deviation considering a t-distribution coefficient for a risk factor of 32%.",
    "molecules": "HCl, P20, surfactant polysorbate 20, NaCl, Tris"
  },
  {
    "caption": "C. Confocal microscopy images of nuclei stained  with DAPI. Scale bar: 10 µm. D. Quantification of the DAPI  area in (C). Data are mean with standard deviation (SD); ***P ≤ 0.001; unpaired two-tailed t-test (n = 3 biological replicates).",
    "molecules": "DAPI"
  },
  {
    "caption": "E. RNA FISH  with a Cy3-labeled  oligo-dT   probe. DAPI  was used for nuclear staining. Scale bar: 10 µm. F. Quantification of mean fluorescence intensity of the Cy3  oligo-dT signal in (E). Data are mean with SD; ****P ≤ 0.0001; Mann-Whitney test (n = 129 cells for HNRNPR+/+ and n = 176 cells for HNRNPR-/-).",
    "molecules": "Cy3, DAPI"
  },
  {
    "caption": "A, B. Western blot  analysis of Ser2-phosphorylated  and total RNA pol II levels in HNRNPR+/+ (A) and -/- (B) cells treated with DMSO  or CHX  for the indicated durations. GAPDH  and α-Tubulin served as loading control.",
    "molecules": "CHX, DMSO"
  },
  {
    "caption": "D, Western blot  analysis of subcellular fractions from cells treated with DMSO  or CHX  for Calnexin, GAPDH  and histone H3 (D) Cyt, cytosol; nuc+org, nuclear  soluble proteins and organelles; chr, chromatin-associated proteins.",
    "molecules": "CHX, DMSO"
  },
  {
    "caption": "E. Western blot  analysis of subcellular fractions from cells treated with DMSO  or CHX  for Cyclin T1 and CDK9  (E). Cyt, cytosol; nuc+org, nuclear  soluble proteins and organelles; chr, chromatin-associated proteins. F. Quantification of relative expression of Cyclin T1 and CDK9  protein levels in subcellular fractions in (E). Data are mean with SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; one-way ANOVA with Tukey's multiple comparisons test (n = 3 biological replicates).",
    "molecules": "CHX, DMSO"
  },
  {
    "caption": "G. Western blot  analysis of  Cyclin T1 levels in HNRNPR+/+ and -/- cells treated with DMSO  or MG132. α-Tubulin served as loading control. H. Quantification of relative expression levels of  Cyclin T1 in (G). Data are mean with SD; ***P ≤ 0.001, ****P ≤ 0.0001, n.s. not significant; two-way ANOVA with Sidak's multiple comparisons test (n = 3 biological replicates).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "TGF-α treatment (10 ng/ml, 24 h) promotes ME180 cell cycle progression. G1, S, and G2 indicate cells in G1 phase, DNA synthesis phase, and the G2/M phase, respectively, of cell cycle. Apo: apoptotic cells.",
    "molecules": "DNA"
  },
  {
    "caption": "Soft agar assay showing the effect of AG1478 and VTPF on colony formation in ME180-MXIV, ME180-YAP and ME180-YAPS127A cells. Scale bar: 500 μm.",
    "molecules": "AG1478, VTPF"
  },
  {
    "caption": "Representative images showing the effect of AREG, EGFR inhibitor (AG1478), and YAP antagonist verteporfin (VTPF) on the growth of ME180 cell in a 3D hanging-drop culture system. ME180 cells were incubated in the 3D hanging-drop culture system for 10 days in the absence or presence of AREG, AG1478 or verteporfin for 8 days. Scale bar: 1.0 mm.",
    "molecules": "AG1478, verteporfin, VTPF"
  },
  {
    "caption": "Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.",
    "molecules": "CHX, cycloheximide, MG132"
  },
  {
    "caption": "HPV16E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16E6, or CHX combined with HPV16E6 for 8 h. Treatment of starved HT3 cells with HPV16E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).",
    "molecules": "CHX"
  },
  {
    "caption": "Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group (red line) and EYFP group (blue line) in C48/80 model (C). Heatmap illustration of fluorescence changes during each corresponding scratching train in GCaMP6s group. n = 8 mice/group. 2 trails for each are shown except for 1 mouse where 1 trial was performed (D). *** P < 0.001. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group.",
    "molecules": "C48/80"
  },
  {
    "caption": "J, Effect of chemogenetic inhibition (J) of dopaminergicA11-SDH neurons on scratching behaviors in response to C48/80 (left) and CQ (right) stimuli. ** P < 0.01, *** P < 0.001. Two-way ANOVA followed by Bonferroni's test. n = 6-8 mice/group.",
    "molecules": "C48/80, CQ"
  },
  {
    "caption": "Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group (red line) and EYFP group (blue line) in DCP model (B).",
    "molecules": "DCP"
  },
  {
    "caption": "F, Effect of chemogenetic inhibition (F) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.",
    "molecules": "DCP"
  },
  {
    "caption": "G Effect of chemogenetic activation (G) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.",
    "molecules": "DCP"
  },
  {
    "caption": "A Effect of SCH23390 and sulpiride on scratching behaviors induced by C48/80 (left) and CQ (right). *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 7-8 mice/group.",
    "molecules": "SCH23390, C48/80, CQ, sulpiride"
  },
  {
    "caption": "C Effect of SCH23390 and sulpiride on DCP-induced scratching behaviors. * P < 0.05. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group.",
    "molecules": "SCH23390, DCP, sulpiride"
  },
  {
    "caption": "D The Drd1 mRNA level in the spinal cord after DCP or vehicle treatment. * P < 0.05. Student's t-test. n = 8 mice/group.",
    "molecules": "DCP"
  },
  {
    "caption": "G Statistical curve shows the firing frequency evoked by step current injection into DRD1+ neurons that incubated with vehicle or SKF38393. *** P < 0.001. Two-way ANOVA. n = 8 neurons/group.",
    "molecules": "SKF38393"
  },
  {
    "caption": "I Statistical shows the resting membrane potential in DRD1+ neurons incubated with vehicle or SKF38393. * P < 0.05. Paired Student's t-test. n = 8 neurons/group.",
    "molecules": "SKF38393"
  },
  {
    "caption": "I Effect of chemogenetic inhibition (left) or activation (right) of spinal DRD1+ neurons on the DCP-induced chronic itch behaviors. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.",
    "molecules": "DCP"
  },
  {
    "caption": "G Representative traces of the whole-cell voltage-clamp show the blue light (470 nm, 2 ms)-evoked EPSCs recorded in a GRPR+ neuron before (black) and after 20 μM DNQX perfusion (gray). H Representative traces show the effect of TTX alone (purple) and the combined presence of 4-AP (gray) superfusion on the blue light-evoked EPSCs (black) recorded in a GRPR+ neuron.",
    "molecules": "4-AP, DNQX, TTX"
  },
  {
    "caption": "I Representative traces show the effect of TTX + 4-AP (gray) on the blue light (470 nm, 2 ms)-evoked EPSCs (black) recorded in a GRPR+ neuron.",
    "molecules": "4-AP, TTX"
  },
  {
    "caption": "I Effect of DNQX, RC-3095, or vehicle on the spontaneous scratching of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO. ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.",
    "molecules": "CNO, DNQX, RC-3095"
  },
  {
    "caption": "J Effect of DNQX, RC-3095, or vehicle on the scratching behaviors of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO in C48/80 model (left) or CQ model (right). * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 6 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.",
    "molecules": "C48/80, CQ, CNO, DNQX, RC-3095"
  },
  {
    "caption": "A Coronal section of E15.5 wild-type (WT) and DKO developing cortices were co-stained with either anti-Sox2 (green) and anti-Pax6 (red) or with anti-Sox2 (green) and anti-Tbr2 (red) and both counter-stained with DAPI (blue). Scale bars are 50 µm. Quantitation of total DAPI+ cells and Sox2+, Pax6+ or Tbr2+ progenitor cells in the SVZ/VZ in the entire image, corresponding to an area of 100 μM x 100 μM are plotted.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, D Coronal sections of WT and DKO developing cortices stained with DAPI (blue) and co-stained with anti-Ctip2 (red) and Satb2 (green) at P2 and P7. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in P2 mice and Layers I-VI in P7 WT mice are indicated. Scale bars are 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C, E Quantitation of Ctip2+ and Satb2+ progenitor cells as a percent of total DAPI+ cells in each of 8 bins (marked on left) within the cortex (dashed lines) from a total area corresponding to 100 μM x 600 μM are plotted.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-D Wild type (WT), single (Ltk-/- and Alk-/-) and double (DKO) knockout embryos were injected with BrdU on day E14.5 and fixed cortices analyzed at E17.5, P2 and P7. A-C Coronal sections of BrdU labeled WT and mutant cortices at the indicated ages. Dotted lines mark the upper and lower cortical limits determined for each section. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in E17.5 and P2 mice and Layers I-VI in P7 WT mice are indicated. Scale bars are 50 µm. B, D Quantitation in E17.5 (B) or P2 and P7 (D) cortices of BrdU-positive cells in the indicated bins (marked on left of images) is plotted. Data are presented as the mean ± SEM from 3 (DKO, Ltk-/-) or 2 (WT, Alk-/-) independent biological experiments (B) or 3 (DKO and WT) independent biological experiments (D). Statistical significance: ****p<0.0001, ***p<0.001, ** p<0.005, *p<0.05 by Student's t-test.",
    "molecules": "BrdU"
  },
  {
    "caption": "D Representative images of WT cortical neurons, treated with the ALK inhibitors, TAE684 and crizotinib or DMSO as a control at 4 h after plating. Neurons were stained for Tau-1 (red) and MAP2 (green). Arrowheads mark axons. : Data are presented as percent of neurons with multiple, single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test.",
    "molecules": "crizotinib, DMSO, TAE684"
  },
  {
    "caption": "E Golgi stains of adult cortices in wild-type, single (Ltk-/- and Alk-/-) and double (DKO) knockout mice show severe defects in neuronal projections and the callosal tract. Representative coronal sections of cortical layers I-V in WT and DKO mice (left), Layers IV-V in WT, single and double knockout cortices (center) and higher magnification images of WT and DKO neurites (right). Examples of disrupted directional growth and extension of projections in pyramidal neurons in knockout mice are marked (red arrowheads).",
    "molecules": "Golgi"
  },
  {
    "caption": "Representative images of cortical neurons fixed at 38 h and stained with Tau-1 (axons, red) and MAP2 (dendrites, green) are shown. Arrowheads mark axons. C, D Treatment of cortical neurons from WT or KO mice with PI3 kinase inhibitors reverses polarity defects caused by loss of Ltk/Alk. Dissociated E16 cortical neurons were treated with LY294002 (10 µM; C), BKM120 (1 µM; D) or DMSO control, 4 h after plating. : In quantitation of the percent of neurons with multiple, single or no axons is plotted as the mean ± SEM of n=150 neurons per condition from , C-WT) or 4 (C-DKO independent biological experiments. Statistical significance: ****",
    "molecules": "BKM120, DMSO, LY294002"
  },
  {
    "caption": "A, B IGF-1R inhibitors rescue the multiple axon phenotype in DKO cortical neurons. Cortical neurons, isolated from WT and DKO mice were treated with 1 µM AG1024 (A), varying concentrations of Picropodophylin (PPP; B) or DMSO as control 4 h after plating, fixed at 38 h, and stained for Tau-1 (axons, red) and MAP2 (dendrites, green). Arrowheads mark axons. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single or no axons is plotted. Data are presented as the mean ± SEM, n ≥ 150 neurons per condition from 4 independent biological experiments.",
    "molecules": "AG1024, DMSO, Picropodophylin, PPP"
  },
  {
    "caption": "C Blocking Igf-1r activation with a neutralizing anti-IGF-1Rα antibody rescued the multiple axon phenotype in DKO cortical neurons. Neurons isolated from WT or DKO mice were treated anti-IGF-1Rα antibody (1.5 µg/ml), BSA as control (1.5 µg/ml), or PPP (200 nM) 4 h after plating. Representative images of Tau-1 and MAP2 stained cells with arrowheads marking axons are shown. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single or no axons is plotted.",
    "molecules": "PPP"
  },
  {
    "caption": "F Inhibition of Igf-1r blocks neuronal migration in vivo. Pregnant E14 WT or DKO mice were injected (i.p) with BrdU and 24 h later were injected with PPP or saline as control. Brains from P2 pups were subjected to staining with anti-BrdU antibody. Quantitation in P2 cortices of BrdU-positive cells in the indicated bins (marked on left) is plotted. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in P2 mice and are indicated. Scale bars are 50 µm. Data in (F) are presented as the mean ± SEM from 3 (DKO) and 2 (WT) independent biological experiments. Statistical significance: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test by Student's t-test (F), where WT vs WT+PPP or DKO vs DKO+PPP were compared.",
    "molecules": "BrdU, PPP, saline"
  },
  {
    "caption": "G Cell-surface expression of Igf-1r is increased in DKO cortical neurons. Cell-surface Igf-1r (NA ppt) is normalized to totals and the fold change relative to WT samples is plotted.",
    "molecules": "NA"
  },
  {
    "caption": "I WT cortical neurons were labeled with biotin and incubated at 37ºC for 15 or 30 min in the presence of CCM or ACM conditioned media. Cell-surface Igf-1r (NA ppt) normalized to totals as the fold change relative to controls at time 0 is plotted.",
    "molecules": "biotin, NA"
  },
  {
    "caption": "(E) In C. elegans, all conserved ciliary cluster genes with available mutants as well as genes of particular interest for which mutants were generated in the course of this study were screened using the dye-fill assay to test for cilium structural integrity. 12 amphid neurons in the head and 4 phasmid neurons in the tail, both featuring non-motile sensory cilia, stain with the lipophilic dye DiI. Failure to take up dye is an indicator of ciliary structural defects. IFT mutants such as osm-5(p813) tend to display dye-fill null phenotypes, while other ciliary mutants present weaker phenotypes. Phenotypes were scored using a scale from 0 (no dye-fill phenotype) to 4 (null).",
    "molecules": "DiI"
  },
  {
    "caption": "(A) Numbers of quantified O-glycopeptides identified with ETD fragmentation with either dimethyl labeling or TMT labeling approach in COSMC KO or WT background, respectively. (B) Numbers of differentially glycosylated peptides in the two approaches, using select filters on quantification values. ",
    "molecules": "TMT, dimethyl, glycosylated peptides, O-glycopeptides"
  },
  {
    "caption": "(C) Numbers of unambiguous unique differential O-glycosites identified with the two different approaches. The stacked bar graph differentiates between glycosites identified on peptides with single and multiple HexNAc/HexHexNAc modifications.",
    "molecules": "HexHexNAc, HexNAc, peptides"
  },
  {
    "caption": "(E) Analysis of differential glycosites identified with the TMT labeling approach using the Qluqore Omics Explorer software. The heatmap represents O-glycosites most significantly contributing to sample variation, using ANOVA with a q value cutoff of 0.1, where each compared sample consists of three individual KO clones. Each horizontal line depicts the median quantification of glycopeptides contributing to the same glycosite. Alternative differentially glycosylated site positions are separated by semicolons on sites derived from glycopeptides with multiple HexNAc/HexHexNAc modifications.",
    "molecules": "HexHexNAc, TMT, glycopeptides, HexNAc"
  },
  {
    "caption": "(L-N) Co-culture of HaCaT WT or GALNT3 KO keratinocytes with N/TERT1 keratinocytes induces cellular differentiation. Cells co-cultured on coverslips were stained for cleaved Notch 1 intracellular domain (L), cytokeratin 10 (M), or GalNAc-T3 (N; inserts depict 2x zoomed in partial images (scale bar - 20 μm)). Green - relevant staining, red - N/TERT1 cells (labeled with CMTMTR), blue - DAPI, scale bar - 20 μm. Boundaries between HaCaT and N/TERT-1 cells in the green panel of (L) are marked with a dashed line.",
    "molecules": "CMTMTR, DAPI"
  },
  {
    "caption": "(b) Flow cytometry-based stratification of cells in different cell cycle phases and in G0  (quiescence, 2n DNA- and low RNA  content), shown for SKOV3  cells in serum  starvation conditions at 8 and 96 hours, respectively. Cells were detached, fixed with ethanol  and stained  with Hoechst 33342 and Pyronin Y prior to flow cytometry  analysis.",
    "molecules": "Hoechst 33342, DNA, ethanol, Pyronin Y, RNA"
  },
  {
    "caption": "(d) Cell confluence  of adherent cell cultures in quiescence-inducing conditions (i.e. serum starvation  and glutamine limitation) was monitored using  time-lapse microscopy  in a plate reader (TECAN Spark 10M) in 1.5-hour intervals. Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach. Data points and shaded areas indicate mean ± standard deviation (SD) over three biological replicates. Approximately 9500, 4500, 19500, 6700, 19500 and 9500 cells/well were seeded for glutamine limitation  and serum starvation  of A549, CCD1070Sk, HCT116, HFL1, MCF7  and SKOV3  cells, respectively.",
    "molecules": "glutamine"
  },
  {
    "caption": "(b) Growth  phenotypic changes induced by TMZ  treatment in A549  and HCT116  cells grown in  full growth  medium (RPMI-1640 with 5% dialyzed FBS). Cell confluence  as a proxy of  cell numbers was monitored using automated time-lapse microscopy  in a plate reader (TECAN Spark 10M). Data points and shaded areas indicate mean ± SD over three biological replicates. (c) Growth  phenotypic changes induced by TMZ  treatment in quiescence-inducing conditions (serum starvation) in A549  and HCT116  cancer cells. Growth  was monitored via  cell confluence as described in panel b. At 96 hours, cells were stimulated to exit quiescence by replacing  serum-free media with  fresh growth  medium without TMZ  (regrowth). Data points and shaded areas indicate mean ± SD over three biological replicates.",
    "molecules": "TMZ"
  },
  {
    "caption": "(d) Viability   of quiescence-induced cells treated with TMZ, or with TMZ   plus 10 µM etomoxir   was assessed using a fluorescence imaging assay involving two fluorescent dyes: Hoechst 33342  (all nuclei) and  propidium iodide   (dead cells). Data points and error bars indicate mean ± SD over three biological replicates.",
    "molecules": "etomoxir, Hoechst 33342, propidium iodide, TMZ"
  },
  {
    "caption": "(e) Dynamic changes in the intracellular levels of citrate  in A549  and HCT116  cells induced to enter quiescence by serum  starvation with or without TMZ. Fold-changes were estimated relative to steady-state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates.",
    "molecules": "citrate, TMZ"
  },
  {
    "caption": "(f) Volcano plot showing TMZ-induced metabolic changes (1 mM, 48 hours) in A549  cells in serum  starvation Shown are differences in log2 fold-change between TMZ  treatment and serum  starvation alone (x-axis), and their statistical significance (y-axis, adjusted p-value, t-test and Benjamini-Hochberg correction).",
    "molecules": "TMZ"
  },
  {
    "caption": "(h) Comparison of relative changes (log2 fold-change) in the abundance of 1791 putatively annotated metabolites upon TMZ  treatment (1 mM, y-axis) against untreated quiescence-induced A549  cells (x-axis). Fold-changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations (Spearman, p < 0.01) with cell death at 96 hours are highlighted in blue and yellow, respectively.",
    "molecules": "TMZ"
  },
  {
    "caption": "(i) Fractions of early apoptotic cells in A549 or HCT116  cancer cell cultures in quiescence-inducing conditions (serum starvation, left panels) or full growth medium (5% dFBS, right panels), treated with TMZ  (0.4 or 1 mM), 5-fluorouracil  (5-FU, 0.5 mM, positive control) or medium  (untreated). Early apoptotic cells  were detected using ApoTracker Green under co-staining with  Hoechst 33342  (nuclei) and propidium iodide  (dead cells). Cell staining  was continuously monitored in 3-hours intervals for 96 hours using automated  fluorescence microscopy  in a TECAN Spark Cyto. Data points and shaded areas indicate mean ± SD over three biological replicates. Approximately 3400 and 7500 cells/well were seeded of A549  and HCT116  cells, respectively.",
    "molecules": "ApoTracker Green, Hoechst 33342, 5-fluorouracil, 5-FU, propidium iodide, TMZ"
  },
  {
    "caption": "Adhesion of control and GPR56 KD K562  cells on   fibronectin-functionalized substrates. Left: Overlay of phase contrast and microinterferometry images of control (above) and GPR56 KD cells (below). The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet, respectively. Right: Comparison of adhesion areas extracted by microinterferometry, reduction of adhesion area by factor 1.9 in GPR56 KD cells (p = 1.0 x 10-10, two-sided Mann-Whitney test, box plots showing medians, quartiles, and outliers according to the Tukey method). Technical replicates Ncontrol = 71, Technical replicates NKD = 104, scale bar 10 µm.",
    "molecules": "fibronectin"
  },
  {
    "caption": "Left: Representative  immunofluorescence (IF) images showing  primary  cilium formation in  RPE  cells upon starvation after infection with shLuc (above) or shGPR56strong (below). The  non-motile primary cilium  is visualized using antibodies against γ-tubulin, which stains the basal body, and the ciliary membrane marker ARL13B. DAPI indicates the  nucleus. Images on the right show selected cells at 3 x magnification. Brightness was increased in all images to enhance visibility of cilia. Right: Percentage of RPE-1  cells with primary cilium in shLuc vs. shGPR56strong RPE-1 cells at different time points after the start of serum starvation. Unpaired t-test, bars and error bars represent mean and stdev of three biological replicate wells. **** p<0.0001, *** p<0.0005, ** p<0.005, * p<0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "C, Immunostaining of endogenous Trnp1 and DAPI in P19 cells and E14 cortical cells at 1 day in vitro (div).",
    "molecules": "DAPI"
  },
  {
    "caption": "E, Representative phase contrast images of in vitro phase separation of the recombinant proteins YFP (upper row) or Trnp1 (lower row) at the indicated concentration in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT with the crowding agent dextran or RNA when depicted.",
    "molecules": "DTT, dextran, RNA, salt"
  },
  {
    "caption": "B, GFP and Trnp1 immunostaining with DAPI labelling in E14 cortical cells transfected for 24 h with plasmids (indicated by arrows) expressing the different truncated/mutant forms of Trnp1 and GFP showing Trnp1 localization.",
    "molecules": "DAPI"
  },
  {
    "caption": "D, WB showing mutCC Trnp1 and ∆1-16 Trnp1 protein turnover in P19 cells 24 h after transfection in the presence of cycloheximide (CHX) for the time indicated below in hours (h). GFP (half live:24h) was used as a loading control.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "F, Representative phase contrast images of in vitro phase separation of the recombinant ∆1-16Trnp1, ∆1−140Trnp1 and ∆95-223Trnp1 proteins at the indicated concentrations in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT plus Dextran or RNA when indicated. G, Violin Dot Plots illustrating quantification of the area of single droplets (N=700) formed by the recombinant Trnp1, ∆1-16Trnp1, ∆1−140Trnp1, ∆95-223Trnp1 proteins at 25 μM concentration in a representative experiment of three repetitions under the conditions shown in F. ",
    "molecules": "DTT, Dextran, RNA, salt"
  },
  {
    "caption": "E-G, Bar and dot plots of the proportion of proliferating (Ki67+) cells (E), the number of NSCs (Pax6+) (F) and the proportion of proliferating (BrdU+) TAPs (Tbr2+)(G) as in A-C showing a significant increase in proliferating NSCs upon Trnp1 IUE compared to control or ∆1-16Trnp1 proteins (E- F) and a significant decrease in proliferating TAPs upon Trnp1 IUE and increase upon ∆1-16Trnp1 IUE compared to control (G) at E15, 2 days after IUE.",
    "molecules": "BrdU"
  },
  {
    "caption": "D, STRING analysis of Trnp1 interactors The disconnected nodules are not shown. Proteins cluster have been color coded (green for chromatin association, red for RNA splicing, blue for proteins involved in nucleolus function and light yellow for the rest).",
    "molecules": "RNA"
  },
  {
    "caption": "G-H Translation assay using OPP-click reaction in P19 cells transfected for 24 hours with GFP, Trnp1-IRES-GFP or ∆1-16Trnp1-IRES-GFP plasmids and analyzed by immunofluorescence (G) or FACs sorting (H) . Graph in (G) is representative from three different biological replicates. Each dot represents a cell. Control=102; Trnp1=126; ∆1-16Trnp1=97. Graph in (H) shows analysis of 2,000 transfected cells from 1 representative experiment with 2 biological replicates for control and 3 biological replicates for Trnp1 and ∆1-16Trnp1 conditons. The same FACs sorting experiment was repeated two more times. The dotted vertical line shows the mean of the OPP click signal for each condition.",
    "molecules": "OPP"
  },
  {
    "caption": "B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (B) Representative bright-field images of spheroids treated with dmPGE2. Scale bars, 200 µm.",
    "molecules": "Iloprost, dmPGE2"
  },
  {
    "caption": "B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM dmPGE2 (average area was 3761 µm2 for 0 nM group; n = 4 images from two independent experiments).",
    "molecules": "Iloprost, dmPGE2"
  },
  {
    "caption": "B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (D) Representative histological sections of spheroids treated with 1 µM dmPGE2 or an equivalent volume of DMSO and stained for hematoxylin. Scale bars, 100 µm.",
    "molecules": "DMSO, Iloprost, dmPGE2"
  },
  {
    "caption": "B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (E) Quantification of the average expression ± s.e.m. of Cldn4mRNA relative to the 0 nM treatment group (n = 3 independent experiments).",
    "molecules": "Iloprost, dmPGE2"
  },
  {
    "caption": "B-F Jejunal spheroids were cultured in differentiation medium containing 0-1,000 nM dmPGE2 or Iloprost. (F) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with 0 nM Iloprost (n = 4 images from two independent experiments).",
    "molecules": "Iloprost, dmPGE2"
  },
  {
    "caption": "A Quantification of the average expression ± s.e.m. of Ptger1, Ptger2, Ptger3, Ptger4, and Ptgir mRNAs in whole-thickness lung, ileum or colon tissues or in jejunal spheroids cultured in stem cell (Stem), WAE (dmPGE2) or enterocyte (EP4i) medium (n = 3 independent experiments). Data are presented as fold change compared to lung tissue. n.d., not detected.",
    "molecules": "dmPGE2"
  },
  {
    "caption": "B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (B) Representative bright field images. Scale bars, 200 µm.",
    "molecules": "DMSO, L-798,106, L-161,982, PF 04418948, dmPGE2, SC 51322"
  },
  {
    "caption": "B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with DMSO alone (n = 4 images from two independent experiments).",
    "molecules": "DMSO, L-798,106, L-161,982, PF 04418948, dmPGE2, SC 51322"
  },
  {
    "caption": "B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (D) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to stem cellspheroids (n = 3 independent experiments).",
    "molecules": "DMSO, L-798,106, L-161,982, PF 04418948, dmPGE2, SC 51322"
  },
  {
    "caption": "G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (G) Representative bright-field images. Scale bars, 200 µm. (H) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from two independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.",
    "molecules": "dmPGE2"
  },
  {
    "caption": "G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (I) average expression ± s.e.m. of Cldn4 mRNA (n = 3 independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.",
    "molecules": "dmPGE2"
  },
  {
    "caption": "J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (J) Representative bright-field images. Scale bars, 200 µm. (K) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from 3 independent donor lines examined over 3 passages each) relative to stem spheroids.",
    "molecules": "dmPGE2"
  },
  {
    "caption": "J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (L) the average expression ± s.e.m. of CLDN4 mRNA (n = 3 independent donor lines) relative to stem spheroids.",
    "molecules": "dmPGE2"
  },
  {
    "caption": "E, F Spheroids were cultured as indicated for 24 hours followed by a 1-hr pulse with EdU to mark the cells undergoing DNA synthesis. (E) Representative images of EdU staining (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm. (F) Quantification of EdU-positive nuclei as a percent of the total nuclei (n = minimum of 15 spheroids counted per sample from 2 independent experiments). ****p<0.0001 by 1-way ANOVA and Tukey's post-test.",
    "molecules": "DNA"
  },
  {
    "caption": "G Graph of the fold change in background-subtracted luminescence ± s.e.m. (relative to 0 hr measurement) of Cdc25A-CBRLuc spheroids (n = 3 independent experiments with 4 technical replicates). ****p<0.0001 for dmPGE2- and EP4i-treated spheroids compared to stem cells by repeated measures 2-way ANOVA (variable = treatment). p<0.001 at the 16 hr, 20 hr and 24 hr time points by Dunnett's post-test comparing dmPGE2-treated and EP4i-treated spheroids to the stem cell control.",
    "molecules": "dmPGE2"
  },
  {
    "caption": "E, F Jejunal spheroids were cultured as in Figure 2B-D. Quantification of the average expression ± s.e.m. of Dpcr1 (E) and Cd55b mRNAs relative to DMSO group (n = 3 independent experiments). *p<0.05, ****p<0.0001 by one-way ANOVA and Dunnett's post-test.",
    "molecules": "DMSO"
  },
  {
    "caption": "A-D Quantification of the average expression ± s.e.m. of Dpcr1 and Cd55b mRNAs (A, C, E) or Fabp1 and Ace2 mRNAs (B, D, F) in jejunal spheroids cultured in differentiation medium containing EP4i or dmPGE2. (A, B) Gene expression analyzed 2, 6, 12 or 24 hours after the start of treatment (n = 3 independent experiments). ***p<0.001, ****p<0.0001 comparing the two treatment groups and †p<0.05, †††p<0.001, ††††p<0.0001 compared to the 2 hr time point of the same medium by 2-way ANOVA and Sidak's multiple comparisons test. (C, D) Gene expression was analyzed after culturing spheroids in EP4i (E) or dmPGE2 (P) for the first 12 hours followed by wash out and re-feeding with EP4i or dmPGE2 for the second 12 hours as shown (n = 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by one-way ANOVA and Dunnett's post-test.",
    "molecules": "dmPGE2"
  },
  {
    "caption": "A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (A) Representative bright-field images. Scale bars, 200 µm.",
    "molecules": "DMSO, Forskolin"
  },
  {
    "caption": "A, B Jejunal spheroids were cultured in differentiation medium containing 10 µM of Forskolin or an equivalent volume of DMSO. (B) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to the DMSO treatment group (n = 3 independent experiments). *p<0.05 compared to DMSO group by unpaired t test.",
    "molecules": "DMSO, Forskolin"
  },
  {
    "caption": "C, D Representative Western blots for β-catenin detected in nuclear (Nuc) and cytoplasmic (Cyt) protein lysates. Lamin A/C and Actin were used as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 3 independent experiments). (C) Spheroids were cultured in stem, enterocyte (EP4i) or WAE (dmPGE2) media. (D) Spheroids were cultured in enterocyte medium with DMSO or 10 µM CHIR 99021.",
    "molecules": "CHIR 99021, DMSO, dmPGE2"
  },
  {
    "caption": "E, F Spheroids were cultured in enterocyte medium with the indicated concentrations of CHIR 99021. Quantification of the average expression ± s.e.m. of Cldn4 (E) and Fabp1 (F) mRNAs shown as fold change relative to 0 µM group (n = 3 independent experiments). **p<0.01, ****p<0.0001 as determined by one-way ANOVA and Dunnett's post-test.",
    "molecules": "CHIR 99021"
  },
  {
    "caption": "G Representative images of Ptger4Δ/Δspheroids cultured in differentiation medium with DMSO or 10 µM CHIR 99021 and stained for β-catenin (green) and F-actin (red). Nuclei are visualized with bisbenzimide (blue). Scale bars, 20 µm.",
    "molecules": "CHIR 99021, DMSO"
  },
  {
    "caption": "H Graph of TOPflash (TOP) to FOPflash (FOP) luciferase reporter ratios ± s.e.m. (n = 3 independent experiments) in transfected 293FT cells treated with conditioned mediums collected from L-WRN cells and diluted as indicated. L-WRN CM had been produced in the presence or absence of the porcupine inhibitor C59 (10-fold dilutions, 10 µM to 1 pM). *p<0.05, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Dunnett's post-test compared to 0% L-WRN CM group.",
    "molecules": "C59"
  },
  {
    "caption": "I, J Spheroids were cultured in enterocyte media, WAE media or WAE media containing 100 pM C59. Quantification of the average expression ± s.e.m. of Cldn4 (I) and Fabp1 (J) mRNAs shown as fold change relative to EP4i group (n = 3 independent experiments). ***p<0.001, ****p<0.0001 as determined by one-way ANOVA and Tukey's post-test.",
    "molecules": "C59"
  },
  {
    "caption": "Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(D) Lapatinib antagonizes ERBB4-PIK3R1 signaling in a dose-dependent manner. Per 96-well, 40,000 split TEV assay cells were incubated with increasing amounts of lapatinib, followed by co-plating 10,000 Nrg1 type I β1a expressing cells. The inset depicts the IC 50 value in µM. After compound/stimulus addition, each assay was incubated for 24h. RLU, relative luciferase units; Fluc, firefly luciferase activity (black line); Rluc, Renilla luciferase activity (grey line); n=6; data are shown as mean, error bars represent SEM.",
    "molecules": "Lapatinib, lapatinib"
  },
  {
    "caption": "Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(B) Graphic visualisation of the primary screen data of the NIH-NCC library set 1. All counts (320 compounds and 64 controls) from the Nrg1-ERBB4-PIK3R1 split TEV compound screen were plotted against the Z-score using the Mondrian programme, with pathway activators displaying high values and inhibitors low values. For the secondary analysis, we selected all candidates that were at least three standard deviations away from the mean. EGFld positive and Lapatinib/CI-1033 negative controls are shown in red.",
    "molecules": "CI-1033, Lapatinib, Spironolactone"
  },
  {
    "caption": "Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type I (C) expressing PC12 cells.",
    "molecules": "Spironolactone, spironolactone"
  },
  {
    "caption": "Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen. (C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type III (D) expressing PC12 cells.",
    "molecules": "Spironolactone, spironolactone"
  },
  {
    "caption": "Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(E) Spironolactone inhibits ERBB4 receptor dimerization. Dose-dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4-NTEV-tevS-GV and ERBB4-CTEV plasmids transfected into PC12 cells. 10 ng/ml EGFld was applied as Nrg1 stimulus. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.",
    "molecules": "Spironolactone"
  },
  {
    "caption": "Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(A) Spironolactone, molecular structure shown left, inhibits ERBB4/PIK3R1 split TEV assay activity (black line), with an IC 50 value of 2.76 µM.",
    "molecules": "Spironolactone, spironolactone"
  },
  {
    "caption": "Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (B) canrenone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.",
    "molecules": "canrenone"
  },
  {
    "caption": "Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment. (B-D) The spironolactone metabolites(C) 7α-thiomethyl-spironolactone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.",
    "molecules": "7α-thiomethyl-spironolactone"
  },
  {
    "caption": "Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (D) eplerenone do not attenuate the ERBB4/PIK3R1 assay activity (black lines). Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.",
    "molecules": "eplerenone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(A) Spironolactone reduces ERBB4 levels. T-47D cells were stimulated with 10 ng/ml EGFld, 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated. Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284.",
    "molecules": "lapatinib, Spironolactone, spironolactone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(B) Quantification of band intensities for phospho-ERBB4 levels (n=4 per condition) shown in (A) using ImageJ. Phosphorylation levels are normalized to protein levels of ERBB4. Data shown as mean, error bars represent SD; t-test, with *, p=0.0356 for p-ERBB4 (Y1056), and **, p=0.0079 for p-ERBB4 (Y1284).",
    "molecules": "Spironolactone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(D) Spironolactone reduces phospho-Erbb4 levels in Nrg1-tg mice. Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis. Lysates were probed with indicated antibodies.",
    "molecules": "Spironolactone, spironolactone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (E) Quantification of band intensities for phospho-Erbb4 and phospho-Limk1 levels shown in (D) using ImageJ. Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1. Data shown as mean, error bars represent SD; t-test, with *, p=0.0330 for p-Erbb4, and p=0.0201 for p-Limk1.",
    "molecules": "Spironolactone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (F) Canrenone (applied as 10 µM) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex (PFC). Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of canrenone.",
    "molecules": "canrenone, Canrenone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(G) Spironolactone (applied as 10 µM) significantly increases frequencies (n=12; p=0.0454) and amplitudes (n=12, p=0.0478) of sIPSCs in pyramidal neurons of PFC. Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of spironolactone.",
    "molecules": "Spironolactone, spironolactone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(H) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0286). Spironolactone (5 µM) was applied in the presence of canrenone (10 µM).For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.",
    "molecules": "canrenone, Spironolactone, spironolactone"
  },
  {
    "caption": "Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(I) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0124). Lapatinib was applied in 5 µM. For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.",
    "molecules": "Lapatinib, lapatinib"
  },
  {
    "caption": "Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(B) Nrg1-tg mice travelled longer distances in the open field arena (effect of genotype F (1,44) =10.53; p=0.0022; 2-way ANOVA). Bonferroni post-hoc analysis revealed a significant genotype-dependent difference between vehicle-treated groups (p=0.0044) but not in spironolactone-treated groups (p=0.3783). Genotype differences were abolished upon spironolactone treatment.",
    "molecules": "spironolactone"
  },
  {
    "caption": "Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(D) There was no significant difference between the genotypes when treated with spironolactone (effect of genotype F (1,22) =2.19; p=0.1535; 2-way ANOVA). However, the interaction of genotype and treatment was significant (F (9,198) =1.94; p=0.0481; 2-way ANOVA).",
    "molecules": "spironolactone"
  },
  {
    "caption": "Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(E) Nrg1-tg mice treated with spironolactone spent more time in the light compartment during the light-dark test (interaction gene × treatment F (1,41) =4.90; p=0.0324; 2-way ANOVA, and p=0.0219, Bonferroni post-test).",
    "molecules": "spironolactone"
  },
  {
    "caption": "Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice. (F) In the Y-maze test, transgenic mice performed less alterations (effect of genotype F (1,44) =11.50; p=0.0015; 2-way ANOVA). The Bonferroni test confirmed this phenotype in vehicle-treated groups (p=0.0011), but not in spironolactone-treated animals (p=0.5950). Spironolactone treatment had a significant effect on the number of alterations (F (1,44) =4.12; p=0.0484; 2-way ANOVA).",
    "molecules": "spironolactone, Spironolactone"
  },
  {
    "caption": "Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(G) Spironolactone treatment significantly enhanced PPI in Nrg1-tg mice (effect of treatment F (1,21) =5.07; p=0.0325; 2-way repeated measures ANOVA). Data are shown as mean, error bars represent SEM. *, p<0.05, **, p<0.01 and ***, p<0.001, Bonferroni test following 2-way ANOVA; Spiro, spironolactone; Veh, vehicle. n=12 per genotype and treatment with an exception of (E) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=10; wt vehicle, n=12; wt Spiro, n=12) and (G) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=12; wt vehicle, n=12; wt Spiro, n=12).",
    "molecules": "spironolactone, Spironolactone"
  },
  {
    "caption": "Gata6-WT and Gata6-KOmye peritoneal macrophages (pMɸ) were unstimulated (-) or stimulated with TLR2L Pam3CSK4 (500 ng.ml-1), TLR3L Poly I:C (1 µg.ml-1), TLR4L purified LPS (100 ng.ml-1), TLR5L Flagellin (100 ng.ml-1), TLR7 and 8L R848 (1 µg.ml-1) or TLR9L CpG ODN1826 (5 µM). Culture supernatants were collected 24 h after the start of stimulation and IL-1β and TNF ELISA were performed. n=5, two-way ANOVA analysis with Tukeys multiple comparison post-test.",
    "molecules": "CpG ODN1826, Flagellin, LPS, Pam3CSK4, Poly I:C, R848"
  },
  {
    "caption": "IL-1β (C) and TNF (D) ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 24 h with the indicated LPS concentrations. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β (E) and TNF (F) ELISA from Gata6-WT and Gata6-KOmye pMɸ stimulated with LPS (100 ng.ml-1) for the indicated times. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or recombinant IL-1 receptor antagonist (rIL-1ra), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or 100 ng.ml-1 recombinant TNF (recTNF), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.",
    "molecules": "LPS"
  },
  {
    "caption": "Il1b and Tnf mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β Western blot protein analysis (D) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.",
    "molecules": "LPS"
  },
  {
    "caption": "Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro-IL-1β+ Gata6-WT and Gata6-KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng.ml-1 LPS. n = at least 3 independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Nlrp3 mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h. Data shown are pooled from 3 independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Western blot protein analysis of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 6 h. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β ELISA of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5). Data shown in (H) are pooled from 5 independent replicates.",
    "molecules": "DMSO, LPS, MCC950"
  },
  {
    "caption": "Western blot protein analysis (I) of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5).",
    "molecules": "DMSO, LPS, MCC950"
  },
  {
    "caption": "Caspase1 (Casp1) mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Western blot protein analysis (K) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or Ac-YVAD-cmk for 24 h. Data shown are pooled of 5 independent replicates.",
    "molecules": "Ac-YVAD-cmk, DMSO, LPS"
  },
  {
    "caption": "IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 µM Nigericin. Data shown are pooled of 5 independent replicates.",
    "molecules": "ATP, LPS, Nigericin"
  },
  {
    "caption": "Representative picture of confocal immunofluorescence analysis of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 µm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ in monoculture or co-cultured (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ co-cultured in the same well (co-culture) or using Transwell system (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.",
    "molecules": "LPS"
  },
  {
    "caption": "Mass spectrometry analysis of 6-keto-PGF1α, TXB2 and PGE2 content of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated for 3 h with 100 ng.ml-1 LPS. n = 6. Two-way ANOVA statistical analysis with Tukey's multiple comparison post-test was performed.",
    "molecules": "6-keto-PGF1α, LPS, PGE2, TXB2"
  },
  {
    "caption": "IL-10 ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS. Data shown are representative of at least 5 independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-10 (B) and IL-1β (C) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, beraprost (1 and 10 µM), cicaprost (1 and 10 µM), Iloprost (1 or 10 nM) or vehicle control (Vh, DMSO). Data shown are representative of at least 3 independent experiments.",
    "molecules": "beraprost, cicaprost, DMSO, Iloprost, LPS"
  },
  {
    "caption": "IL-10 (D) and IL-1β (E) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 10 µM PGE2, 10 µM U46619, 100 µM picotamide or vehicle control (Vh, methyl acetate). Data shown are representative of at least 3 independent experiments.",
    "molecules": "picotamide, beraprost, LPS, methyl acetate, PGE2, U46619"
  },
  {
    "caption": "IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10, 10 µM beraprost or vehicle control (Vh). Data shown are representative of at least 3 independent experiments.",
    "molecules": "beraprost, LPS"
  },
  {
    "caption": "IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 5 µg.ml-1 αIL-10R or isotype antibody. Data shown are representative of at least 3 independent experiments.",
    "molecules": "beraprost, LPS"
  },
  {
    "caption": "IL-1β (H) and IL-10 (I) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM MCC950, 5 µg.ml-1 αIL-10R or isotype antibody All stimulations were performed for 16 h. Data shown are representative of at least 3 independent experiments.",
    "molecules": "LPS, MCC950"
  },
  {
    "caption": "IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10 and 5 mM ATP. Data shown are a pool of 3 independent experiments.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "Flow cytometry analysis of Mitotracker (MT) Green and Red integration in Gata6-WT and Gata6-KOmye naïve pMɸ (gated on F4/80hi/+ Tim4+) in vivo, 30 min after intraperitoneal injection (i.p.) of 1 µM of Mitotracker Green and Red. Data shown are representative of 3 to 5 independent mice of each genotype and are expressed as mean +/- SEM. Student's t test analysis was performed.",
    "molecules": "Mitotracker (MT) Green, Mitotracker Green"
  },
  {
    "caption": "Flow cytometry analysis of Mitotracker green and red staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1), αIL-10R (5 µg.ml-1) or isotype (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 3 independent experiments and normalized to WT unstimulated.",
    "molecules": "LPS, Mitotracker green"
  },
  {
    "caption": "Flow cytometry analysis of MitoSOX staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1) and αIL-10R (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 2 independent experiments and normalized to WT unstimulated.",
    "molecules": "MitoSOX, LPS"
  },
  {
    "caption": "IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 500 µM MitoTempo, 10 mM NAC, 0.5 µM MitoQ for 16 h. Data are representative of at least 3 experiments.",
    "molecules": "LPS, MitoQ, MitoTempo, NAC"
  },
  {
    "caption": "mRNA expression analysis of Il1b, Nlrp3 and Caspase1 (Casp1) of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. Data are representative of at least 3 independent experiments.",
    "molecules": "beraprost, LPS"
  },
  {
    "caption": "Western blot analysis (left) and quantification (right) of Gata6-WT and Gata6-KOmye pMФ stimulated for 6 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of 3 independent experiments.",
    "molecules": "beraprost, LPS"
  },
  {
    "caption": "Caspase1 activity analysis of Gata6-WT and Gata6-KOmye pMФ stimulated for 16 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. n = 5 to 8 individual mice.",
    "molecules": "beraprost, LPS"
  },
  {
    "caption": "Mean fluorescence intensity (MFI) of pro-IL-1β of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype for 16 h or freshly isolated. n = 6 to 13 individual mice. Data were log transformed before performing statistical analysis.",
    "molecules": "beraprost, LPS"
  },
  {
    "caption": "mRNA expression analysis of Il10 and Tnf of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of at least 3 independent experiments.",
    "molecules": "beraprost, LPS"
  },
  {
    "caption": "E-F, Candidate gene regions with neurogenesis-associated de novo methylation (E) and demethylation (F). Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions. Stars indicate significantly differentially methylated CpGs with q < 0.05 (logistic regression with multiple testing correction using SLIM method) and methylation difference greater than 20 %.",
    "molecules": "CpGs"
  },
  {
    "caption": "D, Dnmt3a/b-KO did not affect NPC proliferation. Depicted are data points for every sample with genotype means ± standard errors of the mean (SEM). E, Reduced percentage of dead cells (Annexin V/propidium iodide (PI) double-positive) in KO cultures at 46 h after start of differentiation. Depicted are data points for every sample with genotype means ± SEM. ",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).",
    "molecules": "CpG"
  },
  {
    "caption": "A, No difference in numbers of Ki67-positive, proliferating cells was observed between the subgranular zone (SGZ) of Dnmt3a/b-WT and Dnmt3a/b-KO mice one week after tamoxifen administration. Depicted are data points for every animal with genotype means ± SEM. B, Bright field image of Ki67-positive cells stained using diaminobenzidine method. Scale bar: 100 µm. ",
    "molecules": "diaminobenzidine, tamoxifen"
  },
  {
    "caption": "C, KO of Dnmt3a/b did not affect numbers of new-born neurons in the dentate gyrus. Mice were administered with tamoxifen and analyzed 5 weeks after the first injection. BrdU was injected 3.5 weeks before analysis. Depicted are data points for every animal with genotype means ± SEM. D, Fluorescence image of BrdU/NeuN-positive new-born neurons. Scale bar: 25 µm. ",
    "molecules": "BrdU, tamoxifen"
  },
  {
    "caption": "A-B, Quantification of adult-born (BrdU-positive) cells in the dentage gyrus (DG) of Dnmt3a/b-KO and WT mice after five weeks or three months of environmental enrichment (ENR) or standard housing (STD). BrdU was injected 4.5 weeks before analysis. Depicted p-values correspond to genotype effects from two-way ANOVA. Influences of ENR were significant with p < 0.01. Depicted are data points for every animal with group means ± SEM.",
    "molecules": "BrdU"
  },
  {
    "caption": "G, Percentage of new-born neurons (BrdU/NeuN-positive) that expressed c-Fos after ENR (n = 5 mice, WT; n = 5 mice, KO). H, Representative fluorescent image of c-Fos expression in BrdU/NeuN-positive cells in the DG. Scale bar: 25 µm. ",
    "molecules": "BrdU"
  },
  {
    "caption": "B SDS-PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−).",
    "molecules": "LiCl"
  },
  {
    "caption": "C B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM.",
    "molecules": "erythromycin"
  },
  {
    "caption": "Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Passive transepithelial absorption of FITC‐dextran (B), was used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.",
    "molecules": "dextran"
  },
  {
    "caption": "WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.A Colitis severity was determined in part by weight loss. n = 5 mice/group.",
    "molecules": "DSS"
  },
  {
    "caption": "WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.B, C Colitis scores based on histopathology and gross morphology of the colons were also used as measures of disease. Scale bar = 50 μm. n = 5 mice/group. Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.",
    "molecules": "DSS"
  },
  {
    "caption": "WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.D Colonoscopies were performed in the different groups with a Multi‐Purpose Rigid™ Telescope attached to a TELE PACK X on day 10.",
    "molecules": "DSS"
  },
  {
    "caption": "WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.E Mean relative colonic expression of tight junction‐associated genes in WT mice. n = 5 mice/group.",
    "molecules": "DSS"
  },
  {
    "caption": "WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.F Fecalalbumin levels in WT mice as a measure of intestinal permeability. n = 5 mice/group.",
    "molecules": "DSS"
  },
  {
    "caption": "Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.A Representative plots indicate the frequency of neutrophils in the colons of untreated or DSS‐treated WT (left) and KO mice (right). Empty bars = WT; lined bars = KO; white bars = untreated; purple bars = DSS; red bars = DSS + NCK56; green bars = DSS + NCK2187; blue bars = DSS + SlpA.",
    "molecules": "DSS"
  },
  {
    "caption": "Signr3+/+ (WT) or Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −1 and −3, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times, and immunity was analyzed by flow cytometry at day 10.B, C Colonic DCs and macrophages (MΦs) were analyzed by flow cytometry for the production of IL‐1β (B), and colonic FoxP3+ Tregs were evaluated for co‐expression of RORγt+ (C). n = 5 mice/group. Gray tinted line = isotype control; black = untreated; purple = DSS; red = DSS + NCK56; green = DSS + NCK2187; blue = DSS + SlpA.",
    "molecules": "DSS"
  },
  {
    "caption": "A. Representative low-pass filtered cryo-EM micrographs of MTs decorated with wild-type DCX (WT) or NDC-NDC construct (NN) after 30 sec or 1 h incubation at 37 °C. Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration. The final ratio of WT or NN to tubulin was 50:5 μM, giving dense protein background. WT co-polymerized exclusively 13-PF MTs, while NN both 13- and 14-PF MTs. Rapid (30 sec) MT polymerization in excess of WT produces MTs in an intermediate GTP/GDP.Pi state, whereas long-lived WT-MTs and NN-MTs are all in the GDP state.",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "(G, H) Jacob shRNA knockdown prevents Aβ-induced CREB shutoff. (G) Representative confocal images of hippocampal neurons transfected with Jacob-shRNA or scrambled (scr) shRNA control (both expressing GFP) and treated with oligomeric preparations of Aβ1-42 or Aβ3(pE)-42. (H) Neurons transfected with a Jacob knockdown construct did not display reduction of pCREB immunofluorescence intensity after treatment with Aβ1-42 or Aβ3(pE)-42. Bar plot of mean nuclear pCREB intensity normalized to untreated control. Scale bar: 10 µm. N=29-39 nuclei from 2 independent experiments.",
    "molecules": "Aβ3(pE)-42, Aβ, Aβ1-42"
  },
  {
    "caption": "(J) Representative confocal images of slices immunolabeled against pCREB, co-labeled with NeuN and DAPI. Scale bar: 100 µm. (K) Bar plot of pCREB, N=17-21 slices.",
    "molecules": "DAPI"
  },
  {
    "caption": "(L, M) The quantification of pCREB intensity in NeuN-positive cells revealed a statistically significant decrease in pCREB immunoreactivity in TBA2.1 but not in double transgenic animals (TBA2.1, -/-). (L) Representative confocal images of CA1 cryosections from 13 weeks old mice stained for NeuN, DAPI and pCREB. Scale bar: 100 µm. Data represented as cumulative frequency distribution. (M) Bar plot of average hippocampal pCREB nuclear immunoreactivity normalized to WT. N=34-38 hippocampal sections from 10-11 animals.",
    "molecules": "DAPI"
  },
  {
    "caption": "Treatment of hippocampal primary neurons expressing phosphodeficient Jacob in the nucleus with OA rescues Jacob-induced CREB shutoff. Confocal images of pCREB immunostaining in DIV15 neurons overexpressing ΔMyr-Jacob-S180A-GFP with and without OA treatment. Scale bar: 20 μm. Lookup table indicates the pixel intensities from 0 to 255. N=14-17 nuclei analyzed from two independent cell cultures.",
    "molecules": "OA"
  },
  {
    "caption": "(K) Treatment with staurosporine decreases Jacob phosphorylation level (S180), but increases its association with LMO4-tagRFP. Immunoblot of HEK293T cells extracts transfected with LMO4-tagRFP and ΔMyr-Jacob-GFP or GFP alone. (L) Treatment with staurosporine decreases the association of Jacob with CREB. Immunoblot of HEK293T cells extract transfected with CREB-tagRFP and ΔMyr-Jacob-GFP or GFP as a control. (",
    "molecules": "staurosporine"
  },
  {
    "caption": "Nitarsone disrupts binding of Jacob to LMO4 in a concentration-dependent manner. (H) MBP immunoreactivity normalized to input. N=6-12 independent experiments.",
    "molecules": "Nitarsone"
  },
  {
    "caption": "(N, O) (N) 5 µM Nitarsone reduced Jacob-LMO4 complex formation in Aβ-treated (500 nM) primary neurons as revealed by proximity ligation assay. Scale bar: 5 µm. (O) Bar plot of normalized PLA signal intensity. N=20-24 neuronal nuclei from 3 cultures.",
    "molecules": "Aβ, Nitarsone"
  },
  {
    "caption": "(A, B) Co-application of Nitarsone prevents Aβ-induced CREB shutoff. DIV16 hippocampal cultures were treated with 5 μM Nitarsone, 500 nM Aβ1-42, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h and stained for pCREB, MAP2, and DAPI. (A) Representative confocal images. Scale bar: 10 µm. (B) Nuclear pCREB immunoreactivity normalized to control. N=63-67 nuclei from three independent cultures.",
    "molecules": "Aβ, Aβ1-42, DAPI, Nitarsone"
  },
  {
    "caption": "(E, F) Treatment with Nitarsone rescues Aβ1-42-induced decrease of synaptic GluR1-immunoreactivity within Shank3. DIV16 dissociated, hippocampal cultures were treated with 5 μM Nitarsone, 500 nM Aβ1-42, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h and stained for Shank3, surface GluR1, and MAP2. (E) Representative confocal images of dendritic segments. Scale bar: 5 µm. (F) GluR1-immunoreactivity within Shank3 signal. N=39-61 of dendritic segments from four independent cell cultures.",
    "molecules": "Aβ1-42, Nitarsone"
  },
  {
    "caption": "Nitarsone administration rescues mEPSC amplitude. (G) Analog traces of mEPSCs recorded in DIV16 hippocampal neurons treatd with 500 nM Aβ1-42, 5 μM Nitarsone, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h.",
    "molecules": "Aβ1-42, Nitarsone"
  },
  {
    "caption": "(B, C) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of TBA2.1 mice. (B) Representative confocal images of cryosections from 11 weeks old mice stained for NeuN, DAPI, and pCREB. Scale bar: 10 µm (C) Bar plot of pCREB nuclear staining intensity. N=21-34 hippocampal sections from 6-9 animals.",
    "molecules": "DAPI, Nitarsone"
  },
  {
    "caption": "(D, E) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of 5xFAD mice. (D) Representative confocal images of CA1 cryosections from 18 weeks old mice stained for NeuN, DAPI, and pCREB. Scale bar: 10 µm. (E) Cumulative frequency distribution of pCREB nuclear staining intensity. N=22-27 hippocampal sections from 6-7 animals.",
    "molecules": "DAPI, Nitarsone"
  },
  {
    "caption": "(K, L) Nitarsone reduces number of varicosities in SLM of CA1 of TBA2.1 mice. (K) Representative, confocal images of dendrites filled with biocytin stained from 13 weeks old mice. Arrows indicate varicosities. Scale bar: 1 μm. (L) Number of dendritic swellings per 10 μm. N=19-22 dendrites from 2 animals per genotype.",
    "molecules": "biocytin, Nitarsone"
  },
  {
    "caption": "(A) Nitarsone rescues late CA1-LTP impairment in TBA2.1 mice. Insets show representative fEPSPs analog traces at indicated time points: 1 = baseline, 2 = late LTP. (B) Averaged fEPSP slopes recorded during the last 30 min. N=14-18 slices from 5-6 mice. (C) Nitarsone rescues late CA1-LTP impairment in 5xFAD mice. Insets show representative fEPSPs analog traces at indicated time points: 1 = baseline, 2 = late LTP. (D) Averaged fEPSP slopes recorded during the last 30 min. N=17-18 slices from 6 mice.",
    "molecules": "Nitarsone"
  },
  {
    "caption": "(H-J) Nitarsone rescues short-term memory impairment in Y-maze object recognition task in (I) TBA2.1 N=9-14 and (J) 5xFAD mice. N=9-11",
    "molecules": "Nitarsone"
  },
  {
    "caption": "F. Two-photon live imaging of vessel perfusion using FITC-dextran IV injection in ROSAmT/mG mice (300µm depth stack).",
    "molecules": "dextran IV, FITC"
  },
  {
    "caption": "E. MRC1 immunohistochemistry on 3 weeks growth glioma in ROSAmTmG::Pdgfb-iCre mouse following MHCII-FITC labeled IV injection at 6 or 24h (5µm depth stack). Note the segregation of MRC1 and MHCII cell labeling at 6 hours and the overlapping of these two markers at 24 hours (n=5 mice per group). Statistical analysis: E. t-test. Error bars: E: 50µm.",
    "molecules": "FITC"
  },
  {
    "caption": "E. Two-photon live imaging of vessel perfusion in 4 weeks implanted glioma growth using FITC-dextran IV injection in ROSAmT/mG mice treated with anti-CSF1 or control antibodies. Scale bars: A-B-E: 150µm.",
    "molecules": "dextran IV, FITC"
  },
  {
    "caption": "Anti-CSF1 antibody in combination with temozolomide chemotherapeutic agent induces a wider tumor cell death efficiency with a significant decrease of blood vessel caliber (D.) (50µm depth stack) (n=6 mice per group). Statistical analysis: D.E. one-way ANOVA followed by multiple comparisons Tukey's test. Error bars: meanSD. Scale bars: 300µm",
    "molecules": "temozolomide"
  },
  {
    "caption": "Anti-CSF1 antibody in combination with temozolomide chemotherapeutic agent induces a wider tumor cell death efficiency with an increase in caspase3 positive tumor cells (E.) (50µm depth stack) (n=6 mice per group). Statistical analysis: D.E. one-way ANOVA followed by multiple comparisons Tukey's test. Error bars: meanSD. Scale bars: 300µm",
    "molecules": "temozolomide"
  },
  {
    "caption": "Ten-fold serial dilutions of 1 OD600 unit/mL of the indicated strains were spotted on SD plates containing 0 (C-) or 1 mM  choline (C+) and 0 (I-) or 75 μM inositol (I+) and incubated at 30oC for 3 d. A representative experiment is shown (from n=5).",
    "molecules": "choline, inositol"
  },
  {
    "caption": "Membrane lipid and TAG content, and ergosterolester content (EE, inset) per OD600 unit of the yeast  strains indicated after culture to mid-log phase  in SD with or without 1 mM  choline (C); * p < 0.05, ** p < 0.01, unpaired two-tailed t-test of the indicated bar compared to the C+ condition. Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the  cho2opi3 parent unless indicated otherwise.",
    "molecules": "choline, EE, ergosterolester, lipid, TAG"
  },
  {
    "caption": "Generation of suppressors of choline auxotrophy of  cho2opi3, cho2opi3lro1, and cho2opi3lro1 pLRO1 on choline-free medium as indicated at 30 oC for 7 d.",
    "molecules": "choline"
  },
  {
    "caption": "Growth (30 oC for 6 d) and phospholipid composition of co acc1N/H lro1  pLRO1  cultured in SD C- ura-, containing glucose/galactose mixtures (2%, w/v) as carbon source with the percentage of galactose indicated. Phospholipid composition analyzed by TLC is presented as mean percentage of total phospholipid of 3 biological replicates with the individual values indicated.",
    "molecules": "galactose, glucose, phospholipid, Phospholipid, ura"
  },
  {
    "caption": "(F-G) EM  analysis of cho2opi3 cells that after preculture in SD C+, were transferred to OD600 0.05 and subsequently cultured to  mid-log phase  in SD C+ (F) or SD C- (G) both containing 0.05 μg/mL SorA. CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 500 nm.",
    "molecules": "SorA"
  },
  {
    "caption": "Growth  of co S(2n-1) in the absence of choline is lost when GPT2 or DGA1  is overexpressed, and restored by SorA. Plasmids pHEYg-1-DGA1, pHEYg-1-GPT2 and the empty vector control (pEV) were transformed into the strains indicated. After preculture in SD C+, ten-fold serial dilutions were spotted on SD C+/- with or without 0.1 μg/mL  SorA  and incubated at 30 oC for 3 d.",
    "molecules": "choline, SorA"
  },
  {
    "caption": "Intensity encoded GP-images of the indicated C-Laurdan-stained yeast strains cultured with or without choline, accompanied by the corresponding intensity weighted GP-histogram fitted to a single Gaussian function (light blue line), and transmission image. White arrows point to  plasma membranes exhibiting higher lipid packing than internal, organellar membranes; blue horizontal arrows indicate highly packed lipid  material in co S(2n-1). To deplete the cho2opi3 parent of PC, cells were transferred to SD C- at OD600 0.1, and cultured into mid-log phase. White scale bars correspond to 10 µm; red scale bars in transmission images correspond to 5 µm.",
    "molecules": "choline, C-Laurdan, lipid, PC"
  },
  {
    "caption": "G. Representative time-lapse of cytosolic GCaMP6f before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressed with mRFP and pSuper empty vector or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.",
    "molecules": "ionomycin"
  },
  {
    "caption": "H. Basal GCaMP6f fluorescence (F) normalized to the maximum GCaMP6f fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. I. Average basal GCaMP6f fluorescence (F0) normalized to the max GCaMP6f fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N=2, n=47-50. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.",
    "molecules": "ionomycin"
  },
  {
    "caption": "J. Representative time-lapse of cytosolic R-GECO1 before (F0) and after (Fmax) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. Arrowheads mark presynaptic boutons. Scale bar: 5 µm.",
    "molecules": "ionomycin"
  },
  {
    "caption": "K. Basal R-GECO1 fluorescence (F) normalized to the maximum R-GECO1 fluorescence (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. L. Average basal R-GECO1 fluorescence (F0) normalized to the maximum R-GECO1 fluorescence intensity (Fmax) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1 or GFP-SCRN1-F402A. N=3, n=70-89. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.",
    "molecules": "ionomycin"
  },
  {
    "caption": "B, In vitro bactericidal action of the CP1-WT peptide on E. coli  Top10 cells. B Growth  profile of E. coli Top10  in the presence of increasing concentrations of the CP1-WT peptide. Data information: data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panels B data are presented as mean ± SD values obtained from all the replicates. In panel B, N.T. - No treatment; 4.5% DMSO  (solvent control); CP1-WT (test peptide); CP1-Mut (negative control peptide) and Cip  - 10µg/mL ciprofloxacin  (positive control).",
    "molecules": "Cip, ciprofloxacin, DMSO"
  },
  {
    "caption": "A-D Peptide-induced membrane  damage was studied by confocal microscopy and measured by  propidium iodide  (PI) influx (% PI  uptake) in (A) S. Typhimurium, (B)  A. baumannii, (C) S. aureus and (D) P. aeruginosa. Data information: In panels A-D, n = 5 biological replicates; scale bars: 5µm. Representative images are shown. In the bar graphs, data are presented as mean ± SD values of percentage of  propidium iodide uptake (% PI  uptake), averaged across all the replicates. 3%, 7%, 8% and 3% DMSO, and 80µM, 50µM, 50µM and 80µM of the peptides in the respective DMSO  concentrations were used to treat S. Typhimurium, A. baumannii, S. aureus  and  P. aeruginosa respectively.",
    "molecules": "DMSO, PI, propidium iodide"
  },
  {
    "caption": "E-H Minimum Inhibitory Concentration (MIC) and  Minimum Bactericidal Dose (MBD) estimation of CP1-WT on stationary phase cells of (E) S. Typhimurium, (F)  A. baumannii, (G) S. aureus and (H) P. aeruginosa. No bactericidal action is observed for P. aeruginosa. In each of the panels and its corresponding bar graph, the black lines and black bars represent the MIC  data indicated by their OD600 values, and the red lines and red bars represent the MBD  data indicated by their CFU/mL values. The bar graphs in each panel depict control reactions used for each strain where the cells were treated with DMSO, peptides and ciprofloxacin  (Cip  - postive control; 10µg/mL). The strain of A. baumannii  used was reported to be ciprofloxacin-resistant (Nagarajan et al, 2018) as is observed here (inset in panel F). In panels E-H, n = 5 biological replicates. Data are presented as mean ± SD values, averaged across all the replicates. For the bar graphs in these panels, 7%, 13%, 8% and 4% DMSO, and 80µM, 100µM, 100µM and 50µM of the peptides, were used for S. Typhimurium, A. baumannii, S. aureus  and  P. aeruginosa respectively. N.T- No treatment; WT - CP1-WT peptide; Mut - CP1-Mut peptide (negative control). P values are calculated by Student's unpaired t-test; nsP > 0.05; *** P < 0.001; **** P < 0.0001 Student's unpaired t-test.",
    "molecules": "Cip, ciprofloxacin, DMSO"
  },
  {
    "caption": "A-D Transmission electron micrographs  of the indicated bacterial strains (left) depicting the loss of membrane  integrity upon treatment with the CP1-WT peptide which is time-dependent. Arrows indicate membrane blebs, generalized membrane  damage and the exclusion of cytoplasmic  contents out of the cell. 1h, 2h and 3h indicate different treatment times  of 1, 2 and 3 hours respectively with the CP1-WT and CP1-Mut peptides as indicated in the panels. Untreated cells, used as controls, are also shown. Scale bars: 2μm. Data information: In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO  or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.",
    "molecules": "DMSO"
  },
  {
    "caption": "E-H Mean fluorescence intensities across the three fluorescent channels (FM 4-64FX, FAM  and DAPI  fluorophores) were estimated from  confocal microscopy experiments to understand if the peptide was bound to the membrane  or internalized in the cytosol of the bacteria indicated in each of the panels (top). Triple staining  procedure with FM 4-64FX (membrane-binding  dye; red), FAM-conjugated peptides (green) and DAPI  (nuclear staining  dye; blue) was used and the mean fluorescence intensities across these three fluorescent channels were calculated using ZEN 2.3 software. The bacterial cells were treated with 2% DMSO/ FAM-labelled CP1-WT/ FAM-labelled CP1-Mut for 0 (0hr) or 2 hours (2hr), followed by staining  with FM 4-64FX  and DAPI  consecutively. The 0th hour data represents the data obtained after immediate addition of DMSO  or peptide. In each panel, green circles represent autofluorescence (cells emitting light of wavelength corresponding to that observed for FAM  fluorophore) of DMSO-treated cells at 0 and 2 hours. Data information: In panels E-H, n = 5 biological replicates are used. In each of these panels, the mean ± SD intensity values (averaged across all the replicates) obtained across each fluorescent channel (FM 4-64FX, FAM  and DAPI) are presented for a particular bacterial strain (indicated on top of the panel). In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO  or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.",
    "molecules": "FAM, DAPI, DMSO, FM 4-64FX"
  },
  {
    "caption": "In vivo toxicity determination of the CP1-WT peptide using a murine  model. BALB/c mice  (Five mice  per cohort) were intraperitoneally injected with 1.6% DMSO  or varying peptide doses as indicated and monitored for 14 days. A Changes in body weights   relative to day 0 were noted. The mean percentage changes in body weights for each cohort are plotted across all the days. Data information: n = 2 biological replicates with 5 mice  per cohort were used. In panel A, across the entire experimental tenure of 14 days, data are presented as mean ± SD values for each cohort, averaged across both the replicates for all the five mice (studied in the respective cohort). Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights  across DMSO-treated and the respective peptide treated cohorts (ns P > 0.05).",
    "molecules": "DMSO"
  },
  {
    "caption": "In vivo toxicity determination of the CP1-WT peptide using a murine  model. BALB/c mice  (Five mice  per cohort) were intraperitoneally injected with 1.6% DMSO  or varying peptide doses as indicated and monitored for 14 days. B, After 14 days post peptide treatment, blood  was collected from the animals by retro-orbital puncture and analysed for (B) alanine transaminase (SGPT), alanine aminotransferase  (SGOT), alkaline phosphatase (ALP) Relative to the values obtained for the DMSO  cohort, insignificant changes in serum  levels of these parameters indicate that the peptide shows no significant hepatotoxic (as seen in panel B) Data information: biological replicates with 5 mice  per cohort were used. In the panels B representative data for only one biological replicate across all the cohorts (five mice  per cohort) are presented as mean ± SD values. P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).",
    "molecules": "DMSO"
  },
  {
    "caption": "In vivo toxicity determination of the CP1-WT peptide using a murine  model. BALB/c mice  (Five mice  per cohort) were intraperitoneally injected with 1.6% DMSO  or varying peptide doses as indicated and monitored for 14 days. C After 14 days post peptide treatment, blood  was collected from the animals by retro-orbital puncture and analysed for (C) creatinine  and blood urea nitrogen (BUN) levels. Relative to the values obtained for the DMSO  cohort, insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects (as seen in panel C). Data information: n = 2 biological replicates with 5 mice  per cohort were used. In panels C, representative data for only one biological replicate across all the cohorts (five mice  per cohort) are presented as mean ± SD values. For panels C, P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).",
    "molecules": "blood urea nitrogen, BUN, creatinine, DMSO"
  },
  {
    "caption": "Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. (A) Survival  for symptoms were monitored. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice  per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice  in each cohort. In panel A, percent survival  was compared between untreated cohort (N.T) and the DMSO/peptide/ciprofloxacin-treated cohort using a two-way ANOVA test using Bonferroni correction (ns P > 0.05; * P < 0.01). The notations, namely, N.T denote no treatment; Pep and Cip  denote CP1-WT and ciprofloxacin  treatments respectively.",
    "molecules": "Cip, ciprofloxacin, DMSO"
  },
  {
    "caption": "Action of the CP1-WT peptide in treatment of  S. Typhimurium oral infection. C-E Bacterial loads in  S. Typhimurium infected organs, namely, (C) liver, (D) kidney  and (E) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice  in each cohort. Data information: For all the three infection  models stated above, n = 2 biological replicates with 7 mice  per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice  in each cohort. In panels, (C-E), Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip  denote CP1-WT and ciprofloxacin  treatments respectively.",
    "molecules": "Cip, ciprofloxacin"
  },
  {
    "caption": "Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. F-J Histopathology  analyses with representative H&E-stained liver  sections at different magnifications are shown for S. Typhimurium infected liver  treated with various agents. K Mean pathological scoring from the tissue sections used in histopathology  analyses in panels F-J. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice  per cohort were used. In panels K to assign histopathological  scores, data are collected from five mice  in each cohort, and the representative data for only one biological replicate are presented as mean ± SD values. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip  denote CP1-WT and ciprofloxacin  treatments respectively. For histopathology  analyses in panels (F-J) except for uninfected and peptide-treated mice, pathology sections from infected or treated (DMSO  or ciprofloxacin) mice  showed aggregation of multiple  inflammatory cells  (INF) and edema  (E) in liver parenchyma, central vein congestion (C), distortion of hepatic portal vein  (HPV), hemorrhage  (H), necrosis  (N) and pyknotic nuclei  (arrows in panels H, Scale bar: 50µm in panels (F-J)",
    "molecules": "Cip, ciprofloxacin, DMSO"
  },
  {
    "caption": "Action of the CP1-WT peptide in treatment of A. baumannii  intraperitoneal infections. L-N Bacterial loads in  A. baumannii infected organs, namely, (L) liver, (M) kidney  and (N) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice  in each cohort. Data information: For all the three infection  models stated above, n = 2 biological replicates with 7 mice  per cohort were used. In panels (L-N) representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice  in each cohort. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip   denote CP1-WT and ciprofloxacin  treatments respectively.",
    "molecules": "Cip, ciprofloxacin"
  },
  {
    "caption": "(A) HeLa cells expressing GFP‐tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e.",
    "molecules": "PBS"
  },
  {
    "caption": "(B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green).",
    "molecules": "BAF A1"
  },
  {
    "caption": "(C) Control cells (untransfected, right panel) or cells overexpressing Myc‐UBQLN2 and GFP‐LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc‐UBQLN2 and GFP‐LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double‐membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.",
    "molecules": "gold particles"
  },
  {
    "caption": "(D) Control and UBQLN‐KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN‐KD and control+BAF A were compared with that of controls at the same time points, with ***P0.001, **P0.01 and *P0.05 (paired Student's t‐test).",
    "molecules": "BAF A"
  },
  {
    "caption": "(G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.",
    "molecules": "BAF A"
  },
  {
    "caption": "Figure 1. Helix-7 dynamically interacts with the miRNA seed region. A) Cartoon representation of the Ago2-miRNA crystal structure. Ago2 is colored gray except for helix-7, which is yellow. miRNA guide colored red.C) Close-up view of the unpaired complex shows helix-7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7.",
    "molecules": "miRNA"
  },
  {
    "caption": "B) Crystal structure of the Ago2-miRNA-target RNA ternary complex. Target RNA is colored blue. D) Close-up view of the Ago2-miRNA-target complex shows helix-7 docks into the minor groove of the guide:target duplex, directly contacting base pairs at positions g6 and g7.",
    "molecules": "miRNA"
  },
  {
    "caption": "B) Dissociation of a 32P-labeled target RNA (0.1 nM) from the Ago2-miR122 complex (1 nM) was monitored in the presence of unlabeled target RNA (100 nM). Fraction of the target RNA bound to Ago2-miR122 is plotted as a function of time for WT and Ηhelix-7 Ago2. Average values from at least three independent experiments ± SD were fit to single exponential decays.",
    "molecules": "32P"
  },
  {
    "caption": "C) Representative fluorescence time traces (with a resolution of 100 ms for wt, 300 ms for MI-AA and Ηhelix-7) show docking and dissociation of wild type (top), MI-AA (middle), and Ηhelix-7 (bottom) Ago2-miRNA at single spots in the microfluidic chamber.",
    "molecules": "miRNA"
  },
  {
    "caption": "Figure 5. ΗHelix-7 Ago2 is sensitive to inhibition by off-target RNAs. A) Ago2-miRNA122 (1 nM) was incubated with a 32P-labeled, seed-matched target RNA (0.1 nM) in the presence of increasing concentrations of unlabeled a competitor RNA (top panel). Fraction target RNA bound to Ago2 is plotted as a function of off-target competitor RNA concentration. B) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA. For A-B, average values from at least three independent experiments ± SD are plotted.",
    "molecules": "32P"
  },
  {
    "caption": "Figure 6. Helix-7 mutants display reduced target-binding rates. A) Ago2-miR122 complexes were mixed with a 32P-labeled seed matched target RNA; bound and free RNAs were then separated using a filter-binding apparatus at various times. Representative time course for target RNA (0.1 nM) binding to wild type, MI-AA, and Δhelix-7 Ago2 (1 nM). Average values from at least three independent experiments ± SD are plotted.",
    "molecules": "32P"
  },
  {
    "caption": "X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatment",
    "molecules": "Erlotinib"
  },
  {
    "caption": "Quantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib)",
    "molecules": "Erlotinib"
  },
  {
    "caption": "Tumor size during treatment (n=6 Vehicle, 5 Erlotinib)",
    "molecules": "Erlotinib"
  },
  {
    "caption": "PET summation images (0-90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [11C]Erlotinib distribution in one H2-c-fosLTR/Egfrwt mouse (M125). Anatomical structures are labelled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1cm Concentration-time curves of [11C]Erlotinib in bone tumors in right scapula of three H2-c-fosLTR/Egfrwt mice measured with PET. Broken line indicates threshold for in vitro effect of Erlotinib (1 µM)",
    "molecules": "11C, Erlotinib"
  },
  {
    "caption": "µPET/CT analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates Standardized uptake values (SUV) of the µPET-tracer Na[18F]F in the pelvic osteosarcoma of 4- and 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice. n=6 wt, 4 ∆Ob for 4 month time-point, n=6 wt, 3 ∆Ob for 7 month time-point",
    "molecules": "Na[18F]F"
  },
  {
    "caption": "Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib",
    "molecules": "Erlotinib"
  },
  {
    "caption": "c-fos and c-fostg mRNA expression levels in H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib (10µM) or DMSO as control (n=4 independent cell lines)",
    "molecules": "DMSO, Erlotinib"
  },
  {
    "caption": "Western Blot analysis of starved H2-c-fosLTR/Egfrwt OS cells, pre-treated with DMSO (1:1000), Afatinib (5µM), GSK2233470 (10µM), Rapamycin (10nM) or U0126 (10µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated",
    "molecules": "Afatinib, DMSO, GSK2233470, Rapamycin, U0126"
  },
  {
    "caption": "Western Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatment",
    "molecules": "Erlotinib"
  },
  {
    "caption": "c-fos mRNA expression levels in p53f/f Rb1f/f Osx-Cre OS cells treated for 24h with Erlotinib (10µM) (n=3)",
    "molecules": "Erlotinib"
  },
  {
    "caption": "Western Blot analysis of starved p53f/f Rb1f/f Osx-Cre OS cells, pre-treated with DMSO (1:1000) or Afatinib (5µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated",
    "molecules": "Afatinib, DMSO"
  },
  {
    "caption": "Cell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO)",
    "molecules": "DMSO, Erlotinib"
  },
  {
    "caption": "143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments) 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments)",
    "molecules": "Erlotinib"
  },
  {
    "caption": "LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments) LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experiment",
    "molecules": "Erlotinib"
  },
  {
    "caption": "Western Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment",
    "molecules": "Erlotinib"
  },
  {
    "caption": "(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ.",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": "(C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection.",
    "molecules": "BrdU"
  },
  {
    "caption": "(E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.",
    "molecules": "BrdU"
  },
  {
    "caption": "(E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05.",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": "Binding of recombinant (r)CEACAM1 (CC1)-HIS (10 μg/mL) to (A) a panel of Gram-positive bacteria, namely Enterococcus faecalis, Enterococcus faecium, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Group C Streptococcus (GCS), Group G Streptococcus (GGS), Streptococcus pneumoniae, Staphylococcus aureus, (B) an expanded collection of GBS isolates, with serotype and carriage of bac gene shown. rCC1 binding to live bacteria was quantified by a secondary anti-His-FITC monoclonal antibody (mAb). Mean and standard deviation (SD) values are reported for n = 3 independent experiments. In (B), GBS strains from left to right are A909, BS39, 515, BS22, H36B, BS29, 18RS21, BM110, COH1, SBL3066, NCTC10/84, SB35, SB10, SB20, BS26.",
    "molecules": "FITC"
  },
  {
    "caption": "Inhibition of β-IgSF binding to DB.CC1 by anti-CC1-N mAb (clone CC1/3/5-Sab), but not by anti-CC1-A1B1 mAb (clone B3-17). β-IgSF-biotin was coupled to SPE. Mean and SD values are reported for n = 6 replicates.",
    "molecules": "SPE, biotin"
  },
  {
    "caption": "Binding of β-IgI3 coupled to Streptavidin-PE to DB coated with rCC1-N wildtype or variants. Mutation of CC1-N residues 29, 44, 89, 91 and 95 lead to significant changes in β-IgI3 binding capacity.",
    "molecules": "Streptavidin"
  },
  {
    "caption": "Binding of β-IgI3, R28-IgI3 or human serum albumin (HSA) coupled to PE-conjugated streptavidin (SPE), to DB coated with CC1 (DB.CC1). Mean and SD is shown for n = 6 independent replicates.",
    "molecules": "SPE, streptavidin"
  },
  {
    "caption": "Heatmap of overlapped expressed genes in response to sorafenib-resistance.",
    "molecules": "sorafenib"
  },
  {
    "caption": "D The mRNA expression level of METTL3 in sorafenib-sensitive (n=3) and sorafenib-resistant (n=3) human liver tumors. Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "sorafenib"
  },
  {
    "caption": "F METTL3 RNA levels between naïve HepG-2 cells (n=3) and sorafenib-resistant HepG-2 cells (n=10). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "sorafenib"
  },
  {
    "caption": "G The IC50 of sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2). Data information: In all relevant panels, ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "O2, sorafenib"
  },
  {
    "caption": "H The protein level of METTL3 in sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxia condition (1%O2).",
    "molecules": "O2, sorafenib"
  },
  {
    "caption": "I The global RNA m6A level in naïve HepG-2 and sorafenib-resistant HepG-2 determined by dot-blotting assay under hypoxia condition (1%O2).",
    "molecules": "m6A, O2, RNA, sorafenib"
  },
  {
    "caption": "A-B Clonogenic survival of METTL3-knockdown (A) and METTL3-overexpression (B) in normal liver cell line WRL68 cells for 7 days in normoxia condition (21% O2) and quantification of clusters in A-1 and B-1, respectively. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "O2"
  },
  {
    "caption": "C-D The IC50 of METTL3-knockdown SMMC-7721 cells (C) and Bel-7402 cells (D) after treated with sorafenib for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "O2, sorafenib"
  },
  {
    "caption": "E Overexpression of wild type METTL3 or catalytic mutant METTL3 in sorafenib-resistant HepG-2 cells.",
    "molecules": "sorafenib"
  },
  {
    "caption": "F The global RNA m6A level in sorafenib-resistant HepG-2 cells with wild type METTL3-overexpression or catalytic mutant METTL3-overexpression by dot-blotting assay.",
    "molecules": "m6A, RNA, sorafenib"
  },
  {
    "caption": "G-I Rescue of shRNA-resistant wild type METTL3 but not catalytic mutant METTL3 sensitized METTL3-knockdown SMMC-7721 cells (G), Bel-7402 cells (H) and sorafenib-resistant HepG-2 cells (I) to sorafenib treatment. The IC50 of the cells was measured after treated with sorafenib for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "O2, sorafenib"
  },
  {
    "caption": "J Cell survival assay of sorafenib-resistant HepG-2 cells with wild type METTL3 overexpression or catalytic mutant METTL3 overexpression after treated with sorafenib for 24h under hypoxia condition (1%O2). Scale bar, 1mm.",
    "molecules": "O2, sorafenib"
  },
  {
    "caption": "E Protein level of LC3 I/II in HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia condition for 48h.",
    "molecules": "sorafenib"
  },
  {
    "caption": "F-H Overexpression of shRNA-resistant wild type METTL3 but not catalytic mutant METTL3 rescue autophagy phenotype in METTL3-knockdown SMMC-7721 cells (F), Bel-7402 cells (G) and sorafenib-resistant HepG2 cells (H) by representative immunostaining images of LC3 under hypoxia condition for 48h. Scale bar, 200μm. Quantification of the numbers of GFP-LC3 puncta/cell by Imaris in F-1, G-1 and H-1, respectively. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "sorafenib"
  },
  {
    "caption": "I-K Protein levels of LC3 I/II in METTL3-knockdown SMMC-7721 cells (I), Bel-7402 cells (J) and HepG-2 cells (K) treated with or without 3-MA under hypoxia condition for 24h.",
    "molecules": "3-MA"
  },
  {
    "caption": "L-N The IC50 of sorafenib in SMMC-7721 cells (L), Bel-7402 cells (M) and HepG-2 cells (N) treated with or without 3-MA for 24h under hypoxia condition (1%O2). Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "3-MA, O2, sorafenib"
  },
  {
    "caption": "D Half-life of FOXO3 mRNA in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with actinomycin D.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "E Half-life of FOXO3 protein in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with cycloheximide. Quantification of the protein optical density by Image J in E-1.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "J The m6A-IP sequencing under hypoxia proved the m6A modification participated in regulation of FOXO3. The YTHDF1 binding site locates at the 3'UTR of FOXO3.",
    "molecules": "m6A"
  },
  {
    "caption": "M Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the m6A MeRIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "m6A"
  },
  {
    "caption": "N Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the YTHDF1-RIP analysis. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "m6A"
  },
  {
    "caption": "B Protein level of FOXO3 in naïve HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia for 48h.",
    "molecules": "sorafenib"
  },
  {
    "caption": "D Overexpression of FOXO3 in sorafenib-resistant HepG-2 cells by Western blot.",
    "molecules": "sorafenib"
  },
  {
    "caption": "F Electron micrographs of METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. G Representative Immunostaining images of LC3 in METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in G-1. H Representative Immunostaining images of LC3 in naïve HepG-2 cells and sorafenib-resistant HepG-2 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in H-1. I The IC50 of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "sorafenib"
  },
  {
    "caption": "J The IC50 of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition. Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.",
    "molecules": "sorafenib"
  },
  {
    "caption": "K Cell survival assay of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.",
    "molecules": "sorafenib"
  },
  {
    "caption": "L Cell survival assay of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.",
    "molecules": "sorafenib"
  },
  {
    "caption": "A Depletion of METTL3 in Hepa1-6 cells by CRISPR-Cas9 knockout enhanced sorafenib-resistance in vivo.",
    "molecules": "sorafenib"
  },
  {
    "caption": "D Knockdown of METTL3 slightly increased liver tumor growth treated with sorafenib in orthotopic xenograft mouse model. Stable METTL3-knockdown Bel-7402 cells and control cells were injected into the liver of each NOD/SCID mouse. 3 weeks after injection, livers were separated for pathological analysis. Red circle indicates tumors in the livers.",
    "molecules": "sorafenib"
  },
  {
    "caption": "E Liver tumors in orthotopic xenograft mouse model were confirmed by hematoxylin and eosin staining.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "F Overexpression of FOXO3 rescued sorafenib-resistance mediated by METTL3-knockdown in xenograft liver tumors. Representative xenograft tumors at endpoint, containing 6 groups.",
    "molecules": "sorafenib"
  },
  {
    "caption": "D-G (D) Immunohistochemical (IHC), IF ­and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines (D), 2 month-old IκBβ;IκBε KO (E) and 2 month-old IκBαNES mice (F and G). A minimum of 50 crypts/villus was counted per genotype (3 mice). Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant. Scale bars in D, E, F and G, 50 μm.",
    "molecules": "Alcian Blue"
  },
  {
    "caption": "(B) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO-derived organoids. Scale bars in B 50 μm.",
    "molecules": "Alcian Blue"
  },
  {
    "caption": "(C) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information Scale bars in 50 μm.",
    "molecules": "EPZ-6438"
  },
  {
    "caption": "(D-E) qPCR analysis of the indicated adult and fetal ISC genes (D) and elements of the Notch and IFN pathway (E) (differentially expressed in IκBα KO organoids) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.",
    "molecules": "EPZ-6438"
  },
  {
    "caption": "(F) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor. Data information Scale bars in F, 50 μm.",
    "molecules": "DAPT, dexamethasone"
  },
  {
    "caption": "(G) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.",
    "molecules": "DAPT, dexamethasone"
  },
  {
    "caption": "(A) IF analysis of colonic tissue from control and DSS-treated animals including acute damaged (AD) areas and recovery (R) areas. Data information: Scale bars 100 μm",
    "molecules": "DSS"
  },
  {
    "caption": "(E-F) Representative images of the colonic tissue from 2 months-old DSS-treated WT and IκBα KO mice are shown. Alcian Blue staining was used to identify the mucus-secreting goblet cells in the colonic glands and Ki67 as proliferation marker. Nuclear counterstain is Fast Red. The table shows number of ulcerations present in the intestines of mice analysed (3 WT, 2 IκBα KO). Data information: Scale bars in E and F, 100 μm",
    "molecules": "DSS, Alcian Blue, Fast Red"
  },
  {
    "caption": "A-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bars, 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Brain cortical tissue obtained from Nrf2 (−/−) (6 months old, one male and two female) or wild-type (11 months old, two male and one female) mice were separated into sarkosyl-soluble (SS) and insoluble (SI) fractions as described in the Methods. The level of total tau was analysed by immunoblotting using a monoclonal antibody to total tau (Tau5). The relative molecular masses (kDa) are indicated to the left of each blot. Bar graph represents the relative optical density of sarkosyl-insoluble tau normalized with that of sarkosyl-soluble tau. n=3. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05).",
    "molecules": "sarkosyl"
  },
  {
    "caption": "(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM, SFN) for 12 or 24 h. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01).",
    "molecules": "SFN"
  },
  {
    "caption": "(b) Stably transfected CN1.4 cortical neurons were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN for 12 or 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau was detected with a polyclonal phospho-independent tau antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01).",
    "molecules": "doxycycline, SFN"
  },
  {
    "caption": "Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (a)Mouse brain tissues were homogenized in the lysis buffer, and 10 μg of lysates was incubated with 2 μg of GST-tau protein at 37°C for 30 min in the presence or absence of 1 mM ATP. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies, respectively. GST-tau was detected with a monoclonal GST antibody (GST-tau; Supplementary Fig. 4). Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of duplicated independent experiments and were analysed using Student's t-test.",
    "molecules": "ATP"
  },
  {
    "caption": "Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (b) Mouse brain tissues were homogenized in the phosphatase storage buffer, and phosphatase activity in the lysates was quantified using the serine/threonine phosphatase assay system (Promega) by measuring the dephosphorylation of a phosphopeptide, RRA(pT)VA in the presence or absence of okadaic acid (OA, 20 nM). PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture. n=4. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test.",
    "molecules": "okadaic acid"
  },
  {
    "caption": "CN1.4mousecortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. On the next day (a), the cells were washed twice with PBS and the media were replaced with Dox-deficient media with either vehicle, epoxomicin (12.5 nM, Epox), MG132 (7 μM) or 3-methyladenine (10 mM, 3-MA) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.",
    "molecules": "3-MA, 3-methyladenine, doxycycline, Epox, epoxomicin, MG132"
  },
  {
    "caption": "CN1.4 mouse cortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. (b) Cells were treated with either chloroquine (50 μM, CQ) or bafilomycin A1 (10 nM, Baf A1) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.",
    "molecules": "Baf A1, bafilomycin A1, chloroquine, CQ, doxycycline"
  },
  {
    "caption": "(d) Stably transfected CN1.4 cortical cells were maintained in the presence of Dox (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN (10 μM) for 12 h. The culture medium was exchanged for fresh media containing Dox (1 μg ml−1) but no SFN after washing with PBS. The cells were then incubated for an additional 18 h in the absence or presence of CQ (50 μM). The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau, beclin-1 and LC3 were detected with the appropriate polyclonal antibodies. The relative molecular masses (kDa) are indicated to the left of each blot. (e) Bar graphs represent the relative optical density of phosphorylated tau, tau or LC3-II normalized with that of actin. n=4. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01).",
    "molecules": "CQ, Dox, SFN"
  },
  {
    "caption": "(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM) for 12 h, and the relative mRNA level of each gene was compared with that of cells not treated with SFN using qRT-PCR.",
    "molecules": "SFN"
  },
  {
    "caption": "(d,e: upper panel) Primary cortical neurons (DIV 5) (d) and SH-SY5Y cells (e) were treated with SFN for 24 h, and the expression levels of rat and human NDP52 (rNDP52 and hNDP52) examined by immunoblotting using anti-NDP52 antibody. The relative molecular masses (kDa) are indicated to the left of each blot. (d,e: low panel) Primary cortical neurons (DIV 5) (d) and SH-SY5Y cells (e) transiently transfected with the pGL4.14/human NDP52 promoter (2,173 bp) luciferase reporter plasmid were treated with SFN for 24 h, and assayed for the luciferase activity.",
    "molecules": "SFN"
  },
  {
    "caption": "(f) SH-SY5Y cells were treated with SFN for 12 h, and cell lysates were used for the ChIP assay using anti-Nrf2 antibody as described in the Methods. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01; ***P0.001).",
    "molecules": "SFN"
  },
  {
    "caption": "(c,d) Primary cortical neurons were transduced with a control lentivirus (FIGB) or with one expressing humanNDP52 (hNDP52) at DIV 1. To induce autophagy, trehalose (150 mM) was added at DIV 5 and the neurons incubated for 24 h (DIV 6). Primary cortical neurons were fixed with 4% paraformaldehyde, and stained with the 12E8 or PHF1 antibodies. Scale bar, 20 μm. The optical density of tau phosphorylated at Ser262/Ser356 (12E8). (e) and Ser396/Ser404 (PHF1) (f) in the soma of ~30 neurons randomly chosen was analysed with the ImageJ program. Data were analysed using Student's t-test (***P0.001).",
    "molecules": "trehalose"
  },
  {
    "caption": "(c) Stably transfected CN1.4 cortical cells were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h. Homogenates from CN1.4 cortical cells were used for the co-immunoprecipitation of mouse NDP52 (mNDP52) using a Tau5 antibody. Co-precipitation of mNDP52 was examined by immunoblotting using anti-NDP52 antibody. Input of the homogenates used for co-immunoprecipitation was analysed by immunoblotting using anti-NDP52 or Tau antibody.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(d,e) CN1.4 cortical cells were transfected with the mock or HA-mNDP52 plasmid (d) or with mNDP52 siRNA or scramble RNA as a control (e). The cells transfected with siRNA or scramble RNA were treated with SFN (10 μM) for 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot.",
    "molecules": "SFN"
  },
  {
    "caption": "(c) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl-soluble and insoluble fractions. The levels of total tau and tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 in the sarkosyl-insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Samples were also immunoblotted with a polyclonal to total tau (Tau) antibody. The levels of hNDP52, p62/SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting. The relative molecular masses (kDa) are indicated to the left of each blot. (d-f) The regression graphs show the relative amount of tau phosphorylated at Ser262/Ser356 (12E8) (d), at Ser396/Ser404 (PHF1) (e) or total tau (f) compared to hNDP52 present in the sarkosyl-insoluble fractions.",
    "molecules": "sarkosyl"
  },
  {
    "caption": "A. Ubiquitin-vinyl sulfone (UbVS) assays  of RING2  release by addition of pUbl and  pUb in trans to R0RBR  parkin. Assays were performed with incubation of 2 μM wild-type or W403A R0RBR  purified in a 1:1 complex with  pUb. Crosslinking was initiated by addition of 10 μM   UbVS in the presence of pUbl   or  pUb∆G76.",
    "molecules": "UbVS"
  },
  {
    "caption": "D. Autoubiquitination assays  of parkin ligase activity induced by  pUbl   or   pUb∆G76 in trans. 2 µM  GST-R0RBR in complex with  pUb  was incubated with 50 nM E1, 2 µM  UbcH7, 100 µM  ubiquitin  and 4 mM ATP.",
    "molecules": "ATP"
  },
  {
    "caption": "C. Release of RING2  by Ub-R0RBR chimera. Ubiquitin-vinyl sulfone assays  (10 μM  UbVS) were performed with 3 μM phosphorylated and  non-phosphorylated full-length parkin  and Ub-R0RBR.",
    "molecules": "UbVS"
  },
  {
    "caption": "B.  UbVS assays  of release of RING2  by K6- or  K48-linked (pUb)4 chains. 10 μM  UbVS was added to 2 μM R0RBR in the presence of increasing amount of   K6(pUb)4 or    K48(pUb)4  chains.",
    "molecules": "UbVS"
  },
  {
    "caption": "D. Competition between K6(pUb)4  chains and monomeric pUb. UbVs  (10 μM) was added to 20 nM of WT and K211N R0RBR in the presence of monomeric pUb   and/or K6(pUb)4  chains. Reactions were resolved on SDS-PAGE and visualized with anti-parkin  antibody by  western blotting. The percentage of unmodified parkin  band in each lane is indicated under the gel.",
    "molecules": "UbVs"
  },
  {
    "caption": "Pyruvate was measured in whole tibialis anterior muscle tissue homogenates. The mean fold change ± SEM compared to age-matched WT are represented with *P = 0.019 at 65 days of age (n = 5/genotype) and *P = 0.041 at 105 days of age (n = 4/genotype), two-way ANOVA followed by Fisher's LSD post hoc test. Data shown are representative of two independent experiments having similar results.",
    "molecules": "Pyruvate"
  },
  {
    "caption": "Left: Representative microphotographs of PAS staining from WT and SOD1G86R tibialis anterior cross-sections at 65 and 105 days of age showing glycogen-negative (light pink) and glycogen-positive (dark pink) fibers. Scale bar: 200 μm. Right: Quantification of glycogen-negative (−) and glycogen-positive (+) fibers at 65 and 105 days of age. For (−) fibers: **P-values versus WT: 0.0026 at 65 days and 0.0074 at 105 days, for (+) fibers: ##P-values versus WT: 0.0025 at 65 days and 0.0073 at 105 days (n = 5/genotype at 65 days and n = 4/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).",
    "molecules": "glycogen"
  },
  {
    "caption": "Relative levels of (left) ATP and (right) NADH and NAD+ measured in total tibialis anterior homogenates of WT and SOD1G86R mice. Left: Graphs represent mean fold change ± SEM in ATP from age-matched WT. **P = 0.002 (n = 9 and 12 for WT and SOD1G86R, respectively, at 65 days; n = 10/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test). Right: The amounts of NADH and NAD+ relative to protein content in whole tibialis anterior homogenates are represented as mean ± SEM. *P = 0.027 and **P = 0.001 (n = 5, Student's t-test).",
    "molecules": "NAD+, ATP, NADH"
  },
  {
    "caption": "A-I Relative mRNA levels of (A) Pdk4, (B) Pparβ/δ, (C) Foxo1, (D) Pfk1, (E) Acsf2, (F) citrate synthase, (G) PGC-1α, (H) Mfn2, and (I) Gpx1 were evaluated by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. P-values versus WT: Pdk4 ***P = 0.002 and ##P = 0.0039, Pparβ/δ #P = 0.0178, Foxo1 **P = 0.0033 and ##P = 0.0038, Pfk1 ***P = 0.0002 and ##P = 0.0080, Acsf2 *P = 0.0437, citrate synthase ###P = 0.0004, Pgc-1α **P = 0.0084 and ###P = 0.0009, Mfn2 *P = 0.0272, $P = 0.0145 and ###P = 0.0002 and Gpx1 ***P < 0.0001 and ###P < 0.0001 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).",
    "molecules": "DCA"
  },
  {
    "caption": "Grip strength is represented as mean of percent from T0 for each experimental group ± SEM. ##P = 0.0045 and ***P = 0.0003 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).",
    "molecules": "DCA"
  },
  {
    "caption": "Relative mRNA levels of muscular atrophy markers Murf1 and Atg-1 were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.0079 and ***P = 0.0018 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).",
    "molecules": "DCA"
  },
  {
    "caption": "Relative mRNA levels of denervation markers AChRα, AChRγ, and MuSK were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.015 for AChRα, #P = 0.028 and **P = 0.074 for AChRγ, #P = 0.042 and **P = 0.0029 for MuSK (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).",
    "molecules": "DCA"
  },
  {
    "caption": "LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR-183/96 (N) or antagomir-183/96 (P) stained with Nile red (n=5). Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR-183/96 (O) or antagomir-183/96 (Q). Scale bars: 5 μm.",
    "molecules": "LD, lipid droplets, Nile red"
  },
  {
    "caption": "Concentration of serum triglyceride (TAG) (left) and non-esterified fatty acid (NEFA) (right) in WT and DKO mice after HFD feeding for 2 months (n=5).",
    "molecules": "NEFA, non-esterified fatty acid, TAG, triglyceride"
  },
  {
    "caption": "(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).",
    "molecules": "biotin"
  },
  {
    "caption": "(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.",
    "molecules": "Biotin"
  },
  {
    "caption": "(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.",
    "molecules": "paraformaldehyde"
  },
  {
    "caption": "(E) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.",
    "molecules": "paraformaldehyde"
  },
  {
    "caption": "B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.",
    "molecules": "Biotin"
  },
  {
    "caption": "(E and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.",
    "molecules": "puromycin"
  },
  {
    "caption": "(B) Effects of RNF5 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. The 293-HA-Ub-K11O cells (2×107) were transfected with the indicated RNAi plasmid (10 µg each). Twelve hours after transfection, the cells were selected with puromycin (1 µg/mL) for twenty-four hours and infected with SeV for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies.",
    "molecules": "puromycin"
  },
  {
    "caption": "(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.",
    "molecules": "3-MA"
  },
  {
    "caption": "(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.",
    "molecules": "3-MA, NH4Cl, MG132"
  },
  {
    "caption": "(G) HT29 cells were transfected with GFP-LC3  and with SHP  siRNA or SHP  expression plasmids and incubated in serum-free medium containing 200 μM  oleic acid for 6 h followed by treatment with 50 ng/ml FGF19  for 6 h The control group (Veh) was treated with vehicle for FGF19, control plasmid DNA, and scrambled siRNA. The FGF19  groups were treated with control DNA or SHP  expression plasmid or SHP  siRNA or control scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3  puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 10 μm. G, The mean and SD are plotted. Statistical significance was determined by (G) one-way ANOVA with the Tukey post-test. P-values are indicated",
    "molecules": "oleic acid"
  },
  {
    "caption": "(H) HT29 cells were treated with 50 ng/ml FGF19  with or without 100 nM  bafilomycin A1 for 12 h. LC3 and actin  levels in cell extracts were determined by IB. Quantification of bands is shown at the right (n=3 culture plates). H) The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(A) HT29  cells were transfected with GFP-LC3  and with Atg7  siRNA or control scrambled RNA (siCtl) and incubated in serum-free medium containing 200 μM oleic acid  for 6 h followed by treatment with 50 ng/ml FGF19  for 6 h The control group (Veh) was treated with vehicle for FGF19 and control siRNA. The FGF19  groups were treated with Atg7  siRNA or scrambled siRNA as indicated. Cells were imaged by  confocal microscopy (Zeiss, LSM700). GFP-LC3  puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 20 μm. The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.",
    "molecules": "oleic acid"
  },
  {
    "caption": "D) HT29  cells were transfected with Atg7  siRNA or control scrambled siRNA (siCtl) for 48 h and then treated with  oleic acid for 6 h followed by treatment with 50 ng/ml FGF19  for 12 h. (D) ApoB48  and actin  levels in cell extracts determined by IB. Bands were quantified and the LC3-II/LC3-I ratio is shown at the right (n=3 culture plates). The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.",
    "molecules": "oleic acid"
  },
  {
    "caption": "(A) HT29  cells were transfected with GFP-LC3  and with Tfeb  siRNA or Tfeb  expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19  for 6 h The control group (Veh) was treated with vehicle for FGF19  and transfected with control plasmid DNA and scrambled siRNA. The FGF19  groups were treated with control DNA or TFEB  expression plasmid or with Tfeb  siRNA or scrambled siRNA as indicated. Cells were imaged by  confocal microscopy (Zeiss, LSM700). GFP-LC3  puncta were counted and the number of puncta/cell is shown at right (n = 20 cells). Scale bar, 10 μm. (A, The mean and SD are plotted. Statistical significance was determined by (A) one-way ANOVA with the Tukey post-test, P-values are indicated.",
    "molecules": "oleic acid"
  },
  {
    "caption": "(B) HT29  cells were transfected with siRNA for Tfeb  (siTFEB) or control scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19  or vehicle with or without 100 nM  bafilomycin A1  (BAF) for 12 h. Levels of the indicated proteins determined by IB.  LC3 II/I and p62/actin  ratios are shown at the right (n=3-4 culture plates). B, The mean and SD are plotted. Statistical significance was determined by (B) two-way ANOVA with the Tukey post-test, P-values are indicated.",
    "molecules": "BAF, bafilomycin A1"
  },
  {
    "caption": "B Sedimentation assay to quantify condensation of unmodified TDP-43 versus in vitro phosphorylated TDP-43 (+CK1δ, +ATP) and controls (CK1δ or ATP  only); TDP-43 detected by Western blot  (rabbit anti-TDP-43 N-term). Due to incomplete TEV cleavage, some TDP-43-MBP-His6 remains present and co-fractionates with cleaved TDP-43, due to TDP-43 self-self interaction. C Quantification of band intensities of cleaved TDP-43 shown as means of Supernatant/(Supernatant+Condensate) [S/(S+C)] ratio of three independent experimental replicates (n=3) ± SD. ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt.",
    "molecules": "ATP"
  },
  {
    "caption": "B Representative images of  FRAP experiments at indicated  time-points. Boxes indicate  bleached area (half-bleach of condensate). Bar, 5 µm. C FRAP curves after half-bleach of freshly formed Alexa488-labeled TDP-43 condensates. Values represent means ± SD of three independent experimental replicates (n=3) of ≥ 9 droplets analyzed per condition. ***p < 0.0002 by one-way ANOVA with Tukey´s multiple comparison test for area under the curve (AUC) of individual  droplets.",
    "molecules": "Alexa488"
  },
  {
    "caption": "D Confocal images of Alexa488-labeled TDP-43 aggregates formed in an in vitro aggregation assay (with TEV protease cleavage). Bar, 100 µm. Zoom shows magnified view of aggregates at the 24h  time point. Bar, 20 µm.",
    "molecules": "Alexa488"
  },
  {
    "caption": "A  Hormone-inducible  nuclear  import assay, representative still images of GCR2-EGFP2-TDP-43 Wt, 12D and 12A before and during import triggered by addition of dexamethasone. Images were live  recorded by spinning disc confocal microscopy. Bar, 20 µm. B Quantification of the  hormone-inducible  nuclear import  measured during a total  time course  of 50 min. Values represent the mean fluorescence intensity of GCR2-EGFP2-TDP-43 in the  cytoplasm for three independent replicates ± SEM (≥ 42 cells per condition).",
    "molecules": "dexamethasone"
  },
  {
    "caption": "F SG  recruitment of different  TDP-43-NLSmut variants in intact HeLa  cells in the absence of endogenous TDP-43. After TDP-43 silencing and expression of NLSmut Wt, 12D and 12A variants, SGs were induced by H2O2 treatment and  SG recruitment of TDP-43 was monitored by TDP-43 and G3BP1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei  were stained in DAPI (turquoise), TDP-43 (green) and G3BP1 (red). Bar, 40 µm.",
    "molecules": "DAPI, H2O2"
  },
  {
    "caption": "H Recruitment of TDP-43 into arsenite-induced nuclear bodies (NBs) in HeLa cells. After TDP-43 silencing and expression myc-TDP-43 Wt, 12D and 12A, NBs were induced by sodium arsenite treatment and NB  recruitment of  TDP-43 was monitored by TDP-43 immunostaining. Bar, 20 µm.",
    "molecules": "arsenite, sodium arsenite"
  },
  {
    "caption": "E SG recruitment of  EGFP-TDP-43 NLS mutant variants (Wt, 12D, 12A) in primary hippocampal neurons. SG formation was induced by 1h heat shock at 42°C.  SGs  and TDP-43 were monitored by G3BP1 antibody staining and EGFP fluorescence, respectively. For clarity, signals were converted to grey values in the individual channels (first two columns). In the merge (third column), EGFP-TDP-43  shown in green, G3BP1 in red and nuclei (DAPI staining) in turquoise. Bar, 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Expression  levels of various GPI-APs in different clones of PIGG-KO HEK293 cells. Fluorescence intensities of NT5E  in PIGG-KO clones #4, #10, #17, #19 and wild type cells are 575, 910, 696, 530 and 1548 while those of EphrinA5 in clones #4, #17, #19 and wild type cells are 1431, 2080, 1868 and 3336, respectively. Data information: Each FACS  analysis was performed at least three times.",
    "molecules": "GPI"
  },
  {
    "caption": "B. Expression  levels of GPI-APs in PIGG-KO clone #19 cells stably transfected with PIGG  cDNA compared to levels in empty vector transfected cells. Fluorescence intensities  of NT5E  in PIGG  cDNA and in vector transfected cells are 1332 and 886, respectively. Data information: Each FACS  analysis was performed at least three times.",
    "molecules": "GPI"
  },
  {
    "caption": "A. Expression  levels of different GPI-APs in PIGG-KO HEK293  cells transiently co-transfected with PIGG  cDNA or empty vector. Vertical bars represent % of levels of GPI-APs expression in non-rescued PIGG-KO cells relative to those in PIGG  cDNA rescued PIGG-KO cells. Analysis was repeated 3 to 6 times and SD bars were shown.",
    "molecules": "GPI"
  },
  {
    "caption": "A. Confocal  images (single slice) of Trp53-/- or Trp53-/-;Pten-/- (1.15) spheroids  expressing mNeonGreen-tagged (mNG) biosensors for PI(4,5)P2  (PH-PLCδ1) or PIP3  (CYTH32G/GRP1). Magnified images from boxed regions, max projection of 8 (PH-PLCδ1) or 3 (CYTH32G/GRP1) z-slices, pseudocoloured in FIRE LUT. Arrowheads: red, cell-cell contact; yellow, nucleus; green, protrusion tip. Scale bar, 7μm. Representative of 8 (Trp53-/-) or 10 (Trp53-/-;Pten-/-) spheroids  imaged across n=2 independent experiments (PH-PLCδ1) and 22 (Trp53-/-) or 23 (Trp53-/-;Pten-/-) spheroids  imaged across n=4 independent experiments (CYTH32G/GRP1). B. Intensity profiles for PH-PLCδ1  and PH-CYTH3  from spheroids  shown in (A). Protrusions measured are annotated on images in upper panels, yellow lines. Arrowheads: red, protrusion tips.",
    "molecules": "PIP3, PI(4,5)P2"
  },
  {
    "caption": "D. Western blotting  and quantitation for S6RP  pS235/236, S6RP, GAPDH  (sample integrity control) in Trp53-/-;Pten-/- 1.15 spheroids treated with DMSO  or inhibitors annotated in (B) for 2 days. Representative of n=3 independent lysate preparations. Data, mean ± SD of pS235/236:total S6RP  ratio, normalised to DMSO. P-values, unpaired, 2-tailed t tests, as annotated.",
    "molecules": "DMSO"
  },
  {
    "caption": "Quantitation of ID8 Trp53-/-;Pten-/- spheroids  treated with PI3K isoform specific inhibitors: A66  (PI3Kα), AZD8186  (PI3Kβ), AS605240  (PI3Kγ) or CAL-101 (PI3Kδ), 6hr time  intervals over 72 hrs. (I) Frequency of Spherical  and Hyper-protrusive phenotypes. Heatmap (grayscale) - phenotype proportion (z-score) in control. Heatmap (blue-red) - log2 fold change from control. P-values, bubble size (Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow-Day test, Bonferroni adjustment, non-significant). N=2 independent experiments, 3-5 technical replicates/experiment.",
    "molecules": "A66, AS605240, AZD8186, CAL-101"
  },
  {
    "caption": "K. Confocal  image of Trp53-/-;Pten-/- (1.15) spheroids stained  for PI3Kβ  (green),  F-actin (magenta) and Hoechst  (grey). Magnified images from boxed regions, pseudocoloured in inverted grayscale (F-actin) or FIRE LUT (PI3Kβ). Yellow or white/black arrowheads, enrichment of F-actin  or PI3Kβ  at protrusion tips  respectively. Scale bar, 5μm. Representative of n=5 spheroids.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A-B. Immunofluorescence  and confocal  imaging of Trp53-/-;Pten-/- 1.15 spheroids stained  for α5-integrin  or β1-integrin (grey or FIRE LUT), Hoechst  (blue) and F-actin (magenta). Magnified images from boxed regions shown. Arrowheads, labelling at protrusion tips. Scale bars, 5 μm. Representative of n=3 spheroids imaged. (B) Intensity profiles for integrins (grey) and  F-actin (magenta) from spheroids  in (A). Tip  measured is annotated, ECM  to body, yellow arrow, tip, white arrowhead.",
    "molecules": "Hoechst"
  },
  {
    "caption": "C-D. Immunofluorescence  and confocal  imaging of Trp53-/-;Pten-/- 1.15 spheroids stained  for pFAK (Y379) or pSRC Family Kinases (SFK  pY416) (grey or FIRE LUT), Hoechst  (blue) and F-actin (magenta). Magnified images from boxed regions shown. Arrowheads, positive staining. Scale bars, 5 μm. Representative of n=5 spheroids  imaged. (D) Intensity profiles for active FAK  and Src  (grey) and F-actin (magenta) from spheroids  in (C). Tip  measured is annotated, ECM  to body, yellow arrow, tip, white arrowhead.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Growth inhibitory concentration (GI50) of CY and bendamustine in the NCI60 cell line panel.",
    "molecules": "CY, bendamustine"
  },
  {
    "caption": "Nontoxic dose of Cpd A enhances bendamustine cytotoxicity in cancer cells. Images were taken at 48 h posttreatment.",
    "molecules": "Cpd A, bendamustine"
  },
  {
    "caption": "Cpd A augments DNA damage caused by bendamustine. H1650 cells were treated with 100 μM Bend, 50 μM Cpd A, 100 μM Bend plus 50 μM Cpd A, or 10 μM CY for 12 h, and cell lysates were blotted for γ-H2AX. Actin was used as a loading control.",
    "molecules": "Cpd A, CY, Bend, bendamustine"
  },
  {
    "caption": "Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.",
    "molecules": "CY, bendamustine"
  },
  {
    "caption": "Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs.",
    "molecules": "CY, bendamustine"
  },
  {
    "caption": "CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control.",
    "molecules": "CpdA, CpdB, CY, Bend, bendamustine, SAHA"
  },
  {
    "caption": "Alteration of CY side chain (Cpd B) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage. Cells were treated for 12 h. Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ-H2AX. Actin was used as a loading control.",
    "molecules": "Cpd B, CY"
  },
  {
    "caption": "Summary of lethal dose 50 (LD50) of bendamustine, CY, Cpd A (HDAC inhibition only), and Cpd B (nitrogen mustard only) against 11 human cancer cell lines.",
    "molecules": "Cpd A, Cpd B, CY, bendamustine"
  },
  {
    "caption": "Cpd A enhances the anticancer activity of bendamustine. H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A (nontoxic dose, see Fig1C and Supplementary Fig S2) for 48 h and subjected to MTT viability assays.",
    "molecules": "Cpd A, bendamustine"
  },
  {
    "caption": "CY, but not bendamustine treatment, led to suppression of CBP, TIP60, MORF, and MSL1 in H1650 cells. Cells were treated with DMSO, bendamustine (350 μM), CY (15 μM), or SAHA (10 μM) for 6 h, and mRNA was extracted for qPCR analysis. Data represent mean ± SEM from three independent experiments, and statistical significance was determined by unpaired two-tailed t-test. ***P < 0.01.",
    "molecules": "CY, bendamustine, DMSO, SAHA"
  },
  {
    "caption": "Suppression of CBP, TIP60, MORF, and MSL1 sensitized cells to bendamustine. Y-axis represents relative resistance calculated from results of GFP competition assays, and statistical significance was determined by unpaired two-tailed t-test. Data represent mean ± SEM from two independent experiments. **P < 0.05, ***P < 0.01.",
    "molecules": "bendamustine"
  },
  {
    "caption": "A Determination of maximally tolerated dose (MTD) of CY at 60 mg/kg. Other drugs were used at MTD according to the literature. For each drug, n = 5 and all mice survived treatment. Data represent mean ± SEM.",
    "molecules": "CY"
  },
  {
    "caption": "B CY exhibited enhanced activity against BCR-ABL Arf−/− murine ALL cells in vitro.",
    "molecules": "CY"
  },
  {
    "caption": "C, D CY showed superior antitumor activity compared with bendamustine, SAHA, and other chemotherapeutic drugs in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 5.02E-06) (C), SAHA (P = 4.45E-06), Bend (P = 5.18E-06), NT (P = 1.20E-05) (D), VCR (P = 1.13E-05), Dox (P = 8.08E-06), AraC (P = 7.62E-06), and CTX (P = 1.13E-05). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.",
    "molecules": "CY, Bend, bendamustine, CTX, AraC, Dox, VCR, SAHA"
  },
  {
    "caption": "E Therapeutic effects of CY (weekly dose) and targeted drugs PP242 (daily dose) and dasatinib (daily dose) in mice transplanted with BCR-ABL ALL cells. P-values of CY versus other drugs: NT (P = 3.94E-05), PP242 (P = 1.20E-05), and dasatinib (P = 0.99). Survival statistical analysis was done with the Mantel-Cox (log-rank) test of GraphPad Prism.",
    "molecules": "CY, dasatinib, PP242"
  },
  {
    "caption": "F, G Antitumor activity of CY and bendamustine in nude mice models. Mice were inoculated with human NSCLC cell line H460. When tumor volume reached 200 mm3, mice were treated with single dose of bendamustine (40 mg/kg, MTD) or CY (20, 40, 60 mg/kg). In (G), tumors were dissected out after 15 days posttreatment.",
    "molecules": "CY, bendamustine"
  },
  {
    "caption": "Dose curve of CY, fludarabine, and bendamustine using fresh CLL samples. Data represent mean ± SD.",
    "molecules": "CY, bendamustine, fludarabine"
  },
  {
    "caption": "CY preferentially killed CLL cells over healthy B cells. Data represent mean ± SD",
    "molecules": "CY"
  },
  {
    "caption": "Toxicity of CY190602 in patients harboring deletion of chromosome 17p (including p53 locus) compared to patients without del17p. Viability of samples was normalized to background apoptosis for individual patient samples. For all experiments, cell viability was determined after 72 h of drug treatment. Data represent mean ± SD.",
    "molecules": "CY190602"
  },
  {
    "caption": "Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP, and FLAG-TCHP at N- and C- terminal. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.",
    "molecules": "Red Ponceau"
  },
  {
    "caption": "Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP ∆1) and 2 (TCHP ∆2), as indicated in the scheme. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.",
    "molecules": "Red Ponceau"
  },
  {
    "caption": "Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: Quantification of PCM1 degradation. (one-way ANOVA; *p=0.0180 vs control time 0).",
    "molecules": "CHX, cycloheximide, MG132"
  },
  {
    "caption": "Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: Quantification of GABARAP degradation. (one-way ANOVA; *p =0.0215 vs control time 0).",
    "molecules": "CHX, cycloheximide, MG132"
  },
  {
    "caption": "Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control.",
    "molecules": "CHX"
  },
  {
    "caption": "Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: Quantification of C (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0003 vs control CHX) and D (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0056 vs control CHX).",
    "molecules": "CHX"
  },
  {
    "caption": "Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP).",
    "molecules": "BafA1"
  },
  {
    "caption": "HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).",
    "molecules": "BafA1, Torin-1"
  },
  {
    "caption": "The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.",
    "molecules": "BafA1, Triton X- 100, TX-100"
  },
  {
    "caption": "Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).",
    "molecules": "BafA1"
  },
  {
    "caption": "Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).",
    "molecules": "BafA1"
  },
  {
    "caption": "Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO).",
    "molecules": "BAY, DMSO"
  },
  {
    "caption": "Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO).",
    "molecules": "DMSO"
  },
  {
    "caption": "migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO).",
    "molecules": "BAY, DMSO"
  },
  {
    "caption": "A Young and aged adrenal gland immunolabelled with HSPG2, NG2 and Podocalyxin. Higher magnification insets show cortex (Co) and medulla (Me). Combined box and whiskers, and scatter plot with quantification of vessel density in young and aged adrenal glands. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " B Young and aged ovary immunolabelled with Sca-1, PDGFRβ and Emcn. Higher magnification insets show cortex and medulla. Combo plot with quantification of vessel density in young and aged ovary. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm. ",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " C Young (10-weeks-old) and aged pituitary gland immunolabelled with ESM-1, Collagen IV and Emcn. Insets with higher magnifications of pars distalis and pars nervosa regions. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Pd: Pars Distalis; Pn: Pars Nervosa. Scale bars for tile scan 3D images 200 µm, for insets 50 µm. ",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " D Young and aged testis immunolabelled with Tie-2 and HSPG2. Higher magnifications of cortex and medulla. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Scale bars for tile scan 3D images 200 µm, for insets 50 µm. ",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " E Young and aged pancreas immunolabelled with SM22α and Endoglin. Insets representative of higher magnifications of regions in young and aged pancreas. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Scale bars for tile scan 3D images 200 µm, for insets 50 µm. ",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " F Young and aged thyroid gland immunolabelled with CXCR4, Laminin and Emcn. Combo plot shows quantification of vessel density in young and aged thyroid glands. Insets: periphery and centre of the thyroid gland. The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. ; Pe: Periphery Ct: Center Scale bars for tile scan 3D images 200 µm, for insets 50 µm. ",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " A 3D images (left) with α-SMA and CD31 immunostaining in young and aged human adrenal gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI. ",
    "molecules": "DAPI"
  },
  {
    "caption": " B 3D images of young and aged human ovary immunolabelled with α-SMA, Vimentin and CD31. Images in the middle panel with a surface rendered for all the channels. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI. ",
    "molecules": "DAPI"
  },
  {
    "caption": " C Representative 3D images (left) of young and aged human testis with SM22α and Endoglin immunostaining. Middle panel images surface rendered for all the channels. Arrowheads indicate arteries. Combo plots show quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI. ",
    "molecules": "DAPI"
  },
  {
    "caption": " D 3D images from young and aged human pancreas staining with α-SMA, Vimentin and Endoglin. 3D images (middle) with a surface rendering for all the channels. Arrowheads indicate arteries. Combined box and whiskers, and scatter plots with the quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI. ",
    "molecules": "DAPI"
  },
  {
    "caption": " E Representative 3D images (left) show α-SMA and CD31 immunostaining in young and aged human thyroid gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI. ",
    "molecules": "DAPI"
  },
  {
    "caption": " A Representative 3D images with Insulin and Caveolin-1 immunostaining in pancreatic islets from young mice. Combined box and whiskers, and scatter plots show the vessel density analysis in the pancreatic islets and non-islet areas from young and aged mice. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. ",
    "molecules": "Insulin, TO-PRO-3"
  },
  {
    "caption": " B Representative 3D images with adult pancreatic islets immunolabeled with Ki67 and VE-Cadherin. Inset (*) shows single-cell resolution and surface rendering for all the channels. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. ",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " C Representative tile scan 3D image with Insulin, ESM-1 and Emcn immunostaining in 19-weeks-old mouse pancreas. Higher magnification at the top of the right panel shows the ESM-1+ pancreatic islets. Inset (*) shows single-cell resolution and surface rendering for ESM-1 and Emcn. The white and dashed line in the tile scan image represents the outline of one lobe of the pancreas, and the dashed lines in the inset represents the pancreatic islet. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. ",
    "molecules": "Insulin, TO-PRO-3"
  },
  {
    "caption": "D Representative 3D images show CD31 and Emcn expressions in young pancreas. Insets (* and #) show higher magnifications of capillaries in the pancreatic islets and non-islet areas. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " F Representative 3D images (left) with Insulin, Ki67 and VE-Cadherin immunostaining on young and aged pancreas. 3D images (middle) show CXCR4, Ki67 and VE-Cadherin in young and aged pancreatic islet. Insets (yellow asterisks) show single-cell resolution and surface rendering for Ki67 and CXCR4. Arrowheads represent Ki67+ β-cell. G Combo plot shows the quantification of Ki67+ β-cells normalized to the total number of β-cells per islet in juvenile (3-weeks-old), adult (12-weeks-old) and aged (55-weeks-old) pancreatic islets. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.",
    "molecules": "Insulin, TO-PRO-3"
  },
  {
    "caption": " H Representative 3D images with Insulin, CD31 and Emcn immunostaining in young and aged pancreas. Combo plot shows the CD31+ Emcnhi vessel density in juvenile, adult and aged pancreatic islets. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. ",
    "molecules": "Insulin, TO-PRO-3"
  },
  {
    "caption": " I Representative 3D images of adult (10-weeks-old) and aged pancreas (55-weeks-old) with Insulin and VEGFA immunostaining. Combo plot (middle) shows the VEGFA as determined by ELISA assay in isolated islets from 10-weeks-old adult and 55-weeks-old aged mice. Combo plot (right) shows the percentage of VEGFA per islet (f.u). Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. ",
    "molecules": "Insulin, TO-PRO-3"
  },
  {
    "caption": " A Representative 3D images of pancreas from adult (18-weeks-old) sunitinib-treated and control mice with Insulin, CD31 and Emcn immunostaining. B Combined box and whiskers, and scatter plot (left) shows the quantification of CD31+ Emcnhi vessel density in sunitinib-treated and control pancreatic islets. Combo plot (right) shows ELISA based analysis of Insulin. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. ",
    "molecules": "DAPI, Insulin, sunitinib, TO-PRO-3"
  },
  {
    "caption": " C Representative 3D images (upper panel) show CXCR4, Ki67 and VE-Cadherin in sunitinib-treated and control pancreatic islet. 3D images (bottom) show sunitinib-treated and control pancreatic islets immunolabeled with Insulin and VE-Cadherin. D Combo plot (top) shows the percentage of CXCR4+ β-cells per islet in sunitinib-treated and control pancreatic islet. Combo plot (middle) shows the quantification of Ki67+ β-cells normalized to the total number of β-cell numbers. Combo plot (bottom) shows the percentage of Insulin+ β-cells per islet. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit.",
    "molecules": "DAPI, Insulin, sunitinib, TO-PRO-3"
  },
  {
    "caption": " E Representative 3D images of pancreas from adult Vegfr2i∆EC mutant and control mice with Insulin, CD31 and Emcn immunostainings. F Combo plot (left) with quantification of CD31+ Emcnhi vessel density in Vegfr2i∆EC and control pancreatic islet. Right combo plot with insulin estimation in pancreas by ELISA assay. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit.",
    "molecules": "DAPI, insulin, Insulin, TO-PRO-3"
  },
  {
    "caption": " G Representative 3D images in the upper panel show CXCR4, Ki67 and VE-Cadherin in islets from Vegfr2i∆EC mutant and control mice. 3D images (lower panel) show Vegfr2i∆EC mutant and control islets immunolabeled with Insulin and VE-Cadherin. H Combo plot (top) shows the percentage of CXCR4+ β-cells per islet in Vegfr2i∆EC mutant and control pancreatic islet. Combo plot (middle) shows the quantification of Ki67+ β-cells normalized to the number of β-cells. Combo plot (bottom) shows the percentage of Insulin+ β-cells per islet. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit.",
    "molecules": "DAPI, Insulin, TO-PRO-3"
  },
  {
    "caption": " A Representative tile scan 3D images with α-SMA, Isolectin, FABP4 and Emcn in the pancreas from 17-weeks-old Gja1i∆EC mutant and control mice. Insets show higher magnifications of regions. B Box whiskers, and scatter plot (left) shows the quantification of CD31+ Emcnhi vessel density in Gja1i∆EC mutant and control pancreatic islets. (n=7), P-value, and two-tailed unpaired t-test. Combo plot (right) shows the quantification of P-Histone H3+ EC numbers per islet (f.u) counted on thick murine pancreatic sections. (n=5), P-value, and two-tailed unpaired t-test. C 3D images show CD31 and Emcn immunostaining in the Gja1i∆EC mutant and control pancreas. The scale bars are 50 µm. D Combined violin, scatter and sample distribution plot shows the quantification of β-cell mass (%) in pancreatic islets from Gja1i∆EC and control mice. Data represent mean ± s.d. (n=140 islets from 7 pancreas), P-value, and two-tailed unpaired t-test. E Representative 3D images with immunostaining of α-SMA, Insulin and VE-Cadherin in the pancreatic islets from Gja1i∆EC and control mice. The scale bars are 50 µm. F Combo plot (left) shows insulin measurement in lysate of pancreas by ELISA. Combo plot (right) shows the percentage of Insulin+ β-cells per islet in Gja1i∆EC mutant and control mice. (n=5), P-values, and two-tailed unpaired t-tests. Data information: The dashed lines in panel A represent the outlines of pancreas. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: DAPI Is: pancreatic islet. f.u: fluorescence unit. The line on the right side of the combo plots represent the sample distribution. Scale bars are 200 µm for tile scan images of longitudinal thick sections across the entire glands, and 50 µm for the higher magnification insets images.",
    "molecules": "DAPI, Insulin, insulin"
  },
  {
    "caption": " G Representative 3D images of ESM-1 and Caveolin-1 in pancreatic islets from Gja1i∆EC and control mice. Insets (* and #) show higher magnifications with surface rendering for ESM-1 and Caveolin-1 immunostaining. Combo plot with the quantification of ESM-1+ cell numbers per islet (f.u). (n=5), P-value, and two-tailed unpaired t-test. The scale bars for the 3D images are 50 µm, and 5 µm for the higher magnifications with single-cell resolution images. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. ",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " H 3D images with CXCR4 and Emcn immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. I Representative 3D images show CXCR4, Ki67 and VE-Cadherin immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. J Combo plots show the percentage of CXCR4+ β-cells per islet and Ki67+ β-cells normalized to the total number of β-cells per islet in Gja1i∆EC mutant versus the littermate control mice. (n=5), P-values, and two-tailed unpaired t-tests. K Box whiskers, and scatter plot shows the quantification of CD31+ Emcnhi vessel density in aged Gja1i∆EC mutant and control pancreatic islets. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Combo plot (below) shows the quantification of β-cells on thick murine pancreatic sections. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Data information: The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": " (b) 2D-[1H;15N]-TROSY spectrum of 400 µM 2H,13C,15N-labeled TREM2-TMH (residues R161-H206) in DPC micelles at 37 °C. Assigned resonances are labeled. ",
    "molecules": "micelles, 13C, 2H, DPC, 1H, 15N"
  },
  {
    "caption": " (d) Circular dichroism (CD) spectrum of TREM2-TMH in DPC micelles indicates an α-helical secondary structure content of ~50%. ",
    "molecules": "micelles, DPC"
  },
  {
    "caption": " (e) 3D-15N-edited NOESY spectra show that the transmembrane helix is interrupted at residues 190-192. Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α-helical secondary structure. ",
    "molecules": "15N"
  },
  {
    "caption": "(a, b) Overlay of 2D-[1H,15N]-TROSY spectra of 2H,15N-labeled TREM2-TMH in DPC micelles (black) and in complex with (a) unlabeled DAP12 (red) or (b) TREM2-TMH lacking its positively charged lysine residue (K186A, red).",
    "molecules": "micelles, 2H, DPC, 1H, lysine, 15N"
  },
  {
    "caption": " (a) Paramagnetic relaxation enhancement (PRE) with a free-radical-modified stearic fatty acid in DPC micelles. Low values indicate location inside whereas values close to one suggest a location outside the detergent micelle. Orange bars: TREM2-TMH wt, green bars: K186A variant. The N-terminal residues of TREM2-TMH-wt are located deeper inside the micelles as in the K186A case, whereas the C-terminal end is positioned at the outside for wild-type and inside for the K186A variant. Negative bars indicate missing assignments. ",
    "molecules": "micelle, micelles, DPC, stearic fatty acid"
  },
  {
    "caption": "(b) NOE-cross peaks between backbone amides of TREM2-TMH wt (top) or the K186A variant (bottom) and selected regions of DPC (see color coding of the structure of the DPC molecule on the right). The K186A variant shows strong contacts to the hydrophobic aliphatic regions of the detergent throughout the transmembrane helix whereas no such contacts can be seen at the kink site of the wild-type helix, indicating a location outside of the micelle for this part.",
    "molecules": "micelle, DPC"
  },
  {
    "caption": " (b) Immunoblot detection of ICD accumulation of TREM2 wildtype, K186A, K186P, and K186L variants mediated by γ-secretase cleavage upon overnight incubation of membrane preparations at 37 °C. ICD formation is blocked by 1 µM of the transition state analogue L-685,458. Note that inhibition of ICD formation from wt TREM2 was less efficient. Due to the charge within the TMH wt TREM2 is less efficiently transported to the cell surface (Lanier et al, 1998) and therefore retained within the endoplasmic reticulum, where other proteases not related to γ-secretase and therefore not inhabitable by L-685,458 may cleave. In vitro generation of the ICD of APP (AICD) was used as a positive control. Immunoblot detection of mature nicastrin and the presenilin 1 NTF, which results from presenilin endoproteolysis necessary to activate γ-secretase (Thinakaran et al, 1996), proves comparable amounts of γ-secretase in the individual assays. ",
    "molecules": "L-685,458"
  },
  {
    "caption": "(f) Water exchange peak as observed in a 3D-15N-edited-[1H,1H]-NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond.",
    "molecules": "1H, 15N"
  },
  {
    "caption": "Accumulation of autophagy adaptors NBR1 and SQSTM1 and the phagophore membrane component LC3 is induced in fibroblasts from two controls and a subject with Vici syndrome (subject 4.1) after 12 h of treatment with rapamycin or dual treatment with rapamycin and bafilomycin.",
    "molecules": "bafilomycin, rapamycin"
  },
  {
    "caption": "In control fibroblasts subjected to 6 h of bafilomycin treatment, lysosomal structures were detected by staining with monoclonal antibody to LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, fibroblasts from individuals with Vici syndrome consistently show smaller LC3-positive puncta that only sporadically colocalize with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici syndrome cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar, 5 μm.",
    "molecules": "bafilomycin"
  },
  {
    "caption": "(G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly.",
    "molecules": "NP40, NaCl, salt"
  },
  {
    "caption": "(B) Expression fold changes (ecdysone inducibility) versus measurements without ecdysone and grouped by native core promoter sequences. The colors refer to different core promoter architectures. Three types of line are used to indicate the expression fold change with no increase (y = x; solid line), 2-fold increase (y = x + 1; dotted line) and 4-fold increase (y = x + 2; dashed line). Red vertical dashed line: log2 basal expressions = 2.",
    "molecules": "ecdysone"
  },
  {
    "caption": "(C) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters. Disrupted INR (highlighted with the black dotted line rectangle) had a slightly negative effect on changing the core promoter responsiveness to ecdysone. (~ 2.3-fold reduction on average, Wilcoxon rank-sum test p = 2.1×10-5). Constitutive and developmental promoters highlighted in red and green, respectively.",
    "molecules": "ecdysone"
  },
  {
    "caption": "(E) APP (magenta) and BACE1 (green) colocalisation in dendrites of siCD2AP- and siControl-treated neurons expressing APP-RFP and BACE1-GFP upon DAPT treatment, recorded by time-lapse spinning-disk confocal microscopy for 120 sec (1 fps) (see supplementary movie 1). APP and BACE1 in dendrites at 0 sec are shown and during 120 sec in kymographs (bottom panels). Dotted white lines in kymographs highlight APP vesicles positive for BACE1. Scale bar, 10 µm.(F) Quantification of the colocalisation between APP and BACE1 in dendrites (n=3, NsiControl=18, NsiCD2AP=21; ****P<0.0001, t-test; mean ± SEM)",
    "molecules": "DAPT"
  },
  {
    "caption": "(G) APP (magenta) and BACE1 (green) colocalisation in axons of siBin1- and siControl-treated neurons expressing APP-RFP and BACE1-GFP upon DAPT treatment, recorded by time-lapse spinning-disk confocal microscopy for 120 sec (1 fps) (see supplementary movie 2). APP and BACE1 in axons at 0 sec are shown and during 120 sec in kymographs (bottom panels). Dotted white lines in kymographs highlight APP vesicles positive for BACE1. Scale bar, 10 µm.(H) Quantification of the colocalisation between APP and BACE1 in axons (n=4, NsiControl =17, NsiBin1=27; ****P<0.0001, t-test; mean ± SEM).",
    "molecules": "DAPT"
  },
  {
    "caption": "(A) APP detected with anti-APP (Y188, green) at early endosomes (anti-EEA1, magenta) in dendrites of siCD2AP- and siControl-treated neurons with or without DAPT treatment, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares are magnified on the right panels. Scale bar, 1 µm.(B) Quantification of colocalisation between APP and EEA1-positive dendritic endosomes (n=3, NsiControl DMSO=15, NsiControl DAPT=14; NsiCD2AP DMSO=21, NsiCD2AP DAPT=22; ****P<0.0001 siCD2AP DMSO vs. siControl DMSO, ####P<0.0001 siCD2AP DAPT vs. siCD2AP DMSO, t-test, mean ± SEM).",
    "molecules": "DAPT, DMSO"
  },
  {
    "caption": "(C) APP (green) distribution within enlarged Rab5QL-GFP endosomes (magenta) in dendrites of siCD2AP- or siControl-treated neurons expressing APP-RFP and Rab5QL-GFP upon DAPT treatment, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares indicate a Rab5QL-positive endosome magnified on the right. Scale bar, 1 µm.(D) Qualitative analysis of APP distribution between the lumen and the membrane of Rab5QL-endosomes (n=4, NsiControl=45 (774 endosomes), NsiCD2AP=52 (888 endosomes); *P=0.0368 siCD2AP vs. siControl, t-test, mean ± SEM).",
    "molecules": "DAPT"
  },
  {
    "caption": "(E - F) APP (green) distribution within enlarged Rab5QL-GFP endosomes (magenta) in cell bodies of siCD2AP- (f) or siControl- (e) treated neurons expressing APP-RFP, upon DAPT treatment, analysed by spinning-disk confocal microscopy. APP (green) and Rab5QL (magenta) line intensity profiles along the endosome (see inset line) are shown on the right. Scale bars, 1 µm.(G) Quantification of the amount of APP fluorescence at the limiting endosomal membrane normalized to total APP fluorescence per endosome (n=3, NsiControl=32 endosomes (11 cells), NsiCD2AP=39 endosomes (15 cells); ****P<0.0001, t-test, mean ± SEM).",
    "molecules": "DAPT"
  },
  {
    "caption": "(H) APP detected with anti-APP (Y188, green) at early endosomes (anti-EEA1, magenta) in dendrites of siCD2AP- and siControl-treated neurons upon DAPT treatment, analysed by super-resolution dSTORM imaging. Scale bars, 200 nm.",
    "molecules": "DAPT"
  },
  {
    "caption": "(I) Qualitative analysis of super-resolved APP distribution between the lumen and the membrane of EEA1-positive endosomes of siCD2AP- or siControl-treated neurons upon DAPT treatment (n=3, NsiControl=35, NsiCD2AP=57, **P=0.0012, Mann-Whitney test, mean ± SEM).",
    "molecules": "DAPT"
  },
  {
    "caption": "C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub.",
    "molecules": "DTT, ATP, Thioester"
  },
  {
    "caption": "(K) Control (WT) and TRIP6-KO HEK293A cells were stained for YAP and TRIP6. Merged image shows YAP (green), TRIP6 (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ****P≤0.0001, Fisher's test). Scale bar=20µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) MCF10A cells were infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4) and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue) (quantification of LATS1 localization at cell-cell junctions is shown in Figure EV3D). Scale bar=20µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) MCF10A cells were either not treated or treated separately with Latrunculin B, Blebbistatin, EGTA, serum starvation, and Y27632, and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.",
    "molecules": "Blebbistatin, DNA, EGTA, Latrunculin B, Y27632"
  },
  {
    "caption": "(B) MCF10A cells were either not treated (control) or treated separately by growth to high density, serum starvation, Latrunculin B, Blebbistatin, EGTA, or Y27632 treatment, and stained using anti-vinculin antibodies by immunofluorescence. Scale bar=20µm.",
    "molecules": "Blebbistatin, EGTA, Latrunculin B, Y27632"
  },
  {
    "caption": "(E) Vinculin was knocked down as described in (D) and cells were stained for YAP and vinculin. Merged image shows YAP (green), vinculin (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ***P≤0.001, Fisher's test). Scale bar=20µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Vinculin was knocked down as described (Figure 5D) in MCF10A cells and cells were stained for LATS1 and TRIP6. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(E, F) MCF10A cells were treated separately (or not) with Latrunculin B (E) and by serum starvation (F) and LATS1 immune complexes and lysates were probed by western blotting for vinculin, LATS1, and TRIP6. Quantification of LATS1-TRIP6-vinculin binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).",
    "molecules": "Latrunculin B"
  },
  {
    "caption": "(H) Vinculin was knocked down as described (Figure 5D) in MCF10A cells grown at high density on PDMS membranes. The membranes were stretched at 17% elongation for 2 hours and fixed while under tension and stained for TRIP6. Merged images show TRIP6 (red) and DNA (blue). Scale bar=20µm.",
    "molecules": "DNA"
  },
  {
    "caption": "G, Graph plots showing the fraction of c-Fos expressing Gal-positive (Galc-Fos+) and Gal-negative neurons (DAPIc-Fos+) in the CL, PCN, and CM after pup retrieval test (G, top) or object exposure (G, bottom). Data were analyzed using 2-way ANOVA and expressed as mean ± s.e.m.; n=3; *p < 0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "E, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the PFC of virgin females, 2 hours following CNO/vehicle administration. Masks demonstrating distribution of DAPI + c-Fos (green), DAPI + dTomato (magenta) and their colocalization (white). Scale bars 100 µm, 20× magnification.",
    "molecules": "CNO, DAPI"
  },
  {
    "caption": "F, Fraction c-Fos+ cells of the total DAPI (top) or hM3D-2A-dTomato (bottom) population in the ACC, following CNO/vehicle administration. (n=3). Data were analyzed using student's t-test and are displayed as mean ± s.e.m; * p< 0.05, ** p< 0.01.",
    "molecules": "CNO, DAPI"
  },
  {
    "caption": "B, Representative images showing AAV-DIO-CAG-hM3D-2A-tdTomato and c-Fos expression in the CL, 2 h after CNO administration. Scale bars 200 µm, 10× magnification (left) and 50 µm, 20× magnification (right).",
    "molecules": "CNO"
  },
  {
    "caption": "DFL inhibits HMGB1-induced, but not fMLP-induced cell migration. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers, 1 nM HMGB1 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points with average ± standard deviation (avg ± sd; n=3, each point represents a biological replicate) in a representative experiment.",
    "molecules": "DFL, fMLP"
  },
  {
    "caption": "Histogram showing residue-specific CSPs of 15N-labelled HMGB1 (~0.1 mM) upon addition of ten-fold excess of DFL (helices are schematically represented on the top). Missing residues are prolines or are absent because of exchange with the solvent. Box A and Box B residues with CSP > avg + sd are represented in magenta and blue, respectively.",
    "molecules": "DFL, 15N"
  },
  {
    "caption": "The R23A/R109A HMGB1 double mutant has chemotactic activity but is not inhibited by DFL (the experiment was carried out as described in panel B). The data points represent three biological replicates in one experiment (of 2 performed in different days); the avg ± sd is shown.",
    "molecules": "DFL"
  },
  {
    "caption": "Histogram showing the CSPs of 15N-labelled CXCL12 amides (~0.1 mM) upon addition of ten-fold excess of DFL. Missing residues are prolines. Elements of secondary structure are depicted on top.",
    "molecules": "DFL, 15N"
  },
  {
    "caption": "Top: 1H spectrum of DFL, where the numbered peaks correspond to DFL protons (left). Middle: superimposition of STD spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) and upon addition of 0.2 mM of CXCR41-38sY21 (red line). Bottom: waterLOGSY spectra obtained for 0.5 mM DFL with 0.05 mM CXCL12 (black line) upon addition of 0.2 mM of CXCR41-38sY21 (red line).",
    "molecules": "DFL"
  },
  {
    "caption": "DFL does not inhibit CXCL12 induced chemotaxis. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM CXCL12 or no chemoattractant was added in the lower chamber, together with the indicated concentrations of DFL. Data points (n=3) with avg ± sd in one representative experiment (of 2 performed in different days); the migration in the presence of increasing concentrations of DFL",
    "molecules": "DFL"
  },
  {
    "caption": "DFL inhibits chemotaxis toward the HMGB1/CXCL12 heterocomplex. Data points (n=3) with avg ± sd in one representative experiment (of 3 performed in different days). Migration in the presence or absence of DFL is significantly different (p=0.0037, one-way ANOVA plus Dunnett's post test; ** p<0.01 relative to no DFL addition).",
    "molecules": "DFL"
  },
  {
    "caption": "H-15N HSQC of HMGB1 (0.1 mM) with CXCL12 (0.2 mM) upon addition of (D) 0.2 mM and (E) 1 mM DFL.",
    "molecules": "DFL"
  },
  {
    "caption": "MST measurements of the heterocomplex (preformed using 50 nM HMGB1 and 50 μM CXCL12) in the presence of increasing concentrations of DFL. Decreasing MST signal (from ∼950 to ∼930 a.u.) upon DFL titration indicates that CXCL12 has been displaced from HMGB1. n=3; error bars represent avg ± sd.",
    "molecules": "DFL"
  },
  {
    "caption": "DFL does not affect the cytokine-inducing activities of disulfide HMGB1 (ds-HMGB1). Mouse Bone Marrow Derived Macrophages were activated or not for 3 h with 3 µg/ml ds-HMGB1 (~100 nM) or 10 ng/ml LPS, in the presence of the indicated concentrations of DFL. The data points represent n=3 biological replicates, with avg ± sd, in one representative experiment (of 2 performed in different days). The levels of TNFα mRNA are not significantly different in the presence of increasing concentrations of DFL (one-way ANOVA plus post tests).",
    "molecules": "LPS, DFL"
  },
  {
    "caption": "DFL does not inhibit chemotaxis towards 3S. Mouse 3T3 fibroblasts were subjected to chemotaxis assays in Boyden chambers; 1 nM fully reduced HMGB1 (fr-HMGB1) (green) or 3S (red), or no chemoattractant (black), were added in the lower chamber, together with 1 µg/ml of anti-CXCL12 monoclonal antibody or 30 nM DFL. Data points (n=4) with avg ± sd in one representative experiment (of two performed in different days). Statistics: one-way ANOVA (p<0.0001), followed by Dunnett's post-test. ***, p<0.001 relative to migration towards HMGB1, the migrations towards 3S are not significantly different among each other.",
    "molecules": "DFL"
  },
  {
    "caption": "Quantification of CD45+ cells isolated with immunobeads from injured muscles 6 hours after damage. Before damage mice were treated either with Phosphate Buffered Saline vehicle, Glycyrrhizin or DFL. Two independent experiments. Each point represents a different mouse, avg ± sd are shown (statistics: Kruskal-Willis plus post-tests).",
    "molecules": "Glycyrrhizin, DFL"
  },
  {
    "caption": "B - E HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (B), UNC13D stable knock-down (KD) (D) or knock-out (KO) (C and E) HeLa cells, and their control cells were stimulated with cGAMP (100 nM) for 30 min (B and C) or for the indicated time (D and E) before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -GAPDH, -actin or -tubulin antibody.",
    "molecules": "cGAMP"
  },
  {
    "caption": "F- H HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h (F and G). UNC13D knock-out (KO) and control HeLa cells were stimulated with cGAMP (100 nM) for 30 min (H). Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies (F and G), IgG and anti-STING antibodies (H), followed by immunoblotting (IB) with anti-HA, -Flag antibody or the indicated antibodies.",
    "molecules": "cGAMP"
  },
  {
    "caption": "UNC13D knock-out (KO) HeLa cells and HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. Confocal microscopy was used to determine the localization of endogenous STING (red), ERGIC marker ERGIC-53 (green) and nuclei (blue).",
    "molecules": "cGAMP"
  },
  {
    "caption": "UNC13D knock-out (KO) HeLa cells and HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. The co-localization of STING and ERGIC was analyzed using ImageJ (J).",
    "molecules": "cGAMP"
  },
  {
    "caption": "K, L UNC13D stable knock-down (KD) HeLa cells and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. The aggregation of endogenous STING (red) was examined via confocal microscopy (K). Scale bar: 25 μm. The red fluorescence intensity was determined by ImageJ (L).",
    "molecules": "cGAMP"
  },
  {
    "caption": "A UNC13D knock-out HeLa cells stably expressing FLAG-tagged UNC13D were stimulated with cGAMP (100 nM) or left unstimulated for the indicated time. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with IgG and anti-Flag antibodies, followed by immunoblotting (IB) with the indicated antibodies.",
    "molecules": "cGAMP"
  },
  {
    "caption": "UNC13D knock-out HeLa cells stably expressing FLAG-tagged UNC13D were pretreated with BFA (20 μM) or left untreated for 2 h, followed by stimulation with cGAMP (100 nM) (or left unstimulated) for 30 min. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with IgG and anti-Flag antibodies, followed by immunoblotting (IB) with the indicated antibodies (E).",
    "molecules": "cGAMP, BFA"
  },
  {
    "caption": "H Wild-type HeLa cells were pretreated with BFA (20 μM) or left untreated for 2 h before stimulation with plasmid DNA (1 μg/ml) for 18h or cGAMP (100 nM) for 30 min (or left unstimulated). Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-p-TBK1, -p-IRF3, -STING or -tubulin antibody.",
    "molecules": "cGAMP, BFA"
  },
  {
    "caption": "K, L HEK293T cells were transfected with HA-tagged UNC13D, VN/VC-STING or vector for 24 h, followed by stimulation with cGAMP (100 nM) (or left unstimulated) for 30 min. Nucleus marker DAPI (blue), VN/VC-STING (green) and HA-UNC13D (red) were examined via confocal microscopy (K). The green fluorescence intensity was determined by ImageJ (L). n = 5 biological replicates.",
    "molecules": "cGAMP, DAPI"
  },
  {
    "caption": "D UNC13D knock-out (KO) HeLa cells, UNC13D knock-out HeLa cells stably expressing FLAG-tagged wild-type UNC13D (UNC13D RE) and FHL3 mutation (FHL3 mut), and control cells were stimulated with cGAMP (100 nM) (or left unstimulated) for 30 min. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -Flag, -p-IRF3, -STING or -tubulin antibody.",
    "molecules": "cGAMP"
  },
  {
    "caption": "A-C Viability counts of the indicated V. parahaemolyticus (A-B) or E. coli (C) prey strains before (0 h) and after (4 h) co-incubation with the indicated V. parahaemolyticus attackers on media containing 3% NaCl at 30°C. In A and B, media also contain 0.1% arabinose to induce expression from plasmids. In A, prey strains contain either an empty plasmid (pEmpty) or a plasmid for arabinose-inducible expression of VP1389 (pVP1389). In B and C, prey strains contain an empty plasmid that provides a selection marker, and the attackers are derivatives of a ∆hns mutant (parental). In B, the attackers contain an empty plasmid, or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit. ∆hcp1 was used as a T6SS1- control strain. The experiment was performed at least three times with similar results; results of a representative experiment are shown.",
    "molecules": "arabinose, NaCl"
  },
  {
    "caption": "A VP1388 binds VP1390. Immunoprecipitation using α-FLAG antibodies from E. coli cells co-expressing the indicated C-terminal FLAG- and Myc-tagged proteins from arabinose-inducible plasmids.",
    "molecules": "arabinose"
  },
  {
    "caption": "B VP1388 and VgrG1 co-precipitate with VP1390. Immunoprecipitation using α-FLAG antibodies from V. parahaemolyticus ∆hns/∆hcp1/∆vp1390 derivatives harboring plasmids for the arabinose-inducible expression of FLAG-tagged sfGFP or VP1390. Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids, and 0.1% arabinose. Endogenous VP1388 and VgrG1 were detected using α-VP1388 and α-VgrG1 antibodies, respectively.",
    "molecules": "arabinose, chloramphenicol"
  },
  {
    "caption": "C Expression (cells) and secretion (media) of VP1388, VP1390, and VgrG1 from the indicated V. parahaemolyticus ∆hns-derived strains harboring an empty plasmid (pEmpty) or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Samples were grown in media containing 3% NaCl and supplemented with 0.1% arabinose at 30°C. RNA polymerase β (RNAp) was used as a non-secreted protein loading control. Arrows denote the bands at the expected size of VP1388 or VP1390, as indicated in the appropriate panels.",
    "molecules": "arabinose, NaCl"
  },
  {
    "caption": "A Toxicity of periplasm-targeted proteins in E. coli. E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control), VP1388 or VP1390 fused to an N-terminal PelB signal peptide (perisfGFP, periVP1388, and periVP1390, respectively) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.001% arabinose, to induce protein expression. Plates were incubated over-night at 30°C or 37°C, as indicated.",
    "molecules": "agar, arabinose, glucose, kanamycin"
  },
  {
    "caption": "C VP1390 induces cell lysis in E. coli. Time-lapse microscopy of E. coli cells expressing periplasm-targeted sfGFP, VP1388, or VP1390 (perisfGFP, periVP1388, and periVP1390, respectively) from an arabinose-inducible vector, grown on LB agarose pads supplemented with kanamycin (to maintain the plasmid), 0.2% arabinose (to induce expression), and propidium iodide (PI; pink). Merging of the phase contrast and PI channels are shown. Scale bar = 2 µm.",
    "molecules": "agarose, arabinose, kanamycin, PI, propidium iodide"
  },
  {
    "caption": "Time-lapse microscopy of competition between V. parahaemolyticus ∆hns (T6SS+/VP1390+), ∆hns/∆hcp1 (T6SS-/VP1390+), or ∆hns/∆vp1390 (T6SS+/VP1390-) attackers and V. parahaemolyticus ∆vp1389 prey that express GFP. Attacker and prey were mixed (2:1 ratio) and spotted on LB agarose pads supplemented with propidium iodide (PI; pink). Merging of the phase contrast, GFP (green), and PI (pink) channels, as well as the PI channel alone are shown. Scale bar = 5 µm. The experiment was performed three times with similar results; results of a representative experiment are shown.",
    "molecules": "agarose, PI, propidium iodide"
  },
  {
    "caption": "C) RNA binding-deficient TDP-43 mutants generated by site-directed substitutions in RRM1 (F2L) or in both RRM domains (F4L) and substitution of RRM1 and RRM2 with monomeric GFP (N-GFP-C). Phase separation of the mutants without added RNA (control) or in the presence A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) at 150 mM NaCl (4 µM protein).",
    "molecules": "A(CA)6, A(GU)18, A(GU)6, NaCl"
  },
  {
    "caption": "D) Lysate from HEK 293 cells expressing a GFP-tagged copy of either wild-type (WT) or F147/149/229/231L (F4L) TDP-43 was added to a mixture of recombinant WT or F4L TDP-43 (5.3 μM, 10% Cy3-labeled protein) and A(GU)6 (22.8 μM), A(CA)18 (7.6 μM), A(GU)18 (7.6 μM), or no RNA control at 250 mM NaCl. Droplets were imaged by brightfield and fluorescence microscopy. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm. E) LLPS measured by turbidity in the same experimental conditions as panel D. Mean and SD of 4 biological replicates using 2 recombinant protein preparations. Analyzed by one-way ANOVA (F(7,24)=69.30, P<0.0001). Sidak's multiple comparisons test was used to compare selected groups. *P<0.05, ****P<0.0001. ",
    "molecules": "A(CA)18, A(GU)18, A(GU)6, Cy3, NaCl"
  },
  {
    "caption": "A) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or increasing A(GU)18 RNA concentration at 250 mM salt. Representative images from 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.",
    "molecules": "A(GU)18, Oregon green, salt"
  },
  {
    "caption": "D) Phase separation of TDP-43 (4 μM) at increasing salt concentration without RNA or in the presence of increasing A(GU)18 concentration. Scale bars, 10 μm. Inlay added to top right panel for increased visibility, scale bar 1 μm. E) Phase diagram derived from panel D, denoting either no droplets or droplet formation for each condition. ",
    "molecules": "A(GU)18, salt"
  },
  {
    "caption": "A) TDP-43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or GU-repeat RNA oligonucleotides of increasing length at 250 mM salt. As indicated, RNA concentration varied to maintain a constant total number of TDP-43 binding sites. Scale bars, 10 μm. B) The plot shows droplet area quantified as a function of GU-repeat length. Mean and SD of >300 droplets from 3 biological replicates using 3 different protein preparations. C) TDP-43 concentration in the light phase (Cout) as a function of GU (blue) or CA (red)-repeat length quantified by Bradford protein assay. Mean and SD from >5 biological replicates using 2 different protein preparations. (250 mM NaCl, 4 µM TDP-43) ",
    "molecules": "CA, GU, Oregon green, NaCl, salt"
  },
  {
    "caption": "B) Phase separation of full-length and TDP-43 variants (4 μM) mixed with A(GU)30 RNA (0.8 μM) or no RNA control at 250 mM NaCl observed by brightfield microscopy. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.",
    "molecules": "A(GU)30, NaCl"
  },
  {
    "caption": "A) TDP-43 ALS-linked amino acid substitutions K181E, A315T/E, A321G, Q331K, M337V, and A382T. Boxed in the C-terminal domain is the α-helical structure within the conserved region (a.a. 320-343). Droplets formed by TDP-43 wild-type (WT) and the different mutants (4 μM) observed by brightfield microscopy in the presence of no RNA control or A(GU)18 RNA (3.9 μM) at 250 mM NaCl. Mutations A321G and M337V, showing decreased liquidity of the condensates in the presence of A(GU)18, are boxed in red. Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V, 2 preparations of A321G, Q331K, K181E and one preparation of A315T/E and A382T. Scale bars, 10 μm.",
    "molecules": "A(GU)18, amino acid, NaCl"
  },
  {
    "caption": "B) Phase separation of WT (4 μM, 10% Oregon green-labeled protein) and M337V (4 μM, 10% Cy-3-labeled protein) observed by brightfield and fluorescence microscopy in the presence of A(GU)18 (3.9 μM) at 250 mM NaCl. The middle panel shows mixing of these WT (2 μM) and M337V (2 μM) samples. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.",
    "molecules": "A(GU)18, Cy-3, Oregon green, NaCl"
  },
  {
    "caption": "A) Binding of TDP-43 to GU-rich RNA promotes liquid properties of TDP-43 condensates, as shown by the fusion of small condensates into larger droplets. This fluidity is impaired by the loss of RNA binding affinity or disease-linked mutations that alter phase separation, such as M337V and A321G, which could explain increased TDP-43 aggregation.",
    "molecules": "GU"
  },
  {
    "caption": "E. Validation of individual targets by manually curated in vitro ubiquitylation  experiments. HA-tagged (hemagglutinin) candidate proteins were purified from Hek293T cells via anti-HA immunoprecipitation  (IP) followed by HA-peptide elution. Candidates were incubated with 20 µM His6-Ubiquitin, 170 nM E1, E2s (0.5 µM each of  UbcH5b and Cdc34) and 5 mM  ATP in the presence or absence of 0.1 µM SCFFbxw5 for 2 hours at 37°C. Substrates were detected via SDS-PAGE followed by western blotting using anti-HA  antibodies for detection.",
    "molecules": "ATP, hemagglutinin"
  },
  {
    "caption": "C. RPE-1  cells were transfected with the indicated siRNAs (72 hours total time), split on coverslips 24 hours later and serum-starved for the last 24 hours. Cells were fixed in methanol  and analysed via  immunofluorescence  using the indicated antibodies together with Hoechst staining. Left: Maximum intensity projections (same for all following microscopy images) of representative images. Arrows indicate ODF2  signal. Scale bar = 5 µm. Note: Middle panel shows an example image of siFw5-3. Right: Quantification of  MCAK signals  co-localising with ODF2. Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells. Asterisks indicate p-value of a Mann Whitney test comparing each sample set with the non-targeting control (**P < 0.01, ***P < 0.001).",
    "molecules": "Hoechst, methanol"
  },
  {
    "caption": "A. Fluorescence-based pulse-chase experiment using a monoclonal RPE-1 cell line expressing mNG-MCAK under a doxycycline-inducible promoter. Cells were transfected with the indicated siRNAs (siFw5-1&3 together) and split 24 hours later on Ibidi 8 Well Glass Bottom µ-slides while simultaneously inducing mNG-MCAK expression with 6 ng/ml doxycycline (pulse). 24 hours later, doxycycline was washed out and cells were released into serum-free medium. Cells that have just finished or were about to finish mitosis (distinguishable for example by the midbody signal of mNG-MCAK (arrow heads)) were selected and imaged over 24 hours, taking an image every 20 min (chase). Left: representative images of selected time points. Scale bar = 10 µm. Arrows indicate centrosomal mNG-MCAK signals, arrowheads  mNG-MCAK  signals at the midbody. Right: Quantification of centrosomal mNG-MCAK  signals. Error bars show standard error of the mean of 3 independent experiments with 25 cells in total. P-value of a two-tailed unpaired Students t-test comparing signal intensity of Fbxw5  knockdown over non-targeting control was mostly below 0.01 for time points 0 to 8 hours and below 0.05 for  time points 9h to 20h. Dark and light blue lines indicate the time points at which  mNG-MCAK  signals are reduced by 50% compared to time point  0 for siNT and siFbxw5  samples, respectively.",
    "molecules": "doxycycline"
  },
  {
    "caption": "D. Same samples as in C, except that cells grown on coverslips were fixed in methanol and analysed by  immunofluorescence. Left: Representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Right: Quantification. Error bars show standard error of the mean of 3 independent experiments, covering in total more than 150 cells. Asterisks indicate the p-value of a Mann Whitney test (***P < 0.001).",
    "molecules": "methanol"
  },
  {
    "caption": "B. CHX chase experiment as in A, except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h, released for 9 hours and again arrested for 16h. Following the 2nd release, 50 µg/ml CHX  was added as indicated and samples were analysed as in A. Left: representative blots. Asterisks indicate unspecific bands detected by the Fbxw5  antibody. Right: Quantification of  MCAK/Uba2  signal intensity ratios normalised to time point  zero. Error bars show standard deviation of 6 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).",
    "molecules": "CHX, thymidine"
  },
  {
    "caption": "C. Extracts of RPE-1 cells treated with the indicated siRNAs for 72 hours, grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO (-) or 10 µM MG-132 (+) for the very last 6 hours. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5  antibody. Right: Quantification of MCAK/Uba2 signal ratio normalised to non-targeting control with DMSO  of 6 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test (**P < 0.01).",
    "molecules": "DMSO, MG-132"
  },
  {
    "caption": "A. Polyclonal  RPE-1  cells expressing  mNG-MCAK under a  doxycycline-inducible promoter were seeded on coverslips, induced with 20 ng/ml doxycycline and grown for 24 hours. Subsequently, cells were washed twice with PBS  and incubated for another 24 hours in  serum-free medium without doxycycline. Cells were then fixed in methanol  and subjected to  IF  using the indicated antibodies together with Hoechst staining. Left: Representative images. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).",
    "molecules": "Hoechst, doxycycline, methanol, PBS"
  },
  {
    "caption": "C. RPE-1 cells were treated with the indicated siRNA for 48 hours followed by serum-starvation for another 24 hours. Cells were then fixed in methanol and subjected to  IF  using the indicated antibodies together with  Hoechst staining. Left: Representative images. Note: Left panel shows an example image of siFw5-1. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).",
    "molecules": "Hoechst, methanol"
  },
  {
    "caption": "(a-d) ELISA for IL-1β (a-c), IL-18 (d) and IL-6 (e) in supernatants of resting or LPS-primed bone marrow-derived macrophages (BMMs) stimulated with palmitate conjugated to BSA (PA-BSA) or BSA control.",
    "molecules": "LPS, PA, palmitate"
  },
  {
    "caption": "(e,f) ELISA for IL-1β (e) and IL-18 (f) in supernatants. Resting or LPS-primed BMMs generated from wild-type (WT), Nlrp3−/−, Pycard−/− or Nlrc4−/− mice were stimulated with PA-BSA.",
    "molecules": "LPS, PA"
  },
  {
    "caption": "(g) IL-1β ELISA of supernatants from LPS-primed BMMs stimulated with PA-BSA in the absence or presence of the pan-caspase inhibitor zVAD or caspase-1 inhibitor zYVAD.",
    "molecules": "LPS, PA, zVAD, zYVAD"
  },
  {
    "caption": "(h) ELISA for IL-1β in supernatants. Resting or LPS-primed BMMs generated from WT or Casp1-/- mice were stimulated with PA-BSA, BSA control, ATP or nigericin.",
    "molecules": "ATP, LPS, nigericin, PA"
  },
  {
    "caption": "(i) ELISA for TNF in supernatants. Resting or LPS-primed BMMs generated from WT, Nlrp3-/-, Pycard-/- or Casp1-/- mice were stimulated with PA-BSA as indicated. Values are expressed as mean ± s.d., and the results are representative of three independent experiments.",
    "molecules": "LPS, PA"
  },
  {
    "caption": "Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments.",
    "molecules": "LPS, PA"
  },
  {
    "caption": "(a) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity.",
    "molecules": "APDC, LPS, PA, ROS"
  },
  {
    "caption": "(b) ELISA for IL-1β on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.",
    "molecules": "APDC, LPS, PA"
  },
  {
    "caption": "(c) immunoblotting for caspase-1 p10 and IL-1β p17 on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.",
    "molecules": "APDC, LPS, PA"
  },
  {
    "caption": "(d) ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In a, one of two independent experiments is shown. In each of b-d, the results are representative of three independent experiments.",
    "molecules": "APDC, diphenyleneiodonium, DPI, LPS, N-acetyl-cysteine, NAC, PA, ROS"
  },
  {
    "caption": "(a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h.",
    "molecules": "LPS, PA"
  },
  {
    "caption": "(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (b) ROS production determined using the fluoroprobe DHR.",
    "molecules": "AICAR, LPS, PA, ROS"
  },
  {
    "caption": "(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (c,d) IL-1β (c) and IL-6 (d) in supernatants, measured by ELISA.",
    "molecules": "AICAR, LPS, PA"
  },
  {
    "caption": "(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (e) Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules.",
    "molecules": "AICAR, LPS, PA"
  },
  {
    "caption": "(f) Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. (g) ELISA for IL-1β in supernatants of cells transfected as in f and stimulated with PA-BSA (0.5 mM) for 24 h. In each of a, b and f, one of two independent experiments is shown. In each of c-e and g, the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P 0.05 versus controls.",
    "molecules": "PA"
  },
  {
    "caption": "(a) Immunoblotting for AMPK activation (Thr172 p-AMPK-α) and LC3B (top), and densitometric analysis to quantify ratio of LC3B-II to actin (bottom). BMMs were pretreated with LPS (200 ng/ml) for 3 h, followed by PA-BSA (0.5 mM) treatment for 24 h in the absence or presence of chloroquine (50 μM). Med, medium control.",
    "molecules": "chloroquine, LPS, PA"
  },
  {
    "caption": "(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right).",
    "molecules": "AICAR, LPS, PA"
  },
  {
    "caption": "(e) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator. In each of a and e, one of three independent experiments is shown. In each of b and d, one of two independent experiments is shown. Values are expressed as mean ± s.d. *P 0.05 versus controls.",
    "molecules": "ROS"
  },
  {
    "caption": "(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b).",
    "molecules": "insulin"
  },
  {
    "caption": "(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation.",
    "molecules": "insulin"
  },
  {
    "caption": "(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation",
    "molecules": "insulin"
  },
  {
    "caption": "(g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin.",
    "molecules": "insulin, anakinra"
  },
  {
    "caption": "(h) p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d.",
    "molecules": "anakinra"
  },
  {
    "caption": "(a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(g,h) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (g), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice (h) on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine.",
    "molecules": "Insulin, insulin"
  },
  {
    "caption": "(J) State 3 and maximal respiration driven by pyruvate  + malate  in isolated mitochondria  from mouse heart. EPI  was added in the respiration media at the indicated concentrations (n≥3). Note that EPI  does not affect  maximal respiration  but has a small effect on ATP  synthesis-dependent OCR. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Unpaired t-test versus Ctrl or untreated conditions shows statistical differences depicted by p-value.",
    "molecules": "EPI, ATP, malate, pyruvate"
  },
  {
    "caption": "(C) Representative blot  (left) and quantification (right) showing CV  assembly by BNGE  in mouse heart mitochondria  treated with EPI  as in A. For each biological replicate, technical replicates were averaged. Data represent average ± SEM.",
    "molecules": "EPI"
  },
  {
    "caption": "(F) CV  in gel ATP  hydrolysis in mouse heart mitochondria  where EPI  or oligo  were added in the assay buffer after mitochondrial  complexes  and supercomplexes  were separated by BNGE. CV  in gel ATP  hydrolysis activity is shown after 3hrs incubation (top). Coomassie staining was used as loading control (bottom). For each biological replicate, technical replicates were averaged.",
    "molecules": "EPI, oligo"
  },
  {
    "caption": "Binding  of purified recombinant ATPIF1-GFP  to CV. Mitochondrial membranes  were disrupted by freeze-thaw and exposed to ATPIF1-GFP  at different ratios of mitochondrial  (mt) protein/ATPIF1  purified protein. Binding  was assessed in BNGE  by quantifying ATPIF1-GFP  fluorescence. Binding competition assay  between ATPIF1-GFP  and EPI  to CV. (E) BNGE  assessed for ATPIF1-GFP binding  (top); CV hydrolytic activity (middle) and protein loading (bottom).",
    "molecules": "EPI"
  },
  {
    "caption": "(A-B) PLA  of CV-bound ATPIF1  produces a red fluorescence signal where anti-ATPIF1  and anti-ATP5A1  antibodies are localized  at a distance less than 10nm of each other. Anti-TOMM20  (green) was used to reveal mitochondrial  architecture. (A) Representative high-resolution Airyscan  confocal micrographs  showing control fibroblasts  treated with EPI  for 24hrs. Maximum intensity projection is shown. Scale bars: 20 µm and 5 µm in zoomed image. Note that PLA  reveals the spatial distribution of ATPIF1 binding  along the mitochondrial  length. (B) Analysis of ATPIF1-CV  complexes occurrence quantified as PLA  dots per µm3of mitochondria. Effect of EPI  is shown relative to untreated. (n=2 from >50 replicates per n). In all cases, for each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.",
    "molecules": "EPI"
  },
  {
    "caption": "(E)  mtATP content measured in control and human mitochondrial  disease fibroblasts  treated for 30min with increasing concentrations of EPI  as indicated. Analysis shows the mean intensity of mtATP probe in MTG  area normalized per % of correspondent untreated cells (n=3). In all cases, for each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.",
    "molecules": "EPI, ATP, MTG"
  },
  {
    "caption": "PLA  of CV-bound ATPIF1  (red). Anti-TOMM20  (green) was used to reveal mitochondrial  architecture. (A) Representative confocal micrographs showing fibroblasts  treated with EPI  for 24hrs. Maximum intensity projection is shown. Scale bars: 20 µm and 5 µm zoomed images. (B) Analysis of ATPIF1-CV  complexes occurrence quantified as PLA  dots per µm3of mitochondria  in patient fibroblasts  normalized in % of Ctrl untreated (n=2 from >50 replicates per n). In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.",
    "molecules": "EPI"
  },
  {
    "caption": "PLA  of CV-bound ATPIF1  (red). Anti-TOMM20  (green) was used to reveal mitochondrial  architecture. (C) Effect of EPI  on ATPIF1-CV  complexes occurrence quantified as PLA  dots per µm3 under the indicated conditions. Values are shown as relative to control untreated. In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.",
    "molecules": "EPI"
  },
  {
    "caption": "(E-G) Effect of EPI  on CIII-deficient (BCS1L) cells stable-expressing  ATPIF1WT or  ATPIF1H49K. (E) CV ATP  hydrolytic capacity measured by the acidification rate  and normalized maximal respiration. Maximal respiration was determined using the OCR  channel (Max OCR  on SR). Effect of EPI  is shown as % of control cells, untreated with EPI. Cells were treated for 24hrs with 50nM EPI  (n≥4). (F) mtATP content measured as fluorescence intensity per mitochondria  area and shown as % of control untreated (n=5). Mitochondrial  area was quantified using MTG staining. Cells were treated for 30min. (G) Total cellular ATP  content shown as % of untreated cells. Cells were treated for 24hrs (n≥4). In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.",
    "molecules": "EPI, ATP, MTG"
  },
  {
    "caption": "D2.mdx mice  were treated with EPI  orally twice daily, with doses ranging from 0.5 to 15 mg/kg/day. After 13 days of treatment, mice  were subjected to exercise induced gastrocnemius injury  by stimulating muscle  contraction and the effect of EPI  on muscle  force were measured. (A) Plantarflexor force quantification after  eccentric injury. n=8. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. unpaired t-tests (A, shows statistical differences depicted by p-value.",
    "molecules": "EPI"
  },
  {
    "caption": "D2.mdx mice  were treated with EPI  orally After 13 days of treatment, mice  were subjected to exercise induced gastrocnemius injury by stimulating muscle  contraction and OPA1  cleavage were measured. (E) Levels of cleaved OPA1  in gastrocnemius   homogenate from mdx mice  subjected to the indicated combinations of injury   and EPI  treatment. n=8. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test",
    "molecules": "EPI"
  },
  {
    "caption": "D2.mdx mice  were treated with EPI  (G) Detection and quantification of myofiber  membrane integrity or damage following injury  using Evans Blue dye (EBD) in gastrocnemius  muscle  from vehicle and EPI  treated mdx mice. Damaged fiber membranes allow dye entry that appears as red color. Yellow asterisks indicate damage fibers  (n≥4). unpaired t-tests G) shows statistical differences depicted by p-value.",
    "molecules": "EPI, EBD, Evans Blue dye"
  },
  {
    "caption": "A. GST-pull down assays of purified GST-tagged Elp3 SAM domain (aa72-389) and KAT domain (aa348-557) with untagged full length Elp1 and Elp451-2706. GSH resin and proteins were used as input controls. Lower panel shows 5% of the input and upper panel shows bound fractions. All samples were analyzed by SDS-PAGE and stained by Coomassie. Identities of respective proteins are indicated on the right.",
    "molecules": "GSH"
  },
  {
    "caption": "B. Same as A but tilted view to highlight the active site cavity (left). Close up view of the active site in which all known tRNA binding sites and a region of Elp2 important for tRNA modification activity are highlighted. Individual structures of the subunits are shown in cartoon and surface representation.",
    "molecules": "tRNA"
  },
  {
    "caption": "D. Close up view on the relative spatial orientation of the conserved basic patch in Elp2 and the potential interaction site between Elp456 and a region of Elp2 important for tRNA modification activity.",
    "molecules": "tRNA"
  },
  {
    "caption": "C RNA purified from wild type, abo1Δ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats.",
    "molecules": "ethidium bromide, RNA"
  },
  {
    "caption": "D RNA purified from wild type, and abo1Δ cells was analysed by strand specific RT-PCR. RNA from hip1Δ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats.",
    "molecules": "RNA"
  },
  {
    "caption": "H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.",
    "molecules": "agar, MMS"
  },
  {
    "caption": "A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats.",
    "molecules": "5-FOA, agar, imr"
  },
  {
    "caption": "B Strains carrying the otr::ura4+ were analysed as described for A.",
    "molecules": "otr"
  },
  {
    "caption": "C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test.",
    "molecules": "RNA"
  },
  {
    "caption": "D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student's t-test.",
    "molecules": "RNA"
  },
  {
    "caption": "E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.",
    "molecules": "adenine, agar"
  },
  {
    "caption": "A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.",
    "molecules": "dg, dh, imr"
  },
  {
    "caption": "B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data.",
    "molecules": "dg"
  },
  {
    "caption": "C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.",
    "molecules": "dg"
  },
  {
    "caption": "D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells.",
    "molecules": "dh, imr"
  },
  {
    "caption": "E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data.",
    "molecules": "dh, imr"
  },
  {
    "caption": "F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.",
    "molecules": "dh, imr"
  },
  {
    "caption": "A Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with TBZ (15 μg/ml) and incubated for 3-5 days 30°C. Data are representative of at three independent biological repeats.",
    "molecules": "agar, TBZ"
  },
  {
    "caption": "B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored (red-white) colonies. At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis.",
    "molecules": "adenine, agar"
  },
  {
    "caption": "C Heterothallic (h90) strains were cultured on nitrogen limiting (EMMG) medium for 3 days at 25°C to induce mating and meiosis. The resulting asci were stained with DAPI and visualised by fluorescence microscopy. Data are representative of three biological repeats",
    "molecules": "nitrogen"
  },
  {
    "caption": "D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4-5 days at 30°C. Data are the mean of three independent biological repeats and error bars are ±SEM. P-value was calculated using a two-tailed unpaired t-test.",
    "molecules": "agar"
  },
  {
    "caption": "Neutral lipid staining of the 5DO larval fat body. White arrows (O) indicate nuclear-localized neutral lipid staining, blue signal shows DAPI.",
    "molecules": "DAPI, Neutral lipid, neutral lipid"
  },
  {
    "caption": "GFP, Phalloidin and LipidTOX detect the expression of dTormGFP chimeras (green), cell boundaries (red), and LD (red), respectively, in dTorKO fat body cells.",
    "molecules": "LD, LipidTOX, Phalloidin"
  },
  {
    "caption": "Bars show the fraction (mean ± SEM) of membrane GL and GPL lipids with each fatty acyl chain characteristic. n = 3 MS analyses of 4DO fat body samples for each genotype. MUFA, mono-unsaturated; PUFA, poly-unsaturated. ‡ indicate a significant difference between w- and dTorKO expressing Luciferase RNAi, * indicate a significant difference compared to dTorKO expressing Luciferase RNAi. Two-way ANOVA, Dunnett's multiple comparison test. ‡ p < 0.05 and * p < 0.05.",
    "molecules": "GL, GPL, mono-unsaturated, MUFA, poly-unsaturated, PUFA"
  },
  {
    "caption": "Control and dTorKO 5DO fat body cells expressing UAS GFP fusion proteins with the Cg-Gal4 driver, and labeled with anti-Calnexin (red) and a neutral lipid dye (LipidTOX, magenta). Note that dTorKO cells are smaller. White arrowhead indicates NE enrichment of LipinmGFP.",
    "molecules": "LipidTOX, Cg, neutral lipid"
  },
  {
    "caption": "Control (w-) and dTorKO fat body nuclei labeled with anti-LaminDm0, Megator/Tpr, mAb414, or expressing UAS Nup107GFP or UAS Nup358GFP with the Cg-Gal4 driver.",
    "molecules": "Cg"
  },
  {
    "caption": "The percentage that Lipin, Ctdnep1, and Nep1r1 RNAi normalize the abundance of membrane GL and GPL classes in the dTorKO fat body. 0% "normal" reflects lipid abundance in dTorKO expressing Luciferase RNAi, while 100% "normal" reflects lipid abundance in w- expressing Luciferase RNAi. Percentage normalization was separately calculated for PI, PA, DAG, PC, PE and PS (for dTorKO expressing Lipin, Ctdnep1, and Nep1r1 RNAi). Points show the mean of all lipid class normalization per sample, bars show mean ± SEM for the group.",
    "molecules": "DAG, GL, GPL, PA, PS, PC, PE, PI"
  },
  {
    "caption": "B. Dose-response curves of PeTa cells and control HDFB cells after 6 days of treatment with GSK-LSD1 or ORY-1001. Dose-response curves of 3 other MCC cell lines are displayed in Fig EV1A. n = 4 technical replicates. Data are represented as means ± SD.",
    "molecules": "GSK-LSD1, ORY-1001"
  },
  {
    "caption": " B. Individual tumor growth in GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 22 ",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": " C. Representative picture of mice treated with vehicle or GSK-LSD1 at day 41 after PeTa cell injection. Tumor location is indicated with a circle ",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": " D. Kaplan-Meier curve of GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or the greatest dimension was ≥ 1.5cm. Red arrow: start of therapy, day 22. ****p < 0.0001 (log-rank Mantel-Cox test) ",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": " F. Individual tumor growth in GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 1 ",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": " G. Relative mouse weight (%) during treatment of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice ",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": " H. Kaplan-Meier curve of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or greatest dimension ≥ 1.5cm. Red arrow: start of therapy, day 1. ****p < 0.0001 (log-rank Mantel-Cox test) ",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": "A. Ki-67 staining of PeTa cells treated for 3 days with GSK-LSD1 (1 μM) or ORY-1001 (1 μM). n = 3 biological replicates. Data are represented as means ±SD. ***p < 0.001 (DMSO vs GSK-LSD1 p = 0.0002; DMSO vs ORY-1001 p = 0.0004; unpaired Student's t-test).",
    "molecules": "DMSO, GSK-LSD1, ORY-1001"
  },
  {
    "caption": "B. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells after 6 days of 100 nM GSK-LSD1 or DMSO treatment. C. Quantification of EdU/Propidium iodide staining depicted in Fig 3B. n = 3 biological replicates. Data are represented as means ±SD. **p<0.01; ****p<0.0001; ns, non-significant (G1 phase: DMSO vs GSK-LSD1 p = 0.0059; S phase: DMSO vs GSL-LSD1 p<0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.2338; unpaired Student's t-test). ",
    "molecules": "EdU, DMSO, GSK-LSD1, GSL-LSD1, Propidium iodide"
  },
  {
    "caption": "D. Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment. Etoposide serves as positive control for apoptosis, H3 serves as loading control. c.PARP1, cleaved PARP1.",
    "molecules": "Etoposide"
  },
  {
    "caption": "E. Caspase-3/7 cleavage activity for PeTa cell line after 6 days of indicated treatment. Etoposide serves as a positive control for apoptosis. n = 3 technical replicates. Data are represented as means ±SD. RLU, relative luminescence units.",
    "molecules": "Etoposide"
  },
  {
    "caption": "F. Representative FACS readout of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment after 6 days. G. Quantification of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment at indicated time points. ",
    "molecules": "DMSO, GSK-LSD1, tetramethylrhodamine ethyl ester, TMRE"
  },
  {
    "caption": "H. Representative FACS readout of Annexin V/DAPI staining of PeTa cells after 6 days of 100 nM GSK-LSD1 DMSO treatment. I. Quantification of Annexin V/DAPI staining depicted in Fig 3H. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (Early, Late, Total; unpaired Student's t-test). ",
    "molecules": "DAPI, DMSO, GSK-LSD1"
  },
  {
    "caption": "J. Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK-LSD1 or vehicle treatment. Representative images in Appendix Fig S1B. n = 8, ***p = 0.001 (Vehicle vs GSK-LSD1; unpaired Student's t-test). ROI, region of interest.",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": "K. Top. Representative images of immunofluorescent Ki-67 staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of Ki-67 signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, **p<0.01, ****p<0.0001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0015, unpaired Student's t-test; D10 vehicle vs D10 GSK-LSD1 p<0.0001, unpaired Student's t-test; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.0012, Mann-Whitney test). ROI, region of interest.",
    "molecules": "DMSO, GSK-LSD1"
  },
  {
    "caption": "L. Top. Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of TUNEL signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, ***p<0.001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0009; D10 vehicle vs D10 GSK-LSD1 p = 0.0001; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.001; unpaired Student's t-test with Welch's correction). ROI, region of interest.",
    "molecules": "DMSO, GSK-LSD1"
  },
  {
    "caption": "M. Representative images of hematoxylin and eosin (H&E) staining of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day (D1, top), 10 days (D10, middle) or until experiment endpoint (bottom). Upper right scale bar represents 50 μm, insert scale bar represents 10 μm.",
    "molecules": "DMSO, GSK-LSD1"
  },
  {
    "caption": "A. Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK-LSD1 or DMSO treatment. Scale bar, 100 μm.",
    "molecules": "DMSO, GSK-LSD1"
  },
  {
    "caption": "B. Metagene plots showing the average CUT&RUN signal for H3K4me1, H3K27ac, and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site; TES transcription end site.",
    "molecules": "DMSO, H3K27ac, H3K27me3, H3K4me1"
  },
  {
    "caption": "C. ATAC-seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site",
    "molecules": "DMSO"
  },
  {
    "caption": "D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor. ",
    "molecules": "GSK-LSD1"
  },
  {
    "caption": "D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor. ",
    "molecules": "DMSO, GSK-LSD1"
  },
  {
    "caption": "B. MA-plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling. Significantly up- and downregulated genes (FDR≤0.05; abs(log2FC)≥0.5) are marked in red and blue, respectively. Genes belonging to neuronal differentiation (GO:0048666) are labelled. FC, fold-change; CMP, counts per million.",
    "molecules": "4sU"
  },
  {
    "caption": "D. Volcano plot depicting the protein-protein binding partners of LSD1 depleting in PeTa cells treated with ORY-1001 versus DMSO. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change.",
    "molecules": "DMSO, ORY-1001"
  },
  {
    "caption": "H. Left. Immunoblot confirmation of HMG20B downregulation. H3 serves as nuclear fraction control and α-Tubulin as loading control. Right. Immunoblot quantification. HMG20B protein levels are normalized to H3 and relative to DMSO control.",
    "molecules": "DMSO"
  },
  {
    "caption": "B. RT-qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A. Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control. n = 4 technical replicates. Bar graphs represent mean ± SD.",
    "molecules": "DMSO"
  },
  {
    "caption": "E. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash-out. F. Quantification of EdU/Propidium iodide staining depicted in Fig 7E. n = 3 biological replicates. Data are represented as means ±SD. *p<0.05, **p<0.01; ***p<0.001 (G1 phase: DMSO vs GSK-LSD1 p = 0.0104; S phase: DMSO vs GSK-LSD1 p = 0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.0062; unpaired Student's t-test).",
    "molecules": "EdU, DMSO, GSK-LSD1, Propidium iodide"
  },
  {
    "caption": "G. Quantification of FACS Annexin V/DAPI staining of PeTa cells 8 days after drug wash-out. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (unpaired Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates).",
    "molecules": "PI(3,4,5)P3, PIP2, 4µ8c, dimethyl sulfoxide, DMSO, PI, PIP"
  },
  {
    "caption": "(D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown).",
    "molecules": "4µ8c, DMSO"
  },
  {
    "caption": "(E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure.",
    "molecules": "4µ8c, thiogylcolate, tunicamycin"
  },
  {
    "caption": "(F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).",
    "molecules": "PA"
  },
  {
    "caption": "IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression.",
    "molecules": "PA"
  },
  {
    "caption": "IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "(A-B) BMDMs were treated with PA, steric acid (SA), palmitoleic acid (PAO) or oleic acid (OA) (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for the expression of (A) miR-2137 and (B) sXBP1 (n=3 biological replicates).",
    "molecules": "PA, SA, steric acid, OA, oleic acid, palmitoleic acid, PAO"
  },
  {
    "caption": "(E-F) Apoe-/- mice were fed with chow or WD for 12 weeks and injected with 4µ8c (10 mg/kg/day) or vehicle (DMSO) in the final 6 weeks of the diet (n=5-7 mice per group): (E) RNA lysates from bone marrow were analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group). (F) Total RNA isolated from the aortic root plaques was analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group).",
    "molecules": "4µ8c, DMSO"
  },
  {
    "caption": "(A) BMDMs were transfected with scrambled (Scr) or IRE1-specific siRNA (40 nM) and treated with palmitate (PA, 500 µM) or vehicle for 6 and 9 hours (hr). RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).",
    "molecules": "PA, palmitate"
  },
  {
    "caption": "(B) BMDMs were treated with PA (500 µM) and 4µ8c (100 µM) or vehicle for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (C) BMDM cells were treated with tunicamycin (Tun; 5 µg/ml) and 4µ8c (100 µM) or vehicle (DMSO) for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). ",
    "molecules": "4µ8c, DMSO, PA, Tun, tunicamycin"
  },
  {
    "caption": "(D) IRE1-/- MEFs were transfected with vector (control), WT-IRE1 or K907A-IRE1 (RNase dead mutant) plasmids for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "(E) BMDMs were transfected with scrambled or XBP-1 specific siRNA (70 nM) for 24 hours, followed by treatment with PA (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "(F) XBP1-/- MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed for miR-2137 expression by qRT-PCR (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "(G) IRE1 cleavage assay performed using synthetic pre-miR-2137 (50 nM) as substrate with active, recombinant IRE1 (100 ng) at 37℃ for 2 hours, followed by sample separation in Urea-PAGE and detection with SYBR gold staining. M indicates micro RNA marker.",
    "molecules": "SYBR gold, Urea"
  },
  {
    "caption": "(H-I) BMDMs isolated from WT (C57BL6) and DICER-/- mice were treated with PA (500 µM) for 12 hours. RNA lysates were analyzed by qRT-PCR for (H) miR-2137 and (I) sXBP1 expression (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "(D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "(A-C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section. The ratio of lipids from this analysis is shown for (A) PI(3,4,5)P3 to PIP2.; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP. Data from LC/MS/MS using a Waters Xevo TQ-S MS/MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods. (n=3 biological replicates)",
    "molecules": "PI(3,4,5)P3, PIP2, lipids, PI, PIP"
  },
  {
    "caption": "Heatmaps displaying standardized log2-transformed signal intensities per glycan  for IgA1   (A) and IgA2   (B) in non-IBD, CD  and UC  groups, plotted by hierarchical clustering of Euclidean distance.",
    "molecules": "glycan"
  },
  {
    "caption": "Macrophage-conditioned medium induces MCF10A colony formation in soft agar as compared to control medium. Colonies ≥ 50 μm were counted at 5 weeks. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "agar"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei (clear, partially filled, almost filled and filled). Spheroids stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red).",
    "molecules": "Hoechst 33342, collagen, DNA, Phalloidin"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; 50 spheroids each). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "collagen"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of Ki67 positive nuclei of MCF10A cells. Spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and Ki67 (green).",
    "molecules": "Hoechst 33342, collagen, DNA, Phalloidin"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage-conditioned media. Error bars represent mean ± SEM from 3 independent experiments (20-30 non-empty spheroids per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "collagen"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. NSC23766 (Rac1 inhibitor; 50 μM) reduces the filling of the spheroid lumen with cell nuclei upon macrophage-conditioned medium stimulation compared to control. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled MCF10A spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "collagen, NSC23766"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Invasive protrusions of spheroids into matrigel/collagen mix marked with a white arrow.",
    "molecules": "collagen"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. 3D structures of spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and laminin V (green). Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions. Invasive protrusions are marked with a white arrow.",
    "molecules": "Hoechst 33342, collagen, DNA, Phalloidin"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces invasive protrusions in MCF10A spheroids compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "collagen"
  },
  {
    "caption": "Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24­h. Representative images of organoids in matrigel/collagen mix.",
    "molecules": "collagen"
  },
  {
    "caption": "Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24­h. Macrophage-conditioned medium induces invasive protrusions in organoids as compared to control. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "collagen"
  },
  {
    "caption": "Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24­h. 3D structure of organoids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). Bi-layered structure of internal luminal cells and external basal myoepithelial cells is established for non-invasive organoids.",
    "molecules": "Hoechst 33342, collagen, DNA, Phalloidin"
  },
  {
    "caption": "Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids isolated from ND or HFD mice were cultured in collagen for 2 days. Representative images of organoids cultured in collagen for 2 days. The number of invasive protrusions per organoid is higher for organoids isolated from mice on HFD compared to mice on ND. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).",
    "molecules": "collagen"
  },
  {
    "caption": "Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on high fat diet (HFD) (n=3). Organoids isolated from HFD mice were cultured in collagen for 2 days. 3D structure of organoid stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). α-SMA, myoepithelial cell marker, appeared to be loss at invasive protrusions.",
    "molecules": "Hoechst 33342, collagen, DNA, Phalloidin"
  },
  {
    "caption": "Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium. Representative images of ductal structures cultured with conditioned medium. Ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Macrophage-conditioned medium induces filling of the duct lumen with cell nuclei compared to control. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "collagen, DAPI, DNA, Phalloidin"
  },
  {
    "caption": "Amlexanox inhibits colony formation of MCF10A cells grown in soft agar with macrophage-conditioned medium for 5 weeks compared to controls. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "agar, Amlexanox"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "Hoechst 33342, amlexanox, collagen, DNA, Phalloidin"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "amlexanox, Amlexanox, collagen"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "amlexanox, Amlexanox, collagen"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of spheroids with invasive protrusions. Error bars represent mean ± SEM from 8 independent experiments (n=2 per condition, at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "amlexanox, Amlexanox, collagen"
  },
  {
    "caption": "Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from mice on ND were cultured in matrigel/collagen mix for 2 days and then stimulated with macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of invasive organoids. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "amlexanox, Amlexanox, collagen"
  },
  {
    "caption": "Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. Amlexanox reduces the number of invasive protrusions in organoids isolated from mice on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).",
    "molecules": "Amlexanox, collagen"
  },
  {
    "caption": "Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in presence of amlexanox. Representative images showing the filling of the ductal lumen with cell nuclei. The ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Scale bar: 20 μm. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "amlexanox, collagen, DAPI, DNA, Phalloidin"
  },
  {
    "caption": "Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in presence of amlexanox. Amlexanox inhibits filling of the duct lumen with cell nuclei. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as M1A - M1 activated, M2 activated macrophages.",
    "molecules": "amlexanox, Amlexanox, collagen"
  },
  {
    "caption": "IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. IKKε CRISPR-Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage-conditioned medium for 5 weeks compared to CRISPR-Cas9 control (nontargeting crRNA) cells. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "agar"
  },
  {
    "caption": "IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. IKKε CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "collagen"
  },
  {
    "caption": "Correlation of mRNA levels of the SBP enzyme genes PSAT1 (E), PHGDH (F), PSPH (G) with the immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patient (Curtis et al, 2012).",
    "molecules": "SBP"
  },
  {
    "caption": "NCT502 (10 μM) inhibits colony formation of MCF10A cells grown in soft agar with macrophage-conditioned medium for 5 weeks compared to controls. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 5 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "agar, NCT502"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "Hoechst 33342, collagen, DNA, NCT502, Phalloidin"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "collagen, NCT502"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "collagen, NCT502"
  },
  {
    "caption": "16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the number of spheroids with invasive protrusions (n=2 per condition). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.",
    "molecules": "collagen, NCT502"
  },
  {
    "caption": "Mouse mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice that were either on normal (ND) or high fat diet (HFD) (n=3). Organoids were isolated from mice on ND and cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h in presence of NCT502 (10 μM). NCT502 reduces the number of invasive organoids. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as M1A - M1 activated, , M2A - M2 activated macrophages.",
    "molecules": "collagen, NCT502"
  },
  {
    "caption": "Mouse mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice that were either on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. NCT502 (10 μM) reduces the number of invasive protrusions in organoids isolated from mice that were either on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).",
    "molecules": "collagen, NCT502"
  },
  {
    "caption": "Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in the presence of NCT502 (10μM). Representative images showing the filling of the ductal lumen with cell nuclei. The ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Scale bar: 20 μm. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "collagen, DAPI, DNA, NCT502, Phalloidin"
  },
  {
    "caption": "Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in the presence of NCT502 (10μM). NCT502 inhibits filling of the duct lumen with cell nuclei. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "collagen, NCT502"
  },
  {
    "caption": "PHGDH gene was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. PHGDH CRISPR-Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage-conditioned medium for 5 weeks compared to CRISPR-Cas9 control (nontargeting crRNA) cells. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as , M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "agar"
  },
  {
    "caption": "PHGDH gene was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. PHGDH CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.",
    "molecules": "collagen"
  },
  {
    "caption": "A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Mice were administered amlexanox (17 mM) or vehicle control daily by oral gavage (Reilly et al, 2013). A control group (wild-type mice) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV-PyMT gene. PyMT-MMTV+/- mice on HFD gain more weight compared to ND mice. *p < 0.05 by one-tailed students t-test. Amlexanox does not affect weight gain either in mice on F) ND or G) HFD.",
    "molecules": "amlexanox, Amlexanox"
  },
  {
    "caption": "A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Mice were administered amlexanox (17 mM) or vehicle control daily by oral gavage (Reilly et al, 2013). A control group (wild-type mice) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV-PyMT gene. Mammary tumours are developing earlier in PyMT-MMTV+/- mice on HFD compared to ND mice. Amlexanox does not affect tumour development in PyMT-MMTV+/- mice on ND, but delays tumour formation in J) HFD mice.",
    "molecules": "amlexanox, Amlexanox"
  },
  {
    "caption": "A LOUCY  cells were treated in vitro with the indicated SMs with or without vincristine  (VCR) for 48h. Apoptosis  was determined by  flow cytometry upon staining  with  Annexin-V  and Propidium Iodide (PI). Mean ± SD of 3 independent experiments is shown; ** p <0.01, *** p <0.001 and **** p <0.0001 by unpaired Student's t-test.",
    "molecules": "PI, Propidium Iodide, VCR, vincristine"
  },
  {
    "caption": "B Fresh primary   leukemic cells  from two pediatric patients, one with common B-ALL at relapse (left) and one with T-ALL at diagnosis (right), both with poor response to second line chemotherapy, were treated with LBW242  and VCR  at the indicated concentrations. Apoptosis  was measured after 24 h as described in A.",
    "molecules": "LBW242, VCR"
  },
  {
    "caption": "The SM-drug combination activates caspases LOUCY  cells were incubated with the indicated chemotherapeutic drugs with or without LBW242  for 48 h. Cells were stained  with respective fluorescent substrates for caspase-8 (C) and -9 (D) , and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.",
    "molecules": "LBW242"
  },
  {
    "caption": "The SM-drug combination activates caspases LOUCY  cells were incubated with the indicated chemotherapeutic drugs with or without LBW242  for 48 h. Cells were stained  with an antibody detecting the active  caspase-3 subunit (E), and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.",
    "molecules": "LBW242"
  },
  {
    "caption": "The SM-drug combination induces PARP  cleavage. LOUCY  cells were incubated with the indicated chemotherapeutic drugs with or without LBW242  for 48 h. Cleavage of PARP  was measured by Western blot using Actin  as loading control (F). One representative immunoblot  out 3 independent experiments is shown.",
    "molecules": "LBW242"
  },
  {
    "caption": "A Necrostatin-1 (Nec) does not affect LBW-mediated sensitization to VCR-induced apoptosis. Apoptosis  induction in LOUCY  cells was determined by  flow cytometry 48h after treatment with SM and/or VCR  (left) and/or TNFα (right) in the presence or absence of the RIPK1-inhibitor Nec. Mean ± SD of 3 independent experiments is shown; * p <0.05 and ** p<0.01 by unpaired Student's t-test.",
    "molecules": "LBW, Nec, Necrostatin-1, VCR"
  },
  {
    "caption": "B RIPK1 is dispensable for apoptosis  induction by LBW242  and VCR. LOUCY  cells were transfected with a non-targeting siRNA (neg-siRNA) or a RIPK1-specific siRNA. 6h post transfection, cells were treated with the indicated drugs and apoptosis  was measured 40h thereafter. Mean ± SD of 3 independent experiments is shown; *** p<0.001 by unpaired Student's t-test. Knockdown (KD) was verified by Western blot at the time of apoptosis  measurement and KD efficiency calculated compared to the control. One representative immunoblot  out of 3 biological replicates is shown.",
    "molecules": "LBW242, VCR"
  },
  {
    "caption": "C The SM-drug combination does not activate the canonical NF-κB pathway. Phosphorylation  and degradation of IκBα  upon treatment with VCR  or TNFα and/or LBW242  was determined in LOUCY  cells by Western blot  following treatment with the indicated drugs for 5, 15 or 60 minutes using Actin  as loading control. One representative immunoblot  out of 3 independent biological replicates is shown.",
    "molecules": "LBW242, VCR"
  },
  {
    "caption": "Parental and XIAP  over-expressing  NALM-6 cells (C) were treated with VCR apoptosis  (Annexin-V  / PI) was measured at the indicated  time points (C) by  flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP   protein level was analyzed in parental and XIAP-overexpressing NALM-6 cells by protein immunoassay (Simple Western); Actin  was used as loading control. One representative immunoassay out of 3 independent experiments is shown.",
    "molecules": "PI, VCR"
  },
  {
    "caption": "D Parental and XIAP  over-expressing REH  cells (D) were treated with VCR  or LBW242; apoptosis  (Annexin-V / PI) was measured after 72h (D) by  flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP  protein level was analyzed by protein immunoassay (Simple Western); Actin  was used as loading control. One representative immunoassay out of 3 independent experiments is shown.",
    "molecules": "LBW242, PI, VCR"
  },
  {
    "caption": "F Moderate knockdown of XIAP  sensitizes towards chemotherapy. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. 4 days after injection, mice  were either treated with PBS  (n=4), or with Vincristin  (VCR, n=8), 0.3mg/kg i.v. once a week for 3 consecutive weeks. 7 days after last treatment, mice  were sacrificed, and relative proportion of sh-CTRL and sh-XIAP  subpopulations re-isolated from the BM  was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test with Welch's correction.",
    "molecules": "PBS, VCR, Vincristin"
  },
  {
    "caption": "B) SUMO2 immunoprecipitation using crosslinked SUMO2 antibody beads and liver samples of wild-type C57BL6/J mice in the fasted (16 h) or re-fed (16 h fasted and re-fed 2 h) states. Eluates analyzed by immunoblotting using anti-Prox1 antibodies and ponceau staining was used as loading control. Data information Data: mean ±SEM. Significance was determined by one-way ANOVA with Dunnett´s multiple comparison test relative to samples collected at ZT 12. *** P≤0.001.",
    "molecules": "ponceau"
  },
  {
    "caption": "C) Mouse un-tagged Prox1 (mProx1) was purified from HEK293T cells. Purified mProx1 was incubated with recombinant E1 and E2 enzyme together with either SUMO1 or SUMO2. The enzymatic reactions were started with ATP and incubated for 15, 30 and 60 min. A reaction with the Prox1 K556R mutant (KR) was used as a control. The 0 min sample was incubated without ATP. Samples were analyzed by immunoblotting using anti-Prox1 antibodies.",
    "molecules": "ATP"
  },
  {
    "caption": "A) 6 weeks old C57BL/6N male mice were fed either a 0% cholesterol diet (ctrl) or a 2 % high cholesterol diet (chol 2%) for 6 weeks. B) 6 weeks old C57BL/6N male mice were fed either a standard chow diet or a 60 % high fat diet (HFD) for 8 weeks. C) 6 weeks old C57BL/6N male mice were fed either a standard chow diet or a 45% high fat + 20% w/v fructose diet (HF/hfD) for 12 weeks. (A-C) Liver samples were collected at ZT 15 in the fasted (11 h) and re-fed (fasted 8h and re-fed 3 h) states (n=4-5). Liver lysates were analyzed by immunoblotting using anti-Prox1 antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. *P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001.",
    "molecules": "chol, cholesterol, fructose"
  },
  {
    "caption": "B) 8 weeks old Prox1K556R K.I. (f/f) male mice were injected with a control AAV (Ctrl_AAV), with an AAV to overexpress Cre recombinase (Cre_AAV) or with phosphate-buffered saline (PBS) (n=4). Liver tissue samples were collected 3 weeks later from mice fed ad libitum. Liver lysates were analyzed by immunoblotting using anti-Prox1 and anti-Cre antibodies; Vcp was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown. qPCR data presented as relative fold change normalized to the housekeeping gene TBP. Data information: (B) Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by one-way ANOVA with Tukey´s multiple comparison test between groups. *** P≤0.001, **** P≤0.0001.",
    "molecules": "PBS, phosphate-buffered saline"
  },
  {
    "caption": "A) 8 weeks old Prox1K556R K.I. (f/f) male mice were injected with a control AAV (Ctrl_AAV) or with an AAV to overexpress Cre recombinase (Cre_AAV) and placed on a high-fat (45%) high-fructose (20% w/v) diet for 18 weeks. Liver samples were collected between ZT 16 and 17 in the fasted (8 h) and re-fed (fasted 8 h and re-fed 4-5 h) states (n=7-8). Liver lysates were analyzed by immunoblotting using anti-Prox1 and anti-Cre antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown. Data information Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. * P≤0.05, ** P≤0.01, **** P≤0.0001.",
    "molecules": "fructose"
  },
  {
    "caption": "B) Serum analysis using a colorimetric-based serum analyzer. Levels of total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) are shown. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. * P≤0.05, ** P≤0.01, **** P≤0.0001.",
    "molecules": "cholesterol, HDL, high density lipoprotein, LDL, low density lipoprotein"
  },
  {
    "caption": "C) Equal amounts of serum were pooled and separated by FPLC, serum lipoprotein cholesterol profiles are shown.",
    "molecules": "cholesterol"
  },
  {
    "caption": "C) Heatmap displaying the expression of enriched genes involved in phase I, II and III of bile acid detoxification and bile acid synthesis in the liver of obese mice expressing the Prox1 K556R mutant (Cre_AAV) in comparison to obese mice expressing wild-type Prox1 (Ctrl_AAV) in the fasted state (n=4). The occupation scores according to a published Chip- sequencing analysis on Prox1 Cyp7a1 was used as a reference gene.",
    "molecules": "bile acid"
  },
  {
    "caption": "B Dose response curves for viability of parental cells (in red) or selected clones (in black) upon treatment with NFR for 48h. The green dashed box highlight concentration in the physiologically relevant range. Curves are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 2way ANOVA analysis of variance between parental cells and each individual clone. Bar graph represents the EC50 (half maximal effective concentration) of the dose responses. Data are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 1way ANOVA analysis of variance (P value in red) followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01.",
    "molecules": "NFR"
  },
  {
    "caption": "C The parental population and the NFR resistant clones were analyzed for eEF2K mRNA expression by real-time PCR relative to β-actin (mean and s.e.m. from 3 independent batches of cells are shown). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.",
    "molecules": "NFR"
  },
  {
    "caption": "D The parental population and representative NFR resistant clones were analyzed by WB for eFF2K total protein level. Tubulin is used as loading control. Histogram represents mean and s.e.m of relative eEF2K level quantified from WB of 4 different batches of cells collected at different dates (see also source data). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *** P value ≤ 0.001.",
    "molecules": "NFR"
  },
  {
    "caption": "A Immunoblot analysis of eEF2 Thr56 phosphorylation (P-eEF2) in HeLa cells treated for 6h with increasing doses of NFR, and compared with 10 μg/ml tunicamycin (TM), 200 nM rapamycin (Rapa.) or 1h starvation (Starv.). Tubulin is used as loading control.",
    "molecules": "NFR, Rapa, rapamycin, TM, tunicamycin"
  },
  {
    "caption": "B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, 200 nM rapamycin (Rapa.), 1h starvation (Starv.) or 1 mM of the AMPK activator AICAR, were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 (Thr56).",
    "molecules": "AICAR, NFR, Rapa, rapamycin"
  },
  {
    "caption": "C Three representative NFR-resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR and compared to parental HeLa cells. For treatments, medium was replaced for 6h with medium containing DMSO (Mock), increasing doses of NFR or 10 μg/ml tunicamycin (TM), or for 1h with PBS for starvation (Starv.). Tubulin is used as loading control.",
    "molecules": "DMSO, NFR, TM, tunicamycin"
  },
  {
    "caption": "D Immunoblot analysis of eEF2 Thr56 phosphorylation in HeLa cells treated for 6h with increasing doses of different HIV-PIs as indicated or the NFR metabolite M8 (hydroxy-tert-butylamide). Molecular structures of the different HIV-PIs used are indicated. Each panel is representative of at least 3 independent experiments.",
    "molecules": "M8, NFR"
  },
  {
    "caption": "B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for indicated time in PBS (Starv.), were analyzed by immunoblot using indicated antibodies. Tubulin is used as loading control.",
    "molecules": "AICAR, NFR, Rapa"
  },
  {
    "caption": "C A potent mTOR inhibition does not impair NFR-mediated eEF2 phosphorylation. WT MEFs were treated with the indicated concentrations of Rapamycin or with vehicle (Mock). After 30 minutes, indicated doses of NFR were added and cells were incubated for additional 6h and analyzed for phosphorylated S6R and eEF2. Tubulin is used as loading control.",
    "molecules": "NFR, Rapamycin"
  },
  {
    "caption": "D NFR-mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO. AMPKα1α2 WT and dKO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for 1h in PBS (Starv.), were analyzed by immuoblot with indicated antibodies. * anti-total AMPKα antibody give an unspecific band with a slightly higher molecular weight in dKO cells. Tubulin is used as loading control. Each panel is representative of at least 3 independent experiments.",
    "molecules": "AICAR, NFR, Rapa, Rapamycin"
  },
  {
    "caption": "A-B EEF2K contributes to the decreased translation observed with NFR without impacting translation in presence of TM. A. Quantification of newly synthesized proteins at 0, 2, 4 and 6h after 20 μM NFR (left panels) or 10 μg/ml TM (right panels) treatment in MEF eEF2K +/+ and eEF2K-/-. Treated cells were labeled for 15 minutes with 35S-methionine and visualized by SDS PAGE and subsequent autoradiography. Autoradiographies from four different experiments (see also source data) were quantified and results show percentage of translation compared to untreated cells. The mean and s.e.m of four independent metabolic labeling experiments are shown. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance (in red) followed by Bonferroni posttest (in black). B. eEF2K+/+, eEF2K-/-, eIF2αWT and eIF2αS51A MEFs were treated for 6h with indicated doses of NFR, 200 nM rapamycin, or 10 μg/ml TM, or for 30 min with 10 μg/ml CHX. 35S-methionine incorporation was measured by liquid scintillation counting. Data are shown as the percentage of translation compared to untreated cells. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance followed by Bonferroni posttest.",
    "molecules": "methionine, CHX, NFR, proteins, rapamycin, TM"
  },
  {
    "caption": "C Representative polysome profiles of eEF2K WT and KO MEF treated 6h with NFR or TM. Area under curve for Sub-polysomes (S) and Polysomes (P) used to calculate the P/S ratio were indicated (see also raw data provided). Bar graph represents ratio normalized to untreated cells. OD254 nm is optical density at 254 nm. Data showed mean ±s.e.m. of P/S ratio calculated from 3 independent experiments. P values were calculated using 1way ANOVA analysis of variance followed by Bonferroni posttest; *P value ≤ 0.05, ** P value ≤ 0.01.",
    "molecules": "NFR, TM"
  },
  {
    "caption": "D The ribosome half-transit time in MEFs eEF2K+/+ and eEF2K-/- was determined as described in Materials and Methods. Incorporation of 35S-Methionine into total protein within the PMS and PRS was obtained by linear regression analysis. Presented graphs are representative of two (CHX 1 μg/ml for 30 min and Starvation 30 min in PBS in eEF2K+/+ cells) to four (NFR 20μM for 6h in eEF2K+/+ and eEF2K-/- cells) independent experiments. Indicated values represent the x displacement measurement (in time) between the PMS line at 300sec and the PRS line (see also raw data provided). Histogram represents mean and s.e.m. of the ribosome half transit time from four independent experiments. P values were calculated using two tails unpaired Student's T-tests; *P value ≤ 0.05.",
    "molecules": "Methionine, CHX, NFR"
  },
  {
    "caption": "A MEF eEF2K WT, KO or KO reconstituted with human eEF2K (Flag-eEF2K) were analyzed for cell growth at indicated times. Fold change (means ± s.e.m of triplicate) of the cell number just before treatment are shown. Histogram shows percentage of growth inhibition after 72h of 10 μM NFR. P values are 1way ANOVA with Bonferroni posttest calculated from 3 independent experiments. *** P value ≤ 0.001.",
    "molecules": "NFR"
  },
  {
    "caption": "B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (B,C) eEF2K -/- MEFs show a decrease susceptibly to NFR compared to eEF2K +/+ controls (B) whereas eEF2K reconstitution restores NFR sensitivity (C).",
    "molecules": "NFR"
  },
  {
    "caption": "B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (D-F), HeLa (D), A549 (E) and MCF7 (F) clones with CRISPR-Cas9 generated eEF2K deficiency (CrEEF2K) show a decrease susceptibly to NFR compare to control cells (CrLuci).",
    "molecules": "NFR"
  },
  {
    "caption": "G NFR-mediated toxicity assessed using AnnexinV/PI staining and FACS analysis after 24h treatment. Histogram shows the percentage of dead cells (AV+/PI+). Data are mean ± s.e.m. of 3 independent experiments. P values are 2way ANOVA with Bonferroni posttest. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.",
    "molecules": "NFR"
  },
  {
    "caption": "A Tumor volumes of eEF2K -/- and eEF2K +/+ RasV12 engraft implanted subcutaneously in AGR129mice. Treatment with NFR or vehicle was initiated 6 days post-implantation. Data are mean of tumor volume ± s.e.m. (n = 8 per group). P values were calculated using 2way ANOVA followed by Bonferroni posttest comparing WT tumor with vehicle or NFR treatment and KO tumors with vehicle or NFR treatment; *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. Experiment is representative of 2 performed in same conditions.",
    "molecules": "NFR"
  },
  {
    "caption": "B Usp27x binds a mutant of Bim incapable of binding to anti-apoptotic Bcl-2-proteins. 293FT cells transfected with constructs encoding 3xFLAG-Usp27x and 3xHA-tagged BimEL (see A, 2µg each) or 3xHA-tagged BimEL∆∆(a mutant with two mutations in the BH3-domain, incapable of binding antiapoptotic Bcl-2proteins [50]) were immunoprecipitated from whole cell extracts using anti-HA-resin. Bim and Usp27x were detected with anti-HA- and anti-FLAG-antibodies as indicated; antiapoptotic proteins: Mcl-1, Bcl-XL, Bcl-2. The caspase-inhibitor Q-VD-OPh (QVD) was added to the cultures described in A and B to inhibit Bim-induced apoptosis. Western blots are representative of n=3 independent experiments.",
    "molecules": "Q-VD-OPh, QVD"
  },
  {
    "caption": "C Usp27x interacts with endogenous BimEL independently of its catalytic activity293FT cells either carrying 3xFlag-Usp27x (293FT-TetR-3xFlag-usp27x) or the catalytic inactive mutant 3xFlag-Usp27xC87A under the control of the Tet-repressor were treated for 24h with dox to induce expression of Usp27x or Usp27xC87A. In all conditions PMA (to induce Bim-ubiquitination, 16.2 nM), and Q-VD-OPh (to inhibit apoptosis, 10 µM, see Fig. 3) were added at the time of Usp27x-induction. MG132 (to prevent Bim-degradation, 40 µM) was added in all conditions 4h prior to cell lysis. 3xFlag tagged Usp27x or Usp27xC87A was immunoprecipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL or β-TrCP was detected by Western blotting using anti-Bim or anti-β-TrCP antibodies. Western blots are representative of n≥3 independent experiments.",
    "molecules": "dox, MG132, PMA, Q-VD-OPh"
  },
  {
    "caption": "D Usp27x expression does not inhibit interaction of BimEL to β-TrCP293FT-TetR-3xFlag-Usp27x cells were transfected with pMIG-3xHA-BimEL in the presence of both PMA and QVD. At the same time dox (to induce 3xFlag-Usp27x) was added as indicated and 20h later cells were treated with MG132 [40µM] for additional 4h.Cells were lysed and 3xHA-BimEL was immunoprecipitated as described above. As a control HA-matrix was used without lysate to rule out any unspecific signal that might come from the immobilized anti HA-antibody (beads). Western blots show representative of n=2 independent experiments.",
    "molecules": "dox, MG132, PMA, QVD"
  },
  {
    "caption": "E Binding of Usp27x to BimEL can be blocked by the MEK-inhibitor UO126293FT-TetR-3xFlag-Usp27x cells (see C) were treated for 24h with dox to induce expression of Usp27x. In all conditions PMA [16.2 nM], and Q-VD-OPh [10 µM] were added at the time of Usp27x-induction. As a control, cells were pretreated with UO126 [10µM] for 30 minutes to block the PMA stimulated ERK pathway before the addition of dox plus PMA plus QVD. 3xFlag tagged Usp27x was immuno-precipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL was detected by Western blotting using anti-Bim antibodies. As a control Flag-matrix (beads) was used alongside the IP under same conditions but without addition of protein lysates. Western blots are representative of n=4 independent experiments (see also Appendix Fig. S2B).",
    "molecules": "dox, PMA, Q-VD-OPh, QVD, UO126"
  },
  {
    "caption": "F Binding of Usp27xto BimEL does not require β-TrCP293FT-TetR-3xFlag-Usp27x cells were transfected with control siRNA (siCo3) or siRNA specific for β-TrCP. 48h later cells were stimulated with PMA plus dox plus QVD for additional 24h. After cell lysis, Flag-Usp27x was immuno-precipitated using anti-Flag-matrix, and BimEL and β-TrCP bound to Usp27x were identified by Western blotting. Blots are representative of n=2 independent experiments.",
    "molecules": "dox, PMA, QVD"
  },
  {
    "caption": "G Usp27x binds specifically to BimEL293FT-TetR-3xFlag-Usp27x cells or 293FT-3xFlag-Usp27x cells with a specific deletion of the BimELprotein were treated with dox, PMA and QVD as in C. Cells were lysed, and lysates were subjected to anti-Flagimmunoprecipitation. 3xFlag-Usp27x was detected using anti-FLAG antibodies. Blots are representative of n=3 independent experiments.",
    "molecules": "dox, PMA, QVD"
  },
  {
    "caption": "A Usp27x inhibits the PMA-induced degradation of BimEL in 293FT cells. 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells were treated with dox to induce 3xFlag-Usp27x or Usp27xC87A, or with PMA [16.2 nM] (to induce BimEL degradation), or with the combination of dox, PMA and QVD [10µM] (to inhibit apoptosis) as indicated for 24 h (left) or 48 h (right). Cell lysates were analysed by Western blotting. The blots are representative of n=4 similar experiments. See also Appendix Fig. S5B. * Probably modified form of wild-type 3xFlag-Usp27x (such as dimer, ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner); this form is only seen if high expression levels of catalytically active Usp27x are reached (see Appendix Fig. S5A where Usp27x was transfected into 293FT cells) or if wt Usp27x is enriched during immunoprecipitation.** Probably degradation product of 3xFlag-Usp27x. These forms of Usp27x were not seen in every experiment.",
    "molecules": "dox, PMA, QVD"
  },
  {
    "caption": "C Usp27x stabilizes expression of p-BimEL (Ser69) in Bim-degrading conditions without reducing β-TrCP levels293FT-TetR-3xFlagUsp27x were treated with dox, PMA [16.2 nM], UO126 [10µM] or QVD [10µM] as indicated for 24h, and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho-Bim (Ser69) specific antibody. Scans are from the same membrane and exposure times. Similar results were obtained in n=2 separate experiments.",
    "molecules": "dox, PMA, QVD, UO126"
  },
  {
    "caption": "D UO126 reduces phosphorylation-associated shift of BimEL293FT-TetR-3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting. UO126 was added 30 minutes prior other stimulation. Similar results were obtained in n=3 separate experiments (see Appendix Fig. S5D).",
    "molecules": "UO126"
  },
  {
    "caption": "E Usp27x-expression reduces ubiquitination of Bim293FT-TetR-3xFlagUsp27x cells were transfected with a vector coding for 6His-ubiquitin (left) or GFP (right). After 24h cells were treated with PMA and QVD, and 3xFlag-Usp27x was induced by dox at the same time. After additional 20h cells were treated for 4h with MG132 (40 µM) to block proteasomal degradation of ubiquitinated proteins. Cells were lysed under denaturing conditions and His-ubiquitin-labelled proteins or proteins from only GFP expressing cells were purified by Ni2+-NTAaffinity chromatography. Ubiquitinated Bim (Bim-Ubn) was detected by Western blot using anti-Bim antibodies (left). Similar results were obtained in n=3 separate experiments (another experiment including a Coomassie stain is shown in Appendix Fig. S3E). *Indicates non-His-ubiquitinated BimEL that was bound unspecifically to the Ni2+-agarose beads",
    "molecules": "His, NTA, agarose, dox, MG132, PMA, QVD"
  },
  {
    "caption": "A 293FT-TetR-3xFlag-Usp27x or 3xFlag-Usp27xC87A, a polyclonal derivative where the Bim-locus had been targeted by CRISPR/Cas9- and four single clones obtained by serial dilution from the polyclonal line were treated as indicated (PMA [16.2 nM], QVD [10 µM]). Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=14 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xC87A, and 3xFlag-Usp27xBim2KO clone #14), n=7 (3xFlag-Usp27xBim2KO clone #11) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown.",
    "molecules": "PMA, QVD"
  },
  {
    "caption": "A Lines based on the human melanoma cell line 1205Lu (BRAF-V600E-positive) were made to carry GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 under the control of a doxycycline (dox)-inducible promoter. GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 were induced with dox for 24 or 48h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n=4 separate experiments (left) and n=3 experiments (right). See also Appendix Fig. S6A where 3xFlag-Usp22 is also shown to be unable to increase Bim levels.",
    "molecules": "dox, doxycycline"
  },
  {
    "caption": "C Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the TetR-repressor or carrying in addition either GFP-Usp27x or GFP-Usp27xC87A. Cells were treated with dox for 72h. Bim was detected by Western blotting. Similar results were obtained in n=2 separate experiments. GFP-Usp27x, GFP-Usp27xC87A, or GFP-Usp22 expression (A-C) was detected using an antibody against GFP.",
    "molecules": "dox"
  },
  {
    "caption": "D Caco2 cells carrying a dox inducible HA-BRAF-V600E [37] (left two lanes) or the same cells stably expressing 3xFlag-Usp27x were treated with dox for 48h to induce expression of HA-BRAF-V600E. Bim was detected by Western blotting. The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n=3 separate experiments with varying induction times. See also Appendix Fig. S6D.",
    "molecules": "dox"
  },
  {
    "caption": "A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF-V600ECaco2 cells carrying inducible HA-BRAF-V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27xmRNA) for 24h prior to BRAF-V600E expression with dox for 2h (left) or 4h (right). BimEL was detected by Western blotting. Similar results were also observed in n=2 more experiments after 3h induction (not shown, see Appendix Fig. S6E for siRNA efficacy).",
    "molecules": "dox"
  },
  {
    "caption": "B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT-Usp27xKO (clone 2/10, generated using CRISPR-Cas-9) were treated with PMA [16.2 nM] for 16h or 24h to induce BimEL degradation. Western blots show protein levels of n=3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Appendix Fig. S6G for verification of the Usp27xKO cell line by DNA sequence analysis.",
    "molecules": "PMA"
  },
  {
    "caption": "A 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point.",
    "molecules": "CHX, cycloheximide, dox, PMA, QVD"
  },
  {
    "caption": "B 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).",
    "molecules": "CHX, Cycloheximide, dox"
  },
  {
    "caption": "C HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.",
    "molecules": "CHX, cycloheximide, dox"
  },
  {
    "caption": "A 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 hUO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B.",
    "molecules": "dox, doxycycline, QVD, UO126"
  },
  {
    "caption": "B HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).",
    "molecules": "dox, gefitinib, QVD"
  },
  {
    "caption": "Figure 8 Loss of Usp27xreduces gefitinib-induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x-locus had been targeted for deletion using CRISPR/Cas9 with two different guide RNA against Usp27x (Usp27xKO-1 and Usp27xKO-4), were treated with gefitinib [10µM] for 48h. Apoptosis was measured by staining for active caspase-3 (A) or by staining for activated Bax (antibody: 6A7 clone) followed by flow cytometric analysis. Data shown in A, B (means/SEM) are from n=4 separate experiments. P-values (t-test) for statistically significant differences are shown.",
    "molecules": "gefitinib"
  },
  {
    "caption": "B-C. Graphs showing the relative weight  (B; n= 15 WT, 12  ECko; curve comparison) and the survival  (C; n= 15 WT, 11 ECko; Log-Rank test) of mice  upon tamoxifen  treatment. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "D-F. Representative images (D) and quantifications (E, F) of stainings   for CD31, Lyve1  and DAPI (nuclei) in  small intestinal wholemounts at an early disease stage (blue arrowheads point to defective lacteals, white arrowheads indicate edema) in Casp8ECko mice  (E; n= 7 WT, 6 ECko. F; n= 7 WT, 10 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "G. Representative images of small intestine  sections of mice  injected with a 70kDa fluorescently labeled dextran  and stained  for CD31  at an early disease stage. White arrowheads point to extravasated dextran  in Casp8ECko mice  (n= 5 WT, 5 ECko). Scale bars: 50µm. H. Quantification of extravasated dextran  in small intestine  and brain  lysates in Casp8WT and  Casp8ECko mice  at an early disease stage (n= 4 WT, 4-5 ECko; two-way ANOVA with tukey's multiple comparison). RFU: relative fluorescent units. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.",
    "molecules": "dextran"
  },
  {
    "caption": "A-B. Representative images (A) and quantification (B) of CD31, TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice  at an early disease stage (red dotted lines: epithelium; white dotted lines: endothelium) in (n= 5 WT, 5  ECko, multiple t-tests with Holm-Sidak method). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. ns: not significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "D-E. Graphs showing survival  and relative weight  of Casp8WT/MLKLko and  Casp8ECko/MLKLko mice  upon tamoxifen  treatment. (D; n= 5-8 WT/ MLKLko, 5-8 ECko/MLKLko; Log-Rank test, E; n= 7 WT, 8  ECko; two tailed unpaired Student's t-test).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "F. Representative pictures of intestines  of Casp8WT/MLKLko and Casp8ECko/MLKLko mice  at 30 days after tamoxifen  treatment (n= 8 WT, 8 ECko).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "G. Representative images of stainings  for CD31, Lyve1  and DAPI  in  small intestinal wholemounts from Casp8WT/MLKLko and Casp8ECko/MLKLko mice  at 30 days after tamoxifen  treatment. Scale bars: 100µm.",
    "molecules": "DAPI, tamoxifen"
  },
  {
    "caption": "H-K. Quantification of vessel area (H, I) and lacteal  length (J, K) at two different timepoints  after tamoxifen  treatment in Casp8WT/MLKLko and Casp8ECko/MLKLko mice  (n= 4-6 WT/MLKLko, 5  ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "L. Graph showing quantification of extravasated  70kDa Dextran  from small intestine  tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice  at 30 days after tamoxifen  treatment (n= 7 WT/MLKLko, 6 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.",
    "molecules": "Dextran, tamoxifen"
  },
  {
    "caption": "M-N. Representative pictures (M) and quantification (N) of lymphatic vessel density in  Peyer's Patches of  Casp8WT/MLKLko and Casp8ECko/MLKLko mice  at 140 days after tamoxifen  treatment (n = 8 WT, 7 ECko Peyer's Patches are pooled from 4 mice  per genotype; two-tailed unpaired students t-test). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. ns: not significant.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "O. Representative pictures of H&E stainings  of  small intestine  tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice  (n= 4-6 WT, 4-5  ECko). Scale bars: 100µm. P-Q. Quantification of intestinal pathology  in Casp8WT/MLKLko and Casp8ECko/MLKLko mice  at two different timepoints  after tamoxifen  treatment (enteropathy score; n=4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "C. Survival (n= 5 WT, 6 BECko; Log-Rank test) of Casp8WT and Casp8ECko mice  upon tamoxifen  treatment.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative images of stainings  for CD31, Lyve1  and DAPI  in small intestinal  wholemounts (H) and quantification of vessel density (I) in  Casp8BECko compared to Casp8WT mice (I; n= 5 WT, 6 BECko; two tailed unpaired Student's t-test, Scale bars: 100µm. Data information: Data shown as mean ± SEM. ns: not significant.",
    "molecules": "DAPI"
  },
  {
    "caption": "H. Survival  graph of Casp8WT and Casp8ECko mice  after Enbrel® treatment (n=7 WT, 5  ECko +  IgG, 8 ECko + Enbrel; Log-Rank test).",
    "molecules": "Enbrel"
  },
  {
    "caption": "Representative images of small intestines (I) of  Casp8ECko mice  after Enbrel® treatment Scale bars: 100µm.",
    "molecules": "Enbrel"
  },
  {
    "caption": "Representative images of H&E staining  (J) with histopathological scoring (K) of Casp8WT and Casp8ECko mice   after Enbrel® treatment (n=6 WT, 4 ECko +  IgG; 8  ECko + Enbrel (from which 3 mice  were diseased, 5 mice  healthy); one-way ANOVA with tukey's multiple comparison). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.",
    "molecules": "Enbrel"
  },
  {
    "caption": "analysis of the most prominent bacterial phyla (B; n= 4 Control, 6-8 Antibiotics; multiple t-tests with Holm-Sidak method) by qPCR analysis, In B, the cycle number of 16S rDNA expression  compared to the total number of cycles is shown. Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.",
    "molecules": "Antibiotics"
  },
  {
    "caption": "Graphs showing the relative weight  (C; n= 11 WT, 7 ECko, 10 ECko+ antibiotics  one-way ANOVA with tukey's multiple comparison) upon tamoxifen  treatment in the presence of an antibiotic cocktail.",
    "molecules": "antibiotics, tamoxifen"
  },
  {
    "caption": "Graphs showing the survival  of mice  (D; n= 6 WT, 7 ECko, 7 ECko+ antibiotics; Log-Rank test) upon tamoxifen  treatment in the presence of an antibiotic  cocktail.",
    "molecules": "antibiotic, tamoxifen"
  },
  {
    "caption": "E. Representative images of the gut  of  Casp8WT and Casp8ECko mice  with antibiotic  treatment 30 days after tamoxifen.",
    "molecules": "antibiotic, tamoxifen"
  },
  {
    "caption": "F-H. Representative images of H&E staining  (F), quantification of intestinal pathology  (G) and mucosal hemorrhages  (H) in  Casp8WT and Casp8ECko mice treated with antibiotics  compared to untreated Casp8ECko mice (G; n= 6 WT, 4 ECko+ antibiotics, 5  ECko; one-way ANOVA with tukey's multiple comparison, H; n= 6 WT, 4 ECko+ antibiotics, 4 ECko; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.",
    "molecules": "antibiotics"
  },
  {
    "caption": "Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations. Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl- and 0.2 mM EGTA (top panel), 150 mM gluconate (middle panel), or 2 mM BAPTA (bottom panel).",
    "molecules": "BAPTA, Cl-, EGTA, gluconate"
  },
  {
    "caption": "Representative averaged traces of sAPs of the mHb neurons with the pipette solution (40 mM Cl- and 0.5 mM EGTA) from TMEM16A cKO mice and littermate controls.",
    "molecules": "Cl-, EGTA"
  },
  {
    "caption": "Representative traces showing that the spontaneous firing of mHb neurons from ChAT(+)-cre::hM4D(Gi) mice were reduced when CNO (10 μM) was applied in the ACSF.",
    "molecules": "CNO"
  },
  {
    "caption": "A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV-hM4Di-mCherry before and during CNO treatment (The numbers inside each bar indicate the numbers of recorded cells).",
    "molecules": "CNO"
  },
  {
    "caption": "In the EPM task, the time spent in the open arms of CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that of the PBS-treated controls (CTL). (nCTL=6, ncKO=9, unpaired Student t-test). E. In the LDB task, the time spent in the light zone by CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that spent by the PBS-treated control mice. (nCTL=6, ncKO=9, unpaired Student t-test). F. In the PA task, the entry latency of CNO-treated mice was significantly decreased compared to that of the CTL group. (nCTL=6, ncKO=9, unpaired Student t-test). G. Nestlet shredding behaviors of the CNO-treated ChAT(+)-cre::hM4D(Gi) mice were also decreased. (nCTL=6, ncKO=9, unpaired Student t-test;).",
    "molecules": "CNO"
  },
  {
    "caption": "In the 3-chamber test, the subject mouse of the PBS-treated mice and the CNO-treated mice explored freely the three test chambers. The time spent in each chamber during two sessions was measured. The CNO-treated ChAT(+)-cre::hM4D(Gi) mice exhibited a decreased interest for a stranger mouse (S1) against objects (O) compared to the PBS-treated group (left two panels) (nCTL=6, ncKO=9, unpaired Student t-test). I. When CNO-treated ChAT(+)-cre::hM4D(Gi) mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to the PBS-treated control group (right two panels). The preference index showed decreased sociability of the CNO-treated group in both sessions (nCTL=6, ncKO=9, unpaired Student t-test;).",
    "molecules": "CNO"
  },
  {
    "caption": "B. Pull-down assay of Sec62 domain with different biotin-labeled Sec63 C-terminal peptides. Sec63-C2P denotes di-phosphorylated Sec63-C2.",
    "molecules": "biotin"
  },
  {
    "caption": "F. Pull-down assay of the Sec62 domain and a mutant in the interaction interface (Sec62R51E) with biotin-labeled Sec63-C2P.",
    "molecules": "biotin"
  },
  {
    "caption": "Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines: MCF7 parental or doxorubicin-resistant MCF7/ADR (ADR, 0.5μg/ml) and irradiation-resistant MCF7/IR (IR, 10Gy), MDA-MB-231 parental or doxorubicin-resistant MDA-MB-231/ADR (ADR, 0.5μg/ml) or cisplatin-resistant MDA-MB-231/DDP (1μg/ml), non-small lung cancer cell lines: A549 parental or cisplatin-resistant A549/DDP (1μg/ml).",
    "molecules": "cisplatin, DDP, ADR, doxorubicin"
  },
  {
    "caption": "MCF7, MCF7/ADR and MCF7/IR, MDA-MB-231 and MDA-MB-231/ADR cells were harvested and mRNA levels of BRD7 were determined by real-time PCR (n=3). Values are mean ± SEM and Error bars indicate SEM.",
    "molecules": "ADR"
  },
  {
    "caption": "Western blot analysis of BRD7 protein levels in MDA-MB-468 and MDA-MB-231 cell after treatment with ADR (5 μM) or camptothecin (CPT) (1 μM) for different intervals (n=3).",
    "molecules": "camptothecin, CPT, ADR"
  },
  {
    "caption": "Representative images of endogenous BRD7 (green) and γH2AX foci (red) in paraformaldehyde-fixed MDA-MB-468 cells after treatment with CPT (1 μM) for different intervals. Visualized by immunofluorescence using anti-BRD7 and Alexa Fluor 555 anti-γH2AX antibodies. DNA staining with DAPI; Scale bars, 2 μm.",
    "molecules": "Alexa Fluor 555, CPT, DAPI, DNA, paraformaldehyde"
  },
  {
    "caption": "Silver staining of the BRD7 complex separated by SDS-PAGE. HEK293T cells stably expressing SFB-tagged BRD7 were used for tandem affinity purification (TAP) of protein complexes. BRD7-interacting proteins, including PARP1 and PIK3R2, are indicated.",
    "molecules": "Silver"
  },
  {
    "caption": "HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to Myc (C) conjugated to agarose followed by Western blot with the indicated antibodies (n=3).",
    "molecules": "agarose"
  },
  {
    "caption": "HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to Flag (D) conjugated to agarose followed by Western blot with the indicated antibodies (n=3).",
    "molecules": "agarose"
  },
  {
    "caption": "MDA-MB-231 cells were treated first with Olaparib (10 μM) for 6 h and lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG or PARP1 antibodies, and analysed by Western blot (n=3).",
    "molecules": "Olaparib"
  },
  {
    "caption": "Association of endogenous BRD7 with PARP1 in HeLa cells was performed by co-immunoprecipitation using anti-BRD7 or anti-PARP1 antibody. HeLa cell were treated with CPT (1 μM, 1 h), followed by IP using indicated antibodies, and Western blot was performed. γH2AX was used as a marker of DNA damage induced by CPT (n=3).",
    "molecules": "CPT"
  },
  {
    "caption": "HeLa cells were untreated or treated with CPT (1 μM) for 1 h followed by lysing with RIPA buffer and lysates were then immunoprecipitated using anti-IgG or anti-PAR antibodies and immunoblotted with the indicated antibodies (n=3).",
    "molecules": "CPT, PAR"
  },
  {
    "caption": "HeLa cells were untreated or treated with CPT (1 μM) for 1 h and cellular lysates were immunoprecipitated using anti-IgG or anti-BRD7 antibodies and immunoblotted were performed using the indicated antibodies (n=3).",
    "molecules": "CPT"
  },
  {
    "caption": "HeLa and 293T cells transfected with Myc-BRD7 plasmid for 24 h were lysed with RIPA buffer. Lysates were then immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies. Ribosylation levels of exogenous BRD7 were detected using anti-PAR antibody (n=3).",
    "molecules": "agarose, PAR"
  },
  {
    "caption": "HeLa cells transfected with Myc-BRD7 plasmid. After 24 hours, cells were treated with either CPT (1 μM) or ADR (5 μM) combined with MG132 (10 μM) for indicated times. Cellular lysates were immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies (n=3).",
    "molecules": "agarose, CPT, ADR, MG132"
  },
  {
    "caption": "HeLa PARP1 wild-type and PARP1 knockout cells were transfected with Myc-BRD7 for 24 h, lysates were subjected to immunoprecipitation using anti-Myc agarose and analysed by Western blot (n=3).",
    "molecules": "agarose"
  },
  {
    "caption": "Ribosylation of BRD7 by PARP1 in vitro. Recombinant BRD7 was subjected to in vitro ribosylation either in absence or presence of biotin-labeled NAD+. Recombinant proteins were detected by indicated antibodies, and ribosylated proteins were determined with anti-biotin antibody (n=3).",
    "molecules": "biotin, NAD"
  },
  {
    "caption": "PAR-binding motif of BRD7 is required for its ribosylation by PARP1. Recombinant Myc-BRD7-WT and Myc-BRD7-mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated (n=3).",
    "molecules": "PAR"
  },
  {
    "caption": "HeLa cells were transfected with Flag-PARP1 and Myc-BRD7 for 24 h, then treated with MG132 (10 μM) for additional 4 h, and proteins were detected by Western blot with indicated antibodies (n=3).",
    "molecules": "MG132"
  },
  {
    "caption": "MDA-MB-231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h, cell lysates were analysed by Western blot (n=3).",
    "molecules": "PDD0017273"
  },
  {
    "caption": "MDA-MB-231 cell were treated with 10 μM of PDD0017273 for indicated times, cell lysates were analysed by Western blot (n=3).",
    "molecules": "PDD0017273"
  },
  {
    "caption": "Wild-type and PARP1 knockout HeLa cells were incubated with 10 μg/ml cycloheximide (CHX) for the indicated times. Lysates were harvested and analysed by Western blot. Quantification of BRD7 protein are shown in F (n=3) and results represent mean ± SEM. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells, respectively.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "PARP1 wild-type and knockout HeLa cells were transfected with HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h, cells were lysed with RIPA buffer; followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3).",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "SFB-BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA-ubiquitin (Lys48 or Lys63 only) for 24 h, and MG132 (10 μM) was added for an additional 4 h, lysed with RIPA, subjected to IP using S tag beads followed by Western blot (n=3).",
    "molecules": "Lys, MG132, ubiquitin"
  },
  {
    "caption": "HeLa cells were co-transfected with either wild-type or BRD7 mutant plus HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h and lysed with RIPA buffer, followed by anti-Myc agarose IP and analysed by Western blot with the indicated antibodies (n=3).",
    "molecules": "agarose, MG132, ubiquitin"
  },
  {
    "caption": "HeLa cells were transfected with either scrambled or RNF146 siRNAs for 48 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for the indicated periods of time. Lysates were harvested and analysed by Western blot (n=3). Quantification of BRD7 protein levels from B, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Control and RNF146 stably overexpressing HeLa cells were transfected with Myc-BRD7-WT for 24 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for indicated periods of time. Lysates were subjected to Western blot analysis (n=3). Quantification of BRD7 protein levels from D, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "HeLa cells transfected with either RNF146 or control siRNA for 24 h followed by transfection of HA-ubiquitin for another 24 h; MG132 (10 μM) was added for 4 h and lysed with RIPA, followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3).",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "HeLa cells stably expressing RNF146 were transfected with HA-ubiquitin for 24 h; MG132 (10 μM) was added for an additional 4 h, followed by anti-BRD7 IP and Western blot (n=3).",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "HeLa cells transfected with the indicated plasmids; after 24 h, cells were lysed with RIPA buffer followed by immunoprecipitation (IP) using either anti-Myc or anti-Flag agarose and Western blot with indicated antibody (n=3).",
    "molecules": "agarose"
  },
  {
    "caption": "Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co-IP using anti-RNF146 antibody. Cell lysates were incubated with protein-G agarose beads conjugated with indicated antibodies, and Western blot was performed (n=3).",
    "molecules": "agarose"
  },
  {
    "caption": "HeLa cells with wild-type or PARP1 knockout were transfected with Myc-BRD7-WT for 24 h, their lysates were subjected to IP using anti-Myc agarose and analysed by Western blot with indicated antibodies (n=3).",
    "molecules": "agarose"
  },
  {
    "caption": "HeLa cells stably expressing Flag-RNF146 were transfected with either Myc-BRD7-WT or Myc-BRD7-mutant for 24 h, lysates were subjected to IP using anti-Flag agarose and analysed by Western blot with indicated antibodies (n=3).",
    "molecules": "agarose"
  },
  {
    "caption": "MDA-MB-231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM), CPT (0.5 μM) or VP16 (10 μM) for 72 h, and CCK8 activity of the cells was detected (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test.",
    "molecules": "CPT, ADR, Olaparib, VP16"
  },
  {
    "caption": "MDA-MB-231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM) for 72 h, and their lysates were subjected to IP using anti-BRD7 antibodies and analysed by Western blot with indicated antibodies (n=3).",
    "molecules": "ADR, Olaparib"
  },
  {
    "caption": "MDA-MB-231 cells were transfected twice with BRD7 siRNAs, and these cells were subjected to CCK8 assay after treatment with Olaparib (10 μM) combined with either ADR (0.5 μM) or VP16 (10 μM) for 72 h (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test.",
    "molecules": "ADR, Olaparib, VP16"
  },
  {
    "caption": "Resistance of BRD7-depleted cells to Olaparib was reversed by the expression of wild-type BRD7 but not mutant BRD7. shRNA-resistant BRD7 wild-type and BRD7 mutant plasmids were transduced into BRD7-depleted MDA-MB-231 cells, and cell viability was determined as indicated above (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test.",
    "molecules": "Olaparib"
  },
  {
    "caption": "A. Colony formation assay (CFA) with imatinib with representative images of colonies formed by each cell line within the panel. Error bars represent mean ±S.E.M., n=9, *P < 0.001 by two-tailed unpaired Student's t-test (H1915 P= 0.0004, H2110 P=0.0001).",
    "molecules": "imatinib"
  },
  {
    "caption": "B. Western blot analysis after 1 hour treatment with imatinib.",
    "molecules": "imatinib"
  },
  {
    "caption": "C. MTT assay for NSCLC cell lines and Beas-2B cells with dasatinib or DMSO. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (H1915 P=1.09×10-5, H2110 P=0.009 and A549 P=6×10-6).",
    "molecules": "dasatinib, DMSO"
  },
  {
    "caption": "D. Western blot analysis after 1 h treatment with dasatinib.",
    "molecules": "dasatinib"
  },
  {
    "caption": "A -C. Drug-resistant ABL1-T315I rescues dasatinib- and imatinib-induced cytotoxicity. Western blot analysis of (A) transient and (B) tetracycline-inducible overexpression of ABL1-T315I in H1915 cells treated with dasatinib or imatinib.",
    "molecules": "dasatinib, imatinib, tetracycline"
  },
  {
    "caption": "C. CFA with dasatinib or imatinib for H1915 cells with or without tetracycline-inducible expression of ABL1-T315I. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (control, DMSO vs dasatinib P=4.3×10-12 vs imatinib P=6×10-6; dasatinib, control vs tetracycline, P=9×10-7; imatinib, control vs tetracycline P=9.7×10-5).",
    "molecules": "dasatinib, DMSO, imatinib, tetracycline"
  },
  {
    "caption": "A-C. Average tumor volumes of xenografts derived from H1915 (A), H650 (B) and (C) H1915 cell line with doxycycline inducible ABL1 gate-keeper mutation (H1915-ABL1T315I) in mice treated with either vehicle or imatinib (100mg/kg). Error bars represent ±S.D., n=6-10 mice per group, *P=0.0016 by Mann-Whitney U test.",
    "molecules": "doxycycline, imatinib"
  },
  {
    "caption": "A. Immunofluorescence imaging of endogenous ABL1 in the panel of NSCLC cell lines; scale bars=20 μm. B and C Quantification of nuclear versus cytoplasmic localization of (B) endogenous ABL1 in NSCLC cell lines and (C) tetracycline-inducible expression of ABL1-WT, ABL1-R351W and ABL1-G340L in Beas-2B cells. Error bars represent ±S.D., n=6-10 images, *P<0.005 by two-tailed unpaired Student's t-test (Beas-2B vs H1915 P=6.6×10-5, vs H2110 P=0.0001; WT vs R351W P=0.003, vs G340L P=0.002).",
    "molecules": "tetracycline"
  },
  {
    "caption": "(C) Apamin  and XE991 decrease the size of aberrant aggresomes, confirming enhanced autophagy  degradation (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with the ALSC9orf72 I line. Scale bar: 5 Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.",
    "molecules": "Apamin, XE991"
  },
  {
    "caption": "(E)  Apamin and XE991 increase the  levels  of pCREBS133 in all the  ALSC9orf72 lines (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 10 µm. Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.",
    "molecules": "Apamin, XE991"
  },
  {
    "caption": "PMO   was administered intravenously into adult mdx mice  at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   administration. (B) Immunohistochemistry  for dystrophin expression  in body-wide muscles  from mdx mice  treated with PMO  in saline  (PMO-S), PMO-G(IV) or PMO-G (D)(scale bar=100μm).PMO-G (IV) refers to intravenous injection of PMO   in combination with intravenous injection of 5% glycine  every other day for 3 weeks. PMO-G (D) represents intravenous injection of PMO   supplemented with 1% glycine  orally.",
    "molecules": "glycine, PMO, saline"
  },
  {
    "caption": "PMO   was administered intravenously into adult mdx mice  at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   administration. (C) Western blot  for dystrophin  expression in body-wide muscles from mdx mice  treated with PMO-G (IV), PMO-G (D) or PMO-S. 0.5µg, 2.5 µg, 5µg, 10µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice  were loaded. α-actinin was used as the loading control. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, D-diaphragm  and A-abdominal muscle. (D) Quantitative analysis of dystrophin  expression in body-wide muscles from mdx mice  treated with PMO-G (i.v., n=4), PMO-G (D25 and D50, n=3) and PMO-S (n=3) (*P<0.05,**P<0.001; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G). (A) Muscle function was assessed to determine the physical improvement with grip strength test (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G). (B) Measurement of bodyweight  changes of DKO mice  treated with PMO-G or PMO-S (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G). (C) Measurement of TA  muscle mass  of DKO mice  treated with PMO-G or PMO-S (n = 3, *P < 0.05; one-way ANOVA post-hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G). (D) Immunohistochemistry  for dystrophin expression  in treated DKO mice (scale bar=100 μm). TA-tibialis anterior",
    "molecules": "glycine, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G). (E, F) Western blot and quantitative analysis for the dystrophin  protein in treated DKO mice  (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle and D-diaphragm. 2.5 µg, 5µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice  were loaded.  α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G) and metformin  daily via drinking water (PMO-GM). (B) Measurement of  Ejection Fraction (EF) and Fractional Shortening  (FS) in treated DKO mouse hearts  (n=3) and C57BL/6 mice  (n=6) with echocardiography  (*P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G) and metformin  daily via drinking water (PMO-GM). (C) Morphological  examination of treated DKO mouse hearts  with H&E staining  (scale bar=800μm). Boxed areas are magnified. (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test) Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G) and metformin  daily via drinking water (PMO-GM). Immunohistochemistry  for  immunoglobin G (IgG) in treated DKO mouse hearts  (D, scale bar=100 μm).",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G) and metformin  daily via drinking water (PMO-GM). Muscle function was evaluated to determine the physical improvement with  grip strength (E) or running wheel tests (F) for treated DKO (n=3) or C57BL/6 mice  (n=4) (*P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G) and metformin  daily via drinking water (PMO-GM). (G) Immunohistochemistry  for dystrophin expression  in body-wide muscles from treated DKO mice  (scale bar=100 μm).",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction (PMO-G) and metformin  daily via drinking water (PMO-GM). (H)  Western blot and quantitative analysis for dystrophin  protein in treated DKO mice (n=3). 5µg, 10µg and 20µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice  were loaded. α-actinin  was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (B) Survival rate  of DKO mice  treated with PMO-GM (n=6).The number of untreated DKO controls is 15.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (C) Measurement of kyphosis  index of DKO mice  treated with PMO-GM (scale bar=5mm) and wild-type C57BL/6 mice  (scale bar=8mm)(n=3, *P<0.05; One way- ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (D) Measurement of  tidal volume in treated DKO mice  with a cardio-respiratory gated technique (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (E-F) Examination of EF  and FS  (E) and stroke  volume and cardiac  output (F) in treated DKO mouse hearts  (n=3) and C57BL/6(n=5) with echocardiography  (*P<0.05, **P<0.001; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (G) Masson trichrome's staining for cardiac fibrosis   and quantitative analysis of fibrotic  areas in treated DKO mouse hearts  (scale bar=800 μm). Boxed areas are magnified (n=3, **P<0.001;One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (H) Measurement of serum creatine kinase-MB   (CK-MB) levels in treated DKO mice  (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (I-J)  Muscle function  was assessed to determine the physical improvement with grip strength (I) or running wheel tests (J) in treated DKO (n=4) and C57BL/6 mice  (n=6) (*P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (A) Immunohistochemistry  for dystrophin expression  in treated DKO mice  (scale bar=100 μm).",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (B) Western blot  and quantitative analysis for dystrophin  protein in treated DKO mice  (n=3).5µg, 10µg and 25µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice  were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (C) Re-localization  of DAPC  components in treated DKO mice  to assess dystrophin  function and recovery of normal myoarchitecture (scale bar=50μm). The arrowheads point to identical myofibers.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (D) Measurement of serum CK  levels in treated DKO mice  (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Measurement of serum  indices from treated DKO mice  to reflect liver  (E) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. Measurement of serum  indices from treated DKO mice  to reflect kidney  (F) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": "PMO   was administered intravenously into 3-week old DKO mice  at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine  for 3 consecutive days per week prior to PMO   introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine  for 7 consecutive days per month prior to PMO   introduction, in combination with metformin  daily via drinking water. (G) Morphological  examination of liver  and kidney  from treated DKO mice  (scale bar=50 μm).",
    "molecules": "glycine, metformin, PMO"
  },
  {
    "caption": " In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed. A and B, Top gene ontologies (GOs) and KEGG pathways of the genes downregulated in VM-glia by the SGK1 inhibitor treatment. The purple bar indicates the number of genes under the designated GO term/KEGG pathway. The red bar indicates the p-value, and the negative log of the p-value (bottom) is plotted on the X-axis. ",
    "molecules": "DMSO, GSK-650394"
  },
  {
    "caption": " B, mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the q-PCR analyses in other independent VM-glial cultures treated with the inhibitor (GSK-650394) and sh-Sgk1 and si-Sgk1 (vs. DMSO, sh-control, and si-control, respectively) (B). n=3; Student's t-test. Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++ in graph B. ",
    "molecules": "DMSO, GSK-650394"
  },
  {
    "caption": " C, Immunoblot analysis to assess NLRP3 inflammasome activation. To activate the inflammasome pathway, VM-derived glial cells were treated with LPS (0.25 µg/ml, 3h) and then ATP (2.5 mM, 30 min) in the presence or absence of the SGK1 inhibitor GSK-650394. Two days later, intracellular and released levels of pro- and activated caspase-1 and IL-1β proteins were determined in the culture media and cell lysates, respectively. ",
    "molecules": "ATP, GSK-650394, LPS"
  },
  {
    "caption": " Expression of CGAS-STING pathway genes in the RNA-seq data. The graph represents log2 RC ratios of [GSK-650394-treated/DMSO-treated control]. ",
    "molecules": "DMSO, GSK-650394"
  },
  {
    "caption": " E, Mitochondrial ROS levels estimated using the MitoSox red probe. Fluorescence intensity was measured using ImageJ, and ROS levels are presented as mean fluorescent intensity (MFI). Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0013*, 0.0273# in graph E. ",
    "molecules": "MitoSox red, CCCP, H2O2, ROS"
  },
  {
    "caption": "F, Mitochondrial membrane potential (JC-1). The red fluorescence (JC-1 Aggregate) indicates disrupted mitochondrial membrane potential, and green fluorescence (JC-1 Monomer, insets) indicates intact potential. The inset images were taken from the same microscopic fields of the respective JC-1 aggregate images. Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0006*, 0.0093# in graph F.",
    "molecules": "JC-1, CCCP, H2O2"
  },
  {
    "caption": " G, Intracellular levels of ATP, an indicator of mitochondrial oxidative phosphorylation. Data are expressed as mean ± SEM. n=3 cultures; one-way ANOVA. Significantly different at p= 0.0009*, 0.00004**, 0.04913#, 0.0024+in graph G. ",
    "molecules": "ATP"
  },
  {
    "caption": " Glial cell senescence rescued by SGK1 inhibition. (H) Expression of genes associated with cell senescence in the RNA-seq data (VM-glia with vs. without GSK-650394 treatment). ",
    "molecules": "GSK-650394"
  },
  {
    "caption": " (I) Intracellular ROS levels estimated by DCF-DA. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=0.0074*, 0.012# in graph I. ",
    "molecules": "DCF-DA, ROS"
  },
  {
    "caption": " (M) Glial cell viability following treatment with senolytic compounds (azithromycin, fisetin). Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0073*,0.0018**, 0.0286#, 0.0269##, 0.0045### in graph M. ",
    "molecules": "azithromycin, fisetin"
  },
  {
    "caption": "B, Western blot analysis for detecting SNCA oligomers. Data are represented as mean ± SEM. n=4; one-way ANOVA. Significantly different at p=2.5E-07*, 1.65E-07**, 0.022#, 0.029##, 0.046+, 0.015++, 0.0136&, 0.0016&& in graph B.",
    "molecules": "oligomers"
  },
  {
    "caption": " Glia derived from mouse VM were transduced with sh-Sgk1 (B) or SGK1 (CMV-Sgk1, C), and co-cultured with VM-derived mDA neurons. Seven days after co-culture, cells were exposed to H2O2 (500µM, 3hr) or not (-), and then mDA neuron viability (TH+ cell counts) and neurite degeneration (TH+ fiber lengths) were estimated. Data are represented as mean ± SEM; n=4 culture coverslips. Student's t-test. Significantly different at p=0.033*, 0.0412**, 7.44E-23#,1.39E-23## in graph B and p=0.0489*, 0.0366**, 0.0078## in graph C. ",
    "molecules": "H2O2"
  },
  {
    "caption": " Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner. VM-glia transduced with sh-Sgk1 (or sh-cont as a control) were challenged with H2O2+LPS for 3hr or not. Glial conditioned medium (GCM) was prepared in each glial culture condition and administered to mDA neurons primarily cultured from mouse VM (E) Cell viability of the neuronal cells was estimated using CCK8 assays and TuJ1+ cell counts. Data are represented as mean ± SEM. n=3 cultures; One-way ANOVA. Significantly different at p=0.0002*, 3.29E-08**, 0.0003***, 3.66E-06#, 0.0029## (TuJ1+ cell counting), 0.0127+, 5.98E-06++, 0.0002+++, 0.0005&, 0.0078&& (CCK8 assays) in graph E. ",
    "molecules": "H2O2, LPS"
  },
  {
    "caption": " (B-D) Behaviors of MPTP-PD mice assessed by pole (B), beam (C) at 3-6 weeks post-MPTP injection, and locomotor activity (total distance & average speed, D) tests at 6 weeks post-MPTP injection. n= 20 GSK-650394-treated MPTP-PD mice, 14 vehicle-treated MPTP-PD mice, and 8 MPTP-untreated non-PD mice. Data are represented as mean ± SEM. Significantly different at p=0.0012#, 0.0001*, 3.09E-08##, 8.21E-08** in graph B and p=0.0009#, 0.0035*, 1.03E-06##, 0.00003** in graph C and p=0.0001*, 0.0284**, 0.0101# (total distance), 1.49E-08*, 0.0005**, 0.0001# (average speed) in graph D. ",
    "molecules": "MPTP, GSK-650394"
  },
  {
    "caption": " Therapeutic effects of GSK-650394 in SNCA-PD model mice. Behaviors (L-N) of the SNCA-PD mice were assessed at 6-12 weeks post- SNCA injection Significantly different at p=0.008#, 0.021*, 0.00001##, 0.0009** in graph L and p=0.00002#, 0.0344*, 0.0005##, 0.0067** in graph M and p=0.0057#, 0.0345* (total distance), 0.0001#, 0.0375##, 0.0338* (average speed) in graph N. ",
    "molecules": "GSK-650394"
  },
  {
    "caption": " Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN of the SNCA-PD mice were assessed at 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice. ",
    "molecules": "GSK-650394"
  },
  {
    "caption": " Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN (O-U) of the SNCA-PD mice were assessed 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice. ",
    "molecules": "GSK-650394"
  },
  {
    "caption": " A-C, Quantitative and morphometric analyses of microglia in the SN area of SNCA-PD mice. Shown in A are representative views of Iba1+ microglia neighboring TH+ mDA neurons in the SNs of PD mice treated with GSK-650394 and vehicle. Insets, enlarged views for the boxed areas. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=1.49E-11* in graph B and p=0.000003* in graph C. ",
    "molecules": "GSK-650394"
  },
  {
    "caption": " F and G, mRNA expression of NLRP3-inflammasome components and pro-inflammatory cytokines in MPTP (F) and SNCA (G) PD mice injected with the SGK1 inhibitor or vehicle (control). Each bar represents the mean ± SEM of 3 PCR values from each animal, n=3 mice per group. Significantly different at p=0.0372*, 0.0173**, 0.0003*** in graph F and p=0.002#, 0.0113##, 0.0053###, 0.0001#### in graph G. ",
    "molecules": "MPTP"
  },
  {
    "caption": "(E and F) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with ethanol-d6 (5 mM) or ethanol-1-13C (5 mM) for 0.5 h. Relative acetate m+3 and acetate m+1 levels (E-F, top panels) were examined. Protein levels of p53 and ALDH2 (E-F, bottom panels) were analyzed by western blot.",
    "molecules": "acetate, ethanol-1-13C, ethanol-d6"
  },
  {
    "caption": "(H-J) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with doxorubicin (2 ng/mL) (H) nutlin3α (10 μM) (I) or cyanamide (1 mM) (J) for 24 h. Relative enzymatic activity of ALDH2 was examined (top panel). Protein expression was determined by western blot analysis (bottom panel).",
    "molecules": "cyanamide, doxorubicin, nutlin3α"
  },
  {
    "caption": "(P-R) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h. Relative acetate m+3 levels in mouse liver tissues (P) and mouse serum (Q), and relative hepatic acetyl-CoA (R) were examined.",
    "molecules": "acetate, acetyl-CoA, ethanol-d6"
  },
  {
    "caption": "(S) Mouse hepatocyte isolated from p53+/+ and p53-/- mice and lysates were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were measured. (T) CCC-HEL-1 cells transfected with control siRNA or p53 siRNA were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were analyzed.",
    "molecules": "acetyl-CoA, cyanamide"
  },
  {
    "caption": "(U-X) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were transfected with control siRNA or ALDH2 siRNA and were treated with ethanol-d6 (5 mM) (U) or ethanol-1-13C (5 mM) (X) for 0.5 h. (U) Relative acetate m+3 and acetyl-CoA m+3 levels are shown. (X) Relative acetate m+1 and acetyl-CoA m+3 levels were examined. Protein levels of p53 and ALDH2 were analyzed by western blot (V- W).",
    "molecules": "acetate, acetyl-CoA, ethanol-1-13C, ethanol-d6"
  },
  {
    "caption": "(G) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 mut312-323 in ALDH2-knockout cell lines. The cells were then transfected with control siRNA or p53 siRNA for 48 h and treated with ethanol (5 mM) for 0.5 h and relative ALDH2 enzymatic activities were examined. Protein expression was analyzed by western blot.",
    "molecules": "ethanol, hygromycin"
  },
  {
    "caption": "(I) HepG2 cells infected with lentivirus expressing ALDH2 sgRNA or control sgRNA were treated with ethanol (5 mM) for 0.5 h and cell lysates were treated with disuccinimidyl suberate (DSS) or DMSO for 0.5 h and protein levels of p53 and ALDH2 were examined.",
    "molecules": "DMSO, disuccinimidyl suberate, DSS, ethanol"
  },
  {
    "caption": "(J) Subcellular fractionation of HepG2 cells treated with doxorubicin (2 ng/mL) or nutlin3α (10 μM) or PFTα (10 μM) for 24 h were immunoprecipitated using p53 antibody, and bound proteins were analyzed by western blot.",
    "molecules": "doxorubicin, nutlin3α, PFTα"
  },
  {
    "caption": "(A-B) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA under glucose or pyruvate deprivation and cell lysates were used to measure ALDH2 activities (A, B, top panels) and examine p53 and ALDH2 proteins (A, B, bottom panels).",
    "molecules": "glucose, pyruvate"
  },
  {
    "caption": "(E) CETSAs exhibit the binding affinity of pyruvate to the N186 amino acid in HepG2 cells. Cell lysates were separated by SDS-PAGE and analyzed by western blot. Actin was used as an internal control. Relative ALDH2 band intensities were quantified and plotted against corresponding incubation temperatures.",
    "molecules": "pyruvate"
  },
  {
    "caption": "(F) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2-N186A, E285A, C319A in ALDH2-knockout cell lines. Cells were digested and cell lysates were incubated with the indicated doses of pyruvate for 1 hour on ice and 0.5 hour at room temperature, followed by pronase digestion. ALDH2 proteins were analyzed by western blot. Relative ALDH2/Actin ratios are shown.",
    "molecules": "hygromycin, pronase, pyruvate"
  },
  {
    "caption": "(H-J) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 N186A in ALDH2-knockout cell lines. The cells were treated with pyruvate (1 mM) for 24 h. Relative ALDH2 activity (H, top panel), acetate (I) and acetyl-CoA (J) were measured. The expression of p53 and ALDH2 were analyzed by western blot (H, bottom panel).",
    "molecules": "acetate, acetyl-CoA, hygromycin, pyruvate"
  },
  {
    "caption": "H&E (hematoxylin-eosin) staining (C, top) and oil red O staining (C, bottom) were examined.",
    "molecules": "eosin, hematoxylin, oil red O"
  },
  {
    "caption": "(J) Relative triglyceride level was examined. (K-L) Relative levels of serum ALT (K) and AST (L) were examined. Data in are from n=5 mice per group.",
    "molecules": "triglyceride"
  },
  {
    "caption": "(M-O) WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h as shown in Figure 1N. Relative acetate and acetate m+3 levels in mouse liver tissues (M) and mouse serum (N) were examined. (O) Relative acetyl-CoA and acetyl-CoA m+3 levels in mouse liver tissues were measured.",
    "molecules": "acetate, acetyl-CoA, ethanol-d6"
  },
  {
    "caption": "(C-D) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 1 h. Chromatin fractions of liver tissues were subjected to ChIP-seq assay using H3K9ac antibody (left panel), H3K27ac antibody (right panel). Heat map of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags (C). Heatmap of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags of fatty acid metabolism related genes (D). For ChIP-seq analysis , there were no technical replicates for anti-H3K9ac or anti-H3K27ac.",
    "molecules": "ethanol-d6"
  },
  {
    "caption": "(F) ChIP assays with H3K9ac antibody and H3K27ac antibody or IgG in WT and p53−/− mice injected intraperitoneally with ethanol-d6 (2 g/kg) or saline for 1 h. Bound SCD1 response elements (RE) were analyzed by qRT-PCR. Data in F are from n=3 biological independent samples.",
    "molecules": "ethanol-d6, saline"
  },
  {
    "caption": "WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were fed control diet and ethanol diet as shown in Fig.4A. Relative mRNA level of SCD1 (G, top panel) was analyzed. Protein levels of p53, ALDH2, SCD1, Histone H3 and Ac-H3, caspase 3, cleaved caspase 3, p21 (G, bottom panel) in mice liver tissues were examined. Data in G are from n=5 technical replicates from one of three independent experiments with similar results.",
    "molecules": "ethanol"
  },
  {
    "caption": "WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (C) Protein levels of p53 and SCD1 were examined by western blot. level were examined.",
    "molecules": "ethanol"
  },
  {
    "caption": "WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level(D), relative serum ALT (E, left panel) and AST (E, right panel) level were examined.",
    "molecules": "ethanol, triglyceride"
  },
  {
    "caption": "WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (H) Protein levels of p53 and SCD1 are shown.",
    "molecules": "ethanol"
  },
  {
    "caption": "WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level (I), relative serum ALT (J, left panel) and AST (J, right panel) were examined.",
    "molecules": "ethanol, triglyceride"
  },
  {
    "caption": "e, Shift in phase durations of RPE cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 115",
    "molecules": "aphidicolin"
  },
  {
    "caption": "f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 115",
    "molecules": "aphidicolin"
  },
  {
    "caption": "k, Shift in phase durations of RPE cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; **, P < 1 × 10-10; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 119.",
    "molecules": "NCS"
  },
  {
    "caption": "l, Pairwise correlation between phase durations for cells treated with NCS. Number of cells: NCS, n = 119.",
    "molecules": "NCS"
  },
  {
    "caption": "g, Shifts in phase durations for RPE cells treated with 2 μM CVT-313. A boxplot representing the distributions of durations in untreated cells is underlaid for comparison. *, P < 1 × 10-5, 2-sided Kolmogorov-Smirnov test. (n = 117 cells).",
    "molecules": "CVT-313"
  },
  {
    "caption": "h, Pairwise correlation between cell cycle phase durations upon treatment with CVT-313.",
    "molecules": "CVT-313"
  },
  {
    "caption": "b, Shift in phase durations of U2OS cells treated with NCS. , boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: NCS, n = 114",
    "molecules": "NCS"
  },
  {
    "caption": "c, Pairwise correlation between phase durations for U2OS cells treated with NCS. Number of cells: NCS, n = 114",
    "molecules": "NCS"
  },
  {
    "caption": "e, Shift in phase durations of U2OS cells treated with 50 ng/mL aphidicolin. boxplots representing the distributions of phase durations in untreated cells are underlaid for comparison. *, P < 1 × 10-5; ***, P < 1 × 10-20, 2-sided Kolmogorov-Smirnov test. Number of cells: aphidicolin, n = 153.",
    "molecules": "aphidicolin"
  },
  {
    "caption": "f, Pairwise correlation between cell cycle phase durations under aphidicolin treatment. Number of cells: aphidicolin, n = 153.",
    "molecules": "aphidicolin"
  },
  {
    "caption": "F. Representative hematoxylin and eosin (H&E)-stained images of mouse brain sections with GSC1123 xenografts expressing shC, shL115-1, or shL115-2. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm. G. Quantification of tumor volume in F. ",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "E. RNA pull-down and qRT-PCR assays of LINC00115 binding with miR-200s. GSC1123 cell lysates were incubated with biotin-labeled LINC00115. Data information data are representative of three independent experiments. Error bars, ± SD. p < 0.05, **, p < 0.01, by One-way ANOVA.",
    "molecules": "biotin, RNA"
  },
  {
    "caption": "LINC00115 regulated ZNF596/EZH2/STAT3 signaling. 1123 and 528 GSCs were treated with or without EZH2 inhibitor GSK343 (3 μM) for 6 Data information: data are representative of three independent experiments. Error bars, ± SD.",
    "molecules": "GSK343"
  },
  {
    "caption": "EZH2 inhibitor GSK343 impaired ZNF596 overexpression-reversed and LINC00115 knockdown-inhibited neuro-like sphere formation. 1123 and 528 GSCs with an inducible ZNF596 shRNA were treated with or without Dox (2 μg/ml) and/or EZH2 inhibitor GSK343 (1 μM) for 7 days. Data information: data are representative of three independent experiments. Error bars, ± SD. by two-tailed t-test",
    "molecules": "Dox, GSK343"
  },
  {
    "caption": "Growth kinetics of subcutaneous xenograft tumor with indicated genetic modifications after treatment with or without Dox and/or GSK343. Tumor-bearing mice (five mice per group) were fed with 10% sucrose water with or without 2 mg/ml Dox and combined with treatment with or without GSK343 (5 mg/kg body weight from Monday to Friday via intraperitoneal injection) for three weeks beginning five days post-xenograft transplantation. Data were from three independent experiments with five mice per group. Data information: *, p < 0.05, **, p < 0.01, ***, p < 0.001 , by two-tailed t-test",
    "molecules": "Dox, GSK343, sucrose, water"
  },
  {
    "caption": "(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm. (F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.",
    "molecules": "EdU"
  },
  {
    "caption": "(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). (I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison.",
    "molecules": "L685458"
  },
  {
    "caption": "(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. (G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.",
    "molecules": "CMFDA"
  },
  {
    "caption": "(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). (I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.",
    "molecules": "CFDA"
  },
  {
    "caption": "G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections; blue=DAPI, green = CD68 (TAMs).",
    "molecules": "DAPI"
  },
  {
    "caption": "I-J, Assessment of TAM suppression of T cells was assayed by evaluation of inhibition of T cell proliferation. TAMs were isolated by magnetic bead separation using F480 microbeads and co-cultured at a 1:1 ratio with CFSE labelled CD8+ T cells. CFSE dilution was analysed by flow cytometry to measure proliferation. Percentages of CFSElo T cells were analysed by Kruskal-Wallis with Dunn's multiple comparisons test. Representative histograms are shown (right panel). Experiments were repeated twice for each tumour cell line (n=3 mice/group) *P <0.05 **P <0.01 ***P <0.001.",
    "molecules": "CFSE"
  },
  {
    "caption": "G-H, Immunofluorescent staining of MC38 (G) and KPC (H) tumour sections, blue=DAPI, green = CD8.",
    "molecules": "DAPI"
  },
  {
    "caption": "HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY or the N- or C-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was ob­served by fluorescence micro­­scopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNFY-treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (ΔcnfY) harboring the empty expression vector were used as negative controls.",
    "molecules": "DAPI, FITC, phalloidin"
  },
  {
    "caption": "HEp-2 cells were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNFY, N- or C-terminal deletion variants fused to GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and smaller images are magnified views of boxed areas. White scale bar is 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Nitrocefin (2 mM) was added to the supernatant from 25°C overnight Yersinia cultures expressing the indicated CNFY derivatives to determine β-lactamase activity by measuring changes in absorbance at 390 nm (yellow) and 486 nm (red). The data represent the mean ± SD of three independent experiments, carried out in triplicates.",
    "molecules": "Nitrocefin"
  },
  {
    "caption": "Comparative analysis of recombinant RhoA deamidation by CNFY and the D4-5 fragment. Recombinant RhoA was incubated with purified CNFY or the D4-5 fragment and samples were separated by SDS-PAGE after the indicated times before subjecting to trypsin digestion and quantification of deamidation of Q63 by mass spectrometry. Error bars represent standard deviations of triplicate measurements.",
    "molecules": "trypsin"
  },
  {
    "caption": "(J) IgM-BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs. Anti-endophilin A2 blots detect overexpressed endophilin A2-GFP in streptavidin immunoprecipitates and total supernatants from anti-IgM-biotin-stimulated Ramos and DG75  cells. Representative experiment and quantification from 3 independent pulldowns in WT, BLNK- or GRB2-knockout (KO)  DG75 cells.  Pulldown  intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment. Data show mean and SEM; p values calculated using one-way ANOVA.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(D) Quantification of  B220+Fas+CD38- GC  population as percentage of total   splenic B cells and NP-specific cells as a percentage of the GC population 14 days after NP-CGG  immunization in alum. Data show mean ± SEM from N = 6 mice, p, significance in unpaired t tests.",
    "molecules": "alum"
  },
  {
    "caption": "(F)  IgM degradation following total cell surface biotinylation, measured as loss of  biotin-labelled membrane IgM (mIgM) in cell lysates over time. (G)  Western densitometry quantifying IgM degradation assay. Proportion of biotin detected is normalized to the initial WT condition (left) or to the initial amount for each genotype (right). Data show mean ± SEM from three independent tests. P, statistical significance from two-way ANOVA. ",
    "molecules": "biotin"
  },
  {
    "caption": "(K) Proliferation (CFSE dilution) of OTII CD4 cells in co-cultures with anti-Igκ-OVA pulsed  B cells; and quantification from 6-8  mice. Data show mean ± SEM.",
    "molecules": "CFSE"
  },
  {
    "caption": "Cell cultures with full RPMI media supplemented with 50 μg/ml  FeSO4 or 5 μM hemin chloride. (J) SH3GL1-targeted Ramos cells. N = 4 independent infections. P, statistical significance from two-way ANOVA from both treatments versus RPMI. (K) Control sgRNA-targeted Ramos cells.",
    "molecules": "hemin chloride, FeSO4"
  },
  {
    "caption": "AGS cells were treated with 200 nM synthetic ADP‑heptose for the indicated times. Total cell lysates were analyzed by immunoblotting using the indicated antibodies.",
    "molecules": "ADP, heptose"
  },
  {
    "caption": "AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by H. pylori infection or addition of ADP-heptose (200 nM). Total cell lysates were analyzed by immunoblotting using the indicated antibodies. CagA demonstrated a similar infection rate.",
    "molecules": "ADP, Dynasore, heptose"
  },
  {
    "caption": "AGS cells were pre‑incubated with 100 μM dynamin GTPase inhibitor Dynasore for 60 min followed by addition of ADP-heptose (200 nM). Co‑IP with an anti‑TIFA antibody was performed. Eluates and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk indicates an unspecific band and the arrow indicates the TRAF3 band.",
    "molecules": "ADP, Dynasore, heptose"
  },
  {
    "caption": "In TIFA‑KO cells transiently expressing TIFA‑tdTomato, co-localization of TRAF6 (A), TRAF2 (B), cIAP1 (C) and TRAF3 (D) with TIFA-tdTomato, which formed TIFAsomes upon infection with H. pylori P1 wild‑type strain, were detected by immunofluorescence staining. The nuclei were counterstained with DAPI. Scale bar = 10 µm. TIFA‑KO cells transiently expressing TIFA‑tdTomato were infected with H. pylori P1 strain mutated in the gmhA gene (∆HP0857). Scale bar = 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (A) Induced Cyp1a1 mRNA expression in response to different treatments (n=3 per group).",
    "molecules": "TCDD, CH223191"
  },
  {
    "caption": "Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (B) Assessment of GFP+ stem cell percentages (n=3 replicates per treatment). Bars with different letters are significantly different (p<0.05).",
    "molecules": "TCDD, CH223191"
  },
  {
    "caption": "(D) Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type (Lgr5-GFPCreERT2 X Tomatof/f, GT) and AhR knock out (AhRf/f X Lgr5-GFPCreERT2 X Tomatof/f, HGT) mice. Top left panel shows representative cells from WT negative control animal (no GFP or Tomato expression). Top right panel shows Tomato only control (CDX2P-CreERT2 X Tomatof/f). Middle panels represent DMSO or TCDD treatment in AhR WT mice, respectively. Mice were gavaged every other day, 4 times total, with vehicle or TCDD (25 µg/kg bw) and terminated 1 d later. Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(E) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD (n=4).",
    "molecules": "TCDD"
  },
  {
    "caption": "(B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm.",
    "molecules": "TCDD, DMSO, tamoxifen"
  },
  {
    "caption": "(C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05).",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group).",
    "molecules": "TCDD"
  },
  {
    "caption": "(J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells.",
    "molecules": "TCDD"
  },
  {
    "caption": "(B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group.",
    "molecules": "TCDD"
  },
  {
    "caption": "Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm.",
    "molecules": "DMSO, FDI-6"
  },
  {
    "caption": "(J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K).",
    "molecules": "DMSO, FDI-6"
  },
  {
    "caption": "(L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment.",
    "molecules": "FDI-6"
  },
  {
    "caption": "(M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.",
    "molecules": "DMSO, FDI-6"
  },
  {
    "caption": "(A and B) Proliferating Lgr5+ stem cells marked by EdU in the distal colon from AhR WT (GT) and KO (HGT) mice. Arrowhead and star symbols denote proliferating stem cells (GFP+EdU+) and static stem cells (GFP+EdU-), respectively, during a 2 h EdU pulse, n=3 per treatment.",
    "molecules": "EdU"
  },
  {
    "caption": "(C) Total number of EdU+ cells per crypt in the distal colon of AhR WT and KO (HGT) mice, n=3 mice per treatment. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.",
    "molecules": "EdU"
  },
  {
    "caption": "(D&E) Crypt cell proliferative zone analysis in AhR WT and KO (HGT) mice, presented as the distance from the crypt base to the highest EdU+ cell divided by total crypt length. Red lines between crosses show the measurement of distance. Each symbol represents one crypt, n=3 mice per treatment, scale bar 20 µm. Grey line represents mean.",
    "molecules": "EdU"
  },
  {
    "caption": "(F& AhR KO promoted cell proliferation 5 d post DSS treatment. AhR WT (AhRf/f [H], n=4) and AhR KO (AhRf/f X Villin-Cre [HV], n=4) mice were used.",
    "molecules": "DSS"
  },
  {
    "caption": "G) AhR KO promoted cell proliferation 5 d post DSS treatment. Data represent mean ± SEM, quantified as the ratio of the EdU+ area relative to the DAPI+ area using KeyenceTM software.",
    "molecules": "DSS, EdU, DAPI"
  },
  {
    "caption": "(H) Increased cell proliferation (EdU+ cells) observed in AhR KO vs WT organoids derived from HGC and GC mice, respectively (n=6 from 3 mice per group).",
    "molecules": "EdU"
  },
  {
    "caption": "(J). FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation. Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton (50 mg/kg) for 2 consecutive days, n=80~100 crypts from 4 mice per group. Scale bar, 50 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. Bars with different letters are significantly different (p<0.05).",
    "molecules": "Thiostrepton"
  },
  {
    "caption": "(B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm.",
    "molecules": "DSS, AOM"
  },
  {
    "caption": "(C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group.",
    "molecules": "TCDD, DMSO"
  },
  {
    "caption": "(J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects.",
    "molecules": "TCDD"
  },
  {
    "caption": "(O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05.",
    "molecules": "TCDD, CH223191, DMSO"
  },
  {
    "caption": "(Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.",
    "molecules": "DMSO, FDI-6"
  },
  {
    "caption": "(a) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT-α in HCT116 cells.",
    "molecules": "PFT-α"
  },
  {
    "caption": "(b) Autophagy induced by PFT-α, p53 knockdown, or p53 knockout in HCT116 cells. The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments (mean ± s.e.m.; *P 0.05). Culture in nutrient-free (NF) conditions was used as a positive control.",
    "molecules": "nutrient, PFT-α"
  },
  {
    "caption": "(d) GFP-LC3 puncta induced by PFT-α, p53 knockdown or knockout. WT and p53−/− HCT116 cells were transfected with control or p53-specific siRNAs, re-transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT-α. The percentage of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (mean ± s.d., n = 3; *P 0.05).",
    "molecules": "PFT-α"
  },
  {
    "caption": "(a) PFT-α or p53 siRNA-induced GFP-LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5, Atg10, Atg12, Beclin 1 or hVps34. Culture in nutrient-free (NF) conditions was used as a control of autophagy induction.",
    "molecules": "nutrient, PFT-α"
  },
  {
    "caption": "(b) PFT-α or p53 siRNA-mediated GFP-LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1.",
    "molecules": "PFT-α"
  },
  {
    "caption": "(c) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells. MCF7 cells stably transfected with a tetracycline-repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression, transfected with GFP-LC3 and subjected to p53 knockdown before quantification of GFP-LC3 puncta. Data in a-c are mean ± s.d. of 3 independent experiments.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-α and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).",
    "molecules": "BafA1, bafilomycin A1, PFT-α"
  },
  {
    "caption": "(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-alpha and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).",
    "molecules": "BafA1, bafilomycin A1, PFT-alpha, PFT-α"
  },
  {
    "caption": "(g) Immunoblot detection of LC3-I/II in cells treated with PFT-α and/or leupeptin in WT HCT116 cells (n = 3).",
    "molecules": "leupeptin, PFT-α"
  },
  {
    "caption": "(d) Quantification of GFP-LC3vac cells (means ± s.d., n = 3 separate experiments) in WT HCT116 and p53−/− HCT116 treated with PFT-α and/or rapamycin.",
    "molecules": "PFT-α, rapamycin"
  },
  {
    "caption": "(a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose. Measurements were performed on luciferase-expressing HCT116, after perfusion of the cells with medium first containing then missing glucose.",
    "molecules": "ATP, glucose"
  },
  {
    "caption": "(b) Effect of methylpyruvate (MetPyr) on ATP levels in WT p53−/− HCT116 cells depleted of glucose. ATP levels were measured as in a, in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr.",
    "molecules": "ATP, glucose, methylpyruvate, MetPyr"
  },
  {
    "caption": "(c) Effect of autophagy on the ATP levels of WT and p53−/− HCT116 cells depleted of glucose. Cells were transfected with siRNAs specific for Beclin 1 and AMPKα, or they were treated with rapamycin for 6 h, followed by measurement of ATP levels as in a, before or after glucose withdrawal (means ± s.d. of triplicates, 3 independent experiments).",
    "molecules": "ATP, glucose, rapamycin"
  },
  {
    "caption": "(d) Metabolic stress-induced cell death is attenuated in the absence of p53. HCT116 cells were transfected with control, Atg5-, Atg10-, or AMPKα-specific siRNAs and were subjected 48 h later to metabolic stress (cultured for 48 h in nutrient-free, hypoxic conditions) and stained with DiOC6(3) and PI. The black and white portions of the columns refer to the DiOC6(3)low PI− (dying) and DiOC6(3)low PI+(dead) population, respectively.",
    "molecules": "nutrient"
  },
  {
    "caption": "(f) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion. MCF7 cells that carry a tetracycline-repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it, transfected with a control siRNA or a p53-specific siRNA, subjected to metabolic stress, and finally stained with DiOC6(3)/PI.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(a, b) PFT-α-mediated stimulation of autophagy in cytoplasts. GFP-LC3-transfected HeLa cells were enucleated by density-gradient centrifugation after cytochalasin B treatment and cultured on polylysine-coated coverslips. Mixtures of cells and cytoplasts (arrows, identified by the lack of Hoechst 33342 staining) were then exposed to three different autophagy inducers: PFT-α, nutrient starvation and rapamycin. Results are means ± s.d. n = 3 experiments).",
    "molecules": "cytochalasin B, nutrient, PFT-α, rapamycin"
  },
  {
    "caption": "p53 inhibition induces autophagy by ER stress (a-c) WT HCT116 cells were transiently transfected with GFP-LC3, treated with PFT-α for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP-LC3+ puncta with the ER marker ERp57 (a) or a mitochondrial marker Complex III (b). Note that cells with GFP-LC3+ puncta often contain scarce amounts of ERp57 (white arrows) after prolonged incubation with PFT-α. The percentage of colocalization (means ± s.d., n = 3 experiments with 50 images per experiment) was determined by confocal microscopy (c).",
    "molecules": "PFT-α"
  },
  {
    "caption": "p53 inhibition induces autophagy by ER stress (a-c) WT HCT116 cells were transiently transfected with GFP-LC3, treated with PFT-α for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP-LC3+ puncta with the ER marker ERp57 (a) or a mitochondrial marker Complex III (b). Note that cells with GFP-LC3+ puncta often contain scarce amounts of ERp57 (white arrows) after prolonged incubation with PFT-α. The percentage of colocalization (means ± s.d., n = 3 experiments with 50 images per experiment) was determined by confocal microscopy (c).",
    "molecules": "PFT-α"
  },
  {
    "caption": "(d) eIF2α phosphorylation in WT or p53−/− HCT116 cells treated with PFT-α (n = 5).",
    "molecules": "PFT-α"
  },
  {
    "caption": "(e, f) WT HCT116 cells (e, f) or p53−/− HCT116 cells (f) were transfected with GFP-LC3, plus a control siRNA or the indicated combination of IRE1α- and/or p53-specific siRNA, and treated with PFT-α, thapsigargin, tunicamycin or brefeldin A.",
    "molecules": "brefeldin A, PFT-α, thapsigargin, tunicamycin"
  },
  {
    "caption": "(g) GFP-LC3-transfected WT or ire1α−/− MEF were treated with PFT-α. Columns in e-g show means ± s.d. of 3 independent experiments (*P 0.05).",
    "molecules": "PFT-α"
  },
  {
    "caption": "Kinetics of p53 degradation (a) and induction of GFP-LC3 puncta (b) in WT HCT116 cells cultured in complete medium in the presence of rapamycin (Rapa), tunicamycin (Tn), lithium (Li) or in nutrient-free medium (NF).",
    "molecules": "lithium, nutrient, rapamycin, tunicamycin"
  },
  {
    "caption": "(c) WT HCT116 cells transfected with control or HDM2-targeted siRNAs were cultured in the presence or absence of MG132 for 3 h, followed by the treatment with autophagy inducers for 6 h (Tp, thapsigargin; other abbreviations as in b). The abundance of p53 and HDM2 was determined by immunoblotting analysis and the percentage of cells showing accumulation of GFP-LC3 in vacuoles (GFP-LC3vac) was determined.",
    "molecules": "MG132, thapsigargin"
  },
  {
    "caption": "(d) Effects of HDM2 inhibitors on p53 protein levels and autophagy. WT HCT116 were left untreated or treated with Nutlin-3, RITA and/or the indicated autophagy inducers, followed by the p53 immunoblot or immunofluorescence microscopy to visualize GFP-LC3 redistribution.",
    "molecules": "Nutlin-3, RITA"
  },
  {
    "caption": "(e) Effect of MG132 and HDM2 siRNA on autophagy of WT and p53−/− HCT116 cells.",
    "molecules": "MG132"
  },
  {
    "caption": "(f) Failure of HDM2 inhibitors to prevent autophagy induced by the absence of p53. Nutlin-3 and RITA were added to p53−/− HCT116 cells, followed by quantification of GFP-LC3 puncta.",
    "molecules": "Nutlin-3, RITA"
  },
  {
    "caption": "(g) Effect of WT and ubiquitination-resistant p53 on autophagy. p53−/− HCT116 cells were transfected with GFP-LC3 alone or in combination with different concentrations of pcDNA3.1, WT p53 or p53 lacking the ubiquitination site at amino acids 13-19 (p53ΔI), then treated with rapamycin, tunicamycin or thapsigargin. Thereafter, p53 expression and GFP-LC3 puncta were quantified.",
    "molecules": "rapamycin, thapsigargin, tunicamycin"
  },
  {
    "caption": "(h) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors. In WT HCT116 cells transfected with siRNA targeting Atg5 or Beclin 1, p53 levels were determined by immunoblot analysis after 6 h of treatment with rapamycin, tunicamycin or thapsigargin.",
    "molecules": "rapamycin, thapsigargin, tunicamycin"
  },
  {
    "caption": "Volcano plot showing differences in metabolite levels between 0 and 6&12 weeks of treatment. Metabolites in histidine, alanine, aspartate and glutamine metabolism, and the TCA cycle pathways are annotated at 10% False Discovery Rate (see Dataset EV1 for the whole list of differentially regulated metabolites. Although some other metabolites showed a more pronounced difference, these did not fit into a recognised pathway). Metabolite labels are colour-coded according to the identified KEGG pathways (dark red = "Alanine, aspartate and glutamate metabolism", dark green = "Histidine metabolism" and dark blue = "Citrate cycle (TCA cycle)", with transition colour-code if the metabolite belongs to more than one KEGG pathways).",
    "molecules": "alanine, Alanine, Citrate, histidine, aspartate, glutamate, glutamine, Histidine, TCA"
  },
  {
    "caption": "Volcano plot of acyl-carnitine metabolites between 0 and 6&12 weeks of treatment with highlighted differential metabolites at 10% FDR.",
    "molecules": "acyl-carnitine"
  },
  {
    "caption": "A Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "B Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.",
    "molecules": "JC-1, FCCP, galactose, glucose"
  },
  {
    "caption": "D TMRM live staining of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium. Scale bar corresponds to 10 μm.",
    "molecules": "TMRM, galactose, glucose"
  },
  {
    "caption": "A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP. The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1. Coomassie Blue (CB) staining was used as loading control and the radiolabeled protein (TNT) as reference for quantification. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. Western blot for TIMM50 is shown as cell line control. B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together. ",
    "molecules": "CB, Coomassie Blue, FCCP, trypsin"
  },
  {
    "caption": "C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 in the absence or presence of the uncoupler FCCP The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05. ",
    "molecules": "Coomassie Blue, FCCP"
  },
  {
    "caption": "Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Two biological replicates are shown for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Two biological replicates were obtained for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "D Oxygen consumption measurements in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A ROS production as measured by intracellular oxidation of the cell-permeant probe DCF-DA in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Measurements were made in untreated cells and after treatment with 250μM hydrogen peroxide for 30 min. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.",
    "molecules": "DCF-DA, galactose, glucose, hydrogen peroxide, ROS"
  },
  {
    "caption": "B Steady state levels of ROS related proteins SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. C Quantification of the steady state levels of SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01 grouping both controls together.",
    "molecules": "galactose, glucose, ROS"
  },
  {
    "caption": "D Steady state levels of mitophagy related proteins VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. E Quantification of the steady state levels of VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A Cell size in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "B Growth curves of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cell growth was monitored continuously by the Incucyte live cell imager (Essen Bioscience). One of the three independent experiments carried out is presented. Data are shown as mean of three technical replicates ± SD.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "C Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cells in the late stage of apoptosis are boxed in the top right corner.",
    "molecules": "galactose, glucose, propidium iodine"
  },
  {
    "caption": "D Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 or 4 biological replicates in the case of transduced cells; ∗*∗p < 0.001.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "A Western-blot of whole cell extracts for TIMM50 and GAPDH in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in either glucose or galactose containing medium.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "B Mitochondrial membrane potential in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.",
    "molecules": "JC-1, FCCP, galactose, glucose"
  },
  {
    "caption": "C Western blot for representatives of all five respiratory complexes in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. GAPDH is from the same blot as panel A. Quantification based on two biological replicates for RNASEH1 and ISCU mutant fibroblasts Significant changes in protein levels in RNASEH1 and ISCU mutant fibroblasts compared to controls have been boxed in red.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "D Oxygen consumption measurements in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "E Steady state levels of ROS related proteins SOD2 and ACO2 and mitophagy related proteins VDAC1, p62 and LC3 in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. GAPDH is from the same blot as panel A.",
    "molecules": "galactose, glucose, ROS"
  },
  {
    "caption": "F Quantification of the number of late apoptotic cells in control and TIMM50, RNASEH1 and ISCU mutant fibroblasts grown in glucose or galactose. Data are shown as mean ± SD, n = 4 or more biological replicates; ∗*∗p < 0.001.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "C The  qRT-PCR  analysis of Akt2  mRNA in PEMs  stimulated with  lipo-poly(I:C) (1 μg/mL), lipo-poly(A:T) (1 μg/mL),  lipo-ISD  (3 μg/mL) or HSV-1 (MOI, 1) for 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using one-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).",
    "molecules": "ISD, poly(A:T), poly(I:C)"
  },
  {
    "caption": "G-H PEMs  were pretreated with DMSO or AKT2  inhibitor CCT128930  (10 μM) (G) for 2 hours, or knocked down of  Akt2 with siAkt2-1 (20 nM) for 48 hours (H), then the mRNA level of Ifnb1  was measured by  qRT-PCR after lipo-poly(I:C),  lipo-poly(A:T), lipo-ISD, VSV, or HSV-1  stimulation for another 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).",
    "molecules": "ISD, CCT128930, DMSO, poly(A:T), poly(I:C)"
  },
  {
    "caption": "A-B qRT-PCR detection for the mRNA  expression  of Ifnb1 (A), Ifna4, Cxcl10  and Ccl5 (B) in WT (n=3) and Akt2  KO (n=3)  PEMs stimulated with lipo-poly(I:C), VSV,  lipo-poly(A:T), lipo-ISD or HSV-1 for 6 hours. C The  expression  of Ifnb1 mRNA level by  qRT-PCR  in WT (n=3) and Akt2 KO (n=3)  PEMs treated with poly(I:C) (10 μg/mL) for 6 hours (left panel) or LPS  (1 μg/mL) for 2 hours (right panel). Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent experiments Error bars mean ± SEM",
    "molecules": "ISD, LPS, poly(A:T), poly(I:C)"
  },
  {
    "caption": "H  Zebrafish larvae were overexpressed the indicated protein for 48 hours and challenged with VSV for another 6 hours, then the mRNA levels of  ifn1 in zebrafish larvae were measured by qRT-PCR (left panel). Every dot represents three  zebrafish embryos. Horizontal square bracket shows the statistical analysis of the comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=5;  PBS VSV, n=13; AKT2 VSV, n=7; AKT2-T309A/S474A  VSV, n=9. H&E staining (middle panel) and survival rates (Kaplan-Meier curve) (right panel) were collected from  zebrafish larvae after  VSV micro-injection for 18 hours or longer. The arrows indicated the  VSV-infected eye  and  skeletal muscle in  zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test or log-rank (Mantel-Cox) test Data are from three independent biological replicates Error bars mean ± SD).",
    "molecules": "PBS"
  },
  {
    "caption": "G Immunofluorescent  microscopic imaging for (left panel) and statistics analysis (right panel) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 hour post-VSV  infection. IRF3 (red), Nuclei  (Hoechst, green). The white arrows indicate the nuclei  with IRF3 translocation. Mock, n=2; VSV, n=3. Bar, 20 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are representative of two or three independent biological replicates Error bars mean ± SEM).",
    "molecules": "Hoechst"
  },
  {
    "caption": "D  Immunofluorescence microscopy (top panel) of  AKT2-,  Flag-IRF3- or  Flag-IRF3-T207A-overexpressed  MEF cells after  poly(A:T) stimulation for 6 hours. Flag (IRF3, red) and Hoechst (nuclei, blue). Immunoassay  of nuclear-cytoplasm extractions (bottom panel) from  HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by  VSV  infection for 6 hours.  GAPDH  and  SP1  were used as cytoplasmic  and nucleic protein loading control respectively. Bar, 20 μm. Data information: * Data are representative of three independent experiments",
    "molecules": "Hoechst, poly(A:T)"
  },
  {
    "caption": "G qRT-PCR analysis of the  VSV  copies in overexpressed zebrafish embryos with the VSV  challenge at 18 hours later. Every dot represents three zebrafish embryos. Horizontal vertical square bracket shows the statistical analysis of comparison with 'PBS  mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=15; PBS VSV, n=9; IRF3 VSV, n=16; IRF3-T207A VSV, n=11; AKT2 VSV, n=14; AKT2-T309A/S474A VSV, n=9; IRF3 + AKT2 VSV, n=9; IRF3 + AKT2-T309A/S474A VSV, n=13; IRF3-T207A + AKT2 VSV, n=10. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are representative of three independent experiments Error bars mean ± SD).",
    "molecules": "PBS"
  },
  {
    "caption": "I Survival  rates of WT mice that were intraperitoneal injected with CCT128930 (20 mg/kg) twice every other day before VSV infection (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively",
    "molecules": "CCT128930"
  },
  {
    "caption": "immunoblot  analysis of  IRF3,  LaminB1 and GAPDH in the PIC  nuclear  and cytoplasmic fractions (C) from WT and Akt2 KO mice  treated with TMPD  for 2 weeks. GAPDH and LaminB1 were used as cytoplasmic and  nucleic protein loading control respectively. Data information: * Data are representative of three independent experiments",
    "molecules": "TMPD"
  },
  {
    "caption": "K Immunofluorescence of  IRF3 (Red), AKT2  (Green),  DAPI (Blue, nucleus) in CD14+ cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus  with Image J software. Cells (n=39) were collected 3 patients. Bar, 2 μm. Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using correlation analyses Data are pooled from different individuals or representative of three independent experiments",
    "molecules": "DAPI"
  },
  {
    "caption": "Masson trichrome staining   and immunostaining  of α-SMA  and Collagen 1 in endometrium  of controls and patients  with IUA  (n=5 samples for each group). Scale bars: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.",
    "molecules": "Masson trichrome"
  },
  {
    "caption": "Western blot  analysis of α-SMA  and Collagen 1  in hESCs  treated with Bemcentinib  (1μM) for 1 hour, followed by GAS6  (50ng/mL) for 72h (n=3 technical replicates for each group). Western blot  analysis of α-SMA and Collagen 1 in hESCs  treated with the supernatants of CD301- and CD301+ macrophages  and GAS6  neutralizing antibody (2.5μg/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.",
    "molecules": "Bemcentinib"
  },
  {
    "caption": "Western blot  analysis of p-p65, p65, Collagen 1 and α-SMA expression in hESCs  treated with or without GAS6  neutralizing antibody (2.5μg/mL) or Bemcentinib  (1μM) for 1 hour, followed by GAS6  protein (50ng/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.",
    "molecules": "Bemcentinib"
  },
  {
    "caption": "qRT-PCR analysis of ACTA2  and COL1A1  expression in hESCs  treated with GAS6  (50ng/mL) or JSH23  (10μM) for 72h (n=3 biological replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.",
    "molecules": "JSH23"
  },
  {
    "caption": "Western blot  analysis of Collagen 1  and  α-SMA expression in hESCs  treated with GAS6  (50ng/mL) or JSH23  (10μM) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.",
    "molecules": "JSH23"
  },
  {
    "caption": "Representative images of Masson trichrome staining  and immunostaining  for  Collagen 1 between endometrium  of Sham and IUA mice  (n=5 mice  for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.",
    "molecules": "Masson trichrome"
  },
  {
    "caption": "Representative images of immunostaining  for AXL, Collagen 1  and p-p65  and Masson trichrome staining in the endometria  of Sham, IUA  and IUA+DT mice  (n=5 mice  for each group). Scale bar: 100μm. Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.",
    "molecules": "Masson trichrome"
  },
  {
    "caption": "qRT-PCR analysis of Acta2  and Col1a1  expression in the uterus  of IUA+PBS  and IUA+BEM mice  (n=5 mice  for each group). Data are presented as mean ± SEM. (B, Two-tailed Student's t-test.",
    "molecules": "BEM, PBS"
  },
  {
    "caption": "Representative images of immunostaining  for AXL, Collagen 1, p-p65   and Masson trichrome staining  in the endometrium  of IUA+PBS  group and IUA+BEM  group (n=5 mice  for each group). Scale bar, 100μm. Data are presented as mean ± SEM. C for AXL  and Collagen 1 Two-tailed Student's t-test. (C for p-p65) Mann-Whitney U test. *, P<0.05; **, P<0.01; ***, P<0.001.",
    "molecules": "Masson trichrome, BEM, PBS"
  },
  {
    "caption": " Immunofluorescence analysis of viral antigens in lungs of SARS-CoV-2-infected hACE2 mice.Co-localization of SARS-CoV-2 S protein and hACE2 receptor in hACE2 mouse lungs, the sections were incubated with anti-SARS-CoV-2 S protein antibody, anti-human ACE2 antibody, and DAPI. The lung sections of ACE2-Mock mice (a-d). The lung sections of ACE2-HB-01 mice (e-h). The white arrows showed the viral S protein (f) and hACE2 (g), respectively, the yellow arrow showed the merge of viral S protein and hACE2 (h). White bar=25 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "  (A-J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A-D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A-D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K-O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M-O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N-O).  ",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.",
    "molecules": "DAPI, rapamycin"
  },
  {
    "caption": "(A) The number of AV in neurites decreased significantly in neurons exposed to the SIV-infected microglia supernatant for 3 or 24 hr, however; pretreatment with rapamycin blocked this effect. *** P<0.001, for n = 6 experiments. Scale bar, 20 µm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Total AV counting results show that a significant drop occurs in neurons exposed to TNF-α or NMDA for 3 or 24 hr. *** P<0.001, ** P<0.01 for n = 6 experiments. All values are mean±SEM. (B and C) Distribution of AV number in soma and neurites respectively.",
    "molecules": "NMDA"
  },
  {
    "caption": "(B) The number of AV in neurites decreased significantly in neurons exposed to both TNF-α or NMDA for 3 or 24 hr. *** P<0.001, for n = 6 experiments.",
    "molecules": "NMDA"
  },
  {
    "caption": "(C) The AV number in soma is increased in neurons after exposure to TNF-α for 3 or 24 hr, however it remained unaffected in neurons after exposure to NMDA. ** P<0.01 for n = 6 experiments.",
    "molecules": "NMDA"
  },
  {
    "caption": "(D) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF-α or NMDA at 25 ng/ml and 35 µM respectively. Scale bar, 20 µm.",
    "molecules": "DAPI, NMDA"
  },
  {
    "caption": "(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments.",
    "molecules": "MK-801, rapamycin, Rapamycin"
  },
  {
    "caption": "(B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.",
    "molecules": "3-MA, Baf"
  },
  {
    "caption": "A Analysis of reactive oxygen species (ROS) accumulation in frozen liver sections from 120-day-old (P120) and 240-day-old (P240) PR-SET7ΔHepA mice and control littermates (WT).",
    "molecules": "reactive oxygen species, ROS"
  },
  {
    "caption": "C Trypsin digestion of CD19. The digestion was performed at 37 °C with intact cells. The mature CD19 forms (m and m') can be digested because they are surface expressed, whereas the intracellular precursor form (p) is protected from the digestion.",
    "molecules": "Trypsin"
  },
  {
    "caption": "E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19. Chol: mutations at the cholesterol-binding site of CD81. CD81 constructs and CD19 (300 ng DNA) were co-transfected into the CD81-knockout 293T cells. Top, Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. In presence of wildtype CD81 (wt), most CD19 is in the properly glycosylated mature form (m), and the overglycosylated m' form is not observed. With the EC1 mutants (A4 in particular), however, m' emerges and m shifts to high MW, indicating an alteration in the CD19 glycosylation. The level of intracellular p form is low with the wildtype CD81 or with the mutants. Bottom, Control experiment showing similar expression level of wild-type CD81 and CD81 mutants. Anti-V5 immunoblotting was conducted for the CD81 constructs with a C-V5 tag, and anti-actin immunoblotting was conducted on the same film.",
    "molecules": "Chol, cholesterol"
  },
  {
    "caption": "Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. B) Magnified SMLM images of GFP-BAX and DRP1 assemblies co-localizing up to 30 nm during apoptosis. Scale bar 100 nm. C) Co-localization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n=4 independent experiments with a total of 720 BAX structure.",
    "molecules": "AF647, CF680"
  },
  {
    "caption": "E,F) Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm.",
    "molecules": "GB, STS"
  },
  {
    "caption": "G) BAX/DRP1 interaction compared to BAX/BAX and BCL‑xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001) compared to BAX/DRP1 after apoptotic induction (STS).",
    "molecules": "GB, STS"
  },
  {
    "caption": "A) Confocal microscopy time-series images of the increase in cytosolic Smac-GFP (magenta) in relation to the detection of RA-BAX/GB-DRP1 complexes (green) during apoptosis induction in MEF DRP1 KO cells. Scale bar 10 μm. Images are representative for n=3 independent experiments.",
    "molecules": "GB"
  },
  {
    "caption": "C) Quantification of BAX/DRP1 interaction (% cells with RA-BAX/GB-DRP1 foci normalized to the mito-BFP positive cells, n=100 cells per experiment) during inhibition of effector caspases (Q-VD-OPh), the mitochondrial permeability transition pore (CsA) versus control (DMSO), with or without apoptosis induction (+/- STS). Box plots represent the interquartile (box), median (line) and SD (whiskers). Significance was determined from n=3 independent experiments (with n=100 cells each) by paired two-tailed Student´s t-test (n.s. indicating p > 0.05) compared to DMSO control after apoptotic induction (STS).",
    "molecules": "GB, CsA, DMSO, Q-VD-OPh, STS"
  },
  {
    "caption": "C) Quantification of %CC between DRP1-AF488 and BAX-AF633 in solution (grey), in the membrane (violet), and in the membrane (beige) in presence of excess unlabeled cBID. Box plots represent the interquartile (outer box), mean (inner box), median (line) and range (whiskers). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01) from n=9 measurements in solution, n=46 individually measured GUVs in the membrane or n=17 GUVs in presence of cBID.",
    "molecules": "AF633, AF488"
  },
  {
    "caption": "BAX peptide array to define interaction-site with DRP1. D) Quantification of the relative fluorescence intensity of DRP1-AF488 at the rim of the GUVs for each BAX peptide of n=3 individual experiments (with n=100 vesicles each). Black lines indicate the mean value. Interacting BAX peptides are highlighted in beige.",
    "molecules": "AF488"
  },
  {
    "caption": "B) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX variants and GB-DRP1, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.",
    "molecules": "GB"
  },
  {
    "caption": "C-E) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to BAX wild type (WT).",
    "molecules": "GB"
  },
  {
    "caption": "G) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX and GB-DRP1 variants, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.",
    "molecules": "GB"
  },
  {
    "caption": "H) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Significance was tested using paired two-tailed Student´s t-test (n.s. p > 0.05) compared to DRP1 WT.",
    "molecules": "GB"
  },
  {
    "caption": "C) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation. Confocal microscopy images of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan) were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5-60 min). From top to down, the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal, respectively. Scale bar 20 µm.",
    "molecules": "MitoTracker, MitoTracker Deep Red FM"
  },
  {
    "caption": "E) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization. U2OS BAX/BAK DKO cells were transfected as described in C) and stained with the mitochondrial membrane potential-sensitive dye MitoSpy NIR (cyan). Confocal microscopy images were acquired before (0 min) and after induced dimerization of BAX and DRP1 (10 min). Scale Bar 20 µm. F) Quantification of mitochondrial depolarization normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (* p < 0.05) compared to non-induced control. G",
    "molecules": "MitoSpy NIR"
  },
  {
    "caption": "G-K) Induced dimerization of DRP1 with itself (G), BAX with itself (H), BAX with TOM20 (I) in U2OS BAX/BAK DKO cells, or BAX with DRP1 in HCT OctaKO cells (J,K), transected with FKBP- and FRB-mCherry DRP1 (G, red), FKBP- and FRB-EGFP-BAX (H, green), TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, I), respectively. Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 µm (G-I) and 10 µm (J,K ). All images are representative of n=3 independent experiments.",
    "molecules": "MitoSpy NIR, MitoTracker Deep Red FM"
  },
  {
    "caption": "A Neurosphere assays were performed using mouse E14.5 primary neural progenitors treated with CRH with or without antalarmin (Ant) (n = 3 biological replicates).",
    "molecules": "Ant, antalarmin, CRH"
  },
  {
    "caption": "B Effects of CRH treatment on the stemness of neural progenitor cells at 2 days post-treatment were analyzed by Western blotting using an anti-SOX2 antibody (n = 3 biological replicates).",
    "molecules": "CRH"
  },
  {
    "caption": "G E14.5 Embryonic brains that were electroporated with CRHR1-expression vectors at E13.5 were pulse labeled with EdU and harvested at E15.5. Samples were then visualized for GFP (green), EdU (blue) and Ki67 (red) (n = 4 biological replicates).",
    "molecules": "EdU"
  },
  {
    "caption": "C E14.5 neural progenitor cells were transduced with CRHR1-containing lentiviral vectors. At 2 days post-transduction, cells were treated with 10 µM H89 (PKA inhibitor) or 1 µM KN93 (CaM kinase inhibitor) for 8 h and harvested for Western blot analysis for CREB (top panel). Band intensities were quantified (bottom panel) (n = 3 biological replicates).",
    "molecules": "KN93, H89"
  },
  {
    "caption": "Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (C) qPCR",
    "molecules": "666-15"
  },
  {
    "caption": "Dose-dependent inhibition of endogenous REST expression by a CREB inhibitor, 666-15. E14.5 neural progenitor cells were treated with the indicated concentrations of 666-15 for 48 h and subjected to (D) Western blot analyses",
    "molecules": "666-15"
  },
  {
    "caption": "Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (E) qPCR",
    "molecules": "666-15"
  },
  {
    "caption": "Inhibition of CRHR1-induced REST expression by 666-15. E14.5 neural progenitor cells transduced with a lentiviral vector expressing CRHR1 were treated with 20 nM of 666-15, and after 48 h, cells were lysed and subjected to (F) Western blot analyses (n = 4 biological replicates).",
    "molecules": "666-15"
  },
  {
    "caption": "A. Ncl and Pus7 mRNA levels (normalized to TBP) at different time points after LPS stimulation in c-mycwt/wt and c-myc∆/∆ cells. Data are presented as mean ± SD; n = 3.",
    "molecules": "LPS"
  },
  {
    "caption": "B. To quantify RNA synthesis, we exposed our cultures to a brief pulse of ethyl-uridine (EU) and measured its incorporation into RNA by light microscopy. The scatter plots show the nuclear Area as detected in the 2D plane (x axis, in pixels) versus the EU signal (y axis, as arbitrary units, AU) for each single cell identified by DAPI staining in c-mycwt/wt and c-myc∆/∆ populations at the indicated time points after LPS treatment. One representative experiment out of 5 is shown.",
    "molecules": "ethyl-uridine, EU, DAPI, LPS, RNA"
  },
  {
    "caption": "C. Variation of each expressed mRNA at 4h of LPS treatment (log2FC, relative to time 0) for c-mycwt/wt (x-axis) and c-myc∆/∆ cells (y-axis), as measured by RNA-seq. Regulatory groups 1-6 and Grey Zone (GZ) are as defined in the text.",
    "molecules": "LPS"
  },
  {
    "caption": "D. Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation.",
    "molecules": "LPS"
  },
  {
    "caption": "A. Overlap between Myc ChIP-seq peaks. For each time-point indicated at the bottom, the percentage of peaks overlapping over ≥1 bp with any other time-point (Reference sample) is reported. Peaks showed consistent distributions along the time-course, almost all those called in untreated cells being included in LPS-stimulated samples",
    "molecules": "LPS"
  },
  {
    "caption": "B, C. Distribution of Myc at promoters (B) and distal sited (C) in LPS-treated B-cells (0, 2, 4 8h) and in B-cells in vivo, in either control c-mycwt/wt (C), pre-tumoral Eµ-myc mice (P) or tumors (T) 4[]. Each row represents a promoter-associated Myc peak within a 6 kb genomic interval (centered on the midpoint of the peak). The heatmap includes every annotated promoter in chromosome 1 (as representative of the whole genome) that was called as Myc-associated by ChIP-seq in at least one sample. For the same intervals, we also show the distribution of H3K4me1, H3K4me3, and H3K27ac in the in vivo control sample, and gene annotations (exons in red, introns in pink; + sense, - antisense strand).",
    "molecules": "H3K27ac, H3K4me3, LPS"
  },
  {
    "caption": "D, E. Numbers of Myc peaks at promoters (-2kb, +1kb) (D) and distal sites (E) at different time points after LPS stimulation. The shadings mark the subsets of peaks associated with H3K4me3 (D) or H3K4me1 (E), with or without H3K27ac.",
    "molecules": "H3K27ac, H3K4me1, LPS"
  },
  {
    "caption": "F. Percentage of active promoters (positive for H3K4me3 and H3K27ac) or distal sites (H3K4me1 and H3K27ac) that overlap with at least a Myc peak at the different time points after LPS stimulation (0, 2, 4, 8h) or in vivo (C, P, T1, T2, T3).",
    "molecules": "H3K27ac, H3K4me1, LPS"
  },
  {
    "caption": "G. Quantitative heatmap showing Myc signal intensities (read counts normalized by library size) at each bound region in chromosome 1, ranked based on the signal in the control sample (0h LPS).",
    "molecules": "LPS"
  },
  {
    "caption": "I. Myc enrichment by ChIP-seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs. In the boxplot, the central tendency marks the median, the box ranges mark the upper and lower quartiles, and the whiskers extend to the upper and lower extreme datapoints which are no more than 1.5 times the interquartile range from the box. The Myc ChIP-seq time-course was performed on a unique pool of cells, obtained from > 10 mice.",
    "molecules": "LPS"
  },
  {
    "caption": "A. Scatter plots correlating the variations in Myc share at promoters (averaged between the 2, 4 and 8h time points) and in RNA synthesis (at 8h), relative to untreated cells, for Myc-dependent and independent genes. The Spearman correlation between the two parameters is reported.",
    "molecules": "RNA"
  },
  {
    "caption": "C. Fold-change in Myc levels at each bound promoter (at 4h LPS, relative to time zero) as a function of the initial binding intensity at time zero (expressed as log2 of the coverage in a 200 bp window around the summit of the peak). Peaks containing a canonical E-box are highlighted in black.",
    "molecules": "LPS"
  },
  {
    "caption": "B, C. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for Myc-dependent (B) and Myc-independent (C) induced and repressed genes. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported. The RNAPII ChIP-seq time-course was performed on two biological replicates (each on pools of cells from > 10 mice), and reads were merged after sequencing.",
    "molecules": "LPS"
  },
  {
    "caption": "D, E. Heatmaps showing the variations in RNAPII kinetic rates (Log2FC) on Myc-dependent (D) and Myc-independent (E) genes during the LPS time-course. The grey scale represents the starting level for each parameter in unstimulated cells. The first column on the left reports the changes in Myc share for the same genes. Genes were grouped using hierarchical clustering.",
    "molecules": "LPS"
  },
  {
    "caption": "F. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for genes of clusters 1 and 9. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.",
    "molecules": "LPS"
  },
  {
    "caption": "G. Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.",
    "molecules": "LPS"
  },
  {
    "caption": "C Pyramidal neurons of NB-3+/+ and NB-3−/− mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml). The neurons were co-stained for Ctip2, NB-3, and Rhodamine phalloidin. (C1-C4) High-magnification images of the growth cones (white arrowheads) of the neurons in (C), respectively.D Quantification of neurite length in (C), and the percentage of collapsed growth cones in (C). n.s., not significant, *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group.",
    "molecules": "Rhodamine"
  },
  {
    "caption": "K The effect of FLT3 inhibitor quizartinib (Qui) on the dorsalization and axis-duplication phenotype-induced by FLT3/ITD mRNA injection in zebrafish. Data information: the experiments were performed in triplicates and the data are presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). NS, not significant.",
    "molecules": "Qui, quizartinib"
  },
  {
    "caption": "A-C WISH of fst in FLT3/WT- (A), FLT3/ITD- plasmid DNA injected zebrafish embryos without (B) or with (C) quizartinib treatment (2.5 μM) from 6 to 36 hpf. fst expression was expanded by FLT3/ITD DNA in 86% of embryos (B, arrow, 32/37) which could be effectively blocked by treating with FLT3 inhibitor quizartinib in 83% of embryos (C, 29/35). Data information: Scale bar = 500 μm.",
    "molecules": "quizartinib"
  },
  {
    "caption": "Generation and characterization of FLT3/ITD-transgenic zebrafish. GFP expression was detected by florescent microscopy (F-H) and in blood circulation and thymus by WISH (I and J, blue arrow) in WT sibling and Runx1-FLT3/ITD transgenic zebrafish (F1) embryos at 4 dpf. FLT3/ITD-positive zebrafish (F1) were confirmed by PCR genotyping of GFP and FLT3/ITD using genomic DNA from fin clip of WT siblings and Runx1-FLT3/ITD transgenic zebrafish (F1) at two months old. Fish 4, 5 and 6 showed germline transmission of FLT3/ITD transgene (K). Data information: Scale bar = 500 μm.",
    "molecules": "genomic DNA"
  },
  {
    "caption": "Kidney marrow (KM) was collected from Runx1-FLT3/ITD transgenic zebrafish (F1) at 18 months old. The morphology and hematopoietic composition of KM from WT siblings (n = 6) and Runx1-FLT3/ITD transgenic (n = 6) zebrafish were examined by Giemsa staining (L) and flow cytometry (abbreviation for panel M: M, Myeloid cells; P, Progenitor cells; L, Lymphoid cells; E, Erythroid cells). Data are presented in box plot. **P<0.01 (Student's t-test).",
    "molecules": "Giemsa"
  },
  {
    "caption": "F-H Phosphor-flow analysis of p-CREB in Ba/F3-parental, Ba/F3-FLT3/ITD, and Ba/F3-FLT3/ITD cells-treated with FLT3 inhibitor Quizartinib (Qui in short). Isotype antibody were used as control to calculate the mean fluorescence intensity (MFI) ratio (F-G). The transcription and expression of Fst was detected by RT-qPCR after Quizartinib treatment (10 nM) in Ba/F3-FLT3/ITD cells for one day (H). Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "Qui, Quizartinib"
  },
  {
    "caption": "I-K The expression of FST and phosphorylation of CREB were detected by Western Blotting (I and K) and phospho-flow analysis (J) in MOLM-13 (I) and Ba/F3-FLT3/ITD (K) cells treated with Quizartinib and BRD7389 for one day, respectively. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "BRD7389, Quizartinib"
  },
  {
    "caption": "M The phosphorylation of CREB and FST expression were detected by Western Blotting in Ba/F3-FLT3/ITD cells treated with CREB inhibitor 666-15 for one day. ^: non-specific staining of p-ATF1 protein due to the conserved motif.",
    "molecules": "666-15"
  },
  {
    "caption": "O The growth of Ba/F3-parental (with IL-3), Ba/F3-FLT3/ITD (without IL-3) and Ba/F3-FLT3/ITD (with IL-3) cells was measured after three days treatment of CREB inhibitor 666-15 in vitro. Data information: the experiments were performed in triplicates, and the data were presented as mean ± SEM. **P<0.01, ***P<0.001 (Student's t-test).",
    "molecules": "666-15"
  },
  {
    "caption": "P The rescue effect of CREB inhibitor 666-15 on FLT3/ITD-induced dorsalization and axis duplication in zebrafish embryos at 1 dpf.",
    "molecules": "666-15"
  },
  {
    "caption": "F The knockdown efficiency of different FST-specific antisense oligoes (ASOs) in MOLM-13 cells was detected by RT-qPCR after three days treatment in vitro. The knockdown and RT-qPCR experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).",
    "molecules": "antisense oligoes, ASOs"
  },
  {
    "caption": "G MOLM-13 cell growth was measured after 3 days treatment of FST-ASO in vitro. The ASO treatment experiments were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).",
    "molecules": "FST-ASO, ASO"
  },
  {
    "caption": "H Intraperitoneal injection of FST-ASO (10mg/kg weekly, 6 mice) significantly prolonged the survival of MOLM-13-engrafted NSG mice. The random sequence was used for negative control (Neg-ASO, 6 mice). Data information: survival curves were analyzed by Log-rank test. *P<0.05.",
    "molecules": "FST-ASO, ASO"
  },
  {
    "caption": "Serum FST decreased in CR and increased after relapse in 4 AML patients receiving Quizartinib monotherapy. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).",
    "molecules": "Quizartinib"
  },
  {
    "caption": "N Serum FST continued to rise during disease progression from a patient who did not respond to Quizartinib. Patients in N were recruited in the QUANTUM-R and patient accrual has been completed. data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test).  ",
    "molecules": "Quizartinib"
  },
  {
    "caption": "(d) Cell lysate of Atg7+/+, Atg7+/− and Atg7−/− MEFs was subjected to immunoblot analysis using antibodies specific for LC3 and p62 after rapamycin (Rap) or control solvent (dimethyl sulfoxide, DMSO) treatment for 3 h. Fold changes of the immunoblot band intensities are shown (middle and right). *P0.05, **P0.01, ***P0.001; Student's t-test, n=3.",
    "molecules": "dimethyl sulfoxide, DMSO, Rap, rapamycin"
  },
  {
    "caption": "(f) Tissue lysates were prepared from the liver of fasted Atg7+/+ and Atg7+/− mice 4 h after leupeptin administration, and immunoblotting was done. Fold changes of the immunoblot band intensities are shown (right). *P0.05; Student's t-test, n=3.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(a) Non-fasting blood glucose level was monitored weekly in Atg7+/+, Atg7+/−, Atg7+/+-ob/ob and Atg7+/−-ob/ob mice. ***P0.001; two-way ANOVA.",
    "molecules": "glucose"
  },
  {
    "caption": "(b) IPGTT was done by glucose injection at 16 weeks of age as described in the Methods (left), and AUC calculated (right). *P0.05, **P0.01, ***P0.001; Student's t-test (left) and one-way ANOVA (right).",
    "molecules": "glucose"
  },
  {
    "caption": "(d) ITT was performed by insulin injection at 16 weeks of age as described in the Methods. *P0.05, **P0.01, ***P0.001; Student's t-test.",
    "molecules": "insulin"
  },
  {
    "caption": "(e) Tissue lysates from Atg7+/+-ob/w, Atg7+/−-ob/w, Atg7+/+-ob/ob and Atg7+/−-ob/ob mice were prepared before and 7 min after injection of 5 U kg−1 regular insulin (Ins) into the tail vein, and subjected to immunoblot analysis using antibodies specific for phospho-Akt S473 and total Akt. Numbers below immunoblot bands indicate the fold changes normalized to control bands.",
    "molecules": "insulin"
  },
  {
    "caption": "(b) Liver sections from each type of mouse were subjected to immunohistochemistry using anti-nitrotyrosine antibody. Scale bars, 100 μm.",
    "molecules": "nitrotyrosine"
  },
  {
    "caption": "(c) Tissue lysates were prepared from 18-week-old mice of each genotype, and subjected to immunoblot analysis using a kit detecting carbonylated proteins.",
    "molecules": "proteins"
  },
  {
    "caption": "(d) Tissue lysates were prepared from the liver of fed ob/w or ob/ob mice 4 h after leupeptin administration, and immunoblotting was performed. Numbers below immunoblot bands indicate the fold changes normalized to control bands.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(e) After labelling SK-Hep1 cells with C14-leucine, cells were treated with FFAs (600 μM PA or 1,200 μM OA). Percent release of radioactivity was measured for 3-6 h as described in the Methods. The difference between the percent proteolysis without E64d/pepstatin A/NH4Cl and that with E64d/pepstatin A/NH4Cl was regarded as lysosomal proteolysis. *P0.05, **P0.01; one-way ANOVA, n=3.",
    "molecules": "C14-leucine, NH4Cl, E64d, FFAs, PA, OA, pepstatin A"
  },
  {
    "caption": "(f) Atg7+/+ and Atg7+/− primary MEFs were loaded with a mixture of the indicated concentrations of PA and OA for 48 h, and TG content was estimated by ORO staining. **P0.01, ***P0.001; two-way ANOVA, n=4.",
    "molecules": "PA, OA, TG"
  },
  {
    "caption": "(e) Primary peritoneal macrophages were isolated, and treated with PA of the indicated concentrations in the presence or absence of LPS. ELISA was conducted to determine IL-1β concentrations in the culture supernatant. (BSA, bovine serum albumin) ***P0.001; two-way ANOVA, n=4.",
    "molecules": "PA, LPS"
  },
  {
    "caption": "(f) After incubation of primary peritoneal macrophages with PA of the indicated concentrations with or without LPS for 16 h, NAD+/NADH ratio was determined using a commercial kit. *P0.05, **P0.01, ***P0.001; two-way ANOVA, n=5.",
    "molecules": "NAD+, PA, LPS, NADH"
  },
  {
    "caption": "(g) After the same treatment of peritoneal macrophages as in f, cells were incubated with MitoSOX to measure mitochondrial ROS by fluorescence-activated cell sorting (FACS) analysis.",
    "molecules": "ROS"
  },
  {
    "caption": "(a) Atg7+/− and control Atg7+/+ mice were fed HFD or normal chow diet (NCD), and non-fasting blood glucose levels were monitored. *P0.05, **P0.01; Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "(b) Fasting bloodglucose levels were determined after 18 weeks of HFD. *P0.05; one-way ANOVA.",
    "molecules": "glucose"
  },
  {
    "caption": "(a) One hour after intraperitoneal injection of 30 mg kg−1 leupeptin to C57BL/6 mice, 25 mg kg−1 of imatinib (Ima) was injected intraperitoneally. Three hours later, tissue lysate was prepared from the liver and subjected to immunoblot analysis.",
    "molecules": "imatinib, leupeptin"
  },
  {
    "caption": "(b) Imatinib (25 mg kg−1), trehalose (2 g kg−1) or PBS was injected intraperitoneally into 12-week-old diabetic Atg7+/−-ob/ob mice 3 times a week, and the blood glucose level was monitored. ***P0.001, ###P0.001; two-way ANOVA.",
    "molecules": "glucose, Imatinib, trehalose"
  },
  {
    "caption": "(c) IPGTT were performed after treatment of Atg7+/−-ob/ob mice with imatinib or trehalose for 8 weeks. #P0.05; ##P or **P0.01; ###P or ***P0.001; Student's t-test.",
    "molecules": "imatinib, trehalose"
  },
  {
    "caption": "(d) ITT were performed after treatment of Atg7+/−-ob/ob mice with imatinib or trehalose for 8 weeks. #P0.05; ##P or **P0.01; ###P or ***P0.001; Student's t-test.",
    "molecules": "imatinib, trehalose"
  },
  {
    "caption": "(e) Regular insulin (Ins) was injected into the tail vein of Atg7+/−-ob/ob mice that were treated with imatinib or PBS for 8 weeks. Seven minutes later, tissue lysates were prepared and subjected to immunoblotting.",
    "molecules": "imatinib, insulin"
  },
  {
    "caption": "(f) Tissue lysates prepared from the same mice without insulin injection were subjected to immunoblotting. Numbers below immunoblot bands indicate the fold changes normalized to control bands. ('*' indicates comparison between imatinib and control: '#' indicates comparison between trehalose and control).",
    "molecules": "imatinib, insulin, trehalose"
  },
  {
    "caption": "A. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "A. Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "D. Confocal images of mitochondrial morphology in Drp1-/- 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented and tubular cluster phenotypes are indicated.",
    "molecules": "MitoTracker"
  },
  {
    "caption": "A. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "B. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with either Mfn1-Myc, Mfn2-Myc or OPA1-Myc, stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).",
    "molecules": "MitoTracker"
  },
  {
    "caption": "D. hFis1 interacts with Mfn1, Mfn2 and OPA1 as well as Drp1, but not with Dyn2 at endogenous levels following chemical crosslinking. Wild-type (WT) and Drp1-/- 293T cells were in vivo crosslinked with 1% formaldehyde (FA) and cell lysates were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.",
    "molecules": "FA, formaldehyde"
  },
  {
    "caption": "G, H. Interaction of hFis1 with Mfn1/2 and with OPA1 are independent events. WT 293T cells were treated with control, OPA1 (G) or Mfn1 plus Mfn2 (H) siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.",
    "molecules": "FA"
  },
  {
    "caption": "I. Interactions between Mfn1/2 and OPA1 occur independent of hFis1. WT 293T cells were treated with control or hFis1 siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG (negative control) or mouse anti-OPA1 antibody as indicated, followed by immunoblotting with indicated antibodies.",
    "molecules": "FA"
  },
  {
    "caption": "J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies.",
    "molecules": "FA"
  },
  {
    "caption": "B. Interaction of different hFis1 mutants with Mfns and OPA1. hFis1-/- 293T cells were transfected with the full-length and the truncated hFis1 mutants as indicated, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with anti-Myc or anti-GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies.",
    "molecules": "FA"
  },
  {
    "caption": "A, B. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or with Myc-hFis1 as indicated and then treated with PEG for cell fusion. Mitochondrial fusion is indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (B).",
    "molecules": "PEG"
  },
  {
    "caption": "E, F. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or hFis1-siRNA as indicated and then co-cultured and fused using PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient in three independent experiments and data summarized in (F).",
    "molecules": "PEG"
  },
  {
    "caption": "A, B. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or Myc-hFis1 as indicated and then stimulated with PEG treatment for cell fusion, followed by immunostaining with anti-Myc antibody (green). Mitochondrial fusion was indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1 transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (B).",
    "molecules": "PEG"
  },
  {
    "caption": "E, F. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or with hFis1-siRNA as indicated and then co-cultured and fused by PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (F).",
    "molecules": "PEG"
  },
  {
    "caption": "A, B. GTP hydrolysis activities of Mfn1, Mfn2, Mfn2K109A and OPA1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined by measuring the concentration of free phosphate (Pi) released from GTP. Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti-Myc agarose beads. GTPase activities of immunopurified Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST-hFis1 or GST protein (A). The input levels of immunopurified Mfn1-Myc, Mfn2-Myc and OPA1-Myc (left panel) as well as recombinant GST-hFis1 (~42 kDa) and GST (~26 kDa) (right panel) were assessed by immunoblotting with indicated antibodies (B).",
    "molecules": "GTP, phosphate"
  },
  {
    "caption": "C, D. GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1-/- 293T cells and immunopurified by protein G beads pre-incubated with Drp1 antibody. GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein (C). The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti-Drp1 antibody (D).",
    "molecules": "GTP"
  },
  {
    "caption": "E, F. GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Endogenous Dyn2 in Drp1-/- 293T cells was immunopurified by protein G beads pre-incubated with anti-Dyn2 antibody. GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST-hFis1 or GST protein (E). The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti-Dyn2 antibody (F).",
    "molecules": "GTP"
  },
  {
    "caption": "(A, B)   Murine cardiac endothelial cells  (MCEC) were treated with 2 mM D-β-hydroxybutyrate  (β-OHB) or 2 mM acetoacetate (AcAc) for 24 hours. Quantification of concentrations of TCA  cycle intermediates compared to control treatment. Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.",
    "molecules": "D-β-hydroxybutyrate, β-OHB, AcAc, acetoacetate, TCA"
  },
  {
    "caption": "(D) Quantifications of [13C]-labeled fractions of  amino acids (asparagine, L-aspartate,  L-glutamate, L-proline), putative lipid species (nervonic acid, 1-palmitoylglycerol, ceramide (d18:1/16:0)) and uridine diphosphate (UDP) species (uridine monophosphate, uridine 5'-diphosphate,  UDP-N-acetylglucosamine) in MCEC  treated with 1 mM 13C4-β-hydroxybutyrate  or 1 mM 13C4-acetoacetate  for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.",
    "molecules": "13C4, nervonic acid, 1-palmitoylglycerol, β-hydroxybutyrate, acetoacetate, asparagine, 12C, 13C, ketone bodies, L-aspartate, L-glutamate, L-proline, ceramide (d18:1/16:0, UDP, uridine 5'-diphosphate, uridine diphosphate, UDP-N-acetylglucosamine, uridine monophosphate"
  },
  {
    "caption": "(A, B)   Murine cardiac endothelial cells  (MCEC) were treated with different concentrations of D-β-hydroxybutyrate (βOHB) or acetoacetate  (AcAc) for 24 hours. Relative BrdU absorbance was quantified compared to control treatment (H2O for βOHB and EtOH for AcAc). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.",
    "molecules": "D-β-hydroxybutyrate, βOHB, BrdU, AcAc, acetoacetate, EtOH, H2O"
  },
  {
    "caption": "(I-M) Angiogenic capacity of MCEC in response to  ketone body treatment was analyzed using a spheroid-based sprouting assay.  Spheroids  were treated with media containing 30 mM D-β-hydroxybutyrate  (βOHB), 30 mM acetoacetate (AcAc), 10 mM acetate, 1 mM octanoate, 1 mM butyrate, 30 mM  L-β-hydroxybutyrate (L- βOHB), 1 mM  niacin or the respective controls (final concentration of reagents is diluted to approximately 10 %) and analyzed after 48 hours. The (I, L) average number of  sprouts per spheroid, the (J, M) accumulated total sprout  length and (K) average  sprout length were quantified. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.",
    "molecules": "D-β-hydroxybutyrate, L- βOHB, L-β-hydroxybutyrate, βOHB, acetate, AcAc, acetoacetate, butyrate, ketone body, niacin, octanoate"
  },
  {
    "caption": "(B, Angiogenic capacity of  Oxct1  knockout cells in response to  ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of  sprouts per spheroid was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.",
    "molecules": "D-β-hydroxybutyrate, βOHB, AcAc, acetoacetate, ketone body"
  },
  {
    "caption": "(C, D) Heatmaps showing expression  levels of genes involved in KEGG pathways (C) fatty acid  degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice  that were fed a ketogenic  diet  or control diet for two weeks.",
    "molecules": "fatty acid, ketone body"
  },
  {
    "caption": "(A, B) Representative images of heart  sections of animals kept on a ketogenic diet  or control diet for two weeks stained against  ERG and Ki67  or  EdU, double positive cells are indicated by arrowheads, and quantification of double positive cells in  heart  sections per high power field (HPF). Scale bar: 50 μm. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.",
    "molecules": "EdU"
  },
  {
    "caption": "(d) The oxygen consumption rate (OCR) in primary hepatocytes isolated from control and Tcfeb-LiKO mice was measured with an XF24 analyser (Seahorse) before and after the addition of palmitic acid (0.2 mM) conjugated with BSA. The vertical red line indicates the time at which palmitate was added to cells. Values are mean ± s.d. for 3 independent experiments *P≤0.05.",
    "molecules": "palmitate, palmitic acid"
  },
  {
    "caption": "(e,f) Total FFA (e) and glycerol (f) in the serum isolated from 6-h-fasted Tcfeb-LiKO and control mice. Values are mean ± s.d. (n = 5 mice per group) *P≤0.05 compared with controls.",
    "molecules": "FFA, glycerol"
  },
  {
    "caption": "(g) Total serum ketones in fed and fasted Tcfeb-LiKO and control mice. Bars are mean ± s.d. for n = 10 mice per group. **P≤0.01 compared with fed control mice.",
    "molecules": "ketones"
  },
  {
    "caption": "(b,c) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group; b) and total lipid content in mice with indicated genotype (mean ± s.d. for n = 10; c). *P≤0.05;***P≤0.001 compared with control.",
    "molecules": "lipid"
  },
  {
    "caption": "(e,f) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group ***P≤0.001; e) and total lipid content in mice with the indicated genotype (mean ± s.d. for n = 5 mice per group; ***P≤0.001; NS, not significant; f). Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.",
    "molecules": "lipid"
  },
  {
    "caption": "(c-f) Total serum insulin, leptin, triglyceride and cholesterol levels in control and HDAd-TFEB mice kept on the high-fat diet for 10 weeks. Value are mean ± s.d. n = 10 add per group. *P≤0.05; ****P≤0.0001 compared with control. (g-i) Glucose and insulin tolerance tests in control and HDAd-TFEB mice challenged with the high-fat diet for 10 weeks.",
    "molecules": "cholesterol, Glucose, insulin, leptin, triglyceride"
  },
  {
    "caption": "(g,h) Glucose (g) and serum insulin (h) levels at the indicated time points after glucose challenge. (i) Glucose levels at the indicated time points after insulin challenge. In g-i, values are mean ± s.d. n = 7 mice per group; *P≤0.05;**P≤0.01 compared with control. Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.",
    "molecules": "Glucose, insulin"
  },
  {
    "caption": "(B) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP, at the indicated concentrations, before immunoblotting.",
    "molecules": "DSP"
  },
  {
    "caption": "(C) GFP, CFP-Atg5, GFP-WIPI1a, and GFP-WIPI2b were transiently expressed in HEK293A cells. GFP-tagged proteins were isolated using GFP-TRAP and incubated with in vitro translated 35S-labeled FLAG-Atg16L1 constructs 1-265, 1-242, 1-230, and 1-207 before washing and analysis by autoradiography. Protein expression was validated by immunoblot (bottom panel).",
    "molecules": "35S"
  },
  {
    "caption": "(F) WIPI2 was immunoprecipitated from lysates from WT or FIP200−/− MEFS after treatment with 0.5 mM DSP. Bound Atg16 and Atg12-5 were detected by immunoblotting.",
    "molecules": "DSP"
  },
  {
    "caption": "(D) GFP-Trap from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2b, GFP-WIPI2b R108E, GFP-WIPI2b R125E, or GFP-WIPI2b R108E R125E was mixed with in vitro translated 35S-labeled FLAG-Atg16L1 and analyzed by autoradiography.",
    "molecules": "35S"
  },
  {
    "caption": "(A) GFP, siRNA-resistant GFP-WIPI2b, or GFP-WIPI2b RERE was expressed in HEK293A cells treated for 72 hr with either RISC-free (RF) or WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(B) Statistical analysis of (A). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Tukey's posttest. ∗p < 0.05.",
    "molecules": "BafA"
  },
  {
    "caption": "(C) GFP, siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's posttest. The SEM for LC3 (n = 2) and p62 (n = 4) are shown. ∗p < 0.05.",
    "molecules": "BafA"
  },
  {
    "caption": "(E) siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were starved in EBSS for 2 hr without or with BafA, fixed, and labeled, and LC3 was visualized by confocal microscopy. Scale bars, 10 μm.(F) Quantification of WIPI2 puncta per cell from (E). SEM from 10 cells per condition; ∗∗∗p < 0.001 using one-way ANOVA.(G) Statistical analysis of LC3 puncta from (E) with GFP control (not shown in E). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Dunn's posttest t test. ∗p < 0.05. See also Table S1.",
    "molecules": "BafA"
  },
  {
    "caption": "(A) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium (F), starvation medium (S), or starvation medium with wortmannin (W) for 2 hr before immunoblot analysis.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.",
    "molecules": "wortmannin"
  },
  {
    "caption": "(G) HEK293A cells treated with either RISC-free (RF), Atg16L1 siRNA, or FIP200 siRNA for 72 hr before transfection with HA-WIPI2b-CAAX were incubated in full medium (F), EBSS (S), or EBSS with wortmannin (W) for 2 hr before immunoblot analysis. Please note that all WIPI2 constructs are FTTG mutants. See also Figure S6 and Table S1.",
    "molecules": "wortmannin"
  },
  {
    "caption": "(B) RAW 264.7 macrophages expressing the lipid biosensor for PI(4,5)P2, PH-Plcδ-RFP (in red), were left uninfected or were infected with live GFP-expressing S. aureus USA300 (in green) for 8 h prior to fixation. The macrophage plasmalemma and extracellular bacteria are indicated by Cy5-conjugated wheat germ agglutinin (WGA, in blue). As a control, to demonstrate displacement of PH-Plcδ-RFP from the macrophage membrane, transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation. These images are representative of three independent experiments.",
    "molecules": "CaCl2, ionomycin, lipid, PI(4,5)P2"
  },
  {
    "caption": "(C, D) Internalization of FITC-Dextran by uninfected (top row) and S. aureus (in blue)-infected (bottom row) RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. The quantitation of FITC-dextran uptake by either uninfected or infected RAW 264.7 and primary bone marrow derived macrophages is shown in (D). For each experiment, macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u.) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).",
    "molecules": "concanamycin A, Dextran, dextran, FITC"
  },
  {
    "caption": "(E) Potential for co-localization of FITC-dextran (in green) with phagocytosed S. aureus expressing mCherry (in red). The micrographs depict representative macrophages infected with S. aureus at 10 h post-infection and the white arrows point to mCherry positive bacteria that are co-localizing with dextran. Macrophages were treated with 1 μM concanamycin A prior to imaging live.",
    "molecules": "concanamycin A, dextran, FITC"
  },
  {
    "caption": "(A) Internalization and processing of DQ™ Green BSA by uninfected and S. aureus (in blue) infected RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. (B) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S. aureus (in blue) infected RAW 264.7. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).",
    "molecules": "DQ BSA green, DQ™ Green BSA"
  },
  {
    "caption": "(C) Fluorescence-based proliferation assays were performed in conjunction with the probe DQ™ Green BSA. Processed DQ™ Green BSA could be found co-localizing with intracellular mCherry-expressing S. aureus (in red) and with intracellular S. aureus has replicated (i.e. is proliferation dye negative) at 10h post-infection as depicted here. Data information: Scale bars equal 10 μm.",
    "molecules": "DQ™ Green BSA"
  },
  {
    "caption": "(A) Uptake of Alexa® Fluor647-conjugated transferrin by RAW 264.7 macrophages in the presence and absence of PIK-III and Dynasore. The fluorescence micrographs show transferrin (in blue) and the macrophage plasmalemma demarcated by TMR-conjugated wheat germ agglutinin (in red). The dashed line indicates the region of the cell for which the line scan analysis, shown graphically on the right, was done. These micrographs are representative images obtained from three independent experiments. Line scan analysis was performed to indicate the distribution of fluorescent transferrin (blue line) relative to the plasmalemma (red line) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs. Scale bars equal 10 μm.",
    "molecules": "Alexa® Fluor647, DMSO, Dynasore, PIK-III, TMR"
  },
  {
    "caption": "(B) RAW macrophages were pre-treated with vehicle (DMSO) or the vps34 inhibitor PIK-III prior to the addition of 10 kDa FITC-dextran (100 µg/mL) for 2 hrs. Prior to imaging live, macrophages were also stained with Cy5-conjugated WGA. The representative micrographs depict inhibited dextran uptake indicating PIK-III treatment can perturb fluid phase uptake. Scale bars equal 10 µm.",
    "molecules": "dextran, DMSO, FITC, PIK-III"
  },
  {
    "caption": "(C) The intensity of dextran fluorescence was quantified for RAW cells pre-treated with vehicle, PIK-III (10 µM), Dynasore (100 µM) or PIK-III and Dynasore (10 and 100 µM) respectively. The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments. * indicates p<0.05, as determined by an ordinary one-way ANOVA with a Bonferroni multiple comparisons test.",
    "molecules": "dextran, Dynasore, PIK-III"
  },
  {
    "caption": "(A) The effects of prolonged exposure to gentamicin (300 µg/mL) or vancomycin (300 µg/mL) on the survival of phagocytosed S. aureus USA300 in RAW macrophages is shown. Depicted above the graph is a timeline detailing the treatment regime. The data are presented as the mean fold change in CFU/mL +/- standard deviation for 15 biological replicates of S. aureus, represented by each symbol, from five independent experiments. For each replicate the fold change was calculated by dividing the bacterial count at 10 h post-infection by the counts at 1.5 h post-infection. The dashed line indicates a fold change value equal to 1 (i.e. no change in bacterial burden). **p<0.01 and statistical significance was determined by a one-way ANOVA with a Dunnett's post-hoc test.",
    "molecules": "gentamicin, vancomycin"
  },
  {
    "caption": "(B) The ability to protect S. aureus USA300 from intracellular killing by gentamicin is shown. Here, experiments were performed as in (A) except during the initial gentamicin treatment (100 μg/mL for 1h) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK-III/Dynasore to inhibit fluid phase uptake. At 1.5 h post-infection cells were washed and treated with gentamicin (300 µg/mL) in the presence of either DMSO (vehicle) or PIK-III + Dynasore which were maintained throughout the experiment. The data are presented as the mean number of surviving bacteria in CFU/mL at 10 h post-infection +/- standard deviation. These data derive from a total of 15 biological replicates of S. aureus from at least three independent experiments. *** p<0.001 and statistical significance was determined by an unpaired two-tailed t-test with a Welch's correction.",
    "molecules": "DMSO, Dynasore, gentamicin, PIK-III"
  },
  {
    "caption": "(C) The effect of PIK-III/Dynasore treatment on the ability of S. aureus USA300 to replicate within RAW macrophages is shown. After gentamicin treatment at 1.5 h, RAW 264.7 macrophages having phagocytosed S. aureus were lysed and plated to determine the number of gentamicin protected bacteria. Alternatively, infected samples treated with either DMSO as a vehicle control or with PIK-III/Dynasore which was added at 1.5h post-infection after gentamicin. At 10 h post-infection macrophages exposed to DMSO or inhibitors, that were maintained throughout the experiment, were lysed and plated to determine the bacterial burden. The data shown are the mean fold change in CFU/mL at 10 h where the data are normalized to the bacterial burden obtained at 1.5 h. Each symbol represents a biological replicate (n=8) of S. aureus and derive from three independent experiments. The data presented are the mean ± standard deviation. * indicates p<0.05 as determine by an unpaired two-tailed t-test with a Welch's correction.",
    "molecules": "DMSO, Dynasore, gentamicin, PIK-III"
  },
  {
    "caption": "(D) The effect of PIK-III + Dynasore or DMSO on the growth of S. aureus USA300 in RPMI with 5% (v/v) FBS is shown. The data are the mean OD600nm +/- standard deviation of four biological replicates of S. aureus for each condition and are representative of three independent experiments.",
    "molecules": "DMSO, Dynasore, PIK-III"
  },
  {
    "caption": "(E) Analysis of the effects of PIK-III/Dynasore treatment on S. aureus replication using a fluorescence based bacterial proliferation assay is shown. The micrographs depict S. aureus USA300 expressing GFP that were co-labelled with proliferation dye. The top row of micrographs depict vehicle treated macrophages where GFP-positive, yet proliferation dye-negative bacteria were present. The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive. The macrophage plasmalemma and extracellular bacteria are demarcated by TMR-WGA (in red). Images were acquired at 10 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm.",
    "molecules": "Dynasore, PIK-III, TMR"
  },
  {
    "caption": "(A) Growth of wild-type S. aureus USA300 or a purK mutant. The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI. Additives are indicated on the x axis and were either 5% (v/v) FBS, 50 μM IMP, 10 μM PIK-III and 100 μM Dynasore or an equivalent volume of DMSO. Each symbol represents a biological replicate of S. aureus and the data derive from three independent experiments. Statistical significance was determined by a one-way ANOVA with a Holm-Sidak's multiple comparison test. n.s. indicates not significant, *** indicates p<0.0001.",
    "molecules": "DMSO, Dynasore, IMP, PIK-III"
  },
  {
    "caption": "(B) The graph depicts the fold change in bacterial burden for RAW 264.7 macrophages infected with purK S. aureus. Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK-III and Dynasore which were maintained throughout the infection. The data are the mean ± standard deviation of the burden at 10 h post-infection normalized to the burden at 1.5h post-infection prior to the addition of IMP. Each symbol represents a single biological replicate of S. aureus and derive from three independent experiments. Significance was determined by one-way ANOVA with a Turkeys multiple comparison test. *p<0.05, **p<0.01.",
    "molecules": "Dynasore, IMP, PIK-III"
  },
  {
    "caption": "(C, D) The ability of purK S. aureus to grow in an IMP-dependent manner within primary bone marrow-derived macrophages is shown. (C) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non-replicating bacteria are both GFP and proliferation dye positive. In contrast, replicating bacteria (white arrows) are only GFP positive. The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S. aureus in the presence of 50 μM IMP. Images were acquired at 12 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm. (D) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs. Each data point represents a biological replicate (n=8) and derived from three independent experiments. The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition. ** indicates p<0.01 as determined by an unpaired two-tailed t-test with a Wilcoxon matched pairs test.",
    "molecules": "IMP"
  },
  {
    "caption": "C, D. Oxidative stress  biochemical markers (lipid peroxidation, ROS production, GSH levels) (C) and p-ERK expression (D) in PD and control (CNTR) fibroblasts (n=6). E, F. Oxidative stress  biochemical markers (lipid  peroxidation, ROS production, GSH levels) (E) and p-ERK expression (F) in  PD  and control (CNTR) myoblasts (n=4). In all instances indicators of stress were increased in PD, compared to the respective control samples. Data information: In each experiment at least biological triplicates were analyzed for each cell line or tissue sample; each assay was performed at least in duplicate. Data are presented as mean ± SD. Student's t-test was applied. Statistically significant comparison p-values are indicated. Fi ",
    "molecules": "GSH, ROS"
  },
  {
    "caption": "A, B. Western Blot  analysis of the autophagy marker LC3 (A) and quantitative analyses (B) in untreated control fibroblasts  (CNTR) and in 2 PD fibroblasts cell lines (PD1, PD2). PD cells were untreated (NT) or subjected to different treatments to modulate autophagy (starvation, STAR; rapamycin, RAPA; MK6-83; bafilomycin, BAFI). Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.",
    "molecules": "BAFI, bafilomycin, MK6-83, RAPA, rapamycin"
  },
  {
    "caption": "C. ROS production, lipid peroxidation, and GSH levels in PD fibroblasts (n=3). CNTR (white bars), CNTR after treatment (dark grey bars), PD samples (black bars) and PD after treatment (light grey bars). Physiological or pharmacological enhancement of autophagy resulted in correction of oxidative stress in PD  cells, while bafilomycin treatment further increased stress in CNTR and  PD  cells. Data information: Starvation of cells was performed for 4 hours; treatments with 20 µM rapamycin  or 30 µM MK6-83 or 100nM bafilomycin was performed for 24 hours. Data presented as mean ± SD. P values were calculated with ANOVA Šídák's multiple comparisons test. Statistically significant comparison p-values are indicated.",
    "molecules": "bafilomycin, GSH, MK6-83, ROS, rapamycin"
  },
  {
    "caption": "A, B, C. Correlations between the levels of single stress indicators and correction of GAA activity  by rhGAA in 6 different PD fibroblasts. ROS production and lipid peroxidation inversely correlate with the  GAA levels attained in cells after incubation with rhGAA for 4 hours. The analysis of correlation was calculated, and the coefficient of Pearson is indicated. D ",
    "molecules": "ROS"
  },
  {
    "caption": "A.  Cell viability in control and  PD fibroblasts (3 different cell lines) measured by MTT assay in the presence of increased concentrations (0-100 µM) of either sodium arsenite  (ARS) or  tert-butyl-peroxide (TBP) at different time points. Data presented as a mean ± SD. B. ROS  production,  lipid  peroxidation, and GSH levels in fibroblasts (3 different cell lines) after 6 hours of treatment with 100 µM ARS and or 10 µM TBP. Both oxidative agents induced increases in oxidative stress in control (CNTR) and in  PD cells. Data presented as a mean ± SD. Significance was calculated by one-way ANOVA followed by Šídák's multiple comparisons test. C Data information: statistically significant p-values are indicated.",
    "molecules": "GSH, ROS, ARS, sodium arsenite, TBP, tert-butyl-peroxide"
  },
  {
    "caption": "A. Effect of antioxidants on ROS production,  lipid  peroxidation, and GSH levels in PD fibroblasts (3 different cell lines for each treatment, each cell line assayed at least in ducplicate). Cells were incubated for 24 hours with: idebenone (IDE) 0.5 µM; edaravone (EDA) 50 µM; N-acetylcysteine (NAC) 0.5 mM;  resveratrol  (RESV) 30 µM. Mean of control (CNTR) values are taken as equal to 100. The results are shown as mean ± SD. Data information: To calculate statistical significance one-way ANOVA was applied for all experiments followed by Dunnett's test. Statistically significant p-values are indicated.",
    "molecules": "EDA, edaravone, GSH, IDE, idebenone, N-acetylcysteine, NAC, ROS, RESV, resveratrol"
  },
  {
    "caption": "C. GAA activity in tissues from the Gaa  KO mouse after treatment with rhGAA alone (black bars) (n of mice = 10) and with co-dosing of rhGAA and 2 gr/Kg/day NAC (n of mice  =7, dark grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. D. GAA activity in tissues from the Gaa KO  mouse after treatment with rhGAA alone (black bars) (n of mice =4) and with co-dosing of rhGAA and 100 mg/Kg/day IDE (n of mice =6, light grey bars). Data presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each of the tissues. E ",
    "molecules": "IDE, NAC"
  },
  {
    "caption": "E, F. Western Blot  analyses of  GAA and quantitative analyses of the enzyme in representative tissues from the  Gaa  KO mouse after treatment with rhGAA alone (n of mice =2) or in combination with NAC (n of mice =3) (E) or IDE (n of mice =3) (F). In F Data are presented as mean ± SD.",
    "molecules": "IDE, NAC"
  },
  {
    "caption": "G. Glycogen  assay in tissues from the  Gaa  KO mouse  after treatment with rhGAA alone (n of mice = 3) or in combination with NAC (n of mice  = 3). H. Glycogen  assay in tissues from the Gaa  KO mouse after treatment with rhGAA alone (n of mice =4) or in combination with  IDE  (n of mice = 6). Data information: i data are presented as mean ± SD. Values obtained in tissues treated with rhGAA alone for each treatment are taken as 100. A Student's t-test was applied to compare the results in each tissue.  ",
    "molecules": "Glycogen, IDE, NAC"
  },
  {
    "caption": "(A) Top panel: Scheme illustrating the tetracycline inducible Flp-In 293 system that controls the expression of HA-IKKε or HA-GFP. Bottom panel: Representative western blot showing induced expression of HA-IKKε in Flp-In 293 cells treated with doxycycline (Dox, 50 ng/ml) for 16 hours compared to endogenous IKKε in T47D, MDA-MB-231 and MDA-MB-468 breast cancer cell lines.",
    "molecules": "Dox, doxycycline, tetracycline"
  },
  {
    "caption": "(B) Heatmap and hierarchical clustering of metabolite concentrations in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (Dox, 50 ng/ml, 16 hours) (n=5 technical replicates).",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) metabolite levels were normalised to the internal standard HEPES.",
    "molecules": "HEPES, 13C6-glucose, doxycycline, glucose, glutamine, 15N2-glutamine, Serine, serine"
  },
  {
    "caption": "(E) Contribution of pyruvate and glucose-derived carbon to TCA cyle metabolites in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.",
    "molecules": "HEPES, carbon, doxycycline, glucose, pyruvate, TCA"
  },
  {
    "caption": "(F) Fractional enrichment of serine, malate and citrate 13C-isotopologues in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates).",
    "molecules": "13C, citrate, doxycycline, malate, serine"
  },
  {
    "caption": "(G) Fractional enrichment of the serine 15-N-isotopologue in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). m+1 shows the naturally occurring 13C isotopologue (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.",
    "molecules": "HEPES, 13C, doxycycline, 15-N, serine"
  },
  {
    "caption": "(C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.",
    "molecules": "HEPES"
  },
  {
    "caption": "(E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.",
    "molecules": "HEPES, carbon, citrate, 13C6-glucose, glucose, glutamine, Glycine, glycine, 15N-glutamine, malate, pyruvate, serine, TCA"
  },
  {
    "caption": "(F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells.",
    "molecules": "carbon, 13C6-glucose, glucose, glutamine, glycine, 15N-glutamine, malate, pyruvate, Serine, serine, TCA"
  },
  {
    "caption": "(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells.",
    "molecules": "TMRM, Dox, doxycycline"
  },
  {
    "caption": "(D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(F) Representative western blot showing l­evel of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells.",
    "molecules": "TMRM, Dox, doxycycline, DCA"
  },
  {
    "caption": "(I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry.",
    "molecules": "doxycycline, DCA, dichloroacetate"
  },
  {
    "caption": "(J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry.",
    "molecules": "doxycycline, pyruvate"
  },
  {
    "caption": "(A) Representative western blot showing level of ATF4 in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4 biological replicates)",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(C) Representative western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(D) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in a non-silenced, non-treated control, and normalised to β-Actin (n=5 biological replicates).",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(E) Representative western blot showing levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(A) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A).",
    "molecules": "NCT502"
  },
  {
    "caption": "(B) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C).",
    "molecules": "6-Diazo-5-oxo-L-norleucine, DON"
  },
  {
    "caption": "a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white dots) or either SF1670 (first row), SC-79 (second row), 740 Y-P (third row) or AS1842856 (fourth row, black dots) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=4 independent experiments performed.",
    "molecules": "740 Y-P, AS1842856, SF1670, SC-79"
  },
  {
    "caption": "b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 for 3 days after 4 hours of stimulation with PMA and ionomycin. Numbers in brackets represent total cell numbers c. Statistical analysis of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle, SF1670, SC-79, 740 Y-P or AS1842856 of n=5 experiments performed.",
    "molecules": "740 Y-P, AS1842856, ionomycin, PMA, SF1670, SC-79"
  },
  {
    "caption": "d. Proliferation of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that have been pre-incubated with either vehicle, SF1670, SC-79 or 740 Y-P at day 4 after co-culture at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression at different Treg:Tresp ratios of n=6 experiments performed.",
    "molecules": "740 Y-P, CFSE, SF1670, SC-79"
  },
  {
    "caption": "a. Sorted CD4+CD25hiCD127- were pre-incubated with increasing concentrations of AS1842856, washed and co-cultured with CFSE-labeled responder T cells. Representative histograms of proliferation of Tresp alone (left) or in co-culture with Tregs treated with either vehicle or different concentrations of AS1842856 measured at day 4 at a 1:2 Treg:Tresp ratio. b. Statistical analysis of the suppression of proliferation at different Treg:Tresp ratios (n=6) on Tregs pretreated with vehicle (white dots) or 10 nM (black dots, left diagram) or 50 nM AS1842856 (black dots, right diagram).",
    "molecules": "CFSE, AS1842856"
  },
  {
    "caption": "c. Representative dot plots of the frequency of IFN+Foxp3+ Tregs in the co-cultures in a at a 1:2 Treg:Tresp ratio after adding GolgiStop for the last 6 hours.",
    "molecules": "GolgiStop"
  },
  {
    "caption": "e. Correlation between frequency of IFN+Foxp3+ Tregs in co-cultures and percentage of suppression in Tregs pre-incubated with different doses of AS1842856 at a 1:4 Treg:Tresp ratio.",
    "molecules": "AS1842856"
  },
  {
    "caption": "f. IFNG and TBX21 gene expression on Tregs transduced with a Foxo1 or Foxo3 shRNA as compared to the NT-shRNA control, measured at day 6 after 4 hours of stimulation with PMA and ionomycin.",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "g. Percentage of suppression of CFSE-labeled Tresp proliferation co-cultured with Tregs transduced with a non-target (white dots), Foxo1 (left diagram, black dots) or Foxo3 shRNA (right diagram, black dots) at different Treg:Tresp ratios (n=4). *p < 0.05, **p < 0.005, ***p < 0.0005.",
    "molecules": "CFSE"
  },
  {
    "caption": "Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. b. Representative example of staining on resorted Tregs stimulated with PMA and ionomicin for 4 hours at day 5 (n=4).",
    "molecules": "ionomicin, PMA"
  },
  {
    "caption": "Sorted Tregs were stimulated with anti-CD3, anti-CD28, IL-2 and shRNA specific for AKT1, AKT2, AKT3, or non-target control and resorted based on GFP expression at day 5. d. shRNA transduced cells were co-cultured with CFSE-labeled Tresp and Tresp proliferation was measured after 4 days. e. Statistical analysis of the percentage of suppression by AKT1-, AKT2- and AKT3-silenced Tregs as compared to NT-transduced cells of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.",
    "molecules": "CFSE"
  },
  {
    "caption": "a. Sorted CD4+CD25hiCD127- Tregs were stimulated in the presence of vehicle (white squares) and IL-12 either alone (white circles) or in the presence of LY294002 or MK2206 (black circles) for 3 days and IFNG and TBX21 gene expression was examined every 24 hours. Statistical analysis of n=8 independent experiments performed.",
    "molecules": "LY294002, MK2206"
  },
  {
    "caption": "b. Representative example of the frequency of IFN+Foxp3+ cells in Tregs stimulated in the presence of vehicle or IL-12 either alone or in the presence of LY294002 or MK2206 for 3 days after a 4 hour stimulation with PMA and ionomycin.",
    "molecules": "ionomycin, LY294002, MK2206, PMA"
  },
  {
    "caption": "d. Proliferation measured at day 4 of CFSE-labeled responder T cells alone (left histogram) or co-cultured with Tregs that had been pre-incubated with either vehicle or IL-12 alone or with LY294002 or MK2206 at a 1:2 Treg:Tresp ratio. e. Statistical analysis of the percentage of suppression as in d at different Treg:Tresp ratios of n=6 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.",
    "molecules": "CFSE, LY294002, MK2206"
  },
  {
    "caption": "d. Representative example of the frequency of IFN+Foxp3+ Tregs isolated ex vivo from RRMS patients (lower row) and healthy controls (HC, upper row) and stimulated in the presence of vehicle (left histogram), LY294002 (middle histogram) or MK2206 (right histogram) for 3 days. Staining was performed after a 4-hour stimulation with PMA and ionomycin. e. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in HC (white bars) and RRMS patients (black bars) stimulated in the presence of either vehicle, LY294002 or MK2206 (n=10).",
    "molecules": "ionomycin, LY294002, MK2206, PMA"
  },
  {
    "caption": "f. Representative example of the proliferation of Tresp cells from HC (upper row) and RRMS patients (lower row) either alone (first column) or in co-culture with Tregs that had been previously incubated with either vehicle, LY294002 or MK2206 for one hour and washed to remove drug traces. g. Statistical analysis of the percentage of suppression as in h at different Treg:Tresp ratios of n=8 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.",
    "molecules": "LY294002, MK2206"
  },
  {
    "caption": "C: Solid phase binding assay to test for binding of different  Dam1 complexes to  Bim1. Recombinant GST-Bim1185-344 was immobilized on glutathione sepharose  beads and incubated with either recombinant Dam1WTc or Dam1∆SxIPc. Input and pull down samples were analyzed by SDS-PAGE.",
    "molecules": "glutathione sepharose"
  },
  {
    "caption": "F: Analysis of  cell cycle progression  of yeast  strains with different  Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor  arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated  time points after release from  α factor arrest was analyzed by  FACS  (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C   DNA content, respectively. An intermediate DNA content is detected for cells in S phase.",
    "molecules": "α factor"
  },
  {
    "caption": "E, F: GST-Bim1185-344 pull down assays. Binding of immobilized GST-Bim1185-344 to Duo1-6xFlag was analyzed in context of (E) selective inhibition of Mps1  (mps1-737) or overexpression of Mps1 from a galactose-inducible promotor (F). For inhibition of Mps1, cells were grown at the restrictive  temperature of 37 °C. Overexpression of Mps1 was achieved by growing cells in medium containing  galactose. GST-Bim1185-344 immobilized on  glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions. Binding of Duo1 was analyzed by  western blot. Pgk1  indicates equal protein amounts in all samples.",
    "molecules": "galactose, glutathione sepharose"
  },
  {
    "caption": "D: Serial dilution assay  of yeast strains with different  Duo1 and Ndc80 alleles in an Ndc80-FRB-GFP background. Ndc80WT or Ndc80∆490-510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1∆SxIP allele (at DUO1 locus). Cells were spotted on  YEPD medium without and with 1 µg/ml Rapamycin and incubated at the indicated temperatures.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "B. Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analyzed by pulse-chase experiments coupled to redox state determination. To this end, cells were incubated with 35S-Met for 5 min (pulse). After removal of 35S-Met, cells were left in \"cold\" Met for different chase times. Further oxidation was stopped by addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5.",
    "molecules": "mmPEG24, Met, 35S, thiols, TCA, trichloroacetic acid"
  },
  {
    "caption": "C. Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells. Cells were incubated with 35S-Met for 5 min (pulse), and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells.",
    "molecules": "Met, 35S"
  },
  {
    "caption": "A. Assessment of MIA40/CHCHD4 variant-AIFM1 interaction. The indicated MIA40/CHCHD4-Strep variants were affinity precipitated (AP) under native conditions after stopping thiol-disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR and MIA40/CHCHD4 by reducing SDS-PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40-Strep. Both, wildtype MIA40/CHCHD4 and MTS-MIA40/CHCHD4 but not MIA40/CHCHD4∆1-40 variants coprecipitated AIFM1. All mitochondria-localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4.",
    "molecules": "NEM, Strep"
  },
  {
    "caption": "Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. HEK293 cells stably expressing MIA40/CHCHD4-Strep were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and MIA40/CHCHD4-Strep (B) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 interact over a period of at least 6-8 hours while the two MIA40/CHCHD4 substrates, NDUFB7 and AK2, are quickly released from MIA40/CHCHD4 (B, gray box). AP, affinity precipitation; asterisk, background band.",
    "molecules": "emetine, Emetine, Strep, triton X-100"
  },
  {
    "caption": "C. Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. AIFM1 knockout cells stably expressing AIFM1-HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and AIFM1-HA (C) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6-8 hours AP, affinity precipitation; asterisk, background band.",
    "molecules": "Emetine, emetine, triton X-100"
  },
  {
    "caption": "Eluted fractions were subjected to SDS-PAGE and TCE staining (E). AIFM1 and full-length MIA40/CHCHD4 migrate together indicating formation of a complex. MIA40/CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40/CHCHD4. AIFM1 and ∆1-40 MIA40/CHCHD4 do not migrate together emphasizing the importance of the N-terminal amino acids in MIA40 for interaction wth AIFM1.",
    "molecules": "TCE"
  },
  {
    "caption": "A. Gel filtration analysis to assess the AIFM1-MIA40 complex in HEK293 cells. HEK293 cells, cells lacking AIFM1 (AIFM1 knockout, AIFM1 KO) or AIFM1 knockout cells complemented with AIFM1-HA were lysed under native conditions (triton X-100), and the cleared lysates subjected to gel filtration analysis. Eluted fractions were subjected to TCA precipitation, resuspension in loading buffer containing SDS and DTT, and subsequent immunoblotting against AIFM1 and MIA40. Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa (as judged by comparison to protein markers: apoferritin 443 kDa; β-amylase 200 kDa; alcohol dehydrogenase 150 kDa; bovine serum albumin 66 kDa; carbonic anhydrase 29 kDa). Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1-HA. B. Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (∆1-40 MIA40/CHCHD4-Strep), which constitute the AIFM1 interaction motif. Experiment was performed as described in (A). AIFM1 and endogenous MIA40/CHCHD4 migrate in a complex with a size larger than 150 kDa, while N-terminally truncated MIA40/CHCHD4 migrates as monomer. For number of biological replicates and quantifications, see 5C. C. Quantification of 5A,B and Appendix Figure S9. Quantification of the areas representing the region of monomeric MIA40/CHCHD4 and the AIFM1-MIA40/CHCHD4 complex, respectively was performed. Quantifications consistently show that AIFM1 knockout, siRNA-mediated depletion of AIFM1 and overexpression of MIA40/CHCHD4 all resulted in an increase of monomeric MIA40/CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40/CHCHD4 are tightly regulated. The numbers below the bars indicate the numbers of biological replicates performed. Averages and standard deviations are presented.",
    "molecules": "DTT, apoferritin, SDS, Strep, TCA, triton X-100"
  },
  {
    "caption": "A. Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analysed by pulse-chase experiments coupled to redox state determination. Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132. Conversely, NDUFS5 levels were strongly increased. In AIFM1 knockout cells, large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation-dependent protein import. The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132. N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) The relative fold change of carbon metabolites with or without HU treatment for 60 min. Error bars represent standard deviations (SD) from six independent experiments.",
    "molecules": "HU"
  },
  {
    "caption": "(C) Rad53 phosphorylation in various snf1 alleles in response to HU. The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ. Rad53 phosphorylation was detected as above.",
    "molecules": "HU"
  },
  {
    "caption": "(D) The snf1 kinase-defective mutants are sensitive to HU. Yeast spot assays were performed as in Fig 2A. The phospho-mimetic snf1T210D (a constitutive active mutant) was applied as a control for nonphosphrylable snf1T210A.",
    "molecules": "HU, phospho"
  },
  {
    "caption": "(F, G) HU induces a rapid Snf1 T210 phosphorylation. Exponential cells were treated by 200 mM HU for the indicated time. Protein extracts were prepared as described in the Methods and Materials. Three independent experiments were done. Quantitation of Snf1 T210 and Snf1 signals was performed by Image J. The relative ratio of Snf1 T210/Snf1 was shown in (H). Error bars represent standard deviations (SD) from three biological repeats.",
    "molecules": "HU"
  },
  {
    "caption": "(C) Snap shot of Rfa1 ChIP-Seq at Chromosome VI. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min. The sequencing reads were mapped to the yeast reference genome. The red dashed box indicated the relative read intensity of ARS607.",
    "molecules": "HU"
  },
  {
    "caption": "(E, F) SNF1 is required for efficient recruitment of Rfa1, but not Psf2, to HU-stalled replication forks. Cells were grown and synchronized in G1 by α-factor before release into the fresh medium supplemented with 200 mM HU for the indicated time. Cell extracts were prepared and subjected to MYC-ChIP of Rfa1-13MYC (E) or Psf2-13MYC (F). The amounts of DNA in the precipitates were quantified by qPCR. Error bars represent SD from three biological repeats.",
    "molecules": "HU"
  },
  {
    "caption": "mig1Δ restores Rad53 phosphorylation of the snf1 mutants in the presence of HU. The experiments were done as in Fig 2.",
    "molecules": "HU"
  },
  {
    "caption": "Phospho-mimetic mig1-2D bypasses the role of SNF1 in HU resistance (C)",
    "molecules": "HU, Phospho"
  },
  {
    "caption": "Phospho-mimetic mig1-2D bypasses the role of SNF1 in RPA recruitment (D). (E) The diagram of the strategy to reinforce the Mig1-Ssn6 interaction via a GFP and GBP pair (left panel). The persistent Mig1-Ssn6 association abolishes the phospho-mimicking effect of mig1-2D (right panel).",
    "molecules": "Phospho, phospho"
  },
  {
    "caption": "(C) Rfa1-13MYC ChIP assays after HU treatment. Strains were synchronized in G1 phase by α-factor prior to release into fresh media containing 200 mM HU for the indicated time points. Cell extracts were prepared as in Fig 3. Error bars represent SD from three biological repeats.",
    "molecules": "HU"
  },
  {
    "caption": "(B) The effects of α-KG on the HU sensitivity of snf1Δ and other checkpoint mutants. Yeast spot assays were performed as in Fig 2A.",
    "molecules": "α-KG, HU"
  },
  {
    "caption": "(D) α-KG restores Rad53 phosphorylation in snf1Δ. CBB indicates coomassie bright blue (CBB) staining of the membrane as the loading control.",
    "molecules": "α-KG, CBB, coomassie bright blue"
  },
  {
    "caption": "(E) The effects of α-KG on the HU sensitivity of Rfa1 zinc finger mutants (C486S, C491S, C505S, C508S). Yeast spot assays were performed as in Fig 2A.",
    "molecules": "α-KG, HU"
  },
  {
    "caption": "(a) Morphological analysis. Jurkat T cells were cultured in the presence and absence of the indicated anticancer drugs for 48 h. Drug-treated cells were classified as normal, apoptotic or abnormal, with enlarged and irregular nuclei, with multipolar mitoses or multinucleated. Cultures treated with etoposide (0.25 μM), cisplatin (3 μM), taxol (4 nM) and nocodazole (10 nM) exhibited high levels of cells with abnormal nuclei. None of the treated populations showed a significant fraction of apoptosis (see also Supplementary Fig. S1). Values are means±s.d. for three independent experiments. Right: representative examples of these morphological categories are shown. Scale bar, 10 μM. Similar abnormalities were also seen after treating NB4 and MDA-MA-231 cells with etoposide or cisplatin (Supplementary Fig. S2).",
    "molecules": "cisplatin, etoposide, nocodazole, taxol"
  },
  {
    "caption": "(b) Immunoblotting. Jurkat T cells were treated as indicated and ATG5 (monomeric and ATG12-conjugated), ATG12 (conjugated with ATG5), LC3 (18 and 16 kDa), Beclin 1 and GAPDH detected. Chloroquine (CQ) was employed to block lysosomal proteases. Each immunoblot is representative of at least three independent experiments. The same assays were performed using NB4 and MDA-MA-231 cells (Supplementary Fig. S2). Moreover, as a marker for autophagy onset, increased LC3-II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy (Supplementary Fig. S2). Full-length immunoblots are provided in Supplementary Fig. S8.",
    "molecules": "Chloroquine, cisplatin, etoposide"
  },
  {
    "caption": "(a) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were cultured in the presence and absence of etoposide and cisplatin, respectively. Treated cells frequently developed multiple nuclei, including micronuclei (upper panel), and demonstrated evidence of multipolar spindles and misaligned chromosomes (lower panel). Cells were also much larger than untreated cells (see also Supplementary Fig. S3). Scale bar, 10 μm. (b) Statistical analysis of the data shown above. At least 100 cells were counted for each drug exposure time according to the classification used in Fig. 1. Values are means±s.d. (n=3).",
    "molecules": "cisplatin, etoposide"
  },
  {
    "caption": "c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3).",
    "molecules": "etoposide, rapamycin"
  },
  {
    "caption": "(a) Cell cycle analysis. After lentivirus gene transfer, Jurkat T cells overexpressing ATG5 or GFP were followed for the indicated times and analysed for DNA content by flow cytometry. Untransduced cells were left untreated or treated with etoposide for comparison. Values are means±s.d. (n=3). Right: representative flow cytometry diagrams for the 48 h time point.",
    "molecules": "DNA, etoposide"
  },
  {
    "caption": "(c) Immunoblotting. Jurkat T cells overexpressing ATG5 or GFP were cultured for the indicated times after transduction and analysed. As controls, cells were treated with etoposide or rapamycin. Although rapamycin, similar to etoposide, induced autophagy, no increase in ATG5 expression and no cell cycle arrest were observed. ATG5 overexpression and etoposide treatment resulted in a similar expression pattern for all proteins investigated. Each immunoblot is representative of at least three independent experiments.",
    "molecules": "etoposide, rapamycin"
  },
  {
    "caption": "(a) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer.",
    "molecules": "etoposide"
  },
  {
    "caption": "(b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d.",
    "molecules": "etoposide"
  },
  {
    "caption": "(a) Immunoblotting. Jurkat T cells overexpressing ATG5 or control cells were cultured in the presence and absence of 3-MA for 48 h. 3-MA blocked the formation of LC3-II and p62 degradation, indicating that it indeed inhibited ATG5-induced autophagy. Results are representative of three independent experiments.",
    "molecules": "3-MA"
  },
  {
    "caption": "(b) Cell cycle analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA. Induction of G2- or M-phase arrest was seen as a consequence of ATG5 overexpression independent of treatment with 3-MA, as normal Jurkat T cells exhibited no G2/M arrest following 3-MA treatment. Representative flow cytometry diagrams are shown (n=3). Moreover, induction of autophagy by Beclin 1 overexpression or starvation failed to produce cell cycle arrest (Supplementary Fig. S4).",
    "molecules": "3-MA"
  },
  {
    "caption": "(c) Morphological analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA and analysed. Representative examples of morphology are shown (n=3); large multinucleated cells and cells with abnormal nuclei were seen as a consequence of ATG5 overexpression independent of 3-MA. Neither induction of autophagy by Beclin 1 overexpression nor starvation resulted in such abnormal cells (Supplementary Fig. S4). Scale bar, 10 μm. (",
    "molecules": "3-MA"
  },
  {
    "caption": "(d) Viability assays. Jurkat T cells either overexpressing ATG5 following lentiviral gene transfer or treated with etoposide were cultured in the presence and absence of the indicated pharmacological inhibitors for the indicated times to block autophagy and caspase activity. All values are means±s.d. (n=3).",
    "molecules": "etoposide"
  },
  {
    "caption": "(a) Subcellular fractionation and immunoblotting. Jurkat T cells were cultured in the presence of etoposide for the indicated times. Following etoposide treatment, the ATG5 monomer was newly present in both cytoplasm and nucleus. The exposure of the ATG5 monomer (shown under the dotted line) was longer than that shown above for the ATG5 conjugated to ATG12. Full-length immunoblots are provided in Supplementary Fig. S10.",
    "molecules": "etoposide"
  },
  {
    "caption": "(c) Subcellular fractionation and immunoblotting of the nuclear fraction. Jurkat T cells, either treated with etoposide, or overexpressing ATG5 following lentiviral gene transfer, were cultured in the presence and absence of leptomycin B for 72 h. Leptomycin B increased relative ATG5 and Beclin 1 levels in the nucleus.",
    "molecules": "etoposide, leptomycin B, Leptomycin B"
  },
  {
    "caption": "(a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies. A physical interaction between ATG5 and survivin, absent in untreated or GFP-expressing control cells, was detected reciprocally. Full-length immunoblots are provided in Supplementary Fig. S12.",
    "molecules": "etoposide"
  },
  {
    "caption": "(e) Confocal microscopy. MDA-MA-231 cells were etoposide treated or transduced as indicated (48-h cultures) and stained. In untreated cells, survivin and ATG5 were in the cytosol with no colocalization. ATG5-transduced cells exhibited ATG5/survivin colocalization in the nucleus. ATG5-ΔNES-transduced cells showed survivin, but not ATG5, in the nuclei of cycling cells. Numerical analysis was performed and Pearson's correlation coefficients are indicated in the images. Scale bar, 10 μm. Right: Statistical analysis (analysis of variance) is presented in which the results of ten representative cells within each group were integrated. Values are means±s.d.",
    "molecules": "etoposide"
  },
  {
    "caption": "(g) Confocal microscopy. HeLa cells treated with etoposide or transduced as indicated (48-h cultures) were stained with centromere-specific antiserum and anti-Aurora B (top panels) or antisurvivin antibodies (lower panels). In untreated or ATG5-ΔNES transduced cells, both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres. In contrast, etoposide-treated or ATG5-transduced cells recruited much less Aurora B to prometaphase centromeres. Such recruitment, however, was observed during anaphase. Compared with cells in normal mitosis, survivin, although reduced, was recruited to centromeres in prometaphase, but, similar to Aurora B, remained attached to centromeres during anaphase. Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects. Colocalization images were prepared using Imaris software. Numerical analysis was performed and correlation coefficients are indicated. Scale bar, 2 μm.",
    "molecules": "etoposide"
  },
  {
    "caption": "C, D. HUWE1-knockout 293T (C) and HeLa (D) cells show increased DNA breaks in the absence of exogenous DNA damage treatment. Results from the Alkaline Comet Assay are shown. The \"n\" numbers of comet tails analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs. HUWE1-knockout HeLa cells did not show increases breakage in the Neutral Comet assay (Fig EV1B), indicating that the majority of breaks observed in cycling HUWE1-knockout cells are not double strand breaks, but rather single strand breaks and other types of lesions.",
    "molecules": "DNA"
  },
  {
    "caption": "E, F. Increased S-phase arrest in HUWE1-knockout 293T (E) and HeLa (F) cells. Cycling cells were incubated with BrdU and subjected to BrdU/PI bi-dimensional flow cytometry. Representative flow cytometry profiles are presented in Fig EV1D-F. Bars represent the fold increase in the percentage of cells with S-phase DNA content (between 2N and 4N) but negative for BrdU staining. Bars represent the average of three independent experiments, with error bars showing SD. The p-values are 0.0091 (E) and 0.0007 (F).",
    "molecules": "DNA"
  },
  {
    "caption": "G, H. The DNA fiber assay shows reduced replication tract length in HUWE1-knockout 293T (G) and HeLa (H) cells in the absence of exogenous DNA damage treatment. The \"n\" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-values are 9.8x10-22 (G) and 1.0x10-10 (H).",
    "molecules": "DNA"
  },
  {
    "caption": "C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N).",
    "molecules": "DNA"
  },
  {
    "caption": "C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N). D. Quantification of S-phase cells. Percentage of cells in R1 region is shown. Bars represent the average of three independent experiments. Error bars indicate SD. P-value is 0.0014. E. Quantification of S-phase arrested cells. Bars represent the fold increase in the percentage of cells in R2 region, normalized to siControl-treated cells. The average of three independent experiments is shown. Error bars indicate SD. P-value is 0.0135.",
    "molecules": "DNA"
  },
  {
    "caption": "F. DNA fiber experiment showing reduced replication tract length in HUWE1-depleted HeLa cells in the absence of exogenous DNA damage treatment. The \"n\" numbers of fibers analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs.",
    "molecules": "DNA"
  },
  {
    "caption": "G, H. Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV (G), and HU (H). As controls, both cells transfected with non-targeting siRNA (siControl), and non- transfected cells, were used. Note that the siHUWE1#2 oligonucleotide shows a stronger downregulation of HUWE1, and confers increased sensitivity compared to siHUWE1#1. The average of nine experiments is shown. Error bars represent SEM. P-values (representing the statistical difference between the samples at the highest dose treatment) are: 4.55x10-9 (G) and 1.8x10-8 (H).",
    "molecules": "HU"
  },
  {
    "caption": "B. Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU (2mM for 16h) and UV (40J/m2, analyzed 2h later).",
    "molecules": "HU"
  },
  {
    "caption": "D. Clonogenic assay showing that stable expression of wild-type HUWE1, but not of the PIP box mutant or the catalytic mutant, corrects the HU sensitivity of the HUWE1-knockout 293T cells. Shown is the average of 3 independent experiments -/+SD. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T (for the 0.5mM HU condition). P-values are: 0.0003 (293T vs HUWE1-/-; 0.157 (293T vs HUWE1-/- +WT); 0.0045 (293T vs HUWE1-/- +FF); 0.0006 (293T vs HUWE1-/- +C4341A; 0.047 (HUWE1-/- +WT vs HUWE1-/- +FF).",
    "molecules": "HU"
  },
  {
    "caption": "A, B. The impact of HUWE1 on H2AX phosphorylation and ubiquitination, under normal conditions (A) or upon induction of replication stress (2mM HU for 18h, or 2h after exposure to 40J/m2 UV). Control or HUWE1-knockout HeLa cells were analyzed.",
    "molecules": "HU"
  },
  {
    "caption": ". E. Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1-knockout 8988T cells. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 4 experiments -/+ SD. P-value is 4.4x10-6.",
    "molecules": "aphidicolin"
  },
  {
    "caption": "F. Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress. HUWE1, RNF168, and BMI1 were knocked-down in HeLa cells. The efficiency of the knockdown is shown in Fig EV4. Cells were treated with 600nM aphidicolin for 24h, 2mM HU for 24h, or analyzed 2h after exposure to 40J/m2 UV.",
    "molecules": "aphidicolin, HU"
  },
  {
    "caption": "G. Chromatin immunoprecipitation from HeLa cells, showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 3 experiments -/+ SD. The p-values shown indicate the statistical significance relative to siControl (0.0016, 0.0005, and 0.0009 respectively).",
    "molecules": "aphidicolin"
  },
  {
    "caption": "H. Chromatin fractionation experiments showing that HUWE1-knockout HeLa cells, treated with 2mM HU for 24h, fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA. In contrast, 53BP1 chromatin association is not induced by HU treatment, and not affected by HUWE1 knockout, and thus can serve as loading control. Shown are blots of whole cell extract (WCE) samples, representing input material, and of chromatin pellet samples.",
    "molecules": "HU"
  },
  {
    "caption": " Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated (-) or treated (+) with 5 µM thapsigargin (TG) for 1 h. THBS4 was transiently transfected into cells in the experiment depicted in panel 2. ATF6α was either immunoisolated with anti-V5 (panel 1 and 3) or anti-HA (panel 2). For the experiment in panel 3 the cells were cross-linked prior to immunoisolation. Samples were separated by SDS-PAGE under reducing conditions followed by western blots to determine co-isolation of endogenous PDIR, ERp18 or exogenously expressed THBS4 with ATF6α. HC indicates immunoglobulin heavy chain. ",
    "molecules": "TG, thapsigargin"
  },
  {
    "caption": " Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain (ATF6 DM) were transfected with PDI substrate-trapping mutants. ATF6α was immunoisolated with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions, and ATF6α detected with rabbit anti-HA. ",
    "molecules": "alanine, cysteine"
  },
  {
    "caption": " HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ",
    "molecules": "S1P, TG"
  },
  {
    "caption": " HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µg/ml tunicamycin to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments. ",
    "molecules": "S1P, tunicamycin"
  },
  {
    "caption": " HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 20 µM MG132 to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments. ",
    "molecules": "MG132, S1P"
  },
  {
    "caption": " HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. Samples from (A) were rerun and probed with anti-sera raised against respective PDI enzymes as indicated (D). *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ",
    "molecules": "S1P, TG"
  },
  {
    "caption": " HEK293T cells were co-transfected with the ERp18 substrate-trapping mutant and the indicated ATF6α constructs. Cell were allowed to recover for 24 h post-transfection before being either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. The blot confirms that ATF6α C618A forms a stable complex with ERp18 trapping mutant following ER stress. M, D and O refer to ATF6 monomer, dimer and oligomer respectively. *indicates positions of the ATF6-ERp18 mixed-disulfide complexes. ",
    "molecules": "TG"
  },
  {
    "caption": " HEK293T cells were co-transfected with GFP-ATF6α and ERp18 trapping mutant constructs as indicated. GFP-ATF6α complexes were immunoisolated using GFP-trap. Immunoisolated material was separated by SDS-PAGE under non-reducing conditions and silver stained to detect ATF6α disulfide-linked complexes. MS analyses confirmed that protein bands indicated by black arrows are a complex comprising ATF6α and ERp18, whilst bands marked as monomer (M) and oligomer (O) contain ATF6α only. ",
    "molecules": "silver"
  },
  {
    "caption": " qPCR analysis of UPR target genes in the absence of ER stress. Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type. Error bars represent ± standard deviation for three independent experiments.   qPCR analysis of UPR target genes following ER stress. Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls. Error bars represent ± standard deviation for three independent experiments.   ",
    "molecules": "TG"
  },
  {
    "caption": " Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised BiP levels from (D ) were quantified. Error bars represent ± standard deviation for three independent experiments.   ",
    "molecules": "TG, thapsigargin"
  },
  {
    "caption": " Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised ERp72 levels from (F) were quantified. Error bars represent ± standard deviation for three independent experiments.   ",
    "molecules": "TG, thapsigargin"
  },
  {
    "caption": " A, B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times. Cells were then subject to differential centrifugation to obtain membrane (A) and nuclear fractions (B) which were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of full-length ATF6α (ATF6-FL), cleaved membrane-associated ATF6α (ATF6-P) and nuclear translocated S2P cleaved ATF6α (ATF6-N) are as indicated. HDAC2 was used as a nuclear marker. ",
    "molecules": "TG"
  },
  {
    "caption": " (D, E) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α, either untreated (-) or treated (+) with 30 µM PF429242 (S1P inhibitor) prior to and following induction of ER stress with 10 mM DTT. The positions of ATF6-P, ATF6-M and ATF6-N are as indicated. The unprocessed ER-localised ATF6α (designated ER) and the O-linked glycan modified Golgi-translocated ATF6α (designated G) are indicated. HDAC2 was included as a nuclear marker. The blots in (D, E) confirm that ATF6-P is produced independently of S1P and also the absence of ATF6α processing to ATF6-N in the presence of the S1P inhibitor. ",
    "molecules": "DTT, PF429242, glycan, S1P"
  },
  {
    "caption": " HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor. Cell lysates were prepared, separated by SDS-PAGE and then probed with anti-ATF6α. Blot reveals the presence of O-linked glycan modified Golgi-translocated ATF6 (designated G), which is absent for the D564G mutant, confirming a lack of ER to Golgi trafficking. ",
    "molecules": "DTT, glycan, S1P"
  },
  {
    "caption": " Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated (-) or treated (+) with 5 µg/ml brefeldin A for1 h prior to cell lysis. The anti-ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6-M, only in the brefeldin A treated cells. Nuclear fractions confirm the presence of S2P cleavage product, ATF6-N following brefeldin A treatment. Anti-HDAC2 was used as a loading control for the nuclear fraction. ",
    "molecules": "brefeldin A, S1P"
  },
  {
    "caption": " A-C Wild type cells stably expressing ATF6α (A) or ERp18 KO cells stably expressing ATF6α (B), and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 (RV cells) (C), were pre-treated with 30 µM PF429242 (S1P inhibitor) for 1 h prior to induction of ER stress with 10 mM DTT as indicated. ",
    "molecules": "DTT, PF429242, S1P"
  },
  {
    "caption": " D-F Wild type cells transiently expressing ATF6 C467A (D), ATF6 C618A (E), or ATF6 DM (F) were treated as above. Whole cell lysates were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of unprocessed ER-localised ATF6α (ER) and the O-linked glycan modified Golgi-translocated ATF6α (Gol) are indicated as is ATF6-P. ",
    "molecules": "glycan"
  },
  {
    "caption": "(H) Western blot analysis of  ATXN1  after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.",
    "molecules": "BI-D1870"
  },
  {
    "caption": "(I) Western blot  analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.",
    "molecules": "BI-D1870, SB-747651A"
  },
  {
    "caption": "(E) Representative Hematoxylin and Eosin stained motoneurons  in the coronally sectioned hypoglossal nucleus  (white dashed line) of 36 weeks old mice with different genotypes. The motoneurons are dark purple with pyramidal shape within the nucleus. C:  central canal. Scale bar = 100 μm.",
    "molecules": "Eosin, Hematoxylin"
  },
  {
    "caption": "(D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β‐gal, β‐galactosidase; BafA1, bafilomycin‐A1; CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; GST, glutathione‐S‐transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin‐like modifier 1; TCL, total cell lysate; TM, transmembrane domain.",
    "molecules": "BafA1, CCCP"
  },
  {
    "caption": "Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the 'colocalization' highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA).",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann-Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A) Flow cytometry of reticulocyte‐enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix−/− bone marrow and reconstituted with wt‐Nix and Nix‐W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP.",
    "molecules": "phenylhydrazine"
  },
  {
    "caption": "a, Etoposide-treated Atg5 -/- MEFs were assessed by EM (left) and by Lamp2immunofluorescence (IF; middle). Right: reverse Lamp2 immunofluorescence image merged with the electron microscopy image. Arrows indicate Lamp2-positive autolysosomes. Magnified photos are provided in Supplementary Fig. 5",
    "molecules": "Etoposide"
  },
  {
    "caption": "b, The size of each autophagic vacuole (AV) (n = 30 cells) in MEFs treated with etoposide. The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5-/- cells.",
    "molecules": "etoposide"
  },
  {
    "caption": "c, Percentage autophagic area (n ≥ 20 cells) in MEFs treated with etoposide.",
    "molecules": "etoposide"
  },
  {
    "caption": "d, e, Representative macroautophagy in etoposide-treated Atg5-/- MEFs. Conventional (d) and quick freezing and freeze-substitution (e) techniques were used. The autolysosomes (arrows) contain multilamellar bodies (arrowheads).",
    "molecules": "etoposide"
  },
  {
    "caption": "f, The number of autophagosomes (white columns) and autolysosomes (black columns) in MEFs (n = 10 cells) treated with etoposide.",
    "molecules": "etoposide"
  },
  {
    "caption": "g, Reduction of percentage autophagic area by 3-methyladenine (3-MA; n = 22 cells).",
    "molecules": "3-methyladenine"
  },
  {
    "caption": "i, Inhibition of long-lived protein degradation by bafilomycin A1, protease inhibitor (PI) cocktail and 3-MA (*P 0.05; n = 4).",
    "molecules": "3-MA, bafilomycin A1"
  },
  {
    "caption": "e, Atg5-/- MEFs were incubated without (NT) or with etoposide and immunostained with an anti-Lamp2 antibody (green). Nuclei were counterstained (red). The percentages of cells with punctate Lamp2 immunostaining are shown (means ± s.d., n = 4).",
    "molecules": "etoposide"
  },
  {
    "caption": "f, Microarray analysis of the Atg5-/- MEFs during treatment with etoposide.",
    "molecules": "etoposide"
  },
  {
    "caption": "g, h, Induction of Ulk1 in the etoposide-treated Atg5-/- MEFs was assessed by qPCR (g) and by immunoblotting (h).",
    "molecules": "etoposide"
  },
  {
    "caption": "i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k)",
    "molecules": "etoposide"
  },
  {
    "caption": "i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).",
    "molecules": "etoposide"
  },
  {
    "caption": "i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l) and a beclin 1 plasmid (k) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k).",
    "molecules": "etoposide"
  },
  {
    "caption": "i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l)for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).",
    "molecules": "etoposide"
  },
  {
    "caption": "a-d, Electron micrographs of etoposide-treated Atg5-/- MEFs. a, AVs were observed near the Golgi apparatus (G). Inset, the isolation membrane (I) was extended from the Golgi stack. b, Developing autophagosome. rER, rough endoplasmic reticulum. c, d, Isolation membrane fusing with vesicles containing thick membrane (d) and a complete autophagosome (c) is generated.",
    "molecules": "etoposide"
  },
  {
    "caption": "e, f, Co-localization of Lamp2 with GFP-M6PR, GFP-Stx7 or GFP-Rab9 in etoposide-treated Atg5-/- MEFs. Arrows indicate co-localized dots. Original magnification, × 200.",
    "molecules": "etoposide"
  },
  {
    "caption": "g-i, Atg5-/- MEFs were transfected with the indicated siRNAs for 24 h and treated with etoposide. Induction of macroautophagy was assessed by Lamp2 IF (g) and by EM (h), and the number of isolation membranes was calculated (i). In g, error bars indicate s.d. (n = 4). In h and i, red and blue lines indicate means and s.e.m., respectively; n = 35 cells each.",
    "molecules": "etoposide"
  },
  {
    "caption": "j, No effect of silencing of Rab9 (siRab9) on the production of LC3-II in etoposide-treated WT MEFs.",
    "molecules": "etoposide"
  },
  {
    "caption": "(b) NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 48 h. Atg14L-linked Vps34 was pulled down by an anti-Atg14L antibody and subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (c) Quantification of IPed Vps34 kinase activity (PI(3)P), Vps34 and Vps15 protein versus IPed Atg14L protein. (Data are shown as mean±s.e.m., P values are indicated on the figures, multiple t-test are followed by Bonferroni correction, n=4 (otherwise indicated, n means number of independent experiments; NS, not significant)).",
    "molecules": "PI(3)P"
  },
  {
    "caption": "(a) Starvation-induced long-lived protein degradation is impaired after NRBF2 knockdown. Compared with control (con) siRNA, NRBF2 siRNA decreases long-lived protein degradation (proteolysis rate) in NIH3T3 cells under starvation (ST) condition. This difference is diminished when the starved cells are co-treated with wortmannin (WM, 100 nM). (Data are shown as mean±s.e.m., P values are indicated on the figures, one-way analysis of variance with Dunnett as post hoc test, n=4; NS, not significant).",
    "molecules": "wortmannin"
  },
  {
    "caption": "(b) Rapamycin (Rap)-induced autophagy is impaired in the NRBF2 KO cells. The MEFs were treated with (dimethyl sulfoxide) DMSO or 50 μg ml−1 of Rap for 4 h then the cell lysates were immunoprobed with anti-LC3 and anti-p62 antibodies. (c) LC3-II and p62 levels were quantified and normalized with β-actin. Quantification results show that Rap-induced LC3 lipidation (upper) and p62 degradation (lower) were markedly impaired in the NRBF2 KO MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figures, one sample t-test (WT con versus KO con) or unpaired t-test (WT Rap versus KO Rap)). p62 blots quantification, n=6; LC3 blots quantification, n=3).",
    "molecules": "dimethyl sulfoxide, DMSO, Rap, Rapamycin"
  },
  {
    "caption": "(d,e) The MEFs were subjected to Rap (50 μg ml−1) or starvation (HBSS) treatment then stained with anti-LC3 (d) or anti-WIPI2 (e) antibody. (f) The LC3 and WIPI2 puncta per cell were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from three independent repeats).",
    "molecules": "Rap"
  },
  {
    "caption": "(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry-GFP-LC3B. WT MEFs expressing mCherry-GFP-LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta markedly increases after rapamycin (Rap; 50 μg ml−1, 4 h) or starvation (HBSS, 4 h) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry-GFP-LC3B contain quite fewer red-only puncta and yellow puncta, even after Rap or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were markedly reduced. (Data are shown as mean±s.e.m., P values are indicated on the figures, Mann-Whitney U-test, n=20 randomly selected cells, a representative result from three independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bars, 20 μm or 10 μm (amplified regions).",
    "molecules": "Rap, rapamycin"
  },
  {
    "caption": "WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin(TP) or 1 μM tunicamycin (TN) for 24 or 48 h. (a) Then cells were either imaged under phase-contrast microscope or (b) stained with propidium iodide (PI) and subjected to cell death rate analysis by flow cytometry. The representative images of cell morphology after treatments and quantitative analysis of PI-positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=5). Scale bars, 100 μm; Con, control.",
    "molecules": "DMSO, thapsigargin, TP, TN, tunicamycin"
  },
  {
    "caption": "(c) Re-introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress. NRBF2-CFP stably expressing cells were established in the NRBF2 KO background. Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry. The cell death rate in CFP-positive (+) population (NRBF2-CFP expression) and negative (−) population (no NRBF2-CFP expression) were recorded and analysed, respectively. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=3).",
    "molecules": "DMSO, TP"
  },
  {
    "caption": "(d) ER stress inducers trigger caspase-8 and caspase-3 activation in NRBF2 KO MEFs. MEFs were treated with DMSO, 1 μM TP or 1 μM TN for 24 h, and then immunoprobed with indicated antibodies. Marked accumulation of cleaved caspase-8 (C-Cas 8; p43 and p18 units) and cleaved caspase-3 (C-Cas 3) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN. This is a representative image from three independent experiments.",
    "molecules": "DMSO, TP, TN"
  },
  {
    "caption": "(e) C-Cas 8 preferentially localizes at p62 aggregation foci. MEFs were treated with DMSO or 1 μM TP for 24 h, and then co-stained with an anti-p62 antibody and an anti-cleaved caspase-8 antibody. The C-Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci. Scale bars, 20 μm. This is a representative image from three independent experiments.",
    "molecules": "DMSO, TP"
  },
  {
    "caption": "(D) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in CCCP-treated (see Figure S1) or HCV-infected cells (A) and those treated with 3-MA or BafA1, respectively (see Figure 4A) (mean ± SEM; n = 10 cells; *p<0.01). P values were calculated by using an unpaired Student's t-test.",
    "molecules": "3-MA, BafA1, CCCP"
  },
  {
    "caption": "(A and B) Quantitative analyses of ATF4, Parkin, and PINK1 gene expression in HCV-infected cells. Huh7 cells infected with HCVcc for 5 days or treated with CCCP (10 or 20 µM) for 24 h were used for analysis of ATF4, Parkin, and PINK1 gene expression. (A) Intracellular mRNA levels of ATF4, Parkin, and PINK1 were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin and PINK1 gene expression (mean ± SD; n = 3; *p<0.01, **p<0.05). P values were calculated by using an unpaired Student's t-test.",
    "molecules": "CCCP"
  },
  {
    "caption": "Confocal microscopy showing mitophagosome formation associated with Parkin in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 day post-infection, cells were immunostained with antibodies against HCV E2 (orange), TOM20 (blue), and Parkin (red). In the zoom images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. Nuclei are demarcated with white dot circles. (",
    "molecules": "3-MA, BafA1"
  },
  {
    "caption": "(E) Immunoelectron microscopic analysis showing the formation of mitophagosome in HCV-infected cells. Huh7 cells infected with HCVcc were stained by triple immunogold labeling method, as described in Materials and Methods. The immunogold particles of GFP-LC3 (white arrow, 18 nm), HCV E2 (blue arrow, 12 nm), and TOM20 (red arrow, 6 nm) are shown. Fluorescence image of HCV core identifies HCV-infected cells (red). Nuclei were stained with DAPI (blue). Scale bar = 0.2 µM.",
    "molecules": "gold"
  },
  {
    "caption": "Confocal microscopy showing the formation of mitophagolysosome in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 days post-infection, cells prestained with LysoTracker (red) were immunostained with antibodies against TOM20 (blue) and HCV core (cyan). Nuclei are demarcated with white dot circles. In the zoomed images, the arrows indicate the colocalization of GFP-LC3 puncta (green), lysosome, and mitochondria in HCV-infected cells (white puncta).",
    "molecules": "3-MA, BafA1"
  },
  {
    "caption": "(A) Rescue effect of 3-MA and BafA1 on reduction of mitochondrial complex I enzyme activity caused by HCV infection. Huh7 cells infected with HCVcc were treated with 3-MA (10 mM) or BafA1 (100 nM) for 12 h before harvest. At 3 days post-infection, the activity of mitochondrial complex I enzyme was measured according to manufacturer's instructions (mean ± SD; n = 3; *p<0.01).",
    "molecules": "3-MA, BafA1"
  },
  {
    "caption": "(C) Western blot analysis of mitochondrial respiratory chain complex enzyme expression. NT-KD and P-KD cells were infected with HCVcc and at 3 days post-infection, the expression levels of complex I and IV enzyme were analyzed by immunoblotting with anti-Hu total OxPhos complex antibody. Huh7 cells treated with CCCP (10 µM) for 12 h were also analyzed as a control. β-actin was used as an internal loading control. P values were calculated by using an unpaired Student's t-test.",
    "molecules": "CCCP"
  },
  {
    "caption": "(H,H') TBR2 (green in H), SOX2 (red in H) and NR2F1 (yellow in H') immunofluorescence (IF) of a GW11 section of human neocortex Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.",
    "molecules": "DAPI"
  },
  {
    "caption": "(J-L') NR2F1 (red) IF of human GW14 neocortex, showing expression levels in the CP (K,L) and VZ (K',L') around primary convolutions in the posterior-most cortex High NR2F1 is detected in the neurogenic area of a gyrus (L'). Data information: Nuclei (blue) were stained with DAPI. In Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.",
    "molecules": "DAPI"
  },
  {
    "caption": "(P-R) NR2F1 (red) and HOPX (green) immunostaining Virtually all HOPX+ bRGs are also NR2F1+ (magnification in P'') and their number is greatly increased in gyri (P'') compared to sulci (P'), as quantified in (R). At a single cell level, NR2F1 pixel intensity is higher in oSVZ HOPX+ cells in gyri compared to sulci (Q). n ≥ 4 sections from n = 1 fetal brain. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. Student t-test Q,R) (*P<0.05, ***P<0.001). oSVZ: outer subventricular zone",
    "molecules": "DAPI"
  },
  {
    "caption": "(D,E) PH3 (green; dividing cells) IF on cross-sections of day 4 neurospheres. White arrowheads point at mitotic figures, which are quantified in (E) as percentage of dividing cells over DAPI-stained nuclei (blue). n ≥ 3 neurospheres from n = 2 batches. Data information: data are represented as means ± SEM. Student t-test (E) (**P<0.01, ***P<0.001). Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-D) Ki67 (green; mitotically active progenitors) and Tuj1 (red; post-mitotic neurons) IF at E14.5 (A,A'), E16.5 (B,B') and E18.5 (C,C') of lateral pallia of wild-type (WT) versus mutant (KO) brains. Ki67+ proliferating cells (quantified in D) accumulate in mutant cortices. n ≥ 3 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E-F') Tbr2 (green; IPs) and Pax6 (red; radial glia cells) IF of E13.5 (E,E') and E17.5 (F,F') lateral pallia of WT and KO brains. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.",
    "molecules": "DAPI"
  },
  {
    "caption": "(N-P) GFP (green; expressed under Sox2 promoter) and RFP (red; expressed under Tis21 promoter) IF of E13.5 lateral pallia, 18-hours after IUE. The proportion of single or double positive NPs is shown in pie charts in (P). n = 3 electroporated brains. Data information: Nuclei (blue) were stained with DAPI. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).",
    "molecules": "DAPI"
  },
  {
    "caption": "(Q-R') Ki67 (green; progenitors) and EdU (red) IF of E13.5 WT (Q,Q') and KO (R,R') embryos, injected with EdU at E12.5. Differentiating cells (EdU+Ki67-) are located in the IZ/CP; percentages shown in (Q',R') were obtained from n ≥ 4 sections of n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. Student t-test (Q',R'; E13.5, EdU injected at E12.5; WT/KO: *=0.03967 (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "(E) Cell cycle duration at different embryonic ages (from E10.5 to E16.5) in WT (blue bars) or KO embryos (orange line), as quantified upon a double EdU/BrdU injection protocol in posterior-most LP n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).",
    "molecules": "BrdU, EdU"
  },
  {
    "caption": "(F-G') Ki67 (green; NPs) and EdU (red) IF in WT (F,F') and KO (G,G') after 6 hours consecutive EdU incorporation. Note the higher number of labelled NPs in KO cortices (G') suggesting faster cell cycle progression and/or longer S-phase. Data information: Nuclei (blue) were stained with DAPI.",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "(H) Best linear fit of EdU+Ki67+ NP percentage after 8 hours EdU cumulative labelling in WT (blue line) and KO (orange line) brains. The x- and y-intercept are proportional to the S-phase length and to the number of cycling cells, respectively, while the steepness of the line is proportional to cell cycle duration. WT: y = 11.66x +24.62; KO: y = 13.54x +32.96). n ≥ 2 brains per time point/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).",
    "molecules": "EdU"
  },
  {
    "caption": "(I-K) PH3 (green; dividing NPs) and Pax6 (red; aRGs) IF of E13.5 WT (I) and KO (J) lateral pallia. Strong and weak/punctate PH3 patterns show M-phase and G2-phase cells, respectively. G2-phase NP percentage is quantified in (K). As a result of shorter G1-phase and globally shorter cell cycle time, the percentage of G2-phase cells is increased in the KO NP pool. n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. Student t-test (K; *P<0.05) Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-B') Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A,A') and KO (B,B') lateral pallia. Data information: nuclei (blue) were stained with DAPI. Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.",
    "molecules": "DAPI"
  },
  {
    "caption": "(J-L) GFP (green; electroporated cells) and P21 (red) IF of E14.5 cortex electroporated 48 hours earlier with control PX458 plasmid (J) or CRISPR/Cas9-expressing plasmid directed against Nr2f1 sequence (PX458-αNr2f1; K). Percentage of P21/GFP double positive cells (L). Arrowheads in (J,K) point to P21+ cells. n = 2 electroporated brains. Data information , nuclei (blue) were stained with DAPI. Student t-test (L; *P<0.05",
    "molecules": "DAPI"
  },
  {
    "caption": "(D-E) Paired cell analysis after neurosphere dissociation and 24-hour culture at clonal density. Couples of dividing cells were identified by Map2 (red; N, neurons) and BLBP (green; P, progenitors) IF. Pie charts in (E) show the proportion of P-P (proliferative; D), P-N (asymmetric differentiative; D') or N-N (symmetric differentiative; D'') couples in WT, Nr2f1KO and Nr2f1KO Pax6HET animals. n ≥ 3 samples from n = 2 culture batches. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001 Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F-I) Pax6 (green) and Nr2f1 (red) IF in the posterior-most region of E12.5 cortex, showing Pax6 upregulation in Nr2f1 KO animals (G,G') compared to WT (F',F''). Normal Pax6 levels can be restored by loss of one Pax6 allele (Nr2f1 KO, Pax6 HET; H,H'). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(J) Cell cycle duration at E12.5 in different mutants as indicated, quantified in posterior regions with double EdU/BrdU injection protocol n ≥ 6 sections from n = 2 brains. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001).",
    "molecules": "BrdU, EdU"
  },
  {
    "caption": "(K) Insets showing representative γ-Tubulin (green) and Phospho-Vimentin (red) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs. Pie charts show the percentage of vertical (green), oblique (yellow) and horizontal (red) division planes of mitotic figures in the E12.5 lateral pallium of animals with different genotypes, as indicated. n ≥ 2 brains per genotype. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). cale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D-G) NR2F1 (red) and PAX6 (green) IF in day 40 organoids. Neuroepithelia with high NR2F1 levels show weak PAX6 expression (E,E'), and vice versa (F,F'). Quantification by pixel intensity analysis (G). n ≥ 4 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(I-J) NR2F1 (red) and GFP (green) IF in day 40 human brain organoids upon electroporation of the PX458-αNR2F1 plasmid. In (I,I'), GFP+NR2F1- cells (empty arrowheads) and a GFP+NR2F1+ cell (white arrowhead) are shown; quantification in (J). n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(K-M) TUJ1 (red; differentiating neurons) and GFP (green) IF 7 days after electroporation of control (K) and PX458-αNr2f1 plasmids (L). Co-expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors (NPs) from neurons (Ns), as quantified in (M). Arrowheads in (K,L) point to TUJ1+ GFP+ cells. n ≥ 6 organoids from n = 2 batches. Data information: Nuclei (blue stained with DAPI. the number of positive cells was normalized over the total number of GFP+ cells. In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, C Sucrose density gradient UV-profile after in vitro splitting assays (B) and relative abundance of 80S and subunits as calculated from triplicates and displayed as mean ± SD. (C); SF = splitting factors including Dom34, Hbs1, ABCE1 and eIF6; (+) = 4-fold molar excess of eIF3j; (++) = 20-fold molar excess of eIF3j.",
    "molecules": "Sucrose"
  },
  {
    "caption": "A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively. B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16.",
    "molecules": "PtdIns4P, U18"
  },
  {
    "caption": "K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.",
    "molecules": "PtdInsP2, PtdIns, PtdInsP"
  },
  {
    "caption": "C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions. D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39. ",
    "molecules": "U18"
  },
  {
    "caption": "J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region. K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm. ",
    "molecules": "U18"
  },
  {
    "caption": "C. Left: Confocal images of control (left) and U18-treated (right) tsA201 cells expressing PI4KIIα -GFP. Bottom panels show an enlarged view of the probe centered on a perinuclear region. Right: Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm. Control: n=12; U18: n=14. D. Same as (C) only PI4KIIIβ. Control: n=12; U18: n=15. ",
    "molecules": "U18"
  },
  {
    "caption": "E. Confocal images of control and U18-treated tsA201 cells fixed and stained for PI4KIIα (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of PI4KIIα and TGN46 regions. F. Quantification of PI4KIIα signal in TGN46 regions (PI4KIIα TGN46). Control: n=36; U18: n=36. ",
    "molecules": "U18"
  },
  {
    "caption": "B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.",
    "molecules": "PtdIns4P, U18"
  },
  {
    "caption": "M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.",
    "molecules": "U18"
  },
  {
    "caption": "I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments. J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18 : n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.",
    "molecules": "PtdIns4P, U18"
  },
  {
    "caption": "A. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts expressing P4M-YFP. Black and orange squares show expanded views of PtdIns4P in Golgi and endo/lysosomal compartments respectively.",
    "molecules": "PtdIns4P"
  },
  {
    "caption": "B. Confocal images of tsA201 cells expressing PI4KIIα-GFP. Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. C. Quantification of PI4KIIα-GFP mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. D, E. Same as (B, C) but in tsA-201 cells transfected with PI4KIIIβ-GFP. Control: n=18, U18: n=22, U18+PF: n=23.",
    "molecules": "PF, U18"
  },
  {
    "caption": "Confocal images of tsA201 cells Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. F, G. Same as (B, C) but in tsA-201 cells transfected with P4M-YFP. Control: n=39, U18: n=42, U18+PF: n=32. H, I. Same as (F, G) but in wild-type and SCAP-/- CHO cells. Control: n=12, U18: n=11, SCAP-/- +U18: n=10.",
    "molecules": "PF, U18"
  },
  {
    "caption": "Flow cytometric analysis of splenocytes of mice immunized with cInsulin or control immunization on day 26. Left and middle panel showing germinal center (GC) B cells (GL-7+) pre-gated on B cells (CD19+ B220+). Right panel showing insulin-reactive GC B cells pre-gated on GL-7+ cells in a histogram. Data are representative of two independent experiments with n=4/group.",
    "molecules": "cInsulin, insulin"
  },
  {
    "caption": "Coomassie stained SDS-page showing purified serum IgG of cInsulin immunized (red) and CI mice (grey) under reducing (+ β-ME), and non-reducing conditions. Representative of two independent experiments.",
    "molecules": "Coomassie, cInsulin, β-ME"
  },
  {
    "caption": "Blood glucose levels of intravenously (i.v.) injected wild-type mice at indicated hours post injection. 20 µg of purified serum IgG from cInsulin immunized mice (red, n=6) or CI mice (grey, n=5) was used for injection. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001, ****P < 0.0001.",
    "molecules": "glucose, cInsulin"
  },
  {
    "caption": "Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (red: 0:1, n=10 and green: 100:1, n=5) and control-immunization (CI: CpG only, grey, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, *P < 0.05, ***P < 0.001.",
    "molecules": "Insulin"
  },
  {
    "caption": "Western blot analysis of insulin-specific serum IgG derived from InsA-peptide immunized mice. Top panel (green): 100:1 serum, lower panel (red): 0:1 serum (sInsA:cInsA). Serum of mice was used as primary antibody and detection was done with anti-mouse-IgG-HRP. Black filled arrow: Proinsulin (12 kD), Black non-filled arrow: Insulin (6 kD), β-actin (42 kD, loading control). Representative of two independent experiments.",
    "molecules": "insulin, Insulin, Proinsulin"
  },
  {
    "caption": "Blood glucose levels of InsA-peptide immunized (red: 0:1, n=5 for E and n=10 for F; green: 100:1, n=5 and n=4 for F, d30, d32) and CI (grey, n=5 for E and n=10 for F) mice were assessed at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test (E) and repeated measure ANOVA test (F), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
    "molecules": "glucose"
  },
  {
    "caption": "Urine glucose levels of InsA peptide immunized (red: n=10 for d28, d32 and n=8 for d30; green: n=5 for d28, n=4 for d30, n=3 for d32) and CI (grey, n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. For 100:1: representative data are shown for two independent experiments with total n=10. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001.",
    "molecules": "glucose"
  },
  {
    "caption": "(C, F) Urine glucose levels of InsA peptide immunized (n=3) and CI (n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *P < 0.05. (D, E) Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (day 59, day 78: n=3, day 49: n=7) and control-immunization (CI: CpG only, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test (D) and repeated measure ANOVA test (E). *P < 0.05, **P < 0.01.",
    "molecules": "CpG, glucose, Insulin"
  },
  {
    "caption": "Anti-Insulin affinity of PR-IgM (blue), primary IgM (red) and isotype control (black) measured by bio-layer interferometry. IgM binding to insulin was acquired in pm and used to calculate the dissociation constant shown in the graph (Kd = 1/Ka). Graph is showing antigen-antibody association phase. Data are representative for three independent experiments.",
    "molecules": "Insulin, insulin"
  },
  {
    "caption": "Blood glucose levels of intravenously injected mice with purified IgM from control immunization (CI, n=5), complex InsA peptide (cInsA) immunization day 7 (total IgM d7, n=4) and day 85 (total IgM d85, n=5). Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test showing comparison of red to black line, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
    "molecules": "glucose"
  },
  {
    "caption": "Anti-nuclear-IgM (ANA) of control-immunized (CI, n=3) and complex InsA peptide (cInsA) immunized mice on day 7 (n=3) and day 85 (n=3) analyzed by indirect immunofluorescence. We used (E) total serum (dilutions: 1:20) or (G) Insulin-specific IgM (concentration: 500 ng/mL) purified from serum at indicated days on commercial HEp-2 slides. Scale bar: 10 µm. Green fluorescence indicates IgM bound to nuclear structures. Data are representative for three independent experiments with isotype control: n=3, day 7: n=3, day 85: n=3.",
    "molecules": "Insulin"
  },
  {
    "caption": "Coomassie stained SDS-page showing primary (cInsA d7) and memory (cInsA d85) Insulin-specific purified IgM pre-incubated with Insulin and calf thymus dsDNA. Samples were loaded onto the gel after size exclusion with a cut-off at 10.000 kD (fractions referring to >/< 104 kD). IgM heavy chain (HC): 69 kD, IgM light chain (LC): 25 kD, J-chain: 15 kD. Data shown are representative of three independent experiments.",
    "molecules": "Coomassie, Insulin"
  },
  {
    "caption": "D The effect of increasing levels of 1,2‐dioleoyl‐PS on the ATPase activity of purified ATP8A1 (WT and E191Q)‐CDC50A complex. The Km and Vmax for the WT complex were 59.1 ± 3.5 μM and 38.7 ± 0.8 μmol ATP/min/mg protein, respectively.",
    "molecules": "1,2‐dioleoyl‐PS, ATP"
  },
  {
    "caption": "A The effect of various lipids on the ATPase activity of the purified ATP8A1‐CDC50A complex. The ATPase activity was measured in the presence of 5 mM ATP and 10 mol % of one of the following lipids: 1,2‐dioleoyl‐phosphatidycholine (PC), 1,2‐dioleoyl‐PS, 1,2‐dioleoyl‐PE, 1,2‐dioleoyl‐phosphatidylglycerol (PG), 1,2‐dioleoyl‐phosphatidylinositol (PI), 1,2‐dioleoyl‐phosphatidic acid (PA), sphingomyelin (SM), cholesterol (Chol), or brain polar lipid (BPL) and 90% PC. The activity of the ATP8A1 (E191Q)‐CDC50A in the presence of 1,2‐dioleoyl‐PS was also determined.",
    "molecules": "1,2‐dioleoyl‐PS, ATP, Chol, cholesterol, 1,2‐dioleoyl‐phosphatidic acid, PA, 1,2‐dioleoyl‐phosphatidycholine, PC, 1,2‐dioleoyl‐PE, 1,2‐dioleoyl‐phosphatidylglycerol, PG, BPL, brain polar lipid, 1,2‐dioleoyl‐phosphatidylinositol, PI, sphingomyelin"
  },
  {
    "caption": "B NBD‐labeled PS or PE transport (flippase) activity of ATP8A1 (WT and E191Q)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.",
    "molecules": "NBD, PS, PE"
  },
  {
    "caption": "A His‐tagged EHD1 was mixed with PC‐based liposomes containing the indicated % (mol/mol) of PS or PE. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue.B, C The intensities of individual bands in (A) were quantified with ImageJ, and the percentage of bound proteins was calculated. In (B), the data represent the mean ± SD of n = 3 independent experiments and were analyzed with one‐way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test. *P 0.01; NS, not significant. In (C), each point represents the mean of two independent experiments with an error bar showing the range of values obtained. The sigmoidal fitting was applied.",
    "molecules": "PC, PS, PE"
  },
  {
    "caption": "D, E His‐tagged EHD1 was mixed with PC‐based liposomes (20, 40, or 60% PS) of decreasing size produced by extrusion through filter pores of the size indicated. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue. In (E), the intensities of individual bands were quantified (mean ± SEM of three independent experiments). See Supplementary Fig S8 for the size distributions of the prepared liposomes.",
    "molecules": "PC, PS"
  },
  {
    "caption": "A His‐tagged 2xPH was mixed with liposomes that were composed of 75% (mol/mol) PE, 20% (mol/mol) PC, and 5% (mol/mol) of PS, PA, PI, PIPs, or sulfatide. In the case of PE/PC, liposomes of 80% (mol/mol) PE and 20% (mol/mol) PC were used. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The gels were stained with Coomassie blue.",
    "molecules": "PA, PC, PS, PE, PI"
  },
  {
    "caption": "B The effect of increasing levels of PS on the ATPase activity of ATP8A2 (WT and I376M)‐CDC50A complex.",
    "molecules": "PS"
  },
  {
    "caption": "C NBD‐labeled PS transport (flippase) activity of ATP8A2 (WT and I376M)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.",
    "molecules": "NBD, PS"
  },
  {
    "caption": "D, Representative example of E-Ls generated after i.p. injection of GFP-expressing miiPSCs. H/E, hematoxylin and eosin. The following antigens were detected by immunohistochemistry: Sox2 (ectoderm), Cd34 (mesoderm), Gata4 (endoderm), AFP and CK8 (visceral endoderm of the yolk sac) and Ter119 (nucleated erythroid cells). Scale bars, 500 μm and 100 μm for higher magnifications.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "G, Representative images of EBs derived from miiPSCs that were transiently and simultaneously transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors, and simultaneously treated with Dox to induce miR-203 expression. Scale bars, 500 μm. Plots show the quantification of EB size, percentage of EBs with large cavities, and beating EBs at different time points during differentiation. Data are represented as mean ± s.e.m. (n=3 independent experiments).",
    "molecules": "Dox"
  },
  {
    "caption": "H, The specific differentially methylated regions (DMR) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite-modified DNA. Ten independent clones were sequenced per condition. The quantification of methylated (filled circles) vs. unmethylated (empty circles) CpGs is shown in the histogram in the indicated conditions.",
    "molecules": "bisulphite, methylated"
  },
  {
    "caption": "I, Heatmaps representing the methylation levels at the top Differentially Methylated Regions (DMRs; n=100; upper panel) or the Imprinting Control Regions (ICRs; n=103 different imprinting loci; lower panel) in the indicated samples (referred to panels A,B). Color code and scale of DNA methylation, from 0.0 (red) to 1.0 (green) is applicable to both heatmaps.",
    "molecules": "Methylated"
  },
  {
    "caption": "B, Representative immunofluorescences showing cardiac Troponin T (cTnT, green) and nuclei (Hoechst, blue) staining of in vitro-generated cardiomyocytes derived from wild-type iPSCs transiently transfected with either control mimics, miR-203 mimics or miR-203 mimics + Dnmt3a/b cDNAs. Pictures were taken at day 15 of differentiation. Scale bars, 68 μm. The cTnT-positive area in these cardiomyocytes is shown in the right histogram. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).",
    "molecules": "Hoechst"
  },
  {
    "caption": "B, C. Detection of CST at stalled and active forks with SIRF assay in U2OS (B) and HCT116 (C) cells. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative SIRF images of CTC1, STN1 and PCNA on normal or stalled forks are shown. Scale bars: 10 µm. Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU-SIRF signals. Mean value in each sample is labeled in the graph. N: the number of cells analyzed in each sample.",
    "molecules": "EdU, HU"
  },
  {
    "caption": "CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 3 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. N, the number of cells analyzed in each condition. Relative BrdU fluorescence intensity was quantitated by Image J.",
    "molecules": "BrdU, HU, ssDNA"
  },
  {
    "caption": "DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment.",
    "molecules": "mirin"
  },
  {
    "caption": "DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment.",
    "molecules": "mirin"
  },
  {
    "caption": "DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment.",
    "molecules": "mirin"
  },
  {
    "caption": "ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown.",
    "molecules": "mirin, ssDNA"
  },
  {
    "caption": "(i) Scheme shows the nuclease activity of MRE11 in degrading 5' Cy3-labeled substrates (25 nt + 60 nt with phosphorothioate bonds on both ends). (ii) The 5' Cy3-labeled substrates (80 nM) were incubated with the indicated concentration of MRE11 with or without the Mn2+ cofactor at 37°C for 20 min. Reactions were stopped by SDS and proteinase K and resolved on 27% denatured polyacrylamide gel. (iii) The results are graphed, and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.",
    "molecules": "Cy3, Mn2+, SDS"
  },
  {
    "caption": "DNA binding activity of wild-type CST complex and Δ700N mutant complex. The 5' Cy3- labeled substrates (80 nM) were incubated with the indicated concentrations of CST. Samples were analyzed by 0.8 % agarose gel.",
    "molecules": "Cy3"
  },
  {
    "caption": "MRE11 degradation analysis. 5' Cy3-labeled substrates (80 nM) were pre-incubated with indicated concentrations of CST wild-type (i) or ∆700N mutant variant (ii) at 37°C for 5 min. Reactions were completed by adding MRE11 (200 nM) for an additional 20 min incubation and then stopped by SDS and proteinase K. Samples were resolved in 27% denatured polyacrylamide gel. The results are graphed and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.",
    "molecules": "Cy3, SDS"
  },
  {
    "caption": "Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing vector (V), WT Myc-CTC1 and Myc-∆700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow).Scale bars: 10 μm. Quantification of percent of cells with >3 CTC1/TRF2 colocalization foci were from three independent experiments. In each experiment, >150 cells from each sample were analyzed.",
    "molecules": "HU"
  },
  {
    "caption": "   ∆700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1 or Myc-∆700N, His6- STN1, HA-TEN1 and Flag-RAD51 or vector control (V), and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1.   ",
    "molecules": "HU"
  },
  {
    "caption": "Micronuclei (MN) formation in BRCA2- and STN1-deficient cells. Co-depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1. Representative DAPI staining images are shown. Arrows point to MN. Scale bars: 20 µm. Average percentages of nuclei containing MN from three independent experiments are presented. In each experiment, >300 nuclei were analyzed per sample.",
    "molecules": "DAPI"
  },
  {
    "caption": "Co-depletion of STN1 and BRCA2 significantly impairs DNA replication. Scale bar: 50 µm. Results were from three independent knockdown experiments. In each experiment, >180 nuclei were analyzed per sample.",
    "molecules": "DNA"
  },
  {
    "caption": "Co-depletion of STN1 and BRCA2 increases chromosome instabilities. U2OS cells with siBRCA2 and/or shSTN1 knockdown were treated with HU (2 mM, 3 h). Representative metaphase images show aberrant chromosomes (red arrows). Boxed areas are amplified and shown at the bottom of images. Scale bars: 20 µm. Two independent knockdown and chromosome spread experiments were performed. N, the number of metaphase spreads analyzed in each sample.",
    "molecules": "HU"
  },
  {
    "caption": "RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi-resistant WT but not by ∆700N. Cells were treated with HU (2 mM, 16 h) and stained with anti-RAD51. Representative images are shown. Scale bars: 10 µm. Scatter plot of RAD51 foci number with and without HU treatment is shown. Mean values in each condition are listed at the top of the graph.",
    "molecules": "HU"
  },
  {
    "caption": "A. Raw 264.7 macrophage cells were treated with DMSO (0.05%), 1 μM control antisense oligonucleotide (ASO), or Rab10-directed ASO (1 μM), for four days. Immunoblots are representative of three independent experiments, with similar results obtained. B. Calculated reduction of Rab10 (n=3 biologically independent experiments). Each dot in the plot presents one independent experiment. Group are shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001.",
    "molecules": "DMSO"
  },
  {
    "caption": "C. Raw 264.7 cells were treated with the indicated ASO for four days, or 50 μM EIPA (5-[N-ethyl-N-isopropyl]amiloride) for 20 min, prior to incubation for 60 min with fluorescent (tetramethylrhodamine, TRITC)-labeled dextran (70 kDa, shown as red color), transferrin, or IgG-conjugated agarose beads. Representative photomicrographs (from 30 images analyzed for each condition, from three biologically independent experiments) are shown. Cells were washed, fixed, and stained with DAPI (shown as blue color). Scale bars show 200 μm. D-F. Relative fluorescent signals were calculated as a percent of control-ASO treated cells to indicate (D) dextran or (E) transferrin uptake level. (F) For quantification of phagocytosis, the average number of IgG-agarose beads internalized were calculated as a percent of control-ASO treated cells. 30 images across three independent experiments were quantified for each condition, with each dot in the plot representing results from one image. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P<0.0001, N.S P>0.05. ",
    "molecules": "DAPI, dextran, 5-[N-ethyl-N-isopropyl]amiloride, EIPA, tetramethylrhodamine, TRITC"
  },
  {
    "caption": "G. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male C57BL/6J mice were treated with murine cytokines to polarize cells towards a monocytic lineage (mBMDM), dendritic lineage (mBMDC), or microglia-like lineage (mMicroglia, see Methods section). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. H-J. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the dotted plot representing 1 image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with ****representing P <0.0001, N.S P>0.05. ",
    "molecules": "DAPI, dextran, TRITC"
  },
  {
    "caption": "K. Human monocytes purified from venous blood draws from healthy adult male volunteers were treated with human cytokines towards a monocytic lineage (hMDM), dendritic lineage (hMDC), or microglia-like lineage (hMicroglia). Cells were incubated with TRITC-dextran for 30 min prior to washing, fixing, and staining with DAPI. Representative photomicrographs from 30 images analyzed for each condition from n=3 biologically independent experiments are shown. Scale bars show 200 μm. L-N. Relative fluorescent signals were calculated as a percent of control-ASO treated cells. 30 images across 3 independent experiments were quantified for each condition, with each dot in the plot representing one image quantified. The group means were calculated and shown. Error bars represent ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with **** representing P <0.0001. ",
    "molecules": "DAPI, dextran, TRITC"
  },
  {
    "caption": "A. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain (PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker), or mkate2(N-term)-2xTAPP1-PH domain (PI(3,4)P2 marker). Representative photomicrographs (from 25 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). White bounding boxes show \"High Mag\" panels that magnify representative individual vesicles. The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the \"CoLoc\" panels. The colocalized pixels are shown in white with the same intensity. Scale bars are 10 μm, or 1 μm in \"Hig Mag\" panels and \"CoLoc\" panels. White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker. B. At least 25 cells across three independent experiments were analyzed for each condition (see Materials and Methods section). % of Rab10 vesicles positive with the indicated marker within each cell was quantified. Each dot represents the mean value from one cell analyzed. Group means are given, with error bars representing ± SEM. ",
    "molecules": "PI(3,4,5)P3, PI(3,4)P2, PI3P, PI(4,5)P2"
  },
  {
    "caption": "C. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5)P3 marker) and treated with wortmannin (1 μM) for one hour. Representative photomicrographs (from 25 cells analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). Scale bars are 10 μm, or 1 μm in \"Hig Mag\" panel. D. Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from >30 cells across five independent experiments. Each dot on the plot represents mean values from one experiment. Error bars represent ± SEM and significance was assessed by Mann-Whitney test (owing to non-normal distributions) with ** indicating P<0.01. ",
    "molecules": "PI(3,4,5)P3, wortmannin"
  },
  {
    "caption": "Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (A) TRITC-dextran (70 kDa, shown as red signal) Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in \"High Mag\" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as \"CoLoc.\" Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in \"Hig Mag\" panels and \"CoLoc\" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.",
    "molecules": "dextran, GTP, TRITC"
  },
  {
    "caption": "Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). 24-hours later, cells were incubated with (B) DQ-ovalbumin, for 30 min before washing and fixation. Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in \"High Mag\" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as \"CoLoc.\" Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in \"Hig Mag\" panels and \"CoLoc\" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.",
    "molecules": "GTP"
  },
  {
    "caption": "Raw264.7 cells were transfected with plasmids expressing eGFP(N-term)-WT-Rab10 or Q68L-Rab10 (GTP-locked). (C, D) Cells were further stained for (C) EEA1, or (D) Lamp1, which was detected with Cy5 dye (show as red signal). Representative photomicrographs are from 25 cells each analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are magnified in \"High Mag\" panels that show individual vesicles. Positive co-localization signal (see Methods) is indicated in bounding boxes labeled as \"CoLoc.\" Colocalized pixels are shown in white. Scale bars are 10 μm, or 1 μm in \"Hig Mag\" panels and \"CoLoc\" panels. White arrow heads indicate Rab10 vesicles colocalized with the indicated marker.",
    "molecules": "Cy5, GTP"
  },
  {
    "caption": "A. Raw 264.7 macrophage cells were transfected with Flag(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain(PI(3,4,5)P3 marker), or eGFP(C-term)-SARA-Fyve domain((PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5)P2 marker, epifluorescence, shown in blue) followed by anti-Flag immunofluorescence (shown in green) and pT73-Rab10 antibody (shown in red). To evaluate colocalization of phospho-Rab10 with early endosomes or late endosome/lysosome markers, immunofluorescence detection was performed for endogenous EEA1 or Lamp1 (shown in blue). Raw 264.7 macrophage cells were further fed 0.05 mg mL-1 of 70 kDa TRITC-conjugated dextran (epifluorescence, shown in blue) or 0.05 mg mL-1 BODIPY conjugated ovalbumin for 30min prior to immunofluorescence analysis. Representative images were selected from 25 cells analyzed for each condition from n=3 biologically independent experiments. White bounding boxes are \"High Mag\" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for \"High Mag\". Positive colocalization signal (depicted as white) in the \"CoLoc (pRab10/Rab10)' and 'CoLoc (pRab10/Marker)' columns are indicated. White arrow heads highlight vesicles scored as co-positive between pT73-Rab10 and Rab10 or the indicated marker. B. 25 cells across three independent experiments were analyzed for each condition, with each dot representing the mean of the vesicle analysis across individual cells. The group means of %pT73-Rab10 vesicles positive for the indicated marker are given, with error bars showing ± SEM. ",
    "molecules": "PI(3,4,5)P3, BODIPY, dextran, PI3P, PI(4,5)P2, TRITC"
  },
  {
    "caption": "A. Raw264.7 cells were transfected with Flag(N-term)-LRRK2 and eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells with eGFP(N-term)-Rab10 epifluorescence (shown as green signal), and immuno-stained for pT73-Rab10 (shown as red signal) and LRRK2 (shown as blue signal). White bounding boxes are magnified in \"High Mag\" panels that show individual vesicles. Scale bars represent 10 μm or 1 μm in \"Hig Mag\" panels.",
    "molecules": "MLi2"
  },
  {
    "caption": "B. Raw 264.7 macrophage cells transfected with eGFP(N-term)-Rab10 and Flag(N-term)-LRRK2 were sequentially lysed mechanically into buffer to create a 'soluble' protein fraction, and then insoluble material lysed into triton X-100 buffer to create a \"triton x-100\" fraction. The \"soluble\" and triton X-100 buffer solubilized fractions were analyzed using SDS-PAGE (TGX gels), and representative immunoblots from n=3 independent experiments are shown.",
    "molecules": "triton X-100, triton x-100"
  },
  {
    "caption": "C. The "triton X-100" fraction was used as input for LRRK2 immunoprecipitation, with subsequent detection of total Rab10 and pT73-Rab10 proteins in LRRK2:Rab10 immunocomplexes using SDS-PAGE (TGX gels). To measure the stoichiometry of pT73-Rab10 to total Rab10, the same immunocomplexes were analyzed using phos-tag SDS-PAGE method, with unphosphorylated eGFP(N-term)-Rab10 migrating below the phospho-Rab10 protein. Immunoblots are representative of three independent experiments, with similar results obtained.",
    "molecules": "triton X-100"
  },
  {
    "caption": "A,B. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were pre-incubated with FITC-conjugated dextran (70 kDa, green signal) prior to washing, fixing, and immunostaining with total-Rab10 (shown as red signal), or pT73-Rab10 antibody (shown as red signal). Alternatively, cells were co-stained with antibodies to Rab5, Rab7 or Lamp1 (shown as green signal in separate images). Representative photomicrographs (from >30 images analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are \"High Mag\" panels that show individual vesicles analyzed. Positive colocalization signal (depicted as white) in \"CoLoc\" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker. Scale bars are 10 μm, or 1 μm in \"Hig Mag\" panels and \"CoLoc\" panels.",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "C. Analysis of 20 cells across three independent experiments are given for each indicated co-localization, where % of Rab10 or pT73-Rab10 vesicles positive with indicated markers was calculated for each cell (with signal inside of vesicles analyzed for TRITC-dextran and DQ-ovalbumin). Each dot represents the mean value of vesicles analyzed inside one cell. The group means of % Rab10 or pT73-Rab10 vesicles positive with indicated markers were calculated. Error bars represent ± SEM. Significance was assessed by Mann-Whitney tests for each marker with **** representing p<0.0001 and N.S representing p>0.05.",
    "molecules": "dextran, TRITC"
  },
  {
    "caption": "D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-LRRK2 mBAC transgenic mice were treated with 0.001% DMSO or 100 nM MLi2 (i.e., IC90 concentration) for two hours prior to immunostaining with total Rab10 or pT73-Rab10 antibody (shown as red signal), and anti-Rab5 or anti-Rab7antibody (shown as green signal). Representative photomicrographs (from 20 cells analyzed for each condition from n=3 biologically independent experiments) are shown. White bounding boxes are \"High Mag\" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for \"High Mag\". Positive colocalization signal (depicted as white) in \"CoLoc\" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 and the indicated marker.",
    "molecules": "DMSO, MLi2"
  },
  {
    "caption": "F. Raw 264.7 macrophage cells were transfected with eGFP(N-term)-Rab10 for 24 hours and treated with or without the LRRK2 kinase inhibitor MLi2 (100 nM) for two hours. Representative photomicrographs (from 25 images analyzed for each condition across n=3 biologically independent experiments) are shown with eGFP(N-term)-Rab10 epifluorescence (shown as green signal) and pT73-Rab10 (shown as red signal), with EEA1 (shown as blue signal). White bounding boxes are \"High Mag\" panels that show individual vesicles analyzed. Scale bars represent 10 μm and 1 μm for \"High Mag\". Positive colocalization signal (depicted as white) in \"CoLoc\" panels are given with white arrow heads highlighting vesicles scored as co-positive between pT73-Rab10 Rab10 and the indicated marker. G. Calculated percentage of colocalized vesicles, with each dot representing mean vesicle frequency in each cell analyzed. The group mean of % Rab10 vesicles positive with EEA1 were calculated, and error bars represent ± SEM. Significance was assessed by Mann-Whitney test for each marker with *** representing P<0.005. ",
    "molecules": "MLi2"
  },
  {
    "caption": "A. Raw264.7 cells were transfected with Flag-Rab10 or control empty vector. The transfected cells were lysed in the presence of 2 mM GTPγS or 2 mM GDP. The Flag-Rab10 complex was immunoprecipitated with Flag-resin followed by extensive washing and elution of complexes with 200 μg mL-1 3xFlag-tide. Eluates were analyzed by SDS-PAGE followed by immuno-blotting with anti-Flag-HRP antibody.",
    "molecules": "GDP, GTPγS"
  },
  {
    "caption": "C. Flag(N-term)-Rab10 protein was purified from HEK293-FT cells transfected with Flag(N-term)-Rab10 plasmids and immobilized on Flag-resin in lysis buffer containing 2 mM GTPγS, or 2 mM GDP, as indicated. Beads were mixed with lysates from cells transfected with Myc-mkate2(N-term)-EHBP1L1 to generate Rab10:EHBP1L1 complexes. Total Rab10 on the beads was detected using anti-Flag antibody, and EHBP1L1 immobilized to total Rab10 detected with an anti-Myc antibody. Representative immunoblots from n=3 biologically independent experiments are shown. D. Quantification of relative EHBP1L1 pulled down. Data are from n=3 biologically independent experiments. Error bars represent ± SEM... ",
    "molecules": "GDP, GTPγS"
  },
  {
    "caption": "E. HEK293-FT cells were transfected with Flag(N-term)-Rab10 and Myc(N-term)-LRRK2R1441C to induce Rab10 phosphorylation. Membrane bound Flag(N-term)-Rab10 protein was extracted with Flag-resin to generate 'pRab10 beads,' or extracted from cells treated with MLi2 (100 nM) to generate 'Rab10 beads.' Beads were mixed with lysates from HEK293-FT cells transfected with Myc-mkate2(N-term)-EHBP1L1 to form on-bead Rab10:EHBP1L1 complexes. The total Rab10 and pT73-Rab10 on the beads was detected using anti-Flag antibody and pT73-Rab10 antibody. The EHBP1L1 in the lysates and immobilized on the beads was detected using anti-Myc tag antibody. Representative immunoblots are shown. F. The relative EHBP1L1 pulled down, normalized to total Rab10 protein on-bead, was determined from n=3 biologically independent experiments. Error bars represent ± SEM. Each dot represents one experiment, with significance assessed by one-way ANOVA with **** representing Tukey's post hoc test P<0.0001. ",
    "molecules": "MLi2"
  },
  {
    "caption": "F. BMDM cells were treated with 0.004% DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa-594 conjugated rat anti-mouse CD11b antibody on ice for ten minutes. Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding (shown in red as indicated). Six wells of cells from each condition were further incubated at 37°C to allow rapid internalization of CD11b-antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0.5% acetic acid on ice for one min. Three of the six wells were fixed and surface-stained with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes remaining on the cell surface (shown in green as indicated). Internalized CD11b-antibody complexes are shown in red. The other three wells of cells were further incubated at 37°C again for ten min to allow recycling of internalized CD11b-antibody complex followed by surface staining with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes recycled back to the surface. Representative images from n=3 biologically independent experiments are shown. Scale bars represent 30 μm. G-I. (G) The initial surface CD11b, or (H) internalized CD11b, or (I) recycled CD11b were calculated from >20 images analyzed for each condition from n=3 biologically independent experiments. Each dot represents the mean value from one experiment. Error bars show ± SEM with significance assessed by Mann-Whitney test. * represents P<0.05. ",
    "molecules": "acetic acid, Alexa-488, Alexa-594, DMSO, MLi2"
  },
  {
    "caption": "A. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were treated with DMSO, 1 μM Rab10 anti-sense oligonucleotide for 4 days, 100 nM MLi2 for 12 hours, as indicated, before stimulation with 100 nM CCL5 for the indicated time (hours). Representative immunoblots from n=3 biologically independent experiments are shown. B. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Data shown are group means ± SEM from n=3 biologically independent replicates, with significance assessed by one-way ANOVA with Tukey's post hoc test with treated groups compared to matched control groups, with ** representing P< 0.01. ",
    "molecules": "DMSO, MLi2"
  },
  {
    "caption": "C. BMDM cells, as described in panel (A), were additionally treated with DMSO (control), EIPA, or Akti-1/2(Akt1/2 inhibitor) as indicated, for 1 hour after CCL5 stimulation. Representative immunoblots from n=3 biologically independent experiments are shown. D. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Each dot represents one experiment, with error bars showing ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P< 0.0005, ****P< 0.0001. ",
    "molecules": "Akti-1/2, DMSO, EIPA"
  },
  {
    "caption": "E. BMDM cells, as described in panels (A and C), were seeded in Transwell (Sigma) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers. After two hours, the cells failing to migrate through the matrices were removed with a cotton swab. Migrated cells were counted with DAPI images, with representative images shown at low magnification. Representative images from n=3 biologically independent experiments are shown. F. Relative chemotaxis activity was calculated as fold of naïve cells that migrated without any added CCL5. Each dot represents the mean of images collected from one experiment. Data are group means ± SEM, with significance assessed by multiple t test., with* representing P<0.05. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) smFISH targeting Bmal1, Cry1 and Nr1d1 in wild-type NIH 3T3 cells at 17h, 29h and 25h after synchronisation with Dex, respectively. Nuclei are stained with DAPI (blue). Each fluorescent dot (white) corresponds to a single transcript. Segmented cell boundaries are delineated in grey. The blue and white channels represent maximum z-projections. Scale bar: 20μm.",
    "molecules": "DAPI, Dex"
  },
  {
    "caption": "(B) Number of Bmal1, Cry1, and Nr1d1 transcripts per cell as a function of time after treatment with Dex. These data combine all smFISH hybridizations from the 4h sampled Nr1d1/Cry1 and Bmal1/Cry1 experiment. Each dot shows the average over one replicate from an independent slide (Methods). The solid lines represent fits using a two-harmonic cosinor model (Equation (1), Methods) for each gene individually.",
    "molecules": "Dex"
  },
  {
    "caption": "(D) Distributions of Nr1d1, Cry1 and Bmal1 transcripts at 21h, 29h and 37h after synchronisation with Dex. μ represents the mean of the distribution, CV represent the coefficient of variation (standard deviation / mean), and N is the number of cells at the given time point. Total number of cells analysed for the Nr1d1/Cry1 pair: 21h - 449; 25h - 414; 29h - 521; 33h - 463; 37h - 477; 41h - 429. Total number of cells analysed for the Bmal1/Cry1 pair: 17h - 465; 21h - 490; 25h - 504; 29h - 436; 33h - 407; 37h - 454; 41h - 404.",
    "molecules": "Dex"
  },
  {
    "caption": "(B) Top image: Dual-channel smFISH targeting Bmal1 and Cry1 simultaneously, taken at 17h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Bmal1 transcripts; red, Cry1 transcripts. Bottom image: Dual-channel smFISH targeting Nr1d1 and Cry1 simultaneously, taken at 25h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Nr1d1 transcripts; red, Cry1 transcripts. Scale bar: 20μm.",
    "molecules": "DAPI, Dex"
  },
  {
    "caption": "(C) Bivariate distributions of mRNA counts per cell for dual-channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1. Each dot corresponds to a single cell, and the contours represent KDE estimates of the density. Time represents the number of hours after Dex synchronisation, and R represents the Pearson correlation coefficient.",
    "molecules": "Dex"
  },
  {
    "caption": "PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15-40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section.",
    "molecules": "formaldehyde, peptides, protein, proteins"
  },
  {
    "caption": "PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1-6, Rpn1-3, Rpn5, 7-9, 11-13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel).",
    "molecules": "protein, proteins"
  },
  {
    "caption": "PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1-α7, β1-β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.",
    "molecules": "proteins"
  },
  {
    "caption": "Protein correlation profiling (PCP) analysis using the median profile of the PA28αβ regulator as the reference profileProfiles of the PA28α, PA28β, and the β2i proteins (blue, red and green lines, respectively).",
    "molecules": "proteins"
  },
  {
    "caption": "Plot of the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1.A zoom of the graph in (B) is represented and χ2 coordinates for PA28α, PA28β, and β2i proteins are highlighted as blue, red, and green dots, respectively. Light gray dots represent the χ2 coordinates of the proteins quantified in all the fractions of the gradient. The median profile of the PA28α and PA28β subunits was used as the reference profile for the calculation of the χ2 values.",
    "molecules": "proteins"
  },
  {
    "caption": "A Proteasome complexes were immunopurified from nine formaldehyde-crosslinked human cell lines and analyzed by nano-LC-MS/MS. Protein abundance indexes (PAIs) were used to represent the abundance of proteins in purified proteasome samples. The correlation between two different proteins was quantified using coefficients of determination (R2).",
    "molecules": "formaldehyde, proteins"
  },
  {
    "caption": "For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI (n = 3).",
    "molecules": "protein"
  },
  {
    "caption": "A) Seahorse experiment measuring oxygen consumption rate (OCR) in MCF7 cells upon addition of oligomycin, FCCP and a mixture of antimycin and rotenone. Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values were indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.",
    "molecules": "antimycin, FCCP, oligomycin, rotenone"
  },
  {
    "caption": "C) 13C5 -Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate, malate and citrate in MCF7 cells. Data are presented as mean ± SD of triplicate samples. Isotopologues are represented as M+n where M indicates the mass and n equals number of 13C incorporated",
    "molecules": "13C, 13C5, citrate, fumarate, Glutamine, malate, TCA"
  },
  {
    "caption": "D) Seahorse experiment measuring extracellular acidification rate (ECAR) in MCF7 cells upon addition of glucose, oligomycin and 2-deoxyglucose (2DG). Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.",
    "molecules": "2-deoxyglucose, 2DG, glucose, oligomycin"
  },
  {
    "caption": "F) Normalized lactate concentration upon PYCR1 KO. Data represents mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.",
    "molecules": "lactate"
  },
  {
    "caption": "E-H) Bar plots show percentage of viable cells after treatment with 0.75 mM H2O2 (E), 0.06 µM paclitaxel (F), 0.15 µM doxorubicin (G), 2.5 mM cyclophosphamide (H). Data represent mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05.",
    "molecules": "cyclophosphamide, doxorubicin, H2O2, paclitaxel"
  },
  {
    "caption": "I) Tumor volume measurements for 26 days in MCF7 injected NSG mice. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) are shown. Data represents mean ± SE. Pax: Paclitaxel, Dox: Doxorubicin. Groups were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *p < 0.05, ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.",
    "molecules": "Dox, Doxorubicin, Paclitaxel, Pax"
  },
  {
    "caption": "(E) ATG38-2GFP ATG17-2mCherry cells in wild-type and atg14Δ were cultured at 30°C with or without rapamycin. After 180 min, cells were analyzed by fluorescence microscopy. Bar, 5 µm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(B) Cells with the indicated genotypes were grown in YPD and treated with rapamycin for 3 h at 30°C. Total protein was separated by SDS-PAGE and detected by immunoblotting with anti-API.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Wild-type and atg38Δ cells expressing the indicated GFP fusion proteins and Ape1-mRFP were cultured at 30°C in the presence of rapamycin. After 60 min, cells were analyzed by fluorescence microscopy. Of the total pool of Ape1-mRFP signal, the percentage of Ape1-mRFP colocalizing with GFP was calculated. This percentage is shown as mean ± SD of two independent experiments, with the number of cells counted indicated at the bottom of the graph. (B) Examples of wild-type and atg38Δ cells expressing either Atg14-3 × GFP or Vps34-3 × GFP in A. Bar, 5 µm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(D) Cells expressing Vps34-6HA, Vps30-9myc, Atg38-3FLAG, and Atg14-TAP were lysed in a buffer containing 150 mM NaCl. The Atg14-TAP immunoprecipitate with IgG-Dynabeads from this lysate was split into eight fractions and washed with lysis buffer containing the indicated concentrations of NaCl. The resulting precipitates were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods, although a 150-mM NaCl sample was used as a control and normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.",
    "molecules": "NaCl"
  },
  {
    "caption": "(A) AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.",
    "molecules": "Ade, His, Leu, Trp"
  },
  {
    "caption": "(B) Interactions between the indicated fusion proteins were analyzed as in A. AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.",
    "molecules": "Ade, His, Leu, Trp"
  },
  {
    "caption": "(A) Interactions between the indicated fusion proteins were analyzed as in Fig. 4 A. AH109cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.",
    "molecules": "Ade, His, Leu, Trp"
  },
  {
    "caption": "(A) Chromatin features mapped here displayed differences between CLL (patient CLL1) and an NBC donor (H7) as shown for the TCF4 locus, which encodes for a transcription factor from the E protein family. Based on the increased H3K4me1, H3K27ac and ATAC signal, two predicted enhancer loci were marked that became active in CLL. Note that the y-axis for RNA-seq is scaled differently for CLL (8000) and NBCs (100) to visualize that the TCF4 gene was not completely silenced but lowly expressed also in NBCs as evident also from the H3K36me3 mark. Light grey depicts active chromatin region and dark grey the confined enhancer locus coinciding with an open chromatin region.",
    "molecules": "H3K36me3, H3K27ac"
  },
  {
    "caption": "(A) Left, example of a large PMD on chromosome 2 derived from a consensus of CLL samples (n=11). Right, genome-wide quantification of PMDs across CLL samples (n=11) and NBCs (n=6). Red, methylated DNA; blue, unmethylated DNA.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Correlation function of H3K4me3 ChIP-seq reads. A broadening of H3K4me3 domains in CLL by 1-2 nucleosomes was detected. Error bars represent the SEM.",
    "molecules": "H3K4me3"
  },
  {
    "caption": "(C) Distribution of nucleosome occupancy calculated from histone H3 ChIP-seq averaged over a 1000 bp window within promoters. H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples.",
    "molecules": "H3K4me3"
  },
  {
    "caption": "(H) HDAC activity and its inhibition by panobinostat in B cells from CLL patients (red) in comparison to healthy donors (grey). Error bars indicate standard deviation measured in four biological replicates.",
    "molecules": "panobinostat"
  },
  {
    "caption": "(I) Genome browser view of H3K27ac tracks (in grey) at exemplary genes for NBCs and CLL cells 24 hours after mock and after panobinostat treatment. At genes such as CDKN1A (cell cycle control) and KLF13, reduced H3K27ac signal in CLL was increased upon HDAC inhibition to the level found in NBCs. WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat.",
    "molecules": "H3K27ac, panobinostat"
  },
  {
    "caption": "(J) Heatmap displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h. A general gain of H3K27ac in enhancers upon panobinostat treatment was observed.",
    "molecules": "H3K27ac, panobinostat"
  },
  {
    "caption": "(B) H3K27ac (left) and DNA methylation (right) at NFAT binding sites. CLL cells showed both an H3K27ac enrichment and DNA-hypomethylation at NFAT target sites, suggesting a higher activity of TFs from the NFAT family in CLL.",
    "molecules": "DNA, H3K27ac"
  },
  {
    "caption": "A. ATPase activity of ABCC3  wild-type (WT) and E1451Q in nanodiscs. The data points for WT and E1451Q are fitted with a Michaelis-Menten equation. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.",
    "molecules": "nanodiscs"
  },
  {
    "caption": "B. Substrate-stimulated ATPase activities of ABCC3  WT and E1451Q upon addition of substrate E217βG or DHEAS. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.",
    "molecules": "DHEAS, E217βG"
  },
  {
    "caption": "C. Relative  ATPase activities of ABCC3  WT and mutants in the presence of 2 mM ATP  upon addition of E217βG. The relative activity represents the substrate-stimulated activity of ABCC3  WT or its mutants harboring mutation of residues at the substrate-binding pocket. For (C) the activities of ABCC3  WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.",
    "molecules": "ATP, E217βG"
  },
  {
    "caption": "D. Transport [3H]-E217βG activity assays of ABCC3  WT and mutants. For (D), the activities of ABCC3  WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.",
    "molecules": "E217βG, 3H"
  },
  {
    "caption": "C. Relative  ATPase activities of ABCC3  WT and mutants in nanodiscs  upon addition of DHEAS  and 2 mM ATP. The relative activity represents the substrate-stimulated activity of ABCC3  WT or mutants harboring mutation of residues at the substrate-binding pocket.",
    "molecules": "nanodiscs, ATP, DHEAS"
  },
  {
    "caption": "E. Relative ATPase activities  of ABCC3  WT and mutants K318A, T535A upon addition of E217βG. For (C) and (E), the activities of ABCC3  WT are set as 100%. One-way ANOVA is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.",
    "molecules": "E217βG"
  },
  {
    "caption": "(G) Immunofluorescence  detected subcellular localization  of SMIM26 (green) with mitochondria, F-actin, ER  and Golgi. Nuclei  were stained  with DAPI  (blue), and the colocalization  between red (mitochondria, F-actin, ER, Golgi) and green (SMIM26) was measured. Scale bar, 10 μm. The colocalization  of SMIM26  with mitochondria, F-actin, ER  and Golgi  in cells was measured by fluorescence intensity analysis with ImageJ software. The red line of statistical analysis centers on the  co-location of SMIM26  and indicate cell organelle.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H, I) Mitochondria  were purified and SMIM26  was enriched in mitochondria  fractions (H), SMIM26  is a mitochondrial outer membrane  protein as detected by proteinase K and Triton X-100  assayin HEK293T  cells (I).",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(E) Immunofluorescence staining  of F-actin  (red) in ACHN  and 769-P cells transfected with indicated constructs. Nuclei  were stained  with DAPI  (blue). The intensity of F-actin fluorescence was statistically plotted. Unpaired two-tailed Student's t-test, **P < 0.01, n.s, nonsignificant. Data are shown as mean ± SEM of three biological replicates. Scale bar, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Co-localization of SMIM26  (green) with SLC25A11  (red) in ACHN  was determined by immunofluorescence. Nuclei  were stained  with DAPI  (blue). Scale bar, 10 μm. The red line of statistical analysis centers on the co-location  of SMIM26  and SLC25A11.",
    "molecules": "DAPI"
  },
  {
    "caption": "A) Upper: Overview of the AB tetramer, with each of the monomers depicted in a different colour. The front monomer has been removed for clarity. The black box indicates the region expanded in the lower panel. Lower: An expanded view of the selectivity filter, showing only two monomers as pink and white ribbon cartoons with the selectivity filter residues drawn as sticks in the corresponding colours. Sodium ions are shown as cyan spheres and well-ordered waters as red spheres (at 0.4x their actual radii, for clarity). The final refined 2Fo-Fc electron density maps for the protein, ions and water are shown at 2.0 σ in dark grey mesh and 0.8 σ in light grey , and the initial difference map (without sodiums or waters included in the phase calculation) at 3.0 σ in green mesh.B) Superposition of the sodium ion binding sites found in the crystallographically-distinct AB and CD tetramers and the proposed binding sites from other studies. The tetramers were superposed with the LSQKAB program (Kabsch, 2010) from CCP4 (Winn et al., 2011) using residues 131-220 from all four monomers in each tetramer. Only two monomers from the AB tetramer are shown for clarity, and coloured as in panel A. Sodium ions from the AB tetramer are depicted as cyan spheres, those from the CD tetramer as teal spheres. Predicted sodium ion binding sites from Ulmschneider et al. (2013) are indicated as small black spheres and labelled on the right of the spheres according to the nomenclature (S0 to S4) in that study. Predicted sodium ion binding sites from Payendeh et al. (2011), based on a closed channel structure of the close orthologue NavAb, are indicated on the far right and labelled as per the nomenclature of that study (SiteHFS, SiteCEN and SiteIN). This and subsequent molecular figures were drawn with PyMol (The PyMOL Molecular Graphics System, Version 1.6, Schrödinger, LLC) unless otherwise stated.",
    "molecules": "sodium, Sodium"
  },
  {
    "caption": "A) Top: Crystal structure overview of the wildtype AB tetramer (PDBID 5ZBZ), view as in Figure 2A.Left: Difference (Fo-Fc) electron density map (mutant minus wildtype) contoured at -2.5σ and drawn as red mesh, plotted on the structure of the wildtype (E178) protein, highlighting the consequences of the loss of the δ-carbon atom from residue 178 in the E178D mutant. For reference, the locations of the sodium ion sites present in the wildtype (E178) structure are depicted as cyan spheres. Both the structure and the ions are displayed as faded backgrounds so that the difference map can be seen more clearly.Right: Final refined 2Fo-Fc electron density map for the E178D mutant (PDBID 4X88), contoured at 1.5σ and shown as grey mesh, with the sodium ions depicted as cyan spheres.B) Surface representations of (left) wildtype NavMs pore and (right) the E178D mutant coloured by electrostatic charge calculated by APBS (Callenberg et al, 2010) and drawn with VMD (Humphrey et al., 1996), showing a slice through the middle of the tetramer. Sodium ions are shown as cyan spheres; only 2 are present in the mutant, instead of the 3 seen in the wildtype, with the top sodium missing in the mutant. The lower panels are expanded views of the selectivity filter regions, with the E178 side chains depicted in yellow sticks and the location of the main chain nitrogen of residue 179 indicated by the black circle. The alteration in the protein structure caused by removal of the methylene group of E178 exposes a positive region of the polypeptide backbone adjacent to the site where the top sodium ion is located in the wildtype protein; this likely contributes to why an equivalent sodium ion is not seen adjacent to this position in the mutant.",
    "molecules": "Sodium, sodium"
  },
  {
    "caption": "Left: Sodium ions (cyan) in the selectivity filter of the NavMs sodium channel. Only two monomers (in white and pink) are shown for clarity. Coordinates are from the AB tetramer of PDBID 5BZB.Right: Potassium ions (blue) in the selectivity filter of the KcsA potassium channel. As for the NavMs structure, only two monomers (in grey and maroon) are shown for clarity. Coordinates are from PDBID 1K4C (Zhou et al., 2004).The two crystal structures were first superimposed using SSM (Krissinel and Henrick, 2004), and are drawn using the same scale, and with the correct ratio between the diameters of the ions used, so that the differences in the internal dimensions of their selectivity filters can be seen, thus making it clear how much more space there is available in the NavMs SF for intervening water molecules. In the KcsA channel, the ions are directly coordinated to the polypeptide backbone atoms, whereas in the NavMs SF, the closest atoms (side chains) are obviously at longer distances than direct bonding to the ions, enabling interactions via water molecules.",
    "molecules": "potassium, Potassium, Sodium, sodium, water"
  },
  {
    "caption": "A Representative immunoblots of the indicated proteins in lysates from HeLa  cells treated with indicated concentrations of halofuginone  (HF) for 5 h. B Quantification of ATF4  and P-eIF2α from experiments as in (A). Data are mean ± SD (n = 3 biological replicates). *p ≤ 0.0414, **p ≤ 0.0019, ****p < 0.0001, as determined by one-way ANOVA with Dunnett's multiple comparison test. Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "halofuginone, HF"
  },
  {
    "caption": "C Similar to (A), but with HeLa  cells treated with indicated concentrations of tunicamycin  for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "D Similar to (A), but using lysates from HeLa cells treated with 62.5 nM and 312.5 nM HF  for indicated times. Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "HF"
  },
  {
    "caption": "A Representative immunoblots  of indicated proteins in lysates from mouse embryonic fibroblasts  (MEFs) of indicated genotype after treatment with 12.5 nM or 200 nM HF  for 5 h. Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "HF"
  },
  {
    "caption": "B Similar to (A), but using HeLa  cells untreated or treated with GCN2  siRNA 48 h before treatment with indicated concentrations of HF  for 5 h. C Similar to (A), but with high HF  concentrations. Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "HF"
  },
  {
    "caption": "B Representative immunoblots  of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF. C Similar to (B), but using HeLa  cells untreated or treated with GCN2  siRNA 48 h before treatment with indicated concentrations of HF. Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "HF"
  },
  {
    "caption": "D Representative immunoblots  of lysates from HeLa  cells treated with indicated concentrations of HF  for 5 h with or without the Nedd8-activating enzyme inhibitor MLN4924  (1 μM). Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "HF, MLN4924"
  },
  {
    "caption": "E. Representative immunoblots  of lysates from HeLa  cells treated with indicated concentrations of HF  for 5 h with or without proteasome  inhibitor MG-132 (10 μM). Data information: Representative results of at least three independent experiments are shown in each panel.",
    "molecules": "HF, MG-132"
  },
  {
    "caption": "A Newly synthesized proteins pulse-labeled  with 35S-methionine  for 10 min in HeLa  cells pre-treated with indicated compounds for 2.5h, except (E) (Bottom)  Coomassie-stained  gel.",
    "molecules": "methionine, 35S"
  },
  {
    "caption": "B Newly synthesized proteins in HeLa  cells treated with 2.5 μg/ml Tm  or 50 μg/ml cycloheximide  (CHX).",
    "molecules": "CHX, cycloheximide, Tm"
  },
  {
    "caption": "C Newly synthesized proteins in 4KO or 4KO+GCN2 MEF  cells treated with 12.5 or 200 nM HF.",
    "molecules": "HF"
  },
  {
    "caption": "D Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF  cells treated with 12.5 or 200 nM HF.",
    "molecules": "HF"
  },
  {
    "caption": "E Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h.",
    "molecules": "Tm"
  },
  {
    "caption": "F Newly synthesized proteins in HeLa  cells untreated or treated with GCN2  siRNA 48 h before treatment with indicated concentrations of HF.",
    "molecules": "HF"
  },
  {
    "caption": "G Newly synthesized proteins in HeLa  cells treated with indicated concentrations of HF  with or without proline  supplementation (10 mM).",
    "molecules": "HF, proline"
  },
  {
    "caption": "H Representative immunoblots  of indicated proteins in lysates from HeLa  cells after treatment with indicated concentrations of HF  with or without proline  supplementation (10 mM).",
    "molecules": "HF, proline"
  },
  {
    "caption": "I Newly synthesized proteins in HeLa  cells untreated or treated with GCN2  siRNA 48 h before treatment with indicated concentrations of histidinol (His) or borrelidin  (Bor).",
    "molecules": "Bor, borrelidin, His, histidinol"
  },
  {
    "caption": "G, H Analysis of  IC50 values of HF  on confluency at 72h endpoint of  H441 (G) and H23  (H) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory  dose-response (IC50). Each point represents mean (n = 3 biological replicates) ± SD.",
    "molecules": "HF"
  },
  {
    "caption": "A Representative immunofluorescence for  PH3 (green), combined with DAPI staining (white), of E14.5 wildtype (WT) and 11B mouse dorsolateral neocortex  rostrally. B Representative  immunofluorescence for pVim (yellow), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (50 µm x 65 µm) in (B) indicate areas that are shown at higher magnification in (B'). Arrow, pVim+ BP with basal process (mitotic bRG); notched arrowheads, basal process; arrowhead, pVim+ BP without basal process (mitotic bIP). C-E Quantification of PH3+ APs (C), PH3+  BPs (D) and pVim+ bRG (E left) and  bIPs (E right) in a 200 µm-wide field of E14.5 WT (white) and 11B   (black) mouse dorsolateral neocortex rostrally. Note the difference in ordinate scales in (E). F Data information: Images are single optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0269 (D); P = 0.0302 (E, left); P = 0.0139 (E, right)",
    "molecules": "DAPI"
  },
  {
    "caption": "F Representative immunofluorescence for  Pax6 (cyan) and Tbr2 (magenta), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (25 µm x 25 µm) in (F) indicate areas that are shown at higher magnification in (F'). Outlined by dashed white lines, Pax6+ & Tbr2+ BP; outlined by solid white lines, Pax6+ & Tbr2- BP. G-K Quantification of Pax6+ & Tbr2- cells in VZ  (G), Pax6+ & Tbr2+ cells in VZ  (H), Pax6+ & Tbr2- cells in SVZ and IZ (I),  Pax6+ & Tbr2+ cells in SVZ and IZ (J) and  Pax6- & Tbr2+ cells in SVZ and  IZ  (K) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. L Data information:  Images are Z projections of 4 optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0281 (I); P = 0.0088 (J); P = 0.0014 (K)",
    "molecules": "DAPI"
  },
  {
    "caption": "L-N, Sequential  BrdU-EdU labelling.  BrdU was injected into pregnant mice  at E13.5, i.e. 24 hr before sacrifice at E14.5, and EdU was injected into the same pregnant mice 0.5 hr before sacrifice at E14.5. (L) Representative immunofluorescence for BrdU (magenta), combined with  EdU staining (cyan) and DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. (M, N) Quantification of BrdU+ & EdU+ cells (M) and the percentage of BrdU+ cells that are  BrdU+ & EdU+ (N) in VZ  and SVZ  in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Data information: Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0049 (M, SVZ); P = 0.0323 (N, SVZ).  ",
    "molecules": "BrdU, EdU, DAPI"
  },
  {
    "caption": "H Representative  immunofluorescence for Tbr1 (cyan), Ctip2 (magenta) and Satb2 (green), combined with DAPI staining (white), of E18.5 WT and 11B mouse neocortex at the position where cortical thickness was measured (A, red lines). I Quantification of zone thickness of E18.5 WT (white) and 11B (black) mouse neocortex  at the position where cortical thickness was measured (A, red lines). J Data information:  Images are single optical sections Scale bar, 20 µm Data are the mean of 7 (WT) and 8-12 (11B) littermate embryos, which were derived from 4 separate litters. Error bars indicate SD. Two-way ANOVA, followed by Bonferroni's multiple comparisons test (B-D); two-tailed unpaired Student's t-test P = 0.0287 (I, IZ); P = 0.0039",
    "molecules": "DAPI"
  },
  {
    "caption": "G, H Representative immunofluorescence for Ctip2 (magenta), Satb2 (green) and Brn2 (yellow), combined with DAPI staining (white), of P56 adult WT and 11B mouse neocortex  at the position where cortical thickness was measured (D, red lines). Data information: Images are single optical sections Scale bar, 20 µm",
    "molecules": "DAPI"
  },
  {
    "caption": " (F) Endothelial cell proliferation analyzed by BrdU incorporation (red) of ERG positive (blue) endothelial nuclei. ",
    "molecules": "BrdU"
  },
  {
    "caption": " (A) Proliferation of WT and EVL-/- MLEC was assessed by BrdU incorporation. The number of BrdU positive cells (red) was normalized to the total number of cells (white). Eight fields of view per condition and genotype for each of 5 independent WT and EVL-/- cell batches; error bars represent SEM; **p<0.01, ***p<0.001, #p=0.171 non-significant vs. Basal EVL-/-, one-way ANOVA with Bonferroni's multi comparison test; scale bar = 50 µm. ",
    "molecules": "BrdU"
  },
  {
    "caption": " B. Rif1-∆C594 retains function to control DNA replication. Growth of a cdc7-1 rif1-∆C594 mutant was compared with growth of cdc7-1 RIF1 and cdc7-1 rif1∆ strains at 23ºC (permissive temperature for cdc7-1 allele), 26ºC (mild restrictive temperature) and 30ºC (strict restrictive temperature) ",
    "molecules": "DNA"
  },
  {
    "caption": " A. Specimen overview of Chromosome VI-left region showing results of ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for Chromosome VI coordinates 1-80,000. Y-axis shows enrichment values (linear scale, range is 0 to 3.5). Values below 1 are shown in grey, and values above 1 (i.e. sequences enriched in ChIP samples) are coloured blue (Rif1) and red (Rif1-∆C594). Plots show ChIP analysis results from cells arrested by α-factor (G1), released from α-factor at 16ºC for 60 min and 90 min, or released from α-factor into 0.2 M HU for 60 min at 23ºC ",
    "molecules": "α-factor, HU"
  },
  {
    "caption": " A. ChIP-Seq analysis of Rif1 and Rif1-∆C594 proteins shows tRNA gene and origin binding, with widened peaks at early origins ARS606 and ARS607 in HU block. Plots show Chromosome VI genome coordinates 160,000- 210,000. Plot colours here and in following Figures are as in Fig 2B. Widened peaks are not observed in unperturbed S phase samples, or for Rif1-∆C594 ",
    "molecules": "HU"
  },
  {
    "caption": " B. Association of Rif1-∆C594 at replication origins is enhanced in HU block. ChIP enrichment of Rif1 and Rif1-∆C594 around early origin ARS1426 (left) and late origin ARS1412 (right) ",
    "molecules": "HU"
  },
  {
    "caption": " A. ChIP-qPCR confirmation of Rif1 and Rif1-∆C594 association with replication origins. ChIP was performed using cells arrested in α-factor (open bars) or HU (grey bars), and qPCR analysis performed for early origin ARS1426 (left) and late origin ARS1412 (right). Values shown are 'normalised ChIP efficiencies' obtained by subtracting the value obtained at a control locus (see Materials and Methods). Bars indicate the averages of two biological replicates, with values from each replicate shown by open circles ",
    "molecules": "α-factor, HU"
  },
  {
    "caption": " B. Numbers of early and late origins associated with Rif1 and Rif1-∆C594 peaks. Plot showing numbers of early and late origins bound by Rif1 and Rif1-∆C594 in G1 and HU-blocked cells, based on peak-calling results ",
    "molecules": "HU"
  },
  {
    "caption": " C. Replication timing programme is intact in rif1-∆C594 cells. Replication of selected origins at an HU block analysed by BrdU incorporation. Cells were synchronised by α-factor  and released into the medium containing 0.2 M HU and 1.13 mM BrdU. Plots show the percentage of total ARS607, ARS422.5, or ARS1412 DNA pulled down by IP with anti-BrdU, calculated from two biological replicate samples. Bars indicate the average of two biological replicates and open circles the values from each replicate. Insets in ARS422,5 and ARS1412 panels show the same data with Y-axis scales adjusted to 0 - 0.03% ",
    "molecules": "α-factor, BrdU, HU"
  },
  {
    "caption": "b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells.",
    "molecules": "rapamycin"
  },
  {
    "caption": "c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.",
    "molecules": "rapamycin"
  },
  {
    "caption": "Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a)",
    "molecules": "Rapamycin"
  },
  {
    "caption": "Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b).",
    "molecules": "Rapamycin"
  },
  {
    "caption": "VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d).",
    "molecules": "rapamycin, VPA"
  },
  {
    "caption": "e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.",
    "molecules": "chloroquine, CQ"
  },
  {
    "caption": "f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux.",
    "molecules": "CQ"
  },
  {
    "caption": "h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.",
    "molecules": "rapamycin"
  },
  {
    "caption": "a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta).",
    "molecules": "rapamycin, VPA"
  },
  {
    "caption": "b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux.",
    "molecules": "rapamycin, VPA"
  },
  {
    "caption": "c, Co-treatment with VPA and rapamycin led to increased cell death.",
    "molecules": "rapamycin, VPA"
  },
  {
    "caption": "d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death.",
    "molecules": "CQ, rapamycin, VPA"
  },
  {
    "caption": "e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment.",
    "molecules": "Ex527, rapamycin"
  },
  {
    "caption": "Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death.",
    "molecules": "3-methyladenine, 3MA, BafA, bafilomycin A1, rapamycin, VPA"
  },
  {
    "caption": "Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).",
    "molecules": "amino-acid, CQ, VPA"
  },
  {
    "caption": "(A) Quantification of ss-eGFP-FKBPF36M trafficking in HeLa(M)-C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry-BspF (mCherry-BspF). Rapamycin was added to initiate secretory traffic of ss-eGFP-FKBPF36M and its colocalization with calnexin (ER), ERGIC-53 (ERGIC), GM130  (Golgi), p230  (TGN), or secretory vesicles (SV) was scored over a 60 min time course. Data are means ± SD from n=3 independent experiments. Asterisks indicate statistically significant differences between mCherry- and mCherry-BspF-expressing cells as determined by a two-way ANOVA with Sidak's multiple comparisons test (P<0.05).",
    "molecules": "Rapamycin"
  },
  {
    "caption": "(F) Representative confocal fluorescence micrographs of HeLa  cells co-transfected for 24 h to produce mCherry-BspF and either GFP-Rab6a, GFP-Rab8a or  Arf6-GFP and treated with  Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). (G) Quantification of  localization of GFP-Rab6a, GFP-Rab8a  and  Arf6-GFP on mCherry-BspF-labelled  tubules in transfected HeLa  cells. Data are means ± SD from n=3 independent experiments, in which at least 300 individual cells per experiment were analyzed. ",
    "molecules": "Cytochalasin D"
  },
  {
    "caption": "(A) Representative confocal fluorescence micrographs of HeLa  cells transfected for 24 h to produce either  mCherry or mCherry-BspF  (grayscale panels), incubated on ice with AlexaFluor™488-Cholera Toxin subunit B  (CTxB; green) and shifted to 37˚C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti-GM130 antibody; purple). CTxB accumulation within Golgi  structures appears white in overlays. Scale bars: 10 and 2 µm (insets). (B) Quantification of  CTxB transport to the Golgi apparatus in HeLa  cells producing either mCherry, mCherry-BspF or HA-BspF over a 30 min time course, expressed as percentages of cells in which  CTxB colocalized  with the GM130 Golgi marker, as in (A). Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared to mCherry-producing cells as determined by a two-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( ",
    "molecules": "AlexaFluor™488"
  },
  {
    "caption": "(D) Quantification of CTxB  transport to the Golgi apparatus in BMMs following siRNA-mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a) or Rab6a/a' (siRab6a/a') after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (E) Quantification of  CTxB transport to the Golgi apparatus in BMMs that were either mock-infected or infected with wild type (2308), ΔbspF, complemented ∆bspF (ΔbspF::bspF), ∆bspB  or complemented ∆bspB (ΔbspB::bspB) bacteria for 24 h, incubated for 30 min with  AlexaFluor™488-CTxB  on ice for  binding  followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to mock-infected cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( ",
    "molecules": "AlexaFluor™488"
  },
  {
    "caption": "(B) Representative co-immunoprecipitations of HA-BspF  and myc-ACAP1 in HeLa  cells. HeLa  cells were transfected to either co-produce or individually produce HA-BspF  and myc-ACAP1  and either  HA-BspF or myc-ACAP1 were immunoprecipitated  using either anti-HA-conjugated (upper panel) or anti-myc-conjugated (lower panel) magnetic beads following cross-linking (+DSP) or not (-DSP) with dithiobis[succinimidylpropionate]. Input lysates (10% of the post-nuclear  supernatant) and co-immunoprecipitates  were separated by SDS-PAGE and probed for HA-BspF and myc-ACAP1 by Western blotting.",
    "molecules": "dithiobis[succinimidylpropionate], DSP"
  },
  {
    "caption": "(C) Representative confocal  micrographs of HeLa  cells transfected to produce mCherry-BspF and GFP-ACAP1 and treated with Cytochalasin D  (200 nM) for 30 min and quantification of colocalization between mCherry-BspF and GFP-ACAP1. Arrows indicate areas of BspF and ACAP1 colocalization. Scale bars: 10 µm and 2 µm (insets). Data are means ± SD from n=3 independent experiments in which 10 cells were analyzed per experiment. Pearson's correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc_2 plug-in.",
    "molecules": "Cytochalasin D"
  },
  {
    "caption": "Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion.",
    "molecules": "doxycycline"
  },
  {
    "caption": "The CRPC cell line 22Rv1 was cultured in 2% CSS media and was treated for 72 hours with vehicle (DMSO), doxorubicin (DOX), N9-isopropylolomoucine (N-9), or the combination of DOX and N-9 at different concentrations. Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each. The data were analyzed using Compusyn software, and a normalized isobologram was built. The table shows the Combination Index (CI) for the different drug combinations. CI=1 represents additivity, CI<1 synergism, and CI>1 antagonistic effects.",
    "molecules": "DMSO, DOX, doxorubicin, N-9, N9-isopropylolomoucine"
  },
  {
    "caption": "The non-tumorigenic prostate epithelial cell line RWPE1, the AR-null PC cell line PC3, the androgen dependent cell line LNCaP, and the CRPC cell lines C4-2B and 22Rv1 were treated for 72 hours with vehicle (DMSO), DOX (100 ng/mL [184 nM]), N-9 (200 ng/mL [613 nM]), or the combination of DOX (100 ng/mL [184 nM]) and N-9 (200 ng/mL [613 nM]). C4-2B and 22Rv1 cells were kept in 10% CSS media, and the other cell lines in 10% FBS. Cell confluence was monitored using the Incucyte Zoom System. Data represent two independent experiments, with four to six replicates each, showing the mean ± s.e.m., and normalized to vehicle controls (Kruskal-Wallis test, P value <0.0001, two-tailed). *Significant at a p value < 0.05, ** p value < 0.01, *** p value < 0.001.",
    "molecules": "androgen, DMSO, DOX, N-9"
  },
  {
    "caption": "The non-tumorigenic prostate cell line RWPE-1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle (DMSO) or the combination of DOX and N-9 at 100 ng/mL and N-9 200 ng/mL, respectively. Cell confluence was monitored using the Incucyte Zoom System.",
    "molecules": "DMSO, DOX, N-9"
  },
  {
    "caption": "Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.",
    "molecules": "Torin1"
  },
  {
    "caption": "Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.",
    "molecules": "Torin1"
  },
  {
    "caption": "Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.",
    "molecules": "rapamycin, Torin1"
  },
  {
    "caption": "Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.",
    "molecules": "Torin1"
  },
  {
    "caption": "Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.",
    "molecules": "Torin1"
  },
  {
    "caption": "Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.",
    "molecules": "rapamycin, Torin1"
  },
  {
    "caption": "KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m.",
    "molecules": "xestospongin B, Torin1"
  },
  {
    "caption": "KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 0.00023 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 Tor1); *P = 0.00045 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 xesto); #P = 3.08e−6 (red dots, KO ctrl vs. KO Tor1); ##P = 6.73e−5 (yellow dots, KO+KRIT1 ctrl vs. KO ctrl). The data are expressed as the mean ± s.e.m. of four independent experiments.",
    "molecules": "xestospongin B, Torin1"
  },
  {
    "caption": "Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments.",
    "molecules": "Rapa, rapamycin, Tor1, Torin1"
  },
  {
    "caption": "Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), and actin in KRIT1-KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h. The results are representative of three independent experiments.",
    "molecules": "rapamycin, Torin1"
  },
  {
    "caption": "Formation of capillary-like structures measured by tube formation assays. HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h. Representative phase-contrast microscopy (Scale bar, 100 μm) and calcein-fluorescent (Scale bar, 50 μm) images were reported. All data are presented as percentage ± s.e.m from three different experiments performed in duplicate. *P = 1.29e−11.",
    "molecules": "calcein"
  },
  {
    "caption": "Immunoblot analysis of p62, LC3I/II, and rapamycin in CCM3 wt and CCM3-KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.",
    "molecules": "rapamycin, Torin1"
  },
  {
    "caption": "B) Replication of S. Typhimurium in Atg5-/- MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 (10 nM) or DMSO. Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation (p.i.) following cell lysis.",
    "molecules": "MRT68843, DMSO"
  },
  {
    "caption": "A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:LC3B (A). Where indicated, Wortmannin (Wort) was added at 100 nM.",
    "molecules": "Wort, Wortmannin"
  },
  {
    "caption": "A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with the indicated GFP:WIPI alleles (B). Where indicated, Wortmannin (Wort) was added at 100 nM.",
    "molecules": "Wort, Wortmannin"
  },
  {
    "caption": "A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:DFCP1 (FYVE* denotes a PI(3)P-binding mutant of DFCP1) (D-E). Where indicated, Wortmannin (Wort) was added at 100 nM.",
    "molecules": "PI(3)P, Wort, Wortmannin"
  },
  {
    "caption": "A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:ML1N*2, a probe for PI(3,5)P2 (G). Where indicated, Wortmannin (Wort) was added at 100 nM.",
    "molecules": "PI(3,5)P2, Wort, Wortmannin"
  },
  {
    "caption": "Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded.",
    "molecules": "IPTG"
  },
  {
    "caption": "Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).",
    "molecules": "IPTG"
  },
  {
    "caption": "Transcription (bottom) and translation (top) profiles for lacZ. Profiles are shown for cells in the absence and presence of IPTG (1 mM). Position of genetic parts and gene is shown below the profiles. RBS is omitted from the genetic design due to its size. (E) Measured promoter strength in RNAP/s units, RBS initiation rate in ribosomes/s units, and the transcriptional terminator and translation termination efficiency for lacZ. Data shown for cells in the absence and presence of IPTG (1 mM).",
    "molecules": "IPTG"
  },
  {
    "caption": "Translation profiles for the PK-LacZ construct in cells cultured in the absence (bottom) and presence (top) of IPTG (1 mM). The gene10, middle, and lacZ regions are labelled above the profiles. Shaded region denotes the PK, and dashed lines denote the start codon and stop codons of gene10 and LacZ.",
    "molecules": "IPTG"
  },
  {
    "caption": "Fraction of the total RPFs and mRNA reads in each reading frame for the gene10, PK or middle, and lacZ regions schematically shown below and are of the PK-LacZ construct. Data shown separately for cells cultured in the absence and presence of IPTG (1 mM).",
    "molecules": "IPTG"
  },
  {
    "caption": "Change in expression of chromosomal genes in E. coli cells following induction of PK-lacZ expression (1 mM IPTG). Each point represents a transcript. Differentially expressed genes (mRNA count: P < 0.001 and absolute log2 fold-change > 1.37; translation efficiency: P < 0.01) are highlighted in color and by an alternative point shape (transcriptional regulation: purple cross; translational regulation: orange open circle).",
    "molecules": "IPTG"
  },
  {
    "caption": "Translation initiation rates for all E. coli RBSs in cells harboring the LacZ and PK-LacZ constructs in the absence and presence of IPTG (1 mM). Solid line shows the same initiation rate for both conditions. Dotted lines denote linear regressions for the data with no offset.",
    "molecules": "IPTG"
  },
  {
    "caption": "Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).",
    "molecules": "IPTG"
  },
  {
    "caption": "SDS-PAGE analysis of the binding of Strep-tagged REC114 variants (using a single Strep-tag (WSHPQFEK)) to co-expressed His-MEI41-127 after purification on Strep-Tactin resin. SDS-PAGE analysis of the binding of His-MBP-REC114226-254 to co-expressed His-MEI4 variants after purification on Amylose resin.",
    "molecules": "Amylose, Strep"
  },
  {
    "caption": "Native MS spectrum of co-purified Strep-REC114 and MEI41-127. The main complexes are Strep-REC114:MEI41-127 in ratios 2:1 and 4:2. There are also free Strep-REC114 and 1:1 Strep-REC114-MEI4 complex.",
    "molecules": "Strep"
  },
  {
    "caption": "Sedimentation velocity profiles of the REC114226-254-MEI41-43 complex obtained at 280 nm at three concentrations (0.2 mg/ml, 1 mg/ml and 4.5 mg/ml) in a solution containing 20 mM Tris pH 8.0 and 200 mM NaCl. Expected sedimentation coefficient for the 2:1 complex is 1.13S and for 4:2 is 1.58S. The sedimentation coefficient of the main peak increases with increasing protein concentration: s=1.2S at 0.2 mg/ml, s=1.44S at 1 mg/ml and s=1.64S at 4 mg/ml. These results suggest a fast exchange between the two forms.",
    "molecules": "NaCl, Tris"
  },
  {
    "caption": "E. Pull-down experiments of full-length Strep-tagged REC114 mutants indicated above the lanes with His-MBP-MEI41-43. All proteins were first purified by affinity chromatography and gel filtration. A total of 0.9% of the input (lanes 1-6) and 1% of the eluates (lanes 7-12) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. Control lanes 1-2 and 7-8 show inputs­ and elutions of REC114 and MEI4 alone, respectively. The MEI4 sample contains also free MBP tag. The REC114 sample also contains a degradation product, likely corresponding to its PH domain.",
    "molecules": "coomassie brilliant blue, Strep"
  },
  {
    "caption": "F. Pull-down experiments of Strep-tagged REC114 with His-MBP-MEI41-43 mutants indicated above the lanes. All proteins were first purified by affinity chromatography and gel filtration. A total of 0.9% of the input (lanes 1-4) and 1% of the eluates (lanes 5-8) were analyzed on 15% SDS-PAGE gels stained with coomassie brilliant blue. The MEI4 sample contains also free MBP tag. The REC114 sample also contains a degradation product, likely corresponding to its PH domain.",
    "molecules": "coomassie brilliant blue, Strep"
  },
  {
    "caption": "Superose 6 gel filtration elution profile of His-MBP-IHO11-281 mixed with Strep-REC114-His-MEI41-127. Overlay with individual elution profiles of His-MBP-IHO11-281 and the Strep-REC114-His-MEI41-127 complex",
    "molecules": "Strep"
  },
  {
    "caption": "Superose 6 gel filtration elution profile of His-MBP-IHO124-281 mixed with Strep-REC114-His-MEI41-127, showing lack of binding. Overlay with individual elution profiles of His-MBP-IHO124-281 and the Strep-REC114-His-MEI41-127 complex",
    "molecules": "Strep"
  },
  {
    "caption": "ITC measurement of the interaction affinity between of His-MBP-IHO11-281 and Strep-REC114-His-MEI41-127. ITC measurement of the interaction affinity between of His-MBP-IHO124-281 and Strep-REC114-His-MEI41-127 ITC measurement of the interaction affinity between of His-MBP-IHO11-23 and Strep-REC114-His-MEI41-127.",
    "molecules": "Strep"
  },
  {
    "caption": "ITC measurement of the interaction affinity between of His-MBP-IHO11-23 and the L104D mutant of Strep-REC1141-159.",
    "molecules": "Strep"
  },
  {
    "caption": "SDS-PAGE analysis of fractions 1-13 of Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (upper panel); mixture of His-MBP-ANKRD311808-1857 with Strep-REC114-His-MEI41-127 (middle panel) and His-MBP-IHO11-281 mixed with Strep-REC114 and His-MEI41-127 (lower panel). L indicates input sample loaded onto the column. The red frame highlights the complex of IHO1 bound to REC114 and MEI4 (lower panel) and the lack of this complex in presence of ANKRD31 (upper panel). Orange square shows equivalent elution of ANKRD31, REC114 and MEI4 in presence or absence of IHO1.",
    "molecules": "Strep"
  },
  {
    "caption": "Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (orange); His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (blue) and His-MBP-IHO11-281 mixed with Strep-REC114 -His-MEI41-127 (green). Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (orange); His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (blue) and His-MBP-IHO11-281 (green).",
    "molecules": "Strep"
  },
  {
    "caption": "(B, C) Purified wild-type and RING mutated His-RNF114 were stained by Coomassie Blue (B). Purified wild-type and RING mutated His-RNF114 were subjected to ubiquitination assays together with E1, Ubc4, FLAG-Ub, and ATP. The results show that mutant RNF114 lacked ubiquitination activity (C).",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Mouse zygotes were microinjected with sterile ddH2O (control group) or Ralgps1, Cd74, Tnip1, Psat1, or Tab1 mRNA. During 96-100 h culturing, the percentages of embryos at various stages were counted under microscopy. The results show that overexpression of Tab1 impaired the early embryonic development. Three independent experiment replicates were performed, error bars represent s.d.; *:P<0.05, ***:P<0.001 in unpaired two-tailed t-test.",
    "molecules": "ddH2O"
  },
  {
    "caption": "(C) NF-κB pathway inhibition using the specific inhibitor Bay11-7082 resulted in two cell stage arrest. Three independent experiment replicates were performed, error bars represent s.d.; ***:P<0.001 in unpaired two-tailed t-test.",
    "molecules": "Bay11-7082"
  },
  {
    "caption": "(left) representative photomicrographs of H&E stained tibialis anterior muscle sections from control and iNOS KO mice injected with or without LPS. Scale bars = 100 μm. (right) frequency histogram showing the distribution of muscle fiber minimum feret diameter in the tibialis anterior muscles from saline or LPS treated (top) WT control and (bottom) iNOS KO mice (n = 4). A total of 300 fibers per muscle were used for the analysis. Statistical comparisons, mean, and standard deviation of the mean are shown in the histogram legend.",
    "molecules": "LPS, saline"
  },
  {
    "caption": "(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle. (right) Quantification of the pAMPK to AMPK ratio. Ratios are expressed relative to the saline treated controls.",
    "molecules": "Thr, saline"
  },
  {
    "caption": "Relative concentrations of metabolites involved in glycolysis and TCA cycle. Relative concentration of arginine.",
    "molecules": "arginine"
  },
  {
    "caption": "(left) Western blot analysis of pThr172-AMPK (pAMPK) and total AMPK (AMPK) in quadriceps muscle (n = 3). (right) Quantification of the pAMPK to AMPK ratio relative to the saline treated control (n = 3).",
    "molecules": "Thr, saline"
  },
  {
    "caption": "Representative immunofluorescence imaging for myoglobin and myosin heavy chain (MyHC) in not treated (NT) controls and IFNγ/TNFα (IT) samples treated with or without GW. Nuclei were visualized with DAPI staining (n=4).",
    "molecules": "DAPI, GW"
  },
  {
    "caption": "Western blot analysis for pThr172-AMPK (pAMPK), total AMPK (AMPK), pSer79-ACC (pACC), and total ACC (ACC).",
    "molecules": "Ser, Thr"
  },
  {
    "caption": "Western blot analysis for pThr389-S6K (pS6K), total S6K (S6K), pSer235/236-S6 (pS6), and total S6 (S6) (n=5).",
    "molecules": "Ser, Thr"
  },
  {
    "caption": "Male C57BL/6 wildtype (WT) and iNOS knockout (KO) mice were intraperitoneally injected with 1mg kg-1 LPS or an equivalent volume of carrier solution. Control WT, Control KO, and LPS-treated KO cohorts were pair-fed (PF) to the WT LPS-treated cohorts. After 18h, mice were euthanized, and tibialis anterior muscles were imaged by transmission electron microscopy. (left) Representative micrograph of tibialis anterior muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria. Scale bar = 0.5 µm.",
    "molecules": "LPS"
  },
  {
    "caption": "Male BALB/C mice were injected subcutaneously with C26 cells (1.25x106 cells) or an equivalent volume of saline. After 5 days, and everyday thereafter, saline and C26 injected mice were treated with or without GW (5mg kg-1). After 16 days, mice were euthanized and gastrocnemius muscles were imaged by transmission electron microscopy. (left) Representative micrograph of gastrocnemius muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria mitochondria. Scale bar = 0.5 µm.",
    "molecules": "GW, saline"
  },
  {
    "caption": "E | SARS-CoV-2-infection manifests histological changes in patients' lungs as demonstrated by H&E staining. 1)For comparison, the normal lung of an elderly individual is shown, containing air-filled empty looking alveolar spaces and thin alveolar septae/gas-exchange membranes. Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested. 2) Diffuse (proliferative) alveolar damage showing evidence of cell death. An alveolar space filled with desquamated pneumocytes and macrophages, lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II. The still recognizable epithelial lining is detached. Cells and surfactant are lost creating perforations in the alveolar wall, allowing migration of blood cells and fluid to enter inside the alveolar space. The adjacent interstitial space is significantly widened, showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes. On the top and the lower right, dilated and congested capillaries can be seen (Hematoxylin and Eosin (H&E) stain, 200x). Abbreviations: a- Alveolar structure destroyed by lymphocytes, desquamated epithelium, as well as extravasated blood; The alveoli should be empty, but here it is filled with a combination of degenerated cells and fibrin; b- Next alveoli; c- Expanded pulmonary interstitium. The interstitium should be as thin as 10um, but here it is 100um; d- Extravasation of erythrocytes; e- Lymphocytes; f- Congested capillaries and arterioles; g- Multinucleated alveolocyte/pneumocyte type II; h- Detached epithelium; i- A histiocyte with a kidney-shaped nucleus at the bottom of the circle has ingested erythrocytes (left-handed), representing the first step of cellular elimination. Round inlet with encircled karyopyknotic, karyorrhectic and \"ghost-cell\" figures indicative of apoptosis. 3) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining. The adjacent interstitial space shows analogous changes to 2A1. At the bottom, there is a prominently dilated and congested capillary (H&E, 200x). Abbreviations: a- Alveoli with detached epithelium; b- Next alveoli; c- E Extravasation of erythrocytes; d- Expanded interstitium; e- Lymphocytes; f- Multinucleated pneumocyte type II. 4) Diffuse alveolar damage showing massive extravasation of fibrin (homogeneous eosinophilic material in the center of the alveolar space). The lining pneumocytes are almost all apoptotic/necrotic. The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris (H&E, 200x). Abbreviations: a- Alveola; b- Congested capillaries; c- Fibrin; d- Remnants of degenerated / dying epithelium; e- Expanded interstitium; f- Cellular debris consisting of macrophages, detached epithelial cells and lymphocytes; g- Fully degenerated / lacking epithelial coverage within the alveoli. Round inlet with encircled karyopyknotic, karyorrhectic and \"ghost-cell\" figures indicative of apoptosis. 5) Immunohistochemical stain (IHC) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes/lung interstitium leading to obstruction of the microcirculation (IHC for fibrin, 200x). Abbreviations: a- All small alveolar septal capillaries are filled out with worm-like fibrin thrombi hampering / obstructing circulation; b- Normal interstitium.",
    "molecules": "Eosin, fibrin, Fibrin, Hematoxylin"
  },
  {
    "caption": "D | Elevated hFwe-Lose expression in the nasal swab samples associates with patients' condition severity and respective medical treatment. Box plots illustrate an increased expression of hFwe-Lose in nasal swabs of patients, who were hospitalized within 14 days of disease progression (n = 177), admitted to intensive care unit (ICU) (n = 34), underwent intubation (n = 58), had respiratory rate greater than 30 (GT30) (n = 76), had blood oxygenation level (SpO2) less than 94% (n = 147), and who died within 30 days of disease progression (n = 21) versus patients without respective conditions. Pairwise two-sided Student's t-tests were performed (compared to patients without respective conditions), and p-values are presented on the plot. The vertical axis represents hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.",
    "molecules": "O2"
  },
  {
    "caption": "(A-E) ChIP-seq experiments indicates the localization  of (A) ASCL1, (B) NEUROD1 and (E) RNAPII. ATAC-seq (C) and (D) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t=0 (blue) and t=4h (green) after  lurbinectedin  treatment; -2.0 and +2.0 kb to TSS coordinates of all hg19 genes.",
    "molecules": "lurbinectedin"
  },
  {
    "caption": "(A-D)  Chip-seq  genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1  and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red).",
    "molecules": "lurbinectedin"
  },
  {
    "caption": "(a)  Western blot  showing RNAPII  across the panel of;  A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526  and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin. α-Tubulin  (α-Tub) is shown as loading control; lower panel, Histogram showing the average of RNAPII before (dark blue) and after (light Blue) lurbinectedin treatment in arbitrary units (a.u.) normalized against α-Tub. Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák's multiple comparisons test (n=3 biological replicas). ****P ≤0.0001.",
    "molecules": "lurbinectedin"
  },
  {
    "caption": "(B  Immunofluorescence of  RNAPII (red) before (Ctrl) and after lurbinectedin  treatment.  DAPI  (blue) was used to stain nuclei; scale bar is 65 μm.",
    "molecules": "DAPI, lurbinectedin"
  },
  {
    "caption": "(D)  Affinity purification of Bio-lur  fractions containing elongating RNAPII  Ser2 (lane 4). Input (lanes 1-2) represents 10% of the starting material. Respective molecular weights are shown on the right (kDa).",
    "molecules": "Bio, lur"
  },
  {
    "caption": "(E) Upper panel: GST-pulldown of ubiquitinated  proteins showing hypo- (IIA), -hyper (II0)  phosphorylated RNAPII (lanes 4-5 upper panel) and  Ser2 Phosphorylated RNAPII (Ser2P) (lanes 4-5 lower panel) after lurbinectedin treatment. Empty beads (Mock) (lane 3) as a negative control; Lower Panel: GST-pulldown  of  ubiquitinated proteins Ser2P) (lanes 4-5). Representative smear of successful  ubiquitinated proteins  pulldown (Lanes 4 and 5) as compared to input (lanes 1 and 2). Empty beads (Mock) (lane 3) as a negative control. Each  Western blot is representative of three independent experiments.",
    "molecules": "lurbinectedin, Ser"
  },
  {
    "caption": "(B)  BMDM-polarized M2 macrophages  were transfected with Cy3-labelled CRNDE and  exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, MFC  cells were incubated with Cy3-CRNDE-EXO. Left: Confocal microscopy was used to detect the  Cy3-labelled  CRNDE in MFC cells. The  nuclei were  stained  with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. MFC cells incubated with the control  EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.",
    "molecules": "Cy3, DAPI"
  },
  {
    "caption": "(D) Monocyte-polarized  M2 macrophages were transfected with Cy3-labelled  CRNDE  and  exosomes  were isolated from the medium (Cy3-CRNDE-EXO). Then, SGC7901 cells were incubated with  Cy3-CRNDE-EXO. Confocal microscopy  was used to detect the  Cy3-labelled CRNDE in SGC7901 cells. The  nuclei were stained  with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. SGC7901 cells incubated with the control  EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.",
    "molecules": "Cy3, DAPI"
  },
  {
    "caption": "(E) SGC7901  cells were incubated with chlorpromazine (CPZ, 50 μM),  genistein (GEN, 200 μM), 5-(n-ethyl-n-isopropyl)-amiloride (EIPA, 100 μM), or methyl-β-cyclodextrin (MβCD, 10 mM), following by the incubation of Cy3-CRNDE-EXO.  Confocal microscopy was used to detect the  Cy3-labelled  CRNDE in SGC7901  cells. The nuclei were stained with  DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.",
    "molecules": "chlorpromazine, CPZ, Cy3, DAPI, 5-(n-ethyl-n-isopropyl)-amiloride, EIPA, GEN, genistein, methyl-β-cyclodextrin, MβCD"
  },
  {
    "caption": "(F) SGC7901 cells were incubated with 0, 25, 50, 100 μM CPZ, following by the incubation of  Cy3-CRNDE-EXO.  Confocal microscopy was used to detect the Cy3-labelled  CRNDE  in SGC7901 cells. The nuclei were  stained with DAPI. Right: Flow cytometry  was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.",
    "molecules": "CPZ, Cy3, DAPI"
  },
  {
    "caption": "Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized  M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). Then, MFC cells were treated with indicated exosomes  and cisplatin (CDDP; 1μg/ml) for 48 h. (C) Representative images of colony formation assay performed on MFC cells.",
    "molecules": "CDDP, cisplatin"
  },
  {
    "caption": "Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages  (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and  monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). Then, SGC7901 cells were treated with indicated exosomes and CDDP (1μg/ml). (G) cell proliferation  were measured.",
    "molecules": "CDDP"
  },
  {
    "caption": "3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or  M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (A) Growth curves of MFC  subcutaneous homograft tumors (The homograft tumor sizes were recorded from the day of CDDP  treatment). *P<0.05, **P<0.01 vs. BMDMs-exo; #P<0.05, ##P<0.01 vs. M2-si-control-exo. Data information: Data are expressed as mean ± SD. one-way ANOVA.",
    "molecules": "CDDP"
  },
  {
    "caption": "3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of  mice  (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice  were sacrificed and the homograft tumors were collected. (D) LncRNA CRNDE expression in homograft tumors of  mice. **P<0.01. Data information: Data are expressed as mean ± SD. : one-way ANOVA.",
    "molecules": "CDDP"
  },
  {
    "caption": "(C) SGC7901 cells were transfected with pcDNA-CRNDE or its negative control (Mock) before MG132 treatment (10 µM, 6 h). The Co-immunoprecipitation (Co-IP) assay was performed to examine the combination of PTEN and NEDD4-1.",
    "molecules": "MG132"
  },
  {
    "caption": "(E) The  expressions of PTEN and  NEDD4-1 were measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock at four time-points after cycloheximide (CHX, 100 μg/mL) treatment. GAPDH  was served as an internal control. Left: Representative bands. Right: Quantitative results. The results shown are from three biological replicates and are expressed as mean ± SD. Student's t-test. *P<0.05, **P<0.01 vs. Mock.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Normal, si-control-transfected, or si-PTEN-transfected  SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. (A) The protein levels of  PTEN, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), p-glycoprotein (p-gp), B-cell lymphoma 2 (Bcl-2) were measured. GAPDH was served as an internal control.",
    "molecules": "CDDP"
  },
  {
    "caption": "Normal, si-control-transfected, or si-PTEN-transfected  SGC7901 cells were treated with CDDP  (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. The cell apoptosis in SGC7901  cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.",
    "molecules": "CDDP"
  },
  {
    "caption": "SGC7901  cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with  CDDP. The cell survival rate were measured. Data information: The results shown are from seven biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.",
    "molecules": "CDDP, SF1670"
  },
  {
    "caption": "SGC7901 cells were divided into four groups: M2-si-control-exo,  M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell apoptosis were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.",
    "molecules": "CDDP, SF1670"
  },
  {
    "caption": "A. Histograms of crossovers (blue) per F2 individual in the indicated populations, with the Poisson expectation plotted in red. Mean values are indicated by the black dotted lines. The genome average SNPs/kb for each cross are printed above the plots.",
    "molecules": "Poisson"
  },
  {
    "caption": "A. Representative DAPI-stained spreads of pachytene, diakinesis, metaphase-I, dyad and tetrad meiotic stages, for wild type and msh2, in Col×Ler or Col×CLC hybrid backgrounds.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Male meiocytes immunostained for MSH2 (red) and ASY1 (green), and stained for DAPI (blue) in wild type (Col) or msh2.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. As for B, but immunostaining wild type male meiocytes for MSH2 (red), MSH4 (green) and staining chromatin with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) The  bur1-ΔC mutant exhibits a synthetic growth defect with the  vac17Δ mutant. Plasmids were transformed into a bur1Δ or  bur1Δ vac17Δ mutant containing YCp50 [URA3] BUR1. Plasmids tested were pRS415 [LEU2] (mock), pRS415 BUR1, or pRS415 bur1-ΔC. Transformed colonies were cultured in liquid medium and serial dilutions spotted onto SC+5-FOA or SC-Leu-Ura  plates. Plates were incubated at 24 ̊C for 3 days.",
    "molecules": "5-FOA, Leu, Ura"
  },
  {
    "caption": "(A, B) The  bur1-ΔC  vac17Δ double mutant exhibits a delay in progression  through  G1  phase of the cell cycle. Yeast were grown in  YPD  medium and collected in log phase growth. DNA content was measured using propidium iodide (PI) staining  and assessed by  flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. (C, D) A bur1-267 mutant also exhibits a delay in progression through  G1  phase of the  cell cycle. DNA content was measured using   PI staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. **** (p-value < 1 x 10-4). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "(B) The bur1-∆C vac17∆ double mutant cells have the ability to generate a  vacuole de novo, which suggests that the  cell-cycle delay is due to a defect in Bur1-dependent signaling. WT, vac17Δ, bur1-∆C, and bur1-∆C vac17∆ cells which express  Vph1-GFP from its endogenous locus, were  pulse labeled with the  vacuole specific dye FM4-64. WT and bur1-∆C cells have both FM4-64 and Vph1-GFP  signals in both mother and daughter cells. vac17Δ and bur1-∆C vac17∆ cells have both  Vph1-GFP  and  FM4-64 on the vacuole in mother cells, however the daughter cells solely have a Vph1-GFP labeled vacuole, indicating that the vacuole was generated de novo, rather than inherited from the mother vacuole. Arrowheads; new vacuoles in daughter cells. Scale bar, 2 μm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(C) bur1-∆C shows high rapamycin sensitivity. WT, tor1∆, bur1-∆C,  tor1∆ bur1-∆C or vac17∆ cells were cultured in liquid YPD medium and serial dilutions were spotted onto YPD plates with 0, 2, 4 or 8 ng/ml of  rapamycin added. Plates were incubated at 24 ̊C for 2 days.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(B) In vitro, Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1  itself. pRS425 BUR1-HA and pVT102-Ura BUR2, the essential cyclin for BUR1, were co-expressed in a bur1Δ mutant, immunoprecipitated using anti-HA antibody and protein A  beads, and then incubated with [γ-32P]ATP  and GST, GST-N-Sch9  (1-390) or GST-C-Sch9  (391-824). Proteins were eluted with SDS  sample buffer, separated by  SDS-PAGE, and transferred to  nitrocellulose  membranes. Membranes exposed to x-ray film (top) or stained  with Ponceau S (bottom). Red arrowhead; phosphorylated GST-C-Sch9. Gray arrowhead; auto-phosphorylated Bur1.",
    "molecules": "ATP, nitrocellulose, γ-32P, Ponceau S, SDS, Ura"
  },
  {
    "caption": "(D)  Sch9-2D3E, but not Sch9-3A-2D3E, partially suppressed the rapamycin sensitivity of tor1∆ cells.  tor1∆ which expresses pRS426 (mock), pRS425 TOR1, pVT102-Ura (mock), pVT102-Ura Sch9, pVT102-Ura Sch9-2D3E, or pVT102-Ura Sch9-3A-2D3E, were cultured in liquid SC-Ura  medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2-3 days.",
    "molecules": "rapamycin, Ura"
  },
  {
    "caption": "(E) The  Sch9-2D3E mutant did not suppress the rapamycin sensitivity of bur1-∆C cells. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pVT102-Ura (mock), pVT102-Ura SCH9, pVT102-Ura sch9-2D3E, pVT102-Ura sch9-3A-2D3E, pVT102-Ura sch9-3D/E, or pVT102-Ura sch9-3D/E-2D3E were cultured in liquid  SC-Ura medium and serial dilutions spotted onto  SC-Ura plates with 0 or 8 ng/ml of  rapamycin  added. Plates were incubated at 24 ̊C for 3 days.",
    "molecules": "rapamycin, Ura"
  },
  {
    "caption": "(A) Bur1  fused SV40-NLS does not rescue the rapamycin sensitivity of bur1-∆C compared to Bur1 without the NLS. bur1-∆C which expresses pRS416 (mock), pRS416 BUR1, pRS416 BUR1-mNG, pRS416 BUR1-mNG-NLS, pRS416 bur1-∆C, pRS416  bur1-∆C-mNG-NLS or pRS416 bur1-∆N, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.",
    "molecules": "rapamycin, Ura"
  },
  {
    "caption": "(B) Adding nuclear-export signal (NES) of PKI to Bur1 and/or deletion of the   importin α-dependent NLS  from  Bur1  resulted in higher sensitivity to rapamycin compared to Bur1  without this NES. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pRS426 bur1-∆N, pRS426  BUR1-mNG, pRS426 BUR1-mNG-NES, or pRS426 bur1-∆N-mNG-NES, were cultured in liquid  SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of  rapamycin added. Plates were incubated at 24 ̊C for 2 days.",
    "molecules": "PKI, rapamycin, Ura"
  },
  {
    "caption": "Representative confocal 3D images of LysoTracker (red), DAPI (blue), and BODIPY (green) stained ATM subsets obtained from eWAT of HFD (16 weeks) treated mice (n = 8 mice from two independent experiments). The merged figures are the result of the overlap of all three fluorochromes. Scale bar, 10 μm.",
    "molecules": "BODIPY, DAPI, LysoTracker"
  },
  {
    "caption": "Mito Stress assay of sorted ATM subsets (red, MHCIIlow; green, MHCIIhi; and blue, CD11c+) from 16-week-HFD obese mice showing overall oxygen consumption rate (OCR), maximal respiration, spare capacity respiration, basal respiration, ATP production, and non-mitochondrial respiration. OCR data shown are representative of four independent experiments and expressed as mean ± SD (n= 4 MHCIIlow; n= 7 MHCIIhi; n=5 CD11c+), with statistical significance determined using one-way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference).",
    "molecules": "ATP"
  },
  {
    "caption": "eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part).",
    "molecules": "lipid, DT"
  },
  {
    "caption": "Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters.",
    "molecules": "eosin, hematoxylin, DT"
  },
  {
    "caption": "qPCR showing the relative expression of genes for adipogenesis (Pparγ), lipolysis (Lpl), and lipogenesis (Dgat1), plus other lipid receptor/transporters (Cd36, Abca1) or chaperones (Fabp4) in obese (HFD 16 weeks) eWAT of CD169-DTR (red bars) mice and DT-injected WT controls (blue bars) (upper panel) and obese eWAT of anti-CSF1R antibody-injected mice (green bars) mice and isotype control-injected WT controls (white bars) (lower panel). Mice were under DT or anti-CSF1R antibody treatment for 12 days. (n = 5 pooled mice per group, mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.",
    "molecules": "lipid, DT"
  },
  {
    "caption": "Representative 3D fluorescence imaging of lean and obese (HFD 16 weeks) eWAT samples stained with anti-MHCII (green in lean samples), anti-CD11c (green in obese samples), anti-CD31 (blue), anti-CD169 (pink), and DAPI (white). The two images at the bottom show the images merged for the lean and obese conditions without DAPI. Scale bar: 100 μm (lean) and 70 μm (obese).",
    "molecules": "DAPI"
  },
  {
    "caption": "Two representative images of eWAT dissected from Evans blue-injected obese (HFD 16 weeks) WT and CD169-DTR mice treated for 7 days with DT. Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm (the absorption values were normalized to the tissue weight in grams). A chart showing the normalized mean optical density ± SD (n = 5-7 mice per group). Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.",
    "molecules": "Evans blue, formamide, DT"
  },
  {
    "caption": "Elastin staining showing more pronounced ECM accumulation in eWAT of CD169-DTR (HFD 16 weeks) when compared with ECM accumulation in the WT obese (HFD 16 weeks) control (DAPI, blue; elastin, red; CD31, green). The fourth image in each row shows the three channels merged. Scale bar: 50 μm. Picrosirius red-staining depicts excessive collagen deposits in obese (HFD 16 weeks) eWAT of CD169-DTR mice when compared with collagen deposits in WT controls. Scale bar: 250 μm. Upper panels: brightfield microscopy; lower panels: fluorescence microscopy.",
    "molecules": "Picrosirius red, DAPI"
  },
  {
    "caption": "(A) Extracellular D-serine concentrations in cortex were detected by enzyme-based microelectrode biosensors.",
    "molecules": "D-serine"
  },
  {
    "caption": "(B, C) High performance liquid chromatography was performed to quantify total D-serine and L-serine contents in mouse brain of two genotypes. APP-KO mice were fed with D-serine over one month. One-way ANOVA was used for analysis followed by Bonferroni's multiple comparisons test and n>6 mice per group; * p<0.05, ** p<0.01 as compared to WT.",
    "molecules": "D-serine, L-serine"
  },
  {
    "caption": "(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex of APP-KO mice housed under standard or enriched environment. Note that both groups of mice received D-serine after the second imaging time point (8 d); white and empty arrowheads point to newly formed and eliminated spines, respectively. Scale bar - 10 μm. (B) Spine TOR prior and during continuous D-serine treatment. (C, D) Summary plots of the fraction of spine elimination and formation in APP-KO mice before and after D-serine treatment (8 d and 46 d, respectively). (E) Relative spine densities in D-serine treated APP-KO mice housed under standard and enriched environments. (F, G) Summary plots of the fraction of thin and mushroom spines in control and D-serine treated APP-KO mice. For illustration purpose, the control data from Figure 2 B and C are presented also here. Non-linear regression (F test) has been used for fitting the data points. Two-tailed Student t-test was used in (C-D, F-G) and repeated one-way ANOVA was performed followed by Dunnett test in (B and E). N=5 mice in each group; * p<0.05, ** p<0.01, NS - no significant difference.",
    "molecules": "D-serine"
  },
  {
    "caption": "(D) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies.",
    "molecules": "digitonin"
  },
  {
    "caption": "(D) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%).",
    "molecules": "digitonin"
  },
  {
    "caption": "(E) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies.",
    "molecules": "digitonin, glycerol"
  },
  {
    "caption": "(F, G) Submitochondrial localization of SLP2. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting.",
    "molecules": "sodium carbonate"
  },
  {
    "caption": "(B) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracycline (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting.",
    "molecules": "tetracylcine"
  },
  {
    "caption": "(C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/-MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001).",
    "molecules": "CHX"
  },
  {
    "caption": "(E) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.",
    "molecules": "digitonin"
  },
  {
    "caption": "(A) PARL dependent cleavage of PINK1-HA is reduced in Slp2-/- and Yme1l-/- MEFs. Whole cell extracts of Slp2-/-, Parl-/ -and Yme1l-/- MEFs expressing PINK1-HA were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h.(B) Quantification of the protein ratio (Log2) PINK1-HA 52 kDa/63 kDa (n=3; *, p<0.05; One-way ANOVA). n.s., not significant. Error bars indicate SEM.",
    "molecules": "CCCP, MG132"
  },
  {
    "caption": "(C) PINK1-HA associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using FLAG-, HA- and SLP2-specific antibodies.",
    "molecules": "digitonin"
  },
  {
    "caption": "(D) PINK1 associates with SLP2. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells, depleted of SLP2 by siRNA as indicated, were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using SLP2- and PINK1-specific antibodies.",
    "molecules": "digitonin"
  },
  {
    "caption": "(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(B) OMA1-mediated processing of PGAM5 in depolarized Parl-/-mitochondria. Processing of L-PGAM5 to S-PGAM5 in Parl-/ mitochondria was assessed as in (A) in the presence or absence of CCCP (20 µM), which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) Impaired PGAM5 processing in depolarized Oma1-/- mitochondria lacking PARL. Processing of L-PGAM5 to S-PGAM5 was examined as in (A) after 2 h in wild type (WT) and Oma1-/- mitochondria in the presence or absence of CCCP (20 µM, 2 h), which were depleted of PARL by siRNA as indicated. * unspecific cross-reaction.",
    "molecules": "CCCP"
  },
  {
    "caption": "(D) OMA1 mediates accelerated processing of L-OPA1 in Slp2-/- MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2-/-, Oma1-/-, and Slp2-/-Oma1-/- cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(E) OMA1 interacts with SLP2. OMA1-/-FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).(F) Quantification of immunoprecipitation efficiencies in (E).",
    "molecules": "digitonin"
  },
  {
    "caption": "A Frequency of MN leukemic  cells expressing the immature marker cKit  and mature myeloid  marker Ly6G  in the peripheral blood following 5 days of treatment (n = 9-10 mice/group; mice  with < 2% tumor burden in any condition were censored from the analysis). Data information: data in A are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox  - doxycycline.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "C  Kaplan-Meier survival curve of leukemic mice  (n = 11-12 mice/group, median survival  is 16.5 for vehicle and not reached for AG636  and doxycycline, P < 0.0001 by Log-rank test). Data information: Dox  - doxycycline.",
    "molecules": "AG636, Dox, doxycycline"
  },
  {
    "caption": "D Representative FACS  plots showing differentiation  induced by AG636  and doxycycline. Data information: Dox - doxycycline.",
    "molecules": "AG636, Dox, doxycycline"
  },
  {
    "caption": "E Spleen weights  of MN  tumor-bearing mice  (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox  - doxycycline.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "F Absolute number of MN  cells in the bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox  - doxycycline.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "G Frequency (as percentage of all MN  cells) of LSCs  (CD11blowcKithighFcgR+) and differentiated  cells (CD182+Ly6G+) in the  bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox  - doxycycline.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "H May-Grunwald-Giemsa-stained  cytospins  of sorted MN  cells showing myeloid differentiation. Data information: Dox  - doxycycline.",
    "molecules": "May-Grunwald-Giemsa, Dox, doxycycline"
  },
  {
    "caption": "A Spleen weights  of RUNX1-RUNX1T1  or I1DN tumor bearing mice  treated with AG636  or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1  model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.",
    "molecules": "AG636"
  },
  {
    "caption": "B-C Absolute number of  leukemic cells  in the  bone marrow  (B) and spleens (C) of RUNX1-RUNX1T1  or I1DN tumor bearing mice  treated with AG636 or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1  model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.",
    "molecules": "AG636"
  },
  {
    "caption": "D-E Frequency of  leukemic cells expressing  the immature marker cKit  and mature myeloid  marker CD11b  in the bone marrow and spleens  of RUNX1-RUNX1T1  (D) or I1DN (E) tumor bearing mice  treated with AG636  or vehicle for 4 days (n = 6 mice/group). Data information: n = 5-6 mice/group for RUNX1-RUNX1T1  model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.",
    "molecules": "AG636"
  },
  {
    "caption": "Various bone marrow populations were quantified by flow cytometry  in mice  treated with AG636  or vehicle for 1 or 4 days (acute treatment) or for 4 cycles followed by 4 weeks off treatment (post-recovery). Data information: n = 4 mice/group for acute treatment, n = 5-6 mice  for prolonged treatment and recovery; data are presented as mean ± s.d.; P values were calculated using a two-tailed Student's unpaired t-test; only comparisons that meet the threshold of P < 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001",
    "molecules": "AG636"
  },
  {
    "caption": "Various bone marrow  populations were quantified by flow cytometry in mice  treated with AG636  or vehicle for 1 or 4 days (acute treatment) or for 4 cycles followed by 4 weeks off treatment (post-recovery). Data information: n = 4 mice/group for acute treatment, n = 5-6 mice  for prolonged treatment and recovery; data are presented as mean ± s.d.; P values were calculated using a two-tailed Student's unpaired t-test; only comparisons that meet the threshold of P < 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001",
    "molecules": "AG636"
  },
  {
    "caption": "E-H Peripheral blood red blood cells (E), platelets  (F), myeloid cells (G) and  lymphoid cells (H) quantified in mice  treated with AG636  or vehicle. Grey bars denote treatment.",
    "molecules": "AG636"
  },
  {
    "caption": "RNA sequencing  performed on  cKithighCD11blow MN  cells sorted from AG636, doxycycline  or vehicle treated mice  (n = 3 mice/group). Gene set enrichment analysis showing common and differential enrichment of biological pathways in  gene expression data from AG636  or doxycycline  treated animals. Gene sets are from C2:CGP and Reactome sub-collections in MSigDB database (B).",
    "molecules": "AG636, doxycycline"
  },
  {
    "caption": "C  RNA sequencing performed on  cKit+CD11b- RUNX1-RUNX1T1  or I1DN cells sorted from mice  treated with AG636  or vehicle for 1 day (n = 3 mice/group). Bar code plots showing enrichment of selected pathways.",
    "molecules": "AG636"
  },
  {
    "caption": "E-F qPCR showing down-regulation of genes encoding ribosomal  proteins in MN  cells (E) and human AML  cell lines (F) treated for 24 hours with AG636  (n = 3 biological replicates for each cell line). Data information: data in E, F are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;",
    "molecules": "AG636"
  },
  {
    "caption": "G-H Polysome profiling of MN cells treated in vitro with AG636 or vehicle for 24h. Representative trace (G) and quantification of sub-polysome  and heavy polysome  fractions (H) determined by measuring area under the curve (AUC) (n = 4 biological replicates). Data information: data in are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;",
    "molecules": "AG636"
  },
  {
    "caption": "I Nascent protein synthesis  quantified using the  AHA  incorporation assay  in MN  or MOLM13  cells treated with AG636  for 24 hours or cycloheximide  for 1 hour. Cells that were cultured in the absence of AHA  served as a negative control. (n = 3 biological replicates). Data information: data in I are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; CHX  - cycloheximide.",
    "molecules": "AHA, AG636, CHX, cycloheximide"
  },
  {
    "caption": "C Rank plots of ChIPseq enrichment scores of transcription factors at the promoter regions (+1000 bp to -50 bp from TSS) of genes within the indicated gene sets extracted from ENCODE 3 (n = 24 genes for translation genes, n = 707 genes for AG636 down-regulated genes, n = 4237 for the other genes).",
    "molecules": "AG636"
  },
  {
    "caption": "D ChIPqPCR showing the binding  of YY1  at the promoter regions of selected translation genes in MN  cells treated with AG636  or DMSO. For each replicate enrichment was normalized to YY1 pull-down in DMSO  treated cells (n = 2 biological replicates). Data information: data in D are presented as mean ± s.e.m.; P values were calculated using a two-tailed Student's unpaired t-test in D ; *P < 0.05, ***P < 0.001, ****P < 0.0001.",
    "molecules": "AG636, DMSO"
  },
  {
    "caption": "E  Time-course showing global downregulation of O-GlcNAcylation  and YY1 expression  in MN  cells treated with AG636. The experiment was repeated twice with similar results.",
    "molecules": "AG636"
  },
  {
    "caption": "F Western blot of YY1 expression  in human AML  cell lines treated with AG636  for 24 hours.",
    "molecules": "AG636"
  },
  {
    "caption": "G qPCR showing down-regulation of genes encoding ribosomal  proteins that are putative YY1  targets in Cas9  expressing MN  cells treated with AG636  or DMSO  (left); or transduced with sgRNAs targeting YY1  or control sgRNAs (right) (n = 2 biological replicates). Data information: data in G are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test in G; *P < 0.05, ***P < 0.001, ****P < 0.0001.",
    "molecules": "AG636, DMSO"
  },
  {
    "caption": "G  Proliferative competition assays  in MN  cells transduced with individual sgRNAs and cultured in AG636  or DMSO  (n = 2 biological replicates). Data information: data in G represented as mean ± s.e.m.; P values were calculated using one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "AG636, DMSO"
  },
  {
    "caption": "A-B  Proliferative competition assays  in AML  cells transduced with indicated sgRNAs and cultured in AG636  or DMSO  (n = 2 biological replicates). Data information: data represented as mean ± s.e.m; P values were calculated using one-tailed Student's unpaired t-test in A and B ; *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.",
    "molecules": "AG636, DMSO"
  },
  {
    "caption": "F AML  cells were treated with AG636, Roscovitine  or the combination for 17 days. Cumulative live cell counts are shown (n = 2 biological replicates). Data information: data represented as mean ± s.e.m; P values were calculated using Brown-Forsythe and Welch ANOVA test in F; *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.",
    "molecules": "AG636, Roscovitine"
  },
  {
    "caption": "A, B Single-molecule FISH (smFISH) on mouse hippocampal neurons. (A) Representative pictures of smFISH on BDNF-treated WT (top) and KO (bottom) mouse hippocampal neurons (DIV6) using a probe directed against the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Pictures show a zoom-in on the cell body, since the signal detected in WT neurons was restricted to the nuclei (scale bar: 5μm). (B) Representative pictures of smFISH on mouse wild type hippocampal neuron cultures (DIV6; basal) using a probe specific for the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Top: overview, scale bar: 10μm; bottom: zoom-in, scale bar: 5μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "c, d, Cnih2, Prr7 and Src are direct miR-379-410 target mRNAs. (C) 3'-UTR luciferase reporter gene assays in cultured rat cortical neurons using the indicated 3'UTR reporter genes (wt or seed mutant) together with miRNA mimics. Cnih2 (n=3 per group; wt: **p=0.0020, mut: p=0.9267; mimic: F1,8=19.92, p=0.0021; utr-construct: F1,8=4.760, p=0.0607; mimic x utr-construct: F1,8=12.99, p=0.0069), Prr7 (n=3 per group; wt: ****p<0.0001, mut: *p=0.0103; wt vs. mut mimic miR329: **p=0.0014; mimic: F1,8=153.1, p<0.0001; utr-construct: F1,8=5.631, p=0.0450; mimic x utr-construct: F1,8=38.54, p=0.0003); Src (n=3 per group; wt: *p=0.0110, mut: p=0.4724; mimic: F1,8=16.99, p=0.0033; utr-construct: F1,8=0.6809, p=0.4332; mimic vs. utr-construct: F1,8=3.920, p=0.0831); two-way ANOVA. (D) Same as in (C), but in the presence of antimiRs (pLNAs) in hippocampal neurons after 48h of PTX stimulation. Cnih2 (n=4 per group; wt: p=0.2688; mut: p=0.8668; wt ctr vs. mut ctr: p=0.6463; anti-miRNA: F1,12=3.629, p=0.0810; utr-construct: F1,12=0.7407, p=0.4063; anti-miRNA x utr-construct: F1,12=0.6679, p=0.4297), Prr7 (n=4 per group; wt: *p=0.0123; mut: p=0.9917; wt ctr vs. mut ctr: *p=0.0152; anti-miRNA: F1,12=6.082, p=0.0297; utr-construct: F1,12=5.271; p=0.0405; anti-miRNA x utr-construct: F1,12=8.212, p=0.0142); Src (n=3 per group; wt: *p=0.0370; mut: p=0.3540; wt ctr vs. mut ctr: p=0.9906; anti-miRNA: F1,12=13.43, p=0.0064; utr-construct: F1,12=0.5672, p=0.4730; anti-miRNA x utr-construct: F1,12=1.362, p=0.2768); two-way ANOVA. Data are presented as mean ± s.d.",
    "molecules": "PTX"
  },
  {
    "caption": "A-C Bar chart with individual points showing the levels of propionate acetate and butyrate in wt and mdx mice receiving DFZ or vehicle, measured by NMR or GC-MS. Data are expressed as μg/ml or bin intensity (arbitrary unit - a.u.) D-F Bar chart with individual points showing the levels of pyruvate, succinate and lactate in wt and mdx mice receiving vehicle or DFZ, measured by NMR. G",
    "molecules": "acetate, butyrate, DFZ, lactate, propionate, pyruvate, succinate"
  },
  {
    "caption": "A-B Muscle coordination and strength were measured in 19 weeks old control and mdx mice treated with vehicle (DMSO), NaB (100 mg kg−1/daily) or DFZ (1.2 mg kg−1/daily) for 3 weeks using the rotarod and weight test. Bar charts show the latency to fall or drop the weight of wt and dystrophic mice.",
    "molecules": "DFZ, DMSO, NaB"
  },
  {
    "caption": "A-I Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in the gastrocnemius of control and mdx mice treated with or without NaB and DFZ.",
    "molecules": "DFZ, NaB"
  },
  {
    "caption": "A-B Levels of AEA and 2-AG in plasma samples of wt and mdx mice expressed as pmol mg−1 of wet tissue weight.",
    "molecules": "2-AG, AEA"
  },
  {
    "caption": "A-C Bar charts with individual points showing the mRNA expression levels of Ulk, Pink and Becn1 measured in control and mdx mice treated with rimonabant (0.5 mg kg-1).",
    "molecules": "rimonabant"
  },
  {
    "caption": "A-B Bar chart with individual points showing the mRNA expression levels of Il6 and Cox2 in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).",
    "molecules": "DMSO, LPS, MK1903, rosiglitazone, T007"
  },
  {
    "caption": "A-F Bar chart with individual points showing the mRNA expression levels of Cb1, Daglα, Daglβ, Magl, Napepld, and Faah in control (vehicle, DMSO) and/or GPR109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either MK1903 (1 μM), rosiglitazone (1 μM) or T007 (1 μM).",
    "molecules": "DMSO, LPS, MK1903, rosiglitazone, T007"
  },
  {
    "caption": "G-H Levels of AEA and 2-AG were measured in C2C12 cells exposed to LPS (1 μg/ml) or NaB (3 mM) for 24 h. I Effect of ACEA (1 μM) and rimonabant (1 μM) on autophagosome formation measured in C2C12 cells. D",
    "molecules": "2-AG, AEA, ACEA, LPS, rimonabant, NaB"
  },
  {
    "caption": "C-J Bar chart with individual points showing the expression of selected miRNAs in control and Gpr109A-silenced C2C12 myoblasts exposed to LPS (1 μg/ml) in the presence or absence of either NaB (3 mM), MK1903 (1 μM), rosiglitazone (1 μM). NaB was also tested in the presence or absence of either rosiglitazone (1 μM) or T007 (1 μM).",
    "molecules": "LPS, MK1903, rosiglitazone, NaB, T007"
  },
  {
    "caption": "(G) Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. hBMSCs were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collected.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Immunoblot of Flag-p65-linked ubiquitination promoted by Myc-CDC20. HEK293T cells were co-transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (B) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11. HEK293T cells were transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. ",
    "molecules": "MG132, Ubiquitin"
  },
  {
    "caption": "(F) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co-transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 and Vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.",
    "molecules": "MG132, Ubiquitin"
  },
  {
    "caption": "Crypts of 2‐ to 3‐month‐old LGR5ki mice were cultured and γ‐irradiated with 6 Gy on day 10 of culture. Sixteen hours before irradiation, cells were exposed to the indicated treatment (n = 4 independent experiments per group).A-J Representative FACS plots of indicated treatments are given. NIR, non‐irradiated; IR, irradiated.K-P Flow cytometry analysis of the percentage of LGR5+, LGR5hi, and LGR5lo cells of irradiated cultures (24 h after IR) compared to the percentage in non‐irradiated controls (NIR) set to 100%. (K-M) Note that inhibition of Wnt signaling by DKK1 treatment or R‐spondin reduction (50% Rspo) partially rescued the decrease in LGR5+ cells in irradiated cultures. (N-P) Note that activation of Wnt signaling by 6‐BIO treatment aggravated the decrease in LGR5+ cells in irradiated cultures. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.",
    "molecules": "6‐BIO"
  },
  {
    "caption": "e) Histogram charts showing the distribution of clones at D1-3 following tamoxifen induction according to total number of cells and number of basal cells.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "f) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining. The frequency of larger clones increases over time. Chi square test p=0.0013 for P21 group and p=0.0296 for P42 group. All data are represented as mean ± s.e.m.",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application.",
    "molecules": "TPA"
  },
  {
    "caption": "c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation.",
    "molecules": "TPA"
  },
  {
    "caption": "e) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in back skin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.",
    "molecules": "TPA"
  },
  {
    "caption": "B. Detection of ALDHbr cells by high content screening. The algorithm first detect nuclear region of interest (ROI) in the Hoechst channel (rainbow areas). Then, it computes the averaged ALDH signal in the ROI to identify ALDHbr cells (red nuclear ROIs) and calculate their proportion.",
    "molecules": "Hoechst"
  },
  {
    "caption": "C-D. Examples of dose-dependent ALDHbr cells variation in response to drug treatments. ALDHbr cell proportion in response to Tazemetostat in SUM159 (n=10) (C) and JQ1 in SUM149 (n=10) (D). The horizontal red dashed-line corresponds to a reduction of 50% of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed-line corresponds to the target dose predicted by the K-score. On the bottom panel, corresponding flow charts for the ALDEFLUOR staining. DEAB is an ALDH inhibitor used as negative control.",
    "molecules": "DEAB, Tazemetostat, JQ1"
  },
  {
    "caption": "A. Interaction surface built using the estimated combination index (CI) for the Salinomycin and JQ1 drug combination, in four different breast cancer cell lines (top panels). The CI is represented in 2D color code interaction surface. The color code spans from strong synergism (dark blue, CI<1) to strong antagonism (red, CI>1). The concentrations of each particular drug in the combinations are denoted on each axis. On the bottom panels, neighborhood Z-core matrix estimating the significant synergistic interaction. The color code spans from a statistically significant synergism (dark blue, CI<1, p<0.01) to a statistically significant antagonism (dark red, CI>1, p<0.01).",
    "molecules": "JQ1, Salinomycin"
  },
  {
    "caption": "B. Representative examples of flow chart for the ALDEFLUOR staining following different JQ1/Salinomycin drug combinations. DEAB is an ALDH inhibitor used as negative control. Data represent mean ±SD.",
    "molecules": "DEAB, JQ1, Salinomycin"
  },
  {
    "caption": "E. Bar plots representing the dose-dependent changes in the proportion of apoptotic cells (YO-PRO-3+) in each cell subpopulations, in response to drug treatments (right panel). Cisplatin (CisPt) is used as a positive control. On the right panel, representative fluorescence images obtained in different treatment conditions (n=6). Nucleus are in blue (Hoechst), bCSCs are in green (ALDH), and apoptotic cells in red (YO-PRO-3).",
    "molecules": "Hoechst, Cisplatin, CisPt, YO-PRO-3"
  },
  {
    "caption": "G. Representation of the BFP+/RFP+ cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone, or in combination. Blue bars: BFP+ cells proportion, Red bars: RFP+ cells proportion.",
    "molecules": "JQ1, Salinomycin"
  },
  {
    "caption": "H-I. Representation of the bCSC proportion in the BFP+ (H) and RFP+ (I) progenies in the control cells compared to the JQ1- and Salinomycin-treated cells alone or in combination (n=4). Statistical test used is a student's t-test. JQ1 Vs CTRL and Sal Vs JQ1 p=0.03; JQ1/Sal Vs CTRL p=0.01. Error bars represent mean ± SD.",
    "molecules": "JQ1, Sal, Salinomycin"
  },
  {
    "caption": "B. Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 (n=10) The grey area corresponds to the period of drug treatments.",
    "molecules": "JQ1, Salinomycin"
  },
  {
    "caption": "J-L. Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434. Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs (J). Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal/JQ1 treated mice compared to the control mice (K, CRCM404, p=0.04; L, CRCM434, p=0.02). Statistical test used is a Wilcoxon test. Error bars represent mean ± SD (n=10).",
    "molecules": "JQ1, Sal, Salinomycin"
  },
  {
    "caption": "Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I  and probed with a MAT-distal sequence (yellow box in panel B).",
    "molecules": "DNA"
  },
  {
    "caption": "Representative Southern blots showing DSB repair products in WT, rad59∆, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box).",
    "molecules": "DNA"
  },
  {
    "caption": "(F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D-G) Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 hours. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.",
    "molecules": "z-VAD"
  },
  {
    "caption": "(F-H) C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (F), cell viabilities (G) and Mtb growth rates (H) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.",
    "molecules": "z-VAD"
  },
  {
    "caption": "(D-F) Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb-infected Socs6-/- BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.",
    "molecules": "Z-VAD"
  },
  {
    "caption": "(E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.",
    "molecules": "ATP, Ub, ubiquitin"
  },
  {
    "caption": "(A, B) Two-dimensional tryptic mapping of YFP-p27, YFP-p27T198A and a mixture of both (A). Phosphoamino-acid analysis of spots a and b (B). The migration of phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) is marked with circles in B. The asterisk indicates the origin of migration.",
    "molecules": "phosphothreonine, phosphotyrosine, phosphoserine"
  },
  {
    "caption": "(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).",
    "molecules": "MG132"
  },
  {
    "caption": "(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).",
    "molecules": "MG132"
  },
  {
    "caption": "(F) Western blots of protein extracts from MCF-7 transfected with YFP-p27, YFP-p27T198A or YFP-p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP-p27, YFP-p27T198A or YFP-p27T198D and selected with G418 for three weeks. Data are presented as mean ± s.d. (n = 3).",
    "molecules": "G418"
  },
  {
    "caption": "(d) Western blots of lysates of MCF-7 cells transfected with siRNA before serum-starvation or treatment with LY294002 (LY, 10 μM) or rapamycin (Rap, 100 nM) for 48 h with or without methylpyruvate (MP, 10 mM).",
    "molecules": "LY294002, methylpyruvate, rapamycin"
  },
  {
    "caption": "(e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm.",
    "molecules": "LY294002"
  },
  {
    "caption": "(f) Western blots of protein extracts from MCF-7 cultured with (+) or without (−) glucose for 24 h following RNAi. Fr, fragment; percentage PARP cleavage = 100 × PARPFr / (PARP + PARPFr).",
    "molecules": "glucose"
  },
  {
    "caption": "(c) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild-type LKB1 cultured with or without glucose for 24 h.",
    "molecules": "glucose"
  },
  {
    "caption": "(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e),",
    "molecules": "AICAR"
  },
  {
    "caption": "(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e)",
    "molecules": "AICAR"
  },
  {
    "caption": "(d-f) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min (f).",
    "molecules": "glucose"
  },
  {
    "caption": "(g) Western blot analysis of 3T3L1 cells 72 h post-transfection RNAi with or without glucose starvation for 1 h before analysis. Densitometric quantification of western blots is shown. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.",
    "molecules": "glucose"
  },
  {
    "caption": "(A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h. Data represent mean ± s.d. of three independent experiments.",
    "molecules": "AICAR"
  },
  {
    "caption": "(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).",
    "molecules": "AICAR, glucose"
  },
  {
    "caption": "(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).",
    "molecules": "AICAR, glucose"
  },
  {
    "caption": "(C-G) Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was respectively examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.",
    "molecules": "amino acid"
  },
  {
    "caption": "(C) The identified 9-amino acid sequence is required for the association between STXBP4 and AMOT. HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay.",
    "molecules": "amino acid"
  },
  {
    "caption": "(J) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition. The indicated cells were subjected to serum starvation (treatment with no-serum medium for 12 hours), glucose starvation (treatment with no-glucose medium for 6 hours) or actin inhibition (treatment with 0.5 μg/mL latrunculin B or 5 μM blebbistatin for 30 min). YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.",
    "molecules": "blebbistatin, glucose, latrunculin B"
  },
  {
    "caption": " A TUNEL assay comparing apoptotic cells in Miwi+/- and Miwi-/- testis during diakinesis. Blue, DAPI; Green, TUNEL labeling. Asterisk indicates TUNEL-positive stained cells in the meiotic metaphase stage. Scale bars, 10 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " J Combined RNA-DNA FISH analysis of major satellite transcripts and DNA regions in Miwi heterozygote (Miwi+/-) and knockout (Miwi-/-) diakinesis spermatocytes. Sense major satellite RNA transcripts were first detected using an antisense probe (red signal), and then the major satellite DNA region was further detected using a sense probe (green signal). For RNase A treatment, the slides were incubated with (+RNase A) or without (-RNase A) 100 μg/ml RNase A for 1 hour before adding antisense probe. Nuclear DNA was stained by DAPI (blue). Arrows indicate the same pericentric heterochromatin in each channel per cell. Scale bars represent 2 µm. ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " A Combined immunofluorescence and DNA FISH analysis of the kinetochore inner plate/centromere formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) spermatocytes at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1A. Chromosomes were stained with anti-centromere protein antibody (CREST, purple signal), indicating the kinetochore inner plate/centromere. Major satellite DNA regions were detected using an antisense probe (red signal) which revealed the specific localization of pericentromeres in chromosomes. Nuclear DNA was stained by DAPI (blue). Scale bars represent 2 µm. ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm. E Magnified images of categories of sister kinetochore configurations from D. One arrow indicates overlapping pair. Double arrows indicate split pair. The scale bar represents 1 µm. F Population of the sister kinetochores shown in E. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 93, 89 and 81, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,720, 3,560 and 3,240, respectively. ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " B Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were resolved on a 1% agarose gel. The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites. The blot was re-hybridized with a 28S rRNA-complementary probe to demonstrate equal loading (bottom on the left). ",
    "molecules": "28S rRNA"
  },
  {
    "caption": " D Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were digested with RNase III (RNase III) or nuclease S1 (S1), and further resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite transcripts. The blot was re-hybridized with 18S and 28S-complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion. ",
    "molecules": "28S"
  },
  {
    "caption": "A) In our chemoattraction-assay setup, nematode chemoattraction behavior is quantified using a measurable chemoattraction index (C.I.); the higher the C.I. measured in the assay, the stronger the attraction of the males to the sex pheromone. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value.",
    "molecules": "sex pheromone"
  },
  {
    "caption": "B) After vaporization (VAC) at 4°C for 24 h, the C. remanei female extract failed to attract wild-type C. elegans males. Conversely, a drop of the sex pheromone extract placed on the lid of a 60-mm Petri dish evoked chemoattractive behavior in wild-type C. elegans males (volatility test). Thus, the functional components in C. remanei female sex pheromone are volatile substances. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.",
    "molecules": "sex pheromone"
  },
  {
    "caption": "C) Mutant males with established defects in amphid neurons responded to C. remanei sex pheromone to a lesser degree relative to wild-type, demonstrating that some or all of the amphid neurons are required for sex pheromone perception. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.",
    "molecules": "sex pheromone"
  },
  {
    "caption": "A) Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.",
    "molecules": "pheromone, sex pheromone"
  },
  {
    "caption": "B) AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type C. elegans males responded normally to the sex pheromone and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms.",
    "molecules": "sex pheromone"
  },
  {
    "caption": "B) Mutant males of 4 GPCR signaling cascade components failed to respond to C. remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.",
    "molecules": "pheromone, sex pheromone"
  },
  {
    "caption": "C) In males from both AWA-specific-rescue odr-3 mutant strain and odr-3 rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used.",
    "molecules": "sex pheromone"
  },
  {
    "caption": "A) srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1000 diacetyl and 10 mg/mL pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.",
    "molecules": "diacetyl, sex pheromone, pyrazine"
  },
  {
    "caption": "F) AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.",
    "molecules": "sex pheromone"
  },
  {
    "caption": "G) Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites. Data information: For each transgenic strain, 3 independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Means ± SEM (error bars) are shown.",
    "molecules": "sex pheromone"
  },
  {
    "caption": "A) The GCaMP5 reporter was used to visualize activation of the SRD-1 receptor by the sex pheromone in AWA neurons. To facilitate real-time imaging of the process, the heads of the transgenic worms were immobilized on soft-agar pads permeable to sex pheromone.",
    "molecules": "agar, sex pheromone"
  },
  {
    "caption": "C) Relative fluorescence-intensity change was quantified through calcium imaging. Wild-type males showed receptor excitation by sex pheromones from C. elegans hermaphrodites or C. remanei females. This response was lost in srd-1-mutant-strain males but was restored following AWA-specific rescue of srd-1. Red triangles: time points of sex pheromone addition. Three males were assayed from each strain.",
    "molecules": "calcium, sex pheromone, sex pheromones"
  },
  {
    "caption": "B) Wild-type C. elegans males are less attracted to sex pheromone than wild-type C. remanei males. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  ",
    "molecules": "sex pheromone"
  },
  {
    "caption": "D) srd-1 mutant males rescued with Ce-srd-1 cDNA showed lower responsiveness than those rescued with Cre-srd-1 cDNA, which indicates that sex pheromone perception ability depends primarily on the protein coding region and not the regulatory sequences in the promoter. The sex pheromone perception defect of srd-1 mutants was not rescued in males expressing CT-truncated SRD-1 proteins. The findings show that the CT region is essential for sex pheromone perception. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  ",
    "molecules": "sex pheromone"
  },
  {
    "caption": "E) srd-1 mutant males rescued by using Ce-srd-1 cDNA together with Cre-srd-1 CT cDNA were more responsive to sex pheromone than those rescued using Cre-srd-1 cDNA with Ce-srd-1 CT cDNA. The results of the CT-swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD-1 protein. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.  ",
    "molecules": "sex pheromone"
  },
  {
    "caption": "Immunofluorescence images against CB1 and the following cellular markers: Calbindin for Purkinje cells (E), GFAP for astrocytes (F) and F4/80 for microglia (G) in the cerebellum of WT and ASM-KO mice. DAPI stains cell nuclei. Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells (shown by white arrows, E), astrocytes (F) and microglia (G) expressed as percentage of the values obtained in WT mice. Graphs to the right show the Mander's coefficient that indicates degree of co-localization between CB1 and the cellular markers for Purkinje cells (E), astrocytes (F) and microglia (G) (E: **PFluorecence intensity = 0.0082, *PMander's Coefficient = 0.0158; F: *PMander's Coefficient = 0.0115); n = 5 mice per group, Student's t-test). Scale bar, 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM-KO mice. TOPRO stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (****P < 0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.",
    "molecules": "TOPRO"
  },
  {
    "caption": "Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT and ASM-KO mice (E)",
    "molecules": "SM"
  },
  {
    "caption": "Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM (F).",
    "molecules": "SM"
  },
  {
    "caption": "Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM (**P = 0.0021, n = 5 independent cultures, Student's t-test).",
    "molecules": "SM"
  },
  {
    "caption": "Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM (n = 2 independent cultures, Student's t-test)",
    "molecules": "SM"
  },
  {
    "caption": "Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (***P <0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.",
    "molecules": "SM"
  },
  {
    "caption": "Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle (veh), with 40μM SM (SM), with 0,1μM Bafilomycin (BafA1) or with 40μM SM and 0,1μM Bafilomyecin (SM+BafA1). Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units (n = 2 independent cultures, one - way ANOVA).",
    "molecules": "BafA1, Bafilomycin, Bafilomyecin, SM"
  },
  {
    "caption": "Mean ± SEM SM levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (*P = 0.0157, n = 3 independent cultures, Student's t-test).",
    "molecules": "SM"
  },
  {
    "caption": "Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM-KO mice incubated or not with exogenous SMase. DAPI stains cell nuclei. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (*P = 0.0189, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P<0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "AEA, SM"
  },
  {
    "caption": "Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA or the combination of GW and AEA (***P <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "AEA, GW, SM"
  },
  {
    "caption": "Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test).",
    "molecules": "AEA"
  },
  {
    "caption": "Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF or URB in the presence or absence of AEA (**PJNJ = 0.0013, *PPF = 0.0143, ***PURB = 0.0002, **PJNJ+AEA = 0.0069, **PPF+AEA = 0.0077, ***PURB+AEA <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "URB, JNJ, AEA, PF, SM"
  },
  {
    "caption": "Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF or with SR141716 + PF (*PPF = 0.0109, *PSR+PF = 0.0343, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF, SM, SR, SR141716"
  },
  {
    "caption": "Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF or URB. Scale bar, 50 μm.",
    "molecules": "URB, JNJ, AEA, PF"
  },
  {
    "caption": "Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF or URB (n = 3 independent cultures).",
    "molecules": "URB, JNJ, AEA, H2O2, PF"
  },
  {
    "caption": "Weekly mean ± SEM body weight of WT and ASM-KO mice treated or not with PF (***P <0.0001, n = 7 mice per group, statistics reflect the significant difference between the slope of the data in the PF-treated ASM-KO with respect to the other three groups, Linear Regression test).",
    "molecules": "PF"
  },
  {
    "caption": "Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM-KO mice after two months of vehicle or PF treatment (*PT3 = 0.0488, *PT4 = 0.361, n = 4 mice per group, two - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF"
  },
  {
    "caption": "Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0024, **PWT PF Vs KO Veh = 0.0016, *PKO PF Vs KO Veh = 0.0391, n = 9 mice per group, two - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF"
  },
  {
    "caption": "Mean ± SEM time of immobility in the Tail suspension test of WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0198, **PWT PF Vs KO Veh = 0.0067, n = 6 mice per group, two - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF"
  },
  {
    "caption": "Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0342, n = 7 mice per group, two - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF"
  },
  {
    "caption": "Percentage of survival with respect to weeks of age in WT and ASM-KO mice treated with vehicle or PF (**P = 0.0031, n = 5 mice in WT vehicle, WT PF treated and KO vehicle, 6 mice in KO PF treated, Mantel-Cox)",
    "molecules": "PF"
  },
  {
    "caption": "Mean ± SEM AEA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWT Veh vs KO Veh = 0.0063; ****PWT Veh vs KO PF < 0.0001; ****PWT PF vs KO PF < 0.0001; ****PKO Veh vs KO PF < 0.0001, n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "AEA, PF"
  },
  {
    "caption": "Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (***PWTVeh Vs KO Veh <0.0001, *PKO Veh Vs KO PF = 0.0465), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF, SM"
  },
  {
    "caption": "Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (n = 3 mice per group, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF"
  },
  {
    "caption": "Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0026, *PKO Veh Vs KO PF = 0.0345), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).",
    "molecules": "PF"
  },
  {
    "caption": "Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to CB1, expressed as percentage of values in vehicle-treated mice (n = 4 mice per group, > 20 Purkinje cells per mouse",
    "molecules": "PF"
  },
  {
    "caption": "Immunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graphs show Mander´s coefficient for the colocalization of CB1 with Lamp1 (left) and mean ± SEM lysosomal size (right) (***PWT Veh vs. KO Veh = 0.0002; **PKO Veh vs. KO PF = 0.0052, n = 4 mice per group, >20 lysosomes per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 5 μm.",
    "molecules": "PF"
  },
  {
    "caption": "Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number of calbindin positive cells per area (***PWTVeh Vs KO Veh <0.0001, **PKO PF Vs KO Veh = 0.00642), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 100 μm. Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to GFAP in arbitrary units (***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0083), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm. Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number (left) and area (right) of Iba1-positive cells (Left ***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0034; Right ***PWTVeh Vs KO Veh < 0.0001, PKO PF Vs KO Veh = 0.00123), n = 4 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.",
    "molecules": "PF"
  },
  {
    "caption": "H&E staining of cerebellum, hippocampus, cortex and liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Scale bar, 100 μm.",
    "molecules": "PF"
  },
  {
    "caption": "Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar, hippocampal, cortical and liver extracts from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. (*PKOVeh Vs KO 5 = 0.0304, n = 3 mice per group, one - way ANOVA, Bonferroni post hoc)",
    "molecules": "PF, SM"
  },
  {
    "caption": "Immunofluorescences against the microglia marker Iba1 in the cerebellum, hippocampus, cortex or against the macrophage marker F4/80 in the liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Graphs show mean ± SEM number of microglia per area unit in cerebellum, hippocampus and cortex or mean ± SEM area of macrophages in the liver (Hippocampus **PKOVeh Vs KO 5 = 0.0015, Cortex *PKOVeh Vs KO 5 = 0.0364, Liver *PKOVeh Vs KO 5 = 0.0152, n = 6 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.",
    "molecules": "PF"
  },
  {
    "caption": "Immunofluorescence images against CB1 and Calbindin in the cerebellum of WT and Npc1nmf164 mice. DAPI stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the Purkinje cells (indicated by arrows) expressed in arbitrary units (**P= 0.0085, n = 5 mice per group, Student's t-test). Scale bar, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Graph shows mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n= 2 different cultures)",
    "molecules": "PF, SM"
  },
  {
    "caption": "Graph shows mean ± SEM cholesterol levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n=2 different cultures)",
    "molecules": "cholesterol, PF"
  },
  {
    "caption": "Immunofluorescence images of CB1 and Lamp1 in cultured fibroblasts from control subject and NPC patient treated with vehicle or with PF at 100μM. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (n= 2 different cultures). Scale bar, 5 μm.",
    "molecules": "PF"
  },
  {
    "caption": "Graphs show mean ± SEM SM and cholesterol levels expressed as percentage of the values obtained in the vehicle treated mice in the cerebellum of Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF (n=3 mice)",
    "molecules": "cholesterol, PF, SM"
  },
  {
    "caption": "Immunofluorescence images against the microglia marker F4/80 in the cerebellum of WT and Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF. Graphs show mean ± SEM number (upper) and area (lower) of F4/80 positive cells (upper graph ****P< 0.0001; lower graph ***PWT Veh vs NPC Veh = 0.0008; *PWT Veh vs. NPC PF = 0.0286; ****PWT PF vs. NPC Veh < 0.0001; **PWT PF vs. NPC PF = 0.0022, n = 3 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.",
    "molecules": "PF"
  },
  {
    "caption": "A) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions, as well as SCAN-B whole mRNA.",
    "molecules": "DNA, mRNA"
  },
  {
    "caption": "B-D BSP amyloid aggregation in vitro is pH-dependent in 10 mM EDTA (B), protein concentration-dependent in 10 mM EDTA and at pH 4.5 (C) and strongly inhibited by millimolar Ca2+ at various protein concentrations (D).",
    "molecules": "amyloid, Ca2+, EDTA"
  },
  {
    "caption": "I-J Amyloid aggregation by BSP subconstructs. BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4.5 (I). The conjunction of module 3 with modules 1 and 2 promotes aggregation (J).",
    "molecules": "Amyloid, EDTA"
  },
  {
    "caption": "B Representative fluorescence microscopy images of BSP and its subconstructs (20 μM) at varying pHs. PEG 8K concentration is 2 %. Scale bar, 20 μm.",
    "molecules": "PEG"
  },
  {
    "caption": "E Quantification of droplet area is shown for each protein under various Ca2+ concentration conditions. Each sample is quantified by total area (left) and mean area (right). Each experiment was repeated three times, data shown as mean ± SD.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L+W133V DHFR strains after being grown at 30°C, 37°C, or 42°C (I91L+W133V was grown at 40°C) in M9 medium supplemented with amino acids for 4 hours (see Methods).",
    "molecules": "amino acids, DAPI"
  },
  {
    "caption": "Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. For the boxplots in panels , F the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B.",
    "molecules": "amino acids, arabinose"
  },
  {
    "caption": "(A) and (B) shows abundance of selected nucleotides and amino acids for mutants I91L+W133V and W133V as well as WT strain treated with 0.5μg/ml of Tmp (Trimethoprim) after 4 hours of growth at 40/42°C in M9 minimal medium (condition of no filamentation), Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions. In minimal medium (B) Methionine levels are extremely low Levels of purines (IMP, AMP) are also largely rescued with amino acid supplementation, however dTMP, dTDP and dTTP levels remain extremely low. Error bars represent SEM of at least three biological replicates (see Methods). The dashed line in each plot represents value of 1 for WT.",
    "molecules": "AMP, dTDP, dTMP, dTTP, IMP, Methionine, purines, Tmp, Trimethoprim"
  },
  {
    "caption": "(A) recA (B) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods).",
    "molecules": "amino acids, dTMP, Tmp"
  },
  {
    "caption": "WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint.",
    "molecules": "amino acid, amino acids, GMP, Tmp"
  },
  {
    "caption": "Expression of SOS response genes recA, recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids (black bars) or in the presence of both amino acids and GMP (gray bars) at 42°C (I91L+W133V was grown at 40°C). Error bars represent SD of 2-3 biological replicates.",
    "molecules": "amino acids, GMP, Tmp"
  },
  {
    "caption": "Comparison of growth rates of WT and mutant DHFR strains at 42°C (40°C for I91L+W133V and V75H+I91L+I155A) in minimal medium that is supplemented with amino acids and/or 1mM dTMP. Except for W133V and to a lesser extent for V75H+I155A, the effect of dTMP on growth rates is only modest. Error bars represent SEM of 3 biological replicates.",
    "molecules": "amino acids, dTMP"
  },
  {
    "caption": "in vitro activity of purified Thymidylate Kinase (Tmk) as a function of dTMP concentration shows Michaelis-Menten (MM) like kinetics. ATP concentration is saturating at 1mM. The KM of dTMP is 13μM. The error bars represent SD of three technical replicates.",
    "molecules": "ATP, dTMP"
  },
  {
    "caption": "The green points were acquired during growth of WT, W133V and I91L+W133V mutants in the presence of different concentrations of thymidine, which follow the red curve. The inset plot shows the same graph with selected datapoints for I91L+W133V mutant. The cyan circle shows I91L+W133V mutant in the absence of any metabolite supplementation, while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively.",
    "molecules": "dTMP, thymidine"
  },
  {
    "caption": "Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for (A) Adenylate Kinase (AK) (B) Uridylate Kinase (PyrH) (C) Adenylosuccinate Lyase (PurB) and (D) GAR Transformylase (PurN). AK, PyrH and PurB appear to follow hyperbolic MM kinetics in vivo, while PurN shows Hill-like dependence with cooperativity. In panel (A), the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK (Adkar et al., 2019) as well as DHFR mutants used in the present study, while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP (Adkar et al., 2019). Data points for all other panels (B-D) are derived from mutant DHFR strains in the present study. Since SAICAR, AICAR, GAR and FGAR levels were undetectable in WT, all data points in (C-D) are relative to those observed in I91L+W133V mutant following 4 hours of growth at 40°C. Data points in all panels represent metabolite levels for individual biological replicates without averaging.",
    "molecules": "AMP, amino acid"
  },
  {
    "caption": "C N1E-115 cells treated with siControl or siINPP5Es were cultured for 2 h in growth medium with or without 125 nM bafilomycin A1 (Baf.A1), and then analyzed by immunoblot using anti-p62, anti-LC3, and anti-GAPDH antibodies.",
    "molecules": "Baf.A1, bafilomycin A1"
  },
  {
    "caption": "G Electron micrographsin N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Arrowheads indicate autophagosomes. Bar, 1 µm.",
    "molecules": "INPP5E"
  },
  {
    "caption": "A N1E-115 cells treated with siControl, siINPP5E, or siAtg2a/2b were cultured in growth medium. The post-nuclear fraction (PNS) was separated into LSP, HSP, and HSS fractions, and then analyzed by immunoblots using anti-p62, anti-LC3, and GAPDH antibodies. The subfractions were treated with proteinase K (Pro. K) with or without Triton X-100. Quantitation of protein signal intensities from immunoblots showing LC3-II (left) or p62 (right) levels, following normalization to the control protein GAPDH. **, P < 0.01; *, P < 0.05.",
    "molecules": "proteinase K, Triton X-100"
  },
  {
    "caption": "B NIE-115 cells stably expressing LAMP1-mCherry treated with siControl or siINPP5Es were cultured for 2 h in growth medium containing 200 nM Torin1 with protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d). Because the treatment of protease inhibitors inhibits lysosomal degradation, LC3puncta persist even ifautophagosomes fuse with lysosomes. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.C Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.",
    "molecules": "E-64-d, Pepstatin, protease inhibitors, Torin1"
  },
  {
    "caption": "B N1E-115 cells stably expressing mSt-INPP5E (WT, D477N, ∆CAAX, or 295-644) treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and then analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mSt-INPP5Es used in this study were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B, showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.",
    "molecules": "Baf.A1"
  },
  {
    "caption": "A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg/ml DQ-BSA for 12 h and chased for 2 h. Cells were fixed and stained with anti-LAMP1 antibodies and analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.B Quantitation of signal intensities in A showing colocalization of DQ-BSA with LAMP1 (means ± s.d.; n > 50 cells from three independent experiments). Baf.A1 was used as a control for inhibition of delivery and degradation of DQ-BSA via the endocytic pathway. **, P < 0.01; n.s., non significant.",
    "molecules": "Baf.A1"
  },
  {
    "caption": "A NIE-115 cells stably expressing mSt-2xML1N or -2xPLCδ1PH were cultured in growth medium with or without 200 nM Torin1. Cells were fixed and stained with anti-LAMP1 or anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.",
    "molecules": "Torin1"
  },
  {
    "caption": "C N1E-115 cells were treated with 0.5 µM Latrunculin A (Lat A) in growth medium for 6 h. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Bar, 10 µm. Quantitation of the number of LC3puncta per cell. (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.",
    "molecules": "Lat A, Latrunculin A"
  },
  {
    "caption": "D NIE-115 cells stably expressing LAMP1-mCherry were treated with DMSO or 0.5 µM Lat A in growth medium for 4 h, subsequently cultured with 200 nM Torin1 and protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d) for 2 h (Lat A treatment for total 6h). Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.",
    "molecules": "E-64-d, DMSO, Lat A, Pepstatin A, protease inhibitors, Torin1"
  },
  {
    "caption": "B N1E-115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.",
    "molecules": "Baf.A1"
  },
  {
    "caption": "(B) GST, GST-MCM2N (human) and GST-Spt5N (yeast) were mixed as indicated with yeast histones H3-H4, in the presence of glutathione agarose beads (see STAR method for further details). The input and pulldown samples were analysed by SDS-PAGE before staining with colloidal Coomassie blue. The asterisks denote truncations of GST-Spt5N. (C) Similar experiment to (B) but with histones H2A-H2B. (D) Similar experiment to (B) to compare histone binding by GST-MCM2N and GST-MCM2N-2A (with Y81A Y90A mutations of conserved residues).",
    "molecules": "colloidal Coomassie blue"
  },
  {
    "caption": "(C) Yeast cells were generated that expressed Protein A-tagged versions of wild type Spt5 (full length or the indicated truncations), or full length Spt5-3A, under the control of the GAL1,10 promoter. Cells were grown in medium containing galactose and the ProteinA-tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG. Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting, using antibodies specific to the indicated phosphorylation sites in the C-Terminal Domain repeats of the Rpb1 subunit of Pol II.",
    "molecules": "galactose"
  },
  {
    "caption": "(C) Cells from the end of the experiment described in (A) were fixed and processed for ChIP-Seq, using antibodies specific for phosphorylated Serine 5 in the C-Terminal Domain of the Pol II catalytic subunit Rpb1 (CTD-Ser5P). For each of the three indicated strains, the histograms represent the normalized read density (DNA sequence read count per million reads, or RPM), across a sample gene body.",
    "molecules": "Ser, Serine"
  },
  {
    "caption": "B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test.",
    "molecules": "homocysteine, methionine"
  },
  {
    "caption": "C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.",
    "molecules": "homocysteine"
  },
  {
    "caption": "E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.",
    "molecules": "methionine"
  },
  {
    "caption": "F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.",
    "molecules": "homocysteine"
  },
  {
    "caption": "HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.",
    "molecules": "DHE, HTL, ROS, superoxide"
  },
  {
    "caption": "HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.",
    "molecules": "DHE, HTL, ROS, superoxide"
  },
  {
    "caption": "C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media.",
    "molecules": "Hcy, HTL, superoxide"
  },
  {
    "caption": "D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot.",
    "molecules": "DHE, Hcy, superoxide"
  },
  {
    "caption": "E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4).",
    "molecules": "Hcy, Met, superoxide"
  },
  {
    "caption": "F The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.",
    "molecules": "DHE, HTL, ROS, superoxide"
  },
  {
    "caption": "G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells.",
    "molecules": "Hcy, Superoxide"
  },
  {
    "caption": "H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot.",
    "molecules": "N-Hcy, HTL"
  },
  {
    "caption": "I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot.",
    "molecules": "N-Hcy"
  },
  {
    "caption": "J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N-Hcy (J) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, N-Hcy, HTL"
  },
  {
    "caption": "J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and superoxide (K) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, HTL, superoxide"
  },
  {
    "caption": "C SOD activities were down-regulated by in vitro N-homocysteinylation. Flag-tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2, and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity purified, and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N-Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "N-Hcy, HTL"
  },
  {
    "caption": "D Upon switching N-Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N-Hcy unmodifiable mutants displayed lower specific activity than the wide-type (WT) SOD. SOD1 (WT, K23W, K122W, K128W, and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W, and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity-purified SOD proteins was determined and normalized to protein levels. Wild-type SOD1 and SOD2 activities were set at 100% (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "lysine, tryptophan, N-Hcy"
  },
  {
    "caption": "E, F Hcy and HTL treatment in NE4C cells increased N-homocysteinylation of SODs and decreased their activities. Flag-tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum-free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, HTL"
  },
  {
    "caption": "G N-Hcy SOD levels were down-regulated in MARS/MARS2 knockdown cells. Flag-tagged SOD1 and SOD2 were each over-expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N-Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "N-Hcy"
  },
  {
    "caption": "A Levels of β-catenin in response to Hcy treatment in NE4C cells were detected by western blot (n=3). Representative western blots are shown, and band intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy"
  },
  {
    "caption": "B Levels of β-catenin in response to HTL treatment in NE4C cells were detected. Band intensities were normalized to that of untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "HTL"
  },
  {
    "caption": "C TCF activities were measured with a Topflash/Fopflash luciferase reporter system (n=4). TCF activities responding to either 20 μM Hcy- or 10 μM HTL-treated were measured by quantifying Topflash/Fopflash relative to the untreated groups. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, HTL"
  },
  {
    "caption": "D Levels of β-catenin in untreated and MARS knockdown NE4C cells were detected (n=3) with the presence or absence of 20 μM Hcy in the culture media. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy"
  },
  {
    "caption": "NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of Hcy (E) The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy"
  },
  {
    "caption": "NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of HTL (F). The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "HTL"
  },
  {
    "caption": "G NRX-Flag and DVL1-Myc were co-transfected in untreated or MARS knockdown NE4C cells. The relative DVL1 levels that co-IPed with NRX-Flag were determined and quantified (n=3) for treated and control cells cultured with or without 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy"
  },
  {
    "caption": "Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL. (H) The levels of β-catenin in cells with and without SOD1/2 expression were determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, HTL"
  },
  {
    "caption": "Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with HTL. (I) Relative levels of DVL1 that co-IPed with NRX-Flag were quantified in SOD-expressing and control cells cultured with or without 10 μM HTL (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. ",
    "molecules": "HTL"
  },
  {
    "caption": "B MARS- or MARS2-catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography.",
    "molecules": "Hcy, HTL, AHT"
  },
  {
    "caption": "C MARS and MARS2 activities were determined (n=4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, AHT"
  },
  {
    "caption": "D Relative HTL levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "HTL, AHT"
  },
  {
    "caption": "E AHT (71 or 110 μg/kg per day) was injected into C57BL/6 mice for 6 days. Plasma HTL levels were detected by LC-MS. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "HTL, AHT"
  },
  {
    "caption": "F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, AHT"
  },
  {
    "caption": "NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; N-homocysteinylation levels were detected by western blot.",
    "molecules": "Hcy, AHT"
  },
  {
    "caption": "NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; Band intensities were quantified relative to the untreated group using Image J (H). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, AHT"
  },
  {
    "caption": "I Cellular superoxide levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Superoxide levels were normalized to those of untreated NE4C cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "AHT, superoxide, Superoxide"
  },
  {
    "caption": "J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT-treated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "Hcy, AHT"
  },
  {
    "caption": "K Levels of β-catenin in untreated, Hcy-, or HTL-treated NE4C cells were detected (n=3) with the presence or absence of 70 μM AHT in the culture media. Mean β-catenin levels in untreated NE4C cells were set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. ",
    "molecules": "Hcy, HTL, AHT"
  },
  {
    "caption": "A, B Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS (green) and N-Hcy (red). Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (B). C, D Double immunofluorescent staining (C) for MARS (green) and N-Hcy (red) in E10 heart sections. Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (D).Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "N-Hcy"
  },
  {
    "caption": "E Goss morphology of E10.5 ATRA-treated and untreated mice embryos. The ATRA-treated embryo exhibits an open neural tube (indicated by white arrows) compared to littermate controls. Scale bar: 500 μm.",
    "molecules": "ATRA"
  },
  {
    "caption": "F Examples of normally developed and maldeveloped mice hearts: in the normal heart (i), the ink travelled from the right ventricle (RV) into the pulmonary trunk (PT); however, in ATRA-treated group hearts, ink travelled into the aorta (Ao) (ii, transposition of great arteries (TGA)) or both the PT and Ao (iii, double outlet right ventricle (DORV)). Scale bar: 1000 μm.",
    "molecules": "ATRA"
  },
  {
    "caption": "G Cardiac malformations were analysed by hematoxylin and eosin (H&E) staining of heart sections from E14.5 mice. The cardiac defects observed in ATRA treated mice are as follows: Ventricular septal defect (VSD) and single ventricle defects (SVD). Heart compartments are marked: RA: right atria, RV: right ventricle, LA: left atria, LV: left ventricle. Scale bar: 1000 μm",
    "molecules": "ATRA, eosin, hematoxylin"
  },
  {
    "caption": "H Total protein N-Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses (i). Western blot was used to detect N-Hcy levels (ii). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "N-Hcy"
  },
  {
    "caption": "I Neural tube tissues at E10 were homogenized from litters of untreated, ATRA-treated, and ATRA+AHT co-treated pregnant mice. Neural tube homogenates of each group were mixed from 6 embryos. Endogenous SOD1 and SOD2 were affinity purified from the homogenates. N-Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined. The SOD activities of the untreated group were set at 100%; error bar indicates assay replicates (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "ATRA, N-Hcy, AHT"
  },
  {
    "caption": "J Superoxide (a), apoptosis (b), and Wnt signalling (c, d) were over-activated in ATRA-treated mice neural tubes and restored by AHT or NAC treatment. (a) Whole mount DHE staining of E10.5 mice embryos. Scale bar: 500 μm. Neural tube defect (white arrows) and heart (green arrow) regions show especially higher fluorescence intensities. (b) TUNEL (green) staining detected apoptotic cell death in E8.5 neural tube (E) sections. MARS (red). Scale bar: 300 μm. (c, d) IHC staining for β-catenin (green) in E8.5 (c) and E10 (d) mice neural tube sections. Scale bar: 300 μm (c), 150 μm (d). DAPI (blue).",
    "molecules": "ATRA, DAPI, DHE, NAC, AHT, Superoxide"
  },
  {
    "caption": "K Average (n=3) DHE fluorescence intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "DHE"
  },
  {
    "caption": "N Hcy-treated eggs exhibited NTDs phenotypes at embryonic stage 8 (E8). A normal embryo (a) and neural tube defect phenotypes (b, c) are shown. Exencephaly (white arrows) and spina bifida (blue arrows) are indicated. Scale bar: 500 μm.",
    "molecules": "Hcy"
  },
  {
    "caption": "O Total protein N-Hcy levels of 72 h chicken embryo homogenates from Hcy and Hcy+AHT-treated groups were detected by western blot.",
    "molecules": "Hcy, N-Hcy, AHT"
  },
  {
    "caption": "P Whole mount DHE staining of 72 h chicken embryos from untreated, Hcy-, Hcy+AHT-, and Hcy+NAC-treated groups. Scale bar:2000 μm. Q Quantification of DHE fluorescence intensities in (P) (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.",
    "molecules": "DHE, Hcy, NAC, AHT"
  },
  {
    "caption": "(D) Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs and DAPI (blue) to label DNA.(E) The angle between the Golgi, nucleus and tissue axis was measured and binned as 'left', 'right', 'upstream' or 'downstream'.(F) Six areas in the aorta were analyzed.(G) There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual un-binned angles. Error bars are +1 S.E.M. Scale bars are 500μm. n=3 control and mutant mice for each region except region 2 where n=3 control and n=2 mutant mice.",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Pkd2 localizes to EC cilia in vivo. Adult mouse aortas were stained for Pkd2, acetylated α-Tubulin, and Hoechst to label DNA. Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells. Arrowheads point out cilia. Example cilium highlighted in inset.",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Phosphorylation at Serine 1176 of eNOS was reduced in the aortas of mice lacking EC cilia. Aortas from control and mutant mice were stained for eNOS, p-eNOS-S1176, acetylated α-Tubulin, and Hoechst to label DNA.",
    "molecules": "DNA"
  },
  {
    "caption": "G) Representative images of the EdU staining in freshly isolated YC and OC FAPs following EdU in vivo exposure for 3 days (EdU in red/DAPI in blue). Scale bar = 25 μm. H) Graph showing the percentage of EdU positive cells for the staining shown in (G).",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "I) Representative images of the LMNB1 staining in freshly isolated YC and OC FAPs (LMNB1 in green/DAPI in blue). Scale bar = 25 μm. J) Graph showing the relative quantification of LMNB1 fluorescence intensity normalized for nuclei number,in the staining shown in (I).",
    "molecules": "DAPI"
  },
  {
    "caption": "L) Representative images of the γH2AX and KI-67 staining in freshly isolated YC and OC FAPs (γH2AX in green/KI-67 in red/DAPI in blue). Scale bar = 10 μm. Magnification in the insert (scale bar = 3 μm) shows γH2AX positive CCF in OC FAPs. M) Graph showing the percentage of cells positive for γH2AX positive CCF, for the staining shown in (L). N) Graph showing the percentage of cells positive for KI-67 for the staining shown in (L).",
    "molecules": "DAPI"
  },
  {
    "caption": "D) Representative images of the phospho-NFkB staining in FAPs (pNFkB-p65 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal section of YC, YT, OC and OT mice; Scale bar = 25 μm; E) Box plot showing the percentage of pNFkB-p65 positive FAPs in the staining shown in (D)",
    "molecules": "DAPI"
  },
  {
    "caption": "F) Representative images of the phospho-p38 staining in FAPs (pP38 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; G) Box plot showing the percentage of pP38 positive FAPs in the staining shown in",
    "molecules": "DAPI"
  },
  {
    "caption": "H) Representative images of the phospho-SMAD2/3 staining in FAPs (pSMAD2/3 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; I) Box plot showing the percentage of pSMAD2/3 positive FAPs in the staining shown in (H).",
    "molecules": "DAPI"
  },
  {
    "caption": "(G)Immunoblot  analysis of MEKK2  in lysates of Nedd4l+/+ and Nedd4l-/- MEFs  treated with CHX  (50 μg/ ml) for indicated time. (H)The intensity values were measured by Image J software to calculate half-life of MEKK  protein ( n = 3 biological replicates). Data information: Numbers between two blots  indicate densitometry  of phosphorylated  proteins relative to that of total proteins, respectively. Densitometry  of MEKK2  was relative to actin. All experiments were performed three times, and representative data are shown. Significant differences were tested using a two-tailed Student's t-test. *P<0.05, **P<0.01. NS, no significance.",
    "molecules": "CHX"
  },
  {
    "caption": "(A)Immunoblot  analysis of NEDD4L, MEKK2, and MEKK3 immunoprecipitated  with NEDD4L-specific antibody from lysates of HeLa  cells pre-treated with 20μM MG-132 for 6 h and then treated with IL-17 for the indicated time. Data information: All experiments were performed three times, and representative data are shown.",
    "molecules": "MG-132"
  },
  {
    "caption": "(D)Immunoblot  analysis of MEKK2  and NEDD4L  in Nedd4l+/+, and Neddl4- /- treated with DMSO, 20μM MG-132, or 0.2 μM bafilomycin A1 (Baf A1) for 6 h. Densitometry  of MEKK2  was relative to actin. Data information: All experiments were performed three times, and representative data are shown.",
    "molecules": "Baf A1, bafilomycin A1, DMSO, MG-132"
  },
  {
    "caption": "(F)Immunoblot  analysis of K48-, K63-linked, and total ubiquitination  of MEKK2  following precipitation of MEKK2  with MEKK2-specific antibody from lysates of Nedd4l+/+ and Nedd4l-/- MEFs  treated with  MG-132. Data information: All experiments were performed three times, and representative data are shown.",
    "molecules": "MG-132"
  },
  {
    "caption": "(B) The cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h. Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins. Fractions: WCL, whole cell lysates; Cyto, purified cytoplasm; Mito, purified mitochondria. Organelle markers: VDAC1, mitochondria; GAPDH, cytoplasm.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(A) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells were immunostained with antibodies against HBsAg (white), TOM20 (blue), and Parkin (red). Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin.",
    "molecules": "3-MA, BafA1"
  },
  {
    "caption": "(D) GFP-LC3-expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells prestained with LysoTracker (Lyso, red) were immunostained with antibodies against HBsAg (white) and TOM20 (blue). Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (−) cells are marked. In the zoom images, the arrows denoting white puncta indicate GFP-LC3 puncta (green) colocalized with TOM20 and lysosome. (E) Quantification of the colocalization of GFP-LC3 puncta containing lysosome with mitochondria in the panel (D).",
    "molecules": "3-MA, BafA1"
  },
  {
    "caption": "(A-C) Parkin silencing accelerates HBV-induced mitochondrial apoptotic signaling. HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non-targeting (NT) and Parkin siRNA, respectively, for 72 h. Cells were used for Western blot analysis using antibodies specific to the indicated proteins (A), caspase-3/7 activity assay (B), and TUNEL assay (C)",
    "molecules": "tetracycline"
  },
  {
    "caption": "(E) Confirmation of PARylated proteins with an in vivo PARylation assay. FLAG-tagged candidate proteins were expressed in protoplasts, and immunoprecipitated by α-FLAG antibody after feeding protoplasts with 32P-NAD+. The immunoprecipitated proteins were separated in 10% SDS-PAGE and detected by autoradiography (top panel). The input of FLAG-tagged proteins is shown in an α-FLAGimmunoblot (middle panel), and the protein loading control is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). The experiments were repeated three times with similar results.",
    "molecules": "NAD+"
  },
  {
    "caption": "(A) flg22-induced callose deposition is reduced in ddl-6 and parp1,2. Leaves of four-week-old plants were inoculated with 0.5 µM flg22 for 20 hr or hrcC at OD600=0.2 for 24 hr and stained with aniline blue. Callose deposits were visualized under UV light and quantified by Image J. The data are shown as mean ± SE (n=12). Bar=100 µm.",
    "molecules": "callose, Callose"
  },
  {
    "caption": "(E) flg22-induced ROS in Col-0 and ddl-6. Leaf discs from four-week-old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min. The data are show as mean± SE (n=24).",
    "molecules": "ROS"
  },
  {
    "caption": "(E) In vitro PARylation of DDL by PARP2. Immunoprecipitated DDL-FLAG proteins from Arabidopsis protoplasts were incubated with GST-PARP2 in a PARylation reaction containing Biotin-NAD+. The reactions in lane 4 and 5 contained 1 mM 3-AB or GST-PARG1 respectively. PARylated proteins were detected by streptavidin-HRP antibody (Strep), which recognizes Biotin-labelled PAR.",
    "molecules": "NAD+, 3-AB"
  },
  {
    "caption": "(A) flg22 stimulates DDL PARylation. DDL-FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P-NAD+. Protoplasts were treated without or with 100 nM flg22 for 30 min. PARylated DDL was immunoprecipitated with α-FLAG affinity beads, separated on 10% SDS-PAGE and visualized by autoradiography. PAR(DDL) indicates PARylated DDL proteins. A representative result from three repeats is shown.",
    "molecules": "NAD+"
  },
  {
    "caption": "(B-C) DDL PARylation is reduced in parp1, parp2 and parp1,2 mutants. Protoplasts isolated from parp1or parp2 single mutants (B), or parp1,2 double mutants (C) were used for in vivo PARylation assay with 32P-NAD+. Protoplasts were treated with 100 nM flg22 for 30 min. The experiments were repeated twice with similar results.",
    "molecules": "NAD+"
  },
  {
    "caption": "(B) in vivo PARylation of DDL-N or DDL-C in protoplasts fed with 32P-NAD+. PARylation was visualized with autoradiography.",
    "molecules": "NAD+"
  },
  {
    "caption": "(C) In vitro PARylation of DDL-N or DDL-C. FLAG-tagged DDL-N or DDL-C was expressed and immunoprecipitated from Arabidopsis protoplasts and subjected to in vitro PARylation by GST-PARP2 with Biotin-NAD+. PARylated proteins were detected by streptavidin-HRP antibody.",
    "molecules": "NAD+"
  },
  {
    "caption": "(F) DDL12E mutants substantially diminish its PARylation in vivo. In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P-NAD+.",
    "molecules": "NAD+"
  },
  {
    "caption": "(D) flg22-induced callose deposition in transgenic lines. Leaves of four-week-old plants were inoculated with 500 nM flg22, and callose deposits were stained with aniline blue at 20 hpi. Number of callose deposits was quantified with Image J (mean ± SE, n=10). All experiments were repeated three times with similar results.",
    "molecules": "callose"
  },
  {
    "caption": "B: U2-OS cells were transfected with the indicated siRNAs. The indicated cells were treated with etoposide (50 µM, 90 min. before analysis). Whole-cell extracts were immunoblotted with the indicated antibodies. The result is representative of three biological replicates.",
    "molecules": "etoposide"
  },
  {
    "caption": "A: Nuclear-cytoplasmic fractionation of U2-OS cells. Indicated samples were transfected with siRNAs against TSG101. PARylation was inhibited by olaparib (10 µM, 24 hours before analysis). Cytoplasmic export of p-ATM was induced by etoposide (50 µM, 45 minutes before analysis). Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. The fractionation efficiency was controlled by the respective subcellular marker proteins (PARP1 and LDH-A). PARylation was detected using the Pan ADPr reagent. Note that PARylation is activated by shearing forces during extract preparation (lane 1)",
    "molecules": "etoposide, olaparib, ADPr"
  },
  {
    "caption": "B: U2-OS cells were transfected with non-targeting (-) or TSG101-targeting (+) siRNAs and cells were irradiated (+) or not (-) as indicated (20 Gy) 10 min. before analysis. Whole-cell extracts were immunoblotted with the 10H PAR monoclonal antibody.",
    "molecules": "PAR"
  },
  {
    "caption": "C: siRNA-transfected U2-OS cells as in B were etoposide-treated or not, as indicated. Whole-cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly- and mono-(ADP-ribose). Note that PARylation also occurred during cell lysis in untreated cells.",
    "molecules": "etoposide, ADP-ribose, ADPr"
  },
  {
    "caption": "A: Proximity ligation assay (PLA) assay with unstimulated, etoposide (Eto)-treated (50 µM, 10 min. before analysis), or etoposide plus olaparib-(Olap) co-treated (50 µM of etoposide, 10 min. before analysis and 10 µM of olaparib 24 hours before analysis) U2-OS cells. The red dots represent PARP1-TSG101 interaction. The nucleus is stained with DAPI (blue). Scale bar: 10 µm. As negative controls, PLA was performed without primary PARP1 or TSG101 antibodies. As an additional control, PLA was performed in PARP1 depleted cells to show specificity B: Quantification of PLA dots per nuclei in A. Blind counting was performed from 30 replicates three biologically independent experiments. The conditions were compared with an ordinary one-way ANOVA (ns, p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001).",
    "molecules": "DAPI, Eto, etoposide, Olap, olaparib"
  },
  {
    "caption": "F: In-vitro PARylation reactions were carried out with the indicated recombinant and purified proteins, including 8 ng PARP1 in the presence of sssDNA (sheared salmon sperm DNA) and MgCl2. 100 ng of TSG101 was added to the reaction, as indicated. The PARylation reaction was also carried out using 100 ng of BSA or RNF113A as negative controls.",
    "molecules": "MgCl2"
  },
  {
    "caption": "B: Representative images of PARP1-GFP association with laser-microirradiation sites in untreated (mock), olaparib-treated (10 µM, 24 hours before analysis) or siTSG101-transfected U2-OS cells at indicated times post-irradiation. Scale bar: 7.5 µm. The image is representative of nine replicates from three biologically independent experiments. Go to Movies EV1-3 for further illustration.",
    "molecules": "olaparib"
  },
  {
    "caption": "C: Cells were transfected with non-targeting or TSG101-targeting siRNAs. Immunofluorescence staining was performed with the indicated antibodies (red: cleaved caspase 3; green: γH2AX). Nuclei were stained with DAPI (blue). The image is a representative of 3 biologically independent experiments. Scale bar: 30 µm. See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR-treatment. D: Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. Conditions were compared with an unpaired t-test (*p < 0.05, **p < 0.01).",
    "molecules": "DAPI"
  },
  {
    "caption": "A Immunoblot of endogenous BiP resolved by native-PAGE from lysates of CHO-K1 S21 wild-type (wt) or FICD-/- cells either transiently overexpressing wild-type FICD (high expression level; Hi) or mutant FICDE234G (E/G) or stably expressing recombinant wild-type FICD (low expression level; Lo). The cells in lanes 1-4 were mock transfected. Where indicated cells were exposed to cycloheximide (CHX; 100 µg/mL) for 3 h before lysis. Unmodified ('A') and AMPylated ('B') monomeric and oligomeric (II and III) forms of BiP are indicated. Immunoblots of the same samples resolved by SDS-PAGE report on FICD, total BiP and eIF2α (loading control). Data representative of four independent experiments are shown.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "B Wild-type FICD forms homomeric complexes in vivo. Immunoblots of orthogonally-tagged wild-type and Leu258Asp mutant FICD in the input cell lysate and following recovery by pull-down with streptavidin (recognising the AviTag) or anti-FLAG antibody. Proteins were detected with fluorescently-labelled streptavidin (StrepIR800) or FLAG antibody. Data representative of three independent experiments are shown.",
    "molecules": "StrepIR800, streptavidin"
  },
  {
    "caption": "F Autoradiograph of BiP, AMPylated in vitro by the indicated FICD derivatives, with [α-32P]-ATP as a substrate and resolved by SDS-PAGE. Proteins in the gel were visualised by Coomassie staining. A representative result of three independent experiments is shown. The graph shows the quantified relative BiP-AMP signals and SD bars generated by wild-type (wt) FICD and the indicated monomeric mutants.",
    "molecules": "AMP, ATP, Coomassie, 32P"
  },
  {
    "caption": "A Monomerising FICD mutations inhibit deAMPylation. Shown is a representative plot of data points and fit curves of the time-dependent deAMPylation of a fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) as detected by a change in fluorescence polarisation (FP). DeAMPylation rates calculated from independent experiments",
    "molecules": "FAM, AMP"
  },
  {
    "caption": "B-C Dimer interface mutants both AMPylate and deAMPylate BiP. Shown are representative autoradiographs of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence or absence of the co-substrate BiP (arrow indicates direction of nucleotide migration). The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. Plotted below are mean values ± SD from at least three independent experiments. Unpaired t-tests were performed.",
    "molecules": "AMP, ATP, 32P"
  },
  {
    "caption": "E Detection of the time-dependent accumulation of AMPylated BiPT229A-V461F in radioactive reactions, containing [α-32P]-ATP and the indicated FICD proteins, in the presence of excess trap. At the specified time-points samples were taken and analysed by SDS-PAGE. The autoradiograph (32P) illustrates the radioactive signals, which represent AMPylated BiP. Proteins were visualised by Coomassie staining. The radioactive signals were quantified and presented in the graph below. Mean values ± SD of three independent experiments are shown.",
    "molecules": "ATP, Coomassie, 32P"
  },
  {
    "caption": "A Autoradiographs of in vitro reactions containing varying concentration of wild-type FICD protein and fixed concentrations of BiPT229A-V461F and [α-32P]-ATP as co-substrates, resolved by SDS-PAGE after the indicated incubation times. The proteins were visualised by Coomassie staining of the gel (bottom). The reactions shown on the right were performed in the presence of an excess of S-SFICDA252C-H363A-C421S (trap) to delay de-modification of BiP. Representative gels are shown, and similar results were observed in three independent experiments. B As in (A) but with 0.2 µM of the indicated FICD variant. The radioactive signals were detected by autoradiography, quantified, and normalised to the signal in lane 6. The mean radioactive signals ± SD from three independent experiments are given. The proteins were visualised by staining with Coomassie. C As in (A) but with dilutions of FICDC421S or covalently linked S-SFICDA252C-C421S. Reactions were preceded by a 16 h incubation of FICD in presence or absence of the reducing agent (DTT). Representative gels are shown of three independent experiments.",
    "molecules": "DTT, ATP, Coomassie, 32P"
  },
  {
    "caption": "D Forced dimerisation does not significantly alter deAMPylation rates. Time-dependent deAMPylation of fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) assayed by fluorescence polarisation A representative experiment (data points and fit curves) is shown",
    "molecules": "FAM, AMP"
  },
  {
    "caption": "E Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. AMP signals were normalised to the total nucleotide signal in each sample and the graph below plots mean values ± SD from at least three independent experiments.",
    "molecules": "AMP, ATP, 32P"
  },
  {
    "caption": "C Radioactive in vitro AMPylation reactions containing the indicated FICD proteins, [α-32P]-ATP, and BiPT229A-V461F were analysed by SDS-PAGE. The radioactive BiP-AMP signals were detected by autoradiography and proteins were visualised by Coomassie staining of the gel.",
    "molecules": "AMP, ATP, Coomassie, 32P"
  },
  {
    "caption": "D Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. The graph shows mean AMP values ± SD from three independent experiments.",
    "molecules": "AMP, ATP, 32P"
  },
  {
    "caption": "F A plot of the melting temperature of the indicated FICD proteins in absence (Apo) or presence of nucleotides. Shown are the mean Tm values ± SD of three independent DSF experiments. Monomeric FICDL258D (mFICD) and FICDL258D-E234G (mFICDE/G) as well as dimeric wild-type FICD (dFICD) and FICDE234G (dFICDE/G) were tested. ADP and ATP concentrations in mM are given in parentheses.",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "A BioLayer interferometry (BLI) derived association and dissociation traces of monomeric FICDL258D-H363A (mFICDH363A) or dimeric FICDH363A (dFICDH363A) from immobilised biotinylated BiPT229A-V461F in absence or presence of nucleotides. Unless indicated (*) BiP was saturated with ATP before exposure to FICD variants. A representative experiment of three independent repetitions is shown. B BLI dissociation traces of proteins as in (A). At t = 0 a pre-assembled complex of immobilised, ATP-saturated BiP and the indicated FICD proteins (associated without ATP) were transferred into a solution without or with ATP, as indicated. A representative experiment is shown and the biphasic dissociation kinetics are quantified in (C) and (D). C Graph of the slow dissociation rates (koff,slow) of monomeric FICD from BiP:ATP as shown in (B). Bars represent mean values ± SD of three independent experiments. D The ATP-induced fold change in the percentage of the dissociation phase attributed to a fast dissociation (%Fast), koff,fast, and koff,slow derived from the data represented in (B). Bars represent mean values ± SD of three independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "E (i) BLI assay workflow used to derive FICD dimer off rate data presented in (ii) (ii) Observed dimer off rates under different nucleotide conditions (5 mM, where applicable). ATP, but no ADP, significantly increases the dimer dissociation rate [n.s.: not significant by Tukey test]. Data shown is the mean ± SD of four independent experiments.",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "F Validation of the homo-FRET dimerisation probe. The dimerisation sensitive TMR fluorescence of the labelled FRET dimer (2.5 nM) is de-quenched specifically by equilibration with excess unlabelled dimerisation-competent FICDH363A but not monomeric FICDL258D-H363A. Mean ± SD of three independent experiments.",
    "molecules": "TMR"
  },
  {
    "caption": "G Homo-FRET measurement of nucleotide dependent modulation of the FICD monomer-dimer equilibrium. ATP increases and ADP decreases the proportion of monomeric FICD. * Plateaus were constrained to a shared best fit value. Data shown is the mean ± SD of four independent experiments.",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "Widefield images of RPA-mCherry-ssDNA molecules (magenta) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).",
    "molecules": "ssDNA"
  },
  {
    "caption": "Widefield images of Rad51-ssDNA molecules (unlabeled) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).",
    "molecules": "ssDNA"
  },
  {
    "caption": "Kymograph illustrating the dissociation of GFP-Rdh54 from Rad51-ssDNA; arrowhead highlights the time point at which free GFP-Rdh54 was flushed from the sample chamber.   Graph depicting the dissociation rate of GFP-Rdh54 from Rad51-ssDNA. Error bars represent s.d. of individual ssDNA molecules (N=25).   ",
    "molecules": "ssDNA"
  },
  {
    "caption": "Kymograph showing the association of GFP-Rdh54 with a Rad51-ssDNA molecule; arrowhead highlights the injection time point for GFP-Rdh54.   Association rates of GFP-Rdh54 on individual Rad51 PSCs (N=44). Red lines represent the mean and s.d.   ",
    "molecules": "ssDNA"
  },
  {
    "caption": "Images of Rad51-ssDNA molecules bound by both mCherry-Rdh54 (30 nM; magenta) and GFP-Rad54 (green; 30 nM).",
    "molecules": "ssDNA"
  },
  {
    "caption": "Quantification of bound GFP-Rad54 (10 nM) signal intensity for Rad51-ssDNA molecules with and without bound mCherry-Rdh54 (10 nM). Error bars represent s.d. of individual ssDNA molecules (N = 21).",
    "molecules": "ssDNA"
  },
  {
    "caption": "Images showing increasing concentrations of GFP-Rad54 (green) only or mCherry-Rdh54 (magenta) only bound separately to Rad51-ssDNA molecules.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Images of GFP-Rad54 (green) and mCherry-Rdh54 (magenta) co-bound to the same Rad51-ssDNA molecules.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules in the presence of 30 nM Hed1.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Quantification of the integrated GFP signal intensity for GFP-tagged Rad54 (N = 75), Rdh54NRad54 (N = 46), Rdh54 (N = 50) and Rad54NRdh54 (N = 35; 1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules. The data was fit by non-linear regression and error bars represent s.d. for individual DNA molecules.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Widefield images of Atto565-labeled PSCs (magenta) bound to dsDNA curtains stained with YOYO1 (green). The PSCs were prepared with the indicated combinations of Rad51, Rdh54 and GFP-Rad54.   Number of Atto565-labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only (minus Rad51 ; N = 372), Rad51 plus Rad54 only (N= 117), Rad51 plus Rdh54 only (N = 258), Rad51 plus Rdh54 and Rad54 (N = 303). The error bars represent the s.d. for three independent experiments.  ",
    "molecules": "Atto565, dsDNA, YOYO1"
  },
  {
    "caption": "PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 (N= 181); Rad54 plus Rdh54-K318R (N= 115); Rad54-K341R plus Rdh54 (N = 90); or Rad54-K341R plus Rdh54­-K318R (N = 54), as indicated. Error bars were generated by bootstrapping. The asterisks denote the targeted region of homology on the donor dsDNA molecules.",
    "molecules": "dsDNA"
  },
  {
    "caption": "D-loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 (300 nM), tailed duplex substrate (15 nM) and a pUC19 plasmid dsDNA (0.3 nM).   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.   ",
    "molecules": "dsDNA"
  },
  {
    "caption": "ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Quantification of Rad51 Atto565-tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 (N = 117), Rdh54NRad54 (N = 150) or Rad54NRdh54 (N=175). The error bars represent the s.d. for three independent experiments.",
    "molecules": "Atto565, dsDNA"
  },
  {
    "caption": "Fraction translocating Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 117), Rdh54NRad54 (N = 150), Rad54NRdh54 (N = 0) and Rdh54 (N = 0).",
    "molecules": "Atto565"
  },
  {
    "caption": "Translocation velocities of Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 86) and Rdh54NRad54 (N = 18). The error bars represent the s.d. of the data.",
    "molecules": "Atto565"
  },
  {
    "caption": "Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rad54∆ yeast strains.   Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rdh54∆ yeast strains.   ",
    "molecules": "MMS"
  },
  {
    "caption": "A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Okp1 (A) each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.",
    "molecules": "deuterium, hydrogen-deuterium"
  },
  {
    "caption": "A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Ame1 (B), each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.",
    "molecules": "deuterium, hydrogen-deuterium"
  },
  {
    "caption": "B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. B) of full-length COMA (lane 11) with full-length polyhistidine-tagged Mcm21 (His-Mcm21) and Ctf19, or COMA variants with polyhistidine-tagged Mcm21 D-RWD domain (His-Mcm21_D-RWD; K. lactis Mcm21 residues 108-293) and Ctf19 D-RWD domain (Ctf19_D-RWD; K. lactis Ctf19 residues 107-270) We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.",
    "molecules": "polyhistidine, Ni2+"
  },
  {
    "caption": "B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. C) of Ame1-Okp1 variants with polyhistidine-tagged Okp1 variants (His-Okp1 variants). We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant.",
    "molecules": "polyhistidine, Ni2+"
  },
  {
    "caption": "D) Image of SDS-PAGE gel with fractions of two purified Ctf19-Mcm21-Okp1 variants (after SEC); expected migrating position of non-proteolysed Okp1 variant (compare with its position in SDS-PAGE images of COMA or Ame1-Okp1 variants in B or C) is marked; 2 and 3: SEC-purified fractions of samples from different elution fractions from ion-exchange chromatography. When we co-expressed full-length Okp1 with Ctf19 and Mcm21, which has a coding regions for an N-terminal polyhistidine-tag, we did not co-purify substantial amounts of full-length Okp1 with Ctf19-Mcm21, probably because Okp1 was prone to proteolysis (in the absence of Ame1).",
    "molecules": "polyhistidine"
  },
  {
    "caption": "D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp1 (D) each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.",
    "molecules": "deuterium"
  },
  {
    "caption": "D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp2 (E), each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.",
    "molecules": "deuterium"
  },
  {
    "caption": "A) Plots showing deuterium-exchanged peptides, after deuterium exchange of 10 sec of Okp1 in Ctf19D-RWD-Mcm21D-RWD-Okp1229-336, in full-length COMA, or in Ame11-260-Okp1123-336. Plots for full time course are in Appendix Fig S6C.",
    "molecules": "deuterium"
  },
  {
    "caption": "D) Close-up view of the Okp1 binding site of Ctf19-Mcm21 with Ctf19-Mcm21 binding motif of Okp1; Okp1 residues, and some Ctf19 or Mcm21 sidechains, are shown as sticks (nitrogen: blue, oxygen: red); Ctf19-Mcm21 shown as secondary structure cartoon-diagram.",
    "molecules": "nitrogen, oxygen"
  },
  {
    "caption": "A) Representative images of dilution-series growth assay of haploid S. cerevisiae clones with cnn1Δ, Okp1_fl, Okp1_cmΔ, Okp1_fl cnn1Δ, or Okp1_cmΔ cnn1Δ, grown for two or three days on solid YPD agar or on solid YPD agar with 20 μg ml−1 benomyl. We show two unique clones each for Okp1_fl cnn1Δ or Okp1_cmΔ cnn1Δ. Arrow indicates decreasing cell-density. Spots are from cell samples that we sequentially diluted four fold.",
    "molecules": "benomyl"
  },
  {
    "caption": "(D-E) Developmental rates of (D) control (control-MO injected), Tet1-, Tet2- and Tet3-single KD and Tet-TKD embryos and (E) control (control-MO + Tet2 mRNA co-injected), Tet2-KD2 (Tet2 MO2 injected), Tet2-KD2 + GFP mRNA and Tet2-KD2 + Tet2 mRNA embryos until blastocyst stage. Indicated p-values were calculated using log rank (Mantel-Cox) test against (D) control embryos or (E) Tet2-KD2 embryos or as indicated (ns = non-significant, * = p < 0.05, **** = p < 0.0001; numbers of analyzed embryos for each condition are indicated in parenthesis).",
    "molecules": "MO"
  },
  {
    "caption": "(A-B) Representative images of (A) 5hmC- and (B) 5mC and 5caC-IF of control (control-MO injected), Tet1+2-DKD and Tet-TKD zygotes at 12 hpf. Paternal and maternal pronuclei are indicated, Pb = polar body; Sp = sperm; Scale bar = 20 μm.",
    "molecules": "5caC, 5hmC, 5mC, MO"
  },
  {
    "caption": "(C-E) Quantification of paternal/maternal (C) 5mC-, (D) 5hmC-, and (E) 5caC-signal ratios of 12 hpf derived zygotes normalized against DNA signal. A total of 5-16 zygotes from two to three experiments per condition were analyzed. Significance to control zygotes was calculated using ordinary one-way ANOVA with Tukey's multiple comparisons test (C, E) or Welch ANOVA with Dunnett's T3 multiple comparisons test (D) and significant differences are indicated (* = p < 0.05, ** = < 0.01, *** = p < 0.001, **** = p < 0.0001, data are represented as mean ± SD, with dots representing single zygotes).",
    "molecules": "5caC, 5hmC, 5mC, DNA"
  },
  {
    "caption": "(A) % CpG methylation of 20 kb tiles in 5% bins in control or Tet-TKD 2-cell embryos. All samples from one condition were merged for this visualization.",
    "molecules": "CpG"
  },
  {
    "caption": "(H) Developmental rate of embryos derived from control, Tet2-MO2, Tet2-MO2 + Tet2 full-length mRNA (as shown in Fig. 1E) and Tet2-MO2 + Tet2 truncated mRNA (no catalytical domain) injected oocytes. Indicated p-values were calculated using the log rank (Mantel-Cox) test against Tet2-MO2 KD or as indicated (* = p < 0.05, *** = p < 0.001, **** = p < 0.0001, numbers of analyzed embryos for each condition is indicated in parenthesis).",
    "molecules": "MO"
  },
  {
    "caption": "(A) ARPE-19 cells were incubated in the presence of DMSO (Control), Tunicamycin (5 µg/ml), or starved (EBSS) for 16 h. Cells were fixed, permeabilized and stained with an antibody against TFE3. Scale bar, 10 µm. Data are representative of three independent experiments.",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "(C) ARPE-19 cells incubated with Tunicamycin as indicated in (A) were immunostained with antibody against CHOP. Scale bar, 10 µm. Data are representative of three independent experiments.",
    "molecules": "Tunicamycin"
  },
  {
    "caption": "(D) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or starvation (Strv.) treatments as indicated in (A) (mean ± SD of three independent experiments, one-way ANOVA analysis versus Control treated cells, ***p < 0.001; n>400 cells per condition).",
    "molecules": "Tunicamycin"
  },
  {
    "caption": "(E) Wild-type mouse embryonic fibroblast (MEF) cells were incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "(F) Wild-type MEF cells were incubated in the presence of DMSO (Control) or BFA (2.5 µg/ml) for 16 h. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(G) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or BFA 16 h treatment (mean ± SD of two independent experiments, student's t-test analysis versus DMSO treated cells, ***p < 0.001; n>400 cells per condition).",
    "molecules": "BFA, DMSO"
  },
  {
    "caption": "(H) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO (Ctrl.) or Tunicamycin (Tun.) for 16 h using TFE3, TFEB, Lamp1 (as a lysosomal membrane marker), and H3b (as a nuclear marker). Data are representative of three independent experiments.",
    "molecules": "DMSO, Tun, Tunicamycin"
  },
  {
    "caption": "(A) Immunoblot of protein lysates from wild-type, PERK-KO and GCN2-KO MEF cells incubated in the presence of DMSO (Control), Tunicamycin (0.1 µg/ml), or starved in EBSS for 16 h. Data are representative of three independent experiments.",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "(B-D) Wild-type (B), GCN2-KO (C), and PERK-KO (D) MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) as indicated in (A). Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(E-F) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control), Tunicamycin treatments for 16 h (E), or starvation for 2 h (F) (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated cells, ns= not significant, ***p < 0.001; n>400 cells per condition).",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "(H) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or Torin-1 (250 nM) treatments for 1 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated wild-type cells, ***p < 0.001; n>400 cells per condition).",
    "molecules": "DMSO, Torin-1"
  },
  {
    "caption": "(C) Immunoblots of TFE3-Ser321 phosphorylation state in nuclear fractions of ARPE-19 cells incubated with DMSO (Ctrl.), Tunicamycin (Tun.), or Torin-1. Data are representative of three independent experiments.",
    "molecules": "DMSO, Torin-1, Tun, Tunicamycin"
  },
  {
    "caption": "(D) Immunoblots of protein of a GST pull-down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin (5 µg/ml) (Tun.) for 16 h. Data are representative of three independent experiments.",
    "molecules": "DMSO, Tun, Tunicamycin"
  },
  {
    "caption": "(E) Immunoblots of protein lysates from ARPE-19 cells incubated in the presence of DMSO (Ctrl.), Tunicamycin (5 µg/ml) (Tun.), or starved in EBSS (Strv.) for 16 h. Data are representative of three independent experiments.",
    "molecules": "DMSO, Tun, Tunicamycin"
  },
  {
    "caption": "(F) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Tunicamycin + FK506 treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus Tunicamycin treated cells, ***p < 0.001; n>400 cells per condition).",
    "molecules": "FK506, Tun, Tunicamycin"
  },
  {
    "caption": "(G) Quantification of the percentage of calcineurin depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Starvation (Strv.) treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment, ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001; n>400 cells per condition).",
    "molecules": "Tun, Tunicamycin"
  },
  {
    "caption": "(J) Quantification of the percentage of ATF4 depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h (mean ± SD of two independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment***p < 0.001; n>400 cells per condition).",
    "molecules": "Tunicamycin"
  },
  {
    "caption": "(B) Relative Quantitative Real-Time PCR analysis of MCOLN1, ATP6V1C1, HEXA and UVRAG mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 upon incubation with DMSO (Control) or Tunicamycin (5 µg/ml) for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the same treatment condition in the siNon-Target (siNT) treated cells or versus the DMSO condition in siNon-Target (siNT) treated cells, *p < 0.05, **p < 0.01, ***p < 0.001).",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "(A) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A, or TFE3 upon incubation with DMSO (Control), Tunicamycin (5 µg/ml) or starved in EBSS for 16 h. Data are representative of five independent experiments.(B) Quantification of ATF4, CHOP and GADD34 protein levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, *p < 0.05, **p < 0.01, ***p < 0.001).",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "(A-D) Relative Quantitative Real-Time PCR analysis of ATF4 (A), ASNS (B), CAT1 (C) and xCT (D) mRNA transcript levels in ATF4-depleted ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A upon incubation with DMSO (Control), Tunicamycin (5 µg/ml), or starved in EBSS for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected and siRNA Non-Target (siNT) treated cells, *p < 0.05, ***p < 0.001).",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "(E) ChIP-qPCR analysis of the ATF4 promoter and 2000bp upstream of the region of interest (ATF4-Control) from MEF cells that were untreated (control), starved for 2 h, or treated with Tunicamycin (0.1 µg/ml) for 16 h. Amplification regions are indicated by arrows. Chromatin DNA was immunoprecipitated with antibodies for anti-TFE3. Bar graphs show the amount of immunoprecipitated DNA detected by the real-time PCR assay. Values were normalized to the input and plotted as relative enrichment compared to untreated conditions (means ± SD of three independent experiments, students t-test, *p < 0.05, **p < 0.01).",
    "molecules": "Tunicamycin"
  },
  {
    "caption": "(A) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times. Data are representative of three independent experiments.(B) Quantification of ATF4 protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (A) (mean ± SD of the ATF4 to actin protein ratio from three independent experiments, student's t-test versus the corresponding time point in Null-MEF cells, ns= not significant, **p < 0.01).",
    "molecules": "DMSO, Tun, Tunicamycin"
  },
  {
    "caption": "(C) Cell viability was determined in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times (mean ± SD of the percentage of viable cells compared to control from three independent experiments, one-way ANOVA analysis versus the corresponding time point in Null-MEF cells ***p < 0.001).",
    "molecules": "DMSO, Tun, Tunicamycin"
  },
  {
    "caption": "(D) Chromatin condensation, an indication of cells undergoing apoptosis, was assessed in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342. Scale bar, 20 µm. Data are representative of three independent experiments.",
    "molecules": "DMSO, Tunicamycin"
  },
  {
    "caption": "e, Representative cresyl violet staining  images of midsagittal sections of  cerebellar vermis  of WT and Cdc14  KO mice  at P0, P5 and P9. Fissures  underdeveloped in mutant samples are labeled with arrowheads. Scale bar, 700 μm. The bar plots show the quantification of the  cerebellar vermis  area, the number of folia  and the average length of the fissures, respectively. Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.",
    "molecules": "cresyl violet"
  },
  {
    "caption": "f, Detailed cresyl violet staining  images of midsagittal sections of cerebellar vermis of the same samples. The molecular layer (ML),  internal granule cell layer (IGL) and external granule cell layer  (EGL) are indicated. The width of these layers is shown in the bar plots. Data information: Bars indicate mean ± SEM. *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice. In f, each point represents the average value of 9 different random measurements across distinctive folia  for each mouse.",
    "molecules": "cresyl violet"
  },
  {
    "caption": "g, Representative images of misdigital sections stained  with DAPI  (blue), Calbindin  (green) and GFAP  (red) of cerebellar vermis of wild-type ad Cdc14  DKO mice  at 9 days after birth. Scale bar, 50 μm. Plots show the quantification of Purkinje cell dendritic  length (average distance of 30 random measurements) and the number of cells per 100 μm from 3 different fields from 3 different mice  in each plot. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.",
    "molecules": "DAPI"
  },
  {
    "caption": "a. Confocal microscopy  analysis of wild-type and Cdc14  DKO embryonic stem cells (ESCs) subjected to neural differentiation. Neuronal  marker TUJ1  (green), glial  marker GFAP  (red) and nuclear  marker DAPI  (blue) are stained  and quantified after 18 days in differentiation  media. Each dot represents the ratio between TUJ  or GFAP  and DAPI  area in an image containing at least 100 cells from 4 independent experiments with two different clones. **** p-value<0.0001. Student's t-test. Scale bar 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "c, Confocal microscopy analysis of WT and Cdc14  DKO ESC  subjected to neural differentiation. Neural progenitor marker Nestin  was stained  and quantified after 6 days in differentiation  media. Each dot represents the ratio between Nestin  and DAPI  area in an image containing at least 100 cells. ****, p<0.0001; Students' t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "e, Western blot  analysis of UTF1  expression in WT and Cdc14  DKO ESCs  in absence or presence of the kinase  inhibitors roscovitine  (R) or trametinib  (T).",
    "molecules": "roscovitine, trametinib"
  },
  {
    "caption": "f, Immunoblot  analysis of UTF1  protein stability after transfection of HeLa  cells with UTF1  and GFP  or CDC14B-GFP. Cells were treated with cycloheximide  (CHX) and total proteins were detected at different time points  as indicated. Cyclin B1  and β-catenin  are used as controls.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "g, Western blot  analysis of UTF1  WT or mutants in three (UTF1-3KR) or five (UTF1-5KR) lysines in absence or presence of trametinib  (T) or roscovitine  (R).",
    "molecules": "roscovitine, trametinib"
  },
  {
    "caption": "Gene-trap-based genetic screen in haploid human  cells for regulators of p38     kinase activation (p38 Thr180/Tyr182 phosphorylation) in response to hyperosmotic shock (500 mM Sorbitol, 1 h). Per gene (dots), the frequency of gene-trap insertions in the 'high' phospho-p38    population divided by the frequency of insertions in the 'low' population is plotted as mutation index (y-axis) against the total number of insertions assigned to the gene (x-axis). Significant negative and positive regulators are colored in orange and blue, respectively (two-sided Fisher's exact test, false discovery rate-corrected, p≤0.05).",
    "molecules": "Sorbitol"
  },
  {
    "caption": "Human U2OS  cells and cells deleted for ZAK  (∆ZAK) were treated with anisomycin  (1 h) or 500 mM sorbitol  (1 h). Lysates were analyzed by immunoblotting as indicated.",
    "molecules": "anisomycin, sorbitol"
  },
  {
    "caption": "Unique gene-trap insertions (dots) mapped to the genomic ZAK  locus (x-axis) identified in the low (blue) and high (orange) channel of two individual haploid genetic screens for stress-induced p38  activation and Anisomycin(Brockmann et al., 2017)). The total number of identified insertions was similar for each channel within the individual screens. For visual purposes, insertion dots were spread on the y-axis and exons in the ZAK gene body schematic have been scaled up (compared to introns).",
    "molecules": "Anisomycin"
  },
  {
    "caption": "U2OS/S-HA-ZAKβ  cells were induced for expression with doxycycline (DOX) and subjected to the indicated drugs and treatments (1 h). Lysates were analyzed by immunoblotting  with the indicated antibodies. Sorb., sorbitol; Ani., anisomycin; Ars., arsenite.",
    "molecules": "Ani, anisomycin, Ars, arsenite, DOX, doxycycline, Sorb, sorbitol"
  },
  {
    "caption": "U2OS  cells stably expressing WT and kinase-dead (KD) versions of S-HA-ZAKβ  were pre-treated with ZAK  inhibitor (ZAKi - 0.5 h) and 500 mM sorbitol  (1 h) as indicated. Whole Cell Extracts (WCE) were analyzed by immunoblotting, and strep-pulldown  material was used in a  kinase assay, separated by SDS-PAGE and analyzed by autoradiography.",
    "molecules": "sorbitol, strep"
  },
  {
    "caption": "U2OS  and ∆ZAK cells rescued with S-HA-ZAKβ, were DOX-induced and treated with 500 mM sorbitol  (1 h) as indicated. Lysates were analyzed by immunoblotting  with the indicated antibodies.",
    "molecules": "DOX, sorbitol"
  },
  {
    "caption": "U2OS  and ∆ZAK cells were transfected with the indicated siRNAs and treated with sorbitol  (500 mM, 1 h) as indicated. Lysates were analyzed by immunoblotting  with the indicated antibodies. T1, TAK1; M2, MEKK2.",
    "molecules": "sorbitol"
  },
  {
    "caption": "U2OS  cells stably expressing Strep-HA-ZAKβ  or -ZAKα  were treated with sorbitol  (500 mM, 1 h) as indicated. Cells were fixed, immunostained  with HA  antibody and counter-stained  with DAPI. Data information: scale bars, 10 μm.",
    "molecules": "DAPI, sorbitol, Strep"
  },
  {
    "caption": "U2OS  cells stably expressing ZAKβ-GFP   were treated with sorbitol  (500 mM, 1 h), pre-extracted, fixed and immunostained  with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line. As in (B) except that Hela  cells were transfected with ZAKbeta-GFP  and immunostained  with 53BP1  antibody. Data information: scale bars, 10 μm.",
    "molecules": "sorbitol"
  },
  {
    "caption": "Cells from (B) were imaged by  live cell fluorescence microscopy. Sorbitol  (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.",
    "molecules": "Sorbitol"
  },
  {
    "caption": "U2OS  cells were transfected with constructs from (F) and imaged by  live cell fluorescence microscopy. Sorbitol  (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.",
    "molecules": "Sorbitol"
  },
  {
    "caption": "U2OS  and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ  constructs were treated with sorbitol  (500 mM, 5 min). Lysates were analyzed by immunoblotting  with the indicated antibodies.",
    "molecules": "sorbitol, Strep"
  },
  {
    "caption": "U2OS  and ∆ZAK  cells were embedded in agarose  and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting  with the indicated antibodies.",
    "molecules": "agarose"
  },
  {
    "caption": "U2OS  and ∆ZAK  cells stably expressing the indicated Strep-HA-tagged ZAKβ  constructs were embedded in agarose  and subjected to cyclic compression  for 5 or 30 min. Lysates were analyzed as in (B).",
    "molecules": "agarose, Strep"
  },
  {
    "caption": "C2C12 myoblasts  and myotubes  (6 d differentiation) were pre-treated with ZAK  inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol  (1 h) as indicated. Lysates were analyzed by immunoblotting  with the indicated antibodies.",
    "molecules": "sorbitol"
  },
  {
    "caption": "Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice  were treated with 500 mM sorbitol  (1 h). Lysates were analyzed by immunoblotting  with the indicated antibodies. *, unspecific band.",
    "molecules": "sorbitol"
  },
  {
    "caption": "Heatmap analysis of RNAseq data highlights the reduction of glucose transporters (glut1 and glut2) in yap-/- mutant larvae at 3 dpf",
    "molecules": "glucose"
  },
  {
    "caption": "Glucose tolerance test in 3-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Glucose tolerance test in 12-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m",
    "molecules": "glucose"
  },
  {
    "caption": "Relative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap-/- mutant larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap-/- larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m",
    "molecules": "13C, 13C6, glucose"
  },
  {
    "caption": "Effect of WZB-117 (WZB, 10 μM) treatment from 3-5 dpf in WT and lf:Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf:GFP reporters. Scale bar: 200μm",
    "molecules": "WZB, WZB-117"
  },
  {
    "caption": "Quantitative analysis of the effect of WZB treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>21, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m",
    "molecules": "WZB"
  },
  {
    "caption": "Quantitative analysis of the effect of Trehalose (100 μM) treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>70, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m",
    "molecules": "Trehalose"
  },
  {
    "caption": "Gross morphology of the liver from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 1 cm",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "Immunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 50μm",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "FDG PET imaging of coronal views from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Arrow is pointing to the liver",
    "molecules": "FDG, doxycycline, Tet"
  },
  {
    "caption": "Scintillation analysis of FDG uptake (fraction of injected dose per gram of liver tissue, %ID/g) in dissected liver tissue from WT and TetON Yap transgenic mice, treated with doxycycline for 10 days. n=4, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m",
    "molecules": "FDG, doxycycline, Tet"
  },
  {
    "caption": "A Hypocotyl lengths of wild-type and suc2 seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium with or without 3% sucrose. Representative seedlings are shown in the upper panel. Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 10). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). In the box plots (A-D), the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.",
    "molecules": "sucrose"
  },
  {
    "caption": "C Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown under continuous white light at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium containing either mock or 20 µM of dexamethasone (DEX). ns, not significant (Student's t-test P ≥ 0.05; n > 10). **P < 0.01 (Student's t-test); n > 10. Number indicates the ratio of hypocotyl lengths (Dex/Mock).",
    "molecules": "DEX, Dex, dexamethasone"
  },
  {
    "caption": "A Western blotting with anti-PIF4 antibody shows that PIF4 protein levels are decreased in the tps1-2;GVG::TPS1 mutants compared to wild-type plants. Seedlings were maintained under 12-h light/12-h dark cycles at 20 °C for 4 days and then transferred under continuous light on the 5th day. The growth temperature was increased to 28 °C or kept at 20°C for 4 hours at ZT 20-24 before harvesting for total protein extraction. Equal loading of samples is shown by Ponceau S staining.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "B Western blotting showing the levels of PIF4-Myc protein in PIF4-OX and PIF4-OX;tps1-2;GVG::TPS1 seedlings. Total protein was extracted from the seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Western blotting was probed using an anti-Myc antibody. Ponceau S staining is shown for equal loading.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "B, C Yeast two-hybrid assays showing the direct interaction between PIF4 and KIN10. Yeast clones were grown on the synthetic dropout medium with histidine (+HIS) or without histidine (-HIS) plus 25 mM of 3-amino-1,2,4-triazole (3-AT).",
    "molecules": "3-amino-1,2,4-triazole, 3-AT, HIS, histidine"
  },
  {
    "caption": "E GRIK1-activated KIN10 phosphorylates PIF4. In vitro phosphorylation assays were performed at 30 °C for 30 min using 1 μg of purified recombinant proteins (KIN10, GRIK1, or PIF4). 32P-labeled proteins were visualized by autoradiography. CBB, coomassie brilliant blue-stained gel.",
    "molecules": "CBB, coomassie brilliant blue, 32P"
  },
  {
    "caption": "F T6P inhibits PIF4 phosphorylation by KIN10. This assay was performed with 1 μg of each protein at 30 °C for 30 min in the presence of 1 mM T6P or sucrose (Suc).",
    "molecules": "Suc, sucrose, T6P"
  },
  {
    "caption": "The levels of PIF4-Myc are negatively correlated with the levels of KIN10-GFP. Total proteins were extracted from mesophyll protoplasts transfected with different amounts of KIN10-GFP and PIF4-Myc and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal loading.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal protein loading. Representative western blots are shown in (B).",
    "molecules": "CHX, cycloheximide, MG132, Ponceau S"
  },
  {
    "caption": "KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Quantification of relative PIF4-Myc levels is shown in (C). PIF4-Myc levels are presented as values relative to those at 0 h. In (C), different letters above each point indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 3). Error bars indicate s.d. (n = 3).",
    "molecules": "CHX, cycloheximide, MG132"
  },
  {
    "caption": "Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.",
    "molecules": "bafilomycin A1, chloroquine, MG132"
  },
  {
    "caption": "(B) Wild-type and FIP200−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(A) FAK+/− and FAK−/− MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(A) Wild-type and FIP200−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.",
    "molecules": "bafilomycin A1, DMSO, rapa, rapamycin"
  },
  {
    "caption": "(B) Wild-type and FIP200−/− MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(C) Wild-type and FIP200−/− MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2-ceramide for 2 h.",
    "molecules": "C2-ceramide, lithium chloride"
  },
  {
    "caption": "(C) Phosphatase sensitivity of ULK1. FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na2VO4, 50 mM KF, 15 mM Na4P2O7, and 1 mM EGTA).",
    "molecules": "Na2VO4, Phosphatase, λ phosphatase, EGTA, KF, Na4P2O7"
  },
  {
    "caption": "(D) Change in the expression levels of 17 genes defining anti-cytokine signature; log2 fold change at 24 hours post high SARS-CoV-2 infection from A549-ACE2 cells are shown.",
    "molecules": "cytokine"
  },
  {
    "caption": "(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.",
    "molecules": "Salmeterol"
  },
  {
    "caption": "(J) Overexpression of hTau increased STAT1-DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay (EMSA). *, indicates STAT1/DNA complex.",
    "molecules": "DNA"
  },
  {
    "caption": "(E, F) The representative images of STAT1 and pY-STAT1 in the brain of AD patients probed by co-immunohistochemical staining and quantitative analysis (hematoxylin stains nuclei, purple; DAB stains the target proteins, brown; n=5-6 slices). Arrowheads indicated typical nuclear staining of STAT1/pY-STAT1. Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.",
    "molecules": "DAB, hematoxylin"
  },
  {
    "caption": "Pharmacological inhibition of ERK1 (C, D) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts (C and the nuclear fraction (D measured by Western blotting (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.",
    "molecules": "Tyr"
  },
  {
    "caption": "Pharmacological inhibition of JNK1 (E, F) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts E) and the nuclear fraction F) measured by Western blotting (n=3). The alteration of pS-STAT1 (Ser727) confirms the efficacy of JNK1 inhibitors. Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.",
    "molecules": "Ser, Tyr"
  },
  {
    "caption": "Pharmacological inhibition of JAK2 (G, H) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts (G and the nuclear fraction (H (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.",
    "molecules": "Tyr"
  },
  {
    "caption": "knockdown JAK2 by siRNA (I, J) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts I) and the nuclear fraction J) (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.",
    "molecules": "Tyr"
  },
  {
    "caption": "(h) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3-2A. Histograms show the CFSE dye dilution derived from the indicated samples. Aphidicolin (2 μg/mL) was also used as a positive control.",
    "molecules": "CFSE, Aphidicolin"
  },
  {
    "caption": "Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3-2A cells by SAMHD1 siRNA or by tetracycline (50 nM) treatment, respectively. Expression of Tubulin is shown as a loading control (e: left). Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig. 3e (e: right).",
    "molecules": "tetracycline"
  },
  {
    "caption": "(d) LM-PCR analysis of the AID-induced DNA double-strand breaks in the Sµ region of WT or Samhd1 KO CH12F3-2A cells. Wedges indicate a three-fold increase in the DNA amount. Amplification of Gapdh was used as an internal loading control.",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Gamma-H2AX-ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA (% IP) normalized to the total amount of DNA used for the immunoprecipitation (n=3; mean + sd; two-tailed unpaired Student's t-test; ns = not significant).",
    "molecules": "DNA"
  },
  {
    "caption": "(f) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH/c-Myc translocations in CH12F3-2A cells (n = 2; mean ± sd; Fisher's exact test; ns = not significant; *** = p ≤ 0.001). The concentration dNTPs used for supplementation either individually or in combination were as follows: 2′-deoxyguanosine (dG), 0.15 mM; 2′-deoxyadenosine (dA), 1.0 mM; 2′-deoxycytidine (dC), 12 mM; and 2′-deoxythymidine (dT), 1.6 mM.",
    "molecules": "2′-deoxyadenosine, dA, 2′-deoxycytidine, dC, 2′-deoxyguanosine, dG, 2′-deoxythymidine, dT"
  },
  {
    "caption": "(d) Length of resected DNA at Sμ-Sα junctions obtained from the joining of DSBs with blunt ends. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown. (e) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends.",
    "molecules": "DNA"
  },
  {
    "caption": "(f) Length of resected DNA at Sμ-Sα junctions obtained from the joining of staggered DSBs. Left: Length of Sμ and Sα overhangs were 35 nt and 98 nt, respectively. Right: Length of Sμ and Sα overhangs were 35 nt and 187 nt, respectively. (g) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of staggered DSBs. Statistical significance as evaluated by Fisher's exact test is shown.",
    "molecules": "DNA"
  },
  {
    "caption": "C. Confocal images of MDA-MB-231 cells transfected with   ZsGreen  or ZsRASSF1C  grown in 3D-collagen  matrix. Cells were imaged and stained with Phalloidin-568 (red), pMLCII/Alexa‑633 (false‑coloured green) or imaged for   ZsGreen  (false-coloured white). Scale 20 μm.",
    "molecules": "Alexa‑633, collagen, Phalloidin-568"
  },
  {
    "caption": "D. Single  cell morphology assay  of MDA-MB-231  cells cultured in 3D-collagen and analysed for morphology 24 hours after transient knock-down using either a control sequence (NT, non-targeting) or a sequence targeting the RASSF1 locus. RASSF1 knock-down marks a reduction in rounded cells. Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.",
    "molecules": "collagen"
  },
  {
    "caption": "H. Quantification of rounded  versus elongated cells in single cell morphology assay in 3D‑collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231  cells when RASSF1C  or the described mutants are expressed. I. Representative confocal images show elongated or  rounded  cells per field of view of MDA‑MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D‑collagen and  stained with Phalloidin-568 (red) for  F‑actin and pMLCII/Alexa 633. Scale bars represent 10 μm. ",
    "molecules": "Alexa 633, collagen, Phalloidin-568"
  },
  {
    "caption": "D. Representative confocal images (upper row) and quantification of fluorescent intensity of  ALDH1 expression in MDA-MB-231  cells expressing RASSF1C (black bar in the graph) or after siRNA treatment against RASSF1  (grey bar). Lower row shows merged images with DAPI  (blue), ALDH1 (green) and RASSF1C (red). Scale bars represent 20 μm. Data Information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). Data information: A-C and F-M: Tha-RABV IHC D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C",
    "molecules": "RVC20, RVC58"
  },
  {
    "caption": "(C) A375 nuclei stained with DAPI (blue), Lamin A/C (green) and H3K9me3 (red) in the indicated migration conditions. Images shown represent a maximum projection of z-stacks (Scale bar= 5 µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Maximum projection confocal images of Top-5 and Bottom-5 cells embedded in collagen and stained with DAPI (blue) and Phalloidin (red). (Inset) Zoom in of nucleus of Bottom-5 cell. Top-5 and Bottom-5 zoomed inset scale = 5 μm; Bottom-5 zoomed out image scale = 20 μm.",
    "molecules": "collagen, DAPI, Phalloidin"
  },
  {
    "caption": "(G) Fraction of Control, Top-5 and Bottom-5 cells displaying projections at indicated timepoints after cells were embedded in 3D collagen matrices. Each biological replicate is connected by a line.",
    "molecules": "collagen"
  },
  {
    "caption": "(H) Nuclei of Top-5 (left) and Bottom-5 cells (right) embedded in 3D collagen matrix and stained with DAPI (blue) and Lamin A/C (green). Scale = 5μm. For 3D reconstruction visualization, see Movies EV5 and EV6.",
    "molecules": "collagen, DAPI"
  },
  {
    "caption": "(I) Percent of cells embedded in collagen that had elongated nuclei (Aspect Ratio > 2.5) for A375 Control (n = 54), Top-5 (n = 52) and Bottom-5 (n = 53).",
    "molecules": "collagen"
  },
  {
    "caption": "(J) Cumulative distribution plot of Lamin A/C coefficient of variation for Control (n = 70), Top-5 (n = 78) and Bottom-5 (n = 109) cells embedded in 3D collagen matrices. Kolmogorov-Smirnov test p-values indicated in plot.",
    "molecules": "collagen"
  },
  {
    "caption": "D. Protrusions (Ps) and cell bodies (CB) of cells induced to migrate towards LPA were isolated and analyzed to detect the indicated proteins (by Western blot; left panels) or RNAs (by RT-ddPCR; right panel). Ps/CB enrichment ratios from 2 independent experiments are shown. Bars: mean ± s.e.m..The enrichment of pY397-FAK serves to verify the enrichment of protrusions containing newly formed adhesions in the Ps fraction.",
    "molecules": "LPA"
  },
  {
    "caption": "Active RAB13 (RAB13-GTP) was pulled-down using MICAL-L1 RBD-GST from the indicated PMO-treated cells The amount of endogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted. n=8 (D), n=4 (E). Bars: mean ± s.e.m..",
    "molecules": "GTP"
  },
  {
    "caption": "E. Active RAB13 (RAB13-GTP) was pulled-down using MICAL-L1 RBD-GST from GFP-RAB13 expressing lines The amount of endogenous or exogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted. n=8 (D), n=4 (E). Bars: mean ± s.e.m..",
    "molecules": "GTP"
  },
  {
    "caption": "B. Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines. Note that even though RABIF binds several-fold more to RAB13(T22N) (see Figure 7A, B) it binds similarly to the wild-type and T22N RAB13 RNA, indicating that RABIF binds similarly to nascent RAB13. After translation, it is likely displaced upon GTP-loading of wild type RAB13, while it remains more stably bound to the nucleotide-free (T22N) form. N=6. Bars: mean ± s.e.m.. p-values: *<0.05 by analysis of variance with Dunn's multiple comparisons test.",
    "molecules": "GTP"
  },
  {
    "caption": "C, D. Proximity biotinylation of endogenous vimentin by 2HA-TurboID-RNF26, catalytically inactive point mutant (I382R), or RING domain truncation mutant (∆RING) in HeLa cells following 30 min incubation with biotin prior to lysis. C. Representative immunoblots of neutravidin precipitates and lysate inputs stained against HA and vimentin. D. Quantifications of vimentin biotinylation by 2HA-TurboID-RNF26 and mutants normalized to auto biotinylated HA-tagged RNF26 species and expressed relative to RNF26 WT, n=6 independent experiments performed in U2OS and HeLa cells. Data information: WCL: whole cell lysate. Graphs in D report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.",
    "molecules": "biotin, neutravidin"
  },
  {
    "caption": "E. In vitro co-precipitation of purified recombinant Rhodamine-labeled RING-I382R (304-433) with purified recombinant His-tagged vimentin on Ni-NTA beads. Rhodamine in-gel fluorescent scan and immunoblot against vimentin are shown, representative of three independent experiments.",
    "molecules": "Ni-NTA, Rhodamine"
  },
  {
    "caption": "G. Proximity-based biotinylation of endogenous vimentin by 2HA-TurboID-I382R, -∆RING, and -I382R-∆C lacking amino acids 423-433. Immunoblots of neutravidin precipitates and lysate inputs against HA and vimentin are shown and are representative of three independent experiments. Data information: WCL: whole cell lysate.",
    "molecules": "neutravidin"
  },
  {
    "caption": "H. In vitro peptide binding assay. Rhodamine-linked C-terminal peptide of RNF26 (Rho-RGILQTLNVYL) or free Rhodamine were incubated at 10nM with increasing concentrations of His-vimentin and binding was measured using fluorescence polarization; n=3 independent experiments. Data information H report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.",
    "molecules": "Rho, Rhodamine"
  },
  {
    "caption": "G, H. Effects of vimentin ablation on trafficking of extracellular materials to lysosomes. G. Time-lapse imaging. Representative confocal overlays of parental and vimentin KO#2 U2OS cells, stained with SiR-Lysosome (magenta) to mark late endocytic compartments and incubated with the cell impermeable dye SR101 (green), taken soon after the start (t=5 min) and at the end (t=120 min) of time-lapse are shown. Zoom-ins highlight select PN regions in overlay and single channels. H. Colocalization (Manders') of SiR-Lysosome overlapping SR101 at t=120 minutes following SR101 addition. Graph reports on n Parental = 71, n Vim KO = 69 technical replicates from 3 independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Zoom-ins designate regions encompassing perinuclear and peripheral areas. All scale bars = 10µm. Graphs report mean (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test H error bars indicate mean -/+ SD) ; * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "SiR, SR101"
  },
  {
    "caption": "D, Consequences of RNF26, UBE2J1, and vimentin loss on ERQC formation. D. Representative confocal images of fixed U2OS cells transfected or genetically modified as indicated, treated in the absence (-) or presence (+) of tunicamycin (5ug/mL, O/N), fixed and immunostained against endogenous calnexin (white).",
    "molecules": "tunicamycin"
  },
  {
    "caption": "F, G. Consequences of RNF26, UBE2J1, and vimentin loss on induction of PERK-mediated UPR signaling by tunicamycin. F. U2OS cells, transfected or genetically modified as indicated, were treated in the absence (-) or presence (+) of tunicamycin (2.5µg/mL, 5hrs). Immunoblots of whole cell lysates against endogenous PERK, phospho-eIF2a, ATF4, HERP1, and Vinculin (loading control) are shown, representative of three independent experiments. G. CHOP transcripts isolated from U2OS cells transfected or genetically modified as indicated and treated in the absence (-) or presence (+) of tunicamycin (2.5ug/mL, O/N) were detected by qPCR and normalized to GAPDH; n siC (-)=4, n siC (+)=4, n siRNF26#1 (+)=3, n siRNF26#2 (+)=4, n PARENTAL (+)=4, n Vim KO (+)=4 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test G error bars indicate mean -/+ SEM) p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "C, D. Lysosomal processing of Sec62-HALO-GFP over time in the presence or absence of RNF26 or vimentin. C. Parental, Vim KO#2 or RNF26 KO U2OS cells were transfected with Sec62-HALO-GFP overnight, incubated with 6-chlorohexanol for 15 minutes, washed 3 times with PBS, pulsed with TAMRA HALOtag ligand (200nM) for 1hr, and incubated with 6-chlorohexanol with or without Bafilomycin A1 (100nM) for the indicated times before cell lysis and analysis by SDS-PAGE. Shown is a representative in-gel fluorescence scan with indication of full-length Sec62, protease-resistant HALO fragment and molecular weight markers (MW), as well as a Vinculin blot (loading control) D. Quantification of Sec62 accumulation in LEs from (B). Unprocessed and protease-resistant (lysosomal) band intensities in (B) were calculated with ImageJ and ratios of lysosomal Sec62 versus unprocessed Sec62 were normalized to transfer in parental cells after 10 hours. Error bars show mean +/- SD of n Parental=6, n R26 KO=4, n Vim KO =6 independent experiments. Scale bar = 10µm. Graphs report mean (in D of sample values (open circles). Statistical analyses were performed using Students' T-test (D (paired) ; * p<0.05, ** p<0.01, *** p<0.001).",
    "molecules": "6-chlorohexanol, Bafilomycin A1, PBS, TAMRA"
  },
  {
    "caption": "E, F. Sec62-mediated transfer of ER to lysosomes as a function of RNF26 or vimentin. E. Representative Airyscan images of Parental, RNF26 KO or Vim KO#2 U2OS cells that were transfected with Sec62-HALO-GFP, incubated with 6-chlorohexanol for 15 minutes, washed 3 times with PBS, pulsed with JF646 HALOtag ligand for 1hr, and incubated with 6-chlorohexanol and Bafilomycin A1 for 8hrs before fixation and immunostaining for CD63 to visualize LEs. F. Colocalization (Manders') analysis of CD63 over HALO signals from (D). Plot reports n Parental=106, n R26 KO=69, n Vim KO =68 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Graphs report mean (in F) of sample values (open circles). Statistical analyses were performed using Students' T-test F (unpaired) ; * p<0.05, ** p<0.01, *** p<0.001).",
    "molecules": "6-chlorohexanol, Bafilomycin A1, PBS"
  },
  {
    "caption": "G, H. Sec62-mediated transfer of ER to lysosomes as a function of RNF26 overexpression and activity. G. Representative spinning disc live cell images of RNF26 WT and I382R inactive mutant with Sec62. U2OS cells were transfected overnight with Sec62-HALO-GFP and RFP-RNF26 WT or I382R, incubated for 5hrs with JF646 and imaged by spinning disc microscopy. Images show overlay of GFP (green), JF646 (magenta) and RFP signals, zoom-ins of overlay and single channels that show transfer of RNF26 into LEs (WT RNF26). H. Analysis of Sec62 transfer to proteolytic compartments as a function of RNF26 WT or I382R overexpression. Number of GFP-negative/HALO-positive signals was manually counted from cells in (B) and (G). Plot reports n EV=86, n R26 WT=35, n R26 IR =40 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, median (in H) of sample values (open circles). Mann-Whitney U test (H); * p<0.05, ** p<0.01, *** p<0.001).",
    "molecules": "JF646"
  },
  {
    "caption": "ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δapg8 mutant harboring vector, TK303; Δapg8 mutant harboring APG8 on a centromeric vector, TK301; Δapg8 mutant harboring 3 × HA-tagged APG8 plasmid, TK307.",
    "molecules": "N"
  },
  {
    "caption": "Effect of nocodazole on autophagy. Vegetatively growing cells, YW5-1B and TN124, were treated with 10 μg/ml nocodazole in YEPD medium for three hours. A, YW5-1B cells were transferred to 0.17% yeast nitrogen base without amino acid and ammonium sulfate containing 1 mM PMSF and 10 μg/ml nocodazole, and further incubated for 4.5 h. The accumulation of autophagic bodies was examined under a light microscope. Arrows indicate autophagic bodies. B, TN124 cells were starved in SD(−N) containing 10 μg/ml nocodazole for 4.5 h. ALP activity was measured before (open bars) and after (filled bars) starvation.",
    "molecules": "amino acid, ammonium sulfate, N, nitrogen, nocodazole, PMSF"
  },
  {
    "caption": "B, The wild-type (YW5-1B) cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, 3, 4.5, and 6 h at 30°C. The amount of Apg8p was estimated by immunoblotting with anti-Apg8p antibody. Each lane has 10 μg of total protein.",
    "molecules": "N"
  },
  {
    "caption": "C, YW5-1B cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, and 6 h at 30°C, and mRNA was prepared from each culture as described in Materials and Methods. APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe. Each lane has 4 μg of total mRNA.",
    "molecules": "N"
  },
  {
    "caption": "D, YW5-1B cells were cultured in YEPD medium until 1 × 107 cells/ml at 30°C. Rapamycin was added to the culture at a final concentration of 0.2 μg/ml and the cells were further incubated in YEPD medium for two hours at 30°C. Western blotting was performed with the anti-Apg8p antibody. Each lane has 20 μg of total protein.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "Subcellular fractionation and solubilization of Apg8p. The wild-type cells (YW5-1B) growing logarithmically in YEPD medium or starved in SD(−N) medium for three hours were lysed. A, The lysates (T) were centrifuged at 13,000 g for 15 min to generate pellet (LSP) and supernatant (LSS). LSS fraction was further centrifuged at 100,000 g for one hour and separated to pellet (HSP) and supernatant (HSS). LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates. Equal volume of each sample was applied to each well, and immunoblotting was performed with anti-Apg8p antibody.",
    "molecules": "N"
  },
  {
    "caption": "C, The sonicated lysates were centrifuged at 100,000 g for one hour to generate pellet fraction. The pellet fraction was treated with 2% Triton X-100 (TX-100) for 30 min on ice. The sample was centrifuged at 100,000 g for one hour and separated to supernatant (S) and pellet (P) fractions. The distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Immunofluorescent staining of Δapg8 cells expressing 3 × HA-Apg8p. A, The Δapg8 cells harboring 3 × HA-tagged APG8 plasmid, TK114 cells, were grown until logarithmic phase. The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts. The spheroplasts were permeabilized by 0.5% Triton X-100, and incubated with the anti-HA antibody, 16B12, followed with the FITC-conjugated anti-mouse IgG. Left, Fluorescence image of 3 × HA-Apg8p; right, Nomarski image of the cells. Arrows show punctate signals that are a little larger than tiny dot signals. Bars, 10 μm. B, Immunofluorescent staining of YW5-1B cells before (0 h) and after (3 h) shift to starvation (negative control). Left, Fluorescence image; right, Nomarski image. Bars, 10 μm. C, The cells were shifted to starvation for 0, 0.5, and 3 h. Upper panels, Fluorescence images of 3 × HA-Apg8p; lower panels, Nomarski images of the cell. Bars, 5 μm.",
    "molecules": "formaldehyde, Triton X-100, Zymolyase 100T"
  },
  {
    "caption": "Immunostaining image of a cell containing autophagic bodies in the vacuole. The Δapg8Δpep4 cells expressing 3 × HA-Apg8p, TK116 cells, were shifted to SD(−N) medium for two hours. Cells were immunolabeled with anti-N mAb, 16B12, followed by 10-nm gold-conjugated goat anti-mouse IgG. AB, vacuole; N, nucleus; V, vacuole.",
    "molecules": "gold, N"
  },
  {
    "caption": "Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti-mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.",
    "molecules": "gold, N"
  },
  {
    "caption": "Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B-D) conjugated goat anti-mouse IgG. V, vacuole.",
    "molecules": "gold, gold particles, N"
  },
  {
    "caption": "Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δypt7 cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δapg8 cell. C-E, The representatives of the membrane structures detected in the starved Δapg8 cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.",
    "molecules": "N, Triton X-100"
  },
  {
    "caption": "Colorectal tumor outcomes in WT (n=7), IL-33KO (n=4) and ST2KO (n=3) mice at the completion of the AOM/DSS carcinogenesis protocol. (A) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), Skin tumor outcomes in WT (n=7), IL-33KO (n=6) and ST2KO (n=7) mice at the completion of the DMBA/DNFB carcinogenesis protocol. (D) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero),",
    "molecules": "DNFB, DMBA, AOM, DSS"
  },
  {
    "caption": "H) Quantification of nuclear IL-33+ epidermal and dermal cells in the DNFB- versus acetone-treated WT skin. Dots represent cell counts from three randomly selected high power field (HPF) images per sample (n=5 per group). J) Quantification of nuclear IL-33+ epithelial and stromal cells of colon treated with AOM/DSS versus no treatment. Dots represent cell counts from three randomly selected HPF images per sample (n=4 per group). Data information: Graphs show mean + SD, unpaired t-test. ",
    "molecules": "DNFB, acetone, AOM, DSS"
  },
  {
    "caption": "(H) ChIP-qPCR assay for Smad6 in the presence of IL-33 full length or cytokine domain using an anti-RUNX2 antibody. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-RUNX2 antibody and eluted RUNX2-bound chromatin was used for qPCR with primers against Smad6 promoter region. Anti-IgG ChIP-qPCR results are shown as negative controls (n=4 per group).",
    "molecules": "cytokine"
  },
  {
    "caption": "(D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results. (E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results. (F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results. Data information: GAPDH is used as the control housekeeping protein in D-F). Graphs show mean + SD, NS: not significant, unpaired t-test.",
    "molecules": "cytokine, PBS, poly (I:C)"
  },
  {
    "caption": "(G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group). (H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group). ",
    "molecules": "DMSO, SB431542"
  },
  {
    "caption": "(I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group).",
    "molecules": "DMBA, acetone, TPA"
  },
  {
    "caption": "(B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test).",
    "molecules": "caerulein, PBS"
  },
  {
    "caption": "(D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test).",
    "molecules": "DMBA, caerulein"
  },
  {
    "caption": "A Dose-response viability curves in presence of increasing concentrations of IMT1  for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM.",
    "molecules": "DMSO, IMT1"
  },
  {
    "caption": "B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM  IMT1 treatment. Data are relative to  DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2,  MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001).",
    "molecules": "DMSO, IMT1"
  },
  {
    "caption": "D Volcano plot showing proteomic changes of  RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial  protein are highlighted in red.",
    "molecules": "DMSO, IMT1"
  },
  {
    "caption": "F CRISPR guides enriched (positive hits) in  IMT1-treated  RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1  and VHL pathway-related genes are reported in green and blue, respectively.",
    "molecules": "IMT1"
  },
  {
    "caption": "B-D  Viable  cells count in presence of DMSO, IMT1  alone or in combination with  rapamycin in RKO (B), MiaPaCa-2 (C) and HeLa (D) cells. Data are expressed as mean values ± SD of n=6 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Paired IMT1 vs. IMT1 + RAPA  comparisons: RKO: ***p<0,0001, MiaPaCa-2: **p= 0,0014, HeLa: *p= 0,0238.",
    "molecules": "DMSO, IMT1, RAPA, rapamycin"
  },
  {
    "caption": "F-H Viability  assessment of RKO (F), MiaPaCa-2 (G) and HeLa (H) cells treated with DMSO, IMT1 alone and in combination with FG4592. Data are expressed as mean values ± SD of n=6, 5, 6 independent experiments for  RKO, HeLa  and  MiaPaCa-2, respectively. Statistical significance was calculated with one-way ANOVA. Paired  IMT1 vs IMT1 + FG4592 comparisons: RKO: **p<0,0028, MiaPaCa-2: **p= 0,0011, HeLa: *p= 0,0029.",
    "molecules": "DMSO, IMT1, FG4592"
  },
  {
    "caption": "C Western blot  analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO, IMT1, rapamycin, rapamycin with IMT1, FG4592  and FG4592 with  IMT1. ACTINB is shown as loading control.",
    "molecules": "DMSO, IMT1, FG4592, rapamycin"
  },
  {
    "caption": "F, G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial  function. RKO cells were treated with DMSO, rapamycin (F) or FG4592  (G) in presence or absence of IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. DMSO-treated controls vs: IMT1: p=0,0008; RAPA: p=0,1578; RAPA + IMT1: p= 0,0034; FG4592: p=0,872; FG4592 +  IMT1: p=0,0029. Paired t-test of IMT1  vs RAPA + IMT1: p= 0,034; IMT1  vs  FG4592 +  IMT1: non-significant.",
    "molecules": "DMSO, IMT1, FG4592, RAPA, rapamycin"
  },
  {
    "caption": "A Differences in sensitivity to increasing  IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments.",
    "molecules": "IMT1"
  },
  {
    "caption": "B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control.",
    "molecules": "IMT1"
  },
  {
    "caption": "G Relative mtDNA copy number measured in RKO and  IMT1-resistant cells treated with  IMT1  for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test.  RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028.",
    "molecules": "IMT1"
  },
  {
    "caption": "H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of  mitochondrial  function in RKO and IMT1-resistant RKO cells treated with  DMSO  or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test.  RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of  RKO +  IMT1 vs resistant +  IMT1: p= 0,135.",
    "molecules": "DMSO, IMT1"
  },
  {
    "caption": "A ECAR measured by Seahorse extracellular flux analysis in DMSO  and IMT1-treated  RKO and IMT1-resistant cells. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test for multiple comparisons. RKO DMSO vs RKO + IMT1: **p=0,0019; RKO + IMT1 vs resistant + IMT: *p=0,0214; RKO DMSO vs resistant DMSO: non-significant.",
    "molecules": "DMSO, IMT, IMT1"
  },
  {
    "caption": "D, E IMT1 extracellular  (D) and  extracellular (E) concentrations measured in the medium at 0, 2 and 24 hours of  IMT1 treatment in RKO and IMT1-resistant RKO cells. Mean values ± SEM of n=3 experiments.",
    "molecules": "IMT1"
  },
  {
    "caption": "B Comparison between proteomic analyses  of parental  RKO and resistant cells treated with 1 μM IMT1 for 96 hours (r: sample correlation coefficient). Data are expressed as average log2-fold change of controls (DMSO-treated RKO cells). Mitochondrial proteins are reported in red.",
    "molecules": "DMSO, IMT1"
  },
  {
    "caption": "A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value.",
    "molecules": "IMT1"
  },
  {
    "caption": "C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242.",
    "molecules": "CAP, IMT1"
  },
  {
    "caption": "G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM  #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1  vs TFAM #2 + IMT1: *p=0,0415.",
    "molecules": "DMSO, IMT1"
  },
  {
    "caption": "MBP or the MBP-Stx17 constructs attached to amylose resin were mixed with GST-PGAM5, and the proteins bound to the resin were separated by SDS-PAGE and blotted onto PVDF membranes. The blots were detected by an anti-GST antibody (upper panels) or stained with Coomassie Brilliant Blue R-250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk may represent MBP dimers and degradation products.",
    "molecules": "resin, amylose, Coomassie Brilliant Blue R-250, PVDF"
  },
  {
    "caption": "HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's",
    "molecules": "digitonin, Digitonin, DMSO"
  },
  {
    "caption": "HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg).",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm.",
    "molecules": "3,3'-diaminobenzidine"
  },
  {
    "caption": "293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and then analyzed by IB using the indicated antibodies.",
    "molecules": "CCCP, ethanol"
  },
  {
    "caption": "293T cells transiently expressing FLAG-Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies.",
    "molecules": "CCCP, ethanol"
  },
  {
    "caption": "HeLa cells stably expressing FLAG-Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).",
    "molecules": "CCCP, ethanol"
  },
  {
    "caption": "HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.",
    "molecules": "CCCP, ethanol"
  },
  {
    "caption": "HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).",
    "molecules": "CCCP"
  },
  {
    "caption": "PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.",
    "molecules": "CCCP, ethanol"
  },
  {
    "caption": "GFP-Parkin stably expressing HeLa cells were mock-transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG-DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and FLAG-DFCP1. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).",
    "molecules": "CCCP"
  },
  {
    "caption": "GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).",
    "molecules": "CCCP"
  },
  {
    "caption": "GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.",
    "molecules": "CCCP"
  },
  {
    "caption": "HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.",
    "molecules": "CCCP, ethanol"
  },
  {
    "caption": "GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1.",
    "molecules": "CCCP"
  },
  {
    "caption": "GFP-Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.",
    "molecules": "CCCP"
  },
  {
    "caption": "A. Left, Western blot analysis of H3K27ac in forebrain tissues of Arid1afl/+and cHet mice. Right, relative quantitation of the intensity of H3K27ac normalized to the H3 level (n=3 mice per group).",
    "molecules": "H3K27ac"
  },
  {
    "caption": "B. Left, Western blot analysis of H3K27ac in forebrain tissues of vehicle- or acetate-treated cHet mice. Right, relative quantitation of the intensity of H3K27ac which was normalized to the H3 level (n=4 mice per group).",
    "molecules": "H3K27ac, acetate"
  },
  {
    "caption": "D. Immunofluorescence (IF) staining of H3K27ac(green) and Vgult1(red) in forebrain tissues of Arid1afl/+ and cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac decreased upon the haploinsufficiency of Arid1a in the cortex. Scale Bar, 20μm. Arid1afl/+ (n = 7) and cHet (n = 4) mice.",
    "molecules": "H3K27ac"
  },
  {
    "caption": "E. Immunofluorescence staining of H3K27ac (green) and Vgult1 (red) in cortex tissues of vehicle- or acetate-treated cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac increased after treatment with acetate in the cortex of cHet mice. Scale Bar, 20μm (n=3 mice per group).",
    "molecules": "H3K27ac, acetate"
  },
  {
    "caption": "F-H. Morris water maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. Acquisition task, the latency to find the platform was used to assess learning ability. Latency to the platform was measured during training trials (F). Latency to the platform was measured during probe test (G). (H) Retention task, the times in the target crossings were used to assess memory retention. The times in the target crossings were measured in probe tests, n = 10-15 mice per group.",
    "molecules": "acetate"
  },
  {
    "caption": "I-K. Barnes maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. (I) During the training phase, the time to find the target in Arid1afl/+ and cHet mice that were treated with vehicle or acetate for 28 days. (J) In probe test trials, the time of latency to the platform to find the target in Arid1afl/+ and cHet mice treated with vehicle or acetate. (K) In probe trials, the numbers of target crossings of Arid1afl/+ and cHet mice treated with vehicle or acetate (n = 12 mice)",
    "molecules": "acetate"
  },
  {
    "caption": "E. Representative images of secondary branches of apical dendrites of vehicle- or acetate-treated cHet CA1 pyramidal neurons stained by the Golgi-Cox method (at least 20 neurons from 4 mice for each group). Scale bar, 5 μm. F. Spine density per 10 μm was measured on secondary dendrites in (E).",
    "molecules": "acetate"
  },
  {
    "caption": "A. Representative image of vehicle- and acetate-treated neurons derived from WT or ARID1A KO hESCs on day 40 of neural differentiation Scale Bar, 20μm B. Quantification of total neurite length of hESC-derived neurons after treatment with acetate or vehicle. n = at least 34 neurons for each group from 3 independent experiments. C",
    "molecules": "acetate"
  },
  {
    "caption": "D. Representative images of dendrites (MAP2, green) showing localization of foci of the pre- and postsynaptic protein complexes, synaptophysin (red), and PSD-95 (white) protein in control, acetate treatment of WT and ARID1A KO hESC-derived neurons on day 55 of neural differentiation. Scale Bar, 5μm.",
    "molecules": "acetate"
  },
  {
    "caption": "(C) day 20±2 cerebral organoids derived from controls, patients and  EML1-heKOs stained  for the  neuronal marker  MAPT and the adherens junction marker  NCAD, counterstained  with  DAPI. Ectopic   neural rosettes and neuronal  heterotopia  are highlighted with dotted yellow lines. (D) Quantification of VZ areas with ectopic neural rosettes (3 batches, 3  organoids analyzed per batch, significance based on Kruskal-Wallis-test, P=0.0001, Dunn's post hoc test for multiple comparisons performed to define statistical differences between genotypes, single data points are presented colored by batch). (E) Quantification of heterotopic, disorganized  or organized cortical areas (3 batches, 3 organoids per batch, significance based on Kruskal-Wallis-test, P=0.0001 for \"organized\" and \"heterotopic\"; no significant difference for \"disorganized\", Dunn's post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P-values: ***/###<0.001. VZ:  ventricular zone; Data in graphs are represented as means ± SD. Scale bars: 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for pVim (red) and  TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, yellow dotted line VZ-surface. (D) Quantification of horizontal, vertical and  oblique plane of cell divisions in control and  EML1-deficient   organoids   (day 20±2; 3 batches, 3 organoids each). Data information: Scale bars: 5µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(F)  Electron microscopy  of  primary cilia derived from control or EML1-deficient  cortical progenitors  in DMSO-control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, red arrow:  ciliary pockets). Data information: Scale bars: 0,5µm.",
    "molecules": "DMSO, EpoD, EpothiloneD"
  },
  {
    "caption": "(H) Immunofluorescence staining for p-Vim  in EML1-heKO and control derived  organoids in DMSO,  Verteporfin  or Fluvastatin treated conditions, counterstained with DAPI. Dotted line indicates VZ  areas. (I) Quantification of p-VIM+ cells located at the basal side  of  VZ  areas in control or EML1-heKO-derived organoids under DMSO, Verteporfin or Fluvastatin treated condition (3 batches, 3 organoids  analyzed per batch). Data information: Data in graphs are represented as means ± SD. Significance based on two-way ANOVA (on log10 normalized data) Tukey post hoc test for multiple comparisons were performed to define statistical differences between genotypes. P-values: ***<0.001, **<0.01, *<0.05. Scale bars: (H) 50µm",
    "molecules": "DAPI, DMSO, Fluvastatin, Verteporfin"
  },
  {
    "caption": "D, E The ATP  levels and the NAD/NADH   ratio were measured in LINC00839-overexpressing and LINC00839-KD cells (mean ± SD, Student's t test, n = 3 biological replicates). Data information: Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
    "molecules": "ATP, NAD, NADH"
  },
  {
    "caption": "A, B ATP  levels and NAD/NADH  ratios were measured in SW480  and LoVo  cells. Data information: In A, B, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.",
    "molecules": "ATP, NAD, NADH"
  },
  {
    "caption": "(D, E) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21% to 14% pO2. Magnified PLIM images of osteoclasts under different peripheral oxygen saturation (SpO2, D). The pO2 of each osteoclast in these mice was plotted (E, n = 47 from three mice for each SpO2). Scale bar, 20 μm.",
    "molecules": "oxygen"
  },
  {
    "caption": "(A) μCT analysis of the femurs of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2, n = 5) or ambient air (n = 7), and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2, n = 6) or ambient air (n = 5) for 10 days (axial view of the metaphyseal region). Scale, 0.5 mm. (B) Histological analysis of the proximal tibias of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2) or ambient air for 10 days, and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2 ) or ambient air (toluidine blue staining [arrow, osteoclasts]). Scale, 100 μm. (C) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10-week-old male mice under low oxygen concentration (n = 3) or ambient air (n = 5), and 10-week-old Hif1aRank-/-; Hif2aRank-/- male under low oxygen concentration (n = 3) or ambient air (n = 5) for 10 days.",
    "molecules": "oxygen, toluidine blue"
  },
  {
    "caption": "(A) Protein expression of HIF-1α, HIF-2α, Blimp1, NFATc1, Ctsk and TRAP in control and Hif1aRank-/-; Hif2aRank-/- BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days.",
    "molecules": "oxygen"
  },
  {
    "caption": "(C) Expression of osteoclastogenic and canonical HIF-target genes in wild-type control and Hif1aRank-/-; Hif2aRank-/- BMMs under conditions of 5% or 2% oxygen.",
    "molecules": "oxygen"
  },
  {
    "caption": "(A) Representative intravital image of calvarial bone marrow of Rosa26GO-ATeam/+; Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and FLIM image for ATP changes (middle and right, fluorescence lifetime of EGFP). The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere (14% pO2) or ambient air (right, n = 11 from three mice for each SpO2).",
    "molecules": "ATP, oxygen"
  },
  {
    "caption": "(B) Representative intravital image of calvarial bone marrow of Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and NAD(P)H autofluorescence lifetime images (middle and right). Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration (14% pO2) or ambient air (right, n = 64 from three mice for each SpO2).",
    "molecules": "NAD(P)H, oxygen"
  },
  {
    "caption": "(E) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. (F) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). (G) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 3 days. (H) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 5 days. Control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right).",
    "molecules": "oxygen"
  },
  {
    "caption": "A Representative 3-color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1/2 (presynaptic marker), PI(3)P and Homer (postsynaptic marker). Scale bar, 0.5 µm. PI(3)P-positive structures appear in both pre- and postsynaptic compartments. B Line scan fluorescence intensity profiles of single synapses from the images in A, normalized to maximum value for each channel. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "PI(3)P"
  },
  {
    "caption": "C Representative 3-color confocal  images of cultured hippocampal neurons fixed and  stained  for VGlut1, VGAT  and  PI(3)P. Arrows indicate PI(3)P puncta in VGlut1+ or  VGAT+ presynaptic  terminals. Scale bar, 10 µm. D  PI(3)P  intensity in  VGlut1+ and  VGAT+ presynaptic terminals. PI(3)P is equally abundant in excitatory  and  inhibitory presynaptic terminals. N = 3 independent experiments (≥ 15 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "PI(3)P"
  },
  {
    "caption": "E Relative PI(3)P levels in the  MAP2+ (Soma) and Syp+ (Synapse) areas in  DMSO and VPS34IN1-treated neurons. N = 3 independent experiments (≥ 35 images per condition); Student's t-test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "DMSO, PI(3)P, VPS34IN1"
  },
  {
    "caption": "F Cultured  hippocampal neurons were field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for MAP2, Synaptophysin (Syp), and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm. PI(3)P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification.",
    "molecules": "PI(3)P"
  },
  {
    "caption": "G Relative intensity of PI(3)P in  neuronal somata (MAP2+) or synapses (Syp+) from the images in F. N = 5 independent experiments; (≥ 40 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "PI(3)P"
  },
  {
    "caption": "H Relative intensity of  PI(3)P in neuronal somata (MAP2+) or  synapses  (Syp+) of cultured hippocampal neurons treated with DMSO or Gabazine (10 µM) for 24 h. N = 3 independent experiments (≥ 65 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "DMSO, PI(3)P, Gabazine"
  },
  {
    "caption": "I Cultured  hippocampal neurons treated with AP5  (20 µM), CNQX (10 µM) or TTX (0.2 µM) for 24 h, fixed and  stained  for MAP2, Syp  and PI(3)P. Arrows indicate  PI(3)P  puncta in synapses. Scale bar, 10 µm.",
    "molecules": "AP5, CNQX, PI(3)P, TTX"
  },
  {
    "caption": "J Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in I. N ≥ 4 independent experiments (≥ 60 images per condition); Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "PI(3)P"
  },
  {
    "caption": "K Relative intensity of PI(3)P  in neuronal somata (MAP2+) or synapses (Syp+) of cultured  hippocampal neurons  were treated with  Roscovitine  (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-). N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Fisher's Least Significant Difference Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "PI(3)P, Roscovitine"
  },
  {
    "caption": "A Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%) or the VPS34 inhibitors, VPS34IN1 (3 µM) or SAR405 (20 µM). Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. VPS34 inhibition causes reduced excitatory synaptic transmission down to 46.0 ± 4.1 % and 55.0 ± 4.4 % for VPS34IN1 and SAR405 at the end of 60-min treatment. N = 6 slices per condition from ≥ 4 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001",
    "molecules": "DMSO, SAR405, VPS34IN1"
  },
  {
    "caption": "B Increased paired paired-pulse facilitation (PPF) at  hippocampal CA3-CA1 synapses after VPS34 inhibition. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below, indicating an increased facilitation of the second response in VPS34IN1-treated acute hippocampal slices. PI(3)P depletion causes a significant facilitation of the second fEPSP, indicative of reduced initial release probability. N = 6 slices per condition from 6 mice; Two-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001",
    "molecules": "PI(3)P, VPS34IN1"
  },
  {
    "caption": "C Synaptophysin-pHluorin responses normalized to baseline from neurons stimulated  with 12 consecutive trains of 20 APs (20 Hz, 1 s) with 30 s gap between each stimulus. Neurons are acutely treated with DMSO (0.1 %) or  VPS34 inhibitor1 (50 µM) starting from the onset of 2nd stimulation. The timing of the treatment and the stimulation  trains are indicated. D Quantification of data from C. Net difference of Synaptophysin-pHluorin fluorescence 2 s before and 28 s after each stimulation  (2 s before the next stimulation) to estimate relative surface retention   of SVs. SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1, leading to progressive accumulation of Synaptophysin-pHluorin signal. N = 4 or 5 independent measurements per condition; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001",
    "molecules": "DMSO, VPS34 inhibitor1, VPS34 Inhibitor1"
  },
  {
    "caption": "A Electron micrographs of VPS34IN1- and DMSO-treated synapses following four consecutive trains of 200 AP (40 Hz, 5 s; 90 s gap between each train) stimulation. The postsynaptic  compartment  and the  postsynaptic density are highlighted in green and purple, respectively. Note the reduced SV numbers and plasma membrane-derived endocytic intermediates in VPS34IN1-treated synapses. Scale bars, 500 nm (left) and 200 nm (right). B High magnification views of endocytic intermediates accumulated in stimulated hippocampal neurons treated with VPS34IN1. Scale bar, 200 nm. C 3D-reconstruction of a synaptic  terminal treated with VPS34IN1 and  stimulated as in A. The plasma membrane is colored in turquoise. Synaptic vesicles (yellow), plasma membrane-derived invaginations  (blue), endosome-like vacuoles  (green) and clathrin-coated vesicles (red) are highlighted. Scale bar, 500 nm (top) and 200 nm (bottom). Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001",
    "molecules": "DMSO, VPS34IN1"
  },
  {
    "caption": "D-H Morphometric quantification of data shown in A. Synapses of VPS34IN1-treated stimulated neurons display reduced numbers of SVs/ bouton area in (D) and an accumulation of endocytic membrane invaginations (E), endosome like vacuoles (ELV) (F), clathrin-coated pits (CCP) (G), and clathrin-coated vesicles (CCV) (H). n = 100 (SV) and n = 200 (invagination, ELV, CCP, CCV) profiles per condition; Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001",
    "molecules": "VPS34IN1"
  },
  {
    "caption": "A-D Normalized Synaptophysin-pHluorin responses from neurons treated with VPS34IN1 (10 µM for 1h prior to imaging) and stimulated  with four consecutive trains of 50 APs (10 Hz, 5 s) in A or 200 APs (40 Hz, 5 s) in C. PI(3)P  depletion in VPS34IN1-treated neurons causes the progressive accumulation of Synaptophysin-pHluorin on the neuronal  surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D. Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive stimulation  trains. N ≥ 4 independent experiments; Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "PI(3)P, VPS34IN1"
  },
  {
    "caption": "E Cultured hippocampal neurons pre-treated with DMSO (0.1%) or VPS34IN1 (10 µM, 1 h) were either field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for surface-stranded  Synaptotagmin-1, total Synaptotagmin-1, and vGlut1. Scale bar, 10 µm. F Fraction of endogenous Synaptotagmin-1 stranded on the surface of presynaptic nerve terminals    from the images shown in E. N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "DMSO, VPS34IN1"
  },
  {
    "caption": "G Surface retention  of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405  (20 µM). Data are similar to those shown in C and D. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "SAR405"
  },
  {
    "caption": "H Exogenous supply of membrane-permeant PI(3)P acutely rescues defective Synaptophysin-pHluorin endocytosis  induced by pharmacological inhibition of VPS34-mediated PI(3)P  synthesis in  hippocampal neurons. Data are similar to those shown in C and D; where indicated exogenous PI(3)P-AM (20 µM) was supplied in parallel to  VPS34IN1 (10 µM) for 1 h prior to imaging. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "AM, PI(3)P, VPS34IN1"
  },
  {
    "caption": "A-C The kinetic block of SV endocytosis  at excitatory synapses depleted of PI(3)P depends on prior neuronal activity and Ca2+. VPS34IN1-induced defects in SV endocytosis  are reverted by parallel treatment with  TTX  (0.2 µM) in A and EGTA (2 mM) in B and AP5 (20 µM) in C, in Synaptophysin-pHluorin-expressing neurons N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "AP5, Ca2+, EGTA, PI(3)P, TTX, VPS34IN1"
  },
  {
    "caption": "D Overexpression of dominant-negative Rab5 (Rab5DN), but not constitutively active Rab5 (Rab5CA), rescues the progressive accumulation of Synaptophysin-pHluorin on the surface of VPS34IN1-treated hippocampal neurons stimulated and analyzed as in A-C. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "VPS34IN1"
  },
  {
    "caption": "E Levels of various synaptic proteins in DMSO (0.1%) and  VPS34IN1 (10 µM) treated cultured cerebellar granule neurons analyzed by western blot. VPS34 inhibition results in  p35 processing to hyperactive p25, a reaction blocked by Calpain  inhibitor ALLN  (100 µM).",
    "molecules": "ALLN, DMSO, VPS34IN1"
  },
  {
    "caption": "F-H Calpain inhibition by  ALLN (100 µM) in F or Calpain 2 knockdown via  lentiviral shRNA (shCAPN2) in H rescue the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1. Calpain 1 knockdown via lentiviral shRNA (shCAPN1) in G had no impact on VPS34IN1-induced kinetic delay of SV endocytosis. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001",
    "molecules": "ALLN, VPS34IN1"
  },
  {
    "caption": "B Cdk5 inhibition by Roscovitine (Ros, 10 µM) rescues the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1 (10 µM). N = 4 independent experiments (20 images per condition); Two-way ANOVA; Tukey's Multiple Comparisons Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001",
    "molecules": "Ros, Roscovitine, VPS34IN1"
  },
  {
    "caption": "C Calcineurin inhibition by Cyclosporin A (10 µM) phenocopies VPS34IN1-induced loss of PI(3)P  with respect to the accumulation of Synaptophysin-pHluorin on the neuronal surface after four successive stimulation  trains (40 Hz, 5 s). Data were analyzed as in B. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001",
    "molecules": "Cyclosporin A, PI(3)P, VPS34IN1"
  },
  {
    "caption": "D Basal excitatory synaptic  transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%), Roscovitine (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic  transmission, akin to Roscovitine alone, and in contrast to VPS34IN1 alone N = 6 slices per condition from 6 mice; Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001",
    "molecules": "DMSO, Ros, Roscovitine, VPS34IN1"
  },
  {
    "caption": "E Decreased paired paired-pulse facilitation (PPF) at  hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice; Two-way RM ANOVA. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001",
    "molecules": "Ros, Roscovitine, VPS34IN1"
  },
  {
    "caption": "A Activity-dependent polyspiking  of population spikes (PS) during 1 Hz stimulation  is reduced by VPS34IN1 and facilitated by Roscovitine compared to DMSO-treated control slices. Top, representative traces of the 1st and 30th stimulus responses with polyspikes  indicated by arrows. N = 12 slices per condition from 12 mice. B Quantification of  polyspiking amplitudes in response to the 30th stimulus in the presence of DMSO, VPS34IN1, or Roscovitine. Polyspiking amplitudes are increased by Roscovitine and reduced by VPS34IN1. N = 12 slices per condition from 12 mice; One-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.",
    "molecules": "DMSO, Roscovitine, VPS34IN1"
  },
  {
    "caption": "C The relative changes in the PS and polyspike areas in the presence of picrotoxin (50 µM) and a subsequent treatment with DMSO or VPS34IN1 (10 µM). VPS34IN1 significantly reduced the PS  and polyspike  area compared to DMSO. N = 6 slices per condition from 6 mice. D Mean area of PS  and polyspikes  in picrotoxin-treated slices 40 min post-application of DMSO or VPS34IN1. N ≥ 6 slices per condition from ≥ 6 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.",
    "molecules": "DMSO, picrotoxin, VPS34IN1"
  },
  {
    "caption": "PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.",
    "molecules": "13C2, 13C3, 15N, PS, PE, serine"
  },
  {
    "caption": "(C) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.",
    "molecules": "PS, PE"
  },
  {
    "caption": "PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serine",
    "molecules": "13C3, 15N, PS, PE, serine"
  },
  {
    "caption": "(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.",
    "molecules": "SFA, UFA"
  },
  {
    "caption": "(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.",
    "molecules": "SFA, UFA"
  },
  {
    "caption": "(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.",
    "molecules": "SGal, glucose"
  },
  {
    "caption": "PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.",
    "molecules": "13C2, 13C3, 2H3, 15N, PS, PE, serine"
  },
  {
    "caption": "(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.",
    "molecules": "PS, PE"
  },
  {
    "caption": "SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.",
    "molecules": "cho, choline, ethanolamine, etn, SFA, UFA"
  },
  {
    "caption": "A Electron microscopy analyses to see autophagic bodies in the vacuoles under both growth and nitrogen starvation conditions. Autophagic bodies typically contain cytosolic ribosomes. pep4∆prb1∆ and pep4∆prb1∆atg2∆ cells were grown in SD to mid‐log phase (OD600 = 1) and transferred to SD(‐N) for 5 h. The cells were examined by transmission electron microscopy as described in . Scale bar, 500 nm.",
    "molecules": "nitrogen"
  },
  {
    "caption": "B-D Time‐dependent changes in nucleoside contents under nitrogen starvation (B), rapamycin treatment (C), and carbon starvation (D) conditions. Wild‐type and atg2Δ cells were grown in SD and transferred to SD(‐N), SD medium with 0.2 μM rapamycin, or SD(‐C) at time 0. At the indicated time points, nucleosides were analyzed by LC/MS as described in . Results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicate samples. The error bars represent the standard deviation.",
    "molecules": "carbon, nitrogen, rapamycin"
  },
  {
    "caption": "A Time‐dependent changes in nucleoside contents under nitrogen starvation. Nucleosides in wild‐type, atg2Δ, atg7Δ, atg11Δ, atg17Δ, atg19Δ, atg32Δ, nvj1Δ, and pep4prb1Δ cells under nitrogen starvation. All data are means of triplicates. The error bars represent the standard deviation.",
    "molecules": "nitrogen"
  },
  {
    "caption": "C Detection of free RNA. Wild‐type, atg2∆, rny1∆, and rny1∆atg2∆ cells grown in SD (left) or SD(‐N) for 2 h (right) were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.",
    "molecules": "RNA"
  },
  {
    "caption": "A Time‐dependent change in nucleoside levels after nitrogen starvation. Wild‐type, atg2∆, ubp3∆, and bre5∆ cells were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. Metabolites were analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of the peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.",
    "molecules": "nitrogen"
  },
  {
    "caption": "B Detection of free RNA within cells. rny1∆, rny1∆atg2∆, rny1∆ubp3∆, and rny1∆bre5∆ cells grown in SD(‐N) for 2 h were stained with FM4‐64 and GR Green and observed by fluorescence microscopy. Scale bar, 5 μm.",
    "molecules": "RNA"
  },
  {
    "caption": "(C) shRNA lentiviruses against NFE2L1 increased the levels of aCASP3 in H2O2-treated (100µm for 12 hours) TH+neurons derived from hNES cells differentiated for 8 days (percentage). White arrows indicate double TH+aCASP3+neurons.",
    "molecules": "H2O2"
  },
  {
    "caption": "(D) PBX1 overexpression protected TH+neurons derived from hNES cells from the oxidative stress induced by H2O2 (100µm for 12 hours), resulting in reduced levels of aCASP3 in TH+neurons (percentage).",
    "molecules": "H2O2"
  },
  {
    "caption": "(E) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining (dark-blue/black, indicated by arrowheads) in the nuclei of neuromelanin+cells (NM+, cytosolic brown aggregates) of the SN. In contrast, PBX1 staining was severely reduced in PD patients. (F) Graphical representation of PBX1 intensity levels in the SN of NM+ neurons in all individual controls and PD patients analyzed. Each dot in the graph represents the level of PBX1 in the nuclei of one single NM+neuron.",
    "molecules": "neuromelanin, NM"
  },
  {
    "caption": "(G) Strong immunoreactivity of the PBX1 direct target, NFE2L1, was also detected as very well defined nuclear punctuate structures in NM+cells of the SN in control samples (arrowheads), but were dramatically reduced in PD patients. Yellow asterisks indicate the nucleoli. (H) Percentage of NM+ neurons showing nuclear NFE2L1 staining in the SN of control donors and PD patients. (I) Graphical representation of all individual controls and PD patients analyzed, showing that NM+neurons with NFE2L1+nuclei in the SN of PD patients are less than 10% or are totally absent.",
    "molecules": "NM"
  },
  {
    "caption": "Immunofluorescent staining of CD3, CD8 and CD45 (red) counterstained with DAPI for DNA (blue) using control or shVgll4 LLC tumors from C57BL/6 mice. Statistical analysis of the percentage of CD3, CD8 and CD45 positive cells in the tumors is shown in the right panel respectively. n=3 tumors for each group",
    "molecules": "DAPI"
  },
  {
    "caption": "A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies. Relative IRF2BP2 expression levels were quantified in the right panel",
    "molecules": "MG132"
  },
  {
    "caption": "Lys48-linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells. HEK293T cells were transfected with indicated plasmids, treated with MG132 for 3 h before harvest, and subjected to immunoprecipitation and immunoblot analysis as indicated",
    "molecules": "Lys, MG132"
  },
  {
    "caption": "Lys48-linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells. HEK293T cells were transfected with indicated siRNA and plasmids, and subjected to immunoprecipitation and immunoblot analysis as indicated",
    "molecules": "Lys"
  },
  {
    "caption": "Color-coded time-traces of fluorescence (left panel) averaged for 50 individual cells per strain (mean ± SD) treated with MMC (0.5 μg/ml) for 2 hours (gray shading). Fluorescence induction during 6 hours from MMC exposure (right panel). Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.",
    "molecules": "MMC"
  },
  {
    "caption": "Pearson's correlation between green (F-I) or yellow (J) fluorescence and red fluorescence, measured 3 hours after MMC exposure (203 < n < 314). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (F-I) or a C-term translational reporter of SSBa fused to mCitrinewt (J). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (F and J), transcriptional reporters of ruvC (G) and radA (H), and mCherrydes expressed from a constitutive control promoter (I). Positive correlations between recA and other DNA-damage markers both under normal growth conditions, where spontaneous DNA-damage events occur at the subpopulation level, and in the presence of an alkylating agent that induces double-strand breaks in the entire population, validate recA as a reliable proxy for DNA damage in single cells.",
    "molecules": "MMC"
  },
  {
    "caption": "Representative time-lapse image series of the merodiploid recA reporter strain (P2P1recA-GFPdes_RecA-mCherrywt) cultured in fresh (A) or spent (B) flow medium (see Materials and Methods). Fresh 7H9 medium is unlabeled; spent medium (S), ciprofloxacin 1 µg/ml in fresh (CIP) or spent (S-CIP) medium, and SYTOX 0.5 µM (SX) are labeled. Phase-contrast (gray), GFPdes (green), and mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Light-green background in the first 4 frames (B) is due to the autofluorescence of spent medium. Cells were imaged at 30-min intervals and numbers represent hours. Scale bar, 10 µm.",
    "molecules": "SYTOX, CIP, ciprofloxacin"
  },
  {
    "caption": "Representative time traces of individual lineages of cells fed with fresh medium (C) or spent medium (D). Different styles are used for lineages with different fates: death and lysis (L); SYTOX-positive (SX+); and survival (S). Line breaks represent division events in the same lineage, where a single random daughter cell is tracked for clarity. Vertical bars represent fresh (white) or spent (dark shading) 7H9 medium, CIP (yellow), and SX (blue).",
    "molecules": "SX, SYTOX, CIP"
  },
  {
    "caption": "Comparison of single-cell growth rates prior to CIP exposure for \"D\" cells that die and \"S\" cells that survive and regrow after CIP washout (E), and lag time of regrowth (F). Cells were cultured in fresh (EXP) or spent (STAT) flow medium. Gray and black lines indicate mean ± SD. Significance by Kruskal-Wallis and Dunn's multiple comparison test (E) and unpaired Mann-Whitney test (F): ns, not significant; ****p < 0.0001; 6 < n < 84. The data shown are from 2 independent experiments. Single-cell fate is independent of the growth rate before exposure to the drug. However, cells cultured in spent medium (STAT) grow at significantly slower rates, survive at higher frequencies, and recover faster after CIP washout compared to cells cultured in fresh medium (EXP).",
    "molecules": "CIP"
  },
  {
    "caption": "Single-cell fate as a function of VMR of P2P1recA-GFPdes (I) and RecA-mCherrywt (J) fluorescence prior to CIP exposure. Significance by Chi-square test of independence for cells that die (n = 84) and cells that survive (n = 54). The data shown are from 2 independent experiments. Cells that do not experience DNA damage prior to drug exposure (RecA-non-pulsing cells) survive more than cells that do experience DNA damage (RecA-pulsing cells).",
    "molecules": "CIP"
  },
  {
    "caption": "Representative time-lapse image series of the dual reporter of RecA and rRNA expression in exponential phase. Pre-CIP growing (PG-S) and arrested (PA-S) survivors and resistant (R) cells are indicated by arrows. CIP exposure (1 µg/ml, 4X-MIC) and SX (0.5 µM) death assay (blue line) are indicated. Phase-contrast (gray), Prrn-GFPdes (green), and RecA-mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Cells were imaged at 3-hour intervals and numbers indicate days. Scale bar, 5 µm.",
    "molecules": "CIP"
  },
  {
    "caption": "Representative time traces of cell lineages with different fates. Vertical bars represent fresh 7H9 medium (white), CIP (yellow), and SX (blue).",
    "molecules": "SX, CIP"
  },
  {
    "caption": "Comparison of growth rate (C), cell size averaged over the lifetime of the cell (D), microcolony size (E) before CIP exposure Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.",
    "molecules": "CIP, ciprofloxacin"
  },
  {
    "caption": "fluorescence range over the lifetime of the cell before CIP exposure (F), and mean fluorescence during CIP exposure (G) of cells that died (n = 174) and cells that survived (n = 164). Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.",
    "molecules": "CIP, ciprofloxacin"
  },
  {
    "caption": "Single-cell fate as a function of VMR of RecA-mCherrywt fluorescence prior to CIP exposure. Significance by Chi-square test of independence: cells that died (n = 174), cells that survived (n = 155), and resistant cells (n = 9). The data shown are from 2 independent experiments. More survivors arise from the subpopulation that does not experience DNA damage before drug exposure.",
    "molecules": "CIP"
  },
  {
    "caption": "G The social avoidance behavior in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 14, 10, 10 mice. Interaction F2, 31 = 2.051, P = 0.1457; Target F1, 31 = 2.666, P = 0.1127; Drug F2, 31 = 3.711, P = 0.0359; For Target, Apyrase-ACSF vs Vehicle, P = 0.0387; Apyrase-ATP vs Apyrase-ACSF, P = 0.0099, Two-way ANOVA with Tukey's post-test).",
    "molecules": "ATP"
  },
  {
    "caption": "H The sucrose preference in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 10, 8, 8 mice. Treatment F2, 23 = 3.32, P = 0.0540; Apyrase-ACSF vs Vehicle, P =0.0466, one-way ANOVA with Fisher's LSD test).",
    "molecules": "ATP"
  },
  {
    "caption": "B The ATPase activity of mice exposure to ARL67156 (n = 7, 7, 5, 5 mice. Treatment F3, 20 = 16.81, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0405. One-way ANOVA with Fisher's LSD test).",
    "molecules": "ARL67156"
  },
  {
    "caption": "C The social avoidance behavior of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 9, 9 mice. Interaction F3, 36 = 5.119, P = 0.0047; Target F1, 36 = 0.005, P = 0.9437; Drug F3, 36 = 3.626, P = 0.022. For Target, CSDS - ACSF vs Ctrl -ACSF, P = 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0054. Two-way ANOVA with Tukey's post-test).",
    "molecules": "ARL67156"
  },
  {
    "caption": "D The sucrose preference of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 10, 10 mice. Interaction F1, 38 = 5.352, P = 0.0262; Group F1, 38 = 5.711, P = 0.0219; Drug F1, 38 = 9.703, P = 0.0035; CSDS - ACSF vs Ctrl - ACSF, P = 0.0083; Ctrl - ARL67156 vs CSDS - ACSF, P = 0.0027; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0031. Two-way ANOVA with Tukey's post-test).",
    "molecules": "ARL67156"
  },
  {
    "caption": "The social avoidance behavior (E) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. For social behavior, Interaction F3, 31 = 5.579, P = 0.0035; Target F1, 31 = 0.5121, P = 0.4796; Drug F3, 31 = 8.367, P = 0.0003; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0131.",
    "molecules": "ARL67156"
  },
  {
    "caption": "sucrose preference (F) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. Sucrose, Interaction F1, 30 = 0.3342, P = 0.5675; Group F1, 30 = 17.22, P = 0.0003; Drug F1, 30 = 0.05903, P = 0.8097; CSDS- ACSF vs Ctrl - ACSF, P = 0.0761. Two-way ANOVA with Tukey's post-test).",
    "molecules": "ARL67156, Sucrose"
  },
  {
    "caption": "G The immobility time in the TST of mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 16, 12, 21, 19 mice. Treatment F 3, 64= 13.61, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0059; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0011; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0002. One-way ANOVA with Fisher's LSD test).",
    "molecules": "ARL67156"
  },
  {
    "caption": "H The immobility time in the FST with intra-hippocampal infusion of ARL67156 after CSDS (n = 21, 12, 23, 19 mice. Treatment F 3, 71 = 12.94, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0036; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0428; CSDS - ARL67156 vs CSDS - ACSF, P < 0.0001. One-way ANOVA with Fisher's LSD test).Data information: data are expressed as the means ± SEM. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control.",
    "molecules": "ARL67156"
  },
  {
    "caption": "H The ATP level of mice after knocking down CD39 in the hippocampal (n = 5 mice/group. Treatment F2, 12 = 6.04, P = 0.0153; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0444; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0051. One-way ANOVA with Fisher's LSD test).",
    "molecules": "ATP"
  },
  {
    "caption": "B Immunostaining for BrdU (blue) and DCX (green) in the DG region of control, susceptible and ARL67156 treatment mice. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.",
    "molecules": "BrdU, ARL67156"
  },
  {
    "caption": "C, D Quantification of BrdU+ cells (C) and BrdU+ DCX+ cells (D) in the DG of susceptible mice with ARL67156 compared with vehicle treatment (n = 4 mice/group. For BrdU+ Treatment F2, 9 = 10.07, P =0.0051; CSDS - ACSF vs Ctrl - ACSF, P = 0.0147; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0018; BrdU+ DCX+ Treatment F2, 9 = 9.005, P = 0.0071; CSDS - ACSF vs Ctrl - ACSF, P = 0.0211; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0024, one-way ANOVA with Fisher's LSD test).",
    "molecules": "BrdU, ARL67156"
  },
  {
    "caption": "F Confocal images of BrdU (blue) and DCX (red) in mice treatment with LV-siCD39 in the DG. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.",
    "molecules": "BrdU"
  },
  {
    "caption": "G, H Quantitative of BrdU+ (G) and BrdU+ DCX+ (H) cells in the DG of susceptible mice with LV-siCD39 compared with LV-GFP treatment (n = 3, 6, 6 mice. For BrdU+ Treatment F2, 12 = 15.79, P =0.0004; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0012; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003; BrdU+ DCX+ Treatment F2, 12 = 12.79, P = 0.0011; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.05; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003, one-way ANOVA with Fisher's LSD test).",
    "molecules": "BrdU"
  },
  {
    "caption": "G-J Quantification of total (G), stubby (H), mushroom (I) and thin (J) spine density in DG from control and susceptible mice treatment with ACSF or ARL67156 (n = 11, 13, 10 segments from 2, 4, 2 mice, respectively. For total density: Treatment F2, 31 = 4.474, P = 0.0196; CSDS - ASCF vs Ctrl - ACSF, P = 0.0366; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0086; stubby density: Treatment F2, 31 = 3.327, P=0.0491; CSDS - ASCF vs Ctrl - ACSF, P = 0.1026; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0179; mushroom density: Treatment F2, 31 = 1.986, P = 0.1543; thin density: F2, 31 = 1.475, P = 0.2445. One-way ANOVA with Fisher's LSD test).",
    "molecules": "ARL67156"
  },
  {
    "caption": "A. Representative images of human neutrophils  treated with the FOXM1  inhibitor, FDI-6. Vehicle (DMSO) served as a control. Neutrophils undergoing NET  formation are visualized in green and live neutrophils  are visualized in red. Quantification was performed by normalizing the number of neutrophils  undergoing NET  formation to the number of live neutrophils. Neutrophils  from n=3 different blood  donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). Data presented as mean ± SD. (Scale bar: 100µm).",
    "molecules": "FDI-6, DMSO"
  },
  {
    "caption": "B. Quantification of ROS  levels in human neutrophils  treated with FDI-6 or in combination with N-acetylcysteine (NAC). Phorbol 12-myristate 13-acetate (PMA) treatment served as a positive control. Data presented as mean ± SD. Neutrophils  from n=3 different blood  donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). C. Quantification of NET  formation in human neutrophils  treated with FDI-6 or in combination with NAC. PMA  treatment served as a positive control. Neutrophils  from n=3 different blood  donors were isolated, pooled and peformed in triplicate. Data presented as mean ± SD. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).",
    "molecules": "FDI-6, N-acetylcysteine, NAC, Phorbol 12-myristate 13-acetate, PMA, ROS"
  },
  {
    "caption": "B. Representative pictures of vehicle (IgG isotype control) and ⍺-TREM1  treated diabetic wounds   at day 4 show basal keratin   marker K5, and neutrophil  marker Ly6G, FOXM1   and  citH3. Treatment of wounds  with ⍺-TREM1   resulted in increased FOXM1+ and decreased citH3+ neutrophils  compared to vehicle treated diabetic wounds. (Scale bar: 50µm). Quantification of mean fluorescence intensity was performed with Fiji software. n= 7 diabetic  vehicle wounds  and 6 diabetic  ⍺-TREM1 wounds. Data presented as mean ± SD. *P < 0.05, *P < 0.05, and **P < 0.01 (two-tailed unpaired Student's t-test).",
    "molecules": "cit"
  },
  {
    "caption": "C. Representative images of cit-H3 immunohistochemistry  show increase staining  in nonhealing DFUs  when compared to healing DFUs, which was confirmed by corresponding quantification from healing  (n=3) and nonhealing  (n=3), shown in the graph. Data presented as mean ± SD. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).",
    "molecules": "cit"
  },
  {
    "caption": "MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either DMSO, 5uM RSL3 or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either EtOH, 25uM 4HNE or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. n=3 experiments per group. Exact p‑values are reported in the Appendix Table S1.",
    "molecules": "4HNE, DMSO, EtOH, fer-1, RSL3"
  },
  {
    "caption": "MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes. Isolated protein was assessed by immunoprecipitating phospho-p38 (Thr180/Tyr182) and immunoblotting for 4HNE and phospho-p38 (Thr180/Tyr182). Shown is one replicate of three independent experiments (n=3).",
    "molecules": "4HNE, EtOH, Thr, Tyr"
  },
  {
    "caption": "MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-p38 (Thr180/Tyr182) and total p38. Shown is one replicate of three independent experiments (n=3).",
    "molecules": "4HNE, DMSO, BIRB, EtOH, Thr, RSL3, Tyr"
  },
  {
    "caption": "HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO, EtOH, DMSO, 25uM 4HNE or 5uM RSL3. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).",
    "molecules": "4HNE, DMSO, EtOH, Ser, Thr, RSL3, Tyr"
  },
  {
    "caption": "MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.",
    "molecules": "4HNE, DMSO, KRIBB11, RSL3"
  },
  {
    "caption": "MCF10A cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. Isolated protein was assessed by immunoblotting for prominin2, phospho‑HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).",
    "molecules": "4HNE, DMSO, EtOH, KRIBB11, Ser, Thr, RSL3, Tyr"
  },
  {
    "caption": "Hs578t cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. SF295 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. NCI H1975 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.",
    "molecules": "DMSO, IKE, KRIBB11, RSL3"
  },
  {
    "caption": "Twenty-four hours after the last treatment, tumors were excised, frozen and sectioned. Each tumor was stained with Abs specific for either 4HNE, prominin2 or HSF1. Images were taken at 20x magnification. Higher‑magnification images (1.5x) are shown to the right of its corresponding inset (white box). Scale bar is 50 microns. Shown is one representative image for each condition.",
    "molecules": "4HNE"
  },
  {
    "caption": "A Indicated tail-anchored (TA) proteins were in vitro translated in rabbit reticulocyte lysate (RRL) using RNAs containing a stop codon. Translations were carried out in the presence of 2 μM HisTrx or HisTrx-SGTA (HT-SGTA), followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration, samples resolved by SDS-PAGE and results visualised by phosphorimaging. Sec61β 3R carries three Arg residues within Sec61β transmembrane domain (TMD) which abolish its hydrophobic character and was used as a negative control for SGTA binding. A schematic representation of a TA-protein is also shown. Open circles indicate TA-proteins selectively bound by HisTrx-SGTA. RAMP4 - stress-associated endoplasmic reticulum protein 1; Syb2 - synaptobrevin 2; Synt5 - syntaxin 5. B As for (A) but total translation products (input) were resolved by SDS-PAGE and results visualised by phosphorimaging. When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant. ",
    "molecules": "Cobalt, haemoglobin, imidazole, Arg"
  },
  {
    "caption": "A Full-length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx-SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide (CHX) or released with puromycin (puro). Reactions were incubated with HisPur Cobalt resin, bound proteins eluted at high imidazole concentration, resolved by SDS-PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX-treated samples.",
    "molecules": "Cobalt, CHX, cycloheximide, imidazole, puro, puromycin"
  },
  {
    "caption": "Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin the eluted fractions were subjected to RNaseA treatment (C) -tRNA\" and \"+ tRNA\" indicate tRNA-lacking and tRNA-bound protein species, respectively.",
    "molecules": "Cobalt, tRNA"
  },
  {
    "caption": "D Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide (CTAB) (D) ;-tRNA\" and \"+ tRNA\" indicate tRNA-lacking and tRNA-bound protein species, respectively.",
    "molecules": "CTAB, hexadecyltrimethylammonium bromide, Cobalt, tRNA"
  },
  {
    "caption": "B Variants of PPLssKO-C99 were translated in the presence of 2 μM HisTrx-SGTA using templates lacking a stop codon and terminating either before (\"-TMD\") or at indicated amino acid distance after the TMD. Pull-down with HisPur Cobalt resin was carried out as described in Materials and Methods, eluted material resolved by SDS-PAGE and results visualised by phosphorimaging. tRNA-bound species were verified by treating PPLssKO-C99 variant terminating immediately after the TMD (TMD + 0) with RNaseA (lane 9).",
    "molecules": "Cobalt, tRNA"
  },
  {
    "caption": "translation reactions were carried out using RNAs coding for the indicated TA-proteins and lacking a stop codon. Sec61β 3R was used as a control protein without a functional TMD. Filled circles indicate tRNA-bound TA-protein species recovered with HisTrx-SGTA.",
    "molecules": "tRNA"
  },
  {
    "caption": "E tRNA-bound species of FLAG-tagged Sec61β from experiment shown in panel (D) were quantified and expressed relative to the value obtained for the control-depleted RRL. Shown is the mean with standard error of mean for n=4 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.",
    "molecules": "tRNA"
  },
  {
    "caption": "Sec61β wild-type (WT) and the 3R variant tagged at their N-terminus with the FLAG epitope were translated in RRL from RNAs lacking a stop codon. Ribosome-nascent chain complexes (RNCs) were recovered using anti-FLAG affinity resin, beads were washed, bound proteins eluted with 3xFLAG peptide and RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS-PAGE and immunoblotted for endogenous rabbit SGTA, FLAG-tagged translation products, proteins of the 40S (Rps3) and 60S (Rpl17) ribosomal subunits or analysed by phosphorimaging (35S). The 3xFLAG peptide eluted material (lanes 4 and 5) corresponds to ~10% of the sedimented RNCs (lanes 7 and 8).",
    "molecules": "sucrose, 35S"
  },
  {
    "caption": "A, B PPL-C99-TMD (A) and PPL-C99FL (B) were in vitro translated in RRL from mRNAs lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and ε-TDBA-Lys-tRNA analogue. Reactions were irradiated with the UV light to induce photo-cross-linking, RNCs isolated and adducts immunoprecipitated with mouse anti-SRP54, mouse anti-SGTA or a control antibody as described in Materials and Methods. Samples were resolved by SDS-PAGE and results visualised by phosphorimaging. C-terminal 40 amino acid residues located in the ribosomal exit tunnel are indicated. \"x SRP54\" and \"x HisTrx-SGTA\" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively. Arrows indicate unmodified translation products.",
    "molecules": "ε-TDBA-Lys, tRNA"
  },
  {
    "caption": "C PPL-C99FL variants lacking a stop codon and carrying the indicated Lys to Arg substitutions were in vitro translated in the presence of 2 μM HisTrx-SGTA. Reactions were processed using chicken anti-SGTA antibody for immunoprecipitation of SGTA adducts. D Cross-linking adducts between ribosome-stalled PPL-C99FL lysine mutants and recombinant HisTrx-SGTA shown in panel (C) were quantified, any differences in translation efficiency accounted for and cross-linking efficiency expressed relative to the wild-type (WT) protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.",
    "molecules": "Arg, Lys, lysine"
  },
  {
    "caption": "A, B PPL-C99FL lacking a stop codon was in vitro translated in RRL in the presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε-TDBA-Lys-tRNA analogue. Reactions were carried out in the presence (A) or absence (B) of 2 μM HisTrx-SGTA. Samples were subjected to UV light-induced photo-cross-linking and processed as described for Fig 5. \"x SRP54\" and \"x HisTrx-SGTA\" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively, whilst \"endo. SGTA\" indicates cross-linking adducts with endogenous SGTA. Cross-linking products between stalled PPL-C99FL and SGTA are also indicated with arrowheads, whilst arrows indicate unmodified translation products.",
    "molecules": "ε-TDBA-Lys, tRNA"
  },
  {
    "caption": "C, D chemical cross-linking using DSS reagent was carried out. E DSS-mediated HisTrx-SGTA cross-linking efficiency to PPL-C99FL in the absence or presence of ER-derived microsomes was quantified. For each repeat, intensity of the cross-linked SGTA adduct in the absence of microsomes was set as 100%. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.",
    "molecules": "DSS"
  },
  {
    "caption": "A Indicated precursor proteins were translated in vitro using RRL supplemented with 20 μM HA-tagged ubiquitin and 2 μM of either HisTrx or HisTrx-SGTA. RNCs were isolated, ubiquitinated nascent chains recovered using anti-HA agarose, samples resolved by SDS-PAGE and analysed by phosphorimaging. Ubiquitinated precursor protein species were assigned based on SDS-PAGE electrophoretic mobility and are indicated in red. A schematic diagram of the proteins used is shown and the ~40 residues of the nascent chain located in the ribosomal exit tunnel are indicated. \"ss\" - signal sequence, \"TMD\" - transmembrane domain.",
    "molecules": "agarose, ubiquitin"
  },
  {
    "caption": " A, B Representative images o O-GlcNAc  (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125",
    "molecules": "O-GlcNAc"
  },
  {
    "caption": " C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008 ",
    "molecules": "O-GlcNAc, O-GlcNAcylation"
  },
  {
    "caption": "D, E Regression plots of average immune-staining  scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16) ",
    "molecules": "O-GlcNAc, O-GlcNAcyl"
  },
  {
    "caption": " E Representative images of O-GlcNAc staining in Paneth cells  of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells ",
    "molecules": "O-GlcNAc"
  },
  {
    "caption": " J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J) are shown (n = 4-5). (L: P = 0.049",
    "molecules": "DSS"
  },
  {
    "caption": " J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes i colitis scores (K are shown (n = 4-5). (L: P = 0.049",
    "molecules": "DSS"
  },
  {
    "caption": " J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049",
    "molecules": "DSS"
  },
  {
    "caption": " F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting ",
    "molecules": "O-GlcNAc"
  },
  {
    "caption": " G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylatio were determined",
    "molecules": "Ac45S-GlcNAc, O-GlcNAcyl"
  },
  {
    "caption": " G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 an STAT1 O-GlcNAcylation (H) were determined",
    "molecules": "Ac45S-GlcNAc, O-GlcNAcyl"
  },
  {
    "caption": " A, B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS. Daily changes in body weight (A) and colitis score (B) (n= 5). (B: day6 P = 0.0171, day7 P= 0.0445 ",
    "molecules": "DSS, H2O, TMG"
  },
  {
    "caption": " C Intestinal barrier functional assays measuring serum FITC-dextran before DSS treatment (n = 5, P = 0.0117)",
    "molecules": "FITC, DSS, dextran"
  },
  {
    "caption": "(D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments.",
    "molecules": "IAA"
  },
  {
    "caption": "(B) Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000-26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected.",
    "molecules": "IAA"
  },
  {
    "caption": "(F) Representative images showing ectopic CENP-A on a chromosome spread after induction of CENP-A overexpression in DLD-1 cells. Centromere position is marked using immunofluorescence staining for CENP-B. Yellow dashed lines contour representative chromosomes. Scale bar, 2µm. Schematic to the right illustrate the observed CENP-AmRFP-DHFR localization pattern on a chromosome. Right panel: Comparison of CENP-ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX/TMP treatment. Each dot represents one centromere or a region on the chromosome arms.",
    "molecules": "DOX, IAA, TMP"
  },
  {
    "caption": "(E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments. ",
    "molecules": "EdU, Palbociclib"
  },
  {
    "caption": "(C) Representative IF-FISH images of mitotic spreads in non-treated cell (NT) and following a CENP-AOFF/ON cycle (IAA WO). Chromosomes are contoured by a green (X), magenta (Y) or white (other autosomes) dashed line. The centromeres of chromosome X/Y are highlighted by a green/magenta arrow. Scale bar, 2 µm.",
    "molecules": "IAA"
  },
  {
    "caption": "(G) Representative IF-FISH images showing CENP-C localization on the Y chromosome in cell growing under selective pressure (G418) following a CENP-AOFF/ON cycle. Chromosomes are highlighted by a white dashed contour line. CENP-C was found at the native centromere position (top panel, blue), the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr-Y painting probe staining (second panel, orange) or CENP-C was found on a different location on the Y chromosome and did not overlap with Cen-Y DNA (lower panels, red). Scale bar, 2 µm. (H) Quantification of indicated events depicted in G. Each dot represents one experiment (>20 spreads for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t-test, *p=0.0137 and 0.039. ",
    "molecules": "DNA, G418"
  },
  {
    "caption": "(C) Representative immunofluorescence images showing CENP-A and CENP-C reloading in the indicated conditions. Cells with centromeric CENP-A/CENP-C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. The centromere position is marked using immunofluorescence staining for CENP-B. White dashed lines contour DAPI stained nucleus of representative cells.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP-CmRFP-DHFR in the presence (control) and in the absence (sample) of endogenous CENP-A and CENP-C. Cells were arrested with Colcemid for 3 h prior to spread. Cells with centromeric CENP-C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-C. Chromosomes in inset are highlighted using a white dashed line. (G) Quantification of relative number of cells with eCENP-CmRFP-DHFR in the indicated conditions. Each dot represents one independent experiment with at least 30 cells per condition.",
    "molecules": "Colcemid"
  },
  {
    "caption": "(A) Gating strategy for the sorting of freshly purified resting human CD4+ T cells based on CENP-A expression. Dead cells were excluded (first plot) using Fixable Viability Dye eFluor 780, CENP-Ahigh and CENP-Alow cells (both CENP-B+) were sorted (second plot). Post-sort analysis of each population is then shown (third and fourth plots).",
    "molecules": "eFluor 780"
  },
  {
    "caption": "(D) Tracking of cell division by CFSE dilution. The black line separates cells that have not divided (nº of div=0, CFSE-high) from those that have undergone at least one division (nº of div≥1, CFSE-low). (E) CENP-A expression and CFSE dilution. Black lines were set based on CENP-A isotype control and CFSE maximal staining at day 0 (cells in shaded quadrants are CFSE-high/CENP-A-low). One representative donor is depicted. (F) Absolute number of CFSE-high/CENP-A-low CD4+ T cells (shaded gate in E). Symbols represent individual donors. Error bars represent SEM from 6 independent donors. One-way ANOVA, multiple comparisons, n=6. *p < 0.05; **p < 0.01. (G) Frequency of cells without centromeric CENP-A determined by immunofluorescence microscopy before and post activation. Symbols represent individual donors, n=4. Error bars represent SEM from 6 independent donors. ",
    "molecules": "CFSE"
  },
  {
    "caption": "B Fatty acid composition of B. subtilis WT cells grown in PF medium, and Δbkd grown with MB, IB or depleted for precursor for 90 min (IBPF). For detailed analyses, see Appendix Fig S1B.",
    "molecules": "MB, Fatty acid, IB"
  },
  {
    "caption": "E Fatty acid composition of E. coli WT and fabA(Ts) cells grown at 30°C and shifted to different temperatures for 120 min. For detailed analyses, see Appendix Fig S2B and C.",
    "molecules": "Fatty acid"
  },
  {
    "caption": "A DPH anisotropy of B. subtilis WT or Δbkd cells supplemented either with MB, IB or depleted for precursor (IBPF) for the times indicated. High DPH anisotropy indicates low membrane fluidity. B DPH anisotropy of E. coli WT cells grown steady state at 30°C, 37°C or shifted from 30°C to 37°C followed by immediate measurement. In addition, DPH anisotropy of fabA(Ts) cells grown steady state at 30°C or shifted from 30°C to 37°C followed by measurement at the times indicated. D",
    "molecules": "DPH, MB, IB"
  },
  {
    "caption": "A Images of B. subtilis WT and Δbkd cells co-labelled with the membrane potential-sensitive dye DISC3(5) and the membrane permeability indicator Sytox Green. Membrane properties were assessed for Δbkd cells grown in the presence of MB, IB or washed precursor-free (IBPF) for the times indicated. As controls, WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC (gABC) or pore-forming lantibiotic Nisin. For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3A-C. B Quantification of DISC3(5) fluorescence for cells (n=100-142) depicted in panel A. Median represented by red line. C Images of E. coli WT and fabA(Ts) cells co-labelled with the same indicator dyes as in panel A. Membrane properties were assessed for fabA(Ts) at 30°C and upon transfer to non-permissive 37°C for the times indicated. As controls, WT cells were incubated with the pore-forming antibiotic Polymyxin B (PolyB). For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3D and E. The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background (see Fig EV1). D Quantification of DISC3(5) fluorescence for cells (n=76-141) depicted in panel C. Median represented by red line. Dat",
    "molecules": "Sytox Green, MB, ONPG, DISC3(5), gABC, gramicidin ABC, IB, Nisin, PolyB, Polymyxin B"
  },
  {
    "caption": "A Images of B. subtilis Δbkd cells stained with membrane dye FM 5-95 and expressing GFP fusions of DNA-binding protein HBsu (top), cell division protein FtsZ (middle) or cell elongation protein MreB (bottom). Cells were grown with IB or depleted for precursors for 90 min (IBPF). For further examples and additional time points, see Appendix Fig S4A. B Images of E. coli fabA(Ts) cells stained with FM 5-95 for the outer membrane and with DNA-intercalating dye DAPI (top), or expressing GFP sandwich (SW) fusions to the cell division protein FtsZ (middle) and the cell elongation protein MreB (bottom), respectively. Depicted are cells grown at 30°C or with a temperature shift to 37°C for 120 min. For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery, see Figs EV2, EV3, EV4, and S5. For further examples and additional time points, see Appendix Fig S4B. D",
    "molecules": "FM 5-95, DAPI, IB"
  },
  {
    "caption": "B Images of B. subtilis Δbkd cells grown with IB or without precursor (IBPF). Cells were stained with membrane fluidity-sensitive dye Laurdan and imaged at 460 nm, 520 nm and as the corresponding colour-coded Laurdan GP map. C Images of E. coli fabA(Ts) cells grown at 30°C or shifted to non-permissive 37°C for 120 min. Cells were stained and imaged as described in panel B. For a more pronounced view on domain formation associated with local differences in membrane fluidity, see Appendix Fig S7, showing E. coli fabA(Ts) grown in LB instead of M9 minimal medium with glucose/casamino acids. D Images of B. subtilis Δbkd cells grown and stained with Laurdan (imaged at 460 nm) as in panel B, but additionally expressing WALP23-mCherry. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8A. Co-localisation of membrane dye FM 5-95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C. E Images of E. coli fabA(Ts) cells grown and stained as in panel C, but additionally expressing WALP23-mScarlet-I. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8B. Dat",
    "molecules": "casamino acids, FM 5-95, LB, glucose, IB, Laurdan"
  },
  {
    "caption": "E Fatty acid composition of E. coli fabA(Ts) cells (same cell batch as in D) upon growth at 30°C, upon depletion of UFA by incubation at 37°C for 120 min and upon recovery by oleate supplementation for additional 120 min. For detailed analyses, see Appendix Fig S11B-D.",
    "molecules": "Fatty acid, oleate, UFA"
  },
  {
    "caption": "Shoot regeneration assay using Col and hag1-6 root explants on media with different cytokinin (2IP) to auxin (IAA) ratios. Col and hag1-6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg/ml IAA-containing media supplemented with 0, 500, or 5,000 mg/ml 2IP. Pictures were taken at 27 d after transfer onto each media using representative explants. Scale bar: 2 mm.",
    "molecules": "auxin, cytokinin, IAA, 2IP"
  },
  {
    "caption": "pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red).",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).",
    "molecules": "PI"
  },
  {
    "caption": "pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).",
    "molecules": "PI"
  },
  {
    "caption": "Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B).",
    "molecules": "DEX"
  },
  {
    "caption": "Activation of SCR by DEX treatment of UBI::mCHERRY-GR-SCR in Col and hag1-6.",
    "molecules": "DEX"
  },
  {
    "caption": "Activation of WOX5 and SCR by DEX treatment of 35S::GVG-WOX5 UBI::mCHERRY-GR-SCR in Col and hag1-6.",
    "molecules": "DEX"
  },
  {
    "caption": "(d) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl, anti-miR-212 and anti-miR-132 after phenylephrine (PE, 10 μM) treatment (n=6-9; P-values against Scr+PE).",
    "molecules": "phenylephrine"
  },
  {
    "caption": "(a-c) Heart-to-body-weight ratios (a), cardiomyocyte diameters (b) and cardiac fibrosis (c) in Sham-operated mice and mice treated with intravenous injection of either scrambled control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC (n=4-11). PSR, picrosirius red staining.",
    "molecules": "Ant-132"
  },
  {
    "caption": "(d-f) Echocardiography analysis of cardiac dimensions and function in Sham-operated mice and mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC for (d) fractional shortening, (e) end-diastolic area and (f) end-systolic area (n=4-9).",
    "molecules": "Ant-132"
  },
  {
    "caption": "(g-i) Cardiac FoxO3 protein levels in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.",
    "molecules": "Ant-132"
  },
  {
    "caption": "(g-i) calcineurin activity (h) and Mcip1.4 mRNA levels (i) in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.",
    "molecules": "Ant-132"
  },
  {
    "caption": "Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex. Representative of n=3 independent experiments.   As in A, using a Trs85 truncation containing only the final 198 amino acids. NOTE: Trs85[501-698] is ~25kD in size and now migrates at the same size as some of the core subunits. The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct. Representative of n=3 independent experiments.   As in A/B, using a Trs85 mutant with a truncation of the loop from 575-603. Trs85[∆575-603] appears to dissociate from the core during chromatography, indicating a reduced affinity for the core subunits. Representative of n=3 independent experiments.   ",
    "molecules": "Coomassie"
  },
  {
    "caption": "GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions. With functional Trs85, there is an accumulation of free GFP. Samples are from pre-rapamycin treatment (-) and 1hr after addition of rapamycin (+). Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)   Quantification of A. The average of at least three replicates is plotted with error bars representing the 95% confidence interval. The percent of free GFP for wild-type Trs85 is compared to each mutant using an unpaired, two-tailed Student's t-test with Welch's correction: vs. EV ** p =   0.0022, vs. R618E, R619E, R620E, K621E *** p = 0.0008, vs. R618A, R619A, R620A, K621A *** p = 0.0006, vs. 501-698 *** p = 0.0003, vs. 4A p = 0.3295, vs. 7A *** p = 0.0008, vs. 4E *** p = 0.0005, ns = not significant. (WT = wild-type, EV = empty vector)",
    "molecules": "rapamycin"
  },
  {
    "caption": "E Effect of plant growth hormones (1 mM SA, 1 μM ABA, 10 μM JA and 10 μM 1-aminocyclopropane -1-carboxylic acid (ACC) on CDF4 expression. Three independent experiments were conducted. Values are given as mean ± SD, n=3.",
    "molecules": "ABA, 1-aminocyclopropane -1-carboxylic acid, ACC, JA, SA"
  },
  {
    "caption": "F Effects of abiotic stress treatments on CDF4 expression. Rosette leaves were treated with salt (100 mM NaCl), drought, H2O2 (2mM) or darkness. Three independent experiments were conducted. Values are given as mean ± SD, n=3.",
    "molecules": "H2O2, NaCl"
  },
  {
    "caption": "G Effects of SA, H2O2, and dark treatment on CDF4 expression in the aba2-1 mutant background. AtACT2 was used as an internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3.",
    "molecules": "H2O2, SA"
  },
  {
    "caption": "C Phenotypes of vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 plants, after treatment with 20 µM estradiol for seven days at 18-days-old. Scale bars indicate 1.5 cm.",
    "molecules": "estradiol"
  },
  {
    "caption": "E Analysis of the relative chlorophyll concentration in the sixth and seventh rosette leaves from the vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 lines shown at various development stages. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.",
    "molecules": "chlorophyll"
  },
  {
    "caption": "D The leaf senescence phenotypes of detached rosette leaves in vector control, pER8::CDF4, pER8::CDF4 -RNAi and pER8::amiCDF4 lines before and after treatment with 2 mΜ H2O2 in the dark for four days. Scale bars indicate 0.7 cm.",
    "molecules": "H2O2"
  },
  {
    "caption": "F The chlorophyll concentration in leaves was analyzed. The percentage indicates the chlorophyll content relative to vector control before treatment. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.",
    "molecules": "chlorophyll"
  },
  {
    "caption": "A Measurement of free ABA levels in the third and fourth rosette leaves from 21-day-old transgenic lines with altered CDF4 expression after estradiol induction for three days (once every day). Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.",
    "molecules": "ABA, estradiol"
  },
  {
    "caption": "B, C qPCR analysis of ABA synthesis and signaling-related genes in Col-0 and 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.",
    "molecules": "ABA"
  },
  {
    "caption": "D, E Relative expression of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM β-estradiol or mock treatment for 0, 2, or 4 h. The expression of the corresponding genes in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.",
    "molecules": "β-estradiol"
  },
  {
    "caption": "F, G Relative expression level of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM DEX, 100μM CHX, DEX plus 100μM CHX, or mock. The gene expression in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.",
    "molecules": "CHX, DEX"
  },
  {
    "caption": "E Observation of rosette leaf longevity from the 26-day-old vector control, pER8::CDF4, pER8::CDF4 & nced2, pER8::CDF4 & nced3 and pER8::CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks. Phenotype of detached rosette leaves arranged from oldest to youngest. Scale bars indicate 1.5 cm.",
    "molecules": "estradiol"
  },
  {
    "caption": "(G) Chlorophyll concentration in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.",
    "molecules": "Chlorophyll"
  },
  {
    "caption": "A The senescence phenotype of the vector control, pER8::CDF4 and pER8::CDF4::RNAi detached leaves treated with H2O2 and incubated in the dark for three days. The third leaf in rosettes of 3-week-old plants was detached and incubated in MES buffer (2 mM MES, pH 5.8) treated with 2 mM H2O2 and 20 μM β-estradiol under dark conditions for three days. Picture shows the leaves before and after treatment. Scale bars indicate 1 cm.",
    "molecules": "MES, β-estradiol, H2O2"
  },
  {
    "caption": "(B) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. The brown color represents H2O2 accumulation. Scale bars indicate 1 cm.",
    "molecules": "DAB, β-estradiol, H2O2"
  },
  {
    "caption": "(C) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.",
    "molecules": "β-estradiol, H2O2"
  },
  {
    "caption": "D Chlorophyll concentrations in the leaves shown in A. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.",
    "molecules": "Chlorophyll"
  },
  {
    "caption": "A Measurement of catalase activity in vector control and CDF4RNAi plants. Ten-day-old green seedlings ("Young") and the third and fourth rosette leaves from 36-day-old plants ("Old") were used, respectively. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.",
    "molecules": "catalase"
  },
  {
    "caption": "B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36-day-old plants. The expression of CAT genes in the wild-type plant is given as 1. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.",
    "molecules": "CAT"
  },
  {
    "caption": "(D) Measurement of chlorophyll in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.",
    "molecules": "chlorophyll"
  },
  {
    "caption": "(E) H2O2 concentrations in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.",
    "molecules": "H2O2"
  },
  {
    "caption": "F ChIP-qPCR analysis of the relative binding of CDF4 to the promoter regions of PGAZAT. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 6-week-old pER8::CDF4-HA transgenic plants after estradiol induction. Black columns indicate the enrichment fold changes normalized to ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.",
    "molecules": "estradiol"
  },
  {
    "caption": "A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex.",
    "molecules": "noscapine"
  },
  {
    "caption": "B. Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora-B (ZM447439) in combination with the proteasome inhibitor MG-132 to keep cells arrested in mitosis and in B. with an inhibitor against Plk1 (BI2536). Drugs were added at the same timing as Hoechst, 30 minutes prior to imaging.",
    "molecules": "BI2536, MG-132, Aurora-B, ZM447439"
  },
  {
    "caption": "A. Immunofluorescence images and quantifications of KT-localized Plk1 in U2OS cells after PBIP1 depletion. Cells were either Mock transfected or with PBIP1 siRNA for 72 hours. Cells were treated with nocodazole for 1 hour and fixed. Cells were stained with the centromere marker ACA, Plk1 and DAPI.",
    "molecules": "nocodazole"
  },
  {
    "caption": "B) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells.",
    "molecules": "RNase"
  },
  {
    "caption": "D) Agarose gel electrophoresis of RNA extracted from control, 4hr and 24hr ActD treated K562 cells.",
    "molecules": "ActD"
  },
  {
    "caption": "E) Barplots showing qRT-PCR gene expression levels (mean +/- S.D.) of PTEN, FNDC3B, and STAM genes in control, 4hr and 24hr actinomycin D treated K562 cells. p-value: two-tailed t-test, (*** p < 0.05). The results represent data from 3 technical replicates from 3 independent biological preparations.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for aXL control and RNase A treated cells. The arrows represent similar patterning of long-range interactions in samples treated with RNase after crosslinking.   Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for bXL control and RNase A treated cells. Black arrow indicates the reduction in long-range interactions in the bXL RNase A dataset. The arrows represent perturbed patterning of long-range interactions in samples treated with RNase before crosslinking.  ",
    "molecules": "RNase, RNase A"
  },
  {
    "caption": "C-D) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal components (1st PC) below for aXL (C) and bXL (D) samples. The bXL RNase A sample shows reduced Pearson correlations across the genome.",
    "molecules": "RNase A"
  },
  {
    "caption": "E) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and RNase treated cells before and after crosslinking. The bXL RNase A sample show reduction of the negative eigenvalues, indicative of perturbation of B-type compartments. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.",
    "molecules": "RNase, RNase A"
  },
  {
    "caption": "F) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions. The bXL RNase A samples display reduced B-B type interactions.",
    "molecules": "RNase A"
  },
  {
    "caption": "Interaction heatmaps at 40kb resolution zooming in chr21:19.5-33Mb and showing the TAD structures in aXL and bXL datasets. The aXL and bXL RNase A / control log2 ratios are shown below the heatmaps.",
    "molecules": "RNase A"
  },
  {
    "caption": "C-D) Mean interactions frequencies +/- 1Mb centered on all TAD boundaries for each dataset at 40kb resolution, as well as the log2 ratios of aXL (left panel) or bXL (right panel) samples. The TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking.",
    "molecules": "RNase, RNase A"
  },
  {
    "caption": "A) Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for control and 24hr ActD treated cells. The arrows point out to perturbed long-range interactions at shorter scales.",
    "molecules": "ActD"
  },
  {
    "caption": "B) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal component (1st PC) below for control and 24hr ActD treated cells.",
    "molecules": "ActD"
  },
  {
    "caption": "C) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and ActD treated cells. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.",
    "molecules": "ActD"
  },
  {
    "caption": "B) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.",
    "molecules": "ActD"
  },
  {
    "caption": "E) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells for boundaries that are bound with differing numbers of CTCF binding sites. The reduction of TAD boundary scores upon ActD treatment is independent of CTCF binding. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.",
    "molecules": "ActD"
  },
  {
    "caption": "F) Scaling plot generated by 40kb resolution Hi-C data showing the interaction frequency as a function of genomic distance, with upper distance limit of 3Mb. The 24hr ActD treated samples shows a slower rate of decay at shorter distances, correlating with the reduction of insulation scores.",
    "molecules": "ActD"
  },
  {
    "caption": "B) Proliferation assay of H3122 parental and isogenic drug-resistant cells, treated with the indicated concentrations of crizotinib for 72h. P < 0.0001 was calculated for IC50 shift as indicated in the methods.",
    "molecules": "crizotinib"
  },
  {
    "caption": "C) Gene-set enrichment analysis after RNA-seq of H3122 sensitive vs CrizR1 crizotinib resistant cells. D) Epithelial to mesenchymal transition enrichment plot from the RNA-seq of C), using the HALLMARK gene collection. ",
    "molecules": "crizotinib"
  },
  {
    "caption": "F) Gene expression analysis by RT-qPCR of CrizR1 cells treated with the indicated concentrations of bemcentinib for 48h. LOX: P=0.03; SNAI2: P =0.02; VIM: P =0.03.",
    "molecules": "bemcentinib"
  },
  {
    "caption": "G) Proliferation assay of CrizR1 and CrizR4 cells treated with the indicated concentrations of bemcentinib ± 1uM crizotinib for 72h. *** = P < 0.001 for IC50 shift, as indicated in the methods (n=4).",
    "molecules": "bemcentinib, crizotinib"
  },
  {
    "caption": "H) Annexin V+ apoptotic assay in CrizR1 cells treated with 1uM crizotinib, 2.5uM bemcentinib, or combination. STE-1 parental cells were used as crizotinib drug control. crizotinib: P =0.2; bemcentinib: P =0.4; combination: P=0.3.",
    "molecules": "bemcentinib, crizotinib"
  },
  {
    "caption": "D) Western blot analysis of Ste-1 parental and isogenic Alectinib resistant cell lines for the indicated proteins.",
    "molecules": "Alectinib"
  },
  {
    "caption": "E) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h. P=0.2 (n=4).",
    "molecules": "alvocidib, crizotinib"
  },
  {
    "caption": "F) As above, in a proliferation assay, H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control. H3122 vs DMSO P <0.0001, CrizR1 vs DMSO P=0.1 (n=4).",
    "molecules": "crizotinib, DMSO"
  },
  {
    "caption": "Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR isogenic drug-resistant cell lines, treated with the indicated concentrations of alvocidib(G) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.",
    "molecules": "alvocidib"
  },
  {
    "caption": "H) Proliferation assay of AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of dinaciclib (H) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.",
    "molecules": "dinaciclib"
  },
  {
    "caption": "A) Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR, and AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of THZ1 for 72h. P <0.05 was calculated for IC50 shift, as indicated in the methods.",
    "molecules": "THZ1"
  },
  {
    "caption": "B) Crystal violet staining of EML4/ALK parental and drug-resistant cells. Cells were treated with the indicated drugs until the vehicle control reached confluence, then fixed and stained.",
    "molecules": "Crystal violet"
  },
  {
    "caption": "C) CrizR1 cells were treated with the indicated drugs for 72h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells. Alvocidib: early apoptosis P=0.002, late apoptosis P=0.006, alive P=0.0008; Dinaciclib: early apoptosis P=0.005, late apoptosis P=0.02, alive P=0.002.",
    "molecules": "Alvocidib, Dinaciclib, PI"
  },
  {
    "caption": "A) Indicated cell lines were treated with DMSO, 200nM alvocidib or 25nM dinaciclib for 48h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells (n=2). Alvocidib: CrizR1 P=0.005, AlecR P =0.001, PC9 P =0.05, A549 P =0.002, H1299 P =0.03, H460 P =0.07; P dinaciclib: CrizR1 P=0.02, AlecR P =0.005, PC9 P =0.06, A549 P =0.08, H1299 P =0.09, H460 P =0.01.",
    "molecules": "alvocidib, Alvocidib, DMSO, dinaciclib, PI"
  },
  {
    "caption": "(B) CrizR1 cells were treated with DMSO or 200nM alvocidib for 24h and cell extracts were hybridized to a 43-antibody array and analysed by immunoblotting. Graphs depict the significant changes from two independent experiments.",
    "molecules": "alvocidib, DMSO"
  },
  {
    "caption": "C) CrizR1/CeritR/AlecR cells were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h and cell extracts were analysed by western blotting.",
    "molecules": "alvocidib, DMSO, dinaciclib"
  },
  {
    "caption": "D) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or siRNA for CDK7 or CDK9 for 72h. (Bottom) CrizR1 were treated as above, or with DMSO/alvocidib/THZ1 and cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection (n=2). Annexin siCDK7 P=0.2, siCDK9 P=0.015, alvocidib P=0.0001, THZ1 P =0.004.",
    "molecules": "alvocidib, DMSO, PI, THZ1"
  },
  {
    "caption": "E) H3122 parental and CrizR1 cells were treated with DMSO, 200nM alvocidib or 250ng/ml actinomycin D for 6 hours. RNA was extracted and the mRNA levels of the indicated genes were quantified by RT-qPCR. CDK1: DMSO vs H3122 DMSO P=0.002, Actinomycin vs DMSO P=0.0003, Alvocidib vs DMSO P=0.0005; CDK2: DMSO vs H3122 DMSO P =0.0001, Actinomycin vs DMSO P<0.0001, Alvocidib vs DMSO P=0.0005; CDK6: DMSO vs H3122 DMSO P =0.0005, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO P =0.0004; CDK9: DMSO vs H3122 DMSO P=0.0003, Actinomycin vs DMSO P =0.0002, Alvocidib vs DMSO P =0.0003; CCNB1: DMSO vs H3122 DMSO P=0.02, Actinomycin vs DMSO P=0.0002, Alvocidib vs DMSO P=0.0003; CCNE1: DMSO vs H3122 DMSO P =0.0004, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO",
    "molecules": "Actinomycin, actinomycin D, alvocidib, Alvocidib, DMSO"
  },
  {
    "caption": "F) RT-qPCR analysis of MCL-1 and Survivin expression after treatment of CrizR1 cells with 100nM alvocidib, 25nM dinaciclib and 50 nM THZ1. RNA was extracted after 24h of treatment. MCL-1 alvocidib vs control P=0.0001, dinaciclib vs control P=0.0001, THZ1 vs control P =0.0001; Survivin: alvocidib vs control P =0.01, dinaciclib vs control P=0.01, THZ1 vs control P =0.0001.",
    "molecules": "alvocidib, dinaciclib, THZ1"
  },
  {
    "caption": "G) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin (siBIRC5). (Bottom) Cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection. Annexin: early apoptosis P =0.0003, late apoptosis P =0.5, alive P <0.0001.",
    "molecules": "PI"
  },
  {
    "caption": "A) CrizR1 cells were treated with vehicle control, 200nM alvocidib or 100nM THZ1 for 6 hrs, then chromatin was precipitated with an anti-RNA polymerase II antibody and sequenced. Plotted is the average number of peaks per condition.",
    "molecules": "alvocidib, THZ1"
  },
  {
    "caption": "GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (B) alvocidib Shown on the right is the plot of the "MYC targets"-enriched signature.",
    "molecules": "alvocidib"
  },
  {
    "caption": "GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (C) THZ1 treatment. Shown on the right is the plot of the \"MYC targets\"-enriched signature.",
    "molecules": "THZ1"
  },
  {
    "caption": "E) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h. The graph represents the mean fold change ± SD. MYC: alvocidib vs DMSO P =0.03, THZ1 vs DMSO P <0.0001; CCND1: alvocidib vs DMSO P =0.001, THZ1 vs DMSO P =0.003.",
    "molecules": "Alvocidib, alvocidib, DMSO, THZ1"
  },
  {
    "caption": "H) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib, rapamycin or both. Trametinib: early apoptosis P =0.002, late apoptosis P =0.05, alive P =0.009; rapamycin: early apoptosis P =0.04, late apoptosis P =0.8, alive P =0.003; tram+rapa: early apoptosis P =0.0001, late apoptosis P =0.04, alive P =0.01.",
    "molecules": "rapa, rapamycin, tram, trametinib, Trametinib"
  },
  {
    "caption": "Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib and correspondent tumour weights (P.O. control n=5; I.P. control n=4; crizotinib n=8 Tumour weights, crizotinib P= 0.44",
    "molecules": "crizotinib"
  },
  {
    "caption": "Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to alvocidib and correspondent tumour weights (P.O. control n=5; I.P. control n=4 and alvocidib n=6). Tumour weights, , alvocidib P=0.03.",
    "molecules": "alvocidib"
  },
  {
    "caption": "C) Tumour growth of in vivo xenografts of Ste-1 AlectR cell lines in response to alvocidib and correspondent tumor weights (I.P. control n=6; alvocidib n=4). Tumour weights alvocidib P=0.004.",
    "molecules": "alvocidib"
  },
  {
    "caption": "D) Number of cleaved caspase-3 positive nuclei in alvocidib treated tumours compared to controls.",
    "molecules": "alvocidib"
  },
  {
    "caption": "F) qPCR of Survivin (P=0.0009), MCL-1 (P=0.04), MYC (P=0.007) and BIM (P=0.03) in Ste-1 AlectR cells xenografts treated with alvocidib (Control n=6; alvocidib n=4).",
    "molecules": "alvocidib"
  },
  {
    "caption": "G) Proliferation assay of H3122 parental and isogenic drug-resistant cell lines, treated with DMSO, 100nM lorlatinib, 200nM alvocidib or 50nM dinaciclib for 72h (n=4).",
    "molecules": "alvocidib, DMSO, dinaciclib, lorlatinib"
  },
  {
    "caption": "H) Growth curve of CrizR1 xenograft tumors in response to lorlatinib (Control n=7; lorlatinib n=7).",
    "molecules": "lorlatinib"
  },
  {
    "caption": "(G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 50μm.",
    "molecules": "picrosirius red"
  },
  {
    "caption": "(B) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or phenylephrine (PE) for another 48h. Scale bar: 50μm.",
    "molecules": "PE, phenylephrine, H2O"
  },
  {
    "caption": "(F) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm.",
    "molecules": "PE"
  },
  {
    "caption": "(A) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or PE for another 48h. Scale bar: 50μm. siMEF2a indicates MEF2a siRNA.",
    "molecules": "PE, H2O"
  },
  {
    "caption": "(C) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm. siMEF2d indicates MEF2d siRNA.",
    "molecules": "PE"
  },
  {
    "caption": "(G) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle (DMSO) or PE for another 48h. Scale bar: 50μm.",
    "molecules": "DMSO, PE"
  },
  {
    "caption": "(G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 20μm.",
    "molecules": "picrosirius red"
  },
  {
    "caption": "assay.B, Teicoplanin inhibit the entry of 2019-nCoV and SARS-nCoV. Chemical structure of teicoplanin (Left). A549 cells were seeded in a 96-well plate, and 24 h later, the cells were infected with HIV-luc/2019-nCoV, HIV-luc/SARS-nCoV or HIV-luc/VSVG pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.C",
    "molecules": "teicoplanin, Teicoplanin"
  },
  {
    "caption": ", A549 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.",
    "molecules": "teicoplanin"
  },
  {
    "caption": "types.A, HEK293T cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.B",
    "molecules": "teicoplanin"
  },
  {
    "caption": "software.B, Huh7 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.C",
    "molecules": "teicoplanin"
  },
  {
    "caption": "A549 cells were incubated with dalbavancin (D) at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. dalbavancin",
    "molecules": "dalbavancin"
  },
  {
    "caption": "D, A549 cells were incubated with vancomycin at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. Left: Chemical structure of vancomycin",
    "molecules": "vancomycin"
  },
  {
    "caption": "Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes (D) Binding is reduced in Delta∆β1-2, but not to the Serrate equivalent using liposomes composed of PC:PS:PE-fluoroscein (80:15:5). Notch binding to Drosophila Delta NE3 (E) WT and ∆β1-2 (Delta∆β1-2) variants both bind to Notch, unlike variant with F204A substitution in DSL domain. Comparisons were performed with a two-tailed unpaired t-test. Values are shown as scattered data points with the dark lines representing the means. ns, no significant difference, **P < 0.01; ***P < 0.0001.",
    "molecules": "fluoroscein, PS, PC, PE"
  },
  {
    "caption": "D. A representative gel showing that EXD2 (500 nM) cleaves the Cy5-RNA and Cy5-RDH substrates.",
    "molecules": "Cy5, RNA"
  },
  {
    "caption": "A-F. (A and D) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of the GC-RDH substrate under 1 mM MnCl2 (A) or the RDH substrate under 0.2 mM MnCl2 (D). The solid red lines are the step-fitting results from an automated step-finding algorithm. (B and E) The corresponding TDP of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (B) or RDH under 0.2 mM MnCl2 (E). (C and F) The dwell-time histograms of combined pauses (Total) in the process of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (C) or RDH under 0.2 mM MnCl2 (F). The histogram fittings to a double exponential function are shown in red. The R2 of the double exponential function are 0.94 and 0.95, respectively.",
    "molecules": "MnCl2"
  },
  {
    "caption": "D Immunohistochemistry of 10-12 month old end-stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein (detected by unique antibody to the polySer protein C-terminus, α-SerCT) in cerebellar white matter, brainstem, hippocampus and layer II of the frontal cortex. Representative polySer aggregates are indicated by black arrows. No aggregates are found in age matched non-transgenic (NT) littermates (n=6 for each cohort).",
    "molecules": "polySer"
  },
  {
    "caption": "F Immunohistochemistry of human SCA8 autopsy tissue (n=4-7) shows accumulation of novel polySer RAN protein in cerebellar white matter, brainstem and frontal cortex.",
    "molecules": "polySer"
  },
  {
    "caption": "A Immunohistochemistry (IHC) of end-stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells. Representative polySer aggregates are indicated by black arrows. Inset: higher magnification of molecular layer and white matter.",
    "molecules": "polyGln, polySer"
  },
  {
    "caption": "B IF double staining shows no co-localization of polyGln and polySer in frontal cortex, pons or hippocampus of end-stage SCA8 BAC mice.",
    "molecules": "polyGln, polySer"
  },
  {
    "caption": "C IF double staining of end-stage SCA8 BAC frontal cortex shows exclusive localization of polyGln (mouse α-Gln, red, bottom panel) in neurons (rabbit α-NeuN, green, bottom panel). In contrast, polySer (Rabbit α-SerCT, red, top panel) shows widespread accumulation in the frontal cortex including within neurons (mouse α-NeuN, green, top panel).",
    "molecules": "polyGln, polySer"
  },
  {
    "caption": "D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells (left panels) while polyGln accumulates in Purkinje cells but not cerebellar white matter (right panels).",
    "molecules": "polyGln, PolySer"
  },
  {
    "caption": "A Representative images of the vestibular nucleus (upper panels), cuneate nucleus (middle panels) and motor cortex layers II/III (lower panels) of SCA8 BAC mice at 2 months (left panels), 6 months (middle panels) and end-stage (10 months, right panels) stained with α-SerCT. Representative aggregates are indicated by black arrows. Corresponding quantifications of polySer aggregates for each region on the right (n=3; mean ± SEM; One-way ANOVA with Tukey's post-hoc test; * p<0.025, ** p<0.005, *** p<0.0005, **** p<0.0001)",
    "molecules": "polySer"
  },
  {
    "caption": "A Disease-specific sensitivity to vacuolization after prolonged storage in ethanol shown by H&E in cerebellar white matter and brainstem of 10-12 month old end-stage SCA8 BAC mice but not in age matched NT (n=3) (panels 1&2). Demyelination shown by luxol fast blue staining (LFB) (panel 3) and axonal degeneration shown by α-SMI-32 (panel 4; degenerated axons are indicated by black arrows) observed in sites of polySer accumulation shown by α-SerCT (panel 5; representative aggregates are indicated by black arrows) in deep cerebellar white matter and brainstem in SCA8 BAC mice (lower panels) but not in NT mice (upper panels) (n=3).",
    "molecules": "ethanol, LFB, polySer"
  },
  {
    "caption": "Demyelination shown by luxol fast blue staining (LFB) (panel 1) and axonal degeneration shown by α-SMI-32 (panel 2) observed sites of polySer accumulation shown by a-SerCT2 (panel 3) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains (n=3).",
    "molecules": "LFB, polySer"
  },
  {
    "caption": "E Cell death measured by lactase dehydrogenase (LDH) assay in T98 cells transfected with codon replacement constructs expressing polyGln (light blue) or polySer (dark blue). NT=nontransfected, EV=empty vector (n=5, ** p<0.003; *** p< 0.0003; mean ± SEM; unpaired t test).",
    "molecules": "polyGln, polySer"
  },
  {
    "caption": "C Dot blot detection of polySer expressionin using α-FLAG antibody showing decrease in RAN polySer but not ATG-polySer when HEK293 cells are co-transfected with eIF3F siRNA. D Quantification of polySer detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test). ",
    "molecules": "polySer"
  },
  {
    "caption": "E Detection of polyAla expression using α-HA antibody showing decrease in RAN polyAla but not ATG-polyAla when HEK293 cells are co-transfected with eIF3F siRNA. F Detection of polyGP expression using α-FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co-transfected with eIF3F siRNA.. G Quantification of polyAla and polyGP detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test). ",
    "molecules": "polyGP, polyAla"
  },
  {
    "caption": "Staining of lamellar bodies in BALO with RFP LysoTracker. Scale bars represent 100 μm.",
    "molecules": "LysoTracker"
  },
  {
    "caption": "H-I. Representative scheme and images of day 40 BALO alveoli (H) and airway (I). BALO alveolar-like structures (H) are shown (h') (red arrowheads) in semithin section (0.5 µm) stained with Toluidine blue. Scale bar indicates 100 μm (left). Electron microscopy showing AEC I (h'') ultrastructure within BALO. Scale bars indicate 2500 nm (center) and 1000 nm (right). An airway-like structure (i') is shown with secretory and ciliated cells (red arrowheads) in semithin sections (0.5 µm) longitudinally cut and stained with Toluidine blue. Scale bar indicates 50 μm (far left). Electron microscopy of a bronchiolar-like airway (I) depicting pseudostratified epithelium (i'') with a basal-like cell (BC), not reaching the lumen in which cilia (C) are seen, located between a secretory (SC) and a ciliated cell (Ci). Scale bars indicate 1000 nm (left and right). Mature cilia (i''') in BALO at higher magnification depicting the 9x2+2 structure with central doubled microtubules (insert, red arrowhead), a characteristic for motile cilia. Scale bar indicates 100 nm (far right).",
    "molecules": "Toluidine blue"
  },
  {
    "caption": "Representative images of day 21 BALO and tdTomato+ rMC stained with LipidTOX (green). Scale bar represents 100 μm.",
    "molecules": "LipidTOX"
  },
  {
    "caption": "Fluorescence images of αSMA (green) and neutral lipids (LipidTOX red) staining in WT‑derived rMC and BALO (dotted lines indicate single BALO or insert) at day 21 of culture. Scale bars represent 50 μm (left) and 25 μm (right).",
    "molecules": "LipidTOX red, neutral lipids"
  },
  {
    "caption": "Fluorescence image of αSMA+PDGFRαhigh MYO (yellow arrows) and LipidTOX+PDGFRαlow LIF (red arrows) from sorted PDGFRα-GFP rMC after 21 days of BALO culture. Scale bars represent 50 μm (left) and 25 μm (right).",
    "molecules": "LipidTOX"
  },
  {
    "caption": "Representative transmission and confocal images after LysoTracker staining indicating branching and number of branching points in n=4 BALO 15 days after treatment with 4 μM Scra or mo142-3p (day 21 of co-culture) in n=3 biological replicates.",
    "molecules": "LysoTracker"
  },
  {
    "caption": "c) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z-VAD-fmk. Numbers indicate the results of the quantitative analysis (percentage) of each dot blot.",
    "molecules": "phosphatidylserine, Staurosporine, z-VAD-fmk"
  },
  {
    "caption": "b) Anti-CD95-induced apoptosis of Jurkat cells. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein and a reduction of 33K Atg5 in a time-dependent manner. The 24K anti-Atg5-reactive protein was also visible in Jurkat cells after treatment with actinomycin D (Act. D) or cycloheximide (CHX; 5 h treatment in both cases). GAPDH expression indicates equal loading in each panel. All immmunoblots are representative of at least three independent experiments.",
    "molecules": "actinomycin D, cycloheximide"
  },
  {
    "caption": "(g) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor-mediated apoptosis of neutrophils. In contrast, Atg6 did not show evidence for cleavage and its levels remained unchanged in this system. Results of 7 h cultures are shown.",
    "molecules": "ALLN"
  },
  {
    "caption": "(h) Atg5 is not cleaved in apoptotic HeLa cells (5 h staurosporine treatment) lacking the small subunit of calpain, which was silenced by using specific siRNA.",
    "molecules": "staurosporine"
  },
  {
    "caption": "(i) Calpain resistant Atg5Δ191-196 was unable to sensitize HeLa cells lacking endogeneous Atg5 (due to gene silencing, see Fig. 1c) to staurosporine-induced cell death. Values are means ± s.e.m. of three independent experiments (18 h cultures).",
    "molecules": "staurosporine"
  },
  {
    "caption": "(d) CEM cells that either lacked Bcl-2 or expressed high levels of Bcl-2 received truncated Atg5 or GFP by lentiviral gene transfer. In the absence of Bcl-2, induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days. Apoptosis was largely blocked in cells expressing high levels of Bcl-2. GFP transfection did not affect viability of the cells in this system.",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Staurosporine-induced Atg5 cleavage (5 h treatment) in CEM cells independent of Bcl-2 expression. Blots were stripped and reprobed with anti-GAPDH antibody.",
    "molecules": "Staurosporine"
  },
  {
    "caption": "(e) Jurkat cells were treated with staurosporine for 5 h or left untreated. Staurosporine induced Atg5 cleavage and both full-length and truncated Atg5 were present in total lysates. Truncated but not full-length Atg5 was detected in Bcl-xL immunoprecipitates obtained from apoptotic Jurkat cells. No evidence for physical interaction between Atg5 and Bax was obtained. The lower panels demonstrate that both Bax and Bcl-xL were enriched following immunoprecipitation. The asterisk indicates P 0.05. The scale bars represent 10 μM.",
    "molecules": "staurosporine, Staurosporine"
  },
  {
    "caption": "B-E. Slices were obtained at ZT12  (B-C) or ZT0  (D-E) and pre-incubated with 50 µM CARB  for more than 1h before recording mIPSCs  in the presence of drug. Slices from the same animals were used as controls. CARB  increased mIPSC  frequency at ZT12  (CTL: 7.74 ± 0.55 Hz; CARB: 12.41 ± 1.03 Hz; P = 0.0001; Mann-Whitney U test), but not ZT0  (CTL:11.79 ± 1.00 Hz; CARB: 11.47 ± 1.60 Hz; P = 0.39; Mann-Whitney U test).  mIPSC  amplitude was unchanged at either time  (C, ZT12: CTL: 40.94 ± 2.08 pA; CARB: 40.64 ± 1.66 pA; P = 0.99; E, ZT0: CTL: 34.73 ± 2.14 pA; CARB: 33.61 ± 2.39 pA; P = 0.85; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "CARB"
  },
  {
    "caption": "F-G. mIPSC  frequency  (F, CTL: 9.82± 0.74 Hz; Atr: 7.70 ± 0.57 Hz; P = 0.0009; Wilcoxon signed-rank test) and amplitude (G, CTL: 45.88 ± 2.87 pA; Atr: 43.64 ± 2.40 pA; P = 0.70; Wilcoxon signed-rank test) recorded at ZT0 with acute atropine  (Atr) wash on. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "Atr, atropine"
  },
  {
    "caption": "H-I. Effect of acute wash-on and wash-off of pirenzepine  (Pir) on mIPSC  frequency (H, CTL: 9.00 ± 1.24 Hz; Pir: 7.94 ± 1.07 Hz; Washout: 8.99 ± 1.14 Hz; One-way repeated measures ANOVA, F(2, 20) = 7.483, P = 0.0037; Holm-Sidak's multiple comparison test) and amplitude (I, CTL: 61.23 ± 3.72 pA; Pir: 58.85 ± 4.06 pA; Washout: 58.25 ± 4.20 pA; One-way repeated measures ANOVA, F(2, 20) = 3.197, P = 0.0624). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "Pir, pirenzepine"
  },
  {
    "caption": "J-K. Effect of pirenzepine  or mecamylamine  (MEC) on carbachol-induced elevation in mIPSC  frequency at ZT12 (J, CARB: 10.02 ± 0.64 Hz; CARB+MEC: 10.95 ± 0.78 Hz; CARB+Pir: 7.75 ± 0.79 Hz; One-way ANOVA, F(2, 25) = 4.848, P = 0.0166; Holm-Sidak's multiple comparison test) and amplitude (K, CARB: 39.85 ± 2.60 pA; CARB+MEC: 42.17 ± 1.65 pA; CARB+Pir: 40.67 ± 4.34 pA; One-way ANOVA, F(2, 25) = 0.1501, P = 0.8614). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "CARB, carbachol, MEC, mecamylamine, Pir, pirenzepine"
  },
  {
    "caption": "A1. Diagram illustrating the in vivo two-photon imaging timeline. A2. GCaMP6s   (green) was expressed in V1  of the PV:Ai9 mice  (red:  td-Tomato). Scale: 500 µm. A3. Schematics of the calcium imaging  showing a mouse  that was head-fixed and free running on an air-suspended arena during the imaging  section. Locomotor activity was simultaneously monitored (bottom left) along with Ca2+ imaging  (Top right. Scale: 100 µm). Example spontaneous ΔF/F0 traces (bottom right).",
    "molecules": "Ca2+, calcium"
  },
  {
    "caption": "D1. Schematic showing in vivo dLGN-evoked Ca2+  reponses in V1. D2. Immunofluorescent staining  showing the GCaMP6s   (green) and ChR2-mCherry  (red) expression in V1  and dLGN  respectively. The optical fiber track was also labeled. (Scale: 500 µm). D3. Response profile of an example neurons  for dLGN  stimulation during the light/dark  cycle. Horizontal blue brief lines represent stimulus frequency.",
    "molecules": "Ca2+"
  },
  {
    "caption": "E-F. Top: color-coded calcium  signals of all responsive neurons  (% ΔF/F0). Bottom: The mean  Ca2+ response waveform for individual mouse  (dotted color lines) and all mice  (thick black and gray lines). Trials were aligned to LED onset time  (gray solid lines). Data information: Sample size is indicated as (mice, cells).",
    "molecules": "Ca2+, calcium"
  },
  {
    "caption": "D Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates and exposed to the same RNAi as in (C). *P<0.05, ANOVA with Dunnetts posthoc comparison to the empty RNAi vector control. N=3 trials. Error bars indicate SEM.",
    "molecules": "epoxomicin"
  },
  {
    "caption": "F Quantified epoxomicin-sensitive proteasome activity (as measured through the turnover of fluorescent chymotrypsin substrate) from the same lysates as in (E). No statistical difference (P<0.05 cutoff) was detected by ANOVA. N=4 trials.",
    "molecules": "epoxomicin"
  },
  {
    "caption": "C Survival curves (percentage of live animals assayed each hour after PA14 exposure) for the indicated mutants at 20oC. Plog-rank<0.0001. Median survival time was 90 hours for wildtype (N=304), 72 hours for dop-1(vs100) (N=180), 78 hours for elt-3(gk121) (N=120), 78 hours for pqm-1(ok485) (N=116), 78 hours for nhr-28(gk568) (N=123), and 42 hours for skn-1(tm3411) (N=33). Eight wild-type animals and six dop-1 mutants were censored (the animals crawled up the side of the plate and dessicated). The media contained 50 µg/ml of 5-fluoro-2′-deoxyuridine (FUdR to prevent the growth of egg offspring during the experiment.",
    "molecules": "5-fluoro-2′-deoxyuridine, FUdR"
  },
  {
    "caption": "D Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from either L4+48 hour animals (left half of graph) or L4+72 hour animals (right half of graph) of the indicated genotype. Animals were either continually grown on regular OP50 food (gray bars) or switched to NGM plates containing 1 mM paraquat at the L4 stage (orange bars). ****P<0.0001, ***P<0.001, ANOVA, Tukey's multiple comparison test compared to wild type. #P<0.001 ANOVA, Bonferoni multicomparison as indicated by the bracketed lines. N=20 animals per genotype. Error bars indicate SEM. No UbG76V-GFP fluorescence was detected in wild type at L4+72 hours on either OP50 or switched to NGM plates containing 1mM paraquat.",
    "molecules": "paraquat"
  },
  {
    "caption": "B) For each population, equatorial Z stacks of two representative nuclei are shown in the left panel (chromatin is shown in blue (DAPI staining) and nuclear envelop in white (Sun2 staining). Scale bar: 5 µm. The corresponding 3D reconstructions are presented in the right panel. Convex and non-convex surfaces are shown in blue and yellow, respectively. C) Nucleus-invagination ratio is defined as the ratio of the non-convex area over the total nucleus surface area. Nuclei are larger and deformed in CD33+ (magenta, n=106) than CD38- and CD19+ cells (blue [n=120] and green [n=115], respectively; 3 independent experiments; Bars correspond to mean ± SD; ****: p<0.0001, Mann-Whitney test; n.s: non significant).",
    "molecules": "DAPI"
  },
  {
    "caption": "D) p150 glued associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI, Sun2 and p150 glued . Scale bar 5µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI and BicD2. Scale bar 5µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B) The nucleus is progressively deformed over time in culture. For each time point, equatorial Z stacks of two representative cells are presented. Left panel: inverted image of chromatin (DAPI). Right panel: microtubules are shown in green and chromatin in blue. Scale bar: 5 µm. C) Nucleus volume (left panel) and invagination ratio (right panel) increase to attain levels similar to those of freshly isolated CD33+ cells (24 h, n=165, 4 donors; 48 h, n=143, 3 donors; and 72h, n=192, 4donors; CD33+, n=227, 5 donors; Bars correspond to mean ± SD; n.s: non significant; ***: p<0.001, ****: p<0.0001; Mann Whitney test). ",
    "molecules": "DAPI"
  },
  {
    "caption": "D) For each condition (CTL, untreated cells; +Bleb, blebbistatin-treated cells; +Bleb+NZ, blebbistatin and nocodazole-treated cells; +TX, taxol-treated cells; and +CilioD, ciliobrevin-D-treated cells) Z stacks of 2 representative cells are displayed. Microtubules are shown in green, chromatin in blue, centrosome in white and actin in magenta. Asterisks indicate regions where microtubules are not in contact with the nucleus. Scale bar 5 μm.",
    "molecules": "Bleb, blebbistatin, ciliobrevin-D, CilioD, nocodazole, NZ, taxol, TX"
  },
  {
    "caption": "F) Quantifications of nuclear volume (left panel), invagination ratio (right panel) in the indicated conditions. Non-treated cells at 48 hours (n=137, 3 donors) and 72 hours (n=143, 3 donors); and blebbistatin-treated cells (n= 96/3 donors), blebbistatin and nocodazole treated cells (n=66, 2 donors), taxol-treated cells (n=122, 3 donors), and ciliobrevin-D-treated cells (n=128, 3 donors). Bars correspond to mean ± SD; ****: p<0.0001. **: p<0.01, n.s: non significant; Mann-Whitney test.",
    "molecules": "blebbistatin, ciliobrevin-D, nocodazole, taxol"
  },
  {
    "caption": "A) Spatial distribution of lamin A/C in the nuclei of HSPC (CD38-), myeloid progenitors (CD33+) and lymphoid progenitors (CD19+). Inverted images of equatorial Z plane of DAPI and lamin A/C are shown. Scale bar: 5 μm. B) Line scans of lamin A/C signal along nucleus contour of representative cells are presented for each cell population. The corresponding variations in lamin A/C intensity and nucleus curvature are plotted in blue and in red, respectively. Negative curvature values correspond to local nuclear invaginations. Red asterisk underlines local nucleus invaginations. C) Dispersion of A/C signal along the nucleus periphery is similarly low in CD38- (n=17, 3 donors), CD33+ (n=61, 3 donors) and CD19+ (n=11, 3 donors) indicative of an homogenous distribution of the protein along the nuclear membrane. (Bars correspond to mean ± SD; n.s: non significant; Mann Whitney test). ",
    "molecules": "DAPI"
  },
  {
    "caption": "D) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38- (n=131, 3 donors), and CD19+ (n=74, 3 donors) indicative of a homogenous distribution of the protein along the nuclear membrane. But is significantly increased in CD33+ (n=146, 3 donors). Bars correspond to mean ± SD; ****:p<0.0001; Mann Whitney test. E-F-G) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38- (E), CD33+ (F) and CD19+ (G) cells. Inverted images of the equatorial Z plane of DAPI (left panel) and lamin B (right panel) of a representative nucleus are shown for each population. Scale bar: 5 μm. The variations in lamin B signal intensity and curvature are plotted in blue in red, respectively. Asterisks indicate local invaginations of the nuclear membrane. In the bottom graphs, positive and negative curvatures are shown in grey and red, respectively. The absence of curvature (curvature =0) is highlighted as a dashed line. In CD33+ cells, negative curvature of the nuclear membrane are associated with low lamin B intensities.",
    "molecules": "DAPI"
  },
  {
    "caption": "A) Spatial distribution of H3K9me3 in CD38-, CD33+ and CD19+ cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B) Spatial distribution of H3K9me3 at the indicated time points of differentiation of CD38- cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C) For each cell, the raw DAPI image at the equatorial Z stack is used to extract the nucleus contour and measure H3K9Me3 intensity (in arbitrary units [a.u]) along the contour (lower row).",
    "molecules": "DAPI"
  },
  {
    "caption": "G) Microtubule perturbations impair H3K9me3 redistribution during HSPC differentiation. Microtubule-modifying agents were added to culture medium between 48 and 72 hours. Cells were fixed and analyzed after 72 hours of culture. For each indicated treatment, one representative nucleus is presented. Inverted images of equatorial Z stacks of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm. H) H3K9me3 signal dispersion significantly increases in non-treated cells between 48h (n=139/3 donors) and 72h (n=148/3 donors) of culture. At 72h, the dispersion is similar in non-treated and blebbistatin-treated cells (n=124/ 3 donors). By contrast, H3K9me3 signal dispersion is significantly reduced compared with non-treated cells after treatment with nocodazole and blebbistatin (n=55, 2donors), taxol (n= 109, 3 donors) and ciliobrevin-D (n=144, 3donors) to a level attained by non-treated cells at 48 hours, demonstrating that microtubules affect H3K9me3 redistribution during HSPC differentiation. Bars correspond to mean ± SD; ****:p<0.0001; n.s: non significant; Mann Whitney test. ",
    "molecules": "blebbistatin, ciliobrevin-D, DAPI, nocodazole, taxol"
  },
  {
    "caption": "A) Microtubules perturbations impact the trancriptome of differentiating cells. Volcano-plot representation of down-regulated (blue) and up-regulated genes (red) in non-treated/control cells relative to ciliobrevin-D treated cells. Each circle represents the fold change (FC) of the quantity of mRNA and the significance of the change based on three independent replicates. Expressed genes with a false discovery rate (FDR) lower than 0.05 are shown in grey, whereas genes with a FDR greater than 0.05 are shown in blue or red. Genes significantly mis-regulated i.e. with an absolute value of FC higher than 1.8 are shown as full disks. Names of genes of interest are indicated within the graph. Genes that are in the signature of the myeloid-differentiation pathway are shown in magenta. Genes that are the signature of the megakaryocyte-differentiation pathway are shown in green. B) Heatmap representation and hierarchical clustering of gene expression changes between non-treated and ciliobrevin-D-treated cells collected from 3 donors in each condition. The color code corresponds to a log2 scale of the differential expression levels. ",
    "molecules": "ciliobrevin-D"
  },
  {
    "caption": "(C) Fluorescence confocal images showing in BMDMs treated with Rv2626c (2.5 μg/ml) for 30 min, fixed, immunostained with antibodies for His (Alexa 488) and DAPI. Scale bar, 10 μm.",
    "molecules": "Alexa 488, DAPI"
  },
  {
    "caption": "(D and E) BMDMs from WT mice were treated with LPS (100 ng/ml) or rRv2626c for indicated times (D). BMDMs from WT, TLR2-/-, TLR4-/-, MyD88-/-, TRIF-/-, IRAK1-/-, TRAF6-/-, TBK1-/- mice were treated with 2.5 μg/ml rRv2626c or rVector for 18 h (E). Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml LPS (A for indicated times. (A Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml Pam2CSK4 (B) B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.",
    "molecules": "Pam2CSK4"
  },
  {
    "caption": "(A) Identification of TLR2, IRAK1, RIP1, TRAF6, MAPK11 and TOLLIP as endogenous binding partners of rRv2626c. THP-1 cell lysates were incubated with His tagged-rRv2626c or rVector and then IP with His-agarose beads. Binding partners were confirmed by silver staining and mass spectrometric analysis. The red-colored letters indicate the peptides identified from mass spectrometry analysis.",
    "molecules": "agarose"
  },
  {
    "caption": "(D) Immunostaining of BMDMs treated with 2.5 μg/ml rRv2626c for 30 min. and then immunolabeled with antibody to His-rRv2626c (Alexa Fluor 488) or TRAF6 (Alexa Fluor 568). DAPI (blue) stained nuclei.",
    "molecules": "Alexa Fluor 488, Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(D) 293T cells were co-transfected with Flag-TRAF6, Myc-Rv2626c as indicated doses and HA-ubiquitin (left), HA-K48-linked ubiquitin (middle), HA-K63-linked ubiquitin (right) and then IP with αFlag, followed by IB with αHA. WCLs were used for IB with αFlag , αMyc, or αActin.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(E) BMDMs were pretreated with 2.5 μg/ml rRv2626c for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αTRAF6, IB with ubiquitin, K48-linked ubiquitin, or K63-linked ubiquitin. WCLs were used for IB with αHis, αTRAF6, or αActin.",
    "molecules": "LPS, ubiquitin"
  },
  {
    "caption": "(D) BMDMs were pretreated with rRv2626c-WT, rRv2626c-CA, or rRv2626c-DN for 1 h, and stimulated with 100 ng/ml LPS for 18 h. Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "(E) Myc-Rv2626c-expressing Raw264.7 or THP-1 cells were pretreated with rRv2626c-WT (2.5 μg/ml), rRv2626c-CA (10 ng/ml), rRv2626c-DN (2.5 μg/ml), for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αMyc or αTRAF6, IB with αMyc and ubiquitin. WCLs were used for IB with αMyc, αTRAF6, αHis or αActin.",
    "molecules": "LPS, ubiquitin"
  },
  {
    "caption": "(F) Splenocytes were used for IP with αHis or αTRAF6, followed by IB with αHis or αUb. WCLs were used for IB with αTRAF6, αHis or αActin.",
    "molecules": "Ub"
  },
  {
    "caption": "Western blot analysis of the endogenous Grp78 protein expression levels in LN229, U87 and SNB19 cells following treatment with increasing concentrations of TMZ. Grp78 level was normalized to GAPDH. Data shown in right panel are representative of two experiments, n=3.",
    "molecules": "TMZ"
  },
  {
    "caption": "Induction of Grp78 promoter activity by TMZ in RGD4C/AAVP-Grp78-Luc cells. LN229, U87 and SNB19 cells stably transduced with RGD4C/AAVP-Grp78-Luc or RGD4C/AAVP-CMV-Luc were grown in the presence of TMZ for the indicated times. Results represent the average RLU/1μg protein. Data shown are representative of two experiments, n=3. Two-way ANOVA with Bonferroni correction (GraphPad Prism 6) was used for data analysis.",
    "molecules": "TMZ"
  },
  {
    "caption": "TMZ induction of phospho-eIF2α and ATF6-p90 expression. Human glioblastoma cells transduced with RGD4C/AAVP-Grp78 were analyzed by Western blot following treatment with TMZ (100 μM for LN229 and SNB19, 60 μM for U87). GAPDH was used as a control.",
    "molecules": "TMZ"
  },
  {
    "caption": "RT-PCR analysis of constitutive expression and splicing of XBP1 in glioblastoma cells transduced with RGD4C/AAVP-Grp78, in the presence of TMZ. Sizes of the PCR products were 289 bp for unspliced XBP1 and 263 bp for spliced XBP1, the lower size band is not specific. Time points were selected among those tested in the Western blot in (A), subsequently the image containing 0, 1 and 2 hours was juxtaposed to images of 6, 12 and 24 hours. A white line has been added between the gel pieces that have been juxtaposed.",
    "molecules": "TMZ"
  },
  {
    "caption": "Tumor cell killing in vitro by the HSVtk/GCV approach. Glioblastoma cells stably transduced with RGD4C/AAVP-Grp78-HSVtk or RGD4C/AAVP-CMV-HSVtk were treated with either GCV (10 μM) or TMZ (100 μM for LN229 and SNB19, 60 μM for U87) or combination of both GCV and TMZ. Cells were stained with MitoSOX and analyzed by FACS at day 4 post-treatment. Data shown are representative of three experiments, n=3. Two-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.",
    "molecules": "MitoSOX, GCV, TMZ"
  },
  {
    "caption": "Hematoxylin and eosin staining (H&E) and immunostaining for the proliferation marker protein Ki67 of representative tumor sections from all experimental groups, Scale bars, 200 μm for H&E and 100 μm for Ki67. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "Blood vessel staining with CD31 (red). DAPI staining of the sections is shown in blue. Scale bar, 200 μm. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings.",
    "molecules": "DAPI"
  },
  {
    "caption": "Treatment of chondrocytes, astrocytes, lung and skin fibroblasts with RGD4C/AAVP-Luc vector, alone or in the presence of TMZ. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein. Data shown are representative of two experiments, n=3. Scale bars, 160 μm for chondrocytes and 80 μm for all other cells.",
    "molecules": "TMZ"
  },
  {
    "caption": "F. Microscopic images of the Q97-GFP fluorescence. Bars, 5 µm. G. The percentage of cells containing or lacking detectable aggregates was quantified. Cell boundaries are indicated by dashed lines.",
    "molecules": "Q97"
  },
  {
    "caption": "E. Q97-GFP was expressed in mia40-3 and corresponding wild type cells for the times indicated. Cells were harvested, washed and survivors were counted after plating on glucose plates. Mean values and standard deviations of three replicates are shown.",
    "molecules": "glucose, Q97"
  },
  {
    "caption": "B. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; ev, empty vector.",
    "molecules": "galactose, Q25, Q97"
  },
  {
    "caption": "C. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm.",
    "molecules": "galactose"
  },
  {
    "caption": "E. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown.",
    "molecules": "PolyQ"
  },
  {
    "caption": "B. Still frames after different times of growth indicate a uniform fluorescence in Q25-GFP-expressing cells. Q97-GFP expression in wild type leads to many small and scattered aggregates per cell. In GAL-Mia40 cells, Q97-GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell. The arrow depicts aggregate formation. Bars, 4 µm. C. Quantified signal intensities in cells of the indicated mutants. In top and middle graphs, each curve corresponds to a single cell. Averaged signals (mean + SEM) in bottom graph (N=20 for WT, N=17 cells for GAL-Mia40).",
    "molecules": "Q25, Q97"
  },
  {
    "caption": "B. Q97-GFP was expressed for 4.5 h in wild type and GAL-Mia40 cells in two biological replicates each. Cells were lysed with NP-40 before soluble proteins were quantified. Mitochondrial proteins (mito.) and members of the mitochondrial carrier family (carrier) are visualized in a volcano plot. P-values were derived from a moderated t-test. Mitochondrial (mito.) and carrier proteins are indicated by yellow and red dots, respectively. Rnq1 is indicated in blue.",
    "molecules": "Q97, NP-40"
  },
  {
    "caption": "I. Cells of the newly made strains were grown on galactose for 1 or 7 days, respectively. Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified. Shown are mean values and standard deviations of three independent replicates.",
    "molecules": "galactose"
  },
  {
    "caption": "C. ∆ura3 cells expressing the Oxa1-Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins (Hansen et al., 2018) were transformed with plasmids expressing the proteins TDP-43, the TDP-43Q331K mutant, Q25-GFP, Q97-GFP, α-synuclein, FUS or GFP from a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Note that the expression of all aggregation-prone proteins (indicated by red arrows) allows cells to grow on uracil-deficient plates indicating the cytosolic accumulation of the Oxa1-Ura3 fusion protein.",
    "molecules": "Q25, Q97, uracil"
  },
  {
    "caption": "E. Wild type cells were transformed with plasmids for the expression of the indicated proteins. Q97-GFPnF is a non-fluorescent version of Q97-GFP. Close proximity of the two split GFP parts results in fluorescence which was visualized by microscopy. Shown are mean values and standard deviations of three replicates. Bar, 5 µm.",
    "molecules": "Q97"
  },
  {
    "caption": "B. The open reading frames of the indicated proteins were cloned downstream of a GAL promoter in plasmids and transformed into cells harboring the Q97-GFP expression plasmid. Cells harboring an empty vector (ev) were used for control. Cells were shifted to galactose medium overnight before they were dropped onto glucose or galactose plates. If cells are directly dropped from lactate medium onto galactose plates, only individual cells (indicated by red protein names) escaped the polyQ toxicity (see Fig. EV4C).",
    "molecules": "galactose, glucose, Q97, lactate, polyQ"
  },
  {
    "caption": "D. Cells expressing the indicated carrier proteins from GAL promoters were dropped onto galactose plates. Cell growth was quantified by densitometry from one representative plate. OM refers to carrier proteins that were expressed as fusions with an N-terminal outer membrane anchor. See Fig. EV4D, E for scans of the respective plates.",
    "molecules": "galactose"
  },
  {
    "caption": "B. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (middle row), ∆N-MIA40 (lower row) or a mock-transfected control (upper row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TOM20 staining. TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.",
    "molecules": "Q97"
  },
  {
    "caption": "D. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (upper row) or ∆N-MIA40 (lower row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.",
    "molecules": "Q97"
  },
  {
    "caption": "C Western blots of the lysates of HEK293T cells, resolved on SDS-PAGE and stained with a pan-arrestin antibody. mt = mock transfected. Bpa: Benzoyl-Phenylalanine. Arrestins were equipped with a 3xHA tag at the C-terminus, which increases their size of about 3 kDa compared to the endogenous proteins (βarr1 47 kDa, βarr2 46 kDa). βarr1 and βarr2 were transfected at 1/3 DNA compared to the corresponding TAG-mutants.",
    "molecules": "Benzoyl-Phenylalanine, Bpa, SDS"
  },
  {
    "caption": "Each panel represents immunoblots of whole cell lysates stained with an anti-HA antibody detecting arrestin. In the left panels GPCRs were combined with βarr1 variants, in the right panels with βarr2 variants. Residues replaced by Bpa are indicated above each lane. Covalent arrestin-receptor complexes are expected between 100-200 kDa, considering the size of the receptors (V2R 41.5 kDa, PTH1R 63.8 kDa, CRF1R, 46.3 kDa) and their glycosylation. A PTH1R, agonist PTH(1-34) B CRF1R, agonist Urocortin 1 (Ucn1) C V2R, agonist arginine-vasopressin (AVP) D No receptor. ",
    "molecules": "arginine, Bpa, PTH, Ucn1, Urocortin 1, vasopressin"
  },
  {
    "caption": "Aliquots from agonist-treated samples shown in Figure 2 were deglycosylated with PNGaseF, run on SDS-PAGE and immunoblotted with antibodies detecting arrestin (upper panels, α-HA) and the GPCRs (as indicated, CRF1R is equipped with a N-terminal FLAG, V2R carries a C-terminal 1D4 epitope).",
    "molecules": "SDS"
  },
  {
    "caption": "A SDS-PAGE and immunoblots of immunoprecipitated samples. Sample 1 was immunoprecipitated without previous UV treatment. Sample 2 is the product of irradiation of sample 1. Sample 3 was treated with UV light before immunoprecipitation. All samples were run in parallel on the same gel.",
    "molecules": "SDS"
  },
  {
    "caption": "(E) Western blot of whole cells lysates stained with an α-HA antibody. Residues exchanged with BrEtY are indicated in the upper row, mutations at PTH1R are indicated on the left side. The red arrows indicate the two most prominent signals. Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R.",
    "molecules": "Cys, BrEtY"
  },
  {
    "caption": "A Transcriptome re-analysis of ulp2Δ strains compared with WT (MHY1379) from previous RNA-seq experiments Two-dimensional agglomerative hierarchical clustering shows 2,903 significantly up- or down-regulated genes (P < 0.05) in triplicate RNA samples. Red and blue indicate up and down regulation of genes, respectively, and their intensity represents the relative gene expression changes. B Summery of transcriptome data in (A). White and gray colors indicate up- and down-regulated genes, respectively, in ulp2Δ cells. The pie graph shows the percentages of significantly changed genes, except for 130 ribosomal protein genes (RPs), and are classified by FPKM (Fragments Per Kilobase of Million reads mapped) values in the bar graph. ",
    "molecules": "RNA"
  },
  {
    "caption": "C qRT-PCR analysis of the highly transcribed PMA1, ADH1 and PYK1 genes in ulp2Δ cells. Expression was measured relative to WT cells and data were normalized to SPT15 expression. Error bars indicate the standard deviations (SDs) from three independent RNA preparations. FPKMs of each gene in WT are shown in the bottom graph.",
    "molecules": "RNA"
  },
  {
    "caption": "E ChIP analysis using IgG-Sepharose or anti-Flag-agarose beads in strains expressing TAP-tagged Paf1, Ubc9, or Ulp1 or Flag-tagged Ulp2. An untagged strain (MHY500) was used as a negative control for immunoprecipitation of Ulp2-Flag The qPCR signals of the indicated genes were quantitated and normalized to an internal background control and the input DNA. The primer pairs used are indicated in (D). Quantification presented as fold over background; a value of 1 indicates no signal detected above background signal at a nontranscribed locus, as marked with the horizontal line. Error bars indicate SDs calculated from three independent chromatin preparations. Data information: Asterisks indicate statistically significant differences of Paf1-TAP and Ubc9-TAP with Ulp1-TAP and Ulp2-Flag with No tag in (E) using a two-tailed Student's t test (*, P <0.05; **, P <0.01).",
    "molecules": "agarose, Sepharose, DNA"
  },
  {
    "caption": "F Occupancy of Rpb3 and Ulp2 at CUP1 gene was determined by ChIP in a strain expressing Flag-tagged Ulp2 using anti-Rpb3 antibody and anti-Flag agarose beads Mock' indicates use of Protein G beads without added antibody. For CUP1 induction, cells were harvested at the indicated time points after adding CuSO4. "Pro" and "ORF" represent the positions of PCR fragments 1 and 2 from the CUP1 gene Black bar indicates a value of 1, the background signal. Error bars, SD from four independent experiments. Data information: Asterisks indicate significant differences between uninduced and induced cells in (F) using a two-tailed Student's t test (*, P <0.05; **, P <0.01)",
    "molecules": "Protein G, agarose, CuSO4"
  },
  {
    "caption": "A Sensitivity of the indicated mutants to 6-AU. All strains carried a URA3 plasmid, pRS316, and were spotted on SD-Ura with or without 6-AU (100 μg/ml); plates were incubated for 2-4 days at 30°C. ULP2 and ulp2(C624A) represent ulp2Δ::HIS3 cells containing either pRS314-ULP2-FLAG or pRS314-ulp2(C624A)-FLAG.",
    "molecules": "6-AU, Ura"
  },
  {
    "caption": "A Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD).",
    "molecules": "6-AU"
  },
  {
    "caption": "C ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives-specifically, full-length, NTD (1-403) and CTD (667-1034)-were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT, full length of Ulp2 (*, P <0.05).",
    "molecules": "galactose, Trp"
  },
  {
    "caption": "D Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting (IB) using anti-Myc or anti-Flag antibodies. The upper and middle panels show anti-Myc blotting and anti-Flag blotting, respectively. The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel.",
    "molecules": "agarose"
  },
  {
    "caption": "A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B with anti-Flag (bottom panel) or anti-SUMO (upper panel) antibodies in WT, ulp2Δ, SUMO-all KR or ulp2Δ SUMO-all KR strains carrying empty vector or pRS426-FLAG-HTB1. One, two or three asterisks represent mono, di and polysumoylated histones, respectively. Note that the apparent diSUMO-H2B species (**) in ulp2Δ SUMO-all KR cells migrates more slowly than H2B linked to a SUMO dimer, suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines (lane 8; also see weaker band in lane 6).",
    "molecules": "histone, lysines"
  },
  {
    "caption": "B Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.",
    "molecules": "5-FOA"
  },
  {
    "caption": "D Mutant ulp2Δ bre1Δ strains carrying a pRS426-ULP2 (URA3) cover plasmid and the indicated plasmids were grown on SD-Ura and SD+5-FOA at 30°C for 2-3 days.",
    "molecules": "5-FOA, Ura"
  },
  {
    "caption": "E Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.",
    "molecules": "5-FOA"
  },
  {
    "caption": "A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B using anti-Flag or anti-SUMO antibodies in the indicated mutants, The upper and lower panels show Flag-H2B-SUMO and Flag-H2B-ub, respectively. One, two or three asterisks indicate mono-, di-, and poly-sumoylated histones (upper panel), respectively, and an arrowhead and bullet point represent mono-ubiquitylated H2B, and a non-specific band, respectively.",
    "molecules": "ub"
  },
  {
    "caption": "C Chromatin double immunoprecipitation (ChDIP) of Flag-H2B-ub in the indicated strains expressing Flag-H2B. The first ChIP was performed with anti-Flag agarose, and the eluted samples were immunoprecipitated with an antibody against ubiquitin. PCR signals from the indicated genes were normalized to an internal control and the input DNA. The error bars indicate the SD from three independent chromatin samples. Data information: Asterisks indicate statistically significant differences compared with WT in *, P <0.05; **, P <0.01",
    "molecules": "agarose, DNA, ub, ubiquitin"
  },
  {
    "caption": "D ChDIP of Flag-H2B-SUMO in the indicated strains expressing both Flag-H2B and HA-SUMO. Chromatin samples were sequentially immunoprecipitated with anti-HA agarose followed by anti-Flag agarose. PCR signals were normalized to the input DNA. The error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01",
    "molecules": "agarose, DNA"
  },
  {
    "caption": "B A ulp2Δ single mutant and three isolates of ulp2Δ ctk1Δ with YCplac33-ULP2 were grown on SD-Ura or SD+5-FOA plates at 30°C for 2-3 days.",
    "molecules": "5-FOA, Ura"
  },
  {
    "caption": "F Co-IP analysis of Ctk1 and histone H2B interaction. WT and ulp2Δ strains expressing the indicated tagged proteins were precipitated with IgG-Sepharose. IP and INPUT proteins were subjected to immunoblotting with PAP (Ctk1-TAP) and anti-Flag (Flag-H2B) antibodies. Protein signals were quantified relative to PGK and then normalized to the WT value.",
    "molecules": "Sepharose"
  },
  {
    "caption": "H Sensitivity to 6-AU of indicated htb1-1 htb2-1 (null alleles for both chromosomal H2B genes) strains carrying pRS314 plasmids that express Flag-H2B or 2SUMO-H2B.",
    "molecules": "6-AU"
  },
  {
    "caption": "(D-E) Representative images at 6 dpf of Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae that had been injected at the 1-2-cell stage with transposase mRNA (control) or transposase mRNA + bactin:igfbp1a (bactin:igfbp1a), subjected to β-cell ablation by metronidazole (MTZ) during 3-4 dpf, and subsequently allowed to regenerate for 2 days. The GFP+ heart (arrowhead) visualizes successful integration of the construct. Islets are indicated by white arrows. Scale bars: 100 μm. (F) Quantification of β-cell regeneration at 6 dpf in control (n=23), bactin:igfbp1a-overexpressing (n=13), and bactin:igfbp1b-overexpressing (n=8) Tg(ins:kaede);Tg(ins:CFP-NTR) larvae; ***P=0.0002, ns= nonsignificant (P=0.3106).",
    "molecules": "metronidazole, MTZ"
  },
  {
    "caption": "(A-C) Igfbp1a promotes β cell regeneration, rather than β cell survival. To trace β cells, we exposed Tg(ins:kaede) larvae to UV light (which causes the existing Kaede protein to switch from emitting green fluorescence to emitting red fluorescence) just before ablating the β cells by MTZ treatment from 3-4 dpf. After regeneration, the newly formed β cells are green, whereas the β cells that survived ablation are yellow (overlap of green and red). (A&B) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae; arrows indicate surviving (yellow) β cells. Scale bars: 10 μm. (C) Quantification of β cell regeneration (green bars) and β cell survival (yellow bars) per larva at 6 dpf. ***P<0.001: n=20 larvae in the control group; n=27 larvae in the bactin:igfbp1a-overexpressing group.",
    "molecules": "MTZ"
  },
  {
    "caption": "(D-F) Igfbp1a does not promote δ-cell regeneration. We treated control and bactin:igfbp1a-overexpressing Tg(sst:flag-NTR);Tg(sst:dsRed) larvae with MTZ from 3-4 dpf to ablate δ cells, and then allowed them to regenerate until 6 dpf. (D&E) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing larvae showing comparable number of δ cells after 2 days of regeneration. Scale bars: 15 μm. (F) Quantification of the total number of δ cells per δ-cell ablated larva at 6 dpf compared to the baseline number of δ cells in non-ablated control larvae. P=0.2325. n=13 in the control group, n=7 in the bactin:igfbp1a group.",
    "molecules": "MTZ"
  },
  {
    "caption": "(G) Other Igfbps do not promote β-cell regeneration. We injected Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics with either transposase mRNA (control; n=31) or transposase mRNA + one of six different igfbps, igfbp2a (n=25), igfbp2b (n=25), igfbp3 (n=26), igfbp5a (n=17), igfbp6b (n=34), and igfbp7 (n=17), treated them with MTZ from 3-4 dpf to ablate their β cells, and then quantified their β cells after 2 days of regeneration, at 6 dpf; ns=nonsignificant; (igfbp2aP=0.986, igfbp2bP=0.037, igfbp3P=0.979, igfbp5aP=0.999, igfbp6bP=0.997, and igfbp7P=0.999).",
    "molecules": "MTZ"
  },
  {
    "caption": "(I&J) Free-glucose levels during β-cell regeneration in control (bactin:mCherry) and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) larvae (I), as well as in Tg(ins:kaede);Tg(ins:CFP-NTR) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 (J). We treated larvae with MTZ from 3-4 dpf to ablate their β cells, and monitored their free-glucose levels at 3-7 dpf. Free-glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1-protein injection (red lines) than in controls (black lines) at 7 and 6 dpf, respectively. Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β-cell ablation. n=24 larvae (four pools of six larvae) per data point.",
    "molecules": "glucose, MTZ"
  },
  {
    "caption": "(A-C) Tg(ins:GFP);Tg(tp1:H2B-mCherry);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate the β cells, and were then allowed to regenerate from 4-6 dpf. Representative confocal images at 6 dpf of control (A) and Tg(bactin:igfbp1a) (B) larvae showing a modest number of ins+ tp1+ co-expressing cells, indicated by arrows, after 2 days of regeneration. Scale bars: 15 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells expressing Tg(tp1:H2B-mCherry), i.e., of ductal origin (yellow bars). ****P<0.0001, ns= nonsignificant. n=23 larvae in the control group, n=17 larvae in the Tg(bactin:igfbp1a) group.",
    "molecules": "MTZ"
  },
  {
    "caption": "(G-I) EdU was used as a marker for cell cycle progression. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate their β cells, and subsequently incubated with EdU during regeneration from 4-6 dpf. (G-H) Representative confocal images at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing β cells in green and the β cells that had incorporated EdU in yellow (green and red overlap; arrowheads). Scale bars: 20 μm. (I) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells that incorporated EdU (yellow bars) during β-cell regeneration from 4-6 dpf. **P<0.01, ns= nonsignificant. n=16 in both the control and the Tg(bactin:igfbp1a) group.",
    "molecules": "MTZ"
  },
  {
    "caption": "(A-E) Control and Tg(bactin:igfbp1a)-overexpressing larvae were treated with MTZ from 3-4 dpf to ablate β cells, and analyzed at 6 dpf, after 2 days of regeneration. (A-B) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(ins:dsRed);Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae. A bihormonal glucagon- and insulin-expressing cell (gcg+;ins+) is indicated by an arrow. Scale bars: 20 μm. (C-D) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae stained for Pdx1. Pdx1- and glucagon-expressing cells (pdx1+;gcg+) are indicated by arrows. Scale bars: 20 μm. (E) Quantification of gcg+;ins+ and pdx1+;gcg+ cells in the control and Tg(bactin:igfbp1a) group; P<0.001, P=0.0012; n=18 and 18 in the control groups, n=15 and 10 in the Tg(bactin:igfbp1a) groups, for the gcg+;ins+ and pdx1+;gcg+ quantification, respectively.",
    "molecules": "MTZ"
  },
  {
    "caption": "(A-H) PPP, an IGF1R inhibitor, promotes β-cell regeneration. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4-6 dpf. (A-B) Representative confocal images at 6 dpf of DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap (arrowheads). Scale bars: 20 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and β cells that had incorporated EdU (white bars) from 4-6 dpf during β-cell regeneration. P=0.0003, P=0.8607 respectively. (D) Rate of β-cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.1194. n=18 larvae for the DMSO-treated group, n=17 larvae for the PPP-treated group. (E-H) To examine whether PPP affected β-cell proliferation during regular development, Tg(ins:H2B-GFP) larvae were treated with EdU and DMSO or PPP from 4-6 dpf. (E-F) Representative confocal images of 6 dpf DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap. Scale bars: 20 μm. (G) Quantification of the total number of β cells (green bars) and β cells that had incorporated EdU (white bars) per larva from 4-6 dpf. P=0.9098 and 0.9976, respectively. (H) Rate of β cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.7822. n=16 larvae for DMSO-treated group, 18 larvae for PPP-treated group.",
    "molecules": "DMSO, MTZ, PPP"
  },
  {
    "caption": "(I-K) Activation of the Igf pathway reduces β-cell regeneration. Control and bactin:igf2a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently let to regenerate from 4-6 dpf. (I-J) Representative confocal images of 6 dpf control and bactin:igf2a-overexpressing larva displaying β cells in green. Scale bars: 15 μm. (K) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4-6 dpf. P<0.0001. n=28 larvae for control, n=15 larvae for bactin:igf2a.",
    "molecules": "MTZ"
  },
  {
    "caption": "(L-P) No synergistic effect was observed for igfbp1a and PPP. Control and bactin:igfbp1a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4-6 dpf. (L) Quantification of the total number of β cells per larva. n>10 (n=23, 20, 14, 13). P=0.9546. (M-P) Representative confocal images of 6 dpf control or bactin:igfbp1a overexpressing larvae treated with either DMSO or PPP, displaying β cells after 2 days regeneration. Scale bars: 10 μm. See also Figure EV2 & EV3.",
    "molecules": "DMSO, MTZ, PPP"
  },
  {
    "caption": "(E) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology.",
    "molecules": "DMSO, thalidomide"
  },
  {
    "caption": "(F) In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars denote ± standard deviation (n=3).",
    "molecules": "DMSO, lenalidomide, pomalidomide, thalidomide"
  },
  {
    "caption": "(E) In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide. The purified AGIA-PLZF or CRL4FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2 and HA-ubiquitin in the presence of DMSO or 200 µM lenalidomide. The second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.",
    "molecules": "DMSO, lenalidomide, ubiquitin"
  },
  {
    "caption": "(B) In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001",
    "molecules": "DMSO, Thalidomide, thalidomide"
  },
  {
    "caption": "(D) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. (E) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. Data information: Data in bar graphs in (D) and (E) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100. Error bars donated ± standard deviation (n = 3) and P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01 and ***P < 0.001).",
    "molecules": "5-hydroxythalidomide, DMSO, thalidomide"
  },
  {
    "caption": "(A, B) Immunoblot analysis of AGIA-MmPLZF (A) or -MmSALL4 (B) in FLAG-MmCRBN-WT, -MmCRBN-I391V or -MmCRBN-V380E/I391V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. Data information: Data in bar graphs were calculated as relative Mm) PLZF or SALL4 band intensity with Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).",
    "molecules": "DMSO, Tha, thalidomide"
  },
  {
    "caption": "(C, D) Immunoblot analysis of AGIA-GgPLZF (C) or -GgSALL4 (D) in FLAG-GgCRBN-WT or -GgCRBN-I390V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. (E, F) Immunoblot analysis of AGIA-Mm PLZF (E) or -MmSALL4 (F) in FLAG-MmCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. (G, H) Immunoblot analysis of AGIA-GgPLZF (G) or -GgSALL4 (H) in FLAG-GgCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. Data information: Data in bar graphs were calculated as relative (Gg or Mm) PLZF or SALL4 band intensity with (Gg or Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).",
    "molecules": "5-HT, 5-hydroxythalidomide, DMSO, Tha, thalidomide"
  },
  {
    "caption": "(E) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl (n = 3), forelimb shPLZF (n = 5) or hindlimb shPLZF (n = 6)). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01 and ****P < 0.0001).",
    "molecules": "Victoria blue"
  },
  {
    "caption": "(F) Photographs show E4 chicken embryos treated with HBC (control, n = 10, C1, C3, C5 and C6), 1 µg/µl (3.9 mM) thalidomide (n = 11, T1, T2, T6 and T9) or 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n = 10, H1, H2, H8 and H9) corresponding to immunoblot analysis in Fig 7G. Red arrows show treated regions. Data information Data in bar graphs in (F) were calculated as relative limb bud length with left limb bud length as one. Error bars denote ± standard deviation (F), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, **P < 0.01, and ***P < 0.001).",
    "molecules": "HBC, 5-HT, 5-hydroxythalidomide, thalidomide"
  },
  {
    "caption": "(G) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud. Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC (n =10), 1 µg/µl (3.9 mM) thalidomide (n =11) and 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n =10). Data information: Data in bar graphs in (G) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4, respectively. The band intensity of HBC was 100. Error bars denote ± standard deviation ((E), n = 3 or (G), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, and **P < 0.01).",
    "molecules": "HBC, 5-HT, 5-hydroxythalidomide, thalidomide"
  },
  {
    "caption": "E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS-GgPLZF was treated with HBC or 1 µg/µl (3.9 mM) thalidomide, and the chicken forelimb bud phenotypes were observed in bright field (BF) or fluorescence of enhanced GFP (EGFP). Fgf10/Fgf8 expression was analysed by section by in situ hybridisation. (Q) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in (Q) were calculated as relative limb bud length with limb bud length of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01).",
    "molecules": "HBC, thalidomide"
  },
  {
    "caption": "(B) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin).",
    "molecules": "aldolase, conalbumin"
  },
  {
    "caption": "(C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "ATP, PI"
  },
  {
    "caption": "(D) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition. Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles (0.5 mg/mL) with 100 μM ATP, and Golgi mimic vesicles (0.5 mg/ml, 10% PS, 20% PI, 25% PE, 45% PC) with 10 μM ATP. PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively, with c10orf76 present at 500 nM in both experiments. The data is normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "ATP, lipid, PS, PC, PE, PI"
  },
  {
    "caption": "(E) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N-lobe linker and smaller changes in the helical domain and C-lobe of the kinase domain. The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB (400 nM) in the presence and absence of c10orf76 (400 nM) over all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC). Each dot represents a peptide graphed on the x-axis according to the central residue. The red boxes highlight key regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "deuterons, deuterium"
  },
  {
    "caption": "(G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "deuterons, deuterium"
  },
  {
    "caption": "(B) The N-lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA. Recombinant PKA at different concentrations (0, 7, 34, 168, or 840 ng) was incubated with recombinant (E. coli) wild-type PI4KB (20 μg) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry. Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity (total area) of the phosphorylated vs non-phosphorylated peptide (486-506).",
    "molecules": "ATP, Ser"
  },
  {
    "caption": "(C) PI4KB phosphorylation by PKA alters the affinity for c10orf76. The kinase activity of different variants of PI4KB (15 nM) was measured in the presence of varying amounts of c10orf76 (23 nM-1.5μM) with 100% PI lipid substrate (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "ATP, lipid, PI"
  },
  {
    "caption": "(D) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine. Kinase assay of PI4KB non-phosphorylated, phos-Ser496 or S496A (15 nM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "alanine, ATP, PI, Ser"
  },
  {
    "caption": "(F) RL494EA PI4KB activity is not inhibited by c10orf76. Kinase assays of either wild type or mutant RL494EA PI4KB (40 nM) were carried out with varying concentrations of c10orf76 (3.9 nM-2μM) with 100% PI lipid vesicles (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "ATP, PI"
  },
  {
    "caption": "(G) Wild-type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity. Kinase assays of either wild-type and mutant PI4KB (10 nM) were carried out with 100% PI lipid vesicles (0.5 mg/L), 100 μM ATP, and PI4KB (300 nM) on Golgi-mimic vesicles (0.5 mg/mL) with 10 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "ATP, PI"
  },
  {
    "caption": "(I) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced. Kinase assay of PI4KB (40 nM) and a concentration curve of c10orf76 (3.9 nM-2μM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).",
    "molecules": "ATP, PI"
  },
  {
    "caption": "(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.",
    "molecules": "DAPI, PI4P"
  },
  {
    "caption": "B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.",
    "molecules": "DAPI, GTP"
  },
  {
    "caption": " B) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm. C) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC-Lsp1beta-MI05460 larvae. The OregonR value indicates the background level. The number of cells included in the analysis is displayed on the x-axis label and were quantified from two independent preparation, p-values were estimated after variance analysis with bilateral student test for equal variance.",
    "molecules": "DAPI"
  },
  {
    "caption": " D) Resident hemocytes located around the oenocytes or along the muscles (top and bottom panels, respectively) from WL srp(hemo)-moesin-RFP, that express moesin tagged with RFP in hemocytes. The immunolabelling was done with anti-RFP (in red), anti-Pendulin (Pen, in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, Pen and RFP, the middle panels RFP alone and the right panels Pen alone. Arrow heads in the top panels indicate cells co-expressing RFP and Pen. The scale bars represent 50 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " D) Hemocytes from WL OregonR infested by wasp. The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea (in gray, top panels) and Shot (in gray, lower panels). Phalloidin-FITC (in green) labels the actin filament particularly abundant in lamellocytes (Tokusumi et al., 2009) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, FITC and the lamellocyte markers, the middle panels show the FITC alone and the right panels the lamellocyte markers alone. The scale bars represent 20µm. ",
    "molecules": "DAPI, FITC, Phalloidin"
  },
  {
    "caption": "(C) OTA treatment (n = 6 animals) significantly reduces the  recognition index in mice compared to the PBS injection control (n = 6 animals) mice (* p = 0.0260, Mann-Whitney test). Data shown as mean ± SEM.",
    "molecules": "OTA, PBS"
  },
  {
    "caption": "(B) Representative confocal image of the pSON region stained with synaptophysin (blue), CTB-Alexa 568 (red), and GFP from GABAergic neurons (green). Arrowheads indicates colocalization. Scale bar = 10 μm.",
    "molecules": "Alexa 568"
  },
  {
    "caption": "(a) Primary siRNA screening analysis. MDA-MB-231 cells reverse transfected with siRNA pools against MT1-MMP, TKS5, or each human RAB GTPase were plated on fluorescein-labeled-gelatin coated optical microplates. Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci (similar results were obtained by measuring the area of degradation/number of cells). The inset shows representative images of degraded Oregon green gelatin (upper panels) and merged images with TRITC-phalloidin (red) and DAPI (blue) in scrambled siRNA (NC), or siRNA against RAB28 or RAB42 transfected cells (lower panel). Data are means ± SEM of 3 independent experiments. Bar, 50 µm.",
    "molecules": "fluorescein, gelatin"
  },
  {
    "caption": "(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Cells plated onto fluorescently conjugated gelatin (red) were stimulated with 100 ng/ml HGF overnight in the presence or absence of GM6001, followed by staining with FITC-phalloidin (green) and DAPI (blue) (c). Data are the mean ± SEM (d, error bars; n > 100 cells/experiment in at least 4 independent ones).",
    "molecules": "FITC, gelatin, GM6001"
  },
  {
    "caption": "(a) Doxycycline-inducible empty vector (EV) or RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (-) or presence (+) of doxycycline for three days, and immunoblotted with anti-RAB2A and anti-actin antibodies. The intensity of RAB2A relative to actin is shown on the bottom. Relative density in uninduced EV cells was set to 1.",
    "molecules": "Doxycycline, doxycycline"
  },
  {
    "caption": "(b) Doxycycline-inducible RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days. Invasion of MDA-MB-231 cells through Matrigel-coated transwells toward HGF was assessed after 24 hours. Representative images are shown. Data are the mean ± SD (n=10 field/condition in at least 3 experiments). Bar, 200 µm.",
    "molecules": "Doxycycline, doxycycline"
  },
  {
    "caption": "(c-e) EV and shRAB2AMDA-MB-231 cells were embedded in Spheroid Formation ECMR in the absence (-) or presence (+) of doxycycline and/or GM6001. After 3 days, the cells were transferred into Invasive MatrixR. Phase contrast light microscopy images of representative spheroids are shown after 9 days (c). Dashed circle indicates the size of spheroids at day 0. Bars, 500 µm. Invasive activity into ECM was expressed as total spheroid area (relative to control cells at day 0) quantified by ImageJ (d). The number of cells at the indicated days was quantified by CyQUANTR Cell Proliferation Assay Kit (e). Data are the mean ± SD (n=3 independent experiments).",
    "molecules": "doxycycline, GM6001"
  },
  {
    "caption": "(f-h) Doxycycline-inducible murine RAB2A (mRAB2A)-expression MCF10.DCIS.com cells were transfected with scrambled siRNA or siRNA against human RAB2A (siRAB2A) in the absence (-) or presence (+) of doxycycline. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue) (f). Bar, 50 μm. Data are the mean ± SEM (error bars; n > 100 cells/experiment in 4 independent ones) (g). Silencing of endogenous RAB2A protein (lower band) and ectopic RAB2A expression (upper band) was verified by immunoblotting (h). * P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.",
    "molecules": "Doxycycline, doxycycline, gelatin"
  },
  {
    "caption": "(e-i) Scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A), VPS39 (siVPS39), or VPS41 (siVPS41) were transfected into MCF10.DCIS.com cells. Serum-starved cells were then plated onto fluorescent-conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue). Quantification of gelatin degradation (f) and efficacy of targeted gene silencing verified by qPCR (g-i). Data are the mean ± SEM (error bars; n > 100 cells/experiment in at least 4 independent ones). Bars, 50 µm.",
    "molecules": "gelatin"
  },
  {
    "caption": "(m-q) Doxycycline-inducible murine RAB2A-expression MCF10.DCIS.com cells were transfected with siNC, siRAB2A, or siVPS39 in the absence (CTR) or presence (RAB2A) of doxycycline. Their ECM degradation activities were quantified (n). Data are the mean ± SEM (error bars; n > 100 cells/experiment in 3 independent ones). Total cell lysates were immunoblotted by anti-RAB2A and anti-tubulin antibodies (o). Efficacy of targeted gene silencing was verified by qPCR (p-q). Bar, 50 μm. **, P < 0.01, ***, P < 0.001.",
    "molecules": "Doxycycline, doxycycline"
  },
  {
    "caption": "(a) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in the absence (CTR) or presence (shRAB2A) of doxycycline. Cells were stimulated with 5 ng/ml EGF and 20 ng/ml HGF (EGF+HGF) or left starved (STV) for 9 days. Representative phase contrast light microscopy images are shown. Invasive activity of the cells was expressed by circularity (with a value of 1 representing perfect circularity = no invasion) of each acini. Bar, 100 µm.",
    "molecules": "doxycycline, Doxycycline"
  },
  {
    "caption": "(e) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days, and immunostained with antibodies indicated on the top (green) and DAPI (blue).",
    "molecules": "Doxycycline, doxycycline"
  },
  {
    "caption": "(a, b) Doxycycline-inducible RAB2A-expression MCF10.DCIS.com (a) and MCF10A (b) cells were cultured in the absence (CTR) or presence (RAB2A) of doxycycline, and immunostained with anti-RAB2A (blue), anti-E-cadherin (green), and anti-TGN46 (red) antibodies. Merged images with DAPI (magenta) are shown on the right. Bars, 10 μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSEs. Protein levels were detected by Western blotting. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSE cells.",
    "molecules": "Ser"
  },
  {
    "caption": "Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP/TAZ) hOSE Cells. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) of control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP&si-TAZ) hOSE cells.",
    "molecules": "Ser"
  },
  {
    "caption": "Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control hOSEs (MXIV) and hOSEs expressing wild type YAP (YAP) or constitutively active YAP (YAPS127A). Cells were collected at passage 4. β-Actin was used as a protein loading control.",
    "molecules": "Ser"
  },
  {
    "caption": "Representative images showing expression and location of YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. YAP was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 50 µm.",
    "molecules": "Alexa-488, DAPI"
  },
  {
    "caption": "Representative images showing expression and location of phosphorylated pRB (807/811) in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the 7th passage. Phosphorylated pRB (807/811) was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue).",
    "molecules": "Alexa-488, DAPI"
  },
  {
    "caption": "Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells. Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry. YAP protein was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI.",
    "molecules": "Alexa-488, DAPI"
  },
  {
    "caption": "Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout. YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue).",
    "molecules": "Alexa-488, DAPI"
  },
  {
    "caption": "(C) Cryosections from β′-COP-depleted cells were labeled with anti-Ub antibodies followed by 10-nm protein A-gold. AV, autophagosomes.",
    "molecules": "gold"
  },
  {
    "caption": "AVs in COPI-depleted cells are not degradative. (A) Long-lived protein degradation was assessed 72 h after transfection with control or β′-COP siRNA in 293/GFP-LC3 cells. Cells were either starved of amino acids for 2 h (St) or incubated in fresh growth medium (Fed) for 2 h. The amount of protein degradation is presented as mean ± SEM.",
    "molecules": "amino acids, protein"
  },
  {
    "caption": "(B) Cryosections of β′-COP-depleted cells labeled with anti-GFP (left) or anti-GFP and anti-LAMP2 (right) followed by protein A-gold as indicated. AV, autophagosomes, M, mitochondria.",
    "molecules": "gold"
  },
  {
    "caption": "(C) Control-starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation.",
    "molecules": "methanol"
  },
  {
    "caption": "(D) After siRNA treatment, rhodamine-labeled dextran was internalized for 20 min, followed by a 40-min chase after which the 293/GFP-LC3 cells were fixed and analyzed by confocal microscopy.",
    "molecules": "dextran"
  },
  {
    "caption": "(D and E) 125I-EGF was added to HeLa cells after 48 h of siRNA depletion of β′-COP, or control siRNA for 10 min. The cells were then washed as described in the Materials and methods, and chased for 30, 60, 120, and 180 min (including 10-min incubation). (D) The amount of 125I-EGF internalized was shown as a percentage of the total 125I-EGF taken up in 10 min.",
    "molecules": "125I, 25I"
  },
  {
    "caption": "(E) The amount of degraded 125I-EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I-EGF internalized. The experiment was performed three times in duplicate and the data shown is the mean ± SEM. The significance was determined by Student's t test. *, P ≤ 0.05.",
    "molecules": "125I, TCA"
  },
  {
    "caption": "(A) Picrosirius red (PSR) staining of collagens (red) in PDAC tissue following surgical resection. Scale bar represents 200 µm. (B) Quantification of PSR in low-grade (1-2) and high-grade (3) tumors indicated in panel A as percentage of area. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.",
    "molecules": "Picrosirius red, PSR"
  },
  {
    "caption": "(D) PSR and α-SMA staining of indicated PDAC cell lines which were injected in NSG mice. Scale bar represents 200 µm for PSR and 100 µm for α-SMA images. (E) Quantification of stainings indicated in panel D. Three tumor ROIs of a tumor derived from epithelial cell lines (Capan-2 and AsPC-1) and mesenchymal cell lines (MIA PaCa-2 and PANC-1) are displayed and averages were pooled for analysis. Data information: data are represented as mean±SD, Student's t-test.",
    "molecules": "PSR"
  },
  {
    "caption": "(C) Brightfield images of PS-1 cells exposed to 10 days of treatment with 1 and 10 µM all-trans retinoic acid (ATRA). Scale bar represents 100 µm.",
    "molecules": "all-trans retinoic acid, ATRA"
  },
  {
    "caption": "(C) A validation cohort of PDAC patients (n=21) that underwent direct resection was stained for CSF-1 (scale bar represents 100 µm), PSR (scale bar represents 500 µm) and α-SMA (scale bar represents 200 µm). OK8 is low in CSF-1 expression and high in PSR and α-SMA expression, while the opposite accounts for OK7. (D) Correlation of % of PSR in the tumor and CSF-1 expressing tumor cells, as indicated in panel C. R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples (E) Correlation of % of α-SMA in the tumor and CSF-1 expressing tumor cells, as indicated in panel C, R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples ",
    "molecules": "PSR"
  },
  {
    "caption": "Analysis of LepB94 translation products on SDS-PAGE. Translation products are visualized by the fluorescence of N-terminal Atto655. Pausing intermediates are indicated P1 and P2.",
    "molecules": "Atto655"
  },
  {
    "caption": " Stopped-flow time courses of co-translational LepB insertion into SecYEG (Atto488 donor-in at position 111 of SecY), as monitored by the fluorescence of the Atto655 acceptor placed at the N-terminus of the nascent peptide. Fits (red lines) were obtained by global fitting ",
    "molecules": "Atto488, Atto655"
  },
  {
    "caption": " Stopped-flow time courses for translocon insertion of TM1 of EmrD (black) and EmrD(-) (blue) with mRNA constructs of varying chain length (50 to 135 aa).The FRET acceptor (Atto655) was placed at the N-terminus of the nascent peptide, the donor (Atto488) at position 111 (donor-in) of SecY. The results of global fitting are indicated (red lines). ",
    "molecules": "Atto488, Atto655"
  },
  {
    "caption": "(A) Schematic showing the tamoxifen  injection paradigm for tdTomato-labelling of all IFE  lineages in tail  and back. Co-expression of markers with the lineage-traced cells at 1-month post-tamoxifen. Dlx1-CrER  is shown as example in tail; Scale bar =50 µm (B) Quantification of images like those in (A) showing % tdTomato+ cells expressing indicated markers. Sox6 (black bar) was not analyzed in K14-Cre/EYFP  lineage. Error bars are SDs. P-values calculated by Student's t-test (mixed effects model) from n=2-3 mice  and 5-6 images per replicate.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "A Representative fluorescent images of centrosomes  in RPE1  WT, CEP250  KO and CROCC  KO cells, without and with MT  depolymerization using Nocodazole. PCNT  was used to mark centrosomes  and DNA was stained  with DAPI. The boxes in the right-hand corner show enlargement of the centrosome  signals of the cell in the main panel. White scale bars, 5 μm. Yellow scale bars, 1 μm. B Quantification of cells with separated centrosomes (centrosome  distance > 2 μm) from RPE1  samples (A). N = 50 cells per experiment, n = 3 independent experiments. For WT with Nocodazole, N = 50 cells, n = 2 independent experiments. Bar and error represent mean and SD. Unpaired t-tests. n.s.: not significant p > 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "DAPI, Nocodazole"
  },
  {
    "caption": "C Representative fluorescent images of centrosomes  in HCT116  WT, CEP250  KO, and CROCC  KO, without and with MT  depolymerization using Nocodazole. γ-tubulin was used to mark centrosomes  and DNA was stained  with DAPI. The boxes in the right-hand corner show enlargements of the centrosome  signals of the cell in the main panel. White scale bars, 10 μm. Yellow scale bars, 2 μm. D Quantification of cells with separated centrosomes  (centrosome  distance > 1 μm) from HCT116  samples (C). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-tests. ** p < 0.01, *** p < 0.001, **** p < 0.0001.",
    "molecules": "DAPI, Nocodazole"
  },
  {
    "caption": "E Representative fluorescent images of RPE1  and HCT116  cells. Rootletin  and CEP68  were stained, PCNT  was used as centrosome  marker and DNA was stained  with DAPI. The boxes on top show the centrosome signals of the cell in the main panel. Scale bars in white: 5 μm. Scale bars in yellow: 1 μm. F Quantification of relative Rootletin  and CEP68  intensity at the centrosome  in RPE1  and HCT116  cells. Mean value of relative intensity at the centrosome  from N = 50 in each replicate, n = 4 are shown. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, *** p < 0.001.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-H (A, C, E, G)  Time-lapse images (4 min interval) of RPE1  WT, CEP250  KO, NIN  KO and CROCC  KO cells expressing  γ-tubulin-mRuby2  (red) as marker for the centrosomes  with and without Nocodazole  treatment. Representative still images (top rows) and kymographs  of centrosomes  (bottom rows) are shown. The DNA was marked with SPY-DNA650 (grey). White scale bars, 5 μm. Yellow scale bars, 2 μm. (B, D, F, H) Intercentrosomal distances (μm) from (A), (C), (E) and (G) were plotted over time  (n = 15 cells).",
    "molecules": "SPY-DNA650, Nocodazole"
  },
  {
    "caption": "E-H (E, G) Fluorescent images of centrosomes  in RPE1 CEP250  KO and CEP128  KO cells with the indicated siRNA depletions, without and with MT  depolymerization using Nocodazole. PCNT  or γ-tubulin was used to mark centrosomes  and DNA was stained  with DAPI. The box in the right-hand corner shows the centrosome  signals of the cell in the main panel. Scale bars in white: 5 μm. Scale bars in yellow: 1 μm. (F, H) Quantification of cells with separated centrosomes (centrosome  distance > 2 μm) for RPE1 CEP250  KO (E) and CEP128  KO (G) samples. N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. n.s.: not significant p > 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "DAPI, Nocodazole"
  },
  {
    "caption": "B Fluorescent images of centrosomes  in Dox-inducible Flag-PLK4 RPE1  WT, CEP250  KO, NIN  KO and CROCC  KO cells, with and without Dox  induction. Flag-PLK4  was stained  with Flag  antibodies, PCNT  was used as centrosome  marker and DNA was stained with DAPI. The boxes on top show enlargement of the centrosome  signals of the cell in the main panel. Scale bars in white: 5 μm; scale bars in yellow: 1 μm.",
    "molecules": "DAPI, Dox"
  },
  {
    "caption": "D Fluorescent images of centrosomes  in Dox-inducible Flag-PLK4 RPE1  WT, CEP250  KO, NIN  KO and CROCC  KO cells upon 48 h Dox  induction, without and with MT  depolymerization using Nocodazole. DNA was stained  with DAPI. Scale bars: 5 μm. E Quantification of the diameter of amplified centrosomes  from (D) as outlined in (A). A representative dataset from six replicates (three replicates per experiment) is shown. N = 50 cells. Dot represents the diameter of amplified centrosomes  in each cell with amplified centrosomes. The red lines represent the median. Unpaired t-test. n.s.: not significant p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001.",
    "molecules": "DAPI, Dox, Nocodazole"
  },
  {
    "caption": "H Representative images from live cell imaging  analysis of WT, CEP250  KO, WT PLK4  OE and CEP250  KO PLK4  OE cells expressing γ-tubulin-mRuby2  (red) as a marker for the centrosomes. The DNA was marked by  SPY-DNA650 dye (cyan). Gray arrows highlight chromosome  segregation errors. Scale bars: 5 μm.",
    "molecules": "SPY-DNA650"
  },
  {
    "caption": "I Fluorescent images of RPE1  WT, CEP250  KO, WT PLK4  OE and CEP250  KO PLK4  OE cells. BUB1  and MAD1  were stained   to analyse the signal distribution or accumulation/persistence. CENP-C was used as centromere  marker, α-tubulin  to visualize the mitotic spindle and DNA was stained  with DAPI. Scale bars: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A Fluorescent image of mitotic spindles in Dox-inducible Flag-PLK4 RPE1  WT, CEP250  KO, NIN  KO and CROCC  KO cells upon 48 h Dox  induction (PLK4   OE). CW069  was used to partially inhibit HSET. DMSO  is solvent control. White scale bars: 5 μm. B Quantification of mitotic cells with  multipolar spindle from (A). N = 50 cells per experiment, n = 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. C Quantification of  multipolar spindle/chromosome  mis-segregation of Dox-inducible Flag-PLK4 RPE1  WT and CEP250  KO cells upon 48 h Dox  induction expressing γ-tubulin-mScarlet-I (red) as markers for the centrosomes with or without CW069  treatment. The DNA was marked with SPY-DNA650 (grey). WT PLK4   OE: nDMSO = 28 cells, nCW069 = 40 cells, CEP250  KO PLK4   OE: nDMSO = 26 cells, nCW069 = 31 cells; from 2 independent live cell experiments. Bar represents mean. Unpaired t-test. * p < 0.05.",
    "molecules": "SPY-DNA650, CW069, DMSO, Dox"
  },
  {
    "caption": "D Time-lapse  analysis of RPE1  WT PLK4  OE and CEP250  KO PLK4   OE cells expressing γ-tubulin-mScarlet-I (red) as markers for the centrosomes followed by 30 h siHSET. The DNA was marked with SPY-DNA650 (grey). (D) shows representative still images of live cell imaging  analysis. Scale bars: 10 μm. E Quantification of multipolar spindle/chromosome  mis-segregation from (D) in RPE1  WT PLK4   OE and CEP250  KO PLK4   OE with siControl and siHSET for 30 h. WT PLK4   OE: nsiControl = 59 cells, nsiHSET = 59 cells, CEP250  KO PLK4  OE: nsiControl = 45 cells, nsiHSET = 32 cells; from 3 independent experiments. Bar and error represent mean and SD. Unpaired t-test. * p < 0.05, ** p < 0.01.",
    "molecules": "SPY-DNA650"
  },
  {
    "caption": "(A) Quantification of free cholesterol levels by lipidomics analysis of total homogenates (TH), ER or MAM fractions isolated from WT and PS-DKO cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. Unpaired t-test (n= 6 for TH, n=3 for MAM or ER; * p<0.05).",
    "molecules": "cholesterol, Lipid, lipids"
  },
  {
    "caption": "(B) Quantification of free cholesterol levels in total homogenates of APP-DKO cells transiently expressing C99, C83 or AICD peptides, or treated with 5 µM Aβ42 oligomers for 16h. Cholesterol levels of WT cells are shown as a control. Lipid units are represented as molar mass over total moles of lipids analyzed (mol %). Graphs represent fold change over controls. One-way ANOVA (n = 4; * p<0.05).",
    "molecules": "Aβ42, Cholesterol, cholesterol, Lipid, lipids"
  },
  {
    "caption": "(C) Quantification of HMGCR enzymatic activity in PS-DKO and DAPT-treated WT cells. unpaired t-test (n=3; * p< 0.05).",
    "molecules": "DAPT"
  },
  {
    "caption": "(D) Quantification of cholesterol uptake by incubating WT and PS-DKO cells, treated or untreated with BACE inhibitor IV (BI, 100 nM), with 2.5 µCi/mL 3H-cholesterol for the indicated times. Graph represents cholesterol radioactivity levels in total cell homogenates (TH). Two-way repeated measures ANOVA (Time, Group)(n = 3 independent experiments; * p< 0.05).",
    "molecules": "BACE inhibitor IV, BI, cholesterol, 3H"
  },
  {
    "caption": "(E-F) Quantification of 3H-cholesterol delivery to MAM (E) or bulk ER (F) after the indicated times. Control and BI-treated or untreated PS-DKO cells incubated with 2.5 µCi/mL 3H-cholesterol for the indicated times, before subcellular fractionation (Western blot of isolated MAM fractions shown in Fig. EV1F). Cholesterol delivery to MAM calculated by quantification of radioactivity levels in the isolated fractions. Two-way repeated measures ANOVA (Time, Group)( n=3 independent experiments; * p< 0.05)",
    "molecules": "BI, cholesterol, Cholesterol, 3H"
  },
  {
    "caption": "(G) Measurement of cholesterol uptake in the indicated cells by internalization of a fluorescent cholesterol analog [NBD-cholesterol (2 µM; upper panel, nuclei in blue)]. Endogenous free cholesterol was stained with filipin (lower panel). Graphs on the right represent fluorescence intensity (using ImageJ) and the percentage of cells with filipin punctae. Scale bar = 20 µm. One-way ANOVA (30-50 cells/condition from at least 3 independent experiments; * p<0.05, ** p<0.01, *** p<0.001).",
    "molecules": "filipin, cholesterol"
  },
  {
    "caption": "Representative confocal images of WT and PS-DKO cells treated with the indicated SMase inhibitors (10 µM desipramine or 5 µM GW4869) or DMSO (VEH) for 12-16h. (A) Endogenous levels of lipid droplets (LDs) were visualized by incubation with LipidTox. Graph shows the Lipidtox fluorescence intensity/cell.",
    "molecules": "LipidTox, Lipidtox, GW4869, desipramine, DMSO"
  },
  {
    "caption": "Endogenous levels of free cholesterol revealed by filipin staining of (B) WT cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.",
    "molecules": "filipin, cholesterol"
  },
  {
    "caption": "Endogenous levels of free cholesterol revealed by filipin staining of (C) PS-DKO cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.",
    "molecules": "filipin, cholesterol"
  },
  {
    "caption": "(A) Crude membrane fractions from APP-DKO cells expressing C99WT or C99MUT were treated with 0.2% Triton-X-100, loaded onto continuous density sucrose gradients and centrifuged for 16h. Fractions from these gradients were analyzed by western blot to determine the migration of the indicated proteins [two parallel gels (bold vertical line)].",
    "molecules": "sucrose, Triton-X-100"
  },
  {
    "caption": "(A) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT (in green) and fluorescent markers of mitochondria (MitoDsRed, in red) and ER (Sec61β-BFP, in blue), and treated with DAPT to prevent C99 cleavage. Note the different distribution of C99WT or C99MUT forms. Scale bar = 10 µm. Insets show 5X amplifications of individual (C99 in green, and mitochondria in red) and merged images. Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize. Upon thresholding each channel, a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask, so the positive pixels found in both channels are shown in black.",
    "molecules": "MitoDsRed, DAPT"
  },
  {
    "caption": "(B) Representative immunoblot to reveal C99 levels in total homogenate (TH) and MAM fractions of APP-DKO cells expressing C99WT or C99MUT before (input) and after cholesterol pull-down. Acsl4 was used as a MAM marker. Streptavidin-HRP was used to detect total biotinylation (biotin conjugated to PhotoClick cholesterol). (C) Quantification of pulled-down C99 levels versus input from the experiment in B. Two-way repeated measures ANOVA (Fraction, mutation) (n=5; * p< 0.05, ** p< 0.01). ",
    "molecules": "biotin, cholesterol"
  },
  {
    "caption": "(D) Immunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by western blot (shown in AppendixFig S1E).",
    "molecules": "Cholesterol"
  },
  {
    "caption": "(E) Quantification of free cholesterol levels (FC) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol%). Graphs represent percentage over controls. Dashed line represents control levels. One-way ANOVA. (n=4; * p< 0.05)",
    "molecules": "cholesterol, Lipid, lipids"
  },
  {
    "caption": "(F-G) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H-cholesterol and subsequent analysis of radiolabel incorporation. The dashed line indicates control levels. Graphs represent fold change over controls. Treatment with Sandoz 58-035, a specific ACAT1 inhibitor, caused a ~95% reduction in cholesterol esterification. EV, empty vector. One-sample t-test. (n=3-4; * p< 0.05).",
    "molecules": "cholesterol, Sandoz 58-035, 3H"
  },
  {
    "caption": "(H) Ratio of cholesteryl esters:free cholesterol (CE:FC) in the indicated cells.",
    "molecules": "FC, free cholesterol, CE, cholesteryl esters"
  },
  {
    "caption": "IHC images for collagen The inserts in panel B represent the hematoxylin (nucleus) & eosin (cytoplasm) staining for the same tissue area of the collagen staining In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) The quantification for each staining is shown in the histogram in the right side",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse, n=27 control and n=37 tamoxifen",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor (ER antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative mechanosensing trace of PSC treated with tamoxifen an G coupled-protein estrogen receptor (GPER) antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Histogram shows percentage of gel contraction by PSCs treated with tamoxifen in the presence of indicated antagonist",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Young"s modulus for matrices previously remodeled by PSCs; control n=126 and tamoxifen n=145. Lines and error bars indicate mean±s.e.m. Four experimental repl",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative images of H&E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs (top) or tamoxifen treated PSCs (bottom), scale bar 50 μm AsPC1 invasion quantification, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value, n=30 control and n=40 tamoxifen",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Western blot bands for YAP, pS127 YAP and total protein. (E) Quantification of YAP and pYAP Ser127 normalised to total protein, expressed relative to unstimulated control (n=8)",
    "molecules": "Ser"
  },
  {
    "caption": "IHC images for YAP staining in KPC mice pancreatic tissues (control and treated with 2 and 5 mg tamoxifen), YAP staining is brown. Scale bar: 100 μm",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative images showing hematoxylin-eosin (HE) staining and ANG IHC staining in mild and severe IBD samples.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Ang mRNA expression in colonic tissue from 2.5% DSS-treated WT mice at indicated time point (n = 4 mice/time point). DSS: dextran sulfate sodium",
    "molecules": "dextran sulfate sodium, DSS"
  },
  {
    "caption": "Kaplan-Meier curve of 3.5% DSS-treated Ang-deficient mice (Ang-/-, n = 14) or littermate controls (WT, n = 13).",
    "molecules": "DSS"
  },
  {
    "caption": "Body weight loss of WT and Ang-/- mice with (n = 9) or without (n = 6) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "disease activity index (DAI) of WT and Ang-/- mice with (n = 9) or without (n = 6) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Colon length of colonic section from the WT and Ang-/- mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "serum FITC-Dextran level of colonic section from the WT and Ang-/- mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment. FITC-Dextran: fluorescein isothiocyanate-labeled dextran.",
    "molecules": "DSS, Dextran, dextran, FITC, fluorescein isothiocyanate"
  },
  {
    "caption": "representative HE staining image, and (H) histopathological score of colonic section from the WT and Ang-/- mice on day 9 with (n = 9) or without (n = 6) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Quantitative mRNA expression of cytokine genes in colonic tissue from the WT and Ang-/- mice on day 0 (n = 3) or day 9 (n = 9) during 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Soluble cytokine level in supernatant of cultured colonic tissue isolated from WT and Ang-/- mice on day 0 (n = 3) or day 9 (n = 7) during 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Body weight loss of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "DAI of WT→WT, Ang-/-→WT and WT→Ang-/- chimera mice with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Colon length of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "serum FITC-Dextran level, of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS, Dextran, FITC"
  },
  {
    "caption": "representative HE staining image, and (F) histopathological score of colonic section from these chimera colitis mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Body weight loss of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "DAI of Angfl/fl and LyzMcre;Angfl/fl mice with (n = 7) or without (n = 3) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Colon length of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "serum FITC-Dextran level, of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.",
    "molecules": "DSS, Dextran, FITC"
  },
  {
    "caption": "representative HE staining image, and (M) histopathological score of colonic section from the Angfl/fl and LyzMcre;Angfl/fl mice on day 8 with (n = 7) or without (n = 3) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Body weight loss of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "DAI of the AA9-infected WT mice with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "Colon length of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "serum FITC-Dextran level of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS, Dextran, FITC"
  },
  {
    "caption": "representative HE staining image, and (K) histopathological score of colonic section from the AAV9-infected WT mice on day 8 with (n = 7) or without (n = 5) 2.5% DSS treatment.",
    "molecules": "DSS"
  },
  {
    "caption": "THP-1-derived ANG translocating into HCT116KO through PLXNB2 in co-cultured system as showed by western blotting. Neamine, a blocker of ANG and PLXNB2 interaction; siCtrl: control siRNA; siPLXNB2-1 and siPLXNB2-2",
    "molecules": "Neamine"
  },
  {
    "caption": "Flow cytometry analysis of HCT116KO stained with Annexin V and PI following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylation",
    "molecules": "BAY, PI"
  },
  {
    "caption": "tiRNA and 5'-tiRNAAla production in HCT116KO following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylation",
    "molecules": "BAY"
  },
  {
    "caption": "Caspase activity of HCT116KO following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylation",
    "molecules": "BAY"
  },
  {
    "caption": "Western blotting of ANG in colonic tissue from WT mice (n = 2) at indicated time point in the ANG prevention model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.",
    "molecules": "DSS"
  },
  {
    "caption": "serum FITC-dextran level of colon section from the pre-treated WT colitis mice on day 8 (n = 8).",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "Western blotting of ANG in colonic tissue from WT (n = 2) mice at indicated time point in the ANG treatment model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.",
    "molecules": "DSS"
  },
  {
    "caption": "serum FITC-dextran level of colon sections from the treated WT colitis mice on day 8 (n = 8).",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "A) From the quantitative  proteomics experiment, average abundance of catalytic proteasome  subunits was calculated and normalised to WT means. Trypsin-like (β2), chymotrypsin-like (β3) and  caspase-like (β1) catalytic subunits are shown. The average was calculated from all 24 time points of the proteomics experiment. Mean±SD, 2-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.",
    "molecules": "Trypsin"
  },
  {
    "caption": "D-E) From one  time-course  experiment, ions, cytosolic  proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course  experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker.",
    "molecules": "K+, potassium"
  },
  {
    "caption": "A) WT and CKO cells were treated with 500 nM tunicamycin (TUN) and lysed in RIPA buffer at  time points between 0-24 hours afterwards. Western blots were carried out, probing for  phosphorylated eIF2α, total  eIF2α and actin. Representative blots shown, n=4. B) Quantification of all replicates represented in (A), where phosphorylated eIF2α  is normalised to total eIF2α. Mean±SD. 2-way ANOVA (TWA) with Holm-Sidak's multiple comparisons.",
    "molecules": "TUN, tunicamycin"
  },
  {
    "caption": "C)  Mice were housed in 12h:12h light-dark cycles, with unrestricted feeding. Mouse lungs were collected 7 hours after transition to the light phase, 5 hours after intraperitoneal injection of mice with  bortezomib (BTZ, 2.5 mg/kg) or vehicle (Veh, 1% DMSO in sterile PBS). Tissues were lysed in RIPA buffer and western blots  were performed, probing for phosphorylated STAT3, total STAT3  and actin. Male (left) and female (right) replicates shown, N=2 mice for each condition. D) Quantification of the blots shown in (C), where phosphorylated STAT3 is normalised to total STAT3. Mean±SD. Multiple t-tests, corrected for multiple comparisons with the Holm-Sidak method, *p≤0.05, ***p≤0.001.",
    "molecules": "bortezomib, BTZ, DMSO, PBS"
  },
  {
    "caption": "A) WT  PER2::LUC cells were treated with  epoxomicin  or vehicle control. Blue arrows show the  time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001. C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines). D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.",
    "molecules": "epoxomicin, tunicamycin"
  },
  {
    "caption": "E) WT  PER2::LUC  cells were treated with radicicol  or vehicle control. Blue arrows show the time points  where drug was washed off. Mean (solid lines) ± SD (dotted lines). F) Quantification of damping rate of the   PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.",
    "molecules": "radicicol"
  },
  {
    "caption": "I-J) Representative double-plotted actograms of WT mice  receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h  light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment.",
    "molecules": "DMSO, ixazomib, Ixazomib"
  },
  {
    "caption": "(A) BECN1 expression of microglia from Becn1+/- and wild type mouse pups in full medium without Bafilomycin A1 (Baf) was quantified by Western blotting. Microglia from Becn1+/- mice show a signifcant reduction of BECN1 (48%) compared to microglia from wild type mice; mean ± SEM, wild type n = 3, Becn1+/- n = 7; two-tailed t-test **: p < 0.01. Blot shows representative amounts of BECN1 after various treatments.",
    "molecules": "Baf, Bafilomycin A1"
  },
  {
    "caption": "(B) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 (Baf). LC3 and ACTIN levels were determined by Western blot. LC3-II was significantly reduced in microglia from Becn1+/- mice after Baf treatment in full medium compared to wild type microglia; mean ± SEM, wild type n = 3, Becn1+/- n = 4; two-tailed t-test *: p < 0.05.",
    "molecules": "Baf, Bafilomycin A1"
  },
  {
    "caption": "(C) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 (Baf) and stained for endogenous LC3. Exemplary images and the quantification of LC3-positive vesicles/µm2 cell area show reduced presence of LC3-positive vesicles in microglia from Becn1+/- and induction of autophagy by HBSS; mean ± SEM, wild type n = 4, Becn1+/- n = 4, 3-5 fields per view/n and condition and 73-271 cells/n and condition; two-tailed t-test *: p < 0.05; scale bar: 20 µm",
    "molecules": "Baf, Bafilomycin A1"
  },
  {
    "caption": "(A, B) Microglia isolated from newborn Becn1+/- and wild type mouse pups were either non treated or treated with a pro-inflammatory stimulus (LPS followed by ATP). As additional controls cells treated only with LPS or ATP were used in (A). The concentration of the pro-inflammatory cytokines IL-1beta (A) and IL-18 (B) in the cell supernatant was determined by ELISA. Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS/ATP; mean ± SEM, wild type n (A/B) = 9/2, wild type LPS n = 3/0, wild type ATP n = 3/0, wild type LPS/ATP n = 9/9, Becn1+/- n = 23/5, Becn1+/- LPS/ATP n = 23/23, two-tailed t-test *: p < 0.05.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(C) Microglia isolated from wild type mouse pups were pre-incubated in full medium or starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment. The concentration of the pro-inflammatory cytokine IL-1beta in the cell supernatant was determined by ELISA. Incubation with HBSS reduced IL-1beta release significantly while 3-MA treatment increased it; mean ± SEM, full medium n = 6, full medium LPS/ATP n = 6, full medium LPS/ATP + 3MA n = 3, HBSS n = 6, HBSS LPS/ATP n = 6, HBSS LPS/ATP + 3MA n = 6; two-tailed t-test *: p < 0.05; **: p < 0.01.",
    "molecules": "3-MA, 3MA, ATP, LPS"
  },
  {
    "caption": "(D) Microglia isolated from wild type mouse pups were pre-incubated in starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment, microglia were stained for LC3 and images taken by confocal microscopy. Representative images show a reduction of LC3-positive vesicles in 3-MA treated cells. Scale bar: 20 µm.",
    "molecules": "3-MA, ATP, LPS"
  },
  {
    "caption": "(E) Proteins were extracted from brains of wild type, Becn1+/-, APPPS1 and APPPS1 Becn1+/- mice at the indicated ages. IL-1beta was measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. IL-1beta was significantly increased in the TBS and TX fraction; mean ± SEM, wild type: n (per age group) = 2/6/3, Becn1+/-: n = 2/5/2, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with WT as control group; *: p < 0.05; **: p < 0.01.",
    "molecules": "TBS, TX"
  },
  {
    "caption": "(A) The expression of NLRP3 protein in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced expression of NLRP3; mean ± SEM, wild type n = 4, wild type LPS/ATP n = 9, Becn1+/- n = 4, Becn1+/- LPS/ATP n = 23; ANOVA with Tukey´s post hoc test *: p < 0.05.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(B) The expression of Nlrp3 mRNA in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by qPCR. No significant differences can be detected between wild type and Becn1+/- microglia; mean ± SEM, wild type: n = 7, Becn1+/- : n = 3; ANOVA with Tukey ´s post hoc test, ns for LPS/ATP treated wild type versus Becn1+/-.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(C) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for ASC (red) and imaged by confocal microscopy to investigate the assembly of inflammasomes. In non stimulated cells ASC is distributed diffusely in the cytoplasm (upper panels). However, after stimulation formation of inflammasomes (arrowheads) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis/ejection (arrow). Quantification showed a significantly increased percentage of inflammasomes in or around Becn1+/- microglia; mean ± SEM, wild type LPS/ATP n = 4, Becn1+/- LPS/ATP n = 7; two-tailed t-test *: p < 0.05; scale bar: 50 µm, insert scale bar: 10 µm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(D) The presence of cleaved CASP1 (p10) protein in the supernatant of LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced release of CASP1; mean ± SEM, wild type LPS/ATP n = 8, Becn1+/- LPS/ATP n = 18; two-tailed t-test *: p < 0.05.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(A) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for NLRP3 (green) and LC3 (red) and imaged by confocal microscopy. Stimulation resulted in the appearance of many NLRP3-containing aggregates of different sizes (arrows). In non treated cells, only a few overlapping signals with LC3-stained autophagosomes (arrowheads) are visible. Stimulated microglia however, showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates; scale bar: 7.5 µm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(B) LPS/ATP treated wild type microglia were immunolabeled for NLRP3 (green) and LC3 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates of different sizes coclustering with LC3-positive autophagosomes are clearly visible. 3D volume rendering of large (I) and small (II) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes; scale bar: 10 µm; magnified ROIs: 1 µm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(A) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by confocal microscopy. Colocalizations of NLRP3 aggregates (arrows) and CALCOCO2 (arrowheads) are apparent; scale bar: 10 µm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(B) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates coclustering with CALCOCO2-positive signals are visible. 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2; scale bar: 10 µm; magnified ROIs: 1 µm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(E) Microglia from Becn1+/- mice were transfected with scrambled siRNA or with siRNA for CALCOCO2. 6 d after transfection cells were stimulated with LPS/ATP. The expression of CALCOCO2 protein was determined by Western blot. Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "C PICT assay for the interactions between indicated pairs of proteins tagged to FRB and GFP. Representative images before and after adding rapamycin (RAP) of RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. Scale bar, 5µm.",
    "molecules": "RAP, rapamycin"
  },
  {
    "caption": "C Left, PICT assay for the interaction of Trs85-FRB (TRAPPIII) and Drs2-GFP or drs2-5A-GFP after adding rapamycin (RAP). The Interaction score was normalized to the measurement of Drs2-GFP. Error bars: mean ± SD, n = 3. Asterisk indicates significant difference as determined by a two-tailed Student's t-test (**P<0.01). Right, representative images of the PICT assay. RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. Scale bar, 5µm.",
    "molecules": "RAP, rapamycin"
  },
  {
    "caption": "C Representative images (left) and quantification (right) of the PICT assay for the interaction of Trs85-FRB (TRAPPIII) and Atg9-GFP in cells expressing wild-type Drs2 or drs2-5A after adding rapamycin (RAP). RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. , C Values were normalized to the measurements in cells expressing wild-type Drs2. Error bars: mean ± SD, n = 3 biological replicates. Asterisks indicate significant difference as determined by a two-tailed Student's t-test (**P<0.01). Scale bar, 5µm.",
    "molecules": "RAP, rapamycin"
  },
  {
    "caption": "(B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(B) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.",
    "molecules": "Ficoll"
  },
  {
    "caption": "(C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.",
    "molecules": "nitrogen"
  },
  {
    "caption": "B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.",
    "molecules": "TCA, Triton X-100"
  },
  {
    "caption": "(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(H, I). Secretory vesicles of primary chromaffin cells were examined by transmission electron microscopy after 100 nM AngII stimulation, with or without pre-incubation with 400 nM Compound 7. Scale bars: (H, I), 150 nm. Red arrows indicate morphologically docked LDCVs and green arrows stands for undocked LDCVs. PM represents plasma membrane. (J). Vesicle numbers according to different distances from the plasma membrane were calculated in the presence or absence of 100 nM AngII or 400 nM Compound 7. PM represents plasma membrane. Data information: Data were analyzed using one-way ANOVA and displayed as the mean ± s.e.m. #, P<0.05; ##, P<0.01; ###, P<0.001: cells pre-incubated with PTP-MEG2 inhibitors compared with control vehicles. N.S. means no significant difference. Data were from 6 independent experiments. # indicates P<0.05 and ### indicates P<0.001 compared with control group.",
    "molecules": "Compound 7, AngII"
  },
  {
    "caption": "(D). Phosphorylated NSF interacted with PTP-MEG2. PC12 cells were transfected with FLAG-NSF. After stimulation of the cells with 100nM AngII in the presence or absence of 100μM H2O2 for 5 minutes (H2O2 was exploited to increase the overall tyrosine phosphorylation of the cellular proteins), the potential PTP-MEG2 substrate in cell lysates was pulled down with GST-PTP-MEG2-D470A or GST control.",
    "molecules": "AngII, H2O2, tyrosine"
  },
  {
    "caption": "(B). Relative values of the decreased phosphatase activities of different PTP-MEG2 mutants towards pNPP (p-Nitrophenyl phosphate) compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.",
    "molecules": "Nitrophenyl phosphate, pNPP"
  },
  {
    "caption": "(C). Fold-change decreases in the phosphatase activities of different PTP-MEG2 mutants towards the NSF-pY83 phospho-segment compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.",
    "molecules": "pNPP"
  },
  {
    "caption": "(C). The GST pull-down assay suggested that PACSIN1, MUNC18-1, VAMP7, SNAP25, DYNAMIN1 and DYNAMIN2 directly interact with PTP-MEG2. PC12 cells were transfected with plasmids of candidate substrates, including SYN1, MUNC18-3, PACSIN1, SCAMP1, MUNC18-1, PPP3CA, STX17, VAMP7, SYT7, SYT11, SNAP25, DYNAMIN1, and DYNAMIN2 stimulated with 100 nM AngII. The tyrosine phosphorylation of these proteins was verified by specific anti-pY antibodies (Appendix Figure S8A-E). The potential substrates of PTP-MEG2 in cell lysates were pulled down with a GST-PTP-MEG2-D470A trapping mutant and then detected by Western blot. White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP-MEG2 substrate.",
    "molecules": "AngII, tyrosine"
  },
  {
    "caption": "(D). Interactions of the PTP-MEG2-trapping mutants with the MUNC18-1-Y145 mutants and DYNAMIN2-Y125 mutants. PC12 cells were transfected with FLAG-MUNC18-1-Y145, FLAG-DYNAMIN2-Y125 and different mutations of the FLAG-MUNC18-1-Y145A, Y145H, Y145E or Y145F, FLAG-DYNAMIN2-Y125A, Y125E, Y125F, 24 hours before stimulation with 100 nM AngII, respectively. The cell lysates were then incubated with GST-beads-PTP-MEG2-D470A complex for 2 hours with constant rotation. The potential PTP-MEG2 substrates were pulled down by GST-beads, and their levels were examined by the FLAG antibody with Western blot.",
    "molecules": "AngII"
  },
  {
    "caption": "(B) mitochondrial ATP synthesis in the indicated VSMC types.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Cytosolic ATP synthesis in the indicated VSMC types, measured by incorporation of phosphate-32 (32Pi) into ADP. [32Pi]-ATP was separated from 32Pi using the molybdate method, as explained in the Methods section.",
    "molecules": "ADP, ATP, molybdate, 32Pi, phosphate-32"
  },
  {
    "caption": "(B) Intracellular lactate concentration",
    "molecules": "lactate"
  },
  {
    "caption": "(D) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi/mL calcium-45 (45Ca2+) as a radiotracer.",
    "molecules": "45Ca2+, Calcium, calcium-45"
  },
  {
    "caption": "(E) Magnesium concentration in isolated mitochondria.",
    "molecules": "Magnesium"
  },
  {
    "caption": "(B) Representative time-course of 2 mmol/L phosphate on calcification of LmnaG609G/+ VSMCs (up). Calcification was visualized with Alizarin red. Representative microscopic images (10x; scale bar: 100 µm) showing calcification of treated and untreated LmnaG609G/+ VSMCs (down).",
    "molecules": "Alizarin red, phosphate"
  },
  {
    "caption": "(C-F) Measures of calcium in treated living LmnaG609G/+ VSMCs (C), treated fixed LmnaG609G/+ VSMCs (D), untreated living LmnaG609G/+ VSMCs (E), and untreated fixed LmnaG609G/+ VSMCs (F).",
    "molecules": "calcium"
  },
  {
    "caption": "(G) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells (ΔCa2+).",
    "molecules": "Ca2+, calcium"
  },
  {
    "caption": "(A) Magnesium intake in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).",
    "molecules": "Magnesium"
  },
  {
    "caption": "(B) Plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).",
    "molecules": "magnesium"
  },
  {
    "caption": "(C) Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n=20).",
    "molecules": "Calcium"
  },
  {
    "caption": "(A) ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated or treated LmnaG609G/+ mice.",
    "molecules": "ATP"
  },
  {
    "caption": "(B) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups.",
    "molecules": "calcium"
  },
  {
    "caption": "(C) Magnesium concentration in isolated mitochondria.",
    "molecules": "Magnesium"
  },
  {
    "caption": "(D) Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 µmol/L), 2-thenoyltrifluoroacetone (TTFA; 1 mmol/L), antimycin A (10 µmol/L), potassium cyanide (KCN; 1 mmol/L), or oligomycin (10 µmol/L).",
    "molecules": "TTFA, 2-thenoyltrifluoroacetone, antimycin A, oligomycin, KCN, potassium cyanide, rotenone"
  },
  {
    "caption": "A IMR90 ER:RasV12 primary fibroblasts were cultured in the presence of EtOH  (proliferating) or 100nM 4OHT  (for 6-8days to induce RasV12 expression and oncogene-induced senescence, OIS). mRNA levels of 4OHT  cells, normalised to PUM1  and presented relative to EtOH  control. (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001)",
    "molecules": "4OHT, EtOH"
  },
  {
    "caption": "B, C Cells as in A were analysed by Western blot  for Lamp1, p62  and Cathepsin B and quantified, normalised to GAPDH  and expressed relative to EtOH. (n=3 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data are analysed by 2-tailed, nonpaired Student's t-test (* <0.05, ** <0.01, *** <0.001",
    "molecules": "EtOH"
  },
  {
    "caption": "K, Cells as in A were incubated with DQ-BSA  and TR-BSA  for 2 hours, fixed and imaged by confocal microscopy. Scale bar: 20µm (n=3 independent experimental repeats (at least 240 cells analysed from 8 fields of view per condition).",
    "molecules": "DQ-BSA, TR-BSA"
  },
  {
    "caption": "O, Cells as in A were incubated with 50µg/ml cycloheximide  (CHX) for the timepoints  indicated. Degradation of autophagy receptors, p62  and NDP52  was assessed by Western blot  (n=3 independent experimental repeats).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "A Representative images of GFP-TFEB localisation  in EtOH  and 4OHT-treated fibroblasts, in full nutrient conditions. Scale bar: 20µm.",
    "molecules": "4OHT, EtOH"
  },
  {
    "caption": "B, EtOH  and 4OHT-treated fibroblasts immunostained  for endogenous TFE3  in the conditions indicated; starvation  indicates serum starvation  overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. Scale bar: 20µm; (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat).",
    "molecules": "4OHT, amino acid, EtOH, leptomycin B, LMB"
  },
  {
    "caption": "C EtOH  and 4OHT-treated fibroblasts  in the conditions indicated; starvation  indicates serum starvation  overnight and one-hour amino acid starvation, refeeding was with full nutrient medium (including FCS) for 2 hours. Cells were incubated with leptomycin B (LMB) for 3 hours and where indicated, wash out involved replacement of the media for a further 2 hours. % cells with nuclear TFE3  was quantified. (n=3 independent experimental repeats (at least 150 cells analysed from at least 5 fields of view per repeat). Data information: Violin blots include all datapoints from n=3, lines represent median and upper and lower quartiles; Analysis: one-way ANOVA with Tukey's multiple comparison test * p<0.05; ** p<0.01; *** p<0.001.",
    "molecules": "4OHT, amino acid, EtOH, leptomycin B, LMB"
  },
  {
    "caption": "A Fibroblasts  stably expressing GFP-TFEB  were treated 4OHT  to induce senescence (or EtOH  control). Cells were lysed and subject to  Western blotting for antibodies as indicated. B Quantification of A. (n=4 independent experimental repeats) C Quantification of A. (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. B and C: analysed by 2-tailed, unpaired T-Test * p<0.05; ** p<0.01; *** p<0.001.",
    "molecules": "4OHT, EtOH"
  },
  {
    "caption": "D Western blot of GFP immunoprecipitation  of control (EtOH) and senescent (4OHT) cells expressing GFP  or GFP-CDK4  and FLAG-TFEB.",
    "molecules": "4OHT, EtOH"
  },
  {
    "caption": "H Fibroblasts  stably expressing GFP-TFEB  were transduced with p16  shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. I Quantification of H (n=4 independent experimental repeats). Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)",
    "molecules": "4OHT"
  },
  {
    "caption": "J Fibroblasts   were transduced with p16  shRNA simultaneously with induction of senescence by 4OHT. Cells were lysed and subject to Western blotting. K, L Quantification of J (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)",
    "molecules": "4OHT"
  },
  {
    "caption": "M Fibroblasts  were transduced with p16  shRNA simultaneously with induction of senescence  by 4OHT. Cells were fixed and immunostained  with antibodies against p62  and Lamp2, Scale bar: 20µm.",
    "molecules": "4OHT"
  },
  {
    "caption": "O Fibroblasts  were transduced with p16  shRNA simultaneously with induction of senescence  by 4OHT. Cells were incubated with serum-free media overnight which was collected and subject to ELISA  analysis for  IL-6 (n=3 independent experimental repeats) Data information: All graphs show individual data points, mean and error bars represent standard deviation. All other panels analysed by one-way ANOVA with Tukey's multiple comparison test (* <0.05, ** <0.01, *** <0.001)",
    "molecules": "4OHT"
  },
  {
    "caption": "(b) Effects of C18-Pyr-Cer (C18PC, lanes 2 and 4) on the lipidation of LC3B (LC3B-II) were examined by western blotting in the absence or presence of a pan-caspase inhibitor Z-VAD (lanes 3 and 4) and compared to vehicle-treated controls (lanes 1 and 3). Full blots can be found in Supplementary Figure 13.",
    "molecules": "Cer, Pyr, Z-VAD"
  },
  {
    "caption": "(c) Formation of double-membrane autophagosomal vesicles (black arrows) in the absence (control) or presence of C18PC was visualized by TEM (left and right, respectively). Higher magnification of TEM visualization is shown in lower panels. Scale bars, 10 μm (top) and 500 nm (bottom).",
    "molecules": "C18PC"
  },
  {
    "caption": "(d) Effects of siRNA-mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18PC were determined and compared to effects of transfection with Scr siRNAs (controls) using Trypan blue exclusion assay.",
    "molecules": "C18PC"
  },
  {
    "caption": "(e) Roles of C18PC in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild-type (ATG5+/+) and ATG5−/− knockout mice with C18PC. Control cells were treated with vehicle.",
    "molecules": "C18PC"
  },
  {
    "caption": "(f) Effects of C18PC on caspase-dependent or caspase-independent cell death were determined in MEFs isolated from Bax−/− Bak−/− or Casp3−/− Casp7−/− mice and from wild-type (Bax+/+ Bak+/+) or Casp3+/− Casp7+/− mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P 0.05).",
    "molecules": "C18PC"
  },
  {
    "caption": "(a) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18-Pyr-Cer (C18PC) at 2 h in UM-SCC22A (22A) cells was examined by visualizing the colocalization of MTG (green) and LTR (red) using live-cell imaging and confocal microscopy. Scale bars, 10 μm. Enlarged images are shown in boxed areas.",
    "molecules": "Cer, C18PC, Pyr"
  },
  {
    "caption": "(b) Effects of C18-dihydro-Cer-14-piperidine (dihydro-C18PC) on OCR measured using the SeaHorse alongside those of C18PC- and vehicle-treated controls. Data shown are an average of at least two independent experiments performed in duplicate ± s.d. (*P 0.05).",
    "molecules": "Cer, C18PC, piperidine"
  },
  {
    "caption": "(a) Effects of expression of wild-type (WT) CerS1 and its catalytically inactive V5-tagged mutant (Mut), which cannot generate C18-ceramide, on the formation of LC3B-II were determined by western blotting (+/− tet). Successful induction of wild-type and mutant CerS1 expression was confirmed using anti-V5. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.",
    "molecules": "V5, ceramide"
  },
  {
    "caption": "(b) Generation of endogenous C18- and C18:1-ceramides in response to wild-type CerS1 and mutant CerS1 induction was measured by LC/MS/MS.",
    "molecules": "ceramides"
  },
  {
    "caption": "(c) Effects of wild-type CerS1 and C18-ceramide induction (+ tet) on GFP and LC3B-GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls (− tet). Scale bars, 10 μm",
    "molecules": "C18-ceramide"
  },
  {
    "caption": "(d) The effects of induction of wild-type CerS1 and C18-ceramide and catalytically inactive mutant CerS1 (+ tet) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls (− tet).",
    "molecules": "C18-ceramide"
  },
  {
    "caption": "(e) Targeting mitochondria with autophagolysosomes in the absence (− tet) or presence (+ tet) of wild-type CerS1 and C18-ceramide was visualized by colocalization of MTG and LTR using confocal microscopy. Scale bars, 10 μm.",
    "molecules": "C18-ceramide"
  },
  {
    "caption": "(f) Effects of wild-type and mutant CerS1 (−/+ tet) on ATP generation.",
    "molecules": "ATP, tet"
  },
  {
    "caption": "(g) Effects of wild-type CerS1 (−/+ tet) on ATP generation in the absence (control shRNA) or presence of shRNA-mediated knockdown of LC3B. Data shown are an average of at least three experiments ± s.d. (*P 0.05).",
    "molecules": "ATP"
  },
  {
    "caption": "(a) Subcellular localization of ceramide, generated by wild-type CerS1 induction (+ tet), was visualized in mitochondria by colocalization of ceramides and mitochondria with anti-ceramide and anti-Tom20, respectively, using confocal microscopy.",
    "molecules": "ceramide, ceramides"
  },
  {
    "caption": "(b,c) Mitochondrial (Mito) targeting of LC3B-II and C18-ceramide, generated by induction of wild-type CerS1 (b), and that of the products of the catalytically inactive mutant CerS1 (+ tet) (c) was visualized by colocalization of ceramide (Cer; green), CFP-LC3B (blue) and MTR using anti-ceramide and confocal microscopy. Noninduced cells (− tet) were used as controls. Scale bars, 10 μm.",
    "molecules": "Cer, ceramide"
  },
  {
    "caption": "(a) Binding of Flag-tagged wild-type LC3B and LC3BI35A, LC3BF52A or LC3BG120A with endogenous C18-ceramide (C18-Cer), generated in response to wild-type CerS1 induction (left) or exogenous C18-Pyr-Cer (right), was measured using LC/MS/MS after pulldown using anti-Flag-conjugated beads. Data shown are an average of at least three experiments ± s.d. (*P 0.05).",
    "molecules": "Cer, ceramide, Pyr"
  },
  {
    "caption": "(b) Effects of CerS1 and C18-ceramide induction on the lipidation of Flag-tagged wild-type, LC3BI35A, LC3BF52A and LC3BG120A proteins were examined by western blotting. Noninduced cells (− tet) were used as controls. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.",
    "molecules": "ceramide"
  },
  {
    "caption": "(a,b) Effects of G120A and F52A mutations, which perturbed C18-ceramide binding of LC3B, on targeting of mitochondria by LC3B-containing autophagosomes (visualized by confocal microscopy using anti-Flag and anti-Tom20) (a) or mitochondrial function (measurement of OCR) (b). V, vector.",
    "molecules": "ceramide"
  },
  {
    "caption": "(c) Effects of siRNA-mediated knockdown of p62, NIX and Drp1 on OCR in the absence or presence of CerS1 and C18-ceramide induction were measured using the SeaHorse. Data shown are an average of at least three experiments ± s.d. (*P 0.05).",
    "molecules": "ceramide"
  },
  {
    "caption": "(d) Effects of siRNA-mediated knockdown of Drp1 (lower left and right) on the localization of ceramide within mitochondrial membranes were visualized using the colocalization of anti-ceramide (red) and anti-Tom20 (green) under confocal microscopy, in the absence (upper and lower left) or presence (upper and lower right) of CerS1 and C18-ceramide induction (−/+ tet) compared to Scr siRNA-transfected controls (upper left and right). Scale bars, 10 μm in a and d. Enlarged images are shown in boxed areas in d.",
    "molecules": "ceramide"
  },
  {
    "caption": "(a,b) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a) or UM-SCC-22A xenograft-derived tumor growth (b) in the absence or presence of wild-type CerS1 and C18-ceramide induction (−/+ tet) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice (n = 6 per group), respectively. Data shown are an average of at least three experiments ± s.d. (*P 0.05).",
    "molecules": "ceramide"
  },
  {
    "caption": "Wet-spun yarn containing human myogenic precursors showed progressive cell differentiation over time (arrows in day 3); dashed box enlargement reveals still undifferentiated cells after 15 days of culture (Upper panel). Middle and lower panel showing MHC immunostaining (red) performed on microfiber yarns and bulk structure loaded with human myoblasts upon 15 days of culture, revealing a significant difference in terms of myotube organization between the two hydrogel systems, with the wet-spun constructs greatly outperforming the bulk gels. Such difference is quantified in the polar plots. Nuclei were counterstained by DAPI (Blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Instability of BU-1 expression in timeless cells. FACS plots of wild type and timeless (clone 1) DT40 cells stained with anti-Bu-1 conjugated with phycoerythrin. Each line represents the Bu-1 expression profile of an individual clonal population. Unstained controls are shown in blue.",
    "molecules": "phycoerythrin"
  },
  {
    "caption": "D. The DNA-binding affinity of DBD was measured by fluorescence anisotropy, titrating the DBD protein against Cy3 3'-labelled ssDNA, dsDNA and G4 DNA The top panel shows binding curves for ss- and dsDNA, the bottom panel shows the binding curve for the G4 DNA substrate.",
    "molecules": "G4 DNA, Cy3, dsDNA, ssDNA"
  },
  {
    "caption": "Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. A. ssG4: G4 flanked by single-stranded DNA; ssHP: hairpin flanked by single-stranded DNA; ss: single-stranded DNA ",
    "molecules": "single-stranded DNA"
  },
  {
    "caption": "Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. B. Binding affinity of Timeless-Tipin for a range of G4 DNA sequences Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) and double-stranded (ds20: ss20 annealed to complementary sequence) DNA were also tested as controls.",
    "molecules": "G4 DNA"
  },
  {
    "caption": "A. Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours. PCNA was precipitated from cross-linked chromatin and the immunoprecipitate blotted for FLAG-DDX11.",
    "molecules": "PDS"
  },
  {
    "caption": "A. Growth curves for DT40 wild type, ddx11, timeless and ddx11/timeless cells, with and without 4 μM pyridostatin (PDS). Cells were seeded at 5 x104 cells/ml on day 0 and the viable cells were counted each 24 h for 4 days. Bars represent SD of two independent experiments performed in duplicate. Doubling times (DMSO): WT 13 hours, timeless 18 hours, ddx11 16 hours, ddx11/timeless 24 hours. Doubling times (PDS): WT 13.6 hours, timeless 27 hours, ddx11 25.7 hours, ddx11/timeless 47.5 hours.",
    "molecules": "DMSO, PDS, pyridostatin"
  },
  {
    "caption": "B. DDR signalling detected by phosphorylation of histone H2AX (γ-H2AX) by flow cytometry in untreated cells or cells exposed to 4 μM PDS for 3 days. Pale histogram, untreated; dark histogram, treated; black dotted line, positive control cells treated with 0.1 µM cisplatin, also for 3 days. C. Quantification of γ-H2AX in DT40 wild type, ddx11, timeless and ddx11/timeless cells treated with 4 μM PDS for 3 days. The central band represents the median, the box the 25th - 75th centile and whiskers the minimum to maximum range of three independent experiments performed in duplicate. ",
    "molecules": "cisplatin, PDS"
  },
  {
    "caption": "(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091).",
    "molecules": "E-64d, pepstatin"
  },
  {
    "caption": "(a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058).",
    "molecules": "chloroquine"
  },
  {
    "caption": "(b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine",
    "molecules": "chloroquine"
  },
  {
    "caption": "(c) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0024).",
    "molecules": "chloroquine"
  },
  {
    "caption": "(d) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024).",
    "molecules": "puromycin"
  },
  {
    "caption": "(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c).",
    "molecules": "chloroquine"
  },
  {
    "caption": "(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c). Cell viability was assessed by MTS 24 h following Fas ligand treatment (d; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 1.6×10−4).",
    "molecules": "chloroquine"
  },
  {
    "caption": "(e,f) BJAB and Jurkat cells were transduced with control, Atg5, Atg7 or Vps34 shRNA lentiviruses, followed by three days of puromycin selection. Cells were then treated with Fas ligand (1.25 ng ml−1) or TRAIL (1.25 ng ml−1) for 24 h and viability was assessed by MTS (e; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 1.4×10−6, **P = 2.3×10−5, ***P = 3.9×10−5, §P = 9.2×10−6, §§P = 3.4×10−6, §§§P = 1.7×10−5). Immunoblots demonstrate Atg5, Atg7 and Vps34 knockdown, autophagy inhibition and altered Fap-1 levels. The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other; the Fap-1 blot is not separated to demonstrate the lack of Fap-1 protein in Type II Jurkat cells (f). Fap-1 blots (b,c,f) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.",
    "molecules": "puromycin"
  },
  {
    "caption": "(a,b) BJAB and Jurkat cells expressing the indicated Fap-1 wild-type (WT) and catalytically inactive (ΔCD) constructs were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (BJAB, 1.5 ng ml−1; Jurkat 15 ng ml−1) for 24 h. Cell viability was determined by MTS (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 2.4×10−5, ***P = 0.0024). Cell lysates were blotted and probed with the indicated antibodies (b).",
    "molecules": "chloroquine"
  },
  {
    "caption": "c-e) BJAB cells expressing control or Fap-1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand (12.5 ng ml−1) or TRAIL (25 ng ml−1) for 24 h; cell viability was then determined by MTS (c; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 4.8×10−6, **P = 8.1×10−5, ***P = 8.0×10−4, §P = 0.013, NS P>0.05).",
    "molecules": "chloroquine"
  },
  {
    "caption": "(d) Immunoblots of lysates collected following chloroquine treatment. Long-term growth was assessed in cells from c by re-plating them at low density and allowing them to recover for 5 days, followed by viability assay (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 0.043; e). Fap-1 blots (b,d) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4°C.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(B) Lineage tracing of the anagen matrix from β-actin-CreER:Confetti, 7 days after induction with either a high dose of tamoxifen (TM) (top) or low dose (bottom) for single cell clonal lineages.",
    "molecules": "tamoxifen, TM"
  },
  {
    "caption": "(C) Images of skin sections at various stages of the hair cycle, immunostained for Gata6 (red), CD34 or K14 (green) and DNA (blue).",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2, 4, and 10 days (d) post TM induction. (C) Images of hematoxylin staining of skin section from the anagen induced mice. Quantification shown in Figure EV2D. Scale bars: 30 m.",
    "molecules": "TM"
  },
  {
    "caption": "(B-G) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color.",
    "molecules": "TM"
  },
  {
    "caption": "(A) Hair follicles exhibit immunostaining signal for DNA damage marker H2A.X (red) and apoptosis marker Caspase3 (green) as early as one day after Gata6 iKO induction. Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage. Note that Caspase 3 is not expressed in the H2A.X cells of the epidermis but is found in some iKO hair follicle matrix cells. Right panels show single color images as indicated at top (B) Quantification (average SD) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3+ cells (n=50 follicles from 3 mice per group) (C) Quantification (average SD) of the percentage of DNA damaged cells per matrix as determined by counting of H2A.X+Caspase3- cells (n=50 follicles from 3 mice per group).",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin. Primary keratinocytes were isolated from induced iKO newborn mice, plated on feeder cells, and stained with hematoxylin at day 7 after plating (top). Phase contrast images of cells are shown below. Scale bars: 30 m. (B) Quantification of colony formation from (A), average SD from n=3 mice in each genotype.",
    "molecules": "TM"
  },
  {
    "caption": "(C) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction, blotted with actin and Gata6 antibodies, show a decrease in Gata6 protein levels after 6 hours.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(D) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM (n > 3 per genotype).",
    "molecules": "TM"
  },
  {
    "caption": "(E-H) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A.X (E and G) and comet assay (F and H). Arrowheads in (F) indicate DNA damaged cell nuclei with comet shape. Quantification of H2A.X (G) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . *Unpaired t-test P-values: 6h=0.01 and 24h=0.004. (H) Quantification of comet assay represents average SD (n=3).",
    "molecules": "DNA"
  },
  {
    "caption": "(D) Staining of ROS by DHE. LY83583 treated cells served as positive control with elevated ROS. (E) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT, based on fluorescence intensity of DHE (average SD, n=2).",
    "molecules": "LY83583, ROS, TM"
  },
  {
    "caption": "(J) Quantification of H2A.X in cells stably transfected with pMock, pGata6, or pEdaradd plasmids, followed by induction with TM for 6 hours (average SD, n=4 with > 500 cells counted per condition). *Unpaired t-test P-values = 2x10-4 (iKO pGata6 vs pMock), 2x10-4 (iKO pEdaradd vs pMock).",
    "molecules": "TM"
  },
  {
    "caption": "(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (a) The cell viability was assayed by trypan blue staining. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test). (b) Cell count and viability assay using Muse (Millipore, USA). Data are mean from three independent experiments, with error bars representing s.d. *P0.05 (Student's t-test).",
    "molecules": "ecdysone"
  },
  {
    "caption": "(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 µM ecdysone for 24 h. (c) Caspase activity was measured from total cell lysate on DEVD-AMC, represented as fold increase relative to control RNAi. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).",
    "molecules": "ecdysone"
  },
  {
    "caption": "(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (d) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49. Relative dUTX expression levels following knockdown using the two independent dsRNA are shown (n=3) with error bars representing s.d. *P0.05 (Student's t-test).",
    "molecules": "ecdysone"
  },
  {
    "caption": "(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (a) GST and GST-dUTX-NR containing the nuclear hormone domain were used in pull-down experiments with 35S-labelled EcR and Usp in the presence of ethanol control (−) or 10 μM ecdysone (+). The GST input proteins are shown in Coomassie Brilliant blue-stained gel, GST is labelled with arrow head and dUTX-NR with an asterisk. The EcR and Usp inputs are shown labelled with arrows.",
    "molecules": "35S, ecdysone, ethanol"
  },
  {
    "caption": "(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (b) Immunoprecipitation (IP) of EcR and dUTX in SL2 cells. Cells were transfected with HA-dUTX and the cell lysates were immunoprecipitated using anti-HA or a control IgG isotype antibody. The immunoprecipitates were analysed by western blotting using the anti-EcR and anti-HA antibodies.",
    "molecules": "ecdysone"
  },
  {
    "caption": "(c) Colocalization of dUTX and EcR on salivary gland polytene chromosomes. Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG-dUTX with dUTX (green) and EcR (red), with Hoechst-stained DNA (blue). Arrows indicate examples of colocalized bands. No signal was detected when immunostaining with secondary antibody only. Scale bar represent 5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of dark, dronc, drice and rpr (a)",
    "molecules": "ecdysone"
  },
  {
    "caption": "(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of Atg1, Atg2, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg9, Atg12 and Atg18 (b), expressed relative to the internal control gene Gapdh. Results are shown as the fold enrichment of % input compared with IgG control.",
    "molecules": "ecdysone"
  },
  {
    "caption": "(c) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP. SL2 cells knocked down for dUTX were chromatin-immunoprecipitated with anti-H3K27me3 and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control.",
    "molecules": "ecdysone"
  },
  {
    "caption": "(d) The occupancy of EcR on the promoter regions of dark, dronc and Atg1 by ChIP expressed relative to the internal control gene rp49. Chromatin was immunoprecipitated with anti-EcR-B1 and anti-GFP (IgG control) antibodies from SL2 cell following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).",
    "molecules": "ecdysone"
  },
  {
    "caption": "(b) The occupancy of dUTX on the promoter regions of BR-C and E93 genes detected by ChIP. SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. In the right hand panel abundance of H3K27me3 on the promoters of BR-C and E93 genes as detected by ChIP is shown. SL2 cells knockdown for dUTX (dUTX RNAi) were chromatin-immunoprecipitated with anti-H3K27me3 and rabbit IgG (control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Students t-test).",
    "molecules": "ecdysone"
  },
  {
    "caption": " Transfection of poly (I:C) induces ACE2 expression.HEK293 cells were cultured in six-well plates and transfected with poly (I:C) as indicated above using Lipofectamine 2000. HEK293 cells were then collected for RNA isolation and RT-qPCR analysis at 9 hrs post-transfection. Results in each panel are representative of three independent experiments. ",
    "molecules": "poly (I:C)"
  },
  {
    "caption": "(A and B) RT-qPCR showing the enrichment of cluster C3 subpopulation (CFAP54+) in CCSC1 (A) and CCSC2 (B) spheres treated with the chemotherapeutic agent 5FU or L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on two-tailed Student's t-test. *, p<0.05; **, p<0.01; ***, p<0.001; ns, no significant.",
    "molecules": "5FU, L-OHP"
  },
  {
    "caption": "(C) Representative RNA-FISH images and quantification showing the level of cluster C3 subpopulation CFAP54+ cell (red) levels in L-OHP treated xenografts. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. *, p<0.05.",
    "molecules": "L-OHP"
  },
  {
    "caption": "(E) Fluorescent images and quantification showing AP20187-mediated ablation of cluster C4 (PLAUR+) cells from CCSC spheres carrying PLAUR-iCaspase9-mCherry construct. Scale bar, 40 μm. Three biological replicates were analyzed. Data represent the mean ± SD. P-value was calculated based on two-tailed Student's t test. ***, p<0.001.",
    "molecules": "AP20187"
  },
  {
    "caption": "(I) Representative H&E images and quantification (number of mice with invasive tumors) showing the invasive potential of cluster C4 cells (PLAUR+). CCSC sphere cells carrying PLAUR-iCaspase9-mCherry construct were orthotopically injected into the mice. AP20187 was applied to ablate cluster C4 (PLAUR+, red) cells. Red arrows indicate invasive tumor. Scale bars, left, 100 μm; right, 300 μm.",
    "molecules": "AP20187"
  },
  {
    "caption": "(K) Left: Representative H&E images of liver tissue sections and quantification (number of mice with liver metastasis) showing the metastatic potential of cluster C4 cells (PLAUR+). AP20187 was applied to ablate cluster C4. Yellow arrows indicate liver metastasis. Scale bars, left, 2 mm; right, 50 μm. Right: Quantification of liver metastasis in mice with intrasplenic injection. Error bars denote mean ± SEM of three mice per group. P-value was calculated based on Student's t-test. ***, p<0.001.",
    "molecules": "AP20187"
  },
  {
    "caption": "(D) RT-qPCR analysis of signature genes of each cluster in control (EV) or ATF6 knockdown (shATF6-1 and shATF6-2) under the treatment with L-OHP. Error bars represent SD of three biological replicates. P-value was calculated based on Student's t-test. ***, p<0.001. Diagram without asterisk indicates no statistical difference.",
    "molecules": "L-OHP"
  },
  {
    "caption": "(F) Representative RNA-FISH of cluster C3 signature gene, CFAP54 (up) and immunohistochemistry of transcription factor ATF6 (bottom) showing the influence of ATF6 knockdown (shATF6-1 and shATF6-2) on cluster C3 (CFAP54+, red) population in xenografts. The mice were treated with vehicle or L-OHP after 14 days after CCSC sphere cells were implanted. Scale bars, upper, 40 μm; lower, 80 μm.",
    "molecules": "L-OHP"
  },
  {
    "caption": "(B-C) Ageing leads to significant cardiac-specific accumulation of sphinganine.",
    "molecules": "sphinganine"
  },
  {
    "caption": "(D) Targeted lipidomics confirm age-dependent accumulation of sphinganine in killifish (n = 4; per condition), zebrafish (n = 4, per condition), mouse (n = 4 for young and n=6 for aged samples) and human hearts (n = 1 for young and 2 for aged). In the case of human heart tissue, statistical significance was computed from technical replicates.",
    "molecules": "sphinganine"
  },
  {
    "caption": "Sphinganine induces DNA damage in human cardiomyocytes depicted here (A-C) by γH2A.X+ nuclei in cardiac-specific ACTN2+ cells (n = 3 biological replicates; # of cells quantified = 147 per condition)",
    "molecules": "Sphinganine"
  },
  {
    "caption": "Sphinganine induces DNA damage in human cardiomyocytes depicted here (D) by neutral comet assay of DHS treated hCMs. (E) Quantification of comet length in the indicated conditions.",
    "molecules": "DHS, Sphinganine"
  },
  {
    "caption": "Elevated DHS levels lead to ageing signatures in hCMs shown here by (F-G) SA-β-GAL activity, Respective graphs depict the quantification of each of the ageing signatures are on the right.",
    "molecules": "DHS"
  },
  {
    "caption": "Elevated DHS levels lead to ageing signatures in hCMs shown here by (H) p21 activation (# cells quantified per replicate = 150-200), (I) HP1-α+ nuclear foci (# of cells quantified per replicate = 120-200) and (J) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining (# of cells quantified per replicate = 100-150 per condition). Respective graphs depict the quantification of each of the ageing signatures are on the right.",
    "molecules": "DHS"
  },
  {
    "caption": "High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs (L) and (M) in aged human hearts. (N) Violin plot depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (When not specified, the experiments were conducted in at least 3 biological replicates). Scale bar = 10 µm.",
    "molecules": "H3K56ac, sphinganine"
  },
  {
    "caption": "(A) Pharmacological inhibition of HDAC1 using romidepsin, MS-275 and pyroxamide in human cardiomyocytes cause extensive DNA damage, shown here by γH2A.X staining.",
    "molecules": "MS-275, pyroxamide, romidepsin"
  },
  {
    "caption": "(E) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC-activity assay, shown here as bar graph.",
    "molecules": "DHS1P, Sphinganine"
  },
  {
    "caption": "(F) In vitro HDAC activity assay revealed inhibition of nuclear HDAC's and purified HDAC1 activity by DHS1P and S1P, shown here as bar graphs.",
    "molecules": "DHS1P, S1P"
  },
  {
    "caption": "Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs",
    "molecules": "DHS"
  },
  {
    "caption": "Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications.",
    "molecules": "DHS"
  },
  {
    "caption": "(I) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co-incubation with RNA Pol II inhibitor, triptolide and (J) the respective quantifications of γH2A.X+ CM nuclei represented as bar graph. Scale bars for A,I=10 µm and G = 50 µm. (When not specified, the experiments were conducted in at least 3 biological replicates).",
    "molecules": "DHS, triptolide"
  },
  {
    "caption": "(A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) in hCMs.",
    "molecules": "ABC294640, SLM6031434, DHS"
  },
  {
    "caption": "(D-E) Treatment of hCMs with ABC294640, a specific SPHK2 inhibitor prevents DHS-induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining (~100 cells quantified/ condition).",
    "molecules": "ABC294640, H3K27ac, DHS"
  },
  {
    "caption": "(G) HAT inhibition by curcumin prevents the aberrant increase in H3K56ac and H3K27ac levels.",
    "molecules": "curcumin, H3K27ac"
  },
  {
    "caption": "(I) HAT inhibition prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) on human cardiomyocytes.",
    "molecules": "DHS"
  },
  {
    "caption": "(K) Curcumin-mediated HAT inhibition lead to inactivation of p21 in hCMs.",
    "molecules": "Curcumin"
  },
  {
    "caption": "(E) Co-treatment of curcumin activates DNA repair pathways as depicted here by heatmaps.",
    "molecules": "curcumin"
  },
  {
    "caption": " C) Sera from WT mice was analysed for N protein (N) and glycoprotein (GP)-specific antibodies by ELISA, each data point corresponds to one mouse from the same experiment. ",
    "molecules": "glycoprotein, GP"
  },
  {
    "caption": " G) Anti-N mAb KL53 was electroporated into WT and KO MEFs and subsequent LCMV infection titres were measured by FFA. ",
    "molecules": "mAb KL53"
  },
  {
    "caption": " H) Anti-N mAb KL53 was co-electroporated with recombinant N protein into WT and KO MEFs, and immunoblotting for N was performed after 3 hours. Electroporation of recombinant KL53 expressing the TRIM21 non-binding mutation H433A was unable to mediate N protein degradation. ",
    "molecules": "KL53, mAb KL53"
  },
  {
    "caption": " Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). A) Weights were monitored throughout infection, with final day 8 weights of individual mice presented separately. ",
    "molecules": "mAb KL53"
  },
  {
    "caption": " Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). Viral titres in the spleen, liver, lung and kidney of all mice were determined by FFA day 8pi. Each data point represents one mouse, with results from two repeat experiments combined. ",
    "molecules": "mAb KL53"
  },
  {
    "caption": " A) CD8 T cells in WT mice were depleted by administration of anti-CD8 mAb 1 day prior to infection with 10^5 FFU LCMV. Anti-N mAb KL53 or PBS control was passively transferred IP on days 1 and 3pi. Viral titres in the spleen, lung, liver and kidney of all mice were determined by FFA day 8pi. ",
    "molecules": "mAb KL53, PBS"
  },
  {
    "caption": " B) Spleens from WT and KO mice +/- mAb KL53 day 8pi with 0.5 x 10^5 FFU LCMV were analysed for the presence of LCMV N-specific CTLs by staining with the class I N396-404 tetramer. ",
    "molecules": "mAb KL53"
  },
  {
    "caption": " D) Macrophages were depleted in WT mice by administration of clodronate liposomes 1 day prior to infection with 0.5 x 10^5 FFU LCMV. KL53 was administered on days 1 and 3pi, and N396-specific CTLs in the spleen were measured day 8pi by tetramer staining. ",
    "molecules": "KL53, clodronate"
  },
  {
    "caption": " A) Splenocytes from uninfected CD45.1 mice, either pulsed with 3 concentrations of N peptide and cell trace violet (CTV) or unlabelled control cells, were transfused intravenously into WT and KO mice (CD45.2) that had been infected with 0.5 x 10^5 FFU LCMV 8 days earlier. After 3 hours, spleens from recipient mice were harvested and the proportion of CTV-labelled CD45.1 cells was analysed by flow cytometry. Histograms from single representative uninfected (UI), WT and KO mice are presented, showing the proportion of CD45.1 cells remaining for each of the labelled fractions normalised to mode. Summary data from all individual mice in the same experiment is presented in associated scatter plot, showing the mean ± standard error. B) Labelled splenocytes as for (A) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier, and received mAb KL53 on days 1 and 3pi. Flow cytometry histograms from single representative mice of each genotype. Summary data from all mice in the experiment is presented, showing the mean ± standard error. ",
    "molecules": "cell trace violet, CTV, mAb KL53"
  },
  {
    "caption": "F-K Representative fundus fluorescein angiography images of C57BL6/J (F-H) and Ifnar1-/- (I-K) mice 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in C57BL6/J controls versus Ifnar1-/- mice. Values show mean ± SD. (n = 14-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p < 0.0001).",
    "molecules": "fluorescein"
  },
  {
    "caption": "F-K Representative fundus fluorescein angiography images of control mice (F-H) or IFN-b treated animals (I-K) 3, 7 and 14 days after laser-induced damage.L Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in control mice and IFN-b treated animals. Values show mean ± SD. (n = 15-22 eyes; One-way ANOVA followed by Tukey's post-test: ***p = 0.0004).",
    "molecules": "fluorescein"
  },
  {
    "caption": "G-O Representative fundus fluorescein angiography images of Cx3cr1CreER (G-I), Ifnar1fl/fl (J-L) and Cx3cr1CreER:Ifnar1fl/fl (M-O) mice 3, 7 and 14 days after laser-induced damage.P Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in Cx3cr1CreER, Ifnar1fl/fl and Cx3cr1CreER:Ifnar1fl/fl mice. Values show mean ± SD. (n = 5-12 eyes; One-way ANOVA followed by Tukey's post-test: **p = 0.0032, *p = 0.0247).",
    "molecules": "fluorescein"
  },
  {
    "caption": "(A), pINCO‐NeoR transfected EBV‐B1.11 cells (B), and as control pINCO‐Tyrosinase‐transfected EBV‐B1.11 cells (C), were either left untreated or treated with 5 μM lactacystin or 50 μM AAF‐CMK for 20 h. Following fixation with 0.5% paraformaldehyde, cells were incubated with T cells, and 24 h later GM‐CSF release was determined by ELISA.",
    "molecules": "AAF‐CMK, lactacystin"
  },
  {
    "caption": "(A) RCC1.24‐NeoR incubated with IFN‐γ were either left untreated (w/o) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing. Subsequently cells were fixed with 0.5% paraformaldehyde and tested with 20-4/A4 in a GM‐CSF release assay. EBV‐B1.11 cells transfected with pINCO‐NeoR (B) or pINCO‐Tyrosinase (C) were tested likewise. Both substances abrogated recognition of neoR‐transfected cells, while HLA‐A2‐restricted presentation of the tyrosinase peptide was not affected",
    "molecules": "chloroquine, leupeptin"
  },
  {
    "caption": "(D) ) EBV‐B1.11 cells were incubated with 200 ng/ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in (A). Presentation of exogenous NeoR on HLA‐DP3 is blocked by both substances as well, indicating that NeoR presentation is dependent on lysosomal processing.",
    "molecules": "chloroquine, leupeptin"
  },
  {
    "caption": "(B) Endosomal/lysosomal fractions were prepared from pINCO‐NeoR‐transfected cells 96 h post transfection as in (A). To prevent lysosomal degradation of NeoR, half of the cells were treated with leupeptin for the last 48 h. These fractions were separated on a 27% Percoll gradient and eight different fractions collected. Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp‐1, Rab‐5 and cytochrome c. Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal/lysosomal fractions 7 and 8.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(C) To determine where NeoR gains access to the vacuolar system, 293T cells were transfected with pINCO‐NeoR, treated with leupeptin to prevent NeoR degradation, and the endosomal/lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation. Starting at 16 h post transfection, NeoR becomes detectable in the Rab‐5‐positive fraction 5, and then spreads into late endosomal/lysosomal fractions. At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(B) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR‐GFP fusion construct. Compared to untreated cells, cells treated for 24 h with 7.5 mM 3‐MA show an about threefold increase in mean fluorescence intensity.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(C) Western blot analysis of neoR‐transfected cells. Treatment of cells with 7.5 mM 3‐MA causes a dramatic increase in NeoR protein levels in only 12 h. The ER‐resident protein BiP was used as gel loading control.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(D) This increase in total NeoR protein in 3‐MA‐treated cells is associated with an increase in cytoplasmic, and a decrease in endosomal/lysosomal NeoR protein levels.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(E) Western blot analysis of whole cell lysates prepared from neoR‐transfected cells. Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels. Simultaneous treatment of cells with 3‐MA prevents NeoR degradation. BiP was again used as a gel loading control.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the T cell clone 20-4/A4‐specific T cell clone 20-4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(B) EBV‐B1.11 cells transfected with pINCO‐NeoR (endogenous), or incubated with recombinant NeoR protein (exogenous), were treated with the inhibitors of autophagy, wortmannin and 3‐MA, and HLA‐DP3‐restricted presentation of the NeoR‐derived peptide determined. Inhibition of antigen presentation by leupeptin served as control.",
    "molecules": "3‐MA, leupeptin, wortmannin"
  },
  {
    "caption": "C Confirmatory screening performed as described above. Schematic distribution of compounds based on Z-score values of cells expressing   polyGP-GFP and AUG-RFP (above) and on Z-score values of the fluorescent intensity of the two reporters per each compound (below). Data are from four technical replicates, boxes are the Z-score mean value and whiskers represent ±SD. Baseline of Z-score = 0 indicates that Z-score is identical to the mean score. Asterisk (*) represents the selected compounds: 1 Forskolin (FSK), 2 Erysolin (ERY), 3 Geldanamycin (GELD), 4 Helenin (HLN) and 5 Spironolactone (SPL). Number sign (#) represents cellular stress inducers: #1 Thapsigargin  and #2 Tunicamycin.",
    "molecules": "Helenin, HLN, ERY, Erysolin, Forskolin, FSK, GELD, Geldanamycin, Spironolactone, SPL, Thapsigargin, Tunicamycin"
  },
  {
    "caption": "D, E Dose-response  analysis of ERY, HLN, SPL, FSK and GELD. Cells were co-transfected with  AUG-RFP and polyGP-GFP plasmids and treated with two concentration ranges 0.5, 1, 5 and 10 µM (D) and 20, 40 and 60 µM (E) for 24 h. CHX  used only low dosages. Data are mean ± SD from three biological replicates. 1-way ANOVA followed by Dunnett's multiple comparison tests: *P<0.5; **P<0.01; ***P<0.001",
    "molecules": "HLN, CHX, ERY, FSK, GELD, SPL"
  },
  {
    "caption": "B, C  HEK293T cells were treated for 24 h and general  RNA and  protein synthesis were monitored by the incorporation of 5-ethynyl uridine (EU) and O-propargyl-puromycin (OPP), respectively. ActD  (5 μM) and CHX (350 μM) were used for 3 h as positive control to block RNA and  protein synthesis, respectively. Data are from three biological replicates and represented as mean ± SEM. (B) Two-tailed, unpaired t-test; ***P<0.001, **P = 0.0047. Concentration used: 1 μM GELD, 10 μM ERY, SPL and FSK.",
    "molecules": "5-ethynyl uridine, EU, ActD, CHX, ERY, FSK, GELD, O-propargyl-puromycin, OPP, SPL"
  },
  {
    "caption": "G Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, ****P<0.0001. Concentration used: 1 μM GELD, 10 μM SPL and FSK.",
    "molecules": "FSK, GELD, SPL"
  },
  {
    "caption": "C Relative  expression level of Sqstm1/p62, Lc3, Tfeb, Hspb8 and  Bag3  mRNAs in  NSC34 cells treated for 24 h. Data are mean ± SEM from three biological replicates. 2-way ANOVA followed by Dunnett's multiple comparisons test, ***P<0.001. Dotted lines represent RNA expression in the DMSO control. Concentration used: 1 μM  GELD, 10 μM ERY, SPL, FSK.",
    "molecules": "DMSO, ERY, FSK, GELD, SPL"
  },
  {
    "caption": "G Lysates from  NSC34 cells transfected with scrambled-GPx100, treated for 24 h with SPL and/or 10 mM 3MA and  immunoblotted for scrambled-GP expression. Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001 for SPL vs DMSO and 3MA  vs DMSO, ***P = 0.0008 SPL + 3MA vs SPL. Concentration used: 1 μM GELD, 10 μM SPL",
    "molecules": "DMSO, 3MA, SPL"
  },
  {
    "caption": "B Lysates from HEK293T cells, transfected with 2R or 66R and treated for 24 h, immunoblotted  using antibody for  poly-GA expression (HA-tagged). Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001, **P = 0.0027 H89 vs DMSO, ***P = 0.0004 FSK + H89 vs DMSO, *P = 0.0234 CLZ  vs DMSO, *P = 0.0209 FSK  + H89  vs FSK. Concentration used: 2.5 μM H89, 10 μM  FSK, CLZ.",
    "molecules": "CLZ, DMSO, FSK, H89"
  },
  {
    "caption": "F  Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated with H89 for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, *P = 0.0130 WT  H89  vs WT DMSO, ****P<0.0001, CUG mutated  H89 vs CUG mutated  DMSO. Concentration used: 2.5 μM H89, ",
    "molecules": "DMSO, H89"
  },
  {
    "caption": "A, B Representative graphs showing the climbing ability  flies carrying the UAS-(G4C2)x36 construct in  neurons using the  Elav-gal4 promoter upon treatment with H89  10 µM diluted with 0.1 % of DMSO and 5% sucrose in PBS. The wild-type (w1118) line was used as control. (A) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. (B) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment. ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. Experiments were performed three times, N=15 adult flies were used for each genotype, sex and treatment (N tot=45). C, D Representative graphs showing climbing activity of flies co-expressing the UAS-Pka-RNAi construct (1, v330111) in combination with UAS-(G4C2)x36 in neurons using the Elav-gal4 promoter. The wild-type (w1118) line was used as control. (C) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 9th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. (D) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 7th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. Experiments were performed twice with different RNA interfering sequence, N=15 adult flies were used for each genotype and sex (N tot=30). ",
    "molecules": "DMSO, H89, PBS, sucrose"
  },
  {
    "caption": "A, B Three independent C9 patient iPSC-MN lines (Becker S6, M211R2, C90202) were treated with GELD, SPL, FSK, H89 or DMSO vehicle control and levels of poly-GA   (A) and  poly-GP (B) measured after 7 days. Values are displayed as percentages of mean DMSO signal. No significant changes in either DPR were observed (linear mixed effects model, all not significant, P>0.05; P = 0.1 DMSO vs FSK). Treatments were performed in four technical replicates.",
    "molecules": "DMSO, FSK, GELD, H89, SPL"
  },
  {
    "caption": "C, D Three independent C9  patient iPSC-MN  lines were treated with 10 µM H89 and levels of poly-GA (C) and poly-GP (D) were measured after 14 days. Values are displayed as percentages of mean DMSO signal. H89  significantly reduced  poly-GA *P = 0.039 and poly-GP levels *P = 0.018 (linear mixed effects model). Treatments were performed in four technical replicates.",
    "molecules": "DMSO, H89"
  },
  {
    "caption": "E No increased  LDH release was observed after 14-day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC-MN lines (unpaired t-tests, Becker S6: significant decrease in  LDH, **P = 0.0064; M211R2: not significant, P>0.05). In Becker S6, mean LDH  release was 22% lower after H89 treatment compared to vehicle. Treatments were performed in eight technical replicates. Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01.",
    "molecules": "H89"
  },
  {
    "caption": "F Relative fold changes of C9orf72  variant RNA levels normalised to RPL13A, after 14-day 10 µM H89 treatment vs vehicle treatment (N=1 C9 patient  line, four technical replicates). No significant changes were observed between  H89 and vehicle for any of the isoforms (2-way ANOVA with Šídák's multiple comparisons test, all not significant, P>0.05). Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01. T ",
    "molecules": "H89"
  },
  {
    "caption": "(B) Representative whole‐mount images of single spheroids cultured in either 50% conditioned media (CM) or 5% CM+5 μM DAPT and stained for Muc2 (green) to visualize goblet cells. Bars=20 μm.",
    "molecules": "DAPT"
  },
  {
    "caption": "(C, D) Graph of relative expression (as measured by qRT‐PCR) of Atoh1 (C) and Muc2 (D) mRNAs comparing spheroids grown in either 5% CM or 5% CM+ DAPT to spheroids grown in 50% CM (n=4-5 samples/group). Error bars indicate s.e.m. *P0.05; **P0.01 as determined by a two‐tailed Student's t‐test.",
    "molecules": "DAPT"
  },
  {
    "caption": "(E, F) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5% CM+DAPT and stained for Muc2 (green: E) or with the lectin, UEA (red: F). Bars=20 μm.",
    "molecules": "DAPT"
  },
  {
    "caption": "(B) Colonic epithelial monolayers from wild‐type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β (green) and EEA1 (red). Bars=5 μm.",
    "molecules": "Dynasore"
  },
  {
    "caption": "(E) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated. GAPDH is a loading control. Representative image is shown from n=3 experiments.",
    "molecules": "BafA1, Dynasore"
  },
  {
    "caption": "(F) Images of wild‐type colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 100 μM Dynasore. Bars=20 μm.",
    "molecules": "Dynasore"
  },
  {
    "caption": "(D) LC3 immunoblots of either wild‐type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated. Representative image is shown from n=3 experiments.",
    "molecules": "BafA1"
  },
  {
    "caption": "(A) Colonic epithelial cell spheroids from p22phox−/−, Atg5VC and wild‐type mice. Wild‐type spheroids were treated with vehicle (control), 10 μM DPI (NADPH oxidase inhibitor) or 100 μM Dynasore. Intracellular ROS in live cells were labelled by 5 μM DCF. Bars=100 μm.",
    "molecules": "DPI, Dynasore, ROS"
  },
  {
    "caption": "(C) Quantification of average area of cytoplasmic mucin from wild‐type mice treated with 3% NAC as compared to controls (n=6-7 mice/group). Error bars indicate s.e.m. *P0.05; **P0.01; ****P0.0001 as determined by the Student's t‐test.",
    "molecules": "NAC"
  },
  {
    "caption": "(D) TEM images of colonic epithelial monolayers treated with cerium chloride. The presence of electron dense precipitate is indicative of ROS production. Bar=500 nm.",
    "molecules": "cerium chloride, ROS"
  },
  {
    "caption": "Exogeneous ROS can restore mucingranule accumulation defects. (A) Quantification of fluorescent DCF normalized to controls (n=3 independent experiments/group; 10-30 spheroids quantified/culture). *P0.05 as determined by ANOVA with Tukey's multiple post‐test comparisons",
    "molecules": "ROS"
  },
  {
    "caption": "(B) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (C) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). ***P0.001, ****P.0001 as determined by ANOVA with Tukey's multiple post‐test comparisons.",
    "molecules": "H2O2"
  },
  {
    "caption": "(D) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT-PCR. No statistically significant differences were found in either group as determined by ANOVA.",
    "molecules": "H2O2"
  },
  {
    "caption": "(E, G) Images of p22 mut or Dynasore‐treated colonic epithelial spheroids (mucin is labelled with TRITC‐UEA; red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (F, H) Quantification of average mucin area/goblet cell (n=6 sample/group; 30-60 cells were quantified/sample) corresponding to experiments performed using p22 mut (E) and Dynasore‐treated wild‐type spheroids (G)",
    "molecules": "Dynasore, H2O2"
  },
  {
    "caption": "Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA‐AM (I) or PMA/ionomycin (J). (I, J) Quantification of average mucin area/goblet cell (n=5 sample/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ****P0.0001 as determined by the Student's t‐test (I) or ANOVA with Tukey's multiple post‐test comparisons (J).",
    "molecules": "BAPTA‐AM, H2O2, ionomycin, PMA"
  },
  {
    "caption": "a) Auto-ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST-HHARIRBR, T7-Triad1ΔAri, GST-ParkinRBR, and Flag-BRCA1/BARD1 and either UbcH5WT or UbcH5L104Q as the E2. Products were visualized by western blotting against indicated tags on E3s. Times given are post-ATP addition.",
    "molecules": "ATP"
  },
  {
    "caption": "b) Overlay of (1H,15N)-HSQC-TROSY spectra of 15N-UbcH5-O-15N-Ub in the absence (black) and presence (red) of 0.5mol equiv. HHARI RING1. A subset of UbcH5 peaks, but not Ub peaks, shift and broaden upon HHARI RING1 binding.",
    "molecules": "1H, 15N"
  },
  {
    "caption": "a) Histogram of CSPs between free 15N-UbcH7 and 15N-UbcH7-O-Ub identifies UbcH7 residues affected by conjugation to Ub. Active site (Ser86) is indicated with a star and cross-over helix residues (101-113) are marked with a gray cylinder. INSET. CSPs greater than 1 stdv (> 0.115 ppm) are highlighted in yellow on a surface representation of UbcH7 (PDB 1fbv). The view on the left shows the surface that contains the UbcH7 cross-over helix.",
    "molecules": "15N"
  },
  {
    "caption": "b) Region of 1H-15N-HSQC TROSY spectra containing resonances of the cross-over helix residues S107 and Q106 are overlaid: unconjugated 15N-UbcH7 (blue), 15N-UbcH7-O-Ub (black), and 15N-UbcH7-O-UbI44A (red). Peaks representing S107 and Q106 in the context of UbcH7~Ub-I44A (red) are closer to peaks observed in free UbcH7 (blue) than UbcH7~Ub-WT (black) indicating that I44A of Ub disrupts interactions with the cross-over helix of UbcH7.",
    "molecules": "1H, 15N"
  },
  {
    "caption": "a) Region of 1H-15N-HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid: free 15N-Ub (blue), UbcH7-O-15N-Ub (black), and HHARI RING1-bound UbcH7-O-15N-Ub (red). Black arrow highlights perturbation upon conjugation to UbcH7 (black arrow) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7.",
    "molecules": "1H, 15N"
  },
  {
    "caption": "b) Region of 1H-13C-HSQC spectra of 13C-UbcH7 (blue), 13C-UbcH7-O-Ub (black), and HHARI RING1-bound 13C-UbcH7-O-Ub (red) that includes the methyl (13CH3) resonance of the surface-exposed UbcH7 cross-over helix residue, Ala 110 (black arrow) is shown. The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C-UbcH7-O-Ub (black) and reappears at its position in free UbcH7 in the presence of HHARI RING1, consistent with disruption of closed UbcH7~Ub conformations. Pairwise overlays and larger spectra are provided in Appendix Fig S3.",
    "molecules": "1H, 13C"
  },
  {
    "caption": "c) Regions of 1H-15N-HSQC-TROSY spectra of UbcH7-O-15N-Ub in the absence (black) and presence (red) of a RING1 domain from the RBR E3s HHARI (left) or RNF144 (middle) or a canonical RING domain of BRCA1/BARD1 (right). The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar, while binding of the canonical RING domain of BRCA1/BARD1 has no observable effect on the Ub spectrum. Gray boxes mark area expanded in a)",
    "molecules": "1H, 15N"
  },
  {
    "caption": "b) LEFT. Auto-ubiquitination assays were performed with wild-type HHARIRBR or an active site-dead mutant (C357A- HHARIRBR). Products were visualized by western blotting against GST. The zero time point was taken immediately prior to ATP addition, all other times are post-ATP addition. RIGHT. Identical assays as shown on left were performed with the UBR-hybrid. The active site-dead mutant (C357A) retains substantial auto-ubiquitination activity signifying that UbcH5~Ub is able to transfer Ub directly onto the GST-UBR-hybrid construct, bypassing the RING2 active site Cys.",
    "molecules": "ATP"
  },
  {
    "caption": "a) LEFT. E3 auto-ubiquitination assays were performed using various Ub mutants (I44A, R42A, Q49A, Q49E, and V70A) and the RBR E3s HHARIRBR (left) and ParkinRBR (right). Products were visualized by western blotting against HA-Ub. Samples were analyzed 30 minutes after ATP addition.",
    "molecules": "ATP"
  },
  {
    "caption": "a) Top panel. Regions of 1H-15N-HSQC TROSY spectra of 15N-HHARI RING2 in the absence (black) and presence of excess Ub (red). Middle panel. Identical spectral region, but in the presence of Ub-V70A (red). Bottom panel. Identical spectral region, but spectra are from a truncated HHARI RING2 construct that lacks N-terminal residues 325-335 (HHARI RING2-ΔL).",
    "molecules": "1H, 15N"
  },
  {
    "caption": "e) 1H-15N-HSQC TROSY spectra of 15N-Ub demonstrate binding by WT HHARI RING2, but not by mutant HHARI RING2 constructs that exhibit decreased auto-ubiquitination activity. Overlay of 15N-Ub spectra in the absence (black) and presence of WT-HHARI RING2 (red), W336A-HHARI RING2 (blue), T341N-HHARI RING2 (green), or E352A-HHARI RING2 (purple). HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d).",
    "molecules": "1H, 15N"
  },
  {
    "caption": "100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of Ki-67+ cells (A) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.",
    "molecules": "EdU, paclitaxel"
  },
  {
    "caption": "C) 100 nM paclitaxel treatment (24h) significantly (p= <0.0001 [****]) increased the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of 9 HFs from 3 patients.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "D) 100 nM docetaxel treatment (24h) significantly (p = 0.0004 [***]) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8-9 HFs from 3 patients.",
    "molecules": "docetaxel"
  },
  {
    "caption": "E) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20 µm scale. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "docetaxel, paclitaxel"
  },
  {
    "caption": "The presence of micronucleated cells in the hair matrix in paclitaxel (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 12-13 HFs (paclitaxel) from 3 patients.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "The presence of micronucleated cells in the hair matrix in docetaxel treated (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 8 HFs (docetaxel) from 3 patients.",
    "molecules": "docetaxel"
  },
  {
    "caption": "C) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix (lined) and dermal papilla in untreated (vehicle) human HFs. 20 µm scale.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "D) Paclitaxel treatment (100 nM, 24h) induces the formation of micronucleated bodies, as visualised by Hoechst 33342 staining (arrows), localising to the proliferative region of the hair matrix. i - 20 µm scale; ii - 10 µm scale.",
    "molecules": "Hoechst 33342, Paclitaxel"
  },
  {
    "caption": "E) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10 µm scale. Error bars are standard error.",
    "molecules": "docetaxel"
  },
  {
    "caption": "A) Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24h). 20 µm scale.",
    "molecules": "ethynyl uridine, paclitaxel"
  },
  {
    "caption": "B) Quantitative analysis highlights a significant (p =<0.0001 [****]) decrease in the number of EU+ nuclei following 24h paclitaxel treatment. Welch's t-test performed using N of 11-12 hair follicles (HFs) from 3 patients.",
    "molecules": "EU, paclitaxel"
  },
  {
    "caption": "C) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3+ cell population that accumulates in response to paclitaxel treatment 10 µm scale.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "D) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment. 20 µm scale.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "E) 100 nM paclitaxel treatment significantly (p = 0.0016 [exact] [**]) increases the number of cleaved caspase 3+ cells in the hair matrix after 24h. Mann Whitney U test performed using N of 16-18 HFs from 5 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "Ai) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20 µm scale. Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase 3 positivity (white arrow) within the K15+ bulge. 10 µm scale. pbORS - proximal bulb outer root sheath.",
    "molecules": "Paclitaxel"
  },
  {
    "caption": "B) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments 50 µm scale. EC - extended cultures.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "C) Graph showing significantly (p = 0.029 [**]) increased K15/caspase 3 double+ cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "D) K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double+ cells. Unpaired t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "Paclitaxel"
  },
  {
    "caption": "E) Representative double immunofluorescence images of elevated γH2A.X immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "F) γH2A.X analysis showing a significant (p = 0.065 [**]) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann Whitney U test performed using N of 5-6 HFs from 2 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "DNA"
  },
  {
    "caption": "B) Palbociclib-only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki-67 expression in the hair matrix beyond that seen with just 24h treatment Data also confirms that Ki-67 expression is unaffected by paclitaxel treatment Analysis performed using N of 6-9 HFs per condition from 3 patients. Ordinary one-way ANOVA with multiple comparisons performed. Adjusted p values = 0.0001[***] and 0.0001 [****] respectively. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel, Palbociclib, palbociclib"
  },
  {
    "caption": "C) Data confirming that paclitaxel treatment significantly increases (adjusted p value = 0.0001[***]) the number of pH3+ cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre- and co-incubated with palbociclib. Analysis performed using N of 6-9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel, palbociclib"
  },
  {
    "caption": "D) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment (adjusted p value = 0.0056 [**]). Paclitaxel treated hair follicles pre- and co-incubated with palbociclib do not show enhanced cleaved caspase 3 expression, contrasting with paclitaxel-only treatment. Ordinary one-way ANOVA with multiple comparisons performed using N of 4-5 HFs per condition. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel, Paclitaxel, palbociclib"
  },
  {
    "caption": "E) Immunofluorescence data representing how palbociclib and/or paclitaxel affect (i) Ki-67 expression, (ii) pH3 immunoreactivity and (iii) cleaved caspase 3 expression. 20 µm scale.",
    "molecules": "paclitaxel, palbociclib"
  },
  {
    "caption": "B) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib-only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one-way ANOVA with multiple comparisons performed using N of 8-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel, palbociclib"
  },
  {
    "caption": "C) A trending increase in the number of cleaved caspase 3+ cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel-only treatment significantly increased the number of cleaved caspase 3+ cells in the hair matrix (adjusted p value = 0.0003 [***]). Palbociclib-only treatment did not increase the number of cleaved caspase 3+ cells in the hair matrix following drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel, palbociclib, Palbociclib"
  },
  {
    "caption": "D) HFs treated with paclitaxel alone showed a significant (adjusted p value = 0.0316 [*]) increase in the number of pH3+ cells in the matrix after washout. A significant (adjusted p value = 0.0278 [*]) increase in the number pH3+ cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3+ hair matrix keratinocytes after drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.",
    "molecules": "paclitaxel, palbociclib, Palbociclib"
  },
  {
    "caption": "E) Representative fluorescence images of EdU and pH3/cleaved caspase 3 staining in paclitaxel-only and palbociclib plus paclitaxel dual treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent. HS - hair shaft; \"wo\" - washout. 20 µm scale.",
    "molecules": "EdU, paclitaxel, palbociclib"
  },
  {
    "caption": "B) Paclitaxel treatment did not affect the number of EdU+ cells within the HF outer root sheath (ORS) following washout. In contrast, palbociclib treated HFs lacked EdU incorporation following drug washout (adjusted p value = 0.0001 [***]). The number of EdU+ cells was significantly decreased in the outer root sheath (ORS) of HFs dual treated with both paclitaxel and palbociclib (adjusted p value = 0.0021 [**]). Ordinary one-way ANOVA with multiple comparisons performed using N of 12-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error",
    "molecules": "EdU, Paclitaxel, paclitaxel, palbociclib"
  },
  {
    "caption": "C) HFs treated with paclitaxel showed a significant (adjusted p value = 0.0001 [****]) increase in the number of cleaved caspase 3+ cells in the ORS following drug washout. Palbociclib-only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3+ ORS cells. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error",
    "molecules": "paclitaxel, Palbociclib, palbociclib"
  },
  {
    "caption": "D) Paclitaxel-only treated HFs showed a significant (adjusted p value = 0.0001 [****]) increase in the number of pH3+ ORS cells following drug washout. Palbociclib treatment alone had no significant effect on the number of pH3+ ORS cells. There was a trending, yet non-significant, increase in the number of pH3+ ORS cells in HFs dual treated with palbociclib plus paclitaxel. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error",
    "molecules": "Paclitaxel, paclitaxel, Palbociclib, palbociclib"
  },
  {
    "caption": "E) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout, whereby DNA synthesis has not resumed. ; wo - washout. 50 µm scale;",
    "molecules": "paclitaxel, palbociclib"
  },
  {
    "caption": "F) Representative images of pH3/caspase 3 staining within the ORS of paclitaxel-only and palbociclib plus paclitaxel treated HFs following drug washout. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 50 µm scale",
    "molecules": "paclitaxel, palbociclib"
  },
  {
    "caption": "Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of cleaved caspase 3+ (G) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.",
    "molecules": "paclitaxel, palbociclib"
  },
  {
    "caption": "Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of pH3+ (H) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.",
    "molecules": "paclitaxel, palbociclib"
  },
  {
    "caption": "(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.",
    "molecules": "(5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one, DPPT, (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one, HPFPT, 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile, 4BPPNit, Hoechst"
  },
  {
    "caption": "(E) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).",
    "molecules": "4BPPNit"
  },
  {
    "caption": "(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs. Data information: Intensities of glycosylation are normalized to GAPDH protein expression.",
    "molecules": "4BPPNit"
  },
  {
    "caption": "(B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time.",
    "molecules": "4BPPNit, DMSO"
  },
  {
    "caption": "(C) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).",
    "molecules": "DMSO"
  },
  {
    "caption": "(D) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (E) Quantification of laminin-binding activities, compared with DMSO only controls. Data information: Intensities of laminin-binding activity are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).",
    "molecules": "4BPPNit, DMSO"
  },
  {
    "caption": "(F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.Data information: Intensities of protein expression are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).",
    "molecules": "4BPPNit, DMSO"
  },
  {
    "caption": "(A) DAG1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01",
    "molecules": "4BPPNit, DMSO"
  },
  {
    "caption": "(B) FKRP gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).",
    "molecules": "4BPPNit, DMSO"
  },
  {
    "caption": "(C) LARGE1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01). ",
    "molecules": "4BPPNit, DMSO"
  },
  {
    "caption": "(A) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1-/- mouse brain and BMDMs.",
    "molecules": "nucleic acid"
  },
  {
    "caption": "Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (B) HT29 control (henceforth IRGM+/+) and single allele CRISPR knockout IRGM cells (henceforth IRGM+/-),",
    "molecules": "nucleic acid"
  },
  {
    "caption": "Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (C) HT29 cells stably expressing control shRNA or IRGM shRNA,",
    "molecules": "nucleic acid"
  },
  {
    "caption": "Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (D) Irgm1+/+ and Irgm1-/- mouse brain (n=3 mice).",
    "molecules": "nucleic acid"
  },
  {
    "caption": "(O) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 (300 nM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(P) Western blotting analysis with lysates of control and untreated or chloroquine (50 µM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(Q) The qRT-PCR analysis with RNA isolated from untreated or Bafilomycin A1 (300 nM; 4 h, 8 h, and 12 h) treated control or Flag-IRGM expressing HT-29 stable cells as indicated.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(E) Representative confocal images of control siRNA and IRGM siRNA transfected THP-1 cells processed for IF analysis with Ubiquitin (green) and TOM20 (red) antibodies. Scale bar, 10 µm.",
    "molecules": "Ubiquitin"
  },
  {
    "caption": "(H) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(I) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(K) Representative confocal images of control and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye.",
    "molecules": "CMXRos red dye"
  },
  {
    "caption": "(L) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye (10 nM, 30 mins). (M) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP-1 cells stained with CMXRos.",
    "molecules": "CMXRos, CMXRos red dye"
  },
  {
    "caption": "(N) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP-1 cells stained with JC-1 dye (2 µM, 30 mins). At low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence, whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission.",
    "molecules": "JC-1, JC-1 dye"
  },
  {
    "caption": "(P) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with MitoSox red dye (1µM, 20 min). The percentage of control and IRGM knockdown cells with increased red fluorescence (mitochondrial ROS generation) is depicted.",
    "molecules": "MitoSox red, ROS"
  },
  {
    "caption": "(Q) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with CellRox-green and CellRox-orange dye (1µM, 30 min).",
    "molecules": "CellRox"
  },
  {
    "caption": "(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 (red) and dsDNA (green) antibody.",
    "molecules": "dsDNA"
  },
  {
    "caption": "(B) Representative STED microscopy images of control THP-1 cells processed for IF analysis with cGAS (green), dsDNA (red), and Lamin B1 (cyan) antibodies. (C) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP-1 cells.",
    "molecules": "dsDNA"
  },
  {
    "caption": "(D-E) Representative STED microscopy images of Irgm1-/- mouse BMDMs (D) transfected with mCherry-cGAS and immunostained with dsDNA (green) antibody. The white line depicts the periphery of the cells. Scale bar, 3 µm. (E) The graph depicts the percentage of cells with micronuclei in Irgm1+/+ and Irgm1-/- mouse BMDMs.",
    "molecules": "dsDNA"
  },
  {
    "caption": "(F) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs.",
    "molecules": "DNA"
  },
  {
    "caption": "(G) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control and IRGM siRNA transfected HT-29 cells.",
    "molecules": "DNA"
  },
  {
    "caption": "(H) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.",
    "molecules": "DNA, EtBr"
  },
  {
    "caption": "(I) The qRT-PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.",
    "molecules": "EtBr"
  },
  {
    "caption": "(J) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with DNase I (15 μg, 6hr).",
    "molecules": "DNA"
  },
  {
    "caption": "(L-M) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with (L) dsRNA (green) and Tom20 (red) antibodies. (M) The graph depicts the percentage of Irgm1+/+ and Irgm1-/- mouse BMDMs with dsRNA aggregates.",
    "molecules": "dsRNA"
  },
  {
    "caption": "(N) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with RIG-I (red) and dsRNA (green) antibodies. Line profile: Co-localization analysis using line intensity profiles.",
    "molecules": "dsRNA"
  },
  {
    "caption": "Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (O) Mda5 (red) and dsRNA (green) antibodies Co-localization analysis using line intensity profiles.",
    "molecules": "dsRNA"
  },
  {
    "caption": "Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (P) Mda5 (red), dsRNA (green) and Tia1 (blue) antibodies (Scale bar, 3 µm). Line profile: Co-localization analysis using line intensity profiles.",
    "molecules": "dsRNA"
  },
  {
    "caption": "For SPR measurements, IL-27Rα or GP130  were immobilized on the chip surface by biotin-streptavidin interaction  and IL-27 were flowed across the chip in solution. SPR  data are representative of three biological replicates. Kinetic  charts for IL-27Rα (A) and GP130  (B). Concentrations used are shown on the curves. Data traces were fitted using a 1:1 interaction  model (black) to quantify the kinetics  (kon, koff) and binding  affinity (KD) of the interactions.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "For SPR  measurements, IL-27Rα were immobilized on the chip surface by biotin-streptavidin interaction  and p28  were flowed across the chip in solution. SPR  data are representative of three biological replicates. (C, Equilibrium chart for p28 binding   to IL-27Rα (C, left panel) and curve-fitting to data points generated at various concentrations of p28  (C, right panel).",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "For SPR  measurements, GP130  were immobilized on the chip surface by biotin-streptavidin interaction  and p28  were flowed across the chip in solution. SPR  data are representative of three biological replicates. (D) Equilibrium chart for p28 binding GP130.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(B and C) IC50  values for MPA-response curves using cell viability assay were shown. Human AML  cell lines (with MLL-fusions: MV4;11, MOLM13, NOMO1  and THP1, without MLL-fusions: HL60, Kasumi-1, OCI-AML3 and U937), human cord blood (CB) CD34+ cells, and those expressing  MLL-AF9  (CB-MLL-AF9#1, 2, 3) or MLL-ENL  (CB#MLL-ENL-1, 2), and patient  cells with or without MLL-fusions (AML#1, 2, 3 or AML#4, 5, 6) were treated with titrating doses of MPA  (1-100 μM) for 72 hours in three technical replicates. Data information: All data are shown as mean ± SEM.",
    "molecules": "MPA"
  },
  {
    "caption": "(D) Response curves of FF-10501-01 (1-100 μM) for 72 hours in three technical replicates.",
    "molecules": "FF-10501-01"
  },
  {
    "caption": "(E) FCM  plots of the cell cycle and apoptosis  analyses in MLL-AF9  expressing  CB cells and MV4;11 cells. Cells were incubated with/without 1 μM MPA  and 100 mM Guanosine  for 36 hours. Numbers indicate the frequency of S/G2/M-phase cells  (left) and Annexin V+ cells (right).",
    "molecules": "Guanosine, MPA"
  },
  {
    "caption": "(F and G) Immunophenotypes  and Wright-Giemsa staining  of MLL-AF9  expressing CB cells  after four days of culture with/without 3.3 μM MPA  and 100 μM Guanosine  (Scale bar: 20 mm).",
    "molecules": "Guanosine, MPA"
  },
  {
    "caption": "(A) Immunoblotting  of the  cord blood cells  and those expressing RUNX1-ETO  or MLL-AF9. The cells were incubated with/without 1 μM MPA  (M) and 100 μM Guanosine  (G) for 24 h.",
    "molecules": "Guanosine, MPA"
  },
  {
    "caption": "(B) CB-MLL-AF9#2 cells were transduced with a Non-targeting (NT) control and an shRNA targeting p21  (sh-p21). Cells were incubated with/without 1 μM MPA  (M) and 100 μM guanosine  (G) for 24 hours. Total cell lysates were analyzed by western blotting  using the antibodies for p21  and Tubulin.",
    "molecules": "guanosine, MPA"
  },
  {
    "caption": "(C) CB-MLL-AF9#2 cells transduced with NT or sh-p21  shRNAs were incubated with MPA  at the indicated concentration for 72 hours. Cell viability assays  were performed using  WST-1 in three technical replicates.",
    "molecules": "WST-1, MPA"
  },
  {
    "caption": "(D) CB-MLL-AF9#2 cells were transduced by vector control or p53DD (dominant-negative) and were incubated with/without 1 μM MPA  (M) and 100 μM guanosine  (G) for 24 h. Total cell lysates were analyzed by  western blotting  using the antibodies for p53  and Tubulin. Note the elevated levels of endogenous p53  protein (upper arrow) in p53DD (lower arrow)-expressing cells.",
    "molecules": "guanosine, MPA"
  },
  {
    "caption": "(E) Cells were incubated with MPA  at the indicated concentration with/without 100 μM guanosine  for 72 hours then measured with WST-1  in three technical replicates.",
    "molecules": "WST-1, guanosine, MPA"
  },
  {
    "caption": "(F) Mouse bone marrow c-Kit+ cells derived from Trp53-/- mice  were transduced with MLL-AF9-GFP  and were transplanted into mice  to generate p53-deficient (p53-/-) leukemia  cells. GFP+ MLL-AF9 leukemia  cells  were collected from  bone marrows  of vehicle- or  FF-10501-01-treated wild-type and p53-/- leukemic mice. Expression levels of p53  and Tubulin  were assessed by western blotting 24 hours after the treatment.",
    "molecules": "FF-10501-01"
  },
  {
    "caption": "(H) Kaplan-Meier survival curves of p53-/- MLL-AF9 leukemia mice  treated with vehicle or FF-10501-01or  DS-5272 (n = 6 per group). Statistical significance was evaluated by the log-rank test (left). Frequency of GFP+ leukemia  cells in peripheral blood at day 19 (right, n = 6 per group). A two-tailed unpaired t-test was used for the comparison. Data information: All data are shown as mean ± SEM.",
    "molecules": "FF-10501-01, DS-5272"
  },
  {
    "caption": "(B) The TLR-expressing BaκB cells were stimulated with the corresponding TLR ligands (100 ng/mL Pam3CSK4   for TLR2, 100 ng/mL LPS  for TLR4, 10 ng/mL Flagellin      for TLR5, 100 ng/ml R848  for TLR7) with titrating doses of MPA  (1 - 10 μM). GFP  expression was assessed 24 hours after the stimulation.",
    "molecules": "Flagellin, LPS, MPA, Pam3CSK4, R848"
  },
  {
    "caption": "(C) 293T  cells were transfected with FLAG-tagged TRAF6  and HA-tagged K63-ubiquitin. After 24 hours, these cells were treated with 3 or 10 μM MPA  (M-3 μM or M-10 μM) with/without 100 μM guanosine  (G). IRAK1/4 inhibitor (IRAK1/4 i) was also used as a control. Whole-cell extracts were immunoprecipitated  with anti-FLAG  antibody, and ubiquitinated TRAF6  was detected with anti-HA  antibody.",
    "molecules": "guanosine, MPA"
  },
  {
    "caption": "(D, Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA  alone (D) N=3 (technical replicates) per group. mRNA levels of TNF-α and  IL-1β were measured by qPCR. Two-tailed unpaired t-tests were used for the comparison in (D). Data information: All data are shown as mean ± SEM.",
    "molecules": "MPA"
  },
  {
    "caption": "E) Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA  alone or co-treated with vehicle, 10 μM MPA  (M), 100 ng/mL Pam3CSK4  and 100 μM Guanosine  (G) for 4 hours (E), as indicated. N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β  were measured by qPCR. Ordinary one-way ANOVA was used for the comparison in (E). Data information: All data are shown as mean ± SEM.",
    "molecules": "Guanosine, MPA, Pam3CSK4"
  },
  {
    "caption": "(F) THP1  cells were pretreated with 10 μM MPA  for 2 hours and then treated with 1000 ng/mL Pam3CSK4  for 0, 30, 60, and (120) minutes. Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38, and GAPDH  were evaluated by western blotting.",
    "molecules": "phosphate, MPA, Pam3CSK4"
  },
  {
    "caption": "(G) MLL-AF9-expressing CB  cells (CB-MLL-AF9#1) were pretreated with 10 μM MPA  for 2 hours and then treated with 1000 ng/mL Pam3CSK4  for 0, 30, 60 Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38  were evaluated by western blotting.",
    "molecules": "phosphate, MPA, Pam3CSK4"
  },
  {
    "caption": " Fluorescence intensity of the FRET donor (GFP) is shown before and after photobleaching of the FRET acceptor (mCherry) in ImageJ Smart pseudocolor scheme (for intensity values see bars on the right). FRET is observed as an increase of donor fluorescence after acceptor photobleaching. The indicated GFP- and mCherry-tagged endocytic proteins localize at the endocytic patches on the plasma membrane of live yeast cells. The enrichment of individual endocytic patches (diameter of 50-80 nm) on the plasma membrane, caused by the latA-induced block of their subsequent invagination and/or their higher abundance at incipient buds, resulted in their apparent clustering on the diffraction-limited fluorescence images (pixel size, 178 nm). All raw acquisitions of GFP fluorescence intensities before and after photobleaching are shown ",
    "molecules": "latA"
  },
  {
    "caption": " Ede1, Apl1, Yap1801, Gts1, Bzz1, Lsb3, Las17, and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of LatA-treated cells and fluorescence recovery was followed every 0.5 s for 60 s (40 s in case of the highly dynamic Bzz1 protein). The curves represent means ± 95% confidence intervals (n = 9-17). ",
    "molecules": "LatA"
  },
  {
    "caption": "(B) Example images of brightfield and fluorescently labelled HPG (Alexa Fluor 647) of wild type cells (PN1) assayed for global cellular translation. Scale bars represent 5 µm. (C) Change in HPG labelling signal with different durations of HPG incubation (in PN1), measured by flow cytometry. Population medians of at least 200,000 cells are shown. The red line is the ordinary least square (OLS) linear regression fitted on the medians between 0 and 5 minutes. (",
    "molecules": "Alexa Fluor 647, HPG"
  },
  {
    "caption": "(E) Example images of brightfield and fluorescently labelled EU (Alexa Fluor 488) of hENT1 and hsvTK cells (PN10567) assayed for global cellular transcription. Scale bars represent 5 µm. (F) Change in EU labelling signal with different lengths of EU incubations, measured by flow cytometry (in PN10597). The mean and standard deviation (SD) of the population medians of at least 200,000 cells in experimental triplicates are shown in black. The dark green line is the OLS linear regression fitted on the mean data between 2 and 12 minutes.",
    "molecules": "EU, Alexa Fluor 488"
  },
  {
    "caption": "(D) For each of the 5 length bins boxed in (C), both 1C and 2C medians are normalised to their respective 1C global cellular translation values. The normalised values are represented as dots, each dot corresponding to one of the 5 length bins, the mean and SD of the normalised values are shown for each DNA content. For each DNA content, the normalised values (dots) are in the same order (left to right) as their corresponding length bins in (C). The p-value is calculated using a Welch's unequal variances t-test.",
    "molecules": "DNA"
  },
  {
    "caption": "(E) SH-SY5Y  cells were infected with  HSV-2 (MOI=1) for the indicated time  intervals, and cell lysates were immunoblotted  for p-MLKL  and vinculin. For comparison, we used lysates from THP1  cells treated with  Z-VAD  (Z, 20 μM), TNFα  (T, 100 ng/ml), Birinapant (B, 100 nM), and Necrostain-1 (NEC-1, 10 μM) as indicated.",
    "molecules": "Birinapant, NEC-1, Necrostain-1, Z-VAD"
  },
  {
    "caption": "(B) SH-SY5Y  cells were pre-treated with CASP2  inhibitor  Z-VDVAD  (20 μM) for 1 h and infected with HSV-2  (MOI=1). Lysates were isolated 16 hpi for immunoblot  analysis.",
    "molecules": "Z-VDVAD"
  },
  {
    "caption": "SH-SY5Y cells were treated with ethidium bromide (2 μg/ml) and infected with HSV-2  (MOI=1, 16 h). Lysate were for immunoblotting  for cell death markers GSDME",
    "molecules": "ethidium bromide"
  },
  {
    "caption": "SH-SY5Y  cells were treated with  ethidium bromide (2 μg/ml) and infected with  HSV-2 (MOI=1, 16 h). Lysate  and supernatant  were for immunoblotting  for cell death markers HMGB1",
    "molecules": "ethidium bromide"
  },
  {
    "caption": "SH-SY5Y cells were treated with ethidium bromide (2 μg/ml) and infected with HSV-2 (MOI=1, 16 h). Lysate were for immunoblotting  for cell death markers GSDME SH-SY5Y cells were transfected with  Poly I:C (50 μg/ml) or dsDNA  (20 μg/ml) for 24 h (J).",
    "molecules": "dsDNA, ethidium bromide, Poly I:C"
  },
  {
    "caption": "SH-SY5Y cells were pretreated with TUDCA  (500 ng/ml) for 1 h and infected with HSV-2 (MOI=1). Lysates were isolated after 16 h of  HSV-2 infection and subjected to immunoblotting  for CL-CASP3, CL-PARP, GSDME",
    "molecules": "TUDCA"
  },
  {
    "caption": "SH-SY5Y cells were pretreated with TUDCA  (500 ng/ml) for 1 h and infected with HSV-2 (MOI=1). supernatants (24 h) were analysed for LDH  release (H, ELISA). Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001 two-way ANOVA in H).",
    "molecules": "TUDCA"
  },
  {
    "caption": "G) SH-SY5Y cells were pretreated with APY29  for 1 h and following HSV-2  infection (16 h). Lysate were immunoblotted  for p-JNK.",
    "molecules": "APY29"
  },
  {
    "caption": "(H) SH-SY5Y  cells were pretreated with SP600125  (10 μM) for 1 h infected with HSV-2 (MOI=1, 16 h). Lysates  and supernatant  were immunoblotted  for GSDME,  cytochrome c, HMGB1, and vinculin.",
    "molecules": "SP600125"
  },
  {
    "caption": "B. Quantification of PLAs  between BRCA2  and DPP9  in control HeLa  WT cells treated with non-targeting siRNA (siNT) or silenced with the indicated oligos. 300 ng / mL MMC  was added for 24 h. Each dot represents the number of PLA  events in a single cell, from two to seven biological replicates (siNT-MMC  (n = 6), siNT+MMC  (n = 7), siDPP9-MMC  (n = 4), siDPP9+MMC (n = 5), siBRCA2-MMC  (n = 2), siBRCA2+MMC  (n = 3), siFLNA-MMC  (n = 5), siFLNA+MMC  (n = 6), NgtCntrl-MMC  (n = 3), NgtCntrl+MMC  (n = 6)). The number of foci are shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, **** = p ≤ 0.0001). C. Representative PLA  images showing close proximity between endogenous DPP9  and endogenous BRCA2  in HeLa WT cells. Exposure of cells to MMC  triggers more PLA  events (white). Phalloidin  (green) stains actin filaments, DAPI  (blue) stains  the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071",
    "molecules": "DAPI, MMC, Phalloidin"
  },
  {
    "caption": "D. Quantification of PLAs  between BRCA2  and DPP9  in asynchronous HeLa  WT cells or in cells in  S-Phase. Each dot represents the number of PLA  events in a single cell, from three biological replicates. Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS  (250 ng / mL for 30 min). Cells were sampled prior to NCS  treatment, after NCS  treatment and with 1.5 h and 3 h of recovery after NCS  treatment. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001). E. Representative PLA  images showing close proximity between endogenous DPP9  and endogenous BRCA2  in HeLa  cells. Exposure of cells to NCS  triggers more PLA  events (white), which increase in time. Cells in  S-Phase show a greater fold-increase in the number of PLA  events in comparison to asynchronous cells. Phalloidin  (green) stains actin filaments, DAPI  (blue) stains  the nucleus. Scale bar 10 µm. Antibodies as in (C).",
    "molecules": "DAPI, NCS, Phalloidin, thymidine"
  },
  {
    "caption": "F.  Co-immunoprecipitation assays showing binding  of BRCA2  and DPP9-SWT. HEK293 DPP9  KO + DPP9WT cells, were treated with 1µg/mL Dox  (24 h) to induce the expression of DPP9-FLAG. DNA damage was induced with 300 nM MMC  treatment for 24h. Control cells do not express DPP9  (- Dox). Bound proteins were eluted with a FLAG  peptide and analysed by Western Blotting (anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-FLAG  RRID:AB_262044).",
    "molecules": "Dox, MMC"
  },
  {
    "caption": "B.  Pull-down  assay showing direct binding  of purified recombinant DPP9  to a BRCA2  N-terminal1-40 fragment immobilized on HA-beads. The DPP9  inhibitors 1G244  or SLRFLYEG  compete with BRCA21-40HA for interaction  with DPP9. Representative data of three technical replicates is shown. Anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.",
    "molecules": "1G244, SLRFLYEG"
  },
  {
    "caption": "C. Quantification of PLAs  showing fewer MMC-induced DPP9-BRCA2 PLA  events in HeLa  cells treated with 10µM 1G244. Each dot represents the number of PLA  events in a single cell, from three biological replicates. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001). D. Representative images of DPP9-BRCA2 PLA  in the presence of 1G244  - a competitive inhibitor of DPP9. Control cells were mock treated with DMSO. Phalloidin  (green) stains actin filaments, DAPI  (blue) stains  the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071",
    "molecules": "1G244, DAPI, DMSO, MMC, Phalloidin"
  },
  {
    "caption": "A-B. Representative  Western Blots  and accompanying graph from more than three biological replicates show that MMC  (300 nM, 24 h) induces a rapid turnover  of endogenous BRCA2  in HeLa  WT cells, which is less pronounced in HeLa  WT cells treated with the proteasome  inhibitor MG132  (100µM), in HeLa DPP9  KD cells and in cells transiently silenced for DPP9  (siDPP9). RAD51  stability is not altered in HeLa DPP9  KD cells. Vinculin  is a loading control for BRCA2, Tubulin  is a loading control for RAD51. Shown images originate from one representative cycloheximide  (CHX) chase assay. The ratios of BRCA2  to Vinculin  and RAD51 to Tubulin  are defined as 100% at time  0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test. (*= p ≤ 0.03). Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-Vinculin: RRID:AB_477629, anti-Tubulin: RRID:AB_628412, anti-RAD51: RRID:AB_1142428.",
    "molecules": "CHX, cycloheximide, MMC, MG132"
  },
  {
    "caption": "C-D. Representative  Western Blots and graph summarizing results of more than three biological replicates. CHX  chase assays show that the MMC-induced degradation   of BRCA2  is less pronounced in HEK293 DPP9  KO cells as well as in HEK293 DPP9  KO + DPP9-SS729A cells overexpressing an inactive DPP9-S mutant. Expression of DPP9  was induced (+ Dox, 1µg/ml) simultaneously with MMC  (300 nM), 24 h. Cells that were overexpressing the active variant (DPP9-SWT) show similar levels of MMC-induced BRCA2 degradation. RAD51 stability  is not altered by MMC  and is similar in all cell lines. Vinculin  is a loading control for BRCA2, Tubulin  is a loading control for RAD51. Shown images originate from one representative cycloheximide  (CHX) chase assay. The ratios of BRCA2  to Vinculin  are defined as 100% at time  0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03, **= p ≤ 0.002). Antibodies as described in (A-B).",
    "molecules": "CHX, cycloheximide, Dox, MMC"
  },
  {
    "caption": "F-G. Representative Western Blots  and graphs summarizing CHX  assays from three biological replicates showing that the BRCA2∆MP3-1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21-1000. Cells were co-transfected with a GFP  expressing plasmid, as transfection and loading control. Control cells were transfected with GFP  only (U.T). Image originates from one representative experiment. BRCA2-FLAG  signals are related to the transfection and loading control GFP. BRCA2  levels in relation to GFP  were defined as 100 % at time  0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03). Anti-FLAG: RRID:AB_262044, anti-GFP: RRID:AB_641123.",
    "molecules": "CHX"
  },
  {
    "caption": "A. Representative immunofluorescence  images of γH2AX  signals (white, RRID:AB_309864) in gki MEF DPP9S729A, showing more γH2AX  in gki MEF DPP9S729A cells following removal of Neocarzinostatin  (NCS), quantification in (B). MEF DPP9S729A and control MEF  cells were treated with 250 ng/mL NCS  for 30 min and allowed to recover for the indicated time-points. γH2AX  signals at time  0, reflect 30 min of NCS  and no recovery time. Nuclei  are shown in blue (DAPI). Scale bar 10 µm. B. Quantification of γH2AX  in gki MEF DPP9S729A and control MEF  cells as described in (A). Signals from more than 1700 cells were quantified per condition per experiment. Mean ± SEM from six biological replicates, each in technical duplicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01).",
    "molecules": "DAPI, NCS, Neocarzinostatin"
  },
  {
    "caption": "C. Representative PLA  images of BRCA2-PALB2 PLA  experiments showing more PLA  events (white) in HeLa  cells silenced for DPP9  (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin  (green) stains actin filaments, DAPI  (blue) stains  the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "E. Representative PLA  images of γH2Ax-BRCA2 PLA  experiment showing a reduction in the number of PLA  events (white) between γH2Ax  and BRCA2  in HeLa DPP9  KD cells. Phalloidin  (green) stains actin filaments, DAPI  (blue) stains  the nucleus. Scale bar 10 µm. Anti-γH2Ax: RRID:AB_2118009, anti-BRCA2: RRID:AB_2259370 F. Quantification of PLAs  showing fewer MMC-induced γH2Ax-BRCA2 PLA  events in HeLa DPP9  KD cells, in comparison to HeLa  WT cells. Each dot represents the number of PLA  events in a single cell, from three biological replicates. The technical control samples (NgtCntrl) omitted the γH2Ax  antibody. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).",
    "molecules": "DAPI, MMC, Phalloidin"
  },
  {
    "caption": "A. Representative immunofluorescence  images showing that re-expression of DPP9-SWT leads to an increase in the number of RAD51  foci formed following exposure to MMC  in HEK293 DPP9  KO + DPP9-SWT cells. Expression of DPP9  was induced (+ Dox, 1µg/ml) simultaneously with MMC  (300 nM), 24 h. RAD51  foci are shown in white, nuclei  (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428. B. Graph showing the number of RAD51  foci following induction of DPP9-SWT expression, compared to uninduced HEK293 DPP9  KO + DPP9WT  cells (-Dox). Induction of HEK293 DPP9  KO + DPP9S729A for expression of DPP9-SS729A did not result in more RAD51  foci. Each dot represents the number of RAD51  foci in a single cell, from three biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).",
    "molecules": "DAPI, Dox, MMC"
  },
  {
    "caption": "Representative images (C, and summarizing graph (D showing the number of RAD51  foci per nucleus  in HeLa  WT cells (C, D) Where stated, cells were treated with control siRNA (siNT) or silenced for BRCA2, and transiently transfected with the BRCA21‑3418 or BRCA23-3418 constructs. Both BRCA2  constructs can rescue the RAD51  foci formation phenotype to the control levels in HeLa  WT cells (C, D). Each dot represents the number of RAD51  foci in a single cell, from three (D) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51  foci are shown in white, nuclei  (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; ; anti-BRCA2: RRID:AB_2259370.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative images , E) and summarizing graph F) showing the number of RAD51  foci per nucleus  in HeLa DPP9  KD cells (E, F). In HeLa DPP9  KD cells significantly more RAD51  foci were present in cells transfected with BRCA2∆MP3-3418 compared to BRCA2  silenced cells and cells expressing the untruncated BRCA21-3418 (E, F). Each dot represents the number of RAD51  foci in a single cell, from four (F) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51  foci are shown in white, nuclei  (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; anti-BRCA2: RRID:AB_2259370.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Bar graph representing the normalized average value of the neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with anisomycin (25μM) and on glutamate stimulation with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. **p<0.01, ***p<0.001, ****p<0.0001, n=5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.",
    "molecules": "anisomycin, ADP, ATP, cycloheximide, glutamate"
  },
  {
    "caption": "(C) Bar graph representing the normalized average value of neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with MPEP, on glutamate stimulation with CNQX + D-AP5 and on glutamate stimulation with CNQX + D-AP5 + anisomycin. Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment.. *p<0.05, ***p<0.001, n=4-5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.",
    "molecules": "anisomycin, MPEP, CNQX, ADP, ATP, D-AP5, glutamate"
  },
  {
    "caption": "(F) Representative voltage-clamp traces of miniature EPSCs recorded (Vhold= -70mV) from a neuron in presence of TTX.",
    "molecules": "TTX"
  },
  {
    "caption": "(J) Bar graph representing the normalized average value of synaptic ATP level on the basal condition, on DHPG treatment for 5 minutes, on DHPG treatment with MPEP, on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. *p<0.05, n=8 animals.",
    "molecules": "anisomycin, MPEP, DHPG, ATP, cycloheximide"
  },
  {
    "caption": "(K) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition, on NMDA treatment for 5 minutes and on NMDA treatment with D-AP5. Data presented as mean +/- SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. n=8 independent platings.",
    "molecules": "ATP, D-AP5, NMDA"
  },
  {
    "caption": "(B) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in somatic compartments on the bath application of DHPG and NMDA. Scale bar 10μm. (C) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in dendritic compartments on the bath application of DHPG and NMDA. Scale bar 10μm.",
    "molecules": "PercevalHR, DHPG, ADP, ATP, NMDA"
  },
  {
    "caption": "(D) Average traces depicting the time course of the normalized somatic ATP/ADP ratio on the basal conditions, on DHPG treatment, and on NMDA treatment. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 5-6 cells from ≥3 independent platings.",
    "molecules": "DHPG, ADP, ATP, NMDA"
  },
  {
    "caption": "(E) Average traces depicting the time course of normalized dendritic ATP/ADP ratio on the basal condition, on the basal condition in the presence of anisomycin, on DHPG treatment, on NMDA treatment, on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 6-9 cells from ≥3 independent platings.",
    "molecules": "anisomycin, DHPG, ADP, ATP, NMDA"
  },
  {
    "caption": "(G) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, *p<0.05, **p<0.01, ****p<0.0001 for comparison with 0 minute. ##p<0.01 and ####p<0.0001 for comparison between DHPG + anisomycin. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 5 animals per group.",
    "molecules": "anisomycin, DHPG, ATP"
  },
  {
    "caption": "(H) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, **p<0.01 for comparison with 0 minutes. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 4 animals per group.",
    "molecules": "anisomycin, ATP, NMDA"
  },
  {
    "caption": "(C) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons at various time points following DHPG and NMDA treatment. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(D) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments. Data presented as mean +/- SEM. Data points in all groups were normalized to the average of the basal group. ***p<0.001 n= 21-54 neurons per group from 3 independent platings.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(E) Representative images showing phospho-eEF2 immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(F) Box plot showing phospho-eEF2 normalized intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=31-49 cells per group from 3 independent platings.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(G) Representative immunoblots describing changes in the phospho-eEF2 and total-eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes. Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho/total undefined ratio.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(H) Line graph showing the average value of the synaptic phospho/total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery. Data presented as mean +/- SEM, **p<0.01. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. ***p<0.001, ****p<0.0001, n≥ 5 animals per group.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(A) Representative images showing phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(B) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=30-46 cells per group from 3 independent platings.",
    "molecules": "DHPG, NMDA"
  },
  {
    "caption": "(C) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total ratio of AMPK.",
    "molecules": "anisomycin, DHPG"
  },
  {
    "caption": "(D) Line graph showing the normalized average value of the synaptic phospho/total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. *p<0.05, **p<0.01, ****p<0.0001, n= 5 animals per group.",
    "molecules": "anisomycin, DHPG"
  },
  {
    "caption": "(E) Representative immunoblots depicting the changes in the α-SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin.",
    "molecules": "anisomycin, DHPG"
  },
  {
    "caption": "(F) Line graph showing the normalized average value of the synaptic α-SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. *p<0.05. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, n≥ 4 animals per group.",
    "molecules": "anisomycin, DHPG"
  },
  {
    "caption": "(G) Representative images showing the α-SNAP (cyan) and AMPK (magenta) immunolabeling in low-density cortical neurons. Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes. Scale bar 10μm. Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions. Scale bar 5μm.",
    "molecules": "DHPG"
  },
  {
    "caption": "(H) Box plot depicting the quantification of co-localization through Pearson's correlation coefficient between α-SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.01, n≥35 cells per group from 4 independent platings. Unpaired-sample t-test.",
    "molecules": "DHPG"
  },
  {
    "caption": "(I) Representative images showing the phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α-SNAP siRNA. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.",
    "molecules": "DHPG"
  },
  {
    "caption": "(J) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA, on DHPG treatment for 5 minutes in the presence of scrambled siRNA, on the basal condition in the presence of α-SNAP siRNA, on DHPG treatment for 5 minutes in the presence of α-SNAP siRNA. Data points in all groups were normalized to the average of the scrambled siRNA basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, n=29-35 cells per group from 3 independent platings.",
    "molecules": "DHPG"
  },
  {
    "caption": "(A) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2+. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total AMPK ratio.",
    "molecules": "Ca2+, NMDA"
  },
  {
    "caption": "(B) Line graph showing the normalized average value for the synaptic phospho/total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2+. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, ****p<0.0001, n= 6 animals per group.",
    "molecules": "Ca2+, NMDA"
  },
  {
    "caption": "(C) Representative images depicting intracellular Ca2+ levels through Fluo8 fluorescence (Pseudo-colored) of a cortical neuron plated at low-density before stimulation, 15 sec (immediate) after NMDA treatment, 300 sec (delayed) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2+. Scale bar 20μm.",
    "molecules": "Fluo8, Ca2+, ionomycin, NMDA"
  },
  {
    "caption": "(D) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2+ and D-AP5. Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point. Ionomycin treatment in the presence of 10mM Ca2+ was used to calculate fluorescence maximum for a particular cell.",
    "molecules": "Fluo8, Ca2+, D-AP5, Ionomycin, NMDA"
  },
  {
    "caption": "(E) Box plot showing the distribution of ΔF/F0 across multiple neurons on the basal condition, on NMDA treatment in the presence Ca2+, on NMDA treatment in the absence of Ca2+, on NMDA treatment with D-AP5 and on NMDA treatment along with MK-801. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n≥30 neurons per group from 3 independent platings.",
    "molecules": "Ca2+, D-AP5, MK-801, NMDA"
  },
  {
    "caption": "(B) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (pseudo-colored) in cortical neurons on the basal condition and on DHPG (50μM) treatment for 5 minutes in the presence or absence of AICAR (1mM). MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.",
    "molecules": "DHPG, AICAR"
  },
  {
    "caption": "(C) Box plot showing the normalized phospho-eEF2 intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=17-46 cells per group from 3 independent platings.",
    "molecules": "DHPG, AICAR"
  },
  {
    "caption": "(D) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n=18-57 cells per group from 3 independent platings.",
    "molecules": "DHPG, AICAR"
  },
  {
    "caption": "(E) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (Pseudo-colored) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.",
    "molecules": "Compound C, NMDA"
  },
  {
    "caption": "(F) Box plot showing phospho-eEF2 intensity distribution across multiple neurons on basal condition (Vehicle control), on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=21-36 cells per group from 3 independent platings.",
    "molecules": "Compound C, NMDA"
  },
  {
    "caption": "(G) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=24-52 cells per group from 3 independent platings.",
    "molecules": "Compound C, NMDA"
  },
  {
    "caption": " (A) Fractionation of nuclear and cytosolic compartments using a standard NP-40 lysis protocol and immunoblotting with chaperone specific antibodies ",
    "molecules": "NP-40"
  },
  {
    "caption": "(B) Histone chaperone immunoprecipitations separated by 15% SDS-PAGE and stained with Coomassie. Major bands that could be confidently identified by mass spectrometry are indicated. Due to the high percentage of acrylamide used, many of the higher molecular weight bands are not resolved. Asterisks represent partial degradation. Background-corrected densitometry profiles are shown for the portions of the gels covering H3 and H4. Gel filtration purified recombinant H3-H4 dimer is shown on the right",
    "molecules": "Coomassie, acrylamide"
  },
  {
    "caption": "A Yeast two-hybrid (Y2H) assays showing the interaction between SEU and SCR. The yeast transformants were plated on synthetic defined (SD) media lacking Leu and Trp (SD/-2) or lacking Ade, His, Leu, and Trp (SD/-4) to assess protein-protein interactions. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain.",
    "molecules": "Ade, His, Leu, Trp"
  },
  {
    "caption": "B In vitro pull-down assays showing that SEU directly interacts with SCR but not with SHR. SCR-FLAG was pulled down by SEU-MBP immobilized on amylose resin. Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti-FLAG antibody. The asterisk indicates the position of SEU-MBP.",
    "molecules": "amylose"
  },
  {
    "caption": "B. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm.",
    "molecules": "DAPI, FITC, U0126"
  },
  {
    "caption": "F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm.",
    "molecules": "milrinone"
  },
  {
    "caption": "C. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm. D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group).",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm. F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).",
    "molecules": "hCG"
  },
  {
    "caption": "A. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12). B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17). C",
    "molecules": "ATP, DMSO, U0126"
  },
  {
    "caption": "C. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. E",
    "molecules": "JC-1, DMSO, U0126"
  },
  {
    "caption": "E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.",
    "molecules": "JC-1"
  },
  {
    "caption": "F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.",
    "molecules": "JC-1, Hoechst 33342"
  },
  {
    "caption": "(E) BDR2637 (Pveg-mCherry) (WT, purple) and ET191 (PrrnE-gfp, ∆sigX) (∆sigX, cyan) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from mCherry (purple) and GFP (cyan) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm. Corresponds to Movie EV4.",
    "molecules": "agarose"
  },
  {
    "caption": "(F) ET27 (PsigX-gfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluorescence from GFP (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.",
    "molecules": "agarose"
  },
  {
    "caption": "(D) BDR2637 (Pveg-mCherry) (WT, purple) and ET261 (∆yueB, PsigX-sigX-gfp) (cyan) cells were mixed, infected with SPP1 at 5:1 (phages:bacteria) MOI, placed on an agarose pad and followed by time lapse fluorescence microscopy. Shown are overlay images from mCherry (purple), SigX-GFP (cyan), and phase contrast (grey), captured at the indicated time points post infection. Scale bar 1 μm.",
    "molecules": "agarose"
  },
  {
    "caption": "(B) ET29 (Pveg-mCherry, PIPTG-sigX) (purple) cells were grown in the presence of IPTG and mixed with PY79 (WT) cells. The mixture was infected with SPP1-lysin-yfp 5:1 (phages:bacteria) MOI, placed on an IPTG-containing agarose pad, and followed by time lapse fluorescence microscopy. Shown are overlay images of phase contrast (grey), signal from mCherry labeled cells (purple), and signal from Lysin-SPP1-YFP (cyan), captured at the indicated time points post infection (upper panels). Corresponding signal from Lysin-SPP1-YFP (cyan) is shown separately (lower panels). Arrows highlight the delayed infection of ET29 cells. Scale bar 1 μm.",
    "molecules": "agarose, IPTG, lysin, Lysin"
  },
  {
    "caption": "(E) OF83 (Ppen-lacIΔ11-cfp) (WT) and ET40 (Ppen-lacIΔ11-cfp, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1-delX110lacO64 at 5:1 (phages:bacteria) MOI. The formation of LacI-CFP foci on phage DNA was monitored 10 min post infection. Non-infected OF83 cells were used for comparison. Shown are overlay images of phase contrast (grey) and signal from LacI-CFP (cyan). Scale bar 1 µm. (F) Quantification of the experiment described in (E). Shown is the percentage of LacI-CFP foci 10 min post infection of OF83 and ET40 cells by SPP1, with average values and SD (n ≥ 850 cells for each population).",
    "molecules": "IPTG"
  },
  {
    "caption": "(A) ET28 (PIPTG-sigX) and ET42 (∆dltA, PIPTG-sigX) cells, grown in the presence or absence of IPTG, were infected with SPP1 at 1:20 (phages:bacteria) MOI, and OD600nm was followed at 2 min intervals. Shown is a representative experiment out of 6 biological repeats, with the average values and SD of 8 technical repeats.",
    "molecules": "IPTG"
  },
  {
    "caption": "(F) AR16 (PrrnE-gfp) (WT, cyan) and ET411 (Pveg-mCherry, ∆dltA) (∆dltA, purple) cells were mixed, infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are overlay images from GFP (cyan) and mCherry (purple) signals of the bacterial lawn captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 150 µm.",
    "molecules": "agarose"
  },
  {
    "caption": "(G) ET43 (dltA-yfp) cells were infected with low concentrations (10-8 PFU/ml) of SPP1, placed on an agarose pad, and plaque formation was followed by time lapse confocal microscopy. Shown are fluoresce from DltA-YFP signal (upper panels) and corresponding phase contrast images (lower panels), captured at the indicated time points (hrs). The plaque is seen as a hole formed on the bacterial lawn. Scale bar 100 µm.",
    "molecules": "agarose"
  },
  {
    "caption": "(A) Relaxed DNA (3 nM), condensin (0, 60, 120, 240 nM) and Topo I (1 unit) were mixed and incubated with or without ATP (1 mM) at 30ºC for 30 min. DNA electrophoresis contained 0.1 µg/ml chloroquine. In all gels, N denotes nicked circles; Lkº, the input Lk distribution of relaxed DNA; and LkC, the Lk distribution restrained by condensin. (B) 2D-gel of the preceding samples in lane 1 (relaxed DNA, R), and lanes 5 and 6 (condensin 120 nM ± ATP). Electrophoresis contained 0.1 and 1 µg/ml chloroquine in the first and second dimension, respectively. The histogram shows the relative intensity (i) of individual topoisomers of the Lk distributions resolved in lane 1 (green) and lane 6 (red). Lkº and LkC denote the midpoint of each Lk distribution; and ∆Lk, the difference (Lk units) between them. (C) Experiment conducted as in A, but reducing the concentration of DNA (0.3 nM) and condensin (0, 1.5, 3, 6 nM). DNA electrophoresis contained 0.4 µg/ml chloroquine. (D) 2D-gel of the preceding sample in lane 1(relaxed DNA, R), and lanes 5 and 6 (condensin 3 nM ± ATP). Electrophoresis contained 0.4 and 1 µg/ml chloroquine in the first and second dimension, respectively. The histogram shows relative Lk intensities (i) of lanes 1 and 6, indicating Lkº, LkC and ∆Lk.",
    "molecules": "Relaxed DNA, relaxed DNA, ATP, chloroquine, DNA"
  },
  {
    "caption": "(E) Experiment conducted as in A, but mixing DNA (1 nM) with intermediate condensin concentrations (10 to 100 nM). DNA electrophoresis contained 0.2 µg/ml chloroquine. The length of DNA (bp) available per condensin complex in each reaction is indicated.",
    "molecules": "chloroquine, DNA"
  },
  {
    "caption": "(A) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were incubated at 30ºC for indicated time periods (0 to 90 min) in the presence of different concentrations of ATP (1, 0.25, 0.1 mM).",
    "molecules": "Relaxed DNA, ATP"
  },
  {
    "caption": "(C) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with ATP 1mM for 10 min (lane 2), AMPPNP 2 mM for 10 min (lane 3), and AMPPNP 2mM for 10 min followed by ATP 1 mM for 10 min (lane 4). (D) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with Apyrase for 10 min (lane 2), Apyrase and ATP 1mM for 10 min (lane 3), ATP 1mM for 10 min (lane 4), no nucleotide for 10 min followed by Apyrase for 60 min (lane 5), ATP 1mM for 10 min followed by Apyrase for 60 min (lane 6).",
    "molecules": "AMPPNP, ATP, DNA, nucleotide"
  },
  {
    "caption": "(E) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 20 min (lane 1), with ATP 1mM for 20 min (lane 2), AMPPNP 2 mM for 20 min (lane 3), ATP 1mM for 10 min followed by AMPPNP 2 mM for 10 min (lane 4). (F) 2D-gel of the samples in E (lanes 1 to 4) and histogram of Lk intensities (i) of lanes 1 and 4, indicating Lkº, LkC and ∆Lk.",
    "molecules": "AMPPNP, ATP, DNA"
  },
  {
    "caption": "(D) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I were mixed with/without nuclease P1 and incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min.",
    "molecules": "Relaxed DNA, AMPPNP, ATP, nucleotide"
  },
  {
    "caption": "(E) Experiment conducted as in D, but using (-) supercoiled DNA and without Topo I. Following the incubation with nucleotides for 30 min, nuclease P1 was added for 5 min.",
    "molecules": "supercoiled DNA, nucleotides"
  },
  {
    "caption": "(A) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were mixed with ss-oligos or ds-oligos (100 and 500 nM). Following incubation for 10 min at 30ºC, ATP (1 mM) was added and incubations continued for 10 min. (B) Experiment as in A, but first adding ATP for 10 min and afterwards the oligonucleotides for 10 min. (C) Experiment as in B, but after the 10 min incubation with oligonucleotides, AMPPNP (2 mM) was added and incubations continued for 10 min.",
    "molecules": "ds-oligos, Relaxed DNA, AMPPNP, ATP, oligonucleotides, ss-oligos"
  },
  {
    "caption": "(D) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were incubated at 30ºC for 10 min. Reactions were then split into thirds to which NaCl concentration was raised to 50, 150 and 300 mM. Salt-resistant condensin-DNA complexes were immobilized to His-Tag magnetic beads and the fractions of free (F) and bound (B) DNA recovered. The plot shows the fractions of bound DNA (mean ±SD, 3 technical replicates). (E) As in D, but containing AMPPNP (2 mM). (F) As in D, but containing ATP (1 mM). (G) As in D, but containing ATP (1 mM) for 10 min followed by AMPPNP (2 mM) for 10 min. (H) As in D, but containing ATP (1 mM) for 10 min followed by Apyrase for 10 min.",
    "molecules": "Relaxed DNA, Apyrase, AMPPNP, ATP, DNA, NaCl"
  },
  {
    "caption": "(B) Relaxed DNA (0.3 nM) and condensin (3 nM) were mixed with Topo I or Topo II (1 unit). Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 30 min. (C) DNA, condensin, and Topo II were mixed as in B. Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 10 min. Topo I was added to one half of the mixtures and incubations continued for 10 min. (D) DNA, condensin, and Topo II were mixed as in B. Following incubation at 30ºC for 10 min with or without ATP, DNA was recovered and nicked to reveal the occurrence of DNA knots. K3 to K6 denote knots with 3 to 6 irreducible DNA crossings.",
    "molecules": "Relaxed DNA, ATP, DNA"
  },
  {
    "caption": "(F) Nicked DNA (0.3 nM) was mixed without or with condensin (3 nM) in the presence of ATP (1 mM). Following incubations at 30ºC for 20 min, mixtures were supplemented with T4 DNA Ligase and Topo I (as indicated) and incubation continued for 10 min.",
    "molecules": "Nicked DNA, ATP"
  },
  {
    "caption": "(B) Relaxed DNA (0.3 nM) was mixed with condensin (3 nM). Following incubation in absence or presence of the indicated nucleotides (ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min), TopA was added to one half of each reaction and Topo I to the other half. Incubations continued for 10 min.",
    "molecules": "Relaxed DNA, AMPPNP, ATP"
  },
  {
    "caption": "(D) Experiment conducted as in B but starting with (-) supercoiled DNA instead of relaxed DNA. Following incubation in presence of the indicated nucleotides (as in B) Topo I was added to one half of each mixture and incubations continued for 10 min. (E) Experiment conducted as in D but adding TopA instead of Topo I.",
    "molecules": "relaxed DNA, supercoiled DNA"
  },
  {
    "caption": "(A) Relaxed DNA (0.3 nM), ∆Ycg1 condensin (3 nM) and Topo I were incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min. The plot (mean ±SD, 3 technical replicates) compares the ∆Lk values restrained by the condensin holo-complex and the ∆Ycg1 condensin.",
    "molecules": "Relaxed DNA, AMPPNP, ATP, nucleotide"
  },
  {
    "caption": "(B) Salt resistance of the ∆Ycg1 condensin-DNA complex. Experiments were conducted as in Fig 4D-H. The plot (mean ±SD, 3 technical replicates) compares the fractions of DNA bound to the holo-complex and to the ∆Ycg1 tetramer.",
    "molecules": "DNA"
  },
  {
    "caption": "E, F H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days. The anchorage-independent growth was assessed by a soft agar colony formation assay. The number of colonies was scored, and the data are presented as the relative colony formation ability.",
    "molecules": "agar"
  },
  {
    "caption": "E Western blot analysis of TMPRSS2 expression in cells treated with CH223191 at 10 μM for 48 h. The relative expression level of TMPRSS2 was quantified by normalized with β-actin and are shown at the right panel. : The data shown represent the mean ± SD of three independent experiments. *p < 0.05 and **p< 0.01, and ***p < 0.001, compared with untreated cells. #p < 0.05, compared with PM2.5 treated cells.",
    "molecules": "CH223191"
  },
  {
    "caption": "H1975 cells were infected with sh-TMPRSS2 (sh-TMPRSS2-1 and sh-TMPRSS2-2) or empty vector (Vector). The stable clones of TMPRSS2 knockdown cells were analyzed and their ability to perform anchorage-independent growth in soft agar (C). The data shown in (C) represent the means ± SDs from three independent experiments. (C) p values were determined by one-sample t-test.",
    "molecules": "agar"
  },
  {
    "caption": "(D) Representative images of EdU (red) and DAPI (blue) staining at 5 dpa. (E) The graph represents the mean +/- SD of EdU+ cells over the total number of cells in the spinal cord of 5 to 12 tadpoles. Each data point represents a tadpole, with an average of 9 sections analysed per animal. ",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "(G) Representative images of EdU (green), BrdU (magenta), Sox3 (white) and DAPI (blue) staining at 3 dpa. (H) Quantification of images in (G). The graph represents the mean +/- SD of 6 tadpoles with an average of 13 sections per tadpole analysed. ",
    "molecules": "BrdU, EdU, DAPI"
  },
  {
    "caption": "(C) Representative sections labelled with an anti-PCNA antibody (red) and DAPI (blue) at the indicated day after amputation (dpa). The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1, G2 and M phases. The right panels correspond to the inset indicated as a white box in the middle panels.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Sox3 (green), EdU (magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "(B) Sections of spinal cords at 5 dpa in the stump (non-regen.) or in the regenerate (regen) labelled with anti-Sox3 (green), anti-Acetylated Tubulin (AcTub, magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Myt1 (green), EdU (magenta) and DAPI (blue). (F) Quantification of images shown in (E). The graph represents the mean +/- SD from 3 tadpoles with an average of 12 sections each. ",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "(A) Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild type (WT) 293T cells were treated with 1μM of Thapsigargin (Tg) for different time and the cell lysates were prepared and analyzed by Western-blot.",
    "molecules": "oligomers, Tg, Thapsigargin"
  },
  {
    "caption": "(C) In vitro liposome fragmentation assay. After the injection of recombinant proteins (100μg/100μl) into the chamber (500μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. "ns" means no significance, one-way ANOVA, error bars indicate SEM (n=3).",
    "molecules": "liposome, liposomes"
  },
  {
    "caption": "(D) Quantification of the time between protein addition and liposome fragmentation. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).",
    "molecules": "liposome"
  },
  {
    "caption": "(E, F) Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84-233) or EGFP-FAM134B (Δ84-233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For Each group, at least 30 cells were counted. Scale bars, 10 μm. ***P<0.001, "ns" means no significance, one-way ANOVA, error bars indicate SEM.",
    "molecules": "Baf A1, Bafilomycin A1, DMSO"
  },
  {
    "caption": "(A) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151.",
    "molecules": "serine"
  },
  {
    "caption": "(D) Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).",
    "molecules": "liposome"
  },
  {
    "caption": "(F, G) Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells was engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA) or EGFP-FAM134B (SD) at endogenous levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n=25); For EGFP-FAM134B (WT), n=27; for EGFP-FAM134B (SA), n=29; for EGFP-FAM134B (SD), n=25. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.",
    "molecules": "Baf A1, Bafilomycin A1"
  },
  {
    "caption": "(A, B) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry-FAM134B and BAP31 upon Tg treatment. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. The colocalization was analyzed by Pearson's correlation coefficient (PCC) in (B). For Ctrl, 16 cells were counted(n=16); for Tg treatment, 18 cells were counted (n=18). ***P<0.001, one-way ANOVA, error bars indicate SEM.",
    "molecules": "Tg"
  },
  {
    "caption": "(D) FAM134B S151 phosphorylation in cells treated with CAMK2 activator (100nM EB1089 for 1h) or/and inhibitor (10μM KN93 for 2h). 293T or SKN-SH (a cell line derived from neuroblastoma) cells were treated with drugs as indicated, whole cell lysates were analyzed by phospho-FAM134B (S151) antibody.",
    "molecules": "KN93, EB1089"
  },
  {
    "caption": "(F) In vitro reconstitution of CAMK2B-FAM134B-mediated membrane fragmentation using liposome assay. Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30°C for 10min, the resultant protein mixtures were transferred to chamber coated with liposomes. Liposome fragmentation was monitored by live imaging. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).",
    "molecules": "Liposome, liposomes, ADP, ATP"
  },
  {
    "caption": "(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT) were treated with compounds as indicated. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (H). For vehicle control, 53 cells were counted (n=53), for Ionomycin, n=56; for EB1089, n=54; for KN-93, n=58. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.",
    "molecules": "Ionomycin, KN-93, EB1089"
  },
  {
    "caption": "(I) FAM134B WT or knockout (KO) U2OS cells were treated with CAMK2 activator Ionomycin (Iono), EB1089 or inhibitor KN93 for 24 hours, Western-blot was performed to analyze the proteins as indicated.",
    "molecules": "Iono, Ionomycin, KN93, EB1089"
  },
  {
    "caption": "(J) CAMK2B WT or knockdown (KD) U2OS cells were treated with 1 μM Thapsigargin (Tg) to induce ER stress for different time as indicated. Cells were collected and analyzed for proteins as indicated by Western blot.",
    "molecules": "Tg, Thapsigargin"
  },
  {
    "caption": "(A) Comparison of the self-interaction of FAM134B WT with mutants as indicated using co-IP. SA-G216R was constructed by simultaneously mutating S149, S151 and S153 to alanine (SA) on the basis of FAM134B-G216R.",
    "molecules": "alanine"
  },
  {
    "caption": "(B) In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).",
    "molecules": "liposome"
  },
  {
    "caption": "(C, D) Measurement of the intracellular ER-scission activity of FAM134B WT, G216R, S151A-G216R and SA-G216R. GFP-tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolyosomal degradation of ER fragments labelled by GFP-FAM134B were blocked by Baf A1 or DMSO. GFP-positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.",
    "molecules": "Baf A1, DMSO"
  },
  {
    "caption": "Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP-FAM134B WT (B), G216R(C), S151A-G216R(D), SA-G216R(E), G216R-ΔLIR (F) respectively. Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 (G) Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.",
    "molecules": "DAPI, KN93"
  },
  {
    "caption": " A Western blot analysis of LC3 in SH-SY5Y cells after IU1 treatment (100 μM, 24h). Graphs represent mean± SEM. N= 8 ",
    "molecules": "IU1"
  },
  {
    "caption": " B-D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control (DMSO) or IU1 treated cells. At least 55 cells were analysed from 3 different experiments. Graphs represent mean± SEM ",
    "molecules": "IU1, DMSO"
  },
  {
    "caption": " E Western blot analysis of mitochondrial marker proteins ATP5a, HSP60 and normalised by actin after 24h of DMSO (C) or IU1 (100 μM) treatment. N=3. Graphs represent mean± SEM ",
    "molecules": "IU1, DMSO"
  },
  {
    "caption": " F-I Confocal images of cells transfected with mito-YFP and treated with DMSO (F, control) or IU1 (G) for 24h. Fragmentation index (H) and total mitochondrial volume / cell were measured and represented in (I), mean ±SEM. At least 35 cells were analysed from 3 different experimental conditions. Scale bar 10 μm ",
    "molecules": "IU1, DMSO"
  },
  {
    "caption": " J-L Mito-YFP transfected cells were treated with IU1 (100 μM, 24h), fixed and immuno labelled for LC3. Number of LC3 dots / cell (K) and LC3 dot overlapping mitochondria (L) were counted. At least 35 cells from 3 independent experiments were analysed. Scale bar: 10 μm ",
    "molecules": "IU1"
  },
  {
    "caption": " M-N Cells were transfected with mito-keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis, counting 10,000 cells per experiment. (M) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm (false colour). (N) represents the average intensity of the mito-keima protein when excited at 560 nm and the ratio between 560: 405 nm. Graphs represent mean ± SEM from 3 different sets of experiments",
    "molecules": "IU1, DMSO"
  },
  {
    "caption": " A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test ",
    "molecules": "IU1"
  },
  {
    "caption": " C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated",
    "molecules": "IU1"
  },
  {
    "caption": " E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test; ",
    "molecules": "IU1"
  },
  {
    "caption": " A-D Wild type MEF (A), Hela (B) and PINK1 KO MEF (C) cells were treated with IU1 (48h, 100 μM) and electron microscope images were evaluated for autophagic vesicle formation. (D) bar graphs represents mean± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments; at least 40 cells were analysed from each group",
    "molecules": "IU1"
  },
  {
    "caption": " E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type. Bar graphs represent mean ± SEM. N= at least 3 independent experiments. Student's t test. *P ≤ 0.05, **P ≤ 0.01 ",
    "molecules": "IU1"
  },
  {
    "caption": " A-B SH-SY5Y cells were immunostained for ATP5a (green) and 20S proteasome core (red) after IU1 treatment (6h-24h). Scale bar 10 μm. Colour mapping of the co-localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel. Intensity of the co-localisation is represented in the colour map scale at the bottom of the panel. (B) 20S proteasome subunit and mitochondria co-localisation was quantified by measuring Mander's coefficient. Bar graphs represent mean fold change (mean ± SEM) from 3 different experiments. At least 30 cells were evaluated for the calculations. ANOVA followed by Dunnet's test ",
    "molecules": "IU1"
  },
  {
    "caption": " C Electron microscopy images showing subcellular localization of immuno- gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment. Experiments were repeated twice in two biological replicates with similar results. The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals ",
    "molecules": "IU1"
  },
  {
    "caption": " D-E Protein proximity ligation assay for Prohibitin2 (PHB2) and LC3 after 24h IU1 treatment. Images showing signal are merged with bright field images to count the signal dots (cyan false colour) as depicted in the figure. In the inset only fluorescence signal is shown, without merging with the bright field from the selected regions. Scale bar 10 μm. (E) Bar graphs represent mean ±SEM of the number of signal dots per cell from 3 different experiments. Student's t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 ",
    "molecules": "IU1"
  },
  {
    "caption": "Electron microscope images of mitochondria from SH-SY5Y e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted",
    "molecules": "IU1"
  },
  {
    "caption": "Electron microscope images of mitochondria fro MEF WT, MEF PINK1 K as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted",
    "molecules": "IU1"
  },
  {
    "caption": "Electron microscope images of mitochondria fro Hela an e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted",
    "molecules": "IU1"
  },
  {
    "caption": "Electron microscope images of mitochondria fro Drosophila thoracic muscle, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments For flies, more than 200 mitochondria from each group were counted from 5 different experiments",
    "molecules": "IU1"
  },
  {
    "caption": " D Dopamine content in the fly heads (15 day old) was measured by HPLC and the individual fold change values are represented in the scatter graph. Graphs represent mean± SEM. N= 14, 11 and 11 fly heads for control, PINK1 and PINK1+USP14 KD fly respectively. ANOVA followed by Newman Keuls test ",
    "molecules": "Dopamine"
  },
  {
    "caption": "C Example images of TIRFM endpoint colocalization assays. Top panels show CEN DNA (Top-left panel, blue circles) or CDEIIIMUT CEN DNA (top-right panel, blue circles) visualized in lysates containing Ndc10-mCherry. Middle panels show the visualized Ndc10-mCherry on CEN DNA (middle-left panel) or CDEIIIMUT DNA (middle-right panel) with colocalization shown in relation to blue DNA circles. Bottom panels show overlay of DNA channel (magenta) with Ndc10-mCherry (yellow) on CEN DNA (bottom-left panel) or CDEIIIMUT DNA (bottom-right panel). Scale bars 3μm. Graph shows the quantification of Ndc10 endpoint colocalization on CEN DNA and on CDEIIIMUT CEN DNA (70 ± 7.6%, 1.6 ± 0.3% respectively, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).",
    "molecules": "DNA"
  },
  {
    "caption": "E Example images of TIRFM endpoint colocalization imaging. Top panels show CEN DNA (top-left panel, blue circles) or CDEIIIMUT CEN DNA (top-middle panel, blue circles) visualized in lysates that included Cse4CENP‑A-GFP or CEN DNA in lysates that lacked Scm3HJURP (scm3-AID) (top-right panel, blue circles). Middle panels show Cse4CENP‑A-GFP visualized on CEN DNA (middle-left panel) or CDEIIIMUT CEN DNA (center panel) or on CEN DNA in lysates lacking Scm3HJURP (scm3-AID) (middle-right panel) with colocalization shown in relation to blue DNA circles. Bottom panels show overlay of CEN DNA channel (magenta) with Cse4CENP‑A-GFP (green) on CEN DNA (bottom-left panel) or CDEIIIMUT DNA (bottom-middle panel) or on CEN DNA in lysates lacking Scm3HJURP (scm3-AID) (bottom-right panel). Scale bars 3μm. Graph shows quantification of observed colocalization of Cse4 on CEN DNA and on CDEIIIMUT CEN DNA or on CEN DNA in lysates that lacked Scm3HJURP (47 ± 2.9%, 3.5 ± 3.0% and 0.6 ± 0.4% respectively, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).",
    "molecules": "DNA"
  },
  {
    "caption": "B Estimated survival function plots of Kaplan-Meier analysis of CEN DNA residence lifetimes of Ndc10 (magenta - median lifetime of 102 s, n=3231 over 3 experiments of ~1000 DNA molecules using different extracts) and Cse4CENP‑A (red - median lifetime of 79 s, n=1049 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference between ternary Ndc10 residence lifetime survival plots (***) compared to Cse4CENP‑A (two-tailed p-value of 0 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.",
    "molecules": "DNA"
  },
  {
    "caption": "E Kaplan-Meier analysis of TernaryNdc10 residence lifetimes of Cse4CENP‑A on CEN DNA (purple - median lifetime of 88 s, n=539 over 3 experiments of ~1000 DNA molecules using different extracts) and of Non-TernaryNdc10 Cse4CENP‑A residence lifetimes (teal - median lifetime of 69 s, n=612 over 3 experiments of ~1000 DNA molecules using different extracts). There is a significant difference (**) between TernaryNdc10 and Non-TernaryNdc10 lifetime survival plots (two-tailed p-value of 7.4e-4 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.",
    "molecules": "DNA"
  },
  {
    "caption": "F Longer residence lifetimes are measured for Cse4CENP‑A after Scm3HJURP colocalizes. Estimated survival function plots of Kaplan-Meier analysis of the lifetimes of TernaryScm3 Cse4CENP‑A residences on CEN DNA (purple - median lifetime of 108 s, n=305 over 3 experiments of ~1000 DNA molecules using different extracts) and Non-TernaryScm3 Cse4CENP‑A residences on CEN DNA (green - of 79 s, n=1230 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference (**) between TernaryScm3 and Non-TernaryScm3 lifetime survival plots (two-tailed p-value of 2.1e-3 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.",
    "molecules": "DNA"
  },
  {
    "caption": "B Kinetochores were assembled on beads containing either a 250 bp single-tether, a 250 bp double-tether or a 250 bp CDEIIImut CEN DNA. They were then treated with MNase, and the remaining DNA was visualized on an agarose gel. Black arrow indicates Cse4CENP‑A nucleosome protected DNA (~150 bp); white arrow indicates theoretical location of undigested template DNA (250 bp).",
    "molecules": "agarose, DNA"
  },
  {
    "caption": "C Example images of TIRFM endpoint colocalization assays. Top panels show Cse4CENP‑A/Ndc10 ternary colocalizations visualized on single-tethered CEN DNA (top-left panel) or on double-tethered CEN DNA (top-right panel) with colocalization shown in relation to Ndc10 in yellow circles. Bottom panels show overlay of Ndc10 channel (magenta) with Cse4CENP‑A (green). Scale bars 3 μm. D Quantification of observed ternary colocalization of Cse4CENP‑A with Ndc10 (right) on single-tethered CEN DNA containing Ndc10 (19.9± 2.4%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts) and on double-tethered CEN DNA (7.3 ± 1.1%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).",
    "molecules": "DNA"
  },
  {
    "caption": "F Residence times for Cse4CENP‑A on double-tethered CEN DNAs are shorter than on single-tethered CEN DNAs and equivalent to those on non-functional mutant CEN DNAs. Estimated survival function plots of Kaplan-Meier analysis of ternary residences of Cse4CENP‑A with Ndc10 on single-tethered CEN DNA (blue - median lifetime of 79 s, n=675 over 3 experiments of ~1000 DNA molecules using different extracts), on double-tethered CEN DNA (red - median lifetime of 67 sec, n=3175 over 3 experiments of ~1000 DNA molecules using different extracts) or Cse4CENP‑A lifetimes on CDEIII-80 bp CEN DNA (purple - median lifetime of 71 sec, n=901 over 3 experiments of ~1000 DNA molecules using different extracts). No significant difference (n.s.) between double-tethered 250 bp CEN DNA and 80 bp CDEIII CEN DNA lifetime survival plots (two-tailed p-value of 0.06 as determined by log-rank test). Significant difference (***) between double-tethered 250 bp CEN DNA and 250 bp single-tether DNA lifetime survival plots (two-tailed p-value of 1.4e-10 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.",
    "molecules": "DNA"
  },
  {
    "caption": "C Example images of TIRFM endpoint colocalization assays. Top panels show visualized Cse4CENP‑A-GFP on CEN DNA in extracts from chl4-K13S (top-left panel) or auxin-treated okp1-AID strains (okp1-AID, top-right panel) with colocalization shown in relation to identified CEN DNA in blue circles. Bottom panels show overlay of CEN DNA channel (magenta) with Cse4CENP‑A-GFP (green), Scale bars 3 μm. D Quantification of Cse4CENP‑A endpoint colocalization with CEN DNA in extracts from WT, chl4-K13S, or okp1-AID genetic backgrounds (19 ± 1.1%, 8 ± 0.7%, 5 ± 0.9%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).",
    "molecules": "auxin, DNA"
  },
  {
    "caption": "F Kaplan-Meier analysis of Cse4CENP‑A residence lifetimes on CEN DNA in extracts from WT (blue - median lifetime of 82 s, n=1419 over 3 experiments of ~1000 DNA molecules using different extracts), chl4-K13S (red - median lifetime of 70 s, n=546 over 3 experiments of ~1000 DNA molecules using different extracts) and okp1-AID (purple - median lifetime of 61 s, n=348 over 3 experiments of ~1000 DNA molecules using different extracts) genetic backgrounds. Significant difference (***) between WT extract and chl4-K13S extract residence lifetime plots (two-tailed p-value of 3.4e-5 as determined by log-rank test). No significant difference (n.s.) between chl4-K13S and okp1-AID residence lifetimes in (two-tailed p-value of .40 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.",
    "molecules": "DNA"
  },
  {
    "caption": "B Example images of TIRFM endpoint colocalization assays. Visualized Cse4CENP‑A-GFP on unstable1 CDEII-mutant DNA (top panel) or on v-unstable1 CDEII-mutant DNA (bottom panel) with colocalization shown in relation to identified CEN DNA in blue circles. Scale bars 3 μm. C Quantification of endpoint colocalization of Cse4CENP‑A on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (19.3 ± 1.1%, 13.8 ± 0.6%, 7.8 ± 0.5%, 5.5 ± 1.0%, 5.5 ± 1.1%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).",
    "molecules": "DNA"
  },
  {
    "caption": "E Very unstable CDEII mutants have reduced average Cse4CENP‑A binding when compared to unstable counterparts. Average residences of Cse4CENP‑A per CEN DNA (left) on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (1.03 ± 0.12, 0.88 ± 0.22, 0.58 ± 0.41, 0.61 ± 0.18, 0.41 ± 0.09, avg ± s.e.m. n=3 experiments of ~1000 DNA molecules using different extracts) and average residences of Scm3HJURP per CEN DNA (right) on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (0.57 ± 0.07, 0.35 ± 0.10, 0.52 ± 0.16, 0.53 ± 0.12, 0.46 ± 0.02, avg ± s.e.m. n=3 experiments of ~1000 DNA molecules using different extracts).",
    "molecules": "DNA"
  },
  {
    "caption": "B. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with EdU for 15min, permeabilized, fixed and stained for EdU  incorporation (green). S phase sub-stages from I to IV were evaluated by visual inspection of the cycling population (>300  EdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of each sub-stage from I to IV (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates.",
    "molecules": "EdU"
  },
  {
    "caption": "G. MEFs cells were transfected with GFP-REV3L761-1029 WT PSVVL or ASAVA mutant and fixed with 4% formaldehyde. The distribution of GFP-REV3L constructs was detected by autofluorescence (green), chromocenters  were visualized by HP1α immunostaining (red) and DNA was  counterstained with DAPI. Scale bar= 10 μm",
    "molecules": "DAPI, formaldehyde"
  },
  {
    "caption": "B. Asynchronous Rev3l+/+ and Rev3l-/- MEFs  were fixed with PFA then subjected to in situ proximity ligation assay  (PLA) using 53BP1  and HP1α antibodies then PLA foci were counted in both cell lines (more than 150 nuclei  for each condition were counted). p-values were calculated by Mann-Whitney test ( ****: p<0.0001). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Experiments were repeated three times. Representative images are shown. Scale bar= 5 μm.",
    "molecules": "PFA"
  },
  {
    "caption": "Rev3l+/+ and Rev3l-/- MEFs were incubated with or without 0.23 μM aphidicolin for 24h before metaphase spreading. FISH  was performed using major satellite probe to quantify breaks in pericentromeric regions (F). Representative images of chromosomes showing abnormalities (see arrows) in pericentromeric regions from Rev3l-/- MEFs. Chromosomes were labelled with  DAPI  Error bars indicate standard error of the mean for three independent experiments. Mann-Whitney test (ns: not significant, * p<0.05; *** p< 0.001).",
    "molecules": "aphidicolin, DAPI"
  },
  {
    "caption": "Asynchronous Rev3l+/+ and Rev3l-/- MEFs were pulse labelled for 15 min using 10 μM  EdU then UV-irradiated  at 4 J/m2. Cells pre-treated with CSK buffer were fixed and 53BP1, RPA  and RAD51 were detected by immunofluorescence. Representative images in non-irradiated  cells (NI) and quantification of 53BP1  foci in  EdU positive cells at different time points  are shown Scale bar= 10 μm The  number  of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats). Bars represent the median ± interquartile range",
    "molecules": "EdU"
  },
  {
    "caption": "F-H. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or  REV3L  were processed as in (C). Quantification of 53BP1 foci in  EdU positive cells at different time points is shown (F). Quantification of the intensity of chromatin-bound RPA2 (G) and  RAD51  foci (H) 24 hours after UV-irradiation  was performed. The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats. Bars represent the median ± interquartile range.",
    "molecules": "EdU"
  },
  {
    "caption": "Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF) or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 µmol/L, 15 min); n=6 independent experiments (2-way ANOVA and Bonferroni).",
    "molecules": "ionomycin, Nitrite"
  },
  {
    "caption": "The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF or YD eNOS; n=6 independent experiments (1-way ANOVA and Newman-Keuls).",
    "molecules": "FAD, FMN"
  },
  {
    "caption": "S-nitrosation of PKM2 in human endothelial cells expressing FLAG-tagged WT or YF eNOS and treated with H2O2 (30 µmol/L) for 15 min; n=8 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) was included to demonstrate specificity of the S-NO signal.",
    "molecules": "Dithiothreitol, DTT, H2O2, S-NO"
  },
  {
    "caption": "PKM2 S-nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG-tagged WT PKM2 or a C358S (CS) PKM2 mutant. SNO-FLAG-PKM2 was detected with an anti-FLAG antibody after biotin switch technique; n=4 independent experiments (2-way ANOVA & Bonferroni). DTT treatment was included to demonstrate specificity of the SNO signal.",
    "molecules": "DTT, biotin, SNO"
  },
  {
    "caption": "PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate (PEP) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS. The data were fit with the Michaelis-Menten equation to determine Vmax and Km; n=4 independent experiments (2-way ANOVA & Bonferroni).",
    "molecules": "PEP, phosphoenolpyruvate"
  },
  {
    "caption": "Quantification of pentose phosphate pathway (PPP) intermediates; gluconate-6-P (G6P), ribulose-5-P (Rl5P), ribose-5-P (R5P), sedoheptulose-7-P (S7P), fructose-6-P (F6P) and erythrose-4-P (E4P) in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).",
    "molecules": "G6P, gluconate-6-P, E4P, erythrose-4-P, F6P, fructose-6-P, pentose phosphate, R5P, ribose-5-P, ribulose-5-P, Rl5P, S7P, sedoheptulose-7-P"
  },
  {
    "caption": "NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).",
    "molecules": "GSH, GSSG, NADP, NADPH"
  },
  {
    "caption": "NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells treated with solvent (Sol) or the PKM2 inhibitor shikonin (SKN, 1 µmol/L) for 45 minutes; n=4 independent cell batches (Unpaired Student's t test).",
    "molecules": "GSH, GSSG, NADP, NADPH, shikonin, SKN"
  },
  {
    "caption": "Phenylephrine (PE)-induced contraction of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).",
    "molecules": "L-NAME, LN, PE, Phenylephrine"
  },
  {
    "caption": "acetylcholine (Ach)-induced relaxation (C) of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).",
    "molecules": "acetylcholine, Ach, L-NAME, LN"
  },
  {
    "caption": "Acetylcholine (ACh)-induced vasodilatation of the buffer-perfused hindlimb in situ. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=12-13 animals per group (2-way ANOVA and Bonferroni).",
    "molecules": "Acetylcholine, ACh, L-NAME, LN"
  },
  {
    "caption": "PKM2 S-nitrosation in pulmonary endothelial cells from WT and YF mice; n=5-6 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent bands cropped from the same membranes.",
    "molecules": "Dithiothreitol, DTT, SNO"
  },
  {
    "caption": "Representative images of eNOS-PKM2 interaction (PLA foci, red) in mouse pulmonary endothelial cells; the Golgi apparatus and endosomes were stained with Golph4 (green), the plasma membrane was stained with CD144 and nuclei were highlighted with DAPI (gray). Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs, demonstrating the specificity of the reaction; n=3 independent cell batches. Scale bars: 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Phosphorylation of eNOS on Tyr656 in lung lysates from animals treated for 7 days, n=6-7 mice per group (Unpaired Student's t test).",
    "molecules": "AII, angiotensin II, Tyr"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Systolic blood pressure (SBP) in WT and YF mice treated for 28 days, n=10 mice per group (Unpaired Student's t test).",
    "molecules": "AII, angiotensin II"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Acetylcholine (ACh)-induced relaxation of aortic rings from the same animals as in panel B, n=8 mice per group (2-way ANOVA and Bonferroni).",
    "molecules": "Acetylcholine, ACh, AII, angiotensin II"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. PKM2 S-nitrosation in lungs from WT and YF mice administered AII for 28 days, n=7-8 mice per group (Unpaired Student's t test). DTT treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent lanes from the same membranes.",
    "molecules": "DTT, AII, angiotensin II, SNO"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in lungs from WT and YF mice treated with AII for 28 days; n=8-9 mice per group (Unpaired Student's t test).",
    "molecules": "GSH, GSSG, AII, angiotensin II, NADP, NADPH"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. 3-Nitrotyrosine (3-NT) levels in lungs from WT and YF mice treated with vehicle (Veh) or AII (AII) for 28 days; n=7-9 mice per group (2-way ANOVA and Bonferroni).",
    "molecules": "3-Nitrotyrosine, 3-NT, AII, angiotensin II"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in pulmonary endothelial cells from WT and YF mice treated with AII for 30 min; n=8 cell batches (2-way ANOVA and Tukey).",
    "molecules": "GSH, GSSG, AII, angiotensin II, NADP, NADPH"
  },
  {
    "caption": "Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. H2O2 levels in pulmonary endothelial cells from WT and YF mice after treatment with AII (1 µmol/L) for 30 min; n=8 cell batches (2-way ANOVA and Tukey).",
    "molecules": "AII, angiotensin II, H2O2"
  },
  {
    "caption": "ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Proximity ligation assays showing S-nitrosated PKM2 (yellow) in the RCA and LCA 2 days after ligation. Phalloidin staining (blue) was included to highlight the vessel wall; n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.",
    "molecules": "cholesterol, Phalloidin"
  },
  {
    "caption": "ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.",
    "molecules": "GSH, cholesterol"
  },
  {
    "caption": "ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-nitrotyrosine (3NT, green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.",
    "molecules": "3-nitrotyrosine, 3NT, cholesterol"
  },
  {
    "caption": "ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted LCA 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni). Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted aorta 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni).",
    "molecules": "Acetylcholine, ACh, cholesterol, phenylephrine"
  },
  {
    "caption": "ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.",
    "molecules": "GSH, cholesterol, shikonin, SKN"
  },
  {
    "caption": "ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-Nitrotyrosine (3NT, green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.",
    "molecules": "3-Nitrotyrosine, 3NT, cholesterol, shikonin, SKN"
  },
  {
    "caption": "ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. ICAM1 and VCAM1 (green) expression in the LCA 2 days after ligation. CD31 (red) was included to label endothelial cells and nuclei are highlighted with DAPI (gray). Comparable images were obtained in 4 additional animals in each group. Scale bars: 50 µm.",
    "molecules": "cholesterol, DAPI"
  },
  {
    "caption": "ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. Percentage of neutrophils and monocytes infiltrated into the LCA 7 days after ligation and high cholesterol diet feeding; n=9-10 mice per group (Unpaired Student's t test).",
    "molecules": "cholesterol"
  },
  {
    "caption": "Representative images and quantification of ICAM1 (green) and VCAM1 (red) staining in human endothelial cells pre-treated with the PKM2 inhibitor shikonin (SKN, 1 µmol/L) or solvent (Sol) for 45 minutes and then stimulated with interleukin-1β (IL-1, 20 ng/ml) or solvent (CTL) for 90 min. Nuclei are highlighted with DAPI (gray). n=6 independent cell batches (2-way ANOVA and Bonferroni). Scale bars: 20 µm.",
    "molecules": "DAPI, shikonin, SKN"
  },
  {
    "caption": "Percentage of neutrophils and macrophages infiltrated into the LCA from ApoE-/- mice treated with Veh or SKN (1.2 mg/kg) 7 days after ligation and high cholesterol diet feeding; n=4-6 mice per group (Unpaired Student's t test).",
    "molecules": "cholesterol, SKN"
  },
  {
    "caption": "Plaque burden in ligated carotid arteries from ApoE-/- (-/-) and ApoEYF (YF) mice fed a high cholesterol diet for 21 days; scale bars: 2 mm. Intimal and medial thickness was determined at four specific locations (proximal, middle 1, middle 2, distal) in cryo-sectioned arteries stained with Oil Red O; scale bars: 200 µm. n=7 mice per group (Unpaired Student's t test).",
    "molecules": "cholesterol, Oil Red O"
  },
  {
    "caption": "Oil Red O staining of plaques in aortae from mice fed a Western diet for four (B) or six months (C); scale bars: 3 mm. n=6 (B) or n=12 (C) mice per group (Unpaired Student's t test).",
    "molecules": "Oil Red O"
  },
  {
    "caption": "Tubulin co-condensation with Tau. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) to a Tau-Dy488 solution (25 μM) induces Tau condensation. Addition of tubulin to  Tau:PEG, Tau:RNA (polyA-Cy3), and Tau:PEG:RNA condensates leads to partitioning of tubulin into existing condensates and the occurrence of additional small Tau:tubulin condensates. Condensates with high tubulin-HiLyte647 intensity have low polyA-Cy3 intensity, and vice versa. In Tau:PEG:polyA condensates, individual condensates host RNA- and tubulin-rich domains (arrow heads) at homogeneous Tau-Dy488 intensity. Scale bars=20 μm. Average intensity of RNA (top graph) normalized to Tau intensity in individual condensates. Addition of tubulin reduces the average amount of RNA in condensates. Intensity of tubulin (middle graph) normalized to Tau intensity in individual condensates. The presence of RNA increases the amount of tubulin in the condensates. Data shown as mean±SD. Data +/- tubulin and +/- polyA were compared by two-tailed Student's t-tests. Tubulin and RNA intensity correlate negatively on an individual condensate level (bottom graph). Line represent linear regression to all data. N=3 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Condensate size (are on surface) relative to tubulin (yellow data points) and RNA (pink data points) intensity. Smaller (new) condensates are high in tubulin, larger (old) condensates have more  RNA. Lines represent linear regressions to tubulin (yellow) and RNA  (pink) data.",
    "molecules": "HiLyte647, Dy488, Alexa647, Cy3, PEG, RNA, polyA, RNA (polyA"
  },
  {
    "caption": "MT polymerization out of  Tau:PEG and Tau:PEG:RNA condensates. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) and GTP to Tau-Dy488 (25 μM) diluted in BRB80 polymerization buffer induces MT polymerization. Images taken 30 min after addition of tubulin show thin partially curved MT bundles in Tau:PEG system, and thick bundles attached to persisting Tau:RNA condensates in the Tau:PEG:RNA system. Scale bar=5 μm. Cross-sectional intensity plots (line plots) of MT bundles differ between Tau:PEG (yellow/red line plots) and Tau:PEG:RNA (blue/green line plots) systems. Four line plots at random positions are shown per condition.",
    "molecules": "Dy488, Alexa647, GTP, PEG, RNA"
  },
  {
    "caption": "Nocodazole treatment (120 μM for 30 min) of MTs formed from  Tau:PEG:RNA and Tau:PEG condensates. MT formed in absence of Tau shown as control. Graphs show surface coverage (%) of  fibrillar MT  structures before and after nocodazole treatment. N=3 experiments, 3 images analyzed per experiment and condition. Data shown as mean±SD. Two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar=20 μm.",
    "molecules": "Nocodazole, nocodazole, PEG, RNA"
  },
  {
    "caption": "FRAP experiments of Tau:PEG,  Tau:polyA, and Tau:PEG:polyA  condensates at 1h, 4h, and 24h after LLPS induction. Tau:PEG and Tau:PEG:polyA  condensates show a decrease in recovery rate and mobile fraction over  time. Tau:polyA coacervates maintain fast recovery and a large mobile fraction even after 24h. Time until half maximum recovery: t(y1/2)polyA: 1h≈2s, 4h≈3.5s, 24h≈4.5s; t(y1/2)PEG: 1h≈6s, 4h≈8s, 24h≈11s; t(y1/2)PEG+polyA: 1h≈4.5s, 4h≈9.5s, 24h≈9s). Scale bars=5 μm for Tau:polyA and Tau:PEG, and 10 μm for Tau:PEG:polyA. Data presented as error bars (SEM), N=15 to 20 condensates per condition and time point.",
    "molecules": "PEG, polyA"
  },
  {
    "caption": "Microscopy of Tau:PEG:polyA condensates (Tau-Dylight488 and polyA-Cy3) shows condensates with different Cy3 intensities. The bimodal fluorescence intensity distribution suggests two groups of condensates with high or low  Cy3 fluorescence. Cy3-intensities, normalized to Tau-DyLight488 intensities in the same condensates, were independent of condensate size. Scale bar=10 μm.",
    "molecules": "Dylight488, DyLight488, Cy3, PEG, polyA"
  },
  {
    "caption": "FRAP of Tau-Dylight488 and polyA-Cy3  of 4h-old Tau:PEG:polyA condensates. The mean FRAP of polyA-Cy3  is lower than of Tau-Dy488 (left). Data shown as mean±SEM, n=24 condensates. FRAP data of individual condensates (bottom; #1 to #3) shows a decrease of Tau=Dy488 (middle) and polyA-Cy3 (right) FRAP with an increase of polyA-Cy3 (relative to Tau-Dy488) in the condensates. Scale bar=5μm.",
    "molecules": "Dylight488, Dy488, Cy3, PEG, polyA"
  },
  {
    "caption": "HEK sensor cell Tau aggregation  assay. Cells were treated with 24 h-old condensates or aggregates (1.2 µM Tau in the presence of 0.8% lipofectamine). The number of Tau aggregates was determined from low magnification (20x) images taken in the green channel and normalized to the number of cells (DAPI). Scale bar=100 μm.",
    "molecules": "DAPI, lipofectamine"
  },
  {
    "caption": "Confocal microscopy  of HEK  sensor cells treated with 24h-old Tau condensates (Tau:tRNA) shows different types of intracellular CFP/YFP-Tau accumulations: spherical intranuclear Tau  (NUC), bright cytoplasmic accumulations (CYT) adjacent to the nucleus, and small, dimmer accumulations right at and along the nuclear envelope  (NE). Cells treated with Tau  aggregates (hepAGG) had mainly large  cytoplasmic (CYT) Tau inclusions. The different  Tau  accumulations coexisted in most cells, and also more than one accumulation of the same type could occur. Nuclei  were stained with propidium iodide  (PI, pink). Scale bars=10 μm.",
    "molecules": "hep, PI, propidium iodide, tRNA"
  },
  {
    "caption": "Immunolabeling of phosphorylated Tau (PHF1=pS396/404, AT8=pS199/pS202/pT205, AT180=pT231) in AD brain section. Both 3,3′-Diaminobenzidine (DAB) and fluorescence labeling show Tau accumulating in a layer at the nuclear envelope  (NE) in non-tangle neurons with or without cytosolic (CYT) Tau accumulations. Control brain does not show Tau labeling in neuronal cell bodies. Scale bars= 20 and 10 μm, as indicated.",
    "molecules": "3,3′-Diaminobenzidine, DAB"
  },
  {
    "caption": "FLIM of 1h- and 24h-old CFP-Tau (full-length) condensates and heparin induced CFP-Tau aggregates. Lifetime components are indicated in phasor plots. Graph shows lifetime reduction in condensates and aggregates relative to respective lifetime of soluble CFP-Tau. N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. One-way ANOVA with Tukey post-test. Scale bar=5 μm.­ *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Bottom right: Schematic of lifetime quenching in condensates and aggregates.",
    "molecules": "heparin"
  },
  {
    "caption": "(A) Motility assay of C.c. ΔpseI cells expressing predicted Leg or Pse synthases (LegI or PseI, in yellow or red, respectively) from Plac on pSRK-Gm. Overnight cultures were spotted on PYE soft (0.3%) agar plates with gentamycin and IPTG (0.5 mM) and incubated for 3 days at 30°C. Only predicted PseI-like synthase coding sequences (CDSs) restore motility of the C.c. ΔpseI cells. Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C. jejuni. Motility assays are representative of three independent experiments.",
    "molecules": "gentamycin, IPTG, lac, Leg, Pse"
  },
  {
    "caption": "(A) and (B) Motility assays of WT, ΔlegI (A) and ΔflmG (B) B.s. cells. Overnight cultures of WT and mutants harboring the empty pSRK-Gm vector (+Plac) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG (0.5 mM) and incubated for 7 days at 30°C. Motility assays are representative of three independent experiments. The same original picture was used to generate Figs 3B and EV2C.",
    "molecules": "gentamycin, IPTG, lac"
  },
  {
    "caption": "(A) Motility assays of B.s. ΔlegI cells complemented with LegI-type (yellow) or PseI-type (red) synthases expressed from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.",
    "molecules": "lac"
  },
  {
    "caption": "(A) Motility assays of WT B.s. and ΔBresu_3266 (ΔlegB) cells expressing Bresu_3266 (LegB), Bresu_3267 (LegX) or Bresu_3266-Bresu_3267 (LegB-LegX, Bresu_3266-67) from Plac on plasmid pSRK-Gm. Motility assays are representative of three independent experiments. (D) Motility assay of WT B.s. and ΔBresu_0506 (ΔlegH) cells complemented with a plasmid expressing Bresu_0506 (LegH) from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.",
    "molecules": "lac"
  },
  {
    "caption": "(A) Motility assay of ΔBresu_3267 (ΔlegX) B.s. cells compared to WT B.s. cells harbouring the empty pSRK-Gm vector (+Plac) or a complementing derivative with PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Motility assays are representative of three independent experiments.",
    "molecules": "lac"
  },
  {
    "caption": "(B) Immunoblots probed with anti-FljKCc antibodies from cell lysates (cells) and supernatants (SN) of WT B.s. and ΔBresu_3267 (ΔlegX) cultures harbouring either the empty pSRK-Gm vector (+Plac) or a complementing plasmid expressing different PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Molecular size standards are indicated by the blue lines with the corresponding value in kDa. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of two independent experiments.",
    "molecules": "lac"
  },
  {
    "caption": "(B) Anti-FljKCc immunoblot analyses of whole cell lysates (cells) from C. crescentus (C.c.) mutant cultures expressing fljKBssyn (codon optimized for E. coli) and flmGBs from the replicative pMT463 plasmid, in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). In the presence of pXGFP4-legBs (integrated at the xylX locus) and pMT375-Bresu_3267, FljKBssyn migration is shifted toward higher molecular mass, indicative of glycosylation. Note that in the C.c. Δfljx6 background, the six flagellin-encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn. The legBs synthetic operon (in red) is composed of Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 (legI) and Bresu_3265. Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg. Immunoblots are representative of at least two independent experiments.",
    "molecules": "leg, Leg, Pse"
  },
  {
    "caption": "(C) Anti-FljKCc immunoblot analyses of whole cell lysates from C. crescentus mutant cultures expressing FlmGCc, FlmGBs and FlmGCc-Bschim chimera from the replicative pSRK-Gm plasmid (Plac) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.",
    "molecules": "lac, leg"
  },
  {
    "caption": "(D) Representative Western Blot analysis of increasing amount of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in absence or presence of FLAG-EB1 C-terminal. Colors label the protein as in (c). Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued.",
    "molecules": "Gly"
  },
  {
    "caption": " (F) Representative Western Blot analysis of GST-STIM1 (cytoplasmic domain) pulled down by Cap-Gly-CC1-p150Glued-V5-coated beads, in presence of FLAG-EB1 C-terminal WT or ΔY. Negative control was performed using equal amount of empty GST protein, together with Cap-Gly-CC1-p150Glued. ",
    "molecules": "Gly"
  },
  {
    "caption": "(A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous LAMP-1 (in green) to visualize LEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), rescued for GFP expression alone (shCTL/GFP and shSTIM1/GFP) or for GFP- STIM1 WT (shSTIM1/GFP-STIM1 WT), GFP- STIM1 NN (shSTIM1/GFP-STIM1 NN) or GFP- STIM1 AA (shSTIM1/GFP-STIM1 AA) and stained for endogenous LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": " (A) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting STIM1 (siSTIM1) and stained for endogenous Rab5 (in green) and EEA-1 (in red) to visualize EEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " (A) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), rescued for GFP expression alone (shCTL/GFP and shSTIM1/GFP) or for GFP-STIM1 WT (shSTIM1/GFP-STIM1 WT) or GFP- STIM1 NN (shSTIM1/GFP-STIM1 NN) and stained for endogenous EEA-1 (in red) to visualize EEs and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " (E) Confocal microscopy images of untreated ECs (UT) or treated with Ciliobrevin D (CilioD) and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. ",
    "molecules": "Ciliobrevin D, CilioD, DAPI"
  },
  {
    "caption": " (F) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3 and EV4) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during Ciliobrevin D (CilioD) treatment. Results are from three independent experiments for a total of and 853 early endosomes in 6 untreated (UT) cells (142 ±22 endosomes per cell) and 657 early endosomes in 9 CilioD cells (73 ± 23 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***. ",
    "molecules": "Ciliobrevin D, CilioD"
  },
  {
    "caption": " (G) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in e) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of LAMP-1+ endosomes. Results are from three independent experiments for a total of 432 late endosomes in 12 untreated (UT) cells (36 ± 9 endosomes per cell) and 1184 late endosomes in 18 CilioD cells (66 ± 9 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***. ",
    "molecules": "CilioD"
  },
  {
    "caption": " (C) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting KIFC1 (siKIFC1) or one targeting STIM1 (siSTIM1) or two targeting both (siKIFC1siSTIM1) and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " (A) Confocal microscopy images of untreated (UT) or treated with Ciliobrevin D (CilioD) or AZ82 or both treatments simultaneously (CilioD + AZ82) ECs and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and Draq5 (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. ",
    "molecules": "AZ82, Ciliobrevin D, CilioD, Draq5"
  },
  {
    "caption": " (B) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment in ECs, compared to untreated cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 untreated cells (142 ± 22 endosomes per cell) and 355 early endosomes in 7 AZ82 cells (51±12 endosomes per cell). Data are analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***. ",
    "molecules": "AZ82"
  },
  {
    "caption": " (C) Distribution of retrograde velocity overtime, quantified by image (data correspond to movies EV3, EV4 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during Ciliobrevin D (CilioD D) or AZ82 treatment in ECs, compared to untreated cells (UT). Results are from three independent experiments for a total of 580 EEs in 6 UT cells (97 ± 18 endosomes per cell), 432 EEs in 9 CilioD cells (48 ± 15 endosomes per cell) and from two independent experiments for a total of 223 EEs in 7 AZ82 cells (32 ± 6 endosomes per cell). Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and CilioD treated ECs p≤ 0,01 ** and for UT and AZ82 treated ECs p≤ 0,001 ***. ",
    "molecules": "AZ82, Ciliobrevin D, CilioD"
  },
  {
    "caption": " (D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3, EV5 and EV6) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment (as in b), alone or in combination with CilioD treatment (CilioD + AZ82), compared to untreated EC cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 UT cells (142 ±22 endosomes per cell), 355 EEs in 7 AZ82 cells (51 ± 12 endosomes per cell) and 1106 EEs in 8 CilioD+AZ82 cells (136 ± 42 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 treated ECs p≤ 0,001 *** (orange) and for CilioD and CilioD + AZ82 treated ECs p≤ 0,001 *** (green). ",
    "molecules": "AZ82, CilioD"
  },
  {
    "caption": " (D) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting HOOK1 (siHOOK1) or one targeting HOOK3 (siHOOK3) or two targeting both (siHOOK1siHOOK3) and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " (H) Representative Western Blot analysis of the endogenous P150Glued co-immunoprecipitated with HOOK1 in untreated (UT) and AZ82 treated ECs (left) and its quantification by normalized densitometry (right). Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG. Results are the average ± SD of three independent assays. UT value of each biological replicate was normalized on itself and so AZ82 experimental value. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 p≤ 0,05 *. ",
    "molecules": "AZ82"
  },
  {
    "caption": " (A) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), to which the pH-sensitive dye pHrodo Green was given and then cells were stained for LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. Scale bar = 20 μm. (B) Relative amount of LAMP-1+ LEs positive to the pH-sensitive dye pHrodo (as in a) in shCTL and shSTIM1 ECs. Results are the average ± SEM of three independent experiments for a total of 90 cells (30 cell for experiment) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***. ",
    "molecules": "pHrodo, DAPI"
  },
  {
    "caption": "(A) Confocal microscopy of GFP-TRIM33-overexpressing mESCs  and differentiated cells. Scale bar = 5 µm. (B) Line scans of GFP-TRIM33  and DAPI  fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (C) FRAP  assays showing recovery time  of normalized fluorescence signals for GFP-TRIM33  puncta in mESCs. Photobleaching  occurs at t = 5s. Data are the mean ± S.D. of three biological experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D, G, I, Representative images of co-localization assays with mCherry-PML  and GFP-TRIM33  in mESCs  (D), differentiated cells (G), NaAsO2-treated mESCs  (200 µM for 2 hr) (I) Scale bar = 5 µm. (E, H, J Line scans of GFP-TRIM33  and mCherry-PML fluorescence imaging  in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (F) Column graph of the numbers of GFP-TRIM33  puncta and mCherry-PML NBs  in mESCs.",
    "molecules": "NaAsO2"
  },
  {
    "caption": "K) Representative images of co-localization  assays with GFP-TRIM33  in mESCs Pml  null (K). Scale bar = 5 µm. L) Line scans of GFP-TRIM33 fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (M) Column graph of the numbers of GFP-TRIM33  puncta and DAPI  in mESCs.",
    "molecules": "DAPI"
  },
  {
    "caption": "B FACS analysis of p-S6   in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with DSF  (5 µM) for 2 h (n = 3). Data information: , data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (B)",
    "molecules": "DSF"
  },
  {
    "caption": "C Representative FACS of p-S6  in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with the indicated concentrations of DSF  for 2 h (n = 2). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (C)",
    "molecules": "DSF"
  },
  {
    "caption": "D  Western blot  analysis of the phosphorylation  level of S6  and p70S6K in WT naïve CD8+ T cells  stimulated by α-mouse CD3ε(145-2C11) (1 µgml-1) in the presence of α-mouse CD28  (1 µgml-1) for the indicated times  after pretreatment with DSF  (5 µM) for 2 h. The immunoblot shows normalized expression of p-S6(S235/236) to S6, p-p70S6K  to  p70S6K, p-Zap70 to Zap70, as well as that of p-LCK(Y394) and P-Lck(Y505) to Lck. Data information: , data are representative of 3 independent experiments, , Image J analysis in (D)",
    "molecules": "DSF"
  },
  {
    "caption": "E FACS  analysis of p-CD3ζ(PY142) in mouse  naïve CD8+ T cells treated with DSF  or stimulated with α-mouse CD3ε(145-2C11) in the presence of α-mouse CD28  (1 µgml-1) for 2 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (E)",
    "molecules": "DSF"
  },
  {
    "caption": "F Western blot  analysis of the indicated proteins in Jurkat T cells treated with the indicated concentrations of DSF  for 2 h. The immunoblot  analysis shows normalized expression of p-CD3ζ  to CD3ζ  as well as that of p-Zap70(Tyr319)/Syk(Tyr352), p-LCK(Y394), and p-LAT(Tyr191) to  β-actin. Data information: data are representative of 3 independent experiments, Image J analysis in (F)",
    "molecules": "DSF, Tyr"
  },
  {
    "caption": "G, H Luciferin  assay of NFAT  activity (G) and Nfatc1  mRNA expression  (H) in J76-NFATRE-luc  and TCR052 cells stimulated with α-human CD3  (OKT3) (1 µgml-1) in the presence of α-human CD28  (1 µgml-1) for 4 h or treated with DSF  (5 µM) for 2 h. The levels of NFAT  activity were normalized with TCR052  cells treated with DMSO  (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; two-way analysis of variance (ANOVA) in (G, H)].",
    "molecules": "DMSO, DSF"
  },
  {
    "caption": "A, B MST  analysis determined the Kd of DSF  towards His-LCK(WT) (86.812 nm) (A) or His-LCK(C20/23S) (22.797 µM) (B) labeled with  RED-tris-NTA  2nd Generation dye. Concentration is reported in nanomolar. Data information: , data are representative of 3 (A, B, independent experiments",
    "molecules": "RED-tris-NTA, DSF"
  },
  {
    "caption": "C, D  MS/MS spectra of the Cys20/23-containing human LCK  peptide WMENIDVCENCHYPIVPLDGK (aa 13-33; 2532.87 Da) (LC  retention time  was 49.16 min; a triplet-charged precursor ion m/z 844.7163 (mass, 2531.1284 Da; ΔM of −0.5 ppm; 10logP = 200) was observed) (C), or of the corresponding LCK  peptide after LCK  incubation with DSF, which was modified on  Cys23 (red) by the diethyldithiocarbamate  moiety of DSF  (an increase of 147.0176 Da; LC  retention time was 56.31 min; a triplet charged precursor ion m/z 893.7295 (mass, 2678.1460 Da; ΔM of 7.7 ppm; −10logP = 116.99) was observed (D). Data information: , data are representative of 2 independent experiments",
    "molecules": "Cys, diethyldithiocarbamate, DSF"
  },
  {
    "caption": "E Recombinant human LCK  proteins left untreated (Ctrl) or incubated with ATP  and increasing concentrations of DSF  for 15 min. Enzyme-linked immunosorbent assay (ELISA) of LCK kinase activity was analyzed by using α-p-LCK  (Y394) antibody. The results show the average values of the optical density at 450 nm (OD450) from five replicates (n = 5). Data information: data are representative of 2 independent experiments, mean ± SD. **P<0. 01; ***P<0.001; ns, not significant [one-way ANOVA in (E)",
    "molecules": "ATP, DSF"
  },
  {
    "caption": "F Confocal  analysis of p-Lck(Y394) (red) in naïve CD8+ T cells treated with DSF  (5 µM) or stimulated with α-mouse CD3  and α-mouse CD28  for 2 h (left). The total fluorescence intensity (IntDen) was quantified (right; n= 20 cells). Scale bars, 3 µm. Data information: data are representative of F, independent experiments, mean ± SD. **P<0. 01; ***P<0.001; ns, not significant [one-way ANOVA in (F)].",
    "molecules": "DSF"
  },
  {
    "caption": "G Western blot analysis of the active LCK  enrichment [assessed by p-Lck  (Y394)] in Triton X-100-insoluble fractions after DSF  (5 µM) treatment in naïve CD8+ T cells. Data information: data are representative of independent experiments,",
    "molecules": "DSF, Triton X-100"
  },
  {
    "caption": "H IVP  assay of His-tagged  CD3εCD peptides was phosphorylated  by LCK  in the presence of DSF. CD3ε phosphorylation  was analyzed by immunoblotting  using α-pTyr(PY100). Data information: , data are representative of 2 , H) independent experiments",
    "molecules": "DSF, Tyr"
  },
  {
    "caption": "I Western blot  analysis of p-CD3ζ, LCK  and p-Zap70  in LCK-deficient cells were infected with the lentivirus  collected from human HEK293T  cells transfected with control plasmid (Control) (1 µg), LCK(WT) (1 µg), or LCK(C20/23S) (1 µg) with psPAX2 (1 µg) and pMD2.G (1 µg) for 48 h followed by treatment with DSF  (5 µM) for 2 h stimulated with α-human CD3  and α-human CD28  for 5 min. Data information: data are representative of I) independent experiments",
    "molecules": "DSF"
  },
  {
    "caption": "C Scatter plot comparing global  gene-expression profiles of DMSO  and DSF  (n = 3). Data information: data are representative of 3 independent experiments",
    "molecules": "DMSO, DSF"
  },
  {
    "caption": "D A heatmap of upregulated genes associated with TCR and T cell activation-related genes in DSF  relative to that in DMSO. One column indicates one sample in each condition (n = 3). Data information: data are representative of 3 independent experiments",
    "molecules": "DMSO, DSF"
  },
  {
    "caption": "E ELISA  analysis of the production of  IL-2 in primary OT-I T cell  supernatants treated with DMSO, DSF  (5 µM) or OVA   peptide (5 µgml-1) for 4 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [unpaired t tests for the measurements between the two groups in (E)",
    "molecules": "DMSO, DSF, OVA"
  },
  {
    "caption": "F CFSE  dilution of proliferation  of primary OT-I T cells treated with DMSO  or DSF (5 µM) for 2 h (n = 3). Data information: , data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [unpaired t tests for the measurements between the two groups in (F)",
    "molecules": "CFSE, DMSO, DSF"
  },
  {
    "caption": "G FACS  analysis of IFNγ and TNFα  production in primary OT-I  T cells primed by N4  (SIINFEKL, 0.01 μgml-1) and A4  (SAINFEKL, 0.01 μgml-1) for 2 h followed by treatment with DSF  (5 µM) for 2 h (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. two-way ANOVA in (G)",
    "molecules": "SAINFEKL, DSF, SIINFEKL"
  },
  {
    "caption": "A, Tumor growth [(A), (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared)] (n = 10 mice  per group)] in DSF  (suspended in 0.5% NaCMC)- and Vehicle (equal volume of 0.5% NaCMC)-treated mice  after B16F10 melanoma  inoculation. Data information: , data are representative of 3 independent experiments, ns, not significant two-way ANOVA in (A)",
    "molecules": "NaCMC, DSF"
  },
  {
    "caption": "B Images and tumor weight  of subcutaneous mouse B16F10  tumors extracted from mice  at day 21 (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: , data are representative of 3 independent experiments, ns, not significant [multiple t tests between the two groups in (B)",
    "molecules": "DSF"
  },
  {
    "caption": "survival curves  [(C), (n = 10 mice  per group)] in DSF  (suspended in 0.5% NaCMC)- and Vehicle (equal volume of 0.5% NaCMC)-treated mice  after B16F10 melanoma  inoculation. Data information: data are representative of 3 independent experiments, mean ± SD. * ns, not significant [ , log-rank (Mantel-Cox) test in (C)",
    "molecules": "NaCMC, DSF"
  },
  {
    "caption": "D - Phenotypic analysis of tumor-infiltrating  T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: , data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [multiple t tests between the two groups in (D)",
    "molecules": "DSF"
  },
  {
    "caption": "immunofluorescence  (E) analysis of tumor-infiltrating  T cells at day 21 after melanoma  inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Scale bars, 20 µm. Statistics data are from 40× field of view.",
    "molecules": "DSF"
  },
  {
    "caption": "F Phenotypic analysis of tumor-infiltrating  T cells  at day 21 after melanoma  inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Data information: data are representative of 3 independent experiments, mean ± SD.",
    "molecules": "DSF"
  },
  {
    "caption": "H, I Tumor growth [(H), (n = 5 mice  per group)] and  survival curves [(I), (n = 5 mice  per group)] in Vehicle- and DSF-treated mice  injected s.c. with B16F10-melanoma  cells followed by intraperitoneal (i.p.) injection with PD-1  antibody on days 0 and 4 and then every third day (n = 5). Data information: data are representative of 3 independent experiments, mean ± SD. ns, not significant two-way ANOVA in (H), log-rank (Mantel-Cox) test in (I)].",
    "molecules": "DSF"
  },
  {
    "caption": "A Tumor growth in Vehicle- and DSF-treated mice  injected s.c. with B16F10 melanoma  cells followed by intraperitoneal (i.p.) injection with CD8(2A3) antibody on days −1, 0, and 2 and then every second day (n = 5). Data information: data are representative of 3 independent experiments, mean ± SD. ns, not significant [two-way ANOVA in (A)",
    "molecules": "DSF"
  },
  {
    "caption": "B FACS  analysis of cytotoxicity  of B16F10-OVA  cells after coculture with DMSO- or DSF-treated primary OT-I T cells (n = 3). Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ****P<0.0001. ns, not significant [two-way ANOVA",
    "molecules": "DMSO, DSF, OVA"
  },
  {
    "caption": "C, D Tumor growth [(C), (n = 5 mice  per group)] and survival curves  [(D), (n = 10 mice  per group)] of B16F10-OVA   tumor-bearing mice  after adoptive transfer of Vehicle- or DSF-treated primary OT-I T cells. Data information: data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ****P<0.0001. ns, not significant [two-way ANOVA in (C), log-rank (Mantel-Cox) test in (D), mean ± SD].",
    "molecules": "DSF, OVA"
  },
  {
    "caption": "G. UPR can be induced in atg9∆ single-, and atg9 atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(A) In vitro Methyltransferase activity. Recombinant proteins Nsp10  and Nsp16  wt or Nsp16mut were combined with cap0   RNA and Adenosyl-L-methionine ([methyl-3H] SAM) for 90 min at 37°C. Methylation  of cap0   RNA was quantified using a scintillation counter  (Beckmann).(-) control did not contain any recombinant protein. Results are plotted as mean of triplicate reactions with error bars representing SD. One out of three independent biological repeats is shown. Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.",
    "molecules": "Adenosyl-L-methionine ([methyl-3H] SAM)"
  },
  {
    "caption": "(B)  MTase activity of Nsp16   upon viral infection. Cellular mRNA   was purified 24 h postinfection with SARS-CoV-2 wt or Nsp16mut (triplicate infections, biological replicates). Purified mRNA  was incubated with 100U of the recombinant MTase VACV VP39  (NEB) and  [methyl-3H] SAM for 90 min at 37 °C. Methylation  of mRNA  from SARS-CoV-2 infected cells was quantified using a scintillation counter (DPM3H; Beckmann). (-) control did not contain cellular RNA. Results are plotted as mean of triplicate infections with error bars representing SD. One out of three independent experiments is shown (n=3). Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.",
    "molecules": "mRNA, [methyl-3H] SAM"
  },
  {
    "caption": "(C)  Calu-3 cells lacking MDA5  (MDA5  KO), RIG-I  (RIG-I KO), or control KO cells (Ctrl) were seeded into 96well plates and infected with increasing amounts of SARS-CoV-2 wt (WT) or Nsp16mut in triplicates (biological replicates). Type I IFN release was quantified after 48 h by incubating supernatants with HEK-Blue IFN-α/β reporter cells. SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD. (D) Calu-3 cells (Ctrl) were seeded into 96 well plates and infected with 10 RNA copies/cell of SARS-CoV-2 wt (WT) or Nsp16mut in the presence of increasing amounts of Remdesivir  (RDV). Type I IFN release was quantified after 48 h by IFN bioassay  on  HEK-Blue IFN-α/β and is depicted as mean of quadruplicates with error bars representing SD. In parallel, infectivity  of SARS-CoV-2  wt and Nsp16 in the presence of increasing amounts of RDV  was determined at the indicated time points by RT-qPCR targeting viral polymerase RdRp   transcripts. Data information: Statistical analysis was performed using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).",
    "molecules": "RDV, Remdesivir"
  },
  {
    "caption": "C, Demonstration of single-molecule tracking in a single cell. Cell outline is shown as an ellipse. Left, Imaged FliM-YPet fluorescence. White dots indicate the estimated motor locations. White circles illustrate a 75 nm radius around these locations. Right, Contour map of the normalized sum of probabilities of the localization events of CheY(I95V)-Atto647 (from blue to red is low to high; black is zero probability). White circles show the motors' locations from the left panel.",
    "molecules": "Atto647"
  },
  {
    "caption": "I, CheY interacts with FliM∆N and this interaction is sensitive to chemotactic stimuli. Each curve is the mean of two FRET measurements of cells in response to an attractant stimulus (0.1 mM serine; in the case of FliMwt, a single measurement was performed with serine; another measurement of FliMwt with 1mM aspartate produced similar results). FRET ratio is the ratio of mCherry to YPet fluorescence. CheY-mCherry and mCherry concentrations were ~170 µM in the case of FliM∆N and ~15 µM in the case of FliMwt Strains used: EW677 (FliMwt, red), EW659 (negative control of FliM∆N without CheY, purple), EW637 (FliM∆N, blue), and EW636 (FliM∆N ∆cheA, yellow).",
    "molecules": "aspartate, serine"
  },
  {
    "caption": "C, The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG), to positive stimuli. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM.",
    "molecules": "MeAsp, α-Methyl-DL-aspartate, benzoate, IPTG, leucine"
  },
  {
    "caption": "The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG) Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM. D, negative stimuli.",
    "molecules": "MeAsp, α-Methyl-DL-aspartate, benzoate, IPTG, leucine"
  },
  {
    "caption": "E, Response of tethered fliM∆N ΔcheA cells (strain EW634) to acetate (50 mM, pH 7.0) at different CheY concentrations. Lines and shaded regions are mean ± SEM.",
    "molecules": "acetate"
  },
  {
    "caption": "F, Response of tethered fliM∆N ΔcheZ cells (strain EW635) to acetate.",
    "molecules": "acetate"
  },
  {
    "caption": "G, Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation. The points are the mean clockwise rotation at time segments 0-20 s and 160-180 s. The variants shown are fliM∆N ∆cheZ cells expressing CheY in the absence of presence of acetate (10 mM, pH 7.0) (i.e., CheY~P and CheY~P~Ac; blue and green curves; strain EW635), fliM∆N ∆cheA cells expressing CheY, in the absence of presence of acetate (CheY and CheY~Ac; purple and red, respectively; EW634), and fliM∆N ∆cheA cells expressing CheY(D13K) (burgundy; EW737). Each data point is the average of all experiments at a given CheY concentration, weighted by the sample number of each experiment. The CheY concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used.",
    "molecules": "acetate, Ac"
  },
  {
    "caption": "H, FliMN further activates CheY variants to produce clockwise rotation. The variants shown are fliM∆N ∆cheZ cells expressing FliMN-CheY (i.e., FliMN-CheY~P; blue curve; strain EW697), fliM∆N ∆cheA cells expressing FliMN-CheY, in the absence of presence of acetate (10 mM, pH 7.0) (FliMN-CheY and FliMN-CheY~Ac; purple and red, respectively; EW696), and fliM∆N ∆cheA cells expressing FliMN-CheY(D13K) (burgundy; EW739).",
    "molecules": "acetate, Ac"
  },
  {
    "caption": " I, Response of fliM∆N ∆cheA cells expressing FliMN-CheY (strain EW696) to acetate and benzoate (10 mM each; pH 7.0). FliMN-CheY expression was induced with 800 μM IPTG. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The black arrow indicates the estimated time point at which the stimulus entered the flow chamber. ",
    "molecules": "acetate, benzoate, IPTG"
  },
  {
    "caption": ", Dependence of clockwise rotation on the level of FliMN-CheY~Ac. The red and purple curves are two experimental days in which the clockwise production by fliM∆N ∆cheA cells expressing FliMN-CheY was measured as a function of FliMN-CheY concentration (strain EW696; Concentrations are estimates based on the calibration curves or which a similar CheY expression system was used. ach data point is a mean ± SEM of the data or the second and third experimental days of this strain (in the presence of acetate).",
    "molecules": "acetate"
  },
  {
    "caption": "B, Response of the strain to addition and removal of varying acetate concentrations (pH 7.0). FliMN-CheY concentration was estimated to be ~300 μM. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrows indicate the estimated time points at which acetate entered or left the flow chamber. Black lines mark the linear part of the biphasic response to acetate removal. N is the number of cells.",
    "molecules": "acetate"
  },
  {
    "caption": "A, Effects of FliMN-CheY acetylation and phosphorylation on clockwise rotation of tethered cells containing FliM∆N FliN(A93D) motors. Acetate concentration was 10 mM each (pH 7.0). FliMN-CheY and FliMN-CheY~P were produced by using ∆cheA and ∆cheZ backgrounds (strains EW713 and EW714), respectively. Each data point is the average of all measurements at a given FliMN-CheY concentration, weighted by the sample number of each experiment. The concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used. Concentrations larger than 500 μM were calculated by the linear extrapolation of the calibration curve. B, Distribution of clockwise interval lengths at different average clockwise levels in ∆cheA cells containing FliM∆N FliN(A93D) motors and expressing FliMN-CheY (strain EW713), in the presence of acetate (10 mM, pH 7.0). C, Average clockwise interval length, calculated as the inverse of the rate constants from mono-phasic fits of the distributions in (B).",
    "molecules": "Acetate, acetate"
  },
  {
    "caption": "D, Response of tethered ∆cheZ cells having FliM∆N FliN(A93D) motors to acetate addition and removal (red; strain EW714; FliMN-CheY concentration estimated at ~300 μM). Similar results were obtained with a ∆cheA strain (EW713). Tethered cells having FliM∆N FliNwt motors and containing FliMN-CheY (strain EW696) are shown Acetate concentration was 10 mM (pH 7.0). The data shown are the mean ± SEM. N is the number of cells. Black lines indicate the clockwise-decay rates of FliNwt motors' slow phase and of FliN(A93D) motors following acetate removal.",
    "molecules": "acetate, Acetate"
  },
  {
    "caption": "A, CheY crosslinks with FliM∆N-YPet in vivo. The cytoplasm of fliM∆N-YPet ΔcheZ cells overexpressing CheY (strain EW694) or FliMN-CheY (strain EW697) from a plasmid was crosslinked by glutaraldehyde and resolved by SDS-PAGE. 1, 2, 3 stand for three different experiments that underwent this procedure. The plus sign before ~14 and ~15 stands for +55 kDa of FliM∆N-YPet. Note that gel running suffered from parabolic distortion in band positions. The annotations relate to the lowest positions of the bands. The gel was imaged for FliM∆N-YPet fluorescence. To see the crosslinking products, the intense fluorescence of monomeric FliM∆N-YPet is shown at saturation.",
    "molecules": "glutaraldehyde"
  },
  {
    "caption": "C, Clockwise rotation of tethered fliM(R94S)∆N ∆cheZ cells and fliM(R94L)∆N ∆cheZ cells expressing FliMN-CheY (strains EW731 and EW733, respectively) in the absence or presence of acetate (10 mM, pH 7.0). FliMN-CheY concentration was estimated to be ~4 mM by extrapolation of the calibration curves for which a similar CheY expression system was used. No clockwise rotation was observed when benzoate substituted for acetate. Each data point is the mean of a separate experiment. Black line is the mean of all experiments.",
    "molecules": "acetate, benzoate"
  },
  {
    "caption": "(C) Equal amounts of WT cells and indicated single or double mutants in serial dilutions were incubated on agar plates at the indicated temperatures. The synthetic growth defects of vps4∆ tul1∆ or vps4∆ ubx3∆ double mutants at 26°C and 37°C were complemented with plasmids encoding VPS4 or TUL1 or UBX3. The dashed boxes indicate the non-complemented double mutants.",
    "molecules": "agar"
  },
  {
    "caption": "(A) Vacuolar proteolysis-deficient pep4∆ and tul1∆ pep4∆ cells were labeled to saturation with heavy 13C6,15C2-L-lysine and then chased for 3 hours in presence of light L-lysine, following quantitative proteome analysis by LC-MS (two biological replicates). To analyze Dsc complex-dependent protein turnover, the ratio of H/L ratios of tul1∆ pep4∆ over pep4∆ cells of 2511 proteins quantified in both strains was plotted, and proteins with a ratio of H/L ratios >2 were selected. Red squares: regulated proteins with at least one predicted transmembrane (TM) domain; black triangles: regulated proteins without TM domains.",
    "molecules": "L-lysine"
  },
  {
    "caption": "(C) Left panel: Epifluorescence and phase contrast microscopy of the indicated mutants expressing GFP-Orm2 (green) with mCherry-Cps1 (red, MVB cargo). E indicates class E compartments; Right panel: Equal amounts of the indicated mutants in serial dilutions were incubated on agar plates at the indicated temperatures. Data information: Scale bars = 5 µm.",
    "molecules": "agar"
  },
  {
    "caption": "WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (A) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (C) SEC13 WT and sec13-4 cells were preincubated for 30 minutes at 37°C before CHX was added.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(A) WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP (integral ER membrane protein) treated with MG-132 were fractionated into insoluble membrane (P100) and soluble cytoplasmic (S100) fractions and analyzed by SDS-PAGE and Western blot with the indicated antibodies.",
    "molecules": "MG-132"
  },
  {
    "caption": "WT cells, tul1∆ mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.",
    "molecules": "MG-132"
  },
  {
    "caption": "WT cells and cdc48-3 mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.",
    "molecules": "MG-132"
  },
  {
    "caption": "(D) P100 fractions from MG-132-treated WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP were extracted with Na2CO3 solution at the indicated pH and subsequently fractionated by 100.000 x g centrifugation into soluble (S) and insoluble membrane (M) fractions, subjected to denaturing FLAG-immunoprecipitations and analyzed by Western blotting with the indicated antibodies. The asterisk indicates reactivity of the secondary antibody with IgG heavy chains.",
    "molecules": "MG-132, Na2CO3"
  },
  {
    "caption": "(A) The levels of two major ceramide (Cer) species in lipid extracts from WT (black), tul1Δ (dark gray) or Orm2-K25,33R mutants (light grey) were measured using LC-MS and quantified using spiked non-yeast ceramides as internal standard. Data are normalized to Cer44:0;4 levels of WT cells (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.",
    "molecules": "Cer, ceramide, ceramides, Cer44:0;4"
  },
  {
    "caption": "(B) Autoradiogram of sphingolipid extracts from [3H]serine radiolabelled WT cells and tul1∆ mutants were separated by thin layer chromatography. IPC, Inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; MIP2C, mannosyldiinositolphosphorylceramide. X indicates an unknown lipid species that is insensitive to myriocin treatment.",
    "molecules": "Inositolphosphorylceramide, IPC, lipid, mannosyldiinositolphosphorylceramide, MIP2C, mannosylinositolphosphorylceramide, MIPC, myriocin, serine, sphingolipid, 3H"
  },
  {
    "caption": "SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (E) from WT cells and tul1∆ mutants expressing FLAG-Orm2, FLAG-Orm2-3A or FLAG-Orm2-3D with or without Myriocin treatment (1.5 µM) for one hour.",
    "molecules": "Myriocin"
  },
  {
    "caption": "(A) Left panel: In tor1-1 avo3∆1274-1430 cells rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al., 2015). tor1-1 avo3∆1274-1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG-Orm2 or of the indicated FLAG-Orm2 mutants from total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.",
    "molecules": "CHX, cycloheximide, methanol, rapamycin"
  },
  {
    "caption": "(B) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each timepoint was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.",
    "molecules": "CHX"
  },
  {
    "caption": "(F) The levels of the long chain bases (LCB) C18-dihydrosphingosine (C18-DHS) and C18-phytosphingosine (C18-PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆, Orm2-K25,33R, Orm2-3A and Orm2-3D mutants were measured using LC-MS and quantified using spiked non-yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.",
    "molecules": "Cer, ceramide, ceramides, lipid, C18-PHS, C18-phytosphingosine, C18-DHS, C18-dihydrosphingosine"
  },
  {
    "caption": "(E) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± miR-3679-5p. Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.",
    "molecules": "calcein"
  },
  {
    "caption": "(A) Heat map representation of RNA-seq results. The p65-dependent genes (total 123; defined as genes up-regulated more than 2-fold via TNF-α treatment and down-regulated more than 0.75-fold via BAY 11-7082 treatment) were classified based on the alteration patterns by the siRNA knockdown of KDM7A, UTX, and both, using the algorithm \"HOPACH\".",
    "molecules": "BAY 11-7082"
  },
  {
    "caption": "(G) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± (siKDM7A+siUTX). Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.",
    "molecules": "calcein"
  },
  {
    "caption": "(B) Representative snapshot from the IVM analysis of leukocyte adhesive interactions in the femoral arteries. Margins of vessels are indicated with dashed lines. White spots represent fluorescently labeled leukocytes visualized by the intravenous injection of rhodamine 6G.",
    "molecules": "rhodamine 6G"
  },
  {
    "caption": "BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours E, F (E) Example of a macrophage that interacts with an Mtb aggregate (0h00), fragments it (3h00), redistributes the bacteria in a "bullseye" pattern around the nucleus (12h00), and dies (16h00). Scale bar, 20 μm. (F) Example of a macrophage that stably interacts with an Mtb aggregate without fragmenting it (0h00-09h00) and ultimately dies (12h00). In (E and F), Draq7 staining of the nucleus (in blue) is used as a marker for cell death. Scale bar, 20 μm.",
    "molecules": "Draq7"
  },
  {
    "caption": "BMDMs infected with aggregates of Mtb Erd-tdTomato from axenic culture and imaged by time-lapse microscopy at 1-hour intervals for 60 hours (G-H) BMDMs infected with aggregates of Mtb were imaged by time-lapse microscopy (every 30 min for up to 13.5h) followed by fixation, immunostaining (nuclei stained with Hoechst, membrane staining with anti-CD45 antibody) and imaging by confocal microscopy. White arrows indicate intracellular bacteria, cyan arrows indicate extracellular bacteria. All scale bars, 10 μM. (G.I,H.I) Time-lapse microscopy image-series of macrophages that interacts with Mtb aggregates and fragment (G) or do not fragment (H) them. (G.II,H.II) Max intensity projection of confocal microscopy images of the same macrophages shown in panels I. (G.III,H.III) 3-D reconstruction of the cells imaged in panels II, images are cropped in x or y in the position indicated by the white dotted lines in panels II to show the inside of the cell.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Mouse bone marrow-derived macrophages (BMDMs) infected with Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1- or 2-hour intervals for 132 hours (O) Percentage of macrophages surviving over time after uptake and fragmentation of Mtb aggregates or after contact without fragmentation. Each line represents an independent biological replicate. The vertical dotted line marks the 12h post-interaction time-point. BMDMs were treated with cytochalasin D to prevent bacterial uptake, infected with aggregates of Mtb and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (P) Percentage survival over time for macrophages in contact with Mtb aggregates. Time 0 represents the time when stable contact with an aggregate begins. Each line represents an independent biological replicate (n = 3 replicates with ≥ 100 cells per replicate).",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (B-E) Examples of macrophages infected with low (B), medium (C), or high (D) doses of non-aggregated bacteria or with bacterial aggregates (E). Scale bars, 20 μm.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "BMDMs were treated with cytochalasin D and infected with aggregated or non-aggregated Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. (F) Percentage survival over time for macrophages in contact with increasing doses of non-aggregated bacteria or with bacterial aggregates. Time 0 for cells "in contact" represents the time when stable contact with the bacteria begins. Each line represents a biological replicate (n ≥ 100 cells per condition).",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(A) BMDMs expressing a membrane-targeted tdTomato were treated with cytochalasin D, infected with aggregates of Mtb expressing GFP, and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Example of a BMDM interacting with an extracellular Mtb aggregate. Top panels show the macrophage plasma membrane and the bacterial aggregate. Intimate interaction between the macrophage plasma membrane and the bacterial aggregate begins at 4h00 (indicated by the white arrow). Bottom panels show the Annexin V-647 positive plasma membrane domain that appears at 5 hours 20 minutes and co-localizes with the bacterial aggregate. At 8h00, plasma membrane integrity is lost (top panel) and Annexin V accumulates throughout the dead cell (bottom panel).",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(E-G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erd-tdTomato in the presence of Annexin V-FITC. Selected cells were imaged by time-lapse fluorescence microscopy followed by SEM. Arrows in top panels indicate the cell imaged by SEM. Top left: brightfield image of macrophage. Top left and right: fluorescence image of the Mtb aggregate. Top right: fluorescence image of Annexin V staining. Bottom: correlative SEM image. Scale bars, 20 μm in top panels and 1 μm in bottom panels. (E) Example of a bystander BMDM that is not in contact with a bacterial aggregate. (F) Example of an Annexin V-negative BMDM interacting with a Mtb aggregate. Arrows in bottom panel indicate areas of intact plasma membrane interacting with bacterial aggregates. (G) Example of a BMDM with blebbed Annexin V-positive membrane domains interacting with an Mtb aggregate.",
    "molecules": "FITC, Cytochalasin D"
  },
  {
    "caption": "Cytochalasin D-treated BMDMs were stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize intracellular Ca2+, infected with aggregates of Mtb Erdman WT and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to time 0 for uninfected bystander cells and to the time of first contact with an Mtb aggregate for infected cells. (A) Examples of BMDMs dying with (yellow outline) or without (white outline) interacting with extracellular Mtb aggregates. Cell outlines are based on brightfield images Cell death is indicated by Draq7 nuclear staining. Scale bar, 10 μm. (B) Oregon Green 488 Bapta-1 AM fluorescence over time in dying bystander macrophages (n=15) and in dying macrophages in contact with an Mtb aggregates (n=63). Lines represent median fluorescence values for all cells, error bars represent interquartile ranges. The distributions of the fluorescence values at time of death in bystander and dying macrophages are significantly different, p value <0.0001, calculated using a Welch's t test.",
    "molecules": "Draq7, Ca2+, Cytochalasin D, Oregon Green 488 Bapta-1 AM"
  },
  {
    "caption": "(E) BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. Percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate without (untr.) or with supplementation of BAPTA-AM. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 60 cells per replicate). Bars represent means and standard deviations, P value calculated using a t test.",
    "molecules": "BAPTA-AM, cytochalasin D"
  },
  {
    "caption": "BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided in Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (A) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact (cont.) with an extracellular Mtb aggregate that stain positive for cleaved Caspase-8 (cCasp8). Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 50 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test. (B) Percentage of dead uninfected bystander macrophages (byst.) or dead macrophages in contact with an extracellular Mtb aggregate (cont.) that display ASC-specks. Each symbol represents the percentage of positive dead macrophages for a single biological replicate (n ≥ 80 cells per replicate). Bars represent means and standard deviations. P values were calculated using a t test.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel (C) Representative example of a dead macrophage in contact with an extracellular Mtb aggregate that display a ASC-speck (white arrow). Nuclei stained with Hoechst. Scale bar, 10 μm.",
    "molecules": "Hoechst, cytochalasin D"
  },
  {
    "caption": "BMDMs treated with cytochalasin D were infected with aggregates of Mtb Erd-tdTomato, fixed at 24 hours post infection and processed for immunofluorescence with antibodies targeting cellular markers for cell death pathways. Controls and representative microscopy images for all the antibodies used are provided Macrophages are defined as "in contact" when the body of a macrophage identified in brightfield images overlaps with a bacterial aggregate identified in the fluorescence channel Median fluorescence values for bystander macrophages (byst.) or macrophages in contact with an extracellular Mtb aggregate (cont.) stained with anti-cleaved Caspase-1 antibody (cCasp1), an (E) anti-phosphorylated RIP3 antibody (pRIP3) or (F) anti-phosphorylated MLKL antibody (pMLKL). In (G) BMDMs were treated with MCC950 (NLRP3 inhibitor) or with VX765 (Caspase-1 inhibitor) during the course of the infection. Each symbol represents a single macrophage. Black bars represent median and interquartile range. P-value were calculated using an unpaired Mann-Whitney test; ns, P values > 0.05. E: n=182, 112 respectively; F: n=186, 84 respectively; G: n=117, 121, 111, 129 respectively; n represents the number of individual cells analyzed per condition).",
    "molecules": "cytochalasin D, MCC950, VX765"
  },
  {
    "caption": "(A-D) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains and imaged by time-lapse microscopy at 1-hour intervals for 60 hours. The plots represent the percentage of macrophages that die within the first 12 hours after stable contact with an Mtb aggregate. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test (A,C,D) or t test (B) comparing each strain to the wild-type reference strain (A-D); ns, P values > 0.05. (A) Macrophages in contact with aggregates of Mtb H37Rv wild-type (WT), ∆RD1 mutant and complemented ∆RD1 mutant (∆RD1::2F9). (B) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or PDIM-deficient (fadD26) strains. (C) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espI eccD1, esxA, espA) and the complemented strains (esxA::esxBA, espA::espA). (D) Macrophages in contact with aggregates of Mtb Erdman wild-type (WT) or mutant strains (espB, espA espB) and the espB-complemented strains (espB::espB, espA espB::espB).",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(F) BMDMs treated with cytochalasin D were infected with aggregates of different Mtb strains, incubated with Annexin V and imaged by time-lapse microscopy at 1-hour intervals for 48 hours. Percentage of macrophages that show Annexin V - positive membrane domains within the first 12 hours after entering in contact with Mtb aggregates. Each symbol represents a single biological replicate (n ≥ 3 replicates with ≥ 90 cells per replicate). Bars represent means and standard deviations.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(G) Cytochalasin D-treated BMDMs were infected with aggregates of Mtb Erdman WT, stained with the membrane-permeable dye Oregon Green 488 Bapta-1 AM to visualize cytosolic Ca2+ and imaged by time-lapse microscopy at 20-minute intervals for 24 hours. Oregon Green 488 Bapta-1 AM fluorescence values at each time point were normalized to the time of first contact with an Mtb aggregate for infected cells. Values in the plot correspond to the time of death after first contact or 16 hours post-contact for cells that survive. Each symbol represents a single macrophage. (n=123, 64, 48, 71, 78, 55 respectively). Black bars represent median and interquartile range. P-value were calculated using a one sample Wilcoxon test; ns, P values > 0.05.",
    "molecules": "Ca2+, Cytochalasin D, Oregon Green 488 Bapta-1 AM"
  },
  {
    "caption": "(H) BMDMs were treated as in (A-D). Percentage of macrophages that die within the first 12 hours after interaction with an Mtb aggregate. Infected cells are incubated with different concentration of BTP15 (0, 2, 10, 50 μM) during the course of the experiment. Each symbol represents the percentage of dead macrophages for a single biological replicate (n ≥ 3 replicates with ≥ 70 cells per replicate). Bars represent means and standard deviations. P-values were calculated using a one-way ANOVA test comparing the treated vs. untreated samples.",
    "molecules": "BTP15"
  },
  {
    "caption": "(A) Impact of TRPV2 genetic manipulation in 501mel, WM266.4 and 451Lu on serum-induced migration and matrigel invasion. Representative pictures show crystal violet-stained cells that have migrated through 8 µm pore size membranes. Histograms illustrate the average numbers of migrating/invading cells normalized to control (presented as mean ± SEM of n=3 independent experiments). For 501mel the Mann-Whitney test was used for statistical analysis (**P = 0.0022); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; ***P < 0.001; ****P < 0.0001 ; See appendix Table S2 for exact P values).",
    "molecules": "crystal violet"
  },
  {
    "caption": "Survival of age- and sex-matched (8-10 week old) Spata2+/+ (WT) and Spata2-/- (KO) mice injected intraperitoneally (i.p.) with LPS (20 mg/kg bodyweight). Data pooled from three independent experiments are shown. Statistical analysis was performed using long-rank test.",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA of serum IL-1β, IL-18, TNFα and IL-6 at 12 h after injection of WT or Spata2-KO mice with 20 mg/kg LPS. Circles and triangles represent individual mice. Horizontal bars indicate mean values. **P < 0.01; ****P < 0.0001. Statistical analysis was performed using unpaired two-tailed Student's t-test.",
    "molecules": "LPS"
  },
  {
    "caption": "Immunoblot analysis of the indicated phosphorylated (P) and total proteins in lysates of LPS-stimulated BMDMs derived from WT or Spata2-KO mice. Molecular weights in kDa are indicated to the right.",
    "molecules": "LPS"
  },
  {
    "caption": "qRT-PCR analysis of the indicated mRNAs in untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA of TNFα and IL-6 in the supernatants from untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or stimulated with the NLRC4 inducer (Salmonella infection) or AIM2 inducer ( Poly(dA:dT) transfection) (B).",
    "molecules": "Poly(dA:dT)"
  },
  {
    "caption": "WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were primed with LPS and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm.",
    "molecules": "LPS, Nigericin"
  },
  {
    "caption": "BMDMs from Spata2+/+ (WT) and Spata2-/- (KO) mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 1 h. ASC specks were assessed by Immunofluorescence with antibody against ASC and imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm.",
    "molecules": "LPS, Nigericin, Z-YVAD-FMK"
  },
  {
    "caption": "Immunoblot analysis of ASC in soluble cell lysate and DSS cross-linked insoluble fraction of WT or Spata2-KO BMDMs primed with LPS and stimulated without or with nigericin (Nig) for 1 h. Relative levels of ASC in cross-linked samples from LPS+ Nig treated cells were quantitated and shown below.",
    "molecules": "DSS, LPS, Nig, nigericin"
  },
  {
    "caption": "Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS (G)",
    "molecules": "alum"
  },
  {
    "caption": "Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. levels of IL-1β (H) and IL-6 (I) in the peritoneal fluids were measured by ELISA.",
    "molecules": "alum"
  },
  {
    "caption": "ELISA of IL-1β secretion by WT or CYLD KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers.",
    "molecules": "LPS"
  },
  {
    "caption": "BMDMs from WT and CYLD KO mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 30 min. ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck-positive cells was quantified.",
    "molecules": "LPS, Nigericin, Z-YVAD-FMK"
  },
  {
    "caption": "LDH release from WT and Cyld KO BMDMs primed with LPS and stimulated as indicated.",
    "molecules": "LPS"
  },
  {
    "caption": "Immunoblotting analysis of active caspase-1 p20 and IL-1β in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant (F108A) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h.",
    "molecules": "LPS, Nigericin"
  },
  {
    "caption": "Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2, stained with Spata2 and γ-tubulin antibodies and the nuclear dye DAPI. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Confocal immunofluorescence analysis of Spata2 localization in RAW264.7 cells stably expressing GFP-Spata2, stained with γ-tubulin antibody and DAPI. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Confocal microscopy of HEK293 cells stably expressing GFP-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) that were stained with γ-tubulin antibody and DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunoblotting analysis of the active caspase-1 p20 in cell supernatants (Sup) and pro-caspase-1 and Flag-Spata2 proteins in cell extracts (Cell ext) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization (ΔPUB orΔ51-81). Cells were primed with LPS and stimulated with Nigericin for 1 h. Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.",
    "molecules": "LPS, Nigericin"
  },
  {
    "caption": "WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag-NEK7 were untreated (NT), primed with LPS for 4 h, or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblotting. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.",
    "molecules": "LPS, Nigericin"
  },
  {
    "caption": "iBMDMs stably expressing GFP-PLK4 and parental iBMDMs were unstimulated or stimulated with 0.5 ug/ml LPS for 4h. The PLK4 interaction with endogenous CYLD was analyzed by co-immunoprecipitation with anti-GFP antibody.",
    "molecules": "LPS"
  },
  {
    "caption": "Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP-ASC, pretreated for 2 days with DMSO and two different PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM), primed with LPS for 4 h, and stimulated with Nigericin for 30 min. GFP-ASC specks were imaged and quantified (>500 cells counted). Scale bar, 10 µm.",
    "molecules": "Centrinone, Centrinone-B, DMSO, LPS, Nigericin"
  },
  {
    "caption": "Immunoblot analysis of the indicated proteins in cell supernatants (Sup) and cell extracts (Cell ext) of LPS-primed and Nigericin-stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM) (E)",
    "molecules": "Centrinone, Centrinone-B, LPS, Nigericin"
  },
  {
    "caption": "LPS-primed and Nigericin-stimulated iBMDMs stably expressing a control shRNA or 3 different PLK4-specific shRNAs (F).",
    "molecules": "LPS, Nigericin"
  },
  {
    "caption": "Immunoblot analysis of active caspase-1 (p20) generation in HEK293 cells that were transfected with inflammasome components (Flag-tagged NLRP3, NEK7, ASC, and Caspase-1) along with EGFP, GFP-tagged PLK4, PLK4 kinase dead mutant (D154A), or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post-transfection.",
    "molecules": "Nigericin"
  },
  {
    "caption": "WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were pretreated with DMSO or PLK4 inhibitor Centrinone at a concentration of 150nM for 2 days, primed with LPS, and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified.",
    "molecules": "Centrinone, DMSO, LPS, Nigericin"
  },
  {
    "caption": "Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in iBMDMs stably expressing Flag-NEK7 that were transduced with control or PLK4-specific shRNA. The cells were untreated, primed with LPS, or primed with LPS and stimulated with Nigericin. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblot analysis. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.",
    "molecules": "LPS, Nigericin"
  },
  {
    "caption": "Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with HA-NEK7 and Flag-NLRP3 along with (+) or without (-) GFP-PLK4. The cells were either treated (+) or not treated (-) with the PLK4 inhibitor Centrinone.",
    "molecules": "Centrinone"
  },
  {
    "caption": "iBMDMs stably expressing Flag-tagged NEK7 or NEK7 mutant S204A were treated with LPS. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for the interaction between NEK7 and endogenous NLRP3.",
    "molecules": "LPS"
  },
  {
    "caption": "Immunoblot analysis of active caspase-1 (p20) in supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of iBMDMs stably expressing WT NEK7 or NEK7 mutant (S204A), primed with LPS and stimulated with nigericin.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "iBMDMs stably expressing Flag-tagged WT NEK7 or NEK7 mutant S204A were left untreated or stimulated with LPS for indicated time. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 (P-NEK7).",
    "molecules": "Ser, LPS"
  },
  {
    "caption": "iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.",
    "molecules": "Centrinone, Ser, LPS"
  },
  {
    "caption": "iBMDMs stably expressing GFP-tagged PLK4 and parental iBMDMs were left untreated or stimulated with LPS for 4h. Anti-GFP immunoprecipitates were prepared for immunoblot analysis for the interaction between GFP-PLK4 and endogenous NEK7.",
    "molecules": "LPS"
  },
  {
    "caption": "Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors, treated with either DMSO or 150 nM PLK4 inhibitor Centrinone.",
    "molecules": "Centrinone, DMSO"
  },
  {
    "caption": "WT and Spata2 KO iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.",
    "molecules": "Centrinone, Ser, LPS"
  },
  {
    "caption": "TP53INP1-KO (−/−) and WT (+/+) male mice were subjected to a high-fat diet (HFD, 60% fat) or a control diet (CTRL) for 16 weeks. Mice drank tap water or tap water supplemented with NAC (10 mg/ml or 1%).Curves show mice body weight recorded every week. CTRL: P (−/− versus +/+; t = 8w) = 0.047; P (−/− versus +/+; t = 9w) = 0.023. HFD: P (−/− versus +/+; t = 7w) = 0.039; P (−/− versus +/+; t = 8w) = 0.029; P (−/− versus +/+; t = 9w) = 0.021; P (−/− versus +/+; t = 10w) = 0.014; P (−/− versus +/+; t = 11w) = 0.0046; P (−/− versus +/+; t = 12w) = 0.0028; P (−/− versus +/+; t = 13w) = 0.0025; P (−/− versus +/+; t = 14w) = 0.00051; P (−/− versus +/+; t = 15w) = 0.00027; P (−/− versus +/+; t = 16w) = 0.00013.",
    "molecules": "NAC, water"
  },
  {
    "caption": "At the end of protocol, mice were sacrificed; liver and epididymal and renal fat masses were taken and weighed. Histograms show organ weight. Liver: P (−/− versus +/+; HFD) = 0.014; P (HFD versus CTRL; +/+) = 0.00063; P (HFD versus CTRL; −/−) = 0.0010; P (HFD versus CTRL; +/+ NAC) = 0.034; P (HFD versus CTRL; −/− NAC) = 0.027; P (NAC versus no NAC; −/− HFD) = 0.014. Epididymal fat mass: P (−/− versus +/+; HFD) = 0.011; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000017; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0054; P (NAC versus no NAC; +/+ CTRL) = 0.037; P (NAC versus no NAC; −/− HFD) = 0.0025. Renal fat mass: P (−/− versus +/+; HFD) = 0.0041; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000013; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0078; P (NAC versus no NAC; −/− HFD) = 0.0047.",
    "molecules": "NAC"
  },
  {
    "caption": "A, B Histograms show bloodglucose (A) or plasmainsulin (B) levels of 6-h-fasted mice at the beginning (Week 0) and/or at the end of the protocol (Week 16). Fasting bloodglucose week 16: P (−/− versus +/+; HFD) = 0.0052; P (CTRL versus HFD; −/−) = 0.000081; P (NAC versus no NAC; −/− HFD) = 0.0019; P (w16 versus w0; +/+ CTRL) = 0.012; P (w16 versus w0; −/− CTRL) = 0.050; P (w16 versus w0; −/− HFD) = 0.0023. Fasting plasmainsulin: P (−/− versus +/+; HFD) = 0.043; P (CTRL versus HFD; +/+) = 0.028; P (CTRL versus HFD; −/−) = 0.013; P (CTRL versus HFD; +/+ NAC) = 0.011; P (CTRL versus HFD; −/− NAC) = 0.0015; P (NAC versus no NAC; −/− HFD) = 0.038.",
    "molecules": "glucose, insulin, NAC"
  },
  {
    "caption": "C Glucose tolerance test (GTT) was performed on 6-h-fasted mice during 120 min after injection of 1 g glucose/kg of body weight. Curves on the left show blood glucose level monitored after injection of glucose. Histograms on the right show area under curve (AUC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 0 min) = 0.045; P (−/− versus +/+; CTRL; t = 15 min) = 0.013; P (−/− versus +/+; CTRL; t = 30 min) = 0.016; P (−/− versus +/+; CTRL; t = 60 min) = 0.030; P (−/− versus +/+; CTRL; t = 90 min) = 0.017; P (−/− versus +/+; HFD; t = 60 min) = 0.041; P (−/− versus +/+; HFD; t = 90 min) = 0.043; P (−/− versus +/+; HFD; t = 120 min) = 0.034; P (HFD versus CTRL; +/+; t = 15 min) = 0.0076; P (HFD versus CTRL; +/+; t = 30 min) = 0.0067; P (HFD versus CTRL; +/+; t = 60 min) = 0.00058; P (HFD versus CTRL; +/+; t = 90 min) = 0.0010; P (HFD versus CTRL; +/+; t = 120 min) = 0.023; P (HFD versus CTRL; −/−; t = 60 min) = 0.032. AUC: P (−/− versus +/+; CTRL) = 0.023; P (−/− versus +/+; HFD) = 0.035; P (HFD versus CTRL; +/+) = 0.042.D Insulin tolerance test (ITT) was performed on 6-h-fasted mice during 150 min after injection of 0.70 U insulin/kg of body weight. Curves on the left show blood glucose level monitored after injection of insulin. Histograms on the right show area above curve (AAC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 15 min = 0.012; P (−/− versus +/+; CTRL; t = 30 min) = 0.022; P (−/− versus +/+; HFD; t = 0 min) = 0.027; P (−/− versus +/+; HFD; t = 15 min) = 0.011; P (−/− versus +/+; HFD; t = 30 min) = 0.0037; P (−/− versus +/+; HFD; t = 60 min) = 0.0028; P (−/− versus +/+; HFD; t = 90 min) = 0.041; P (−/− versus +/+; HFD; t = 120 min) = 0.0032; P (−/− versus +/+; HFD; t = 150 min) = 0.0025; P (HFD versus CTRL; +/+; t = 15 min) = 0.0082; P (HFD versus CTRL; +/+; t = 30 min) = 0.033; P (HFD versus CTRL; +/+; t = 90 min) = 0.047; P (HFD versus CTRL; +/+; t = 150 min) = 0.028; P (HFD versus CTRL; −/−; t = 15 min) = 0.026; P (HFD versus CTRL; −/−; t = 30 min) = 0.0095; P (HFD versus CTRL; −/−; t = 60 min) = 0.0031; P (HFD versus CTRL; −/−; t = 90 min) = 0.033; P (HFD versus CTRL; −/−; t = 120 min) = 0.0068; P (HFD versus CTRL; −/−; t = 150 min) = 0.0082. AAC: P (−/− versus +/+; HFD) = 0.030; P (HFD versus CTRL; −/−) = 0.037.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "A, B (A, B) Immunocytofluorescent staining of TP53INP1 (red) and insulin (green) in mouse pancreatic sections (A) and single human islet beta cell (B). Scale bars represent 50 μm (A) and 10 μm (B).",
    "molecules": "insulin"
  },
  {
    "caption": "Immortalized MEFs (MEFi) deficient (−/−) or not (+/+) for TP53INP1 were left untreated (NT) or treated with 3-methyladenine (3-MA, 5 mM) during 4 h.Histograms show mt ROS level measured by flow cytometry upon MitoSox staining. P (−/− versus +/+; NT) = 0.019; P (−/− versus +/+; 3-MA) = 0.025; P (3-MA versus NT; +/+) = 0.031.Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker staining. P (−/− versus +/+; NT) = 0.012; P (3-MA versus NT; +/+) = 0.036.Histogram shows MitoSox fluorescence normalized with MitoTracker fluorescence. P (−/− versus +/+; NT) = 0.041; P (−/− versus +/+; 3-MA) = 0.030; P (3-MA versus NT; +/+) = 0.045.",
    "molecules": "3-MA, 3-methyladenine, ROS"
  },
  {
    "caption": "After 4 h recovering in normal media, H2O2 (1 h, 100 μM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were observed by transmission electron microscopy (TEM). N = nucleus; white arrow = mitophagic vacuoles. Scale bar represents 0.5 μm.Mean size of mitochondrion (area), number of mitochondria and mitophagic vacuoles normalized by cytoplasmic surface area were quantified. Size: P (−/− versus +/+; H2O2) = 0.000027; P (H2O2 versus NT; +/+) = 0.0070; P (H2O2 versus NT; −/−) = 0.013. Nb mito.: P (−/− versus +/+; NT) = 0.035; P (−/− versus +/+; H2O2) = 0.016. Nb vacuoles.: P (−/− versus +/+; NT) = 0.047.",
    "molecules": "H2O2"
  },
  {
    "caption": "After 4 h recovering in normal media, TCLs from H2O2- (1 h, 100 μM), NAC- (24 h, 10 mM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were analyzed by immunoblotting for PGC-1α, PINK1, PARKIN, BNIP3, BNIP3L/NIX, VDAC1 and β-tubulin.",
    "molecules": "H2O2, NAC"
  },
  {
    "caption": "A-D High-resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient (−/−, black bars) or not (+/+, white bars) using glucose (A), lipid-related substrates (C) or complex IV substrate (D). High-resolution respirometry was also performed on intact MEFi (B). For different respiratory state details (routine, 3, leak, and ETS capacity), please refer to the Materials and Methods section. Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A): P (−/− versus +/+; State 3) = 0.0079; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (B): P (−/− versus +/+; routine) = 0.0159; P (−/− versus +/+; leak) = 0.0159; P (−/− versus +/+; ETS) = 0.0159; n = 5 in each group. In (C): P (−/− versus +/+; State 3) = 0.029; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (D): P (−/− versus +/+; ETS) = 0.0259; n = 5 in each group. *TP53INP1−/− versus TP53INP1 +/+; 1 character: P < 0.05; 2 characters: P < 0.01.",
    "molecules": "complex IV substrate, glucose, lipid"
  },
  {
    "caption": "MEFi deficient (−/−) or not (+/+) for TP53INP1 were seeded in media containing or not 10 mM NAC. Forty-eight hours later, MEFi were treated or not (NT) with 100 μM hydrogen peroxide (H2O2) during 1 h in serum free media. Cells were left to recover for 4 h in normal media containing or not NAC (10 mM), GW-9662 (PPARγ inhibitor, 10 μM) or PNU-74654 (Wnt/β-catenin pathway inhibitor, 50 μM) before being harvested. Cells were observed by TEM.Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure. Scale bar represents 0.5 μm. Results are expressed as the mean ± SEM and are representative of three independent experiments. *P (−/− versus +/+; NT) = 0.00086; #P (H2O2 versus NT; +/+) = 0.013.",
    "molecules": "PNU-74654, GW-9662, H2O2, hydrogen peroxide, NAC"
  },
  {
    "caption": "Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell  stimulation was performed through  CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human  pan T cell RNA was isolated and cell culture supernatant was sampled. A mRNA expression  of CD4+  cytokines  Il2, IL4,  IL8 and IL22 relative to endogenous controls, n=13/11/10/8 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "glucose, beta-hydroxybutyrate, BHB"
  },
  {
    "caption": "Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose  (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell  stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8.  Human pan T cell RNA was isolated and cell culture supernatant was sampled. D mRNA expression of CD8+ cytokines IFNγ,  PRF1,  TNFα, GZMB and CTLA4  in stimulated human T cells relative to internal controls, n=15/14/10/14/9 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "glucose, beta-hydroxybutyrate, BHB"
  },
  {
    "caption": "Human peripheral blood mononuclear cells  (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell  stimulation was performed through CD3/CD28 Dynabeads  at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. F  Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative  Foxp3  histogram plot (right side), n=7/11 biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "glucose, beta-hydroxybutyrate, BHB"
  },
  {
    "caption": "Human peripheral blood mononuclear cells  (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl  glucose  (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell  stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled. G  IL10 and TGFβ1 mRNA and protein expression of  human Treg cells, n=8/9 (IL10), 5/9 (TGFβ1) biological replicates. Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate. ",
    "molecules": "glucose, beta-hydroxybutyrate, BHB"
  },
  {
    "caption": "Human PBMCs were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate  (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan T cells, CD4+ and CD8+ T cells were isolated through magnetic cell separation. Mitochondrial  metabolism was analyzed for each subpopulation using a Seahorse XF96  Analyzer. (A-C) OCR, maximum respiration,   spare respiratory capacity  and basal respiration were measured in A pan  T cells, B CD4+ T cells and C CD8+ T cells, n=9 (pan T), n=3/4 (CD4 unstimulated), n=7/8/9 (CD4 stimulated), n=5 (CD8 unstimulated) and n=8/7/7 (CD8 stimulated) individual experiments. Data depicted as mean ± SEM (OCR) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate. ",
    "molecules": "Seahorse XF96, glucose, beta-hydroxybutyrate, BHB"
  },
  {
    "caption": "Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose  (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. A Quantification of cellular reactive oxygen species (ROS) using CellROX   dye, indicated by  mean fluorescence intensity   (MFI)  FITC in  human  pan/CD4+/CD8+ T cells, n=8/6/6 biological replicates. B Mitochondrial superoxide production quantified through  MitoSOX staining, depicted as MFI Phycoerythrin (PE) in human pan/CD4+/CD8+ T cells, n=8/14/14 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "CellROX, MitoSOX, FITC, glucose, beta-hydroxybutyrate, BHB, PE, Phycoerythrin, reactive oxygen species, ROS, superoxide"
  },
  {
    "caption": "Human peripheral blood mononuclear cells  (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl  glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. C Confocal microscopy images  of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.",
    "molecules": "glucose, MitoTracker green, beta-hydroxybutyrate, BHB"
  },
  {
    "caption": "Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Flow cytometry analyses were performed using the indicated dyes and antibodies identifying  T cells and CD4+/CD8+ T cell subsets. K Flow cytometric  quantification of memory T cells:  CD4+/CD8+ T cells were stained  for CCR7 (PE+/-) and subsequently defined memory phenotype for  CD45RO+ (Pacific Blue+) and  CD45RA- (PerCP-) staining. Histogram plots depicting the exemplary change of  CD45RO and CD45RA distribution for CD8+ T cells  following memory cell differentiation (left side; NC=black, BHB=red). Fractions of CCR7+CD45RA-CD45RO+ central memory (CM) and CCR7-CD45RA-CD45RO+ effector memory (EM) CD4+/8+ T cells (right side), n=12/11/8/7 biological replicates. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate. ",
    "molecules": "glucose, Pacific Blue, PerCP, beta-hydroxybutyrate, BHB, PE"
  },
  {
    "caption": "Healthy volunteers conducted a three week KD with a limited carbohydrate  consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell  stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. A IL2, IL4, and CTLA4 mRNA expression in stimulated CD4+ T cells, n=17/18/17 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "carbohydrate"
  },
  {
    "caption": "Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood  was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated.  T cell  stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. B Tbet and GATA3 mRNA expression in CD4+ T cells, n=7 individual human  subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "carbohydrate"
  },
  {
    "caption": "Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell  stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. C Flow cytometric quantification of  Th1/Th2 cells and respective ratio of Th1/Th2 cells, n=7 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "carbohydrate"
  },
  {
    "caption": "Healthy volunteers conducted a three week KD with a limited  carbohydrate consumption of <30g per day. Blood  was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads  at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. D IL10 mRNA expression and flow cytometric quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg), n=19/9individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "carbohydrate"
  },
  {
    "caption": "Healthy volunteers conducted a three week KD with a limited  carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell  stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. E IFNγ GZMB, PRF1 and CTLA4 mRNA expression in CD8+ T cells, n=16/18/17/17 individual human subjects. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "carbohydrate"
  },
  {
    "caption": "Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1).  PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling. F relative CD8+ cell lysis activity as measured by calcein-fluorescence of isolated T cells, n=4 individual experiments. Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate. ",
    "molecules": "calcein, carbohydrate"
  },
  {
    "caption": "44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day.  Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs  were isolated.  T cell  stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial  metabolism was analyzed for each subpopulation using a Seahorse XF96  Analyzer. (A, B) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A CD4+ and B CD8+ T cells, n=14 individual experiments, each performed in three technical replicates. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range , crosses indicating mean Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "Seahorse XF96, carbohydrate"
  },
  {
    "caption": "44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through  CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial  metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. E Western Blot of mitochondrial oxidative phosphorylation proteins in CD4+/CD8+ T cells as indicated, representative of five individual experiments.",
    "molecules": "Seahorse XF96, carbohydrate"
  },
  {
    "caption": "44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood  was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through  CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. F Confocal microscopy images  of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.",
    "molecules": "Seahorse XF96, carbohydrate, MitoTracker green"
  },
  {
    "caption": "44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer. H TruCulture IFNγ, IL4, IL6,  IL8, IL12 subunit p40, IL23 and TNFα protein quantification of LPS-stimulated whole blood samples, n=11/12/11/10/11/10/11 individual human subjects. Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.",
    "molecules": "Seahorse XF96, carbohydrate, LPS"
  },
  {
    "caption": "Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm.",
    "molecules": "DMSO, R406"
  },
  {
    "caption": "RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3.",
    "molecules": "DMSO, R1, R406"
  },
  {
    "caption": "RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3.",
    "molecules": "R406, FK506"
  },
  {
    "caption": "Metabolite abundance of SAM, SAH in samples treated with DMSO and R406. n = 6.",
    "molecules": "DMSO, R406, SAH, SAM"
  },
  {
    "caption": "Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm. Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm.",
    "molecules": "HA, NAC, R406"
  },
  {
    "caption": "RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.",
    "molecules": "HA, NAC"
  },
  {
    "caption": "Fluorescence images of DCFH-DA staining for ROS levels in cells treated with DMSO, HA, NAC, and R406 on d12 (E) and the quantification of the ROS levels (F). n = 4. Scale bar, 100 μm. MDA quantification in cells treated with DMSO, HA, NAC, and R406 on d12. n = 9.",
    "molecules": "DMSO, DCFH-DA, HA, MDA, NAC, R406, ROS"
  },
  {
    "caption": "(B) Contribution of  somatic mutational signatures per tumor  and tumoroid  model. SBS = single base substitution, TMZ  = temozolomide, T = tumor, O = tumoroid.",
    "molecules": "temozolomide, TMZ"
  },
  {
    "caption": "(C)  Dose-response curve of TP53  WT and KO cells treated with the Chk-1 inhibitor prexasertib. Thin lines with numbers indicate individual biological replicates (n = 3) while thick lines indicate fitted lines over all replicates. The statistical significance of the differences in fitted IC50  values between WT and KO were obtained using a two-sided t-test (p = 0.008).",
    "molecules": "prexasertib"
  },
  {
    "caption": "B. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-laser irradiated (10 µM TMP, 2 h followed by UVA laser irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.",
    "molecules": "DAPI, TMP"
  },
  {
    "caption": "C. Confocal microscopy images of PrimPol  and γH2AX  (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated cells following control or RPA2 downregulation. Nuclear DNA is  counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.",
    "molecules": "DAPI, TMP"
  },
  {
    "caption": "D. Confocal microscopy images of PrimPol  and γH2AX  (damage control)  immunofluorescence staining in control (UVA) or TMP-UVA-treated PRIMPOL KO cells stably expressing V5-tagged WT or  RPA binding domain mutant (RBDm) PrimPol  versions. Nuclear DNA is  counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of two assays (n ≥100 cells per condition in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.",
    "molecules": "DAPI, TMP"
  },
  {
    "caption": "E.  Confocal microscopy images  of  PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated WT and BLM KO cells upon downregulation of TOP3A, RMI1 and RMI2 (siTR) when indicated. Nuclear  DNA is counterstained with DAPI. UVA laser path  is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥250 cells per condition). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test.",
    "molecules": "DAPI, TMP"
  },
  {
    "caption": "H. Immunoblots showing the levels of BLM, FANCD2 and PrimPol after BLM  or FANCD2 downregulation in WT and PRIMPOL KO cells for experiments in (G). Ponceau-S  is shown as loading control.",
    "molecules": "Ponceau-S"
  },
  {
    "caption": "C. Representative confocal microscopy images  of  EdU staining in WT and PRIMPOL KO cells, 8 h after  UVA (control) or TMP-UVA treatment (2 µM TMP, 2 h followed by 5 s irradiation).  Nuclear DNA is  counterstained with  DAPI. When indicated, V5-PrimPol  was reintroduced into KO cells. Scale bar, 25 µm. Dot plots indicate the distribution of nuclear EdU intensity, and the mean values are indicated by horizontal red lines. Data from the combination of three replicates (n ≥200 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated.",
    "molecules": "EdU, DAPI, TMP"
  },
  {
    "caption": "A. Confocal microscopy images  of FANCD2 immunofluorescence staining in control (UVA) or TMP-UVA-treated (2 µM TMP, 2 h followed by 5 s irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained  with DAPI. Scale bar, 25 µm.",
    "molecules": "DAPI, TMP"
  },
  {
    "caption": "A. Kaplan-Meier survival curves of WT and PRIMPOL  KO  mice after the administration of a single  dose of MMC (7.5, 10 or 15 mg/kg). Dashed vertical lines represent median  survival values for WT (blue) and KO (orange)  mice. Statistical analysis was conducted with Log-rank (Mantel-Cox) test; p-value for the highest dose of MMC (15 mg/kg) is indicated.",
    "molecules": "MMC"
  },
  {
    "caption": "B. Haematoxylin-eosin (H&E) and Ki-67 immunohistochemistry (IHC) stainings of bone marrow  sections derived from WT and PRIMPOL KO mice following the administration of 10 mg/kg  MMC. Control mice (PBS) were sacrificed when MMC-injected mice reached the humane endpoint. Scale bar, 2.5 mm. In each  image, a relevant area is shown at higher magnification (10x).",
    "molecules": "eosin, Haematoxylin, MMC, PBS"
  },
  {
    "caption": "E. IF detection of FANCD2 foci in WT and  PRIMPOL  KO cells 48 h after TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation) with or without MCM3 downregulation. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.",
    "molecules": "DAPI, TMP"
  },
  {
    "caption": "G. Cell survival assays of WT and PRIMPOL KO cells with or without MCM3 downregulation in the presence of increasing amounts of MMC  or TMP (followed by 90 sec UVA irradiation) for 72 h. The same control curves without MCM3 downregulation are also shown as part of Figure EV6D. Each point in the curve represents the percentage of surviving cells (average and SD of three assays for MMC and two assays for TMP). Statistical analysis was conducted with two-way ANOVA followed by Bonferroni post-test. p-values of comparisons between KO and KO-siMCM3 are indicated.",
    "molecules": "MMC, TMP"
  },
  {
    "caption": "A  Cell cycle  analysis by flow cytometry (EdU vs. TO-PRO-3) in complete medium (ENRAD) or in the absence of A83-01 (24 hours).",
    "molecules": "EdU, A83-01, TO-PRO-3"
  },
  {
    "caption": "D Representative stereoscopic images of  mouse prostate organoids cultured in the absence of A83-01, with or without Follistatin  (Fst, 500 ng/mL, 6 days), or with or without the  Activin A  neutralizing antibody MAB3381  (1 μg/mL, 6 days). Scale bar = 1 mm",
    "molecules": "A83-01, Activin A"
  },
  {
    "caption": "E Western blot  (WB) analysis in mouse prostate organoids  for selected canonical and non-canonical Tgf-β  signalling mediators, and the cell cycle inhibitor p21  (Activin A, 50 ng/mL; Tgfb1, 500 ng/mL; 24 hours).",
    "molecules": "Activin A"
  },
  {
    "caption": "G Representative stereoscopic images of  mouse prostate organoids following treatment with  Takinib (Tak1 inhibitor; 5 μM, 6 days), SB203580 (p38α/β inhibitor; 10 μM, 6 days) or the structurally unrelated Ralimetinib (p38α/β inhibitor; 1 μM, 7 days). Scale bar = 1 mm",
    "molecules": "Ralimetinib, SB203580, Takinib"
  },
  {
    "caption": "H Nuclear/Cytoplasmic fractionation and western blot  analysis in mouse prostate organoids  for selected signalling mediators in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 hours).",
    "molecules": "SB203580"
  },
  {
    "caption": "I Gene set enrichment analysis (GSEA) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor  (TNF) signalling in mouse prostate organoids cultured without A83-01 (24 hours) versus complete medium (ENRAD).",
    "molecules": "A83-01"
  },
  {
    "caption": "J Nuclear/Cytoplasmic fractionation and  western blot  analysis in  mouse prostate organoids for selected cell cycle regulators, in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 or 48 hours).",
    "molecules": "SB203580"
  },
  {
    "caption": "A Diagram depicting the expansion of mouse prostate organoid  cultures in the absence of the  Tgf-β  ligand inhibitor A83-01 (n = 3 biological replicates per lobe-specific  organoid  culture; arrow = continuous expansion; dot = passage; cross = culture loss).",
    "molecules": "A83-01"
  },
  {
    "caption": "B Representative stereoscopic  images of C57BL/6J  DLP #1 and #3  mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mm",
    "molecules": "A83-01"
  },
  {
    "caption": "D Gene set enrichment analysis (GSEA) plot displaying significant enrichment for activation of ATR signalling in  mouse prostate organoid  lines (C57#1 DLP, top; C57#3 DLP, bottom) adapted to grow in the absence of A83-01 vs. normal control  organoids cultured in complete medium (ENRAD).",
    "molecules": "A83-01"
  },
  {
    "caption": "E Western blot  analysis in C57#1 and C57#3  DLP mouse prostate organoid  lines adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD).  Immunoblots are displayed for Chek1 (ATR  signalling mediator), Trp53 and p21.",
    "molecules": "A83-01"
  },
  {
    "caption": "F Representative spectral karyotype (SKY) images of metaphase spreads obtained from C57#1  DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread genomic instability is observed following adaptation. Scale bars = 5 µm. G-H Representative telomere FISH images - and quantification (n = 2 biological replicates, 20 metaphases per condition) - in C57#1 DLP mouse prostate organoids  cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread telomeric instability is observed following adaptation (arrows indicate telomere doublets, One-way ANOVA, scale bars = 10 µm).",
    "molecules": "A83-01"
  },
  {
    "caption": "(h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies.",
    "molecules": "forskolin, FRSK, rapamycin"
  },
  {
    "caption": "(i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.",
    "molecules": "forskolin"
  },
  {
    "caption": "(a) Pseudocolour-coded images of EPACI-camps FRET from real-time imaging of wild-type MEFs transfected with EPACI-camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments.",
    "molecules": "forskolin, FRSK"
  },
  {
    "caption": "(c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.",
    "molecules": "forskolin, H89"
  },
  {
    "caption": "(d) Representative images of the effect of H89 on mitochondrial morphology on starvation. Wild-type and Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. (e) Morphometric analysis. Experiments were carried out as in d. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition).",
    "molecules": "H89"
  },
  {
    "caption": "(f) Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (g) Morphometric analysis of mitochondrial shape. Experiments were carried out as in f. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).",
    "molecules": "H89"
  },
  {
    "caption": "(h) Representative confocal micrographs of mitochondrial morphology of Drp1−/− MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (i) Morphometric analysis of mitochondrial shape. Experiments were carried out as in h. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).",
    "molecules": "H89"
  },
  {
    "caption": "(e) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0.5 μM wortmannin (Wortm.). Lysates from 2.7×105 cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.",
    "molecules": "wortmannin"
  },
  {
    "caption": "(a,b) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype. Where indicated, cells were starved for 5 h before TMRM loading. Where indicated (arrows), 2.5 μg ml−1 oligomycin and 2 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added. Data represent mean ± s.e.m. of seven independent experiments.",
    "molecules": "FCCP, oligomycin"
  },
  {
    "caption": "(c) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "(d) Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments and are normalized to the initial value.",
    "molecules": "ATP"
  },
  {
    "caption": "(g) MEFs of the indicated genotype were starved for 2.5 h. Where indicated, cells were treated with 20 μM H89. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.",
    "molecules": "H89"
  },
  {
    "caption": "(j,k) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.",
    "molecules": "oligomycin"
  },
  {
    "caption": "(a) Blue-native electrophoresis analysis of ATPase dimerization and activity. Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4% digitonin and proteins were separated by blue-native PAGE. ATPase activity was measured in-gel (top) and ATPase levels were measured by immunoblotting for the indicated antibody (bottom). (b,c) Quantitative analysis of the ratio of levels (b) and activity (c) between dimeric and monomeric forms of ATPase, assessed by densitometric analysis of gels from experiments performed as in a. Data represent mean ± s.e.m. of five independent experiments. (d) Experiments were carried out as in a, but with cells of the indicated genotype.",
    "molecules": "digitonin"
  },
  {
    "caption": "The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test).",
    "molecules": "BrdU, eosin, hematoxylin"
  },
  {
    "caption": "Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm.",
    "molecules": "4-OHT"
  },
  {
    "caption": "The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001.",
    "molecules": "m6A"
  },
  {
    "caption": "Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test.",
    "molecules": "m6A"
  },
  {
    "caption": "Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test.",
    "molecules": "m6A"
  },
  {
    "caption": "MeRIP-qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).",
    "molecules": "m6A"
  },
  {
    "caption": "Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing TCF7L2. Scale bar, 250 μm. Quantification of differentiated versus undifferentiated organoids from (H). Data are represented as mean ± SEM. **p<0. 01, ***p<0. 001 (3 biological replicates, t-test).",
    "molecules": "4-OHT"
  },
  {
    "caption": "Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction.",
    "molecules": "AOM, DSS, TAM"
  },
  {
    "caption": "Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test).",
    "molecules": "AOM, DSS, TAM"
  },
  {
    "caption": "Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.",
    "molecules": "TAM"
  },
  {
    "caption": "Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).",
    "molecules": "TAM"
  },
  {
    "caption": "(D) Patients suffering from anorexia and admitted to treatment show a blunted ACTH response and hypercortisolemia, which resolves within 6-24 months after weight normalization- data from (Gold et al, 1986a). However, 3-4 weeks after weight normalization, cortisol dynamics are normal whereas ACTH dynamics are blunted. Pregnancy is associated with elevated cortisol levels due to CRH secretion by the placenta. 3 weeks after delivery, cortisol levels and dynamics return to normal, whereas ACTH dynamics are blunted- data from (Magiakou et al, 1996). After 12 weeks, ACTH dynamics normalize as well, while cortisol dynamics remain normal. Individuals recovering from alcohol abuse show hypercortisolemia and blunted ACTH response after admission - data from (von Bardeleben et al, 1989). After 2-6 weeks, these individuals show normal cortisol dynamics, but blunted ACTH responses persist. In all panels, control patient data are denoted by thin gray line (Anorexia: N=13. Pregnancy: N was unspecified. Alcohol abuse disorder: N=11), and case data by a thicker black line (Anorexia: left panel, N=9, center panel, N=5, right panel, N=6. Pregnancy: N=17. Alcohol abuse disorder: N=20). Shown are mean ± SEM for all panels.",
    "molecules": "cortisol"
  },
  {
    "caption": "C. Generation of sFn14 is sensitive to the γ-secretase  inhibitor DAPT. HEK293E  cells were transfected with either Fn14  or C99  (C-terminal fragment of APP), both containing a N-terminal HA-tag and C-terminal double FLAG-tag. One day after transfection, cells were treated with γ-secretase  inhibitor DAPT  (1 μM), broad-spectrum metalloprotease  inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO  as indicated. The conditioned media and the lysates  were blotted  with anti-HA  antibody. Shown are representative blots   from N=4 experiments.",
    "molecules": "DAPT, DMSO, TAPI-1"
  },
  {
    "caption": "A. Human breast cancer  cell line MDA-MB-231 shows cellular accumulation of Fn14  upon γ-secretase inhibition. The cells were treated overnight with  γ-secretase inhibitor DAPT  (1 μM), broad spectrum metalloprotease  inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO  as indicated. Lysates were blotted  for Fn14  with an antibody that targets the C-terminal end of the protein, or against calnexin  as loading control. The asterisk labels an N-terminally truncated form of Fn14. B. Quantification of blots  from panel A. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels B the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.",
    "molecules": "DAPT, DMSO, TAPI-1"
  },
  {
    "caption": "C. Human ovarian cancer  cell line  SKOV-3 shows cellular accumulation of Fn14  upon  γ-secretase  inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT  (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or corresponding amount of vehicle DMSO  as indicated. Lysates were blotted  for Fn14  with an antibody that targets the C-terminal end of the protein, or against calnexin  as loading control. The asterisk labels an N-terminally truncated form of Fn14. D. Quantification of blot  from panel C. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels D, the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.",
    "molecules": "DAPT, DMSO, TAPI-1"
  },
  {
    "caption": "A. MDA-MB-231 cells were transfected with an siRNA pool against human Fn14  or non-targeting control (Ctrl) siRNA. A day after transfection, the cells were treated with  γ-secretase inhibitor DAPT  (1 μM) or vehicle overnight. The lysate was collected and blotted  against Fn14  (C-terminal antibody) or calnexin  as loading control. Shown are representative blots  from N=4 experiments. B. Quantification of blot  in panel A. The control condition where the cells were only treated with vehicle DMSO  and non-targeting siRNA (DMSO  + siCtrl) was used as baseline, and its average normalized to 1. N=4 experiments. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against control (DMSO  + siCtrl) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.",
    "molecules": "DAPT, DMSO"
  },
  {
    "caption": "A. Cellular Fn14  in ex vivo glioblastoma  samples increased upon  γ-secretase inhibition. Primary cells from four different glioblastomas  were treated with DAPT  (1 μM) or vehicle overnight. Lysates were blotted  against Fn14  and calnexin  as loading control. B. Quantification of the blot  in panel A. For each glioblastoma, the relative (rel.) mean intensity of the normalized Fn14  vehicle condition was used for normalization. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against their corresponding control (DMSO) condition using two-tailed unpaired t-tests. The p-values that are above 0.05 have not been included into the panels. For all the panels, three biological replicates were performed.",
    "molecules": "DAPT, DMSO"
  },
  {
    "caption": "D. Serum  samples from 10 patients  with refractory   multiple myeloma were collected pre- and post-treatment  with 25 mg LY3039478  administered in 3 daily doses over 5 days. sFn14 levels in these samples are displayed (N=10). Data information: For panel D, ratio-paired t-test was used to compare the sFn14 levels pre- and post-treatment.",
    "molecules": "LY3039478"
  },
  {
    "caption": "E) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-GFP and α-HA or α-FLAG antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores. Bottom panel. GFP::ZNFX-1 and HA::TagRFP::ZNFX-1 expressing animals were crossed and germlines of trans-heterozygous progeny from this cross were fixed and stained and imaged by STED as described above. Scale bar, 0.5μm.",
    "molecules": "CF430, RFP, Alexa 488"
  },
  {
    "caption": "(B-D) Micrographs of H9C2  cells corresponding to (B) expressions of single species: MuRF1-mCherry (red),  titin EGFP-A168-TK (green),  Venus-Nbr1 (yellow) or Cerulean-p62 (cyan); (C) Co-expression of titin EGFP-A168-TK  with mCherry-MuRF1. Neither Nbr1 nor p62  were co-transfected. The titin fragment and MuRF1 co-localize forming filamentous formations, which is in agreement with their known complexation; (D) Co-expression  of  MuRF1-mCherry  (red), titin EGFP-A168-TK  (green) and  Venus-Nbr1  (yellow) or Cerulean-p62 (cyan) or respective truncated variants of the latter lacking the ubiquitin binding domain (∆UBA). (Throughout, cell nuclei are stained with DAPI; blue). (Scale bar is 10 μm in all cases).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. The L. monocytogenes EGD-e wild-type strain (WT), the ΔinlK mutant (ΔinlK) and the complemented strain (ΔinlK+pPL2 inlK) 104 CFU were inoculated i.v into BALB/c mice. Animals were euthanized 24 h, 48 h, 72 h or 96 h after infection and organs were recovered, homogenized, and homogenates serially plated on BHI. The number of bacteria able to colonize liver (left panel) and spleen (right panel) is expressed as log10 CFU. Four animals per bacterial strain, per time points and per experiment were used. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.",
    "molecules": "BHI"
  },
  {
    "caption": "B. Detection by immunofluorescence microscopy of InlK over-expressing in L. monocytogenes EGD-e (WT), ΔinlK, WT+pADc-inlK, ΔinlK+pPRT-inlK and the ΔsrtA mutant over-expressing inlK (ΔsrtA+pPRT-inlK) grown in BHI medium using the rabbit polyclonal anti-InlK antibody. InlK was detected at the surface of InlK over-expressing bacteria (WT+pADc-inlK and ΔinlK+pPRT-inlK), whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over-expressing inlK.",
    "molecules": "BHI"
  },
  {
    "caption": "C. Detection of InlK by Western blot on total lysates of L. monocytogenes EGD-e (WT), ΔinlK and ΔinlK+pPRT-inlK grown in BHI using the rabbit polyclonal anti-InlK antibody. Decreased concentrations of recombinant purified InlK were used as a positive control.",
    "molecules": "BHI"
  },
  {
    "caption": "D. Detection of secreted InlK in the supernatant of ΔsrtA mutants over-expressing InlK. Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA+pPRT-inlK culture (OD600 = 1) supernatants using the rabbit polyclonal anti-InlK antibody.",
    "molecules": "trichloroacetic acid"
  },
  {
    "caption": "A. InlK and ActA expression in Listeria strains used for survival assays. Total lysates of L. monocytogenes EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) grown in BHI were immunoblotted using anti-ActA and anti-InlK antibodies.",
    "molecules": "BHI"
  },
  {
    "caption": " A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA -Dendra2- full length nucleolin; Ncl ΔGAR - HA-Dendra2- nucleolin with a GAR domain deletion; Ncl GAR(N) - HA-Dendra2- nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN - Dendra2. Note that HA input blots for HA-Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE. B. Quantification of (A). n = 3 independent biological repeats; means ± SEM; **** p < 0.0001, ANOVA with Tukey's post-test.",
    "molecules": "arginines, asparagines"
  },
  {
    "caption": " D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm. E. Quantification of (D). n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test a. u. - arbitrary units. ",
    "molecules": "TAMRA"
  },
  {
    "caption": " G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl FL), HA-Dendra2-nucleolin with a GAR domain deletion (Ncl ΔGAR) or HA-Dendra2-nucleolin with all 10 arginines in the GAR domain mutated to asparagines (Ncl GAR(N)) and processed as in (F). H. Quantification of (G). Levels of nucleolin in pulldowns were normalized to input levels and expressed as % relative to full-length HA-Dendra2-nucleolin. n = 4 independent biological repeats; means ± SEM; **** p<0.0001, ANOVA with Dunnett's post-test.",
    "molecules": "arginines, asparagines"
  },
  {
    "caption": " A. GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons, transduced with the peripheral neuron specific AAV-PHP.S vector expressing full length HA-Dendra2-nucleolin (Ncl FL) or respective ΔGAR mutant (Ncl ΔGAR) upon seeding and analyzed by epifluorescence microscopy 9-10 days later. Shown are representative Dendra2 fluorescence images collected from the non-activated (green) emission line. Hoechst 33342 (10 µM) was used to outline nuclei. Superimposed phase contrast and Dendra2 fluorescence images are shown on the right. Scale bar - 25 µm. B. Quantification of the nucleolar/nuclear intensity ratios in DRG cells shown in (A). n = 38-54 cells per sample in 3 independent biological repeats; means ± SEM; **** p<0.0001, Mann-Whitney test ",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": " C. Time-lapse live imaging of nuclei in DRG neurons transduced with AAV-PHP.S HA-Dendra2-nucleolin (full length and ΔGAR mutant), after a hypotonic challenge with double-distilled water (ddH20). Shown are epifluorescence images (Dendra2 green emission line) taken at indicated times after medium replacement with ddH20. Scale bar - 10 µm. D. Quantification of nucleolar Dendra2 signal in (C) with intensity values immediately before medium replacement set as 100%. n = 17-24 cells per sample in 3 biological repeats; means ± SEM; Two-way ANOVA with Sidák's post-test detected a significant time-dependent reduction of nucleolar signal (p < 0.0001) and no difference between full length and ΔGAR mutant nucleolin (p > 0.05) ",
    "molecules": "ddH20, double-distilled water"
  },
  {
    "caption": " A, B. Representative maximum projections of exposure matched confocal images of immunofluorescence for nucleolin and neurofilament (NF) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR+/- mice shown in (A). Upper panels show total nucleolin stain. Middle panels show merged image of nucleolin (gray), NF and Tuj1 (magenta), and DAPI (blue). Lower panels show nucleolin overlaps with NF and Tuj1 signals as the "axon only" signal. B shows quantification of nucleolin immunofluorescence with approximately a 50% reduction in nucleolin in the axons of GAR+/- mice in vivo. n = 3 WT, n = 4 GAR+/-; means ± SEM; * p < 0.05, unpaired Student's t-test. Scale bar - 10 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " C-F. Representative, exposure matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR+/- mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled \"axon only\" signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR+/- mice. n = 3 WT, n = 3 GAR+/-; means ± SEM; ** p < 0.01, *** p < 0.001, ****p < 0.0001 unpaired Student's t-test. Scale bar - 10 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " D. Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48h with 10 µM AS1411 or 10 µM control aptamer, replated and grown for 18h. FISH signal is shown in gray, cell somata (left) and axons (right) are visualized by neurofilament immunostaining (magenta). Scr - scrambled FISH probe served as a negative control. Scale bar - 10 µm. ",
    "molecules": "AS1411"
  },
  {
    "caption": " C. Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV-PHP.s expressing HA-Dendra fused with the wild type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were re-plated 9-10 days after AAV infection. 24h after re-plating cells were fixed and stained with anti-HA (grey) and anti-NFH (magenta) antibodies. Scale bar - 100 μm. D. Quantification of total axonal outgrowth and mean process length in HA-positive cells in (C). Outgrowth measurements were based on NFH staining. n = 6 independent biological repeats; means ± SEM; * p < 0.05, *** p < 0.001 (Student's-test). ",
    "molecules": "arginines, asparagines"
  },
  {
    "caption": "D Western blot  of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1  cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol  for 6 h. Pgk1 served as a loading control.",
    "molecules": "estradiol"
  },
  {
    "caption": "E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1  cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.",
    "molecules": "DTT, estradiol"
  },
  {
    "caption": "A Sec63-mNeon images of cells of the indicated genotypes harboring the inducible system. Cells were treated with 800 nM estradiol  for 6 h. Two examples of each phenotypic class are shown.",
    "molecules": "estradiol"
  },
  {
    "caption": "C Sec63-mNeon and Rtn1-mCherry images of ∆lnp1  and ∆sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h.",
    "molecules": "estradiol"
  },
  {
    "caption": "C Quantification of peripheral  ER  structures in WT and ∆ice2  cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the  times indicated. Bars are the mean percentage of cell cortex  covered by tubules  (purple) or  sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of  tubules  and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.",
    "molecules": "estradiol"
  },
  {
    "caption": "C, D Fluorescence  images of  cortical  sections of WT and Δice2 cells expressing Sec63-mNeon  and  Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).",
    "molecules": "DTT"
  },
  {
    "caption": "D  Growth assays on solid media of WT, ∆hac1, Δice2, and Δhac1 Δice2  cells (SSY1404, 2356, 2805, 2806) in the absence or presence of 0.2 µg/ml tunicamycin. For each series, cells were diluted fivefold from one step to the next.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "Original single-cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons (baseline pHi 7.21, 7.13 and 7.11, respectively). Superfusion with CO2/HCO3−-free HEPES solution (upper horizontal bars) evoked an intracellular alkalinization, which in P14 WT was large and suppressed by 100 μM acetazolamide (AZ, lower horizontal bar), indicating the presence of CA activity (see Supplementary Materials and methods). The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide (a poorly permeant CA inhibitor). Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT (scale bar 10 μm).",
    "molecules": "acetazolamide, benzolamide, HEPES"
  },
  {
    "caption": "Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2/HCO3− (horizontal bar, HEPES) in slices from >P35 WT, CAVII KO and CA II/VII KO mice. The rate of rise and the amplitude of the alkaline shift are identical in WT (P39, baseline pHi 7.01) and CAVII KO neurons (P40, 7.06), whereas alkalinization develops much more slowly in the CA II/VII KO (P46, 7.04)",
    "molecules": "HEPES"
  },
  {
    "caption": "Excitatory GABAergic response in WT, but not in CAVII KO neurons. Picrotoxin blocked the biphasic response (PiTX 90 μM; n=3/genotype). The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO (13.53±1.27 versus 6.39±0.96 mV/s, P<0.001, Student's t-test). The resting membrane potential of WT and CAVII KO neurons did not differ (−71.0±1.1 versus −72.3±1.4 mV, respectively; n=15 neurons for both genotypes, P=0.47, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)",
    "molecules": "Picrotoxin, PiTX"
  },
  {
    "caption": "Microinjection of GABA (5 mM for 100 ms, sr/slm border in the presence of CGP55845/TTX, arrowheads) induced a pronounced depolarization in WT neurons (black) that was abolished in the absence of CO2/HCO3− (grey). The depolarization was smaller in CA VII KO (red). Bar diagram illustrates GABAergic mean peak depolarization in P12-16 WT and CAVII KO (13.3±0.5 versus 8.3±0.6 mV, P<0.001, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D).",
    "molecules": "CGP55845, GABA, TTX"
  },
  {
    "caption": "In the presence of AP5/CNQX/CGP55845, microinjection of GABA (5 mM, sr/slm border) triggered SR 95531-sensitive field potential spikes in WT slices already upon 8-ms injection, while even 16-ms injection evoked hardly any spikes in CAVII KO (left). Mean number of spikes was plotted against GABA puff duration in P12-16 WT and CAVII KO (P<0.01 and P<0.001, Student's t-test; right). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)",
    "molecules": "CNQX, CGP55845, GABA, AP5, SR 95531"
  },
  {
    "caption": "After P35, depolarizations upon GABA microinjection (arrowheads) in the presence of CGP55845/TTX were indistinguishable in WT (black), CA VII KO (red) and CA II KO (blue) but smaller in CA II/VII KO (green). The depolarization in WT was abolished in the absence of CO2/HCO3− (grey, n=5). Bar diagram summarizes mean peak depolarization for each genotype (right; ANOVA, Bonferroni). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)",
    "molecules": "CGP55845, GABA, TTX"
  },
  {
    "caption": "Diazepam (DZP, 150 μg/kg) reduced the latency to seizure onset in P14 rat pups (P<0.05). There was no statistical difference between 50 μg/kg and saline-injected animals. DZP 2.5 mg/kg completely blocked the seizures. DZP was given intraperitoneally 15 min before hyperthermia (HT) onset.",
    "molecules": "Diazepam, DZP"
  },
  {
    "caption": "Diazepam given at 150 μg/kg did not affect the baseline breath rate, and HT induced an analogous increase in the breath rate in saline- and diazepam-injected rats (P>0.05). At a higher concentration (2.5 mg/kg), DZP suppressed breathing significantly (P<0.01) and HT caused only a small increase in the breath rate.",
    "molecules": "Diazepam, diazepam, DZP"
  },
  {
    "caption": "In slice preparation, DZP (1 μM) potentiated the high-frequency stimulation (40 pulses at 100 Hz, horizontal bar) -evoked GABAAR-mediated depolarization. Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum-moleculare was given in the presence of AP5, CNQX and CGP 55845. The bar diagrams summarize the effects of DZP on the duration of the depolarization (time to half-maximum) and the number of action potentials associated with the depolarizing phase. P-values (*P<0.05, **P<0.01 and ***P<0.001) are based on ANOVA, Bonferroni (A, B) or Student's t-test (C), and error bars denote s.e.m. The number of animals (A, B) or cells (C) is indicated in bar diagrams.",
    "molecules": "CGP 55845, CNQX, DZP, GABA, AP5"
  },
  {
    "caption": "(H) Representative hematoxylin and eosin (H&E) and p-Fak1 immunohistochemical (IHC) staining of LUAD tumor sections 21 weeks after infection with control sgTom (grade 1 depicted), sgNf1.1 (grade 3 depicted), sgNf1.2 (grade 3 depicted), or sgNf1.3 (grade 3 depicted) (n = 50 tumors each). H&E scale bars (low-magnification top row = 100 μm, high-magnification bottom row = 250 μm); p-Fak1 IHC scale bars (low-magnification top row = 250 μm, high-magnification bottom row = 500 μm).",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates).",
    "molecules": "DOXO, doxorubicin"
  },
  {
    "caption": "(B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates).",
    "molecules": "DOXO"
  },
  {
    "caption": "(D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates).",
    "molecules": "DOXO"
  },
  {
    "caption": "(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates).",
    "molecules": "DOXO"
  },
  {
    "caption": "(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).",
    "molecules": "DOXO"
  },
  {
    "caption": "(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).",
    "molecules": "DOXO"
  },
  {
    "caption": "(A) Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).",
    "molecules": "Glucose, lactate"
  },
  {
    "caption": "(B) Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).",
    "molecules": "Glutamine"
  },
  {
    "caption": "(C) Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units) (n = 4 biological replicates).",
    "molecules": "GPNA"
  },
  {
    "caption": "(D) Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates).",
    "molecules": "glutamine"
  },
  {
    "caption": "Relative viability of KP and KPΔNF1 clones after CB-839 treatment (IC50: 18 nM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).",
    "molecules": "CB-839"
  },
  {
    "caption": "Relative viability of KP and KPΔNF1 clones after AOA treatment (IC50: 6 µM) for 72 h assayed via CellTiter-Glo (relative luminescence units). All data points are relative to vehicle-treated controls (n = 4 biological replicates).",
    "molecules": "AOA"
  },
  {
    "caption": "(D) Cumulative population doublings of KP and KPΔNF1 cells incubated with vehicle, 18 nM CB-839, or 6 µM AOA after 6 d in culture (n = 4 biological replicates). For bar charts, data presented as means with error bars representing standard deviations (SDs).",
    "molecules": "AOA, CB-839"
  },
  {
    "caption": "(E) Relative viability of the indicated human KRAS-mutant lung cancer cell lines by trypan blue exclusion assay. Cells were incubated with vehicle, 18 nM CB-839, or 6 µM AOA for 72 h (n = 4 biological replicates). All data points are normalized to vehicle-treated cell lines. For bar charts, data presented as means with error bars representing standard deviations (SDs).",
    "molecules": "AOA, CB-839"
  },
  {
    "caption": "Quantification by micro-CT of mean tumor volumes in (B) CB-839-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (B)",
    "molecules": "CB-839"
  },
  {
    "caption": "Quantification by micro-CT of mean tumor volumes in (C) AOA-, or vehicle-treated KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom or sgNf1.3 (n = 21 mice and 21 mice, respectively). Vehicle-treated KP mice are the same control group for (C).",
    "molecules": "AOA"
  },
  {
    "caption": "Quantification by micro-CT of total tumor volume per mouse in (D) CB-839-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (D)",
    "molecules": "CB-839"
  },
  {
    "caption": "E) Quantification by micro-CT of total tumor volume per mouse in , (E) AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection. Vehicle-treated KP mice are the same control group for (E).",
    "molecules": "AOA"
  },
  {
    "caption": "Relative responses of KP mice tumors treated with (F) CB-839, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "CB-839"
  },
  {
    "caption": "Relative responses of KP mice tumors treated with (G) AOA, or vehicle starting from week 8 post-infection and measured until week 20. Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "AOA"
  },
  {
    "caption": "(H) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in CB-839-, AOA-, or vehicle-treated KP mice after infection with control sgTom or sgNf1.3 at 20 weeks post-infection. Vehicle-treated KP mice are the same control group in both boxplots. For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.",
    "molecules": "AOA, CB-839"
  },
  {
    "caption": "Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, (B) CB-839, starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "CB-839"
  },
  {
    "caption": ") Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, C) AOA starting from day 12 and measured until day 27 (n = 6 tumors per cell line per treatment) Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "AOA"
  },
  {
    "caption": "Relative responses of subcutaneous KP and KPΔNF1 tumors treated with vehicle, CB-839, or AOA (D) the final masses of these tumors. or boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.",
    "molecules": "AOA, CB-839"
  },
  {
    "caption": "Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (E) CB-839 starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.",
    "molecules": "CB-839"
  },
  {
    "caption": "Relative responses of orthotopic KP and KPΔNF1 tumors treated with vehicle, (F) AOA starting from day 12 and measured until day 27 (n = 24 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.",
    "molecules": "AOA"
  },
  {
    "caption": "Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle, (G) CB-839, with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "cDNA, PIP2, CB-839, DOX, doxycycline"
  },
  {
    "caption": "Relative responses of subcutaneous KP-ix tumors harboring doxycycline (DOX)-inducible gain-of-function (GOF)-Fak1 cDNA treated with vehicle (H) AOA with or without DOX/PIP2 (n = 36 mice per treatment group). Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "cDNA, PIP2, AOA, DOX, doxycycline"
  },
  {
    "caption": "Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (I) CB-839 starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "CB-839"
  },
  {
    "caption": "Relative responses of subcutaneous patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle (J) AOA starting from day 12 and measured until day 30 (n = 6 tumors per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle.",
    "molecules": "AOA"
  },
  {
    "caption": "Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (K) CB-839 starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.",
    "molecules": "CB-839"
  },
  {
    "caption": "Relative responses of orthotopic patient-derived NF1-mutant (PDKN1 and PDKN2) and NF1-WT (PDK) LUAD tumors treated with vehicle, (L) AOA starting from day 12 and measured until day 27 (n = 12 mice per cell line per treatment). Relative response = average tumor volume with treatment / average tumor volume with vehicle. Quantification of tumor growth by photon flux luminescence after orthotopic transplantation with KP or KPΔNF1 cells transduced with a luciferase vector.",
    "molecules": "AOA"
  },
  {
    "caption": "pH-dependent dissociation of E and M. TBEV preparations treated at pH 8 (left) and pH 6 (right) were solubilized with Triton X-100 and subjected to sucrose gradient centrifugation. The fractions were analyzed by SDS-PAGE and densitometry. Representative examples of at least two independent experiments are shown. Positions of E dimers (D) and E trimers (T) in the gradients are indicated.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Difference in deuterium uptake by sE404r and sE419r trimers (D404-D419). Left panel, deuteration difference for each peptide reported against the 1-p value obtained via Student's t-test between the two different constructs. In the right panel, peptides are reported as short horizontal lines, with length and position corresponding to the peptide length and location along the protein sequence (x-axis) and with their deuteration delta on the y-axis. In both panels, red (positive) and blue (negative) indicate changes above the Student's t-test threshold, and in grey those not reaching this threshold. Four peptides, termed HDX-a, HDX-b, HDX-c and HDX-d, were identified as showing a strong increase in deuteration in sE404r compared to sE419r. The same pattern was observed in the comparison between sE404r and sE428r, whereas no difference deemed \"significant\" by the same test was seen when comparing sE419r with sE428r (see Fig EV3).",
    "molecules": "deuterium"
  },
  {
    "caption": "(a) The exonic SNP rs10065172, which is in strong linkage disequilibrium with the 20-kb insertion/deletion polymorphism upstream of IRGM, can be used to distinguish IRGM transcripts that arise from the deletion haplotype from IRGM transcripts that arise from the reference haplotype. This makes it possible to measure the relative expression of the two haplotypes in heterozygotes by measuring the relative abundance of the two rs10065172 alleles in cDNA. (Yellow and green rectangles indicate transcribed sequence; green rectangles indicate protein-coding sequence.) (b-g) Differential expression of IRGM from the two structural haplotypes in heterozygotes. The relative abundance of the C and T alleles of rs10065172 in cDNA (colored circles) and genomic DNA (black squares) from human tissues and cell lines were measured. Gray squares indicate control measurements for genomic DNA from 48 HapMap YRI individuals, identifying the three reference genotype classes CC, CT and TT. Genomic DNA from all other samples was heterozygous for the C and T alleles (black squares); HeLa genomic DNA (b,c) showed a 2:1 allelic ratio (CCT) reflecting HeLa's triploid 5q karyotypic status. In e, cDNA and genomic DNA from lymphoblastoid cell lines from ten different heterozygous individuals were analyzed: the ten different colors represent the ten individuals; technical replicates from the same cell line are connected by line segments. HeLa, SNU182 and the lymphoblastoid cell lines expressed the C allele of rs10065172 more strongly than the T allele (b-e); HCT116 and primary bronchial cells expressed the T allele much more strongly than the C allele (f,g).",
    "molecules": "DNA"
  },
  {
    "caption": "A. Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO (Ctrl) for 1 h before fixation and immunostained with Hoechst (blue), anti-CHMP4B (green), anti-GAL3 (red) and anti-LAMP1 (white) are presented. Cells treated with LLOMe show increased recruitment of ESCRT-III protein to lysosomes when compared to Ctrl cells. Number of foci per cell were quantified and are indicated as mean ± SD. Data from >86 cells per condition from three independent experiments. CHMP4B, GAL3, LAMP1 foci per cell (Ctrl versus LLOMe treatment): p=0.0398, p=0.0033, p=0.2400 (Student\"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)",
    "molecules": "Hoechst, LLOMe, DMSO"
  },
  {
    "caption": "B. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 treated as in (A) and stained with CD63 antibody are shown. Number of foci per cell were quantified and are indicated as mean ± SD. Data is quantified from >87 cells per condition from three independent experiments. CHMP4B, GAL3, CD63 foci per cell (Ctrl versus LLOMe treatment): p=0.0111, p=0.0050, p=0.3793 (Student\"s t‐test), respect Data information: Scale bars: 10 μm and 1 μm (inset)",
    "molecules": "LLOMe"
  },
  {
    "caption": "To screen for ESCRT proteins that are involved in the endolysosomal repair process, cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence. TSG101, a component of the ESCRT-I complex, and EAP30, an ESCRT-II protein, are clearly recruited to the sites of damage compared to the DMSO control (Ctrl). As shown, the ESCRT-III complex together with ALIX is recruited to damaged endolysosomes. In contrast, HRS, a component of the ESCRT-0 complex, and HD-PTP show no recruitment to damaged endomembranes. Number of foci per cell was quantified from >85 cells per condition from three independent experiments and are presented as mean ± SD. Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student's t‐test, the p-values for which are: HRS p=0.9257, GAL3 p=0.0050; TSG101 p=0.0243, GAL3 p=0.0489 ; EAP30 p=0.0006, GAL3 p=0.0307; CHMP2A p=0.0284, GAL3 p=0.0260; CHMP4B p=0.0028, GAL3 p=0.0414; VPS4A p=0.0309, GAL3 p=0.0461; ALIX p=0.0249, GAL3 p=0.0471; HD-PTP p=0.4898, GAL3 p=0.0105. Data information: Scale bars: 5 μm and 1 μm (inset). ",
    "molecules": "LLOMe, DMSO"
  },
  {
    "caption": "HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against: (A) HR 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system. A. Depletion of HRS does not alter the dynamics of CHMP4B-eGFP recruitment as compared to siCtrl during treatment with LLOMe Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)",
    "molecules": "LLOMe"
  },
  {
    "caption": "HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (B) TSG10 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system B. Downregulation of TSG101, with two independent siRNAs causes a delay in CHMP4B-eGFP recruitment as compared to siCtrl, indicating an important role of the ESCRT-I complex in recruiting the downstream components Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)",
    "molecules": "LLOMe"
  },
  {
    "caption": "HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs agains (C) CHMP2 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system C. CHMP2A depletion accumulates and stabilizes CHMP4B-eGFP positive foci Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)",
    "molecules": "LLOMe"
  },
  {
    "caption": "HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (D) ALI 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system D. siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)",
    "molecules": "LLOMe"
  },
  {
    "caption": "HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against (E) both TSG101 and ALIX 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system E. Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity)",
    "molecules": "LLOMe"
  },
  {
    "caption": "A. HeLa cells stably expressing CHMP4B-eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND-99 and fixed at different time points as indicated. After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm",
    "molecules": "LLOMe, Lysotracker, Lysotracker DND-99"
  },
  {
    "caption": "B. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker positive foci per cell at different time points. Error bars correspond to 95% confidence intervals",
    "molecules": "Lysotracker"
  },
  {
    "caption": "C. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101 and ALIX depleted cells. HeLa cells stably expressing CHMP4B-eGFP were co-transfected with siRNAs against TSG101 and ALIX. 48 h post-transfection, cells were pre-treated for 20 min with 75 nM Lysotracker Deep-Red, which was used as a read-out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live-cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.",
    "molecules": "Lysotracker"
  },
  {
    "caption": "Representative movie montage of a live-cell imaging experiment illustrating the dynamics of CHMP4B-eGFP and mCherry-Galectin3 recruitment at different time points before and after LLOMe treatment. Using the perfusion system, 250 μM LLOMe was added to HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 and images were acquired every 20 sec for a period of 20 min. As shown in the representative movie montage and the quantification graph, CHMP4B recruitment precedes Galectin3, which is an established marker for detection of damaged endolysosomal membranes. The quantification graph is normalized to the area occupied by the cells. Data were obtained from >40 cells per condition from three independent experiments. Error bars correspond to 95% confidence intervals. Data information: Scale bars: 5 μm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "A. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were incubated with 250 μM LLOMe, fixed at different time points as indicated and stained for ubiquitin. As presented, CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments",
    "molecules": "LLOMe, ubiquitin"
  },
  {
    "caption": "B. HeLa cells stably expressing CHMP4B-eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1-positive foci appears stable independently of LLOMe treatment Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments",
    "molecules": "LLOMe"
  },
  {
    "caption": "A. HeLa cells were transfected with different siRNAs as indicated. 48 h post-transfection cells were treated with 250 μM LLOMe for 3, 6, 10 hours harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co-transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001",
    "molecules": "LLOMe"
  },
  {
    "caption": "B. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "A. HeLa cells stably expressing CHMP4B-eGFP were transfected with the mCherry-Galectin3 plasmid. 24 h later, cells were infected with WT Coxiella burnetti. Live-cell imaging started 24 h post-infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella-containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B-eGFP and mCherry-Galectin3. Data information: Scale bars: 50 μm",
    "molecules": "DRAQ5"
  },
  {
    "caption": "B. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then cells were infected with mCherry-Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. 72 h later, infected cells were fixed, DAPI stained and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one-way ANOVA test. **p-value≤ 0.01 and ***p-value≤ 0.001.  ",
    "molecules": "DAPI"
  },
  {
    "caption": "b, HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector.",
    "molecules": "DAPI"
  },
  {
    "caption": "e, VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector.",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) Giant unilamellar vesicles (GUVs) incubated with mCherry-Atg19. The membrane was marked by incorporation of an Oregon Green labelled lipid. (b) GUV coated with GFP-Atg8 incubated with mCherry-Atg19. (c) Schematic representation showing the proteins and reaction resulting in GFP-Atg8 conjugation to the GUV shown in b. (d) GUVs containing nickellipids incubated with GFP-Atg8-6×His and wild-type mCherry-Atg19.",
    "molecules": "nickel"
  },
  {
    "caption": "(c) Quantification of the percentage of GUV-associated eGFP-Ape1-coated beads showing detectable membrane bending (N = 3; 40 GUVs per condition). The experimental set-up is shown in b. The density of Atg8-6×His on the membrane was titrated by different amounts of nickel lipids in the GUV membrane. The graph is based on at least three independent experiments. Shown are the averages and s.d. (",
    "molecules": "nickel"
  },
  {
    "caption": "(a) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Rap., rapamycin; Log.,",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b) Representative electron micrographs of ypt7Δ, atg19Δ yeast cells grown under cytoplasm-to-vacuole targetting (Cvt) conditions expressing the indicated proteins labelled with an anti-Myc antibody. The white arrowheads indicate the isolation membrane. The dashed line indicates the circumference of the prApe1 oligomer. Gold particles, 10 nm. Scale bars, 200 nm. See Supplementary Fig. 5 for full images.",
    "molecules": "Gold"
  },
  {
    "caption": "Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively",
    "molecules": "CCCP"
  },
  {
    "caption": "representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows.",
    "molecules": "CCCP"
  },
  {
    "caption": "F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively",
    "molecules": "CCCP"
  },
  {
    "caption": "Representative dual-channel kymographs showing that Rheb enhances mitophagy in CCCP-treated axons.",
    "molecules": "CCCP"
  },
  {
    "caption": "quantitative analysis showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, were quantified",
    "molecules": "CCCP"
  },
  {
    "caption": "D,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E).",
    "molecules": "CCCP"
  },
  {
    "caption": "F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H).",
    "molecules": "CCCP"
  },
  {
    "caption": "F,G Mitophagic accumulation at Aβ-enriched synapses in AD mouse brains. Note that the levels of mitophagic/autophagic proteins (red box) along with Aβ (green box) are increased in synaptic fractions isolated from AD mouse brains. Equal amounts (10 μg) of synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP/Aβ, Rheb, LC3, p62, and Rab7, as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH (F). Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls. Data were quantified from five repeats (G).",
    "molecules": "Aβ"
  },
  {
    "caption": "Representative images showing increased mitochondrial association with Rheb in AD axons, relative to that of WT littermate controls. Note that CCCP treatment enhances Rheb localization to mitochondria. The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP",
    "molecules": "CCCP"
  },
  {
    "caption": "quantitative analysis The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified, respectively",
    "molecules": "CCCP"
  },
  {
    "caption": "C- Representative dual-channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons. mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified",
    "molecules": "CCCP"
  },
  {
    "caption": "quantitative analysis , the numbers of Mito, AVs, and Mito-AVs, and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified, respectively",
    "molecules": "CCCP"
  },
  {
    "caption": "quantitative analysis Relative motility of AVs and Mito-AVs/mitophagosomes in WT or mutant hAPP axons with and without CCCP were quantified",
    "molecules": "CCCP"
  },
  {
    "caption": "F,G Quantitative analysis (F) and representative blots (G) showing that ∆ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains. Note increased localization of Rheb along with LC3-II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment (red box). Data were quantified from three independent repeats. P: post-nuclear supernatants; M: mitochondria-enriched membrane fractions; S: cytosolic supernatants.",
    "molecules": "CCCP"
  },
  {
    "caption": "Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls",
    "molecules": "CCCP"
  },
  {
    "caption": "representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons, which is exacerbated after dissipating ∆ψm. Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls",
    "molecules": "CCCP"
  },
  {
    "caption": "Representative dual-channel kymographs (A) and quantitative analysis (B showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, reduces mitophagic accumulation within AD axons incubated with CCCP.",
    "molecules": "CCCP"
  },
  {
    "caption": "Representative quantitative analysis showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, enhances retrograde transport of mitophagosomes within AD axons incubated with CCCP.",
    "molecules": "CCCP"
  },
  {
    "caption": "D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin-L99K were quantified and normalized to those of mutant hAPP controls. Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment.",
    "molecules": "CCCP"
  },
  {
    "caption": "D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative TEM images of normal and injured acinar cells. Scale bar, 2μm. Right (insets)- higher magnification images of boxed regions from left show emergence of double-membraned autophagic vesicles engulfing mature granules, mitochondria, and rough ER 24h after cerulein (CER) injury. Scale bar, 500nm. Arrowheads mark degradation events without double membranes. Graph of the number of acinar cell granules per cell in vehicle vs. 24h CER treatment. Each data point=total granules in a cell in a single cell counted from one mouse. Red line indicates mean. Graph of the number of degradation events scored in vehicle vs. 24h CER treatment. Each data point=total events in a cell in a single cell counted from one mouse. Red line indicates mean.",
    "molecules": "CER, cerulein"
  },
  {
    "caption": "Top - Representative RAB7 expression in base of stomach units (ZCs) in vehicle or 24h HDT by immunohistochemistry. Arrowheads point to RAB7 vesicle aggregates. Scale bar 20µm. Bottom - Representative ATF3 expression in base of stomach units (ZCs) by immunohistochemistry. ATF3 appears in nuclei following 24h high-dose tamoxifen (HDT). Scale bar 20µm. Stomach unit base outlined by dashed black line.",
    "molecules": "HDT, tamoxifen"
  },
  {
    "caption": "Western blot of ATF3 expression over HDT and CER timecourses with β-Actin loading control.",
    "molecules": "CER, HDT"
  },
  {
    "caption": "Top­ - Representative RAB7 staining in pancreatic acinar cells. Arrowheads indicate RAB7 vesicles. Scale bar 20µm. Bottom - Representative ATF3 expression in pancreatic acinar cells. Nuclei express ATF3 after 24h cerulein (CER) injury. Scale bar 20µm.",
    "molecules": "CER, cerulein"
  },
  {
    "caption": "ChIP-PCR on mouse stomach tissue at vehicle and 6 hour HDT. Chromatin probed with ATF3, Histone H3 antibodies, or a Rabbit IgG control. Also included is the 2% chromatin input and water to control for DNA contamination. Rab7b amplicon is from a conserved, putative ATF3-binding site in the first intron. Amplicon from Atf3 is a positive control from a previously characterized ATF3-binding site; negative control is from a site in Rab9a lacking putative ATF3 binding motifs.",
    "molecules": "HDT"
  },
  {
    "caption": "Top - Immunofluorescence for RAB7 (red) and DAPI (blue, nuclei) in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cells. Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue. Bottom - Immunofluorescence for ATF3 (purple) and Cathepsin D (CTSD, green, lysosomes) in AGS cells. Scale bar, 20µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C (PGC, red, granules) and MAP1LC3B (green, autophagy marker). Top row is wild-type, bottom row is Atf3−/−. Scale bar, 50μm. Stomach unit base outlined by dashed white line.",
    "molecules": "HDT"
  },
  {
    "caption": "Transmission electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or HDT treatment in WT (top) and Atf3−/− mice (bottom). Scale bar, 1μm. Arrowheads indicate cell components being degraded. Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3−/−mice as shown in panel 3D. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (data point colors indicate cells from a single mouse). Red line indicates mean across all the mice.",
    "molecules": "HDT"
  },
  {
    "caption": "Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line.",
    "molecules": "BRDU, DAPI, HDT, tamoxifen"
  },
  {
    "caption": "Representative immunohistochemistry of Cleaved-Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein (CER) treatment. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Apoptosis quantified following 24h CER injury by the number of Cleaved-Caspase 3+ cells from a 40x field. Each data point is mean of positive cells in 4-5 40x fields in an individual mouse, n=3 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.",
    "molecules": "CER, cerulein"
  },
  {
    "caption": "Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Quantification of proliferation at peak ADM as counted by the number of BrdU+ cells from a 20x field following 7D cerulein treatment. Plotting and statistical analysis as per panel F.",
    "molecules": "BrdU, cerulein"
  },
  {
    "caption": "Serial sections of human tissue. Representative immunohistochemistry of GSII (top; spasmolytic polypeptide-expressing metaplasia [SPEM] marker and neck cells) counterstained with hematoxylin (blue) and eosin (pink). Representative staining of ATF3 (bottom) counterstained with eosin (pink). Tissue was taken from regions adjacent to gastric adenocarcinoma. Scale bars left - 50μm right - 20μm. Arrowheads indicate strongly expressing ATF3 cells and the corresponding region in the same gland labeled with GSII in a serial tissue section. GSII at the base of gastric glands in the body of the stomach indicate transition to SPEM.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(N-P) CSCs  were treated with Mdivi-1  (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Scale bar 20 mm. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "Mdivi-1"
  },
  {
    "caption": "(R) CSCs  were treated with DAPT  (10 μM) and analysed for  sphere formation  and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "DAPT"
  },
  {
    "caption": "(T) CSCs  with or without transfection of Notch1  ICD were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "Mdivi-1"
  },
  {
    "caption": "(A-B) Lysosomal DNA  was identified with anti-LAMP1  and anti-DNA  antibodies in non-CSCs  and CSCs. Scale bar 5μm. Representative and mean±SEM with 50 cells from 5 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "DNA"
  },
  {
    "caption": "(D) CSCs  were treated with Mdivi-1 (50 μM) and analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "Mdivi-1"
  },
  {
    "caption": "(J-K) CSCs  treated with EtBr  (50 ng/ml) were analysed for lysosomal mtDNA, TLR9, and MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "EtBr"
  },
  {
    "caption": "(L) CSCs  transfected with DNase II  expression vector or the control were analyzed for lysosomal DNA  plus MyD88  protein. Mean±SEM from 6 patients. (M) CSCs  transfected with  DNase II shRNA or the control were analyzed for lysosomal DNA  plus MyD88  protein. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) CSCs  were stimulated with  CpG ODN (10 μg/ml) or control and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(G) CpG  promoted  tumor growth  of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG"
  },
  {
    "caption": "(H) TLR9  inhibitor chloroquine  abrogated the effect of CpG  on tumor growth in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "chloroquine, CpG"
  },
  {
    "caption": "(I) CpG  treatment increased the frequency of CD133-expressing CSCs  in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG"
  },
  {
    "caption": "(J) Chloroquine  impeded the function of CpG  in elevating CD133-expressing CSCs  of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "Chloroquine, CpG"
  },
  {
    "caption": "(B) CSCs  treated with or without EtBr  (50 ng/ml) were analyzed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "EtBr"
  },
  {
    "caption": "(D) CSCs  with or without CpG  treatment were analysed for interaction  between TLR9 and Notch1. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG"
  },
  {
    "caption": "(E) CSCs  were treated with CpG ODN (10 μg/ml) in the presence or absence of DAPT  (10 μM), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN, DAPT"
  },
  {
    "caption": "(F) CSCs  were transfected with 2 different Notch1  shRNAs or control, treated with CpG ODN  (10 μg/ml), and analyzed for sphere formation  plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "PDX chimeras treated by CpG  with or without DAPT  were analysed for tumor growth Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG, DAPT"
  },
  {
    "caption": "PDX chimeras treated by CpG  with or without DAPT  were analysed for CD133-expressing CSCs. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG, DAPT"
  },
  {
    "caption": "(B) CSCs  were treated with  2-DG (10 mM) or Malonate  (10 mM), and assayed for  sphere formation plus tumorgenicity. Mean±SEM from 6 patients. Data information: ANOVA test with Turkey's method (B). **p < 0.01, ***p < 0.001.",
    "molecules": "2-DG, Malonate"
  },
  {
    "caption": "(C) CSCs  treated with DAPT  (10 μM) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.",
    "molecules": "DAPT"
  },
  {
    "caption": "(E) CSCs  treated with EtBr  (50 ng/ml) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.",
    "molecules": "EtBr"
  },
  {
    "caption": "(G) CSCs  treated with chloroquine  (50 μM) were analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.",
    "molecules": "chloroquine"
  },
  {
    "caption": "(A) CSCs  stimulated with or without  CpG ODN (10 μg/ml) were analyzed for expression  of phosphor-AMPKα. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(B) CSCs  were stimulated with CpG ODN (10 μg/ml) in the presence or absence of DAPT  (10 μM), and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "CpG ODN, DAPT"
  },
  {
    "caption": "(C) CSCs  transfected with Notch1  shRNA or control were stimulated with  CpG ODN (10 μg/ml) and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(D) CSCs  were treated with DAPT  (10 μM) in the presence or absence of A769662  (10 μM), and analysed for mitochondrial OCR. Ctrl represents CSCs  without any treatment. Mean±SEM from 6 patients. Data information: ANOVA with Turkey method (D), **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "A769662, DAPT"
  },
  {
    "caption": "(E) CSCs  were stimulated with or without CpG ODN (10 μg/ml) and analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(F) CSCs  were stimulated with CpG ODN (10 μg/ml) in the presence or absence of Compound C (10 μM) and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "CpG ODN, Compound C"
  },
  {
    "caption": "(G) CSCs  were transfected with 2 different AMPKα  shRNA or the control, stimulated with  CpG ODN (10 μg/ml), and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(A) CSCs  transfected with or without Notch1  ICD were analyzed for AMP/ATP  ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "AMP, ATP"
  },
  {
    "caption": "(B) CSCs  treated with or without DAPT  (10 μM) were analyzed for AMP/ATP  ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "AMP, ATP, DAPT"
  },
  {
    "caption": "(C) CSCs  treated with or without CpG ODN (10 μg/ml) were analysed for interactions  between Notch1  and AMPK. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(D) CSCs  transfected with LKB1  shRNA（right）or the control (left) were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(E) CSCs  transfected with TAK1  shRNA or the control were treated with CpG ODN  (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(F) CSCs  transfected with LAMTOR1  shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "(G) CSCs  transfected with AXIN1  shRNA or the control were treated with CpG ODN  (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.",
    "molecules": "CpG ODN"
  },
  {
    "caption": "A. Representative FACS plots of freshly isolated epidermal cells (Epi d0) and day 14 (d14) cultures of epidermal cells in different growth conditions and media. KGM 2D: 2D in basal KGM; 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. CD34+α6+ cells were quantified by flow cytometry from d14 as in (A) (mean ±SD; n=3-5; *p ≤ 0.05; ***p ≤ 0.001, Kruskal-Wallis/Dunn's post test). 3C 2D: 2D in KGM + Y27632 + VEGF-A + FGF-2; Y: 3D-Matrigel in KGM + Y27632; YV: Y + VEGF-A; YF: Y + FGF-2; 3C: Y + VEGF-A + FGF-2. C. Absolute numbers of CD34+α6+ cells from Epi d0 and d14 cultures as in (A-B). Fold enrichment over Epi d0 is shown (mean ±SEM, n=3-5; *p ≤ 0.05, Mann-Whitney U test).",
    "molecules": "Y27632"
  },
  {
    "caption": "D. Epidermal cells grown for 14 days in the indicated conditions. 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. 3C 2D: 2D in 3C medium. Scale bars 30µm.",
    "molecules": "Y27632"
  },
  {
    "caption": "A. Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells (Epi d0; control) (mean ±SEM; n=5; *p ≤ 0.05, Mann-Whitney U-test). 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2.",
    "molecules": "Y27632"
  },
  {
    "caption": "A-B. Inhibitor treatment scheme (A) and FACS plots (B) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin (mean ±SD; n=3). C. Quantification of experiments as in (B) shows enrichment of HFSCs upon cyclopamine (Cyclo) treatment, whereas dorsomorphin (Dorso) treatment leads to depletion of HFSCs (mean ±SD; n=3; *p ≤ 0.05, Kruskal-Wallis).",
    "molecules": "Cyclo, cyclopamine, Dorso, dorsomorphin"
  },
  {
    "caption": "D. Heatmap of significantly (padj value<0.05) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h. HFSC identity genes (triangles) and genes implicated in HFSC lineage progression (circles) are indicated.",
    "molecules": "Cyclo, Dorso"
  },
  {
    "caption": "E. RT-qPCR analysis of 3C cultures treated as in (D) confirms that Id 1,2 and 3 are significantly upregulated in Cyclo treated cells (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).",
    "molecules": "Cyclo"
  },
  {
    "caption": "F. RT-qPCR analysis of 3C cultures treated as in (A) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).",
    "molecules": "cyclopamine, dorsomorphin"
  },
  {
    "caption": "Control (siLuc) and Chk1-depleted (siChk1) U20S cells were labeled with CldU and IdU for 10 and 30 min, respectively. Mean (+SEM) track lengths (top) and CldU/IdU ratios (bottom) are shown. Data come from 6 independent experiments and a total of 579 (siLuc) and 590 (siChk1) fibers were scored.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "Western Blot of phospho-serine-296-Chk1 (p-Chk1) in U2OS cells. Cells were treated 1.5 h with the Chk1 inhibitor Gö6976 (Chk1i) or DMSO, irradiated with 100J/m2 UV to induce high levels of p-Chk1, and collected 1.5 h afterwards. Actin was used as a loading control.",
    "molecules": "DMSO, Gö6976, serine"
  },
  {
    "caption": "U2OS cells treated with DMSO (control) or Chk1i were labeled with CldU and IdU for 10 and 30 min, respectively. Data come from 3 independent experiments and a total of 304 (DMSO) and 312 (Chk1i) fibers were scored.",
    "molecules": "CldU, IdU, DMSO"
  },
  {
    "caption": "U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. (A) Labelling scheme and representative DNA fibers. Scale bars: 5 μm.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. IdU track length after 10-80 min. Theoretical IdU track lengths are shown in red; the theoretical 10-min points were considered equal to the experimental values obtained for siLuc or siChk1, and the following theoretical points were calculated proportionally. Data were calculated as the mean (+SD) of mean IdU track lengths (B) n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "U2OS cells were labeled with CldU for 10 min, followed by increasing IdU labelling times and subjected to DNA stretching. Fork speed for each IdU labelling time. Data were calculated as the mean (+SD) of mean fork speeds (C). n=4 for IdU times 20-60 min or n=3 for IdU times 10 and 80 min; for each experiment and condition 60-100 fibers were scored.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "DNA combing from HCT116 cells labeled with CldU and IdU for 20 min. The upper left panel shows the labelling scheme. The upper right panel shows representative sister replication forks that emanate from the same origin (ORI=origin of replication); a pair of sister forks is considered asymmetric if their lengths (CldU+IdU) differ in more than 33%. The percentage of asymmetric forks (lower left panel; a Western Blot of Chk1 and Ku80, loading control, performed in parallel to the combing experiments, is shown) and the lengths of each pair of forks (lower right panel; points outside of the dotted lines correspond to asymmetric forks) are shown. Data (siLuc, n=131; siChk1, n=117) come from 2 independent experiments; the numbers indicate the mean (+SD) percentage of asymmetric forks.",
    "molecules": "CldU, IdU"
  },
  {
    "caption": "Labelling protocol used here and in all figures that follow, except otherwise indicated. The lower panels show representative images and schemes of an ongoing fork and of origins that fired during the CldU (1st) or IdU (2nd) pulses. Gray dotted lines represent unlabeled DNA. Percentage of origins was calculated as: (1st+2nd pulse origins)/total number of fibers.",
    "molecules": "CldU, IdU, DNA"
  },
  {
    "caption": "IdU track lengths from U2OS cells (Rosc, roscovitine). 180-200 DNA fibers obtained from 2 independent experiments were measured for each condition. The upper panel shows a Western Blot of Polη, 48 h after transfection with siRNA. Actin was used as a loading control.",
    "molecules": "IdU, Rosc, roscovitine"
  },
  {
    "caption": "IdU track lengths from U2OS cells transfected with GFP (-) or GFP-Polη-WT/S687A/S687D. >180 fibers obtained from 2 independent experiments were measured for each condition.",
    "molecules": "IdU"
  },
  {
    "caption": "IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells treated or not with a CDC7 inhibitor (CDC7i). >380 (track length) and >600 (origin frequency) fibers obtained from 4 (track length) or 3 (origin frequency) independent experiments were measured for each condition.",
    "molecules": "IdU"
  },
  {
    "caption": "IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with siCDT1. Track lengths from >290 fibers obtained from 3 independent experiments were measured for each condition. To calculate origin frequency >700 DNA fibers from 3 independent experiments were scored.",
    "molecules": "IdU"
  },
  {
    "caption": "IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells supplemented or not with nucleosides (NS). >195 (track length) and >350 (origin frequency) fibers obtained from 2 independent experiments were measured for each condition.",
    "molecules": "IdU, NS, nucleosides"
  },
  {
    "caption": "IdU track lengths and percentage of origin firing (mean+SD) from U2OS cells transfected or not with GFP-Polη. >180 (track length) and >400 (origin frequency) DNA fibers obtained from 2 independent experiments were measured for each condition.",
    "molecules": "IdU"
  },
  {
    "caption": "Percentage of γH2AX-positive U2OS cells (mean+SD) after 5 h treatment with the Chk1 inhibitor Gö6976 (Chk1i), in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >900 (DMSO) or >2000 (Chk1i) cells/sample were analyzed in 2 independent experiments. Total γH2AX was used to perform the statistics. Representative images of data shown in (B). Scale bar: 50 μm.",
    "molecules": "DMSO, Gö6976, NS, roscovitine"
  },
  {
    "caption": "DSB accumulation measured by neutral comet assay after 8 h treatment with Chk1i, in the presence of 5 μM NS, siCDC7 or 10 μM roscovitine. 85-170 cells/sample were analyzed in 2 independent experiments. Representative images of data shown in (D). Scale bar: 25 μm.",
    "molecules": "NS, roscovitine"
  },
  {
    "caption": "Percentage of parental ssDNA-positive U2OS cells (mean+SD) after 5 h treatment with Chk1i, in the presence of 5 μM NS, 1,25 μM CDC7i or 10 μM roscovitine. >450 cells/sample were analyzed in 2 independent experiments. The induction of ssDNA on control samples was null. Representative images of data shown in (F). Scale bar: 25 μm.",
    "molecules": "NS, roscovitine, ssDNA"
  },
  {
    "caption": "Sensitivity of U2OS cells to Chk1 inhibition, roscovitine (5 μM) and Polη depletion. Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 3 independent experiments.",
    "molecules": "Rosc, roscovitine"
  },
  {
    "caption": "Western Blot of CDC45 and Chk1 in U2OS cells after an extraction with CSK buffer to separate the insoluble and soluble fractions. Roscovitine (25μM) was added 2 h before harvesting. H2B was used as a loading control.",
    "molecules": "Roscovitine"
  },
  {
    "caption": "IdU track lengths from U2OS cells. 200 DNA fibers obtained from 2 independent experiments were measured for each condition.",
    "molecules": "IdU"
  },
  {
    "caption": "IdU track lengths from U2OS cells. >170 DNA fibers obtained from 2 independent experiments were measured for each condition. For comparison, the data showing that Polη is required for roscovitine-dependent rescue of fork elongation in Chk1-depleted cells (Fig 3B) is shown on the right. The lower panel shows the corresponding Western Blot of Chk1, Polη and CDC45. Actin was used as a loading control.",
    "molecules": "IdU, roscovitine"
  },
  {
    "caption": "Sensitivity of U2OS cells to Chk1 inhibition and roscovitine (5 μM). Cell number was determined 4 days after a 24 h treatment (5 days in total) with Chk1i±Rosc. Data represent the mean (+SD) of 2 independent experiments. Representative image of data shown in (G). Scale bar: 100 μm.",
    "molecules": "Rosc, roscovitine"
  },
  {
    "caption": "(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.",
    "molecules": "PIP2"
  },
  {
    "caption": "(E-H) Terminal cells expressing the membrane reporter CD4::mIFP together with markers for vesicles of the endocytic pathway. (E) Rab5::YFP, red arrowheads point to a Rab5-positive vesicle; the white arrowhead shows the same vesicle once it lost the Rab5 signal. (F) FYVE::GFP, a PI3P reporter and Spin::mRFP , a lysosomal marker. Black arrowheads point to small FYVE::GFP vesicles and the squared area shows large ones at the tip. (G) Par6::mCherry and endogenously labelled Rab7::YFP. (H) Percentage of CD4::mIFP vesicles that carry the indicated markers. Data are plotted as mean +/-SD. For each marker we analyzed at least 20 minutes of cell growth with 10-20 timepoints. Number of cells analyzed: Par3, n=2; Rab5, n=3; FYVE::GFP, n=4; FYVE::GFP-Spin::RFP (which includes single- and double-positive vesicles), n=3; dLamp1, n=2; Rab7, n=5.",
    "molecules": "PI3P"
  },
  {
    "caption": "C. Cathepsin activity  assay in WT and Tpl2[D270A] BMDMs 0.5 h after uptake of  latex beads. BMDMs were stained with the Magic Red cathepsin L substrate (red). Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40-51 cells). E. pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with  latex beads. BMDMs were stained  with the LysoTracker Red DND-99 dye  (red). Average  fluorescence intensity of the  Lysotracker Red probe per cell was quantified (n = 95-126 cells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test.",
    "molecules": "Lysotracker Red, LysoTracker Red DND-99 dye"
  },
  {
    "caption": "G. Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye. Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80-110 cells) Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test.",
    "molecules": "Deep Red, Deep Red dye, latex beads, ROS, Reactive oxygen species"
  },
  {
    "caption": "Experiments were performed using murine BMDMs. (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted  for the indicated antigens.",
    "molecules": "LPS"
  },
  {
    "caption": "Experiments were performed using  human primary  monocyte-derived macrophages. (K) Cell extracts from LPS-stimulated, inhibitor-treated primary human macrophages were immunoblotted for p-ERK1/2,  ERK1/2, p-p38, p38 and HSP90.",
    "molecules": "LPS"
  },
  {
    "caption": "A. TPL-2-dependent phosphoproteome following phagocytosis of latex beads  (0.5 h) was determined by TMT mass spectrometry. Volcano plot representing the significance (-log10 P-values after Welch's t-test) vs. phosphorylation  fold change (Welch difference ratios) between WT and Tpl2[D270A] BMDMs. Five biological replicates were analysed per genotype (n = 5). Three of the most highly and significantly downregulated phospho-sites in Tpl2[D270A]  BMDMs relative to WT, as well as unaltered DMXL1 phospho-sites, are shown.",
    "molecules": "latex beads"
  },
  {
    "caption": "BMDMs of the indicated genotypes were  infected  with YFP-S. aureus  (MOI 10) for 1 h. (B) Representative  images  of  YFP+ BMDMs labelled with Magic Red cathepsin L substrate (red) and DAPI  (nuclear stain) 1h post-infection. (C) Quantification of Magic Red  cathepsin L staining from panel (A). Average fluorescence intensity of Magic Red  cathepsin L  substrate per cell relative to fluorescence intensity of YFP-S.aureus (n = 26-29 cells). (D) Quantification of Magic Red  cathepsin L staining co-localised with YFP-S. aureus from panel (A) (n = 26-29 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "BMDMs of the indicated genotypes were infected with YFP-S. aureus  (MOI 10) for 1 h. (E) Representative images  of YFP+ BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1h post-infection with YFP-S. aureus (green).(F) Quantification of LysoTracker Red signal from panel (D). Average  fluorescence intensity of LysoTracker Red per cell relative to fluorescence intensity  of YFP-S. aureus  (n = 66-98 cells). (G) Quantification of LysoTracker Red co-localisation with YFP-S. aureus from panel (D) (n = 66-98 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.",
    "molecules": "DAPI, LysoTracker Red, LysoTracker Red DND-99 dye"
  },
  {
    "caption": "BMDMs of the indicated genotypes were infected with YFP-S. aureus  (MOI 10) for 1 h. (H) Representative  images  of BMDMs stained with an anti-EEA1  antibody (red) and DAPI, 1h post-infection with YFP-S. aureus  (green). (I) Quantification of EEA1+ S. aureus-containing phagosomes from panel (G) (n = 27 cells). (J) Representative  images of BMDMs stained  with an anti-LAMP-1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (K) Quantification of LAMP-1+ S. aureus phagosomes from panel (I) (n = 25 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "E. BMDMs  of the indicated genotypes were  infected with GFP-C. rodentium (MOI 2) for 0.5h. C. rodentium CFU were assessed at 2 h after infection and normalised to CFU at 0.5 h (n = 12; 2 biological and 6 technical replicates per condition per experiment). As a control, WT cells were treated with bafilomycin A. Data information: One representative experiment out of three shown. Error bars represent SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant (ns). one-way ANOVA.  ",
    "molecules": "bafilomycin A"
  },
  {
    "caption": "F. Representative images of GFP+ BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1h post-infection with  GFP-C. rodentium (green). G. Quantification of LysoTracker Red co-localisation  with GFP- C. rodentium from panel (F) (n = 54-63 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant (ns). P one-way ANOVA.  ",
    "molecules": "DAPI, LysoTracker Red, LysoTracker Red DND-99 dye"
  },
  {
    "caption": "Asynchronously growing human U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, and stained for 53BP1 and DNA content for cell cycle resolved quantification of 53BP1 foci in individual cells by QIBC.",
    "molecules": "DNA"
  },
  {
    "caption": "Cells were treated with 0.5 Gy of IR in absence or presence of 0.4 M sorbitol, fixed 1h later, and 53BP1 assembly at DSBs was analyzed by QIBC. Treatments as in (C); RIF1 accumulation at DSBs was analyzed by QIBC.",
    "molecules": "sorbitol"
  },
  {
    "caption": "GFP-53BP1 cells were treated with 25 ng/ml NCS to induce DNA breaks and imaged at 30 minutes intervals. Examples of GFP-53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown.",
    "molecules": "NCS"
  },
  {
    "caption": "53BP1-RFP cells, in which the endogenous 53BP1 gene locus had been engineered by CRISPR/Cas9 to express 53BP1-mScarlet from the natural promoter, were treated with NCS (25 ng/ml) and imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown on the left and in higher magnification on the right.",
    "molecules": "NCS"
  },
  {
    "caption": "GFP-53BP1 cells were left untreated, or treated with APH (0.5 μM) or ATRi (1 μM) to induce replication stress-associated heritable DNA lesions, and cells were imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) are shown.",
    "molecules": "APH, DNA"
  },
  {
    "caption": "Western blot of p53 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M Sorbitol.",
    "molecules": "Sorbitol"
  },
  {
    "caption": "qPCR of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol. Mean ± standard deviation is indicated.",
    "molecules": "sorbitol"
  },
  {
    "caption": "Western blot of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol.",
    "molecules": "sorbitol"
  },
  {
    "caption": "QIBC cell cycle analysis based on DNA content and nuclear Cyclin A levels upon DNA damage with and without 0.4 M sorbitol. Cells were irradiated with 2 Gy in absence or presence of 0.4 M sorbitol. After 1h of recovery, sorbitol-containing medium was exchanged with fresh medium, and cells were allowed to recover for an additional 7h. Percentages in G1 and S/G2 are provided.",
    "molecules": "sorbitol, DNA"
  },
  {
    "caption": "A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control.",
    "molecules": "zVAD.fmk, BV6, PI"
  },
  {
    "caption": "D HT‑29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα after pre-incubation with 30 µM Nec1-s and 20 µM GSK'872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells.",
    "molecules": "zVAD.fmk, GSK'872, BV6, Nec1-s, PI"
  },
  {
    "caption": "F HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining.",
    "molecules": "zVAD.fmk, BV6, PI"
  },
  {
    "caption": "A HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for the indicated time points. Detection of indicated proteins was carried out by Western blotting. GAPDH served as a loading control.",
    "molecules": "zVAD.fmk, BV6"
  },
  {
    "caption": "B HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Detection of indicated proteins was carried out by Western blotting. β-Actin served as a loading control.",
    "molecules": "zVAD.fmk, BV6"
  },
  {
    "caption": "C HT‑29 control and USP22 KO cells were incubated with 30 µM Nec1-s or 20 µM Dab for 18 h, as indicated. Cell were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 5 h. 100 μg of each lysate were incubated with 400 U/μl λ-phosphatase for 30 min at 30 °C. Protein expression of RIPK3 was monitored by Western blotting. β-Actin was used as loading control. High molecular weight RIPK3 'smears' were quantified after λ-phosphatase treatment and normalized to total RIPK3 and β-Actin levels.",
    "molecules": "zVAD.fmk, Dab, BV6, Nec1-s"
  },
  {
    "caption": "A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band.",
    "molecules": "Dox"
  },
  {
    "caption": "B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei.",
    "molecules": "zVAD.fmk, Dox, BV6, PI"
  },
  {
    "caption": "C HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4 and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control.",
    "molecules": "zVAD.fmk, Dox, BV6"
  },
  {
    "caption": "D HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti‑Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.",
    "molecules": "zVAD.fmk, Dox, BV6"
  },
  {
    "caption": "E USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.",
    "molecules": "zVAD.fmk, BV6"
  },
  {
    "caption": "A HT-29 control, USP22 KO and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Poly-ubiquitinated proteins were enriched by GST-TUBE pull-down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. β-Actin served as loading control. Ponceau-staining was used to confirm equal loading of GST-TUBE.",
    "molecules": "zVAD.fmk, BV6"
  },
  {
    "caption": "B HT-29 control and USP22 KO cells were transfected with His-ubiquitin for 24 h, as indicated. Cells were pre‑stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre-treatment, 1 ng/ml TNFα was added for 4 h. His-ubiquitin was immunoprecipitated using Ni-NTA beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas His-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.",
    "molecules": "zVAD.fmk, BV6, ubiquitin, Ni-NTA"
  },
  {
    "caption": "C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre-treatment, 10 ng/ml TNFα were added for 3 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-levels served as loading control for immunoprecipitated ubiquitin.",
    "molecules": "zVAD.fmk, Dox, BV6, ubiquitin"
  },
  {
    "caption": "D HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR or 3xKR were pre‑incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI-positive nuclei.",
    "molecules": "zVAD.fmk, Dab, Dox, BV6, NSA, PI"
  },
  {
    "caption": "E HeLa cells expressing Dox-inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.",
    "molecules": "Dox"
  },
  {
    "caption": "A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β-Actin was used as a loading control.",
    "molecules": "Dab, Dox, NSA"
  },
  {
    "caption": "B HeLa cells expressing Dox-inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3 and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β-Actin served as loading control.",
    "molecules": "zVAD.fmk, Dox, BV6"
  },
  {
    "caption": "C HeLa cells expressing Dox-inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control. D HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control.",
    "molecules": "zVAD.fmk, Dox, BV6, SDS"
  },
  {
    "caption": "E HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K518R or 3xKR were incubated overnight with 1 µg/ml Dox and pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep-RIPK3 was immunoprecipitated by using anti-Strep-beads. Co-immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β-Actin served as a loading control.",
    "molecules": "zVAD.fmk, Dox, BV6"
  },
  {
    "caption": "F HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.",
    "molecules": "zVAD.fmk, Dox, BV6, ubiquitin"
  },
  {
    "caption": "A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI-positive nuclei.",
    "molecules": "zVAD.fmk, Dox, BV6, PI"
  },
  {
    "caption": "B HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β-Actin served as a loading control.",
    "molecules": "zVAD.fmk, Dox, BV6"
  },
  {
    "caption": "(C) hematoxylin-eosin staining  with scale bar of 50 µm of the back skin   of ILEIind and K5-ILEIind mice  kept on normal or doxycycline  diet and treated with acetone  or TPA  for 5 days (n=2-5; 3 independent experiments).",
    "molecules": "acetone, doxycycline, eosin, hematoxylin, TPA"
  },
  {
    "caption": "(D) mean epidermal  thickness ±SEM of the  back skin  of ILEIind and K5-ILEIind mice  kept on normal or doxycycline  diet and treated with acetone  or TPA  for 5 days (n=2-5; 3 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; ***p,0.001).",
    "molecules": "acetone, doxycycline, TPA"
  },
  {
    "caption": "(E) Representative images of ILEI immunohistochemistry  on sections of back skin  of ILEIind and K5-ILEIind mice  kept on normal or doxycycline  diet upon 5 days of treatment with acetone  or TPA. Scale bar 50 µm. Inlets show a magnification of the marked regions, scale bar 20 µm.",
    "molecules": "acetone, doxycycline, TPA"
  },
  {
    "caption": "(A) Representative images of Ki67 immunohistochemistry  on back skin  sections of K5-ILEIind and ILEIind mice  kept on normal or doxycycline  diet and treated with acetone  or TPA  for 5 days. Scale bar: 100 µm. (B) Percentage of Ki67  positive cells in the epidermis  shown as mean ±SEM and quantified on samples described in panel A (n=1-5; 3 independent experiments). Data information: Statistical significance was determined by one-way ANOVA (B at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).",
    "molecules": "acetone, doxycycline, TPA"
  },
  {
    "caption": "(C) Immunofluorescence  for Keratin 5  (K5; red) and  Keratin 10  (K10; green) expression  on TPA-treated skin  sections of mice  described in panel A. Nuclei  were counterstained  with DAPI  (blue). Scale bar: 50 µm. Inlets show a magnification of the marked regions. Scale bar: 20 µm.",
    "molecules": "DAPI, TPA"
  },
  {
    "caption": "(D) Keratin 16 (K16)  western blot  analysis (left) and quantification (right) of proteins extracted from back skin  of ILEIind and K5-ILEIind mice  kept on doxycycline  and treated with acetone  or TPA  for 5 days (n=3). Vinculin  was used as loading control. Data information: Statistical significance was determined by one-way ANOVA at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).",
    "molecules": "acetone, doxycycline, TPA"
  },
  {
    "caption": "Mean fold change ±SEM in mRNA expression  of Tnfa, Il1α  and Cxcl1  (E) in freshly sorted keratinocytes  enriched for the inter-follicular epithelium (IFE) and hair follicles  (HF) from acetone  and TPA  treated back skin in mice  supplemented with doxycycline  and treated with acetone  or TPA  for 96 hours (n=3 (E), independent experiments). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.",
    "molecules": "acetone, doxycycline, TPA"
  },
  {
    "caption": "(G-I) Mean fold change ±SEM in mRNA expression  of (G) Tnfa, (H) Il1α  and (I) Cxcl1  in  primary keratinocytes treated with acetone  and TPA  and with increasing concentrations (100ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml) of murine  recombinant ILEI  (mILEI) or dimerization-disabled ILEI   (mILEICA) or with empty-vector (empty) for 72 hours (n=3, standing for independent keratinocyte  cultures from three mice). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.",
    "molecules": "acetone, TPA"
  },
  {
    "caption": "(A-C) Representative images of (A) Phospho-STAT3  (Tyr727), (B) Phospho-Erk1/2 and (C) Phospho-Akt immunofluorescence  on thin sections of acetone  and TPA  treated back skin of  ILEIind and K5-ILEIind mice  kept on doxycycline  diet. Scale bar, 50 µm.",
    "molecules": "acetone, doxycycline, TPA"
  },
  {
    "caption": "(E) Mean STAT3  (Ser727 and Tyr705), Akt  and Erk1/2 phosphorylation  levels ±SEM over time  in primary keratinocytes upon TPA  and recombinant ILEI  treatment as described in panel A (n=3, standing for independent keratinocyte  cultures, each a pool of isolates from two mice).",
    "molecules": "TPA"
  },
  {
    "caption": "(F) Western blot  analysis of STAT3  (Ser727 and Tyr705), Akt  and Erk1/2 phosphorylation  levels in primary Dox-induced K5-ILEIind keratinocytes  treated with acetone  or TPA  for 4 hours in the presence of the inhibitors STATTIC  (10µM), LY92004  (10µM) and UO126  (10µM). DMSO  was used as vehicle control. Vinculin  was used as loading control. Lanes are from non-continuous parts of the same gel.",
    "molecules": "acetone, DMSO, Dox, LY92004, TPA, STATTIC, UO126"
  },
  {
    "caption": "(B) hematoxylin-eosin staining  with a scale bar of 50 µm and (C) mean epidermal  thickness ±SEM of the back skin  of ILEIfl/fl and ILEI∆Ep mice  treated with acetone  or TPA  for 5 days (n=3-8; 3 independent experiments). Data information: In C statistical significance was determined one-way ANOVA with Tukey multiple comparison test (C marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.",
    "molecules": "acetone, eosin, hematoxylin, TPA"
  },
  {
    "caption": "(F-H) (F) Representative images of MPO+ microabscesses   and neutrophils, (G) mean number ±SEM of MPO-positive microabsesses/cm skin  section and (H) mean number of neutrophils  (MPO+ cells)/mm2 epidermal-dermal  area ±SEM on thin sections of acetone  and TPA  treated back skin of ILEIfl/fl and  ILEI∆Ep mice  (n=6-8 (G) and n=2-8 (H); 3 independent experiments). Scale bar, 100 µm. Data information: In G and H, statistical significance was determined one-way ANOVA with Tukey multiple comparison test H) or by Student's t-test (G) and marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.",
    "molecules": "acetone, TPA"
  },
  {
    "caption": "Mean fold change ±SEM in mRNA expression  of uPA  and uPAR  (D) in freshly sorted keratinocytes  enriched for the interfollicular epithelium from acetone  and TPA  treated back skin, (E) in primary keratinocytes isolated from ILEIind and K5-ILEIind mice, supplemented with doxycycline  and treated with acetone  or TPA  for 96 hours (n=3 (D), n=6 (E 1-2 independent experiments). Data information: In D,E Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "acetone, doxycycline, TPA"
  },
  {
    "caption": "Mean fold change ±SEM in mRNA expression  of uPA  and uPAR  (F) in primary wild type keratinocytes  treated with acetone  and TPA  and with murine  recombinant wild type ILEI  (mILEI) for 8 and 96 hours n=6 F); 1-2 independent experiments). Data information: Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).",
    "molecules": "acetone, TPA"
  },
  {
    "caption": "(L) ILEI Western blot  analysis of conditioned media harvested from  primary keratinocytes of ILEIind and K5-ILEIind mice  after 48-hours doxycycline  induction and treatment with TPA  and DMSO  or indicated concentrations of UK371804. Loading was normalized to cell count, numbers indicate relative intensities. Lanes are from non-continuous parts of the same gel.",
    "molecules": "DMSO, doxycycline, TPA, UK371804"
  },
  {
    "caption": "B Representative AIY calcium activity over 60 seconds for the indicated genotypes. C-D Quantification of the peak frequency and amplitude of AIY::GCaMP6s in for the indicated genotypes.",
    "molecules": "calcium"
  },
  {
    "caption": "F, Representative immunoblot and quantification of pFAK levels in 5% LPDS (-LDL) and upon increasing  LDL loading times. Mean ± SD, n = 3, paired student's t-test (*p < 0.05).",
    "molecules": "LDL"
  },
  {
    "caption": "G, Representative confocal images of NPC1, internalized integrin β1 and pFAK localization. Stably NPC1-GFP expressing cells were loaded with 2 μg/mL integrin β1 antibody for 30 min before fixation and  stained with pFAK antibody followed by secondary antibodies. Dashed line indicates the cell edge. H, Quantification of G using Mander's colocalization for the indicated markers. Mean ± SD, n = 5 cells. Cells were treated with 5 % LPDS for 1 day and then without (-) or with (+) 50 μg/ml  LDL for 1h. ",
    "molecules": "LDL"
  },
  {
    "caption": "D, After 1 day  LDL depletion in 5 % LPDS, cells were loaded with 50 μg/ml LDL for the indicated  times and  intracellular cholesterol  was detected with D4H as in C by confocal microscopy. CM, complete medium. Dashed lines indicate cell outlines. E, Quantification of D. Mean ± SD, n = 25-26 cells pooled from 2 independent experiments. ",
    "molecules": "cholesterol, LDL"
  },
  {
    "caption": "F, Cells were treated for 1 day in 5 %  LPDS followed by 50 μg/ml LDL  loading for the indicated  times. Representative confocal images of cells triple labeled with  intracellular accessible  cholesterol  (D4H), NPC1-GFP  and internalized integrin β1. Dashed lines indicate cell outlines. G, Quantification of  D4H  area positive for NPC1 or internalized  integrin β1 as in F. H, Fraction of D4H area overlapping with NPC1 or internalized integrin β1 in a  peripheral region toward the leading edge (exemplary box shown as inset in F). Mean ± SD, n = 27-30 cells pooled from 2 independent experiments. Student's t-test. ",
    "molecules": "cholesterol, LDL"
  },
  {
    "caption": "A, After 1 day in 5 % LPDS,  NPC1-GFP expressing cells were loaded with 50 μg/ml LDL with or without 10 μM PF228 for the indicated times, and stained for intracellular D4H. Representative confocal images of  intracellular D4H, NPC1-GFP and internalized integrin β1 labeling. Dashed lines indicate cell outlines. B, Fraction of D4H positive area in the cell as in A. C, Fraction of D4H area overlapping with  NPC1 in a peripheral region toward the leading edge (exemplary box shown as inset in A). D, Quantification of D4H area positive for integrin β1 in a peripheral region toward the leading edge. Mean ± SD, n = 26-30 cells pooled from 2 independent experiments. Student's t-test. ",
    "molecules": "PF228, LDL"
  },
  {
    "caption": "I, After 1 day 5 %  LPDS, cells were loaded with 50 μg/ml  LDL with or without 10 μM PF228 for the indicated times and endomembrane PI(4,5)P2 was stained with antibody and imaged by confocal microscopy.",
    "molecules": "PF228, LDL, PI(4,5)P2"
  },
  {
    "caption": "J, Quantification of mean PI(4,5)P2 immunoreactivity in cells. Mean ± SD, n = 16-26 cells pooled from 2 independent experiments. Student's t-test.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "C, Confocal image of endo-GFP-ORP2 and pFAK immunofluorescence staining in saponin pre-permeabilized cells loaded with LDL for 2 h before fixation. GFP  antibody was used to enhance endo-GFP-ORP2 signal. Insets show examples of \"no contact\", \"contact\" and \"overlap\" of organelles. Dashed line indicates cell edge. D, Quantification of pFAK, integrin β1, and NPC1 association with endo-GFP-ORP2 in C. n = 380 pFAK; 353 integrin β1; 232 NPC1 organelles in 2 cells. ",
    "molecules": "LDL, saponin"
  },
  {
    "caption": "F, Schematic outline for plasma membrane D4H labeling in combination with ORP2 degron system, and representative widefield epifluorescent images of co-plated cells after 4 h of LDL loading. Asterisk indicates ORP2-depleted degron cells. Dashed lines separate ORP2  expressing and depleted cells. G, Quantification of F. Mean ± SD, n = 20 fields of cells pooled from 2 independent experiments. Student's t-test. ",
    "molecules": "LDL"
  },
  {
    "caption": "A, Control and degron ORP2 cells were co-plated, and after 1 day incubation in 5 %  LPDS, cells were loaded with  LDL (+LDL) and IAA (+IAA; ORP2 depletion) for the indicated  times. Intracellular cholesterol was labeled with D4H  and imaged by widefield epifluorescence microscopy. Asterisk indicates region of ORP2-depleted cells. CM, complete medium. Dashed lines separate ORP2 expressing and depleted cells. B, Quantification of A. Mean ± SD, n = 15-27 cells pooled from 2 independent experiments. Student's t-test. ",
    "molecules": "cholesterol, IAA, LDL"
  },
  {
    "caption": "C, E, Control and degron ORP2 cells were co-plated, and after 1 day in 5 % LPDS, treated with 50 μg/ml LDL and IAA for the indicated times. Asterisk indicates an  ORP2-depleted cell. Double labeling of intracellular D4H and  Lamp1 (C) or intracellular D4H  and internalized integrin β1 (E). Images were acquired by confocal microscopy. Red box indicates a peripheral region toward the leading edge in the control cell, and blue box a corresponding region in the ORP2-depleted cell.",
    "molecules": "IAA, LDL"
  },
  {
    "caption": "B, Cells stably expressing NPC1-mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP-ORP2 or -ORP2-mHHK (PI(4,5)P2 binding-deficient mutant) for 1 day, stained with  PI(4,5)P2 antibody and imaged by confocal microscopy. For FAK  inhibition, GFP-ORP2 transfected cells were treated with 10 μM PF228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5)P2  and NPC1. C, Quantification of B. Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA/Vector ctrl. Mean ± SD, n = 20-43 cells pooled from 2 independent experiments. Student's t-test. ",
    "molecules": "PF228, PI(4,5)P2"
  },
  {
    "caption": "D, E, Cells stably expressing NPC1-mCherry were transfected with GFP-ORP2 or  GFP-ORP2-mHHK for 1 day and were indicated, treated with 10 μM PF228 for 4 h. For degron  ORP2  cells,  NPC1-mCherry was transiently transfected for 2 days and cells incubated without (-IAA; ORP2 present) or with IAA  (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest  NPC1 tubule per cell was measured. Mean ± SD, n = 102-122 cells. Students's t-test. Please see also ctrl (MovieEV4) and PF228 treated cell (MovieEV5), both videos 1 min recordings with 1 s frame rate.",
    "molecules": "PF228, IAA"
  },
  {
    "caption": "A, B, Control and degron ORP2 cells were co-plated, and after 1 day 5 % LPDS, treated with 50 μg/ml LDL and  IAA (+IAA; ORP2 depletion) for the indicated  times and  immunostained for pFAK to quantify its intensity. For rescue experiments, degron ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-∆ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2  were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous  ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20-23 cells pooled from 2 independent experiments. Student's t-test.",
    "molecules": "IAA, LDL"
  },
  {
    "caption": "C, Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol+membranes) is presented ± SD. For no IAA n=13 and for  IAA n= 11, 2 independent experiments. Student's t-test.",
    "molecules": "IAA, LDL"
  },
  {
    "caption": "(C) Representative images of  single molecule mRNA FISH  (smFISH) staining  of  S. pombe using probes against GFP  (red). DNA was stained  with DAPI  (blue). The gamma-value was adjusted to make the cytoplasm  visible; cell shapes are outlined in blue. (D) Frequency distribution of mRNA numbers per cell determined by smFISH; n = number of cells. Curves show fit to a Poisson distribution.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) mRNA abundances by qPCR  following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for non-instantaneous labeling by 4tU. Act1+ and ecm33+ were used as long and short half-life controls, respectively; qPCR  was performed for the endogenous mRNAs. Half-lives (95 % confidence interval): mad1+ 5.6 min (4.3 - 8.4), mad2+ 7.7 min (6.2 - 10.4), mad3+ 5.2 min (4.3 - 6.9), act1+ 61.8 min (37.2 - 172.3), ecm33+ 5.0 min (4.5 - 5.7).",
    "molecules": "4tU"
  },
  {
    "caption": "(E) Protein abundances after translation shut-off with cycloheximide  (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one-phase exponential decay. Cdc2  and Cdc13  were used as long and short half-life controls, respectively.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(E) Time course  of RNA abundances by qPCR  following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life  estimates are included with 95 % bootstrap confidence intervals in brackets.",
    "molecules": "4tU"
  },
  {
    "caption": "(C)  Time course of RNA abundances by qPCR  following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark =  ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets.",
    "molecules": "4tU"
  },
  {
    "caption": "(C) Top: Anti-GFP immunoprecipitation  (IP) and Strep pull-down (PD) from extracts of diploid cells expressing Mad1-GFP  and Mad1-Strep  from the two endogenous loci; membrane probed with anti-Mad1; in = input (7 % of extract for IP/PD), sup = supernatant after IP/PD. Bottom: as in (A), 2 experiments each. The experiment shown at the top and two more GFP-IPs  from the same strain were unquantifiable, because no second band was visible in the IP.",
    "molecules": "Strep"
  },
  {
    "caption": "(D) In vitro translation  (IVT) of Mad1-flag-His  and untagged Mad1  in the presence of 35S-labelled Methionine  and Cysteine, followed by Mad1 immunoprecipitation  (IP) or His pull-down (PD); in = input (9.5 % of extract for IP/PD), sup = supernatant after IP/PD. An IVT with only untagged Mad1     was used to check for specificity of the His PD  (right side). Shown is the autoradiograph  after  SDS-PAGE  with quantification of the Mad1-flag-His  to untagged Mad1  ratio in select lanes.",
    "molecules": "Cysteine, Methionine, 35S"
  },
  {
    "caption": "Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG.",
    "molecules": "DMOG"
  },
  {
    "caption": "Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 expression with siRNA confirms antibody specificity (scale bars: 200um).",
    "molecules": "formalin, paraffin"
  },
  {
    "caption": "Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).",
    "molecules": "formalin, paraffin"
  },
  {
    "caption": "gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR).",
    "molecules": "O2"
  },
  {
    "caption": "Expression of an siRNA-resistant GPRC5A cDNA rescues hypoxic GPRC5A-depleted cells from apoptosis. Upper: doxycycline-induced expression GPRC5Asi1R rescues increases caspase-3/PARP cleavage induced by GPRC5A depletion in hypoxia. Lower: generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Caspase inhibitor QVD prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.",
    "molecules": "QVD"
  },
  {
    "caption": "The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments).",
    "molecules": "verteporfin"
  },
  {
    "caption": "(B) Nuclear fractions from U2-OS cells were harvested at indicated time points post IR (20 Gy) as in (A). Top panel: NF-κB DNA binding was analyzed by EMSA. Lower panels: Western blot of indicated NF-κB subunits and PARP1 as loading control. The two phases of nuclear translocation",
    "molecules": "DNA"
  },
  {
    "caption": "(G) Skin sections as in (E) analyzed by immunofluorescence with IκBα (red) or IL-6 antibody (green) and nuclear DAPI staining (blue). Dotted lines delineate hair follicles, Bu, bulge region, HS, hair shaft, IFE, interfollicular epidermis, SG, sebaceous gland. Scale bar: 50 μm. (H) Quantitation of (G) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for IκBα or IL-6 were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. * = p < 0.05, *** = p < 0.001. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) NFKBIA knockdown by dox-inducible shRNA in U2-OS cells. Cells were pre-treated with Dox to induce knockdown. For knockdown efficiency see Fig. S2F. Cells were irradiated (20 Gy) or not, as indicated and described above. NF-κB activity was analyzed by EMSA. ns, non-specific band. A representative gel from n = 3 biological replicates is shown.",
    "molecules": "dox, Dox"
  },
  {
    "caption": "(B) Expression of SASP targets of NF-κB obtained from RNA-seq analysis (Dataset EV1B), was quantitated using RT-qPCR from the shRNA (RELA) stably expressing U2-OS cells. For knockdown efficiency see Appendix Fig S4A. Cells were treated with doxycycline (Dox) to induce knockdown of p65. Heatmap represents targets normalized to the untreated Scrambled control. Expression is shown as log2 change with p values < 0.05. Statistical analysis performed using ANOVA with Bonferroni correction for multiple testing. First-phase and second-phase samples were irradiated (20 Gy) 1.5h and 7 Days prior to harvest, respectively. Analysis based on n = 3 biological replicates.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(C) Cell duplication was measured at time points indicated in U2-OS cells bearing either scrambled control or Dox-inducible shRNA against RELA (n = 3 biological replicates). Treatment with Dox was initiated at two days prior to IR (20 Gy) or after IR. Statistical significance in total duplication number between groups at day 6 was determined by ANOVA with Tukey multiple comparisons test. SD shown. * = P < 0.05, *** = p < 0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "(B) U2-OS cells treated with dox to deplete endogenous p65, were irradiated (20 Gy) and transfected with plasmids encoding p65, p65-S276A or p65-S468A, as indicated. Nuclear (N) and cytoplasmic (C) lysates were analyzed by SDS-PAGE at day seven post IR. Representative gel from n = 3 biological replicates is shown. PARP1 and αTubulin serve as fractionation and loading controls.",
    "molecules": "dox"
  },
  {
    "caption": "(F) U2-OS cells were left untreated or exposed overnight to 10 mM LiCl, with or without prior irradiation, as indicated, and analyzed as in (E). Representative data from n = 3 experiments are shown.",
    "molecules": "LiCl"
  },
  {
    "caption": "(G) RT-qPCR of U2-OS cells irradiated (20 Gy) either 1.5h or 7 days prior to harvest. CHIR denotes treatment with GSK3 inhibitor CHIR-99021 (10nM overnight). DMSO overnight treatment was used as control. Quantitation was performed and statistical significance obtained from n = 3 replicates, using ANOVA with Tukey multiple comparisons test. SD shown. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.",
    "molecules": "DMSO, CHIR, CHIR-99021"
  },
  {
    "caption": "(D) U2-OS cells were exposed to IR at indicated time points prior to harvest and analyzed by western blotting for levels of IκBα, NIK, IL-1α and PARP1. Treatment with proteasomal inhibitor MG132 (10 μM) started 4 hours prior to harvest. NIK serves as positive control for efficient proteasome inhibition. Quantitation from n = 4 biological replicates and SD is shown for IκBα (right panel). Brown-Forsynthe ANOVA test: p = 0.0005.",
    "molecules": "MG132"
  },
  {
    "caption": "Representative fluorescence polarization binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 with or without Ca2+ added.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ with or without Ca2+ added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch's t-test. ns, not significant.",
    "molecules": "Ca2+"
  },
  {
    "caption": "A A neutral red-stained mouse pancreas is excised and digested with collagenase. Islets are then separated by hand picking and further digested into single islet cells. Scale bars: 10 mm (left image); 200 µm (middle image); 60 µm (right image).",
    "molecules": "collagenase, neutral red"
  },
  {
    "caption": "D Immunofluorescence stainings of CD31 / α-SMA and CD31 / PDGFR-β in MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 20 µm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "A Neutral red and trypan blue stainings of FI, CI, PI and PI+MVF. CI incubated for 24 h with 0.2% H2O2 served as control. Scale bar: 100 µm.",
    "molecules": "H2O2, Neutral red, trypan blue"
  },
  {
    "caption": "B Representative flow cytometric scatterplots of propidium iodide / annexin V-stained cells from PI+MVF and CI+H2O2 (positive control). C Quantitative analysis of propidium iodide / annexin V-stained cells from FI, CI, PI, PI+MVF and CI+H2O2 subdivided in necrotic, necroptotic, apoptotic and vital cells in % of total cell number (n = 3 each). ",
    "molecules": "H2O2, propidium iodide"
  },
  {
    "caption": "D Schematic illustration of the mitochondrial ROS-sensor mito-roGFP2-Orp1. The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels, as shown by representative fluorescence images of FI. Scale bar: 70 µm.",
    "molecules": "H2O2, ROS"
  },
  {
    "caption": "E Quantitative analysis of the ROS sensor ratio (405 nm / 488 nm) of FI, CI, PI and PI+MVF (n = 25 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.",
    "molecules": "ROS"
  },
  {
    "caption": "H Representative immunofluorescence stainings of HO-1 / insulin and HO-1 / CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 µm.",
    "molecules": "Hoechst 33342, insulin"
  },
  {
    "caption": "A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI, CI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 60 µm. B-D Quantitative analysis of insulin- (B), glucagon- (C) and somatostatin-positive cells (D) in FI, CI, PI and PI+MVF in % of all islet or organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. ",
    "molecules": "Hoechst 33342, glucagon, insulin, somatostatin"
  },
  {
    "caption": "E Western blot analysis of β-actin and insulin in whole cell extracts from FI, CI, PI and PI+MVF.",
    "molecules": "insulin"
  },
  {
    "caption": "Insulin secretion (µU/mL) (normalized to DNA content) of FI, CI, PI and PI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test as well as an unpaired Student's t-test were used for statistical analysis. *P < 0.05 vs. insulin secretion at 1.1 mM glucose within each individual group; +P < 0.05 vs. insulin secretion of PI at 16.5 mM glucose.",
    "molecules": "glucose, Insulin, insulin"
  },
  {
    "caption": "A Representative immunofluorescence stainings of insulin and CD31 in CI, FI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm. B Quantitative analysis of CD31-positive cells in FI, CI, PI and PI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. ",
    "molecules": "Hoechst 33342, insulin"
  },
  {
    "caption": "F Representative immunofluorescence staining of Ki67 and CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark endothelial cells expressing Ki67. Scale bar: 100 µm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "H Flow cytometric analysis of BrdU-positive cells from PI+MVF treated with DMSO (vehicle) or linsitinib during the entire fusion process (n = 5 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.",
    "molecules": "BrdU, DMSO, linsitinib"
  },
  {
    "caption": "I Representative Western blot analysis of pAkt, Akt, pERK1/2 and ERK1/2 from MVF cell extracts. MVF were treated with vehicle or insulin for 3 h. Quantitative analysis of pERK1/2 / ERK1/2 (J) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.",
    "molecules": "insulin"
  },
  {
    "caption": "Quantitative analysis of pAkt / Akt (K) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.",
    "molecules": "insulin"
  },
  {
    "caption": "L Representative bright field image of angiogenic sprouting from PI+MVF on 3 day of the sprouting assay. Scale bar: 150 µm. Insert: Immunofluorescence staining of insulin (red) and CD31 (green) of sprouted PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 30 µm.",
    "molecules": "Hoechst 33342, insulin"
  },
  {
    "caption": "C Representative immunohistochemical stainings of insulin and CD31 in hFI, hCI, hPI and hPI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm.",
    "molecules": "Hoechst 33342, insulin"
  },
  {
    "caption": "D-G Quantitative analysis of insulin-(D), glucagon-(E), somatostatin-(F) and CD31-positive cells (G) in hFI, hCI, hPI and hPI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.",
    "molecules": "glucagon, insulin, somatostatin"
  },
  {
    "caption": "J Quantitative analysis of C-peptide secretion (pg/mL) (normalized to DNA concentration) of hFI and hPI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD.",
    "molecules": "C-peptide, glucose"
  },
  {
    "caption": "B Observation window of a dorsal skinfold chamber with 8 neutral red-stained transplanted FI on day 0. Scale bar: 5 mm.",
    "molecules": "neutral red"
  },
  {
    "caption": "D Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 0 and 14. FITC-labeled dextran is used for the visualization of blood-perfused microvessels. Grafts are marked by broken lines. Scale bar: 150 μm.",
    "molecules": "FITC, dextran"
  },
  {
    "caption": "G Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 14. Rhodamine 6G is used to visualize blood perfused vital endocrine cells (bright signals). Scale bar: 150 µm.",
    "molecules": "Rhodamine 6G"
  },
  {
    "caption": "H Quantitative analysis of the rhodamine 6G-positive area (% of islet size) within transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14. Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.",
    "molecules": "rhodamine 6G"
  },
  {
    "caption": "A Representative immunofluorescent stainings of insulin / glucagon, insulin / somatostatin and insulin / CD31 in FI, CI, PI and PI+MVF on day 14 after transplantation. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark intra-islet endothelial cells. Scale bar: 70 µm. B-E Quantitative analysis of insulin- (B), glucagon- (C), somatostatin- (D) and intra-islet CD31-positive cells (E) within FI, CI, PI and PI+MVF on day 14 after transplantation (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. ",
    "molecules": "Hoechst 33342, glucagon, insulin, somatostatin"
  },
  {
    "caption": "D Quantitative analysis of blood glucose level (mg/dL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day -8 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.",
    "molecules": "glucose"
  },
  {
    "caption": "E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day 0 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.",
    "molecules": "glucose"
  },
  {
    "caption": "F Quantitative analysis of blood glucose level (mg/dL) according to the IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls to the indicated time points (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI.",
    "molecules": "glucose"
  },
  {
    "caption": "H Plasma insulin level (µU/mL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls at 180 min after IPGTT (n = 4 each). Mean ± SD.",
    "molecules": "insulin"
  },
  {
    "caption": "I Representative immunofluorescent stainings of insulin / CD31 in FI and PI+MVF on day 28 after islet transplantation. Scale bar: 50 µm.",
    "molecules": "insulin"
  },
  {
    "caption": "J Quantitative analysis of insulin- (β-cells), glucagon- (α-cells), somatostatin- (δ-cells) and CD31-positive (endothelial) cells within transplanted FI and PI+MVF on day 28 after islet transplantation. Data are expressed in % of total islet cells (n = 25). Mean ± SD.",
    "molecules": "glucagon, insulin, somatostatin"
  },
  {
    "caption": "A. Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s, recorded in a standard bath solution containing 2 mM Ca2+. B. Pan-neurexin deletion suppresses synaptic transmission. Summary graphs show the amplitude (left) and charge transfer (right) of the first EPSC recorded in response to the paired stimuli. C. Pan-neurexin deletion more than doubles the paired-pulse ratio (PPR), suggesting a large decrease in release probability. D. Pan-neurexin deletion decelerates the EPSC time course. Summary graphs show the rise time (left) and decay time constants (right) of the first EPSC in response to the paired stimuli. ",
    "molecules": "Ca2+"
  },
  {
    "caption": "A-D. Same as Fig 2A-D, except that ACSF contained only 1 mM Ca2+, which is close to the estimated physiological concentration of extracellular Ca2+ (Forsberg et al., 2019).",
    "molecules": "Ca2+"
  },
  {
    "caption": "E-H. Same as Fig 2A-D, except that the slice were incubated with 0.2 mM EGTA-AM for 30 mins before recording, which introduces EGTA into the presynaptic terminals.",
    "molecules": "EGTA, EGTA-AM"
  },
  {
    "caption": "A. Example traces of presynaptic Ca2+-currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV. B. Summary graphs of the I-V relationship for the peak Ca2+-current amplitude (left) and of the peak Ca2+-current density (right).",
    "molecules": "Ca2+"
  },
  {
    "caption": "C. Example traces (left) and summary graphs (right) of the Ca2+-current activation and deactivation kinetics. Currents were induced by a 50 ms step depolarization from -80 mV to +10 mV.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D. Example traces of Ca2+-currents evoked by an action potential-equivalent depolarization (APe, from -80 mV to +17 mV for 1 ms; left), and summary graph of the APe-evoked Ca2+-current peak amplitude and charge (right).",
    "molecules": "Ca2+"
  },
  {
    "caption": "E. Example traces of presynaptic Ca2+-currents induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q-type Ca2+ channel blocker.",
    "molecules": "Ca2+, agatoxin, Agtx"
  },
  {
    "caption": "F. Summary graph of the relative contribution of P/Q-type Ca2+ channels to the total presynaptic Ca2+ currents, as quantified by the relative reduction in Ca2+ currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.",
    "molecules": "Ca2+"
  },
  {
    "caption": "B. Example traces of the presynaptic capacitance and Ca2+-currents at the calyx terminals induced by a 20 ms step depolarization from −80 mV to +10 mV with an extracellular solution containing 2 mM Ca2+, and an internal pipette solution containing a standard 0.05 mM BAPTA. C. Summary graphs of the depolarization-induced capacitance jump that monitors synaptic vesicle exocytosis (left), and the peak Ca2+-current density (right). D, E. Same as (B, C), except that 0.5 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. F, G. Same as (B, C), except that 1.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. H, I. Same as (B, C), except that 10.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. ",
    "molecules": "BAPTA, Ca2+, EGTA"
  },
  {
    "caption": "J, K. Summary plots of the relationship between the total capacitance jump (J) or between the Ca2+-current density (K) and the concentration of EGTA in the patch pipette.",
    "molecules": "Ca2+, EGTA"
  },
  {
    "caption": "A. Representative traces of Ca2+-currents and 4-AP insensitive K+-currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12-P14. B. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion. C. Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in (B) from currents shown in (A). D. Summary graphs of the capacitance, Ca2+-current density, and BK-current density. ",
    "molecules": "4-AP, Ca2+, iberiotoxin, IbTx, K+"
  },
  {
    "caption": "(E) Western Blot analysis interaction of CREBH and HRD1 after immunoprecipitates of Flag-agarose in transfected HEK293T.",
    "molecules": "agarose"
  },
  {
    "caption": "(G) Western Blot analysis ubiquitination of CREBH after immunoprecipitates of Flag-agarose in transfected HEK293T.",
    "molecules": "agarose"
  },
  {
    "caption": " (A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm. ",
    "molecules": "cisplatin, doxorubicin"
  },
  {
    "caption": "(D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.",
    "molecules": "doxorubicin, 3‐MA, 3‐methyladenine"
  },
  {
    "caption": "(A-C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05).",
    "molecules": "cisplatin, doxorubicin"
  },
  {
    "caption": "(D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. *P0.05 compared with control shRNA group.",
    "molecules": "doxorubicin, E64D, pepstatin A"
  },
  {
    "caption": "(E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5+/+ and ATG5−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z‐VAD‐FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, *P0.05).",
    "molecules": "doxorubicin, Z‐VAD‐FMK"
  },
  {
    "caption": "(F) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)‐specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, *P0.05; shControl+Doxo compared with shUVRAG+Doxo).",
    "molecules": "doxorubicin"
  },
  {
    "caption": "(A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co‐immunoprecipitation (RIPA or CHAPS buffer) or western blotting.",
    "molecules": "CHAPS"
  },
  {
    "caption": "(E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2‐associated X protein; CCD, coiled‐coil domain; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate; GST, glutathione‐S‐transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.",
    "molecules": "doxorubicin"
  },
  {
    "caption": " UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A-C) and activation of BAX (D-F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P0.05. AU, arbitrary units; BAX, BCL2‐associated X protein; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene. ",
    "molecules": "doxorubicin"
  },
  {
    "caption": "Maximum projection (2 z-slices) of HeLa cells immunostained for F-actin (Phalloidin), Tubulin, Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase. Scale bar- 10µm Quantification of F-actin (Phalloidin) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in (A), showing the increase in F-actin around centrosomes at early anaphase. Mean actin metaphase = 1 ± 0.03996, n=92, mean actin anaphase = 1.875 ± 0.08895, n=121, Student t-test, ****p<0.0001;",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "Stills from time-lapse of HeLa cells expressing GFP-alpha-tubulin and RFP-lifeact arrested at prometaphase with STLC (t=0) and forced to exit mitosis with Cdk1 inhibition (Ro3306) imaged every 90 seconds. Scale bar = 10µm and for zoom = 4µm. n = 21 cells from 4 independent experiments.",
    "molecules": "Ro3306, STLC"
  },
  {
    "caption": "HeLa cells expressing GFP-centrin1 arrested at prometaphase or forced mitotic exit (Ro3306 5') and stained with phalloidin. Scale bar- 5µm and for zoom-2µm.",
    "molecules": "Ro3306, phalloidin"
  },
  {
    "caption": "The level of actin around the centrosome in (C) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit. STLC arrest 1 ± 0.01317, n=210, STLC+Ro-5mins=1.251 ± 0.02441, n=144, Unpaired t-test with Welch correction, ****p<0.0001. Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor. Tubulin intensity decreses during forced mitotic exit. STLC-DMSO- 1 ± 0.02407, n=180, STLC-RO-3306- 0.6516 ± 0.02337, n=136. P<0.0001, Welch's t test.",
    "molecules": "Ro, RO-3306, DMSO, STLC"
  },
  {
    "caption": "Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments.",
    "molecules": "CK666, DMSO"
  },
  {
    "caption": "Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin.",
    "molecules": "CK666, DMSO, phalloidin"
  },
  {
    "caption": "Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001.",
    "molecules": "RO-3306, CK666, DMSO, STLC"
  },
  {
    "caption": "Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation.",
    "molecules": "CK666, DMSO"
  },
  {
    "caption": "Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.",
    "molecules": "CK666, DMSO"
  },
  {
    "caption": "Levels of p34-Arc around the centrosome (3um area) were quantified relative to metaphase and increase during forced mitotic exit. STLC arrested = 1 ± 0.01576, n=153, STLC+RO-3306= 1.268 ± 0.01748, n=191, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.",
    "molecules": "RO-3306, STLC"
  },
  {
    "caption": "Quantification of p34-Arc levels around the centrosome in (C), relative to metaphase, showing its increase during forced exit. STLC arrested = 1 ± 0.00779, n=93, STLC+RO-3306= 1.249 ± 0.02446, n=134, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.",
    "molecules": "RO-3306, STLC"
  },
  {
    "caption": "Time-lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time. Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in (E) over an area of 2 μm, (white circle), showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit. N=2 isolations, 5 independent experiments, n=16 STLC and n=19 STLC RO.",
    "molecules": "STLC"
  },
  {
    "caption": "Color switch experiment using green- and red-labelled actin. Red actin was used first, followed by Capping protein, then green-labelled actin. The accumulation of green in the centre indicates that actin is nucleated at the centrosome. Scale bar = 10µm. Quantification of (G) over the time. N=2 isolations, 2 independent experiments, n=19 STLC and n=14 STLC RO. Error bars represent standard deviation.",
    "molecules": "STLC"
  },
  {
    "caption": "Time-lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre-treated with DMSO or CK666. Scale bar = 10µm. Quantification of (I), showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre-treated with CK666. N=2 isolations, 2 independent experiments, n = 17 DMSO, 26 Ro CK666 24 STLC DMSO, 23 STLC CK666. Error bars represent standard deviation.",
    "molecules": "Ro, CK666, DMSO, STLC"
  },
  {
    "caption": "Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.",
    "molecules": "Ro3306, STLC"
  },
  {
    "caption": "Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm.",
    "molecules": "Ro3306"
  },
  {
    "caption": "Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA.",
    "molecules": "RO-3306, phalloidin, STLC"
  },
  {
    "caption": "WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with hematoxylin and eosin. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).",
    "molecules": "eosin, hematoxylin, PBS"
  },
  {
    "caption": "WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with anti-CD45. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).",
    "molecules": "PBS"
  },
  {
    "caption": "(B) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to three 40-bp DNAs: 1-40 (black circles), 31-70 (Site 1; green squares), and 81-120 (Site 2; orange triangles). Fit Kd and Hill coefficient for each DNA is shown. Error bars indicate standard deviation from three technical replicates.",
    "molecules": "DNA, DNAs"
  },
  {
    "caption": "(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; wild-type or indicated point mutants) to the Site 2 DNA (bases 81-120 in panel A). Fit Kd and Hill coefficient for each DNA is shown.",
    "molecules": "DNA"
  },
  {
    "caption": "(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; full-length (black circles) or NTD(green squares)) to the Site 2 DNA. WT Kd = 0.30 +/- 0.08 μM, Hill coefficient = 2.0 +/- 0.7; no binding detected for CapHNTD). Error bars indicate standard deviation from three technical replicates. (E) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to the Site 1 (bases 31-70) and Site 2 (bases 81-120) DNAs. Wild-type CapH binding Site 1 is shown in orange triangles, and binding Site 2 is shown in blue diamonds. CapH(I99M) binding Site 1 is shown in black circles, and binding Site 2 is shown in green squares. Fit Kd and Hill coefficient for each combination is shown.",
    "molecules": "DNA, DNAs"
  },
  {
    "caption": "(B) In vitro cleavage of purified MBP-CapH-GFP (yellow arrowhead) into N-terminal and C-terminal products (white arrowheads) by CapP is stimulated by DNA. For both ssDNA and dsDNA, the highest concentration is 350 ng/μL, followed by three 5-fold dilutions. Red asterisk indicates the band that was analyzed by Edman degradation.",
    "molecules": "dsDNA, ssDNA"
  },
  {
    "caption": "(E) In vitro cleavage of MBP-CapH-GFP (wild-type or R83A mutant) by CapP, in the presence of 10 μM ssDNA.",
    "molecules": "ssDNA"
  },
  {
    "caption": "(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type CapP after exposure to zeocin (100 μg/mL). Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. α-RNAP: anti-RNA polymerase loading control.",
    "molecules": "zeocin"
  },
  {
    "caption": "(B) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to zeocin (100 μg/mL). α-RNAP: anti-RNA polymerase loading control. (C) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to mitomycin C (1 μg/mL). α-RNAP: anti-RNA polymerase loading control.",
    "molecules": "mitomycin C, zeocin"
  },
  {
    "caption": " A. In vivo genetic complementation of the E. coli MC4100 wild-type (WT) and secB or/and tig knock-out derivatives by either an empty vector or one carrying the secB or tig (encoding TF) genes or derivatives (see "Plasmid" table in Appendix), as indicated. Serial dilutions of a culture (OD600= 0.5) were spotted (12μl) on LB-Ampicillin plates containing or not anhydrotetracycline (AHT; 5 ng/ml) and grown at 16°C or 30°C (as indicated). n= 3-7 biological replicates. ",
    "molecules": "Ampicillin, AHT, anhydrotetracycline"
  },
  {
    "caption": " C. SecA ATPase activity determination in solution (basal; B), or plus SecYEG IMVs; (membrane; M) or plus proOmpA (pre-treated with 10 mM DTT; 5 mM EDTA; 20 min; 4°C; translocation; T). n= 21 biological replicates. Mean values (± SEM) shown. ",
    "molecules": "DTT, EDTA"
  },
  {
    "caption": " A. TF-proOmpA physical interaction in solution. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) with the indicated molar excess of TF2. Soluble proteins were analyzed on 10% native-PAGE (4 mA; 16h; 4°C) and Coomassie-blue stained. A representative experiment is shown; n= 4 biological replicates ",
    "molecules": "urea"
  },
  {
    "caption": " C. TF solubilizes proOmpA*. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) alone or in the presence of TF. Proteins in the soluble fraction were analyzed on 15% SDS-PAGE and Coomassie-blue stained. Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 5 biological replicates ",
    "molecules": "urea"
  },
  {
    "caption": " D. proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity. proOmpA (0.5 μM) was either diluted directly (lane 1) or after preincubation alone (10 min; buffer D; < 0.2 M urea; either at 4°C or 37°C) into reactions containing SecYEG-SecA alone or together with TF2 or SecB4 (2 and 4 x molar excess over proOmpA, respectively). T/M ratios (as in Fig. 1D) are shown (n= 8 biological replicates; mean values ± SEM; Unpaired parametric t-test, 95% confidence interval: **: p = 0.0017; ****: p < 0.0001) ",
    "molecules": "urea"
  },
  {
    "caption": " E. proOmpA pre-incubated at 37oC does not interact with TF. proOmpA1 (5 μM) was preincubated (Buffer D; < 0.2 M urea; 37°C; 10 min) or not (lane 3), prior to addition of the indicated molar excess of TF2 (analyzed as in A). Representative experiment shown; n= 4 biological replicates ",
    "molecules": "urea"
  },
  {
    "caption": " A. SecB maintains soluble translocation-competent states of proOmpA at 37°C. proOmpA1 (0.5 µM) was incubated with a range of SecB4 concentrations (0 - 2 µM; Buffer D; < 0.2 M urea; 10 min, 37°C). Soluble samples (20,000 x g; 10 min; 4°C) were added to translocation ATPase reactions (as in Fig. 1C). Normalized T/M ratios were plotted (n= 2-8 biological replicates; mean values ± SEM) and an apparent solubilization constant (Ksol) determined (as in Fig. 1F)",
    "molecules": "urea"
  },
  {
    "caption": " C. SecB physically interacts with proOmpA. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 4°C; 60 min), with the indicated molar excess of SecB4 (filled arrow), analyzed by native-PAGE as in Fig. 2A, and stained as indicated. brace: higher order proOmpA species. Representative experiment shown; n= 5 biological replicates ",
    "molecules": "urea"
  },
  {
    "caption": "A,C) After 24, 48, and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner. Untreated cells were measured as controls (n = 12, mean ± S.D.).",
    "molecules": "bortezomib"
  },
  {
    "caption": "B,D) Dynamic proliferation curves for Cal-78 and SW-1353 cells in the presence of 0 (red), 2.5 nM (green), 5 nM (blue), and 10 nM (purple) bortezomib. Data shown are representatives from three independent experiments (n = 3, measured in biological quadruplicates).",
    "molecules": "bortezomib"
  },
  {
    "caption": "A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1).",
    "molecules": "Bortezomib"
  },
  {
    "caption": "B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.",
    "molecules": "bortezomib"
  },
  {
    "caption": "A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h (n = 10).",
    "molecules": "bortezomib"
  },
  {
    "caption": "D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.",
    "molecules": "bortezomib"
  },
  {
    "caption": "A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC50 concentrations for 48 h. Untreated control cells served as reference value (ratio = 1).",
    "molecules": "bortezomib"
  },
  {
    "caption": "A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.",
    "molecules": "bortezomib"
  },
  {
    "caption": "B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.",
    "molecules": "bortezomib"
  },
  {
    "caption": "C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II.",
    "molecules": "E64d, pepstatin A"
  },
  {
    "caption": "A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm).",
    "molecules": "E64d, bortezomib, DAPI, pepstatin A"
  },
  {
    "caption": "B) Cal-78 and SW-1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A (Inh; striated bars) and cell viability was analysed by the MTS assay. Untreated cells were measured as controls (light grey; n = 8, mean ± S.D.).",
    "molecules": "E64d, bortezomib, pepstatin A"
  },
  {
    "caption": "Representative immunofluorescence  images (left) of GAPDH  (red) in HeLa  cells untreated (Utr), or treated with IR, etoposide  (ETO), adriamycin  (ADR), and camptothecin  (CPT) as indicated. DNA was stained  with DAPI  (blue). Quantitative analysis (right) of nuclear GAPDH. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001",
    "molecules": "camptothecin, CPT, DAPI, ADR, adriamycin, ETO, etoposide"
  },
  {
    "caption": "HeLa  cells were pretreated with or without 10 μM PP2  for 24 h and exposed to 4 Gy IR. After 4 h of recovery, nuclear  and cytoplasmic  fractions were separated and subjected to immunoblot  with indicated antibodies (left). Quantification of nuclear GAPDH  is in the right panel. β-Tubulin  and Lamin B1  served as indicators for the cytoplasmic  and nuclear  fractions. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001",
    "molecules": "PP2"
  },
  {
    "caption": "E-F. Immunoblot   of anti-GAPDH immunoprecipitates from HEK293T  treated with 4 Gy IR  and recovered for 4 h (E), or with 5 μM ADR  for 2 h (F).",
    "molecules": "ADR"
  },
  {
    "caption": "Cell viability analysis of HeLa  cells stably expressing the sh-NC and sh-GAPDH  were treated with the indicated concentration of ADR  for 48 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "ADR"
  },
  {
    "caption": "Left, representative immunofluorescence  images of γH2AX  (C, green) in HeLa  cells transfected with scramble siRNA or si-GAPDH  after 4 Gy IR  exposure and recovered for 4 h. DNA was stained  with DAPI  (blue). Right quantification of γH2AX  foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "DAPI"
  },
  {
    "caption": "Left, representative immunofluorescence  images of 53BP1  (D, red) in HeLa  cells transfected with scramble siRNA or si-GAPDH  after 4 Gy IR  exposure and recovered for 4 h. DNA was stained  with DAPI  (blue). Right, quantification of 53BP1  foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunoblot  (left) and quantification (right) of RAD51  in HeLa  cells with or without GAPDH  knockdown treated with 50 μg/mL CHX  at the indicated time points. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "CHX"
  },
  {
    "caption": "Immunoblot  (left) and quantification (right) of RAD51  in GAPDH  knockdown HeLa  cells and control cells treated with or without MG132  (10 μM) for 10 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "MG132"
  },
  {
    "caption": "Immunoblot  (left) and quantification (right) of RAD51  in HeLa  cells at different durations of 50 μg/mL CHX  administration with or without TSA  (10 μM, 12 h) treatment. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "CHX, TSA"
  },
  {
    "caption": "Immunoblot  of cell lysates or anti-RAD51 immunoprecipitates  from HeLa  cells with scramble siRNA or si-GAPDH  transfection treated with MG132  (10 μM) for 2 h.",
    "molecules": "MG132"
  },
  {
    "caption": "Immunoblot  of cell lysates or anti-Flag immunoprecipitates  from HEK293T  cells transfected with indicated Flag-RAD51  mutations and then exposed to 10 μM MG132. Immunoblot  of cell lysates or anti-Flag immunoprecipitates  from HEK293T  cells transfected with HA-Ubiquitin   and indicated Flag-RAD51  mutations and then exposed to 10 μM MG132.",
    "molecules": "MG132, Ubiquitin"
  },
  {
    "caption": "Left, GAPDH  knockdown HeLa  cells transfected with Flag-HDAC1  were treated with 4 Gy IR, fixed after 4 h, and immunostained  with anti-RAD51  (red). DNA was stained  with DAPI  (blue). The number of RAD51  foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "DAPI"
  },
  {
    "caption": "Left, GAPDH  knockdown HeLa  cells transfected with Flag-HDAC1  were treated with 4 Gy IR, fixed after 4 h, and immunostained  with anti- γH2AX  (green). DNA was stained  with DAPI  (blue). The number of γH2AX  foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Light/heavy ratio of tau peptides in label-free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau. Light peptides represent endogenous tau peptides. Data are mean ± SEM, n=3 biological repeats, ***p<0.001.",
    "molecules": "15N"
  },
  {
    "caption": "D. Western blot of HEK293T intracellular lysates show the DnaJC5/Hsc70/tau complex is disrupted by the addition of increasing amounts of YM-01 (0, 10, 30, 100 µM) in a dose-dependent fashion. DnaJC5 was detected by FLAG antibody.",
    "molecules": "YM-01"
  },
  {
    "caption": "F. Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM-01. Quantification of extracellular tau levels are mean ± SEM, n=6, *p<0.05 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. Intracellular levels shown by Western blot. DnaJC5 was detected on Western blot by FLAG antibody.",
    "molecules": "YM-01"
  },
  {
    "caption": "G. Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM-01 (10µM). FLAG-DnaJC5 was transduced into neurons using AAV9. Quantification of extracellular tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected on Western blot by FLAG antibody.",
    "molecules": "YM-01"
  },
  {
    "caption": "(A) Example traces of inside-out patch clamp  recordings of acutely isolated FDB myofibers  of WT (black, top left), SUR2-STOP475 (purple, top right), and skeletal muscle dominant negative (SkM-DN; magenta, bottom left) mice. Patches were voltage clamped at -50 mV and excised at time-points indicated by arrows. ATP  (5 mM) was administered to the cytoplasmic  face of membrane patches as indicated by horizontal bars. Dashed lines denote the zero KATP   current level.",
    "molecules": "ATP"
  },
  {
    "caption": "(B) Maximal ATP-sensitive current  levels from excised patches. Bars show mean (± SEM) with individual recordings from each myofiber  shown as dots/squares (cells from ≥ 3 mice; WT n = 10 recordings; STOP n = 8; SkM-DN n = 11; Dunn's test following Kruskal-Wallis omnibus test p = 1.0 x 10-4 for WT vs STOP and 0.014 for WT vs SkM-DN ; * denotes p value < adjusted α = 0.0167).",
    "molecules": "ATP"
  },
  {
    "caption": "(D) Left - Picrosirius red stained TA  muscle from WT and SkM-DN mice. Right - quantification of red stained  area (WT n = 4; SkM-DN n = 4; p = 0.028 according to Mann Whitney U test). Bars show mean (± SEM) with individual measurements shown as dots/squares. (E) Left - TA  muscle for WT and SkM-DN mice  showing Pax7  positive immunolabelled satellite cells  (red arrows). Right - quantification of Pax7+ cells (WT n = 4; SkM-DN n = 4; p = 0.028 according to Mann Whitney U test). Bars show mean (± SEM) with individual measurements shown as dots/squares. Data information: * denotes p value < α = 0.05 for all panels. Scale bars show 50 µm in each panel.",
    "molecules": "Picrosirius red"
  },
  {
    "caption": "(A) Survival  of WT and SUR2-STOP475 mice  administered with verapamil  in drinking water (n = 7 [4 male, 3 female] for WT administered 0.9 g/l verapamil; n = 14 [8 male, 6 female] for SUR2-STOP475 administered 0.9 g/l verapamil; n = 6 [3 male, 3 female] for SUR2-STOP475 administered 0.3 g/l verapamil). Verapamil  induced deaths were observed in both male  and female mice  (7 of 8 male  and 4 of 6 female SUR2-STOP475 mice  died within 28 days of 0.9 g/l verapamil). (B) Survival  of WT and SUR2-STOP1149 mice  administered with verapamil  in drinking water (n = 10 [3 male, 7 female] for SUR2-STOP1149 mice  administered 0.9 g/l verapamil).",
    "molecules": "verapamil, Verapamil"
  },
  {
    "caption": "(D) Heart  rates calculated from ECG  recordings from WT and SUR2-STOP mice  administered with no verapamil  (0 Verap) or 0.9 g/l verapamil  in drinking water.",
    "molecules": "verapamil"
  },
  {
    "caption": "(F) Cumulative durations of inversion in multi-trial inverted screen test for WT mice  (black circles) and SkM-DN mice without (magenta squares) or with 0.9 g/l verapamil  (pink triangles) administration. Data from WT and SkM-DN without verapamil  Bars show mean ± SEM (WT 0 verapamil  n = 9 [6 male; 3 female]; SkM-DN 0 verapamil  n = 16 [12 male; 4 female]; SkM-DN with 0.9 g/l verapamil  n = 6 [4 male; 2 female]; Dunn's test p = 0.006 for WT 0 verapamil  vs SkM-DN 0 verapamil  and 0.027 for WT 0 verapamil  vs SkM-DN 0.9 g/l verapamil  following Kruskal-Wallis omnibus test; * denotes p value < adjusted α = 0.017).",
    "molecules": "verapamil"
  },
  {
    "caption": "(A) Left - Average performance in the  inverted screen test  of mice  homozygous for the ncDHPR   allele alone (ncDHPR) or homozygous for both the ncDHPR and SUR2-STOP475 alleles (STOP475/ncDHPR). Right - Summary of inverted screen test data. Data shown as mean ± SEM (WT n = 9 [4 male; 5 female]; ncDHPR n = 15 [male  6; female  9]; STOP475 n = 15 [5 male; 10 female]; STOP475/ncDHPR n = 12 [4 male; 12 female]; Dunn's test p = 4.0 x 10-4 for WT vs STOP475, 6.9 x 10-4 for WT vs STOP475/ncDHPR, 0.001 for ncDHPR  vs STOP475/ncDHPR verapamil, following Kruskal-Wallis omnibus test). * denotes p value < adjusted α = 0.0083.",
    "molecules": "verapamil"
  },
  {
    "caption": "(F) Summary of unstimulated force  (normalised by physiological cross-sectional area) at 60 s for each experiment. Bars show mean ± SEM with individual biological replicates shown as dots/squares/triangles (WT without glibenclamide  n = 6 [3 male; 3 female]; WT with glibenclamide  n = 10 [5 male; 5 female]; ncDHPR  without glibenclamide  n = 6 [3 male; 3 female]; ncDHPR with glibenclamide  n = 6 [3 male; 3 female]; Dunn's test p = 7.0 x 10-4 for WT without glibenclamide  vs WT with glibenclamide  and 0.002 for ncDHPR without glibenclamide  vs ncDHPR with glibenclamide, following Kruskal-Wallis omnibus test; * denotes p value < adjusted α = 0.0083).",
    "molecules": "glibenclamide"
  },
  {
    "caption": "A) Example genomic region of MSL2 MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. DNA binding is defined as sub-nucleosomal fragments (10 - 130 bp), while chromatin binding as mono-nucleosomal fragments (130 - 220 bp). Called peaks/regions are represented as boxes above the tracks and HAS below. Late-appearing regions are marked by a plus (+) sign. Graph represents pooled IP/Input signal of 3 biological replicates.",
    "molecules": "DNA"
  },
  {
    "caption": "B) Average composite plots of sub-nucleosomal MSL2 MNase-ChIP-seq (DNA) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos. Total number of PionX = 56 and HAS = 247. Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.",
    "molecules": "DNA"
  },
  {
    "caption": "B) Average composite plots of MSL2 [Chromatin] profiles (stage 5-8, orange and stage 13-15, purple) centered at MSL2 DNA peaks which do not overlap (DNA) or overlap MSL2 chromatin regions (DNA + Chromatin), and MSL2 chromatin regions which do not overlap MSL2 DNA peaks (Chromatin). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.",
    "molecules": "DNA"
  },
  {
    "caption": "A) Example genomic region of DNA binding by MSL2 (DNA), and chromatin binding of MSL2 (Chrom.), MOF and H4K16ac MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. Called peaks/regions are represented as boxes above the tracks. Graph represents pooled IP/Input signal of 3 biological replicates.",
    "molecules": "DNA"
  },
  {
    "caption": "C) Log10 distance between the middle of a gene and its nearest functional MSL2 DNA peak (cluster 1-3). Red line indicates median distance to the nearest peak.",
    "molecules": "DNA"
  },
  {
    "caption": "A Levels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.actin, Western blot",
    "molecules": "O2"
  },
  {
    "caption": "C Levels of ULK1 in MEFs treated with FCCP (20 μM) for the indicated times.actin",
    "molecules": "FCCP"
  },
  {
    "caption": "E MEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 6 h and then fixed by 4% paraformaldehyde. Cells were stained with anti‐TIM23 (mouse) and anti‐ULK1 (rabbit) primary antibodies, then Alexa Fluor‐488‐labeled donkey anti‐rabbit IgG and Alexa Fluor‐555‐labeled donkey anti‐mouse IgG secondary antibodies before analysis by immunofluorescence microscopy. Scale bar, 10 μm.",
    "molecules": "FCCP, O2"
  },
  {
    "caption": "F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia (1% O2) for 12 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1",
    "molecules": "O2"
  },
  {
    "caption": "G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP (20 μM) for 6 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1",
    "molecules": "FCCP"
  },
  {
    "caption": "CMEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 3 h, followed by immunoprecipitation using anti‐ULK1 antibody.",
    "molecules": "FCCP, O2"
  },
  {
    "caption": "D The expression of FUNDC1 is induced by 10 ng/ml tetracycline (Tet) in FUNDC1‐inducible HeLa cells. Cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N) for 24 h. Then, the cell lysates were prepared for immunoblotting using indicated antibodies.",
    "molecules": "tetracycline"
  },
  {
    "caption": "G Western blotULK1, FUNDC1, and the phosphorylated FUNDC1 in MEFs exposed to hypoxia (1% O2) for the indicated times.",
    "molecules": "O2"
  },
  {
    "caption": "HWestern blot analysis of the kinetics of ULK1, FUNDC1, and the phosphorylated in MEFs treated with FCCP(20 μM) for the indicated times.actin",
    "molecules": "FCCP"
  },
  {
    "caption": "KMEFs were transfected with FUNDC1‐Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA. 36 h after transfection, cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1) and then lysed for immunoblotting with the indicated antibodies.",
    "molecules": "BAF1, bafilomycin A1"
  },
  {
    "caption": "L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.",
    "molecules": "FCCP, O2"
  },
  {
    "caption": "AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.",
    "molecules": "BAF1, bafilomycin A1, O2"
  },
  {
    "caption": "B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.",
    "molecules": "BAF1, bafilomycin A1, FCCP"
  },
  {
    "caption": "C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.",
    "molecules": "BAF1, bafilomycin A1"
  },
  {
    "caption": "D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted.",
    "molecules": "BAF1, bafilomycin A1, O2"
  },
  {
    "caption": "EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.",
    "molecules": "BAF1, bafilomycin A1, FCCP"
  },
  {
    "caption": "H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted.",
    "molecules": "BAF1, bafilomycin A1"
  },
  {
    "caption": "I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit.",
    "molecules": "BAF1, bafilomycin A1"
  },
  {
    "caption": "A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.",
    "molecules": "BAF1, bafilomycin A1, O2"
  },
  {
    "caption": "B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.",
    "molecules": "BAF1, bafilomycin A1, FCCP"
  },
  {
    "caption": "C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.",
    "molecules": "FCCP, O2"
  },
  {
    "caption": "Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs and MEFs. TDG and TDG-SUMO conjugates before (2i, 0 h) and during differentiation of TDG wildtype (TDGwt) and TDG deficient (TDGnull) mESC and MEFs (5 µM all-trans retinoic acid (RA, 24 h, 48 h). Percentages of TDG-SUMO relative to total TDG signal are indicated.",
    "molecules": "all-trans retinoic acid, RA"
  },
  {
    "caption": "Immunoblot analyses of TDG, TDG-SUMO in whole cell extracts of mESCs TDG and TDG-SUMO before and after H2O2 treatment (10 mM, 15 min).",
    "molecules": "H2O2"
  },
  {
    "caption": "Immunoblot analyses of XRCC1 and , XRCC1-SUMO, in whole cell extracts of mESCs and MEFs. XRCC1 and XRCC1-SUMO conjugates before (2i, 0 h) and during RA-induced differentiation (24 h, 48 h) in mESCs and MEFs. Percentages of XRCC1-SUMO relative to total XRCC1 signal are indicated.",
    "molecules": "RA"
  },
  {
    "caption": "Immunoblot analyses of , XRCC1 and XRCC1-SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 (5 mM, 15 min).",
    "molecules": "H2O2"
  },
  {
    "caption": "Immunoblot analyses of TET1 isoforms in whole cell extracts of mESCs and MEFs. TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs.",
    "molecules": "RA"
  },
  {
    "caption": "Immunoblot analysis of interactions of XRCC1 and XRCC1-SUMO with endogenous TDG. Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1-SUMO was coupled to Ni-NTA agarose and incubated with mESC extracts. Input (I), last wash (LW) and elution (E) fractions.",
    "molecules": "agarose, his, Ni, NTA"
  },
  {
    "caption": "0.5 µM TDGwt, were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) and equimolar amounts of XRCC1, POLβ and LIGIII proteins.",
    "molecules": "G•5caC DNA, G•C DNA, 5caC"
  },
  {
    "caption": "Schematic of biotin-5caC DNA substrate pulldown experiment. 0.5 µM TDGwt or TDGcat (TDG(N140A)) incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1-SUMO (>50% SUMOylated), POLβ and LIGIII.",
    "molecules": "5caC DNA, G•5caC DNA, G•C DNA, 5caC, biotin"
  },
  {
    "caption": "1 µM biotinylated 5caC substrate (G•5caC DNA) and 10 µM homoduplex competitor DNA (G•C DNA) were incubated with XRCC1-SUMO (50% SUMOylated) and BER proteins as indicated.",
    "molecules": "G•5caC DNA, G•C DNA, 5caC"
  },
  {
    "caption": "0.5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate (G•5caC DNA), 0.5 µM SUMO and 10 µM homoduplex competitor DNA (G•C DNA) as indicated. Shown is an immunoblot analysis of TDG and free SUMO.",
    "molecules": "G•5caC DNA, G•C DNA, 5caC"
  },
  {
    "caption": ""Far Western" blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1-SUMO. TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1-SUMO (probe:XRCC1 or XRCC1SUMO) where indicated. TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated.",
    "molecules": "his"
  },
  {
    "caption": "Schematic of biotin-5caC DNA substrate pulldown experiment. Immunoblot analysis of purified TDGwt or TDG SUMO-binding mutant (TDGE310Q), 6hisXRCC1/XRCC1-SUMO and 6his BER proteins bound to Streptavidin (St) Biotin-5caC DNA substrate. 0.5 µM TDGwt or TDG SUMO-binding mutant (TDGE310Q) were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1, POLβ, APE1 and LIGIII proteins. Grey ovals, BERosome.",
    "molecules": "5caC DNA, G•5caC DNA, G•C DNA, 5caC, biotin, Biotin, his, Streptavidin"
  },
  {
    "caption": "Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA.",
    "molecules": "G•AP, DNA"
  },
  {
    "caption": "Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMOylation assay as in (A) with SUMO provided as XRCC1-SUMO instead of free SUMO.",
    "molecules": "G•AP, DNA"
  },
  {
    "caption": "Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMO transfer as depicted in (B) with additional APE1, POLß, LIGIII, and TDG.",
    "molecules": "G•AP, DNA"
  },
  {
    "caption": "Immunoblot analysis of TDG and TDG-SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H2O2 as indicated.",
    "molecules": "H2O2"
  },
  {
    "caption": "Top: Schematic of the SUMO-mediated TDG-BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double-strands in presence of dynamic SUMO-conjugation/de-conjugation. Left panels lanes 3-14: BER reconstitution with XRCC1 or XRCC1-SUMO as indicated. Right panels lanes 15-21: BER reconstitution with free SUMO or XRCC1-SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1-2 CG/CG HpaII methylation sensitive restriction site, cleavable by HpaII; lane 3-6, 8-14, 15-17, 21 HpaII methylation sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII; lane 7 and 20 repaired Product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single stranded DNA.",
    "molecules": "5caC, DNA"
  },
  {
    "caption": "Bright field captures of TDGwt, TDGcat and TDGsnm mESCs at indicated stages of differentiation. DAPI, Alexa Fluor® 680 phalloidin actin staining at the bottom. Scale bars 25µM, 50µM, 500µM as indicated. Quantification of neural cells with neurites of total differentiating TDGwt, TDGcat and TDGsnm mESCs. n ≥ 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.",
    "molecules": "Alexa Fluor® 680 phalloidin, DAPI"
  },
  {
    "caption": "LC-MS/MS analysis of global cytosine-modification levels in DNA from ground-state mESCs (2i) and EBs. Means with standard deviation (SD) of 5mC, 5hmC, 5fC and 5caC per 106 bases). n ≥ 3. *: P≤0.05, **: P<0.01, ***: P<0.0001 by one-way ANOVA, Dunnett's multiple comparisons test.",
    "molecules": "5hmC, 5caC, 5fC, 5mC, cytosine, DNA"
  },
  {
    "caption": "Alkaline comet assay with pluripotent (0 h) and differentiating (24 h 5 µM RA) mESC as indicated. Olive Tail Moments and % DNA in tail. n ≥ 3. Medians with standard deviation (SD). *: P≤0.05; **: P≤0.01, ***: P≤0.0001 by one-way ANOVA, Sidak's multiple comparisons test.",
    "molecules": "RA, DNA"
  },
  {
    "caption": "Cell proliferation assay. Relative viability of Talazoparib treated to untreated cells. n = 3. Means with standard deviation (SD), P≤0.05, **: P<0.01, by one-way ANOVA, Dunnett's multiple comparisons test.",
    "molecules": "Talazoparib"
  },
  {
    "caption": "ChIP-qPCR analysis of TET1, TDG and XRCC1 at gene promoters in mESCs (24 h 5 µM RA). Relative enrichments to an unbound control region (Chr2neg). n ≥ 3. Means with standard deviation (SD). *: P≤0.05; **: P<0.001; ***: P≤0.0001, by two-tailed Mann-Whitney test.",
    "molecules": "RA"
  },
  {
    "caption": "Representative images of mESC neuronal differentiation as in Fig 4B. Shown are XRCC1wt and XRCC1null (XRCC1null1, XRCC1null2) mESCs in 2i medium supplemented with LIF (2i LIF) and in ESC medium supplemented with LIF (ESCM LIF), EBs in ESCM LIF and 5µM RA (EB 4d ESCM RA) and dissociated neuron-like cells in B27 (B27). Scale bars 25µM, 50µM, 500µM as indicated. Neurite grids were observed for XRCC1wt; heterogeneous cell populations formed with the XRCC1null (XRCC1null1, null2) clones. Quantification (below) of neural cells with neurites of total differentiating XRCC1wt, XRCC1null mESCs. n = 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.",
    "molecules": "RA"
  },
  {
    "caption": "Dox-induced prolongation of Nanog expression in Nanogtg embryos up to E9.5 results in lack of blood (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-mount in situ hybridization for Hbb-bh1 (in embryos with intact yolk sacs) and for the long non-coding RNA Redrum. Asterisks mark the aorta-gonad-mesonephros region (AGM) and arrows the tailbud. Embryos of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm.",
    "molecules": "Dox, dox"
  },
  {
    "caption": "Endomucin staining of vessels in control (-dox) or treated (+dox) E9.5 Nanogtg embryos. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm.",
    "molecules": "dox"
  },
  {
    "caption": "Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox-treated E9.5 Nanogtg embryos. Quantification of the CD71+ Ter119+ population in controls (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. ***P. < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD).",
    "molecules": "dox"
  },
  {
    "caption": "Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls (-dox) and Nanog expressing (+dox) E9.5 Nanogtg embryos. Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 embryos. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. Horizontal line represents mean values and error bars SD.",
    "molecules": "dox"
  },
  {
    "caption": "Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71+ Ter119+ population of control (-dox; n=7) and Nanog-expressing (+dox; n=4) E9.5 embryos. **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.",
    "molecules": "dox"
  },
  {
    "caption": "Freshly dissected dox-treated Nanogtg E10.5 embryos without (control) and with (chimera) contribution of wt-ESGFP cells (left, brightfield; right, GFP). Arrows mark the presence of blood in chimeric embryos that is absent from controls. Scale bar, 500 µm.",
    "molecules": "dox"
  },
  {
    "caption": "Representative FACS plots showing of red blood cell maturation as determined by CD71/Ter119 staining in single dox-treated E10.5 control (left) and chimeric (right) embryos.",
    "molecules": "dox"
  },
  {
    "caption": "Quantification of the CD71+ Ter119+ population in single dox-treated E10.5 embryos (D; control, n=18; chimera, n=10), untreated embryos (E; control, n=7; chimera, n=12), and in GFP- cells (not derived from wild type ES cells) from dox-treated embryos (F; control, n=18; chimera, n=10). ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.",
    "molecules": "dox"
  },
  {
    "caption": "Representative FACS plots showing the distribution of different populations distinguished by CD71/Ter119 staining in whole bone marrow from untreated (-dox) or treated (+dox) adult mice. S0 (double negative cell), S1 (proerythroblast), S2 (basophylic erythroblast), S3 (polychromatic erythroblast), and S4 (orthochromatic erythroblast) are different stages of blood maturation. Quantification of the S1-S4 erythroid populations (-dox, n=4; +dox, n=5). *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.",
    "molecules": "dox"
  },
  {
    "caption": "Representative FACS plots showing the distribution of CD16/32 and CD34 hematopoietic precursors sorted from the cKit+Sca1-LIN- bone marrow of untreated (-dox) or treated (+dox) adult Nanogtg mice.",
    "molecules": "dox"
  },
  {
    "caption": "Quantification of precursor populations based on CD16/32 and CD34 sorting, as total number of cells per individual femur (-dox, n=5; +dox, n=6). *P < 0.05, **P < 0.005; Student's t-test. Horizontal line represents mean values and error bars SD.",
    "molecules": "dox"
  },
  {
    "caption": "RT-qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte-erythroid progenitors (MEP) (-dox, n=8; +dox, n=5). *P < 0.05, ****P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.",
    "molecules": "dox"
  },
  {
    "caption": "Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before (left) and after (right) dox treatment. Percentage of host derived cells (CD45.1+) are shown in black, and of donor derived cells (CD45.1/CD45.2 double +) in red. Individual mice are indicated on the x-axis (n=7).",
    "molecules": "dox"
  },
  {
    "caption": "E6.5 Nanogtg embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm.",
    "molecules": "dox, doxycycline"
  },
  {
    "caption": "RT-qPCR quantification of the relative expression of Nanog, Tal1, and Klf1 in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.",
    "molecules": "dox"
  },
  {
    "caption": "Whole mount in situ hybridization of Tal1 in E7.5 untreated (-dox) or in utero treated (+dox) Nanogtg embryos. Scale bar, 100 µm.",
    "molecules": "dox"
  },
  {
    "caption": "Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones (Nanogtg;dTal1del#1 and Nanogtg;dTal1del#2) where the binding site for NANOG distal to Tal1 has been deleted (left). On the right, relative expression of Tal1 determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; Nanogtg and Nanogtg;dTal1del#1, n=8; Nanogtg;dTal1del#2, n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to Nanogtg ESC. *P < 0.05, **P < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).  ",
    "molecules": "dox"
  },
  {
    "caption": "Activated GFAP+ astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 (arrows). Photomicrographs are obtained from case 9 (A) and case 5 (B) listed in Table EV1. Data are representative for all 10 MS patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC); original magnification ×400; scale bars correspond to 50 µm.",
    "molecules": "Cy3, FITC"
  },
  {
    "caption": "Normal CNS architecture in OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Myelination is normal in the brain and spinal cord of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. CV-LFB staining; original magnification ×50; scale bars correspond to 100 µm. All photographs are representative of three mice per group.",
    "molecules": "CV, LFB"
  },
  {
    "caption": "In an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse, OTUB1 is strongly expressed by NeuN+ neurons in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC). Single NogoA+ oligodendrocytes in the brain and spinal cord (arrows) of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse express OTUB1. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488). GFAP+ astrocytes are of normal morphology and distribution in the brain and spinal cord of a non-immunized OTUB1fl/fl mouse and a GFAP-Cre OTUB1fl/fl mouse. Note that GFAP+ astrocytes of an OTUB1fl/fl mouse do not express OTUB1. GFAP+ astrocytes of a GFAP-Cre OTUB1fl/fl mouse are also OTUB1-negative and represent a negative control for OTUB1 staining. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC).",
    "molecules": "Alexa Fluor 488, Cy3, FITC"
  },
  {
    "caption": "Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk). At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk). At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse. All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 µm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining.",
    "molecules": "CV, Cy3, FITC, LFB"
  },
  {
    "caption": "Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids. Twenty-four hours after transfection, cells were treated with cycloheximide (CHX, 100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were transfected with SOCS1-MYC plasmids or cotransfected with SOCS1-MYC + OTUB1-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (upper panel). The lower panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 3 for both groups) (mean + SEM).",
    "molecules": "CHX"
  },
  {
    "caption": "Primary astrocytes from GFAP-Cre OTUB1fl/fl mice were cotransfected with SOCS1-MYC and OTUB1-GFP/OTUB1-∆N-GFP/OTUB1-C91S-GFP plasmids. Twenty-four hours after transfection, cells were treated with CHX (100 μg/ml) for indicated times. SOCS1 protein levels were analyzed by WB (left panel). The right panel shows the relative protein levels of SOCS1 normalized to Tubulin (n = 4 for both groups). Data represent mean + SEM.",
    "molecules": "CHX"
  },
  {
    "caption": "Primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were stimulated with IFN-γ (10 ng/ml) for four hours. Thereafter, cells were treated with CHX (100 μg/ml) for indicated times. Whole cell lysates were analyzed by WB with indicated antibodies.",
    "molecules": "CHX"
  },
  {
    "caption": "(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(G) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen, scale bar 100 μm",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(C) Expression of LOX family genes obtained from RNA-seq data in control and tamoxifen treated PSCs (n = 3 experimental replicates). Expression value was normalized by tubulin family genes",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(F) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 50 μm. (G) Quantification of LOX-L2 for images in F. n ≥ 4 mice per condition, and n> 10 sections per animal",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(J) Quantification of average forces applied by PSCs on pillars. BBI = blebbistatin. In the box and whisker plot, the central box represents values from the lower and upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum. ***P<0.001 Three experimental repeats",
    "molecules": "BBI, blebbistatin"
  },
  {
    "caption": "(A) Images of matrigel collagen gels previously remodelled by PSCs, second harmonic generation signal for fibrillar collagen (green) and F-actin (red), scale bar 100 μm. (B) Fiber thickness colour-code map in a represented through the BoneJ plugin where larger spheres fit along fibers represent greater thickness, scale bar 100 μm. (C) SHG fibrillar collagen images used for calculation of alignment through the FFT algorithm. Insets show FFTs of fibrillar collagen-I images, representing alignment with respect to the elliptical distribution of the FFT central maxima. Circular behaviour (values approaching 1) represent no aligned orientation and lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to the orientation direction. Scale bar 20 μm (D-F) Quantification of fiber thickness, length, and alignment for images in A-C. (G) Quantification of collagen fiber thickness using the BoneJ algorithm for images in B,H. n≥6 matrices per condition. In the scattered plots each point represents a section. In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value. Histogram bars represent mean±s.e.m. ***P<0.001 (t-test for D, E, F and ; Anova and Tukey"s post hoc test for G). Three experimental replicates for all p",
    "molecules": "collagen, matrigel"
  },
  {
    "caption": "(G) MMP-2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography; above signal intensity of the representative bands used for the quantification presented in the plot below",
    "molecules": "tamoxifen"
  },
  {
    "caption": "quantification of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 100 μm (n=4 animals per condition and n≥ 5 sections per animal)",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Immunohistochemistry image of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxife",
    "molecules": "tamoxife"
  },
  {
    "caption": "(C) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs",
    "molecules": "collagen, matrigel"
  },
  {
    "caption": "(D) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs",
    "molecules": "collagen, matrigel"
  },
  {
    "caption": "(E) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control, and 2mg, and 5mg of tamoxifen. (n=4 animals per condition, and n ≥ 5 sections per animal). (F) FN fiber thickness colour-code map in E represented through the BoneJ plugin",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control, and 2mg of tamoxifen, scale bar is 100 μm. Upper panels: Ki67 staining is used as a surrogate of proliferation. White arrows indicate Ki67 positive nuclei in epithelial cells. Lower panels: Cc3 staining shows the cells undergoing caspase-mediated apoptosis. Tamoxifen panels show higher levels of yellow staining, which indicates higher percentage of apoptotic epithelial cells. (B) Quantification of staining in panel A (n=4 animals per condition, and n≥ 5 sections per animal, two experimental repetitions). Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test",
    "molecules": "tamoxifen, Tamoxifen"
  },
  {
    "caption": "(A, E, F) Immunofluorescence images of control and tamoxifen treated suit-2 cells, scale bar is 20 μm. Panel A represents HIF-1A staining in hypoxia and non-hypoxia conditions, panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase-mediated apoptosis, respectively. Panel E: Red - F-actin, green - Ki67, blue - nuclei. Tamoxifen negatively regulates HIF-1A in hypoxia and non-hypoxia conditions. (B,C,D) Quantification of immunofluorescence staining in panels A, E, F. For quantification, eight fields of view (n>50 cells) per condition. Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test. All panels include data collected during 3 independent experiments",
    "molecules": "tamoxifen, Tamoxifen"
  },
  {
    "caption": "(F) HeLa cells were transfected with vector, wild-type HA-Bim, or HA-BimEE. Lysates were probed with the indicated antibodies. HeLa cells treated with staurosporine (STS) for 5 hr were a positive control. Note that wild-type Bim causes apoptosis, which reduces its levels compared to BimEE.",
    "molecules": "staurosporine, STS"
  },
  {
    "caption": "(G) Vector, wild-type Bim(EL), or Bim(EL) L152E F159E (BimEE) was transfected into HeLa cells. Transfection efficiency was >90%. After 20 hr, cell viability was determined by measurement pf ATP levels. Data are shown as mean ± SD. ∗∗∗p < 0.001.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001.",
    "molecules": "Baf, Bafilomycin A1"
  },
  {
    "caption": "(B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001.",
    "molecules": "Baf"
  },
  {
    "caption": "(B) T cells from Bim+/+ and Bim−/− mice were cultured with/without Baf for 4 hr preceding harvest, immunoblotted, and probed with the indicated antibodies.",
    "molecules": "Baf"
  },
  {
    "caption": "(B) Panel i: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were starved in HBSS for 2 hr. Blots were probed as indicated. LC3-II/tubulin ratio of siRNA-transfected cells is set as 1 (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant. Panel ii: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were treated with Baf for 2 hr; one set of transfections were starved and treated with Baf for 2 hr. Blots were probed as indicated and analyzed as in (Bi). ∗∗p < 0.01; NS, not significant.",
    "molecules": "Baf"
  },
  {
    "caption": "(B) Time constants of decay, τdecay, of the STC isolated pharmacologically in WT and FHM2 KI mice. Top, superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by a single pulse stimulation (indicated by the arrow) in a WT slice, before (trace a) and after (trace b) application of a saturating concentration of the GluT inhibitor TFB-TBOA (TBOA). The STC was obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a-b); the decay of the STC was best fitted by a single exponential function with τdecay = 6.53 ms (in red). The bar plot shows the average values of τdecay of the STC isolated pharmacologically in cortical slices (n=13) from P22-23 WT (N=7) and KI mice (n=9; N=3). STC τdecay is 17% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.003). Hereafter, n indicates the number of slices and N indicates the number of mice.",
    "molecules": "TBOA, TFB-TBOA"
  },
  {
    "caption": "(C) τdecay of the STC isolated using an exponential waveform approximating the average TBOA-insensitive current in WT and KI mice. Top trace, average normalized TBOA-insensitive current obtained by pooling the normalized TBOA-insensitive currents recorded in 16 WT and KI cells. This current was best fitted by an exponentially rising function (1-exp (-t/τrise)) with τrise= 2.35 ms (in green). The STC was obtained by subtracting from the total current elicited in the astrocyte (a: same representative trace as in (B)) the exponential function A(1-exp (-t/τrise)) with τrise = 2.35 ms and A equal to the maximal current measured in the astrocyte at about 60 ms after stimulation (trace c); the decay of the STC (trace a-c) was best fitted by a single exponential function with τdecay = 6.49 ms (in red). The bar plot shows the average values of τdecay of the STC isolated as shown in the top panel in cortical slices from P22-23 WT (n = 28; N=11) and KI mice (n = 27; N=9). STC τdecay is 21% higher in KI compared to WT astrocytes (unpaired t-test: P < 0.0001).",
    "molecules": "TBOA"
  },
  {
    "caption": "(A) Isolation of the STC elicited by the last pulse of a high-frequency train of action potentials. Left: superimposed representative traces of the inward current evoked in an astrocyte (held at -90 mV) by extracellular stimulation with a train of 10 pulses (trace a: black) and a train of 9 pulses (trace b: brown) at 50 Hz in a WT cortical slice. The inward current elicited by the 10th pulse was obtained by subtracting the current elicited by 9 pulses from that elicited by 10 pulses (trace a-b). Right: The STC elicited by the 10th pulse (trace a-b-c) was obtained by subtracting the exponential function that simulates the TBOA-insensitive current elicited by a single pulse (trace c, obtained as in Figure 1C) to the 10-9 pulses difference current (trace a-b). The decay of the STC elicited by the 10th pulse was best fitted by a single exponential function with τdecay = 8.04 ms (in red).",
    "molecules": "TBOA"
  },
  {
    "caption": "(A) Distribution of GLT-1agold particles in astrocytic processes contacting asymmetric synapses (perisynaptic astrocytic processes: PAP) in SI of 5 WT and 5 KI (2 male, 3 female) mice (P34-35). In cortical PAP of KI mice the density (particles/µm2) of total and membrane-associated gold particles (arrowheads) are reduced compared to WT mice, whereas the density of cytoplasmicgold particles (arrows) is comparable to that in WT (cf Table I for numerical values and statistics).",
    "molecules": "gold"
  },
  {
    "caption": "(B) Distribution of GLT-1agold particles in excitatory axon terminals forming asymmetric synaptic contact (AxT) in SI of P34-35 WT and KI mice. In cortical AxT of KI and WT mice, GLT-1a density is comparable (arrowheads: membrane-associated gold particles; arrows: cytoplasmicgold particles) (cf Table I).",
    "molecules": "gold"
  },
  {
    "caption": "Left, superimposed representative traces of the normalized inward current evoked in a WT (black trace) and a KI corticalastrocyte (blue trace) by extracellular stimulation with a train of 10 pulses at 50 Hz in cortical slices. The inset shows in an expanded time scale the portion of the traces indicated by the dotted line. The slowly decaying current (τdecay = 2.00 s and 3.04 s for the WT and KI trace, respectively) is largely a [K+]e-dependent K+current (IK) whose decay kinetics provide a measure of the rate of K+ clearance by astrocytes (see text). Right, time constant of decay of IK elicited by trains of 10 pulses at 50 Hz in corticalastrocytes from P22-23 WT (n = 21; N=12) and KI mice (n = 20; N=8). IK τdecay is 22% higher in KI compared to WT astrocytes (unpaired t-test: P = 0.001).",
    "molecules": "K+"
  },
  {
    "caption": "(A) Images of a cortical slice before (t = 0) and after (t = 15.6 s) pressure ejection of a high KCl pulse that elicits a CSD (top panels), showing that the propagating CSD is associated with a change in light transmittance. Scale bar: 500 µm. The traces below show representative changes in intrinsic optical signal (IOS) relative to background measured in a WT and a KI cortical slice during CSD propagation at increasing times and distances from the KCl puff, as indicated in color code in the right image on the top. The velocity of CSD propagation, obtained from the rate of horizontal spread of the change in IOS, in these two representative WT and KI slices is 3.11 and 4.14 mm/min, respectively.(B) Stimulation threshold for CSD induction (CSD threshold) and rate of CSD propagation (CSD velocity) in WT (n = 24; N=3) and KI (n = 27; N=8) cortical slices from P22-23 mice. CSD threshold is expressed as duration of the first KCl pulse eliciting a CSD. CSD threshold is 28% lower (Mann Whitney U test, P < 0.0001) and CSD velocity 21% higher (unpaired t-test: P< 0.0001) in KI compared to WT mice.",
    "molecules": "KCl"
  },
  {
    "caption": "(A) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days. GLT-1a levels are significantly increased in mice treated with Cef (Cef WT, N = 4) compared to control saline-injected mice (Ctr WT, N = 4) (Mann-Withney U test: P = 0.028).",
    "molecules": "Cef"
  },
  {
    "caption": "(B) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in sections from SI of P45-46 WT mice that were treated with saline (Ctr WT) or Cef (Cef WT). Arrows point to some GLT-1a/VGLUT1 related puncta. Framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment increased significantly the size of GLT-1a+ puncta overlaying VGLUT1 (142 and 186 GLT-1a+ puncta analyzed from 4 Ctr WT and 4 Cef WT mice, respectively; 3 sections/animal) (Mann-Withney U test: P < 0.0001). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.",
    "molecules": "Cef"
  },
  {
    "caption": "(C) CSD threshold and CSD velocity in cortical slices from P30-33 KI mice that were injected with saline (Ctr KI, n = 38; N=7) or Cef (Cef KI, n = 31; N=6). CSD threshold is 12% higher (Mann-Withney U test: P = 0.02) in Cef-treated compared to saline-treated KI mice. CSD velocity is not altered by Cef treatment (P = 0.90).",
    "molecules": "Cef"
  },
  {
    "caption": "(A) Distribution of GLT-1agold particles in PAP of saline-injected (Ctr KI, N=4) and Cef-treated KI mice (Cef KI, N=4) (P45-46). Cef treatment does not modify the density of total, cytoplasmic (arrows) and membrane-associated (arrowheads) gold particles in PAP of KI mice (cf Table II).",
    "molecules": "gold, Cef"
  },
  {
    "caption": "(B) Distribution of GLT-1agold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II).",
    "molecules": "gold, Cef"
  },
  {
    "caption": "(C) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment. GLT-1a levels are similar in Cef-treated (Cef KI, N=4) and and saline-injected (Ctr KI, N=4) (Mann-Withney U test: P = 0.83).",
    "molecules": "Cef"
  },
  {
    "caption": "(D) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in KI mice that received saline (Ctr KI) and in Cef-treated KI mice (Cef KI) (P45-46). Arrows point to some GLT-1a/VGLUT1 related puncta; framed regions are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment does not increase the size of GLT-1a+ puncta overlaying with VGLUT1 (160 and 175 GLT-1a+ puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post-embedding electron microscopy analysis; 3 sections/animal) (Mann-Withney U test: P = 0.31). Scale bar: 3.5 µm for left and right panels and 1 µm for enlarged framed areas. All microscopic fields are from layers II/III.",
    "molecules": "Cef"
  },
  {
    "caption": "(A) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 2.5 µM DL-TBOA (TBOA 2.5) (n = 4; N=2) (right panel). The traces on the left are the corresponding average normalized STCs, isolated as in Figure 1C. The STC τdecay in TBOA 2.5 is 32 ± 2 % higher than in Ctr WT (paired t-test: P = 0.002). The left top panel shows the time course of τdecay of the transient component (due to the STC) of the current recorded in an astrocyte during a representative experiment in which TBOA 2.5 was applied at the time indicated by the horizontal bar. The steady-state effect was reached within 10-15 minutes from the beginning of the drug perfusion.",
    "molecules": "DL-TBOA, TBOA"
  },
  {
    "caption": "(B) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 2.5 µM DL-TBOA (TBOA 2.5: n = 25; N = 8). CSD threshold in TBOA 2.5 is 36 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 20% higher (unpaired t-test: P < 0.0001).",
    "molecules": "DL-TBOA, TBOA"
  },
  {
    "caption": "(C) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 1.5 µM DL-TBOA (TBOA 1.5) (n = 4; N=4). The STC τdecay in TBOA 1.5 is 22 ± 3 % higher than in Ctr WT (paired t-test: P = 0.004).",
    "molecules": "DL-TBOA, TBOA"
  },
  {
    "caption": "(D) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 1.5 µM DL-TBOA (TBOA 1.5: n = 18; N = 6). CSD threshold in TBOA 1.5 is 23 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 13 % higher (unpaired t-test: P = 0.003).",
    "molecules": "DL-TBOA, TBOA"
  },
  {
    "caption": "U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, anti-human centromere autoantibody (ACA), and antibodies for Bub1 and TOP2A. Example images are shown (A). The immunofluorescence intensity ratios of centromeric TOP2A/ACA (B), centromeric TOP2A/arm TOP2A (C), and arm TOP2A/DNA (D), were determined on around 600 (B and C) or 400 (D) chromosomes in 20 cells. ns, no statistical significance. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, and antibodies for Bub1 and TOP2A. Lysates of nocodazole-arrested mitotic cells were immunoblotted with the indicated antibodies (E). ns, no statistical significance.",
    "molecules": "DAPI, nocodazole"
  },
  {
    "caption": "U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. Example images of the immunofluorescence staining are shown (H).",
    "molecules": "AZ3146, dimethyl sulfoxide, DMSO, MG132, Reversine, STLC"
  },
  {
    "caption": "U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric Bub1/ACA (I) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.",
    "molecules": "AZ3146, dimethyl sulfoxide, DMSO, MG132, Reversine, STLC"
  },
  {
    "caption": "U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric TOP2A/ACA (J), and centromeric TOP2A/arm TOP2A (K) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.",
    "molecules": "AZ3146, dimethyl sulfoxide, DMSO, MG132, Reversine, STLC"
  },
  {
    "caption": "U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA, and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A (A). Data information: Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA Lysates of nocodazole-arrested mitotic cells were immunoblotted (B). * represents non-specific bands.",
    "molecules": "nocodazole"
  },
  {
    "caption": "U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. Example images are shown (C). Data information: Scale bars, 10 μm.",
    "molecules": "BAY 1816032, DMSO"
  },
  {
    "caption": "U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. The immunofluorescence intensity ratios of centromeric H2ApT120/ACA (D), centromeric TOP2A/ACA (E), and centromeric TOP2A/arm TOP2A (F) were determined on around 450 chromosomes in 15 cells. Means and error bars representing S.D. are shown (D-F; unpaired t test).",
    "molecules": "BAY 1816032, DMSO"
  },
  {
    "caption": "C U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining with DAPI, and antibodies for TOP2A and H2ApT120 or H3pS10. Example images of interphase cells are shown. The cells in the top line, which is H3pS10-positive, are in early prophase/late G2. Data information: Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C, D HeLa and the Sgo1-K492A cells were exposed to nocodazole for 4 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (C). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 150 chromosomes in 10 cells (D). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.",
    "molecules": "nocodazole"
  },
  {
    "caption": "G, H HeLa and the Sgo1-K492A cells were transfected with siRNA, and treated with nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 280 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.",
    "molecules": "nocodazole"
  },
  {
    "caption": "B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins, and then exposed to nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm.",
    "molecules": "DAPI, nocodazole"
  },
  {
    "caption": "HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP-TOP2A/ACA (D), centromeric EGFP-TOP2A/arm EGFP-TOP2A (E), and arm EGFP-TOP2A/DNA (F), were determined on around 160 chromosomes in 8 cells. Data information: Means and error bars representing S.D. are shown unpaired t-test).",
    "molecules": "DNA"
  },
  {
    "caption": "U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Example images are shown (I). Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. The relative enrichment of EGFP-TOP2A at the LacO repeats was quantified in 130 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Cell lysates were immunoblotted (K).",
    "molecules": "DAPI"
  },
  {
    "caption": "A HeLa cells were transfected with siRNA and plasmids encoding the indicated proteins, and then exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm.",
    "molecules": "DAPI, nocodazole"
  },
  {
    "caption": "U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (B). The relative enrichment of EGFP signal at the LacO repeats was quantified in 30 cells (C). Arrows point to the transgene loci. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (I). Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. The relative enrichment of anti-Myc staining signal at the centromere region versus that in the nearby nuclear region was quantified on around 200 chromosomes in 10 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t test).",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Cell lysates were immunoblotted (K).",
    "molecules": "DAPI"
  },
  {
    "caption": "HeLa cells with or without transient expression of H2B-GFP or EGFP-TOP2A were exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI and antibodies for GFP, Sgo1 and ACA. Example images are shown (A). The immunofluorescence intensity ratio of centromeric Sgo1/ACA was determined on 400 chromosomes in 20 cells (B). The means and S.D.s from three independent experiments with the Sgo1/ACA ratio determined on around total 1200 chromosomes in 60 cells are shown (C). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.",
    "molecules": "DAPI, nocodazole"
  },
  {
    "caption": "HeLa cells with or without transient expression of H2B-GFP or EGFP-TOP2A were exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and Lysates of asynchronous cells were immunoblotted (D). * represents non-specific bands.",
    "molecules": "nocodazole"
  },
  {
    "caption": "U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531), were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1. Example images are shown (E). Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531), were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1. The relative enrichment of EGFP-TOP2A (F) and Sgo1 (G) at the LacO repeats was quantified in 120 cells. Data information: Means and error bars representing S.D. are shown unpaired t test).",
    "molecules": "DAPI"
  },
  {
    "caption": "U2OS-LacO cells transiently expressing EGFP-LacI-Bub1-K together with VSV-Sgo1 or a control vector were subjected to immunofluorescence staining with DAPI and antibodies for TOP2A and VSV-tag. Example images are shown (I). The relative enrichment of TOP2A (J) and VSV-Sgo1 (K) at the LacO repeats was quantified in 50 cells. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "HeLa cells were transfected with siRNA and subjected to immunofluorescence staining with DAPI, ACA and the anti-PICH antibody. Example images of anaphase cells are shown (A). The percentage of PICH-positive cells was determined in at least 339 anaphase cells in each condition. Means and S.D.s from three independent experiments are shown (B). Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining Example images of anaphase cells are shown (D). Scale bars, 10 μm.",
    "molecules": "BAY 1816032, DMSO, thymidine"
  },
  {
    "caption": "E HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining The percentage of anaphases with PICH was determined (E).",
    "molecules": "BAY 1816032, DMSO, thymidine"
  },
  {
    "caption": "HeLa cells were arrested in a prometaphase-like state with nocodazole treatment for 5hr, and then BAY 1816032 or DMSO was added for 1 hr in the continued presence of nocodazole. Then, nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO. After 1 hr, cells were fixed and subjected to immunofluorescence staining. Example images are shown (F). The percentage of anaphases with PICH was determined (G). Arrows point to the UFBs. Scale bars, 10 μm.",
    "molecules": "BAY 1816032, DMSO, nocodazole"
  },
  {
    "caption": "HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO and then immunostained with the indicated antibodies. Example images of anaphase cells are shown (H). The percentage of anaphases with PICH was determined in at least 256 cells in each condition. Means and S.D.s from three independent experiments are shown (I). Example images of prometaphase cells immunostained with the indicated antibodies are shown (J). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.",
    "molecules": "BAY 1816032, DMSO"
  },
  {
    "caption": "HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO Lysates of nocodazole-arrested mitotic cells were immunoblotted (K).",
    "molecules": "BAY 1816032, DMSO, nocodazole"
  },
  {
    "caption": "HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH, GFP and CENP-C. Example images of anaphase cells are shown (B). The percentage of anaphases with PICH was determined in at least 152 cells in each condition. Means and S.D.s from three independent experiments are shown (C). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.",
    "molecules": "DAPI, thymidine"
  },
  {
    "caption": "HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells Lysates of nocodazole-arrested mitotic cells were immunoblotted (D). * represents non-specific bands.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "E, F HeLa cells transiently expressing CB-GFP or CB-TOP2A-GFP were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining as in (B). Example images of anaphase cells are shown (E). The percentage of anaphases with PICH was determined in at least 142 cells in each condition. Means and S.D.s from three independent experiments are shown (F). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.",
    "molecules": "BAY 1816032, DMSO, thymidine"
  },
  {
    "caption": "HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. Example images of anaphase cells are shown (G). Arrows point to the UFBs. Scale bars, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. The percentage of anaphases with PICH was determined in at least 174 cells in each condition. Means and S.D.s from three independent experiments are shown (H). Data information: Means and error bars representing S.D. are shown unpaired t test).",
    "molecules": "DAPI"
  },
  {
    "caption": "D, E  Immunostaining  of MCT2  and SMCT1 in tumors of KIC  mice treated with  βOHB (100mg/kg/day, i.p.) or 0.9% NaCl  (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel)  stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and  SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm.",
    "molecules": "βOHB, NaCl"
  },
  {
    "caption": "B-D [U-13C]Glucose (n=3 mice) (B), [U-13C]Glutamine (n=5 mice) (C) and [U-13C]Acetate (n=6  mice) (D) tracing into  βOHB in poorly differentiated PDA   explants from KIC  mice. Data are expressed as mean ± SEM.",
    "molecules": "βOHB, Acetate, 13C, Glucose, Glutamine"
  },
  {
    "caption": "D, E Quantification of  volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of  tumor volume or  weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.",
    "molecules": "βOHB, NaCl"
  },
  {
    "caption": "H [U-13C]βOHB  tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.",
    "molecules": "βOHB, 13C, citrate, glucose"
  },
  {
    "caption": "(B) Box-and-whisker plots of    ketone bodies   (KB) metagene score defined as first component of   PCA  of genes in primary tumors (n=728), in all metastases  (n=76), and in   liver metastases specifically (n=35). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined at 1.5x IQR. Significance was defined by Student's t-test.",
    "molecules": "KB, ketone bodies"
  },
  {
    "caption": "D-F Effect of  βOHB treatment on  liver metastatic incidence, size and status. Representative  HPS staining  of liver lobes from  metastatic mice treated with βOHB (100mg/kg/day, i.p.) or 0.9%  NaCl (i.p.) (n=8  mice/group). Metastatic areas are separated from  liver by yellow dotted lines. Scale bar: 1mm and 500 µm for insets (D). Number and size of metastasis   per lobe and classified in small, medium and large size (E). Pathological status of metastasis  from mice treated with βOHB or NaCl (n=13 or 5 lobes/group, respectively). Data are expressed as percentage of total metastasis   in all liver lobes presenting metastasis  (F).",
    "molecules": "βOHB, NaCl"
  },
  {
    "caption": "(c) Shown are the responses of the glucose sensor promoter (PgluA7), a negative control lacking the CRP operator (PgluA7*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of glucose",
    "molecules": "glucose"
  },
  {
    "caption": "(d) Shown are the responses of the oxygen sensor promoter (PfnrF8), a negative control lacking the FNR operator (PfnrF8*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of oxygen",
    "molecules": "oxygen"
  },
  {
    "caption": "Shown are the schematics and responses for the glucose sensor The response functions (center) are shown for each sensor (black circles) compared to promoter variants where the operators are removed (PgluA7*, PfnrF8*) (open circles) text). Representative cytometry florescence distributions for Figures 1f",
    "molecules": "glucose"
  },
  {
    "caption": "Shown are the schematics and responses for th oxygen sensors Horizontal error bars in the PfnrF8 response reflect one standard deviation of three DO measurements Representative cytometry florescence distributions for Figure 1g",
    "molecules": "oxygen"
  },
  {
    "caption": "Shown are the schematics and responses for th acetate sensors For the acetate sensor, the response of the sensor is shown in unmodified E. coli MG1655 with glnL intact (open diamonds). The dynamics of induction are shown (right graph) where cells are induced at the time indicated by the dashed line (see text). Representative cytometry florescence distributions for Figure 1h are shown i",
    "molecules": "acetate"
  },
  {
    "caption": "The responses of the glucose sensors during growth in shake flasks are shown",
    "molecules": "glucose"
  },
  {
    "caption": "The responses of th e, oxygen sensors during growth in shake flasks are shown",
    "molecules": "oxygen"
  },
  {
    "caption": "The responses of th acetate sensors during growth in shake flasks are shown",
    "molecules": "acetate"
  },
  {
    "caption": "(b) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth (Methods). The inducers are either 1 mM IPTG (sRNA) or 25 μM DAPG (sgRNA, mf-LON). sgRNA and mf-LON are co-transcribed on a single transcript, processed by ribozymes (Appendix Figure S13). (c) The dynamics of repression by each of the mechanisms is shown. Empty circles are uninduced and black circles are induced (1 mM IPTG or 25 µM DAPG) at the 2-hour time point (dashed line) (Methods)",
    "molecules": "DAPG, IPTG"
  },
  {
    "caption": "(f) The impact on acetate production for a double mutant where poxB is complemented on a BAC (Methods). Note that complementation causes a ~3-fold reduction in acetate compared to WT in part d. N-ter and E170 refer to the location of the SuMMV tag. (g) Reduction of acetate production by the expression of SuMMV from an DAPG-inducible promoter (- no inducer, + 25 µM). The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the genome",
    "molecules": "DAPG, acetate"
  },
  {
    "caption": "(b) The transcription of poxB over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/acetate sensors will be on based on metabolite measurements. It should be noted that these are right-shifted compared to Figure 2 due to slower growth of the tested strains",
    "molecules": "acetate, glucose"
  },
  {
    "caption": "(c) The production of acetate is shown over time for E. coli MG1655∆glnL∆pta poxB::E170 (black) as compared to the same strain containing the complete circuit (green). A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆pta containing an untagged poxB as a control (red). Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB",
    "molecules": "acetate"
  },
  {
    "caption": "(e) The transcription of pta over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/oxygen sensors will be on based on metabolite measurements",
    "molecules": "glucose, oxygen"
  },
  {
    "caption": "(f) The production of acetate is shown over time for E. coli MG1655∆glnL∆poxB pta::pdt3 as compared to a strain containing the complete circuit (green) tested in same strain. A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆poxB containing an untagged pta as a control (red) Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB",
    "molecules": "acetate"
  },
  {
    "caption": "(H, I) Enzymatic activity of FBL is not required for cell proliferation. Colony formation assays were conducted to investigate the role of enzymatic activity in cell proliferation. FBL knockdown HCT116 or A549 cells with ectopic expression of FBL or the catalytically inactive mutant (FBL-E191A/ D236A) were used, with a bar graph showing the relative number of colonies per well (mean ± SD). All cells were allowed to form colonies for 10 days and representative images of colonies in plates stained with Giemsa were displayed. The bar graphs present data as mean ± SD from three independent experiments. The statistical significance was determined using one-way ANOVA. ns denotes no statistical significance, * indicates P < 0.05, *** indicates P < 0.001.",
    "molecules": "Giemsa"
  },
  {
    "caption": "(A, B) Knockdown of FBL results in increased cellular sensitivity to DNA damage treatment. Cell viability analysis was performed on control (shNC) and shFBL-HCT116/shFBL-A549 cells treated with indicated concentrations of 5 μM MMC using CCK-8 assays. Each data point represents the mean ± SD from three replicates. The statistical significance was determined using Student's t-test. ***p < 0.001.",
    "molecules": "MMC"
  },
  {
    "caption": "(D-F) Immunofluorescence staining showed the formation of γH2AX and BRCA1 foci in shFBL and shNC HCT116 cells treated with 5 μM MMC or mock treatment. The scale bar represents 10 μm. The (E) and (F) panel displays quantification of the (D) panel: indicating the number of γH2AX or BRCA1 foci per cell (n≥100 cells). Data are shown as the mean ± SD. The statistical significance was determined using one-way ANOVA, with ns indicating no statistical significance, *** indicating P < 0.001.",
    "molecules": "MMC"
  },
  {
    "caption": "(B) HCT116 cells were treated with or without 5 μM MMC overnight or 25 μM cisplatin for 2 hours, cells were then harvested and separated into nuclear and cytoplasmic fractions. The protein level of YBX1 in each fraction was detected by Western blotting assays. GAPDH and Histone 3 (H3) were used as loading controls for cytoplasmic and nuclear fractions, respectively.",
    "molecules": "cisplatin, MMC"
  },
  {
    "caption": "(C) Subcellular localization of YBX1/FBL was confirmed by immunofluorescence staining following MMC (5 μM, overnight) or cisplatin (25 μM, 2 hours) treatment in shFBL and shNC cells. Scale bar, 10 μm.",
    "molecules": "cisplatin, MMC"
  },
  {
    "caption": "(D) The endogenous association between FBL and YBX1 in HCT116 cells was examined by co-immunoprecipitation assays with 5 μM MMC treatment. Cells were lyzed with NETN300 buffer, and nuclear lysates were examined by IP and western blot with indicated antibodies. GAPDH was used as the protein-loading control.",
    "molecules": "MMC"
  },
  {
    "caption": "(E) The distribution of YBX1 proteins in the nuclear and cytoplasmic fractions of control or FBL KD HCT116 cells after 5 μM MMC incubation overnight were determined by western blotting assays. H3 was used as a nuclear control. GAPDH was used as a cytoplasmic control.",
    "molecules": "MMC"
  },
  {
    "caption": "(F) The protein contents of YBX1 in the nuclear and cytoplasmic fractions in FBL KD HCT116 cells reconstituted with wild-type FBL or deletion mutants of FBL with 5 μM MMC treatment overnight were detected by western blotting assays with indicated antibodies.",
    "molecules": "MMC"
  },
  {
    "caption": "(C) HE staining of tumor tissues. Scale bar, 50 µm. (D Representative images of immunohistochemical staining showing BRCA1 and γH2AX expression levels in MMC-treated and untreated xenograft tumors driven by control or FBL KD HCT116 cells. panel displays quantification of the (D) panel: showing BRCA1 and γH2AX expression levels. Scale bar, 25 µm. Graphs represent the mean ± SD (n = 3 mice in each group). The statistical significance was determined using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "MMC"
  },
  {
    "caption": "The rapZ  mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ  gene in (b) Northern blotting experiment detecting GlmZ  (top panel) and 5S rRNA (bottom panel; loading control) to assess the degree of GlmZ  cleavage triggered by the RapZ  variants. Processed GlmZ  is indicated with an asterisk. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ  variants",
    "molecules": "arabinose, glucose"
  },
  {
    "caption": "The rapZ  mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ  gene in (c) GlmS  protein (MW = 66.9 kDa) levels in the same cultures as revealed by  SDS-PAGE  of total protein extracts followed by Coomassie blue staining. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ  variants",
    "molecules": "arabinose, glucose"
  },
  {
    "caption": "The rapZ  mutants were expressed from plasmids under control of the PBAD  promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ  gene in (d)  β-Galactosidase activities produced by the same cultures and replicates (mean values and standard deviations are shown). Cells carry a  glmS'-lacZ  fusion on the chromosome, which requires base-pairing with sRNA GlmZ  for high expression. Strains Z8 (wild-type columns 1, 2) and Z28 all other columns) were used. Strain Z28 carried plasmid pBAD33 columns 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ  variants",
    "molecules": "arabinose, glucose"
  },
  {
    "caption": "Inhibition of proteasomal degradation leads to CP110 stabilization on the m-centriole and impaired ciliogenesis. RPE-1 cells on cover-slips were either grown in media with serum, serum starved for 4 h in the absence of MG132, or serum starved for 4 h in the presence of MG132 prior to fixation and immunostaining with antibodies for acetylated tubulin (to detect cilia) and anti-CP110 antibodies. (E) Representative micrographs showing MG132 treatment impedes removal of CP110 from the m-centriole and impairs ciliogenesis. Bar, 10 μm. (F) Graph quantifying percent of cells displaying primary cilia. Normal distribution was determined by the D'Agostino and Pearson normality test.",
    "molecules": "MG132"
  },
  {
    "caption": "(H-I) EHD1 depletion prevents CP110 degradation upon serum starvation. (H) Mock-transfected RPE-1 cells or EHD1 siRNA-transfected RPE-1 cells were grown in media containing serum, or serum starved for 4 h in the absence or presence of MG132, and then lysed, separated by SDS-PAGE and immunoblotted with anti-CP110 antibodies (upper panel) or anti-actin antibodies (loading control; lower panel). Validation of EHD1 depletion is shown in the right panel. (I) Graph displaying densitometric analysis of the intensity of the CP110 bands shown in H normalized to actin (means and p-values are representative of 3 independent experiments). Normal distribution was determined by the Shapiro-Wilk normality test.",
    "molecules": "MG132"
  },
  {
    "caption": "(A-B) PCM1-depletion impedes CP110 ubiquitination upon serum starvation. (A) Mock-transfected RPE-1 cells were maintained in media with serum, and both mock-transfected and PCM1 siRNA-transfected cells were serum starved for 4 h in the presence of MG132 prior to lysis and immunoblotting with anti-CP110 antibodies (second panel from top), or first immunoprecipitated with anti-CP110 antibodies and then immunoblotted with antibodies to detect ubiquitinated CP110 (top panel). Bottom panels depict the efficacy of PCM1 depletion upon PCM1 siRNA-transfection, with actin as a loading control. (B) Graph displaying densitometric analysis of ubiquitinated CP110 levels (from the upper panel of A) normalized to total CP110 immunoprecipitated. Normal distribution was determined by the Shapiro-Wilk normality test.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Validation of siRNA depletion. RPE-1 cells were mock-transfected or transfected with siRNA oligonucleotides to impair translation of Cullin3, HERC2, or MIB1. 72 h later, the cells were lysed, subjected to SDS-PAGE and immunoblotted. Immunoblotting with anti-actin antibodies was done as a loading control.",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "(B) HERC2 or MIB1 depletion impairs CP110 ubiquitination. RPE-1 cells were grown in serum (first lane from left), or with MG132 in the absence of serum for 4 h (second lane from left), transfected with Cullin3 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (third lane from left), transfected with HERC2 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (fourth lane from left), or transfected with MIB1 siRNA-oligonucleotides and treated with MG132 in the absence of serum for 4 h (fifth lane from left). Cells were lysed and immunoprecipitated with anti-CP110 antibodies, and the resultant immunoprecipitates were immunoblotted with anti-ubiquitin to identify ubiquitinated CP110 (upper panel). The blot was then stripped and re-immunoblotted with anti-CP110 to reveal the amount of CP110 pulled-down from the lysate (lower panel).",
    "molecules": "MG132, oligonucleotides, ubiquitin"
  },
  {
    "caption": "(A) EHD1 is required for the interaction between HERC2 and CP110. RPE-1 cells were mock-transfected or transfected with EHD1 siRNA oligonucleotides for 72 h. Cells were lysed and immunoprecipitated with anti-CP110 or beads only (control) and separated by SDS-PAGE along with 7% of the lysate (input). After transfer to nitrocellulose, antibodies were used to detect CP110 (lower panel) and HERC2 (upper panel).",
    "molecules": "nitrocellulose, oligonucleotides"
  },
  {
    "caption": "(A-D) RPE-1 cells on coverslips were mock-transfected (A), transfected with CP110 siRNA-oligonucleotides (B) transfected with HERC2 siRNA-oligonucleotides (C), or transfected with both CP110 and HERC2 siRNA-oligonucleotides (D). Cells were fixed and immunostained with acetylated tubulin to detect primary cilia (yellow arrows). Bars, 10 μm. : Graphs show standard deviation and p-values from 3 independent experiments. The micrographs (A-D) are representative ones from the 3 individual biological experiments. More than 50 cells were quantified in each experiment, although fewer ciliated cells were observed and measured under the HERC2 knock-down condition.",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "H Evaluation of murine peritoneal permeability six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily by TRITC-dextran dye extravasation assay. For each group, n = 8. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (H), P values are determined by Student's t-test",
    "molecules": "TRITC-dextran"
  },
  {
    "caption": "E ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test;",
    "molecules": "SCH772984"
  },
  {
    "caption": "H Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984. Scale bar, 100 µm. For each group, n = 4. Data information: All data are shown as mean ± SD. for (H) P values are determined by one-way ANOVA.",
    "molecules": "SCH772984"
  },
  {
    "caption": "I The levels of Ras, c-Raf, pMEK, ERK, pERK  and p-P90RSK in  Caco-2  cells treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984 by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. for (I), P values are determined by one-way ANOVA.",
    "molecules": "SCH772984"
  },
  {
    "caption": "A The normalized fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2  cells were incubated with Control-Fc or Spike RBD-Fc after 24 hours. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-test",
    "molecules": "TRITC-Dextran"
  },
  {
    "caption": "E Immunofluorescence analysis of VE-cad expression and subcellular localization in Caco-2 cells that were treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab by confocal microscopy. VE-cad staining in green and nuclear staining in blue. Scale bar, 20 µm.",
    "molecules": "Bevacizumab, SCH772984"
  },
  {
    "caption": "F Fluorescence intensity of TRITC-Dextran  dye from coculture systems in which  Caco-2 cells were incubated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Data are from five technical replicates with similar results from three biological replicates. Data information: All data are shown as mean ± SD. ; for (F) , P values are determined by one-way ANOVA.",
    "molecules": "Bevacizumab, SCH772984, TRITC-Dextran"
  },
  {
    "caption": "G Immunohistochemical staining shows levels of VE-cad and pVE-Cad  (731) in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. for (G), P values are determined by one-way ANOVA.",
    "molecules": "Bevacizumab, SCH772984"
  },
  {
    "caption": "A ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti-VEGF antibody. SCH772984, ERK inhibitor; Bevacizumab, anti-VEGF antibody. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.",
    "molecules": "Bevacizumab, SCH772984"
  },
  {
    "caption": "B Evaluation of  intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay. For each group, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.",
    "molecules": "Bevacizumab, Evans Blue, SCH772984"
  },
  {
    "caption": "C, D Representative  H&E images (C) and the degrees of inflammation (D) of intestinal tissues from animals treated with SCH772984 or Bevacizumab. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.",
    "molecules": "Bevacizumab, SCH772984"
  },
  {
    "caption": "(A) p53-dependent dephosphorylation of Ulk1 at Ser637. p53+/+ and p53−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells assessed by propidium iodide (PI) staining. α-Tubulin was used as a loading control. A semiquantitative analysis of protein expression is shown in Fig. EV1.",
    "molecules": "etoposide"
  },
  {
    "caption": "(B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells.",
    "molecules": "etoposide"
  },
  {
    "caption": "(B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+MEFs and PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells. (C) Semiquantitative analysis of protein expression is shown (n = 3; mean ± SD). *p < 0.05, **p < 0.01, NS: not significant. (D-I) Suppression of etoposide-induced autophagy in PPM1D−/−MEFs.",
    "molecules": "etoposide"
  },
  {
    "caption": "(D, E) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then immunostained with an anti-LC3 antibody (green). Representative images are shown in (D). LC3 puncta are observed in etoposide-treated PPM1D+/+MEFs. (E) The proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.",
    "molecules": "etoposide"
  },
  {
    "caption": "(F, G) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then analyzed using electron microscopy. Representative images are shown in (F). Many autophagic vacuoles (arrows) can be seen in etoposide-treated PPM1D+/+cells (upper panel). \"N\" indicates the nucleus. Bar = 2 µm. A representative autophagosome (AP) and autolysosome (AL) are shown in the insets. (G) The number of autophagosomes and autolysosomes in each cell were counted (n > 8 cells). Red lines indicate the mean value. *p < 0.05.",
    "molecules": "etoposide"
  },
  {
    "caption": "(H, I) Analysis of autophagic flux. The indicated MEFs were treated with etoposide (10 µM) for 6 hr in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses of protein expression (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.",
    "molecules": "bafilomycin A1, etoposide"
  },
  {
    "caption": "(A, B) Effect of stable expression of PPM1D in PPM1D−/−MEFs. PPM1D−/−MEFs and PPM1D-transfected PPM1D−/−MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. (B) Semiquantitative analyses of protein expression in (A) (n = 3; mean ±  SD). *p < 0.05, **p < 0.01, NS: not significant.",
    "molecules": "etoposide"
  },
  {
    "caption": "(C, D) Effect of a PPM1D inhibitor on etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) in the presence or absence of GSK2830371 (30 µM), and the expression of each protein was examined by western blotting. (D) Semiquantitative analysis of protein expression in (C) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.",
    "molecules": "etoposide, GSK2830371"
  },
  {
    "caption": "(E, F) Effect of the transient overexpression of PPM1D. PPM1D+/+MEFs were transfected with a PPM1D-Flag plasmid in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. (F) Semiquantitative analyses of protein expression in (E) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(A) Physical interaction between endogenous Ulk1 and endogenous PPM1D. PPM1D+/+MEFs were treated with etoposide (10 µM) or left untreated for 6 hr. Cells were then lysed and immunoprecipitated with an anti-Ulk1 antibody or a control IgG. Immune complexes and total lysates (1.8% input) were analyzed by western blotting using anti-PPM1D and anti-Ulk1 antibodies.",
    "molecules": "etoposide"
  },
  {
    "caption": "(D) The presence of cytosolic PPM1D puncta in etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with an anti-PPM1D antibody, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (green; upper panels) and Hoechst 33342 (blue; lower panels) are shown. Arrowheads indicate cytoplasmic PPM1D puncta.",
    "molecules": "etoposide"
  },
  {
    "caption": "(E) Colocalization of endogenous PPM1D and endogenous Ulk1 in etoposide-treated PPM1D+/+MEFs. PPM1D+/+MEFs were treated with etoposide (10 µM) for 6 hr, immunostained with anti-PPM1D and anti-Ulk1 antibodies, and their nuclei stained with Hoechst 33342 (50 ng/ml). Representative images of anti-PPM1D (red; left), anti-Ulk1 (green; middle), and a merged image (right) are shown. Magnified images of the areas within the dashed squares are shown at the bottom. Arrowheads indicate cytoplasmic PPM1D colocalized with Ulk1.",
    "molecules": "etoposide"
  },
  {
    "caption": "(F-J) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide-induced autophagy. (F-H) Ulk1/Ulk2 DKO MEFs that were stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM). Then, the cell lysates were collected time-dependently, and the expression of each protein was examined by western blotting. Asterisks in the Ulk1 blots are nonspecific bands. Semiquantitative analyses are shown in Fig. EV2E.",
    "molecules": "etoposide"
  },
  {
    "caption": "(F-J) The crucial role of Ulk1 dephosphorylation at Ser637 in etoposide-induced autophagy. (I, J) The indicated MEFs were treated with or without etoposide (10 µM) for 6 hr, followed by immunostaining with an anti-LC3 antibody. Representative images are shown in (I). LC3 puncta are markedly observed in DKO MEFs transfected with wild-type HA-Ulk1. Puncta were absent and weakly observed in MEFs expressing the mutants S637D and S637A, respectively. (J) The population of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.",
    "molecules": "etoposide"
  },
  {
    "caption": "(A-D) Ulk1 puncta formation was induced by etoposide in a PPM1D-dependent manner. The indicated MEFs were treated with etoposide (10 µM) with or without GSK2830371 (20 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images are shown in (A). Ulk1 puncta are observed in etoposide-treated PPM1D+/+MEFs. (B-D) The proportion of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01, NS: not significant.",
    "molecules": "etoposide, GSK2830371"
  },
  {
    "caption": "(E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (E, F) Cells were immunostained with an anti-Ulk1 antibody (E), and the population of cells with Ulk1 puncta was calculated (n > 100 cells in each experiment) (F). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01.",
    "molecules": "etoposide"
  },
  {
    "caption": "(E-I) Role of the dephosphorylation of Ulk1 at Ser637 on etoposide-induced Ulk1 puncta formation and Atg13 phosphorylation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were treated with etoposide (10 µM) for the indicated times. (G-I) Cell lysates were collected in a time-dependent manner, and Atg13 protein levels and its phosphorylation at Ser317 were examined by western blotting. Semiquantitative analyses are shown in Fig. EV3D.",
    "molecules": "etoposide"
  },
  {
    "caption": "(J) EGFP-DFCP1-expressing PPM1D+/+MEFs were treated with etoposide (10 µM) for the indicated times, followed by immunostaining with an anti-Ulk1 antibody. Representative images of EGFP-DFCP1 (green; left), anti-Ulk1 (red; center), and a merged image (right) are shown. The nuclei were stained with Hoechst 33342 in the merged image. A magnified image of the area within the dashed square is also shown. Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta.",
    "molecules": "etoposide"
  },
  {
    "caption": "(K, L) The role of Ulk1 dephosphorylation at Ser637 on etoposide-induced DFCP1 puncta formation. Ulk1/Ulk2 DKO MEFs stably transfected with HA-Ulk1 or its mutants, S637D and S637A, were transfected with EGFP-DFCP1 and treated with etoposide (10 µM) for the indicated hours, followed by immunostaining with an anti-HA antibody. Representative merged images of EGFP-DFCP1 (green), anti-HA (red), and Hoechst 33342 (blue) are shown in (K). Arrowheads indicate DFCP1 puncta localized close to Ulk1 puncta. The number of DFCP1 puncta in each cell was calculated and is shown in (L) (n > 8 cells in each experiment). Red lines indicate the mean values. **p < 0.01, NS: not significant.",
    "molecules": "etoposide"
  },
  {
    "caption": "(B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (F) Analysis of autophagic flux. The indicated thymocytes were X-rayirradiated (5 Gy) in the presence or absence of chloroquine (120 µM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. Semiquantitative analyses are shown in Figure EV4D.",
    "molecules": "chloroquine"
  },
  {
    "caption": "C. The number  of PGCs labeled with PkH26 (red) that  migrated to the genital ridge in a frozen section, the number of PGCs  labeled with  PkH26 (red) was counted using Image J software. Scale bar: 70 µm (Data are shown as mean ± SEM, n = 6 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).",
    "molecules": "PkH26"
  },
  {
    "caption": "Enrichment levels of  m6A in LncBMP4 were detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).",
    "molecules": "m6A"
  },
  {
    "caption": "H and I. The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA.).",
    "molecules": "m6A"
  },
  {
    "caption": "A. The influence of  m6A on the stability of LncBMP4 was detected by qRT-PCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).",
    "molecules": "m6A"
  },
  {
    "caption": "D. The enrichment level of m6A, for LncBMP4 variants with the progressive accumulation of RRACH site mutations, was detected by RIP-qPCR   (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, one-way ANOVA).",
    "molecules": "m6A"
  },
  {
    "caption": "E. The ability of LncBMP4, modified with different levels of  m6A, to bind gga-mir-12211 was detected by the  luciferase  reporting system (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).",
    "molecules": "m6A"
  },
  {
    "caption": "C. Effects of  m6A on small peptide expression, assessed by western blot  (Up), Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).",
    "molecules": "m6A"
  },
  {
    "caption": "D and E. Western blot(Up) was used to detect gga-mir-12211 and  m6A affect the ability of LncBMP4  to translate EPC5. Image J software was used for  gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001,**** p < 0.0001, one-way ANOVA.).",
    "molecules": "m6A"
  },
  {
    "caption": "(B) Tumor growth curves of 3LL-tumor bearing mice treated with recombinant FKN (rFKN), the CX3CR1 inhibitor AZD8797, a non-relevant protein (IgG2a) or saline buffer as vehicle control (WT). Arrows indicate the time of intraperitoneal injections of rFKN (blue) and AZD8797 (black). Data are expressed as means ± SD (n=6 mice per group). Statistical comparisons by ANOVA followed by Tukey´s pairwise comparison tests are provided.",
    "molecules": "AZD8797, saline"
  },
  {
    "caption": "(I)  L929  Cells were infected with MHV-A59 for 4h, then  cyclohexamide (CHX) was added to block further viral protein synthesis. After 5h, cells were electroporated with anti-N or anti-GFP  antibodies and left for a further 3h before being western blotted for cellular N protein levels (*denotes a non-specific band). Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.",
    "molecules": "CHX, cyclohexamide"
  },
  {
    "caption": "Immunoblot analysis of p-SMAD and t-SMAD2 in A549-VIM-RFP cells pretreated with eliglustat for 4 or 6 days and then treated with vehicle control or TGF-β for 1 h. GAPDH: loading control. Quantification of the p-SMAD2 level in A549-VIM-RFP cells as shown in (A). The levels were normalized to that of GAPDH, and fold changes were then further normalized to the level of p-SMAD2 in control cells without TGF-β treatment.",
    "molecules": "eliglustat"
  },
  {
    "caption": "Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers N-cadherin and vimentin in A549-VIM-RFP cells treated with eliglustat for 4 or 6 days and/or with vehicle control and/or TGF-β for 2 days. Tubulin: loading control.",
    "molecules": "eliglustat"
  },
  {
    "caption": "mCherry-labeled A549 cells were pretreated with eliglustat (2 μM) for 4 days and were then injected into ducts of Cuvier of zebrafish embryos. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. SB group zebrafish were treated with the inhibitor SB505124 (1 μM) in the egg water together with eliglustat for 4 days after injection with daily refreshment of the treatments.",
    "molecules": "eliglustat, SB, SB505124"
  },
  {
    "caption": "Lysates from NMuMG control cells (Cas9-expressing cells) and Ugcg KO cells were subjected to sucrose density gradient ultracentrifugation. The expression levels of flotillin-1, β1-integrin, EEA1, and TβRI in the sucrose gradient fractions were analyzed by immunoblotting. Fractions 3, 4, and 5 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from Ugcg KO NMuMG and control cells. Isolation of lipid rafts from other cellular components in A549-VIM-RFP cells treated with the UGCG inhibitor eliglustat (2 μM) for 6 days using sucrose density gradient ultracentrifugation and measurement of flotillin-1, β1-integrin, EEA1, and TβRI levels in the sucrose gradient fractions using immunoblot analysis. Fractions 3-6 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from A549-VIM-RFP cells treated with eliglustat (2 μM) for 6 days.",
    "molecules": "eliglustat"
  },
  {
    "caption": "Ubiquitination of TβRI was detected by immunoprecipitation (IP) of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without eliglustat (2 μM) treatment for 6 days. All groups were treated with MG132 (5 μM) for 6 h.",
    "molecules": "eliglustat, MG132"
  },
  {
    "caption": "Immunoblot analysis of TβRI expression levels in control and eliglustat (2 μM)-treated A549-VIM-RFP cells treated with 50 μg/mL CHX for the indicated times. Vinculin: loading control.   Quantification of TβRI expression levels with normalization to the t=0 controls.  ",
    "molecules": "CHX, eliglustat"
  },
  {
    "caption": "HEK293T cells transfected with Myc-tagged caTβRI, (HA-Ub, and nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti-Myc antibody and immunoblot analysis. All groups were treated with MG132 (5 μM) for 6 h.",
    "molecules": "MG132"
  },
  {
    "caption": "Ubiquitination of TβRI was detected by IP of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without ST3GAL5 overexpression. All groups were treated with MG132 (5 μM) for 6 h.",
    "molecules": "MG132"
  },
  {
    "caption": "Western blot analysis of TβRI expression in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with CHX for the indicated times. Vinculin: loading control.",
    "molecules": "CHX"
  },
  {
    "caption": "A,B, No difference in voltage dependent Ca2+-currents (A) and fractional activation of ICa-channels (B) between OtofI515T/I515T IHCs (n=16) and IHCs of Otof+/+ littermates (n=13).",
    "molecules": "Ca2+"
  },
  {
    "caption": "C, Exocytosis was recorded by measures of changes in membrane capacitance (ΔCm, lower panel) in response to depolarization (left, 20ms; right, 100ms) to the voltage where maximum Ca2+-currents were elicited (upper panel), typically -14mV.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D, Upper panel, while for stimuli up to 20ms exocytosis from OtofI515T/I515T (n=13) and Otof+/+ (n=11) IHCs was similar, sustained exocytosis, representing most likely the release of replenished vesicles, was significantly reduced in OtofI515T/I515T IHCs (t-test). Lower panel, Ca2+-current integrals were of similar size.",
    "molecules": "Ca2+"
  },
  {
    "caption": "E, Flash photolysis of caged Ca2+ elicited a smaller exocytic response in OtofI515T/I515T IHCs.",
    "molecules": "Ca2+"
  },
  {
    "caption": "K, Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps. 5 OtofI515T/I515T mice (pink, mean ±SEM red) avoided drinking less efficiently than 2 Otof+/+ mice (grey, mean ±SEM black) for shorter gap durations. See also Figs S2/S3.",
    "molecules": "water"
  },
  {
    "caption": "D-G, Magnified synaptic ribbons (R) and postsynaptic afferent boutons of SGNs (Aff), displaying otoferlin immunogold labelling at active zone membranes (pink arrowheads) but not around the synaptic ribbon, using saponin permeabilization (D-F), or Triton-X-100 treatment (G).",
    "molecules": "saponin, Triton-X-100"
  },
  {
    "caption": "A RT-qPCR reveals that miR-210 was increased in lungs of mice with PH triggered by various conditions: VHL−/− as compared with VHL+/+ mice (N = 4/group), ***P < 0.0001 (first graph); hypoxia + SU5416 (Hyp + SU5416) (N = 6/group) as compared with normoxia + SU5416 (Norm + SU5416) (N = 7/group), **P = 0.0015 (second graph); Il6 transgenic versus littermate control mice (N = 4/group), **P = 0.0097 (third graph); and S. mansoni-infected mice (N = 4) compared with non-infected control mice (N = 5), ***P < 0.0001 (fourth graph).",
    "molecules": "SU5416"
  },
  {
    "caption": "C Representative ISH stain of miR-210 in < 100 μm pulmonary vessels of mice (bottom micrographs) exposed to Hyp + SU5416 compared with Norm + SU5416 (α-smooth muscle actin stain from serial sections, top row of micrographs).",
    "molecules": "SU5416"
  },
  {
    "caption": "F From animals in (C) immunohistochemistry (IHC) revealed that the miR-210 targets ISCU1/2 were reciprocally down-regulated in miR-210-enriched remodeled pulmonary vessels-namely in PH mice exposed to Hyp + SU5416 (F, ***P = 0.0002).",
    "molecules": "SU5416"
  },
  {
    "caption": "H, I miR-210 expression (H) was increased (***P = 0.0003), and ISCU1/2 expression (I) was decreased (***P < 0.0001) in PECAM+pulmonary vascular endothelial cells isolated from PH mice (Hyp + SU5416) as compared with control (Norm + SU5416) (N = 4/group, left bars).",
    "molecules": "SU5416"
  },
  {
    "caption": "After lentiviral delivery of GCN4 or GRX2 sensor genes to human PAECs, cellular fluorescence was measured by flow cytometry. Unlike control GCN4 sensors that homodimerized independent of Fe-S levels and induced consistent fluorescence, fluorescence derived from the GRX2 Fe-S-dependent sensors decreased in hypoxia, as displayed in representative flow cytometric plots (left) and by quantification of percentage of positive cells [(cell number in M2 gate)/(total cell number) × 100]. Immunoblotting revealed consistent expression of either GRX2 or GCN4 sensors (FLAG-tagged) in hypoxia (Hyp) compared with normoxia (Norm) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1848 for GCN4).In contrast to consistent fluorescence from control GCN4 sensors, Fe-S-dependent GRX2 sensor fluorescence was decreased by siRNA knockdown of ISCU1/2 (siISCU) as compared with control (siCont) (N = 3, ***P < 0.0001 for GRX2; N = 3, NS P = 0.1790 for GCN4).",
    "molecules": "Fe-S"
  },
  {
    "caption": "After lentiviral delivery of sensor genes, Fe-S-dependent GRX2 sensor fluorescence was decreased in PECAM-positive cells from PH mouse lung (Hyp + SU5416) as compared with control non-PH mouse lung (Norm + SU5416, N = 3, ***P < 0.001).",
    "molecules": "Fe-S, SU5416"
  },
  {
    "caption": "By electron paramagnetic resonance (EPR) spectroscopy (representative VHL−/− versus control lung), Fe-S cluster signal was decreased (left graph) in VHL−/− mice lung tissue (VHL−/−, N = 3) as compared with WT control lung tissues (WT, N = 5), ***P = 0.0003.",
    "molecules": "Fe-S"
  },
  {
    "caption": "By EPR, Fe-S-specific signal was decreased (right graph) in human PH-diseased lung tissue harvested at lung transplantation (PH, N = 3, Supplementary Table S3) compared with non-PH control donor lung tissues (control, N = 3), *P = 0.048.",
    "molecules": "Fe-S"
  },
  {
    "caption": "B By immunohistochemistry (IHC), ISCU1/2 was unchanged in < 100 μm pulmonary vessels of miR-210−/−mice exposed to Hyp + SU5416 (N = 6) versus Norm + SU5416 (N = 7), NS P = 0.2833.",
    "molecules": "SU5416"
  },
  {
    "caption": "C Fe-S-dependent Complex I-specific activity was decreased in WT PH mice as compared with control (N = 7/group, left bars, ***P < 0.0001), but activity was preserved in the lungs of miR-210−/−mice in either condition (N = 8/group, right bars), NS P = 0.3693.",
    "molecules": "Fe-S"
  },
  {
    "caption": "E 3-nitrotyrosine (3-NT) was increased in pulmonary vessels of WT PH mice but was reduced in miR-210−/− mice in either condition (N = 6/group) (***P < 0.0001, NS P = 0.4087).",
    "molecules": "3-nitrotyrosine, 3-NT"
  },
  {
    "caption": "I Unlike WT mice (black bars) demonstrating increased right ventricular systolic pressure (RVSP) after Hyp + SU5416 (N = 10) versus Norm + SU5416 (N = 11), hemodynamic dysregulation was significantly alleviated in miR-210−/− mice (white bars, N = 11) (***P < 0.0001, *P = 0.0258, **P = 0.0059).",
    "molecules": "SU5416"
  },
  {
    "caption": "J Compared with WT controls (N = 9), miR-210−/− mice (N = 8) displayed a blunted increase of the Fulton index (RV/LV + S) under PH versus baseline conditions (expressed as a ratio of RV/LV + S under Hyp + SU5416 versus Norm + SU5416, *P = 0.031).",
    "molecules": "SU5416"
  },
  {
    "caption": "G Endothelin-1 was decreased in lung tissue from mice treated with anti-miR-210 during Hyp + SU5416 exposure (N = 6/group), *P = 0.0181.",
    "molecules": "SU5416"
  },
  {
    "caption": "A Following the schema of Fig5A in the presence of hypoxia but the absence of SU5416, intravenous delivery of anti-miR-210 ameliorated the elevation of RVSP compared with anti-miR-Control (N = 6/group, ***P = 0.001).",
    "molecules": "SU5416"
  },
  {
    "caption": "B, C Pharmacologic inhibition of ISCU (N = 8/group) did not influence miR-210 expression in the lungs either after Norm + SU5416, NS P = 0.8634 (B) or Hyp + SU5416, NS P = 0.9786 (C).",
    "molecules": "SU5416"
  },
  {
    "caption": "D As compared with control siRNA (siCont) where ISCU1/2 expression was decreased after exposure to Hyp + SU5416 (white bars), lung delivery of siRNA specific for ISCU1/2 (siISCU) via Staramine-mPEG nanocomplexes (Polach et al, 2012) down-regulated ISCU1/2 in < 100-μm pulmonary vessels in Norm + SU5416 and Hyp + SU5416 (N = 5/group, ***P = 0.0006, **P = 0.0019 for Hyp + SU5416, **P = 0.0029 for siCont).",
    "molecules": "Staramine-mPEG, SU5416"
  },
  {
    "caption": "E By electron paramagnetic resonance (EPR) spectroscopy (representative siCont versus siISCU lung), Fe-S cluster signal was decreased (quantitative graph, right) in siISCU mouselung tissue (N = 4 mice) compared with siCont (N = 5 mice), *P = 0.0143.",
    "molecules": "Fe-S"
  },
  {
    "caption": "G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased pulmonary vascular remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).",
    "molecules": "SU5416"
  },
  {
    "caption": "G-I In the absence (left bars) and presence (right bars) of Hyp + SU5416 for 2 weeks, siISCU (black bars) induced elevations in RVSP (***P < 0.0001, **P = 0.0031) (G) and increased lung remodeling in < 100-μm pulmonary vessels (H, I) as compared with siCont (N = 5/group, ***P = 0.0003, *P = 0.0114 for H; ***P = 0.0006, **P = 0.0098 for I).",
    "molecules": "SU5416"
  },
  {
    "caption": " (A) N-glycan composition of PD-1 protein expressed from 293T cells by ESI-MS analysis. N-glycans of representative peaks from ESI-MS were identified by MS/MS analysis. H, mannose; N, N-acetylglucosamine; F, fucose. Structural formulas: blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose. ",
    "molecules": "fucose, galactose, mannose, N-acetylglucosamine, N-glycan, N-glycans"
  },
  {
    "caption": " (D) SPR analysis of the binding characteristics of camrelizumab with PD-1 expressed in 293T cells, insect cells or E. coli cell, which enabled different levels of glycosylations. The binding affinity is presented as KD. The data presented here is a representative of two independent experiments with similar results. ",
    "molecules": "camrelizumab"
  },
  {
    "caption": " (A) Gel filtration profiles of PD-1 (orange), camrelizumab-scFv (blue), and the PD-1/camrelizumab-scFv complex (red) are analyzed by size-exclusion chromatography as indicated. The SDS-PAGE analyses are shown with peak 1 of camrelizumab-scFv, peak 2 of camrelizumab-scFv/PD-1 complex, and peak 3 of PD-1. ",
    "molecules": "camrelizumab"
  },
  {
    "caption": "(E) SPR analysis of the binding of camrelizumab with N58A mutated PD-1 expressed in 293T cells.",
    "molecules": "camrelizumab"
  },
  {
    "caption": "(F) The SPR analysis of the binding of camrelizumab with WT (upper) or PNGase F treated PD-1 proteins (lower) expressed in 293T cells. ",
    "molecules": "camrelizumab"
  },
  {
    "caption": "(G The SPR analysis of the binding of nivolumab with WT (upper) or N58A mutated (lower) PD-1 expressed in 293T cells",
    "molecules": "nivolumab"
  },
  {
    "caption": " H) The SPR analysis of the binding of pembrolizumab with WT (upper) or N58A mutated (lower) PD-1 proteins. ",
    "molecules": "pembrolizumab"
  },
  {
    "caption": " (A,B) The blocking of the binding of WT PD-1-mFc (A) or N58A mutated PD-1-mFc (B) to PD-L1 transiently expressed on the surface of 293T cells are analyzed with varied concentrations of camrelizumab. The PD-L1 expressing 293T cells stained with PBS alone is used as negative controls, whereas the cells stained with WT or N58A mutated PD-1-mFc proteins are used as a positive control. The frequency of PD-1-mFc staining positive cells in PD-L1-GFP positive cells is labeled in the upright corner. The reduced frequency of the PD-L1-mFc-staining-positive subpopulation compared with positive control indicates the blockage of the PD-1/PD-L1 interaction. The density of events at a given position in the plot is color-coded, with red representing the highest number of events at that point in the graph, while yellow, green, and blue represent progressively lower event densities. The results presented here are representative of three independent experiments with similar results. ",
    "molecules": "camrelizumab"
  },
  {
    "caption": "(C) (upper panel) LC-MS/MS analysis was performed to measure 15N-glutamine-labeled purine synthesis intermediates in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs. (lower panel) LC-MS/MS was used to analyze metabolites labeled with 13C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs(three biological replicates). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.",
    "molecules": "13C, glutamine, glycine, 15N"
  },
  {
    "caption": "(D) RNA and DNA with incorporated 14C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs were examined using LC-MS/MS(three biological replicates). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.",
    "molecules": "14C, DNA, glycine, RNA"
  },
  {
    "caption": "(F) UHMK1 silencing significantly decreased SGC7901 cell proliferation, while ATIC overexpression or purine supplementation markedly reversed this inhibition. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. . Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.",
    "molecules": "purine"
  },
  {
    "caption": "(G) Treatment with the ATIC inhibitor significantly reversed the proliferation of BGC823 cells induced by UHMK1 overexpression. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.",
    "molecules": "ATIC inhibitor"
  },
  {
    "caption": "(A) (Ai) Western blot analysis was used to confirm UHMK1 overexpression and ATF4 knockdown in BGC823 cells. (Aii) Silencing ATF4 significantly decreased the levels of the indicated metabolites in BGC823 cells overexpressing UHMK1(three biological replicates). (Aiii) Silencing ATF4 significantly decreased the levels of RNA and DNA containing U-14C-glycine in BGC823 cells overexpressing UHMK1(three biological replicates). (B) LC-MS/MS was used to examine the metabolites in SGC7901 cells with or without UHMK1 knockdown or ATF4 reintroduction. The data are shown in the heatmap. (C) Silencing ATF4 significantly decreased the levels of the indicated genes in BGC823 cells overexpressing UHMK1. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. (D) (Di) qRT-PCR was used to measure the expression of ATF4 in BGC823 cells overexpressing UHMK1. (Dii) UHMK1 overexpression enhanced the nuclear translocation of ATF4 in BGC823 cells. (Diii) ChIP assays on the ATIC and PPAT promoters were performed in BGC823 cells with the indicated antibody. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.",
    "molecules": "14C, DNA, glycine, RNA"
  },
  {
    "caption": "(A) GST-UHMK1 full-length (FL), GST-UHMK1 ΔN (N-terminal truncated, 1-310aa), and GST-UHMK1 ΔC (C-terminal truncated, 311-419aa) were constructed (upper panel). GST labeled FL, ΔN and ΔC protein fragments were incubated with His-NCOA3. Pull-down, Coomassie blue staining, and Western blotting were performed (lower panel).",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "(C) We performed an in vitro kinase assay by incubating purified NCOA3 FL or its protein fragments with purified UHMK1 kinase. These proteins were visualized using Coomassie blue staining. The phosphorylation of the substrates is shown in the autoradiograph.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "(D) Immunoblotting of UHMK1, NCOA3 phosphorylated at S1062/T1067, NCOA3, and ATF4 in the samples (E) An ATF4 luciferase reporter assay was carried out in BGC823 cells with or without H. pylori infection. Data were presented as mean ± standard deviation from three replicates. (F) (Fi) The domain architecture of UHMK1 and the positions of human GC-associated mutations. (Fii and Fiii) IP kinase assay. Briefly, UHMK1 from HEK293 cells expressing WT-UHMK1 or its mutants was immunoprecipitated with anti-HA antibodies. The immunoprecipitated proteins were mixed with a synthetic peptide of the CATS protein (a known substrate of UHMK1) and [32P] ATP. A phosphocellulose paper assay was used to measure kinase activity. The results were normalized to a value of 1.0 for WT-UHMK1(three biological replicates). (G) The effects of human GC-associated UHMK1 mutations on the proliferation and colony formation of GC cells (three biological replicates). (H) The effects of human GC-associated UHMK1 mutations on GC cell invasion and migration (three biological replicates). Data information: Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data represent mean ± SD. *P< 0.05, **P< 0.01,. ",
    "molecules": "ATP, 32P"
  },
  {
    "caption": "B. Immunohistochemical analyses of lamin B1-high cells. The lamin B1-high cells express Sox2 or PSA-NCAM, but not Prox1. Arrowheads indicate Prox1 negative lamin B1-high cells in the SGZ.",
    "molecules": "PSA"
  },
  {
    "caption": "B-D. Quantification of lamin B1 levels using immunofluorescent signals. Lamin B1 levels in Sox2-positive, PSA-NCAM-negative-ANSPCs were selectively downregulated at 5.5 months of age (ANPCs, P = 1.02129e-19; Neuroblasts, P = 0.09, DGCs, P = 6.4011e-15, ANOVA). ** P < 0.001, ANOVA followed by Tukey-Kramer test. (3 animals, 30 cells per conditions)",
    "molecules": "PSA"
  },
  {
    "caption": "B, C. Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment. Note that the numbers of DCX-positive cells are increased. Boxes indicate magnified regions in C.",
    "molecules": "TAM"
  },
  {
    "caption": "D, E, F. Quantification three weeks after TAM treatment. Among EYFP-positive cells, the density of RGL-ANSCs is not different (P = 0.65, t-test) (D). The density of ANPCs is significantly decreased (P = 0.0037, t-test) (E) whereas the density of DCX-positive cells is markedly increased (P = 0.014, t-test) (F). (n = 4 for Control, n =3 for cKO). Open arrowheads indicate respective cells. G, H, I. Quantification two months after the induction of KO (n = 3 for Control, n = 4 for cKO). The density of RGL-ANSCs is not significantly different (P = 0.29, t-test) (G). The density of ANPCs is significantly decreased. (P = 0.0092, t-test) (H). The density of neuroblasts is not significantly different (P = 0.76, t-test) (I). ",
    "molecules": "TAM"
  },
  {
    "caption": "J, K, L. Quantification of proliferating cells three weeks after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.027, Mann Whitney test) (J), and that of Ki67+ANPCs is also significantly decreased. (P = 0.034, t-test) (K). The density of Ki67+ neuroblasts is significantly increased (P = 0.0011, t-test) (N) (n = 4 for Control, n = 4 for cKO) (L). M, N, O. Quantification of proliferating cells two months after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.0086, t-test) (M), and densities of Ki67+ANPCs and Ki67+neuroblasts are also significantly decreased. (P = 0.023, P = 0.025, t-test) (n = 3 for Control, n = 3 for cKO) (N, O). ",
    "molecules": "TAM"
  },
  {
    "caption": "P. Declined return to RGL-ANSCs. Left, experimental scheme. Analysis was performed 3 weeks after BrdU injections. Middle, a representative image of BrdU+GFAP+ RGL-ANSCs. Right, Quantification of BrdU+GFAP+RGL-ANSCs (P = 0.019, n = 4 for Control, n = 4 for cKO).",
    "molecules": "BrdU"
  },
  {
    "caption": "Representative confocal images of the SGZ 6.5 months after the treatment with TAM.",
    "molecules": "TAM"
  },
  {
    "caption": "C. Representative confocal images of the SGZ 6.5 months after the treatment with TAM.",
    "molecules": "TAM"
  },
  {
    "caption": "(E) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 1 day after GT intoxication. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. GT - glutamate; noninj - noninjured control.",
    "molecules": "glutamate"
  },
  {
    "caption": "(A) Representative micrograph of an eye from day 7 after glutamate intoxication, from a mouse that was i.v.-injected with monocytes, showing monocyte localization to the SRS after this mode of application, and indicating a potential homing route for the cells (IL-10, red; CX3CR1-GFP, green).",
    "molecules": "glutamate"
  },
  {
    "caption": "(B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (D, E) Representative micrographs of GT-intoxicated eyes that were i.v.t.-injected with monocytes, showing the expression of an array of cytokines and neurotrophic factors (red) by the injected monocytes (CX3CR1-GFP, green). The injected cells were found in the vitreous and in proximity to injured RGCs (left panels), as well as in the SRS, adjacent to the RPE (right panels). Insets show higher magnification of representative cells (scale bar, 100 µm; inset scale, 20 µm). n = 4-5 per group. GCL - ganglion cell layer; GT - glutamate; mon - monocytes; RGC - retinal ganglion cell; RPE - retinal pigment epithelium; SRS - subretinal space; vit - vitreous.",
    "molecules": "glutamate"
  },
  {
    "caption": "(B) Representative emission spectra of mixtures containing ECFP-D4H, mVenus-Lact-C2, and liposomes containing indicated compositions of lipids. Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%) and that FRET signal was decreased when sphingomyelin (SM) (25%) was additionally incorporated into the liposomes. Asterisks indicate positions of emission maximum of ECFP (*477 nm) and FRET (**525 nm). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; SM, sphingomyelin (N-oleoyl-D-erythro-sphingosylphosphorylcholine). (C) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm (FRET/ECFP) (see Materials and Methods) from mixtures as shown in (D) (mean ± SEM, n = 4 independent experiments; Dunnett's multiple comparisons test, **P < 0.0001).",
    "molecules": "1,2-dioleoyl-sn-glycero-3-phospho-L-serine, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, Chol, cholesterol, lipids, phosphatidylserine, phosphatidylcholine, N-oleoyl-D-erythro-sphingosylphosphorylcholine, SM, sphingomyelin"
  },
  {
    "caption": "(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing mCherry-GRAM1b. Cells were pre-incubated with either control imaging buffer (for control) or imaging buffer containing purified ECFP-D4H proteins (3 µM) (for masking accessible PM cholesterol) for 30 min at 37°C and imaged with the same buffer conditions. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 32 cells (Control), n = 35 cells (Pre-incubation w/ ECFP-D4H); data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].",
    "molecules": "cholesterol, SMase"
  },
  {
    "caption": "(E) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing EGFP-GRAM1b together with either mCherry for control or mCherry-Lact-C2 for PS masking. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 53 cells (mCherry), n = 32 cells (mCherry-Lact-C2 OE); data are pooled from four independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].",
    "molecules": "PS, SMase"
  },
  {
    "caption": "(C-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (K161A, R191A, and K161A/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 3 independent experiments for all conditions in phosphatidylserine (PS) sensitivity, co-incidence detection, and anionic lipid selectivity assays; n = 12 independent experiments for WT, n = 3 independent experiments for K161A and R191A, and n = 6 independent experiments for K161A/R191A in the cholesterol sensitivity assay). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; PA, phosphatidic acid (1,2-dioleoyl-sn-glycero-3-phosphate); PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI, L-α-phosphatidylinositol.",
    "molecules": "1,2-dioleoyl-sn-glycero-3-phospho-L-serine, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, Chol, cholesterol, L-α-phosphatidylinositol, PI, lipids, 1,2-dioleoyl-sn-glycero-3-phosphate, PA, phosphatidic acid, phosphatidylserine, phosphatidylcholine, phosphatidylinositol 4-phosphate, PI(4)P, phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2"
  },
  {
    "caption": "(B) Sterol specificity of the GRAM domain of GRAMD1b (GRAM1b) as assessed via liposome sedimentation assays. Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as indicated. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 6 independent experiments for liposomes containing only DOPC, n = 3 independent experiments for the rest). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine).",
    "molecules": "1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, lipids, phosphatidylcholine, Sterol"
  },
  {
    "caption": "(D-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (R189W, R189W/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all conditions in the co-incidence detection assay; n = 3 independent experiments for R189W and R189W/R191A, n = 6 independent experiments for WT in phosphatidylserine (PS) sensitivity assay; n = 3 independent experiments for R189W and R189W/R191A, n = 5 independent experiments for WT in cholesterol sensitivity assay]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol.",
    "molecules": "1,2-dioleoyl-sn-glycero-3-phospho-L-serine, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, Chol, cholesterol, lipids, phosphatidylserine, phosphatidylcholine"
  },
  {
    "caption": "(F) Confocal images of live GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-tagged GRAM domain of GRAMD1b (EGFP-GRAM1b) constructs as indicated. Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Anionic lipid sensing defective mutants (K161A, R191A, and K161A/R191A), a cholesterol sensing defective mutant (R189W), and a cholesterol/anionic lipid sensing defective mutant (R189W/R191A) are shown. Note the absence of (R189W, R189W/R191A, and K161A/R191A) or significantly reduced (K161A, R191A) PM recruitment of the mutant versions of EGFP-GRAM1b compared to the strong PM recruitment of wild-type EGFP-GRAM1b (WT). Scale bars, 10 µm.",
    "molecules": "cholesterol, lipid, SMase"
  },
  {
    "caption": "(G) Quantification of the ratio of PM EGFP-GRAM1b signals to the cytosolic EGFP-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b with or without SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (F) and Appendix Fig S1B (mean ± SEM, n = 40 cells for WT, and n = 30 cells for other conditions; data are pooled from four independent experiments for WT and three independent experiments for the rest; two-tailed unpaired Student's t-test, **P < 0.0001. n.s. denotes not significant).",
    "molecules": "SMase"
  },
  {
    "caption": "(H) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b constructs as indicated. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 126 cells (WT), n = 38 cells (K161A), n = 33 cells (R191A), n = 43 cells (K161A/R191A), n = 46 cells (R189W), n = 44 cells (R189W/R191A); data are pooled from six independent experiments for WT and two independent experiments for the rest; Dunnett's multiple comparisons test, **P = 0.0002 (WT vs. K161A) and **P < 0.0001 for the rest].",
    "molecules": "SMase"
  },
  {
    "caption": "(C) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer (FRET) between DHE and DNS-PE in LPM. A series of liposomes with different DHE mol% were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred (see Appendix Fig S2D and Materials and Methods). Right: Values of DHE transfer rate of GRAMD1b (WT), GRAMD1b (R189W), and GRAMD1b (RW/RA), as estimated by the FRET-based lipid transfer assay [mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for R189W and RW/RA",
    "molecules": "DNS, DHE, Methyl-β-cyclodextrin, PE, MCD"
  },
  {
    "caption": "(B) Confocal images of live GRAMD1 TKO HeLa cells stably expressing EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor, mCherry-tagged GRAM domain of GRAMD1b (mCherry-GRAM1b). Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note the very weak PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b WT, compared to the strong PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b R189W mutant (R189W) or EGFP-GRAMD1b R189W/R191A mutant (RW/RA). Scale bars, 10 µm.",
    "molecules": "cholesterol, SMase"
  },
  {
    "caption": "(C) Quantification of the ratio of PM mCherry-GRAM1b signals to the cytosolic mCherry-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing mCherry-GRAM1b with SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (B) and Fig EV3B (mean ± SEM, n = 20 cells for each condition; data are pooled from two independent experiments; Dunnett's multiple comparisons test, **P < 0.0001. n.s. denotes not significant).",
    "molecules": "SMase"
  },
  {
    "caption": "(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells stably expressing either EGFP or EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry-GRAM1b. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 31 cells (EGFP), n = 28 cells (EGFP-GRAMD1b WT), n = 23 cells (EGFP-GRAMD1b R189W), n = 27 cells [EGFP-GRAMD1b R189W/R191A (RW/RA)]; data are pooled from two independent experiments for each condition; Dunnett's multiple comparisons test, **P < 0.0001 (EGFP vs. WT and WT vs. R189W), **P = 0.0002 (WT vs. RW/RA)].",
    "molecules": "cholesterol, SMase"
  },
  {
    "caption": "(E) Wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)], were cultured in the medium supplemented with 10% lipoprotein-deficient serum (LPDS) and mevastatin (50 µM) for 16 hours and then treated with SMase (100 mU/ml) for 3 hours at 37°C. Top: Lysates of the cells were processed for SDS-PAGE and IB with anti-SREBP-2 and anti-actin antibodies. Arrows indicate precursor (P) and cleaved (C) forms of SREBP-2. Bottom: The response rate was obtained by normalizing the ratio of the band intensity of the cleaved SREBP-2 over the total band intensity of cleaved and precursor forms of SREBP-2 from the cells with SMase treatment by the one from the cells without SMase treatment for each condition. Note that the suppression of SREBP-2 cleavage is attenuated in GRAMD1 TKO HeLa cells compared to wild-type control HeLa cells. Note also the rescue by expression of wild-type EGFP-GRAMD1b (WT) but not by mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] [mean ± SEM, n = 4 lysates (independent experiments) for each condition; Dunnett's multiple comparisons test, **P = 0.0035 (Control + EGFP vs. TKO + EGFP), **P < 0.0001 (TKO + EGFP vs. TKO + WT), n.s. denotes not significant].",
    "molecules": "lipoprotein, mevastatin, SMase"
  },
  {
    "caption": "(F) Amphotericin B resistance of SMase-treated wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)]. Left: Cells that had been pre-treated with SMase (100 mU/ml) for 3 hours at 37°C were treated with indicated concentration of Amphotericin B for 20 min at 37ºC. After overnight recovery in culture media, cell viability was measured by detecting ATP present in each well via luminescence (see Materials and Methods). The same number of cells were seeded in each well before SMase treatment. Note the reduced viability of cells with increasing amount of Amphotericin B. Right: Quantification of cell viability with increasing amount of Amphotericin B. Note the resistance of wild-type control and GRAMD1 TKO cells that stably expressed EGFP-GRAMD1b (WT) compared to GRAMD1 TKO cells or GRAMD1 TKO cells that stably expressed mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] (mean ± SEM, n = 3 independent experiments for each condition).",
    "molecules": "Amphotericin B, ATP, SMase"
  },
  {
    "caption": "(D) Confocal images of live wild-type (control) and GRAMD1 TKO HeLa cells, expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L), with or without SMase treatment (100 mU/ml for 1 hour at 37°C). Insets show at higher magnification the regions indicated by white dashed boxes. Note the recruitment of mutant EGFP-GRAM1b (G187L) to the PM even in wild-type HeLa cells at rest, which was further enhanced by SMase treatment. Scale bars, 10 µm.",
    "molecules": "SMase"
  },
  {
    "caption": "(E) Quantification of the ratio of PM signals to the cytosolic signals of wild-type EGFP-GRAM1b (WT) and mutant EGFP-GRAM1b (G187L), as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells, expressing indicated constructs, with or without SMase treatment (100 mU/ml for 1 hour at 37°C), as shown in (D) (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment).",
    "molecules": "SMase"
  },
  {
    "caption": "(F) A confocal image of live GRAMD1 TKO HeLa cells expressing mutant EGFP-GRAM1b (G187L). Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours to deplete accessible cholesterol before imaging. An inset shows at higher magnification the region indicated by a white dashed box. Note the absence of PM recruitment (compare to (D)). Scale bars, 10 µm.",
    "molecules": "cholesterol, lipoprotein, mevastatin"
  },
  {
    "caption": "(G) Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO (TKO) HeLa cells expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L) as indicated. Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours before imaging. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 36 cells (WT), n = 37 cells (G187L), data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P = 0.0003]. Note the rapid PM recruitment of G187L mutant compared to WT.",
    "molecules": "lipoprotein, mevastatin, SMase"
  },
  {
    "caption": "(H, I) Liposome sedimentation assays of wild-type GRAM1b (WT) and mutant GRAM1b (G187L). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all the conditions; (H) Left (10% PS): Holm-Sidak t-test for multiple comparisons, *P = 0.022679 at 40% cholesterol, **P = 0.009195 at 50% cholesterol. Right (20% PS): Holm-Sidak t-test for multiple comparisons, *P = 0.023163 at 30% cholesterol, **P = 0.000327 at 40% cholesterol, *P = 0.017194 at 50% cholesterol. (I) Holm-Sidak t-test for multiple comparisons, n.s. denotes not significant, **P < 0.000001 at 50% DOPC, 50% cholesterol, *P = 0.011271 at 40% DOPC, 60% cholesterol]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol.",
    "molecules": "1,2-dioleoyl-sn-glycero-3-phospho-L-serine, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, Chol, cholesterol, lipids, phosphatidylserine, PS, phosphatidylcholine"
  },
  {
    "caption": "(A) Effects of mutation of G187 on the property of the GRAM domain of GRAMD1b (GRAM1b) to sense transient expansion of the accessible pool of PM cholesterol. Indicated EGFP-tagged mutant versions of GRAM1b were expressed in GRAMD1 TKO HeLa cells and imaged under confocal microscopy with or without SMase treatment (100 mU/ml for 1 hour at 37°C) or with SMase treatment followed by 5 min treatment with Methyl-β-cyclodextrin (MCD) (5 mM) to assess their PM recruitment. The mean values (n = 20 cells from two independent experiments for untreated and SMase-treated conditions; n = 10 cells from one experiment for SMase & MCD-treated conditions) of the ratio of PM signals to the cytosolic signals, as assessed by line scan analysis, are presented for each condition as a heatmap. Amino acids are ranked according to Goldman, Engelman and Steitz (GES) hydrophobicity scale with the most hydrophobic amino acid on the top.",
    "molecules": "cholesterol, Methyl-β-cyclodextrin, MCD, SMase"
  },
  {
    "caption": "(C) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b G187L proteins (G187L), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Experiments and data analysis were performed as in Fig 4. Right: Values of DHE transfer rate of wild-type GRAMD1b (WT) and mutant GRAMD1b (G187L), as estimated by the FRET-based lipid transfer assay (mean ± SEM, n = 3 independent experiments; two-tailed unpaired Student's t-test, **P < 0.0014).",
    "molecules": "DHE, Methyl-β-cyclodextrin, MCD"
  },
  {
    "caption": "(D) Confocal images of live GRAMD1 TKO HeLa cells that stably expressed either EGFP control or EGFP-GRAMD1b constructs as indicated. Cells were stained with recombinant mCherry-D4H proteins (10 mg/ml) (accessible PM cholesterol biosensor) for 15 min at room temperature before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note that expression of EGFP-GRAMD1b-WT, but not EGFP-GRAMD1b-R189W, reduced binding of mCherry-D4H to the PM, in GRAMD1 TKO HeLa cells. Further reduction in mCherry-D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-WT. Scale bars, 10 µm. (E) Values of mCherry-D4H signals at the PM after background subtraction, as assessed by confocal microscopy and line scan analysis of GRAMD1 TKO HeLa cells that stably expressed either EGFP or indicated EGFP-GRAMD1b constructs as shown in (D) [mean ± SEM, n = 30 cells for all the conditions; data are pooled from three independent experiments; Dunnett's multiple comparisons test, **P < 0.0001].",
    "molecules": "cholesterol"
  },
  {
    "caption": "Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.",
    "molecules": "puromycin"
  },
  {
    "caption": "Passage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.",
    "molecules": "puromycin"
  },
  {
    "caption": "Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.",
    "molecules": "ATP"
  },
  {
    "caption": "Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.",
    "molecules": "etoposide"
  },
  {
    "caption": "Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).",
    "molecules": "DMSO, ABT-888"
  },
  {
    "caption": "Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).",
    "molecules": "ABT-888"
  },
  {
    "caption": "Plots showing the RT-qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands. Results are expressed as the mean ± s.d (n=10 for PBS, n=5 for WT and n=4 for CV2) of the 2-ΔΔCt relative expression values of the indicated interleukins; the value from each individual animal was calculated from three technical replicates. The values obtained in the PBS group were used as control for normalization of gene expression (=1). Statistical analysis was performed using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test: (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005).",
    "molecules": "interleukins, PBS"
  },
  {
    "caption": "(A, B) Mature S. aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions, or culture supernatants of the CV2 or the CV2-DispB strains (A) or the WT or the WT-DispB strain (B). Biofilm presence was assessed by crystal violet staining and included a negative staining control (i.e., crystal violet without biofilm). The results are expressed as mean ± s.d. of OD 595 nm absorbance values obtained from three different biological replicates (n=3) and statistically compared by one-sided ANOVA followed by the post-hoc Fisher's PLSD test: *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005; ****P ≤ 0.00005",
    "molecules": "crystal violet, polystyrene"
  },
  {
    "caption": "A) ELISA quantification of PD-L1,  pTau 181 and  Aβx-42 in the CSF  of control and AD patients (n=10 +/-SEM, Student's t-test, * p<0.05, *** p<0.001, PD-L1: t=2.47 df = 18,  Aβx-42: t=4.83 df=18, pTau 181: t=5,768 df=18).",
    "molecules": "Aβx-42"
  },
  {
    "caption": "B, C) (B) Colocalization of PD-L1 and  GFAP in AD and (C) in control patients (MX-O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar=30 µm). D) Immunohistochemical  detection of PD-L1 in 9-month-old female APP/PS1 mice (bar = 50 µM). E) Immunohistochemical  detection of PD-L1 in 4-month-old female  APP/PS1 mice  (bar = 20 µm). ",
    "molecules": "MX-O4, amyloid"
  },
  {
    "caption": "A) C6 cells expressing PD-L1-myc treated with the  γ-secretase  inhibitor DAPT for 18 h. PD-L1 was detected using a myc-antibody.",
    "molecules": "DAPT"
  },
  {
    "caption": "D) 18 h 1 μM PMA  treatment of C6  cells expressing PD-L1-myc. Secretion of PD-L1 in the conditioned medium (CM) was detected using antibody AF1019.",
    "molecules": "PMA"
  },
  {
    "caption": "E) Mouse astrocytes were incubated for 48 h with 1 μM PMA  and 10 μg/ml TNFα / 100 U/μl  IFNγ  and lysates and the CM were  immunoblotted using antibody AF1019.",
    "molecules": "PMA"
  },
  {
    "caption": "A) Colocalization of PD-1 and CD11b (MX-04=methoxy-XO4, stains amyloid  deposits) in AD by  immunohistochemistry (bar=50 µm). B) Colocalization of PD-1 and Iba1 or PD-1 and  Aβ in a female APP/PS1 mouse at 9 months of age by  immunohistochemistry (bar=20 µm). ",
    "molecules": "methoxy-XO4, MX-04, amyloid"
  },
  {
    "caption": "E) ELISA analysis of TNFα secretion by wild type (wt) and PD-1-/- microglia induced by 0.5 µM Aβ1-42 for 6 h in vitro (n=3 biological replicates, one-way ANOVA (DF = 1, F = 8.29, p= 0.021), Tukey's HSD, * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "F-H) Microglia from methoxy-XO4-injected, 8-month-old female, wild type (wt) and APP/PS1 mice  were analyzed (F) for PD-1 (n=5 biological replicates per group, one-way ANOVA (DF = 2, F = 13.99, p=0.0013), Tukey's HSD, ** p<0.01), (G) for CD11b (n=5 biological replicates per group, one- way ANOVA (DF = 2, F = 30.06, p=5.47x10-05), Tukey's HSD, *** p<0.001) and (H) CD45 by flow cytometry (n=5 biological replicates per group, one-way ANOVA (DF = 1, F = 24.96, p=0.00013), Tukey's HSD, *** p<0.001) and normalized to the expression of wild type  mice (represented by dashed lines). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.",
    "molecules": "methoxy-XO4"
  },
  {
    "caption": "A, B)  ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for  Aβx-40 (biological replicates with n=6 for APP/PS1  and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction  (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001).",
    "molecules": "Aβx-40, SDS"
  },
  {
    "caption": "A) In vitro phagocytosis of  FAM-Aβ1-42 by wild type or PD-1-/- microglia  for up to 6 h (mean +/-SEM of a technical quadruplicate, Student's t-test, ** p<0.01, *** p<0.001, representative result of an n=2 biological replicate). B) In vitro phagocytosis  of FAM-Aβ1-42 by wild type  microglia cultured in astrocyte-conditioned medium (ACM) from wild type or PD-L1-/- astroglial cultures (mean +/-SEM of a technical quadruplicate, Student's t-test, * p<0.05, *** p<0.001, representative result of an n=2 biological replicate) ",
    "molecules": "FAM, Aβ1-42"
  },
  {
    "caption": "D) Quantification of microglial in vivo phagocytosis  in female  APP/PS1  and APP/PS1 PD-1-/- mice at 8.5 month of age after intraperitoneal injection of methoxy-XO4 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, Student's t-test (t = 2.1808, df = 6.9829), * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.",
    "molecules": "methoxy-XO4"
  },
  {
    "caption": "E-G) Relative expression of the cell surface marker (E) CD36 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, ANOVA (DF=1, F= 0.002, p=0.94), Tukey's HSD, * p<0.05, *** p<0.001), (F) CD11b (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) and (G) CD45 on methoxy-XO4+ versus methoxy-XO4- microglia in APP/PS1  and APP/PS1 PD-1-/- mice normalized to methoxy-X04-negative cells from  APP/PS1 mice (biological replicates with n=5 for  APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.",
    "molecules": "methoxy-X04, methoxy-XO4"
  },
  {
    "caption": "(B) Tumor growth curves from BRAFi-persister cells (monitored during 5 months) V for Vem-resistant cells and PB for PLX8394-resistant cells.",
    "molecules": "PB, PLX8394, Vem"
  },
  {
    "caption": "(G) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib) on BRAFi-resistant cells (SKMel28R). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib; BRAFi). n=3 biologically independent experiments. Each histogram represents the mean + s.d. (H) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib, BRAFi) on BRAFi-resistant cells (Me1402). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib). n=3 biologically independent experiments. Each histogram represents the mean + s.d.",
    "molecules": "dimethylsulfoxide, DMSO, vemurafenib"
  },
  {
    "caption": "(O) The chemical inhibition of SMAD3 by SIS3 (SMAD3i) improved current therapy effect (BRAFi+MEKi; Vem 5µM and Cobi 1µM) on BRAFi-resistant cells (SKMel28R, M229R & M238R). Cells have been treated",
    "molecules": "Cobi, SIS3, Vem"
  },
  {
    "caption": "(B) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction. 501Mel cells AhR wild-type or knock-out have been exposed to exogenous and endogenous AhR ligands; TCDD (5nM) or ITE (10µM) or the solvent (DMSO) during 10 days. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d. (C) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction. 501Mel cells have been treated as described in B. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d.;",
    "molecules": "TCDD, ITE, DMSO"
  },
  {
    "caption": "(e) In gel-activity assays for MRC complexes II and IV in DDM-solubilized mitochondria from RNAi (act5c-gal4>UAS-CG7630RNAi) and control larvae (act5c-gal4>+).",
    "molecules": "DDM"
  },
  {
    "caption": "(b) Relative quantification (RQ) of CG7630 and COX7B transcripts in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock). N/D = not detected.",
    "molecules": "neo"
  },
  {
    "caption": "(c) Immunoblot analysis of stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). The CG7630 transcript was knocked-down (KD) in cells by a dsRNA targeting CG7630 and compared with cells treated with a mock control dsRNA (mock). Samples were probed with an antibody anti HA tag and with an antibody against Hsp70 as an endogenous control.",
    "molecules": "neo"
  },
  {
    "caption": "(d) Kinetic enzyme activity of COX normalized by citrate synthase activity (CS) in stable S2R+ cell lines carrying the empty expression vector (EV:GFP;neo), and expression vectors carrying HA-tagged forms of human COX7B (hCOX7B-HA;neo) and CG7630 (CG7630-HA;neo). Cells were treated with a dsRNA targeting CG7630 (striped bars, CG7630 KD) and a mock control dsRNA (solid bars, mock).",
    "molecules": "neo"
  },
  {
    "caption": " Total carbonyl-bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E. coli and D. radiodurans with and without irradiation. The left axis is the number of sequence-unique proteins detected as carbonylated. The right axis is the number of sites in total detected as carbonylated (red) or not oxidized (black) in peptides bearing at least one carbonyl. Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples. ",
    "molecules": "carbonyl, carbonylated"
  },
  {
    "caption": "Volcano plots for relative protein abundance changes measured by mass spectrometry in E. coli (left) and D. radiodurans (right) after irradiation using the same biological replicates as in Fig 2A. Black-circled points are those proteins with significant changes (paired, 2-sided t-test p-value < 0.05) of >2-fold or <0.5-fold. Red points are proteins with at least one carbonylated peptide detected. Fold change and p-value cutoffs considered for significance are indicated by dashed lines.",
    "molecules": "carbonylated"
  },
  {
    "caption": "Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism. Ratios are given above each pair of bars. All proportions are significantly different between each RKPT and their respective carbonylation state by two-tailed z-test of two proportions (p-values < 0.01; see Materials and Methods), meaning carbonylated proportions are not determined simply by relative prevalence of RKPT. See also Appendix Fig S1.",
    "molecules": "carbonylated"
  },
  {
    "caption": "Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes. BMDMs were untreated or pretreated with rapamycin (Rap) for 1 h and kept throughout the infection. BMDMs were infected with L. pneumophila JR32 or the leg A9 mutant for (A) 1 or (B) 24 h with an MOI of 0.5. CFUs were scored at these time points.",
    "molecules": "rapamycin (Rap)"
  },
  {
    "caption": "H Violin plots showing the association of different B-cell signatures with the response (R, N=15) and no response (NR, N=42) to Pembrolizumab in the Kim cohort. p values calculated using the student's t-test;",
    "molecules": "Pembrolizumab"
  },
  {
    "caption": "Blistered samples at P1. (B) Hematoxylin and eosin (H&E, top) and alkaline phosphatase (AP, bottom) staining. Hair follicles (HFs) remaining in the dermis (indicated by arrows). Scale bar: 500 μm. (C) α6 Integrin (ITGA6, indicated by arrowheads) and type IV collagen (COL4, arrows) labeling (left). Laminin 332 (L332, arrows) staining (right). Scale bar: 100 μm. (D) Ultrastructural findings of blistered skin (left image: blister roof, right image: blister bottom) . Hemidesmosomes (white arrowheads) and lamina densa (arrows) are indicated. Scale bar: 1 μm.",
    "molecules": "alkaline phosphatase, AP, eosin, Hematoxylin"
  },
  {
    "caption": "BrdU labeling of blistered samples at P2. Scale bar: 100 μm. BrdU-positive cells are indicated by arrows. Blisters are indicated by stars. (Bottom) Quantification of BrdU-positive cells in the epidermis (left) and HFs (right) (n=4 biological replicates). The data are shown as the mean ± SE. *0.01 test, followed by Tukey's test. NS, no significance. ",
    "molecules": "BrdU"
  },
  {
    "caption": "BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance.",
    "molecules": "BrdU"
  },
  {
    "caption": "K10/K14 (low magnification, left) and K14 (high magnification, middle and right) labeling of Col7a1-/- mouse (bottom) and littermate control (top) blistered skin at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=55 HFs from three control and 143 HFs from four Col7a1-/- mice). The data are shown as violin plots. Student's t-test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=244 (control, L), and 132 (Col7a1-/-, L) cells from four mice, respectively) and in the surrounding intact epidermis (basal cells; n=200 (control, NL), 299 (Col7a1-/-, NL), from four mice, respectively). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.",
    "molecules": "BrdU"
  },
  {
    "caption": "K10/K14 (left) and K14 (high magnification, right) labeling of WT blistered skin treated with CaCl2 (middle and bottom) or PBS (top) at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=83 (PBS), 95 (1.8 mM CaCl2), and 97 (9.0 mM CaCl2) HFs from four mice). One-way ANOVA test, followed by Tukey's test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=433 (PBS, L), 451 (1.8 mM CaCl2, L), and 425 (9.0 mM CaCl2, L) cells from four mice) and in the surrounding intact epidermis (basal cells; n=311 (PBS, NL), 279 (1.8 mM CaCl2, NL), 302 (9.0 mM CaCl2, NL) cells from four mice). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.",
    "molecules": "BrdU, CaCl2"
  },
  {
    "caption": "A Treatment scheme of experimental timeline and  Immunofluorescence staining of telogen backskin hair follicles  (HF) for Integrin α6  (Itgα6, red), active  Caspase 3  (aCas3, green) and DAPI (blue) in control (Ctr) and Bcl-2 inhibitor (ABT-199) treated mice. B: bulge, CH: club hair, DP: dermal papilla, HF: hair follicle, HG:  secondary hair germ. Scale bar, 50 µm B Quantification of  backskin HF with aCas3+ cells in bulge, hair germ and dermal papilla of Ctr and ABT-199 treated mice (n=4 mice biological replicates; mean ± standard deviation (SD); *** p<0.001, ns=not significant, two-way ANOVA test). nd=not detected. ",
    "molecules": "DAPI, ABT-199"
  },
  {
    "caption": "C,D Treatment scheme, FACS  plot (C) and quantification (D) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution (Ctr) or ABT-199 (n=6 biological replicates; mean ± standard deviation (SD); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal  (bBSC, CD34+/Itgα6high) BSCs and n-B (non-bulge epidermal cells). ** p<0.005.",
    "molecules": "ABT-199"
  },
  {
    "caption": "B Treatment scheme of experimental timeline and histology of backskin  sections of control (Ctr) and ABT-199 treated mice. Scale bar, 200 µm.",
    "molecules": "ABT-199"
  },
  {
    "caption": "D qRT-PCR  for  mRNA expression of the transcription factors Lef1  and Runx1 in epidermis of ABT-199 treated and control (Ctr) mice at P77 (n=3 biological replicates; mean ± standard error of mean (SEM); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test).",
    "molecules": "ABT-199"
  },
  {
    "caption": "D  Immunofluorescence staining of  telogen backskin HF for BrdU  incorporation (1 h pulse, green) and DAPI (blue) in 8 week old control (Ctr) and Bcl-2EOE mice. Scale bar, 50 µm.",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": "B Immunofluorescence staining of  epidermal tail  whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice,  stained for keratin 15 (Krt15, green), activated Caspase 3   (aCas3, red) and DAPI (blue). Note increased number of aCas+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge,  HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D  Immunofluorescence staining of epidermal tail  whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), BrdU (1 h pulse, red) and DAPI (blue). Note increased  number  of BrdU+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": "(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n≥3); *P0.05.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase‐1 and IL‐1β was determined by immunoblotting.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(D) LPS‐pretreated BMMs were exposed to alum (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "alum, LPS"
  },
  {
    "caption": "(E) LPS‐pretreated BMMs were exposed to silica (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "LPS, silica"
  },
  {
    "caption": "(F) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to nigericin (20 μM) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(H) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to silica (250 μg/ml) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "LPS, silica"
  },
  {
    "caption": "(I) Colocalization of IL‐1β with the basal autophagic machinery factor LC3. Fluorescence: LC3 (green, Alexa488); IL‐1β (red, Alexa568). BMMs were from GFP-LC3 knock‐in mice, treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL‐1β.",
    "molecules": "LPS"
  },
  {
    "caption": "(A, B) LPS‐pretreated BMMs were treated with 20 μM nigericin (Nig) and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β (A) and IL‐18 (B) were measured. Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "bafilomycin A1, LPS, Nig, nigericin"
  },
  {
    "caption": "(C) LPS‐pretreated BMMs were treated with 250 μg/ml of silica and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β were measured. Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "bafilomycin A1, LPS, silica"
  },
  {
    "caption": "(D) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), and cathepsin B (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were treated with LPS and then analysed for immunofluorescence.",
    "molecules": "LPS"
  },
  {
    "caption": "(F) LPS‐pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA‐074 Me (10 μM), with (Starvation) or without (Full) autophagic induction, for 1 h and secreted IL‐1β was measured. Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "CA‐074 Me, LPS, nigericin"
  },
  {
    "caption": "(G) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting. Figure source data can be found in Supplementary data.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(A) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization.",
    "molecules": "LPS"
  },
  {
    "caption": "(D) BMMs were transfected with siRNAs against Rab8a or scramble (Scr) control. At 24 h after the first transfection, cells were transfected again with siRNA, treated with LPS and the day after subjected to nigericin in full medium for 1 h, and IL‐1β secretion measured.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(F) RAW 264.7 macrophages were transfected with GFP‐tagged Rab8a constructs (WT, wild type; S22N, dominant‐negative mutant), treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation. IL‐1β secretion was measured by ELISA. Data represent mean values±s.d. (n≥3); *P0.05. Figure source data can be found in Supplementary data.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(A) BMM cells were transfected with scramble (Scr) control siRNA or siRNA against GRASP55. After 48 h of transfection, cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS, and secreted IL‐1β was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Inset: Immunoblot analysis of GRASP55 knockdown.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(B) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution. LC3 (green, Alexa488), GRASP55 (red, Alexa568). BMMs were pretreated overnight with 100 ng/ml LPS and either not stimulated (Ctrl) or stimulated (Nig) for 30 min with the inflammasome agonist nigericin (20 μM) in full medium.",
    "molecules": "LPS, Nig, nigericin"
  },
  {
    "caption": "(A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(a) Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, towards PI(4,5)P2 and PI(3,4,5)P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean±SEM, Paired t-test, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "PI(3,4,5)P3, PI(4,5)P2"
  },
  {
    "caption": "(b) Representative confocal image of HDMEC stained for Paladin (green), Vascular Endothelial Cadherin (VEC, red) and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(c) HDMEC stained for VEGFR2 (green), Paladin (red) and nuclei (DAPI, blue) and with or without stimulation of 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (d) Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "DAPI"
  },
  {
    "caption": "(e) HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE-cadherin (red) and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (f) Quantification of (e), the number of PLA Paldin-VEGFR2 complexes per cell at 0, 2 and 10 minutes after VEGF-A stimulation (top). Quantification of (e), average distance for the PLA complexes to the nearest VE-cadherin positive junction at 0, 2 and 10 minutes after VEGF-A stimulation (bottom). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "DAPI"
  },
  {
    "caption": "(g) HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2 or 10 minutes, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. (h) Quantification of (g), EEA1/Paladin double positive particles per field of view at 0, 2 and 10 minutes after VEGF-A stimulation. Mean±SEM, One-way ANOVA. n= 3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin. (b) Quantification of data in (a). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC. n=4 for each time point, biological replicates, Mean±SEM. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "biotin, SA, streptavidin"
  },
  {
    "caption": "(c) Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin and actin. (d) Quantification of data in (c). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean±SEM, unpaired t-test for indicated time points, normalized to cntrl siRNA sample. n=3 for each time point, biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "biotin, SA"
  },
  {
    "caption": "(e) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2 and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm. (f) Quantification of (e), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean±SEM, Two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "DAPI"
  },
  {
    "caption": "(g) HDMEC stained for PI(4,5)P2 (cyan), VE-cadherin (red) and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2 or 10 minutes (50 ng/ml). DAPI in blue. Scale bar: 10 µm (h) Quantification of (g), Total PI(4,5)P2 signal (top) or intracellular PI(4,5)P2 not overlapping with VE-cadherin (bottom). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.",
    "molecules": "DAPI, PI(4,5)P2"
  },
  {
    "caption": "(e-f) Immunoblotting on total heart lysates from adult Pald1+/+ and Pald1-/- mice, tail-vein injected with VEGF-A (0.25µg/g body weight) or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, Erk1/2 and β2-microglobulin (β 2-MG, loading control). (f) Quantification of pVEGFR pY1173 normalized to total VEGFR2 (n=3), total VEGFR2 levels normalized to total loading control (n=4), pT202/pY204 Erk1/2 normalized to Erk1/2 (n=5). Mean±SEM, multiple t-test with Holm-Sidak correction (total VEGFR2), two-way ANOVA (others). Data information: *p < 0.05, **p < 0.01.",
    "molecules": "PBS"
  },
  {
    "caption": "(l) Quantification of relative radial expansion in vehicle (n=2 litters, 5 Pald1+/+ and 4 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=4 litters, 7 Pald1+/+ and 5 Pald1-/- pups). MEK inhibitor/vehicle was administered twice at 12h interval at P4 and eyes collected at P5. Each dot is one mouse. Mean±SEM, one-way ANOVA, n=4-7. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "U0126"
  },
  {
    "caption": "(m) Quantification of the tip cell number in vehicle- (n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=3 litters, 5 Pald1+/+ and 4 Pald1-/- pups). MEK inhibitor/vehicle administered twice at P5 at 2-h intervals, and eyes collected 2 h after the second injection. Each dot is one mouse. Mean±SEM, one-way ANOVA. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "U0126"
  },
  {
    "caption": "(a) Eyes from Pald1+/LacZ mice collected at P17 from untreated animals or animals with oxygen-induced retinopathy (OIR). Pald1-promoter driven LacZ expression and X-gal staining generated signals in capillaries, veins and arteries in the OIR retina at P17 compared to the normoxia control with predominantly arterial LacZ expression. Scale bar: 1 mm.",
    "molecules": "oxygen, X-gal"
  },
  {
    "caption": "(c) Eyes from adult Pald1LacZ/+ mice collected at 72 h after single-bolus intravitreal injection of 1 μg VEGF-A, and PBS in the contralateral eye, followed by X-gal staining. Arrow indicates Pald1 promoter activity in veins (outlined by dashed line) specifically after VEGF-A treatment. A, artery; V, vein. Scale bar: 100 μm.",
    "molecules": "PBS, X-gal"
  },
  {
    "caption": "F PC3-LN4 CRISPR-Ctr and Pim1 KO cells were treated with PIMi (PIM447, 3μM), AKTi (GSK690693, 5μM), or the combination for 24 h. Extracts were subjected to Western blotting with the indicated Abs.",
    "molecules": "GSK690693, PIM447"
  },
  {
    "caption": "J Western blot analysis of proteins extracted from PC3-LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies.",
    "molecules": "AZD5363, DMSO, Pim447"
  },
  {
    "caption": "Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK-p25 animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte.",
    "molecules": "phosphotyrosine"
  },
  {
    "caption": " Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. ",
    "molecules": "phosphotyrosine"
  },
  {
    "caption": " Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. (E) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals. ",
    "molecules": "phosphotyrosine"
  },
  {
    "caption": "(F-G) Relative abundances across all tissues for phosphotyrosine peptides: (F) SVyTEIK (Siglec-F pY561); (G) LyDFVK (Inpp5d pY868). Bars indicate mean ± 95% confidence interval (CI); n = 3-8 samples; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.",
    "molecules": "phosphotyrosine"
  },
  {
    "caption": "(D-E) IF staining of Siglec-F and Iba1 in (D) 5XFAD 2 month hypothalamus (HY) and (E) 5XFAD 6 month dentate gyrus (DG). Yellow = Aβ from methoxy X04 (D) or D54D2 (E). Scale bars = 50 μm.",
    "molecules": "methoxy X04, Aβ"
  },
  {
    "caption": " (F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (G) Percent area coverage of Siglec-F localized to Iba1 compared to total Aβ plaque volume in each field of view across all analyzed images from 5XFAD and non-Tg control mice. Linear regression R2 value is shown for images with ≥10 μm2 total amyloid volume. ",
    "molecules": "Aβ plaque"
  },
  {
    "caption": " (H) Cumulative distribution function (CDF) of the distance between Siglec-F+ and Iba1+ mask voxels and nearest Aβ plaques in 6 month 5XFAD mice. Each value in the CDF represents the mean distance calculated from a single z-stack of a ROI containing ≥ 1 plaque of size ≥10 μm2. n = 55 images (4 animals). ",
    "molecules": "Aβ plaques"
  },
  {
    "caption": "(A) Flow cytometry quantification of BV-2 Siglec-F expression levels. Cells were cotreated with 0-10 ng/mL IFNγ and 0-10 μM tofacitinib. Values are geometric mean fluorescence intensities (gMFI). Bars indicate mean ± 95% CI; n = 2-4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.",
    "molecules": "tofacitinib"
  },
  {
    "caption": " (B) IF staining of iMGLs treated with IFNγ. Siglec-8 fluorescence is only shown on non-nuclear (Hoechst 33342-) regions. Images are 60x super-resolution max z-stack projections. Colors are: Red = Iba1, Green = Siglec-8, Blue = 33342. Scale bars = 20 μm. ",
    "molecules": "33342, Hoechst 33342"
  },
  {
    "caption": "(C) Quantification of Siglec-8 on iMGLs treated with PBS or 25 ng/mL IFNα, IFNβ, or IFNγ. Values are mean Siglec-8 intensity values from Iba1+;33342- regions. Bars indicate mean ± 95% CI; n = 8 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.",
    "molecules": "33342, PBS"
  },
  {
    "caption": "(D) Quantification of Siglec-8 on iMGLs treated ± 25 ng/mL IFNγ and 5 μM tofacitinib. Legend is same as (C). Bars indicate mean ± 95% CI; n = 5-20 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.",
    "molecules": "tofacitinib"
  },
  {
    "caption": "(B) Flow cytometry quantification of cell live / dead markers Annexin-V and propidium iodide on BV-2 cells with stable retrovirus expression of Siglec-F constructs. % parent values are shown for early apoptotic (Annexin-V+;PI‑), late apoptotic (Annexin-V+;PI‑), and necrotic (Annexin-V-;PI+) populations. Bars indicate mean ± SD; n = 8 replicates.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": " (C) Confluency doubling times quantified from BV-2 cells with dox-inducible expression of Siglec-F constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Bars indicate mean ± SD; n = 8 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. ",
    "molecules": "dox"
  },
  {
    "caption": "(F) Expression levels of C1qa, IL-1β, and β-actin by qPCR analysis in BV-2 cells with inducible Siglec-F expression after 48 hours of treatment with 500 ng/mL doxycycline. Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B-C) Volcano plots showing phosphotyrosine peptides that were (B) increased or (C) decreased after Siglec-F expression. Plots show Siglec-F compared to 2xY->F mutant data integrated across all three runs. Protein names are shown for altered peptides. Labels are only shown for peptides with maximum directional change from each protein. Labels colored according to GO terms: magenta = signal transduction, red = endosome, cyan = cell adhesion, yellow = cytoskeleton, green = metabolism.",
    "molecules": "phosphotyrosine"
  },
  {
    "caption": "(G) Western blot quantification of myc, SHP-2, and SHP-1 from myc co-IP eluates. Co-IP lysates were prepared from BV-2 cells stably expressing Siglec-F and optionally treated with 1 μM SHP099.",
    "molecules": "SHP099"
  },
  {
    "caption": "(H) Heatmap showing phosphosites that were perturbed by SHP099 treatment. Color scale is same as (D).",
    "molecules": "SHP099"
  },
  {
    "caption": "(B-C) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec-F expression for (B) Siglec-F and (C) Siglec-F 2xY->F. Siglec-expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates.",
    "molecules": "Aβ"
  },
  {
    "caption": "Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ, Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.",
    "molecules": "Aβ"
  },
  {
    "caption": " Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 10,000 MW Dextran Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. ",
    "molecules": "Dextran"
  },
  {
    "caption": " Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. ",
    "molecules": "FluoSpheres"
  },
  {
    "caption": "(G) pHrodo Dextran uptake in BV-2 cells with stable expression of Siglec-F estimated by Incucyte measurements. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.",
    "molecules": "pHrodo, Dextran"
  },
  {
    "caption": " (H) pHrodo dextran uptake in BV-2 cells stably expressing Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). ",
    "molecules": "pHrodo, dextran, SHP099"
  },
  {
    "caption": " (I) Confluency doubling times for BV-2 cells with stable expression of Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). Bars indicate mean ± 95% CI; n = 4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated 2xY->F group. ",
    "molecules": "SHP099"
  },
  {
    "caption": "D. Representative flow cytometry plots revealing the effects of prostaglandin E2 (PGE2) and EP1-4 antagonists on mouse bone marrow monocyte dendritic cell (DC)/macrophage differentiation (n = 3). E. The frequencies of F4/80+CD11C− macrophages and F4/80−CD11C+ DCs under varying treatments as analyzed by flow cytometry analysis (n = 3). ",
    "molecules": "PGE2, prostaglandin E2"
  },
  {
    "caption": "G. Representative flow cytometry plots revealing the effects of PGE2 and EP1-4 antagonists on mouse bone marrow derived-MDSC differentiation. Upper subpanel: vehicle group and PGE2 group. Lower subpanel: ONO-8711 (EP1 antagonist) group, PF-04418948 (EP2 antagonist) group, L-798106 (EP3 antagonist) group, and E7046 (EP4 antagonist) group. (n = 3). H. The frequencies of Ly6C+Ly6G− mMDSC and Ly6CmidLy6G+ PMN-MDSCs under varied treatments as analyzed by flow cytometry analysis (n = 3). ",
    "molecules": "E7046, L-798106, ONO-8711, PF-04418948, PGE2"
  },
  {
    "caption": "B. Dose-effect curve of TP-16 in GloSensor™ cAMP assay in EP4- expressing 293 cells (n=3).",
    "molecules": "TP-16"
  },
  {
    "caption": "C. Dose-effect curves of TP-16 in PGE2-induced calcium flux assay (n=3).",
    "molecules": "TP-16, PGE2"
  },
  {
    "caption": "D. The Schild plot of PGE2 in the presence of varying concentrations of TP-16. TP-16 shifted the dose-response curve of PGE2-induced intracellular cAMP levels in a dose-dependent manner (n=3). E. The pA2 value and slope of the Schild plot. ",
    "molecules": "TP-16, cAMP, PGE2"
  },
  {
    "caption": "B. The antitumor activities of E7046 (150 mg/kg) and TP-16 (37.5, 75, and150 mg/kg) in CT26 tumor-bearing BALB/c mice (n = 11 per group). Tumor growth curves in the CT26 model (left panel). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.",
    "molecules": "TP-16, E7046"
  },
  {
    "caption": "C. Representative graph showing that the antitumor activity of TP-16 is lost in CT26 tumors engrafted into BALB/c nude mice (left panel) (n = 8). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.",
    "molecules": "TP-16"
  },
  {
    "caption": "D. Single-cell suspensions of tumor tissues from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).",
    "molecules": "TP-16"
  },
  {
    "caption": "F. Single-cell suspensions of tumor tissues from MC38 tumor-bearing C57BL/6 mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).",
    "molecules": "TP-16"
  },
  {
    "caption": "A, B. Tumors from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were harvested, and tumor single-cell suspensions were analyzed for tumor-associated myeloid cells by flow cytometry analysis. Representative graphs and quantification of F4/80+MHC-II+ immunosuppressive myeloid cells (IMCs)-M1 macrophages (A) and F4/80+CD206+ IMCs-M2 macrophages (B) gated on CD45+CD11b+ myeloid cells (n = 5).",
    "molecules": "TP-16"
  },
  {
    "caption": "M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 50 ng/mL mouse recombinant IFN-γ (A) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M1 markers (Cxcl10 and Tnfa) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).",
    "molecules": "TP-16, PGE2"
  },
  {
    "caption": "B. M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 20 ng/mL mouse recombinant IL-4 (B) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M2 markers (Arg-1 and Ym-1) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).",
    "molecules": "TP-16, PGE2"
  },
  {
    "caption": "C. Bone marrow cells were cultured with 40 ng/mL GM-CSF/IL-6 or co-stimulated with PGE2 ± varying concentrations of TP-16 for 6 days. Representative quantification of Ly6C+Ly6G− monocytic myeloid-derived suppressor cells (mMDSCs) and Ly6CmidLy6G+ PMN-MDSCs (n = 3).",
    "molecules": "TP-16, PGE2"
  },
  {
    "caption": "D. Bone marrow cells were treated with GM-CSF/IL-6 for 6 days to induce MDSCs and thereafter, with PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected MDSC markers (Arg-1, Ptgs2, Il-4, and Il-10) were measured by qPCR. Data are normalized to β-actin expression levels (n=3).",
    "molecules": "TP-16, PGE2"
  },
  {
    "caption": "E, F. In vitro T cell suppression activity of CT26-tumor infiltrating CD11b+ myeloid cells collected at 2 weeks post-treatment with vehicle or 75 mg/kg TP-16. CD8+ T cells were labeled with CFSE and then stimulated with CD3/CD28 antibodies for 3 days in the absence or presence of tumor-infiltrating CD11b+ myeloid cells from vehicle- or 75 mg/kg TP-16-treated CT26 tumors. Representative flow cytometry histograms (E) and percentages (F) of proliferating CD8+ T cells when plated in a ratio of 2:1, 1:1, and 1:2 CD8+ T cells to CD11b+ myeloid cells (n = 3).",
    "molecules": "CFSE, TP-16"
  },
  {
    "caption": "A. The growth of CT26 tumor volume when treated with vehicle, TP-16 (75 mg/kg, p.o., daily), anti-PD-1 antibody (50 μg, i.p., twice weekly) or their combination (left) and percent change in tumor volumes between days 0 and 20 (right) (n=12).",
    "molecules": "TP-16"
  },
  {
    "caption": "B. BALB/c mice with subcutaneous CT26 tumors were treated with vehicle, TP-16 (75 mg/kg, p.o., daily), PD-1 antibody (50 μg, i.p., twice weekly) or combined therapies for 2 weeks. Animal survival (time to tumor burden reaching 2000 mm3) was analyzed by the Kaplan-Meier method using GraphPad Prism (n=10). P values were calculated using the Log-rank test.",
    "molecules": "TP-16"
  },
  {
    "caption": "C. Representative immunofluorescence staining images of tumor sections from CT26 tumor-bearing mice stained for CD8 and DAPI. Scale bars, 50 μm. D. Representative immunostaining images of tumor sections from CT26 tumor-bearing mice stained for p-STAT3, p-AKT and PD-L1. Scale bars, 100 μm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Representative hematoxylin and eosin (H&E) staining and immunostaining for CD8, p-STAT3, p-AKT, and ARG-1 of tumors treated with vehicle, TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined. Scale bars, 500 μm (left images), 50 μm (right images).",
    "molecules": "TP-16"
  },
  {
    "caption": "H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by Relative Fluorescence Intensity (RFI, normalized to T0; left) and percentage (right) on UT (n=3), edited (GFP+) or unedited (GFP-) HD or Pt CD4+ T cells from (D) (n=9 HD, 1 Pt). Longitudinal comparisons between HD GFP+ vs GFP- were performed with an LME model, accounting for multiple donors and separately for RFI and %CD40L+ cells (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to 8 hours time-point (****p<0.0001 and *p=0.0450, respectively). #measured on the small fraction of CD40L+ cells. Median ± IQR.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "I Representative plots showing CD40L and GFP expression in UT or bulk edited (Tretated) CD4+ T cells derived from male HD or Pt from (D) at 8 hr after PMA/Ionomycin stimulation.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "K Left: IgG positive spots resulting from B-T cell co-culture. Right: IgG+ secreting B cells, evaluated by ELISPOT assay. B cells were isolated from PB of HD and co-cultured with male HD or Pt sorted GFP+, GFP- and UT T cells, resting (R) or stimulated with beads (B) or PMA/Ionomycin (PI). B cells cultured alone (-) or in presence of sCD40L (+) were used as negative and positive controls, respectively (n=1 for each group).",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "C Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by RFI (normalized to T0) on UT (n=2), edited (NGFR+) or unedited (NGFR-) HD or Pt derived CD4+ T cells (n=4 HD, 1 Pt). Median ± IQR.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "D Representative plots showing CD40L and NGFR expression in UT or bulk edited (Treated) CD4+ T cells derived from male HD or Pt from (C) before and 8 hr after Pma/Ionomycin stimulation.",
    "molecules": "Ionomycin, Pma"
  },
  {
    "caption": "H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleens of mice from (F) (n=1 for each group, except for NGFR- unedited group, n=3). Median ± IQR.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "J Time course of expression of WT-CD40LG mRNA or CO-CD40LG mRNA, measured as fold change (FC) on IPO8 housekeeping gene and normalized to T0. Sorted edited (+) and sorted unedited (-) CD4+ T cells were analyzed before and 3, 6, 12 hr after Actinomycin D treatment (n=1 for each group).",
    "molecules": "Actinomycin D"
  },
  {
    "caption": "B Representative plots showing hEGFRt expression in bulk edited CD4+ T cells derived from male HD before and 8 hr after Pma/Ionomycin stimulation. Cells were edited with the three constructs depicted in (A).",
    "molecules": "Ionomycin, Pma"
  },
  {
    "caption": "H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleen of mice from (D) (n=4 NGFR+, 4 NGFR-). Mean ± SEM.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "B Total counts of recipient CD3+ cells gated within CD45.2+ cells in PB of experimental mice (n=4 CPA 300, 9 NC). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 1 (****p<0.0001). Mean ± SEM.",
    "molecules": "CPA"
  },
  {
    "caption": "D, E Percentage of donor CD4+ and CD8+ cells gated within CD3+CD45.1.2+ cells in PB of CPA treated (D) or NC mice (E) from (B). Mean ± SEM.",
    "molecules": "CPA"
  },
  {
    "caption": "F TNP-KLH specific IgG in sera of transplanted mice collected at the times indicated in (A) (n=7 CPA 300, 13 NC for first boost and 4 CPA 300, 9 NC for second boost). Sera from vaccinated WT (n=5) and CD40lg-/- (n=5) mice were used as positive and negative controls, respectively. Two independent experiments. Comparisons of first boost data were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.",
    "molecules": "TNP, CPA"
  },
  {
    "caption": "G Average area of PNA+ foci in splenic sections of experimental mice from (B), calculated as ratio between total PNA+ area and number of PNA+ foci/cells. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test (n=9 CD40LG, 6 WT, 18 NC, 8 CPA300). P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0149, **p=0.0065 and ****p<0.0001). Mean ± SEM.",
    "molecules": "CPA"
  },
  {
    "caption": "H Detection of splenic TNP-KLH specific IgM (left) and IgG (right) -secreting cells by ELISPOT assay (n=7 NC, 4 CPA300, 3 WT, 2 CD40LG). Spots were counted by an ELISPOT Reader using a size range of 0.005-1 mm. Median ± IQR.",
    "molecules": "TNP, CPA"
  },
  {
    "caption": "B Total counts of recipient CD3+ CD45.2+ cells in PB of mice NC (n=13) or pre-conditioned with 300 mg/kg CPA (n=19), 200 mg/kg CPA (n=6), anti-lymphocyte serum (ALS; n=5) or anti-CD4 antibody (n=6). Three independent experiments. Comparisons at day 3\4 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0009 and ****p<0.0001 in all comparisons). Mean ± SEM.",
    "molecules": "CPA"
  },
  {
    "caption": "C Total counts of engrafted donor CD45.1+ cells in PB of mice from (B). Three independent experiment. Comparisons at day 18\24 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. Mean ± SEM.",
    "molecules": "CPA"
  },
  {
    "caption": "E TNP-KLH specific IgG concentration in sera of mice from (B) collected before (day 35; pre) and after (day 49; post) the first boost (n=13 NC, 19 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4; three independent experiments) and at day 81 (pre) and 89 (post) for the second boost (n=4 NC, 4 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4). Sera from vaccinated WT (n=5) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. For early challenge data, comparisons were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. WT group, not indicated in the figure, is significantly different from all groups (p<0.0001 in all comparisons). Of note, regarding the comparisons between time-points, the groups CPA 300 and WT show a significant increase between pre and post values (p<0.0001 for both), while all other groups show a significant decrease between pre and post values (p<0.0001 for all except p=0.0022 for ANTI-CD4). Median ± IQR.",
    "molecules": "TNP, CPA"
  },
  {
    "caption": "F Total counts of recipient CD3+ cells gated within CD45.2+ cells after transfer of primed donor T cells in PB of mice treated with 300 mg/kg CPA (n=11), 200 mg/kg CPA (n=11) or NC (n=6). Longitudinal comparisons were performed by LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 4 (****p<0.0001 in all comparisons). Mean ± SEM.",
    "molecules": "CPA"
  },
  {
    "caption": "G Total counts of engrafted donor CD45.1+ cells in PB of mice from (F). Longitudinal comparisons were performed with an LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 25. At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 (****p<0.0001 for both). Mean ± SEM.",
    "molecules": "CPA"
  },
  {
    "caption": "H TNP-KLH specific IgG concentration in sera of mice from (F) collected before (day 36, pre) and after (day 50, post) the first boost (n=6 NC, 11 CPA 300, 11 CPA 200) and at day 212 (pre) and 219 (post) for the second boost (n=6 NC, 11 CPA 300, 9 CPA 200). Sera from vaccinated WT (n=11) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. Comparisons were performed with an LME model followed by an appropriate post-hoc analysis, separately for each challenge data (Appendix Supplementary Statistical Methods). Reported statistical comparisons refer to the overall difference among groups, for early challenge data (*p=0.0282 and **p=0.0037), while to differences between time-points within each group, for late challenge data (**p=0.0018 and ****p<0.0001 in both comparisons). Of note, in the late challenge data, no significant overall differences were observed among the groups. Median ± IQR.",
    "molecules": "TNP, CPA"
  },
  {
    "caption": "C TNP-KLH specific IgG concentration in sera of mice from (A) collected seven days before (pre) and after (post) TNP-KLH vaccination (n=13 100% WT, 13 25% WT, 14 10% WT, 6 1% WT, 14 0% WT). Three independent experiments. Comparisons were performed with an LME model, accounting for multiple experiments, followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.",
    "molecules": "TNP"
  },
  {
    "caption": "E Quantitation of P. murina rRNA in lung homogenate of mice from (A) transplanted with different ratios of WT HSPC cells (n=8 100% WT, 7 25% WT, 7 10% WT, 8 0% WT) and infected with the pathogen. Results are expressed in P.murina/HPRT RNA copies. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (**p=0.0028). Median ± IQR.",
    "molecules": "rRNA"
  },
  {
    "caption": "H Quantitation of P. Murina rRNA in lung homogenate of mice adoptively transferred (n=9 CD40LG + T cells) or not (n=8 CD40LG) with in vivo primed CD4+ T cells in absence of conditioning and infected with the pathogen. Results are expressed in P.Murina/HPRT RNA copies. Mann-Whitney test. ***P-value of the comparison = 0.0003. Median ± IQR.",
    "molecules": "rRNA"
  },
  {
    "caption": "(B) Reporter gene expression in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA)) of netrin-1+/lacZ+ adult (P60) mice incubated with S-Gal. (Scale bar, 2000 µm). Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel.",
    "molecules": "S-Gal"
  },
  {
    "caption": "(A) Representative images of dopamine neurons (TH-positive) in the substantia nigra (SN) (left panel) and in the SN and striatum (right panel top, and bottom respectively) by immunofluorescence, six weeks after the intranigral injection of control (CTL) (AAV6-GFP) or Cre (AAV6-GFP Cre) adenovirus vectors in Netrin-1fl/fl mice (left panel: green-GFP, red-TH; right panel: Cy-5-TH) (Scale bar, 1000 μm). Filled arrows indicate SN TH dopamine neurons.",
    "molecules": "Cy-5"
  },
  {
    "caption": "(C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01.",
    "molecules": "Cy5"
  },
  {
    "caption": "(B) Amphetamine-induced rotations at two and six weeks after lesion. Individual values and Mean ± SD are shown, n=11 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05 compared to WT.",
    "molecules": "Amphetamine"
  },
  {
    "caption": "(B) Cumulative amphetamine-induced ipsilateral rotations. Progression of amphetamine-induced ipsilateral rotations over time (right panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, **** P <0.0001 compared to the initial rotation score at week 2. Comparison of the treatment's effect between groups (left panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group.",
    "molecules": "amphetamine"
  },
  {
    "caption": "(C) Dopamine fibres in the striatum of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the striatum showing TH IHC staining (left panel), intact side on the left and 6-OHDA lesioned side on the right. (Scale bar, 2000 µm). Optical density measurement of TH-positive fibres in the lesioned side of the striatum (bar graph, right panel). Individual values and mean ± SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom",
    "molecules": "Dopamine, 6-OHDA"
  },
  {
    "caption": "(D) Dopamine cell bodies in the SN of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the SN (lesioned side) showing TH-positive cell bodies stained by IHC (left panel) (Scale bar, 2000 µm). TH-positive cell count in the SN (right panel). Individual values and mean + SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.",
    "molecules": "Dopamine, 6-OHDA"
  },
  {
    "caption": " Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test ",
    "molecules": "LPS"
  },
  {
    "caption": "(B, Sequential dissociation of EI24 and BiP from IRE1 elicits a two-phase activation of IRE1 in response to ER  stress. Cells were treated with 4 μM tunicamycin (TM) for the indicated times. The  interactions of  EI24  and BiP with  IRE1 were analyzed by immunoprecipitation",
    "molecules": "TM, tunicamycin"
  },
  {
    "caption": "(D)  EI24  knockout promotes the activation of IRE1, but not  PERK or ATF6. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were treated with 4 μM  TM for the indicated  times. Protein expression  was analyzed by immunoblotting.",
    "molecules": "TM"
  },
  {
    "caption": "(G) Expression of  Flag-EI24 in EI24 knockout cells inhibits IRE1 activity. Flag-Metap2 served as the control. Cells transfected with the indicated cDNAs were treated with or without 4 μM TM for 2 hr.  Protein expression  was analyzed by  immunoblotting.",
    "molecules": "TM"
  },
  {
    "caption": "(B)  EI24 persistently  localizes on the ER regardless of the presence or absence of ER stress. HeLa cells stably expressing  Flag-EI24 were treated with or without 4 μM  tunicamycin  (TM) for 2 hr. Colocalization  of EI24 with ER was analyzed. KDEL-mCherry, ER marker. Scale bar, 10 μm.",
    "molecules": "KDEL, TM, tunicamycin"
  },
  {
    "caption": "(B, C)  EI24 knockout causes resting-state  ER calcium  reduction (B) and cytosolic calcium elevation (C) under ER  stress. Control (sgGFP) and EI24 knockout (sgEI24)  HeLa cells were first perfused in  TM-free buffer, and switched to the buffer containing 4 μM  TM to induce ER stress where indicated. Resting state calcium levels in the  ER (B) and cytosol (C) with or without  TM are displayed in columns. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.",
    "molecules": "calcium, TM"
  },
  {
    "caption": "(G) Knockdown of IP3R1 prevents  calcium mobilization  from the  ER to the  cytosol. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.",
    "molecules": "calcium"
  },
  {
    "caption": "(I) Wild-type  EI24, but not the TM34- or C-terminus-deleted mutant, preserves  ER calcium  stores in EI24 knockout cells. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.",
    "molecules": "calcium"
  },
  {
    "caption": "(B) Knockdown of IP3R1 prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by  flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test",
    "molecules": "TM"
  },
  {
    "caption": "(D) Knockdown of  IP3R1 prevents ER stress-induced apoptosis. Cells were treated with 4 μM TM  for the indicated times. Protein expressions were analyzed by  immunoblotting.",
    "molecules": "TM"
  },
  {
    "caption": "(F-H) Reintroduction of full-length EI24, but not the TM34- or C-terminus-deleted mutant (F, G) or the C-terminus alone (H), inhibited ER stress-induced apoptosis in EI24  knockout cells. Cells treated with or without  TM were analyzed for apoptosis  by  flow cytometry (F, H) or by  immunoblotting (G). Arrows in (H) denote decreased or increased apoptotic levels. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test for F), one-tailed Student's t-test for (H).",
    "molecules": "TM"
  },
  {
    "caption": "(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). miR-122 signals were detected by Northern blotting and position of the 32P-labelled Oligos that served as size markers are shown in the lane (A). U6 snRNA was used as loading control.",
    "molecules": "amino acids"
  },
  {
    "caption": "(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). Cellular CAT-1 levels were measured by qRT-PCR using GAPDH mRNA as control (B). A scheme of experiment is shown in the top panel in B.",
    "molecules": "amino acids"
  },
  {
    "caption": "(E) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real-time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869 treated cells.",
    "molecules": "DMSO, GW4869"
  },
  {
    "caption": "(F) CAT-1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells. qRT-PCR techniques was adopted for quantification using 18S rRNA values for normalization (mean +/- s.e.m., n=3).",
    "molecules": "DMSO, GW4869"
  },
  {
    "caption": "(L) Effect of thapsigargain (TG) treatment of Huh7 cells on cellular and EV associated miR-122 level. A schematic representation of the experiment (upper panel). miR-122 levels were measured by qRT-PCR in total cellular RNA and in EVs released by the treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean+/- s.e.m., n=3) (lower panels) for cellular and EV associated RNA respectively.",
    "molecules": "TG, thapsigargain"
  },
  {
    "caption": "(M) Effect of TG on cellular level of eIF2-a and its phosphorylated form, and Ago2 and CD63 in EVs. b-Actin was used as loading control for cellular samples.",
    "molecules": "TG"
  },
  {
    "caption": "(G) Effect of GW4869 on EV associated content of three different miRNAs in HA-HuR expressing Huh7 cells quantified by qRT-PCR (mean+/- s.e.m., n=5). EVs from DMSO treated cells were used as control.",
    "molecules": "DMSO, GW4869"
  },
  {
    "caption": "(H) Increase in ubiquitinated form of HuR in Starved Huh7 cells. Levels of ubiquitinated HuR in Fed and Starved (5h) HA-Ub expressing Huh7 cells treated either with ethanol or MG132 (20µM) for same duration. The lysates were immunoprecipitated with HuR and western blotted for HA and HuR. Position of ubiquitinated bands were marked by arrows. The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non-ubiquitinated form were measured and plotted (mean+/- s.e.m., n=3).",
    "molecules": "ethanol, MG132"
  },
  {
    "caption": "(G and H) HeLa cells were synchronized to G1 or S phase, then the degradation rates of endogenous (G) and exogenous (H) BRCA1 were analyzed by western blot. The quantification of BRCA1 relative levels according to GAPDH level are labeled below the BRCA1 band. CHX, Cycloheximide that inhibits nascent protein production. SFB-BRCA1, exogenous BRCA1 with flag tag in N-terminal.",
    "molecules": "CHX, Cycloheximide"
  },
  {
    "caption": "(A and B) Western blot analysis of RBM10 phosphorylation in the indicated cell phases (A) or G1/S phase (B). In (B), cells were treated with the indicated CDK inhibitors (10 μM) for 6 h. CDK1i, Cucurbitacin E. CDK2i, SB1317. CDK4/6i, Palbociclib.",
    "molecules": "Cucurbitacin E, Palbociclib, SB1317"
  },
  {
    "caption": "γH2AX levels in RBM10-KD #4 cells under the treatments as indicated. Representative images were shown in (A). CPT, camptothecin. BLM, bleomycin. IR, ionizing radiation.",
    "molecules": "bleomycin, BLM, camptothecin, CPT"
  },
  {
    "caption": "(H and I) HeLa cells were transfected with RBM10 or/with 53BP1 siRNA. Immunofluorescence analysis of γH2AX after CPT (H) and cisplatin (I) treatment.",
    "molecules": "CPT, cisplatin"
  },
  {
    "caption": "Colony formation assay (n = 3) for the determination of the viability of SK-OV-3 (F), HeLa (G) cells treated with a combination of SB1317 and Olaparib. SB1317, CDK2 inhibitor.",
    "molecules": "Olaparib, SB1317"
  },
  {
    "caption": "WB analysis (G) of MUC5AC in HT29 cells transduced with sh-RNAs against trypsin (T1, T2) or chymotrypsin (C1, C2). Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed",
    "molecules": "chymotrypsin, trypsin"
  },
  {
    "caption": "(A) m6A dot blot assay using total RNA from NK92-siCTRL or NK92-siFTO cells.",
    "molecules": "m6A"
  },
  {
    "caption": "(C) m6A methylated RNA immunoprecipitation & quantitative PCR (MeRIP-qPCR) analysis using a m6A antibody in NK92-siFTO (upper) or NK92+FTO (lower) cells compared to NK92-siCTRL or NK92+MOCK cells.",
    "molecules": "m6A"
  },
  {
    "caption": "(a-c) At 12-16 h post-transfection with GFP-LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2-4 h (a) or with 2 μM rapamycin for 1-3 h (b, c). Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 10 μm (a). The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01; **P 0.05) (b, c). At 60 h post-transfection with control siRNA or cFLIP siRNA, MEF cells were transfected with GFP-LC3 (c).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(e, f) NIH3T3 vector and NIH3T3-KSHV-vFLIP cells (e) or TREX-BCBL-Vector and TREX-BCBL-vFLIP cells (f) were treated with Hank's solution or 2 μM rapamycin for 2-4 h, respectively, followed by IB with anti-LC3 and anti-actin antibody.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(g) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL-vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 2 μM rapamycin for an additional 12 h. GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 5μm. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01).",
    "molecules": "doxycycline, rapamycin"
  },
  {
    "caption": "(b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a-g).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a) TREX-BCBL vector cells were treated or untreated with 50 nM rapamycin for 6 days in the presence of doxycycline and subjected to scanning electron microscopy. The morphologies of more than 100 dead cells were examined to quantify the levels of apoptosis, autophagic death, apoptosis/autophagic death, and others forms of cell death.",
    "molecules": "doxycycline, rapamycin"
  },
  {
    "caption": "(b) At 12-16 h post-transfection with GFP-LC3, doxycycline-treated TREX-BCBL vector, TREX-BCBL-vFLIP and TREX-BCBL-vFLIP mutant cells were incubated with or without 50 nM rapamycin for 6 days (top) or 2 μM rapamycin for 12 h (bottom). The percentage of cell death (trypan blue staining) and apoptosis level (annexin V staining) of rapamycin-treated cells was quantified as mean ± s.d. of three independent experiments. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P<0.01.",
    "molecules": "doxycycline, rapamycin"
  },
  {
    "caption": "(c) BCBL1 cells were transfected with control siRNA or Beclin1 siRNA and treated with or without rapamycin for 5 days. The percentage of cell death was determined by trypan blue staining. Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.05.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(d) TREX-BCBL vector and TREX-BCBL-vFLIP cells were mock-treated or treated with 50 nM rapamycin for the indicated periods of time. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.001.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(e) HEK293 cells carrying KSHV or KSHVΔvFLIP were treated with 500 nM rapamycin for 6 days. The results were quantified as mean ± s.d. of the combined results from three independent experiments; *P 0.01. Trypan blue staining and a Beckman Coulter Z2 Particle Count and size analyzer (BC Z2 CS analyser) were used to determine cell death and cell growth analysis (b-e).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h. Trypan blue staining was then used to determine cell death (as a percentage). Data are mean ± s.e.m.; n = 200-300; three independent experiments; *P 0.01; **P 0.05.",
    "molecules": "doxycycline, rapamycin"
  },
  {
    "caption": "(c) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 30 μM of the TAT, K-α2 or K-α4 peptide (top left) or the TAT, C-α2, or C-α4 peptide (bottom left) for an additional 12 h. Subsequently, autophagy was quantified as means ± s.d. of the combined results from three independent experiments (2 μM rapamycin treatment was included as a control). TREX-BCBL vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 0, 30, or 50 μM of the TAT, K-α2, or K-α4 peptide (top right) or the TAT, C-α2, or C-α4 peptide (bottom right) for an additional 12 h and GFP-LC3 puncta were subsequently detected using an inverted fluorescence microscope. Scale bar, 5 μm.",
    "molecules": "doxycycline, rapamycin"
  },
  {
    "caption": "(A-B) Mitochondria isolated from (A) hTim8aKO HEK cells with and without hTim8aFLAG re-expression, or (B) hTim8bKO HEK cells with and without hTim8bFLAG re-expression were compared to mitochondria isolated from control HEK293 cells by SDS-PAGE and immunoblot. Protein expression of the hTim8 protein was induced with tetracycline (Tet) for the indicated time. Representative of n=2 biological replicates.",
    "molecules": "Tet, tetracycline"
  },
  {
    "caption": "Affinity enrichment mass spectrometry. Mitochondria isolated from (C) hTim8aKO HEK cells expressing hTim8aFLAG, (D) hTim8bKO HEK cells expressing hTim8bFLAG, (E) hTim8aMUT SH cells expressing hTim8aFLAG, (F) hTim8bKO SH cells expressing hTim8bFLAG were treated with DSP crosslinker prior to immunoprecipitation with anti-FLAG resin. The log2 fold change in mean LFQ intensity is plotted against unpaired Student's t-test p-value (n=3 biological replicates). Curve indicates significantly enriched proteins. FDR<0.01; (C) s0=5.5, (D) s0=4.5, (E) s0=2, (F) s0=5, Functional annotations manually curated and coloured: TIMM family proteins (purple), Complex I subunits and assembly factors (orange), Complex IV subunits and assembly factors (green), MIA import substrates (blue), significantly enriched (black).",
    "molecules": "DSP"
  },
  {
    "caption": "(F) Intact cells or mitochondria isolated from hTim8bKO HEK and control HEK293 cells were subject to ICP-MS analysis to measure copper (Cu) and iron (Fe) content. Abundance is shown as mean ±S.D for metal content per gram of protein. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.01; 'ns' indicates not significant, p>0.05.",
    "molecules": "copper, Cu, Fe, iron"
  },
  {
    "caption": "(G) Recombinant COA6, hTim8a and hTim8b were purified and incubated, in increasing concentrations, with Cu(I) and bathocuproine disulfonic acid (BCS). Protein-Cu(I) binding outcompetes BCS2-Cu(I) binding and the decrease in BCS2-Cu(I) is shown by absorbance, particularly at 482 nm. n=1 biological replicates.",
    "molecules": "bathocuproine disulfonic acid, BCS, Cu(I)"
  },
  {
    "caption": "(A) hTim8aKO HEK, hTim8bKO HEK and control HEK293 cells were pulsed with [35S]-Met/Cys for 2 h in the presence of anisomycin and chased for the indicated times. Isolated mitochondria were analysed by SDS-PAGE, auto-radiography and immunoblot. n=1 biological replicates.",
    "molecules": "anisomycin, Cys, Met, 35S"
  },
  {
    "caption": "[35S]-COX6C and [35S]-COX7C were incubated with mitochondria isolated from (B) hTim8aMUT SH control SH-SY5Y Import proceeded in the presence or absence of mitochondrial membrane potential (ΔΨ) for the indicated times. Samples were analysed by BN-PAGE and autoradiography. CBB = Coomassie brilliant blue staining. Representative of n=2 biological replicates.",
    "molecules": "CBB, 35S"
  },
  {
    "caption": "[35S]-COX6C and [35S]-COX7C were incubated with mitochondria isolated from (C) hTim8bKO HEK and control HEK23 cells. Import proceeded in the presence or absence of mitochondrial membrane potential (ΔΨ) for the indicated times. Samples were analysed by BN-PAGE and autoradiography. CBB = Coomassie brilliant blue staining. Representative of n=2 biological replicates.",
    "molecules": "CBB, 35S"
  },
  {
    "caption": "(D) Mitochondria isolated from hTim8bKO HEK cells with and without hTim8aFLAG expression compared to mitochondria isolated from control HEK293 cells by BN-PAGE. hTim8aFLAG expression induced by tetracycline for 8 h. Native protein complexes detected by immunoblot. COX4I1-containing complexes by immunoblot indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Amount of COX4I1 present in mature monomeric Complex IV (CIV) was quantified as mean ±S.D percentage of control HEK293 mitochondria. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.01; 'ns' indicates not significant, p>0.05.",
    "molecules": "tetracycline"
  },
  {
    "caption": "A Immunofluorescence staining   of KANK1  (red) and  α-tubulin (blue) in Strep-GFP-CEP170B  knock-in HeLa  cells. Lower panels show enlargements of the peripheral (1) and central (2) cortical  regions boxed in the upper panels. Data information: Scale bars, 5 μm.",
    "molecules": "Strep"
  },
  {
    "caption": "C Immunofluorescence staining  of KANK1  in Strep-GFP-CEP170B  knock-in HeLa  cells treated with nocodazole. Lower panels show enlargements of boxed areas in the upper panels. D Immunofluorescence staining  of CEP170B  and α-tubulin in MCF7  cells. Lower panels show enlargements of boxed areas in the upper panels. Data information: Scale bars, 5 μm.",
    "molecules": "nocodazole, Strep"
  },
  {
    "caption": "G Immunofluorescence staining  of KANK1, KANK2, or  liprin-α1  in control Strep-GFP-CEP170B  knock-in HeLa  cell line or indicated knockout (KO) cell lines that were generated based on the parental Strep-GFP-CEP170B  knock-in HeLa  cell line. Data information: Scale bars, 5 μ",
    "molecules": "Strep"
  },
  {
    "caption": "B Streptavidin pull-down assay with extracts of HEK293T  cells expressing Bio-GFP-tagged  liprin-α1 full length (FL) or its indicated fragments, analyzed by Western blotting with indicated antibodies. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin.",
    "molecules": "Bio, Streptavidin"
  },
  {
    "caption": "D Streptavidin pull down  assay with extracts of HEK293T  cells expressing Bio-GFP-tagged wild-type (WT) liprin-α1 fragment N1 or its NR/AA mutant, analyzed by  Western blotting with indicated antibodies. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin. E Streptavidin pull down  assay with extracts of HEK293T  cells expressing Bio-GFP-tagged WT liprin-α1 N1 fragment or its NR/AA mutant (bait) together with GFP-CEP170B   (prey), analyzed by Western blotting with GFP  antibody.",
    "molecules": "Bio, Streptavidin"
  },
  {
    "caption": "J, K Immunofluorescence staining  of α-tubulin  (blue) in  liprin-α1 KO/Strep-GFP-CEP170B  knock-in double-engineered HeLa  cell line transiently transfected with mCherry-tagged liprin-α1 FL (J), fragment N1 (K), or their NR/AA mutant counterparts (J, K). Data information: Scale bars, 5 μm.",
    "molecules": "Strep"
  },
  {
    "caption": "A TIRF microscopy time-lapse images on the left show the accumulation of CEP170B  along the distal segments of MT  lattices in the cortical  region of Strep-GFP-CEP170B  knock-in HeLa  cell line transiently transfected with EB3-TagRFP. Representative kymographs   on the right show an MT  plus end undergoing very short alternating growth and shrinking episodes (corresponding to the one marked as \"1\" in the images; upper right panel) and an MT  plus end undergoing longer depolymerization followed by rescues (corresponding to the one marked as \"2\" in the images; bottom right panel ). Scale bars: horizontal, 2 μm; vertical, 30 s. Data information: Scale bars, 5 μm.",
    "molecules": "Strep"
  },
  {
    "caption": "G Caco-2  cells were cultured in Matrigel  for 5 days, and subsequently treated with  Cholera toxin  for 12 hours. Phase-contrast images of thus  formed control or CEP170B  KO Caco-2 cysts  were shown. H, I Representative images and quantification of four categories of lumen  formation for experiments shown in (G). n = 100 cysts  from three technical replicates. J Quantification of the diameter of control and CEP170B  KO Caco-2 cysts. n = 30 cysts  from three technical replicates. Data information: Data represents the mean ± SD. ***, P < 0.001; two-tailed t test. Scale bars: 25 μm",
    "molecules": "Cholera toxin"
  },
  {
    "caption": "A Images and corresponding kymographs  showing that KIF2A-SNAP  labelled with  Alexa Fluor 647  (shown here as green) tracks growing MT minus ends  at 0.5-2.5 nM but induces depolymerization from minus-end  at 5 nM. Quantification of MT minus-end (B) or plus-end (C)  catastrophe frequency  in the presence of KIF2A-SNAP  at indicated concentrations. N.A., not available. n = 20-39 MTs  from 2-3 independent experiments. Data information: Data represent mean ± SD. Scale bars: horizontal, 2 μm; vertical, 1 min.",
    "molecules": "Alexa Fluor 647"
  },
  {
    "caption": "Treatment of HeLa cells with DFP (1mM for 24 h) reduces the conjugation of SUMO-2/3 (n=5, biological replicates; **, p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change global SUMO-1-ylation (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).  ",
    "molecules": "DFP"
  },
  {
    "caption": "Treatment of HeLa cells with DFP (1mM for 24 h) leads to increased levels of SENP3 (n=8, biological replicates; **p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change levels of SENP5 (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).  ",
    "molecules": "DFP"
  },
  {
    "caption": "DFP treatment increases SENP3 stability. DFP (1mM for 24 h)-treated HeLa cells were treated with cycloheximide (CHX; 50μg/ml) for the indicated durations in a chase experiment. Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated.",
    "molecules": "CHX, cycloheximide, DFP"
  },
  {
    "caption": "Treatment of HeLa cells with DFP (1mM for 24 h) decreases levels of CHIP (n=8, biological replicates; ***p<0.001; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of CHIP mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).  ",
    "molecules": "DFP"
  },
  {
    "caption": "Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP. Histogram shows relative levels of cellular CHIP upon four different conditions (n=3, biological replicates; ****p<0.0001; *** p<0.001; repeated measures analysis of variance followed by Tukey's multiple comparisons test).",
    "molecules": "DFP, MG132"
  },
  {
    "caption": "CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells. Nsi or CHIPi (Nsi, non-specific siRNA; CHIPi, CHIP siRNA; concentration, 20 nM) was transfected into HeLa cells for 48 h. Lysate samples were blotted as indicated.   CHIP knockdown prevents the DFP-induced increase in SENP3 levels. Nsi, SENP3i or CHIPi (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; CHIPi, CHIP siRNA; siRNA concentration, 20nM) was transfected into HeLa cells for 48 h. Two days post-transfection the cells were treated with DFP for a further 24 h.  ",
    "molecules": "DFP"
  },
  {
    "caption": "SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).",
    "molecules": "DFP"
  },
  {
    "caption": "Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test).",
    "molecules": "CQ, DFP"
  },
  {
    "caption": "SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).",
    "molecules": "pHfluorin, SEP, DFP"
  },
  {
    "caption": "Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test).",
    "molecules": "DFP"
  },
  {
    "caption": "Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test).",
    "molecules": "DFP"
  },
  {
    "caption": "Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).",
    "molecules": "pHfluorin, SEP, DFP"
  },
  {
    "caption": "Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated.",
    "molecules": "DFP"
  },
  {
    "caption": "SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).",
    "molecules": "Cyan, SEP, DFP"
  },
  {
    "caption": "D: Representative images results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 100 μm. Arrows indicate polar body-1 (PB1)",
    "molecules": "hCG"
  },
  {
    "caption": "E: Confocal microscopy results of oocytes collected from oviducts of WT and Cnot6l-/- mice at 16 h after hCG injection. scale bar, 20 μm. Arrows indicate spindle poles",
    "molecules": "hCG"
  },
  {
    "caption": "G: Quantification of preimplantation embryos derived from WT and Cnot6l-/- females that develop to the indicated stages after hCG administration and mated with adult WT males. The numbers of analyzed embryos are indicated (n)",
    "molecules": "hCG"
  },
  {
    "caption": "H: Representative images of embryos collected from the oviducts or uteri of WT and Cnot6l-/- female at indicated time points after hCG administration. Scale bar, 100 μm. Data information: Error bars, s.e.m. **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-signif",
    "molecules": "hCG"
  },
  {
    "caption": "A: Rates of germinal vesicle breakdown (GVBD) and PB1 emission in oocytes cultured in vitro. Fully-grown GV oocytes were collected from PMSG-primed (44 h) WT and Cnot6l-/- mice. PB1: polar body-1. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)",
    "molecules": "PMSG"
  },
  {
    "caption": "G: Pericentrin immunofluorescence showing MTOCs in cultured WT, Cnot6l-/- and Btg4-/- oocytes at MI and MII stages. Spindle and DNA were labeled by microtubule nucleation factor (TPX2) and DAPI, respectively. Scale bar, 20 μm",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "C: SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l-/- oocytes at 16 h after culture. Centromeres and DNA were labeled by CREST and DAPI, respectively. Scale bar, 5 μm",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "E: PB1 emission rates in WT and Cnot6l-/- oocytes cultured with or without MPS1 inhibitor reversin (5 μm). Reversin is added at 9 hours after culture. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)",
    "molecules": "reversin, Reversin"
  },
  {
    "caption": "Immunofluorescenc of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001",
    "molecules": "HPG, L-homopropargylglycine"
  },
  {
    "caption": "quantification (D) of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001",
    "molecules": "HPG, L-homopropargylglycine"
  },
  {
    "caption": "fluorescence microscopy results (B) showing the translation activities of the Cnot6l 3\"-UTR (WT and CPE mutated) in GV- (maintained by 2 μM milrinone) or MII- (released from milrinone) arrested oocytes. GFP signal indicated translational activation of Cnot6l 3\"-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive control. Scale b",
    "molecules": "milrinone"
  },
  {
    "caption": "fluorescence microscopy results (E) showing the expression of GFP-fused Cnot6l 3"-UTR in GV and MII oocytes with or without U0126 treatment (20 μM). Scale bar, 1",
    "molecules": "U0126"
  },
  {
    "caption": "e expression of GFP-fused Cnot6l 3\"-UTR in GV and MII oocytes with or without U01 F: Relative intensity of GFP signa after normalization by mCherry signal in the same oocyte The numbers of analyzed oocytes were indicated (n)",
    "molecules": "U01"
  },
  {
    "caption": "G: Rates of PB2 emission and normal spindle assembly in oocytes cultured with or without U0126. Fully grown GV oocytes were microinjected with mRNAs encoding CNOT6L, CNOT7 and/or BTG4 and are released from milrinone at 12 h after microinjection. Then the oocytes were further culture for 24 h with or without adding U0126 to the medium. Error bars, s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student"s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicate",
    "molecules": "milrinone, U0126"
  },
  {
    "caption": "C Percentage of pollen tubes with normal reception at the female gametophyte (black bars; representative image middle centre of figure) and with overgrowth (grey bars; representative image lower centre) as assessed by aniline blue staining. 15 self-pollinated stage 16 flowers from wild-type, herk1, anj and herk1 anj were analysed. Legend scale bars = 50 µm. *** p<0.001 (χ-square tests).",
    "molecules": "aniline blue"
  },
  {
    "caption": "D Aniline blue staining of pollen tube reception in reciprocal crosses between wild-type and herk1 anj plants with at least two siliques per cross. Legend as per (C). *** p<0.001 (χ-square tests).",
    "molecules": "Aniline blue"
  },
  {
    "caption": "B Localisation of NTA in the synergid cell of wild-type and herk1 anj plants before (upper panels) and after (lower panels) pollen tube arrival. In green, NTA localisation as shown by pMYB98::NTA-GFP fluorescence. In magenta, callose of the filiform apparatus and pollen tube stained with SR2200. From left to right, images shown are DIC, merged fluorescence images, and merged images of DIC and fluorescence. White and red dotted lines delineate the pollen tube and synergid cells, respectively. Scale bars = 25 µm. M, micropyle.",
    "molecules": "SR2200"
  },
  {
    "caption": "B Quantification of H2DCF-DA staining of ROS in ovules from wild-type, herk1 anj, lre-5 and fer-4 plants at 20 HAE. Categories are listed in the legend Ovules dissected from at least five siliques per line. *** p<0.001 (χ-square tests).",
    "molecules": "H2DCF-DA, ROS"
  },
  {
    "caption": "B Y2H assays with the extracellular domains of HERK1, ANJ and FER (HERK1-ECD, ANJ-ECD and FER-ECD, respectively). Ø represents negative controls where no sequence was cloned into the activating domain (AD) or DNA-binding domain (BD) constructs. -L-W-H, growth medium depleted of leucine (-L), tryptophan (-W) and histidine (-H).",
    "molecules": "histidine, leucine, tryptophan"
  },
  {
    "caption": "D Co-immunoprecipitation of FER with HERK1-GFP in Arabidopsis seedlings expressing pFER::HERK1-GFP. Numbers indicate molecular weight marker sizes in kDa. Assays were performed twice with similar results. CBB refers to Coomassie Brilliant Blue staining of total proteins.",
    "molecules": "CBB, Coomassie Brilliant Blue"
  },
  {
    "caption": "A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA1 antibody in H1299 cells. Pulled-down protein complexes were analyzed. Benzonase (B) was added in cell lysate. B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA2 antibody in H1299 cells. Pulled-down protein complexes were analyzed.",
    "molecules": "Benzonase"
  },
  {
    "caption": "G HEK293T cell lysates were incubated with GST or GST-HMGA1 sepharose beads. Pulled-down protein complexes were analyzed.",
    "molecules": "sepharose"
  },
  {
    "caption": "J HEK293T cell lysates were incubated with GST or GST-HMGA1 deletion mutants sepharose beads. Recombinant proteins from pull-down assays visualized by silver staining and western blot.",
    "molecules": "sepharose, silver"
  },
  {
    "caption": "A The in vivo therapy scheme. Eight-day-old mice were injected intraperitoneally with AAV9-mCherry-anti-miR-59 (5x1011 viral particles). The mCherry signals were detected 30 days post-injection (DPI) in LmnaG609G/G609G mice versus PBS-injected control (dorsal).",
    "molecules": "PBS"
  },
  {
    "caption": "B Expression of the mCherry reporter in different organs at 30DPI in LmnaG609G/G609G mice versus PBS-injected control. Hrt, Mscl, Ver, Kid, Liv: heart, Muscle, vertebrae, kidney, liver. AAV9-mCherry-anti-miR-59 was injected into 8-day-old mice and detected at different points.",
    "molecules": "PBS"
  },
  {
    "caption": "Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle (B) The boxes and arrows denoted the aortic adventitia.",
    "molecules": "Masson"
  },
  {
    "caption": "Masson staining showed the adventitial fibrosis (blue areas) and the adventitial width of aorta (C). The boxes and arrows denoted the aortic adventitia.",
    "molecules": "Masson"
  },
  {
    "caption": "Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM",
    "molecules": "SM"
  },
  {
    "caption": "Time-lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies.",
    "molecules": "SM, propidium iodide"
  },
  {
    "caption": "immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM",
    "molecules": "SM"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre-incubated with MCC950 (10 μM) 20-30 min prior to TNF/TAK1i stimulation.",
    "molecules": "MCC950, KCl"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and SM (250 nM; 6 h) and mixed supernatant and cell extracts were analysed by immunoblot",
    "molecules": "SM"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and SM LDH release in the cell culture supernatant were quantified at the indicated time points.",
    "molecules": "SM"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and SM",
    "molecules": "SM"
  },
  {
    "caption": "HEK 293T cells were transfected with doxycycline-inducible DmrB-caspase-8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB-caspase-8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase-8. Mixed supernatant and extracts were analysed by immunoblot.",
    "molecules": "B/B homodimerizer, doxycycline"
  },
  {
    "caption": "Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF (100 ng/ml) and SM for 6 h and LDH release was quantified.",
    "molecules": "SM"
  },
  {
    "caption": "Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h and LDH release was quantified.",
    "molecules": "LPS, LCL161"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified Where indicated, cells were treated with the inhibitors Nec-1s (50 μM)",
    "molecules": "Nec-1s"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors MCC950 (10 μM), GSK'872 (1 μM), 20-30 min prior to cell stimulation. KCl (50 mM) was added together with TNF and TAK1i. To induce necroptosis, BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and Q-VD-OPh (10 μM) was added at the last 20-30 min of priming and stimulated with SM (500 nM) for 4 h.",
    "molecules": "SM, LPS, GSK'872, MCC950, KCl, Q-VD-OPh"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors GSK'872 (1 μM), 20-30 min prior to cell stimulation.",
    "molecules": "GSK'872"
  },
  {
    "caption": "BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors probenecid (1 mM) 20-30 min prior to cell stimulation. To activate the NLRP3 inflammasome, BMDM were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.",
    "molecules": "LPS, nigericin, probenecid"
  },
  {
    "caption": "BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with SM (0.5 μM) for a further 4 h. Probenecid (1 mM) was added 20-30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot.",
    "molecules": "SM, LPS, Probenecid"
  },
  {
    "caption": "BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and (G) mixed supernatant and extracts were analysed by immunoblot",
    "molecules": "SM"
  },
  {
    "caption": "BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and LDH release was quantified.",
    "molecules": "SM"
  },
  {
    "caption": "BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.",
    "molecules": "ABT-737, S63845"
  },
  {
    "caption": "BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.",
    "molecules": "ABT-737, S63845"
  },
  {
    "caption": "BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified",
    "molecules": "ABT-737, S63845"
  },
  {
    "caption": "BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) mixed supernatant and extracts were analysed by immunoblot at 6 h",
    "molecules": "ABT-737, S63845"
  },
  {
    "caption": "BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified",
    "molecules": "ABT-737, S63845"
  },
  {
    "caption": "BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and mixed supernatant and extracts were analysed by immunoblot at 6 h",
    "molecules": "ABT-737, S63845"
  },
  {
    "caption": "BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and mixed supernatant and extracts were analysed by immunoblot",
    "molecules": "LPS, ABT-737, S63845"
  },
  {
    "caption": "BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and IL-1β in cell-free supernatant were quantified by ELISA",
    "molecules": "LPS, ABT-737, S63845"
  },
  {
    "caption": "A. Northern blot analysis of the expression of VcdRP and Spot42 sRNA monitored at low cell density (OD600 of 0.1) and high cell density (OD600 of 2.0), in the absence (-) or presence (+) of cAMP (5mM f.c.) supplemented externally in LB medium. The solid triangle indicates the band corresponding to the full-length primary transcript. Probing with 5S rRNA confirmed equal loading.",
    "molecules": "cAMP"
  },
  {
    "caption": "B. Western blot analysis (top) of CTX levels detected in secreted protein fractions of V. cholerae ΔhapR cells grown under AKI conditions and carrying the indicated plasmids. Coomassie-stained SDS gel (bottom) confirmed equal loading of the protein fractions. The normalized band intensities are indicated for each expression plasmid relative to pCtrl, which was set to 100%.",
    "molecules": "Coomassie"
  },
  {
    "caption": "A. Immunoprecipitation of chromosomally tagged GltA::HA combined with either an empty vector control (pCtrl) or SPA-tagged vcdP over-expression plasmid, grown in LB medium to exponential phase (OD600 of 0.5). The SPA epitope contains the 3×FLAG and the calmodulin binding peptide sequences separated by a TEV protease cleavage site (Zeghouf et al, 2004). Therefore, protein samples corresponding to the total input and cell lysates before and after subjecting to immunoprecipitation with anti-Flag antibody were loaded on a SDS-PAGE gel. Western blotting with anti-HA and anti-Flag antibodies confirmed the interaction of GltA with VcdP in vivo. RNAP served as loading control. The solid triangles indicate the corresponding protein sizes.",
    "molecules": "calmodulin"
  },
  {
    "caption": "C. In vitro ubiquitination  assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1  and His-Ub  in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST. Data information: the experiments were performed at least three times independently with similar results.",
    "molecules": "ATP"
  },
  {
    "caption": "Western blot analysis of HEK293T  cells transfected with empty Flag  vector (-) or Flag-TRIM27  after treatment with or without 10 μM MG132  for 4 hours (E). Data information: the experiments were performed at least three times independently with similar results.",
    "molecules": "MG132"
  },
  {
    "caption": "F.  Fluorescence microscopy image of MEFs  with stable expression of mCherry-ULK1  and GFP-TRIM27  after treating cells with DMSO (MOCK) or 10 μM MG132  for 4 hours. Scale bar, 10 μm. Data information: the experiments were performed at least three times independently with similar results.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "HA-ULK1  levels in 50 μg/mL cycloheximide  (CHX)-treated HEK293T  cells with or without co-expression of Flag-TRIM27 (G, H) Western blots  against HA-ULK1  and actin  control (G, were subjected to densitometric  quantitation (H, Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests (H , **P < 0.01.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "endogenous ULK1  levels (I, J) in 50 μg/mL cycloheximide  (CHX)-treated HEK293T  cells or without TRIM27  RNAi (I, J). and actin  control I) were subjected to densitometric  quantitation J). Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests J), **P < 0.01.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Ubiquitination  assays were performed in HEK293T  cells after co-transfection with double (K568/K571R) or single (K568R, K571R) lysine  substitution mutants (L) in the presence or absence of Flag-TRIM27. Data information: the experiments were performed at least three times independently with similar results.",
    "molecules": "lysine"
  },
  {
    "caption": "O. ULK1/2 knockout MEFs  stably expressing the indicated HA-ULK1  constructs were subjected to  Western blot  analysis against HA  or actin  before (0h) and after treatment with CHX  for 12 hours. Data information: the experiments were performed at least three times independently with similar results.",
    "molecules": "CHX"
  },
  {
    "caption": "A. Western blot  analysis of autophagy  marker proteins (p62  and Ser318 phosphorylated ATG13) in WT HeLa  cells and two independent TRIM27  KO clones. ULK1  and TRIM27  levels were measured, along with actin  as a loading control.",
    "molecules": "Ser"
  },
  {
    "caption": "C. LC3  conversion was measured by  Western blot in WT or TRIM27  KO HeLa  cells with or without pretreatment with 200 nM bafilomycin A1 (BAF A1).",
    "molecules": "BAF A1, bafilomycin A1"
  },
  {
    "caption": "L.  Western blot analysis of p62  or LC3  levels in HeLa  cells with or without STK38L  knockout, the cells were treated with starvation  for the indicated time. Bafilomycin A1 (BAF A1) was used to inhibit the fusion between autophagosomes  and lysosomes.",
    "molecules": "BAF A1, Bafilomycin A1"
  },
  {
    "caption": "B. Phosphorylation  of HA-ULK1  in HeLa STK38L  knockout cells with or without expression of Flag-STK38L. HA-ULK1  was immunoprecipitated  and then analyzed by western blot using anti-phosphoserine  or anti-phosphothreonine, and the cell lysates were analyzed using anti-HA  and anti-Myc.",
    "molecules": "phosphothreonine, phosphoserine"
  },
  {
    "caption": "E. Phosphorylation  of HA-ULK1  in HEK293T  cells co-expressing Myc-STK38L  with or without CIP  treatment. HA-ULK1  was immunoprecipitated  with anti-HA  and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).",
    "molecules": "CIP, Ser"
  },
  {
    "caption": "F. Phosphorylation  of HA-ULK1 or HA-ULK1  S495A in HEK293T  cells co-expressing Myc-STK38L. HA-ULK1  or HA-ULK1  S495A was immunoprecipitated  with anti-HA  and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).",
    "molecules": "Ser"
  },
  {
    "caption": "G. In vitro kinase assay using GST-STK38L, or GST-STK38L  K119A (a kinase-inactive mutant) and HA-ULK1   as substrate. GST-STK38L  or GST-STK38L K119A was  pulled down using glutathione Sepharose  beads. HA-ULK1  was immunoprecipitated  from HEK293T  using anti-HA  and treated with lambda phosphatase, the reaction product was analyzed by western blot  using p-ULK1-S495 (Ser495).",
    "molecules": "Sepharose, glutathione, Ser"
  },
  {
    "caption": "L. Ubiquitination  analysis of Myc-STK38L  K1-38R mutant (All lysines  were mutated to arginines), or Myc-STK38L-R181/215/224/432/434K (All lysines were mutated to arginines except the indicated five lysine  residues) mutant in HEK293T  cells co-expressing HA-TRIM27  and His-Ub.",
    "molecules": "arginines, lysine, lysines"
  },
  {
    "caption": "M. Phosphorylation  of HA-ULK1  S495(Ser495) in HEK293T  cells co-expressing Myc-STK38L  WT, or Myc-STK38L-5KR mutant in K. The cells were cultured in fed  or starved  conditions as indicated and HA-ULK1  was immunoprecipitated  using anti-HA, the immunoprecipitates  and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-Myc, anti-HA, or anti-actin.",
    "molecules": "Ser"
  },
  {
    "caption": "N. Phosphorylation  of HA-ULK1  in HeLa  cells with or without TRIM27-knockout, cells were cultured in fed  or starved  conditions as indicated, HA-ULK1  was immunoprecipitated  using anti-HA, the immunoprecipitates  and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-HA, or anti-actin.",
    "molecules": "Ser"
  },
  {
    "caption": "Pulmonary surface nodules   and hematoxylin  and eosin  (H&E) staining  in Trim27+/- PyMT mice  and Trim27-/- PyMT mice  J). Scale bar, 1mm. (K). Bar chart showing quantification for the number of metastatic nodules  per lung lope (Trim27+/-, n=6 mice; Trim27-/- n=6 mice), when compared at the same final timepoint. Each symbol represents a mouse; Data are presented as mean ± s.d. from three independent experiments; ***P<0.001 (unpaired student's t-test).",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "CAC was induced in control (n = 7) and ColVIcre-Kif3aflx/flx (n = 7) female mice according to the timeline shown in Figure 2A. (E) Representative images of hematoxylin and eosin staining of paraffin-embedded colon sections. Areas with high-grade dysplasia are delimited by black lines.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(H) Elevated numbers of F4/80+ macrophages in colons of DSS-treated ColVIcre-Kif3aflx/flx mice. Representative images for F4/80 staining in areas with crypt loss are shown. At least 5 fields in the regions of crypt loss were analyzed from each colon of control (n=6) and ColVIcre-Kif3aflx/flx (n=6) mice. Mean cell numbers were scored as low (<500 cells/mm2) or high (>500 cells/mm2).",
    "molecules": "DSS"
  },
  {
    "caption": "Elevated numbers of IL-6-expressing F4/80+ macrophages in ColVIcre-Kif3aflx/flx mice treated with DSS as described in Figure 4A. At least 5 fields in the regions of crypt loss were analyzed. Scale bar represent 100 µm. *p<0,05 was calculated by chi-squared test.",
    "molecules": "DSS"
  },
  {
    "caption": "Elevated IL-6 expression in colonic epithelial cells and fibroblasts of PC-deficient mice upon DSS-treatment as described in Figure 4A. Representative images and corresponding quantification of vimentinpositive fibroblasts (in green) and vimentinnegative epithelial cells (identified by histological criteria) labeled for IL-6 (upper panel) and p-STAT3 (lower panel) in red. Nuclei were stained with DAPI (in blue). White arrow heads indicate IL-6 (upper panel) and nuclear staining for pSTAT3 (lower panel) for vimentinpositive fibroblasts, blue arrow heads the respective labeling for epithelial cells. Scale bars represent 100 µm. *p<0.05, ****p<10-4, n.s. (not significant) was calculated by chi-squared test on absolute numbers and is presented as % of cells positive for the indicated marker within vimentinpositive and epithelial cells, respectively.",
    "molecules": "DAPI, DSS"
  },
  {
    "caption": "(A) Representative image of vimentin+ fibroblasts (in green) expressing PC (identified using Arl13b, in red) in a normal colon. Nuclei were stained with DAPI (in blue). Most PC are detected on vimentin+ cells (arrow), and few on vimentin-negative epithelial cells (arrow head). (B,C) Quantitative analysis of PC presence on tumoral and peri-tumoral regions of colons from CRC patients (n=28) at tumor (T) stages 1 to 4. The panels represent quantification of PC expression (B) of vimentinpositive cells per mm2 and (C) per vimentinpositive cells as arbitrary unit (AU, see material and methods). Images were acquired on an Andor Dragonfly Spinning Disk Confocal microscope and analyzed with the Imaris software. Quantification of PC and Vimentin+ fibroblasts was done on at least 5 random fields (about 1mm2 each) per sample using ImageJ software.",
    "molecules": "DAPI"
  },
  {
    "caption": "NE activates mTORC2 in vitro via cAMP, PI3K and Epac1. (A) Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins.",
    "molecules": "NE, norepinephrine"
  },
  {
    "caption": "(B) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin (Rapa), Torin or Wortmannin (Wrtm) for the indicated proteins.",
    "molecules": "NE, Rapa, Rapamycin, Torin, Wortmannin, Wrtm"
  },
  {
    "caption": "(C) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Rapa, Torin or Wrtm for the indicated proteins.",
    "molecules": "8-Br-cAMP, Rapa, Torin, Wrtm"
  },
  {
    "caption": "(D) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins.",
    "molecules": "NE, ESI-09, H89, Wrtm"
  },
  {
    "caption": "(E) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. All experiments were performed in triplicates, a representative replicate is presented.",
    "molecules": "8-Br-cAMP, ESI-09, H89, Wrtm"
  },
  {
    "caption": "NE and cold activate mTORC2 in vivo. (A) Immunoblot analysis of BAT from control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).",
    "molecules": "NE, norepinephrine"
  },
  {
    "caption": "(B) Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).",
    "molecules": "NE, norepinephrine"
  },
  {
    "caption": "(B) Non-esterified fatty acids (NEFAs) in plasma of AdRiKO and control mice (n=6/group).",
    "molecules": "NEFAs, Non-esterified fatty acids"
  },
  {
    "caption": "(C) Glycerol in plasma of AdRiKO and control mice (n=6/group).",
    "molecules": "Glycerol"
  },
  {
    "caption": "(E) Triglycerides (TGs) in BAT of AdRiKO and control mice housed at 22°C or 4°C for 8h (n=6/group).",
    "molecules": "Triglycerides"
  },
  {
    "caption": "(F) NEFAs in BAT of AdRiKO and control mice (n=6/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.",
    "molecules": "NEFAs"
  },
  {
    "caption": "(G) Oxygen consumption rate (OCR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).",
    "molecules": "Oxygen"
  },
  {
    "caption": "mTORC2 in adipose tissue is required for cold-induced glucose uptake and glycolysis (A) 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=6/group). (",
    "molecules": "2-Deoxyglucose-6-phosphate, 2DG6P, glucose"
  },
  {
    "caption": "(B) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 4°C for 4h (n=8/group).",
    "molecules": "2-Deoxyglucose-6-phosphate, 2DG6P"
  },
  {
    "caption": "(H) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 4°C for 4h (n=7 (control AAV8-null), n=6 (AdRiKO AAV8-null), n=6 (control AAV8-AktS474D), n=6 (AdRiKO AAV8-AktS474D)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between viruses were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.",
    "molecules": "2-Deoxyglucose-6-phosphate, 2DG6P"
  },
  {
    "caption": "C, Phosphorylation of  TOC1  is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12h/12h light/dark cycles were subject to heat  (30ºC 20min), pyrophosphatase (PP) and NaF/Na3VO4  (phosphatase  inhibitors) treatments. TOC1-GFP at different phosphorylation states were separated by 8% SDS-PAGE  (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP.",
    "molecules": "acrylamide, bisacrylamide, NaF, Na3VO4"
  },
  {
    "caption": "B, Measurement of TOC1-GFP protein turn-over in  TOC1, 5X, T135A, S175A native promoter lines by cycloheximide  (CHX) treatment. 10-d-old seedlings grown in 12h/12h light/dark cycles were subject to cycloheximide (CHX) or  ethanol (Mock) treatment for indicated  time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves. Data information: TOC1  protein was detected by α-GFP, ADK  was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK  bands using ImageJ.",
    "molecules": "CHX, cycloheximide, ethanol"
  },
  {
    "caption": "A Detection of the RBPJ/L3MBTL3 interaction using the yeast two-hybrid (Y2H) assay. In this Y2H experiment, RBPJ is fused to the GAL4 DNA-binding (DB) domain and L3MBTL3 is fused to the GAL4 activation domain (AD). The DB-RBPJ and AD-L3MBTL3 fusion proteins interact with each other, leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine. The six Y2H controls were previously described (Dreze et al, 2010). The experiment was independently replicated thrice.",
    "molecules": "adenine, histidine"
  },
  {
    "caption": "C Western blot  analysis of ACE2  in  Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. Data are representative of three biological replicates.",
    "molecules": "VitD3, VitK1, VitB1, VitB12, VitB6, VitC"
  },
  {
    "caption": "D Western blot analysis of ACE2  in HEK293T, 2fTGH, Caco-2  and A549  cells treated with VitC  at indicated concentrations for 24 hrs. Data are representative of three biological replicates.",
    "molecules": "VitC"
  },
  {
    "caption": "G Western blot  analysis of ACE2, IRF3  and STAT1  in A549  cells treated with VitC  at indicated concentrations for 24 hrs. Data are representative of three biological replicates.",
    "molecules": "VitC"
  },
  {
    "caption": "H Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP  viruses in  Caco-2-N cells pretreated with VitC  (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP  (MOI = 0.1) viruses for 24 hrs. Data are representative of three biological replicates. Scale bar: 100 µm.",
    "molecules": "VitC"
  },
  {
    "caption": "A  RT-qPCR  analysis of Ace2  mRNA in 2fTGH  cells treated with VitC  (5 mM) as indicated. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant (two-tailed unpaired Student's t-test).",
    "molecules": "VitC"
  },
  {
    "caption": "F IP-IB  analysis of ubiquitination  types of Myc-ACE2  in HEK293T  cells cotransfected with Myc-ACE2  and different types of HA-Ub, and then treated with VitC  (5 mM) for 12 hrs. The red dashed contour represents the group of K48-linked ubiquitination  we focused on studying here. Data are representative of three biological replicates.",
    "molecules": "VitC"
  },
  {
    "caption": "H IP-IB  analysis of K48-Ub  of endogenous ACE2  in 2fTGH  cells treated with VitC  (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.",
    "molecules": "VitC"
  },
  {
    "caption": "G  Western blot  analysis of ACE2  in HEK293T  cells transfected with Flag-USP50  and then treated with CHX  (50 μM) as indicated. Data are representative of three biological replicates.",
    "molecules": "CHX"
  },
  {
    "caption": "K Fluorescence microscopy  of the VSV  virus with a SARS-CoV-2-S gene and a GFP  gene (VSV-Spike) in Usp50+/+ and Usp50-/- HEK293T   cells pretreated with or without VitC  (5 mM) for 12 hrs, followed by infection  with the VSV-Spike  virus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. The average intensity each unit area was shown as mean and s.d. of three-unit areas (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).",
    "molecules": "VitC"
  },
  {
    "caption": "C Western blot analysis of Myc-ACE2  in HEK293T  cells transfected with Myc-ACE2  (WT or K788R) and then treated with CHX  (50 μM) as indicated. Data are representative of three biological replicates.",
    "molecules": "CHX"
  },
  {
    "caption": "D IP-IB  analysis of K48-Ub  of Myc-ACE2  in HEK293T  cells transfected with Myc-ACE2  (WT or K788R) and then treated with VitC  (5 mM) for 12 hrs, by a specific anti-K48-Ub  antibody. Data are representative of three biological replicates.",
    "molecules": "VitC"
  },
  {
    "caption": "G ACE2-KO  Caco-2 cells transfected with ACE2  (WT or K788R) were treated with VitC  (5 mM) for 12 hrs, and then were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA  levels. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).",
    "molecules": "VitC"
  },
  {
    "caption": "A IP-IB  analysis of the interaction  between Flag-USP50  and Myc-ACE2  in HEK293T  cells transfected with these two constructs and then treated with VitC  (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.",
    "molecules": "VitC"
  },
  {
    "caption": "D Flag-USP50  proteins were obtained as (C). Flag-USP50  and r-hACE2  proteins were mixed, together with increasing amounts of VitC  or with VitC-Na+ (20 mM). HCl  is a pH control (pH = 4). After 2 hrs incubation, ACE2  proteins were analyzed by immunoblotting  by a specific anti-ACE2  antibody. Data are representative of three biological replicates.",
    "molecules": "HCl, Na, VitC"
  },
  {
    "caption": "D Western blot analysis of hACE2  levels in lung  tissues of hACE2 mice  (n = 6) administrated with VitC  as (B). Data are representative of three biological replicates.",
    "molecules": "VitC"
  },
  {
    "caption": "G  RT-qPCR  analysis of the SARS-CoV-2-Spike  mRNA levels in lung, kidney, liver  and spleen  tissues of hACE2 mice  treated with VitC  and the VSV-Spike  viruses as (F). All graphs show the mean ± SEM for five individual mice  (n = 5). ***p < 0.001 (two-tailed unpaired Student's t-test).",
    "molecules": "VitC"
  },
  {
    "caption": "K, L. Representative images of double  immuno-staining  for insulin (green) and  BrdU  (red) of pancreas from PbkWT/WT (K) or PbkKI/KI (L) mice with 5 weeks HFD  feeding. Islet area was circled with white dashed line. Nuclei  were labeled by DAPI (blue). Scale bar: 50 μm.",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": "J, K. The effect of T5224 on the binding of JunD,  menin, HDAC3, and acetylated histone3 at the Pbk  locus in PIME  cells (J) or  INS-1 cells (K). Three independent experimets (n=3). *P = 0.0490 (J, JunD), *P = 0.0479 (J, Menin), *P = 0.0154 (J, HDAC3), **P = 0.0029 (J, Ac-H3), *P = 0.0104 (K, JunD), **P = 0.0024 (K, Menin), ***P = 0.0002 (K, HDAC3), ***P = 0.0001 (K, Ac-H3), (two-tailed unpaired student's t-test). ns, not statistically significant difference.",
    "molecules": "T5224"
  },
  {
    "caption": "F.  Mouse primary  islets treated with MI-503 for 3 days were subjected ChIP assay to detect the  binding of menin, HDAC3, JunD, as well as histone acetylation  level at the  Pbk locus. Three independent experimets (n=3). *P = 0.0218 (Menin), *P = 0.0145 (HDAC3), *P = 0.0184 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.",
    "molecules": "MI-503"
  },
  {
    "caption": "G. MI-503 increases  proliferation  of  menin-expressing PIME cells but not menin-null PIME cells. Three independent experimets (n=3). ***P < 0.0001 (Two-way ANOVA).",
    "molecules": "MI-503"
  },
  {
    "caption": "K-M.  5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets  (donor number is four). qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference. ",
    "molecules": "MI-503"
  },
  {
    "caption": "E-L. Representative  images  of insulin (green) and Ki67 (red) double staining on mouse islets with 8 wks MI  treatment (E-H), and insulin  (green) or Pbk  (red)  double staining  on mouse islets (I-L).  Nuclei were labeled by  DAPI (blue). Islet area was circled by white dashed line.  Ki67 and Pbk staining are denoted by white arrows. Scale bars: 50 μm.",
    "molecules": "DAPI, MI"
  },
  {
    "caption": "D, E. Double staining for  insulin  (green) and BrdU (red) of mice islets after 12 weeks of vehicle (D) or  MI (E) administration. Islet area was circled by white dashed line.  Nuclei were labeled by DAPI (blue). BrdU staining is indicated by arrows. Scale bar:50 μm. F, G. Quantification of BrdU positive beta cells (F) and  beta cell mass (G) of MI-463 treated HFD-fed mice or control  mice. Five mice for each group, and at least 10  islet images per  mouse were analyzed. **P = 0.0059, *P = 0.0103 (two-tailed unpaired student's t-test). ",
    "molecules": "BrdU, DAPI, MI, MI-463"
  },
  {
    "caption": "H, I.  Double staining for insulin (green) and Pbk (red) of mice islets after 12 weeks of vehicle (H) or  MI  (I) administration. Islet  area was circled by white dashed line. Nuclei  were labeled by DAPI (blue).  Pbk staining is indicated by arrows. Scale bar: 50 μm. J. Quantification of  Pbk staining-positive  β-cells. Five mice for each group, and at least 10 islet images per mouse were analyzed. *P = 0.0120 (two-tailed unpaired student's t-test). ",
    "molecules": "DAPI, MI"
  },
  {
    "caption": "K. Quantification of the percentage with  pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean values of five mice from 5 sections for each mouse were presented.",
    "molecules": "BrdU"
  },
  {
    "caption": "O, P. GTT data for MI-463 or vehicle treated PbkKI/KI or PbkWT/WT mice (n=3 for each group) on 8 weeks HFD  (O) (*P = 0.0133 (Two way ANOVA), ns, not statistically significant difference.) and AUC of GTT data (P) (*P = 0.0442 (two-tailed unpaired student's t-test), ns, not statistically significant difference.).",
    "molecules": "MI-463"
  },
  {
    "caption": "A Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels (V Low, V Med, and V High, respectively) and treated with VEGF-Trap or saline after 10 and 17 days. Analysis was performed after 4 days of treatment (2 and 3 weeks, respectively). All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes, whereas aberrant structures induced by high VEGF were covered with α-SMA-positive smooth muscle cells. Asterisks indicate muscle fibers, which were mostly sectioned in longitudinal orientation, although some cross sections are visible depending on the orientation of muscle bundles. Scale bar = 25 μm.B, C Quantification of vessel length density (VLD) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF, but not until 17 days for those induced by medium VEGF, despite similar pericyte coverage, while aberrant structures induced by high VEGF always regressed. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group; 2 weeks (B): V Low saline, n = 9; V Med saline, n = 13; V High saline, n = 8; V Low TRAP,n = 28; V Med TRAP,n = 22; V High TRAP,n = 30; 3 weeks (C): V Low saline, n = 9; V Med saline, n = 11; V High saline, n = 19; V Low TRAP,n = 43; V Med TRAP,n = 14; V High TRAP,n = 19; *P < 0.05 and ***P < 0.001 (all versus Ctrl) by one-way ANOVA with Bonferroni multiple comparisons test; 2 weeks (B): V Low TRAP versus Ctrl P < 0.0001; 3 weeks (C): V Low TRAP versus Ctrl P < 0.0001; V Med TRAP versus Ctrl P = 0.0158.",
    "molecules": "VEGF-Trap"
  },
  {
    "caption": "A-C Mice, implanted with myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated on days 4 and 6 after cell implantation with intramuscular injections of Sema3A-Fc (0.1 or 10 mg/kg of average muscle tissue weight), or with control Fc protein. (A) Quantification of the number of NEM/cm of vessel length in sites of new angiogenesis after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 5 muscles/group: V Low Fc, n = 21; V Low 0.1 mg/kg, n = 22; V Low 10 mg/kg, n = 28; V Med Fc, n = 20; V Med 0.1 mg/kg, n = 30; V Med 10 mg/kg, n = 25; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low Fc versus V Low 10 mg/kg P = 0.0027; V Med Fc versus V Med 0.1 mg/kg P = 0.001; V Med Fc versus V Med 10 mg/kg P < 0.0001. (B) Quantification of vessel length density (VLD) in treated muscles after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 8; V Low 0.1 mg/kg, n = 20; V Low 10 mg/kg, n = 18; V Med Fc, n = 8; V Med 0.1 mg/kg, n = 23; and V Med 10 mg/kg, n = 27. Data were subjected to Kruskal-Wallis analysis with Dunn's multiple comparisons test, and no significant differences were detected. (C) Vascular stabilization rate was determined 2 weeks after cell implantation by measuring vessel length density after abrogation of VEGF signaling by VEGF-Trap. Treatment with 10 mg/kg Sema3A-Fc did not affect the stabilization of angiogenesis induced by low VEGF, but significantly increased the resistant fraction of vessels induced by medium VEGF. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 32; V Low Sema3A, n = 14; V Med Fc, n = 28; V Med Sema3A, n = 33; *P < 0.05 by one-way ANOVA with Bonferroni multiple comparisons test: V Med Fc versus V Med Sema3A P = 0.0458.",
    "molecules": "VEGF-Trap"
  },
  {
    "caption": "(J) PKA R1α/β is enriched at mitochondria (WT: 1.10± 0.266, KO: 2.73± 0.515; p=0.0082, two-tailed unpaired t-test) and synapses (WT: 0.52± 0.131, KO: 1.14± 0.221; p=0.0234, two-tailed unpaired t-test) of KO mice. Values display the mean number of gold particles counted per structure in nWT=21 and n KO=22 pictures per genotype.",
    "molecules": "gold"
  },
  {
    "caption": "(A) Western Blot analysis of PKA R1α protein levels in NSC34 cells, treated with 250 nM Torin1 or deprived of amino-acids and serum for 16 hours. Lysosomal degradation was blocked in the last 4 hours using 67 nM of Bafilomycin A (BafA). (B) Starvation significantly reduced PKA R1α protein levels in NSC34 cells (vehicle set to 100%, starvation: 31.19± 5.085%, p<0.0001, one-tailed unpaired t-test). n=5. (C) Application of 67 nM BafA for 4 hours before harvesting was sufficient to stabilize the PKA R1α protein levels in starved NSC34 cells (starvation set to 100%, starvation+BafA: 155.5± 13.93%, p=0.002, one-tailed unpaired t-test).",
    "molecules": "BafA, Bafilomycin A, Torin1"
  },
  {
    "caption": "16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2- positive cultured primary neurons (Vehicle: 212.4±5.619, Starvation: 255.1±5.715; p<0.0001, two-tailed paired t-test). nVehicle=314, nStarvation=306 neurons from n=3.",
    "molecules": "cAMP"
  },
  {
    "caption": "16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2- positive cultured primary neurons (Vehicle: 212.4±5.619, Starvation: 255.1±5.715; p<0.0001, two-tailed paired t-test). nVehicle=314, nStarvation=306 neurons from n=3.",
    "molecules": "cAMP"
  },
  {
    "caption": "(F, G) Phosphorylation state of proteins containing PKA substrate RRXS/T motif was increased upon 16 hours of amino-acid and serum starvation in primary cortico-hippocampal neurons at DIV14 and was suppressed by 1 µM H89 supplementation in the media (Vehicle set to 100%, Starvation: 326.60±94.53%, H89: 214.80±56.87%, p=0.033, one-way ANOVA with Dunn's multiple comparison test). n=4. The pPKA substrate levels were normalized to the total protein amount stained with Ponceau S.",
    "molecules": "H89, Ponceau S"
  },
  {
    "caption": "Treatment of NSC34 cells with BafA (67nM) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO-treated group set to 100% (BafA: 198.9± 28.44%, p=0.003, one-tailed unpaired t-test).",
    "molecules": "BafA, DMSO"
  },
  {
    "caption": "Treatment of NSC34 cells with BafA (67nM) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO-treated group set to 100% (BafA: 198.9± 28.44%, p=0.003, one-tailed unpaired t-test). n=6.",
    "molecules": "BafA, DMSO"
  },
  {
    "caption": "(J) Western blot analysis of purified autophagosomes (AVs) (50μg AVs/lane) with and without Proteinase K (PK) treatment. TX-100 (1% final) was used as a positive control for the activity of PK. Synaptosome lysates (Syn, 30μg/lane) were used as a positive control for the signal of antibodies.",
    "molecules": "TX-100"
  },
  {
    "caption": "(M, N) Representative fluorescent images and subsequent analysis of PKA R1- α/p62 colocalization (Pearson's correlation coefficient) in primary cortico-hippocampal neurons, which have undergone 16 hours amino-acids and serum starvation and were additionally treated with BafA (67nM) to visualize the lysosomes (control: 0.68± 0.019, starvation: 0.58± 0.015, starvation+BafA: 0.83± 0.014; pcontrol vs starvation<0.0001, pcontrol vs starvation+BafA<0.0001, pstarvation vs starvation+BafA<0.0001 Ordinary one-way ANOVA with Tukey's multiple comparison test). ncontrol=36, nstarvation=38, nstarvation+BafA=36 neurons from n=3.",
    "molecules": "BafA"
  },
  {
    "caption": "(A-B) Western Blot analysis of pCREB protein levels in Atg5flox:CAG-CreTmx WT/ KO cultured neurons at DIV14, treated with forskolin (WTDMSO set to 100%, WTForskolin: 214.17± 48.87%, KODMSO: 63.26± 15.85%, KOForskolin: 33.50± 10.84%, pWT DMSO/WT Forskolin=0.042, pWT Forskolin/KO Forskolin=0.002, two-way ANOVA with Tukey's multiple comparisons). n=4. The same lysates were run in parallel to detect pCREB and CREB and normalized to their respective loading control before calculating the pCREB/CREB ratio.",
    "molecules": "DMSO, forskolin, Forskolin, Tmx"
  },
  {
    "caption": "(I, J) Representative fluorescent images and subsequent analysis of primary cortico-hippocampal neurons containing nuclear pCREB either under DMSO-treated condition or under condition when amino acids and serum starvation was induced for 16 hours in the absence or presence of 1µM of PKA inhibitor H89. Neurons were identified by their polarized morphology in DIC. White rectangular boxes indicate panel magnified to the right. In the inserts, LUT gradient was added to highlight the pCREB intensity in the nucleus. Scale bar: 50 µm. DMSO: 52.50±2.757, starvation: 70.00±2.697, starvation+H89: 53.92±3.147; pDMSO/starvation<0.0001, pstarvation/starvation&H89=0.0004, one-way ANOVA with Tukey's multiple comparisons test, n=30 images from n=3 independent experiments.",
    "molecules": "DMSO, H89"
  },
  {
    "caption": "(A-B) Western Blot analysis of PKA substrates carrying RRXS/T motif in primary cortico-hippocampal Atg5flox:CAG-CreTmx WT/KO neurons at DIV14, treated with Forskolin (WTDMSO set to 100%, WTForskolin: 291.85± 17.02%, KODMSO: 152.97± 25.26%, KOForskolin: 182.56± 31.89%, pWTDMSO/WTForskolin: 0.001, pWTForskolin/KOForskolin: 0.033, two-way ANOVA with Tukey's multiple comparisons).",
    "molecules": "DMSO, Forskolin, Tmx"
  },
  {
    "caption": "(G) Representative fluorescence images of Atg5flox:CAG-CreTmx WT/KO neurons transfected with eGFP or eGFP-ATG5 and immunostained for GLUR1, PSD95 and GFP. (H) The overexpression of eGFP-ATG5 in Atg5flox:CAG-CreTmx KO neurons diminished the of GLUR1/PSD95 colocalization compared to the eGFP-overexpressing KO neurons (KOeGFP: 70.07±1.473%, KOeGFP-ATG5: 65.42±1.721%,; p=0.048, two-tailed unpaired t-test). nKO+GFP=42, nKO+GFP-ATG5=50 images from n=5. (I, J) The coexpression of eGFP-PKA R1α and eGFP-PKA R1β in cultured Atg5flox:CAG-CreTmx WT neurons results in significantly increased colocalization between GLUR1 and PSD95 compared to the eGFP-expressing neurons (WT: 42.30±2.453%, KO: 51.32±2.624%, p=0.014, two-tailed unpaired t-test). nWT+GFP=46, nWTGFP+PKA R1 OE=46 images from n=3. Analysis in H, J is performed using a unbiased custom-written Plugin for ImageJ.",
    "molecules": "Tmx"
  },
  {
    "caption": "(K-M) GCAMP7f responses to tetanic stimulation (four tetani, 100 APs at 100Hz, 3s interval) in cell bodies of CamKIIα-Cre-transduced primary cortico-hippocampal Atg5wt:wt/Ai9 (WT) and Atg5flox:flox/Ai9 (KO) neurons expressing mCamKIIα-jGCaMP7f and treated either with DMSO (K) or with 10 µM CNQX for 5 min (L). Autophagy deficient cells showed significantly increased facilitation of GCAMP7f signal to electrical stimulation (ΔF/Fpeak1) compared to the WT, a phenotype which was blocked by the application of CNQX (WTDMSO: 0.28± 0.12, WTCNQX: -0.07± 0.03, KODMSO: 3.74± 0.86, KOCNQX: -0.014± 0.13, p WT DMSO/ KO DMSO<0.0001, pKO DMSO/KO CNQX<0.0001, two-way ANOVA with Tukey's multiple comparisons test). nWT DMSO= 25, nWT CNQX= 27, nKO DMSO= 33, nKO CNQX= 30 neurons from n=3.",
    "molecules": "CNQX, DMSO"
  },
  {
    "caption": "C, Growth curves for E. coli 113/3 in defined media and in the presence or absence of B12 ( and o, r­­espectively). When challenged with 40 nM isolated R-domain ( ), cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A (▫) a­­pproached that of the no colicin control, consistent with OmpF threading causing the disengagement of ColE9 from BtuB. Error bars represent the standard deviation across three biological replicates.",
    "molecules": "colicin, B12"
  },
  {
    "caption": "ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin. A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.",
    "molecules": "CCCP, Hoechst, trypsin, Trypsin"
  },
  {
    "caption": "C, Box and whisker plots showing mean pixel intensities for ColE9* K469CAF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. From left-to-right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469CAF647 cell-associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469CAF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469CAF647 fluorescence. n, number of cells used, typically from 2-4 biological replicates. ****, indicates a t value <0.05 in a student T-test as a statistically significant result, ns indicates no significant difference as determined by student T-test.",
    "molecules": "CCCP, trypsin"
  },
  {
    "caption": "B, Overnight plate assays comparing the cytotoxic activity of wild-type ColE9 with ColE9 K469C labelled with AF488, AF568 or AF647 against a lawn of E. coli with either OmpF (JW2203) or OmpC (JW0912) in the outer membrane (shown as +OmpF and +OmpC, respectively). Each plate was spotted with a serial dilution of the colicin (10 µM - 2.4 pM, 4-fold dilution per spot). Wild-type ColE9 was more active against E. coli cells with OmpF in the outer membrane than OmpC. Labelling ColE9 K469C with fluorophores reduced colicin activity in cells with OmpF in the outer membrane but largely abolished colicin activity in cells with OmpC.",
    "molecules": "AF488, AF568, AF647, colicin"
  },
  {
    "caption": "C, Widefield fluorescence microscopy images of E. coli JW2203 (+OmpF) and JW0912 (+OmpC) cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst stain (20 µM) for 30 min at 37 °C with or without trypsin treatment. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and fluorescence of ColE9* K469CAF647 (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. Data show that following trypsin-treatment, significant ColE9* K469CAF647 fluorescence remains associated with ompF-expressing E. coli whereas little or no fluorescence remains associated with ompC-expressing E. coli. Scale bar, 1 µm. D, Box and whisker plots of E. coli JW2203 and E. coli JW0912 cells labelled with ColE9* K469CAF647 with and without trypsin treatment: whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. Microscopy data were collected as in c. The mean pixel intensity of ColE9* K469CAF647 per cell was measured for each cell condition. n, number of cells, typically from 3 or 4 biological replicates. Data show that ColE9* K469CAF647 translocates across the OM through OmpF and that this is impeded when E. coli has OmpC in the outer membrane. ",
    "molecules": "Hoechst, trypsin"
  },
  {
    "caption": "(A) Hematoxylin and Eosin (H&E) staining of WT and K14ΔNLef1 HF from back skin at 11.5 weeks (early anagen). White arrowheads: ectopic SG and epidermal cysts. Data information Scale bars: 50 μm.",
    "molecules": "Eosin, Hematoxylin"
  },
  {
    "caption": "(B) Tail epidermal whole-mounts from WT and K14ΔNLef1 mice labelled with Fabp5, Krt15 and counterstained with Dapi. Data information: Scale bars: 50 μm.",
    "molecules": "Dapi"
  },
  {
    "caption": "(A) Sections of WT embryonic skin at different HF stages stained for Gata6, Lef1, Sox9 and counterstained with Dapi. Black asterisks indicate overexposed areas of nonspecific Lef1 staining in the suprabasal epidermis. Quantification of the percentage of cells labelled for both Gata6 and Lef1, or Gata6 and Sox9 in stages 4-5 HF are shown (upper right panel). Data are means ± SD and were obtained from 9 HF from 3 mice. (B) Sections of E18.5 WT and Lef1-/- mouse skin stained for Lef1 and Gata6. Deletion of Lef1 does not impair Gata6 expression. Data information: Scale bars: 50 μm.",
    "molecules": "Dapi"
  },
  {
    "caption": "(C) Sections of WT and K14ΔNβcateninER (K14ΔNβ-catER D2 and D4 strains) adult dorsal skin stained with antibodies against Krt14 and Gata6. Topical treatment with 1 or 6 doses of 4OHT activates β-catenin leading to anagen induction and ectopic hair follicles but not Gata6 expression. Data information: Scale bars: 50 μm.",
    "molecules": "4OHT"
  },
  {
    "caption": "(B) Schematic representation of plasmid constructs used for in utero lentiviral infection. Whole-mounts or sections of adult infected epidermis with empty vector (EV) or Gata6-ires-GFP (G6OE) lentivirus were stained for GFP and Fasn. In vivo overexpression of Gata6 leads to ectopic Fasn expression in the HF/SG unit. White dotted lines define SG, and yellow dotted lines define a cyst. Note that the cyst is mostly negative for Fasn in agreement with its SD-like phenotype. White arrows indicate GFP-positive infected cells. These cells are stained with Fasn only in G6OE condition. H&E-stained skin from G6OE mice shows a cyst with SG elements in the HF unit (black arrows). Staining for Gata6 (both endogenous and exogenous) shows that Gata6 expression occurs in a limited number of cells (representative image in upper right panel). Bottom left graph shows quantification of the percentage of clones labelled for both Fasn and GFP in the SG, HF and IFE compartments. Data are means ± SD and originate from 4 EV mice and 8 G6OE mice (average of 11 clones per mouse). (C) Whole-mounts adult infected epidermis with EV or G6OE lentivirus stained with LipidTOX. Ectopic Gata6 cells are not stained with LipidTOX indicating an incomplete sebaceous maturation. Data information: Scale bars: 50 μm.",
    "molecules": "LipidTOX"
  },
  {
    "caption": "(D) Lineage tracing experiments in Gata6EGFPCreERT2:Rosa26-fl/STOP/fl-tdTomato (Gata6creER ROSA-dTom) mice. A single dose of 4OHT was injected in pregnant females at E16.5. Tail skin from pups was collected at P13. Representative example of whole-mount epidermis showing tdTomato labelled cells counterstained with Dapi (top left panel). Right panels show the different Z-stacks related to this whole-mount. Gata6 progenies are mainly found in the upper SG/JZ. Localization of Gata6 progenies (dTomato+) was quantified in 20-26 pilosebaceous units per mouse (N=4 mice) (bottom left panel). Data are means ± SD. As a control, quantification was also performed in Rosa26-fl/STOP/fl-tdTomato (ROSA-dTom) mice that were treated similarly to Gata6creER ROSA-dTomato mice. Data information: Scale bar: 25 μm.",
    "molecules": "Dapi, 4OHT"
  },
  {
    "caption": "(A) Sections of mouse skin tumors were stained with antibodies to Gata6 and Krt14. A papilloma tumor found in WT mice treated with DMBA and TPA (left panel), is compared with sebaceous tumors found in K14ΔNLef1 mice (right panels). Data information: Scale bars: 100 μm.",
    "molecules": "DMBA, TPA"
  },
  {
    "caption": "(B) Tumor burden and tumor incidence in DMBA-treated K14ΔNLef1 (n=11) and K14ΔNLef1:cKO (n=8) mice. (***) p-value < 0.001; Wilcoxon matched-pairs signed rank test. Data information: Data are means ± SEM.",
    "molecules": "DMBA"
  },
  {
    "caption": "(C) MSI analysis performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice (5 mice in each group). Representative microsatellite profiles of K14ΔNLef1 and K14ΔNLef1:cKO tumors. Peak heights were normalized to the highest peak in each microsatellite profile to obtain the relative frequency of each allele for 5 different markers. \"0\" indicates the position of the highest peak in bp. (*) p-value < 0.05; (**) p-value < 0.001; paired t-test.",
    "molecules": "DMBA"
  },
  {
    "caption": "(D) Representative immunohistochemistry stainings against Mlh1 performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice. 4 mice were included in each group. A technical control is displayed (without primary antibody incubation). Semi-quantitative analysis of Mlh1 staining and statistical analysis (χ2 test) were performed. Data information: Scale bar: 250 μm.",
    "molecules": "DMBA"
  },
  {
    "caption": "A, Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces. Cross-sections along the direction of migration (yellow) and perpendicular to the direction of migration (blue, 1-4) are shown below.",
    "molecules": "phalloidin"
  },
  {
    "caption": "Representative current traces from oocytes expressing wild-type (WT) and double cysteine mutant NMDARs before and after DTE treatment. For each mutant, traces are normalized (in height, not in current amplitude) to the maximal response obtained after DTE treatment on wild-type (WT) receptors",
    "molecules": "cysteine, DTE"
  },
  {
    "caption": "Summary of the DTE-induced potentiation of WT and double cysteine mutants",
    "molecules": "cysteine, DTE"
  },
  {
    "caption": "Immunoblots from Xenopus oocytes expressing either wt or mutant subunits. M1 indicates the GluN1 monomer (~110 kDa); M2 the GluN2B monomer (~180 kDa); D1/1 the GluN1 homodimer (~220 kDa); and D1/2 the GluN1/GluN2B heterodimer (~290 kDa). Lower panels: immunoblots in reducing conditions (+ β-mercaptoethanol). N.I., non-injected oocytes",
    "molecules": "β-mercaptoethanol"
  },
  {
    "caption": "Assessment of receptor channel activity using MK-801 inhibition kinetics. Representative current traces from oocytes expressing either wild-type (WT) or GluN1-E698C/GluN2B-L795C mutant receptors in response to 10 nM MK-801 during agonist application. Responses were scaled to the current amplitude obtained before MK-801 application. Inset, mono-exponential fits of MK-801 wash-in and wash-out. Note the strikingly faster kinetics in mutant receptors, both at the onset and offset of MK-801",
    "molecules": "MK-801"
  },
  {
    "caption": "Relative MK-801 inhibition on- and off-rate constants (kon and koff). All values were normalized to the value obtained for WT GluN1/GluN2B receptors",
    "molecules": "MK-801"
  },
  {
    "caption": "Zinc, ifenprodil and pH dose-response curves of wild-type (WT) GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors. For comparison, zinc and ifenprodil dose-response curves of GluN1/GluN2B receptors lacking the whole GluN2B NTD (GluN1 WT/GluN2B-delNTD) are also shown (dashed lines; data from Rachline et al, 2005)",
    "molecules": "ifenprodil, Zinc, zinc"
  },
  {
    "caption": "Spermine (200 µM, pH 6.3) potentiation of WT GluN1/GluN2B and mutant GluN1-E698C/GluN-L795C receptors. Spermine (200 µM, pH 6.5) sensitivity is also shown for WT GluN1/GluN2B receptors and receptors lacking either the GluN1 (GluN1-delNTD/GluN2B WT) or GluN2B (GluN1 WT/GluN2B-delNTD) NTD (data from Mony et al, 2011)",
    "molecules": "Spermine"
  },
  {
    "caption": "Glutamate and glycine dose-response curves of WT GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors",
    "molecules": "Glutamate, glycine"
  },
  {
    "caption": "MK-801 inhibition kinetics. On-rate constants (kon) of inhibition by 10 nM MK-801 on wild-type (WT) and mutant receptors. All values are normalized to that obtained for WT GluN1/GluN2B receptors",
    "molecules": "MK-801"
  },
  {
    "caption": "(D) T6BP  silencing affects the formation of stable MHC-peptide complexes. HeLa-CIITA  cells were transfected as above and pulse-labeled with 35S-Met/Cys  for 30 min, washed and chased for 4h at 37°C. MHC-II  molecules were then immunoprecipitated  using TÜ36  antibody and analyzed on SDS-PAGE  after incubation of the immunoprecipitated protein complexes with SDS at 95°C (B: boiled) or room temperature  (NB: non-boiled) to visualize α, β and Ii chains and SDS  resistant αβ dimers, respectively. The bands corresponding to α, β and Ii chains are indicated. The arrow indicates the SDS-resistant stable HLA αβ heterodimers. This gel is representative of two independent experiments.",
    "molecules": "Cys, Met, SDS, 35S"
  },
  {
    "caption": "(A) MHC-II  and T6BP expressions  were assessed using confocal microscopy. HeLa-CIITA  cells were transfected with control and T6BP-silencing siRNAs. 48h post-treatment, MHC-II  and T6BP  were detected using L243 and anti-T6BP  antibodies, respectively, and revealed with species specific secondary antibodies. Nuclei  were stained  using DAPI. (B) Quantitative analysis using in-house ImageJ script displaying distance of each MHC-II+ vesicles  to the nucleus  and number of vesicles  per cell. At least 20,000 vesicles  from 160 cells corresponding to five biological replicates were analyzed. Right panel, quantification in the siCTRL cells of the colocalization  between MHC-II+ and T6BP+ dots using Pearson's coefficient where the dotted lines (at 0.5) indicate the limit under which no significant  co-localization is measured (number of cells = 54). Data information: In graphs representing the number of vesicles  per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) As in A, adding Lysotracker staining. (F) As in B, quantitative analysis of Lysotracker+ vesicles: localization  to the nucleus  and number of vesicles  per cell. Colocalization  of Lysotracker+ vesicles  with MHC-II+ puncta was analyzed using JACoP plugin (scales start at 0.5 above which the % of  co-localization  is considered significant), number of vesicles  > 2000 from at least three biological replicates were analyzed corresponding to 20 cells. Data information: In graphs representing the number of vesicles  per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.",
    "molecules": "Lysotracker"
  },
  {
    "caption": "(D) CD74  expression is partially recovered by blocking lysosomal  acidification. As in (A), following siRNA transfection of Hela-CIITA  cells, CD74  expression was assessed. The last 16h prior to harvesting, cells were treated with chloroquine  (CQ) or epoxomicin  (Epo). Left panel a representative experiment is shown, membranes were blotted  using anti-T6BP, -CD74  and -actin  antibodies. The different degradation fragments of CD74  are indicated (Iip33, Iip22 and Iip16). Right panel, expression levels of Iip33 (top) and Iip16 (bottom) were quantified in three biological replicates using ImageJ and are presented as mean of ratios of each fragment to actin  (+/-SD). Data information: (D) One-way Anova statistical test combined with Bonferronis multiple comparison test was applied. Dotted lines indicate statistically significant differences between conditions.",
    "molecules": "chloroquine, CQ, Epo, epoxomicin"
  },
  {
    "caption": "(E Analysis of CD74 proteolysis. HeLa-CIITA  cells were transfected with siCTRL and siT6BP. 48h post treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed and chased for 1, 2 and 4h. HLA-DR/CD74  complexes were first immunoprecipitated  using Tü36 antibody (E) Samples were boiled and analyzed using  SDS-PAGE. The bands corresponding to CD74  isoforms (Iip41 and Iip33) and the cleavage products (Iip16) are indicated. (E right panel) Iip16 expression was quantified using ImageJ and presented as a percentage of Iip16 normalized to the highest quantity of Iip16 detected after 1h of chase in the control condition. Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control.",
    "molecules": "Cys, Met, 35S"
  },
  {
    "caption": "F). Analysis of CD74 proteolysis. HeLa-CIITA  cells were transfected with siCTRL and siT6BP. 48h post treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed and chased for 1, 2 and 4h.  HLA-DR/CD74  complexes were immunoprecipitated  using VICY1 antibody (F). Samples were boiled and analyzed using SDS-PAGE. The bands corresponding to CD74  isoforms Iip33) Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control.",
    "molecules": "Cys, Met, 35S"
  },
  {
    "caption": "(C) CANX interacts  with T6BP  using proximity ligation assay (PLA). 48h post-transfection with the indicated siRNAs, HeLa-CIITA  cells were fixed, stained  with anti-T6BP  and anti-CANX  antibodies and proximity revealed using PLA  (Duolink). Nuclei  were stained  using DAPI. Top panel, two representative fields are shown. Bottom panel, quantitative analysis using ImageJ displaying the number of PLA  per cell. The continuous line indicates the mean (+/-SEM). PLA  were quantified in at least 130 cells corresponding to three biological replicates. Scale bars: 10μm. CTRL: control; Mann-Whitney's test; ***: p<0.0003.",
    "molecules": "DAPI"
  },
  {
    "caption": "H. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; LSL-ROCK2:ER (RKPC) mice without (n=21) or with conditional ROCK activation with tamoxifen citrate (n=19). Survival p value determined by Log-Rank test.",
    "molecules": "tamoxifen citrate"
  },
  {
    "caption": "A. H&E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor. Means ± SEM (n=9 for untreated, n=8 for H1152), p value by unpaired t test.",
    "molecules": "H1152"
  },
  {
    "caption": "C. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER-fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.",
    "molecules": "H1152, EtOH, 4HT"
  },
  {
    "caption": "D. H&E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPflC cells treated with 1 µM 4HT. Means ± SEM (n=6), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.",
    "molecules": "4HT"
  },
  {
    "caption": "H. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on uncoated plastic surfaces. Means ± SEM (n=3).I. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on collagen1-coated surfaces. Means ± SEM (n=3).",
    "molecules": "4HT"
  },
  {
    "caption": "A. Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate (FDR) < 5%, with log10 fold-change (FC) in expression in 4HT treated ROCK1:ER expressing cells relative to GFP:ER expressing cells (left) or 4HT treated ROCK2:ER expressing cells relative to GFP:ER expressing cells (right) versus log10 adjusted p value (n=3). ROCK1:ER+4HT vs GFP:ER+4HT; 3828 genes. ROCK2:ER+4HT vs GFP:ER+4HT; 4481 genes.",
    "molecules": "4HT"
  },
  {
    "caption": "E. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.",
    "molecules": "H1152, EtOH, 4HT"
  },
  {
    "caption": "A. Confocal microscope images of ROCK1:ER-expressing cells co-stained for F-actin, MMP10 (left) or MMP13 (right) and DAPI following treatment with vehicle or 1 µM 4HT for 24 h. Multiple z-planes were used to generate x-z and y-z images. Scale bar = 5 µm.",
    "molecules": "4HT"
  },
  {
    "caption": "B. Representative immunoblot of MMP13 and MMP10 in conditioned media, as well as MMP10 and GAPDH in cell lysates. Cells were treated with vehicle (-) or 1 µM 4HT for 48 h.",
    "molecules": "4HT"
  },
  {
    "caption": "C. Representative stained gel of ultracentrifuge-enriched microvesicle protein from GFP:ER or ROCK1:ER-expressing cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h (top). Absolute arbitrary unit values for total microvesicle protein levels in stained gels (bottom). Means ± SEM (n=4), p value by ratio paired t-test.",
    "molecules": "protein, 4HT"
  },
  {
    "caption": "D. Representative immunoblot of MMP10 and caveolin in ultracentrifuge-enriched microvesicle proteins from cell-conditioned media following treatment with vehicle (-) or 1 µM 4HT for 24 h as well as MMP10, caveolin and GAPDH in cell lysates (top left). Absolute arbitrary unit values for MMP10 levels in western blots of ultracentrifugation-enriched microvesicle proteins (bottom left). Means ± SEM (n=4), p value by ratio paired t test. Ratios of MMP10 to caveolin (bottom center) or to total microvesicle proteins (bottom right). Means ± SEM (n=4), p value by ratio paired t test.",
    "molecules": "4HT"
  },
  {
    "caption": "D. Transmission electron microscopy of ultracentrifuge-enriched microvesicles (top center), and immunogold labeling of MMP10 in ROCK1:ER 4HT-treated microvesicles, indicated by red arrows (top right).",
    "molecules": "4HT"
  },
  {
    "caption": "A. Representative fluorescence images of cells treated with 1 µM 4HT in the absence or presence of 10 µM H1152 for 18 h on Collagen1-FITC (Col1-FITC). Co-staining for F-actin, MMP10 (red) and DAPI (blue). Scale bar = 10 µm.B. Quantification of collagen degradation by cells treated with 1 µM 4HT or 4HT + 10 µM H1152 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.",
    "molecules": "H1152, 4HT"
  },
  {
    "caption": "C. Quantification of collagen degradation by cells treated with 1 µM 4HT, 4HT + DMSO vehicle, 4HT + 50 µM Blebbistatin or 4HT + 10 µM GM6001 for 16 h. Means ± SEM (n=15), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.",
    "molecules": "Blebbistatin, DMSO, GM6001, 4HT"
  },
  {
    "caption": "A. H&E stained sections of cell invasion into collagen matrix after 8 days, in the absence (top) or presence of 10 µM GM6001 (bottom). Scale bar = 100 µm.B. Invasion index of KPflC cells. Means ± SEM (n=4), p value by unpaired t test.C. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=20), p value by unpaired t test.",
    "molecules": "GM6001"
  },
  {
    "caption": "D. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.E. Ki67 positive cell percentages at the collagen matrix surface. Means ± SEM (n=20; n=19 for GFP:ER/vehicle, n=18 for GFP/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Ki67 positive cell percentages in collagen matrix. Means ± SEM (n=20; n=8 for GFP:ER/ vehicle, n=10 for GFP:ER/GM6001, n=17 for ROCK1:ER/GM6001, n=15 for ROCK2:ER/GM6001), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.",
    "molecules": "GM6001"
  },
  {
    "caption": "G. The increase in viable cell numbers of cells plated on uncoated plastic surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µm GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.H. The increase in viable cell numbers of cells plated on collagen1 (Col1) coated surfaces and treated with 1 µM 4HT for 24 h was not affected by 10 µM GM6001. Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.",
    "molecules": "GM6001, 4HT"
  },
  {
    "caption": "A. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; (KPC) mice without (n=13) or with Fasudil treatment (n=13). Survival p-value determined by Gehan-Breslow-Wilcoxon test.",
    "molecules": "Fasudil"
  },
  {
    "caption": "B. Representative images of picrosirius red stained endpoint tumors from KPC mice. Scale bar = 1 mm.C. Picrosirius red percentage positive staining area (top) or average staining intensity of positive area (bottom) in pancreatic tumors from vehicle (n=8) and Fasudil (n=7) treated mice. Box plots with exact p value by Mann-Whitney test.",
    "molecules": "Fasudil"
  },
  {
    "caption": "Nanoconjugate internalization in CXCR4 overexpressing (CXCR4+) SW1417 CRC cells after 1 hour exposure at 1µM, as measured by fluorescence emission using flow cytometry. (N=3 experiments in duplicate) Significant difference at p= 0.002 between T22-GFP-H6-FdU and AMD3100+T22-GFP-H6-FdU groups",
    "molecules": "AMD3100, FdU"
  },
  {
    "caption": "Intracellular trafficking of T22-GFP-H6-FdU in CXCR4+ SW1417 cells by confocal microscopy after exposure at 1µM for 24 h. The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye (CellMask™), whereas cell nucleus was stained in blue with hoescht. The insets show detail of the intracellular localization of nanostructured, fluorescent entities, in an isosurface representation within a three-dimensional volumetric x-y-z data field",
    "molecules": "red dye, FdU, hoescht"
  },
  {
    "caption": "Linearized T22-GFP-H6-FdU dose-response trend line representation compared with unconjugated free oligo-FdU exposure. Antitumor effect was measured as CXCR4+ SW1417 cell viability by MTT after 72 h exposure as the described concentrations. (N=3 experiments in duplicate)",
    "molecules": "MTT, FdU"
  },
  {
    "caption": "Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22-GFP-H6-FdU for 48 h, as compared to T22-GFP-H6 or free oligo-FdU. (N=3 experiments in duplicate; Scale bar, 100µm).",
    "molecules": "FdU"
  },
  {
    "caption": "Selective T22-GFP-H6-FdU nanoconjugate biodistribution in subcutaneous CXCR4+ SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose, as measured by fluorescence emission using IVIS Spectrum 200 (N=5/group). Biodistribution is similar to that achieved by the T22-GFP-H6 targeting vector and undetectable after Buffer or free oligo-FdU treatment. (N=5 mice/group)",
    "molecules": "FdU"
  },
  {
    "caption": "Co-localization (yellow merged) of the T22-GFP-H6-FdU (green) and the CXCR4 receptor (red) and release of T22-GFP-H6-FdU into the cytosol in CXCR4+ tumor cells 5h after a 100ug dose of nanoconjugate, as measured by dual anti-GFP/anti-CXCR4 immunofluorescence (IF). DAPI (blue nuclear staining). Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area (A)±s.e.m (µm2) (N=10, 2 tumor fields x 5 mice; 200X). Note the significant (P=0.003) increase in the area occupied by the green dots (nanoconjugate realeased to the cell cytosol) in T22-GFP-H6-FdU-treated tumors, compared to free oligo-FdU-treated control tissues. Scale bar, 50 µm.",
    "molecules": "DAPI, FdU"
  },
  {
    "caption": "Administration of the CXCR4 antagonist AMD3100 completely blocks T22-GFP-H6-FdU tumor biodistribution, as measured by fluorescence emission. Fluorescence is not detected in Buffer or free oligo-FdU controls (N=5 tumor fields/group).",
    "molecules": "AMD3100, FdU"
  },
  {
    "caption": "The uptake of T22-GFP-H6-FdU observed in CXCR4+ SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration, as quantified using the anti-GFP IHC H-score. (N=5 tumor fields/group).",
    "molecules": "AMD3100, FdU"
  },
  {
    "caption": "Representative images of T22-GFP-H6-FdU uptake and AMD3100 competition by anti-GFP inmunostaining, which quantitation is reported in panel E. Scale bar, 50 µm",
    "molecules": "AMD3100, FdU"
  },
  {
    "caption": "Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N=5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6 and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase 3 activation (measured with anti-ɣH2AX or anti-cleaved caspase 3 by IHC) 5h post-administration (middle panels). Apoptotic induction (Hoescht staining, 24h post-administration, lower panels). Note the higher number of cells positive for DSBs, Caspase 3 activation and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells. Quantitation of the number of cells containing DSBs or active Caspase-3 in IHC-stained tumor sections 5h post-treatment and the number of condensated or disaggregated nuclei (by Hoescht staining) 24h post-treatment in tumor sections of 10 high-powder fields (400x magnification) using the Cell D software. (N=50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean±s.e.m.",
    "molecules": "DNA, FdU, Hoescht"
  },
  {
    "caption": "T22-GFP-H6-FdU depletes CXCR4+ cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration. Note the reduction in CXCR4+ cell fraction in the tumor 24h after injection, their almost complete elimination at 48h and the re-emergence of CXCR4+ cells 72h post-administration, using anti-CXCR4 IHC. In contrast, the CXCR4+ cancer cell fraction (CXCR4+ CCF) in tumor tissue remains constant along time after free oligo-FdU treatment. The three day time-lapse for CXCR4+ tumor cell re-appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect. (N=5: 5 mice/group; 1 samples/mouse). Scale bar, 50 µm. Data expressed as mean±s.e.m",
    "molecules": "FdU"
  },
  {
    "caption": "Significant reduction in the number of spheroid formed (C. optical microscope) and their bioluminescence emission (D, IVIS Spectrum 200), generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates), obtained from CXCR4+ luciferase+ SW1417 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. D. Quantitation of the bioluminescent signal (BLI) expressed as Average Radiant Intensity, obtained using the IVIS spectrum 200 equipment (N=2 plates/group).",
    "molecules": "FdU"
  },
  {
    "caption": "Reduction in the number of formed spheroids (optical microscope) generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates) obtained from CXCR4+ M5 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. (N=8; 2 mice/group; 4 plates/mouse). (Scale bar, 100µm).",
    "molecules": "FdU"
  },
  {
    "caption": "Reduction in tumor re-initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice (N=4 tumors/group) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days , as compared to Free oligo-FdU-treated or Buffer-treated mice. Recording of the number and size of positive tumors. (N=4; 2 mice/group, 2 injection points/mouse).",
    "molecules": "FdU"
  },
  {
    "caption": "T22-GFP-H6-FdU-induced reduction in the number of intravasated tumor emboli in peri-tumoral vessels of the M5-orthotopic primary tumor (E. optical images; F. emboli number quantitation) and reduction of CXCR4 expression in these emboli (G), treated 7 days after tumor cell implantation with 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. Tumor emboli counting in 10 high powder field at 200x magnification in H&E stained sections from each tumor. (N=5/group).",
    "molecules": "FdU"
  },
  {
    "caption": "T22-GFP-H6-FdU prevents metastases in the CXCR4+ patient-derived M5 model by potently reducing the total and mean number of liver, lung and peritoneal mets, as recorded in H&E stained histology sections at the end of treatment, in comparison to free oligo-FdU o Buffer-treatment. In contrast, the number of LN mets is not reduced after T22-GFP-H6-FdU or free oligo-FdU administration (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m.",
    "molecules": "FdU"
  },
  {
    "caption": "T22-GFP-H6-FdU induces a higher reduction of CXCR4+ cancer cell fraction (CXCR4+ CCF) in liver, lung and peritoneal metastatic tissue, at the end of treatment, than free oligo-FdU, as measured by anti-CXCR4 IHC. In contrast, T22-GFP-H6-FdU or free oligo-FdU does not reduce the CXCR4+ CCF in LN mets or primary tumor tissue after therapy. (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m",
    "molecules": "FdU"
  },
  {
    "caption": "Representative CXCR4 IHC images of the reduction in CXCR4+ CCF induced by T22-GFP-H6-FdU (or its absence in free oligo-FdU mice) at the end of treatment, in the M5 patient-derived CRC model, which quantitation is reported in panel B. In the M5 model, the highest reduction in foci number and size occurs in liver metastases, which show the highest reduction in CXCR4+ CCF.",
    "molecules": "FdU"
  },
  {
    "caption": "Undetectable T22-GFP-H6-FdU emitted fluorescence in normal tissues, except for a transient accumulation 5h after a 100μg dose in the liver, which disappears at 24 h. Liver emitted fluorescence is transient and significantly lower than the one registered in tumor tissue. Tumor/Liver ratio= 7.5 (see tumor intensity in Fig 2B, which was registered in the same experiment) (N=5 mice/group). Scale bar, 1 cm.",
    "molecules": "FdU"
  },
  {
    "caption": "Representative images depicting the level of DNA double strand break (DSB) induction in histologically normal bone marrow 5h after treatment, as measured by anti-ɣ-H2AX, which is higher in free oligo-FdU-treated mice than in T22-GFP-H6-FdU (P=0.047). Low level of cells containing DSBs in histologically normal kidney after T22-GFP-H6-FdU or free oligo-FdU treatment, a finding occurring in all normal tissues analysed. (N=50, 5 mice/group; 10 fields/mouse).",
    "molecules": "DNA, FdU"
  },
  {
    "caption": "Representative images showing lack of histopathological alterations in H&E stained tissue or apoptotic induction in H&E stained samples of CXCR4+ (bone marrow) and CXCR4- (brain, kidney, liver, lung and heart) normal tissues 24h after the administration of a 100 μg dose of T22-GFP-H6-FdU or an equimolecular dose of free oligo-FdU (N=5/group). Note that the transient nanoconjugate distribution to liver or the DNA damage induced in bone marrow do not lead to cytoxicity on these no-tumor tissues. (N=50, 5 mice/group; 10 fields/mouse)",
    "molecules": "FdU"
  },
  {
    "caption": "Lack of differences in body weight among groups registered along time in the SW1417-derived CCR model and the regression of metastases protocol (N=10 mice/group). Lack of differences in body mouse weight among groups registered along time in the SW1417 cell line-derived model and the prevention of metastasis protocol (Buffer (N=11; Free oligo-FdU (N=12), T22-GFP-H6-FdU (N=12)). Lack of differences in body mouse weight among groups registered along time in the M5 patient-derived model and the prevention of metastasis protocol (Buffer (N=6); Free oligo-FdU (N=17); T22-GFP-H6-FdU (N=8). Scale bar, 100 μm",
    "molecules": "FdU"
  },
  {
    "caption": "Fluorescence microscopy images of smFISH probes labelled with CAL-Fluor 610 (grey) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.",
    "molecules": "CAL-Fluor 610, Hoechst 33342"
  },
  {
    "caption": "Fluorescence microscopy images of smFISH probes CAL-Fluor 610 (grey) overlayed with Hoechst 33342 staining (blue) at 3 h after 10 Gy IR or the indicated target genes in A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.",
    "molecules": "CAL-Fluor 610, Hoechst 33342"
  },
  {
    "caption": "Chk2 inhibition with the small molecule BML-277 induces transient p53 dynamics with only one pulse after 10 Gy IR . A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and addition of 10 µM BML-277 by immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).",
    "molecules": "BML-277"
  },
  {
    "caption": "Sequential treatment with Nutlin-3 converts pulsatile p53 dynamics into sustained nuclear levels. A schematic illustration of the experimental setup and quantification of p53 levels in A549 wild type cells after irradiation with 10 Gy IR and sequential treatment with 0.75 µM Nutlin-3 at 2.5 h, with 2.25 µM at 3.5 h and 4 µM at 5.5 h post IR based on immunofluorescence staining (see Methods section for details) are shown as box plots (see Data Visualization section).",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "We quantified promoter activity of MDM2 (E, transient archetype) and CDKN1A (F, transient archetype) before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after irradiation with 10 Gy IR and sequential Nutlin-3 treatment. Left panel: The percentage of cells with active TSS, subdivided into populations with strong (> 75% of TSS, solid colors) and weak (<75% of TSS, shaded colors) activity, is shown as stacked bar graphs, the mean fraction of active promoters is indicated above each bar. Right panel: Distributions of calculated transcription rates at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The relative fraction of active promoters strongly increased, changing transient to sustained archetypes. The transcription rate increased as well both compared to basal levels and to previous experiments with pulsatile p53 dynamics (inset, fold change relative to IR alone for each time point).",
    "molecules": "Nutlin-3"
  },
  {
    "caption": "Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western Blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML-277, central lanes) and sustained p53 (10 Gy IR + Nutlin-3, right lanes). See Figure 3 and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western Blot and normalized to the abundance 3 h post IR. Means and propagated standard errors from 3 independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12.",
    "molecules": "BML-277, Nutlin-3"
  },
  {
    "caption": "E. RT-qPCR of Axin2 in bEnd.3 cells treated with NDP (200 ng/ml), isotype control or F4L5.13 (1200 ng/ml) and transfected with control, Tspan12 or Fzd4 siRNAs. Data are presented as mean ± SD, n=3 technical replicates. Data is representative of two independent experiments. F. Time course of phosphorylated Disheveled-3 (p-DVL3) and ßcatenin protein levels in bEnd.3 cells treated with 30nM of F4L5.13 or NDP. Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß-Tubulin measured by densitometry of independent experiments. Data are presented as mean ± SEM, n=4-6 (*p<0.05 as compared with NT). Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared with NT). ",
    "molecules": "F4L5.13"
  },
  {
    "caption": "A. Volcano plot of gene expression changes in bEnd.3 cells following 24h of treatment with 30nM F4L5.13. Genes with significant changes in expression level are highlighted in blue and genes co-regulated by treatment with 30nM NDP for 24h are highlighted in red. Statistical analysis was performed using the DESeq2 R package and adjusted p-value (Wald test) threshold less than 0.1 was used. Labelled genes are associated with enriched GO biological processes indicated below.",
    "molecules": "F4L5.13"
  },
  {
    "caption": "B. Immunofluorescence of ZO-1, CLDN3 and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1h. ZO-1 is shown in green, CLDN3/5 in red and DAPI in blue. Scale bars: 15 µm. C. Quantification of ZO-1, CLDN3 and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to VEGF treatment). D ",
    "molecules": "F4L5.13, DAPI"
  },
  {
    "caption": "C. IB4-stained retinal cross sections show the restored retinal vasculature and the absence of misdirected angiogenesis after F4L5.13 treatment. Boxed areas are shown enlarged in the panels on the right. Scale bars: 500 µm. D. Quantification of the vascularized area in the outer plexiform layer. 10 mg/kg F4L5.13 or isotype control antibody was injected every 48h from P5 to P20. Data are presented as mean± SD, n=5 retinas (2-4 fields of view per retina) from 4-5 mice per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test. E ",
    "molecules": "F4L5.13"
  },
  {
    "caption": "E. F4L5.13 rescues barrier function defects in Tspan12-/- mice as shown by an increased expression of the tight junction component CLDN5 and decreased expression of the EC fenestration component PLVAP. IB4-Alexa647 was used to stain ECs. Scale bars: 100 µm.",
    "molecules": "F4L5.13, Alexa647"
  },
  {
    "caption": "D. Flat-mounted retinas of adult Tspan12-/- mice injected systemically with sulfo-NHS-biotin show that F4L5.13 partially restored BRB function. PECAM was used to stain endothelial cells. Images are representative of 4 retinas per group. Scale bar: 500 µm.",
    "molecules": "F4L5.13, sulfo-NHS-biotin"
  },
  {
    "caption": "E. Maximum intensity projection of adult Tspan12-/- retinas after mice were injected with F4L5.13 or isotype control. Endothelial cells were stained with IB4-Alexa 647. Images are representative of 4 retinas per group. Scale bar: 100 µm.",
    "molecules": "F4L5.13, Alexa 647"
  },
  {
    "caption": "A. Schematic diagram of the OIR model (top) and representative images of P17 OIR retinas are shown (bottom). Neonatal mice were exposed to 75% oxygen from P7 to P12 to induce vessel loss. Then mice received intravitreal injections of PBS vehicle, mouse-specific anti-VEGF (as a positive control) delivered at 0.1 μg/μl, or F4L5.13v2 (a second-generation F4L5.13 modality, in which the N-terminal FZD4-specific diabody was replaced with a FZD4-specific Fab) at 50nM or 500nM target vitreous concentrations. Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17. At P17, retinas from injected mice were collected, dissected as flat‐mounts and stained with Isolectin B4. Scale bar = 500 µm. B. Quantification of the ratio of neovascular and avascular area on total area of vascularization. Data are presented as mean ± SEM, n=11-13 retinas per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (indicated p-values are in comparison to Vehicle). E ",
    "molecules": "F4L5.13, F4L5.13v2, oxygen"
  },
  {
    "caption": "G)  HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1  and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained  72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei  were  stained with DAPI (blue). Scale bar is 50 μm. Total nuclei  (>450 nuclei  per experimental condition) and  IRF3-positive nuclei  were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.",
    "molecules": "DAPI"
  },
  {
    "caption": "A) ADAR1-knockout HEK293 cells (clone 1) were modified with a  lentiviral-based inducible system to express FLAG-LGP2 WT or a FLAG-LGP2 RNA binding mutant (K138E/R490E/K634E, denoted as \"KRK mutant\") in a  doxycycline-regulated manner. Cells were treated 72h with  doxycycline  (dox). Protein lysates were analyzed by SDS-PAGE  and  immunoblotting  using the indicated antibodies (n=3). iEV = inducible empty vector; iLGP2  = inducible LGP2.",
    "molecules": "dox, doxycycline"
  },
  {
    "caption": "C) Cells generated in (A) were plated on coverslips and treated with or without  doxycycline  for 72h. Cells were fixed, permeabilized and stained with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained  with  DAPI  (blue). Scale bar is 50 μm. Total  nuclei  (>500  nuclei per experimental condition) and  IRF3-positive nuclei  were counted using semi-automated software analysis and plotted as percentage IRF3-positive  nuclei  of total nuclei per field of view (a representative of two biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analysis was performed using a Kruskal-Wallis test with a Dunn's post hoc test for multiple comparisons. ns, not significant; ****, p<0.0001.",
    "molecules": "DAPI, doxycycline"
  },
  {
    "caption": "B) CAL27 cells transduced with doxycycline-inducible shRNAs targeting  ADAR1 or GFP (negative control) were treated with doxycycline  and transfected with two independent siRNAs targeting LGP2  (siLGP2 #1 or #2) or a control siRNA (siCtrl). To determine  cell confluency at 120h post-transfection, cells were fixed, stained  with Crystal Violet, and imaged. Images of a representative experiment are shown (n=3). C) Crystal Violet was extracted from stained cells (B) and the dye intensity was quantified using a colorimetric assay (OD590). OD590 values of doxycycline-treated cells were normalized to the OD­590 values of untreated cells. Quantification of data from three independent experiments is shown as mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.",
    "molecules": "Crystal Violet, doxycycline"
  },
  {
    "caption": "A) HT29  cells were treated with or without 300 nM  5-AZA-CdR  for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the  type I IFN  response was analyzed by RT-qPCR  analysis of  IFN-β, IFIT1, and  ISG15 transcript  expression, normalized to  ACTB. B) LIM1215 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed as in (A). C) LIM1215 cells were treated with 250 nM palbociclib or a DMSO  control for 7 days. Three days after treatment initiation, cells were transfected with the indicated siRNAs and cultured for an additional 72h. The type I IFN  response was monitored as in (A). Data information: Data from three biological independent experiments are shown with mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. ",
    "molecules": "5-AZA-CdR, DMSO, palbociclib"
  },
  {
    "caption": "S values measured in different environments. In all cases the standard error is below 5%. Color scale: purple in absence of uracil (-Ura), white in the presence of uracil (+Ura) and orange in the presence of 5FOA (+5FOA). At 37°C, the heat stress together with the inability to fold Ura3p have such a strong fitness effect that the strains barely grew in absence of uracil, impeding the measurement of S. A bar of purple-white diagonal lines indicates a presumed value of S at 37°C without uracil.",
    "molecules": "5FOA, Ura, uracil"
  },
  {
    "caption": "E. Confocal microscopy image of URA3agg incubated in the presence of 5FOA after 18 hours at 30°C. F. Schematic showing that in our system, the phase separation-promoting region can modulate Ura3p activity and thus its phenotypic effect in a cell. G. Schematic highlighting that higher/lower level of free protein inside the cell results in higher/lower Ura3p activity leading to the manifestation of a stronger/weaker phenotype. H. Boxplots of the cytoplasmic fluorescence normalized by the cell area (FCYTOagg/cell area) for individual cells of the URA3agg population in 5FOA. Light green box, cells with foci containing at least 70% of the total fluorescence (big foci). Dark green box, cells with foci containing less than 70% of the total fluorescence (small foci). I-J. Cell cycle arrest phenotype. Density plots showing the fluorescence distribution of individual cells from the URA3agg population stained with propidium iodide to measure their DNA content. The cells were incubated for 18 hours at 30°C without (I) and with 5FOA (J). ",
    "molecules": "5FOA, DNA, propidium iodide"
  },
  {
    "caption": "After 2 hour starvation, E. coli cultures were pulse fed 10 µM glucose at varying frequencies using the spin flask and plate reader systems, and optical density (OD) was measured over time (inset example figures). Grey dots are OD measurements and the black lines are an empirical fit (see Methods). For separate experiments (n = 18), the lag time is plotted against the frequency, represented as the time-integrated (TI) feedrate f (mmol glucose/g dry cell weight/hour). An empirical fit (grey solid line, see Methods) was used to separate the lag (non-dividing) and dividing phases",
    "molecules": "glucose"
  },
  {
    "caption": "Immobilized cells in the microfluidic experiment divided after a lag time that decreased with increasing glucose pulse frequency. The labeled times indicate the period, time between pulses, for a given experiment",
    "molecules": "glucose"
  },
  {
    "caption": "Time course of the distribution of cellular DNA content. Sampled cells were stained with SYBR Green I and measured with flow cytometry over the course of a pulsing experiment (f = 0.6 mmol/g/h) The DNA content distribution over time is shown on the left side, and three specific time points are shown on the right. Within the first time point (t = 0), the highest distribution is taken to be high DNA content (2N), and the distribution at half of the 2N average was taken (1N) as medium DNA. DNA distributions were separated into medium (1N) and high DNA (2N). Distribution-specific estimated counts (see Methods) over time (f = 0.6 mmol/g/h) suggested that net division from high to medium DNA cells can explain the increase in cell counts and OD increase",
    "molecules": "DNA, SYBR Green I"
  },
  {
    "caption": "The spin flask system for glucose pulsing was connected to a real-time metabolomics platform. Traces of exemplary ions are shown that correspond to hexose phosphate, guanine, phenylalanine, and hypoxanthine for pulsing at non-division supporting frequencies of 0.06, 0.12, and 0.18 mmol/g/h. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Glucose pulses are indicated by the grey bars, and the pink region shows a no pulse control. Dots are ion intensity measurements. Solid lines are a moving average filter of the measured ion intensity. For clarity, dots are not shown for f = 0 mmol/g/h condition A metabolic scheme describing the propagation of fed glucose. Pulsed glucose is hypothesized to pass through central carbon metabolism and then be converted to downstream pathways including amino acid synthesis and nucleic acid synthesis. For nucleic acid synthesis, glucose is converted to the intermediate PRPP, which then can combine with nitrogen bases to form nucleotides for nucleic acid synthesis. Different pathways can be blocked with antibiotics",
    "molecules": "glucose, PRPP, Glucose, guanine, hexose phosphate, hypoxanthine, phenylalanine"
  },
  {
    "caption": "Influence of antibiotics that inhibit macromolecular synthesis at the non-division TI feedrate of 0.18 mmol/g/h. Antibiotics were added one minute after the second pulse (yellow region). Four different ions are shown corresponding to glutamate, phenylalanine, guanine, and thymine. Chloramphenicol (blue) inhibits protein biosynthesis, rifamycin (orange) inhibits RNA polymerase, and azidothymidine (AZT; red) inhibits DNA synthesis",
    "molecules": "Chloramphenicol, glutamate, guanine, phenylalanine, rifamycin, thymine, azidothymidine, AZT"
  },
  {
    "caption": "Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non-dividing cells. After 6 hours of pulsing uniformly labeled 13C-glucose, cultures were lysed, and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers. Labeling data are presented as the mean ± standard error of independent biological replicates (n = 3, all pairwise P < 0.02 as determined by one sided Student\"s t-",
    "molecules": "glucose, deoxyribose, 13C, threonine"
  },
  {
    "caption": "Pulsing experiment was repeated in the presence of protease inhibitor (PI) that reduced the lag time for a given TI feedrate (f = 0.28 mmol/g/h). The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line",
    "molecules": "glucose, PI, protease inhibitor"
  },
  {
    "caption": "Cells expressing a nearly functional FtsZ-mVenus fluorescent fusion as their sole copy of ftsZ were monitored after transition into carbon starvation. Two low motile MG1655 strains, one (wild-type) containing clpX and one without (ΔclpX), were grown in M9 glucose to steady-state. At time zero, M9 media without glucose was flushed through device. Cells rapidly ceased elongation as measured by phase contrast imaging at 2 minute intervals. Fluorescent images were taken every hour throughout to measure both total Ftsz-mVenus per cell and localization patterns Thin individual lines show the total fluorescent per cell in a single lineage (wild-type in solid blue, n = 154. ΔclpX in dotted red, n = 144). Thick solid lines are the time average across individual lineages for each subset (wild-type in blue, ΔclpX in red). After starvation, cells containing clpX degrade FtsZ faster than cells which do not. Fluorescent signals are normalized to the average value for their respective subset at time 0 Distributions of cellular fluorescence for each subset at times corresponding to the top panel. Cellular fluorescence varies widely as total FtsZ per cell is a function of cell size; a typical size distribution is shown at left. FtsZ concentration is roughly constant at steady-stat Student\"s t-test P value shown for when distributions differ with significance level α = 0.01. Representative images from cells in a single lineage with and without clpX. Wild-type strains may contain the characteristic FtsZ ring at midcell after shift down but it dissipates after several hours. ΔclpX strains display an abnormal FtsZ localization pattern even in steady-state. After shift down, the FtsZ may disassemble and reform along the cell body in distinct puncta. Image timing corresponds to the abscissa in the top",
    "molecules": "glucose, carbon"
  },
  {
    "caption": "Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate (f = 0.38 mmol/g/h). Induction level corresponds to the amount of doxycycline (max - 50 ng/uL, half - 10 ng/uL, and zero 0 ng/uL) added at the onset of starvation. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line. Lag time reduced with synthesis levels of titrated FtsZ in the wild-type strain at f = 0.31-0.32 mmol/g/h. Lag time increased with titrated synthesis of ClpX in wild-type cells at f = 0.43-0.45 mmol/g/h",
    "molecules": "glucose, doxycycline"
  },
  {
    "caption": "D Maximum intensity projections of smFISH fluorescence microscopy z-stacks of basal Jurkat T cells stained for the indicated genes. Nfkbia, Tnfaip3, Tnf, and  Il6 were labeled with Quasar 670, and Il8  and Csf2 were labeled with  fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm.",
    "molecules": "Quasar 670, fluorescein"
  },
  {
    "caption": "A Maximum  intensity projections of  smFISH fluorescence microscopy z-stacks of Jurkat T cells  stained for the indicated genes after 1-hour (Nfkbia, Tnfaip3, Tnf,  Il8) or 4-hour (Il6, Csf2) treatment with 20 ng/mL TNF. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8  and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm. B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes. Cells were combined from three replicates (Nfkbia 1h; Tnfaip3 1h; Tnf  1h, 2h; and  Il6 2h, 4h) two replicates (Nfkbia 2h) or one replicate (Tnfaip3 2h; Il8 1h, 2h; Csf2 2h, 4h). Basal data are same as in Fig. 1E. Error bars indicate bootstrapped 95% CIs for the samples in (C). Significant differences indicated by non-overlapping CIs. C ",
    "molecules": "Quasar 670, fluorescein"
  },
  {
    "caption": "A Enrichment of histone H3,  AcH3, total RNPII, ser5-p RNPII,  ser2-p  RNPII, and NELF-E in the basal state (0 hours) and after treatment (2 and 4 hours) with 20 ng/ml TNF  quantified using  ChIP  and shown as % input (non-IP control). Data are presented as mean ± standard error of the mean (s.e.m.) of three biological replicates. Significance calculated by Dunnett's multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).",
    "molecules": "ser"
  },
  {
    "caption": "C Bar graphs of mean mRNA level for basal condition, 1h TNF, 1h TNF + 4h pretreatment with 300nM A-485, and 1h TNF+62.5nM JQ1 cotreatment measured by smFISH for  Tnfaip3 (left) and Tnf (right). Cells were combined from two replicates (Tnfaip3 A-485, JQ1; Tnf A-485) or one replicate (Tnf JQ1). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant. D Change in enrichment of total and Serine-5-phosphorylated RNAPII  after 20ng/mL TNF treatment for 1 hour with 62.5nM  JQ1 measured by  ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean for two biological replicates. E ",
    "molecules": "A-485, JQ1, Serine"
  },
  {
    "caption": "e-h, Oxygen consumption rate (OCR) of unstimulated (e) and stimulated CD8 Tn (f) by 2ug/ml of anti-CD3 and 1ug/ml of anti-CD28 for 24 hours. The CD8 Tn were isolated from 45-wk-old mice. OCR of unstimulated (g) and stimulated CD8 Tn (h) by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each OCR measurement panel, representation plots following Mito stress test. Right of each panel, quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin (Oligo), Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) or Rotenone and Antimycin (AA/Rot) was added.",
    "molecules": "AA, Antimycin, FCCP, phenylhydrazone, Oligo, Oligomycin, Rot, Rotenone, Carbonyl cyanide-4, trifluoromethoxy"
  },
  {
    "caption": "i-k, Extracellular acidification rate (ECAR) of unstimulated and stimulated CD8 Tn by anti-CD3 and -CD28 antibodies for 24 hours (i). The CD8 Tn were isolated from 45-wk-old mice. ECAR of unstimulated (j) and stimulated (k) CD8 Tn by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each ECAR measurement panel, representation plots following Glycolysis stress test. Right of each panel, quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions. The dashed line indicates the time when the corresponding modulator glucose, Oligomycin (Oligo) or 2-deoxy-glucose (2-DG) was added.",
    "molecules": "2-deoxy-glucose, 2-DG, glucose, Oligo, Oligomycin"
  },
  {
    "caption": "The C-terminal domains of IFT52 and IFT46 co-purify with the IFT81460-C/74460-C/27/251-136 protein complex (C) The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.",
    "molecules": "Coomassie"
  },
  {
    "caption": "The C-terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623-C of IFT81 and 615-C of IFT74 (D). The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.",
    "molecules": "Coomassie"
  },
  {
    "caption": "(G) Coomassie stained SDS PAGE gel of a GST-pulldown using the CrIFT88His-GST/70/521-430/46 complex immobilized on GST beads as bait and the CrIFT811-387-GFP/74135-420 complex used as prey. The band highlighted by an * corresponds to pulled down CrIFT74135-420. CrIFT811-387-GFP runs on top of IFT70 and is not visible in the pull-down.",
    "molecules": "Coomassie"
  },
  {
    "caption": "(I) Distribution of number of nascent RNAs at the transcription site determined by smFISH. The distribution of UASwt does not fit a Poisson distribution (grey line), supporting that GAL3 is not constitutively transcribed, but is transcribed in bursts. Example image in shown, yellow arrows indicate TSs. Scale bars: 5 μm. n = 11,839 cells. (J) for cells with UASmut. The distribution of UASmut fits a Poisson distribution, indicating that GAL3-UASmut is transcribed with random initiation of individual polymerases, similar to constitutive genes. n = 10,616 cells.",
    "molecules": "nascent RNAs"
  },
  {
    "caption": "(A) Competitive binding experiments were performed to determine relative affinity of Gal4 to UASwt (green) and UASmut (blue) motifs. Gal4 occupancy on UASwt Cy3/Cy5 DNA was determined by measuring protein induced fluorescence enhancement (PIFE) on 51 bp oligos containing either Gal4 UASwt or UASmut site 1 bp away from Cy3 fluorophore).",
    "molecules": "Cy3, Cy5, DNA"
  },
  {
    "caption": "(B) Titrating unlabeled UASwt or UASmut competitor DNA reduces UASwt Cy3/Cy5 DNA occupancy. IC50UASwt = 4.0 ± 0.6 nM, IC50UASmut = 17.3 ± 3.5 nM. n = 3. Error bars indicate SD.",
    "molecules": "competitor DNA, Cy3, Cy5, DNA"
  },
  {
    "caption": "(C) Comparison between relative affinities of Gal4 UASwt versus UASmut in naked or nucleosomal DNA shows 4.3 ± 1.1x difference in KD from naked DNA and 6.8 ± 1.7x from nucleosomal DNA. n = 3. Error bars indicate SD.",
    "molecules": "DNA"
  },
  {
    "caption": "(D) Experimental setup for smFRET experiments to measure Gal4 binding at nucleosomal DNA. Gal4 binds to site 8 bp into nucleosome. A FRET pair in the entry/exit region provides readout on binding events (one fluorophore on DNA, one on histone). In the absence of Gal4, nucleosomes are in the high FRET state. A Gal4 binding event traps nucleosome in low FRET state.",
    "molecules": "DNA, histone"
  },
  {
    "caption": "(F) Example smFRET traces showing Gal4 binding to UASwt in nucleosomal DNA at two different Gal4 concentrations. States are determined using HMM fit. (G) Same as F but for UASmut. ",
    "molecules": "DNA"
  },
  {
    "caption": "(J) Histogram of Gal4 dwell time to nucleosomal DNA containing UASwt and UASmut sequences. n = 11 for UASwt, n = 8 for UASmut. Error bars indicate SE.",
    "molecules": "DNA"
  },
  {
    "caption": "(K) Scatter plot showing the average number of nascent RNA at TS (from smFISH) vs affinity of Gal4 to different UAS sequences in nucleosomal DNA (from in vitro measurements). In vivo transcription levels correlate with in vitro affinity of Gal4, but transcription saturates above wildtype sequence. UAS sequences are shown in the box. For mean number of nascent RNA from smFISH, n = 2 for UASconsensus, UAS-2C, UAS-8T and n = 8 for UASwt, UASmut and errors indicate SEM. For in vitro affinity measurements, n = 3 and errors indicate SD.",
    "molecules": "DNA, nascent RNA"
  },
  {
    "caption": "Profiles of nucleosome midpoint positions by MNase-seq experiments. Samples were digested with the indicated MNase concentrations in both raffinose (raf) and galactose (gal) containing media. Midpoints of nucleosomes are smoothed by 31 bp. In galactose the stable nucleosomes move away from the Gal4UAS, creating space for an additional fragile nucleosome (indicated by arrow).",
    "molecules": "gal, galactose, raf, raffinose"
  },
  {
    "caption": "(A) Simultaneous imaging of Gal4 binding kinetics in vivo using single-molecule tracking and RNA imaging of the GAL10 target gene. Gal4 is tagged with a HALO-tag, which covalently binds to the dye JF646. Transcription of the target gene GAL10 is visualized by PP7-loops.",
    "molecules": "dye JF646"
  },
  {
    "caption": "(D) Survival probability of the duration of Gal4 tracks (after displacement thresholding) at 200 ms interval from cells grown in raffinose (n = 258 tracks in 30 cells) or galactose (n = 346 tracks in 25 cells). Lines show bi-exponential fit, indicating 2 Gal4 populations. Inset shows data in semi-logarithmic plot. (E) Residence time of the fast and slow component of the fits from (D). The slow component changes between conditions. Error bars indicate 95% CI. ",
    "molecules": "galactose, raffinose"
  },
  {
    "caption": "(A) Example trace of PP7-GAL10 transcription in 2% galactose.",
    "molecules": "galactose"
  },
  {
    "caption": "(B) Boxplot of the onset of the first transcription event in different doses of galactose. The box indicates quartiles, the horizontal line inside the box indicates the median, the whiskers indicates 1.5 times the interquartile range of the box. Data points outside the whiskers are indicated with circles (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells).",
    "molecules": "galactose"
  },
  {
    "caption": "(C) The autocorrelation was used to interpret the burst duration and burst frequency changes. Burst duration is measured by the intersect of the two linear fits. When burst duration is constant, the amplitude is inversely related to the burst frequency. (D) Burst duration from autocorrelation is constant across 4 different galactose concentrations (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM. (E) Amplitude of autocorrelation decreases (burst frequency increases) at higher doses of galactose. (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM. ",
    "molecules": "galactose"
  },
  {
    "caption": "(F) Example traces of two single cells exposed to two different doses of galactose. (G, H) Same-cell dose response shows same burst duration (G) but lower amplitude (H) at higher galactose concentration, indicating that galactose levels regulate burst frequency (n = 13 cells). Error bars indicate SEM. ",
    "molecules": "galactose"
  },
  {
    "caption": "B Vero E6 cells were inoculated with SARS-CoV-2, fixed with paraformaldehyde after two days, stained with 30 nM of the indicated Alexa Fluor 488 (AF488)-labeled anti-RBD VHH antibodies, and imaged by  confocal laser scanning microscopy (CLSM). C Immunofluorescence (IF) staining  as in B, but with the indicated AF568-labeled anti-S1ΔRBD VHH antibodies. ",
    "molecules": "AF488, Alexa Fluor 488, AF568, paraformaldehyde"
  },
  {
    "caption": "A Vero E6  cells were infected with SARS-CoV-2, pre-incubated with indicated concentrations of the neutralizing VHH-72  Cells were fixed two days after inoculation,  stained with sets of anti-RBD (green) and anti-S1ΔRBD (red) nanobodies and analyzed by CLSM. We used a cocktail of such fluorophore-labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody.",
    "molecules": "fluorophore"
  },
  {
    "caption": "A  HeLa cells were transiently transfected to express the SARS-CoV-2 Spike protein. Following fixation, cells were  stained  for 1 hour with  fluorophore-labeled  Re10B10  (5 nM, green) and  Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled  nanobodies. The weakly binding competitors  Re5A08, Re9F06, Re6B06, and, Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated.",
    "molecules": "fluorophore"
  },
  {
    "caption": "RAW 264.7 mouse macrophages were treated with either oxLDL (50 µg/ml) or TG (300 nM) for 6 hours. Protein lysates were treated with λ Phosphatase (PPase) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=6 biological replicates).",
    "molecules": "oxLDL, TG"
  },
  {
    "caption": "Control- or IRE1-siRNA transfected HEK293T cells were stimulated by either PA (500 µM) or TG (600 nM) for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).",
    "molecules": "PA, TG"
  },
  {
    "caption": "MEF cells were transfected with either empty vector, EGFP-FMRP or 3xFLAG-IRE1 plasmids then pre-treated either with vehicle (dimethyl sulfoxide, DMSO) or AMG-18 (25 µM; 1 hour) followed by TG (600 nM) stimulation for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).",
    "molecules": "AMG-18, dimethyl sulfoxide, DMSO, TG"
  },
  {
    "caption": "Purified FMRP and IRE1 kinase (activated) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP, IRE1 and FMRP (n=3 biological replicates) and with LC-MS/MS. Identified IRE1 kinase-mediated FMRP phosphorylation sites (bottom).",
    "molecules": "ThioP"
  },
  {
    "caption": "Fmr1+/+ and Fmr1-/- mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n=7 mice per group; Scale bar = 50 µm).    Apoe-/- mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG-18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n=5 mice per group; Scale bar = 50 µm).  ",
    "molecules": "AMG-18, DMSO, Oil Red O, ORO"
  },
  {
    "caption": "RCT experiment (I) plasma cholesterol levels after 24 and 48 hours, (J) liver cholesterol levels after 48 hours, and (K) feces cholesterol levels after 48 hours (n=12 mice per group).",
    "molecules": "cholesterol"
  },
  {
    "caption": "A-E In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80+ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)+ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1- or control-siRNA and incubated CFSE-labeled AC for the indicated hours (n=4 biological replicates). (B) Fmr1+/+ and Fmr1-/- BMDMs were treated with PA (500 µM) for 6 hours and then incubated with CFSE-labeled ACs for 4 hours (n=4 biological replicates). (C) Fmr1+/+ and Fmr1-/- mice were fed WD (16 weeks) and injected intraperitoneally with CFSE-labeled AC (1.5 hours), followed by PM elicitation (n=4-5 mice per group). (D) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour then incubated with CFSE-labeled ACs for 4 hours (n=3 biological replicates). (E) C57BL/6 mice were injected with AMG-18 (30 mg/kg) or vehicle (DMSO) for 8 hours, followed by intraperitoneal injection with CFSE-labeled ACs for 1.5 hours and PM elicitation (n=4 mice per group).",
    "molecules": "carboxyfluorescein succinimidyl ester, CFSE, AMG-18, DMSO, PA"
  },
  {
    "caption": "F-G In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80+ (red), AC were labeled with CFSE (AC-1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80+, CFSE+ and Violet+ (triple positive) cells to total F4/80+ and CFSE+ (double positive) cells. (F) Fmr1+/+ and Fmr1-/- BMDM were incubated with AC-1 for 2 hours, and after 2 hours interval, incubated with AC-2 for 2 more hours (n=4-3 biological replicates). (G) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour, incubated with CFSE-labeled AC-1 for 2 hours, followed by incubation with Violet-labeled AC-2 for 2 hours and PM collection (n=4 biological replicates).",
    "molecules": "CFSE, AMG-18, DMSO"
  },
  {
    "caption": "RNA lysates from Fmr1+/+ and Fmr1-/- BMDM that were treated with PA (500 µM; 6 hours) were fractionated using a 10%-50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n=3 biological replicates). (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47 and Rac1 mRNA in polysome to NTR fraction (n=3 biological replicates).",
    "molecules": "PA, sucrose"
  },
  {
    "caption": "D Fmr1-/- MEF cells were transfected with EV, WT-FMRP or STSA-FMRP plasmids followed by PA treatment (500 µM; 6 hours). Protein lysates were analyzed by western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP and β-Actin and fold inductions relative to β-Actin are depicted above the blots (n=5 biological replicates).",
    "molecules": "PA"
  },
  {
    "caption": "Lesion area calculated from en face aorta, stained with ORO (n=12-13 mice per group; Scale bar = 5 mm).",
    "molecules": "ORO"
  },
  {
    "caption": "Lesion area calculated from en face aorta, stained with ORO (n=9 mice per group; Scale bar = 5 mm).",
    "molecules": "ORO"
  },
  {
    "caption": "Lesion area calculated from en face aorta, stained with ORO (n=5 mice per group; Scale bar = 5 mm).",
    "molecules": "ORO"
  },
  {
    "caption": "Lesion area calculated from en face aorta, stained with ORO (n=6 mice per group; Scale bar = 5 mm).",
    "molecules": "ORO"
  },
  {
    "caption": "D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samples",
    "molecules": "lysine"
  },
  {
    "caption": "C. Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples",
    "molecules": "lysine"
  },
  {
    "caption": "F. Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples",
    "molecules": "lysine"
  },
  {
    "caption": "A Detection of the activation of IRE1α, PERK and ATF6 UPR branches by immunoblotting in control (shCtrl) and Fam20C stable knockdown (shFAM20C) HepG2 cells treated with or without 5 μM Tg for 30 min. ATF6-FL, full-length ATF6; ATF6-N, N-terminal cleavage product of ATF6; Asterisk, unspecific background bands. Fam20C knockdown was verified by protein immunoblotting of Concanavalin A-Sepharose (Con A)-enriched culture medium. Ponceau staining was shown as a loading control.",
    "molecules": "Ponceau, Tg"
  },
  {
    "caption": "B (Left) Detection of spliced XBP1 (S) and unspliced XBP1 (U) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).",
    "molecules": "Tg"
  },
  {
    "caption": "C (Left) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi-resistant codon-altered Fam20C wild-type (WT) or its inactive D478A mutant (DA) treated with or without 5 μM Tg for 1 h. Fam20C expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).",
    "molecules": "Tg"
  },
  {
    "caption": "(Left) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells (D) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).",
    "molecules": "Tm"
  },
  {
    "caption": "Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA (E) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).",
    "molecules": "Tm"
  },
  {
    "caption": "F Volcano plot depicting the log2 of fold change versus - log10 (p-value) for Fam20C-interacting proteins in Tg-treated HepG2 cells. The red dots represent proteins showing statistically significant increase of binding with Fam20C (log2 fold change ≥ 1; - log10 (p-value) ≥ 1.3); the blue dots represent proteins showing statistically significant decrease of binding with Fam20C (log2 fold change ≤ - 1; - log10 (p-value) ≥ 1.3); the green dot is Fam20C.",
    "molecules": "Tg"
  },
  {
    "caption": "G HepG2 cells expressing FLAG-tagged Fam20C were treated with or without 5 μM Tg for 30 min. FLAG-immunoprecipitates were analyzed by PDI immunoblotting.",
    "molecules": "Tg"
  },
  {
    "caption": "A Representative MS/MS fragmentation spectrum of tryptic phosphorylated PDI peptide (Ile351-Lys370) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min.",
    "molecules": "Tg"
  },
  {
    "caption": "C Time-dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C. Phosphorylated PDI was determined by anti-pS357-PDI immunoblotting on both regular and phostag gels.",
    "molecules": "phosphate"
  },
  {
    "caption": "Detection of pS357-PDI in HepG2 cells treated with 5 μM Tg (E) for indicated times.",
    "molecules": "Tg"
  },
  {
    "caption": "Detection of pS357-PDI in HepG2 cells treated with 2.5 μg/ml Tm (F) for indicated times.",
    "molecules": "Tm"
  },
  {
    "caption": "Detection of pS357-PDI in HepG2 cells treated with 1 mM DTT (G) for indicated times.",
    "molecules": "DTT"
  },
  {
    "caption": "(Left) Detection of p-PDI in WT and PDI KO HepG2 cells (H) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of three (H) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).",
    "molecules": "Tg"
  },
  {
    "caption": "(Left) Detection of p-PDI in WT and Fam20C KO HeLa cells (I) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of four (I) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).",
    "molecules": "Tg"
  },
  {
    "caption": "A Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red). The cells were treated with DMSO (Upper) or 5 μM Tg (Middle) for 30 min. After Tg treatment, the cells were washed with PBS and then returned to culture medium for 60 min (Lower). GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.",
    "molecules": "DMSO, Tg"
  },
  {
    "caption": "C Detection of pS357-PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times.",
    "molecules": "Tg"
  },
  {
    "caption": "D HepG2 cells were treated with 25 ng/μl CHX for indicated times and analyzed by immunoblotting for pS357-PDI and UPR signaling. 5 μM Tg or DMSO was added 30 min before cell harvest.",
    "molecules": "CHX, DMSO, Tg"
  },
  {
    "caption": "E Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red) pretreated with 25 ng/μl CHX for 7.5 h, followed by introduction of DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.",
    "molecules": "CHX, DMSO, Tg"
  },
  {
    "caption": "F Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple-KDEL (red) treated with DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. Scale bar = 10 μm.",
    "molecules": "DMSO, Tg"
  },
  {
    "caption": "G HepG2 cells were transfected with empty vector (-), FLAG-tagged Fam20C or Fam20C-KDEL followed by introduction without or with 5 μM Tg for 30 min, respectively. PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels. p-PDI percentage was quantified based on band intensities on phostag gels.",
    "molecules": "Tg"
  },
  {
    "caption": "C (Left) ANS fluorescence spectra of PDI WT and ­­S357E. Controls with ANS alone was shown. A.U., arbitrary units. (Right) Enhancement factor were quantified. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).",
    "molecules": "ANS"
  },
  {
    "caption": "E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min, and in some aliquots of cells Tg was removed for 30 min. Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis.",
    "molecules": "Tg"
  },
  {
    "caption": "B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 μm.",
    "molecules": "TMR"
  },
  {
    "caption": "C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 μM Tg for 30 min. Scale bar = 10 μm.",
    "molecules": "P1, DMSO, Tg, TMR"
  },
  {
    "caption": "D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (-), PDI WT, S357E or S357A. Scale bar = 10 μm.",
    "molecules": "P1, TMR"
  },
  {
    "caption": "E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 μm.",
    "molecules": "P1, TMR"
  },
  {
    "caption": "F (Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (-), PDI WT, S357E or S357A. Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey's HSD test).",
    "molecules": "crystal violet, Tg"
  },
  {
    "caption": "A (Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05 (two-tailed Student's t-test).",
    "molecules": "Tg"
  },
  {
    "caption": "B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, the post hoc Tukey's HSD test).",
    "molecules": "Tg"
  },
  {
    "caption": "C Detection of IRE1α, PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min. Asterisk indicates unspecific background band.",
    "molecules": "Tg"
  },
  {
    "caption": "D HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.",
    "molecules": "Tg"
  },
  {
    "caption": "E HepG2 cells expressing FLAG-tagged IRE1α were treated without or with 5 μM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG-immunoprecipitates were analyzed by PDI and BiP immunoblotting.",
    "molecules": "Tg"
  },
  {
    "caption": "F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.",
    "molecules": "Tg"
  },
  {
    "caption": "J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h, and then used to pulldown His-IRE1αNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "C (Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).",
    "molecules": "Tm"
  },
  {
    "caption": "Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 μM Tg for 2 h (D)",
    "molecules": "Tg"
  },
  {
    "caption": "Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.5 μg/ml Tm for 2 h (E).",
    "molecules": "Tm"
  },
  {
    "caption": "Detection of Bip (F), Pdi (G), Erdj4 (H) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).",
    "molecules": "Tm"
  },
  {
    "caption": "Detection of Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).",
    "molecules": "Tm"
  },
  {
    "caption": "L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).",
    "molecules": "Tm"
  },
  {
    "caption": "M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed.",
    "molecules": "eosin, Hematoxylin, Tm"
  },
  {
    "caption": "Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.A Immunolabeling results (Kcnq1 labeled in green) in WT mice.B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/−mice, middle (B) and apical (C) turns, respectively.D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/−mice (black bars, right). Data are given as mean ± SD (n = 6).E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/−mice (F) are compared.Data information: Scale bars represent approximately 50 μm.",
    "molecules": "phalloidin, rhodamine"
  },
  {
    "caption": "B-B2 Largely non‐overlapping distribution of AVP+, oxytocin+ and secretagogin+ (sgcn+) neurons in the magnocellular PVN. The mousesupraoptic nucleus (SON) harbored vasopressin+ and oxytocin+ but not secretagogin+neurons. Open arrowheads pinpoint single‐labeled neurons. Solid arrowhead denotes AVP/secretagogin dual‐labeling. lv, lateral ventricle; PVNm, magnocellular part of the paraventricular nucleus; scgn, secretagogin.B3, B4 Secretagogin+neurons had smaller somatic diameters than AVP+ or oxytocin+neurons yet without a difference in their diameter quotient, a measure of ovoid profiles (*P 0.05, Student's t‐test).",
    "molecules": "vasopressin"
  },
  {
    "caption": "A Secretagogin (scgn) distribution at the ultrastructural level as revealed by pre‐embedding silver‐enhanced immunogold labeling. Secretagogin (arrowheads) was localized to membranous organelles in the perikarya (A), particularly the plasmalemma (A1) and endoplasmic reticulum (ER) in neuronal soma (s; A2). Open rectangles in (A) denote the location of insets. Semi‐transparent shading is used to visually dissociate subcellular compartments in (A-C).B Pre‐embedding secretagogin labeling (arrowheads) was also seen in dendrite (d) segments.C In axo‐dendritic terminals (ax), secretagogin was closely associated with synaptic vesicles along the plasmalemma (arrowheads).D Quantitative analysis of subcellular secretagogin distribution upon electron microscopy detection of silver‐enhanced gold particles. Particles were considered as membrane bound when they were at 50 nm of a membrane (plasma membrane or endomembrane; i.e., secretory vesicle, Golgi or endoplasmic reticulum). In the soma of PVNneurons, significantly more particles were found in the cytosol as along the plasma membrane (**P 0.01). In contrast, membrane association predominated in axonal nerve endings in the median eminence (*P 0.05). Note that a significant proportion of particles was found adjacent to endomembranes in all subcellular compartments studied.",
    "molecules": "gold"
  },
  {
    "caption": "B, B1 Large axon terminals in the median eminence were immunopositive for secretagogin with silver‐intensified immunogold particles (open arrowheads) associated with axonal membrane and dense‐core vesicles (see Fig D for quantitative data). Solid arrowheads denote silver deposit particles proximal to the plasmalemma.",
    "molecules": "silver"
  },
  {
    "caption": "C Immunoprecipitation using an anti‐secretagogin antibody in Ca2+‐free and Ca2+‐loaded conditions was subtractively used to decipher the Ca2+‐dependent protein interactions. Silver‐staining gel is shown.C1 Ontology classification of the 99 protein hits based on primary function assignments. Unbiased MALDI‐TOF proteomics was used to identify interacting proteins. The most abundant hits (Supplementary Table S3 is referred to for details on individual proteins) are proteins implicated in vesicle fusion, trafficking, transport and formation and the regulation of vesicle exocytosis. \"Other\" refers to a group of proteins without known function.",
    "molecules": "Ca2+"
  },
  {
    "caption": "B, B1 Stress induced by subcutaneous injection of formalin‐triggered co‐expression of c‐fos and CRH in secretagogin+ paraventricular neurons (arrowheads). Scale bars: 300 μm (B), 25 μm (B1).",
    "molecules": "formalin"
  },
  {
    "caption": "C, D In vivo siRNA‐mediated silencing of secretagogin expression in the PVN occluded the stress‐induced surge of serum ACTH levels (C) and significantly reduced the increase in plasma corticosterone (D). *P 0.05 versus control.",
    "molecules": "corticosterone"
  },
  {
    "caption": " B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows",
    "molecules": "Silver nitrate"
  },
  {
    "caption": "F. Violin plots depicting transcript levels of androgen-responsive genes in luminal-C cells of sham and CTX Pten(i)pe-/- mice. P-values were determined by Wilcoxon rank sum test.",
    "molecules": "androgen"
  },
  {
    "caption": "I. Western blot analysis of CC3 in protein lysates of organoids generated from prostates of Pten(i)pe-/- and Pten/Hif1a(i)pe-/- mice at 3 months AGI, and treated with enzalutamide (Enz) for 24 hours at the indicated concentrations. β-actin was used as a loading control.",
    "molecules": "Enz, enzalutamide"
  },
  {
    "caption": "D. Representative western blot analysis of CPARP and CC3 in human C4-2B PCa cells treated for 24 h with PX-478, increasing concentrations of enzalutamide (Enz), or a combination of both. Beta-actin was used as loading control.",
    "molecules": "PX-478, Enz, enzalutamide"
  },
  {
    "caption": "F. Representative H&E staining of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). Scale bar: 100 µm. N=3-4 mice/condition.",
    "molecules": "PX-478"
  },
  {
    "caption": "H. Gland areas of DLP of sham and CTX Pten(i)pe-/- mice, treated with vehicle or PX-478, as depicted in (E). N=3-4 mice/condition. Data presented are mean ± SEM. Five glands per mouse were quantified,",
    "molecules": "PX-478"
  },
  {
    "caption": "A Immunohistochemistry for SOX9 (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. SOX9 positive bile ducts (bd) and hepatocytes (arrowheads) indicated.",
    "molecules": "BDL, CCl4"
  },
  {
    "caption": "C Co-localization by immunohistochemistry (top panel) and in situ hybridization (lower panel) for SOX9 (red) and αSMA (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis.",
    "molecules": "BDL, CCl4"
  },
  {
    "caption": "D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control.",
    "molecules": "CCl4"
  },
  {
    "caption": " A-J Representative images and quantification shown for olive oil treated (n=6) or chronic CCl4 (n=5 Sox9fl/fl;RosaCreER-; n=8 Sox9fl/fl;RosaCreER+) induced fibrosis or following sham operation (n=5) or BDL (n=7 Sox9fl/fl;RosaCreER-; n=5 Sox9fl/fl;RosaCreER+)) induced fibrosis. A Picrosirius red (PSR) staining (collagen deposition in red) counterstained with fast green (top row) and immunohistochemistry for α-SMA (brown staining bottom row; activated HSC / myofibroblast marker) in olive oil treated (left) or chronic CCl4 induced fibrosis (right) in control and Sox9-null mice. Size bar = 200 μm. ",
    "molecules": "CCl4"
  },
  {
    "caption": "D PSR staining (red; top row) and immunohistochemistry for α-SMA (brown; middle row) and CK19 (brown; bottom row) in control and Sox9-null mice following sham operation (left) or BDL induced fibrosis (right). Size bar = 500 μm.",
    "molecules": "BDL"
  },
  {
    "caption": "G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).",
    "molecules": "CCl4"
  },
  {
    "caption": "G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).",
    "molecules": "bilirubin, BDL"
  },
  {
    "caption": "I Sox9-loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4. Olive oil treated mice had no bridging fibrosis, in line with a non-fibrotic liver histology in (A).",
    "molecules": "CCl4"
  },
  {
    "caption": "J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in (D) is reduced in Sox9-null mice compared to control following BDL. All mice were treated with tamoxifen (Tam) which did not induce ectopic expression of SOX9 in non-fibrotic livers (Figure 2A, E and Appendix Figure S3). All experiments are n≥5 as indicated. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated.",
    "molecules": "BDL, tamoxifen"
  },
  {
    "caption": "A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).",
    "molecules": "BDL, CCl4"
  },
  {
    "caption": " A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A). ",
    "molecules": "BDL, CCl4"
  },
  {
    "caption": "A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).",
    "molecules": "BDL, CCl4"
  },
  {
    "caption": "F F4/80 immunohistochemistry (brown staining) in control and Sox9-null mice livers following 4-weeks CCl4 induced fibrosis. Size bar = 100μm. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated. All experiments are n=4.",
    "molecules": "CCl4"
  },
  {
    "caption": "C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin (VP) in activated mouse HSCs expressed relative to DMSO control (n=3).",
    "molecules": "verteporfin"
  },
  {
    "caption": "D Immunohistochemistry for SOX9 (brown) following CCl4 or BDL induced fibrosis in mice treated with DMSO (CCl4 n=4, BDL n=7) or verteporfin (CCl4 n=3, BDL n=5).",
    "molecules": "BDL, CCl4, verteporfin"
  },
  {
    "caption": "a) Cells expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to abolish expression of endogenous VPS35. Each dish of cells was lysed in either HEPES lysis buffer (H) or PBS lysis buffer (P). Following centrifugation, equal portions of each lysate were combined to generate a mixed lysate (H/P). Each lysate was incubated with anti-GFP to recover the respective GFP-tagged VPS35 protein.",
    "molecules": "HEPES"
  },
  {
    "caption": "a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 × 10−6 (DMSO) and 3.81 × 10−5 (Baf) by 2-tailed Student's t-test.",
    "molecules": "bafilomycin A1, DMSO"
  },
  {
    "caption": "(h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(a) HeLa cells were transfected with pEGFP vector or GFP-FAM21 tail for 48 h and subsequently treated with bafilomycin A1 as in Fig. 4. Endogenous LC3-II and tubulin levels were assessed by western blot. (b) Quantification of the representative experiment in triplicate shown in a, of two independent experiments. *P=0.02 (DMSO) and 0.08 (Baf) by 2-tailed Student's t-test.",
    "molecules": "bafilomycin A1, DMSO"
  },
  {
    "caption": "(c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with pEGFP vector or GFP-FAM21, and subsequently treated with bafilomycin A1, lysed and subjected to western blot as in a. (d) Quantification of the representative experiment in triplicate shown in c, of two independent experiments. **P=0.008 by 2-tailed Student's t-test.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(e) Cells were depleted of WASH1, treated with bafilomycin A1 and examined for endogenous LC3-II, tubulin, WASH1 and GAPDH levels. A representative experiment of six independent experiments is shown. (f) Quantification of the representative experiment shown in e. **P=0.002 (DMSO) and P=0.0028 (Baf) by 2-tailed Student's t-test.",
    "molecules": "bafilomycin A1, DMSO"
  },
  {
    "caption": "(i) HeLa cells depleted of FKBP15 were subsequently transfected with a GFP-Q74 construct for 24 h and fixed in PFA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of two experiments, each in triplicate with min 220 cells counted per replicate",
    "molecules": "PFA"
  },
  {
    "caption": "(a) SH-SY5Y cells were depleted of WASH1, treated with bafilomycin A1 and examined for LC3-II, tubulin, WASH1 and GAPDH levels. Blots shown are representative of two independent experiments in triplicate. (b) Quantification of the representative experiment in triplicate shown in (a), of two independent experiments. Error bars indicate s.e.m. **P=0.0058 by 1-tailed Student's t-test.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated.",
    "molecules": "Coomassie blue"
  },
  {
    "caption": "Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm.",
    "molecules": "calcofluor white, CW"
  },
  {
    "caption": "Subcellular localizations of HST:GFP expressed from the UBQ10 promoter (pUBQ) in Arabidopsis roots (i). Distinct planes of the same root cells imaged by confocal microscopy are shown (ii-iii). PI: propidium iodide staining. Scale bars: 50µm (i); 10µm (ii-iii).",
    "molecules": "PI"
  },
  {
    "caption": "Subcellular localizations of hst-3:GFP expressed from pUBQ in root cells. Distinct planes of the same root cells imaged by confocal microscopy are shown (i-ii). PI: as in (A). Scale bars: 10µm.",
    "molecules": "PI"
  },
  {
    "caption": "miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions.",
    "molecules": "SO4"
  },
  {
    "caption": "Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm.",
    "molecules": "Basic fuchsin"
  },
  {
    "caption": "(B, Characterization of DONSON's chromatin  association. Sperm nuclei  (5,000/μL) were incubated for the indicated times  in 20 μL of interphase egg extract. Chromatin  fractions were isolated and analyzed by immunoblotting. His-p27  (100 μg/mL, p27  (CDKi)), PHA-767491 (50 μM, Cdc7i) were supplemented as indicated. Similar experiments with (B) were performed in triplicate and DONSON  signal intensity was quantified by Image J.",
    "molecules": "PHA-767491"
  },
  {
    "caption": "C, Characterization of DONSON's chromatin  association. Sperm nuclei  (5,000/μL) were incubated for the indicated times  in 20 μL of interphase egg  extract. Chromatin  fractions were isolated and analyzed by immunoblotting. aphidicolin  (10 μg/mL, aph), His-geminin  (50 μg/mL, gem) were supplemented as indicated. Similar experiments with (C) were performed in triplicate and DONSON  signal intensity was quantified by Image J.",
    "molecules": "aph, aphidicolin"
  },
  {
    "caption": "E) Characterization of DONSON's chromatin  association. Sperm nuclei  (5,000/μL) were incubated for the indicated times  in 20 μL of interphase egg  extract. Chromatin  fractions were isolated and analyzed by immunoblotting. NMS-873 (100 μM, p97i), and MLN-4924 (20 μM, Culi) were supplemented as indicated.",
    "molecules": "NMS-873, MLN-4924"
  },
  {
    "caption": "(B) DONSON  requirement for the replisome  assembly. Sperm nuclei  (5,000/μL) were incubated in 20 μL of the mock- and DONDON-depleted extracts as prepared in (A) for 60 min in the presence of 10 μg/mL aphidicolin. The extracts (0.5 μL) and isolated chromatin  fractions were analyzed by immunoblotting.",
    "molecules": "aphidicolin"
  },
  {
    "caption": "(A) DONSON  requirement for DNA replication. Sperm nuclei  were incubated for 60 min in egg  extract with no IgG (non), control IgG (α-control), or one of the two kinds of anti-DONSON  antibodies (α-DNS  #1, α-DNS  #2) in a final concentration of 14 μg/mL. Cy3-dCTP was added to all of the reactions at 1 μΜ to visualize the DNA replication. After incubation, the nuclei  were fixed, stained  with Hoechst 33258, and observed under a fluorescent microscope. Representative images are shown above. Scale bar; 50 µm. The relative Cy3  intensity of >100 nuclei  was quantified using Image J and is shown in the box plot below. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.",
    "molecules": "Cy3, dCTP, Hoechst 33258"
  },
  {
    "caption": "(B) DONSON  requirement for DNA replication. Sperm nuclei  (5,000/μL) were incubated for 60 min in 10 μL of the untreated (control), mock- (mock), and DONDON-depleted (ΔDNS) extracts with 2 μM Cy5-dUTP. Aphidicolin  (aph) was added to the mock-depleted extract (2.5 μg/mL) as a negative control. Various versions of the recombinant Xenopus DONSON  (1-6) were added to the DONSON-depleted extracts at about 0.06 μM. 1; untagged DONSON, 2 and 3; GST-DONSON, 4 and 5; GST-DONSON  (residues 155-579) (ΔN154), 6; His-DONSON. In the reactions corresponding to lanes 3 and 5, 3C protease was added to separate the GST  from DONSON. After incubation, the genomic DNA was isolated and subjected to 0.8% TAE agarose gel electrophoresis, followed by SYBR Gold staining. The Cy5  and  SYBR Gold fluorescent signals show replicated DNA  and total DNA, respectively. The same experiment was performed in triplicate; the relative Cy5  intensities to the control reaction were quantified using Image J, as shown in the bar graph below. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.",
    "molecules": "SYBR Gold, aph, Aphidicolin, Cy5, DNA, dUTP"
  },
  {
    "caption": "(A) Salt-sensitivity of chromatin-bound DONSON. Sperm nuclei  (5,000/μL) were incubated in 20 μL of egg  extract under each of the conditions as indicated, and the chromatin  fractions were isolated with normal buffer plus 0 (-), 0.1, or 0.2 M NaCl, and analyzed by immunoblotting. In addition, 0.5 μL of each extract (ext) was analyzed for comparison.",
    "molecules": "NaCl"
  },
  {
    "caption": "(E) (left) The isolated genomic DNA  was analyzed in the same manner as in Figure 3B. (right) The same experiment was repeated six times. The relative Cy5  intensities to the control reaction (normal α-C) were quantified using Image J, as shown in the bar graph. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.",
    "molecules": "Cy5, DNA"
  },
  {
    "caption": "(B) Requirement of GINS- and Polε-interacting motifs within DONSON  for replication activity. The same experiment procedure was used as in Figure 3A for the mock- and DONSON-depleted extracts with the same recombinant proteins as in Figure 5E. Xenopus proteins were added at 0.06 μM each while human  proteins were added at 0.3 μM. The relative Cy3  intensity of >100 nuclei  was quantified using Image J and is shown in the box plot. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.",
    "molecules": "Cy3"
  },
  {
    "caption": "(C) (above) Requirement of DONSON  for CDK  and DDK function in the replisome  assembly. Sperm nuclei  (5,000/μL) were incubated for 50 min in 120 μL of egg  extract with control IgG (α-cont) or anti-DONSON  antibody #1 (α-DNS). Each part of this first reaction (1st) was divided equally into six parts, and the nuclear  fractions were isolated and transferred to 20 μL of flesh extract with control IgG (α-cont) or anti-DONSON  antibody #1 (α-DNS) in the presence or absence of CDK  inhibitors (Ci: 100 μM roscovitine, 100 μg/mL His-p27) and Cdc7  inhibitor (7i: 50 μM PHA-767491). Aphidicolin  was added to the first and second extracts at 10 μg/mL to suppress replication termination. After incubation for 40 min in the second reaction (2nd), the chromatin  fractions were isolated and analyzed by immunoblotting. In addition, the extracts (0.5 μL) were analyzed (ext). (below) It is summarized whether or not replisome  assembly was allowed under each condition, where DONSON, CDK, and Cdc7  were either functional (+) or non-functional (-) in the 1st and 2nd incubations.",
    "molecules": "Aphidicolin, PHA-767491, roscovitine"
  },
  {
    "caption": "(C) Fluorescent microscopy demonstrates that downregulating ERMES contacts (by growing GALp-MDM34 strains in glucose) indeed caused expansion of vCLAMP (GFP-Vps39). However, this expansion was diminished in a Δlam6 background. Scale bar represents 5 μm (see also Figure S4).",
    "molecules": "glucose"
  },
  {
    "caption": "Dose-response curves representing 72-h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib. Ranges of estimated IC50 and Emax for the selected lines are shown. Data are represented as mean ± SD.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "B, C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (B) Dose-response profiles for apoptosis induction with vemurafenib and JNK-IN-8 combination.",
    "molecules": "JNK-IN-8, vemurafenib"
  },
  {
    "caption": "C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (C) Excess over the predicted Bliss independence (EOBI) calculated for different combined doses of vemurafenib and JNK-IN-8.",
    "molecules": "JNK-IN-8, vemurafenib"
  },
  {
    "caption": "E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of vemurafenib",
    "molecules": "vemurafenib"
  },
  {
    "caption": "E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of selumetinib (F)",
    "molecules": "selumetinib"
  },
  {
    "caption": "Time-dose-response plots indicating changes in RPPA measurements for p27 versus p-histone H3 for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.",
    "molecules": "DMSO, PLX4720"
  },
  {
    "caption": "B-D Time-dose-response plots indicating changes in RPPA measurements for pERK(Thr202/Tyr204) versus pAKT(Ser473) for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.",
    "molecules": "DMSO, PLX4720"
  },
  {
    "caption": "Time-dose-response plots indicating changes in RPPA measurements for pS6(Ser235/236) versus total c-Jun levels for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.",
    "molecules": "DMSO, PLX4720"
  },
  {
    "caption": "Covariate single-cell analysis of Ki-67 (left) and pRb(Ser807/811) (right) versus p-cJun(Ser73) in WM1552C cells before and 24 h after exposure to 0.8 μM vemurafenib. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The vertical lines were used to gate p-cJunHigh versus p-cJunLow cells. The horizontal lines were used to gate Ki-67High versus Ki-67Low cells, and pRbHigh versus pRbLow cells.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "Analysis of drug dose-dependent changes in proportion of pRbLow/p-cJunHigh and pRbHigh/p-cJunHigh subpopulations in four melanoma cell lines (WM115, WM1552C, LOXIMVI, COLO858) after exposure to vemurafenib for 24 h. These subpopulations were gated as shown in (A). Data are represented as mean ± SD for two replicates",
    "molecules": "vemurafenib"
  },
  {
    "caption": "Covariate single-cell analysis of pRb(Ser807/811) versus pS6(Ser235/236) following 24-h treatment of WM1552C cells with different doses of vemurafenib alone (top) and vemurafenib and JNK-IN-8 together (bottom). Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The horizontal and vertical lines were used to gate pS6High versus pS6Low cells, and pRbHigh versus pRbLow cells, respectively.",
    "molecules": "JNK-IN-8, vemurafenib"
  },
  {
    "caption": "Selected immunofluorescence images of pS6(Ser235/236) and Hoechst staining in WM1552C cells in a DMSO-treated control and 24 h after exposure to 5 μM of vemurafenib, JNK-IN-8, and their combination.",
    "molecules": "DMSO, JNK-IN-8, vemurafenib"
  },
  {
    "caption": "Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines (LOXIMVI, WM115 and WM1552C) as a function of drug concentration for single-drug vemurafenib and JNK-IN-8 treatments and their combination treatment. Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. pS6High population of cells was gated as indicated in (C). Data are represented as mean ± SD for two replicates. Data comparison between vemurafenib treatment and vemurafenib/JNK-IN-8 combined treatment was made by using two-way analysis of variance (ANOVA).",
    "molecules": "JNK-IN-8, vemurafenib"
  },
  {
    "caption": "Fraction of c-JunHighcells (as measured by single-cell (immunofluorescence microscopy) after 48 h JUN knockdown followed by 24-h treatment with vemurafenib. Fold changes are shown relative to control-treated cells. Data are presented as mean ± SD.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "Single-cell pS6(Ser235/236) levels in the c-JunHigh and c-JunLow fractions of cells (as measured by single-cell multiplex immunofluorescence microscopy) after 48 h of JUN knockdown and 24-h treatment with 0.32 μM vemurafenib. Single-cell pS6 data are presented as box-and-whisker plots with median signal intensities and interquartile ranges; bars extending to 1.5× the interquartile range are shown for each condition as a measure of variance. P-values were calculated using a two-sided nonparametric Mann-Whitney U-test.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "Comparison of the level of 1-h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720.",
    "molecules": "PLX4720, SB590885"
  },
  {
    "caption": "Comparison of the 72-h measured relative viability AUC for 8/10 cell lines treated with SB590885 versus PLX4720",
    "molecules": "PLX4720, SB590885"
  },
  {
    "caption": "Pairwise Spearman's correlation between the 24-h p-cJun levels as measured by immunofluorescence microscopy and 72-h relative viability after treatment with PLX4720 for 8 cell lines. p-cJun levels are averaged over five doses (0.1-5 μM), and viability is represented by the AUC of the dose-response curves.",
    "molecules": "PLX4720"
  },
  {
    "caption": "Comparison of the level of 24-h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720.",
    "molecules": "PLX4720, SB590885"
  },
  {
    "caption": "Adult mouse back skin sections immunostained for LYVE1 (red), αSMA (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. LV, lymphatic vessels; apm, arrector pili muscle.",
    "molecules": "DAPI"
  },
  {
    "caption": "Back skin sections of P55 mouse skin immunostained for LYVE1 (red), Tenascin-C (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. TNC, Tenascin-C.",
    "molecules": "DAPI"
  },
  {
    "caption": "Adult mouse back skin sections from different postnatal (P) days immunostained for LYVE1 (red), Lhx2 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.",
    "molecules": "DAPI"
  },
  {
    "caption": "Back skin sections of P49 mouse skin immunostained for LYVE1 (red), CD34 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. White arrowheads denote the proximity of lymphatic capillaries to HFSC.",
    "molecules": "DAPI"
  },
  {
    "caption": "Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.",
    "molecules": "DAPI, mifepristone"
  },
  {
    "caption": "Distribution of LV and HF at the mouse embryo stage E15.5 (A), E16.5 (B) and E17.5 (C). Maximum projection images of whole mount immunofluorescence analyses using LYVE1 (red) as lymphatic endothelial marker and counterstained with DAPI (blue). Red arrowheads denote HF placodes. n= 3 - 4 embryos. Scale bar, 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Adult back skin sections from different postnatal (P) days immunostained for LYVE1 (red) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 10 μm. White arrowheads denote LV membrane protrusion and fenestrated areas. LV, lymphatic vessels; HF, hair follicle.",
    "molecules": "DAPI"
  },
  {
    "caption": "H&E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. epi, epidermis; der, dermis.",
    "molecules": "Clodronate"
  },
  {
    "caption": "LYVE1 immunofluorescence (red) counterstained with DAPI (blue) (D) in back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.",
    "molecules": "Clodronate, DAPI"
  },
  {
    "caption": "Histogram of the caliber (μm) of LV (E) in back skin sections from mice treated intradermally with Control or Clodronate liposomes.",
    "molecules": "Clodronate"
  },
  {
    "caption": "Back skin sections of Controls and K14ΔNβ-cateninER+/T mice immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.",
    "molecules": "DAPI"
  },
  {
    "caption": "Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.",
    "molecules": "Cilostazol"
  },
  {
    "caption": "H&E staining (A) and histogram of the HF length (B) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal Diphtheria Toxin (CSA+DT) starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.",
    "molecules": "CSA, tamoxifen"
  },
  {
    "caption": "Ki67 immunostaining (C) and histogram of the number of Ki67+ cells per HF (D) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle; and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.",
    "molecules": "CSA, tamoxifen"
  },
  {
    "caption": "LYVE1 immunostaining (E) and histogram of the LV caliber (F) analyzed in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). Scale bar, 10 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.",
    "molecules": "CSA, tamoxifen"
  },
  {
    "caption": "H&E staining (G) and histogram of the HF length (H) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Cleaved caspase 3 immunostaining (I) and histogram of the number of cleaved caspase 3+ cells per HF (J) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). Scale bar, 50 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.",
    "molecules": "tamoxifen"
  },
  {
    "caption": " C). Western blot of LMF1-partners after DSP crosslinking and affinity tag purification using LMF1's C-terminal His tag. Tetracycline induces LMF1 expression. Loading dye with 50mM DTT was used to break the disulfide bonds between LMF1 and its interacting partners. LMF1 complexes are labeled with asterisks and an arrow points to LMF1 ",
    "molecules": "DTT, DSP, disulfide, Tetracycline"
  },
  {
    "caption": " D). Western blot of LMF1-partners after pBPA photo-crosslinking and affinity tag purification. The DMSO panel does not include pBPA and serves as a negative control. Fractions were eluted with increasing concentrations of imidazole. An arrow points to LMF1 ",
    "molecules": "pBPA, DMSO, imidazole"
  },
  {
    "caption": " E). SYPRO orange protein stain of DSP crosslinked samples. Samples without tetracycline but with DSP and DTT (negative control), and with tetracycline, DSP and DTT were sent for LC MS/MS. LMF1 complexes are labeled with asterisks and an arrow points to LMF1 ",
    "molecules": "DTT, DSP, tetracycline"
  },
  {
    "caption": " F). SYPRO orange stained gel of pBPA samples. An arrow points to LMF1 ",
    "molecules": "pBPA"
  },
  {
    "caption": " A). A Western blot against the C-terminal V5 tags of both lipases shows that LPL, but not PL, forms intermolecular disulfide bonds in cells. Lipase monomers are marked with a single arrow, and aggregates are marked with double and triple arrows. The LPL aggregates are not present when samples are treated with DTT. There is no PL in the pellet fraction because PL does not aggregate ",
    "molecules": "DTT, disulfide"
  },
  {
    "caption": " C). Co-translational LPL folding was carried out in the presence of SP HEK293 or HEK293∆LMF1 cells. At the indicated time points, AMS was added to reactions to differentiate reduced (R) and oxidized (O) LPL. The position of the stacker layer is indicated ",
    "molecules": "AMS"
  },
  {
    "caption": "Differential sensitivity of HEK293 cells containing and lacking LMF1 (A) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.",
    "molecules": "DTT, BSO, tunicamycin"
  },
  {
    "caption": "Differential sensitivity of cld/cld and wt/cld MEFs (B) to drugs that perturb cellular redox homeostasis. Cells were treated with BSO, DTT, or tunicamycin as described in materials and methods. Treated cell and untreated cells were counted, and the fraction of surviving cells was calculated. Three wells were counted and averaged for each data point, and three independent trials were carried out per condition. Significance was determined by a 2-tailed student's t-test.",
    "molecules": "DTT, BSO, tunicamycin"
  },
  {
    "caption": "). Secretion of LPL-V5 from HEK293∆LMF1 cells transiently transfected with LMF1 bearing the indicated cysteine to alanine mutations. Lipase levels in the lysate and pellet, and LMF1 levels in the pellet (α-HIS), are also probed. GAPDH serves as a loading control.",
    "molecules": "alanine, cysteine"
  },
  {
    "caption": "α-His Western blot to detect the redox state of LMF1. In the first two lanes samples were untreated and resolved in reducing or non-reducing loading dye. The next 4 lanes were subject to the indicated treatments prior to cell lysis then treated with AMS prior to non-reducing SDS-PAGE.",
    "molecules": "AMS"
  },
  {
    "caption": "α-His Western blot to detect acid trapped partners of LMF1. Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control.",
    "molecules": "NEM, TCA"
  },
  {
    "caption": "Samples were TCA precipitated, treated with NEM, and LMF1 was purified via its C-terminal His tag. Samples were resolved by reducing or nonreducing SDS-PAGE. The lanes marked -LMF1 are HEK293∆LMF1 cells included as a negative control. Samples were probed with an antibody for TRX.",
    "molecules": "NEM, TCA"
  },
  {
    "caption": "B Antiproliferative activities of XMU-MP-5 and crizotinib against EML4-ALK Ba/F3 and parental Ba/F3 cells, respectively. Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay. Each concentration was performed in triplicate. Data were presented as Mean ± SEM from three independent experiments (n = 3) .",
    "molecules": "XMU-MP-5, crizotinib"
  },
  {
    "caption": "C XMU-MP-5 downregulates ALK signaling pathways in EML4-ALK Ba/F3 cells. Cells were treated with XMU-MP-5 or crizotinib for 4 h, and then analyzed by immunobloting.",
    "molecules": "XMU-MP-5, crizotinib"
  },
  {
    "caption": "C XMU-MP-5 suppressed ALK signaling pathway in H3122 cells.",
    "molecules": "XMU-MP-5"
  },
  {
    "caption": "E Comparison of tumor volume of different treatment groups in 11 days. BALB/c nude mice bearing H3122 xenograft tumors were treated with vehicle, XMU-MP-5 and crizotinib by tail vein injection once daily. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test. N.S., P = 0.1063; **P = 0.0097.",
    "molecules": "XMU-MP-5, crizotinib"
  },
  {
    "caption": "A Cell viability was measured by MTS assay. Each concentration was performed in triplicate. The exact IC50 value was presented as Mean ± SEM from three independent experiments (n = 3) and shown in Appendix Table S6. IC50 fold changes of wild type and mutant EML4-ALK Ba/F3 cell lines to parental Ba/F3 cell line of XMU-MP-5 and crizotinib were determined.",
    "molecules": "XMU-MP-5, crizotinib"
  },
  {
    "caption": "C, D In vivo activity of XMU-MP-5 against L1196M xenograft mouse model. BALB/c nude mice bearing L1196M Ba/F3 xenograft tumors were treated with vehicle, 30 mg/kg or 60 mg/kg XMU-MP-5 by tail vein injection once daily. Tumor volume (C) and tumor weight (D) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (C), N.S., P = 0.1482; *P = 0.037. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (D), N.S., P = 0.2162; *P = 0.0122.",
    "molecules": "XMU-MP-5"
  },
  {
    "caption": "E, F Efficacy of XMU-MP-5 in G1202R Ba/F3 xenograft mouse model. BALB/c nude mice bearing G1202R Ba/F3 cells xenograft tumors were administrated with vehicle or XMU-MP-5 by tail vein injection twice daily. Lorlatinib was used as a positive control by oral administration. Tumor volume (E) and tumor weight (F) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (E), Vehicle vs XMU-MP-5 30 mg/kg, *P = 0.0433; Vehicle vs XMU-MP-5 30 mg/kg BID, **P = 0.0093; Vehicle vs Lorlatinib, **P = 0.0029. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (F). Vehicle vs XMU-MP-5 30 mg/kg, ***P = 0.0008; Vehicle vs XMU-MP-5 30 mg/kg BID, ***P = 0.0003; Vehicle vs Lorlatinib, ***P = 0.0002.",
    "molecules": "XMU-MP-5, Lorlatinib"
  },
  {
    "caption": "A Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10A. Mice were treated with 30 mg/kg XMU-MP-5 twice daily for 3 weeks. H: heart; arrow indicated tumor. Scale bars, 200 μm. B IHC analyses of lungs from EML4-ALK transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.",
    "molecules": "XMU-MP-5"
  },
  {
    "caption": "C Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK(L1196M) tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10B. Mice were treated with 40 mg/kg XMU-MP-5 twice daily for 1 week. H: heart; arrow indicated tumor. Scale bars, 200 μm. D IHC analyses of lungs from EML4-ALK(L1196M) transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.",
    "molecules": "XMU-MP-5"
  },
  {
    "caption": "(B) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at 6 h.p.i. Cycloheximide (Cy.) was added to mock infected cells at a final concentration of 50 µg/mL, for 4 hours. Cells were then pre-treated with 0.1 µM bafilomycin A1 (Baf.A1) for 14 hours prior to infection and kept under treatment throughout infection. MG132 was used at a final concentration of 20 µM and added to the media at the time of infection. The blot shown is representative of three independent experiments.",
    "molecules": "bafilomycin A1, Cycloheximide, MG132"
  },
  {
    "caption": "(B) Cells were treated with 10 mM 3-MA for 12 h, then infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i. and lysates were used for immunoblots.",
    "molecules": "3-MA"
  },
  {
    "caption": "Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell (A) or MOI of 50 pfu/cell (B). Cells were pre-treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection. Cell-associated virus was collected at the indicated times post-infection, and virus titers were determined by plaque assay. Parallel infections were collected for Western blots of LC3 (A) or p62 (B) In (B) cell-associated virus was collected at 6 h.p.i. for plaque assay.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(C) Infections were performed as in (A), then 10 µg/mL each of E64d and Pepstatin A (Pep.A) were added to the media at the time of infection. Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH. (D) Infection at an MOI of 50 pfu/cell, with E64/Pep.A as in (C).",
    "molecules": "E64d, Pepstatin A"
  },
  {
    "caption": "(A) Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, and cell-associated virus was collected at the indicated times post-infection. NH4Cl (20 mM) was added to the media at the time of infection (solid line). Virus titers were then determined by plaque assay. (B) Infection as in (A), carried out to 16 h to represent a multiple-cycle infection.",
    "molecules": "NH4Cl"
  },
  {
    "caption": "(E) Cells were pre-treated for 14 hours with 0.1 µM bafilomycin A1 (Baf.A1) and kept under treatment throughout infection (MOI = 0.1 pfu/cell). Cell-associated virus was collected at 6 or 12 h.p.i. and virus titers were determined by plaque assay. * p<0.05, ** p<0.01, *** p<0.0001.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(A) H1-HeLa cells were infected with PV at an MOI of 50 pfu/cell. Cells were pulsed at the indicated h.p.i with 35S-labeled methionine for 1 h then lysed. Lysates were run on SDS-PAGE. Expected viral proteins are labeled according to recognized banding patterns.",
    "molecules": "methionine, proteins"
  },
  {
    "caption": "(B) Triplicate plates of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, virus RNA and host GAPDH RNA were measured by qRT-PCR. Virus RNA levels were normalized to GAPDH levels using the delta-Ct method. NH4Cl treatment was as described in Figure 4, and Guanidine HCl (2 mM) was added to the media at the time of infection. The data shown are pooled from three replicate experiments, and the titer of cell-associated virus collected at 6h.p.i. from each replicate was determined by plaque assay.",
    "molecules": "NH4Cl, Guanidine, HCl, RNA"
  },
  {
    "caption": "(C) Triplicate plates of 293T cells were treated with 20 mM 3-MA for 2 hours prior to infection, and kept under treatment throughout infection. PV infections were done at an MOI of 0.1 pfu/cell. RNA levels and virus titers were analyzed as in (B). ** p<0.01, *** p<0.0001.",
    "molecules": "3-MA, RNA"
  },
  {
    "caption": "(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with NH4Cl. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 or 6 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. Representative gradients from three independent experiments are shown.",
    "molecules": "NH4Cl, 35S-Methionine, sucrose"
  },
  {
    "caption": "(C) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE, and the 35S-Methionine labeled bands were visualized. The bands are labeled according to expected relative migration pattern, and VP2 is identified by its absence in the 75S peak. The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units. * p<0.05, ** p<0.01, *** p<0.0001.",
    "molecules": "35S-Methionine"
  },
  {
    "caption": "(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with bafilomycin A1 as described in Figure 4. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 h.p.i., and lysates were then separated on a 15-30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction.",
    "molecules": "bafilomycin A1, 35S-Methionine, sucrose"
  },
  {
    "caption": "(C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells.",
    "molecules": "BafA1, Bafilomycin A1, DMSO"
  },
  {
    "caption": "(D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells).",
    "molecules": "BafA1, Bafilomycin A1, DMSO"
  },
  {
    "caption": "(B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D) Wild-type and indicated K562 knock-out cells were treated The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4). (E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant",
    "molecules": "Trypsin"
  },
  {
    "caption": "(B) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. (B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate. (E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A. Four full-thickness excisional wounds were produced on each of CD18-/- or WT mice by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or PBS control. Each wound region was digitally photographed at the indicated time points, and wound areas were analyzed using Adobe Photoshop. Depicted are representative macroscopic pictures of PBS injected or AT-MSCs injected WT or CD18-/- wounds at days 0, 3, 5, 7 and 9 post-wounding. B. Quantitative analysis of 20 wound areas per group, expressed as percentage of the initial wound size at day 0. The line in each group represents the mean value of 20 wounds from 5 mice. *p<0.05, **p<0.01, ***p<0.001 by Student's t-test with Welch's correction. ",
    "molecules": "PBS"
  },
  {
    "caption": "A. AT-MSCs or PBS were intradermally injected to WT or CD18-/- wounds and the wound tissues were harvested at days 2, 5, 7 post-wounding. Depicted are representative photographs of wound cryosections at days 2 and 5 post-wounding stained for human-specific β2M (green). Nuclei were stained with DAPI (blue). Scale bars: 100 µm.",
    "molecules": "DAPI, PBS"
  },
  {
    "caption": "A. Cryosections of PBS or AT-MSCs injected CD18-/- wounds at days 5 and 7 post-wounding were immunostained for α-SMA (red). Nuclei were stained with DAPI (blue). The dashed lines separate epidermis and wound bed. e, epidermis; wb, wound bed. Scale bars: 100 µm. B. Quantification of α-SMA immunofluorescence shown in (A) ",
    "molecules": "DAPI, PBS"
  },
  {
    "caption": "A-F. Representative photographs of immunofluorescent staining for α-SMA (red) and human β2M (green) on cultured human AT-MSCs (A), murine primary dermal fibroblasts (mFB) (B), mFB treated with 2 ng/ml recombinant human TGF-β1 (C), and co-cultures of mFB with AT-MSCs (D) or TGF-β1 siRNA transfected AT-MSCs (E) or control siRNA transfected AT-MSCs (F) after 48 h culture. Nuclei were counterstained with DAPI (blue). G. Quantification of α-SMA immunofluorescence shown in (A-F)",
    "molecules": "DAPI"
  },
  {
    "caption": "H. Four full-thickness excisional wounds were produced on each of CD18-/- mouse by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or human dermal fibroblasts (HDF) or TGF-β1 siRNA transfected AT-MSCs or control siRNA transfected AT-MSCs or PBS. Each wound region was digitally photographed at days 0, 3, 5, 7 and 10 post-wounding, and wound areas were analyzed using Adobe Photoshop. The depicted result is the quantitative analysis of all wound areas per group, expressed as percentage of the initial wound size at day 0.",
    "molecules": "PBS"
  },
  {
    "caption": "A-C. 2.5x105 of TGF-β1 siRNA or control siRNA transfected AT-MSCs were intradermally injected around each of CD18-/- murine wound. PBS mock injection served as negative control. Wound tissue were harvested at day 2, 5, and 7 post-wounding for quantification of human TGF-β1 mRNA (A) at day 2 by qPCR, total TGF-β1 (B) and active TGF-β1 (C) protein at day 5 by ELISA.",
    "molecules": "PBS"
  },
  {
    "caption": "A. AT-MSCs were cultured in the labelling media containing [35S]-Met/Cys and increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for overnight. Supernatants were harvested and incubated with TGF-β1 capture antibody. After washing the captured TGF-β1 was transferred to glass filter membrane, and the radioactivity was counted with a scintillation counter (black bars). The supernatants were incubated with new TGF-β1 capture antibody, and the radioactivity of captured TGF-β1 was counted again (white bars). The data is given as mean ± SD of 5 repeated measurements. CPM, count per minute.",
    "molecules": "Cys, Met, 35S"
  },
  {
    "caption": "A. AT-MSCs were pretreated with a TGFβRI inhibitor SB431542 at 10 µM, or control DMSO for 1 h, and subsequently exposed to r.h. TGF-β1 at indicated concentrations. The cells were cultured for another 24 h and harvested for miR-21 assay. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.",
    "molecules": "DMSO, SB431542"
  },
  {
    "caption": "K-L. Full-thickness excisional wounds were produced on CD18-/- mice by 6 mm biopsy punches. One day after wounding, each wound received intradermal injection of 2.5x105 AT-MSCs that had been transfected with 50 nM miR-21 inhibitor or control inhibitor 2 days after transfection. CD18-/- wounds received mock injection with PBS served as control. Each wound region was digitally photographed at days 0, 3, and 6 post-wounding, and expressed as percentage of the initial wound size at day 0 (K). Wound tissues were harvested on day 6 post-wounding for quantification of total TGF-β1 protein by ELISA (L). Data is given as mean ± SEM; n = 5 wounds per group; *, p<0.05; **, p<0.01, by one-way ANOVA with Tukey's test. MSC_miR-21-IN, miR-21 inhibitor transfected AT-MSCs; MSC_ctrl-IN, control inhibitor transfected AT-MSCs.",
    "molecules": "PBS"
  },
  {
    "caption": "A: Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs.",
    "molecules": "ATP"
  },
  {
    "caption": "G-I: qPCR analysis of indicated gliosis genes in ATP-treated EGCs.",
    "molecules": "ATP"
  },
  {
    "caption": "J: qPCR analysis of IL-6 in msEGCs that were treated for 6h with ATP.",
    "molecules": "ATP"
  },
  {
    "caption": "K: IL-6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP.",
    "molecules": "ATP"
  },
  {
    "caption": "A: Effect of P2 receptor antagonism on ATP induced IL-6 release. Cells were treated with P2-antagonist suramin (1, 10 and 100µM) alone or together with ATP (100µM) for 24h.",
    "molecules": "ATP, suramin"
  },
  {
    "caption": "B: ATP induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP-and ATPɣS for 24h.",
    "molecules": "ATPɣS, ATP"
  },
  {
    "caption": "C: Effects of P2X antagonists on ATP induced IL-6 release. Cells were treated for 24h alone or together with ATP (100µM) in absence or presence of P2X2, P2X4 and P2X7-antagonists PSB-1011, 5-BD-BD and A740003, respectively.",
    "molecules": "PSB-1011, 5-BD-BD, A740003, ATP"
  },
  {
    "caption": "D: P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP and RCAN by qPCR in msEGCs. Cells were treated with the P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (10µM) for 6h.",
    "molecules": "PSB-1011, ATP"
  },
  {
    "caption": "F: P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100µM) treatment for 6h.",
    "molecules": "ATPɣS"
  },
  {
    "caption": "G: P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10µM, 100µM) for 6h.",
    "molecules": "ATPɣS"
  },
  {
    "caption": "A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.",
    "molecules": "ATP"
  },
  {
    "caption": "G: FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle. CD45, Ly6C and CX3CR1 were used to distinguish resident macrophages (CD45+/Ly6C-/CX3CR1+), infiltrating monocytes (CD45+/Ly6C+/CX3CR1-) and infiltrated monocyte-derived macrophages (CD45+/Ly6C+/CX3CR1+).",
    "molecules": "ambroxol"
  },
  {
    "caption": "H: Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle.",
    "molecules": "ambroxol"
  },
  {
    "caption": "E: IL-6 protein release in hEGC cultures upon stimulation with ATP (200µM) or ATPɣS (100µM) after 6h and 24h treatment (n=4-6, hEGCs).",
    "molecules": "ATPɣS, ATP"
  },
  {
    "caption": "F: IL-6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations; n=4-6, hEGCs.",
    "molecules": "ARL67156"
  },
  {
    "caption": "H: Effect of the P2X2 antagonism on ATP-induced IL-6 release. ELISA measurement of IL-6 in hEGCs upon treatment with P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (200µM) treated for 24h (n=6, hEGCs).",
    "molecules": "PSB-1011, ATP"
  },
  {
    "caption": "I: PSB-1011 (20μM) treatment inhibited ATP-triggered calcium responses in HEK cells transfected with P2X2. Data are represented as ΔF/F0 +SEM; n=102 HEK cells. J: The P2X2 receptor antagonist PSB-0711 (20μM) nearly abolished the ATP-triggered calcium response in HEK cells transfected with P2X2. n=219 HEK cells.",
    "molecules": "PSB-0711, PSB-1011, ATP, calcium"
  },
  {
    "caption": "K: Ambroxol blocks ATP-induced IL-6 release in hEGCs; Protein release measurement by ELISA of IL-6 in hEGCs. Cells were treated with ATP (200 µM) alone or together with ambroxol (20 µM) for 24h; n=6, hEGCs.",
    "molecules": "Ambroxol, ambroxol, ATP"
  },
  {
    "caption": "L-N: qPCR analysis of several gliosis and ATP-target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol (20µM). (n=7, 4 human patients).",
    "molecules": "ambroxol, ATP"
  },
  {
    "caption": "(E, F) c-Maf  quantification by qPCR (E) and cytokine concentration (F) in the supernatants of adult and cord blood ILC2s  unstimulated, stimulated for 3h with PMA-Ionomycin  (3h stim) and stimulated for an overnight with  IL-33 and then 3h with PMA-Ionomycin  (o.n. stim). ILC2s  were sorted from 3 different donors. (E) Multiple T tests, unstimulated: p=0.000101; 3h stim: p=0.0036. (F) Multiple T tests, IL-13: p=0.022;  IL-5: p=0.015 (*p<0.05). Bars represent mean ± SEM.",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "(A) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector or the indicated TRIB1-FLAG construct (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. See also Appendix Fig S1B for western blots showing the expression levels of the constructs used.",
    "molecules": "Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(C) Representative images of COS7 cells showing GFP-COP1 (green) and TRIB1-FLAG (red) colocalizing in punctate nuclear structures. Insets show enlarged images of these nuclear puncta. Cells were stained with anti-FLAG and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(D) Representative images of COS7 cells showing that GFP-COP1 (green) and TRIB1-HA (red) are diffuse in the nucleoplasm when coexpressed with C/EBPα (cyan). Cells were stained with anti-HA (Santa Cruz), anti-C/EBPα (Cell Signaling), anti-mouse Alexa Fluor 405, and anti-rabbit Alexa Fluor 680. Scale bar, 10 µm. See also Fig EV2C for images of cells expressing each construct alone or in pairs.",
    "molecules": "Alexa Fluor 680, Alexa Fluor 405"
  },
  {
    "caption": "(A) Representative images of COS7 cells expressing COP1-3xFLAG (green) co-transfected with empty vector, GFP, or the indicated GFP-TRIB1 constructs (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(C) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector, TRIB1-FLAG, TRIB2-FLAG, or TRIB3-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(C) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 4A (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and empty vector or TRIB1-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(A) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 NESmut (V238A/L242A) (green) and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling) and anti-rabbit Alexa Fluor 680. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "Alexa Fluor 680, DAPI"
  },
  {
    "caption": "(B) Representative images of COS7 cells transfected with GFP-COP1 WT (green), the indicated TRIB1-HA constructs (red), and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling), anti-HA (Sigma), anti-rabbit Alexa Fluor 680, and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "Alexa Fluor 680, Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(E) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.",
    "molecules": "Alexa Fluor 568, DAPI"
  },
  {
    "caption": "(B) Genotyping of Brd4fl/fl mice with ERT2-Cre (Cre) or without ERT2 -Cre (+/+). Tamoxifen treatment deleted 2.2 kb Brd4fl/fl fragment from Cre/+ and Cre/Cre mice (top). Cre/+, Cre/Cre and +/+ mice were confirmed by the presence of ERT2 -Cre band (Cre), or its absence (WT) (bottom).",
    "molecules": "Tamoxifen"
  },
  {
    "caption": "(C) Flow cytometry profiles of BrdU incorporation and Ki67, ARG1 expression in peritoneal cells (gated for F4/80+, CD19- macrophages) from IL-4C treated WT and KO mice (left). The numbers of cells positive for BrdU, Ki67 and ARG1 (right). The numbers represent the average of three experiments +/- S.D.",
    "molecules": "BrdU"
  },
  {
    "caption": "(B) Flow cytometry profiles of CD11b+, F4/80+ macrophage population harvested on Day 7 from untreated (WT) or tamoxifen treated (KO) cultures (left). Histogram depicting F4/80 expression levels in WT and KO macrophages (middle). Total number of macrophage yields calculated from > 15 independent experiments. Values represent the average of 21 WT and 24 KO experiments +/- S.D.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(C) MA plots of RNA-seq data from untreated (UT) or LPS treated (4h) WT and KO macrophages. Differentially expressed genes were identified by a cut off line of log2FC>1 (FDR < 0.1). (D) Venn diagram depicting the number of LPS induced genes in WT and KO macrophages.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Heat maps of BRD4 occupancy on the promoter region (TSS +/- 5kb), aligned by the degree of BRD4 signal intensity in UT and LPS treated macrophages.",
    "molecules": "LPS"
  },
  {
    "caption": "(D) Immunoblot detection of BRD4 protein levels in UT and LPS treated WT macrophages. Forty μg of whole cell extracts from UT or LPS treated (1h or 4h) macrophages using antibody for BRD4 or β-actin (loading control).",
    "molecules": "LPS"
  },
  {
    "caption": "(E) Binding of BRD4 and PolII and distribution of histone marks (H3K27ac and H3K9ac) on BRD4 dependent and independent genes (from -5kb of TSS to +5kb of transcription end site (TES)) in UT and LPS treated WT and KO macrophages. Normalized ChIP-seq signals (rpm/bp) are shown on the Y axis. Arrows show reduced signals in Brd4 KO macrophages.",
    "molecules": "H3K27ac, LPS"
  },
  {
    "caption": "(A) BRD4 bound enhancers within 12.5kb were stitched together and ranked by BRD4 ChIP-seq signals (rpm) in untreated (UT, left) and LPS treated (middle) WT macrophages. The dotted line distinguished SEs from TEs. Gene names with the ranking in parenthesis are among genes closest to SEs. Venn diagram (right) showing nearest genes common in UT and LPS treated macrophages.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Genome-wide gain and loss of BRD4 SEs upon LPS stimulation. Based on SE coordinates (rpm from LPS treated samples/rpm in untreated samples), changes greater or less than 2 folds (Log2) were plotted as gain or loss.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) Examples of nearest genes that gained or lost SEs upon LPS treatment. Gene tracks for gained and lost loci (near Tnf and Csf1r) showing clusters of BRD4, Pol II and H3K27ac signals. Red bar represents SE length.",
    "molecules": "H3K27ac, LPS"
  },
  {
    "caption": "(D) Meta-gene alignment of BRD4 signals on BRD4 ranked SEs. Pol II and H3K27ac ChIP-seq reads were aligned centering on BRD4 SEs of LPS treated WT macrophages.",
    "molecules": "H3K27ac, LPS"
  },
  {
    "caption": " (E) Enhancer plots obtained by the H3K27ac-ranking in LPS stimulated WT and KO macrophages. ",
    "molecules": "H3K27ac, LPS"
  },
  {
    "caption": "B. NLK phosphorylates YAP in vitro. Flag-NLK-WT, or Flag-NLK-KM was immunoprecipitated with anti-Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST-YAP in the presence of [32P-ATP]. Phosphorylation of GST-YAP and autophosphorylation of NLK were examined by autoradiography (top panel), and Coomassie Blue staining showed similar levels of GST-YAP and Flag-NLK (bottom panel).",
    "molecules": "ATP"
  },
  {
    "caption": " (B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection. ",
    "molecules": "ampicillin"
  },
  {
    "caption": " (A) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla::Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect, pBR322 and pBR322 bla::Im7 L53A I54A were transformed into strains containing in-frame deletion mutants of each of the pst genes (pstS, pstC, and pstA, respectively). The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla::Im7 L53A I54A, but did not shower higher ampicillin resistance in the presence of pBR322. ",
    "molecules": "ampicillin"
  },
  {
    "caption": " (D) To test if levels of the Im7 L53A I54A protein itself, in the absence of the fusion, were also higher in the pstA mutant, expression of the Im7 L53A I54A protein from the IPTG-inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0-5 mM. Periplasmic fractions were isolated and examined by SDS-PAGE and subsequent Coomassie staining to determine overall protein expression patterns. Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7. MBP was used as a loading control. Im7 L53A I54A was increased in the pstA strain relative to the WT control strain. Coomassie staining revealed that a number of periplasmic proteins, including UgpB, were also induced in the pstA strain. ",
    "molecules": "IPTG"
  },
  {
    "caption": " (A) MG1655 WT, single mutants of pstA and ugpB, and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322-based folding biosensor constructs. The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations. Strains containing pBR322 bla::Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation (compare uppermost left and right panels). This resistance is completely lost upon transducing in a ugpB mutation (compare next panels down), showing that ugpB is responsible for the resistance gained by the pstA mutation. ",
    "molecules": "ampicillin"
  },
  {
    "caption": " (B) Ampicillin sensitivity conferred by a ugpB deletion mutant (CL543) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB (pCL1). ",
    "molecules": "Ampicillin, arabinose"
  },
  {
    "caption": " (C) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein. Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain (CL543) from pCDFTrc and pTrc99a vectors, respectively, both of which have an IPTG-inducible promoter. Periplasmic fractions were isolated and examined by Western blotting. "-" indicates strains containing empty vector and "+" indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB. MBP was used as a loading control. Data information: These experiments were repeated twice with similar results and one representative is shown. ",
    "molecules": "IPTG"
  },
  {
    "caption": "(A) UgpB overexpression confers bile salt resistance, and a ugpB deletion mutant is slightly CHO sensitive.",
    "molecules": "bile salt, CHO"
  },
  {
    "caption": " (B) CHO-induced MDH protein aggregation analyzed by monitoring light scattering. ",
    "molecules": "CHO"
  },
  {
    "caption": " (C) CHO-induced MDH protein aggregation analyzed by a solubility assay using centrifugation (cfg) at 16,000 g for 20 min. The supernatant (Sup) represents the soluble fraction and the pellet the insoluble fraction; 12 µM MDH was used as a model aggregation-prone substrate (see Methods). Addition of increasing quantities of UgpB increasingly solubilizes MDH. The molar ratios of MDH:UgpB that were used (left to right) are as follows: 1:0, 1:0.1, 1:0.2, 1:0.5, and 1:1. 5-fold more of the pellet fraction was loaded relative to the supernatant fraction. ",
    "molecules": "CHO"
  },
  {
    "caption": " (E) 12 µM of MDH alone and a 1:1 ratio of either MDH:UgpB or MDH:MBP were incubated at 37°C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above (C) . The pellet fraction was concentrated 5-fold. In some samples (+G3P), 120 µM of G3P was added prior to the addition of any other protein. Addition of G3P inactivates UgpB's ability to inhibit protein aggregation. ",
    "molecules": "CHO, G3P"
  },
  {
    "caption": " (F) 800 μg of E. coli total lysate and 60 µM of UgpB/MBP were used for the supernatant/pellet assay. 600 µM of G3P was added before adding proteins. The pellet fraction was concentrated 10-fold. As before, addition of UgpB inhibited aggregation, but only in the absence of G3P. ",
    "molecules": "G3P"
  },
  {
    "caption": " (G) The anti-aggregation activity of tryptophan single mutant (W169S) and double mutant (W169S/W172S) was examined by CHO-induced MDH aggregation assay. Light scattering values were monitored. ",
    "molecules": "CHO, tryptophan"
  },
  {
    "caption": " (H) Solubility of MDH is also monitored by supernatant/pellet assay. Tryptophan mutants exhibit constitutively active chaperone function independent of the presence of G3P. The pellet fraction was concentrated 5-fold. 12 µM of MDH and 1:1 ratio of MDH:UgpB proteins and 120 µM of G3P were used.",
    "molecules": "G3P, Tryptophan"
  },
  {
    "caption": " (I) The anti-aggregation activity of the UgpB variants (E66A and R374V) was examined by CHO-induced MDH aggregation assay. CHO-induced MDH protein aggregation analyzed by monitoring light scattering. ",
    "molecules": "CHO"
  },
  {
    "caption": " (J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used. 15 mM CHO were used for all the aggregation and supernatant/pellet assays. ",
    "molecules": "CHO, G3P"
  },
  {
    "caption": "(A) Acid denatures UgpB. The secondary structure of 2 µM UgpB was monitored by circular dichroism (CD) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7.",
    "molecules": "sodium phosphate"
  },
  {
    "caption": " (B) G3P binds to acid-stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum, either at pH7 or after transition from pH 2 to 7 (see Methods). Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift, indicating G3P binding. ",
    "molecules": "G3P, tryptophan"
  },
  {
    "caption": " (C) The anti-aggregation activity of UgpB is monitored by CHO-induced MDH aggregation assay. Light scattering values were monitored. The anti-aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P (120 µM) can again inactivate its chaperone activity. ",
    "molecules": "CHO, G3P"
  },
  {
    "caption": " (D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity . 12 µM of MDH and a 1:1 ratio of UgpB:MDH were used in a CHO-induced aggregation assay. At 30 min, the indicated concentrations of G3P were added to solutions containing MDH, and MDH aggregation was monitored by light scattering. Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan fluorescence",
    "molecules": "CHO, G3P, tryptophan"
  },
  {
    "caption": "D) Expression of the WT or R527A mutant GFP-KNL2 in KNL2 conditional knock out chicken DT40 cells (cKO-KNL2). Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition in cKO-KNL2 cells (None). GFP-fused KNL2 WT or R527A mutant was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.",
    "molecules": "tet, tetracycline"
  },
  {
    "caption": "E) Localization analyses of GFP-fused KNL2 WT or R527A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or R527A mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.",
    "molecules": "tet"
  },
  {
    "caption": "D) Expression of GFP-KNL2 WT or CENP-A C-terminal tail binding mutants in cKO-KNL2 cells. Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition to the cKO-KNL2 cells (None). GFP-fused KNL2 WT, F535A, W536A or F535A/W536A was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.",
    "molecules": "tet, tetracycline"
  },
  {
    "caption": "E) Localization analyses of GFP-fused KNL2 WT and F535A, W536A or F535A/W536A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or each CENP-A C-terminal tail binding mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.",
    "molecules": "tet"
  },
  {
    "caption": "A) Immunostaining with anti-Mis18α antibody in AID-based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h. Clear colocalization of KNL2 signals with Mis18α was observed (Insets). DNA was stained with DAPI. Scale bar indicates 10 µm.",
    "molecules": "DAPI, IAA"
  },
  {
    "caption": "B) AID-based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP-tagged CENP-A were used. After addition of IAA, SNAP-CENP-A signals were visualized by TMR-Star staining. CENP-T was stained as a kinetochore marker. Representative images were presented. Mitotic centromere localization of each KNL2 mutant was shown as a merged image (Insets): SNAP-CENP-A (red) and each KNL2 mutant (green). DNA was stained with DAPI. Scale bar indicates 10 µm.",
    "molecules": "DAPI, IAA"
  },
  {
    "caption": "A) KNL2 (red) localization in CENP-C conditional knockout chicken DT40 cells (cKO-CENP-C) with or without CENP-C expression. DNA was stained with DAPI (blue). CENP-T was stained as a kinetochore marker (light blue). Scale bar indicates 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D SHP1IP in the presence and absence of SHP1 tail phosphorylation. SHP1 was immunoprecipitated from J.CaM1.6LCK‐Tet cells. Where indicated, LCK expression was induced with doxycycline prior to the experiment or Src‐kinase inhibitor PP2 was used to abolish residual SHP1‐pY564. The upper panel of immunoblots shows the effect of LCK activity on SHP1‐pY564 levels. The red arrowhead indicates residual pY564. Isolated protein complexes are shown in the lower panels.",
    "molecules": "doxycycline, PP2"
  },
  {
    "caption": "C Poly‐caspase activity in THEMIS KD 1G4‐CD8 cells. Cells were stimulated for 24 h with NY‐ESO‐1 pMHC tetramers. Camptothecin was used as a positive control for caspase activation. A FAM‐FLICA detection probe was used to assess poly‐caspase activity by flow cytometry. Data shown are a representative example of three independent experiments.D Quantification of poly‐caspase activity of cells from (C). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01; ns, not significant.",
    "molecules": "Camptothecin"
  },
  {
    "caption": "Sucrose gradient UV profile of 60S/40S/IAPV-IRES reaction mixture after an overnight run. The peaks corresponding to 80S and 40S were used for RNA extraction and UREA-PAGE shown in the inset.",
    "molecules": "Sucrose, UREA"
  },
  {
    "caption": "(B) Disintegration reaction (1 hour) performed by RAG1 aa 216-1008 R848M/E649V, RAG2 aa 1-361 and full length human HMGB1 with Mg2+ or Mn2+ at the indicated temperatures. Denaturing gel displays the fluorophore-labeled DNA strand from the RAG STC before (16 nt band, lane 2) and after (37 nt) the disintegration reaction. Lane 1, fluorophore-labeled 37 nt DNA marker. Representative of 2 independent experiments.",
    "molecules": "DNA, Mg2, Mn2+"
  },
  {
    "caption": "Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.",
    "molecules": "Glucose, tamoxifen"
  },
  {
    "caption": "Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.",
    "molecules": "Food, tamoxifen"
  },
  {
    "caption": "Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.",
    "molecules": "cholesterol, fatty acid, FFA, tamoxifen"
  },
  {
    "caption": "H, I WT and lincRNA-EPS-/- iBMMs were infected with VSV (MOI 0.1) for 6 h or 10 h (H) or transfected with polyI:C (1 μg/mL) for 4 h or 8 h (I), phosphorylation and total protein expression were analyzed by Western blot with α-tubulin and β-Actin as loading controls.",
    "molecules": "polyI:C"
  },
  {
    "caption": "I RNA-pulldown using biotinylated lincRNA-EPS from cell lysates of polyI:C (1 and 2.5 μg/mL) transfected iBMMs for 4 h, PKR protein was detected by Western blot and β-Actin was shown as a loading control.",
    "molecules": "biotinylated, polyI:C"
  },
  {
    "caption": "A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Scale bar: 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L) Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Scale bar: 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.",
    "molecules": "methylcellulose"
  },
  {
    "caption": "E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Binding of Annexin V Alexa Fluor 488 to control (Scr) and MDGI-silenced (shMDGI1) BT12 cells was measured by flow cytometric analyses five days after silencing. The analysis was performed by gating the cells into two populations: P1 (small, granular) and P2 (normal-sized cells) (30 000 analysed events/cell line, n = 3). B. Geometric mean fluorescence (FL-1) relative to the controls (Scr) that was set as 1 in P1 and P2 in MDGI-silenced (shMDGI1) cells. Data are represented as mean ± SD. *P < 0.05, two-tailed, nonparametric Mann-Whitney's U-test.",
    "molecules": "Alexa Fluor 488"
  },
  {
    "caption": "C, D Representative micrographs of non-treated control BT12 cells (C) and cells treated with 2 mM LLOMe for 4 h (D) before staining with the anti-galectin-1 antibodies (LGALS1) (green). Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.",
    "molecules": "DAPI, LLOMe"
  },
  {
    "caption": "E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "G, H Representative micrographs of LGALS1-stained (green) BT13 control (G) and MDGI-silenced cells (H) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "L. Number of live cells in MDGI-silenced BT12 and BT13 cells incubated without (sh1) or with (sh1+K777) the pan-cathepsin inhibitor (K777) after 1, 3 and 5 days of incubation. Data shows the percentage of live cells compared to the control (Scr) cells set as 100 %. Data are represented as mean ± SD. *P < 0.05; **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.",
    "molecules": "K777"
  },
  {
    "caption": "A. Proportions (mol %) of saturated (Sat), monounsaturated (Mono), diunsaturated (Di; total of 2 double bonds in the acyl chains), and polyunsaturated (Poly; total of 3-7 double bonds in the acyl chains) lipid species from the two main phospholipid classes (phosphatidylcholine PC and phosphatidylethanolamine PE) of the lysosomal fractions of the BT12 and BT13 control (Scr) and MDGI-silenced (shMDGI) cells. A representative graph of two independent experiments is shown.",
    "molecules": "lipid, PC, phosphatidylcholine, PE, phosphatidylethanolamine, phospholipid"
  },
  {
    "caption": "B. Fold change of the main PC and PE species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) cells (Log2, by mol % data). Abbreviations: [Lipid class] [total number of acyl chain carbons]:[total number of double bonds in the chains]. A representative heatmap of two independent experiments is shown.",
    "molecules": "carbons, Lipid, PC, PE"
  },
  {
    "caption": "C. Profiles of ceramide (Cer) species (mol %) in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. Abbreviations: Cer [number of acyl chain carbons]:[number of double bonds in the acyl chain]; all principal species containing a sphingosine chain. A representative graph of two independent experiments is shown.",
    "molecules": "carbons, Cer, ceramide, sphingosine"
  },
  {
    "caption": "D. Ratio of the 24:1 to 24:0 (mol %.mol %) Cer species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. A representative graph of two independent experiments is shown.",
    "molecules": "Cer"
  },
  {
    "caption": "A. Measurement of the BT12, BT13, and ZH305 glioblastoma cell viability using the MTT-assay at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.",
    "molecules": "clemastine"
  },
  {
    "caption": "B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.",
    "molecules": "clemastine"
  },
  {
    "caption": "C. Representative micrographs of BT12, BT13, and ZH305 glioblastoma cells treated with 1 µg/ml of clemastine for 24 h and stained with anti-galectin-1 (LGALS1) antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 20 µm.",
    "molecules": "clemastine, DAPI"
  },
  {
    "caption": "D. Representative micrographs of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and kidney cells (HEK293T) treated with 1 µg/ml clemastine for 24 h and stained with anti-LGALS1 antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.",
    "molecules": "clemastine"
  },
  {
    "caption": "A. Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells), and daily treated with saline vehicle (Ve) or 50 mg/kg clemastine (Cle, 100 mg/kg single dose administered at day 1) for 12 days starting at day 15 after tumour implantation. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), ZH305 (Ve n = 10, Cle n = 10), and BT13 (Ve n = 10, Cle n = 9). Human glioma cells were visualised with an anti-human vimentin (hVim, red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.",
    "molecules": "Cle, clemastine, DAPI"
  },
  {
    "caption": "B, C Quantification of the number of the invading single BT12 wt glioblastoma cells (B) and distance of the invaded cells from the primary tumour (C) of vehicle (black bars, Ve) and clemastine-treated (white bars, Cle) animals (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.",
    "molecules": "Cle, clemastine"
  },
  {
    "caption": "D. Quantification of the number of secondary tumours (diameter >300 µm) in the murine brain for the indicated treatments and tumour models. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), and BT13 (Ve n = 10, Cle n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.",
    "molecules": "Cle"
  },
  {
    "caption": "E. Quantification of the number of angiotropic BT12 cells that have co-opted existing blood vessels in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.",
    "molecules": "Cle"
  },
  {
    "caption": "Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.",
    "molecules": "Cle"
  },
  {
    "caption": "G. Representative micrographs of consecutive brain sections (separating distance: 9 µm) showing xenografts treated with vehicle or clemastine and labelled with the anti-human vimentin (red in left panels) and TUNEL (red in right panels). Nuclei were visualised by using DAPI (white). Scale bar: 50 µm.",
    "molecules": "clemastine, DAPI"
  },
  {
    "caption": "H. Kaplan-Meier survival curve of the BT12 xenografted animals treated with vehicle (green line, n = 15) and clemastine (black line, n = 12). Survival of the animals has been measured and pooled from two independent experiments. P = 0.044, Mantel-Cox Log-rank test.",
    "molecules": "clemastine"
  },
  {
    "caption": "BrdU incorporation in the Kit+ IAHC upon cumulative administration. Multistack reconstruction of confocal images. Representative picture of a short BrdU pulse (1h). Scale bars: 50μm (overview), 10μm (magnification). (F) Quantification of the percentage of Kit+ cells within IAHCs incorporating BrdU using confocal images. All cells (Labelling Index=100%) can be labelled after 8h, indicating the lack of quiescent cells inside the IAHCs. Graph represents mean±SE.",
    "molecules": "BrdU"
  },
  {
    "caption": "Quantification and classification of IAHC containing recombined cells in TAM-induced VeCadCreER:Confetti embryos after IgG or αDll4 treatment. Embryos treated with αDll4 contain more IAHC with colored cells. Quantification of confocal images (n=7 embryos).",
    "molecules": "TAM"
  },
  {
    "caption": "(A) Bar chart showing the relative abundance (signal intensity) of the phosphorylated tau species identified using an exploratory IP-MS approach using HT7 antibody on TBS-soluble control and Alzheimer's disease (AD) pools. Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP-MS: mono-phosphorylated p-tau231 and double phosphorylated p-tau(231+235).",
    "molecules": "TBS"
  },
  {
    "caption": "d, Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to PCNA+ CMs; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles).",
    "molecules": "2-DG, DCA"
  },
  {
    "caption": "e) Immunostaining of heart sections for MEF2 N2.261 and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to N2.261+ CMs; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles).",
    "molecules": "2-DG, DCA"
  },
  {
    "caption": "Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(f AFOG staining of heart sections from 60 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "g) AFOG staining of heart sections from 60 dpci animals and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(a, b) Staining for Ki67, CTNI and DNA (DAPI) in control, PDHA1STA OE or PDK3 OE RNCMs cultured in growth (a) or non-growth (b) medium; arrowheads point to Ki67+ RNCMs; percentage of Ki67+ RNCMs shown on the right (n = 4 biological replicates for each condition).",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(c) Staining for CTNI and DNA (DAPI) in scratch assay using control, PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch; magnified view of area in white boxes shown below; arrowheads point to membrane protrusions of RNCMs; quantification of CM membrane protrusions shown on the right (n = 4 biological replicates for each condition).",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(B) Islet graft survival of mice receiving vehicle (PBS) or IL-33 was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001 vs islet+vehicle. (C) Blood glucose level of mice treated with IL-33 (the horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection). Each line represents one mouse.",
    "molecules": "glucose, PBS"
  },
  {
    "caption": "C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (A, B) Representative FACS analysis showing the proportion of Tregs (CD4+Foxp3+) in the CD4+ T cell compartment from the spleens (A) and kidneys (B) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6). ",
    "molecules": "PBS"
  },
  {
    "caption": "C57BL/6 mice were treated with mouse recombinant IL-33 or PBS daily for 5 consecutive days. (C, D) Representative FACS analysis showing the proportion of ILC2s (Lin-GATA-3+) in the CD45+ leukocyte compartment from the spleens (C) and kidneys (D) at day 3 after treatment in C57BL/6 mice receiving PBS (n=4) or IL-33 (n=6).",
    "molecules": "PBS"
  },
  {
    "caption": "(E, F) proportion of Tregs (E) and ILC2s (F) in the spleen and kidney in PBS-injected controls (n=4) and at weeks 1-12 after IL-33 treatment (n=4-6 per IL-33-treated group). Data information: Data shown are the mean ± SEM; Statistical analysis was performed with an unpaired t-test, **P<0.01, ***P<0.001.",
    "molecules": "PBS"
  },
  {
    "caption": "(I) Representative confocal microscopy images of immunostaining for CD127, ST2, CD3 and insulin in islet grafts. Scale bar = 50 µm.",
    "molecules": "insulin"
  },
  {
    "caption": "Proportion of CD4+Foxp3+ Tregs (B) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.",
    "molecules": "PC61"
  },
  {
    "caption": "Proportion of Lin-GATA-3+ ILC2s (C) from the spleens and kidneys of islet transplant mice receiving vehicle, IL-33, IL-33/DT, IL-33/PC61, or IL-33/DT/PC61 at day 5 post-islet transplantation. Data shown are the mean ± SEM (n=4-5 per group) and a one-way ANOVA was performed; NS: non significant, ***P<0.001.",
    "molecules": "PC61"
  },
  {
    "caption": "(D) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. *P<0.05, **P<0.01.",
    "molecules": "glucose"
  },
  {
    "caption": "(E) Data are shown as blood glucose measurement in islet transplant mice treated with IL-33/DT, IL-33/PC61, or IL-33/DT/PC61. The horizontal black line indicates a BGL of 16 mmol/liter, the threshold for rejection. Each line represents one mouse.",
    "molecules": "PC61, glucose"
  },
  {
    "caption": "(C) IL-10 reporter C57BL/6 mice were treated with PBS, IL-33 alone or IL-33 and IL-2C daily for 5 consecutive days. Proportion of lin-CD127+ST2+IL-10-GFP ILC210 from the kidneys at day 3 after treatment in IL-10 reporter C57BL/6 mice. Data shown are the mean ± SEM (n=4-6 per group) and a one-way ANOVA was performed; ***P<0.001.",
    "molecules": "PBS"
  },
  {
    "caption": "(G) The kidney ILC2s were cultured with Medium, IL-33/IL-2C, or IL-33/IL-2C and STAT5 inhibitor (STAT5-IN) for three days. The secreted cytokine IL-10 was analyzed via ELISA. Data shown are the mean ± SEM (n=6 per group) and a one-way ANOVA was performed; ***P<0.001.",
    "molecules": "STAT5 inhibitor, STAT5-IN"
  },
  {
    "caption": "(C) Transfused ILC2s (CFSE labeled, Green) were observed in islet graft at day 5 post islet transplantation. Scale bar = 50 µm. The numbers of CFSE-labeled ILC2s in the islet graft were counted. Data shown are the mean ± SEM and a one-way ANOVA was performed (n=4 per group). NS, not significant.",
    "molecules": "CFSE"
  },
  {
    "caption": "(E) Islet graft survival of four groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.",
    "molecules": "glucose"
  },
  {
    "caption": "(B) Islet graft survival of five groups of mice was assessed by monitoring blood glucose and calculated using the Kaplan-Meier method. Cumulative data from two independent experiments are shown. Statistical analysis was performed with a log-rank test. ***P<0.001.",
    "molecules": "glucose"
  },
  {
    "caption": "D) Representative images of SYTO 9 and propidium iodide (PI) double stained M. florum cells, harvested from an exponential or death-phase culture, observed by widefield fluorescence microscopy. The brightness of each channel was adjusted equally between conditions. Scale bar: 5 µm.",
    "molecules": "SYTO 9, PI, propidium iodide"
  },
  {
    "caption": "A) Circular representation of the M. florum L1 chromosome enhanced with 5'-RACE data generated in this study. Outer to inner circle: genomic coordinates (kbp); genes encoded on the positive (blue for coding sequences, turquoise for RNAs) and negative (red for coding sequences, fuchsia for RNAs) DNA strands; raw 5'-RACE signal (0-1,000 read starts scale) observed at each genomic position for the positive (black) and negative (gray) DNA strands; putative transcription start sites (TSSs) identified on the positive (green) and negative (orange) DNA strands from significant 5'-RACE peaks.",
    "molecules": "RNAs"
  },
  {
    "caption": "(B) Reduced-minus-oxidized difference spectra of strains described in (A). Heme peaks are indicated for Cor1WT in WT background.",
    "molecules": "Heme"
  },
  {
    "caption": "Steady-state protein levels of strains as described in (A) cultivated on YP media containing glycerol",
    "molecules": "glycerol"
  },
  {
    "caption": "Steady-state protein levels of strains as described in (A) cultivated on YP media containing CM media with galactose as carbon source (D, E).",
    "molecules": "carbon, galactose"
  },
  {
    "caption": "(A) Growth analysis of strains expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Growth curves and calculated doubling times from the exponential phase (insert) are presented. Cells were cultivated in media containing either glucose or glycerol as carbon source.",
    "molecules": "carbon, glucose, glycerol"
  },
  {
    "caption": "(B) Determination of chronological lifespan via propidium iodide (PI) staining of cells described above.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "(D,E) Analysis of competitive fitness as described in (C). Cor1WT strains harbored a chromosomally-integrated hygromycin selection cassette, whereas Cor1** contained a clonNAT selection cassette (D). Subsequently, selection markers were switched as a control (E).",
    "molecules": "hygromycin, clonNAT"
  },
  {
    "caption": "(C,D) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH (C) or succinate + glycerol-3-phosphate (G3P; D) in isolated coupled mitochondria of indicated strains. Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.",
    "molecules": "ADP, NADH, KCN, G3P, glycerol-3-phosphate, succinate"
  },
  {
    "caption": "(E) Respiratory control ratio calculated from polarographic measurements of KCN-sensitive oxygen consumption driven by NADH and succinate + glycerol-3-phosphate (G3P) in isolated coupled mitochondria of indicated strains.",
    "molecules": "NADH, KCN, G3P, glycerol-3-phosphate, succinate"
  },
  {
    "caption": "(F,G) Polarographic measurement of KCN-sensitive oxygen consumption driven by NADH in mitoplasts in the absence (endog. Cyt c) or presence (+ exog. Cyt c) of exogenous oxidized Cyt c. NADH oxidation (E) and the calculated ratio +Cyt c/- Cyt c of substrate oxidation (F) are visualized.",
    "molecules": "NADH, KCN"
  },
  {
    "caption": "(A) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH in isolated coupled mitochondria. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Thereby, strains either overexpressed cytochrome c (Cyt cOE) or harbored the empty plasmid as a control (Empty). Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.",
    "molecules": "ADP, NADH, KCN"
  },
  {
    "caption": "Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing glucose (B) as carbon source.",
    "molecules": "carbon, glucose, hygromycin, clonNAT"
  },
  {
    "caption": "Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing galactose (C) or glycerol (D) as carbon source.",
    "molecules": "carbon, galactose, glycerol, hygromycin, clonNAT"
  },
  {
    "caption": "HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 (grey bars), 48 (pink bars), or 72 (yellow bars) hours and cell proliferation was assessed. HCT-116",
    "molecules": "sodium butyrate"
  },
  {
    "caption": "(C, E) and HT-29 (D, F) cells were treated with the indicated concentrations of NaB for 24 h. C.D. The percentage of apoptotic cells in HCT-116 (C) and HT-29 (D) cells was quantified in three independent experiments using an annexinV/PI assay (annexinV+/PI-) by flow cytometry and expressed as mean ± SD. One-way ANOVA was used to compare between the control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. E.F. Representative Western blots of full-length and cleaved PARP products in HCT-116 (E) and HT-29 (F) cells.",
    "molecules": "NaB"
  },
  {
    "caption": "HCT-116 (A, C) and HT-29 (B, D) cells were treated with 2mM sodium butyrate (NaB) for 24 h and then stained with acridine orange (AO; A-B) or monodansylcadaverine (MDC; C-D).",
    "molecules": "NaB, sodium butyrate"
  },
  {
    "caption": "Flow cytometry measured the percentage of AO+ cells and the results of three independent AO fluorescence experiments for HTC-116 (E) and HT-29 (F) cells were expressed as mean ± SD. One-way ANOVA was used to compare control cells and NaB treatments. *p<0.05, ** p<0.01 when compared to control.",
    "molecules": "NaB"
  },
  {
    "caption": "HCT-116 (A) or HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative blots showed expression of Beclin1, ATG3, and LC3 as well as GAPDH, the loading control.",
    "molecules": "NaB, sodium butyrate"
  },
  {
    "caption": "C. Electron microscopy images of control and NaB (2mM) treated HCT-116 cells. ER, endoplasmic reticulum; Mt, mitochondrion; AL.",
    "molecules": "NaB"
  },
  {
    "caption": "HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (A) HCT-116 cells treated with 3-MA and NaB;",
    "molecules": "3-MA, NaB, sodium butyrate"
  },
  {
    "caption": "HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (B) HT-29 cells treated with 3-MA and NaB",
    "molecules": "3-MA, NaB, sodium butyrate"
  },
  {
    "caption": "HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of null and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group. (C) HCT-116 cells treated with CQ and NaB;",
    "molecules": "chloroquine, CQ, NaB, sodium butyrate"
  },
  {
    "caption": "HCT-116 or HT-29 cells were treated with 5mM 3-MA or 5μM chloroquine (CQ) for 30 min, and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of LC3-I and LC3-II expression were quantified by densitometry and normalized to GAPDH (ratio of LC3:GAPDH). The fold change from control is indicated as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. ** p<0.01, compared to the respective control group.(D) HT-20 cells treated with CQ and NaB.",
    "molecules": "chloroquine, CQ, NaB, sodium butyrate"
  },
  {
    "caption": "  HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative Western blots show the expression of IRE-1a, BIP, PDI, and CHOP. GAPDH was used as the loading control. ",
    "molecules": "NaB, sodium butyrate"
  },
  {
    "caption": "  HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showed the expression of IRE-1a, BIP, PDI, and GADPH (loading control) in HCT-116 (A) or HT-29 (C) cells. Protein expression was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group. ",
    "molecules": "cycloheximide, NaB, sodium butyrate"
  },
  {
    "caption": "  HCT-116 (A, B) or HT-29 (C,D) cells were treated with 0.1μM mithramycin for 30 min followed by sodium butyrate (NaB) for 24 h. Representative Western blots showing the expression of IRE-1a, BIP, and PDI in HCT-116 (A) or HT-29 (C) cells are shown. GAPDH was used as loading control. Protein expression (IRE-1a, BIP, and PDI) was quantified by densitometry and normalized to GAPDH (ratio of protein:GAPDH). The fold change from control for each protein is expressed as mean ± SD of three independent experiments for HCT-116 (B) and HT-29 (D) cells. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group. ",
    "molecules": "mithramycin, NaB, sodium butyrate"
  },
  {
    "caption": "HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (A-B) HCT-116 cells treated with cyclohexamide; (E-F) HT-29 cells treated with cyclohexamide;",
    "molecules": "cyclohexamide, cycloheximide, mithramycin, NaB, sodium butyrate"
  },
  {
    "caption": "HCT-116 or HT-29 cells were treated with 10 μg/mL cycloheximide or 0.1μM mithramycin for 30 min and then with 2mM sodium butyrate (NaB) for 24 h. Representative Western blots of the expression of LC3-II are shown. The level of LC3-II expression was quantified by densitometry and normalized to GAPDH (ratio of LC3-II:GAPDH). The fold change from control cells is shown. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the control group. (C-D) HCT-116 cells treated with mithramycin;  (G-H) HT-29 cells treated with mithramycin. cancel ",
    "molecules": "mithramycin, NaB, sodium butyrate"
  },
  {
    "caption": "  HCT-116 cells were transfected with BIP or CHOP specific siRNAs for 48 h and then treated with or without 2mM sodium butyrate (NaB) for 24 h. Negative control (NC) scramble siRNA was used the negative control for the transfection. Representative Western blots are shown for BIP siRNA (A) and CHOP siRNA (C). The expression level of each protein was determined by densitometry and normalized to GAPDH (ratio of protein:GAPDH). (B) Normalized expression levels of BIP and LC3-II in HCT-116 cells treated with BIP specific siRNA. (C) Normalized expression levels of CHOP and LC3-II in HCT-116 cells treated with CHOP specific siRNA. Means and standard deviation (SD) of three independent experiments are shown. One-way ANOVA was used for statistical analysis. * P<0.05, ** p<0.01, compared to the respective control group.  ",
    "molecules": "NaB, sodium butyrate"
  },
  {
    "caption": "  HCT-116 (A) and HT-29 (B) cells were exposed to 5mM 3-MA or 5μM chloroquine (CQ) for 30 min and then treated with 2mM sodium butyrate (NaB) for 24 h. The percentage of apoptotic cells (annexinV+/PI-) was quantified by flow cytometry using an annexinV/PI assay and expressed as mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis to compare control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. (C.D.) Cells were treated as described above and the proteins were harvested for Western blot analysis. Expression of full-length and cleaved PARP in HCT-116 (C) or HT-29 (D) cells was compared to GAPDH, the loading control. The ratio of cleaved PARP to total PARP was calculated and the fold change in normalized PARP expression relative to cells treated with 2mM NaB is shown as the mean ± SD of three independent experiments. One-way ANOVA was used for statistical analysis. *P<0.05, ** p<0.01, compared to the 2mM NaB group. ",
    "molecules": "3-MA, chloroquine, NaB, sodium butyrate"
  },
  {
    "caption": "Immunostaining  of  H3K27ac in mouse germinal vesicle (GV) oocytes  (n = 8), metaphase II  (MII) oocytes  (n = 7), zygotes  (n = 7), early 2-cell (2C) (n = 7) and late 2C (n = 9) embryos. Right panel is the quantification of H3K27ac immunostaining  relative signal intensities at different stages. Scale bar: 20 μm. Data information: For boxplots in A the central band represents the median. The lower and upper edges of the box represent the first and third quartiles, respectively. The whiskers of the boxplot extend to 1.5 times inter-quartile range (IQR).",
    "molecules": "H3K27ac"
  },
  {
    "caption": "Immunostaining  of H3K27ac in A-485 treated embryos  versus control (DMSO  treated) at different time points. Scale bar: 20 μm.",
    "molecules": "A-485, DMSO"
  },
  {
    "caption": "Developmental rate of embryos  treated with  A-485  versus control.",
    "molecules": "A-485"
  },
  {
    "caption": "Immunostaining  of H3K27ac in TSA  treated embryos  under different concentrations versus control (DMSO  treated) at 28 hpi. Scale bar: 20 μm.",
    "molecules": "DMSO, TSA"
  },
  {
    "caption": "Developmental rate of embryos  treated with TSA  at different time points (0-20 hpi and 8-28 hpi) versus control.",
    "molecules": "TSA"
  },
  {
    "caption": "IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue).",
    "molecules": "Alexa-488, Alexa-647, Dapi, DNA"
  },
  {
    "caption": "Human KZFPs KRAB domain A-boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme (residues sharing common physicochemical properties display the same color: http://www.jal- view.org/ help/html/colourSchemes/zappo.html). This figure also displays a zoom-in on the variant KRAB domain cluster (right). On this zoom-in, the tested vKZFPs, whose interactomes were defined in our study, were marked by an asterisk. D. Box plot representing KAP1FC values in function of KZFPs age. On a superimposed swarm plot, the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts, by blue dots. Mann-Whitney two-sided rank test.",
    "molecules": "amino acid"
  },
  {
    "caption": "Representative images of Sirius-red-stained heart sections (scale bar: 1mm) (J)",
    "molecules": "Sirius-red"
  },
  {
    "caption": "Quantification of cardiomyocyte area (B), N=6 to 8 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1, FBS: fetal bovine serum, PE: phenylephrine.",
    "molecules": "endothelin-1, ET-1, PE, phenylephrine"
  },
  {
    "caption": "Quantification of protein/DNA ratio (C), N=9 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.",
    "molecules": "DNA, PE, phenylephrine"
  },
  {
    "caption": "Quantification of Acta1 mRNA transcript abundance (D), N=3 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. ET-1: endothelin-1 PE: phenylephrine.",
    "molecules": "endothelin-1, ET-1, PE, phenylephrine"
  },
  {
    "caption": "Quantification of cell death with a 7-AAD assay (E), N=7 samples/group in NRCM transduced with Ad.Con or Ad.TIP30 and stimulated as indicated. PE: phenylephrine.",
    "molecules": "PE, phenylephrine"
  },
  {
    "caption": "(C) Confocal microscopy images of neonatal rat cardiomyocytes (NRCM) stained with antibodies for endogenous TIP30 (green) and endogenous eEF1A1 (red). DAPI: blue. (Scale bar: 20µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Microscopy images of isolated neonatal rat cardiomyocytes and subsequent Proximity Ligation Assay (PLA). Red: endogenous TIP30/eEF1A1 interaction; Blue: DAPI. Control cells were stained for TIP30/Myc-Tag interaction (scale bar: 50µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Western blot analysis of co-immunoprecipitation (IP) for endogenous TIP30 and eEF1A1 protein in NRCM after stimulation with phenylephrine for 24 h. IgG HC - High chain of IgG molecule.",
    "molecules": "phenylephrine"
  },
  {
    "caption": "(D) Microscopy images of NRCM after adenoviral transduction with Ad.TIP30 or control virus (Ad.Con) and stimulation with phenylephrine (PE) and subsequent Proximity Ligation Assay (PLA). Red: eEF1A1-eEF1B2 interaction; Blue: DAPI. (scale bar: 50µm). (E) Quantification of eEF1A1-eEF1B2 interaction in conditions described in (D) ",
    "molecules": "DAPI, PE, phenylephrine"
  },
  {
    "caption": "(F) Microscopy images of adult mouse cardiomyocytes isolated from hearts 3 days (d) after TAC or sham surgery and subsequent PLA. Red: eEF1A1-eEF1B2 interaction; Blue: DAPI (scale bar: 100µm). (G) Quantification of eEF1A1-eEF1B2 interaction in adult mouse cardiomyocytes isolated from hearts 3 days after TAC or sham surgery and subsequent PLA (N=4 to 6 mice/group). *",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Western blot analysis of isolated neonatal rat cardiomyocytes (NRCM) after adenoviral transduction either with control adenovirus (Con) or Ad.TIP30 (TIP30) followed by stimulation with phenylephrine (PE, for 3 hours) and puromycin incorporation (for 30 min). (B) Quantification of the Western blot shown in (A) (N=3 samples/group). ",
    "molecules": "PE, phenylephrine, puromycin"
  },
  {
    "caption": "(C) Quantification of cell surface area of isolated neonatal mouse cardiomyocyte of Tip30 Het and WT mice treated with endothelin-1 (ET-1) and Narciclasine (Narci) or without stimulation as indicated (N=6 samples/group).",
    "molecules": "endothelin-1, ET-1, Narci, Narciclasine"
  },
  {
    "caption": "Quantification of HW/TL ratio (E), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.",
    "molecules": "Narciclasine"
  },
  {
    "caption": "Quantification of , echocardiographic wall thickness (F), N=6 to 10 mice/group, in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.",
    "molecules": "Narciclasine"
  },
  {
    "caption": "Quantification of cardiomyocyte area (G), N=5 mice/group in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.",
    "molecules": "Narciclasine"
  },
  {
    "caption": "Quantification of fractional area change (FAC, H, N=6 to 10 mice/group) in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated.",
    "molecules": "Narciclasine"
  },
  {
    "caption": "(I) Western blot analysis of puromycin incorporation in hearts of Tip30 Het and WT mice 3 days after TAC or sham surgery and daily Narciclasine (Narci) injection and their quantification (N=2 to 4 mice/group). Puromycin was injected 3 hours prior to sacrifice.",
    "molecules": "Narci, Narciclasine, puromycin, Puromycin"
  },
  {
    "caption": "(D-E) Measurements of ciliary cAMP  levels using cilia-Pink Flamindo. (D) Exemplary images are shown for cilia  (cilia-bPAC   or cyto-bPAC   cells) expressing the cilia-Pink Flamindo biosensor after light   stimulation. Scale bars are indicated; cAMP  levels are color-coded using a high-low look-up table. (E) Quantification of data exemplified in (D). The cAMP  level is determined as the mean ciliary  fluorescence intensity during the first 60 s after light  stimulation (measurement interval 10 s); p value for a  Kolmogorov-Smirnov test  is indicated. Each data point represents an individual cilium. Bars display median and interquartile range; cilia-bPAC: 40 cilia  (biological replicates) from n = 14 independent experiments; cyto-bPAC: 31 cilia  (biological replicates) from n = 31 independent experiments.",
    "molecules": "cAMP"
  },
  {
    "caption": "(A) Wild-type (WT) mIMCD-3  cells cultured in a 3D matrix during continuous exposure to DMSO  (control) or 10 µM of Forskolin. Exemplary images are shown (n = 5 biological replicates). Quantification of cyst   number and area is shown on the right Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment, p values calculated using an unpaired, two-sided Student's t-test are indicated.",
    "molecules": "DMSO, Forskolin"
  },
  {
    "caption": "(D) mIMCD-3 cells stably expressing cilia-bPAC  or cyto-bPAC, cultured in a 3D matrix in the dark, during light  exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) or in the dark  and incubated with 10 µM of Forskolin. Exemplary images are shown (n > 3). (E) Quantification of the data exemplified in (D). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using an unpaired, two-sided Student's t-test are indicated.",
    "molecules": "Forskolin"
  },
  {
    "caption": "(D) Immunoblotting  of lysates from mIMCD-3 wild-type (WT) cells and mIMCD-3  cells stably expressing cilia-bPAC  for S6 ribosomal protein (S6RP) phosphorylation  at  Ser235/236 (pS6RP). Levels of total S6RP  protein (S6RP) and beta-Tubulin  have been determined as controls. Cilia-bPAC  cells were treated with DMSO  (control) or with 10 nM of Rapamycin. All cells were stimulated with light (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²) (E) Quantification of the pS6RP/S6RP  ratio. Data are shown as mean ± S.D., n > 3; p values were calculated using a paired, two-sided Student's t-test. Each data point shows an independent experiment",
    "molecules": "DMSO, Ser, Rapamycin"
  },
  {
    "caption": "(G) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark, during light  exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubation with 10 nM rapamycin. Light  exposure started 2 d later than incubation with 10 nM rapamycin. Exemplary images are shown (n = 3) (H) Quantification of the data exemplified in (G). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using a paired, two-sided Student's t-test are indicated",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A)  mIMCD-3  cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark  or during light  exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubated with DMSO  (control), 250 µM IBMX, a ubiquitous PDE inhibitor, or 10 µM Rolipram, a PDE4-specific inhibitor. Exemplary images are shown (n = 3). Scale bars are indicated. (B) Quantification of data exemplified in (A). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual experiments.",
    "molecules": "IBMX, DMSO, Rolipram"
  },
  {
    "caption": "(C)  mIMCD-3  cells stably expressing cyto-bPAC, cultured in a 3D matrix during light  exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO  or 10 µM rolipram. Exemplary images are shown (n = 4-5). Scale bars are indicated. (D) Quantification of data exemplified in (C) Data are shown as mean ± S.D., p values calculated using an unpaired Student's t-test are indicated. Data points show individual experiments.",
    "molecules": "DMSO, rolipram"
  },
  {
    "caption": "(H)  mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during light  exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO  (0 µM), 3 µM, or 10 µM of the Pde4-long-isoform activator MR-L8. Exemplary images are shown (n = 3). Scale bars are indicated. (I) Quantification of data exemplified in (H). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data have been normalized to the Light, 0 µM  MR-L8 condition (Set to 100%) within each experiment. Data points show individual experiments.",
    "molecules": "MR-L8, DMSO"
  },
  {
    "caption": "(D) 3D culture of mIMCD-3  WT cells continuously exposed to acetone  (control) or 100 nM PGE2. Exemplary images are shown (n = 3 biological replicates). (E) Quantification of images exemplified in (D). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual biological replicates. (F) Quantification of the cyst  area in PGE2-stimulated  mIMCD-3  WT  cells in the presence of the PDE4  long isoform-specific activator MR-L8. Data were normalized to PGE2- and control-treated cells and are shown as mean ± S.D. p values calculated using a one-sample t-test compared to 100% are indicated, n = 4 biological replicates.",
    "molecules": "MR-L8, acetone, PGE2"
  },
  {
    "caption": "(G) 3D culture of  mIMCD-3 WT cells continuously exposed to acetone  (control) or 100 nM PGE2, and DMSO  (control) or 10 µM of the EP4  inhibitor L161,982. PGE2  treatment started 2 d after seeding. Exemplary images are shown (n = 3). Scale bars are indicated. (H) Quantification for images exemplified in (G), including addition of 3 µM AH6809, an EP2  inhibitor Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated (p value of n/a indicates that the test is not applicable because both conditions showed no variance). Data points show individual experiments and have been normalized to addition of 100 nM PGE2  (set to 100%) within each experiment.",
    "molecules": "AH6809, acetone, DMSO, L161,982, PGE2"
  },
  {
    "caption": "(I) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO  (control) or the EP4-inhibitor L161,982 in the dark  or during light  exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO  or L161,982). Exemplary images are shown (n = 3). (J) Quantification of images exemplified in (I). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments and have been normalized to the light/DMSO  condition (Set to 100%) within each experiment.",
    "molecules": "DMSO, L161,982"
  },
  {
    "caption": "(F) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO  (control) or 20 µM Celecoxib, a COX-2-inhibitor, in the dark  or during light  exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO  or Celecoxib). Exemplary images are shown (n = 3). Scale bars are indicated. (G) Quantification of images exemplified in (F) and from 3D cultures incubated with 1 μM celecoxib. Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments.",
    "molecules": "Celecoxib, celecoxib, DMSO"
  },
  {
    "caption": "(A) Wild-type (WT), cilia-bPAC, or cyto-bPAC  (magenta)  mIMCD-3  cells were stimulated by light  (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²), and treated with DMSO  (control) or 50 µM Ciliobrevin-D, a dynein-inhibitor. Cells were labeled with DAPI  (blue) to label the DNA, an ARL13B  antibody (white) to label cilia, and a phospho-specific (Ser133) CREB  antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. B) Quantification of the ciliary  pCREB signal in cilia-bPAC and cyto-bPAC  cells. Data are shown as mean ± S.D., n > 3. Each data point represents an independent experiment and corresponds to the median of > 73 cilia  (biological replicates); p values for an unpaired, two-sided Student's t-test with Welch's correction are indicated.",
    "molecules": "Ciliobrevin-D, DAPI, DMSO"
  },
  {
    "caption": "(C) mIMCD-3 Ift27-/- cells with or without stable cilia-bPAC  expression (magenta) were stimulated with light  (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI  (blue) to label the DNA, an ARL13B  antibody (white) to label cilia, and a phospho-specific (Ser133) CREB  antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. (D) Quantification of the ciliary  pCREB  signal. Data are shown as mean ± S.D., n > 3. Each data point represents the median of > 90 cilia  from the same experiment; p values are indicated for an unpaired, two-sided Student's t-test with Welch's correction.",
    "molecules": "DAPI, Ser"
  },
  {
    "caption": "(A-C) Tissue lysates from (A) spleen, (B) liver  and (C) kidney  from 6- to 9-day old indicated mice  were immunoblotted  to determine cleavage of  caspase-1. Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs  stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from caspase-1-/- mice.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(D-G) Tissue lysates from (D, F) spleen  and (E, G) liver  from 6- to 9-day old indicated mice  were immunoblotted  to determine pro-IL-1β cleavage to  IL-1β (D-E) or GSDMD  cleavage to its p30   and p20  fragments (F-G). Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs  stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from (D-E) caspase-1-/- or (F-G) Gsdmd-/- mice.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "(B) Fstl1 induced phosphorylation of ERK1/2 was ablated by pretreatment with MEK inhibitor PD98059. NRCFbs were treated by PD98059 (5nM) for 30min and then stimulated with recombinant Fstl1 protein (50ng/ml) for 15min. Error bars represent mean ± SEM. Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test (n=3 for each time point). Two independent experiments were performed.",
    "molecules": "PD98059"
  },
  {
    "caption": "(D) Fstl1-stimulation of cell migration was reversed by PD98059. Endogenous Fstl1 in NRCFbs was ablated by Fstl1 siRNA. Serum-deprived NRCFbs were treated with PD98059 (5nM) for 30 min and then stimulated by Fstl1 (50ng/ml) or vehicle. Cell migration was assessed at 6 hours after Fstl1 stimulation. Error bars represent mean ± SEM (n=7-9 for each group). Statistical analysis was performed by Kruskal-Wallis test and Dunnett's T3 test. Two independent experiments were performed.",
    "molecules": "PD98059"
  },
  {
    "caption": "(E) Morphological changes of NRCFbs after recombinant Fstl1stimulation was assessed by immunocytochemistry. 24 hours serum starved NRCFbs was stimulated by Fstl1 (50ng/ml) or vehicle for 3 hours. PD98059 (5nM) or DMSO was added 30min prior to Fstl1 stimulation. Cells were stained with Alexa Fluor 488-conjugated phalloidin antibody and nuclei were stained by DAPI. Arrow shows lamellipodium of the cell. Scale bar indicates 50μm.",
    "molecules": "PD98059, DMSO"
  },
  {
    "caption": "(C) Fstl1 promoted fibroblasts proliferation was diminished by PD98059. PD98059 (5nM) was added 30 min prior to recombinant Fstl1 stimulation (50ng/ml). The cells were cultured for 48 hours, and EdU was added into media at 4 hours before harvest. Error bars represent mean ± SEM (n=4-10, per treatment group). Statistical analysis was performed by one-way ANOVA and Tukey multi comparison test. Two independent experiments were performed.",
    "molecules": "PD98059"
  },
  {
    "caption": "A. In the Discovery cohort, Kaplan-Meier estimates of disease-free survival (DFS) in years (y) in patients with low (red) versus high (blue) nuclear PRMT5 expression (upper panel) and in 2 groups of patients according to their treatment. Patients treated with tamoxifen (Tam, middle panel) and with aromatase inhibitors (AIs, lower panel). P-values are calculated with the logrank test. B. The same analyses were performed in the Validation cohort.",
    "molecules": "Tam, tamoxifen"
  },
  {
    "caption": "A. GST and GST-PRMT5 fusion proteins were incubated with in vitro-translated ERα, the interaction was then visualized by Western blotting using an anti-ERα antibody. The corresponding Coomassie-stained gel is shown in the right panel.",
    "molecules": "Coomassie"
  },
  {
    "caption": "C. The same experiment was performed for MCF7 cells treated or not with tamoxifen (Tam) for the indicated times. The scale bar is 10µM.",
    "molecules": "Tam, tamoxifen"
  },
  {
    "caption": "D. Proximity Ligation Assay (PLA) to detect interactions between ERα and PRMT5 in MCF7 cells treated with E2, Tam 1 µM or both for 6 h. After fixation, PLA experiments were performed to evaluate the interactions between ERα/PRMT5 using specific antibodies. The detected dimers are represented by red dots. The nuclei were counterstained with mounting medium containing DAPI (blue) (Obj:X60). The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.",
    "molecules": "DAPI, Tam"
  },
  {
    "caption": "F. Paraffin-embedded sections from a fresh tumor were incubated with or without G595, and PRMT5 and SDMA expression were assessed by IHC.",
    "molecules": "G595, SDMA"
  },
  {
    "caption": "C. MCF7 cells were treated with or without E2, Tam or both for 6 h. ERα methylation was determined by PLA using an anti-ERα and a pan methyl recognizing symmetrical dimethylation (SDMA). The scale bar is 10µM. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.",
    "molecules": "SDMA, symmetrical dimethylation, Tam"
  },
  {
    "caption": "D. The same experiment was performed with G595 to verify that ERα methylation was PRMT5-dependent. Red dots reflect ERα methylation. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. The sale bar is 40µM. Western blotting was performed to assess ERα, PRMT5 expression and the global profile of methylation using a pan SDMA antibody.",
    "molecules": "G595, SDMA"
  },
  {
    "caption": "E. MCF7 cells were treated as in C and lysates were immunoprecipitated with a pan SDMA antibody and immunoblotted with an anti-ERα antibody. ERα, SDMA and GAPDH expression was evaluated in the input.",
    "molecules": "SDMA"
  },
  {
    "caption": "B. MCF7 cells were treated with Tam for 6 h in the presence or absence of G595, cell extracts were then subjected to ChIP assay using anti-ERα or anti-SMRT antibodies. The precipitated DNA fragments were used for qPCR analysis using specific primers for the indicated promoters. The results are expressed relative to the signal obtained from input chromatin. The mean ± SEM of at least three experiments is shown. P-values were determined using a Student t-test.",
    "molecules": "G595, Tam"
  },
  {
    "caption": "C. MCF7 cells were treated with Tam for 6 h and total RNA was prepared and cDNAs were analyzed by RT-QPCR with specific primers for GREB1. The values were normalized against 28S mRNA and represent the ± SEM of three experiments.",
    "molecules": "Tam"
  },
  {
    "caption": "B. MCF7 cells were treated with Tam for 6h in the presence or in absence of G595, then ERα interaction with SIRT2 and SAP18 was determined by PLA using specific antibodies against both proteins. The scale bar is 10µM. Quantification was performed as in Figure 2. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. Western blotting was performed to assess ERα, SIRT2, SAP18 and GAPDH expression and the global profile of SDMA.",
    "molecules": "G595, SDMA, Tam"
  },
  {
    "caption": "A. ERα methylation SDMA was studied by PLA, and PRMT5 and MEP50 expression were analyzed by IHC on formalin-fixed MCF7 xenografts treated or not with Tam (Obj: X40). The scale bar is 50 µM. Histogram quantifying the number of dots/cells and the H-scores for nuclear PRMT5 and MEP50 are presented on the right. The mean ± SEM of 7 tumors is shown. P-values were determined using a Student t-test.",
    "molecules": "formalin, SDMA, Tam"
  },
  {
    "caption": "B. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed PDX model HBCx-34 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 3 tumors is shown. P-values were determined using a Student t-test.",
    "molecules": "formalin, SDMA, Tam"
  },
  {
    "caption": "C. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed the PDX model HBCx-22 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 7 tumors is shown.",
    "molecules": "formalin, SDMA, Tam"
  },
  {
    "caption": "A. For each tumor, we analyzed the level of ERα SDMA methylation by PLA. The H-score for nuclear PRMT5 expression and the number of dots/cell are listed under the figures. The scale bar is 20µM.",
    "molecules": "SDMA"
  },
  {
    "caption": "F Minimal inhibitory concentrations of DHA treated wild-type, heterozygous (YME1/yme1) and homozygous (yme1/yme1) mutant strains. Plates were imaged after 72h.",
    "molecules": "DHA"
  },
  {
    "caption": "D Competitive growth assay of DHA resistant single cell clones. Wild-type (mCherry+_Cre_Puro) and knockout (GFP+_Puro) sister clones were derived from mutant (retrovirus - intron RE, darker shading, Tol2 transposon - intron T2, lighter shading) resistant colonies, treated with DHA, analyzed with flow cytometry and Sanger sequenced for insertion site mapping.",
    "molecules": "Puro, DHA"
  },
  {
    "caption": "A, B Cell survival of dihydroartemisinin treated ESCs in combination with (A) the Ppox inhibitor acifluorfen or upon (B) 5-ALA administration. Alamar Blue staining was used to assess viability after 72h of treatment.",
    "molecules": "Alamar Blue, 5-ALA, acifluorfen, dihydroartemisinin"
  },
  {
    "caption": "C, D Cell survival of DHA treated primary human glioblastoma cells from cancer patients and in combination with (C) the Ppox inhibitor acifluorfen (BTL90 cells) or upon (D) 5-ALA administration (VBT12 cells). Viability was assessed using CellTiter-Glo after 72h. Experiments were performed in triplicate.",
    "molecules": "5-ALA, acifluorfen, DHA"
  },
  {
    "caption": "E, F (E) ROS levels (DHE staining, PE 582/15 nm - MFI) and (F) cell survival of DHA (0.5μM), 5-ALA (0.25mM) or Ppox inhibitor (10μM) treated human neuroblastoma cells (SHSY5Y). Fluorescence of DHE (ROS levels) and cell numbers (survival) were assessed by flow cytometry and automated cell counting. The experiments were performed two times, in triplicate each.",
    "molecules": "5-ALA, DHA, DHE, PE, ROS"
  },
  {
    "caption": "C, D (C) Fluorescence and (D) brightfield images of DMSO (control), 5-ALA (0.0625mM), DHA (1μM), or 5-ALA and DHA (0.0625mM and 1μM) treated tumor organoids (CNS-PNET-like, c-MYC overexpression). Organoids were imaged on day 1, day 5 and day 7. One experiment is shown, representative of four independent experiments.",
    "molecules": "5-ALA, DHA, DMSO"
  },
  {
    "caption": "G ROS/ DHE staining and flow cytometry analysis of dissociated tumor organoids (PE, 582/15 nm). Mean fluorescence intensities (MFI) of DHE stained GFP+ tumor cells and GFP- control cells are indicated. n=2, values are mean ± SD.",
    "molecules": "DHE, PE, ROS"
  },
  {
    "caption": "A Fluorescence (left panel) and brightfield (right panel) images of 5-ALA and DHA treated human cerebral tumor organoids (glioblastoma-like). Organoids were monitored on day 1, day 5, day 8 and day 11. One experiment is shown, representative of two independent experiments. Scale bar 500μm. B, C Image analysis and quantification of GFP-positive tumor areas of the treated organoids. Four organoids per group were analyzed on day (B) d5 and (C) d11, normalized to day 1. Data is shown as box plots (25th-75th percentiles, median) n=4. D",
    "molecules": "5-ALA, DHA"
  },
  {
    "caption": "A Representative brightfield images of 5-ALA and DHA treated patient-derived glioblastoma spheroids (VBT92). Images were taken on day 3 of culture left untreated or exposed to the indicated treatments. Scale bar 500μm.",
    "molecules": "5-ALA, DHA"
  },
  {
    "caption": "B Re-plating of spheroids after 3 days of 5-ALA and DHA treatment (duplicates). Representative brightfield images are shown for glioblastoma VBT92.",
    "molecules": "5-ALA, DHA"
  },
  {
    "caption": "C Quantification of the re-adhesion capacity of re-plated VBT92 spheroids as assessed by crystal violet staining and absorbance measurement. Data is shown as mean ± SD. Experiments were performed in quadruplicates.",
    "molecules": "crystal violet"
  },
  {
    "caption": "D Lipid peroxidation in VBT92 cells treated with 5-ALA and DHA. Lipid peroxidation was measured using BODIPY™ 581/591 C11 staining and green (from red) fluorescence shift was determined using flow cytometry. Experiments were performed in triplicate; values are shown as mean ± SD.",
    "molecules": "BODIPY, 5-ALA, DHA"
  },
  {
    "caption": "B In vivo efficacy comparison of solvents (control), 5-ALA, artesunate (ARS) and combined treatment in an orthotopic glioblastoma model. 11-14 days after stereotactic intracerebral injection of 2 x 105 VBT529 cells, mice received the following treatments: Control (solvents), 5-ALA (80 mg/kg), ARS (40 mg/kg), or 5-ALA plus ARS 5 times per week via intraperitoneal injection (n ≥ 5).",
    "molecules": "5-ALA, ARS, artesunate"
  },
  {
    "caption": "C, D Quantification and representative images of the VBT529 flank tumor luminescence signal. 6-7 days after injection of 1.5 x 105 VBT529 cells into the flank, mice received the following treatments: Control (solvents) or 5-ALA plus ARS 4-5 times per week for 5 weeks via intraperitoneal injection (n = 6). After 4 days of treatment, concentrations of 5-ALA (100 mg/kg) and ARS (50 mg/kg) were increased to 120 mg/kg and 60 mg/kg, respectively. The 20% dosage increase of both drugs was used as the body-weight, behaviour and overall health of the mice were not affected by both, the lower as well as higher dosages. E, F Quantification and representative images of VBT529 brain tumor luminescence signals. 6-7 days after stereotactic intracerebral injection of 1.5 x 105 VBT529 cells, mice received the treatments as described in (C and D) (n = 6).",
    "molecules": "5-ALA, ARS"
  },
  {
    "caption": "G Kaplan-Meier survival curves of mice injected with VBT529 glioblastoma cells into the flank and brain (same mice as in C-F; n = 6). Mice were treated with solvents (control) or 5-ALA plus ARS (same mice as in C-F).",
    "molecules": "5-ALA, ARS"
  },
  {
    "caption": "(d) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5-mC labeling of P3 skin. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.",
    "molecules": "5-mC, DNA"
  },
  {
    "caption": "(h) Hematoxylin and Eosin (H&E) staining, immunolabeling  for KERATIN 10 (KRT10), LORICRIN (LOR) and KERATIN 6 (KRT6) expression of dorsal skin sections (P5) of control and Dnmt1Δ/Δep mice.",
    "molecules": "Eosin, Hematoxylin"
  },
  {
    "caption": "Immortalized  human keratinocytes (NHEK  SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (c, d) PicoGreen staining of PBS- and DAC-treated keratinocytes for 48h detected micronuclei  (arrow), which were analyzed in a blinded manner, and compared using two-tailed t-test. Data are mean ± SEM, * p≤0.05 (n=3 biological replicates) (d). Data information: Scale bars, 20 µm",
    "molecules": "5-aza-2′-deoxycytidine, DAC, PBS, PicoGreen"
  },
  {
    "caption": "Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (e) Co-labeling of  DNA by PicoGreen and by  cGAS  in  PBS and DAC  treated  keratinocytes. Within enlarged sections on the right arrows point at  DNA/cGAS double-positive blebs and micronuclei. (f) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC  for 45 h or 72 h using one way-ANOVA with post hoc Dunnett multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001 (n=4 biological replicates). Data information: Scale bars, , 50 µm",
    "molecules": "5-aza-2′-deoxycytidine, DAC, PBS, PicoGreen"
  },
  {
    "caption": "(C, E and G) WT PEMs expressing GFP-FAK were incubated with either ΔinvG Salmonella (C), LPS-coated beads (E) or E. coli (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) ΔinvG Salmonella (D), LPS-coated beads (F) and E. coli (H) was quantified. At least 100 bacteria were counted per condition; N = 3.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) WT PEMs were incubated with LPS-coated beads for 0-5 hours before immunoblotting for the indicated antibodies.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies.",
    "molecules": "PF-228"
  },
  {
    "caption": "(C-D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0-5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) WT PEMs were incubated with Akt inhibitor AKTV/triciribine (10 µm) for 30 minutes before infection with S. typhimurium strain ΔinvG for 5 hours. Lysates were immunoblotted with the indicated antibodies. (",
    "molecules": "triciribine"
  },
  {
    "caption": "(E) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 (300 ng) before immunoblotting with the indicated antibodies. Vertical white line indicate non-contiguous lanes generated from a single exposure.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "G) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin (4 µm) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours. Cells were lysed before immunoblotting with the indicated antibodies.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(H) WT and FAK−/− PEMs were pretreated with rapamycin (4 µm), Akt inhibitor AKTV/triciribine (10 µm) or left untreated (-Tx) or before infection with S. typhimurium strain ΔinvG for a further 5 hours. WT macrophages were also pretreated with the FAK inhibitor PF228 (0.5 µm) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours. Cells were then assessed for the percentage of ΔinvG Salmonella co-localizing with LC3. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05. N.s. not significant.",
    "molecules": "PF228, rapamycin, triciribine"
  },
  {
    "caption": "A) WT and FAK−/− PEMs were incubated with LPS-coated beads (50∶1; beads∶cell) for 1 hour before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei. Bars represent 10 µm. The percentage of LPS-beads co-localizing with LC3 is displayed to the right. At least 100 beads were counted per condition. Values are means ± SEM, N = 3.",
    "molecules": "LPS"
  },
  {
    "caption": ". (C-D) FAK−/− PEMs treated with Atg5, ULK1 or control siRNA for 48 hours were incubated with LPS-coated beads or ΔinvG Salmonella for 1 or 5 hours before analysis with antibodies recognizing LC3. The percentage of LPS-beads or Salmonella co-localizing with LC3 is displayed to the right. At least 100 beads or bacteria were counted per condition. Values are means ± SEM, N = 3.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) WT and FAK−/− PEMs were pretreated with IFN-γ (50 ng/ml), rapamycin (4 µm), or left untreated before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 6, *p<0.05.",
    "molecules": "rapamycin"
  },
  {
    "caption": "A, Misreading (A) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes (n=8 misreading, mutant A1555G mitohybrid ribosomes (n=8 misreading, E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). Mistranslation was determined by assessing the misreading at His245 (CAC codon) of the F-luc gene that was replaced by the near-cognate codon CGC or the non-cognate codon AGA, both encoding for Arg. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.",
    "molecules": "Arg"
  },
  {
    "caption": "C Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA. Following immunoprecipitation, the MT-CO1 band was quantified by autoradiography, wild-type mitohybrid ribosomes were set as 1 (n=3), error bars indicate SEM; ****p<0.001 (Student's t-test).",
    "molecules": "Cysteine, Methionine, 35S"
  },
  {
    "caption": "D Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation in the presence of tobramycin using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA; the MT-CO1 band was quantified by autoradiography.",
    "molecules": "Cysteine, Methionine, 35S, tobramycin"
  },
  {
    "caption": "E Misreading of wild-type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin. Misreading was assessed by dual luciferase assay, and results derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.",
    "molecules": "tobramycin"
  },
  {
    "caption": "F In-vitro translation of MT-CO1-TGA mRNA and MT-CO1-AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). Autoradiography of immunoprecipitated MT-CO1 proteins; top: 35S-Met labelling, bottom: 14C-Lys labelling. In-organello translated 35S-Met labelled MT-CO1 was used as a marker. * MT-CO1, ** MT-CO1 extended by read-through, MT-CO1 AGA-polyA and MT-CO1 TGA-polyA constructs used for in-vitro translation are schematized.",
    "molecules": "14C, Lys, Met, 35S, tobramycin"
  },
  {
    "caption": "G Ratio of 14C-Lys and 35S-Met labelled immunoprecipitated MT-CO1 proteins. In-vitro translation using MT-CO1-TGA mRNA, MT-CO1-AGA mRNA, and mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). The corresponding MT-CO1 band was quantified by autoradiography.",
    "molecules": "14C, Lys, Met, 35S, tobramycin"
  },
  {
    "caption": "A Autoradiography of in-organello mitochondrial translation products derived from HEK293 wild-type cells in the presence of tobramycin, incorporation of 35S-Met and 35S-Cys in MT-CO1 protein. Following in-organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in-organello translation in the presence of 1000 µM tobramycin. MT-CO1 bands were quantified and ratio of 35S-Cys / 35S-Met calculated, the 35S-Cys / 35S-Met ratio in the absence of tobramycin was set as 1 (n=3), error bars indicate SEM.",
    "molecules": "Cys, Met, 35S, tobramycin"
  },
  {
    "caption": "B Ratios of 35S-Cys / 35S-Met labelled MT-CO1 and MT-CO2 proteins synthesized in in-organello translation and effect of tobramycin. Following in-organello translation in the presence of tobramycin (1000 µM, 2000 µM) proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 and MT-CO2 bands were quantified and ratio of 35S-Cys / 35S-Met incorporation calculated (n=3), error bars indicate SEM; *p<0.05, ns, not significant (Student's t-test).",
    "molecules": "Cys, Met, 35S, tobramycin"
  },
  {
    "caption": "Ratio of 35S-Cys and 35S-Met labelled MT-CO1 protein synthesized in in-organello translation using mitochondria from HEK293 MRPS5 wild-type cells and HEK MRPS5 mutant V336Y cells. Following in-organello translation, proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 bands quantified and ratios calculated. The ratio of the MRPS5 wild-type was set as 1 (n=8 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).",
    "molecules": "Cys, Met, 35S"
  },
  {
    "caption": "D Autoradiography of in-organello mitochondrial translation products derived from HEK293 MRPS5 wild-type cells in the presence of tobramycin (0-750 µM). Proteins were 35S-Met labeled and loaded either directly on SDS-PAGE (total, top) or following poly-lysine immunoprecipitation (bottom). * MT-CO1 and ** MT-CO1 with poly-lysine extension.",
    "molecules": "Met, poly-lysine, 35S, tobramycin"
  },
  {
    "caption": "E Quantification of MT-CO1 proteins in HEK293 MRPS5 wild-type cells and HEK293 MRPS5 mutant V336Y cells. In-organello mitochondrial translation in the presence of 35S-Met and 750 µM tobramycin. Proteins were loaded either directly on SDS-PAGE (total) or following poly-lysine immunoprecipitation and analyzed by autoradiography. For total protein samples the MT-CO1 band was quantified, for poly-lysine immunoprecipitated samples the extended MT-CO1 band was quantified and the ratio of extended MT-CO1/total MT-CO1 calculated. The ratio of MRPS5 wild-type was set as 1 (MRPS5 wild-type, n=5 clones, MRPS5 V336Y, n=7 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).",
    "molecules": "Met, poly-lysine, 35S, tobramycin"
  },
  {
    "caption": "B Whole cell in-vivo mitochondrial translation as determined by 35S-Met incorporation (n=4 clones, ± SEM).",
    "molecules": "Met, 35S"
  },
  {
    "caption": "C Autoradiography of in-organello translation using 35S-Met labelling.",
    "molecules": "Met, 35S"
  },
  {
    "caption": "Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. C State 3 OCR (after addition of ADP), effect of age (p<0.001) and genotype (p<0.0001).",
    "molecules": "ADP"
  },
  {
    "caption": "Oxygen consumption rate (OCR) was measured in freshly isolated mitochondria from wild-type and mutant mouse cortex. D State 3 uncoupled OCR (state 3u after addition of FCCP), effect of genotype (p<0.0001) and age-genotype interaction (p<0.0001).",
    "molecules": "FCCP"
  },
  {
    "caption": "F ATP levels.",
    "molecules": "ATP"
  },
  {
    "caption": "G Superoxide anion radicals.",
    "molecules": "Superoxide"
  },
  {
    "caption": "D Immunofluorescence images showing colocalization of Flag-PHGDH (green) and mitochondria (red) acquired with a laser confocal microscope system in PLC cells. Nuclei were stained with DAPI (blue), and mitochondria were stained with MitoTracker Red. Scale bar, 10 μm (left panel), 2 μm (right panel).",
    "molecules": "DAPI, MitoTracker Red"
  },
  {
    "caption": "H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 (pH 11.5) for 30 min at 4 ℃. The soluble component (Supernatant/Sup.) and membrane component (Pellet) were separated by centrifugation prior to western blot analysis. COX2, ATP8, and ANT2 are markers for the membrane compotent; CYTC and TFAM are markers for the soluble component.",
    "molecules": "Na2CO3"
  },
  {
    "caption": "A The oxygen consumption rate (OCR) was measured by successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot) in PLC cells infected with viruses expressing nontargeting control (NTC) shRNA or shPHGDH. Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.",
    "molecules": "AA, antimycin A, ATP, FCCP, oligo, oligomycin, Rot, rotenone"
  },
  {
    "caption": "B Western blot analysis of mtDNA-encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector (EV) or PLVX-PHGDH. β-Actin was used as the loading control.",
    "molecules": "mtDNA"
  },
  {
    "caption": "C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX-PHGDH prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.",
    "molecules": "mtDNA"
  },
  {
    "caption": "D PLC cells expressing NTC or shPHGDH were treated with 100 μg/ml cycloheximide (CHX) for 20 min prior to puromycin treatment for 15 min. Purified mitochondrial lysates were subjected to a SUnSET assay with an anti-puromycin antibody to measure the actively translated polypeptide chains. Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs. β-Actin was used as the loading control.",
    "molecules": "CHX, cycloheximide, puromycin"
  },
  {
    "caption": "F PLC cells with or without PLVX-Flag-PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol (CAP) in combination with 50 μM thiamphenicol (TAP) for 24 h prior to SUnSET assays. The assays were performed using whole-cell lysate.",
    "molecules": "CAP, chloramphenicol, DMSO, TAP, thiamphenicol"
  },
  {
    "caption": "G PLC cells with or without PLVX-PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.",
    "molecules": "mtDNA, CAP, DMSO, TAP"
  },
  {
    "caption": "H The translation efficiency of mtDNA-encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV, wild-type PHGDH, or the catalytically inactive PHGDH mutant PHGDHR236E as indicated. β-Actin was used as the loading control.",
    "molecules": "mtDNA"
  },
  {
    "caption": "I mtDNA-encoded proteins were analysed by western blotting in PLC cells overexpressing EV, wild-type PHGDH, or PHGDHR236E. β-Actin was used as the loading control.",
    "molecules": "mtDNA"
  },
  {
    "caption": "I PLC cells overexpressing PLVX-PHGDH were further infected with viruses expressing NTC or shANT2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.",
    "molecules": "mtDNA"
  },
  {
    "caption": "K PLC cells overexpressing pSin-3xFlag-PHGDH were further infected with viruses expressing NTC or shmtEFG2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.",
    "molecules": "mtDNA"
  },
  {
    "caption": "D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. mtDNA-encoded proteins were analysed by western blot. β-Actin was used as the loading control.",
    "molecules": "mtDNA"
  },
  {
    "caption": "F Binding of mtDNA-encoded mRNA by endogenous MRPL44 (representing mitochondrial ribosomes) was evaluated by RNA immunoprecipitation (RIP) in PLC cells with PHGDH knockdown.",
    "molecules": "mtDNA"
  },
  {
    "caption": "G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient. After centrifugation, all fractions were subjected to western blot analysis of MRPS35, MRPL44, and MRPL48 proteins. RNA concentrations of ND6 were measured.",
    "molecules": "CAP, sucrose, TAP"
  },
  {
    "caption": "A PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2. The OCR was measured after successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot). Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.",
    "molecules": "AA, antimycin A, ATP, FCCP, oligo, oligomycin, Rot, rotenone"
  },
  {
    "caption": "B PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were constructed by cell counting with trypan blue exclusion (right).",
    "molecules": "trypan blue"
  },
  {
    "caption": "C PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were determined by cell counting with trypan blue exclusion (right).",
    "molecules": "H2O, tigecycline, trypan blue"
  },
  {
    "caption": "G-I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice (n = 5 in each group). Mice were intraperitoneally injected with 100 mg/kg tigecycline daily beginning 14 days after inoculation. Tumour sizes were measured beginning 14 days after inoculation (G). At the end of the experiment, the tumours were excised (H), and PHGDH expression in tumour lysates was analysed by western blotting (I).",
    "molecules": "tigecycline"
  },
  {
    "caption": "WT or SLC15A4feeble BMDCs non-transduced (C) were infected with flagellin-expressing STm  and fixed C. Cells were stained  for endogenous ASC  and p62, labeled with DAPI  and analyzed by fluorescence microscopy. Representative images showing ASC  speck (red) relative to p62  (green) in two infected WT and SLC15A4feeble DCs  each. Note p62 staining  surrounding ASC  specks in SLC15A4feeble DCs. D. Quantification of perinuclear  (within a radius of 3 μm from the nucleus) ASC  specks in at least 30 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC  images show nuclear  position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "SLC15A4feeble BMDCs  transduced with SLC15A4-GFP  (E were infected with flagellin-expressing STm  and fixed (E, Cells were stained  for endogenous ASC, labeled with DAPI  and analyzed by fluorescence microscopy. Representative images showing ASC  speck (red) and SLC15A4-GFP  (green) in SLC15A4feeble DCs  together with non-transduced SLC15A4feeble DCs. Note the perinuclear  positioning of ASC  specks in the transduced cells (E). N, nucleus. Dotted white lines, cell outlines. Corresponding DIC  images show nuclear  position. Scale bar, 8 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "WT or SLC15A4feeble BMDCs  transduced with constitutively active RagB  (RagBQ99L) or metap2  (control) H, I) and mcherry-LC3  (H, I), (H, I). Cells were stained  for endogenous ASC, labeled with DAPI  and analyzed by fluorescence microscopy. Representative images showing ASC  speck (green) and LC3  (red) in SLC15A4feeble DCs  (H). Quantification of perinuclear  (within a radius of 3 μm from the nucleus) in at least 30 cells per cell type in each of three independent experiments (I). N, nucleus. Corresponding DIC  images show nuclear  position. Scale bar, 8 μ Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of the artificial substrate by wild-type (WT)/inactive (S201A) GlpG encoded on pBAD33 with/without arabinose (Ara.). rhomboid substrates that are uncleaved, cleaved by GlpG are marked by black, red, arrows, respectively. Controls, empty pBAD33 (empty)",
    "molecules": "Ara, arabinose"
  },
  {
    "caption": "C Western blot analysis to detect cleavage of the artificial substrate by wild-type (WT)/modified (S133A or H187A) Rhom7 encoded on pBAD33 with/without arabinose (Ara.). , rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Controls, empty pBAD33 (empty) and wild-type S. sonnei (Ss).",
    "molecules": "Ara, arabinose"
  },
  {
    "caption": "Western blot analysis (probing with an anti-FLAG mAb) to detect cleavage of HybO with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). homboid substrates that are uncleaved, are marked by black, rrows, ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.",
    "molecules": "alanine, histidine, serine"
  },
  {
    "caption": "Western blot analysis to detect cleavage of HybA with (+)/without (-) chromosomal (native) or pBAD33-encoded rhomboids (plasmid). rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.",
    "molecules": "alanine, histidine, serine"
  },
  {
    "caption": "Western blot analysis to detect cleavage of HybAG296F by endogenous or pBAD33-encoded rhomboid. Wild-type S. sonnei, Ss. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, re marked by black, red, and blue arrows, espectively. ild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.",
    "molecules": "alanine, histidine, serine"
  },
  {
    "caption": "Western blot analysis to detect cleavage of HybAP300A by endogenous or pBAD33-encoded rhomboid. rhomboid substrates that are uncleaved, cleaved by GlpG, or by Rhom7, are marked by black, red, and blue arrows, respectively. Wild-type S. sonnei, Ss. Wild-type (WT)/inactive (SAHA: alanine substitution of the catalytic serine and histidine residues) enzymes were pBAD33-encoded (plasmid) in S. sonneiΔglpGΔrhom7.",
    "molecules": "alanine, histidine, serine"
  },
  {
    "caption": "A Bacterial two-hybrid analysis with HybA and/or HybB fused chromosomally with the T25 and T18 domains of B. pertussis CyaA, respectively in the absence of endogenous CyaA. Bacteria were grown on LB agar containing 20 µg/ml X-gal at 37°C for 14 hours in the presence/absence of O2. Scale bar, 1 cm.",
    "molecules": "O2, X-gal"
  },
  {
    "caption": "D Western blot analysis to detect GlpG-mediated cleavage of native tagged HybA at indicated times after blocking protein translation by the addition of chloramphenicol at T0 in the absence of HybB (-). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.",
    "molecules": "chloramphenicol"
  },
  {
    "caption": "E Western blot analysis to detect GlpG-mediated cleavage of C-terminally sfCherry-3xFLAG tagged HybA at times after blocking protein translation by the addition of chloramphenicol at T0 in the presence of HybB (+). , HybA that is uncleaved or cleaved by GlpG is marked by black and red arrows, respectively.",
    "molecules": "chloramphenicol"
  },
  {
    "caption": "F Growth of bacteria lacking hydrogenases (ΔhyaA-F ΔhycE +/- ΔhybO-G) or GlpG (ΔglpG), or expressing uncleavable HybB (hybAG296F) in 5% H2",
    "molecules": "H2"
  },
  {
    "caption": "G H2 consumption by S. sonnei. Bacteria were grown aerobically in LB overnight then diluted into M9 minimal media supplemented with 0.5% fumarate, 12.5 μg/ml nicotinic acid and 0.2% casamino acids in a sealed glass chromatography vial. The headspace was purged with 10% H2/90% argon and cultures were incubated at 37 °C with shaking at 180 rpm for 9 hours. H2 in the headspace was sampled and measured by gas chromatography.",
    "molecules": "argon, H2, fumarate, nicotinic acid"
  },
  {
    "caption": "H S. sonnei growth in 10% H2 with plasmid expressed wild-type or non-functional (pglpGSAHA) GlpG.",
    "molecules": "H2"
  },
  {
    "caption": "Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdoH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed chromosomally (native) or from pUC19 (plasmid) without FdoI , +/- exposure to 400 µM CuCl2 for 30 min. : Rhomboid substrates that are uncleaved, cleaved by GlpG, are marked by black, red, arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.",
    "molecules": "CuCl2"
  },
  {
    "caption": "F Degradation of N-terminally V5-tagged wild-type (WT) or modified (P259A) FdnH in S. sonneiΔrhom7 with wild-type (+) or inactive (S201A, SAHA) GlpG expressed from pUC19 (plasmid) without FdnI (-), , +/- exposure to 400 µM CuCl2 for 30 min. Rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, and blue arrows, respectively. Degradation products post rhomboid cleavage are marked by green arrows.",
    "molecules": "CuCl2"
  },
  {
    "caption": "Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1. These phosphoproteomic analyses were conducted in the presence of 0.02% MMS for 2 hours.",
    "molecules": "MMS"
  },
  {
    "caption": "Immunoblot analysis of Sgs1 N-terminus (amino acids 1-647) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and/or MEC1. This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments.",
    "molecules": "MMS"
  },
  {
    "caption": "Immunoblots showing co-immunoprecipitation between Dpb11 and Sgs1 in the presence of 0.04% MMS in either wildtype, mec1Δ, tel1Δ or mec1Δ tel1Δ cell lines. DPB11-3HA was tagged at its endogenous locus and co-immunoprecipitated with SGS11-647-FLAG tagged and truncated at its endogenous locus.",
    "molecules": "MMS"
  },
  {
    "caption": "Dilution assay of wild-type or rev3Δ cells expressing BRCT3/4-SGS1 in the presence of 0.0025% MMS.",
    "molecules": "MMS"
  },
  {
    "caption": "A-C. Pins-myr-GFP and myr-RFP show a similar localization pattern to Pins:Tom during mitosis. Representative pictures are shown (A, B); a heatmap lookup table (A', B') emphasizes the relative enrichments of Pins-myr-GFP and myr-RFP along the cortex. Quantification (Pins-myr: 4 cells, myr-RFP: 2 cells) (C).",
    "molecules": "myr"
  },
  {
    "caption": "D-F. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full-length Pins or Pins-myr-GFP. Representative pictures (D, E) and quantification (F) are shown. Scale bars = 5 microns. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001.",
    "molecules": "myr"
  },
  {
    "caption": "D. Confocal microscopy images of live non-infected PD myotubes (left) or PD myotubes infected for 72 h with Ad-TFEB (right) show a dramatic reduction in the amount of accumulated glycogen in TFEB-treated cells. The cells were incubated with the fluorescent glucose (2-NBDG; green), extensively washed, and analysed by confocal microscopy. Bar: 10 µm for all panels.",
    "molecules": "2-NBDG, glucose, glycogen"
  },
  {
    "caption": "A. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected musclesglycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test).",
    "molecules": "glycogen"
  },
  {
    "caption": "B. PAS staining of TFEB-treated muscle shows a reduction of lysosomalglycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×.",
    "molecules": "glycogen"
  },
  {
    "caption": "A. EM images of muscle injected with either AAV-GFP (untreated) or AAV-TFEB (TFEB-treated). Asterisks indicate glycogen-containing lysosomes. Bar: 1.5 µm (upper panels) and 0.45 µm (lower panels). Higher magnification images (lower panels) show that glycogen particles are less densely packed in TFEB-treated muscle. Black arrows indicate autophagosome profiles; the white empty arrow shows remnants of mitochondria engulfed by the lysosome.B. Graphical presentations of lysosomal length (average ± SE; n = 100 lysosomes; p = 6.31 × 10−5), the number of lysosomes per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields; p = 4.80 × 10−3), and the number of autophagosomes flanking glycogen-containing lysosomes (average ± SE; n = 100 lysosomes; p = 4.39 × 10−5 < 0.001). Student's t-test was used for each comparison.C. Graphical presentations of mitochondrial size (average ± SE; n = 100) and the number of mitochondria per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields). The differences were not significant by Student's t-test (p = 3.65 × 10−1 and 4.27 × 10−1, respectively).",
    "molecules": "glycogen"
  },
  {
    "caption": "(A) Masson's trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation.",
    "molecules": "PBS, bleomycin"
  },
  {
    "caption": "(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05)",
    "molecules": "Bleo"
  },
  {
    "caption": "(E) CD68 mRNA quantification in Bleo and Bleo-MCT rats",
    "molecules": "Bleo, MCT"
  },
  {
    "caption": "(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph.",
    "molecules": "Bleo, MCT"
  },
  {
    "caption": "(A-F) Representative PAS-stained sections of the pons  (A) and hippocampal (C) regions of the brain and heart (E) of one year old HOIL-1[C458S] and WT  mice are shown. Scale bar = 100 μm. Arrow heads indicate  α-amylase-resistant   PAS-positive   polyglucosan deposits. Graphs showing  α-amylase resistant  PAS  scores of the  pons (B) and  hippocampal  (D) regions of the  brain and the  heart (F) of HOIL-1[C458S] (red) and WT (blue) mice aged 0.5, 1.0 and 1.5 years. The number of biological replicates analysed at each age is indicated. The word zero highlighted in blue indicates that no α-amylase resistant, PAS-positive material could be detected in the WT  mice. The error bars show mean + SEM. Statistical significance between the genotypes was calculated by using two-way ANOVA and Šidák's multiple comparison's test. **** denotes p<0.0001.",
    "molecules": "PAS, polyglucosan"
  },
  {
    "caption": "(A)  HOIL-1 activity towards purified  bovine liver glycogen was measured using Cy5-labelled ubiquitin. Control assays lacking ATP, UBE2L3,  HOIL-1  and  glycogen are indicated, as are subsequent treatments with 50 μg/mL human salivary α‑amylase  and 1.5 M  hydroxylamine (NH2OH). The gel has been  stained using the periodic acid-Schiff  (PAS) method to visualise  glycogen (purple-magenta) and Coomassie Blue stain to visualise protein.",
    "molecules": "PAS, periodic acid-Schiff, ATP, Cy5, glycogen, hydroxylamine, NH2OH, ubiquitin"
  },
  {
    "caption": "(B) Prior to  PAS  and Coomassie staining, the gel shown in (A) was  stained with   Flamingo fluorescent protein  stain  (shown in red) and  glycogen ubiquitylation observed by visualising the fluorescent  Cy5-ubiquitin signal (shown in green).",
    "molecules": "Flamingo fluorescent protein  stain, PAS, Cy5, glycogen, ubiquitin"
  },
  {
    "caption": "(D) In vitro ubiquitylation of maltoheptaose by  HOIL-1. Assays were treated with α‑amylase or hydroxylamine  as indicated. Reaction products were detected by  Coomassie staining.",
    "molecules": "hydroxylamine, maltoheptaose"
  },
  {
    "caption": "(F)  HOIL-1 activity  against the indicated maltosaccharides  (2 mM) was assayed and visualised by staining  with  Coomassie protein stain. Where indicated, assays were treated with 1.5 M  hydroxylamine. Met1-linked  ubiquitin tetramer was also included in these assays.",
    "molecules": "maltosaccharides, hydroxylamine, Met, ubiquitin"
  },
  {
    "caption": "(H) In vitro ubiquitylation  of the indicated  cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.",
    "molecules": "Met, ubiquitin"
  },
  {
    "caption": "(A) 1 μg of each of the indicated recombinant proteins was incubated with amylose resin for 60 min, the beads were pelleted by centrifugation, and proteins in the  pellet  (P) and supernatant (S) were compared to the protein input (I) by means of western blotting with the appropriate antibodies.",
    "molecules": "amylose"
  },
  {
    "caption": "(B)  Bovine liver glycogen  and potato starch-derived amylopectin were incubated for 60 min with the indicated recombinant proteins, centrifuged for 90 min at 100,000 × g, and protein in the resulting supernatants  (S) and pellets  (P) analysed by western blotting with the appropriate antibodies. Protein without polysaccharide  was used as a negative control (Buffer).",
    "molecules": "amylopectin, glycogen, polysaccharide, starch"
  },
  {
    "caption": "(C)  Amylose binding assays of full-length (1-1072) and truncated HOIP protein, visualised by immunoblotting. Shown on the right are schematic representations of the truncated HOIP constructs used. Images not drawn to scale. PUB, peptide  N-glycanase and UBA- and UBX-containing protein domain; ZF, zinc finger domain; NZF, Npl4 zinc finger domain; UBA, ubiquitin-associated domain; RBR, RING-in-between-RING domain; LDD, linear ubiquitin chain determining domain.",
    "molecules": "Amylose"
  },
  {
    "caption": "(D) Amylose binding assays  of full-length (1-387) and truncated Sharpin mutants along with schematic representations of the Sharpin constructs used. Images not shown to scale. PH, pleckstrin homology domain; LTM, LUBAC-tethering motif; UBL, ubiquitin-like domain.",
    "molecules": "Amylose"
  },
  {
    "caption": "(A)  Maltoheptaose ubiquitylation  by HOIL-1 was assayed at 30°C in the presence of Met1- or  Lys63-linked ubiquitin dimers for the indicated times  and visualised by Coomassie staining.",
    "molecules": "Lys, Maltoheptaose, Met, ubiquitin"
  },
  {
    "caption": "(B) Maltoheptaose ubiquitylation  by HOIL-1 was assayed at 30°C in the presence of  Met1- or  Lys63-linked  ubiquitin tetramers  for the indicated  times and visualised by Coomassie staining.",
    "molecules": "Lys, Maltoheptaose, Met, ubiquitin"
  },
  {
    "caption": "(C) HOIL-1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers  and tetramers. These mutations have been reported to impair the  binding  of  ubiquitin to the NZF domain and their approximate location in HOIL-1 is indicated (schematic not to scale).",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(D) Assay of HOIL-1[Δ194-222] lacking the NZF core domain at 30°C in the absence or presence of the indicated  ubiquitin oligomers.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(E) Allosteric activation of HOIL-1[233-510] (lacking the LTM, UBL and NZF domains at the N‑terminus) by  ubiquitin oligomers.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(B) Time-course  assay performed at 30°C featuring wild-type  HOIL-1  in the absence or presence of the indicated  ubiquitin oligomers, visualised by Coomassie staining.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(A) Requirements for en bloc  transfer of linear  ubiquitin  dimer to  maltoheptaose  were investigated by leaving out the reaction components indicated. Reaction products were detected by  staining  with Coomassie.",
    "molecules": "Coomassie, maltoheptaose, ubiquitin"
  },
  {
    "caption": "(B) Requirements for  en bloc transfer of  Met1-linked ubiquitin tetramer to maltoheptaose were investigated by leaving out the reaction components indicated.",
    "molecules": "maltoheptaose, Met, ubiquitin"
  },
  {
    "caption": "(C) Time-course assay performed at 30°C in the presence of Met1-linked ubiquitin oligomers in which the two most C-terminal residues were either  glycine (normal ubiquitin) or  alanine  to prevent conjugation to substrates (GG/AA).",
    "molecules": "alanine, glycine, Met, ubiquitin"
  },
  {
    "caption": "(D)  Maltoheptaose ubiquitylation was assayed for 60 s at 30°C in the presence of the indicated  ubiquitin tetramers.",
    "molecules": "Maltoheptaose, ubiquitin"
  },
  {
    "caption": "(B) The concentration of productively signalling TCRs   (CN) over the antigen  concentration for antigens  with the indicated zero-force off-rates  (koff). Shown is the effect of forces  at 100 pN (left) and 10 pN (right). The horizontal dashed line is the threshold value of CN  required for activation in the model (λ). The vertical dashed line shows the antigen concentration (P) required to elicit activation by the antigen with the largest value of koff.",
    "molecules": "antigen, antigens"
  },
  {
    "caption": "(C) The antigen  concentration required to elicit activation  (P) over koff  for the indicated molecular forces  on the TCR/pMHC interaction. (D) Antigen  sensitivity quantified as the value of P from (C) over the applied force  for a higher-affinity antigen  with koff  = 0.5 s-1. (E) Antigen  discrimination quantified by the discrimination power (α) over the applied force. Discrimination power is defined as the ratio of the logarithm of the fold-change in P over the fold-change in koff  for a higher-affinity ligand (koff  = 0.5 s-1) and a lower-affinity ligand (koff  = 101.5 s-1) from panel (C).",
    "molecules": "antigen, Antigen"
  },
  {
    "caption": "(F) Experimental value of P (symbols) over antigen  affinity fitted by the kinetic proofreading model with force  (dashed and solid lines). The value of P is defined as the concentration of pMHC  required for 15% upregulation of CD69  above baseline and is shown as the mean with SEM from 4 independent experiments. (G) The fitted value of the applied force  with SEM. (H) The fitted value of λ with SEM. Data information: An F-test is used to produce a p-value for the null hypothesis that the applied force  or λ is the same across the three conditions. All data fitting (F-H) used N = 2.46 and kp = 2.15 s−1 (average of CD58  and ICAM-1 conditions) taken",
    "molecules": "antigen"
  },
  {
    "caption": "(F) Pheromone sensitivity halo assay with PGAL1-3HA-CLB5 sic1Δ strains carrying either wild-type FAR1, far1Δ, far1(L135A) or far1(RxL). Different concentrations of α-factor were pipetted on the paper discs. On glucose plates, α-factor triggers cell cycle arrest. CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing, which is rescued by the presence of α-factor, which leads to Far1-depedent inhibition of excess Clb5-Cdk1 activity. The experiment was repeated twice, a representative example is shown.",
    "molecules": "α-factor, glucose"
  },
  {
    "caption": "(B) The degradation of Far1(1-150)-GFP with either wild-type or all serine-based phosphorylation sites and the indicated docking motifs was measured in time-lapse microscopy. Plot shows mean±SEM of GFP fluorescence intensities from Start.",
    "molecules": "serine"
  },
  {
    "caption": "(C IHC labeling for MEIS2 (C) of LRCs positive for BrdU only and neuroblasts marked by DCX.",
    "molecules": "BrdU"
  },
  {
    "caption": "D) IHC labeling for MCM6 (D) of LRCs positive for BrdU only and neuroblasts marked by DCX.",
    "molecules": "BrdU"
  },
  {
    "caption": "Dot plots depicting the abundance of LRCs (BrdU+, Ki67-, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.",
    "molecules": "BrdU"
  },
  {
    "caption": "Dot plots depicting the abundance of aNSCs and TAPs (BrdU-, Ki67+, DCX-) in the SEZ 7 days after aCSF or AntimiR204 injection.",
    "molecules": "BrdU"
  },
  {
    "caption": "(F, G) Micrographs depicting the elbow - the most ventral region of the RMS (F) and OB neuronal layers (G) upon injection of artificial CSF (control) or AntimiR204. (H) Dot plot showing the density of BrdU+ cells (progeny of NSCs) in the RMS and in the OB 7 days after artificial CSF or AntimiR204 injection.",
    "molecules": "BrdU"
  },
  {
    "caption": "(G) Micrographs of transmission electron microscopy imaged extracellular vesicles (EVs) isolated from sham treated (upper row) and GW4869 treated animals (lower row). (G'' and G''') are magnifications of boxed areas in overview images to the left. (H) Dot plot depicting miR-204 levels in the CSF isolated 2 h after the ventricular injection of GW4869 inhibitor.",
    "molecules": "GW4869"
  },
  {
    "caption": "(C) Dot plot showing the proportion of neural stem cells (GFAP+) out of DAPI+ cells in primary SEZ/ChP co-culture with or without treatment with GW4869 inhibitor or AntimiR204.",
    "molecules": "DAPI, GW4869"
  },
  {
    "caption": "(D-G) Micrographs depicting the cellular compositions of SEZ cultures after co-culturing with control treated, GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating. Boxed areas correspond to the higher magnification images in adjacent panels (D', E', F', G').",
    "molecules": "GW4869"
  },
  {
    "caption": "Micrographs depicting immunoreactivity for MEIS2 (E, F) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR-204 interference.",
    "molecules": "BrdU"
  },
  {
    "caption": "Micrographs depicting immunoreactivity for MCM6 (H, I) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MCM6+ LRCs. Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 (J) after ChP specific miR-204 interference.",
    "molecules": "BrdU"
  },
  {
    "caption": "(K, L) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control (K) and miR-204 specific TuD (L). Note the reduction of primed LRCs (BrdU+ only, white arrows in K and L) upon TuD-204 injection. (M) Dot plot depicting the number of LRCs (BrdU+ only) in the SEZ 7 days after miR-204 inhibition. ",
    "molecules": "BrdU"
  },
  {
    "caption": "(C, D) Reduced synaptic width in USH1C pigs at an age of 1 year (1y). (C) Fluorescent microscopic analysis of the cone synapse phenotype. Left panel: Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre-synaptic marker PSD-95 (green) and by fluorescent peanut agglutinin (PNA, red) for cone synaptic pedicles (white arrows) and counter-stained by DAPI for nuclear DNA (blue). Scale bar 10µm. Middle panel: Higher magnification of a PNA-stained cone synaptic pedicle. Synapse width was determined as the maximum extension of consistent PNA signals. Scale bar 1µm. Right panel: Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA-stained sections indicated reduced synaptic width (2 pigs, 1y, 1 retina each, number of examined synapses indicated, error bars represent SD, two-tailed Student's t tests, ***p<0.001). (D) Determining cone synaptic pedicles width by TEM. Left panel: Representative images of retinal cross sections. Scale bar 500nm. Right panel: Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs. (1 pig, 1y, 1 retina each, number of examined synapses indicated,",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-C) Ciliary length measured by fluorescence microscopic analysis at 1 year of age (1y). (A) Representative immunostaining of the ciliary marker Gt335 (green) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas. Counterstaining by DAPI, scale bar 10µm. (B) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining. Scale bar 500nm. (C) Length of connecting cilia (CC) appear elongated in USH1C when applying a Fiji script to immunostained histological sections (2 pigs, 1y, 1 retina each, number of examined connecting cilia is indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).",
    "molecules": "DAPI"
  },
  {
    "caption": "Length of primary cilia in dermal fibroblasts. (F) Staining of ciliary structures in fibroblasts stained with ARL13B (ciliary shaft, green) and PCNT2 (ciliary base, red), DAPI (nucleus, blue). Scale bar upper panel 5 µm, lower panel 2.5 µm. (H) Similarly, ciliary length of fibroblasts from a human USH1CR31X/R80fs patient are elongated, compared to a healthy control (7 TR, number of cells examined indicated, KS-test, ****p<0.0001).",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Efficacy of AAV gene therapy was examined in vivo by sub-retinal injection of Anc80 virus capsid expressing harmonin_a1 under a ubiquitous promoter into a USH1C pig (see also Figure EV5). Left panel: Sub-retinal injection reconstituted harmonin expression in the AAV-treated eye but not in the sham-treated eye (3 TR). Protein extract from a WT retina served as positive control, actin staining as loading control. The 72kD band correlates with harmonin_a isoforms while higher MW indicates putative dimer formation. Middle panel: Immunofluorescence reveals absence of harmonin in the retina of the PBS-injected control eye and harmonin abundance (green) in the retina of the AAV-treated eye. (Representative image from a single animal. Blue: DAPI, scale bar 25 µm). Right panel: Photopic gfERG reveal increased a- and b-wave response to single flash and flicker stimulation in the AAV-treated (red), compared to the sham-treated (black) eyes of two USH1C pigs.",
    "molecules": "DAPI, PBS, ubiquitous"
  },
  {
    "caption": "HDAC6 KO MEF cells are defective in aggregate clearance but not in autophagosome induction or targeting. (A) Filter‐trap analysis of MG132‐induced, SDS‐insoluble ubiquitinated aggregates generated in wild‐type (WT), HDAC6 KO, and KO MEFs reconstituted with different HDAC6 constructs as indicated. The signal intensity from the ubiquitinimmunoblot (bottom panel) was quantified and presented as the average of the means from three independent experiments along with the standard deviation (top). Note the significant accumulation of ubiquitin‐positive aggregates in HDAC6 KO MEFs and KO MEFs stably expressing HDAC6‐CD and ΔBUZ mutants. WT, HDAC6 KO, and HDAC6 KO MEFs stably expressing human HDAC6 (hHDAC6 WT), HDAC6 CD (catalytically inactive mutant), or HDAC6 ΔBUZ (ubiquitin‐binding‐deficient mutant) were analysed for the level of HDAC6 using an anti‐human HDAC6 antibody. Mouse endogenous HDAC6 (mHDAC6) and actin levels were determined by each corresponding antibody.",
    "molecules": "MG132, SDS"
  },
  {
    "caption": "(B) WT and HDAC6 KO MEFs were treated with MG132 and subjected to Western blot analysis for LC3, actin, and HDAC6.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Cells were treated with MG132 as described under Materials and methods and immunostained with antibodies to ubiquitin (green) and LC3 (red). Arrows indicate ubiquitin‐positive aggregates that colocalize with LC3‐positive autophagosomes. Scale bar, 10 μm.",
    "molecules": "MG132"
  },
  {
    "caption": "(D) WT and ATG5 KO MEFs are treated with 2.5 μM MG132 for 1 day and incubated with normal growth media for 18 h. MEFs are immunostained with anti‐LC3 (red) and anti‐ubiquitin antibody (green).",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Long‐lived protein degradation in wild‐type and HDAC6 KO MEF cells. The degradation of [14C]‐valine labelled long‐lived protein was measured in the presence or absence of 3‐methyl adenine (3MA, inhibits the formation of autophagic vacuoles). The average of percentage degradation from three independent experiments is presented. The error bar represents the standard deviation.",
    "molecules": "[14C]‐valine, 3MA, protein"
  },
  {
    "caption": "(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1.",
    "molecules": "MG132"
  },
  {
    "caption": "(B, C) Wild‐type and HDAC6 KO MEFs were transfected with mCherry‐GFP‐LC3, followed by treatment with LatA (100 nM) or nocodazole (250 nM) for 6 h and analysed as described in Figure 3A.",
    "molecules": "LatA, nocodazole"
  },
  {
    "caption": "(D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01.",
    "molecules": "LatA, latrunculin"
  },
  {
    "caption": "(E) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at *P0.05. The number of total fusion events/total number of vesicles for each condition was as follows: 169/1250; 87/972; 190/1120; and 175/1165.",
    "molecules": "LatA, latrunculin"
  },
  {
    "caption": "(F) Autophagosomes (APGs) and lysosomes (Lys) isolated from HDAC6 KO MEFs were treated or not with latrunculin (LatA) as indicated and subjected to in vitro fusion assay.",
    "molecules": "LatA, latrunculin"
  },
  {
    "caption": "(B) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed cells were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at **P0.01.",
    "molecules": "LatA, latrunculin"
  },
  {
    "caption": "(C) WT and ATG5 KO MEFs were treated with MG132 and immunostained with antibodies to ubiquitin (green) as indicated. F‐actin was detected by phalloidin (red). The arrows indicated ubiquitin‐positive protein aggregates.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin (red), ubiquitin (green), and phalloidin for F‐actin (blue) as indicated. The arrows indicated ubiquitin‐positive aggregates that were colocalized with F‐actin and cortactin.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) Wild‐type MEFs were transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for Lap‐1 (red, to label lysosome), or ubiquitin (green) and phalloidin for actin (blue). Note that F‐actin staining at protein aggregates was lost, but lysosomes remained concentrated in cortactin knockdown cells (arrow).",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Wild‐type MEFs were transfected with control or cortactin siRNA, treated MG132 2.5 μM for 18 h, and subjected to filter‐trap assay using a ubiquitin antibody. The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Heatmap of genes in the livers  of HFD-feeding induced NAFL mice  and WD/CCl4-treatment induced NASH mice. n=5. The color gradient represents log2(Fold change).",
    "molecules": "CCl4"
  },
  {
    "caption": "(E) Correlations between KLF10  and NAS, inflammation  score, fibrosis  scores, and hepatic triglyceride  contents (TG) from NAFL mice  (n=7) and NASH mice  (n=8). Data information: * P <0.05, ** P <0.01-. Results are shown as mean ± SD. Spearman's correlation (E",
    "molecules": "TG, triglyceride"
  },
  {
    "caption": "(F) H&E staining, Sirius red staining, and KLF10 immunofluorescence staining  of liver  sections from NAFL  and NASH patients. n=3.",
    "molecules": "Sirius red"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (B) Protein levels  of KLF10  in the livers  (left) and its expression  was normalized to the β-actin (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (C) H&E staining  of liver  sections.",
    "molecules": "CCl4"
  },
  {
    "caption": "8-week-old male C57BL/6J mice were administered with AAV-GFP  or AAV  -Klf10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (D) CD68 immunofluorescence staining  of liver  sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (E) Body weight (BW), liver/body weight  ratio, serum AST   and ALT   levels, and hepatic TG  contents of two groups of mice. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4, TG"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (F) Sirius red staining  of liver  sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "Sirius red, CCl4"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (G) Relative mRNA levels of genes involved in the hepatic inflammation  and fibrosis. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (H) Protein levels of TNF-α  in the livers  (left) and its expression  was normalized to the β-actin  (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through tail vein  injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (M) Body weight (BW), liver/body weight  ratio, serum AST  and ALT  levels, and hepatic TG  contents. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "TG"
  },
  {
    "caption": "8-week-old male C57BL/6J mice  were administered with AAV-GFP  or AAV  -Klf10  through  tail vein injection, and then kept on HFD-feeding for 12 weeks. n=5 per group. (N) Sirius red staining  of liver  sections (left) and its quantitation (right). Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "Sirius red"
  },
  {
    "caption": "(F) Protein levels  of zDHHC7  in the livers  of HFD mice  or WD/CCl4 mice  The protein level  of zDHHC7  was normalized to the β-actin. n=5. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "(I)  Chromatin immunoprecipitation  analysis of KLF10 binding  activity at the zDHHC7  promoter. Soluble chromatin  was prepared from the livers  of WD/CCl4  treatment-induced NASH mice  and immunoprecipitated  with antibodies against KLF10  or against IgG. The final DNA extractions were amplified using pairs of primers that cover the regions of zDHHC7  and GAPDH  gene promoters. (J) Quantitative real-time RCR results of ChIP  assays. IgG, immunoglobulin G. n=3. Results represent three independent experiments. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test J).",
    "molecules": "CCl4"
  },
  {
    "caption": "The liver  tissues were collected from WD/CCl4-treated mice  expressing KLF10  or control (A) Representative immunofluorescence staining  of CD36  and β-catenin. The arrows represent the colocalization  of CD36  and  β-catenin.",
    "molecules": "CCl4"
  },
  {
    "caption": "The liver  tissues were collected from WD/CCl4-treated mice  expressing KLF10  or control (B) Quantitative results of CD36 colocalization  rate. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test (B",
    "molecules": "CCl4"
  },
  {
    "caption": "The liver  tissues were collected from WD/CCl4-treated mice  expressing KLF10  or control (C) Expression  of CD36  in whole livers  and plasma membrane fractions.  Protein levels were normalized to the β-actin or ATP1a1, respectively. n=5. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "The liver  tissues were collected from WD/CCl4-treated mice  expressing KLF10  or control (D) Protein levels  of palmitoylated CD36  in mouse livers. (E) Quantitative results of CD36 colocalization  rate. n=3. Results represent three independent experiments. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001. Results are shown as mean ± SD. Student's t test E,",
    "molecules": "CCl4"
  },
  {
    "caption": "HepG2  cells were treated with  2-BP and/or lentivirus  vectors expressing KLF10. n=3. Results represent three independent experiments. (D) Protein levels  of KLF10, and its normalization to   β-actin. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "2-BP"
  },
  {
    "caption": "HepG2  cells were treated with 2-BP and/or lentivirus  vectors expressing KLF10. n=3. Results represent three independent experiments. (E)  Protein levels  of palmitoylated CD36. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "2-BP"
  },
  {
    "caption": "HepG2  cells were treated with 2-BP  and/or lentivirus  vectors expressing KLF10. n=3. Results represent three independent experiments. (F) CD36 protein expression the  plasma membrane fractions, and its normalization to ATP1a1. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "2-BP"
  },
  {
    "caption": "HepG2  cells were treated with  2-BP and/or lentivirus  vectors expressing KLF10. n=3. Results represent three independent experiments. (G) BODIPY staining  showing the  lipid droplets.",
    "molecules": "2-BP, BODIPY"
  },
  {
    "caption": "HepG2  cells were transfected with lentiviruses  expressing KLF10  and/or shRNA targeting zDHHC7. n=3 independent experiments. (K) BODIPY staining  for  lipid droplets.",
    "molecules": "BODIPY"
  },
  {
    "caption": "HepG2 cells were transfected with lentiviruses  expressing KLF10  and/or shRNA targeting zDHHC7. n=3 independent experiments. (L) Cellular  TG  contents. Data information: ** P < 0.01. Results are shown as mean ± SD. 1-way ANOVA , L).",
    "molecules": "TG"
  },
  {
    "caption": "6-week-old male CD36  wild-type (WT) or knockout mice  (CD36KO) were administered with AAV-GFP  (Ctrl) or AAV-KLF10  (KLF10OE) through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (A) Protein levels of KLF10  and CD36  in the liver  (left), and their normalization to β-actin (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA (A",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  wild-type (WT) or knockout mice  (CD36KO) were administered with AAV-GFP  (Ctrl) or AAV-KLF10  (KLF10OE) through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (B) Representative immunofluorescence staining  of KLF10  and CD36  (left), and the quantitative results (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  wild-type (WT) or knockout mice  (CD36KO) were administered with AAV-GFP  (Ctrl) or AAV-KLF10  (KLF10OE) through tail vein  injection, and then kept on  WD/CCl4  for 12 weeks. n=5 per group. (C) H&E and Sirius red staining  of liver  sections. (D) Quantitative results of Sirius red staining. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "Sirius red, CCl4"
  },
  {
    "caption": "6-week-old male CD36  wild-type (WT) or knockout mice  (CD36KO) were administered with AAV-GFP  (Ctrl) or AAV-KLF10  (KLF10OE) through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (E)  Body weights and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  wild-type (WT) or knockout mice  (CD36KO) were administered with AAV-GFP  (Ctrl) or AAV-KLF10  (KLF10OE) through  tail vein  injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (F) Hepatic TG  contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4, TG"
  },
  {
    "caption": "6-week-old male CD36  wild-type (WT) or knockout mice  (CD36KO) were administered with AAV-GFP  (Ctrl) or AAV-KLF10  (KLF10OE) through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (G) Serum AST  and ALT  levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  knockout mice  were administered with AAV-TBG-wildtype-CD36, wildtype-CD36  plus KLF10, palmitoylation site mutant CD36, mutant-CD36  plus KLF10  through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (A) Serum AST  and ALT  levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  knockout mice  were administered with AAV-TBG-wildtype-CD36, wildtype-CD36  plus KLF10, palmitoylation site mutant CD36, mutant-CD36  plus KLF10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (B) Hepatic TG  contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4, TG"
  },
  {
    "caption": "6-week-old male CD36  knockout mice  were administered with AAV-TBG-wildtype-CD36, wildtype-CD36  plus KLF10, palmitoylation site mutant CD36, mutant-CD36  plus KLF10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (C) Body weights  and liver/body weight  ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  knockout mice  were administered with AAV-TBG-wildtype-CD36, wildtype-CD36  plus KLF10, palmitoylation site mutant CD36, mutant-CD36  plus KLF10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (D) H&E and Sirius red staining  of liver  sections (left), and quantitation of Sirius red staining  (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "Sirius red, CCl4"
  },
  {
    "caption": "6-week-old male CD36  knockout mice  were administered with AAV-TBG-wildtype-CD36, wildtype-CD36  plus KLF10, palmitoylation site mutant CD36, mutant-CD36  plus KLF10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (E) Immunofluorescence staining  of CD36  and β-catenin. (F) Quantitation of CD36  positive area. (G) Co-localization rate of CD36  and β-catenin. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  knockout mice  were administered with AAV-TBG-wildtype-CD36, wildtype-CD36  plus KLF10, palmitoylation  site mutant CD36, mutant-CD36  plus KLF10  through  tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (H) Protein levels of palmitoylated CD36  in mouse livers  (left), and its quantitative results (right). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "6-week-old male CD36  knockout mice  were administered with AAV-TBG-wildtype-CD36, wildtype-CD36  plus KLF10, palmitoylation site mutant CD36, mutant-CD36  plus KLF10  through tail vein injection, and then kept on WD/CCl4  for 12 weeks. n=5 per group. (I) CD36 protein expression in the plasma membrane fractions, and its normalization to  ATP1a1. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. 1-way ANOVA",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (A-B)  Protein levels  of KLF10, zDHHC7  and  TNF-α (A), and their normalization to β-actin (B). Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (C)  Body weights. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male  KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (D) Liver weights  and liver/body weight ratio. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with  KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (E) Hepatic TG  contents. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4, TG"
  },
  {
    "caption": "Albumin-cre mice  were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male  KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (F) Serum ALT  and AST  levels. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with  KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (G) H&E staining  and  Sirius red staining  of liver  sections.",
    "molecules": "Sirius red, CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with  KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male  KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (H) Quantitative results of  Sirius red staining. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "Sirius red, CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (I) Immunofluorescence staining  of CD36  and β-catenin. The arrows represent the colocalization  of CD36  and β-catenin.",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (J) Quantitative results of co-localization  rate of CD36  and β-catenin. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with  KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (K) Protein levels of palmitoylated CD36  in mouse livers. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with  KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male  KLF10hep-/- mice  and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (L) CD36 protein expression in  plasma membrane fractions from mouse livers, and its normalization to  ATP1a1. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "Albumin-cre mice  were crossed with KLF10flox/flox to generate hepatocyte-specific KLF10  knockout mice  (KLF10hep-/-). Then, 8-week-old male KLF10hep-/- mice   and KLF10flox/flox were treated with WD/CCl4  for 12 weeks. KLF10flox/flox mice  were used as the control (Ctrl). n=5 per group. (M) Relative mRNA level of genes involved in the liver inflammation  and fibrosis. Data information: * P <0.05, ** P <0.01, *** P <0.001. Results are shown as mean ± SD. Student's t test",
    "molecules": "CCl4"
  },
  {
    "caption": "(B) Hierarchical analysis of genes exhibiting a >2-fold change in expression level in bak1-3 plants after Pep2 application for 10 h in a whole-genome microarray analysis. Using Genevestigator v3, these genes were separately cross-referenced to public databases to show their expression responses to salicylate (SA), methyl-jasmonate (JA), or ethylene (ET).",
    "molecules": "methyl-jasmonate, ethylene, SA, salicylate"
  },
  {
    "caption": "(C, D and E) Immunoblot analysis of PEPR1-FLAG in 10-day-old seedlings following Pep1 or flg22 application. (C and D) Plants were exposed to 1 M Pep1 or flg22 for the indicated times. (E) Plants were pre-exposed to MG132 (50 µM), Wortmannin (Wm) (30 µM) and Tyrphostin A23 (TyrA23) (100 µM) for 1 h before being exposed to Pep1 for 10 h. The numbers below the immunoblots represent relative intensities of the PROPEP-Venus (B) and PEPR1-FLAG (C-E) bands normalized to the backgrounds in the Ponceau S-stained loading controls (bottom), with the band/background values in WT plant extracts 24 h (upper) and 0 h (middle) after Pep2 application, respectively, set as 1.0.",
    "molecules": "MG132, Tyrphostin A23, Wortmannin"
  },
  {
    "caption": "(C) Total SA levels were determined in the rosette leaves of 4-week-old plants with or without 1 µM flg22 application for 24 h. Data are averages (±SD) of material from three independent plants. The experiments were repeated twice, with similar results. (▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)",
    "molecules": "SA"
  },
  {
    "caption": "(G) Immunostaining of pS6 (Ser240/244) in skin lesions from control and LL37-induced mice. Bottom right panels, magnified images of boxed areas. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. Right panel, the quantification of relative fluorescence intensity for pS6 (Ser240/244) in control and LL37 group (n=5 for each group). Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test was used.",
    "molecules": "DAPI, Ser"
  },
  {
    "caption": "(I) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle. Scale bar: 50 μm. (J) Dermal infiltrating cells were quantified (n = 6 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used. ",
    "molecules": "RAPA"
  },
  {
    "caption": "(G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM.",
    "molecules": "DAPI, RAPA"
  },
  {
    "caption": "(E) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC-labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed, then live cells were analyzed by fluorescent microscope. Scale bar: 50 μm.",
    "molecules": "PKH26, FITC, PBS"
  },
  {
    "caption": "Topical rapamycin treatment twice daily for 4 weeks showed an obvious therapeutic effect in rosacea patients. (C) The change of patients' CEA score showed in scatter histogram at 0 week and 4th week. (D) The change of patients' IGA score showed in scatter histogram at 0 week and 4th week. \"The Change value < 0\" indicates that skin lesions show positive improvement after treatment; \"The Change value > 0\" indicates skin lesions show exacerbation after treatment; \"The Change value = 0\" indicates that skin lesions show no change after treatment; Data represent the mean ± SEM, and two-tailed unpaired Student's t-test was used (C, D).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(C) Quantification of cell number after each target knockdown, calculated as the mean fraction of DAPI+ cells relative to the scrambled non-targeting siRNAs (i.e. the latter was normalized to 1). Colors of dots and P value are interpreted in the same way as in (B).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups: control (no virus or remdesivir treatment), virus (SARS-CoV-2-infected), virus+remdesivir (SARS-CoV-2-infected treated by remdesivir), remdesivir (remdesivir treatment alone without virus). There are 3 replicates in each group.",
    "molecules": "remdesivir"
  },
  {
    "caption": "I Picrosirius red staining of histology sections of hearts from WT and TAZ mutant mice.",
    "molecules": "Picrosirius red"
  },
  {
    "caption": "A Mass spectrometric analysis of Cardiolipin and Monolysocardiolipin in WT and TAZG197V mouse heart. Mean ± SEM, n=3, unpaired t test: p < 0.05 *, p < 0.01 * *.",
    "molecules": "Monolysocardiolipin, Cardiolipin"
  },
  {
    "caption": "B Western blot analysis of BN-PAGE separted protein complexes from digitonin solubilized heart mitochondria (12-week-old mice).",
    "molecules": "digitonin"
  },
  {
    "caption": "E and F Oxygen consumption rate of isolated heart mitochondria from 12-week-old mice using succinate E and pyruvate F as substrates. Mean ± SEM, n=3, unpaired t test: p < 0.001 * * *, p < 0.0001 * * * G Flow cytometric analysis of isolated mitochondria using MitoSOX Red staining. Mean ± SEM, n=3, unpaired t test: p < 0.05 *.",
    "molecules": "MitoSOX Red, pyruvate, succinate"
  },
  {
    "caption": "F Western Blot analysis of BN-PAGE separated complexes from digitonin solubilized heart mitochondria (12-week-old mice).",
    "molecules": "digitonin"
  },
  {
    "caption": "D Real time respirometry. Left, Oxygen consumption measured as basal, and upon addition of oligomycin, CCCP, and antimycin/rotenone using palmitate-BSA as substrate, ETO, etomoxir. Right, quantification of basal and maximal respiration. Mean ± SEM, n=4, unpaired t test: p < 0.01* *, p < 0.0001* * * *",
    "molecules": "etomoxir, antimycin, CCCP, ETO, palmitate, oligomycin, rotenone"
  },
  {
    "caption": "E Extracellular acidification rate of control and iTAZ iPSC-cardiomyocytes. Basal state and upon sequential addition of glucose, oligomycin and 2 deoxyglucose (2-DG). Right, quantification of glycolytic capacity. Mean ± SEM, n=6, unpaired t test: p < 0.001* * * *",
    "molecules": "2 deoxyglucose, 2-DG, glucose, oligomycin"
  },
  {
    "caption": "C Glycolytic and mitochondrial ATP production measured using a real time ATP rate assay. Mean ± SEM, n=3, unpaired t test: p < 0.001* * *.",
    "molecules": "ATP"
  },
  {
    "caption": "D Western blot analysis of steady state AMPK and pAMPK protein levels of starved and/or A769662 (AMPK activator) treated cells as indicated.",
    "molecules": "A769662"
  },
  {
    "caption": "F Gene expression addressed by qPCR of ANP (left) and BNP (right) in 24-week-old WT and TAZG197V mice heart upon 6-week treatment with A769662. Mean ± SEM, n=4, unpaired t test: ns (non-significant)",
    "molecules": "A769662"
  },
  {
    "caption": "G Quantification of steady state levels of AMPK normalized to beta tubulin in tissue lysates from 24-week-old WT and TAZG197V mice heart from untreated control or following a 6-week treatment with A769662. The mean of the WT values was set to 1. Mean ± SEM, n=7 (control); 5 (treated), unpaired t test: p < 0.01 * *",
    "molecules": "A769662"
  },
  {
    "caption": "I Real time respirometry, oxygen consumption rate, of isolated heart mitochondria from 24-week-old TAZG197V mice, from untreated control or following a 6-week treatment with A769662, driven by palmitoyl/carnitine/malate/ADP. The OCR of the untreated WT samples was taken as 100%. Mean ± SEM, n=3, unpaired t test: p < 0.01 * *",
    "molecules": "A769662, ADP, carnitine, malate, palmitoyl"
  },
  {
    "caption": "The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from CpG-stimulated mice. Black, CTR MBMC; red, Ifnar1-TST MBMC (red) Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.",
    "molecules": "CpG"
  },
  {
    "caption": "The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from CpG-stimulated mice. Black, CTR MBMC pale blue, Ap3b1-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.",
    "molecules": "CpG"
  },
  {
    "caption": " (A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated fro CpG-stimulated (B) MBMC of each indicated type",
    "molecules": "CpG"
  },
  {
    "caption": "Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from CpG-stimulated (B) MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.",
    "molecules": "CpG"
  },
  {
    "caption": "(A) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Sec63 were transformed with a centromeric plasmid expressing BFP-Ape1. Cells were grown in SGC-LEU, shifted to SG-N for the indicated time, and analyzed by fluorescence microscopy. Images are shown as single planes. Scale bar, 5 µm. Arrows indicate Ykt6 dots colocalizing with Ape1. (B) Percentage of Ykt6 puncta simultaneously co-localizing with both Ape1 and Sec63. The data were quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Ape1 dots (n≥50) were quantified.",
    "molecules": "nitrogen, LEU"
  },
  {
    "caption": "(C) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Snx41 (endosomes), Sec63 (ER), Vac8 (vacuole), or Mnn9 (Golgi) were were grown in SGC-LEU, then shifted to SG-N for 2 h, and analyzed by fluorescence microscopy. Single imaging planes are shown. Scale bar, 5 µm. Arrows indicate Ykt6 dots that colocalize with marker proteins.",
    "molecules": "nitrogen, LEU"
  },
  {
    "caption": "Ykt6 localizes to the isolation membrane during starvation. (D) Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying the Ape1 encoding plasmid pRS315-CUP1pr-BFP-APE1 were grown in SGC medium containing CuSO4 and starved for 1 h, before to be analyzed by microscopy. Scale bar, 5 µm. (E) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view. Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying plasmid pRS315-CUP1pr-BFP-APE1, cells , images were analyzed by Imaris. Scale bar, 5 µm. Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 (D,E).",
    "molecules": "CuSO4"
  },
  {
    "caption": "(A) Effect of ykt6 and dsl1 complex ts mutants on autophagosome-vacuole fusion. Cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 24°C in SGC and then shifted to SG-N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4-64 and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset is 0.6 µm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "(B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP.",
    "molecules": "TCA"
  },
  {
    "caption": "(C) Protease protection assay of ykt6-11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 37°C and subjected to the protease (PK)-protection",
    "molecules": "protease"
  },
  {
    "caption": "(D) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12-1 ts mutant. Cells carrying a CEN plasmid expressing GFP-Atg8 and encoding 3xmCherry-tagged Atg1 were grown at 24°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Arrows in images indicate dots colocalization of Atg1 with Atg8 (D)",
    "molecules": "nitrogen"
  },
  {
    "caption": "(E) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Dsl1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (F) Percentage of Ykt6 puncta co-localizing with Dsl1. The data was quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Dsl1 dots (n≥50) were quantified. Arrows in images indicate dots colocalization of Ykt6 with Dsl1 (E).",
    "molecules": "nitrogen"
  },
  {
    "caption": "(G) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP-tagged Dsl3 and mCherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Scale bar, 5 µm.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes. Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. Vacuoles were stained by CMAC and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar in inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 and Atg8 (A)",
    "molecules": "CMAC"
  },
  {
    "caption": "(C) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions. tip20-5 cells expressing GFP-tagged Ykt6 and 3xmCherry-tagged Sec63 or Sec13 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 with Sec63 (C).",
    "molecules": "nitrogen"
  },
  {
    "caption": "(D) Effect of dsl mutation on autophagosome-vacuole fusion in vitro. The vam3∆ or dsl ts strain expressing Atg9-3xFLAG and GFP-Atg8 were starved for 3 h at 30°C before purifying autophagosomes, which was sufficient to induce the ts phenotype. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Localization of Atg8 relative to Atg9 during nitrogen starvation. dsl3 cells expressing mCherry-tagged Atg9 and carrying a CEN plasmid expressing GFP-Atg8 were grown at 23°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(D) Atg9 localization in dsl3 cells during starvation. dsl3 ts cells expressing mCherry-tagged Atg9, and carrying a CEN plasmid expressing GFP-Atg8 and plasmid pRS315-CUP1pr-BFP-APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure, and starved for 1 h. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.",
    "molecules": "CuSO4"
  },
  {
    "caption": "(B) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Atg1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Right, percentage of Ykt6 puncta co-localizing with Atg1. Error bars represent standard deviation of three independent experiments. Cells (n≥200) and Atg1 dots (n≥100) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg1 (B)",
    "molecules": "nitrogen"
  },
  {
    "caption": "(C) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex. Purified Ykt6 was incubated with the Atg1-Atg13 complex in the presence or absence of ATP, and Ykt6 band was cut out and analyzed by mass spectrometry.",
    "molecules": "ATP"
  },
  {
    "caption": "(D) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions. Wild-type and atg1D211A cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual focal planes are shown. Scale bar, 5 µm. (E) Percentage of Ykt6 puncta co-localizing with Atg8. Numbers in the column indicates the percentage. Cells (n≥300) and Atg8 dots (n≥60) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg8 (D).",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A) Growth test of Ykt6 wild-type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild-type Ykt6 and pRS413-GAL1pr-YKT6-GFP (wild-type or mutant) were spotted as serial dilutions on SGC-HIS to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to force the loss of the plasmid coding for wild-type Ykt6.",
    "molecules": "5-Fluoroorotic acid, 5-FOA"
  },
  {
    "caption": "(D) Effect of Atg1-Atg13 subcomplex on autophagosome-vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutant), and expressing Atg9-3xFLAG and GFP-Atg8, were starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1-Atg13 subcomplex at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.",
    "molecules": "ATP"
  },
  {
    "caption": "(B-C) Representative images (B) and quantification (C) of Oil Red O staining of hypoxia- and normoxia-cultured cells, 9 days after induction of adipogenesis. n=5 biologically independent replicates.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "(D-E) Representative images (D) and quantification (E) of Alizarin Red S staining of hypoxic, normoxic and reversed hypoxic (R_2% O2) cells, 12 days after induction of osteogenesis. Cells were exposed to 21% O2 for 7 days and then moved back to 2% O2, where osteogenesis was induced after 4 days. n=3 for hypoxic cells and n=5 biologically independent experiments for normoxic and reversed-hypoxic cells and merged results are shown in (E).",
    "molecules": "Alizarin Red S, O2"
  },
  {
    "caption": "(C-D) Representative images (C) and quantification (D) of Mean Fluorescence Intensity (MFI) after immunostaining against H3ac and H3 of hypoxic and normoxic cells. Mean Fluorescence Intensity (MFI) of histone H3 was used as internal control, for normalization. Nuclei were stained with DAPI. Quantification of H3ac/H3 MFI from n=67 hypoxic and n=56 normoxic individual cells from a representative experiment out of 3 biologically independent experiments is shown in D. Distribution of data points in (D) is shown as a violin plot, where the mean is indicated by a solid line and the quartiles are indicated with dashed lines.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-B) Representative images after immunostaining of hypoxia- and normoxia-cultured cells against acetyl-Lysine and TOMM20 (A), and assessment of the acetyl-Lysine signal localization, after manual assignment into three categories: exclusively nuclear, exclusively mitochondrial and nuclear/mitochondrial (B). Nuclei were stained with DAPI. n=3 biologically independent experiments. Results are shown as mean ± S.E.M and statistical significance was determined using two-sided unpaired t-test. In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.",
    "molecules": "DAPI, acetyl-Lysine"
  },
  {
    "caption": "(B-C) Representative images (B) and quantification (C) of Nile Red staining of hypoxic control and BTA-treated cells and of normoxic control and acetate-treated cells. Treatments were done for three days with 1mM BTA or 5mM acetate.",
    "molecules": "acetate, BTA, Nile Red"
  },
  {
    "caption": "Representative images after immunostaining of hypoxic control and BTA-treated cells and normoxic control and acetate-treated cells against acetyl-Lysine and TOMM20 (D), assessment of the acetyl-Lysine signal localization, as described above (E) Nuclei were stained with DAPI. In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.",
    "molecules": "acetate, BTA, DAPI, acetyl-Lysine"
  },
  {
    "caption": "Representative images after immunostaining of hypoxic control and BTA-treated cells quantification of nuclear acetyl-lysine MFI (F). Nuclei were stained with DAPI. Quantification of nuclear acetyl-Lysine MFI from n=67 hypoxic, n=71 hypoxic_BTA, n=44 normoxic and n=63 normoxic_acetate individual cells from a representative experiment out of 3 biologically independent experiments is shown in (F). In magnified insets intensity of the acetyl-Lysine signal was adjusted similarly to all samples, for visualization purposes.",
    "molecules": "acetate, BTA, DAPI, acetyl-lysine, acetyl-Lysine"
  },
  {
    "caption": "(G-H) Representative images and quantification of Alizarin Red S staining of control hypoxic and normoxic cells and of normoxic acetate-treated cells, 12 days after induction of osteogenesis. Merged results of n=5 biologically independent experiments for hypoxic and normoxic cells and n=4 biologically independent experiments for acetate-treated normoxic cells are shown in (H).",
    "molecules": "acetate, Alizarin Red S"
  },
  {
    "caption": "(A) Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "(B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for one hour and then immediately injected. Right, H&E staining of representative lung sections. N=6 mice/group.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "(C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "(A,B) LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nm rapamycin and subjected to the migration assay. N = 5 inserts/group.",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "(G) Widefield images showing RelA localization in unstimulated EL4 cells and EL4 cells stimulated by SLB formed on and off grids. EL4 were fixed 30 min after addition to IL-1-functionalized SLBs and stained for RelA (magenta); DAPI stained nuclei (blue). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) TIRF images of fixed EL4-MyD88-GFP cells off grids and stained with antibodies against K63-Ub, M1-Ub, pIKK and pp65. Cells were activated on IL-1 functionalized SLBs for 30 min before fixation. Scale bar, 5 µm. (B) A magnified view of the large patch-like Myddosome clusters from the highlighted region of interest in panel A (yellow box on merge images). Scale bar, 1 µm.",
    "molecules": "Ub"
  },
  {
    "caption": "(C) Structured illumination microscopy images of Myddosome clusters stained with anti-pIKK. Top row right, insets show detail of Myddosome staining with anti-pIKK. Inset taken from regions of interest overlaid the merge image (yellow boxes 1 and 2). Bottom row, x-z view slice taken from yellow line overlaid on the merge image (top row). Myddosome and pIKK staining localize the cell-SLB interface. Blue dashed line defines the nucleus volume determined from the DAPI stain. Scale bar in main image and Z projection, 1 µm; scale bar inset, 0.5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A and B) Top, TIRF images of fixed EL4-MyD88-GFP cells incubated with IL-1 functionalized SLBs for 30 minutes, and stained with anti-K63-Ub (A) or anti-M1-Ub (B). Scale bar, 5 µm. Region of interest (red box, merge image) shows an example of MyD88-GFP puncta that is colocalized with K63-Ub (A) or M1-Ub (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (A) or M1-Ub (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms). Blue shaded region on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88-GFP channel and associated K63-Ub (A) and M1-Ub (B) channel (K63/M1-Ub images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88-GFP image (numbered red arrows). Scale bar, 1 µm.",
    "molecules": "Ub"
  },
  {
    "caption": "(E and F) 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (E) or M1-Ub (F) staining intensity on 2.5 µm and 1 µm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient of linear fit labeled on 2D histograms).",
    "molecules": "Ub"
  },
  {
    "caption": "(G and H) Quantification of mean K63-Ub (G) or M1-Ub (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 µm grids. The normalized mean intensity for clustered, single Myddosomes, and MyD88 puncta on 2.5 and 1 µm grids are the following: for K63-Ub 0.444 ± 0.030, 0.257 ± 0.025, 0.113 ± 0.015 and 0.104 ± 0.008; for M1-Ub 0.520 ± 0.020, 0.183 ± 0.008, 0.174 ± 0.022 and 0.153 ± 0.016 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. p values are **<0.01, ***<0.001. Bars represent mean ± SEM (n = 3 - 4 biological replicates for K63, with 14571, 27494 and 24026 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates; n = 3 - 4 biological replicates for M1, with 3114, 6091 and 1844 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates). Statistical significance is determined using unpaired two-tailed Student's t-test.",
    "molecules": "Ub"
  },
  {
    "caption": "D Quantitative measurement of H2O2  in tomato roots  after infection by the WT and FolSrpk1p::FolSrpk1-GFP  strains at 0-5 days post incubation (dpi). Data are presented as mean ± SD. The presence of different letters above the mean values of three replicates indicates a significant difference between different samples (P < 0.05, ANOVA).",
    "molecules": "H2O2"
  },
  {
    "caption": "F Association of FolSrpk1 with FolAha1 under ROS  stress. After treatment with 10 mM H2O2  for 0, 1 and 2 h, proteins pulled down with GFP-Trap beads were probed with anti-GFP  and anti-Flag  (top). Input proteins were shown by Western blotting with anti-GFP  and anti-FLAG  antibodies (bottom). The amount of immunoprecipitated FolSrpk1-Flag  and  FolAha1-GFP  at 0 h was each set as 1. Data information: For , F each gel shown is a representative experiment carried out at least three times.",
    "molecules": "H2O2, ROS"
  },
  {
    "caption": "G Fluorescence microscopy analysis of FolSrpk1-GFP  in mycelia   of the FolSrpk1p::FolSrpk1-GFP  strain treated with 10 mM H2O2  for 0, 1 and 2 h. Arrows indicate the nucleus. Scale bars = 5 µm.",
    "molecules": "H2O2"
  },
  {
    "caption": "I Subcellular fractionation analysis of  FolSrpk1-GFP  in mycelia  of the  FolSrpk1p::FolSrpk1-GFP  strain treated with 10 mM H2O2  for 0, 1 and 2 h. The amount of nuclear FolSrpk1-GFP  at 0 h was set as 1. Data information: For I, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin  (cytosol) and Coomassie brilliant blue (CBB) staining.",
    "molecules": "H2O2"
  },
  {
    "caption": "I K304 acetylation  of WT and mutant FolSrpk1 proteins in the indicated strains after treatment with 10 mM H2O2  for 0, 1 and 2 h. The amount of acetylated  and total  FolSrpk1-GFP  at 0 h was each set as 1. Data information: For each gel shown is a representative experiment carried out at least three times.",
    "molecules": "H2O2"
  },
  {
    "caption": "A K304 acetylation  (top) and amount (middle) of FolSrpk1-GFP  and FolSrpk1K304Q-GFP  in WT and FolArd1-OE strains treated with 10 mM H2O2  for 2 h. The over-expressed FolArd1-Flag  was detected by anti-Flag  (bottom). The amount of acetylated  and total FolSrpk1 in the WT was each set as 1. Data information: each gel shown is a representative experiment carried out three times.",
    "molecules": "H2O2"
  },
  {
    "caption": "C Determination of H2O2  content in tomato roots  infected by the indicted strains at 0-5 dpi. The experiments were carried out as in Fig 1. Data are presented as mean ± SD of three biological replicates.",
    "molecules": "H2O2"
  },
  {
    "caption": "F Relative density of DAB staining  in E (four biological replicates). Data information: For F the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.",
    "molecules": "DAB"
  },
  {
    "caption": "A. Detection of HYL1 phosphorylation in Col-0, free1-ctmut or cpl1-8. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies. The numbers in the first row indicate the relative grayscale intensities of total HYL1, the numbers in the second row indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the intensity in WT was arbitrarily set to 1.00.",
    "molecules": "polyacrylamide, SDS"
  },
  {
    "caption": "F. Proteins extracted from 7-day-old Arabidopsis seedlings treated with CHX (0.5 mM) in dark for 0h, 2h, 4h were used for immunoblotting analysis with indicated antibodies. The numbers represent the relative intensities of each band detected with anti-HYL1 and anti-Actin, and the first lane was arbitrarily set to 1.00.",
    "molecules": "CHX"
  },
  {
    "caption": "F. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels followed by immunoblotting analysis with HYL1 antibody. The numbers indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the first lane was arbitrarily set to 1.00.",
    "molecules": "polyacrylamide"
  },
  {
    "caption": "Sample recordings from 7-week-old  hESC-derived Cerebral Organoids (COs). Sample traces show visible differences in local field potential, with WWOX-KO  COs (Red) showing increased activity compared to WT (Blue) in baseline condition (Left) and in the presence of 100µM 4AP (Right).",
    "molecules": "4AP"
  },
  {
    "caption": "(A) mNG-PilH  fluorescence profiles in chpA  mutants after 2h surface growth. To avoid negative effects of low cAMP  level on localization  of PilH, cpdA  was deleted in all displayed strains to rescue cAMP  level to WT levels Solid lines, mean normalized fluorescence profiles across biological replicates. Shaded area, standard deviation across biological replicates.",
    "molecules": "cAMP"
  },
  {
    "caption": "(A) mNG-PilH  fluorescence profiles in ΔpilG after 2h surface growth (low cAMP).",
    "molecules": "cAMP"
  },
  {
    "caption": "(C) mNG-PilH  fluorescence profiles in ΔpilG ΔcpdA  after 2h surface growth (rescued cAMP).",
    "molecules": "cAMP"
  },
  {
    "caption": "(A) mNG-PilG  fluorescence profiles in pilH  mutants after 2h surface growth. We included ΔcpdA for comparison since both pilH  mutations result in high cAMP  levels. PilG  is more polar and asymmetric in ΔpilH  and pilHLOF. (A Solid lines, mean normalized fluorescence profiles across biological replicates. Shaded area, standard deviation across biological replicates.",
    "molecules": "cAMP"
  },
  {
    "caption": "D. A. tumefaciens antibacterial activity assay against E. coli. The strains of A. tumefaciens were co-cultured at a ratio of 30:1 with E. coli DH10B (+ pRL662) on LB agar. The survival of target E. coli cells was quantified by counting CFUs on gentamicin-containing LB agar plates. Data represent mean ± SEM of 6 biological replicates from three independent experiments. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Two groups with significant differences (P=0.0010053) are indicated with different letters (a and b).",
    "molecules": "agar, gentamicin"
  },
  {
    "caption": "C. Antibacterial activity assay of wild type and mutants of 12D1, 1D1108 and 15955 was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in three independent experiments.",
    "molecules": "agar, gentamicin"
  },
  {
    "caption": "C. Antibacterial activity assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in two independent experiments.",
    "molecules": "agar, gentamicin"
  },
  {
    "caption": "B Identification of sec homozygotes. LB represents the left border primer of the T-DNA insertion. LP and RP represent the left and right genomic primers, respectively.",
    "molecules": "T-DNA"
  },
  {
    "caption": "B ChIP-qPCR assay of H3K4me2 levels of indicated regions at FLC chromatin. C ChIP-qPCR assay of H3K4me3 levels of indicated regions at FLC chromatin. D ChIP-qPCR assay of H3K36me3 levels of indicated regions at FLC chromatin. E ChIP-qPCR assay of H3K27me3 levels of indicated regions at FLC chromatin .Data information: For ChIP analysis, 12-d-old plants were collected and three independent experiments were conducted. Each bar represents the mean ± s.d. of three independent experiments, n=3. The relative abundance was normalized to the input.",
    "molecules": "H3K36me3, H3K27me3, H3K4me3"
  },
  {
    "caption": "A Detection and conformation of O-GlcNAc modification of His-ATX1ΔN catalyzed by His-SECΔN in vitro. His-ATX1ΔN (expressing residues 592-952 of the C-terminus) and His-SECΔN (expressing residues 801-1062 of the C-terminus) were recombinantly expressed and affinity purified separately. O-GlcNAcylation of His-ATX1ΔN was detected by anti-CTD110.6 antibody, and further conformed by β-elimination analysis.",
    "molecules": "O-GlcNAc"
  },
  {
    "caption": "B His-SECΔN activates His-ATX1ΔN in vitro. Histone methyltransferase activity of ATX1ΔN was detected with or without recombinant SECΔN. H3K4A: mutated H3 in which the fourth amino acid, a lysine (K), was replaced with alanine (A).",
    "molecules": "H3K4A, alanine, lysine"
  },
  {
    "caption": "C Analysis of ATX1 O-GlcNAc modification in wild-type and sec-5 plants. Total soluble protein extracts from 12-d-old seedlings were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. CBB: Coomassie brilliant blue staining, showing relative protein loading amount.",
    "molecules": "CBB, O-GlcNAc"
  },
  {
    "caption": "D Loss of SEC function reduced ATX1 activity in Arabidopsis. Nuclear proteins were extracted and ATX1 was immunoprecipitated with anti-ATX1 antibody from wild-type and sec-5 mutant plants, respectively, and then used for histone H3K4 methyltransferase activity analysis with recombinant H3 as catalyzing substrate. Band intensities were quantified with Image J. The H3 signal was first normalized by input signal and then was used for H3K4me3 signal normalization.",
    "molecules": "H3K4me3"
  },
  {
    "caption": "A Mutation of either S947 alone or all 12 serine and threonine residues in the SET domain reduced O-GlcNAc modification level of ATX1ΔN.",
    "molecules": "O-GlcNAc, serine, threonine"
  },
  {
    "caption": "B Site mutation of S947 or all 12 serine and threonine residues in the ATX1 SET domain inhibited the activation of ATX1 by SEC; moreover, mutation of five conserved amino acids in the SEC functional domain inhibited SEC activity.",
    "molecules": "serine, threonine"
  },
  {
    "caption": "A Flowering phenotypes of Col-0, atx1-2, atx1-2 35S::ATX1-FLAG, and atx1-2 35S::ATX1m-FLAG (with Ser947 replaced with alanine) plants under LD conditions. Scale bar: 1 cm. Two independent lines are shown for each transformation.",
    "molecules": "alanine, Ser"
  },
  {
    "caption": "(A) Epifluorescence microscopy of live cells containing a subgenomic replicon with a GFP-tagged NS5A. Huh7-Lunet cells were transfected with replicon RNA and seeded onto carbon-patterned sapphire discs. Twenty-four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM. (a) Fluorescence image; (b) enlarged fluorescence image of the cell of interest; (c) merge of bright field and fluorescence images. Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells. White squares in a and c enclose the cell shown in b. (B) EM micrograph of the cell boxed in panel Ab overlapped with the fluorescence image. Areas marked with a green dotted line indicate regions of intense fluorescence. Note that the images do not match perfectly because the fluorescence image corresponds to the complete cell whereas the EM image represents one 60 nm ultrathin section of the same cell. (C) Higher magnification images of the two different regions, labeled 1 and 2 in panel B, corresponding to regions with intense fluorescence (1) or a region corresponding to the intersection of high to low fluorescence (2). Region 1 (top panel) corresponds to a DMV-containing area residing in close proximity of the ER; region 2 (bottom panel) corresponds to a LD-enriched area containing DMVs in very close proximity. Areas marked with white squares in the left images are magnified in the corresponding right panels. LD, lipid droplet; ER, endoplasmic reticulum; DMV, double membrane vesicle; m, mitochondrium; if, intermediate filaments.",
    "molecules": "RNA"
  },
  {
    "caption": "Immuno-EM localization of NS5A and NS3 in HCV-infected cells.Huh7.5 cells were infected with 100 TCID50/cell of Jc1 and fixed 16 h post-infection. (A) Cells shown in panels b, c and e to g were post-fixed with 0.1% OsO4 providing best membrane preservation, especially in case of DMVs (f and g) and LDs (e). Both NS5A and NS3 localize predominantly to the ER (b and d) and 50-70 nm diameter single-membrane vesicles (SMVs; subpanel a, c and d). NS5A also localizes to lipid droplets (e).",
    "molecules": "OsO4"
  },
  {
    "caption": "(B) Amount of gold particles per µm2 in Jc1-infected versus mock-infected cells after immunolabeling with NS3- and NS5A-specific primary antibodies. Note the higher immunolabeling with samples prepared without OsO4, but also the lower membrane preservation under this condition.",
    "molecules": "gold, OsO4"
  },
  {
    "caption": "(C) Relative labeling distribution of NS3 and NS5A. Thawed cryosections of cells post-fixed or not with OsO4 were labelled with NS3- or NS5A-specific antibodies by using two different blocks and 3 different labeling experiments. Per labeling experiment two grids were considered, counting ∼100-200 gold particles per grid and attributing the particles to the indicated structures. In the case of uninfected cells only background labeling in the cytoplasm, on mitochondria and undefined structures was seen. Numbers refer to the percent of total gold particles counted per sample. ER, endoplasmic reticulum; SV, small vesicles; Cyto, cytosol; Mito, mitochondria; n.d., non-defined structures; NE, nuclear envelope; EE/LE, early/late endosomes; PM, plasma membrane; DMV, double membrane vesicle; LD, lipid droplet.",
    "molecules": "gold, OsO4"
  },
  {
    "caption": "Correlation between HCV RNA replication and appearance of double membrane vesicles.(A) Time course of spread of HCV infection in Huh7.5 cells infected with 100 TCID50/cell of Jc1. Infected cells were detected by immunofluorescence using an NS5A-specific antiserum (upper panels). The graph shown below represents the result of counting ∼200 cells for each time point to determine the percentage of infected cells. Scale bars represent 50 µm.",
    "molecules": "RNA"
  },
  {
    "caption": "(B) Time course of accumulation of intracellular HCV RNA in infected Huh7.5 cells. The graph shows the result of two independent experiments (3 replicas each). Whiskers indicate the minimum and maximum values.",
    "molecules": "RNA"
  },
  {
    "caption": "(C) Colocalization of dsRNA and NS5A in cells infected with Jc1 (10 TCID50/cell). Cells were fixed at time points specified in the left of each panel row and NS5A and dsRNA were detected by indirect immunofluorescence microscopy. DNA was stained with DAPI (blue). Boxed areas in the left panels indicate areas that are shown as enlargements in the corresponding right panels. The quantification of the degree of colocalization (Pearson's correlation coefficient) is given in the enlarged pictures. Scale bars represent 10 µm and 2 µm (left and right panels, respectively).",
    "molecules": "dsRNA"
  },
  {
    "caption": "(D) Time course of accumulation of DMVs and MMVs. For each time point, 10 cellular profiles were counted. The Mann-Whitney (non-parametric) test was applied to determine statistical significance. Error bars refer to the standard deviation. Note the striking correlation between the increase of intracellular HCV RNA and DMV number between 16 and 24 hpi.",
    "molecules": "RNA"
  },
  {
    "caption": "Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data was normalized to the reads of control PC3 cells.",
    "molecules": "aspartate, glutamate, leucine"
  },
  {
    "caption": "Endogenous levels of aspartate, asparagine, glutamate and glutamine in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization.",
    "molecules": "asparagine, aspartate, glutamate, glutamine"
  },
  {
    "caption": "IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified-aspartate (6mM) or esterified-glutamate (6mM), and esterified-leucine (6mM) as a control. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001. ",
    "molecules": "aspartate, glutamate, leucine"
  },
  {
    "caption": "cell lines were transduced with either control (sgNon-targeting) or sgSLC1A3. Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines. Leucine uptake level was used for normalization. The numbers above the control column denote the basal aspartate uptake capacity. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.",
    "molecules": "Aspartate, aspartate, Leucine"
  },
  {
    "caption": "RT-qPCR was used to determine the relative mRNA levels (to GAPDH) of aspartate/glutamate transporter genes (SLC1A1, SLC1A2, SLC1A3, SLC1A6 and SLC1A7) in LNCaP, BT549 and SUM159PT cells. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.",
    "molecules": "aspartate, glutamate"
  },
  {
    "caption": "Immunofluorescence staining of the V5-tagged SLC1A3 in MCF7 and DU145 cells using anti-V5 antibody. Green staining indicates the plasma membrane localization of V5-SLC1A3 and blue DAPI staining marks the nuclei. Scale bar stands for 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Relative aspartate uptake levels in control and V5-SLC1A3-expressed MCF7 and DU145 cells. Leucine uptake level was used for normalization.",
    "molecules": "aspartate, Leucine"
  },
  {
    "caption": "DU145 cells were supplemented with cell-permeable aspartate (6mM, esterified) or glutamate (3mM, esterified) following ASNase treatment, with esterified leucine (6mM or 3mM) as control.",
    "molecules": "aspartate, glutamate, leucine"
  },
  {
    "caption": "PC3, DU145 and V5-SLC1A3-DU145 cells were subjected to ASNase and TFB-TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay.",
    "molecules": "TFB-TBOA"
  },
  {
    "caption": "PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions. ASNase (0.3 U/ml), TFB-TBOA (5 μM).",
    "molecules": "TFB-TBOA"
  },
  {
    "caption": "DU145 and V5-SLC1A3-DU145 cells were treated under indicated conditions with ASNase (0.2 U/ml) or TFB-TBOA (20μM) or both, and subjected to IncuCyte analysis for apoptotic cell counts.",
    "molecules": "TFB-TBOA"
  },
  {
    "caption": "PC3 cells were treated under ASNase (0.3 U/ml), or TFB-TBOA (5 μM) conditions for 3 days and cell lysates were extracted and intracellular contents of aspartate, asparagine, glutamate and glutamine were determined by liquid-chromatography mass spectrometry (LC-MS). Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "asparagine, aspartate, glutamate, glutamine, TFB-TBOA"
  },
  {
    "caption": "key metabolites involved in urea cycle, pyrimidine synthesis, TCA cycle, oxidation, glycolysis and carnitines metabolism were determined. The NAD+/NADH ratio of the indicated conditions was calculated and normalized to control (mean± SEM). Dash line indicates indirect effect. TCA cycle: tricarboxylic acid cycle; OAA: oxaloacetic acid; UMP: uridine monophosphate; CMP: cytidine monophosphate; PEP: phosphoenolpyruvate; NADH: nicotinamide adenine dinucleotide (reduced form); NAD+: nicotinamide adenine dinucleotide (oxidized form); NADPH: nicotinamide adenine dinucleotide phosphate (reduced form); NADP+: nicotinamide adenine dinucleotide phosphate (oxidized form); FAD: flavin adenine dinucleotide; GSSG: glutathione disulfide; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA. Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "3-hydroxy-3-methylglutaryl-CoA, HMG-CoA, CMP, cytidine monophosphate, FAD, flavin adenine dinucleotide, glutathione disulfide, GSSG, NAD+, nicotinamide adenine dinucleotide, NADH, NADP+, nicotinamide adenine dinucleotide phosphate, NADPH, OAA, oxaloacetic acid, PEP, phosphoenolpyruvate, UMP, uridine monophosphate"
  },
  {
    "caption": "A-C. PC3 (A), DU145 (B) and DU145-V5-SLC1A3 (C) cells were treated with ASNase (0.3 U/ml in A; 0.2 U/ml in B and C), TFB-TBOA (5 μM) for 3 days as indicated and subjected to transcriptome analysis. Bioinformatics pathway or gene ontology (GO) biological process analysis was performed on the sets of genes that were upregulated or downregulated when PC3 cells were treated with ASNase and TFB-TBOA compared to mock. Transcriptome analysis was based on one biological replicate for each cell line and validated by real-time PCR experiments in Figures EV4A-C. Heatmap presents row scaled normalized read counts and the biological signaling pathway enrichment analysis was performed by ToppGene online program",
    "molecules": "TFB-TBOA"
  },
  {
    "caption": "SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice. Once SUM159PT tumors reached 250mm3 volume, mice were treated with mock or ASNase (60U per day) for 5 consecutive days (n=3). Following treatment, mice were sacrificed, and blood, mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level. Essential amino acids were used for the raw data normalization. Data are presented as mean ± SD. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "asparagine"
  },
  {
    "caption": "tumors were surgically removed once reached a volume of ~500mm3 and collected and subjected to LC-MS to determine the levels of asparagine, aspartate, glutamine and glutamate. Leucine level was used as a control. Results are based on five tumor samples and presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "asparagine, aspartate, glutamate, glutamine, Leucine"
  },
  {
    "caption": "A) IL-8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cells (Hoechst in blue, IL-8 in red)",
    "molecules": "Hoechst, βHBP, digitonin"
  },
  {
    "caption": "after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cell B) Quantification of IL-8 a",
    "molecules": "βHBP, digitonin"
  },
  {
    "caption": "with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat C) TIFA oligomerization at 6 h in TIFA-GFP-expressing HeLa cells treate",
    "molecules": "βHBP, digitonin"
  },
  {
    "caption": "with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat at 6 h in TIFA-GFP-expressing HeLa cells treate D) Quantification of TIFA oligomerizatio",
    "molecules": "βHBP, digitonin"
  },
  {
    "caption": "with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat E) TIFA oligomerization at 30 min in cells treate",
    "molecules": "βHBP, digitonin"
  },
  {
    "caption": "F) Kinetics of TIFA-GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S. flexneri lysate",
    "molecules": "βHBP, digitonin"
  },
  {
    "caption": "B) TIFA-GFP oligomerization at 30 min in HeLa cells treated with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC)",
    "molecules": "digitonin"
  },
  {
    "caption": "with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strain (pHldA/C means plasmid-encoded HldA and HldC) C) TIFA-GFP oligomerization at 6 h in cells treate",
    "molecules": "digitonin"
  },
  {
    "caption": "with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC) D) IL-8 production at 6 h in cells were treate",
    "molecules": "digitonin"
  },
  {
    "caption": "E) TIFA-GFP oligomerization in cells treated for 30 min with digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrations",
    "molecules": "ADP-L-β-d-heptose, digitonin"
  },
  {
    "caption": "with digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrations. F) IL-8 production after 6 h in cells treate",
    "molecules": "ADP-L-β-d-heptose, digitonin"
  },
  {
    "caption": "C) TIFA-GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway (MOI 10)",
    "molecules": "LPS"
  },
  {
    "caption": "of the LPS biosynthesis pathway (MOI 10). D) TIFA-GFP oligomerization after 4 h of infection with the S. flexneri mutant",
    "molecules": "LPS"
  },
  {
    "caption": "A) ADP-heptose-induced TIFA-GFP oligomerization depends on ALPK1. After siRNA transfection, HeLa cells were transfected with empty pCMV or pCMV-ALPK1 as indicated. They were then infected with wt S. flexneri (MOI 10) or treated with digitonin and ADP-heptose (10-5 M) for 30 min. As control, cells were left untreated (mock) or treated with digitonin",
    "molecules": "ADP-heptose, digitonin"
  },
  {
    "caption": "ADP-heptose-induced cytokine secretion is ALPK1-dependent. HeLa cells were transfected with control or ALPK1-targeting siRNAs and infected with wt S. flexneri (MOI 2) or treated with ADP-heptose (10-5 M). As control, cells were left untreated (mock) or treated with digitonin. Cytokines were measured in cell culture supernatants after 8 h",
    "molecules": "ADP-heptose, digitonin"
  },
  {
    "caption": "D FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4-/- organ of Corti. Top shows OHC plane and bottom shows IHC plane.",
    "molecules": "FM1-43"
  },
  {
    "caption": "G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured.",
    "molecules": "DAPI"
  },
  {
    "caption": "H 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Confocal images of representative eye cryosections immunostained with anti-Lamp1 antibody from 2-month-old miR-211-/- and control mice sacrificed 3h after light on, at 10 AM (diurnal condition). Enlarged boxes highlight Lamp1-positive structures (white arrowheads) in the RPE. Autofluorescence from lipofuscin granules are visible in all cryosections of miR-211-/- compared to control littermates. Nuclei were counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Representative confocal images of eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old WT mice sacrificed 3h after light on at 10 AM (diurnal condition - LIGHT) and 3h after light off at 10 PM (nocturnal condition - DARK), nuclei were counterstained with DAPI (blue). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. (B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005. ",
    "molecules": "DAPI, DMSO, NSC668394"
  },
  {
    "caption": "(C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005.",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(D) Representative images of RFP-GFP-LC3 assay in ARPE-19 cells transiently transfected with RFP-GFP-LC3 and treated with DMSO or NSC668394. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. (E) Box plots showing quantitative analysis of RFP+GFP+ puncta (Autophagosome) and RFP+GFP- puncta (Autolysosome ) (n ≥ 100 cells) from 3 independent experiments. Box limits represent 25th percentile and 75th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t-test (NSC668394 vs DMSO) *p ≤ 0.05.",
    "molecules": "DAPI, DMSO, NSC668394"
  },
  {
    "caption": "(F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.",
    "molecules": "Magic Red, cresyl violet, DMSO, NSC668394"
  },
  {
    "caption": "(H) Cathepsin B (CTSB) activity in lysates from NSC668394-treated and DMSO-control ARPE-19 cells. Graph showing the percentage of values ± s.e.m. of CTSB activity from 4 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(A) Representative images from HC assay of HeLaTFEB-GFP cells transfected with control (siCTRL) or siPPP3CB, and subjected to the indicated conditions. Analysis of TFEB nuclear translocation in HeLaTFEB-GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394, silenced for EZRIN (siEZR) or serum-starved (stv). Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB-GFP cells. Starvation (stv) was used as control. Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB. Scale bar 5 μm.",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(B) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared to control; n=3 independent experiments were performed. Mann and Whitney test (NSC668394 and stv vs DMSO; siEZR vs siCTRL) *p ≤ 0.05.",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(E) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis. Torin treatment was used as control.",
    "molecules": "Torin"
  },
  {
    "caption": "(F) Representative traces of ML1-GCaMP3 normalized fluorescence recorded in transiently transfected ARPE-19 cells. During time-lapse recording, cells were stimulated with ML-SA1 (20 µM) after addition (arrowhead) of DMSO or NSC668394. Where indicated, the specific ML1 inhibitor ML-SI3 (10 µM) was added. Bar graph reports the mean values ± s.e.m. of the time required by fluorescence to decay to half of the ML-SA1-induced peak. n = 50 cells from 3 independent experiments. ***p<0.005 by Mann and Whitney test.",
    "molecules": "ML-SI3, DMSO, ML-SA1, NSC668394"
  },
  {
    "caption": "(G) Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is reduced after Ca2+ chelator BAPTA treatment. Scale bar 5 μm.",
    "molecules": "BAPTA, Ca2+"
  },
  {
    "caption": "H) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared with DMSO under Ca2+ chelator BAPTA treatment. At least 3 independent experiments were performed. Mann and Whitney test (stv, stv+BAPTA, BAPTA, NSC668394, NSC668394+BAPTA vs DMSO) *p ≤ 0.05.",
    "molecules": "BAPTA, Ca2+, DMSO, NSC668394"
  },
  {
    "caption": "(I) qRT-PCR analysis for TFEB target genes (TRPML1, BECLIN, MAPLC3B and LAMP1) was performed on ARPE-19 cells treated with DMSO or NSC668394. Bar graphs represent mean values ± s.e.m. of at least 3 independent experiments. Mann and Whitney *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(A) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) /anti-ZO-1 (red), anti-Ezrin-pT567 (green) /anti-ZO-1 (red), anti-Lamp1 (green) and anti-Sqstm1/p62 (green) antibodies from 6-month-old miR-211-/- sacrificed five months after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.",
    "molecules": "DAPI, DMSO, NSC668394"
  },
  {
    "caption": "(B) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6-month-old miR-211-/- mice. Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1, Ezrin, Ezrin-pT567, Sqstm1/p62 and LC3. The plots show the quantification of the indicated proteins normalized to the β-Actin loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice). Mann and Whitney test (DMSO vs WT; NSC668394 vs DMSO), *p ≤ 0.05, ***p≤0.005.",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05. (D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(A) Representative images of conventional TEM analysis of RPE of WT, DMSO-treated miR-211-/- mice and NSC668394-treated miR-211-/- three-month-old mice. NSC668394-treated miR-211-/- mice show rescue of accumulation of double-membrane phagolysosome-like structures containing poorly processed POS (red arrows) compared to DMSO-treated miR-211-/- mice. Scale bar 1 mm. Data information: (OS) outer segment (RPE) Retinal Pigment Epithelium.",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(B) Confocal images of representative eye cryosections immunostained with anti-Rhodopsin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment, which are sacrificed 3h after light on at 10 AM (diurnal condition). Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. The enlarged box highlights rhodopsin accumulation in the RPE (green spots). Scale bar 5 μm. Autofluorescence from lipofuscin granules from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Representative images of retina cryosections immunostained with anti-Cone Arrestin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). N = at least 6 mice per group. Scale bar 100 μm. Data information: (OS) outer segment; (ONL) outer nuclear layer; (RPE) Retinal Pigment Epithelium.",
    "molecules": "DAPI, DMSO, NSC668394"
  },
  {
    "caption": "(C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice).",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(D) Graphs show cone percentage (cones/area) from the retina of mice treated as in (B). Error bars represent s.e.m. Mann and Whitney test (DMSO vs WT and NSC668394 vs DMSO treated mice) ***p ≤ 0.005 (n=6 mice).",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "(E) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394-treated miR-211-/- mice (green lines) compared to DMSO-treated miR-211-/- control mice (red lines). WT mice were used as a control (black lines). Flicker recordings were performed with light intensities ranging from 10−4 to 15 cd s/m2 in steps of 0.6 logarithmic units at 6 Hz frequency. (F) Flicker responses, plotted as a function of stimulus intensity, from WT (black lines), DMSO-treated miR-211-/- (red lines) and NSC668394-treated miR-211-/- (green lines) mice, at three months of age. The amplitude of the recordings from NSC668394 miR-211-/- treated mice is significantly rescued compared to DMSO-treated miR-211-/- mice. WT mice were used as a control. Error bars represent s.e.m. ANOVA test (DMSO vs WT and NSC668394 vs DMSO treated mice) **p ≤ 0.01, ***p ≤ 0.005. ",
    "molecules": "DMSO, NSC668394"
  },
  {
    "caption": "Representative immunoblot of WT, LUZP1 KO, and Venus-LUZP1-expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [WT, 1.00 (control) vs. 1.40 ± 0.06 (calyculin A) vs. 1.14 ± 0.33 (washout); KO, 0.09 ± 0.04 (control) vs. 1.49 ± 0.06 (calyculin A) vs. 0.81 ± 0.99 (washout); REV, 2.06 ± 1.78 (control) vs. 1.82 ± 1.50 (calyculin A) vs. 1.80 ± 1.14 (washout)]. n = 3. Bars and error bars represent the mean ± SD.",
    "molecules": "calyculin A"
  },
  {
    "caption": "In vitro MLC phosphorylation assay using 1 μg GST-PP1c β/δ in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the di-phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 up-regulated ppMLC/MLC levels in a dose-dependent manner [1.00 (1st lane, control) vs. 1.27 ± 0.33 (2nd lane) vs. 1.76 ± 0.68 (3rd lane) vs. 2.53 ± 1.65 (4th lane) vs. 2.93 ± 2.45 (5th lane)]. n = 3 or 6. **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.   In vitro Merlin phosphorylation assay using 1 μg GST-PP1c β/δ, 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), and 5 μg GST-LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 up-regulated pMerlin/Merlin levels [0.23 ± 0.15 (1st lane) vs. 1.00 (2nd lane, control) vs. 0.32 ± 0.17 (3rd lane) vs. 0.97 ± 0.42 (4th lane) vs. 1.25 ± 0.39 (5th lane)]. n = 4 or 9. *p < 0.05, **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 3rd lane)]. Bars and error bars represent the mean ± SD.   ",
    "molecules": "ATP"
  },
  {
    "caption": "Box plots with dot density plots showing the ratio of the apical area/basal area in co-cultures of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [REV, 0.65 ± 0.16 (control) vs. 0.90 ± 0.18 (nocodazole) vs. 0.64 ± 0.16 (washout); KO, 1.30 ± 0.17 (control) vs. 1.07 ± 0.13 (nocodazole) vs. 1.32 ± 0.19 (washout)]. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range. Representative confocal micrographs of co-cultures of LUZP1-expressing wild-type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di-phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down-regulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [WT, 21.43 ± 6.96 arbitrary units (a.u.) (control) vs. 17.67 ± 5.40 a.u. (nocodazole) vs. 20.84 ± 7.19 a.u. (washout); KO, 8.74 ± 1.71 a.u. (control) vs. 8.67 ± 1.89 a.u. (nocodazole) vs. 7.96 ± 2.35 a.u. (washout)]. n = 3. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). Bars and error bars represent the mean ± standard deviation (SD).   ",
    "molecules": "nocodazole, Nocodazole"
  },
  {
    "caption": "In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, 1 μg GST-protein phosphatase 1c β/δ (PP1c β/δ), and 0-5 μg GST-LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1-mediated inhibition of PP1c β/δ [1.00 (1st-lane, control) vs. 1.42 ± 0.59 (2nd-lane) vs. 1.72 ± 0.76 (3rd-lane) vs. 1.99 ± 0.56 (4th-lane) vs. 1.14 ± 0.37 (5th-lane) vs. 2.87 ± 1.51 (6th-lane) vs. 2.74 ± 1.19 (7th-lane) vs. 2.50 ± 0.88 (8th-lane)]. n = 6. *p < 0.05 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.",
    "molecules": "ATP"
  },
  {
    "caption": "(a) NOX2 activity in Slamf1+/+ and Slamf1-/- BALB/c peritoneal macrophages stimulated for 0-100 min with E. coli F18, S. aureus or PMA, assessed with lucigenin. Data are representative of five independent experiments (mean ± s.d.).",
    "molecules": "PMA"
  },
  {
    "caption": "(c) NOX2 activity in primary macrophages in response to LPS, purified OmpC, peptidoglycan (PGN) or PMA, assessed with lucigenin. TLR4-KO, TLR4-deficient (strain del/Jtht; C3H). Data are representative of five independent experiments (mean ± s.d.).",
    "molecules": "LPS, peptidoglycan, PGN, PMA"
  },
  {
    "caption": "(a,b) Production of phagosomal PtdIns(3)P in E. coli-containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP-tagged PX domain of p40phox (p40 PX-eGFP) and treated with DsRed-expressing E. coli (E. coli-DsRed; a) or beads coated with E. coli outer membrane extract (b). Numbers in bottom right corners indicate time (in min). Original magnification, ×60. Right, quantification of fluorescence in microscopy at left.",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": "(c) HPLC analysis of the production of PtdIns(3)P in primary macrophages labeled with 3H-tagged myoinositol and treated with beads coated with E. coli outer membrane extract.",
    "molecules": "myoinositol, PtdIns(3)P"
  },
  {
    "caption": "(d) PtdIns(3)P production in RAW264.7 cells stably expressing SLAM or a mock construct, transfected with eGFP-tagged p40 PX and treated with beads coated with E. coli outer membrane extract. Data are from three combined experiments (a) or are representative of at least three independent experiments (b,d) or two independent experiments (c) with at least 100 beads or bacteria per experiment (error bars, s.d.).",
    "molecules": "PtdIns(3)P"
  },
  {
    "caption": " (C, D) Standard replication assays on undamaged (UD), thymine glycol (Tg), and cyclobutane pyrimidine dimer (CPD) templates in the presence (C) and absence (D) of Pol η, Pol ζ, Rev1, Rad6-Rad18, Uba1, and ubiquitin (TLS machinery). Reactions contained 2.5 nM Pol δ. ",
    "molecules": "CPD, cyclobutane pyrimidine dimer, Tg, thymine glycol, ubiquitin"
  },
  {
    "caption": " (B) Oligonucleotide competition assay on UD and Tg templates. S; scrambled oligonucleotide. (C) Lane profiles of lanes 4-6 in (B) normalised to the run off product. FL; FL-lead, OR; oligonucleotide restart, S; Stall, RO; Run off, OF; Okazaki fragments. ",
    "molecules": "oligonucleotide, Tg"
  },
  {
    "caption": " (D) Pulse chase reaction on UD and Tg templates performed as shown. Where present, Pol δcat (10 nM) was added directly after addition of the chase. ",
    "molecules": "Tg"
  },
  {
    "caption": "(E) Standard replication reaction performed in the absence and presence of RFC/PCNA on the Tg template.",
    "molecules": "Tg"
  },
  {
    "caption": " (B) Pulse chase reactions on the Tg template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel. ",
    "molecules": "Tg"
  },
  {
    "caption": " (D) Pulse chase reaction on the Tg template in the absence and presence of 10 nM Pol δ analysed on a urea polyacrylamide gel. ",
    "molecules": "Tg, urea"
  },
  {
    "caption": " (E) Titration of Pol δ in a pulse chase reaction on the Tg template. Pol δ was added after the 0 min time point was taken. (F) Quantification of bypass 5 min after addition of the chase for the data in (E). ",
    "molecules": "Tg"
  },
  {
    "caption": " (G) Titration of Pol δ or Pol ε (1, 2.5, 5, 7.5, 10 nM)­­ into a primer extension assay on a Tg template in the presence of PCNA and RFC. The primer/template sequence and location of the lesion are shown above. ",
    "molecules": "Tg"
  },
  {
    "caption": " (B) Mapping the 3ʹ-end of the stalled nascent leading strand. Pulse chase reaction on Tg and CPD templates. Where present, Pol δ (10 nM) was added immediately after the 0 min time point. Products were cleaved with SwaI and BamHI and resolved alongside a sequencing ladder corresponding to the nascent strand. dd; dideoxy. ",
    "molecules": "CPD, Tg"
  },
  {
    "caption": " (A) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP, dCTP, dGTP, or dTTP. ",
    "molecules": "dATP, dCTP, dGTP, dTTP, Tg"
  },
  {
    "caption": " (C) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in (B). Three individual experiments were performed, two in the presence and one in the absence of Pol δ (10 nM), giving 99, 97, and 95 successful sequencing reads respectively. ",
    "molecules": "Tg"
  },
  {
    "caption": "(A) Standard replication assay on undamaged (UD), 8-oxoguanine (8oxoG), and thymine glycol (Tg) templates in the absence of TLS components.",
    "molecules": "8-oxoguanine, 8oxoG, Tg, thymine glycol"
  },
  {
    "caption": " (B) Pulse chase reaction on UD and 8oxoG templates. Where present, Pol δcat (10 nM) was added directly after addition of the chase. ",
    "molecules": "8oxoG"
  },
  {
    "caption": " (C) Pulse chase reactions on the 8oxoG template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel. ",
    "molecules": "8oxoG"
  },
  {
    "caption": "(C-D) Diverse outcome for  tumor  cells following interactions with CTLs. OT-I CTLs and OVA+ Eµ-myc B cells were labeled with the calcium-sensitive dye Indo-1, cultured together in the presence of  propidium iodide (PI) and subjected to live imaging. Representative time-lapse images (C) and corresponding fluorescence quantification (D) showing the three distinct outcomes for CTL-tumor cell  contacts (left, calcium response in T cells, and right, calcium and PI signal in targets): no detectable damage (no calcium signal in the target cells), reversible damage (transient calcium  elevation and low uptake of PI), and irreversible damage (prolonged calcium elevation and bright PI staining). CTLs are outlined by white dashed lines. White arrowheads indicate tumor cells. Red arrowheads indicate tumor cells with internalized PI. Scale bar, 5 µm.",
    "molecules": "calcium, Indo-1, PI, propidium iodide"
  },
  {
    "caption": "(A-B)  Calcium  responses were recorded over  time in (A) unconjugated tumor cells or in (B)  tumor cells forming a conjugate with a CTL  (during the duration of the contacts). Each line corresponds to an individual tumor cell and each square to a  time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.",
    "molecules": "Calcium, calcium"
  },
  {
    "caption": "(A) Representative time-lapse images showing tumors without  CTLs (upper panels) or with OT-I   CTLs (lower panels).  Tumor cells with low and high intracellular  calcium appear in blue and red, respectively. CTLs appear in white. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm. Data information: Data were pooled of four independent experiments. (*p<0.05, ***p<0.001, unpaired t‑test).",
    "molecules": "calcium"
  },
  {
    "caption": "(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B)  tumor cells forming a conjugate with a CAR T cell. Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events  (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.",
    "molecules": "Calcium, calcium"
  },
  {
    "caption": "(A) Representative time-lapse images showing tumors without  CAR T cells (upper panels) or with  CAR T Cells (lower panels).  Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CAR T cells appear in light green. Red arrows indicate  tumor cells with elevated calcium. Scale bar, 5 µm.",
    "molecules": "calcium"
  },
  {
    "caption": "(A) Top: SERA6 architecture. Positions of the papain-like domain, catalytic triad residues, SUB1 cleavage sites and epitopes for the antibodies used are indicated. Positions of the inserted mTAP or myc3 epitope tags (between codons Asn886-Val887) introduced in SERA6-mTAP:loxP parasites are shown. Bottom: time-course analysis of egress by western blot, showing processing of SERA6-mTAP into multiple protein species (representative of 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2-mediated arrest. Nomenclature for each protein species was based on apparent molecular mass or predicted composition. Note that the predicted mass of the p40 species is ~27 kDa, but its migration on SDS PAGE may be aberrant due to its acidic nature; the predicted pI of sequence between SUB1 site 1 and the central papain-like domain (Asp371-Lys605) is ~4.4 (see https://web.expasy.org/compute_pi/).",
    "molecules": "Asn, Asp, C2, Val, Lys"
  },
  {
    "caption": "(C) Giemsa-stained thin films and western blot of DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites showing that cleavage of host RBC β-spectrin (red arrowhead) does not occur unless a functional copy of SERA6 is present, and is tightly associated with egress (reproducible in 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2. Scale bar, 5 μm. Also see Fig EV2.",
    "molecules": "Giemsa, RAP, Ala, C2, Cys, DMSO"
  },
  {
    "caption": "(F) Representative IFA images confirming loss of MSA180-HA3 expression following RAP-treatment (reproducible in 2 independent experiments). Mature C2-arrested cycle 0 schizonts were co-stained with anti-HA (red) and anti-SERA5 (green). Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 5 μm.",
    "molecules": "RAP, C2, 4,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "(G) Western blots showing MSA180 is a predominantly soluble PV protein (representative of 2 independent experiments). Mature C2-arrested MSA180-HA3:loxP schizonts were sampled prior to (Total) and following saponin fractionation to separate RBC cytosol plus soluble PV proteins (Supernatant) from other parasite proteins and membrane-associated proteins (Pellet). The same samples were iprobed with anti-SERA5 and anti-MSP1 (mAb 89.1) as positive controls for soluble PV proteins and merozoite surface parasite proteins respectively. The presence of MSA180 p45-HA3 is likely a post-lysis artefact, perhaps due to SUB1 release during sample manipulation. Note the less efficient saponin-mediated release of MSA180-HA3 compared to SERA5, indicating limited membrane association of a fraction of MSA180. See also Fig EV3.",
    "molecules": "C2, saponin"
  },
  {
    "caption": "(A) Replication of DMSO- and RAP-treated MSA180-HA3:loxP parasites over three erythrocytic cycles. Parasitaemia values are averages from replicates in different blood sources. The similar growth rate of DMSO-treated parasites and the parental B11 clone shows that the genetic modifications to generate MSA180-HA3:loxP parasites did not affect parasite viability. Error bars, ± S.D (B11: n=3; DMSO- and RAP-treated MSA180-HA3:loxP: n=6 each).",
    "molecules": "RAP, DMSO"
  },
  {
    "caption": "(B) Stills from simultaneous time-lapse DIC and fluorescence microscopic examination of DMSO- and RAP-treated MSA180-HA3:loxP cycle 0 schizonts following removal of C2-arrest (representative of 2 independent experiments). Note the defect in RBCM rupture and merozoite release in RAP-treated parasites despite normal PVM rupture (indicated by a sudden increased visibility and motility of merozoites) and RBCM poration (green ring indicating access and binding of phalloidin to the host RBC cytoskeleton). Scale bars, 5 μm.",
    "molecules": "RAP, C2, DMSO, phalloidin"
  },
  {
    "caption": "(D) Time-course comparing fate of SERA6 from DMSO- and RAP-treated MSA180-HA3:loxP parasites (representative of 2 independent experiments). Schizonts were sampled at the indicated times following removal of C2-arrest, or arrested with E64-d (50 μM). RAP-treated SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites were included in the blot to allow comparison with processing of catalytically-dead SERA6 (SERA6-Cys644Ala-myc3). Note the accumulation of SERA6 p65 and poor conversion to SERA6 p40 in the RAP-treated (ΔMSA180) parasites.",
    "molecules": "RAP, Ala, E64-d, C2, Cys, DMSO"
  },
  {
    "caption": "(A) Representative IFA images demonstrating the dynamic changes in localisation of SERA6-mTAP during egress (reproducible in 4 independent experiments). SERA6-mTAP:loxP schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green; ankyrin is a major component of the RBC cytoskeleton). Scale bar, 5 μm.",
    "molecules": "C2, DAPI"
  },
  {
    "caption": "(C) Representative IFA images demonstrating localisation of MSA180-HA3 during egress. MSA180-HA3 [B11] schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green). Scale bar, 5 μm. See also Fig EV5.",
    "molecules": "C2, DAPI"
  },
  {
    "caption": "(B) E64-d, MMV676881 and Compound 31 inhibit RBCM rupture and merozoite release despite normal PVM rupture, indicated by loss of a continuous ring of EXP2-mNeon signal (green) and RBCM poration (red ring indicating access and binding of phalloidin to the host RBC cytoskeleton) (reproducible in 2 independent experiments). P. falciparum EXP2-mNeon schizonts were allowed to reach the point of egress in the presence of C2 (1 µM), E64-d (10 µM), MMV676881 (2 µM), Compound 31 (2 µM) or DMSO (0.1% v/v; control) then examined by DIC and fluorescence microscopy. Scale bar, 5 μm.",
    "molecules": "Compound 31, E64-d, C2, MMV676881, DMSO, phalloidin"
  },
  {
    "caption": "(C) Western blot comparing dose-response of E64-d, MMV676881 and Compound 31-mediated inhibition of maturation of SERA6-mTAP from SERA6-mTAP:loxP parasites (reproducible in 3 independent experiments).",
    "molecules": "Compound 31, E64-d, MMV676881"
  },
  {
    "caption": "b, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl and IFT20− MEFs (top, **P = 0.0003, n = 7), and in WT and Ift88−/− KECs (bottom, *P = 0.035, n = 9). PI, protease inhibitors; S, serum. c",
    "molecules": "protease inhibitors"
  },
  {
    "caption": "a, GFP-LC3 puncta pattern (left) and quantification (right) in MEFs treated with purmorphamine (Purmo; top, *P = 0.028, n = 4), in Ptc−/− MEFs (middle, *P = 0.015, n = 4) and after myc-GLI1 overexpression (bottom, *P = 0.012, n = 3).",
    "molecules": "purmorphamine"
  },
  {
    "caption": "b, Autophagic flux by mCherry-GFP-LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum. Yellow, autophagosomes; red only, autophagolysosomes; total, both compartments. (*P = 0.0323, 40 fields).",
    "molecules": "purmorphamine"
  },
  {
    "caption": "c, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl (**P = 0.001) and MEFs knocked down for SMO (SMO−) (**P = 0.0001) upon purmorphamine treatment (*P = 0.009, n = 4).",
    "molecules": "purmorphamine"
  },
  {
    "caption": "d, Autophagic flux by mCherry-GFP-LC3 puncta quantification in WT and Ift88−/− KECs upon purmorphamine treatment (*P = 0.046, **P = 0.008, 40 fields).",
    "molecules": "purmorphamine"
  },
  {
    "caption": "f, Autophagic flux by mCherry-GFP-LC3puncta quantification in MEFs treated with cyclopamine upon serum removal (*P = 0.011, **P = 0.013, 25 fields). Scale bars, 10 µm. Mean ± s.d. in c, f and mean ± s.e.m. in other panels.",
    "molecules": "cyclopamine"
  },
  {
    "caption": "(A) Coomassie stained SDS-PAGE showing purified T. thermophila tubulin.",
    "molecules": "Coomassie"
  },
  {
    "caption": "(B) A representative cryo-electron micrograph of paclitaxel-stabilized T. thermophila MTs.",
    "molecules": "paclitaxel"
  },
  {
    "caption": "(D) Plot showing the time course of T. thermophila cell growth in the presence of CA4 (20µM) or parabulin (50µM; error bars corresponding to the standard deviation of triplicate biological measurements). Control experiments are done with DMSO alone.",
    "molecules": "CA4, DMSO, parabulin"
  },
  {
    "caption": "(A) Plot showing quantification of the T. thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs. Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments. For growth rates and growth lengths, n=41(DMSO), n= 4 (0.04uM parabulin), n= 34 (0.04 uM colchicine). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P<0001 (****); DMSO vs colchicine p = 0.40 (n.s., not significant). (B) T. thermophila MT growth rates in the presence of the indicated drugs (40 μM). Error bars represent the mean +/- 95% CI (n=4). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P = 0.25; DMSO vs colchicine P = 0.20 (n.s., not significant).",
    "molecules": "colchicine, DMSO, parabulin"
  },
  {
    "caption": "(D) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt-MTs.",
    "molecules": "colchicine, parabulin"
  },
  {
    "caption": "(B) Replication of GFP-expressing T. gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging. Since vacuoles containing more than eight tachyzoites are not always easy to score, we tallied vacuoles above eight as being 8+ in size. At 36 hours, both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites (three or more doublings). Vacuoles in parabulin-treated cultures contain significantly fewer parasites, indicating that replication is slowed. The graph shows three biological replicates with values normalized to 100% for each condition and each replicate, error bars represent standard error of the mean between experiments.",
    "molecules": "parabulin"
  },
  {
    "caption": "(C) The parabulin EC50 value for T. gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions. Error bars represent standard error of the mean. The results represent the average of 18 readings (6 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.",
    "molecules": "parabulin"
  },
  {
    "caption": "(D) Phase contrast of extracellular parasite cultures. Serial passage of T. gondii in 40-80 μM parabulin induces accumulation of aberrant tachyzoite forms. These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding.",
    "molecules": "parabulin"
  },
  {
    "caption": "(A) Size exclusion chromatography  profile of NS1 pull-down experiments showing a clear shift after incubation in complete or heat-inactivated human serum  (solid and dotted black lines, respectively) from the same healthy donor compared to the NS1  protein alone (blue line). NS1  protein interaction  partners were identified by  SDS-PAGE and N-terminal sequencing as the  Apolipoprotein B scaffold of the  low-density lipoproteins (LDL) in the first SEC elution peak, and the ApoA-I  protein scaffold of the high-density lipoproteins (HDL) in the second elution peak.",
    "molecules": "HDL, high-density lipoproteins, LDL, low-density lipoproteins"
  },
  {
    "caption": "(B) Biolayer interferometry (BLI) profiles corresponding to the binding  of NS1  at various concentrations respective to human HDL  (left panel) and human LDL  (central panel) particles. The concentration-dependence of the steady-state signal corresponding to the binding  of NS1  to HDL  (black dots) and LDL  (white dots) is shown on the right-hand side panel. The measurements were replicated at least three times using novel biosensors and samples. Data points and error bars correspond to the mean ± SD.",
    "molecules": "HDL, LDL"
  },
  {
    "caption": "(C) Typical sedimentation  coefficient distribution of NS1  or human HDL  alone or pre-incubated together (mixture of NS1  and HDL  at a 1:1 or 5:1 mass ratio) monitored using an interferometric  detector. Peaks were integrated for all the detectors (interference and absorbance at 280nm). The calculated stoichiometries are indicated for each peak. Solutions were equilibrated at 20°C for 2h before sedimentation  velocity analysis.",
    "molecules": "HDL"
  },
  {
    "caption": "(A, B)  Electron microscopy observations from left to right: a representative image, followed by the three most representative classes of (A) purified HDL particles and (B) NS1-HDL  complexes. White bar: 50 nm, Black bar: 20 nm.",
    "molecules": "HDL"
  },
  {
    "caption": "(D) Differential scanning calorimetry (DSC) of NS1  alone (blue line) or of an NS1-HDL  mixture at a 2:1 molar ratio (orange). Of note, the HDL  particles alone did not generate any signal in the temperature  range tested.",
    "molecules": "HDL"
  },
  {
    "caption": "(E) Binding   inhibition  of hexameric NS1  to HDL  with anti-ApoA-I polyclonal antibodies (anti-ApoA-I Ab) measured by BLI.",
    "molecules": "HDL"
  },
  {
    "caption": "(A-D) Human primary macrophages  were incubated for 24h with the different potential effectors (NS1, HDL, mix NS1-HDL) or with control suspensions (PBS  buffer, LPS, mix LPS-HDL). LPS  stimulation was used as a positive control in the presence or absence of HDL   and provided values consistent between experiments.  Phosphate buffer  used in the SEC  purification step was used as a negative control. Cell culture supernatants were clarified and tested with a  Luminex assay  to quantify the amount of (A) TNF-α, (B) Il-6, (C) Il-1ß and (D)  Il-10 released in the extracellular medium. Data reported on the graphs correspond to biological replicates of macrophages  isolated from four blood  donors (n=9 for TNF-α, Il-6 and Il-1ß, n=5 for Il-10). Data represent mean +/- SEM. A Mann-Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants. Not significant: ns, * p < 0.05, *** p < 0.001.",
    "molecules": "HDL, LPS, PBS, Phosphate buffer"
  },
  {
    "caption": "levels of (B) NS1, (C) NS1-ApoA-I, (D) NS1-ApoE and (E) NS1-ApoB NS1-ApoA-I  and NS1-ApoB  complexes are representative of NS1-HDL  and NS1-LDL  complex species, respectively, while the NS1-ApoE-positive complexes remain to be fully characterized. Errors bars indicate SEM.",
    "molecules": "HDL, LDL"
  },
  {
    "caption": "levels of (F) total cholesterol, (G) HDL-cholesterol and (H) triglycerides. Errors bars indicate SEM.",
    "molecules": "cholesterol, HDL, triglycerides"
  },
  {
    "caption": "E) Critically short telomeres arise after 90 population doublings in the absence of telomerase (tlc1). Genomic DNA was extracted at indicated population doublings and digested with XhoI. A radioactive probe was used to recognize telomeric DNA in a southern blot. Abbreviations: PD: population doubling.",
    "molecules": "Genomic DNA"
  },
  {
    "caption": "B) Npl3 associates to TERRA. Cross-linked samples from the indicated strains were subjected to Npl3-TAP RIP. Enrichment of the indicated RNAs was determined by quantitative PCR on pulled-down reverse-transcribed RNA. Data represents mean % input +/- SEM n=3 (unpaired t-test two tailed *p<0.05, **p<0.01).",
    "molecules": "RNA, RNAs"
  },
  {
    "caption": "Npl3 associates strongly to telomeres in the absence of Rat1. C) The Rat1-AID tagged protein is strongly degraded following 1h of 1mM IAA treatment. Protein levels are determined using anti-FLAG (Rat1-AID) and anti-actin antibodies. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.",
    "molecules": "Auxin, IAA, indole-3-acetic acid"
  },
  {
    "caption": "Npl3 associates strongly to telomeres in the absence of Rat1. D) TERRA levels increase in cells expressing the Rat1-AID variant after 1h treatment with 1mM IAA. TERRA levels were determined by quantitative PCR on reverse-transcribed RNAs after phenol-chloroform extraction. RNA levels are normalized to 7S ncRNA levels. Data represents fold increase to corresponding -IAA samples n=2. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.",
    "molecules": "Auxin, chloroform, IAA, indole-3-acetic acid, phenol, RNA, RNAs"
  },
  {
    "caption": "Npl3 associates strongly to telomeres in the absence of Rat1. E) Npl3-TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1-AID. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.",
    "molecules": "Auxin, IAA, indole-3-acetic acid"
  },
  {
    "caption": "F-G) Npl3 associates to telomeres in an R-loop dependent manner. E) RNH1 was overexpressed from a galactose-inducible promoter. Protein levels are determined using anti-HA and anti-actin antibodies. F) Npl3-TAP associates to telomeres in an R-loop-dependent manner. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: eV: empty vector control, oE: overexpression",
    "molecules": "galactose"
  },
  {
    "caption": "A-B) Npl3 associates to telomeres in early S. A) Cell cycle profiles of indicated strains. Cells were synchronized in G1 with alpha-factor for 2.5h and released into the cell cycle at 30°C in pre-warmed medium supplemented with the indicated HU concentrations. B) Npl3-TAP associates strongly to telomeres in early S (250 mM HU). Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to cells arrested in alpha factor n=3 (paired t-test two tailed *p<0.05, **p<0.01 Abbreviations: ns: not significant HU: hydroxyurea. *p<0.05, **p<0.01",
    "molecules": "alpha factor, alpha-factor, HU, hydroxyurea"
  },
  {
    "caption": "C) NPL3 over expression stabilizes telomeric R-loops. The indicated strains were grown on 1% raffinose 2% galactose to induce NPL3 over expression. Cross-linked samples were subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to 250 mM HU eV n=3 (paired t-test two tailed *p<0.05, **p<0.01). Abbreviations eV: empty vector control, oE: over expression HU: hydroxyurea. *p<0.05, **p<0.01",
    "molecules": "galactose, HU, raffinose"
  },
  {
    "caption": "E-G) NPL3 stabilizes R-loops. E) R-loop dot blot. The indicated strains were grown on 1% raffinose 2% galactose for 72h and subsequently released into 1% raffinose 2% galactose or 2% glucose medium for 6 PDs. R-loop levels and dsDNA levels were determined using the S9.6 antibody and anti-dsDNA antibodies. Specificity of the S9.6 antibody was confirmed by treatment with RNase H. Loaded genomic DNA content is indicated at the bottom of the figure. F) R-loop dot blot quantification. Data represents R-loop signal as the S9.6 antibody signal relative to corresponding dsDNA signal n=3 (unpaired t-test two tailed *p<0.05). G) Serial dilutions of indicated strains were assayed on 1% raffinose 2% galactose media. Plates were imaged after 72h incubation at indicated temperatures. Abbreviations: V: empty vector control, oE: over expression, ds: double-strand,",
    "molecules": "genomic DNA, dsDNA, galactose, glucose, raffinose"
  },
  {
    "caption": "Fluorescent images and tracings of wild type control (top) and lect-2(gk846764) hypomorphic animals (bottom) fed on plates with 300µm swainsonine  vs a DMSO  control. PVD  is visualized by the wyIs581 transgene. The  cell body  is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Vesicular gut  autofluorescence is visible as white circular staining. Scale bars represent 20μm. Quantification of the number of full tertiaries in denoted genetic backgrounds (aman-2 null is gk248486). Black data points indicate DMSO  and orange data points show swainsonine treated animals. Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n = 12 for all genotypes and are biological replicates. Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine. Animals treated with swainsonine  show a significant decrease in quaternary branch number, Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001). n = 12 for each experiment and are biological replicates.",
    "molecules": "DMSO, swainsonine"
  },
  {
    "caption": "Western blot against GFP  in C. elegans lysate expressing no transgenes (N2) and expressing DMA-1::GFP  (qyIs369), after precipitating with anti-GFP  antibody. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The downwards size shift reveals that N-glycan structures are present on DMA-1. Ladder is marked in kilodaltons (kDa). The GFP  tag contains no N-glycosylation sites. The experiment was repeated four times with biological replicates.",
    "molecules": "N-glycan"
  },
  {
    "caption": "Western blot  against GFP  in C. elegans lysate  DMA-1::GFP  (qyIs369), after precipitating with anti-GFP  antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The upward size shift in the mutant reveals that the loss of aman-2  alters the identity of N-glycan structures on DMA-1. The experiment was repeated four times with biological replicates.",
    "molecules": "N-glycan"
  },
  {
    "caption": "Western blot  against GFP  in C. elegans lysate DMA-1::GFP  (qyIs369), after precipitating with anti-GFP  antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The red boxed +F indicates that the lysate is treated with the PNGase F glycosidase, while the green boxed +H corresponds to the Endo H glycosidase, which cleaves high-mannose  and hybrid type N-glycans. For complementary experiments using the Endo D glycosidase, which cleaves paucimannose  type N-glycans, Size shifts indicate that some hybrid/high-mannose  structures are present on DMA-1  (left), and that the  aman-2  mutant results in only hybrid/high-mannose  structures on DMA-1 (right). The experiment was repeated four times with biological replicates.",
    "molecules": "mannose, N-glycans, paucimannose  type N-glycans"
  },
  {
    "caption": "Quantification of the number of quaternary branches in  DMA-1::2xFLAG control animals and animals with combinations of DMA-1::2xFLAG N-glycan  attachment sites mutated. Note that  DMA-1::2xFLAG control animals display a slightly reduced number of quaternary dendrites  compared to wild type animals Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.",
    "molecules": "N-glycan"
  },
  {
    "caption": "Fluorescent images (left) and tracings (center) of PVD  in animals of denoted backgrounds (aman-2(null) is gk248486). S4 corresponds to the endogenous alteration of  N-glycan attachment site N386 of  DMA-1. PVD  is visualized by the dzIs117 transgene. The cell body  is marked with an asterisk. Scale bars represent 20μm.",
    "molecules": "N-glycan"
  },
  {
    "caption": "E. Schematics of astrocytic calcium imaging and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus in OB. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively. F-H. Quantification of amplitude (F), frequency (G), and area under curve (AUC) (H) of ∆F/F GCaMP6f signal events in OB. Amplitude and AUC of Daam2-deficient astrocytes are significantly increased in OB. Data is presented as mean ± SEM. Total number of cells is n=16, 15 (F-H) from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05. ",
    "molecules": "calcium"
  },
  {
    "caption": "F-J. Representative images of GFAP immunostaining (F) and quantification (G-J) in controls, Daam2 icKO, Slc4a4 icKO, and double icKO 6 weeks after tamoxifen injection. Scale bar: 20 μm. Data is presented as mean ± SEM. n=10-24 images, N=3-4 mice per genotype. Scale bar: 20 μm. Two-way ANOVA is used for statistics. [*] and [#] indicates comparison with control and double icKO, respectively. **p<0.01, p***<0.001, ##p<0.01.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "RT‐PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam‐treated mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/flmice (right lane); the Syk‐ and Hprt‐specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control.",
    "molecules": "Tam"
  },
  {
    "caption": "Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl (right lane) mice both treated with Tam as described; blots were probed with anti‐Syk and anti‐GAPDH Ab, GAPDH being used as a loading control.",
    "molecules": "Tam"
  },
  {
    "caption": "Genomic DNA analysis of Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/flmice (as indicated). Upper row: amplification of floxed (fl) and wt (+) alleles. Middle row: amplification of the deleted (d) allele. Lower row: loading control (TC21, a gene unaffected by the deletion of Syk). Established control DNA was used to visualize the individual bands (as indicated).",
    "molecules": "Tam"
  },
  {
    "caption": "A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/flmice (right) treated with Tam as described in the Materials and Methods section. The BM cells were stained with anti‐IgM and anti‐B220, and the SP cells with anti‐CD19 and anti‐CD93 or anti‐CD23 and anti‐CD21. The gated regions in the dot blots correspond to individual B‐cell populations: (A) Bone marrow: gate P (B220+IgM−) pro‐/pre‐B cells, gate I (B220loIgM+) immature B cells, gate M (B220hiIgM+) mature B cells; (B) spleen: gate T (CD19+ CD93+) transitional B cells (T), gate M (CD19+ CD93−) mature B cells; in CD93−splenic B cells, gate M (CD23hiCD21int CD93−) mature follicular B cells and gate MZ (CD23lo CD21hi CD93−) marginal zone B cells. The numbers in the dot plots indicate the mean relative frequency of cells in the gate.C-E Statistical analysis of absolute cell numbers per Tam‐treated mouse: filled circles indicating cells obtained from mb1‐CreERT2 control mice and open circles from mb1‐CreERT2;Sykfl/flmice. (C) Statistical analysis of the absolute cell numbers of pro‐/pre‐, immature and mature recirculating B cells in the BM. (D) Absolute cell numbers of transitional, M and MZ B cells found in the SP. (E) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1‐a and B1‐b. An asterisk (*) marks statistically significant differences (P 0.05), two asterisks (**) indicate P 0.01, P‐values were obtained using two‐tailed Student's t‐test. Cell numbers of four to five mice per group are shown, with each dot representing an individual animal.",
    "molecules": "Tam"
  },
  {
    "caption": "A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/flmice (solid line).",
    "molecules": "Tam"
  },
  {
    "caption": "B Flow cytometric analysis of tyrosine phosphorylation (pY) in splenic B cells from Tam‐treated mb1‐CreERT2 and mb1‐CreERT2;Sykfl/flmice stimulated with medium (dotted line) or 50 μM pervanadate (dashed and solid lines).",
    "molecules": "Tam, pervanadate"
  },
  {
    "caption": "C, D Flow cytometric analysis of the intracellular Ca2+ influx in purified splenic B cells derived from Tam‐treated mice. mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl were treated with (C) 10 μg/ml anti‐Kappa F(ab′)2 fragments or (D) 1 μM ionomycin.",
    "molecules": "Ca2+, ionomycin, Tam"
  },
  {
    "caption": "Ex vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). Purified splenic B cells were either left unstimulated (medium) or were stimulated with the indicated stimuli. The cells were analyzed by flow cytometry after 24 h. Shown are CD19 versus CD86 dot plots. 7‐AAD was included to distinguish dead from viable cells. The numbers in the quadrants indicate the relative frequencies of cells in the gate.",
    "molecules": "Tam"
  },
  {
    "caption": "Ex vivo proliferation assay with mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). The cells were incubated with the indicated stimuli for 90 h.",
    "molecules": "Tam"
  },
  {
    "caption": "A Flow cytometric analysis of splenic B cells from Rag2−/−;γc−/− mice. CD19 versus CD3ε dot plots are shown. The mice were injected i.v. with 5 × 106 splenic (CD19+ CD93−) B cells and (CD3ε+) T cells from Tam‐untreated mb1‐CreERT2 control or mb1‐CreERT2;Sykfl/fl mice. The recipient mice were treated with Tam beginning 1 day after transfer as described in the Materials and Methods section.B Quantitative analysis of B and T cells from Rag2−/−;γc−/− mice repopulated with mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl splenocytes. Each symbol represents an individual mouse. Filled circles and squares represent Syk+/+ B and T cells, respectively; open circles and squares represent Sykfl/fl B and T cells.",
    "molecules": "Tam"
  },
  {
    "caption": " Growth was tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) on BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. (A) BW25113 ΔrelA derivatives harboring plasmids pKT2(ycbB), pKT8(relA') and the vectors pHV6 and pHV7 used to construct these plasmids, respectively. Expression of β-lactam resistance requires induction of both ycbB and relA'.",
    "molecules": "β-lactam, ceftriaxone, IPTG, L-arabinose"
  },
  {
    "caption": " Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.",
    "molecules": "2,4-diacetylphloroglucinol, DAPG, ceftriaxone, IPTG, L-arabinose"
  },
  {
    "caption": " The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance. ",
    "molecules": "ceftriaxone, IPTG, L-arabinose, L-rhamnose"
  },
  {
    "caption": " Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. ",
    "molecules": "ceftriaxone, IPTG, L-arabinose, L-rhamnose"
  },
  {
    "caption": " (B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53. ",
    "molecules": "L-arabinose, L-rhamnose"
  },
  {
    "caption": " (B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB). ",
    "molecules": "chloramphenicol, L-arabinose, L-rhamnose"
  },
  {
    "caption": " (A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB). ",
    "molecules": "chloramphenicol, L-arabinose, L-rhamnose"
  },
  {
    "caption": " (A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives. ",
    "molecules": "ampicillin, ceftriaxone, IPTG, L-arabinose"
  },
  {
    "caption": " C, Dose-response curves of BEAS-2B and BEAS-2B-KRAS cells treated for 72 hours with inhibitors of FGFR (AZD4547, BGJ398) and PLK1 (BI2536, BI6727), alone or combination. Values of Fa and CI are shown underneath, with CI<1.0 indicating synergistic effect. Data were from three independent experiments (n=3), error bar: SD ",
    "molecules": "AZD4547, BGJ398, BI2536, BI6727"
  },
  {
    "caption": " C, Clongenic assay of KRAS-mutant lung and pancreatic cells (MIA PaCa-2, H2122, H2009, H23, H358 and A549) treated with control (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination. ",
    "molecules": "AZD4547, BI2536, DMSO"
  },
  {
    "caption": " D, Apoptotic assay of KRAS-mutant cancer cells (H358, H441, A549, and SW620) and KRAS-WT lung cancer cells (EBC-1) treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 48h. Data are presented as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "AZD4547, BI2536, DMSO"
  },
  {
    "caption": " D, Clonogenic assay of H358, A549 and MIA PaCa-2 cells treated with AZD4547, BI2536, and NAC, alone or in combination as indicated. ",
    "molecules": "AZD4547, BI2536, NAC"
  },
  {
    "caption": " E, Immunoblots of H358 and A549 cells treated for 24 h with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of NAC (5 mM). ",
    "molecules": "AZD4547, BI2536, DMSO, NAC"
  },
  {
    "caption": " clonogenic assay of H358 cells treated for 48 h with vehicle, AZD4547 (5 µM) and BI2536 (5 nM), in the presence or absence of Q-VD-OPh (20 µM). Data shown as mean ± SD (n=3). ***P<0.001 and ****P<0.0001 by two-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "AZD4547, BI2536, Q-VD-OPh"
  },
  {
    "caption": " B, C, Immunoblots (B) and viability assay (C) of H358 and A549 cells preincubated overnight with SP600125 (2 µM) and SB203580 (1 µM) and subsequently treated with AZD 4547 (5 µM) and BI2536 (5 nM) for 24 hours. Data were shown as mean ± SD (n=3); ***P<0.001, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "SP600125, AZD 4547, BI2536, SB203580"
  },
  {
    "caption": " D, E, Immunoblots (D) and viability assay (E) of H358 and A549 cells transfected with E2F1-specific or control siRNAs followed by treatment (48 h post transfection) with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536(5 nM) for additional 24 hours. Data were shown as mean ± SD (n=3). **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "AZD 4547, BI2536, DMSO"
  },
  {
    "caption": " F, H358 cells transfected with E2F1-specific or control siRNAs for 24h were subsequently treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or combination for 48h before apoptotic assay. Data were shown as mean ± SD (n=3). *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "AZD4547, BI2536, DMSO"
  },
  {
    "caption": " B, A549 cells stably expressing mCHerry-GFP-LC3B were exposed to vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or in combination for 24h. Cell were then fixed and processed for immunofluorescence, scale bar=10 µm. ",
    "molecules": "AZD4547, BI2536, DMSO"
  },
  {
    "caption": " E, Flow cytometry-based ROS measure in H358, A549 and MIA PaCa-2 cells treated with vehicle (DMSO), AZD4547 (5 µM) and BI2536 (5 nM), alone or the combination, in the presence or absence of HCQ (10 µM) for 24h. Quantification of relative ROS levels was shown to the right. Data are presented as mean ± SD (n=3). *P<0.05, **P<0.01, ****P<0.0001 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. ",
    "molecules": "AZD4547, BI2536, DMSO, HCQ, ROS"
  },
  {
    "caption": " F, Immunoblots of H358 cells transfected with ATG5-specific or control siRNAs for 48h and before treated for 24h with vehicle (DMSO) or combined AZD 4547 (5 µM)/BI2536 (5 nM). ",
    "molecules": "AZD 4547, BI2536, DMSO"
  },
  {
    "caption": " A, Growth curve of a KRAS-mutant lung cancer PDX (BE564T) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, ***P<0.001 and P＞0.05 (ns) by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD ",
    "molecules": "AZD4547, BI6727, HCQ"
  },
  {
    "caption": " F-K, Growth curves (F, I) of KRAS-mutant lung cancer PDXs (PF139, PF563) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (4 mice/group); error bar: SD Relative tumor volume (G, J) and weights (H, K) of the PDX tumors after the treatment. *P<0.05, **P<0.01, ***P<0.001 and P＞0.05 (ns) by unpaired two-tailed t-test. Data are the mean ± SD (error bar) of tumor weights of each group (4 mice/group). ",
    "molecules": "AZD4547, BI6727, HCQ"
  },
  {
    "caption": "A, B Organotypic slices of Xkr8 WT and cKO hippocampus with Thy1::GFP neurons (green) were labelled with PtdSer-binding fluorescently tagged Annexin V (magenta) at 16-19 DIV. The fluorescence of bound Annexin V was quantified on ImageJ by measuring the integrated density of fluorescent structures within the same volume for each slice and expressing the value as fluorescence units (f.u.; two-tailed Student's t-test, each dot represents an individual slice preparation, n = 4).",
    "molecules": "PtdSer"
  },
  {
    "caption": "Genetic inhibition of autophagy in the germline prevents DNA fragmentation. Confocal micrographs of stage 13/14 egg chambers after TUNEL staining. (A) Wild-type (wt) stage 13 egg chamber contains nurse cell nuclei exhibiting fragmented DNA (positive TUNEL staining; arrows). (B) Wild-type stage 14 egg chambers do not contain nurse cells. Arrows point to the respiratory appendages (RA). (C-E) Stage 14 atg1−/− (C), atg13−/− (D), and vps34−/− (E) germline mutant chambers have persisting nurse cell nuclei that do not contain fragmented DNA (negative TUNEL staining; arrows). Nurse cells are outlined with a white line. Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "dBruce is required for nurse cell survival by controlling DNA fragmentation during oogenesis. (A) Confocal micrographs of dBrucee00984 mutant ovarioles stained for TUNEL and DNA. Arrows point to degenerating stage 8/9 egg chambers that are TUNEL positive.",
    "molecules": "DNA"
  },
  {
    "caption": "(B and C) Confocal micrographs of vps34/dBruceE81 and atg, dBruceE81 double mutant egg chambers stained for TUNEL (green and red, respectively) and DNA. (B) vps34−/−GLCs; dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei (outlined; arrow). (C) atg1−/−GLCs, dBruceE81/dBruceE81 stage 14 mutant egg chamber contain persisting TUNEL-positive nurse cell nuclei. atg1 mutant nurse cells are identified by the lack of GFP staining (outlined; arrow). Draq5/Hoechst staining (blue) was performed to visualize the nuclei. PhC, phase contrast. Bars, 50 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression",
    "molecules": "DOX"
  },
  {
    "caption": "(E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated.",
    "molecules": "DOX, doxycycline, Galunisertib"
  },
  {
    "caption": "(G) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A-pIND20-HPN pL6-TGFβ1 cells with (DOX+) or without (DOX-) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns).",
    "molecules": "DOX"
  },
  {
    "caption": "(H) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM).",
    "molecules": "Silver"
  },
  {
    "caption": "(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry",
    "molecules": "Coomassie, DOX"
  },
  {
    "caption": "(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels.",
    "molecules": "DOX"
  },
  {
    "caption": "(D) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N-terminal fragment of fibronectin (lower panel). Full-length fibronectin (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin.",
    "molecules": "Silver"
  },
  {
    "caption": "(E) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) and without (DOX-) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels (N = 3 biological repeats).",
    "molecules": "DOX"
  },
  {
    "caption": " Immuno-EM of SipB-transfected COS-2 cells. COS-2 cells were transfected with a plasmid expressing SipB-YFP, and 24 h after transfection, cells were fixed and prepared for immuno-EM using an antibody specific for GFP and a gold-labeled anti-rabbit antibody. SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria (A). Mitochondria were often seen apparently fusing to the SipB-induced membranous structure (B, see arrows in inset on top; C, inset to the right). Mitochondria were often seen contained with the SipB-induced membrane structures (D). Bars, 250 nm. ",
    "molecules": "gold"
  },
  {
    "caption": " The S. typhimurium SPI-1 TTSS-secreted protein SipB localizes to the mitochondria of infected macrophages. (A) Bone marrow-derived macrophages were infected with either wild-type S. typhimurium or the TTSS translocation-defective sipD mutant strain with a multiplicity of infection of 50. 3 h after infection, macrophages were collected and processed for immuno-EM using gold-labeled antibodies. SipB could be readily detected in macrophages infected with wild-type (A and B), but not in macrophages infected with the sipD mutant strain (C). Bars, 100 nm. ",
    "molecules": "gold"
  },
  {
    "caption": " The S. typhimurium-induced macrophage death does not require caspase activity. BMDPM were infected with wild-type S. typhimurium or the TTSS-defective invG mutant in the presence or absence of 50 μM of the pan-caspase inhibitor Z-VAD, and cell death was measured as indicated in the legend to Fig. 1. ",
    "molecules": "Z-VAD"
  },
  {
    "caption": " Determination of the interaction of PIE1-Flag, MBD9-Myc, and CHR11-HA by in vitro pull down assays. PIE1-Flag, MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. Anti-Flag agaroses were independently used in the pull down assays. ",
    "molecules": "agaroses"
  },
  {
    "caption": " C) Determination of the interaction of MBD9-Myc, and CHR11-HA by in vitro pull down assays. MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. anti-HA (C) agaroses were independently used in the pull down assays. ",
    "molecules": "agaroses"
  },
  {
    "caption": " D) he effect of the pie1 and mbd9-1 mutations on the formation of the SWR1 complex as determined by IP-MS. Transgenic plants expressing Flag-tagged MBD9, PIE1, and CHR11 in the WT and mutant backgrounds were subjected to the IP-MS experiment with anti-Flag agarose. ",
    "molecules": "agarose"
  },
  {
    "caption": "(A) Increasing amounts of CHR11 (left panel) or PIE1 (right panel) were incubated with end-positioned (lane 1-4 and 9-12) or center-positioned (lane 5-8 and 13-16) mono-nucleosomes in the presence of ATP. The sliding results were then visualized on native PAGE gels by EB staining.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Immunocytochemistry analyses show that glutamate stimulation (Glut, 100 µM 5 min + 25 min washout) increased PRR7 in the nucleus. Cycloheximide (100 µM) added 30 min prior to glutamate stimulation did not affect this increase (Cont=100±12.5%, Glut=202.1±15.2%; +cycloheximide Cont=104.9±3.6, Glut=231.4±10.9) (n=~30-50 cells).",
    "molecules": "Cycloheximide, cycloheximide, glutamate"
  },
  {
    "caption": "(B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test.",
    "molecules": "Epoxomicin, epoxomicin, glutamate"
  },
  {
    "caption": "(C) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons. Treatments (APV [50 µM, APV-(2R)-amino-5-phosphonovaleric acid], CNQX [40 µM, 6-cyano-7 nitroquinoxaline-2,3-dione], TTX [1 µM, tetrodotoxin], NMDA [50 µM, N-Methyl-D-Aspartate]) as indicated. Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 (108.1 ± 19%). Statistical significance was determined using one-way ANOVA in conjugation with Sidak post-hoc test.",
    "molecules": "APV, APV-(2R)-amino-5-phosphonovaleric acid, 6-cyano-7 nitroquinoxaline-2,3-dione, CNQX, glutamate, N-Methyl-D-Aspartate, NMDA, tetrodotoxin, TTX"
  },
  {
    "caption": "(D) PRR7-GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment. Depicted are representative averaged confocal images (30x30 µm2) from optical sections (300 nm step size) of PRR7-GFP expressing neurons (DIV17) before and after bleaching. Prior to stimulation there was substantial PRR7-GFP in the nucleus due to basal neuronal activity (upper panel). This was reduced by incubation overnight with MK801 (10 µM) and TTX (1 µM) (lower panel). NMDA (50 µM) stimulation, but not MK-801, led to recovery of nuclear PRR7-GFP fluorescence following bleaching of nuclear ROIs at t0`(blue arrow in E).(E) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0` and corrected for bleaching. No significant differences in PRR7-GFPFRAP were found in control (MK801) conditions. GFP intensities are displayed via look-up-table (LUT). Data are represented as mean±SEM; n=9 cells (NMDA) and n=5 cells (MK801). Analysis was done using one-way ANOVA using Bonferroni post-hoc correction.",
    "molecules": "MK-801, MK801, NMDA, TTX"
  },
  {
    "caption": "(F) PRR7-mEos translocates from distal dendrites to the nucleus upon NMDA treatment. (Left panels) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7-mEos before and after photoconversion of distal dendrites (ROIs) through the image z-stack from from green (488nm) to red (568nm) using a UV laser (405 nm). (Right panels) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7-mEos (568nm) in the nucleus following NMDA stimulation (50 µM- 30min).(G) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7-mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment. (Average increase; NMDA=617.8±47.7%, n=11 cells, p<0.0005; MK801=206.1±13.6%, n=5 cells, n.s.). Statistical significance was determined by two-way ANOVA using Sidak's correction for multiple comparisons.",
    "molecules": "MK801, NMDA"
  },
  {
    "caption": "(E) Co-IPs of PRR7 with GluN1 from high-density primary neuronal cultures following treatment with NMDA (100µM, 30 min, representative of n=4).",
    "molecules": "NMDA"
  },
  {
    "caption": "(B) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% vs control 68.7 ± 7.9%) and 240 minutes (83.6 ± 5.2% vs control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n=4 gels for each group performed blind). T-tests between similar time points were used to analyze results.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(A) Western blots of phospho-c-Jun in primary neuronal cultures. Quantification of phospho-c-Jun represents a ratio of phospho-c-Jun to total c-Jun and all ratios were normalized to NT. PRR7 knockdown (KD) decreased, while PRR7 overexpression (OE) increased phospho-c-Jun levels (black bars). Glutamate stimulation (gray bars) increased phospho-c-Jun/c-Jun ratio in NT but not in KD or OE neurons (n=7 gels for all groups). Significance was determined using two-way ANOVA with Sidak post-hoc test.",
    "molecules": "Glutamate"
  },
  {
    "caption": "(E) NMDA treatment (25 min, 100 µM followed by 14h chase) caused 83.6±3.5% cell death in control neurons compared to 60.3±4.6% cell death in neurons with PRR7 expression knocked down. A c-Jun inhibitory peptide reduced NMDA-mediated cell death to 66.2±6.0% in control neurons and to 55.6±5.3% in neurons knocked down for PRR7, but this difference was not statistically significant (n=5 independent experiments). Two-way ANOVA was used in conjugation with Tukey post-hoc test. (Left) Representative images showing transduced neurons (green- GFP) and PI staining (red).",
    "molecules": "NMDA"
  },
  {
    "caption": "(F) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c-Jun or a dominant negative c-Jun4A (4A). Overexpression of WT c-Jun caused extensive neuronal cell death (84.8±2.4%) that was reduced in neurons knocked down for PRR7 (43.8±9.2). Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity (50.8±7.9%) but was not additive with PRR7 knockdown (42.3±8.1%) (n=3 independent experiments). Significance was determined using two-way ANOVA in conjugation with Tukey post-hoc test. Images are representative 10X widefield micrographs showing transduced neurons (green- GFP) and PI staining (red). Data is presented as mean±sem. All % values represent comparisons to control.",
    "molecules": "NMDA"
  },
  {
    "caption": "(B) Peritoneal macrophages from the mice were subjected to the ESI-MS/MS method to quantify endogenous LPA. Individual molecular species of LPA and total LPA (inset graph) were compared. The data are shown as mean ± SEM (n=3/group) *P < 0.05 (one-tailed unpaired t-test).",
    "molecules": "LPA"
  },
  {
    "caption": "(C and D) Thioglycolate-elicited peritoneal immune cells harvested from the mice were immediately fixed for transmission electron microscopy to visualize the appearance of macrophages (C) and DCs (D). Scale bar indicates 1 μm.",
    "molecules": "Thioglycolate"
  },
  {
    "caption": "Lipid rafts from peritoneal macrophages (E, F) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Caveolin or Flotillin-1.",
    "molecules": "sucrose"
  },
  {
    "caption": "Lipid rafts from mouse macrophage Raw264.7 cells treated with Atx inhibitor (Atx-i) PF8380 (50 μM, 30 min) or vehicle (Veh., DMSO 0.1%) (G) were fractionated into 10 fractions though sucrose density gradient ultracentrifugation at 100,000g for 17 h. Protein was purified and concentrated with the 3K cut-off filter, followed by Western blot analysis with antibody recognizing the lipid raft marker proteins Flotillin-1.",
    "molecules": "DMSO, PF8380, sucrose"
  },
  {
    "caption": "(H) Peritoneal macrophages were stained with Alexa Fluor 594-cholera toxin subunit B (Red). The plasma membrane was examined with FV10i confocal scanning microscopy. Inset areas were magnified at the right panel. Each scale bar indicates 10 μm.",
    "molecules": "Alexa Fluor 594"
  },
  {
    "caption": "Peritoneal macrophages (A) were stimulated with LPS (20 ng/mL, 20 min) Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.",
    "molecules": "LPS"
  },
  {
    "caption": "Raw264.7 cells (B) were stimulated with LPS (20 ng/mL, 20 min) in the presence/absence of ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%). Cell lysates were subjected to co-immunoprecipitation (IP) with CD14 antibody, followed by immunoblotting (IB) with TLR4 antibody.",
    "molecules": "DMSO, LPS"
  },
  {
    "caption": "The physical interaction of TLR4-CD14 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP (yellow) and CD14-ECFP (blue) (C) followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.",
    "molecules": "DMSO"
  },
  {
    "caption": "The physical interaction of TLR4-TLR4 was examined by FRET analysis. HEK293 cells were transfected with TLR4-EYFP and TLR4-ECFP (D), followed by ATX inhibitor (50 μM, 30 min) or vehicle treatment (Veh, DMSO 0.1%). Cells were washed with PBS and fixed, then visualized with filter sets for CFP and YFP. FRET images were visualized with the FRET filter set, expressed as corrected FRET efficiency and displayed in quantitative pseudocolor indicating the distance between proteins (arbitrary linear units of fluorescence intensity). Presented are the representative images from at least three independent experiments. Scale bar indicates 5 μm.",
    "molecules": "DMSO"
  },
  {
    "caption": "(A and B) Peritoneal macrophages from Atx+/+ (A) and Atx∆ΜΕ/∆ΜΕ (B) mice were stimulated with LPS (20 ng/mL, 20 min), followed by fixation in 4% paraformaldehyde for 15 min at room temperature and staining with TLR4-anti rabbit FITC (Green), early endosomal marker EEA1-anti mouse Dylight405 (Blue), and Alexa Fluor 594-CTXB (Red). TLR4 internalization was examined with confocal microscopy. Presented are the representative from 3 independent experiments in which more than 95% of the cells exhibited similar results. Scale bar, 10 μm.",
    "molecules": "Alexa Fluor 594, FITC, LPS, paraformaldehyde"
  },
  {
    "caption": "The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.",
    "molecules": "DMSO, LPS"
  },
  {
    "caption": "The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Raw264.7 cells were stimulated by LPS with the ATX inhibitor (50 μM, 30 min) or vehicle (Veh, DMSO 0.1%) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.",
    "molecules": "DMSO, LPS"
  },
  {
    "caption": "The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated with LPS (20 ng/mL, 20 min) The cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting (IB) analysis with the antibodies indicated. Presented is the representative from three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min : The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2, were used for a loading control. Presented are the representative image from at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "The macrophages from Atx∆ΜΕ/∆ΜΕ and Atx+/+ mice were stimulated by LPS [20 ng/mL, 20 min The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. regular Akt were used for a loading control. Presented are the representative image from at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "Atx-heterozygous (Atx-Het) mice and wild type littermates (C) were stimulated by LPS indicated time point The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. RasGAP levels were used for a loading control. Presented are the representative image from at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "(D) Raw264.7 cells were stimulated by LPS with the ATX inhibitor. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. , β-Actin were used for a loading control. Presented are the representative image from at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with TLR2 ligand Peptidoglycan (PGN, 30 ng/mL) (E) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.",
    "molecules": "Peptidoglycan, PGN"
  },
  {
    "caption": "The macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates were activated with Pam3CSK4 (1 μg/mL) (F) for the indicated time points. The cell lysates were subjected to immunoblotting analysis with antibodies as indicated. Regular ERK1/2 were used for a loading control. Presented are the representative image from at least three independent experiments.",
    "molecules": "Pam3CSK4"
  },
  {
    "caption": "Peritoneal macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates (A) were stimulated with LPS (50 ng/mL, 8 h) The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "Raw264.7 cells (B) were stimulated with LPS (50 ng/mL, 8 h) in the presence/absence of the ATX inhibitor (50 μM, 30 min). The culture medium was collected for ELISA to measure the level of secreted cytokines. All assays were performed in triplicate, and data are shown as mean ± SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.",
    "molecules": "endotoxin, LPS"
  },
  {
    "caption": "Peritoneal macrophages were stimulated with LPS or vehicle (endotoxin free water), after which intracellular cytokine production was measured through FACS analysis. With the scatter dot plot, the gate was set so that the viable cells were selected to analyze the intensity of the fluorescence signal by flow cytometry. The overlay plot represents the cytometry data.",
    "molecules": "endotoxin, LPS"
  },
  {
    "caption": "(F) To induce sepsis via LPS injection (Shirey et al., 2013; Voss et al., 2016), age (9-10 weeks)- and sex-matched Atx∆ΜΕ/∆ΜΕ mice (n = 9) and Atx+/+ littermates (n = 21) were injected with LPS (i.p. 25 mg/kg). Survival was monitored for up to 16 h, and analyzed by the Kaplan-Meier method (Log-rank P=0.0004).",
    "molecules": "LPS"
  },
  {
    "caption": "(G) Blood samples were collected from Atx∆ΜΕ/∆ΜΕ (n = 7) and Atx+/+ (n = 6) mice 10 h after LPS injection, after which the serum TNFα protein level was measured. The data are shown as mean ± SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a higher(A)",
    "molecules": "DAPI, LPS, Phalloidin"
  },
  {
    "caption": "Confocal laser scanning micrographs of the macrophages from Atx∆ΜΕ/∆ΜΕ mice and Atx+/+ littermates. Cells were incubated with IgG-opsonized latex beads in the absence/presence of LPS (20 ng/mL, 2 h) and stained with Phalloidin (F-actin) and DAPI. Internalized beads were examined under a lower magnification (B) to quantify % phagocytosis by dividing latex bead-positive cell numbers by the total number of DAPI-positive cells (n=11 - 16 per group). The data are analyzed with results accumulated from three independent experiments and shown as mean ± SEM (B). **P < 0.01 (Mann-Whitney U test).",
    "molecules": "DAPI, LPS, Phalloidin"
  },
  {
    "caption": "(C) LPS-stimulated macrophages were co-incubated with IgG-opsonized latex beads for 40 min. Phagocytic cups were visualized by F-actin staining with Phalloidin-iFluor 647. The arrow indicates the phagocytic cup formed in the plasma membrane. Scale bar is 10 μm.",
    "molecules": "iFluor 647, LPS, Phalloidin"
  },
  {
    "caption": "After LPS (20 ng/mL, 4 h) treatment, LPS-induced iNos, Arginase-1 (Arg-1), and Ym-1 mRNA expression were evaluated by semi-quantitative PCR (D).",
    "molecules": "LPS"
  },
  {
    "caption": "With LPS stimulation (20 ng/mL, 8 h), iNOS protein production was examined by immunoblotting analysis (E).",
    "molecules": "LPS"
  },
  {
    "caption": "(A) Confocal live cell imaging of HeLa cells overexpressing GFP-Bax (green) and mitochondria marked with TMRE 100 nM (magenta) show cytosolic GFP-Bax distribution and healthy mitochondria. Lower panels are zoomed images corresponding to the white rectangle in the upper panel and represent the individual and merged channels. Time shows the minutes after apoptosis induction with Staurosporine (STS) 1 µM. Scale bars, 5µm (overview) and 2µm (zooms)(B) HeLa cells shown in A at longer times after STS treatment. Images show sequential GFP-Bax translocation (green), which correlates with mitochondria depolarization (TMRE, magenta). Scale bars, 5 µm(C) HeLa cells overexpressing GFP-Bax mutant (1-2/L-6) (green) and mitochondria marked with TMRE 100 nM (magenta). Images show constitutive GFP-Bax localization to healthy mitochondria without apoptosis induction. Right panels are zoomed images corresponding to the white rectangle in the left panel and represent the individual and overlaid channels. Scale bars, 5µm (overview) and 2 µm (zooms)(D) Standard deviation of the fluorescence intensity of GFP-Bax blue) versus the corrected total cell fluorescence (CTCF) of TMRE (purple) in individual cells (N=4). CTCF= Integrated Density - (Area of selected cell X Mean fluorescence of 3 background readings). Time 0 corresponds to the normalized time when both events cross in each cell. Black line represents the average of the individual cells.",
    "molecules": "Staurosporine, STS"
  },
  {
    "caption": "(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HeLa cells stained with AF647-anti-GFP-nanobodies. Image was acquired on fixed cells 3 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Magnified reconstructed superresolution image corresponding to the white rectangle in A showing the shapes of GFP-Bax WT structures (white arrows). Scale bar, 500 nm.(C) Gallery of typical GFP-Bax WT structures during apoptosis found in all the analyzed cells. Scale bars, 100 nm.(D) Overview (left) and 3 enlarged insets (right) of HeLa cells overexpressing GFP-Bax 1-2/L-6 stained with AF647-anti-GFP-nanobodies. Figure shows reconstructed superresolution images acquired without apoptosis induction. Scale bars, 5 µm (overview) and 500 nm (insets).",
    "molecules": "STS"
  },
  {
    "caption": "(A) Overview of a reconstructed superresolution image of GFP-Bax overexpressing HCT116Bax/Bak -/- cells stained with AF647-anti-GFP-nanobodies Image was acquired on fixed cells 7 h after apoptosis induction with STS. Dotted line shows the cell shape (see Fig EV3). Scale bar, 5 µm.(B) Gallery of typical GFP-Bax WT non-random structures during apoptosis on HCT116Bax/Bak -/- cells. Scale bars, 100 nm.",
    "molecules": "STS"
  },
  {
    "caption": "(C) Comparison of confocal (left) and STED (right) images of GFP-Bax overexpressing HeLa cells 2h after STS treatment. STED reveals a notable increase in resolution. Scale bars, 500 nm.(D) Representative GFP-Bax non-random structures found with STED (lines, arcs, rings, double lines). Scale bars, 100 nm.",
    "molecules": "STS"
  },
  {
    "caption": "(A) Representative TEM pictures of HeLa wild-type cells. Bottom panels show healthy mitochondria. Scale bars, 500nm (overview) and 200 nm (zooms).(B) Representative TEM pictures of apoptotic HeLa wild-type cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms)(C) Representative TEM pictures of apoptotic HeLaGFP-Bax transfected cells 4h after STS treatment (1µM). Black arrowheads point to defects on the mitochondrial outer membrane. Scale bars, 500nm (overview) and 200 nm (zooms).",
    "molecules": "STS"
  },
  {
    "caption": "(A) Left panel shows the individual channels and overlaid images of confocal live cell imaging of HeLa cells overexpressing GFP-Bax (green) and mito-dsRed (magenta) at 0 and 85 minutes after apoptosis induction with STS 1µM. Right panel shows zoomed overlaid images of HeLa cells following GFP-Bax translocation (green) and mitochondrial fragmentation (magenta) during apoptosis induction with STS 1µM. Scale bars, 10 µm (left) and 2 µm (right)(B) Reconstructed dual-color superresolution image of GFP-Bax overexpressing HeLa cells. GFP-Bax was imaged by immunosstaining with AF647-anti-GFP-nanobodies and mitochondria were immunostained with CoxIV (primary) and cf680 (secondary) antibodies. Image was acquired 3h after STS treatment. Left panel shows the cell overview (scale bar, 5 µm), right panel up shows the overview inset (scale bar, 500nm) and right panel down shows a non-random linear GFP-Bax assembly at mitochondria (scale bar, 100nm).(C) Reconstructed dual-color superresolution image of HeLa cells co-overexpressing mMaple-mito and GFP-Bax and stained with AF647-anti-GFP-nanobodies. Image was acquired 3h after STS treatment. Lower panel shows enlarged insets corresponding to the white rectangles in the upper panel. Scale bars, 1 µm (overview) and 100 nm (insets)",
    "molecules": "STS"
  },
  {
    "caption": "(C) 3D AFM topography of a Bax arc (left) and ring (right). Both images reveal a circular dark hole that spans the lipid membrane (green). Bax molecules around the pore rim (magenta and white) protrude 3,97 ± 1,02 nm above the membrane plane, as confirmed by the height profiles shown below each image (corresponding to the white line in the 2D image insets). The topography of the arc structure reveals a pore only partially surrounded by Bax molecules, while lipids alone form the rest of the pore rim. Images are shown in a 42° tilted representation.",
    "molecules": "lipids"
  },
  {
    "caption": "A-C Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight and (B, C) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "liraglutide, RM-493"
  },
  {
    "caption": "A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A, B) energy expenditure. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "liraglutide, RM-493"
  },
  {
    "caption": "A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C) respiratory exchange ratio (RER). Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "liraglutide, RM-493"
  },
  {
    "caption": "A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (D) locomotor activity. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "liraglutide, RM-493"
  },
  {
    "caption": "A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E) cumulative food intake, (F) meal number and (G) meal size. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "food, meal, liraglutide, RM-493"
  },
  {
    "caption": "A, B Treatment-induced changes in hypothalamic gene expression in DIO mice treated for 2 (A) or 5 (B) days with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Compounds were administered by daily subcutaneous injections, and the last injection was provided 2 h prior to tissues sampling. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, §P < 0,001, comparison between treatment and vehicle control. #P < 0.05, ##P < 0.01, ###P < 0.001, comparison to liraglutide. +P < 0.05, comparison to RM-493.",
    "molecules": "liraglutide, RM-493"
  },
  {
    "caption": "A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Fasted blood glucose levels. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "Glucose, liraglutide, RM-493"
  },
  {
    "caption": "A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered).(B, C) glucose tolerance. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "Glucose, glucose, liraglutide, RM-493"
  },
  {
    "caption": "A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) fasted insulin levels,. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "insulin, liraglutide, RM-493"
  },
  {
    "caption": "A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (E, F) glucose-induced insulin secretion. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "glucose, insulin, liraglutide, RM-493"
  },
  {
    "caption": "A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (G) HOMA-IR. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "Glucose, liraglutide, RM-493"
  },
  {
    "caption": "A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (H) insulin sensitivity. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "Glucose, insulin, liraglutide, RM-493"
  },
  {
    "caption": "A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (I) hepatic phosphorylation of AKT (p-AKTSer473) were analyzed. Compounds were administered by daily subcutaneous injections. To assess insulin-stimulated p-AKTSer473, insulin (n = 5) or saline (n = 3) was injected 10 min prior to liver sampling. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "insulin, liraglutide, RM-493"
  },
  {
    "caption": "A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Plasma lipoprotein fractions. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "Cholesterol, lipoprotein, liraglutide, RM-493"
  },
  {
    "caption": "A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (B) liver cholesterol levels. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "Cholesterol, cholesterol, liraglutide, RM-493"
  },
  {
    "caption": "A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (C) expression of genes implicated in hepatic cholesterol and lipoprotein uptake. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "Cholesterol, cholesterol, lipoprotein, liraglutide, RM-493"
  },
  {
    "caption": "A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "bile acid, cholesterol, Cholesterol, liraglutide, RM-493"
  },
  {
    "caption": "A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "liraglutide, RM-493"
  },
  {
    "caption": "A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (B) cumulative food intake. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "food, liraglutide, RM-493"
  },
  {
    "caption": "A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C, D) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "liraglutide, RM-493"
  },
  {
    "caption": "A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E, F) glucose tolerance. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.",
    "molecules": "glucose, liraglutide, RM-493"
  },
  {
    "caption": "Total CoQ levels (CoQ9 + CoQ10) in kidneys (C) and muscle (D) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.",
    "molecules": "Q10H2, ubiquinol-10"
  },
  {
    "caption": "Levels of SQR protein in kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.",
    "molecules": "Q10H2, ubiquinol-10"
  },
  {
    "caption": "Quantification of sulfide levels in cerebrum and kidneys of Coq9+/+, Coq9R239X and Coq9Q95X mice (A).",
    "molecules": "sulfide"
  },
  {
    "caption": "Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9+/+ and Coq9R239X mice (B).",
    "molecules": "hydrogen sulfide"
  },
  {
    "caption": "Total GSH in cytosol and mitochondria of cerebrum of Coq9+/+ and Coq9R239X mice(A).",
    "molecules": "GSH"
  },
  {
    "caption": "Levels of L-Glutamate (L-Glu), N-Acetyl-Glutamate (NacGlu), L-Tryptophan (L-Trp), 5HIAA, N-Acetyl-Tryptophan (NALT), L-Tyrosine (L-Tyr) in cerebrum of Coq9+/+ and Coq9R239X mice (E).",
    "molecules": "5HIAA, L-Glu, L-Glutamate, L-Trp, L-Tryptophan, L-Tyr, L-Tyrosine, N-Acetyl-Tryptophan, N-Acetyl-Glutamate, NacGlu, NALT"
  },
  {
    "caption": "SQR (A) and TST (B) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137.",
    "molecules": "GYY4137"
  },
  {
    "caption": "TST protein level in kidneys (C) and cerebrum (D) of Coq9+/+ mice supplemented with the H2S donor GYY4137.",
    "molecules": "GYY4137"
  },
  {
    "caption": "Levels of neurotransmitters in cerebrum of Coq9+/+ mice supplemented with the H2S donor GYY4137 (E).",
    "molecules": "GYY4137"
  },
  {
    "caption": "B, shRNA PTPN3-mediated reduction of endogenous TβRI level is restored by MG132. HaCaT cells expressing shRNA PTPN3-1, shRNA PTPN3-2 or control shRNA were treated with MG132 (20 μM) for 4 h. Cell lysates were analyzed by Western blotting with anti-TβRI, anti-PTPN3 and anti-GAPDH antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "E, PTPN3 blocks Smurf2-mediated ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids, and treated with MG132 (20 μM, 4 h) before harvest. TβRI IP was performed with the anti-HA antibody, and TβRI ubiquitination was determined by Western blotting with the anti-Myc antibody. Protein levels were examined by Western blotting with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "E, Knockdown of endogenous PTPN3 promotes the Smad7-TβRI association. HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 µM) for 4 h before harvested. SFB-Smad7 proteins were retrieved with the Streptavidin pulldown. Levels of TβRI and other proteins were examined by Western blotting with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "F, PTPN3 and PTPN3 (D811A) abolish the interaction between TβRI and Smurf2 in HEK293T cells. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h, followed by IP and Western blotting.",
    "molecules": "MG132"
  },
  {
    "caption": "D, PTPN3 (L232R) does not block the interaction between TβRI and Smurf2. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h and analyzed by IP-Western blotting.",
    "molecules": "MG132"
  },
  {
    "caption": "E, Ectopic expression of PTPN3 wildtype and PTPN3 (D811A), but not PTPN3 (L232R), completely abolishes the Smad7-TβRI interaction. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 µM) for 4 h, followed by IP and Western blotting.",
    "molecules": "MG132"
  },
  {
    "caption": "C. McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice. Arrows show representative positive cells for McIdas (green) and FoxJ1 (red) in the GemC1WT/WT mice. DNA was stained with Draq-5 (blue). Scale bars, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "D. Transverse sections from P0 GemC1WT/WT (upper) or GemC1KO/KO (lower) trachea, immunostained with antibodies against Keratin 5 (Krt5, basal cell marker, green), and acetylated y-tubulin (ACT, cilia marker, red). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section on the left. Higher magnification images are shown on the right. Scale bars, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "E. Transverse sections from P0 GemC1WT/WT (left two images) or GemC1KO/KO (right two images) trachea, immunostained with antibodies against Muc5ac (marker for mucus-expressing cells, red) and E-cadherin (marker for cell boundaries, green). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section (left). Higher magnifications from the same section are shown on the right. Scale bars, 10 µm.",
    "molecules": "DNA, mucus"
  },
  {
    "caption": "Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4.",
    "molecules": "SSP, staurosporine"
  },
  {
    "caption": "Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively. Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n.",
    "molecules": "NBQX, AMPA, MK801, Glut, Glutamate, glutamate, HC, NMDA"
  },
  {
    "caption": "Filter trap binding assays show that ISR proteins are enhanced by all stressors. Significant increases following cellular stress treatments are seen in the ratio of phospho-eif2α to total eif2α as well as phospho-PERK and ATF4 levels compared to untreated controls. Changes in ISR proteins are normalized to GAPDH and shown relative to non-stressed or DMSO-only treated cells (CTRL) depending on the vehicle compounds were dissolved in. n = 4.",
    "molecules": "DMSO"
  },
  {
    "caption": "Fluorescent intensity measurements show the levels of two ISR-related proteins, PERK and relative phospho-eif2α (phospho-eif2α / total eif2α), are increased following different excitotoxic stress treatments in cortical neurons. Representative images indicate fluorescent intensity differences for phospho-eif2α and phospho-PERK levels 24 h post treatment with different excitotoxic stressor paradigms. Blocking of AMPA and NMDA receptors using known antagonists significantly reduces ISR-related protein levels, which parallels reduced DPR levels (Figure 3B). Changes in ISR proteins are relative to non-stressed cells (CTRL).",
    "molecules": "AMPA, NMDA"
  },
  {
    "caption": "Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when stressor challenged NSC34 cells were treated with compounds inhibiting ISR-related or translation-targeting pathways. Dibenzoylmethane (1,3DBM; 20 µM), which inhibits eif2α-P activity, had the most significant decreases in DPR production. Trazodone (Traz; 20 µM) showed similar significant reduction in DPR levels as the PERK inhibitor, GSK2606414 (500 nM).",
    "molecules": "1,3DBM, Dibenzoylmethane, GSK2606414, Traz, Trazodone"
  },
  {
    "caption": "Relative fluorescent intensity quantification shows a significant decrease in C9 DPR reporter levels when glutamate stressed primary cortical neurons were treated with the compounds Trazodone (Traz; 20 µM) or Dibenzoylmethane (1,3DBM; 20 µM). Similar to the treatments in NSC34, 1,3DBM shows the most significant decrease in DPR production.",
    "molecules": "1,3DBM, Dibenzoylmethane, glutamate, Traz, Trazodone"
  },
  {
    "caption": "(B) The concentration-effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone-based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM. (C) Concentration-dependent increase in PARP1-DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD+, and the ΔFA values represent the changes in PARP1-DNA binding. Data from four biological replicates are presented as mean ± SEM. (",
    "molecules": "thioparib, DNA, NAD, olaparib, talazoparib"
  },
  {
    "caption": "(F) and (G) Effects of thioparib on BRCA1-deficient MDA-MB-436 (F) and BRCA2-deficient Capan-1 (G) xenografts. Mice bearing MDA-MB-436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice bearing Capan-1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks (n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors.",
    "molecules": "thioparib, olaparib, talazoparib"
  },
  {
    "caption": "(C) The IC50 values of thioparib in six acquired PARPi-resistant cell lines. Numbers 1-6 represent the PARPi-resistant Capan-1/OP, Capan-1/TP, MDA-MB-436/OP, MDA-MB-436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC50 values are expressed as the mean ± SD from three separate experiments.",
    "molecules": "thioparib, OP, TP"
  },
  {
    "caption": "(E) Efficacy of thioparib in the PDX model BR-05-0028 (BRCA1-deficient). Mice bearing BR-05-0028 tumors derived from a patient with breast cancer (n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. ***P < 0.0001.",
    "molecules": "thioparib, olaparib"
  },
  {
    "caption": "(B) Concentration-dependent effects of thioparib on the viability of malignant hematologic cells. Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay. Data from three independent experiments were presented as mean ± SEM.",
    "molecules": "thioparib"
  },
  {
    "caption": "(D) Kaplan-Meier survival curve of M-NSG mice transplanted with JeKo-1 cells treated with 10 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (n = 10). Statistical analysis was performed by log-rank (Mantel-Cox) test. ***P < 0.0001, ns: P = 0.3526.",
    "molecules": "thioparib, talazoparib"
  },
  {
    "caption": "(B) Western blotting of γH2AX and phosphor-RPA32 in cells exposed to thioparib, talazoparib, or olaparib for 12 h.",
    "molecules": "thioparib, olaparib, talazoparib"
  },
  {
    "caption": "(E) and (F). Thioparib caused changes in apoptosis-related proteins in JeKo-1 or THP-1 cells, as determined by western blotting. Cells were exposed to thioparib, talazoparib, olaparib, or Cpd 391 for 24 h and then subjected to western blotting.",
    "molecules": "Cpd 391, Thioparib, thioparib, olaparib, talazoparib"
  },
  {
    "caption": "(G) and (H). Depletion of PARP1 in HT-29 and Capan-1/TP cells resulted in a decrease in DNA damage and thioparib resistance. The upper panel shows the changes in γH2AX and phospho-RPA32 in HT-29 PARP1 knockout (KO) clones (G) or Capan-1/TP PARP1 KO clones (H) and their parental cells after exposure to thioparib for 24 h. The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells. Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments. Data are presented as the mean ± SD. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391.",
    "molecules": "Cpd 391, thioparib, Thioparib, Olaparib, OP, Talazoparib, TP"
  },
  {
    "caption": "(A) HR repair assays in U2OS-DR-GFP cells treated with increasing concentrations of thioparib. Cells were incubated with the indicated drugs at the time of I-SceI introduction for 48 h. GFP-positive cells were analyzed after I-SceI transfection for 72 h by flow cytometry. The ATR inhibitor VE-821 was used as a positive control. Three independent experiments were performed and data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, and all groups were compared with control. ***P < 0.0001, *P = 0.0205, 0.0101 (from left to right).",
    "molecules": "thioparib, VE-821"
  },
  {
    "caption": "(H) HR repair assay. U2OS-DR-GFP (WT) and PARP1 KO cells (KO1 and KO2) were treated with the indicated drugs for 48 h and collected after 72 h of I-SceI transfection as in (A). VE-821 served as a positive control. PARP1 knockout efficiency was evaluated by western blotting. Data from three biological replicates are presented as mean ± SD. Statistical analysis was performed by one-way ANOVA, and each group was compared with the control. ***P < 0.0001, *P = 0.0143, 0.0483, ns: P = 0.3061, 0.8269, 0.9997, 0.3906 (from left to right).",
    "molecules": "VE-821"
  },
  {
    "caption": "(C) Effects of thioparib on CXCL9, CXCL10, and IL15 mRNA in JeKo-1 cells. Data from three independent experiments are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Left panel: *P = 0.0367, **P = 0.0011; median panel: ***P = 0.0002, < 0.0001, < 0.0001; right panel: ***P = 0.0005, 0.0009, 0.0001 (from left to right).",
    "molecules": "thioparib"
  },
  {
    "caption": "(D) The protein levels of p-STAT1, p-TBK1, γH2AX, and phospho-RPA32 in HT-29 parent, PARP1−/− (#KO1), and PARP7−/− cell lines after 24 or 48 h thioparib treatment.",
    "molecules": "thioparib"
  },
  {
    "caption": "(F) Western blot analysis of p-STAT1, p-TBK1, and γH2AX in HT-29 parent, STING−/−, and TBK1−/−cells after 24 or 48 h thioparib treatment.",
    "molecules": "thioparib"
  },
  {
    "caption": "(A-D) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Flow cytometry results for annexin V single positive (A and C) and annexin V and zombie dye double positive (B and D) cells. Percentage of positive cells are depicted at 12 hpi or 24hpi.",
    "molecules": "zombie"
  },
  {
    "caption": "(A) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of the full-length gasdermin D. Equal loading was confirmed by beta actin. One representative blot of five independent repeats is shown. (B) Quantification of band intensity for the gasdermin D full-length isoform. The mean ± standard deviation five independent experiments is shown. Statistical analysis was performed by the paired two‑tailed Student's t test., The asterisk (*) marks a significant difference when compared to the mock group ** p < 0.01. ",
    "molecules": "nig, nigericin"
  },
  {
    "caption": "(C) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of cleaved gasdermin D N-terminal and C-terminal isoforms by immunoblot in supernatants; one representative experiment of three independent repeats is shown. (D) Quantification of band intensity for N-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. (E) Quantification of band intensity for C-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. ",
    "molecules": "nig, nigericin"
  },
  {
    "caption": "(F) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Pro-caspase-1 and active caspase-1 were detected by immunoblot in cell lysates (cell, upper panels) and supernatants (sup, lower panels) from the same experiment. One representative experiment of three independent repeats is shown. (G) Quantification of band intensity for active caspase-1 band. Five independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test. * p < 0.05. (H) Levels of total pro-caspase-1 were quantified on pellets and respective supernatants. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test. ",
    "molecules": "nig, nigericin"
  },
  {
    "caption": "B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.",
    "molecules": "RA"
  },
  {
    "caption": "D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).",
    "molecules": "RA"
  },
  {
    "caption": "D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).",
    "molecules": "RA"
  },
  {
    "caption": "Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.",
    "molecules": "miRNA inhibitors"
  },
  {
    "caption": "Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.",
    "molecules": "SSEA1"
  },
  {
    "caption": "E Representative images of the proximity ligation assay (PLA) using Cos7  cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI  images. Arrowheads, the plasma membrane. Scale bar, 10mm. F The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.",
    "molecules": "DAPI"
  },
  {
    "caption": "A The  nickel-pull down assay using  myc/HIS-tagged ENTREP along with HA-tagged ubiquitin. Arrows indicate  HA-ubiquitin-incorporating ENTREP. Arrowhead indicates ENTREP without ubiquitination. Data shown are representative of three independent experiments.",
    "molecules": "nickel, ubiquitin"
  },
  {
    "caption": "B Representative images of the proximity ligation assay (PLA) using Cos7  cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Scale bar, 10mm. C The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.",
    "molecules": "DAPI"
  },
  {
    "caption": "F The nickel-pull down assay using indicated vectors along with HISx6-tagged wild type ubiquitin vector. HA-tagged DsRed was used as a control for HA-CXCR4DD-DsRed. Arrowhead,  FLAG-tagged ITCH (wild or C830A); arrow, FLAG-ENTREP  wild cyt. Data shown are representative of at least two independent experiments. G Co-localization of ENTREP  and CXCR4 in the  endosome.  Cos7  cells were transfected with ENTREP-FLAG, CXCR4-DsRed and Halo-ITCH wild vectors and treated with  CXCL12. ENTREP-FLAG and CXCR4-DsRed co-localized in the RAB7-positive  endosome. Inserts, the high-magnification images of the marked area indicating the  colocalizaion of ENTREP-FLAG, CXCR4-DsRed and RAB7. Scale bar 10mm. The image is representative of at least two independent experiments.",
    "molecules": "nickel, ubiquitin"
  },
  {
    "caption": "A Dot plot presentation of the chemotaxis  analyses using the Boyden chamber. CXCL12 (+) and (-) indicate the presence and absence of CXCL12  as an attractant in the bottom chambers. ENTREP-knockout enhanced the chemotaxis toward CXCL12, which was blocked by pre-treatment with AMD3100 (upper panel). The doxycyclin (DOX)-induced  ENTREP expression suppressed the chemotaxis  of mouse 4T1-Luc  cells toward  CXCL12 (lower panel). Ratio of  migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non-treated cells (n=7, upper panel, and n=9, lower panel, from at least two independent experiments, respectively). P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. Insert, the  immunoblot  analysis of the DOX-induced ENTREP expression in 4T1-Luc cells. NS, not significant.",
    "molecules": "DOX, doxycyclin, AMD3100"
  },
  {
    "caption": "B Dot plot presentation of  mammosphere assay (left panel) and the images of  mammosphere (right panel). Each mark in dot plot presentation indicates the number of  mammosphere  from 3,000 cells per well in an ultra-low attachment plate from two independent experiments. To estimate the effect of CXCL12/CXCR4 pathway, 100 ng/ml of  CXCL12  and 12 μM of  AMD3100 were applied as indicated. For statistical analysis, variance was assessed using two-way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison. NS, not significant. Scale bar in the images of mammosphere 100 mm.",
    "molecules": "AMD3100"
  },
  {
    "caption": "(D) Serum lactate levels of WTFlx and Ndufs3-smKO animals at different ages. Ndufs3 smKO male mice (filled black squares) showed increased levels compared to matched WTFlx males (empty black squares). Error bars represent SEM.",
    "molecules": "lactate"
  },
  {
    "caption": "(G) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1/18S. Inset shows the appearance of the quadriceps, reflecting normalization of mitochondrial levels after replacement of NDUFS3.",
    "molecules": "DNA"
  },
  {
    "caption": "(I) Plasma lactate levels were determined as described in methods. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test.",
    "molecules": "lactate"
  },
  {
    "caption": "(H) Total mtDNA levels were determined by qPCR. MtDNA levels in KO-NDUFS3 mice were decreased when compared to KO-GFP mice and not different from the levels in WTFlx mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.",
    "molecules": "DNA"
  },
  {
    "caption": "(I) The serum lactate levels were similar to WTFlx in KO-NDUFS3 samples and significantly decreased when compared to KO-GFP levels. The individual KO-NDUFS3 animals are color coded. Green triangles represent the animals injected with rAAV9-eGFP whereas black triangles represent animals which did not receive an rAAV9-eGFP injection. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.",
    "molecules": "lactate"
  },
  {
    "caption": "(A-F) TNF-α, Cxcl1  and Cxcl2 (A, C, and E respectively) were measured at 24 h after stimulation of  bone-marrow-derived neutrophils (5 x 105 cells) with increasing concentrations of bacterial nucleic acids (1, 5, 10 and 20 µg of DNA + RNA in equal proportions), HK-bacteria (1, 5, 10 or 20 μg/ml) or live bacteria (MOIs of 2, 5, 10 and 20). LPS (1, 10, 100 and 1000 ng/ml) was used as a positive control stimulus. (B, D and F), Effect of FPR antagonist Boc-2 on  GBS-induced TNF-α, Cxcl1  and Cxcl2 production, respectively. Bone-marrow-derived neutrophils were exposed to  Boc-2  (50 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.",
    "molecules": "DNA, LPS, RNA, Boc-2"
  },
  {
    "caption": "Bone-marrow-derived neutrophils were exposed to Boc-2 (10 µM) or WRW4 (1, 5 and 10 µM) for 1 hour and then stimulated with  HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.",
    "molecules": "Boc-2, WRW4"
  },
  {
    "caption": "(A-F) Cxcl1 and Cxcl2 concentrations in 24-h culture supernatants of  neutrophils (A and B), CSF-polarized (C and D) or GM-CSF-polarized (E and F) BMDMs (ΜΦ) from WT or Fpr-deficient mice stimulated with live  GBS (MOI of 2, 5, and 10) or HK-GBS  (10 µg/ml). LPS (100 ng/ml) was used as a positive control stimulus. Means + SD of data from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs WT  mice, determined by unpaired t-test.",
    "molecules": "LPS"
  },
  {
    "caption": "(C and D), Cxcl2 release in neutrophil  cultures stimulated with combinations of HK-bacteria, GBS  fMet peptides or TLR  agonists. Neutrophils were exposed to a 50 µM concentration of Fpr agonists (fMIFL, WKYMVM  or  fPep8  and  fPep10) for 1 h before the addition of the indicated TLR  agonists, which included HK-GBS (10 μg/ml), LPS  (1 µg/ml) and RNA (1 µg /ml). In some cases, neutrophils were exposed to graded doses (10, 20 and 50 µM) of the indicated  Fpr  agonists alone. Data information: Data are expressed as means + SD from three independent experiments, each conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.",
    "molecules": "fMIFL, fPep10, fPep8, LPS, RNA, WKYMVM"
  },
  {
    "caption": "(A), Effect of actinonin on Cxcl2  production by neutrophils. Neutrophils were stimulated with live  GBS  (MOI 5) in the presence or absence of the  formyl peptidase inhibitor actinonin (32µg/ml). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.",
    "molecules": "actinonin"
  },
  {
    "caption": "(D), Effect of treatment with ROS  scavengers/inhibitors on Cxcl2  production. Neutrophils were treated with the singlet oxygen scavenger MnTBAP (100 μM), the hydrogen peroxide scavenger  DMTU (10 mM), the NADPH oxidase inhibitor DPI (20 μM) or the ASK1  inhibitor GS-444217 (1 μM) for 1 h before the addition of live  GBS   (MOI of 5). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.",
    "molecules": "GS-444217, DPI, hydrogen peroxide, MnTBAP, DMTU, ROS, singlet oxygen"
  },
  {
    "caption": "(C), Effect of p38MAPK inhibition on the activation of AP-1   family members in neutrophils stimulated with live  GBS. Neutrophils were pretreated with SB202190  (5μM), stimulated for 90min with live GBS (MOI 5), lysed and assayed for  AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001, as determined by unpaired t-test.",
    "molecules": "SB202190"
  },
  {
    "caption": " B. MCF-7 cells were transfected with a control vector or FLAG-TRIM46 for 40 h and treated with DMSO or MG132 (2μM) for 10h. Cellular lysates were then collected for western blotting with antibodies against the indicated proteins. ",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": " F. MCF-7 cells were transfected with empty vector or FLAG-TRIM46 for forty-eight hours and then treated with 50 μg/mL cycloheximide (CHX) for the indicated hours. DMSO was added to cells 10 h before cellular proteins were collected for western blotting (left). On the right panel, quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1. G. MCF-7 cells were treated as described in F, except that MG132 (10 μM) was added to cells 10 h before cellular proteins were collected for western blotting (left). Quantitation was performed on the right panel as in F. H. MCF-7 cells were treated with control siRNA or TRIM46 siRNA. Forty-eight hours after transfection, cells were treated with 50 μg/mL CHX for the indicated hours before cellular proteins were extracted for western blotting analysis (left). Quantitation was performed on the right panel as in F. ",
    "molecules": "CHX, cycloheximide, DMSO, MG132"
  },
  {
    "caption": "  In vivo ubiquitination assays performed in MCF-7 cells. (B) Stable shCTR- or shTRIM46-expressing MCF7 cells were transfected with His-Ub for 40 h. Cells were then treated with MG132 (10 μM) for 12 h before Ni-NTA bead precipitation followed by IB with anti-HDAC1. ",
    "molecules": "MG132, Ni, NTA, Ub"
  },
  {
    "caption": " (C) and (D) MCF-7 cells were co-transfected with the indicated plasmids. Forty-eight hours after transfection, cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni-NTA bead precipitation followed by IB with anti-HDAC1. ",
    "molecules": "MG132, Ni, NTA"
  },
  {
    "caption": " B. EdU assays were performed in MCF-7 cells treated with control siRNA, TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA for 72 h, or MCF-10A cells treated with vector, FLAG-TRIM46 or FLAG-TRIM46 and FLAG-HDAC1 for 48 h. Representative images from FACS analysis are shown on the left panel, the percentage numbers of cells in S phase was quantified by FlowJo software on the right panel. ",
    "molecules": "EdU"
  },
  {
    "caption": " G. Accumulation of γH2AX in cells depleted of TRIM46 or HDAC1 after cisplatin stimulation. MCF-7 cells were treated with 4 μM cisplatin for 8 h before harvesting. Cell lysates were analyzed by western blotting. ",
    "molecules": "cisplatin"
  },
  {
    "caption": "D. Control MCF-7 cells or MCF-7 cells depleted with TRIM46 were treated with different concentrations of cisplatin (cis), VP16, or adriamycin (ADM) for 72 h, and the proportions of viable cells were examined. E. Control MCF-10A cells or MCF-10A cells overexpressed TRIM46 were treated with different concentrations of cisplatin, VP16, or adriamycin for 72 h, and the proportions of viable cells were examined.",
    "molecules": "cis, cisplatin, ADM, adriamycin, VP16"
  },
  {
    "caption": "5 ng/ml TGF-β and DMSO were applied to stimulate SW-1116 -Vector or SW-1116 -LOC113230 cells for 24 and 48 hrs, respectively. Cell migration was detected by transwell assays. Scale bar represents 100 μM.",
    "molecules": "DMSO"
  },
  {
    "caption": "SW-1116 cells bearing stable expression or knockdown of LOC113230 were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub), ASS1 and α-Flag antibodies, respectively.",
    "molecules": "MG132, Ub, ubiquitin"
  },
  {
    "caption": "SW-1116 cells stably expressing ASS1WT, or ASS1 K234R and LOC113230 or not were treated with MG132 (5 μM) for 12 hrs. Cell lysates were immunoprecipitated (IP) assays were performed with α-Flag antibody. The co-eluted proteins were detected by western blot assays with ubiquitin (Ub) and α-Flag antibodies, respectively.",
    "molecules": "MG132, Ub, ubiquitin"
  },
  {
    "caption": "Arginine production from each group was determined by a commercially available Arginine assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs. Urea production for each group was determined by a commercially available urea assay kit. The cells were stimulated with 5 ng/ml TGF-β or DMSO for 24 hrs.",
    "molecules": "Arginine, DMSO, Urea, urea"
  },
  {
    "caption": "The interaction of endogenous TRAF2, ASS1 and LRPPRC was detected in SW-1116 cells by co-IP assays. GST-TRAF2, HA-LRPPRC, His-ASS1 and Biotin-LOC113230 were expressed in E. coli BL21 cells, and were used for in vitro binding assays.",
    "molecules": "Biotin"
  },
  {
    "caption": "SW-1116-LOC113230 cells transiently transfected with siRNAs to LRPPRC or TRAF2 or scrambled control (SiRNA-NC) were treated with MG132 (5 µM) for 12 hrs. Cell lysates were immunoprecipitated with α-Flag antibody. The precipitate proteins and input proteins were analyzed by immunoblotting. NC: Negative control.",
    "molecules": "MG132"
  },
  {
    "caption": "SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data.",
    "molecules": "resveratrol, spermidine"
  },
  {
    "caption": "SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1.",
    "molecules": "bafilomycin A1, spermidine"
  },
  {
    "caption": "(D-F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.",
    "molecules": "EX527, resveratrol, spermidine"
  },
  {
    "caption": "The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2. (A-E) EGFP-Atg8p was ectopically expressed in wild-type (WT) or Δsir2 S. cerevisiae undergoing chronological aging on small synthetic 2% glucose media with or without (Co, control) supplementation of 4-mM spermidine (Spd). (A) Representative images. EGFP-Atg8p localization (bottom) was visualized by fluorescence microscopy. Yeast cells undergoing autophagy (in which EGFP-Atg8p exhibits a prominent vacuolar localization) are indicated by arrows. Yeast morphology was monitored by differential interference contrast (DIC; top).",
    "molecules": "glucose, spermidine"
  },
  {
    "caption": "(B) Representative immunoblots against EGFP. Free EGFP indicates the vacuolar degradation of EGFP-Atg8p fusion, thereby representing the autophagic flux. Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine-mediated autophagy induction.",
    "molecules": "spermidine"
  },
  {
    "caption": "(E) Quantification of reactive oxygen species. Bars indicate the percentages of cells exhibiting the reactive oxygen species-mediated conversion of dihydroethidine (DHE) into ethidium (Eth; n = 4). Data represent means ± SEM; *, P < 0.001 as compared with untreated cells of the same genotype. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RFU, relative fluorescence unit.",
    "molecules": "dihydroethidine, ethidium, reactive oxygen species"
  },
  {
    "caption": "The life-extending and autophagy-inducing effects of spermidine in C. elegans are not mediated by Sir2. (A) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity. Two representative pictures of wild-type (WT) and sir-2.1 embryos untreated (Co, control) or treated with 0.2-mM spermidine (Spd) supplementation of food are shown. (B) Quantification of autophagic activity through the measurement of DsRed::LGG-1 pixel intensity from images of WT animals shown in A. Data represent means ± SEM (n = 3) with ≥25 images processed for each trial.",
    "molecules": "food, spermidine"
  },
  {
    "caption": "C) Survival of WT C. elegans during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.005). (D) Survival of sir-2.1 C. elegans (ok434 phenotype) during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.01). P-values were calculated using the log-rank test as described in Materials and methods.",
    "molecules": "food, spermidine"
  },
  {
    "caption": "Convergent alterations in the phosphoproteome status after resveratrol and/or spermidine treatment. (A-C) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle (Co, control), 100-µM resveratrol (Resv), and 100-µM spermidine (Spd), alone or in combination (Resv + Spd). (A) Representative phosphoprotein arrays are shown.",
    "molecules": "resveratrol, spermidine"
  },
  {
    "caption": "(C) Representative immunoblots of selected kinases whose phosphorylation status was unaffected (PRKAA1, RPS6KB1, and acetyl-CoA carboxylase a [ACACA]) or affected by resveratrol or/and spermidine treatment (PTKB, AKT1, MAPK8, and CDKN1B), validating phosphoprotein array data.",
    "molecules": "resveratrol, spermidine"
  },
  {
    "caption": "(D) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle or the indicated dose of resveratrol or spermidine, alone or in combination, for 2 h. Quantitative data. Bars depict the percentages (means ± SD; n = 3; *, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.",
    "molecules": "resveratrol, spermidine"
  },
  {
    "caption": "Convergent acetylproteome modification after resveratrol or spermidine treatment. (A-C) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes. Cells were treated with 100-µM resveratrol (Resv) or spermidine (Spd) for 2 h, fractioned into cytoplasmic, nuclear, and mitochondrial extracts, processed for acetyl lysine peptide enrichment, and analyzed by MS. (A) Hierarchical clustering of drug-specific organellar distributions of all acetylated sites quantified in at least one fraction. Fold changes are calculated relative to untreated cells. Only sites regulated >1.5-fold were included in statistical analyses. (B) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way. n = 560.",
    "molecules": "peptides, resveratrol, spermidine"
  },
  {
    "caption": "Significant motifs among sites undergoing acetylation. (A) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Consensus acetylation motifs identified upon resveratrol (left) or spermidine (right) treatment are depicted using the MotifX algorithm (Schwartz and Gygi, 2005). Among sites that were hyperacetylated in response to both agents, the K(F/Y) motif is significantly enriched when tested against the whole proteome (P < 0.00001). (C) When testing against the largest acetylation site dataset from Choudhary et al. (2009) (acetylation background dataset [ABD]), the SxK motif is significant (P < 0.0001) for sites whose acetylation increased upon spermidine treatment. No general consensus motifs were found for sites whose acetylation increased in response to both agents.",
    "molecules": "resveratrol, spermidine"
  },
  {
    "caption": "Significant motifs among sites undergoing deacetylation. (A) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Among sites that were hypoacetylated in response to both agents, the KP motif is significantly enriched when tested against ABD (P < 0.001). (C and D) When tested against the ABD (C) or the whole proteome (D), the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment (P < 0.0001). No general consensus motifs were found for sites whose acetylation decreased in response to both agents.",
    "molecules": "resveratrol, spermidine"
  },
  {
    "caption": "Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3-transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac).",
    "molecules": "resveratrol, rapamycin, spermidine"
  },
  {
    "caption": "Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo. (A) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle (Co, control) or the indicated dose of resveratrol (Resv) or spermidine (Spd), alone or in combination, for 2 h. (top) Quantitative data. Bars depict the percentages (means ± SD; n = 3; **, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (bottom) Representative immunoblots showing endogenous LC3 lipidation.",
    "molecules": "resveratrol, spermidine"
  },
  {
    "caption": "B) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 (BafA1). (C-E) Transgenic C57BL/6 mice expressing a GFP-LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations. 3 h later, mice were killed, and tissues were processed for immunofluorescence microscopy determinations of GFP-LC3vac.",
    "molecules": "bafilomycin A1, resveratrol, spermidine"
  },
  {
    "caption": "D, E Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment (CM from PaSC, or from CAF, or from CAF ± SOM230, SOM230) as a percentage of the respective gemcitabine-untreated cells (= 100%) (n = 4; from left to right: **P = 0.007, ##P = 0.006 in D; *P = 0.032, #P = 0.041, #P = 0.047 in E).",
    "molecules": "gemcitabine, SOM230"
  },
  {
    "caption": "F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti-cleaved caspase-3 or anti-PARP antibody (representative of n = 3).",
    "molecules": "gemcitabine"
  },
  {
    "caption": "Immunoblotting of protein extracts from PaSCs or CAFs treated (+) or not with SOM230 (10−7 M) for 48 h, using the anti-puromycin antibody (representative of n = 3).",
    "molecules": "SOM230, puromycin"
  },
  {
    "caption": "Polysomes profiles of CAFs treated or not with SOM230 for 48 h (representative of n = 3).",
    "molecules": "SOM230"
  },
  {
    "caption": "Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from PaSCs or CAFs treated (+) with SOM230 for 30 min (representative of n = 3).",
    "molecules": "SOM230"
  },
  {
    "caption": "Immunoblotting of protein extracts from CAFs treated (+) or not with SOM230 (10−7 M) for the indicated times, using anti-P-Akt, anti-P-S6 or anti-4E-BP1 antibody (representative of n = 3).",
    "molecules": "SOM230"
  },
  {
    "caption": "Protein concentration in untreated or SOM230-treated CAF-CM or extracts normalized per 1 × 106 cells. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; from left to right: ##P = 0.008, ##P = 0.007).",
    "molecules": "SOM230"
  },
  {
    "caption": "Immunoblotting of equal amounts of protein from siCTR- or si4E-BP1-transfected CAFs treated (+) or not with SOM230 for 48 h, using the anti-puromycin antibody (representative of n = 3).",
    "molecules": "SOM230, puromycin"
  },
  {
    "caption": "Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with the siCTR or si4E-BP-1. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; **P = 0.003, §§P = 0.002).",
    "molecules": "gemcitabine, SOM230"
  },
  {
    "caption": "Caspase-3 and PARP cleavage induced by gemcitabine in Panc-1 was evaluated by Western blot using the respective antibodies (representative of n = 3). Arrow indicates cleaved forms of PARP.",
    "molecules": "gemcitabine"
  },
  {
    "caption": "H, I Immunoblotting of protein extracts from siCTR- or sisst1-transfected CAFs treated (+) or not with SOM230 for 48 h (sst1, puromycin and GAPDH as loading control, H) or for 30 min (P-Akt, P-S6, 4E-BP1 and GAPDH as loading control, I) (representative of n = 3).",
    "molecules": "SOM230, puromycin"
  },
  {
    "caption": "J Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with siCTR or si-sst1. Results (mean ± SD) are presented as the percentage of the untreated cells (= 100%) (n = 3; **P = 0.003, §§P = 0.002).",
    "molecules": "gemcitabine, SOM230"
  },
  {
    "caption": "A-D Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs pre-treated (+) or not overnight with PTX (100 ng/ml) (A), gallein (10 μM) (B), NSC87877 (10 μM) (C), Src inhibitor (5 μM) (D), treated (+) or not with SOM230 (10−7 M) for 30 min (representative of n = 3).",
    "molecules": "gallein, NSC87877, SOM230"
  },
  {
    "caption": "E, F Immunoblotting using anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAF siCTR- or siSHP2- (E) or siPTPε- (F) transfected CAF treated or not with SOM230 for 30 min (representative of n = 3).",
    "molecules": "SOM230"
  },
  {
    "caption": "G, H Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs treated with the indicated molecules (PDGF receptor inhibitor, 5-10 μM; Jak1/2 inhibitor ruxolitinib, 5 or 10 μM; EGFR inhibitor, 150 or 300 nM and recombinant PDGF, 5 μg/ml) (representative of n = 3).",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "A-C MIA PaCa-2-GLuc cells were injected with or without CAFs into the pancreas of nude mice. Mice were treated with each indicated treatment (SOM230-LAR at day 7 and gemcitabine at days 7, 10, 14 and 17), and the plasmatic luciferase activity was measured (mean ± SD) at days 7, 14 and 21 (n = 5) (P = 0.003 for gemcitabine + SOM230LAR versus untreated) (A). Tumours were excised, photographed (scale bars are 1 cm) (B), weighted (P = 0.0008) (C) and paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (E).",
    "molecules": "gemcitabine, SOM230"
  },
  {
    "caption": "D Human tumours were subcutaneously xenografted in nude mice and tumour volumes (mean ± SD) measured (at day 37, P = 0.0009 for gemcitabine + SOM230LAR versus untreated and P = 0.021 for gemcitabine + SOM230LAR versus gemcitabine). Mice were treated with the indicated treatments (SOM230-LAR at days 3 and 31, and gemcitabine at day 3 and twice a week thereafter).",
    "molecules": "gemcitabine, SOM230"
  },
  {
    "caption": "A Membrane antibody array assay using CM from SOM230-treated or not CAFs (representative of n = 3). Controls are circled in blue dashed line and IL-6 in red square.",
    "molecules": "SOM230"
  },
  {
    "caption": "B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).",
    "molecules": "SOM230"
  },
  {
    "caption": "B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).",
    "molecules": "SOM230"
  },
  {
    "caption": "I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).",
    "molecules": "SOM230"
  },
  {
    "caption": "A-C Immunohistofluorescence using an anti-α-SMA or anti-IL-6 antibody in paraffin-embedded sections from MIA PaCa-2-Gluc and CAF cells orthotopic co-xenografted tumours from Fig5A (A) or from human tumour subcutaneously xenografted from Fig5D (B) in nude mice treated or not with gemcitabine + SOM230 (representative of five different tumours). Representative co-localization of α-SMA with IL-6 in human PDAC samples (C) (n = 15). Scale bar = 100 μm.",
    "molecules": "gemcitabine, SOM230"
  },
  {
    "caption": "piRNAs derived from HMR_-L1s are enriched in adenine at their 10th nucleotide. HMR_-L1s generating more than 40 unique piRNAs were analyzed (678 HMR_-L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_-L1. See A for the explanation of box plots.",
    "molecules": "adenine, nucleotide"
  },
  {
    "caption": "DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 DMR in E12.5 whole embryos is shown. E12.5 whole embryos were obtained by the crosses shown in the panel. Two independent bisulfite-treated DNA sample were examined for Homo13 embryo.",
    "molecules": "DNA"
  },
  {
    "caption": "e. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted and averaged within these fields. Cell counts as indicated.",
    "molecules": "DAB"
  },
  {
    "caption": "BES1 accumulation is independent of brassinosteroid levels. Six day-old 35S::BES-GFP seedlings were treated overnight with 100 nM epi-brassinolide (BL) or 0.5 µM brassinazole (BRZ). Material was collected at the indicated time points and analyzed by western blot hybridization with anti-GFP and -RPT5 antibodies",
    "molecules": "BL, epi-brassinolide, brassinazole, BRZ, brassinosteroid"
  },
  {
    "caption": "Col-0 (black), PIF4-OX (red) and pifq (blue) seedlings were grown for 6 days under short days and samples harvested at the indicated time points. Total RNA was used for qRT-PCR quantification of PIF+BES-UP and the BR biosynthetic BES-DOWN transcripts, using PP2A as endogenous control. Error bars represent SD of three technical replicates. Dark periods are shown in grey. Relative expression of the XTR7, PRE5, IAA19 and BR6ox2 genes in PIF4-OX lines are referred to the right y axis of the graph. The experiment was repeated three times with similar results.",
    "molecules": "BR"
  },
  {
    "caption": "Col-0 and pifq seedlings were grown under short days at 22ºC and 28ºC, and samples were collected at the indicated time points. Expression of BR biosynthetic genes was analyzed by qRT-PCR, using the constitutive PP2A gene as internal control. Error bars represent SD of three technical replicates. The experiment was repeated twice with similar results",
    "molecules": "BR"
  },
  {
    "caption": "Increased levels of de-phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis. Col-0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC, on half strength MS media (mock) or media supplemented with 0.5 µM BRZ. BES1 was analyzed by western blot hybridization with an anti-BES1 antibody. RPT5 was used as a loading control.",
    "molecules": "BRZ, BR"
  },
  {
    "caption": "(A) Actual PER2::LUC levels (dark symbols (3-hour moving average, n=3 ±SEM, 4 outliers removed)) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours, compared with parallel longitudinal co-recordings from cells in the presence of 0.1 mM luciferin (light lines (n=6, mean ±SEM)).",
    "molecules": "luciferin"
  },
  {
    "caption": "(B) PER2::LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 (10 µM, applied at the arrow) (n=3, mean ±SEM). (C) Quantification of relative PER2::LUC induction upon proteasome inhibition, n=3, mean (solid) ±SEM (dashed).",
    "molecules": "MG132"
  },
  {
    "caption": "(D) Phase-dependent PER2::LUC half-life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one-phase exponential decay curve (n=3, mean ±SEM). Left image depicts the timing of cycloheximide (CHX, 10 µM) pulses (labelled I (PER2 levels going up) and II (PER2 levels going down)), plotted on PER2::LUC bioluminescence traces of control cells (dark colours). A representative trace of CHX-treated cells at time point I is shown in light colours. Right image shows quantifications, p-values were calculated by unpaired t test.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(A) Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CK1δ/ε inhibitor PF670462 (0.3 µM; PF).",
    "molecules": "PF, PF670462"
  },
  {
    "caption": "Period analyses of WT and CKO PER2::LUC cells in the presence or absence of GSK3 inhibitor CHIR99021 (5 µM; CHIR).",
    "molecules": "CHIR, CHIR99021"
  },
  {
    "caption": "Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CRY inhibitor KL001 (1 µM).",
    "molecules": "KL001"
  },
  {
    "caption": "Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in "Materials and Methods". Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, **p < 0.01.",
    "molecules": "3-NPA, DMSO, PicoGreen"
  },
  {
    "caption": "Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01.",
    "molecules": "3-NPA, 8-oxo-dG, DAPI, DMSO, H2O2"
  },
  {
    "caption": "Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-way ANOVA, N.S., not significant, **p < 0.01.",
    "molecules": "3-NPA, DMSO, H2O2"
  },
  {
    "caption": "HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies, then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, Data are presented as mean ± SD (n = 30), statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparisons test, ***p < 0.001.",
    "molecules": "3-NPA, DMSO, H2O2"
  },
  {
    "caption": "Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm.",
    "molecules": "H2O2"
  },
  {
    "caption": "Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.",
    "molecules": "3-NPA"
  },
  {
    "caption": "Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test , N.S., not significant, **p < 0.01.",
    "molecules": "3-NPA, 8-oxo-dG, DAPI"
  },
  {
    "caption": "PINK1 KO HeLa cells stably expressing mito-Keima were infected with control or shATAD3B. Five days later, cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were then analyzed and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561nm/458nm) of the cells described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.",
    "molecules": "3-NPA, DMSO, H2O2"
  },
  {
    "caption": "HeLa cells were treated with DMSO, 4 mM 3-NPA, 8 mM 3-NPA, or 10 µM CCCP for 2 h, and cell lysates were then analyzed by Western blotting using the indicated antibodies.",
    "molecules": "3-NPA, CCCP, DMSO"
  },
  {
    "caption": "PINK1 KO HeLa cells stably expressing ATAD3A-Flag (3A-Flag) or ATAD3B-Flag (3B-Flag) were mixed with control PINK1 KO cells respectively, and incubated for 24 h. Cells were treated with 4 mM 3-NPA (D) or 200 µM H2O2 (F) for 2 h, and then washed with fresh medium and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and anti-Flag antibodies and analyzed by confocal microscopy. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (E) or H2O2 (G) was quantified by ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by individual two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. Scale bar, 10 µm.",
    "molecules": "3-NPA, 8-oxo-dG, H2O2"
  },
  {
    "caption": "Control (GFP+) and shATAD3B (GFP-) PINK1 KO HeLa cells were mixed and incubated for 24 h. Cells were then treated with 4 mM 3-NPA (H) or 200 µM H2O2 (J) for 2 h, and washed with fresh medium, and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and DAPI, then analyzed by confocal microscopy. Control or shATAD3B cells are circled by white dashed lines. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (I) or H2O2 (K) was quantified using ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, **p < 0.01. Scale bar, 10 µm.",
    "molecules": "3-NPA, 8-oxo-dG, DAPI, H2O2"
  },
  {
    "caption": "293T cells were treated with H2O2 for 2 h, and cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with rabbit IgG (control) or anti-ATAD3 antibody, followed by Western blotting with anti-LC3B or anti-ATAD3 antibodies.",
    "molecules": "H2O2"
  },
  {
    "caption": "HeLa cells were treated with 200 µM H2O2 for 2 h, and cell lysates were incubated with the indicated GST-ATAD3A or GST-ATAD3B protein fragments (expressed in E.coli) coupled to glutathione agarose beads (Pierce) for GST pull-down assay. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.",
    "molecules": "glutathione, H2O2"
  },
  {
    "caption": "The indicated GST-ATAD3A or GST-ATAD3B protein fragments and His-LC3B were expressed in E.coli and purified. GST pull-down assay was performed using glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using anti-GST or anti-His antibodies.",
    "molecules": "glutathione"
  },
  {
    "caption": "The indicated GST-ATAD3B(WT) or GST-ATAD3B (LIR mutations) and His-LC3 were expressed in E.coli and purified. GST pull-down assay was performed using the glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.",
    "molecules": "glutathione"
  },
  {
    "caption": "HeLa expressing GFP-LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B. Five days later, cells were treated with DMSO, or 200 µM H2O2 for 2 h. Cells were fixed and immunostained with anti-Tom20 and imaged by confocal microscopy. The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 (mitochondria). Scale bar, 10 µm.",
    "molecules": "DMSO, H2O2"
  },
  {
    "caption": "293T cells transiently transfected with control or ATAD3B-Flag were treated with or without 200 µM H2O2 for 2 h. Cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with anti-Flag antibody, followed by Western blotting with anti-Flag or anti-LC3B antibodies. Relative protein levels of LC3B and ATAD3B-Flag were further evaluated by densitometry",
    "molecules": "H2O2"
  },
  {
    "caption": "Mic10 KO COS7 cells stably expressing ATAD3A-Flag or ATAD3B-Flag were treated with DMSO or 200 µM H2O2 for 2 h, and then fixed and immunostained with anti-Tom20 and anti-Flag antibodies, and analyzed by confocal microscopy. Scale bar, 10 µm.",
    "molecules": "DMSO, H2O2"
  },
  {
    "caption": "293T cells treated with DMSO (B) or H2O2 (200 µM, 2 h) (D), were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix). Relative protein levels of proteins in (B) or (D) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from DMSO- or H2O2-treated cells were shown (C and E). Data are presented as mean ± SD (n = 3 independent experiments).",
    "molecules": "DMSO, H2O2"
  },
  {
    "caption": "Control, ATAD3A KO, ATAD3B KO, or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min, and then were analyzed by Western blotting with the indicated antibodies.",
    "molecules": "EDC, DMSO"
  },
  {
    "caption": "293T cells were treated with DMSO, 200 µM H2O2, 10 mM NAC, or 200 µM H2O2 plus 10 mM NAC for 2 h. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies. Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in (F). Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.",
    "molecules": "EDC, DMSO, H2O2, NAC"
  },
  {
    "caption": "143B, 143B ρ0 cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies.",
    "molecules": "EDC, DMSO"
  },
  {
    "caption": "293T cells were treated with DMSO or ddC (50 µM) plus EB (0.5 µg/ml) for 10 days. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37℃, and then were analyzed by Western blotting with the indicated antibodies.",
    "molecules": "EDC, ddC, DMSO, EB"
  },
  {
    "caption": "ρ0206_A and ρ0206_B cells were stained with mitoSOX and analyzed by confocal microscopy. DIC, Differential Interference Contrast. Scale bar, 25 µm. Quantification of the MitoSOX fluorescence intensity described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by student's t-test, **p < 0.01.",
    "molecules": "mitoSOX, MitoSOX"
  },
  {
    "caption": "Mitochondria carrying the 3243A>G mutation in the tRNA Leu(UUR) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206_A (100% wild-type mtDNA) and ρ0206_B (~90% m.3243A>G mtDNA) cell lines. ρ0206_A and ρ0206_B cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.",
    "molecules": "Leu, tRNA"
  },
  {
    "caption": "ρ0206_A, ρ0206_B, and ρ0206-B cells pretreated with ddC (50µM) plus EB (0.5µg/ml) for 10 days, and then harvested and lysed. Cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-GAPDH antibodies.",
    "molecules": "ddC, EB"
  },
  {
    "caption": "ρ0206_B cells were infected with control, ATAD3A-Flag, ATAD3B-Flag, or ATAD3B (mLIR)-Flag. After two weeks, the cellular ATP levels were measured. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.",
    "molecules": "ATP"
  },
  {
    "caption": "B Immunofluorescence analysis of fibronectin (green) and collagen (red) fibers on de-cellularized ECM produced by human fibroblasts. Fibers orientation was quantified using ImageJ software. Percentages indicate oriented fibers accumulated in a range of ± 21° around the modal angle. Data is represented as mean ± s.d. (n=10 random fields from 2 independent determinations)..Scale bar, 50 µm. A representative image of picrosirius red staining from 10 analyzed is shown for each condition.",
    "molecules": "picrosirius red"
  },
  {
    "caption": "D, E Quantification of proliferation of 1205Lu (D) and SKMEL5 (E) cells plated for 48 h on plastic, Coll-1 or the indicated fibroblast-derived ECMs prior a 96 h treatment with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi. Cells were counted by Hoechst-labeled nuclei staining. Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments. (D) **P=0.015, ***P=0.0009, ****P<0.0001 and (E) *P=0.0204, ***P=0.0006, ****P<0.0001. Kruskal-Wallis followed by Dunn's multiple comparisons test.",
    "molecules": "Hoechst"
  },
  {
    "caption": "D Immunoblotting of protein extracts from 1205Lu, SKMEL5 and MM099 cells plated for 48 h on FRC-derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib (7 µM for 1205Lu, 10 µM for SKMEL5 and MM099) or DDR1-IN-1 (1 µM for 1205Lu, 5 µM for SKMEL5, 3 µM for MM099), using antibodies against the indicated proteins. HSP60, loading control.",
    "molecules": "DDR1-IN-1, Imatinib"
  },
  {
    "caption": "E Kaplan-Meier survival curves of mice treated with vehicle, Imatinib, BRAFi or BRAFi plus Imatinib. Median time to progression was 18, 20, 36 and 48 days, respectively. Log rank (Mantel-Cox) for BRAFi vs BRAFi/Imatinib mesylate. ****P<0.0001 and hazard ratio (log rank): 0.2403 (95% CI of ratio, 0.08123 to 0.7106).",
    "molecules": "Imatinib"
  },
  {
    "caption": "A Sections of 1205Lu xenografts from Fig. 6B were stained with picrosirius red and imaged under transmission light (upper panels) or polarized light (middle panels) (scale bar, 500 µm) or imaged by second harmonic generation (SHG) microscopy (lower panels) (scale bar, 50 µm) to examine collagen fiber network upon the mono or combined regimens.",
    "molecules": "picrosirius red"
  },
  {
    "caption": "F, G Immunoblot analysis of protein extracts obtained from SKMEL5 (F) or MM099 (G) cells plated on FRC- or MAF-derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1-IN-1 (5 µM for SKMEL5 and 3 µM for MM099) or 10 µM NIK inhibitor (NIKi) for 96 h. Antibodies against P-ERK1/2, ERK2, P-Rb, Rb, Survivin, Caspase 3, cleaved Caspase 3, RelB, p100/p52 and HSP60 as loading control were used.",
    "molecules": "DDR1-IN-1"
  },
  {
    "caption": "A, B Dot plots of FACS cell cycle analyses of WT and SirT7-/- MEFs in passage 3 (P3) and 6 (P6) using EdU incorporation and 7AAD (A). Percentages of cells in Sub G1 (apoptotic cells, left square) and cells with DNA content above 4N (polyploid, right square). (B) Quantitation of experiment shown in (A) (mean ± SEM; 3 samples per genotype).",
    "molecules": "DNA"
  },
  {
    "caption": "A Quantitation of neutral comet assays, using passage 3 WT and SirT7-/- primary MEFs, showing (left) the amount (%) of DNA in the tail and (right) the tail moment (see Fig S3A for representative images).",
    "molecules": "DNA"
  },
  {
    "caption": "B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7-/- primary fibroblasts after IR (8Gy). ATM inhibitor (ATMi) KU-55933 was added 30 min prior to irradiation where indicated. One representative blot from 4 independent experiments is shown.",
    "molecules": "KU-55933"
  },
  {
    "caption": "G FACS quantitation of WT and SirT7-/- MEF cells in S-phase after insult with 10 mM hydroxyurea (HU) for 24 hours. Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS (mean ± SEM; 5 samples per genotype).",
    "molecules": "HU, hydroxyurea"
  },
  {
    "caption": "H, I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7-/- MEFs. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ± SEM; 3 samples per genotype per condition).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.",
    "molecules": "DNA"
  },
  {
    "caption": "G Same as (F), using 293T-REX cells treated with doxycycline or vehicle to induce SIRT7-HA expression.",
    "molecules": "doxycycline"
  },
  {
    "caption": "H ChIP-on break assay. (Top) Schematic of I-SceI substrate construct introduced into HT1080 cells, which contains a single I-SceI site located within a puromycin resistance cassette. Shown along the top are the coordinates for amplicons probed by Q-PCR. (Bottom) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells. Q-PCR measurements were normalized to input DNA and non-I-SceI treated samples (No Cut) (mean ± SEM; one of two independent experiments shown). (Bottom Right) western blot demonstrating efficient knockdown of SirT7, with Histone H3 as a loading control.",
    "molecules": "puromycin"
  },
  {
    "caption": "K, L Class-switch recombination in splenic B cells from WT and SirT7-/- mice stimulated with lipopolysaccharides (LPS) and interleukin 4 (IL4). (K) The switching from IgM to IgG1 (left) and to IgG3 (right) was measured by FACS. (L) Quantitation of (K) showing mean ± SEM of 3-4 samples per genotype. One representative experiment from two is shown.*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.",
    "molecules": "lipopolysaccharides, LPS"
  },
  {
    "caption": "F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min.",
    "molecules": "KU-55933, DMSO, Olaparib"
  },
  {
    "caption": "A-H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3-WT, H3-K18Q or H3-K18R (naive, acetylated or deacetylated H3K18 residues, respectively).A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette (mean number of puromycin resistant colonies normalized by plating efficiency ± SEM; One representative transfection from 3 is shown).",
    "molecules": "puromycin"
  },
  {
    "caption": "B MAPKKK5-C interacts with PBL27 in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 2 mM 3-aminotriazole (3-AT) indicates a positive interaction.",
    "molecules": "3-aminotriazole, 3-AT, histidine, leucine, tryptophan, uracil"
  },
  {
    "caption": "C Chitin-induced MAPK activation was analyzed by immunoblots with α-pMAPK.",
    "molecules": "Chitin"
  },
  {
    "caption": "D Complementation of chitin-induced MAPK activation in mapkkk5 mutants by expression of MAPKKK5-FLAG. MAPK activation was analyzed by immunoblots with α-pMAPK.",
    "molecules": "chitin"
  },
  {
    "caption": "A Chitin-induced callose deposition in the mapkkk5 mutants. Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Representative pictures from the biological replicates are presented. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).",
    "molecules": "callose, Chitin, chitin"
  },
  {
    "caption": "B Leaf discs were treated with 10 μM (GlcNAc)7 in a solution containing 500 μM L-012 and 10 μg/ml horseradish peroxidase. ROS production was quantified using a luminescence microplate reader. Data are means ±SD calculated using three biological replicates, where each biological replicate consisted of two technical replicates.",
    "molecules": "L-012, (GlcNAc)7, ROS"
  },
  {
    "caption": "D Analysis of chitin-induced transcriptional reprogramming in mapkkk5-1. Total RNA was extracted from seedlings treated with mock or 40 μM chitin for 3 hours and analyzed by RNA-seq. Genes that are significantly induced or suppressed by chitin in the wild type were selected (q-value < 0.05; 12,992 genes). The log2 fold changes of the selected genes compared to mock in the wild type and mapkkk5-1 were plotted. Yellow and blue dots indicate genes that show reduced induction or suppression (506 genes) or enhanced induction or suppression (151 genes) in mapkkk5-1 compared to the wildtype, respectively. The slope of the linear regression line (red) indicates that the overall transcriptional response is weakened in mapkkk5-1 compared to the wild type. As a comparison, the line y=x (black) is shown. For more details, see the Methods.E A heatmap showing MAPKKK5-dependent genes. Genes showing reduced induction or suppression in mapkkk5-1 compared to the wild type were selected as described in the Methods (339 genes). The log2 fold changes of the selected genes compared to mock were subjected to hierarchical clustering analysis. Yellow indicates positive values, blue indicates negative values and black indicates zero: see the color scale.",
    "molecules": "chitin"
  },
  {
    "caption": "F Expression patterns of representative MAPKKK5-dependent genes. qRT-PCR analysis of defense-related genes in 8-day-old seedlings exposed to 40μM (GlcNAc)7 for 3h. Data are shown as the average of three independent biological replicates ± SD. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).",
    "molecules": "(GlcNAc)7"
  },
  {
    "caption": "C Arabidopsisprotoplasts expressing PBL27-HA and MAPKKK5-GFP were treated with 30 μM MG132 for 3 h and subjected to immunoblots with α-HA and α-GFP.",
    "molecules": "MG132"
  },
  {
    "caption": "F The disassociation of PBL27-MAPKKK5 interaction after chitin treatment in Arabidopsis protoplasts. Protoplasts were co-transfected with MAPKKK5K375M-GFP and PBL27-HA, stimulated with or without 10μM chitin or 1 μM flg22 for 10 min and subjected to the co-immunoprecipitation assay. Experiments (A) - (F) were performed three times with similar results.",
    "molecules": "chitin"
  },
  {
    "caption": "A PBL27 phosphorylates MAPKKK5-C in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. CBB, Coomassie brilliant blue.",
    "molecules": "γ-ATP"
  },
  {
    "caption": "E PBL27 does not phosphorylate MAPKKK5-C6xAin vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.",
    "molecules": "γ-ATP"
  },
  {
    "caption": "F PBL27 interacts with MAPKKK5-C6xA in yeast two-hybrid experiments. The growth of yeast colonies on plates (-ULWH) lacking uracil (U), leucine (L), tryptophan (W), and histidine (H) with 10 mM 3-AT indicated a positive interaction.",
    "molecules": "3-AT, histidine, leucine, tryptophan, uracil"
  },
  {
    "caption": "A Chitin-induced MAPK activation in a mapkkk5 mutant (Line 1) expressing MAPKKK56xA. MAPK activity was detected using immunoblots with α-pMAPK.",
    "molecules": "Chitin"
  },
  {
    "caption": "B Chitin-induced callose deposition in the MAPKKK56xA plant (Line 1). Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student's t-test (P < 0.05).",
    "molecules": "callose, Chitin, chitin"
  },
  {
    "caption": "C CERK1 does not phosphorylate the C-terminal domain of MAPKKK5 in vitro. The in vitro kinase assay was performed with recombinant proteins of GST-PBL27, MAPKKK5-C and the intracellular kinase domain of CERK1 (GST-CERK1:IC). The protein loading control was shown by staining with Coomassie Brilliant Blue. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. Asterisks indicate artificial bands.",
    "molecules": "γ-ATP"
  },
  {
    "caption": "E The kinase activity of GST-PBL273xA was analyzed by the in vitro kinase assay using [32P]γ-ATP.",
    "molecules": "γ-ATP"
  },
  {
    "caption": "B MAPKKK5-KD phosphorylates MKK4 and MKK5 in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.",
    "molecules": "γ-ATP"
  },
  {
    "caption": "EdU incorporation (green, 2 hours) was visualized by confocal imagery in GSC#1 or by FACS in GSC#9 transfected with sic or siMALT1. In GSC#1 the percentage of EdU-positive cells was quantified. Nuclei (DAPI) are shown in blue. n> 240 cells per replicate. Scale bar: 10 μm. Data are presented as the mean + s.e.m. on 3 independent experiments.",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": "FACS analysis of propidium iodide (PI) staining in GSC #1 and #9 transfected with non-silencing duplexes (sic) or MALT1 siRNA duplexes (siMALT1) and analyzed 72 hours later.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "Linear regression plot of in vitro limiting dilution assay (LDA) for GSC#9 treated with MALT1 inhibitor, mepazine (MPZ, 20 μM, 14 days). DMSO vehicle was used as a control. Data are representative of n=2.",
    "molecules": "DMSO, mepazine, MPZ"
  },
  {
    "caption": "Stem cell frequency was calculated from LDA in GSCs #1, #4, and #12 treated with MPZ treatment (20 μM, 14 days). Data are presented as the mean + s.e.m. on 2 independent experiments.",
    "molecules": "MPZ"
  },
  {
    "caption": "Tumorspheres per field of view (fov) were manually counted in GSCs #1, #4, #9, and #12 in response to MPZ (20 μM) and vehicle (DMSO), and in GSC#9 treated with Z-VRPR-FMK (75 µM) and vehicle (H2O) for 4 days. Data are presented as the mean + s.e.m. on 4 independent experiments for MPZ and 3 independent experiments for Z-VRPR-FMK.",
    "molecules": "DMSO, H2O, MPZ, Z-VRPR-FMK"
  },
  {
    "caption": "The expression of the stemness markers SOX2 and NESTIN was evaluated by western-blot and immunofluorescence (SOX2 in red NESTIN in green) in MPZ (+, 20 μM, 16 hours) and vehicle (-, DMSO, 16 hours) treated GSC#9. GAPDH served as a loading control. Scale bar: 10 μm.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Cell viability was measured using Cell TiterGlo luminescent assay in GSCs #1, #4, #9, and #12, human brain endothelial cells (endo), human astrocytes (astro), and human neuron-like cells (neuron) treated for 48 hours with DMSO or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "FACS analysis of EdU staining was performed on GSC#1 treated overnight with MPZ (10 μM). Data are presented as the mean + s.e.m. on 3 independent experiments.",
    "molecules": "EdU, MPZ"
  },
  {
    "caption": "FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (20 μM).",
    "molecules": "DMSO, MPZ, PI, propidium iodide"
  },
  {
    "caption": "Cell viability was measured using Cell TiterGlo luminescent assay in differentiated GSC#1 #4, and #9 (DGCs) treated for 48 hours with vehicle (DMSO) or MPZ (20 μM). Data were normalized to their respective DMSO-treated controls and are presented as the mean + s.e.m of 3 independent experiments. Morphology of GSCs #1, #4, #9 and DGCs #1, #4, #9 was shown using brightfield images.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Heatmap of cell viability of GSC#9 using increasing doses (0, 5, 10, 20, 40 μM) of phenothiazines: mepazine (MPZ), fluphenazine (FLU), cyamemazine (CYAM), chlorpromazine (CHLO), pipotiazine (PIPO), alimemazine (ALI), promethazine (PRO), and doxylamine (DOXY). Data were normalized to their respective DMSO-treated controls.",
    "molecules": "CHLO, chlorpromazine, CYAM, cyamemazine, DMSO, DOXY, doxylamine, FLU, fluphenazine, mepazine, MPZ, phenothiazines, PIPO, pipotiazine, PRO, promethazine, ALI, alimemazine"
  },
  {
    "caption": "Nude mice were implanted with GSC#9 (106 cells) in each flank and randomized cages were treated with either vehicle (DMSO) or MPZ (8 mg/kg) daily i.p., for 14 consecutive days, once tumors were palpable. Tumor volume was measured from the start of treatment until 1 week after treatment was removed. Graph of tumor volume on day 21 post-treatment is presented. Data are presented as the mean + s.e.m. n=10/group.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Cryosections from GSC-xenografted tumors were stained for the endothelial marker PECAM1 (red) and tumor marker NESTIN (green). Nuclei (DAPI) are shown in blue. Scale bar: 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Jurkat T-cells, GSC#1 and GSC#9 were stimulated with PMA (20 ng/mL) and Ionomycin (Iono, 300 ng/mL) for 30 minutes. Total protein lysates were analyzed by western-blot for CYLD (full length FL and cleaved c'd), HOIL1 (full length FL and cleaved c'd), p-IκBα and IκBα. MALT1 and BCL10 served as loading controls.",
    "molecules": "Iono, Ionomycin, PMA"
  },
  {
    "caption": "Jurkat T-cells, GSC#1 and GSC#9 were treated with vehicle (DMSO) and mepazine (MPZ, 20 μM) for 4 hours. PMA/Ionomycin mixture was also administered to Jurkat cells for the last 30 minutes. Total protein lysates were analyzed by western-blot for CYLD (full length FL and cleaved c'd). MALT1 served as a loading control.",
    "molecules": "DMSO, Ionomycin, mepazine, MPZ, PMA"
  },
  {
    "caption": "(Left) Western-blot analysis of CYLD, ROQUIN1/2, MALT1, and BCL10 in total protein lysates from GSC#9 treated for 4 hours with vehicle (H2O) or Z-VRPR-FMK (75 μM). GAPDH served as a loading control. (Right) Densitometric analysis of c'd/FL was performed for ROQUIN1/2 and CYLD. Data are presented as the mean + s.e.m. on 3 independent experiments. Schematic drawing of MALT1 structures highlighting the C464A substitution in the protease-dead version. DD: Death Domain, C-like D: Caspase-like Domain, Ig: Immunoglobulin domain.",
    "molecules": "H2O, Z-VRPR-FMK"
  },
  {
    "caption": "Transmission electron microscopy of GSC#9 treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. ER: endoplasmic reticulum; MVB: multivesicular bodies; lys: lysosome; mit: mitochondria; nuc: nucleus. Red stars denote lysosomes, blue stars vacuoles.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Confocal analysis of LAMP2 staining (red) at 0, 1, 2, 4, and 6 hours post-MPZ (20 μM) treatment. Nuclei (DAPI) are shown in blue. Scale bar: 10 μm.",
    "molecules": "DAPI, MPZ"
  },
  {
    "caption": "Western-blot analysis was performed in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or MALT1 targeting siRNA duplexes (siMALT1). Alternatively, western-blot analysis of LAMP2, CTSD, and MALT1 was done in total protein lysates from GSC#9 treated for 16 hours with MPZ (20 μM) or Z-VRPR-FMK (75 µM). DMSO was used as vehicle. GAPDH served as a loading control.",
    "molecules": "DMSO, MPZ, Z-VRPR-FMK"
  },
  {
    "caption": "Confocal analysis of LAMP2 staining (red) in GSCs #1, #4, #12 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM). Additionally, cells were transfected with non-silecing duplexes (sic) or MALT1 and BCL10 targeting siRNA duplexes (siMALT1 and siBCL10). Alternatively, lysotracker staining (red) was used to track for lysosomes in either GSC#9 expressing either wild-type (WT) or C464A FLAG-MALT1 (green). Scale bar: 10 μm.",
    "molecules": "DMSO, lysotracker, H2O, MPZ, Z-VRPR-FMK"
  },
  {
    "caption": "Cryosections from GSC#9-xenografted tumors in vehicle and MPZ-challenged animals (as described in Fig 2J) and assessed for LAMP2 staining (green). Nuclei (DAPI) are shown in blue. Scale bar: 10 μm. Western-blot analysis of LAMP2 was performed in tumor lysates. GAPDH served as a loading control.",
    "molecules": "DAPI, MPZ"
  },
  {
    "caption": "Confocal analysis of lysotracker staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (upper panel) or transfected with sic and siMALT1 (bottom panel). As indicated, number of lysotracker-positive puncta and lysotracker pixel intensity (arbitrary unit, AU) were quantified per cell. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.",
    "molecules": "DAPI, DMSO, lysotracker, H2O, MPZ, Z-VRPR-FMK"
  },
  {
    "caption": "(Left) Confocal analysis of LAMP2 (red) and LC3B (green) in GSC#9 treated for 4 and 16 hours with vehicle (DMSO) and MPZ (20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. (Right) Super-resolution imaging (SIM, Structured Illumination Microscopy) of LAMP2 (red) and LC3B (green) staining in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 µM).",
    "molecules": "DAPI, DMSO, MPZ"
  },
  {
    "caption": "Western-blot analysis of LC3B and P62 in total protein lysates from GSC#9 at 0, 2, 4, 6, and 16 hours post-MPZ treatment (20 µM). GAPDH served as a loading control.",
    "molecules": "MPZ"
  },
  {
    "caption": "Western-blot analysis of LC3B in total protein lysates from GSCs #1 and #9 at 72 hours post-transfection with sic or siMALT1, and subsequently treated 4 hours with vehicle (DMSO) or chloroquine (CQ, 20 µM). Knockdown was verified by MALT1 blotting and GAPDH served as a loading control.",
    "molecules": "chloroquine, CQ, DMSO"
  },
  {
    "caption": "Confocal analysis of LC3B (green) in GSC#9 treated for16 hours with vehicle (DMSO) and MPZ (20 µM) with or without chloroquine (CQ, 20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.",
    "molecules": "chloroquine, CQ, DAPI, DMSO, MPZ"
  },
  {
    "caption": "GSC#9 were transfected with LC3B reporters (wild type WT or G120A mutant, which cannot be lipidated), treated 24 hours later with vehicle (DMSO) or MPZ (20 µM) for 6 more hours. Ratios of WT/mutant luciferase signals are presented as the mean + s.e.m of 3 independent experiments.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Confocal analysis of P62 staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either transfected with sic or siMALT1 (middle) or treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (bottom). Quantification of P62 staining pixel intensity on GSC#9 treated for 16 hours with vehicle (DMSO or H2O), MPZ (20 µM) or Z-VRPR-FMK (75 µM) or sic and siMALT1. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30.",
    "molecules": "DMSO, H2O, MPZ, Z-VRPR-FMK"
  },
  {
    "caption": "Cell viability was measuring using Cell TiterGlo in GSCs #1 and #9 pre-treated for 1 hour with vehicle (DMSO) or QVD (20 μM), and treated for 72 hours more with the indicated doses of MPZ. Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 4 independent experiments.",
    "molecules": "DMSO, MPZ, QVD"
  },
  {
    "caption": "FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with QVD (20 μM). (Left) Percentage of PI positive cells, normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments. (Right) Histogram plots for representative experiment (DMSO in red and MPZ in blue).",
    "molecules": "DMSO, MPZ, PI, propidium iodide, QVD"
  },
  {
    "caption": "CSTD ELISA was performed on culture media from GSC#9 treated for 8 hours with vehicle (DMSO) or MPZ (20 µM). Alternatively, cells were transfected with sic or siMALT1 and analyzed 72 hours later. Data are presented as the mean + s.e.m of 3 independent experiments.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "(Left) Cell viability was measured using Cell TiterGlo luminescent assay in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (10 μM), following a 30 minute-pre-treatment with the following drugs: Bafilomycin A1 (Baf, 100 nM), Pepstatin A (Pep, 1 μg/mL), or CTS inhibitor 1 (Ctsi, 1 μM). Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate, stars refer to comparison to Vehicle + MPZ group (blue squares). (Right) FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with Baf, Pep and Ctsi. Percentage of PI positive cells normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments.",
    "molecules": "Baf, Bafilomycin A1, DMSO, Ctsi, MPZ, Pep, Pepstatin A, PI, propidium iodide"
  },
  {
    "caption": "Heatmap of differentially expressed genes obtained from RNAseq analysis of GSC#9 treated for 4 hours with vehicle (DMSO) or MPZ (20 μM), from three biological replicates.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Volcano plot of differentially expressed genes in RNAseq analysis of GSC#9, expressed as fold changes between vehicle (DMSO) and MPZ-treated cells.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "GSEA (gene set enrichment analysis) plot showing enrichment of "Bilanges serum and rapamycin sensitive genes" signature in vehicle (DMSO) versus MPZ-treated triplicates.",
    "molecules": "DMSO, MPZ, rapamycin"
  },
  {
    "caption": "qRT-PCR was performed on total RNA from GSC#9 treated for 4 hours with vehicle (DMSO) or MPZ (20 μM). Histograms showed changes in RNA expression of indicated targets. Data were normalized to two housekeeping genes (ACTB, HPRT1) and are presented as the mean + s.e.m of technical triplicates.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Western-blot analysis of LC3B, CSTD and TFEB in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or siRNA duplexes targeting TFEB (siTFEB) and treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. GAPDH served as a loading control.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Western-blot analysis of p-ULK1, p-AKT, p-S6, and p-p70S6K in GSC#9 treated for 1 hour with MPZ (20 μM) or rapamycin (RAPA, 50 nM). Total ULK, AKT, S6, and p70S6K served as loading controls. DMSO was used as a vehicle.",
    "molecules": "DMSO, MPZ, RAPA, rapamycin"
  },
  {
    "caption": "Western-blot analysis of p-AKT, p-S6, and p-p70S6K in total protein lysates from GSC#9 treated for 1 hour with vehicle (DMSO) or 20 μM of phenothiazine compounds (MPZ, FLU, CHLO, and CYA). Total AKT, total S6 and total p70S6K served as loading controls.",
    "molecules": "CHLO, CYA, DMSO, FLU, MPZ, phenothiazine"
  },
  {
    "caption": "Confocal analysis of LAMP2 (red) and mTOR (green) staining in GSC#9 treated with vehicle (DMSO) or MPZ (20 μM), Z-VRPR-FMK (75 μM), FLU (20 μM), CHLO (20 μM), and CYA (20 μM). Alternatively, cells were transfected with sic or siMALT1. Nuclei (DAPI) are shown in blue. Arrows point to LAMP2-positive area. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10.",
    "molecules": "CHLO, CYA, DAPI, DMSO, FLU, MPZ, Z-VRPR-FMK"
  },
  {
    "caption": "Total protein lysates (input) from GSC#9 treated with vehicle (-, DMSO) or MPZ (+, 20 μM, 1 hour) or with vehicle (-, H2O) or Z-VRPR-FMK (+, 75 μM, 4 hours), were processed for control immunoglobulins (Ig) or anti-QKI antibodies immunoprecipitation (IP). Western-blots were performed with indicated antibodies. Western-blots were performed with indicated antibodies.",
    "molecules": "DMSO, H2O, MPZ, Z-VRPR-FMK"
  },
  {
    "caption": "Confocal analysis of Lysotracker (green) or FLAG (red) in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue.",
    "molecules": "DAPI, Lysotracker"
  },
  {
    "caption": "Confocal analysis of LAMP2 (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue. Quantification of LAMP2 staining pixel intensity on GSC#9 transfected with mock and FLAG-QKI. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>15.",
    "molecules": "DAPI"
  },
  {
    "caption": "Confocal analysis of mTOR (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or Flag-QKI. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 16 hours with vehicle (DMSO) or MPZ (10 μM). Total protein lysates were processed for western-blots against LAMP2, CTSD, QKI, and LC3B expression, as indicated. GAPDH served as a loading control.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "Confocal analysis of mTOR (green) and LAMP2 (red) in GSC#9 transfected with sic or siQKI, and treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10.",
    "molecules": "DAPI, DMSO, MPZ"
  },
  {
    "caption": "GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 1 hour with vehicle (DMSO) or MPZ (20 μM). Total protein lysates were processed for western-blots against QKI and p-S6. TUBULIN and Total S6 served as loading controls.",
    "molecules": "DMSO, MPZ"
  },
  {
    "caption": "FACS analysis of EdU staining was performed on GSC#9 cells transfected with non-silencing RNA duplexes (sic, pink), QKI targeting siRNA duplexes (siQKI, light purple), MALT1 targeting siRNA duplexes (siMALT1, blue) or double transfected with siQKI and siMALT1 (purple).",
    "molecules": "EdU"
  },
  {
    "caption": "FACS analysis of propidium iodide (PI) staining in GSC#9 transfected with non-silencing RNA duplexes (sic), QKI targeting siRNA duplexes (siQKI), MALT1 targeting siRNA duplexes (siMALT1) or double transfected with siQKI and siMALT1 and analyzed 72 hours later. Percentage of PI-positive cells normalized to vehicle treated controls are presented as the mean + s.e.m. on 3 independent experiments.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12)  human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin, marked by arrows. Cells were co-immunostained with Ki67 and DAPI (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Arrowheads mark the AIS. Scale bar = 10µm in overview, 2µm in zooms.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Typical examples of Centrinone-B treated or control human iPSC-derived NSCs immunostained for  Pericentrin and Centrin. Inserts represent centriole(s). Scale bar = 5µm in overview, 2µm in inserts. B. Quantifications of the percentage of cells with 0, 1 or 2 centrioles per cell after 0 (control), 2 or 5 days Centrinone-B treatment and prior to neuronal induction. n = 48-51 cells in 2 independent experiments. C Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05",
    "molecules": "Centrinone-B"
  },
  {
    "caption": "C. Typical examples of Centrinone-B treated or control human iPSC-derived neurons (12-15 days) immunostained for AnkG, Trim46, MAP2 and Centrin. Arrowheads mark  AIS structures, arrows mark centrosomes. Inserts represent  centrosomes, zooms on the right represent AIS structures. Scale bar = 20µm in overview, 2µm in insert, 10µm in zooms.",
    "molecules": "Centrinone-B"
  },
  {
    "caption": "M. AP threshold, amplitude and after-hyperpolarization recorded in  Centrinone-B treated (n = 43 cells in 3 independent experiments) and control human iPSC-derived neuron cultures (n = 59 cells in 4 independent experiments). Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Mann Whitney test after-hyperpolarization Student's t test (M: threshold, AP amplitude). *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05",
    "molecules": "Centrinone-B"
  },
  {
    "caption": "D. Representative images of fan-like growth cones at day 5 of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions. Growth cone  visualized by immunostaining for phalloidin. Scale bar = 5µm. E. Quantifications of the average area (µm²) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different  time points. n = 25-83 growth cones in 3 independent experiments. F Data information: Data represent mean ± SEM. One-way ANOVA including Tukey's post-hoc analysis *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05",
    "molecules": "Centrinone-B, nocodazole, phalloidin"
  },
  {
    "caption": "F. Representative images of different  growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B  treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of  growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different  time points. n = 5-55 growth cones in 3 independent experiments. Da Data information: Chi-square-test including post-hoc analysis with Bonferroni correction (G), One-way ANOVA including Sidak's post-hoc analysis (H). *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05",
    "molecules": "Centrinone-B, nocodazole"
  },
  {
    "caption": "A. Example stills from a spinning-disk time-lapse recording of a  neurite transfected with mRFP and GFP-MT+TIP. The top panel is a still of a neurite in mRFP, showing neurite morphology. The other panels show moving GFP-MT+TIP comets pointing in either an anterograde direction (green arrowheads) or retrograde direction (blue arrowheads). P indicates the proximal direction and D the distal direction of the neurite. Timestamp in minutes:seconds given on bottom right. Scale bar = 5 µm. B. Kymographs and schematic representations of time-lapse recordings  of the distal axon  as shown in (A), for different time points (day 7; day 13) and conditions (control; Centrinone-B). Scale bar = 5 µm. C ",
    "molecules": "Centrinone-B"
  },
  {
    "caption": "J. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. Red line denotes the location of LS. Scale bar = 5 µm. K. Kymographs and schematic representations of  time-lapse recordings  of the distal axon as shown in (J) following  LS, for different time points (day 7; day 13) and conditions (control; Centrinone-B). Red line and red arrowhead denote location and time of LS. Scale bar = 5 µm. L ",
    "molecules": "Centrinone-B"
  },
  {
    "caption": "(B) Effect of ND-011992 on the intracellular ATP level in M. bovis BCG. The bacteria were treated with DMSO (blue triangle), 100 nM Q203 alone (green triangle), 20 μM ND-011992 alone (brown square), and a dose-range of ND-011992 in the presence of Q203 (red squares) for 15 hours before quantification of intracellular ATP levels.",
    "molecules": "ND-011992, ATP, DMSO, Q203"
  },
  {
    "caption": "(C) Oxygen consumption assay in M. bovis BCG using methylene blue as an oxygen sensor. (D) Oxygen consumption assay in M. bovis BCG using the Seahorse XFe96 Extracellular flux analyser. 12 μM ND-011992 was injected alone (green triangles), 100 nM Q203 (blue circles), or in combination with Q203 (red squares). For each condition, OCR readings were normalised to the last basal OCR reading before drug injection. OCR: Oxygen Consumption Rate. The dotted lines indicate the timepoints of drug injection. ",
    "molecules": "methylene blue, ND-011992, oxygen, Q203"
  },
  {
    "caption": "(E) Dose-dependent inhibition of M. bovis BCG OCR by ND-011992 (in the presence of 100 nM Q203) measured on a Seahorse XFe96 analyser platform. For each condition, OCR readings were normalised to the last basal OCR reading before drug injection.",
    "molecules": "ND-011992, Q203"
  },
  {
    "caption": "(F) Effect of ND-011992 on the OCR of M. smegmatis IMVs using the Oroboros O2k fluorespirometer. IMVs OCR from the parental strain (green triangles), ∆cydAB knockout (blue circles), and ∆cydAB complemented with M. tuberculosis CydABDC+ (red squares) energized with NADH were determined. Q203 was used at 1 μM. 100% OCR was defined as the OCR of the untreated samples for each strain.",
    "molecules": "ND-011992, NADH, Q203"
  },
  {
    "caption": "(A) Differential gene expression analysis of H37Rv treated with Q203, ND-011992, or combination (H37Rv-Combo), and ∆cydAB treated with Q203 compared to untreated controls. The log2-fold differences in gene expression of various conditions relative to the untreated control is indicated using a sliding scale where higher expression is reflected in red and lower expression is reflected in blue, with a midpoint signifying no difference in white.",
    "molecules": "ND-011992, Q203"
  },
  {
    "caption": "(C) Absence of the Cyt-bcc:aa3 sensitized H37Rv to ND-011992. M. tuberculosis H37Rv wildtype (Blue circles), ∆ctaE-qcrCAB (red squares) and complemented strain (green triangles) were treated with ND-011992. Growth inhibitory potency (MIC50) was recorded after 2 to 3 weeks of incubation.",
    "molecules": "ND-011992"
  },
  {
    "caption": "(D) The reduced minus oxidized difference spectrum of M. smegmatis ΔqcrCAB inverted membrane vesicles (IMVs). The green line represents the spectrum without treatment, while the blue line represents the spectrum of the IMVs in the presence of 10 μM ND-011992.",
    "molecules": "ND-011992"
  },
  {
    "caption": "(A) Combined potency of ND-011992 + Q203 against the drug-resistant clinical isolate M. tuberculosis 123-20-0047.",
    "molecules": "ND-011992, Q203"
  },
  {
    "caption": "(C) Growth inhibition assay in M. bovis BCG. 106 bacteria were plated onto 7H10 agar supplemented with DMSO, 25 nM Q203, 3 µM ND-011992, or 25 nM Q203 + 3 µM ND-011992.",
    "molecules": "ND-011992, agar, DMSO, Q203"
  },
  {
    "caption": "(D) Fluctuation analysis in M. tuberculosis H37Rv. M. tuberculosis ΔcydAB was plated on 7H10 containing 100 nM Q203 (red). Parental H37Rv was plated on 7H10 with 100 nM Q203 and 6 µM ND-011992 or 2 µg/mL rifampicin (blue). The corresponding values of the median frequency of resistance is indicated in the graph.",
    "molecules": "ND-011992, rifampicin, Q203"
  },
  {
    "caption": "(A) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in replicating M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, and Q203 at 100 nM. Inoc.: starting inoculum; DMSO: vehicle control. Bacteria viability was determined by enumerating colony-forming units (CFU) on nutrient-agar plates after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0. *P < 0.05; **P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0346, 0.0143, 0.00016, 0.000052. (B) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in nutrient-starved M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, isoniazid was used at 1 μM, Q203 was used at 100 nM, and ND-011992 was used at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony-forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0. **P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0098, 0.000027, 0.000067. ",
    "molecules": "ND-011992, agar, BDQ, Bedaquiline, DMSO, isoniazid, Q203"
  },
  {
    "caption": "(C) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in M. tuberculosis H37Rv under hypoxia. BDQ was used at 500 nM, isoniazid at 1 μM, metronidazole (MTZ) at 200 μM, Q203 at 100 nM, and ND-011992 at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony-forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0.**P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0092, 0.00010, 0.00079.",
    "molecules": "ND-011992, BDQ, isoniazid, metronidazole, MTZ, Q203"
  },
  {
    "caption": "(D) Efficacy of ND-011992-Q203 combination treatment in a mouse model of acute tuberculosis. Bacterial load (CFU) were enumerated in the lungs of mice before treatment, and after 5 days of treatment with either the vehicle control (brown squares), Q203 at 5 mg/kg (red squares), ND-011992 at 25 mg/kg (green triangles), or ND-011992 at 25mg/kg with Q203 at 5 mg/kg (orange diamonds). **P = 0.0006 unpaired Student's t-test, two-tailed; n = 4; comparing Q203 alone vs Q203 + ND-011992.",
    "molecules": "ND-011992, Q203"
  },
  {
    "caption": "B HSATIII ASO-mediated depletion of nSBs. Thermal stress-exposed HeLa cells (42°C for 2 h and recovery for 1 h at 37°C) were visualized by HSATIII-FISH and immunofluorescence using an anti-SAFB antibody or anti-HNRNPM antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D Thermal stress-induced expression of HSATIII in CHO (His9) cells. The cells were visualized by HSATIII-FISH (green), and the nuclei were stained with DAPI (blue). Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E Ectopic HSATIII-induced nSB assembly in CHO (His9) cells. The nSBs (yellow arrowheads) were visualized by HSATIII-FISH and immunofluorescence using an anti-SRSF1 antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Colocalization of novel nSB proteins with HSATIII. Endogenous TRA2B (E) were stained with anti-TRA2B HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Colocalization of novel nSB proteins with HSATIII. THRAP3 (F), were stained with anti-THRAP3, HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Colocalization of novel nSB proteins with HSATIII. FLAG-tagged PPHLN1 (G) were stained with anti-FLAG antibodies HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Re-translocation of FLAG-CLK1 to nSBs after thermal stress removal. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and then exposed to thermal stress (42°C for 2 h) with or without recovery (37°C for 1 h). FLAG-CLK1 was visualized with an anti-FLAG antibody. Nuclear stress bodies and nuclei were visualized by HSATIII-FISH and DAPI staining, respectively. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Recovery phase-specific colocalization of CLK1 and SRSF9. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and exposed to thermal stress (42°C for 2 h) followed by recovery (37°C for 1 h). FLAG-CLK1 and SRSF9 were co-stained using anti-FLAG and anti-SRSF9 antibodies, respectively, and the nuclei were stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "H The re-phosphorylation status of SRSF9 within nSBs. Phosphorylated (arrows) and hypo- and de-phosphorylated (arrowheads) SRSF9 in the nSB fractions (lanes 4, 6, and 8) were analyzed by western blotting on Phos-tag gels. KH-CB19 (10 μM) or DMSO as a control was administrated during the recovery period. The lower panel shows a shorter exposure image of the phosphorylated forms. The asterisks indicate bands different from band (a) or (d). I Quantification of the data shown in (H). Relative band intensities were calculated as the ratios of each band to total SRSF9 detected by western blotting of a standard SDS-PAGE, and were normalized to those at 42°C.",
    "molecules": "DMSO, KH-CB19"
  },
  {
    "caption": "(b) LC/MS analysis of VCP after 1 h incubation at 20 °C with 1% DMSO (top) or 100 μM NMS-859 (bottom), showing a 313-Da increase corresponding to the addition of NMS-859 lacking the Cl atom.",
    "molecules": "NMS-859, DMSO"
  },
  {
    "caption": "(a) Activity of VCP inhibitors NMS-249 and NMS-873 on wild-type VCP and mutants. Data represent mean values of at least three independent experiments.",
    "molecules": "NMS-249, NMS-873"
  },
  {
    "caption": "(b) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP in the presence of different allosteric inhibitors or ADP.",
    "molecules": "ATTO 590, ADP"
  },
  {
    "caption": "(c) Binding of the EDA-ADP-ATTO 590 probe to wild-type (WT) VCP and the K615V and N616F mutants in the presence of NMS-249. Minor differences in the mP values are due to the use of different enzyme batches.",
    "molecules": "ATTO 590, NMS-249, ADP"
  },
  {
    "caption": "(d) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP and Walker A mutants in the presence of NMS-249. Data shown are representative results of one of three independent experiments: each determination was done in triplicate, data points denote the average value, and error bars represent s.d.",
    "molecules": "ATTO 590, NMS-249, ADP"
  },
  {
    "caption": "(a) Western blot analysis of biomarker modulation in the HCT116 cell line. HCT116 cells were treated with the indicated doses of inhibitors for 8 h (top) or 24 h (bottom), and protein cell extracts were resolved on SDS-PAGE gel. Filters were probed with the indicated antibodies. C, control; B, bortezomib (0.01 μM); T, thapsigargin (0.1 μM); Mg, MG132 (0.1 μM).",
    "molecules": "bortezomib, MG132, thapsigargin"
  },
  {
    "caption": "(b) NMS-873 compound-mediated stabilization of Mcl-1 in live cells. Top, confocal time-lapse images of live stable, inducible T-REx-293 cells expressing a bicistronic construct of mCherry-BimS(2A)ΔC (red) with eGFP-Mcl-1 (green) after doxycycline treatment. Cells were treated with cycloheximide (CHX, 50 μg ml−1) ± 2.5 μM of NMS-873 for 6 h. Bottom, total intensity of eGFP-Mcl-1 and mCherry-BimS(2A)ΔC was measured for time-lapse series shown at top. A decrease in eGFP-Mcl-1 and mCherry-BimS(2A)ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control. WT, wild type. Scale bar, 10 μm.",
    "molecules": "NMS-873, cycloheximide, DMSO, doxycycline"
  },
  {
    "caption": "(D) Boxplot/Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2, DPPA4 and DUX ChIP, and in a random shuffle of peaks.",
    "molecules": "GC nucleotide"
  },
  {
    "caption": "(B) NTERA-2 cells were transduced with either an empty vector (WT) or the same vector containing an shRNA against KAP1 (KD) prior to puromycin selection and transduction with increasing doses of GFP reporter vectors. GFP expression was analysed 72 hours post reporter transduction. A representative experiment of 2 experiments is shown. Two-tailed unpaired t test p-values: PBS-LTR-GFP: 0.0148; PBSChr15-LTR-GFP: 0.0377",
    "molecules": "puromycin"
  },
  {
    "caption": "(B) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following 5-AZA treatment of HeLa cells and PBMCs (day 6 post transduction). A representative experiment of two is shown in each case. Two-tailed unpaired t tests were performed. HeLa cell p-values: ISG56: 0.005; CCL5: 0.0006; CXCL10: 0.0007; Mxa: 0.0001; IFNb: 0.02. For repeat expression (left panel), p values not available as N=2. PBMC p-values: HERVK14C: 0.1; SVA D VNTR: 0.003; ISG56: 0.001; CCL5: 0.012; CXCL10: 0.012; Mxa: 0.02; IKba: 0.08. Results were normalized to GAPDH and B2M (GAPDH results shown). See also Fig S2C for results in 293T cells. All error bars show SD.",
    "molecules": "5-AZA"
  },
  {
    "caption": "(C) DNA methylation at endogenous SVAs was measured over 18 CpGs in the stated treatment groups (day 5 post transductions, or day 2 post 5-AZA treatment). Each point represents the average methylation state of one sequence with at least 10 sequences analyzed per group. Mann-Whitney tests were performed to compare shControl to shKAP1 (p=0.6593) and DMSO to 5-AZA (0.0027). 5-AZA was added to all experiments at 7uM. All numbers above bars depict fold changes compared to control cells (to one decimal place).",
    "molecules": "5-AZA, DMSO"
  },
  {
    "caption": "(A) H3K9me3 ChIP-PCR where error bars show SD of 3 independent IPs. Results show the IP enrichment relative to the total input normalized to GAPDH as a negative control region. IgG control IPs gave only background enrichments, shown.",
    "molecules": "H3K9me3"
  },
  {
    "caption": "(B) 293T cells were transduced with the stated shRNAs and cells harvested 4 days after puro selection for Western Blot to verify KAP1 and SETDB1-depletion (left) and ChIP (right). Cell numbers were normalized per treatment group before sonication and IPs were performed in triplicate. Results show IP relative to total input and GAPDH normalized. IgG control IPs gave only background enrichments shown. One representative experiment of two is shown. Unpaired t tests were performed. P values: ZNF239: KAP1, 0.0292; SETDB1, 0.0137; HERVK14C_1: KAP1, 0.0227; SETDB1, 0.0082; HERVK14C_2: SETDB1, 0.0462; SVA D VNTR, SETDB1, 0.0064. The inlay shows qRTPCR expression analysis of HERVK14C in the same experiment, normalized to B2M or GAPDH as stated. All error bars show SD.",
    "molecules": "puro"
  },
  {
    "caption": "(C) Venn diagram showing H3K9me3 enriched sites (18271) as those identified in both ChIP replicates in the human cell line K562 (left). Right: % of ISGs or random genes intersecting a H3K9me3 or KAP1 ChIP-seq peak. 5kb upstream and downstream of each gene was also included. N=437 genes per group.",
    "molecules": "H3K9me3"
  },
  {
    "caption": "Immunofluorescence staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples Two representative microscopy images from six separate images show: (A) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques. Scale bar = 50 μm. (B) Colocalization of (red) apoE23 and (green) FH around brain capillaries. The blue nuclei were detected using DAPI staining. (C) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques from a biopsy sample obtained from an iNPH patient diagnosed with Alzheimer's clinical syndrome (ACS). Colocalization of apoE on Aβ is shown with an arrow. The negative staining control is shown. (D) ApoE-FH colocalization analysis of all detected colocalized foci in six microscope images from the apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient biopsy samples. Six microscope images from Alzheimer's clinical syndrome (ACS) biopsy sample are included in the apoE44 dataset (red dots).",
    "molecules": "Aβ, DAPI"
  },
  {
    "caption": "Immunofluorescence staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples (I) MST showing binding of NT647-labeled full-length FH to different apoE isoforms, (J) NT647-labeled apoE2(1-165) fragment to increasing concentrations of FH402Y and FH402H variants and FH domains 1-4 and 19-20 and (K) NT647-labeled recombinant N-terminal apoE2(1-165) to increasing concentrations of FH5-7 402Y and FH5-7 402H fragments.",
    "molecules": "NT647"
  },
  {
    "caption": "(B) Different mixtures of apoE isoforms, FH, FH19-20 and Aβ1-42 were incubated for 72 h, and the presence of stable complexes was analyzed by running the sample in Tris-glycine PAGE for (left and middle) 90 or (right) 60 min at 100 V in the presence of 1 Bolt™ LDS Sample Buffer (Thermo Fisher). Gels were subjected to silver staining (left) or WB using anti-Aβ antibody (middle and right). ApoE isoform and incubation time are shown on the top. The presence of each protein is indicated with arrows. Aβ1-42 aggregate and the stable complexes formed between apoE, Aβ1-42 and FH are shown near the top of the gel, while apoE2/Aβ1-42 formed in the absence of FH are shown in several bands below apoE/Aβ1-42/FH complexes.",
    "molecules": "Aβ, Aβ1-42, silver"
  },
  {
    "caption": "(C) Single-molecule TIRF micrograph of Hylite 488 labeled Aβ1-42 and Alexa Fluor 568 C5 Maleimide labeled apoE2. Scale bar = 5 μm. (D) Jitter plot of proportion of colocalized Aβ1-42/apoE2 and Aβ1-42/apoE3 over multiple fields of view in the presence and absence of FH. Statistics was calculated using Student's t-test from multiple images (n=12) in each sample. Error bars indicate SD values. (",
    "molecules": "Hylite 488, Alexa Fluor 568, Aβ1-42"
  },
  {
    "caption": "(E) Jitter plot of Aβ1-42 foci stoichiometry in number of fluorophores colocalized or not with apoE2 and apoE3 and in the presence and absence of FH. Statistics of multiple images was calculated using two-sided Student's t-test. Statistics was calculated using Student's t-test from all detected colocalized foci in multiple images (n=12) in each sample. Error bars indicate SD values.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(A and B) Immunofluorescence staining of apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples showing localization of FH (green) with Iba-1 stained cells (red) and clustering around cells and brain capillaries. Presence of (white) Aβ plaques in apoE44 samples and (arrow) FH in both samples are shown.. C-E (C) Iba-1, (D) Aβ and (E) FH intensity analysis was calculated from all detected foci in six microscope images from the apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient biopsy samples.",
    "molecules": "Aβ"
  },
  {
    "caption": "(F) Flow cytometric experiments showing binding of (left) 405-labeled apoE4 and (right) 405-labeled apoE2 to activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH, FH fragments 19-20 and 5-7, BSA and Aβ1-42.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(I) Binding of 488-labeled Aβ1-42 to (left) activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH19-20, BSA, with combinations of apoE4 and FH, apoE2 and FH or apoE3 and FH. The fluorescence intensities of 488-Aβ1-42 in each experiment was normalized against activated U937 empty cells sample.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(A) Immunofluorescence staining of iNPH patient biopsy samples (apoE33) showing (arrow) colocalization of complement activator, (red) C1q, and complement regulator (green) FH. (B) (arrow) Colocalization of (green) apoE and (red) C1q on (white) Aβ plaques. Colocalization analysis is calculated from colocalized foci in two separate microscope images.",
    "molecules": "Aβ"
  },
  {
    "caption": "(D) C3a formation in supernatant of C3, factor B (fB), factor D (fD) and factor I (fI) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH complexes. Beads with488-Aβ1-42/BSA complexes shows the level of C3a formation without inhibitor. Release of C3a is calculated as relative C3a increase % compared to the absorbance measured from 488-Aβ1-42/FH sample.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(G) (left) Phagocytosis of complement C3, fB, fD and fI (C) or only buffer (no C) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA (oligomerized Aβ = OAβ) or with non-oligomerized 488-Aβ1-42 (monomeric Aβ = MAβ). Also, samples without apoE or FH were included in the assay (buffer). (right) Cell permeability of C3, fB, fD and fI incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA complexes (OAβ) or with MAβ. Also, cells incubated with only buffer were included in the assay (buffer). Cell permeability is calculated by flow cytometry as % of DAPI stained cells in the cell population. The effect of CR3 expression in the phagocytosis was measured using PMA activated U937 cells with (U937 CR3 cells) or without (U937 empty cells) CR3 expression.",
    "molecules": "MAβ, monomeric Aβ, Aβ1-42, OAβ, oligomerized Aβ, DAPI, PMA"
  },
  {
    "caption": "(A) 488-Aβ1-42 phagocytosis in the presence of FH and apoE isoform. Flow cytometry data are presented as mean fluorescence intensity of the cell population of the sample.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(a) Top: immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of changes in total LRRK2 (n = 6 independent experiments). SH-SY5Y cells were untreated or incubated with 5 μM lactacystin (Lact), 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin (NL) for 9.5 h.",
    "molecules": "3MA, NH4Cl, lactacystin, leupeptin"
  },
  {
    "caption": "(b) Top: representative immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of LRRK2 relative to that in untreated samples (n = 3 or 4 independent experiments). Brain slices from mouse cortex, midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 °C.",
    "molecules": "3MA"
  },
  {
    "caption": "(c) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes. Values are percentage of the degradation without LRRK2 (n = 5 or 6 independent experiments).",
    "molecules": "proteins"
  },
  {
    "caption": "(e,f) Association of LRRK2 and RNase A (e) or LRRK2 and GAPDH (f) with PI-treated rat liver lysosomes incubated with increasing concentrations of GAPDH (e) or RNase A (f) or ovalbumin (Ov). Left: representative immunoblots. Right: protein association expressed as a percentage of the value when incubated with lysosomes alone (n = 5 or 6 independent experiments). Inp: RNase A (0.4 μg), LRRK2 (0.04 μg) or GAPDH (1 μg).",
    "molecules": "ovalbumin"
  },
  {
    "caption": "(a) Top: immunoblot for GFP of tet-on HEK293 cells expressing GFP-tagged WT or G/S LRRK2 pulsed 24 h with doxycycline and, 48 h later (no longer undergoing de novo synthesis), incubated without additions (−) or with 20 mM NH4Cl and 100 μM leupeptin (NL), 5 μM MG132 (MG) or 10 mM 3MA. Bottom: percentage inhibition (n = 3 independent experiments).",
    "molecules": "3MA, NH4Cl, doxycycline, leupeptin, MG132"
  },
  {
    "caption": "(a) Top: immunoblot for GFP of tet-on HEK293 cells expressing GFP-tagged WT or G/S LRRK2 pulsed 24 h with doxycycline and, 48 h later (no longer undergoing de novo synthesis), (b) Same cells as in a, untreated (Ctr) or treated with RNA interference for LAMP-2A (L2A(−)), pulsed as in a, sequentially collected and immunoblotted. Top left panels: representative immunoblots for GFP. Actin is shown as a loading control. Bottom left panels, quantification, corrected by levels of knockdown and loading control (n = 3 or 4 independent experiments). Top right: immunoblot for LAMP-2A. Bottom right: percentage of LRRK2 degraded per hour in the same samples.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(a) Proteolysis in HEK293 cells stably transfected with an empty plasmid (Ctr) or plasmids expressing WT or G/S LRRK2 under the control of a tet promoter, treated with doxycycline to activate protein expression and then labeled with [3H]leucine for 48 h. After extensive washing, protein degradation at the indicated times was measured, in cells maintained either in the presence (+) or absence (-) of serum, as the amount of acid-precipitable radioactivity (amino acids and small peptides) released into the media. Values are expressed as percentage of radioactivity in proteins (acid precipitable) at time 0 (n = 4 independent experiments, each in triplicate).",
    "molecules": "[3H]leucine, doxycycline, proteins"
  },
  {
    "caption": "(a) Immunoblots of rat liver lysosomes incubated with WT or G/S LRRK2 alone or with 1 mM GTP and/or GAPDH. Duplicate samples are shown. Bottom: quantification of lysosome-bound LRRK2 expressed as a multiple of the amount bound without additions (n = 6 independent experiments).",
    "molecules": "GTP"
  },
  {
    "caption": "(d) Co-IP of LAMP-2A with anti-GFP in tet-on HEK293 cells expressing GFP-LRRK2 proteins and pulsed for 24 h with doxycycline. Inputs (Inp), one-quarter of material added.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(b-d) Degradation of radiolabeled proteins (b), binding of GST-α-syn (c) and levels of endogenous α-syn (d) in intact brain lysosomes (Lysos) from non-transgenic (Ctr) and G/S LRRK2 transgenic mice. Bottom panel in c shows lower exposure to visualize monomers of α-syn. Homog, homogenates; olig, oligomers; I, input (n = 3 independent experiments).",
    "molecules": "proteins"
  },
  {
    "caption": "(B) Patterns of DNA methylation for each cytosine context in an endogenous Hi locus calculated from WGBS data. Vertical dashed lines indicate the termini of Hiun.",
    "molecules": "cytosine"
  },
  {
    "caption": "(A) Immunoblot analysis of caspase-1 and gasdermin D in WT or Nlrp3-/- unprimed and LPS-primed BMDMs left untreated [Medium alone (Med.)] or assessed 20 hr after stimulation with the supernatant (Sup.) of or assessed 20 hr after infected with WT C. perfringens (WT C. per), ΔpfoA C. perfringens (ΔpfoA), Δcpa C. perfringens (Δcpa, lacking lecithinase) or ΔpfoAΔcpa C. perfringens (ΔpfoAΔcpa) at MOI 100.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) Immunoblot analysis of caspase-1, gasdermin D and GAPDH (loading control) in WT or mutant BMDMs left untreated or LPS-primed and assessed 3 hr after stimulation with lecithinase (Lec.).",
    "molecules": "LPS"
  },
  {
    "caption": "(J) Survival of WT mice administered with either PBS (n = 20) or MCC950 (n = 18), followed by intraperitoneal administration of 0.312 Units/ml of lecithinase (Lec.) as in H.",
    "molecules": "MCC950, PBS"
  },
  {
    "caption": "(A) Confocal microscopy analysis of CD11b (green) and lecithinase (red) in unprimed WT BMDMs left untreated [Medium alone (Med.)] or assessed 1 hr after stimulation with Alexa Fluor 568 labelled lecithinase (AF568-Lec.) in the absence or presence of cytochalasin D (Cyto D; 50 μM) or methyl-β-cyclodextrin (MCD; 5 mM).",
    "molecules": "AF568, Alexa Fluor 568, Cyto D, cytochalasin D, MCD, methyl-β-cyclodextrin"
  },
  {
    "caption": "(D) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 3 hr after stimulation with perfringolysin O (PFO), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of methyl-β-cyclodextrin (MCD; 5 mM). (E) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in D. (F) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 4 hr after infection of with S. Typhimurium (S. Tm.; MOI, 5), or 30 min after stimulation with nigericin in the absence or presence of cytochalasin D (Cyto D; 50 μM). (G) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in F. (H) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 4 hr after infection of with S. Typhimurium (S. Tm.; MOI, 5), or 30 min after stimulation with nigericin in the absence or presence of latrunculin B (Lat B; 1 μg/ml). (I) Release of IL-1β (left) and IL-18 (middle), and death (right) of WT BMDMs after treatment as in H.",
    "molecules": "Cyto D, cytochalasin D, Lat B, latrunculin B, LPS, MCD, methyl-β-cyclodextrin, Nig, nigericin"
  },
  {
    "caption": "(B) Confocal microscopy analysis of LAMP1 (green) and lecithinase (red) in WT BMDMs left untreated or assessed 1 hr after stimulation with AF568-lecithinase. (C) Three-dimensional surface rendering of LAMP1 (green) and lecithinase (red) shown in B. (D) Line scan values for LAMP1, AF568-lecithinase, and DAPI taken from the white dashed line in the gold-colored inset in B. These values were plotted and fitted with a single Gaussian equation (Arbitrary Unit (AU)).",
    "molecules": "AF568, DAPI"
  },
  {
    "caption": "(A) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated [Medium alone (Med.)] or assessed 3 hr after stimulation with lecithinase (Lec.), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of bafilomycin A (Baf A; 100nm). (C) Immunoblot analysis of caspase-1 and GSDMD of unprimed or LPS-primed WT BMDMs left untreated or assessed 3 hr after stimulation with lecithinase (Lec.), or 16 hr after infection with F. novicida (F. novi., MOI 100), or 30 min after stimulation with nigericin (Nig.) in the absence or presence of ammonium chloride (NH4Cl; 20 μM).",
    "molecules": "ammonium chloride, NH4Cl, Baf A, bafilomycin A, LPS, Nig, nigericin"
  },
  {
    "caption": "(F) Flow cytometry analysis of WT BMDMs stained with acridine orange (1μg/ml) and pre-treated with various inhibitors (MCD, Cytochalasin D, Bafilomycin A, ammonium chloride) for 30 min, and assessed 60 min after stimulation with lecithinase (Lec.). WT BMDMs stained with acridine orange were also assessed 60 min after stimulation with heat-treated lecithinase (Heat).",
    "molecules": "acridine orange, ammonium chloride, Bafilomycin A, Cytochalasin D, lecithinase, MCD"
  },
  {
    "caption": "(I) Release of IL-1β (top), IL-18 (middle) and death (bottom) of WT or Ninj1-/- BMDMs left untreated or LPS-primed and assessed for a range of time points after stimulation with lecithinase (Lec.) or 16 hr after transfection with LPS (LPS trans.).",
    "molecules": "LPS"
  },
  {
    "caption": "(J) Immunoblot analysis of caspase-1, gasdermin D and NINJ1 of WT or Ninj1-/- BMDMs left untreated or LPS-primed and assessed 3 hr after stimulation with lecithinase (Lec.) or 16 hr after transfection with LPS (LPS trans.).",
    "molecules": "LPS"
  },
  {
    "caption": "F HEK293A  cells at steady state (upper) or upon cyclic 0.1Hz 200mbar  hydrostatic pressure (lower) were analysed by immunofluorescence. Cells are labelled for Hoechst  (blue) and YAP  (green). Scale bar = 20µm. Graph on the right depict quantification of nuclear-to-cytoplasmic  (Nucl/Cyt) ratio of YAP  in cells as shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001. G 143B  cells at steady state (upper) or upon cyclic 0.1Hz 200mbar  hydrostatic pressure (lower). Graph on the right show quantification of nuclear-to-cytoplasmic  (Nucl/Cyt) ratio of YAP  in 143B  from images as those shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001.",
    "molecules": "Hoechst"
  },
  {
    "caption": "C Representative immunofluorescence  images showing Phalloidin-labelled actin  (red) and Hoechst  (blue) in Latrunculin B (LatB) (0.5µM) or Cytochalasin D (CytD) (2µM)-treated HEK293A  cells compared to control across Hippo pathway genome-edited cells as shown. Scale bar = 20μm. D Average change in  cell volume obtained by DHM  in response to hydrostatic pressure with 2µM Cytochalasin D  (CytD) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. **p=0.0044 (WT con vs CytD), p*=0.0409 (Y/T DKO con vs CytD), p>0.9999 (LATS1/2 DKO con vs CytD), ***p=0.0003 (WT con vs Y/T DKO con), ***p<0.001 (WT con vs Y/T DKO CytD), ***p<0.001 (Y/T DKO CytD  vs LATS1/2 DKO CytD). E Fold difference in average change in  cell volume in response to dynamic hydrostatic pressure from (H) normalised against untreated cells. Each dot represents a single cell and error bars are 95% CI. Mann-Whitney U test. **p=0.0069 (WT vs. Y/T DKO), **p=0.0031 (WT vs LATS1/2 DKO). F Average change in cell volume in response to hydrostatic pressure with 0.5µM  Latrunculin B (Lat B) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. ***p<0.001 (all comparisons), p=0.0972 (LATS/12 DKO con vs LatB), *p=0.0216 (Y/T DKO LatB  vs LATS1/2 DKO LatB). G Fold difference in average change in cell volume in response to cyclic 0.1Hz 100mbar  hydrostatic pressure from (F) normalised against control. Each dot represents a single cell and error bars represent mean ± 95% CI. Mann-Whitney U test. p=0.1472 (WT vs. Y/T DKO), **p=0.0015 (WT vs LATS1/2 DKO).",
    "molecules": "Hoechst, CytD, Cytochalasin D, Lat B, LatB, Latrunculin B, Phalloidin"
  },
  {
    "caption": "A Confocal IF  images labelled with Hoechst  (Blue) and internalised Transferrin   (Trf). Cells were allowed to internalize  for ten minutes, fixed and acid stripped to remove surface bound Trf. Hydrostatic pressure (H.P.) promotes transferrin  uptake in WT and LATS1/2 DKO cells. Scale bar = 20µm. B Quantification of transferrin  uptake in WT, Y/T DKO and LATS1/2 DKO cells treated with  hydrostatic pressure  compared to steady state. Each dot represents a single cell and error bars represent mean ± 95% CI. Data from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. Steady-state statistical analysis is shown in blue, transferrin  uptake induced by pressure is shown in black. ***p<0.001 (WT con vs +H.P.), p=0.2375 (Y/T DKO con vs +H.P.), ***p<0.001 (LATS1/2 DKO con vs +H.P.), p=0.2764 (WT con vs Y/T DKO con), p=0.0808 (WT con vs LATS1/2 DKO con), ***p=0.0004 (Y/T DKO con vs LATS1/2 DKO con).",
    "molecules": "Hoechst"
  },
  {
    "caption": "C Confocal IF   images myc  tagged AP180C   (green) cells showing internalised Trf  (red) and stained  with Hoechst (blue). AP180C  inhibits transferrin uptake in WT and Y/T DKO cells. Scale bar = 20µm. D Quantification of transferrin  uptake in AP180C  positive cells compared to vector control from images as in C. Each dot represents a single cell from four independent experiments and error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001 (for both comparisons).",
    "molecules": "Hoechst"
  },
  {
    "caption": "E Confocal IF  images of HEK293A WT cells showing subcellular localisation  of YAP  (red) in AP180C  (green) negative and positive WT and LATS1/2 DKO cells. Cells also stained  with Hoechst (blue). F Quantification of cytoplasmic-to-nuclear  ratio of YAP  in AP180C  positive WT and LATS1/2 DKO cells compared to vector control from images as in E. Each dot represents a single cell pooled from four independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p=0.0008 (WT), p=0.9318 (LATS1/2 DKO).",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) DAB staining of H2O2 accumulation in the indicated genotypes.",
    "molecules": "H2O2"
  },
  {
    "caption": "(B-C) DAB staining of H2O2 accumulation (C) in the indicated genotypes.",
    "molecules": "H2O2"
  },
  {
    "caption": "(B) Phosphorylation of CRCK3G390R by MPK4. MPK4 was immunoprecipitated from wild type or MPK4::MPK4-FLAG. After incubation with [γ-32P] ATP and the immunoprecipitated MPK4-FLAG protein in protein kinase buffer, CRCK3G390R was separated by SDS-PAGE. The autoradiograph of the gel is shown in the top panel, and immunoblot analysis of MPK4-FLAG levels is shown in the bottom panel. Myelin basic protein (MBP) was used as a positive control. This experiment was repeated three times with similar results.",
    "molecules": "[γ-32P] ATP"
  },
  {
    "caption": "A, Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU (3 mM) for 2 hrs prior to pre-extraction, fixation and imaging.",
    "molecules": "HU"
  },
  {
    "caption": "D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl.",
    "molecules": "DNA"
  },
  {
    "caption": "A, DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n= 3 biological replicas.",
    "molecules": "DNA"
  },
  {
    "caption": "B, Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in A. Error-bars, SD; n = 6 biological replicas. Two-tailed t test, ** p<10-2.",
    "molecules": "DNA"
  },
  {
    "caption": "E, Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes.",
    "molecules": "histones"
  },
  {
    "caption": "G, Analysis of histone acetylation in BRPF3 depleted cells. (left) Western blot of siRNA treated U-2-OS cells. TSA treatment (1 hr) was included as a positive control. 2x, double amount of extract loaded in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates.",
    "molecules": "TSA"
  },
  {
    "caption": "A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann-Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown.",
    "molecules": "DNA"
  },
  {
    "caption": "D, Scatter plot of chromatin-bound GFP-RPA1 intensity per cell. GFP-RPA1 and RFP-PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN-01 (300 nM) prior to pre-extraction and fixation. GFP-RPA1 levels were measured in PCNA positive cells. Lines represent medians. n > 150. t-tests: **** P < 10-4, * P < 0.05; a.u., arbitrary unit.",
    "molecules": "UCN-01"
  },
  {
    "caption": "A, ChIP-seq analysis of BRPF3 binding on the human genome. BRPF3 enrichment signal obtained by ChIP-seq with asynchronous (AS), synchronized (S) and released into HU (S+HU) RKO cells, on the MCM4 (left) and Lamin B2 (right) replication origins. Signals were compared to the published enrichment signals for ORC1 in HeLa cells ((Dellino et al, 2013), GSE37583), HBO1 in RKO cells (Avvakumov et al., 2012, GSE33221), and H3K14ac in IMR90 cells (GSM521881). The negative control IgG signal shown is from the S+HUchromatin.",
    "molecules": "HU"
  },
  {
    "caption": "A, Western blot of DNA damage signalling in BRPF3 depleted cells. siRNA transfected U-2-OS cells were treated with HU for 2 h or 24 h. One representative experiment out of two biological experiments is shown.",
    "molecules": "HU"
  },
  {
    "caption": "B-C, Recovery from replication stress. siRNA treated cells were treated for 24 hrs with HU (0 h), and released into normal medium for the indicated times (see also Fig. S6A, B). (B) DNA replication rate measured by EdU incorporation. Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells. n > 1000. Mann-Whitney: **** P < 10-4. (C) Cell cycle progression measured by FACS analysis of DNA content. One representative experiment out of two independent biological replicas is shown.",
    "molecules": "HU, DNA"
  },
  {
    "caption": "(A) Quantification of CXCR4+ cells by flow cytometry and Sox17+ cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH. Average and standard deviation (SD) of eight independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(C) Immunostaining for Ecadherin (left) and FoxA2 (right) at day 7 of differentiation in presence of 3 µM CH.",
    "molecules": "CH"
  },
  {
    "caption": "(D) Quantification of CXCR4+ by flow cytometry over a seven-day differentiation time course in the presence of 3 µM CH. Average and SD of three independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(E) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH. Scale bars = 200 µM",
    "molecules": "CH"
  },
  {
    "caption": "(A) Assay of Tcf7l1 mRNA by RT-PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH. Average and SD of three independent experiments. Day2 *p= 0.048, Day3 *p=0.036.",
    "molecules": "CH"
  },
  {
    "caption": "B) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence (+) or absence (-) of 3 μM CH.",
    "molecules": "CH"
  },
  {
    "caption": "(C) Assay of FoxA2 mRNA by RT-PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. **p=5x10-7.",
    "molecules": "CH"
  },
  {
    "caption": "(F) ChIP for Tcf7l1 followed by qPCR. Analysis was performed in wild-type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. FoxA2 *p = 0.049.",
    "molecules": "CH"
  },
  {
    "caption": "(A) Assay of FoxA2 mRNA by RT-PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH. Average and SD of three independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(B) Gene expression analysis of endodermal associated genes by RT-PCR in control wild-type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(C) FoxA2 (green) and Oct4 (red) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH. DAPI staining in blue.",
    "molecules": "CH"
  },
  {
    "caption": "(D) Assay of CXCR4, Hex and Sox17 mRNA by RT-PCR in control wild-type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(A) Assay of FoxA2 mRNA by RT-PCR in FoxA2-Tet ES cell lines 48 hr after treatment with the indicated dose of DOX. Average and SD of two independent experiments from 2 independent lines.",
    "molecules": "DOX"
  },
  {
    "caption": "B) FoxA2 immunostaining of FoxA2-Tet ES cells 48 hr after DOX treatment. Left, no DOX control. Middle and right, 10ng/ml DOX. DAPI staining shown in blue. Right panel shows middle image without DAPI signal.",
    "molecules": "DOX"
  },
  {
    "caption": "(C) Assay of FoxA2 and CXCR4 mRNA by RT-PCR on day 3 of differentiation with DOX at the indicated dose. Average and SD of two experiments from two independent cell lines.",
    "molecules": "DOX"
  },
  {
    "caption": "(D) Assay of Sox17 mRNA by RT-PCR on day 7 of differentiation with addition of 3 µM CH and/or 10 ng/ml DOX at the indicated time points (in days). Average and SD of two independent experiments from two independent cell lines.",
    "molecules": "CH, DOX"
  },
  {
    "caption": "(E) Sox17 immunostaining of FoxA2-Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng/ml DOX and the presence and absence of 3 µM CH as indicated. Far right panel shows image to its left without DAPI.",
    "molecules": "CH, DOX"
  },
  {
    "caption": "(A) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng/ml (W100) or 200 ng/ml (W200) recombinant Wnt3a plus 500 ng/ml recombinant RSpondin1. Relative luciferase units are normalised to FOPflash. Average and SD of three independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(B) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a (200 ng/ml plus 500 ng/ml RSpondin1(WR)). Average and SD of three independent experiments. ** p<0.01.",
    "molecules": "CH"
  },
  {
    "caption": "(C) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH. Average and SD of three independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(D) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH. Results from two independent, clonally-derived Ctnnb1 null ES cell lines are shown. Tubulin was used as a loading control.",
    "molecules": "CH"
  },
  {
    "caption": "(B) Immunoblot for total cMyc and phospho Ser62 (pSer62) cMyc during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH.",
    "molecules": "CH"
  },
  {
    "caption": "C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.",
    "molecules": "CH"
  },
  {
    "caption": "C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.",
    "molecules": "CH"
  },
  {
    "caption": "(E) Assay of Tcf7l1 mRNA by RT-PCR in wild-type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc (cMyc Inhibitor II; Myci). Treatment with Myc inhibitor 10058-F4 (Sigma) generated similar results (data not shown). Average and SD of three independent experiments. **p=0.0002.",
    "molecules": "10058-F4, cMyc Inhibitor II, Myci"
  },
  {
    "caption": "(F) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in cMyc null/nMyc heterozygous cells (C-/-;N+/-) and floxed parental cells (cMycfl/fl;nMycfl/fl (control)) at day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of four or six independent experiments. Tcf7l1 *p=0.01, FoxA2 *p=0.034.",
    "molecules": "CH"
  },
  {
    "caption": "(G) FoxA2 immunostaining of control and cMyc null/nMyc heterozygous (cMyc-/-;nMyc+/-) cells at day 3 of differentiation in the presence of 3 µM CH.",
    "molecules": "CH"
  },
  {
    "caption": "(H) Assay of cMyc and Tcf7l1 mRNA by RT-PCR in cMycT58A inducible cell lines (icMycT58A) 24 hr after DOX treatment. Average of 2 independent experiments.",
    "molecules": "DOX"
  },
  {
    "caption": "(I) Assay of Tcf7l1 mRNA by RT-PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng/ml DOX. Average and SD of 3 independent experiments. **p=0.007.",
    "molecules": "CH, DOX"
  },
  {
    "caption": "(J) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng/ml.",
    "molecules": "DOX"
  },
  {
    "caption": "(B) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the absence or presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, *p=0.028.",
    "molecules": "CH"
  },
  {
    "caption": "(C) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058-F4. Average and SD for three independent experiments, *p=0.026.",
    "molecules": "10058-F4, CH"
  },
  {
    "caption": "(D) Assay for Miz1 mRNA by RT-PCR for the first three days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments.",
    "molecules": "CH"
  },
  {
    "caption": "(E) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, **p=0.0015.",
    "molecules": "CH"
  },
  {
    "caption": "(E) Peripheral blood leukocyte populations (by lineage) between genotypes at day 0 and day 28. ; R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, spleen and thymic analysis at day 28 post tamoxifen treatment",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(G) Differential analysis of leukocyte populations in the BM. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM analysis at day 28 post tamoxifen treatment",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(H) BM erythroid cells. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, analysis at day 28 post tamoxifen treatment",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(J) Differential analysis of leukocyte populations in the spleen. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). spleen analysis at day 28 post tamoxifen treatment;",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(K) Thymic cellularity. (L) Differential analysis of thymocyte populations in the thymus. ( R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). thymic analysis at day 28 post tamoxifen treatment",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(N) qPCR (SYBR green) based analysis of indicated gene expression in BM. Data expressed as mean +/- SEM gene expression related to Ppia expression. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4).",
    "molecules": "SYBR green"
  },
  {
    "caption": "(E) Viability of cell lines with and without tamoxifen treatment (isogenic pairs) over 14 days of treatment (n=3 independent lines per genotype; biological replicates).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(F) qPCR (SYBR green) analysis of Ifit1 (left) and Irf7 (right) expression on day 14 of analysis.",
    "molecules": "SYBR green"
  },
  {
    "caption": "(H) qPCR (SYBR green) of Ifit1 (left) and Irf7 (right) expression in the cell lines collected at 3 or 24 hours post-treatment with 50 or 1000 U/mL of IFNβ. No statistical difference between genotypes in Ifit1 (left) and Irf7 (right) expression.",
    "molecules": "SYBR green"
  },
  {
    "caption": "(A-C) DA level (pictograms per head) in males of the indicated genotypes. n = 3, 3, 3 (A); n = 4, 5, 3, 3 (B); n = 3, 3, 3 (C) times (100 heads per genotype).",
    "molecules": "DA"
  },
  {
    "caption": "(E) DA level (pictograms per head) in the head of males of the indicated genotypes. n = 4 (100 heads per genotype).",
    "molecules": "DA"
  },
  {
    "caption": "B, C. WISH of runx1/c-myb(B) or notch1b(C) after treatment with DMSO (left), the VEGF receptor inhibitor SU5461 (middle) and respectively SU5461 after injection of UAS:mEvi1 (inducing endothelial evi1 expression) in Tg(fli.1:Gal4FF;UAS:RFP) embryos (right).",
    "molecules": "DMSO, SU5461"
  },
  {
    "caption": "A. Expression of runx1/c-myb in the VDA after treatment with DMSO vehicle control (left), the PI3K/AKT inhibitor Wortmannin (WM, middle) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased runx1/c-mybexpression for each condition.",
    "molecules": "DMSO, WM, Wortmannin"
  },
  {
    "caption": "B. Expression of notch1b after treatment with DMSO vehicle control (left) or Wortmannin (WM, middle). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.",
    "molecules": "DMSO, WM, Wortmannin"
  },
  {
    "caption": "U2OS cells were transfected, synchronized at the G1/S-border by thymidine for 24 hours and then either released into nocodazole for 16 hours (unperturbed mitotic entry) or released for 7 hours, treated with 0.5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine (checkpoint recovery). Mitotic index was scored based on the percentage of Histone H3-pS10 positive DAPI-nuclei and normalized to the untransfected controls. Black diamonds indicate individual siRNA-targeted genes from the library, light grey diamonds indicate positive (Wip1-depleted) and negative (untransfected and GAPDH-depleted) controls and dark grey diamonds indicate the hits based on the two screens. Dotted lines indicate selection criteria for recovery-specific genes.",
    "molecules": "caffeine, Adriamycin, nocodazole, thymidine"
  },
  {
    "caption": "B) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels.",
    "molecules": "Adriamycin, thymidine"
  },
  {
    "caption": "C) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted.",
    "molecules": "thymidine"
  },
  {
    "caption": "D) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized and damaged in G2. Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated.",
    "molecules": "caffeine"
  },
  {
    "caption": "E) Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release, damaged with 0.5 μM Adriamycin for one hour. After a 16-hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS. Error bars represent SD, n=4. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, *** for P≤0.001).",
    "molecules": "caffeine, Adriamycin, thymidine"
  },
  {
    "caption": "F) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt or FLAG-Tlk2-D613A were thymidine synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated.",
    "molecules": "tetracycline, Tetracycline, thymidine"
  },
  {
    "caption": "G) Caffeine induced recovery assay of cell lines shown in 2F. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01, *** for P≤0.001).",
    "molecules": "Caffeine"
  },
  {
    "caption": "A) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release. Cells were fixed at the indicated times after adriamycin-induced DNA damage and stained for DAPI and γH2AX. Scale bar indicates 10 μm. B) Quantification of cells in 3A. Each dot represents the total signal of γH2AX in the nucleus of a single cell. Bars indicate the mean and error bars indicate the SD of the data points in each condition. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (NS for P>0.05, **** for P≤0.0001).",
    "molecules": "DNA, adriamycin, thymidine"
  },
  {
    "caption": "C) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin-induced DNA damage with or without Tlk2-depletion. Samples were analyzed by western blotting for the indicated proteins.",
    "molecules": "adriamycin"
  },
  {
    "caption": "F) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels. Lanes were all present on the same blot but pasted together for comparison as indicated by the lines.",
    "molecules": "Adriamycin, thymidine"
  },
  {
    "caption": "G) Cells were synchronized in S- or G2-phase by a single thymidine block. Damage was induced by 0.5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards. Samples were fixed at the indicated cell cycle stages and stained for Asf1A-pS166. Nuclear fluorescence intensity was measured relative to DAPI. Whiskers represent 5-95% of data points.",
    "molecules": "Adriamycin, thymidine"
  },
  {
    "caption": "H) Cells were treated and analyzed as in 2E. Induction of FLAG-Asf1A-4D was induced using doxycycline. Error bars represent SD, n=5. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).",
    "molecules": "doxycycline"
  },
  {
    "caption": "I) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0.5 μM Adriamycin for one hour and after the indicated time points were pre-extracted and fixed. Cells were stained for total levels of Histone H3 and quantified relative to DAPI. Scale bar indicates 10 μm. Whiskers represent 5-95% of data points.",
    "molecules": "Adriamycin, thymidine"
  },
  {
    "caption": "J) FLAG-Asf1A-4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (**** for P≤0.0001).",
    "molecules": "doxycycline"
  },
  {
    "caption": "C) U2OS cells were synchronized in G2 and harvested or synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.",
    "molecules": "adriamycin"
  },
  {
    "caption": "F) U2OS cells were transfected with the indicated siRNA's, arrested in G2 with Adriamycin and induced to recover by addition of caffeine. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).",
    "molecules": "caffeine, Adriamycin"
  },
  {
    "caption": "G) U2OS cells were synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.",
    "molecules": "adriamycin"
  },
  {
    "caption": "H) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt were thymidine synchronized, damaged in G2 and harvested 24 hours afterwards. Tetracycline was either absent (off), added from the start of the experiment (early) or for 16 hours after induction of DNA damage (late).",
    "molecules": "tetracycline, Tetracycline, thymidine"
  },
  {
    "caption": "I) Cells were treated as in 5G and recovery was induced by addition of caffeine for 8 hours. Mitotic index was analyzed by FACS and normalized to the control. Error bars represent SD of three independent experiments. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).",
    "molecules": "caffeine"
  },
  {
    "caption": "E The representative  images of  IF-IHC staining  of E14.5 embryonic brains with antibodies against  Iba-1  (green) and DAPI (blue). Scale bar, 200 μm. F Quantification of numbers  of Iba-1+ cells on the meningeal  regions. Mock n=5; ZIKV n=7 embryos. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.",
    "molecules": "DAPI"
  },
  {
    "caption": "G The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 50 μm. H Quantification of total number of Iba-1+ cells per ChP  area. I Percentage of Iba-1+ cells on the surface of ChP. J Percentage of round-shaped Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM. Mock n=5; ZIKV n=6 biological replicates",
    "molecules": "DAPI"
  },
  {
    "caption": "K The representative images of  IF-IHC staining  of  choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green), CD68 (red) and DAPI (blue). Scale bar, 50 μm. L Percentage of  CD68+; Iba-1+ cells out of total  Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM. ",
    "molecules": "DAPI"
  },
  {
    "caption": "J Representative confocal images of CTB488-labeled  tdTomato-positive neurons in the  vHIP. White arrowheads indicate CTB488 single positive neurons. Scale bars: 200 μm. K Quantification of the percentage of tdTomato;CTB488 double-positive cells out of total CTB488-positive cells. Mock, n= 7 mice; ZIKV, n=7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise. ",
    "molecules": "CTB488"
  },
  {
    "caption": "1]. (F) H3K27Ac read density in NPCs or GPCs around DNA-regions bound by SOX3 in NPCs. SOX3 DNA-regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci. Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II loci",
    "molecules": "K27Ac"
  },
  {
    "caption": "(B) Samples from (A) were separated on a native agarose  gel and visualized by autoradiography  Aberrant Replication  Products (ARPs) are also indicated, as are the different species of catenated dimers: nicked-nicked catenanes, 'n-nCats'; nicked-supercoiled catenanes, 'n-scCats'; supercoiled-supercoiled catenanes 'sc-scCats'. (C) Quantification of total DNA  synthesis from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (D) Quantification of total DNA  synthesis from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (E) Quantification of supercoiled circular monomers ('scCMs') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (F) Quantification of scCMs  from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. (G) Quantification of linears  ('Lins') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (H) Quantification of Lins  from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Data information: For (E)-(H), abundance of the indicated species was calculated as a percentage of total lane signal (% of total).",
    "molecules": "agarose"
  },
  {
    "caption": "(A) Plasmid DNA  was replicated  in the presence of [α-32P]dATP and vehicle control, etoposide, or doxorubicin. Purified DNA  intermediates were digested with XmnI, which cuts the plasmid template once, to allow for identification of DNA breaks. (B) Samples from (A) were separated on a native agarose  gel and visualized by autoradiography. (C) Quantification of DNA breaks  from (B). Mean ± SD, n=3 independent experiments. Abundance of breaks  was calculated as a percentage of total lane signal (% of total). (D) Quantification of linears  ('Lins') from (B). Mean ± SD, n=3 independent experiments. Abundance of linears  (AU) was normalized to the maximum recorded signal within each individual experiment.",
    "molecules": "agarose, dATP, doxorubicin, etoposide, 32P"
  },
  {
    "caption": "(F) Samples from (E) were separated on native agarose  gels and visualized by autoradiography. (G) Quantification of DPC-containing molecules from (F). Mean ± SD, n=3 independent experiments. Abundance of the DPC-containing molecules was calculated as a percentage of total lane signal (% of total). (H) Quantification of the amount of  supercoiled circular monomers ('scCMs') from '+ProK' samples from (F). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers (AU) was normalized to the maximum recorded signal within each individual experiment.",
    "molecules": "agarose"
  },
  {
    "caption": "(A) Plasmid DNA was replicated  in Mock- or TOP2α-immunodepleted extracts in the presence or absence of vehicle control, etoposide, or doxorubicin. In mock-depleted extracts (ΔMock), the final replication  product are supercoiled circular monomers ('scCMs'). In TOP2α-depleted extracts (ΔTOP2α), the final replication  product are catenanes ('Cats') because TOP2α   is required for DNA  unlinking. (B) Samples from (A) were separated on a native agarose  gel and visualized by autoradiography. Linear  molecules, arising from DNA breaks, are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes. (C) Quantification of scCMs  from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (D) Quantification of Cats  from (B) lanes 16-30. Mean ± SD, n=3 independent experiments. Abundance of catenanes  was calculated as a percentage of total lane signal (% of total).",
    "molecules": "agarose, doxorubicin, etoposide"
  },
  {
    "caption": "(F) Samples from (E) were separated on native agarose  gels and visualized by autoradiography. Linear  molecules are indicated with a red arrow. (G) Quantification of scCMs from + ProK  gel in (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of  supercoiled monomers  was calculated as a percentage of total lane signal (% of total). (H) Quantification of DPC Containing Molecules from (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of DPC-containing molecules was calculated as a percentage of total lane signal (% of total).",
    "molecules": "agarose"
  },
  {
    "caption": "(A) Plasmid DNA  was replicated   in the presence or absence of etoposide  and p97-i, which inhibits removal of replisomes  (green) from DNA. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (B) Proteins from (A) were recovered and analyzed via  Western blotting. Input corresponds to 1% of total reaction. Modified TOP2α species are also indicated. (C) Quantification of TOP2α  (both modified and unmodified species) from (B) lanes 1-8. (D) Quantification of TOP2α  (both modified and unmodified species) from (B) lanes 9-16. (E) Quantification of CDC45  from (B) lanes 1-8. (F) Quantification of CDC45  from (B) lanes 9-16. Data information: (C-F Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.",
    "molecules": "etoposide"
  },
  {
    "caption": "(G) Plasmid DNA  was replicated  in the presence or absence of etoposide  and aphidicolin  (Aph), which prevents DNA synthesis but still permits unwinding by replisomes. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (H) Proteins from (G) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. (I) Quantification of RPA30  from (H) lanes 9-16. (J) Quantification of RPA30 from (H) lanes 1-8. (K) Quantification of TOP2α  (both modified and unmodified species) from (H) lanes 1-4 and 9-12. (L) Quantification of TOP2α  (both modified and unmodified species) from (H) lanes 5-8 and 13-16. Data information: I-L) Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.",
    "molecules": "Aph, aphidicolin, etoposide"
  },
  {
    "caption": "(A) Plasmid DNA  was replicated  in the presence of [α-32P]dATP and vehicle control, aclarubicin, or doxorubicin. (B) Samples from (A) were separated on a native agarose  gel and visualized by autoradiography. (C) Quantification of supercoiled circular monomers from (B). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total).",
    "molecules": "aclarubicin, agarose, dATP, doxorubicin, 32P"
  },
  {
    "caption": "(E) Samples from (D) were separated on a native agarose  gel and visualized by autoradiography. (F) Quantification of fork  merger from (E) lanes 1-8. Mean ± SD, n=3 independent experiments. (G) Quantification of  fork  merger from (E) lanes 9-17. Mean ± SD, n=3 independent experiments. Fork  merger was calculated as a percentage of total lane signal (% of total) for (F)-(G).",
    "molecules": "agarose"
  },
  {
    "caption": "(E) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester.",
    "molecules": "ATP-γ-S"
  },
  {
    "caption": "F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.",
    "molecules": "dsDNA, ssDNA"
  },
  {
    "caption": "B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.",
    "molecules": "DNA"
  },
  {
    "caption": "D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.",
    "molecules": "DNA"
  },
  {
    "caption": "E, H460 and A549 cells were cultured in 5% Matrigel for 10 days. Representative images of the 3D Matrigel culture are shown in the top panel, and immunofluorescence staining with anti-laminin5 and DAPI is shown in the bottom panel (laminin5, red; DAPI, blue). Scale bars, 200 μm. The right panel shows the percentages of colonies with a diameter ≥ 50 μm and colonies with a diameter < 50 μm. The mean ± SD represents 5 visual fields from one experiment. Three independent experiments were performed. ****p < 0.0001, unpaired two-tailed Student's t test.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, Immunoblot showing the expression of PKA substrate phosphorylation, PKA and Actin in the indicated cells and genotypes and the indicated cells treated with H89.",
    "molecules": "H89"
  },
  {
    "caption": "BrdU incorporation assay (B) of the indicated cells after treatment with H89. Scale bars, 200 μm. Values shown are means ± SD obtained from one experiment. Three independent experiments were performed. ns, nonsignificant, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.",
    "molecules": "H89"
  },
  {
    "caption": "D, Representative images and quantification of patient-derived organoids treated with 20 μM H89 and controls. The mean ± SD represents 5 visual fields from one experiment. Scale bars, 20 μm. ns, nonsignificant, ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.",
    "molecules": "H89"
  },
  {
    "caption": "E Michaelis-Menten kinetic  parameters as calculated by saturation curves using differential concentrations of myristoylated peptide  or NAD+. n=3 technical replicates (TR); error bars=SD.",
    "molecules": "NAD+"
  },
  {
    "caption": "A, B Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones  were incubated with purified SIRT6  and 5 mM NAD+, then resolved by SDS-PAGE  and analyzed by immunoblotting  with acetyl-specific histone  antibodies. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisks indicate p<0.05 compared to WT, color corresponds to allele. H3K9ac, H3K18ac  and H3  are blots, SIRT6  loading Coomassie stain HeLa histone  preps were also probed",
    "molecules": "H3K18ac, H3K9ac, acetyl, NAD+"
  },
  {
    "caption": "D Whole cell  histone H3 acetylation  levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate  dosage required for equivalent SIRT6   protein abundance was determined by Western blot, and administered accordingly to respective cell lines Cells were incubated with appropriate cumate  dose for 48hrs prior to harvest, with PQ-treated cells receiving PQ  24hrs after initial cumate  induction.",
    "molecules": "Cu, cumate, Cumate, paraquat, PQ"
  },
  {
    "caption": "E-H Relative abundance of H3K9ac at SIRT6-dependent  NF-κB target genes. Anti-H3K9ac  antibodies were used to perform ChIP  in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6  expression. Samples were normalized using 10% input. n=3 BR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition. Red asterisk indicated p<0.05 compared to +Cu  WT.",
    "molecules": "H3K9ac, Cu, cumate"
  },
  {
    "caption": "I, J Relative abundance of H3K18ac at  satellite repeats associated with SIRT6   deacetylase activity. Anti-H3K18ac  antibodies were used to perform ChIP  in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6   expression. Samples were normalized using 10% input. n=3 TR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu  condition.",
    "molecules": "H3K18ac, Cu, cumate"
  },
  {
    "caption": "A Self-ribosylation  of SIRT6  using biotin-labeled NAD+. Recombinant SIRT6  was incubated with NAD+ conjugated with a biotin  residue and then run on an SDS-PAGE. Each allele was assessed relative to its 0 hr  time point  and normalized to SIRT6  total protein loading controls. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05. Black asterisks indicate significance over 0 hr control. Red asterisks indicated significance over time-matched WT SIRT6.",
    "molecules": "biotin, NAD+"
  },
  {
    "caption": "B Self-ribosylation  of SIRT6  with titration  of NAD+. mADPr-specific antibody was used to detect ribosylated wild type SIRT6  and centSIRT6 proteins. Plot was fit the Michaelis-Menten best-fit with GraphPad software. Km NAD was 142+29 for centSIRT6 and 106+45 for wild type SIRT6  and maximal signal was ~2x greater for centSIRT6  compared to the wild type SIRT6. Two separate batches of recombinant protein of each SIRT6  allele were prepared and assayed. n=3 TR; error bars=SD.",
    "molecules": "mADPr, NAD, NAD+"
  },
  {
    "caption": "C Activation of PARP1  by SIRT6  variants. SIRT6  protein was incubated with human PARP1  protein and then analyzed by immunoblotting  with poly-ADPr antibody. Poly-ADPr  activity of PARP1  results in a wide range of product size. Activity was assessed by quantifying  poly-ADPr signal in whole lanes for each sample. n=3 TR; error bars=SD. Two-way ANOVA. Asterisk indicate p<0.05. Black asterisks indicate significance over HPRT  control. Red asterisks indicated significance over wild type SIRT6.",
    "molecules": "poly-ADPr, Poly-ADPr"
  },
  {
    "caption": "A  qRT-PCR  analysis of LINE1  expression in cumate-inducible SIRT6 fibroblasts. Primers assessed both 5' (ORF1) and 3' (ORF2) LINE1  sequences from the L1HS family of evolutionarily active LINE1  retrotransposons. Assessment of both regions was conducted to mitigate contributions from partial insertion sequences in coding genes. Asterisk indicate p<0.05. n=3 TR; error bars=SD. Students t-test, two tailed.",
    "molecules": "cumate"
  },
  {
    "caption": "F Basal γH2AX foci in cumate-inducible SIRT6 fibroblasts. Foci were scored in at least 80 cells per condition. Asterisk indicate p<0.05. n=3 BR; error bars=SD. Students t-test, two tailed.",
    "molecules": "cumate"
  },
  {
    "caption": "G DNA repair kinetics in cumate-inducible SIRT6 fibroblasts. Cells were grown on slides and irradiated with 2 Gy gamma radiation, followed by immunostaining  for γH2AX. Irradiation  was conducted when the cells were at 75% confluency on slides. Cells were fixed and foci scored at t=0.5 hr, 2 hr, 4 hr, 6 hr, and 24 hr post-irradiation. Foci were scored in at least 80 cells per genotype per time point. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6   allele. n=3 BR; error bars=SD. Students t-test, two tailed.",
    "molecules": "cumate"
  },
  {
    "caption": "H, I Oxidative stress resistance. Cumate-inducible SIRT6 fibroblasts  were induced for SIRT6   expression and exposed to paraquat for 24 hours. Resistance was determined by apoptosis staining  48 hours after exposure. Asterisk indicate p<0.05. Asterisks indicate significance over WT SIRT6. Color of the asterisk corresponds to the SIRT6   allele. n=3 BR; error bars=SD. Students t-test, two tailed.",
    "molecules": "Cumate, paraquat"
  },
  {
    "caption": "B, C Apoptosis staining  of cancer cell lines 48 hours after transfection. Cells were stained  with Annexin V/PI  and analyzed by flow cytometry. Asterisk indicate p<0.05. Red asterisk indicates significance over control, black asterisk indicates significance over WT SIRT6. n=3 BR; error bars=SD. Two-way ANOVA.",
    "molecules": "PI"
  },
  {
    "caption": "B IP  experiments on lysates from cumate-induced fibroblasts  expressing wild type or centSIRT6 alleles with antibodies to SIRT6, LMNA, and mADPr. SIRT6   expression was induced 48 hours prior to IP. n=3 BR. One representative set of IPs  is shown.",
    "molecules": "mADPr, cumate"
  },
  {
    "caption": "C CentSIRT6 shows stronger interaction with LMNA  compared to the wild type SIRT6. Quantification of the IP  experiment shown in (B). SIRT6 IP  followed by  Western blot  with antibodies to LMNA. D LMNA  shows enhanced interaction  with centSIRT6 compared to the wild type. Quantification of the IP  experiment shown in (B). LMNA IP  from cumate-inducible SIRT6 fibroblasts  followed by Western blot  with antibodies to SIRT6. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05",
    "molecules": "cumate"
  },
  {
    "caption": "E centSIRT6  shows enhanced mADPr. Quantification of the IP experiment shown in (B). SIRT6 IP  from cumate-inducible SIRT6 fibroblasts  followed by Western blot with antibody to mADPr  residues F LMNA  shows enhanced mADPr  signal in cells expressing centSIRT6. Quantification of the IP  experiment shown in (B). IP  with mADPr  antibody using extract from cumate-induced SIRT6 fibroblasts, followed by Western blot  with antibodies to LMNA. Data information: n=3 BR; error bars=SD. Students t-test, two tailed. Asterisk indicate p<0.05",
    "molecules": "mADPr, cumate"
  },
  {
    "caption": "Down regulation of SREBP target genes in Cideb-deficient liver. Microarray heatmap of fatty acids/cholesterol synthesis genes that were down-regulated in Cideb-/- mice and Scap liver-specific knockout mice, comparing to the respective WT controls (WT VS Cideb-/-, WT VS L-Scap-/-, n = 2 per group). The expression level of each gene was linearly normalized to range from -1 to 1 before plotting.",
    "molecules": "cholesterol, fatty acids"
  },
  {
    "caption": "Decreased SREBP processing and maturation in Cideb deficient liver. Immune blotting (IB) of the full length, precursor SREBP-1/2 (SREBP FL) and the cleaved, active SREBP-1/2 (SREBP N) from WT and Cideb-/- mice liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting.",
    "molecules": "fat, carbohydrate"
  },
  {
    "caption": "Defective SREBP transcriptional response to refeeding in Cideb-deficient liver. Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb-/- liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting (n = 3 per group).",
    "molecules": "fat, carbohydrate, cholesterol"
  },
  {
    "caption": "Liver morphology of WT and Cideb-/- liver under chow diet and HFLF diet. H/E staining, oil red-O staining, immunohistochemistry and ultra-structure (Electron Microscope) were performed. Scale bar represented 50 µm in the upper three rows of images, and 2 µm in the bottom row of images. LD: lipid droplet, ER: endoplasmic reticulum, M: mitochondrial, N: nucleus.",
    "molecules": "oil red-O"
  },
  {
    "caption": "Total liver TAG levels (B) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.",
    "molecules": "TAG"
  },
  {
    "caption": "Liver cholesterol ester levels (C) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.",
    "molecules": "cholesterol ester"
  },
  {
    "caption": "Serum fed glucose levels (G) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.",
    "molecules": "glucose"
  },
  {
    "caption": "Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A-Flag-BirA*. Cells were cultured in sterol-depleted medium and treated with 80 µM biotin, prior to pulldown with streptavidin-conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS-PAGE.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "Cleaved SREBP bypasses Cideb deficiency. WT and Cideb-/- mice were injected with adenovirus expressing GFP, SREBP-1c full length (FL) or SREBP-1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP-1 target genes in the indicated groups were determined (n=5 per group). Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.",
    "molecules": "TAG"
  },
  {
    "caption": "Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immuno-precipitated from primary hepatocytes under normal medium, 25-HC depletion or addition conditions. And the presence of Cideb in the immune-complex was detected by IB. SREBP-1 was detected as the positive control. Grp94 was detected as the negative control.",
    "molecules": "25-HC"
  },
  {
    "caption": "Sterol-dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag-tagged Insig-1 or Cideb was co-expressed with HA-tagged SREBP-2 and SCAP or SCAP-Y298C in CHO-K1 cells in sterol-depleted medium. When indicated, 10 µg/ml 25-HC were supplemented before cells were immuno-precipitated with an anti-Flag antibody, and the levels of co-immunoprecipitated proteins were detected by IB following SDS-PAGE. The dotted blue vertical line was drawn to separate the result between SCAP-WT and SCAP-Y298C.",
    "molecules": "25-HC, Sterol, sterol"
  },
  {
    "caption": "Cideb relieves sterol-dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag-Cideb were cultured in sterol deprived condition. When indicated, cells were treated with different doses of 25-HC before subjected to IB with the indicated antibodies following SDS-PAGE. Quantification of IB data from 3 independent experiments. Data represents Mean ± SEM.",
    "molecules": "25-HC, sterol"
  },
  {
    "caption": "Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP-SCAP alone or with HA-Cideb were cultured in sterol deprived conditions or treated with 1 µg/ml 25-HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 µm (Merge), and 2 µm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi-positive region per cell (left) and quantification of the percentage of cells containing Golgi-localized GFP-SCAP (right) under indicated conditions, from 3 independent experiments; Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.",
    "molecules": "25-HC, sterol"
  },
  {
    "caption": "Co-localization of SCAP and Cideb at the ER exit sites. HepG2 cells transfected with GFP-SCAP and HA-Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours, then fixed with methanol and stained with antibodies against Sec12 or HA. 3D reconstruction was obtained by processing 3D-original image with Imaris. Scale bars represent 10 µm (Merge), and 1 µm (Inlay and 3D reconstruction).",
    "molecules": "BFA, methanol, Nocodazole"
  },
  {
    "caption": "Localization of Cideb at the ER exit sites. AML12 cells knocked-in with a GFP tag at the C-terminus of Cideb genome was treated with 10 µM BFA for 1 hour, then fixed with methanol and stained with antibodies against Sec12. Scale bars represent 10 µm (Merge), and 1 µm (Inlay).",
    "molecules": "BFA, methanol"
  },
  {
    "caption": "Lysine 128 of Cideb is required for the interaction with Sec12. 293T cells transfected with HA-Sec12 and the indicated Flag-tagged Cideb constructs were subjected to immunoprecipitation with an anti-Flag antibody, and levels of the co-immunoprecipitated HA-Sec12 were detected with an anti-HA antibody.",
    "molecules": "Lysine"
  },
  {
    "caption": "Cideb enriches SCAP to ERESs via interacting with Sec12. CHO-K1 cells transfected with GFP-SCAP, HA-Cideb or HA-Cideb-K128A were arrested with 5 µM Nocodazole for 30 min, then fixed with methanol and stained with antibodies against Sec12 and HA. Scale bars represent 10 µm (left), and 2.5 µm (Inlay).",
    "molecules": "methanol, Nocodazole"
  },
  {
    "caption": " Figure 1b. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Expanded View Fig. 1a. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively. ",
    "molecules": "Ethidium bromide"
  },
  {
    "caption": " Figure 1d. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3-MB-PP1 for indicated times. Long and short exp.= long (4-14 min) and short (10-60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from 3 replicates is shown. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 1e, f. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT-qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (e) and WT CDK12 HCT116 (f) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n=3 replicates, error bars indicate standard error of the mean (SEM). ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation . Flow cytometry profiles of control (-3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig. 2a. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor the 24 h time point is also shown. n=3 replicates; representative result is shown. Figure 2c. Quantification of cells (%) in individual cell cycle phases based on flow cytometry profiles of the representative replicate in Fig. 2b.",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 2f, g. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (f) and BrdU (g) labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig. 2e. CTRL in Fig. 2g = control sample without 3-MB-PP1. n=3 replicates, representative result is shown. ",
    "molecules": "BrdU, 3-MB-PP1, propidium iodide"
  },
  {
    "caption": " Figure 2h. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 µM camptothecin. A representative Western blot from 3 replicates is shown. FUS is a loading control. ",
    "molecules": "camptothecin, CPT"
  },
  {
    "caption": " Figure 3d. Validation of RNA-seq for select DNA replication genes by RT-qPCR. Graph shows relative levels of mRNAs of described genes in serum arrested and released (0 h G0/G1) AS CDK12 HCT116 cells either treated (3-MB-PP1) or not (CTRL) with the inhibitor for indicated times after the release. mRNA levels were normalized to B2M mRNA expression and mRNA levels for each gene at the time of release (0 h) was set as 1. n=3 replicates, error bars indicate SEM. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 3e. Protein levels of core DNA replication factors are dependent on the CDK12 kinase activity. Western blot analyses of protein expression by the depicted antibodies in serum synchronized and released (0 h) cells either treated or not with 3-MB-PP1 for the indicated times after the release. FUS is a loading control. A representative Western blot of 3 replicates is shown. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 3f. CDK12 inhibition affects loading of CDC6 and CDT1 DNA replication factors to chromatin. Western blotting analyses of chromatin association of the indicated DNA replication factors in serum synchronized and released AS CDK12 HCT116 cells treated or not with 3-MB-PP1 for the indicated times. Histone H2A serves as a loading control of chromatin fractions. A=asynchronous cells, 0 h=time of release. A representative Western blot of 3 replicates is shown. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT-qPCR of replication gene mRNA RT-qPCR were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Removal of CDK12 inhibitor in early G1/S rescues cell cycle progression. Western blotting of , protein levels Western blotting were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Removal of CDK12 inhibitor in early G1/S rescues replication and cell cycle progression. flow cytometry analyses of cell cycle progression, flow cytometry analyses were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 4e. Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3-MB-PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated. A representative image of three replicates is shown. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown. ",
    "molecules": "BrdU, 3-MB-PP1"
  },
  {
    "caption": "Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).",
    "molecules": "3-MB-PP1, hydroxyurea"
  },
  {
    "caption": " Figure 5b Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3´ ends Metagene analyses of RNAPII ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have higher relative occupancy of P-Ser2 in their gene bodies. Metagene analyses of P-Ser2 ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 5d Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for MED13 genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance in control and CDK12 inhibited samples, respectively. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 5d e. Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for UBE3C genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance (see Fig. 7d and 7e and corresponding text) in control and CDK12 inhibited samples, respectively. ",
    "molecules": "3-MB-PP1"
  },
  {
    "caption": " Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs. Figure 8c. Proposed model. Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes. The situation in cells with normal and aberrant CDK12 kinase activity is depicted. CDK12 (green oval) phosphorylates (P) unknown substrate(s) (orange oval), possibly including the CTD (blue line), which results in optimal elongation and processivity (blue arrow) of RNAPII (blue oval) for CDK12-sensitive genes. Full length, functional mRNAs are synthesized (upper panel). Inhibition of CDK12 leads to hyperphosphorylation (capital P) of Ser2 (S2) in bodies of CDK12-sensitive genes which is associated with slower elongation and premature termination. Shorter, aberrant mRNAs are made (lower panel). mRNAs are depicted as black lines. ",
    "molecules": "DRB, 3-MB-PP1"
  },
  {
    "caption": "Hindbrain vessel morphology. IsolectinB4 (IsoB4; white), ERG (red) and EdU incorporation (green). EdU signal was masked with ERG signal. Scale bar, 50 µm.",
    "molecules": "EdU"
  },
  {
    "caption": "IsolectinB4 (IsoB4; white), ERG (red), EdU (green) and double-positive cells (yellow). Lower panel shows ERG/EdU merged staining alone with EdU signal masked by ERG signal. Scale bar, 50 µm; error bars: SD; unpaired T test, *p < 0.05. n=5-8 retinas, one retina/mouse.",
    "molecules": "EdU"
  },
  {
    "caption": "VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes in isolated (i)ECs. E. PLA for VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes (black puncta) in iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 show nuclei (grey). Scale bar, 20 µm. F, G. Fold-increase over PBS-treated sample; each dot represents the mean of 6 fields. 2-way ANOVA p=0.0206 (F), p=0.0368 (G); Sidak's multiple comparison test, * p <0.05. n=3 experiments.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "phosphoERK1/2 and phosphoAkt immunostaining (green) of iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 (blue) shows nuclei. Arrows indicate nuclear accumulation of phosphoERK1/2 and phosphoAkt, respectively. Scale bar, 20 µm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "Proliferation of C57Bl/6 and FVB iECs. CD31 (white), ERG (red), EdU (green) and double-positive cells (yellow) in C57Bl/6 (F) and FVB (G) iECs. EdU signal was masked with ERG signal.",
    "molecules": "EdU"
  },
  {
    "caption": "Rescue of Ec proliferation. Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. ERG (red), EdU (green) and double-positive cells (yellow; arrows).",
    "molecules": "EdU"
  },
  {
    "caption": "Rescue of Ec proliferation. Quantification of EdU/ERG double positive ECs normalized to total ECs. Scale bar, 50 µm. Error bars: SD; unpaired T test, *** p <0.001, **** p <0.0001. n=3-7 explants, one explant/embryo.",
    "molecules": "EdU"
  },
  {
    "caption": "(B) Purified GST and GST-tagged DID proteins (50 nM) were mixed with biotin-labeled DADs (WT; R1204X; and M1190D, 50 nM) in binding buffer. After rotation, streptavidin-coupled magnetic beads were added to the solution, and the mixture was agitated. The material absorbed to the beads was eluted in Laemmli's sample buffer, and were subjected to SDS-PAGE, followed by immunoblotting using an HRP-conjugated GST Ab. Comparable levels of input (GSTproteins) are confirmed in the right panel. Representative of 5 experiments.",
    "molecules": "biotin"
  },
  {
    "caption": "Loss of LRRC8D causes resistance to carboplatin and cisplatin, but not to oxaliplatin. Survival of parental, vector‐transduced, or LRRC8D‐deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin, carboplatin, and oxaliplatin. The corresponding IC50 values and 95% confidence interval (CI) are given in Appendix Table S2. Data are presented as mean ± SEM.",
    "molecules": "carboplatin, cisplatin, oxaliplatin"
  },
  {
    "caption": "A, BVRAC currents (ICl,vol) of the HAP1 (A) and KBM7 (B) haploid cell lines. Left panels, example current traces of ICl,vol fully activated by hypotonic cell swelling measured with the voltage‐clamp protocol shown in (A). Dashed lines indicate zero current. Right panels, averaged current/voltage relationships of maximally activated ICl,vol. Consistent with VRAC currents, they needed hypotonic swelling for activation, displayed an I− > Cl− permeability sequence, and were blocked by DCPIB (Appendix Fig S2A-H). The difference in current inactivation between HAP1 and KBM7 cells can be explained by the fact that KBM7 cells hardly express LRRC8E (Fig EV1) which accelerates VRAC inactivation (Voss et al, 2014). At potentials > +100 mV, also KBM7 currents inactivated (Appendix Fig S2I). Data are presented as mean ± SEM; n = 5-10.C",
    "molecules": "DCPIB"
  },
  {
    "caption": "CDependence of regulatory volume decrease (RVD) of HEK cells on LRRC8 genes. Cells were exposed to hypotonic medium starting at t = 0, and intracellular calcein fluorescence was followed over ˜1 h as semiquantitative measure of cell volume. Data are presented as mean values ± SEM from sixteen wells.",
    "molecules": "calcein"
  },
  {
    "caption": "LRRC8 subunit‐ and osmolarity‐dependent caspase induction in HCT116 cellsA, BCisplatin‐induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions (A), or after 1 h exposure to 200 μM cisplatin under iso‐ and hypotonic conditions (B), was followed over time in WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells. Results from HAP1−/− and LRRC8D−/− were obtained with two different clonal cell lines each and averaged.CCaspase activation after 1‐h exposure to 4 μM staurosporine under iso‐ or hypotonic conditions of WT, HAP1−/−, LRRC8D−/−, and LRRC8 −/−HCT116 cells.Data information: Data are presented as mean ± SEM, n = 3-6. *P < 0.05; **P < 0.01; and ***P < 0.001. Similar results were obtained in three independent experiments. Fold change in (A) refers to t = 0. Control experiments indicated that hypotonicity per se had no effect (Appendix Fig S4).",
    "molecules": "Cisplatin, cisplatin, staurosporine"
  },
  {
    "caption": "Activation of LRRC8 channels by pro‐apoptotic stimuliA-CCisplatin‐induced iodide influx into WT (black ■), but not HAP1−/− (green ▼) HEK cells indicates VRAC halide current activation during apoptosis. Cells expressing an iodide‐sensitive YFP variant were exposed to 200 μM cisplatin for periods of 0.5 h (A), 4.5 h (B), or 8.5 h (C) before adding extracellular I− (50 mM final). The difference in slopes of YFPfluorescence quenching between control and cisplatin‐treated cells semiquantitatively reflects VRAC current activation. Note that increased YFP quenching with cisplatin preincubation is not due to large non‐specific leaks resulting from cell morbidity. Such leaks should lead to a fast component of YFP quenching in WT, but not HAP1−/− cells after the pipetting artifact that immediately follows addition of iodide (indicated by arrows).DSwelling‐induced iodide influx into WT (black ■) and HAP1−/− (green ▼) HEK cells for comparison. Iodide (50 mM final) was added in isotonic or hypotonic (230 mOsm final) solution at the time indicated by arrow.E, FTime course of VRAC activation by 200 μM cisplatin (E) or 4 μM staurosporine (F) determined as in (A-C). Averaged maximal slopes of YFP quenching from eight wells (E) or 16 wells (F) each were evaluated to estimate iodide influx rates. WT (black ■) and HAP1−/− (green ▼). Data are presented as mean ± SEM.",
    "molecules": "Cisplatin, cisplatin, iodide, staurosporine"
  },
  {
    "caption": "Carboplatin uptake into control KBM7 cells and two LRRC8D‐deficient clones (n = 6).",
    "molecules": "Carboplatin"
  },
  {
    "caption": "Hypotonicity‐stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone (CBX), a non‐specific blocker of VRAC (n = 3).",
    "molecules": "carbenoxolone, cisplatin"
  },
  {
    "caption": "Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long‐term isotonic (C) or 200 μM cisplatin in short‐term hypo‐ and isotonic (D) conditions as function of time (n = 3). Similar results were obtained in HCT116 cells (Fig EV4).",
    "molecules": "Cisplatin, cisplatin"
  },
  {
    "caption": "Cisplatin uptake (200 μM) into HEK cells of indicated genotypes. LRRC8(B,C,E) −/− and LRRC8(B,D,E) −/− cells express only LRRC8A and LRRC8D, and LRRC8A and LRRC8C, respectively (n = 3 for WT, 6 for LRRC8D −/−, 9 for LRRC8A −/− in E; n = 3 in F).",
    "molecules": "Cisplatin"
  },
  {
    "caption": "Ratio of LRRC8‐dependent swelling‐activated cisplatin uptake (60 min) to mean ICl,vol (as in G) as function of genotype.Data information: Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 (for (C, E) compared to HAP1−/− cells). Dotted lines in (D) and (F) highlight that there is no significant difference in isotonic 60‐min uptake between the genotypes.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Cisplatin-DMSO inhibition of ICl,vol depends on the LRRC8D subunitUpper panel, example current traces (as in Fig 3A) of fully activated ICl,vol in HEK cells exposed to hypotonic solution containing vehicle (0.3% DMSO) or 200 μM cisplatin in 0.3% DMSO. Dashed lines indicate zero current. Lower panel, ICl,vol current densities (at −100 mV and 100 mV) of WT HEK cells treated with different cisplatin concentrations.No effect of 200 μM cisplatin/DMSO on ICl,vol in LRRC8D−/− HEK cells.Data information: Data are presented as mean ± SEM; the number of experiments is given for each bar; *P < 0.05; **P < 0.01.",
    "molecules": "cisplatin, DMSO"
  },
  {
    "caption": "Swelling‐induced efflux of 3[H]‐taurine from WT and LRRC8D −/− HEK cells (A, B) and partial rescue by transient transfection of LRRC8D (B). Rescue is incomplete due to low transfection/expression efficiency of LRRC8D (Voss et al, 2014).",
    "molecules": "taurine, 3[H]"
  },
  {
    "caption": "C-ESwelling‐induced efflux of 3[H]‐taurine from LRRC8(B,C,E) −/− (C), LRRC8(B,D,E) −/− (D), or LRRC8(B,C,D) −/− (E) HEK cells compared to WT cells.FCisplatin‐induced taurine efflux (over 30 min) from WT and HAP1−/−HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h, or without cisplatin (0 h). Carbenoxolone (CBX; 100 μM) blocks taurine efflux from WT cells treated for 12 h with cisplatin, excluding taurine flux through unspecific leaks.Data information: Data are presented as mean ± SEM. Dashed lines in (A-E) represent isotonic efflux from the same individual experiments. Red arrows, change to hypotonic solution. Each panel shows the average of two independent experiments (n = 4 for each experiments, i.e., n = 8 in total).",
    "molecules": "Carbenoxolone, Cisplatin, cisplatin, taurine, 3[H"
  },
  {
    "caption": "(B) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.",
    "molecules": "acridine orange"
  },
  {
    "caption": "(C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm.",
    "molecules": "TMP"
  },
  {
    "caption": "(B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control).",
    "molecules": "cycloheximide, puromycin, TMP"
  },
  {
    "caption": "(A) Boxplots of mRNA half-lives (hrs) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP comparing (Tiana et al., 2020). Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values but not further than 1.5× the inter-quartile range (IQR). (B- H) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.",
    "molecules": "actinomycin D, TMP"
  },
  {
    "caption": "(B) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.",
    "molecules": "actinomycin D, TMP"
  },
  {
    "caption": "(B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.",
    "molecules": "acridine orange, propidium iodide, TMP"
  },
  {
    "caption": "(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.",
    "molecules": "TMP"
  },
  {
    "caption": "(C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.",
    "molecules": "Poly(A), poly(A)"
  },
  {
    "caption": "Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (A) Age-matched control Atg7 littermates (Atg7flox/+;nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4) or Atg7 KO (Atg7flox/flox;nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3) are shown.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (B) Control Atg5 littermates (Atg5flox/+;nestin-Cre; denoted +) at 16 (open circles; n = 4) and 26 wk (open diamonds; n = 3) or Atg5 KO (Atg5flox/flox;nestin-Cre; denoted −) at 16 (closed circles; n = 4) and 26 wk (closed diamonds; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(B) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line, m5-7, as measured by the decreased levels of phosphatidylethanolamine-modified LC3 (LC3-II).",
    "molecules": "dox"
  },
  {
    "caption": "(D) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells. The ASI is the ratio of the relative increase in green and red fluorescence, as assessed by two-color flow cytometry, in response to dox. In control experiments, the ASI of two soluble, nonaggregating proteins, htt(Q25)-GFP and chFP, was 1. In contrast, the ASI of the aggregation-prone protein, htt(Q47)-GFP, compared with chFP after autophagy shutoff (+dox; 100 h) was 2. Data represent three independent experiments performed on different days. **, P ≤ 0.01; ***, P ≤ 0.005.",
    "molecules": "dox"
  },
  {
    "caption": "(E) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff (+dox). The degree of cell death correlated directly with increased htt(Q47)-GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt(Q47)-GFP compared with htt(Q25)-GFP (***, P ≤ 0.005). Data represent three independent experiments performed on different days.",
    "molecules": "dox"
  },
  {
    "caption": "(F) AQUA using heavy isotope-labeled GFP standard in addition to the routine Ub standards after P2UBA pull-down of soluble and insoluble lysates from htt(Q)-GFP-IRES-chFP bicistronic expressing cells before (−dox) and after autophagy shutoff (+dox). The experimental GFP peptide was only detected in two of the four experiments, and the data shown are representative of these. In the other two experiments, the experimental GFP peptide was below the threshold of detection.",
    "molecules": "dox"
  },
  {
    "caption": "(H) Sqa, but not Atg1, directly phosphorylated Sqh in vitro. Flag-Atg1, Flag-Atg1‐KR, HA-Sqa and HA-Sqa‐KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [γ‐32P]ATP and bacterially expressed recombinant wild‐type Sqh or SqhA20A21. As shown on the autoradiogram (top panel), wild‐type but not the kinase‐deficient Atg1 (Atg1‐KR) and Sqa (Sqa‐KA) was autophosphorylated. No phosphorylation was seen with SqhA20A21. The equal input of His‐fusion proteins is shown on the Coomassie staining. Anti‐Flag and anti‐HA immunoblottings (IBs) were used as controls to quantify the amount of proteins precipitated.",
    "molecules": "[γ‐32P]ATP"
  },
  {
    "caption": "(A, B) Activation of myosin II on nutrient deprivation. The larval fat body of denoted genotypes under fed or starved conditions were dissected, lysed, and subjected to western blot analysis using antibodies specific for phospho‐myosin regulatory light chain (MRLC) and total MRLC. The Rheb, SqaRNAi, Sqa‐T279A, spaghetti‐squash (SqhA20A21), and Atg7RNAi transgenes were expressed under the control of hs-GAL4 driver (B). For quantification, the relative phosphorylation levels of MRLC were quantified as in Figure 3D. Data are represented as mean±s.e. of triplicates.",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) Myosin II was activated upon nutrient deprivation in MCF7 cells. MCF7 cells were cultured in serum containing DMEM medium (nutrient‐rich condition) or in Earle's balanced salt solution (EBSS; starved condition) for 2 h. The myosin II activity was significantly downregulated after EBSS‐starved cells were replenished with fresh DMEM medium containing 10% FBS for 2 h. The myosin II activity was quantified and expressed as a fold changes compared with the DMEM controls. Each value represents mean±s.e. of triplicates.",
    "molecules": "FBS, nutrient"
  },
  {
    "caption": "(D) MCF7 cells stably infected with lentivirus expressing control (shLuc), Ulk1 or ZIPK shRNA were cultured in nutrient‐rich DMEM medium (F) or EBSS (S) for 2 h. Effects of Ulk1 and ZIPK knockdown on starvation‐induced myosin II activation were analysed by immunoblotting with antibodies as indicated. Data are mean±s.e. of triplicates.",
    "molecules": "nutrient"
  },
  {
    "caption": "(A) MCF7/GFP-LC3 cells stably infected with lentivirus expressing control (shLuc), ZIPK, or non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) shRNA were cultured in nutrient‐rich medium (DMEM) or starvation medium (Earle's balanced salt solution; EBSS) in the presence or absence of lysosomal inhibitor bafilomycin A1 (BafA1) for 2 h. Depletion of ZIPK and NMHC‐IIA markedly inhibited starvation‐induced GFP-LC3 puncta formation. Quantification of the number of GFP-LC3 dots per cell (lower panel) was shown (data are represented as mean±s.e. of 100 cells, ***P0.001).",
    "molecules": "BafA1, bafilomycin A1, nutrient"
  },
  {
    "caption": "(B) Cells as in (A) were cultured in nutrient‐rich medium (F) or EBSS (S) with or without BafA1 for 2 h. Effects of ZIPK and NMHC‐IIA knockdown on starvation‐induced GFP-LC3 conversion were assessed by immunoblotting with anti‐LC3, anti‐ZIPK, anti‐NMHC‐IIA, and anti‐tubulin antibodies. The relative ratio of LC3II/LC3I is shown at the right panel. Data are mean±s.e. of triplicates.",
    "molecules": "BafA1, nutrient"
  },
  {
    "caption": "(A) The fat body of spaghetti‐squash (sqhAX3); sqh-GFP larva under fed or starved conditions were dissected and subjected to immunofluorescence analysis. Sqh-GFP and phospho‐myosin regulatory light chain (MRLC) were enriched in cell-cell junction under nutrient‐rich (fed) condition. Under starvation conditions, Sqh-GFP and phospho‐MRLC were localized to both cell-cell junction and perinuclear region. Actin was stained with TRITC‐labelled phalloidin (red) and nucleus was stained with DAPI (blue). Bar, 20 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(B) Starvation induced redistribution of myosin II in MCF7/GFP-LC3 cells. Both phospho‐MRLC and MRLC were localized in peripheral region of cells in nutrient‐rich medium (DMEM), whereas they redistributed to the perinuclear region and co‐localized with GFP-LC3 under starvation conditions (Earle's balanced salt solution; EBSS). Bar, 10 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) MCF7/GFP-LC3 cells cultured in nutrient‐rich (DMEM) or starved (EBSS) conditions were homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by high‐speed centrifugation into membrane pellet and cytosol. Proteins were resolved by SDS-PAGE and immunoblotted with anti‐TGN46 antibody as a control for membrane‐association proteins, anti‐caspase‐3 as a control for cytosolic proteins. The levels of phospho‐MRLC and MRLC in each fraction were quantified using ImageJ and plotted relative to their amounts in PNS (n=3). Each value represents mean±s.e. of three experiments. *P0.05, **P0.01.",
    "molecules": "nutrient"
  },
  {
    "caption": "(A) Immunofluorescence analysis of GFP-mAtg9 and phospho‐myosin regulatory light chain (MRLC) in MCF7/GFP-mAtg9 cells. GFP-mAtg9 was enriched in the trans‐Golgi network (TGN) labelled with anti‐TGN46 antibody and phospho‐MRLC localized to cell peripheral in nutrient‐rich condition (DMEM). GFP-mAtg9 redistributed and co‐localized with phospho‐MRLC in starvation condition (Earle's balanced salt solution; EBSS).",
    "molecules": "nutrient"
  },
  {
    "caption": "(B) mAtg9 interacted with non‐muscle myosin heavy chain‐IIA (NMHC‐IIA) under starvation conditions. 293T cells (right panel) or 293T cells stably infected with lentivirus expressing control (shLuc), UNC‐51‐like kinases (Ulk1) or zipper‐interacting protein kinase (ZIPK) shRNA (left panel) were transfected with V5‐tagged mAtg9 and GFP-tagged NMHC‐IIA. At 48 h after transfection, cells were incubated in either serum‐containing medium (DMEM) or EBSS for 2 h with or without the treatment of 50 μM ML‐7 and blebbistatin (Blebb). Cell lysates were immunoprecipitated with anti‐V5 antibody and immunoblotted with indicated antibodies.",
    "molecules": "Blebb, blebbistatin, ML‐7"
  },
  {
    "caption": "(f) Wild-type and A20 deficient BMDMs were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for RANK, A20, TRAF6, HOIP and Sharpin.",
    "molecules": "sepharose, glutathione"
  },
  {
    "caption": "(e) BMDMs isolated from control (A20WT), A20ZnF7, A20ZnF4ZnF7 and A20DUB mice were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.",
    "molecules": "sepharose, glutathione"
  },
  {
    "caption": "(f) Wild-type BMDMs were pretreated for 1h with HOIPIN-8 (50µM) or left untreated and stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.",
    "molecules": "sepharose, glutathione, HOIPIN-8"
  },
  {
    "caption": "H. Endogenous levels of Stim1 in testes  from C57BL/6 wt and splice deficient 10A  mice, using an antibody detecting the N-term of  STIM1, detecting STIM1A  (A, red arrow), STIM1 and its  glycosylated  (wt) and unglycosylated  (wt gl.) form.",
    "molecules": "glycosylated, unglycosylated"
  },
  {
    "caption": "A. Traces showing average changes (mean±s.e.m.) in  intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar in MEF Stim1/Stim2-/- cells transfected with Stim1- (black trace, n=111), Stim1A (red trace, n=116), the combination Stim1 with Stim1A (blue trace, n=86) or with vector only (grey trace, n=79). B. Quantification of changes in ratio of resting,  influx rate (Δratio/time), Δpeak and Δplateau measured in A. *** p<0.001, Kruskal-Wallis Anova. C. Traces showing average changes (mean±s.e.m.) in   intracellular Ca2+  (Fura 2 ratio) in  HEK293 cells co-transfected with  Orai1    and either Stim1- (black trace, n=119) or Stim1A  IRES-mCherry (red trace, n=118). D. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in C. ** p<0.01 *** p<0.001 Mann-Whitney test. E. Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.",
    "molecules": "Ca2+, Fura 2, Fura2"
  },
  {
    "caption": "I. Traces showing average changes (mean±s.e.m.) in  intracellular Ca2+ (Fura 2 ratio) in primary astrocytes  from C57BL/6 wt (CTRL,black trace, n=49) and splice deficient C57BL/6 10ACon mice  (10ACon, red trace, n=82). J. Quantification of parameters determined in [I]. *** p<0.001 Mann-Whitney test. Data information: D were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.",
    "molecules": "Ca2+, Fura 2"
  },
  {
    "caption": "C. Images showing representative HEK293 cells expressing Orai1-GFP (green = donor molecule) or either STIM1-mCherry (upper panel = acceptor molecule) or  STIM1A-mCherry  (lower panel = acceptor molecule) after stimulation with TG. Also shown is the overlay of the two channels, as well as the FRET  signal.",
    "molecules": "TG"
  },
  {
    "caption": "B. Traces showing average changes (mean±s.e.m.) in  intracellular Ca2+ (Fura2 ratio) over  time  in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKO1.  STIM1  (black, n=145), STIM1A  (red, n=157) and STIM1A_D503A (n=185). C. Relative fluorescence intensities of mCherry-tagged constructs measured in (B). D. Quantification of changes in resting ratio, TG  peak (∆ ratio) and SOCE parameters measured in B. E. Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.",
    "molecules": "Ca2+, Fura2, TG"
  },
  {
    "caption": "F. Traces showing average changes (mean±s.e.m.) in  intracellular Ca2+ (Fura2 ratio) over  time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKS1/S2-/-. G. Quantification of the relative reduction of ratio/time  or ratio for cells (135 H Data information: , ***P<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test Data points (total n) were obtained from three biological replicates with each three technical replicates (with multiple cells) and shown as mean±s.e.m for traces (F) and as scatter plots with the underlying boxes showing the means for individual parameters (G).",
    "molecules": "Ca2+, Fura2"
  },
  {
    "caption": "H,I. Average traces (mean±s.e.m.) showing  whole- cell current density (CD) over  time extracted at -80 mV in in HEKS1/S2-/- cells co-transfected either with STIM1  (black) or STIM1A (red) and ORAI1 (H) or with ORAI1  R77E (I) and recorded using extracellular solution containing 2 mM  Ca2+. J. Average maximum  CDs recorded from cells measured in [H,I] (n within bars). Data information: I , *P<0.05, **P<0.01, unpaired T test with Welch's correction. For patch-clamp experiments, number in bars indicate measured cells, which were from three independent transfections.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D. Ratio  nuclear NFAT-GFP  vs. cytosolic GFP  intensity normalized to t=0 (mean±s.e.m.) after transfection of SH-SY5Y S1-/- cells with STIM  (n=42), STIM1A (n=82), STIM1A_D503A (n=49) or vector only (ØSTIM, n=15) IRES mCherry and induction of SOCE after stimulation with 1 µM TG. Data (total n as indicated) from at least three independent transfections.",
    "molecules": "TG"
  },
  {
    "caption": "A. Time course  of translocation (Nuclear NFAT-GFP signal/cytosolic signal normalized to TG addition at t=0, mean±s.e.m, for each time point) after transfection of indicated constructs and preincubation with 1 µM PF-04957325 (analyzed cells: STIM1 + PF n = 75, STIM1A + PF  n = 50, ØSTIM + PF n = 9) Data (total n as indicated) from at least three independent transfections. * p< 0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test. B. PF-04957325 induced difference of the  nuclear NFAT-GFP signal vs. cytosolic signal from (A) to mean of control without the blocker (see Fig. 6D). * p< 0.05, **p<0.01, ***p<0.001,Mann-Whitney test of differences.. C Data information: *p<0.05, **p<0.01, ***p<0.001, 2way Anova Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.",
    "molecules": "PF, PF-04957325, TG"
  },
  {
    "caption": "C. Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment, shown as mean±s.e.m , ØSTIM + 6Bnz-cAMP/8-pCPT-cAMP n = 15/14 cells; STIM1  +  6Bnz-cAMP/8-pCPT-cAMP: n = 87/66, STIM1A + 6Bnz-cAMP : n = 78/67; from three independent transfections. Data information: *p<0.05, **p<0.01, ***p<0.001, , Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.",
    "molecules": "8-pCPT-cAMP, 6Bnz-cAMP, PF"
  },
  {
    "caption": "G. Normalized fluorescence signal change (mean±s.e.m) of  R-FlincA, SH-SY5Y S1-/- cells expressing STIM1 (n=20) or  STIM1A  (n=22) IRES  GFP variants and the indicator were stimulated by TG (1 μM). To avoid photobleaching, values were only recorded after 5 and 30 min, respectively The mutant R-Flinc (n=15) was measured in SH-SY5Y  wt cells after TG stimulation. Data information: *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.",
    "molecules": "TG"
  },
  {
    "caption": "Cells containing Rfa1-TAP were synchronized at G1, S, and G2/M phase and used for the TAP purification. Rfa1-TAP associated protein complexes were analyzed by Western blot using CBP, Rfa2, and Rtt105 antibodies (left panel). DNA content was monitored by flow cytometry (right panel).",
    "molecules": "DNA"
  },
  {
    "caption": "MBP-Rtt105 pulled down recombinant RPA, resolved on SDS-PAGE gels and visualized by Coomassie Brilliant Blue (CBB) staining. MBP was used as a negative control.",
    "molecules": "CBB"
  },
  {
    "caption": "GST-tagged full length (FL) and Rtt105 deletion mutants (Schematic of the Rtt105 truncations is shown in the upper panel) were purified and used to pull down Rfa1 protein. GST proteins were used as a negative control. Isolated protein complexes were resolved on 15% SDS-PAGE gels and visualized by CBB staining.",
    "molecules": "CBB"
  },
  {
    "caption": "Snapshots of Rfa1 ChIP-Seq peak at ARS305 from WT and rtt105∆ cells released into HU-medium. The average Rfa1 ChIP-Seq read density from cells released into HU-medium around ACS sites. ACS: ARS consensus sequence.",
    "molecules": "HU"
  },
  {
    "caption": "NLS-RFA1-GFP rescues the nuclear localization of Rfa1 in rtt105∆ mutant cells. The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence (NLS) at its 5'-end and a GFP gene at its 3' end to obtain a construct, driven by the RFA1 promoter to express NLS-RFA1-GFP fusion protein. The engineered construct was then transformed into WT or rtt105∆ mutant yeast cells to replace its endogenous RFA1 expression. The resulting yeast cells were then visualized under microscope, and GFP signals enriched in the nuclei were scored. RFA1-GFP lacking the NLS sequence was transformed into rtt105∆ rfa1∆ mutant cells as a control. DAPI staining indicates nuclear DNA.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105∆ cells. The percentage of Rfa2 ChIP-DNA over the total input DNA was calculated. The mean and standard error (SE) of three biological replicates were shown. Statistical significance was evaluated based on Student's t-tests (*: 0.01≤P value<0.05).",
    "molecules": "DNA"
  },
  {
    "caption": "An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105∆ cells. The percentage of Rfa2 ChIP-DNA over the total input DNA was calculated. The mean and standard error (SE) of three biological replicates were shown. Statistical significance was evaluated based on Student's t-tests (*: 0.01≤P value<0.05).",
    "molecules": "DNA"
  },
  {
    "caption": "EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105-RPA complex. Cy3-labeled 30nt ssDNA was used as the substrate, and reaction mixtures were resolved on native PAGE gels. Quantification of the RPA-bound ssDNA is shown in (C).",
    "molecules": "ssDNA"
  },
  {
    "caption": "Recombinant Rtt105-RPA complex was mixed with a 30nt-ssDNA in a 2:1 ratio, and the reaction product were subjected to gel filtration chromatography. Equal volume of fraction 5-10 were resolved on SDS-PAGE for detecting proteins by Ruby staining (upper panel) or were resolved by native agarose gel for detection of Cy3-ssDNA signal (lower panel).",
    "molecules": "agarose, ssDNA"
  },
  {
    "caption": "GST-RPA pull down Rtt105 with increased amount of 30nt ssDNA, and the bound proteins were resolved on SDS-PAGE and visualized by silver staining.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Kymograms showing most ssDNA molecules two distinct stretching patterns of RPA-ssDNA molecules. Pattern 1, slow stretching; Pattern 2, fast stretching. The counts of the two patterns in each reaction were plotted in (C). Please note a fraction of individual ssDNA shows both patterns. Rtt105 increases the rate of ssDNA stretching upon RPA binding. Stretching rate was defined as the length change of ssDNA upon binding to RPA-mCherry in unit time as shown in (B, white arrow). Rtt105 increases the extent of ssDNA stretching upon RPA binding. Length increment = DNA length at 8 minutes / DNA length at 3 minutes.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium containing HU for 45 min. The average BrdU IP-Seq read density around fired ACS from cells released into YPD medium containing HU for 45 min sites. Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium at 16°C for 72 min. The average BrdU IP-Seq read density around ACS sites from cells released into 16°C for 72 min.",
    "molecules": "HU"
  },
  {
    "caption": "Rad53 phosphorylation was analyzed in WT and rtt105∆ cells by immunoblotting of protein extracts with anti-Rad53 antibodies. Ponceau S (Pon S) staining was applied as a loading control.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "Ten-fold serial dilutions WT or rtt105∆ cells in two different backgrounds were assayed on normal growth media (YPD) and on media containing the indicated DNA damaging agents, methyl-methane sulfonate (MMS), camptothecin (CPT), bleomycin (Bleo), and hydroxyurea (HU).",
    "molecules": "Bleo, bleomycin, camptothecin, CPT, HU, hydroxyurea, methyl-methane sulfonate, MMS"
  },
  {
    "caption": "A. Tunicamycin (Tn, 2 µg/ml) increased Nox4 protein levels in H9c2 cells. Tubulin was used as a loading control. n=4-6/group. *, significant compared to baseline.",
    "molecules": "Tn, Tunicamycin"
  },
  {
    "caption": "B. Thapsigargin (Tp, 1 µg/ml) increased Nox4 protein levels in H9c2 cells. n=4-6/group. *, significant compared to baseline.",
    "molecules": "Thapsigargin, Tp"
  },
  {
    "caption": "C. Effect of Nox4 on the unfolded protein response. Nox4 was depleted in H9c2 cells by shRNA-mediated knockdown (Ad.shNox4) or cells were treated with a control adenovirus (Ad.Ctl). In cells with Nox4 knockdown, tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94, Grp78 and calreticulin than in conrol cells. Nuclear protein levels of ATF4 were substantially lower in Nox4-depleted cells than control cells but the levels of cleaved ATF6 (ATF6c) were similar. Histone was used as a loading control. The relative mRNA levels of Xbp1-s (a readout of IRE1 signaling) were unaltered after Nox4 knockdown. Mean data are shown in Appendix Fig S1E. Similar results were obtained with an independent siRNA approach (Appendix Fig S2A).D. Effect of adenoviral-mediated overexpression of Nox4 (Ad.Nox4) or a control β-galactosidase protein (Ad.β-Gal) on tunicamycin responses of H9c2 cells. Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin-induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels. Mean data are shown in Appendix Fig S2B.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "E,F. Effect of Nox4 knockdown or overexpression, respectively, on the tunicamycin-induced changes in mRNA levels of ATF4 target genes. n=4/group. Psat1, phosphoserine aminotransferase; Phgdh, 3-phosphoglycerate dehydrogenase; Asns, asparagine synthetase; Slc6a9, glycine transporter 1.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "G. Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin-treated H9c2 cells. Scrambled siRNAs were used as a control (Ctl). Representative immunoblots shown to the top (captions at bottom of bar graphs refer also to the immunoblots); tubulin was used as a loading control. n=4/group. *, significant compared to baseline; #, significant comparing siATF4 versus corresponding siCtl.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "A. The knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin-induced eIF2a phosphorylation in H9c2 cells, with no change in phospho-Thr980-PERK (PERK-P) levels. GADD34 levels were significantly decreased after Nox4 knockdown while there was no change in PP1 protein levels.B. Overexpression of Nox4 in H9c2 cells caused prolongation of tunicamycin-induced eIF2a phosphorylation, with minimal change in phospho-PERK levels.C,D. Mean levels of phosphorylated eIF2a relative to total eIF2a protein in tunicamycin-treated cells after Nox4 knockdown or overexpression, respectively. n=3/group. *, significant compared to baseline; #, significant comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).",
    "molecules": "tunicamycin"
  },
  {
    "caption": "E,F. Effect of Nox4 knockdown or overexpression, respectively, on okadaic acid-resistant Ser/Thr phosphatase activity in membrane fractions of tunicamycin-treated H9c2 cells. n=4/group. *p<0.05, **p<0.01 cf. baseline; #, p<0.05, ##, p<0.01 comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).",
    "molecules": "okadaic acid, tunicamycin"
  },
  {
    "caption": "I. Nox4-/- MEF cells (KO) showed blunted tunicamycin-induced increases in levels of phospho-eIF2a, ATF4 and ER chaperones as compared to wild-type (WT) MEFs, a response that was rescued by re-introduction of Nox4 (KO+Nox4). The latter had no effect on GS-P or H3-P levels.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "A. Sub-cellular localization of Nox4. Tunicamycin (Tn 2 µg/ml, 6h) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy (scale bar, 10 µm).3D SIM images (scale bars, 2 µm) showed localisation of Nox4 (green) to the ER, which was labeled with an anti-KDEL antibody (red). At higher magnification (right), yellow dots denote co-localization of Nox4 and KDEL signals. Cell nuclei were stained with DAPI (blue). 1 Z slice from 3D stack is shown.",
    "molecules": "Tn, Tunicamycin"
  },
  {
    "caption": "B. Progressive enrichment of GADD34, PP1, eIF2α and Nox4 in membrane fractions of tunicamycin-treated H9c2 cells.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "C. After sucrose gradient fractionation of lysates of tunicamycin-treated H9c2 cells, GADD34, PP1 and Nox4 co-eluted in fractions 12 and 13 (F12, F13).",
    "molecules": "tunicamycin"
  },
  {
    "caption": "G. Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto, respectively, in tunicamycin-treated MEF cells. Redox-insensitive mutant probes were used as negative controls and to exclude pH changes. Extracellular H2O2 (200 nM) was added as a positive control. The pseudocolor scale is shown along the left vertical edge of each image. KO = Nox4-/-. Scale bars, 2 µm.",
    "molecules": "H2O2, ROS, tunicamycin"
  },
  {
    "caption": "A. Recombinant PP1 was inhibited by H2O2 (0.2 mM) and activity was not restored by glutathione (GSH), cysteine (Cys) or dithiothreitol (DTT). A Cys127Ser/Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild-type PP1. Values above bars denote level of significance for the inhibitory effect of H2O2.",
    "molecules": "Cys, cysteine, dithiothreitol, DTT, glutathione, GSH, H2O2"
  },
  {
    "caption": "B. Ascorbate (Asc) dose-dependently restored PP1 activity. #, significant effect of Asc compared to H2O2 alone.",
    "molecules": "Asc, Ascorbate, H2O2"
  },
  {
    "caption": "C. EPR spectra of PP1 incubated with ascorbate (1 mM) alone (a) or PP1 exposed to H2O2 followed by catalase treatment, then incubation with ascorbate (b). Panel b shows a typical spectrum for the ascorbyl radical (hyperfine splitting constant, aH = 1.8G), similar to the positive control obtained by exposing ascorbate to H2O2 (c). Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1, prior to ascorbate addition.",
    "molecules": "ascorbate, H2O2"
  },
  {
    "caption": "D. Cartoon representation of the active site of H2O2-treated PP1 as in Fig EV2D. 2mFo-DFc electron density map at the 2.2 Å resolution is shown in yellow at the 1.1 level. H2O2-treatment causes an overall shrinkage of the PP1 coordination sphere by 0.12 Å compared to ascorbate-treated crystals consistent with the oxidation of the dinuclear center. This increases the energy barrier for the catalytic steps involving -OH- attack on the phosphorous center of the bridging phosphate and rupture of the P-O scissile bond with the assistance of H125 (black arrows). Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit. See also Appendix Table S1.",
    "molecules": "H2O2"
  },
  {
    "caption": "F. Effect of ascorbate (Asc, 0.5 mM) on phosphatase inhibition in tunicamycin-treated H9c2 cells with overexpression or knockdown of Nox4 (Ad.Nox4 and Ad.shNox4, respectively). In control cells, ascorbate enhanced tunicamycin-stimulated increases in phosphatase activity. Phosphatase activity was lower in Nox4-overexpressing than control cells but was normalized by ascorbate to the same level as in control cells. In Nox4 knockdown cells, tunicamycin-induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect.",
    "molecules": "Asc, ascorbate, tunicamycin"
  },
  {
    "caption": "A. H9c2 cells treated with tunicamycin (2 µg/ml, 12h) showed significantly lower survival when endogenous Nox4 was silenced (siNox4) as compared to cells treated with a scrambled siRNA (siCtl). Cell survival was restored by treatment with either guanabenz (Gbz, 5 µM) or salubrinal (Sal, 50 µM) but was unaffected by clonidine (Cld, 5 µM). n=3/group.",
    "molecules": "Cld, clonidine, Gbz, guanabenz, Sal, salubrinal, tunicamycin"
  },
  {
    "caption": "B. Nox4-depleted cells had lower levels of phospho-eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz (Gbz).",
    "molecules": "Gbz, guanabenz"
  },
  {
    "caption": "D. Hearts from Nox4 knockout (KO) mice and WT controls were subjected to global ischemia followed by aerobic reperfusion (I/R). Infarct size assessed by triphenyltetrazolium chloride (TTC) staining was greater in Nox4 KO hearts compared to WT and was significantly reduced by guanabenz (Gbz). In the representative heart sections shown at the top, white denotes infarct area and red the viable area. Scale bars, 1 mm. Numbers of hearts are indicated within the bars.",
    "molecules": "Gbz, guanabenz"
  },
  {
    "caption": "E. Immunoblotting of heart homogenates after I/R showed lower levels of phospho-eIF2a, ATF4 and ER chaperones, and higher levels of cleaved caspase-12, in Nox4 KO compared to WT. Tubulin was used as a loading control. Treatment with guanabenz (Gbz) reversed these changes (blots shown to the right).",
    "molecules": "Gbz, guanabenz"
  },
  {
    "caption": "A. Plasma urea levels were elevated to a greater extent in tunicamycin-treated Nox4 KO mice than WT. Co-treatment with guanabenz (Gbz) reduced urea levels in both groups. Numbers of animals are indicated within bars.",
    "molecules": "Gbz, guanabenz, tunicamycin, urea"
  },
  {
    "caption": "B. 48 hours after systemic tunicamycin treatment, kidneys of Nox4 KO mice showed a marked surface pallor (bottom right).",
    "molecules": "tunicamycin"
  },
  {
    "caption": "C. TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin-treated Nox4 KO mice. n=4/group.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "D. Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase-12 and cleaved PARP levels in tunicamycin-treated Nox4 KO mice compared to WT.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "E. Survival curves showed that a very high proportion of Nox4 KO mice died after AKI. Guanabenz (Gbz) treatment dramatically improved survival in tunicamycin-treated KO mice. Number of animals as indicated. Levels of significance by Kaplan Meier analysis are reported to the right.",
    "molecules": "Gbz, Guanabenz, tunicamycin"
  },
  {
    "caption": "C. After 1, 8, 24 and 48 h of 4-hydroxytamoxifen treatment, genomic DNA was extracted and quantified by semi-quantitative PCR (see Appendix Figure S6). Error bars correspond to the standard deviation for PCR duplicates. The percentages of cells with the large 1+3 and loxP_LE (1) + βA-globin+β-actin (3) or β-actin (1+3) excision at each time point are shown for different cell lines.",
    "molecules": "4-hydroxytamoxifen"
  },
  {
    "caption": "A, B. Clones 1+2+3, 1+3 (A) and loxP_LE (1) + βA-globin+β-actin (3) (B) were used to assess chromatin accessibility  within the transgenes. The positions of the amplicons used for quantification in each shifting construct individually (thick black lines; 5' 2xFIV-1 or -3,   β-actin pro-1 or -3, BsR gene, PuroR gene, LoxP site), in both shifting constructs (βA  pro 1 and 2, IL2R gene, 3' 2xFIV1-3) or in the GFP reporter construct (βA-GFP, GFP gene, h.K7) are shown. The insertion sites on the wt chromosome  (insertion sites 1, 2 and 3) and a genomic region located 5 kb downstream from the insertion site (insertion site +5kb) were used as controls for the targeted genomic region. The endogenous active MED14  promoter was analyzed as a control. Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Quantifications by  real-time qPCR  of total chromatin  (input) extracted from two 1+2+3, 1+3 and loxP_LE (1)+ βA-globin+β-actin (3) clonal cell lines, after digestion with micrococcal nuclease (MNAse, 2.5 U, 10 U, 40 U, 160 U/mL) and size selection were shown. Error bars indicate the standard deviation for qPCR triplicates, for two independent clones. Data are presented as total chromatin  input relative to the condensed  genomic regions (cond1 and cond2) used for the normalization. ",
    "molecules": "MNAse"
  },
  {
    "caption": "A, B.  UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites (genomic positions: chr1:69,000,000-74,200,000; 5.2 Mb (A) and chr1: 175,300,000-180,500,000; 5.2 Mb (B); galGal5). RT  weighted average (WA) values obtained after cells sorting into four S-phase fractions, BrdU pulse-labeled nascent strands (NS) immunoprecipitation and sequencing  are shown. Early-replicated regions are represented in orange and late-replicating regions in blue. Below, track of eigenvector 1 values corresponding to A/B compartments at 100 kb resolution after Hi-C analysis in wt cells, track of replication origins determined in (Massip et al, 2019) (Ori peaks) and annotated genes are reported. The three sites chosen for insertion are indicated with red arrows and dotted lines (mid-late, late 1 (A) and late 2 (B) insertion). At the bottom, an enlargement of late insertion sites (chr1: 69,500,000-71,500,000 (A), chr1: 177,000,000-179,000,000 (B); galGal5), displaying genes, mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in (Massip et al, 2019) (Ori peaks) is shown. Tracks of RefSeq genes are represented in blue, tracks of Ensembl genes in red, tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black. Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions. ",
    "molecules": "BrdU"
  },
  {
    "caption": " F. CMTs visualized by MAP4-GFP in root epidermal cells at the TZ, the EZ and the DZ prior (0) and 60 min after treatment with 1 µm oryzalin. Scale bar 10 µm. ",
    "molecules": "oryzalin"
  },
  {
    "caption": " G. Quantification of the MAP4-GFP CMTs reporter signal in wild-type root epidermal cells at the TZ, the EZ and the DZ zone treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and cytokinin (CK; 10 µM 6-benzylaminopurine, BAP) and oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min. ",
    "molecules": "CK, cytokinin, DMSO, 6-benzylaminopurine, BAP, oryzalin"
  },
  {
    "caption": " A. Arabidopsis root expressing the dual reporter TCSn::dndT-tNOS DR5v2:3nGFP that is sensitive to cytokinin (CK) and auxin. CK (red), auxin (green) and overlay of both signals detected in nuclei of cells at the root tip (top, scale bar 50 µm). Green, pink, and blue arrowheads point at epidermal cells located in the meristematic (MZ), transition (TZ) and elongation (EZ) zones, respectively. Magnification of the TZ shown (bottom, scale bar 10 µm). ",
    "molecules": "auxin, CK, cytokinin"
  },
  {
    "caption": " C-F. CMTs visualized with MAP4-GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK (C), CK and oryzalin (D), auxin (E) and CK and auxin (F) treatments, and in DZ cells at time point 0 and 60 min after auxin treatment (E). As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively, and 1 µM oryzalin. For the double (CK/Oryzalin and CK/Auxin) treatments, roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds. Scale bar 10 µm. ",
    "molecules": "NAA, Auxin, auxin, CK, BAP, Oryzalin, oryzalin"
  },
  {
    "caption": " H. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 sec in epidermal cells without (mock, DMSO) and with CK, auxin or CK and auxin treatment (60 min). Scale bars 10 µm. I. CMT plus-end growth rates (µm/min) measured by tracking of EB1b-GFP reporter over 20-30 min in epidermal cells of different growth zones treated for 60 min with mock (DMSO), or media supplemented with CK (30 or 60 min), auxin and CK plus auxin. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot. Statistical significance evaluated by two-way ANOVA (n = 3-5 cells per root growth zone with 3-5 roots per replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions. Scale bar 0.5 µm. Hormone concentrations and treatment conditions (H, I) were as described (C, E and F). ",
    "molecules": "auxin, CK, DMSO"
  },
  {
    "caption": " A-D. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant at the transition (TZ) and the elongation (EZ) zones at time point 0 and 60 min after mock (DMSO) (A), cytokinin (CK) (B), auxin (C) and CK plus auxin (D) treatments. As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively. For the double CK plus auxin treatment roots were pretreated for 60 min with CK, whereafter they were transferred to medium supplemented with both compounds. Scale bar 10 µm. ",
    "molecules": "NAA, auxin, CK, cytokinin, DMSO, BAP"
  },
  {
    "caption": " F, G. Analysis of the CMT plus-end growth with the EB1b-GFP reporter in cre1-12. F. Z-stack maximum image projection of EB1b-GFP tracked for 30 seconds (on the left) and single trajectories of EB1b-GFP signal tracked over 60 sec (on the right) in root epidermal cells of the EZ in the wild type and cre1-12 treated with CK for 60 min. Scale bars 10 µm and 0.5 µm, respectively. G. CMT plus-end growth rates (µm/min) quantified from the EB1b-GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock (DMSO) or CK. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. (ns = non-significant, *** P < 0.001 by Student's t-test, n= 7-9 cells with 7 roots per condition in 3 independent experiments). Concentrations and treatment conditions (F, G) were as for (A, B). ",
    "molecules": "CK, DMSO"
  },
  {
    "caption": " H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm. J. Quantification of the MAP4-GFP CMTs reporter signal in cre1-12 root epidermal cells at different root zones treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and CK (10 µM BAP) plus oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min. The center lines show the medians and the box limits indicate the 25th and 75th percentiles; whiskers span the minimal to maximal values, individual data points are represented by dots. Ratio close to 1 (segmented line) corresponds to the unchanged MAP-GFP signal for 60 min. (* P < 0.05, **** P < 0.0001 by Student's t-test, n= 3-10 cells per root growth zone with 5-8 roots per condition in 4 independent replicates). ",
    "molecules": "CK, DMSO, BAP, oryzalin"
  },
  {
    "caption": " A. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on mock (DMSO; continuous line) or with dexamethasone (DEX 10 µM; dashed line) supplemented medium. Five days old seedlings (Day 0) were monitored for 3 days. Mean ± s.d.; ** P < 0.01, **** P < 0.0001 by Student's t-test, referred to wild-type - mock. n = 12-14 roots. ",
    "molecules": "DEX, dexamethasone, DMSO"
  },
  {
    "caption": " B. Immunostaining of α-tubulin in epidermal cells of the transition (TZ) and the elongation (EZ) zone of 35S::ARR1∆DDK-GR roots after non-, 3h or 16h of treatment with dexamethasone (DEX 10 µM). Scale bar 10 µm. ",
    "molecules": "DEX, dexamethasone"
  },
  {
    "caption": " D, E. Analysis of root sensitivity to oryzalin in wild-type (Col-0) and 35S::ARR1∆DDK-GR. Seedlings were grown for 5 days on mock (Murashige and Skoog) medium and then transferred to medium supplemented with 1 µM oryzalin, CK with oryzalin (10 µM BAP and 1 µM oryzalin), with or without DEX 10 µM, for 3 days. For the double CK and oryzalin treatment, seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds. Representative images; white arrowheads indicate root length at day of transfer. Scale bar 1 mm (D). Quantifications calculated as percentage (%) of root tips exhibiting swelling (dark grey bars), and weak and strong resistance to oryzalin (grey and white bars, respectively). On the right representative images of root phenotype categories. n=10-25 roots per treatment were evaluated. (E). ",
    "molecules": "CK, DEX, BAP, oryzalin"
  },
  {
    "caption": " F. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on oryzalin 1µM (continuous line) or on oryzalin 1µM and DEX 10 µM (dashed line) supplemented media monitored for 3 days. Day 0, day of transfer. Mean ± s.d.; n = 12-14 roots. ",
    "molecules": "DEX, oryzalin"
  },
  {
    "caption": " A. Time lapse imaging of Arabidopsis root expressing the plasma membrane marker (PM) pUB10::EYFP-NPSN12 (wave line W131Y)(Geldner et al, 2009). Root growth recorded for 2 min and overlay of root tip images at time point 0 (magenta) and 2 min (cyan) used for the calculation of the root growth rate (RGR) and the cell elongation rate (CER). Scale bar 25 µm. B. RGR of wild type (grey bars) and cre1-12 (blue bars) treated for 1 and 4 hours with mock (DMSO), CK (10 µM BAP) or auxin (0.1 µM NAA). Mean ± s.d., (* P < 0.05and **** P < 0.0001 by the Student's t-test, n= 5-8 roots per condition in 3 independent replicates). ",
    "molecules": "NAA, auxin, CK, DMSO, BAP"
  },
  {
    "caption": " C-E. Analysis of CK and auxin effects on CER of epidermal cells. Epidermal cells of the TZ and the EZ in the wild type and cre1-12 with PMs visualized with the YFP-NPSN12 marker. Overlay of images of epidermal cells at time 0 (magenta) and 2 min (cyan) is presented. Scale bar 10 µm (C). CER (µm/min) of root epidermal cells of the TZ (D) and the EZ (E) of the wild type (grey bars) and cre1-12 (blue bars) mutant. Hormone concentrations and conditions of treatment (D, E) were as described for (C). Mean ± s.d., (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by Student's t-test, n=15-20 cells per root growth zone in 3-6 roots per condition, in 3 independent). ",
    "molecules": "auxin, CK"
  },
  {
    "caption": " A. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry plus-end marker before (dark grey) and after (light grey) treatment with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin) and auxin (0.1 µM NAA). MT plus-end growth rates were monitored for 10 min before and for 10 min after each treatment. The number of MT plus-end events (EB3-mCherry) tracked per cell was 100-200/min. Mean ± s.d., (*** P < 0.001 by Student's t-test, n = 1-3 cells per condition in 4 independent replicate). B. Kymograph representation of individual EB3-mCherry comets tracked for 30 sec in leukocytes cells, before and after treatment with mock (DMSO) or CK (10 µM BAP). Scale bar 2 µm.",
    "molecules": "NAA, auxin, CK, DMSO, BAP, trans-zeatin"
  },
  {
    "caption": " C-E. Immunostaining of α-tubulin (green) in leukocytes mounted in media containing DAPI (blue, nuclear marker) and quantification of immunolabeled MTs. Cells were not or treated with 300 nM nocodazole (N) for 10 min (C). Leukocytes pretreated for 30 min with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin), adenine (10 µM) or auxin (10 µM NAA) prior to a 10-min treatment with 300 nM nocodazol (D). Scale bar 50 µm (C, D). E. Relative fluorescence intensity of MTs in leukocytes treated with nocodazole, or nocodazole and hormones as described for (D) compared to untreated (black bar) cells. Mean ± s.d., (P < 0.05 by two-way Anova, n = 18-33 cells per condition in 3 independent replicates). ",
    "molecules": "NAA, adenine, auxin, CK, DAPI, DMSO, BAP, nocodazol, nocodazole, trans-zeatin"
  },
  {
    "caption": "(B) Coomassie and  ProQ Diamond-stained gels on   mChRZZSF (4 µM) treated with the indicated kinases (1 µM) for 15 hours at 20ºC. Rev = reversine, used at 10 µM.",
    "molecules": "ProQ Diamond, Coomassie, Rev, reversine"
  },
  {
    "caption": "(D) Levels of Zwilch  at kinetochores  of HeLa  cells that had been previously synchronized in G2 phase with 9 μM RO3306  for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 500 nM  reversine, 3.3 µM nocodazole, and 10 µM MG132  for 1 hour and imaged while in mitosis. CREST   serum  was used to visualize kinetochores  and DAPI  to stain  DNA. Scale bar: 10 µm. Three biological replicates were performed. (E) Cells treated as for panel D were treated for visualization of Spindly. Scale bar: 10 µm.",
    "molecules": "DAPI, MG132, nocodazole, reversine, RO3306"
  },
  {
    "caption": "(F) Scatter dot plots representing normalized total area of the Zwilch  and Spindly  signals, normalized to the reversine-untreated control, in the indicated number of cells from the experiment shown in panels C-D. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.",
    "molecules": "reversine"
  },
  {
    "caption": "(G) Representative images showing the effects of a knockdown of the endogenous Spindly  in HeLa  cells. RNAi treatment was performed for 48 h with 50 nM siRNA. Before fixation, cells were synchronized in  G2 phase with 9 μM RO3306  for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM nocodazole  for an additional hour. CREST  serum  was used to visualize kinetochores  and DAPI  to stain  DNA. Scale bar: 10 µm. Three biological replicates were performed.",
    "molecules": "DAPI, nocodazole, RO3306"
  },
  {
    "caption": "(E) The phosphorylation  state of RZZ and SpindlyF was monitored by ProQ Diamond  after SDS-PAGE  separation of reactions. Data information: Two technical replicates for panels , E",
    "molecules": "ProQ Diamond"
  },
  {
    "caption": "(A) Size-exclusion chromatography and SDS-PAGE  of elution fractions of the indicated samples. Each Spindly construct was incubated with RZZ  in absence (dotted lines) or in presence (continuous lines) of farnesyl transferase and farnesyl pyrophosphate. Elution shifts of  SpindlyF and of the resulting  RZZSF complexes is indicative of successful interaction. Two technical replicates were performed.",
    "molecules": "farnesyl pyrophosphate"
  },
  {
    "caption": "(D) Representative images of fixed HeLa  cells electroporated  with full-length mChSpindly and mChSpindly354-605 constructs in cells depleted of endogenous Spindly  by RNAi. Spindly localization  was detected with an antibody against the C-terminal region of Spindly. Corona   expansion or lack thereof were monitored through CENP-E. CREST serum was used to visualize kinetochores, DAPI  to stain  for DNA. Scale bar: 10 µm. Experiments were replicated three times.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Survival rates of wild-type (n=33) and Angptl2-/- (n=33) mice supplied with drinking water containing 2.5 % DSS for 6 days and then untreated water for 6 more days, from two different experiments. ***p < 0.001, by log-rank test.",
    "molecules": "DSS, water"
  },
  {
    "caption": "B. Representative images of colons from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).C. Colon length of wild-type (n=7) and Angptl2-/- (n=6) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).",
    "molecules": "DSS, water"
  },
  {
    "caption": "D, E, F. Representative images of colon crypts as assessed by H&E (D), Ki-67 (E), and pH3 (F) staining in wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 100 μm.G, H. The number of regenerating crypts as quantified using Ki-67 (G) and pH3 (H) staining. Each data point represents the number of Ki-67-positive viable crypts or the number of pH3-positive cells in a single field of view. Multiple areas in at least 6 mice per genotype were quantified. Numbers below panels represent the average.",
    "molecules": "DSS, water"
  },
  {
    "caption": "I. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 50 μm.",
    "molecules": "DSS, water"
  },
  {
    "caption": "J. mRNA levels of indicated genes in isolated crypts from wild-type (n=4) and Angptl2-/-(n=4) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).",
    "molecules": "DSS, water"
  },
  {
    "caption": "D. Representative staining for P-Smad1/5 in crypts from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Dashed lines indicate half of the crypt and arrowheads show P-Smad1/5-positive cells in lower halves of crypts. Scale bar = 50 μm.E. The number of P-Smad1/5-positive cells in the crypt base (n=60) from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Multiple areas in 3 mice per genotype were quantified.",
    "molecules": "DSS"
  },
  {
    "caption": "C, D. mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild-type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors. n=3. U0126, ERK inhibitor; LY294002, PI3K inhibitor; BAY11-7085, NF-κB inhibitor.",
    "molecules": "BAY11-7085, U0126, LY294002"
  },
  {
    "caption": "Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, P*** = 0.0003).",
    "molecules": "oil, 4-OHT"
  },
  {
    "caption": "The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.",
    "molecules": "PS"
  },
  {
    "caption": "Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005). Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).",
    "molecules": "DMSO, PLX3397"
  },
  {
    "caption": "Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).",
    "molecules": "PS"
  },
  {
    "caption": "Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090).",
    "molecules": "PBS, PS"
  },
  {
    "caption": "Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).",
    "molecules": "4-OHT"
  },
  {
    "caption": "Cholesterol remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). Cholesterol content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).",
    "molecules": "Cholesterol"
  },
  {
    "caption": "triglyceride remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). triglyceride content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).",
    "molecules": "triglyceride"
  },
  {
    "caption": "MRI measures of total body weight, percentage lean mass, and percent fat mass prior to and after 6 weeks on the HFD (males, n=5). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).",
    "molecules": "fat"
  },
  {
    "caption": "A, B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).",
    "molecules": "Oil Red O"
  },
  {
    "caption": "D-F Serum levels of free fatty acids (FAA), insulin, and leptin in WT and KO after 25 weeks on NC or HFD (males, n=8-10). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).",
    "molecules": "FAA, free fatty acids, leptin"
  },
  {
    "caption": "Oxygen consumption (VO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons). ",
    "molecules": "Oxygen"
  },
  {
    "caption": "carbon dioxide production (VCO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons). ",
    "molecules": "carbon dioxide"
  },
  {
    "caption": "respiratory exchange ratio VCO2/VO2 (RER) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).",
    "molecules": "O2, CO2"
  },
  {
    "caption": "C Representative images of 3T3-L1 adipocytes transfected with the indicated si-RNAs prior to differentiation and stained with Oil Red O on day 10. Scale bar = 100 μm.",
    "molecules": "Oil Red O"
  },
  {
    "caption": "D Western blot for adiponectin (ADPOQ) released into the media during differentiation by 3T3-L1 adipocytes transfected with the indicated si-RNAs. Ponceau stained image provides loading control.",
    "molecules": "Ponceau"
  },
  {
    "caption": "Representative bright-field images showing lipid accumulation at 10-days differentiation of Ing-svf cells treated with the indicated siRNA. Scale bar = 50 μm.",
    "molecules": "lipid"
  },
  {
    "caption": "G Transcript levels for Adrenoceptor beta 3 (Adrb3), uncoupling protein 1 (Ucp1), PPARγ coactivator 1 alpha (PGC1α), and deiodinase type II (Dio2) Ing-svf cultures transfected with control siRNA (C) or one targeting Rcan1 (KD) then maintained in growth media (G), or differentiated for ten-days (D) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting (D+ISO) (n=2 in triplicate). * = p < 0.05; ** = p < 0.01, two-way ANOVA with multiple comparisons. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD",
    "molecules": "isoproterionol"
  },
  {
    "caption": "B, C Correlation of Rcan1 expression in gonadal adipose and liver with body weight, plasma insulin and plasma triglycerides, in a data set comparing backgrounds susceptible (BTBR background) or resistant (C57BL/6 background) to diabetes when carrying the leptinob/ob (ob) mutation. Measures are reported as the ratio of the mean log10 intensity (ml ratio). Regression line (black), r = linear regression, p = p-value.",
    "molecules": "triglycerides"
  },
  {
    "caption": "(E-F) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group.",
    "molecules": "doxycycline, Doxycycline"
  },
  {
    "caption": "Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by either 1 μM doxorubicin (D-E) with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown (E Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.",
    "molecules": "etoposide, TSA"
  },
  {
    "caption": "Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown G) as the mean ± SD, n=3 biological replicates. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.",
    "molecules": "etoposide, TSA"
  },
  {
    "caption": "Knockdown of endogenous CSAG2 in HCT116 (H-I) cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown (I as the mean ± SD, n=3 biological replicates.",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins.",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown K) as the mean ± SD, n=3 biological replicates.",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "(L-M) Re-expression of CSAG2 rescued p53 ac-K382 levels in CSAG2 knockdown stable cell lines. Myc-CSAG2 was transfected in HCT116 (L) or A375 (M) CSAG2 knockdown stable cell lines. Cells were treated with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr and then blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown as the mean ± SD, n=3 biological replicates.",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "(A) Knockdown of SIRT1 abolished CSAG2-mediated decrease in ac-p53 K382. CSAG2 over-expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin/TSA treatment for 6 hr. Cell lysates were blotted for the indicated proteins.",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "(B) Inhibition of SIRT1 enzymatic activity blocked CSAG2-induced decrease in p53 ac-K382. CSAG2 over-expressing HCT116 cells were treated with 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cell lysates were blotted for the indicated proteins.",
    "molecules": "doxorubicin, EX-527, TSA"
  },
  {
    "caption": "Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins (C)",
    "molecules": "doxorubicin, EX-527, TSA"
  },
  {
    "caption": "Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins and quantitated (D; n=3 biological replicates). Data shown as mean ± SD.",
    "molecules": "doxorubicin, EX-527, TSA"
  },
  {
    "caption": "(G) Inhibition of SIRT1 activity abolished CSAG2-induced anchorage-independent growth. Soft agar colony formation assays were performed in control or CSAG2 over-expressing HCT116 cells with or without EX-527 treatment. Data are represented as the mean ± SD, n=3 biological replicates.",
    "molecules": "EX-527"
  },
  {
    "caption": "(J) CSAG2 Δ37 mutant does not regulate p53 ac-K382 levels. Cells were transfected with the indicated Myc-CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin/ 0.4 μM TSA. Cell lysates were blotted for the indicated proteins.",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, doxorubicin"
  },
  {
    "caption": "Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, H2O2"
  },
  {
    "caption": "shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, doxorubicin"
  },
  {
    "caption": "shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, H2O2"
  },
  {
    "caption": "(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).",
    "molecules": "doxorubicin, TSA"
  },
  {
    "caption": "(G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, H2O2"
  },
  {
    "caption": "(H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, H2O2, EX-527"
  },
  {
    "caption": "Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, H2O2"
  },
  {
    "caption": "Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, doxorubicin"
  },
  {
    "caption": "shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, H2O2"
  },
  {
    "caption": "shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, doxorubicin"
  },
  {
    "caption": "Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, H2O2"
  },
  {
    "caption": "Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).",
    "molecules": "alamarBlue, doxorubicin"
  },
  {
    "caption": "(E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity.",
    "molecules": "Nicotinamide"
  },
  {
    "caption": "(G) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates.",
    "molecules": "NAD"
  },
  {
    "caption": "(H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.",
    "molecules": "Histone, TSA"
  },
  {
    "caption": "(J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.",
    "molecules": "Histone"
  },
  {
    "caption": "(C) Maximum intensity projection of 3D confocal stack obtained from the dissected thyroid gland of Tg(tg:nls-EGFP) animal and labeled with DAPI. (D) Confocal scan of a transverse section across the dissected thyroid gland from Tg(tg:nls-EGFP) animal at 3 mpf. Sections were stained with DAPI to visualize cells surrounding thyroid follicles. (E) Boxplot depicting the proportion of thyrocytes present in transverse sections obtained from three Tg(tg:nls-EGFP) animals at 3 mpf. Each dot represents a transverse section. The Tukey boxplot marks the 25th percentile, median and 75th percentile with whiskers extending from smallest to largest values. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(F , G) Representative FACS plot of single cells from Tg(tg:nls-mVenus-T2A-NTR) animals at 2 mpf (F) and 8 mpf (G). Calcein (Pacific Blue) labels live cells, while green fluorescence (FITC) labels thyrocytes. Percentage values represent proportion of calcein+ thyrocytes within total calcein+ cells.",
    "molecules": "Calcein, calcein, FITC, Pacific Blue"
  },
  {
    "caption": "(A-D) Images show immunofluorescence labeling of thyroid gland from adult zebrafish. Transverse sections were utilized for imaging. The organ was isolated from tissue-specific transgenic lines to allow marking of a particular cell-type adjacent to the thyroid follicle. Blood vessels were marked using Tg(kdrl:EGFP) (A), macrophages using Tg(mpeg1.1:mCherry) (B) and stroma using Tg(col1a2:mCherry) (C). Thyrocytes were labeled with pax2amKO2 expression in (A) and Tg(tg:nls-EGFP) expression in (B - C). (D) NFE was labeled using antibody against TP63 in sections of the thyroid gland isolated from Tg(tg:nls-EGFP) animals. Gills are marked based on their morphological appearance. DAPI labels nuclei. Scale bars: 10 µm (A - B), 50 µm (C - D).",
    "molecules": "DAPI"
  },
  {
    "caption": "(D - F) Confocal microscopy imaging of a sagittal section of a 55 hpf pax2amKO2 (red); Tg(tg:nls-EGFP) (green) embryos immunostained with PAX2A antibody (cyan) showing co-localization of mKO2 and pax2a in the pronephros (D), thyroid gland (E) and mid-hindbrain barrier (F). In the thyroid gland, mKO2 (red), PAX2A-Ab (cyan) and thyrocyte-specific GFP (green) show co-localization. Scale bar: 50 µm.",
    "molecules": "cyan"
  },
  {
    "caption": "(A-D) Analysis of 3 mpf thyroid gland from pax2amKO2 zebrafish shows heterogeneity in pax2a reporter expression. (A) Whole mount confocal imaging of a 3 mpf pax2amKO2 thyroid labelled with mKO2 (red), E-cadherin (cyan, not shown in 'A' for clarity reasons) and DAPI (dark blue) for nuclear localisation. (B - D) Optical sections of three follicles, with mKO2-Low cells labelled with arrows. E-cadherin (B' - D') and DAPI (B'' - D'') staining shows that absence of mKO2 signal does not correspond to an absence of cells. Anterior to the bottom of the pictures.",
    "molecules": "cyan, DAPI"
  },
  {
    "caption": "(E,F) (E) Confocal image of thyroid gland section from Tg(tg:nls-EGFP) at 4 mpf stained with PAX2A antibody and DAPI. The dotted region is displayed at high magnification in (F). Arrows marks thyrocytes displaying low PAX2A staining. Notably, PAX2A-Low thyrocytes display tg-driven EGFP expression, demonstrating their differentiated status.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A - C) Cells from the thyroid gland of 5 mpf Tg(tg:nls-GFP); pax2amKO2 animals were labelled with calcein (live cell marker) and analysed using FACS. (A) A FACS plot showing calcein on Y-axis and GFP on X-axis. The box encompassing the GFP+ cells represents the thyrocyte population, which was gated for further analysis. (B) Histogram showing the distribution of GFP intensity in thyrocytes. (C) Histogram showing the distribution of mKO2 intensity in thyrocytes. Thyrocytes were selected by gating for GFP+ population. Horizontal lines indicate the mKO2-Low and mKO2-High expression level, with percentage values representing proportion of thyrocytes with mKO2-Low and mKO2-High expression.",
    "molecules": "calcein"
  },
  {
    "caption": "Dose-response curve of HF and DWN12088 in WI-26 VA4 cells. The IC50 value for collagen level was measured as a representative of chemical efficacy. The cells were starved of serum for 6 hr and then incubated with the indicated concentrations of compounds in the presence of TGF-β (2 ng/ml) for 72 hr. The secreted pro-collagen I levels were determined by ELISA. The CC50 values were determined by CellTiter-Glo assay. Dose-response curves were determined by combining at least 2 biologically independent experiments. The IC50 values were calculated using GraphPad Prism 7.0 (Collagen level, n = 5 from three independent experiments (singlicate for one experiment and duplicate for two experiments; mean ± SEM); Celltiter-Glo assay, n = 4 from two independent experiments (duplicate for each experiment; mean ± SEM). ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg;",
    "molecules": "DWN12088, HF"
  },
  {
    "caption": "In vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin (D). The saturation of percutaneous oxygen (SpO2) (E) was determined as a measurement of lung function. BLM, bleomycin; ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg",
    "molecules": "bleomycin, BLM, O2, oxygen, DWN12088, HF"
  },
  {
    "caption": "In vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin The collagen level was determined by hydroxyproline assay (G) BLM, bleomycin HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg;",
    "molecules": "bleomycin, BLM, DWN12088, HF"
  },
  {
    "caption": "In vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin (D). Masson's trichrome staining of lung tissues. ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg; b, bronchiole; v, blood vessel.",
    "molecules": "Masson's trichrome, bleomycin, DWN12088, HF"
  },
  {
    "caption": "WI-26 VA4 cells were incubated in the presence of the indicated concentrations of HF and DWN12088 for 72 hr. The chemical concentrations were determined based on the IC50 for collagen level (HF, 15 nM; DWN12088, 2 μM). The levels of proteins were determined by immunoblot assay. The proline content of the proteins based on Uniprot amino acid sequence is shown on the right side of the immunoblot images.",
    "molecules": "DWN12088, HF, proline"
  },
  {
    "caption": "WI-26 VA4 cells were incubated with the indicated concentrations of HF and DWN12088 in the presence of 2 ng/ml TGF-β for 15 hr. The chemical concentrations were determined based on the IC50 values for collagen levels (HF, 15 nM; DWN12088, 2 μM). The activation of the TGF-β pathway was determined by monitoring the phosphorylation of SMAD2. Immunoblot images are representative of three biologically independent experiments.   The band intensities of the immunoblot images in (C) were quantified using ImageJ (n = 3; One-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001; mean ± SEM).  ",
    "molecules": "DWN12088, HF"
  },
  {
    "caption": "The IC50 values of the indicated compounds were determined for collagen level (HF and DWN12088, n = 8 from four independent experiments (duplicate for each experiment); DWN11748, n = 12 from six independent experiments (duplicate for each experiment); DWN11761 and DWN11251, n = 6 from three independent experiments (duplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for cytotoxicity (n = 4 from two independent experiments (duplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for TNT-luciferase activity (n = 6 from two independent experiments (triplicate for each experiment); only for DWN11761, n = 9 from three independent experiments (triplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for puromycin incorporation (HF, n = 12 from four independent experiments (triplicate for each experiment); DWN11748, DWN12088, and DWN11251, n = 9 from three independent experiments (triplicate for each experiment); DWN11761, n = 8 from three independent experiments (triplicate for two experiments and duplicate for one experiment); mean ± SEM).",
    "molecules": "DWN11251, DWN11748, DWN11761, DWN12088, HF"
  },
  {
    "caption": "The binding of HF and DWN12088 to in vitro purified PARS1 WT and F1097A/E1123A/R1152L (FA/EA/RL) mutant was determined via microscale thermophoresis (MST). Kd values are listed in the top left part of the graph (technical replicates n = 3; mean ± SD).",
    "molecules": "DWN12088, HF"
  },
  {
    "caption": "Catalytic activities of PARS1 WT and FA/EA/RL mutant were determined by prolylation assay in the presence of [3H]proline. To monitor the effect of PARS1 RA/EA/RL mutant on the activity of PARS1 WT, the same concentrations of the proteins were pre-incubated for 10 min and then used for the assay. The formation of Pro-tRNAPro was measured using liquid scintillation counter (technical replicates n = 3; mean ± SEM).",
    "molecules": "Pro-tRNAPro, proline, 3H"
  },
  {
    "caption": "293T cells were transfected with the indicated plasmid DNAs for 24 hr. The cell lysates were prepared and pulled down with Strep bead for 2 hr. Co-precipitated proteins were determined by immunoblot assay. WT, PARS1 wild type; MT, PARS1 FA/EA/RL.",
    "molecules": "Strep"
  },
  {
    "caption": "G, H. WI-26 VA4 cells stably expressing each of EV (empty vector) or PARS1 FA/EA/RL mutant were incubated with the indicated compounds for 72 hr. Dose-response curves of the compounds for collagen levels and cytotoxicity were determined and relative ratio of CC50 to the IC50 of collagen for HF (G) and DWN12088 (H) were calculated. The IC50 values were calculated using GraphPad Prism 7.0 (collagen ELISA, n = 12 from four independent experiments (triplicate for each experiment); cytotoxicity assay, n = 8 from four independent experiments (duplicate for each experiment); Welch's t test; ***P < 0.001; mean ± SEM).",
    "molecules": "DWN12088, HF"
  },
  {
    "caption": "K. WI-26 VA4 cells stably expressing each of EV or PARS1 FA/EA/RL mutant were incubated with the indicated compounds in the presence of TGF-β for 72 hr. The levels of the indicated proteins were determined by immunoblotting. The percentage of proline in polypeptide sequences is shown on the right side of immunoblot images.",
    "molecules": "proline"
  },
  {
    "caption": "(A) Maximal intensity projection of a confocal  z-stack of 24hpf Tg(piwil:egfp), Tg(piwil:egfp) injected with kif26ab MO   and Tg(piwil:egfp) injected with control MO embryos. (B) Quantitative analysis of PGCs  in 24hpf Tg(piwil:egfp) (n = 11), Tg(piwil:egfp) injected with kif26ab MO  (n = 12) and Tg(piwil:egfp) injected with control MO  (n = 13) embryos. Data shown are mean ± SEM (n = the number of embryos  analyzed).",
    "molecules": "MO"
  },
  {
    "caption": "(C) Maximal intensity projection of a confocal  z-stack of  4-cell stage embryos injected with GFP-Buc  mRNA + RFP  mRNA, GFP-Buc  mRNA + RFP mRNA + Control MO  and GFP-Buc  mRNA + RFP  mRNA + kif26ab MO. White arrowheads point to cleavage furrows of  4-cell embryos.",
    "molecules": "MO"
  },
  {
    "caption": "(D) Relative GFP-Buc  density at the cleavage furrow  of 4-cell stage embryos injected with GFP-Buc  mRNA + RFP  mRNA (n = 11), GFP-Buc  mRNA + RFP  mRNA + Control MO  (n = 11) and GFP-Buc  mRNA + RFP  mRNA + kif26ab MO  (n = 10). Data shown are mean ± SEM (n = the number of embryos analyzed).",
    "molecules": "MO"
  },
  {
    "caption": "(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (F)  Western blots for GFP-Buc  expression in 4-cell stage embryos injected with GFP-Buc  mRNA + RFP  mRNA, GFP-Buc  mRNA + RFP  mRNA + Control MO  and GFP-Buc  mRNA + RFP  mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.",
    "molecules": "MO"
  },
  {
    "caption": "(I) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (J) Western blots for GFP-Buc  expression in MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc  mRNA + RFP  mRNA, GFP-Buc  mRNA + RFP  mRNA + Control MO  and GFP-Buc mRNA + RFP  mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.",
    "molecules": "MO"
  },
  {
    "caption": "C, 8 μm skin  section of Cre- control (CT) and  Alk1EndoKO mice stained  for Cdh5  (red), Ki67  (green), Erg  (white), and Dapi  (blue). Examples of Cdh5/Ki67  double positive cells were enlarged in the white dotted inset for visualization purposes.",
    "molecules": "Dapi"
  },
  {
    "caption": "D-G, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice  per group, and n = 9-16 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used as statistical test and individual mouse  was treated as random effect to take into the account of mouse-to-mouse  variability. D, Quantification of number of ECs  in HPuHG  area from C. E, Percentage of proliferative ECs  (percentage of Ki67+ Cdh5+ cells in total Cdh5+ cells) in HPuHG  from C. F, Percentage of proliferative ECs  (percentage of Ki67+/Cdh5+ cells in total skin Cdh5+ cells) from C G, Percentage of proliferative ECs  (percentage of BrdU+ Cdh5+ cells in total Cdh5+ cells) in total skin  from PD17-24 BrdU-pulsed CT and Alk1EndoKO mice",
    "molecules": "BrdU"
  },
  {
    "caption": "A-C, Immunofluorescence staining  images of 12 μm skin  section from PD17 induced PD20 telogen  CT, PD25 early anagen  CT, and PD25 telogen Alk1EndoKO mice stained  for Endomucin  (EMCN, red), CD31  (green), and Dapi  (blue). HPuHG  region was outlined in yellow dotted lines. D-F, Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice  per group, and n = 8-9 images per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse  was treated as random effect to take into the account of mouse-to-mouse  variability. D, Quantification of EMCN+/CD31+ area from images like those in A-C, shown as percentage of BV  in the HPuHG. E, Quantification of absolute EMCN+ area (e.g. BV  area) in HPuHG  in images like those in A-C. F, Quantification of EMCN-/CD31+ area from images like those in A-C as LV  area in the HPuHG.",
    "molecules": "Dapi"
  },
  {
    "caption": "G-J, Maximum projection through optical Z-stacks of 60 μm skin  sections from mice TM-induced at PD17 and sacrificed at PD20 telogen  CT, PD25  early anagen  CT, PD32 Anagen  CT, and PD25 telogen Alk1EndoKO mice stained  for VEGFR3  (red), LYVE1  (green), and Dapi  (blue). Lymphatic capillaries  were outlined in white dotted lines. K, Quantification of lymphatic capillary caliber from images like those in G-J in PD20 telogen CT, PD25 early anagen CT, and PD25 telogen Alk1EndoKO mice  Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one  lymphatic capillary. N = 3 mice  per group, and n = 9-25 measurements per mouse. Restricted maximum likelihood (REML) analysis with unbounded variance components was used to calculate p-values and individual mouse  was treated as random effect to take into the account of mouse-to-mouse  variability.",
    "molecules": "Dapi, TM"
  },
  {
    "caption": "B, Immunofluorescence  image of 12 μm skin  section from mice  treated as shown in A, stained  for VEGFR3  (green) and Dapi  (blue). C, Quantification of tdTomato+/VEGFR3+ area from images like those in B, shows high induction efficiency. Error bars represent standard deviation. Each bar represents one individual mouse, and each dot represents measurement from one image. N = 3 mice, and n = 7-8 images per mouse.",
    "molecules": "Dapi"
  },
  {
    "caption": "G, Immunofluorescence  images of 12 μm skin  section of PD17 induced PD25 CT and Alk1LymphKO mice stained  for Ki67  (red), Itga6  (green) and Dapi  (blue) showing representative hair follicle. Hair follicles were outlined with yellow dotted lines. Note Ki67+ cells in the hair germ (HG) area indicative of HFSC  activation. Telogen hair follicle  is defined as no Ki67  in the hair germ. Anagen I is defined as ≤7 Ki67+ cells in the unenlarged hair germ. Anagen II  is defined as >7 Ki67+ cells in the hair germ or enlarged hair germ  with changed shape that starts to enclose the DP. Experiments were repeated at least 2 times with at least 3 skin  sections/each mouse  analyzed with N = 6/4 mice/group. See Table 1 for quantification detail.",
    "molecules": "Dapi"
  },
  {
    "caption": "B, Immunofluorescence  image of 12 μm skin  section from CT and  Alk1EndoKO mice  used for RNA-seq stained  for Ki67  (red) and Dapi  (blue) show that HFSCs  are in quiescence.",
    "molecules": "Dapi"
  },
  {
    "caption": "C, Images of 20 μm skin  section of PD20 and PD25 BMP4-LacZ; Alk1f/f; Cdh5-CreERT2 negative, injected with tamoxifen TM  at PD17 (labelled as CT), and PD20/PD25 Cdh5CreERT2; Alk1f/f; BMP4-LacZ mice  (labelled as Alk1EndoKO) TM  injected at PD17, all stained  for CD31  (IHC  method, brown) and X-gal  (blue). Hair follicles  (HF) were outlined with black dotted lines. Arrows point to X-Gal+ vascular  structure in HPuHG  area.",
    "molecules": "X-gal, X-Gal, tamoxifen, TM"
  },
  {
    "caption": "G-J, Immunofluorescence  images of 12 μm skin  section of PD17 induced stained  for (Top panel) Ki67  (red), Itga6  (green), and Dapi  (blue) and (Bottom panel) CD31  (red) and Dapi  (blue). HPuHG  region was outlined with white dotted lines.",
    "molecules": "Dapi"
  },
  {
    "caption": "B.  Western blot  analysis of the   dose-dependent  expression of ZBP1  in HT29  / TO-ZBP1WT and HT29  / TO-ZBP1Zα1α2mut cells treated with indicated concentrations of Dox  for 24 h. Blots  are representative of three biological repeats.",
    "molecules": "Dox"
  },
  {
    "caption": "L. Transwell migration of primary neutrophils  towards conditioned media from HT29  / TO-ZBP1WT cells treated with DMSO  or 500 ng/mL Dox  for 24 hours, or HT29  chemotaxis buffer (control buffer) containing equal volume and amount of DMSO  or Dox  as the conditioned media. Data is presented as individual values with grand mean of the migrated percentage induced by control buffer containing DMSO  or Dox  (n = 10 biological replicates), or conditioned media from DMSO-treated cells (n = 11 biological replicates) or Dox-treated cells (n = 12 biological replicates), where the number of biological replicates is defined by the total number of independent cell cultures tested on primary neutrophils  from four donors. An unpaired t-test was used to test for statistical differences between indicated conditions. **** p < 0.0001.",
    "molecules": "DMSO, Dox"
  },
  {
    "caption": "M. Transwell migration of primary neutrophils towards conditioned media from HT29  / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox  in combination with DMSO  or 20 µM zVAD  + 10 μM Nec1s  for 24 h. Data is presented as individual values with mean and S.E.M. (n = 3 biological replicates of conditioned media). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.4129); *** p = 0.0004.",
    "molecules": "DMSO, Dox, Nec1s, zVAD"
  },
  {
    "caption": "C. Transwell migration of primary neutrophils towards conditioned media from HT29  / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox  in combination with indicated inhibitors for 24 h. Data is presented as individual values with grand mean (n = 3 biological replicates of conditioned media from GSK'872  and NSA-treated wells, n = 6 biological replicates of conditioned media from other conditions). Two-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. **** p < 0.0001; ns = not significant (p > 0.96).",
    "molecules": "Dox, GSK'872, NSA"
  },
  {
    "caption": "D. CXCL8  concentration in the culture media of HT29  / TO-ZBP1WT cells transfected with siRNA targeting mismatch sequence (MM) or RIPK3  and treated with Dox  at indicated concentrations for 24 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Unpaired t-tests were used to test for statistical differences between indicated conditions. ** p = 0.0079. Cell lysates from one biological replicate were analyzed by Western blotting.",
    "molecules": "Dox"
  },
  {
    "caption": "E. Cytokine concentration  in the culture media of HT29  / TO-ZBP1WT or HT29  / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0, 50 or 500 ng/mL Dox. data is plotted as mean with S.E.M. (n = 6 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. Cell lysates were analyzed by Western blotting for ZBP1  levels. Blots  are representative of six biological replicates. ns = not significant (p > 0.2); *** p = 0.0003 for CXCL8, p = 0.0008 for CXCL1; **** p < 0.0001.",
    "molecules": "Dox"
  },
  {
    "caption": "A. Western blot  analysis of anti-FLAG  (ZBP1) immunoprecipitation  from HT29  / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox  for 16 h. Blots  are representative of three biological replicates. Immunoprecipitated  material was treated or not with USP21  (1 μM) for deubiquitination. Input loaded was 5% for ZBP1  and 1% for co-immunoprecipitants. Asterisk indicates antibody heavy chain signal in IP  samples.",
    "molecules": "Dox"
  },
  {
    "caption": "B. Enrichment of Ub-conjugates by GST-1xUBA   for analysis of ubiquitination  status of RIPK1, ZBP1  and RIPK3  in HT29  / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox  for 16h. Blots  are representative of at least three biological replicates. After enrichment, samples were treated or not with USP21  (1 μM) for deubiquitination. Asterisk indicates unspecific bands. Arrows indicate RIPK3  signal.",
    "molecules": "Dox, Ub"
  },
  {
    "caption": "C. Enrichment of Ub-conjugates for analysis of the ubiquitination  status of RIPK1  and ZBP1  using GST-1xUBA or linkage-specific SUBs  in HT29  / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox  for 16 h.",
    "molecules": "Dox, Ub"
  },
  {
    "caption": "D. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination  status of RIPK1  and ZBP1  in HT29, HT29  / TO-ZBP1WT and HT29  / RIPK1-KO aA3 / TO-ZBP1WT cells treated with 500 ng/mL Dox  for 16 h. Blots  are representative of four biological replicates. Asterisk indicates antibody background signal.",
    "molecules": "Dox, Ub"
  },
  {
    "caption": "D-F. Western blot  analysis of anti-FLAG  (ZBP1) immunoprecipitation  from HT29  or HT29  / TO-ZBP1WT or HT29  / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox  for 16 h. Asterisk indicates antibody heavy chain signal in IP  samples. Blots  are representative of at least (D) two and (E-F) three biological replicates.",
    "molecules": "Dox"
  },
  {
    "caption": "B. Western blot analysis of HCT116  / RIPK3-2xFV cells (FL and ∆C) treated with 100 nM dimerizer. Blots  are representative of two biological replicates.",
    "molecules": "dimerizer"
  },
  {
    "caption": "D. CXCL8  concentration in the culture media of HCT116  / RIPK3-2xFV cells (WT and RHIMmut) treated with 100 nM dimerizer  for 24 h. In RIPK3-RHIMmut three key residues, VQV, of the RHIM region was changed to AAAA. Data is plotted as mean with S.E.M. (n = 5 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated time points. ** p = 0.0011; ns = not significant (p = 0.9301). Inset: Western blot analysis to determine RIPK3  expression levels in one biological replicate. Line indicates that image was cut and spliced to remove non-relevant lanes from the scanned blot.",
    "molecules": "dimerizer"
  },
  {
    "caption": "H. Relative mRNA levels of CXCL8 in HCT116  / RIPK3-2xFV cells pretreated with 100 nM CpA  for 0.5 h before treatment with 0 or 100 nM dimerizer  for 3 h. Data is presented as mean with S.E.M. (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO+dimerizer-treated condition. **** p < 0.0001. (H) Cell lysates were analyzed by Western blotting for cIAP1  levels. Blots  are representative of two biological replicates.",
    "molecules": "dimerizer, CpA, DMSO"
  },
  {
    "caption": "J. Relative CXCL8  mRNA levels in WT or HOIP-knockout HCT116  / RIPK3-2xFV cells treated with 0 or 100 nM dimerizer  for 3 h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for the statistical difference as indicated. *** p = 0.0010. Cell lysates were loaded for Western blot  analysis.",
    "molecules": "dimerizer"
  },
  {
    "caption": "K. Relative CXCL8  mRNA levels in HCT116  / Tet-On-GFP-K63-SUB  / RIPK3-2xFV, HCT116  / Tet-On-GFP-M1-SUB  / RIPK3-2xFV and HCT116  / Tet-On-GFP  / RIPK3-2xFV cells treated with 0 or 100 ng/mL Dox  for 48 h before stimulated with 0 or 100 nM dimerizer  for 3 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.9997); ** p = 0.0030; * p = 0.0366. Cells were analyzed by Western blotting  for the inducible-expression levels of GFP  (control) and GFP-SUBs. Blots are representative of three biological replicates.",
    "molecules": "dimerizer, Dox"
  },
  {
    "caption": "M. Relative CXCL8  mRNA levels in HCT116  / RIPK3-2xFV cells stably knocked down against mismatch (shMM) or two different sites of  caspase-8 (shCASP8-A and shCASP8-B), pretreated with DMSO, 10 µM Nec1s, 10 µM GSK'872 or 1 µM NSA  for 1h followed by treatment with 0 or 100 nM dimerizer  for 3h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparison tests were used to test for statistical differences between indicated conditions. *** p = 0.0001; ** p = 0.0035. Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase-8 levels.",
    "molecules": "dimerizer, DMSO, GSK'872, Nec1s, NSA"
  },
  {
    "caption": "(C, D) Representative images of interphase NBs in fixed wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains expressing Polo-GFP from the native promoter and stained for DAPI and Spd2 (C). Asymmetric indexes of Polo-GFP in interphase NBs wor>lacZ (control, top panels) and HA-Spd2WT-OE larval brains (D). 38 and 39 NBs (n) from at least three brains were analysed in each line. The asymmetric distribution of Polo-GFP at interphase centrosomes was unaffected by HA-Spd2WT overexpression.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Representative images of interphase NBs in fixed mCh-Spd2WT-RES (top panels) and mCH-Spd2DK-RES larval brains and stained for DAPI, γTub and Cnn. Both multichannel and single-channel images of mCh-Spd2, γTub and Cnn are shown. Dotted yellow squares highlight centrosomes and their magnified images are shown in insets. Similar to Spd2DK-RES NBs, in mCh-Spd2CONS-RES NBs, Spd2 and γTub were more symmetrically accumulated at the two centrosomes than in mCh-Spd2WT-RES NBs, but Cnn was still asymmetrically distributed.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Kaplan-Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, Log-rank (Mantel-Cox) test). \"Normal\" represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1 and TFRC; \"Dysfunctional iron homoeostasis\" represents the patients with altered expression of iron homoeostasis-related genes.",
    "molecules": "iron"
  },
  {
    "caption": " (D) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination. ",
    "molecules": "ubiquitin"
  },
  {
    "caption": " (G) Half-life analysis of IREB2 in wild-type (WT) and OTUD1─/─ NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for western blot analysis (Up), the relative protein level of IREB2 was assessed by ImageJ software (Down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student's t-test). ",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": " (H) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC (50 μM) and Hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student's t-test). ",
    "molecules": "AFC, Hemin"
  },
  {
    "caption": " (I) Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1─/─ NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student's t-test). ",
    "molecules": "DFO"
  },
  {
    "caption": " Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates) ",
    "molecules": "iron"
  },
  {
    "caption": " Intracellular iron concentration was measured in wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) ",
    "molecules": "iron"
  },
  {
    "caption": " (D) Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1─/─ mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student's t-test). ",
    "molecules": "ferrous gluconate, iron"
  },
  {
    "caption": " Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. ",
    "molecules": "iron"
  },
  {
    "caption": " mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. ",
    "molecules": "iron"
  },
  {
    "caption": " mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. ",
    "molecules": "iron"
  },
  {
    "caption": " , red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. ",
    "molecules": "iron"
  },
  {
    "caption": " hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times. ",
    "molecules": "HB, hemoglobin, iron"
  },
  {
    "caption": " (I) Iron concentration of mock, OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry (n = 3 biological replicates, mean ± s.e.m., *P = 0.0237, **P = 0.0098, two-tailed unpaired Student's t-test). ",
    "molecules": "Iron"
  },
  {
    "caption": " (B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test). ",
    "molecules": "H2O2, PI"
  },
  {
    "caption": " (C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0119, **P = 0.0015, ***P < 0.001, two-tailed unpaired Student's t-test). ",
    "molecules": "Fer-1, H2O2, nec-1, PI, Z-VAD"
  },
  {
    "caption": " (D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student's t-test). ",
    "molecules": "Erastin, PI, RSL3"
  },
  {
    "caption": " (E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P < 0.001, two-tailed unpaired Student's t-test). ",
    "molecules": "Erastin, PI, RSL3"
  },
  {
    "caption": " F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates). ",
    "molecules": "DCFDA, Erastin, AFC, H2O2, ROS"
  },
  {
    "caption": " (G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity. ",
    "molecules": "DCFDA, ROS"
  },
  {
    "caption": " (I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates ",
    "molecules": "NP, VE"
  },
  {
    "caption": " Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity. ",
    "molecules": "DCFDA, NP, VE"
  },
  {
    "caption": " Flow cytometric analysis of the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. ",
    "molecules": "NP, VE"
  },
  {
    "caption": " CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test). ",
    "molecules": "ATP, RSL3"
  },
  {
    "caption": " wild-type (WT) or OTUD1─/─ NCM460 cells were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test). ",
    "molecules": "ATP, RSL3"
  },
  {
    "caption": " (E) ATP level in tumor interstitial fluid (TIF) of mock or OTUD1 overexpressing CT26 cells (n = 5 mice, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test). ",
    "molecules": "ATP"
  },
  {
    "caption": " (A) Representative pictures of colon tumors in AOM/DSS-induced Colitis-Associated Cancer (CAC) model (n = 6 biological replicates). ",
    "molecules": "DSS, AOM"
  },
  {
    "caption": " (D) Representative H&E (Hematoxylin-eosin) staining pictures of colon tumors (The scale bars represent 1000 μm). ",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": " (E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test). ",
    "molecules": "DSS, AOM"
  },
  {
    "caption": " (F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score. ",
    "molecules": "DSS, AOM"
  },
  {
    "caption": " (H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test). ",
    "molecules": "DSS, AOM"
  },
  {
    "caption": " (I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity. ",
    "molecules": "DCFDA, DSS, AOM, ROS"
  },
  {
    "caption": " (J) ATP level in tumor interstitial fluid (TIF) of mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., *P = 0.0292, two-tailed unpaired Student's t-test). ",
    "molecules": "ATP, DSS, AOM"
  },
  {
    "caption": "(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm.",
    "molecules": "K‐acetate, PMSF"
  },
  {
    "caption": "(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997).",
    "molecules": "[35S]methionine, nitrogen"
  },
  {
    "caption": "(C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).",
    "molecules": "K‐acetate, nitrogen"
  },
  {
    "caption": "Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.",
    "molecules": "K‐acetate"
  },
  {
    "caption": "Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.",
    "molecules": "K‐acetate"
  },
  {
    "caption": "Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.",
    "molecules": "K‐acetate"
  },
  {
    "caption": "(B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6).",
    "molecules": "NaCl"
  },
  {
    "caption": "Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.",
    "molecules": "benomyl"
  },
  {
    "caption": "(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm.",
    "molecules": "K‐acetate, PMSF"
  },
  {
    "caption": "(B) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm.",
    "molecules": "nitrogen, PMSF"
  },
  {
    "caption": "(a) Representative confocal microscopy images showing HACE1 (green) localizing to the Ub+ protein aggregates (red) induced by puromycin treatment in HA-HACE1 MEF. Arrows indicate co-localized puncta. Scale bar, 10 μm.",
    "molecules": "puromycin"
  },
  {
    "caption": "(b) Representative confocal micrographs of Hace1−/− and Hace1+/+ NCM treated with puromycin for 4 h (puromycin), puromycin followed by 8 h recover (puromycin+recover). Vehicle (PBS)-treated cells were used as control. Hace1 deficiency in cardiomyocytes (Hace1−/− NCM) blocks ubiquitinated protein aggregate degradation. Scale bars, 10 μm.",
    "molecules": "puromycin"
  },
  {
    "caption": "(c,d) WT and Hace1−/− MEF as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1were subjected to protein aggregates clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Cells were fixed and stained for ubiquitinated proteins to visualize aggregates. Vehicle (PBS)-treated MEFs were used as control. (c) Representative confocal micrographs of images used for quantification of the efficiency of aggregates clearance in d (percentage of cells that cleared all aggregates by 8 h after puromycin removal). At least 10 images from each group were used for the calculation, error bars represent s.e.m., P0.001 (one-way analysis of variance). Scale bars, 10 μm.",
    "molecules": "puromycin"
  },
  {
    "caption": "(a) Diagrams representing GFP-tagged WT HACE1 and mutant HACE1 (HACE1Δ HECT and HACE1Δ ANK) constructs transiently transfected into Hace1−/− MEF for protein aggregate clearance assay. (b-d) Representative confocal micrographs of (b) WT, (c) ΔHECT and (d) ΔANK MEF subjected to protein aggregate clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Vehicle-treated MEFs were used as control. Scale bars, 10 μm.",
    "molecules": "puromycin"
  },
  {
    "caption": "(a) Fluorescence microscopy images showing HACE1 (green) co-aggregating with p62 (red) in the perinuclear space on puromycin treatment (8 h) in Hace1−/− NCM transiently expressing HACE1-GFP. p62 was visualized by immunofluorescent staining. Arrows indicate co-localized puncta.",
    "molecules": "puromycin"
  },
  {
    "caption": "(b) Representative confocal images showing HACE1 (red) localizes to LC3+ puncta (green) induced by MG132 treatment (8 h) in Hace1−/−GFP-Lc3Tg NCM cells transiently expressing HACE1-RFP. Arrows indicate co-localized puncta.",
    "molecules": "MG132"
  },
  {
    "caption": "(c) Primary NCMs from Hace1+/+Lc3Tg and Hace1−/−Lc3Tg newborn pups were treated with or without MG132 for 8 h and the cell lysates were subjected to western blot with anti-GFP antibodies. Gapdh was used as a loading control.",
    "molecules": "MG132"
  },
  {
    "caption": "(d) Fluorescence microscopy images showing co-localization of HACE1 (green) with Lamp1 (red) on MG132 treatment (8 h) in Hace1−/−NCM cells transiently expressing HACE1-GFP. Lamp1 was visualized by immunofluorescent staining. Scale bar, 10 μm in all images.",
    "molecules": "MG132"
  },
  {
    "caption": "(a,b) Primary NCMs from WT and Hace1−/− newborn pups stably transfected with WT HACE1 and C876S-mutant HACE1, were transiently transfected with a tandem tagged mRFP-GFP-LC3 reporter (tfLC3) plasmid, followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h, and were subjected to confocal microscopy. DMSO treatment only was used in the control group. (a,c) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d. At least 10 images from each group were used for the calculation; error bars represent s.e.m., P-value as indicated (one-way ANAVO). Scale bars, 10 μm. Autophagosomes show both GFP and mRFP-LC3 signals (yellow) while autolysosomes exhibit mRFP-L3 signals only (red).",
    "molecules": "Baf, DMSO, MG132"
  },
  {
    "caption": "(c,d) WT and Hace1−/− MEF, as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1, were transiently transfected with a tandem tagged mRFP-GFP-LC3 reporter (tfLC3) plasmid, followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h, and were subjected to confocal microscopy. DMSO treatment only was used in the control group. (a,c) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d. At least 10 images from each group were used for the calculation; error bars represent s.e.m., P-value as indicated (one-way ANAVO). Scale bars, 10 μm. Autophagosomes show both GFP and mRFP-LC3 signals (yellow) while autolysosomes exhibit mRFP-L3 signals only (red).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": " A mRNA expression in FACS-isolated Lin(Ter119/CD31/CD45)-Sca1+ progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 µM cPGI2 or vehicle for the indicated time, as determined by expression profiling (n=3, E-MTAB-3693)  ****P=3*10-6 (Day 2), ****P=4*10-7 (Day 4), ****P=1*10-6 (Day 6) in 2x2 ANOVA with Bonferroni posttests (cPGI2 vs. vehicle)",
    "molecules": "cPGI2"
  },
  {
    "caption": " B mRNA expression in MACS-isolated Lin-Sca1+ progenitor cells  from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT-PCR (n=4) ****P=1*10-10 (Day 1 and 2), **P=0.001, in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle)",
    "molecules": "Rosi"
  },
  {
    "caption": " C mRNA expression in female Lin-Sca1+ cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT-PCR (n=3) t-test Cited4-/- vs. Cited4+/+ (Rosi) *P=0.013 (Ucp1), **P=0.004 (Cpt1b), *P=0.026 (Dio2)",
    "molecules": "Rosi"
  },
  {
    "caption": " D-F mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D-F), as determined by qRT-PCR at the indicated time points (n=5 patients). ♀/♂ represent individual data (D) ****P=3*10-5 (Day 2), ****P=3*10-6 (Day 6), ****P=4*10-9 (Day 10), ****P=3*10-7 (Day 14), in 2x2 ANOVA with Bonferroni posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient (r) and P-value are shown",
    "molecules": "Rosi"
  },
  {
    "caption": " G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT-PCR (n=3) ***P=0.0002 (CITED4), **P=0.002 (UCP1), *P=0.02 (UCP1), *P=0.035/0.026 (PPARG), ***P=0.0006 (SLC2A4), **P=0.002 (ADIPOQ) in one-way ANOVA with Tukey posttests (vs. siCtrl)",
    "molecules": "Rosi"
  },
  {
    "caption": " A-C Quantitative fluorescence microscopy of LipidTOX- and DAPI-stained female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days (n=5). **P=0.002 in t-test (Cre vs. Ctrl). Scale bar is 10 µm ",
    "molecules": "DAPI, LipidTOX, Rosi"
  },
  {
    "caption": " D, E Ucp1 expression in female Cited4F/F Lin-Sca1+ progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days, as determined by Western blot with VCP as loading control (n=3) ",
    "molecules": "Rosi"
  },
  {
    "caption": " F Cellular respiration in female Lin-Sca1+ progenitor cells differentiated in the presence of 100 nM Rosi for 8 days. The extracellular oxygen consumption rate (OCR) was determined upon injection of the indicated substances and normalized to DNA content. CL: CL-316243 ",
    "molecules": "CL-316243, oxygen, Rosi"
  },
  {
    "caption": " A, B mRNA expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by qRT-PCR t-test Cited4-/- vs. Cited4+/+ (Rosi), (A) n=5/5/6/6, **P=0.003 (Ucp1), **P=0.008 (Cidea), **P=0.007 (Cyc1), *P=0.048 (Ndufb3), (B) n=5/4/5/5, *P=0.022 (Ucp1), *P=0.037 (Cidea)",
    "molecules": "Rosi"
  },
  {
    "caption": " C, D Ucp1 expression in scWAT of mice treated as in A, as determined by Western blot with VCP as loading control t-test Cited4-/- vs. Cited4+/+ (Rosi), females: n=5/5/6/6, *P=0.011, males: n=5/4/5/5",
    "molecules": "Rosi"
  },
  {
    "caption": " E mRNA expression in scWAT of female mice treated with  CL-316,243 (CL) (1 mg/kg/day via Alzet minipumps) or vehicle for 10 days (n=5/7/13, t-test Cited4-/- vs. Cited4+/+ (CL)) ",
    "molecules": "CL, CL-316,243"
  },
  {
    "caption": " A Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 2.5 weeks, determined by indirect calorimetry and normalized to body weight (n=9/10). Grey bars represent the dark phase ",
    "molecules": "Oxygen, Rosi"
  },
  {
    "caption": " B Oxygen consumption rate of female mice shown in (A) adjusted for body weight by ANCOVA. 3-day averages of VO2 were calculated for each mouse (n=9/10). *P=0.025 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+) ",
    "molecules": "Oxygen"
  },
  {
    "caption": " C Oxygen consumption rate of female mice fed a diet with 0.0075% Rosi for 3.5 weeks and injected with 1 mg/kg CL-316,243 (CL) at the indicated time point, as determined by indirect calorimetry (n=9/10). *P=0.013 (-1 h), *P=0.045 (3 h), *P=0.015 (5 h), *P=0.016 (6 h), *P=0.019 (8 h), *P=0.046 (10 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+) ",
    "molecules": "CL-316,243, Oxygen, Rosi"
  },
  {
    "caption": " D Maximal CL-induced oxygen consumption rate of female mice shown in (C) adjusted for body weight by ANCOVA. Averages of VO2 throughout 3-8 hours post CL injection were calculated for each mouse (n=9/10). *P=0.049 in ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+) ",
    "molecules": "CL, oxygen"
  },
  {
    "caption": " E Oxygen consumption rate of male mice treated as in (A), determined as in (C) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+) ",
    "molecules": "Oxygen"
  },
  {
    "caption": " F Oxygen consumption rate of male mice treated as in (C), determined as in (C) (n=9/10). *P=0.044 (9 h), *P=0.013 (12 h) in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+) ",
    "molecules": "Oxygen"
  },
  {
    "caption": " G Maximal CL-induced oxygen consumption rate of male mice shown in (F) and calculated as in (D) (n=9/10). ANCOVA with Bonferroni posttest (Cited4-/- vs. Cited4+/+) ",
    "molecules": "CL, oxygen"
  },
  {
    "caption": " A, B mRNA expression in scWAT of mice fed a high-fat diet (HFD) for 11 weeks followed by 5 weeks of HFD with 0.0075% Rosi (qRT-PCR). t-test Cited4-/- vs. Cited4+/+, (A) n=8/7, *P=0.029 (Ucp1), **P=0.009 (Elovl3), **P=0.006 (Cpt1b), **P=0.004 (Cyc1), *P=0.016 (Ndufb3), **P=0.003 (Cd36), **P=0.009 (Gyk), (B) n=7/8, *P=0.026 (Pck1) ",
    "molecules": "Rosi"
  },
  {
    "caption": " E, F Blood glucose during insulin tolerance test (ITT) with 1 U insulin per kg body mass on female mice after 12 weeks of HFD (n=8) (E) or 16 weeks of HFD and 4 weeks of HFD+Rosi (n=7) (F), expressed as % of the 0-time point value. **P=0.003 in repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). G Area under the curve (AUC)  of blood glucose during the ITT (0-120 min) in (E,F). 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi) ",
    "molecules": "glucose, insulin, Rosi"
  },
  {
    "caption": " H, I Blood glucose during  
insulin tolerance test (ITT) on male mice as in (E,F), n=8 (H), n=7/8 (I). Repeated measures 2x2 ANOVA with Holm-Sidak posttests (Cited4-/- vs. Cited4+/+). J Area under the curve (AUC) of blood glucose during the ITT in (H,J). **P=0.001, ***P=0.00006 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi) ",
    "molecules": "glucose, Rosi"
  },
  {
    "caption": " K, L Fasting serum non-esterified fatty acids (NEFA) in female (K) or male (L) mice after 10 weeks of HFD (\"Before Rosi\") or 11 weeks of HFD and 2.5 weeks of HFD+Rosi (\"After Rosi\"), n=7/8/8/8 (K), n=6/6/6/8 (L). **P=0.001 in 2x2 ANOVA with Holm-Sidak posttests (After Rosi vs. Before Rosi) ",
    "molecules": "non-esterified fatty acids (NEFA), Rosi"
  },
  {
    "caption": "C. qRT-PCR data showing induction of each gene in the iniBAC operon in WT Mtb cultured in low iron and in the virR mutant strain cultured in high iron, expressed as fold change relative to WT Mtb cultured in iron sufficient conditions. RNA extracted from biological triplicates was analyzed. The data are presented as means +/- SEM. **P≤ 0.01 ***P ≤ 0.001 (Student's t-test).",
    "molecules": "iron"
  },
  {
    "caption": "A. MEV quantitation based on protein and lipid content in vesicle preparations isolated from the same number of Mtb cells treated with INH 50 ng.mlL-1 for 24 and 48 h relative to untreated cells. Three independent cultures were analyzed. Data are presented as mean ± SEM ***P ≤ 0.001 (Student's t-test).",
    "molecules": "INH, lipid, protein"
  },
  {
    "caption": "B. Immunodot blot assay detecting MEVs obtained from three independent cultures (1, 2, 3) and derived from the same number of Mtb exposed to 50 ng.mlL-1 INH for 24 and 48 h. A polyclonal antiserum raised against purified MEV (Prados-Rosales et al., 2014a) was used as a primary antibody.",
    "molecules": "INH"
  },
  {
    "caption": "E. Protein and lipid content in vesicle preparations isolated from the same number of WT and Msm overexpressing iniACMtb (Msm iniACMtb). Three independent cultures were analyzed. Data are presented as mean ± SEM *P≤ 0.05; *** P≤ 0.001 (Student's t-test).",
    "molecules": "lipid, Protein"
  },
  {
    "caption": "H. The graph shows the avarage of nanoparticle size distribution in three independent vesicle preparations derived from WT (black) and Msm iniACMtb (grey).",
    "molecules": "nanoparticle"
  },
  {
    "caption": "A. Quantitation of MEVs released by Mtb strains in low iron cultures based on vesicle protein and lipid content normalized to CFUs. Three biological replicates were processed. Data are presented as mean ± SD *P ≤0.05, **P≤ 0.005 (Student's t-test).",
    "molecules": "iron, lipid, protein"
  },
  {
    "caption": "C. Protein in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant grown in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SEM *P ≤ 0.05. ***P≤ 0.001 (Student's t-test)",
    "molecules": "hyg, iron, INH, Protein"
  },
  {
    "caption": "D. Lipid in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant cultured in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SD *P ≤ 0.05 (Student's t-test)",
    "molecules": "hyg, iron, INH, Lipid"
  },
  {
    "caption": "B. Mycobactin synthesized by Mtb WT and iniA::hyg grown in low iron conditions. Data are presented as the mean of mycobactin extracted from two independent cultures.",
    "molecules": "hyg, iron, Mycobactin, mycobactin"
  },
  {
    "caption": "C. The Mtb siderophore synthesis mutant (ST142) was cultured in low iron MM (medium) plain or supplemented with MEV preparations derived from equal number of Mtb WT, iniA::hyg and complemented strains grown in low iron MM. ST142 growth was determined based on the number of viable cells detected by the resazurine cell viability assay. MEV obtained from three independent cultures were tested.",
    "molecules": "hyg, iron, resazurine, siderophore"
  },
  {
    "caption": "A. Representative Immunodot-blot of bacterial antigens associated with EVs isolated from the culture supernatants of THP-1 cells infected with Mtb WT, iniA:hyg and complemented strains versus uninfected cells (UI). Anti-MEV specific antiserum was used for detection. The decreasing black bar indicates that increasing dilutions were spotted onto the membrane. The experiment was repeated three times with independent cultures.",
    "molecules": "hyg"
  },
  {
    "caption": "B. Number of nanoparticles per mL determined by Zeta View NTA in three EV preparations derived from the culture supernatans of THP-1 cells infected with Mtb WT, iniA:hyg and iniA::hyg complemented strains.",
    "molecules": "hyg, nanoparticles"
  },
  {
    "caption": "C. Representative Western blot of total EVs isolated from the CSF of THP-1 cells infected with Mtb WT, iniA::hyg and complemented strains versus uninfected cells (UI) detecting the exosome marker CD9. The experiment was repeated twice with similar results.",
    "molecules": "hyg"
  },
  {
    "caption": "(D) HaloTag pulse-chase analysis in WT MEF monitoring fluorescent Halo-AATNNN during up to 6 h of chase in the presence of BafA1. The fluorescent JF646-Halo ligand and the polymer-specific 2C1 antibody stains are shown. (E) Same as (D) for fluorescent Halo-ATZNNN. (F) Quantification of accumulation of fluorescent Halo-AATNNN and Halo-ATZNNN within LAMP1-positive endolysosomes. Mean ± SEM, n=20, 11, 10, 13 for Halo-AATNNN, n= 18, 12, 12, 13 for Halo-ATZNNN. Two-way ANOVA and Sidak's multiple comparison test, ns P>0.05, *** P<0.001. ",
    "molecules": "BafA1"
  },
  {
    "caption": "A Confocal laser scanning microscopy analyses in cells treated with BafA1. Total HA-ATZNNN (red) or HA-ATZNNN polymers (magenta) in LAMP1-positive endolysosome (green) in WT MEF. (C) Same as (A) in Cnx-KO MEF. (D) Same as (A) in WT MEF exposed to kifunensine (KIF). (E) Same as (A) in WT MEF exposed to Castanospermine (CST). (F) Same as (A) in sCNX MEF. (G) Same as (A) in Uggt1-KO MEF. ",
    "molecules": "BafA1, Castanospermine, CST, KIF, kifunensine"
  },
  {
    "caption": "(B-I) Confocal laser scanning microscopy analyses (as in Fig. 2) in WT MEF treated with BafA1. (J, K) (J) Quantification of ATZxxx-positive endolysosomes and (K) of ATZxxx polymers-positive endolysosomes (mean, n= 18, 30, 24, 14, 27, 22, 27, 18 cells for HA stain and n= 12, 14, 14, 11, 14, 14, 17, 11 cells for 2C1 stain of panels B-I, respectively). One-way ANOVA and Dunnett's multiple comparison test, ns P>0.05, **** P<0.0001. ",
    "molecules": "BafA1, polymers"
  },
  {
    "caption": "(G) Confocal laser scanning microscopy in Flp-In 3T3 cells stably expressing ATZNNN in the absence of BafA1 (to allow clearance of misfolded model proteins delivered within endolysosomes). Co-localization with Calnexin (CNX). (H) Same as (G) for BiP co-localization. (I) Same as (G) for ATZQQQ. (J) Same as (H) for ATZQQQ. ",
    "molecules": "BafA1"
  },
  {
    "caption": "(D) Clonogenic assay  of iHCC1-1 cells treated with a  MEK inhibitor (trametinib) and/or a CDK4/6 inhibitor (palbociclib); Representative images were shown based on three biological replicates",
    "molecules": "palbociclib, trametinib"
  },
  {
    "caption": "(F) Clonogenic assays (Top) of iHCC1-1 cells treated with XL413. The data are the representative of three biological replications. (Bottom) Growth curves of   iHCC in xenografts  (n=5) treated with PBS or XL413  at 50 mm3 tumor volume. P=0.0486 (XL413  vs. PBS  group) by two-way ANOVA with Sidak's multiple comparison test at Day24, *P≤0.05. Data are presented as the mean ± SD. N-numbers refer to biological replicates.",
    "molecules": "PBS, XL413"
  },
  {
    "caption": "HeLa  cells were transfected with a WT or mutant Parkin  plasmid (FLAG), shown in blue, along with reporters for either PA  or DAG  shown in green. DMSO  or 10 µM CCCP  was added for the indicated periods of time. Mitochondria  were visualized via anti-Tom20 staining  (red). (A) PA  accumulated on Parkin-positive mitochondria  after 1 hour of 10uM CCCP  treatment, with a more dramatic increase by 3hr and 18 hr. Scale bar = 25 µM, and zoom is 9x. (B) Quantification of imaging experiments shown in A and B. Bars represent mean with SEM from three biological independent experiments; two-way ANOVA analysis was performed for statistical analysis (***P<0.0001). (C) DAG  (diacylglycerol) accumulated on Parkin-positive mitochondria  after 5.5 hr of 10uM CCCP  treatment. Arrowheads and arrows point to the DAGR  concentration at the Golgi  and mitochondria, respectively. Scale bar = 25 µM, and zoom is 9x.",
    "molecules": "CCCP, DAG, diacylglycerol, DMSO, PA"
  },
  {
    "caption": "(B) HEK-293T  cells were transfected with Parkin-FLAG  and DAGR-YFP  followed by CCCP  treatment for indicated times. Mitochondrial  and cytosolic  fractions were purified and analyzed by Western blots by indicated antibodies. Citrate synthetase  and α-tubulin were used as a mitochondrial  and cytosolic  marker, respectively. Note that PLD2  and Parkin  levels were elevated in the mitochondrial  fraction in response to CCCP  treatment.",
    "molecules": "CCCP, DAG"
  },
  {
    "caption": "(C) Hela  cells were co-transfected with mCherry-Parkin  and the PA  reporter, followed by CCCP  treatment alone or with a PLD2  inhibitor VU 0364739 (3 µM) for 5.5 hrs. Line scan analysis (Image J software) corresponding to the line drawn in the images indicate colocalization  between the PA  reporter (green) and mitochondria  (red). Note that VU 0364739 suppressed mitochondrial PA  accumulation. Scale bar = 10 µM. (D) Quantification of the numbers of cells with the PA  reporter positive mitochondria  shown in (C). Asterisks indicate statistical significance (***P<0.001, Student's t-test) from three biological independent experiments. The bars indicate mean± SEM.",
    "molecules": "CCCP, PA, VU 0364739"
  },
  {
    "caption": "(E) Hela  cells were transfected with a lipin1  siRNA, followed by the DAG  reporter and mCherry-Parkin, treated with CCCP  (10 µM) for 9 hrs and then subject to image analysis. Scale bar = 25 um, and zoom is 4x. Lipin 1 KD reduced mitochondrial DAGR, but not Golgi-DAGR, accumulation (arrowheads) (F) The numbers of cells with DAG-reporter positive mitochondria  shown in (E) and in Lipin-1 knockdown cells transfected with siRNA-resistant wildtype (WT) and catalytically dead (CD; D712E;D714E) mutant cDNA. Note that Lipin1  KD reduced mitochondrial DAG  accumulation, which can be significantly restored by the re-expression of WT, but not CD mutant, Lipin-1. The graph shows the means with SEM (error bars) from three biological independent experiments. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001).",
    "molecules": "CCCP, DAG"
  },
  {
    "caption": "(A-B) Hela  cells were transfected with a Lipin1  or control (cKD) siRNA followed by a GFP-Parkin  expression plasmid. Transfected cells were treated with CCCP  at 10 µM for 18 hrs, and subject to immuno-staining. (B) Quantification of Parkin-positive cells in (A) that have lost a majority of mitochondria  (marked by TOM20, arrows). Note that mitochondrial  clearance is reduced in Lipin-1  knockdown cells (arrowheads in (A)). Scale bar = 25 µM.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C). Control or lipin1  knockdown Hela  cells stably expressing parkin-mCherry  were treated with DMSO  or CCCP  (10 µM for 18 hrs) and Bafilomycin A1  (1μM, lysosomal inhibitor) treatment, followed by immunoblotting  with indicated antibodies and quantified by the Image J. software. Note that lipin1  silencing suppressed CCCP-induced mitochondrial  protein degradation. Lipin-1-mediated mitochondrial DAG  production is required for mitophagosome  production.",
    "molecules": "Bafilomycin A1, CCCP, DAG, DMSO"
  },
  {
    "caption": "(D-F). Hela  cells were transfected with control or a Lipin1  siRNA followed by the expression plasmids of GFP-Parkin  and RFP-DAGR. Transfected cells were treated with DMSO or CCCP  at 10 µM for 9 hr, with or without further incubation with 1,2-Dipalmitoyl-sn-glycerol  (DPG) at 100 µM, as indicated. Autophagosome formation was assessed by an LC3  antibody (pseudocolored in white in single-channel and blue in the overlay images). Arrows point to DAGR concentration at the Golgi  under basal conditions. The number (E) and size (F, arbitrary unit) of LC3 vesicles  were quantified. n=4 biological replicates. The bars indicates mean± SEM. Asterisks indicate statistical significance by one-way ANOVA (*P< 0.05, **P<0.01). Scale bar = 25 µM and zoom is 5x. Note that both the number and size of LC3-vesicles  in Lipin-1 knockdown cells were much smaller than those in WT cells, and these defects were corrected by DPG  treatment.",
    "molecules": "1,2-Dipalmitoyl-sn-glycerol, DPG, CCCP, DAG, DMSO"
  },
  {
    "caption": "(A) E3 ligase-deficient Parkin  mutants (T240R and T415N) have no effect on PA  accumulation (green) on depolarized, Parkin-positive mitochondria. The expression of WT and Parkin  mutants were determined by Immunoblotting. Scale bar = 25 µM, and zoom is 3x. (B) DAG  accumulation depends upon intact Parkin E3 ligase  activity. Scale bar = 25µm, and zoom is 3x. (C) Quantification of imaging experiments shown in (A) and (B). Bars represent mean with SEM; two-way ANOVA analysis was performed for statistical analysis(***P<0.001). n=3 biological replicates.",
    "molecules": "DAG, PA"
  },
  {
    "caption": "(D) Hela  cells were transfected with control or optineurin- and NDP52- siRNAs and expression plasmids of FLAG-Parkin  and YFP-DAGR. These cells were treated with DMSO  or 10 µM CCCP  for 5.5 hrs, as indicated. Note that OPTN/NDP52  double knockdown prevented mitochondrial YFP-DAGR accumulation. (Scale bar = 25 µM and zoom is 3x) (E) Hela OPTN/NDP52  knockdown cells were transfected with a siRNA-resistant wildtype or ubiquitin-binding deficient E478G OPTN  mutant, as indicated. The percentage of cells with mitochondrial YFP-DAGR following CCCP  treatment was quantified. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001) from three biological independent experiments. The bars indicate mean± SEM. Bottom panel: Expression of OPTN, OPTN  E478 and NDP52  was determined by immunoblotting.",
    "molecules": "CCCP, DAG, DMSO, ubiquitin"
  },
  {
    "caption": "(F-G) Hela OPTN/NDP52  knockdown cells were incubated with 1,2-Dipalmitoyl-sn-glycerol (DPG) followed by CCCP  treatment for 18 hrs. Mitophagy  efficiency was assessed by TOM20 staining  and quantified in (G) (n = 2 biological replicates). Note that DPG  significantly restored mitophagy  in OPTN/NDP52  knockdown cells. Scale bar = 10 µM.",
    "molecules": "1,2-Dipalmitoyl-sn-glycerol, DPG, CCCP"
  },
  {
    "caption": "(B) Mitochondrial  and cytosolic  fractions obtained from control and CCCP  treated cells, were immunoblotted  for EndoB1  and OPTN, as indicated.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C). Cytosolic  and mitochondrial  fractions purified from control and OPTN/NDP52  knockdown cells treated with CCCP  were immunoblotted  with indicated antibodies. Note that mitochondrial EndoB1  levels were reduced in OPTN/NDP52  knockdown cells",
    "molecules": "CCCP"
  },
  {
    "caption": "(D-E) Hela  cells were transfected with an EndoB1  siRNA, followed by the expression plasmids for mCherry-Parkin  and YFP-DAGR, and CCCP  treatment (10 µM) for 9 hrs. Cells with mitochondrial YFP-DAGR were quantified in (E) (*P<0.05, Student's t-test, n=3 biological replicates). Scale bar = 25 µM and zoom is 5x. Note that knockdown of EndoB1  suppressed mitochondrial DAG  production.",
    "molecules": "CCCP, DAG"
  },
  {
    "caption": "(A) WT BMDM were incubated for 1 h without (-) or with (+) the TAK1 inhibitor NG25  (2 μM), the  IKKβ inhibitor BI605906 (5 μM) or the IKKβ  inhibitor PS1145  (10 μM). The cells were then co-stimulated for 30 min with 100 ng/ml LPS and 4 mM ATP. Cell lysates (10 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. (B) As in A, except that the cells were co-stimulated for 1 h with 100 ng/ml  LPS and 5 μM nigericin. Similar results were obtained in three independent experiments in A and B. ",
    "molecules": "ATP, BI605906, LPS, NG25, nigericin, PS1145, SDS"
  },
  {
    "caption": "(A, B) The  Mouse macrophage J774A.1 cell line was transfected with control siRNA or siRNA against IKKβ (A) or  IKKα  (B). 72 h post-transfection, the cells were co-stimulated for 60 min without (-) or with (+) 100 ng/ml LPS  and/or 5 μM nigericin. Cell lysates (10 μg) were denatured in SDS, subjected to SDS-PAGE  and  immunoblotted with the antibodies indicated. Similar results were obtained in three (A) or two (B) independent experiments.",
    "molecules": "LPS, nigericin, SDS"
  },
  {
    "caption": "(C, D) As in (A, B) except that BMDM from  IKKβ-LysM-Cre (flox/flox) (IKKβ (fl/fl)) or WT control mice were co-stimulated without (-) or with (+) 100 ng/ml LPS  and/or 4 mM ATP (C) or with 100 ng/ml  LPS and/or 5 μM nigericin (D). Similar results were obtained in two independent experiments.",
    "molecules": "ATP, LPS, nigericin"
  },
  {
    "caption": "(A, B) WT BMDM were incubated for 1 h without (-) or with (+), the  IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or  PS1145  (10 μM), the TAK1 inhibitor NG25  (2 μM) or the  NLRP3  inhibitor  MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP  (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was  precipitated (see Methods), dissolved in  SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL)  gasdermin D (GSDMD), the p20  and p10 fragments of caspase-1  and IL-18. Similar results were obtained in two independent experiments. (C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments. ",
    "molecules": "TPCA-1, ATP, BI605906, LPS, MCC950, NG25, nigericin, PS1145, SDS"
  },
  {
    "caption": "(A) WT  BMDM  were incubated for 1 h without (-) or with (+) 5 μM BI605906, then stimulated for 1 h with (+) 100 ng/ml  LPS and/or 5 μM nigericin or left unstimulated. The cells were lysed in buffer containing 1% (v/v)  Triton-X-100, and the Triton-X-100-soluble and Triton-X-100-insoluble fractions were prepared as in Methods. The Triton-X-100-insoluble fraction was dissolved SDS  subjected to  SDS-PAGE and immunoblotted with the antibodies indicated. The nuclear protein Histone H3B (H3B) was used as a loading control in the Tritin X-100 insoluble fraction. Similar results were obtained in three independent experiments. (B) As in A, except that the Triton-X-100-insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2.0 mM DSS. ",
    "molecules": "BI605906, DSS, LPS, nigericin, SDS, Tritin X-100, Triton-X-100"
  },
  {
    "caption": "(A-H) WT  BMDM were incubated for 1 h without or with the IKKβ  inhibitors TPCA-1  (5 μM) (C, G) or BI605906 (5 μM) (D, H). The cells were then stimulated for 1 h with 100 ng/ml  LPS (E) or 5 μM  nigericin (F, G, H), or co-stimulated with 100 ng/ml LPS and 5 μM nigericin  (B, C, D) or left unstimulated (control) (A). The cells were fixed and processed for  PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3. A  PLA  signal is only if the NLRP3  antibody and the TGN38 antibody come into close proximity. (A-H) the panel images show the  PLA signal (red) and  DAPI staining for nuclei (blue). Images were acquired by sequential  laser scanning on a confocal microscope. Similar results were obtained in three independent experiments and representative images are shown. Scale bar = 50 μm.",
    "molecules": "TPCA-1, BI605906, DAPI, LPS, nigericin"
  },
  {
    "caption": "(I-L) As in A-H, except that IKKβ  inhibitors were omitted and iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl)) or WT mice  were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated (control). Scale bar = 50 μm.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(A-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown Data information: In all panels, scale bar = 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ  (fl/fl) or WT  mice  were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments. Data information: In all panels, scale bar = 50 μm. ",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(H)  Eosin-hematoxylin-stained  sections of the lung and liver of a  Pax5Jak2/+ tumor mouse.  Infiltrating and blasting tumor cells  are indicated by an arrow.",
    "molecules": "Eosin, hematoxylin"
  },
  {
    "caption": "(G) Kaplan-Meier survival  analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey)  mice. Statistical analysis of the  survival  curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.",
    "molecules": "neo"
  },
  {
    "caption": "(H)  Flow-cytometric analysis of  B220 and CD19 expression  in  B-ALL  tumor cells from the  lymph node  of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression  in these B-ALL  tumor cells (black line) and control  Pax5+/+ lymph node B cells (grey filled) was determined by intracellular  Pax5 staining (right).",
    "molecules": "neo"
  },
  {
    "caption": "(D, Tumor progression in  mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with  ruxolitinib or vehicle, and the  tumor mass was monitored by bioluminescence   measurements. Images of three representative mice  with or without  ruxolitinib treatment are shown",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "(F)  Kaplan-Meier survival  analysis of the transplanted  mice  upon prolonged treatment with  ruxolitinib  (black) and vehicle (grey). Statistical analysis of the survival  curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "(B) Genome-wide  binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice  at the age of 3 weeks (expressing  Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding  pattern of full-length  Pax5  was determined by  Bio-ChIP-seq  analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an  IRES‐BirA gene insertion in the 3' untranslated region of  Pax5  Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative  binding patterns of Pax5 and Pax5-Jak2 in the three B cell  types are shown for a selected genomic region, with horizontal bars indicating Pax5  or Pax5-Jak2  peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and  Pax5-Jak2  (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.",
    "molecules": "Bio, biotin"
  },
  {
    "caption": "(C) Specificity of the anti-H3Y41ph antibody, as shown by  immunoblot  analysis. One  phosphorylated (pY41) or non-phosphorylated peptide was coupled to  ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1  cell supernatant. Following treatment with  calf intestinal alkaline phosphatase   (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody.",
    "molecules": "Ubi, ubiquitin"
  },
  {
    "caption": "(D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control  Pax5Etv6/+ Cdkn2ab+/- B-ALL   cells as well as from the human HEL, TMD8 and  K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to  ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "BrdU incorporation reflecting DNA synthesis and SA-b-gal activity as markers of CTX-induced senescence in Em-myc transgeniccontrol;Bcl2 versus Suv39h12;Bcl2lymphomas in vivo. Numbers represent mean percentages of positive cells6s.d. (n55 each).",
    "molecules": "CTX, DNA"
  },
  {
    "caption": "b, Representative whole-body FLT- and FDG-PET scans of an individual control;Bcl2 lymphoma-bearing mouse at tumour manifestation (untreated) and at day 6 after a single dose of CTX. Red circles, areas of specific tracer retention; green circles, matching areas with lost retentions. Specific tracer uptake was assessed by calculation of tumour-to-background ratios. Data represent mean ratios ± s.d. (n = 5 mice for FLT-PET scans and n = 6 mice for FDG-PET scans). Insets show representative Ki67 staining and colour-calibrated (red, highest intensities; blue, lowest intensities) bioluminescence images of glucose levels in tissue sections of the respective groups.",
    "molecules": "CTX, glucose, tracer"
  },
  {
    "caption": "c, Intracellular glucose concentrations in untreated and ADR-treated control;Bcl2 (that is, senescent) versus Suv39h1-;Bcl2 (that is, non-senescent) lymphomas in vitro (as in d-g). Results represent means ± s.d. (n = 5 each).",
    "molecules": "ADR, glucose"
  },
  {
    "caption": "d, Extracellular levels of the specified metabolite-to-glucose ratios in the indicated lymphoma cells groups relative to untreated control;Bcl2 lymphoma cells after 18 h of cultivation. Data represent mean ratios ± s.d. (n = 4 each, measurements were carried out in duplicate). Note that glucose-6-phosphate is not detectable (n.d.) in the medium. Insets show (ADR-treated relative to untreated) intracellular levels of the respective metabolites after labelling with 13C6-glucose for one representative lymphoma from each group. Data represent mean ratios (see Supplementary Fig. 5 for an assessment of the heterogeneity of the TIS-associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas).",
    "molecules": "glucose-6-phosphate, ADR, glucose, 13C6"
  },
  {
    "caption": "e, Immunoblot analyses of phosphorylated AMP-activated protein kinase (pAMPK), total AMPK, pyruvate kinase isoforms M1 and M2 in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells in vitro (n = 3 each; α-tubulin as a loading control. Gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).",
    "molecules": "ADR"
  },
  {
    "caption": "f, Mean OCRs in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells in vitro. Results represent means ± s.d. (n = 5 each).",
    "molecules": "ADR"
  },
  {
    "caption": "g, Intracellular ATP concentrations in cells as in f. Results represent means ± s.d. (n = 5 each). *P<0.05.",
    "molecules": "ATP"
  },
  {
    "caption": "a-e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro. All results in this figure represent means ± s.d. (n = 5 each). a, Inhibition of glucose metabolism by phloretin, cytochalasin B, and sodium oxamate.",
    "molecules": "sodium oxamate, cytochalasin B, ADR, glucose, phloretin"
  },
  {
    "caption": "b, Exposure to 2DG or switch to glucose-free medium with or without glucose replenishment.",
    "molecules": "2DG, glucose"
  },
  {
    "caption": "c, Lentiviral knockdown of hexokinase 2 (Hk2) by short hairpin RNA (shHk2). d, Inhibition of AMPK by compound C. e, Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir. f, Inhibition of the electron transport chain by antimycin A. *P  0.01, except b (n = 8 each) and e, in which *P  0.05.",
    "molecules": "antimycin A, compound C, etomoxir, fatty acid"
  },
  {
    "caption": "Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig. 2, or versus SASP-attenuated TISlymphoma cells. a, Global protein synthesis rate, measured by incorporation of green-fluorescent methionine. Mean fluorescent intensities reflect newly synthesized protein within 2 h (DAPI used as a counterstain). Data represent means ± s.d. (n = 5 each). b, Rate of global protein synthesis (measured as in a) in ADR-senescent relative to untreated (ut) control;Bcl2 lymphoma cells; either empty vector-infected cells or cells stably expressing the NF-kB super-repressor IkBaDN(SR, ref. 18). Results represent means ± s.d. (n = 5 each). c, Relative intracellular glucose concentrations in cells as in b.",
    "molecules": "ADR, glucose, methionine, protein"
  },
  {
    "caption": "d, Relative OCR in cells as in b. e, Relative intracellular ATP concentrations in cells as in b",
    "molecules": "ATP"
  },
  {
    "caption": "g, Global protein ubiquitination, expression of p62/SQSTM1 (whose transcript levels were also selectively induced in TIS cells by approximately 4.8-fold (data not shown)) and V-ATPase subunit A1, and conversion of MAP1-LC3-I to MAP1-LC3-II by immunoblot analysis, demonstrating autophagosome formation in lymphoma cells as in f (α-tubulin was used as a loading control, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). *P<0.05.",
    "molecules": "protein"
  },
  {
    "caption": "a, Cellular viability of super-repressor- or empty-vector-infected control;Bcl2 lymphomas treated with ADR or untreated for 5 days, then exposed for 2 days to the inhibitors 2DG or bafilomycin A1, compared to no inhibitor (ut) in vitro. Results represent means ± s.d. (n = 5 each).",
    "molecules": "2DG, bafilomycin A1, ADR"
  },
  {
    "caption": "b, Immunoblot analyses of ATF4, global protein ubiquitination, p62/SQSTM1, and MAP1-LC3-I to MAP1-LC3-II conversion in control;Bcl2 lymphomas with no ADR treatment, in TIS cells, and in TIS cells after exposure to 2DG or bafilomycin A1in vitro; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). c, Cleavage (activation) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control;Bcl2 lymphomas as in b; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).",
    "molecules": "2DG, bafilomycin A1, ADR, protein"
  },
  {
    "caption": "d, Cellular viability of ADR-treated control;Bcl2 lymphoma cells infected with short-hairpin RNAs against either caspase 12 (shCasp12) or caspase 3 (shCasp3) compared to empty vector-infected lymphoma cells treated as in a. Results represent means ± s.d. (n = 4 each).",
    "molecules": "ADR"
  },
  {
    "caption": "e, TIS cells in primary human acute myeloid leukaemia (AML) samples by SA-b-gal staining of treated (0.1 µg ml−1 ADR for 5 days) versus untreated cells in vitro. Data represent means ± s.d. (n = 5 each).",
    "molecules": "ADR"
  },
  {
    "caption": "f, Intracellular glucose (left) and ATP (right) concentrations in senescent AML cells as in e.",
    "molecules": "ATP, glucose"
  },
  {
    "caption": "h, Colour-coded whole-body fluorescence imaging to visualize green fluorescent protein (GFP)-tagged tumour burden in time-courses of three representative mice harbouring CTX-senescent control;Bcl2 lymphomas at day 5 (see Figure 1a), and two days later, after the administration of 2DG (n = 3 mice), bafilomycin A1 (n = 4 mice) or mock treatment (n = 4 mice) starting from day 5 post CTX (red, highest intensities; blue, lowest intensities). TdT-mediated dUTP nick end labelling (assay) (TUNEL) staining of lymph node sections in situ to visualize apoptosis at day 7. Numbers represent means ± s.d. (n = 3).",
    "molecules": "2DG, bafilomycin A1, CTX, protein"
  },
  {
    "caption": "i, Overall survival of 5 day-CTX-treated control;Bcl2 or Suv-39h1-;Bcl2 lymphoma-bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h (control;Bcl2: n = 12 and Suv39h1-;Bcl2: n = 10), presented as matched pair analyses (P<0.001 for the control; Bcl2 comparison; P = 0.221 for the Suv39h1-;Bcl2 comparison). *P<0.05.",
    "molecules": "bafilomycin A1, CTX"
  },
  {
    "caption": "e, Olomoucine triggers differentiation of WT NPCs into TUJ1-positive neurons under non-differentiating conditions. Error bars are s. e. m. *** p<0.001, Ordinary Two-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments. 298 cells for untreated, 151 cells for DMSO and 146 cells for olomoucine, 25 μM.",
    "molecules": "DMSO, Olomoucine, olomoucine"
  },
  {
    "caption": "c, VX680 (0.5 μM, a concentration that will specifically allow neuronal differentiation) was used to inhibit Aurora A activity. Bar diagrams below show quantifications. Compared to DMSO-treated control NPCs, a higher proportion of VX680 treated NPCs are ciliated (***p<0.001), exhibiting longer cilia (***p<0.001) and show Tuj1 positive neurons (***p<0.001). Error bars are s. e. m. For ciliated cells and cilium length: n = 3 independent experiments. 164 cells for DMSO-treated and 77 cells for VX680-treated. For NPC differentiation: n = 4 independent experiments. 286 cells for DMSO-treated and 118 cells for VX680-treated. Scale bar 1 μm.",
    "molecules": "DMSO, VX680"
  },
  {
    "caption": "d, Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours. Inhibition of Ofd1 (*p<0.05), Nde1 (**p<0.01) and Aurora A (***p<0.001) caused significantly reduced incorporation of EdU.",
    "molecules": "VX680"
  },
  {
    "caption": "(H-J) Immuno-gold localization of Snap29 in cryo-sections of S2 cells in pro-metaphase. Pseudo-coloring in red identifies chromosomes (Ch), in yellow membrane organelles and in green MTs. The white arrows in H-I indicate examples of signal in the nucleoplasm that is not associated to membranes, but rather, to electron-dense material. MVB: multivesicular body; NE: nuclear envelope; PM: plasma membrane.",
    "molecules": "gold"
  },
  {
    "caption": "(E) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA. (F) Averaged spindle pole length based on >20 cells with bipolar spindles/sample. The P value is obtained by two-tailed paired t-test.",
    "molecules": "DNA"
  },
  {
    "caption": "(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.",
    "molecules": "Nocodazole, thymidine"
  },
  {
    "caption": "(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.",
    "molecules": "Nocodazole, thymidine"
  },
  {
    "caption": "(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.",
    "molecules": "Nocodazole, thymidine"
  },
  {
    "caption": "(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.",
    "molecules": "Nocodazole, thymidine"
  },
  {
    "caption": "Representative images of combined IF for H3K27me3 or H2AK119ub (green) with RNA FISH for Xist (red) in Xist-TetOP lines (for clone 1 of each mutant type) upon D2 in DOX conditions; Blue - DAPI staining; Scale bar: 10 µm.",
    "molecules": "DAPI, DOX, RNA"
  },
  {
    "caption": "Top 20 protein hits from the ChIRP-MS of Xist FL; The ranking was based on fold-enrichment of Xist FL DOX versus Xist FL noDOX; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 (out of 5) were excluded; Fold-enrichment for Xist ΔB+C is also displayed for comparison; Light green boxes correspond to proteins as Xist interactors [13]; protein in red (RNF2/RING1B) represents a protein not found in the Xist ΔB+C interactome; Protein in light brown (TRIM71) is less enriched in Xist ΔB+C than in Xist FL.",
    "molecules": "DOX"
  },
  {
    "caption": "Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB+C for the 74 out of 81 Xist-interactors with a minimum of fold-change of 2.5 in Xist FL or Xist ΔB+C; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions; proteins retrieved by both Xist FL and Xist ΔB+C ChIRPs with a proposed role in XCI such as SPEN, RBM15, WTAP, YTHDC1 and hnRNPU are indicated; light brown dots mark proteins more represented in Xist FL than in Xist ΔB+C ChIRPs, while red dots display proteins which are only retrieved by Xist FL ChIRP.",
    "molecules": "DOX"
  },
  {
    "caption": "Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB+C with upon DOX induction at day 2 (D2) of differentiation; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition; Black line is a loess regression on all windows; Xist locus is represented by a blue long line, active genes by green lines.",
    "molecules": "DOX"
  },
  {
    "caption": "Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions, active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB+C cell lines at D2 upon DOX induction; Shown is the log2 fold change of DOX vs noDOX conditions; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; n = indicates the number of regions/genes analyzed; p-values were calculated using paired Wilcoxon test, comparing Xist FL and Xist ΔB+C cell lines.",
    "molecules": "DOX"
  },
  {
    "caption": "Average plots showing the mean enrichment of H3K27me3 (top) and H2AK119ub (bottom) over all X-linked initially active transcriptional start sites (TSS); Shown is the mean of normalized log2 enrichment of DOX vs noDOX in both Xist FL and Xist ΔB+C cell lines.",
    "molecules": "DOX"
  },
  {
    "caption": "Graph represents the mean % + S.E.M. of Xist-coated chromosomes presenting an active Pgk1 or Lamp2 gene as determined by RNA FISH (as represented in B) at D2 in the presence of DOX (Xist FL was also used in noDOX conditions) in the different Xist-TetOP mutants; each bar represents the mean from to 2-to-4 independent experiments; A minimum of 59 Xist-coated chromosomes were counted per experiment; For Xist FL noDOX a minimum of 100 cells (which do not have Xist-coated chromosome) were counted; Only p-values corresponding to significant differences comparing mutants (or Xist FL noDOX) to Xist FL DOX are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test; dashed line marks the mean percentage of silencing for the Lamp2 gene in Xist FL DOX.",
    "molecules": "DOX"
  },
  {
    "caption": "Representative RNA FISH images for Xist (red) and nascent-transcript of Pgk1 (green) in Xist-TetOP lines at day 4 of differentiation in the presence of DOX (Xist FL is also shown in noDOX conditions); DNA stained in blue by DAPI; Numbers represent % of Xist-coated X-chromosomes ± S.E.M. with active Pgk1 gene (except for Xist FL noDOX, where numbers represent % of cells with Pgk1 active gene); The values represent 2-to-4 independent experiments, where a minimum of 50 Xist-coated chromosomes were counted per experiment; Significant differences compared with Xist FL (DOX) are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test.",
    "molecules": "DAPI, DNA, DOX"
  },
  {
    "caption": "Clustering analysis of the normalized RNA-seq counts on the X chromosome (chrX) for all the duplicates of Xist FL, Xist ΔA and Xist ΔB+C in DOX and noDOX conditions.",
    "molecules": "DOX"
  },
  {
    "caption": "Plots displays the log2(fold-change) in the expression of X-linked genes along the chrX comparing DOX versus noDOX samples for Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; red dots correspond to genes which are differently expressed in DOX vs noDOX (p < 0.05, Limma t-test), while black dots represent genes which are not differentially expressed between the two conditions (p ≥ 0.05).",
    "molecules": "DOX"
  },
  {
    "caption": "Violin plots displaying the average log2(fold-change) in gene expression between DOX and noDOX conditions on the chrX in Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; p-values were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.",
    "molecules": "DOX"
  },
  {
    "caption": "Box plots displaying the log2(DOX/noDOX) fold-change in expression of X-linked genes in Xist FL and Xist ΔB+C categorized according to the enrichment of H3K27me3 and H2AK119ub marks at promoters in Xist ΔB+C upon DOX induction (with no or little accumulation vs accumulation); the horizontal band of the boxplot is the median of the values, the lower and upper hinges correspond to the 25th and 75th percentiles, the upper whisker extends from the hinge to the largest value not further than 1.5 interquartile range from the hinge, the lower whisker extends from the hinge to the smallest value at most 1.5 interquartile range of the hinge; p-values between samples were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.",
    "molecules": "DOX"
  },
  {
    "caption": "Genome browser plots showing RNA-seq reads, H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL (left) and Xist ΔB+C (right) at day 2 of differentiation in both DOX and noDOX conditions.",
    "molecules": "DOX"
  },
  {
    "caption": "B, C, Representative images of pPLT3::erCFP (cyan) expressing and PI-stained (red) Arabidopsis roots in Col or wox5 background, respectively. D, Mean fluorescence intensities of the pPLT3::erCFP roots summarized in box and scatter plots. The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 %. D, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. B, C, Scale bars represent 10 µm. PI = propidium iodide CFP = cyan fluorescent protein.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "E, F, Representative images of pPLT3::PLT3-YFP (yellow) expressing and FM4-64-stained (red) Arabidopsis roots in Col or wox5 mutant background, respectively. g, Mean fluorescence intensities of the pPLT3::PLT3-YFP expressing roots summarized in box and scatter plots. The mean fluorescence intensity of the YFP signal in Col roots was to set to 100%. G, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. , E, F, Scale bars represent 10 µm. YFP = yellow fluorescent protein",
    "molecules": "FM4-64"
  },
  {
    "caption": "A-F, Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3 and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. E', F', Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. E'', F'', Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G, Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H, Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. G, H Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. (G, H) Kruskal-Wallis ANOVA analysis with subsequent Dunns test (G) or one-way ANOVA and post-hoc Holm-Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localisation signal.",
    "molecules": "FM4-64"
  },
  {
    "caption": "A, Schematic representation of a longitudinal section of an Arabidopsis RM. QC cells are marked in red, CSCs are marked in dark blue, CCs in light blue. Combined mPSPI  (grey) and EdU (red) staining for 24 hours (SCN staining) to analyse the CSC (A-I) and QC division phenotype (J-R) within the same roots are shown. B-H, Representative images of the SCN staining in Col, and the indicated single, double, and triple mutant roots. QC positions are marked by yellow arrowhead. I, Analyses of the SCN staining for CSC phenotypes. Frequencies of roots showing 0, 1, 2, or 3 CSC layers are plotted as bar graphs. J, Schematic representation of a transversal section of an Arabidopsis RM. QC cells are marked in red, CEI initials are marked in turquoise. K-Q, Representative images of transversal sections with QC cells outlined in yellow. R, Analyses of the SCN staining for QC division phenotypes. Frequencies of roots showing 0, 1, 2, 3 or ≥4 dividing QC cells are plotted as bar graphs. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype. QC = quiescent center, CSC = columella stem cell, CEI = cortex endodermis initial, SCN = stem cell niche, mPSPI = modified pseudo-Schiff propidium iodide, EdU  = 5-ethynyl-2'-deoxyuridine, scale bars represent 5 µm.",
    "molecules": "modified pseudo-Schiff propidium iodide, mPSPI, 5-ethynyl-2'-deoxyuridine, EdU"
  },
  {
    "caption": "The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC  layer and QC division phenotypes. Number of CSC layers are shown on the y axis and the QC  division phenotype is shown on the x axis. The darker the colour, the more roots show the respective phenotype (see colour gradient top right indicating the frequencies in percent). Col wild-type roots show one layer of CSCs and no EdU stained cells (no QC division) after 24 h EdU staining. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype.",
    "molecules": "EdU"
  },
  {
    "caption": "Fig 1: Urinary triclosan levels are elevated in TC mothers following 6 months of exposure. Summaries are provided as the interquartile range with median of triclosan molecules in a picogram/microliter (pg/L). Urinary triclosan measurements are available for 38 mothers (17 TC, 21 nTC) and 33 infants (15 TC, 18 nTC). The p-value for the mothers in Figure 1 (represented by ***) is p=5.07e-5 (Mann-Whitney U test).",
    "molecules": "TC, triclosan"
  },
  {
    "caption": "Fig 3: TC randomization does not decrease gut microbial diversity in infants or mothers. Shannon diversity measures are plotted as the interquartile range with median for each TC exposure class and time point grouping for infants and mothers.",
    "molecules": "TC"
  },
  {
    "caption": "(A) Differentially abundant taxa between nTC and TC households. Values left of the grey line indicate an enrichment in nTC household and values to the right indicate an enrichment in TC households. (A) Analyses are separated by mothers and infants for all samples across the 3 time points (FDR adjusted p-value < 0.01).",
    "molecules": "TC"
  },
  {
    "caption": "LDH release assay from indicated wildtype, GBP1 knockout (∆GBP1) or GBP1 reconstituted (∆GBP1+Tet-GBP1) cells infected with type I or type II Tg for 24 hours. Cells were untreated or treated with IFNγ or additionally treated with doxycycline (Dox) as indicated.",
    "molecules": "Dox, doxycycline, Tet"
  },
  {
    "caption": "Realtime propidium iodide (PI) uptake assay from indicated THP-1 cells infected with type I or type II Tg, and untreated or treated with IFNγ and/or Dox as labelled. AU, arbitrary units.",
    "molecules": "Dox, PI, propidium iodide"
  },
  {
    "caption": "LDH release assay from wildtype THP-1 or ∆GBP1 cells stably reconstituted with Dox-inducible expression plasmids of the indicated mutants of GBP1. Cells were pre-treated with IFNγ and Dox and infected with either type I or type II Tg for 24 h.",
    "molecules": "Dox"
  },
  {
    "caption": "Propidium iodide (PI) uptake in indicated wildtype THP-1 or ∆CASP-1, ∆CASP-4, ∆ASC cells, or THP-1 stably expressing GSDMD-targeting miRNA (GSDMDmiR) infected with the indicated strains of Toxoplasma gondii (Tg) for 24 h. Cells were untreated or treated with IFNγ as indicated. Mean area under the curve (AUC) ± SEM from n = 3 experiments are shown. ns, not significant, from two-way ANOVA following adjustment for multiple comparisons.",
    "molecules": "PI, Propidium iodide"
  },
  {
    "caption": "Immunoblots from primary MDMs infected with the indicated Tg strains for 12 h. Cells were untreated or pre-treated with IFNγ before infection. Treatment with TNFα and cycloheximide (CHX) served as positive control for apoptosis. Images are representative of n = 4 biologically independent experiments.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Representative images (top) from IFNγ-primed THP-1 WT infected with type I Tg for 8 hours and stained for ASC and active caspase-8. Blue, Nuclei; Red, ASC; Green, active caspase-8 (IETD-FITC) and Grey, Tg. Scale bar, 5 μm or 2 μm in the magnified images. Quantification of ASC specks (bottom) in Tg-infected THP-1 WT or ∆CASP1 treated with IFNγ or left untreated at 8 hours post infection, plotted as mean ± SEM from n = 3 independent experiment.",
    "molecules": "FITC"
  },
  {
    "caption": "Representative images (A) and quantification (B) from IFNγ-primed THP-1 ∆GBP1 stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or monocyte derived macrophages (MDM) stained for GBP1. Cells were infected with type I or type II Toxoplasma gondii (Tg) for 2 h. Blue, Nuclei; Grey, Tg; Red, mCH-GBP1 or Magenta, GBP1. Scale bar, 5 μm. Graph in (B) shows mean percentage of Tg vacuoles targeted by GBP1 from n = 4 experiments.",
    "molecules": "Tet"
  },
  {
    "caption": "Propidium iodide (PI) uptake assay from IFNγ-primed primary MDMs transfected with non-targeting control siRNA (CTRL) or siRNA against GBP1 (left) or indicated lines of THP-1 (right) infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.",
    "molecules": "PI, Propidium iodide"
  },
  {
    "caption": "PI uptake assay from IFNγ-primed primary MDMs transfected with indicated siRNA or non-targeting control (CTRL) and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 4 independent experiments.",
    "molecules": "PI"
  },
  {
    "caption": "PI uptake assay from IFNγ-primed or untreated THP-1 ΔCASP-1 cells transfected with indicated siRNA and infected with STm-GFP for 4 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows overview of the pathway leading to caspase-1 and caspase-4 activation.",
    "molecules": "PI"
  },
  {
    "caption": "LDH release assay from indicated THP-1 cells treated with IFNγ and Dox and infected with STm-GFP for 4 h. Shown is the mean ± SEM of n = 3 experiments.",
    "molecules": "Dox"
  },
  {
    "caption": "Representative images from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm.",
    "molecules": "Tet"
  },
  {
    "caption": "quantification (G) from IFNγ-primed THP-1 ∆GBP1 cells stably reconstituted with Tet-mCH-GBP1, mCH-GBP1K51A, mCH-GBP1C589A or mCH-GBP1∆589-592 variants or primary MDMs stained for GBP1. Cells were infected with STm-GFP for 2 h. Blue, Nuclei; Grey, STm; Red, mCH-GBP1 or Magenta GBP1. Scale bar, 5 μm. Graph in (G) shows percentage of STm vacuoles targeted by GBP1.",
    "molecules": "Tet"
  },
  {
    "caption": "Representative images from THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S. Cells were infected with STm for 2 h. Blue, Nuclei & STm (Hoechst dye); Red, mCH-GBP1; Green, YFP-CASP4C258S. Scale bar, 5 μm.",
    "molecules": "Hoechst, Tet"
  },
  {
    "caption": "Quantification of THP-1 ∆GBP1+Tet-mCH-GBP1 cells stably expressing YFP-CASP4C258S and infected with STm for 2 h. Graph shows percentage of STm vacuoles targeted by GBP1, CASP4 or both from n = 3 experiments. n.d. not detected.",
    "molecules": "Tet"
  },
  {
    "caption": "Propidium iodide (PI) uptake assay from MDMs transfected with non-targeting control (CTRL) or GBP1 siRNA and then left untreated (UT), treated with LPS or transfection reagent (Lipofectamine) only or transfected with LPS for 4 h. Area under the curve (AUC) from real-time assays plotted as mean ± SEM from n = 4 independent experiments. Schematic on right shows an overview of pathways leading to caspase-4 activation.",
    "molecules": "Lipofectamine, LPS, PI, Propidium iodide"
  },
  {
    "caption": "C LC-MS/MS analysis of m6A/A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO (KO-6B) mESCs.",
    "molecules": "m6A"
  },
  {
    "caption": "D PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1832 site and A1825 site (for input control) in 18S rRNA of WT and Mettl5 KO (KO-6B) mESCs, respectively.",
    "molecules": "m6A"
  },
  {
    "caption": "E LC-MS/MS analysis of d3-m6A/A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro. No enzyme group was used as a negative control.",
    "molecules": "m6A"
  },
  {
    "caption": "E Immunostaining analysis of the expression of pluripotency markers (POU5F1, SOX2 and NANOG) in WT and Mettl5 KO mESCs, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Histogram showing the threshold cycles of qPCR (qPCRCT) for detecting m6A1832 site in 18S rRNA during the indicated time points of EB induction. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *, P<0.05.",
    "molecules": "m6A"
  },
  {
    "caption": "C Immunostaining analysis of the expression of pluripotency marker POU5F1(left), endoderm marker FOXA2 (middle) and neuroectoderm marker NESTIN (right) in control and Mettl5 KO EBs at different time points, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 100 μm. D Quantification of the percentages of positive cells shown in (C). Quantitative analysis was based on at least three independent experiments. Data are represented as mean ± SD. Student's t test, two-tailed. **, P < 0.01; ***, P<0.001. ",
    "molecules": "DAPI"
  },
  {
    "caption": "D RT-qPCR analysis of the expression of pluripotency and lineage-specific markers in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "E Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "G RT-qPCR analysis the expression of pluripotency and lineage-specific markers in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "H Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.",
    "molecules": "Dox"
  },
  {
    "caption": "(C) Detection of decoy antibody ACE2-Fc and Spike S1-Fc chimeric homodimer formation in nonreducing Coomassie Brilliant Blue staining.",
    "molecules": "Coomassie Brilliant Blue"
  },
  {
    "caption": "(E, F) Deglycosylation of ACE2-Fc and Spike 1-674-Fc by PNGase F. PNGase F digested ACE2-Fc (500 ng) and Spike 1-674-Fc (500 ng) were subjected to Coomassie Brilliant Blue staining (E) and Western blot analysis by anti-human IgG Fc antibody (F).",
    "molecules": "Coomassie Brilliant Blue"
  },
  {
    "caption": "(A) Competitive ELISA. ACE2-Fc can compete with ACE2-Fc-Biotin for Spike 1-674 binding in a dose-dependent manner. The competition effects of ACE2-Fc on ACE2-Spike protein binding was normalized to PBS control. Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. **P < 0.01, ***P < 0.001.",
    "molecules": "Biotin"
  },
  {
    "caption": "(C) Confocal microscopy of H1975-Spike-overexpressing cells. Spikes on H1975 cells were stained with anti-Spike antibody and Alexa Fluor® 594-conjugated secondary antibody on ice for 1 hr. After that, ACE2-Fc-FITC was incubated for another 1 hr. Scale bars equal to 20 µm. DAPI was used as a nuclear counterstain.",
    "molecules": "Alexa Fluor® 594, DAPI"
  },
  {
    "caption": "(E) Inhibition of angiotensin II (Ang II)-induced TNF-α production by ACE2-Fc. Ang II was preincubated with indicated amounts of ACE2-Fc or IgG for 30 minutes. After that, the mixtures were added to RAW264.7 cells for 12 hr. The concentration of TNF-α in the culture medium was determined by ELISA. Error bars represent the standard deviation (SD), n=3. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. The dotted line represents the mean value of TNF-α in control group.",
    "molecules": "Ang II, angiotensin II"
  },
  {
    "caption": "(F) Inhibition of Ang II-induced ADAM17 (a disintegrin and metalloprotease 17) phosphorylation by ACE2-Fc. The protein extracts from (E) were immunoblotted with the indicated antibodies (left panel). β-actin served as the loading control. The signal intensity was normalized to cells only (right panel). Data are representative of three independent experiments, and the values are expressed as the mean ± SD (right panel). Error bars represent the standard deviation (SD), n=2. Statistical analysis was performed by unpaired two tail t-test. *P < 0.05. IgG represents the human normal IgG control.",
    "molecules": "Ang II"
  },
  {
    "caption": "(B) Confocal microscopy images of the airway organoids. The green fluorescence represents the specific staining of indicated monoclonal antibodies and Alexa Fluor 488-conjugated secondary antibodies. DAPI was used as a nuclear counterstain. Hematoxylin and eosin (H&E) stain: hematoxylin stained the nuclei in blue color; eosin stained the cytoplasm in pink color. Scale bars equal to 20 µm.",
    "molecules": "eosin, Hematoxylin, hematoxylin, Alexa Fluor 488, DAPI"
  },
  {
    "caption": "(A) Inhibition of SARS-CoV-2 infection by ACE2-Fc in the plaque assay. Mixtures of ACE2-Fc and SARS-CoV-2 were incubated for one hour before adding to Vero E6 cells for another 1 hr at 37°C. The ACE2-Fc and SARS-CoV-2 premixtures were removed, and the cells were washed once with PBS and overlaid with methylcellulose with 2% FBS for 5-7 days before being stained with crystal violet. Those results showed increase of plaque formation was regarded as no inhibition of plaque formation. TPCK-treated trypsin: N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin.",
    "molecules": "crystal violet, methylcellulose, N-tosyl-L-phenylalanine chloromethyl ketone, TPCK, trypsin"
  },
  {
    "caption": "Human placental syncytiotrophoblast cells express markers of cellular senescence, p16 Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (A) p16 Green label: the syncytiotrophoblast marker βHCG, red label: p16 Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells βHCG+/p16+ (A) Scale bar, 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Human placental syncytiotrophoblast cells express markers of cellular senescence, p21. Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (B) p21. Green label: the syncytiotrophoblast marker βHCG, red label: p21. Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells , βHCG+/p21+ (B). Scale bar, 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (A) Representative T1-weighted image of a pregnant WT mouse and a selection of four different placentas (outlined by colored lines). ",
    "molecules": "biotin, BSA-GdDTPA"
  },
  {
    "caption": "DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (B) Representative SI dynamics of a single WT placenta (marked in A), following biotin-BSA-GdDTPA administered for 60 min. SI parameters of Initial Enhancement and Recovery are illustrated.",
    "molecules": "biotin, BSA-GdDTPA"
  },
  {
    "caption": "DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (C) Representative SI dynamics of a single WT placenta (marked in A) over time. Scale bar, 2.5 mm.",
    "molecules": "biotin, BSA-GdDTPA"
  },
  {
    "caption": "DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (D−G) Representative SI plots of Cdkn1a-/- (D), p53-/- (E), Cdkn2a-/- (F), and Cdkn2a-/-;p53-/- (G) placentas, compared to their WT or HET littermates. (H−I) Quantification of the SI Initial Enhancement (H) and SI Recovery (I) in WT, Cdkn1a-/-, p53-/-, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas. MRI experiments were repeated at least three times for each murine genotype (n>=3 placental SI measurements from each genotype). Values are means +SEM; Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.",
    "molecules": "biotin, BSA-GdDTPA"
  },
  {
    "caption": "Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. On days 1 and 5 after seeding, cell fusion was monitored by staining for phalloidin (F-actin) (green) and DAPI (blue). Scale bar, 50 μm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(A,B) HEK 293T cells transfected with denoted plasmids were (A) deprived of nutrients for 90 min, and then replenished with DMEM for 30 min or (B) stimulated with 50 ng/ml epidermal growth factor (EGF) for 30 min. The S6K immune complexes were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel).",
    "molecules": "nutrients"
  },
  {
    "caption": "(C) HEK 293T cells transfected with control siRNA, ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min, and then the phosphorylation and protein levels of endogenous S6K (top two panels) and S6 (bottom two panels) were determined.",
    "molecules": "nutrients"
  },
  {
    "caption": "(D) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min, and then replenished with DMEM for 30 min. The phosphorylation and protein levels of endogenous S6K were examined. For quantification, the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop, and normalized to the S6K protein levels. Results are expressed as a fold change compared with the first indicated sample.",
    "molecules": "nutrients"
  },
  {
    "caption": "c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.",
    "molecules": "carbon, galactose, glucose"
  },
  {
    "caption": "e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.",
    "molecules": "carbon, galactose, glucose"
  },
  {
    "caption": "h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.",
    "molecules": "geranylgeranyl alcohol, palmitic acid, myristic acid, protein"
  },
  {
    "caption": "C. Identification of autophagic vesicles in extracellular tachyzoites by immuno-electron microscopy. Extracellular tachyzoites expressing GFP-TgAtg8 were starved for 8 hours in HBSS medium, fixed, cryosectioned and stained with an anti-GFP antibody, then with gold-conjugated secondary antibody and observed. Gold particles can be seen around cytosol-sequestrating membranous structures resembling autophagosomes (Ap), as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). Scale bar = 200 nm.",
    "molecules": "gold, Gold particles"
  },
  {
    "caption": "A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control.",
    "molecules": "PE"
  },
  {
    "caption": "B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFP",
    "molecules": "urea"
  },
  {
    "caption": "C. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction.",
    "molecules": "DOC, urea"
  },
  {
    "caption": "D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).",
    "molecules": "ethanolamine, PE, 3H, urea"
  },
  {
    "caption": "A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM.",
    "molecules": "glycine"
  },
  {
    "caption": "C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.",
    "molecules": "glycine, PE, urea"
  },
  {
    "caption": "C. Southern blot verification of the TgAtg3 gene replacement. ∼4 µg of genomic DNA from mutant (A) and parental (B) tachyzoites were digested by HindIII/AvrII, transferred on a nylon membrane and probed with 32P-labeled 5′ and 3′ DNA probes located as schematised in A.",
    "molecules": "DNA, genomic DNA, 32P"
  },
  {
    "caption": "A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast.",
    "molecules": "ATc"
  },
  {
    "caption": "C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control.",
    "molecules": "PE, urea"
  },
  {
    "caption": "A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p<0.005, Student's T test). Data are mean from n = 3 independent experiments ±SEM.",
    "molecules": "ATc"
  },
  {
    "caption": "C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM.",
    "molecules": "ATc"
  },
  {
    "caption": "TgAtg3-depleted parasites show a defect in mitochondrion morphology.Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI.",
    "molecules": "ATc"
  },
  {
    "caption": "Altered mitochondrial ultrastructure in TgAtg3-depleted tachyzoites observed by electron microscopy.Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal.",
    "molecules": "ATc"
  },
  {
    "caption": "(A) Cellomics images (top) of GFP-LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP-LC3 spots (red spots) in FM and EL. Enlarged GFP-LC3 images in FM and EL (bottom right). Scale bars=10 μm.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(C-E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM.",
    "molecules": "leupeptin, LiCl, Resveratrol, Torin1"
  },
  {
    "caption": "(C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP-LC3 (green, bottom) in HEK293 and GFP-LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm.",
    "molecules": "amino‐acid"
  },
  {
    "caption": "(B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively.",
    "molecules": "BafilomycinA1"
  },
  {
    "caption": "(B) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation. Anti‐Myc, ‐Actin and ‐LC3 blot after transfection with pcDNA or Myc-FEZ1 in HEK293 cells. Quantification of LC3II/actin; error bars represent s.e.m. (n=3); pcDNA ES versus Myc-FEZ1 ES, P=0.0318. Anti‐GFP, ‐Actin and ‐p62 blot after transfection with GFP or FEZ1-GFP and treatment with ES or ES plus Bafilomycin (EB) for 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); GFP FM versus GFP ES, P=0.0077; GFP FM versus GFP EB, P=0.0212; GFP ES versus FEZ1-GFP ES, P=0.048.",
    "molecules": "Bafilomycin"
  },
  {
    "caption": "(D) FEZ1 interacts with SCOC in vitro. GST‐pulldown assays using in vitro translated 35S‐labelled Myc-FEZ1 (right) or 35S‐labelled Myc‐control (left) and GST or GST-SCOC (asterisk in bottom panel). Bound proteins were detected by autoradiography following SDS-PAGE (top), protein loading by coomassie blue (bottom).",
    "molecules": "35S"
  },
  {
    "caption": "(E) The FEZ1-SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled‐coil domain of FEZ1 as demonstrated in GST‐pulldown assays with in vitro translated 35S‐labelled WT or mutant Myc‐FEZ1 and GST or GST‐SCOC. Autoradiographs of 10% input and bound proteins (top panels) and Coomassie blue‐stained gels of bead‐bound GST and GST‐SCOC proteins used (bottom panels).",
    "molecules": "35S"
  },
  {
    "caption": "(A) FEZ1 interacts with both the kinase domain (1-278) and the proline-serine rich spacer of ULK1 (279-828). GFP‐ or Myc‐tagged ULK1 fragments were in vitro translated with 35S‐methionine and subjected to GST‐pulldown assays using GST and GST-FEZ1 purified from E. coli (bottom). Bound proteins were detected by autoradiography following SDS-PAGE (top).",
    "molecules": "35S‐methionine"
  },
  {
    "caption": "(C) The FEZ1-GFP and Myc-ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1-GFP and Myc-ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody.",
    "molecules": "amino‐acid"
  },
  {
    "caption": "(G) SCOC can reduce the interaction of ULK1 with MBP-FEZ1. Immobilised MBP-FEZ1 was incubated with in vitro 35S‐methionine‐labelled Myc-ULK1. Addition of increasing amounts of in vitro translated 35S‐methionine SCOC reduced the amount of Myc-ULK1 bound. Top and bottom panels are as described in (A).",
    "molecules": "35S‐methionine"
  },
  {
    "caption": "(E) UVRAG can interact with both FEZ1 and SCOC in vitro. GST, GST-FEZ1 or GST-SCOC were immobilised and incubated with 35S‐labelled Myc-UVRAG as indicated. Arrowheads indicate GST-FEZ1 (left) and GST-SCOC (right).",
    "molecules": "35S‐"
  },
  {
    "caption": "(D) WAC depletion accelerates degradation of a UPS reporter. Anti‐p62, ‐GFP, ‐Actin, ‐LC3, ‐WAC and ‐Atg16 blots after siRNA treatment in UbG76V-YFP MelJuSo cells and incubation conditions FM, ES or FM with 10 μM MG132 (MG) for 2 h. Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC‐03 siRNA treatment (n=3) and compared with ATG16 siRNA treatment (n=2). Error bars represent s.e.m.: RF FM versus siWAC‐03 FM, **P=0.0082.",
    "molecules": "MG132"
  },
  {
    "caption": "(E) Soluble HttQ25-CFP degradation is increased after WAC depletion. Anti‐WAC, ‐Actin and ‐GFP blots after indicated siRNA treatment (siWAC duplexes ‐02, ‐03, ‐04) of HttPolyQ‐mutant HeLa cell lines and incubation conditions DMSO or DOX (Doxycyline) for 3 days. The insoluble fraction was obtained by pelleting the HttQ103 lysates (see Materials and methods). Quantification of normalised average HttQ25-CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment; error bar represents s.e.m. (n=3): HttQ25-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0350; soluble HttQ103-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0380.",
    "molecules": "Doxycyline, DMSO"
  },
  {
    "caption": "(D) Caspase-8-like (IETDase) activity assay in the unbound soluble fraction from the DISC IP An untreated control (0) was used for each cell line to show basal caspase-8 activity.",
    "molecules": "IETD"
  },
  {
    "caption": "(D) Caspase-8 (IETDase) and -3/7 (DEVDase) activity assay in the unbound soluble fraction",
    "molecules": "DEVD, IETD"
  },
  {
    "caption": "(E) Western blot analysis of FLIP, Caspase 8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or flag tagged FLIP L (FL) treated with 2.5µM MS-275 for 48 hours, followed by a 90 minute DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L).",
    "molecules": "MS-275"
  },
  {
    "caption": "(G) Caspase-8 (IETDase) activity assays of the unbound soluble fraction",
    "molecules": "IETD"
  },
  {
    "caption": "(F) NanoBiT® assay of U20S cells transiently co-transfected with caspase 8 (LgBiT) and either WT FADD smBiT or FADD (smBiT) constructs with a point mutation at either phenylalanine 25 (F25A) or histidine 9 (H9G) for 48 hours. Western blot analysis was used to assess the expression level of each construct.",
    "molecules": "histidine, phenylalanine"
  },
  {
    "caption": "(C) Caspase-8 (i) and -3/7 (ii) activity assays and high content microscopy assessment of cell death (iii) in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 6 hours.",
    "molecules": "isoleucine"
  },
  {
    "caption": "(D) Caspase-8 (i) and -3/7 (ii) activity assays in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 24 hours. (iii) Flow cytometry assessment of cell death induction in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with IZ-TRAIL for 24h.",
    "molecules": "isoleucine"
  },
  {
    "caption": "B. In vitro exoribonuclease assay using 5′ radiolabeled RNA substrate and immunopurified wild-type (WT) or catalytic mutant (CM) FLAG-dmDis3l2 (mutation indicated in Figure 1C), incubated for the indicated time and separated on a 15 % polyacrylamide gel followed by phosphorimaging. Quantification of degraded substrate in percent is indicated.C. Change in abundance of 256 different substrate RNAs as determined by high-throughput sequencing of substrates in experiment shown in (B).",
    "molecules": "RNA"
  },
  {
    "caption": "D. Decay rates of 256 different substrate RNAs. Data shown in (C) was normalized to overall decrease in substrate abundance as determined by phosphorimaging (as shown in B) and fit to the indicated model for exponential decay. Apparent decay rate of all substrates, as determined by phosphorimaging is indicated in red.E. Selected examples for individual substrates shown in (D). Values report the observed decay rate (kobs) in min-1.F. Overview of decay rates (kobs) of all 256 different substrate RNAs, as determined in (D). Error represents SEM of curve fit.",
    "molecules": "RNAs"
  },
  {
    "caption": "G. Impact of RNA secondary structures, as determined by RNAfold (Gruber et al, 2008), and reported as effective free energy (EFE, kcal/mol), on decay rates (kobs) are shown. Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value was determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured substrates used for subsequent analysis are indicated (EFE groups 4 to 6).",
    "molecules": "RNA"
  },
  {
    "caption": "H. Nucleotide content of single-stranded RNA substrates, ranked from slow (top) to fast (bottom) decay by dmDis3l2.I. Cumulative distribution of decay rates for RNA substrates with the indicated nucleotide at each one of four randomized position at the 3′ end of the substrate RNA. P-Value was determined by Mann-Whitney test comparing individual nts against all.",
    "molecules": "Nucleotide, nucleotide, RNA"
  },
  {
    "caption": "J. Functional coupling between two base positions within the randomized 3′ end of substrate RNA. Red squares show average increase in decay rate compared to all substrates. Black squares show no or negative effect.",
    "molecules": "RNA"
  },
  {
    "caption": "K. Boxplot decay rates of substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median decay rates SEM of fit are indicated.L. Decay rate of all substrates, or substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median and quartile ranges are indicated. P-values were determined by Mann-Whitney test. Substrate containing 4 uridines (4U) is indicated.",
    "molecules": "RNA"
  },
  {
    "caption": "B, C, F and G. Lysate of S2 cells (as characterized in A and E) were incubated with -32P-UTP (B and F) or -32P-ATP (C and G) for the indicated time followed by RNA extraction, denaturing polyacrylamide gel electrophoresis and phosphorimaging.",
    "molecules": "ATP, RNA, UTP"
  },
  {
    "caption": "B, C, F and G. Lysate of S2 cells (as characterized in A and E) were incubated with -32P-UTP (B and F) or -32P-ATP (C and G) for the indicated time followed by RNA extraction, denaturing polyacrylamide gel electrophoresis and phosphorimaging.",
    "molecules": "ATP, RNA, UTP"
  },
  {
    "caption": "A. Protein/RNA co-immunopurification experiments. FLAG-tagged versions of indicated proteins were expressed in S2 cells, followed by immunopurification and RNA isolation. RNA was visualized by CIP/PNK treatment using -32P-ATP followed by denaturing polyacrylamide gel electrophoresis and phosphorimaging. Star indicates non-specific contaminant.",
    "molecules": "ATP, RNA"
  },
  {
    "caption": "B. Meta-analysis of 3′ end counts in dmDis3l2CM-IP mapping to the indicated region around the annotated 3′ end of 68 tRNA loci in the Drosophila melanogaster genome. Cumulative relative distribution of 3′ end signal, relative to the annotated 3′ end, is reported (bottom). The average genome thymine (T)-content for the same regions is displayed (top).",
    "molecules": "tRNA"
  },
  {
    "caption": "C. Northern hybridization experiment using total RNA from adult whole male flies using probes against 3′ trailer or mature tRNAAla(TGC). Wild-type flies (w1118), or flies bearing a homozygous frame-shift mutation in the first coding exon (dmDis3l2-/-) or a homozygous amino acid-exchange mutation in the catalytic site (dmDis3l2CM) in the endogenous dmdis3l2 locus in the same genetic background were used. Probes against 2S rRNA served as loading control.D. Quantification of four independent biological replicates of experiment shown in C. P-Value determined by Student's t-test.",
    "molecules": "RNA, tRNAAla(TGC)"
  },
  {
    "caption": "E. Exoribonuclease assay using immunopurified dmDis3l2 and in vitro transcribed, 5′ radiolabeled mature tRNA, containing a CCA modification or 3′ trailer-containing tRNA. Reactions were incubated for the indicated time and subjected to polyacrylamide gel electrophoresis and phosphorimaging.",
    "molecules": "tRNA"
  },
  {
    "caption": "A. Impact of RNA secondary structures on RNA decay rates (kobs) of 256 different RNA substrates subjected to E.coli RNase R (ecoRNaseR)- or Drosophila melanogaster Dis3l2 (dmDis3l2)-directed RNA decay assay as described in Fig. 2. RNA secondary structures were determined by RNAfold (Gruber et al, 2008) and reported as effective free energy (EFE). Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured RNA used for subsequent analysis are indicated (EFE groups 4 to 6).",
    "molecules": "RNA"
  },
  {
    "caption": "B. Nucleotide content of randomized 3′ end sequence among the indicated number of substrate RNAs, ranked according to decay kinetics from slow (left) to fast (right). Fold-change in abundance for individual nucleotides is reported (bottom). Only unstructured RNA substrates (see A) were considered.",
    "molecules": "Nucleotide, RNA, RNAs"
  },
  {
    "caption": "C. Decay rate of the indicated number of sequences, grouped according to individual nucleotide content in the randomized 3′ end sequence. Data is represented as boxplot, outliers are not shown. P-Value reports significant differences compared to all substrates as determined by Mann-Whitney test. Grey area represents the inner quartile range and white line the median of all substrates.",
    "molecules": "nucleotide"
  },
  {
    "caption": "A549 AT2  cells were infected with Delta SARS-CoV-2 variant. Proteasome  inhibitor Bortezomib  (BZ, 1μM) was added to cells 18h post-infection. Cells were lysed 24h post-addition of inhibitor and cyclins and viral proteins were detected by western blot. (N) nucleocapsid. Densitometry  analysis of western blots  for D-cyclins (normalized to actin) in A549 AT2  cells. Plots are average of 2 (cyclin D1), 3 (cyclin D3) biological replicates. Bars indicate mean with SD. Statistical analysis was performed using ordinary two way ANOVA; ns, non-significant; ∗p < 0.1.",
    "molecules": "Bortezomib, BZ"
  },
  {
    "caption": "A459 AT2  cells were infected with Delta SARS-CoV-2 variant. Proteasome  inhibitor Bortezomib  (BZ, 1μM) was added to cells 8h post-infection. Cells were fixed and stained  24h post-addition of inhibitor. (N) nucleocapsid.Scale bars: 40μm. Contour lines represent outlines of infected cells.",
    "molecules": "Bortezomib, BZ"
  },
  {
    "caption": "(A) 293T  cells were contransfected with HA  - cyclin D3 and Strep-tag-SARS-CoV-2 E or nsp9, and control plasmid (EV). Immunoprecipitation  was performed using anti-HA  antibody. The immunoprecipitates  were blotted  with anti-Strep, anti-HA, and cyclin D3 antibodies.",
    "molecules": "Strep"
  },
  {
    "caption": "(B) 293T  cells were contransfected with HA  - cyclin D3 and Strep-tag-SARS-CoV-2 E, M, both E and M or nsp9. Immunoprecipitation  was performed using anti-HA  antibody. The immunoprecipitates  were blotted  with anti-Strep, anti-HA  antibodies. WCL, whole cell lysate.",
    "molecules": "Strep"
  },
  {
    "caption": " A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm. B) Lamplight-driven response to increasing intensity 1s 405nm light C) 405nm irradiance response curve D) Response to 1s 405nm (blue line) and 525nm light (green line) E) Response to 405nm (blue line) followed by 525nm light of different intensities (green line) ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Immunoblotting of NSC34 cells expressing AR-24Q or AR-97Q (NSC24Q and NSC97Q, respectively) that were treated with DHT for Dnmt1, Dnmt3a and Dnmt3b, and quantification of the signal intensities of bands corresponding to Dnmt1, Dnmt3a and Dnmt3b (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(C) Protein levels of Dnmt1, Dnmt3a and Dnmt3b in DHT-treated and -untreated NSC97Q cells by Western blotting analysis.",
    "molecules": "DHT"
  },
  {
    "caption": "(D) RT-qPCR analysis of Dnmt1 in DHT-treated and -untreated NSC97Q cells (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(G) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST-8 assay (n = 3 per group).",
    "molecules": "DHT, RG108"
  },
  {
    "caption": "(H) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1, Dnmt3a and Dnmt3b.",
    "molecules": "DHT, RG108"
  },
  {
    "caption": "(I, J) Cell viability analysis with siRNA-mediated knockdown of Dnmts in DHT-untreated NSC97Q (I) and DHT-treated NSC24Q (J).",
    "molecules": "DHT"
  },
  {
    "caption": "(A-D) Grip power (A), body weight (B), rotarod performance (C) (Two-way ANOVA with Tukey's test) and survival rate (D) (Kaplan-Meier analysis and log-rank test) of AR-97Q mice treated with or without RG108 (DMSO, n =20; 0.5 mg/dL, n = 20; 2.0 mg/dL, n = 20). All parameters were significantly improved in AR-97Q mice treated with 2.0 mg/dL RG108 compared with those treated with DMSO: *P < 0.05 (grip); *P < 0.05 (body weight); *P < 0.05 (rotarod); and *P < 0.05 (survival).",
    "molecules": "DMSO, RG108"
  },
  {
    "caption": "(E) Representative photograph of a 13-week-old AR-97Q mouse treated with DMSO (left) and an age-matched AR-97Q mouse treated with 2.0 mg/dL RG108 (right). AR-97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO-treated AR-97Q mouse of same age. The white compact masses (arrow) of mice head are the cement covering the surgical site.",
    "molecules": "DMSO, RG108"
  },
  {
    "caption": "(F,G) Immunoblots (F) and relative intensities (G) of the Dnmt1-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3 per group).",
    "molecules": "RG108"
  },
  {
    "caption": "(H,I) Dnmt1 immunohistochemistry in spinal cord sections from AR-97Q mice treated with or without RG108 (n = 3 per group).",
    "molecules": "RG108"
  },
  {
    "caption": "(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody. (B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group).",
    "molecules": "DMSO, polyglutamine, RG108"
  },
  {
    "caption": "(C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR. (D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3).",
    "molecules": "DMSO, RG108"
  },
  {
    "caption": "(E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3).",
    "molecules": "RG108"
  },
  {
    "caption": "(H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).",
    "molecules": "RG108"
  },
  {
    "caption": "(C, D) Immunoblots for Dnmts of SH97Q with or without DHT treatment (C) and quantification data (D) (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(F) The relative mRNA levels of CDC25B, GFRA3, NPY, HES5, SCTR, LEF1-AS1 and CABS1, normalized to beta-2 microglobulin, in DHT-treated SH24Q and SH97Q cells were measured using RT-qPCR (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(G) RT-qPCR analysis indicated the decrease of Hes5 mRNA level in DHT-treated SH97Q cells compared with DHT-untreated SH97Q cells (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(H) Methylation-specific PCR of SH24Q and SH97Q cells that were treated with DHT.",
    "molecules": "DHT"
  },
  {
    "caption": "DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108.",
    "molecules": "DHT, RG108"
  },
  {
    "caption": "HES5 mRNA levels of SH97Q cells treated with DHT and RG108.",
    "molecules": "DHT, RG108"
  },
  {
    "caption": "DNA methylation levels of the Hes5 promoter region in DHT-treated NSC97Q cells treated with or without RG108.",
    "molecules": "DHT, RG108"
  },
  {
    "caption": "mRNA levels of Hes5 (n = 3) in DHT-treated NSC97Q cells treated with or without RG108.",
    "molecules": "DHT, RG108"
  },
  {
    "caption": "(E) Hes5 mRNA levels in the spinal cords of AR-97Q mice treated with or without RG108 (n = 3).",
    "molecules": "RG108"
  },
  {
    "caption": "(F, G) Hes5 immunoreactivity of spinal motor neurons in AR-97Q mice treated with or without RG108 (n = 3).",
    "molecules": "RG108"
  },
  {
    "caption": "(A) Viability of DHT-treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST-8 assay (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(B) Immunoblots of DHT-administrated NSC97Q cells treated with Hes5 siRNA (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(C) Quantitative cell viability analysis revealed that siRNA-mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT (n = 3), as determined by the WST-8 assay.",
    "molecules": "DHT, RG108"
  },
  {
    "caption": "Cell viability measured with the WST-8 assay of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "Cell viability measured with immunoblotting of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(F) Quantification of LDH release from DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 4).",
    "molecules": "DHT"
  },
  {
    "caption": "(G) Immunoblots of DHT-administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR. (H) AR aggregation as quantified by densitometry (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(D) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment (n = 3).",
    "molecules": "DHT"
  },
  {
    "caption": "(A) RAW264.7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(B) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(C) Macrophages were pre-treated with either 1 μM GSK inh or 0.1 μM MLi-2 and then treated with 1 mM LLOMe for 30 min. Rab8A and Rab8A pT72 levels were analysed by Western blot.",
    "molecules": "LLOMe, MLi-2"
  },
  {
    "caption": "(D-F) RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (D) Endogenous LRRK2 and (E) Rab8A recruitment to LAMP-1 positive compartments was visualised by immunofluorescence. Scale bar = 10 μm. (F) Quantification of D-E. Data represents the mean ± SEM of three independent biological experiments.",
    "molecules": "LLOMe"
  },
  {
    "caption": "LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of LRRK2 per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.",
    "molecules": "LLOMe, GSK2578215A"
  },
  {
    "caption": "LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.",
    "molecules": "LLOMe, GSK2578215A"
  },
  {
    "caption": "(A) RAW264.7 macrophages were electroporated with EGFP-Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin-3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(B) Macrophages were electroporated with EGFP-Rab8A and RFP-Galectin-3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(C) RAW264.7 macrophages were treated with 1 mM of LLOMe and LRRK2, Rab8A, CHMP4B, Galectin-3 and LC3B foci over time was analysed by high-content imaging. Data shows the mean ± SEM of five biological replicates.",
    "molecules": "LLOMe"
  },
  {
    "caption": "RAW264.7 macrophages were pre-treated with 10 μM BAPTA-AM for 1 hr, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm.",
    "molecules": "LLOMe, BAPTA-AM"
  },
  {
    "caption": "RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.",
    "molecules": "LLOMe, GSK2578215A"
  },
  {
    "caption": "RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. Galectin-3 vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of Galectin-3 positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.",
    "molecules": "LLOMe, GSK2578215A"
  },
  {
    "caption": "Live cell imaging of LysoTracker positive spots in WT, LRRK2 KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.",
    "molecules": "LLOMe, LysoTracker"
  },
  {
    "caption": "Live cell imaging of LysoTracker positive spots in WT, Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.",
    "molecules": "LLOMe, LysoTracker"
  },
  {
    "caption": "WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.",
    "molecules": "LLOMe, GSK2578215A"
  },
  {
    "caption": "WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.",
    "molecules": "LLOMe, GSK2578215A, ubiquitin"
  },
  {
    "caption": "(C) Representative images of LC3B/LAMP-1 double positive endolysosomes in control and LLOMe treated macrophages. Scale bar = 10 μm. (D) Analysis of the % of LC3B/LAMP-1 double positive endolysosomes in LLOMe treated macrophages. Data shows mean ± SEM of three independent biological experiments. *p≤0.05, ***p≤0.001 by Student's t-test. ",
    "molecules": "LLOMe"
  },
  {
    "caption": "Monocyte-derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2-3) or R1441C (Donor 1) LRRK2 mutation were pre-treated with 0.1 μM MLi-2 and treated with 1 mM LLOMe for 30 min. (A) Rab8A and Rab8A pT72 levels were analysed by Western blot. ",
    "molecules": "LLOMe, MLi-2"
  },
  {
    "caption": "A) Fluorescence analysis of RPE-1 cells expressing endogenously tagged γ-tubulin-EGFP. Cells were synchronized in G2, using RO-3306, to allow for a clear distinction of the two centrosomes present at this cell cycle stage. Panels illustrate sum projections of the stacks from an original picture (left), a whole cell mask (middle) and a centrosome mask (right); both masks are based on intensity thresholding. Bar: 10 μm.",
    "molecules": "RO-3306"
  },
  {
    "caption": "E Golden's trichrome staining illuminating howship's lacunae (arrow) of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice were performed along with double-calcein bone labeling and confocal imaging to assess bone formation. Scale bar, 10 μm. F Quantification of MAR and BFR as assessed in 3-month-old male wild-type and Zeb1ΔM/ΔM mice (n = 6). D",
    "molecules": "Golden's trichrome, calcein"
  },
  {
    "caption": "H, I After a 6-day culture atop bone slice, phalloidin staining (red) was performed in wild-type versus Zeb1ΔM/ΔM osteoclasts (I), and actin ring area per cell quantified (H). Scale bar, 20 μm.",
    "molecules": "phalloidin"
  },
  {
    "caption": "A Phosphocreatine (PCr) and creatine (Cr) level, as well as PCr/Cr ratio of wild-type or Zeb1ΔM/ΔM osteoclasts cultured atop plastic substrata as determined by LC/MS analysis (n = 3).",
    "molecules": "Cr, creatine, PCr, Phosphocreatine"
  },
  {
    "caption": "E PCr/Cr ratio of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts cultured on plastic substrata as determined by LC/MS analysis (n = 3).",
    "molecules": "Cr, PCr"
  },
  {
    "caption": "J, K Mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type pre-osteoclasts were cultured atop bone slices for 3 days, stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (J). The actin ring area per cell and resorption pit area were quantified (K; n = 6). Scale bar, upper 20 μm, lower 100 μm.",
    "molecules": "DAB, phalloidin"
  },
  {
    "caption": "G, H siCTRL- or siZEB1-transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (G). Scale bar, upper 20 μm, lower 100 μm. The actin ring area per cell, resorption pit area, and the number of TRAP+ MNCs were quantified (H; n = 6).",
    "molecules": "DAB, phalloidin"
  },
  {
    "caption": "I-L Calvaria isolated from wild-type and Zeb1ΔM/ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine, and stained for phalloidin (red) and Vpp3 (green) (I). Scale bar, 10 μm. The TRAP activity of whole cell lysates, actin ring area per cell, and supernatant CTX-I were quantified (J-L; n = 6).",
    "molecules": "cyclocreatine, phalloidin"
  },
  {
    "caption": "(A-D) Isolated epidermal LCs  (A, n = 2), isolated vaginal LCs  (B-C, both n = 3), or vaginal  explants (D, n = 5) were stimulated O/N with  P. timonensis (PT) and exposed to  HIV-1 (SF162; MOI 0.5) for five days in presence or absence of HIV-1 replication inhibitors (Inh) zidovudine  (AZT), tenofovir  (TFV), iamivudine  (3TC) or indinavir  (IDV). HIV-1 levels were determined by intracellular staining  for HIV-1 p24  using  flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.",
    "molecules": "IDV, indinavir, 3TC, iamivudine, tenofovir, TFV, AZT, zidovudine"
  },
  {
    "caption": "(G) Isolated epidermal LCs  (n = 3) were treated with T20, Maraviroc  (MAR), neutralizing antibodies against CD4  (αCD4) and isotype control (Iso), followed by O/N stimulation with P. timonensis (PT). Next, LCs  were exposed to HIV-1 for five days (SF162; MOI 0.5). Intracellular  HIV-1 p24  levels were determined using flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.",
    "molecules": "T20, MAR, Maraviroc"
  },
  {
    "caption": "Ex vivo skin  explants were stimulated O/N with L. crispatus  (LC), M. elsdenii (ME), P. timonensis (PT) or poly(I:C) and at day 3 post-inoculation, emigrated LCs  were collected, washed and activation phenotype was determined by flow cytometry. Cells were analysed for CD1a expression  and the absolute number of CD1a-positive cells, i.e. LCs, migrated  from the epidermis  was determined using counting beads, the graph shows the migration  relative to the untreated condition (F, n = 5). CD1a-positive cells, i.e. LCs, were analysed using flow cytometry and the graphs show combined experiments (MFI expression  relative to untreated condition) of CD80  (G, n = 5) and CD86  (H n = 5) surface expression. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "Isolated vaginal LCs  were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure. (A) Intracellular HIV-1 detection was determined by  confocal microscopy (Hoechst  in blue, HIV-1 p24  in green), scale bar represents 5 µm (representative donor).",
    "molecules": "Hoechst"
  },
  {
    "caption": "(B) Isolated epidermal LCs  were stimulated O/N with  P. timonensis (PT) followed by a 4-hour exposure to HIV-1. HIV-1 p24  was determined by ELISA  after trypsin  treatment and lysis of LCs  (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.",
    "molecules": "trypsin"
  },
  {
    "caption": "(C) Isolated epidermal LCs  were stimulated O/N with  P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure for 3 days. After 3 days, LCs  were treated with PBS  or trypsin  and HIV-1 p24  was determined by flow cytometry (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.",
    "molecules": "PBS, trypsin"
  },
  {
    "caption": "(F) Isolated epidermal LCs  were stimulated O/N with P. timonensis  (PT) followed by  HIV-1  exposure (SF162; MOI 0.5) in presence or absence of anti-langerin  (10E2) or mannan  (MAN). HIV-1 p24  content was determined by ELISA  after trypsin-treatment and subsequent lysis of LCs (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.",
    "molecules": "MAN, mannan, trypsin"
  },
  {
    "caption": "(G) Isolated epidermal LCs  were stimulated O/N with P. timonensis (PT) followed by treatment with Rapamycin  (µM) and exposure to HIV-1 (SF162). After 3 days  HIV-1 p24  was measured by ELISA  after trypsin  treatment and lysis of LCs  (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.",
    "molecules": "Rapamycin, trypsin"
  },
  {
    "caption": "C) Ex vivo skin  explants were stimulated O/N with  L. crispatus (LC), M. elsdenii  (ME), or P. timonensis  (PT) and inoculated with HIV-1 (JRCSF; MOI 0.5) in presence or absence of tenofovir. At day 2 post-inoculation, emigrated LCs  were collected, washed and co-cultured with  U87.CD4.CCR5  cells for 3 days. pooled data of  HIV-1   positive cells (%) of the CD1a  negative  U87.CD4.CCR5  cells (C, n = 4). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.",
    "molecules": "tenofovir"
  },
  {
    "caption": "(D) Ex vivo skin  explants were stimulated with P. timonensis (PT), pre-treated with T20  or maraviroc  (MAR) and exposed to HIV-1 (JRCSF; MOI 0.5) for 2 days, after which the migratory fraction was co-cultured with U87.CD4.CCR5  cells. HIV-1 positive cells (%) of the CD1a-negative fraction, i.e. U87.CD4.CCR5 cells, were determined using flow cytometry (n = 5). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.",
    "molecules": "T20, MAR, maraviroc"
  },
  {
    "caption": "(A, B) Isolated epidermal LCs  were stimulated by different Prevotella spp. and Bacteriodus spp. followed by T/F variant (CH058; MOI 0.25) exposure in presence or absence of AZT  (A, n = 3) or tenofovir  (B, n = 2). A lower MOI was used due to higher susceptibility of LCs  for CH058  than for laboratory strains. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05.",
    "molecules": "tenofovir, AZT"
  },
  {
    "caption": "(C)  HIV-1  uptake was determined by flow cytometry. Ex vivo skin  explants (n = 3) were stimulated O/N with P. timonensis (PT) followed by pre-treatment with tenofovir  (TFV). After pre-treatment, skin  explants were inoculated with lab-adapted strain (SF162; MOI 0.25) or  HIV-1 T/F strain (CHO58; MOI 0.25). After 2 days the migratory fraction was collected and co-cultured with U87.CD4.CCR5 cells. Cells were analysed for CD1a expression  and p24  content in U87.CD4.CCR5  cells by  flow cytometry. A lower MOI was used due to higher susceptibility of LCs  for CH058  than for laboratory strain SF162. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05.",
    "molecules": "tenofovir, TFV"
  },
  {
    "caption": "B Proliferation of adoptively transferred OT-I T cells in C57BL/6mice (n = 3/group) immunized with vehicle (PBS), NLDC:pOVA, NLDC:pOVA plus CpG, fd, fd/sc-αDEC or LPS-free ovalbuminprotein. Percentage of CFSE-labeled proliferating OT-I T cells was evaluated after three days on Vα2+CD8+-gated cells. The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.",
    "molecules": "CpG, LPS"
  },
  {
    "caption": "D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6 mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.",
    "molecules": "LPS"
  },
  {
    "caption": "IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/−mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/−mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.",
    "molecules": "CpG, ODN, LPS"
  },
  {
    "caption": "IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.",
    "molecules": "CpG, ODN, LPS"
  },
  {
    "caption": "IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.",
    "molecules": "LPS"
  },
  {
    "caption": " [A] The same MVs from GB03 caused opposed impacts on plants grown in different medium. The petri dishes contain plastic partitions (red dotted lines) that separate different medium. 0.5 MS and 0.05 MS indicate 1/2-strength (nutrient-sufficient) and 1/20th-strength (nutrient-deficient) Murashige & Skoog medium. Images were taken at 11 days after treatments (DAT).",
    "molecules": "MVs"
  },
  {
    "caption": " [D] Cell death visualization by trypan blue staining of 11 DAT leaves. Bar= 1mm ",
    "molecules": "trypan blue"
  },
  {
    "caption": " [B] Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots ",
    "molecules": "5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, BCIP"
  },
  {
    "caption": "Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.",
    "molecules": "anthocyanin, Pi"
  },
  {
    "caption": "Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-induction of IPS1 gene expression (D) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.",
    "molecules": "Pi"
  },
  {
    "caption": " [E] Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant. [F] A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.",
    "molecules": "Pi"
  },
  {
    "caption": " [A] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22. ",
    "molecules": "acetoin, ATN, agar, 2,3-butanediol, BTDL, DA, diacetyl, 2-methyl-1-propanol, MP"
  },
  {
    "caption": "anthocyanin hyper accumulation (B) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "anthocyanin, DA, Pi"
  },
  {
    "caption": "PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "DA, Pi"
  },
  {
    "caption": "Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) triggered by DA. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "anthocyanin, DA"
  },
  {
    "caption": "Compared to the wild type plants, phr1phl1 showed substantially decreased PSR gene hyper induction (E) triggered by DA. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "DA"
  },
  {
    "caption": " [A] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "DA, Pi, SA"
  },
  {
    "caption": " [B] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "DA, Pi, SA"
  },
  {
    "caption": " [C] Images of plants grown with different treatments using DA (9.7 µg per mL free space) or SA (100 µM in plant growth medium). ",
    "molecules": "DA, SA"
  },
  {
    "caption": "Exogenous application of SA mimics DA-induced anthocyanin accumulation (D) patterns in Pi-deficient plants. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "anthocyanin, DA, Pi, SA"
  },
  {
    "caption": "Exogenous application of SA mimics DA-induced IPS1 gene induction (E) patterns in Pi-deficient plants. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "DA, Pi, SA"
  },
  {
    "caption": "Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition, as shown by plant images (F)",
    "molecules": "DA, Pi"
  },
  {
    "caption": "Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition and quantification of anthocyanin accumulation levels (G). Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.",
    "molecules": "anthocyanin, DA, Pi"
  },
  {
    "caption": " [A] Clustering of DA-regulated immunity-related genes in Arabidopsis with sufficient Pi, as revealed by RNAseq analysis. ",
    "molecules": "DA, Pi"
  },
  {
    "caption": " [B] DA suppresses plant ROS burst induced by flg22 or elf18. ROS accumulation was represented in relative luminescence units (RLU). Data bar indicate mean ± SE (n=24) readings for 60 min. Three independent experiments were performed and similar results were observed. **p < 0.01, student t-test. ",
    "molecules": "DA, ROS"
  },
  {
    "caption": " [C] DA induces gene expression of AZI1 in Arabidopsis. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. Bar indicates mean ± SE (n=3), two biological replicates were analyzed with similar results. ",
    "molecules": "DA"
  },
  {
    "caption": " [D] flg22-induced MPK3/MPK6 phosphorylation in Arabidopsis is not decreased by DA. Kinase assays were performed at 5 min after flg22 treatment. Three independent experiments were performed and similar results were observed. ",
    "molecules": "DA"
  },
  {
    "caption": " [E] Plant resistance to the pathogen Pst DC3000 is not reduced by DA. The severity of pathogen infection was measured as colony forming units (CFUs) per mg FW plants at 0 and 3 days after inoculation. Data bar indicate mean ± SE (n=8), two independent experiments were performed. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. ",
    "molecules": "DA"
  },
  {
    "caption": " [F] Root colonization of GB03 in wild type Arabidopsis (Col-0) and the mutants/transgenic lines npr1, NahG, and rbohd. *** p < 0.001compared with Col-0 without DA treatment, n= 9 biological replicates, student t-test. ",
    "molecules": "DA"
  },
  {
    "caption": " [G] Bacteria chemotaxis with different concentrations of DA. Data point indicates mean± SE (n=6). Two independent experiments were performed and similar results were observed. *** p < 0.001, student t-test. ",
    "molecules": "DA"
  },
  {
    "caption": "(A-D) A. castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of PflaA (Ptac-mCherry-PflaA-gfp) and mCherry constitutively, (B) GFP under the control of PralF (PralF-gfp) and stained with DAPI, (C) GFP under the control of PsidC (PsidC-gfp) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post-infection, analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP-positive) emerge at the colony cluster periphery only after bacterial replication has ceased (red fluorescent Timer).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-E) Representative live cell microscopy images of D. discoideum infected with PflaA-GFP-producing L. pneumophila. D. discoideum Ax3 producing cytosolic mCherry (pDM1042) was infected (MOI 5) with (A, B) wild-type L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp), (C, D) JR32 (Ptac-mCerulean-PflaA-gfp) or (E) ∆flaA (Ptac-mCerulean-PflaA-gfp) and embedded in agarose for 48 h. PflaA-GFP-positive L. pneumophila (A) escapes the LCV (0 sec), followed by other bacteria, which are contained in the cytosol and localize to the plasma membrane, where a PflaA-GFP-positive L. pneumophila first leaves the ruptured host cell (185 sec), or (B) triggers host cell death (loss of cytosolic mCherry) upon exit of the amoeba. PflaA-GFP-positive L. pneumophila (C) escape the LCV after 75 sec and spread within the cytosol for several minutes, and (D) are the first to exit the host cell, leading to the loss of cytosolic mCherry and host cell death. (E) L. pneumophila ∆flaA lyses the LCV and host cell less vigorously. Scale bars: 5 µm.",
    "molecules": "agarose"
  },
  {
    "caption": "(B) L. pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae. A. castellanii amoebae were infected (MOI 5, 52 h) with L. pneumophila JR32, ∆flaA or ∆lqsA harboring Ptac-mCherry-PflaA-gfp and embedded in 0.5% agarose/PYG medium. The fluorescence images are representative for the L. pneumophila strains indicated: JR32 (n = 25), ∆flaA (n = 13) or ∆lqsA (n = 19). Scale bars: 20 µm.",
    "molecules": "agarose"
  },
  {
    "caption": "(C-F) Role of the Lqs system for LCV escape and cytosolic localization of L. pneumophila. (C, D) A. castellanii or (E, F) D. discoideum was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, fixed and treated with an anti‐ubiquitin antibody. Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy. Representative images of (C) A. castellanii (scale bars: 10 µm) or (E) D. discoideum (scale bars: 5 μm) infected with L. pneumophila JR32 escaping or not from the LCV are shown. The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells: (D) JR32: n = 482; ΔlqsA: n = 834; ΔlqsR: n = 572; ΔlqsS: n = 729; ΔlqsT: n = 1469; ΔlqsS-ΔlqsT: n = 693; (F): JR32: n = 136; ΔlqsA: n = 201; ΔlqsR: n = 303; ΔlqsS: n = 183; ΔlqsT: n = 534; ΔlqsS-ΔlqsT: n = 180.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(G, H) D. discoideum producing the LCV/PtdIns(4)P probe P4C-GFP and cytosolic-mCherry was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, ∆lqsS-∆lqsT, or ∆flaA harboring Ptac-mCerulean. Representative images of (G) D. discoideum infected with L.-pneumophila JR32 in intact cells with intact LCV (18.5%), lysed cells and lysed LCVs (60.2%), intact cells with lysed LCVs (15.5%) and lysed cells with intact LCVS (5.8%) are shown (scale bars: 5 µm). (H) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane: JR32: n = 426; ΔlqsA: n = 468; ΔlqsR: n = 344; ΔlqsS: n = 325; ΔlqsT: n = 287; ΔlqsS-ΔlqsT: n = 289; ΔflaA: n = 248.",
    "molecules": "PtdIns(4)P"
  },
  {
    "caption": "D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and fixed and treated with an anti‐ubiquitin antibody, LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (A) Representative images of amoebae harboring L. pneumophila confined within LCVs or released from the LCV to the cytosol are shown (scale bars: 5 μm).",
    "molecules": "PtdIns(4)P, ubiquitin"
  },
  {
    "caption": "(A) Raw 264.7 cells were pre-treated with DMSO or the EGFR inhibitor gefitinib (10 µM) before cGAMP transfection; after 5h, IFNβ mRNA was measured by qRT-PCR (n=3).",
    "molecules": "cGAMP, DMSO, gefitinib"
  },
  {
    "caption": "(B) WT and EGFR knockdown Raw 264.7 cells were transfected with cGAMP; 4.5h post treatment, IFNβ mRNA was measured (n=3).",
    "molecules": "cGAMP"
  },
  {
    "caption": "(C) WT and EGFR-/- BMDMs were transfected with cGAMP; 5h post transfection, IFNα mRNA induction was measured (n=4).",
    "molecules": "cGAMP"
  },
  {
    "caption": "(D) WT and EGFR-/- HeLa cells were transfected with cGAMP; after 5h IFNβ mRNA was measured (n=3).",
    "molecules": "cGAMP"
  },
  {
    "caption": "MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (E) RNA-seq analysis (n=2). For RNA-seq analysis, poly T enriched cDNA library was prepared and 30 M single-end 50 bp reads were analyzed. The heat map for some of the genes is shown.",
    "molecules": "cGAMP, gefitinib, poly T"
  },
  {
    "caption": "MEF cells were pre-treated with gefitinib for 1h and then transfected with cGAMP; 4.5h post treatment, total RNA was harvested for analysis. (G) The RNA-seq analysis results were confirmed by qRT-PCR analysis of a gefitinib-sensitive and an insensitive mRNA (n=3).",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(H) MEF cells were pretreated with gefitinib for 1h and followed with cGAMP or Poly (I:C) transfection; 4.5h post treatment, total RNA was harvested and IFNα mRNA induction was measured (n=3).",
    "molecules": "cGAMP, gefitinib, Poly (I:C)"
  },
  {
    "caption": "(A) MEF cells were pre-treated with gefitinib or DMSO for 1 hour, and then infected with HSV1 at m.o.i. of 1. 4 hours post infection (h.p.i.), IFNβ mRNA induction was monitored by qRT-PCR (n=3). Uninfected cells were used as negative control (U).",
    "molecules": "DMSO, gefitinib"
  },
  {
    "caption": "(B) DNA was extracted from gefitinib-treated or untreated infected MEFs 0 and 16 h.p.i. and viral DNA was measured by qPCR (n=3).",
    "molecules": "gefitinib"
  },
  {
    "caption": "(D) Survival of uninfected mice treated with gefitinib and HSV1-infected mice treated or untreated with gefitinib.",
    "molecules": "gefitinib"
  },
  {
    "caption": "(A) EGFR was not required for ligand-induced STING dimerization: MEFs were transfected with HA-tagged STING and treated with gefitinib or DMSO before cGAMP transfection, 4h later STING was immunoprecipitated with anti-HA antibodies and analyzed by Western blot.",
    "molecules": "cGAMP, DMSO, gefitinib"
  },
  {
    "caption": "(B) EGFR was required for the activation of IRF3, but not TBK1: MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; cell lysate were subjected to Western blot analysis using the indicated antibodies.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(C) Nuclear translocation of IRF3, but not NFκB p65, required EGFR: MEFs were pre-treated with gefitinib (+) or DMSO (-) for 1 hour before transfecting cGAMP. Cells were harvested and nuclei were isolated at the indicated time points; total cell extracts (T) and nuclear extracts (N) were analyzed by Western blot.",
    "molecules": "cGAMP, DMSO, gefitinib"
  },
  {
    "caption": "MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; (D) IRF3, but not TBK1, binding to STING required EGFR cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(E) STING-IRF3 interaction in human cells required EGFR: WT and EGFR-/- (KO) HeLa cells were transfected with cGAMP, 2h later cells were lysed, immunoprecipitated with anti-STING antibody and analyzed by Western blot.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(F) STING and IRF3 co-localized in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours then treated with cGAMP for 1h. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(G) EGFR was not required for ligand-induced phosphorylation of STING Ser366: EGFR-/- HeLa cells or gefitinib-treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(H) Kinetics of STING and IRF3 co-localization in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours and treated with cGAMP for the indicated time. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies and DAPI. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.",
    "molecules": "cGAMP, DAPI"
  },
  {
    "caption": "(A) STING was Tyr-phosphorylated in cGAMP-treated cells. Raw 264.7 cells were tranfected with cGAMP; after 60 min, cell lysates were immuno-precipitated with anti-pTyr antibodies and then subjected to anti-STING Western blot.",
    "molecules": "cGAMP, Tyr"
  },
  {
    "caption": "(B) STING Tyr-phosphorylation was inhibited by gefitinib. MEF cells were pre-treated with gefitinib before cGAMP-stimulation for 60 min. Cell lysates were immuno-precipitated with anti-pTyr antibody and then analyzed by anti-STING Western blot. NTC: no treatment control.",
    "molecules": "cGAMP, gefitinib, Tyr"
  },
  {
    "caption": "(C) STING Tyr-phosphorylation required EGFR. Raw 264.7 cells expressing EGFR shRNA (KD) or non-target shRNA (CO) were transfected with cGAMP for 2 hours; cell lysates were analyzed by Western blot before or after immunoprecipitation with anti-pTyr antibodies.",
    "molecules": "cGAMP, Tyr"
  },
  {
    "caption": "(D) cGAMP stimulation triggered EGFR-binding to STING. Raw 264.7 cells were transfected with cGAMP for 1h; cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(E) STING co-localized with EGFR after cGAMP stimulation. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours and stimulated with cGAMP for 1h, cells were fixed and labeled with EGFR (Red) and Calnexin (Purple) antibodies. Co-localization was analyzed by Fuji and co-localized dots are shown in white. Scale bars represent 10 µ m.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(F) STING-EGFR interaction was transient. Raw 264.7 cell lysates were prepared at indicated time points after cGAMP transfection, immunoprecipitated with anti-EGFR antibodies and subjected to Western blot.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(G) Gefitinib did not block, but prolonged STING-EGFR interaction. Raw 264.7 cells were pre-treated with gefitinib or DMSO for 1 hour before cGAMP stimulation. Cell lysates were prepared at indicated time points, immuno-precipitated with anti-STING antibody and subjected to Western blot.",
    "molecules": "cGAMP, DMSO, Gefitinib, gefitinib"
  },
  {
    "caption": "(H) Poly I:C did not activate EGFR. STING-/- MEF cells were transfected with cGAMP or Poly (I:C); after 3h, p-EGFR were detected by Western blot.",
    "molecules": "cGAMP, Poly (I:C), Poly I:C"
  },
  {
    "caption": "(I) Kinetics of EGFR activation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were analyzed for p-EGFR via Western blot.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(K) Kinetics of phosphorylation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were subjected to Western blot using the indicated antibodies.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(L) Poly I:C did not trigger Tyr or Ser phosphorylation of STING. HeLa cells were transfected with cGAMP or Poly (I:C); after 3h, cell lysates were analyzed by Western blot using the indicated antibodies.",
    "molecules": "cGAMP, Poly (I:C), Poly I:C, Ser, Tyr"
  },
  {
    "caption": "(A) In the absence of EGFR, after cGAMP stimulation, STING translocated out of the ER. STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h; cells were fixed and labeled with Calreticulin (ER marker, Red) antibody. Co-localization (Col) is indicated by white dots.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (B) STING was present in the ERGIC of stimulated cells in the presence or the absence of gefitinib. , the cells were labeled with P58 (ERGIC marker, Red) antibody.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (C) STING translocated to the autophagosomes in the absence of EGFR. the cells were labeled with LC3 (Autophagosome marker, Red) antibody. (E) In gefitinib-treated cells, STING was detected in the autophagosomes as early as 0.5h. The procedures were as in C.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(F) STING translocation to late endosomes required EGFR activity. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time periods; cells were fixed and labeled with CD63 (late endosome marker, Red) antibody.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(C) LC-MS/MS analysis of STING identifies phosphorylated Y245. STING-GFP was purified from cGAMP-stimulated cells, subjected to protease digestion and LC-MS/MS analysis. Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified. The MS/MS spectra for the phosphorylated peptide VYSNSIpYELLENGQR is shown. This doubly charged peptide has an observed m/z of 932.9266 Da and is within -0.96 ppm of the expected mass. The mass difference between the y8 and y9 ions is consistent with phosphorylation at Y245.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(E) Gefitinib pretreatment inhibits phosphorylation of Y245. STING-GFP-293XL cells were pretreated with gefitinib and then treated with cGAMP. LC-MS/MS analysis was done, as above, for detecting pY245 (n=3).",
    "molecules": "cGAMP, Gefitinib, gefitinib"
  },
  {
    "caption": "(F) EGFR is required for STING Tyr phosphorylation. STING-GFP was expressed in 293XL cells in which EGFR expression had been knocked down. STING phosphorylation was analyzed by LC-MS/MS",
    "molecules": "Tyr"
  },
  {
    "caption": "(I) TBK1, but not IRF3, can bind to Y245F STING and be phosphorylated in cGAMP-stimulated cells. Lysates from cells expressing the WT or the mutant STING were analyzed by Western blot after (upper panel) or before (lower panel) GFP pull down.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(J) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL cells expressing WT or mutant STING.",
    "molecules": "cGAMP"
  },
  {
    "caption": "(K) Inhibition of EGFR by gefitinib induced more LC3II after 1.5h cGAMP treatment of L929 cells.n",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with LC3 (autophagosome marker, Red) antibody. Co-localization (Col) is indicated by white dots.",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with CD63 (late endosome marker).",
    "molecules": "cGAMP, gefitinib"
  },
  {
    "caption": "C, D ImmunoFISH images of CAF-1 bodies and HIV-1 genomic DNA within J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were acquired by treating with TNFα.",
    "molecules": "DNA, DMSO"
  },
  {
    "caption": "G-I GFP-tagged CHAF1A-IDR and CHAF1A-mIDR were purified in vitro. Ten micromole of each protein was incubated with gradient droplet formation buffer. The droplets of CHAF1A-IDR appeared in NaCl concentration of 125 mM or lower (G). No CHAF1A-mIDR droplet was detected within any NaCl concentration (I). The volumes of CHAF1A-IDR droplets increased with the increase of protein concentration (H). Left subpanels within each panel represented images of protein droplets in different buffers. Right subpanels within each panel represented statistical analysis results of droplets areas and numbers.",
    "molecules": "NaCl"
  },
  {
    "caption": "D The amounts of intracellular HIV-1 RNAs of latently infected primary CD4+ T cells which were isolated from PBMCs of HIV-1-infected individuals. αCD3/αCD28/IL-2 treatment was performed as positive control. shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A. shluc lentiviruses treatment was performed as negative control. SAHA was added as LRA supplement.",
    "molecules": "SAHA"
  },
  {
    "caption": "Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in L6 rat myoblasts with siRNA-mediated knockdown of endogenous STIM1 (siSTIM1) or transfected with scramble control siRNA (Control). Cells were treated with the AMPK activator A-769662 (+) or vehicle (-) for 30 min. Total STIM1 was used to demonstrate STIM1 knockdown efficiency. Total ACC and 14-3-3 were used as loading controls.",
    "molecules": "A-769662"
  },
  {
    "caption": "Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK β1/β2 knockout (AMPKβ KO) mouse endothelial fibroblasts were isolated and treated with A-769662 (+) or vehicle (-) for 30 min. AMPKβ was used to demonstrate knockout, and AMPKɑ was used to demonstrate specificity for AMPKβ. Total ACC and STIM1 were used as loading controls.",
    "molecules": "A-769662"
  },
  {
    "caption": "Representative Western blots for phosphorylated ACC (S79) and STIM1 (S257 and S521) in response to a 15 min treatment with increasing doses of thapsigargin (.01, .1, 1 or 10 µM) or .5 mM AICAR. DMSO was used as a vehicle control. 14-3-3 was used as a loading control. n=3 independent experiments.",
    "molecules": "AICAR, DMSO, thapsigargin"
  },
  {
    "caption": "Western blots for phosphorylated ACC (S79) and STIM1 (S257 or S521) in wild type (WT) or AMPK KO mouse endothelial fibroblasts were isolated and treated with 2 µM thapsigargin (+) or vehicle (-) for 15 min.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "Time correlated single photon counting was used to estimate the fluorescence lifetime of mClover2. STIM1 OASF expressed in live L6 myoblasts before (-) or after (+) treatment with A-769662.",
    "molecules": "A-769662"
  },
  {
    "caption": "L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Representative microscopy images of GCaMP6s signal indicating SOCE in L6 myoblasts,",
    "molecules": "Ca2+"
  },
  {
    "caption": "L6 myoblasts were transfected with the genetic cytosolic Ca2+ sensor GCaMP6s. Cells were also transfected with non-targeted siRNA (Control) or siRNA directed at endogenous STIM1 (siSTIM1). Some cells with endogenous STIM1 knockdown were also transfected with full-length wild type (WT) or mutant (S257A, S257E or L251S) STIM1-mRuby3. Ca2+ levels over time (fold of 0 mM Ca2+ + EGTA basal) and (C) quantification of SOCE in in L6 myoblasts incubated in buffer containing 1 or 0 mM Ca2+ and treated with 2 µM thapsigargin during the indicated times.",
    "molecules": "Ca2+, EGTA, thapsigargin"
  },
  {
    "caption": "(E) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LiCor.",
    "molecules": "LxxAxA, LxxIxE"
  },
  {
    "caption": "(G) YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments.",
    "molecules": "LxxIxE"
  },
  {
    "caption": "(B) C2C12 myotubes were treated with 1 pM agrin and/or 250 pM CTGF for 6 h. Lrp4 was immunoprecipitated (IP) with an anti-Lrp4 antibody, and phosphorylated Lrp4 was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody.",
    "molecules": "p-Tyr, phosphotyrosine"
  },
  {
    "caption": "(A) C2C12 myotubes were treated with indicated concentrations of agrin-mycAP, CTGF-mycAP, and/or CTGF-∆CT-mycAP for 3 h. Total MuSK was immunoprecipitated (IP) with an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. CTGF-mycAP, but not CTGF-∆CT-mycAP, enhanced agrin-mediated MuSK phosphorylation.",
    "molecules": "p-Tyr, phosphotyrosine"
  },
  {
    "caption": "(B) C2C12 myotubes were treated with 5 pM agrin-mycAP, 250 pM CTGF-mycAP, and/or 250 pM CTGF-∆CT-mycAP for 18 h. BSA was added to the control medium. AChRs and the plasma membrane were visualized with Alexa594-conjugated α-bungarotoxin (red) and CellMask (green), respectively. Scale bar = 20 µm. Arrowheads point to the AChR clusters with an axis length of 4 μm or more. CTGF enhances agrin-mediated AChR clustering.",
    "molecules": "CellMask, Alexa594"
  },
  {
    "caption": "(C) C2C12 myotubes were infected with lentivirus expressing shControl, shCtgf-1, or shCtgf-2. Doxycycline was added for 2 days to induce shRNA expression. BSA or agrin was added to the medium at 10 pM. Total MuSK was immunoprecipitated (IP) by an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. Ctgf knockdown suppressed MuSK phosphorylation.",
    "molecules": "Doxycycline, p-Tyr, phosphotyrosine"
  },
  {
    "caption": "(D) Ten pM agrin was added to the medium. AChRs were visualized with Alexa594-conjugated α-bungarotoxin (red signals) in the infected myotube (green GFP signals). Ctgf knockdown decreased the number and length of AChR clusters. Scale bar = 10 µm.",
    "molecules": "Alexa594"
  },
  {
    "caption": "A Immunofluorescence staining of HK2 with an AlexaFluor488-conjugated antibody in HeLa cells expressing mitochondria-targeted RFP. Yellow signals in the merge analysis indicate mitochondrial localization of HK2. Scale bar: 15 µm.",
    "molecules": "AlexaFluor488"
  },
  {
    "caption": "B Immunofluorescence staining of HK2 with a secondary AlexaFluor555-conjugated antibody in HeLa cells expressing both mitochondria-targeted YFP and ER-targeted CFP. The merged white signal indicates MAM localization of HK2 and is quantified in the bar graph on the right (n=24). Image magnifications are shown in the lower part of the panel; arrows indicate HK2 dots in mito-ER contact sites. Scale bar: 15 µm.",
    "molecules": "AlexaFluor555"
  },
  {
    "caption": "C Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts (SPLICSL) on HeLa cells; HK2 is revealed with a secondary AlexaFluor555-conjugated antibody, the merged signal is white. Scale bar: 15 µm.",
    "molecules": "AlexaFluor555"
  },
  {
    "caption": "Effect of cl-HK2pep on glucose phosphorylation by human recombinant HK2 (I) where both total hexokinase activity and HK1/HK2 specific activities are measured.",
    "molecules": "glucose"
  },
  {
    "caption": "Effect of cl-HK2pep on glucose phosphorylation in 4T1 cell extracts (J), where both total hexokinase activity and HK1/HK2 specific activities are measured.",
    "molecules": "glucose"
  },
  {
    "caption": "Effect of cl-HK2pep on cellular Ca2+ dynamics ER Ca2+ levels are measured by the FRET-based, D4ER fluorescent probe expressed in the lumen of ER (A) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).",
    "molecules": "D4ER, Ca2+"
  },
  {
    "caption": "Effect of cl-HK2pep on IP3 levels. IP3 levels are assessed with the GFP-PHD probe; histamine (100μM) is used as a positive control for IP3 generation; data are reported as mean of fluorescent signals±SEM (n=3 independent experiments; B) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here [",
    "molecules": "IP3, histamine, Xe-C, Xestospongin C"
  },
  {
    "caption": "Effect of cl-HK2pep on cellular Ca2+ dynamics changes in mitochondrial Ca2+ levels are recorded (C; scale bar: 20µm) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Effect of cl-HK2pep on cellular Ca2+ dynamics changes in mitochondrial Ca2+ levels are recorded and quantified using the GCAMP6f sensor (in D, as mean of 475/410 nm ratio; signal±SEM of at least 5 independent experiments and more than 20 cells analized) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here",
    "molecules": "Ca2+, Xe-C, Xestospongin C"
  },
  {
    "caption": "Effect of cl-HK2pep on cellular Ca2+ dynamics (E) as percentage of cells with increased Ca2+ in mitochondria, with a threshold 475/410 ratio for positivity >3; baseline mean ratio=1.84±0.54) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here [",
    "molecules": "Ca2+, Xe-C, Xestospongin C"
  },
  {
    "caption": "Effect of cl-HK2pep on cellular Ca2+ dynamics with mitochondria-targeted aequorin (F, where data are reported as mean of [Ca2+]±SEM of 3 independent experimental days). Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).",
    "molecules": "Ca2+"
  },
  {
    "caption": "Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments (G, single cell analysis, n=216 cells analyzed in at least 10 independent experiments",
    "molecules": "TMRM"
  },
  {
    "caption": "Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments H, representative field; scale bar: 20µm) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+ free medium plus 500 µM EGTA with or without 10 µM BAPTA-AM",
    "molecules": "BAPTA-AM, Ca2+, EGTA, TMRM"
  },
  {
    "caption": "Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. quantified (I and J; TMRM fluorescence is normalized to initial value and expressed in percentage for each time point, with a depolarization threshold placed at 40% of initial value). Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+ free medium plus 500 µM EGTA with or without 10 µM BAPTA-AM; Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here",
    "molecules": "BAPTA-AM, Ca2+, EGTA, TMRM, Xe-C, Xestospongin C"
  },
  {
    "caption": "Effects of the PTP desensitizers CsA or C63 (5 µM each, 1 h preincubation) on mitochondrial Ca2+ levels recorded with GCAMP6f (A) in cells treated with cl-HK2pep. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)",
    "molecules": "C63, Ca2+, CsA"
  },
  {
    "caption": "Effects of the PTP desensitizers CsA or C63 (5 µM each, 1 h preincubation) on mitochondrial membrane potential assessed with TMRM (B) in cells treated with cl-HK2pep. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)",
    "molecules": "C63, CsA, TMRM"
  },
  {
    "caption": "cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each) data are presented as mean±SD of at least 3 independent experiments. *** p<0.001 with a Student's t test",
    "molecules": "7-AAD, FITC"
  },
  {
    "caption": "cell death induction by cl-HK2pep; in the cytofluorimetric analyses in the kinetic experiment shown in (D; scale bar: 50µm) viable cells are double negative for Annexin V-FITC and 7-AAD and TMRM positive. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)",
    "molecules": "7-AAD, FITC, TMRM"
  },
  {
    "caption": "cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated, the caspase inhibitor Z-VAD-fmk or the calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment.",
    "molecules": "PD150606, 7-AAD, FITC, Z-VAD-fmk"
  },
  {
    "caption": "cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each) where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. data are presented as mean±SD of at least 3 independent experiments. p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).",
    "molecules": "PD150606, 7-AAD, FITC"
  },
  {
    "caption": "Effect of calpain inhibition on mitochondrial membrane potential assessed with TMRM (G, normalized fluorescence signal mean±SD; 3 different experiments including 3 technical replicates each) in cells treated with cl-HK2pep Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. data are presented as mean±SD of at least 3 independent experiments. p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).",
    "molecules": "PD150606, TMRM"
  },
  {
    "caption": "Effect of calpain inhibition on mitochondrial Ca2+ levels recorded with GCAMP6f (H) in cells treated with cl-HK2pep . Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment.",
    "molecules": "PD150606, Ca2+"
  },
  {
    "caption": "Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. HK2-expressing B-CLL cells (B; GAPDH and respiratory complex III subunit UQCRC2 are cytosol and mitochondrial loading controls, respectively) Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls.",
    "molecules": "PD150606"
  },
  {
    "caption": "Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. HK2-expressing B-CLL cells undergo mitochondrial depolarization assessed by TMRM staining (C) Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. two-way ANOVA: p<0.0001 (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments) Student's t test; ***p<0.01, *p<0.05.",
    "molecules": "PD150606, TMRM"
  },
  {
    "caption": "ffect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. In C, D, F and G data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. wo-way ANOVA: p<0.0001 (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); tudent's t test; ***p<0.01, *p<0.05.",
    "molecules": "PD150606, 7-AAD, FITC"
  },
  {
    "caption": "Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls.",
    "molecules": "PD150606, 7-AAD, FITC"
  },
  {
    "caption": "Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. Bonferroni post-test in graph ***p<0.001(cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep) Student's t test; ***p<0.01, *p<0.05.",
    "molecules": "PD150606, 7-AAD, FITC"
  },
  {
    "caption": "Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. Bonferroni post-test in graph ***p<0.001(cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep) Student's t test; ***p<0.01, *p<0.05.",
    "molecules": "PD150606, 7-AAD, FITC"
  },
  {
    "caption": "Effect of HK2 targeting on in vitro tumorigenicity. Treatment with cl-HK2pep (50 µM, 6 h; C, scale bar: 100 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.",
    "molecules": "Trypan Blue"
  },
  {
    "caption": "Effect of HK2 targeting on in vitro tumorigenicity. Treatment with uncleaved HK2pep (2 µM, 48 h; D, scale bar: 200 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.",
    "molecules": "Trypan Blue"
  },
  {
    "caption": "B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT (WT#1), UP∆44 (UP∆44#3) and ∆44 edited clones (UP∆44#3.1 and UP∆44#3.5) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) HEK293 cells were starved of glucose (4 h) as indicated, endogenous Ulk1 was immunoprecipitated and an autophosphorylation assay was performed. Proteins were resolved by SDS-PAGE and visualized with autoradiography (top) or western blotting (WB; bottom).",
    "molecules": "glucose"
  },
  {
    "caption": "(b) Cells were incubated in glucose-free medium (4 h) as indicated and lysed. Lysates were incubated with lambda phosphatase (λ PPase) as indicated. Endogenous Ulk1 mobility was examined by western blotting.",
    "molecules": "glucose"
  },
  {
    "caption": "(c) HA-Ulk1 was transfected into HEK293 cells together with wild-type (WT) AMPKα1 or a kinase-dead (DN) mutant. Cells were starved of glucose (4 h; Glu) or amino acids (-A.A) and treated with compound C (20 μM, C.C) as indicated. Ulk1 mobility as well as phosphorylation levels of ACC and S6K were determined by western blotting.",
    "molecules": "amino acids, compound C, glucose"
  },
  {
    "caption": "(d) HA-Ulk1 proteins were immunopurified from transfected HEK293 cells, which had undergone glucose starvation (4 h) as indicated. The HA-Ulk1 proteins were treated with λ PPase, and in vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography , HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.",
    "molecules": "glucose"
  },
  {
    "caption": "(e) HA-Ulk1 was immunopurified from transfected HEK293 cells under glucose-rich media and treated with AMPK in the presence of cold ATP for 15 min, followed by kinase assays as described in d. In vitro kinase assays were performed in the presence of GST-ATG13. Proteins were resolved by SDS-PAGE; phosphorylated proteins were visualized with autoradiography, HA-Ulk1 by western blotting and GST-Atg13 by Coomassie staining.",
    "molecules": "ATP, glucose"
  },
  {
    "caption": "(f) AMPK wild-type (WT) and α1/α2 double knockout (DKO) MEFs were incubated with or without glucose (4 h). Endogenous Ulk1 was immunoprecipitated and autophosphorylation was measured (mean ± s.d., n = 3). Autophosphorylation activity was normalized to Ulk1 protein level; relative activity is calculated by normalization to Ulk1 activity from AMPK wild-type MEFs in glucose-rich conditions.",
    "molecules": "glucose"
  },
  {
    "caption": "(g) HA-Ulk1 was transfected into HEK293 cells together with vector (Vec) or an AMPKα1 kinase-dead mutant (DN). The cells were starved of glucose (-Glu) or amino acids (-A.A), or treated with 50 nM rapamycin (Rapa) for 3 h before lysis. Left: autophosphorylation activity was assessed and normalized as in f (mean ± s.d., n = 3). Right: fold induction in Ulk1 autophosphorylation, compared with Ulk1 autophosphorylation from cells under nutrient-rich conditions. Uncropped images of blots are shown in Supplementary Fig. S5.",
    "molecules": "amino acids, glucose, rapamycin"
  },
  {
    "caption": "(b) Determination of AMPK phosphorylation sites in Ulk1. The indicated recombinant GST-Ulk1 mutants were expressed and purified from Escherichia coli, and used as substrates for in vitro phosphorylation by AMPK. Deletion analyses indicated that two Ulk1 fragments in the S/T domain, 279-425 and 769-782, were highly phosphorylated by AMPK in vitro. Mutation of Ser 317 abolished the majority of phosphorylation in the Ulk1 fragment 279-425. Within the fragment 769-782, mutations of five serine residues (Ser 774, Ser 777, Ser 778, Ser 779 and Ser 780) to alanine, denoted as (769-782) 5SA, completely abolished the phosphorylation by AMPK. Reconstitution of Ser 777 in this mutation background, (769-782) 4SA-S777, but not any of the other four residues, restored the phosphorylation by AMPK. GST and GST-TSC2F (TSC2 fragment 1300-1367 containing AMPK phosphorylation site at Ser 1345) were used as negative and positive controls for AMPK reaction, respectively. Phosphorylation was determined by 32P-autoradiograph and the protein levels were detected by Coomassie staining.",
    "molecules": "32P"
  },
  {
    "caption": "(c) Ser 317/Ser 777 are required for glucose-starvation induced Ulk1 phosphorylation in vivo. HA-Ulk1 and mutants were transfected into HEK293 cells. Cells were starved for glucose for 4 h as indicated. HA-Ulk1 was immunoprecipitated and examined by western blot for mobility.",
    "molecules": "glucose"
  },
  {
    "caption": "(a) AMPK wild-type or DKO MEFs were starved of glucose (4 h) as indicated. Total cell lysates were probed for Ulk1 protein and phosphorylation.",
    "molecules": "glucose"
  },
  {
    "caption": "(b) Time course of Ulk1 Ser 317 and Ser 777 phosphorylation in response to glucose starvation/re-addition. MEFs were starved of glucose (-Glu) for the indicated times. After 3 h starvation, the culture was switched to glucose-containing (25 mM) medium and samples were harvested (Re-Glu). In parallel, cells were treated with amino-acid-free (-A.A) medium or 50 nM rapamycin (Rapa) for 3 h.",
    "molecules": "amino-acid, glucose, rapamycin"
  },
  {
    "caption": "(c) Phosphorylation of Ulk1 Ser 317 and Ser 777 correlates with AMPK activity. MEFs were starved of glucose (4 h) as indicated in the presence or absence of 20 μM compound C (C.C). In parallel, cells were treated with 2 mM Metformin (Met, 2 h) in glucose-rich medium. Phosphorylation of ACC S79 was tested as a positive control for AMPK activation.",
    "molecules": "compound C, glucose, Metformin"
  },
  {
    "caption": "(d) Ulk1 is highly phosphorylated at Ser 317 and Ser 777 by glucose starvation in vivo. To determine the Ulk1 phosphorylation level in vivo, immunopurified HA-Ulk1 protein was phosphorylated by AMPK in vitro (100% represents full phosphorylation of Ulk1 by AMPK). In vitro phosphorylated HA-Ulk1 was diluted as indicated, and was immunoblotted along with the immunoprecipitated HA-Ulk1 from cells grown in either glucose-rich (+ Glu) or glucose-free (- Glu, 4 h) medium. The density of the bands was then quantified. By this measurement, approximately 50% of Ulk1 isolated from glucose-starved cells was phosphorylated on Ser 317 and Ser 777.",
    "molecules": "glucose"
  },
  {
    "caption": "(e) The indicated HA-Ulk1 proteins were immunopurified from transfected HEK293 cells grown in high-glucose medium, and then incubated with AMPK in the presence of cold ATP for 15 minin vitro. After the reaction, AMPK was removed by extensive washing, the resulting Ulk1 immuno-complexes were assayed for kinase activity in the presence of 32P-ATP.",
    "molecules": "ATP, glucose"
  },
  {
    "caption": "(f) HA-Ulk1 proteins (wild type or S317/777A mutant) were immunoprecipitated from the transfected HEK293 cells, which were incubated with or without glucose (4 h) before lysis. An in vitro kinase reaction was performed in the presence of GST-ATG13 and FIP200. Uncropped images of blots are shown in Supplementary Fig. S5.",
    "molecules": "glucose"
  },
  {
    "caption": "(c) Rheb inhibits the Ulk1-AMPK interaction. HA-AMPKα, Flag-Ulk1 and Myc-Rheb were co-transfected into HEK293 cells as indicated. Cells were treated with or without rapamycin (50 nM Rapa) for 1 h before lysis. Flag-Ulk1 was immunoprecipitated and co-immunoprecipitates of AMPKα were determined by western blot.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(d) Rapamycin treatment enhances the interaction of endogenous Ulk1 and AMPK. Endogenous Ulk1 proteins were immunoprecipitated from either Ulk1 or AMPK wild-type and knockout (single-knockout; KO or double-knockout; DKO) MEFs. Treatment with 50 nM rapamycin for 1 h is indicated (Rapa). Co-immunoprecipitation of endogenous AMPKα protein was determined by western blot. The arrow indicates AMPKα protein.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "(e) Phosphorylation by mTORC1 inhibits the ability of Ulk1 to bind AMPK in vitro. CBP/SBP-Ulk1 was purified from transfected HEK293 cells by streptavidin beads and the Ulk1-bead complex was incubated with mTORC1, which was prepared by Raptor immunoprecipitation, in the presence of cold ATP, as indicated. The resulting Ulk1 complex was incubated with the cell lysates containing AMPK, then extensively washed. The Ulk1 and associated AMPKα were detected by western blot. Uncropped images of blots are shown in Supplementary Fig. S5.",
    "molecules": "ATP"
  },
  {
    "caption": "(c) Rheb increases Ulk1 Ser 757 phosphorylation. HA-Ulk1 wild type and the S757A mutant were immunoprecipitated from transfected HEK293 cells. Co-transfection with Rheb and rapamycin (Rapa) treatment are indicated. Ulk1 Ser 757 phosphorylation was determined by western blot.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(e) Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho-Ulk1 Ser 757 antibody. Uncropped images of blots are shown in Supplementary Fig. S5.",
    "molecules": "glucose, rapamycin"
  },
  {
    "caption": "(a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA-AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitatedHA- AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1-AMPK interaction in vitro. CBP/SBP Ulk1 proteins (wild type or S757C) were prepared from transfected HEK293 cells, which were pre-incubated with 50 nM rapamycin (Rapa, 1h) as indicated. The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1-bead was incubated with the bacterial purified AMPKα/β/γ complex. AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody. L.E.; long exposure.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(c) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777.",
    "molecules": "glucose"
  },
  {
    "caption": "(d) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1. HA-Ulk1, AMPKα kinase-dead mutant (DN), and Myc-Rheb were co-transfected into HEK293 cells as indicated. The cells were incubated with glucose-free medium (-Glu, glucose), in which either 20 μM compound C (C.C.) or 50 nM Rapamycin (Rapa) was added. Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317, Ser 777, Ser 757, and HA, as indicated.",
    "molecules": "compound C, glucose, Rapamycin"
  },
  {
    "caption": "(e) Rheb inhibits glucose starvation-induced Ulk1 activation. HA-Ulk1 and Myc-Rheb was transfected into HEK293 cells, which were incubated with glucose-free (-Glu), amino-acid-free (-A.A) medium, or 50 nM rapamycin (Rapa) for 4 h before lysis. HA-Ulk1 was immunoprecipitated and kinase assays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA-Ulk1 and GST-Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5.",
    "molecules": "32P, amino-acid, glucose, rapamycin"
  },
  {
    "caption": "(a) Ser 317/Ser 777 is required for Ulk1 to protect cells from glucose starvation. Viability (24 h, mean ± s.d., n = 4; top) and PARP cleavage (8 h; western blot, middle; quantification, n = 2, bottom) was examined in Ulk1+/+ (WT), Ulk1−/− (KO), Ulk1−/− re-expressing wild-type Ulk1 (KO-WT), and Ulk1−/− re-expressing Ulk1 S317/777A mutant (KO-S317/777A) MEFs. Arrows in western blots indicate non-cleaved and cleaved PARP.",
    "molecules": "glucose"
  },
  {
    "caption": "(b) The Ulk1 S317/777A mutant is compromised in LC3 lipidation in response to glucose starvation. ULK1MEFs were cultured in MEFs-free medium for the indicated times. LC3-II level was determined by western blotting and the LC3-II accumulation was normalized by α-tubulin and quantified (bottom, n = 3, mean ± s.d.). A representative western blot was shown. The LC3 antibody used in this experiment seemed to preferentially recognise the lipid-modified form of LC3-II, which migrated faster on the gel.",
    "molecules": "glucose"
  },
  {
    "caption": "(c) The Ulk1 S317/777A mutant is defective in autophagosome formation. The indicated MEFs were starved of glucose (4 h) and the formation of GFP-LC3-positive autophagosomes was examined by confocal microscopy. GFP-LC3; green and DAPI; blue. Scale bar, 20 μm.",
    "molecules": "glucose"
  },
  {
    "caption": "SDS-PAGE of recEna1B, treated with β-mercaptoethanol or TEV protease (to remove N-terminal 6xHis tag) as indicated. Bands with apparent MW of ~13 and ~15 kDa correspond recEna1B monomer with (Ena1B6His) and without (Ena1BC) the 6xHis tag and TEV recognition site, respectively. In the uncleaved sample, a band running at ~ 30 kDa (labeled *) corresponds to a non-physiological disulfide bound S-Ena1B dimer. Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS-resistant high molecular weight complexes stuck in the stacking gel.",
    "molecules": "disulfide, β-mercaptoethanol, SDS"
  },
  {
    "caption": "Negative stain TEM images of rec1Ena1B oligomers formed after refolding, but prior to TEV removal of the N-terminal 6xHis tag. Close-up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S-Ena fiber (model - right). Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs.",
    "molecules": "oligomers"
  },
  {
    "caption": " A MOAP-1 co-localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition. HCT116 cells were transfected with plasmid encoding GFP-MOAP-1 or GFP only control. 14 hours later, cells were treated with the proteasome inhibitor MG132 (5 µM) for 8 hours and analyzed by immunofluorescence (IF) with anti-GFP (in green) and anti-p62 (in red) antibodies. Data information: , nuclei were counterstained with DAPI (blue) ",
    "molecules": "DAPI, MG132"
  },
  {
    "caption": " B Endogenous MOAP-1 resides at the p62 bodies upon proteasome inhibition. HCT116 cells were treated with MG132 (5 µM) for 8 hours and analyzed by IF with anti-MOAP-1 (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue) ",
    "molecules": "DAPI, MG132"
  },
  {
    "caption": " C MOAP-1 is recruited to the p62 bodies upon exposure to cellular stresses. LO2 hepatocytes were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were then treated with MG132 (5 µM), arsenic trioxide (As2O3, 10 µM) or diethylnitrosamine (DEN, 200 µM) for 8 hours each. Cells were then processed for IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue) ",
    "molecules": "DAPI, arsenic trioxide, As2O3, MG132, DEN, diethylnitrosamine"
  },
  {
    "caption": " D MOAP-1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines, HepG2, Huh-1, JHH5 and JHH7. The liver cancer cell lines were transfected with the plasmid encoding Myc-MOAP-1. 14 hours later, transfected cells were subjected to IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue) ",
    "molecules": "DAPI"
  },
  {
    "caption": " E Absence of the aggregated patterns of MOAP-1 in the p62 deficient cells. WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc-MOAP-1 for 14 hours and the cells were subjected to IF analysis as in (D). Data information: , nuclei were counterstained with DAPI (blue) ",
    "molecules": "DAPI"
  },
  {
    "caption": " A MOAP-1 downregulates the levels of the diethylnitrosamine (DEN)-induced p62 bodies in a time-dependent manner. LO2 cells were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were treated with DEN (200 µM) for the indicated period of times, before being subjected to IF with anti-Myc (in green) and anti-p62 (in red) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. Data information: nuclei were counterstained with DAPI (blue) ",
    "molecules": "DAPI, DEN, diethylnitrosamine"
  },
  {
    "caption": " B MOAP-1 downregulates the p62 bodies in the resting HepG2 liver cancer cells. HepG2 were transfected with plasmid encoding Myc-MOAP-1. At the indicated time post-transfection, transfected cells were subjected to IF as in (A). Data information: nuclei were counterstained with DAPI (blue) ",
    "molecules": "DAPI"
  },
  {
    "caption": " C Loss of MOAP-1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to IF with anti-p62 antibody (in red). p62 KO cells were included as a negative control. Insets in the upper panels are enlarged in the lower panels. D Quantification of the p62 bodies in the WT and MOAP-1 KO LO2 cells. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples. (Right panel) size of p62 bodies. n = 442 and 1014 bodies detected in the WT and MOAP-1 KO LO2 cells, respectively, from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)",
    "molecules": "DAPI"
  },
  {
    "caption": " E Re-expression of MOAP-1 reduces levels of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 for 24 hours and subjected to IF with anti-p62 (in red) and anti-Myc (green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. F Quantification of the p62 bodies in the MOAP-1 KO LO2 cells re-introduced with Myc-MOAP-1. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples of MOAP-1 KO cells transfected with empty vector control (Ctrl) or plasmid encoding Myc-MOAP-1. (Right panel) size of p62 bodies. n = 178 and 69 bodies detected in the Ctrl and Myc-MOAP-1 expressing cells, respectively, from three independently plated samples. Data information: , nuclei were counterstained with DAPI (blue)",
    "molecules": "DAPI"
  },
  {
    "caption": " G Increased level of p62 bodies in MOAP-1 KO MEFs upon treatment with arsenic trioxide. WT and MOAP-1 KO MEFs were treated with arsenic trioxide (As2O3, 10 µM) for the indicated durations and subjected to IF with anti-p62 antibody (in red). H Quantification of the p62 bodies in the WT and MOAP-1 KO MEFs treated with arsenic trioxide. (Left panel) number of p62 bodies per cell. n ≥ 6 independently plated samples. (Right panel) size of p62 bodies. n = 10 (WT, basal), 17 (MOAP-1 KO, basal), 104 (WT, As2O3 2h), 137 (MOAP-1 KO, As2O3 2h), 605 (WT, As2O3 4h), 977 (MOAP-1 KO, As2O3 4h), 721 (WT, As2O3 6h) and 487 bodies (MOAP-1 KO, As2O3 6h) from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)",
    "molecules": "DAPI, arsenic trioxide, As2O3"
  },
  {
    "caption": " J MOAP-1 deficiency does not affect total protein levels of p62 in MEFs under basal and As2O3-treated conditions. Western blotting analysis of p62 and MOAP-1 in the WT and MOAP-1 KO MEFs treated with As2O3 as indicated. MOAP-1 was immunoprecipitated from the lysates. Actin as loading control. ",
    "molecules": "As2O3"
  },
  {
    "caption": " K Loss of MOAP-1 does not alter protein turnover of p62 in the LO2 cells. WT and MOAP-1 KO LO2 cells stably expressing Halo-tagged p62 were subjected to TMR labelling and harvested at the time points post-labelling as indicated. TMR signals of Halo-p62 were visualized by a Bio-rad Imager at 565 nM wavelength, whereas the total Halo-p62 protein was detected by Western blotting using anti-Halo or anti-p62 antibodies. L Quantification of the TMR signals in the WT and MOAP-1 KO LO2 cells by densitometric analysis. Average integrated density values of the TMR signals from three independent experiments were presented, relative to the levels at 0 hour. ",
    "molecules": "TMR"
  },
  {
    "caption": " A Overexpression of MOAP-1 downregulates levels of p62 bodies in the ATG5-depleted cells. HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 (shATG5) or scramble control (shCtrl) for 48 hours before being transfected with Myc-MOAP-1 or empty vector (EV) control for 24 hours and subjected to IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. B Quantification of the p62 bodies in the ATG5-depleted cells transfected with Myc-MOAP-1 or empty vector. (Upper panel) number of p62 bodies per cell. n ≥ 3 independently plated samples. (Lower panel) size of p62 bodies. n = 135, 133 and 65 bodies in the shCtrl+EV, shATG5+EV and shATG5+Myc-MOAP-1 cells, respectively, from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)",
    "molecules": "DAPI"
  },
  {
    "caption": " D Inhibition of the auto-lysosomal degradation pathway does not prevent MOAP-1-mediated reduction of the p62 bodies. MOAP-1 KO LO2 cells were pre-treated with auto-lysosome inhibitors chloroquine (CQ) (10 µM) or BafA1 (1 µM) for 3 hours, before being transfected with Myc-MOAP-1 for 24 hours in the presence of CQ or BafA1 and subjected to immunofluorescence using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. E Quantification of the p62 bodies in the MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 before being transfected with Myc-MOAP-1 or empty vector control. (Upper panel) number of p62 bodies per cell. n = 5 independently plated samples. (Lower panel) size of p62 bodies. n = 362 (Ctrl, BafA1), 226 (Myc-MOAP-1, BafA1), 544 (Ctrl, CQ) and 266 bodies (Myc-MOAP-1, CQ), from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue), ",
    "molecules": "BafA1, chloroquine, CQ, DAPI"
  },
  {
    "caption": " F Western blotting of lysates from MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 and transfected with Myc-MOAP-1. ",
    "molecules": "BafA1, CQ"
  },
  {
    "caption": " A MOAP-1 associates with p62 and their interaction is enhanced by DEN-mediated stress. LO2 cells transfected with Myc-MOAP-1 were treated with or without diethylnitrosamine (DEN) (200 µM) for 12 h and subjected to Duolink proximity ligation assay using anti-p62 and anti-Myc antibodies. Red dots represent MOAP-1/p62 interaction. ",
    "molecules": "DEN, diethylnitrosamine"
  },
  {
    "caption": " B Alanine substitution of positively charged residues in the KYKKLR sequence of MOAP-1 abolishes its interaction with p62. Replacement of the positively charged residues in the KYKKLR sequence of MOAP-1 with alanine (referred to as the M3 mutant) disrupts MOAP-1/p62 interaction, whereas alanine substitution in the neighboring region of positively charged KRRR (M1 mutant) and negatively charged EEE (M2 mutant) has no effect. LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or the indicated alanine mutants together with plasmid encoding Flag-p62. The cells were then subjected to co-IP assay with anti-Myc antibody. ",
    "molecules": "alanine, Alanine"
  },
  {
    "caption": " A Increased recruitment of Keap1 to p62 bodies in the MOAP-1 deficient LO2 cells. WT and MOAP-1 KO LO2 cells were subjected to IF analysis with anti-Keap1 (green) and anti-p62 (red) antibodies. Insets in the upper panels are enlarged in the lower panels. Nuclei were counterstained with DAPI (in blue). Scale bar: 5 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " D Diminished Keap1/Nrf2 interaction in the absence of MOAP-1. WT and MOAP-1 KO LO2 cells were subjected to Duolink PLA using anti-Keap1 and anti-Nrf2 antibodies. Red dots represent Keap1/Nrf2 interaction. Nuclei were counterstained with DAPI (in blue). Scale bar: 10 µm. E Quantification of the Duolink signals depicted in (D). Duolink signals, which appear as red dots, were quantified by ImageJ analysis and normalized to the number of nuclei. ",
    "molecules": "DAPI"
  },
  {
    "caption": " A High level of p62 bodies in the liver of the MOAP-1 KO mice subjected to acute diethylnitrosamine (DEN) treatment. Eight-week-old male WT and MOAP-1 KO mice were injected with an acute dose of DEN (100 µg/g body weight). Mice were sacrificed at 48 hours post-injection. Livers were harvested for immunohistochemistry (IHC) analysis with anti-p62 antibody (in red). Nuclei were counterstained with DAPI (in blue). Insets in the upper panels are enlarged in the lower panels. Scale bar: 20 µm. ",
    "molecules": "DAPI, DEN, diethylnitrosamine"
  },
  {
    "caption": " B Loss of MOAP-1 results in higher levels of Nrf2 and its downstream target protein, HMOX-1, in liver upon acute DEN treatment. Lysates of the livers harvested from mice subjected to acute DEN treatment as described in (A) were prepared in RIPA lysis buffer containing 1% SDS and analyzed by Western blotting. Actin as loading control. ",
    "molecules": "DEN"
  },
  {
    "caption": " C MOAP-1 deficiency elevates levels of p62 protein in the detergent-insoluble fractions of the liver lysates from mice subjected to acute DEN treatment. Livers from mice injected as described in (A) were lysed in 1% Triton-X lysis buffer, separated into detergent-soluble (S) and insoluble (I) fractions and analyzed by Western blotting. ",
    "molecules": "DEN"
  },
  {
    "caption": " D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm. E Quantification of the p62 bodies in the WT and MOAP-1 KO tumors. (Left panel) number of p62 bodies per cell. n = 7 tumors from the livers of WT and MOAP-1 KO mice. (Right panel) size of p62 bodies. n = 456 and 1211 bodies detected in the WT and MOAP-1 KO tumors, respectively, from three independent samples. Error bar represents S.E.M. *P<0.05, ***P<0.001, student's t-test. ",
    "molecules": "DAPI, DEN"
  },
  {
    "caption": " F Increased levels of Nrf2 and its downstream target protein, HMOX-1, in the MOAP-1 deficient tumors. While p62 protein is upregulated in the tumors compared to the adjacent non-tumor tissues in both WT and MOAP-1 KO livers, no significant difference is observed in the p62 protein levels between the WT and MOAP-1 KO tumors. Lysates of the tumors and non-tumor parts of the livers from mice injected with DEN as described in (D) were prepared in RIPA lysis buffer containing 1% SDS and subjected to Western blotting. Actin as loading control. P, PBS injected control; N, non-tumor; T, tumor. ",
    "molecules": "DEN"
  },
  {
    "caption": " G Upregulation of mRNA levels of HMOX-1 and NQO1 in the MOAP-1 deficient tumors. Livers harvested from the mice injected with PBS or DEN as described in (D) were subjected to RT-PCR analysis. n = 3 WT and 3 MOAP-1 KO mice injected with the PBS control, and 8 WT and 9 MOAP-1 KO mice injected with DEN. Error bar represents S.E.M. **P<0.01, student's t-test. ",
    "molecules": "DEN"
  },
  {
    "caption": " H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm. I Measurements of the number, maximum diameter of the tumor nodules and total liver weight from WT and MOAP-1 KO mice (n=13 and 14 respectively) injected with DEN as described in (H). Error bar represents S.E.M. **P<0.01, ***P<0.001, student's t-test. ",
    "molecules": "DEN"
  },
  {
    "caption": " MOAP-1 deficiency promotes tumor initiation. measurements of the number, diameter of the largest nodules and total liver weight of the 4.5-month-old WT and MOAP-1 KO mice (n=18 and 23 respectively) injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. ",
    "molecules": "DEN"
  },
  {
    "caption": "Morphology of engrafted GFP-labeled cells in lung parenchyma by direct fluorescence. Blue color indicates nucleus DAPI staining. Scale bar, 200 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nucleus DAPI staining. Scale bar, 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) (Left) representative immunofluorescence  micrograph of postnatal hippocampal  sections immunostained  for GFAP  (green), BrdU  (red), NeuN  (white) and nuclear DNA  (blue) at 30 dpi of scrambled (Control) or GapmeR1 against Mili  (Mili  KD); (right graphs) percentages of NeuN+BrdU+ (white arrowheads in the images), or GFAP+BrdU+ (yellow arrowheads in the images) double-positive cells over total BrdU+ cells. Data information: data are expressed as mean ± SEM, n = F) biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. GCL, granular cell layer, SGZ, subgranular zone. The scale bars represent 100 µm F).",
    "molecules": "BrdU, DNA"
  },
  {
    "caption": "(C) Representative  fluorescence microscopy images (left) and quantification (right) of control or Mili  KD neuroblasts  48h after spontaneous differentiation, immunostained  with anti-BrdU  (white) and Ki67  (purple) antibodies. (Right) Percentage of BrdU+ and Ki67- cells over BrdU+ cells. Data information: data are expressed as mean ± SEM, n = C) biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm C)",
    "molecules": "BrdU"
  },
  {
    "caption": "(A) Cell count-based comparison of cellular proliferation   of T-ALL  cells after treatment with dexamethasone  (Dexa). Individual and mean values of three biological replicates ± SD analyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide  (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "Dexa, dexamethasone, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(B) Flow cytometric  analysis of  cell death by AnnexinV staining  in T-ALL cell lines upon treatment with indicated Dexa  concentrations. Cell death is represented by the number of AnnexinV  positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells. Mean values of n≥4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "Dexa"
  },
  {
    "caption": "(C) Expression  levels of human GRα   and LRH-1 mRNA in human T-ALL  cell lines were determined by probe-based real-time quantitative PCR, calculated as relative expression  compared to  beta-actin and used to determine the GR:LRH-1 ratio (right). Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM-C7  and GC-resistant cell lines were determined by one-way ANOVA.",
    "molecules": "GC"
  },
  {
    "caption": "(D) Immunoblots  of GR, BimEL, BimL  and BimS  and Tubulin  from  T-ALL  cells treated for 24 h with 20 µM 3d2  or 30 µM Dexa. Representative results from n=2 biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "3d2, Dexa, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(E) Immunoblots  of GR, PARP  (nuclear  loading control) and Tubulin  (cytoplasmatic  loading control) from cytoplasmic  (CP) and nucleoplasmic  (NP) lysates of  T-ALL  cells treated with Dexa. Note: Nuclear  extracts were more highly concentrated (approx. 6-fold) than cytosolic fractions. Representative results from two biological replicates are shown.",
    "molecules": "Dexa"
  },
  {
    "caption": "(F) GR  activity in human T-ALL  cells transfected with a control luciferase  reporter plasmid (pGL3) (F) GR  responsive (GRE) luciferase  reporter and treated with 3d2  and/or Dexa.  β-galactosidase  (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal  activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat  and CEM-C7  whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide  (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "3d2, Dexa, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(G) LRH-1  activity in human T-ALL cells transfected with a control luciferase  reporter plasmid (pGL3) and (G) LRH-1 responsive (5xRE) luciferase  reporter and treated with 3d2 β-galactosidase (bGal) was co-transfected as an internal transfection control.  Luciferase reporter activity was normalized to bGal  activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat  and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for  MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "3d2, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(H) Flow cytometry-based quantification of BiFC  in Jurkat   cells transiently co-transfected with  YFP1-LRH-1 and GR-YFP2 and treated for indicated  time periods  with Dexa. YFP+ cells are shown as a percentage (%) of viable  cells. Individual and mean values ± SD of three biological replicates analyzed by one-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0h) was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.",
    "molecules": "Dexa, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(A) Daily cell counting-based comparison of  cellular proliferation   of T-ALL cells after (A) treatment with 3d2  Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.",
    "molecules": "3d2, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(C) Cell cycle distribution of  T-ALL cells treated for 24 h with indicated concentrations of 3d2  as measured by propidium iodide flow cytometry. Representative results from n=3 biological replicates are shown. Stacked bars represent the mean of technical triplicates ± SD. Data information: ns = not significant, * p < 0.05, *** p < 0.001.",
    "molecules": "3d2, propidium iodide"
  },
  {
    "caption": "(D) Simultaneous  flow cytometric analysis of  cell cycle distribution by EdU/DAPI staining  and cell death by AnnexinV staining  in MOLT-4 cells upon treatment with 3d2. Dead cells are represented by the number of AnnexinV  positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells and cell cycle phase  distribution as % of viable, AnnexinV  negative (AnnexinV-) cells. Representative results from three biological replicates are shown. Bars represent the mean of technical triplicates ± SD and differences in AnnexinV+ cells were analyzed by one-way ANOVA. Data information: ns = not significant, * p < 0.05, *** p < 0.001.",
    "molecules": "3d2, EdU, DAPI"
  },
  {
    "caption": "(E) Flow cytometric  analysis of cell death by AnnexinV staining  in T-ALL cell lines upon treatment with indicated 3d2  concentrations. Mean values of n=4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.",
    "molecules": "3d2"
  },
  {
    "caption": "(F)  Flow cytometric  analysis of 3d2-induced cell death by AnnexinV staining  in wild-type (WT), Bcl-2 overexpressing (Bcl-2) and Z-VAD-FMK (zVAD)-treated  CEM-C1. Mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD analyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.",
    "molecules": "3d2, Z-VAD-FMK, zVAD"
  },
  {
    "caption": "(G) Immunoblots  of full-length and cleaved (cl.) PARP, P-p38,  c-Myc, Caspase 3 (Casp. 3), cl. Casp. 3, BimEL, BimL  and BimS  and Tubulin  from MOLT-4 cells treated with 40 µM 3d2  for indicated time periods. Representative results from two biological replicates are shown. Data information: For all displayed experiments  dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.",
    "molecules": "3d2, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(A) Immunoblots  of full-length and cleaved (cl.) PARP, BimEL, BimL  and  BimS  and Tubulin  from MOLT-4  cells treated for 24 h with 10 µM dexamethasone  (Dexa) and 3d2. Representative results from three biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.",
    "molecules": "3d2, Dexa, dexamethasone, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(B) Flow cytometric analysis of  cell death by AnnexinV staining  in MOLT-4 cells treated with 3d2, 30 µM Dexa  and/or 50 µM pan caspase  inhibitor Z-VAD-FMK (zVAD). Individual and mean values of technical triplicates of one representative out of n=3 biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control. Two-way ANOVA (B ns = not significant, ** p < 0.01, *** p < 0.001.",
    "molecules": "3d2, Dexa, dimethyl sulfoxide, DMSO, Z-VAD-FMK, zVAD"
  },
  {
    "caption": "(C) Flow cytometric  analysis of cell death  by AnnexinV staining  in control or Bim  knockout (Bim  KO) CEM-C7  cells treated with 3d2  and 30 µM Dexa. Mean values of technical triplicates of one representative of n=4 biological replicates ± SD are shown. Data information: Two-way ANOVA C, ns = not significant, ** p < 0.01, *** p < 0.001.",
    "molecules": "3d2, Dexa"
  },
  {
    "caption": "(E) Flow cytometric  analysis of  cell death by AnnexinV staining  in control or glucocorticoid receptor knockout (GR  KO) MOLT-4  cells treated with 3d2  and 30 µM Dexa. Mean values of n=3 biological replicates ± SD are shown. Data information: Two-way ANOVA E); ns = not significant, ** p < 0.01, *** p < 0.001.",
    "molecules": "3d2, Dexa"
  },
  {
    "caption": "(F/G) mRNA expression  levels of human GR (F) and Bim (G) in shcontrol and shLRH-1 CEM-C1 cells were determined by  real-time quantitative PCR after treatment with 1 µM Dexa  for indicated times. Fold induction relative to the untreated shcontrol sample (0h) was calculated after normalization to beta-actin. Individual values of technical triplicates of one representative of two biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.",
    "molecules": "Dexa, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(H) Immunoblots  of full-length and cleaved (cl.) PARP, GR  and phospho-AMPK  (P-AMPK), c-Myc, full-length and cl. Caspase 3, BimEL, BimL  and BimS  and Tubulin  from MOLT-4 cells treated with 40 µM 3d2  and/or 10 µM Dexa. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide  (DMSO; represented by Ø) was used as a solvent control.",
    "molecules": "3d2, Dexa, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "(A) Ex vivo response of human T cell acute lymphoblastic leukemia (T-ALL) patient-derived xenografts   (PDX), co-cultured on hTERT-immortalized primary  bone marrow MSCs, to treatment with indicated concentrations of 3d2  and/or dexamethasone  (Sample 1, 3 and 4: 1 nM; Sample 2: 0.1 nM). Cell viability  was quantified by fluorescent live cell staining using CyQuant combined with automated image analysis and calculated as percentage (%) of the  dimethyl sulfoxide treated control. Data information: All displayed experiments display one biological replicate.",
    "molecules": "3d2, dexamethasone, dimethyl sulfoxide"
  },
  {
    "caption": "(B) Synergy scores (Z-scores) between 3d2  and dexamethasone  of human T-ALL patient-derived xenografts  (PDX) were calculated from the viability  curves of PDX samples treated with increasing concentrations of 3d2  and/or dexamethasone  using SynergyFinder tool. Z-scores ≥ 0 indicate additivity or synergism and < 0 antagonism. Data information: All displayed experiments display one biological replicate.",
    "molecules": "3d2, dexamethasone"
  },
  {
    "caption": "(E) In vivo leukemia  progression of  human T-ALL PDX transplanted into NSG mice  treated for 3 weeks with vehicle (PBS  control), 40 mg/kg 3d2  and or 10 mg/kg Dexamethasone  starting 3 days post transplantation. Engraftment  was calculated as % of human  vs total (human+mouse) CD45  positive (CD45+) cells as assessed by  flow cytometric analysis of peripheral blood. Data information: All displayed experiments display one biological replicate.",
    "molecules": "3d2, Dexamethasone, PBS"
  },
  {
    "caption": "A. H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represents 30-50 counts from three biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA analysis. ",
    "molecules": "DAPI"
  },
  {
    "caption": "C. H2228 cells expressing endogenous EML4-ALK V3 and HEK293 transfected with YFP-EML4-ALK V3 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. D. Violin plots showing the number of EML4-ALK V3 cytoplasmic foci per cell. Data represents 30-50 counts from four biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V3 KD by one-way ANOVA analysis. ",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.",
    "molecules": "DAPI"
  },
  {
    "caption": "C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 hrs and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-Hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5-10 counts from 3 independent experiments (n=3). Error bar represents SD of three biological replicates. ****P< 0.0001 in comparison to YFP-EML4-ALK V3 WT DMSO by one-way ANOVA analysis. ",
    "molecules": "DMSO, 1,6-hexanediol, 1,6-Hexanediol, Hexanediol"
  },
  {
    "caption": "E. Representative western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 hrs. Cells were treated with either 5% or 10% 1,6-Hexanediol for 5 minutes. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n=2 experiments.",
    "molecules": "DMSO, 1,6-Hexanediol"
  },
  {
    "caption": "A, B, C, D. HEK293 cells were transfected with EML4-ALK V1 WT or V3 WT or Kinase Dead (KD) for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.",
    "molecules": "DAPI, DMSO"
  },
  {
    "caption": "B. HEK293 cells were transfected with EML4-ALK V1 deletion 12N blade for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.",
    "molecules": "DAPI, DMSO"
  },
  {
    "caption": "D. HEK293 cells were transfected with EML4-ALK V3 ∆JM 1058-1100 for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. E. Violin plot representing the number of cytoplasmic foci counted per cell from D. Data represent counts from at least 30 cells, n=3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA. ",
    "molecules": "DAPI, DMSO"
  },
  {
    "caption": "A-F HEK293 cells were transfected with A. YFP-EML4-ALK V3 K1150M, C. R1275Q or E. F1174L constructs and treated with ALK inhibitors or DMSO for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B, D, F. Violin plots representing the number of cytoplasmic foci counted per cell from A, C and E. Data represent counts from at least 30 cells, n=3 or 4. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.",
    "molecules": "DAPI, DMSO"
  },
  {
    "caption": "B. HEK293 cells transfected with YFP-EML4-GyrB and either untreated (DMSO) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP-EML4-GyrB. Data represent counts from >30 cells, n= 4. ****P< 0.0001 in comparison to DMSO by unpaired t test. ",
    "molecules": "Coumermycin A1, DAPI, DMSO"
  },
  {
    "caption": "(B)  Western blot analysis of the generated isogenic cell lines described in A after inducing GFP-TDP-43 expression for 48 h showing the tightness of the doxycycline  (DOX)-modulated expression system. Note also the different protein levels of the expressed variants. endTDP-43: endogenous TDP-43.",
    "molecules": "DOX, doxycycline"
  },
  {
    "caption": "(C) Representative images of widefield fluorescence microscopy of the isogenic cell lines depicted in B. GFP  brightness is adjusted in each condition for optimal visualization of GFP-TDP-43 localization. Original intensity values are represented in the right column using grayscale. Cell nuclei  are stained  with DAPI. Scale bar: 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) GFP-TDP-43 expression was induced with DOX  for 24 h before cycloheximide  (CHX) treatment for the indicated times  and western blot analysis. (E) Quantification of the GFP  signal from D. N=3 independent experiments. Two-way ANOVA with Tukey's multiple comparisons post hoc test.",
    "molecules": "CHX, cycloheximide, DOX"
  },
  {
    "caption": "(B) Representative maximum intensity Z-projections from confocal  fluorescence imaging of human neurons  transduced with TDP-43-HA  variants and immunolabeled  for the HA  tag and the neuron-specific marker MAP2. Scale bar: 10 µm. Cell nuclei  are stained  with DAPI",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) GFP-TDP-43 expression was induced with doxycycline (DOX) for 4 h before nucleocytoplasmic  fractionation and subsequent analysis of GFP-TDP-43 and endogenous TDP-43 (endTDP-43) levels by  western blot. T: total lysate, C: cytoplasmic  fraction, N: nuclear  fraction. (E) Quantification of the GFP  signal from D. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test. Cyt: cytoplasm, Nuc: nucleus.",
    "molecules": "DOX, doxycycline"
  },
  {
    "caption": "(F) HEK293  cells were treated with ActD  to inhibit transcription or subjected to protein-protein cross-linking with DSG  followed by nucleocytoplasmic  fractionation and  western blot  analysis. T:  total lysate, C: cytoplasmic  fraction, N: nuclear  fraction, H3: histone H3. (G) Quantification of endTDP-43 signal from F. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Dunnett's multiple comparisons post hoc test.",
    "molecules": "DSG, ActD"
  },
  {
    "caption": "(H) Representative images of widefield fluorescence microscopy of HEK293  cells treated with ActD  for 4 h and immunolabeled  for TDP-43. Scale bar: 20 µm. (I) Quantification of nucleocytoplasmic levels of endTDP-43 in the immunocytochemistry  images shown in H. N=3 independent experiments. Unpaired two-tailed t-test. Cell nuclei  are stained  with DAPI",
    "molecules": "ActD, DAPI"
  },
  {
    "caption": "(J) Representative images of  confocal fluorescence microscopy  of human neural  cultures treated with ActD  and ivermectin  (IVM) and immunolabeled  for TDP-43 and the neuron-specific marker MAP2. Scale bar: 20 µm. (K) Quantification of nucleocytoplasmic levels of endTDP-43 in the immunocytochemistry  images shown in J. Kruskal-Wallis test with Dunn's multiple comparisons post hoc test. N=23-48 fields corresponding to a total of 351-569 neurons  per condition. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Graph bars represent mean ± SD. Violin plots show mean and quartiles. Cell nuclei  are stained  with DAPI",
    "molecules": "ActD, DAPI, ivermectin, IVM"
  },
  {
    "caption": "(A) Representative maximum intensity Z-projections from confocal  fluorescence imaging (thickness of ~10 µm, in steps of 0.21 µm) of HEK293  cells after mock or  1,6-hexanediol (1,6-HD) treatment for 15 min stained  for endogenous TDP-43 (endTDP-43). Scale bar: 5 µm. (B) Quantification of three-dimensional (3D) reconstructions from the images of the conditions shown in A depicting the number of  nuclear  condensates per cell. N=14-23 cells. Unpaired two-tailed t-test.",
    "molecules": "1,6-HD, 1,6-hexanediol"
  },
  {
    "caption": "(C)  Fluorescence microscopy  images of 10 μM purified full-length TDP-43  and its oligomerization-deficient counterpart showing different abilities to undergo LLPS  and its disruption by 1,6-HD treatment for 10 min. Scale bar: 10 μm. (D) Quantification of the number of condensates in the conditions shown in C per 4000 µm2 field. N=10 independent experiments. Kruskal-Wallis test with Dunn's multiple comparisons post hoc test.",
    "molecules": "1,6-HD"
  },
  {
    "caption": "(H) GFP-TDP-43  expression was induced with DOX  for 4 h before crosslinking protein-protein interactions with DSG  and subsequent analysis by  western blot. *, ** and *** indicate GFP-TDP-43 monomers, dimers and trimers, respectively. o, oo, ooo and oooo indicate endTDP-43 monomers, dimers, trimers and tetramers. ­­­(I) Quantification of GFP-TDP-43 dimer/monomer ratio based on the GFP  signal from H. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test. * p<0.05, ** p<0.01, **** p<0.0001. Graph bars represent mean ± SD.",
    "molecules": "DSG, DOX"
  },
  {
    "caption": "(A) HEK293  cells were treated with actinomycin D  (ActD) for 4 h to inhibit transcription before treatment with the protein-protein cross-linker DSG  and  western blot  analysis. o, oo and oooo indicate endogenous TDP-43 (endTDP-43) monomers, dimers and trimers, respectively. (B) Quantification of the endTDP-43  signal from A. N=3 independent experiments. Paired two-tailed t-test.",
    "molecules": "DSG, ActD, actinomycin D"
  },
  {
    "caption": "(C) Representative image of confocal fluorescence microscopy of HEK293  cells and neurons  treated with 5 μg/ml ActD  for 4h. Scale bar: 5 µm. (D) Single-plane quantification of the number of nuclear  condensates per neuron  in the conditions described in C. N=25-26 cells. Mann-Whitney U test.",
    "molecules": "ActD"
  },
  {
    "caption": "(F) PLA  using a monoclonal anti-GFP  antibody reveals the localization   of GFP-TDP-43  dimers in the isogenic cell lines upon protein expression with doxycycline  (DOX) for 48 h. Note the absence of dimers in the oligomerization-deficient variants (6M and 6M&RRMm). Scale bar: 5 µm.",
    "molecules": "DOX, doxycycline"
  },
  {
    "caption": "(G) Tripartite GFP  complementation assay involving the co-transfection of a pair of N-terminally T10- and T11-tagged  TDP-43 constructs in HeLa  cells subjected to arsenite  stress for 30 min and incubated with recombinant  GFP1-9 after fixation to label T10- and T11-TDP-43 dimers. TriFC: trimolecular fluorescence complementation. Scale bar: 10 µm. (H) Quantification of the  trimolecular fluorescence complementation  (triFC) signal of GFP  in the TIA-1-marked SGs  from the images shown in G. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test.",
    "molecules": "arsenite"
  },
  {
    "caption": "(I) Expression of GFP-TDP-43 mutNLS variants was induced with doxycycline  (DOX) for 4 h before crosslinking protein-protein interactions with DSG  and subsequent analysis by western blot. * and ** indicate GFP-TDP-43 monomers and dimers, respectively. (J) Quantification of the GFP  signal from I. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test.",
    "molecules": "DSG, DOX, doxycycline"
  },
  {
    "caption": "(K) After expression of GFP-TDP-43 mutNLS variants for 48 h, the isogenic lines were treated with DSG  to cross-link protein-protein interactions  before performing nucleocytoplasmic fractionation and  western blot analysis. * and ** indicate GFP-TDP-43 monomers and dimers, respectively.",
    "molecules": "DSG"
  },
  {
    "caption": "(A) Representative  confocal microscopy images of the isogenic GFP-TDP-43  lines after 48 h of expression, treated with the proteasome  inhibitor MG132  for the last 24 h and stained  for lamin B  to mark the nuclear envelope. Note the different localization  of  TDP-43 inclusions in the oligomerization- (6M and 6M&RRMm) versus RNA binding-deficient (RRMm) variants. Scale bar: 20 µm (5 µm for inset). (B) Quantification of the localization  of GFP-TDP-43 inclusions after MG132  treatment for the different variants in the isogenic HEK293  lines as shown in A. Represented values are averages from N=3 independent experiments, with N=189-497 cells quantified per condition and replicate.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Representative maximum intensity Z-projections from confocal  fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of human neurons  transduced with TDP-43-HA  variants and treated overnight with the proteasome  inhibitor MG132. Scale bar: 10 µm. (D) Quantification of the differentially localized TDP-43-HA  inclusions in human neurons  as described in C. Represented values correspond to the quantification of N=85-97 neurons from two independent experiments. Nuclei  are stained  with DAPI  The neuron-specific marker MAP2  is shown in magenta or cyan",
    "molecules": "DAPI, MG132"
  },
  {
    "caption": "(E) Representative maximum intensity Z-projections from confocal  fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of the same experimental conditions as shown in A, with the addition of a DMSO  control for MG132  treatment. GFP-TDP-43-expressing cells were double immunolabeled  for TDP-43 phosphorylated at the S403/404 and S409/410 epitope. Note the absence of phospho-S409/410 immunopositivity in all conditions and positivity for phospho-S403/404 in cytoplasmic  inclusions and a subset of nuclear GFP-TDP-43  RRMm inclusions (appointed by arrowheads). Scale bar: 10 µm. Nuclei are stained  with DAPI",
    "molecules": "DAPI, DMSO, MG132"
  },
  {
    "caption": "(F-G) Representative maximum intensity Z-projections from confocal  fluorescence imaging (thickness of 4 µm, in steps of 1 µm) of the same experimental conditions as shown in C, with the addition of a DMSO  control. TDP-43-HA  expressing human neurons  were stained  for either phospho-S403/404 (F) or phospho-S409/410 (G). Scale bar: 10 µm. Note the re-localization  of the phospho-S403/404 signal from neuronal nuclei  to inclusions (F) and the presence of phospho-S409/410 signal in only a subset of aggregate-bearing neurons  (G). Nuclei  are stained  with DAPI  The neuron-specific marker MAP2  is shown in magenta or cyan",
    "molecules": "DAPI, DMSO"
  },
  {
    "caption": "(B) Representative fluorescent  confocal microscopy images of FRAP  experiments of the GFP-TDP-43 aggregates formed upon MG132  treatment as described in Figure 7A. FRAP  was performed in the areas highlighted in magenta. Numbers in images indicate the experimental time point in seconds ('') of MG132  treatment. Scale bar: 5 µm. Bottom panel: Measured GFP  intensity values are expressed as a fraction of the average pre-bleach  fluorescence levels.",
    "molecules": "MG132"
  },
  {
    "caption": "(D) Representative  confocal microscopy  images of the isogenic GFP-TDP-43 lines at the endpoint (24 h) of MG132  treatment of the experimental conditions described in Figure 7A and stained  for vimentin. Scale bar: 5 µm. Cell nuclei are visualized with DAPI",
    "molecules": "DAPI, MG132"
  },
  {
    "caption": "(E) Representative confocal microscopy images of the isogenic GFP-TDP-43  lines at the endpoint (24 h) of MG132  treatment of the experimental conditions described in Figure 7A and stained  for p62. Cell nuclei  are visualized with DAPI",
    "molecules": "DAPI, MG132"
  },
  {
    "caption": "(a) PC12 cells stably expressing A53T mutant α-synuclein were induced for 24 h, after which expression was switched off by removing doxycycline from the medium for the next 24 h. Transgene product clearance can be inferred by assessing levels at various times after switching off expression after an initial induction period, as expression levels decay when synthesis is reduced or stopped. Cells were treated with EHNA or AMP-PNP or left untreated during the switch-off period. Blots were probed with antibody to HA to detect the transgene and antibody to actin as a loading control.",
    "molecules": "AMP-PNP, doxycycline, EHNA"
  },
  {
    "caption": "(c) Immunofluorescence images of cells expressing Q74-GFP after 48 h of switch-off after treatment with (EHNA) or without (Control) EHNA.",
    "molecules": "EHNA"
  },
  {
    "caption": "(d) Quantification of aggregates and cell death in COS-7 cells transiently transfected with Q74-GFP either with (EHNA) or without (Control) EHNA (added during the last 33 h before fixation).",
    "molecules": "EHNA"
  },
  {
    "caption": "(e) Western-blot analysis of HeLa cells stably expressing GFP-UbG76V treated with lactacystin (Lac), EHNA or AMP-PNP or left untreated (Control) using antibody to GFP. ***P 0.0001; **P 0.001.",
    "molecules": "AMP-PNP, EHNA, Lac, lactacystin"
  },
  {
    "caption": "(c) Immunocytochemistry analysis (using antibody to HA) of COS-7 cells cotransfected for 48 h with HA-Q74 (red) and pEGFP or p50 (green). Panels on left are magnifications of boxed areas in second panels. The boxes are the same size. ***P 0.0001; **P 0.001. Examples of fragmented nuclei in cells overexpressing p50 and nuclei in cells overexpressing p50 but treated with Z-VAD-fmk (broad-spectrum caspase inhibitor) are shown in Supplementary Figure 4 online.",
    "molecules": "Z-VAD-fmk"
  },
  {
    "caption": "(b) Western-blot analysis of COS-7 cells treated with EHNA or left untreated using antibodies to LC3 and to actin. Quantification of the band intensity from two independent experiments is shown.",
    "molecules": "EHNA"
  },
  {
    "caption": "Relative gene expression of selected DEGs measured by qRT-PCR in Rv compared with Rv treated with CHP (g) and Rv as compared with Rv∆aosR under oxidative conditions (h). Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates.",
    "molecules": "CHP, 16s rRNA"
  },
  {
    "caption": "WCLs of Rv, Rv∆aosR, and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane, and probed with indicated antibodies.",
    "molecules": "nitrocellulose membrane, CHP"
  },
  {
    "caption": "The promoter of sigA and mec was fused to luciferase, and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=6)",
    "molecules": "CHP"
  },
  {
    "caption": "Rv::pN and Rv::pN-F-sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-AosR. 50 µg WCLs were probed with α-FLAG,α-AosR, and α-SigA as controls.",
    "molecules": "CHP"
  },
  {
    "caption": "Expression of CysO, CysM, AosR and GroEL1 (control) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h.",
    "molecules": "CHP"
  },
  {
    "caption": "5-7.5 x 104 end labelled 100 bp double-stranded oligonucleotide was incubated with no protein (lanes 1 and 6) or 4 μΜ BSA (lanes 2 and 7) or 2 μΜ His-AosR + 2 μΜ BSA (lanes 3 and 8) or , 2 μΜ His-SigH + 2 μΜ BSA (lanes 4 and 9) or 2 μΜ His-AosR + 2 μΜ His-SigH (lanes 5 and 10). The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols, respectively, and resolved on 8% PAGE gel. Protein-DNA complexes (bound) and free probe are indicated by arrows. To confirm the specificity of DNA-protein interaction, EMSA reaction was performed in the presence of 5, 10, 25, 50, and 100 molar excess of unlabelled 100 bp double-stranded oligonucleotide.",
    "molecules": "diamide, DTT, double-stranded oligonucleotide, thiols"
  },
  {
    "caption": "Msm::FLAG-SigH, Msm::FLAG-SigH-AosR-HA and Msm::FLAG-SigH-AosRAxxxA-HA were either subjected to none or 50 µM of CHP for 3 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-HA. 50 µg WCLs were probed with α-FLAG, α-HA and α-SigA as controls.",
    "molecules": "CHP"
  },
  {
    "caption": "Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified. Data presented is representative of one of two biological replicates, each performed in triplicates (n=3). Y-axis represents µM thiol /µg of WCL (mean ± SD). Metabolite levels of reduced mycothiol, MSH ergothioneine, ERG were measured in Rv, Rv∆aosR, and Rv∆aosR::aosR in normal and CHP stressed cells using LC-MRM MS/MS analysis. The absolute values obtained under normal conditions were normalized to 100% for each strain and the relative values were calculated for samples processed from CHP treated cells. The data indicates relative levels of metabolites (mean percent ± SD) of four replicates (n=4).",
    "molecules": "CHP, ERG, ergothioneine, MSH, mycothiol, thiol"
  },
  {
    "caption": "qRT-PCR measurements for the log2 fold induction (mean ±SD, n=3) for indicated genes in Rv∆aosR and Rv∆aosR treated with 1mM L-cysteine as compared with Rv. All strains were treated with 50 μM CHP. Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates.",
    "molecules": "CHP, L-cysteine"
  },
  {
    "caption": "(A) Summary of HMCES recovery upon TAP-MS analyses performed with 293T cells stably expressing SFB-PCNA. Cells were treated with CPT: camptothecin (1 μg/ml for 6 h); HU: hydroxyurea (2 mM for 16 h), UV: ultraviolet light irradiation (10 mJ/cm2 harvested after 6 h post-irradiation) or Thymidine (100 mM for 6 h). Recovered peptide-spectrum matches (PSMs) for PCNA and HMCES are indicated for each condition.",
    "molecules": "camptothecin, CPT, HU, hydroxyurea, Thymidine"
  },
  {
    "caption": "(E) aniPOND-Western blotting analysis revealed the presence of HMCES on replication fork. Cells were pulsed with EdU for 10 mins, and click-reaction was performed. In thymidine chase samples, cells were incubated with thymidine for another 30 mins prior to click-reaction.",
    "molecules": "EdU, thymidine"
  },
  {
    "caption": "(F) Analysis of HMCES level throughout the cell cycle. Cells were synchronized by double thymidine block and released for the indicated time points. CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases.",
    "molecules": "thymidine"
  },
  {
    "caption": "(D) Colonogenic survival assay of 293A wild-type control and HMCES KO cells treated with various concentrations of hydroxyurea (HU) was shown. Seeding density with respect to control was indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate.",
    "molecules": "HU, hydroxyurea"
  },
  {
    "caption": "(E) Quantification showing survival fraction of wild-type control (WT) and HMCES KO cells upon HU treatment. Data are presented as mean±s.e.m (n=3, each performed in duplicates) and student's t-test were performed for statistical analysis (ns, not significant; *, p<0.05).",
    "molecules": "HU"
  },
  {
    "caption": "(G, H) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony-forming assay upon exposure to H2O2. Experiments were repeated three independent times, each time performed in duplicates and data are presented as mean±s.e.m. Statistical significance for the corresponding experiment was analyzed by student's t-test (*, p<0.05).",
    "molecules": "H2O2"
  },
  {
    "caption": "(I) Bar graph showing sensitivity of WT, HMCES KO and stably reconstituted WT cells towards H2O2 treatment. Experiment was repeated two independent times with technical repeats.",
    "molecules": "H2O2"
  },
  {
    "caption": "(A, B) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild-type (WT) 293A cells and HMCES knockout (HMCES KO) cells (A) and quantification of results were shown (B). Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug-dose response curve. Seeding density with respect to control is indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate. (B) Data are presented as mean±s.e.m. and student's t-test was performed for statistical analysis (****, p<0.0001).",
    "molecules": "pemetrexed"
  },
  {
    "caption": "(E) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III (APEi). Results are presented from two independent experiments and data are presented as mean±s.e.m.",
    "molecules": "AP Endonuclease inhibitor III"
  },
  {
    "caption": "(F) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi. Data are presented as mean±s.e.m (n=2 each with technical repeats).",
    "molecules": "pemetrexed"
  },
  {
    "caption": "G-K Increase in phosphatidylserine (PS) exposure in the ONL during photoreceptor degeneration. (G) At P18, PS immunopositivity is near absent in the ONL, but increased significantly in ONLsomata at P21-23, before decreasing at P30. (H) Quantitation of PS exposure by image analysis (by fractional area of PS immunopositivity within the ONL (top), and the mean intensity of PS staining in the ONL (bottom)) demonstrated a transient increase at P22. (I) Co-immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods (inset shows at high magnification the co-labeling of PS and rhodopsin in multiple rodsomata). (J) Conversely, immunolabeling of cones with conearrestin demonstrates the sparse co-localization of PS in cones (inset shows close juxtaposition but no colocalization of PS and arrestin labeling). (K) Scoring of rhodopsin+rods and arrestin+cones for PS co-labeling demonstrates that a large majority of rods, but only a small minority of cones, showed PS exposure (two-sided unpaired t-test, n = 3 animals at P22). Scale bars = 20 μm.",
    "molecules": "phosphatidylserine, PS"
  },
  {
    "caption": "Retinalmicroglia were depleted in rd10/CreDTAmice by the oral administration of tamoxifen (in corn oil) to activate microglia-specific Cre-mediated recombination and diphtheria toxin expression; control CreDTA littermates were administered corn oil without tamoxifen.A-D Depletion of retinalmicroglia in the rd10 retina. Representative retinal section from a P28 control animal demonstrates Iba1+microglia in the retina, including those infiltrating the ONL (A), while a tamoxifen-administered littermate (B) was substantially depleted of retinalmicroglia. Scale bar, 40 μm. Microglial cell counts in the entire retina (C) and in the ONL only (D) confirmed efficient depletion of infiltrating microglia following tamoxifen administration (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).E-J Effect of microglial depletion on retinal degeneration at P28-29. ONL atrophy and thinning in control animals (E) was significantly more advanced relative to microglia-depleted littermates (F). Scale bar, 40 μm. Quantification of mean ONL thickness (G) and mean number of layers of ONLnuclei (H) at P28-29 demonstrate significantly greater ONL preservation in depleted retinas; the degree of ONL preservation correlated with the extent of microglia depletion (I). (J) The mean density of TUNEL+nuclei in the ONL was not significantly decreased in depleted vs. control animals (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).K-N Continuation of microglial depletion until P37-39 resulted in the persistence of morphological rescue (K-M), with a significant reduction in TUNEL+nuclei density (N) (n = 6 control and nine depleted animals from two litters, two-sided unpaired t-test).O-R Similar rescue effects as in (K-N) remained apparent when depletion was sustained until P50 (O-R), a time when rod degeneration in the rd10 model is relatively complete (n = 9 control and 10 depleted animals from three litters, two-sided unpaired t-test).S, T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark- (S) and light-adapted (T) responses in ERG testing in depleted animals (green lines) relative to control animals (black lines), in both a- and b-wave amplitudes across multiple flash intensities (n = 9 control and nine depleted animals, *P < 0.05 in one-way ANOVA with Sidak's multiple comparison test).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Depletion of infiltrating microglia in the rd10 retina decreases IL-1β levels. Cytokine levels in rd10/CreDTA mouse retinas were assayed following tamoxifen-induced microglial depletion (from P21 to P37-50, green bars; n = 9 animals) and compared with their untreated littermate controls (white bars, n = 8 animals; values normalized to control animals in the same litter). IL-1β protein levels were significantly lowered following microglial depletion, but IL-6, CCL2, or TNFα were not significantly changed.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Angiogenic sprouting of primary human umbilical vein endothelial cell (HUVEC) spheroids embedded in a collagen-based matrix was induced by the application of EGFL7 (E7), fibronectin (Fn), or a combination of both (upper row). Co-application of an integrin α5β1 blocking antibody (anti-α5β1) reduced sprouting in all cases but control (lower row). Scale bar represents 100 µm.",
    "molecules": "collagen"
  },
  {
    "caption": "Treatment of glioma-bearing NOD SCID mice with temozolomide (TMD) as a chemotherapeutic agent and a combination of anti-EGFL7 and anti-VEGF antibody increased the median survival time (Combo; 87 d; n = 5; log-rank test) as compared to anti-VEGF alone (80 d; n = 5; log-rank test) or isotype-treated controls (69 d; n = 5; log-rank test). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.",
    "molecules": "temozolomide, TMD"
  },
  {
    "caption": "Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A). Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.",
    "molecules": "formic-acid, SDS"
  },
  {
    "caption": "Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.",
    "molecules": "Proteostat, DAPI"
  },
  {
    "caption": "Bar plots depicting the quantification of chymotrypsin-like (CT-L) activity from native gels calculated for doubly capped (30S) or singly capped (26S) proteasomes. n >=5 per sample group; error bars indicate standard error of the mean. * p < 0.05, ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of activity in young samples (5 wph) was set to 100%.",
    "molecules": "chymotrypsin-like, CT-L"
  },
  {
    "caption": "Proteasome inhibition induces proteasome activators (PSME4), assembly factors (POMP) ubiquitin ligases (UBE3C), mediators of autophagosome formation (ATG2B), heat shock proteins and chaperones in killifish brain. Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples (DMSO) set to 1, n=10 per sample group. Adj. p < 0.05 for all the displayed proteins. Error bars indicate mean +/-SD.",
    "molecules": "DMSO, Proteasome, proteasome"
  },
  {
    "caption": "Bortezomib treatment affects the stoichiometry of a subset of protein complexes. The number of affected proteins (adj. p < 0.25) for each protein complex is indicated. Only protein complexes that had at least two members affected are shown.",
    "molecules": "Bortezomib"
  },
  {
    "caption": "Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment. Relative protein abundances (normalized to the mean of the protein complex to which they belong) are shown. The mean value of vehicle control samples (DMSO) was set to 1. n=10 per sample group. Adj. p < 0.25 for all the displayed proteins. Error bars indicate mean +/-SD.",
    "molecules": "Bortezomib, DMSO"
  },
  {
    "caption": "(A m6A dot blot of the P493-6 cells treated with Tet for 0h or 72h (A) equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments.",
    "molecules": "methylene blue, m6A, Tet"
  },
  {
    "caption": "B) m6A dot blot of Raji cells that expressed NTC or MYC shRNAs (B); equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments.",
    "molecules": "methylene blue, m6A"
  },
  {
    "caption": "(C) Quantification of m6A abundance by HPLC-MS. *** P<0.001 as compared to corresponding high MYC group (mean ± SD, n = 3 biological replicates, Student's t‐test).",
    "molecules": "m6A"
  },
  {
    "caption": "(D) Metagene profiles of m6A peak distribution along a normalized transcript composed of three rescaled non-overlapping segments: 5′UTR, CDS, and 3′UTR in P493-6 cells treated with Tet for 0h or 72h.",
    "molecules": "m6A, Tet"
  },
  {
    "caption": "(F) IGV graph showing location of m6A peaks on representative genes.",
    "molecules": "m6A"
  },
  {
    "caption": "(G) m6A-RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding high MYC group, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).",
    "molecules": "m6A, Tet"
  },
  {
    "caption": "(H Western blot analysis for protein levels in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (H) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.",
    "molecules": "Tet"
  },
  {
    "caption": "(A Western blot analysis for protein levels of m6A methyltransferases (METTL3 and METTL14) and demethylases (ALKBH5 and FTO) in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (A) β-actin serves as loading controls. Data are representative of at least three independent experiments.",
    "molecules": "m6A, Tet"
  },
  {
    "caption": "B) Western blot analysis for protein levels of m6A methyltransferases (METTL3 and METTL14) and demethylases (ALKBH5 and FTO) in Raji cells that expressed NTC or MYC shRNAs (B). β-actin serves as loading controls. Data are representative of at least three independent experiments.",
    "molecules": "m6A"
  },
  {
    "caption": "(C) RT-qPCR analysis of the mRNA levels of methyltransferases and demethylases in P493-6 cells treated with Tet for 0h or 24h. Data were presented as mean (±SD), n = 3 biological replicates. * P<0.05 relative to corresponding -Tet group (Student's t‐test).",
    "molecules": "Tet"
  },
  {
    "caption": "(D) IGV graph showing location of MYC binding peaks on ALKBH5 and FTO from published ChIP-seq datasets (Walz et al, 2014; Data ref: Eilers M, 2014). In this dataset, high levels of MYC were induced with 1µg/ml doxycycline for 30h and ChIPed by MYC antibody. Red triangle indicates the E-box.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(E) RIP assay, using ALKBH5, FTO or IgG antibody to detect the binding to MRGs (SPI1 and PHF12) in P493-6 cells treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding IgG group (mean ± SD, n = 3 biological replicates, Student's t‐test).",
    "molecules": "Tet"
  },
  {
    "caption": "(F Western blot analysis for protein levels in P493-6 cells that overexpressed empty vector (EV), ALKBH5, or FTO and were then treated with Tet or not (F) HPRT1 and β-actin served as negative and loading controls, respectively. Data are representative of at least three independent experiments.",
    "molecules": "Tet"
  },
  {
    "caption": "(H) m6A RIP assay in P493-6 cells that overexpressed EV, ALKBH5, or FTO and were then treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups. ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).",
    "molecules": "m6A, Tet"
  },
  {
    "caption": "(I, Western blot analysis for protein levels in P493-6 cells that expressed NTC or ALKBH5 shRNAs and were then treated with Tet or not (I) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.",
    "molecules": "Tet"
  },
  {
    "caption": "(K) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups (mean ± SD, n = 3 biological replicates, Student's t‐test).",
    "molecules": "m6A"
  },
  {
    "caption": "(A) RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 relative to indicating groups (mean ± SD, n = 3 biological replicates, Student's t‐test).",
    "molecules": "Tet"
  },
  {
    "caption": "(B, Western blot analysis for protein levels in P493-6 cells that expressed NTC or YTHDF3 shRNAs and were then treated with Tet or not (B) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.",
    "molecules": "Tet"
  },
  {
    "caption": "(F) Click-iT AHA (L-azidohomoalaine) experiments were performed using IgG, anti-SPI1 or anti-PHF12 antibody. P493-6 cells expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug/mL AHA. The translated proteins were detected by Western blot. Arrow indicates translated MRGs.",
    "molecules": "AHA"
  },
  {
    "caption": "(I) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).",
    "molecules": "m6A"
  },
  {
    "caption": "(A) Trypan blue counting was used to analyze growth curves for P493-6 cells that expressed NTC or SPI1 shRNA or PHF12 shRNA. ** P<0.01 as compared between indicated groups. (mean ± SD, n = 4 biological replicates, Student's t‐test)",
    "molecules": "Trypan blue"
  },
  {
    "caption": "(B, C) Trypan blue counting was used to analyze growth curves for P493-6 cells overexpressing ALKBH5 and further infected with viruses expressing SPI1 (B) or PHF12 (C). ** P<0.01 as compared between indicated groups (mean ± SD, n = 4 biological replicates, Student's t‐test).",
    "molecules": "Trypan blue"
  },
  {
    "caption": "B. Representative image of  primary microglia simultaneously treated with Aβ  oligomer-bound SN  (oAβ-SN; in pHrodo red (magenta)) and control SN  (Ctrl-SN; in pHrodo deep red (cyan)). Scale bar, 50 μm.",
    "molecules": "pHrodo deep red, pHrodo red, Aβ"
  },
  {
    "caption": "F. Time-lapse images of primary Homer1-eGFP neurons  (green) treated with 50 nM oAβ versus vehicle control. Yellow arrows indicate increasing  PSVue550 (magenta) signal on dendritic spines  with Aβ  oligomer treatment over 45 min. Scale bar, 2 μm.",
    "molecules": "Aβ"
  },
  {
    "caption": "G. (Top panel) Representative traces of spontaneous GCaMP7  signals at 48 h post-treatment with Aβ  oligomer in dendritic spines from neuron-only culture (cyan), neuron-microglia  co-culture (magenta), and neuron-microglia  co-culture treated with AnnxV  (yellow). (Bottom panel) number of calcium  transients per minute before (0 h), at 24 h and 48 h post-treatment of Aβ  oligomer. ∼10 spines  per neuron, ∼10 neurons  per experiment, from 3 independent experiments. Data information: Kruskal-Wallis test followed by Dunn's multiple comparisons test (G) P-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ",
    "molecules": "Aβ, calcium"
  },
  {
    "caption": "A. Trem2  CV and Trem2  R47H KI primary microglia treated simultaneously with oAβ-bound synaptosomes  (SN) conjugated with pHrodo red  (magenta) and control SN, conjugated with pHrodo deep red (cyan). Scale bar, 50 μm. B. pHrodo  fluorescence with time shown as AUC at 3 h. AUC of oAβ-SN  is higher compared to Ctrl-SN  in Trem2  CV but not in Trem2  R47H KI microglia. ∼40 microglia  per ROI, 2 ROIs per well, 2-3 wells per experiment, n=3 independent experiments. C. Quantification of the percent of C1qa  area covered normalized to WT showing an increase in the  NL-F but not NL-F KI; Trem2  R47H KI. N=3-4 animals per genotype. Data information: Data shown as mean ± SEM. Each shaded point represents 1 ROI, and each open point represents the mean of each independent experiment. Two-way ANOVA followed by Bonferroni's post-hoc test. P-values shown as ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001.",
    "molecules": "pHrodo, pHrodo deep red, pHrodo red, Aβ"
  },
  {
    "caption": "D. Number of spontaneous calcium  transients per minute at 48 h post-treatment of Aβ  oligomer in neuron-only culture (cyan), neuron- Trem2  CV microglia  co-culture (magenta) or neuron- Trem2  R47H KI microglia  co-culture (purple). ∼10 spines  per neuron, ∼10 neurons  per experiment, from 3-4 independent experiments. Data information: Data shown as mean ± SEM. Each shaded point represents 1 ROI, and each open point represents the mean (or median for GCaMP  studies) of each independent experiment. Central bands of the violin plot (D) represent median and quartiles. Kruskal-Wallis test followed by Dunn's test (D), P-values shown as ns P>0.05; *P<0.05; **P<0.01, ****P<0.0001.",
    "molecules": "Aβ, calcium"
  },
  {
    "caption": "(A) Cells were cultured in the presence or absence of BODIPY-αGalCer at 4oC or 37oC as indicated. Flow cytometry profiles and percentages of BODIPY-αGalCer+ DC or ILC3 from the indicated tissues incubated at 4oC (white bars) or 37oC (grey bars) are shown (n=3).",
    "molecules": "αGalCer"
  },
  {
    "caption": "(B) WT mice were intravenously injected with BODIPY-αGalCer or PBS (control); lipid uptake was analysed by flow-cytometry in the depicted populations from the spleen 16 h after lipid injection (n=3). Numbers indicate percentage of cells in the depicted gates.",
    "molecules": "αGalCer"
  },
  {
    "caption": "(C) IL-2 secretion by DN32.D3 cells co-cultured with ILC3 sort-purified from mLN or spleen as indicated, pulsed (+) or not (-) with αGalCer and with or without antiCD1d-blocking antibody.",
    "molecules": "αGalCer"
  },
  {
    "caption": "(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (E) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort-purified from spleen, mLN or SI-LP of mice injected with αGalCer-loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort-purified from mice injected with control ILC3s (n=3-5).",
    "molecules": "αGalCer"
  },
  {
    "caption": "(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (F) Intracellular cytokine staining (left) and percentage of cytokine+ cells (right) in spleniciNKT cells from ILC3-recipient mice (n=3). Numbers indicate percentage of cells in the depicted gates. **p<0.01, two-tailed unpaired t-test.",
    "molecules": "αGalCer"
  },
  {
    "caption": "(H) WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenicILC3s was assessed by flow-cytometry as CD69 up-regulation at 6 h or 16 h after injection. Grey filled profile= ILC3 from αGalCer-injected mice; Empty profile= ILC3 from PBS-injected mice; Dotted line= CD45-cells. Right graph, percentage of CD69+ILC3s 6 h after injection of αGalCer (grey) or PBS (white). (n=3) *p<0.05 two-tailed unpaired t-test.",
    "molecules": "αGalCer"
  },
  {
    "caption": "(I-J) Fold change in mRNA expression for the indicated cytokines in ILC3s sort-purified from spleen (I) or SI-LP (J) 6 h after intravenous (I) or oral (J) αGalcer administration. Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort-purified from PBS-injected mice (n=3). *p<0.05, **p<0.01 two-tailed unpaired t-test.",
    "molecules": "αGalcer"
  },
  {
    "caption": "F Clonogenic growth assay of indicated HPAF-II cells left untreated or in the presence of 150nM LGK974 for 10 days; representative of three independent replicates.",
    "molecules": "LGK974"
  },
  {
    "caption": "G Immunoblot of cytoplasmic βcatenin expression from lysates of HPAF-II cells from indicated genotype, treated with vehicle or 100nM LGK974 for 48h; representative of three biological replicates.",
    "molecules": "LGK974"
  },
  {
    "caption": "B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF-II wild-type and FBXW7-/- cells , representative blot of 3 independent replicates. C Quantification of cycloheximide chase in B, mean ± SEM of three independent replicates.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "G Immunoblot of lysates following cycloheximide treatment of HEK293T cells expressing wild-type or degron-mutated CCNL1, representative blot of 3 independent replicates. H Quantification of cycloheximide chase in G, mean ± SEM of three independent replicates.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "B Immunoblot of HPAF-II wild-type and FBXW7-/- released from overnight nocodazole treatment at indicated time points,",
    "molecules": "nocodazole"
  },
  {
    "caption": "D Representative images of GFP-tubulin labeled wild-type, FBXW7-/-, and CCNL1OE HPAF-II cells monitoring cellular progression through cytokinesis following arrest in prometaphase using monastrol (150μM for 18 hours). E Quantification of cytokinetic timing, n=20, 25, 26 (wild-type, FBXW7-/, CCNL1OE ) pooled from three independent experiments F Immunoblot of wild-type, FBXW7-/- and CCNL1OE HPAF-II cell lines demonstrating varying levels of CCNL1.",
    "molecules": "monastrol"
  },
  {
    "caption": "A Clonogenic growth assays for HPAF-II wild-type and FBXW7-/- polyclonal, and FBXW7-/- clones in presence of various OTS964 doses for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595 and B Clonogenic growth assays for HPAF-II wild-type and CCNL1OE polyclonal, and CCNL1OE clones in presence of various doses of OTS964 for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595",
    "molecules": "crystal violet, OTS964"
  },
  {
    "caption": "C Clonogenic growth assay of C33A, Caski, and SiHa cells in presence of various doses of OTS964 for 14 days. Representative images of 3 independent replicates, quantified by crystal violet absorbance at A595",
    "molecules": "crystal violet, OTS964"
  },
  {
    "caption": "B Average seizure score of KA-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h. * p < 0.05 compared to WT mice (two-way Repeated Measures ANOVA; interaction effect of time and genotype; F1,666 = 10.05, p = 0.0016). KA-treated WT mice (n=16), KA-treated Fxr2 KO mice (n=12). Data are presented as mean ± Standard Error of the Mean (SEM). D Average seizure score of pilocarpine-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h (two-way Repeated Measures ANOVA). Pilocarpine-treated WT mice (n=6), pilocarpine-treated Fxr2 KO mice (n=6). Data are presented as mean ± SEM.",
    "molecules": "KA, pilocarpine, Pilocarpine"
  },
  {
    "caption": "A Bar plot showing phospho-ERK1/2 over total ERK1/2 levels in vehicle- and KA-treated WT (closed and open black circles, vehicle-treated WT n=6; KA-treated WT n=6) and Fxr2 KO mice (closed and open magenta circles, vehicle-treated Fxr2 KO n=5; KA-treated Fxr2 KO n=6). *** p < 0.001 comparison between vehicle- and KA-treated WT (two-way ANOVA: interaction effect between genotype and treatment: F1,19 = 24.66, p < 0.001). Data are presented as mean ± SEM. B Phospho-ERK1/2 levels in vehicle- and pilocarpine-treated WT (n=4-6) and Fxr2 KO mice (n=4-6). Both genotypes responded with long-lasting seizures upon pilocarpine administration * p < 0.05 (two-way ANOVA: main effect for treatment: F1,16 = 10.98, p < 0.01). Data are presented as mean ± SEM. ",
    "molecules": "KA, pilocarpine"
  },
  {
    "caption": "A-C.  Immunodetection of BES1-CFP from heat-treated  plants. Plants pre-grown at 21°C were (A) exposed for 60 minutes to the indicated temperatures, (B) to 45°C for the indicated period of  time, or (C) to 45°C and, following sampling after 30 minutes (lane 2) and 60 minutes (lane 3), returned to 21°C for 3 hours (lane 4). CBB, Coomassie Brilliant Blue stained  gel.",
    "molecules": "CBB"
  },
  {
    "caption": "B.  Immunodetection of BES1-CFP from heat-stressed plants, treated with epiBL or BRZ. 35S:BES1-CFP plants were treated for 24 hours with  DMSO (control), 1 μM 24-epiBL or 5 μM  BRZ  and then heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes; lane 4: 45°C for 60 minutes and recovery at 21°C for 180 minutes.",
    "molecules": "24-epiBL, epiBL, BRZ, DMSO"
  },
  {
    "caption": "A.  Immunodetection of BES1-CFP from  heat-stressed plants, treated with ABA or Fluridone (Flu). 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 5 μM ABA or 10 μM  Fluridone and heat-stressed  as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.",
    "molecules": "ABA, DMSO, Fluridone"
  },
  {
    "caption": "C. Immunodetection of native  BES1 from heat-stressed and epiBL-treated abitM plants. abitM and wild-type plants were grown on ½ MS medium either containing  DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.",
    "molecules": "epiBL, DMSO"
  },
  {
    "caption": "A, B. Constitutive and BR-responsive  growth  of bes1-DxhsfA1qM seedlings. Hypocotyl  elongation of 7-day-old dark-grown (A) and light-grown (B)  seedlings on medium containing either 1 μM  epi-BL, 1 μM  BRZ or DMSO as a control (c). Left: Mean and SD of 30 plants. Significant difference at P≤0.05 of results is indicated with different letters and was determined with a Student's t-test. Right: photos of representative plants.",
    "molecules": "epi-BL, BR, BRZ, DMSO"
  },
  {
    "caption": "C. Expression of BR-regulated genes in bes1-DxhsfA1qM, as compared to its parents and respective wild-types analyzed by  qPCRs. Data show the mean ±SD. n=3 biological repeats, each measured in 3 technical replicates, normalized to  UBC. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.",
    "molecules": "BR"
  },
  {
    "caption": "B Segregation defects upon inhibition of transcription in luciferase and lds RNAi. Graph depicts mean ± s.e.m. of the frequencies of defects scored in 11 (luciferase RNAi, H2O and lds RNAi, α-amanitin) or 8 (lds RNAi, H2O and Luc RNAi, α-amanitin) independent embryos; a total of ~800 nuclei were scored per condition.",
    "molecules": "α-amanitin, H2O"
  },
  {
    "caption": "B, C Loading of EGFP-Top (B) and Lds-EGFP (C) (labelled in green) on mitotic chromatin of metaphase arrested embryos (UbcH10C114S injection), upon subsequent injection of DMSO/ICRF-193; DNA is marked with H2AvD-mRFP1 (magenta); times (min:sec) are relative to time of injection. Graphs depict integrated intensities normalized to the first time point, represented as mean (dots) ± SD (grey area); n=54 (Lds+ ICRF-193, Lds+DMSO, Top2 + ICRF-193) or 47 (Top2 + DMSO), derived from 8-9 independent embryos per experimental condition. Data information: Scale bar is 10 µm and applies to all images.",
    "molecules": "DMSO, ICRF-193"
  },
  {
    "caption": "E-F FRAP analysis with and without ICRF-193 treatment for EGFP-Top2 (E) and Lds-EGFP (F). Metaphase-arrested embryos were microinjected with DMSO/ICRF-193 and an entire metaphase plate was bleached 10 min after. Graphs depict recovery of fluorescence signal over time. Dots represent average and grey areas SD; Top2: n= 14 (DMSO) and 18 (ICRF-193) metaphases; Lds: n= 11 (DMSO) and 9 (ICRF-193) metaphases, derived from 3-7 independent embryos Data information: Scale bar is 10 µm and applies to all images.",
    "molecules": "DMSO, ICRF-193"
  },
  {
    "caption": "A, Representative images of mitotic nuclei in prophase (1 min before NEBD) and metaphase (1 min before anaphase onset), upon RNAi for luciferase/lds, microinjected with water (A) Scale bars are 5 µm and refer to all images.",
    "molecules": "water"
  },
  {
    "caption": "B Representative images of mitotic nuclei in prophase (1 min before NEBD) and metaphase (1 min before anaphase onset), upon RNAi for luciferase/lds ICRF-193 (B). Scale bars are 5 µm and refer to all images.",
    "molecules": "ICRF-193"
  },
  {
    "caption": "(C) Quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry trafficking into cortical neurons obtained from WT (n= 753 vesicles/ 81 axons), HTTSA (n= 812 vesicles/ 81 axons), or HTTSD (n= 787 vesicles/ 82 axons) homozygous knock-in mice in which S421 of HTT was replaced by an alanine (HTTS421A/S421A or HTTSA), mimicking the absence of phosphorylation, or by an aspartic acid (HTTS421D/S421D or HTTSD), mimicking constitutive phosphorylation (3 independent experiments; one-way ANOVA test with Tukey's multiple comparisons). Data information: , ***p < 0.001, ****p < 0.0001, ns = not significant. Data are presented as the means ± SEM.",
    "molecules": "alanine, aspartic acid"
  },
  {
    "caption": "(A) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h, and quantification of pS421 HTT (n=9 per group). Data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test.",
    "molecules": "FK506"
  },
  {
    "caption": "(B) Representative kymographs showing axonal trafficking of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h. Scale bar = 20 µm.",
    "molecules": "FK506"
  },
  {
    "caption": "(C) Quantification of anterograde, retrograde, mean velocity, linear flow rate and number of BDNF-mCherry-containing vesicles from the data in (A) (siMecp2+Vehicle: n=801 vesicles/95 axons; siMecp2+FK506: n=1020 vesicles/116 axons; siCtl+Vehicle: n=780 vesicles/83 axons; siCtl+FK506: n=1029 vesicles/102 axons). Data are presented as the mean ± SEM of at least three independent experiments (one-way ANOVA with Tukey's comparison). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.",
    "molecules": "FK506"
  },
  {
    "caption": "(A) Western blot of HTT S421 phosphorylation. We treated 30-day-old Mecp2 KO mice intraperitoneally with FK506 (5 mg/kg) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting, 2 h after administration, using an anti-phospho-HTT-S421 specific antibody. The D7F7 antibody recognizes total HTT. The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio (KO FK506 n = 4, KO Vehicle n = 4). Data are presented as means ± SEM, *p < 0.05, Mann Whitney test.)",
    "molecules": "FK506"
  },
  {
    "caption": "We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (B) FK506-treated KO mice lived longer than vehicle-treated KO mice and FK506-treated KO HTTSA mice (Kaplan-Meier survival test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (C) Body weight of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (D) Frequency of apnea of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (E) Motor coordination of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice on the accelerating rotarod test at P30 and P50 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (F) Forelimb strength of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice assessed by the grip strength test at P40 and P60 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.",
    "molecules": "FK506"
  },
  {
    "caption": "A. (Left) Representative single confocal  z-section of CellROX-DeepRed fluorescence of ESC  treated with 0.2M sorbitol  for the indicated time  from 2 biological replicates. Scale bar: 10µm. (Right) Quantification of CellROX-DeepRed  fluorescence intensity from single z-sections from 2 biological replicates. Two-tailed Mann-Whitney tests were performed. Boxes indicate the range between the first and third quartile, the line indicates the median and the whiskers display the spread from 5 to 95% of the data. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.",
    "molecules": "CellROX-DeepRed, sorbitol"
  },
  {
    "caption": "B. Percentage of 2CLCs  obtained by FACS  after the indicated sorbitol  treatment with addition of ROS  scavenger N-AcetylCysteine  (NAC) during either the sorbitol  treatment or recovery time. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.",
    "molecules": "sorbitol, N-AcetylCysteine, NAC, ROS"
  },
  {
    "caption": "A. Percentage of 2CLCs  obtained by FACS  after the indicated sorbitol  treatment with addition of ROS  scavenger N-AcetylCysteine (NAC) or ATR  inhibitor. Shown are the mean ± s.d from 5 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. B. Percentage of 2CLCs  obtained by FACS  after the indicated NaCl  treatment with the addition of ROS  scavenger N-AcetylCysteine (NAC) or ATR  inhibitor. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.",
    "molecules": "sorbitol, N-AcetylCysteine, NAC, ROS, NaCl"
  },
  {
    "caption": "C. Percentage of 2CLCs  obtained by FACS  after the indicated treatments. Shown are the mean ± s.d from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Individual and combinatorial treatments performed with 0.2M of sorbitol, 24mM of sodium acetate, 2.5nM of PlaB spliceosome  inhibitor and 0.53µM of retinoic acid. Unpaired two-tailed t-tests were performed. Unpaired two-tailed t-tests were performed against each individual treatment in the combinatorial experiments. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.",
    "molecules": "sorbitol, PlaB, retinoic acid, sodium acetate"
  },
  {
    "caption": "(E) Subcellular localization of GmLMM1-GFP and GmLMM1L407H-GFP in N. benthamiana. PI4P indicates the membrane localization. Scale bar, 10 μm. GFP, green fluorescent protein. Data information: The experiments were performed three times as biological replicates, with similar results.",
    "molecules": "PI4P"
  },
  {
    "caption": "(F) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro. The proteins purified from E. coli were subjected to pMIAGO phosphorylation detection assay (upper panel) and were detected using western blot (lower panel) with BIK1-HIS as a control. GmLMM1K564E contains a mutation at the predicted ATP binding site. Data information: The experiments were performed two times (F), as biological replicates, with similar results.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) ROS accumulation in Gmlmm1-1 and Gmlmm1-2 mutant plants. Soybean leaves (aged 3 weeks) were subjected to ROS accumulation examination by DAB staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.",
    "molecules": "DAB, ROS"
  },
  {
    "caption": "(B) ROS accumulation upon P. sojae treatment. The leaves (aged 3 weeks) were inoculated with P. sojae isolate P7076, and DAB staining was performed at 16 hpi. The left panel was photographed under bright field (scale bar, 1 cm). The middle and right panels were photographed under a microscope (scale bar, 50 μm). Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.",
    "molecules": "DAB, ROS"
  },
  {
    "caption": "(A) Flg22-induced ROS burst in Gmlmm1-1, Gmlmm1-2 and Williams 82. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the peak relative luminescence unit (RLU) value was recorded (Mean ± SEM, n ≥ 12, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.",
    "molecules": "Flg22, flg22, ROS"
  },
  {
    "caption": "(B) Flg22-induced ROS burst in the CRISPR lines C9-24-13 and DN50. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the relative RLU value was recorded (Mean ± SEM, n ≥ 9, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.",
    "molecules": "Flg22, flg22, ROS"
  },
  {
    "caption": "(C) Suppression of flg22-induced ROS production in Arabidopsis by GmLMM1. The GmLMM1 gene was introduced into Arabidopsis WT (Col-0) plants by Agrobacterium-mediated transformation. Two independent T2 lines were subjected to flg22-induced ROS examination (Mean ± SD, n ≥8, n represents sample number, ***, p < 0.001, Student's t-test). The protein expression is shown. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.",
    "molecules": "flg22, ROS"
  },
  {
    "caption": "Suppression of flg22-induced (D) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.",
    "molecules": "ATP, flg22, ROS"
  },
  {
    "caption": "E) Suppression of chitin-induced (E) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.",
    "molecules": "ATP, chitin, ROS"
  },
  {
    "caption": "(F) Inhibition of XEG1-induced cell death in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed in N. benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector. The cell death phenotypes were visualized 5 days later. Protein expression of GmLMM1-HA and GmLMM1K564E-HA is shown in the lower panel.",
    "molecules": "HA"
  },
  {
    "caption": "(A) Effect of flg22 on the NbFLS2-GmLMM1 interaction. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days, and the leaves were treated with flg22 (1 μM) or water for 10 min before being subjected to Co-IP assay. Data information: The experiments were performed three times , as biological replicates, with similar results.",
    "molecules": "flg22, water"
  },
  {
    "caption": "(B) Regulation of NbBAK1-GmLMM1 interaction by flg22. Data information: The experiments were performed three times as biological replicates, with similar results.",
    "molecules": "flg22"
  },
  {
    "caption": "(C) Suppression of flg22-induced NbFLS2-NbBAK1 interaction by GmLMM1. NbFLS2 and NbBAK1 were co-expressed in N. benthamiana along with GmLMM1 or GmLMM1L407H, treated with H2O or flg22 and subjected to co-IP assays. Data information: The experiments were performed three times as biological replicates, with similar results.",
    "molecules": "flg22, H2O"
  },
  {
    "caption": "(D Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1a (D) Data information: The experiments were performed two times (D as biological replicates, with similar results.",
    "molecules": "flg22"
  },
  {
    "caption": "E) Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1b (E). Data information: The experiments were performed two times E), as biological replicates, with similar results.",
    "molecules": "flg22"
  },
  {
    "caption": " EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1. EdU images are shown in red, DAPI images in blue. (A) Left: nucleus of an infected cell at 48 h p.i. displays three large and bright EdU foci (colored arrows), as well as small and dim punctae (grey arrows). Center: Magnified views have colored borders matching the colors of the arrows. 3D images show that EdU foci are located within the nucleoplasm (Movie EV1, Appendix Fig. S2). Right: nucleus of an uninfected cell displays no EdU signal. (B) Graph shows EdU intensity profiles along lines shown in A, with colors matching the corresponding arrows. ",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": " (C) Dual-color image of an infected cell with EdU (red) and immunolabeled lamin (grey). Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line. The EdU intensity peaks do not coincide with lamin enrichment. See also Fig. EV1A,B. ",
    "molecules": "EdU"
  },
  {
    "caption": " (D) Multi-color images of an infected cell showing EdU (red) with CA (yellow) and integrase (green). The colocalization of nuclear EdU foci with CA and IN (arrows) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci. See also Appendix Fig. S3. (E,F) vDNA foci are much larger than the predicted size of single viral genomes. ",
    "molecules": "EdU"
  },
  {
    "caption": " (E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17). ",
    "molecules": "EdU"
  },
  {
    "caption": " Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6. ",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": " (D) Boxplots show Pearson correlations r between vDNA (EdU) and CPSF6, NEAT1 or SC35 in 17-19 regions of interest (ROIs). Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts. Highly significant (p<0.01) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs; highly significant negative correlations between vDNA and NEAT1 are found in 19/19 ROIs, and highly significant positive correlations between vDNA and SC35 are found for 16/17 ROIs. Data are from one experiment. For CPSF6, the experiment was repeated with similar results. ",
    "molecules": "EdU"
  },
  {
    "caption": " (E) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above. ",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": " (A) Image of a ThP1 cell infected with an integration-deficient HIV-1 carrying the mutation D116A in the catalytic site of IN. The EdU-labeled vDNA is shown in red and the vRNA detected by RNA-FISH in green. The nucleus (DAPI) is shown in blue. See Appendix Fig. S12 for a larger image region. ",
    "molecules": "EdU, DAPI"
  },
  {
    "caption": " (D) ThP1 cells were infected with HIV-1 at an MOI of 20, in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Red: vDNA (EdU). (E) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters. ",
    "molecules": "EdU, DAPI, nevirapine, NVP"
  },
  {
    "caption": " (F) Image of a ThP1 cell infected by HIV-1 with EU labeled RNA in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Grey: EU-labeled vRNA. See Appendix Fig. S19 for a larger image region. (G) Intensity profiles of EU and RNA-FISH along the dotted lines in panel F (top). ",
    "molecules": "EU, DAPI, nevirapine, NVP"
  },
  {
    "caption": " The left panel shows the timeline of drug exposure experiments. (A-D) Images show vRNA and vDNA in an infected ThP1 cell (MOI 20) for each of four experimental conditions. See Appendix Fig. S15-S17 for images of larger regions. (A,B) ThP1 cells were exposed to NVP for 48 h (A) or 72 h after infection (B), then NVP was washed out, and cells were fixed for click chemistry 24 h later. (C) Cells were exposed to PF74 for 72 h after infection, then fixed for click chemistry. (D) Cells were exposed to NVP for 24 h after infection, then NVP was washed out, and cells were exposed to PF74, before being fixed for click chemistry 24 h later. (E,F,H) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p-values. Replicates of the four experiments yielded similar results. (G) qPCR measures DNA synthesis (left) and nuclear import (right) at 72 h p.i. in absence of drug treatment, or after exposure to NVP or PF74. Bars and error bars define mean and standard deviation, respectively. Samples were analyzed in triplicate and two biological replicates were performed. ",
    "molecules": "NVP, PF74"
  },
  {
    "caption": " The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Percentage of GFP positive cells at 3 and 7 days post-infection analyzed by FACS in absence of EdU. (A) Uninfected control cells. (B) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (C) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (D) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. (E-H) Multicolor images of ThP1 cells infected with a GFP-reporter virus. vRNA (RNA-FISH) is labeled in red, GFP in green, and nuclei (DAPI) in blue. (E) Uninfected control cells. (F) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (G) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (H) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. The experiment was repeated three times. ",
    "molecules": "EdU, DAPI, NVP, PF74"
  },
  {
    "caption": " The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Multicolor images of vDNA, vRNA, DAPI and/or GFP in monocyte derived macrophages (MDMs) from two different donors, infected with HIV1. Cells from donor 1 were infected with VSV-G-pseudotyped HIV-1ΔEnv Vpx carrying a GFP reporter (A) and cells from donor 2 were infected with a VSV-G-pseudotyped HIV-1ΔEnv without Vpx (B-D). (A,B) Infected cells were left untreated and fixed at 6 d p.i. (C) Infected cells were treated with 10 µM Nevirapine (NVP) throughout the experiment and fixed at 6 d p.i. (D) Infected cells were exposed to NVP for 3 d p.i., then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p.i. Arrows in (A-D) show the position of selected vRNA foci. (E) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p.i. (B), measured by the FWHM of n=24 foci. The red line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (F) Boxplot shows Pearson's r, measuring colocalization of DNA with RNA foci in the same cells (n=20). Center line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. ",
    "molecules": "DAPI, Nevirapine, NVP"
  },
  {
    "caption": " (G) FACS analysis of Gag positive cells from donor 2 in different conditions. Red: uninfected cells. Blue: untreated infected cells at 7 d p.i. as in B. Orange: cells were treated with NVP for 7 d as in C. Green: infected cells were exposed to NVP for 3 d, then NVP was washed out and cells were cultured for another 4 d as in D. Median of fluorescence intensities (MFI) for each sample are shown in the table. ",
    "molecules": "NVP"
  },
  {
    "caption": "A, B Regression plot showing the association between baseline AV45 PET (x-axis) and annual AV45 PET change (y-axis) across the entire sample (N=300). The vertical dashed line represents the Aβ-positivity threshold of AV45 PET SUVR > 0.079 (A). Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels (B).",
    "molecules": "AV45"
  },
  {
    "caption": "Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in GE180 PET (A) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).",
    "molecules": "GE180"
  },
  {
    "caption": "B Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in the rate of change in Florbetaben PET (B) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).",
    "molecules": "Florbetaben"
  },
  {
    "caption": "C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline, i.e. at 5 months of age (C). The shaded area corresponds to the 95% CI of the regression line.",
    "molecules": "Florbetaben, GE180"
  },
  {
    "caption": "A-C Coronal (top row) and axial (bottom row) slices of average 18-F GE-180 TSPO (microglia) tracer and 18F-florbetaben amyloid-PET split up by group (A - C): C57BL/6 (A), APPNL-G-F mice subgroup with a low (< median) GE-180 PET (B), and the APPNL-G-F mice subgroup with a high (> median) GE-180 PET (C). APPNL-G-F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months (B) compared to those at high baseline level of GE-180 PET (C).",
    "molecules": "18F-florbetaben, Florbetaben, 18-F GE-180, GE-180, GE180"
  },
  {
    "caption": "Effects of sTREM2 on AV1451-PET-assessed tau pathology stratified by Braak-stages shown as boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range).",
    "molecules": "AV1451"
  },
  {
    "caption": "(E) Representative images of 1205Lu  shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) melanoma spheroids  after 72 h invasion  in collagen. Live cells   are shown in green. Scale bar: 100 µm. (F) Quantification of 1205Lu  stable  MCUA_KD spheroid core size (n=6 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU  cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).",
    "molecules": "collagen"
  },
  {
    "caption": "(L) Representative images of 451Lu  wild-type (grey frame), 451Lu-BR3 (lighter green frame), WM983B  wild-type (grey frame) and WM983B-BR (darker green frame) melanoma spheroids   after 72 h invasion  in collagen. Live cells  are shown in green. Scale bar: 100 µm. (M-N) Quantification of 451Lu  versus 451Lu-BR3 (M) and WM983B  versus  WM983B-BR (N)  spheroid core size   (n≥9 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU  cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).",
    "molecules": "collagen"
  },
  {
    "caption": "(A-C) Mitochondrial hydrogen peroxide  (H2O2) measurement in 1205Lu  and WM3734  with and without stable  MCUA_KD using mito-HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (A). Scale bar: 10 µm. Quantification of mito-HyPer  ratio in 1205Lu  (B) and WM3734  (C) under resting state (1205Lu  ─ shCTRL: n=217 cells from 6 biological replicates; shMCU_1: n=197 cells from 7 biological replicates; shMCU_2: n=212 cells from 7 biological replicates; WM3734  ─ shCTRL: n=104 cells from 6 biological replicates; shMCU_1: n=107 cells from 7 biological replicates; shMCU_2: n=114 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+  statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.",
    "molecules": "Ca2+, H2O2, hydrogen peroxide"
  },
  {
    "caption": "(D-F) Cytosolic hydrogen peroxide (H2O2) measurement in 1205Lu  and WM3734  with and without stable MCUA_KD using HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (D). Scale bar: 10 µm. Quantification of HyPer  ratio in 1205Lu  (E) and WM3734  (F) under resting state (1205Lu  ─ shCTRL: n=232 cells from 6 biological replicates; shMCU_1: n=290 cells from 6 biological replicates; shMCU_2: n=290 cells from 6 biological replicates; WM3734 ─ shCTRL: n=443 cells from 9 biological replicates; shMCU_1: n=297 cells from 9 biological replicates; shMCU_2: n=233 cells from 9 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM  Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.",
    "molecules": "Ca2+, H2O2, hydrogen peroxide"
  },
  {
    "caption": "(I-K) Mitochondrial glutathione redox potential, measured with mito-Grx1-roGFP2 in 1205Lu  and WM3734  with and without stable MCUA_KD. Representative ratiometric images (F385 nm/F475 nm) are shown for all conditions (I). Scale bar: 10 µm. Quantification in 1205Lu  (J) and WM3734  (K) under resting state (1205Lu ─ shCTRL: n=231 cells from 10 biological replicates; shMCU_1: n=176 cells from 7 biological replicates; shMCU_2: n=164 cells from 7 biological replicates; WM3734  ─ shCTRL: n=127 cells from 7 biological replicates; shMCU_1: n=142 cells from 7 biological replicates; shMCU_2: n=116 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.",
    "molecules": "Ca2+, glutathione"
  },
  {
    "caption": "(G-I) Mitochondrial ATP, measured using mito-ATEAM  in 1205Lu  and WM3734  with and without stable MCUA_KD. Exemplary ratiometric images (FRET/CFP) are shown for all conditions (G). Scale bar: 10 µm. Quantification of basal levels in 1205Lu  (H) and WM3734  (I) (1205Lu  ─ shCTRL: n=141 cells from 14 biological replicates; shMCU_1: n=143 cells from 12 biological replicates; shMCU_2: n=143 cells from 13 biological replicates; WM3734  ─ shCTRL: n=122 cells from 12 biological replicates; shMCU_1: n=103 cells from 12 biological replicates; shMCU_2: n=99 cells from 13 biological replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).",
    "molecules": "ATP"
  },
  {
    "caption": "(J) Exemplary confocal microscope  images of the   mitochondrial network  (blue: DAPI staining  of the nucleus; green: TOMM20 staining  of mitochondria); scale bar: 50 µm. (K-O)  Electron microscopy of mitochondria  of stable  MCUA_KD cell lines. (K) Exemplary images of stable  MCUA_KD cells' mitochondria. Scale bar: 500 nm. (L-O) Quantification of mitochondrial  diameter (L and N) and intercristae  distance (M and O) of 1205Lu  (L and M) and WM3734  (N and O) cells with and without stable MCUA_KD, presented as boxplot. The box presents the 25 %- quartile, median and 75 %-quartile, the X represents the mean and the whiskers the minimum and maximum, outliers are represented as dots (mitochondrial  diameter: 1205Lu  ─ shCTRL: n=47 technical replicates; shMCU_1: n=44 technical replicates; shMCU_2: n=40 technical replicates; WM3734  ─ shCTRL: n=35 technical replicates; shMCU_1: n=52 technical replicates; shMCU_2: n=38 technical replicates; intercristace  distance: 1205Lu  ─ shCTRL: n=330 technical replicates; shMCU_1: n=285 technical replicates; shMCU_2: n=255 technical replicates; WM3734  ─ shCTRL: n=352 technical replicates; shMCU_1: n=433 technical replicates; shMCU_2: n=357 technical replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).",
    "molecules": "DAPI"
  },
  {
    "caption": "A, Top: Strategy for EdU pulse and animal fixation. Bottom: Confocal images of EdU-stained atm and control RNAi animals. B, Quantification by area of EdU+ cells from A. Circles represent individual animals. Inset, expanded scale showing compressed data below. Data includes two independent experiments.",
    "molecules": "EdU"
  },
  {
    "caption": "D, Confocal images of phosphohistone H3 (pH3)-stained RNAi animals after radiation exposure. Blue, DAPI. E and F, Quantification by area of pH3 cells in radiated (E) or unirradiated (F) animals after atm (solid line) and control (dashed line) RNAi.",
    "molecules": "DAPI"
  },
  {
    "caption": "RNA-seq  heatmaps of the indicated pluripotency gene sets in control and INK128 (200 nM) treated mESCs in  2iL.   RNA-seq heatmaps of indicated pluripotency gene sets in Rptor WT and KO mESCs  in 2iL.  ",
    "molecules": "INK128"
  },
  {
    "caption": "Images of mESCs transduced with Larp1  shRNAs in 2iL, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm.   Relative  proliferation of mESCs as in (E) (mean values ± SD). n = 3 biological replicates.  ",
    "molecules": "INK128"
  },
  {
    "caption": "OCR analysis of INK128 (200 nM) treated mESCs in 2iL.",
    "molecules": "INK128"
  },
  {
    "caption": "qPCR for mtDNA copy number in mESCs treated with INK128  or transduced with Rptor shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.",
    "molecules": "INK128"
  },
  {
    "caption": "(F) Hematoxylin and eosin (H&E)-staining of lung sections were from EphA2+/+ and EphA2−/− mice intranasally infected with reovirus (4×104 PFU per mouse) on day 0, day 1 and day 3. Scale bars represent 200μm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "(I) H&E staining and PAS staining of lung sections from wild-type (WT) and EphA2-knockout (KO) mice sensitized with ova. Scale bars represent 200μm.",
    "molecules": "PAS"
  },
  {
    "caption": "(E) Colocalization of endogenous EphA2 and NLRP3 in AEC cells. AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis. EphA2 was stained with rabbit anti-EphA2 (1:500), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red), while NLRP3 was stained with mouse anti-NLRP3 (1:500), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 20μm. The Pearson's correlation coefficient and the Mander's overlap coefficient in the upper red box are 0.5, and 0.95 respectively, and the lower red box 0.78, and 0.94 respectively.",
    "molecules": "Alexa Fluor 488, Alexa Fluor 594, 4′,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc.",
    "molecules": "Tyr"
  },
  {
    "caption": "(B) Immunoblot analysis of p-Tyr of NLRP3 precipitated with anti-NLRP3 antibody in AECs without (Ctrl) or with LPS stimulation or reovirus (Reo) infection.",
    "molecules": "LPS, Tyr"
  },
  {
    "caption": "(C) The indicated plasmids of Myc-tagged wild-type NLRP3 or its mutants Y132F, Y164F, Y251F, Y570F and Y589F were respectively transfected into HEK293T cells along with GFP-tagged EphA2, and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-HA antibody and anti-Myc antibody.",
    "molecules": "Tyr"
  },
  {
    "caption": "(A-B) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection (MOI) of 20 for 3h. AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control. ASC was stained with mouse anti-ASC (1:200), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.",
    "molecules": "Alexa Fluor 488, ATP, DAPI, LPS"
  },
  {
    "caption": "(C-D) Confocal microscopy analysis of HEK293T cells transfected with flag-ASC and Myc-tagged vector or Myc-tagged NLRP3 or Myc-tagged NLRP3 Y132F. Cells were stained with anti-flag (1:200), anti-Myc (1:200), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (green) or Alexa Fluor 594 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.",
    "molecules": "Alexa Fluor 488, Alexa Fluor 594, DAPI"
  },
  {
    "caption": " b In HEK293 cells, recombinantly expressed GluN1/GluN2A and GluN1/GluN2A(N615S) channels were activated by fast glutamate application (1 mM; in the continuous presence of the co-agonist glycine, 10 µM) at holding potentials from -100 to +40 mV in different extracellular Mg2+ concentrations. NMDAR-mediated peak currents were normalized to those obtained at +40 mV. Data points represent mean ± SEM for n = 4-7 different HEK293 cells. Representative current traces evoked in 0 mM Mg2+ at - 60 mV, with 20 and 600 ms applications, are shown below the IV plots and were used to determine the current kinetics (Supplementary Table 1). ",
    "molecules": "glycine, glutamate, Mg2+"
  },
  {
    "caption": " a (left) In the absence of extracellular Mg2+ the synaptic AMPA/NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a+/S and Grin2aS/S mice compared to control littermates. (right) In the presence of extracellular Mg2+ the strong reduction of the AMPA/NMDA ratio in Grin2aS/S mice relates to the increased NMDA currents at CA1 synapses (Tukey's test). Example traces are depicted to the right of each bar graph. Data from the same experimental group were pooled across animals and are presented as mean ± SEM (see also ref. 35) with p < 0.05 being designated as statistically significant. Numbers in bar graphs indicate the number of slices. ",
    "molecules": "Mg2+"
  },
  {
    "caption": " c Memantine i.p. injection in Grin2aS/S mice, 3 h before tone exposure, rescued AGS susceptibility in Grin2aS/S mice. The rescue effect could still be observed in four out of eight, and in two out of eight, mice 27 and 51 h after memantine treatment, respectively. ",
    "molecules": "Memantine"
  },
  {
    "caption": " d Increased c-Fos immunofluorescence (in grayscale) in the medial amygdala (MeA) and the VMH of a Grin2aS/S animal with AGS (saline injection) compared to a memantine rescued Grin2aS/S littermate. In the paraventricular nucleus of the thalamus (PVT), the hippocampus (HPC) and cortex (Cx) there was no difference in c-Fos expression between memantine-injected and saline-injected animals (for fluorescence images, see Supplementary Fig. 5). ",
    "molecules": "memantine"
  },
  {
    "caption": "e As in Fig. 4c but now MK-801 and saline are used for pre-treatment. The MK-801 effect was longer lasting compared to memantine and one out of four animals showed resistance to tone exposure even 4 days after MK-801 injection.",
    "molecules": "MK-801, memantine"
  },
  {
    "caption": " f Decreased c-Fos DAB immunosignal in an MK-801 AGS-rescued Grin2aS/S animal (bottom) in the medial amygdala (MeA), the VMH and the PVT when compared to saline-injected Grin2aS/S littermate (top). Scale bars are in mm (for statistics: Supplementary Statistics to Fig. 4). ",
    "molecules": "MK-801"
  },
  {
    "caption": " C-Fos expression 120 min after i.p. injection of MK-801. a C-Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated. The original images, including the cortical regions and results of PBS injected animals, are presented in Supplementary Fig. 6. The number of mice analyzed is given in brackets. b For quantitative comparisons the total numbers of c-Fos-positive cells in the cellular layers of the hippocampus were counted. C-Fos-positive cells in extra-hippocampal regions are given per mm2. Slices from six mice per genotype were used for the statistical analyses (two-tailed t-tests) and error bars represent mean ± SEM. CA1 and CA3 cornu ammonis regions 1 and 3, DG dentate gyrus, PC piriform cortex, RSC retrosplenial cortex, Neo neocortex, MTN midline thalamic nuclei, BLA basolateral amygdala. All scale bars: 0.2 mm. ",
    "molecules": "MK-801"
  },
  {
    "caption": "H1299 cells transfected with WT USP36 or the indicated mutants and His-SUMO2 were subjected to Ni2+-NTA agarose beads pull down (PD) followed by IB using anti-SUMO2/3 antibody.",
    "molecules": "agarose, Ni2+, NTA"
  },
  {
    "caption": "USP36 SUMOylates PARP1 in vitro. Recombinant T7-PARP1 protein (0.1 μM) was incubated with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the presence of USP361-800 (50 nM) and/or ATP (2.5 mM) at 30ºC for 5 hours and then assayed by IB.",
    "molecules": "ATP"
  },
  {
    "caption": "USP36 SUMOylates Nhp2 and Nop58 in vitro. In vitro SUMOylation assays were performed by incubating recombinant Nhp2 (F, G) or Nop58 (H) (0.1 μM) with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the absence or presence of USP361-800 (50 nM, 1x; 100 nM, 2x) and/or ATP (2.5 mM) at 37ºC for two hours or the indicated times. The reactions were assayed by IB using anti-Nhp2 and anti-Nop58 antibodies, respectively.",
    "molecules": "ATP"
  },
  {
    "caption": "Knockdown of USP36 inhibits translation. HeLa cells infected with scr or USP36 shRNA lentiviruses were incubated with O-propargyl-puromycin (OPP) followed by Click-iT OPP protein synthesis assays. Representative images (C) and quantification from three independent experiments (D). Data were presented as mean ± SD, n=3 biological replicates. ***P<0.001, compared to scr control (Student's t-test). Scale bar = 50 μM.",
    "molecules": "O-propargyl-puromycin, OPP"
  },
  {
    "caption": "(B) MSD, MPS‐IIIA and WT MEFs cells loaded with dextran (alexafluor‐594 conjugated) were labelled with anti‐LAMP1 antibody and the percentage of dextran co‐localizing with LAMP1 was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of dextran co‐localizing with LAMP1 measured in 15 different cells of triplicate experiments.",
    "molecules": "dextran"
  },
  {
    "caption": "(C) The rate of lysosome fusion with autophagosomes was monitored in MSD, MPS‐IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 ( Kimura et al, 2007). The rate of autophagosome maturation reflected the percentage of the LC3 'unfused' (green/red fluorescence ratio) at each time (1 and 3 h) after bafilomycin removal (T0). The percentage of LC3 'unfused' was displayed versus the value at T0 (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B, C): WT versus MSD and WT versus MPS‐IIIA at each time point. Scale bar: 10 μm (B, C).",
    "molecules": "bafilomycin"
  },
  {
    "caption": "(A) Cholesterol levels were measured in the indicated endolysosomal membrane samples containing equal amount of proteins and expressed as ng of cholesterol per μg of protein (see Materials and methods section for details).",
    "molecules": "Cholesterol, cholesterol"
  },
  {
    "caption": "(B) Filipin staining showing cholesterol accumulation in the endolysosomal compartment of MSD and MPS‐IIIA MEFs (arrowheads and enlarged images).",
    "molecules": "cholesterol"
  },
  {
    "caption": "(C, D) Total lipids extracted from the indicated endolysosomal membrane samples (30 μg of proteins) were either (C) subjected to a phosphate assay to quantify the bulk of phospholipids or (D) separated by TLC. Phospholipids and cholesterol on TLC plates were revealed by molybdenum blue staining. CHOL, cholesterol; LBPA lysobisphosphatidic acid; PC, phosphatidylcoline; PE phosphatidylethanolamine; PI phosphatidyinositol; SM sphingomyelin. (A, C) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus MSD and WT versus MPS‐IIIA. Scale bar, 10 μm (B).",
    "molecules": "cholesterol, lysobisphosphatidic acid, phosphatidylcoline, phosphatidylethanolamine, phosphatidyinositol, phospholipids, sphingomyelin"
  },
  {
    "caption": "Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD",
    "molecules": "Cholesterol, cholesterol"
  },
  {
    "caption": "Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar",
    "molecules": "cholesterol"
  },
  {
    "caption": "Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).",
    "molecules": "MβCD, cholesterol, dextran"
  },
  {
    "caption": "Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).",
    "molecules": "MβCD"
  },
  {
    "caption": "Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).",
    "molecules": "MβCD, dextran"
  },
  {
    "caption": "(A) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were treated with 1% Triton X‐114 and loaded on a sucrose gradient. Immunoblots with Flotillin‐1 identified DRMs in fractions 2, 3 and 4 (arrows). The fractions at the bottom of the gradient (12 and 13) correspond to high‐density detergent soluble fractions, whereas the remaining ones were defined as intermediate fractions (intermediate‐I: 5, 6, 7 8; intermediate‐II: 9, 10 and 11). The percentage of Flotillin‐1 in DRMs was calculated from the densitometric quantification of immunoblots.",
    "molecules": "Triton X‐114"
  },
  {
    "caption": "(B) Immuno‐EM of GM1 lipid was carried out in WT, MSD and MPS‐IIIA MEFs by staining cells with anti‐cholera toxin B antibodies (see Materials and methods section). The number of GM1‐positive dots was measured in 25 cells from three independent experiments and displayed as fold to WT.",
    "molecules": "GM1"
  },
  {
    "caption": "(C) Endolysosomal membranes from MSD, MPS‐IIIA and WT MEFs were stained with C‐laurdan and subsequently analysed by fluorescence spectrophotometry to calculate the GP value (see Materials and methods section for details). Distribution of cholesterol was also measured throughout the gradient ad expressed as percentage of total cholesterol in raft (DRMs) and soluble fractions.",
    "molecules": "cholesterol"
  },
  {
    "caption": "(A) VAMP7, Vti1b and syntaxin 7 distributions in endo‐lysosomal membranes from MSD, MPSIIIA and WT MEFs was evaluated by immonoblotting analysis of gradient fractions. To simplify the analysis the DRM, the intermediate‐I, the intermediate‐II and soluble fractions of the gradient were pooled separately and then subjected to immunoblotting. SNARE distribution was also analysed after loading WT cells with cholesterol and after MβCD treatment. In MSD and MPS‐IIIA MEFs, all analysed SNAREs abnormally accumulate in DRMs of lysosomal membranes. Cholesterol modulation results in a change of SNARE distribution.",
    "molecules": "MβCD, cholesterol, Cholesterol"
  },
  {
    "caption": "(C, E) Immunoblots and relative quantification showing SNARE protein levels along with LAMP1 protein levels in (C) endolysosomal membranes and (E) total cell lysates from WT (untreated and cholesterol treated) and MSD MEFs (untreated and MβCD treated). In the graphs, the protein levels were displayed as relative amount versus WT.",
    "molecules": "MβCD, cholesterol"
  },
  {
    "caption": "(A) SDS‐resistant complexes containing Vti1b were detected by immunoblotting analysis of nonboiled samples corresponding to total, detergent insoluble (DRM) and detergent soluble (Sol.) endo‐lysosomal membrane fractions derived from MSD and WT MEFs. The SDS‐resistant complexes were also visualized after loading WT MEFs with cholesterol and after treating MSD MEFs with MβCD. Immunoblots revealed the presence of low molecular weight complexes (*, 50-60 kDa) and high molecular weight complexes (**, >80 kDa). The percentage of Vti1b in SDS‐resistant complexes in total endolysosomal membranes (bottom‐left chart) and the amount of Vti1b‐containing SDS‐resistant complexes in DRM and soluble fractions (bottom‐right chart) were calculated by the densitometric quantification of the correspondent immunoblots (ImageJ). Values represent the mean±s.e.m. values of three independent measurements. *P0.05, Student's t‐test: WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD (bottom‐left chart); WT versus MSD, WT versus WT+cholesterol and MSD versus MSD+MβCD for each fraction (bottom‐right chart).",
    "molecules": "MβCD, cholesterol, SDS"
  },
  {
    "caption": "(B) Syntaxin 7 and VAMP7 were co‐immunoprecipitated with Vti1b using anti‐Vti1b antibodies in WT (untreated or cholesterol treated) and in MSD (not treated or MβCD treated) MEFs. The amount of Vti1b precipitated in each cell line is also shown.",
    "molecules": "MβCD, cholesterol"
  },
  {
    "caption": "(C) SDS‐resistant complexes are decorated by anti‐syntaxin 7 antibodies in total endolysosomal membrane fraction from WT and MSD MEFs.",
    "molecules": "SDS"
  },
  {
    "caption": "(D) Membrane‐associated α‐SNAP and its release in the cytosol were evaluated by western blot analysis on total cell lysates (total), intracellular membranes recovered after centrifugation from a post‐nuclear supernatant fraction (membrane associated) and cell lysates devoid of membranes (cytosolic released) derived from MSD (untreated or MβCD treated) and WT (untreated or cholesterol treated) MEFs.",
    "molecules": "MβCD, cholesterol"
  },
  {
    "caption": "(B) Co‐localization of Vti1b with epsinR was quantified by double‐labelling experiments in MSD and MPS‐IIIA (untreated and MβCD treated) and in control WT (untreated and cholesterol treated) MEFs. The chart displays merge values (means±s.e.m.) that represent the percentage of Vti1b co‐localizing with epsinR measured in 15 different cells.",
    "molecules": "MβCD, cholesterol"
  },
  {
    "caption": "(C) Vti1b trafficking was monitored by FRAP analysis in WT (untreated and cholesterol treated) and MSD (untreated and MβCD treated) MEFs transfected with GFP-Vti1b (see Materials and methods section for details). FRAP data are displayed as percentage of recovery with respect to the fluorescence before bleach (100%) and are representative of 10 recordings from different cells.",
    "molecules": "MβCD, cholesterol"
  },
  {
    "caption": "(B) Upper graph shows percentage of regenerating axons of DIV 1-2 adult DRG neurons in the presence of p110 inhibitors, 2 h after injury. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: LY294002, 20 μM. A66 5 μM. XL-147 5 μM. TGX221 500 nM. IC-87114 10μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance analysed by Fishers exact (upper graph) or Kruskal-Wallis test (lower graph).",
    "molecules": "A66, TGX221, IC-87114, Idelalisib, LY294002, XL-147"
  },
  {
    "caption": "(F) Upper graph shows percentage of regenerating axons in microfluidic chambers. p110α or δ inhibitors were applied to either soma or axons. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: A66 5 μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance as measured by Fishers exact test (upper graph) or by Kruskal-Wallis test (lower graph).",
    "molecules": "A66, Idelalisib"
  },
  {
    "caption": "(A) DIV 3 cortical neurons immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (B) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (C) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (D) Somatic PIP3 quantification in the soma at increasing days in vitro. Data are shown as the mean +/- SEM. P values are significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (E) Growth cone PIP3 quantification at increasing days in vitro. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones. ",
    "molecules": "PIP3"
  },
  {
    "caption": "(A) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (B) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (C) Quantification of PIP3 immunofluorescence in the soma. Data are shown as the mean +/- SEM. P values indicate significance as measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (D) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones. ",
    "molecules": "PIP3"
  },
  {
    "caption": "C) Retinal sections from AAV-injected mice, immunolabelled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image.",
    "molecules": "DAPI"
  },
  {
    "caption": "G) Representative images of CTB-labelled RGC axons 4 weeks after optic nerve crush in wild type (c57bl/6), Rosa26-p110αH1047R, and Rosa26-p110δ transgenic mice injected with AAV2.Cre.GFP. Dashed line indicates crush site.",
    "molecules": "CTB"
  },
  {
    "caption": "(A) Retinal sections from mice injected with AAV2 viruses as indicated, immunolabeled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image. (B) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2.shScramble.GFP or AAV2.shPTEN.GFP. (C) Number of pS6-postive RGCs of mice injected with AAV2.GFP or AAV2.p110δ.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) CTB-labelled RGC axons 4 weeks after optic nerve crush. Boxes i. to iv. show regenerating axons. Dashed line indicates crush site. White arrows mark the end of axons. Graph shows quantification of regenerating axons at different distances distal to the lesion sites.",
    "molecules": "CTB"
  },
  {
    "caption": "Treatment of HCT116 cells with siRNA targeting Ku70 or control (A-I) combined or not with a treatment with doxorubicin (Doxo) (positive control for phosphorylation of ATM and p53) (B) followed by western blot analysis.",
    "molecules": "Doxo, doxorubicin"
  },
  {
    "caption": "Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with wortmannin (WTN) (D) followed by western blot analysis.",
    "molecules": "wortmannin, WTN"
  },
  {
    "caption": "Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with NU-7441 (E,F) followed by western blot analysis.",
    "molecules": "NU-7441"
  },
  {
    "caption": "Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), XRCC4 (F) or control (A-I) combined or not with a treatment with NU-7441 (E,F), followed by western blot analysis.",
    "molecules": "NU-7441"
  },
  {
    "caption": "Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with MG132 (H) followed by western blot analysis (A-F, H-I).",
    "molecules": "MG132"
  },
  {
    "caption": "Treatment of HCT116 cells with siRNA targeting Ku70 (A-I), or control (A-I) combined or not with a treatment with cycloheximide (CHX) (I) followed by western blot analysis (A-F, H-I).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(C) UV cross-linking of recombinant (Ku70/Ku80) with the in vitro transcribed 5'p53 with alpha 32P-GTP or alpha 32P-UTP (i). UV cross-linking of recombinant Ku70/Ku80 with the in vitro transcribed 5'p53 and the Ctrl (5'p53 antisense) with alpha 32PGTP (ii).",
    "molecules": "32P, GTP, UTP"
  },
  {
    "caption": "(A) Western blot analysis after treatment with Ku70 or control siRNA, followed by incubation with DMSO, etoposide (ETO) or bleomycin (BLEO) for 16 h (i). p53 levels after Ku70 depletion normalized to siRNA control for each condition (ii, n=3). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.",
    "molecules": "BLEO, bleomycin, DMSO, ETO, etoposide"
  },
  {
    "caption": "(B) Western blot analysis as in A, except that tricostatin A (TSA) was used after Ku/control knockdown (n=4). All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.",
    "molecules": "tricostatin A, TSA"
  },
  {
    "caption": "(C) Western blot in cells treated either with DMSO, TSA, or ETO for 16 or 24 h. All the experiments were performed with HCT116 cells. GAPDH: loading control in A, B, C. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.",
    "molecules": "DMSO, ETO, TSA"
  },
  {
    "caption": "(D) RNA affinity chromatography using the 5'p53RNA and total extracts from cells treated with TSA, ETO or BLEO for 24 h, followed by western blot analysis of Ku70 quantified and normalized to the input (ii, n=3). All the experiments were performed with HCT116 cells. Statistical analysis by unpaired t-test (*P<0.05; **P<0.01; *** P<0.001; NS, non significant). All error bars reflect SEM.",
    "molecules": "BLEO, ETO, TSA"
  },
  {
    "caption": "A. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs than in CFs. The expression level in resting conditions of the PDFs was set as 1. Differences between knockdown and control with p values ≤ 0.05 (Student's t test) are indicated with an asterisk. B. as in A. for patient VII1.",
    "molecules": "LPS"
  },
  {
    "caption": "B. ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS-stimulated PDFs than in LPS-stimulated-CFs. The binding activity at the IL1B promoter was set as 1. Differences between knockdown and control with p value= 0.00436 (Student's t test) are indicated with an asterisk.",
    "molecules": "LPS"
  },
  {
    "caption": "C. NF-kB inhibitor BOT-64 blocked the inducible mRNA expression of the inflammatory cytokines. Differences between knockdown and control with p values (Student's t test) are indicated with an asterisks and exact values.",
    "molecules": "BOT-64"
  },
  {
    "caption": "B. qPCR showed that ppp1r13l-knocked down murine cardiomyocytes (using two alternative shRNA sequences) expressed higher level of pro-inflammatory cytokine mRNAs after 4 hrs of LPS stimulation. The expression in ppp1r13l-knocked down murine cardiomyocytes was set as 1. Genome wide expression analysis is presented in Figure 1S. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks and exact values.",
    "molecules": "LPS"
  },
  {
    "caption": "A. LPS (5mg/Kg for 2 hrs) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts. The results are presented as fold induction of untreated controls. The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts, while of Mybpc3 and Sgcg was unchanged. For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7. Differences between knockdown and control with p values =0.04998 (Student's t test) are indicated with an asterisk.",
    "molecules": "LPS"
  },
  {
    "caption": "B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS (1mg/Kg).",
    "molecules": "LPS"
  },
  {
    "caption": "B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mousehypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mousehypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star).",
    "molecules": "SDS"
  },
  {
    "caption": "H. Effect of oligo#5 on food uptake in wild-type mice. C57BL/6 wild-type mice received a guide canulae and did not have access to food for 16 hours. After injection of 2 µg oligonucleotides, they could freely access food. The food consumption was measured by weighing the food used. A total of 52 animals were used, p=0.001 for the difference between oligo#5 and control oligonucleotides. Each of the six individual experiments showed statistically significant differences between oligo#5 and control (p<0.01 - 0.001). Control oligo: an oligonucleotide against human SMN2.",
    "molecules": "food"
  },
  {
    "caption": "I. Effect of oligo#5 on food uptake in ob/ob mice. Ob/obmice received oligo#5 through ICV injection, similar to panel F. However, the mice could freely access food at all times (p=0.04, n=3).",
    "molecules": "food"
  },
  {
    "caption": "C. Effect of oligo#5 delivered through a carotid artery catheter on food uptake in wild-type mice. C57BL/6 wild-type mice with a pre-implanted cranial dosing carotid catheter received 50 or 100 µg of the oligonucleotides indicated.",
    "molecules": "food"
  },
  {
    "caption": "(C) Activity assay for the catalytic subunits of the proteasome using the pan activity-based probe (ABP) MV151, the β1/LMP2 specific LW124, and the β5/LMP7 specific MVB127 ABPs in three PolgA+/+ and three PolgAmut/mut cell lines. Resolution of β5/LMP7 in the SDS gel is hampered as both subunits are of similar molecular weight and thus do not separate in our gels. Total protein staining was used as a loading control.",
    "molecules": "SDS"
  },
  {
    "caption": "Senescence-associated heterochromatin foci formation (SAHF) in A549 cells with either shCtrl or shGUARDIN. Representative IF staining with H3K9me3 antibodies (green) and nuclear Hoechst counterstaining (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "SDS-PAGE of RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells indicating putative GUARDIN-binding proteins (left); protein identities with high probabilities were determined by mass spectrometry (right).",
    "molecules": "biotin"
  },
  {
    "caption": "Subcellular localization of GUARDIN and its co-localization with LRP130. RNA FISH for GUARDIN (red) and IF for LRP130 (green) in A549 cells with either shCtrl or shGUARDIN. Nucleus were counterstained with Hoechst (blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells. GUARDIN levels were measured by RT-PCR and co-precipitated LRP130 and PGC1α detected by Western blotting. BRCA1 and β-actin served as positive and negative controls, respectively.",
    "molecules": "biotin"
  },
  {
    "caption": "Half-life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg/ml of actinomycin (ActD) for the indicated times.",
    "molecules": "ActD, actinomycin"
  },
  {
    "caption": "qPCR assays for GUARDIN expression in A549 cells treated with increasing doses rapamycin for 24h",
    "molecules": "rapamycin"
  },
  {
    "caption": "qPCR assays for GUARDIN expression in A549 cells treated with 100nM of rapamycin for the indicated times (B).",
    "molecules": "rapamycin"
  },
  {
    "caption": "Western blotting assays for p53, LRP130, FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle (DMSO) for 48h.",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h. Western blotting was used to measure p21 levels (left) while conducting SA-β-gal staining in parallel (middle) with the percentage of senescent cells calculated from the SA-β-gal staining (right).",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells treated with DMSO or 100nM rapamycin. Samples were subjected to the luciferase activity assays.",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "SIX4 and FOSL2 mRNA (left) and protein levels (right) measured by qPCR and Western blotting, respectively in A549 cells treated with DMSO or 100nM rapamycin. TBP served as a control.",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "(a,b) Changes in HEK293T cell cytoplasmic (a) and mitochondrial (b) [Ca2+] in response to ionomycin (2.5 μM) were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. Each bar represents one target gene silenced with pooled siRNA.",
    "molecules": "Ca2+, ionomycin"
  },
  {
    "caption": "(g,h) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic (green) and mitochondrial (red) [Ca2+] before, during and after ionomycin exposure. Scale bar, 20 μm.",
    "molecules": "Ca2+, ionomycin"
  },
  {
    "caption": "(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HEK293T (i-l) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (i) Wild-type HEK293T cells. (j) Cells expressing negative shRNA. (k) Clone shHK5 (n = 4). (l) Quantification of peak rhod-2 fluorescence. **P0.01 (mean±s.e.m.).",
    "molecules": "Ca2+, histamine, ionomycin"
  },
  {
    "caption": "(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HeLa (m-p) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n = 3). (p) Quantification of peak rhod-2 fluorescence. *P0.05, **P0.01 (mean±s.e.m.).",
    "molecules": "Ca2+, histamine, ionomycin"
  },
  {
    "caption": "(q) [Ca2+]c and [Ca2+]m signals evoked by ATP (100 μM) and thapsigargin (Tg, 2 μM) were monitored simultaneously using fura-2/AM and mitopericam (ipcam490), respectively in control (left) and MCUR1-knockdown (right) HeLa cells. [Ca2+]c is calibrated in nanomolar concentrations (black), whereas mitopericam fluorescence is inversely normalized to baseline (F0/F; red).",
    "molecules": "ATP, Ca2+, thapsigargin"
  },
  {
    "caption": "(r) Summary mean [Ca2+]c and [Ca2+]m peaks during ATP stimulation (negative shRNA n = 29; MCUR1 knockdown n = 36 cells). *P0.05 (mean±s.e.m.).",
    "molecules": "ATP, Ca2+"
  },
  {
    "caption": "(s) Increase in bath [Ca2+](Rfura2) and [Ca2+]m (Ripcam) signals in response to CaCl2 (1 μM) and InsP3 (IP3, 7.5 μM) addition in permeabilized cells. Uncropped images of blots/gels are shown in Supplementary Fig. S6.",
    "molecules": "InsP3, CaCl2, Ca2+"
  },
  {
    "caption": "Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca2+ indicator Fura2FF, to which pulses of 10 μM Ca2+ were added before the addition of the mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). (a-e) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in cells expressing negative shRNA (Neg shRNA; a), clone shHe1 (b), clone shHe2 (c), clone shHe2 re-expressing MCUR1 (d) and HeLa cells stably overexpressing MCUR1 (e). Under similar conditions, 1 μM Ru360 was added before 10 μM Ca2+ pulses until the addition of CCCP.",
    "molecules": "Ru360, Ca2+, carbonyl cyanide m-chloro phenyl hydrazone, CCCP, Digitonin, thapsigargin"
  },
  {
    "caption": "(f-i) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in negative shRNA cells (f), MCUR1-knockdown clone shHe1 (g) and shHe2 (h), and in control cells overexpressing MCUR1 (i).",
    "molecules": "Ca2+"
  },
  {
    "caption": "(j,k) Negative shRNA (j) and MCUR1-overexpressing (k) HEK293T cells were permeabilized with digitonin in intracellular-like medium containing thapsigargin and bath [Ca2+] indicator Fura2FF, and then pulsed with 10 μM Ca2+. After mitochondrial clearance of bath Ca2+, Ru360 caused an elevation of bath [Ca2+], indicating that steady-state bath [Ca2+] after the pulse was maintained by a balance of MCU-mediated Ca2+ uptake and CGP37157 (10 μM)-sensitive Na+-Ca2+ exchanger-mediated extrusion. CCCP was added as indicated. The solid line is the mean; shaded areas are ±s.e.m. (n = 3).",
    "molecules": "CGP37157, Ru360, Ca2+, CCCP, digitonin, Na+, thapsigargin"
  },
  {
    "caption": "(l) [Ca2+]m efflux rate derived from j and k during the initial 60 s following Ru360 addition (NS; not significant; n = 3).",
    "molecules": "Ru360, Ca2+"
  },
  {
    "caption": "(d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma-membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified. Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1.",
    "molecules": "proteinase K"
  },
  {
    "caption": "(b) [Ca2+]m responses to histamine (100 μM) in HeLa cells stably overexpressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA, and in stable MCUR1-overexpressing HeLa cells transfected with MCU siRNA. After 48 h of siRNA transfection, cells were loaded with rhod-2 and [Ca2+]m responses were visualized by confocal microscopy. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (c) Quantification of peak rhod-2 fluorescence following histamine stimulation. *P0.05, ***P0.001 (mean±s.e.m.; NS, not significant; n = 3).",
    "molecules": "Ca2+, histamine"
  },
  {
    "caption": "(e) [Ca2+]m responses to histamine (100 μM) in wild-type and MCUR1 (shHe2)-knockdown HeLa cells overexpressing MCU. Negative shRNA (Neg shRNA) and MCUR1-shHe2 cells were used as controls. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (f) Quantification of peak rhod-2 fluorescence following histamine stimulation. ***P0.001 (mean±s.e.m.; n = 3).",
    "molecules": "Ca2+, histamine"
  },
  {
    "caption": "(a) AMP/ATP ratios in stable HeLa cell lines stably expressing negative shRNA (Neg shRNA), MCUR1 shRNA (clone shHe2) or shHe2 with MCUR1 re-expressed. **P0.01 (mean±s.e.m.; n = 3).",
    "molecules": "AMP, ATP"
  },
  {
    "caption": "(b) O2 consumption rates (OCR) in stable HeLa cells expressing irrelevant shRNA, clone shHe2, and clone shHe2 re-expressing MCUR1, exposed sequentially to oligomycin (i), FCCP (ii) and rotenone plus myzothiazol (iii).",
    "molecules": "myzothiazol, FCCP, oligomycin, O2, rotenone"
  },
  {
    "caption": "(c) Basal and maximal O2 consumption rates in cells as described in b. *P0.05 (mean±s.e.m.; n = 3).",
    "molecules": "O2"
  },
  {
    "caption": "(f,g), The same as in d and e. Activation of AMPK (f) and autophagy (**P0.001; mean±s.e.m.; n = 3) (g) in the absence and presence of InsP3R inhibitor xestospongin B (XeB). *P0.05, **P0.001, NS, not significant (mean±s.e.m.; n = 3). Uncropped images of blots/gels are shown in Supplementary Fig. S6.",
    "molecules": "xestospongin B"
  },
  {
    "caption": "Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion. Mitochondria from HeLa FBXL4-FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X-100 buffer. Different mitochondrial preparations were then digested with Protease K or trypsin.",
    "molecules": "Triton X-100, trypsin"
  },
  {
    "caption": "Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction. Purified mitochondria from HeLa FBXL4-FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions. S: supernatant; P: pellet.",
    "molecules": "alkaline carbonate"
  },
  {
    "caption": "Immunoprecipitation analysis of the ubiquitination of BNIP3. FLAG-BNIP3 (knockin), FBXL4-KO, HA-ubiquitin (Ub) HeLa cells were rescued with WT or ∆F FBXL4, and treated with DMSO or MG132 (20 μM) for 8 hours.   Immunoprecipitation analysis of the ubiquitination of NIX. FLAG-NIX (knockin), FBXL4-KO, HA-Ub HeLa cells were treated the same as (F).  ",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Immunoprecipitation analysis of the integrity of the SCF-FBXL4 complex. Upper: FBXL4-KO HeLa cells expressing vector, WT or mutant FBXL4-FLAG were crosslinked with DSP and subject to anti-FLAG immunoprecipitation; lower: quantification of the ratio of Cullin1/FLAG band intensities.",
    "molecules": "DSP"
  },
  {
    "caption": "Viability of the indicated mice. n: number of mice analyzed.   Immunoblot analysis of organs from the indicated mice at P0 of age. Elevated BNIP3 and NIX levels are highlighted by red boxes; reduced mitochondrial proteins are highlighted by blue boxes. Three mice for each genotype were analyzed. Protein samples from HeLa cells treated with DMSO or Actinomycin D (ActD) were used as positive control for PARP cleavage.  ",
    "molecules": "ActD, Actinomycin D, DMSO"
  },
  {
    "caption": "C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography.",
    "molecules": "DMSO, SnPP, CoPP, NaCl"
  },
  {
    "caption": "C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Total leukocyte and red blood cell count in PB. CoPP increases the number of all WBC populations. Granulocyte and monocyte percentage are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP.",
    "molecules": "DMSO, SnPP, CoPP, NaCl"
  },
  {
    "caption": "C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box).",
    "molecules": "DMSO, SnPP, CoPP, NaCl"
  },
  {
    "caption": "C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6 and IL-5. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 5 mice per group.",
    "molecules": "DMSO, SnPP, CoPP, NaCl"
  },
  {
    "caption": "C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP decreases percentage of Ly6G+ macrophages in the BM",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP increases relative spleen weight.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. viSNE maps of CD11b+ CD11c- blood cells, colored by SSC value. Three independent experiments were performed and the results were pooled. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 7 mice per group.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. (A) Representative flow cytometry plots show higher granularity (SSC) and Ly6G expression in CoPP-mobilized granulocytes than in G-CSF mobilized granulocytes.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in blood of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in BM (C) of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF.",
    "molecules": "DMSO, CoPP, NaCl"
  },
  {
    "caption": "C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. The percentage of cells isolated from C57BL/6xFVB mice treated with G-CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli.",
    "molecules": "DMSO, CoPP, reactive oxygen species, NaCl"
  },
  {
    "caption": "CoPP mobilizes higher numbers of HSC (KLS CD48-CD150+), MPP (KLS CD48-CD150-), HPC-1 (KLS CD48+CD150-) and HPC-2 (KLS CD48+CD150+) cells than G-CSF to the PB.",
    "molecules": "CoPP"
  },
  {
    "caption": "Treatment with CoPP increases percentage of KLS cells in BM, what is related to increase in HPC populations, but not in LT-HSC fraction.",
    "molecules": "CoPP"
  },
  {
    "caption": "The number of KLS- (c-Kit+Lin-Sca-1-) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage committed progenitors: GMP (granulocyte-macrophage progenitors, KLS- CD48+CD150-CD34+) and MEP (megakaryocyte-erythroid progenitors, KLS- CD48+CD150+CD34-) were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS- CD48-CD150-CD34-).",
    "molecules": "CoPP"
  },
  {
    "caption": "Both G-CSF and CoPP treatments decrease the KLS- percentage in the BM, however the decrease after CoPP is smaller. G-CSF and CoPP decrease percentages of MEP and EP, but only G-CSF decreases the percentage of GMP. Results are shown as mean + individual values, one-way Anova with Bonferroni post-test, n = 7 mice per group.",
    "molecules": "CoPP"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. (A) Effect of G-CSF and CoPP-induced mobilization in C57BL/6-Tg(UBC-GFP)30Sch/J donor mice (mean + individual values, one-way Anova with Bonferroni post-test, n = 16 mice per group): Donor mice treated with CoPP had the highest number of mobilized granulocytes (i) and KLS cells (ii), compared to control or G-CSF-treated donor mice",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest number of WBC, PLT and the highest hematocrit values 2 weeks after PBMC transplantation, compared to G-CSF-mobilized and control PBMC recipients (mean + individual values, one-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest chimerism among CD45+ cells, granulocytes and B cells 2, 4 and 18 weeks after transplantation. Chimerism among T cells was the highest in recipients of G-CSF-mobilized PBMC 2 weeks after transplantation, but 18 weeks after transplantation it was the highest in CoPP-mobilized PBMC recipients (mean + SEM, two-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. The majority of primary recipients transplanted with G-CSF and CoPP-mobilized PBMC have detectable GFP chimerism among BM KLS cells.",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Fraction of secondary recipient mice with chimerism in PB CD45+ cells exceeding 1% tends to be higher after CoPP-mobilized PBMC transplant than G-CSF-mobilized PBMC transplant.",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. PB chimerism in secondary recipients is higher in CoPP group than in the control group (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Percentage of secondary recipient mice with GFP chimerism in BM KLS (G) and KLS-(H) cells higher that 1% is similar in G-CSF and CoPP groups.",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. GFP chimerism in KLS (I) and KLS- (J) cells in the BM of secondary recipients (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).",
    "molecules": "CoPP, NaCl"
  },
  {
    "caption": "Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. G-CSF and IL-6 concentration in plasma",
    "molecules": "CoPP"
  },
  {
    "caption": "Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. CD45+ cells, granulocytes and HSC numbers in PB measured by flow cytometry.",
    "molecules": "CoPP"
  },
  {
    "caption": "HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Total leukocyte counts in PB (HO-1+/+: n = 5, HO-1-/- DMSO: n = 6, HO-1-/- CoPP: n = 4 mice per group). Granulocyte percentage among PB leukocytes.",
    "molecules": "DMSO, CoPP"
  },
  {
    "caption": "HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Cytokine and growth factor concentrations in plasma (E - HO-1+/+ n = 5, HO-1-/- DMSO: n = 5, HO-1-/- CoPP: n = 3 mice per group). Results are shown as mean + SEM, two-way Anova with Bonferroni post-test.",
    "molecules": "DMSO, CoPP"
  },
  {
    "caption": "Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). CD45+ cell numbers in PB measured by flow cytometry. Granulocyte percentage among PB leukocytes.",
    "molecules": "DMSO, CoPP"
  },
  {
    "caption": "Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). Cytokine and growth factor concentrations in plasma",
    "molecules": "DMSO, CoPP"
  },
  {
    "caption": "Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (A) Morphology of the ENCC-derived neurospheres on days 2 and 4. (B-C) The proliferation of ENCC-derived neurospheres is represented by neurosphere diameter (B) and change in diameter on day 4 compared with day 2 (C).",
    "molecules": "BAC"
  },
  {
    "caption": "Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (D) ENCC viability assessed by CCK-8 assay.",
    "molecules": "BAC"
  },
  {
    "caption": "Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (E) ENCCs number analyzed by nuclear staining with DAPI.",
    "molecules": "BAC, DAPI"
  },
  {
    "caption": "Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (F) Migration of ENCCs evaluated by transwell plate assay and stain with crystal violet,",
    "molecules": "BAC, crystal violet"
  },
  {
    "caption": "Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls. (G) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry.",
    "molecules": "BAC, FITC, PI"
  },
  {
    "caption": "Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (A-D) Cluster analysis at the (A) phylum and (C) genus levels. A column accumulation diagram of the top 10 most abundant (B) phyla and (D) genera.",
    "molecules": "BAC"
  },
  {
    "caption": "Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. Inter-group diversity analysis to compare the differences in (H) intestinal microbiota The species indicated in red are linked to SCFAs production.",
    "molecules": "BAC, SCFAs"
  },
  {
    "caption": "Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. metabolic mass spectrometry to compare the differences (I) SCFAs metabolites between the hypoganglionosis and sham rats.",
    "molecules": "BAC"
  },
  {
    "caption": "Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (J) Representative HE staining and quantification to assess the intestinal mucosal injury.",
    "molecules": "BAC"
  },
  {
    "caption": "Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (K) Representative IHC stain and quantification of CRP and TNF-α levels.",
    "molecules": "BAC"
  },
  {
    "caption": "Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively. (L) Representative immunofluorescence stain and quantification of β-catenin levels. Nuclei were stained blue with DAPI.",
    "molecules": "BAC, DAPI"
  },
  {
    "caption": "Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (C-D) Migration of ENCCs using transwell plate assay and stain with crystal violet (C), scale bar, 200 µm. (D) Quantification of C. (E-F) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (E). (F) Quantification of E.",
    "molecules": "crystal violet, FITC, PI"
  },
  {
    "caption": "Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. (H) Laparoscopic analysis indicating that the same segments were treated with BAC in all the groups.",
    "molecules": "BAC"
  },
  {
    "caption": "Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. Comparison of neuronal markers. (J) Immunofluorescence stain and number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell.",
    "molecules": "DAPI"
  },
  {
    "caption": "Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat. Comparison of neuronal markers. (K) Representative immunofluorescence images of Tuj1 and GFP localization at the injection and adjacent sides. Asterisks and triangles indicate the Tuj1+/GFP+ cells and Tuj1+/GFP- cells, respectively. Nuclei were stained blue with DAPI. (L-M) Number of Tuj1+, Tuj1+/GFP+ cells (L) and Tuj1+/GFP- cells(M).",
    "molecules": "DAPI"
  },
  {
    "caption": "Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (A-D) Cluster analysis at the (A) phylum and (C) genus. Column accumulation diagrams of the top abundant microbiota at the (B) phylum and (D) genus levels.",
    "molecules": "Antibiotic, antibiotics, short-chain fatty acids"
  },
  {
    "caption": "Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (G) SCFAs subsets in the feces and intestinal extracts, analyzed by targeted mass spectrometry.",
    "molecules": "Antibiotic, antibiotics"
  },
  {
    "caption": "Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. (H) IL-10, IL-17, and TNF-α levels in serum and intestinal extracts, assessed by ELISA.",
    "molecules": "Antibiotic, antibiotics"
  },
  {
    "caption": "Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups. Correlation analysis of acetate, propionate or butyrate with IL-17 in the intestinal extracts.",
    "molecules": "acetate, Antibiotic, antibiotics, butyrate, propionate"
  },
  {
    "caption": "Phenotype analysis of ENCCs treated with SCFAs. (B-C) Migration of ENCCs evaluated using transwell plate assay and stain with crystal violet (B); Scale bars represent 200 µm. (C) Quantitative analysis of B.",
    "molecules": "crystal violet"
  },
  {
    "caption": "Phenotype analysis of ENCCs treated with SCFAs. (G-H) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (G). (H). Quantification of G.",
    "molecules": "FITC, PI"
  },
  {
    "caption": "(C-D) Representative immunofluorescent images (C) and (D) number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) ENCC viability assessed by CCK-8 assay after intervention with MEK1/2 inhibitor (U0126, 50 μM), p38 inhibitor (SB203580, 1 μM), JNK inhibitor (SP600125, 50 μM) and MER5 inhibitor (BIX 02189, 10 μM).",
    "molecules": "SP600125, BIX 02189, SB203580, U0126"
  },
  {
    "caption": "migration of ENCCs evaluated using transwell plate assay and stain with crystal violet on day 2 (G, H) show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration.",
    "molecules": "crystal violet, U0126"
  },
  {
    "caption": "migration of ENCCs evaluated using transwell plate assay show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration.",
    "molecules": "U0126"
  },
  {
    "caption": "(C) Subcellular localization of WT Lint-O and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Subcellular localization of WT L(3)mbt-L and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(I) Subcellular localization of WT Lint-O and its 8CA and ∆PHD mutants (green). All proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. B. Band intensities of Kap -βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.",
    "molecules": "Coomassie blue, SDS"
  },
  {
    "caption": "A, B. Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC. SEC (Superdex 200) elution profiles of the (A) Kap114pRanQ69L and (B) Kap114pyTBP complexes. Peak fractions were analyzed by SDS-PAGE and stained with Coomassie blue. The void volume (Vo) of the peak fraction and absorbance (a.u.) at 280 nm for each complex is indicated.",
    "molecules": "Coomassie blue, SDS"
  },
  {
    "caption": "B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.",
    "molecules": "Coomassie blue, SDS"
  },
  {
    "caption": "G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran. ",
    "molecules": "Coomassie blue, SDS"
  },
  {
    "caption": "I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein. ",
    "molecules": "Coomassie blue, SDS"
  },
  {
    "caption": "B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).",
    "molecules": "FAM, TGTATGTATATAAAAC, DNA, oligonucleotide"
  },
  {
    "caption": "C. Indicated concentrations (0.25 to 16 μM) of purified recombinant Kap114p were incubated with yTBP (4 μM) and FAM-labeled DNA (5 nM), followed by native PAGE analysis. Kap114p (16 μM) was incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).",
    "molecules": "FAM, DNA"
  },
  {
    "caption": "D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).",
    "molecules": "FAM, DNA"
  },
  {
    "caption": "E. Indicated concentrations (0.06 to 4 μM) of purified Kap114p were added to the reaction containing 0.5 μM of TFIIA, yTBP and FAM-labeled DNA (TGTATGTATATAAAAC), before conducting native PAGE analysis. TFIIA and Kap114p (4 μM) were incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). Electrophoretic bands containing protein-DNA complex and free DNA are indicated.",
    "molecules": "FAM, TGTATGTATATAAAAC, DNA"
  },
  {
    "caption": "A. Wild type and KAP114 knockout strains were diluted serially and spotted onto YPD plates in the presence of 1.5 M NaCl.",
    "molecules": "NaCl"
  },
  {
    "caption": "B. The KAP114 knockout strain was transformed with vector control (lane 2) and a plasmid containing wild type KAP114, KAP114 (Δ347-371), or KAP114 (Δ899-956) (lanes 3-5). Wild type and KAP114 knockout strains containing plasmids were serially diluted and grown in minimal medium plates supplemented with 1.5 M NaCl. Growth temperatures are indicated.",
    "molecules": "NaCl"
  },
  {
    "caption": "C. A heatmap generated by three independent biological replicates shows gene expression profiles of wild type and KAP114 knockout strains grown in the presence of 1.5 M NaCl. The Log2 (fold-change) values and the color scale are shown.",
    "molecules": "NaCl"
  },
  {
    "caption": "A) RT-PCR (30 cycles) showing skipping of exon 23 for PMO and PS49 oligos. No skipping was visible in saline treated mice.",
    "molecules": "PMO, PS49, saline"
  },
  {
    "caption": "B) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration. M is molecular ladder. WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle. NT is saline treated mdx mice.",
    "molecules": "saline"
  },
  {
    "caption": "C) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset (logFC WT vs mdx at week 12).",
    "molecules": "PMO, PS49"
  },
  {
    "caption": "D, E) Histograms comparing the distribution of selected genes across different mice groups (PMO, PS49 and saline in the MoTreat dataset, WT and mdx at week 12 in the MoLong dataset).",
    "molecules": "PMO, PS49, saline"
  },
  {
    "caption": "F) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al. (2012).",
    "molecules": "steroid"
  },
  {
    "caption": "G) Scatter plot showing genes for which a significant effect of treatment with steroids. The group effects at baseline are plotted on the x-axis, while the effects of steroids are plotted on the y-axis.",
    "molecules": "steroids"
  },
  {
    "caption": "A Affinity measurements by  biolayer interferometry  (BLI) of bipNbs  NM1267 and NM1268. NM1267  and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD derived from SARS-CoV-2 B.1  (RBDB.1). Global 1:1 fits are illustrated as dashed lines. Global 1:1 fits are illustrated as dashed lines and  binding  affinity (KD), association (kon) and dissociation constant (koff) determined for the individual bipNbs are summarized.",
    "molecules": "NM1267, NM1268"
  },
  {
    "caption": "A-P  Affinity  measurements by BLI  of bipNb NM1267  and NM1268 on recently identified RBD variants  B.1.1.7 (Alpha) (A, I), B.1.351 (Beta) (B, J), P1 (Gamma) (C, K), B.1.617.2 (Delta) (D, L), B.1.429 (Epsilon) (E, M), P3 (Theta) (F, N), B.1.617.1 (Kappa) (G, O) and A.23.1 (H, P). NM1267 and  NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD variants. Global 1:1 fits are illustrated as dashed lines.",
    "molecules": "NM1267, NM1268"
  },
  {
    "caption": "A-F Neutralization potency of  NM1267 and NM1268 were analyzed in Caco‑2  cells using the SARS-CoV-2 B.1 (A, D) SARS-CoV-2 B.1.351 (Beta) (B, E) and  SARS-CoV-2 B.1.617.2 (Delta) (C, F). Infection normalized to virus-only infection control is illustrated as percent of infection (infection [%]). Data are presented as mean ± SEM of three (n = 3) biological replicates.",
    "molecules": "NM1267, NM1268"
  },
  {
    "caption": "C Hemizygous  K18-hACE2 mice  were treated intranasally with 20 µg of NM1251 (n = 15) or NM1267 (n = 12) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.  survival were monitored for 14 days. , ****P < 0.0001, by log-rank test",
    "molecules": "NM1251, NM1267"
  },
  {
    "caption": "Histopathological analysis of mice, which were intranasally treated with bipNb  NM1267 or control Nb NM1251 and subsequently infected with 3*103 PFU SARS-CoV-2 B.1. A-B Serial tissue sections revealed severe inflammation (H&E) and numerous widespread SARS-CoV-2 RNA positive  alveolar epithelia cells and macrophages (in situ hybridization (ISH)) in  lungs  of infected, control-treated mice. In infected and NM1267-treated animals no inflammation or only small focal areas with inflammation and a few SARS-CoV-2 RNA positive cells were observed. Scale bars represent 1 mm and 200 µm, respectively.",
    "molecules": "NM1251, NM1267"
  },
  {
    "caption": "B, Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or  NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta).  Weight loss were monitored for 14 days. Dashed line indicates humane endpoint and symbols represent mean ± SEM ****P < 0.0001 in orange between NM1251  and NM1267  and in purple between  NM1251 and  NM1268, *P < 0.1 in black between  NM1267  and NM1268, by two-way ANOVA with Sidak's multiple comparison test",
    "molecules": "NM1251, NM1267, NM1268"
  },
  {
    "caption": "C Hemizygous K18-hACE2 mice  were treated intranasally with 20 µg of NM1251 (n = 8), NM1267  (n = 9) or  NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2  (Delta). survival were monitored for 14 days. ***P < 0.001 in orange between NM1251 and NM1267 and ****P < 0.0001 in purple between NM1251 and  NM1268, by log-rank test",
    "molecules": "NM1251, NM1267, NM1268"
  },
  {
    "caption": "Two top-ranked SplashRNA-designed shRNAmirs targeting  human or  mouse surface protein  FAS  were cloned into Venus-expressing  miR-AB lentiviral  vectors which harbor different promoters to drive the shRNAmir expression. The packaged  lentiviruses were used to infect the indicated human or mouse cell lines. A shRNAmir targeting human or mouse CD4 driven under hCMV  promoter was used as control. After >96h of infection,  FAS  protein levels in the transduced  Venus-positive cells were determined by surface  staining and shown in histograms (top panels) and quantified by MFI of  APC (bottom panels).",
    "molecules": "APC"
  },
  {
    "caption": "293T cells were co-transduced with four  miR-AB lentiviruses  at 1:1 ratio, which carries a neutral shRNAmir targeting CD4 or CD19, with Azurite, GFP, Ametrine or mOrange as reporter respectively. Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated. Representative contour FACS  plots cells on day 4 and day 22 shown (top panels). Cell numbers of untransduced, single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4, and plotted over time (bottom panel). Data are shown as mean ± s.d. and n=4. NS indicates no significant difference (P>0.05, two-tailed unpaired Student's t-test).",
    "molecules": "Ametrine, Azurite"
  },
  {
    "caption": "CD8+ T cells were activated for 16h and then co-transduced with  miR-AB retroviruses at 1:1 ratio, which carries a SplashRNA-designed shRNAmir, targeting surface protein CD127,  CD90, CD44  or  CD8, with  Azurite, GFP,  Ametrine or mCherry2 as fluorescent reporters respectively for 4h. miR-AB retroviruses  expressing CD19-specific shRNAmirs with same fluorescent reporters were used as controls. Surface staining was performed 72h after transduction. FACS data were shown as overlaid histograms of control shRNAmirs (shCD19s) and target shRNAmirs (shCD127, shCD90, shCD44 or shCD8). Cells were categorized as sixteen populations expressing single, double, triple or quadruple fluorescent proteins or not (none) and fluorescent reporter-positive plots were highlighted with thick frames and white background. Each population's surface  staining   profile was shown on the right. The data was representative of two biologically independent experiments.",
    "molecules": "Ametrine, Azurite"
  },
  {
    "caption": "(a) Western blot analysis of CALM, actin, GAPDH and LC3-II in several cell lines (HeLa, HEK, CAD and murine embryonic fibroblast (MEF)) where CALM was knocked down using shRNA or siRNA, as indicated, or knockout with rescue experiment. In all experiments in this paper, we used a scramble siRNA or a luciferase shRNA as controls. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (BC, basal conditions; SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin or GAPDH ratio is shown in Supplementary Fig. 1A. *Not specific.",
    "molecules": "Baf A1"
  },
  {
    "caption": "(d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P0.01; two-tailed t-test). Scale bars, 5 μm.",
    "molecules": "Baf A1"
  },
  {
    "caption": "(e) In vitro fusion assay of post-nuclear supernatant from HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1 in control and CALM knockdown conditions. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control for the reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles is represented. n=numbers of vesicles scored per field (a minimum of five fields were analysed per condition). Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test).",
    "molecules": "ATP"
  },
  {
    "caption": "(b) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells where VAMP2 was knocked down, as indicated. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure.) Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; NS, not significant, two-tailed t-test).",
    "molecules": "Baf A1"
  },
  {
    "caption": "(f) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells stably expressing VAMP2-HA wild type or VAMP2-HA mutated in the CALM-binding site at different levels (wild-type clone 6, wt Cl6: low level; wild-type clone 11, wt Cl11: high level; mutant clone 11, AA Cl11: low level; mutant clone 12, AA Cl12: high level). The cells were treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).",
    "molecules": "Baf A1"
  },
  {
    "caption": "(e) In vitro fusion assay of post-nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control of reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles (white) is represented. Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test). n=numbers of vesicles scored per field.",
    "molecules": "ATP"
  },
  {
    "caption": "(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafish larvae where ATG7 was downregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II in zebrafishlarvae where CALM-HA was expressed, as indicated. The larvae were treated with ammonium chloride (NH4Cl), as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).",
    "molecules": "ammonium chloride, NH4Cl"
  },
  {
    "caption": "(b) Modulation of autophagy alters Dendra-tau clearance dynamics. The fluorescence intensity of each individual cell was quantified at each timepoint (n≥ 31 cells, ≥9 larvae per treatment group) and mean cell intensity values for each drug treatment at each timepoint were calculated. Images were taken immediately after photoconversion and at 24, 30 and 48 h intervals thereafter. Rapamycin treatment significantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatment significantly decreases the rate of Dendra-tau clearance (**P0.01, ***P0.001, one-way analysis of variance (ANOVA)). Error bars are ±s.e.m.",
    "molecules": "Ammonium chloride, NH4Cl, Rapamycin"
  },
  {
    "caption": "(c) Dendra-tau clearance in the presence of CALM: representative images of CALM with mosaic expression of Dendra-tau and full-length Dendra-tau taken immediately after photoconversion and at 6, 24, 30, 48 and 54 h after conversion. The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co-expressing Dendra-tau and either full-length CALM or Δ-ANTH CALM (larvae-ANTH-HA) constructs (n≥ 100 cells, ≥9 larvae per treatment group) at each time point was calculated. (i) Expression of full-length HA significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM (***P0.001, one-way ANOVA). One graph, representative of three independent experiments, is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-CALM: 18 cells, 8 fishes). Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii) Treatment with CALM alters the dynamics of CALM clearance. Expression of full-length CALM significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM, as observed in i (P0.001, one-way ANOVA). However, treatment of Δ-ANTH CALM injected CALM with CALM slows the CALM clearance by 70% to a level comparable to that observed in larvae injected with full-length Dendra-tau. Ammonium chloride treatment of Dendra-tau injected with full-length null results in a modest (22%) decrease in Dendra-tau clearance, suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect (n≥25 cells, n≥9 Dendra-tau per treatment group). Error bars are mean ±s.d. Note that i and ii are distinct experiments.",
    "molecules": "ammonium chloride, Ammonium chloride"
  },
  {
    "caption": "(a) rho::GFP-tau fishes were incubated from 3 to 9 d.p.f. in either dimethyl sulphoxide (DMSO) or rapamycin (i) or from 3 to 7 d.p.f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin (ii). Images through the central retina at 9 d.p.f. (top panel; (i)) reveal rod degeneration (arrow) throughout the retina in control (DMSO-treated) larvae, whereas rod photoreceptors are present throughout the retina, particularly in the central region following treatment with rapamycin (arrow). Images taken through the central retina at 7 d.p.f. (bottom panel; (ii)) show normal photoreceptors in the marginal zones (arrows) and only limited numbers in the central region. NH4Cl exacerbates degeneration-photoreceptors are absent from the central retina and reduced/absent from marginal zones (arrows). Sections were stained with anti-rhodopsin (1D1) antibody. GFP labels whole rod photoreceptors, whereas rhodopsin is present in the rod outer segment. GFP co-localizes with the red rhodopsin label in all experimental conditions. Scale bars, 50 μm. Quantification of rodphotoreceptor degeneration (n=10 larvae per group; ***P0.001; ###P0.001, two-tailed unpaired t-test). Error bars are mean ±s.d.",
    "molecules": "ammonium chloride, NH4Cl, dimethyl sulphoxide, DMSO, rapamycin, Wortmannin"
  },
  {
    "caption": "(c) NH4Cl treatment of rho::GFP-tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control (non-electroporated) side but did not alter the degeneration caused by CALM electroporation. Rapamycin treatment of Rapamycin::GFP-tau immediately after unilateral CALM electroporation rescued photoreceptors on the control (non-electroporated) side but not on the CALM-electroporated side. To demonstrate that loss of GFP corresponds to loss of photoreceptors, sections were stained with anti-rhodopsin (1D1) antibody. Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish; Mann-Whitney test was used to compare drug treatment in the right eye of different fishes. *P0.05. Scale bars, 50 μm. Error bars are mean ±s.e.m.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "C. Confirmation of sensor localization to different cellular compartments. Green, HyPer7; blue, DAPI stain",
    "molecules": "DAPI"
  },
  {
    "caption": "D.-H. Response of  HyPer7 probes targeted to indicated compartments to incubation with antimycin A (a  complex III inhibitor, red curve and data points) or ethanol  as control (black line and data points). HEK293 cells were grown either with glucose or galactose as carbon  source as indicated. Solid line represents mean, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. **, p ≤ 0.01, ***, p ≤ 0.001",
    "molecules": "antimycin A, ethanol, galactose, glucose"
  },
  {
    "caption": "A-D. Response of HyPer7  probes targeted to indicated compartments to reoxygenation  after hypoxia (red curve and data points) or to continued hypoxia (black line and data points). HEK293 cells were grown either with glucose or galactose as  carbon source. Solid line represents mean, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. E. Model. Cells grown on galactose-containing medium exhibit increased production of H2O2 and thus also increased detection of cytosolic H2O2  indicating H2O2 gradients of different steepness around mitochondria. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs.***, p ≤ 0.001",
    "molecules": "galactose, glucose, H2O2"
  },
  {
    "caption": "C. Response of  HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing HEK293  cells with 2 mM  D-Ala (cells generated with the Flp-In T-REX-system). HEK293  cells were grown either with  glucose (black curve and data points) or galactose  (red curve and data points) as carbon  source. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001",
    "molecules": "D-Ala, galactose, glucose"
  },
  {
    "caption": "A. Response of HyPer7 probes targeted to indicated compartments to treatment with increasing amounts of exogenous H2O2 (2, 4 and 8 µM  H2O2; 20 mM DTT). HEK293 cells were grown either with glucose (black curve and data points) or galactose (red curve and data points) as carbon  source. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001",
    "molecules": "DTT, galactose, glucose, H2O2"
  },
  {
    "caption": "A. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by quantitative mass spectrometry (n = 4 biological replicates Levels of antioxidative enzymes (especially peroxiredoxins, PRDX  in blue) are not changed except for  cytosolic thioredoxin reductase (TXNRD1).",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "B. Protein levels in cells grown on glucose  and galactose. Cell lysates were analyzed by  immunoblot  (n = 2 biological replicates). Cytosolic thioredoxin reductase  (TXNRD1) is increased by more than two-fold in cells grown on galactose compared to glucose-grown cells.The dotted line depicts 1, meaning no change in protein levels.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "B. Response of HyPer7  probes targeted to indicated compartments to incubation with antimycin A (AntA, red curve and data points) or ethanol as control (black curve and data points). HeLa  cells were grown with  galactose as carbon source. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001",
    "molecules": "AntA, antimycin A, ethanol, galactose"
  },
  {
    "caption": "D. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1  KO; blue, PRDX2  KO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001",
    "molecules": "D-Ala, glucose"
  },
  {
    "caption": "E. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala  (black, wild type; red, PRDX1/2 DKO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: Statistical analysis was only performed to compare the PRDX1/2 DKO with the wildtype. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001",
    "molecules": "D-Ala, glucose"
  },
  {
    "caption": "(B) Growth inhibition upon overexpression of HOS3-NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1. 10-fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids, were spotted onto SC-Glu and SC-Gal medium (to activate the GAL1 promoter) and incubated at 25 °C for 3 days. Note that HOS3-NLS (under the control of the native HOS3 promoter) does not affect cell growth.",
    "molecules": "Gal, Glu"
  },
  {
    "caption": "(C) esa1-ts and gcn5Δ esa1-ts mutants have bud emergence defects. (Top) Cells of the indicated strains were arrested in G1 by treatment with ɑ-factor for 2.5 h at 25 ºC, shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC. Cells were fixed at the indicated times and the presence of buds was assessed by microscopy. Mean and SEM are derived from n = number of independent experiments. At least 200 cells were scored for each strain and time point. (Bottom) Bright field images of the indicated strains 60 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.",
    "molecules": "ɑ-factor"
  },
  {
    "caption": "(D) Inactivation of ESA1 and GCN5 delays DNA replication. Cells of the indicated genotypes were synchronised as in panel C, and DNA content was evaluated by flow cytometry. Numbers indicate time in minutes after the release. This experiment was repeated three times with similar results; one experiment is shown.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60. (Top) Nup60-GFP was immunoprecipitated from extracts of the indicated strains, and its acetylation state probed with anti-AcLys antibodies. (Bottom) Total extracts probed with anti-HA antibodies to verify KAT overexpression.",
    "molecules": "AcLys"
  },
  {
    "caption": "(B) nup60-KN partially rescues the budding defect of esa1-ts cells. (Left) Budding of cells of the indicated strains were determined as in Figure 1C. At least 200 cells were scored for each strain and time point. Data from 3 independent experiments is represented as mean and SEM (esa1-ts, esa1-ts nup60-KN). (Right) Bright field images of the indicated strains 40 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.",
    "molecules": "ɑ-factor"
  },
  {
    "caption": "(B) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3-NLS overexpression. 10-fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC-Glu and SC-Gal medium and incubated at 25 °C for 3 days.",
    "molecules": "Gal, Glu"
  },
  {
    "caption": "(D) Overexpression of Hos3-NLS promotes nuclear accumulation of mRNA. (Left) Cultures of the indicated strains were treated with galactose overnight to induce HOS3-NLS expression, cells were fixed, and FISH was performed using Cy3-Oligo(dT). (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions. Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.",
    "molecules": "Cy3, DAPI, galactose, mRNA, Oligo(dT), polyadenylated RNA"
  },
  {
    "caption": "(E) Inactivation of Esa1 impairs export of poly(A) RNA. (Left) Cultures of the indicated strains were incubated at 37 ºC. (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.",
    "molecules": "DAPI, mRNA, poly(A) RNA, polyadenylated RNA"
  },
  {
    "caption": "(F) nup60-KN mutation partially rescues the mRNA export defects of esa1-ts. Cells of the indicated strains were processed Data information: arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.",
    "molecules": "DAPI, mRNA, polyadenylated RNA"
  },
  {
    "caption": "(B) Inactivation of Esa1 decreases Sac3 nuclear levels. Wild-type (WT) and esa1-ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC, shifted to 37 ºC, released from the mitosis block in fresh medium at 37 ºC, and imaged by time-lapse microscopy. Fluorescence levels were quantified in G1 Data information: arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(C) Rapamycin-dependent dimerisation abolishes Sac3 mother/daughter asymmetries. NUP60-mCherry-FKBP SAC3-GFP-FRB cells were incubated with rapamycin (RAPA) to trigger FRB-FKBP heterodimerization, or with DMSO as control. Fluorescence levels were quantified in G1 cells 15 to 30 minutes after addition of the drug. Data information arrowheads point to daughter cells, boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.",
    "molecules": "DMSO, RAPA, Rapamycin, rapamycin"
  },
  {
    "caption": "(D) Sac3 anchoring to the nuclear basket advances Start in esa1-ts daughter cells. Composite of bright field and Whi5-mGFP (top) and quantification of Whi5 nuclear export timing (bottom) in wild-type (WT) and esa1-ts mother (M) and daughter (D) cells treated with either rapamycin (RAPA) or DMSO and expressing Nup60-FRB and Sac3-mCherry-FKBP. Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells (DMSO vs RAPA, p > 0.05, Log-rank Mantel-Cox test), but it advances Whi5 export in esa1-ts daughters (p = 0.0001). Whi5 export efficiency was slightly improved also in mother cells (p = 0.0374). 8 z-confocal slices spaced 0.4 µm were acquired every 3 min; maximum projections are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. Data information: arrowheads point to Whi5 export.",
    "molecules": "DMSO, RAPA, rapamycin"
  },
  {
    "caption": "(A) Time Lapse microscopy of WT and nup60-KN cells expressing GAL1pr:sfGFP and Nup60-mCherry at the indicated times of galactose induction. Scale bar, 4 µm.",
    "molecules": "galactose"
  },
  {
    "caption": "(B) Depletion of Hos3, and expression of acetyl-mimic Nup60 (nup60-KN) enhances GAL1 expression. WT, hos3Δ, nup60-KN and nup60-KN hos3Δ cells were shifted to galactose and imaged by time-lapse microscopy to monitor GAL1pr:sfGFP expression during 7 hours. Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60-mCherry was quantified from sum projections of whole-cell Z-stacks at the indicated times. At least 200 cells were scored for each strain and time point. Shaded areas indicate the SEM.",
    "molecules": "galactose"
  },
  {
    "caption": "(C) mRNA levels of GAL1 were determined for wild type (WT) and nup60-KN cells at the indicated times after galactose addition. One of two independent experiments with similar results is shown (mean and SEM from 3 technical replicates).",
    "molecules": "galactose"
  },
  {
    "caption": "(D) The GFP intensity of mother/daughter pairs for cells in B, at 5-minute intervals after galactose addition (left) and 425 min after galactose addition (right). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.001; ns, p > 0.05, two-tailed paired t-test. n = number of cells. One of two independent experiments with similar results is shown.",
    "molecules": "galactose"
  },
  {
    "caption": "(E) Expression of GAL1pr:sfGFP induced with galactose (left) or ß-estradiol (right), in the presence of the ß-estradiol-dependent transactivator Gal4-ER-VP16. Smooth lines show GFP fluorescence intensity (left x axis); lines with circles show the difference in GFP intensity between the indicated strains (right x axis). The difference between wild type and hos3∆, and between wild type and nup60-KN, increases continuously over time in response to galactose, but not to ß-estradiol.",
    "molecules": "ß-estradiol, galactose"
  },
  {
    "caption": "(A) Cells of the indicated strains were incubated with galactose in the presence of either rapamycin (RAPA) to induce FRB-FKBP heterodimerization, or DMSO as control. GAL1pr:sfGFP expression was monitored over time",
    "molecules": "DMSO, galactose, RAPA, rapamycin"
  },
  {
    "caption": "(B) (Top) Representative images of the indicated cells in rapamycin or DMSO, 250 min after galactose addition. Scale bar 4 µm. (Bottom) Mother/daughter pairs were quantified as in Figure 6D at 250 min after galactose addition (pink shaded area in A). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. **, p ≤ 0.01; *, p ≤ 0.05; ns, p > 0.05, two-tailed paired t-test for M-D comparisons, unpaired for comparisons between strains. n = number of cells. One of two independent experiments with similar results is shown.",
    "molecules": "DMSO, galactose, rapamycin"
  },
  {
    "caption": "(F) Representative image analyses of autophagy markers LC3 and LAMP2 with DAPI in HSCs after culture under pH 7.4 or pH 6.9 conditions (Scale bar = 5μm)",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Relative levels of spermidine and spermine in pH 6.9 and pH 7.4 conditions with respect to untreated samples. n=3 biological replicates. *p < 0.05 and exact p values are mentioned between groups.",
    "molecules": "spermidine, spermine"
  },
  {
    "caption": "(E) Effect of DFMO treatment on pHi of HSCs., n= 5 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.",
    "molecules": "DFMO"
  },
  {
    "caption": "(G) Pictures of HSCs after culture at pH 7.4 or pH 6.9 and in presence or absence of DFMO and SPN for 40 h. Data are representative of 6 biological replicates. Scale bar = 100μm (H) Cell volume of HSCs cultured at pH 7.4 in the presence of DFMO or in combination with SPN. Data are from >80 cells per group, representative of 3 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.",
    "molecules": "DFMO, SPN"
  },
  {
    "caption": "(I,J) Reconstitution level of donor derived -Ly5.2+ cells, -lineage distribution in peripheral blood and BM in primary (I) and secondary (J) recipients from 100 HSCs cultured at pH 7.4 in presence of DFMO or in combination of SPN. Data in figure I,J are from n= 9-12 recipient mice from 2 independent biological transplantation experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed unpaired Student's t-test.",
    "molecules": "DFMO, SPN"
  },
  {
    "caption": "(K) Representative images of autophagy markers LAMP2 with DAPI in untreated HSCs or after culture under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO (Scale bar = 5μm). Bar graphs represent quantification of LAMP2 levels in cells. Data are from > 50 cell representing 3 biological replicates. ***p < 0.001, ****p < 0.0001 using two-tailed unpaired Student's t-test.",
    "molecules": "DAPI, DFMO"
  },
  {
    "caption": "(L) Reconstitution level of donor derived Ly5.2+ cells or human derived cells in peripheral blood in primary recipients from either untreated 100 murine or human HSCs or after culture in serum free medium STEMSpan under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO. n= 7-9 mice from 2 biological transplantation experiments for murine HSC data. For human HSPC transplants, n= 6 recipient per condition from 3 biological transplantation experiments. *p < 0.05, or exact p value mentioned between groups. Statistic significance was calculated using two-tailed unpaired Student's t-test.",
    "molecules": "DFMO"
  },
  {
    "caption": "(B) Representative IF images in CHME3 stably expressing Flag alone control or the Flag-tagged EB1 constructs depicted in A co-stained for Tyrosinated-MTs (Tyr-MTs), and the nucleus (Hoechst). Scale bar, 10 μm.",
    "molecules": "Hoechst, Tyr, Tyrosinated"
  },
  {
    "caption": "Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells either treated with control or CLIP170 siRNAs (A) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing ∼ 9% (Control siRNA) and 3% (CLIP170 siRNA) shift (A) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.",
    "molecules": "CCF2"
  },
  {
    "caption": "Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells expressing Flag control or Flag-CLIP170 (D) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing no difference (D) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.",
    "molecules": "CCF2"
  },
  {
    "caption": "(I) The induction of Ac-MTs is blocked in HIV-1-VSV-luc (Virus) infected, but not in uninfected (NI) or Mock infected CHME3 depleted of EB1, but not CLIP170 at 6 h.p.i. Tyrosinated MTs (Tyr-MTs) are not affected. Representative fields are shown. Similar results were obtained in three independent experiments. Scale bar, 50 μm.",
    "molecules": "Ac, Tyr, Tyrosinated"
  },
  {
    "caption": "(H) Representative images (n=3) of CHME3 expressing Flag-CLIP170 or Flag-CLIP170ΔCAPgly stained for Flag and Tyr-MTs. Scale bar, 10 μm.",
    "molecules": "Tyr"
  },
  {
    "caption": "HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH, as shown by WB analysis (F)",
    "molecules": "Ac"
  },
  {
    "caption": "HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH or imaging at 6 h.p.i (G). Scale bar, 15 μm. Representative fields are shown. Results are representative of three experimental replicates.",
    "molecules": "Ac"
  },
  {
    "caption": "FRhK4 cells infected with SIV-VSV-luc (F) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i",
    "molecules": "Hoechst, Ac, Tyr"
  },
  {
    "caption": "Rat2 cells infected with MuLV-VSV-luc (G) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i,",
    "molecules": "Hoechst, Ac, Tyr"
  },
  {
    "caption": "B. Melanoma  cells were treated with increasing concentrations of THZ1 as indicated for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. IC50 for each cell line is indicated. Melanocytic-type (MITF-High, proliferative) melanoma cells are shown in red, while mesenchymal-like (MITF-low, invasive) melanoma cells are shown in blue. Data information: data are presented as mean values + Standard Deviation (SD) for three replicates (n=3).",
    "molecules": "DMSO, THZ1"
  },
  {
    "caption": "J. MM029 (left) and MM099 (right) were pre-treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of  THZ1 for 72h. Mean growth   is shown relative to vehicle (DMSO)-treated cells. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant",
    "molecules": "DMSO, THZ1"
  },
  {
    "caption": "C-D.  MM099 (c) and  MM029 (d) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. E-F. MM099 (e) and MM029 (f) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing doses  of Vemurafenib for 72h. Mean growth  is shown relative to vehicle (DMSO)-treated cells. D Data information: data are presented as mean values + SD for three replicates (n=3). IC50 for each cell line is indicated.",
    "molecules": "DMSO, THZ1, Vemurafenib"
  },
  {
    "caption": "C-E. qRT-PCR  analysis showing average  TBP-normalized fold expression of MITF (C), SOX10 (D) and GATA6 (E) in 501mel treated either with DMSO/THZ1 (50nM) (upper) or DMSO/JQ1  (10μM) (lower) for 24h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.",
    "molecules": "DMSO, JQ1, THZ1"
  },
  {
    "caption": "D. MM099MITF-SOX10-PAX3 expressing inducible  MITF-SOX10-PAX3  genes was treated or not with Doxycycline (1μg/ml) for 24h and protein lysates were immuno-blotted  for the indicated protein. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity  using ImageJ software, and normalized relative to VINCULIN.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "E. qRT-PCR  analysis showing average TBP-normalized fold expression of GATA6, ABCG2, AMIGO2 or  SERPINE1  in  MM099MITF-SOX10-PAX3 treated or not with Doxycycline (1μg/ml) for 24h. Data information: data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "A. Rab25 expression decreases nutrient withdrawal (24 h) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay (left); cells were cultured in complete media (containing 5% FBS), serum free media (SF), complete media plus 2DG, serum and glucose free media (SF-glu), or in amino acid, glucose and SF Earls buffer salt solution (EBSS). (a) p < 0.01 versus control and (b) p < 0.05 versus pcDNA. (Right) Percentage of apoptotic cells (sub-G0 population) by flow cytometry in A2780 cells following glucose and serum withdrawal (Glu/SF). (a) p < 0.001 versus A2780pcDNA control and (b) p < 0.05 A2780Rab25 versus A2780pcDNA control.",
    "molecules": "2DG, amino acid, glucose, nutrient"
  },
  {
    "caption": "B. Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death. Expression of Rab25 was down-regulated by siRNA or shRNA specific to Rab25.",
    "molecules": "nutrient"
  },
  {
    "caption": "C. Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions. Western blot for the 16kD LC3-II fragment, indicative of autophagy activity, in A2780 cells after 4 and 6 h of serum and glucose withdrawal.",
    "molecules": "glucose"
  },
  {
    "caption": "D. Electron microscopy of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal (upper panel, low magnification). High magnification images of boxed areas with arrowheads depicting autophagic vacuoles (lower panel, inset; N: nucleus). Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two-independent experiments.",
    "molecules": "glucose"
  },
  {
    "caption": "E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.E. RPPA detection of time-dependent activation of AMPK after nutrient withdrawal.",
    "molecules": "glucose, nutrient"
  },
  {
    "caption": "E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.F. WB analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in HEY ovarian cancer cells (upper panel). WB of phospho-ACC levels in A2780, IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal (lower panel).",
    "molecules": "glucose, nutrient"
  },
  {
    "caption": "E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.G. RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose.",
    "molecules": "glucose"
  },
  {
    "caption": "E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.H. Effect of Rab25 down-regulation on AMPK (left panel) and ACC (right panel) phosphorylation. Total cellular protein, isolated from A2780pcDNA and Rab25 expressing cells 72 h post-transfection with either non-target (NT) RNAi or Rab25 specific RNAi, was separated by polyacrylamide gel electrophoresis (PAGE) followed by WB analysis. (a) p < 0.001 versus NT RNAi control and (b) p < 0.001, pcDNA versus Rab25.",
    "molecules": "glucose"
  },
  {
    "caption": "Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29htpcDNA cells, 6.21−10 moles/mg for IOSE29htRab25 cells, 8.14−11 moles/mg for IOSE80htpcDNA cells, 1.05−10 moles/mg for IOSE80htRab25 cells, 2.85−10 moles/mg for A2780pcDNA cells, 3.81−10 moles/mg for A2780Rab25 cells, 7.23−10 moles/mg for HEYpcDNA cells and 9.82−10 moles/mg for HEYRab25 cells.",
    "molecules": "ATP"
  },
  {
    "caption": "Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA.",
    "molecules": "ATP, glucose"
  },
  {
    "caption": "Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control.",
    "molecules": "ATP, glucose"
  },
  {
    "caption": "Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control.",
    "molecules": "glycogen"
  },
  {
    "caption": "Decrease in cellular glycogen content after glucose and serum withdrawal *p < 0.001 Rab25 versus pcDNA.",
    "molecules": "glucose, glycogen"
  },
  {
    "caption": "Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group.",
    "molecules": "Bay U6571, ATP, GPi, glucose, oligomycin"
  },
  {
    "caption": "Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment.",
    "molecules": "Bay U6571, ATP, GPi, glycogen"
  },
  {
    "caption": "Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death *p < 0.05 versus shRNA control.",
    "molecules": "2DG, GPi, glycogen"
  },
  {
    "caption": "B. WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103, GPi and synthase kinase 3 inhibitor (GSK3i) in HEY cells.",
    "molecules": "GPi, PI103, GSK3i"
  },
  {
    "caption": "C. Inhibition of GSK3 activity increases total cellular glycogen levels. pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times. Total cellular glycogen content was measured and normalized with total protein content. (a) p < 0.001 versus no GSK3i treated pcDNA cells.",
    "molecules": "glycogen, GSK3i"
  },
  {
    "caption": "D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.D. There is an increase in glycogen content during recovery phase (5% FBS) and a decrease in glycogen when cells are subject to a second nutrient stress (15 min SF).",
    "molecules": "ATP, glucose, glycogen, nutrient, PI103"
  },
  {
    "caption": "D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.E. Utilization of glycogen to produce ATP.",
    "molecules": "ATP, glucose, glycogen, nutrient, PI103"
  },
  {
    "caption": "F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF.",
    "molecules": "glycogen, AKTi, MK2206"
  },
  {
    "caption": "G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. *p < 0.001 shRNA Rab25 versus shRNA control.",
    "molecules": "3BrPy, glucose, LND, AKTi, PI103"
  },
  {
    "caption": "A. Rab25 over-expression increases H3-labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment.",
    "molecules": "H3, 2DG"
  },
  {
    "caption": "B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT.",
    "molecules": "glucose, MK2206"
  },
  {
    "caption": "C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25.",
    "molecules": "glucose, AKTi, MK2206"
  },
  {
    "caption": "E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.E. AKT inhibition increases sensitivity to 2DG induced cell death. *p < 0.001 versus HEYpcDNA cells.",
    "molecules": "2DG, MK2206, PI103"
  },
  {
    "caption": "E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.F. Down-regulation of AKT by siRNA specific to AKT.",
    "molecules": "2DG, MK2206, PI103"
  },
  {
    "caption": "E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. *p < 0.001 versus RNAi control cells.",
    "molecules": "2DG, MK2206, PI103"
  },
  {
    "caption": "Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.",
    "molecules": "ATP, glycogen"
  },
  {
    "caption": "A. Rab25 expression correlates with glycogen levels in patienttumours. Total RNA and total cellular extracts, isolated from 31 ovarian cancer patient specimens, were subjected to Rab25 gene expression and glycogen content analysis using qPCR and glycogen assay, respectively.",
    "molecules": "glycogen"
  },
  {
    "caption": "
 (A) Stopped-flow assay measuring relative rates of lactic acid transport into reconstituted vesicles containing wild-type or mutant transporter (green, control with empty vesicles; red, wild-type; blue, K35A; cyan, F90A; magenta, F94A; yellow, K177A; olive green, H230A; and gray, H230N). (B) Relative rates of lactic acid transport based on reswelling rates of proteoliposomes. Each data point is an average of five different light scattering data sets. Error bars depict standard deviation for these five light scattering data. All of the transport rates from the mutant transporters are significantly different compare from that of wild-type PfFNT (K35A, P = 2.8 x 10-3; F90A, P = 4.4 x 10-7; F94A, P = 4.9 x 10-6; K177A, P = 1.2 x 10-3; H230A, P = 4.5 x 10-7; H230N, P = 2.0 x 10-6; student's t-test) (green, control with empty vesicles; red, wild-type; blue, K35A; cyan, F90A; magenta, F94A; yellow, K177A; olive green, H230A; and gray, H230N). 
",
    "molecules": "lactic acid"
  },
  {
    "caption": "Human red blood cells (RBCs) were incubated with 400 nM pigment or control ΔcylE extract, and kinetics of K+ and Hb release was monitored. Data shown are the average and SEM of six independent experiments. The time to 50% K+ and 50% Hb release with pigment was 4.8 min and 8.4 min, respectively; n = 6, P < 0.0001, extra sum-of-squares F test.",
    "molecules": "pigment, Hb, K+"
  },
  {
    "caption": "Role of osmoprotectants in pigment-treated RBC. HumanRBCs were pre-incubated with GBSpigment for 2 min at RT, centrifuged, and resuspended in the presence and absence of 30 mM osmoprotectant with hydrodynamic radius of 0.40 nm (PEG200), 0.56 nm (PEG400), 0.89 nm (PEG1000), 1.1 nm (PEG1500), or 1.6 nm (PEG3000), respectively. Release of Hb was measured after 1 h of incubation at 37°C. Data shown are the average and SEM of three independent experiments.",
    "molecules": "pigment, Hb, PEG"
  },
  {
    "caption": "Characteristics of membrane permeabilization by the GBSpigment in artificial lipid bilayers. Lipid bilayers were generated using diphytanoylphosphatidylcholine (DPhPC) and treated with either 2 μM pigment or an equivalent amount of the control ΔcylE extract. In the pigment-treated sample, channel conductance indicating disruption of the membrane is seen within 45 s. Erratic and non-discrete fluctuations in current are observed, suggesting the formation of multiple, small membrane defects. The bilayer eventually breaks at 120 s. In lipid bilayers treated with the control ΔcylE extract, the mean ionic current trace remains constant at 0 pA, showing no membrane disruption. Data shown are representative of three independent experiments.",
    "molecules": "pigment, diphytanoylphosphatidylcholine, DPhPC"
  },
  {
    "caption": "C, D PBMC-derived macrophages primed with 100 ng/ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D). (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.",
    "molecules": "pigment, LPS"
  },
  {
    "caption": "C, D WT THP-1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).(C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.",
    "molecules": "pigment"
  },
  {
    "caption": "C, D The shRNA THP-1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release in supernatants was measured by Luminex assay (D). Pigment-mediated cytotoxicity is dependent on the NLRP3 inflammasome components, suggesting that pigment is inducing a programmed cell death. Data shown are the average of at least three independent experiments performed in triplicate.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (C) n = 3, for 4 μM pigment: **P = 0.0063 (vector versus shNLRP3), **P = 0.0035 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3), **P = 0.009 (scrambled versus shASC); for 2 μM pigment: **P = 0.0088 (vector versus shNLRP3), **P = 0.0066 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3 or shASC); for 1 μM pigment: *P = 0.02 (vector versus shNLRP3), *P = 0.01 (vector versus shASC). Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.99. (D) n = 3, ****P < 0.0001, *P = 0.015.",
    "molecules": "pigment, Pigment"
  },
  {
    "caption": "A, B THP-1macrophages proficient for NLRP3 (transfected with scrambled control, A) or deficient for NLRP3 (shNLRP3, B) were treated with 1 μM pigment or ΔcylE extract for 20 min, and propidium iodide (PI) was added during the final 10 min. PI uptake was measured by flow cytometry, and data shown are representative of two independent experiments.",
    "molecules": "pigment"
  },
  {
    "caption": "C, D Intracellular potassium concentration was measured by ICP-AES. THP-1macrophages transfected with the scrambled control (C) or shNLRP3 (D) were treated with GBSpigment (1 μM) or an equivalent amount of the ΔcylE extract. At various time points, cells were lysed and intracellular [K+] was measured relative to untreated cells (see bars and left y-axis), and percent cell death was quantified by alamar blue (see squares, dotted connecting lines and right y-axis). Both NLRP3-proficient and NLRP3-deficient macrophages initially lose intracellular K+ due to GBSpigment (compare t = 0 min to t = 30 min), but the NLRP3-deficient cells (shNLRP3) are able to recover, while the scrambled control do not, demonstrating that initial K+ loss occurs independently of NLRP3. Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett's multiple comparison test following ANOVA; all data were compared to control at t = 0, error bars ± SEM. (C) n = 3, ***P = 0.0002, *P = 0.019, **P = 0.0032 for 120 min, **P = 0.0072 for 180 min. (D) n = 3, **P = 0.0043, *P = 0.031.",
    "molecules": "pigment, K+, potassium"
  },
  {
    "caption": "(E) WT THP-1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride, and cytotoxicity was measured by alamar blue assay. The addition of potassium chloride is able to protect the macrophages from cytolysis, demonstrating that K+ efflux is essential for this process. Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM (n = 3, ****P < 0.0001, **P = 0.0036).",
    "molecules": "pigment, potassium chloride, K+"
  },
  {
    "caption": "THP-1macrophages were treated with GBSpigment, and caspase 1 activation was measured by flow cytometry using a FLICA reagent. Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line, demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome. Results are representative of three independent experiments.",
    "molecules": "Pigment, pigment"
  },
  {
    "caption": "WT THP-1macrophages were treated with the caspase 1 inhibitor Z-YVAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or DMSO only prior to treatment with the GBSpigment. YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment, demonstrating that caspase 1 is required for GBSpigment-mediated cell death, characteristic of pyroptosis, while DEVD had no effect. Data are average of three independent experiments, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA (n = 3, ****P < 0.0001, ***P = 0.0002, *P = 0.014).",
    "molecules": "DMSO, pigment, Z-DEVD-FMK, Z-YVAD-FMK"
  },
  {
    "caption": "D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.",
    "molecules": "Shield-1"
  },
  {
    "caption": "A Caspase 3/7 activity measured in HCT116 Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression in the absence of apoptosis stimuli. Caspase activity is displayed in relative fluorescence units (RFU). pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA are displayed.B Staurosporine (STS, 1 µM)-induced caspase 3/7 activity of Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression displayed in relative fluorescence units (RFU). Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA. BaxTBak activities with and without Bcl-xL expression revealed no significant difference (n.s.), while in the presence of Bcl-xL overexpression BaxTBak activity is significantly higher than Bax, Bak or BakTBax activities (p < 0.001).",
    "molecules": "Staurosporine"
  },
  {
    "caption": "C Analysis of the active Bax conformation in HCT116 Bax/Bak DKO cells expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL overexpression (light grey bars) by the monoclonal antibody 6A7 (Sigma) detecting the active Bax protein fold by fluorescence imaging. Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan-caspase inhibitor qVD. Data are represented as % of the expressing cell population ± SEM. n = 4.",
    "molecules": "qVD, STS"
  },
  {
    "caption": "D HCT116 Bax/Bak DKO cells ectopically expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL (light grey bars) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c. Data are represented as % of the expressing cell population ± SEM. n = 4.",
    "molecules": "qVD, STS"
  },
  {
    "caption": "E Flow cytometry analysis of Annexin V-staining of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in the absence (red line) or the presence of Bcl-xL overexpression (black line), following STS-treatment. The percentage of gated cells is displayed in the color of the corresponding graph. Data represent averages ± SD. n = 4.",
    "molecules": "STS"
  },
  {
    "caption": "F Colony formation of Bax/Bak DKO cells transfected with pcDNA, Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression. STS (1 µM) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment.",
    "molecules": "STS"
  },
  {
    "caption": "G Quantification of colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression after STS treatment (Fig. 5F). Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA. BaxTBak expressing cells with or without Bcl-xL expression showed no significant difference (n.s.).H Quantification of the colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in presence of Bcl-xL overexpression without apoptosis stimulation. Data represent averages ± SEM. n = 5. p-values according to One Way ANOVA.",
    "molecules": "STS"
  },
  {
    "caption": "F STS-induced caspase 3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3.",
    "molecules": "STS"
  },
  {
    "caption": "G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as control for PARP cleavage and actin is the loading control. n = 3.",
    "molecules": "STS"
  },
  {
    "caption": "(A) BH3‐only BIK displaces a subset of BCL‐2 complexes at the ER. Purified LM from H1299 HA‐BCL‐2b5 cells either mock infected or infected with Ad‐BIKb5 was subjected to cross‐linking with BMH and visualized by immunoblot. Asterisks (*) denote BCL‐2 cross‐linked products, which are displaced by BIK. Bold arrow denotes region containing cross‐linked NAF‐1/BCL‐2.",
    "molecules": "BMH"
  },
  {
    "caption": "(D) Endogenous NAF‐1 cross‐links with BCL‐2 at the ER. LM purified from H1299 neo and HA‐BCL‐2b5 cells were subjected to cross‐linking by BMH and immunoprecipitation with anti‐NAF‐1 antibody. Precipitates were analysed by immunoblot with anti‐BCL‐2.",
    "molecules": "BMH"
  },
  {
    "caption": "(A) H1299 neo and HA‐BCL‐2b5 cells treated with control (CTRL) or NAF‐1 shRNA were either mock infected or infected with Ad‐BIK in the absence or presence of 50 μM zVAD‐fmk. Cell lysates were analysed by immunoblot.",
    "molecules": "zVAD‐fmk"
  },
  {
    "caption": "(C) Prolonged BIK expression and caspase inhibition induces autophagy, which is enhanced by knockdown of NAF‐1. H1299 neo and HA‐BCL‐2b5 cells treated with CTRL or NAF‐1 shRNA were infected with Ad‐BIK in the presence of zVAD‐fmk for the indicated periods of time. Cell lysates were analysed by immunoblot. Levels of LC3 II were normalized to actin levels by densitometry analysis. Graph depicts normalized LC3 II levels of each lane.",
    "molecules": "zVAD‐fmk"
  },
  {
    "caption": "(A) Effect of NAF‐1 knockdown on starvation‐induced autophagy. H1299 cells infected with CTRL or NAF‐1 shRNA were starved for 4 h in EBSS with DMSO (vehicle) or Baf A1 (100 nM). Cell lysates were analysed by immunoblot.",
    "molecules": "Baf A1, DMSO"
  },
  {
    "caption": "(A) H1299 neo and HA‐BCL‐2b5 cells treated with either CTRL or NAF‐1 shRNA were loaded with Fura‐2AM, and ER Ca2+ stores were measured as the difference in cytoplasmic Ca2+ concentration before and after addition of TG (2 μM). Shown are representative traces of Fura‐2AM fluorescence measured at 340/380 nm excitation wavelength ratio at 510 nm wavelength emission. Arrow indicates time at which TG was added, delta values indicate TG‐sensitive ER Ca2+ stores.",
    "molecules": "Ca2+, TG"
  },
  {
    "caption": "(B) Differences in ER Ca2+ stores are shown as the average±s.e.m. of three independent experiments as described in (A).",
    "molecules": "Ca2+"
  },
  {
    "caption": "A microfluidic device for quantitative recovery time measurements. Each PDMS-fabricated chip consists of six independent channels, one of which is shown, from top view (top row). Green circles indicate media inflow; black circle indicates outflow. Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers (gray circles), the height of which ensures that bacteria are imaged in a monolayer (bottom row). Growth conditions (e.g. antibiotic dose profile) are controlled via programmable syringe pumps. Fluorescent images (bottom row) show representative growth of a monolayer of E. coli BW25113 cells constitutively expressing GFP over four hours.",
    "molecules": "antibiotic"
  },
  {
    "caption": "Representative time series fluorescence data showing dose-dependent population recovery in response to transient streptomycin treatment. Here, time zero corresponds to the end of treatment (120 minutes). Trajectories show mean and standard deviation for five replicates; colors indicate increasing antibiotic concentration (2, 4, 6, 8, 10, 12 μg/mL). Fluorescence values are normalized to those at time zero. Dotted line indicates a 2-fold increase, corresponding to the recovery time for each population.",
    "molecules": "streptomycin"
  },
  {
    "caption": "Recovery time increases with total antibiotic exposure. Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations (as in panel C). When plotted against total antibiotic exposure (calculated as ), all recovery time values collapse onto a single exponential function (black line, R2 = 0.83). Recovery time was measured in response to streptomycin concencentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL, and treatment durations 30, 60, 90, and 120 minutes.",
    "molecules": "streptomycin"
  },
  {
    "caption": "Streptomycin treatment (closed data points, R2 = 0.83) results in significantly longer recovery times than chloramphenicol treatment (open data points). Here, the difference between two responses is statistically significant (p<0.05 by ANOVA).",
    "molecules": "chloramphenicol, Streptomycin"
  },
  {
    "caption": "The addition of efflux pump inhibitor (CCCP) (closed data points, R2 = 0.91) increases population recovery time in response to chloramphenicol treatment (open data points, R2 = 0.80). Here, CCCP was added at subinhibitory concentrations (3μg/mL); in the absence of antibiotic treatment, CCCP alone did not inhibit population recovery. The CCCP-mediated increase in recovery time is statistically significant (p < 0.01).",
    "molecules": "CCCP, chloramphenicol"
  },
  {
    "caption": "The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0.01% w/v (closed data points) to 0.05% w/v (open data points). Faster synthesis resulted in lower recovery times (p<0.001) in response to streptomycin treatment. In each of these panels, the color scheme indicates increasing antibiotic concentration, as in Figure 1C.",
    "molecules": "casamino acids, streptomycin"
  },
  {
    "caption": "Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop. Antibiotics are as follows: penicillin G (PenG), spectinomycin (Spec), gentamicin (Gent), streptomycin (Str), chloramphenicol (Cm), tetracyclin (Tet), and carbenicillin (Carb). Antibiotic concentrations used have been scaled by their respective IC50 values (solid points and open circles show response with no efflux inhibition and 2μg/mL CCCP, respectively). Colors correspond to the motifs of action shown in Figure 4A.",
    "molecules": "gentamicin, carbenicillin, CCCP, chloramphenicol, penicillin, spectinomycin, streptomycin, tetracyclin"
  },
  {
    "caption": "Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C; each dot represents a strain-condition replicate as in panel B. The conditions maltose, glycerol, and NaCl are highlighted.",
    "molecules": "glycerol, maltose, NaCl"
  },
  {
    "caption": "Changes in deletion phenotypes for growth on glycerol and maltose. The top heatmap contains the MAL genes, while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains. Significant growth phenotypes are marked with "-".",
    "molecules": "glycerol, maltose"
  },
  {
    "caption": "lipids content (F) per gram of feces.",
    "molecules": "lipids"
  },
  {
    "caption": "G. Free-fatty acids and H. Triglycerides content in serum of male mice after 17 weeks in HFD. n=10.",
    "molecules": "Free-fatty acids, Triglycerides"
  },
  {
    "caption": "C. Western blot analysis of phosphorylated PKD2 in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=3.",
    "molecules": "CRT0066101, water"
  },
  {
    "caption": "F. WB of Apolipoprotein A4 (APOA4) in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. Quantification of bands normalized to GAPDH and relative to control (water) n=6.",
    "molecules": "CRT0066101, water"
  },
  {
    "caption": "G. Body weight of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=6 for control and n=5 for CRT0066101.",
    "molecules": "CRT0066101, water"
  },
  {
    "caption": "A. Body weight gain of male C57BL/6 mice that, after 7 weeks in HFD, started receiving treatment with CRT0066101 or water. HFD continued along with treatment. n=8.",
    "molecules": "CRT0066101, water"
  },
  {
    "caption": "F. Western blot analysis of PKD phosphorylation in proximal jejunum of morbidly obese female patients. Samples are loaded according to fasting triglyceride levels (lower to higher). n=7.",
    "molecules": "triglyceride"
  },
  {
    "caption": "D. Trisomic and wild-type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.",
    "molecules": "Cycloheximide, MG132"
  },
  {
    "caption": "A. Real-time fatty acid oxidation (FAO) profile of WT and Tfcp2l1_KO  2i-ESCs using the FAO stress test kit in Seahorse XF. Oxygen consumption rate  (OCR) were measured. Error bars indicate SEM; n = 5 biological repeats.  Palm-BSA, palmitate conjugated BSA, a substrate of FAO; Oligomycin, an inhibitor of ATP synthase (complex V); FCCP,  Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, a proton gradient uncoupler; Rotenone and Antimycin, a complex I inhibitor and a complex III inhibitor, respectively.",
    "molecules": "Antimycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, FCCP, Palm-BSA, palmitate, Oligomycin, Oxygen, Rotenone"
  },
  {
    "caption": "F. Real-time FAO profile using the  FAO Stress Test  in Seahorse XF. n = 4 biological repeats; error bars indicate SEM; **, p-value <0.01; NS, not significant; t-test.",
    "molecules": "Seahorse XF"
  },
  {
    "caption": "D. Live-cell propidium iodide staining to measure cell death in WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs after 5 mM 2-DG treatment for 24 hours. n=3 biological repeats; error bars indicate SEM; p-value were calculated using two-sample t-test.",
    "molecules": "2-DG"
  },
  {
    "caption": "F. Relative levels of metabolites  in  glutamine metabolism. n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.",
    "molecules": "glutamine"
  },
  {
    "caption": "D. 0.5 million of  2i-ESCs with indicated genotypes were seeded and treated with 200 nM INK-128 on day0. Cell numbers were counted on day2, 4, and 6. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.",
    "molecules": "INK-128"
  },
  {
    "caption": "E. Immunoblotting of WT and  Tfcp2l1_KO 2i-ESCs untreated or treated with 200 nM INK-128.",
    "molecules": "INK-128"
  },
  {
    "caption": "F.  Real-time PCR showing Cpt1a mRNA levels in WT and  Tfcp2l1_KO 2i-ESCs in the presence and absence of 200 nM INK-128. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.",
    "molecules": "INK-128"
  },
  {
    "caption": "D. Kaplan-Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK-128. n, number  of  blastocysts of each genotype. p-value was calculated using the log rank test. E. Kaplan-Meier curve of ex vivo WT  blastocysts cultured in the M2 medium in the presence of 200 nM of INK-128 with or without 50 µM of etomoxir (Eto) and/or 100 µM of  octanoate, as indicated. p-values were calculated using the log rank test. ",
    "molecules": "Eto, etomoxir, octanoate, INK-128"
  },
  {
    "caption": "Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) purified GST, wild type GST-LC3s, GST-GABARAPs, GST-Ubiquitin (Ub), GST-Tetra-Ubiquitin, A representative Ponceau staining is shown.",
    "molecules": "Ponceau, Ubiquitin"
  },
  {
    "caption": "Lysates of HEK293T cells transiently transfected with HA-tagged wild-type and wild-type or ∆LIR mutant FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) A representative Ponceau staining is shown.",
    "molecules": "Ponceau"
  },
  {
    "caption": "Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) using GST-LC3B and GST- LC3B F52A-V53A as baits. A representative Ponceau staining is shown.",
    "molecules": "Ponceau"
  },
  {
    "caption": "Immunofluorescence images of U2OS expressing wild-type or ∆LIR mutant FLAG-HA-FAM134 proteins after 24hrs doxycycline induction under different conditions (basal=DMSO, Baf.A1=2h 200nM Bafilomycin A1, EBSS=2h starvation in EBSS) Scale bar: 10μm; staining against FAM134 (HA; green) and endogenous LC3B (red).",
    "molecules": "Baf.A1, Bafilomycin A1, DMSO, doxycycline"
  },
  {
    "caption": "Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8.",
    "molecules": "DMSO, Torin1"
  },
  {
    "caption": "Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3",
    "molecules": "Baf.A1, Bafilomycin A1, Dox, Torin1"
  },
  {
    "caption": "Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "A-C qRT-PCR detection of mRNA levels of  IL-1β, TNFα and IL-6 mRNA levels in  CCDC50-WT and CCDC50-KO THP-1 cells primed with LPS and then treated with ATP and Nigericin (A), or infected with Listeria monocytogenes at a multiplicity of infection (MOI) of 10 and Salmonella typhimurium (MOI of 10) for 6 h (B) or MSU (40 μg/ml ) and SiO2  (40 μg/ml) for 6 h (C) (n = 3 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; mRNA results are normalized to GAPDH  and relative to untreated wild-type cells Data are representative of three biological replicates and shown as mean with SEM (A-C); **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.",
    "molecules": "ATP, LPS, Monosodium urate, MSU, Nigericin, SiO2"
  },
  {
    "caption": "D Enzyme-linked immunosorbent assays (ELISA) of IL-1β production in supernatants of  THP-1 cells treated as in (A-C) (n = 2 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; ; data are representative of two biological replicates (D). **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.",
    "molecules": "Monosodium urate, MSU"
  },
  {
    "caption": "E-G  CCDC50-WT and  CCDC50-KO  THP-1 cells were stimulated as in (A-C). The cells were collected for immunoblot analysis of pro-caspase-1, and the supernatants were subjected to cleaved  caspase-1 and  IL-1β analysis. The expression levels of cleaved-caspase-1 and cleaved-IL-1β were quantitated using ImageJ software. Data information: L.m, Listeria monocytogene; S.t,  Salmonella typhimurium; MSU, Monosodium urate; Casp1,  caspase-1; actin  was used as a loading control.",
    "molecules": "Monosodium urate, MSU"
  },
  {
    "caption": "C qRT-PCR detection of Il6, IL-1β and Tnfα mRNA levels in Ccdc50+/+ and Ccdc50-/- BMDMs primed with LPS for 3 h and then induced with ATP and Nigericin (A),  L.m and  S.t (B), or MSU  and  SiO2 (C) for indicated time points (n = 3 biological replicates). D-F Ccdc50+/+ and  Ccdc50-/- BMDCs treated as BMDMs  were collected and subjected to qRT-PCR analysis (n = 3 biological replicates). Data information: mRNA data are normalized to GAPDH  and relative to untreated wild-type cells; Data are representative of three biological replicates and are shown as the mean with SEM *P <0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.",
    "molecules": "ATP, LPS, MSU, Nigericin, SiO2"
  },
  {
    "caption": "C  Immunoprecipitation (IP) analysis of the interaction  of  NLRP3  with endogenous  CCDC50 and ASC in THP-1 cells activated by  LPS  plus ATP. IgG was used as a negative control. Data information: actin was used as a loading control. All the experiments were repeated at least once.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "D Representative endogenous  colocalization analysis of CCDC50  and NLRP3  in PMA-induced THP-1  cells treated with LPS and ATP. Scale bar, 10 μm.",
    "molecules": "ATP, LPS, PMA"
  },
  {
    "caption": "E Representative  confocal microscopy image of HCT116 cells overexpressing GFP-CCDC50 and stimulated with  LPS as well as  ATP. Scale bar, 10 μm.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "H Immunoblot  analysis of lysates from CCDC50-KO and CCDC50-WT  THP-1  cells induced with  LPS  plus  ATP and then treated with cycloheximide (CHX) for indicated  time points. The expression  levels of NLRP3 and  Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.",
    "molecules": "ATP, CHX, cycloheximide, LPS"
  },
  {
    "caption": "I  HEK293  cells were co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h and then treated with MG132, NH4Cl, 3-MA, CQ or DMSO  for another 6 h. Data information: actin was used as a loading control. All the experiments were repeated at least once.",
    "molecules": "3-MA, NH4Cl, CQ, DMSO, MG132"
  },
  {
    "caption": "J Immunoblot analysis of NLRP3 in CCDC50-WT and CCDC50-KO THP-1 cells stimulated with LPS  in combination with  ATP. Data information: actin was used as a loading control. All the experiments were repeated at least once.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "A Denature-IP (with anti-Flag) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293  cells transfected with Flag-NLRP3, HA-Ub (ubiquitin) as well as HA- CCDC50 or empty vector for 24 h.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "B Denature-IP analysis (with anti-Flag) of K63-linked polyubiquitination  of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h.",
    "molecules": "CQ"
  },
  {
    "caption": "C The  colocalization  analysis of K63-Ub (Red),  Flag-NLRP3 (Purple) and GFP- CCDC50 in  HeLa cells; scale bars, 5 μM.",
    "molecules": "Ub"
  },
  {
    "caption": "D,E Images of HeLa  cells transfected with CCDC50 (red) (D) or K63-Ub (red) (E) and Flag-NLRP3  (Purple) and GFP-LC3B for 24 h and then induced with  LPS plus ATP; scale bars, 5 μM.",
    "molecules": "ATP, LPS, Ub"
  },
  {
    "caption": "F The colocalization  analysis of NLRP3 and LC3B  in CCDC50-WT and CCDC50- KO  HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were  stained with DAPI; data are representative of three individual experiments.",
    "molecules": "ATP, DAPI, LPS"
  },
  {
    "caption": "G The  oligomerization analysis  of  NLRP3 in CCDC50 wild-type and knockout THP-1  cells stimulated with  LPS as well as ATP. LPS-primed (1 μg/ml, 3 h)  THP-1 cells were stimulated with  ATP (5 mM) for another 30 min and then were collected and lysed. The cell lysates were centrifuged and separated into soluble  and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by  native-PAGE.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "H The  oligomerization analysis  of NLRP3 in HEK293  cells transfected with plasmids expressing Flag-NLRP3, HA-CCDC50  or empty vector for 24 h, and then treated with  DMSO or CQ for 6 h. After treatment, the cells were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The  insoluble fraction was analyzed by  native-PAGE.",
    "molecules": "CQ, DMSO"
  },
  {
    "caption": "I Co-IP (with anti-HA) and  immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h, and treated with or without CQ for 6 h.",
    "molecules": "CQ"
  },
  {
    "caption": "J Confocal microscopy analysis of colocalization  between NLRP3  and ASC  in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus  ATP; scale bars, 5 μM. Data information: The  nuclei  were stained  with DAPI; data are representative of three individual experiments.",
    "molecules": "ATP, DAPI, LPS"
  },
  {
    "caption": "K Confocal microscopy  analysis of ASC speck formation in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei  were stained with DAPI; data are representative of three individual experiments.",
    "molecules": "ATP, DAPI, LPS"
  },
  {
    "caption": "(B) Light microscopy images of LGCs, FBGCs and osteoclasts stained with Giemsa staining (upper panel). Representative Hoechst dye images acquired by ImageStream (lower panel) of sorted cells showing mononuclear (mono) and multinucleated (multi) populations for each cell type.",
    "molecules": "Hoechst, Giemsa"
  },
  {
    "caption": "(C) MRC1 surface marker expression (red) acquired by ImageStream in mononuclear (mono) and multinucleated (multi) LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) represent normalized MRC1 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(E) The effect of lysosomotropic agents (hydroxychloroquine, hydroxy; ammonium chloride, NH4Cl) and v-ATPase inhibitors (Bafilomycin A1, Baf A1; Concanamycin A, Con A) on fusion and multinucleation in LGCs (upper panel), FBGCs (middle panel) and osteoclasts (lower panel). To test the effect of lysosome dysfunction on cellular iron, FeCl3 was supplemented. Error bars are median with interquartile range; significance tested by one-way Anova followed by Sídák's multiple comparisons on log transformed data; n=4 donors; *, P < 0.05; **, P < 0.01; *** P < 0.001; ****, P < 0.0001.",
    "molecules": "ammonium chloride, NH4Cl, Baf A1, Bafilomycin A1, Con A, Concanamycin A, hydroxy, hydroxychloroquine, iron, FeCl3"
  },
  {
    "caption": "(E) B7-H3 surface marker expression (red) acquired by ImageStream in mononuclear and multinucleated LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) show normalized B7-H3 mean fluorescence intensity (MFI), measured by ImageStream; n=4 donors. Error bars are mean ± SD; significance tested by paired t-test; **, P < 0.01; ns, non-significant.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Volcano plots representing transcript down- (red) or up-regulation (yellow) after 48 hours PTX in the somatic and process compartment. Representative transcripts are highlighted and labelled. Linear model was used to fit transcriptome data and subsequently contrasted using likelihood ratio testing for PTX-treatment effects across compartments.",
    "molecules": "PTX"
  },
  {
    "caption": "(A) Real-time quantitative PCR (RT-qPCR) of transcripts changing differentially between compartments using the compartmentalized cultures after 48 hours PTX-treatment. n=8 independent biological replicates (except for Plk2 (n=7), where one measurement couldn't be performed due to insufficient cDNA); PTX-effect was assessed by three-way ANOVA followed by Tukey's post-hoc multiple comparison test; *p < 0.05; **p < 0.01.",
    "molecules": "PTX"
  },
  {
    "caption": "(B) Representative images of single-molecule FISH (smFISH) in either control or 48 h PTX-treated rat hippocampal neurons (DIV20) using probes specific for Add2, Dnajc6 and Sort1 (green). MAP2 immunostaining (red) was used to visualize neuronal somata and dendrites. Inserts at higher magnification illustrate PTX-dependent changes in dendritic RNA puncta. Scale bar = 10 μm. (C) Quantification of B. (n=3-4 independet biological replicates with 8-10 cells averaged per condition and replicate; two-sample Student's t-test; *p < 0.05; **p < 0.01). B",
    "molecules": "PTX"
  },
  {
    "caption": "(D) Representative plots of genes changing after PTX at the transcript and protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4). Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5x interquartile range (IQR).",
    "molecules": "PTX"
  },
  {
    "caption": "(A) Representative pictures of nascent Camk2a-peptides in mock- and PTX-treated neurons. Upper Panels: Representative overview images with merged channels (grey: MAP2-staining, red: Duolink-PLA signal, blue: Hoechst nuclear staining). Middle Panels: Magnifications of boxed areas marked in (A). MAP2 signal was used to outline somata and dendritic processes (white lines). Lower Panels: Negative control of puromycin-untreated cells with representative Duolink-signal in dendrite and soma, respectively. (Scale bars: in overview images 20 μm, in inserts of higher magnification 5 μm). (B) Same as A with nascent Synapsin1-peptides. (",
    "molecules": "Hoechst, PTX, puromycin"
  },
  {
    "caption": "Figure 3 | DND1 co-localizes with NANOS2 in P-bodies. A−C, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (green) and DCP1a (red). D−F, Squash preparation of a male gonocyte from E16.5embryoimmunostained with antibodies against DND1 (red) and DCP1a (green). Arrowheads indicate co-localization of DND1 and DCP1a. G−I, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (red) and NANOS2 (green). Arrowheads indicate co-localization of DND1 and NANOS2. J−M, NIH3T3 cells transfected with HA-tagged Dnd1 and FLAG-tagged Nanos2 were then immunostained with antibodies against DND1 (J) (magenta), NANOS2 (K) (red), and DCP1a (L) (green). N−Y, Biomolecular fluorescence complementation assay. NIH3T3 cells transfected with VENUS-C-fused Dnd1 and VENUS-N-fused Nanos2 (N−U) or Nanos2 (C61A, C96A) (V-γ) were immunostained with antibodies against DND1 (N, S, V), NANOS2 (O, W, α) or DDX6 (R, Z). Then, the signals of VENUS fusion protein were visualized (P, T, X, β). Arrowheads in Z indicate P-bodies. DNA was labeled with DAPI (blue). Scale bars: 50 m in A for A−C; 10 m in D for D-F, G for G−I, J for J−M, N for N−γ. Insets in A-C and J-γ show an enlarged vision of each picture to better depict localization of each protein. See also Fig. EV2T-Y.",
    "molecules": "DNA"
  },
  {
    "caption": "A, Western blotting analyses of proteins in testes from E13.5 to E16.5 embryos of Dnd1flox/flox or Dnd1flox/flox_Tg(Oct4PE-CreERT2) each administered with tamoxifen at E13.5 and E15.5 embryos of Nanos2+/- or Nanos2-/- with the indicated antibodies.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "B-P, Sections of testes from Dnd1flox/flox (B, E, H, K, N), Dnd1flox/flox_Tg(Oct4PE-CreERT2) (C, F, I, L, O) or Nanos2-/- (D, G, J, M, P) embryos were prepared at E16.5 and then immunostained with antibodies against pH3 (B-D), STRA8 (E-G), SYCP3 (H-J), activated-Caspase3 (K-M) or LAMININ (N-P) (green). Germ cells were immunostained with TRA98 (B-D) or DAZL (N-P) (red), and DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in B for B−D and K-P, 50 m in E for E−J. See also Fig. EV3.",
    "molecules": "DNA, Tamoxifen"
  },
  {
    "caption": "A-L, Sections of testes from Dnd1flox/flox (A, B, C, G, H, I, M, N, O) and Dnd1flox/flox_Tg(Oct4PE-CreERT2) (D, E, F, J, K, L, P, Q, R) embryos were prepared at E16.5 and then immunostained with antibodies against NANOS2 (A, D) (green), DCP1a (G, J) (green), DDX6 (M, P) (green) and TRA98 (B, E, H, K, N, Q). DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in A for A−F; 50 m in G for G-L; 50 m in M for M-R. Insets show an enlarged vision of each picture to better depict localization of NANOS2, DCP1a and DDX6.",
    "molecules": "DNA, Tamoxifen"
  },
  {
    "caption": "C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary IF microscopy images of the indicated fission yeast strains are shown. Homothallic strains were incubated on SPAS plates. DNA and tubulin were stained with DAPI and anti-TAT-1, respectively. Missegregating chromosome is indicated with a yellow arrow; D Quantification of meiotic chromosome segregation phenotypes. The data for clr4+ and clr4F449Y is the same as in Fig 3B. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.",
    "molecules": "DAPI, DNA, H3K9me2"
  },
  {
    "caption": "E Comparison of co-segregation of centromere I (CenI) during MI in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown. Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis. Heterozygous CenI-GFP was used to follow the segregation pattern during meiosis. The GFP-signal is additionally displayed in greyscale for better visibility F Quantification of CenI-GFP segregation during meiosis. The data for clr4+ and clr4F449Y is the same as in Fig 3D. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.",
    "molecules": "H3K9me2"
  },
  {
    "caption": "D Western blot of FLAG IPs in 3xFLAG-clr4+ cdc2-as cells, treated with 2 µM 1-NM-PP1 for the indicated amount of time. The blot was first probed with the phospho-specific anti-pS458 antibody, stripped, and reprobed with an anti-FLAG antibody.",
    "molecules": "1-NM-PP1"
  },
  {
    "caption": " H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN. ",
    "molecules": "DIO"
  },
  {
    "caption": "D, In vitro GST pull-down assays were performed with bacterially expressed and purified GST  or GST-tagged AR  immobilized onto GSH  beads. Immobilized GST  protein beads were used to pull down  purified his-MBP  (left) or his-MBP-tagged TRIM33  (right).",
    "molecules": "GSH"
  },
  {
    "caption": "A-D, AR  transcript levels were measured by  RT-qPCR on LNCaP  cells (A and C) or VCaP  cells (B and D) transfected with siTRIM33  or the TRIM33  expression construct and treated with EtOH  or DHT. Protein lysates were harvested at 72 h after transfection and subjected to western blot  analysis with the indicated antibodies. Data information: In (A-D, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).",
    "molecules": "DHT, EtOH"
  },
  {
    "caption": "E and F, LNCaP  cells transfected with siTRIM33 or NC were treated with cycloheximide  (CHX). Protein lysates were harvested at the indicated time points for  western blot analysis. AR  protein levels were quantified and normalized to β-actin and then plotted relative to 0 h. Data information: In F, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "G, LNCaP  cells transfected with siTRIM33  or NC were treated with DMSO  or MG132  for 8 h. Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "HA-Ub   was expressed in LNCaP  cells co-transfected with siTRIM33 (H) The cells were treated with MG132  for 4 h before harvesting. Endogenous AR  was immunoprecipitated  from cell lysates, and interacting  proteins were detected by immunoblotting  with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "I, HA-Ub  was expressed in LNCaP  cells co-transfected with the TRIM33  expression construct (I). The cells were treated with MG132  for 4 h before harvesting. Endogenous AR  was immunoprecipitated  from cell lysates, and interacting  proteins were detected by immunoblotting  with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "Q, AR pull-down was performed on protein extracts from MG132-treated HEK293T  cells overexpressing myc-AR, HA-Ub, myc-Skp2, and various flag-tagged TRIM33  constructs. AR ubiquitination  and specific protein levels were detected by  western blot  analysis with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(D) Chromatographic profile of GALC in selected brain slices and spinal cord (SC) tissue of untreated (UT) WT and LV.hGALC-treated Krabbe NHP (JT02). Extracts were run through a Sephadex S-300 gel filtration column. Fractions (0.2 ml) were assayed for enzyme activity using MUGAL substrate in the presence () or absence of 11µM AgNO3 (●).",
    "molecules": "MUGAL, AgNO3"
  },
  {
    "caption": "F Visualization of infected region. E. coli FM15  was inoculated at the center of a semi-solid agar plate and the reporter phage M13-sfGFP was inoculated 1-cm away from the center. Fluorescence images were captured after overnight incubation using FITC  channel and an exposure time of 200 ms. Scale bar represents 1 cm. G Relative fluorescence intensity at the center and edge positions of the infection  zone as shown in (F). The relative intensity was obtained by dividing the detected values with the maximum value of green fluorescence intensity on the plate. Data represent mean values ± s.d. for three values of the center and six values of the edge from three biological replicates. Two-tailed t-test was used to compare two groups. ***, p = 0.0005.",
    "molecules": "FITC"
  },
  {
    "caption": "(A and B) One hundred human keratinocytes  (YF29, passage V) were cultured in duplicate for twelve days in individual Thermo Scientific Heracell 150 incubators with the temperature  set at 35oC, 36oC, 37oC, or 38oC. Temperature  of each incubator was monitored with independent thermo probes In parallel, the temperature  of the culture medium was recorded every ten seconds using immerged RFID temperature  sensors Sharp spikes resulted from wireless communication artifacts. (B) Cells were fixed and stained  with 1%  Rhodamine B. Temperature  did not affect colony-forming efficiency  (CFE) but impacted colony size; colonies were smaller when temperatures  were below 37oC and larger at 38oC. Asterisks indicate thermo probes for monitoring the temperature.",
    "molecules": "Rhodamine B"
  },
  {
    "caption": "(F and G) YF29  cells were cultured for seven days at 37oC in a Nuaire 8700E culture incubator. Cells were then maintained at 37oC or switched to an adjacent incubator with temperature  set at 32oC. Temperature  was monitored with independent thermo probes in the presence or absence of rapamycin  (100 nM), or a cold agonist (menthol  - 500 μM), a warm agonist (2-APB - 200 μM) and the vehicle (control - ethanol  70%) for 30 and 60 minutes. (F) mTORC1 kinase activity decreases in the presence of rapamycin  and when the temperature  is switched from 37oC to 32oC. (G) mTORC1 kinase activity decreases in the presence of menthol  and increases in presence of 2-APB, indicating that ThermoTRP and mTOR signaling are connected. Rapamycin  (Rapa), menthol, and 2-APB were added in the keratinocyte  cultures 1 h before starting the temperature  changes.",
    "molecules": "2-APB, ethanol, menthol, Rapa, rapamycin, Rapamycin"
  },
  {
    "caption": "(A) Colony-forming efficiency  (CFE) of keratinocytes  was unaltered when keratinocytes  were cultured at 35oC or in the presence of 100 nM rapamycin. However, the mean area of colonies was smaller when compared to cells cultured at 37oC. (B) Culture at 35oC and treatment with rapamycin  decreased keratinocyte proliferation  as shown by the lower total number of cells after 7 days of culture compared with in the control condition. (C) Culture at 35oC and treatment with rapamycin  also reduced cell growth as shown by a smaller mean diameter of keratinocytes  compared with that in the control condition after 7 days of culture. Data information: In B and C, data are presented as the mean ± SD (n = 4 biological replicates). * p < 0.05 (Two-tailed Student's t-test). The data shown were confirmed in two technical replicates.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(D) CHO  cells engineered to transiently express SNAP-mTOR, SNAP-RAPTOR, and SNAP-RICTOR  (Gautier et al., 2008) were incubated with the nuclear  export inhibitor  leptomycin B (LMB) (6.4 ng/mL) for six hours at 37°C. Cells were labeled with TMR-Star (1 μM) for 30 min, washed and then imaged. This experiment demonstrated that mTOR, RAPTOR, and RICTOR  accumulated in the nucleus  after LMB  treatment. Scale bars, 10μm.",
    "molecules": "TMR-Star, leptomycin B, LMB"
  },
  {
    "caption": "A genome-wide expression screen was performed in parallel on normal human epidermal keratinocytes  (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin  (100 nM). (C)  Quantitative PCR analysis of epidermal  differentiation-associated genes. Data are presented as the mean ± SD (n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA with Dunnett's multiple comparisons test vs 37oC).",
    "molecules": "rapamycin"
  },
  {
    "caption": "Comprehensive miRNA expression  was performed in parallel on normal human epidermal keratinocytes  (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin  (100 nM). (B) The MA plot showing all miRNAs without low expression  or log2FC = 0. miRNAs indicated in pink: upregulated (≥ 1 log2(FC)), blue: downregulated (≤ -1 log2(FC)), grey: others (-1 < log2(FC) < 1) and black: miRNAs related to epidermal   differentiation. log2(FC) values are calculated using the edgeR package.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(B) Cultured human keratinocytes  (YF29  passage III) treated with rapamycin  maintained a basal phenotype as shown by the expression of several basal (KRT14, BNC1) and terminal differentiation markers (SPINK5, IVL, KRT1, KRT10) using  quantitative PCR (left panel) and Western blotting (right panel). Data are presented as the mean (n = 2 biological replicates).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(E) Rapamycin-treated keratinocytes  (YF29  passage IV) can fully regenerate an epidermis  when transplanted onto SCID mice; grafts were harvested after 71 days and immunostained  with an HLA-1 antibody to identify the human  cells (white dotted line indicates the boundary between mouse  and human epidermis). Scale bar, 100 μm.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "Determination of serum testosterone (B) and 17β-estradiol levels, in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).",
    "molecules": "17β-estradiol, testosterone"
  },
  {
    "caption": "H. The BCAA serum levels quantified enzymatically, as described in the materials and methods section.",
    "molecules": "BCAA"
  },
  {
    "caption": "A. Serum testosterone levels in the adult male and CtM Hanwoo (n = 10 in each group). The scattered dots in the bar graph represent the individual replicates.",
    "molecules": "testosterone"
  },
  {
    "caption": "B. The intestinal branched-chain amino acid (BCAA) levels in the adult male and CtM Hanwoo (n = 10 in each group). BCAA levels in the luminal contents of the ileum, cecum, and colon were quantified by an enzymatic method using a BCAA assay kit. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates.",
    "molecules": "BCAA, branched-chain amino acid"
  },
  {
    "caption": "B. Dressed body weight, thickness of the dorsal subcutaneous fat, total weight of the extramuscular fat (posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat), and serum levels of the branched-chain amino acids (BCAAs).",
    "molecules": "BCAAs, branched-chain amino acids"
  },
  {
    "caption": "F, G. The relative abundances of intramuscular metabolites (F) and β-hydroxybutyrate (3-HB) (G) in the male and CtM samples.",
    "molecules": "3-HB, β-hydroxybutyrate"
  },
  {
    "caption": "B. Serum testosterone levels.",
    "molecules": "testosterone"
  },
  {
    "caption": "C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon, shown according to the different diets and antibiotic treatment.",
    "molecules": "antibiotic"
  },
  {
    "caption": "E. The serum branched-chain amino acid (BCAA) levels quantified enzymatically, as described in the Materials and Methods section.",
    "molecules": "BCAA, branched-chain amino acid"
  },
  {
    "caption": "B. The serum testosterone levels in the Sham-R and castrated male (CtM)-R mice.",
    "molecules": "testosterone"
  },
  {
    "caption": "B. Body weight gain in response to the different levels of dietary BCAAs.",
    "molecules": "BCAAs"
  },
  {
    "caption": "D. The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high-fat diet (HFD) with or without BCAAs.",
    "molecules": "BCAAs"
  },
  {
    "caption": "E. The serum BCAA levels in the mice fed HFD with or without BCAAs.",
    "molecules": "BCAA, BCAAs"
  },
  {
    "caption": "(B) Representative confocal images show the distribution of the P4M2x reporter in HEK293-AT1 cells before and after the addition of GSK-A1 (30 nM). Note the signal disappearing from the PM and strongly highlighting the endosomes and Golgi. Scale bars are 20 µ",
    "molecules": "GSK-A1"
  },
  {
    "caption": "(C) Representative result from a BRET experiment detecting PI4P in various endosomes (Rab7, Rab5 and Rab11) following addition of GSK-A1. The graph shows normalized BRET values where the averages of triplicate measurements of A1-treated cells were divided by the averages of triplicates obtained in DMSO-treated controls. Note that these signals originate from thousands of cells.",
    "molecules": "DMSO, A1, GSK-A1, PI4P"
  },
  {
    "caption": "(D) Similar BRET experiment showing the Rab7-pool of PI4P both in control HEK293-AT1 cells and in two clones of cells with PI4K2A deletion (#20 and #26). Means ± S.E.M. are shown from three experiments performed in triplicates.",
    "molecules": "PI4P"
  },
  {
    "caption": "(A) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K/O cells after expression of HA-tagged PI4K2A or its kinase-dead mutant. Means ± S.E.M. are shown from three experiments performed in triplicates.",
    "molecules": "PI4P"
  },
  {
    "caption": "(B) Representative confocal pictures showing these changes in live cells. Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin (100 nM) for 10 min. Note that the Rab7 recruiter itself is dissociating from the membranes after PI(4,5)P2 generation. Left panels show some cells enlarged. Scale bars: 20 µm.",
    "molecules": "PI(4,5)P2, rapamycin"
  },
  {
    "caption": "(C) Quantification of the changes in BRET experiments. Here the BRET sensor contained the PLCδ1PH-fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co-transfected with the Rab7-targeted FRB (tagged with iRFP) and the FKBP12-fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence (CFP*). Recruitment of the PIP5Kγ but not its kinase-dead version caused an increase in the BRET signal indicating the increased PI(4,5)P2 in this compartment (means ± S.E.M., from three separate experiments performed in triplicates).",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "(D) PI(4,5)P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP-locked form of Rab7 (Q67L) and was negligible with the GDP-locked form of Rab7 (N125I) in the BRET construct. Also note that the Rab7 wild-type-based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs (means ± S.E.M., from three separate experiments performed in triplicates).",
    "molecules": "PI(4,5)P2, GDP, GTP"
  },
  {
    "caption": "(F) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P = 0.0478 and 0.0287 for #20 and #26 clones, respectively).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Representative confocal images of HEK293-AT1 cells, which show that treatment with OSW1 (20 nM), a drug that inhibits the cholesterol-PI4P transport protein, OSBP, causes accumulation of PI4P in the Golgi and in the endosomes (upper panels). Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K/O cells. Scale bars: 20 µm",
    "molecules": "cholesterol, OSW1, PI4P"
  },
  {
    "caption": "(B) Quantification of these changes by BRET analysis. The PI4P-Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 (20 nM) for the indicated times. The BRET ratios were expressed relative to those of DMSO treated cells. Data information: Means ± S.E.M. are shown from three experiments performed in triplicates.",
    "molecules": "DMSO, OSW1, PI4P"
  },
  {
    "caption": "(C) Detection of endosomal PI(4,5)P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5)P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5)P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5)P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm.",
    "molecules": "PI(4,5)P2, AngII, Angiotensin II, OSW1"
  },
  {
    "caption": "(D) Quantification of the same changes by BRET analysis in wild-type cells using the PI(4,5)P2 sensor with Rab7-Q67L-targeting. Note the transient signal increase after AngII stimulation only in cells treated with OSW1. (E) This increase is barely detectable in the PI4K2A K/O cells. D Data information: Means ± S.E.M. are shown from three experiments performed in triplicates. The values in (D) and (E) were measured in the same 96-well plate at the same time.",
    "molecules": "PI(4,5)P2, AngII, OSW1"
  },
  {
    "caption": "(A) BRET experiment in parental HEK293-AT1 cells in OSW1-pretreated cells after RNAi mediated knock-down of PIP5Kβ or PIP5Kγ. (means ± S.E.M. from three experiments, each performed in triplicates). Note the reduced response in the PIP5Kγ knock-down cells and the slight reduction in the case of PIP5Kβ knock-down. (B) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P=0.0241; **P=0.0088). ( ",
    "molecules": "OSW1, rapamycin"
  },
  {
    "caption": "(C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells. (D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct. D Data information Means ± S.E.M. are shown from three experiments performed in triplicates.",
    "molecules": "DMSO"
  },
  {
    "caption": "(C) Percentage of SNT‐1‐deficient animals that survive near‐lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia.",
    "molecules": "sodium azide"
  },
  {
    "caption": "(E) Percentage of UNC‐57‐deficient animals that survive after the hypoxia‐inducing treatment with sodium azide.",
    "molecules": "sodium azide"
  },
  {
    "caption": "(C) Percentage of unc‐116 animals that survive after treatment with sodium azide.",
    "molecules": "sodium azide"
  },
  {
    "caption": "(E) unc‐104 mutant animals are more resistant to hypoxic death induced by sodium azide.",
    "molecules": "sodium azide"
  },
  {
    "caption": "Single molecule fluorescent in situ hybridization (smFISH) showing her6 (green), elavl3 (magenta) and dapi nuclear staining (blue) obtained from hindbrain (r6) sections of wild type embryo at 34hpf; head arrows indicates examples of co-existence of transcriptional active sites for her6 and elavl3; scale bar 3μm.",
    "molecules": "dapi"
  },
  {
    "caption": "A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).",
    "molecules": "4OHT"
  },
  {
    "caption": "C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm. D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments).",
    "molecules": "doxycycline"
  },
  {
    "caption": "E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm. F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).",
    "molecules": "PBS, tamoxifen"
  },
  {
    "caption": "B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. *P<0.05. Two-way paired ANOVA.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A Immunofluorescence showing gammaH2AX foci in Trf2F/F MEFs at the indicated time points following 4OHT treatment and consequent TRF2 knockout and treated with DMSO or ATMi. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).",
    "molecules": "4OHT, DMSO, ATMi"
  },
  {
    "caption": "C Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce telomere uncapping) or vehicle and injected with the indicated ASOs or PBS as control. Scale bar, 10 µm. D RT-qPCR detection of Ace2 mRNA levels in livers of mice treated as in C (n = 5-8 mice per group).",
    "molecules": "ASOs, PBS, tamoxifen"
  },
  {
    "caption": "E Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 200 µm.",
    "molecules": "ASOs"
  },
  {
    "caption": "F Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive cells in lungs of age-matched wild type and G3 Terc-/- mice, treated with the indicated ASOs (n = 4-9 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.",
    "molecules": "ASOs"
  },
  {
    "caption": "(B) CohesinHalo488 loaded onto DNA in the presence of Scc2-Scc4 was incubated in the buffer with or without TEV protease. After high-salt washing, CohesinHalo488 intensities on DNA were measured. Red bars denote the median, lower, and upper quartile values (n ≥ 98; *p < 0.0001, two-tailed Mann-Whitney test).",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Kymograph of a single DNA-interacting cohesinHalo488 particle. CohesinHalo488 is loaded onto DNA in the presence of Scc2-Scc4 and, after high-salt washing, single cohesin particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 2 μm.(D) Histogram of the net displacement (xfinal - xinitial) of cohesin particles treated as in (C). The magenta curve represents the Gaussian fit.",
    "molecules": "ATP, DNA, KCl"
  },
  {
    "caption": "(E) CohesinHalo488 was loaded and washed in high salt as in (C), and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl. Kymograph of DNA-associated cohesin particles under high-salt conditions (750 mM KCl, right) and the mean square displacement (MSD) versus time (left) are shown. Dotted lines are regression lines used to estimate the diffusion coefficient (D). Scale bar, 2 μm.",
    "molecules": "ATP, DNA, KCl"
  },
  {
    "caption": "(F) MSD versus time for DNA-bound cohesinWT-Halo488 or cohesinKA-Halo488 particles. CohesinWT-Halo488 or cohesinKA-Halo488 was loaded onto DNA in the presence of Scc2-4 and ATP. After high-salt washing, the cohesin particles on DNA were observed in the presence of 1 mM AMP-PCP or ATP and 100 mM KCl. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).",
    "molecules": "AMP-PCP, ATP, DNA, KCl"
  },
  {
    "caption": "(A) Kymograph of CohesinHalo488 in the presence or absence of Wapl-Pds5. CohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with or without Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 1 μm.(B) MSD versus time of cohesin particles in (A) are shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).",
    "molecules": "ATP, DNA, KCl"
  },
  {
    "caption": "(C) Acetylated- or unacetylated-cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).",
    "molecules": "ATP, DNA, KCl"
  },
  {
    "caption": "(D) Acetylated cohesinHalo488 was treated as in (C), the DNA-bound cohesin was further incubated with buffer, Wapl-Pds5, or Wapl-Pds5 plus Sororin, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).",
    "molecules": "ATP, DNA, KCl"
  },
  {
    "caption": "(A) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5 plus Sororin, and then treated with buffer, Plk1, or Plk1 plus BI4834. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).",
    "molecules": "BI4834, ATP, DNA, KCl"
  },
  {
    "caption": "(B) Acetylated cohesinHalo488 was treated as in (A), or treated with CDK1 or Aurora B rather than Plk1. Sororin intensity on DNA was measured after immunofluorescence microscopy with anti-Sororin antibody (n > 100, *p < 0.0001, two-tailed Mann-Whitney test).",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Acetylated cohesinHalo488 was treated as in (A) but treated with Aurora B rather than Plk1. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).",
    "molecules": "ATP, KCl"
  },
  {
    "caption": "(A) Stretched DNA was incubated in the HSS in the presence or absence of geminin. After washing the HSS, endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Scale bar, 2 µm.(B) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in (A). (22 DNAs per condition, n = 3, mean ± s.e.m. *p < 0.0001, two-tailed Mann-Whitney test).",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Chromatin formed in the HSS in the presence of geminin, and cohesinHalo488 (green) was loaded onto the chromatin with Scc2-Scc4. Chromatin was detected by immunostaining of histone H3 (magenta). DNA was counterstained with SYTOX (gray). The distributions of cohesin particles and histone H3 are shown as line-scan plots (bottom). Scale bar, 5 μm.(D) Cohesin particles observed in (C) were categorized as indicated (> 37 particles per condition. n = 6, mean ± s.e.m.).(E) Cohesin particle numbers for each condition. Red bars denote the median, lower, and upper quartile values (n ≥ 12, *p < 0.0004, unpaired t-test).(F) Colocalization of Mcm2 with Smc3 was quantified (50 Mcm2 particles per condition, n = 3, mean ± s.e.m.)",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Stretched DNA was incubated in the HSS. Endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Colocalization of Smc3 and Mcm2 was quantified (bottom, n = 3, mean ± s.e.m.). Scale bar, 2 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Fluorescently labeled Xenopus cohesins (xCohesinHaloTMR) were loaded onto DNA in the HSS, and DNA-bound cohesin particles were observed in the HSS with or without 5 mM AMP-PNP supplementation. On the kymograph, the magenta lines indicate the duration of DNA binding of each cohesin particle. DNA was counterstained with SYTOX. Scale bar, 5 μm.",
    "molecules": "AMP-PNP, DNA"
  },
  {
    "caption": "(C) Kymographs of DNA-bound xCohesinHaloTMR (green) in HSS. After time-lapse imaging of xCohesinHaloTMR, nucleosomes were immunostained by anti-H3 antibody (magenta). Result of H3 immunostaining was merged with the kymograph. DNA was counterstained with SYTOX. Scale bar, 5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(E) Kymographs of DNA-bound xCohesinHaloTMR in mock-, XEco2-depleted, or hEsco1-added XEco2-depleted HSS. Scale bar, 2 μm.(F) MSD versus time of cohesin particle in the experiment in (E). D indicates the diffusion coefficient (n = 15, mean ± s.e.m.).",
    "molecules": "DNA"
  },
  {
    "caption": "(B) Kymographs of examples for cohesin motion during DNA replication. xCohesinHaloTMR (magenta) was loaded onto DNA in the HSS, and NPE was introduced to allow DNA replication. DNA replication was monitored by xFen1D179A-PAGFP (green). Thirthy-four particles were categorized into five groups. Arrowheads indicate the time points when cohesin was dissociated from chromatin. Scale bar, 2μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(a) Ten-month-old adult mice and 20-month-old aged mice were treated with an intraperitoneal injection of 5 mg kg-1 CCCP and their hearts were collected 12 h later. Representative electron micrographs of hearts from CCCP-treated adult and aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents an autophagic vacuole containing mitochondria; original magnification × 12,000; scale bar, 500 nm. Mitochondria incorporated into an autophagic vacuole in baseline and CCCP-treated conditions were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).",
    "molecules": "CCCP"
  },
  {
    "caption": "(b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments.",
    "molecules": "CCCP, DOX"
  },
  {
    "caption": "(d) Parkin was overexpressed in young and senescent MEFs using adenovirus-mediated transduction for the in vitro cell-based bioassay to assess the ability to clear damaged mitochondria. Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM. Results from four independent experiments performed in duplicate are shown. MEFs at passage 3 and passage 9 were used as young and senescent MEFs, respectively.",
    "molecules": "CCCP"
  },
  {
    "caption": "(e) Mitochondrial DNA content was assessed by real-time PCR. Results are shown from six-independent experiments.",
    "molecules": "DNA"
  },
  {
    "caption": "(f) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone, GRP75 (matrix protein) and complex I subunit, NDUFA9 (inner membrane protein). MEFs were treated with 100 nM bafilomycin-A1, (Baf-A1, an inhibitor of the vacuolar-type proton ATPase) to inhibit autophagy. Representative immunoblots are shown from three independent experiments.",
    "molecules": "Baf-A1, bafilomycin-A1"
  },
  {
    "caption": "(g) Representative images of GFP-LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti-TOM20 (red); original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm. Line scans below the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the arrows drawn in the images. WHL indicates whole-heart lysate. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) The senescence-associated molecules p53, p21, p16, H-Ras 12V and Rb were overexpressed in MEFs by retrovirus-mediated transduction. After puromycin selection, mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus-mediated Parkin expression. Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM. Results are shown from four independent experiments performed in duplicate.",
    "molecules": "CCCP, puromycin"
  },
  {
    "caption": "(d) Mitochondrial content of WT and p53−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. WT and p53−/− MEFs were transfected with control or p53-specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin-α before mitophagy induction. Results are shown from four independent experiments performed in duplicate.",
    "molecules": "pifithrin-α"
  },
  {
    "caption": "(e) Murine HL-1 cardiac myocytes were infected with retrovirus vectors encoding p53, p21 or p16 and after puromycin selection, underwent mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown.",
    "molecules": "puromycin"
  },
  {
    "caption": "(f) HL-1 cells were transfected with control, p53, p21 or p16-specific siRNAs, followed by treatment with 0.02 μM DOX and mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate.",
    "molecules": "DOX"
  },
  {
    "caption": "(g) CCCP-induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX-treated WT, p53−/− and p21−/− mice were determined by immunoblotting of the heartmitochondria-rich fraction. Representative immunoblots are shown from four independent experiments. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).",
    "molecules": "CCCP, DOX"
  },
  {
    "caption": "(a) Whole-cell lysates of HL-1 cells treated with 0.02 μM DOX and transfected with FLAG-tagged Parkin or the FLAG-empty vector were immunoprecipitated with the anti-Flag-M2 antibody and blotted with anti-p53 and Flag antibodies.",
    "molecules": "DOX"
  },
  {
    "caption": "(b) Whole-cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti-Parkin antibody.",
    "molecules": "DOX, H2O2"
  },
  {
    "caption": "(d) Schematic representation of N-terminally FLAG-tagged WT Parkin and various mutants of Parkin. (e) The association of endogenous p53 with various Parkin mutants in DOX-treated HeLa cells. The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53.",
    "molecules": "DOX"
  },
  {
    "caption": "(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments.",
    "molecules": "CCCP, DOX"
  },
  {
    "caption": "(c,d) Representative images of YFP-Parkin overexpressing p53−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(e-g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300-400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P0.05, compared with the corresponding control (two-tailed unpaired Student's t-test).",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) Representative electron micrographs of DOX-treated hearts. Arrows indicate abnormal mitochondria defined by marked swelling, vacuolation (loss of electron density) or disrupted cristae; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents abnormal mitochondria (blue line) and an autophagic vacuole containing mitochondria (red line); original magnification × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).",
    "molecules": "DOX"
  },
  {
    "caption": "(d) The 8-hydroxy-2′-deoxyguanosine (8-OHdG), popular marker for oxidative DNA damage, in mitochondria DNA from DOX-treated hearts. Results are shown from 6-8 hearts analysed in duplicate.",
    "molecules": "8-OHdG, DNA, DOX"
  },
  {
    "caption": "(e) Maximum dP/dt was examined using cardiac catheterization under graded dobutamine infusion in PBS-treated (control) and DOX-treated mice (n=6-8 per group). DKO indicates double knockout. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).",
    "molecules": "dobutamine, DOX"
  },
  {
    "caption": "We used 20-month-old heterozygous littermates surviving grossly tumor free. (a,b) The sequential state III oxygen consumption rate of heart isolated mitochondria (300 μg) was monitored with a fibre-optic system. Substrates and inhibitors were added sequentially at the final concentrations indicated as follow. FCCP, 1 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone; M/P, 5 mM malate+5 mM pyruvate; Rot, 100 nM rotenone; Suc, 5 mM succinate; AA, 50 nM antimycin A; TMPD/Asc, 0.4 mM N,N,N′,N′-tetramethyl-p-phenylenediamine +1 mM ascorbate; KCN, 5 mM potassium cyanide. Representative results are shown (a) and oxygen consumption by each complex was calculated (n=5-8 per group, b).",
    "molecules": "N,N,N′,N′-tetramethyl-p-phenylenediamine, TMPD, antimycin A, ascorbate, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, FCCP, malate, oxygen, KCN, potassium cyanide, pyruvate, rotenone, succinate"
  },
  {
    "caption": "(e) H2O2 production with 0.5 mM NADH, complex I-linked substrate, was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red (n=5-8 per group).",
    "molecules": "H2O2, NADH, rotenone"
  },
  {
    "caption": "(f) Myocardial high-energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC (n=5 per group).",
    "molecules": "dobutamine"
  },
  {
    "caption": "(g) Maximum dP/dt was examined using cardiac catheterization with graded a dobutamine infusion (n=5 per group).",
    "molecules": "dobutamine"
  },
  {
    "caption": "(h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section. HT indicates heterozygous; DHT double heterozygous. Data are shown as the means±s.d. *P0.05; **P0.01; ***P0.001 (two-tailed unpaired Student's t-test)",
    "molecules": "SA-β-gal"
  },
  {
    "caption": "(d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e).",
    "molecules": "TMPD, antimycin A, ascorbate, FCCP, NADH, oxygen, rotenone, succinate"
  },
  {
    "caption": "(d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e).",
    "molecules": "TMPD, antimycin A, ascorbate, FCCP, NADH, oxygen, rotenone, succinate"
  },
  {
    "caption": "(f) NADH-driven H2O2 production was determined using Amplex Red. Results are shown from five hearts analysed in duplicate.",
    "molecules": "H2O2, NADH"
  },
  {
    "caption": "(g) Maximum dP/dt was examined using cardiac catheterization with graded dobutamine infusion in WT and Parkin Tg aged littermates (n=8 per group).",
    "molecules": "dobutamine"
  },
  {
    "caption": "The impact of individual Bmp ligands on the growth of freshly established intestinal organoids. (After 96 hours in 500 ng/ml of indicated Bmp. Brightfield images. Scale bar, 200 μm) Proliferation of freshly established intestinal organoids cultivated with indicated Bmp for 96 hours determined by quantification of EdU incorporation (% of EdU positive cells). Each dot represents one cultured organoid. Average value for each treatment indicated by line, error bars show sd.",
    "molecules": "EdU"
  },
  {
    "caption": "Expression of Cd73 (Nt5e gene) (green) and Fabp2 (red) upon the Bmp2 and Bmp4 treatment in the intestinal organoids. Immunocytochemistry, DAPI (blue) counterstains nuclei, timepoint: 48 hours. (Scale bar,100 μm).",
    "molecules": "DAPI"
  },
  {
    "caption": "Quantification of Cd73 (Nt5e gene) and Fabp2 immunostainings determined as the ratio of specific signal to DAPI ratio. (Each dot represents one organoid, timepoint: 48 hours, n= at least 20 organoids, graphs show: min.,1st quartile, median, 3th quartile, max., p values counted using t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "Four days treatment with LDN-193189-4HCl (50 mg/kg) reduces the expression of Ada (green) protein at the villus tips. (Immunohistochemistry, DAPI (blue) counterstains nuclei. E-cadherin (white) marks epithelial cells, scale bar, 100 μm).",
    "molecules": "DAPI, LDN-193189-4HCl"
  },
  {
    "caption": "Changes in the expression of villus tip and villus center genes after LDN-193189-4HCl (150 mg/kg) treatment determined by qRT-PCR normalized to β-actin (β-act). (Each dot represents one technical replicate of one representative experiment, error bars show sd.).",
    "molecules": "LDN-193189-4HCl"
  },
  {
    "caption": "Induction of shRNA against Smad4 upon the doxycycline (+Dox) treatment (3 days) followed by Bmp2 treatment for additional 24 hours. shRNA activation is visualised by the expression of coupled TdTomato (red), scale bar, 200 μm.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "Expression of E-cadherin (red) and Bmp2, Bmp4, Wnt5a, Bmp2 + Wnt5a treatment in the intestinal organoids. Immunocytochemistry, DAPI (blue) counterstains nuclei. (Immunocytochemistry, timepoint: 24 hours, 500 ng/ml each protein, scale bar, 200 μm).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n (number of animals per group)=6.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n (number of animals per group)=5.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) Tumor volume in SCIDmice treated or not with LPS n (number of animals per group)=5.",
    "molecules": "LPS"
  },
  {
    "caption": "(D) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n (number of animals per group)=6.",
    "molecules": "LPS"
  },
  {
    "caption": "(E) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n (number of animals per group) ≥ 4. NT, control; DT, diphtheria toxin treated animals to deplete DCs; LPS, mice treated with LPS. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.",
    "molecules": "LPS"
  },
  {
    "caption": "(A,B) Absolute numbers of IFN-γ+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics.",
    "molecules": "FTY720, LPS"
  },
  {
    "caption": "(C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme.",
    "molecules": "FTY720, LPS"
  },
  {
    "caption": "(D) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.",
    "molecules": "FTY720, LPS"
  },
  {
    "caption": "(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cel ingress in the lymph node from blood. Where indicated mice were challenged with LPS.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Absolute numbers of IFN-γ+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration.",
    "molecules": "LPS"
  },
  {
    "caption": "(D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN-γ antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown.",
    "molecules": "LPS"
  },
  {
    "caption": "(E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment.",
    "molecules": "LPS"
  },
  {
    "caption": "(F) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope. The sequence of images shows the interaction between an NK cell (red) and a DC (green). In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back. In the right lower panel the stable contact DC-NK cell is clearly visible. (B) Time duration of the DC-NK contacts in steady state (-LPS) and inflammatory (+LPS) conditions. Bars indicate the mean of the distribution. The percent of cells showing long interaction times (≥ 15 min) are shown. (C) NK cell 3D velocities. 3D velocities of NK cells taking contacts with DCs before (black) and 5 h (green) after LPS treatment.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures.",
    "molecules": "LPS"
  },
  {
    "caption": "(C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS-activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "Time-of-addition study with selected FDA-approved drugs.Five FDA-approved drugs were analyzed by time-course experiments to determine the stage in the MERS-CoV life cycle that is inhibited by the drugs. Vero cells were infected at a multiplicity of infection (MOI) of 5 with MERS-CoV, and FDA-approved drugs were administered at six-time points prior to or post-infection as indicated. Drug concentrations were higher than the 90% inhibitory concentration (IC90) values of the drugs, and chloroquine was used as an early stage infection inhibitor control.",
    "molecules": "chloroquine"
  },
  {
    "caption": "Chl-HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection  in N. benthamiana epidermal cells. Ratiometric images (F488/405 nm) of fluorescence excitation at 488 and 405 nm show the oxidized state of  chloroplast-targeted HyPer2, respectively. The false-blue color indicates the  chloroplasts. Scale bars, 30 μm. Data information: In (A), data are representative of at least three independent experiments.",
    "molecules": "hydrogen peroxide"
  },
  {
    "caption": "Quantification of the  BSMV-dependent changes in Chl-HyPer2 fluorescence at 0 to 6 dpi. The percentage of  ROS-positive  chloroplasts (white) among all chloroplasts  (blue and white) per visual field. Data information: In (B), error bars indicate mean ± SEM (n = 5).",
    "molecules": "ROS"
  },
  {
    "caption": "BSMV accumulation in  N. benthamiana leaves transiently expressing  chloroplast ROS  scavenging proteins. Agrobacterium harboring plasmids expressing various proteins are indicated above the panels. BSMV  was agroinfiltrated into the same  leaves at 1.5 dpi. Three days later, Western blot analyses were conducted with anti-TGB1  or anti-Flag  antibodies.  RbcL served as the loading control. 2-CP, 2-Cys Prx. Data information: representative data are shown, at least three independent replicates were performed with similar results.",
    "molecules": "ROS"
  },
  {
    "caption": "Competitive  GST pull-down assays showing the effects of γb  on interactions between  NbNTRC  and 2-Cys Prx. GST-NbNTRC and 2-Cys Prx-His were incubated with increasing amounts (5, 10, 20, and 40 μg) of γb and its mutant derivatives. Glutathione-Sepharose beads were washed and Western blot  analysis was performed using anti-GST and anti-His antibodies. Data information: representative data are shown, and three independent experiments had similar results.",
    "molecules": "Glutathione-Sepharose"
  },
  {
    "caption": "Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in  N. benthamiana epidermal cells. The pCB301 empty vector (EV) was used as a negative control. Scale bars, 30 μm. The false-blue color indicates the  chloroplasts.   Quantification of  LRSV-dependent changes in Chl-HyPer2 fluorescence at 3 dpi.   Data information: , data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.",
    "molecules": "hydrogen peroxide"
  },
  {
    "caption": "Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N. benthamiana epidermal cells. Scale bars, 30 μm. The false-blue color indicates the  chloroplasts.   Quantification of the  TYMV-dependent changes in Chl-HyPer2 fluorescence at 5 dpi.   Data information: data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.",
    "molecules": "hydrogen peroxide"
  },
  {
    "caption": "(A)  C-circle assay  for tumor samples. The presence of C-circles was tested in 22 osteosarcomas  by Rolling Circle Amplification  (RCA) assay using Φ29 DNA polymerase. Φ29 DNA polymerase  negative controls and Φ29 DNA polymerase-based reactions starting respectively with 75, 37.5, and 18.75 ng of DNA   were applied to dot blots  and C-circles were detected by hybridization  with a 32P-(CCCTAA)4 telomeric  probe. U2OS  and HeLa  cells correspond to positive and negative control samples, respectively.",
    "molecules": "32P"
  },
  {
    "caption": "(B) Representative images of telomere FISH  (red) and PML IF  (green) colocalizations  (APBs) in osteosarcoma  tumor samples. DNA is counterstained  blue with DAPI. APBs  are indicated by white arrows. Scale bars are 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Representative image of TeSLA Southern Blot of two representative samples of ALT-positive (left) and telomerase-positive (right) osteosarcomas. Nine TeSLA PCRs  (30 pg each reaction) were done for each DNA  sample. DIG-labelled MW ladder has been added a posteriori. (D) Plot of cumulative TeSLA  fragment sizes (in kb) in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Points and error bars (± SEM) represent cumulative percentage of TeSLA  fragments from all the TeSLA PCRs  (n=9 per tumor sample) from all the patients  in both groups (n=16 in ALT-positive group, and n=6 in ALT-negative group). The Kolmogorov-Smirnov test was applied to identify statistical differences in TeSLA  fragments distributions (p=0.0016). (E) Individual TeSLa  fragment lengths in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Each dot represents a TeSLA  fragment, bars represent the median value for individual patient's  samples.",
    "molecules": "DNA, DIG"
  },
  {
    "caption": "(B) Tissue sections of two representative high-grade osteosarcomas  with HES (hematoxylin eosin saffron) staining  (left panel), anti-ATRX  (HPA064684) immunochemistry  (middle panel), and anti-DAXX  (HPA008736) immunochemistry  (right panel), showing ATRX protein expression in two ALT-positive samples. In the top panel, intratumoral ATRX  expression is negative (positive control osteoclasts  are shown). In the bottom panel, intratumoral ATRX  expression is high. Scale bars are 250 μm.",
    "molecules": "saffron, eosin, hematoxylin"
  },
  {
    "caption": "(B) ATSA assay assessing ALT-mediated telomeric DNA synthesis (telomere  in red, EdU  in purple) in PML  (in green) according to TOP3A  KD. EdU  signal at APB  (telomere/PML) are indicated by white arrows. Approximately 150 cells were divided into five groups (0,1-2, 3-4, 5-6n and ≥7) based on the number of EdU+APBs. Scale bars are 5 μm.",
    "molecules": "EdU, DNA"
  },
  {
    "caption": "Ni-NTA His-ubiquitin pulldown in control transfected or HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pull down. Relative ubiquitination of the representative immunoblot was calculated using undefined for normalization.",
    "molecules": "Ni, NTA, ubiquitin"
  },
  {
    "caption": "Ni-NTA His-ubiquitin pulldown K48 or K63 in control and HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization.",
    "molecules": "Ni, NTA, ubiquitin"
  },
  {
    "caption": "Ni-NTA His-ubiquitin pulldown in control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.",
    "molecules": "Ni, NTA, ubiquitin"
  },
  {
    "caption": "Immunoblot of USP28 and ∆Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR-619 for 24 hours. Relative protein abundance was calculated ACTIN as loading control.",
    "molecules": "DMSO, PR-619"
  },
  {
    "caption": "Inducible depletion of USP28 in A-431 upon treatment with doxycycline (1µg/ml) for 96h, western blot (left, VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN (right) was performed.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Tandem ubiquitin binding entity (TUBE) pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon DOX depletion of USP28. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.",
    "molecules": "DOX"
  },
  {
    "caption": "Cycloheximide (CHX) chase assay (100µg/ml) of control or inducible sh-USP28 A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot (left, VINCULIN as loading control) of USP28 and ∆Np63 and quantification of relative protein abundance (right).",
    "molecules": "Cycloheximide, dox, EtOH"
  },
  {
    "caption": "Doxycycline induced murine USP28 overexpression (EtOH or 1ug/ml dox for 96 hours) in A-431 cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63. For qPCR, human USP28 and murine USP28 (mUSP28) primers were used. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping).",
    "molecules": "Doxycycline, EtOH"
  },
  {
    "caption": "TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon overexpression of mUSP28 for 96 hours (EtOH or 1µg/ml dox). Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.",
    "molecules": "dox, EtOH"
  },
  {
    "caption": "CHX chase assay (100µg/ml) of control or inducible mUSP28 overexpressing A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot analysis of USP28 and ∆Np63 as well quantification of relative protein abundance (VINCULIN as loading control).",
    "molecules": "dox, EtOH"
  },
  {
    "caption": "Doxycycline induced mUSP28 overexpression (EtOH or 1µg/ml dox for 96 hours) in LUDLU-1adh cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28, mUSP28 and ∆Np63. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping for the analysis).",
    "molecules": "dox, Doxycycline, EtOH"
  },
  {
    "caption": "Representative haematoxylin and eosin (H&E) staining of tumour bearing animals 12 weeks post intratracheal infection. Boxes indicate highlighted tumour areas in (C) (a, b, a' and b'). Scale bar = 2000µm; nKPL= 6 and nKPLU = 5. H=heart.",
    "molecules": "eosin, haematoxylin"
  },
  {
    "caption": "Representative haematoxylin and eosin (H&E) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells/animal. KPSCC sh-NTC (n=3); KPSCC sh-USP28 (n=3), Scale bar = 5000µm",
    "molecules": "eosin, haematoxylin"
  },
  {
    "caption": "Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.) for 24 hours. USP28 Upper band: Active USP28; USP28 lower band: Inactive USP28, VINCULIN as loading control.",
    "molecules": "DMSO, AZ1"
  },
  {
    "caption": "Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control. TP63 and USP28 half maximal inhibitory protein abundance (IC50) was calculated. Graphs are represented as mean ± SD (n=3).",
    "molecules": "DMSO, AZ1"
  },
  {
    "caption": "TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours. VINCULIN served as loading control for relative Ubiquitination.",
    "molecules": "DMSO, AZ1"
  },
  {
    "caption": "Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG-132 (20µM) or DMSO. VINCULIN served as loading control.",
    "molecules": "DMSO, MG-132, AZ1"
  },
  {
    "caption": "A-431 cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. Scale bar = 250µm. n= 30 fields analysed",
    "molecules": "Hoechst, DMSO, AZ1"
  },
  {
    "caption": "Immunoblot of endogenous USP28, ∆Np63 and KRT14 in LUDLU-1adh, H1299, Ca Ski, Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control",
    "molecules": "DMSO, AZ1"
  },
  {
    "caption": "LUDLU-1ADH, H1299, Ca Ski, Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Scale bar for LUDLU-1adh and H1299 = 500µm. Scale bar for Ca Ski, Hela and SiHa = 250µm. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63. Number of cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. n=30 fields analysed",
    "molecules": "Hoechst, DMSO, AZ1"
  },
  {
    "caption": "Immunofluorescence (IF) staining against endogenous ∆Np63 in KP and KPL cell lines (blue: DAPI [staining of nuclei] and red/magenta: ∆Np63), Scale bar = 32µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "GFP Fluorescent images of lungs treated with either PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Tumour cells are positive for GFP (green arrows). n=3 Quantification of % tumour area (normalised to total lung area) in control (n=5), 125 mg/kg AZ1 (n=4) and 375 mg/kg AZ1 (n=4) treated animals.",
    "molecules": "DMSO, Tween80, AZ1"
  },
  {
    "caption": "Representative staining of H&E and IHC for USP28 and ∆Np63 of SCC tumours from mice treated with PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Boxes indicate highlighted tumour areas. First line Scale bars = 500µm; Lower scale bars = 20µm. n=3   ∆Np63 staining intensity of lung tumours treated with PBS/DMSO/0,1%Tween80 or indicated concentrations of AZ1. nControl = 33712 cells ; nAZ1 125mg/kg = 7382 cells; nAZ1 375mg/kg = 4967 cells. Cells were analysed from three independent animals.   ",
    "molecules": "DMSO, Tween80, AZ1"
  },
  {
    "caption": "(A)  Huh7.5  cells were pre-incubated with neutralizing antibody to ACE2  and SARS-CoV-2  pseudovirus was pre-incubated with patient  isolated mAb COVA1-18, COVA1-21 and COVA2-15 (10 µg/mL) or UF heparin  (250 IU/mL) for 30 min at 37°C. SARS-CoV-2  pseudovirus alone or with blocks was added to the cells for 4h at 4°C and binding was determined by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way ANOVA with Tukey multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001(n=3)",
    "molecules": "heparin"
  },
  {
    "caption": "(D) Control and  EXT1-/- XG1  cells were exposed to SARS-CoV-2  pseudovirus or SARS-CoV-2 pseudovirus pre-treated with 250 IU/mL UF  heparin for 30 min at 37°C. After incubation for 4h at 4°C, cells were lysed and binding  was measured by  ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using two-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=3).",
    "molecules": "heparin"
  },
  {
    "caption": "(B) SARS-CoV-2 pseudovirus was pre-incubated for 30 min at 37°C with UF heparin (250 IU/mL) or LMWHs tinzaparin (250 IU/mL) or dalteparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) or nadroparin  (250 IU/mL). Huh7.5  were exposed to the SARS-CoV-2  pseudovirus, alone or treated with different LMWHs for 4 hours at 4°C, washed, lysed and binding  was determined by  ELISA. (C) SARS-CoV-2  pseudovirus was pre-incubated for 30 min at 37°C with UF  heparin (250 IU/mL) or LMWHs  tinzaparin (250 IU/mL) or dalteparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) or nadroparin (250 IU/mL).  Huh7.5 cells were infected with SARS-CoV-2  pseudovirus in presence or absence of different LMWHsSARS-CoV-2  and infection was determined after 5 days by luciferase reporter activity. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (B) ordinary one-way ANOVA with Tukey's multiple-comparison test (n=3 donors measured in monoplo), (C) ordinary one-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=3 donors measured in triplicate)",
    "molecules": "dalteparin, enoxaparin, nadroparin, heparin, tinzaparin"
  },
  {
    "caption": "(C, D) SARS-CoV-2 binding was measured in polarized Caco-2 (C) and Calu-3 (D) cells in presence or absence of antibodies against  ACE2 (cell incubation), and UF heparin  (250 IU/mL) or LMWH enoxaparin (250 IU/mL) (virus incubation). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3 Caco-2 donors measured in triplicate), (D)(n=3 Calu-3 donors measured in triplicate)",
    "molecules": "enoxaparin, heparin"
  },
  {
    "caption": "(A) Namalwa cells ectopically expressing either Syndecan 1 or 4 were exposed to either SARS-CoV-2  pseudovirus alone or SARS-Cov-2  pseudovirus pre-treated with UF heparin  (250 IU/mL) or LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Binding was measured after 4 hours at 4°C by ELISA. (B) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-incubated with LMWH  enoxaparin (250 IU/mL) for 30 min at 37°C. Namalwa  cells expressing Syndecan 1 and 4 were exposed to either SARS-CoV-2 isolate (100 TCID/mL) or SARS-CoV-2 isolate (100 TCID/mL) pre-treated with LMWH LMWH enoxaparin (250 IU/mL) for 4 hours at 4°C and binding was determined by  quantitative real-time PCR. Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) 2way-ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n = 7), (B) 2way-ANOVA with Sidak's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n = 3 measured in triplicate).",
    "molecules": "enoxaparin, heparin"
  },
  {
    "caption": "(B) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated for 30 min at 37°C with either LMWH enoxaparin (250 IU/mL) or one of the following neutralizing antibodies (COVA1-18, 1-21 and 2-15), a non-neutralizing antibody (COVA1-27) or a human IgG1 isotype control, all at the concentration of 1 pg/mL. SARS-CoV-2 isolate alone or with blocks was added at a concentration of 100 TICD/mL. Detection of virus  binding to Syndecan 1 expressing  Namalwa was measured by  quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using ordinary one-way with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01 (n=2 measured in duplicates).",
    "molecules": "enoxaparin"
  },
  {
    "caption": "(F, G) DCs (F) and LCs  (G) were pre-incubated with SARS-CoV-2  pseudovirus for 4 hours at 37°C in presence or absence of UF  heparin (250 IU/mL) or LMWH enoxaparin  (250 IU/mL), extensively washed and co-cultured with Huh7.5 cells. Transmission by  DCs or LCs  to Huh7.5 cells was determined by luciferase reporter activity. Data information: Data show the mean values and error bars are the SEM. (F) ordinary one-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=4 measured in triplicate), (G) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=3 measured in triplicate). DCs: Dendritic cells, LCs:  Langerhans cells, RLU: relative light units",
    "molecules": "enoxaparin, heparin"
  },
  {
    "caption": "(H, I) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-treated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. DCs  (H) and LCs (I) were exposed to either the untreated or pre-treated SARS-CoV-2 isolate (100 TCID/mL) for 24h, washed thoroughly and co-cultured with Huh7.5 cells. Quantification of viral RNA  was measured by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. (H, I) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (H) (n=3 in duplicates), (I) (n=4 in duplicates). DCs: Dendritic cells,  LCs: Langerhans cells",
    "molecules": "enoxaparin"
  },
  {
    "caption": "(B) ACE2, Syndecan 1  and Syndecan 4 cell surface expression on nasal epithelial cells, compared to polarized epithelial Calu-3 was confirmed by quantitative real-time PCR. (C, D) Nasal epithelial cells were exposed to SARS-CoV-2  isolate (hCoV-19/Italy, 100 TCID/mL) either directly or after pre-treatment with antibodies against ACE2 cell surface receptors (1 hour at 37°C )or after pre-treatment with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Detection of viral binding  after 4 hours at 4°C (C) and persistently-infected cells lysed after 24 hours at 37°C (D) was determined by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (C, D) ordinary on-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3), (D) (n=3 in duplicates).",
    "molecules": "enoxaparin"
  },
  {
    "caption": "B. Dsl1pVN•ε-COPVC BiFC foci showed different subcellular distribution depending on the growth status of cells. Cells were cultured in PM+ura medium with or without 2 % glucose at 30 °C for 90 min, and imaged. BiFC signals dispersed under glucose starvation, resulting in an ER exit site-like fluorescence pattern. Scale bar 10 µm.",
    "molecules": "glucose"
  },
  {
    "caption": "C. The effect of carbon source depletion on COPI•Dsl BiFC signal polarization. Cells carrying Dsl1pVN•ε-COPVC were starved in glucose-free PM+ura medium for 90 min. Then the medium was replaced by PM Glc+ura with 2 % glucose, while imaging the cells continuously at 1 min intervals. Two exemplary cells are shown (upper and lower panel). Starvation-induced dispersed BiFC signals reversibly returned to polarized distribution within minutes after addition of glucose-containing medium. Arrowheads mark the point of signal repolarization, arrows indicate resumed bud outgrowth. Scale bar 5 µm.",
    "molecules": "glucose"
  },
  {
    "caption": "Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.I. β'-COPmGFPfluorescence foci in Dsl1p-depleted GAL-DSL1 cells after incubation in glucose-containing medium (YEPD) for 4 h at 30 °C.",
    "molecules": "glucose"
  },
  {
    "caption": "A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (A) Upon disruption of actin filaments with 1,25 μM latrunculin A, COPI•Dsl BiFC signals dispersed. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm.",
    "molecules": "latrunculin A"
  },
  {
    "caption": "A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (B) Microtubuli disruption through 24 μg/ml nocodazole did not affect polarization of the COPI•Dsl BiFC signals. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm.",
    "molecules": "nocodazole"
  },
  {
    "caption": "A-B. Immuno-electron micrographs showing the buds and the neck region of two β'-COPVN•Dsl3pVCcells. Both parts contain an overview image (left) and two zoomed details (right). Slides were decorated with antibody against COPI and visualized with protein A-conjugated 10 nm gold particles. Gold particles mark electron-dense areas in apposition to membranes and/or vesicular structures, which are localized at the side of the bud and the bud neck of the mother cell. Scale bar 100 nm.",
    "molecules": "gold"
  },
  {
    "caption": "D. COPI-positive, electron-dense structures in close apposition to perinuclear ER. 10 nm gold particles mark COPI. Scale bar 100 nm.",
    "molecules": "gold"
  },
  {
    "caption": "E. Accumulation of COPI-positive vesicles. 10 nm gold particles mark COPI. PM: plasma membrane, nuc: nucleus. Scale bar 100 nm.",
    "molecules": "gold"
  },
  {
    "caption": "A Pancreatic  sections (20X magnification) from 10-months old WT and Glo1KD mice stained  with hematoxylin  and eosin (H&E), anti-insulin, anti-glucagon   and anti-F4/80  antibodies, are representative of sections from 7 mice  each group.",
    "molecules": "eosin, glucagon, hematoxylin, insulin"
  },
  {
    "caption": "H, I and J Insulin  and glucagon IS, calculated as the intensity score multiplied with percentage of positive cells (H, I) and the percentage of islets  area stained  with F4/80 (J) (n=7 mice  for each genotype). Data information: box plots show the minimum and maximum values (ends of the whiskers), interquartile range (length of the box), median (line through the box) and mean (+) values. *p≤0.05, **p≤0.01 (Mann-Whitney test).",
    "molecules": "glucagon, Insulin"
  },
  {
    "caption": "C Whole cell protein lysates of INS-1 832/13 β-cells  analysed by  Western Blot  with anti-p53  and anti-p21  antibodies; anti-vinculin  antibody was used as loading control (n=9 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the  densitometric analysis  of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO  vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO  (Mann-Whitney test",
    "molecules": "MGO"
  },
  {
    "caption": "D Nuclear protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-HMGB1  antibody; anti-histone H3 antibody was used as loading control (n=5 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO  vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO  (Mann-Whitney test",
    "molecules": "MGO"
  },
  {
    "caption": " A. RT-PCR of CNPase (left) and MBP (right) mRNAs expression in MO3.13 precursors under control conditions and following 10-200 μM D-Asp exposure for 3 days. Graphs show quantification of ratio of CNPase, and MBP to L19 Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, left panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control (n=3); A, right panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P<0.05 versus control (n=3 ",
    "molecules": "D-Asp"
  },
  {
    "caption": " B. Western blotting (left) and densitometric analysis (right) of MBP expression in absence or in presence of 10-200 μΜ D-Asp exposure for 5 days Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); B, one-way ANOVA P=0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control (n=3",
    "molecules": "D-Asp"
  },
  {
    "caption": " C. Cell growth analysis of human MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 1-5 days. The density of M03.13 oligodendrocytes was daily recorded through trypan blue dye exclusion. Mean of daily measurements were recorded. The data of each experimental group were normalized to the density of cells plated at day 0 and expressed as percentage of ctrlday0 Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, for each day, two-tailed Student's t test, *P<0.05 versus control at 3 days (n=5)",
    "molecules": "D-Asp, trypan blue"
  },
  {
    "caption": " D. FACS-based cell cycle distribution analysis after PI incorporation of MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 3 days. Representative FACS plots of biological replicates are shown (n=5 independent experimental sessions) ",
    "molecules": "D-Asp, PI"
  },
  {
    "caption": " E. FACS-based cell cycle distribution analysis after PI incorporation of rat primary OPC in absence or in presence of 100 μM D-Asp for 1 day. Representative FACS plots of biological replicates are shown (n=3 independent experimental sessions) ",
    "molecules": "D-Asp, PI"
  },
  {
    "caption": " F. Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin-594 (a-b and c-d, respectively) in rat primary OPC cultured in absence or in presence of 100 μM D-Asp for 4 days. (e), Single representative MBP++ cell. Scale bars: 50 μm in a-d; 10 μm in e. (f-g); Quantitative analysis of Alexa-594-phalloidin-positive oligodendrocytes (f) or MAG+ and MBP++ oligodendrocytes (g) scored in control and D-Asp exposed cells. Alexa-594-phalloidin-positive oligodendrocytes were scored in 4 categories, according to their morphological complexity. In each category, data were normalized on the total number of oligodendrocytes Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each category two-tailed Student's t test, *P<0.05 versus control (n=3)",
    "molecules": "594, Alexa-594, D-Asp, phalloidin, Phalloidin"
  },
  {
    "caption": " B. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in 7 DIV cerebellar organotypic slices cultured in absence (b-d) or in presence of 100 μM D-Asp (f-h). Panels d and h display colocalized points (white). Panels a and e show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a and e: 200 μm; in b-d and f-h: 50 μm ",
    "molecules": "D-Asp"
  },
  {
    "caption": " C. Scatter plot histogram analysis of the myelination index in 7 DIV cerebellar slices cultured in absence or in presence of 100 μΜ D-Asp Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in C, two-tailed Student's t test, *P<0.05 versus control (n=4 animals, 3-4 slices per group ",
    "molecules": "D-Asp"
  },
  {
    "caption": " D. Western blotting analysis of MBP protein levels from homogenates of organotypic cerebellar slices culture in absence or in presence of 100 μΜ D-Asp. Data were normalized on the basis of β-actin and expressed as percentage of controls Data information: The values represent the means ± S.E.M. Level of significance was determined by using D, two-tailed Student's t test, *P<0.05 versus controls (n=3",
    "molecules": "D-Asp"
  },
  {
    "caption": "F. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 6 days after LPC exposure (dpl) in absence (f-h) or in presence 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h, and j-l: 50 μm",
    "molecules": "D-Asp, LPC"
  },
  {
    "caption": " G. Western blotting and quantitative analysis of MBP protein levels from homogenates of organotypic cerebellar slices at 6 dpl cultured in absence or in presence of 100 μΜ D-Asp Data information: The values represent the means ± S.E.M. Level of significance was determined by usin G, one-way ANOVA P=0.003 followed by Bonferroni post hoc test, *P<0.05 versus control, ˄P< 0.05 versus LPC (n=3",
    "molecules": "D-Asp, LPC"
  },
  {
    "caption": " H. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 6dpl after LPC exposure cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); H, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group)",
    "molecules": "D-Asp, LPC"
  },
  {
    "caption": " I. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 10 dpl in absence (f-h) or in presence of 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h,and j-l: 50 μm",
    "molecules": "D-Asp"
  },
  {
    "caption": " J. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 10 dpl cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control Data information: The values represent the means ± S.E.M. Level of significance was determined by usin J, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group)",
    "molecules": "D-Asp, LPC"
  },
  {
    "caption": " A. RT-PCR of CNPase mRNA expression in oligodendrocyte MO3.13 progenitors under control conditions and following 200 μM D-Asp exposure for 3 days, in absence or in presence of 10 μM MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel). Graphs show quantification of ratio of CNPase to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, left, one-way ANOVA P=0.009 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); A, right, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3 ",
    "molecules": "D-Asp, BED, MK-801, YM-244769"
  },
  {
    "caption": " B. RT-PCR of MBP mRNA expression in MO3.13 cells under control conditions and following D-Asp exposure for 3 days, in absence or in presence of 10 μΜ MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel) Graphs show quantification of ratio of MBP to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin B, left, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); B, right, one-way ANOVA P=0.0004 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3",
    "molecules": "D-Asp, BED, MK-801, YM-244769"
  },
  {
    "caption": " C. (a-j); Confocal images displaying the expression of NG2 and MAG proteins in rat primary OPC cultured in presence of D-Asp for 3 days, in absence (a-c) or in presence of 10 μM MK-801 (d-f), or 30 nM YM-244769 (g-i) or 100 nM BED (j-l) C. (m-n); Quantitative analysis of NG2+ and MAG+ cells in rat primary OPC cultured in presence of D-Asp for 3 days, in absence or in presence of 10 μM MK-801, 30 nM YM-244769 or 100 nM BED Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using C, m and C, n, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3)",
    "molecules": "D-Asp, BED, MK-801, YM-244769"
  },
  {
    "caption": " D. RT-PCR of NCX3 mRNA expression under control conditions and following 10-200 μM D-Asp exposure or 100 nM PMA for 3 days Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i D, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3)",
    "molecules": "PMA, D-Asp"
  },
  {
    "caption": " E. RT-PCR of NCX3 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using E, one-way ANOVA P=0.0083 followed by Tukey's post hoc test, *P<0.05 versus control, ˄P< 0.05 versus D-Asp (n=3), n.s, not significant",
    "molecules": "D-Asp, MK-801"
  },
  {
    "caption": " F. RT-PCR of NCX1 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801. Graphs show quantification of ratio of NCX1 and NCX3 to L19 ",
    "molecules": "D-Asp, MK-801"
  },
  {
    "caption": "left panels. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells (A in absence or in presence of 10 μM MK-801 or 150 μM APV right panels. Quantification of the oscillation index in MO3.13 cells (A in absence or in presence of 10 μM MK-801 or 150 μM APV Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 10 μM MK-801 or 150 μM AP in M03.13 cells (a MK-801 and APV were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A and a, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus control (basal value), ˄P<0.05 versus D-Asp. Data are reported as mean of 23-30 cells in each group, n=3 biological replicate ",
    "molecules": "AP, APV, D-Asp, [Ca2+]i, MK-801"
  },
  {
    "caption": "left panels. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected i primary OP (B) in absence or in presence of 10 μM MK-801 or 150 μM APV right panels. Quantification of the oscillation index i primary OPC (B) in absence or in presence of 10 μM MK-801 or 150 μM APV Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 10 μM MK-801 or 150 μM APV -i primary OPC (b). MK-801 and APV were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using B and b, one way-ANOVA, P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus control (basal value), ˄P< 0.05 versus D-Asp. Data are reported as mean of 20-23 cells in each group, n=3 biological replicate",
    "molecules": "APV, D-Asp, [Ca2+]i, MK-801"
  },
  {
    "caption": "left panels; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells (C in absence or in presence of 30 nM YM-244769 or 100 nM BED right panels; Quantification of the oscillation index in MO3.13 cells (C in absence or in presence of 30 nM YM-244769 or 100 nM BED d, Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 30 nM YM-244769 or 100 nM BED in M03.13 cells (c YM-244769 or BED were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using C and c, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *p< 0.05 versus control (basal value), ˄ P< 0.05 versus D-Asp. Data are reported as mean of 19-30 cells in each group, n=3 biological replicate",
    "molecules": "D-Asp, BED, [Ca2+]i, YM-244769"
  },
  {
    "caption": "left panels; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected i primary OPC (D) in absence or in presence of 30 nM YM-244769 or 100 nM BED D, right panels; Quantification of the oscillation index i primary OPC (D) in absence or in presence of 30 nM YM-244769 or 100 nM BED Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 30 nM YM-244769 or 10 nM BED -i primary OPC (d). YM-244769 or BED were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin D and d, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus control (basal value), ˄P< 0.05 versus D-Asp. Data are reported as mean of 18-20 cells in each group, n=3 biological replicate",
    "molecules": "D-Asp, BED, [Ca2+]i, YM-244769"
  },
  {
    "caption": " E. left panel; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells in presence of siCtl or sincx3 silencing. E, right panel; Quantification of the oscillation index in MO3.13 cells in absence or in presence of sincx3. e, Quantification of the initial [Ca2+]i increase elicited by D-Asp and measured as ∆% of peak versus basal values in absence or in presence of sincx3 Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin E and e, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus sictl, ˄P< 0.05 versus D-Asp + sictl. Data are reported as mean of 25-30 cells in each group, n=3 biological replicate",
    "molecules": "D-Asp, [Ca2+]i"
  },
  {
    "caption": " F. left panel. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in primary OPC obtained from wild-type ncx3+/+, heterozygous ncx3 +/-, and knock-out ncx3-/- mice. F. right panel. Quantification of the oscillation index elicited by D-Asp in primary mouse OPC obtained from ncx3+/+, ncx3 +/-, and ncx3-/- mice. f, Quantification of the initial [Ca2+]i increase measured as ∆% of peak versus basal values Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using F and f, one way-ANOVA P<0.001 and P=0.003, respectively, followed by Bonferroni post hoc test, *P< 0.05 versus basal value, ˄P< 0.05 versus ncx3+/+ and ncx3+/-. Data are reported as mean of 10-19 cells in each group, n=3 biological replicates",
    "molecules": "D-Asp, [Ca2+]i"
  },
  {
    "caption": " A. Representative inward current traces elicited by 10μM AMPA in human oligodendrocyte MO3.13 progenitor cells in the absence or in the presence of 10μM DNQX. The concentration-dependent curve of AMPA (1-100 μM) on inward currents is showed at the bottom of panel A Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus 1μM AMPA, n=3 biological replicate ",
    "molecules": "AMPA, DNQX"
  },
  {
    "caption": " B. Representative inward current traces elicited by 100μM D-Asp application in MO3.13 cells in the absence or in the presence of 10μM DNQX ",
    "molecules": "DNQX, D-Asp"
  },
  {
    "caption": " C. Representative traces of the inward current (upper panel) and the corresponding quantification (bottom panel) observed in human oligodendrocyte MO3.13 progenitors after: (I) the first application of 10μM AMPA alone; (II) the second application of 10μM AMPA alone; (III) the third application of 10μM AMPA in the presence of 10μM DNQX; (IV) the fourth application of 10μM AMPA alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin i one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus versus AMPA(I). Data are reported as mean of at least 25 cells in each group, n=3 biological replicate",
    "molecules": "AMPA, DNQX"
  },
  {
    "caption": " D. Representative traces of the inward current (upper panel) and the corresponding quantification (bottom panel) observed in human oligodendrocyte MO3.13 progenitors after: (I) the first application of 100μM D-Asp alone; (II) the second application of 100μM D-Asp alone; (III) the third application of 100μM D-Asp in presence of 10μM DNQX; (IV) the fourth application of 100μM D-Asp alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus D-Asp (I). Data are reported as mean of at least 25 cells for each group, n=3 biological replicates",
    "molecules": "DNQX, D-Asp"
  },
  {
    "caption": " E. Representative inward current traces elicited by 10μM AMPA in rat primary OPC in the absence or in the presence of 10μM DNQX ",
    "molecules": "AMPA, DNQX"
  },
  {
    "caption": " F, Representative inward current traces elicited by 100μM D-Asp in rat primary OPC in the absence or in presence of 10μM DNQX or 10μM DNQX + 20μM PDC ",
    "molecules": "DNQX, D-Asp, PDC"
  },
  {
    "caption": " G. Representative traces of the inward current (upper panel) and the corresponding quantification (lower panel) observed in rat primary OPC after: (I) the first application of 10μM AMPA alone; (II) the second application of 10μM AMPA alone; (III) the third application of 10μM AMPA in the presence of 10μM DNQX; (IV) the fourth application of 10μM AMPA alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i G, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus versus AMPA(I). Data are reported as mean of at least 25 cells in each group, n=3 biological replicate",
    "molecules": "AMPA, DNQX"
  },
  {
    "caption": " H, Representative traces of the inward current (upper panel) and the corresponding quantification (lower panel) observed in rat primary OPC after: (I) the first application of 100μM D-Asp alone; (II) the second application of 100μM D-Asp alone; (III) the third application of 100μM D-Asp in presence of 10μM DNQX; (IV) the fourth application of 100μM D-Asp in presence of 10μM DNQX + 10μM MK801; (V) the fifth application of 100μM D-Asp in presence of 10μM DNQX + 10μM MK-801+ 20μM PDC; (VI) the sixth application of 100μM D-Asp alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i H, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus D-Asp (I). Data are reported as mean of at least 25 cells for each group, n=3 biological replicates",
    "molecules": "DNQX, D-Asp, MK-801, MK801, PDC"
  },
  {
    "caption": " B-C. Beam crossing latency during daily training (left panel, averaged across 3 trials per day) and average latency to cross the beam over 3 consecutive days (right panel), recorded in control (open circles), cuprizone- (filled black circles) and in cuprizone + D-Asp-treated mice (filled red triangles) during 3 weeks (B), and 5 weeks (C) of cuprizone feeding The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOVA P=0.0013 (B, 1d), P=0.00 followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOVA P=0.0001 (B), P<0.0001 (C ), followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " D. Beam crossing latency during daily training (left panel, averaged across 3 trials per day) and average latency to cross the beam over 3 consecutive days (middle and right panels) recorded after 2.5 weeks of remyelination in control (open circles), vehicle- (filled black circles) and in D-Asp- treated mice. D-Asp (I) (filled grey triangles) refers to the group of mice which received D-Asp only for 2.5 weeks after cuprizone withdrawal; D-Asp (II) (filled red triangles) refers to the group of mice which received D-Asp during the last week of cuprizone feeding and for 2.5 additional weeks after cuprizone withdrawal The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOV P=0.0027 (D, 3d), P=0.0025 (D, 2d followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV P<0.0001 (D, for both middle and right panels followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " E-F. Latency to fall during daily training in accelerating rotarod test (left panel, averaged across 3 trials per day) and average over 3 consecutive days (right panel) recorded in control (open circles), cuprizone- (filled black circles) and D-Asp- treated (filled red triangles) mice at 3 weeks (E), and 5 weeks (F) of cuprizone feeding The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by usin left panels (daily training), for each day (d) one-way ANOV P<0.0001 (E, 3d), P=0.0082 (F, 3d followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV ), P=0.0002 (E), P<0.0001 (F) followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " G. Latency to fall during daily training in accelerating rotarod test (left panel, averaged across 3 trials per day) and average over 3 consecutive days (right panel) recorded after 2.5 weeks of remyelination (REM) in control, vehicle- and in D-Asp- treated mice. D-Asp (II) refers to the group of mice which received D-Asp during the last week of cuprizone feeding and 2.5 additional weeks after cuprizone withdrawal The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOV P<0.0001 (G, 1d), P=0.0044 (G, 2d), P=0.0038 (G, 3d), followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV P<0.0001 (G), followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " A. Representative confocal double immunofluorescence images displaying MBP (red) and NF200 (green) distribution and their coexpression (white) in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 20 μm. (j), Densitometric analysis of MBP immunofluorescence signal and (k) quantification of MBP-NF200 colocalized points in the middle corpus callosum of septostriatal sections from control mice, and from mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp Data information The values represent the means ± S.E.M. Level of significance was determined by using in: A, j, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group); A, k, one-way ANOVA P=0.0162 followed by Newman-Keuls post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group ",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " B. Western blot and densitometric analysis of MBP levels in corpus callosum lysates from control and cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively. Data were normalized on the basis of α-tubulin and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i B, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " C. Representative confocal double immunofluorescence images displaying GFAP (green) and Iba1 (red) distribution in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 50 μm. (j-k) Quantification of GFAP+ and Iba1+ cells in the middle corpus callosum of septostriatal sections of control mice and of mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp. Data were normalized to the total cell number (Hoechst signal) and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i C, j-k, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group",
    "molecules": "cpz, D-Asp, cuprizone, Hoechst"
  },
  {
    "caption": " D. Western blot of GFAP and Iba1 protein levels in corpus callosum lysates obtained from control or cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively Data information The values represent the means ± S.E.M. Level of significance was determined by using i D middle panel, one-way ANOVA P=0.0146 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group); D right panel, one-way ANOVA P= 0.0026 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group)",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " A. Confocal double immunofluorescence images depicting Olig2+ (a-e) and Olig2+-CC1+ (f-j) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice at 5 weeks, in absence (cpz) or in presence of D-Asp (cpz + D-Asp), and 2.5 weeks after cuprizone withdrawal, in absence (vehicle) or in presence of D-Asp (D-Asp). (k, left panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice, and cuprizone+D-Asp-treated mice at 5 weeks. (k, right panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, vehicle, and D-Asp-treated mice 2.5 weeks after cuprizone withdrawal. Arrows point to Olig2+-CC1+ cells. Arrowheads point to Olig2+-CC1+ cells. Scale bar in a-j: 50 μm Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, k left panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus cpz (n=3 mice for each group). A, k right panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus vehicle (n=3 mice for each group ",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " B. Representative electron microscopy images of middle corpus callosum sections from control mice (a), cuprizone treated-mice for 5 weeks in the absence (b) or in the presence of D-Asp (c), and from mice 2.5 weeks after cuprizone withdrawal in the absence (d, vehicle) or in presence of D-Asp (e). Scale bars in a-e: 1 μ ",
    "molecules": "D-Asp, cuprizone"
  },
  {
    "caption": " C. Percentage of myelinated axons in the middle corpus callosum of cuprizone (blue) and cuprizone + D-Asp-treated mice (red) for 5 weeks Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, two-tailed Student's t test, *P<0.05 versus cpz (n=4 mice for each group",
    "molecules": "cpz, D-Asp, cuprizone"
  },
  {
    "caption": " D. Scatter plot analysis of myelin thickness, expressed as individual G-ratios against axon diameters, after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue circle) or D-Asp-treated mice (red triangle). Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters ",
    "molecules": "D-Asp, cuprizone"
  },
  {
    "caption": " F. Percentage of myelinated axons after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue) or D-Asp- treated mice (red). Abbreviations: DEM, demyelination; REM, remyelination Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group)",
    "molecules": "D-Asp, cuprizone"
  },
  {
    "caption": "A Proliferation of melanoma cell lines treated with LDHA inhibitor GSK-2837808A (10 μM) for the indicated duration under hypoxic (1% O2) and normoxic conditions. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (A) statistical comparison for the treatments that showed a significant change.",
    "molecules": "GSK-2837808A, O2"
  },
  {
    "caption": "B, C Glucose (B) and glutamine (Gln, C) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.",
    "molecules": "Glucose, Gln, glutamine"
  },
  {
    "caption": "D Medium Gln levels after transfection of melanoma cells with non-targeting (NT) or LDHA-specific siRNA for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.",
    "molecules": "Gln"
  },
  {
    "caption": "E GC-MS-based quantification of intracellular Gln levels in cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.",
    "molecules": "Gln"
  },
  {
    "caption": "J Immunoblotting of SLC1A5 in MeWo cells mock (NT-siRNA + DMSO) or si-SLC1A5-treated for 24 hr and then treated with LDHAi for 48 hr.",
    "molecules": "DMSO"
  },
  {
    "caption": "K Medium Gln levels (left), intracellular Gln levels (right) of MeWo cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.",
    "molecules": "Gln"
  },
  {
    "caption": "N Immunoblotting of SLC1A5 and cleaved caspase-3 in melanoma cells subjected to mock (NT-siRNA + DMSO) or si-SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.",
    "molecules": "DMSO"
  },
  {
    "caption": "H Proliferation of melanoma cells treated with LDHAi, m-TORC1 inhibitor (Rapamycin, 100 nM), or a combination of the two. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Rapamycin"
  },
  {
    "caption": "GC-MS-based quantification of intracellular asparagine (Asn) and alanine (Ala) levels in MeWo and 1205Lu cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Ala, alanine, Asn, asparagine"
  },
  {
    "caption": "GC-MS-based quantification of intracellular Asn and Ala levels in MeWo cells mock-transfected or transfected with si-ATF4 (K) for 24 hr and then treated with LDHAi for 48 hr. and shared Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Ala, Asn"
  },
  {
    "caption": "GC-MS-based quantification of intracellular Asn and Ala levels in MeWo cells mock-transfected or si-SLC1A5 (L) for 24 hr and then treated with LDHAi for 48 hr. and shared Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Ala, Asn"
  },
  {
    "caption": "O 3H-Glutamine uptake in MeWo cells transfected with si-GCN2 and treated 48 hr later with LDHAi for 6 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Glutamine, 3H"
  },
  {
    "caption": "P Gln levels in the medium after treatment of MeWo cells with mock- or si-GCN2 for 24 hr and subsequent incubation with LDHA-i for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Gln"
  },
  {
    "caption": "Q GC-MS-based quantification of intracellular Gln and essential amino acid levels in MeWo cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Gln"
  },
  {
    "caption": "A GC-MS-based quantification of intracellular Asp (upper) and Ser (lower) levels in melanoma cell lines treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Asp, Ser"
  },
  {
    "caption": "B (upper) GC-MS-based 13C6-glucose fractional isotope labeling of Ser and Asp in melanoma cells treated for 72 hr with LDHAi. (lower) GC-MS-based 13C5-glutamine fractional isotope labeling of Asp in melanoma cell lines treated for 72 hr with LDHAi. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "13C5-glutamine, 13C6-glucose, Asp, Ser"
  },
  {
    "caption": "GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in DMEM (C) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Asp, Ser"
  },
  {
    "caption": "GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in MEM supplemented with Ser (400 μM; D) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Asp, Ser"
  },
  {
    "caption": "GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells treated with LDHAi for 1 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Asp, Ser"
  },
  {
    "caption": "F GC-MS-based quantification of intracellular Asp levels in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Asp"
  },
  {
    "caption": "G Immunoblotting of ATF4 in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with GSK-2837808A (10 μM) for 1, 2, or 24 hr.",
    "molecules": "Asp, GSK-2837808A"
  },
  {
    "caption": "H Immunoblotting of ATF4 in MeWo cells pre-treated with Ser (400 μM), Asp (10 mM), or Ser (400 μM) + Asp (10 mM) for 6 hr and then treated for 1 hr with LDHAi (10 μM).",
    "molecules": "Asp, Ser"
  },
  {
    "caption": "A Relative NAD+/NADH ratio in MeWo cells treated with LDHAi, nicotinamide mononucleotide (NMN, 1 mM), or a combination of the two for 24 hr. Cells were pretreated with nicotinamide mononucleotide for 6 hr and then treated with LDHAi for 24 hr in the combination setting. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "NAD+, NADH, nicotinamide mononucleotide, NMN"
  },
  {
    "caption": "B GC-MS-based quantification of total intracellular levels of Ser and Asp and their 13C6-glucose fractional isotope labeling in MeWo cells pretreated with nicotinamide mononucleotide (NMN, 1 mM) for 6 hr and then treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "13C6-glucose, Asp, nicotinamide mononucleotide, NMN, Ser"
  },
  {
    "caption": "D GC-MS-based quantification of intracellular Ala levels in MeWo cells treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Ala"
  },
  {
    "caption": "E GC-MS-based 13C6-Glc (M+2) and 13C5-Gln (M+4) isotopologue levels of the key TCA cycle metabolites and Asp in MeWo cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "13C5-Gln, 13C6-Glc, Asp"
  },
  {
    "caption": "F GC-MS-based 13C6-Glc (left) and 13C5-Gln (right) isotopologue levels of Asp in MeWo cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "13C5-Gln, 13C6-Glc, Asp"
  },
  {
    "caption": "G GC-MS-based quantification of intracellular Ala (left) and Asp (right) levels in si-GPT2-treated MeWo cells incubated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001",
    "molecules": "Ala, Asp"
  },
  {
    "caption": "A (left) Immunoblotting of LDHA and ATF4 in A375 cells expressing inducible (Tet-On) sh-LDHA expression vector treated with doxycycline (Dox) for 48 hr. (right) qRT-PCR (n=3) analysis of the ATF4 target genes in A375 cells treated with doxycycline (0.5 μg/ml) for 24 hr. Statistical analysis was performed by unpaired t-test. Data are shown as the mean ± SEM , ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.",
    "molecules": "Dox, doxycycline, Tet"
  },
  {
    "caption": "B Proliferation of inducible (Tet-On) sh-LDHA expression vector harboring A375 cells (n=3) treated with Dox (0.5 μg",
    "molecules": "Dox, Tet"
  },
  {
    "caption": "C Athymic nude mice were injected subcutaneously with A375 melanoma cells expressing inducible (Tet-On) sh-LDHA expression vector. Starting day 5, animals were treated on alternate days with 1 g/L doxycycline, 4 mg/kg rapamycin, or with a combination of the two (n=8 in each group). The tumor growth was determined by measuring the tumor volume. Statistical analysis was performed using Welch's t-test (two-tailed). Data are shown as the mean ± SEM ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.",
    "molecules": "doxycycline, rapamycin, Tet"
  },
  {
    "caption": "A qRT-PCR analysis of LDHA mRNA levels in A375 cells treated with BRAF inhibitor (PLX-4032, 1 μM) for 24 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.",
    "molecules": "PLX-4032"
  },
  {
    "caption": "GC-MS-based quantification of (B) intracellular 3-PG in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.",
    "molecules": "3-PG, PLX-4032"
  },
  {
    "caption": "GC-MS-based quantification of (C) lactate levels in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.",
    "molecules": "lactate, PLX-4032"
  },
  {
    "caption": "D Immunoblotting of the indicated proteins in A375 cells treated with PLX-4032 (1 μM) for 2, 6, or 24 hr.",
    "molecules": "PLX-4032"
  },
  {
    "caption": "Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (A) HUVEC had been transfected prior to the experiment with control or PECAM-1-specific siRNA. Cell lysates were analyzed for expression levels of PECAM-1 and tubulin (below). Relative VE-cadherin internalization efficiency is given on the right.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (B) PECAM-1 siRNA treated HUVEC were transduced with WT PECAM-1-EGFP or PECAM-1-Y663,686F-EGFP. Expression levels of transduced PECAM-1 forms and of EGFP and VE-cadherin are shown in immunoblots (on the right). Relative VE-cadherin internalization efficiency is given on the right.",
    "molecules": "Hoechst"
  },
  {
    "caption": "C VE-cadherin-catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated (no SDS) or treated with 0.2% SDS for 30 min at room temperature, followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30°C. Precipitates were analyzed in immunoblots for pY731-VE-cadherin, plakoglobin, β-catenin and VE-cadherin. D Quantification of pY731 blot signals of three independent experiments (from C), with the signal intensity for untreated VE-cadherin set as 1. Molecular weight markers are indicated in kDa. ",
    "molecules": "SDS"
  },
  {
    "caption": "A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator (MAPTAM) for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin.",
    "molecules": "MAPTAM, Ca2+, DMSO"
  },
  {
    "caption": "B TNFα-activated bEnd.5 cells were treated with DMSO or MAPTAM, prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%.",
    "molecules": "MAPTAM, DMSO"
  },
  {
    "caption": "A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin. C Transmigration assays treatment with Blebbistatin.",
    "molecules": "Blebbistatin, Ca2+, DMSO"
  },
  {
    "caption": "B TNFα-activated bEnd.5 cells were treated with DMSO prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%. treatment with Blebbistatin.",
    "molecules": "Blebbistatin, DMSO"
  },
  {
    "caption": "D Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS. Cells were either exposed to flow and PMNs or to flow alone, fixed using 4 % PFA and washed with PBS. FLIM measurements were performed at sites of transmigration (PMNs) or at junctions without PMNs (Flow).",
    "molecules": "PFA, PBS"
  },
  {
    "caption": "E Representative images of HUVEC expressing VE-cad FL at a site of PMN transmigration (left micrographs) or without PMN (right micrographs). Maximum intensity projection of a Z-stack of YPet fluorescence (VE-cadherin in green) and CellTracker DeepRed (PMN in red) is shown for the upper micrographs. The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue. The amplitude averaged lifetime of YPet per pixel (FLIM measurement) of the same cells is shown in the bottom micrographs.",
    "molecules": "CellTracker DeepRed"
  },
  {
    "caption": "F HUVEC were grown on collagen-coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa and 20 kPa, and stimulated with TNFα for 17 h prior to adding HL60-derived neutrophils for 20 min. VE-cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens. Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE-cadherin are shown on the right",
    "molecules": "collagen"
  },
  {
    "caption": "Three sex chromosome axes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) was performed in 2-month-old WT and XUsp26+XUsp26-Y spermatocytes. Nuclei were stained with DAPI (blue).The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome.",
    "molecules": "DAPI"
  },
  {
    "caption": "F The deletion of Usp26 causes pachytene and meiotic division arrest. Representative PAS-hematoxylin staining in 6-month-old Usp26+/Y and Usp26-/Y seminiferous tubules. Paraffin sections from Usp26+/Y and Usp26-/Y testes were stained with PAS-hematoxylin. P: pachytene spermatocyte, D: diplotene spermatocyte, rST: round spermatid, eST: elongating spermatid, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions.",
    "molecules": "PAS, hematoxylin"
  },
  {
    "caption": "Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MRE11 (red) (G); was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.",
    "molecules": "DAPI"
  },
  {
    "caption": "Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of ; SYCP3 (green), γH2AX (red) (H); SYCP3 (green), ATR (red), p-ATM (pink) (I) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.",
    "molecules": "DAPI"
  },
  {
    "caption": "L Crossover on sex chromosomes was impaired in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MLH1 (red), and ATR (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "D Representative TUNEL results in Usp26+/Y and Usp26-/Y testes. Paraffin sections from 6-month-old Usp26+/Y and Usp26-/Y testes were stained with TUNEL (green) and DAPI (blue) to show dead cells in stage IV, VIII and XII tubules with pachytene and metaphase spermatocytes, respectively. The arrowheads indicate the laggard chromosomes. P: pachytene spermatocytes, rSt: round spermatid, spz: spermatozoa, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions, arSt: apoptotic round spermatid.",
    "molecules": "DAPI"
  },
  {
    "caption": "F Usp26-deficient mice produced XY aneuploid round spermatids. FISH analysis of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y round spermatids. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. G Quantification of different types of round spermatids in 2-month-old Usp26 +/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y (n= 3 independent experiments), Usp26-/Y mice (n= 4 independent experiments). P=0.0026 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. P=0.0216 for O spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. H Usp26-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. I Quantification of different types of spermatozoa in 2-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y, Usp26-/Y mice (n= 5 independent ex experiments). P=0.0195 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. Data are presented as means ± SD.",
    "molecules": "DAPI"
  },
  {
    "caption": "The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of SYCP3 (green), TRF1 (red), ATR (white) (D); SYCP3 (green), MLH1 (red), ATR (white) (E) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome.",
    "molecules": "DAPI"
  },
  {
    "caption": "C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in fertile men with or without the USP26 mutated haplotype. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.",
    "molecules": "DAPI"
  },
  {
    "caption": "B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel).",
    "molecules": "auxin"
  },
  {
    "caption": "(E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain.",
    "molecules": "NaCl"
  },
  {
    "caption": "Retinal sagittal cross-sections from 6-7 week old mice stained with hematoxylin and eosin. NFL = Nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = Rods and cones. Scale bars 30 µ",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "B Transverse semithin sections of spinal cords of WT and NYKO mice of the indicated age. Sections were stained with toluidine blue. Orange arrows indicate degenerating neurons (Myelin dense bodies, MDBs). Scale bars 25 µm.",
    "molecules": "toluidine blue"
  },
  {
    "caption": "F, G Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 39 axonal segments were analyzed for WT control neurons and 37 axonal segments for Yme1l-/- neurons at DIV7. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.",
    "molecules": "Mitotracker"
  },
  {
    "caption": "H, I Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.",
    "molecules": "Mitotracker"
  },
  {
    "caption": "A, B Oxygen consumption rates of spinal cord mitochondria isolated from WT (n = 4) and NYKO (n = 4) mice in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates + ADP), state 4 (+ oligomycin), uncoupled (+ FCCP). Unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns=not significant. Data are means ± SEM.",
    "molecules": "Oxygen, ADP, FCCP, glutamate, malate, oligomycin, pyruvate, succinate"
  },
  {
    "caption": "E Transverse semithin sections of spinal cords of 6-7 week old WT, NOKO and NYOKO mice. Sections were stained with toluidine blue. Orange arrows point to MDBs indicating degenerating neurons. Scale bars 25 µm.",
    "molecules": "toluidine blue"
  },
  {
    "caption": "F Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Arrowheads indicated tdTomato+ periosteal cells. Two representative periosteal regions were magnified (a and b). GP: Growth plate. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm. G Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice 7 days after fracture. Aggrecan (green), tdTomato (red), DAPI (blue). Arrowheads indicated tdTomato+ periosteal cells. Two representative regions were magnified (a and b). Scale bars were 100 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D Flow cytometry analysis of BrdU incorporation in uncultured Lepr-Cre+Notch3+/- BMSCs (n=4 independent experiments). Eight-week-old Lepr-Cre; tdTomato mice were given a single intraperitoneal injection of BrdU (100 mg/kg body mass) and maintained on 0.5 mg/ml of BrdU in the drinking water for 14 days.",
    "molecules": "BrdU"
  },
  {
    "caption": "E Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Sinusoid (a), arteriole (b), artery (c) and avascular bone marrow (BM, d) regions were shown. Arrowheads indicated Notch3+ tdTomato+ adventitial cells closely associated with ECs. Arrows indicated Notch3- tdTomato+ reticular cells in the bone marrow that were not associated with ECs. Scale bars were 10 µm. F Localization quantification of Lepr-Cre+Notch3+ cells relative to bone marrow ECs (n=5 biological replicates from three independent experiments). Cells within 5 µm diameter of ECs were considered adjacent. G",
    "molecules": "DAPI"
  },
  {
    "caption": "G Representative flow cytometry plots of long bone periosteum in 8-week-old Lepr-Cre; tdTomato mice. The percentage of DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+/- cells were shown.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B Representative oil red staining after 7 days of adipogenic differentiation in Lepr-Cre+ subsets (A) and qPCR analysis of Adipoq and Pparg expression (B) (n=3 independent experiments). Scale bars were 100 µm.",
    "molecules": "oil red"
  },
  {
    "caption": "C, D In vitro chondrogenic differentiation of sorted periosteal cells. Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection (C). Scale bars were 100 µm. Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression (D) (n=3 independent experiments).",
    "molecules": "alcian blue"
  },
  {
    "caption": "E, F In vitro osteogenic differentiation of sorted periosteal cells. Representative alizarin red staining after osteogenic differentiation for 14 days. Scale bars were 200 µm. Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression (F) (n=3 independent experiments).",
    "molecules": "alizarin red"
  },
  {
    "caption": "C Upper panel: senescence curve  of a representative est2∆ clone. DNA samples were prepared at the indicated time points  to analyze telomere length (middle panel) and t-circles production (lower panel and graph) during  senescence and cell immortalization. Telomeres  were analyzed by  Southern blot  of the XhoI    cut DNA hybridized with a  TG1-3  probe. The presence of t-circles was monitored by RCA and analyzed by TG1-3 probed dot blot. The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units). Similar results were obtained with at least 16 other est2∆ clones.",
    "molecules": "TG1-3"
  },
  {
    "caption": "E) Dot blot  of the RCA   product either treated or not with the Exonuclease VII that degrades linear single-stranded DNA in both the 3' to 5' and 5' to 3' directions (upper panel). The membrane was hybridized with a telomeric TG1-3  probe. The signal intensities of the dots are shown (lower graph).",
    "molecules": "TG1-3, single-stranded DNA"
  },
  {
    "caption": "B Senescence  curve of a representative est2∆ clone (upper panel). DNA samples were prepared at the indicated  time points to analyze  telomere length by Southern blot (middle panel) and  t-circles production by  RCA assay performed in the absence of dCTP and dATP.(lower panel). The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units).",
    "molecules": "dATP, dCTP"
  },
  {
    "caption": "C Detection of the t-circles co-immunoprecipitated  with  Nic96-RFP from the extracts obtained from several type II survivors. The DNA co-immunoprecipitated with Nic96-RFP was amplified by RCA  assay, loaded on a dot blot  and detected with a  telomeric TG1-3  probe. The numbers above the blot  refer to the  growth curves in (A) and correspond to  type II survivors produced in independent cultures. The lower panel shows the efficiency of Nic96-RFP IP that was evaluated by western blot with anti-mCherry antibody.",
    "molecules": "TG1-3"
  },
  {
    "caption": "C Detection of the  C-circles co-immunoprecipitated with Ada2-Myc   in type II survivors. The DNA co-immunoprecipitated  with Ada2-Myc was amplified by RCA, loaded on a dot blot  and detected with a telomeric TG1-3 probe. The numbers above the blot  refer to clones in (A) and correspond to type II survivors   produced in independent cultures. The lower panel shows the efficiency of Ada2-Myc IP analyzed by western blot  with anti-Myc  antibody.",
    "molecules": "TG1-3"
  },
  {
    "caption": "C Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS-PAGE/immunoblotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control.",
    "molecules": "acetylated-lysine, AcK, NAM, NAD+"
  },
  {
    "caption": "D Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS-PAGE/immunoblotting or Cl--dependent 86Rb+ uptake under hypotonic conditions.",
    "molecules": "Cl, NAM, 86Rb"
  },
  {
    "caption": "E Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS-PAGE/immunoblotting.",
    "molecules": "H20"
  },
  {
    "caption": "F Oocytes were injected with either WT, K114R or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in D.",
    "molecules": "NAM, NAD+"
  },
  {
    "caption": "A, B KCC4 activity (A), and protein levels (B) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD+. Non-injected oocytes (UI) were used as a control.",
    "molecules": "NAD+"
  },
  {
    "caption": "D Oocytes were injected with either KCC4 WT, K114R or K114Q for 48 h and then incubated with vehicle (V), NAD+ or NAM for 24 h, and KCC4 and SIRT7 protein levels were analyzed by SDS-PAGE/immunoblotting.",
    "molecules": "NAM, NAD+"
  },
  {
    "caption": "E Oocytes were injected with either KCC4 WT, K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle (V) or NAM for 4 h. After lysis, KCC4 was immunoprecipitated using an anti-KCC4 antibody, and the acetylation status of KCC4 was determined by SDS-PAGE/Western blotting using and anti-acetyl lysine antibody.",
    "molecules": "acetyl lysine, NAM"
  },
  {
    "caption": "F, G FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ (F) or NAM (G) and then analyzed by SDS-PAGE/immunoblotting using an anti-KCC4 or anti-SIRT7 antibody. Non-transfected cells (UT) were used as a control.",
    "molecules": "NAM, NAD+"
  },
  {
    "caption": "H FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ and NAM. After lysis, KCC4 immunoprecipitation using an anti-FLAG antibody was performed, and the interaction with SIRT7 was determined by SDS-PAGE/Western blotting.",
    "molecules": "NAM, NAD+"
  },
  {
    "caption": "I HEK293 cells expressing KCC4 were incubated with NAD+ 200 μM and cycloheximide (CHX) for 0, 3, 6 and 9 h. KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA (+SIRT7) or a shRNA against SIRT7 (-SIRT7).",
    "molecules": "CHX, cycloheximide, NAD+"
  },
  {
    "caption": "A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4. After lysis, KCC4 was immunoprecipitated using an anti-FLAG antibody, and then analyzed by SDS-PAGE/Western blotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-transfected cells (UT) were used as a control.",
    "molecules": "acetylated-lysine, AcK"
  },
  {
    "caption": "A, B Sirt7 mRNA (A) and protein (B) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau, respectively. Enrichment for each segment was previously validated by immunoblotting against segment-specific markers as indicated in material and methods (Spirli et al, 2019).",
    "molecules": "Ponceau"
  },
  {
    "caption": "C Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis (SIRT7, red; ATPase, green; DAPI, Blue). Scale bar: 20 μm. Controls of the specificity of antibodies using kidney of SIRT7-deficient mice are provided in Appendix Fig S4.",
    "molecules": "DAPI"
  },
  {
    "caption": "C Immunolocalization of KCC4 and H-ATPase in the kidneys of SIRT7+/+ and SIRT7-/- mice under normal conditions (Control) or with metabolic acidosis (NH4Cl). (KCC4, red; H-ATPase, green). Scale bar: 20 μm. Cell count was performed automatically using the Gen5 software to report the percentage of KCC4+ cells among H-ATPase+ cells (lower panel).",
    "molecules": "NH4Cl"
  },
  {
    "caption": "a, Expression levels of ISX, TWIST1, Snail1, Fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (p< 0.001 compared to time point 0h; Student's t‐test) of 3 independent experiments, each performed in triplicate.",
    "molecules": "DOX, Tet"
  },
  {
    "caption": "b, Western blotting analysis of the protein levels of ISX, HIF1α, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), Fibronectin, N-cadherin (N-Cad.), Vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8hs after DOX induction.",
    "molecules": "DOX"
  },
  {
    "caption": "e, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in DOX-inducible ISX expression system A549 cells treated with TH1834, Garcinol, C646, MB-3 for 8 h after DOX induction. Data information: Each experiment was repeated at least three times.",
    "molecules": "C646, TH1834, DOX, Garcinol, MB-3"
  },
  {
    "caption": "f, Proximity ligation assay of ISX and BRD4 interaction in A549 cells treated with Garcinol. Red foci indicate close proximity of the two proteins. Blank arrows indicate interactions of ISX with BRD4 in the cells. NC: negative control. Data information: Each experiment was repeated at least three times.",
    "molecules": "Garcinol"
  },
  {
    "caption": "g, Western blotting analysis of the protein levels of ISX, PCAF, and EMT markers in A549 cells with PCAF knockdown and DOX-inducible ISX expression system at 8 h time point after DOX induction. Data information: Each experiment was repeated at least three times.",
    "molecules": "DOX"
  },
  {
    "caption": "Cell migration (wound healing, h) ssay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times. ",
    "molecules": "Garcinol"
  },
  {
    "caption": "i, Cell invasion (transwell, i) assay of A549 lung cancer cells treated with Garcinol. V, vehicle. Data are presented as mean ± SD in graph (***, p < 0.001 , Student's t‐test) of 3 independent experiments, each performed in triplicate. Scale bar, 100μm. Data information: Each experiment was repeated at least three times.",
    "molecules": "Garcinol"
  },
  {
    "caption": "c, Recombinant PCAF acetylates His6-ISX at lysine residue 69 by in vitro acetylation assay. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.",
    "molecules": "lysine, Lysine"
  },
  {
    "caption": "d and e, The protein levels of GFP-tagged WT or mutant ISX, PCAF and BRD4 were determined in cytosol, nuclei, and anti-GFP immunoprecipites of A549 cells by Western blotting. Acetylated ISX was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.",
    "molecules": "Lysine"
  },
  {
    "caption": "j. Tumor xenografts metastasis activity of constitutively expressing RFP A549 cells transfected with wild type or ISX AC3 mutant cDNA were imaged by IVIS imaging system at fifth weeks Data information: Each experiment was repeated at least three times.",
    "molecules": "cDNA"
  },
  {
    "caption": "c, Recombinant PCAF acetylates His6-BRD4 at lysine residue 332. Acetylated BRD4 was detected by anti-acetyl Lysine antibody. Data information: Each experiment was repeated at least three times.",
    "molecules": "lysine, Lysine"
  },
  {
    "caption": "i, The cell invasion (transwell) activity were determined in A549 cells co-transfected with cDNA coding for GFP-tagged ISX and mCherry-tagged BRD4 mutants. Data are presented as mean ± SD in bar graph (p < 0.001, Student's t‐test) of 3 independent experiments, each performed in triplicate. Data information: Each experiment was repeated at least three times.",
    "molecules": "cDNA"
  },
  {
    "caption": "a, IHC staining with ISX (brown) or PCAF (brown) antibody in lung tumors from patients with lung cancer. N, normal tissue; T, tumor mass; Nuclei, hematoxylin (blue); Blue and red arrow, cytosol and nuclear ISX (PCAF). Scale bar, 500μm (400X); Scale bar, 2mm (100X).",
    "molecules": "hematoxylin"
  },
  {
    "caption": "b, Confocal immunofluorescence detection of ISX (green) and BRD4 (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.",
    "molecules": "DAPI"
  },
  {
    "caption": "c, Confocal immunofluorescence detection of ISX (green) and PCAF (red) in lung tumors from patients with NSCLC. Cell nuclei were visualized by DAPI (blue); Yellow arrows indicate co-localization; IgG, negative control. N, normal tissue; T, tumor mass.",
    "molecules": "DAPI"
  },
  {
    "caption": "Histograms showing the distribution of M/L quantitation ratios of monoglycosylated peptides identified in (A) HepG2SCΔT1, (B) ΔT2 and (C) +T3. Glycopeptides with M/L ratio < -1 are colored red and glycopeptides with a M/L ratio > +1 are colored green. (D) Venn diagram showing the distribution of candidates for isoform-specific sites among HepG2SCΔT1, T2 and +T3 applying a log10 (+/-1) cut-off (excluding sites identified in both TCL and SEC for each isoform), and (E) TCL alone and (F) SEC alone.",
    "molecules": "glycopeptides, Glycopeptides"
  },
  {
    "caption": "(A) Pictures of plants treated with BLE. Plants were grown vertically on 1/2 MS medium with or without 4 μM BLE for 9 days . Scale bar = 1 cm. (B) The relative root length of the indicated plants. The data are represented as means ± SD (n = 10 plants) relative to the values of Col-0 under the control condition. The statistical significance was determined using two-way ANOVA analysis. ****, P < 0.0001. The experiments were repeated three times with similar results.",
    "molecules": "BLE"
  },
  {
    "caption": "(A-F) (A, C, and E) Representative images of CFP-PAF1 expressed in N. benthamiana leaves (A), Arabidopsis protoplasts (C), and transgenic Arabidopsis (E). The N. benthamiana leaves and the transgenic seedlings were treated with 40 μM BLE for 4 h before imaging. The Arabidopsis protoplasts were treated with 20 μM BLE for 2 h before imaging. Scale bars in (A and C) = 2.5 μm. Bars in (E) = 10 μm. (B, D, and F) The percentage of cells with CFP-PAF1 foci in N. benthamiana leaves (B), Arabidopsis protoplasts (D), and transgenic Arabidopsis (F). The data are represented as means ± SEM (n = 3 biological replicates). For each sample, more than 35 cells were scored. The statistical significance was determined using two-tailed Students' t-test.",
    "molecules": "BLE"
  },
  {
    "caption": "(G and H) In vitro pull-down assays. The Dextrin beads coupled with MBP, MBP-SMC5, or MBP-SMC6B were incubated with His-PAF1, respectively. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies.",
    "molecules": "Dextrin"
  },
  {
    "caption": "The PAF1 foci are dependent on SMC5 and SMC6B. (A) CFP-PAF1 was transfected alone or co-transfected with the RNAi construct of SMC5 (SMC5-Ri) into Col-0 protoplasts. (C) CFP-PAF1 was transfected into the protoplasts of Col-0 or smc6b, respectively. The protoplasts were treated with 20 μM BLE for 2 h before imaging. The percentage of cells with foci. The data were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 32 cells were scored. The statistical significance was determined using two-way ANOVA analysis. **, P < 0.01; ***, P < 0.001.",
    "molecules": "BLE"
  },
  {
    "caption": "(G and H) eChIP-qPCR assays. (G) The SMC5-Ri construct was transfected into the protoplasts of SSDIS/CFP-PAF1 transgenic plants. (H) The 35S:CFP-PAF1 construct was transfected into the protoplasts of SSDIS/smc6b or SSDIS/Col-0 transgenic plants. The protoplasts were treated with 100 μM DEX for 4 h to induce DSBs. The eChIP assays were carried out using an anti-GFP antibody. The immunoprecipitated DNA (eChIP) and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD (n = 3 technical replicates). The statistical significance was determined using two-tailed Students' t-test. ns, not significant; ****, P < 0.0001.",
    "molecules": "DEX"
  },
  {
    "caption": "(G and H) In vitro pull-down assays. The Dextrin beads coupled with MBP or MBP-UBC2 were incubated with His-CDC73 or His-VIP5. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies.",
    "molecules": "Dextrin"
  },
  {
    "caption": "(I and J) The UBC2 foci are dependent on VIP5. UBC2-mCherry and/or VIP5-YFP were transfected into the vip5 protoplasts. The protoplasts were treated with 20 μM BLE for 2 h before imaging. Scale bars = 2.5 μm. The data in (J) were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 30 cells were scored. The statistical significance was determined using two-way ANOVA analysis. ****, P < 0.0001. All experiments were repeated at least three times with similar results.",
    "molecules": "BLE"
  },
  {
    "caption": "(C and D) In vitro pull-down assay. The Dextrin beads coupled with MBP, MBP-HUB1, or MBP-HUB2 were incubated with His-UBC2. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies. The experiments were repeated three times with similar results.",
    "molecules": "Dextrin"
  },
  {
    "caption": "(E-J) The HUB1/2 foci are dependent on UBC2. VIP5-CFP, HUB1/2-YFP, and/or UBC2-mCherry were transfected into the ubc1 ubc2-c1 protoplasts. The protoplasts were treated with 20 μM BLE for 2 h before imaging. Bars = 2.5 μm. The data in (F, G, I, and J) were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 60 cells were scored. The statistical significance was determined using two-way ANOVA analysis. ns, not significant; **, P < 0.01.",
    "molecules": "BLE"
  },
  {
    "caption": "(A, C, E, and G) Pictures of plants treated with BLE. Plants were grown vertically on 1/2 MS medium with or without 6 μM BLE (A) or 4 μM BLE (C, E, and G) for 8 days. Scale bar = 1 cm. (B, D, F, and H) The relative root length of the indicated plants. The data are represented as means ± SD (n = 10 plants) relative to the values of Col-0 under the control condition. The statistical significance was determined using two-way ANOVA analysis. ns, not significant; ***, P < 0.001; ****, P < 0.0001. The experiments were repeated three times with similar results.",
    "molecules": "BLE"
  },
  {
    "caption": "(I and J) eChIP-qPCR assays. (I) The SSDIS/Col-0 transgenic Arabidopsis was mock-treated or treated with 100 μM DEX for 4 h. (J) The SSDIS/Col-0 and SSDIS/ddrm4-1 transgenic Arabidopsis were treated with 100 μM DEX for 4 h. The eChIP assays were carried out using an anti-H2Bub antibody. The immunoprecipitated DNA (eChIP) and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD (n = 3 technical replicates). (I) The data are relative to the values of Mock. (J) The data are relative to the values of UBQ5 in Col-0. The statistical significance was determined using two-tailed Students' t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The experiments were repeated at least twice with similar results.",
    "molecules": "DEX, ub"
  },
  {
    "caption": "B) Histological sections of 3 IDHwt PDXs (P3, T434, P8) and 3 IDHm PDXs ((T394, E478, T186) showing a large ROI (boxed rectangle, >500 pixels) within the tumor area applied for targeted quantification of selected metabolites. Left rectangle in P3 PDX was used as control contralateral brain (CB) for quantifications. Middle panel shows tissue distribution of D-2-hydroxyglutarate (D2HG) by MSI, which is exclusively detected in IDHm PDXs (D2HG in IDHwt tumours is below detection limit). Right panel shows tissue distribution of a key metabolite (m/z 778.51) presenting strong accumulation in IDHm lower grade gliomas (LGG) versus glioblastomas (GBM) independent of IDH status. An intensity-dependent color code indicates the relative amount of a specific compound (defined by m/z value) throughout the tissue section.",
    "molecules": "D-2-hydroxyglutarate, metabolite, D2HG"
  },
  {
    "caption": "C) Quantification of several high mass metabolites differentially present in IDHm PDXs (IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey). Box plots represent log values of metabolite intensities measured within a large ROI (>500 pixels). Many metabolites in this mass range (m/z 700-900) have been putatively assigned to phospholipids (Tables EV2-3). m/z represents the mass over charge ratio of ionized metabolites, as measured by the mass spectrometer. Except where otherwise indicated, statistical significance was calculated for IDHm (n=3) versus IDHwt (n=3) PDXs using T test. pv: p-values.",
    "molecules": "high mass metabolites, phospholipids"
  },
  {
    "caption": "A) MALDI imaging of indicated metabolites in IDHwt PDX (P3), IDHm PDX (E478) and control brain (CB). Hex-P: hexose-phosphate; AMP, ADP, ATP: adenosine mono-, di- and triphosphate respectively.",
    "molecules": "ADP, AMP, ATP, hexose-phosphate"
  },
  {
    "caption": "B) LC-MS quantification of indicated metabolites in 6 PDXs. Relative quantities of metabolites are expressed in log scale and normalized to tissue quantity used for extract preparation. IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey. Sdh7-P: sedoheptulose-7-phosphate.",
    "molecules": "sedoheptulose-7-phosphate"
  },
  {
    "caption": "C) Energetic charge defined as the ratio of (ATP+1/2ADP)/(ATP+ADP+AMP) was calculated at each pixel to present its distribution throughout the tumor.",
    "molecules": "ADP, AMP, ATP"
  },
  {
    "caption": "C) In vivo flux analysis of 13C-labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX (P3) and one IDH1m PDX (E478) (n=3/ sample). The percentage of glucose-derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms (13C0 - 13C6 as indicated by color code). Note the absence of 13C2 label of αKG and D2HG in IDHm tumours, while the label was detectable in IDHwt PDX and in contralateral brain of both tumors (lower panel).",
    "molecules": "D2HG, αKG, 13C, glucose"
  },
  {
    "caption": "D) Quantities of 13C3 and 13C2 labeled metabolites from glucose tracer experiment in (C). D2HG in IDH1m PDX shows a detectable 13C2 signature here, but represents a minor fraction of the total cellular D2HG pool in this tumor. Hex-P: hexose phosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate, Cit: citrate, Mal: malate, Asp: aspartate, Glu: glutamate, Gln: glutamine, α-KG: α-Ketoglutarate, D2HG: D2-Hydroxyglutarate. Indicated statistical significant group differences are based on T test, pv: p-values.",
    "molecules": "D2-Hydroxyglutarate, D2HG, α-Ketoglutarate, aspartate, 13C, citrate, glucose, glutamate, glutamine, hexose phosphate, malate, phosphoenolpyruvate, pyruvate"
  },
  {
    "caption": "B) Quantification of metabolite distribution by MSI in 6 PDX with IDH1wt and IDH1m status (n=3 / group) and in contralateral control brain (CB). No significant differences between IDH groups were detected, although significant differences are found between tumors and control brain (glutamate, apsartate). Color code as in Fig. 1, NAA: N-acetylaspartic acid; NAAG: N-acetylaspartylglutamic acid.",
    "molecules": "NAAG, glutamate, NAA"
  },
  {
    "caption": "A) NADPH levels are reduced in IDHm compared to wildtype tumors, resulting in a reduced NADPH/NAD+ ratio (n=3/ sample). p-value from T test: *<0.05. NADPH, NADP: reduced, oxydized nicotinamide adenine dinucleotide phosphate, NADH, NAD: reduced, oxydized nicotinamide adenine dinucleotide.",
    "molecules": "NAD+, NAD, NADH, NADP, NADPH"
  },
  {
    "caption": "B) MALDI images for glutathione (GSH) and cystathionine on IDHwt (P3) and IDHm (E478) PDXs.",
    "molecules": "cystathionine, glutathione, GSH"
  },
  {
    "caption": "C) Quantification of MSI signals for GSH and cystathionine from 6 PDXs (n=3 / group).",
    "molecules": "cystathionine, GSH"
  },
  {
    "caption": "D) LC-MS quantification of independent samples for GSH, GSSG (oxidized GSH), GSSG/GSH ratio and cystathionine from 6 PDXs (n=3/ sample / group). Statistical significance between IDH groups calculated by T test, pv: p-value.",
    "molecules": "GSSG, cystathionine, GSH"
  },
  {
    "caption": "(A) Fed rat liver lysosomes were extracted with 1% of the indicated detergents and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were subjected to SDS-PAGE and immunoblotted for LAMP‐2A and flotillin‐1. Stars indicate the aliquots of the gradient of translucent appearance (highly enriched in lipids).",
    "molecules": "sucrose"
  },
  {
    "caption": "(B) The distribution of LAMP‐2A through the sucrose gradient after extraction with 1% Triton X‐114 was calculated from the densitometric quantification of immunoblots as the one shown here. Values are expressed as percentage of the total lysosomal levels and are mean+s.e. of four experiments.",
    "molecules": "sucrose, Triton X‐114"
  },
  {
    "caption": "(A) Lysosomes from livers of fed (Fed) or 24 h starved rats (Stv) were extracted with 1% Triton X‐114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent‐resistant (DR) fraction, and anything below this fraction was pooled, as the solubilized fraction (Sol) (B). Lanes 5-6 are one‐tenth of the amount of lysosomes loaded in the gradient.",
    "molecules": "Triton X‐114"
  },
  {
    "caption": "(C) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11-12; heavy: aliquots 13-14).",
    "molecules": "Triton X‐114"
  },
  {
    "caption": "(D) Fed ratliverlysosomes were incubated with MBCD or filipin, as described under Material and methods, and then processed as in (C). The mean value of the densitometric quantification of the LAMP‐ from two experiments is shown at the bottom in (C) and (D).",
    "molecules": "MBCD, filipin"
  },
  {
    "caption": "(A) Lysosomalmembranes from fed ratlivers and their corresponding 1% Triton X‐114‐resistant regions, isolated as described under Material and methods, were subjected to one (left)‐ or two‐dimensional (right) electrophoresis and stained with Sypro Ruby.",
    "molecules": "Triton X‐114"
  },
  {
    "caption": "LAMP‐2A localizes in cholesterol‐ and GM‐1‐enriched intracellular membrane domains. Mouse fibroblasts grown on coverslips in the presence (+) or absence (−) of serum were fixed and labeled with filipin (red) (A) or incubated with Texas‐Red‐cholera toxin B subunit to label GM‐1‐enriched membrane regions (red) and then fixed.",
    "molecules": "cholesterol, GM‐1"
  },
  {
    "caption": "(B) Both groups of cells were then subjected to indirect immunofluorescence for LAMP‐2A (green). Where indicated, cells were treated with MBCD (25 mM) for 30 min before fixation. The merged images of both fluorophores are shown on the right. Insets show vesicles at higher magnification. Bar: 10 μm.",
    "molecules": "MBCD"
  },
  {
    "caption": "(A, B) Regions of the lysosomalmembrane resistant to 1% Triton X‐114 recovered from the sucrose gradients were fixed and processed for electron microscopy (A) or visualized without fixation by electron microscopy (B). Arrows point to the membrane bilayer.",
    "molecules": "sucrose, Triton X‐114"
  },
  {
    "caption": "(C) Intact ratliverlysosomes (top) and DR regions of the lysosomal membranes (bottom) were fixed and subjected to immunogold labeling for LAMP‐2A and LAMP‐2B, and a gold‐conjugated secondary antibody as described under Material and methods. Top right panels show 'clusters' of gold particles (black arrows) in individual lysosomes at higher magnification. Graph shows the labeling density (gold particles per μm) in the indicated fractions for both proteins. Values are mean+s.e. from the quantification of 15 different micrographs.",
    "molecules": "gold, gold particles"
  },
  {
    "caption": "(A) Fed ratliverlysosomes were incubated in an isotonic buffer without additions, or in the presence of ratlivercytosol (25 μg), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (25 μg), GST‐hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X‐114 extraction, apyrase density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP‐2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3-6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP‐2A in lysosomes remained constant throughout the incubation.",
    "molecules": "ATP, protease inhibitors, Triton X‐114"
  },
  {
    "caption": "(B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP‐2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments.",
    "molecules": "paraquat"
  },
  {
    "caption": "(A) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples.",
    "molecules": "NH4Cl, MBCD, leupeptin"
  },
  {
    "caption": "(B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA).",
    "molecules": "3MA, MBCD"
  },
  {
    "caption": "(C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples.",
    "molecules": "MBCD, 14C, 3H"
  },
  {
    "caption": "(D) Lysosomal-mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal-mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.",
    "molecules": "14C, cholesterol, sucrose, Triton X‐114"
  },
  {
    "caption": "(A) Lysosomes from mouse fibroblasts transfected with wild‐type hLAMP‐2A and maintained in the presence (Serum+) or absence (serum−) of serum were collected by centrifugation and subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP‐2A. Lane 1: homogenate.",
    "molecules": "sucrose, Triton X‐114"
  },
  {
    "caption": "(F) Proteolysis of a pool of 3[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild‐type (WT) or the indicated mutant forms of hLAMP‐2A. Values are the mean+s.e. of two experiments with triplicate samples.",
    "molecules": "3[H]"
  },
  {
    "caption": "(G) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.",
    "molecules": "MBCD"
  },
  {
    "caption": "(A) The Triton X‐114‐resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous (10-80%) sucrose density gradient, SDS-PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100-200 kDa); fraction 3 (200-400 kDa); fraction 4 (400-600 kDa); fraction 5 (>600 kDa).",
    "molecules": "sucrose, Triton X‐114"
  },
  {
    "caption": "(B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot.",
    "molecules": "CaCl2, protease inhibitors, sucrose, Triton X‐114"
  },
  {
    "caption": "(C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS-PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a).",
    "molecules": "MBCD"
  },
  {
    "caption": "(D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments.",
    "molecules": "CaCl2, sucrose, Triton X‐114"
  },
  {
    "caption": "J Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) after the L4 stage. Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.",
    "molecules": "Ub"
  },
  {
    "caption": "L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) and the indicated genotypes after the L4 stage, as per (J). *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Error bars indicate SEM.",
    "molecules": "Ub"
  },
  {
    "caption": "K,L Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. *P<0.001 using ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point, with N=20 animals per genotype and time point. Data has been normalized to wild type. Error bars indicate SEM.",
    "molecules": "Ub"
  },
  {
    "caption": "B A separate SDS-PAGE and Western blot of the same lysates from (A), probed with antibodies recognizing ubiquitin or actin as a loading control. The position of molecular weight markers is shown on the left of each blot. The position of UbG76V-GFP protein, as well as UbG76V-GFP with the indicated number of additional ubiquitin moieties, based on molecular weight, is indicated to the right of each blot. Endogenous poly-ubiquitinated proteins are also detected in the 80-200 kDa range, as indicated by the bracket. Twenty animals were loaded per lane for each indicated genotype.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": " D Abundance of the indicated free ω6 PUFA-derived eicosanoid, normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ***P<0.001, **P<0.01, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM. E Abundance of the indicated free ω3 PUFA-derived eicosanoid, as in (D), normalized to total protein, for each indicated genotype. Red, gray, and blue stacked bars indicate hydroxides, epoxide, and epoxide diol forms of eicosanoids, respectively. ****P<0.0001, ANOVA with Dunnett's posthoc comparison to wild type. N=3 trials. Error bars indicate SEM ",
    "molecules": "epoxide diol, epoxide, hydroxides, eicosanoid, eicosanoids, ω3 PUFA, ω6 PUFA"
  },
  {
    "caption": "C Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4+48 hours. ****P<0.001, ANOVA with Dunnett's posthoc comparison to the wild-type control equivalent time point. N=20 animals per genotype and time point. Error bars indicate SEM.",
    "molecules": "Ub"
  },
  {
    "caption": "A) Single dose AR-42 plasma and tissue pharmacokinetic study. Tumor-free CD2F1 mice were administered a single dose of 10, 20 or 50 mg/kg AR-42 (n=3) and plasma (dashed) and gastrocnemius (solid) tissue were analyzed for AR-42 content at different times using LC-MS/MS analyses according to the Materials and Methods (Mean±SD).",
    "molecules": "AR-42"
  },
  {
    "caption": "B) AR-42 dose-response. Starting six days after C-26 cell injection, animals received vehicle or AR-42 orally at 1, 3, 10 or 20 mg/kg daily or 50 mg/kg every other day for 13 days. Individual animal terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor-free controls. Groups: 1mg/kg (n=8), 3mg/kg (n=8), 10mg/kg (n=13), 20mg/kg (n=6), 50mg/kg (n=5). Dashed reference line (100%) represents the mean tumor-free control value, solid line represents non-linear fit of dose-response data.",
    "molecules": "AR-42"
  },
  {
    "caption": "Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. C): Terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) Terminal tumor volumes (mean±SD).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. E) Terminal gastrocnemius (mean±SD) and F) quadriceps muscle mass (mean±SD) compared to tumor-free controls.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S2A-D, one-way ANOVA followed by Tukey's multiple comparison test.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "E) Survival analyses of Study 2 animals performed as outlined in the Materials and Methods. Statistics: V, G indicate significant differences versus tumor-bearing vehicle-treated and tumor-bearing GTx-024-treated groups, respectively. p=0.0005 for Combo vs. V, p=0.0064 for Combo vs. G, Logrank (Mantel-Cox) test using Bonferroni corrected threshold for multiple comparisons.",
    "molecules": "GTx-024"
  },
  {
    "caption": "Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD) .",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S3A-D, one-way ANOVA followed by Tukey's multiple comparison test.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).",
    "molecules": "DHT, AR-42, TFM-4AS-1"
  },
  {
    "caption": "Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.",
    "molecules": "DHT, AR-42, TFM-4AS-1"
  },
  {
    "caption": "Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, T, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing TFM-4AS-1-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S4A-D, Tukey's multiple comparison test.",
    "molecules": "DHT, AR-42, TFM-4AS-1"
  },
  {
    "caption": "A) Gene expression of multiple cachexia-associated markers in gastrocnemius muscles of individual animals from Study 1 (n=5 for tumor-bearing groups; n=6 for tumor-free groups). Expression was determined by qRT-PCR and presented as described in the Materials and Methods (Geometric Mean ± Geometric STD). B) Androgen receptor (AR) mRNA expression in gastrocnemius muscles from Study 1. Statistics: p values provided in Appendix Table S5A-B. Dunnett's multiple comparison test. CEBPδ, n=4, insufficient sample to analyze all tumor-bearing GTx-024-treated animals.",
    "molecules": "GTx-024"
  },
  {
    "caption": "A) ELISA analysis of serum IL-6 levels in terminal samples from Study 2. Tumor-free groups (n=6), tumor-bearing receiving Vehicle (n=7), GTx-024 (n=10), AR-42 (n=8) or Combination (n=9). ND, not detected.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "B and C) Phospho(p)STAT3 (Y705)/total (t)STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 (B) and Study 3 (C). Black circle - tumor-free, blue square - tumor-free/GTx-024, black triangle - tumor-bearing, blue diamond - tumor/GTx-024, red triangle - tumor/AR-42, green circle - tumor/combo.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "D) Spleen weights normalized to tumor-corrected terminal body weights of mice from Study 2. Tumor-free groups (n=6), tumor-bearing Vehicle (n=7), tumor-bearing receiving GTx-024 (n=10), AR-42 (n=9) or Combination (n=9).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "E) ELISA analysis of pSTAT3(Y705)/tSTAT3 within C-26 tumors from Study 2. Tumor-bearing Vehicle-treated (n=7), tumor-bearing receiving GTx-024, AR-42 or Combination (n=8).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "A) Effect of GTx-024 and AR-42 monotherapies on cachexia-related differentially regulated genes (DEGs) from RNA-seq analyses of Study 1 gastrocnemius muscles. All three panels consist of individual genes plotted with respect to their log2 fold change and -log10 Benjamini-Hochburg adjusted p-values from the comparison of cachexia vs tumor-free controls. Colors of the points reflect the DEG status of each gene for the given comparison.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "D) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D.",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "A) Volcano plot from RNA-seq analyses of Study 1 gastrocnemius muscles for tumor-bearing combination-treated mice versus tumor-bearing controls. Genes not differentially expressed in this comparison are indicated with blue. The remaining genes (red and green) are DEGs in the combination-treated versus tumor-bearing control comparison. The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR-42-treated tumor-bearing mice versus tumor-bearing controls, suggesting these genes are responsive to only the combination therapy. Log2-transformed fold change (FC) in expression is plotted on the x-axis and -log10 transformed Benjamini-Hochburg adjusted p-values are plotted on the y-axis.",
    "molecules": "AR-42"
  },
  {
    "caption": "D) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor-free control comparisons. GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": "E) mRNA expression of WNT effectors upstream of β-catenin. Data are presented as mean±SD of log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).",
    "molecules": "GTx-024, AR-42"
  },
  {
    "caption": ", B. survival of Ssrp1fl/fl; CreERT2+/+ mice  of different  ages (weeks) after the start of tamoxifen (TMX) treatment. B.  Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other  male  groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.",
    "molecules": "tamoxifen, TMX"
  },
  {
    "caption": "C. Normalized  blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "A, B. Effect of tamoxifen on replication in the spleen (A) and  intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice.  EdU was given to  mice three days after the end of tamoxifen  treatment (1 hour before tissue collection). Organ sections were  stained  for total DNA  (Hoechst), EdU, and SSRP1. Scale bars - 100 μM. C. Quantitation of  EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed. D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice  were analyzed. Data information: P-values for C and D were calculated using the unpaired t-test.",
    "molecules": "Hoechst, EdU, tamoxifen"
  },
  {
    "caption": "D. Fold-change in the proportion of cells in each cluster shown in panel A in samples from  tamoxifen-treated versus vehicle-treated  mice. Cell types are identified by color as in panel B.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "A, B. Effect of 4-OHT administration on colony formation by  bone marrow cells  of different genotypes. A. Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4-OHT. B. Quantitation of bone marrow colony assay. Mean colony number  per plate ± SD, n = 3 plates per  mouse. P values < 0.05 are shown (unpaired t-test). 2  mice of each genotype were analyzed. C, D. Growth of intestinal organoids in the presence or absence of 4-OHT. C. Photographs of small intestine organoids on different days after plating. D. Number of organoids in the presence and absence of 4-OHT. Mean fold change between number of organoids in control and 4-OHT treated wells ± SD, n = 4 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids  were isolated from one mouse of each genotype in two independent experiments.",
    "molecules": "4-OHT"
  },
  {
    "caption": "F, G. Effect of  4-OHT  administration on  intestinal organoids of different genotypes kept in the presence (undifferentiated) or absence (differentiated) of R-spondin-1. F. The  viability of organoids assessed by the resorufin  assay. Mean ± SD, n = 3 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from two  mice  of each genotype. G. IF images of  intestinal organoids from Ssrp1fl/fl; CreERT2+/+ mice kept in the presence or absence of R-Spondin-1. The  organoids  were stained  with antibodies to  LGR5 and SSRP1  and counterstained  with Hoechst (DNA).",
    "molecules": "Hoechst, 4-OHT, resorufin"
  },
  {
    "caption": "Effect of 4-OHT administration on  MSCs and their differentiated counterparts. H. Methylene blue viability assay  for undifferentiated MSCs or MSCs differentiated into  osteocytes  or adipocytes  for 21 days and then treated with vehicle or 4-OHT. Mean ± SD, n = 3 plates per  mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from two mice  of each genotype.",
    "molecules": "4-OHT, Methylene blue"
  },
  {
    "caption": "A. EU incorporation measured by  flow cytometry. Histograms of EU  fluorescent signals from two biological replicates for each condition (except no EU). B.  SSRP1 staining of the cells in A.",
    "molecules": "EU"
  },
  {
    "caption": "H. Comparison of Hoechst 33342 binding to live control and 4-OHT-treated MSCs from different genotypes. Data are presented as the median fluorescence intensity ± SD, n = 4 biological replicates. P < 0.05 is shown (unpaired t-test).",
    "molecules": "Hoechst 33342, 4-OHT"
  },
  {
    "caption": " A) Oocytes were fixed for chromosome spreads 8 hours after GVBD, and stained for PP2A-c (red), Sgo2 (green), CREST (blue) and DNA (Hoechst, shown in grey). Spreads were classified into metaphase I and anaphase I, depending on whether chromosome segregation had taken place, or not. Arrowhead indicates fused sister kinetochores, arrows indicate separated sister kinetochores. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " B) Example of a whole mount oocyte staining, in early anaphase I after cold-treatment to visualize only cold-stable microtubule fibers, stained with anti-tubulin antibody (green). Kinetochores are stained with CREST serum (red) and chromosomes with Hoechst (blue). Shown is an overlay of 20 z-sections. Arrows indicate separated sister kinetochores. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " C) Metaphase I (6 hours after GVBD) and metaphase II (16-20 hours after GVBD) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin (pink) and the centromere (green), respectively. Chromosomes were stained with Hoechst (blue). Below, schematic representation of a bivalent in metaphase I and a dyad in metaphase II. Enlargement shows the centromere region and a region that we call "chromatid junction" with the corresponding scheme. ",
    "molecules": "Hoechst"
  },
  {
    "caption": " E) Chromosome spreads as in A), stained for Sgo2 (red), CREST (green), and DNA (Hoechst, in blue). Spreads in early anaphase I and late anaphase I - telophase I were classified, depending on whether dyads of two chromosome sets had been found close to each other as one group, or were scattered apart into two groups. In early anaphase I, Sgo2 is found inbetween sister kinetochores that are already separated. F) Quantification of E) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair (dot plot on the left), and distance between sister kinetochores (dot plot on the right) at the indicated stages of meiosis I. Meta: metaphase, ana: anaphase, telo: telophase. Each dot represents one kinetochore pair; the number of kinetochore pairs analysed is indicated. Below: scheme of Sgo2 signal (in red) measurements. ",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": " A) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Sgo2 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). On the right, schemes illustrate the chromosome figures observed, and the corresponding Sgo2 staining (in red). ",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": " B) Dyads of metaphase II chromosome spreads of control, Bub1KD, Mps1∆N and Reversine-treated oocytes (from GVBD onwards) stained with Sgo2 antibody (red), CREST serum (green) and Hoechst (blue). ",
    "molecules": "Hoechst, Reversine"
  },
  {
    "caption": " C) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards, corresponding to the time of anaphase I onset. PBE: Polar Body Extrusion. ",
    "molecules": "Reversine"
  },
  {
    "caption": " D) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Mps1 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later, respectively. E) Quantification of D) showing Mps1 signal relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Mps1 signal averaged per oocyte (right dot plot) at the indicated time points. The number of kinetochore pairs (kinetochores in anaphase II) and oocytes analyzed is indicated. The scheme of measurements is shown on the right. ",
    "molecules": "Hoechst, DNA, Strontium"
  },
  {
    "caption": " F) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst. Where indicated, oocytes were treated with Reversine from GVBD onwards, and treatment was renewed every 8 hours. Early CSF corresponds to 8 hours after GVBD (Reversine treated oocytes undergo anaphase I on average 4-5 hours after GVBD), and late CSF corresponds to 36 hours after GVBD (>29 hours in CSF arrest). PSCS: precocious sister chromatid segregation. ",
    "molecules": "Hoechst, Reversine"
  },
  {
    "caption": " (A and D) Oocytes in meiosis I (6 hours after GVBD) or meiosis II (left and right panels, respectively) were treated with STLC for 2.5 hours and fixed for whole mount immunofluorescence. Cold stable microtubules were stained with an antibody against tubulin (green), centromeres with CREST (red) and DNA with Hoechst (blue). Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed. ",
    "molecules": "Hoechst, DNA, STLC"
  },
  {
    "caption": " (B and E) Chromosome spreads at metaphase and anaphase of meiosis I (6 and 9 hours after GVBD) or meiosis II (left and right panels, respectively), in the presence or absence of STLC (for 2.5 hours) and Reversine (+Rev., for 1 hour) as indicated. Spreads were stained with CREST serum (red) and Hoechst (grey). Single chromosomes are present in anaphase I and single sister chromatids in anaphase II. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed. ",
    "molecules": "Hoechst, Rev, Reversine, STLC"
  },
  {
    "caption": " (C and F) Chromosome spreads exactly as described in B and E), except that spreads were additionally stained with an antibody against Rec8 (green). The CREST signal appears in red and DNA in blue. Rev: Reversine. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed. ",
    "molecules": "DNA, Rev, Reversine"
  },
  {
    "caption": " B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated. C) Same as B), except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards. In (B and C) n indicates number of oocytes analyzed.",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": " D) Metaphase-to-anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated, in GV. The schemes indicate the chromosome figures observed upon activation. Chromosome spreads are stained with CREST serum (green), and Rec8 antibody (red). DNA was stained with DAPI (blue). In (D) n indicates the number of oocytes with the shown phenotype (e.g., upon successful activation) and the total number of oocytes analyzed. ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " A) Sep+/+ or Sep-/- oocytes were matured in vitro until metaphase II. Sep-/- oocytes were injected with wild-type separase or cleavage mutant separase encoding mRNAs in metaphase II. Oocytes were either fixed in metaphase II or activated and fixed in anaphase II. Spreads were stained with Rec8 antibody (red), CREST serum (green), and DAPI (blue) to label DNA. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " B) Representative image of kinetochore individualization in Sep-/- oocytes injected with wild-type separase in metaphase II and fixed after activation, from A). Shown is staining with CREST serum (green), and DAPI (blue) to label DNA. Arrows indicate separated sister kinetochores. The scheme on the left illustrates the individualization of sister kinetochores (green arrows) at anaphase II. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed Scale bars: 10 µm, except bottom panel in B), where the scale bar is 2 μm. ",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": " B) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP-Sgo2 expressing oocyte. Spreads were stained with Rec8 antibody (pink), CREST serum (green), Hoechst for DNA (blue), GFP-Sgo2 was visualized by GFP fluorescence (grey). The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms. In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed ",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": " C) Metaphase I (4hrs after GVBD) and II spreads after GFP-Sgo2 expression (from GV onwards). Spreads were stained with CREST serum (red), Hoechst for DNA (grey), GFP-Sgo2 was visualized by GFP fluorescence (green). Arrowheads mark separated sister kinetochores. On the right, graph showing percentage of metaphase II spreads containing bivalent chromosomes. In (A-C) n indicates the number of control non-injected and GFP -Sgo2-expressing oocytes analyzed The number of analysed oocytes in C)",
    "molecules": "Hoechst, DNA"
  },
  {
    "caption": " D) Chromosome spreads of oocytes expressing GFP-Sgo2 (from GV onwards) in metaphase II, and upon activation. Spreads were labeled with Rec8 antibody (shown in red, far-red secondary antibody was used), CREST serum (pseudo-colored in green), and Hoechst (blue). GFP direct fluorescence was imaged for GFP-Sgo2 detection. Below the corresponding chromosome figures are represented in the schemes. E) Quantification of Rec8 signal found around and in between CREST signals that were close to each other. Below: scheme illustrating how Rec8 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines). Meta: metaphase, ana: anaphase. D), the number of oocytes with the shown phenotype and the total number of oocytes analyzed. On each graph, mean is shown, error bars are +s.e.m. in C) and and ± SD in E), asterisks indicate significant difference (**** = p<0,0001) according to Mann-Whitney U test. AU: arbitrary units. The number of analysed kinetochore pairs in E) is indicated.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Identified acetylated histone peptides plotted against their log2 SILAC ratio (1h after 16 J/m2 UV/mock), ranked by SILAC ratio",
    "molecules": "acetylated, histone"
  },
  {
    "caption": "(C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated",
    "molecules": "CTK7A, histones, TSA, CPTH2"
  },
  {
    "caption": "(D) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV-irradiation (16 J/m2). Blots were stained with α-acetyl-lysine (top panels, high and low exposure), α-histone H4 (middle panel) and α-tubulin (bottom panel)",
    "molecules": "acetyl, lysine"
  },
  {
    "caption": "(F) Representative western blots of HeLa WCE from different time points after UV-C irradiation (16J/m2) using the indicated histone modification specific antibodies. Histone H4 and tubulin were used as loading controls",
    "molecules": "histone"
  },
  {
    "caption": "(A) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation (16 J/m2, left panel), α-amanitin (second panel, 100 µg/ml), THZ1 (third panel, 1 μM) or flavopiridol (right panel, 1 μM) treatment. Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)",
    "molecules": "histone, acetyl, α-amanitin, flavopiridol, lysine, THZ1"
  },
  {
    "caption": "(C) Representative western blots of histone acetylation levels of HeLa cells pre-treated with transcription inhibitors (mock (left), THZ1 (middle, 1 μM) or flavopiridol (right, 1 μM) an hour before UV irradiation and lysed at the indicated time points after UV (16J/m2). Blots were stained with the indicated antibodies",
    "molecules": "histone, flavopiridol, THZ1"
  },
  {
    "caption": "(A) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation (16 J/m2), HU/AraC treatment (100 mM/10 μM) or MMC treatment (10 µg/ml). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)",
    "molecules": "acetyl, AraC, histone, HU, lysine, MMC"
  },
  {
    "caption": "(C) Representative Western blots, stained with the indicated antibodies of HeLa cells pre-treated o/n with an S-phase inhibitor (PHA 767491 hydrochloride, 10 μM) and lysed at indicated time points after UV (16 J/m2)",
    "molecules": "PHA 767491 hydrochloride"
  },
  {
    "caption": "A HEK 293 cells were transfected with mCherry-tagged Parkin and FLAG-tagged UBQLN2 for 24 h, and then were treated with 5 µg/mL Antimycin A and Oligomycin A (A/O) for 4 h and 24 h. The cells were stained with anti-FLAG (green) antibody and anti-TOM20 (gray), and then were visualized using confocal microscopy. Scale bars, 10 µm. Insets were higher magnifications of the dashed box area. Scale bars, 2.5 µm. B, C The dashed square is magnified as shown in the boxed region in A with corresponding line scan indicating Parkin and UBQLN2 co-localization on the depolarized mitochondria (the line scan indicates pixel intensity of Parkin and UBQLN2).",
    "molecules": "Antimycin A, Oligomycin A"
  },
  {
    "caption": "A HEK 293T cells expressing mCherry-Parkin were pre-treated with proteasome inhibitor (20 µM MG132) for 12 h, and treated with 1 µg/mL A/O for 2 h. Then the cells were fractionated and proteins were immunoblotted for relative antibodies. B The level of Mfn2 (OMM protein), TOM20, COXIV and HSP60 was quantified and normalized relative to GAPDH, and was shown in A. The data from three independent experiments were presented as mean± SD., ns, not significantly different; **, p<0.01; ***, p<0.001, one-way ANOVA.",
    "molecules": "MG132"
  },
  {
    "caption": "HEK 293T cells were transfected with indicated siRNAs for 48 h. C The cells were re-transfected with mCherry-Parkin for 12 h and were pre-treated with or without 20 µM MG132 for 12 h and treated with1 µg/mL A/O for 2 h. The cell lysates were subjected to immunoblot with indicated antibodies. The data from three independent experiments were presented as mean± SD., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA.",
    "molecules": "MG132"
  },
  {
    "caption": "HEK 293T cells were transfected with indicated siRNAs for 48 h. H The cells were re-transfected with HA-tagged UBQLN2-WT, along with mCherry-Parkin for 12 h. Then the cells were pretreated with 20 µM MG132 for 12 h and treated with 5 µg/mL A/O for 4 h. Mitochondria isolated from cell lysates were treated with or without 10 µg/mL proteinase K (Pro.K) and then were subjected to immunoblot with indicated antibodies. Mito: the isolated mitochondria; Mito (Pro.K): mitochondria after Pro.K treatment. COXIV level in \"Mito (Pro.K)\" group was quantified and normalized relative to HSP60 in \"Mito group\". The data from three independent experiments were presented as mean± SD., ** p<0.01, ***, p<0.001, one-way ANOVA.",
    "molecules": "MG132"
  },
  {
    "caption": "HEK 293T cells were transfected with indicated siRNAs for 48 h. I The cells were re-transfected with HA, HA-UBQLN2-WT or P497H, along with GFP-Parkin and FLAG-PHB2 for 20 h, and then were treated with or without 5 µg/mL A/O for 4 h. Images of situ PLA using rabbit anti-LC3B and mouse anti-FLAG antibodies. Blue: nuclei (DAPI); white dots: PLA positive puncta. Scale bar, 10 µm. J Over 500 cells were counted for each sample in I. The data from three independent experiments were presented as mean± SD., *, p<0.05; ** p<0.01, ***, p<0.001, one-way ANOVA.",
    "molecules": "DAPI"
  },
  {
    "caption": "Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. B The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h and then were treated with 1 µg/mL A/O for 48 h. The neurons were stained with DAPI, anti-UBQLN2 (green) and anti-HSP60 (gray) antibodies and visualized. Scale bars, 10 µm. Yellow arrows indicate uncleared mitochondria.",
    "molecules": "DAPI"
  },
  {
    "caption": "Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. F The neurons were infected with lentivirus carrying mCherry-Parkin for 36 h and were treated with 100 ng/mL Antimycin A (Antimycin A alone, AA) and 1 µM DFP for 10 h. The neurons were subjected to immunofluorescent assay using anti-TOM20 (magenta) and anti-LAMP 2 (gray) antibodies. Scale bars, 10 µm. Right panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm. Yellow arrows indicate the co-localization between mitochondria and lysosomes.",
    "molecules": "AA, Antimycin A, DFP"
  },
  {
    "caption": "Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. H The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h. Then, the neurons were treated with 100 ng/mL Antimycin A and 1 µM DFP for another 10 h and subjected to immunofluorescent assay using anti-MAP2 antibody. Scale bars, 10 µm. I, J The neurite length (I) and cell death (J) in more than 30 neurons were quantified from each group in H. The data from three independent experiments were presented as the means ± SD., *, p<0.05; ***, p<0.001, one-way ANOVA.",
    "molecules": "Antimycin A, DFP"
  },
  {
    "caption": "B HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 4 independent experiments. Student's t-test, *** = p<0.001.",
    "molecules": "IPTG"
  },
  {
    "caption": "C HeLa cells were infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for p62. The scale bar represents 5 μm. Graph represents mean % ± SEM of Shigella responding to IPTG without (-) or with (+) p62 from at least 4 independent experiments. Student's t-test, *** = p<0.001.",
    "molecules": "IPTG"
  },
  {
    "caption": "D Control (CTRL) or isopropanol-treated x-light Shigella GFP were induced with IPTG for 30 min, then labelled with SYTOX Orange for 10 min. The scale bar represents 5 μm.",
    "molecules": "isopropanol, IPTG"
  },
  {
    "caption": "E HeLa cells were infected for 1 h 30 min, treated with ethanol (CTRL) or erythromycin (EM) for 2 h prior to fixation, and then labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella in SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.",
    "molecules": "erythromycin, ethanol"
  },
  {
    "caption": "A HeLa cells were labelled with MitoTracker Red CMXRos, treated with cytochalasin D for 30 min, then fixed and labelled for endogenous SEPT7 for confocal microscopy. The scale bar represents 0.5 μm. See also Fig EV3A.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "B HeLa cells were treated with Drp1 siRNA for 72 h, and then infected with x-light Shigella mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from 3 independent experiments. Student's t-test, *** = p<0.001.",
    "molecules": "IPTG"
  },
  {
    "caption": "B. Normalized fitness functions of E. coli BW25113 in the absence of tet(B) and their corresponding model fits in the presence of varying concentrations of antibiotics TET, DOX, and MCN in the media ([S]M). Normalized quadruplicate data is shown for each antibiotic.",
    "molecules": "DOX, MCN, tet, TET"
  },
  {
    "caption": "B. Normalized fitness functions of cells expressing wild type tet(B) and their corresponding model fits in the presence of TET, DOX and MCN. Triplicate data is shown for each antibiotic",
    "molecules": "DOX, MCN, TET"
  },
  {
    "caption": "B. Model fits for E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT and seven variants in TET, DOX, and MCN. Our model fit triplicate fitness function data for all strains (Figure S2). At low drug concentrations for all antibiotics, tet(B)G366C exhibits much slower growth rates than wild-type. Compared to wild-type, strains expressing tet(B)L9F exhibit similar growth rates at low and high drug concentrations but have faster growth rates at intermediate drug concentrations.",
    "molecules": "DOX, MCN, TET"
  },
  {
    "caption": "C. Fitness landscapes with mutants overlaid for TET=35 µg/mL, DOX=10 µg/mL, and MCN=2.5 µg/mL corresponding to the dashed lines in 3B.",
    "molecules": "DOX, MCN, TET"
  },
  {
    "caption": "B. The calculated mean and error of γ/κ for the sampled parameter fittings shown in Figure S2. The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $\frac{\gamma}{\kappa}\ \approx k_{1}\frac{{2P}_{\text{tot}}}{D_{2}}$.",
    "molecules": "MCN"
  },
  {
    "caption": "A. Changes in drug binding affinity Δk1 and pumping efficiency Δr for TetB variants relative to wild type in TET, DOX and MCN. Error bars are comprised from a propagation of errors from A and B global parameter modeling and in vivo protein level determination.",
    "molecules": "DOX, MCN, TET"
  },
  {
    "caption": "A. Triplicate normalized fitness functions (*) for E. coli BW25113 strains harboring plasmid copies of tet(B)WT, tet(B)K183E,A290V,T326I (variant 6) and tet(B)F151I,F179L,T326A (variant 7) in TET and their corresponding least squares model fits (solid line) where both variants display decreased fitness in TET compared to WT.",
    "molecules": "TET"
  },
  {
    "caption": "Hematoxylin and Eosin (HE) staining of representative transverse sections of GC muscles from 3-month-old HSA-Cre and control mice. The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration. Scale bars: 40 μm",
    "molecules": "Eosin, Hematoxylin"
  },
  {
    "caption": "Phosphatidic Acid (PA) levels in HSA-Cre and control GC muscles.",
    "molecules": "PA, Phosphatidic Acid"
  },
  {
    "caption": "Phospholipidome in HSA-Cre and control GC muscles. Proportions of indicated phospholipid classes in total phospholipids are represented. PC; Phosphatidylcholine, PE; Phosphatidylethanolamine, PI; Phosphatidylinositol, PS; Phoshpatidylserine, PA; Phosphatidic Acid, PG; Phosphatidylglycerol.",
    "molecules": "Phoshpatidylserine, PS, PA, Phosphatidic Acid, PC, Phosphatidylcholine, PE, Phosphatidylethanolamine, PG, Phosphatidylglycerol, Phosphatidylinositol, PI, phospholipid, Phospholipidome, phospholipids"
  },
  {
    "caption": "Amounts of indicated phospholipid classes normalized by protein content in HSA-Cre and control GC muscles. (D) Total phospholipid (PL) amount normalized by protein content in HSA-Cre and control GC muscles.",
    "molecules": "phospholipid, PL"
  },
  {
    "caption": "Distribution of the total carbon number in acyl chains of PC in HSA-Cre and control GC muscles.",
    "molecules": "carbon, PC"
  },
  {
    "caption": "Distribution of the number of double bonds in acyl chains of PC in HSA-Cre and control GC muscles.",
    "molecules": "PC"
  },
  {
    "caption": "Distribution of the total carbon number in acyl chains of PE in HSA-Cre and control GC muscles.",
    "molecules": "carbon, PE"
  },
  {
    "caption": "Distribution of the number of double bonds in acyl chains of PE in HSA-Cre and control GC muscles.",
    "molecules": "PE"
  },
  {
    "caption": "HE, Oil Red O (ORO), Cytochrome c Oxidase (COX) and Succinate Deshydrogenase (SDH) staining of representative frozen sections of TA muscle from 3-month-old HSA-Cre and control mice.",
    "molecules": "Oil Red O, ORO"
  },
  {
    "caption": "Quantification of triacylglycerol (TAG) levels in GC muscles from 3-month-old HSA-Cre and and control mice.",
    "molecules": "TAG, triacylglycerol"
  },
  {
    "caption": "Content of TAG assessed by MS in HSA-Cre and control GC muscles.",
    "molecules": "TAG"
  },
  {
    "caption": "Cholesterol, (E) Cholesteryl ester (CE), (F) DAG was assessed by MS in HSA-Cre and control GC muscles.",
    "molecules": "Cholesterol, CE, Cholesteryl ester, DAG"
  },
  {
    "caption": "Fold change of phospholipid precursors in GC muscles of 4-month-old HSA-Cre and control mice measured by liquid-chromatography (LC) tandem mass spectrometry (MS/MS) (n=7-4, *P<0.05).",
    "molecules": "phospholipid"
  },
  {
    "caption": "Fold change of Lactate/Pyruvate, Pyruvate/AcetylCoA, Pyruvate/Citrate, ATP/AMP, and ATP/ADP ratios in GC muscles of 4 month-old HSA-Cre and control mice (n=7-4, *P<0.05).",
    "molecules": "AcetylCoA, AMP, ADP, ATP, Citrate, Lactate, Pyruvate"
  },
  {
    "caption": "Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies.",
    "molecules": "TUDCA"
  },
  {
    "caption": "Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.",
    "molecules": "TUDCA"
  },
  {
    "caption": "Histological analysis of TA muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm.",
    "molecules": "neutral lipids, Oil Red O, TUDCA"
  },
  {
    "caption": "TAG level measurement in GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.",
    "molecules": "TUDCA, TAG"
  },
  {
    "caption": "In vivo force measurements performed on GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.",
    "molecules": "TUDCA"
  },
  {
    "caption": "Representative staining of IgG positive fibers on frozen sections of TA of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.",
    "molecules": "IgG, TUDCA"
  },
  {
    "caption": "Representative immunoblots from protein extract of GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice, with the indicated antibodies.",
    "molecules": "Bezafibrate"
  },
  {
    "caption": "Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.",
    "molecules": "Bezafibrate"
  },
  {
    "caption": "Histological analysis of TA muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice by Oil Red O staining to detect the neutral lipids.",
    "molecules": "Bezafibrate, neutral lipids, Oil Red O"
  },
  {
    "caption": "TAG level measurement in GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.",
    "molecules": "Bezafibrate, TAG"
  },
  {
    "caption": "In vivo force measurements performed on GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.",
    "molecules": "Bezafibrate"
  },
  {
    "caption": "Representative staining of IgG positive fibers on frozen sections of TA of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.",
    "molecules": "Bezafibrate, IgG"
  },
  {
    "caption": "A, HeLa  cells were treated with H2O2 (5 mM, 1 min) or PR-619 (10 µM, 10 min) and analysed by immunoblotting  for endogenous p62, NDP52  and OPTN  in either reducing  (2.5%  β-mE) or non-reducing  conditions. Data information: #, non-specific band or a post-translational modification.",
    "molecules": "H2O2, β-mE, PR-619"
  },
  {
    "caption": "B, NDP52  and OPTN  are reducible by the thioredoxin antioxidant system. HeLa  cells were pre-treated with TrxR  inhibitors, curcumin  (50 μM) or auranofin  (5 μM) for 30 min, then treated with H2O2  (500 μM) at different time points as indicated. Immunoblotting  was performed to detect NDP52, OPTN  and  PRX-3 (dimer formation reflecting oxidation of the protein) in non-reduced  conditions and PRX-SO3 (marker of oxidative stress and actin  in reduced  conditions.",
    "molecules": "auranofin, curcumin, H2O2"
  },
  {
    "caption": "C, D, Immunoblotting  for NDP52  in either reducing  or non-reducing  conditions of whole cell samples (C) and mitochondrial   fractions (D) from HeLa  cells treated with the combination of 4 µM antimycin A1  and 10 µM oligomycin  (AO) in the presence or absence of 400 nM bafilomycin A1  (Baf) for 2 h. NDP52  DLC in mitochondrial  fraction was quantified (D). Data are mean ± s.e.m (D). P values were calculated by one-way ANOVA followed by Sidak test on four independent experiments (D). ***, p<0.001; ns (non-significant). Data information: #, non-specific band or a post-translational modification.",
    "molecules": "antimycin A1, Baf, bafilomycin A1, oligomycin"
  },
  {
    "caption": "A-D, Single (A), double (B), triple (C), and quadruple (D) cysteine mutant NDP52  plasmids were generated and transfected into HeLa PentaKO cells. Cells were treated with 20 µM  PR-619  for 10 min and analysed by immunoblotting  for NDP52  in either reducing  or non-reducing conditions. The constructs highlighted in red were subjected to the next round of Cys-Ala screen or further analyses.",
    "molecules": "PR-619"
  },
  {
    "caption": "B, Recombinant proteins of NDP52  WT and C18,153,163,321S mutant (Mut) were exposed to the indicated concentrations of H2O2  for 5 min and subjected to SDS-PAGE and CBB gel staining.",
    "molecules": "H2O2"
  },
  {
    "caption": "A, B, Fluorescence microscopy images (A) and quantification (B) of mitophagy  of HeLa  wild-type (WT),  PentaKO + empty vector, PentaKO + NDP52  WT or NDP52  C18,153,163,321S (Mut) cells, stably expressing YFP-Parkin    and mt-mKeima    were pre-treated with or without MitoQ for 22 h and treated with 4 µM / 10 µM AO for the indicated times. Data information Data are mean ± s.e.m. (B or displayed as cell popular violin plots (B). P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments (B *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm (A",
    "molecules": "MitoQ"
  },
  {
    "caption": "C, HeLa PentaKO + NDP52  WT or NDP52  Mut cells stably expressing YFP-Parkin  and mt-mKeima  were treated with 4 µM / 10 µM AO for 2 h in the absence or presence of 10 μM MG132  (MG) and 400 nM Baf, followed by a mitochondrial  fractionation and immunoblotting  for NDP52  in non-reducing conditions. The mitochondrial  protein UQCRC2  was used as a loading control. Data information: #, non-specific band or a post-translational modification.",
    "molecules": "Baf, MG132"
  },
  {
    "caption": "D, HeLa PentaKO + NDP52  WT cells stably expressing YFP-Parkin  and mt-mKeima  were treated with 1 mM DFP  for 24 h (D), or pre-treated with 500 nM MitoQ  for 22 h and treated with 4 µM / 10 µM AO for 2 h (D Cells were then analysed by MitoSOX staining  (D) Data information: #, Data are mean ± s.e.m. D) P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments D). *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm D).",
    "molecules": "MitoSOX, DFP, MitoQ"
  },
  {
    "caption": "E, HeLa PentaKO + NDP52  WT cells stably expressing YFP-Parkin  and mt-mKeima  were treated with 500 nM MitoQ  for 22 h and treated with 4 µM / 10 µM AO for 2 h E) in the presence or absence of 400 nM Baf  (E). immunoblotting  for NDP52  (E). Data information: #, non-specific band or a post-translational modification.",
    "molecules": "Baf, MitoQ"
  },
  {
    "caption": "A, Fluorescence microscopy  images and quantification of mitophagy  in HeLa PentaKO + NDP52  WT cells stably expressing YFP-Parkin    and mt-mKeima    were treated with 4 µM rotenone  (R), 4 µM A, 10 µM O, or the combination of RO or AO for 3h. Data information Data are mean ± s.e.m. (A or displayed as cell popular violin plots (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated (A, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm (A",
    "molecules": "rotenone"
  },
  {
    "caption": "B, Fluorescence microscopy  images and quantification of MitoSOX staining  in HeLa PentaKO + NDP52  WT cells in the same conditions as (A). Data information Data are mean ± s.e.m. B, or displayed as cell popular violin plots B, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated B, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm B,",
    "molecules": "MitoSOX"
  },
  {
    "caption": "HeLa PentaKO + NDP52  WT cells stably expressing YFP-Parkin  and mt-mKeima  (C, were pre-treated with or without 10 µM S1QEL  or 20 µM S3QEL  for 30 min and treated with AO for 2 h or with RO for 3 h, followed by mitophagy  measurement (C) Data information Data are mean ± s.e.m. C, or displayed as cell popular violin plots C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated C, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm",
    "molecules": "S3QEL, S1QEL"
  },
  {
    "caption": "HeLa PentaKO + NDP52  WT cells (D) were pre-treated with or without 10 µM S1QEL  or 20 µM S3QEL  for 30 min and treated with AO for 2 h or with RO for 3 h, followed by MitoSOX staining  (D) Data information Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm , D",
    "molecules": "MitoSOX, S3QEL, S1QEL"
  },
  {
    "caption": "HeLa PentaKO + NDP52  WT cells stably expressing YFP-Parkin  and mt-mKeima  E) were pre-treated with or without 10 µM S1QEL  or 20 µM S3QEL  for 30 min and treated with AO for 2 h or with RO for 3 h, followed by immunoblotting  for NDP52  in non-reducing conditions (E). Data information: #, non-specific band or a post-translational modification.",
    "molecules": "S3QEL, S1QEL"
  },
  {
    "caption": "F, HeLa  PentaKO + NDP52  WT stably expressing YFP-Parkin  and mt-mKeima  were pre-treated with or without 10 mM DTT  for 30 min and treated with AO for 2 h followed by mitophagy  measurements (F) Data information Data are mean ± s.e.m. F) or displayed as cell popular violin plots F). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated F). *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm F).",
    "molecules": "DTT"
  },
  {
    "caption": "G, HeLa PentaKO + NDP52  WT stably expressing YFP-Parkin  and mt-mKeima  were pre-treated with or without 10 mM DTT  for 30 min and treated with AO for 2 h followed by immunoblotting  for NDP52 in non-reducing conditions (G). Data information: #, non-specific band or a  post-translational modification.",
    "molecules": "DTT"
  },
  {
    "caption": "HeLa PentaKO + NDP52  WT or NDP52  Mut cells stably expressing YFP-Parkin  and mt-mKeima  were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunofluorescence  analyses (A) The number of foci of the indicated proteins colocalised  with NDP52, or foci of NDP52 colocalised  with Parkin, was quantified. Data information: Data are mean ± s.e.m. (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments (A *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant). Scale bars: 20 μm (A,",
    "molecules": "Baf"
  },
  {
    "caption": "HeLa PentaKO + NDP52  WT or NDP52  Mut cells stably expressing YFP-Parkin  and mt-mKeima  were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunoblotting  for LC3   and NDP52  in mitochondrial  fraction (B) or  whole cell lysate (C) in reducing conditions Data information: Data are mean ± s.e.m. B, C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments B, C *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).",
    "molecules": "Baf"
  },
  {
    "caption": "HeLa PentaKO + NDP52  WT or NDP52  Mut cells stably expressing YFP-Parkin  and mt-mKeima  were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by co-immunoprecipitation  assay to analyse the interaction  of NDP52  with FIP200, ULK1  and ATG13  (D). Data information: Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments D, *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).",
    "molecules": "Baf"
  },
  {
    "caption": "E, MEFs  stably expressing YFP-Parkin, mt-mKeima  and empty, human NDP52  (hNDP52) WT or hNDP52 Mut were pre-treated with or without 500 nM MitoQ  for 21 h and treated with AO for 3 h followed by mitophagy  measurement. Data information: Data are mean ± s.e.m. E) or displayed as cell popular violin plots (E). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments E). *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm E).",
    "molecules": "MitoQ"
  },
  {
    "caption": "A. BMDMM  were treated as indicated with either 60ng/mL ultra-pure (UP) LPS, 50ng/mL human  derived recombinant GM-CSF, 40µM WEHD-FMK for 16 hours or 5µM nigericin  for the last hour. Supernatants were then analysed for  IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.",
    "molecules": "LPS, nigericin, WEHD-FMK"
  },
  {
    "caption": "C. HoxB8 macrophages  of the indicated genotypes were treated as indicated with 60ng/mL UP-LPS, 1%  GM-CSF containing supernatant (approximately 2ng/mL GM-CSF), 40µM WEHD.FMK, 0,5µM  CRT-0066101  for 16 hours, 5µM nigericin was added for the last hour. Supernatants were analysed by ELISA  for IL-1β. Inlay panels show western blots for levels of IL-1β  expression in the indicated knockouts. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.",
    "molecules": "CRT-0066101, LPS, nigericin, WEHD.FMK"
  },
  {
    "caption": "D. HoxB8 cells were treated with 60ng/mL of UP-LPS, 1%  GM-CSF supernatant, 50mM KCl, and 5µM nigericin  as indicated for 16 hours before supernatants were analysed for  IL-1β by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.",
    "molecules": "LPS, nigericin, KCl"
  },
  {
    "caption": "A. BMDMM  (left panel) and HoxB8 macrophages  (right panel) were treated as indicated with either LPS  or LPS+ GM-CSF. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2. Shown is the densitometric  quantification of Nrf2  protein level from three independent experiments. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.",
    "molecules": "LPS"
  },
  {
    "caption": "C. HoxB8 macrophages were treated for 12 hours with LPS  or LPS+GM-CSF and RNA extracted and analysed by qPCR  for the indicated targets. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.",
    "molecules": "LPS"
  },
  {
    "caption": "D. Wild type and Tnf-/- HoxB8  cells were treated as indicated. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2  and HMOX1. For wild type cells,  MitoTEMPO (50µM) was also added as indicated. Shown are the densitometric  quantifications of NRF2  and HMOX1  protein levels of three independent experiments (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.",
    "molecules": "MitoTEMPO"
  },
  {
    "caption": "E. HoxB8  cells of the indicated genotypes were treated with LPS  for 8 hours followed by staining  with mitoSOX red. The fluorescent signal was analyzed using flow cytometry. Shown are average geometric mean fluorescence intensity of mitoSOX red. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.",
    "molecules": "mitoSOX red, LPS"
  },
  {
    "caption": "F. Tnf-/- HoxB8 macrophages  were treated with LPS  with or without 50ng/ml recombinant TNF  for 16 hours. Cells were harvested and proteins extracted and analysed by western blot  for levels of NRF2  and HMOX1  (upper panel). Shown are the densitometric  quantifications of indicated protein levels of three independent experiments (lower panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.",
    "molecules": "LPS"
  },
  {
    "caption": "A. HoxB8 macrophages  were seeded in an 8-well Ibidi chamber and treated as indicated for 12 hours. Cells were fixed and stained  for ASC  (Red) and with DAPI  (blue). For nigericin  treatments nigericin  was added for only 30 minutes. Example cells containing ASC  Specks are shown with white arrows. Scale bars represent 50µM. Data information: Microscopy  pictures are representative images from 3 biological replicates.",
    "molecules": "DAPI, nigericin"
  },
  {
    "caption": "B. HoxB8  cells were treated as indicated with LPS  or LPS+GM-CSF. The cross linker DSS  was added to the cells as described in methods. Samples were analysed by western blot  for ASC oligomerization. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel. Data information: westerns  are representative images from 3 biological replicates.",
    "molecules": "DSS, LPS"
  },
  {
    "caption": "D. HoxB8 macrophages  were treated as indicated with LPS  or LPS+GM-CSF for 12 hours. Cells were lysed and NLRP3  was  immune precipitated using a magnetic bead coupled antibody. As a negative control, beads without NLRP3  antibody were used. Beads were boiled and analysed for recruitment of NLRP3, and caspase-1. * indicates light chain from the antibody. Molecular weight markers shown in middle of NLRP3  blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: westerns  are representative images from 3 biological replicates.",
    "molecules": "LPS"
  },
  {
    "caption": "E. HoxB8 macrophages  were incubated with combinations of LPS, GM-CSF, nigericin  +/- Z-VAD.FMK as indicated. Proteins were extracted and analysed by western blot  for caspase-1 processing. Data information: westerns  are representative images from 3 biological replicates.",
    "molecules": "LPS, nigericin, Z-VAD.FMK"
  },
  {
    "caption": "F. HoxB8 macrophages  were seeded in a 96 well microplate and co-treated with either 10µM or 50µM  z-VAD with LPS+GM-CSF  for 16 hours. Then 100nM of Sytox Green  was added to the cells. The number of sytox green+ cells per image was quantified. Data information: Significance was calculated using one way ANOVA with multiple comparisons. Every dot represents one biological replicate. P values are shown.",
    "molecules": "Sytox Green, sytox green, LPS, z-VAD"
  },
  {
    "caption": "A. HoxB8 macrophages  were treated for 16 hours with LPS  +/- GM-CSF as indicated. Proteins were extracted and analysed for levels of  IL-1β by western blot.",
    "molecules": "LPS"
  },
  {
    "caption": "B. HoxB8 macrophages  were treated with LPS  or LPS+GM-CSF as indicated for 16 hours. Cells were lysed in buffer containing FLAG  tagged K63 TUBE  and K63 ubiquitylated  proteins were precipitated using anti FLAG pull down. Pulled down  proteins were analysed for  IL-1β and ubiquitin   by western blot.",
    "molecules": "LPS, ubiquitin"
  },
  {
    "caption": "C. IL-1β-/- hoxB8 macrophages  were infected with lentivirus  expressing IL-1β-FLAG-6xHis or the indicated mutants from the  IL-1β promoter. Cells were treated as indicated with LPS or LPS+GM-CSF or LPS+Nigericin and levels of secreted  IL-1β were analysed by ELISA  (top panel). GM-CSF treated cells were also lysed in urea buffer and nickel  NTA affinity pull down was performed. Nickel  NTA pull down was also performed on supernatants (bottom panel). Pulled down proteins form the His tag pull down were analysed for the presence of  IL-1β by western blot. Molecular weight markers shown in middle of blot  were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.",
    "molecules": "LPS, nickel, Nickel, Nigericin, urea"
  },
  {
    "caption": "D. HoxB8 macrophages  were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated. Proteins were extracted and analysed for  IL-1β by  western blot.",
    "molecules": "mitoTEMPO, NAC"
  },
  {
    "caption": "E. Wild type and TNF-/- HoxB8 macrophages  were treated as indicated with LPS  or LPS+GM-CSF. Proteins were extracted and analysed for  IL-1β by  western blot. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel.",
    "molecules": "LPS"
  },
  {
    "caption": "F. HoxB8 macrophages  were treated as in A but with the addition of 25µM Punicalagin  throughout the 16 hour time course. Proteins were extracted and analysed by western blot (left panel). Supernatants were also analysed by ELISA for secreted IL-1β (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.",
    "molecules": "Punicalagin"
  },
  {
    "caption": "B. HoxB8 macrophages  were seeded in an 8-well Ibidi chamber and treated for 14 hours with LPS  and  GM-CSF. Cells were fixed and stained  for GasderminD  (green),  IL-1β (red) and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope Data information: shown. Microscopy  pictures are representative images from 3 biological replicates. Scale bars in microscopy  images represent 10µM.",
    "molecules": "DAPI, LPS"
  },
  {
    "caption": "C. HoxB8 macrophages were co-stimulated with three different PEG  3mM for 16 hours as indicated. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.",
    "molecules": "PEG"
  },
  {
    "caption": "D. HoxB8 macrophages  were seeded in 96 well microplate and treated as indicated. Before treatment, sytox green was added and cells were imaged in an incucyte incubator microscope  every hour for 16 hours. Shown is the sytox green signal normalized to the total cell area. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.",
    "molecules": "sytox green"
  },
  {
    "caption": "(D) Using the dual color labeling system (esg-Gal4>UASp-FRT-H3-eGFP-FRT-H3-mCherry), old and new H3   distribution in prophase  and prometaphase ISCs  shows large, separable domains of H3-eGFP  (old) and H3-mCherry  (new). Note: all figure panels in this work are maximum intensity projection images to show signals from all Z-stacks. (E) Using the UASp-FRT-histone-eGFP-T2A-histone-mCherry-PolyA  transgene that co-expresses H3-eGFP    and H3-mCherry, significant overlap between the two signals were detected in ISCs  during prophase  and prometaphase. (F) Quantification of the colocalization  between old and new H3, as well as between the co-expressed eGFP- and mCherry-tagged H3    in prophase  and prometaphase ISCs. Pearson's correlation coefficients were measured, where 1 represents complete colocalization  and 0 stands for no colocalization. Old and new H3   showed significantly less colocalization  (Pearson's correlation coefficient for H3   = 0.44 + 0.02, n = 81 ISCs) when compared to the co-expressed eGFP- and mCherry-tagged H3   (Pearson's correlation coefficient for H3  co-expressed = 0.86 + 0.01, n = 19 ISCs). Individual data points and mean values are shown. Error bars represent SEM. **** p < 0.0001; unpaired t test to compare two individual datasets to each other. Data information: n for individual ISCs. Scale bar in (D), (E): 5 µm.",
    "molecules": "PolyA"
  },
  {
    "caption": "(H) Distribution of the  Dl-nLacZ-positive (magenta) cells in an H3-expressing midgut, which are well spaced and interspersed within the  intestinal epithelium. (I) Distribution of the Dl-nLacZ-positive (magenta) cells in an H3T3A-expressing midgut, which are unevenly distributed in clusters with two or more Delta-positive cells. DAPI: white in (H-I). Data information: Scale bar in (H) and (I): 50 µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Wild-type primary B cells were treated with 5 μg/mL of anti-kappa antibody (a-k chain, top) or 1 µM LatrunculinA (LatA, bottom). Intracellular calcium flux was measured by flow cytometry in the presence of Src-inhibitor (PP2), Syk-inhibitor (BAY 613606) or vehicle control.",
    "molecules": "calcium, LatA, LatrunculinA, BAY 613606, PP2"
  },
  {
    "caption": "(B) Wild-type primary B cells were treated with vehicle control (-), 5 μg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Lyn (Y507) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the respective unstimulated sample, set as 1.",
    "molecules": "LatA"
  },
  {
    "caption": "(C and D) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-Syk (pSyk), phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.",
    "molecules": "LatA"
  },
  {
    "caption": "(E and F) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-PLCγ2 (pPLCγ2) and total ERK as loading control. The intensity of phosphorylated proteins, normalized to ERK, was referred to the respective unstimulated sample, set as 1.",
    "molecules": "LatA"
  },
  {
    "caption": "(G and H) Wild-type primary B cells were treated with vehicle control (-), 5 µg/mL anti-kappa antibody or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (pCD22), phospho-SHP1 (pSHP1), and CD22 or total ERK as loading controls. The intensity of phosphorylated proteins, normalized to CD22 or ERK, was referred to the respective unstimulated sample, set as 1.",
    "molecules": "LatA"
  },
  {
    "caption": "(I) Wild-type and Cd22-/- primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.",
    "molecules": "calcium, LatA"
  },
  {
    "caption": "(J) Wild-type and Cd22Y2,5,6F primary B cells were treated with 5 µg/mL anti-kappa antibody or 1 µM LatA and intracellular calcium flux was measured by flow cytometry.",
    "molecules": "calcium, LatA"
  },
  {
    "caption": "(K and L) Wild-type, Cd22-/- and Cd22Y2,5,6F primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (pCD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin as loading control. The intensity of phosphorylated proteins, normalized to actin, was referred to the unstimulated sample of the wild type cells, set as 1.",
    "molecules": "LatA"
  },
  {
    "caption": "(A) Determining the number and density of CD22, CD19, IgM and IgD on the surface of wild-type primary B cells. Cells were stained with PE-conjugated antibody against CD22 (blue profile), CD19 (red profile), IgM (red profile) or IgD (red profile) and their fluorescence profiles compared to those of standard beads (grey profiles), which each possess a well-defined number of binding sites (st0=blank, st1-st4=increasing number of binding sites) against the primary antibody. For IgM, where the range of expression is large, the low and high 25 percentiles were calculated as in Mattila et al. 2013 and shown in brackets.Data are representative of three independent experiments.",
    "molecules": "PE"
  },
  {
    "caption": "Figure 3. CD22 is highly clustered on the surface of resting B cells(A-C) dSTORM analysis of CD22, CD19, IgM and IgD. Wild-type primary B cells were stained with Alexa647-conjugated antibody against CD22, Cy5-conjugated antibody against CD19 or IgD, or Cy5-conjugated Fab fragments against IgM and settled onto nonstimulatory coverslips. Cells were then fixed, imaged with dSTORM and analysed.(A) 2D (top and middle) and pseudo 3D (bottom) dSTORM images were reconstructed from single molecule localizations. The white dashed square is shown in magnification (middle) and pseudo 3D (bottom).(B and C) Quantification of the distribution of CD22, CD19, IgM and IgD with H function and Hopkins index. Error bars (Hopkins index) and thin lines (H function) denote mean ± SEM. Data are pooled from at least three experiments per receptor.*P < 0.001, **P < 0.0001 (Student's t-test).",
    "molecules": "Cy5"
  },
  {
    "caption": "(G) Wild-type primary B cells were labelled with Atto633-conjugated Fab fragments against CD22, settled onto nonstimulatory coverslips and treated with vehicle control (DMSO) or 1µM LatA. Single particle trajectories were then analysed. Bars and numbers indicate the median. Data are pooled from two experiments.*P < 0.0001 (Student's t-test).",
    "molecules": "DMSO, LatA"
  },
  {
    "caption": "(K) Flow cytometric analysis of F-actin by intracellular phalloidin staining of unstimulated or LatA treated wild-type B cells for the indicated length of time.",
    "molecules": "LatA"
  },
  {
    "caption": "(L) Flow cytometric analysis of CD22 surface expression in resting or stimulated B cells with 10µg/ml anti-µ chain (Jackson ImmunoResearch), 1µM or 10µM LatA for 5 and 15 minutes. Error bars denote mean ± SD.",
    "molecules": "LatA"
  },
  {
    "caption": "(G and H) CD45-deficient primary B cells were labelled with Atto633-conjugated Fab fragments against CD22 or IgM, settled onto nonstimulatory coverslips, treated with vehicle control (DMSO) or 1µM LatA and imaged. Single particle trajectories were then analysed and diffusion coefficients calculated.(G) Diffusion coefficients of 300 representative CD22 and IgM particles. Bars and numbers indicate the median.(H) Confinement analysis. Bars and numbers indicate the median.",
    "molecules": "DMSO, LatA"
  },
  {
    "caption": "(K and L) Wild-type and CD45-deficient primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated times. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD22 (p-CD22) and total CD22 as loading control. The intensity of p-CD22, normalized to CD22, was referred to the unstimulated sample of the wild-type cells, set as 1.",
    "molecules": "LatA"
  },
  {
    "caption": "(H) Wild-type and Cd22R130E primary B cells were treated with 1 µM LatA and intracellular calcium flux was measured by flow cytometry.",
    "molecules": "calcium, LatA"
  },
  {
    "caption": "(I-K) Wild-type and Cd22R130E primary B cells were treated with vehicle control (-) or 1 µM LatA for the indicated time. Cells were lysed and analysed by SDS-PAGE followed by immunoblotting with phospho-CD19 (p-CD19), phospho-Akt (pAkt), phospho-ERK (pERK) and actin and total ERK as loading controls. The intensity of phosphorylated proteins, normalized to actin or ERK, was referred to the unstimulated sample of the wild type cells, set as 1.Data are representative of at least two experiments.",
    "molecules": "LatA"
  },
  {
    "caption": "(a) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis. The increase in cell number following each mitosis is shown by an overlaid curve (in orange; mean and s.d. of n=3).",
    "molecules": "PI"
  },
  {
    "caption": "(d) The measured concentration of CTP in synchronized cells shown in red (mean and s.d. of n=5; ) in Equation 7; the deconvoluted concentration dynamics, in case of no synchronization loss (green; in Equation 7); and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization, matching the measured concentrations (black; Equation 7). The measured concentrations converge towards the steady-state concentrations measured in non-synchronized cells (horizontal blue line).",
    "molecules": "CTP"
  },
  {
    "caption": "Figure 2: Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells. The figure shows metabolites found to significantly oscillate throughout the cell cycle (concentrations normalized per metabolite; maximal concentration in red; minimal concentration in blue). The amplitude of the oscillations is shown on the right. Metabolites are color-coded according to metabolic pathways: Energy/redox cofactors (purple); amino acids (blue); glycolytic metabolites (red); TCA cycle metabolites (green).",
    "molecules": "metabolite, Metabolites, metabolites"
  },
  {
    "caption": "(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).",
    "molecules": "13C, glucose"
  },
  {
    "caption": "(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).",
    "molecules": "13C, glucose"
  },
  {
    "caption": "(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).",
    "molecules": "13C, glucose"
  },
  {
    "caption": "(f) Oscillations in citrate m+4 labeling after feeding [U-13C]-glutamine (mean and s.d. of n=3).",
    "molecules": "13C, citrate, glutamine"
  },
  {
    "caption": "(g) Oscillations in the total citrate concentration throughout the cell cycle (mean and s.d. of n=5).",
    "molecules": "citrate"
  },
  {
    "caption": "(h) The measured lactate secretion flux in synchronized cells shown in red (mean and s.d. of n=5; in Equation 9;); the deconvoluted secretion flux dynamics, in case of no synchronization loss (green; in Equation 10); and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization, matching the measured fluxes (black; Equation 10).",
    "molecules": "lactate"
  },
  {
    "caption": "Oscillations in aspartate (a)concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).",
    "molecules": "13C, glutamine, aspartate"
  },
  {
    "caption": "Oscillations in malate (b) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).",
    "molecules": "13C, glutamine, malate"
  },
  {
    "caption": "(c) Uniform malate m+4 labeling throughout the cell cycle (combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase).",
    "molecules": "malate"
  },
  {
    "caption": "(d) Oscillations in pyrimidines m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing de novo pyrimidine biosynthesis).",
    "molecules": "13C, glutamine"
  },
  {
    "caption": "(e) Oscillations in lactate m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing malic enzyme activity).",
    "molecules": "13C, glutamine, lactate"
  },
  {
    "caption": "(f) Oscillations in malate m+3 throughout the cell cycle when feeding [U-13C]-glucose (representing pyruvate carboxylase activity).",
    "molecules": "13C, glucose, malate"
  },
  {
    "caption": "(g) Oscillations in citrate m+5 when feeding [U-13C]-glutamine throughout the cell cycle (representing reductive IDH flux).",
    "molecules": "13C, citrate, glutamine"
  },
  {
    "caption": "(h) Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glucose, representing oxidative glucose metabolism.",
    "molecules": "acetyl-CoA, 13C, glucose"
  },
  {
    "caption": "Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glutamine, representing reductive glutamine metabolism (i). For measurements of metabolite concentrations (a-b), red marks represent mean and s.d. of n=5. For measurements of fractional isotopic labeling (c-i), red marks represent mean and s.d. of n=3.",
    "molecules": "acetyl-CoA, 13C, glutamine"
  },
  {
    "caption": "Figure 5: Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle. (a-i) Oscillations in metabolic flux throughout the cell cycle (in mM/h), computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling (red and green marks represent optimal estimates of transient flux with 95% c.i.). Blue lines represent average fluxes inferred in a non-synchronized cell population. As shown, glucose-derived flux into TCA cycle peak in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase.",
    "molecules": "glucose, glutamine"
  },
  {
    "caption": "(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m+2 (a) from isotopic glucose (representing glycolytic flux into TCA cycle).",
    "molecules": "citrate, DCA, glucose"
  },
  {
    "caption": "(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl-CoA m+2 (b) from isotopic glucose (representing glycolytic flux into TCA cycle).",
    "molecules": "acetyl-CoA, DCA, glucose"
  },
  {
    "caption": "It further inhibits oscillations in citrate m+5/m+4 ratio (c) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).",
    "molecules": "citrate, glutamine"
  },
  {
    "caption": "It further inhibits oscillations in acetyl-CoA m+2 (d) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).",
    "molecules": "acetyl-CoA, glutamine"
  },
  {
    "caption": "(e) The fraction of cells in G1, S, and G2/M phases in synchronized HeLa cells after 3 hour treatment with DCA (normalized by measurements in untreated control cells).",
    "molecules": "DCA"
  },
  {
    "caption": "(f) The fraction of cells in G1, S, and G2/M phases in non-synchronized HeLa and HCT-116 cells after 24 hour treatment with DCA (normalized by measurements in untreated control cells). As shown, DCA significantly increases the fraction of cells in S phase, inhibiting cellular progression into G2 phase; showing mean and s.d. of n=3 for all isotopic labeling forms and FACS measurements (a-f). Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two-tailed, unequal variance t-test (e-f).",
    "molecules": "DCA"
  },
  {
    "caption": "A-D Proportional distribution of chymotrypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Proportional distribution of trypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Graphical representation of color code of tissues analyzed.",
    "molecules": "chymotrypsin, trypsin"
  },
  {
    "caption": "Chymotrypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.",
    "molecules": "Chymotrypsin"
  },
  {
    "caption": "trypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.",
    "molecules": "trypsin"
  },
  {
    "caption": "E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed.",
    "molecules": "chymotrypsin, Ub"
  },
  {
    "caption": "J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine. K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord. ",
    "molecules": "chymotrypsin"
  },
  {
    "caption": "A-D Comparison of chymotrypsin-like activity from female (n=9) and male (n=10) mice in young (3-5 months old) or older (10-15 months old, female (n=6) and male (n=7)) groups. Each point indicates a single mouse. A) Tissues that did not show significant decline, B) Tissues that show significant decline in females only, C) Tissues that show significant decline in males only or D) in both males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=9 young females, n=6 older females, n=10 young males, n=7 older males). P values obtained by unpaired two-tailed students t-test comparing young and older samples are indicated.",
    "molecules": "chymotrypsin"
  },
  {
    "caption": "B) Western blot analysis of Ub-K48 proteins in the soluble fractions of intestine lysates from mice plus or minus heat shock. C) Quantification of panel B. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group). Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.",
    "molecules": "Ub"
  },
  {
    "caption": "D) Western blot analysis of Ub-K48 proteins in the insoluble fraction of intestine lysates from mice plus or minus heat shock. E) Quantification of D. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.",
    "molecules": "Ub"
  },
  {
    "caption": "F) Western blot analysis of Ub-K48 proteins in the soluble fractions of spinal cord lysates from mice plus or minus heat shock. G) Quantification of F. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.",
    "molecules": "Ub"
  },
  {
    "caption": "H) Western blot analysis of Ub-K48 proteins in the insoluble fraction of spinal cord lysates from mice plus or minus heat shock. I) Quantification of H. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=3 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.",
    "molecules": "Ub"
  },
  {
    "caption": "K) Western blot of Ub-K48 proteins in the insoluble fraction of protein lysates of SOD1-G93A mouse spinal cord plus or minus heat shock. L) Quantification of panel K. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group) Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.",
    "molecules": "Ub"
  },
  {
    "caption": "M) Chymotrypsin-like proteasome activity in spinal cord lysates from SOD1-G93A mice. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 biological replicates per group). Bordered points indicate biological replicates and un-bordered points indicate technical replicates. Data information: * indicates p<0.05, ** indicates p<0.005, and *** indicates p<0.0005 by unpaired two tailed students t-test comparing male and female samples or samples with or without heat shock.",
    "molecules": "Chymotrypsin"
  },
  {
    "caption": "FLMycER cells, primed or not with OHT  (48h), were treated with 135 nm  IACS-010759 (IACS) for 48 hours (B) H2O2  quantification, expressed as fold-increase of the 405/488 nm fluorescence ratio in treated vs. untreated  FLMycER cells, expressing either the cytoplasmic  (left) or mitochondrial  (right) roGFP2-ORP1  biosensor. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.",
    "molecules": "IACS, IACS-010759, OHT, H2O2"
  },
  {
    "caption": "FLMycER cells, primed or not with OHT  (48h), were treated with 135 nm IACS-010759 (IACS) for 48 hours (C) Superoxide anion O2•-  production in treated vs. untreated FLMycER cells, based on dihydroethidium staining. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B and C is from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.",
    "molecules": "IACS, IACS-010759, OHT, dihydroethidium, Superoxide anion O2•-"
  },
  {
    "caption": "FLMycER cells were primed with100 nM OHT  (48h) and/or treated with 135nM IACS-010759, as indicated. (B) glutathione  quantification in FLMycER cells, measured after 40 hours of IACS-010759 treatment. Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).",
    "molecules": "IACS-010759, OHT, glutathione"
  },
  {
    "caption": "(C) Cell viability, as determined by PI staining  after 48 hours of IACS-010759 treatment, with or without addition of 10 mM N-acetyl-cysteine  (NAC). (D) Same as (C), with 50 µM buthionine-sulfoximine (BSO). Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).",
    "molecules": "IACS-010759, N-acetyl-cysteine, NAC, BSO, buthionine-sulfoximine"
  },
  {
    "caption": "(F) Immunoblot  on lysates from FLMycER cells treated as indicated, with the addition of 10 mM NAC  concomitantly with IACS-010759.",
    "molecules": "IACS-010759, NAC"
  },
  {
    "caption": "FLMycER  cells were primed with 100 nM OHT  and treated with 135 nM  IACS-010759, as indicated. (B) NADPH/NADP redox state in  FLMycER cells, assayed after 40 hours of IACS-010759 treatment. n=3 biological replicates. ** p ≤ 0.01; **** p ≤ 0.0001 (one-way ANOVA).",
    "molecules": "IACS-010759, OHT, NADP, NADPH"
  },
  {
    "caption": "FLMycER cells were primed with 100 nM OHT  and treated with 135 nM  IACS-010759, as indicated. (E-G) Cell viability after 40 hours of IACS-010759 treatment in the presence of (E) 50 µM dehydroepiandrosterone  (DHEA), (F) 10 µM 6- aminonicotinamide  (6AN) or (G) 1 mM  2-deoxyglucose (2DG). See Supplemental Figure3F for detailed statistical analysis. Data information: In E,G) n=6; in (F) n=9; all biological replicates; error bars: SD.",
    "molecules": "IACS-010759, 2-deoxyglucose, 2DG, 6- aminonicotinamide, 6AN, OHT, dehydroepiandrosterone, DHEA"
  },
  {
    "caption": "(A) Viability  of FLMycER and BaFMycER cells primed or not with 100 nM OHT  and treated with 135 nM IACS-010759 for 48h and/or with ascorbate  at the indicated concentration for 6h. n=3 biological replicates; error bars: SD.",
    "molecules": "IACS-010759, OHT, ascorbate"
  },
  {
    "caption": "(C) Cell viability of FL5.12cells at the end of treatment (48h  IACS-010759,6h Asc), in the presence or absence of 50 µM deferoxamine  (DFX, added 1 hour before Asc). n=3 biological replicates; error bars: SD.",
    "molecules": "IACS-010759, Asc, deferoxamine, DFX"
  },
  {
    "caption": "(D) Quantification of   lipid peroxides  in FL5.12 cells treated with 135 nM IACS-010759  for 24h and/ or 400 µM Asc  for 3 hours. ** p ≤ 0.01; * p ≤ 0.05 (one-way ANOVA).",
    "molecules": "lipid peroxides, IACS-010759, Asc"
  },
  {
    "caption": "The DLBCL  cell lines DoHH2, SU-DHL-6, Karpas 422 and HBL-1 were treated with the indicated concentrations of IACS-010759 and ascorbate, and cell viability  determined after 24 hours. (A) Live cell counts. n=3 biological replicates; error bars: SD.",
    "molecules": "IACS-010759, ascorbate"
  },
  {
    "caption": "The indicated lymphoma  cell lines and PDX samples were xenografted  in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with  IACS-010759 (IACS) by oral gavage  and/or ascorbate  (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (A, B) Tumor progression  (mm3) in CD1 nude mice  bearing subcutaneous Ramos  (A) or DoHH2  tumors (B). Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.",
    "molecules": "IACS, IACS-010759, Asc, ascorbate"
  },
  {
    "caption": "The indicated lymphoma  cell lines and PDX samples were xenografted  in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS-010759 (IACS) by oral gavage and/or ascorbate  (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (C, D) Tumor progression  in NSG mice  injected with the luciferase-positive DHL-derived PDX lines DFBL-20954 (C) or  DFBL-69487 (D), as determined by in vivo imaging and quantification of bilateral femur radiance efficiency. Examples from the control and double-treated groups are shown at the bottom. Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.",
    "molecules": "IACS, IACS-010759, Asc, ascorbate"
  },
  {
    "caption": "H, I Lipid species profiles of cardiolipin (H) and monolysocardiolipin (I) in isolated cardiac mitochondria analyzed by mass spectrometry. n= 4 per genotype.",
    "molecules": "cardiolipin, monolysocardiolipin"
  },
  {
    "caption": "J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues.",
    "molecules": "cardiolipin, monolysocardiolipin"
  },
  {
    "caption": "B OCR rates of cardiac mitochondria after administration of pyruvate, malate and ADP, n= 7 per genotype.",
    "molecules": "pyruvate, ADP, malate"
  },
  {
    "caption": "A Mitochondrial membranes, isolated from cardiac tissue, were solubilized in 1% digitonin and analyzed by BN-PAGE and Western- blotting with the indicated antibodies.",
    "molecules": "digitonin"
  },
  {
    "caption": "C Cardiac mitochondria were solubilized in 0.4% DDM and complexes were separated by BN-PAGE and analyzed by Western- blotting with the indicated antibodies.",
    "molecules": "DDM"
  },
  {
    "caption": "E Quantification of ROS production over time in cardiac mitochondria using the ROS-specific fluorescence-based sensor H2DCFDA. One representative measurement is shown. Analyses were performed in triplicates and confirmed for three animals per genotype. Significance was calculated using Student's T-TEST. sh, shTAZ.",
    "molecules": "H2DCFDA, ROS"
  },
  {
    "caption": "A Enzymatic activity of ubiquinone-cytochrome c oxidase in mitochondria isolated from cardiac tissue. The ubiquinone analogue decylubiquinone was used in this assay. Data were standardized to malate-dehydrogenase activity and presented as percent of wild-type, n= 4 per genotype.",
    "molecules": "decylubiquinone"
  },
  {
    "caption": "B Mitochondrial membranes were solubilized in 1% digitonin and analyzed by BN-PAGE and Western-blotting using antibodies against SDH components SDHA and SDHB (upper panel). For a loading control, samples were separated on SDS gel and analyzed by Western-blotting (lower panel).",
    "molecules": "digitonin"
  },
  {
    "caption": "G  Quantitative PCR  analysis of Ptpn11 mRNA levels in the  IMQ-treated or non-treated  dorsal  back of C57BL/6  mice  at day 5 (n=6/group). Data were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.",
    "molecules": "IMQ"
  },
  {
    "caption": "H Representative  histological  sections of  IMQ-treated or non-treated dorsal  back of C57BL/6  mice at day 5. Scale bar: 100 μm.",
    "molecules": "IMQ"
  },
  {
    "caption": "C57BL/6 mice (n=6/group) were treated with indicated dose of SHP099  or vehicle for 4 days. A Phenotypic presentation (top),  H&E staining (middle) and statistical data (bottom) (mean ± SEM) of dorsal skin. Scale bar: 100 μm. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.",
    "molecules": "SHP099"
  },
  {
    "caption": "Quantitative PCR analysis of mRNA encoding  IL-23/IL-17A axis cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099  or vehicle for 4 days. Results were normalized to  Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.",
    "molecules": "SHP099"
  },
  {
    "caption": "ELISA  quantification of protein levels of cytokines in mouse serum. Quantitative PCR analysis of mRNA encoding  psoriasis-related cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099  or vehicle for 4 days. Results were normalized to Gapdh  expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.",
    "molecules": "SHP099"
  },
  {
    "caption": "Representative images (A and E), histological  sections of dorsal back (B and F) and clinical scores (C and G) and quantitative PCR  analysis of mRNA levels in dorsal skin  (D and H) from wild-type (n=6) and M-Shp2-/- mice  or DC-Shp2-/- mice (n=6) treated with IMQ for 4 days. Scale bar: 100 μm. Left: H&E  (hematoxylin and eosin) staining data; right: statistical data (mean ± SEM). Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values were calculated by Tukey multiple comparison test (B, D, F, H) or Bonferroni multiple comparison test (C, G). *P<0.05, **P<0.01, ns, not significant.",
    "molecules": "eosin, hematoxylin, IMQ"
  },
  {
    "caption": "A Heat map showing mRNA expression  in peritoneal macrophages derived from wild-type and M-Shp2-/- mice after IMQ treatment based on RNA sequencing (n=3/group). Colors represent high (red) and low (blue) intensity. Genes labeled in green indicate they are in the NF-κB signaling.",
    "molecules": "IMQ"
  },
  {
    "caption": "D BMDMs and PMs derived from wild-type and M-Shp2-/- mice were stimulated by IMQ  (10 µg/ml) for indicated times. Whole cell lysates were subjected to Western blotting.",
    "molecules": "IMQ"
  },
  {
    "caption": "F PMA-differentiated THP-1 cells were infected with vector or SHP2 lentivirus  and then treated with or without R848  (10 µg/ml). Subcellular fractionation was performed, and cytoplasmic and cell membrane proteins were probed by respective antibodies.",
    "molecules": "PMA, R848"
  },
  {
    "caption": "G, H  Immunoblot  analysis of TLR7  and SHP2 in the isolated ER, Golgi and endosome from PMA-differentiated THP-1  cells with either SHP2 overexpressed (G) and SHP2  deficiency(H).",
    "molecules": "PMA"
  },
  {
    "caption": "G HEK293T  cells transfected with NF-κB-luciferase  reporter and indicated TLR7  mutant vectors and then treated with  IMQ (10 µg/ml) for 24 h were harvested for luciferase assay. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test **P<0.01, ns, not significant.",
    "molecules": "IMQ"
  },
  {
    "caption": "Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female  mice (n=6) and  Tlr7ki/ki female  mice (n=5) were treated with indicated dose of IMQ for 4 days. A Phenotypic presentation (top) and H&E staining (bottom) of dorsal skin. Scale bar: 100 μm. Left: H&E staining data; right: statistical data (mean ± SEM). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.",
    "molecules": "IMQ"
  },
  {
    "caption": "Tlr7wt/wt female mice  (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ  for 4 days. D Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines and other psoriasis-related cytokines in the dorsal skin. Results were normalized to  Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.",
    "molecules": "IMQ"
  },
  {
    "caption": "Tlr7wt/wt female  mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female  mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ  for 4 days. E Representative p-p65 staining of the dorsal skin. Scale bars: 100 μm.",
    "molecules": "IMQ"
  },
  {
    "caption": "(G) Tumour growth (in mm3) of FBXW7+/+ and FBXW7-/- xenografts in nude mice (n=10 animals per group). Treatment with either vehicle, paclitaxel (1,5mg/kg) or tigecycline (50mg/kg) started at day 6 post-tumour-injection, and was administered three times per week. Error bars indicate SEM. (H) Representative images of the xenografts defined in (G) at day 15.",
    "molecules": "paclitaxel, tigecycline"
  },
  {
    "caption": "A, The indicated cells co-transfected with p5HRE-Luc and either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (empty vector: EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay. B, U2OS cells co-transfected with p5HRE-Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "C, HCT116 p53-/- cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 (at two concentrations: + and ++) or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies.",
    "molecules": "oxygen"
  },
  {
    "caption": "D, HCT116 p53+/+ cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 R175H, R248W, R273H, or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) either pcDNA3/p53 (p53) or pcDNA3 (EV) (C) with the indicated siRNA were cultured under < 0.1% O2 conditions for 24 hours and subjected to qRT-PCR for the indicated mRNA",
    "molecules": "O2"
  },
  {
    "caption": "The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (H) were cultured under < 0.1% O2 (H) for the indicated periods and subjected to the in vitro cell proliferation assay.",
    "molecules": "O2"
  },
  {
    "caption": "The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) transiently transfected with siScr or siZBTB2 (I) the indicated oxygen conditions (I) for the indicated periods and subjected to the in vitro cell proliferation assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "A, The indicated cells transiently transfected with pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies.",
    "molecules": "oxygen"
  },
  {
    "caption": "The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "The indicated cells transiently transfected with pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated (B,D,E,F) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "I,J, The indicated cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT or pcDNA4/LgBiT and either pcDNA4/ZBTB2-SmBiT or pcDNA4/SmBiT, as indicated, were cultured under < 0.1% oxygen (I) or the indicated oxygen conditions (J) and subjected to the split luciferase complementation assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "K, HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 (ZF2 or ZF3-deletion) were cultured under < 0.1% oxygen conditions, and subjected to immunoprecipitation with anti-myc antibody. Coprecipitated DNA was subjected to the qRT-PCR experiment using primers against HRE regions of the STC1 promoter.",
    "molecules": "oxygen"
  },
  {
    "caption": "B, HCT116 p53-/- cells co-transfected with pG5H1bLuc, pcDNA6/Gal4 DBD-HIF-1α TAD P564A, and either pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[8-11] (ZBTB2 del[8-11]), pEF6/ZBTB2 del[51-54] (ZBTB2 del[51-54]), or pEF6/myc-His B (EV), as indicated, were cultured under < 0.1% oxygen conditions for 24 hours and subjected to the luciferase assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "D, HCT116 p53-/- (left) cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT (ZBTB2), pcDNA4/ZBTB2 4A-LgBiT 4A (4A), or pcDNA4/LgBiT (-) and either pcDNA4/ZBTB2-SmBiT (ZBTB2), pcDNA4/ZBTB2 4A-SmBiT (4A), or pcDNA4/SmBiT (-), as indicated, were cultured under < 0.1% oxygen and subjected to the split luciferase complementation assay.",
    "molecules": "oxygen"
  },
  {
    "caption": "G, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.",
    "molecules": "DNA, double stranded DNA, RNA, ribonucleotide"
  },
  {
    "caption": "A. Validation of Rnaseh2b-/- MEF lines. RER activity (DRD:DNA) is undetectable in a Rnaseh2b-/- line consistent with complete inactivation of the Rnaseh2b gene (Reijns et al, 2012). Mean of three independent experiments; error bars, SEM, **** = p<0.0001, two-tailed t-test. Rnaseh2b-/- and Rnaseh2b+/+ control MEFs on a C57BL/6 p53-/- background.",
    "molecules": "DNA"
  },
  {
    "caption": "A. Overexpression of RNase H1 in Rnaseh2b-/- cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wildtype levels, while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b-/- MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n=3 independent experiments ± SEM.",
    "molecules": "DNA, RNA"
  },
  {
    "caption": "A. Immunoblots of subcellular fractions from HeLa cells exposed to normoxia. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.B. Immunoblots of subcellular fractions from HeLa cells exposed to hypoxia (1% O2) for 5 h. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.C. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 under normoxic or hypoxic conditions. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ** P < 0.01.",
    "molecules": "O2"
  },
  {
    "caption": "D. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Arrows indicate gold particles.",
    "molecules": "gold, O2"
  },
  {
    "caption": "E. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-TOM20 antibody (12 nm gold particles). Bar = 500 nm.",
    "molecules": "gold, O2"
  },
  {
    "caption": "F. Quantification of the MAM:mitochondrion ratio of FUNDC1 immunogold particles from D under normoxic and hypoxic (1% O2) conditions; Data (mean ± s.e.m.) are from 3 independent experiments. n = 60 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria), ** P < 0.01.",
    "molecules": "O2"
  },
  {
    "caption": "G. Quantification of the MAM:mitochondrion ratio of TOM20 immunogold particles from E under normoxic and hypoxic (1% O2) conditions. Data (mean ± s.e.m.) are from 3 independent experiments. n = 50 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria). NS means not significant.",
    "molecules": "O2"
  },
  {
    "caption": "J. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-FACL4 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate FACL4-gold particles.",
    "molecules": "gold, O2"
  },
  {
    "caption": "L. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-DRP1 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate DRP1-gold particles.",
    "molecules": "gold, O2"
  },
  {
    "caption": "F. Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia (1% O2) for 5 h. PNS: post-nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.G. Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA (si-CNX) and exposed to hypoxia (1% O2) for 5 h. PNS: post nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.H. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si-CNX and exposed to hypoxia (1% O2) for 5 h. Data are presented as mean ± s.e.m. from 3 independent experiments; *** P < 0.001.",
    "molecules": "O2"
  },
  {
    "caption": "A. HeLa cells were transfected with the indicated si-RNAs. 24 h post-transfection, cells were exposed to hypoxia (1% O2) for 12 h and then immunostained by anti-TOM20. Bar = 50 µm. Boxed regions in the left panels are enlarged in the right panels.C. Quantification of mitochondria morphology (fragmented, intermediate and elongated mitochondria) in 100 cells from (A). Data represent mean ± s.e.m. (*** P<0.001).",
    "molecules": "O2"
  },
  {
    "caption": "B. Electron microscopy analysis of HeLa cells treated with the indicated si-RNAs for 24 h and then exposed to hypoxia (1% O2) for 12 h. Bar = 1 μm. Boxed regions in the left panels are enlarged in the right panels.",
    "molecules": "O2"
  },
  {
    "caption": "B. Immunogold electron microscopy of control HeLa cells (top) or HeLa cells transfected with si-FUNDC1 (bottom) and then exposed to hypoxia (1% O2) for 12 h. DRP1 was detected with anti-DRP1 (12 nm gold particles). Blue stars indicate mitochondria. Yellow arrows indicate gold particles. Red arrows indicate double-membraned autophagosomes.",
    "molecules": "gold, O2"
  },
  {
    "caption": "F. HeLa cells were exposed to hypoxia (1% O2) for the indicated times and cell lysates were immunoprecipitated using anti-FUNDC1 antibody. Endogenous DRP1, Calnexin, LC3 and FUNDC1 were detected by anti-DRP1, anti-Calnexin, anti-LC3, and anti-FUNDC1 antibodies.",
    "molecules": "O2"
  },
  {
    "caption": "B and C. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h. Cells were then treated with or without 50 nM BAF A1 (Bafilomycin A1) for 6 h before harvesting. Cell lysates were immunoblotted with the indicated antibodies.",
    "molecules": "BAF A1, Bafilomycin A1"
  },
  {
    "caption": "C, Image sequence of an L1 synchronized grh-1::aid animal (HW2418) grown at 20°C on a 250 μM auxin-containing plates. A lethargic animal was transferred to an agar-pad containing microscopy slide and images were collected every 1 sec, using DIC, 100x magnification. Selected images of Movie EV1 are shown. Time stamp (min:sec) is indicated. Arrows indicate phenotypic features: loosening of the cuticle (0:00); back-and-forth movements (0:05); inflation of the cuticle (2:46); vesicles underneath loosened cuticle (6:11); rupturing of the cuticle (6:20, 6:40).",
    "molecules": "agar, auxin"
  },
  {
    "caption": "A, Quantification of the percentage of grh-1::aid animals constitutively expressing luciferase (HW2434) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated.",
    "molecules": "auxin"
  },
  {
    "caption": "B, Boxplot showing the duration of M1, M2 and M3 of animals treated with indicated concentrations of auxin. Animals that failed to develop beyond M3 in (A) were excluded. The horizontal line represents the median, hinges extend to first and third quartiles, and the whiskers extend up to the most extreme value within 1.5*IQR (interquartile range) of the median. Significant differences relative to 0 nM auxin are indicated. P-values were determined by a non-parametric Wilcoxon rank sum test that does not assume normal distribution.",
    "molecules": "auxin"
  },
  {
    "caption": "A-D, Heatmaps showing trend-corrected luminescence (Lum, arbitrary units) of grh-1::aid animals constitutively expressing luciferase (HW2434). T = 0h corresponds to time of plating embryos, which subsequently hatch at different times. Arrow indicates time point when 250 μM auxin was added, i.e. prior to the first molt (A; M1), M2 (B), M3 (C) or M4 (D) larval stage. Note that for technical convenience in (A), auxin was provided at time of plating. Animals are sorted by entry into M1 (A), M2 (B), M3 (C), M4 (D), respectively.",
    "molecules": "auxin"
  },
  {
    "caption": "D, Western blot revealing rapid GRH-1 depletion in the grh-1::aid strain (HW2434) upon addition of 250 µM auxin. A synchronized culture of animals was grown in liquid at 20°C. After 21 h (denoted t=0 h in the figure), the culture was split in two and either auxin or vehicle were added as indicated. Cultures were sampled hourly and protein lysates probed by Western blotting using anti-FLAG and anti-actin antibodies as indicated.",
    "molecules": "auxin"
  },
  {
    "caption": "E. Proliferating H9-OCT4-mCherry hESCs were imaged for 24 h, fixed, and stained for DNA content with DAPI. The probability density of DAPI ratio (blue) and OCT4-mCherry ratio (red) between sister cells that divided ≤1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function. A two-sample Kolmogorov-Smirnov test was used to determine significance. * P < 0.05, *** P < 0.0005, two-sample Kolmogorov-Smirnov test; ns, not significant.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(J) In vitro RNA pull-down assays were performed using biotin-labeled TGFβR1 3' UTR segments in A549 and H1299 cells. After pull-down, endogenous QKI-5 enrichments were detected by western blot analyses. β-actin was used as the protein control.",
    "molecules": "biotin"
  },
  {
    "caption": "F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l-/- females at the indicated time points after hCG injection. Scale bar = 100 μm.",
    "molecules": "hCG"
  },
  {
    "caption": "E Poly(A) tail assay results showing poly(A)-tail length of indicated transcripts in WT and Pabpn1l-deleted oocytes and embryos. The poly(A) tails of Actin are used as an internal control. Each sample was prepared from 100 oocytes or embryos. Plots show the averaged relative signal intensity (y-axis) and length of the PCR products based on mobility (x-axis).",
    "molecules": "poly(A)"
  },
  {
    "caption": "C 5-Ethynyl uridine (EU) fluorescence (green) showing RNA transcription in WT and Pabpn1l♀−/♂+ 2-cell embryos. Some WT embryos were treated with α-amanitin as early as the zygote stage and cultured to the 2-cell stage. The phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) (red) is co-stained to label the RNA polymerase II activity. Nuclei are labeled by DAPI (blue). Scale bar = 20 μm.",
    "molecules": "5-Ethynyl uridine, EU, α-amanitin, DAPI"
  },
  {
    "caption": "D Quantification of EU and pS2 signals in (A). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.",
    "molecules": "EU"
  },
  {
    "caption": "F Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 h and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm.",
    "molecules": "hCG, polyadenylated"
  },
  {
    "caption": "Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm).",
    "molecules": "adenosine, poly(A)"
  },
  {
    "caption": "Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm).",
    "molecules": "adenosine, poly(A)"
  },
  {
    "caption": "B HeLa cells were transfected with plasmids expressing HA-BTG4 and FLAG-PABPN1L for 12 h and then treated by cycloheximide (CHX, 10 μM). Cells were harvested at the indicated time points for western blots. DDB1 is a loading control.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(D) Northern blot showing the 5' NGD-IM derived from the R(CGN)12 reporter in ski2∆ cells expressing the indicated Hel2 mutant proteins. 5' NGD-IMs were detected with a DIG-labelled GFP probe.",
    "molecules": "DIG"
  },
  {
    "caption": "(E) Primer extension mapping of 5' ends of 3' NGD-intermediates in Hel2-WT or Hel2(1-315) mutant cells at nucleotide resolution. . The primer extension samples were analysed using 5% TBE-Urea-PAGE and detected by fluorescence. Non-specific reverse transcription (ReTr) products are indicated by asterisks.",
    "molecules": "Urea"
  },
  {
    "caption": "(A) Northern blot analysis demonstrating that K63-linked ubiquitination was required for an endonucleolytic cleavage by R(CGN)12. The ski2∆UB-WT and ski2∆ub-K63R cells were transformed with the R(CGN)12 reporter, and total RNA samples were separated by 2% agarose/MOPS gel. 5'NGD-IMs in the cells were detected as in Fig 1D.",
    "molecules": "agarose, MOPS"
  },
  {
    "caption": "(F-H) Northern blot analysis of NGD-cleavage sites in the absence of uS10 ubiquitination or RQT complex components: The full-length GFP-R(CGN)12-FLAG-HIS3 mRNA and 5' NGD-intermediates (5' NGD-IM) or 3' NGD-intermediates (3' NGD-IM) were detected in the indicated mutant cells by Northern blotting with DIG-labelled probes. 5' NGD-intermediates were detected by DIG-labelled GFP probe and 3' NGD-intermediates were detected by the DIG-labelled HIS3 probe. SCR1 was used as loading control. FL = full-length. Note the upstream shift of NGD cleavage sites in lanes F4, G4, H6, and H8.",
    "molecules": "DIG"
  },
  {
    "caption": "Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (C) Northern blot for the 5' NGD-IM derived from the (CGA-CCG) reporter in ski2∆ cells. 5'-NGD-IMs were detected with a DIG-labelled GFP probe. SCR1 was used as a load control.",
    "molecules": "DIG"
  },
  {
    "caption": "(E) Sucrose gradient fractions (top) and western blot analysis (bottom) of the (CGA-CCG) dicodon-stalled RNC samples. After the translation reaction, the RNCs were affinity-purified as indicated in (C). The eluate was loaded on a 10-50 % sucrose gradient and fractionated. Each collected fraction was analysed using anti-HA antibody to detect uS10-HA. Note that disomes are preferentially polyubiquitinated over monosomes.",
    "molecules": "sucrose, Sucrose"
  },
  {
    "caption": "(C) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination: Both copies of eS7 (eS7A and eS7B) were deleted and transformed with a plasmid containing HA-tagged eS7A (eS7-WT) or eS7A mutated in the four potential ubiquitination sites (4KR). Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 (hel2Δ and not4Δ). Note that the four mutated lysine residues were responsible for Not4-dependent monoubiquitination.",
    "molecules": "lysine"
  },
  {
    "caption": "Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (C) Cells expressing HA-tagged eS7A or the eS7A-4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5'-NGD intermediates by Northern blotting. Note that the four mutated lysine residues in eS7 were responsible for NGDRQC- in the R(CGN)12 reporter.",
    "molecules": "lysine"
  },
  {
    "caption": "Gdf15 gene expression of differentiated C2C12 muscle cells treated with vehicle control (Ctrl) or chemical mitochondrial uncoupler (FCCP, 1µM vs. 5µM) for 5hrs (n=3 biological replicates)",
    "molecules": "FCCP"
  },
  {
    "caption": "Representative traces of oxygen consumption rate (OCR) during substrate-uncoupler-inhibitor-titration (SUIT) protocol for mitochondrial respiratory capacity in permeabilized mouse soleus muscle fibers, PM (Pyruvate+Malate; LEAK respiration), ADP (OXPHOS capacity), Cyt c (cytochrome c, integrity of outer mt-membrane), G (glutamate;), S (succinate), U (Uncoupler, FCCP), Rot (rotenone), Ama (antimycin A; less than 2% residual oxygen consumption, ROX).",
    "molecules": "ADP, Ama, antimycin A, FCCP, glutamate, Malate, Pyruvate, Rot, rotenone, succinate"
  },
  {
    "caption": "Plasma FGF21 levels from male mice at 20wks of age (n=10 per genotype). Plasma Leptin levels from male mice at 20wks of age (n=5 per genotype). Post-absorptive plasma insulin levels at 20wks of age (WT n=8, TG n=9, TG n=9). : Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.",
    "molecules": "insulin"
  },
  {
    "caption": "Blood glucose (I) and insulin levels (J) with total area under the curve (AUC) of insulin (K) during oral glucose tolerance test (oGTT) at 17wks of age (WT n=8, TG n=11, TG n=11). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(a) Silver-stained gel image of the affinity-purified complexes of PSPC1, SFPQ and NONO. The respective proteins are marked along with AcTEV protease used for the elution of purified proteins.",
    "molecules": "Silver"
  },
  {
    "caption": "(a) Silver-stained gel image of the affinity-purified complexes of ARID1A and ARID1B. The bait and a few other SWI/SNF subunits are marked as per their molecular weight.",
    "molecules": "Silver"
  },
  {
    "caption": "(F) Comparison of protein degradation efficiency using different degrons. Cells were incubated for indicated time periods with Asv or DOX/IAA  or both (DIA, see methods) then collected for western blot analysis. Doxycycline  was added 2 hours before addition of  IAA or Asv/IAA. To determine protein degradation, either  cyclin A2 or cyclin B1 were probed as indicated. Anti-myc antibody was used to check OsTIR1-myc expression. Asterisks indicate non-specific protein crossreacting with the cyclin B1  antibody.",
    "molecules": "Asv, DOX, Doxycycline, IAA"
  },
  {
    "caption": "A)  Cyclin B2 knock-out and induced degradation by immunoblotting. Indicated cell lines were analysed 24 hours after mock or Dox/IAA/Asv (DIA) treatment using the indicated antibodies to confirm homozygous gene tagging and efficiency of  protein degradation. ( ",
    "molecules": "Asv, Dox, IAA"
  },
  {
    "caption": "(D) Cell proliferation of A2dd, CycB1dd and B1dd/B2ko following mock or DIA treatment. 1000 cells were plated in each well (diameter 3.5cm) and incubated for 10 days before methanol fixation and Crystal Violet staining.",
    "molecules": "Crystal Violet, methanol"
  },
  {
    "caption": "(I) Cyclin A2  siRNA depletion causes endoreplication. Following 72 hours of siRNA transfection MCF7 and MCF10A cells were analysed by  PI staining and  FACS. The histograms show the changes in  DNA  content (PI Int.) towards >4N following cyclin A2 depletion.",
    "molecules": "DNA, PI"
  },
  {
    "caption": "(C) Representative images from  live-cell imaging of SiR-Tubulin labelled (red) mitotic B1dd/B2ko cells expressing FusionRed-H2B (green), Mis12-GFP (white); time in mins, scale bar = 5µm. Cells were imaged four hours after DIA treatment.",
    "molecules": "SiR-Tubulin"
  },
  {
    "caption": "(F) Cyclin A2  levels were assessed by immunoblotting after 18 hours in 100ng/ml Nocodazole and/or 5µM STLC. 6µM proTAME was added to the indicated samples for the final six hours.",
    "molecules": "STLC, Nocodazole, proTAME"
  },
  {
    "caption": "(G) Upper panel, confirmation of  Cdc27  depletion by  immunoblotting 72 hours after siRNA transfection in  B1dd/B2ko cells. Lower panel, following 40-hour siRNA transfection B1dd/B2ko cells were treated for 24 hours with Thymidine, released for 10 hours and treated with proTAME and Apcin for additional four hours. At this point, the cells were fixed and  stained with tubulin and pericentrin for  immunofluorescence  analysis.",
    "molecules": "Apcin, proTAME, Thymidine"
  },
  {
    "caption": "(H) Representative images of mitotic spindles in control- and DIA-treated B1dd/B2ko cells following arrest in mitosis by proTAME/Apcin treatment Tubulin staining  is shown in white, pericentrin in green and  DAPI in red, scale bar = 5µm.",
    "molecules": "Apcin, DAPI, proTAME"
  },
  {
    "caption": "(L) Immunofluorescence  images from mitotic Ctr and DIA treated  B1dd/B2ko cells after P/A synchronisation stained with anti-alpha-tubulin (green), anti-gamma-tubulin (red) antibodies and DAPI  (blue), scale bar = 5µm. Cells were exposed to ice-cold medium and either fixed immediately after cold exposure, or incubated for 5,10, or 20 minutes at 37° C before fixation.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Time-lapse  images of PCNA-mRuby tagged  A2dd cells. The imaging sequence was started at the time of Doxycycline addition, and degron activity was triggered three hours later by addition of  IAA  and Asv or PBS (indicated by dashed line). Time is shown as hh:min, scale bar equals 10µm. For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase.",
    "molecules": "Asv, Doxycycline, IAA, PBS"
  },
  {
    "caption": "(D) Images from time-lapse sequence of mitosis showing cell division  in controls and after addition of 0.5µM PD-166285 in DIA treated  A2dd labelled with SiR-DNA (time  is indicated as hh:min, the scale bars represent 20µm).",
    "molecules": "SiR-DNA, PD-166285"
  },
  {
    "caption": "(I) Immunoprecipitation of YFP from extract from   A2dd following induction of YFP (Y), CycB1-YFP (BY) and CycB1-YFP-NLS (BYN). The samples in the odd lanes (IAA/Asv-) were treated with 1µg/ml  doxycycline alone, while samples in even lanes (IAA/Asv+) were treated with Auxin and  Asv to induce cyclin A2 depletion. Total cell extract and  immune-precipitates  were probed by immunoblotting with the indicated antisera.",
    "molecules": "Asv, Auxin, doxycycline, IAA"
  },
  {
    "caption": "(G) Representative images from  live-cell imaging  of DIA-treated SiR-Tubulin labelled (red) B1dd/B2ko cells expressing FusionRed-H2B (green) and AurB-GFP  (white); time is indicated in minutes, scale bar = 5µm. Bottom panel shows frequencies of aberrant AurB localisation in P/A synchronised B1dd/B2ko cells.",
    "molecules": "SiR-Tubulin"
  },
  {
    "caption": "(E) Dynamics of Greatwall, PP1, and Cdc27 dephosphorylation   following Cdk1 inhibition in proTAME/Apcin treated mitotic cell extracts from Ctr or DIA treated cells. Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol  and analysed by immunoblotting",
    "molecules": "Flavopiridol, Apcin, proTAME"
  },
  {
    "caption": "A) Representative images from live-cell imaging of siRNA transfected B1dd/B2ko cells (FusionRed-H2B in red, SiR-Tubulin  in white, scale bar = 10µm). Bar-plot panels on the right show single-cell analysis of 10 cells manually scored for length mitosis pre-NEBD and post-NEBD. Entry into  prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the  nucleus.",
    "molecules": "SiR-Tubulin"
  },
  {
    "caption": "(B) Widefield Imaging. DIC (Grey), SiR-DNA  (b/w), of ENSA/ARPP19 siRNA transfected DIA treated B1dd/B2ko cells. Time is indicated in hh:min, scale bar = 10µm.",
    "molecules": "SiR-DNA"
  },
  {
    "caption": "(F) Skin stratification in WT and cKO  skin was determined by immunofluorescence staining with different antibodies as indicated. Krt14:  Keratin 14; Krt10: Keratin 10; β4: β4-integrin; DAPI for nucleus staining. The dashed line denotes the basement membrane that separates dermis  and epidermis (Epi).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A)  EdU staining of WT or Mettl14 cKO skin  after pulse-chase labeling. Skin samples were counterstained with antibody against β4-integrin. Note reduced EdU label-retaining cells in cKO  skin epidermis. Arrows indicate  Edu-positive cells. (B) Label-retaining cells in WT or Mettl14 cKO skin were quantified and shown as box plots. The plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). n=6 (biological repeats), P<0.05 (Student's t-test). n=19, P<0.01 (Student's t-test). ",
    "molecules": "EdU, Edu"
  },
  {
    "caption": "(I) Fluorescence microscopy demonstrates different  survival capability of WT and Mettl14  inducible KO cells with or without Tamoxifen (TAM) treatment. (J) Ratio of WT and Mettl14  inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with  TAM  treatment compared with WT cells or KO cells without TAM stimulation at both Day 7 and 14. Error bar represents S.D. ",
    "molecules": "TAM, Tamoxifen"
  },
  {
    "caption": "B) Mutations in the predicted  m6A  modification sites can significantly reduce  Pvt1 methylation.n=3. Error bar represents S.D.",
    "molecules": "m6A"
  },
  {
    "caption": "H)  Fluorescence microscopy  demonstrates different survival capability of WT and Pvt1 inducible KO cells with or without Doxycycline  (Dox) treatment.",
    "molecules": "Dox, Doxycycline"
  },
  {
    "caption": "I) Ratio of WT and Pvt1  inducible KO cells in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells with Dox treatment compared with WT cells or KO cells without Dox stimulation at both Day 7 and 14. Error bar represents S.D.",
    "molecules": "Dox"
  },
  {
    "caption": "A) Interaction between  Pvt1 and MYC was determined by immunoprecipitation followed with RT-PCR. Note significant decrease in Pvt1  and MYC interaction upon  FTO treatment (left panel), calcium shift-induced differentiation (middle panel, Hi: high calcium), and Pvt1 methylation site mutations (right panel). n=3, P<0.01 (Student's t-test). Error bar represents S.D.",
    "molecules": "calcium"
  },
  {
    "caption": "Undifferentiated WT and Mettl14 KO cells (C) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to  immunoblotting with different antibodies as indicated.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "undifferentiated WT and  Pvt1 KO cells (D) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to  immunoblotting with different antibodies as indicated.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "WT cells before and after calcium  shift-induced differentiation (E) were treated with cycloheximide. Cell lysates were collected at 0, 30, 60, 90, 120, 180, and 300 minutes post treatment and subjected to immunoblotting with different antibodies as indicated.",
    "molecules": "calcium, cycloheximide"
  },
  {
    "caption": "(J) Ratio of WT and Mettl14 KO cells with inducible expression of  MYC in the co-culture model was quantified and shown as dot plots. n=8, P<0.01 (Student's t-test) for KO cells without Dox treatment (no  MYC expression) compared with WT cells or KO cells with Dox stimulation (exogenous MYC expression) at both Day 7 and 14. Error bar represents S.D.",
    "molecules": "Dox"
  },
  {
    "caption": "(A) Hybridization Chain Reaction (HCR) detection of CGG repeat  RNA   along with  immunocytochemistry (ICC) to detect SRSF1-FLAG in U2OS cells. Expanded view of merged channels shows the  CGG RNA foci (arrowhead) and an example of SRSF1-CGG RNA   foci co-localization (arrow). DAPI marks the nucleus. Scale bars are 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Anti-FLAG immunoblot of DMSO and SRPIN340 pre-treated HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. β-Actin is used as a loading control. To prevent signal saturation, AUG-nLuc lysate was diluted 1:3 in sample buffer prior to loading (n=3 biological replicates). Schematics of the AUG-nLUC-3xFLAG and +1CGG(100)-nLuc-3xFLAG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. To prevent over-exposure, the AUG-nLuc lysate was diluted 1:3 in sample buffer.",
    "molecules": "DMSO, SRPIN340"
  },
  {
    "caption": "(D) Anti-FLAG immunoblot  of DMSO  and SRPIN340 pre-treated HEK293T cells expressing GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top. Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.",
    "molecules": "DMSO, polyA, SRPIN340"
  },
  {
    "caption": "(E) Immunoblot of DMSO and SPHINX31 pre-treated  HEK293T cells expressing AUG-nLuc-3xFLAG control or CGG-nLuc-3xFLAG RAN translation reporters. (n=3 biological replicates). (F) Anti-FLAG of DMSO and SPHINX31 pre-treated HEK293T cells expressing  GGGGCC-nLuc-3xFLAG (GA70) and +2CGG-nLuc-3xFLAG (FMRpolyA) RAN translation reporters (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Statistical analysis was performed using two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. ",
    "molecules": "DMSO, polyA, SPHINX31"
  },
  {
    "caption": "(C, D) Expression of GGGGCC-nLuc-3xFLAG (C) and +2CGG-nLuc-3xFLAG (D) RAN translation reporters in HEK293T  cells treated with 2 μM TG (for stress induction) analyzed by immunoblot  (n=3 biological replicates). To evaluate effects of  SRPK1 inhibition cells were pre-treated with  DMSO or SRPIN340 before reporter transfection. Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.",
    "molecules": "DMSO, SRPIN340, TG"
  },
  {
    "caption": "(E) SRPK1 inhibition by SRPIN340 did not alter  eIF2α phosphorylation in response to 2 μM  TG (for stress induction) in HEK293T cells by immunoblot (n=3 biological replicates). (F) SPRK1 inhibition suppressed  eIF2α phosphorylation in  HEK293T  cells treated with 2 μM TG (for stress induction) in the presence of the +1CGG reporter. Cells were pre-treated with DMSO or SRPIN340 before reporter transfection (n=3 biological replicates). Data information: Error bars represent mean +/- SD. Two-tailed Student's t test with Welch's correction. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. ",
    "molecules": "DMSO, SRPIN340, TG"
  },
  {
    "caption": "(B)  ICC images of  FLAG-tagged  SRSF1 after  SRPIN340 treatment compared to vehicle (DMSO). Quantification shows the ratio of nuclear and cytoplasmic intensity of SRSF1  signal Error bars indicate mean +/- 95% CI (n= 85-126 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.",
    "molecules": "DMSO, SRPIN340"
  },
  {
    "caption": "(C) Nucleocytoplasmic distribution CGG repeat  RNA after SRPIN340  treatment compared to vehicle (DMSO) as detected by  HCR. Quantification shows the ratio of  nuclear  and  cytoplasmic  intensity of CGG RNA signal as parts of whole. Error bars indicate mean +/- 95% CI (n= 124-151 cells/condition). Statistical analysis was performed using t test with Welch's correction, ∗∗∗∗p < 0.0001.",
    "molecules": "DMSO, SRPIN340"
  },
  {
    "caption": "(D) Anti-FLAG immunoblot blot  of DMSO  and SRPIN340 pre-treated HEK293T cells transfected with in vitro transcribed CGG-nLuc-3xFLAG reporter RNA. β-Actin is used as a loading control. Error bars represent mean +/- SD (n=6 biological replicates). Statistical analysis was performed using Student's t test with Welch's correction. ∗p < 0.05",
    "molecules": "DMSO, SRPIN340"
  },
  {
    "caption": "(A, B) Pharmacological targeting of SPRK1 with (A) 40 μM SRPIN340  or (B) 8 μM SPHINX31 reduced the cumulative risk of death in +1(CGG)100-EGFP (encoding  FMRpolyG) expressing neurons. n= # of  neurons quantified for each condition; Cox proportional hazard analysis, ∗∗∗p < 0.001.",
    "molecules": "polyG, SPHINX31, SRPIN340"
  },
  {
    "caption": "(A-C) Representative images of Cellular localization of TLR2-p and scrTLR2-p peptides in BM-derived macrophages were observed using confocal microscopy. (A) Cells were probed with anti TLR2 antibody following by staining with APC-labeled secondary antibody or (B) with TLR1 antibody following by staining with FITC-labeled secondary antibody (left panels, red). For membrane staining, cells were labelled with lipid dye, DiD (C). Then rhodamine-labeled fluorescent peptide was added to the cells (middle panels, green). Merged images are shown in the right panel. Scale bars 10µm. The mean Pearson correlation coefficient observed of TLR2-p or scrTLR2-p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S.EM (n=19-61).",
    "molecules": "APC, rhodamine"
  },
  {
    "caption": "(A) Biochemical analyses of peptide interaction with TLR. BM-derived macrophages were incubated with 1µM fluorescently labeled rhodamine (Rho)-TLR2-p or its mutant Rho-ScrTLR2-p peptide. After 1h of incubation at 370C the cells were lysated. Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies. Proteins samples were resolved by SDS-PAGE and the presence of the labeled peptide was detected with a fluorescent scanner. Subsequently, the gel was transferred to a membrane and subjected to Western blotting for TLR6,1,2 and 4 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. The results are presented as the mean of fluorescent intensity from two independent experiments. Non-specific binding of the peptides to G protein beads was subtracted. The results are mean± S.E.M of at least two independent experiments.",
    "molecules": "rhodamine"
  },
  {
    "caption": "(B) Proteins analysis of secreted pro-inflammatory cytokines, IL-6 and TNF-α from primary CD115+ cells. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with LTA (5μg/ml), PAM3CSK (0.5μg/ml) or LPS (0.2μg/ml) at 370C for 5h (for TNF-α detection) and 22h (for IL-6 detection). The results are mean± S.E.M of two independent experiments (n=8).",
    "molecules": "PAM3CSK, LPS, LTA"
  },
  {
    "caption": "(B-C) Represented images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer (FRET) using ImageStreamX. Scale bars 10μm. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with 500ng/ml LTA for another 0.5h at 370C. Cells were probed with anti TLR6-PE or anti F4/80-PE conjugated antibody (donor) and anti TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreanX imaging flow cytometer. The FRET intensity shown are mean ± S.E.M of two independent experiments (n=2579-15567).",
    "molecules": "APC, PE, LTA"
  },
  {
    "caption": "(D) Represented image of ERK1,2 phosphorylation levels in CD115+BM monocytes cells upon LTA exposure. Cells were pre-treated for 0.5h with 20µM of TLR2-p peptide, scrTLR2-p peptide or untreated, then washed and incubated with 500ng/ml LTA for the indicated times. ERK1,2 phosphorylation levels and total ERK1,2 levels were detected by western blotting. Equal loading was detected by measuring anti tubulin. Results are representative data of 2 independent experiments. The bands intensity was quantified for p-ERK levels after 15min of LTA exposure. Results are normalized to tubulin levels and are the mean of 2 experiments ± S.D.M.",
    "molecules": "LTA"
  },
  {
    "caption": "(B) Representative colonoscopy images of the indicated treatments (day 7). C57BL mice were treated with 2% of dextran sulfate sodium (DSS) in drinking water for 7 days. An amount of 5mg/Kg of TLR2-p and scrTLR2-p peptides were injected IP every following day from day one.(C) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only, DSS with TLR2-p peptide, DSS with scrTLR2-p peptide and untreated mice (n= 19, 21, 22 and 19 respectively). Results represent 3 independent experiments, scale bars 100µm and 1000μm.",
    "molecules": "dextran sulfate sodium, DSS, water"
  },
  {
    "caption": "(A) Flow cytometry analysis of colonic lamina propria CD11c- CD11b+LY6Chi MHCII-monocytes from steady-state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocytes infiltrates in to the colitic colon. Plots were pre gated on live CD45+ cells. Results are representative of three independent experiments (n=3 per group). (B) Graphical summary of monocytes presented as % out of CD11b + cells.",
    "molecules": "DSS"
  },
  {
    "caption": "(B) Weight loss of DSS challenged mice on day 6.",
    "molecules": "DSS"
  },
  {
    "caption": " A The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos) ",
    "molecules": "Mitotracker CMXRos, NAD(P)H"
  },
  {
    "caption": " B 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%) ",
    "molecules": "NAD(P)H"
  },
  {
    "caption": "a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel for mouse cortical (C neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests",
    "molecules": "R, L, NAD(P)H, NADH, NADPH"
  },
  {
    "caption": "D a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel fo human (D) neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests",
    "molecules": "R, L, NAD(P)H, NADH, NADPH"
  },
  {
    "caption": "Wild type (WT) mouse cortical neurons cultured for 10 days in vitro (A were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test",
    "molecules": "R, L, NAD(P)H, Torin1"
  },
  {
    "caption": "human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells (B) were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test",
    "molecules": "R, L, NAD(P)H, Torin1"
  },
  {
    "caption": " A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 hours. Next, amino acids (R+L), insulin, insulin + R+L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later cellular reactive oxygen species (ROS), ATP levels and oxygen consumption were measured. The data were collected from 3 independent assays in which each experimental condition contained 6-8 replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test",
    "molecules": "oxygen, ATP, R, L, reactive oxygen species, ROS, NaN3"
  },
  {
    "caption": " B MP-PAM imaging of wild type (WT) mouse cerebral cortex through an open-skull window 1 hour after topical application of amino acids (R+L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from 3 mice , each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m",
    "molecules": "O2, oxygen, R, L"
  },
  {
    "caption": " C MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R+L was applied to the open skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from 4 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R+L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m",
    "molecules": "O2, oxygen, R, L, rapamycin"
  },
  {
    "caption": " B Mouse cortical neurons starved in Hank's balanced salt solution for 2 hours were treated with AβOs, insulin or amino acids (R+L) for 30 minutes, and then were fixed and double labeled with anti-mTOR, and either anti-LAMP1 or Mitotracker CMXRos. Error bars represent ± s.e.m. Data are representative of 3 independent assays ",
    "molecules": "Mitotracker CMXRos, AβOs, R, L"
  },
  {
    "caption": "A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3-hour pulse of the thymidine analog, EdU. Cells were labeled additionally with the mitochondrial marker, Mitotracker CMXRos, and with an antibody to the neuron marker, MAP2",
    "molecules": "DNA, Mitotracker CMXRos, thymidine"
  },
  {
    "caption": "B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit ­mtDNA replication by ~60%, an effect that was blocked by AβOs. Error bars represent ± s.e.m. Statistical analyses were performed using Student"s two-tailed unpaired t-test and are representative of 3 independent a",
    "molecules": "mtDNA, AβO"
  },
  {
    "caption": "C WT mouse cortical neurons expressing either Flag-Raptor H-Ras25 or Flag-Raptor Rheb15 were serum-starved in AβO-containing Hank\"s balanced salt solution for 2 hours. Next, the cells were pulse-labeled for 3 hours with EdU. Note that forcing mTORC1 to the plasma membrane (Flag-Raptor H-Ras25) yielded an ~80% increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin+R+L. In contrast, EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes (Flag-Raptor-Rheb15). Error bars represent ±",
    "molecules": "mtDNA, AβO, AβOs, R, L"
  },
  {
    "caption": " A Unmodified ReNcell VM human neuronal progenitor cells were co-plated with comparable Ren-GFP or Ren-APPSL cells into 3-dimensional Matrigel matrices, and differentiated into neurons for 60 days. Cytosolic GFP is produced by both Ren-GFP and Ren-APPSL cells, and the latter also produce pathogenic human APP with the Swedish and London mutations, and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation, 2P-FLIM was used to monitor NiMA in the ReNcell VM neurons, which were discriminated from Ren-GFP and Ren-APPSL neurons because they lack GFP. Note that NiMA was observed in ReNcell VM neurons when they were co-cultured with Ren-GFP neurons, but not when they were co-cultured with Ren-APPSL neurons. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test",
    "molecules": "Aβ"
  },
  {
    "caption": " B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures. Subsequent AβO exposure for 16 hours blocked NiMA, but expression of Flag-Raptor-Rheb15, which forces mTORC1 to lysosomes, prevented NiMA inhibition by AβOs. Each colored line refers to a single field of view containing 5-15 cells, and each identically colored pair of solid and dotted lines refers to the same field of view. Flag-Raptor -Rheb15 expression was confirmed by western blot­ting. Statistical analyses were performed using Student's two-tailed unpaired t-test. Western blots are representative of 3 independent assays",
    "molecules": "AβO, AβOs"
  },
  {
    "caption": " B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin was immediately added, and the cells were then imaged again 30 minutes later. Note that NiMA occurred in the absence or presence of AβOs, which inhibited NiMA in wild type human neuron Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test",
    "molecules": "AβO, AβOs"
  },
  {
    "caption": "Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test",
    "molecules": "R, L"
  },
  {
    "caption": "(E) Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immun­o­fluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test ",
    "molecules": "Flag"
  },
  {
    "caption": "(B) Upper panel: dissolved oxygen curve over 120 h time course showing controlled depletion (1), sustained hypoxia (2) and controlled reaeration (3). Points represent the average of three biological replicates and were measured via fiber optic technology that non-invasively probes oxygen levels in the culture (PreSens Precision Sensing GmbH). Lower panel: expression profiles (RNA-seq) of desA1 and desA2 over the time course and oxygen levels. Error bars show the standard deviation from three biological samples.",
    "molecules": "oxygen"
  },
  {
    "caption": "Boxplots representing RPKM values from RNA-seq of MSM with inducible overexpression of MSMEG_0916. Significant log2 fold change (FC) between uninduced (-ATc) and induced (+ATc) samples for MSMEG_0916 (log2 FC = 4.99), desA1 (log2 FC = -1.32) and desA2 (log2 FC = -1.8) with multiple hypothesis adjusted P-values < 0.0001. Data is from three biological samples (induced) and nine biological samples (uninduced).",
    "molecules": "ATc"
  },
  {
    "caption": "(A) Serial ten-fold dilutions of MTB H37Rv wild type (wt) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc.",
    "molecules": "agar, ATc"
  },
  {
    "caption": "(B) Argentation TLC of 14C-labeled methyl esters of mycolic acids (MAMEs) obtained from apolar lipids and delipidated cell wall fractions of MSM wt and MSM with inducible overexpression of MSMEG_0916. The α, αʹ, epoxy (e) and cyclopropanated α- (X1) MAMES species are labeled. Faster-migrating species that co-migrated with α-MAMES and accumulate with induced MSMEG_0916 overexpression are indicated as X2 and X3.",
    "molecules": "cyclopropanated α- (X1) MAMES, MAMEs, methyl esters of mycolic acids, α-MAMES, 14C, epoxy, apolar lipids"
  },
  {
    "caption": "(C) BCG wt and BCG with inducible overexpression of Rv0472c cultures, labeled with 14C-acetate, were induced (+ATc) or uninduced (-ATc) for 4 h or 8 h. The total FAMEs and MAMEs were extracted and analyzed by autoradiography-TLC using equal counts (15,000 cpm) for each lane.",
    "molecules": "MAMEs, acetate, ATc, 14C, FAMEs"
  },
  {
    "caption": "(A) Quantitative analysis of the Aβ1-40 protein in the TBS (*p=0.0212), Triton-X (p=0.0544) and SDS (**p=0.0063) fractions of brain homogenates from male (n=7) and female (n=10) APP23 mice. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.",
    "molecules": "Aβ1-40, SDS, Triton-X"
  },
  {
    "caption": "(C) Fluorescence intensity based analysis of pFTAA-stained Aβ plaques in the cortex of male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0011.",
    "molecules": "pFTAA, Aβ"
  },
  {
    "caption": "(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in male (n=10) and female (n=8) APP23 mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, ***p=0.0001.",
    "molecules": "Congo Red"
  },
  {
    "caption": "(E) Native filter assay analysis of TBS (p=0.2453), Triton-X (p=0.0604), SDS (*p=0.0196) and formic acid (FA) (**p=0.0057) fractions from male (n=8) and female (n=7) APP23 mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test between the same fractions. Data points were taken from graphs analysing APP23p40+/+ against APP23p40-/- mice.",
    "molecules": "FA, formic acid, SDS, Triton-X"
  },
  {
    "caption": "(A) Mesoscale analysis for the Aβ1-40 protein in the TBS (p=0.2298), Triton-X (p=0.1329) and SDS (p=0.7184) fractions of brain homogenates from male APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice. Total protein concentration of each sample was used as an internal reference. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.",
    "molecules": "Aβ1-40, SDS, Triton-X"
  },
  {
    "caption": "(C) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0051.",
    "molecules": "pFTAA"
  },
  {
    "caption": "(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=10) and APP23p40-/- (n=8) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, **p=0.0070.",
    "molecules": "Congo Red"
  },
  {
    "caption": "(E) Native filter assay analysis of TBS (p=0.7124), Triton-X (p=0.3170), SDS (p=0.4833) and formic acid (FA) (p=0.8144) fractions from APP23p40+/+ (n=8) and APP23p40-/- (n=8) mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test between the same fractions.",
    "molecules": "FA, formic acid, SDS, Triton-X"
  },
  {
    "caption": "(A) Mesoscale analysis for the Aβ1-40 protein in the TBS (*p=0.0208), Triton-X (*p=0.0440) and SDS (p=0.0540) fractions of brain homogenates from APP23p40+/+(n=7) and APP23p40-/- (n=8) mice. Total protein concentration of each sample was used as an internal reference. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test.",
    "molecules": "Aβ1-40, SDS, Triton-X"
  },
  {
    "caption": "(C) Fluorescence intensity based analysis of pFTAA-positive area covered in the cortex of APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images for each genotype (right), scale bar = 1 mm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.3406.",
    "molecules": "pFTAA"
  },
  {
    "caption": "(D) Stereological analysis of cortical area covered by Congo Red-positive plaques in APP23p40+/+ (n=8) and APP23p40-/- (n=10) mice (left) and representative images (right), scale bar = 500 µm. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test, p=0.4542.",
    "molecules": "Congo Red"
  },
  {
    "caption": "(E) Native filter assay analysis of TBS (p=0.6823), Triton-X (p=0.1146), SDS (p=0.0508) and formic acid (FA) (p=0.6603) fractions from APP23p40+/+ (n=7) and APP23p40-/- (n=7) mouse brain homogenates. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-tests between the same fractions.",
    "molecules": "FA, formic acid, SDS, Triton-X"
  },
  {
    "caption": "(E) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D/PepA. Data are representative of three independent experiments.",
    "molecules": "E64D, PepA, Torin"
  },
  {
    "caption": "(I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.",
    "molecules": "PEG 1000"
  },
  {
    "caption": "M. Whole-mount retina stained with anti-ADAM17 (green) and phalloidin to mark the photoreceptor membranes (red).",
    "molecules": "phalloidin"
  },
  {
    "caption": "N. Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia, n=10 fly retinas. Scale bars: 10μm. ",
    "molecules": "phalloidin"
  },
  {
    "caption": "D-J. Fluorescent images of 1 day old fly retinas stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and the photoreceptor membranes respectively; (D) wild type; (E) ADAM17-/- mutant; (F) ADAM17-/ Deficiency; (G) knockdown of ADAM17 throughout the retina; (H) knockdown in glial cells; (I) knockdown in neurons; and (J) kuz-/-; n=10 fly retinas. Scale bars 10μm",
    "molecules": "BODIPY, FM-dye"
  },
  {
    "caption": "Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.",
    "molecules": "BODIPY, lipid"
  },
  {
    "caption": "Quantitation of the BODIPY signal shown as normalised LD size of lipid droplets for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.",
    "molecules": "BODIPY"
  },
  {
    "caption": "A-D. Fluorescent images of 1 day old retinas, stained with BODIPY (green) and FM-dye (red) to stain lipid droplets and the photoreceptor membranes respectively; (A) wild type; (B) ADAM17-/- mutant; (C) overexpression of lipase in glial cells of ADAM17-/- mutant; (D) overexpression of lipase in neurons of ADAM17-/- mutant. Scale bars: 10μm.",
    "molecules": "BODIPY, FM-dye"
  },
  {
    "caption": "Quantitation of the BODIPY signal shown as (E) LD numbers; (F) normalised LD size for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.",
    "molecules": "BODIPY"
  },
  {
    "caption": "Quantitation of the BODIPY signal shown as (G) integrated total lipid, for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.",
    "molecules": "BODIPY, lipid"
  },
  {
    "caption": "A. Alkaline phosphatase (AP)-shedding assays performed for Eiger in S2R+ cells expressing either GFP, Drosophila ADAM17, or activity dead mutants of Drosophila ADAM17, in the presence of either PMA, or PMA and TAPI-1 (DMSO is used as control; see methods); n=5. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.",
    "molecules": "DMSO, PMA, TAPI-1"
  },
  {
    "caption": "B-C. Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI-1, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively. Scale bars: 10μm.",
    "molecules": "BODIPY, DMSO, FM-dye, TAPI-1"
  },
  {
    "caption": "D. Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI-1 starting from larval stages. n=10 fly retinas. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.",
    "molecules": "DMSO, lipid, TAPI-1"
  },
  {
    "caption": "E-H. Fluorescent images of 1week old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (E) wild type; (F) eiger-/-; (G) grindelwald-/-; (H) wengen-/-. Scale bars: 10μm.",
    "molecules": "BODIPY, FM-dye"
  },
  {
    "caption": "I. Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above. n=10 fly retinas. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.",
    "molecules": "BODIPY, lipid"
  },
  {
    "caption": "A-D. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A, B) wild type; (C, D) ADAM17-/- mutants, reared either on DMSO or AD4. Scale bars: 10μm.",
    "molecules": "BODIPY, DMSO, AD4, FM-dye"
  },
  {
    "caption": "E. Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above; n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.",
    "molecules": "lipid"
  },
  {
    "caption": "F-I. TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17-/- mutants grown on either DMSO or AD4. Scale bars: 10μm.",
    "molecules": "DMSO, AD4"
  },
  {
    "caption": "K-N. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) ADAM17-/- ; (L) glial specific SOD2 over-expression in an ADAM17-/- mutant; (M) glial specific SOD1 over-expression in an ADAM17-/- mutant; (N) neuronal specific SOD2 overexpression in ADAM17-/- mutant. Scale bars: 10μm.",
    "molecules": "BODIPY, FM-dye"
  },
  {
    "caption": "O. Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above, n=10 for each. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01, *p<0.05.",
    "molecules": "BODIPY, lipid"
  },
  {
    "caption": "A-B. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A) wild type; (B) ADAM17-/-, both reared in the dark. Scale bars: 10μm.",
    "molecules": "BODIPY, FM-dye"
  },
  {
    "caption": "G. Quantitation of the BODIPY signal shown as integrated total lipid from 1 day old retinas corresponding to the genotypes described in A-B, all reared in the dark. n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.",
    "molecules": "BODIPY, lipid"
  },
  {
    "caption": "I. Measurement of MDA levels (in nmoles) from 1 day old retinas of wild type or ADAM17-/- , reared either in light or dark. Four biological replicates with 3 technical replicates for each genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.",
    "molecules": "MDA"
  },
  {
    "caption": "J. mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17-/- mutant retinas. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.",
    "molecules": "AD4"
  },
  {
    "caption": "K-L. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) heterozygous mutant of bsk alone; or (L) in combination with ADAM17-/-. Scale bars: 10μm.",
    "molecules": "BODIPY, FM-dye"
  },
  {
    "caption": "M. Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes mentioned in K-L, along with w1118 and ADAM17-/-. n=10 flies per genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.",
    "molecules": "BODIPY, lipid"
  },
  {
    "caption": "A-C. Fluorescent images of human iPSC derived microglia cells, labelled with 493/503 BODIPY (green) and FM-dye (red) to mark lipids and membranes respectively. iPSC derived microglia cells were obtained from 3 different donors, and treated with either DMSO, GW, TAPI-1 or GI. Scale bars: 10μm",
    "molecules": "493/503 BODIPY, DMSO, GI, GW, lipids, FM-dye, TAPI-1"
  },
  {
    "caption": "Quantitation of the BODIPY signal shown as integrated total lipid for cells corresponding to each treatment and donor n=15 cells for each treatment. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.",
    "molecules": "BODIPY, lipid"
  },
  {
    "caption": "Quantitation of the BODIPY signal shown as normalised LD size (normalised to DMSO) for cells corresponding to each treatment and donor mentioned =15 cells for each treatment. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.",
    "molecules": "BODIPY, DMSO"
  },
  {
    "caption": "F-G. Q-PCR analysis of mRNA transcripts of lipogenic genes FASN and LDLR in pooled data obtained from 3 different donors of human iPSC derived microglia treated with either DMSO, GW or GI for 24 hours. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.",
    "molecules": "DMSO, GI, GW"
  },
  {
    "caption": "H-J. Fluorescent images of Mitotracker and MitoSOX labelling of cells from Donor number 2 treated for 24hours with either DMSO, GW or GI. Scale bars: 10μm",
    "molecules": "MitoSOX, DMSO, GI, GW, Mitotracker"
  },
  {
    "caption": "K. Quantitative measurements of MitoSOX intensity normalised to that of Mitotracker in iPSC derived microglia cells from three different donors, treated with either DMSO, GW or GI; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.",
    "molecules": "MitoSOX, DMSO, GI, GW, Mitotracker"
  },
  {
    "caption": "L-N. Fluorescent images of C-11 BODIPY treated microglia cells from Donor no.1, treated for 24 hours with either DMSO, GW or GI. These representative images display relative amounts of peroxidated and non-peroxidated lipids. Scale bars: 10μm",
    "molecules": "C-11 BODIPY, DMSO, GI, GW, lipids"
  },
  {
    "caption": "O. Quantification of BODIPY C-11 staining, shown as ratios of peroxidated versus non-peroxidated lipids, depicted as normalised intensity; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.",
    "molecules": "BODIPY C-11, lipids"
  },
  {
    "caption": "A, B. Under transcription inhibition with Actinomycin D, cells with EXOSC10 knockdown (EXO10 KD) showed more stable expression of the repeat RNA compared to cells with control knockdown with non-targeting siRNA (Ct KD).",
    "molecules": "Actinomycin D"
  },
  {
    "caption": "C, D. Degradation of FAM-labeled synthetic (G4C2)8 repeat RNA with recombinant human EXOSC10 (hEXO10) (Upper left). 3' blocked FAM-labeled G4C2 repeat RNA resisting 3' exoribonuclease activity is not degraded by recombinant EXOSC10 (Lower left). Lack of recombinant EXOSC10 (Upper right) or Mg2+ ion which is essential co-factor of EXOSC10 (Lower right) abolished the RNA degradation activity. 3 or 4 independent experiments. Quantification of the top bands are shown in (D).",
    "molecules": "FAM, Mg2"
  },
  {
    "caption": "A. In situ hybridization. G4C2 RNA foci were detected in a C9 patient-derived fibroblast but not in a control-derived fibroblast with non-targeting siRNA treatment (left panels, control/si Ct. middle panels, C9/si Ct). Knockdown of EXSOC10 increased RNA foci (right panels, C9/siEXOSC10#5). Nuclei were stained with DAPI (blue). White arrows indicate nuclear RNA foci. The green arrows show cytoplasmic RNA foci.",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. ",
    "molecules": "DAPI"
  },
  {
    "caption": "C-F. HA-tagged poly-GA, poly-GP, poly-GR or poly-PR (red) and EXOSC10 (green) were immunostained with anti-HA and anti-EXOSC10 antibodies. Nuclei were stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "G. EXOSC10 (green) were immunostained with anti-EXOSC10 antibody in cells transfected with control mock plasmid. Nuclei were stained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": " (F) Co-immunoprecipitation of PARP1 with endogenous KLF4 is independent on DNA. The DNA binding inhibitor EtBr was added to the MDA-MB-231 cell lyses followed by immunoprecipitation of KLF4 complex. ",
    "molecules": "EtBr"
  },
  {
    "caption": " (J) KLF4 was poly(ADP-ribosyl)ated. KLF4 was immunoprecipitated and detected by anti-PAR and anti-KLF4 antibodies in MDA-MB-231 cells. ",
    "molecules": "PAR"
  },
  {
    "caption": " (K) The PARylation of KLF4 was increased after exposure to DNA damage. KLF4 complex was purified using immunoprecipitation from MDA-MB-231 cells that were treated with 5 µM doxorubicin and were then collected at different times (1h, 2h, 4h and 8h). PARylation was detected by using antibody against PAR. ",
    "molecules": "doxorubicin, PAR"
  },
  {
    "caption": " (M) PARP1 inhibitors decrease KLF4 PARylation. U2OS cells were pretreated with 10 µM various PARP1 inhibitor niraparib, Olaparib and rucaparib for 1hr followed by exposure to10 Gy radiation for 4 hours. KLF4 was pulled down and the PARylation was detected. ",
    "molecules": "niraparib, Olaparib, rucaparib"
  },
  {
    "caption": "(D). Identification of amino acid stretch 411- 441 on the Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T cells.. PARylation for Flag tagged KLF4 deletion mutants were analyzed, respectively, by pulldown using PAR antibody followed by Western blotting using antibody against Flag.",
    "molecules": "PAR"
  },
  {
    "caption": "(B) The effect of YYR motif mutations on KLF4 PARylation. Constructs of KLF4-Zinc 1-YKH/AAA mutation, KLF4-Zinc 2-YYR/AAA mutation and KLF4-Zinc-2-Y451A mutation were co-transfected with Myc-PARP1 into 293T cells, respectively, and then pulled-down using M2 agarose followed by measuring KLF4 PARylation with anti-PAR antibody.",
    "molecules": "PAR"
  },
  {
    "caption": " (C) KLF4-Zinc 2-YYR/AAA mutation has only effect on KLF4 PARylation but not on other types posttranslational modifications such as protein methylation, ubiquitylation, acetylation and sumoylation in 293T cells. The FLAG-tagged wild-type or mutant KLF4 were immunoprecipitated with FLAG and detected by anti-SYM10 (methylation), anti-SUMO (sumoylation), Anti-ac-lysine (acetylation), anti-Ub (ubiquitination) and anti-PAR (PARylation). ",
    "molecules": "ac-lysine, SYM10, PAR, Ub"
  },
  {
    "caption": " Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. While the activation of PARP1 in response to genotoxic stress enhances chromatin recruitment of KLF4 ORC2 were the loading control for chromatid fraction. Ponceau S were the control of nuclear soluble fraction. ",
    "molecules": "Ponceau S"
  },
  {
    "caption": " E) Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. , inhibition of PARP1 by Olaparib attenuates the KLF4 accumulation in chromatin fraction H3 were the loading control for chromatid fraction. PARP1 were the control of nuclear soluble fraction. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " (G). Confocal analysis of PAR and FLAG-tagged KLF4 showing that KLF4-Zinc 2-YYR/AAA mutation significantly reduces KLF4 PARylation in U2OS cells. Scale bars, 10 μm. ",
    "molecules": "PAR"
  },
  {
    "caption": " (I) The effect of PARP1 mutations (H909A or T824A) on KLF4 PARylation, confirming the critical role of H909 in mediating KLF4 PARylation in 293T cells. Myc-PARP1 wildtype or mutations (H909A or T824A) and FLAG-KLF4 were co-transfected into 293T cells and then the FLAG-KLF4 was pulldown by anti-FLAG antibody, the PARylation modification of KLF4 was blotted with anti-PAR antibody, and the binding PARP1 was blotted with anti-myc antibody. ",
    "molecules": "PAR"
  },
  {
    "caption": " (J). Replacement of this histidine by alanine on PARP1 or replacement of the YYR motif of KLF4 by triple alanines leads to the attenuation of KLF4 PARylation. In PARP1-/- MEFs, wild-type or mutant PARP1 (H909A) were co-transfected with FLAG-tagged wild-type or KLF4-Zinc 2-YYR/AAA mutation followed by immunoprecipitation using M2-agarose and Western blotting by anti-PAR antibody. ",
    "molecules": "alanine, alanines, histidine, PAR"
  },
  {
    "caption": " (P) ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA-MB-231 at -.3K positions relative to the TSS in untreated and Olaparib (10µM for 8h) or 5Gy radiation treat cells. No significant difference of KLF4 binds to BRCA1 promoter between untreated and Olaparib or radiation treat cells. n=3, p=0.80, one-way ANOVA assay. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " (A) KLF4+/+, KLF4-/- MEFs, KLF4-/- MEF with wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant were treated with 5-10 µM cisplatin for 48 hours followed by measuring the expression of PARP1, p21 and Bax. Loss of PARylation on KLF4 impairs KLF4-mediated inhibition of apoptosis in the presence of genotoxic stress. ",
    "molecules": "cisplatin"
  },
  {
    "caption": " (B) The apoptotic response in U2OS cells with expression of wild-type and mutant KLF4. U2Os cells with wildtype (pLenti-tet-on-KLF4WT) or mutant (pLenti-tet-on-KLF4YYR/AAA) KLF4 were incubated with 10 ng/ml doxycycline for 24h and then treated with 10uM doxorubicin (Dox) or cisplatin (CDDP) for additional 24h. While expression of wild-type KLF4 decreases Bax expression and inhibits drug-induced PARP1 cleavage, expression of PARylation-deficient KLF4 leads to failure in inhibiting genotoxic-induced Bax expression and PARP1 cleavage. ",
    "molecules": "CDDP, cisplatin, Dox, doxorubicin, doxycycline, tet"
  },
  {
    "caption": "KLF4 sensitizes cell to Olaparib. The asterisk in the panels represents the significant difference (p<0.05). (G) The representative of MEFs clonogenic assay.",
    "molecules": "Olaparib"
  },
  {
    "caption": "KLF4 sensitizes cell to Olaparib. The asterisk in the panels represents the significant difference (p<0.05). (G) The representative of MEFs clonogenic assay. (H) is the summary of G.",
    "molecules": "Olaparib"
  },
  {
    "caption": " (I) Disruption of KLF4 PARylation reduces KLF4 effect on Olaparib efficacy. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " (J-K) While elevated KLF4 inhibits PARP1 inhibitor efficacy in killing MDA-MB-231 cells, disruption of KLF4 PARylation partially rescue the KLF4-mediated resistance to Olaparib. J) The representative of MEFs clonogenic assay. (K) is the summary of J. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " (L) Elevated KLF4 expression increases resistance to Olaparib in MDA-MB-468 (BRCA-proficient) cells, while disruption of KLF4 PARylation attenuates KLF4-driven resistance to Olaparib. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines HCC1937 cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in HCC1937 cells. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines MDA-MB-436 cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in MDA-MB-436 cells. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " Modulating KLF4 by knockdown or overexpression affects cellular response of MDA-MB-231 to Olaparib in clonogenic assay. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " Modulating KLF4 by knockdown or overexpression affects cellular response of MCF7 to Olaparib in clonogenic assay. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Doxorubicin in MDA-MB-231(Q) Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4. ",
    "molecules": "Doxorubicin, Olaparib"
  },
  {
    "caption": " Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Cisplatin in MCF7 Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4. ",
    "molecules": "Cisplatin, Olaparib"
  },
  {
    "caption": " (A) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells (4T1). 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB/c Nude mice. Drug treatment was started at 10th day. Placebo or Olaparib at the dose of 100 mg/kg was administrated daily for twelve days. Tumor volumes were measured every other day. Tumor volumes were measured every other day. n=9 per group, p values were labeled in figure, one-way ANOVA assay. ",
    "molecules": "Olaparib"
  },
  {
    "caption": " The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (F). The image of MDA-MB-231 xenograft tumors ",
    "molecules": "dasatinib, DMSO, ABT-263, Olaparib, PEG400"
  },
  {
    "caption": " The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (G). The tumors were weighed and summarized (H). Placebo (n=7), ABT263 (n=9), Dasatinib (n=8), Olaparib (n=6), Olaparib+ABT263 (n=9), Olaparib+Dasatinib (n=10), p values were labeled in figure, one-way ANOVA assay. ",
    "molecules": "Dasatinib, dasatinib, DMSO, ABT-263, ABT263, Olaparib, PEG400"
  },
  {
    "caption": "(I) Staining of H & E, KLF4, PARP1, p21 and Bax in MDA-MB-231 xenograft tumors. Scale bars, 100 μm. Combination of ABT-263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression, but increase Bax expression in xenograft tumors.",
    "molecules": "dasatinib, ABT-263, Olaparib"
  },
  {
    "caption": "B, CPP score during the pre-test and CPP test. Two-way ANOVA with Sidak's multiple comparisons test. Test of ASE-sdC mice, t = 1.306, p = 0.3586 versus Pre-test of ASE-sdC mice; Test of ACE-sdC mice, t = 10.91, p < 0.0001 versus Pre-test of ACE-sdC mice. ASE-sdC group, n = 18 mice; ACE-sdC group, n = 24 mice. Data information: ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using Two-way ANOVA with Sidak's multiple comparisons test or unpaired t-tests. N.S., p > 0.05, ##, p < 0.01 versus baseline (pre-test CPP score); N.S., p > 0.05, **, p < 0.01 versus ASE-sdC mice.",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "A, The number of c-Fos-positive neurons after CPP test in mPFC and its subregions of Cg1, PrL, IL. The c-Fos (red) and NeuN (green) are used as markers for evaluating cell activity and labeling neuron, respectively. Two-tailed unpaired t test. mPFC, t = 2.89, p = 0.0161; Cg1, t = 2.912, p = 0.0155; PrL, t = 2.311, p = 0.0435; IL, t = 0.8092, p = 0.4372. n = 6 mice per group. Scale bar, 400 μm/100 μm. Data information: Cg1, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex; ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood.",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "B, The correlation of number of mPFC c-Fos-positive neurons with the corresponding ΔCPP score in ACE-sdC mice. mPFC, r = 0.9048, p = 0.0132; Cg1, r = 0.9206, p = 0.0092; PrL, r = 0.8646, p = 0.0262; IL, r = 0.4181, p = 0.4094. n = 6 mice. Data information: Cg1, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex ; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05 versus ASE-sdC mice.",
    "molecules": "cocaine"
  },
  {
    "caption": "E, Quantification of ΔF/F. ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05 versus ASE-sdC mice.",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "A, Volcano plot illustration showed the upregulated, downregulated and unchanged genes in response to cocaine administration in mPFC. Each dot represents one gene. Red, blue and gray dots represent the upregulated, downregulated and not changed genes, respectively.",
    "molecules": "cocaine"
  },
  {
    "caption": "C, Dusp1 mRNA levels on P74. Two-tailed unpaired t test. t = 0.3691, p = 0.7307. n = 3 biological replicates per group. Data information: ASE, adolescent saline-exposed mice during adulthood; ACE, adolescent cocaine-exposed mice during adulthood The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE mice",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "D, DUSP1 protein levels on P74. Two-tailed unpaired t test. t = 1.251, p = 0.2792. n = 3 biological replicates per group. Data information: ASE, adolescent saline-exposed mice during adulthood; ACE, adolescent cocaine-exposed mice during adulthood; The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE mice",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "I, Mean gray value of DUSP1 in CaMKII-positive neurons. Two-tailed unpaired t test. t = 5.751, p = 0.0045. n = 3 mice per group. Scale bar, 400 μm/100 μm. Data information ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE-sdC mice.",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "J, Levels of p-ERK/ERK, p-ERK and ERK proteins on P78. Two-tailed unpaired t test. p-ERK to ERK: t = 3.338, p = 0.0289; p-ERK: t = 3.143, p = 0.0348; ERK1/2: t = 1.030, p = 0.3611. n = 3 biological replicates per group. Data information: ASE-sdC, adolescent saline-exposed mice with subthreshold dose of cocaine treatment during adulthood; ACE-sdC, adolescent cocaine-exposed mice with subthreshold dose of cocaine treatment during adulthood. The data were presented as the mean ± SEM. Statistical analyses were performed using unpaired t-tests. N.S., p > 0.05, *, p < 0.05, **, p < 0.01 versus ASE-sdC mice.",
    "molecules": "cocaine, saline"
  },
  {
    "caption": "F) Bar graphs comparing bacterial burden (CFU) in the lung  and spleen  at 48h, 72h and 7d post-S. pneumoniae infection  in unvaccinated  and BCG vaccinated mice. N = 3 - 5 mice/group. Data information: Data presented in F) show mean ± SEM of biological replicates. Statistical analysis for F) were two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01; ****p < 0.0001.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "B) Dot plots of gated  mononuclear cells from the BAL  of control and  BCG vaccinated mice. Analysis was performed utilizing default FlowJo V.10 software settings. C) Line graphs comparing absolute neutrophil cell counts in the airways at 18 and 36h post-S. pneumoniae infection in  BCG vaccinated  and unvaccinated  hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (C, were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "D) Dot plots of gated mononuclear cells from the airways of control and  BCG vaccinated  hosts. Analysis was performed utilizing default FlowJo V.10 software settings. Line graphs comparing absolute AM cell counts in the airways at 18 and 36h post-S. pneumoniae infection in BCG vaccinated   and unvaccinated  hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "G) Line graphs comparing the levels of neutrophilic  chemokines in the  BAL  fluid at 18 and 36h post-S. pneumoniae infection in BCG vaccinated  and unvaccinated  hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05; ***p < 0.001.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "A) Dot plots of gated mononuclear cells from the lung  of control and  BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. B) Line graphs comparing absolute neutrophil  cell counts in the lung  at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (B, were two-way ANOVA with Sidak's multiple comparisons test.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "C) Dot plots of gated mononuclear cells from the lung  of control and  BCG vaccinated hosts. Analysis was performed utilizing default FlowJo V.10 software settings. D - Line graphs comparing absolute AM, cell counts in the lung  at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "Line graphs comparing absolute IM  and MDM  cell counts in the lung  at 18 and 36h post-S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 - 4 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis were two-way ANOVA with Sidak's multiple comparisons test.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "E) Line graphs comparing changes in body weight  following  S. pneumoniae  infection in  BCG vaccinated and unvaccinated hosts. N = 4 mice/group. Data information: Data presented represent mean ± SEM of technical replicates. Statistical analysis for (E) was two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "G) Bar graphs comparing bacterial counts (CFU) in the lung  and spleen  at 72h post-S. pneumoniae infection  in BCG vaccinated  and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented represent mean ± SEM of technical replicates. Statistical analysis for G) were two-tailed unpaired t-tests, *p < 0.05.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "B) Line graphs comparing changes in  body weight  following S. pneumoniae  infection in BCG vaccinated and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented in (B represent mean ± SEM of biological replicates. Statistical analysis for (B were two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01; ****p < 0.0001.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "C) Bar graphs comparing bacterial counts (CFU) in the lung  and spleen  at 72h post-S. pneumoniae infection  in BCG vaccinated  and unvaccinated hosts. N = 4 - 5 mice/group. Data information: Data presented in C) represent mean ± SEM of biological replicates. Statistical analysis for C) were two-way ANOVA with Sidak's multiple comparisons test, (C) was two-tailed unpaired t-test. *p < 0.05; **p < 0.01; ****p < 0.0001.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "B) Line graphs comparing changes in body weight  following S. pneumoniae infection in BCG vaccinated and unvaccinated hosts. N = 3 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (B was two-way ANOVA with Sidak's multiple comparisons test *p < 0.05; **p < 0.01.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "D) Bar graphs comparing bacterial counts (CFU) in the lung  and spleen  at 48h post- S. pneumoniae infection  in  BCG vaccinated and unvaccinated hosts. N = 3 mice/group. Data information: Data presented represent mean ± SEM of biological replicates. Statistical analysis for (D) was one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05; **p < 0.01.",
    "molecules": "BCG vaccinated"
  },
  {
    "caption": "(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31",
    "molecules": "calcium, chloroquine, histamine"
  },
  {
    "caption": "(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31 (J) and 5HT1F agonist Ly344864 (K).",
    "molecules": "calcium, Ly344864"
  },
  {
    "caption": "(C-F) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour. All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control (AviliDTR) or Sst-Cre::AviliDTRmice. Diphtheria toxin injection in control mice (AviliDTR) had no effect on scratching behavior evoked by histamine (C), chloroquine (D), IL-31 (E) or 5HT1F agonist Ly344864 (F) but significantly reduced responses to IL31, or Ly344864 in Sst-Cre::AviliDTRmice. n-numbers indicated in brackets, asterisk denotes p<0.05, two way RM ANOVA, Holm-Sidak multiple comparison. Error bars indicate SEM.",
    "molecules": "chloroquine, histamine, Ly344864"
  },
  {
    "caption": "(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 97 traces as in (C) fit with a Gaussian model (black curve).",
    "molecules": "ADP•BeFx"
  },
  {
    "caption": "(E) As in (C), but in the presence of ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (257 traces).",
    "molecules": "ATP"
  },
  {
    "caption": "(B) Traces as in (A) obtained in the presence of different nucleotides, were used to determine the number of states best fit by the Markov model. (C) Transition density plot of idealized ATP FRET states obtained in (B).",
    "molecules": "ATP"
  },
  {
    "caption": "(D) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1500 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (E) As in (D), but with high FRET (1656 high FRET states).",
    "molecules": "ATP"
  },
  {
    "caption": "(F) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (G) Distribution of FRET values determined from 168 traces as in (D) fit with a Gaussian model (black curve).",
    "molecules": "ATPγS"
  },
  {
    "caption": "(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 197 traces as in (B) fit with a Gaussian model (black curve).",
    "molecules": "ADP•BeFx"
  },
  {
    "caption": "(E) As in (C), but with ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (228 traces). ",
    "molecules": "ATP"
  },
  {
    "caption": "(A) FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. (B) Transition density plot of idealized ATP FRET states obtained in (A).",
    "molecules": "ATP"
  },
  {
    "caption": "(C) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve).",
    "molecules": "ATPγS"
  },
  {
    "caption": "(E) Comparison of high and low FRET state occupancy in ADP•Pi and ADP•Vi for the clamp and THF.",
    "molecules": "ADP•Pi, ADP•Vi"
  },
  {
    "caption": "(F) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (G) As in (F), but with high FRET (1773 high FRET states).",
    "molecules": "ATP"
  },
  {
    "caption": "Cryo-TEM images demonstrate the lipid bilayer EV purified from Molm-14 and U-937 cells. Scale bars are 100nm.",
    "molecules": "lipid"
  },
  {
    "caption": "Live-cell microscopy of KSL cells (left panel) LT-HSC cells (right panel). Cells were sorted from control mice (top), Molm-14-mGFP (middle) and U-937-mGFP (bottom), stained with Cell Mask (red) and nuclear stain Hoechst (blue); imaged using the GE/API Deltavision (DV) widefield microscope (60X objective) to show the uptake of the GFP+ EV (green). Scale bars are 5μm.",
    "molecules": "Cell Mask, Hoechst"
  },
  {
    "caption": "Cell-cycle histograms of KSL (upper panel) and LT-HSC (lower panel) from Molm-14-xenografts or control using Hoechst-33342 staining.",
    "molecules": "Hoechst-33342"
  },
  {
    "caption": "Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: Molm-14-xenografts, red n=6 vs. non-engrafted controls, black n=6,",
    "molecules": "O-propagyl-puromysin, OPP"
  },
  {
    "caption": "Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,43), AML patients plasma (Orange, n=6), or human CD34+ cells (blue, n=3) normalized to vehicle-injected contralateral femurs after subtracting the background fluorescence.",
    "molecules": "O-propagyl-puromysin, OPP"
  },
  {
    "caption": "Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP, or (G) pS6RP in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR-scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates.",
    "molecules": "OPP"
  },
  {
    "caption": "Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, (J) Cells were nucleofected with miR-Scramble (CTRL) or miR-1246 mimic (n=3) for 72 hours,",
    "molecules": "Hoechst-33342"
  },
  {
    "caption": "Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, Cells were nucleofected with Anti-miR-Scramble (CTRL) or Anti-miR-1246 (n=5) and 1-hour later co-treatment with Molm-14 EVs for 72 hours.",
    "molecules": "Hoechst-33342"
  },
  {
    "caption": "CFU-C assay showing the relative number of colonies (left panel) and the colony subtypes (right panel) from KSL cells derived from Molm-14-xenografted mice (red, n=6) vs. non-engrafted control mice (black, n=5) and in vitro cultured for 7 days in methylcellulose media. Experiments were performed in technical triplicates. "G" Granulocyte, "M" Monocyte, "E" Erythrocytes.",
    "molecules": "methylcellulose"
  },
  {
    "caption": "LT-HSC assessment after repopulation, recipient female mice (n=4 per group) were sacrificed after 21 days and their LT-HSC were compared using P53, pS6RP and OPP flow cytometric assays. (D-E) Evaluation of the in vivo long-term repopulation capacity.",
    "molecules": "OPP"
  },
  {
    "caption": "DNA-damage assessment in KSL from primary recipients and after two turns of replating. (D, F, H) Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: (D) Primary recipient KSL, n=211 and 321, All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W1 plating, n=218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software. Scale bars are 2μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W2 plating, n=770 and 853. All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.",
    "molecules": "Hoechst"
  },
  {
    "caption": "E. Representative immunofluorescence  images of HT29  and HCT116  cells stained  for endogenous PCIF1 protein (green). Nuclei  were stained  with DAPI  (blue). Scale bar, 30 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. LC-MS/MS  identification and quantification of m6Am  or m6A  levels in mRNA from control and PCIF1-depleted HCT116  cells. Data are presented as the enrichment of methylated  vs unmethylated mRNA. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.",
    "molecules": "m6Am, m6A"
  },
  {
    "caption": "B. Metagene plots analysis of   m6Am enrichment around the transcription start site (TSS) of all expressed genes in control and PCIF1-depleted HCT116  cells.",
    "molecules": "m6Am"
  },
  {
    "caption": "F. Genome browser views of FOS   with m6Am  sites. Read coverage of input sample and IP  sample are shown in blue and red, respectively, and green rectangle indicates the m6Am  peaks located near the TSS.",
    "molecules": "m6Am"
  },
  {
    "caption": "G.  m6Am-exo-qPCR  analysis of m6Am enrichment in FOS  mRNAs from the indicated HCT116  cells. GAPDH  served as a negative control. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.",
    "molecules": "m6Am"
  },
  {
    "caption": "H. qRT-PCR analysis of FOS  mRNA stability in control and PCIF1-depleted HCT116  and HT29  cells treated with actinomycin D for the indicated times. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01 by Student's t-test.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "A. Confocal images show Iba1-immunoreactive microglia and macrophages in RPE/choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Laser spots of vehicle-injected SIGLEC11 transgenic (tg) and wild type control animals revealed strong accumulation of activated microglia and macrophages, which was effectively decreased in polySia avDP20-injected SIGLEC11 tg animals and to a lower extent in wild type controls. Representative images out of at least three independent experiments are shown. Scale bar: 50µm.B. Quantification of the average pixel intensity of Iba1-positive area in laser spots reflects accumulation of reactive microglia/macrophages on RPE/choroid whole mounts. In comparison to the accumulation of reactive microglia/macrophages in control animals, there is a reduced pixel intensity found in polySia avDP20-treated SIGLEC11 tg animals. Notably, treatment with a high polySia avDP20 dose (3 µg) also reduced accumulation of microglia/macrophages in laser spots of wild type animals. Data show mean +/- SD. * p< 0.05, ** p< 0.01, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 24 spots), WT 0.2 µg Sia (n = 37 spots), WT 3 µg Sia (n = 20 spots), SIGLEC11 tg PBS (n = 25 spots), SIGLEC11 tg 0.2 µg Sia (n = 35 spots), SIGLEC11 tg 3 µg Sia (n = 31 spots), WT PBS vs WT 3 µg Sia p = 0.0147, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0046, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0071, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0.0001.",
    "molecules": "polySia avDP20"
  },
  {
    "caption": "C. Confocal images of retinal whole mounts show Iba1-immunoreactive microglial cells. Retinal microglia of polySia avDP20-treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS-vehicle treated mice. Interestingly, high polySia avDP20 dose (3 µg) also exerted a weak therapeutic effect in wild type animals. Representative images out of at least three independent experiments are shown. Scale bar: 20 µm.D. Percentage of retina showing activated microglial cells within the laser spots was quantified. PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose (3 µg) also in wild type controls. Data show mean +/- SEM. * p< 0.05, ** p< 0.01, ***p<0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 6 retinas), WT 0.2 µg Sia (n = 8 retinas), WT 3 µg Sia (n = 6 retinas), SIGLEC11 tg PBS (n = 8 retinas), SIGLEC11 tg 0.2 µg Sia (n = 7 retinas), SIGLEC11 tg 3 µg Sia (n = 6 retinas),WT PBS vs WT 3 µg Sia p = 0.0052, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0005, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0159, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.001.",
    "molecules": "polySia avDP20"
  },
  {
    "caption": "A. Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Late stage (10-11 minutes after fluorescein injection) fundus fluorescein angiography revealed that PBS-treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20-treated SIGLEC11 transgenic (tg) mice had lower levels of vessel leakage compared to PBS-injected wild type or SIGLEC11 tg mice. High polySia avDP20 dose reduced vascular leakage also in wild type animals. Representative images out of at least eight independent experiments are shown.B. Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software (NIH). Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted. PolySia avDP20-treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS-injected wild type control and SIGLEC11 tg mice. A reduction in vascular leakage was also observed in high dose polySia avDP20-injected wild type control mice. Data show mean +/- SD. * p< 0.05, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 20 eyes), WT 0.2 µg Sia (n = 17 eyes), WT 3 µg Sia (n = 9 eyes), SIGLEC11 tg PBS (n = 16 eyes), SIGLEC11 tg 0.2 µg Sia (n = 20 eyes), SIGLEC11 tg 3 µg Sia (n = 15 eyes), WT PBS vs WT 3 µg Sia p = 0.0193, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.0002.",
    "molecules": "fluorescein, polySia avDP20"
  },
  {
    "caption": "C. Anti-C5b-9 immunostaining of RPE/choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle-injected controls. PolySia avDP20 treatment reduced MAC formation in a dose-dependent fashion independent of SIGLEC11 presence. Scale bar: 100 µm.D. Quantification of C5b-9 fluorescence signal intensity in the laser lesions. In comparison to the high amount of MAC deposition in vehicle-injected controls, the pixel intensity is reduced in polySia avDP20-treated eyes in a dose-dependent fashion and regardless of SIGLEC11 presence. Data show mean +/- SD. *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 12 laser spots), WT 0.2 µg Sia (n = 12 laser spots), WT 3 µg Sia (n = 7 laser spots), SIGLEC11 tg PBS (n = 6 laser spots), SIGLEC11 tg 0.2 µg Sia (n = 10 laser spots), SIGLEC11 tg 3 µg Sia (n = 12 laser spots); all statistical comparisons p < 0.0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0.2 µg Sia p = 0.0004.",
    "molecules": "polySia avDP20, PolySia avDP20"
  },
  {
    "caption": "A. Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. The levels of gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=7 and p< 0.0001, PolySia avDP20 1.5 µM n=4 and p=0.0002, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=0.009, LPS/PolySia avDP20 1.5µM n=5 and p=0.002. Siglec11/16 KO: no treatment n=5 and p=0.01, PolySia avDP20 n=4 and p=0.122, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.",
    "molecules": "LPS, polySia avDP20, PolySia avDP20"
  },
  {
    "caption": "B. Analysis of relative TNFSF2 protein release in human control and SIGLEC11/16 knockout THP1-macrophages. The released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=8 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=0.002, LPS/PolySia avDP20 1.5µM n=4 and p=0.0003. Siglec11/16 KO: no treatment n=6 and p<0.001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=1.0, LPS/PolySia avDP20 1.5µM n=5 and p=1.0.",
    "molecules": "LPS, polySia avDP20, PolySia avDP20"
  },
  {
    "caption": "C. Analysis of relative VEGFA gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. Gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout macrophages. Data show mean +/- SEM. *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=6 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.0001, LPS n=5, LPS/PolySia avDP20 0.15µM n=5 and p=0.0002, LPS/PolySia avDP20 1.5µM n=5 and p<0.0001. Siglec11/16 KO: no treatment n=5 and p=0.022, PolySia avDP20 n=3 and p=0.063, LPS n=4, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.",
    "molecules": "LPS, polySia avDP20, PolySia avDP20"
  },
  {
    "caption": "D. Analysis of relative VEGFA protein release in human control and SIGLEC11/16 knockout THP1-macrophages. Released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human macrophages. Data show mean +/- SEM. * p < 0.05, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=9 and p<0.0001, PolySia avDP20 1.5 µM n=6 and p=0.009, LPS n=9, LPS/PolySia avDP20 0.15µM n=6 and p=0.043, LPS/PolySia avDP20 1.5µM n=7 and p=0.0001. Siglec11/16 KO: no treatment n=9 and p=0.349, PolySia avDP20 1.5 µM n= 6 and p=0.249, LPS n=7, LPS/PolySia avDP20 0.15µM n=6 and p=1.0, LPS/PolySia avDP20 1.5µM n=7 and p=1.0.",
    "molecules": "LPS, polySia avDP20, PolySia avDP20"
  },
  {
    "caption": "E. Quantification of human control and SIGLEC11/16 knockout THP1-macrophages having ingested cellular debris. PolySia avDP20 (1.5 µM) reduced the percentage of phagocytic cells having ingested drusen-like debris. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. *p ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0.028, debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0.00012.",
    "molecules": "PolySia avDP20, polySia avDP20"
  },
  {
    "caption": "F. Prevention of superoxide release in activated human control and SIGLEC11/16 knockout THP1-macrophages by polySia avDP20. Cultured human THP1-macrophages were stimulated with RPE cell debris or co-stimulated with debris and polySia avDP20. Addition of debris stimulated the production of superoxide. 1.5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. p* ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Untreated WT macrophages vs debris treated WT macrophages p = 0.037, debris treated WT macrophage vs debris plus polySia avDP20 1.5 µM treated WT macrophages p < 0.001, debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0.001,",
    "molecules": "polySia avDP20, superoxide"
  },
  {
    "caption": "G. PolySia avDP20 (0.15 to 50 µM) was added to normal human serum (NHS) to evaluate any interference with activation of the classical complement pathway by IgM. Complement activation was determined by C3b deposition. PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway. Data show mean +/- SEM. n = 3, n.s.= not significant.",
    "molecules": "PolySia avDP20"
  },
  {
    "caption": "H. PolySia avDP20 inhibits the alternative complement pathway. PolySia avDP20 (0.15 to 50 µM) was added to NHS and activation of the alternative pathway was induced by LPS. C3b deposition was monitored by ELISA. Data show mean +/- SEM (n = 4. * p < 0.05, ** p < 0.01, p***<0.001, ANOVA followed by Bonferroni).",
    "molecules": "LPS, PolySia avDP20"
  },
  {
    "caption": "Imaging flow cytometry analysis on Zscan4+ siArg2 and Zscan4+ siCtrl cells. Samples were stained by labelling histone H3 protein. The intensity of H3 fluorescent signal was measured in the nuclear region by creating a mask defined by DAPI staining. The texture feature of fluorescent signal was analysed by using the modulation method (for details see Materials and Methods). Two cell populations (R1 and R2) correspond to two different patterns of fluorescent signal: homogenous and clustered distribution, respectively.",
    "molecules": "DAPI"
  },
  {
    "caption": "representative SeaHorse time courses of OCR and ECAR respectively; where are indicated the following compounds added: oligomycin (olig.), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone plus antimycin A (R/A), glucose (Glu), 2-deoxyglucose (2DG). Each point in the OCR and ECAR time-courses is the average ± S.D. of four technical replicates.",
    "molecules": "2-deoxyglucose, 2DG, antimycin A, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, FCCP, Glu, glucose, olig, oligomycin, rotenone"
  },
  {
    "caption": "OCR/ECAR ratio; (4 technical replicates each); Basal, (resting OCR)/(ECAR +Glu); Max, (OCR+FCCP)/(ECAR+Olig.)",
    "molecules": "FCCP, Glu, Olig"
  },
  {
    "caption": "Cellular NAD+ and NADH content and NAD+/NADH.",
    "molecules": "NAD, NADH"
  },
  {
    "caption": "Confocal microscopy analysis of the mitochondrial membrane potential DYm. Pictures on the right, representative imaging of cells loaded with tetramethylrhodamine, ethyl ester (TMRE); histogram on the left, averaged fluorescence intensity/cell ± SD of n=3 independent biological replicates; *, P < 0.05.",
    "molecules": "tetramethylrhodamine, ethyl ester, TMRE"
  },
  {
    "caption": "Representative confocal microscopy analysis of cells loaded with MitoTracker Green (pictures on the left); digital enlargements and false colors rendering of details shown as white contoured squares (pictures on the right).",
    "molecules": "MitoTracker Green"
  },
  {
    "caption": "Representative confocal microscopy analysis of cells loaded with dichlorofluorescein-diacetate converted in dichlorofluorescein (DCF) (pictures on the left); digital enlargements and false-color rendering of details shown as white contoured squares (pictures on the right); histogram on the left, averaged fluorescence intensity/cell ± SEM of n=3 independent biological replicates.",
    "molecules": "DCF, dichlorofluorescein, dichlorofluorescein-diacetate"
  },
  {
    "caption": "Basal R/A-sensitive OCR measured as in Fig. 2A in the absence and in the presence of a cocktail of inhibitors (BPTES + etomoxir + UK5099); End. Sub., OCR measured with endogenous substrates; Exo. Sub., OCR measured with glutamine (Gln), fatty acids (FA) and glucose (Glc) supplemented in the assay medium. The superimposed bars are means ± SEM of n=3 independent biological replicates (3 technical replicates each);",
    "molecules": "BPTES, etomoxir, FA, fatty acids, Glc, glucose, Gln, glutamine, UK5099"
  },
  {
    "caption": "Measurement of dependency and flexibility of the OCR to either of Gln, FA, Glc; the assay was carried out according to the MitoFuel protocol (see Material and Methods); the superimposed bars (whose sum corresponds to the capacity) are means ± SEM of n=3 independent biological replicates (3 technical replicates each);",
    "molecules": "FA, Glc, Gln"
  },
  {
    "caption": "LC-MSMS analysis of the cellular content of free aminoacids; the symbols of aminoacids are indicated in x-axis; the amount expressed as μmol/μg protein is indicated in y-axis. The inset shows the content of ornithine, citrulline and arginine.",
    "molecules": "arginine, citrulline, ornithine"
  },
  {
    "caption": "(D)LC-MSMS analysis of the cellular content of acylcarnitines; the length of the acyl moiety is indicated by the number of carbon atoms in abscissa; bars values are means ± SEM of n=3 independent biological replicates; The inset shows the content of carnitine (C0) and acetylcarnitine (C2).",
    "molecules": "carbon, carnitine, acetylcarnitine, acylcarnitines"
  },
  {
    "caption": "C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind epitopes of antigenic site II (Mota and 47F) or antigenic site IV (101F). mAs specific of hMPV F bind epitopes of antigenic site II (MF9, MF12, MF14 and MF15) or antigenic site IV (MF16).",
    "molecules": "Mota"
  },
  {
    "caption": "Binding of Mz and 47F Fabs to the immobilized proteins indicated at left. Sensorgrams of eight different concentrations are shown in each panel. The calculated binding parameters are shown in the Table. RUs, response units.",
    "molecules": "Mz"
  },
  {
    "caption": "C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind to epitopes of antigenic site Ø (D25), antigenic site II (Mz and 47F) or antigenic site IV (101F and 107F). mAs specific of hMPV F bind to epitopes of antigenic site II (MF14), antigenic site IV (MF11, MF16 and MF29) or an epitope shared by hRSV F and hMPV F (MPE8).",
    "molecules": "Mz"
  },
  {
    "caption": "(F and G) WT and TEX264-KO HeLa  cells (with or without the indicated TEX264  mutants with a C-terminal FLAG  tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline  for 24 h to induce the reporter. After doxycycline  was removed, cells were cultured in  starvation  medium  lacking amino acids and serum   for 9 h (F). The band intensities of RFP  and RFP-GFP  were quantified and the ratio of RFP:RFP-GFP  (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A and B) MEFs stably expressing TEX264-GFP  were cultured in  starvation  media  with or without  bafilomycin A1 (Baf A1) and immunostained  with anti-pTEX264 and anti-LC3  antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264  puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).",
    "molecules": "Baf A1, bafilomycin A1"
  },
  {
    "caption": "(C) HeLa  cells were treated with 250 nM  Torin 1  and  Baf A1  for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.",
    "molecules": "Baf A1, Torin 1"
  },
  {
    "caption": "(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa  cells were cultured in  starvation  media  lacking  amino acids and serum  with or without Baf A1  for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264  to HSP90  and total TEX264  during starvation   are shown (E and F). Data represent the mean ± SEM of three independent experiments.",
    "molecules": "Baf A1"
  },
  {
    "caption": "(A) HeLa  cells were cultured with 20 nM CK1  inhibitor (D4476) or CK2  inhibitor (CX4945) for 3 h, followed by starvation  (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting  using the indicated antibodies.",
    "molecules": "D4476, CX4945"
  },
  {
    "caption": "(B) Recombinant TEX264-FLAG  was incubated with or without CK2  and the CK2  inhibitor (CX4945) in the presence of 200 µM ATP  for 1 h.",
    "molecules": "ATP, CX4945"
  },
  {
    "caption": "(D and E) HEK293T  cells transiently expressing WT or TEX264-FLAG  mutants were subjected to immunoprecipitation  (IP). Inputs (20% of the lysates) and immunoprecipitates  (from 80% of the lysates) were analyzed by immunoblotting  using the indicated antibodies (D). The band intensities in the IP  fractions were quantified, and the phospho-serine:FLAG  ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).",
    "molecules": "serine"
  },
  {
    "caption": "(H and I) WT HeLa  cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline  was removed, cells were cultured with 20 nM CK2  inhibitor for 3 h followed by starvation  (with the CK2  inhibitor) for 6 h (H). The band intensities of RFP  and RFP-GFP  were quantified and the ratio of RFP:RFP-GFP  (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG  mutants) HeLa  cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline  for 24 h to induce the reporter. After doxycycline  was removed, the cells were cultured in starvation  medium lacking amino acids and serum  for 9 h (E). The band intensities of RFP and RFP-GFP  were quantified and the ratio of RFP:RFP-GFP  (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).",
    "molecules": "doxycycline"
  },
  {
    "caption": " A Whole mount FISH for piwi-1 and immunostaining for BrdU at 1-day, 1-week and 5-weeks post-BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm. ",
    "molecules": "BrdU"
  },
  {
    "caption": " B Quantification of the percentage of BrdU label-retaining neoblasts. Data are presented as mean ± s.d. (n = 4-6 animals per time point). ",
    "molecules": "BrdU"
  },
  {
    "caption": " C Double pulse experiment showing colocalization of BrdU after a 2-week chase period with EdU after a 1-day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm. ",
    "molecules": "BrdU, EdU"
  },
  {
    "caption": " A Dissociated cells from unirradiated, 1 dpi or 7 dpi animals (60 Gy of irradiation) were stained with Hoechst (DNA) and Pyronin Y (RNA) and sorted by FACS to reveal an RNAlow population. Percentages indicate the proportion of events within each gate. ",
    "molecules": "Hoechst, DNA, Pyronin Y"
  },
  {
    "caption": " B Cell size and nucleus:cytoplasm ratio measured by CellMask (cytoplasm) and DAPI (nucleus) staining of FACS sorted cells at homeostasis (n ≥ 47 per sample). Representative images are shown, with dashed lines to indicate the area measured. Scale bars, 5 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": " C EdU pulse-chase time course of label-retention in sorted RNAlow and S/G2/M neoblasts (n ≥ 3 individual experiments per time point). Red and blue p values indicate the statistical significance of EdU dilution from 2 dpp to 14 dpp in S/G2/M or RNAlow neoblasts, respectively. Black p values indicate the statistical significance of EdU retention in RNAlow neoblasts compared to S/G2/M neoblasts. ",
    "molecules": "EdU"
  },
  {
    "caption": " D Maximum projection confocal images showing examples of cells from the beginning and end of the EdU label retention timecourse in C. Red boxes denote examples of EdU- cells. Scale bars, 5 µm. ",
    "molecules": "EdU"
  },
  {
    "caption": " A Representative images of RNAlow, G1 and S/G2/M cells stained with CellMask and DAPI in intact animals and at 6 and 48 hpa. All images shown are from samples processed in the same experiment. Intact images are duplicated from Figure 2B. White arrows mark projections. Percentage of cells displaying projections in each condition is noted (n > 100 cells counted). Scale bars, 5 µm. B Average projection length in RNAlow, G1 and S/G2/M cells from intact animals and at 6 and 48 hpa (n ≥ 13). C-E Quantification of the number of projections per cell (blue data) and the sum total projection length per cell (orange data) in RNAlow (C), G1 (D) or S/G2/M (E) cells isolated from intact animals and at 6 and 48 hpa (n ≥ 23 for projections/cell, n ≥ 10 for Σ projection length/cell). ",
    "molecules": "DAPI"
  },
  {
    "caption": " B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment. ",
    "molecules": "Hoechst, NDZ, Pyronin Y"
  },
  {
    "caption": " F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate. ",
    "molecules": "Hoechst, nocodazole, Pyronin Y"
  },
  {
    "caption": "Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B).",
    "molecules": "His, Leu"
  },
  {
    "caption": "(D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.",
    "molecules": "3MA, His, Leu, Rap, rapamycin"
  },
  {
    "caption": "Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least).",
    "molecules": "Gly, 3MA, His, Leu, rapamycin, Val"
  },
  {
    "caption": "Immunolocalization of Aβ in AVs from L/APP cells and PS1/APPbrains and γ-secretase components (PS1 and nicastrin) in L/APP cells. Immunogold localization of Aβ40 (A), Aβ42 (B), PS1 (C), nicastrin (D), and in the absence of primary antibody (E) in L/APP cells grown for 6 h in the absence of Leu and His. (F) Quantification of gold particle frequency in AV or tubulovesicular compartments (TBV), which comprise 27.0 ± 11.0 and 19.2 ± 7.2%, respectively, of the total cell area. Error bars represent SEM.",
    "molecules": "His, Leu"
  },
  {
    "caption": "(C) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L/APP cells grown in −serum media (left) or −Leu/−His media (right). PNP, postnuclear pellet.",
    "molecules": "Leu, His"
  },
  {
    "caption": "(D and E) Proportions of the total recovered cell γ-secretase activity in different subcellular fractions after serum deprivation (D) or in uninduced (+serum) or induced (−Leu/−His) cells (E). Comparison of +serum versus −Leu and −His conditions shows the redistribution of γ-secretase activity from the tubulovesicular to AV fractions after macroautophagic induction. G, Golgi; A, AV.",
    "molecules": "His, Leu"
  },
  {
    "caption": "Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. A) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against alpha-tubulin. B) Immunofluorescence images of mitotic cells ­­­with CENP‑T∆C‑dCas9‑EGFP targeting after nocodazole treatment. C) Length measurements of individual EGFP foci in µm. Example measurements are indicated on zooms in A-B) in orange. Data is ≥20 cells per condition, from 1 experiment. Lines = Mean ± SEM. * = p<0.05, ** = p<0.01,**** = p<0.001 (Kruskal-Wallis with multiple comparison correction). Images in A,B are maximum intensity projections taken across the depth of the EGFP foci. In A,B) Orange bars = measured lengths in C.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).",
    "molecules": "PA"
  },
  {
    "caption": "(b) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulphate (SDS) and analysed by immunoblotting with indicated antibodies.",
    "molecules": "SDS, sodium dodecyl sulphate, Triton X-100, TX100"
  },
  {
    "caption": "(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).",
    "molecules": "PA"
  },
  {
    "caption": "(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).",
    "molecules": "PA, Rap, rapamycin"
  },
  {
    "caption": "(g,h) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 h and examined under a live confocal microscope (g). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified (h; n=7). Scale bar, 5 μm. All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.",
    "molecules": "PA, Rap"
  },
  {
    "caption": "HepG2 cells were treated with bovine serum albumin (Con or (−)), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 9 h. (a-d) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM (a,b) or Fura-2-AM (c,d). Calcium levels were visualized by laser confocal microscopy (a) or by dual fluorescent microscopy (c, 340/380 nm ratio image) and quantified (b,d; n=8 and 30, respectively).",
    "molecules": "Calcium, PA, Nic, nicardipine, Ver, verapamil"
  },
  {
    "caption": "(e-h) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analysed by immunoblotting (e,f) and quantified (g,h; n=3).",
    "molecules": "SDS, Triton X-100"
  },
  {
    "caption": "(i-l) Cells with indicated treatments were subjected to immunostaining with indicated antibodies (i,k). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified (j; n=10). Co-localization between LAMP1 and LC3 was quantified (l; n=4). Boxed areas in fluorescence images are magnified in right-most panels (i,k). Scale bar, 10 μm (a), 20 μm (c), 5 μm (i,k). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.",
    "molecules": "DNA"
  },
  {
    "caption": "Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet (LFD)-kept mice (n=5) of same age were used as a negative control. (a) Body weight was daily monitored during injection period. (b) Daily food consumption was measured during injection period.",
    "molecules": "food, Ver, verapamil"
  },
  {
    "caption": "(k,l) Calcium-induced CaMKII autophosphorylation in livers was analysed by immunoblotting (k) and quantified (l).",
    "molecules": "Calcium"
  },
  {
    "caption": "(m) Primary hepatocytes from 2-month-old C57BL/6 mice kept on LFD were treated with bovine serum albumin (Con), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 12 h. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified (n=17). Scale bar, 1 cm (c), 200 μm (i). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.",
    "molecules": "Calcium, PA, Nic, nicardipine, Ver, verapamil"
  },
  {
    "caption": "Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a-d) Livers were subjected to solubility fractionation. 1% Triton X-100-soluble (a) and -insoluble (b) fractions were analysed by immunoblotting (a,b) and quantified (c,d).",
    "molecules": "Triton X-100, Ver, verapamil"
  },
  {
    "caption": "Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a,b) Levels of LC3-II from 1% Triton X-100-insoluble fraction of livers were analysed by immunoblotting (a) and quantified (b).",
    "molecules": "Triton X-100, Ver, verapamil"
  },
  {
    "caption": "Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice of same age (n=5) were used as a negative control. (a,b) Liver sections were subjected to F4/80 immunostaining, which visualizes macrophage infiltration and hematoxylin counterstaining (a). F4/80-positive areas were quantified (b).",
    "molecules": "Ver, verapamil"
  },
  {
    "caption": "(g) Serum insulin levels were measured from indicated mice before (Basal) and 10 min after (Glucose stimulated) glucose injection (n=4).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "(a-g) Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=4) for 10 days. Systolic blood pressure (a), LV wall mass (b), diastolic LV volume (c), systolic LV volume (d), ejection fraction (e, % EF), stroke volume (f, SV) and cardiac output (g, CO) were analysed by tail-cuff method (a) or echocardiography (b-g). All data are shown as mean±s.e.m. *P0.05 (Student's t-test).",
    "molecules": "Ver, verapamil"
  },
  {
    "caption": "(D, E) Extra-mitochondrial  puncta formation of CHCHD2T61I in Neuro2a  cells. Neuro2a  cells were transfected with the CHCHD2WT-HA  and CHCHD2T61I-HA  plasmids for 4 hr, then cultured with medium containing 2% FBS  and 10 µM retinoic acid for neuronal  differentiation. At 28 hr after transfection, cells were fixed and stained  with anti-HA  and anti-Tom20  antibodies. In (D), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Representative images at lower magnification are shown (E) Quantification of cells displaying mitochondrial CHCHD2   and  extra-mitochondrial CHCHD2   puncta (n ≥ 100 cells in each experiment). In E, data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01",
    "molecules": "retinoic acid"
  },
  {
    "caption": "Neuro2a   cells were transfected with the CHCHD2WT-HA  and  CHCHD2T61I-HA  plasmids for 4 hr, and then cultured in medium containing 2% FBS  and 10 µM retinoic acid to induce neuronal  differentiation. At 28 hr after transfection, cells were fixed and stained  with an anti-HA  antibody and ProteoStat  protein aggregation dye (A, B), anti-HA  and anti-Nefl  antibodies (C, D), anti-HA and anti-p-Nefl473 antibodies (E, F), In (A, C, E, representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized  puncta between CHCHD2T61I-HA  and aggresomes  or the indicated proteins. Representative images at lower magnification are shown (B, D, F, The amount of aggresomes (B), Nefl (D), p-Nefl473 (F) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In (B, D, F, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01",
    "molecules": "ProteoStat, retinoic acid"
  },
  {
    "caption": "Neuro2a  cells were transfected with the CHCHD2WT-HA  and CHCHD2T61I-HA  plasmids for 4 hr, and then cultured in medium containing 2% FBS  and 10 µM retinoic acid to induce neuronal  differentiation. At 28 hr after transfection, cells were fixed and stained  with anti-HA  and anti-p-α-Synuclein129 (p-α-Syn129) antibodies (G, H). In G), representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized  puncta between CHCHD2T61I-HA  and the indicated proteins. Representative images at lower magnification are shown H) The amount of p-α-Syn129 (H) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In H, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01",
    "molecules": "retinoic acid"
  },
  {
    "caption": "(D-I) Neuro2a  cells were transfected with the CHCHD2T61I-HA  plasmid together with the indicated siRNAs, and were differentiated into neuronal  cells after 4 hr. At 28 hr after transfection, cells were fixed and stained  with anti-HA, anti-p-Nefl473, and anti-p-α-Syn129 antibodies, or ProteoStat aggresome  detection dye. Similar experiments were performed by the addition of PF-670462  (10 µM) after 4 hr of transfection instead of gene silencing. In (D, F, H), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Arrowheads indicate colocalized  puncta between CHCHD2T61I-HA  and the indicated phosphorylated  proteins or aggresomes. In (E, G, I), the amount of p-Nefl473 (E), p-α-Syn129 (G), and aggresomes (I) was measured as the fluorescence intensity per cell (n = 30 cells in each experiment). Red bars indicate mean values. In E, G, I), comparisons were performed using one-way ANOVA followed by the Tukey post-hoc tests or unpaired two-tailed Student t-test. *p < 0.05; **p < 0.01. NS: not significant",
    "molecules": "ProteoStat, PF-670462"
  },
  {
    "caption": "Reduction in TH  signals in the SNpc  of knock-in mice. In (F), cryosections of the SNpc  were immunostained  with anti-Chchd2  and anti-TH  antibodies. Arrowheads indicate Chchd2T61I puncta in TH-positive cells. Dashed lines indicate cell shapes. (G-K) Extra-mitochondrial aggresome  formation by p-Nefl473, p-α-Syn129, and Csnk1e/d in the SNpc  of knock-in mice. Cryosections of the SNpc  were immunostained  with anti-Chchd2  and anti-Ant1/2 (G), anti-p-Nefl473 (H), anti-p-α-Syn129 (I), and anti-Csnk1e/d antibodies (J), and with ProteoStat  dye (K). Ant1/2 are mitochondrial membrane proteins and arrowheads indicate extra-mitochondrial Chchd2T61I (G). In (H-K), arrowheads indicate the colocalization  of puncta with  Chchd2T61I and the indicated proteins or aggresomes. Dashed lines indicate cell shapes.",
    "molecules": "ProteoStat"
  },
  {
    "caption": "(F-J) The same experiments as Fig. 5G-K were performed using PF-670462-infused and vehicle-infused Chchd2T61I knock-in mice.",
    "molecules": "PF-670462"
  },
  {
    "caption": "CHCHD2T61I iPSC-derived DA neurons were treated with  PF-670462 (10 µM) for 20 hr and analyzed by immunofluorescence  using anti-CHCHD2  and anti-p-α-SYN129 antibodies (G), and ProteoStat aggresome  dye (H). Arrowheads indicate puncta showing colocalization  of CHCHD2T61I with p-α-SYN129 G) or aggresomes  H). Dashed lines indicate cell shapes.",
    "molecules": "ProteoStat, PF-670462"
  },
  {
    "caption": "F. Akt phosphorylated WT (lane 10), 1-898 (lane 12) and 675-1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675-1065, ∆727-896, 1-857, or 1-822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.",
    "molecules": "amino acid"
  },
  {
    "caption": "A. PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), but not 3A INVS (top panel, lane 6). PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively).",
    "molecules": "GSK690693, LY294002, MK2206"
  },
  {
    "caption": "(A) MEFs were transfected with GFP-tagged wild-type (WT) or mutant Parkin expression plasmid followed by an 18-h treatment of CCCP. Cells are immunostained with a Tom20 antibody to visualize mitochondria (red). GFP-parkin-transfected cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of mitochondria-free cells from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) MEFs were transfected and treated with CCCP as described in A, followed by an immunoblotting analysis with antibodies for cytochrome c, actin, and parkin. Note that levels of parkin R275W and R42P mutant were lower, as previously reported (Wang et al., 2005).",
    "molecules": "CCCP"
  },
  {
    "caption": "CCCP-induced parkin-mitochondrial aggregate formation is an intermediate step for mitophagy. (A) MEFs expressing WT GFP-parkin were treated with CCCP or CCCP and nocodazole (NOC, 10 µM) for 8, 16, and 24 h as indicated. Cells were immunostained with anti-Tom20 to visualize mitochondria. GFP-parkin-positive cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 25 µm. (B) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01.",
    "molecules": "CCCP, NOC, nocodazole"
  },
  {
    "caption": "(A) MEFs were transfected with GFP wild-type (WT), A240R, and T415N mutant parkin followed by CCCP treatment for 8 h. Cells were immunostained with antibodies for cytochrome c (red) and ubiquitin (blue). Arrows indicate mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of ubiquitin-positive mitochondrial aggregates from three independent experiments from A are presented with standard deviation as error bar.",
    "molecules": "CCCP"
  },
  {
    "caption": "(C and D) MEFs were transfected and treated with CCCP as described in A. Cells were subjected to the fractionation to isolate crude mitochondria. Cytoplasmic and mitochondrial fractions were subjected to immunoblotting analysis using antibodies for ubiquitin, parkin, cytochrome c, and cytosolic Hsp90.",
    "molecules": "CCCP"
  },
  {
    "caption": "Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar.",
    "molecules": "CCCP"
  },
  {
    "caption": "Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar.",
    "molecules": "CCCP"
  },
  {
    "caption": "(E) Wild-type and HDAC6 knockout (KO) MEFs were transfected with parkin-GFP or cotransfected with a Flag-tagged HDAC6 followed by CCCP treatment for 16 h as indicated. Cells are immunostained with Tom20 (red) and Flag (blue) antibodies. Bar, 25 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(F) Wild-type MEFs were transfected with control or HDAC6 siRNA and parkin-GFP, and treated with or without CCCP for 16 h. Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20, parkin, HDAC6, and actin.",
    "molecules": "CCCP"
  },
  {
    "caption": "MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated. (A) Cells were subjected to immunostaining with antibodies for Tom20 and parkin. Bar, 10 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated.(B) Cell lysates were subjected to immunoblotting with antibodies for Tom20, parkin, cortactin, and actin.",
    "molecules": "CCCP"
  },
  {
    "caption": "(G) Sorted human monocytes  were incubated with control medium or SARS-CoV-2  viruses at an MOI of 1 for 1 h, followed by washing away extracellular viruses and further culture for 1 h. Cells were fixed and stained with the indicated antibodies. Otherwise, monocytes were challenged with 1 μg/ml LPS  for 3 h and 10 μM  nigericin for 30 min, followed by fixing and staining  with the indicated antibodies. Treatment with medium from uninfected Vero  cells served as a control. Scale bar: 5 μm. Data information: results were repeated for three times with similar results.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Supernatants from control or SARS-CoV-2 infected  CD14+ monocytes  with or without LPS and nigericin stimulation were collected and subjected to an ELISA  assay for IL-1β levels (H). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: , Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Cell viability in cell pellets were examined using a cell viability assay through checking ATP levels inside cells (I). The percentage of cells undergoing pyroptosis was calculated through checking released  LDH in the supernatants (J). Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: Student's t-test was used and data were shown as means±SD of three technical replicates. *, P<0.05; **, P<0.01; ***, P<0.001; NS, non-significant. results were repeated for three times with similar results.",
    "molecules": "ATP"
  },
  {
    "caption": "(B, C) THP-1  cells stably expressing the indicated SARS-CoV-2 genes were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were collected for an  ELISA  assay to determine supernatant  IL-1β  levels (B) and cell pellets were subjected to a cell viability assay  to verify the percentage of viable cells through checking cellular ATP  levels (C). For B-C, Student's t-test was used and data were shown as means±SD of three technical replicates. NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.",
    "molecules": "ATP, LPS, nigericin"
  },
  {
    "caption": "(D-F) THP-1 cells stably expressing the nucleocapsid gene or control vector were stimulated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants and cell pellets were collected and  immunoblotted  with antibodies against the indicated proteins (D). The percentage of viable cells was determined through checking cellular ATP  levels in cell pellets (E). The percentage of cells undergoing pyroptosis  was calculated through checking released  LDH in the supernatants (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Data information: control vector used here was empty vector corresponded to the nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Similar results were observed for at least three times.",
    "molecules": "ATP, LPS, nigericin"
  },
  {
    "caption": "(D) GSDMD+/+ and GSDMD-/- THP-1  cells were transfected with plasmids encoding nucleocapsid for 24 h, followed by cell fixation, permeabilization and stain  with antibodies against nucleocapsid  and GSDMD. The nucleus was counterstained  with DAPI. Scale bar: 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E, F) THP-1  (E) or   human CD14+ monocytes  (F) were transfected with plasmids encoding  FLAG-tagged nucleocapsid  for 36 h, followed by pretreatment of 1 μg/ml  LPS  for 3 h and 10 μM nigericin stimulation for 30 min. Cells were lysed and  immunoprecipitated with antibody against FLAG or isotype IgG. Input and precipitates were immunoblotted  as indicated.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(I)  GST-GSDMD  and control GST  were mixed with HEK293T  cell lysate containing FLAG-tagged full-length nucleocapsid  or truncated nucleocapsid lacking amino acids 70-160 and 290-360, followed by a GST-pulldown assay. Input and precipitates were immunoblotted  as indicated.",
    "molecules": "amino acids"
  },
  {
    "caption": "(A, B) THP-1  cells stably expressing  nucleocapsid or control vector were stimulated with 10 μM  nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted  with antibodies against the indicated proteins (A) or stained with propidium iodide (PI) (B, left). Scale bar: 20 μm. The percentage of PI positive cells was calculated (B, right). Student's t-test was used and data were shown as means±SD of three technical replicates. ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the  nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.",
    "molecules": "LPS, nigericin, PI, propidium iodide"
  },
  {
    "caption": "(H, I) THP-1  cells stably expressing full-length  nucleocapsid  or mutant lacking amino acids 70-160 and 290-360 were stimulated with 10 μM nigericin  for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Cells were either immunoblotted  with antibodies against the indicated proteins (H). Cells were also stained with  PI  and the percentage of  PI  positive cells was calculated (I). Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; NS, non-significant. Results were repeated for three times with similar results. Data information: control vector used here was empty vector corresponded to the  nucleocapsid-containing vector. Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Similar results were observed for at least three times.",
    "molecules": "amino acids, LPS, nigericin, PI"
  },
  {
    "caption": "GSDMD-/- THP-1  cells rescued with wild-type or linker substituted GSDMD were transfected with plasmids encoding  nucleocapsid  or control vector and then stimulated with 10 μM nigericin  for 30 min following pretreatment with 1 μg/ml  LPS for 3 h. Cells were  immunoblotted  with antibodies against the indicated proteins (A)",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(E, F) GSDMD-/- THP-1 cells rescued with wild-type or linker substituted GSDMD  were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Otherwise, cells were stimulated with 10 μM nigericin  for 30 min following pretreatment with 1 μg/ml LPS for 3 h. Supernatants were subjected to an ELISA assay to determine  IL-1β levels (E). Otherwise, cell viability in cell pellets was examined using a cell viability assay  through checking  ATP levels inside cells (F). For E, F, Student's t-test was used and data were shown as means±SD of three technical replicates. **, P<0.01; ***, P<0.001; NS, non-significant. Results were repeated for three times with similar results.",
    "molecules": "ATP, LPS, nigericin"
  },
  {
    "caption": "(G) Sorted  CD14+ monocytes  from donors were infected with SARS-CoV-2 at an MOI of 1 for 1h, followed by washing away extracellular viruses and further culture for 3 h. Cells were  immunoblotted with antibodies against the indicated proteins. CD14+ monocytes from healthy donors were treated with 10 μM nigericin for 30 min following pretreatment with 1 μg/ml LPS for 3 h and served as a positive control. Data information: experiments were repeated for three time with similar results.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "B Measurement of 2-NBDG uptake in different cell lines by flow cytometry (n=4 biological replicates).",
    "molecules": "2-NBDG"
  },
  {
    "caption": "G and H Glycolysis stress test of HaCaT cells upon UK5099 (G, n=4 biological replicates) or PC siRNA (si-PC, H, n=5 biological replicates) in 1mM glucose condition. ECAR and OCR were normalized to the amount of protein.",
    "molecules": "glucose, UK5099"
  },
  {
    "caption": "C Violin plot of glutamine consumption, glutamate secretion, and cystine consumption of HaCaT cells upon different treatments (n=12 biological replicates).",
    "molecules": "cystine, glutamate, glutamine"
  },
  {
    "caption": "G Cellular and extracellular levels of metabolites were detected by targeted metabolomics using C13 glutamine tracing protocol in a time course (n=3 biological replicates).",
    "molecules": "C13, glutamine"
  },
  {
    "caption": "C Quantitative RT-PCR detection of Has-miR-31-5p expression levels in HaCaT cells exposed to a range of glucose (n=4 biological replicates). RNU6-1 was used as house control gene.",
    "molecules": "glucose"
  },
  {
    "caption": "D Western blot analysis of GS in HaCaT cells with miR-31 overexpression under different concentrations of glucose or 2-DG treatment, and Tubulin α was used as a loading control.",
    "molecules": "2-DG, glucose"
  },
  {
    "caption": "G and H FACS analysis of Th17 differentiation upon TFB-TBOA treatment (G, n=4 biological replicates) or AOA and iGOT1 treatments (H), n≥3 biological replicates in each group.",
    "molecules": "AOA, TFB-TBOA"
  },
  {
    "caption": "(a) Overall structure of GAL8 with bound Galbi peptide (cyan) and NAD (purple) in the asymmetric unit. The other pair of crystallographic dimer is coloured grey. The first β-strand forming unique interaction is labelled as β1. (b) A topology diagram of GAL8. The β-strands in each N-CRD and C-CRD are numbered sequentially with prime (′) in the C-CRD. The β-strands with a solid line indicate a carbohydrate-binding side in each CRD and those of with a dashed line indicate an opposite side. The elongated purple and cyan ovals indicate NAD- and NDP52-binding sites, respectively. (c) Overall view of Galbi peptide (cyan) bound to the C-CRD of GAL8 (β-strands in yellow and residues in gray).",
    "molecules": "NAD"
  },
  {
    "caption": "(a) Magnified view showing the detailed interaction around Leu374(p). (b) Magnified view showing the detailed interaction around Tyr380(p). (c) Magnified view showing the importance of main-chain hydrogen bonds of the Galbi peptide. (a-c) Red dashed lines designate hydrogen bonds.",
    "molecules": "hydrogen, Leu374, Tyr380"
  },
  {
    "caption": "(a) Sequence alignment of N-CRD and C-CRD of GAL8. The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles, respectively. (b) Detailed view of the peptide-binding site of C-CRD superposed with N-CRD. The residues for the Galbi peptide (cyan) interaction are labelled and the side chains of the corresponding N-CRD residues (magenta) are shown. (c) A detailed view of the NAD-binding site of N-CRD superposed with C-CRD. The residues for NAD (purple) interaction are labelled and the side chains of corresponding C-CRD residues (yellow) are shown.",
    "molecules": "NAD"
  },
  {
    "caption": "(a) Three GAL8 structures, including both CRDs, are available from PDB (4FQZ-lactose bound in only N-CRD; 3VKL-lactose bound in both N-CRD and N-C-CRD; 3VKM-sialylated lactose in N-CRD and N-CRD in C-CRD). The C-CRDs of these Gal8 structures were superposed with that of our Gal8. For clarity, only Cα-traces of each structure (our GAL8-yellow; 4FQZ-red; 3VKM-blue; and 3VKL-cyan) are shown. The large rotation of the N-CRD domain relative to GAL8 compared with the other NDP52 structures is observed in the GAL8-NDP52 complex. A representative angle between CRD of our Gal8 and that of the 4FQZ structure is indicated in degrees. (b) A 90° rotation of panel (a) along the horizontal axis showing a top view. Arg59, which is a unique residue of Gal8CRD that provides carbohydrate specificity, is used as a standard for measuring the distance of domain movement.",
    "molecules": "lactose"
  },
  {
    "caption": "Effects of ubiquitin surface mutants based on the UbcH7-Ub:R0RBR:pUb model for the ubiquitin loading and off-loading potential of pParkinCH (pParkinC431S,H433A). Coomassie stained gels (top) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species. Anti-HA blots (bottom) depict the ubiquitination of HA-tagged Miro1181-579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species. The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin.",
    "molecules": "Ub, ubiquitin"
  },
  {
    "caption": "Auto-ubiquitination assay for parkin using ARJP and non-ARJP substitutions in parkin observed near the interface with UbcH7-Ub. Assays were done in the absence and presence of pUb. Experiments with PINK1 were done by treating parkin:pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination. Assays were monitored using a DyLight-labelled Ub protein and measuring fluorescence at 800 nm.",
    "molecules": "Ub"
  },
  {
    "caption": "Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe. The different stages of activation using parkin, parkin:pUb, pParkin:pUb and pParkin:pUb in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-10 minutes) and visualized by SDS-PAGE.   Relative percentages of pParkin and the pParkin-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of pParkin -Ub, parkin and UbVS/pUb bands. Error bars represent standard deviation from the average for duplicate measurements.  Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe. The different stages of activation using R0RBR, R0RBR:pUb, R0RBR:pUb:pUbl and R0RBR:pUb:pUbl in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-60 minutes) and visualized by SDS-PAGE   Relative percentages of R0RBR and the R0RBR-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of the R0RBR-Ub, R0RBR and UbVS/pUb/pUbl bands.   ",
    "molecules": "SDS, Ub"
  },
  {
    "caption": "Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3).",
    "molecules": "CHX"
  },
  {
    "caption": "Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9.",
    "molecules": "thymidine"
  },
  {
    "caption": "Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX. Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX.",
    "molecules": "CHX"
  },
  {
    "caption": "Immunofluorescence confocal analysis of Ser1490-phosphorylated LRP6 and CLTC co-localization in mitotic HeLa cells; white arrows indicate the colocalization. Scale bars represent 5 μm.",
    "molecules": "Ser"
  },
  {
    "caption": "Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC, an inhibitor of clathrin-mediated-endocytosis.",
    "molecules": "DMSO, MDC"
  },
  {
    "caption": "A representative image of soft agar oncogenic colony formation assays for stable knockdown cells.",
    "molecules": "agar"
  },
  {
    "caption": "Immunoprecipitation analysis of the transiently-expressed BCL9-MF interaction in mitotic cells treated with RO3306.",
    "molecules": "RO3306"
  },
  {
    "caption": " (A) HeLa cells stably expressing GFP-Parkin (HeLa-GFP-Parkin cells) were treated with 1 µM valinomycin and immunostained with anti-NDP52 and anti-Tom20 antibodies. Boxed areas in the images are shown in the next panels on the right. Colocalization of GFP-Parkin, NDP52, and Tom20 were determined using Line scan. Fluorescence intensities of each channels were measured along the dotted white arrow. Scale bars, 10 µm. Images are representative of three independent experiments",
    "molecules": "valinomycin"
  },
  {
    "caption": " (B) HeLa-GFP-Parkin cells were transfected with siNDP52 or scrambled siRNA. Seventy-two hours after transfection, cells were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments ",
    "molecules": "valinomycin"
  },
  {
    "caption": " (C) Mitophagy flux assay using Mtphagy dye. Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment. Results are a summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test ",
    "molecules": "Mtphagy dye, valinomycin"
  },
  {
    "caption": "( Images of HeLa GFP-Parkin cells expressing mCherry-NDP52 (A under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta, mCherry-NDP5 Scale bars, 10 µm. Images are representative of five independent experiments",
    "molecules": "valinomycin"
  },
  {
    "caption": "(B) Time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin treatment. The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures",
    "molecules": "valinomycin"
  },
  {
    "caption": "Images o HeLa GFP-Parkin cells expressin mCherry-OPTN (C) under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta OPTN. Scale bars, 10 µm. Images are representative of five independent experiments",
    "molecules": "valinomycin"
  },
  {
    "caption": "(D) mCherry-NDP52 or OPTN signals colocalized with Parkin-ring structures and existing inside of Parkin-ring structures (irrupted) were quantified. Data were from five independent experiments in cells incubated with valinomycin for 2 h. Values are the means ± SEM. **p < 0.01 compared with mCherry-OPTN expressing cells, determined with one-way ANOVA followed by the Student\"s t",
    "molecules": "valinomycin"
  },
  {
    "caption": "(E) Mitophagic flux in NDP52 KD cells expressing siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05, ††p < 0.01 vs empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test",
    "molecules": "valinomycin"
  },
  {
    "caption": "(F) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": "(G) Images of HeLa GFP-Parkin cells expressing mCherry-NDP52∆LIM-L or mCherry-NDP52∆SKICH treated with valinomycin for 2h. The images are from Movie EV 4. Cyan, GFP-Parkin; magenta, mCherry-NDP52 mutant. Scale bars, 10 µm. Images are representative of three independent experiments",
    "molecules": "valinomycin"
  },
  {
    "caption": "(B) HeLa-GFP-Parkin cells transfected with siNDP52, siOPTN, siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times. Cell lysates were subjected to SDS-PAGE or Phos-tag-PAGE prior to western blotting using the indicated antibodies. Data are representative of two independent experiments. The position of phosphorylated forms of NDP52 or OPTN are indicated by arrowheads",
    "molecules": "valinomycin"
  },
  {
    "caption": "(C) Images of TBK1 KD cells expressing mCherry-NDP52 or mCherry-OPTN under valinomycin treatment from Movie EV 5. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm",
    "molecules": "valinomycin"
  },
  {
    "caption": "(C) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": "(D) HeLa-GFP-Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments",
    "molecules": "valinomycin"
  },
  {
    "caption": "(E) Mitophagic flux in cells transfected with siNDP52, siMTPAP and combinations of siRNAs. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test",
    "molecules": "valinomycin"
  },
  {
    "caption": "(F) Images of MTPAP KD cells expressing mCherry-NDP52 under valinomycin treatment. The images are from Movie EV 7. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm",
    "molecules": "valinomycin"
  },
  {
    "caption": "(A) HeLa-GFP-Parkin cells were incubated with valinomycin for 0 or 2 h. Immunostaining was performed with anti-MTPAP and anti-Tom20 antibodies. Images are representative of two independent experiments. Scale bar, 10 µm",
    "molecules": "valinomycin"
  },
  {
    "caption": "(B) Mitochondrial-rich fraction prepared from HeLa-GFP-Parkin cells treated with or without valinomycin and epoxomicin for 2 h, was incubated with various concentrations of trypsin (0-1000 µg/ml) for 30 min on ice. Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins (Tom20 for OMM, Tim23 for intermembrane space, UQCRC1 and TFAM for mitochondrial matrix). Data are representative of five independent experiments",
    "molecules": "epoxomicin, trypsin, valinomycin"
  },
  {
    "caption": "(C) HeLa-GFP-Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0-3 h in the presence or absence of 10 µM epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of five independent experiments. (D) Quantification of MTPAP co-immunoprecipitated with NDP52 in (C). Results are a summary of five independent experiments. Values are the means ± SEM. **p < 0.01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test",
    "molecules": "bafilomycin A1, epoxomicin, valinomycin"
  },
  {
    "caption": "(E) HeLa-GFP-Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0, 4 and 8 h in the presence or absence of epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments",
    "molecules": "bafilomycin A1, DFO, epoxomicin"
  },
  {
    "caption": "(F) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments",
    "molecules": "bafilomycin A1, DFO, valinomycin"
  },
  {
    "caption": "(G) Immunoelectron micrographs of HeLa-GFP-Parkin cells expressing FLAG-NDP52. Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin, were processed for immune-electron microscopy with anti-FLAG antibody. Images are representative of ten cells. Arrow, immunogold labeled FLAG-NDP52. Mito, mitochondrion; MP, mitophagosome. Scale bars, 200 nm",
    "molecules": "bafilomycin A1, epoxomicin, valinomycin"
  },
  {
    "caption": "(H) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": "(A, B) mCherry-LC3 and MTS-TagBFP were expressed in MTPAP KD and control cells. Cells were incubated with valinomycin for the indicated times and immunostained with anti-NDP52 antibody. Images are representative of two independent experiments. Yellow arrows indicate co-localization of MTS-TagBFP, mCherry-LC3 and NDP52. White arrows indicate co-localization of MTS-TagBFP and NDP52 (not mCherry-LC3). Scale bars, 10 µm. Co-localization of mCherry-LC3 and NDP52 on mitochondria is quantified in (B). At least ten cells from two independent experiments were quantified. Values are the means ± SEM. **p < 0.01 vs control, determined with one-way ANOVA followed by the Student"s t",
    "molecules": "valinomycin"
  },
  {
    "caption": "MTPAP KD and control cells expressed mCherry-NDP52 or empty vector (C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": "MTPAP KD and control cells expresse mCherry-LC3 (D). Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": "(E) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h. Cell lysates were subjected to immunoprecipitation using an anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": "(F) GST-NDP52 conjugated with glutathione-Sepharose resin was incubated with purified recombinant LC3 and MTPAP∆MTS∆C. The pulled down complex was subjected to western blotting. Data are representative of three independent experiments",
    "molecules": "glutathione, Sepharose"
  },
  {
    "caption": " (A) HeLa-GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments ",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": " (B) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9. Cyan, GFP-Parkin; magenta, mCherry-NDP52 D439R/C443K. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm",
    "molecules": "bafilomycin A1, valinomycin"
  },
  {
    "caption": "b-d, MEFs were cultured in nutrient-rich medium (fed) or Hanks' buffer (starved) for 1 h. Total cell lysates were prepared, and blotted with the indicated antibodies (b).",
    "molecules": "nutrient"
  },
  {
    "caption": "f, Degradation of long-lived proteins in MEFs. Wortmannin is an autophagy inhibitor. The results shown are mean1s.d. Statistical significance (P value) was determined by the Student's t-test.",
    "molecules": "Wortmannin"
  },
  {
    "caption": "a, Cytokine production from macrophages stimulated with LPS (100 ng ml-1) for 24 h. Statistical significance was determined by the Student's t-test. *P  0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "c, IL-1b production from LPS-primed macrophages infected with S. typhimurium (multiplicity of infection, m.o.i., 1), or stimulated with ATP or MSU for 1 h.",
    "molecules": "ATP, LPS, MSU"
  },
  {
    "caption": "e, LPS-induced production of IL-1b from macrophages with the indicated phenotype.",
    "molecules": "LPS"
  },
  {
    "caption": "g, Effect of the ROS scavenger FeTPPS (25 mM), N-acetyl-L-cysteine (NAC; 25mM) or P2X7 receptor antagonist oxidized ATP (250 mM) on IL-1b production.",
    "molecules": "FeTPPS, N-acetyl-L-cysteine, oxidized ATP, ROS"
  },
  {
    "caption": "h, ROS in LPS-stimulated macrophages were detected by CM-H2DCFDA staining.",
    "molecules": "LPS, ROS"
  },
  {
    "caption": "a, Macrophages were stimulated with LPS (100 ng ml-1) for the indicated time period. The number of endogenous LC3 dots within each cell was counted. The results shown are mean + s.d. (n > 100).",
    "molecules": "LPS"
  },
  {
    "caption": "c, IL-1b and IL-6 production by wild-type or Atg7-deficient macrophages stimulated with LPS. Statistical significance was determined by the Student's t-test. *P,0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "d, Macrophages were pre-treated with or without 10 mM 3MA and then stimulated with the indicated ligands.",
    "molecules": "3MA"
  },
  {
    "caption": "a, b, Fetal liver chimaeric mice were given 5% DSS in drinking water for 7 days. The survival (a) and weight loss (b) of each mouse genotype were plotted. The results shown are mean ± s.d. Statistical significance was determined by the Students t-test.",
    "molecules": "DSS, water"
  },
  {
    "caption": "c, Typical distal colon appearance 6  days after the initiation of DSS administration. The results shown are mean ± s.d. (n = 3, each group).",
    "molecules": "DSS"
  },
  {
    "caption": "e, f, Atg16L1-deficient chimaeric mice given 5% DSS in drinking water were intraperitoneally injected with both anti-IL-1b and anti- IL-18 neutralizing antibodies (squares; n55) or isotype control IgG (circles;n55) at days 1, 3, 5 and 7. The survival (e) and weight loss (f) of each mouse genotype were plotted.",
    "molecules": "DSS, water"
  },
  {
    "caption": "A OT-I CD8+ CTLs were conjugated with 10 nM OVA257-264-pulsed EL-4 cells for 30 min, fixed, and stained with anti-SKAP55 (red), anti-LFA-1 (green) and Hoechst (blue).",
    "molecules": "OVA"
  },
  {
    "caption": "B, C WT and SKAP55−/−OT-ICD8+CTLs were generated from WT or SKAP55−/−OT-I Tg mice, then incubated with 10 nM OVA257-264-pulsed EL4 targets for 4 h to assess the in vitro cytotoxicity at different Effector:Target ratios (B; mean of triplicates ± SD); surface expression and the mRNA levels of PD-1 (C). Graphs are representative of at least three independent experiments.",
    "molecules": "OVA"
  },
  {
    "caption": "D WT and SKAP55−/− OT-I CD8+ CTLs (3 × 106) were injected into C57BL/6 mice, followed by injection of non-pulsed (CFSEhi) or 10 nM OVA257-264-pulsed (CFSElo) splenocytes (5 × 106) to measure in vivo cytotoxicity (mean ± SD, n = 3 mice per group). Representative data of three independent experiments.",
    "molecules": "OVA"
  },
  {
    "caption": "E WT and SKAP55−/− OT-I CD8+ CTLs were pretreated with anti-PD-1 antibody or IgG control, followed by incubation with OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD). Graphs are representative of three independent experiments.",
    "molecules": "OVA"
  },
  {
    "caption": "F CD8+ CTLs were transfected with plasmids expressing SKAP55-GFP or EGFP, then treated with non-pulsed or 10 nM OVA257-264-pulsed EL4 cells to examine surface PD-1 expression (left panel) or the in vitro killing assay (mean of triplicates ± SD) (right panel).",
    "molecules": "OVA"
  },
  {
    "caption": "A OT-I CD8+ CTLs were conjugated with CFSE-labeled OVA257-264-pulsed or non-pulsed EL-4 cells for 30 min, fixed, and stained with anti-ADAP (red).",
    "molecules": "OVA"
  },
  {
    "caption": "B OT-I CD8+ CTLs were transfected with plasmids expressing ADAP or GFP, then stimulated with 10 nM OVA257-264-pulsed EL-4 cells to detect surface PD-1 expression. Representative of three independent experiments.",
    "molecules": "OVA"
  },
  {
    "caption": "C, D WT and ADAP−/− OT-I CD8+ CTLs were stimulated with 10 nM OVA257-264-pulsed or unpulsed EL-4 cells for 4 h to examine surface expression and the mRNA levels of PD-1 (C), in vitro cytotoxicity at different Effector:Target ratios (D; mean of triplicates ± SD). Graphs are representative of at least three independent experiments.",
    "molecules": "OVA"
  },
  {
    "caption": "E WT and ADAP−/− OT-I CD8+ CTLs were pretreated with 10 μg/ml anti-PD-1 antibody or IgG control, followed by incubation with 10 nM OVA257-264-pulsed EL4 cells to examine in vitro killing ability (mean of triplicates ± SD).",
    "molecules": "OVA"
  },
  {
    "caption": "A-C WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were stimulated with OVA257-264-pulsed or unpulsed EL4 cells to detect the levels of NFATc1 expression, activation and nuclear entry by immunoblotting (A) or immunostaining (B). Alternatively, nuclear extracts from these CTLs were incubated with the N1 DNA probes containing NFAT-binding sites for an EMSA. The unlabeled competitor probe (Comp. N1) and the mutant probe that contains the same sequence except for carrying a mutated NFAT-binding site (Comp. mutN1) were included as controls (C).",
    "molecules": "OVA"
  },
  {
    "caption": "D WT, ADAP−/−, or SKAP55−/− OT-I CD8+ CTLs were pretreated with DMSO or 5 μM CsA, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).",
    "molecules": "CsA, DMSO, OVA"
  },
  {
    "caption": "E WT, ADAP−/−, or SKAP55−/− OT-I CTLs were pretreated with the DMSO control, 10 μM PP2 or 75 μM 2APB, then stimulated with OVA257-264-pulsed EL-4 cells for 4 h to examine surface PD-1 expression (mean of triplicates ± SD).",
    "molecules": "2APB, DMSO, OVA, PP2"
  },
  {
    "caption": "F GFP, ADAP, or SKAP55 was overexpressed with pGL3-NFAT Luciferase reporter plasmid into Jurkat cells. Cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of PP2 or 2APB for 6 h, followed by measuring luciferase readings (mean of triplicates ± SD).",
    "molecules": "2APB, PP2"
  },
  {
    "caption": "A, B WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by two injection at day 7 and day 14 of OVA257-264-pulsed DCs or the PBS control respectively (n ≥ 5). The growth of tumors under skin was measured every three days according to tumor diameter (mean ± SEM, n ≥ 5 mice per group) (A). Surface expression levels of PD-1 were checked on OVA257-264-specific Vα2high/Vβ5+ CD8+ effector cells at day 30 (mean ± SD, n ≥ 5 mice) (B).",
    "molecules": "OVA"
  },
  {
    "caption": "C WT and SKAP55−/− mice were s.c. injected with MO5 melanoma cells followed by s.c. two injection of OVA257-264-pulsed DCs at day 7 and day 14. anti-PD-1 monoantibody or IgG controls were i.v. injected three times at day 7, day 10, and day 14 (50 μg/injection). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 5 mice).",
    "molecules": "OVA"
  },
  {
    "caption": "D The WT recipient mice were s.c. injected with MO5 melanoma cells followed by injection of 10 nM OVA257-264-stimulated WT or SKAP55−/− CTLs at day 8 (n ≥ 8). The diameter and volume of tumors were measured every three days (mean ± SEM, n ≥ 8 mice).",
    "molecules": "OVA"
  },
  {
    "caption": "A, B During generation of OVA257-264 peptide-specific CTLs, naïve OT-I splenocytes were stimulated with 10 nM OVA257-264 with or without CsA (10 nM) for 3 days, then washed, and further cultured in the RPMI growth medium until day 6. The CsA-treated or untreated OVA257-264 peptide-specific CTLs were incubated with OVA257-264-pulsed EL-4 cells for 4 h to assess the in vitro cytotoxicity (A, mean of triplicates ± SD) or i.v. injected into WT mice to measure in vivo cytotoxicity (B, mean ± SD, n = 4 mice).",
    "molecules": "CsA, OVA"
  },
  {
    "caption": "C WT mice were s.c. injected with B16-MO5 melanoma cells followed by injection of the CsA-treated or untreated OVA257-264 peptide-specific CTLs at day 9 (n ≥ 12). The diameter and volume of tumors were measured every 3 days (mean ± SEM, n ≥ 12 mice).",
    "molecules": "CsA, OVA"
  },
  {
    "caption": "(E) Estradiol levels in LNs (A: axillary, B: branchial, C: cervical, and I: inguinal) in sham-operated and OVX mice (n=5 mice per group).",
    "molecules": "Estradiol"
  },
  {
    "caption": "(A) Lta mRNA expression in BMDCs developed from bone marrows of WT and ERα-/- mice 3 h after treatment with LPS (100 ng/ml) with vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h (n=4 samples per group). (B) Il1b and Ifnb mRNA expression 3 h after treatment with LPS (100 ng/ml) with vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h (n=4-5 samples per group).",
    "molecules": "PPT, LPS"
  },
  {
    "caption": "(C) Representative Western blot of TRAF3 and HSC70 in WT BMDCs and ERα-/- BMDCs. TRAF3 expression in BMDCs following vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h was evaluated by normalization of HSC70 (n=5-7 per group).",
    "molecules": "PPT"
  },
  {
    "caption": "(F) Nuclear localization of IRF3 in BMDCs treated with vehicle or with LPS for 2 h prior upon a vehicle or PPT (1 µM) pre-treatment for 18 h. The results are presented as a ratio of the nuclear IRF3 signal to the cytosol IRF3 signal (n=5-9 biological samples)",
    "molecules": "PPT, LPS"
  },
  {
    "caption": "Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents (C)",
    "molecules": "α-factor, DNA"
  },
  {
    "caption": "Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for immunofluorescence using anti-tubulin antibodies in order to score metaphase and anaphase spindles (D). Budding and nuclear division were scored on the FACS samples.",
    "molecules": "α-factor"
  },
  {
    "caption": "F: Wild type and mps1 mutant cells bearing the TetO/TetR-GFP system to mark the centromere of chromosome V [43] and expressing the SPBs marker Spc97-mCherry were synchronised as in (C-D) and arrested in anaphase through the temperature-sensitive cdc15-2 allele. At 120 and 150 minutes, cells were fixed for scoring chromosome V segregation (n≥172). Arrowheads indicate the sister chromatids of chromosome V. Error bars: SD. N=3. Representative images of cells for each genotype are shown on the right. Scale bar: 5 μm.",
    "molecules": "Tet"
  },
  {
    "caption": "G: Wild type and mps1-3 mutant cells carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for 1 hour before filming. Cells were filmed at 34°C every 2 minutes by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.",
    "molecules": "Tet"
  },
  {
    "caption": "A: Wild type and mps1 mutant cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.",
    "molecules": "α-factor, DNA, nocodazole"
  },
  {
    "caption": "B: Cells were treated as in (A) and after 2 hours from the release α-factor was re-added to prevent cells from undergoing a second cell cycle. Cell samples were collected at the indicated time points for western blot analysis of the indicated proteins. Pgk1 was used as loading control. Cyc: cycling cells.",
    "molecules": "α-factor"
  },
  {
    "caption": "C: Top: in vitro kinase assays with recombinant Mps1 and Mps1-3 purified from E. coli and incubated at 34°C for the indicated times in the presence of radioactive ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate. Bottom left: kinase activity was quantified on the autoradiographs by Image J and normalized to the levels of the full length protein on the Coomassie Blue-stained gel (right). Bottom right: 1μg of the recombinant Mps1 kinases used for kinase assays was loaded on SDS page and stained with Coomassie Blue for normalisation of kinase assays.",
    "molecules": "ATP, Coomassie Blue, SDS"
  },
  {
    "caption": "D: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole. Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S2). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.",
    "molecules": "α-factor, formaldehyde, nocodazole"
  },
  {
    "caption": "E: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.",
    "molecules": "α-factor, DNA"
  },
  {
    "caption": "D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C in presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.",
    "molecules": "α-factor, DNA, nocodazole"
  },
  {
    "caption": "F: Cells with the indicated genotypes were synchronised at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.",
    "molecules": "DNA"
  },
  {
    "caption": "G-H: Cells with the indicated genotypes carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for one hour before filming. Cells were filmed at 34°C every 2 or 4 minutes by time lapse fluorescence microscopy. Chromosome V segregation errors are reported in the table (H). Representative cells are shown as examples in the montages (G). Representative montages for wild type and mps1-3 cells are shown in Fig. 1F. DIC: differential interference contrast. Scale bar: 5 μm.",
    "molecules": "Tet"
  },
  {
    "caption": "I: Wild type, mps1-3, mps1-3 spc105-18, and mps1-3 GLC7-24 cells were synchronised in G1 with α-factor at 25°C and then released at 32°C in the presence of nocodazole (note that the presence of 3HA tags at the C-terminus of Mps1-3 slightly decreases the maximal temperature of suppression). Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S5). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.",
    "molecules": "α-factor, formaldehyde, nocodazole"
  },
  {
    "caption": ": Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (A) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.",
    "molecules": "α-factor, nocodazole"
  },
  {
    "caption": "Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (B) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.",
    "molecules": "α-factor, nocodazole"
  },
  {
    "caption": "Cells with the indicated genotypes expressing Bub1-GFP and the kinetochore marker Mtw1-Tomato were grown in SD glu 2% and then shifted to 34°C for 1 hour before filming; they were then filmed every 4 minutes by time lapse fluorescence microscopy at 34°C. Note that BUB1-GFP is synthetic lethal with mps1-3. Thus, we used mps1-3 GALs-MPS1 BUB1-GFP cells that were grown in -His RG medium at 30°C; glucose was added to the culture for 30 minutes to shut off GALs-MPS1, followed by shifting cells to SD medium at 34°C for 1 before imaging in the same medium. Montages show representative cells (C). Arrowheads indicate Bub1-GFP signals at kinetochores. DIC: differential interference contrast. Scale bar: 5 μm.",
    "molecules": "glu, glucose, His"
  },
  {
    "caption": "Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (E-F) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.",
    "molecules": "α-factor, DNA, nocodazole"
  },
  {
    "caption": "Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (G-H) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents (F, H). Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.",
    "molecules": "α-factor, DNA, nocodazole"
  },
  {
    "caption": "D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C. Cells were collected at the indicated time points for FACS analysis of DNA contents.",
    "molecules": "α-factor, DNA"
  },
  {
    "caption": "(A) Representative photomicrographs of MAP2 (green) and cleaved caspase-3 (red) immunocytochemistry of embryonic cortical neurons from wild type (upper panels) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h. Scale bar 15µm.",
    "molecules": "Aβ"
  },
  {
    "caption": "(B) Quantification of active (cleaved) caspase-3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers (2.5, 5, or 10 µM). Data are expressed as percentage cleaved caspase-3 and MAP2 double positive cells over the total number of MAP2 positive cells.",
    "molecules": "Aβ"
  },
  {
    "caption": "(C) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers (2.5, or 10 µM). Data are expressed as mean neurite length in individual neurons, relative to wild type vehicle (set to 100 units).",
    "molecules": "Aβ"
  },
  {
    "caption": "ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers (G) refers to the soluble fraction after Tris-buffered saline extraction",
    "molecules": "Tris-buffered saline, Aβ, Aβ1-42"
  },
  {
    "caption": "ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers (H) to the soluble fraction after RIPA buffer extraction of the Tris-buffered saline pellet,",
    "molecules": "RIPA buffer, Tris-buffered saline, Aβ, Aβ1-42"
  },
  {
    "caption": "ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils (I) to the soluble fraction after formic acid treatment of the RIPA pellet.",
    "molecules": "RIPA, Aβ, Aβ1-42, formic acid"
  },
  {
    "caption": "(G) MitoSox staining, a mitochondrial superoxide indicator, and DAPI in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 60μm. (H) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows MitoSox mean fluorescence intensity in arbitrary units (mean ± SEM, N=5 mice per group).",
    "molecules": "MitoSox, MitoSOX, DAPI, superoxide"
  },
  {
    "caption": "(C) Western blot analysis of soluble APP alpha (sAPPα) in the soluble Tris-buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH. (D) Quantification (mean ± SEM) normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs. 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P<0.05 5xFAD/ΔDD vs. 5xFAD/KO. N=9 mice per group.",
    "molecules": "Tris-buffered saline"
  },
  {
    "caption": "(A) Internalization of triple mutant hAPP in wild type, p75NTR knock-out (KO), ΔDD and C259A hippocampal neurons (15min time point). Counterstaining for p75NTR and DAPI are also shown. Scale bar, 10μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) Internalization of endogenous p75NTR in wild type hippocampal neurons (30min time point). Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) Internalization of hAPP/p75NTR PLA signals in hippocampal neurons of wild type and p75NTR mutant mice. Live neuron cultures were fed with anti-mouse p75NTR and anti-hAPP antibodies on ice, washed and plates placed at 37°C for different periods of time to allow internalization. Internalization was stopped by acid wash, followed by fixation and PLA reaction. Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Micrographs of triple mutant hAPP after 15min internalization (red), BACE1 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main images. Scale bar, 5μm. (B) Quantification of the proportion of BACE1 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % BACE co-localized with hAPP. N=3 independent experiments, each performed in duplicate; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (C) Quantification of the proportion of internalized hAPP that co-localized with BACE1 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with BACE1. N=3 independent experiments, each performed in duplicate; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test).",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Micrographs of p75NTR after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (E) Quantification of the proportion of Rab11 that co-localized with p75NTR after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with p75NTR. n=4; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (F) Quantification of the proportion of internalized p75NTR that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized p75NTR co-localized with Rab11. n=4; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). ",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Micrographs of triple mutant hAPP after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (H) Quantification of the proportion of Rab11 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with hAPP. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test). (I) Quantification of the proportion of internalized hAPP that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with Rab11. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test). ",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalase. (",
    "molecules": "ROS"
  },
  {
    "caption": "b) Measurement of intracellular glutathione redox potential with Grx1-roGFP2.",
    "molecules": "glutathione"
  },
  {
    "caption": ". (e) Immunoblot of precipitated p47phox probed with an anti-phosphoserine or anti-p47phox antibody.",
    "molecules": "phosphoserine"
  },
  {
    "caption": "(f) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor.",
    "molecules": "ROS"
  },
  {
    "caption": "g) Extracellular ROS production was assessed using Amplex-red dye.",
    "molecules": "ROS"
  },
  {
    "caption": "(h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+.",
    "molecules": "calcium, Mn2+"
  },
  {
    "caption": "(i) Intracellular RNS generation was measured using DAF-FM. Bars represent average±s.e.m. from n=15 individual fibres for each condition in (a,b,f,g,i",
    "molecules": "RNS"
  },
  {
    "caption": ",j). Markers of autophagy were analysed in isolated fibres (incubated with or without PP2) from FDBs.",
    "molecules": "PP2"
  },
  {
    "caption": "(d) Effect of gp91 ds and PP2 on LC3-LAMP1 colocalization was analysed using confocal microscopy. Representative images from independent biological experiments (n=3) are shown (scale bar=140 and 40 μm for red box areas).",
    "molecules": "PP2"
  },
  {
    "caption": "(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn2+. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition.",
    "molecules": "calcium, Mn2, ROS"
  },
  {
    "caption": "(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP.",
    "molecules": "CCCP"
  },
  {
    "caption": "(A) Live confocal micrographs of siRNA‐transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(D) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA‐transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; siRNA, short interfering RNA.",
    "molecules": "Antimycin A, Oxygen"
  },
  {
    "caption": "(A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA‐transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour.",
    "molecules": "CCCP, MG132"
  },
  {
    "caption": "(B) Quantification of 52‐kDa PINK1 from A (with MG132), n=3; *P0.05; **P0.001 compared with NT siRNA.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Mitochondria from siRNA‐transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C.al hour.",
    "molecules": "CCCP, dimethylsulphoxide"
  },
  {
    "caption": "(A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK).",
    "molecules": "CCCP, proteinase K"
  },
  {
    "caption": "(B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.",
    "molecules": "CCCP, Na2CO3"
  },
  {
    "caption": "(A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification).",
    "molecules": "CCCP"
  },
  {
    "caption": "(C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP.",
    "molecules": "CCCP"
  },
  {
    "caption": "(D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.",
    "molecules": "CCCP"
  },
  {
    "caption": "C, Average Ca2+ response following stimulation in terminals with or without a mitochondrion (average of ΔF measurement taken for a t=20-30s of B), ΔF/F0 = 1.9 0.3 with and 3.5 0.4 without mitochondria, paired t-test, ***p<0.0001.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D & E, However, there is no significant difference in the presynaptic Ca2+ response in terminals with and without a mitochondrion in response to single action potentials. D, Example trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5. 1 ms stimulation pulses were applied every 5 s. The red trace represents an average of the terminals occupied with a mitochondrion in this neuron, whereas the black trace represents the averaged response of terminals without a mitochondrion. E, Maximal fluorescence intensity in terminals without a mitochondrion (left) or with a mitochondrion (right) within the same axon (dots connected by a line) (n=8 neurons, 44 terminals, paired t-test, p=0.55).",
    "molecules": "Ca2+"
  },
  {
    "caption": "A, Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min. Neurons were stimulated using field stimulation for 10s at 10 Hz. Red trace = average of the terminals occupied with a mitochondrion, black trace = averaged response of terminals without a mitochondrion. B, as in A, but after 30 min treatment with 10 µM Ru360.",
    "molecules": "Ru360"
  },
  {
    "caption": "C, Summary bar graph of the control neurons (corresponds to the traces shown in panel A). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 3.5 0.4) and unoccupied terminals (ΔF/F0 = 4.9 0.5, n=15 neurons, 96 terminals, ***p<0.001, paired t-test). D, Summary bar graph of the Ru360-treated neurons (corresponds to the traces shown in panel B). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 4.1 0.5) and unoccupied terminals (ΔF/F0 = 3.8 0.9, n=13 neurons, 79 terminals, p=0.53, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.",
    "molecules": "Ca2+, Ru360"
  },
  {
    "caption": "A, C & E, Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN-GFP and C, myc-Miro1 (Miro OE) or E, myc-Miro1ΔEF (Miro ΔEF). Neurons are either non-treated (NT), TTX treated (1 M, 48h), PTX treated (100 M, 48h), or DMSO treated (1:2000, as PTX). Scale bar, 10 m. B, D & F, Fraction of SYN-GFP clusters co-localising with mitochondria in cultures in B, control conditions, DMSO 28.7% 3.9, TTX 16.6% 3.1, PTX 50.0% 7.2, ANOVA **p<0.001, n=8-9 neurons, D, with the expression of myc-Miro1, DMSO 33.9% 4.7, TTX 35.9% 5.4, PTX 46.7% 4.0, ANOVA, *p<0.05, n=8-9, F, with the expression of myc-Miro1ΔEF, ANOVA p>0.05, n=9-12. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.",
    "molecules": "DMSO, PTX, TTX"
  },
  {
    "caption": "A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed or myc-ΔEF-Miro1-IRES-MtDsRed (ΔEF Miro) before and during 10 Hz field stimulation with and without TTX treatments. Scale bar, 10 m. B, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 2.5 0.5), n=10 neurons (62 terminals) and non-treated terminals (black trace, ΔF/F0 = 1.3 0.6), n=11 neurons (69 terminals) co-transfected with MtDsRed, *p<0.05, t-test. C, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 1.8 0.3) in n=7 neurons (33 terminals) and non-treated terminals (black trace, ΔF/F0 = 2.3 0.6) in n=7 neurons (32 terminals) co-transfected with myc-ΔEF-Miro1-IRES-MtDsRed; p=0.5, t-test. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.",
    "molecules": "TTX"
  },
  {
    "caption": "E The odorant habituation results shown as the ratio of odor sniffs/water of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice. These odorants included male urine (1:50), female urine (1:50), isopentyl acetate (50 μM), citral (50 μM), 2-heptanone (50 μM) and propyl propionate (50 μM) (n=6 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test).",
    "molecules": "citral, 2-heptanone, isopentyl acetate, propyl propionate"
  },
  {
    "caption": "D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the EdU+ cells and the yellow arrows indicate the cells positive for both EdU and Ki67. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the numbers of costained Ki67+ and EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR-200b/a KD mice compared with their NC controls [the cell cycle exit index was calculated using the following formula: (EdU+ Ki67− cells/total EdU+ cells)×100; n=3 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test]. ",
    "molecules": "EdU"
  },
  {
    "caption": "G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the BrdU+ cells, and the yellow arrows indicate the cells positive for both BrdU and EdU. G' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). G: Scale bars, 20 μm; G': Scale bars, 2 μm.",
    "molecules": "BrdU, EdU"
  },
  {
    "caption": "H The S phase length of the GBCs in the MOE of the NC and miR-200b/a KD mice (the S phase length was calculated using the following formula: 3 h×BrdU+/BrdU+EdU-; n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).",
    "molecules": "BrdU, EdU"
  },
  {
    "caption": "I Total cell cycle length of the GBCs in the MOE of the NC and miR-200b/a KD mice (total cell cycle length was calculated using the following formula: 20 h+(Ts×EdU+BrdU-/Edu+); n=3 mice each group; p=0.9785; data represent the mean±SEM; Student's t-test).",
    "molecules": "BrdU, EdU, Edu"
  },
  {
    "caption": "F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Sox2 and EdU. F' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). F: Scale bars, 20 μm; F': Scale bars, 2 μm. G Quantification of the number of Sox2+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; p=0.7988; Student's t-test). ",
    "molecules": "EdU"
  },
  {
    "caption": "H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Mash1 and EdU. H' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). H: Scale bars, 20 μm; H': Scale bars, 2 μm. I Quantification of the number of Mash1+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). ",
    "molecules": "EdU"
  },
  {
    "caption": "D Representative IF costaining with GAP43 and BrdU antibodies in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d. The white arrows indicate the cells positive for both GAP43 and BrdU. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the ratio of the number of BrdU+ GAP43+ cells and the number of total GAP43+ cells in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d (n=3 mice each group; data represent the mean±SEM; ****p<0.0001; Student's t-test). ",
    "molecules": "BrdU"
  },
  {
    "caption": "L Representative IF staining of 5mC in the MOE of the NC and miR-200b/a knockdown mice. L' is shown at higher magnification of the boxed region. L: Scale bars, 20 μm; L': Scale bars, 2 μm.",
    "molecules": "5mC"
  },
  {
    "caption": "M Quantification of the number of 5mC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).",
    "molecules": "5mC"
  },
  {
    "caption": "N Representative IF staining of 5hmC in the MOE of the NC and miR-200b/a knockdown mice. N' is shown at higher magnification of the boxed region. N: Scale bars, 20 μm; N': Scale bars, 2 μm.",
    "molecules": "5hmC"
  },
  {
    "caption": "Quantification of the number of 5hmC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).",
    "molecules": "5hmC"
  },
  {
    "caption": "B. Blue native-PAGE loading of protein extracts from nrc2/3/4 knockout N. benthamiana plants after immunoprecipitation with anti-Flag antibody. Co-migration of Rpi-amr3-HF and AVRamr3-V5 are indicated (*, red). Same samples were loaded twice on one blue native-PAGE gel, transferred onto one membrane, and then the membrane was cut into two and immunoblotted separately. GUS-V5, β-glucuronidase fused with V5 tag. C. NRC2EEE-Myc does not alter association between Rpi-amr3 and AVRamr3. Samples of Fig 1B were SDS-boiled and loaded on SDS-PAGE. Input samples were taken prior to immunoprecipitation to show expression of all proteins.",
    "molecules": "SDS"
  },
  {
    "caption": "A. NRC2EEE-Myc is oligomerized upon effector-dependent activation of Rpi-amr3. Protein lysates from Fig 1B, C were loaded on blue native-PAGE. SDS-boiled protein lysate samples serve as control for actual size of NRC2EEE-Myc. Oligomerized NRC2EEE-Myc is indicated (*, red). B. NRC2EEE-Myc oligomerizes upon effector-dependent activation of Rpi-amr1. Protein lysates from nrc2/3/4 knockout N. benthamiana plants were loaded on blue native-PAGE. Oligomerized NRC2EEE-Myc is indicated (*, red).",
    "molecules": "SDS"
  },
  {
    "caption": "C. Samples from Fig 2B were SDS-boiled and loaded on SDS-PAGE. Protein accumulation of Rpi-amr1-Flag, NRC2EEE-Myc, AVRamr1-V5 and AVRamr3-V5 are shown.",
    "molecules": "SDS"
  },
  {
    "caption": "B. Representative spinning disk confocal live-cell imaging time series images of S-trityl-L-cysteine (STLC)-treated U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red). Note that MT-flux is abrogated in presence of taxol (lower panel). Scale bars, 10 µm. Time, min:sec.",
    "molecules": "taxol, S-trityl-L-cysteine, STLC"
  },
  {
    "caption": "Quantification of MT-flux in bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments): - bipolar spindles: siControl (49, 5), STLC (31, 3), siKIF15 (39, 3), siKID (32, 3), siKIF4A (28, 3), siKIF2A (36, 3), siCLASPs (21, 2), GSK923295 (44, 3)",
    "molecules": "GSK923295, STLC"
  },
  {
    "caption": "Quantification of MT-flux in monopolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments) monopolar spindles: siControl (35, 3), siKIF15 (44, 3), siKID (38, 3), siKIF4A (44, 3), siKIF2A (38, 3), siCLASPs (12, 2), GSK923295 (33, 3).",
    "molecules": "GSK923295"
  },
  {
    "caption": "A. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec.",
    "molecules": "SiR-DNA, doxycycline"
  },
  {
    "caption": "B. Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants. The anti-KIF4A antibody was used, together with anti-vinculin antibody as a loading control.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A. Representative spinning disk confocal time series of MT-flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes (MUGs). U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), labeled for chromosomes with SiR-DNA (grey) are shown, with 5 mM caffeine used as a control. Scale bar, 10 µm. Time, min:sec. B. Quantification of the MT-flux rates from indicated conditions. MT-flux values with mean ± SD are plotted. N (number of cells, number of independent experiments): Caffeine-only control (30, 3) and MUGs (34, 3).",
    "molecules": "SiR-DNA, caffeine, Caffeine"
  },
  {
    "caption": "C. Representative point-scanning confocal maximum-intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions, immunostained with antibodies against KIF4A and α-tubulin. DNA was counterstained with DAPI. KIF4A in cyan and α-tubulin in red in merged image. Scale bar, 10 µm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "B. Quantification of the impact of stable end-on KT-MT attachments on MT-flux in NDC80-depleted cells under indicated conditions and in early prometaphase cells with and without the CENP-E inhibitor GSK923295. MT-flux values are plotted with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siNDC80 (59, 6), siNDC80 + siKIF4A (41, 3), siNDC80 + STLC (bipol.) (28, 3), siNDC80 + siKIF15 (36, 4), siNDC80 + siCLASPs (33, 3), siNDC80 + siKIF2A (38, 4), siNDC80 + GSK923295 (38, 3), early prometaphase (23, 3), early prometaphase + GSK923295 (28, 3).",
    "molecules": "GSK923295, STLC"
  },
  {
    "caption": "A. Representative maximum-intensity projected coherent-hybrid STED (CH-STED) images of mitotic spindles in U2OS cells treated with control and NDC80 siRNAs and stained for DNA (DAPI), KTs (CENP-C, magenta), α-tubulin (red) and CENP-E (green). Scale bar, 10 µm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "C. CH-STED analyses of HeLa cells stably expressing CENP-E-GFP illustrating bipolar spindles in prometaphase, metaphase and just after sister-chromatid separation at anaphase onset. Approximate x-y resolution is 70 nm. The expected/described localization of CENP-E-GFP (green) at KTs is shown. In addition, CENP-E-GFP was found associated with interpolar MTs (ipMTs), including regions of overlapping anti-parallel MTs, and k-fibers. Chromosomes (confocal mode only) were revealed in the larger panels with DAPI (cyan) and MTs (magenta) were detected with an anti-α-tubulin antibody. Arrows indicate examples of clear MT bundles, including regions of overlapping anti-parallel MTs. Scale bar in all panels is 5 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, C. Quantification of MT-flux in prometaphase (B) and metaphase (C) bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. siNDC80, siNDC80 + GSK923295, early prometaphase, early prometaphase + GSK923295, siControl, and siKIF4A N (number of cells, number of independent experiments): siNDC80 + KIF15 + STLC (33, 5), siNDC80 + KIF15 + STLC + GSK923295 (25, 3), early prometaphase + KIF15 + STLC + GSK923295 (11, 2), siKIF15 + STLC (29, 3), siKIF4A + siKIF15 + STLC (10, 3).",
    "molecules": "GSK923295, STLC"
  },
  {
    "caption": "D. Representative images from spinning disk confocal live-cell time series of U2OS PA-GFP-α-tubulin/mCherry-α-tubulin cells under indicated treatments, with SiR-DNA-labelled DNA. Microtubules are shown in red and DNA in cyan in the merged image. Scale bar: 10 µm.",
    "molecules": "SiR-DNA, DNA"
  },
  {
    "caption": "E. Quantification of spindle lengths at metaphase following indicated treatments. Graph represents the spindle length of individual cells with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siMCAK (34,3), siKIF4A + siKIF15 + STLC (46, 6), siKIF4A + siKIF15 + STLC + siMCAK (43, 3), siKIF4A + siKIF15 + STLC + siKIF2A (22, 3), siCLASPs (64, 6), siCLASPs + siMCAK (33, 3), siCLASPs + siKIF2A (32, 3).",
    "molecules": "STLC"
  },
  {
    "caption": "U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls.",
    "molecules": "nocodazole"
  },
  {
    "caption": "U2OS cells stably expressing GFP-PHF6 were laser-irradiated and cells were analyzed by time-lapse imaging. In (B), the PARP inhibitor Olaparib was added 30 min before laser-induced damage. At least 60 cells were analyzed in three individual experiments and error bars represent the SD (lower panel). Scale bar is 5 μM.",
    "molecules": "Olaparib"
  },
  {
    "caption": "GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later genomic DNA was extracted and repair of I-SceI cut-sites analyzed through junction analysis (n= independently derived sequences, see materials and methods for details).",
    "molecules": "DNA"
  },
  {
    "caption": "(G) RT-qPCR of ZIKV RNA in hNSCs incubated with vehicle or 100 nM rapamycin, 20 μM chloroquine, 10 nM bafilomycin A1, or 5 mM 3-methyladenine for 24 h. Mean ± SEM of 3 biological replicates. *p < 0.05, ***p < 0.001 by Student's t test.",
    "molecules": "3-methyladenine, bafilomycin A1, chloroquine, RNA, rapamycin"
  },
  {
    "caption": " (H) GFP-LC3 puncta in ZIKV with or without rapamycin and 3-MA treated hNSC cells. Cells were treated with or without rapamycin (100 nM) or 3-methyladenine (5 μM). Box plots show the mean and the smallest; largest values in whiskers represent the 10th and 90th percentiles in 50 randomly selected cells per group; and solid horizontal line indicates median. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test. ",
    "molecules": "3-MA, 3-methyladenine, rapamycin"
  },
  {
    "caption": " (I) RT-qPCR of ZIKV RNA in infected hNSCs transfected with siRNAs targeting LC3, BECN or ATG9A 48 h post infection. Mean ± SEM of 3 biological replicates. *p < 0.05 by Student's t test. ",
    "molecules": "RNA"
  },
  {
    "caption": " Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the SVZ of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone. ",
    "molecules": "chloroquine, DAPI, rapamycin"
  },
  {
    "caption": " Immunostaining and quantification of neural progenitor marker SOX2 (red) and ZIKV envelope flavivirus group antigen (ZIKVE, green) in the hippocampus of uninfected or ZIKV Paraiba-infected wild-type mice treated with vehicle, rapamycin, or chloroquine at 6 days post-infection. n=10 sections/group, mean ± SEM, *p < 0.05, **p < 0.001. Nuclei were stained with DAPI (blue). Right-most column shows enlargements of the boxed regions. STR = striatum, CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, SVZ = subventricular zone. ",
    "molecules": "chloroquine, DAPI, rapamycin"
  },
  {
    "caption": "(K) Analysis of E2F4 ChIP-seq datasets (Encode) showing direct chromatin-binding interaction between E2F4 and FA pathway genes in HeLa (GEO: GSM935365, Farnham lab), K562 (GEO: GSM935600, Farnham lab), and MCF10A (GEO: GSM: 935400, Struhl lab) cell lines. H3K9ac and H3K4me3 datasets identify promoter regions. The genomic region is displayed in log scale with the scale range identified in each panel (0-30). The summed interaction (Sum) across all three cell types is represented as a heatmap.",
    "molecules": "H3K9ac, H3K4me3"
  },
  {
    "caption": "Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunostaining of neural progenitor marker SOX2 (green) and ZIKV envelope flavivirus group antigen (ZIKVE, red) in the SVZ of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Right-most column in (A) shows enlargements of the regions. CC = cortical cortex, GCL = granular cell layer, LV = lateral ventricle, SGZ = subgranular zone, STR = striatum, SVZ = subventricular zone. Scale bars, 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Ifnar-/- mice. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.",
    "molecules": "RNA"
  },
  {
    "caption": "(H) RT-qPCR analysis of relative ZIKV RNA in different brain regions of Fancc KO neonatal mice six days post-infection. mean±SEM of n=3 biological replicates, **p<0.005, ***p<0.001 by Student's t test.",
    "molecules": "RNA"
  },
  {
    "caption": "(A) Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the hippocampus of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). Bottom row shows enlargements of the hippocampus region. GCL = granular cell layer, SGZ = subgranular zone, DG = dentate gyrus, InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunostaining of immature neuronal marker DCX (green) and ZIKV envelope (ZIKVE, red) in the olfactory bulb of uninfected or ZIKV Paraiba-infected Ifnar-/- mice six days post-infection. Nuclei were stained with DAPI (gray). InGr = internal granular layer, Me = medulla, OB = olfactory bulb. Scale bars, 50 and 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E-F) Serum levels of T (E) and LH (F) in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.",
    "molecules": "LH"
  },
  {
    "caption": "(B) Serum levels of A-dione in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.",
    "molecules": "A-dione"
  },
  {
    "caption": "(B) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT. Values are normalized to protein expression.",
    "molecules": "DHT"
  },
  {
    "caption": "(C) Western blot on cytoplasmic, nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT. Both panels represent the same blot. The blot was cut in two parts and antibodies against HSP90 and Lamin A/C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins. For AR visualization (upper panel), longer exposure time was used. # = residual AR expression (lower panel).",
    "molecules": "DHT"
  },
  {
    "caption": "(B) Confocal fluorescence microscopy images of AR-WT-BirA* and AR W752R-BirA*expressing HEK293 cells treated in the presence of 50 µM biotin with 100 nM DHT or vehicle (ethanol) and with or without 0.03 µg/ml TET as indicated. AR-BirA*s were detected with anti-AR (red) and biotinylated proteins with fluorescently labeled streptavidin (green). Nuclei were visualized using DAPI.",
    "molecules": "DHT, biotin, DAPI, ethanol, TET"
  },
  {
    "caption": "MPK4 and MPK6 were immunoprecipitated from extracts of Col‐0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (−), extracts were incubated with agarose beads without antibodies. IPs were incubated with His6‐PAT1, His6‐PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS‐PAGE. Autoradiogram (top panel), Coomassie‐stained gel for loading control (bottom).",
    "molecules": "agarose, His6"
  },
  {
    "caption": "Confocalmicroscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(A) THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/mL LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1β were determined in supernatants.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(C) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(E) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(F) Levels of IL-1β were determine in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/mL LPS for overnight and then with 0.25mM of Silica, Alum, or monosodium urate (MSU).",
    "molecules": "Alum, LPS, Silica, monosodium urate, MSU"
  },
  {
    "caption": "(G) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data, means ± SEM; n≥5 (except for B where a representative data set from 3 repeats). *P < 0.05 (t test or ANOVA).",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(A and B) Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(C) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(F) Confocal microscopy of THP-1 cells treated with LPS and LLOMe, and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Pearson correlation coefficient was calculated using SlideBook as described previously. Scale bars, 5 μm.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(G) In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (-) of flag-ULK1 and cold ATP, GST pulldowns were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by PAGE and autoradiography. Amounts of GST fusion proteins are shown in coommassie brilliant blue (CBB)-stained gels. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (t test or ANOVA).",
    "molecules": "ATP"
  },
  {
    "caption": "(A and B) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation. HeLa cells reconstituted with flag-pro-IL-1β and myc-pro-Caspase-1 were treated with 1mM LLOMe for 1h, and lysates were subjected to differential centrifugations at 3,000xg (3k), 25,000xg (25k) and 100,000xg (100k) as depicted in the cartoon. Arrows, two ways the membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B-D, or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(C) IL-1β levels in 25k membrane fractions (pellets) from HeLa cells and their CRISPR knockout derivatives (TRIM16KO) that were reconstituted for IL-1β secretion and treated with 1mM LLOMe for 1 h.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(H) Confocal images of LC3 in HeLa cells expressing GFP-Sec22b treated with LLOMe or untreated (control). Arrowheads, colocalization. Scale bars, 5 μm. (I) GFP-Sec22b and LC3 overlapping area obtained by high content microscopy.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(K) Co-IP analysis of endogenous proteins, TRIM16 and Sec22b, in lysates from THP-1 cells treated with LLOMe.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(A and B) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then (A) with LLOMe or (B) starved in EBSS.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(C) IL-1β levels in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C) or starved in EBSS (B).",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C).",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(C) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(E) Levels of ferritin were determine in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe.",
    "molecules": "LLOMe, LPS"
  },
  {
    "caption": "(F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm;",
    "molecules": "LLOMe"
  },
  {
    "caption": "F NALCN  knockdown suppresses invadopodia  formation reported by Cy3-fluorescent gelatin  degradation (arrows). Confocal  images (left) of Cy3  (red) and DAPI  (blue; nuclei) fluorescence in control (shCTL) and shNALCN  48 hours after plating the cells on gelatin. Bar diagram plot: NALCN  suppression causes significant decrease of the number of gelatin  degradation puncta counted within 48 h of incubation. Data information: The images: scale bar is 100 µm. Bar diagram plots show the degradation puncta per cell (F) mean±S.E.M. for N=5 (F) biological replicates per condition. **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test.",
    "molecules": "Cy3, DAPI, gelatin"
  },
  {
    "caption": "C Top: temporal profiles of self-normalised fluorescence intensity (∆F/F0) of fluo-4  (green), averaged within outlined region (inset: left, green outline), and mCherry  (red), averaged within 14 circles (inset: right, red outlines). Middle: galleries show confocal  images of fluo-4  and mCherry  fluorescence captured during two periods of interests, POIs (blue bars): for POI1- every image, for POI2 - every 100th image. Bottom left: confocal  images of Dynamin2-mCherry  fluorescence captured before and 3 min after stimulation with 10% FBS. Bottom middle and right: plots relate the dynamics of [Ca2+]c changes (Fluo-4) to formation of dynamin  puncta (Dynamin2-mCherry) at two regions of interests (ROIs) (insets); the galleries (below) show every 40th image (after 90° rotation) during the POIs (plots: blue bars).",
    "molecules": "Ca2+, fluo-4, Fluo-4"
  },
  {
    "caption": "D Top the overlay of NALCN-GFP  and Cal-590  images is related to the gallery of self-normalized rainbow-coded Cal-590 images showing [Ca2+]c response to 10% FBS. Bottom: 3 rainbow-coded Cal-590 images are superimposed on grey-coded NALCN-GFP  image and rotated by 90°. Arrows: NALCN-enriched structure.",
    "molecules": "Cal-590, Ca2+"
  },
  {
    "caption": "A FBS-induced transient [Ca2+]c elevations (Fluo-4, top left) are associated with transient decreases of Ca2+  concentration in endoplasmic reticulum ([Ca2+]ER) (Mag-Fluo-4, bottom left). Corresponding galleries (right) show every 100th image during the highlighted POIs (I and II, respectively). Visualization of the ER  with pDsRed2-ER  confirms the ER  origin of Mag-Fluo-4 signal (inset bottom left: confocal  images of cell fragment).",
    "molecules": "Mag-Fluo-4, Ca2+, Fluo-4"
  },
  {
    "caption": "C-D [Ca2+]c oscillations reported by  Fluo-4  fluorescence in FBS-exposed  PC-3 cells pre-treated with siCTL (C) or siNALCN (D) for 72 hours. Top: sample traces from 8 different cells. Middle: POIs (top: grey background) are presented on enlarged plots to emphasize that NALCN  suppression abolishes \"pacemaker events\" observed in control (fitted red curves). Bottom: the galleries show every 3rd image during the POIs (middle: orange background). Scale bars: 10 μm.",
    "molecules": "Ca2+, Fluo-4"
  },
  {
    "caption": "A-D Cytosolic Na+ concentration ([Na+]c) changes reported by ratiometric Na+  indicator SBFI  following (A-C) switch of extracellular Na+ concentration ([Na+]o) from 0 to 130 mM or (D) application of 1 µM thapsigargin  (TG). The effects of: (A) NALCN  knockdown, (B) extracellular Ca2+ concentration ([Ca2+]o) and (C) Ca2+  store depletion. Data information: Data show mean±S.E.M. The cartoons highlight experimental design and the plots show mean±S.E.M. traces (n=45-501) of SBFI  (olive) and Fura-2 (wine) fluorescence with the axes (∆R/R0) presented in corresponding colour.",
    "molecules": "Ca2+, Fura-2, Na+, SBFI, TG, thapsigargin"
  },
  {
    "caption": "E-H Store-operated Ca2+  entry (SOCE)-induced changes of [Na+]c (SBFI) and [Ca2+]c (Fura-2). The effects of: NALCN  knockdown (E), the reverse-mode plasmalemmal Na+/Ca2+ exchanger (RM-NCX) inhibitor KB-R7943 (1 µM) either at [Na+]o=130 mM (F) or upon [Na+]o switch from 0 to 130 mM following SOCE activation (G), and knockdown of the mitochondrial Na+/Ca2+ exchanger, siNCLX (H). Data information: Data show mean±S.E.M. The cartoons highlight experimental design and the plots show mean±S.E.M. traces (n=45-501) of SBFI  (olive) and Fura-2 (wine) fluorescence with the axes (∆R/R0) presented in corresponding colour.",
    "molecules": "Ca2+, Fura-2, KB-R7943, Na+, SBFI"
  },
  {
    "caption": "I SOCE augments NALCN-mediated Na+ influx  (top) by addressing NALCN  to plasma membrane (bottom). Data information: Plots (I, top) compare mean rates of Na+ influx  induced by [Na+]o-switch (n=283-360) and SOCE (n=180-216), and their shNALCN-sensitive fractions. Immunoblotting  (I, bottom) compares NALCN expression  in total cell lysates and biotinylated fractions before and after SOCE induction. Numbers show (in corresponding colour) mean values (n=3) of NALCN  protein levels (band intensity) normalized to N-Cadherin, Calnexin  and β-actin.",
    "molecules": "Na+"
  },
  {
    "caption": "J-K The effect of KB-R7943  (1 µM) on FBS-induced [Ca2+]c responses (Fura-2, ∆R/R0) and inward current  (perforated patch, Vh=-80 mV), respectively. Data information: The bar diagram plots show mean signal temporal densities (J, n=20) and mean current  densities (K, n=6-8) during initial transient (cyan) and oscillations (orange). n= number of cell **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test.",
    "molecules": "Ca2+, Fura-2, KB-R7943"
  },
  {
    "caption": "A Top: visualisation of mitochondria  with  MitoTracker Green confirms mitochondrial  origin of Rhod-2 signal. Middle: FBS-induced [Ca2+]c oscillations (Fluo-4) are associated with [Ca2+]mito oscillations (Rhod-2). Bottom: every 2nd image from the highlighted (orange background) period. B Top: FBS-induced [Ca2+]c oscillations (Fluo-4) are associated with oscillations in O2-  production by mitochondria  (MitoSOX Red). Bottom: every 3rd image from the highlighted (orange background) period. Data information: Cartoons (left) highlight experimental design and assessed signaling pathways.",
    "molecules": "MitoSOX Red, Ca2+, Fluo-4, MitoTracker Green, Rhod-2, O2-"
  },
  {
    "caption": "C O2- metabolite, H2O2, suppresses FBS-induced [Ca2+]c oscillations (Fluo-4). Data information: In C, mean signal temporal density during POI2 was normalized to that during POI1 in control (n=58) and in 50 µM of H2O2  (n=74), and compared. Bar diagram plots: mean±S.E.M. n= number of cell, ***P<0.001, unpaired two-tailed Student's t-test. Cartoons (left) highlight experimental design and assessed signaling pathways.",
    "molecules": "Ca2+, Fluo-4, H2O2, O2-"
  },
  {
    "caption": "D Suppression of NCLX  with siNCLX attenuates [Ca2+]c oscillations (Fluo-4). Data information: In D, mean signal temporal densities during highlighted periods were compared in control (n=18) and following 48 h pre-treatment with siNCLX (n=17). Bar diagram plots: mean±S.E.M. n= number of cell, ***P<0.001, unpaired two-tailed Student's t-test. Cartoons (left) highlight experimental design and assessed signaling pathways. Note that Na+ delivered to the cytosol  by NALCN, on the one hand, activates RM-NCX, while on the other hand, is exchanged by NCLX  to Ca2+ thus restricting elevation of [Ca2+]mito and, hence, production of superoxide  (O2-) and H2O2, which is known to inhibit SERCA, RM-NCX and SOCE elements.",
    "molecules": "Ca2+, Fluo-4, H2O2, Na+, O2-, superoxide"
  },
  {
    "caption": "E Plot: mean rates of secretion calculated as signal mass (cyan, left) per second. Data information: Data (E): mean±S.E.M. for siCTL (n=35), siNALCN (n=40), Control (n=45), EGTA-AM  (n=35) and BAPTA-AM  (n=62). n= number of cell, ***P<0.001; and n.s., not significant, unpaired two-tailed Student's t-test.",
    "molecules": "BAPTA-AM, EGTA-AM"
  },
  {
    "caption": "C) Cellular oxygen consumption rates (OCR) in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data were obtained using Seahorse XF24 analyzer and shows mean±S.D. n=4 technical repeats (representative of 2 independent experiments);",
    "molecules": "oxygen"
  },
  {
    "caption": "D) Representative 3D EM pictures of senescent (20 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts with or without CCCP. Mitochondria are in red, the nucleus in blue;",
    "molecules": "CCCP"
  },
  {
    "caption": "F) Quantification of ROS levels (DHE fluorescence), Sen-β-Gal positive cells and Senescence-associated heterochromatin foci (SAHF) observed by DAPI in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "ROS"
  },
  {
    "caption": "A) Representative images of colony assays of wild-type and PGC-1β -/- MEFs grown at 3% or 21% O2 (10 days after seeding 5,000 cells/well). Data are representative of 3 independent experiments;",
    "molecules": "O2"
  },
  {
    "caption": "B) Effect of 3% or 21% O2 and X-ray irradiation (at 3% O2) on the percentage of Ki67 (at day 6) and Sen-β-Gal positive cells (at day 10) and number (N) of 53BP1 foci (at day 6) in wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "O2"
  },
  {
    "caption": "E) Effects of overexpression of PGC-1β on the percentage of Ki67 and Sen-β-Gal positive cells, number (N) of 53BP1 foci and percentage change of mitochondrial mass (NAO intensity) in proliferating and senescent (2 days after 10Gy X-ray) MEFs cultured at 3% O2. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "O2"
  },
  {
    "caption": "B) Representative western blots of the mitochondrialproteins TOMM20, NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MT-CO1 (complex IV) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5fibroblasts. Data are representative of 4 independent experiments;",
    "molecules": "Rapamycin"
  },
  {
    "caption": "C) Effect of 100nM rapamycin on mitochondrial mass (measured by NAO fluorescence) 2-4 days following replication exhaustion (RS) or genotoxic stress (generated by X-ray irradiation, Etoposide, Neocarcinostatin (NCS), H2O2 or telomere dysfunction (TRF2ΔBΔM)) in a variety of cell lines. Data are mean from 3 independent experiments per cell line or treatment;",
    "molecules": "Etoposide, H2O2, NCS, Neocarcinostatin, rapamycin"
  },
  {
    "caption": "D) (top) Representative transmission electron microscopy (T.E.M.) micrographs of proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. Mitochondria are labeled in pink. Scale bar=2µm; (bottom left and middle) Quantification of mitochondrial volume fraction (%Vv) and mitochondrial number per cross section in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. T.E.M. mitochondrial analysis are mean±S.E.M of 24 electron micrographs per condition; (bottom right) mtDNA copy number analysis by q-PCR in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "rapamycin, Rapamycin"
  },
  {
    "caption": "E) Representative western blots showing expression of phosphorylated p70S6K (T389) and AKT (S473), the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis (AT) at different time points after 20Gy X-ray. Data are representative of 3 independent experiments (ATM inhibitor) and 1 experiment (AT patient);",
    "molecules": "KU55933"
  },
  {
    "caption": "F) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A.X, AKT phosphorylation and p21 expression in MRC5fibroblasts after 20Gy X-ray. Data is from one experiment;",
    "molecules": "KU55933"
  },
  {
    "caption": "G) Effect of rapamycin and/or ATM inhibitor (KU55933) on mitochondrial mass (NAO intensity) in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "KU55933, rapamycin"
  },
  {
    "caption": "A) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci after mTOR knockdown (72 hours) in proliferating and senescent (2 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "ROS"
  },
  {
    "caption": "B) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin and/or 2.5mM of the antioxidant NAC. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "NAC, ROS, Rapamycin"
  },
  {
    "caption": "C) (top) Scheme illustrating the experimental design: human MRC5 fibroblasts were irradiated with 20Gy X-ray and treated at day 3 after IR with 10µM of the ATM inhibitor (ATMi) KU55933 and/or 100nM Rapamycin (Rap); (bottom) Effect of single or combined inhibition of ATM and mTORC1 on the mean number (N) of γH2A.X foci, Sen-β-Gal activity and p21 expression in proliferating and senescent (10 days after 20Gy X-ray) MRC5fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Western blots are representative of 3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "KU55933, Rap, Rapamycin"
  },
  {
    "caption": "D) ROS levels (DHE intensity) in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β-/- MEFs (top) and PGC-1β overexpressing MEFs (bottom). Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "ROS"
  },
  {
    "caption": "E) (top) Representative images and quantification of immunofluorescence staining against FLAG-PGC-1β (red) and 53BP1 (green) in proliferating and senescent (2 days after 10Gy X-ray) PGC-1β overexpressing MEFs cultured at 3% O2, with or without 250µM of the antioxidant Trolox (scale bar=10µm). Data are mean±S.E.M, n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "O2, Trolox"
  },
  {
    "caption": "F) (top) Effect of overexpression of mutated Rheb (N153T) and (bottom) effect of 100nM rapamycin on the mean number (N) of 53BP1 foci in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments (at least 125 cells were analyzed per condition). Asterisks denote statistical significant P<0.05 One-way ANOVA.",
    "molecules": "rapamycin"
  },
  {
    "caption": "C) Effect of 4 months of rapamycin supplemented diet on PGC-1β expression in liver tissue of 16 months old mice. Data are from n=3 mice per group;",
    "molecules": "rapamycin"
  },
  {
    "caption": "D) (top) Quantification of mitochondrial number per cross section and mitochondrial volume fraction (%Vv) in hepatocytes from 16 months old mice with or without 4 months rapamycin diet. Data are mean±S.E.M of n=3 mice per group (at least 20 cells were analyzed per mouse); (bottom) Representative electron micrographs of hepatocytes from 16 months old mice with or without 4 months rapamycin diet, (mitochondria are labelled in pink). Scale bar=5µm;",
    "molecules": "rapamycin"
  },
  {
    "caption": "E) mtDNA copy number (measured by q-PCR) in mice liver tissue at 3, 12, 16 months and at 16 months after 4 months rapamycin diet. Data are mean±S.E.M of n=3-4 mice per group; Asterisks denote statistical significant P<0.05 using One-way ANOVA.",
    "molecules": "rapamycin"
  },
  {
    "caption": "F) Oxygen consumption rates (OCR) in isolated liver mitochondria from 16 months old mice fed with or without rapamycin for 4 months, in the presence of pyruvate/malate. State III was induced by injection of ADP. State IV was induced by inhibition of the ATP synthase with oligomycin and uncoupled respiration rates were determined by injection of FCCP. Antimycin A (AA) was used to determine background, non-mitochondrial OXPHOS, OCR. Data are mean±S.E.M of n=5 mice per group;",
    "molecules": "ADP, Antimycin A, FCCP, malate, oligomycin, pyruvate, rapamycin"
  },
  {
    "caption": "G) (top) Representative immuno-TeloFISH images of hepatocytes from 3 and 15 months old mice with or without rapamycin (12 months diet). Co-localizing foci are amplified in the right panel (amplified images are from single Z-planes where co-localization was found); (bottom) Dot plot graph of Telomere-associated foci (TAF) in 3, 15 and 16 months old mice (15 and 16 months old mice were fed with rapamycin for 12 and 4 months respectively). Data are from n=3-9 mice per group (at least 50 cells were analyzed per mice);",
    "molecules": "rapamycin"
  },
  {
    "caption": "H) (top) 4 months old and 15 months old micelivers [control (-) or rapamycin (+)] stained with Sen--β-Gal solution (Sen--β-Gal activity is evidenced by blue staining). Data are from n=3 mice per group; (bottom) Representative image showing Sen--β-Gal staining (Sen--β-Gal activity is evidenced by blue staining) in hepatocytes and corresponding immuno-TeloFISH (arrows represent co-localizing foci); Asterisks denote statistical significant P<0.05 using One-way ANOVA.",
    "molecules": "rapamycin"
  },
  {
    "caption": "I) Representative western blot showing the effect of 4 months rapamycin feeding on p21 expression in 16 months old mice. Data are from n=3 mice per group;",
    "molecules": "rapamycin"
  },
  {
    "caption": "j) Effect of 4 months rapamycin feeding on mRNA expression of the SASP components CXCL1, CXCL5 and Inhibin A in liver tissue of 16 months old mice with or without 4 months rapamycin treatment. Data are from n=5 mice per condition; Asterisks denote statistical significant P<0.05 using Two-tailed t-test.",
    "molecules": "rapamycin"
  },
  {
    "caption": "C. Confocal images of 8-day-old wild type WT, bri1-116 and pRP5SA:BRI:YFP;bri1-116 roots stained with PI. Green lines label the end of the transition zone and the yellow lines label the first root hair (end of elongation zone). The inset shows pRP5SA expression domain of an 8-day-old pRP5SA:BRI:YFP;bri1-116 seedling. Green line labels the end of the meristematic zone, which coincides with the end of pRP5SA expression domain. Scale bars correspond to 100 microns.",
    "molecules": "PI"
  },
  {
    "caption": "Simulation results are depicted in gray for Ruler, pink for Timer and red for Sizer models (n=28 each). Data from cortex tissue in Col-0 grown with 1µM PAC is in green (n=28, day 6 post germination). Parameter values for the models (Table EV3) selected such that no statistical significance is found between model (for each model) and cortex data in any of the five phenotypic traits depicted in A (Wilcoxon-rank sum test, p-value >0.01, Table EV4). For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rates, the meristematic activities and the initial cell length. The parameter values are the same for the three models except for the threshold for cell elongation termination, which has relative variability of 16% (Ruler), 2% (Timer) and 14% (Sizer). A Boxplots for phenotypic traits: elongation factor rEZ , mature cell length (length of the EZ cell closest to the DZ) lmax, length of the elongation zone LEZ , number of cells in the elongation zone NEZ and root growth rate. For the cortex data, the first four phenotypic traits are all measured in the same root files (n=28, day 6 post germination), while the root growth rate is measured on a different set of roots (n=40, Methods).",
    "molecules": "PAC"
  },
  {
    "caption": "a) Rapamycin (Rap) treatment or nutrient deprivation attenuated the expression of the Wnt reporter TopFlash in HEK-293T cells. (n = 3; two asterisks, P 0.01). DMSO, dimethylsulphoxide.",
    "molecules": "dimethylsulphoxide, DMSO, nutrient, Rapamycin"
  },
  {
    "caption": "(b) Expression analysis of Wnt target genes by quantitative RT-PCR after 4 h of treatment with rapamycin. (n = 3; asterisk, P 0.05; two asterisks, P 0.01). Wnt3a CM, Wnt3a conditioned medium.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(e) The continued degradation of Dvl2 in Atg5−/− cells is proteasome-dependent. MEFs were starved for 2, 4 or 8 h with MG132 (1 μM) or BFA1 (0.1 μM) for different durations, followed by anti-Dvl2 immunoblotting to detect endogenous Dvl2 protein levels. Tubulin served as a loading control. Uncropped images of blots are shown in Supplementary Information, Fig. S8.",
    "molecules": "BFA1, MG132"
  },
  {
    "caption": "(a) Immunoelectron microscopy analysis of HeLa cells transfected with Flag-Dvl2 under nutrient-rich or starvation conditions. The arrowheads indicate Flag-Dvl2. Scale bars, 1 μm (left); 250 nm (right).",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) Localization of endogenous Dvl proteins (red) and GFP-LC3 (green) in GFP-LC3 stable HeLa cells in nutrient-rich medium or under starvation (starv.) conditions with BFA1 for 4 h. The right panel shows the percentage co-localization between Dvls and GFP-LC3-positive dots. Scale bar, 5 μm.",
    "molecules": "BFA1, nutrient"
  },
  {
    "caption": "(d) Dvl2-LC3 interaction in vivo. IB, immunoblotting; IP, immunoprecipitation; S, starvation; WCL, whole-cell lysate; N, nutrient-rich.",
    "molecules": "nutrient"
  },
  {
    "caption": "(b) Ubiquitylation of wild-type Dvl2 and its variants. Transfected HEK-293T cells were treated with BFA1 (0.2 μM) for 6 h. Ubiquitylated proteins were precipitated with Ni-nitrilotriacetate (Ni-NTA) beads, followed by anti-HA immunoblotting. Ub, ubiquitin.",
    "molecules": "BFA1"
  },
  {
    "caption": "(d) Starvation elevates Dvl2-Cul2 interaction. After co-transfection with Myc-Cul2 and Flag-Dvl2 for 36 h, HEK-293T cells were cultured in nutrient-rich medium or starved with BFA1 (0.1 μM) for 4 h. Cells were harvested for anti-Myc immunoprecipitation followed by anti-Flag immunoblotting. Total protein expression was confirmed by immunoblotting with whole-cell lysates.",
    "molecules": "BFA1, nutrient"
  },
  {
    "caption": "(B) Western blots for Rab11a and Rab11b using small intestinal lysates prepared from adult wild type (WT, lane 1), Rab11aFl/Fl;Villin-CreER (aKO, lane 2) 2 days after tamoxifen treatment, Rab11b-/- (bKO, lane 3), and Rab11aFl/Fl;Rab11b-/-;Villin-CreER (DKO) mice 1-day (lane 4), 2-day (lane 5), and 3-day (lane 6) after tamoxifen treatment. All mice were given a single tamoxifen injection. β-actin was used as loading control. Results represent more than 3 independent experiments.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(C) H. & E. staining of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice, before and after tamoxifen injection. Images represent jejunum tissues collected 1, 2, and 3 days following tamoxifen injection. Experiments were repeated over 5 times using independent litters (n>10). Scale bars, 50 μ",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(D) Alkaline phosphatase (AP) staining was performed on intestinal tissue sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice that were treated with corn oil or with tamoxifen. Results represent at least 3 independent experiments. Scale bars, 50 μm.",
    "molecules": "corn oil, tamoxifen"
  },
  {
    "caption": "(F) Alcian blue staining was performed on adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before or 1 to 3 days after tamoxifen injection. Results represent at least 3 independent experiments. Scale bars, 100 μm. (G) Quantification of the number of alcian blue positive cells per crypt-villus axis in mice of various genotypes. Data represent average values of approximately 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "Alcian blue, alcian blue, tamoxifen"
  },
  {
    "caption": "(H) Immunofluorescent staining for lysozyme and E-Cad was performed on adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (I) Quantification of lysozyme fluorescent signal abundance per crypt in mice of various genotypes. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(B) Immunohistochemistry for Ki67 staining was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (C) Quantification of ratio of Ki67+ cells over total epithelial cells per crypt-villus axis in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(D) Immunofluorescent staining for pHH3 and E-Cad was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (E) Quantification of ratio of pHH3+ cells over total IECs per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(G) Representative Western blots for Olfm4 and pHH3 in intestinal tissue lysates of DKO mice before and after tamoxifen injection.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(H) Immunohistochemistry for Olfm4 was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (I) Quantification of Olfm4+ cell number per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(J) Immunofluorescent staining for Sox9 and E-Cad in DKO mice before and after tamoxifen treatment. Scale bars, 50 μm. (K) Quantification of Sox9+ cell number per crypt in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(L) Immunohistochemistry for c-Myc was performed on intestinal sections of adult Rab11aFl/Fl;Rab11b-/-;Villin-CreER mice before and after tamoxifen injection. Experiments represent 3 independent replicates. Scale bars, 50 μm. (M) Quantification of c-Myc+ cell number per crypt-villus axis in DKO mice before and after tamoxifen treatment. Data represent approximately 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(F) High magnification images were taken from CIdU/IdU labeled WT and DKO mouse crypt regions to visualize double positive cells. White arrows point to shedding CIdU cells. Scale bars, 50 μm. (G-H) Quantification of IdU+ cells, and shedding CIdU+ cells per crypt-villus axis in WT and DKO mice. Data represent 10-15 different microscopic fields taken from 3 mice per condition.",
    "molecules": "CIdU, IdU"
  },
  {
    "caption": "(I) Immunofluorescent staining for KIf11 (green) and E-Cad (grey) in WT and DKO mouse intestines. Yellow arrows point to Kif11+ spindles. Images represents at least 10 images per mouse and 5 mice for each genotype. Scale bars, 50 μm. (J) WT and DKO mice were injected with IdU 3 hrs before sacrifice to label new dividing cells. Immunofluorescent staining for KIf11 (green), E-Cad (grey), and IdU (red) were performed. Yellow arrows point to abnormal Kif11 localization in DKO cells. Scale bars, 50 μm.",
    "molecules": "IdU"
  },
  {
    "caption": "(G) Quantification of co-immunoprecipitated KIF11 was normalized to input KIF11 from cells treated by different inhibitors. DMSO data was quantified for 5 experimental replicates and all other treatments were quantified for 4 experimental replicates.",
    "molecules": "DMSO"
  },
  {
    "caption": "(H) HEK293T cells were transiently transfected with 3×Flag-Rab11a, treated with nocodazole (100 ng/ml) or vehicle overnight, and lysates were used for Flag immunoprecipitation followed by immunoblot for KIF11. Quantification of co-immunoprecipitated KIF11 was normalized to input KIF11 from control (n=6) and nocodazole-treated (n=9) cells.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(B) Propidium iodide (PI) staining of WT, BKO, and DKO enteroids 3 days after 4-OHT treatment ex vivo. Note DKO enteroids had increased PI staining along the epithelial edge. Vehicle treated DKO enteroids were used as controls. Scale bars, 200 μm. (C) PI-stained areas were normalized to total enteroid area for each enteroid of different genotypes.",
    "molecules": "4-OHT, PI, Propidium iodide"
  },
  {
    "caption": "Immunofluorescent staining for Kif11 (red) and E-cad (green) were performed on enteroids of various genotypes, all of which were treated with 4-OHT. Same experiments were done for DKO enteroids treated with vehicle or 4-OHT. White arrows point to Kif11+ spindles. Scale bars, 20 μm.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(I-J) Numbers of pHH3+ or Lyz1+ cells were counted per enteroid of vehicle (n=11 for pHH3; n=8 for Lyz1) or 4-OHT treated DKO (n=9 for both pHH3 and Lyz1) enteroids from biological replicates.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(K-L) Representative confocal fluorescent images of pHH3 (red) or Lyz1 (red) stained with E-Cad (green) from vehicle or 4-OHT treated DKO enteroids. Scale bars, 50 μm.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(A) Immunohistochemistry for cleaved caspase 3 (CC3) was performed on TAM-treated enteroids of different genotypes. Black arrows point to CC3+ cells at the epithelial lining of DKO enteroids. Scale bars, 50 μm.",
    "molecules": "TAM"
  },
  {
    "caption": "(I) Immunofluorescent staining for CC3 (green) was performed on WT and Rab11 DKO intestinal epithelial tissues before and after tamoxifen treatment. Scale bars, 50 μm. (J) Quantification of CC3 positive cells per crypt-villus axis in WT and Rab11 DKO intestines. Results were obtained from 10-15 independent microscopic images taken from 4-5 mice for each genotype.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(A) Yeast two-hybrid assays to test for interaction between Munc18-1 and ERK1/2. pGAL4-Munc18-1 (M18) and pGAL4-Empty (control) were used as baits and ERK1 and ERK2 as prey. Both ERK1 and ERK2 bind Munc18-1 and allow growth on selection plates (minus Histidine (-HIS) and minus Histidine and Adenosine (-ADE)).",
    "molecules": "ADE, Adenosine, HIS, Histidine"
  },
  {
    "caption": "(B) In vitro kinase assays showing Munc18-1 phosphorylation by ERK. Radioactive phosphoblot (32P) and immunoblot (IB) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT, Munc184A (M18T78A,S158A,S241A,T574A), Munc183A (M18S158A,S241A,T574A), and Munc18AA (M18S241A,T574A) from HEK293T cells incubated with recombinant ERK2. The phosphoblot shows phosphorylated Munc18-1 (pMunc18-1) and auto-phosphorylated ERK2 (pERK2).(C) Quantification of phospho-Munc18-1 intensity in wild-type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18-1 normalized to total Munc18-1 levels. Phosphorylation of Munc18WT was set to 100%. (Munc18WT: 100±3%; Munc18AA: 24±2%, n=3, * p = 0.01).",
    "molecules": "32P"
  },
  {
    "caption": "(D) ERK1 in vitro kinase assay showing ERK-dependent 32P-incorporation in Munc18WT immuno-precipitated from HEK293T cells. Western Blot stained with Munc18-1 antibody shows total precipitated Munc18-1 levels were identical in all conditions.",
    "molecules": "32P"
  },
  {
    "caption": "(A) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 (10 M) and DMSO, the GABAA receptor blocker GABAzine (GBZ, 10 M) or the voltage-gated sodium channel blocker TTX (2 M) for 4 h. Blots were stained for pMunc18S241, Munc18-1 and tubulin as loading control.(B) Quantification of (A). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.4%; GBZ: 1.73 ± 0.12; TTX: 0.052 ± 0.002, n = 4, * p<0.05, ** p<0.01).",
    "molecules": "DMSO, MG132, GABAzine, GBZ, TTX"
  },
  {
    "caption": "(C) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 (10 M) and MEK inhibitor PD98059 (10 M) for 4 h. Proteasome block increases the levels of phosphorylated Munc18-1 and ERK1/2, which can be prevented by MEK inhibition. Blots were stained for pMunc18S241, Munc18, pERK1/2 and ERK1/2.(D) Quantification of (C). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.8%; MG132: 375 ± 14.2%; MG132 + PD98059: 24 ± 3%, n = 3, ** p<0.01).",
    "molecules": "PD98059, DMSO, MG132"
  },
  {
    "caption": "(E) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 (1 M) for 5 min or BDNF (100ng/ml) for 48 h in the presence of 2 M TTX and stained for pMunc18S241, Munc18, pERK and tubulin as loading control.",
    "molecules": "LY379268, TTX"
  },
  {
    "caption": "(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(F) Mean synaptic Munc18-1 intensity in cultures treated with GABAA receptor antagonist Bicuculline (40 μM, 48 h). (M18WT: 761 ± 121 a.u., n = 26; M18S241A: 1482 ± 150 a.u., n = 31, ** p < 0.01).",
    "molecules": "Bicuculline"
  },
  {
    "caption": "(H) Western Blot of lysates from high-density neuronal cultures from munc18-1 null mutant mice rescued with M18WT, M18S241A or M18S241D. Prior to lysis, cells were treated for 6 h with MG132 (10 M) or DMSO as control. Western Blots were stained for Munc18-1 and tubulin as loading control.(I) Quantification of (H). Munc18-1 levels were normalized to tubulin levels. Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant (M18WT: 1.62 ± 0.41; M18S241A: 1.26 ± 0.16; M18S241D: 7.53 ± 1.17, n = 3; **p<0.01).",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "(J) Ubiquitination of different Munc18-1 mutants in HEK293T cells. Western Blot of denatured Munc18-1 immuno-precipitations from cells expressing Munc18-1 mutants and HA-tagged ubiquitin. Prior to lysis, cells were treated for 8 h with MG132 (10 M). Ubiquitin covalently attached to Munc18-1 can be observed as a smear above the characteristic 68 kD Munc18-1 band on Western blots stained for HA-tag.(K) Quantification of immuno-precipitation in (J). Level of Munc18-1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18-1 levels. M18WT was set to 1 (M18WT: 1 ± 0.06; M18S241A: 0.84 ± 0.57; M18S241D: 2.70 ± 0.07, n = 3; **p<0.01). All data are expressed as mean ± SEM.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Western blot from whole cell lysate of munc18-1 null mutant neurons rescued with M18WT and incubated with WIN55,212-2 (5 M) for 30 s. Cells were harvested after the indicated time. CB1R activation leads to a rapid and transient increase in pERK activity.",
    "molecules": "WIN55,212-2"
  },
  {
    "caption": "(B-D) Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A. CB1R agonist WIN55,212-2 (5 M) was applied at t = 60 s for 30 s.(B) Amplitude of excitatory synapse response normalized to t=0 s. Neurons were stimulated by a single action potential every 30 s for 4 min (M18WTn = 21, M18S241An = 24, *p<0.05). Note the broken y-axis.(C) Ratio of the frequency of spontaneous release events after (t = 180 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 58.0 ± 2.3 %, n = 21; M18S241A: 84.2 ± 5.6 %, n = 24, * p < 0.05).(D) Ratio of amplitude of spontaneous release events after (t=240 s) and before (t=0 s) WIN55,212-2 application (M18WT: 85.0 ± 2.7 %, n = 21; M18S241A: 83.1 ± 2.6 %, n = 24).",
    "molecules": "WIN55,212-2"
  },
  {
    "caption": "(E-G) Autaptic neurons of munc18-1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55,212-2 (1 M), WIN55,212-2 (1 M) plus AM251 (1 M) or vehicle only (DMSO).(E) Amplitude of excitatory synapse response normalized to t=0 s. CB1R agonist WIN55,212-2 (1 M) or vehicle only (DMSO) were applied at t=60 s for 30 s. (WT + WIN n = 13; WT + DMSO n = 16, * p < 0.05). Note the broke Y-axis.(F) Paired pulse ratio (20 ms interval) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55,212-2 (1 M) at t = 60 s for 30 s. WIN55,212-2 application increases PP ratios indicative of a reduction of synaptic release probability (t = 0 s: 0.94 ± 0.1, n = 13; t = 240 s: 1.36 ± 0.2, n = 16, * p < 0.05).(G) Amplitude of excitatory synapse response normalized to t = 0 s. CB1R agonist WIN55,212-2 (1 M) was applied together with the CB1R antagonist AM251 (1 M) at t = 60 s for 30 s. (WT + WIN n = 13, same trace as 6F; WT + WIN + AM251 n = 15, * p < 0.05). Note the broke Y-axis.",
    "molecules": "AM251, DMSO, WIN55,212-2"
  },
  {
    "caption": "(H-J) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with M18WT or M18S241A. mGluR2/3 agonist LY379268 (1 M) was applied by bath-perfusion for 4 min.(H) Ratio of amplitude of evoked release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 55.0 ± 4.7 %, n = 14; M18S241A: 81.1 ± 3.6 %, n = 16, * p < 0.05).(I) Ratio of frequency of spontaneous release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 63.0 ± 5.7 %, n = 14; M18S241A: 85.1 ± 3.4 %, n = 16, * p < 0.05).(J) Ratio of the amplitude of spontaneous release events after (t = 240 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 81.0 ± 1.3 %, n = 14; M18S241A: 82.1 ± 3.3 %, n = 16).",
    "molecules": "LY379268, WIN55,212-2"
  },
  {
    "caption": "(A-D) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre-treatment with PD98059 (PD; 10 µM) compared to 0.1% DMSO (control).(A) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 196.36 +/- 16.51 pA, n = 10; PD: 196.42 +/- 18.61 pA, n = 11, p = 0.9).(B) DSE in neurons of DMSO pre-treated slices, induced by a 10s depolarization to 0 mV (arrow). Inset: example traces of 2 averaged EPSCs before (black) and after depolarization (grey).(C) DSE in slices pre-treated with PD. Insets: example traces of 2 averaged EPSCs before (red) and after depolarization (grey).(D) Comparison of DSE between control and PD. PD pre-treatment limits amplitude and duration of DSE. ** = p < 0.01; unpaired T-test with Welch's correction. Numbers in bars represent the number of neurons in each group.",
    "molecules": "PD98059, DMSO"
  },
  {
    "caption": "(E-H) ERK-pathway inhibition by pre-treatment with PD98059 (PD; 10 µM) blocks the effect of WIN55,212-2 (WIN; 2 µM) on the amplitude of EPSCs recorded in CA1 pyramidal neurons.(E) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 295.69 +/- 28.73 pA, n = 6; PD: 270.86 +/- 32.14 pA, n = 6, p= 0.6).(F) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of 0.1% DMSO (control) pre-treated slices. Inset: example traces of averaged EPSCs of baseline (black) and the last 5 min (grey).(G) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of PD pre-treated slices. Inset: example traces of averaged EPSCs of baseline (red) and the last 5 minutes (grey).(H) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference. * = p < 0.05; unpaired T-test. Numbers in bars represent number of slices in each group.",
    "molecules": "PD98059, DMSO, WIN55,212-2"
  },
  {
    "caption": " D PCR validation of 4 somatic, neoplasia-specific TE insertions. Primers were designed to target regions flanking the insertion sites. Yellow arrowheads indicate PCR products containing an insertion amplified in the clonal DNA but not in the neighboring gut tissue (non-clonal), head or thorax for the same fly. Short wild-type amplicon was detectable in all samples. Thorax DNA sample was not available for sample P15. ",
    "molecules": "DNA"
  },
  {
    "caption": " B TE families with tissue-specific, singleton TSD-bearing insertions detected in pooled gut or head DNA. ",
    "molecules": "DNA"
  },
  {
    "caption": "Splenocytes from C57BL/6 mice were freshly harvested. Resting and activated T cells, monocytes, macrophages, DCs and B cells were obtained as in Figure 2. The resting and activated immune cells were stained with biotinylated TAPBPL-Ig or control Ig，followed by streptavidin PE, as well as anti-CD4, CD8, CD11b, F4/80, CD11c, B220 or CD19 antibody to identify immune cells. statistical analysis showing the binding TAPBPL-Ig or control Ig to freshly harvest and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with control Ig. ** P<0.05 compared with resting cells.",
    "molecules": "PE, streptavidin"
  },
  {
    "caption": "Splenic cells were labelled with CFSE and cultured in 96-well plates that were precoated with anti-CD3 antibody and hTAPBPL-Ig or control Ig for 3 days as in (A). The cells were analyzed for CFSE levels by CD4+ and CD8+ T cells. statistical analysis of (D) CD4 and (E) CD8 T cell proliferation.",
    "molecules": "CFSE"
  },
  {
    "caption": "Purified human T cells were cultured with plate-bound anti-human CD3 antibody (1 µg/ml) in the presence of graded doses of hTAPBPL-Ig (1.5 and 3 µg/ml) or control Ig protein (1.5 and 3 µg/ml) for 3 days. Cell proliferation was measured by [3H] thymidine incorporation. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.",
    "molecules": "[3H] thymidine"
  },
  {
    "caption": "f, Kaplan-Meier analysis comparing PDAC-free survival of KrasG12D/+ p53−/− mice that were chloroquine-treated (CQ, red), vehicle-treated (PBS, black) or untreated (blue). a, e, f, The blue curve in all Kaplan-Meier plots represents the same colony of mice (KrasG12D/+, p53−/−, Atg7+/+, Atg5+/+). A Mantel-Cox test was used for statistics. b, d, Scale bars: 100 µm (b), 200 µm (d). *P 0.05, **P 0.01.",
    "molecules": "chloroquine"
  },
  {
    "caption": "c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR,",
    "molecules": "oxygen"
  },
  {
    "caption": "c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m.",
    "molecules": "oxygen"
  },
  {
    "caption": "e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m.",
    "molecules": "2-deocyglucose, 2-DG"
  },
  {
    "caption": ", g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates.",
    "molecules": "glucose, lactate"
  },
  {
    "caption": "h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype.",
    "molecules": "2-DG, 2-DG-6-phosphate"
  },
  {
    "caption": "i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row).",
    "molecules": "FDG"
  },
  {
    "caption": "H&E images of paraffin-embedded liver sections from 9-week-old mice (n=4 per group, 3 independent repeats).",
    "molecules": "paraffin"
  },
  {
    "caption": "Protein levels of Sel1L (left panel) and mRNA levels of Fgf21 (right panel) in primary mouse hepatocytes isolated from the tamoxifen-inducible Sel1L-knockout Sel1LERCre mice (2 independent repeats). Hsp90 , loading control for Western blot analysis. Ribosomal L32, loading control for qPCR analysis.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Blood glucose levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).",
    "molecules": "glucose"
  },
  {
    "caption": "serum insulin levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).",
    "molecules": "insulin"
  },
  {
    "caption": "serum triglyceride (TG) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).",
    "molecules": "TG, triglyceride"
  },
  {
    "caption": "serum cholesterol (CHOL) levels in 9-week-old mice after 6 hr fasting (n=6-10 per group).",
    "molecules": "CHOL, cholesterol"
  },
  {
    "caption": "Western blot analysis of Crebh protein in 9-week-old mice (n=3 per group, 2 independent repeats). WT mice injected i.p. with tunicamycin (Tm, 1.5 µg/g body weight) for 72 hours were included as a control. Hsp90 loading controls for Western blot analysis.",
    "molecules": "Tm, tunicamycin"
  },
  {
    "caption": "Western blot analysis of Crebh in the livers of 9-week-old mice after NP40-detergent fractionation into NP40 soluble (NP40S) and pellet (NP40P) (n=3 per group, 2 independent repeats). Hsp90 H2A loading controls for Western blot analysis.",
    "molecules": "NP40"
  },
  {
    "caption": "Western blot analysis of Crebh protein half-life in transfected WT and HRD1-/- HEK293T cells treated with cycloheximide (CHX) for indicated times. The decay of protein from one experiment is shown below. Hsp90, loading control for Western blot analysis.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Western blot analysis and quantitation of Crebh in Crebh-transfected WT (B) and HRD1-/- (C) HEK293T cells pre-treated with the proteasomal inhibitor bortezomib (BTZ) or lysosomal inhibitor chloroquine (CHQ) for 2 hours and then with CHX for additional 1 hour (n=2 per group, 2 independent repeats). Hsp90, loading control for Western blot analysis.",
    "molecules": "bortezomib, BTZ, chloroquine, CHQ, CHX"
  },
  {
    "caption": "Western blot analysis of Crebh ubiquitination following immunoprecipitation (IP) of Crebh-Flag and Crebh-N-Flag in HEK293T cells transfected with indicated plasmids. Samples were boiled with SDS before IP for denaturing IP and not so for native IP. These cells were treated with proteasomal inhibitor BTZ for the last 6 hr prior to immunoprecipitation. Hsp90, loading control for Western blot analysis.",
    "molecules": "BTZ, SDS"
  },
  {
    "caption": "A. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD-N) (left panel). Scale bar 1 µm. Right panel- Quantification of of cells with or without GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001) (ns- not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n=446), Δcho2 (n=272), Δopi3 (n=272)), SD-N (WT (n=398), Δcho2 (n=224), Δopi3 (n=213)).",
    "molecules": "FM4-64, nitrogen"
  },
  {
    "caption": "A, B. WT and Δopi3 cells expressing GFP-Atg8 were grown to log phase in SD-URA, and shifted to SD-N for 4 h, choline (1 mM) was not supplemented (-choline) or supplemented to SD and SD-N (+choline SD, SD-N) or only to SD-N (+choline, SD-N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel- Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns-not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD-N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared to WT (ns-not significant, *, p≤0.05, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.",
    "molecules": "choline"
  },
  {
    "caption": "C. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT and Δopi3 cells expressing GFP-Atg8 were grown in SD-URA and shifted to SD-N. Choline (1mM) was either excluded (-choline), added to growth and starvation medium (+choline SD, SD-N) or added only to starvation medium (+choline, SD-N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 µm. Right panel- Quantification of cells with GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; -Choline: WT (n=487), Δopi3 (n=316), Choline SD, SD-N: WT (n=251), Δopi3 (n=361), Choline SD-N: WT (n=320), Δopi3 (n=267). D. PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP-Atg8, with or without choline (1 mM) supplementation to SD-N. Samples were taken after 4 h in SD-N medium.",
    "molecules": "Choline, choline, FM4-64, PC"
  },
  {
    "caption": "C. Representative images of mNG-tagged Atg8 colocalized with mScarletI-PXVam7. WT, Δopi3, Δatg1 and Δopi3Δatg1 cells on the background of Δymr1, expressing mScarletI-PXVam7 under the CUP1 promoter from the knocked-out VPS38 locus, were grown to log phase in SD medium in the presence of 10 µM copper sulfate, and shifted to SD-N in the presence of 10 µM copper sulfate. Images were taken during SD-N (1-3 h) by widefield microscopy. D. Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 (C) by ImageJ. Statistical analysis was done by student's t-test, unpaired, two sided (ns- not significant), error bars represent SEM of at least three independent experiments. Number of cells analyzed for each strain, WT (n=51), Δopi3 (n=28).",
    "molecules": "copper sulfate"
  },
  {
    "caption": "E. Representative images of Δypt7, Δopi3Δypt7 cells expressing GFP-Atg8 (projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, with supplementation of choline (1 mM) to SD and SD-N as indicated. Images were taken during growth and starvation by confocal microscopy (left panel). Scale bar 1 µm. Right panel- quantification of cell with more than two GFP-Atg8 puncta, in Δypt7 and Δopi3Δypt7 at growth (SD) or starvation (SD-N) in the presence (+choline: SD, SD-N) or absence (-choline) of choline (1 mM). Incidence were normalized to Δypt7 (SD-N) control. Statistical analysis was done by Anova multiple comparisons test- Sidak's (***, p≤0.001). Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and condition, SD: Δypt7 (n=209), Δopi3Δypt7 (n=111), SD-N: Δypt7 (n=118), Δopi3Δypt7 (n=103), SD+Choline: Δypt7 (n=107), Δopi3Δypt7 (n=120), Δypt7 (n=84), Δopi3Δypt7 (n=74).",
    "molecules": "choline, Choline"
  },
  {
    "caption": "(A) 293/GFP-LC3 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium (FM), full medium with leupeptin (FM Leu), EBSS (ES), or EBSS with leupeptin (ES Leu) for 2 h. FM Leu (data not shown) is identical to FM alone. Quantification of GFP-LC3‐positive autophagosome number was performed by counting in a blinded experiment. Bars=5 μm (data are represented as mean±s.e.m. of 60 cells, ***P=0.0002, Students t‐test).",
    "molecules": "leupeptin"
  },
  {
    "caption": "(E) HEK293A cells were transfected with HA-p38IP or myc-ULK1 C‐terminal domain (CTD). After 24 h, cells were labelled with [14C]valine as described in Material and Methods section, and incubated in either full medium or EBSS for 2 h. Cells were then collected and analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS mock versus EBSS HA-p38IP (***P=0.036); EBSS mock versus ULK1 CTD (***P=0.0001); Students t‐test).",
    "molecules": "[14C]valine"
  },
  {
    "caption": "(A) 293/GFP-LC3 cells were treated with 10 μM anisomycin for 30 min, or exposed to UV irradiation for 3 min followed by a 40‐min recovery and incubation in full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells were then fixed and visualized by confocal microscopy. GFP-LC3‐positive structures per cell were quantified as in Figure 3A. Bars=5 μm. Data are represented as mean±s.e.m. n=60 cells, mock versus anisomycin EBSS (***P=0.0001); mock versus UV EBSS (***P=0.0001); mock versus anisomycin EBSS with leupeptin (***P=0.0001); mock versus EBSS with leupeptin UV (***P=0.0001). All analysed using Student's t-test.",
    "molecules": "anisomycin, leupeptin"
  },
  {
    "caption": "(B) HEK293A cells were pretreated with 10 μM anisomycin for 30 min, or UV irradiation for 3 min followed by a 40‐min recovery, before incubation in EBSS for 2 h. Images were analysed as in Figure 2E. Bars=5 μm. Data are represented as mean±s.e.m. of 60 cells, EBSS mock versus EBSS anisomycin (*P=0.0142); EBSS mock versus EBSS UV (*P=0.0150); Student's t‐test.",
    "molecules": "anisomycin"
  },
  {
    "caption": "(A) 293/GFP-LC3 cells were pre‐treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS. Cell lysates were analysed by SDS-PAGE for GFP-LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐β tubulin. The GFP-LC3 lipidation was quantified as the amount of GFP-LC3II/GFP-LC3I (data are represented as mean±s.e.m. of triplicates, P=0.0091, Student's t‐test).",
    "molecules": "anisomycin"
  },
  {
    "caption": "(B) HEK293A cells labelled with [14C]valine, as described in Material and Methods section, were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium, EBSS, or EBSS with leupeptin for 2 h. Cells were then analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS untreated versus anisomycin treated (P=0.0363); EBSS untreated versus leupeptin treated (P=0.0001), Student's t‐test).",
    "molecules": "[14C]valine, anisomycin, leupeptin"
  },
  {
    "caption": "(C) HEK293 cells were transfected with control siRNA or siRNA for p38IP. After 24‐h incubation, control or p38IP‐depleted cells were pre‐treated with anisomycin, followed by a 2‐h incubation with full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin. Cell lysates were analysed with anti‐p38α or anti‐LC3 antibodies. LC3II/LC3I levels were quantified and presented normalized to control, untreated cells incubated in EBSS. Data are representative of two experiments.",
    "molecules": "anisomycin, leupeptin"
  },
  {
    "caption": "(D) HEK293A cells transiently transfected with HA‐p38IP and RFP‐mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated. 5% of the lysates or the immunoprecipitates were probed with anti‐HA and anti‐mAtg9 antibodies (data is representative of 3 experiments, *P=0.0332).",
    "molecules": "anisomycin"
  },
  {
    "caption": "(C) HEK293A cells were transfected with Flag-p38α for 24 h. Cells were incubated in either full medium, EBSS, or EBSS plus leupeptin for 2 h, lysed, and analysed for endogenous LC3 lipidation using an anti‐LC3 antibody, and immunoblotted with anti‐Flag antibody. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean±s.e.m. of triplicates, n=2 experiments, EBSS control versus EBSS with Flag‐p38α, ***P=0.0026).",
    "molecules": "leupeptin"
  },
  {
    "caption": "(A) 293/GFP-LC3 cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS with leupeptin for 2 h, then fixed and visualized by confocal microscopy. Bars=5 μm (data are represented as mean±s.e.m. n=60 cells, EBSS control versus p38α siRNA (***P=0.0001); EBSS with leupeptin control versus p38α siRNA (***P=0.0001), Student's t‐test).",
    "molecules": "leupeptin"
  },
  {
    "caption": "(B) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS containing leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐actin and anti‐p38α antibodies. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean ±s.e.m. n=5, full medium control versus p38α siRNA (**P=0.0056); EBSS control versus p38α siRNA (**P=0.048); EBSS with Leu control versus p38α siRNA (**P=0.04), Student's t‐test).",
    "molecules": "leupeptin"
  },
  {
    "caption": "(E) 72 h cells were transfected with control, p38α siRNA or both p38α siRNA, and p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐p38α. LC3 lipidation was quantified as the amount of LC3II/LC3I. Data are representative of two experiments.",
    "molecules": "leupeptin"
  },
  {
    "caption": "Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.",
    "molecules": "DAPI"
  },
  {
    "caption": "Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Indicated regions (orange squares) are shown as magnifications in (b). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.",
    "molecules": "DAPI"
  },
  {
    "caption": "Fluorescence stainings for YAP, CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH. Arrows indicate BC. Note the enrichment of apical YAP at 0.5 and 1.5d post PH.",
    "molecules": "DAPI"
  },
  {
    "caption": "EM image of immunogold-labelled YAP on liver tissue sections at 1.8 d post PH. Image (A) shows a BC in between two hepatocytes. Indicated regions (black squares) of the apical (a') and the basolateral (a'') membrane of the hepatocyte are shown as magnification. Arrows indicate gold particles.",
    "molecules": "gold"
  },
  {
    "caption": "Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.",
    "molecules": "gold, phalloidin"
  },
  {
    "caption": "Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h. Indicated areas (dashed rectangle) are shown as magnifications in insets.",
    "molecules": "DAPI, DCA, deoxycholic acid, DMSO, Y27"
  },
  {
    "caption": "Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO (control), Y27, DCA or DCA+Y27 for ~18 h as representatively shown in (A). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n = 7; DCA vs. DMSO p=8.98*10-5; DCA+Y27 vs. DMSO, p=0.05; Y27 vs. DMSO, p=0.55 (n.s.); DCA+Y27 vs. DCA p = 0.05",
    "molecules": "DCA, DMSO, Y27"
  },
  {
    "caption": "Fluorescence stainings of primary hepatocytes for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue). Cells were treated with DMSO (control), Y27, SMIFH2 or Cytochalasin D (CytoD) for 6 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Note the dilation of canaliculi (asterisks) and fragmentation of F-actin (arrows) upon CytoD treatment.",
    "molecules": "Cytochalasin D, CytoD, DAPI, DMSO, SMIFH2, Y27"
  },
  {
    "caption": "Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO, Saline, Y27, Fasudil, SMIFH2, CK666, CytoD or Latrunculin A (LatA) for 6 h. Saline serves as control for Fasudil, DMSO serves as control for all other conditions. Inhibitors affecting similar actin processes are displayed in the same grey level. Saline, Fasudil, CK666 and LatA conditions are not shown in (d). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n=3-5; DMSO vs. Y27, p=0.002; DMSO vs. CytoD, p=5.11*10-7; DMSO vs. LatA, p=0.01; DMSO vs. CK666, p=0.15 (n.s.); DMSO vs. SMIFH2, p= 0.11 (n.s.); Saline vs. Fasudil, p= 0.92.",
    "molecules": "CK666, CytoD, DMSO, Fasudil, LatA, Latrunculin A, SMIFH2, Y27"
  },
  {
    "caption": "Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH, treated with saline (control) or the Rho kinase inhibitor Fasudil for 1 h. Shown are maximum projections of 50 µm stacks in the PV area. Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) from mice at 2 d post sham OP (green) or PH (red), treated with saline (control, solid line) or Fasudil (dashed line). Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. BC diameter of sham operated mice treated with saline vs. Fasudil, p= 6.59*10-8; BC diameter of PH mice treated with saline vs. Fasudil, p=2.31*10-14.",
    "molecules": "Fasudil"
  },
  {
    "caption": "Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH, treated with saline (control) or Fasudil for 1 h. Indicated regions (dashed rectangle) are shown as magnifications in insets. Quantification of the mean nuclear YAP intensity within 10 zones along the CV-PV axis, (zone 1, peri-central; zone 10, peri-portal) from IF images as representatively shown in (c). Data was normalized to untreated animals. Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. Nuclear YAP intensity of sham operated mice treated with saline vs. Fasudil, p=0.13 (n.s.) ; nuclear YAP intensity of PH mice treated with saline vs. Fasudil, p=0.0004.",
    "molecules": "DAPI, Fasudil"
  },
  {
    "caption": "High power single plane confocal  images of EdU  labeled control and TRIM71-expressing organoids. An EdU  pulse was given on day 8 and  EdU incorporation  (green) was analyzed 1.5 hours later. JAG1 immunostaining  (magenta) marks supporting cells/prosensory cells, mCherry  (red) marks transduced cells, Hoechst  labels cell nuclei  (blue). Scale bar= 25µm. Percentage of EdU+ mCherry+ cells per organoids in (I); (n = 5, two independent experiments). P-values were calculated using two-tailed, unpaired t test. **P < 0.01. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ±standard deviation (SD) of biological replicates.",
    "molecules": "Hoechst, EdU"
  },
  {
    "caption": "High power single plane confocal  images of EdU  (red) and Hoechst  (blue) labeled WT and TRIM71  KO organoids. A single EdU  pulse was given on day 7 and  EdU incorporation was analyzed 1.5 hours later. Scale bars = 25µm. Percentage of EdU+ cells in (F) (n=15 in WT, n=11 in Trim71  KO, three independent experiments). Two-tailed, unpaired t test was used to calculate P values, n.s. not significant. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.",
    "molecules": "Hoechst, EdU"
  },
  {
    "caption": "Cochlear epithelial cells  isolated from P5 wild type were transduced with control (Ctrl) or TRIM71-expressing lentivirus  and expanded as organoids. MCherry (red) marks transduced cells. Hoechst staining  (blue) labels cell nuclei. High power single plane confocal images of control (Ctrl) and TRIM71-expressing organoids immuno-stained  for progenitor/supporting cell marker JAG1  (magenta) and progenitor  marker HMGA2  (green) after 10 days of expansion. Scale bars=25µm. Quantification of JAG1+ mCherry+ HMGA2+ cells per organoid in (A) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. ***P < 0.001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Cochlear epithelial cells  isolated from P5 wild type were transduced with control (Ctrl) or TRIM71-expressing lentivirus  and expanded as organoids. MCherry  (red) marks transduced cells. Hoechst staining  (blue) labels cell nuclei. High power confocal   images of control (Ctrl) or TRIM71-expressing organoids immuno-stained  for progenitor/supporting cell marker JAG1  (magenta) and supporting cell  marker S100A1  (green) after 10 days of expansion. Scale bars=25µm. Quantification of JAG1+ mCherry+ S100A1+ cells per organoid in (C) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. **P < 0.01. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Cochlear epithelial cells isolated from Atoh1-nGFP  transgenic mice  (E, F) were transduced with control (Ctrl) or TRIM71-expressing lentivirus  and expanded as organoids. MCherry  (red) marks transduced cells. Hoechst staining  (blue) labels  cell nuclei. High power single plane confocal  images of control (Ctrl) or TRIM71-expressing organoids immuno-stained  for transcription factor NFIB  (magenta) after 2 days of differentiation. Atoh1-nGFP  (green) marks nascent hair cells. White arrowheads mark Atoh1-GFP+ cells with low NFIB  expression (NFIBlow). Scale bars=25µm. Quantification of NFIBlow, NFIBhigh, NFIBlowAtoh1-nGFP+, NFIBhigh Atoh1-nGFP+ cells in control (Ctrl) and TRIM71-expressing organoids (n=4, two independent experiments). Two-way ANOVA with Tukey's correction was used to calculate P values. ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A Robust immunofluorescence detection of ubiquitination on LAMP1-positive compartments with pan-ubiquitin FK2 or K48 chain-specific antibodies. HeLa cells were treated with 250 µM LLOMe or EtOH (untreated) for 3 h, fixed and stained with indicated antibodies. Confocal microscopy was followed by automated image analysis. Graphs represent data from three independent experiments with ≥ 50 cells per condition (mean ± SD). Scale bar: 20 µm.",
    "molecules": "LLOMe, EtOH, ubiquitin"
  },
  {
    "caption": "C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes. HeLa cells were seeded in 384-well plates, transfected with a siRNA library in quadruplicates for 72 h. Cells were LLOMe-treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope. Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined. Robust z-scores were calculated for LLOMe-treated samples for each depletion against the values of the whole plate. Samples exceeding the threshold of -2 of robust z-score (highlighted in red) were considered for the secondary screen.",
    "molecules": "LLOMe"
  },
  {
    "caption": "E Damage-induced translocation of UBE2QL1 to lysosomes. The three best candidates (from D) were transiently expressed with N-terminal HA-tag in HeLa cells. Cells were treated with LLOMe or EtOH (untreated) for 3 h, fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy. Scale bar: 10 µm. F Automated quantification of (E). Shown are Pearson correlation coefficients (P.C.C) of HA and LAMP1 signals. Graph represents data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test). ",
    "molecules": "LLOMe, EtOH"
  },
  {
    "caption": "A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h, and LLOMe or control-treated (untreated) for 3 h. Cells were fixed and immuno-stained for LAMP1 along with antibodies specific for K63 or K48-linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope. Note the strong reduction of damage-induced K48 ubiquitination and decreased intensity of the K63 signal. Scale bar: 20 µm. B, C Automated image quantification of (A). Shown are percentages of K48 or K63-positive LAMP1 vesicles per cell. Graphs represent data from three independent experiments with ≥ 70 cells per condition (mean ± SD). **P<0.01; ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).",
    "molecules": "LLOMe, ubiquitin"
  },
  {
    "caption": "A HeLa cells expressing UBE2QL1-HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy. Cells were stained with antibodies specific for LAMP1 (not shown), and combinations of HA with K48 chains, K63 chains or p62 as indicated. Note the delayed co-emergence of K48 chains and UBE2QL1 vesicles, compared to the earlier appearance of K63 chains and p62. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 10 µm, 2 µm for inlays. B Automated quantification of (A). Shown are the Pearson correlation coefficients (P.C.C.) representing colocalization of UBE2QL1-HA, K48 chains, K63 chains or p62 with LAMP1-positive vesicles. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD). C Automated quantification of (A). P.C.C.'s representing colocalization of K48 chains, K63 chains or p62 with UBE2QL1-HA in cells treated with LLOMe or vehicle alone (untreated) for 180 min. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ",
    "molecules": "LLOMe"
  },
  {
    "caption": "Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (A) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 (A) p62 (10 nm gold particles) localizes as a cap (arrowheads) to the outer limiting membrane of lysosomes (L). Scale bars: 200 nm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (B) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 and K48. (B) K48 ubiquitin chains (15 nm gold particles, arrows) are found in the lumen of lysosomes, that also contain p62 (10 nm gold particles, arrowheads) at their outer membrane. Scale bars: 200 nm.",
    "molecules": "LLOMe, ubiquitin"
  },
  {
    "caption": "Immuno-electron microscopy of LLOMe-treated HeLa cells. (C) Overexpressed UBE2QL1-GFP (10 nm gold particles) is found in late endosomes (LE, Inset) and lysosomes. M = mitochondrion. Scale bars: 200 nm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "D, E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram (D) and displayed high correlation coefficients (E).",
    "molecules": "LLOMe, biotin, EtOH, H2O2, phenol, streptavidin"
  },
  {
    "caption": ", F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). (F) Results from the four experiments are summarized in a volcano plot. Horizontal dotted line represents the significance threshold (p > 0.05). The vertical lines indicate the fold change cutoff (log2 (H/L) ≥ 1.5). Red dots indicate proteins above the significance and fold-change thresholds. Protein names are color-coded in blue (endosome/lysosome-associated), green (endolysosomal damage/autophagy-associated) and orange (VCP/p97 and its cofactors).",
    "molecules": "LLOMe, biotin, EtOH, H2O2, phenol"
  },
  {
    "caption": "A HeLa cells stably expressing p97-GFP were control or UBE2QL1-depleted for 48 h and expression of p97-GFP was induced for the last 24 h. Cells were LLOMe or control treated for 1 h, fixed after a chase of 2 h, and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated. Note the reduction of the K48 and p97-GFP signals on damaged lysosomes in UBE2QL1-depleted cells. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 10 µm for inlays.",
    "molecules": "LLOMe, ubiquitin"
  },
  {
    "caption": "C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.",
    "molecules": "LLOMe"
  },
  {
    "caption": "G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h, treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A/B. GAPDH was probed as loading control.",
    "molecules": "LLOMe, Bafilomycin A1"
  },
  {
    "caption": "A Gal3 puncta assay for damaged lysosomes. HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone (untreated) for 1 h. Cells were fixed at indicated times after washout, stained with DAPI and Gal3 and LAMP1 antibodies, and processed for confocal microscopy. Note increased number of Gal3 puncta in untreated UBE2QL1-depleted cells and their persistence 10 h after LLOMe-induced damage. Scale bars: 10 µm, 2 µm for inlays. Arrows indicate Gal3 and LAMP1 colocalizing vesicles.",
    "molecules": "LLOMe, DAPI, EtOH"
  },
  {
    "caption": "C Survival assay. Control or UBE2QL1-depleted HeLa cells (48 h) were treated with increasing concentrations of LLOMe as indicated. Cell viability was measured with the MTS assay. Graph represents data from one experiment including three replicates (mean ± SD). *P<0.05; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).",
    "molecules": "LLOMe"
  },
  {
    "caption": "A Control or UBE2QL1-depleted (for 60 h) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy. Note nuclear translocation of TFEB upon loss of UBE2QL1. Scale bar: 20µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "F Western blot analysis of lysates of cells treated As a positive control, cells were treated with the mTOR-inhibitor Torin1 (1 µM) for 2 h. GAPDH was probed as loading control.",
    "molecules": "Torin1"
  },
  {
    "caption": "(b) IRGMC mRNA was significantly enriched in miR-196B complexes. Extracts of cells expressing FLAG-tagged-AGO1 and transfected with biotinylated miR-196B or control miR-20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification (immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads). IRGM mRNA variants were quantified using quantitative RT-PCR; results are presented as the ratio between miR-196B and miR-20 (non-relevant miRNA) pull-downs and the mean of three independent experiments ± standard deviation (s.d.). IP strep, immunoprecipitation straptavidine.",
    "molecules": "biotinylated"
  },
  {
    "caption": "(a) The basal flux of autophagy is affected by IRGM expression level. HEK293 cells transfected with an IRGM-expressing plasmid, miR-196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti-LC3B. (b) Quantification of LC3-II relative to actin (mean of three independent experiments ± s.d.).",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "a, TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2µm left, 1µm right;",
    "molecules": "melanin"
  },
  {
    "caption": "d, Lysosomes can be exchanged, rarely, between PhNT-connected cells. Lysosomes were labelled with SiR-Lyso and their movements were analysed by TrackMate software. Images show deconvolution of 3D reconstructions of lysosomes (surface; red) and cGFP (volume; green) from time-lapse live imaging series of Nrl.Gfp+/+ photoreceptor cultures (green); the position of a transferred lysosome is marked as1 (t = 0), 2 (t = 3'), 3 (t = 6'), 4 (t = 9'); N.B. Deconvolution shows a segment-like process extending from cell \"A\" and a PhNT connecting cells \"A\" and \"B\"; Scale bar = 2 µm;",
    "molecules": "SiR-Lyso"
  },
  {
    "caption": "e, Mitochondria can be exchanged, rarely, between PhNT-connected cells. Mitochondria movements determined by TrackMate software. Images show deconvolution of 3D reconstructions of mitochondria (surface; red) and cGFP(volume; green) from time-lapse live imaging series; position of transferred mitochondrion is marked as 1 (t = 0), 2 (t = 6'), 3 (t = 12'), 4 (t = 18'); Mitochondria labelled with mito-Tracker-Orange; Scale bar = 5 µm.",
    "molecules": "mito-Tracker-Orange"
  },
  {
    "caption": "d, MIP image of wt retina transplanted with P8 Nrl.Gfp+/+ x myrRFP/+ photoreceptors; showing wt host ONL photoreceptor labeled with cGFP (green) but not with myrRFP (red); Scale bar = 50µm; e, quantification and statistical analysis of material transfer events following subretinal transplantation of live (cGFP = 3775 ± 804, myrRFP = 436 ± 145) or UV-treated (cGFP = 1.3 ± 2.5, myrRFP = 4.3 ± 5.7) Nrl.Gfp+/+x myr-Rfp+/+ photoreceptors into wt hosts (N = 8 and N = 4 retinae, respectively); One-way ANOVA, parametric, two tail, Tukey's multiple comparisons; Graph shows mean ± S.D. ",
    "molecules": "myr"
  },
  {
    "caption": "(A) Average plots of Med8 cleavages around the TSSs of SAGA- and TFIID-dependent genes in Kin28AS cells treated with DMSO (-NA-PP1) or 6 μM NA-PP1 (+NA-PP1).",
    "molecules": "NA-PP1, DMSO"
  },
  {
    "caption": "(B) Signal tracks of Med8-MNase cleavages at the upstream regions of several genes previously shown to have increased core promoter association of Mediator by ChIP-qPCR following NA-PP1 treatment of a Kin28AS strain (Wong et al., 2014). All time points for a given treatment were concatenated to generate a combined track. TSSs are indicated by arrows.",
    "molecules": "NA-PP1"
  },
  {
    "caption": "(B) Average plots of Med8 cleavages around the TSSs of genes upregulated and downregulated ≥2 fold in SM (Saint et al., 2014) in WT and med15Δ. Control cleavages were subtracted from SM cleavages at each base position.",
    "molecules": "SM"
  },
  {
    "caption": "(B) Average plots of Taf1 cleavages around the TSSs of SAGA- and TFIID-dependent genes ± rapamycin to deplete Med14-FRB.",
    "molecules": "rapamycin"
  },
  {
    "caption": "Average plots of Med8 cleavages around the TSSs of SAGA- and TFIID-dependent genes ± rapamycin to deplete Taf1-FRB.",
    "molecules": "rapamycin"
  },
  {
    "caption": "A-C. Scanning electron microscopy (SEM) images of (A) nanowires (VNw; scale - 5 µm) and (B) crude nanosheets (VSh; scale - 200 nm), (C) ultrathin-nanosheets (Vs), scale - 20 µm.",
    "molecules": "nanosheets, Sh, nanowires, Nw"
  },
  {
    "caption": "C. Michaelis-Menten plot with the variation of H2O2 (0-400 µM) in the presence of Vs (20 ng/µL), GSH (2 mM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.",
    "molecules": "Vs, GSH, H2O2, NADPH, phosphate"
  },
  {
    "caption": "D. Michaelis-Menten plot with the variation of GSH (0-7 mM) in the presence of Vs (20 ng/μL), H2O2 (200 µM), NADPH (0.2 mM), GR (1.7 units) in phosphate buffer (100 mM, pH 7.4) at 25°C.",
    "molecules": "Vs, GSH, H2O2, NADPH, phosphate"
  },
  {
    "caption": "G, H. U1 Grx1-roGFP2 cells were supplemented with BSO (G) or GSH (H) for 16 h to deplete or replenish GSH, respectively. Following this, cells were treated with Vs for 15 min, exposed to H2O2, and the ratiometric response was measured.",
    "molecules": "BSO, Vs, GSH, H2O2"
  },
  {
    "caption": "E. U1-Grx1-roGFP2 cells were serum starved for 30 min in the presence or absence of Vs and sodium selenite (0.5 nM), and the biosensor response was measured. Data were compared to serum starved control cells (C).",
    "molecules": "sodium selenite, Vs"
  },
  {
    "caption": "F. U1 cells were either serum-starved or supplemented with Se (0.5 nM) or Vs (0.62 ng/μL) and HIV reactivation was measured at 6 h post starvation by gag RT-PCR.",
    "molecules": "Vs, Se"
  },
  {
    "caption": "Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. (C Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.",
    "molecules": "Vs, PMA"
  },
  {
    "caption": "Total RNA isolated from untreated (UT), PMA-treated, Vs-treated and Vs+PMA treated U1 was examined by NanoString technology to assess the expression of genes responsive to oxidative stress and HIV. D) Heat map showing functional categories of DEGs under PMA/UT, Vs/UT and PMA+Vs/UT comparisons. mRNA counts were normalised using the internal control β2 microglobulin (B2M), and fold change (FC) was calculated using the nSolver 4.0 software. Genes showing an absolute FC >1.5, and P <0.05 were considered as significantly altered.",
    "molecules": "Vs, PMA"
  },
  {
    "caption": "F. Primary CD4+ T cells purified from human PBMCs (3 healthy donors) were activated, pre-treated with 25 ng/μL Vs for 15 min, and infected with 0.05 moi of HIV-1 NL-4.3. Virus released in supernatant was quantified by p24 ELISA. Vs treatment was repeated every 48 h.",
    "molecules": "Vs"
  },
  {
    "caption": "B. Expanded CD4+ T cells from 3 patients were cultured in presence of IL-2 and ARVs, with and without 25 ng/µL Vs for 21 days. Vs treatment was given for 15 min every 3rd day. HIV transcripts were quantified by RT-qPCR at day 14, day 21, and at 24 h post-stimulation of cells cultured for 21 days by prostratin. Limit of detection for RT-qPCR was 3 viral transcripts per million cells.",
    "molecules": "Vs, prostratin"
  },
  {
    "caption": "C. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT-qPCR. Reduction in viral stimulation in Vs treated samples are represented as percentage values. ND - non determined. D. Aggregate plot for 3 patients from data (C). ",
    "molecules": "Vs, prostratin"
  },
  {
    "caption": "(C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %.",
    "molecules": "dithiothreitol, DTT, IAA, iodoacetamide"
  },
  {
    "caption": "(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot.",
    "molecules": "dithiothreitol, DTT, 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid, AMS, IAA, iodoacetamide"
  },
  {
    "caption": "(B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS.",
    "molecules": "DTT, AMS, cysteine, IAA"
  },
  {
    "caption": "(C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space.",
    "molecules": "sucrose, Triton X-100"
  },
  {
    "caption": "(B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.",
    "molecules": "IAA, iodoacetamide"
  },
  {
    "caption": "(C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot.",
    "molecules": "DTT, IAA"
  },
  {
    "caption": "(D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot.",
    "molecules": "PEG-PCMal, DAM, diamide, DTT"
  },
  {
    "caption": "Radiolabeled [35S]COA7 (A) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.",
    "molecules": "IAA, iodoacetamide"
  },
  {
    "caption": "Radiolabeled [ 35S]TIMM8A (B) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.",
    "molecules": "IAA, iodoacetamide"
  },
  {
    "caption": "Radiolabeled [35S]COA7 (F) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.",
    "molecules": "IAA, iodoacetamide"
  },
  {
    "caption": "Radiolabeled [ [35S]TIMM8A (G) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.",
    "molecules": "IAA, iodoacetamide"
  },
  {
    "caption": "(E) Equal amounts of wildtype and mutant radiolabeled [35S]COA7 precursors were imported into mitochondria isolated from HEK293 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to wildtype COA7 at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.",
    "molecules": "IAA, iodoacetamide"
  },
  {
    "caption": "(B) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot.",
    "molecules": "CHX"
  },
  {
    "caption": "(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and/or MG132 for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot. CHX, cycloheximide.",
    "molecules": "CHX, cycloheximide, MG132"
  },
  {
    "caption": "(F) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132. The samples were analyzed by reducing SDS-PAGE and Western blot.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Cellular protein extracts were isolated from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot.",
    "molecules": "bortezomib, carfilzomib, MG132"
  },
  {
    "caption": "(B) Cellular fractions were prepared from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.",
    "molecules": "bortezomib, carfilzomib, MG132"
  },
  {
    "caption": "(B) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient-derived skin fibroblasts treated with MG132 (1 μM), bortezomib (20 nM), or carfilzomib (50 nM) for 12 h and recovered for another 6 h. The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.04 [DMSO vs MG132], **p=0.0025 [DMSO vs Bortezomib], *p=0.02 [DMSO vs Carfilzomib]).",
    "molecules": "bortezomib, Bortezomib, carfilzomib, Carfilzomib, DMSO, MG132"
  },
  {
    "caption": "(C) Immortalized patient-derived skin fibroblasts transiently expressing wildtype or mutant COA7 (COA7-Y137C and COA7-Ex2∆) for 48 h were treated with bortezomib (20 nM) during the last 12 h. The fibroblasts were harvested, and complex IV activity was measured in digitonized cellular extracts. The results of three biological replicates were quantified and normalized to empty vector-bortezomib treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.01 [Empty vector vs COA7- Wt], *p=0.005 [Empty vector vs COA7 - Y137C]).",
    "molecules": "bortezomib"
  },
  {
    "caption": "(D) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.",
    "molecules": "digitonin"
  },
  {
    "caption": "(E) Immortalized patient-derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7-Y137CHIS were solubilized in DDM buffer and analyzed by 4-13 % gel BN-PAGE and Western blot.",
    "molecules": "DDM"
  },
  {
    "caption": "(F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.",
    "molecules": "bortezomib, digitonin"
  },
  {
    "caption": "HEK293 cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO served as vehicle control. Transfection with pCS2-NICD vector served as positive control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following inhibitor treatment and the top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01 and ns=not significant.   ",
    "molecules": "DRB, DMSO, LY411575, MLN4924, Roscovitine, XAV939"
  },
  {
    "caption": "iPS cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO has been used as vehicle control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following Inhibitor treatment and the top band disappears following λ phosphatase treatment indicative that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (C) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01, ***p≤0.001 and ns= not significant.   ",
    "molecules": "DRB, DMSO, LY411575, MLN4924, Roscovitine, XAV939"
  },
  {
    "caption": "HEK293 cells were treated with the inhibitors NICD phosphorylation status was analysed by a Phos-tag assay. DMSO served as vehicle control. NICD phosphorylation profile varies following Roscovitine, DRB, XAV939 or MLN4924 treatment. Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms.",
    "molecules": "DRB, DMSO, MLN4924, Roscovitine, XAV939"
  },
  {
    "caption": "NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student t-test was used to determine p values, with **p≤0.01.   ",
    "molecules": "DMSO, MLN4924"
  },
  {
    "caption": "Roscovitine treatment reduced the NICD-FBXW7 interaction. 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.   ",
    "molecules": "MLN4924, Roscovitine"
  },
  {
    "caption": "Phosphorylation of serine 2513, but not serine 2516, appears to be essential for the NICD-FBXW7 interaction. hNICD-GFP phospho-mutant peptides encoding non-phosphorylatable residues at S2513 and/or 2516 (serine to alanine) were expressed in HEK293 cells. The exogenously expressed protein was subsequently immunoprecipitated with anti-GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody. Wild-type hNICD-GFP and GFP only vectors were included as positive and negative controls, respectively. Western blot using GFP antibody served as immunoprecipitation efficiency control. β-Actin has been used as loading control for the input lanes.",
    "molecules": "alanine, phospho, serine"
  },
  {
    "caption": "Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here. At both 1 and 10µM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1µM) inhibits more than 94% of CDK2 activity. At both 1 and 10 µM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor Database",
    "molecules": "GSK650394A, Purvalanol B, Roscovitine"
  },
  {
    "caption": "HEK293 cells were treated with three highly selective CDK2 inhibitors (10 µM of Roscovitine, 0.1µM of Purvalanol B and 10µM of GSK650394A) for 3 hours. Endogenous levels of NICD were detected by western blot. β-Actin served as loading control.   Quantification of the density of western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed, with *p≤0.05, **p≤0.01, and ns= not significant.   ",
    "molecules": "GSK650394A, DMSO, Purvalanol B, Roscovitine"
  },
  {
    "caption": "HEK293 cells were treated with RO-3306, a specific CDK1 inhibitor, for 3 hours at 10 µM. Levels of NICD, FBXW7, phospho-Histone H3 and Histone H3 were detected by western blot. β-Actin was used as loading control.   Quantification of the density of western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.   ",
    "molecules": "DMSO, RO-3306"
  },
  {
    "caption": "Cell cycle profile for HEK293 cells released from synchronization after double thymidine block. Cells were released and harvested at the indicated time points (AS= asynchronous). Analysis of cell cycle arrest and release was performed using propidium iodide (PI) staining and flow cytometry. A representative experiment of three performed is shown.",
    "molecules": "thymidine"
  },
  {
    "caption": "iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU (marked in red within the NICD panel). EdU incorporation occurs during S phase (marked by yellow in the NICD panel). Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase. M phase cells are distinguishable by their chromatin condensation, observable in the DAPI panel (dividing cells are indicated with white in the NICD panel). G1 cells do not have any of the features described above (blue in NICD panel). Scale bars are 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Purvalanol B treatment reduced the NICD-FBXW7 interaction. 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0.1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin has been used as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.   ",
    "molecules": "MLN4924, Purvalanol B"
  },
  {
    "caption": "E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of Purvalanol B (1 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin was used as loading control.",
    "molecules": "DMSO, Purvalanol B"
  },
  {
    "caption": "E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of RO-3306 (10 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin has been used as loading control.",
    "molecules": "DMSO, RO-3306"
  },
  {
    "caption": "Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the "-" explant. n=18. Scale bar is 100 µm.",
    "molecules": "Purvalanol B"
  },
  {
    "caption": "Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 10 µM of RO-3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. RO-3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the "-" explant. n=10. Scale bar is 100 µm.",
    "molecules": "RO-3306"
  },
  {
    "caption": "The mean log2 fold change in successive somite length at position +1 (last formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.0437 for RO-3006 and p= 0.0524 for PurB, compared to DMSO. n=14.   The mean log2 fold change in successive somite length at position +2 (penultimate formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.6409 for RO-3006 and p= 0.4065 for PurB, compared to DMSO. n=14.   ",
    "molecules": "DMSO, PurB, RO-3006"
  },
  {
    "caption": "(A) HEK cells transfected with STIM1-CFP, Orai1-mCherry, and with (red) or without (black) untagged ANO8 were used to measure CRAC current with pipette solution (cytoplasmic buffer) containing 3 mM EGTA. The columns show the averaged effect of ANO8 on current density and on the slop of slow Ca2+-dependent inactivation (SCDI). The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "Ca2+, EGTA"
  },
  {
    "caption": "(B) Fura2-loaded HEK cells transfected with YFP (control, black) or ANO8-YFP (red) were used to measure store-mediated Ca2+ influx. Stores were depleted by treatment with the SERCA inhibitor CPA and Ca2+ influx was measured by Ca2+ add-back. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "Ca2+, CPA, Fura2"
  },
  {
    "caption": "(A) FRET ratio was measured with HEK cells transfected with STIM1-CFP, Orai1-YFP (black) and co-transfected with untagged ANO8 (red) before and after store depletion with 25 µM CPA in Ca2+-free solution. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "Ca2+, CPA"
  },
  {
    "caption": "(A, B) Knockdown of ANO8 (siA8) reduced CRAC current in cells transfected with Orai1 (O1) and STIM1 (S1) and buffered with 3 mM EGTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.",
    "molecules": "EGTA"
  },
  {
    "caption": "(C) Knockdown of ANO8 reduces the native store-dependent Ca2+ influx measured in store depleted cells by Ca2+ add-back. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(F, G) Current was measured with pipette solution contacting the fast and strong Ca2+ buffer 10 mM BAPTA in HEK cells transfected with STIM1, Orai1, and with (red) or without ANO8 (black). Panel (G) shows the increase in current density at peak current. Note the prominent current inactivation in the presence of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.",
    "molecules": "BAPTA, Ca2+"
  },
  {
    "caption": "(H) Knockdown of SARAF (red) in wild-type cells had no effect on current inactivation in the presence of 10 mM BAPTA. (I) Knockdown of SARAF did not prevent the ANO8-dependent current inactivation in the presence of 10 mM BAPTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test. ",
    "molecules": "BAPTA"
  },
  {
    "caption": "(A, B) Depletion of plasma membrane PI(4,5)P2 with the FRB/FKBP system prevented the ANO8-mediated increase in STIM1-Orai1 current measured in pipette solutions containing 3 mM EGTA (A) or 10 mM BAPTA (B). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "BAPTA, EGTA, PI(4,5)P2"
  },
  {
    "caption": "(C) HEK cells transfected with ANO8-YFP, STIM1-CFP Orai1-HA and FRB and mCherry-FKBP PI(4,5)P2 depleting constructs were treated with 0.2 µM rapamycin (rapa) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "CPA, PI(4,5)P2, rapa, rapamycin"
  },
  {
    "caption": "(D) Mutating ANO8 R948, R950, R951 in RPRRP, a region that is predicted to include an ANO8 PI(4,5)P2 binding site, prevented increased STIM1-Orai1 current and inactivation. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "(A) Plot of the slope of inactivation as a function of current density in the presence (red) and absence of ANO8 (black). Increasing current density was obtained by varying external Ca2+ between 2-50 mM. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(B, C) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA (b) or 10 mM BAPTA (c). SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution (green in c). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "IP3, BAPTA, CPA, EGTA"
  },
  {
    "caption": "(F, G) HEK cells expressing STIM1, Orai1, and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with (red) or without (black) 100 µM IP3 or 10 mM of the ER Ca2+ chelator TPEN (green). The current density (F) and the normalized current (G) are shown, illustrating the delay in current inactivation by IP3 and TPEN. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.",
    "molecules": "IP3, BAPTA, Ca2+, TPEN"
  },
  {
    "caption": "(A, B): ER Ca2+ content was measured with ER-GECO1 in the periphery (A) and the center (B) or cells transfected with M3 receptors and with (red, green) or without (black, blue) ANO8. Receptor mediated store depletion was initiated by stimulating the cells with 0.5 mM carbachol in Ca2+-free solution. Cell stimulation was terminated with 10 µM atropine and ER Ca2+ uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2+. The fluorescence was measured as F/F0 and normalized to the initial fluorescence in the presence of ANO8. Panel (C) shows traces of ER Ca2+ uptake at expanded time scale in the periods marked by rectangular in (A and B) and the averaged slopes of ER Ca2+ influx and (D) shows the averaged slope of Ca2+ release at the cell periphery (Peri) and cell center (Center). In (C) the traces were aligned along the Y axis to better show the difference in uptake rate. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.",
    "molecules": "GECO1, atropine, Ca2+, carbachol"
  },
  {
    "caption": "(H, I) Simultaneous measurements of ER Ca2+ with ER-GECO1 and PI(4,5)P2 hydrolysis with PHPLCδ-GFP in response to cell stimulation of M3 receptors. Traces are shown in (h) and the average slopes are in (i). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.",
    "molecules": "GECO1, Ca2+, PI(4,5)P2"
  },
  {
    "caption": "(A-D) HeLa cells transfected with GFP (black) or ANO8 (red) were stimulated with 0.5 and then 1 µM ATP to activate the native P2Y2 receptors. The oscillations were analyzed in terms of the % of responding cells (B), oscillation frequency (C), and the amplitude of the Ca2+ signal (D). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.",
    "molecules": "ATP, Ca2+"
  },
  {
    "caption": "(E-H): HeLa cells were treated with scrambled siRNA (black) or siANO8 (red) and were stimulated with 1 and then 5 µM ATP. The oscillations were analyzed in terms of the % of responding cells (F), oscillations frequency (G), and the amplitude of the Ca2+ signal (H). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.",
    "molecules": "ATP, Ca2+"
  },
  {
    "caption": "(A-C) Representative confocal imaging  of CFSE-stained apoptotic bodies (green) incubated with secreted  NimB4-RFP (B, C) or SPvkg-RFP (A). Apoptotic bodies were pre-incubated in absence (A, B) or presence (C) of annexin V (25 μg/mL). Scale bar = 10 μm. (D) Quantification of the  colocalization of NimB4-RFP or SPvkg-RFP with the apoptotic bodies in presence or absence of Annexin V, as measured by Pearson's correlation coefficient. Values from at least four independent experiments are represented as mean ± SD (** P<0.01 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). ",
    "molecules": "CFSE"
  },
  {
    "caption": "(A) Representative  confocal imaging of wild type (w1118), NimB4 sk2 and draper macrophages incubated with fluorescently-labeled apoptotic cells (red, CellTraceTM Red) on ice for 1h and stained  with AlexaFluorTM 488 phalloidin  (green). Scale bar: 10 μm. (B) Quantification of the  number  of  apoptotic cells (red)  binding to the  macrophages  (green). Values from three independent experiments are represented as mean ± SD (*** P<0.001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). ",
    "molecules": "AlexaFluorTM 488, CellTraceTM Red, phalloidin"
  },
  {
    "caption": "(D) Ex vivo phagocytosis assay  using Alexa555 fluorescent apoptotic bodies. Wild type, NimB4sk2, NimB4 genomic rescue (NimB4sk2, [NimB4]), draper and NimC11,  eater1 macrophages from L3 wandering were incubated with Alexa555 fluorescent apoptotic bodies for 30, 60 or 120 min at room temperature. Phagocytosis  was quantified by flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05,** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests ns: not significant). (E) Ex vivo phagocytosis assay  using  AlexFluor488 S.aureus Bioparticles. Wild type, NimB4sk2 and NimC11, eater1 macrophages from L3 wandering were incubated with  bioparticles for 30, 60 or 120 min at room temperature. Phagocytosis  was quantified by  flow cytometry. Data are represented as mean ± SD from three independent experiments (**** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). ",
    "molecules": "bioparticles, Bioparticles, AlexFluor488, Alexa555"
  },
  {
    "caption": "(A) Representative fluorescence microscopy images of wild type (w1118),  NimB4sk2, draper, eater1 and croquemort△ third instar larvae macrophages stained with LysoTracker Red (live imaging). Overlay of fluorescence and differential interference contrast microscopy  (DIC). Scale bar = 10 μm. (B) Mean fluorescence intensity after  staining  with LysoTracker Red (live confocal imaging). Values from at least three independent experiments are represented as mean ± SD ( ** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). ",
    "molecules": "LysoTracker Red"
  },
  {
    "caption": "(E) Representative confocal imaging of Rab7EYFP immunostaining in wild type (w1118) and NimB4 sk2 macrophages. Tissues were stained with anti-GFP (red, Rab7), counterstained with  phalloidin (gray) and  DAPI  (blue). The arrows indicate the enlarged vesicles  are decorated with Rab7EYFP in the NimB4sk2 macrophages. Scale bar = 10 μm.",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": "(A) Representative confocal imaging of localization  of  Lamp1-mcherry and LysoTracker Green in wild type (w1118), NimB4 sk2 and draper△5 macrophages (live imaging). The arrows indicate the colocalization (w1118) or the clustering (NimB4sk2 and draper△5) of  Lamp1-mcherry and LysoTracker Green signals. Scale bar: 10 μm. (B) Quantification of the  colocalization of Lamp1-mcherry with LysoTracker Green, as measured by Pearson's correlation coefficient between the two signals. Values from at least five independent experiments are represented as mean ± SD (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests). ",
    "molecules": "LysoTracker Green"
  },
  {
    "caption": "(C) Ex vivo phagocytosis assay using apoptotic cells labelled with  pHrodo™ Red. Wild type (w1118), NimB4sk2 and draper△5 macrophages from L3 wandering larvae were incubated with pHrodo™ Red apoptotic cells for 60 min at room temperature.  Phagocytosis was quantified by  flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). (D) Ex vivo phagocytosis assay using pHrodoTM Red S.aureus BioparticlesTM conjugates. Wild type, NimB4sk2 and NimC11, eater1 Hml∆-Gal4>UAS-GFP macrophages from L3 wandering  larvae were incubated with  pHrodoTM Red S. aureus BioparticlesTM for 30, 60 or 120 min at room temperature. Phagocytosis was quantified by  flow cytometry. Data are represented as mean ± SD from three independent experiments (* P<0.05by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). ",
    "molecules": "Bioparticles, pHrodoTM Red, pHrodo™ Red"
  },
  {
    "caption": "(A-B) Representative fluorescence microscopy images of wild type (A) or NimB4sk2 (B) macrophages expressing Rab5  (middle panel) or  Rab7 (right panel) driven by Hml∆-Gal4 and stained with the  LysoTracker Red  (live imaging). Overlay of fluorescence and DIC. Scale bar = 10 μm. (C) Quantification of the mean fluorescence intensity of  LysoTracker in macrophages from  larvae (confocal live imaging). Values from at least three independent experiments are represented as mean ± SD (** P<0.01, **** P<0.0001 by ANOVA test followed by post hoc Dunnett's multiple comparison tests. ns: not significant). ",
    "molecules": "LysoTracker, LysoTracker Red"
  },
  {
    "caption": "Addition of NID1&FBLN2 to the Matrigel® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition.",
    "molecules": "Matrigel, PBS"
  },
  {
    "caption": "(F) The overexpression of GFP (green) and eggpl in Sf9 cell line in vitro. The anti-eggpl (red) was used to detect the eggpl protein, and Hoechst (blue) was used to label the nucleus. The scale bar is 10 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Immunofluorescence staining with anti-BrdU (red). The scale bar is 10 μm.",
    "molecules": "BrdU"
  },
  {
    "caption": "(e) Binding of NDP52 to GST-LC3 and GST-ubiquitin. Beads carrying the indicated purified GST fusion proteins were incubated with lysates of E. coli expressing NDP52. Bound proteins were eluted with glutathione and stained with Coomassie blue after SDS-PAGE.",
    "molecules": "glutathione"
  },
  {
    "caption": "Logarithm of the relative viability  after treatment with Resiquimod  (A, n=169), IL4  (B, n=174) stratified by   trisomy 12, and in A, also by IGHV  status. P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.",
    "molecules": "Resiquimod"
  },
  {
    "caption": "(D - I) Examples of drug-stimulus interactions, for each category. Plots show the natural logarithm of the relative viability  of 192 CLL  samples after treatment with the respective drug and stimulus. Each line represents one patient  sample linked across treatments. Black horizontal lines in each single treatment indicate the expected viability  based on linear modelling. Black and blue horizontal lines in the combinatorial treatment indicate the expected viability  based on the additive effect of the drug and stimulus (blue), and the expected viability  accounting for the additive effect and the interaction (black). (D+E) Ibrutinib, a clinically used BTK  inhibitor, is blocked by IL4  and IFNγ. (F) The JAK  inhibitor pyridone-6 inhibits the pro-survival effect of sCD40L  + IL4  stimulation. (G) The p38  inhibitor ralimetinib  and IFNγ  show a synergistic pro-survival effect not observed in either single treatment. (H) TLR  agonists, including CpG ODN (shown) increase sensitivity to BTK  inhibition by ibrutinib, despite increasing viability  as single treatments. (I) Soluble anti-IgM  sensitises CLL  samples to HSP90  inhibition by luminespib.",
    "molecules": "pyridone-6, Ibrutinib, ibrutinib, luminespib, ralimetinib"
  },
  {
    "caption": "Beeswarm-boxplots of the natural logarithm of the relative viability  of 169 CLL  samples, for (C) fludarabine  + CpG ODN and combinatorial treatments, faceted by IGHV  status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.",
    "molecules": "fludarabine"
  },
  {
    "caption": "Beeswarm-boxplots of the natural logarithm of the relative viability  of 169 CLL  samples, for (D) ibrutinib  + IL4  single and combinatorial treatments, faceted by IGHV  status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.",
    "molecules": "ibrutinib"
  },
  {
    "caption": "(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm",
    "molecules": "DOX"
  },
  {
    "caption": "(C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments",
    "molecules": "DOX"
  },
  {
    "caption": "A representative nuclease assay with MR, MBP-NBS1-his (denoted MBP-NBS1), MRN and pCtIP on 5'-end labeled 70-bp dsDNA with all ends blocked with streptavidin. Samples were separated on 15 % denaturing polyacrylamide gel. Quantitation of nuclease assays such as in (A). Averages shown; n ≥ 3; error bars, SEM.",
    "molecules": "dsDNA, polyacrylamide"
  },
  {
    "caption": "Nuclease assays with MR and various NBS1 fragments (50 nM) on oligonucleotide-based 5'-end labeled dsDNA. The reaction buffer contained 60 mM NaCl and reactions were incubated for 2 h.   Quantitation of experiments such as shown in (A). Averages shown; n ≥ 3, error bars, SEM.   ",
    "molecules": "dsDNA, oligonucleotide, NaCl"
  },
  {
    "caption": "In vivo resection assay using BrdU staining under native conditions to directly detect ssDNA at sites of DSBs. Shown are representative images.",
    "molecules": "ssDNA"
  },
  {
    "caption": "In vivo resection assay using RPA2 staining as a marker for ssDNA present at sites of DSBs. Shown are representative images.",
    "molecules": "ssDNA"
  },
  {
    "caption": "Quantitation of the single strand annealing (SSA) reporter assay in U2OS  cells. All cells were treated with DMSO; where indicated, both PFM03  (50 μM) and PFM39  (50 μM) MRE11  inhibitors were added. Averages shown; n = 4, error bars, SEM. Insert, western blot analysis of NBS1  and CtIP protein levels upon depletion with siRNA.",
    "molecules": "PFM03, PFM39, DMSO"
  },
  {
    "caption": "Nuclease assays with MR and pCtIP on 3'-end labeled dsDNA, incubated for 2 h.   Quantitation of experiments such as shown in (A). Averages shown; n = 2; error bars, range.   ",
    "molecules": "dsDNA"
  },
  {
    "caption": "Nuclease assays with MRN, MR and various concentrations of pCtIP (mock-treated) or λCtIP on 5'-end labeled dsDNA, incubated for 2 h.",
    "molecules": "dsDNA"
  },
  {
    "caption": "Phosphorylated CtIP was mock-incubated (pCtIP, lane 2) or treated with λ phosphatase (λCtIP, lane 3). The proteins were separated on a 10% polyacrylamide gel and stained with Coomassie brilliant blue.",
    "molecules": "polyacrylamide"
  },
  {
    "caption": "Nuclease assays with MR, MRN, pCtIP or pCtIP T847A, as indicated, on 5'-end labeled dsDNA. Reactions were incubated for 2 h.   Quantitation of experiments such as shown in (H). Averages shown; n = 4; error bars, SEM.   ",
    "molecules": "dsDNA"
  },
  {
    "caption": "Representative nuclease assays with MRE11, MR, MRN and pCtIP, as indicated, on 5'-end labeled dsDNA with a single streptavidin-blocked end (S). The substrate contained 5 phosphorothioate bonds (PTO) at both 3'-ends to prevent exonucleolytic DNA degradation. Reactions were incubated for 2 h.",
    "molecules": "dsDNA, phosphorothioate"
  },
  {
    "caption": "Representative nuclease assays as in (A), but with a substrate containing a nick in the top oligonucleotide (20 nt away from the 3'-end, see cartoon).",
    "molecules": "oligonucleotide"
  },
  {
    "caption": "A. Left: Snapshots of actin  comet tails  assembled in Assembly  conditions: 4.5 µm polystyrene  beads coated with 400 nM Strep-SNAP-WA; 3 µM actin, 6 µM profilin, 90 nM Arp2/3 complex, 15 nM capping protein. Right: Tracking of the comet shown in the snapshots. Time  is encoded in color. B. Left: Snapshots of actin  comet tails  assembled in Disassembly conditions: same as assembly  conditions with 200 nM ADF/cofilin  added. Right: Tracking of the comet  shown in the snapshots. Time  is encoded in color. C. Left: Snapshots of actin  comet tails assembled in Recycling  conditions: Disassembly  conditions with 400 nM Cyclase Associated Protein (CAP) added. Right: Tracking of the comet  shown in the snapshots. Time  is encoded in color.",
    "molecules": "polystyrene, Strep"
  },
  {
    "caption": "A. Left: Snapshots of  actin  comet tails in recycling conditions with the indicated ATP  concentrations. Right: Tracking of the comet  shown in the snapshots. Time  is encoded in color.",
    "molecules": "ATP"
  },
  {
    "caption": "D. Test of  actin  monomer aging. Actin  monomers were prepared in salt-free motility buffer (Ca-ATP  monomers, see Methods) or in salt-free motility buffer with EGTA  and MgCl2  (Mg-ATP  monomers, see Methods) and left on the bench at room temperature overnight. The day after, aged actin  monomers were added to a fresh mixture (without  actin  monomers) in presence of fresh beads  . Velocity of beads was measured in the different conditions. Composition of the reaction mix: 3 µM actin, 6 µM profilin, 90 nM Arp2/3, 15 nM  Capping protein, 200 nM cofilin, 400 nM Cyclase Associated Protein  (CAP). Ca-ATP actin  monomers fresh: N = 1, n = 50 comets. Ca-ATP actin  monomers (old) N = 1, n = 46 comets. Mg-ATP actin  monomers  (fresh): N = 2, n = 121 comets. Mg-ATP actin  monomers (old): N = 2, n = 134 comets. Individual points for each comet  are represented with mean and standard deviation superimposed.",
    "molecules": "ATP, Ca, EGTA, Mg, MgCl2"
  },
  {
    "caption": "(F) In situ hybridization of PLCγ1 in mDA neurons (scale bar = 50 μm) (green: TH, violet: DIG-labeled probe).",
    "molecules": "DIG"
  },
  {
    "caption": "(G) Representative images of the CC axon bundles in control (left) and a Plcg1f/f;Nes-Cre (right) mice (n = 4). The tip of the CC was triangular in the control mouse, but oval in the Plcg1f/f;Nes-Cre mouse. The areas delineated by the white boxes are magnified in the insets (red: neurofilament, green: glial fibrillary acidic protein, blue: Hoechst; scale bar = 1 mm).",
    "molecules": "Hoechst"
  },
  {
    "caption": "Western blot analysis of Fxr1 during (A), TTX (48h treatment) inducedupscaling (n=6 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.",
    "molecules": "TTX"
  },
  {
    "caption": "Western blot analysis of Fxr1 during (B), BIC (48h treatment) induced downscaling (n=8 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.",
    "molecules": "BIC"
  },
  {
    "caption": "C, D, Western blot analysis of (C), Fxr2 (Veh n=8, TTX n=8) and (D), Fmrp (Veh n=10, TTX n=12) during upscaling, Student's T-test *p<0.05.",
    "molecules": "TTX"
  },
  {
    "caption": "H, Western blot analysis for Fxr1, pGsk3α/β, Gsk3α/β and GAPDH in neuronal cultures treated with 1µM TTX (TTX) or 1mM LiCI (Li) or 1mM NaCI (Ctrl) for 48h.",
    "molecules": "Li, LiCI, NaCI, TTX"
  },
  {
    "caption": "I, Expression of Fxr1 protein in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "Li, TTX"
  },
  {
    "caption": "J, Expression of pGsk3β/Gsk3β or pGsk3α/Gsk3α in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "Li, TTX"
  },
  {
    "caption": "B, Western blot analysis of total GluA1 expression in Ctrl or Fxr1 overexpression (Fxr1) condition during upscaling (Ctrl/Veh n=5, Ctrl/TTX n=5, Fxr1/Veh n=6, Fxr1/Veh n=6). One way Anova with Dunnett's Multiple Comparison Test ***p<0.001.",
    "molecules": "TTX"
  },
  {
    "caption": "D, RT-qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling (Ctrl/Veh n=3, Ctrl/TTX n=3, Fxr1/Veh n=3, Fxr1/Veh n=3). One way Anova with Bonferroni's Multiple Comparison Test ***p<0.001.",
    "molecules": "TTX"
  },
  {
    "caption": "G, Immunostaining for surface GluA1 in GFP (Ctrl) or GFP-Fxr1 (Fxr1 over) infected cultures after treatment with Veh, TTX or BIC for 48h.",
    "molecules": "BIC, TTX"
  },
  {
    "caption": "H, Percentage of surface GluA1 relative to the mean of Ctrl/Veh condition (Ctrl condition: Veh n=63, TTX n=34, BIC n=29, Fxr1 condition: Veh n=56, TTX n=30, BIC n=29 ). One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "BIC, TTX"
  },
  {
    "caption": "A, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure (Ctrl/Veh n=16 and Ctrl/TTX n=17). B, A linear fit of Ctrl/TTX and Ctrl/Veh amplitudes. C, The degrees of overlap between Ctrl/TTX and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant p-value was obtained with 1.47 scaling factor. D, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Ctrl/TTX and Ctrl/TTX divided by scaling factor 1.47, which yielded the maximum overlap with Ctrl/Veh data. ",
    "molecules": "TTX"
  },
  {
    "caption": "Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (E) Fxr1overexpressing neurons (Fxr1/Veh n=10 and Fxr1/TTX n=8)",
    "molecules": "TTX"
  },
  {
    "caption": "Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (F) Gsk3 KO neurons (Gsk3KO/Veh n=10 and Gsk3KO/TTX n=11)",
    "molecules": "TTX"
  },
  {
    "caption": "Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (G) Fxr1 KO neurons (Fxr1KO/Veh n=16 and Fxr1KO/TTX n=8). H, A linear fit of Fxr1 KO/Veh and Ctrl/Veh amplitudes. I, The degrees of overlap between Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.27 scaling factor. J, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Fxr1 KO/Veh and Fxr1 KO/Veh divided by scaling factor 1.27, which yielded the maximum overlap with Ctrl/Veh data. ",
    "molecules": "TTX"
  },
  {
    "caption": "K, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical Gsk3 and Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure (Gsk3/Fxr1KO/Veh n=8 and Gsk3/Fxr1KO/TTX n=11). L, A linear fit of Gsk3/Fxr1 KO/Veh and Ctrl/Veh amplitudes. M, The degrees of overlap between Gsk3/Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.68 scaling factor. N, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Gsk3/Fxr1 KO/Veh and Gsk3/Fxr1 KO/Veh divided by scaling factor 1.68, which yielded the maximum overlap with Ctrl/Veh data. ",
    "molecules": "TTX"
  },
  {
    "caption": "O, mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure. One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "TTX"
  },
  {
    "caption": "P, mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure.",
    "molecules": "TTX"
  },
  {
    "caption": "(A) Expression levels of NAT4 analyzed by qRT-PCR using total RNA extracted from a wild-type (BY4741) and Pste5-NAT4 strain grown in 2% (NCR), or 0.1% glucose (CR). NAT4 levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM (Standard Error of the Mean) of 3 independent experiments. ** p ≤ 0.01; calculated by unpaired two-tailed Student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "(D) Gene expression analysis showing fold induction levels (Log2) of selected genes comparing BY4741 wild-type and STE5p-NAT4 strains under NCR (2% glucose) and CR (0.1% glucose) conditions. Expression levels were normalized to TAF10, whose expression remains unchanged.",
    "molecules": "glucose"
  },
  {
    "caption": "(A) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot. The negative isotype control (mGO53) and a commercially available anti-LAM antibody (positive control) are shown for comparison.",
    "molecules": "LAM"
  },
  {
    "caption": "(B) Anti-BCG specificity of the LAM-reactive antibody TB24PB037 (black) compared to E.coli as determined by ELISA with whole bacteria (left) and flow cytometry (right).",
    "molecules": "LAM"
  },
  {
    "caption": "(C) Anti-LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations. The mean ±SD of the absorbance was calculated.",
    "molecules": "LAM"
  },
  {
    "caption": "(D) Antibody TB24PB037 binds to MTB-LAM but not to LAM from M. smegmatis. ELISA performed as described in (C).",
    "molecules": "LAM"
  },
  {
    "caption": "(C) Absolute bacterial counts (CFUs) in human THP1 macrophages 1 h after infection with MTB pre-incubated with the LAM-reactive IgA1 (left) or IgG1 (right) antibody TB24PB037 or the respective isotype control antibody as indicated. Mean and SEM are indicated.",
    "molecules": "LAM"
  },
  {
    "caption": "Histological examinations of tumor types in (F) K versus KrasG12D; NanogL/L (KN) Tumor tissues collected form indicated mice analyzed using Haematoxylin Eosin (H&E) staining. Representative H&E images are shown. Scale bar, 500 μm (top), 50 μm (bottom).",
    "molecules": "Eosin, Haematoxylin"
  },
  {
    "caption": "(G, H) Statistical analysis of NICD (G) and HES1 (H) expression in lung sections of KLN mice following vehicle or LY-411575 treatment. The numbers of tumors analyzed over vehicle and LY-411575 groups were 22, 19 for NICD, 22, 22 for HES1. Results were shown as mean ± SD. Significance was calculated by two-tailed unpaired Student's t-test with Welch's correction. ****p<0.0001.",
    "molecules": "LY-411575"
  },
  {
    "caption": "(I) H&E and IHC staining of NICD and HES1 in lung tumor tissues of vehicle and LY-411575 groups. Scale bar, 50 μm. (J) H&E, AB and PAS staining in lung tumors of vehicle and LY-411575 groups. Scale bar, 50 μm. ",
    "molecules": "LY-411575"
  },
  {
    "caption": "(I, J) Statistical analysis of Ki67 (I) and CC3 (J) activity in ADC and IMA lung sections of vehicle and phenformin (Phen) groups. The numbers of tumors analyzed over vehicle group and phenformin groups for ADC and IMA were 17, 8, 9 for Ki67, 10, 10, 7 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0010 (I), ***p=0.0009 (I), *p=0.0427(J).",
    "molecules": "Phen, phenformin"
  },
  {
    "caption": "(E, F) Statistical analysis of Ki67 (E) and CC3 (F) reactivity in lung sections of KLN mice following vehicle, LY-411575 treatment or a combinational therapy of phenformin and LY-411575. The numbers of tumors analyzed over vehicle, phenformin and combinational therapy groups were 20, 30, 13 for Ki67, 14, 11, 9 for CC3. Results were shown as mean ± SEM. Significance was calculated by one-way ANOVA with Dunnett's multiple comparisons test. **p=0.0014 (E), ***p=0.0001 (F).",
    "molecules": "LY-411575, phenformin"
  },
  {
    "caption": "a, alpha-KG extends the lifespan of adult worms in the metabolite longevity screen. All metabolites were given at a concentration of 8 mM.",
    "molecules": "alpha-KG"
  },
  {
    "caption": "c, Dose-response curve of the α-KG effect on longevity.",
    "molecules": "α-KG"
  },
  {
    "caption": "d, e, α-KG extends the lifespan of worms fed bacteria that have been ampicillin arrested, mean lifespan (days of adulthood) with vehicle treatment (mveh) = 19.4 (n = 80 animals tested), mα-KG = 25.1 (n = 91), P  0.0001 (log-rank test) (d); or γ-irradiation-killed, mveh = 19.0 (n = 88), mα-KG = 23.0 (n = 46), P  0.0001 (log-rank test) (e). OP50, E. coli OP50 strain.",
    "molecules": "α-KG, ampicillin"
  },
  {
    "caption": "f alpha-KG does not further extend the lifespan of ogdh-1 RNAi worms, mveh = 21.2 (n = 98), mα-KG = 21.1 (n = 100), P = 0.65 (log-rank test).",
    "molecules": "alpha-KG"
  },
  {
    "caption": "a, DARTS identifies ATP5B as an alpha-KG-binding protein. Red arrowhead, protected band.",
    "molecules": "alpha-KG"
  },
  {
    "caption": "b, DARTS confirms alpha-KG binding specifically to ATP5B. IB, immunoblot.",
    "molecules": "alpha-KG"
  },
  {
    "caption": "c, Inhibition of ATP synthase by alpha-KG (released from octyl alpha-KG; Supplementary Notes). This inhibition was reversible (data not shown).",
    "molecules": "alpha-KG, octyl alpha-KG"
  },
  {
    "caption": "d, e, Reduced ATP levels in octyl alpha-KG-treated normal humanfibroblasts (**P = 0.0016, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (d)",
    "molecules": "octyl alpha-KG, ATP"
  },
  {
    "caption": "d, e, Reduced ATP levels in octyl alpha-KG-treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance) (e). RLU, relative luminescence units.",
    "molecules": "octyl alpha-KG, ATP"
  },
  {
    "caption": "f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated cells (***P = 0.0004, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (f)",
    "molecules": "octyl alpha-KG"
  },
  {
    "caption": "f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated worms (P  0.0001; by t-test, two-tailed, two-sample unequal variance) (g).",
    "molecules": "octyl alpha-KG"
  },
  {
    "caption": "h, alpha-KG, released from octyl alpha-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl alpha-KG- (3.1 ± 0.6) versus vehicle-treated mitochondria (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). Oligo, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; AA, antimycin A.",
    "molecules": "alpha-KG, octyl alpha-KG, antimycin A, FCCP, oligomycin"
  },
  {
    "caption": "i, Eadie-Hofstee plot of steady-state inhibition kinetics of ATP synthase by alpha-KG (produced by in situ hydrolysis of octyl alpha-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl alpha-KG. alpha-KG (produced from octyl alpha-KG) decreases the apparent Vmax (53.9 to 26.7) and Km (25.9 to 15.4), by nonlinear regression least-squares fit. c-i, Results were replicated in two independent experiments. Mean ± standard deviation (s.d.) is plotted in all cases.",
    "molecules": "alpha-KG, octyl alpha-KG, ADP, ATP, octanol"
  },
  {
    "caption": "a-g, Effect of alpha-KG on the lifespan of mutant or RNAi worms. a, atp-2 RNAi, mveh = 22.8 (n = 97), mα-KG = 22.5 (n = 94), P = 0.35; or RNAi control, mveh = 18.6 (n = 94), mα-KG = 23.4 (n = 91), P  0.0001. b, daf-2(e1370), mveh = 38.0 (n = 72), mα-KG = 47.6 (n = 69), P  0.0001. c, eat-2(ad1116), mveh = 22.8 (n = 59), mα-KG = 22.9 (n = 40), P = 0.79. d, let-363 RNAi, mveh = 25.1 (n = 96), mα-KG = 25.7 (n = 74), P = 0.95; or gfp RNAi control, mveh = 20.2 (n = 99), mα-KG = 27.7 (n = 81), P  0.0001. e, daf-16(mu86), mveh = 13.4 (n = 71), mα-KG = 17.4 (n = 72), P  0.0001; or N2, mveh = 13.2 (n = 100), mα-KG = 22.3 (n = 104), P  0.0001. f, pha-4(zu225), mveh = 14.2 (n = 94), mα-KG = 13.5 (n = 109), P = 0.55. g, hif-1(ia4), mveh = 20.5 (n = 85), mα-KG = 26.0 (n = 71), P  0.0001; or N2, mveh = 21.5 (n = 101), mα-KG = 24.6 (n = 102), P  0.0001. P values were determined by the log-rank test. Number of independent experiments: RNAi control (6), atp-2 (2), let-363 (3), N2 (5), daf-2 (2), eat-2 (2), pha-4 (2), daf-16 (2), hif-1 (5).",
    "molecules": "alpha-KG"
  },
  {
    "caption": "a, Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl alpha-KG or oligomycin. Similar results were obtained in HEK-293 cells, normal human fibroblasts and mouse embryonic fibroblasts (data not shown). P, phospho.",
    "molecules": "octyl alpha-KG, oligomycin"
  },
  {
    "caption": "b, Increased autophagy in animals treated with alpha-KG or RNAi for atp-2 or let-363. Photographs were taken at ×100 magnification. c, GFP::LGG-1 puncta quantified using ImageJ (Methods). Data show results of 2-3 independent experiments. Bars indicate the mean. ****P  0.0001; NS, not significant (t-test, two-tailed, two-sample unequal variance).",
    "molecules": "alpha-KG"
  },
  {
    "caption": "d, alpha-KG levels are increased in starved worms. **P  0.01 (t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted.",
    "molecules": "alpha-KG"
  },
  {
    "caption": "A 3-D reconstructions of propidium iodide (PI)-stained cell walls (red) and BCECF-stained vacuoles (green) of epidermal atrichoblasts in the early and late meristem and in the early and late elongation zone. Scale bars: 5 µm.",
    "molecules": "BCECF, PI, propidium iodide"
  },
  {
    "caption": "A Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and pUBQ10::VAMP711 (yellow) depict cell wall and tonoplast, respectively. Seedlings were treated with DMSO (solvent control) or 5 µM FC (Fusicoccin) for 2.5 h in liquid medium (n=24). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.",
    "molecules": "DMSO, FC, Fusicoccin, PI"
  },
  {
    "caption": "B Left: 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Right: Boxplot depicts vacuolar occupancy of the cell treated with the solvent DMSO or 5 µM FC for 2.5 h in liquid medium (n = 11). Student´s t-test (*p < 0.05). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "DMSO, FC, BCECF, PI"
  },
  {
    "caption": "C Cell wall and vacuolar membrane was visualized with PI (green) and MDY-64 (yellow). pER8::GFP-SAUR19 seedlings were treated with DMSO (n=60) or 10 µM β-estradiol (n=56) for 6 h in liquid medium. Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.",
    "molecules": "β-estradiol, DMSO, MDY-64, PI"
  },
  {
    "caption": "D Left: 3-D cell reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells in pER8::GFP-SAUR19 lines. Right: Boxplot depicts vacuolar occupancy of the cell. Seedlings were treated with the solvent control DMSO (n=11) or 10 µM β-estradiol (n=8) for 6 h in liquid medium. Student´s t-test (**p < 0.01). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "β-estradiol, DMSO, BCECF, PI"
  },
  {
    "caption": "E Cell wall and vacuolar membrane was visualized with PI (green) and pUBQ10::VAMP711 (E) (yellow). Col-0 wild type seedlings were treated for 3 h in liquid medium adjusted to pH 5.7 (n=44) or pH 4.5 (n=40). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.",
    "molecules": "PI"
  },
  {
    "caption": "F Left: 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Right: Boxplot depicts vacuolar occupancy of cell. Seedlings were treated for 3 h in liquid medium adjusted to pH 5.7 or pH 4.5 (n=11). Student´s t-test (***p < 0.001). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "BCECF, PI"
  },
  {
    "caption": "A Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Seedlings were treated with DMSO solvent control or 50 µM EGCG for 22 h on solid medium (n=40). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.",
    "molecules": "EGCG, DMSO, MDY-64, PI"
  },
  {
    "caption": "B 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of the cell. Seedlings were treated with DMSO (n=16) or 50 µM EGCG (n=15) for 22 h on solid medium. Student´s t-test (**p < 0.01). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "EGCG, DMSO, BCECF, PI"
  },
  {
    "caption": "C Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Seedling roots were grown on the surface (n=28) or into the matrix (n=28) of 2 % agar-containing solid medium. Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.",
    "molecules": "MDY-64, PI"
  },
  {
    "caption": "D 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of surface (n=10) and into the medium (n=11) grown seedling roots. Student´s t-test (**p < 0.01). Data information: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "BCECF, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 (n=64) and fer-4 (n=60). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "MDY-64, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green). Boxplot depicts vacuolar occupancy of Col-0 control (n=12) and fer-4 (n=10) mutant cells. Mann Whitney U test (**p < 0.01). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "BCECF, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (C) Col-0 (n=52) and fer-4 (n=44) seedlings were treated with solvent DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "EGCG, DMSO, MDY-64, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=28) and fer-4 (n=28) mutant seedling roots were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "agar, MDY-64, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 control (n=52) and lrx3/4/5 triple mutants (n=48). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "MDY-64, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of the cell in Col-0 control (n=11) and lrx3/4/5 (n=10). Student´s t-test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "BCECF, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (C) Col-0 (n=40-44) and lrx3/4/5 (n=36) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.01, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "EGCG, DMSO, MDY-64, PI"
  },
  {
    "caption": "Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=40-48) and fer-4 (n=28-32) seedlings were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "agar, MDY-64, PI"
  },
  {
    "caption": "A Rosette phenotype of 3-weeks-old Col-0, fer-4, lrx3/4/5 and fer-4/lrx3/4/5 (upper panel). Vacuolar morphology (MDY-64-stained) of late meristematic atrichoblast cells of Col-0, fer-4, lrx3/4/5 and fer-4/lrx3/4/5 (lower panel). Scale bars: 1 cm (upper row) and 5 µm (lower row).",
    "molecules": "MDY-64"
  },
  {
    "caption": "B Representative images and quantification of vacuolar morphology of late meristematic cells of Col-0 and 35S::LRR4-cit. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Col-0 (n=36) and 35S::LRR4-cit (n=32-36) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). Data information: 5 µm (B). Boxplots: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.",
    "molecules": "EGCG, DMSO, MDY-64, PI"
  },
  {
    "caption": "Four to six-week-old C57BL/6 mice were injected i.p. with 104-107 PFU of WNV-poly(A), 104 PFU of WT WNV or PBS (negative control) in a volume of 200 μL. Survival (n=5) in  infected   mice were monitored daily Data information: Log-rank test was used",
    "molecules": "PBS, poly(A)"
  },
  {
    "caption": "Four to six-week-old C57BL/6  mice were injected i.p. with 104-107 PFU of  WNV-poly(A), 104 PFU of WT  WNV  or PBS (negative control) in a volume of 200 μL. Histological analysis  of mouse brain. Scale bars represent 100 μm and 50 μm for left and right panels, respectively. Severe vacuolization (black arrows) and  inflammation (yellow arrow) in brain small vessels as well as degenerative changes in neurons with  cytoplasmic rarefaction (blue arrow) and  necrosis  with nuclear compaction (red arrow) were indicated. Scale bars represent 100 μm and 50 μm for left and right panels, respectively",
    "molecules": "PBS, poly(A)"
  },
  {
    "caption": "E Viral load and  replication kinetics in different tissues of C57BL/6 mice. Six-week-old female C57BL/6 mice  were inoculated subcutaneously with 106 PFU of WT WNV or WNV-poly(A) in the  left rear footpad, and tissues  were harvested from two mice  at the indicated times post-inoculation. Viral loads in brain,  spleen, left lymph node, right lymph node and  liver  were measured by  plaque assay and expressed as PFU per gram of tissue. n=3, values are mean ± sd, the dotted line corresponds to the limit of detection (LOD).",
    "molecules": "poly(A)"
  },
  {
    "caption": "G-H T-cell  responses in vaccinated  mice were measured through ELISpot assay. Two groups of C57BL/6 mice (n = 4 / group) were i.p. inoculated with 107 of WNV-poly(A) or equal volume of PBS.   ELISpot assay  specific for IFN-γ in  splenocytes was performed at 7 days post-immunization. Representative images were shown in (G) and the numbers of antibody secreting cells (ASCs) were counted (The values represent mean ± sd in each group, each symbol represents a mouse) (H). Data information: Mann-Whitney test was used for H. *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant",
    "molecules": "PBS, poly(A)"
  },
  {
    "caption": "C Comparison of growth kinetics between WT WNV and WNV-poly(A) viruses in MEF cells. MEF  cells were infected with either WT WNV  or WNV-poly(A) virus at an MOI of 0.01. Values indicate mean ± sd from two replicates of one representative experiment. Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.",
    "molecules": "poly(A)"
  },
  {
    "caption": "D Survival rate  of IFNAR-/- mice infected with different dosages of WNV-poly(A)  (n=5). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.",
    "molecules": "poly(A)"
  },
  {
    "caption": "E Cell morphology  changes of MEF cells infected with WT WNV and WNV-poly(A). Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.",
    "molecules": "poly(A)"
  },
  {
    "caption": "Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (E) Survival  of mice infected with 107 PFU of WNV-poly(A)-P50. Data information: For statistical analysis, one-way ANOVA analysis and Log-rank test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.",
    "molecules": "poly(A)"
  },
  {
    "caption": "Virulence and efficacy of the passaged WNV-poly(A) (WNV-poly(A)-P50) in mice (n=5). (F) Antibody titers of mice infected with WNV-poly(A)-P50 at different days post immunization. (For the values represent mean ± sd in each group (n=5) and the horizontal dotted line represents the limit of detection). Data information: For statistical analysis, Mann-Whitney test were used **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant.",
    "molecules": "poly(A)"
  },
  {
    "caption": "(D) Immunoblotting of proteins extracted from HeLa cells that express TFEB-3 × FLAG treated with DMSO, chloroquine (CQ) or SalA, subjected to nuclear/cytosolic fractionation and blotted with antibody against FLAG to detect TFEB. H3 and tubulin were used as nuclear and cytosolic markers, respectively. Blots are representative of triplicate experiments.",
    "molecules": "SalA, chloroquine, CQ, DMSO"
  },
  {
    "caption": "(B) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB-3 × FLAG HeLa cells starved in amino acid‐free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(C) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB-GFP HeLa cells were seeded in 96‐well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin‐Elmer). Scale bars represent 30 μm.",
    "molecules": "U0126, Rapamycin, Torin 1, Torin 2"
  },
  {
    "caption": "(D) Dose-response curves of the effects of ERK and mTOR inhibitors on TFEBnuclear translocation. TFEB-GFPHeLa cells were seeded in 384‐well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details).",
    "molecules": "U0126, Rapamycin, Torin 1, Torin 2"
  },
  {
    "caption": "(E) Immunofluorescence of HEK‐293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM.",
    "molecules": "DMSO, Torin 1"
  },
  {
    "caption": "(F) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB-3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh‐) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(G) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB-3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(A) Torin 1 induces S142 dephosphorylation. HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p‐S142 phosphoantibody and with anti‐FLAG antibody. Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(B) mTORC1 in‐vitro kinase assays. Highly purified FLAG-S6K1, TFEB-3 × FLAG, or TFEBS142A-3 × FLAG were incubated with radiolabelled ATP without kinase, with purified mTORC1 or with mTORC1+Torin 1, and analysed by autoradiography. The lower panel shows a FLAG immunoblot of the substrates.",
    "molecules": "ATP, Torin 1"
  },
  {
    "caption": "(C) Time‐lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB-GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1‐induced lysosomal accumulation of TFEB-GFP. Time intervals are in minutes.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(D) Immunofluorescence of HEK‐293T cells expressing TFEB-3 × FLAG, treated with DMSO (top) or Torin1 (bottom) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).",
    "molecules": "DMSO, Torin1"
  },
  {
    "caption": "(E) FRAP analysis of TFEB-GFP‐positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(F) Time‐lapse of photobleaching and fluorescence recovery of TFEB-GFP‐positive lysosomes from control‐treated MEFs (top) or MEFs treated with Torin 1 (bottom). Red arrowheads indicate time of photobleaching. Time intervals are in seconds.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(G) Torin 1 increases binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG along with HAGST‐Rap2A or HAGST‐RagsDN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG-Metap2 served as negative control.",
    "molecules": "Torin 1, Torin1"
  },
  {
    "caption": "(H) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (top) or the RagsDN mutants (bottom), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(A-C) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with a control GTPase or the indicated Rag mutants. Cells were deprived of amino acids (top) or deprived and then stimulated (bottom) for the indicated times and stained for FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).",
    "molecules": "amino acids"
  },
  {
    "caption": "(G) Quantification of the number of cells with nuclear TFEB from DMSO‐ and CQ‐treated fields in (E) and (F). In all fields, scale bars represent 10 μm. In all histograms, each value represents mean±s.d. from three independent fields with N=300.",
    "molecules": "CQ, DMSO"
  },
  {
    "caption": "(A) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes. Primary hepatocytes isolated from 2‐month‐old Tcfebflox/flox (control) and Tcfebflox/flox;Alb‐Cre(Tcfeb−/−) mice were left untreated, or treated overnight with Torin 1, U0126, or Chloroquine. Subsequently, cells were lysed and protein extracts were immunoblotted with the indicated antibodies.",
    "molecules": "U0126, Chloroquine, Torin 1"
  },
  {
    "caption": "(B, C) TFEB mediates the transcriptional response to chloroquine and Torin 1. Quantitative PCR (qPCR) of TFEB target genes in primary hepatocytes from control (flox/flox) and Tcfeb−/− mice. Cells were treated with Chloroquine (left) or Torin 1 (right). The graphs show the relative increased expression in the treated versus the corresponding untreated samples. Values represent means±s.d. of three independent hepatocyte preparations (three mice/genotype). Student's t‐test (two tailed) *P‐value ⩽0.05.",
    "molecules": "chloroquine, Chloroquine, Torin 1"
  },
  {
    "caption": "D. Western blots of the DNA-bound fraction eluted off the beads in a DNA-affinity purification assay. Super core promoter 1 (SCP1) was used a positive control to bind TFIIA-β-FLAG (top panel). DRE promoter pools of varying lengths were assayed for their ability to bind TFIIA. Sonicated salmon sperm DNA was used as competitor DNA and titrated from 100ng to 1.6ug per reaction. Histone H3 was used as an abundant non-specific DNA interacting protein for loading control. From top to bottom we have used promoter fragments ranging at 121bp, 350bp and 1kb in length.",
    "molecules": "DNA"
  },
  {
    "caption": "F. Western blot of anti-FLAG antibody of TBP-AID, Trf2-AID and a double tagged TBP-AID + Trf2-AID cell lines from a multi-day time course of auxin treatment showing prolonged depletion.",
    "molecules": "auxin"
  },
  {
    "caption": "J. qPCR on an auxin treatment time course of TBP and Trf2 dependent genes upon individual depletion of TBP or Trf2 and a double depletion of both. Error bars represent the standard deviation across three biological replicates.",
    "molecules": "auxin"
  },
  {
    "caption": "F-G, drug tolerant rate of (F) Du145/Du145TXR (20 nM  taxol/20 nM taxol plus 50 μM verapamil) or (G)  MCF-7/MCF-7ADR (200 nM adriamycin/200 nM  adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug tolerant rate  defined by comparing cell viability followed treatment, RMCs  VS. MCs  or  CCs  VS. PeCs, > 1 representing  drug tolerant. Induction  Time indicates the time  of entering DTP state; Sustaining Time indicates the  time of maintaining DTP state. Blue colors represent control groups; Red colors represent  MDR  cancer cells. Mean with ± SD.",
    "molecules": "adriamycin, taxol, verapamil"
  },
  {
    "caption": "A-C, flow cytometric analysis of ROS levels in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 and 36 hours. (A) Representative images and quantification of ROS  in (B) Du145TXR or (C) MCF-7ADR cells are shown. One-way ANOVA was used to analyze statistical differences. Mean with ± SD Data information: Results are representative of three independent experiments.",
    "molecules": "ROS"
  },
  {
    "caption": "C-D, endocytosis  analysis of (C) Du145TXR or (D) MCF-7ADR cells treated with indicated agents for 8 hours, followed by staining  with 250 μg/mL FITC-DEX for 4 hours. Scale bars, 10 μm. Data information: Results are representative of three independent experiments.",
    "molecules": "DEX, FITC"
  },
  {
    "caption": "A. Survival assay of Myc/Bcl2+GMP- and DN2-derived leukemia cells that were isolated from diseased mice and treated for 24 h with 0.15 μM daunorubicine or 1 μM doxorubicine in liquid culture. Cell death was measured by FACS of 7-AAD+cells and was normalized to the solvent controls. The graphs show the mean+SEM of three different DN2- and five GMP-leukemias. **=p≤0.01.",
    "molecules": "daunorubicine, doxorubicine"
  },
  {
    "caption": "F. Proliferation curves of 3 Myc/Bcl2+ DN2- and 3 GMP-derived leukemia lines during treatment with 0.5 μM ruxolitinib. 1.5105 cells were seeded in culture and counted at the indicated time points. Results are shown as fold growth of the starting cell number which was set to 1. *=p≤0.05, **=p≤0.01, Student's t-test.",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "G. Western blot indicating active Jak/Stat signaling in DN2-derived leukemia with Stat3 as well as pStat3 protein expression, that diminishes after treatment with the Jak-inhibitor ruxolitinib.",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "(E) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice folding structure. Asterisks indicate gyrus and arrows indicate sulcus structures. A1, A2, B1and B2 (DAPI) are high-magnification images. Scale bars, 200μm(left); 100μm(right). (F) Quantification of GI of WT and hFOXM1-cKI mice pups at P0. The quantification of the GI was analyzed in pre-selected hFOXM1-cKI mice with folding. (n = 6 each group; two-tailed u unpaired t-test, P= 0.0010).",
    "molecules": "DAPI"
  },
  {
    "caption": "(L) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1-cKI mice. White arrows represent BrdU and EdU double-positive cells. Scale bars, 50μm(left);20μm(right). (M) Graph shows the percentage of BrdU and EdU double-positive cells in E15 WT and hFOXM1-cKI mice (n = 6 each group; two-tailed unpaired t-test, P=0.0413). D",
    "molecules": "EdU, BrdU"
  },
  {
    "caption": "(A) Super-resolved average intensity (bar: high 8 to low 0 density), average diffusion coefficient (bar: Log10D high 1 to low -5 mobility) and trajectory maps (16000 frames) of BoNT/Aiwt-At647N on the plasma membrane (uPAINT) and following endocytosis (sdTIM).",
    "molecules": "At647N"
  },
  {
    "caption": "B, C Number of BoNT/Aiwt-At647N and mutant trajectories (B) on the plasma membrane (uPAINT) and (C) following endocytosis (sdTIM). N = 17 technical replicates/ condition from 5-6 independent biological replicates",
    "molecules": "At647N"
  },
  {
    "caption": "(E) EM analysis of endocytosed HRP-tagged BoNT/Ai. Representative images show cytochemically stained HRP precipitate (arrowheads). Endo/lysosomes, multivesicular bodies (MVB), structures with the morphology of autophagosomes (AP), and (i) presynaptic (Ps) and (ii) axonal (Ax) plasma membrane (PM). Postsynaptic density (asterisks), non-stained endocytic compartments (arrows) and background binding on the PM (arrows in ii) are indicated. Scale bars 100 nm.",
    "molecules": "HRP"
  },
  {
    "caption": "F, G (F) Percentage of total BoNT/Aiwt-HRP and indicated mutants localized on the PM or in the indicated endocytic structures, and (G) the average sectional area of the HRP-stained endocytic compartments. (F,G) n = 60 electron micrographs/ condition from 3 independent biological replicates.",
    "molecules": "HRP"
  },
  {
    "caption": "A, B Low resolution images and the corresponding super-resolved uPAINT average intensity (bar: high 8 to low 0 density), average diffusion coefficient (bar: Log10D high 1 to low -5 mobility) and trajectory maps of SV2Awt-pH/At565nb in (A) control conditions (high K+; Ctrl), and (B) following treatment with 100 pM BoNT/Aiwt-At647N (supplemented in high K+).",
    "molecules": "At647N, nb, At565, K+"
  },
  {
    "caption": "(C) Scatter plots of the average diffusion coefficient (µm2 s-1) single-molecule mobility of SV2Awt-pH/At565nb and SV2AT84A-pH/565nb in the control condition (high K+; Ctrl) and following treatment with 100 pM BoNT/Aiwt-At647N or BoNT/AiSV2-At647N (supplemented in high K+). (D) Scatter plots of the average diffusion coefficient (µm2 s-1) single-molecule mobility of Syt1wt-pH/At565nb, Syt1K362A,K328A-pH/565nb and Syt1K52A-pH/At565nb in the control condition (high K+; Ctrl) and following treatment with 100 pM BoNT/Aiwt-At647N (supplemented in high K+).",
    "molecules": "At647N, nb, 565, At565, K+"
  },
  {
    "caption": "A-C (A) Representative low-resolution image of SV2Awt-pH (green), and corresponding dual-colour super-resolved uPAINT trajectories of SV2Awt-pH/At565nb (cyan) imaged simultaneously with BoNT/Aiwt-At647N (magenta). Regions (i-ii) are magnified below, and (iii) is magnified in (B) showing a representative time-lapse sequence of the single molecules of SV2Awt-pH/At565nb (cyan) and BoNT/Aiwt-At647N (magenta) detected coincidentally in a neuronal projection. The 10 x 10 pixel boxed region (iv) is magnified in (C), showing frame-by-frame coincidental detection of BoNT/Aiwt-At647N and SV2Awt-pH/At565nb obtained using a 50 Hz (20 ms exposure time) 16,000 frame acquisition as indicated.",
    "molecules": "At647N, nb, At565"
  },
  {
    "caption": "H, I Representative images of NASTIC nanocluster analysis of SV2Awt-pH/At565nb in (H) the control condition (high K+; Ctrl) and (I) following high K+ stimulation in the presence of 100 pM BoNT/Aiwt-At647N, with 2D kernel density estimation (KDE, high density in red), instantaneous Log10 diffusion coefficient map (low mobility in red), and nanoclusters with their respective trajectories (white) and centroids (orange) shown. Boxed areas are magnified on the right, with arrowheads indicating nanoclusters.",
    "molecules": "At647N, nanoclusters, nb, At565, K+"
  },
  {
    "caption": "J-N Quantification of the (J) SV2Awt-pH/At565nb, (K) SV2AT84A-pH/At565nb, (L) Syt1wt-pH/At565nb, (M) Syt1K326A,K328A-pH/At565nb and (N) Syt1K52A-pH/At565nb nanoclustering in control conditions (high K+; Ctrl) and following BoNT/Aiwt-At647N treatment (diluted in high K+). (J-N) N = 11-19 technical replicates from 3-5 independent biological replicates,",
    "molecules": "At647N, nb, At565, K+"
  },
  {
    "caption": "(A) Representative confocal Z-stack sum projections of neurons transduced with TagBFP2-expressing (blue) control (ctrl; non-targeting sgRNA) and CRISPRi Syt1 KD (Syt1 sgRNA1-3) lentiviruses. Neurons were treated for 30 min with 10 units of AbobotulinumtoxinA, and immunostained for endogenous Syt1 (yellow) and BoNT/A-cleaved SNAP-25 (SNAP-25/A; magenta). Boxed regions (i-iv) are magnified on the right, with neuronal somas outlined by dashed lines for clarity. B, C Quantification of mean fluorescence intensity (MFI) of endogenous (B) Syt1, and (C) cleaved SNAP-25 (SNAP-25/A), in control and Syt1 sgRNA1-3 KD neurons following AbobotulinumtoxinA treatment. (B-C) n = 21 technical replicates/ condition from 2 independent biological replicates. Statistical significance was assessed using a non-parametric Kruskal-Wallis multiple comparison test",
    "molecules": "AbobotulinumtoxinA"
  },
  {
    "caption": "H, I Quantification of (H) Syt1 and (I) SNAP-25/A MFI in non-transduced neurons (neurons negative for TagBFP2) with and without pLenti6.3-Syt1wt-pH expression following AbobotulinumtoxinA treatment (10 units, 30 min.) n = 3 technical replicates/ condition from one biological experiment.",
    "molecules": "AbobotulinumtoxinA"
  },
  {
    "caption": "(A) Representative patch-clamp spontaneous miniature inhibitory postsynaptic current (mPSC) recordings in naïve neurons, and in control (non-targeting) and Syt1 KD (CRISPRi sgRNA1) neurons following a 30 min treatment with AbobotulinumtoxinA (10 units). B, C Quantification of mPSC (B) peak amplitudes and (C) frequencies for indicated conditions. Error bars are shown as standard error of the mean (±SEM). N = 5-12 technical replicates/ condition from 2 independent biological replicates in (B, C) statistical significance was assessed using a non-parametric Kruskal-Wallis multiple comparison test (B), an ordinary one-way ANOVA multiple comparison test (C)",
    "molecules": "AbobotulinumtoxinA"
  },
  {
    "caption": "(D) EM analysis of endocytosed BoNT/Aiwt-HRP following lentiviral control (ctrl; non-targeting sgRNA) and Syt1 sgRNA1 CRISPRi KD transduction. HRP precipitate in synaptic vesicles (SVs; arrowheads) and tubular structures (arrows) in presynapses (Ps) are indicated. The postsynaptic density is marked with an asterisk. (E) Percentage of total BoNT/Aiwt-HRP localized in SVs, endo/lysosomes (E), tubular structures (T), multivesicular bodies (MVB), structures resembling autophagosomes (AP) and on the plasma membrane (PM) in the indicated conditions. n = 56 regions of interest/ condition from 2 independent biological replicates (E) two-way ANOVA multiple comparison test (E).",
    "molecules": "HRP"
  },
  {
    "caption": "(a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells.",
    "molecules": "Ca, CCK"
  },
  {
    "caption": "(c-e) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.",
    "molecules": "CCK"
  },
  {
    "caption": "(e) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1-/-acini stimulated with 100 pM CCK. The results are the mean±s.e.m of 3 experiments with acini obtained from 3 wild-type and 3 Trpml1-/-mice.",
    "molecules": "CCK"
  },
  {
    "caption": "(c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line.",
    "molecules": "caerulein"
  },
  {
    "caption": "(d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages.",
    "molecules": "CCK"
  },
  {
    "caption": "(e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity.",
    "molecules": "ceramide, CCK"
  },
  {
    "caption": "(f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.",
    "molecules": "CCK"
  },
  {
    "caption": "(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.",
    "molecules": "Ca, carbachol"
  },
  {
    "caption": "(b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.",
    "molecules": "isoproterenol, pilocarpine"
  },
  {
    "caption": "(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.",
    "molecules": "isoproterenol"
  },
  {
    "caption": "(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.",
    "molecules": "isoproterenol, pilocarpine"
  },
  {
    "caption": "(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.",
    "molecules": "Ca, KCl"
  },
  {
    "caption": "(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).",
    "molecules": "Glutamate, glutamate, KCl"
  },
  {
    "caption": "(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.",
    "molecules": "glutamate, KCl"
  },
  {
    "caption": "B-C. Wound healing assays were performed using IBIDI® chambers to evaluate the role of the AhR  on cell migration. Images of the wound  were captured using an Axio Vert.A1 inverted microscope (Carl Zeiss®) at 5x magnification. The histogram represents the mean ± s.d. Wound  closure was determined by measuring the distance between the edges of the wound  at time  0 and 15 h (n = 3 independent technical experiments for each cell lines or conditions) and compared using unpaired t-tests with the Sidak-Bonferroni method. B. Results obtained with BRAFi-sensitive or resistant SK28  cells KO for the AhR  in the absence of treatment. (n = 3 independent technical experiments for each cell lines). C. Results obtained for the migration assay  (0 to 15 h) for SK28  R cells KO or not for the AhR  after treatment or not with 10 μM CH-223191. (n = 3 independent technical experiments for each cell lines or conditions, mean ± s.d.). Statistical analysis using unpaired t-tests with the Sidak-Bonferroni method has been performed between the mean of the 3 independent experiments.",
    "molecules": "CH-223191"
  },
  {
    "caption": "D. Three-dimensional spheroid growth  of BRAFi-sensitive or resistant SK28  cells KO before or after knockout of AhR  by CRISPR/Cas9  in the absence of treatment. Images were captured four days after implantation of the spheroids  into collagen  gel. (n = 4 independent technical experiments, mean ± s.d. E. Three-dimensional spheroid growth  of BRAFi-resistant SK28  cells KO before or after knockout of AhR  by CRISPR/Cas9  after daily treatment with the specific AhR  inhibitor CH-223191 (5 μM) for one week or in the absence of treatment. Images were captured four days after implantation of the spheroids  into collagen  gel. (n = 3 independent technical experiments, mean ± s.d.). Statistical analysis using unpaired t-tests method has been performed with the Sidak-Bonferroni method, (p<0.01 ##, **; p<0.001 ###,***).",
    "molecules": "CH-223191"
  },
  {
    "caption": "B. Protein levels of the AhR, p-SRC  (Y416), SRC, p-FAK  (Y576/577), and FAK  in the four different melanoma  cell lines (n=3). The level of BRAFi resistance corresponds to our measure of IC50  (Vemurafenib) for the different cell lines 501Mel  (0,23uM), SKMel28  (0,29uM), M229  (0,89uM) and M238  (2,16uM). These cell lines correspond to the sensitive parental cells.",
    "molecules": "Vemurafenib"
  },
  {
    "caption": "D. Protein levels of AhR, p-SRC  (Y416), and SRC, in the SK28R cell line after treatment with different AhR  ligands for 24h (5 μM BaP, 5 μM indirubin, 5 μM ITE, 10 nM TCDD, and 5 μM FICZ).",
    "molecules": "TCDD, BaP, FICZ, indirubin, ITE"
  },
  {
    "caption": "E. Protein levels of AhR, p-SRC  (Y416), and SRC, in the  SK28R cell line after 24 h of treatment with increasing doses of ITE.",
    "molecules": "ITE"
  },
  {
    "caption": "B. Three-dimensional spheroid growth  of a 50%/50% mix of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with SRC  inhibitors: bosutinib  (1 µM) or dasatinib  (1 µM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.",
    "molecules": "bosutinib, dasatinib, DMSO"
  },
  {
    "caption": "C. Three-dimensional spheroid growth   of SK28R WT (in red) and KO cells (in green) over five days. Cells were treated or not with dasatinib  (1 µM) in combination with ITE  (5 μM) or TCDD  (20 nM) every two days (n = 3 independent technical experiments, mean ± s.d.). Comparisons with control (DMSO) were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.05 *, p<0.01 **. Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.",
    "molecules": "TCDD, dasatinib, DMSO, ITE"
  },
  {
    "caption": "D. Protein levels of AhR, p-SRC  (Y416), and SRC  in the  SK28R cell line after 24 h of treatment with ITE  (5 μM) or SRC  inhibitor (dasatinib) (1 μM).",
    "molecules": "dasatinib, ITE"
  },
  {
    "caption": "F. Expression  heatmap of the median of  gene expression  for the various signatures (left) and AhR   target genes (right) from RNAseq  datasets for  SK28R cells treated or not for 24 h with SRC  inhibitors: bosutinib  (Bos, 1 μM) or dasatinib  (Das, 1 μM). The scale corresponds to the Z scores.",
    "molecules": "Bos, bosutinib, Das, dasatinib"
  },
  {
    "caption": "B. PDX  tumor volumes were measured every 2 days until reaching 1500mm3. Values correspond to the mean +/- sem. Statistical analysis (2way ANOVA) has been performed between the different experiments (Das  or Das  before combo vs Dab) at different times. Stars represent time  from when  tumor size is significantly lower than group treated with BRAFi alone (Dabrafenib) p<0.05 *, p<0.01 **.",
    "molecules": "Dab, Dabrafenib, Das"
  },
  {
    "caption": "(a) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7.5mg/kg cisplatin. Arrows identify caspase 3 positive cells. Scale bars = 50 µm. (b) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells (Mann-Whitney, n = 3 vs 5).",
    "molecules": "cisplatin"
  },
  {
    "caption": "C, Comparison of wild-type colonies with phytochrome and  hox-deletion strains in the presence of 0.12 mM menadion (MD), 7 mM  H2O2 or 2 mM tert-butyl hydroperoxide (tBooH). The strains were incubated for 5 days at 28°C in the dark.",
    "molecules": "H2O2, MD, menadion, tBooH, tert-butyl hydroperoxide"
  },
  {
    "caption": "B, HoxA-GFP and CitA-GFP  (ca. 170 amino acids from the N-terminus of citrate synthase) were expressed over night at 30°C in minimal medium with 2%  threonine and 0.2% glucose. Mitochondria were incubated with 100 μg/ml  proteinase K (PK) for 20 min on ice. Crude extract (CE), supernatant 1 (S1) containing  mitochondria, pellet 1 (P1), supernatant 2 (S2) and pellet 2 (P2) with mitochondria were analyzed by Western Blot using anti GFP  antibodies. CitA-GFPc = cytoplasmic form; CitA-GFPi = imported version.",
    "molecules": "amino acids, glucose, threonine"
  },
  {
    "caption": "A,  BiFC  (split-YFP) analysis of HOs and phytochrome. Strains (SChS29-33) were grown over night at 28°C minimal medium with 2%  threonine  and 0.2% glucose. MitoTracker Red CMOXRos was used for  mitochondrial staining. The mitotracker signal was observed in the  RFP and the split-YFP signal in the YFP  channel of the fluorescent microscope. Pictures in both channels were overlaid (merge). The left pictures show the same hyphae in differential interference contrast  (DIC). The scale bar represents 10 µm.",
    "molecules": "glucose, mitotracker, MitoTracker Red CMOXRos, threonine"
  },
  {
    "caption": "C, Co-expression of the HOXs along with the photosensory domain of A. nidulans FphA (PGP) over night at 20°C. PGP was purified via the Strep-tag system and the eluate concentrated (upper panel). The lanes were loaded as follows: negative control before induction (-), 20 h after induction (20 h) crude extract (CE), supernatant (S), pellet (P), flow through (F) and eluate (E). The fractions were analyzed by Western Blot  using the Anti-His antibody (lower panel).",
    "molecules": "Strep"
  },
  {
    "caption": "A, B,  Biolayer interferometry analysis with Apo-PGP A and holo-PGP B. Proteins were immobilized at a streptavidin sensor tip. Free binding sites were blocked with biotin. Association and dissociated kinetics were recorded at the indicated HoxA concentrations. Kd values were calculated with the manufacturers software using a global fit for both experiment",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(A, B) CMH2DCFDA staining (A) and data quantification (B) showing H2O2 level in different aged fly brains with and without treatment with the indicated RET inhibitors (n=5 per group).",
    "molecules": "CMH2DCFDA, H2O2"
  },
  {
    "caption": "(I) Dose-dependent effect of CPT on RET-ROS in mitochondria isolated from 45 days old flies and respiring under RET condition (n=4).",
    "molecules": "CPT, ROS"
  },
  {
    "caption": "(K) CMH2DCFDA staining showing H2O2 level in young flies treated with DES or DES + CPT (n=5 per group).",
    "molecules": "CMH2DCFDA, CPT, DES, H2O2"
  },
  {
    "caption": "(E-H) Survival curves comparing control flies and flies fed with food containing 5 µM CPT (E), 10 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (F), or 20 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (G) from 35 days onwards. Bar graphs (H) show quantification of mean and maximal lifespan (n=3 groups, 20-25 flies per group), using single factor ANOVA with Scheffe's analysis as a post hoc test. : Data are representative of at least three repeats. H) Data are shown as mean ± Survival curves were analyzed with logrank test along with Wang Allison test for maximum lifespan. Asterisks indicate statistical significance (**p< 0.01, *p< 0.05). Dashed lines mark time points of 50% survival.",
    "molecules": "CPT, food"
  },
  {
    "caption": "(C) Images and data quantification showing accumulation of protein aggregates in the brains of aged flies with or without CPT treatment (n=5).",
    "molecules": "CPT"
  },
  {
    "caption": "(D) Images and data quantification showing DA neuron mitochondrial morphology in the brains of aged flies with or without CPT treatment (n=5).",
    "molecules": "CPT"
  },
  {
    "caption": "Images (E) showing 4-HNE levels within or near TH-positive DA neurons in young flies, old flies, and old flies treated with CPT (n=4 sets, 5 samples per set). Arrows point to HNE signals co-labelled with TH- positive DA neurons.",
    "molecules": "4-HNE, HNE, CPT"
  },
  {
    "caption": "(D) CMH2DCFDA staining of H2O2 and quantification of signal intensity in the muscle tissues of 50-day old control flies and flies with NDUFS2 or NDUFS3 knockdown (n=5 per group).",
    "molecules": "CMH2DCFDA, H2O2"
  },
  {
    "caption": "(F) Survival curves of wild type flies with or without NMN treatment from 35 days onwards (**p<0.01 logrank, **P<0.01 Wang Allison) (n=3 groups, 20-25 flies per group).",
    "molecules": "NMN"
  },
  {
    "caption": "(A, B) Survival curves showing effect of neuronal dSirt2 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (C, D) Survival curves showing effect of muscle dFoxo knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (E, F) Survival curves showing effect of neuronal ATG1 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group).",
    "molecules": "CPT"
  },
  {
    "caption": "(H) Images and data quantification in single factor ANOVA with Scheffe's analysis as a post hoc test showing lysotracker staining in the muscle of young flies, old flies, and old flies treated with CPT (n=5 per group).",
    "molecules": "CPT, lysotracker"
  },
  {
    "caption": "(A, B) Survival curves showing effect of CPT treatment on the lifespan of Mhc>APP.C99 flies (**p<0.01 logrank, **p<0.01 Wang Allison) (A) and elav>APP.BACE flies (**p<0.01 logrank, **p<0.01 Wang Allison) (B) (n=3 groups, 20-25 flies per group).",
    "molecules": "CPT"
  },
  {
    "caption": "(E) Immunofluorescence images and data quantification showing effect of CPT on the formation of ubiquitin- and 6E10-positive amyloid aggregates in the brain of elav>APP.BACE flies (n=5).",
    "molecules": "amyloid, CPT"
  },
  {
    "caption": "(G) Quantification of the effect of CPT on DA neuron number in the PPL1 cluster of elav>APP/BACE fly brain (n=4 sets, 5 brains per set).",
    "molecules": "CPT"
  },
  {
    "caption": "(A, B) Quantification of effect of CPT on ROS level as detected by staining with the H2O2 dye CMH2DCFDA (A), and on NAD+/NADH ratio (B) in control, FAD, and DS iPSC-derived neurons.",
    "molecules": "CMH2DCFDA, CPT, H2O2, NAD, NADH, ROS"
  },
  {
    "caption": "(D) Survival curves showing effect of CPT on lifespan of PS19 tauopathy mice (*p<0.05 logrank) (n=9 in vehicle group, 8 in CPT group).",
    "molecules": "CPT"
  },
  {
    "caption": "(a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue). The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times. The cells shown are representative of the majority of cells in each sample.",
    "molecules": "DNA, thymidine"
  },
  {
    "caption": "(c) DNA histograms of the same cells at the indicated time points.",
    "molecules": "DNA"
  },
  {
    "caption": "(d) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin (green), VHR (red) and DNA (DAPI; blue).",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Anti-VHR immunoblot of synchronized cells from G1, S, G2 and M phase treated for 30 min with 50 μg ml−1 of the protein synthesis inhibitor cycloheximide. Note that only cells in G1 showed a rapid loss of VHR protein. The scale bars represent 5 μm in a and 2 μm in b.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(d) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA. The data represent the mean ± sd. (n = 3).",
    "molecules": "Thymidine"
  },
  {
    "caption": "(a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4-5 days. The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture.",
    "molecules": "DNA"
  },
  {
    "caption": "(b) Confocal microscopy of the same HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI, blue). Control siRNA-treated cells and cells treated with VHR are shown. The bottom panels represent merges of the 3 images above",
    "molecules": "DNA"
  },
  {
    "caption": "(e) Confocal microscopy of control or VHR siRNA-transfected HeLa cells stained for VHR (green) and phospho H3 (red) to indicate M-phase entry, and DNA (DAPI, blue). The bottom panels represent merges of the images above.",
    "molecules": "DNA"
  },
  {
    "caption": "(C, D) Oxygen consumption rates (OCRs) of MEFs were measured on an XF24 Analyzer. (C) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin (2 μg/mL) (Oligo), FCCP (2 μM), and rotenone (1 μM) plus actinomycin (2 μM) (R & A). (D) OCR (pmol O2/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.",
    "molecules": "actinomycin, FCCP, Oligo, oligomycin, O2, rotenone"
  },
  {
    "caption": "(E) ATP production (pmol/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.",
    "molecules": "ATP"
  },
  {
    "caption": "(F) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype. MEFs were treated with tunicamycin (5 μg/ml) or vehicle control in the presence or absence of bafilomycin A1 (400 nM), and endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin in the absence of bafilomycin, which were normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.",
    "molecules": "bafilomycin, bafilomycin A1, tunicamycin"
  },
  {
    "caption": "(G) Survival rate of MEFs treated with tunicamycin (5 μg/ml), thapsigargin (1 μM), or hydrogen peroxide (100 μM). Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.",
    "molecules": "hydrogen peroxide, thapsigargin, tunicamycin"
  },
  {
    "caption": "MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (A) Absolute [Ca2+]m changes in MEFs of indicated genotype in response to bradykinin (2.5 μM). (B) Basal and peak values of Ca2+ transients (μM). Error bars represent ±SD from six independent experiments. Data information: For graph P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "bradykinin, Ca2+"
  },
  {
    "caption": "MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (C) Peak values of Ca2+ transients in MEFs transfected with IP3R or shRNA against IP3R, or treated with 2-AP (20 mM). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "2-AP, Ca2+"
  },
  {
    "caption": "MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (D) Peak values of Ca2+ transients in MEFs transfected with VDAC1 or shRNA against VDAC1. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "Ca2+"
  },
  {
    "caption": "(G) Peak values of Ca2+ transients in MEFs transfected with synthetic tethering protein (TOM-mRFP-ER) to induce artificial tethering of the ER and mitochondria. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "Ca2+"
  },
  {
    "caption": "(C) Peak values of Ca2+ transients in MEFs transfected with deletion constructs of LRRK2(G2019S). Error bars represent ±SD from six independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05",
    "molecules": "Ca2+"
  },
  {
    "caption": "Peak values of Ca2+ transients in MEFs transfected with ligase-active MARCH5(WT), MULAN(WT), and Parkin(W403A) [Parkin(WA)] or dominant-negative forms of MARCH5(H43W) [MARCH(HW)], MULAN(C339A) [MULAN(CA)], Parkin(C431A) [Parkin(CA)] (E) Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05",
    "molecules": "Ca2+"
  },
  {
    "caption": "Peak values of Ca2+ transients in MEFs transfected with active USP30 or inactive USP30(C77S) [USP(CS)] (F). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05",
    "molecules": "Ca2+"
  },
  {
    "caption": "(A) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2+/+ and LRRK2(G2019S)-expressing MEFs treated with vehicle (Control) or tunicamycin (1 μg/ml). Endogenous PERK, MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Data represent the ratio of phosphorylated to total protein levels. Error bars represent ±SD from independent experiments. Data information: For graph A the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(B) Immunoprecipitation/immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin (5 μg/ml). LRRK2(G2019S) MEFs were also pretreated with LRRK2-IN-1 (1 μM). MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the Myc epitope, phosphoserine or mitofin2 as indicated to the right of each panel. Mfn2: mitofusin 2, Ub: ubiquitin. Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin. Error bars represent ±SD from four independent experiments. Data information: For graph B, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "LRRK2-IN-1, phosphoserine, tunicamycin, biquitin, Ub, ubiquitin"
  },
  {
    "caption": "(A) Peak values of Ca2+ transients in MEFs treated with LRRK2-IN-1 (1 μM). Error bars represent ±SD from six independent experiments. Data information: For graph A , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "Ca2+, LRRK2-IN-1"
  },
  {
    "caption": "(B)(Upper panel) Diagram showing full-length PERK Myc at the N-terminus and FLAG at the C-terminus (M-PERK-F). S1P recognition sequence R33SLL is mutated to A33SLL (M-PERK(R33A)-F). (Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against FLAG. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right.",
    "molecules": "tunicamycin"
  },
  {
    "caption": "(C) In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase and [γ-32P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were with antibody against Myc, FLAG or phosphoserine. [γ-32P]ATP-incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ).",
    "molecules": "ATP, phosphoserine, 32P"
  },
  {
    "caption": "(D) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA-tagged Ubl, and His-tagged mitofusin2 in the presence of E1 enzyme, UbcH7 and ATP. E3 ubiquitin ligases were initially phosphorylated PERK or PERK(K618R) in vitro. Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination. itofusin 2 with Ni-NTA subjected to SDS/PAGE. Blots were with antibody against HA or mitofusin2. Mfn2: mitofusin 2, Ub: ubiquitin.",
    "molecules": "Ni-NTA, ATP, Ub, ubiquitin, Ubl"
  },
  {
    "caption": "(E) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)-expressing MEFs transfected with the increasing amounts of LRRK2-d1-V5 (1, 5, 25 μg/106 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti-mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ±SD from independent experiments. Data information: For graph E, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01",
    "molecules": "LRRK2-d1-V5, tunicamycin"
  },
  {
    "caption": "(B) In vitro kinase assay for auto-phosphorylated LRRK2 using isolated LRRK2 mutants and [γ-32P]ATP with or without LRRK2-IN-1 (1 μM). Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS/PAGE. Blots were with antibody against V5 or phosphoserine. [γ-32P]ATP-incorporated LRRK2 visualized by autoradiography.",
    "molecules": "ATP, LRRK2-IN-1, phosphoserine, 32P"
  },
  {
    "caption": "(B) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments.",
    "molecules": "Ca2+"
  },
  {
    "caption": "A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.",
    "molecules": "ADP-ribose"
  },
  {
    "caption": "B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.",
    "molecules": "PAR"
  },
  {
    "caption": "B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).",
    "molecules": "Olaparib"
  },
  {
    "caption": "D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).",
    "molecules": "doxo, doxocyclin"
  },
  {
    "caption": "B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.",
    "molecules": "H3K9me3, DAPI"
  },
  {
    "caption": "Depletion of neutrophils using anti-Ly6G led to impaired fracture healing. Representative section stained with Masson's trichrome at day 14 comparing fracture healing in mouse treated with isotype control (top) versus anti-Ly6G (bottom). Black and white images in the right column are identical to the color images in the left column; they have been labeled to clearly demonstrate the anatomical structures. Control section shows advanced mineralized callus, while treatment section shows a large immature unmineralized callus. Muscle fibers: red; collagen, bone, and mineralized callus: green. Scale bar, 1 mm.",
    "molecules": "collagen"
  },
  {
    "caption": "Treatment with CCR2 antagonist, INCB3344, led to impaired fracture healing compared to vehicle control as shown by the reduced % callus mineralization at day 28 after operation. Data are presented as mean ± SEM. *P = 0.011 unpaired two-sided t-test. Representative micro-CT images are shown. Scale bar, 2 mm.",
    "molecules": "INCB3344"
  },
  {
    "caption": "C MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were intravenously injected with poly(I:C) (200 μg/mouse). Serum samples were collected at 2 hpi and secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). Percentage of surviving mice (A, log-rank test, **P < 0.01)",
    "molecules": "LPS"
  },
  {
    "caption": "MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). body weight changes (B) in each groups.",
    "molecules": "LPS"
  },
  {
    "caption": "C MARCH2+/+ (n = 4) or MARCH2-/- (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6mm.",
    "molecules": "LPS"
  },
  {
    "caption": "D MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, CCL-5 in serum from mice in each groups were measured at12 hpc by ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "E, F MARCH2+/+ (n = 5) or MARCH2-/- (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR.",
    "molecules": "LPS"
  },
  {
    "caption": "D, E BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with viruses or treated with poly(I:C) (80 µg/ml) or poly (dA:dT) (1 µg/ml). The concentration of secreted IFN-β (D) and IL-6 (E) in the supernatants was measured in an ELISA.",
    "molecules": "poly (dA:dT), poly(I:C)"
  },
  {
    "caption": "J, K BMDMs (J) or PMs (K) isolated from MARCH2+/+ or MARCH2-/- mice were infected with Salmonella typhimurium or L. monocytogenes, or treated with LPS or zymosan. The concentration of IL-6 and IL-12 in supernatant was analyzed in an ELISA.",
    "molecules": "zymosan, LPS"
  },
  {
    "caption": "L, M RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with PR8-GFP (MOI = 1), VSV-GFP (MOI = 0.5), poly (I:C) (20 µg/ml) or HSV-GFP (MOI = 1) poly (dA:dT) (1 µg/ml) and IFN-β (L), and IL-6 (M) levels in the supernatant were measured in an ELISA.",
    "molecules": "poly (dA:dT), poly (I:C)"
  },
  {
    "caption": "N RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with S. typhimurium, or L. monocytogenes, or treated with LPS (100 ng/ml) or zymosan (100 µg/ml) and IL-6 secretion into the cell supernatant was measured in an ELISA",
    "molecules": "zymosan, LPS"
  },
  {
    "caption": "HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter , plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for RIG-I 2CARD, RIG-I, MDA-5, MAVS or stimulated with poly (I:C), for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).",
    "molecules": "poly (I:C)"
  },
  {
    "caption": "Interaction between MARCH2 and NEMO in response to viral infection. HEK293T cells (E) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "Interaction between MARCH2 and NEMO in response to viral infection. Raw264.7 cells (F) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "G Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.",
    "molecules": "MG132"
  },
  {
    "caption": "Interaction between MARCH2 and NEMO in response to bacterial infection. Raw264.7 cells were infected with L. monocytogenes (H) or S. typhimurium (I) in a time-dependent manner in the presence of MG132. Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "J Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment (100 μg/ml) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.",
    "molecules": "zymosan, MG132"
  },
  {
    "caption": "C HEK293T cells transfected with a Strep-tagged empty vector or MARCH2 together with GST-tagged NEMO were treated with MG132 (10 μM), chloroquine, NH4Cl, or Z-VAD for 6 h before harvest. Whole cell lysates were subjected to immunoblotting with indicated antibodies.",
    "molecules": "NH4Cl, chloroquine, MG132, Z-VAD"
  },
  {
    "caption": "D HEK293T cells were transfected with different doses of Strep-tagged MARCH2 together with and GST-tagged NEMO plasmid. MG132 (10 μM) was added to cells for 6 h prior to harvest. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "E Confocal microscopy was used to examine colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence or absence of MG132 (10 μM). Scale bar, 10μm. Arrow indicates the co-localized NEMO and MARCH2 protein.",
    "molecules": "MG132"
  },
  {
    "caption": "F HEK293T cells transfected with Strep-tagged empty vector or MARCH2 with GST-tagged NEMO and each different HA-tagged ubiquitin mutants (indicated lysine (K) only, other lysines (K) mutated to arginines (R) were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to pull down with GST beads, followed by immunoblotting with an anti-HA antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.",
    "molecules": "arginines, lysine, lysines, MG132"
  },
  {
    "caption": "G HEK293T cells transfected with His-tagged ubiquitin and HA-tagged NEMO WT or NEMO lacking the CC1 domain together with Strep-tagged empty vector or MARCH2 were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-His antibody. WCL were subjected to immunoblotting with anti-HA, anti-Strep, or anti-β-actin antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "B HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants together with Strep-tagged MARCH2 in the presence of MG132. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-K48 linkage specific polyubiquitin antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "Electron micrographs showing the immunogold localization of α-amylase in attached cotyledons cells (A-E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti-α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti- α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti-α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti- α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti-α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.",
    "molecules": "Gold particles, gold particles"
  },
  {
    "caption": "Electron micrographs showing ultrastructures of cotyledon cells of germinatedV. mungo seeds (A-G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B-E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti-α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.",
    "molecules": "Gold particles"
  },
  {
    "caption": "Five representative images of MT repair sites in HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. From left to right, control cell, CLASPs&GCC185 KD cell, CAMSAP2 KD cell, AKAP450 KD cell and Centrinone-B treated cell. Grey: α-tubulin; red: GTP-tubulin. Scale bar: 20 μm. Box-whisker plot presents intensity analysis of MT repair sites in HRPE cells under combinative KDs. (1 representative of 3 independent experiments and n= 20 cells). ***p < 0.001, unpaired t-test.",
    "molecules": "Centrinone-B, GTP"
  },
  {
    "caption": "Conventional image (left) and STORM image (right) of MTs and MT repair sites. Grey: α-tubulin; yellow: GTP-tubulin; green: Golgi. Scale bar: 5 μm.",
    "molecules": "GTP"
  },
  {
    "caption": "MT repair sites on GaMTs and non-GaMTs extracted from C. Red: GaMTs; Green: non-GaMTs; yellow: GTP-tubulin. MT repair sites on GaMTs and non-GaMTs separately presented in detail. White arrows indicates GTP-tubulin labeling sites. Scale bar: 5 μm. Box-whisker plot presents the ratio of MT repair sites on GaMTs and non-GaMTs separately. (1 representative of 3 independent experiments and n= 8 cells). ***p < 0.001, unpaired t-test.",
    "molecules": "GTP"
  },
  {
    "caption": "Representative time series show one fast cargo moving on a GaMT and slowing down at the MT repaire site. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Representative time series show one cargo moving fast between two MT repair sites and slowing down at the MTrepair sites. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Pause (I) and reverse (J) events of cargos on MT repair segment and non-MTs repair segment, respectively. (Data were pooled from 2 independent experiments and n= 22 cells). ***p <0.001, ns, no significant difference, unpaired t-test.",
    "molecules": "GTP"
  },
  {
    "caption": "(F) Hematoxylin and eosin staining of sections of seminiferous tubules from ~4 weeks old Bbs4+/+ (n=2 mice) and Bbs4GT/GT (n=4) males. Scale bar, 100 µm. Representative images are shown.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "(K) Leptin concentration in blood plasma taken from young adult (7-8 weeks) or mid-age (14-20 weeks) mice. Young adult mice: Bbs4+/+ (n=4 mice), Bbs4KO/KO (n=3), analyzed in two independent experiments. Mean+SEM. Mid-age adult mice: Bbs4+/+ (n=7 mice), Bbs4KO/KO (n=5), Bbs4GT/GT (n=3), analyzed in 4 independent experiments. Kruskal-Wallis tests was used for the statistical analysis. Mean+SEM.",
    "molecules": "Leptin"
  },
  {
    "caption": "Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (E) An alternative analysis of the experiment shown in (D). Geometric mean fluorescence intensity (MFI) of BV421 (IgM) on IgD+ B cells was determined. Mean of MFI values for each genotype per experiment was quantified, and obtained values were normalized to Bbs4+/+ (=1) for each experiment. Two-tailed One Sample Wilcoxon Signed Rank Test was used for the statistical analysis.",
    "molecules": "BV421"
  },
  {
    "caption": "(A,B) Splenocytes isolated from Bbs4+/+ (n=4 mice) and Bbs4GT/GT (n=3) B-cell transgenic B1-8 littermates were activated with 4-hydroxy-3-nitrophenylacetic acid succinimide ester-loaded T2-Kb cells in three independent experiments. Percentage of activated B cells (gated as CD69+ B220+ IgLλ+ viable cells) in the samples without T2-Kb (negative control), and in the samples with T2-Kb added in ratios 1:10 or 1:3 was determined by flow cytometry. (A) Representative mice are shown. (B) Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.",
    "molecules": "4-hydroxy-3-nitrophenylacetic acid succinimide ester"
  },
  {
    "caption": "(D) CFSE-loaded T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were incubated with DDAO-labeled WT splenocytes loaded with OVA peptide or with the indicated altered peptide ligands for 20 min. Percentage of T cells conjugated with the APCs was determined by flow cytometry. Four biological replicates were performed. Mean+SEM. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all peptides.",
    "molecules": "DDAO, CFSE"
  },
  {
    "caption": "500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (E) Glucose level in the urine of mice was monitored on a daily basis. The mouse was considered diabetic when it had urine glucose level ≥ 1000 mg/dL for two consecutive days. Statistical significance was calculated by Log-rank (Mantel-Cox) test.",
    "molecules": "Glucose, glucose"
  },
  {
    "caption": "500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (F) Glucose concentration in blood on day 7 post-infection. 500 OT-I ctrl (n=13 mice), 500 OT-I Bbs4 cKO (n=13), 1000 OT-I ctrl (n=9), 1000 OT-I Bbs4 cKO (n=11), analyzed in four independent experiments, mean is shown. Statistical significance was calculated using two-tailed Mann-Whitney test.",
    "molecules": "Glucose"
  },
  {
    "caption": "Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters Mean cell volume, Hemoglobin n=39 for parameters Red Blood cells, Mean cell Hemoglobin and red cell distribution width (RDW). Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in",
    "molecules": "Hemoglobin"
  },
  {
    "caption": "qRT-PC  of mGPDH, myogenin and developmental myosin heavy chain (myh8, myl4 and myh3) in gastrocnemius (GA) muscle from C57BL/6J mice at indicated day after CTX intramuscular injection",
    "molecules": "CTX"
  },
  {
    "caption": "immunoblo (B) of mGPDH, myogeni in gastrocnemius (GA) muscle from C57BL/6J mice at indicated day after CTX intramuscular injection",
    "molecules": "CTX"
  },
  {
    "caption": "Activity assay of mGPDH in GA muscle from C57BL/6J mice at day 0 and 7 after CTX injection",
    "molecules": "CTX"
  },
  {
    "caption": "Representative images of the H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (D), distribution of the fibers cross-section area (CSA) (E), percentage of myofibers with central nuclei (F) in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection",
    "molecules": "CTX"
  },
  {
    "caption": "immunofluorescence staining of desmin (green) (G) in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection",
    "molecules": "CTX"
  },
  {
    "caption": "Muscle weight (H in GA muscle from WT and mGPDH-/- mice at day 14 post CTX injection",
    "molecules": "CTX"
  },
  {
    "caption": "trichrome staining (I) in GA muscle from WT and mGPDH-/- mice at day 14 post CTX injection. Quantification represents the fibrotic areas",
    "molecules": "CTX"
  },
  {
    "caption": "qRT-PCR (J for mGPDH, myogenin and myh3 in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection",
    "molecules": "CTX"
  },
  {
    "caption": "immunoblot (K) for mGPDH, myogeni in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection",
    "molecules": "CTX"
  },
  {
    "caption": "Mitochondrial DNA (A) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation",
    "molecules": "DNA"
  },
  {
    "caption": "Immunoblots of p-AMPK, p-ACC and PGC1α and corresponding quantifications represent p-AMPK, p-ACC and PGC1α protein levels (G in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation",
    "molecules": "CC, compound C"
  },
  {
    "caption": "mitochondrial DNA (H in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation",
    "molecules": "DNA, CC, compound C"
  },
  {
    "caption": "nuclear-encoded OXPHOS genes combined by NDUFS8, SDHb, Uqcrc1, COX5b and ATP5a1 (I) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation",
    "molecules": "CC, compound C"
  },
  {
    "caption": "NAD+/NADH ratio (J in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "immunoprecipitation analysis for PGC-1α acetyl-lysine (Ac-Lys) level (K) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation",
    "molecules": "Ac-Lys, acetyl-lysine"
  },
  {
    "caption": "Immunoblot of c-myc and myogenin (L in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,M after differentiation",
    "molecules": "CC"
  },
  {
    "caption": "corresponding quantifications represent c-myc and myogenin protein levels (M) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,M",
    "molecules": "CC"
  },
  {
    "caption": "representative images of MyHC immunofluorescence (N), fusion index (O) and the distribution of nuclei per myotube (P) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC a 72 h (N-P) after differentiation",
    "molecules": "CC"
  },
  {
    "caption": "Immunoblots of p-AMPK, p-ACC, PGC1α and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhibitor STO-609 at 24 h after differentiation. Quantifications represent p-AMPK, p-ACC, PGC1α and myogenin protein levels",
    "molecules": "STO-609"
  },
  {
    "caption": "Immunoblots of p-AMPK and p-ACC in C2C12 myocytes transfected with mGPDH plasmid with the Ca2+ chelator BAPTA-AM at 24 h after differentiation. Quantifications represent p-AMPK and p-ACC protein levels.",
    "molecules": "Ca2+, BAPTA-AM"
  },
  {
    "caption": "qRT-PCR of mGPDH in GA muscle of indicated mice at days 0 and 3 post CTX intramuscular injection",
    "molecules": "CTX"
  },
  {
    "caption": "Experimental setup (upper of E), qRT-PCR of mGPDH, myogenin and myh3 (bottom of E in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection",
    "molecules": "CTX"
  },
  {
    "caption": "), H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (F), distribution of the fiber CSA (G) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection",
    "molecules": "CTX"
  },
  {
    "caption": "percentage of myofibers with central nuclei (H) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection",
    "molecules": "CTX"
  },
  {
    "caption": "Experimental setup (upper of I qRT-PCR of mGPDH, myogenin and myh3 (bottom of I) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injection",
    "molecules": "CTX, STZ"
  },
  {
    "caption": "H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (J) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injection",
    "molecules": "CTX, STZ"
  },
  {
    "caption": "distribution of the fibers CSA (K), percentage of myofibers with central nuclei (L in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injection",
    "molecules": "CTX, STZ"
  },
  {
    "caption": "Experimental setup (uppe and muscle weight (bottom of M) in GA muscle from AAV-mGPDH-treated STZ-treated mice at da 14 (M) post CTX intramuscular injection",
    "molecules": "CTX, STZ"
  },
  {
    "caption": "A) Time-dependent GSK-3 inhibition of VP2.51 at 1 and 2 μM.",
    "molecules": "VP2.51"
  },
  {
    "caption": "B) Kinetic GSK-3 inhibition data determined for VP2.51. ATP concentrations in the reaction mixture varied from 1 to 50 μM. Compound concentrations used are depicted in the plot, and the concentration of GS-2 was kept constant at 12.5 μM. Each point is the mean of two different experiments, each one analyzed in duplicate.",
    "molecules": "GS-2, VP2.51, ATP"
  },
  {
    "caption": "C) Kinase profiling of VP2.51 on human recombinant kinases. The percentage (%) of kinase activity after the treatment with a fixed concentration (10 μM) of the compound is shown.",
    "molecules": "VP2.51"
  },
  {
    "caption": "A) Evolution of rat weight of different treatment groups (12 animals in each group): control, L-BMAA (300 mg/kg/day during 5 consecutive days treated at weaning), VP2.51 (2,5 mg/kg during 15 days) with a group treated at PT1 and another at PT30, L-BMAA (with the same treatment as the group of L-BMAA) + VP2.51 (with the same treatment groups VP2.51) with a group treated at PT1 and another at PT30.",
    "molecules": "VP2.51, L-BMAA"
  },
  {
    "caption": "B). Evolution of neurological evaluation in the treatment assay with VP2.51 (12 animals in each group). The control group and the groups treated exclusively with VP2.51 (at PT1 and PT30) are not shown because its assessment coincides with the horizontal axis. The time when the inhibitor is added is indicated with arrows.",
    "molecules": "VP2.51"
  },
  {
    "caption": "A) Analysis of LC3-II and P-mTOR by western-blot in the motor cortex of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, ** Indicate differences of p-value <0.05 <0.01 with the control group, respectively. #, ### indicate differences of p-value <0.05 <0.001 with the group of animals treated with L-BMAA, respectively.",
    "molecules": "L-BMAA"
  },
  {
    "caption": "B) Analysis of LC3-II and P-mTOR by western-blot in the lumbar spinal cord of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, *** Indicate differences of p-value <0.05 <0.001 with the control group, respectively. ## Indicate differences of p-value <0.01 with the group of animals treated with L-BMAA.",
    "molecules": "L-BMAA"
  },
  {
    "caption": "E) Representative immunofluorescence stainings of HOBIT (red), CD3 (green), dystrophin (purple) and Hoechst 33342 (blue) performed on muscle tissue for patient 6 (IBM), patient 3 (DM) and 4 (ASyS). Images were acquired using a LSM 880 confocal without Airyscan microscope (63x oil objective). Scale bar=20μm. The white arrows show HOBIT+ T cells. DM: DermatoMyositis; ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body Myositis",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm. F)",
    "molecules": "galactose, MitoTracker™Green, MTG, TMRE"
  },
  {
    "caption": "A) BN-PAGE and subsequent immuno-blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1. Monomers (M), dimers (D) and oligomers (O) were detected. Left panel: Coomassie-stained gel. Right panels: Immunoblotting and detection of ATP synthase and IF1. WT: wild type HeLa cells; IF*: cells expressing IF1-H49K. Stars indicate oligomeric IF1 forms. I: complex I; III: complex III.",
    "molecules": "Coomassie"
  },
  {
    "caption": "D) Assembly of sEcGFP-tagged subunits CoxVIIIa-sEcGFP (CIV#) into complex IV, and assembly of subunits γ-sEcGFP (SUγ#) and SU e-sEcGFP (Sue#) into ATP synthase. Proteins were separated by BN-PAGE and immunoblotted. Lanes #1-#10 were cut into three pieces for further evaluation. Lanes 1-2: complex IV assembly in WT and CoxVIIIa-sEcGFP-expressing cells, detection of CIV with anti-Cox1. Lanes 3-4: complex V assembly in WT and SUγ-sEcGFP-expressing cells, detection with anti-SU γ. Lanes 8-10: assembly of CIV# into CIV, SU γ-sEcGFP (SUγ#) into CV, and SU e-sEcGFP (SUe#) into CV. At lane #6 in the Coomassie-stained gel, the positions of molecular weight markers (M) are shown. SC: respiratory supercomplexes; CIV2: complex IV dimers; CIV: complex IV monomer; s1: F1-c8 subcomplexes; s2: subcomplexes of CV containing subunits b, e and g He et al., 2018(). Blue **: dimeric F1F0 ATP synthase; blue*: monomeric F1F0 ATP synthase. Red asterisks: respiratory supercomplexes",
    "molecules": "Coomassie"
  },
  {
    "caption": "B) Diffusion coefficients for ATP synthase under control conditions (5.6 mM glucose), and under OXPHOS conditions. The probability density function (PDF) and cumulative density function (CDF) of -log D is plotted. Number of trajectories analyzed: 8622 (Glc) and 10333 (Gal) from mitochondria in at least 5 different cells.",
    "molecules": "Gal, Glc, glucose"
  },
  {
    "caption": "C) Images showing matrix pH equilibration with extracellular pH. Shown are ratiometric fluorescence images of MPP-sEcGFP before and after addition of the uncouplers and inhibitors FCCP, nigericin and oligomycin (plus 0.002 % w/v Triton X-100 where indicated). False color images (λ511/λ464) indicate ratio values. D) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH=8, pH=7.4 and pH=6.5. Scale bars = 10 µm. E",
    "molecules": "FCCP, nigericin, oligomycin, Triton X-100"
  },
  {
    "caption": "B) Mitochondrial pH values at SU e, SU γ and in the matrix bulk in hyper-glycolytic cells (high glucose: HGlc, 25 mM). C) pH at SU e , SU γ and in the matrix in respiring cells (Gal, 10 mM). D) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain. Da",
    "molecules": "Gal, glucose, HGlc, hyper-glycolytic"
  },
  {
    "caption": "A) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions. Where indicated, 5 µg/mL oligomycin was added to inhibit CV activity. B) Mitochondrial pH at CV SU e in IF1-KO cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. C) Mitochondrial pH at CV SU e in IF1-OE cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. D)",
    "molecules": "Gal, glycolytic, HGlc, oligomycin"
  },
  {
    "caption": "F) Effect of inhibiting the respiratory chain at complex III (CIII) with antimycin A (AA) on the local pH at SU e in WT cells (glycolytic conditions). G) Local pH at SU e in IF1-KO cells after inhibiting CIII. H) Local pH at SU e in IF1-OE cells after inhibiting CIII. Da",
    "molecules": "AA, antimycin A, glycolytic"
  },
  {
    "caption": "A) Mitochondrial pH at CV SU e is compared for HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.",
    "molecules": "glycolytic"
  },
  {
    "caption": "D) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.",
    "molecules": "glycolytic"
  },
  {
    "caption": "A) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined with an automatic flux analyzer (Seahorse/Agilent). After monitoring basal respiration, oligomycin (1 µM), and rotenone plus antimycin A (Rot; 0.5 µM; AA, 0.5 µM) were added sequentially to determine glycolytic and mitochondrial ATP production rates. ~12 technical replicates per measurement, N=4 independent assays.",
    "molecules": "antimycin A, ATP, glycolytic, oligomycin, Rot, rotenone"
  },
  {
    "caption": "Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Cross-sectional linear regressions between A) centiloid and p-tau181 and B) centiloid and sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.",
    "molecules": "amyloid, Aβ"
  },
  {
    "caption": "Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Longitudinal linear regressions between C) centiloid and change in p-tau181 and D) centiloid and change in sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.",
    "molecules": "amyloid, Aβ"
  },
  {
    "caption": "B) T-value projection of the association between sTREM2 on FDG-PET, stratified by group. FDG-PET z-scores were derived by referencing FDG-PET SUVRs to cognitively normal controls (i.e. n=131; Aβ CSF−/PET−).",
    "molecules": "FDG, Aβ"
  },
  {
    "caption": "(B) Hoechst (DNA) staining and IF for Tbr2 to calculate the thickness (white bars) of the VZ, SVZ and intermediate zone + cortical plate (IZ+CP) in control and DKO E13.5 neocortex. Scale bar 20 μm. (C) Quantification of VZ, SVZ and IZ+CP thickness in control and DKO E13.5 neocortex. Each zone is represented as percentage of total thickness (measured in µm) of the neocortex. Columns are means ± SEM (n=6 embryos, 4 litters). ( ",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Triple-IF for BrdU, Pax6 and Tbr2 (not shown) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice. (B) Quantification of the proportion of Pax6+Tbr2- cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs. controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters). (C) Average percentage of APs (Pax6+Tbr2-) (from B) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM. (D) Co-IF for Tbr2 and BrdU (double positive cells depicted by white arrowheads) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice. (E) Quantification of the proportion of Tbr2+ cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters). (F) Average percentage of BPs (Tbr2+) (from E) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM.",
    "molecules": "BrdU"
  },
  {
    "caption": "(G) Co-transfection of myc-tagged GSK3β and Flag-Sox4 in 293T cells. BIO treatment (0.5 µM) performed for 1 hour prior to harvest. Lower band (65kDa) represents non-phosphorylated Sox4. High and low exposure (exp) blots shown to better visualize non-phosphorylated Sox4. Numbers above blot are quantification of Sox4 protein levels normalized to GFP and are expressed as fold change vs. controls. The sum of upper and lower bands was quantified (n=8 biological replicates).",
    "molecules": "BIO"
  },
  {
    "caption": "(E) IF for Tuj1 to identify newly formed neurons in NPCs after seven-day culture in differentiation medium. Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm. (F) Quantification of Tuj1+ cells in control, control + CHIR (1 µM, 3 µM), DKO, and DKO + CHIR NPC cultures. Data are means ± SEM (experiment performed twice in triplicates, representative experiment is shown). Numbers below asterisks show fold change.",
    "molecules": "CHIR"
  },
  {
    "caption": "Boxplots depicting the change in temperature from baseline to 24 hours after the second 1μg dose of each of a variety of Escherichia coli lipopolysaccharide (LPS) and peptidoglycan (PGN) preparations, with and without alum, and combinations compared with 1/5 of the human dose of 4CMenB. Boxplots represent the median with interquartile ranges. Vaccine group medians at each timepoint were compared with baseline for that vaccine using a Wilcoxon signed-rank test. * < 0.05, ** < 0.01, **** < 0.0001. N= 6 mice per vaccine group.",
    "molecules": "alum, lipopolysaccharide, LPS, peptidoglycan, PGN"
  },
  {
    "caption": "B In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species.",
    "molecules": "biotinylated, ubiquitin"
  },
  {
    "caption": "C Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS-PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species.",
    "molecules": "biotin"
  },
  {
    "caption": "C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins.",
    "molecules": "arginine, biotinylated, ubiquitin"
  },
  {
    "caption": "A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis.",
    "molecules": "H3K9me2"
  },
  {
    "caption": "D Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines.",
    "molecules": "H3K4me2, H3K9me2, lysine, lysines"
  },
  {
    "caption": "B Heterochromatic silencing assays of wild-type and mutant Hht1-FH strains. Silencing at the mating-type Kint2::ura4+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), and medium containing 5-FOA (5-FOA).",
    "molecules": "5-FOA, Ura, uracil"
  },
  {
    "caption": "E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants. Silencing at the mating-type kint2::ura4+ was evaluated",
    "molecules": "lysine"
  },
  {
    "caption": "In vitro HMTase assays. Recombinant H3N-GST (WT or K9R) pre-ubiquitylated by the CLRC was purified and used in the HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (left) and autoradiography (right).",
    "molecules": "CBB, Coomassie Brilliant Blue"
  },
  {
    "caption": "B Recombinant proteins were resolved by 10% SDS-PAGE and visualized by CBB staining.",
    "molecules": "CBB"
  },
  {
    "caption": "C In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed",
    "molecules": "H3K14CUb"
  },
  {
    "caption": "D In vitro pulldown assays of GST-Clr4 proteins with biotinylated ubiquitin. Bound ubiquitin was analyzed by Western blotting with HRP-conjugated streptavidin (left). Precipitated GST proteins were analyzed by SDS-PAGE and CBB staining (right).",
    "molecules": "biotinylated, CBB, streptavidin, ubiquitin"
  },
  {
    "caption": "(B) U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110-130 fibers for each experiment (right). Experiments were repeated four times for each sample. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.",
    "molecules": "CldU, IdU, HU"
  },
  {
    "caption": "(D) U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.",
    "molecules": "Dox"
  },
  {
    "caption": "(C) U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.",
    "molecules": "Dox"
  },
  {
    "caption": "(E) U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.",
    "molecules": "HU"
  },
  {
    "caption": "(D) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.",
    "molecules": "Dox"
  },
  {
    "caption": "(E) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal (left). U2OS (EGFP-STGC-1731) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were infected by lentivirus expressing I-SceI. The percentage of EGFP positive cells by I-SceI induction was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.",
    "molecules": "HU"
  },
  {
    "caption": "(F) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.",
    "molecules": "Dox"
  },
  {
    "caption": "(G) FLAG-PIF1 was stably expressed in U2OS (EGFP-Flex1-STGC-1541) cells by lentiviral infection. Enrichment of FLAG-PIF1 at Flex1 site or GAPDH site with or without HU (2 mM, 10 hr) treatment was calculated by anti-FLAG ChIP (left). When PCNA was depleted by shRNA using vector as a control (Ctrl), enrichment of FLAG-PIF1 at Flex1 site was calculated by anti-FLAG ChIP (right). ChIP value in cells without HU treatment is set up as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.",
    "molecules": "HU"
  },
  {
    "caption": "(C) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.",
    "molecules": "APH"
  },
  {
    "caption": "(I) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells, or PIF1-KO cells expressing PIF1-WT, and E307Q and L319P mutants before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05. ",
    "molecules": "APH"
  },
  {
    "caption": "25HC inhibits membrane fusion mediated by SARS-CoV-2 S (F) 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding SARS-CoV-2 S (F) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.",
    "molecules": "25HC, EtOH"
  },
  {
    "caption": "25HC inhibits membrane fusion mediated by , SARS-CoV S (G, left), and MERS-CoV S (G, right). 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding , SARS-CoV S (G, left), and MERS-CoV S (G, right) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. Scale bar, 50 μm. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.",
    "molecules": "25HC, EtOH"
  },
  {
    "caption": "(B-C) 25HC depletes accessible cholesterol on the plasma membrane. (B) Calu-3 cells were treated with indicated concentrations of 25HC for 1h, and subsequently incubated with ALOD4 for 30 min. Cell were washed twice with PBS, lysed, and the amounts of bound ALOD4 were examined by western blotting. (C) Calu-3 cells were treated with 5 µM 25HC for 1h, and subsequently incubated with AF488 labeled ALOD4 for 30 min. Cells were washed twice and examined by fluorescence microscopy.",
    "molecules": "25HC, AF488, cholesterol, PBS"
  },
  {
    "caption": "(D) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC-treated cells. Calu-3 cells were pretreated with ethanol or 5 µM 25HC for 16h. Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h. After further incubation with ALOD4, cells were washed with PBS twice and lysed. The amount of bound ALOD4 was examined by western blotting.",
    "molecules": "25HC, Cholesterol, cholesterol, ethanol, PBS"
  },
  {
    "caption": "Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. Entry of SARS-CoV-2 pseudovirus (E and F) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.",
    "molecules": "25HC, cholesterol, ethanol, PBS"
  },
  {
    "caption": "Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus, and MERS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. , SARS-CoV pseudovirus (G), and MERS-CoV pseudovirus (H) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.",
    "molecules": "25HC, cholesterol, ethanol, PBS"
  },
  {
    "caption": "(C) SZ58-035 rescues surface accessible cholesterol in 25HC-treated cells. Calu-3 cells were treated with vehicle or 40 µM SZ58-035 for 16 h, prior to treatment with ethanol or 2.5 µM 25HC for 1h. Cells were then incubated with ALOD4 for 30 min. The amount of bound ALOD4 was examined by western blotting.",
    "molecules": "25HC, cholesterol, ethanol, SZ58-035"
  },
  {
    "caption": "F) Representative immunoblots of co-immunoprecipitation (co-IP) experiments in eNPCs (top panels) and smNPCs (bottom panels). Co-IPs were performed with antibodies against BAF170 (left) and BRG1(right). Inputs are homogenates from eNPCs (left) and smNPCs (right). IgG indicates IgG-bound Sepharose beads (as controls), while IP indicates immunoprecipitated samples after elution from the beads. Arrow head indicates BCL7A band at the correct MW.",
    "molecules": "Sepharose"
  },
  {
    "caption": "I) Immunofluorescence staining of BCL7A (purple), EdU (green) and NeuN (white) in embryonic (E17.5) and adult (PD120) hippocampal brain sections. EdU was used to label proliferating NPCs, whereas NeuN was used as a neuronal marker.",
    "molecules": "EdU"
  },
  {
    "caption": "A) Immunoblot and immunofluorescence staining of BCL7A (red) in eNPCs isolated from Bcl7awt/wt (wt) and Bcl7ako/ko (ko) embryos (E13.5). Nestin (green) was used as NPC marker, whereas DAPI (blue) was used to stain nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "E) EdU experiments in adult mice. Animals were injected with EdU twice daily for three days and sacrificed 21 days thereafter. Images show immunofluorescence stainings for EdU (green), doublecortin (DCX, white), and S100β (red) in hippocampal brain sections from Bcl7awt/wt; Nestin-Cretgt/wt (as control) and Bcl7afl/fl; Nestin-Cretg/wt mice. Representative EdU+ DCX+ (yellow triangles) or EdU+ S100β+ (yellow asterisk) double-labelled cells are indicated in the insets. Scale bar= 50 μm and 20 μm (for insets).",
    "molecules": "EdU"
  },
  {
    "caption": "F) Quantification of EdU+ (right panel), EdU+ DCX+ (middle panel) and EdU+ S100β+ (left panel) cells within the hippocampal dentate gyrus (DG) of adult control (n=4) and Bcl7afl/fl; Nestin-Cretg/wt (n=5) animals.",
    "molecules": "EdU"
  },
  {
    "caption": "B-C) Representative Ca2+ measurements in (B) wt vs BCL7A KO cortical neurons (CN, 10 days in vitro) and in (C) smNPC-derived parental and BCL7A KO neurons. Ca2+ changes were detected using Fluo-4 and upon treatment with (B) 50 μM glutamate (final concentration) and (C) 100 μM glutamate (final concentration). Upper right panels report Ca2+ peaks (F) relative to baseline (F0). In B, wt: n=18 cells; ko: n=23 cells (n=2 independent experiments). In C, P: n=27 cells; KO1: n=20 cells; KO2: n=19 cells (n=2 independent experiments).",
    "molecules": "Ca2+, Fluo-4, glutamate"
  },
  {
    "caption": "E) RT-PCR analysis of FOS expression upon glutamate (100 μM for 10 min) stimulation in smNPC-derived parental and BCL7A KO neurons (n=5-6 biological replicates).",
    "molecules": "glutamate"
  },
  {
    "caption": "F) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs in presence of 1 μM DAPT or 3 μM CHIR99021.",
    "molecules": "CHIR99021, DAPT"
  },
  {
    "caption": "G) Immunofluorescence staining of eNPCs undergoing spontaneous differentiation for 7 days. BCL7A KO eNPCs were incubated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with DMSO, 1 μM DAPT, 3 μM CHIR99021 or a combination of 1 μM DAPT + 3 μM CHIR99021. Quantification of the β-III tubulin-positive or GFAP-positive cells is shown on the right (n=3-6 technical replicates from 3 independent experiments).",
    "molecules": "CHIR99021, DAPT, DMSO"
  },
  {
    "caption": "I) mRNA expression levels of TUBB3 and GFAP at 15 and 25 days of spontaneously differentiating BCL7A KO compared parental smNPCs. BCL7A KO smNPCs were treated with DAPT, CHIR99021, DAPT + CHIR99021 or DMSO (as control= ctr) during the initial 7 days of spontaneous differentiation (n=3-7 biological replicates).",
    "molecules": "CHIR99021, DAPT, DMSO"
  },
  {
    "caption": "A) OCR measurements in wt and BCL7A KO eNPCs treated with 3 μM of CHIR99021 or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 5-7 biological replicates).",
    "molecules": "FCCP, CHIR99021, DMSO"
  },
  {
    "caption": "B) OCR measurements in wt and BCL7A KO eNPCs following 48h of spontaneous differentiation. BCL7A KO eNPCs were treated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with CHIR99021 or DMSO. The average maximal respiration upon FCCP treatment is shown on the right (n= 4 biological replicates).",
    "molecules": "FCCP, CHIR99021, DMSO"
  },
  {
    "caption": "C) Immunoblot analysis of differentiated BCL7A KO eNPCs exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (rot). Antibodies against β-III tubulin and actin were used.",
    "molecules": "CHIR99021, rot, rotenone"
  },
  {
    "caption": "D) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 3 μM CHIR99021, in presence or absence of 20 nM rotenone. Scale bar= 50 μm. Statistics are shown on the right (n=3-6 technical replicates from 3 independent experiments).",
    "molecules": "CHIR99021, rotenone"
  },
  {
    "caption": "E) Sholl analysis of β-III tubulin-positive immature neurons following 7 days of spontaneous differentiation. BCL7A KO eNPCs were exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (n=3 biological replicates).",
    "molecules": "CHIR99021, rotenone"
  },
  {
    "caption": "F) Representative OCR measurements in BCL7A KO eNPCs treated with 5 and 10 μM of pioglitazone or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 3 biological replicates).",
    "molecules": "FCCP, DMSO, pioglitazone"
  },
  {
    "caption": "G) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 5 μM pioglitazone. The percentage of β-III-tubulin-positive cells is shown on the right (n=3-8 technical replicates from 2 independent experiments).",
    "molecules": "pioglitazone"
  },
  {
    "caption": "B) Localization of the GFP-tagged PE synthesizing enzyme (Psd) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM choline. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs); LD, lipid droplets; MM, mitochondrial matrix. A scheme depicting each chimeric Psd construct is shown under the corresponding microscopy image. C) Localization of the GFP-tagged PC synthesizing enzyme (Pmt) targeted to the indicated organelles in choppΔ cells, grown in SD medium supplemented with 10 mM ethanolamine. Images shown are either a single Z-slice (ER construct) or a maximum intensity projection of several Z-sections (other constructs). A schematic depicting each chimeric Pmt construct is shown under the corresponding microscopy image.",
    "molecules": "choline, ethanolamine"
  },
  {
    "caption": "B) Five-fold serial dilutions of strains of the indicated genotypes (wt cells and choppΔ cells harboring empty vectors or choppΔ cells expressing chimeric Psd and Pmt enzymes). Cells were grown on SD-HIS-LEU (-HL) medium (KennedyOFF) or SD-HL containing ethanolamine and choline (KennedyON).",
    "molecules": "choline, ethanolamine, HIS, LEU"
  },
  {
    "caption": "C) Thin layer chromatography (TLC) analysis of steady-state phospholipid profiles of cells of the indicated genotypes. Where Psd and/or Pmt are missing (-), choppΔ cells were grown in the presence of ethanolamine and/or choline. Bands corresponding to the phospholipids PA, PS, PE, PI and PC are indicated.",
    "molecules": "choline, ethanolamine, PA, PS, PC, PE, PI, phospholipid, phospholipids"
  },
  {
    "caption": "C) VPS13 is required in KennedyOFF conditions when chimeric enzymes are targeted to mitochondria or endosomes. Volcano plots show the fold change of number of transposon insertions per gene of libraries grown in KennedyOFF versus ON conditions. This comparison included all six libraries where the enzymes are targeted to endosomes and/or mitochondria (top left panel) or six libraries with enzymes targeted to other organelles (bottom left panel). Data-points for VPS13 are highlighted in red. Schematic illustrating Vps13-mediated lipid transport at endosomes and mitochondria contact sites (right panel).",
    "molecules": "lipid"
  },
  {
    "caption": "B) Requirement for mitochondrial lipid transport and biosynthesis genes in rewired libraries. Left panel, schematic depicting PE and cardiolipin (CL) synthesis in mitochondria. The inner mitochondrial putative lipid transporter Mdm31 is depicted in yellow. Grey arrows indicate the need for lipid transport from the ER or other organelles to mitochondria and transport between the MOM and MIM. Right panel, transposon numbers (normalized to wt libraries) for the indicated (set of) libraries for MDM31, PGS1, and CRD1.",
    "molecules": "cardiolipin, CL, lipid, PE"
  },
  {
    "caption": "B) Five-fold serial dilutions of strains of the indicated genotypes grown at 30°C on SD medium supplemented with 10 mM ethanolamine and/or 10 mM choline. Growth assays are representative images of biological replicates.",
    "molecules": "choline, ethanolamine"
  },
  {
    "caption": "C) Five-fold serial dilutions of strains of the indicated genotypes grown at indicated temperatures, in the presence or absence of 10 mM ethanolamine (etn).",
    "molecules": "ethanolamine, etn"
  },
  {
    "caption": "E) Bar graphs depicting the fraction of more saturated species (defined as fatty acid chains with one double bond for ≤30 carbon chains or with two double bonds for ≥32), and less unsaturated species (defined as fatty acid chains with zero double bond for ≤30 carbon chains or with one double bond for ≥32) for each phospholipid class with different head groups. Bars represent the means of two or three biological replicates (individual measurements are shown as points and triangles). p-values are calculated with a two-sample t-test.",
    "molecules": "phospholipid, saturated, unsaturated"
  },
  {
    "caption": "C. Western blot for NHP2 in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines treated with DMSO or 10 μM MG132 for 6 h. β-Actin served as loading control. Quantifications are shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=5). Statistical analyses were performed with Student's two-tailed unpaired t-test. Data information: Data are presented as mean ± SD.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "D. Western blot in U2OS and U2OS-hTR cells treated with or without Hydroxyurea (2 mM for 24 h), for the indicated proteins. β-Actin  and Vinculin served as loading controls.",
    "molecules": "Hydroxyurea"
  },
  {
    "caption": "G. Western blot analyses in the indicated cell lines cells transfected for 48 h with siLuc or sihTR, with the indicated antibodies. U2OS  cells treated with Hydroxyurea (2 mM for 24 h) were used as positive controls.",
    "molecules": "Hydroxyurea"
  },
  {
    "caption": "A. Representative images for fragile telomeres using the CO-FISH assay. Lagging strands (C-probe) and leading strands (G-probe) were marked with green PNA  probes and red LNA probes, respectively. White circles and arrows indicate \"smeary\" or \"doublet\" telomeric signals, respectively. Quantification of fragile  telomeres is shown on the right as the percentage of total telomeres counted (n=932-1073). Comparison of fragile to non-fragile telomere percentage was performed using Χ2-test. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.",
    "molecules": "LNA, PNA"
  },
  {
    "caption": "D. Representative images for TIF quantification, as in (A), in U2OS or U2OS-hTR cells, treated as indicated with DMSO or 5 μM ATRi or 5 μM ATMi for 20 h. White arrows indicate TIF. Data information: All scale bars: 5 μm. Data are shown as mean ± SD.",
    "molecules": "ATRi, DMSO, ATMi"
  },
  {
    "caption": "C. Representative images for co-localization between EdU (green) and telomeres (red FISH probe) within PML bodies (cyan) in U2OS and U2OS-hTR cells. White arrows indicate  co-localization events. D, E. Quantification of images shown in (C).  Co-localization events between EdU foci and APBs were analyzed (D) and the percentage of APBs that are actively engaged in G2/M telomeric  synthesis was calculated (E). A total of at least 50 nuclei were scored from three independent experiments. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution. ",
    "molecules": "EdU"
  },
  {
    "caption": "F. Representative images for co-localization between RPA32 (green) and telomeres (red FISH probe) in the indicated cell lines treated with either DMSO or 10 μM  NU7026 DNA-PK-inhibitor (DNA-PK-i) for 48 h. White arrows indicate  co-localization  events. Quantifications are shown on the right. A total of 53-84 nuclei were scored from three independent experiments. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. ",
    "molecules": "NU7026, DMSO"
  },
  {
    "caption": "D. Representative images for TIF quantification: co-localization between 53BP1  (green) and telomeres (red FISH probe), measured 72 h post-transfection with siLuc or siNHP2 in cells further treated with either DMSO or Doxorubicin (DOXO, 1.5 μM) for 2 h. White arrows indicate TIF. Quantification of TIF is shown on the right. A total of at least 100 nuclei were scored from two independent experiments. Each dot represents the number of TIF per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons. Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value.",
    "molecules": "DMSO, DOXO, Doxorubicin"
  },
  {
    "caption": "B β-gal staining revealed the increased senescent cell population in ATRX-deleted U87 cells. Upper panel, micrographs of control and ATRX-deleted U87 cells processed for ATRX immunofluorescence (IF) and β-gal senescence assay. Scale bar, 200 µm. D, DAPI. Lower panel, percentage of senescent cells. Data are expressed as means ± s.d., N = 3, unpaired t test.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labelled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT positive control.",
    "molecules": "Genomic DNA, Genomic DNAs, EtBr, oligonucleotide, 32P"
  },
  {
    "caption": "E 2D gel analysis revealed increased T-circle formation in ATRX-deleted U87-T cells without or with Ad-Cre infection. Shown are gels stained with EtBr and blots hybridized with a TelG probe. Indicated are linear (lin) and open (oc) T-circle forms of telomeric DNA.",
    "molecules": "DNA, EtBr"
  },
  {
    "caption": "B Western blots of extracts from U87 cells infected with Lenti-CRISPR(puro) encoding individual ATRX-targeted sgRNAs. The extracts were isolated after puromycin selection, and assayed for ATRX depletion levels using antibody against ATRX or ACTIN control. The asterisks (*) denote the two sgATRXs (sgA#10 and sgA#19) used in following experiments.",
    "molecules": "puro, puromycin"
  },
  {
    "caption": "C ATRX immunofluorescence staining of U87 cells infected with lentiviral control sgRosa, sgA#10, or sgA#19. D, DAPI. Scale bar, 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C, D RPA/TRF2 co-immunofluorescence (C) revealed strongly increased telomeric replication stress in U87 cells at day 30 but not day 10 post lentiviral sgA#10 infection (D). Lentiviral control and sgA#10 infected U87 cells at indicated time post-infection were released following thymidine (Thy) synchronization and processed for RPA and TRF2 co-staining. Scale bar, 10 µm. (D) Percentages of cells with > 4 RPA/TRF2 co-localized foci are expressed as means ± s.d., N = 3, unpaired t test.",
    "molecules": "Thy, thymidine"
  },
  {
    "caption": "A, B ATAC-seq analyses revealed progressive increased telomeric DNA accessibilities in sgA#10 transduced IMR90 cells. (A) The graph plots show the cumulative percentage of total reads that contained at least indicated number of TTAGGG (or CCCTAA) tandem repeats. (B) The percentage of total reads that contains > 3 tandem telomeric repeats are expressed as means ± s.d., N = 2, unpaired t test.",
    "molecules": "DNA"
  },
  {
    "caption": "C, D ChIP assays (C) show reduced nucleosome densities in sgA#10 transduced IMR90 cells (D). Input and ChIP DNAs processed against indicated antibodies were assayed by dot-blotting and hybridized with 32P-labeled probes specific for TelG. (D) The relative enrichment was calculated after normalization of ChIP DNA signals to the respective input DNA signals. Data are expressed as means ± s.d., N = 3, paired t test.",
    "molecules": "DNAs, 32P"
  },
  {
    "caption": "B: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:2)2, 4 µg TAZ, and 0.2 mg MBP or 0.2 mg Alb. C: CL formation was measured as in B with different amounts of MBP or Alb. ",
    "molecules": "Alb, CL, PC, MLCL"
  },
  {
    "caption": "G: CL formation was measured in the presence of 40 nmol PC(18:2)2, 10 nmol MLCL(18:2)3, 8 µg TAZ, 1250 nmol Triton X-100 (TX), and 5.6 nmol bacteriorhodopsin (BR). H: PC formation was measured in the presence of 180 nmol CL(18:2)4, 20 nmol LPC16:0, 8 µg TAZ, 20 mM Ca2+, and 5.6 nmol BR. ",
    "molecules": "Ca2+, CL, LPC, PC, MLCL, Triton X-100, TX"
  },
  {
    "caption": "J: Transacylations were measured as in I in the presence of 0.9 g BR/ g lipid. The ratio of total linoleoyl transfer over total oleoyl transfer was calculated.",
    "molecules": "linoleoyl, lipid, oleoyl"
  },
  {
    "caption": "B. Coronal section of a P3 spheroid  embedded with paraffin  and stained  with DAPI  (blue), LDHA  (green), and LDHB  (red). Magnification boxes show different areas as depicted in the main image. Quantification of LDHA  and LDHB staining  was performed on the spheroid  areas as indicated in the graphs. Scale bar: 100 µm. Data are represented as mean ± s.d. (5-8 fields per area of interest, one representative spheroid  of 3 independent  invading spheroids)",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Western blot  analysis of LDHA  and LDHB  from P3  cells upon exposure to 21% or 0.1% O2  for 6, 24, 48, and 72 h. The graphs represent densitometry  quantification of immunoblots  normalized to tubulin (n = 4 independent experiments). Data are represented as mean ± s.d. and analyzed using One-Way ANOVA following Dunnett's multiple comparisons tests. LDHA: O2  21% vs O2  0.1% 6h, P>0.99; O2  21% vs O2  0.1% 24h, P=0.15; O2  21% vs O2  0.1% 48h, P=0.0009; O2  21% vs O2  0.1% 72h, P<0.0001. LDHB: O2  21% vs O2  0.1% 6h, P>0.99; O2  21% vs O2  0.1% 24h, P>0.99; O2  21% vs O2  0.1% 48h, P=0.97; O2  21% vs O2  0.1% 72h, P=0.88.",
    "molecules": "O2"
  },
  {
    "caption": "A.  Western blot  analysis of LDHA  and LDHB  expression in P3 cells transduced with CRISPR-Cas9 lentiviral  vectors with scramble sequence (sgControl) or against LDHA  (sgLDHA), LDHB  (sgLDHB) or both (sgLDHA/B). Knockout (KO) cells were exposed to 21% or 1% O2  for 48 h.",
    "molecules": "O2"
  },
  {
    "caption": "D. Left: P3 spheroid growth  recorded over one week at 1% O2  (n = 3 independent experiments, one experiment including 16 spheroids per condition). Data are represented as mean and analyzed using Kruskal-Wallis test followed by Dunn's multiple comparisons test. Spheroid growth: sgCont vs sgLDHA, P=0.0003; sgCont vs sgLDHB, P=0.78; sgCont vs sgLDHA/B, P<0.0001. Dead area: sgCont vs sgLDHA, P=0.19; sgCont vs sgLDHB, P<0.0001; sgCont vs sgLDHA/B, P<0.0001. Right: Viability  of spheroids  at one week, incubated with calcein (green) or ethidium homodimer-1  (red). Scale bar: 200 µm.",
    "molecules": "calcein, O2, ethidium homodimer-1"
  },
  {
    "caption": "E. P3 spheroid invasion  in collagen  I   gel, incubated 24 h at 21% or 0.1% O2. Invasion rate is expressed as fold change from controls (n = 4 independent experiments, one experiment including 6-8 spheroids  per condition). Data are represented as mean and analyzed using Two-way ANOVA following by Tukey's multiple comparisons test: sgCont 21% vs sgLDHA 21%, P=0.003; sgCont 0.1% vs sgLDHA  0.1%, P<0.0001; sgCont 21% vs sgLDHB  21%, P<0.0001; sgCont 0.1% vs sgLDHB 0.1%, P<0.0001; sgCont 21% vs sgLDHA/B 21%, P<0.0001; sgCont 0.1% vs sgLDHA/B 0.1%, P<0.0001. Images of representative of control or LDHA/B KO spheroids. Scale bar: 100 µm.",
    "molecules": "O2"
  },
  {
    "caption": "F-G. A first cohort of mice  was sacrificed when one mouse  reached a limit point, brains  was extract, snap frozen in liquid nitrogen, sliced and stained. For the second cohort, each mouse was sacrificed when it reached a limit point allowing the draw of survival curves. Left: Immunofluorescence staining  of Nestin  (top and middle) and CD31  (bottom) of control and LDHA/B KO P3 tumor section. Scale bars: 2 mm (top), 250 µm (middle) and 100 µm (bottom). Right: Graphs represent tumor core and invasion  area of control and  LDHA/B KO P3 tumors in mm2 (n = 5 mice  per group, average of 5-6 sections per tumor). Data are represented as mean. For F, data are analyzed using One-way ANOVA following by Dunnett's multiple comparisons test. Core: sgCont vs sgLDHA, P=0.48; sgCont vs sgLDHB, P=0.99. Invasion: sgCont vs sgLDHA, P=0.40; sgCont vs sgLDHB, P=0.81. For G, data are analyzed using unpaired t test. Core: sgCont vs sgLDHA/B, P=0.005. Invasion: sgCont vs sgLDHA/B, P=0.01.",
    "molecules": "nitrogen"
  },
  {
    "caption": "A. Western blot  analysis of mitochondrial respiratory chain subunits from LDH KO P3 cells upon exposure to 21% or 0.1% O2. Roman numbers indicate the respiratory complex number. The diagram below represents densitometry  quantification of the immunoblots  normalized to vinculin  and expressed as log2 foldchange to control cells in 21% and 0.1% O2  (n = 3 independent experiments).",
    "molecules": "O2"
  },
  {
    "caption": "B. Epifluorescence  (top) and quantitative image analysis (bottom) of immune stained mitochondria  (TOM20, green) from LDH KO P3 cells (Phalloidin, red; DAPI, blue) upon exposure to 0.1% O2  (n = 2 independent experiments, 28-32 cells per group). Data are represented as mean and analyzed using One-way ANOVA following by Tukey's multiple comparisons test. Mitochondrial  mass: sgCont vs sgLDHA/B, P=0.005; sgLDHA/sgLDHB vs sgLDHA/B, p=0.006. Network aspect ratio: sgCont vs sgLDHA/B, P=0.03; sgLDHA vs sgLDHA/B, P=0.02; sgLDHB vs sgLDHA/B, p=0.04. Mitochondrial  mass corresponds to the area covered by the mitochondria  relative to the entire cell area. Network aspect ratio is the ratio of the major axis to minor axis of the  mitochondrial network. Scale bar: 10 µm.",
    "molecules": "DAPI, O2, Phalloidin"
  },
  {
    "caption": "E. Kaplan-Meier survival curves of xenotransplanted mice  with LDHA/B KO (red) or control (blue) P3 cells, treated with phenformin  or irradiated  with 10 Gy (n = 6-8 mice  per group). Data are analyzed using Log-rank (Mantel-Cox) test: sgCont Vehicle vs sgCont Phenformin, P=0.019; sgCont Vehicle vs sgCont Irradiation, P=0.0002; sgCont Irradiation vs sgLDHA/B Irradiation, P=0.024.",
    "molecules": "phenformin, Phenformin"
  },
  {
    "caption": "D. Left: P3 spheroid growth  recorded over 72 h during incubation with stiripentol  at 0.1% O2  (n = 3 independent experiments, one experiment including 8 spheroids  per condition). Data are represented as mean ± s.d. and growth  at 72 h are analyzed using unpaired t test: P<0.0001. Right: Viability  of spheroids  at 72 h, incubated with calcein (green) or ethidium homodimer-1 (red). Scale bar: 100 µm. E. Left: P3 spheroid invasion  in collagen  I  gel, incubated 24 h with 500 µM stiripentol  at 0.1% O2. Invasion rate  is expressed as fold change from control (n = 3 independent experiments, one experiment including 8 spheroids  per condition). Data are represented as mean and analyzed using unpaired t test: P<0.0001. Right: Representative images of invasive spheroids  for each condition. Scale bar: 100 µm.",
    "molecules": "calcein, O2, ethidium homodimer-1, stiripentol"
  },
  {
    "caption": "F.  Kaplan-Meier survival curves  of xenotransplanted mice  with P3 cells. Mice were treated either with vehicle (blue) or stiripentol  (red) (n = 15-16 mice  per group). Data are analyzed using Log-rank (Mantel-Cox) test: P=0.002",
    "molecules": "stiripentol"
  },
  {
    "caption": "Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) models. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.",
    "molecules": "pilocarpine"
  },
  {
    "caption": "(G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10).",
    "molecules": "pilocarpine"
  },
  {
    "caption": "(H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times.",
    "molecules": "Mg2+, Pic, picrotoxin"
  },
  {
    "caption": "(J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). a-tubulin (a-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student's t test.",
    "molecules": "Mg"
  },
  {
    "caption": "(A-C) Whole-cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons. A, representative traces showing the neuronal activity under the indicated treatments. Ctrl: extracellular solution. Cyclo: cyclopamine. B, the expanded view of a single burst (arrow) from A; C, statistics of the percentage of neurons showing epileptiform activity (left, n=6-7) and the burst frequency (right, n=18-19) shown in A. Cells were incubated in the presence of Cyclo in extracellular solution for 30 min, then transferred to 0Mg plus Cyclo (0Mg+Cyclo) and incubated for another 30 min.",
    "molecules": "Cyclo, cyclopamine, Mg"
  },
  {
    "caption": "(D,E) The effects of Sant-1, robotnikinin (Robot) or 5E1 on the percentage of neurons showing epileptiform activity (D, n=6-10) and the burst frequency (E, n=15-22). (F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF).",
    "molecules": "Robot, robotnikinin, Sant-1"
  },
  {
    "caption": "(F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF). G, the expanded view of a single burst (arrow) in F. Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min, then transferred to picrotoxin in aCSF (Pic) or with Cyclo (Pic+Cyclo) and recorded for 30 min. H, statistics of the spontaneous burst frequency of F from 10 slices (7 rats).",
    "molecules": "Cyclo, Pic, picrotoxin"
  },
  {
    "caption": "(I,J) Left are representative traces showing the D-CPP-sensitive currents recorded from cultured hippocampal neurons (I) or slices (J) incubated in 0Mg or Pic, respectively, with or without Cyclo. Black lines: application of D-CPP. Right are statistics of D-CPP-sensitive current density from 18-26 cells (I) or 16-18 slices (15 rats) (J). Unless stated, 10 µM cyclopamine was used. Data were mean + SEM. *p<0.05,**<0.01,***p<0.001 vs. Ctrl; #p<0.05,##<0.01,###p<0.001 vs. 0Mg or Pic; &p<0.05 vs. 0Mg + IgG. C-E used one-way ANOVA. H-J used student's t test.",
    "molecules": "D-CPP, Cyclo, cyclopamine, Mg, Pic"
  },
  {
    "caption": "(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6.",
    "molecules": "Glutamate"
  },
  {
    "caption": "Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10)",
    "molecules": "3H, glutamate"
  },
  {
    "caption": "Statistics of 3H-glutamate uptake by neurons transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1.",
    "molecules": "3H, glutamate"
  },
  {
    "caption": "Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18).",
    "molecules": "Asp, Aspartate"
  },
  {
    "caption": "(G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9.",
    "molecules": "3H, glutamate"
  },
  {
    "caption": "(H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student's t test.",
    "molecules": "3H, Asp, glutamate"
  },
  {
    "caption": "Effects of Cyclo (A-C, n=14) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.",
    "molecules": "Cyclo"
  },
  {
    "caption": "Effects of ablation of Smo in CaMKIIα-positive neurons (G-I), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23.",
    "molecules": "Cyclo, pilocarpine"
  },
  {
    "caption": "(N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student's t test.",
    "molecules": "Cyclo, pilocarpine"
  },
  {
    "caption": "E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0. Data information: the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed.",
    "molecules": "DNA, IgG"
  },
  {
    "caption": "(A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm.",
    "molecules": "3MA, chloroquine"
  },
  {
    "caption": "DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm.",
    "molecules": "chloroquine, rapamycin"
  },
  {
    "caption": "(A) DOR gain‐of‐function accelerates the degradation of proteins of middle-long half‐life. Untreated HeLa cells and those transiently transfected with DOR were incubated in DMEM or HBSS. *Significant effects of starvation, P0.01; τsignificant effects caused by DOR overexpression, P0.01.",
    "molecules": "proteins"
  },
  {
    "caption": "(E) Scramble or DOR siRNA1 C2C12 cells were incubated in DMEM with and without 10 mM 3MA. *Significant effects of DOR knockdown, P0.01; τsignificant effects caused by 3MA, P0.01. 3MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; LC3, microtubule‐associated protein 1A/1B‐light chain 3.",
    "molecules": "3MA"
  },
  {
    "caption": "Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25-35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)",
    "molecules": "Meth"
  },
  {
    "caption": "(B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25-35, mean ± SEM, Student's two-tailed t-Test).",
    "molecules": "Meth"
  },
  {
    "caption": "(C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30-90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)",
    "molecules": "Meth"
  },
  {
    "caption": "(D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm.",
    "molecules": "Clq, Meth"
  },
  {
    "caption": "(E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.",
    "molecules": "Clq, Meth"
  },
  {
    "caption": "(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300-400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001).",
    "molecules": "Clq, Meth"
  },
  {
    "caption": "(B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300-400 cells, mean ± SEM, ***p < 0.0001).",
    "molecules": "Meth"
  },
  {
    "caption": "(C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.",
    "molecules": "Clq, Meth"
  },
  {
    "caption": "(A) Cells were incubated in indicated concentrations of Meth or Clq for 2 h, and then IgG coated erythrocytes were added. Extracellular uningested EIgG were lysed and removed. Phagocytic index (PI) was quantified and inhibition is indicated as percent of control. Data were collected from four to seven experiments (n = 300 cells, mean ± SEM, ***p < 0.0001, two-tailed ANOVA).",
    "molecules": "Clq, Meth"
  },
  {
    "caption": "(B) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment. Arrows denote phagocytosed erythrocytes. Scale bar, 10 μm.",
    "molecules": "Clq, Meth"
  },
  {
    "caption": "(C, D) J774.16 cells were exposed to PBS, chloroquine, or methamphetamine for 2 h followed by incubation with Cn or Ca. The phagocytic indices (ratio of intracellular yeast to the number of macrophages counted) were determined after 1 h or 30 min for Cn and Ca, respectively (C). (Statistics n = 300, mean ± SEM, *p < 0.05, two-tailed ANOVA.) CFU determinations were preformed after 24 h incubations (D). The values are the mean and standard deviation from determinations made in triplicate wells (n = 300 cells, mean ± SEM, *p < 0.05, two-tailed ANOVA.). Experiments were repeated three times with similar results.",
    "molecules": "chloroquine, methamphetamine"
  },
  {
    "caption": " (G-H) Exemplar STCs in response to SGN illumination with all µLEDs in a CatCh-transduced, kanamycin-deafened gerbil before (G) and after (H) sacrifice. ",
    "molecules": "kanamycin"
  },
  {
    "caption": "(B) Immunoblot analysis of the immunoprecipitated HA-SOX9 or FLAG- FBW7α and their pull-down products. The corresponding Western blotting from the whole cell extract (WCE) of the transfected HEK293 showed the levels of exogenous HA-SOX9 or FLAG-FBW7α proteins in the total input. Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation. Blots are representative from 3 independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α, β or γ. HEK293 cells were transfected with 20 nM of non-targeting scramble RNA or those specifically targeting FBW7α, β or γ for 72 hours prior to chase with addition of 100 ng /mL cycloheximide. Immunoblot of Cyclin E, an established SCF-FBW7 substrate, was used to assess the efficacy of RNAi-mediated depletion of FBW7 protein with GAPDH served as protein loading control.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(D) Expression of FLAG-FBW7α enhanced HA-SOX9-WT protein turnover, which can be attenuated with addition of 10 µM MG-132 (MG) in HEK293 cells. By contrast, forced expression of FLAG-FBW7α did not enhance HA-SOX9-T236A and -T236A/T240A protein turnover over the course of 4 hours protein chase. Protein loading for each sample was indicated by the level of β-actin protein. The set of blots shown is representative from 4 independent experiments.",
    "molecules": "MG, MG-132"
  },
  {
    "caption": "(E) Treatment of HEK293 cells with increasing concentration of BIO, a GSK3α/β inhibitor, increased HA-SOX9-WT protein level in dose-dependent manner. HA-SOX9-WT and FLAG-FBW7α were co-expressed in HEK293 overnight prior to treatment with various concentration of BIO for 4 hours. Whole cell lysates were collected for Western blotting with protein loading for each sample adjusted to GAPDH protein level. The set of blots shown is representative from 2 independent experiments.",
    "molecules": "BIO"
  },
  {
    "caption": "(F) The RNAi depletion of GSK3ß decreased FBW7α-induced HA-SOX9-WT turnover in HEK293 cells. The HA-SOX9-WT protein turnover was examined in the presence of 100 ng/mL cycloheximide, 48 hours following si-GSK3ß (20 nM) transfection, 24 hours after co-expression of HA-SOX9-WT and FLAG-FBW7α. Immunoblots of GSK3ß demonstrated depletion of GSK3ß protein with the siRNA. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(G) Forced FLAG-FBW7α-WT expression promoted poly-ubiquitylation of endogenous SOX9 in HEK293, which accumulated further in the presence of 10 µM MG132. Expression of non-functional FBW7α lacking the F-box domain (ΔF), or those containing R465A mutation did not induce SOX9 poly-ubiquitylation in vivo. Ubiquitylation assay was performed under denaturing condition to disrupt non-covalently linked ubiquitin as described in the Materials & Methods. Expression of various FLAG-FBW7α constructs and endogenous SOX9 protein were examined in the whole cell extract (WCE). GAPDH protein was used to indicate total protein loading with protein loading. The set of blots shown is representative from 4 independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Overall survival analysis of mice bearing Daoy cell line expressing SOX9 with doxycycline (dox) -inducible FBW7α ( Daoy-SOX9/T-FBW7α). The dox-preconditioned or the control cells (105cells) were orthotopically xenografted to the cerebellum of 6-weeks old Athymic Nude-Foxn1nu mice, which were continuously fed with either the dox-containing chow (red line) or the regular chow food (green line), respectively. Micexenografted with dox-preconditioned Daoy-SOX9/T-FBW7α and fed continuously with dox-containing chow (red line) survived longer (p= 0.0285) than the control group (green line)",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C) Representative bright field images of migrating Daoy-SOX9/T-FBW7α in the absence or presence of Dox. Daoy-SOX9/T-FBW7α cells were either maintain in regular culture or pre-conditioned with dox prior to experiments. The cells (2 x 105cells / insert) were seeded and let to migrate across 5 μm filter pore toward the laminin-coated trans-well for 4 hours prior to fixation and staining with crystal violet. Histogram quantification of cell migration were performed by measuring the crystal violet absorbance of the stained-migrating cells and presented as mean + standard deviation from 2 independent experiments each containing 2 technical replicates.",
    "molecules": "Dox"
  },
  {
    "caption": "(F) Transwell migration of SOX9-expressing MIC with dox-inducible FBW7α or dominant negative ∆F FBW7α. The cells were either maintain in regular culture or pre-conditioned with dox prior to assessment of in vitro transwell migration as described in (C). Data are mean + standard deviation from 2 independent experiments each containing 2 technical replicates.",
    "molecules": "dox"
  },
  {
    "caption": "(E) Quantitative PCR (qPCR) of SNAI2 (SLUG), VIM and POU3F1 (OCT6) (normalized to GAPDH levels) in MB002 cells stably transduced with EV (set to 1), SOX9-WT and SOX9-T236/240A. Cells were induced for 8 or 24 hours with doxycycline.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(F) Western blot of SOX9, HA-SOX9, pSOX9-T236, SNAI2 (SLUG), VIM and POU3F1 (OCT6) in MB002 cells stably transduced with EV, SOX9-WT and SOX9-T236/240A. Cells were induced for 24 and 48 hours with doxycycline and GAPDH were used as a loading control.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A) Cisplatin dose response curves of (i) MB002, (ii) Daoy, and (iii) MIC in the absence (EV) or presence of SOX9 by Alamar blue. Cells were pre-conditioned with doxycycline to induce expression of SOX9 (or EV) prior to treatment with increasing concentrations of cisplatin. The IC50 were calculated following 5 (MB002 and MIC) or 3 days (Daoy) of treatment. Data are mean + standard deviation from 3 independent repeats, each containing 5 technical replicates.",
    "molecules": "Cisplatin, cisplatin, doxycycline"
  },
  {
    "caption": "(B) Cisplatin dose response curves of SOX9-expressing (i) Daoy and (ii) MIC in the absence or presence of FBW7α. Experiments and data analysis were performed as described in (A)",
    "molecules": "Cisplatin"
  },
  {
    "caption": "(C) Overall survival analysis of mice bearing Daoy or Daoy-expressing dox-inducible SOX9 treated with cisplatin. The dox-preconditioned cells (105 cells) were orthotopically xenografted to Nude-Foxn1nu mice and left for 1 week to prior to being treated with vehicle control or cisplatin (2mg/kg) intraperitoneally for every other day for a total of 6 doses.",
    "molecules": "cisplatin, dox"
  },
  {
    "caption": "(D) Heat map of the row-wise z-scores of 11 genes associated with cisplatin resistance in MB002 expressing Sox9-WT or Sox9-T236/T240A. Heat map was generated using the GenePattern software.",
    "molecules": "cisplatin"
  },
  {
    "caption": "(E) Quantitative analysis of ATP7A, DUSP2, and TTK mRNAs in MB002 following expression of SOX9-WT or SOX9-T236/240A. Total RNA were collected 24 hours following doxycycline treatment, from which cDNA were generated for qPCR. Data are mean mRNA level (normalized to B2M transcript) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2-way ANOVA with Bonferroni's post-test.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(F) Time course western blotting of HA-SOX9, ATP7A, DUSP2, ERK1/2 pThr202/Tyr204 and total ERK1/2 in MB002 cells following doxycycline induction of either EV, SOX9-WT or SOX9-T236/240A. GAPDH was used as a loading control.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A) Western blotting of pT236- and total SOX9 following treatment of Daoy with a panel of inhibitors targeting PI3K/AKT/mTOR pathway. The whole cell lysates were collected for gel electrophoresis following 6 hours treatment with 1 μM of AZD5363, 8186, or 2014. The set of blots shown is a representative from 3 independent repeats.",
    "molecules": "AZD5363"
  },
  {
    "caption": "(B) Western blotting of endogenous SOX9 protein turnover in the presence of PI3K/mTOR dual inhibitor, AZD2014. Daoy were transfected with either siScr or siFBW7 for 48 hours prior to further treatment with 1 µM AZD2014. The cycloheximide chase of SOX9 protein turnover was examined 2 hours following the inhibitor treatment. Immunobloting of Cyclin E protein was used to indicate the efficacy of siFBW7 transfection. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.",
    "molecules": "AZD2014, cycloheximide"
  },
  {
    "caption": "(C) Quantification of resazurin-based Daoy and MB002 cell viability following treatment with Cisplatin (blue bars), AZD2014 (red bars), or their combination (green bars). Daoy were treated for 72 hours, while MB002 were subjected to 5 day course of treatment. The cellviability were calculated relative to the vehicle-treated cells. The synthetic lethality combination index (CI) for each treatment was calculated from the mean of 3 independent experiments using the Compusyn software. Error bars indicate standard deviation.",
    "molecules": "AZD2014, Cisplatin"
  },
  {
    "caption": "(D) Time course western blotting of endogenous SOX9 and apoptotic markers including cleaved PARP and caspase 3 following MB002 treatment with cisplatin, AZD2014, or their combination. β-actin were used as a loading control. The set of blots shown is representative from 3 independent experiments.",
    "molecules": "AZD2014, cisplatin"
  },
  {
    "caption": "(E) Western blotting profiling of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 48 hours treatment with cisplatin, AZD2014, and their combination. The set of blots shown is representative from 3 independent experiments.",
    "molecules": "AZD2014, cisplatin"
  },
  {
    "caption": "(F) Quantification of cell viability of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 5 days treatment with either of 10 μM cisplatin (black bars), 0.5 µM AZD2014 (blue bars), or their combination (red bars). Data are mean + standard deviation from 3 independent experiments, from which statistical significance were analysed by 2-way ANOVA multiple comparisons with Bonferroni's post-test.",
    "molecules": "AZD2014, cisplatin"
  },
  {
    "caption": "(G) Immunoblots of ATP7A and DUSP2 protein following doxycycline treatment of Daoy-SOX9/T-FBW7α. The cells were either untreated or incubated with 5 µg/mL doxycycline for 72 hours prior to being harvested for gel electrophoresis. The set of blots shown is representative from 2 independent experiments.",
    "molecules": "doxycycline"
  },
  {
    "caption": "NIX and BNIP3 are required for mitophagy induced by MLN4924 and other HIF1α stabilisers. Parental HeLa cells and NIX/BNIP3 double knockout (NIX/BNIP3 DKO) HeLa cells expressing mt-Keima were treated with DFP (1 mM), MLN4924 (0.5 μM), or DMOG (10 nM) for 24 hours and analysed by live-cell confocal microscopy. The emission signals obtained after excitation with the 458 nm laser (neutral pH) or 561 nm laser (acidic pH) are shown in green and magenta, respectively. Quantification of mitophagy shown in panel A. Mitophagy is represented as the ratio of mt-Keima 561 nm fluorescence intensity to mt-Keima 458 nm fluorescence intensity for individual cells normalised to the mean of the untreated condition. In B , the measurements from individual cells are represented as translucent grey dots, and the mean ratio from each independent experiment is represented by coloured circles. The centre lines and bars depict the mean of the averaged independent replicates +/- standard deviation. N=4. A minimum of 50 cells were analysed for each condition within each individual replicate experiment, and over 300 cells were analysed for each condition in total. P values were calculated based on the mean ratio values from independent experiments using one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001).",
    "molecules": "DFP, DMOG, MLN4924"
  },
  {
    "caption": "Analysis of NIX and BNIP3 protein levels after MLN4924, DMOG, or DFP treatments. HeLa cells or HeLa NIX/BNIP3 DKO cells were treated with MLN4924, DMOG, or DFP for 24 hours. Total-cell lysates corresponding to conditions in panel A were subject to immunoblotting.",
    "molecules": "DFP, DMOG, MLN4924"
  },
  {
    "caption": "Inhibition of HIF1α with echinomycin prevents DFP-induced mitophagy but not MLN4924-induced mitophagy. U2OS cells expressing mt-Keima were treated with the indicated drugs for 24 hours and analysed by live-cell confocal microscopy. Quantification of mitophagy shown in panel D. In F, the measurements from individual cells are represented as translucent grey dots, and the mean ratio from each independent experiment is represented by coloured circles. The centre lines and bars depict the mean of the averaged independent replicates +/- standard deviation. N=4. A minimum of 50 cells were analysed for each condition within each individual replicate experiment, and over 300 cells were analysed for each condition in total. P values were calculated based on the mean ratio values from independent experiments using one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001).",
    "molecules": "DFP, MLN4924, echinomycin"
  },
  {
    "caption": "Inhibition of HIF1α with echinomycin prevents the increase of NIX and BNIP3 in response to DFP, but not MLN4924. U2OS cells were treated with DFP or MLN4924 over a time course. Where indicated, cells were also treated for 24 hours with echinomycin. Total-cell lysates were subject to immunoblotting as shown.",
    "molecules": "DFP, MLN4924, echinomycin"
  },
  {
    "caption": "Re-expression of FBXL4 into FBXL4-defective CRISPR lines reduces the levels of NIX and BNIP3 in FBLX4 deficient clones. FBXL4-deficient 2G10 and FBXL4-deficient 1D4 cell lines were stably transduced with a doxycycline-inducible FBXL4-HA construct. Cells were treated with doxycycline for the indicated times prior to immunoblotting with the specified antibodies.",
    "molecules": "doxycycline"
  },
  {
    "caption": "FBXL4 localises to the mitochondrial outer membrane. Cells transiently transfected with FBXL4-HA-C were treated with DFP for 24 h. Cells were stained with an anti-HA antibody (to recognise FBXL4) and either TOM20 (an outer mitochondrial membrane protein) or TIM50 (an inner mitochondrial membrane protein). The line scan intensity profiles for FBXL4 (green) and TOM20/TIM50 (magenta) represent fluorescence intensity (y-axis) plotted against distance (x-axis). Scale bars = 5 μm.",
    "molecules": "DFP"
  },
  {
    "caption": "FBXL4 is a proximity interactor of NIX and BNIP3. Cells expressing inducible BirA-BNIP3, BirA-NIX and BirA control were transduced with a lentiviral vector expressing FBXL4, as indicated. Cells were treated with doxycycline for 48 hours (to induce BirA-bait protein expression), biotin for 24 hours (for the biotinylation reaction), and, where indicated, MLN4924 for 24 hours (to stabilise NIX and BNIP3). Streptavidin-coupled beads were used to capture the biotinylated proteins. FBXL4 was specifically detected in the eluate from BirA-BNIP3 and BirA-NIX compared with BirA-alone.",
    "molecules": "biotin, doxycycline, MLN4924, Streptavidin"
  },
  {
    "caption": "Loss of FBXL4 leads to an increase in mitophagy, which can be reduced by re-expression of FBXL4. U2OS mt-Keima FBXL4 KO clones (2G10 and 1D4) expressing doxycycline-inducible wild-type FBXL4-HA were treated with doxycycline for 72 hours. The emission signals obtained after excitation with the 458 nm laser (neutral pH) or 561 nm laser (acidic pH) are shown in green and magenta, respectively.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Hyperstable NIX deletion mutants increase mitophagy compared with wild-type NIX. Hela Flp-in NIX KO mt-Keima cells stably expressing inducible NIX(WT), FLAG-NIX∆151-170, or FLAG-NIX∆171-184 were treated with doxycycline for 48 hours Mitophagy was visualised as in A and quantified as in B. Hyperstable BNIP3 deletion mutants increase mitophagy compared with wild-type NIX. Hela Flp-in NIX/BNIP3 DKO mt-Keima cells stably expressing BNIP3 deletion mutants were treated with doxycycline for 48 hours translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicate means +/- standard deviation. N=3. At least 200 cells were analysed per condition. P values were calculated based on the mean values using a one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001). Scale bars = 20 μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "FBXL4 patient-derived variants exhibit reduced efficiency compared to wild-type FBXL4 in mediating the downregulation and destabilization of NIX and BNIP3. U2OS FBXL4 KO (2G10) cells were rescued with constructs expressing wild-type FBXL4-HA, FBXL4(F-box mut), FBXL4(∆MTS), or specified patient variants. Cells were treated with cycloheximide for 3 hours prior to harvesting. Samples were lysed, and immunoblotting was performed. GFP serves as a marker of transduction efficiency/transgene expression. EV = empty vector.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "FBXL4-Arg482Trp and FBXL4-Gly568Ala patient variants are less efficient than FBXL4-wild-type at assembling into a complex with SKP1 and CUL1. FBXL4-KO cells expressing wild-type FBXL4-HA or FBXL4 variants were harvested and lysed. Whole-cell extracts were subjected to immunoprecipitation (IP) with anti-HA agarose beads and immunoblotting, as indicated.",
    "molecules": "agarose"
  },
  {
    "caption": "(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23.",
    "molecules": "MG132"
  },
  {
    "caption": "(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (B) Degradation of model proteins containing other UbL domains or ubiquitin. The graphs plot the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.",
    "molecules": "MG132"
  },
  {
    "caption": "(B) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail, and affected yeast growth under selective condition (+3-AT, -his). Replacing UbL domains with DHFR domains rescued the growth defect. Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. Plates were incubated at 30 ˚C for 3 daysfor imaging.",
    "molecules": "3-AT, his"
  },
  {
    "caption": "(D) Steady-state accumulation of endogenous Mdy2 in S. cerevisiae, with deleted (get4∆) or endogenous (wt) Get4 in the presence or absence of the proteasome inhibitor bortezomib. Protein levels were determined by western blotting for the HA-tag, Flag-tag, Mdy2 or Scs2 of SDS-PAGE gels of S. cerevisiae protein extracts. Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.",
    "molecules": "bortezomib"
  },
  {
    "caption": "C) Degradation of Ubp6 proteinsin vivo. The steady-state accumulation of N-terminally HA-tagged Ubp6 and mutants in S. cerevisiae. Ubp6 and Ubp6ΔN are stable in yeast and not further stabilized by bortezomib. Attachment of a Su9 tail to the Cterminus lead to the degradation of Ubp6 and Ubp6ΔNproteins and the proteins were stabilized by bortezomib treatment.",
    "molecules": "bortezomib"
  },
  {
    "caption": "(D) The steady-state accumulation of endogenous Ubp6 and Ubp6 derivatives with short (19 amino acid) or long (54 amino acid) tails at their C-termini. The 19 amino-acid tail destabilized Ubp6 and the 54-amino-acid tail lead to complete depletion of Ubp6. Protein levels were determined by western blotting for the HA-tag, Ubp6 or Scs2 in SDS-PAGE gels of S. cerevisiae protein extracts. The proteasome was inhibited by 100 µM bortezomib as indicated. Protein loading levels in each lane were normalized in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.",
    "molecules": "amino acid, amino-acid, bortezomib"
  },
  {
    "caption": "(d-g) Gold-enhanced immunogold electron microscopy revealed that GFP-DFCP1 localized to ER-IM complexes in both control (d, e) and Atg4BC74A-expressing cells (f). Immunogold particles are indicated by small arrows. No specific labelling was observed on ER-IM complexes in cells expressing GFP (g). Large arrows and arrowheads indicate the ER and the IM, respectively. Scale bars, 200 nm in a and d-g, an d 10 μm in c.",
    "molecules": "Gold"
  },
  {
    "caption": "Atg4BC74A-expressing cells (for Atg16L1) or control cells (for PDI, COP II and β-COP) were labelled with specific antibodies against the indicated markers. The left and right anti-Atg16L1 and anti-PDI images are from two different ER-IM complexes. Arrows, arrowheads and open arrowheads indicate the ER, IMs and VTCs, respectively. Small arrows in the image from the anti-COP II experiment indicate immunogold particles. Scale bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities. E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D). ",
    "molecules": "Cy3"
  },
  {
    "caption": "(A-B) Representative Hematoxylin and Eosin (H&E) staining, and immunohistochemistry of phosphorylated FAK on tyrosine (Y) 397 (pY397 FAK), alpha-Smooth Muscle Actin (α-SMA), and Fibroblast Activated Protein alpha (FAP-α) in serial sections of human normal pancreas (Ct1), of human normal adjacent PDAC tissue (Ct2) and of human PDAC tissues (PDAC1-3); scale bar, 100 μm (A), and 300 µm (B).",
    "molecules": "tyrosine"
  },
  {
    "caption": "(B) Representative images from primary tumour stained for H&E, cytokeratin 19 (CK19), α-SMA, Sirius Red and polarized Sirius Red. Scale bar, 100 μm.",
    "molecules": "Sirius Red"
  },
  {
    "caption": "(C) Quantification of fibrillar collagen I (Yellow-Orange) and III (Green) based on polarized Sirius Red images from B. Each dot represents a mouse (n=11 per group).",
    "molecules": "Sirius Red"
  },
  {
    "caption": "(E) Impact of soluble factors secreted by CAFs on macrophage migration. Left: schematic of BMDM derived M1 or M2-like macrophages. Right: Representative pictures and quantification of M1 and M2 macrophage migration at 24 and 48h towards CM secreted by CAFs (previously treated or not with FAK inhibitor for 48h). Cells were stained using Cristal Violet and percentage of cristal violet positive area quantified using Cell Observer videomicroscope (Zeiss) and analyzed with ImageJ.",
    "molecules": "Cristal Violet, cristal violet"
  },
  {
    "caption": "(H) Left: Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine (Y) 397 and of CD206 in serial sections of human PDAC tissues. Scale bar: 200µm Middle: Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206+ cell number within human PDAC samples. Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining. Spearman r = 0.4145, p=0.0019. Right: Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score.",
    "molecules": "tyrosine"
  },
  {
    "caption": "(B) Representative images of ECM deposition (stained using Alexa Fluor 488 succinimidyl ester (NHS)) generated by FAK-WT or -KD activated fibroblasts during migration.",
    "molecules": "succinimidyl ester (NHS), Alexa Fluor 488"
  },
  {
    "caption": "(I) Normalized peptide abundance for glycoproteins from hCAFs treated or not with FAK-I.",
    "molecules": "glycoproteins"
  },
  {
    "caption": "(J) Heatmap of the glycoprotein modifications upon FAK-I in hCAFs.",
    "molecules": "glycoprotein"
  },
  {
    "caption": "(L) Left: Representative pictures of β1 integrin activation (red) of tumour cells, upon adhesion to either ECM deposited from non-treated CAFs or treated with FAK-I for 7 days, analyzed by IF. Merge: activated β1 integrin (red), phalloidin (green), dapi (blue). Right: quantification of the number of integrin β1 activated cluster per cells and size of those clusters.",
    "molecules": "dapi, phalloidin"
  },
  {
    "caption": " (A) Confocal images of ALIX (red), Asl (white), α-tubulin (green) and Hoechst (blue) in wild type (w1118), Sas-6c02901, DSpdG02143, cnnHK21 and γ-Tub23CPI NBs (scale bars, 5 μm). The insets show higher magnification of the boxed centrosomes",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) GST or GST-ALIX were incubated with in vitro translated 35S-labelled Cnn or DSpd-2 proteins. Binding was detected by autoradiography (upper panel), and expression of GST and GST-ALIX was visualized by Ponceau S-staining (lower panel)",
    "molecules": "35S, Ponceau S"
  },
  {
    "caption": "*. (E) Control, Cep192 or Cep215 siRNA-transfected HeLa cells were immunostained with anti-ALIX (red), anti-α-tubulin (green) and Hoechst (blue). Confocal micrographs are presented and the insets show the indicated centrosomes (arrows) at higher magnification (scale bars, 5 μm)",
    "molecules": "Hoechst"
  },
  {
    "caption": " (A) Brains were dissected from w1118, alix1, alix-l/CyO; alix1 and mud4 larvae and stained with anti-Bazooka (red), anti-Cnn (white) and Hoechst (blue). Representative confocal images are shown. Scale bars, 5 μm. The graph summarizes the average variation of the relative spindle angles in w1118 (n=74), alix1 (n= 96), alix-l/CyO; alix1 (n= 78) and mud4 (n=32) NBs (four separate experiments) (±S.E). Compared to w1118 or alix-l/CyO; alix1 NBs both  alix1 and mud4 NBs showed greater variation in MS orientation (***p<0.001, ANOVA) ",
    "molecules": "Hoechst"
  },
  {
    "caption": " (A) Ovaries of adult female flies (w1118, alix1 and alix-l/CyO; alix1) were stained with rhodamine-phalloidin (red), anti-α-tubulin (green), anti-Cnn (white) and Hoechst (blue). Typical confocal micrographs of metaphase FECs are presented and the apical membrane facing the egg chamber lumen is indicated with *. Scale bar, 5 μm. The MS orientation was measured and the graph shows the average percentage of FECs (w1118, n=45, alix1, n=45, alix-l/CyO; alix1, n=39, from three independent experiments) with the indicated relative spindle angle (±S.E). Statistically significant difference is indicated as ***p<0.001 (ANOVA)",
    "molecules": "phalloidin, rhodamine, Hoechst"
  },
  {
    "caption": "(D) The MS orientation was determined in metaphase cells in organoids of Caco-2 cells transfected with control (n=75), ALIX (n=88) or NuMA (n=79) siRNA (from three independent experiments). The graph shows the average percentage of Caco-2 cells with the indicated relative spindle angle (±S.E). Greater spindle angle variation was observed in ALIX- and NuMA-depleted cells compared to control cells (***p<0.001, ANOVA). Representative confocal images are shown where the cells are stained with rhodamine-phalloidin (red), α-tubulin (green) and Hoechst (blue). The apical membrane facing the lumen of the organoid is marked (*). Scale bar, 5 μm",
    "molecules": "phalloidin, rhodamine, Hoechst"
  },
  {
    "caption": " (A) The MARCM strategy was used to generate alix mutant NBs clones (FRT82B, alix1) or genetic equivalent control clones (FRT82B con) in the larval brain. Dissected brains were immunostained with anti-α-tubulin (red) and Hoechst (blue). Recombinant NBs are positively marked with GFP (green) and outlined. The presence or lack of astral MTs is indicated with arrows or asterisks, respectively. Scale bars, 5 μm. The average intensity of astral, total and astral/total MT (±S.E) in GFP-positive relative to GFP-negative control cells (set to 100%, indicated as dotted line in the graph) was calculated from three separate experiments (including 13 GFP-negative FRT82B con NBs, 11 GFP-positive FRT82B con NBs, 10 GFP-negative FRT82B, alix1 NBs and 8 GFP-positive FRT82B, alix1 NBs) (*p<0.05 and **p<0.01, Student's t-test) ",
    "molecules": "Hoechst"
  },
  {
    "caption": "(B) HeLa cells depleted of ALIX or not were immunostained with anti-α-tubulin (red), anti-Pericentrin (green) and Hoechst (blue). Representative confocal images are presented. Scale bars, 5 μm. The graph shows MT intensities as percentage of control cells (±S.E) obtained from 99 control and 90 ALIX siRNA transfected cells (data obtained from six separate experiments). Significant differences are noted as *p<0.05 and ***p<0.001 (Student's t-test)",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C, D) Regrowth of MTs after cold-induced depolymerization was performed using brains of w1118 and alix1 larvae, stained with anti-α-tubulin (red), anti-Bazooka (green) and Hoechst (blue) (C Typical confocal images showing MT-regrowth at given time points are shown (scale bars, 5 μm). The graphs show the time-dependent MT intensities relative to the respective 0''-samples (±S.E) calculated from three independent experiments. In (C) 46, 64, 65 and 48 w1118 NBs and 73, 37, 44 and 49 alix1 NBs from 0, 30, 60 and 300'' were analyzed, respectively (*p<0.05)",
    "molecules": "Hoechst"
  },
  {
    "caption": "(E) Brains dissected from control (FRT82B con) or alix1 (FRT82B, alix1) larvae were immunostained with anti-γ-tubulin (red) and Cnn (white, left panel) or anti-Dgrip91 (red) and α-tubulin (white, right panel), as well as Hoechst (blue). Recombinant NBs are positively marked with GFP (green). The confocal images show typical accumulation of centrosomal γ-tubulin or Dgrip91, and the insets zoom in at the indicated centrosomes. Scale bars, 5 μm. The intensities of γ-tubulin, Dgrip91 and Cnn in control NBs (GFP-negative, set to 100 %) and GFP-positive, recombinant NBs are presented in the table (±S.E), also indicating the number of NBs quantified (n) from three separate experiments (*p<0.05)",
    "molecules": "Hoechst"
  },
  {
    "caption": "(G) GST or GST-ALIX were incubated with in vitro translated 35S-methionine-labelled γ−tubulin or Dgrip91. The upper panel shows the autoradiography indicating interaction between GST-ALIX and radiolabelled γ-tubulin or Dgrip91. The lower panel shows the Ponceau S-stained membrane visualizing the expression of GST and GST-ALIX. The experiment was performed at least three times and a representative result is presented",
    "molecules": "35S-methionine, Ponceau S"
  },
  {
    "caption": "(H) HeLa cells depleted of ALIX or not grown on coverslips were stained with anti-γ−tubulin (white), anti-Pericentrin (red) and Hoechst (blue) before being examined by confocal fluorescence microscopy. The confocal images and insets show the centrosomal accumulation of γ-tubulin and Pericentrin. Scale bars, 5 μm. The graph presents the average centrosomal intensities of γ-tubulin and Pericentrin relative to control cells (set to 100%) calculated from three independent experiments (10 cells per condition/experiment) (±S.E). Significant difference is noted as **p<0.01 (Student's t-test)",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) Dissected brains of w1118, alix1, γ−Tub23CPI and γ−Tub23CPI;alix1 larvae were subjected to MT regrowth assay and stained with anti-α-tubulin (red), anti-Cnn (white) and Hoechst (blue). Representative images of NBs after 0 and 60 seconds regrowth are presented. Scale bars, 5 μm",
    "molecules": "Hoechst"
  },
  {
    "caption": "(E) The relative spindle angle between the polarity axis perpendicular to the apical phalloidin-stained F-actin surface and the MS was determined in metaphase Caco-2 cysts cells transfected with control, ALIX and/or γ-tubulin siRNA (29, 29, 18 and 32 metaphase cells, respectively, were analyzed from three separate experiments). Compared to control cells, depletion of either ALIX or both ALIX and γ-tubulin, induced a significantly increased randomization of the relative spindle angles (**p<0.01 and ***p<0.001, ANOVA). Caco-2 cells depleted of ALIX+γ-tubulin showed a greater variation in the spindle angles compared to depletion of ALIX or γ-tubulin (*p<0.05, ANOVA). ALIX-depleted cells showed greater spindle angle-variation than γ-tubulin-depleted cells (*p<0.05, ANOVA)",
    "molecules": "phalloidin"
  },
  {
    "caption": " (A) HeLa cells transfected with control or ALIX siRNA were fixed, permeabilized and stained with anti-MAP1S (red), anti-γ-tubulin (white) and Hoechst (blue). Interphase cells are shown and the insets show close ups of the centrosomes. Scale bars, 5 μm. (B) The fluorescence intensity of centrosomal MAP1S was determined and the graph shows the average of three independent experiments (± S.E), in which in total 52 control and 36 ALIX siRNA cells were analyzed. Significant difference is indicated as *p<0.05 (Student's t-test) ",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D, E) Cells treated as in C were immunostained with anti-α-tubulin (white), Hoechst (blue) (D Scale bars, 5 μm. The intensities of astral MTs relative to total MTs were determined from confocal microscopy images and the graph in (D) shows the average of four separate experiments (10 cells per condition/experiment) (±S.E). *p<0.05 and **p<0.01, Student's t-test",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D, E) Cells treated as in C were immunostained wit anti-acetylated tubulin (green) (E) The relative intensity of acetylated MT relative to total MT was determined in (E) The average of four separate experiments (10 cells per condition/experiment) is graphically presented (±S.E). Significant differences are indicated as *p<0.05 (Student's t-test)",
    "molecules": "acetyl"
  },
  {
    "caption": "(G) HeLa cells transfected with control or ALIX siRNA grown on coverslips were transiently transfected with pEYFP-C1 (green), pEYFP-MAP1S (green), pEGFP-C1 (green) or pEGFP-CLASP2α and immunostained with anti-α-tubulin (white), anti-Pericentrin (red) and Hoechst (blue). Scale bars, 5 μm",
    "molecules": "Hoechst"
  },
  {
    "caption": "Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available.",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3).",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": " Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1. ",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3).",
    "molecules": "APR-246, DNA, MK-571, propidium iodide"
  },
  {
    "caption": "OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3).",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1.",
    "molecules": "APR-246, Reversan"
  },
  {
    "caption": "Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B.",
    "molecules": "APR-246"
  },
  {
    "caption": "Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.",
    "molecules": "APR-246"
  },
  {
    "caption": "Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.   Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of >1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.   ",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint (5 days) or post treatment / late timepoint (>22 days) after treatment initiation with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Dots indicate individual tumors.",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": " Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post treatment / late timepoint (>22 days after treatment initiation) with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). ",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Growth suppression determined by the ATP-based CTG assay in colorectal cancer patient-derived organoids (colo-PDO) after treatment with APR-246 +/- MK-571 (n = 3).",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "IC50 values (µM) APR-246 +/- 20 μM MK-571 in indicated PDOs. *p = 0.04, paired t test (n = 3 except colo-PDO2 and eso-PDO3 where n = 2).",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Organoid area (in black, left y-axis) and PI intensity (in red, right y-axis) of colo-PDO1 and colo-PDO2 as determined by image analysis 72h after treatment with APR-246 +/- MK-571 (n = 3).",
    "molecules": "APR-246, MK-571, PI"
  },
  {
    "caption": "14C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3.",
    "molecules": "APR-246, 14C, MK-571"
  },
  {
    "caption": "14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure).",
    "molecules": "APR-246, 14C, MK-571, Reversan"
  },
  {
    "caption": "14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.",
    "molecules": "APR-246, 14C"
  },
  {
    "caption": "APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Glutathione-conjugated MQ (GS-MQ) in OVCAR-3 cells after 24h treatment with APR-246 +/- MK-571 (n = 2). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.",
    "molecules": "MQ, APR-246, Glutathione, GS, MK-571"
  },
  {
    "caption": "Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.",
    "molecules": "MQ, GS, NAC"
  },
  {
    "caption": "Amount of GS-MQ by LC-MS (green line, left axis) and NAC-MQ (purple line, right axis) over time after incubation of GS-MQ with NAC (n = 2). Values on y-axes are not comparable because GS-MQ and NAC-MQ have different response signals on MS. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.",
    "molecules": "MQ, GS, NAC"
  },
  {
    "caption": "Mean IC50 (µM) values of APR-246 in 17 different cell lines (n ≥ 3) as shown by WST-1. *p = 0.03, Mann Whitney test. Mean IC50 (µM) values for APR-246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.",
    "molecules": "APR-246"
  },
  {
    "caption": "Box-and-whisker plot of Pearson correlations between PRIMA-1 area-under-the-curve (AUC) and the levels of 225 different metabolites from the DepMap portal. Central band indicates median, boxes indicate 25th and 75th percentile, and whiskers show 2.5th and 97.5th percentile outlier metabolites. High GSH and GSSG (in red) correlate with low PRIMA-1 sensitivity. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.",
    "molecules": "GSH, GSSG, PRIMA-1"
  },
  {
    "caption": " Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. ",
    "molecules": "doxycycline, glutathione, GSH, GSSG, tet"
  },
  {
    "caption": " Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. ",
    "molecules": "glutathione, GSH, GSSG"
  },
  {
    "caption": "Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.",
    "molecules": "Thiol"
  },
  {
    "caption": " Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. ",
    "molecules": "APR-246, 14C"
  },
  {
    "caption": " 14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. ",
    "molecules": "APR-246, 14C, doxycycline"
  },
  {
    "caption": " Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5. ",
    "molecules": "APR-246, doxycycline"
  },
  {
    "caption": " Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n. ",
    "molecules": "glutathione, GSH, GSSG, MK-571"
  },
  {
    "caption": "Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3).",
    "molecules": "APR-246, glutathione, GSSG, MK-571"
  },
  {
    "caption": " Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3). ",
    "molecules": "APR-246, glutathione, GSH, MK-571"
  },
  {
    "caption": " Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h. ",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": "Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h.",
    "molecules": "APR-246, MK-571"
  },
  {
    "caption": " Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3). ",
    "molecules": "APR-246, CySS, cystine, MK-571"
  },
  {
    "caption": " Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3). ",
    "molecules": "APR-246, Cys, cysteine, MK-571"
  },
  {
    "caption": " 14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D. ",
    "molecules": "APR-246, 14C, MK-571"
  },
  {
    "caption": " Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C. ",
    "molecules": "APR-246"
  },
  {
    "caption": " Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3). ",
    "molecules": "glutathione, GSH, GSSG"
  },
  {
    "caption": " Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots). ",
    "molecules": "glutathione, GSH, GSSG, MK-571, SSZ, sulfasalazine"
  },
  {
    "caption": " 14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D. ",
    "molecules": "APR-246, 14C, MK-571, SSZ, sulfasalazine"
  },
  {
    "caption": " Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots) ",
    "molecules": "APR-246, MK-571, SSZ, sulfasalazine"
  },
  {
    "caption": " OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3). ",
    "molecules": "APR-246, MK-571, SSZ, sulfasalazine, sulfasalzine"
  },
  {
    "caption": "K. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media (PM) after isolation. L. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media (DM). hiPSC donor 1.",
    "molecules": "Vtn"
  },
  {
    "caption": "(A) HeLa cells transfected with His-tagged HTTex1p (46Q) or 46QP-K6,9,15R (3R) along with GFP-SUMO-1 or GFP-SUMO-2, lysed under denaturing conditions, and nickel purified (Ni-NTA). Unmodified HTT-46Q is indicated by the arrow and SUMO-modified HTT by the boxed region. The lysine mutant (3R) serves as a negative control. Ni-NTA represents nickel-purified His-tagged HTT, and WC TCA represents 10% of the whole-cell lysate expression of HTT and myc-actin (transfection control). HTT is modified by SUMO-1 (left) and SUMO-2 (right).",
    "molecules": "nickel"
  },
  {
    "caption": "(C) Titration of SUMO-1. Denaturing nickel purification of HTTex1p (46QP-H4) following transfection with decreasing amounts of SUMO-1 reduces the amount of SUMO-modified HTT to undetectable levels. The Ni-NTA blot displayed in the gray scale shows purified HTTex1 and SUMO-modified HTTex1 using HTT antibody. Note that 0.5 μg of SUMO-1 (¼ the amount of SUMO-1 cDNA ) was used for identifying the SUMO-1 E3 ligase for HTTex1p. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100. Note that all experiments were performed in triplicate, and a representative figure is shown.",
    "molecules": "nickel"
  },
  {
    "caption": "(C) Left panel is the autoradiography results of a GST pull-down assay showing that radiolabeled human PIAS1 interacts with HTTex1p. Right panel is a phosphorimager analysis of GST pull-downs, performed in triplicate, showing the percentage of 35S-labeled PIAS protein that bound the GST proteins: GST alone, unexpanded HTT with and without the proline-rich region (20QP and 20Q), expanded HTT with and without the proline-rich region (51QP and 51Q), and the proline-rich region alone (Pro). Error bars were calculated as an estimate of SE = STDEV(n)/SQRT(n-1).",
    "molecules": "35S"
  },
  {
    "caption": "(C) Bait plasmids (HTT-586-25Q or HTT-586-73Q aa) were transformed into the L40ccua MATa yeast strain. Yeast clones encoding bait proteins were individually mated against a matrix of MATα yeast clones encoding 16,888 prey proteins (with Gal4 activation domain fusions) using pipetting and spotting robots. Diploid yeasts were spotted onto SDIV (-Leu-Trp-Ura-His) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β-galactosidase assays. After 5-6 days of incubation at 30ºC, digitized images of the agar plates and nylon membranes were assessed for growth and β-galactosidase activity using the software Visual Grid (GPC Biotech).",
    "molecules": "agar, Leu, Trp, Ura"
  },
  {
    "caption": "(E) HeLa cells overexpressing either expanded 586 aa-HTT or the phosphomimetic (S13,16D = DD) with SUMO-2 plus and minus PIAS1. HTT was purified by IP using hydrazide beads (Bioclone) crosslinked to HTT (Enzo) antibody and subjected to western analysis using anti-HTT (MAB5490). Arrows indicate SUMO-2-modified HTT.",
    "molecules": "hydrazide"
  },
  {
    "caption": "(F) IP of HTT with hydrazide-linked beads shows that both unexpanded and expanded HTT 586 aa phosphomimetics (S13, 16D-586 aa) are modified by SUMO-2. Arrows indicate SUMO-conjugated HTT. Note that all experiments including the Y2H assay were done in triplicate; representative experiments are shown. Arrows indicate SUMO-2-modified HTT.",
    "molecules": "hydrazide"
  },
  {
    "caption": "(A) Western analysis of whole-cell lysates from HeLa cells transfected with His-SUMO-1 or SUMO-2 and/or 97Q-HTT exon 1 and treated with 5 μM MG132 for 18 hr. Lysates were separated using differential centrifugation into a detergent-soluble fraction (SOLUBLE) with 1% Triton X-100 and a detergent-insoluble fraction (INSOLUBLE) with 4% SDS. Western blot probed with anti-HTT shows full-length endogenous HTT in the SOLUBLE fraction (upper arrow) and 97Q-HTTex1 (lower arrow) (left panel). In the INSOLUBLE fraction, HTT HMW species are indicated by the bracket and asterisks (right panel).",
    "molecules": "MG132, SDS, Triton X-100"
  },
  {
    "caption": "(B) MG132 and SUMO-2 cause mutant HTT to accumulate as HMW species (bracke and asterisk). Western blot showing IP with HTT antibody crosslinked beads from the detergent-insoluble fraction probed with the anti-HTT antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Mutant HTT (97Q-Httex1) fibrils are detected with anti-HTT in the insoluble fraction with treatment of MG132 or addition of exogenous SUMO-2.",
    "molecules": "MG132"
  },
  {
    "caption": " (B) Expression of S protein on the plasma membrane of Jurkat-S cells assessed by surface biotinylation and followed by immunoprecipitation with anti-T2A, SDS-PAGE under reducing conditions and western blot with streptavidin-peroxidase. Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control. The arrow indicates the position of the reduced S protein. (C) Expression of the S protein in native form was assessed by surface biotinylation of Jurkat-S and Jurkat cells followed by immunoprecipitation with anti-T2A followed by SDS-PAGE under non-denaturing conditions (i.e. without reducing agents and without boiling). The nitrocellulose membrane was blotted with streptavidin-peroxidase. Arrows point at different oligomerization forms of the S protein. ",
    "molecules": "peroxidase, streptavidin"
  },
  {
    "caption": " (D) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells was measured by flow cytometry by analyzing the percentage of cells that become double positive for CTFR and CFSE markers. The Bar plot to the right shows the effect of different doses of HepG2 cells on the formation of syncytia with a fixed number of Jurkat. Parental Jurkat cells (not expressing S protein) are considered negative controls. Data represent the mean±SD of triplicated datasets. ",
    "molecules": "CTFR, CFSE"
  },
  {
    "caption": " (E) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells, in an experiment as in D, was confirmed by confocal microscopy after sorting cells double positive for CTFR and CFSE. Nuclei were identified by DAPI staining. Red arrowhead in the DAPI image indicates a nucleus of Hep-G2 origin; the green arrowhead the nucleus of Jurkat-S origin. ",
    "molecules": "CTFR, CFSE, DAPI"
  },
  {
    "caption": " (F) Overlay plot of Jurkat-S cells that were incubated with anti-EGFR mAb conjugated to Bv421 and serum from either donor #15 or from a pre-COVID-19 donor and followed by a secondary anti-human IgG1 antibody conjugated to PE. ",
    "molecules": "Bv421"
  },
  {
    "caption": "(A) Survival curves of C57BL/6J naïve and adrenalectomized (Adx) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 30 µg (naïve) or 2 µg (Adx) TNF, solved in sterile PBS, per 20 g body weight. No deaths occurred later than 72 h after TNF injection. Results represent combined data of 3 experiments.",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(B) Corticosterone levels in the serum of C57BL/6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water (N=6 per group).",
    "molecules": "Corticosterone, ZnSO4"
  },
  {
    "caption": "(C) Survival curves of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 50 µg (GRWT) or 12.5 µg (GRDim) TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 50 h after TNF injection. Results represent combined data of 2 experiments.",
    "molecules": "ZnSO4"
  },
  {
    "caption": "Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of C57BL/6J naïve and Adx mice (D, N=3-6),",
    "molecules": "Zinc, ZnSO4"
  },
  {
    "caption": "Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRWT and GRDim mice (E, N=6-9).",
    "molecules": "Zinc, ZnSO4"
  },
  {
    "caption": "(F) Agtpbp1 gene-expression measured with RT-qPCR in the ileum of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water (N=3 per group).",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(G) Survival curves of GRfl/fl and GRVillKO mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 35 µg TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 150 h after TNF injection. Results represent combined data of 6 experiments.",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(I) Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRfl/fl and GRVillKO mice (N=9 per group).",
    "molecules": "Zinc, ZnSO4"
  },
  {
    "caption": "(D) Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT and GRDim mice, as measured by RNA-seq in ileum samples. All genes, induced in ileum by DEX in GRWT with LFC>0.8, p<0.05 are considered, while these genes with p<0.05 in GRDim were considered. The slope of the correlation curve as well as Pearson correlation coefficient were calculated by Graphpad Prism.",
    "molecules": "DEX, ZnSO4"
  },
  {
    "caption": "(E) C57BL/6J mice received antibiotics (AB) in the drinking water for 3 weeks, followed by 1 week 25 mM ZnSO4 in the drinking water. During this week AB administration was continued by oral gavage. Mice were challenged i.v. with 12.5 µg TNF solved in sterile PBS and survival recorded. Combined data of 2 experiments.",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(F) Zinc levels measured in serum after the antibiotics and ZnSO4 protocol (N=5-8).",
    "molecules": "Zinc, ZnSO4"
  },
  {
    "caption": "Mice housed in germ-free (GF) or specific pathogen free (SPF) conditions received 25 mM ZnSO4 in the drinking water for 1 week and were then challenged i.p. with 35 µg TNF, dissolved in sterile PBS, per 20 g bodyweight. Combined data of 2 experiments.",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(H) displays the blood zinc levels in SPF and GF mice treated for 1 week with water or ZnSO4 (N=3/4 per group).",
    "molecules": "zinc, ZnSO4"
  },
  {
    "caption": "(I) Ileum Slurry (IS) was isolated from C57BL/6J mice put on 25 mM ZnSO4 or control water for 1 week and 750 µL of this IS was injected i.p. in C57BL/6J GF mice.",
    "molecules": "ZnSO4"
  },
  {
    "caption": "Transcriptional downregulation of ISRE/IRF genes in the intestinal epithelium by zinc part 1. RNA-seq experiment of GRWT and GRDim mice which received normal drinking water or drinking water containing 25mM ZnSO4 for 1 week. Ileum was sampled and RNA-seq performed (N=5-6 per group). Genes downregulated by zinc compared to water control animals (LFC<0.8 and p<0.05) were considered. (C) Heat map of a selection of the 48 genes of Fig 3B.",
    "molecules": "zinc, ZnSO4"
  },
  {
    "caption": "(A-B) Expression levels (RT-qPCR data) of Mlkl and Zbp1, from the collection of 48, in ileum biopsy samples, showing the reducing effects of zinc in both GRWT and GRDim mice, and the higher expression in GRDim compared to GRWT (N=4-6/group).",
    "molecules": "zinc"
  },
  {
    "caption": "RT-qPCR data of Mlkl , in ileum biopsy samples of C57BL/6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week, either or not pretreated with antibiotics (AB) for three weeks. (N=5 per group)",
    "molecules": "ZnSO4"
  },
  {
    "caption": "RT-qPCR data of Zbp1, in ileum biopsy samples of C57BL/6J mice treated with normal drinking water or 25 mM ZnSO4 in the drinking water for 1 week, either or not pretreated with antibiotics (AB) for three weeks. (N=5 per group)",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only. 6h later, sections through ileum were made and stained with (A) Hematoxylin & Eosin and MMP7, a Paneth cell marker. Pictures at magnification of 20x were taken and one representative picture of each condition is shown. Arrows point to crypts and their (ab)normal occurrence (see text). Scale bars: 50 μm",
    "molecules": "Eosin, Hematoxylin, ZnSO4"
  },
  {
    "caption": "(C) Transmission electron microscopy (TEM) images of ileum crypts show protective effects of zinc therapy on the level of TNF induced effects on Paneth cells. C57BL/6J mice (N=2 /group) were put on normal drinking water or on 25 mM ZnSO4 water for 7 days. Mice were given 50 µG TNF or PBS and 8h later, ileum samples were taken and prepared for TEM. Figure shows ileal crypts at a magnification of 1000x. In each of the 4 conditions, 2 Paneth cells are selected in red area",
    "molecules": "zinc, ZnSO4"
  },
  {
    "caption": "(A-B) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days and were injected i.p. with 50 µg TNF, solved in 200 µL sterile PBS, per 20 g bodyweight or with PBS only (N=5). 18h after TNF challenge, spleen and MLNs were isolated and lysates prepared and plated on TSA plates. Colonies were counted and expressed as bacterial colonies per ml of lysate. 40 colonies per plate were prepared for Maldi Tof identification and the relative amounts of bacterial species found on the plates are mentioned underneath the figure. Data are shown as means +/- SD. p-value was analyzed by means of a Student's t-test (unpaired, two-tailed).",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(E) Impact of zinc concentration in bacterial TSA plates on the growth of colonies of 4 bacterial species commonly found in spleen and MLNs of mice injected with TNF (see A-B). Pure colonies of Staphylococcus nepalensis (orange), Staphylococcus sciuri (red), Eschericchia coli (purple) and Enterococcus faecalis (blue) were grown in LB medium, then diluted and plated on TSA plates containing increasing concentrations of ZnCL2. 24h later, the amounts of colonies were counted, and the amounts found in the 0 mM ZnCl2 condition expressed as 100%. Data are shown as mean +/- SD pooled from three independent experiments.",
    "molecules": "zinc, ZnCL2, ZnCl2"
  },
  {
    "caption": "Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri or 108 S. nepalensis (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected, and 24h later body temperature (insert in F) and lethality (F) recorded and estimated by one-way ANOVA and Chi2-tests resp. Survival significance was tested towards the H20 control group.",
    "molecules": "H20, ZnSO4"
  },
  {
    "caption": "Monocolonization experiment. Mice (N=5 all groups) were treated with H20 or ZnSO4 for 7 days, after which the ZnSO4 was removed and replaced by H20. 12h and 24h later, the mice were transplanted with PBS, 108 Staphylococcus sciuri (in 100 ul), and 3h later a lethal dose of TNF (35 µg/mouse) was injected In a second experiment (G-I) (N=4 all groups) with S. sciuri colonization, Zbp1 and Stat1, two key ISRE genes, and Mt2, a Zn-induced gene, were measured in ileum biopsies by RT-qPCR.",
    "molecules": "ZnSO4"
  },
  {
    "caption": "(A) C57BL/6J mice received 25 mM ZnSO4 or control water for 7 days. Mice were injected i.p. with 100, 200 or 500 µg dexamethasone (Dex, rapidexon), solved in sterile PBS, 30 min before a 50 µg TNF/20 g bodyweight i.p. challenge. STAT1+/+ and STAT1-/- mice were challenged with equal TNF dose, as controls. No mice died later than 120 h after TNF injection. Data show combined results of 2 experiments.",
    "molecules": "Dex, dexamethasone, rapidexon, ZnSO4"
  },
  {
    "caption": "B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG-tag alone (FLAG) were either not crosslinked (-), UV crosslinked (UV) or treated with 5-azacytidine (5-AzaC). The protein-RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and exposed to an X-ray film.",
    "molecules": "5-AzaC, 5-azacytidine, RNA"
  },
  {
    "caption": "G. 5-AzaC crosslinking was performed and RNA associated with wildtype NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA analysed by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.",
    "molecules": "tRNA, 5-AzaC"
  },
  {
    "caption": "A. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosyl methionine as a methyl group donor and in vitro transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me).",
    "molecules": "EtBr, ethidium bromide, tRNA"
  },
  {
    "caption": "B. The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC crosslinking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar.",
    "molecules": "tRNA, 5-AzaC"
  },
  {
    "caption": "D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long).",
    "molecules": "tRNA"
  },
  {
    "caption": "E. In vitro methylation assay of in vitro transcribed mt-tRNAMet and chemically synthesized mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A).",
    "molecules": "tRNA"
  },
  {
    "caption": "B. In vitro methylation assays using [3H-methyl]-labelled S-adenosyl methionine, the in vitro transcripts of the mt-tRNAMet mutants described in (A) and recombinant His14-MBP-NSUN3. RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr), dried and exposed to an X-ray film to detect methylated transcripts (3H-Me).",
    "molecules": "EtBr, ethidium bromide, tRNA"
  },
  {
    "caption": "B. HEK293 cells expressing ABH1-HisPrcFLAG (ABH1) or the HisPrcFLAG-tag alone (FLAG) were UV crosslinked (UV) and protein-RNA complexes were affinity purified. Co-precipitated RNA was isolated and analysis by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.",
    "molecules": "tRNA"
  },
  {
    "caption": "A. In vitro transcribed mt-tRNAMet was methylated at C34 using recombinant NSUN3 and 3H-labelled S-adenosyl methionine as a methyl group donor. Radiolabelled mt-tRNAMet was re-extracted and then subjected to oxidation assays without protein (-), with maltose binding protein (MBP), with the dioxygenase FTO or using wildtype (ABH1) or mutant (R338A, D233A) His14-MBP-ABH1. Besides ABH1 controls lacking a-ketoglutarate (KG) or Fe2+ ions, all samples contained a-ketoglutarate and Fe2+ ions. After oxidation, RNA was precipitated and tritium released upon oxidation of radiolabelled mt-tRNAMet was quantified in the supernatant and counts per minute (CPM) are shown for experiments performed in triplicate with error bars indicating ± SD (upper panel). Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X-ray film to analyse the tritium retained (3H-Me).",
    "molecules": "Fe2+, tRNA, a-ketoglutarate"
  },
  {
    "caption": "C. Anion exchange HPLC analysis was performed on synthetic m5C containing ASL (20nt) before and after oxidation by ABH1. The small shift in retention time indicates formation of f5C modified RNA. The ABH1 oxidation product was then treated with NaBH4 to generate hm5C-modified RNA. All three samples were analysed by ESI-MS and the molecular weight (m.w.) is indicated on the HPLC trace. Only the f5C-containing RNA was labelled efficiently with 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate (TMI).",
    "molecules": "1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate, TMI, NaBH4"
  },
  {
    "caption": "A. MTIF2-dependent reading of initiation codons AUG, AUA or AUU in the P site of the ribosome by unmodified (unmod.) or C34-modified [14C]Met-tRNAMet. Binding was determined by nitrocellulose filtration and [14C]Met-tRNAMet retrieved on the membrane was quantified by scintillation counting. Binding in the absence of ribosomes (no 70S) or mRNA (no mRNA) served as controls. Data from three independent experiments are presented with error bars indicating ± SEM. The statistical significance of the results was analysed by T-test and is indicated by the asterisks in the graph.",
    "molecules": "tRNA, mRNA"
  },
  {
    "caption": "B. Chemically synthesised f5C modified mt-tRNAMet and total RNA from wild-type (WT) cells or those transfected with siRNAs as in (A) were treated with TMI to specifically label f5C residues. Primer extension, using a radiolabelled antisense primer, was performed under limited dNTP conditions. Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager.",
    "molecules": "TMI, tRNA, RNA"
  },
  {
    "caption": "C. Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B. Stops corresponding to position C34 in mt-tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD.",
    "molecules": "tRNA, RNA"
  },
  {
    "caption": "D. RNA from wild-type HeLa cells and cells treated with siRNAs against NSUN3 or ABH1 (as in A) was either first reduced with NaBH4 or directly treated with bisulfite. After deamination and desulfonation, mt-tRNAMetRNAs were reverse transcribed, amplified, cloned and sequenced. The proportions of thymine (grey) generated by bisulfite-conversion or non-converted cytosine (black) at position 34 of mt-tRNAMet are shown. Note that for sequences from non-reduced samples, thymine can originate from unmodified or f5C-containing mt-tRNAMet, while in reduced samples it originates from unmodified cytosine.",
    "molecules": "tRNA, RNA, NaBH4"
  },
  {
    "caption": "A. (Top) USP44 transcript levels across CD4+ T cell populations. Suspension of the lymph node and spleen cells of 6-8 week old female C57BL/6 mice (n= 5 experiments) were obtained, and naïve (CD62Lhigh/CD25-) CD4+ T cells and nTregs (CD4+/CD25high) were obtained by FACS. The indicated effector T cell populations were generated by activating 1x10^6 naïve CD4+ T precursors with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days under the indicated in vitro skewing conditions (described in the Methods section). After harvesting RNA by Trizol reagent and generating cDNA, the Usp44 mRNA levels expressed by the resulting Teff and iTreg, as well as freshly isolated nTreg cells were determined by qRT-PCR. (Bottom) Suspension of lymph node and spleen cells of 6-8 week old female C57BL/6 mice were obtained, and naïve (CD62LhighCD25-) CD4+ T cells and nTregs (CD4+/CD25high) were sorted by FACS. iTregs were generated by activating 1x10^6 naïve CD4+ T cells with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days in the presence of IL-2 (100u/ml) and TGF-β (5ng/ml) before the cells were harvested for SDS-PAGE. Panels A and B depict the mean mRNA expression normalized to the housekeeping gene GAPDH.",
    "molecules": "Trizol"
  },
  {
    "caption": "D. Murine iTregs generated in vitro as above were activated in the presence of recombinant TNFα, IL-6, or IL-1β (each at a 10 ng/ml concentration), or LPS (1µg/ml) for 24 hours. Treated Tregs were then harvested and USP44 protein levels were determined as above.",
    "molecules": "LPS"
  },
  {
    "caption": "A. HEK293T cells were transfected with MYC-FOXP3 and FLAG-USP44 encoding expression constructs using Polyethylenimine. 48hrs post-transfection, cells were harvested, lysed, and anti-FLAG or anti-MYC antibody coated beads were used to immunoprecipitate the given labeled protein along with its binding partner. Co-IP'ed proteins were subjected to SDS PAGE followed by immunoblot analysis. Antibodies recognizing FLAG or MYC tags were used to probe for USP44 and FOXP3, respectively.",
    "molecules": "Polyethylenimine"
  },
  {
    "caption": "D. Naïve murine CD4+ T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl/fl CD4Cre+ mice and that of their wild type littermates (USP44fl/fl CD4Cre- mice; n=2-3/group/experiment). iTreg cells were generated from these mice as described for Fig. 1 before incubation on a microscope slide pre-coated with poly-L lysine for 1h. Adhered cells were then fixed by PFA for 0.5 followed by blocking with 1% BSA for 1h, then incubation with the specified antibodies. Representative confocal microscopy images (40X) were visualized for endogenous USP44 (red) and FOXP3 Baxter et al(). DAPI was used to visualize cell nuclei (blue); scale bar 50μm.",
    "molecules": "DAPI, PFA, poly-L lysine"
  },
  {
    "caption": "A. HEK293T cells were transfected with expression constructs encoding MYC-FOXP3, His-Ubiquitin, and either a wild type or catalytically inactive (C282S, "cs") version of USP44 tagged with FLAG. Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis. Pull-down of His-labeled proteins using Ni-NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC.",
    "molecules": "MG132, Ubiquitin"
  },
  {
    "caption": "B. Effect of USP44 knock down on FOXP3 ubiquitination. Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells. After selected for 7d, cells were transfected with MYC-FOXP3 and His-Ubiquitin, then treated with 20 μM MG132 for 4 hrs before harvest and lysis. Ubiquitinated FOXP3 proteins were visualized as in A.",
    "molecules": "G418, MG132, Ubiquitin"
  },
  {
    "caption": "C. Levels of polyubiquitinated FOXP3 in the presence or absence of USP44. As in Fig 2b, murine iTregs were generated from naïve CD4+ precursors isolated from wild type and mice globally deficient in USP44-/- mice (n=3/group/experiment). Ubiquitinated proteins were extracted from iTreg lysates with anti-ubiquitin antibodies (anti-Ubi) prior to resolution by SDS PAGE and immunoblot analysis, probing for FOXP3.",
    "molecules": "Ubi, ubiquitin"
  },
  {
    "caption": "D, E. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, FLAG-USP44 (either wild type USP44, "wt" or the C282S mutant). These cells were subsequently treated with cycloheximide (CHX; 5μg/ml) for the indicated time points before harvested and cell lysis. FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti-MYC antibodies.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "C. Effect of USP44 and USP7 coexpression on FOXP3 ubiquitination. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, either FLAG-USP44 or FLAG-USP7 or both, His-ubiquitin. Cells were treated with 20 μM MG132 for 4 hrs before being harvested, lysed, and incubated on Ni-NTA beads to pull down ubiquitinated FOXP3, which was visualized by immunoblotting using antibodies specific for MYC.",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "A. Jurkat T cells were transfected with the indicated combinations and relative doses of expression constructs encoding MYC-FOXP3, FLAG-USP44 along with an IL-2 promoter-driven firefly luciferase reporter. After 8 hours stimulation with PMA/Inomycin, cells were lysed and luciferase activity, which was observed and normalized to renilla luciferase activity.",
    "molecules": "Inomycin, PMA"
  },
  {
    "caption": "C, D. Analysis of the in vitro suppressive function of Tregs isolated from WT mice or age- and sex-matched USP44-/- mice (n=3/group/experiment). Tregs were mixed with CFSE-labeled responder naïve T cells from CD45.1+ C57BL/6 mice (n=3/experiment) for three days. CFSE dilution in CD45.1+ T cells was observed by flow cytometry and the percent suppression of responder cell proliferation by the co-cultured Tregs was determined.",
    "molecules": "CFSE"
  },
  {
    "caption": "B. Representative photomicrographs of the distal colon of Rag2-/- mice after T cell transfer. 10 weeks after transfer, mice were euthanized, and colons were harvested, fixed in 10% buffered formalin, and processed for standard H&E staining prior to histological analysis. (i)- (iv) present bright-field micrographs (100x).",
    "molecules": "formalin"
  },
  {
    "caption": "G. CD4+ T cells were recovered from suspensions of lamina propria-infiltrating leukocytes. These lymphocytes were stimulated ex vivo by PMA/Ionomycin cocktail in the presence of brefeldin A and IFN-γ and IL-17 production by these lymphocytes was analyzed by ELISA.",
    "molecules": "brefeldin A, Ionomycin, PMA"
  },
  {
    "caption": "E, F. Frequencies of CD4+ and CD8+cells producing the proinflammatory cytokine IFNγ were found among the tissues of tumor-bearing mice in the MC38 modelby intracellular cytokine staining Kralovics et al(, 2005) and flow cytometry after ex vivo re-stimulation of recovered leukocytes by PMA and ionomycin in the presence of brefeldin A.",
    "molecules": "brefeldin A, ionomycin, PMA"
  },
  {
    "caption": "(A) Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels2) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.",
    "molecules": "oleate"
  },
  {
    "caption": "HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM.",
    "molecules": "Dynole 31-2, MiTMAB, DMSO, Dynasore, Dynole 34-2, oleate"
  },
  {
    "caption": "(C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01.",
    "molecules": "4-hydroxy-tamoxifen"
  },
  {
    "caption": "(E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure.",
    "molecules": "leupeptin"
  },
  {
    "caption": "(F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.",
    "molecules": "DMSO, Dynasore, leupeptin"
  },
  {
    "caption": "Dyn2 knockdown results in the formation of enlarged autophagic structures. (A-D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (<1 µm) electron-dense lysosomes and autolysosomes (arrowheads). Under conditions in which Dyn2 expression is suppressed (B-D), far fewer small lysosomes are observed. Instead, the cells are populated by larger autolysosomes (≥1 µm) with aberrant morphologies and containing putative LDs (*). (E) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells. Values represent the fold-increase in the number of autophagic structures of a given diameter (measured in microns) observed over siNT-treated control cells. Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition.",
    "molecules": "oleate"
  },
  {
    "caption": "Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A-D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A-D): 20 µM; (A′-B′) 2 µM; (C′-D′) 10 µM. (E-G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.",
    "molecules": "DMSO, Dynasore"
  },
  {
    "caption": "(A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5. Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8-27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria).",
    "molecules": "Dynasore"
  },
  {
    "caption": "(C) Equilibrium dissociation curves of FITC-RBBP6  from eVP30130-272 in the presence of increasing concentrations (0.13-500 μM) of the PPxPxY containing peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation.",
    "molecules": "FITC"
  },
  {
    "caption": "(B) Equilibrium dissociation curves of  FITC-RBBP6 from eVP30130-272 as it is outcompeted by increasing concentrations (0.13-500 μM) of RBBP6 alanine mutants. Fluorescence polarization  was determined with constant concentrations of FITC-RBBP6 and eVP30130-272 at 0.50 µM and 3.8 µM respectively. Experiments were performed in two independent replicates. Error bars represent the standard deviation. ",
    "molecules": "alanine, FITC"
  },
  {
    "caption": "(A)  HeLa cells were transfected with scrambled siRNA (SCR si) or siRNAs targeting  hnRNP L, hnRNPUL1  or PEG10, 48 h post-transfection, cells were challenged with EBOV-EGFP at an MOI of 0.5. 24 h post-infection, cells were fixed, stained  for nuclei with Hoechst dye and imaged. Scale bar is 200 µm.The graph (right) depicts relative number of infected cells as determined by calculating the ratio of  number of infected cells to  nuclei  and shown relative to SCR siRNA. The data shows a representative result of two independent experiments with the mean ± S.D. calculated from three independently treated wells. Statistical significance was calculated relative to the values obtained in SCR siRNA-treated cells using ANOVA with Dunnett's multiple comparisons test. ***p<0.0001, ***p<0.001, **p<0.01",
    "molecules": "Hoechst dye"
  },
  {
    "caption": "(D) Immunoblots to assess the levels of VP30, pVP30  and NP in EBOV infected lysates upon knockdown of NPC1 or  RBBP6. HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6. 48 h post-transfection cells were infected with  EBOV  at MOI=0.1. At 24 h post-infection, cells were lysed in TRIzol. Protein was extracted from the TRIzol  reagent and analyzed by Western blotting to determine levels of VP30, pSer29  VP30 and  NP.",
    "molecules": "TRIzol"
  },
  {
    "caption": "LPS-primed WT and Vdr-/- BMDMs  were infected with 50 MOI S. Typhimurium  for 2 h. Culture supernatants  (SN) and cell lysates(Lysate) were collected and immunoblotted  with the indicated antibodies（A）.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed WT and Vdr-/- BMDMs  were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) were collected and (B-E) IL-1β  secretion(B), IL-18  secretion (C) and TNF-ɑ secretion (D) and LDH  release(E) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed WT and Vdr-/- BMDMs  were infected with 50 MOI S. Typhimurium for 2 h. (F) HBVLV-Vdr  expression in Vdr-/- BMDMs  by lentivirus-mediated transduction primed with LPS  and infected with 50 MOI S. Typhimurium for 2 h, cell lysates, and culture supernatants  (SN) were collected and immunoblotted  with the indicated antibodies.",
    "molecules": "LPS"
  },
  {
    "caption": "(G) LPS-primed WT and Vdr-/- BMDMs  were treated with 1 µg/ml FLIC  (LFn-Flagellin  and PA) or PrgJ  （1 µg/ml LFn- PrgJ  and PA）for 1 h, total mixtures( culture supernatants  and cell lysates) were collected and immunoblotted  with the indicated antibodies.",
    "molecules": "LPS"
  },
  {
    "caption": "(H) LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs  were treated with FLIC  for 1 h, Culture supernatants  (SN) and cell lysates  were collected and immunoblotted  with the indicated antibodies.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs  were treated with FLIC  for 1 h, Culture supernatants (SN) were collected and (I-L) IL-1β secretion(I),  IL-18 secretion (J) and TNF-ɑ secretion (K) and LDH  release(L) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs  were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants  (SN) and Cell lysates were collected and immunoblotted   with the indicated antibodies(A).ELISA",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs  were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants  (SN) were collected antibodies(A).ELISA  detected  TNF-ɑ(B)andIL-1β (C) in supernatants. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs  were infected with S. Typhimurium Bacterial burden was detected in spleens(D), PCF(E), and blood  (F)of WT, Vdr-/-, NLRP3-/-, and NLRP3-/- Vdr-/- mice  24 h after S. Typhimurium infection. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.",
    "molecules": "LPS"
  },
  {
    "caption": "A LPS-primed WT and Vdr-/- BMDMs  were infected with  S. Typhimurium at an MOI of 50 for 2 h. Endogenous ASC  specks (arrows) were represented and quantified by immunofluorescence  images. The data show representative results from three independent experiments: scale bar, 20 μm. Data are presented as the mean ± SEM; determined by Student's t-test ; ***,p < 0.001.",
    "molecules": "LPS"
  },
  {
    "caption": "B ASC oligomerization  induced by the indicated stimuli in WT and Vdr  −/− BMDMs  primed with LPS.",
    "molecules": "LPS"
  },
  {
    "caption": "LPS-primed BMDMs  were infected with S. Typhimurium at an MOI of 50 for 2h. Lysates were immunoprecipitated  (IP) using an anti-VDR  antibody and were immunoblotted  with the indicated antibodies(A). Whole-cell lysates were shown as the input. Data are representative of three independent experiments.",
    "molecules": "LPS"
  },
  {
    "caption": "(D) Purified GST-VDR  was incubated with purified His-NLRC4  for 2 h. His-NLRC4- bound to GST-VDR  was pulled down by glutathione  beads and subjected to immunoblot  analysis. Data are representative of three independent experiments.",
    "molecules": "glutathione"
  },
  {
    "caption": "(F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ-32P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate was used as a positive control.",
    "molecules": "[γ-32P] ATP"
  },
  {
    "caption": "Cytoplasmic chromatin fragments in senescent cells. (A) Cytoplasmic chromatin fragments (CCFs) in senescent cells are strongly positive for histone H3. Yellow arrow marks CCF. (B) Increased proportion of cells with CCFs in RS or OIS. Mean ± SEM, n = 3; P < 0.01 (OIS), P < 0.002 (RS). Assays were performed 10 d after induction with tamoxifen (OIS) or 4 wk after the last passage and >90% SA β-gal+ (RS).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(C) Isolated nuclei of senescent cells are permeable to both 70- and 500-kD dextrans, indicating impairment of the barrier function of nuclear envelope. Dark nuclei exclude fluorescent dextran. Bars, 10 µm. (D) Quantitation of results from C. Mean ± SD, n = 4; P < 0.000001 for both 70- and 500-kD dextrans.",
    "molecules": "dextran, dextrans"
  },
  {
    "caption": "(E) Representative OIS SAHF+ nucleus showing dextran permeability. Bars, 10 µm. (F) Quantitation of dextran 70 uptake in SAHF+ and SAHF− OIS cells. P < 1.9 × 10−5. (G) In individual nuclei, chromatin extrusion and dextran intrusion occurred in close physical proximity. Right-hand panels are expanded from yellow boxed areas on the left. Bars: (left) 10 µm; (right) 2 µm.",
    "molecules": "dextran"
  },
  {
    "caption": "Senescence-associated loss of histones is V-ATPase dependent. (A) Bafilomycin A1 (BafA1) blocks the loss of nuclearhistone H3content in cells undergoing OIS. BafA1 was added to cells at 50 nM on day 5 after RASG12V induction. Cells were harvested 24 h later, and stained for histone H3. Bars, 10 µm. (B) Quantitative histone H3immunofluorescence in cells from A. Single representative experiment out of three repeats.",
    "molecules": "BafA1, Bafilomycin A1"
  },
  {
    "caption": "(C) RASG12V-induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot. LC3 I/II has been used as a control for BafA1 activity. Lysates from 10,000 cells were loaded per well.",
    "molecules": "BafA1"
  },
  {
    "caption": "(D) BafA1 blocks the accumulation of H3cs.1 in cells undergoing OIS.",
    "molecules": "BafA1"
  },
  {
    "caption": "(E) BafA1 and concanamycin A (Con A) block the accumulation of H3cs.1 in RS cells.",
    "molecules": "BafA1, concanamycin A"
  },
  {
    "caption": "Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Upper 2 blots; n= 6 mice at each time-point for each group). 18S rDNA was used as loading control. DNA isolated from Lrpprc and Mterf4 knockout hearts used as controls. Western blot analyses of VDAC and Histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Bottom 2 blots; ; n= 6 mice at each time-point for each group). VDAC represents mitochondria loading and Histone H3 was used as a loading control for nucleus DNA.",
    "molecules": "DNA, mtDNA"
  },
  {
    "caption": "Southern blot analyses of mtDNA to obtain 7S DNA in 8-week-old control and Tefm knockout mice. Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA. Equal amounts of mtDNA shown on the top of the gel, were loaded as the endogenous control. This experiment has performed three times.",
    "molecules": "mtDNA"
  },
  {
    "caption": "Volcano plot showing TEFM-BioID. TEFM-BirA*-HA was stably expressed in HEK 293 cells, followed by streptavidin affinity purification and mass spectrometry analysis to detect the associated proteins. Mitochondrial targeted BirA* (MTS-BirA*) was used as a control. The significantly enriched proteins are presented above the horizontal dashed line with 5% false discovery rate (FDR) significance. The blue dots highlight proteins involved in mtDNA transcription and the green dots represent proteins involved in RNA metabolism. All other proteins are in grey.",
    "molecules": "mtDNA, streptavidin"
  },
  {
    "caption": "XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.",
    "molecules": "Fe"
  },
  {
    "caption": "DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 Cells were then fed with Ova-Fe and exposed to reporter T cells as in (B). Error bars show SD of duplicate wells. * p < 0.05 using two-way ANOVA. Representative plot of 3 independent experiments.",
    "molecules": "dox, Fe"
  },
  {
    "caption": "MHC-II presentation of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe. The cells were then exposed to MF2.2D9-Luc Reporter CD4 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control β2m using two-way ANOVA. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m. Representative plot of 3 independent experiments",
    "molecules": "Fe"
  },
  {
    "caption": "DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with Ova-Fe and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m using two-way ANOVA. Representative plot of 3 independent experiments",
    "molecules": "dox, Fe"
  },
  {
    "caption": "MHC-I Presentation of SIINFEKL-H2-Kb complexes on siRNA treated DC3.2 NS-Ova (C) or DC3.2 UbS8L (D) cells. 48 hours after siRNA transfection, the indicated amounts of dox was added to the cells to induce Ova expression. After 2 hours at 37oC, RF33.70-Luc CD8 T cells were added along with Brefeldin A (Golgiplug) to a final concentration of 1:1000. The cells were incubated overnight for reporter T cell luciferase expression. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control I-Ab using two-way ANOVA. None of the dox concentrations are p < 0.05 for Rab39a siRNA vs I-Ab. Representative plot of 3 independent experiments",
    "molecules": "Brefeldin A, dox, Ub"
  },
  {
    "caption": "Phagocytosis assay after 48 hours of siRNA knockdown. DC3.2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads. The mixture was then placed in media at 37oC for 1 hour. Cells were harvested and incubated on ice with 1 mg/ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry. As a control, some cells were incubated with 10 µM Cytochalasin D during the feeding period. Shown are percentages of cells that have taken up beads. Representative plot of two independent experiments.",
    "molecules": "Alexa Fluor 647, Cytochalasin D, Trypan Blue"
  },
  {
    "caption": "Rab39a was induced in DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) via overnight incubation with 1µg / ml dox. KO and rescued cells were then harvested and assayed as in (A) for phagocytosis. Representative plot of two independent experiments.",
    "molecules": "dox"
  },
  {
    "caption": "DC3.2 Rab39a KO cells were transduced with dox inducible mcherry-tagged Rab39a. Cells were plated in 35 mm glass bottom dishes and induced with 1 µg/ml dox. The next day, 1 µm latex beads conjugated with Alexa 488-NHS (1 bead/cell) were added. After 2 hours at 37oC, cells were imaged under a Leica TCS SP5 confocal microscope. Green = Alexa 488 labeled beads. Magenta = mcherry-Rab39a. Arrows highlight internalized beads surrounded by Rab39a. Scale bar = 10 µm. Representative image of two independent experiments.",
    "molecules": "Alexa 488, dox, NHS"
  },
  {
    "caption": "5x105 of DC3.2-Rab39a cells were induced to express Rab39a overnight with 1 µg/ml dox in a 6 well plate. Cells were fed at 1 bead/cell of biotinylated-6µm magnetic beads for the indicated lengths of time. Magnetic-bead-phagosomes were isolated using the listed protocol. Phagosomes were washed and stained for Rab39a (HA-tag). Representative data for 3 independent experiments.",
    "molecules": "biotinylated, dox"
  },
  {
    "caption": "1.25 x 105 of DC3.2-Rab39a cells were incubated in a 12 well plate overnight ± dox (1µg/ml) to induce Rab39a. Cells were fed at 4 beads/cell of biotinylated-ova-6µm-magnetic-beads for 3 hours with or without 1:1000 BFA (golgiplug, BD). Phagosomes were isolated using the listed protocol. Isolated phagosomes were washed, permeabilized and stained. Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes. Representative plot of two independent experiments.",
    "molecules": "biotinylated, BFA, dox"
  },
  {
    "caption": "XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments",
    "molecules": "iron oxide"
  },
  {
    "caption": "XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts o Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments",
    "molecules": "iron oxide"
  },
  {
    "caption": "DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. * p < 0.05 between no dox and + dox using two-way ANOVA. Representative plot of 3 independent experiments",
    "molecules": "dox"
  },
  {
    "caption": "DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. Representative histograms shown in (B). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2-Ld (dox/no dox) was calculated. Three independent experiments were combined for analysis and error bars show the SD between experiments. Open H2-Ld, but not Closed H2-Ld was statistically significant (p<0.05) between KO and rescued cells using two-tailed ratio-paired Student's t-test.",
    "molecules": "biotinylated, dox"
  },
  {
    "caption": "DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. H2-Ld cell surface levels of KO and induced cells as in (A).",
    "molecules": "biotinylated, dox"
  },
  {
    "caption": "DC3.2 Rab39a-H2-Ld cells were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours in the presence or absence of BFA. Isolated phagosomes were stained for H2-Ld. Representative of two independent experiments.",
    "molecules": "biotinylated, BFA, dox"
  },
  {
    "caption": "DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours with the indicated inhibitors. Magnetic-bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open and Closed (30-5-7s) forms of H2-Ld. Representative histograms of two independent experiments shown.",
    "molecules": "biotinylated, dox"
  },
  {
    "caption": "Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours. Phagosomes were isolated and stained for Ova and Open H2-Ld. Representative of 3 independent experiments.",
    "molecules": "biotinylated, dox"
  },
  {
    "caption": "Quantification of Ova remaining in bead-phagosomes (C) and Nox2 staining (D). Representative Histogram shown. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target protein (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.",
    "molecules": "dox"
  },
  {
    "caption": "Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were incubated on ice with 4 beads/cell of biotinylated-Ova-beads for bead attachment. Cells were then transferred into 37oC media containing 5 µM CellROX Deep Red. After 4 hours of incubation, unfixed phagosomes were extracted and analyzed via flow cytometry for CellROX fluorescence. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.",
    "molecules": "CellROX, CellROX Deep Red, biotinylated, dox"
  },
  {
    "caption": "Cells as in (A) were fed at 1 bead per cell with pHrodo conjugated beads that increase fluorescence at low pH. After 4 hours of incubation, whole cells were harvested and run through flow cytometry to measure fluorescence of eaten beads. For analysis, the background fluorescence of uneaten beads was subtracted from the resulting gMFI and the fold change of dox/no dox was calculated. Three independent experiments were combined. p<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments. Also shown are histograms of whole cells compared to background fluorescence of pH beads.",
    "molecules": "pHrodo, dox"
  },
  {
    "caption": "DC3.2R cells were treated with the indicated siRNA. 48 hours after transfection, cells were fed with Ova-Fe (2 µg Ova) with or without 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.",
    "molecules": "Fe, leupeptin"
  },
  {
    "caption": "5x104 DC3.2-Rab39a-H2-Ld cells were fed with Ova-Fe (1µg Ova) with or without 1 µg dox and/or 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.",
    "molecules": "dox, Fe, leupeptin"
  },
  {
    "caption": "Rab39a expression in splenocyte subsets was measured using an FDG assay. Shown are relative FDG fluorescence of Rab39a KO cells/WT background. Summary of two independent experiments.",
    "molecules": "FDG"
  },
  {
    "caption": "Splenic DCs were expanded in Rab39a KO and WT mice by injection of B16-Flt3L tumor. 10 days post tumor implantation, DCIR2+ (Cd8α-) or XCR1+ (Cd8α+) dendritic cells were isolated from spleens via magnetic positive selection. 5 x 104 cells were plated in a 96 well plate and incubated with the indicated amounts of Ova-Fe and 1:1 of Reporter CD8 or CD4 T-cells overnight before measuring luciferase activity. Error bars indicate SD of 2 replicate wells. Shown are representative graphs of 3 independent experiments. * p < 0.05 using two-way ANOVA.",
    "molecules": "Fe"
  },
  {
    "caption": "CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm biotin-ova-magnetic beads for 4 hours. Phagosomes were extracted, permeabilized and stained for the indicated proteins. For ROS measurements, phagocytosis was performed with 5 µM CellROX and the phagosomes were not permeabilized. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target (dox/no dox) was calculated. Shown are three independent experiments combined. Error bars show SD between experiments. p values were calculated using two-tailed ratio-paired Student's t-test.",
    "molecules": "CellROX, biotin, dox, ROS"
  },
  {
    "caption": "CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to pHrodo. After 4 hours, cells were harvested for flow cytometry to measure fluorescence of eaten beads due to phagosomal pH. For analysis, the background fluorescence of unstained beads was subtracted from the resulting gMFI and the fold change (dox/no dox) was calculated. Shown are three independent experiments with error bars indicating the SD. p values were calculated using two-tailed ratio-paired Student's t-test.",
    "molecules": "pHrodo, dox"
  },
  {
    "caption": "CD11b+ CD11c+ dendritic cells were enriched from WT or Rab39a KO spleens via magnetic negative selection. Cells were fed with 3 µm magnetic beads conjugated to Alexa fluor dye. After 4 hours, cells were harvested and resuspended in 1 mg/ml Trypan blue to quench fluorescence of uneaten beads. Cells were run for flow cytometry to measure phagocytosis. The ratio of KO cells with beads / WT cells with beads was then obtained. Graphs show three independent experiments and p values were calculated using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.",
    "molecules": "Alexa fluor, Trypan blue"
  },
  {
    "caption": "Rab39a KO and WT mice were injected intravenously with 1 x 105 A20-Ova cells. The next day, CFSE labeled OTI-splenocytes were injected i.v.. 3 days post OT-I transfer, spleens were harvested and assayed for OT-I proliferation. Three independent experiments (WT vs KO) were performed. To combine the data, the OT-I proliferation in each individual mouse was normalized to the average proliferation of the WT controls in that experiment. Error bars show SD between individual mice and p value was derived from two-tailed student's t-test. n = 14 (WT) or 9 (KO).",
    "molecules": "CFSE"
  },
  {
    "caption": "C. Representative image of 5-mC immunofluorescence in the sperm of saline- and 5-Aza-treated mice (ST and AT, respectively). Scale bar: 100 μm. D. Quantification analysis of 5-mC immunofluorescence in individual sperm cells from STO (n=1,673 sperm from 4 male mice) and ATO (n=2,675 sperm from 4 male mice).Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.",
    "molecules": "5-Aza, AT, ATO, 5-mC, saline"
  },
  {
    "caption": "F. Overall USV number in STO and ATO. Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.",
    "molecules": "ATO, STO"
  },
  {
    "caption": "(A) SPT activity and Western blot (WB) analysis of S1PR1 in S1pr1f/f and S1pr1ECKO endothelial cells after 4-OHT (1μM, 72h) treatment. (n=8/group from 3 independent EC isolations/group; 4 mice/EC isolation).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(A,B) (A) WB analysis of ORMDLs, NOGO-B, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and (B) relative ORMDLs quantification (n=7 biological replicates).",
    "molecules": "S1P"
  },
  {
    "caption": "(C) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of C16:0-ceramide (300nM, 30'), with or without SKI II 1µM, 1h pre-treatment). (D) SPT activity in HUVEC in absence or presence of SEW2871 (300nM, 15') (n=7 biological replicates).",
    "molecules": "ceramide, C16:0, SEW2871, SKI II"
  },
  {
    "caption": "(E,F) (E) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of SEW2871 (300nM, 15' and 30') and (F) relative ORMDLs quantification (n=6 biological replicates).",
    "molecules": "SEW2871"
  },
  {
    "caption": "(I) WB analysis of ORMDLs, P-p38, p38, P-JNK, JNK, P-ERK, ERK, P-AKT, and AKT in HUVEC lysates in absence or presence of the indicated inhibitors, with or without S1P (300nM, 30').",
    "molecules": "S1P"
  },
  {
    "caption": "(A) WB analysis of ORMDL3 (HA), SPTLC1 and SPTLC2 in HUVEC lysates expressing HA-ORMDL3 and treated with cycloheximide (CHX, 10µM) for the indicated period of time.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and with or without (C) DMOG (1mM, 1h pre-treatment), (D) Eeyarestatin 1 (EER1, 10µM, 1h pre-treatment),",
    "molecules": "DMOG, EER1, Eeyarestatin 1, S1P"
  },
  {
    "caption": "WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') (E) MG132 (10µM, 1h pre-treatment).",
    "molecules": "MG132, S1P"
  },
  {
    "caption": "(F) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A in HUVEC lysates in absence or presence of S1P (300nM, 30'), DMOG (1mM, 1h), EER1 (10µM, 1h), and MG132 (10µM, 1h).",
    "molecules": "DMOG, EER1, MG132, S1P"
  },
  {
    "caption": "(G,H) (G) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A, SPTLC1 and SPTLC2 in HUVEC lysates treated with CHX (10µM) for the indicated period of time, and (H) relative quantification",
    "molecules": "CHX"
  },
  {
    "caption": "SPT activity in S1pr1,3f/f and S1pr1,2ECKO mEC , in absence or presence of S1P (300nM, 30') (B) relative WB analysis for S1PR1, ORMDLs, P-AKT and AKT.",
    "molecules": "S1P"
  },
  {
    "caption": "(G) WB analysis of ORMDLs and S1PR1 in HUVEC lysates in absence or presence of P-FTY720 (100nM, 30') and",
    "molecules": "P-FTY720"
  },
  {
    "caption": "(A-C) (A) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-IR (Y1150/1151), IR, P-AKT (S473), AKT, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2') or Insulin (1U/mL, 2') and (B-C) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).",
    "molecules": "Insulin"
  },
  {
    "caption": "(D,E) (D) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2'), and in absence or presence of Myriocin (300nM) for the indicated time and (E) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).",
    "molecules": "Myriocin"
  },
  {
    "caption": "Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a primary healthy colon (C) DAPI was used to stain cell nuclei. Scale bars:50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a malignant lymphoma (D). DAPI was used to stain cell nuclei. Scale bars:50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1 or RFP control. Confocal images of transfected cells pulsed with EU for 16 hours were collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1-transfected cells are indicated with arrowheads.",
    "molecules": "EU, Alexa 488, DAPI"
  },
  {
    "caption": "Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against NPM1 (B) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against UBF1 (C) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm). A magnification of the merged channel images is provided for UBF1 staining.",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Agarose/formaldehyde gel separation of neotranscribed rRNAs collected from LacZ and PNRC1-expressing cells pulsed for 15 minutes with 3H-uridine and chased for the reported time points (minutes). Samples were loaded according to the amount of incorporated 3H, quantified by liquid scintillation. FU was included as a control of a blocked rRNA processing.",
    "molecules": "FU, Agarose, formaldehyde, 3H, uridine"
  },
  {
    "caption": "C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody in absence or presence of 1mg/ml RNaseA.",
    "molecules": "RNaseA"
  },
  {
    "caption": "E. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing a shRNA-resistant GFP-DCP1α in combination with RFP or RFP-PNRC1 transgenes (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing an shRNA-resistant GFP-DCP1α in combination with RFP, RFP-PNRC1WT or RFP-PNRC1W300A mutant (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Confocal images of a Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1W300A pulsed with EU for 16 hours and collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads.",
    "molecules": "EU, Alexa 488, DAPI"
  },
  {
    "caption": "Figure 3: Infiltrating leukocyte subsets in human glioma. Paraffin sections of healthy human brain (n=3), low grade glioma (n=12), anaplastic astrocytoma (n=7) and glioblastoma (n=9) were stained for IBA1 (macrophages/microglia), CD15 (granulocytes) and CD3 (T-cells) (A). Quantification of leukocyte subsets in human glioma specimens (B). For statistical analysis Kruskall-Wallis (Dunn´s post test) was applied.* p < 0.05, ** p < 0.01; data are mean ± SEM",
    "molecules": "CD15"
  },
  {
    "caption": "Figure 4: Dual anti-Ang-2 and anti-VEGF therapy acts synergistic on vascular normalization and macrophage infiltration in experimental GBM. Immunofluorescence staining of CD31 and desmin in GL261 glioblastoma after single and dual treatment with anti-Ang-2 (AMG386) and anti-VEGF (aflibercept) (A). Corresponding quantitative analysis of microvessel densities and pericytes numbers (B; control n=24; AMG386 n=10; aflibercept n=12; AMG386 + aflibercept n=13).",
    "molecules": "aflibercept, AMG386"
  },
  {
    "caption": "Quantification of necrotic areas in GL261 glioblastoma sections after anti-angiogenic treatment is shown as percent of whole tumor area (E; control, aflibercept, AMG386 + aflibercept n=4; AMG386 n=3).",
    "molecules": "aflibercept, AMG386"
  },
  {
    "caption": "Double-immunofluorescence stainings of macrophages (F4/80) and tumor vessels (vWF) in mouse GBM after treatment with AMG386, aflibercept or the combination of both are shown in (G)",
    "molecules": "aflibercept, AMG386"
  },
  {
    "caption": "Quantitatve analysis of tumor infiltrating leukocytes (F4/80+, CD3+, Ly-6G+) following anti-angiogenic treatment is displayed in (H; control n=28; AMG386 n=12; aflibercept n=11; AMG386 + aflibercept n=12).",
    "molecules": "aflibercept, AMG386"
  },
  {
    "caption": "Kaplan-Meier survival analysis of GL261 tumors grown in C57BL/6 mice following anti-angiogenic treatment (I; control n=38; AMG386 n=13; aflibercept n=12; AMG386 + aflibercept n=13).",
    "molecules": "aflibercept, AMG386"
  },
  {
    "caption": "Double-immunofluorescence stainings with anti-F4/80 and anti-CD206 in brain tumor sections of mice treated with anti-Ang-2 (AMG386), anti-VEGF (aflibercept) or the combination of both are shown in (J). Corresponding quantitative analyses of tumor infiltrating F4/80+ cells and CD206+ cells, and the ratio of CD206+ vs. F4/80+ upon anti-angiogenic therapy are displayed in (K; control n=21; AMG386 n=13; aflibercept n=9; AMG386 + aflibercept n=4).",
    "molecules": "aflibercept, AMG386"
  },
  {
    "caption": "Flow cytometry of tumor infiltrating macrophages following enzymatic dissociation of mouse GL261 brain tumors plus/minus anti-angiogenic therapy. Percent of CD206+ (L) and MHC class IIHI cells (M) among CD45+CD11b+GR1-F4/80+ cells are displayed (L,M; control n=4; AMG386 n=4; aflibercept n=5; AMG386 + aflibercept n=5). Statistical analyses were performed using one-way ANOVA and Tukey's multiple comparison except for Log-rank and Wilcoxon (I) and Kruskall Wallis (B). * p < 0.05, ** p < 0.01, *** p < 0.005, # p < 0.005 of aflibercept + AMG386 vs aflibercept; data are mean ± SEM (A-K)",
    "molecules": "aflibercept, AMG386"
  },
  {
    "caption": "Figure 5: Anti-VEGF therapy led to decreased infiltration of CD68+ macrophages in human GBM. Anti-CD68 (A), anti-CSFR1 (B) and anti-CD206 (C) immunohistochemistry of patient samples derived from treatment-naive GBM (n=24), post-radiochemotherapy (S/RTx/CTx) (n=7) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) (n=29) therapy is shown.",
    "molecules": "bevacizumab"
  },
  {
    "caption": "Figure 6: Bevacizumab therapy leads to reduced vessel density and increased Ang-2 expression. Immunohistochemistry stainings with antibodies directed against CD31 (A) and Ang-2 (B) in treatment-naive GBM, post-radiochemotherapy (S/RTx/CTx) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) therapy are displayed.",
    "molecules": "Bevacizumab, bevacizumab"
  },
  {
    "caption": "Western Blot and corresponding quantification of CD31 and Ang-2 in control and aflibercept treated mice (F).",
    "molecules": "aflibercept"
  },
  {
    "caption": "(A) 12-lead ECG of patient 1. (B) 12-lead ECG of patient 2. (C) ECG of patient 2 during epinephrine test (A- represents baseline ECG; B- ECG at 17 min and 47 s of the beginning of epinephrine infusion (0.2 μg/kg of epinephrine) and C- ECG after the end of epinephrine infusion).",
    "molecules": "epinephrine"
  },
  {
    "caption": "(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz. (B) Time to reach 50% of [Ca2+]i transient amplitude, t1/2; [Ca2+]i concentrations during systole and diastole (C) Amplitude and tau decay of the [Ca2+]i transient.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz, in the presence of caffeine (caff) or caffeine and NiCl2. (B) Amplitude of [Ca2+]i in the presence of caffeine and NiCl2and fractional SR release in hiPSC-CMs.",
    "molecules": "Ca2+, caffeine, NiCl2"
  },
  {
    "caption": "(C) Rate constants of SERCA-, NCX- and slow mechanisms-based [Ca2+]i decay in hiPSC-CMs. (D) Relative contribution of SERCA, NCX and slow mechanisms to [Ca2+]i extrusion in hiPSC-CMs.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(A) Representative AP traces and (B) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the absence of NA. (C) Representative AP traces and (D) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the presence of NA.",
    "molecules": "NA"
  },
  {
    "caption": "(A) Representative AP trace of a TECRLHom-hiPSC-CM in the presence of NA alone or NA and flecainide. (B) Effect of flecainide on AP parameters of hiPSC-CMs. (C-D) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC-CMs. Note that the effect of flecainide on triggered activity of TECRLHet- and TECRLHom-hiPSC-CMs is more pronounced than its effect on their spontaneous activity.",
    "molecules": "flecainide, NA"
  },
  {
    "caption": "(a) Representative photographs of Bax/Bak−/− MEFs treated with etoposide. Bax/Bak−/− MEFs were treated with 20 μM etoposide in the presence of PI (2 μM) for the indicated times, and were analysed under a phase-contrast and fluorescence (PI) microscope.",
    "molecules": "etoposide"
  },
  {
    "caption": "(b, c) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide. WT (circles) and Bax/Bak−/− (squares) MEFs were treated with 20 μM etoposide in the presence (closed symbols) or absence (open symbols) of 100 μM zVAD-fmk. Then cell viability was measured by the PI staining (b) and CTB assay (c), and expressed as a percentage of the initial value without etoposide. Data are shown as mean ± s.d. (n = 4).",
    "molecules": "etoposide, zVAD-fmk"
  },
  {
    "caption": "(d) Reduced viability of etoposide-treated Bax/Bak−/− MEFs as assessed by proliferation assay. Bax/Bak−/− MEFs were either not treated (0 h) or were treated with 20 μM etoposide for 12 and 24 h, then all cells were recovered and 5,000 cells were reseeded. Viable cell numbers were measured on the indicated days by the CTB assay. Results were normalized by adjusting the day 0 value. n = 4.",
    "molecules": "etoposide"
  },
  {
    "caption": "e) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine, as assessed by clonogenicity assay. MEFs were treated with etoposide (20 μM) or staurosporine (1 μM) at the indicated times, collected, and 2,000 cells were seeded in the normal medium. After 1 week, colonies were counted. n = 4",
    "molecules": "etoposide, staurosporine"
  },
  {
    "caption": "f-i) Induction of autophagy in Bax/Bak−/− MEFs by etoposide. (f) Electron micrograph (×14,800) of Bax/Bak−/− MEF treated with etoposide (20 μM) for 18 h. Representative features of Bax/Bak−/− MEFs that have received no treatment (NT; that is, are healthy) are also shown (NT: ×14,000).",
    "molecules": "etoposide"
  },
  {
    "caption": "(h, i) Punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs treated with etoposide. GFP-LC3-transfected cells were incubated with and without etoposide (20 μM) for 24 h (h) or the indicated times (i), and then examined by fluorescent microscopy. (h) Representative photographs of healthy (NT) and etoposide-treated Bax/Bak−/− MEFs are shown. (i) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-positive cells. Data are shown as mean ± s.d. (n = 4).",
    "molecules": "etoposide"
  },
  {
    "caption": "(a, b) Bax/Bak−/− MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3-MA for 24 h, and then were examined by phase-contrast microscopy (a) and electron microscopy (×8,400) (b). Autolysosomes/autophagosomes were hardly observed. (",
    "molecules": "3-MA, etoposide"
  },
  {
    "caption": "c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).",
    "molecules": "3-MA, etoposide"
  },
  {
    "caption": "(c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).",
    "molecules": "3-MA, etoposide"
  },
  {
    "caption": "(e-g) Inhibition of etoposide- and staurosporine-induced cell death by 3-MA. Bax/Bak−/− MEFs were treated with 20 μM etoposide (e-g) or 1 μM staurosporine (g) in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA for the indicated time. Cell viability was measured by PI staining (e), CTB assay (f) and clonogenicity assay (g) (as in Fig. 1e). Upper panels in g: viable cells were visualized by staining with calcein-AM. n = 4.",
    "molecules": "3-MA, etoposide, staurosporine"
  },
  {
    "caption": "(a, b) Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex (a), Beclin 1 (b) and VDAC (loading control) was analysed by Western blot analysis.",
    "molecules": "etoposide"
  },
  {
    "caption": "(c) Inhibition of etoposide-induced autophagy by silencing of Beclin 1. Bax/Bak−/− MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy (×8,500).",
    "molecules": "etoposide"
  },
  {
    "caption": "d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were transfected with GFP-LC3 together with the indicated siRNAs, were incubated without (NT) or with 20 μM etoposide for 24 h, and then examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).",
    "molecules": "etoposide"
  },
  {
    "caption": "(e, f) Inhibition of etoposide- and staurosporine-induced cell death by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide (e, f) or 1 μM staurosporine (f) for the indicated time. Cell viability was measured by CTB assay (e) and clonogenicity assay (f). n = 4.",
    "molecules": "etoposide, staurosporine"
  },
  {
    "caption": "(a, b) APG5-dependent punctate GFP-LC3 fluorescence in Bcl-2-transfected MEFs after etoposide treatment. APG5+/+ and APG5−/− MEFs that were transfected with both GFP-LC3 and the indicated plasmids (Bcl-2 or vector) were incubated without or with 20 μM etoposide for 24 h, then examined by confocal fluorescent microscopy (a). The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells (b).",
    "molecules": "etoposide"
  },
  {
    "caption": "(c) Accumulation of Beclin 1 and APG5-APG12 complex in Bcl-2-transfected MEFs after etoposide treatment. WT MEFs were transfected with the indicated plasmids, and were incubated without (NT) or with 20 μM etoposide for 24 h.",
    "molecules": "etoposide"
  },
  {
    "caption": "(d, e) Occurrence of etoposide-induced 3-MA-inhibitable non-apoptotic death in Bcl-xL-overexpressing APG5+/+ MEFs, but not APG5−/− MEFs. APG5+/+ (open columns) and APG5−/− (closed columns) MEFs were transfected with the indicated plasmids (2 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay (d) and apoptotic nuclear morphology (e). Data are shown as mean ± s.d. (n = 4).",
    "molecules": "3-MA, etoposide, zVAD"
  },
  {
    "caption": "(f) Overexpression of Bcl-xL does not sensitize Beclin 1-silenced MEFs to etoposide-induced 3-MA-inhibitable non-apoptotic death. WT MEFs with silencing of control GFP (open columns) or Beclin 1 (closed columns) were transfected with the indicated plasmids (1 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay. n = 3.",
    "molecules": "3-MA, etoposide"
  },
  {
    "caption": "(a) Enhancement of etoposide-induced non-apoptotic death by overexpression of human Bcl-2 and Bcl-xL in Bax/Bak−/− MEFs in a Beclin 1-dependent manner. Bax/Bak−/− MEFs with silencing of control Bax or Beclin 1 were transfected with the indicated plasmids (1 μg). After 24 h, the transfected cells were incubated with 20 μM etoposide for 24 h, and cell viability was assessed by the CTB assay. Data are shown as mean ± s.d. (n = 3).",
    "molecules": "etoposide"
  },
  {
    "caption": "(c-e) Inhibition of etoposide-induced autophagy in Bax/Bak−/− cells treated with siRNA for Bcl-x. (c) Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide for 24 h, then examined by phase-contrast microscopy.",
    "molecules": "etoposide"
  },
  {
    "caption": "(d) Bax/Bak−/− MEFs that were transfected with siRNA for Bcl-x and then treated with etoposide for 18 h were analysed by electron microscopy (×8,500)",
    "molecules": "etoposide"
  },
  {
    "caption": "(e) Reduction of punctate GFP-LC3 fluorescence in etoposide-treated Bax/Bak−/− MEFs by silencing of Bcl-x. Bax/Bak−/− MEFs that were transfected with both GFP-LC3 and the indicated siRNAs were incubated without (NT) or with etoposide for 24 h, and then were examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. n = 4.",
    "molecules": "etoposide"
  },
  {
    "caption": "f) Inhibition of etoposide-induced cell death by silencing of Bcl-x. Bax/Bak−/− MEFs were transfected with siRNA (circles, siRNA for Bax; squares, siRNA for Bcl-x) were treated with 20 μM etoposide in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA, and cell viability was assessed by the CTB assay at the indicated times. n = 4.",
    "molecules": "3-MA, etoposide"
  },
  {
    "caption": "(g) Clonogenicity assay of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine. Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h. After collection, 2,000 cells were seeded in the normal medium. After one week, colonies were counted.",
    "molecules": "etoposide, staurosporine"
  },
  {
    "caption": "h, i) Inhibition of APG5-APG12 induction by silencing of Bcl-x or Beclin 1. Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex, Beclin 1 and GAPDH/VDAC (loading control) was analysed by Western blot analysis.",
    "molecules": "etoposide"
  },
  {
    "caption": "A, Reconstructed  MSOT images (800 nm) without and with linear unmixing data in the neck  and the upper and lower abdominal areas, showing the ROIs for the  liver, kidney, iBAT, rpWAT,  aorta, and SV. Unmixing result: blue for  Hb, red for HbO2, yellow for  lipid. The colour bar shows the colour coding of MSOT  a.u. from 0 to maximum (bottom to top) (maximum value neck/upper abdominal/lower abdominal: Hb: 2.5/3.5/0.9; HbO2:2.9/4.9/2.5; Lipid: 24000/19000/13000) Scale bar: 4 mm.",
    "molecules": "Hb, HbO2, lipid, Lipid"
  },
  {
    "caption": "A, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb,  HbO2, and lipid from  chow  and HFD-fed  mice. The colour bar shows the colour coding of  MSOT a.u. from 0 to maximum (bottom to top) (maximum value   chow/HFD: Hb: 1.3/0.6; HbO2: 1.8/0.9; Lipid: 27000/27000). Scale bar: 4 mm.",
    "molecules": "Hb, HbO2, lipid, Lipid"
  },
  {
    "caption": "D, Tissue oxygenation (sO2) and total blood volume (TBV) results from  liver,  kidney, iBAT, and rpWAT, Each dot represents data from one animal, in total 8 animals (n = 8). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units. ",
    "molecules": "sO2"
  },
  {
    "caption": "A,  Oil-red-O staining  of control (healthy) and steatotic liver. Scale bar: 100 µm.",
    "molecules": "Oil-red-O"
  },
  {
    "caption": "B, Reconstructed MSOT image (800 nm) with linear unmixing and difference value of  lipid in  lower abdominal section. Unmixing result: blue for  Hb, red for  HbO2, yellow for lipid, jet for 700 nm - 930 nm difference. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: Hb: 1.1/0.5;  HbO2: 1.9/0.8; Lipid: 21000/24000; 700-900nm: 3000/3000) Scale bar: 4 mm.",
    "molecules": "Hb, HbO2, lipid, Lipid"
  },
  {
    "caption": "E, Reconstructed  MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow  and  HFD-fed mice. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value chow 1 month/ chow 2 month/ chow 3 month/HFD 1 month/HFD 2 month/HFD 3 month: Hb: 2.5/1.9/2.9/2.7/1.8/1.3; HbO2: 2.8/2.6/2.8/3.1/2.1/1.9; Lipid: 27000/19000/29000/ 32000/26000/27000). Scale bar: 4 mm.",
    "molecules": "Hb, HbO2, lipid, Lipid"
  },
  {
    "caption": "F, Longitudinal track of  lipid in mouse livers. Each line represents data from one animal. Each data point is from a single measurement of each animal at each  time point. All animals have grade 3  steatosis at the endpoint confirmed by histology.",
    "molecules": "lipid"
  },
  {
    "caption": "A, Reconstructed MSOT image (800 nm) with linear unmixing data of ICG from  mice with and without  steatosis. The colour bar shows the colour coding of MSOT  a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: 0.07/0.02) Scale bar: 4 mm.",
    "molecules": "ICG"
  },
  {
    "caption": "A HeLa cells were transfected with constructs for a non-targeting shRNA (NT), USP17 shRNA1, or USP17 shRNA2. Cells were stained using an anti-LAMP1 antibody (green) and the nuclei counterstained with DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. B HeLa and MDA-MB-231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position (see C). C",
    "molecules": "DAPI"
  },
  {
    "caption": "A HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 48 hours post transfection the cells were either serum starved (upper panels), or treated with serum free medium containing 100ug/ml EGF (lower panels) for 16 hours prior to staining for LAMP1 (green) and DAPI (blue). Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line. Scale bar represents 25μm. B The distribution of at least 300 LAMP1 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicates not significant, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.",
    "molecules": "DAPI"
  },
  {
    "caption": "A 48 hours post transfection the cells were washed with Ca2+ and Mg2+ free ice cold HBSS. The different cell populations were split into three aliquots. One aliquot was placed into fresh medium (Left panels). SLO was added to other two aliquots and then the toxin-containing medium was removed and the aliquots were either placed into Ca2+ and Mg2+ free HBSS (Middle panels), or EBSS containing Ca2+ and Mg2+ (Right panels) prior to staining with propidium iodide and analysis by flow cytometry.",
    "molecules": "Ca2+, Mg2+, propidium iodide"
  },
  {
    "caption": "A HeLa cells were transfected with negative control siRNA and RNF26 siRNA as well as constructs coding a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2, as indicated. 48 hours post transfection cells were stained for LAMP-1 (green) and DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. Scale bar represents 25μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "C HeLa cells were transfected with expression constructs for HA-tagged p62 and FLAG-tagged ubiquitin as well as empty vector, USP17 or USP17CS. After 48hrs cell lysates were prepared and HA tagged proteins were pulled down using anti-HA agarose. Pull downs, and lysates, were immunoblotted with anti-HA, anti-FLAG, and anti-USP17 antibodies, as indicated, to confirm the presence of ubiquitinated p62.",
    "molecules": "ubiquitin, ubiquitinated"
  },
  {
    "caption": "A HeLa cells were transfected with constructs for EEA1-GFP and RAB5-GFP in addition to non-targeting scrambled (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 72 hours post transfection the cells were stained with DAPI. The scale bar represents 25μm. B The distribution of at least 400 EEA1 or RAB5 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups. E",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Enhanced migration of CD68 positive, PGRN deficient cells in an ex vivo model. Immunofluorescence analysis of cultured Grn+/+ and Grn-/- organotypic brain at 7 DIV immunostained using microglial marker CD68. Nuclei were counterstained using DAPI. Images of boxed regions in left panels are shown at higher magnifications in right panels. Scale bars: 500 µm (left panels), 250 µm (right panels). Quantitative analysis reveals that Grn-/- microglia migrate larger distances than Grn+/+. Data are shown as mean ± SD (n=3 independent experiments) Quantification displays an increased number of migrating Grn-/- cells compared to Grn+/+ cells. Data are shown as mean ± SD (n=3 independent experiments)",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Enhanced microglial clustering around amyloid plaques in the absence of PGRN. Left: IBA1-stained microglia clustering around X-34-positive amyloid plaque cores in 4-month-old APPPS1/Grn+/+ mice. Right: Age-matched APPPS1/Grn-/- mice display microglial hyper-clustering around amyloid plaques compared to APPPS1/Grn+/+. Dotted white boxes indicate the area that is shown at higher magnification. Scale bars indicate 10µm and 3µm in inset. Immunohistochemical quantification of IBA1 positive microglial cell number per plaqu",
    "molecules": "X-34"
  },
  {
    "caption": "FDG-µPET in rodents: Axial slices (as indicated in Fig. 4A) of averaged %-FDG-µPET differences between Grn-/-or Trem2-/- mice and wt indicate decreased glucose metabolism in the cerebrum of Grn-/- and Trem2-/- (both cold color scale) when compared to wt. Scatter plot depicts single FDG-PET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=6 at 11 months; wt n=10 at 8-11 months).",
    "molecules": "FDG, glucose"
  },
  {
    "caption": "E. Subcellular localization of WT Yb and deletion mutants ∆Hel-C and ∆eTud. Myc-Yb is shown in green. Endogenous Armi is shown in red. DAPI shows nuclei (blue). Scale bar: 5 μm. Western blotting (right) showing WT Yb and deletion mutants ∆Hel-C and ∆eTud expressed in Yb-lacking OSCs.",
    "molecules": "DAPI"
  },
  {
    "caption": "F. CLIP shows that WT Yb and ∆Hel-C bind RNA in vivo but ∆eTud does not.",
    "molecules": "RNA"
  },
  {
    "caption": "A. Rescue experiments showing that ∆Hel-C but not ∆eTud restored piRNA biogenesis attenuated by endogenous Yb depletion. Flag-Piwi was expressed upon endogenous Yb depletion.",
    "molecules": "piRNA"
  },
  {
    "caption": "C. Mapping of flam-piRNAs produced in the presence and absence of the Hel-C domain in OSCs. D. Mapping of tj-piRNAs produced in the presence and absence of the Hel-C domain in OSCs. ",
    "molecules": "piRNAs"
  },
  {
    "caption": "E. Scatterplot showing changes in the abundances of transposon-repressing piRNAs and non-transposon-repressing piRNAs in the presence and absence of the Hel-C domain. Orange and blue dots indicate the number of normalized piRNA reads mapped uniquely to transposons in the reverse orientation and coding genes in the sense orientation, respectively.",
    "molecules": "piRNA, piRNAs"
  },
  {
    "caption": "F. The relative enrichments of RNAs coimmunopurified with WT Yb and ∆Hel-C. Bars and error bars represent means ± SEM values of three independent experiments. In addition to tj, three genic piRNA sources that were characterized in previous studies (dm/Myc [59], c-Fos/kay [60], and CG9257 [34]) are also shown. rp49 was used as a negative control. p value was calculated by t-test. ***: p < 0.001.",
    "molecules": "piRNA"
  },
  {
    "caption": "A. Yb bodies (Yb in green) disappeared after 1,6-hexanediol treatment of OSCs, as did P bodies (Dcp1 in red). Nuclei are shown in blue. Scale bar: 5 μm.",
    "molecules": "1,6-hexanediol"
  },
  {
    "caption": "D. Yb Y23A and F129A mutants failed to produce flam-piRNAs. miR-310 was used as a loading control.",
    "molecules": "piRNAs"
  },
  {
    "caption": "E. Mapping of flam- and tj-piRNAs produced in the flamKG mutant and heterozygote ovaries.",
    "molecules": "piRNAs"
  },
  {
    "caption": "A HeLa cells expressing LAMP1-RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin-3 (Gal3) antibodies, and analyzed by confocal microscopy.",
    "molecules": "LLOMe"
  },
  {
    "caption": "C Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).",
    "molecules": "LLOMe"
  },
  {
    "caption": "D Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout and stained for endogenous Gal3. Quantification of the number of Gal3-positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥30 cells quantified per condition (one-sided Welch's unequal variances t-tests comparing the siCtrl with sip97).",
    "molecules": "LLOMe"
  },
  {
    "caption": "E Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM) or the p97 inhibitor NMS-873 (5 µM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition (one-sided Welch's t-tests).",
    "molecules": "NH4Cl, bortezomib, DMSO, LLOMe, NMS-873"
  },
  {
    "caption": "F p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments.",
    "molecules": "LLOMe"
  },
  {
    "caption": "A Impaired clearance of damaged lysosomes in cells expressing a p97 disease-mutant. Wild-type and p97R155H/+MEFs were LLOMe-treated for 1 h and released for indicated times. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD, Student's unpaired t-test). Scale bar, 25 µm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "B Stable U2OS cell lines were doxycycline-induced to express myc-tagged p97 wild-type or disease-mutants as indicated. Induced parental cells served as control (U2OS -). Cells were treated with LLOMe for 1h and chased for indicated time before fixation. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments. Comparable expression levels were verified (see Figure EV2A).",
    "molecules": "doxycycline, LLOMe"
  },
  {
    "caption": "C Stable U2OS cell lines described in (B) were treated with LLOMe for 1 h and chased for indicated time. LC3 and GAPDH levels were analyzed by immunoblotting. Quantification is shown in Figure EV2B.",
    "molecules": "LLOMe"
  },
  {
    "caption": "C YOD1, UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes. Cells expressing indicated tagged proteins were LLOMe-treated for 1 h or with vehicle alone (control), fixed 2 h after washout, and stained for endogenous LAMP1 and, in the case of PLAA, with Alexa-488 conjugated anti-HA antibodies. See Figure EV3B for quantification.",
    "molecules": "LLOMe"
  },
  {
    "caption": "D An ATP-stabilized complex of p97 containing both UBXD1 and PLAA. Stable HEK293 cell lines were doxycycline-induced to express p97 wild-type (wt) or the ATPase-mutant E578Q (EQ) at near endogenous level. Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies. Arrowheads indicate endogenous (lower band) and induced p97 (upper band).",
    "molecules": "ATP, doxycycline"
  },
  {
    "caption": "A K63- and K48-ubiquitination of damaged lysosomes. Untransfected HeLa cells, or cells expressing mCherry-Gal3 (Ch-Gal3) were LLOMe-treated for 1 h. 2 h after washout, cells were stained with antibodies specific for K63 or K48 chains, and LAMP1 or p62 as indicated. Percentage of K63- or K48-ubiquitinated LAMP1 vesicles and percentage of p62-positive K63 or K48 vesicles were quantified from three independent experiments (mean ± SD, Student's unpaired t-test for LAMP1). * = p < 0.05.",
    "molecules": "LLOMe"
  },
  {
    "caption": "F ELDR components target K48-conjugates on lysosomes. Detection of GFP-YOD1-CS or UBXD1-GFP alone, or co-transfected with PLAA-HA and GFP-p97-EQ, 2 h after LLOMe washout and co-stained with indicated antibodies.",
    "molecules": "LLOMe"
  },
  {
    "caption": "A Time course of K63- and K48-ubiquitination. Control-depleted cells were fixed at indicated time points after LLOMe washout and stained with chain-specific antibodies and LAMP1. Automated quantification of LAMP1 vesicles positive for K63 or K48 chains. Data represent mean ± SD of three independent experiments.B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes. Experiment and quantification as in (A) in cells treated with p97 or YOD1 siRNA oligos.",
    "molecules": "LLOMe"
  },
  {
    "caption": "D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site. LLOMe-treated cells expressing GFP-YOD1-CS or the double mutant GFP-YOD1-CS-MutS2 were chased for 2 h and stained for K48 chains and LAMP1. Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined.",
    "molecules": "LLOMe"
  },
  {
    "caption": "A Largely mutually exclusive localization of LC3 and K48 chains. GFP-LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains. Correlation coefficients were calculated for colocalization of LC3 with K63 or K48. The whiskers represent 10th and 90th percentile. Points below and above the whiskers are drawn as individual dots. The single line indicates the median. *** = p < 0.001 (Mann-Whitney-U test).",
    "molecules": "LLOMe"
  },
  {
    "caption": "E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation. Cells were transfected with control (Ctrl), p97 or YOD1 siRNA oligos, treated with LLOMe, fixed after 12 h chase and stained for endogenous LC3 and K48 chains. Quantification of the number of K48-positive vesicles per cell and number of LC3-positive K48 vesicles per cell was determined. Data represent mean ± SD of three independent experiments. Scale bars, 10 µm.",
    "molecules": "LLOMe"
  },
  {
    "caption": "(D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.",
    "molecules": "Blue Coomassie, HEPES, MgCl2, SDS, Thr72, Thr73"
  },
  {
    "caption": "(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.",
    "molecules": "GDP, GTP, GTPγS, phosphatase, Thr72"
  },
  {
    "caption": "(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).",
    "molecules": "phospho-peptide, phosphopeptide, phosphatase, Thr72, Thr73"
  },
  {
    "caption": "(F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.",
    "molecules": "GTPγS, malachite green, Thr72"
  },
  {
    "caption": "(C) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa.",
    "molecules": "Blue Coomassie"
  },
  {
    "caption": "(D) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.",
    "molecules": "GTPγS, malachite green, Thr72, Thr73"
  },
  {
    "caption": "(C) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins (25 nM for the protein substrate, 50 nM for the peptide substrate) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS). The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. (D) In vitro malachite green assay against 32 μM pThr72-Rab8a peptide (left) and pThr73-Rab10 peptide mimic (right). The experiments were repeated 3 times. The error bars represent standard error (SE) of mean of the technical replicates.",
    "molecules": "GTPγS, malachite green, Thr72, Thr73"
  },
  {
    "caption": "(E, F) (E) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system. Left, each lane represents cell extract obtained from a different dish of cells (two biological replicates per condition without MLi-2 treatment, one biological replicate per condition with MLi-2 treatment). Right, the ratio of phospho-Rab8a/total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild-type expressing cells. (F) As in (E) assessing phospho-Rab10 levels. For quantitation the two replicates from the presented blots were used. The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates.",
    "molecules": "MLi-2"
  },
  {
    "caption": "(B) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.",
    "molecules": "GTPγS, malachite green, Thr72, Thr73"
  },
  {
    "caption": "(C) 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie.",
    "molecules": "Blue Coomassie"
  },
  {
    "caption": "(E) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS) using the malachite green assay time course. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Quality of purified proteins (right panel) is shown using the protocol in (C).",
    "molecules": "GTPγS, malachite green, Thr72"
  },
  {
    "caption": "A) Overview of immunofluorescence staining  for centrosomes  (TUBG1) and cilia  (ARL13B) in control (Con) (left) and EPZ  treated sections (right). Dotted lines mark either ventricular   (bottom part) or pial   (top part) surface of the tissue . Data information: Scale bars in all panels: 10 µm.",
    "molecules": "EPZ"
  },
  {
    "caption": "B) Left: quantification of total centrosome  numbers in control and EPZ  treated sections in 100 µm. Right: number of centrosomes  in ventricular zone (VZ),  subventricular zone (SVZ), and cortical plate (CP) expressed as percentage of total per condition. C) Left: quantification of total cilia  numbers in control and EPZ  treated sections in 100 µm. Right: number of cilia  in VZ, SVZ, and CP  expressed as percentage of total per condition. Data information: T-test, two-tails was performed for all quantifications, *p<0.05, ***p<0.001, error bars represent SD.",
    "molecules": "EPZ"
  },
  {
    "caption": "D) Overview of VZ  showing TUBG1  and ARL13B immunofluorescence staining  in control (top) and EPZ  (bottom) condition. Squared parentheses indicate the ventricular  and abventricular   compartment. Dotted lines mark ventricular  surface of tissue slide. Data information: Scale bars in all panels: 10 µm.",
    "molecules": "EPZ"
  },
  {
    "caption": "A) Immunostaining  of control and EPZ  treated sections showing SOX2  and EOMES expression  and EdU  labelling. Scale bar: 20 µm. Dotted lines mark ventricular   surface of the tissue. B) Quantification of SOX2-, EOMES-, EdU- positive cells, and co-labelled cells normalised to 100 μm of apical surface. T-test was performed, *p<0.05, **p<0.01, error bars represent SEM. Total n of independent experiments for EdU  labelling: 9 (n=4 control samples, n=5 EPZ-treated samples). Total n of independent experiments for SOX2, EOMES, and double stainings: 6 (n=3 control samples, n=3 EPZ-treated samples).",
    "molecules": "EdU, EPZ"
  },
  {
    "caption": "E) Immunofluorescence staining  showing overview of CTIP2-positive cells in control and EPZ  treated sections. Scale bars: 20 µm. Dotted lines mark either ventricular  or pial  surface of the tissue. F) Quantification of total CTIP2-positive cells, normalised to 100 µm of apical surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). Error bars represent SEM.",
    "molecules": "EPZ"
  },
  {
    "caption": "G) Immunofluorescence staining  showing SOX2-, EOMES-, and CTIP2-positive cells in control and EPZ  treated sections. Scale bars: 10 μm. Dotted lines mark the ventricular   surface of the tissue. Magnifications on the right show exemplarily a CTIP2  positive cell (arrowhead) that is negative for SOX2  and positive for EOMES  in EPZ  treated condition. H) Quantification of CTIP2-positive cells in the VZ, normalised to 100 µm of apical  surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). T-test was performed, **p<0.01, error bars represent SEM.",
    "molecules": "EPZ"
  },
  {
    "caption": "A) Microinjected cells in the VZ  (magenta) in sections treated with DMSO  (Con) (upper panel) or EPZ  (lower panel) showing Dx-A555 labelled cells after 24 h in slice culture. White arrow indicates the apical  process contacting the ventricular    surface. White arrowheads indicate the apical-directed process of delaminated cells. Scale bars: 10 µm. B) Immunofluorescence staining  of microinjected sections showing the progeny of microinjected cells (a daughter cell pair) and expression  of EOMES  and TUBB3. White arrows indicate the apical-directed processes. Note that in the top panel the apical-directed process contacts the  ventricular   surface (dotted white line) (EOMES-, TUBB3-negative), while in the bottom panel it does not contact the  ventricular   surface, indicating that the cell has delaminated (EOMES-, TUBB3-positive). Scale bars MIP: 10 µm; insets: 5 µm. Data information: VZ  -  Ventricular Zone, SVZ  - Subventricular Zone, CP  - Cortical Plate. Scale bars in all main panels: 20 µm, and all insets: 5 µm.",
    "molecules": "Dx-A555, DMSO, EPZ"
  },
  {
    "caption": "C) Quantification of microinjected cells found as single-cell or two-cells clusters, expressed as percentage of total Dx-A555-positive cells per condition. D) Quantification of microinjected cells with or without contact to the apical  surface of the organotypic slice expressed as percentage of total  Dx-A555-positive cells per condition. Total n of independent microinjection experiments: 6 (n=3 wild type embryos, n=3 in Tis21-GFP embryos). Total number of cells scored: Con, 156 cells; EPZ, 287 cells. E) EOMES-positive and EOMES-negative cells plotted as a percentage of total cells scored per condition. F) TUBB3-positive and TUBB3-negative cells in control and EPZ  conditions plotted as a percentage of total cells scored per condition. Data information: Cell scoring based on TUBB3  or EOMES expression  are from three independent experiments (n=3; Con: 70 cells, EPZ: 113 cells). Fisher's exact test was performed for all quantifications, ***p<0.001, error bars represent SEM. MIP - maximum intensity projection.",
    "molecules": "Dx-A555, EPZ"
  },
  {
    "caption": "A) Microinjected cells in the VZ  (magenta) of control (Con) and EPZ  treated slices 24 h after microinjection and slice culture. Left: daughter cell pair in control section with asymmetric GFP expression, with cell 1 (basal  location) being mild GFP-positive, and cell 2 (apical  location) GFP-negative. Right: EPZ  treated section showing a daughter cell pair with symmetric  mild GFP-positive expression. Scale bars: 5 µm. Dotted shapes indicate the contour of Dx-A555 cell soma. B) Graph showing proportion of labelled cells expressing GFP-positive, -mild positive, or -negative, expressed as percentage of total cells per condition. Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. VZ  - ventricular zone. The following number of cells were scored: B) TIS21-GFP  - 267 total cells were quantified (Con: 86 cells, EPZ: 181 cells).",
    "molecules": "Dx-A555, EPZ"
  },
  {
    "caption": "C) Symmetry  or asymmetry  in daughter cells' apical  contact (yes/yes: both cells have apical  contact; yes/no: one cell has apical  contact and the other not; no/no: both cells lack apical  contact). D) Symmetry  (pos/pos, neg/neg) or asymmetry  (pos/neg) in GFP expression  in daughter cells expressed as percentage of total cells per condition. E) Symmetry  or asymmetry  in TUBB3 expression  in daughter cells plotted (pos/pos: both cells express TUBB3; pos/neg: one cell expresses TUBB3  and the other not; neg/neg: none of the cells expresses TUBB3). Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. The following number of cells were scored: C) Apical  contact - 85 total daughter cell pairs (Con: 31 pairs, EPZ: 54 pairs). D) TIS21-GFP  - 58 total daughter cell pairs (Con: 21 pairs, EPZ: 37 pairs). E) TUBB3  - 50 total daughter cell pairs (Con: 17 pairs, EPZ: 33 pairs).",
    "molecules": "EPZ"
  },
  {
    "caption": "F) Phalloidin staining  of control and EPZ  treated sections showing cells during mitosis. Scale bar: 5 µm. G) Pie chart showing the proportion of cells with different angles of the cleavage plane in respect to the ventricular   surface, divided in groups with increments of 30° . H) Percentage of cells undergoing symmetric  or asymmetric   cell division, defined by the cleavage plane bypassing or bysecting the apical plasma membrane, in control and EPZ  treated samples. Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. VZ  - ventricular zone. The following number of cells were scored: G) For control condition 41 cells from four independent samples, 5 sections and 15 different images were assessed. EPZ  treated quantifications were performed on a total of 26 cells from 3 independent samples, 5 sections and 16 images. H) For control condition a total of 37 cells from four independent samples, 5 sections and 15 different images were assessed. EPZ  treated quantifications were performed on a total of 23 cells from 3 independent samples, 5 sections and 16 images.",
    "molecules": "Phalloidin, EPZ"
  },
  {
    "caption": "C) Immunostaining  showing ASNS-positive cells in the VZ, SVZ  and CP  of control and EPZ  treated sections; and signal intensity for each condition. Scale bars: 50 µm. Dotted lines mark either ventricular  or pial  surface of the tissue .",
    "molecules": "EPZ"
  },
  {
    "caption": "D) ChIP-seq and ATAC-seq  tracks showing distribution of peaks for selected chromatin  marks on the Asns  locus for NPCs  and cerebral cortex  of E12.5 and E14.5 mice. Tracks for control and EPZ  conditions are compared in NPCs. Indicated is the TSS of Asns.",
    "molecules": "EPZ"
  },
  {
    "caption": "F) ChIP-qPCR  analysis for DOT1L  and IgG (n=7; biological replicates) at the TSS on Asns  genomic locus in in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. **p<0.01. G) ChIP-qPCR  analyses for EZH2  (n=5; biological replicates) and H3K27me3 (n=9; biological replicates) at the TSS on Asns  genomic locus in control and EPZ  treated condition of in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. *p<0.04.",
    "molecules": "EPZ"
  },
  {
    "caption": "A) A representative autoradiograph from a GST-pulldown of CENP-C to map the CENP-C binding domain of M18BP1-1. Radiolabeled Myc-M18BP1-1 truncations (amino acids indicated at the top) were mixed with recombinant GST-CENP-C1191-1400. Material bound to glutathione agarose was resolved by SDS-PAGE and visualized by autoradiography C) Quantification of (A). Bound material as a fraction of the input was calculated from autoradiographs. The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1-1161-580, the CENP-C binding domain.",
    "molecules": "agarose, glutathione"
  },
  {
    "caption": "C) Cdk inhibition with flavopiridol prevents M18BP1-1161-580 localization at metaphase centromeres. Representative images showing Myc-M18BP1-1161-580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors. Inhibitor treatment is indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.",
    "molecules": "flavopiridol"
  },
  {
    "caption": "A) Representative images showing G1 localization of human M18BP1WT or human M18BP1SANTA in sfGFP-AID-M18BP1 DLD1 cells treated with 1 mM IAA for 24 h to remove endogenous M18BP1. Centromeric localization is indicated by localization with ACA (α-centromere autoantibody serum), early G1 cell cycle state is indicated by midbody staining in the tubulin channel. M18BP1 species indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X.",
    "molecules": "IAA"
  },
  {
    "caption": "(C) Confocal images of formaldehyde fixed GFP+ S. flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***, P ≤ 0.001. Scale bars equal 5 µm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis.",
    "molecules": "Alexa-Fluor647, formaldehyde, GTP"
  },
  {
    "caption": "(B) Confocal time-lapse microscopy was used to image 10 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP+ S. flexneri. Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points.",
    "molecules": "Alexa-Fluor647, formaldehyde, GTP"
  },
  {
    "caption": "(C) Images were taken at 45 min after addition of 10 µM Alexa-Fluor647-hGBP1F to formaldehyde fixed GFP+ S. flexneri in the presence of indicated nucleotides (GTP, natural substrate; GppNHp, non-hydrolysable GTP analog; GTPγS, slowly hydrolysable GTP analog; GDP·AlFX, GTP transition state analog). hGBP1-associated bacteria after 45 min were quantified. Combined data from two independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.",
    "molecules": "Alexa-Fluor647, AlF, formaldehyde, GDP, GTP, GTPγS, GppNHp"
  },
  {
    "caption": "(A) Polymerization of 10 µM hGBP1F and hGBP1FR584-586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis.",
    "molecules": "GTP"
  },
  {
    "caption": "(B) Scanning-electron micrographs of live S. flexneri incubated with no protein or with 5 µM of either hGBP1F, hGBP1FR584-586A, or hGBP1FR48A (non-polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces.",
    "molecules": "GTP"
  },
  {
    "caption": "(C) Confocal time-lapse microscopy frames of formaldehyde fixed GFP+ S. flexneri following admixture of 10 µM Alexa-Fluor647-labeled hGBP1F or hGBP1FR584-586A and 2 mM GTP. Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by by unpaired t-tests, two-tailed. ***, P ≤ 0.001; ****, P ≤ 0.0001.",
    "molecules": "Alexa-Fluor647, formaldehyde, GTP"
  },
  {
    "caption": "(A) Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F to formaldehyde fixed gram-negative and gram-positive bacteria expressing GFP (S. enterica Typhimurium [ST], L. monocytogenes [Lm]), or RFP (S. flexneri [Sf], uropathogenic E. coli [UPEC]), or dsRed (L. pneumophila [Lp], S. aureus [Sa]).",
    "molecules": "dsRed, Alexa-Fluor488, Alexa-Fluor647, formaldehyde, GTP"
  },
  {
    "caption": "(B) (Left panel) Confocal time-lapse microscopy frames of Alexa-Fluor488-LPS-O55:B5 after addition of 5 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP. (Right panel) Confocal images taken of 5 µM Alexa-Fluor488-LPS-O55:B5 20 min after addition of 5 µM Alexa-Fluor647-hGBP1F or -hGBP1FR48A supplemented with 2 mM GTP. Graphs depict average aggregate area of Alexa-Fluor488-LPS-O55:B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant. Mean area ± SEM of combined data from three independent experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test.. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.",
    "molecules": "Alexa-Fluor488, Alexa-Fluor647, GTP, LPS"
  },
  {
    "caption": "(C) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS-O55:B5 was monitored by absorption spectroscopy at 350 nm. The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points. Maximal hydrolysis rates (turnover numbers) were determined for hGBP1F variants in the presence and absence of 5 µM LPS-O55:B5. Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.",
    "molecules": "GTP, LPS"
  },
  {
    "caption": "(D) Formaldehyde fixed GFP+ S. flexneri supplemented with varying LPS-O55:B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP. After 60 min hGBP1F-enclosed S. flexneri were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.",
    "molecules": "Formaldehyde, GTP, LPS"
  },
  {
    "caption": "(E) Confocal images of formaldehyde fixed GFP-, RFP-, or dsRed-expressing gram-negative bacteria 60 min after addition of 5 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS-O55:B5. After 60 min hGBP1F-enclosed gram-negative bacteria were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.",
    "molecules": "Alexa-Fluor488, Alexa-Fluor647, formaldehyde, GTP, LPS"
  },
  {
    "caption": "(B) Time-lapse microscopy of fixed GFP+ co-isogenic S. flexneri wild type and rfaL mutant after adding 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP. Bacteria bound by hGBP1F at 5 min and hGBP1F-enclosed bacteria after 60 min were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; ****, P ≤ 0.0001.",
    "molecules": "Alexa-Fluor647, GTP"
  },
  {
    "caption": "(C) Scanning-electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces",
    "molecules": "GTP"
  },
  {
    "caption": "(E) Confocal images of formaldehyde fixed, co-isogenic GFP+ wild type and mutant S. flexneri strains harboring rfb regions of E. coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP.",
    "molecules": "Alexa-Fluor647, formaldehyde, GTP"
  },
  {
    "caption": "(A) Following incubation for 1.5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP, live bacteria were treated with antibiotics (2.5 µg/ml PMB or 5 mg/ml lysozyme) for 30 min or left untreated. The number of viable bacteria was subsequently determined by colony forming unit (CFU) counts. Graphs show mean CFUs ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.",
    "molecules": "lysozyme, GTP, PMB"
  },
  {
    "caption": "(B) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A supplemented with 2 mM GTP. Following 60 min of incubation hGBP1F-enclosed UPEC were quantified. Graphs depict mean ± SEM of combined data from three independent experiments. Significance was determined by unpaired t-test, two-tailed. ****, P ≤ 0.0001.",
    "molecules": "Alexa-Fluor488, Formaldehyde, GTP"
  },
  {
    "caption": "(C) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A and 2 mM GTP. Samples were fixed after 60 min incubation and stained with an antibody against the inner core/ lipid A fragment of E. coli LPS (anti-LPS). The frequency of anti-LPS stained UPEC was quantified. Graphs show mean ± SEM of combined data from three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.",
    "molecules": "Alexa-Fluor488, Formaldehyde, GTP, lipid A, LPS"
  },
  {
    "caption": "(B Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (B) Unprimed HeLa cells were infected with wild type or rfaL S. flexneri. Total and unipolar localization of IcsA as well as the actin tail formation were quantified at 1 hpi. Significance was determined by unpaired two-tailed t-tests. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01.",
    "molecules": "poly-D-lysine"
  },
  {
    "caption": "Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (C- mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for IcsA, N-WASP Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.",
    "molecules": "poly-D-lysine"
  },
  {
    "caption": "Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. D) mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for Arp2, and F-actin at 2.5 hpi. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.",
    "molecules": "poly-D-lysine"
  },
  {
    "caption": "Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (E) IFNγ-primed and unprimed wild type and hGBP1-KO HeLa cells were infected with S. flexneri ΔipaH9.8 and subcellular localization of IcsA, N-WASP, Arp2, and F-actin was assessed and quantified at 2.5 hpi. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.",
    "molecules": "poly-D-lysine"
  },
  {
    "caption": "(A) Immunohistochemistry  of Bcl6 (red), Cux1  (green), and Satb2  (blue) in P2 Bcl11aF/F;Emx1IRESCre and control neocortex. Nuclei  are stained  with Dapi  (white). (B) Relative quantification of Bcl6+, Satb2+, and Cux1+ cells in Bcl11aF/F;Emx1IRESCre and control neocortex  (n = 4). (C) Numbers of Cux1+ or Satb2+ cells that coexpress Bcl6  are reduced in Bcl11aF/F;Emx1IRESCre compared to control neocortex  (n = 4). Data information: All graphs represent the mean ± s.e.m.; Student's t test; *** p < 0.001. Scale bar, 50 µm.",
    "molecules": "Dapi"
  },
  {
    "caption": "(C-E) Immunohistochemistry  of electroporated P2 Bcl11aF/F neurons  in superficial cortical  layers with GFP  (green) and Bcl6  (red, C), Cux1  (red, D), or Satb2  (red, E) antibodies. Bcl6 expression  is specifically downregulated Bcl11aF/F neocortex  upon electroporation of CAG-CreGFP in comparison to CAG-CtlGFP control plasmid. Nuclei  are stained  with Dapi  (white). Data information: White arrowheads point at GFP+ cells that also express Bcl6, Cux1, Satb2  indicate cells expressing only GFP+. Scale bars, 20 µm (C-E),",
    "molecules": "Dapi"
  },
  {
    "caption": "G)  Protein decay analysis of HP1 interaction-defective  Setdb1  mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated  times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).",
    "molecules": "CHX, MG132"
  },
  {
    "caption": "D)  Electron microscopy  images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT  for 4 days (right). Boxes represent enlarged images.",
    "molecules": "4OHT"
  },
  {
    "caption": "E) Evaluation of HP1β  mutants for maintaining H3K9me2/3 and ensuring protein stability of  H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in  HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT  treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).",
    "molecules": "4OHT"
  },
  {
    "caption": "C) Evaluation of  HP1β mutants for ensuring protein stability of Jmjd1a/b. Protein levels of Jmjd1a/b in  HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after  4OHT  treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).",
    "molecules": "4OHT"
  },
  {
    "caption": "In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.",
    "molecules": "Ub, Ubiquitin"
  },
  {
    "caption": "Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.",
    "molecules": "Ponceau S"
  },
  {
    "caption": "(C) Representative example of immunofluorescence staining of N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue), cell mask (red). Scale bar represents 50µM.",
    "molecules": "DAPI"
  },
  {
    "caption": "Fold induction of (F) interferons (IFNβ, IFNλ1 and IFNλ3) pro-inflammatory mediators (IL-6 and CCL5) each overlaid with SARS-CoV-2 E (qRT-PCR).",
    "molecules": "interferons"
  },
  {
    "caption": "Peak SARS-CoV-2 replication precedes innate immune activation. (A-I) (A,C) Representative images of NF-κB p65 (A) (red) and IRF3 (C) (red) nuclear localisation in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. SARS-CoV-2 N protein (green. (E and G) Representative images of IL-6 mRNA (E) detected by FISH (red) and N protein (green) , or IFIT1 mRNA (G) (green) with N protein (red), both with nuclei (DAPI, blue) in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. (B, D, F, H, I) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF-κB p65 (B), IRF3 (D) , or overall integrated intensity for IL-6 (F) or IFIT1 (H) mRNA over time in N protein positive cells and N protein negative cells (I). n=2. Kruskal-Wallis test with Dunn's multiple comparison. * (p<0.05), **** (p<0.0001). Scale bar represents 50µM.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E-H) Measurement of (E) viral genomic and subgenomic E RNA (F) fold induction of CXCL10, (G) IFIT2 and (H) and IL-6 at 24 hpi, of Calu-3 cells with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) with Remdesivir treatment (5μM) prior to, at the time of, or 8 h post-infection. Mean +/- SEM, n=3, One way ANOVA with Dunnett's multiple comparisons test to compare to untreated infected condition ('mock'), ** (p<0.01), *** (p<0.001), **** (p<0.0001).",
    "molecules": "Remdesivir"
  },
  {
    "caption": "(I) Representative example of immunofluorescence staining of dsRNA (red) and N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue). Scale bar represents 50µM.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G-H) Viral genomic and subgenomic E RNA at 24 hpi (RT-qPCR) with DMSO or TPCA (G) or Rux (H) treatment. Data information: (A-H) Mean +/- SEM, n=3, statistical comparisons are made by unpaired t test comparing inhibitor-treated to mock-treated SARS-CoV-2 infected conditions at each MOI and each timepoint. * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001).",
    "molecules": "DMSO, Rux, TPCA"
  },
  {
    "caption": "(B-J) Calu-3 cells were transfected with siRNA targeting MAVS or non-targeting control (siCtrl) (B-D) or treated with DMSO vehicle or inhibitors 10 μM TPCA-1 (E-G) or 2 μM Ruxolitinib (Rux) (H-J) as shown, and were mock-infected or infected with SARS-CoV-2 at MOI 0.04 TCID50VERO/cell. Virus containing conditioned media (CoM) was harvested at 48 hpi. MDM were treated with Calu-3 virus containing CoM for 6 hpi, before washing and measuring MDM gene expression (B, E, H), and MDM activation markers by flowcytometry 48 h later (C,D,F,G,I,J) , plotting relative median fluorescent intensity (MFI) compared to mock-infected siCtrl (C, D) or mock-infected DMSO control (F, G, I, J). Legends in (B), (E) and (H) apply to (C,D), (F,G) and (I,J) respectively. The inhibitors in (E) and (H) were tested side-by-side with the same mock condition. Mean +/- SEM shown, data from 4-6 independent MDM donors is shown. Statistical comparison by two-tailed paired t-test comparing MDM exposed to control infected CoM to siMAVS/inhibitor treated infected CoM. * (p<0.05), ** (p<0.01), *** (p<0.001).",
    "molecules": "DMSO, Rux, Ruxolitinib, TPCA-1"
  },
  {
    "caption": "(C) The levels of trehalose compared to a DMSO treated control are shown for both wild-type yeast and gpr1 strains treated with predicted antagonists for GPR1. The bar height indicates mean values, and error bars indicate the standard deviation of the mean. Asterisks indicate a p-value of less than 0.05 from a two-sided student's T-test (n = 4 biological replicates) when comparing The WT and gpr1 reponses to the drug. Results are representative of 3 independent experiments.",
    "molecules": "DMSO, trehalose"
  },
  {
    "caption": "(C) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated compounds at a concentration of 20 µM across four biological replicates. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).",
    "molecules": "DMSO"
  },
  {
    "caption": "(D) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated concentrations of ibuprofen. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).",
    "molecules": "DMSO, ibuprofen"
  },
  {
    "caption": "(E) The average relative amount of trehalose in ibuprofen-treated cells compared to a DMSO treatment is shown. The bar height indicates mean values, error bars indicate the standard deviation of the mean, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing to the WT control (n = 8 biological replicates). Results are representative of 3 independent experiments.",
    "molecules": "DMSO, ibuprofen, trehalose"
  },
  {
    "caption": "(A) Assessment of vacuole (arrows) formation in neutrophils after stimulation with PMA (II) and rapamycin (III), compared with control cells (I: treated with PBS and serum) (magnification 1000×).",
    "molecules": "PMA, rapamycin"
  },
  {
    "caption": "(B) (I) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin (RAP) compared with control cells (PBS) and cells treated with PMA and 3‐MA (magnification 400 and 1000×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. Data are representative of six independent experiments and are presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05: PMA (bar 2: 86.16±6.49%), rapamycin (bar 3: 65.83±8.47%) treated cells, cells treated with PMA and 3‐MA (bar 4: 9.17±0.75%) and cells treated with rapamycin and 3‐MA (bar 5: 8.5±1.87%) compared with control conditions (bar 1: 15.67±3.50%). The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3‐MA (bar 4) or rapamycin and 3‐MA (bar 5) was significantly lower (p<0.05) compared with cells treated with PMA (bar 2) and rapamycin (bar 3), respectively.",
    "molecules": "3‐MA, PMA, rapamycin"
  },
  {
    "caption": "(C) Analysis of LC3B‐I to LC3B‐II conversion in cells treated with PMA (lane IV) and rapamycin (lane II), as observed by LC3B immunoblotting. Effect of 3‐MA in both PMA (lane V) and rapamycin (lane I)‐treated cells. Lane III: control cells, and lane VI: cells treated only with 3‐MA. One representative out of six independent experiments is shown.",
    "molecules": "3‐MA, PMA, rapamycin"
  },
  {
    "caption": "(D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown.",
    "molecules": "LY294002, PMA"
  },
  {
    "caption": "(E) Immunofluorescence confocal microscopy staining of LC3B. (I) Control cells treated with PBS and serum, (II) PMA‐treated cells. 1. DAPI: blue, 2. LC3B: red and 3. merge. (III) Percentage of PMN with LC3B aggregates. Control conditions (bar 1: 17.7±2.4%), 3‐MA (bar 2: 3±1.01%), PMA (bar 3: 69±2.1%), PMA and 3‐MA (bar 4: 4±1.9%), rapamycin (bar 5: 56±1.6%). Data are representative of five independent experiments and presented as mean±SD; Student's t‐test, *p<0.01 compared to the control (bar 1) or the indicated comparision (bar 3 vs 4).",
    "molecules": "3‐MA, PMA, rapamycin"
  },
  {
    "caption": "ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.",
    "molecules": "3‐MA, PMA, rapamycin"
  },
  {
    "caption": "(A) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)−treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched−pairs test, *p<0.05, n.s.=not significant.",
    "molecules": "LPS, LTA, loxorubin, poly I:C, R848"
  },
  {
    "caption": "(B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown.",
    "molecules": "LPS, LTA, loxoribin, R848"
  },
  {
    "caption": "(C) LC3B−II/LC3B-I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched−pairs test, *p<0.05.",
    "molecules": "LPS, LTA, loxorubin, R848"
  },
  {
    "caption": "(D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium−treated control cells. Dotted line represents LC3B mRNA levels of medium-treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.",
    "molecules": "LPS, LTA, R848"
  },
  {
    "caption": "(E) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium-treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.",
    "molecules": "LPS"
  },
  {
    "caption": "(A) Effect of 3‐MA on neutrophil phagocytic capacity of opsonized E. coli as observed by flow cytometry. Percentage of phagocytosing neutrophils after 3‐MA pretreatment (III) compared with cells incubated with opsonized bacteria (II). Data are representative of six independent experiments and are presented as mean±SD, (I: control unstimulated group), (IV) MFI representation (bar 1: control: 1±1.9, bar 2: E. coli: 387.7±33.3, bar 3: E. coli+3‐MA: 25.13±18, Wilcoxon matched‐pairs test, *p<0.05; bar 2 compared with bar 1 and bar 3 compared to bar 2).",
    "molecules": "3‐MA"
  },
  {
    "caption": "(C) LC3B conversion analysis in E. coli‐phagocytosing neutrophils (lane II), control neutrophils (lane I) and E. coli‐phagocytosing neutrophils after 3‐MA pretreatment (lane III). One representative out of six independent experiments is shown.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(D) Effect of 3‐MA administration to E. coli‐phagocytosing neutrophils 15 min after the initiation of phagocytosis (lane III) as detected by LC3B conversion analysis. Lane II represents E. coli‐phagocytosing neutrophils and lane I control neutrophils. One representative out of four independent experiments is shown.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(E) (I) Percentage of neutrophils with LC3B punctuated structures. E. coli (bar 1: 84±2.2%) compared with E. coli+3‐MA (bar 2: 4±1.95%). Data are representative of six independent experiments and presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (II) Formation of LC3B puncta in phagocytosing neutrophils and (III) in cells treated with 3‐MA before incubation with opsonized E. coli, as observed by immunofluorescence confocal microscopy (1. LC3B: red; 2. FITC conjugated E. coli: green; 3. DAPI: blue; and 4. merge). Solid line arrows point the colocalization of LC3B puncta with E. coli bacteria. Dashed line arrows point LC3B aggregates that are not colocalized with E. coli.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(A) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E. coli stimulation (lane I: PMA, lane II: PMA+BHA, lane III: E. coli, lane IV: E. coli+BHA, lane V: control cells). One representative out of four independent experiments is shown.",
    "molecules": "BHA, PMA, ROS"
  },
  {
    "caption": "(B) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E. coli−phagocytosing neutrophils (lane I: PBS, lane II: E. coli, lane III: E. coli+DPI). One representative out of four independent experiments is shown.",
    "molecules": "DPI"
  },
  {
    "caption": "(C) (I) Effect of NADPH‐oxidase inhibition with DPI in the formation of acidified autophagosomes in E. coli‐phagocytosing neutrophils. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli−phagocytosing PMN (77.17±8.33%) and PMN treated with DPI before incubation with opsonized E. coli (21.67±4.37%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05",
    "molecules": "DPI"
  },
  {
    "caption": "(D) (I) Assessment of formation of acidified autophagosomes in neutrophils pretreated with BNP before stimulation with peptidoglycan (PGN), compared with cells treated with PGN. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. PGN‐treated neutrophils (74.83±9.09%) and neutrophils pretreated with BNP before the addition of PGN (17.33±2.07%) compared with control conditions (14.67±3.20%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05, n.s.=not significant:",
    "molecules": "BNP"
  },
  {
    "caption": "(E) Alterations in LC3B conversion in BNP pretreated, peptidoglycan‐stimulated neutrophils (lane III), (lane I: medium‐treated cells, lane II: peptidoglycan‐treated cells). One representative out of four independent experiments is shown.",
    "molecules": "BNP"
  },
  {
    "caption": "(F) Effect of PI3K inhibition with 3‐MA in the percentage of PMA‐induced oxidizing cells. PMA (99.41±0.33%) and PMA+3‐MA (15.82±3.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.",
    "molecules": "3‐MA, PMA"
  },
  {
    "caption": "A. 293T cells were co-transfected with CMV-renilla, 8X-TBS (eight repeats of the TEAD-binding-sequence) luciferase reporter, Flag-YAP, HA-TEAD2 and wild-type (WT) or catalytically inactive (CS) USP9X-V5. One day after transfection, luciferase activity was measured and normalized with respect to renilla activity. The value for Flag-YAP/HA-TEAD-transfected cells (2nd column) was adjusted to 1. Shown below is a representative Western blot showing that the expression levels were comparable between samples (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).",
    "molecules": "TBS"
  },
  {
    "caption": "B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student's t-test).",
    "molecules": "TBS"
  },
  {
    "caption": "E. RPE cells were transfected with the indicated siRNAs for 24 hours, and then co-transfected with CMV-renilla and 8X-TBS luciferase. One day after the latter transfection, the cells were re-seeded to either sparse or dense conditions, and reporter activity was measured at 24 hours after re-seeding (n=3). Error bars indicate the S.E.M. (**p < 0.01; paired Student's t-test).",
    "molecules": "TBS"
  },
  {
    "caption": "B. The SBP-AMOTL2 coiled-coil domain was expressed in 293T cells, subjected to tandem affinity purification, fractionated by SDS-PAGE and stained with Coomassie blue. Bands corresponding to the AMOTL2 coiled-coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites (K-G-G linkages). Shown are MS/MS spectra recoded using an LTQ-Orbitrap mass spectrometer for the doubly charged peptide, IEK*LESEIQR (MH+ = 1358.72, z = 2+; upper), and the triply charged peptide, LASK*TQEAQAGSQDMVAK (MH+ = 1992.96, z = 3+; lower). These peptides contain the K347 and K408 ubiquitination sites, respectively (marked by asterisks). Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry.",
    "molecules": "peptide"
  },
  {
    "caption": "B, C. 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti-Flag antibody or pulled down with streptavidin-binding beads.",
    "molecules": "streptavidin"
  },
  {
    "caption": "D, E. 293T cells were transfected as indicated and immunoprecipitated with an anti-HA antibody. LATS2 immune complexes were subjected to a kinase assay with His-YAP and cold ATP.",
    "molecules": "His, ATP"
  },
  {
    "caption": "F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.",
    "molecules": "Zeocin"
  },
  {
    "caption": "A Left, kymograph from sequential epifluorescence (20×) images showing cytosolic Ca2+ oscillations in speed‐fluctuating immature DC after slow motility phases (upper and lower panel) and a change in direction (lower panel). DCs were loaded with the Ca2+ dye Oregon GreenBAPTA 1‐AM, introduced in micro‐channels, and imaged every 10 s. Right, sequential epifluorescence (20×) images (1 image/20 s is shown). Instantaneous velocities and intracellular calcium oscillations were measured as described in Materials and Methods. Scale bar: 10 μm.",
    "molecules": "BAPTA 1‐AM, Ca2+, calcium"
  },
  {
    "caption": "B, C Mean cell velocity of immature DCs (n > 125 cells from two independent experiments) (B) or CD8+T lymphocytes (n > 40 cells from one experiment) (C) migrating in micro‐channels with or without BAPTA. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.",
    "molecules": "BAPTA"
  },
  {
    "caption": "D Mean intracellular calcium concentration in speed‐fluctuating (ΔV/V0 > 0.3; ΔV/V0 relative speed variation divided by the global median velocity) and non‐speed‐fluctuating (ΔV/V0 0.3) immature DCs (n = 187 cells from more than three independent experiments). P‐values were calculated using a Mann-Whitney test.",
    "molecules": "calcium"
  },
  {
    "caption": "E, F Velocity fluctuations (ΔV/V0) (E) or mean Ca2+ activities (F) displayed by DCs migrating in micro‐channels in the presence of 100 ng/ml CCL3. P‐values were calculated using a Mann-Whitney test, with respect to non‐treated DCs (NT).",
    "molecules": "Ca2+"
  },
  {
    "caption": "G Mean Ca2+ activities during phases of fast (> 50% of max velocity) and slow ( 50% of max velocity) motility in speed‐fluctuating DCs (Supplementary Fig S2F) (n = 64 cells, from three independent experiments). Activities were normalized to the mean Ca2+ activity shown by each cell during the entire movie. The P‐value was calculated using a Mann-Whitney test.",
    "molecules": "Ca2+"
  },
  {
    "caption": "H Quantification of the mean cell velocity of shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels in the presence of 2 mM BAPTA. shScramble‐infected DCs were used as a control (gray) (n > 70 cells from two independent experiments). P‐values were calculated using a Kruskal-Wallis test.",
    "molecules": "BAPTA"
  },
  {
    "caption": "A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.",
    "molecules": "ML7"
  },
  {
    "caption": "B, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10-90 percentiles plus the median. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.",
    "molecules": "DMSO, ML7"
  },
  {
    "caption": "D, E Analysis of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.",
    "molecules": "ML7"
  },
  {
    "caption": "Hematoxylin and eosin sections of BAT from WT and CPEB2-KO female mice at 4 and 12 mo old. Scale bars, 1 mm",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "Lipid droplet size and cell number  (i.e., number of nuclei) in WT and KO BAT (representative images shown in 4D) were quantified and displayed as arbitrary units. Three tissue sections from each BAT and 3 mice per group were analyzed",
    "molecules": "Lipid"
  },
  {
    "caption": " CPEB2-WT and -KO mice were transduced with adeno-associated virus (AAV) expressing full-length (AAV_CP2) or RNA-binding domain (AAV_RBD) of CPEB2. Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV_CP2 for 2 weeks, with Hoechst staining of nuclei. Scale bar, 10 µm. See more images in Fig EV4D ",
    "molecules": "Hoechst"
  },
  {
    "caption": " BAT temperature in WT and KO male mice, and of AAV_CP2-transduced WT and KO male mice (n = 7 mice per group) before and after i.p. injection of CL316243 (CL). * p < 0.05, repeated-measures two-way ANOVA ",
    "molecules": "CL316243"
  },
  {
    "caption": "A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.",
    "molecules": "BFA"
  },
  {
    "caption": "Brdu incorporation (E and F) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (E) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments. Scale bars represent 20μm in (E)",
    "molecules": "Brdu"
  },
  {
    "caption": "E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.",
    "molecules": "pentose phosphate, PPP, TCA, tricarboxylic acid"
  },
  {
    "caption": "F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.",
    "molecules": "Glu, glucose, Oligo, oligomycin"
  },
  {
    "caption": "H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.",
    "molecules": "2-NBDG, glucose"
  },
  {
    "caption": "glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells.",
    "molecules": "glucose, lactate"
  },
  {
    "caption": "basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells.",
    "molecules": "2-DG, ATP, oligomycin"
  },
  {
    "caption": "L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.",
    "molecules": "ATP, lactate"
  },
  {
    "caption": "E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.",
    "molecules": "MK2206, Ruxolitinib, U0126"
  },
  {
    "caption": "H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.",
    "molecules": "2-NBDG"
  },
  {
    "caption": "I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).",
    "molecules": "2-BNDG, Ruxolitinib"
  },
  {
    "caption": "C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1",
    "molecules": "2-DG"
  },
  {
    "caption": "F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.",
    "molecules": "2-DG, cis-platinum, DDP"
  },
  {
    "caption": "(C) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-hour treatment with the EGFR inhibitor cetuximab (50μg/ml). Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *p=0.0153, ***p=0.0001, ****p<0.0001; AS901 *p=0.0201.",
    "molecules": "cetuximab"
  },
  {
    "caption": "(A) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab (50μg/ml). In the plot, each box represents values from 25th to 75th percentiles, the central band indicates the mean, and the whiskers expand from maximum to minimum values of individual cetuximab-treated experimental points (n≥9), normalized to the average value of untreated cells within each group. The statistical significance across three independent experiments, each comprising ≥3 technical replicates, was verified by unpaired t-test multiple comparisons of cetuximab-treated and untreated conditions, per each group: ***p=0.0002; ****p<0.0001.",
    "molecules": "cetuximab"
  },
  {
    "caption": "(B) The viability of AS43, in presence (or absence) of cetuximab (50μg/ml), was assessed over the indicated time course, compared to values at T0. The graph shows the mean ± SD of two independent experiments (with quadruplicate technical replicates each). The statistical significance was verified through multiple comparisons by two-way ANOVA with Bonferroni correction of cetuximab-treated and untreated samples, per each time point: ****p<0.0001.",
    "molecules": "cetuximab"
  },
  {
    "caption": "(D) The invasive capacity of AS906 cells (overexpressing WT or G842C-mutated PlxnB2, or mock transfected) was assessed as in Fig. 7E, in the presence or absence of cetuximab 50μg/ml. Images show representative results (scale bar: 100μm), while the graph on the right shows the mean ± SD of n=3 independent experiments. The statistical significance across replicates was verified by one-way ANOVA with Bonferroni correction: **p= 0.0010; ***p=0.0005; ****p<0.0001.",
    "molecules": "cetuximab"
  },
  {
    "caption": "C) Serine 74 substitution to a phosphomimetic aspartate amino acid (stn1-S74D) is sufficient to confer ssu72∆ telomere defects. Telomere length epistasis analysis of ssu72∆ and stn1-S74D mutants were performed by Southern blotting of ApaI digested genomic DNA using a telomeric probe.",
    "molecules": "aspartate, Serine"
  },
  {
    "caption": "D) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants. Multi-copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells.",
    "molecules": "thiamine"
  },
  {
    "caption": "C) hSSU72 down-regulation results in multi-telomeric signals (MTS) that are dependent on DNA replication. Visualization of mitotic spreads of HT1080 hSSU72 shRNA cells treated with aphidicolin and colcemid. Telomere FISH was carried out using a PNA-telomere probe. MTS are represented by arrows. Quantification of MTS per metaphase: n=4; **p ≤0.01 ****p ≤0.0001 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).",
    "molecules": "colcemid, aphidicolin"
  },
  {
    "caption": "B Interaction of MXL-1 with TDPT-1 by yeast two-hybrid assays. The reporter yeast strain L40u was co-transformed with expression vectors encoding LexA DBD-MXL-1 and GAL4 AD-TDPT-1 as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5-aminotriazole for 4 days.",
    "molecules": "5-aminotriazole, histidine"
  },
  {
    "caption": "C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation.",
    "molecules": "PMA"
  },
  {
    "caption": "B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3).",
    "molecules": "RNA"
  },
  {
    "caption": "G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2).",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "H The RBM15 5'UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Volcano plot of phosphorylation changes in iKras PDAC cells in response to KrasG12D induction. Phospho- to total proteomic changes were quantified from three independent biological replicates of Dox treated vs. untreated cells, via TMT (Dataset EV8). 872 phosphorylations were found to be significantly increased, while 82 phosphorylations were significantly decreased (FDR < 0.05). Significantly changing phosphorylations on RBPs whose RNA-binding activity was significantly increased (red) or decreased (blue) as per Fig 1C are highlighted on the plot.",
    "molecules": "Dox"
  },
  {
    "caption": "(E) KrasG12D induction enhances phosphorylation of CK2α (T360/S362), in an Erk1/2-dependent manner. IKras PDAC cells were grown in the absence of Dox for 48 hrs, before its addition to the indicated cells, with or without Trametinib (10 nM), for a further 24 hrs. Cells were then lysed and analyzed by immunoblotting (IB) with the indicated antibodies. The red triangle marks the main phospho band which overlaps with the total CK2α.",
    "molecules": "Dox, Trametinib"
  },
  {
    "caption": "(G) Analysis of the RNA-binding activity of WT, Phospho-defective (S4A), and phospho-mimicking (S4D) mutants of Ncl by OOPS. IKras PDAC cells were transiently transfected with myc-tagged WT, S4A, and S4D Ncl constructs. Cells were subsequently grown in the absence of Dox for 48 hrs, followed by Dox addition for 24 hrs to induce KrasG12D expression. Cells were then subjected to OOPS to isolate the interface (RNA-bound proteins), or whole cell lysis (total lysate), followed by immunoblotting with anti-Ncl antibody. For comparison, Gapdh, which also binds RNA but does not show a change in its RNA-binding activity (Dataset EV1), was also blotted for. A fraction of each interface or total lysate sample was also subjected to RNA extraction, which was resolved and quantified by capillary electrophoresis (CE) as loading control.",
    "molecules": "Dox"
  },
  {
    "caption": "(B) Immunofluorescence analysis of WT, S4A, and S4D Myc-Ncl subcellular localization. IKras PDAC cells ectopically expressing either WT, S4A, or S4D Myc-Ncl were fixed and immunostained with anti-Myc-tag antibody, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker, and Hoechst, followed by confocal microscopy analysis.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) Distribution of WT, S4A, and S4D Myc-Ncl crosslink sites across the annotated 47S pre-rRNA genomic region. Peak heights represent mean crosslink intensities from three independent iCLIP replicate experiments.",
    "molecules": "47S pre-rRNA"
  },
  {
    "caption": "(D) Quantification of the percentage of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Percentage of crosslinks relative to 47S total were quantified from three independent biological replicate iCLIP experiments. (E) Quantification of the proportional density of crosslinks in each region of the 47S pre-rRNA for WT, S4A, and S4D Myc-Ncl. Proportional density was calculated from three independent iCLIP experiments by normalizing the number of crosslinks in each region to the sequence length of that region.",
    "molecules": "47S, 47S pre-rRNA"
  },
  {
    "caption": "(A) Nascent RNA imaging in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, grown for 48hrs in presence or absence of Dox, prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody (green) to visualize nascent RNA, along with anti-Fibrillarin (Fbl) antibody as a Nucleolar marker (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 182-220 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).",
    "molecules": "Hoechst, FUrd, Dox"
  },
  {
    "caption": "(C) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 4 biological replicate experiments were quantified (***: P < 0.001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).",
    "molecules": "pre-rRNA, Dox"
  },
  {
    "caption": "(E) Assessment of the overall protein synthesis rates in control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D, by puromycinylation. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, and grown for 48hrs in presence or absence of Dox, before pulse labelling with Puromycin (10 μg/ml) for 15 min to label nascent proteins. Cells were subsequently lysed and analyzed by immunoblotting with the indicated antibodies. (F) Quantification of puromycin incorporation from (E), as an indicator of the overall protein synthesis rate. Total Erk1/2 levels were used as loading control. A total of 4 independent biological replicate experiments were quantified. Error bars depict SD. (**: P < 0.01; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). (",
    "molecules": "Dox, Puromycin, puromycin"
  },
  {
    "caption": "(G) Nascent RNA imaging of WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox. Cells were then subjected to pulse labeling with FUrd, fixation, and immunostaining with anti-FUrd antibody (green), anti-Myc-tag antibody (red), and Hoechst (blue), followed by confocal microscopy analysis. Scale bar = 10 µm. (H) Quantification of FUrd levels in Myc-positive nucleoli from (E). FUrd fluorescence densities in single nucleoli were quantified from 160-281 individual cells per condition, combined from three independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test). (",
    "molecules": "Hoechst, FUrd, Dox"
  },
  {
    "caption": "(I) RT-qPCR analysis of 5'ETS-containing pre-rRNA transcript levels in WT, S4A, and S4D Myc-Ncl expressing iKras PDAC cells, in presence or absence of KrasG12D. Vectors encoding Myc-tagged WT, S4A, and S4D Ncl were transiently transfected into iKras PDAC cells, before reseeding and growing the cells for 48hrs in presence or absence of Dox, followed by RT-qPCR analysis with a specific probe against the mouse 5'ETS region. A probe against mouse Actb mRNA was used as loading control for normalization. A total of 5 to 8 biological replicate experiments per condition were quantified (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).",
    "molecules": "pre-rRNA, Dox"
  },
  {
    "caption": "(A) Colony formation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, followed by clonogenic assay for 7 days in presence or absence of Dox. Colonies were visualized by Crystal Violet staining. (B) Quantification of Crystal Violet staining levels from (A). A total of 6 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). (",
    "molecules": "Dox"
  },
  {
    "caption": "(C) 3D proliferation of control and Ncl depleted iKras PDAC cells, in presence or absence of KrasG12D. Cells were transfected with a non-targeting control siRNA, or two independent siRNAs against Ncl, before being reseeded onto 3D Collagen-I gels, with or without Dox, and allowed to grow for 48 hrs. Cells were subsequently imaged live by phase contrast microscopy. Scale bar = 200 µm. (D) Analysis of the relative percentage of viable cells in 3D collagen-I cultures from (C). Cells were subjected to luminescence-based viability assay by CellTiter-Glo to quantify the percentage of viable cells. A total of 3 biological replicate experiments were quantified. Error bars depict SD. (****: P < 0.0001; n.s.: not significant - calculated from two way ANOVA with Šídák's multiple comparisons test). (",
    "molecules": "Dox"
  },
  {
    "caption": "(A) Dose response analysis of CX-5461 impact on nascent rRNA expression. IKras PDAC cells were grown in the absence of Dox for 48hrs. Cells were subsequently treated with or without Dox for 24 hrs to induce KrasG12D expression, followed by pre-treatment with the indicated concentrations of CX-5461 for 30 mins prior to pulse labeling with FUrd. Cells were then fixed and immunostained with anti-FUrd antibody to visualize nascent RNA (green), anti-Ncl antibody to reveal the Nucleolus (red), and Hoechst (blue) as the Nuclear stain, followed by confocal microscopy analysis. (B) Quantification of Nucleolar FUrd levels in images from (A). FUrd fluorescence densities in single nucleoli were quantified from 251-479 individual cells per condition, combined from 2 independent biological replicate experiments (****: P < 0.0001; n.s.: not significant - calculated from one-way ANOVA with Šídák's multiple comparisons test).",
    "molecules": "CX-5461, Hoechst, FUrd, Dox"
  },
  {
    "caption": "(G) MRI imaging of orthotopic iKras tumors in untreated or CX-5461 treated mice. IKras PDAC cells were engrafted into the pancreas of Dox-fed nude mice and allowed to form tumors for 4 days. Animals were then divided into two groups, with the first group treated by daily oral administration of CX-5461 (50mg/kg) for a further 10 days, whilst the second group was left untreated for the same period. T2 scans were taken on the indicated days, post-engraftment. Green areas mark the tumors.",
    "molecules": "CX-5461, Dox"
  },
  {
    "caption": "(K) H&E analysis of the extracted pancreas tissues from (I). Representative pancreatic tissue images from the untreated and CX-5461 treated mice, showing typical PDAC histology with malignant ductal structures surrounded by stroma in the untreated, but a largely normal pancreas histology with a small malignant (M) component (marked by the dashed line) in the treated mice.",
    "molecules": "CX-5461"
  },
  {
    "caption": "(a) Immunostaining of TCF20, NESTIN, and merged with DAPI in E13 and E16 embryonic brain sections. TCF20 is widely expressed in the brain. Data information: Scale bar in (A) e13, 50 μm, e16 100 μm",
    "molecules": "DAPI"
  },
  {
    "caption": "(b) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. Scale bar: 50 μm.",
    "molecules": "BrdU"
  },
  {
    "caption": "(f) A diagram of the BrdU labeling time point in electroporation experiments. (g) Immunostaining of BrdU. White arrows represent BrdU GFP double-positive cells. (h) Percentage of BrdU and GFP double-positive cells in GFP-positive cells n=3 brains for each group Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**), p<0.001(***). Scale bar: f) 25 μm",
    "molecules": "BrdU"
  },
  {
    "caption": "(c-f) MeDIP-qPCR showed that 5fC, 5caC, and 5mC are increased in the TCF-4 promoter, while 5hmC is decreased when TCF20 is insufficient. The MeDIP assay measure the region -500 bp from the TCF-4 promoter. (n=4 biological repeats) Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). ",
    "molecules": "5hmC, 5caC, 5fC, 5mC"
  },
  {
    "caption": "(g) NSCs were isolated from C57B6/J E13.5 embryonic brains and then were infected with a lentivirus containing a TCF20 shRNA or with a control plasmids. Immunostaining of 5fC shows that TCF20 depletion increased 5fC levels. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm (h)Statistical analysis of 5fC fluorescence level. n=20 cells, and P<0.0001 Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). ",
    "molecules": "5fC"
  },
  {
    "caption": "(i) Immunostaining of 5caC shows that TCF20 depletion increased 5caC level. White dashed lines show the cells infected with lentivirus. Scalebar 25 μm. (j) Statistical analysis of the 5caC fluorescence level. n=20 cells, and P<0.0001. Data information: Error bars represent the means ± S.E.M. Two tailed unpaired T-tests were used to analyze the data, n.s. (no significant difference), p<0.05(*), p<0.01(**). and p<0.001(***). ",
    "molecules": "5caC"
  },
  {
    "caption": "MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).",
    "molecules": "BSA, palmitate"
  },
  {
    "caption": "TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis.",
    "molecules": "MitoSOX, BSA, DMSO, MitoTracker green, MitoTracker Green, C2-ceramide, palmitate, SH-BC-893, TMRE"
  },
  {
    "caption": "NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).",
    "molecules": "NAD(P)H, SH-BC-893"
  },
  {
    "caption": "Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.",
    "molecules": "SH-BC-893"
  },
  {
    "caption": "NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).",
    "molecules": "NAD(P)H, SH-BC-893"
  },
  {
    "caption": "TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).",
    "molecules": "MitoTracker Green, MitoTracker, TMRE"
  },
  {
    "caption": "MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).",
    "molecules": "MitoSOX, MitoTracker Green, MitoTracker"
  },
  {
    "caption": "Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.",
    "molecules": "SH-BC-893"
  },
  {
    "caption": "Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.",
    "molecules": "SH-BC-893"
  },
  {
    "caption": "Average daily distance run by HFD-fed mice treated with vehicle (n=8) or SH-BC-893 (120 mg/kg, n=6) on Mondays, Wednesdays, and Fridays over 4 weeks when housed with a running wheel.",
    "molecules": "SH-BC-893"
  },
  {
    "caption": "Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm.",
    "molecules": "SH-BC-893"
  },
  {
    "caption": "Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E).",
    "molecules": "glucose"
  },
  {
    "caption": "Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.",
    "molecules": "Insulin, SH-BC-893"
  },
  {
    "caption": "SHAPE analysis of the complete sfRNAs of ZIKV (D) Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (B) Nucleotides which SHAPE reactivity was not determined are labeled in gray in (B)",
    "molecules": "sfRNAs"
  },
  {
    "caption": "SHAPE analysis of the complete sfRNAs of WNV (E). Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (C). Nucleotides which SHAPE reactivity was not determined are labeled in gray in (C).",
    "molecules": "sfRNAs"
  },
  {
    "caption": "(A)-(D) The scattering profiles (A), pair distance distribution functions (PDDFs) (B), dimensionless Kratky plots (C) and Porod-Debye plots (D) of the complete sfRNAs of DENV2 (cyan line), ZIKV (red line) and WNV (blue line). The fine features of P1 in the scattering profiles in (A) arise from helical interstrand pair-distance correlation. The PDDF profiles in (B) were calculated using GNOM (qmax=0.3). The dimensionless Kratky plot and Porod-Debye plot for ZIKV xrRNA1 (red dashed line) were included in (C) and (D) for comparison, indicating that the structures of sfRNAs are more extended and open than that of ZIKV xrRNA1.",
    "molecules": "sfRNAs"
  },
  {
    "caption": "(D) The progeny virus RNA in cultured supernatants of ZIKV and the mutants were detected by qRT-PCR in three technical replicates. Two-way ANOVA and Dunnett's multiple comparison test was used for statistical analysis. The error bars represent the standard deviation. *** p<0.001, **** p<0.0001.",
    "molecules": "RNA"
  },
  {
    "caption": "(A)-(C) The fitting chi2 are plotted against the ensemble size for complete sfRNAs of DENV2 (A), ZIKV (B) and WNV (C), suggesting optimized minimal structural ensembles with 3, 3 ,3 conformers, respectively. The respective insets show the distribution of the minimal ensembles and the initial pools of 10000 random conformers as a function of Rg.",
    "molecules": "sfRNAs"
  },
  {
    "caption": "(B) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI. Acquisition settings were kept constant for all samples. Scale bar: 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Confocal tile-scans of retinal cross sections immunolabelled for eGFP and Hoechst. Scale bar: 500 μm",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D-E) Immunolabelled cross sections of mouse retina infected with AAV2.GL and AAV2.NN respectively, with focus on the ONL and INL, stained for eGFP, recoverin and DAPI. Scale bar: 50 μm ONL, outer nuclear layer; INL, inner nuclear layer.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Confocal scans of retinal cross sections, with focus on the ONL, immunolabelled for DAPI, eGFP and recoverin. The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images (noted with asterisk *) with merged DAPI, eGFP and recoverin signal. Acquisition settings were kept constant for all samples. Scale bar: 50 μm. ONL, outer nuclear layer.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B-C) Confocal scans of immunolabelled retinal cross sections (14 μm) stained for GFP and DAPI at 6 WPI time point. : DAPI was used a nuclear marker. GFP=eGFP; RPE, retinal pigmented epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Confocal scans of day 10 tissue sections immunolabelled for eGFP and Hoechst. Scale bar: 50 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(F) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 12 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N = 3.",
    "molecules": "Atto 488, Lipid"
  },
  {
    "caption": "(I) Lipid mixing was monitored by the increase of Atto 488 fluorescence for 60 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. Error bars = s.e.m., N =3.",
    "molecules": "Atto 488, Lipid"
  },
  {
    "caption": "Autaptic munc18-1 null hippocampal neurons were rescued with a phosphomimetic Munc18-1 mutant, Y473D or wild-type Munc18-1 as control and assessed on morphology and synaptic transmission using whole-cell patch clamp electrophysiology (D) Left: typical responses to hyperosmotic sucrose application (500 mM, 3.5 sec.) used to assess the RRP. Average RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473D: 0.08 ± 0.02 nC, n=10, t-test with Welch correction, p = 0.0274).",
    "molecules": "sucrose"
  },
  {
    "caption": "Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(D) A hypertonic sucrose solution (500 mM, 3.5 sec.) was applied to release the RRP before (naive (n)) or two seconds after (a) HFS (100 pulses at 40 Hz).",
    "molecules": "sucrose"
  },
  {
    "caption": "Synaptic transmission was assessed during and immediately after sustained stimulation in autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473D.(F) PDBu application (1 µM) enhanced EPSC size in neurons expressing M18Y473D.",
    "molecules": "PDBu"
  },
  {
    "caption": "(D) Lipid mixing was monitored by the increase of Atto 655 fluorescence for 30 minutes. Data were normalized to the maximum fluorescence after liposome lysis by detergent. The minimum fluorescence was set to 0%. Error bars = s.e.m., N = 3.",
    "molecules": "Atto 655, Lipid"
  },
  {
    "caption": "Synaptic transmission in autaptic excitatory munc18-1 neurons expressing M18WT or a non-phosphorylatable Munc18-1 variant, M18Y473F, was tested with whole-cell patch clamp electrophysiology.(G) PDBu was bath applied during 0.05 Hz stimulation. Maximum potentiation of EPSC amplitude (M18WT: 2.56 ± 0.48 fold, n=7; M18Y473F: 2.82 ± 0.98 fold, n=8, t-test with Welch correction, p = 0.8168).",
    "molecules": "PDBu"
  },
  {
    "caption": "(B-G) Munc18-1 null hippocampal neurons were rescued with wildtype Munc18-1a or a Munc18-1 mutant, Y473A, in which Y473 was replaced with alanine.(E) Release by hyperosmotic sucrose application (500mM, 3.5sec.) was used to assess the RRP. Left: RRP charge (M18WT: 1.68 ± 0.64 nC, n=13; M18Y473A: 0.27 ± 0.08 nC, n=13, t-test with Welch correction, p = 0.0487). Typical responses are depicted in the middle. Right: vesicular release probability per neuron (EPSC charge / RRP charge). Mean Pves (M18WT: 6.75 ± 0.77%, n=13; M18Y473A: 1.98 ± 0.72%, n=13, t-test, p = 0.0001).",
    "molecules": "sucrose"
  },
  {
    "caption": "Autaptic excitatory munc18-1 neurons expressing M18WT or M18Y473A were subjected to stimulation trains of 100 pulses at different frequencies.(E) A single action potential or sucrose application (500 mM, 3.5 sec.) was given before (naive) or two seconds after HFS (100 pulses at 40 Hz). Average EPSC charge M18WT (Naive: 70.9 ± 19.5 pC, after HFS: 75.4 ± 17.4 pC, n=12, Paired t-test, p = 0.4049). Average EPSC charge M18Y473A (naive: 10.3 ± 4.5 pC, after HFS: 46.2 ± 14.5 pC, n=13, Wilcoxon matched-pairs signed-ranks test, p = 0.0005). Average RRP size M18WT (naive: 1.73 ± 0.69 nC, after HFS: 1.34 ± 0.54 nC, n=12, paired t-test, p = 0.0557) Average HFS size M18Y473A (naive: 0.268 ± 0.075 nC, after HFS: 0.439 ± 0.107 nC, n=13, paired t-test, p = 0.0100). Average Pves M18WT (EPSC charge / RRP charge * 100) (Naive: 7.07 ± 0.76 %, after HFS: 10.6 ± 1.6 %, n=12, paired t-test, p = 0.0201). Average Pves M18Y473A (naive: 1.98 ± 0.72 %, 12.2 ± 2.8 %, n=13, paired t-test, p = 0.0021).",
    "molecules": "sucrose"
  },
  {
    "caption": "U2Os cells co-transfected with YFP-FKBP and PCM1F2-Ce3-FRB, PACT-Ce3-FRB, or Ce3-FRB-Centrin2 were treated with rapamycin (100 nM). The translocation of YFP-FKBP onto PCM1F2- Ce3-FRB-labeled centriolar satellites, the PACT-Ce3-FRB-labeled pericentriolar matrix, and the Ce3-FRB-Centrin2-labeled centriolar lumen was monitored. The normalized fluorescence intensity of YFP-FKBP accumulation at centrosomes upon rapamycin (100 nM; blue) and DMSO (0.1%, vehicle control; red) treatment. Data are shown as the mean ± S.E.M. The graphs show immediate translocation of YFP-FKBP after rapamycin induction at centriolar satellites (left, n = 24 cells), the pericentriolar matrix (middle, n = 20 cells), and the centriolar lumen (right, n = 23 cells) from seven independent experiments.",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "U2Os cells co-transfected with each YFP-FKBP-labeled varisized probe (YFP-FKBP, YFP-FKBP-Grp1, YFP-FKBP-Luciferase, YFP-PSD95-FKBP, YFP-FKBP-β-Gals, YFP-FKBP-β-Galm, YFP-FKBP-ΔNβ-Gal, and YFP-FKBP-β-Gal) and Ce3-FRB with labels specific for centrosomal sub-compartments were treated with rapamycin (100 nM). The images of cerulean channel show centrosome sub-compartment from the untreated cells. Arrowheads indicate sites of centrosomes. Insets show higher-magnification images of the centrosomal regions. Dotted lines indicate the cell boundaries. The normalized fluorescence intensity of each probe at centriolar satellites (left, n = 14, 19, 17, 13, 11, 22, 14, and 9 cells from small to large probes), pericentriolar matrix (middle, n = 20, 19, 18, 16, 10, 18, 10, and 12 cells from small to large probes), and centriolar lumen (right, n = 10, 13, 13, 6, 3, 19, 13, and 9 cells from small to large probes) from 3-6 independent experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "Depolymerization of microtubules by Nocodazole (10 µM) treatment. U2Os cells that expressed PACT-Ce3-FRB (blue, a marker of centrosomes) were incubated with antibodies against α-tubulin to label microtubules (red) in the absence (0.1% DMSO) or presence of Nocodazole treatment for 20 min. Lower panels show magnified images of the centrosomal regions. The normalized intensity of α-tubulin around centrosomes in the absence or presence of Nocodazole treatment for 20 min. The circular ROIs in diameter of 3 µm, centering PACT-labeled centrosomes, were used for measurement of the level of microtubule around centrosomes. Data (black) are shown as the mean ± SD. (n = 196 and 156 cells from left to right; 3 independent experiments).",
    "molecules": "DMSO, Nocodazole"
  },
  {
    "caption": "U2Os cells expressing PACT-Ce3-FRB (blue, a marker of centrosomes) were incubated with phalloidin to label the F-actin (red) with DMSO (0.1%), CK666 (0.4 mM) for 2 hr, or Latrunculin A (LatA, 0.5 mM) for 1 hr. Lower panels show the magnified images of the centrosomal regions. The normalized intensity of actin filaments around centrosomes in the absence or presence of CK666 or LatA treatment. The circular ROIs in diameter of 3 µm, centering PACT-labeled centrosomes, were used for measurement of the level of actin filaments around centrosomes.",
    "molecules": "phalloidin, CK666, DMSO, LatA, Latrunculin A"
  },
  {
    "caption": "HeLa cells co-transfected with H2B-RFP (a chromosome marker), PACT-Ce3-FRB, and YFP-FKBP-Luciferase were sequentially incubated with thymidine (2 mM) for 16-18 hr and RO3306 (2.5 ng/mL) for 12 hr to synchronize cells in G2/M phase (Interphase cells in left panel). The probe trapping experiment in interphase cells was performed right after RO3306 washout. Anaphase cells were chosen for probe trapping experiment 45-60 min after RO3306 washout. Transfected cells in interphase or anaphase were treated with 100 nM rapamycin (Rapa). The centrosome regions are shown in lower panels. The normalized fluorescence intensity of YFP-FKBP-Luciferase accumulation at centrosomes in interphase (Green, n = 12 cells) or anaphase (Red, n =10 cells) cells upon rapamycin (100 nM) treatment.",
    "molecules": "RO3306, Rapa, rapamycin, thymidine"
  },
  {
    "caption": "HeLa cells co-transfected with H2B-RFP (a chromosome marker), PACT-Ce3-FRB, and YFP-FKBP-Luciferase were synchronized in anaphase with or without CK666 treatment (0.4 mM). Rapamycin (100 nM) was added for 5 min to trigger the YFP-FKBP-Luciferase trapping at centrosomes. The right panel is the enlarged images of centrosome regions and arrows indicated centrosome sites. The percentage of cells in D exhibiting probe translocation. Data are shown as mean ± S.E.M. (n = 47 and 31 cells in DMSO and CK666 treated groups, respectively; 3-4 independent experiments).",
    "molecules": "CK666, DMSO, Rapamycin"
  },
  {
    "caption": "U2Os cells were incubated with antibodies against 𝑟-tubulin (green; a marker of 𝑟-TuRC) or Pericentrin (red; a marker of centrosomes) in the absence (0.1% DMSO) or presence of CK666 treatment (0.4 mM) for 2 hr. The right panel shows magnified images of the indicated region of interest (ROI). S The normalized intensity of 𝑟-tubulin at centrosome in control (0.1% DMSO, 2 hr) or CK666 (0.4 mM, 2 hr)-treated cells. Individual data points and the mean ± SD (in red) are shown (n = 150 and 150 cells in DMSO and CK666-treated group, respectively; four independent experiments).",
    "molecules": "CK666, DMSO"
  },
  {
    "caption": "U2Os cells transfected with GCP4-GFP were treated with or without CK666 (0.4 mM, 2 hr) and then observed for the indicated time after photobleaching. The mobile fraction percentage (left) and mobile recovery half-time (right) of GCP4- GFP in FRAP experiments (c). Data (red) are shown as mean",
    "molecules": "CK666"
  },
  {
    "caption": "U2Os cells expressing EB1-mNeon were incubated with 0.1% DMSO or 0.4 mM CK666 for 2 hr. Images were captured over 150 sec and were overlaid to show the overall tracks of the microtubules. The insets show enlarged images of the centrosomal regions. F, G. The number of microtubule tracks that emanate from centrosomes (F) and the microtubule elongation rate (G) in the absence and presence of CK666 treatment (0.4 mM). Individual data points and the mean ± SD (in red) are shown (n = 78449 and 54283 microtubule tracks from 27 and 22 cells in the DMSO- and CK666-treated groups, respectively; three independent experiments).",
    "molecules": "CK666, DMSO"
  },
  {
    "caption": "WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5).",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5).",
    "molecules": "DEX, dextran, FITC, LPS"
  },
  {
    "caption": "RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts.",
    "molecules": "LPS"
  },
  {
    "caption": "WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "WT mice (n=3-5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated 6 hours after LPS challenge. (A) p-MLKL and MLKL protein levels and (B) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control. Expression levels were quantified using FIJI and normalized to ACTIN levels. P-values were calculated using 1-way ANOVA.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "Lysozyme expression (in red) was detected via IHC, with Hoechst counterstaining. Scale bars: 50µm. Quantification of lysozyme expression. For every picture (n=3-4 per treatment), a selection of 10 crypts was made. Lysozyme positive signal was quantified in this region of interest (ROI) using an automated script in QuPath. Values are the positive area relative to total area of ROI.",
    "molecules": "Hoechst"
  },
  {
    "caption": "TNFR1+/+ (n=9) and TNFR1-/- mice (n=11) were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS"
  },
  {
    "caption": "TLR4fl/fl mice (n=7) and TLR4VillinKO (n=9) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS"
  },
  {
    "caption": "TNFR1fl/fl mice (n=13) and TNFR1VillinKO (n=10) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS"
  },
  {
    "caption": "TNFR1+/+ and TNFR1-/- mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.",
    "molecules": "LPS"
  },
  {
    "caption": "TNFR1fl/fl and TNFR1VillinKO mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.",
    "molecules": "LPS"
  },
  {
    "caption": "GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS"
  },
  {
    "caption": "GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.",
    "molecules": "LPS"
  },
  {
    "caption": "GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "GRfl/fl (n=8) and GRVillinKO mice (n=10) were treated with 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS"
  },
  {
    "caption": "GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n= 4). Mice were treated with 5 mg/kg LPS and blood was drawn 2 hours later.",
    "molecules": "LPS"
  },
  {
    "caption": "TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n=4). Mice were treated with PBS or LPS (12.5 and 5 mg/kg for GRfl/fl and GRVillinKO mice respectively) with or without pre-treatment with 10 mg/kg DEX for 30 minutes. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "GRVillinKO mice were treated with PBS (n=8) or a neutralizing P55 (TNFR1) antibody (n=9) 2 hours before and 8 hours after 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS"
  },
  {
    "caption": "GRfl/fl (n=4) and GRVillinKO mice (n=4) were treated with 5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR.",
    "molecules": "LPS"
  },
  {
    "caption": "Left: LPS dose-response curves of GRwt/wt (n=3-7 per LPS dose) and GRdim/dim mice (n=3-7 per LPS dose) treated with 1.5 mg/kg LPS. Right: LD50 values of LPS are depicted on top of each bar for each group.",
    "molecules": "LPS"
  },
  {
    "caption": "GRdim/dim mice were treated with PBS (n=5) or 10 mg/kg DEX (n=5) before 1.5 mg/kg LPS and survival was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "TNF measurement in the serum of GRwt/wt (n=5) and GRdim/dim mice (n= 5). Mice were treated with 1.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.",
    "molecules": "LPS"
  },
  {
    "caption": "TNF measurement in the serum of GRwt/wt and GRdim/dim mice (n=4) treated with PBS or LPS (12.5 and 1.5 mg/kg in GRwt/wt and GRdim/dim mice respectively) with or without pretreatment with 10 mg/kg DEX. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "GRdim/dim mice were treated with PBS (n=6) or a neutralizing P55 (TNFR1) antibody (n=8) 2 hours before and 8 hours after 1.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS"
  },
  {
    "caption": "Tofacitinib (n=6) or vehicle (n=7) was given orally to GRdim/dim mice. After 1.5 mg/kg LPS injection survival was monitored and p-values were analyzed with a Fisher's exact test.",
    "molecules": "LPS, Tofacitinib"
  },
  {
    "caption": "GRwt/wt (n=4) and GRdim/dim (n=4) mice were treated with 1.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.",
    "molecules": "LPS"
  },
  {
    "caption": "GRdim/dim mice (n=4) were treated with PBS or 1.5 mg/kg LPS with or without 10 mg/kg DEX pretreatment. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.",
    "molecules": "DEX, LPS"
  },
  {
    "caption": "(D) RipAC overexpression inhibits flg22-triggered MAPK activation in Arabidopsis. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points. Immunoblots were analysed using anti-pMAPK and anti-RipAC antibodies. Coomassie brilliant blue (CBB) staining and a non-specific band were used as loading control. Molecular weight (kDa) marker bands are indicated for reference.",
    "molecules": "CBB, Coomassie brilliant blue"
  },
  {
    "caption": "(E) flg22-triggered MAPK activation in pub4 T-DNA mutants. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots. Immunoblots were analyzed using anti-pMAPK and anti-FLS2 antibodies. Coomassie brilliant blue (CBB) staining was used as loading control. Molecular weight (kDa) marker bands are indicated for reference.",
    "molecules": "CBB, Coomassie brilliant blue"
  },
  {
    "caption": "(A) PUB4 has auto-ubiquitination activity in vitro. PUB4 autoubiquitination assay using His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2) and MBP-PUB4 or MBP-PUB4 Cys239Ala inactive mutant reconstituted in bacteria. Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography (IMAC). Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates autoubiquitinated PUB4.",
    "molecules": "acrylamide gels, Ubiquitin"
  },
  {
    "caption": "(B) PUB4 ubiquitinates non-active BIK1, and does not ubiquitinate active BIK1. Operon containing His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2), and MBP-PUB4 were co-transformed with GST, GST-BIK1, or GST-BIK1 kinase dead (BIK1-KD) in the presence of PUB4. Ubiquitinated products were purified under denaturing conditions by IMAC, and modified proteins further enriched by GSH chromatography. Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates the ubiquitination products.",
    "molecules": "acrylamide gels, GSH, Ubiquitin"
  },
  {
    "caption": "(A) Analysis of BIK1-HA protein accumulation in BIK1-HA+/-, pub4-/- BIK1-HA+/-, and PUB4-FLAG+/- BIK1-HA+/- in 4- to 5-week-old plants. Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132, 50 mM CHX, and for 10 min with water (as mock) or 1 mM flg22Paer. Proteins were extracted in SDS buffer and analysed by western blot. BAK1 was used as a loading control. Numbers correspond to the quantitation of the BIK1-HA band, normalized to the quantitation of the BAK1 control band in the same sample.",
    "molecules": "CHX, MG132, SDS, water"
  },
  {
    "caption": "(E) HCT116 cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1B.",
    "molecules": "Embelin"
  },
  {
    "caption": "(F) HCT116 XIAP KO cells were treated with Embelin as indicated. LC3‐II accumulation was determined by western blot analysis with anti‐LC3 antibody. The data are representative of three biological replicates.",
    "molecules": "Embelin"
  },
  {
    "caption": "(G) HCT116 cells were treated with Leupeptin and Embelin in the indicated combinations. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1D.",
    "molecules": "Embelin, Leupeptin"
  },
  {
    "caption": "(I) HCT116XIAP WT and XIAP KO cells were treated with 20 ‐μM Z‐VAD‐FMK or DMSO for 2 h. Cell lysates were then analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S1F.",
    "molecules": "Z‐VAD‐FMK"
  },
  {
    "caption": "(F) HCT116 XIAP WT and XIAP KO cells treated with 10 μM Nutlin3 for 6 h. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S2H.",
    "molecules": "Nutlin3"
  },
  {
    "caption": "(G) HCT116 XIAP KO cells were treated with the indicated amounts of Nutlin for 8 h. Cell lysates were analysed by western blotting with anti‐LC3 antibody. The data are representative of three biological replicates.",
    "molecules": "Nutlin"
  },
  {
    "caption": "(C) HCT116 cells were treated with XIAP‐specific or control siRNAs. Twenty‐four hours later, cells were cultured in the presence of 50 μg ml−1 cycloheximide for the indicated periods of time, and subsequently analysed by western blotting. We should mention that amounts of cell lysates were adjusted to achieve similar expression levels of Mdm2 at time 0, while the same amounts of cell lysates were used to examine levels of XIAP and actin. The data are representative of three biological replicates. The ratio of Mdm2 to actin is presented in Supplementary Figure S3B.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(E) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins and total cell lysates (TCLs) were analysed by western blot with anti‐Mdm2 antibody. The data are representative of three biological replicates.",
    "molecules": "Ni2+, MG‐132"
  },
  {
    "caption": "(H) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins were analysed by western blotting with anti‐p53 antibody. The data are representative of three biological replicates.",
    "molecules": "MG‐132"
  },
  {
    "caption": "(C) HCT116 XIAP WT and XIAP KO cells were treated with 1 μM API‐2 for 6 h. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6C.",
    "molecules": "API‐2"
  },
  {
    "caption": "(G) p53−/−Mdm2−/− MEF cells were transfected with HA‐Mdm2 plus Flag‐XIAP, Flag‐XIAP S87A or Flag‐XIAP S87D. Twenty‐four hours later, cells were treated with 20 μM MG-132 for another 4h. Cell lysates were subjected to immunoprecipitation with anti‐HA antibody. Immunoprecipitates were analysed by western blotting. The data are representative of two biological replicates.",
    "molecules": "MG-132"
  },
  {
    "caption": "To verify the specificity of Cx43-CreERT mice for astrocytes, mice were crossed to a tdTomato reporter line to induce Cre-dependent tdTomato expression after tamoxifen administration. TdTomato fluorescence was enhanced with an antibody against red fluorescent protein, and astrocytes were stained with antibodies against GFAP or S100β (not shown). Merged images and quantification show efficient and widespread tdTomato expression specific for astrocytes (arrowheads) in 8-month and 11-month old animals. Expression was small in neurons (stained with NeuN, not shown) and negligible in NG2 cells and oligodendrocytes. Scale bars, 250 µm (upper panel) and 50 µm (lower panel).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "in postmortem brain sections from AD patients, we detected nuclear pStat3 (arrows) in the majority of peri-plaque reactive astrocytes (identified by GFAP) in the cortex and hippocampus (plaques were stained with methoxy-XO4, arrowheads). Scale bar, 50 µm.",
    "molecules": "methoxy-XO4"
  },
  {
    "caption": "Plaque density, assessed by staining brain sections with thioflavin (yellow), remained unchanged in APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice (Mann-Whitney test; scale bar, 250 µm).",
    "molecules": "thioflavin"
  },
  {
    "caption": "Electrochemiluminescence ELISA after sequential extraction from whole-brain homogenates using RIPA and SDS buffer revealed that soluble Aβ1-42 and Aβ1-40 were significantly reduced (Mann-Whitney test), whereas there was a nonsignificant trend towards reduced levels of insoluble Aβ1-42 and Aβ1-40 (Mann-Whitney test).",
    "molecules": "Aβ1-40, Aβ1-42, SDS"
  },
  {
    "caption": "Internalization of Aβ (stained with IC16 antibody or methoxy-XO4) was assessed using an engulfment assay, in which glial and Aβ structures were surface-rendered and Aβ volumes co-localized with glial volumes were quantified. Scale bars, 10 µm.",
    "molecules": "methoxy-XO4"
  },
  {
    "caption": "Microglia (left Y axes) from APP/PS1 mice internalized significantly more Aβ positive for methoxy-XO4 when Stat3 was deleted in astrocytes (Mann-Whitney test), whereas no changes were seen in astrocytes (right axes; APP/PS1-Stat3WT, n = 8 (4 female and 4 male) mice; APP/PS1-Stat3KO, n = 11 (5 female and 6 male) mice; age, 11 months; Mann-Whitney test).",
    "molecules": "methoxy-XO4"
  },
  {
    "caption": "Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri-plaque reactive astrocytes (arrows; plaques were visualized with methoxy-XO4, arrowheads; scale bars, 50 µm; APP/PS1-Stat3WT, n = 6 (2 female and 4 male) mice; APP/PS1-Stat3KO, n = 6 (3 female and 3 male) mice; age, 8 months; Mann-Whitney test).",
    "molecules": "methoxy-XO4"
  },
  {
    "caption": "For in vivo two-photon imaging, astrocytes (arrows) and neurons (arrowheads) were labeled with the calcium indicator OGB-1, and astrocytes were co-labeled with sulforhodamine 101 (SR101; arrows). Aβ plaques were labeled with the intravital dye methoxy-XO4 (open arrowheads). Scale bar, 50 µm.",
    "molecules": "calcium, methoxy-XO4, OGB-1, SR101, sulforhodamine 101"
  },
  {
    "caption": "Calcium imaging of anesthetized animals showed that the hyperactivity of astrocytes in APP/PS1-Stat3KO mice was reduced to levels comparable to WT-Stat3WT mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; n = 6 mice (3 female and 3 male) for each group; age, 8 months).",
    "molecules": "Calcium"
  },
  {
    "caption": "Similarly, neuronal activity was also reduced to levels comparable to WT-Stat3WT mice in APP/PS1-Stat3KO mice, but significantly increased in APP/PS1-Stat3WT mice (One-Way ANOVA followed by Bonferroni's multiple comparison test; same mice as in B). The cumulative distributions of neuronal calcium transients in APP/PS1-Stat3KO mice were not different from those of WT-Stat3WT mice (p = 0.31, Kolmogorov-Smirnov test), but significantly different from those of APP/PS1-Stat3WT mice (p = 0.001, Kolmogorov-Smirnov test).",
    "molecules": "calcium"
  },
  {
    "caption": "Two-photon imaging of these mice confirmed that astrocytic hyperactivity were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).",
    "molecules": "MRS2179"
  },
  {
    "caption": "Two-photon imaging of these mice confirmed that propagating calcium waves were reduced by MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test).",
    "molecules": "calcium, MRS2179"
  },
  {
    "caption": "MRS2179 (APP/PS1-Stat3WT, n = 6 (4 female and 2 male) mice; APP/PS1-Stat3KO, n = 6 (2 female and 4 male) mice; age, 8 months; Mann-Whitney test). This network normalization induced a reduction of activated Stat3 (pStat3) in astrocytes assessed in fixed brain sections of the same mice (Mann-Whitney test). Scale bars, 50 µm.",
    "molecules": "MRS2179"
  },
  {
    "caption": "APP/PS1 mice were systemically treated with SH-4-54 for 6 weeks and compared to age-matched APP/PS1 mice treated with vehicle. In the Morris Water Maze test, treatment with SH-4-54 led to significantly faster latencies to reach the hidden platform on the last training day (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test).",
    "molecules": "SH-4-54"
  },
  {
    "caption": "(E) To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (Mann-Whitney test).",
    "molecules": "calcium"
  },
  {
    "caption": "SH-4-54 significantly decreased plaque growth, as assessed by IC16 immunohistochemistry, while plaque load remained unchanged.",
    "molecules": "SH-4-54"
  },
  {
    "caption": "SH-4-54 significantly There was also no significant change in dystrophic neurite area during the treatment time (Mann-Whitney test for all comparisons; scale bars, 500 µm).",
    "molecules": "SH-4-54"
  },
  {
    "caption": "D. 10-fold serial dilutions of indicated strains grown in medium permissive (-URA) or restrictive (5-FOA) of the retention of a URA3/NUP116 plasmid. Images acquired after incubation for 3 days (left) or 6 days (right) at 23°C.",
    "molecules": "5-FOA, URA"
  },
  {
    "caption": "TNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours subjected to the Caspase 3 activity assays.",
    "molecules": "CHX, DEVD-AFC"
  },
  {
    "caption": "TNF-induced Caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF (100ng/ml) with or without Cycloheximide (CHX) (1µg/ml) or z-VAD (20µM). Representative data from three independent experiments, n=4.",
    "molecules": "CHX, Cycloheximide, z-VAD"
  },
  {
    "caption": "Immunoblotting of TNF-induced cleavage of PARP and caspase 3 in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) for indicated times. Representative of three independent experiments. H ",
    "molecules": "CHX"
  },
  {
    "caption": "TNF-dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD-AFC. MEFs treated with hTNF (100ng/ml) alone, or with CHX (1µg/ml) for 4 hours (H) or 2 hours (I) subjected to the Caspase 3 activity assays. A representative data of two independent experiments, n=4.",
    "molecules": "CHX, DEVD-AFC"
  },
  {
    "caption": "TNF-induced Caspase 8 activity in immortalized MEFs of the indicated genotype. Luminol-dependent activity of Caspase 8 in immortalized MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) . A representative data of three independent experiments, n=4.",
    "molecules": "CHX"
  },
  {
    "caption": "(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Left: micrographs show the SOD1G85R-YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of headneurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of SOD1G85R-YFP (n = 30). Right: a decrease in the protein levels of SOD1G85R-YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. (B and C) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of TDP-43-c25-YFP (n = 30) or PolyQ-YFP (n = 12) as in (A).",
    "molecules": "PolyQ"
  },
  {
    "caption": "(C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12-21 h).",
    "molecules": "cycloheximide"
  },
  {
    "caption": ". (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6).",
    "molecules": "Suc-LLVY"
  },
  {
    "caption": "(D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.",
    "molecules": "3-methyladenine"
  },
  {
    "caption": "(A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions.",
    "molecules": "CP-31398, Tenovin-1"
  },
  {
    "caption": "(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.",
    "molecules": "DMSO, TEN1, Tenovin-1"
  },
  {
    "caption": "IL-2 quantification by ELISA in supernatants collected at 5 hours post activation of naïve CD4 T cells with PMA/Ionomycin (n=7). Data is shown as means+/-SEM, Two-Way ANOVA with Sidak's multiple comparison test   IL-2 quantification by ELISA in supernatants collected at 72hrs following activation of CD4 T cells with anti-CD3/CD28 without exogenous IL-2 or TGF-β (n=3). Data is shown as means+/-SEM, One-Way ANOVA with Tukey's Multiple comparison test.  ",
    "molecules": "Ionomycin, PMA"
  },
  {
    "caption": "(L) Larval NSCs of control (ctrl; yw), pr-set7716/Df, and UAS-pr-set7 induced with insc­-Gal4 at 72h ALH were stained for Pr-set7, Mira and DNA. (M, N) Quantifications of NSC diameters (M) at 24h ALH and EdU incorporation (N) for genotypes in (O). In (M), n=152 for control; n=129 for pr-set7716/Df. In (N), for control, n=601, 13 BL at 24h ALH; n=988, 14 BL at 48h ALH; n=813, 9 BL at 72h ALH and n=829, 10 BL at 96h ALH. For pr-set7716/Df, n=566, 12 BL at 24h ALH; n=808, 13 BL at 48h ALH; n=1032, 10 BL at 72h ALH and n=825, 9 BL at 96h ALH. Data information: Quantification data are presented in as mean ± SD M, N). n numbers in N) were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. White arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann-Whitney test M, N). ***P < 0.001. Scale bars, 5 μm L),",
    "molecules": "EdU, DNA"
  },
  {
    "caption": "(C) Larval NSCs of control (ctrl; yw), pr-set7716/Df, pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4, and pr-set7716/Df with pr-set7-BAC at 24h ALH were stained for H4K20me1, Dpn, Mira and DAPI. Control: n=108; pr-set7716/Df: n=87; pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4: n=167 and pr-set7716/Df with pr-set7-BAC: n=104. Data information: White arrows point to NSCs. Scale bars, 5 μm",
    "molecules": "BAC, DAPI"
  },
  {
    "caption": "(E) Larval NSCs with FUCCI constructs induced with insc-Gal4 at 24h were stained for GFP, RFP, Dpn, and Prospero (Pros). (F) Quantifications of quiescent NSCs in different cell-cycle stages from FUCCI flies (induced with insc-Gal4) in control (and insc-Gal4>FUCCI) and pr-set7716/Df. Quiescent NSCs of insc-Gal4>FUCCI: 76.1%±1.5% in G2, 1.7%±0.2% in S and 22.2%±1.3% in G0 (n=343). Quiescent NSCs of insc-Gal4>FUCCI; pr-set7716/Df: 74.4%±1.0% in G2, 0.9%±0.2% in S and 24.7%±0.8% in G0 (n=344). Data information: Quantification data are presented in as mean ± SD Statistical analyses were done comparing two different genotypes using Mann-Whitney test ns: non-significant. White arrows point to NSCs. Scale bars, 5 μm",
    "molecules": "FUCCI"
  },
  {
    "caption": "(H) Larval brains of control (ctrl; yw), cdk1-Myc overexpression under insc­-Gal4, pr-set7716/Df(3R)ED5644 (716/Df), and pr-set7716/Df with cdk1-Myc induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU. (I, J) Quantifications of EdU incorporation (I) and NSC diameters (J) for genotypes in (H). In (I), n=1334, 16 BL for control; n=1299, 15 BL for insc>cdk1; n=1221, 18 BL for pr-set7716/Df and n=1372, 16 BL for pr-set7716/Df with cdk1-Mycinduced with insc-Gal4. In (J), n=153 for control; n=152 for insc>cdk1; n=156 for pr-set7716/Df and n=159 for pr-set7716/Df with cdk1-Myc induced with insc-Gal4.Data information: Data were presented in quantification graphs as mean ± SD I, J). n numbers in I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA ***P < 0.001. Scale bars, 30 μm H). Control: insc­-Gal4/+ UAS-dicer2",
    "molecules": "EdU"
  },
  {
    "caption": "(A) Larval NSCs of control (ctrl; yw) and ebd1240 at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira). (B) Quantifications of cellular extensions for genotypes in (A). Control: n=886, 11 BL; ebd1240: n=864, 12 BL. (C) Larval brains of control (ctrl; yw) and ebd1240 at 24h ALH were labelled with Dpn and EdU. (D) Quantifications of NSC without EdU incorporation for genotypes in (C). Control: n=1034, 12 BL; ebd1240: n=1139, 16 BL. Data information: Data are presented in (B, D as mean ± SD. n numbers in (B, D, were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, D ns: non-significant; *P < 0.05; ***P < 0.001.",
    "molecules": "EdU"
  },
  {
    "caption": "(H) Larval brains of control (ctrl; yw), ebd1-Flag overexpression under insc­-Gal4, pr-set7716/Df(3R)ED5644 (716/Df) and pr-set7716/Df with ebd1 induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU. (I) Quantifications of NSCs without EdU incorporation for larval brains in (H). Control: n=1162, 13 BL; insc>ebd1: n=997, 11 BL; pr-set7716/Df: n=928, 13 BL and pr-set7716/Df with ebd1 induced with insc-Gal4: n=1284, 16 BL. Data information: Data are presented in I) as mean ± SD. n numbers in I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA I). ns: non-significant; *P < 0.05; ***P < 0.001.",
    "molecules": "EdU"
  },
  {
    "caption": "(A) Mice bearing established A375 were treated with vehicle (n=7), PLX4720 (n=5) bevacizumab (n=7) or COMBO (n=5). Tumor growth is expressed as % change of the initial tumor, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720 P = 0.0049; bevacizumab P = 0.0003; P = 4.86E-10) ŦP < 0.05 compared to PLX4720 (P = 0.015).(B) Mice bearing established COLO205 were treated with vehicle (n=7), PLX4720 (n=8) bevacizumab (n=7) or COMBO (n=8). Tumor growth is expressed as % change of the initial tumor, ***P < 0.001 versus vehicle (PLX4720 P = 2.75E-05; bevacizumab P = 2.96E-05; COMBO P = 2.13E-07) Ŧp < 0.05 compared to PLX4720 (P = 0.039).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(C) Melanoma MC-1 cells (5 x 105) were injected into the tail vein of CD1 mice. Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs. The mice analyzed were: start point, n=6; vehicle, n=7; PLX4720, n=6; bevacizumab, n=6; COMBO, n=7, *P < 0.001 versus vehicle (P = 0.031).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(D) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated. Bar graphs indicate the Ki67+ area/tumor area (n=4 tumors), ***P < 0.001 versus vehicle (PLX4720 P = 2.83E-08; bevacizumab P = 4.25E-13; COMBO P = 6.16E-08).(E) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated. Bar graphs indicate the TUNEL+ area/tumor area (n=3 tumors). ***P < 0.001 versus vehicle (P = 6.2.7E-10).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(A) Representative images of vasculature stained by an antibody anti-CD31 in A375 xenografts treated as indicated. Bar graphs indicate quantitative micro vessel density (MVD) and micro vessel area (MVA) analysis (n=5 tumors), ***P < 0.001 versus vehicle (MVD: bevacizumab P = 5.94E-25; COMBO P = 1.84E-04) (MVA: PLX4720 P = 4.98E-09; bevacizumab P = 1.16E-19; COMBO P = 3.00E-08).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(B) Representative images of vessel lumen in A375 xenografts treated as indicated. Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts (n=3 tumors), *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 0.0078; COMBO P = 0.0012) (COLO205: PLX4720 P = 2.01E-06; COMBO P = 1.33E-07).",
    "molecules": "PLX4720"
  },
  {
    "caption": "(C) Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated. Bar graphs indicate the % of CD31+ vessels co-stained with fluorescent beads (n=4 tumors), **P < 0.01 versus vehicle (PLX4720 P = 0.0031; COMBO P = 0.0033).",
    "molecules": "PLX4720"
  },
  {
    "caption": "(D) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated. Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts (n=5 tumors), **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 6.94E-12; COMBO P = 3.34E-12) (COLO205: PLX4720 P = 0.0015; bevacizumab P = 0.0004; COMBO P = 0.0003).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(D) GSEA enrichment plots for \"Reactome Cytokine Signaling in Immune System\" and \"Reactome Innate Immune System\" (upper panels) and \"Reactome Extracellular Matrix Organization\" and \"Reactome Collagen Formation\" (lower panels) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments (vehicle, PLX4720, bevacizumab).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(E) Heatmap representation of gene expression changes within the \"Reactome Innate Immune System\" (left panel) and \"Reactome Extracellular matrix Organization\" (right panel) gene set. Genes in heatmaps are shown in rows and samples are shown in columns. Expression level is represented as a gradient from high (red) to low (blue). V, P, B and C respectively indicate vehicle, PLX4720, bevacizumab and COMBO.",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(A) Real-time quantitative PCR of the indicated genes (M1-like and M2-like macrophages markers) of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for housekeeping gene TBP (n=3 tumors), (*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720: m-Arg1 P = 5.65E-06) (bevacizumab: m-Ccl5 P = 0.025) (COMBO: m-Ccl5 P = 0.0070; m-Cd40 P = 0.00099; m-Cxcl10 P = 0.043; m-Cxcl9 P = 0.046; m-Il1b P = 0.0367; m-Stat1 P = 0.049; m-Tlr2 = 0.00182; m-Arg-1 P = 0.0013).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(C) Graphs showing the quantification of FACS analysis of infiltrating F4/80+ tumor macrophages and average percentage of median fluorescence intensity (MFI) of TNFα+ or INOS+ on F4/80+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.037; INOS = 0.038).",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(D) Graphs showing the quantification of FACS analysis of infiltrating CD11b+Ly6C+ tumor monocytes and average percentage of MFI of TNFα+ or INOS+ on CD11b+Ly6C+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.048; INOS = 0.038).",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(A) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 2.93E-06).(B) Representative images of CAF density determined by α-SMA immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate α-SMA+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 0.0006).(C) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the LOX+ area/tumor area (n=3 tumors), *P < 0.05 versus vehicle (bevacizumab P = 0.033; COMBO P = 0.040).",
    "molecules": "bevacizumab"
  },
  {
    "caption": "(E) Quantitative Real-time PCR of h-TGFB1 and m-Tgfb1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for the housekeeping gene TBP (n=3 tumors), *P < 0.05 versus vehicle (P = 0.043).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(A) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors. Tumor growth is expressed as % change of the initial tumor. Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab (n=5 mice/group), *P < 0.05, ***P < 0.001 versus vehicle (vehicle Clodronate P = 0.049, COMBO P = 5.72E-06) ŦP < 0.05 compared to COMBO (P = 0.033).",
    "molecules": "bevacizumab, clodronate, PLX4720"
  },
  {
    "caption": "(D) Waterfall plots showing the percent change in volume (relative to the initial tumor volume) for the individual A375 xenografts in each treatment group (vehicle, PLX4720, bevacizumab and COMBO) from week 1 to week 6.",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(G) Real-time quantitative PCR of h-TGFB1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP (n=3 tumors) ***P < 0.001 versus vehicle (P = 0.0006).",
    "molecules": "bevacizumab, PLX4720"
  },
  {
    "caption": "(B) Representative spinning disc confocal microscopy images of iDCs cultured in 2D suspension (left) or 3D collagen (middle) 8h post seeding. Scale bars = 20µm. Cells were stained with phalloidin-Atto390 to visualize F-actin and cell morphology. The dotted lines indicate which area are shown in higher magnification in the panel on the right. Scale bars = 10µm.",
    "molecules": "Atto390, phalloidin"
  },
  {
    "caption": "Comparison of the volume (G) of iDCs derived from four donors (n = 1559 to n = 4675 each, mean +/-SD) cultured either in 2D suspension or 3D collagen and measured with RT-DC at a flow rate of 0.12 µl/s. A significant increase in deformability of iDCs when cultured in collagen was found , whereas no significant difference in cell volume (G) For statistical significance testing, linear mixed model analysis was performed to calculate ANOVA p-values; *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "collagen"
  },
  {
    "caption": "(I-J) Two days post culturing in 2D suspension or 3D collagen, RNA was extracted and the transcriptome was determined by Clariom™ S Pico assay. The left panels show the heatmap related to the comparison of the expression between 2D suspension and 3D collagen profile of the untreated (mock, top) and LPS treated cells (bottom) (adjusted p-value < 0.05). Inside each cluster, two subclusters identify the 2D suspension and the 3D collagen cells. The right panels show the set of statistically enriched pathways identified with the genes in the heatmap using metascape.",
    "molecules": "LPS"
  },
  {
    "caption": "(B) Representative spinning disc confocal microscopy images of iDC cultured in 2D suspension (left) or 3D collagen (right) at 4 h.p.i. Cells were stained with Phalloidin-Atto390 to visualize F-actin. Vpr-mRuby2 particles are shown in red. Scale bar = 20µm. Arrow heads highlight virus particles in 3D collagen. The dotted lines indicate which area are shown in higher magnification. Scale bars = 2µm (C) Vpr signal per cell. Graphs depict means values +/- SD for cells from four donors *** p< 0.001 calculated with 2way ANOVA test.",
    "molecules": "Atto390, Phalloidin"
  },
  {
    "caption": "Cells were infected with HIV-1 R5 Vpr-Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11°C to prevent particle fusion during the staining process. For the indicated sample, T20 was added at the moment of infection. (E) Percentage of positive cells for the presence of the CFF2 product at 4h p.i. ** p<0.01calculated with 2way ANOVA test.",
    "molecules": "CCF2, CFF2, T20"
  },
  {
    "caption": "Cells were infected with HIV-1 R5 Vpr-Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11°C to prevent particle fusion during the staining process. For the indicated sample, T20 was added at the moment of infection. (F-G) Percentage of positive cells for the presence of the CFF2 product at 4h p.i. (left) and 48h p.i. (right) *p<0.05 calculated with 2way ANOVA test. The graphs show mean values +/- SD for cells from five and three donors, respectively.",
    "molecules": "CCF2, CFF2, T20"
  },
  {
    "caption": "(B) Representative dot plots of FACS analysis to quantify the number of productively infected cells. Shown is the CD3-PerCp-Cy5-5 fluorescence intensity against intracellular p24-FITC after gating on live cells. The gate denotes the productive infected p24+ cell population with the percentage of p24+ CD4 T cells indicated. (C) Percentage of p24+ cells at 2 d.p.i. (top) and 4 d.p.i. (bottom). Graphs depict mean values +/- SD for cells from four donors.",
    "molecules": "Cy5-5, FITC, PerCp"
  },
  {
    "caption": "(C) Representative dot plots of FACS analysis to quantify the number of productively infected cells that upregulated CD86. Shown is CD86-PerCp-770 fluorescence intensity against intracellular p24-FITC. The gates indicate the percentage of p24+ and p24- cells together with the percentage of CD86+ and CD86- cell. (D) Ratio between the percentage of CD86 positive and CD86 negative cells for HIV-1 R5 infected iDC in the presence or absence of Vpx and for LPS treaded iDCs, as positive control. The graph shows mean values +/- SD for cells for five donors. n.s calculated with 2way ANOVA and Sidak's multiple comparison test.",
    "molecules": "PerCp-770, FITC, LPS"
  },
  {
    "caption": "(C-E) Graphs show the fold change increase, over uninfected control, for infection with HIV-1 R5 in the presence of Vpx (C), HIV-1 R5 (D) or HIV-1 R5 in the presence of Vpx and Raltegravir control (E) of the 16 selected genes with antiviral properties for cells from three donors. Bars represent the mean +/- SD * p< 0.05 calculated with 2way ANOVA with Sidak test.",
    "molecules": "Raltegravir"
  },
  {
    "caption": "iDCs were spin-transduced with VLPs carrying Vpxmac239 one day prior to infection. Cells were incubated with anti-DC-SIGN and anti-MMR blocking antibody or isotype control for 30 min, infected with HIV-1 R5 Vpr-Blam for 4 hours to asses fusion or for 48 hours to asses productive infection. (I) Percentage of positive cells for the presence of the CFF2 product at 4 hours p.i. (J) Percentage of p24+ cells at 48 hours p.i. The graphs show mean values +/- SD for cells from three donors. *p<0.05 calculated with 2way ANOVA test.",
    "molecules": "CFF2"
  },
  {
    "caption": "(G) Luciferase-expressing A549 cells overexpressing miR-515-5p or a non-targeting control (miR-NC) were injected in the tail-vein of SCID mice and the development of tumor burden monitored by whole animal-imaging in response to luciferin administration at week 1 and 4 following injection. (G-left panel) Representative imaging of the same animals for the indicated conditions at week 1 and 4. (G-Right panel) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1. Data represented are representative of three independent experiments. (E-G) P values were calculated by t-test (*, P<0.05; **, P<0.01; ***, P<0.001).",
    "molecules": "luciferin"
  },
  {
    "caption": "KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P-Ser465/467) (E and H-I), STAT3 (P-Tyr705) (F and J-K) and expression of IL-6R (G) was detected by immunohistochemistry (n = 4/group).",
    "molecules": "Ser, Tyr"
  },
  {
    "caption": "Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control.",
    "molecules": "LPS"
  },
  {
    "caption": "Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A).",
    "molecules": "Ser, LPS"
  },
  {
    "caption": "Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Conditioned media (CM) was collected mouse IL-6 ELISA (B-C).",
    "molecules": "LPS"
  },
  {
    "caption": "KIC (mPLRB9), KPC (KPC-M09) and KPfC (BMFA3) cell lines were treated with normal DMEM (CTRL), CM from NIH 3T3 (CM), CM from TGFβ-treated NIH 3T3 (TGFβ-CM), CM from TGFβ-treated NIH 3T3 + 2G8 (TGFβ-CM + 2G8) normal DMEM + TGFβ (TGFβ), CM from TGFβ-treated NIH 3T3 + IL-6 neutralizing antibody (TGFβ-CM + IL-6 Ab). Cell lysates were harvested and blotted for P-STAT3 (P-Tyr705), STAT3, P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (J).",
    "molecules": "Ser, Tyr"
  },
  {
    "caption": "3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).",
    "molecules": "HEMA"
  },
  {
    "caption": "3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).",
    "molecules": "HEMA"
  },
  {
    "caption": "An in vitro NK cell cytotoxicity assay was performed. Human NKL cells were used as effector cells, and mouse pancreatic cancer BMFA3 cells were used as target cells in normal DMEM (control), conditioned medium collected from TGFβ (30 ng/ml)-treated human pancreatic CAFs (CAF-PC2; CAF TGFβ-CM), CAF TGFβ-CM + IL-6 antibody (100 ng/ml) or normal DMEM + TGFβ (TGFβ). Living cells were labeled with CFSE, and dead cells were labeled with 7-AAD. Samples were analyzed by flow cytometry. Cytotoxicity percentage was calculated using the formula (7-AAD positive cells %) / (7-AAD positive cells % + CFSE positive cells %) x 100%. n = 4/group, P values by t test are shown.",
    "molecules": "CFSE, 7-AAD"
  },
  {
    "caption": "Human pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice. After tumor establishment, mice were randomized to receive rat IgG Mac84 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 6/group) for 3 weeks. For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Tumors were harvested for analysis and metastatic burden was determined by histologic evaluation of H&E-stained liver tissue. Ten sections of the anterior lobe of the liver (n = 6 per group) were scored for lesions.",
    "molecules": "Asialo-GM1"
  },
  {
    "caption": "3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture).",
    "molecules": "HEMA"
  },
  {
    "caption": "3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). Control IgG, 2G8 and IL-6 neutralizing antibody (each 100 ng/ml, n = 5/group). Scale bars = 50 μm. P values by t test are shown (C).",
    "molecules": "HEMA"
  },
  {
    "caption": "Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.",
    "molecules": "Asialo-GM1"
  },
  {
    "caption": "Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.",
    "molecules": "Asialo-GM1"
  },
  {
    "caption": "Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.",
    "molecules": "Asialo-GM1"
  },
  {
    "caption": "(A) NSCLC expressing single site mutants of EGFR, PC9 (3X106) or H3255 (8X106), were seeded in 10-cm dishes. On the next day, complete media were replaced with media containing serum (1%) and the cells were treated for 24 hours with different EGFR-specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). Thereafter, cells were washed with cold saline and extracted. Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (60 minutes), and treatment with Clarity™ Western ECL Blotting Substrates (Bio-Rad). ECL signals were detected using the ChemiDoc™ Imaging System (Bio-Rad) and images were acquired using the ImageLab software. Signals (relative to Control) were quantified and normalized to the signals of GAPDH (numbers shown below each lane).",
    "molecules": "afatinib, cetuximab, erlotinib, nitrocellulose, osimertinib, trastuzumab"
  },
  {
    "caption": "with different EGFR-specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). (C) PC9 cells were seeded on coverslips and treated for 24 hours Cells were washed in acidic buffer, fixed in paraformaldehyde (4%) and incubated with specific primary antibodies, followed by an Alexa Fluor 555-conjugated secondary antibody (pseudo colored in green). DAPI (blue) was used to stain nuclei. Images were captured using a confocal microscope (40X magnification).",
    "molecules": "afatinib, Alexa Fluor 555, cetuximab, DAPI, erlotinib, osimertinib, trastuzumab"
  },
  {
    "caption": "PC9 cells (3X106/mouse) were subcutaneously implanted in the flanks of CD1-nu/nu mice. When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, 0.2 mg/mouse/injection), once every three days, or with erlotinib (50 mg/kg/day), once per day. Alternatively, mice were treated with combinations of erlotinib (50, 20, 10, 5 or 1 mg/kg) and the two monoclonal antibodies. Following 60 days of treatment reduced the frequency of erlotinib administration to once every other day (underneath dotted line). animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3.",
    "molecules": "cetuximab, erlotinib, trastuzumab"
  },
  {
    "caption": "PC9 cells (exon 19 deletion) were subcutaneously implanted in the flank of CD1-nu/nu mice (3X106/mouse). When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, each at 0.1 mg/mouse/injection) once every three days, or daily with different TKIs: osimertinib (5 mg/kg), afatinib (5 mg/kg) or imatinib (100 mg/kg), either alone or in combination with the two antibodies. Following 60 days of treatment, the frequency of TKI administration was reduced to once every other day (underneath dotted lines), while mAb treatment remained unaltered. animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3.",
    "molecules": "afatinib, cetuximab, imatinib, osimertinib, trastuzumab"
  },
  {
    "caption": "CD1-nu/nu mice carrying PC9 xenografts were divided in groups (2-3 mice/group) and treated for seven days with different TKIs: erlotinib (50 mg/kg or 20 mg/kg), osimertinib (5 mg/kg) or afatinib (5 mg/kg), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection). when singly applied used erlotinib at 50 mg/kg. (B) After treatment, all mice were sacrificed and tumors extracted. Protein extracts were resolved by means of electrophoresis and transfer to nitrocellulose membranes, which were later incubated overnight with the indicated antibodies. This was followed by incubation with peroxidase-conjugated secondary antibodies. Signals were detected using ChemidocTM (from Bio-Rad), quantified and normalized to the signals of GAPDH or tubulin (numbers shown below each lane).",
    "molecules": "afatinib, cetuximab, erlotinib, nitrocellulose, osimertinib, trastuzumab"
  },
  {
    "caption": "A, B PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of CD1-nu/nu mice. When tumors reached a volume of approximately 500 mm3, mice were treated daily with erlotinib (50 mg/kg, blue area; panel A) or with osimertinib (5 mg/kg, grey area; panel B) using oral gavage. Initially, all tumors displayed stable disease or they regressed, but eventually all started relapsing. Once relapsing tumors reached 800 mm3, while under erlotinib or osimertinib treatment, we supplemented the treatment with a combination of 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection; twice a week) plus either erlotinib (50 mg/kg; A, red area) or osimertinib (5 mg/kg; B, orange area). Tumor volumes of individual mice are shown.",
    "molecules": "cetuximab, erlotinib, osimertinib, trastuzumab"
  },
  {
    "caption": "A, B NSG mice were pre-implanted with tumor fragments derived from two different PDX models: (A) TM00199 (L858R-EGFR; from The Jackson Laboratory) or (B) TM00193 (E746_A750 del-EGFR; from The Jackson Laboratory). Once tumors reached approximately 300 mm3, mice were treated with monoclonal antibodies (2XmAbs, cetuximab plus trastuzumab, 200μg/injection) twice a week, or with EGFR TKIs, erlotinib (50mg/kg) or osimertinib (10mg/kg) daily. Alternatively, mice were treated with a combination of 2XmAbs and either erlotinib or osimertinib, for either 32 days (A) or 42 days (B). Tumor growth was monitored twice a week. the antibodies were injected into the peritoneum and the TKIs were delivered orally. The figure shows tumor volumes of individual mice and the respective survival curves (right hand panels). The grey areas mark time windows of animal treatment.",
    "molecules": "cetuximab, erlotinib, osimertinib, trastuzumab"
  },
  {
    "caption": "(B) For RNAseq analyses, PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of 13 CD1-nu/nu mice. When tumors reached 500 mm3, mice were treated daily with erlotinib (50 mg/kg). Once tumors that initially responded to erlotinib monotherapy started relapsing and reached 800 mm3, we switched to a combination of 2XmAbs plus erlotinib (50 mg/kg). The respective tumor volumes were sacrificed when tumor volumes reached approximately 500 mm3 Mice responding to erlotinib (N=3) were sacrificed after one week of treatment Mice resistant to erlotinib (N=3) were sacrificed when tumor volumes reached 800 mm3 whereas mice responding to erlotinib+2XmAbs (N=2) were sacrificed after one week of treatment RNA was extracted from all 13 tumors and utilized for RNA-seq analysis. Differentially expressed (DE) genes in the Erlotinib (Er)+2XmAbs arm compared to the Control group are presented in the heatmap.",
    "molecules": "Er, erlotinib, Erlotinib"
  },
  {
    "caption": "B Hops−/− mouse genotyping and protein expression. Genotyping by PCR using genomic DNA showed a 242bp band for wild type (Hops+/+) allele and a 331bp band for mutant (Hops−/−) allele. NTC is Null Template Control (left panel). Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops−/− mice (middle panel). Protein extracts from Hops+/+ and Hops−/− MEFs were analysed by Western Blot using anti-HOPS antibody and anti-α-tubulin antibody as loading control (right panel). Data information: in B data are presented as mean ±SD.",
    "molecules": "genomic DNA"
  },
  {
    "caption": "A Reduced caspase-3 activation in Hops−/− mice upon apoptotic stimulus. Hops+/+ and Hops−/− mice were treated with etoposide for the indicated times and thymus, spleen, testis and liver were harvested and Tissue-Tek® O.C.T. embedded. Sections were immunostained with anti-cleaved caspase-3 antibody followed by appropriate secondary antibody labelling (green). The graphs display the relative positive cells number. Scale bars, 10µm. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In A data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.",
    "molecules": "etoposide"
  },
  {
    "caption": "A Thymocytes from Hops+/+ and Hops−/− mice were treated with dexamethasone (Dex) and the apoptotic cells were measured by flow cytometry. The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right. Data information: the experiments were performed three times in primary cells from three different mice per condition. In A data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.",
    "molecules": "Dex, dexamethasone"
  },
  {
    "caption": "B HOPS was pivotal for apoptosis induction. Hops+/+ and Hops−/− MEFs were subjected to etoposide (Eto) and apoptosis was analysed by flow cytometry (left) and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.",
    "molecules": "Eto, etoposide"
  },
  {
    "caption": "D HOPS affects p53 levels following stress in vitro. Hops+/+ and Hops−/− MEFs were treated with etoposide and harvested at the indicated time points. The p53 protein levels were semi-quantified using α-tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1. Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown.",
    "molecules": "etoposide"
  },
  {
    "caption": "A HOPS loss and p53 half-life. Hops+/+ and Hops−/− MEFs untransfected or transfected with HOPS, were analysed by immunoblotting following cycloheximide treatment for the indicated time (left panel). The p53 protein levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100% (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In A data are presented as mean ±SD.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "B p19Arf-independent HOPS control of p53 half-life. p19Arf−/− MEFs untransfected (CTRL), transfected with empty vector (pSGV) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti-p53, anti-HOPS and anti-GAPDH antibodies (left panel). The p53 protein levels were semi-quantified as above (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In B data are presented as mean ±SD.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "C HOPS and p53 ubiquitination. p53, HDM2, His-tagged ubiquitin were co-expressed in H1299 cells with HOPS or HOPS-G176A or HOPS-K126A mutants and treated with MG132. p53 ubiquitination rate was evaluated by elution using Ni2+-NTA His-bind resin followed by immunoblotting with anti-p53 and the indicated antibodies.",
    "molecules": "MG132, Ni2+, NTA, ubiquitin"
  },
  {
    "caption": "D HOPS depletion and HOPS-G176A mutant affect p53 ubiquitination. Hops+/+ and Hops−/− MEFs untransfected and Hops−/− MEFs transfected with HOPS or HOPS-G176A mutant were co-expressed with p53, HDM2 and His-tagged ubiquitin. Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above. Immunodetection of ubiquitinated p53 was performed using anti-p53 antibody and the samples were tested with the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "E HOPS-G176A mutant ability to stabilize p53. Hops−/− MEFs were transfected with HOPS-G176A, subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1. Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD.",
    "molecules": "FITC, DAPI, DNA, etoposide"
  },
  {
    "caption": "B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.",
    "molecules": "etoposide"
  },
  {
    "caption": "C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.",
    "molecules": "etoposide"
  },
  {
    "caption": "A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm.",
    "molecules": "DAPI, MitoTracker"
  },
  {
    "caption": "C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls.",
    "molecules": "etoposide"
  },
  {
    "caption": "D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls.",
    "molecules": "etoposide"
  },
  {
    "caption": "E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1. Data information: assays involving animals were performed three times in three different mice per condition. In E data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.",
    "molecules": "etoposide"
  },
  {
    "caption": "F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel). Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.",
    "molecules": "JC-1, etoposide"
  },
  {
    "caption": "A HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel). Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD.",
    "molecules": "DAPI"
  },
  {
    "caption": "HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal. Data information: all the experiments detailed above were performed three times, and representative panels are shown.",
    "molecules": "etoposide"
  },
  {
    "caption": "D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies. Data information: all the experiments detailed above were performed three times, and representative panels are shown.",
    "molecules": "Lysine, nicotinamide, trichostatin A"
  },
  {
    "caption": "B, Immunoblot  showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (B) JPX-0750  The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4  cytokine addition. HSC70  was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.",
    "molecules": "JPX-0750"
  },
  {
    "caption": "C Immunoblot  showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (C) IQDMA. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4  cytokine addition. HSC70  was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.",
    "molecules": "IQDMA"
  },
  {
    "caption": "Real-Time apoptosis/necrosis  assay in the (D, SeAx  and (F, Myla  cell lines treated with 2.5µM JPX-0750 RLU, Apoptosis  [RLU] = relative luminescence; Necrosis  [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.",
    "molecules": "JPX-0750"
  },
  {
    "caption": "Real-Time apoptosis/necrosis  assay in the E) SeAx  and G) Myla  cell lines treated with 2.5µM IQDMA. RLU, Apoptosis  [RLU] = relative luminescence; Necrosis  [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.",
    "molecules": "IQDMA"
  },
  {
    "caption": "H-I The effect of (H) JPX-0750 and (I) IQDMA  on the viability  of CTCL  cells in comparison to viability  of primary  juvenile fibroblasts (JF) and keratinocytes  (HaCat) at a low dose range (0.7-3µM). One experiment, in triplicates, was performed with JF  and HaCat  cells, and three experiments, in triplicates with the CTCL  cells. Error bars represent mean +/-SD. Statistical significance was calculated by two-way ANOVA with Tukey's multiple comparisons test. P-value: <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****).",
    "molecules": "JPX-0750, IQDMA"
  },
  {
    "caption": "B Subcellular fractions of SeAx  cells treated with IQDMA  for 24h and immunoblotted  for pY-STAT5  and total STAT5. The cells were collected 30 min after 1 ng/ml IL-2  cytokine addition.  α-Tubulin and Histone H3 were used as loading controls for cytoplasmic  and nuclear  fractions, respectively. The normalized levels of phospho- and total STAT5  in the nucleus  and cytoplasm, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown.",
    "molecules": "IQDMA"
  },
  {
    "caption": "D-F Scatter plot depicting the profile of SeAx  cells treated with (D) 1µM IQDMA, (E) 2.5µM IQDMA  and (F) 10µM IQDMA. Protein abundances were determined using TMT-based quantification mass spectrometry Significant changes were assessed by a modified t-test as implemented in the limma package, with the negative log10 p values on the x-axis, and log2 fold change shown on the y-axis.",
    "molecules": "IQDMA"
  },
  {
    "caption": "(D) Heatmap showing MSA scores of the combination of FRAx597  with the listed drugs. One representative out of two independent experiments is shown.",
    "molecules": "FRAx597"
  },
  {
    "caption": "E Heatmap showing the IC50  values upon treatment of primary PBMCs  isolated from L-CTCL patients  with JPX-0750, IQDMA  and FRAx597. One experiment performed in triplicates using CellTiter-Glo viability assays  upon 48h drug treatment is shown. The relative STAT3/5 log2 ratio value is depicted in grey.",
    "molecules": "JPX-0750, FRAx597, IQDMA"
  },
  {
    "caption": "C-D Myla-derived tumor weight  upon experiment termination, after 20 days of treatment with 5 mg/kg of (C) JPX-0750 (n = 5) and IQDMA  (n = 6), or vehicle (n = 7), and (D) FRAx597  (n = 5) or vehicle (n = 4), with n representing the number of analyzed tumors  per group. Statistical significance was calculated using two-tailed paired t-test. P- value: <0.05 (*). Error bars represent mean +/-SEM.",
    "molecules": "JPX-0750, FRAx597, IQDMA"
  },
  {
    "caption": "H&E analysis of Myla-derived tumors treated with  JPX-0750 (n = 6), IQDMA  (n = 6) or vehicle (n = 6), and FRAx597  (n = 5) or vehicle (n = 4), stained  with (E, F) H&E with n representing the number of analyzed tumors  per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the perimeter of the annotated tumor cell-infiltration/expansion region into the intradermal and subcutaneous region of the skin  Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.",
    "molecules": "JPX-0750, FRAx597, IQDMA"
  },
  {
    "caption": "IHC  analysis of Myla-derived tumors treated with  JPX-0750 (n = 6), IQDMA  (n = 6) or vehicle (n = 6), and FRAx597  (n = 5) or vehicle (n = 4), stained  with (G, H) Ki67, as a marker for proliferation  with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the quantification of the percentage of Ki67+ cells in the tissue. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.",
    "molecules": "JPX-0750, FRAx597, IQDMA"
  },
  {
    "caption": "IHC  analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA  (n = 6) or vehicle (n = 6), and FRAx597  (n = 5) or vehicle (n = 4), stained  with (I, J) CD31, as a tumor vessel  marker, with n representing the number of analyzed tumors  per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. CD31 staining  was quantified as raw counts of vessels  with lumen. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.",
    "molecules": "JPX-0750, FRAx597, IQDMA"
  },
  {
    "caption": "Ub-PA activity-based probe assay for wild-type (WT) and catalytic Cys-to-Ala mutants (CA). Strong, Cys-dependent reactivity is indicated with asterisks.",
    "molecules": "Ala, Cys"
  },
  {
    "caption": "Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations. Note that BurkOTU displays an increase in fluorescence polarization, indicative of noncovalent binding.",
    "molecules": "TAMRA, Ub"
  },
  {
    "caption": "Heatmap representation of DUB activity against the Ub-KG(TAMRA) substrate shown in G., including the WT enzyme and Ala substitutions at the predicted catalytic Cys, general base His, or acidic position. Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants.",
    "molecules": "TAMRA, Ala, Cys, His, Ub"
  },
  {
    "caption": "Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate.",
    "molecules": "TAMRA, Ala, Cys, Ub"
  },
  {
    "caption": "Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration.",
    "molecules": "Ub"
  },
  {
    "caption": "Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided wMelOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for reference.",
    "molecules": "TAMRA, Ub"
  },
  {
    "caption": "Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided EschOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for clarity.",
    "molecules": "TAMRA, Ub"
  },
  {
    "caption": "Ub-PA activity-based probe assay for WT vOTU and sequence-permutated vOTUP. Strong reactivity is indicated with asterisks.",
    "molecules": "PA, Ub"
  },
  {
    "caption": "Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence-permutated vOTUP.",
    "molecules": "TAMRA, Ub"
  },
  {
    "caption": "(B) Kinase assays of CDKA;1-SDS, -TAM, and -CYCA3;1 complexes using ASY1 puried from baculovirus-infected insect cells as a substrate. The upper panel shows the autoradiograph. The control reaction without CDKA;1-cyclin complex indicates a background activity co-purified from insect cells. The lower panel indicates protein loading by coomassie briliant blue (CBB) staining. Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay. Numbers indicate the relative intensities of ASY1 bands.",
    "molecules": "CBB, coomassie briliant blue"
  },
  {
    "caption": "(D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays.",
    "molecules": "Ade, glucose, His, Leu, Trp"
  },
  {
    "caption": "(B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies. (C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed.. ",
    "molecules": "Triton X-100"
  },
  {
    "caption": "A WT and Ccnc-/- MEF cultures were treated with 0.4 mM H2O2 as indicated, extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax. The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C. Whole cell extracts (WCE) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "H2O2"
  },
  {
    "caption": "C Mitochondrial relocalization of cyclin C is required for Bax activation. The experiment described in (A) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin-µ (PFT) for 24 h as indicated. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "PFT, pifithrin-µ, H2O2"
  },
  {
    "caption": "D Cyclin C is required for efficient Bax-Bcl-XL disassociation. Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 (0.4 mM, 4 h) were immunoprecipitated with active conformation Bax antibodies. The immunoprecipitates were subjected to Western blot analysis probing with Bcl-XL antibodies. Western blot analysis of WCE was performed as input concentration controls. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "H2O2"
  },
  {
    "caption": "E The mitochondrial fission complex is required for cyclin C-dependent Bax activation. Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 (0.4 mM, 4 h). The extracts were treated as in A. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "H2O2"
  },
  {
    "caption": "F Drp1 is required for cyclin C-Bax interaction. Extracts were prepared from wild type or Drp1-/- MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "H2O2"
  },
  {
    "caption": "A Wild type MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. Zoom panels (4X) are indicated by the boxes in the merge panels. Green arrows indicate cyclin C and mitochondrial co-localization. Bar indicates 10 µM.",
    "molecules": "DAPI, MitoTracker Red"
  },
  {
    "caption": "MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. S-HAD peptide activity requires cyclin C. The experiment described in A was repeated with Ccnc-/- MEF cultures.",
    "molecules": "DAPI, MitoTracker Red"
  },
  {
    "caption": "E Bax activation was monitored in wild-type MEF cultures treated with S-HAD (10 µM) for the times indicated. The anti-oxidant N-acetyl cysteine (NAC) was added (1 mM) 2 h prior to S-HAD treatment. Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated. Bax, cyclin C and ß-actin levels were monitored in extract preparations as input controls. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "N-acetyl cysteine, NAC"
  },
  {
    "caption": "A Caspase activation was measured by fluorescent cell analysis in wild-type MEF cells treated with S-HAD (10 µM, 24 h) or cisplatin (CP, 30 µM, 16 h). n = 3. Error bars indicate SD with * and ** indicate p <0.05 and p<0.01, respectively using Student's T test.",
    "molecules": "cisplatin"
  },
  {
    "caption": "B PARP cleavage was not induced by S-HAD treatment. PARP cleavage was monitored by Western blot analysis in Hela cells treated with S-HAD or cisplatin (CP) as described in (A). The blot was stripped and reprobed with ß-actin antibodies as a loading control. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "cisplatin"
  },
  {
    "caption": "C Wild type and Ccnc-/- MEF cultures were treated with S-HAD and cisplatin (CP) as just described. Cells were imaged and examined for a \"rounding up\" phenotype diagnostic for death.",
    "molecules": "cisplatin"
  },
  {
    "caption": "D Cyclin C is required for normal Bax oligomerization in response to cisplatin. Whole cell extracts prepared from DSP crosslinked wild type and CCNC-/- MEF cells exposed to cisplatin (30 µM, 16 h) were probed for total Bax. Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "DSP, cisplatin"
  },
  {
    "caption": "F S-HAD treatment sensitizes Hela cells to cisplatin. Hela cultures were treated with cisplatin (30 µM), S-HAD (10 µM) and N-acetyl cysteine (NAC, 1 mM) as indicated. Fluorescent cell analysis was used to quantitate the apoptotic (Annexin V positive) and necrotic (PI positive) population percentages, respectively. n = 3 independent cultures. Error bars indicate SD. Asterisk indicates p <0.05 (Student's T-test).",
    "molecules": "cisplatin, N-acetyl cysteine, NAC, PI"
  },
  {
    "caption": "A S-HAD treatment induces mitochondrial ROS. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 24 h), rotenone or vehicle control as indicated. Mitochondrial ROS was measured by MitoSox staining (4 µM, 15 min) and quantitative confocal imaging. Error bars indicate SD. n=3, asterisk indicates p<0.05 (Student's T-test), n.s., not significantly different.",
    "molecules": "ROS, rotenone"
  },
  {
    "caption": "B Quenching mitochondrial ROS reduces cyclin C-Bax interaction. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 16 h) and 100 nM mitochondrial-specific reactive oxygen scavenger MitoTempol. Western blot analysis was conducted as described in Fig. 1. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "MitoTempol, ROS"
  },
  {
    "caption": "C Insulin treatment stimulates S-HAD induced Bax activation. Wild type and Ccnc-/- MEF cultures were grown with or without insulin (10 µg/ml) then treated with S-HAD (10 µM, 2 h). Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins. Atp5A and ß-actin levels control for mitochondrial and cytosolic loading, respectively. The two blots were prepared, processed and developed identically. Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "Insulin, insulin"
  },
  {
    "caption": " SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm) ",
    "molecules": "DAPI"
  },
  {
    "caption": " (A) Filipin staining of SKOV3-OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0, 30 minutes, and 12 hours in serum free (SF) medium",
    "molecules": "Filipin, MBCD"
  },
  {
    "caption": "(B) Western blotting of SKOV3-OPCML  cells treated with MBCD and separated into the detergent resistant \"R\"  membrane fraction and the detergent soluble \"S\" fraction upon Gas6  stimulation",
    "molecules": "MBCD"
  },
  {
    "caption": "(D) MBCD treatment of SKOV3-EMPTY and SKOV3-OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot. GAPDH was used as loading control",
    "molecules": "MBCD"
  },
  {
    "caption": "(E,F) Signalling kinetics are presented for pAXL and pERK in SKOV3-EMPTY "E" and SKOV3-OCPML "O", treated with MBCD and no treatment control, normalised to GAPDH, from (D)",
    "molecules": "MBCD"
  },
  {
    "caption": "(G) PLA assay of AXL-OPCML interaction (red) upon Gas6 stimulation in both MBCD treated and non-treated SKOV3-OPCML cells. Scale bar = 50μm",
    "molecules": "MBCD"
  },
  {
    "caption": "(B) Western Blotting of SKOV3-Empty \"E\" and SKOV3-OPCML \"O\" cells pre incubated with either no drug (DMSO only control), 1.09 μM R428, or 2.84 μM R428 for an hour and stimulated with Gas6 for the indicated times. GAPDH was used as loading control",
    "molecules": "R428"
  },
  {
    "caption": "(C) Immunofluorescence staining of pERK (red) and OPCML (magenta) in SKOV3-Empty \"E\" and SKOV3-OPCML \"O\" cells treated with R428 and stimulated with Gas6 for the indicted time points. Scale bars = 50μm",
    "molecules": "R428"
  },
  {
    "caption": "(A, B) Transmission electron micrographs of negatively stained 2F (A) and 3F (B) fibrils before sonication and injection into mice. The two fibril polymorphs were grown in vitro from synthetic Aβ40 and show the morphological differences originally reported Fibrils are negatively stained with uranyl acetate. Bars represent 100 nm.",
    "molecules": "fibril, fibrils, Fibrils, Aβ40, uranyl acetate"
  },
  {
    "caption": "(C) PK resistance profile of 2F and 3F fibrils. Values are expressed as the mean densitometric value at each PK concentration (technical triplicates) ± standard error. Statistical analyses were performed by student's t-test (*p<0.05).",
    "molecules": "fibrils"
  },
  {
    "caption": "D) Seeding assays using 100 pM of 2F (blue) and 3F (green) fibrils as seeds as described in Materials and Methods. Curves were generated by measuring ThT emission values at different time points. Reaction kinetics were statistically different as assessed by using the multiple comparisons method to compare pairs of curves (***p<0.0001). Data in graphs is represented as averages depicting standard errors of five technical replicates.",
    "molecules": "fibrils, ThT"
  },
  {
    "caption": "F-I) 2F and 3F fibrils were tested for their reactivity against a variety of luminescent conjugated thiophenes able to discriminate among conformational variants of misfolded proteins. Stock solutions of LCOs (1.5 mM) were diluted in distilled water to 15 μM and added to the wells to a final concentration of 0.3 μM. All wells contained 10 μM of either 2F or 3F fibrils. The samples were incubated at 37°C and the emission spectrum of each probe was collected after 30 minutes by exciting the samples at 440 nm (HS-68) or 535 nm (HS-194, HS-208 and HS-212).",
    "molecules": "fibrils"
  },
  {
    "caption": "(A-H) Representative images of hippocampi from mice treated with 2F-, 3F- and Tg2576- derived seeds after visualization for Aβ deposits using the 4G8 antibody (A-D) or ThS (E-H). CA, cornu ammonis 1; DG, dentate gyrus. Scale bar in (B), (D), (F) and (H) represents 500 µm and applies to all panels. Arrows represent regions of interest (alveus, DG).",
    "molecules": "Aβ, ThS"
  },
  {
    "caption": "(I, J) Aβ (I) and ThS (J) burden quantification in hippocampus. Statistical analyses were performed using One-way ANOVA (*p<0.05, **p<0.001). Data in graphs is represented as averages depicting standard errors. Measurements considered five tissue slices per animal, and 4-7 animals were included per group.",
    "molecules": "Aβ, ThS"
  },
  {
    "caption": "(O-R) Correlations between Aβ burden and glial burden were calculated in the alveus for mice injected with 2F (O) and 3F (P) fibrils. In the same way, the relationship between glial activation and Aβ burden in the hippocampus was calculated for mice treated with 2F (Q) and 3F (R) aggregates. Statistical analyses were performed by Pearson correlation coefficients test in which p<0.05 was considered significant.",
    "molecules": "fibrils, Aβ"
  },
  {
    "caption": "A) Panels showing representative pictures of the dentate gyrus of mice treated with experimental and control Aβ seeds and stained with both 4G8 (green) and anti-Iba-1 (red) antibodies. The third column of panels from left to right depict merged images. Right panels are insets obtained from the merged images (white punctuated squares). : Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as \"4G8\", \"Iba-1\" and \"Merge\". Scale bars on the right images represent 50µm. ; g, granular layer; h, hilar region; m, molecular layer;",
    "molecules": "Aβ"
  },
  {
    "caption": "A) 1D 13C ssNMR spectra of fibrils that were prepared in vitro by seeded growth from amyloid-containing extracts of mouse brain homogenates. Mice had been treated with 2F-, 3F-, or Tg2576-derived seeds. Aβ40 was 15N,13C-labeled at F19, V24, G25, S26, A30, I31, L34, and M35. Vertical dashed lines indicate some of the ssNMR peaks that vary among the three samples. Asterisk indicates a peak from residual sodium dodecyl sulfate in the 2F sample.",
    "molecules": "fibrils, amyloid, Aβ40, 13C, 15N, sodium dodecyl sulfate"
  },
  {
    "caption": "MT regrowth experiments of WT cells mixed with either WT (as a control), akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO or pc-c5-2KO cells. Samples were fixed 3 min after NZ washout and stained for EB1 (green) and the indicated protein in the red channel. Quantification of EB1 signal intensity around the centrosome",
    "molecules": "NZ"
  },
  {
    "caption": "IF images of WT and pcnt KO RPE-1 cells treated with NZ for 3h and triple stained for AKAP450 (green), CDK5Rap2 (red) and GMAP210 as a Golgi marker (blue). At right, quantification of fluorescence co-localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells­.",
    "molecules": "NZ"
  },
  {
    "caption": "Control and centrinone-treated cells were subjected to a 3-min MT regrowth assay, fixed and labelled for EB1 (green) and giantin (red). Insets show magnified images of boxed regions.",
    "molecules": "centrinone"
  },
  {
    "caption": "RPE-1 cells were treated wi­­th centrinone for either 7 days (left) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation (right). Representative images of a triple IF staining with EB1 (green), γ-tubulin (red) and giantin (blue) as a Golgi marker are shown. Insets show MT nucleation from Golgi elements (left) or from multiple centrioles (right) and are enlarged at right. Arrowheads indicate the position of Golgi elements.",
    "molecules": "centrinone"
  },
  {
    "caption": "Quantification of the percentage of cells exhibiting Golgi-nucleating activity in centrinone-treated cells 48h after the washout of the drug. Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63. At least 100 cells from two independent experiments were analyzed in each condition.",
    "molecules": "centrinone"
  },
  {
    "caption": "Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR).",
    "molecules": "centrinone, DAPI, DNA"
  },
  {
    "caption": "IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces.",
    "molecules": "centrinone, NZ"
  },
  {
    "caption": "MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads).",
    "molecules": "centrinone, NZ"
  },
  {
    "caption": "Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3).",
    "molecules": "Centrinone, NZ"
  },
  {
    "caption": "MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.",
    "molecules": "centrinone"
  },
  {
    "caption": "MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments.",
    "molecules": "centrinone, NZ"
  },
  {
    "caption": "Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels).",
    "molecules": "Centrinone"
  },
  {
    "caption": "Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies.",
    "molecules": "centrinone"
  },
  {
    "caption": "MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue).",
    "molecules": "centrinone, NZ"
  },
  {
    "caption": "MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.",
    "molecules": "centrinone, DMSO, gatastatin, NZ"
  },
  {
    "caption": "Confocal images showing MT network in centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO backgrounds. Cells containing extra centrosomes induced by centrinone washout were also quantified (bottom panels).",
    "molecules": "centrinone"
  },
  {
    "caption": "Quantification of α-tubulin fluorescence intensity (as a measure of MT mass polymer, C), in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.",
    "molecules": "centrinone"
  },
  {
    "caption": "Quantification of number of EB1 comets per cell (D) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.",
    "molecules": "centrinone"
  },
  {
    "caption": "Quantification of α , cell area (E) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.",
    "molecules": "centrinone"
  },
  {
    "caption": "Quantification of MT density (MT mass polymer/cell area) (F) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.",
    "molecules": "centrinone"
  },
  {
    "caption": "(A and B) THP1-derived macrophages were treated with the TLR ligands Pam3, lipopolysaccharide (LPS), or FSL-1 and analyzed by RNA-seq 8 h later. Circos plots show genome-wide differential expression of protein-coding genes (A) and non-protein-coding genes (B) between untreated and treated macrophages. The outermost to innermost circles show downregulated (blue) or upregulated (red) genes in cells stimulated with FSL-1, LPS, and Pam3, respectively. Chromosomes are indicated by the numbers 1-22 and X and Y.",
    "molecules": "FSL-1, lipopolysaccharide, LPS, Pam3"
  },
  {
    "caption": "(F) RT-qPCR validation of differentially expressed lncRNAs and mRNAs in THP1-derived macrophages stimulated for 8 h with flagellin (FLA), FSL-1, imiquimod, LPS, Pam3, or poly(I:C). Heat map shows the fold change in expression of 13 mRNAs (left) and lncRNAs (right).",
    "molecules": "FSL-1, imiquimod, LPS, Pam3, poly(I:C)"
  },
  {
    "caption": "Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.",
    "molecules": "gentamicin"
  },
  {
    "caption": "Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 IL-6 ELISA quantification (C) of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. ELISA were performed at 10 h post-infection.",
    "molecules": "gentamicin"
  },
  {
    "caption": "Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-1α (D). RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.",
    "molecules": "gentamicin"
  },
  {
    "caption": "Overexpression of lnc-MARCKS decreases MARCKS expression RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. Lnc-MARCKS and MARCKS expression (F)",
    "molecules": "Pam3, PMA"
  },
  {
    "caption": "Overexpression of lnc-MARCKS enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. IL-6 expression (G).",
    "molecules": "Pam3, PMA"
  },
  {
    "caption": "lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect lnc-MARCKS (H)",
    "molecules": "antisense oligo, Pam3"
  },
  {
    "caption": "lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS (I),",
    "molecules": "antisense oligo, Pam3"
  },
  {
    "caption": "lnc-MARCKS regulates IL-6 in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect IL-6 expressions (J).",
    "molecules": "antisense oligo, Pam3"
  },
  {
    "caption": "(A) ROCKI is recruited to the MARCKS promoter in Pam3-stimulated THP1-derived macrophages. ChIRP analysis of THP1-derived macrophages treated with Pam3 for 8 h. ChIRP assays were performed using a non-specific lacZ probe or probes specific for lnc-MARCKS. Specificity of ROCKI probes (left), and enrichment at the MARCKS promoter (right).",
    "molecules": "Pam3"
  },
  {
    "caption": "(E) ROCKI associates with APEX1. Left: RT-qPCR analysis of ROCKI present in anti-APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3-treated THP1-derived macrophages. Right: APEX1 western blot of the same immunoprecipitates.",
    "molecules": "Pam3"
  },
  {
    "caption": "ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA (NC) or ROCKI shRNA and treated with Pam3 for 8 h. The y-axis shows the percentage of enrichment in normalized to input. An unrelated region of the ACTB2 promoter was amplified as a control.",
    "molecules": "Pam3"
  },
  {
    "caption": "Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter. ChIP-qPCR analysis of H3K27ac was performed using an anti-H3K27ac antibody.",
    "molecules": "H3K27ac"
  },
  {
    "caption": "RT-qPCR validation of inflammatory gene expression in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages stimulated with Pam3 for 8 h. E.V., empty vector; NC, control shRNA.",
    "molecules": "Pam3"
  },
  {
    "caption": "MARCKS is a negative regulator of Ca2+ signaling. Fluo-4 fluorescence of MARCKS, ROCKI, or NC (control) shRNA-expressing THP1-derived macrophages before and after treatment with PMA and Pam3. Triton X-100 was added to obtain the maximal [Ca2+] signal. Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence (ΔF/Fmax).",
    "molecules": "Ca2+, Fluo-4, Pam3, PMA, Triton X-100"
  },
  {
    "caption": "(A) QTL signals for ROCKI (LINC01268) gene expression in whole blood (top, black), H3K27ac promoter signal in monocytes (middle, green), and H3K4me1 signal in monocytes (bottom, orange) in a 400 kb window around the MARCKS/ROCKI locus. The signals were strongly co-localized (Pshared=.97, Pshared=.98) suggesting they represent the same signal.",
    "molecules": "H3K4me1"
  },
  {
    "caption": "(A) Cell lysates prepared from HeLa cells were solubilized with Triton X-100 and subjected to immunoprecipitation using preimmune sera (lane 1) or anti-Beclin antibodies (lanes 2 and 3). The immunoprecipitates were incubated with phosphatidylinositol, [γ-32P]ATP and 60 μM cold ATP in the presence of Mn2+ (lanes 1 and 2) or Mg2+ (lane 3) for 5 min at 30°C.",
    "molecules": "γ‐32, ATP, Mg2+, Mn2+, phosphatidylinositol, Triton X–100"
  },
  {
    "caption": "(B) Immunoprecipitates of anti‐Beclin antibodies prepared as described in (A) were assayed for PI 3‐kinase activity in the presence of wortmannin at concentrations of 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4). The labeled lipids were extracted and separated by thin layer chromatography, followed by detection by autoradiography using a bioimaging analyzer BAS2000 (Fuji Film).",
    "molecules": "wortmannin"
  },
  {
    "caption": "(C) Cell lysates prepared from HeLa cells were solubilized with Triton X‐100 and incubated with protein A‐immobilized anti‐Beclin (lane 2) or anti‐PtdIns 3‐kinase antibodies (lane 4). As controls, preimmune sera of the respective antibodies (lanes 1 and 3) were used. Retained proteins were separated by SDS-PAGE and detected by immunoblotting with anti‐PtdIns 3‐kinase (upper panels) and anti‐Beclin (lower panels) antibodies.",
    "molecules": "Triton X‐100"
  },
  {
    "caption": "(A) Total cell lysates were treated with DSP (lanes 3, 4, 7 and 8) or its solvent, dimethylsulfoxide (lanes 1, 2, 5 and 6), at 4°C for 2 h. Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A‐immobilized anti‐PtdIns 3‐kinase (PtdIns 3‐K) (lanes 1-4) or Beclin (lanes 5-8) antibodies. Immunoprecipitates were eluted with 1× SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, a trace amount of Bromophenol blue). After treatment with 5% 2‐mercaptoethanol, proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti‐Beclin (upper panels) and anti‐PtdIns 3‐kinase (lower panels) antibodies.",
    "molecules": "dimethylsulfoxide, DSP, SDS"
  },
  {
    "caption": "(C) Membrane (lanes 1-4; 100 000 g, 30 min, pellet) and soluble fractions (lanes 5-8; 100 000 g, 30 min, supernatant) prepared from HeLa cells were treated with DSP (lanes 3, 4, 7 and 8) or dimethylsulfoxide (lanes 1, 2, 5 and 6). Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A-immobilized anti-PtdIns 3-kinase antibodies. Immunoprecipitates were separated by SDS-PAGE under a reducing condition, followed by detection by immunoblotting with Beclin antibodies. F and E indicate flow-through and elute fracions, respectively.",
    "molecules": "dimethylsulfoxide, DSP, SDS"
  },
  {
    "caption": "Beclin localizes to the TGN. HeLa (A, D, E and F), BNL CL.2 (B) and H‐4‐II‐E cells (C) were grown on cover slips. Cells were permeabilized with digitonin before (C, D and E) or after (A, B and F) fixation with 3% paraformaldehyde. Cells were double‐labeled with anti‐Beclin (A-F, left panels) and either anti‐syntaxin 6 (A and B, middle panels), anti‐TGN38 (C, middle panel), anti‐EEA1 (D, middle panel), anti‐LBPA (E, middle panel) or anti‐TfR (F, middle panel) antibodies. In the merged images (right panels), yellow indicates co‐localization. Bar, 10 μm.",
    "molecules": "digitonin, LBPA"
  },
  {
    "caption": "Immunofluorescence localization of PtdIns 3‐kinase. HeLa (A and C) and H‐4‐II‐E cells (B) were permeabilized, fixed, and then subjected to immunostaining. Cells were double‐labeled with anti‐PtdIns 3‐kinase (A-C, left panels) and either anti‐syntaxin 6 (A, middle panel), anti‐TGN38 (B, middle panel) or anti‐LBPA (C, middle panel) antibodies. Bar, 10 μm.",
    "molecules": "LBPA"
  },
  {
    "caption": "(A) Heat map indicates which TFs significantly affects rosette area, growth rate, shoot biomass, flowering time and seed yield and whether the effects of TFs are dependent on salt treatment. TFs are hierarchically clustered using Euclidean distance.",
    "molecules": "salt"
  },
  {
    "caption": "(C) Rosette area of mutant allele of BPC4 from day 7 to day 21 post-germination. Linear model for two-way ANOVA considers AGI, salt treatment, day post-germination and their interactions. The AGI and AGI:Salt Condition terms are statistically signification (P < 0.001, P < 0.01, respectively). Heat map under line plot indicates which term in linear model is statistically significant (P < 0.05) using two-way ANOVA for each day from 9 biological replicates per condition. Solid lines, Col-0; dashed line, bpc4; circle, control condition; triangle, 50 mM NaCl.",
    "molecules": "NaCl, salt, Salt"
  },
  {
    "caption": "E The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. (Left panel) GB18 was treated with AGK2 (10 μM) or DMSO. (Right panel) Secondary sphere formation of GB18 was examined in the absence of AGK2. Bars indicate mean ± s.d. of 8 wells.",
    "molecules": "AGK2, DMSO"
  },
  {
    "caption": "D The effect of AK7 on the deacetylation of acetyl-tubulin (Left panel) and the proliferation of GB2 cells (Right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl-tubulin/tubulin were performed on day 3 in Right panel. Bars indicate mean ± s.d. (n = 3).",
    "molecules": "acetyl, AK7"
  },
  {
    "caption": "E Ten days after intracranial transplantation of GB2 cells (1.0 × 104 cells), AK7 was intraperitoneally administrated for 4 weeks (15mg/kg, twice/week). After eight weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA was quantified by qRT-PCR.",
    "molecules": "AK7"
  },
  {
    "caption": "A Cells transfected with FLAG-tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.",
    "molecules": "acetylated lysine, AGK2, DMSO"
  },
  {
    "caption": "Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.",
    "molecules": "acetylated lysine, AGK2, DMSO"
  },
  {
    "caption": "E HEK293T cells transfected with FLAG-tagged TAp73α were treated with 20 μM AGK2 (pre-AGK2 +) or DMSO (pre-AGK2 -) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM) and/or AGK2 (3.5 μM) as indicated.",
    "molecules": "AGK2, DMSO, NAD"
  },
  {
    "caption": "(A) Flag‐tagged DAPK (100 ng) was incubated with GST (900 ng) or GST-beclin‐1 (750 ng) in the presence of Ca2+, calmodulin and [γ‐33P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X‐ray film exposure, and GST/GST-beclin‐1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples.",
    "molecules": "γ‐33P, ATP, Ca2+"
  },
  {
    "caption": "(B) Flag‐tagged DAPK (60 ng) was incubated with Flag‐tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin‐1‐bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X‐ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney.",
    "molecules": "LiCl, KCl"
  },
  {
    "caption": "(C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot).",
    "molecules": "ATP, Ca2+"
  },
  {
    "caption": "(C) Cleavage  of supercoiled (SC)  plasmid DNA substrates using purified Cas12l  nuclease and respective sgRNA variants. Efficient full length linearization (FLL) of the substrate resulting from a complete double-strand break was only observed when using sgRNAs bearing the 5' most end of the tracrRNA (variants 1 and 3). Moreover, the terminator-like Hairpin 3 is not required for target cleavage (variant 1). OC - open circular; FLL - full length linear; SC- supercoiled.",
    "molecules": "plasmid DNA"
  },
  {
    "caption": "(B) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l. wt - wildtype.",
    "molecules": "dsDNA"
  },
  {
    "caption": "(C) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition. The molar ratio of RNP to substrate was kept low (5:1) to increase reaction stringency. Asp2Cas12l predominantly recognizes a 5'-CCY-3' PAM and Asp3Cas12l a 5'-CCB-3' PAM. N=3. Data information: In (C), individual data points are plotted, where n = 4 replicates from independent experiments (Asp2Cas12l) and n = 3 replicates from independent experiments (Asp3Cas12l). The data points were fitted to a single exponential association curve (solid lines).",
    "molecules": "dsDNA"
  },
  {
    "caption": "(A) Asp2  and Asp3Cas12l RNP complexes degrade M13 ssDNA  in the presence of single-stranded (ss) DNA  or dsDNA  (including PAM) activators with target sequences complementary to the gRNA spacer. NS activator - non specific activator (ssDNA oligonucleotide or dsDNA duplex) with no sequence complementarity to the spacer of the gRNA.",
    "molecules": "ssDNA oligonucleotide, dsDNA, dsDNA duplex, single-stranded (ss) DNA, ssDNA"
  },
  {
    "caption": "(B) Asp2Cas12l RNP complexes  activated with ss or dsDNA  degrade quenched fluorescent ssDNA  or ssRNA  probes. Background-subtracted traces from fluorescent reporter assays with favored ssDNA  (5'-CCCCCCCC-3') or ssRNA  (5'-CCCCCCCC-3') probes. RFU - relative fluorescence units. (C) Background-subtracted traces of fluorescent reporter cleavage  using ssDNA  or ssRNA  probes. (D) Rates of trans-degradation with ssRNA  or ssDNA  reporters activated with either ssDNA  or dsDNA  targets. Collateral ssDNAse activity is about 3-fold higher than the rate of ssRNA degradation. Fluorescence intensities in B-C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters. Data information: In (B-D), individual data points from n = 3 replicates from independent experiments are plotted. In (D), bars represent the mean.",
    "molecules": "ssRNA, dsDNA, ssDNA"
  },
  {
    "caption": "A) Visualisation of the pathology load at 9M of age of TAUwt, TAUtg and APPtg mice at 9M of age. Immunofluorescent staining for X34 (a fluorescent derivative of Congo Red), Iba1 (microglia) and TO-PRO-3 (nuclei) has been pseudocolored. Scale bar = 100μm.",
    "molecules": "X34, Congo Red, TO-PRO-3"
  },
  {
    "caption": "C: Surface expression of TRAP mutants on fixed but not permeabilized sporozoites. Differential interference contrast (DIC) and immunofluorescence images of representative examples of hemolymph derived sporozoites of the indicated parasite lines stained with anti-TRAP antibodies shown in red Hoechst was used to reveal the sporozoite nuclear DNA shown in blue. Cytoplasmic GFP or staining with anti-CSP antibodies was used to validate the integrity of sporozoites. Scale bar: 5 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "D: Percentage of gliding and non-gliding control hemolymph sporozoites (HLS) at increasing concentrations of DTT. n indicates the number of observed sporozoites. Floaters are included in the non-motile fraction. Each condition represents pooled data from 2 biological replicates. Shown is the mean +/- SEM. E: Percentage of gliding hemolymph sporozoites (HLS) expressing the open TRAP I domain at increasing concentrations of DTT. n indicates the total number of observed sporozoites. Floaters are included in the non-motile fraction. Shown are pooled data from 3 (0mM DTT), 2 (50mM DTT), and 3 biological replicates for concentrations of 100mM DTT in the S210C/Q216C line. For concentrations of 200mM and 330mM DTT in the S210C/Q216C line, data shown represent pooled data from 1 biological replicate. Several comparable screening experiments previously performed showed no productive motility at these concentrations. Shown is the mean +/- SEM.",
    "molecules": "DTT"
  },
  {
    "caption": "F: Maximum projections (1 min time lapse, 3 s per frame) of single S210C/Q216C sporozoites in presence or absence of 50 mM DTT. A projection from a motile sporozoite of the cFluo line treated with 50 mM DTT is shown as control. Scale bar: 5 µm.",
    "molecules": "DTT"
  },
  {
    "caption": "(F) In vivo epifluorescence microscopy following neosynthesized alcAp-UapA-GFP in a single hypha at 120, 130, 140, 150, 160 and 170 min, under overexpressing (derepression/ethanol-induction) conditions, as described in Materials and methods. Notice that UapA labels the perinuclear ER rings (white arrows).",
    "molecules": "ethanol"
  },
  {
    "caption": "(B) Key endogenous genes controlling Golgi (sedV, geaA, hypB) or post-Golgi trafficking (rabE, ap1σ, claH) were genetically replaced by versions transcribed under the highly repressible thiAp promoter via targeted homologous recombination. In the absence of thiamine from the growth medium (derepressed conditions) the relative proteins are expressed, while upon addition of thiamine at the onset of conidiospore germination (ab initio repression) the expression of these proteins is tightly repressed. Proteins are detected by a standard western blot analysis using either anti-FLAG or anti-GFP antibodies for Golgi and Post-Golgi proteins. Equal loading and protein steady state levels are normalized against the amount of actin, detected with a specific antibody.",
    "molecules": "thiamine"
  },
  {
    "caption": "(C) In the absence of thiamine from the growth medium (derepressed conditions) the corresponding strains grow nearly as an isogenic wild-type control, although a delay in growth is observed for thiAp-sedV and less so for thiAp-ap1σ (upper row). In the presence of thiamine to the growth medium most cells do not form colonies, except for thiAp-hypB which forms a compact slow growing colony (lower row).",
    "molecules": "thiamine"
  },
  {
    "caption": "(D) Microscopic examination of the corresponding strains under thiamine (repressing conditions) shows that, in most cases, the apical region of germlings is enlarged and growth is arrested. This morphological phenotype, taken as a strong indication of blocked secretion, is more evident in thiAp-sedV and thiAp-rabE but concerns all strains, except for thiAp-hypB. Scale bar: 5 μm.",
    "molecules": "thiamine"
  },
  {
    "caption": "(E) Subcellular localization of UapA, after 6-8 hours of initiation of transcription, via its native uapA promoter, while conventional sedV, geaA or hypB transcription is repressed by thiamine ab initio. Scale bars: 2 μm. Notice that UapA-GFP translocates in the PM of germlings in all cases. Results shown are confirmed by quantification (right panel) of UapA-GFP PM/cytosolic intensity ratios for the four strains (for details see Materials and methods). Mean PM/cytosolic intensity ratios for wild-type, thiAp-sedV, thiAp-geaA and thiAp-hypB are 0.53±0.02, 0.52±0.02, 0.52±0.02 and 0.52±0.02, respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). No statistical significance was found between the wild-type and each of the mutant strains. Biological/technical replicates: 3/15 for wild-type, 3/18 for thiAp-sedV, 3/15 for thiAp-geaA and 3/18 for thiAp-hypB.",
    "molecules": "thiamine"
  },
  {
    "caption": "(A) Growth test showing that Sec24 expression is essential for growth as its transcriptional repression by thiamine (+thi) leads to absence of colony formation, despite initial germination The strains shown are isogenic except for the sec24 locus. thiAp-sec24 signifies the strain where the endogenous sec24 promoter was replaced by the thiAp promoter.",
    "molecules": "thi, thiamine"
  },
  {
    "caption": "(B) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec24 transcription is ab initio derepressed (upper panel) or repressed by thiamine (lower panel). o/n (overnight) means addition of thiamine from the onset of germination. Germination of conidiospores takes place until full repression of Sec24 is achieved (10-12 h). Notice the total lack of PM-associated signal of UapA under conditions of Sec24 repression. Scale bar: 5 μm. Quantification: In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when sec24 is derepressed (-thi) or repressed (+thi). Mean PM/cytosolic intensity ratios are 0.55±0.02 and 0.32±0.02 respectively. To test the significance of differences, an unpaired t-test was performed, which verified the significant difference (****P<0.0001) in the presence of thiamine. Biological/technical replicates: 2/15 for each condition.",
    "molecules": "thi, thiamine"
  },
  {
    "caption": "(C) Epifluorescence microscopy analysis of the subcellular localization of the apical marker chitin synthase (GFP-ChsB) under sec24 derepressed or repressed conditions. Notice the loss of apical depositioning and the concurrent labeling of cytoplasmic foci and membranous structures under repression conditions. Scale bar: 5 μm. Quantification: GFP-ChsB PM/cytosolic intensity ratios are plotted in cases where sec24 is derepressed (-thi) or repressed (+thi). Mean values are 0.56±0.03 and 0.12±0.02 respectively. The fluorescence intensity of GFP-ChsB is statistically lower (****P<0.0001) when the expression of sec24 is repressed (statistical analysis as in B). Biological/technical replicates: 2/15 for each condition.",
    "molecules": "thi"
  },
  {
    "caption": "(E) Epifluorescence microscopy analysis of the subcellular localization of UapA-GFP under conditions where sec13 transcription is ab initio derepressed (left panel) or repressed by thiamine (right panel). The inserts in the upper right corner in both panels reflect growth tests showing that Sec13 repression leads to arrest in growth and absence of colony formation. Scale bars: 5 μm. All images reflect practically identical results obtained in several experiments. Quantification: UapA-GFP PM/cytosolic intensity ratios are quantified when sec13 is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.31±0.02, respectively. There is a significant difference on UapA PM fluorescence intensity (****P<0.0001) in the absence of the COPII outer coat protein (statistical analysis as in B). Biological/technical replicates: 2/15 for each condition.",
    "molecules": "thi, thiamine"
  },
  {
    "caption": "(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made UapA-GFP, after 6-8 hours of initiation of transcription in strains where the expression of RabE, AP-1σ or ClaH (thiAp-rabE, thiAp-ap1σ or thiAp-claH) has been repressed ab initio (o/n) by addition of thiamine. Notice that when rabE or ap1σ is repressed UapA-GFP still reaches the PM. In contrast, repression of claH abolishes labeling of the PM and leads to the cytosolic puncta and a membranous network. Scale bars: 5 μm. Quantification: UapA-GFP PM/cytosolic intensity ratios are plotted on the right. Mean ratios for wild-type, thiAp-rabE, thiAp-ap1σ and thiAp-claH are 0.55±0.02, 0.53±0.03, 0.54±0.02 and 0.29±0.02 respectively. For the statistical analysis, Tukey's Multiple Comparison test was performed (One-way ANOVA). After 6-8h of transcriptional derepression, UapA-GFP fluorescence to the PM does not change when rabE or ap1σ are repressed. On the other hand, in the absence of ClaH, UapA does not reach the PM, as its fluorescence intensity there is statistically lower (****P<0.0001) in comparison to that of the wild-type strain. Biological/technical replicates: 2/15 for each strain.",
    "molecules": "thiamine"
  },
  {
    "caption": "(A) Time course of treatment with the actin polymerization drug latrunculin B for 2, 5, 10, 30 or 50 minutes of a strain expressing neosynthesized UapA-GFP under conditions of derepression compared to an untreated strain included as control (150 min). In all cases latrunculin B was added at 100 min of UapA derepression, so that total time of UapA-GFP expression was 102, 105, 110, 130, or 150 minutes in the different samples. Notice the abolishment of sorting of UapA to the PM after 130 or 150 min of expression when latrunculin B was present for the last 30 or 50 min, respectively. Scale bar: 5 μm.",
    "molecules": "actin, latrunculin B"
  },
  {
    "caption": "(B) Time course of treatment of strains expressing neosynthesized alcAp-UapA-GFP and mCherry-TubA with the anti-microtubule drug benomyl for 5, 55, or 95 minutes. In all cases benomyl was added at 95 min of UapA derepression, so that total time of UapA-GFP expression was 100, 150 or 190 minutes Benomyl abolished the thread-like appearance microtubules in all samples added, evident by the diffuse cytoplasmic signal of mCherry-TubA. Notice that UapA reaches normally the PM (at 190 min of derepression), similarly to the untreated control (right panel). Scale bar: 5 μm.",
    "molecules": "benomyl, Benomyl"
  },
  {
    "caption": "(C) Subcellular localization of neosynthesized alcAp-GFP-ChsB in the absence (left panel) or presence (≈40 min) of benomyl (middle panel) or latrunculin B (right panel), after ≈2h of derepression. Notice the abolishment of proper polar localization of ChsB at the apical tip in both cases. Scale bars: 5 μm.",
    "molecules": "benomyl, latrunculin B"
  },
  {
    "caption": "(D) Effect of latrunculin B or benomyl on COPII vesicle formation, followed through the subcellular localization Sec24-GFP. Notice that 40 min of latrunculin B led to loss of the wild-type punctuate appearance of Sec24 cytoplasmic puncta and the appearance of a diffuse fluorescent haze in the cytoplasm. In contrast, benomyl treatment did not affect the wild-type punctuate localization of Sec24. Scale bar: 5 μm.",
    "molecules": "benomyl, latrunculin B"
  },
  {
    "caption": "(B) Subcellular localization of neosynthesized UapA-GFP (8 hours after transcriptional depression) while ssoA transcription is ab initio repressed. Notice the localization of UapA in the ER-membranous network, often at very close proximity with the PM. Scale bar: 5 μm. In the scatter plot on the right, UapA-GFP PM/cytosolic intensity ratios are quantified when ssoA is derepressed (-thi) or repressed (+thi), with mean values being 0.54±0.02 and 0.30±0.02 respectively. There is statistically lower fluorescence intensity to the PM (****P<0.0001) when SsoA is absent from the cell, as confirmed by an unpaired t-test. Biological/technical replicates: 2/15 for each condition.",
    "molecules": "thi"
  },
  {
    "caption": "(A) Epifluorescence microscopy analysis examining the subcellular localization of de novo made alcAp-AzgA-GFP and alcAp-FurA-GFP, after 6-8 hours transcriptional derepression in strains where the expression of Sec24, SedV, GeaA, HypB, RabE, AP-1σ and SsoA was repressed ab initio (o/n) by addition of thiamine. Images showing that sorting of AzgA or FurA to the PM does not require early (SedV, GeaA), late (HypB) or post-Golgi (RabE, AP-1σ ) key proteins. On the other hand, COPII coat protein Sec24 and PM t-SNARE SsoA are essential for proper localization of the two transporters to the PM. Scale bar: 5 μm.",
    "molecules": "thiamine"
  },
  {
    "caption": "(B) Epifluorescence microscopy analysis of the localization of de novo made AzgA and FurA transporters (see text for details) in the absence or presence of Benomyl or Latrunculin B after derepression of the relevant genes for the time indicated. Notice that addition of Benomyl, has no effect on translocation of AzgA and FurA to the PM, whereas Latrunculin B abolishes their PM localization, as seen also for UapA. Scale bar: 5 μm.",
    "molecules": "Benomyl, Latrunculin B"
  },
  {
    "caption": "Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon & Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk.",
    "molecules": "32P"
  },
  {
    "caption": "A: HIF-1α levels in HT29 primary tumors. Left: vehicle-treated controls Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB) Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Inset, high-magnification image of the region marked with a green asterisk (*). Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-1α+ area. F(5,12) = 8.791 and P = 0.001 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.046 vs vehicle; #, P< 0.0001 vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. ",
    "molecules": "capecitabine, CPB, CTX, cyclophosphamide"
  },
  {
    "caption": "A: Effect of LDM and MTD chemotherapy on HT29 liver metastatic nodule size. Left: Histogram of pooled cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. A decreasing linear trend for median metastatic diameter was observed (left to right, slope = -143.2, P = 0.043 by post-test for trend). Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Liver nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 6.80; P= 0.2361 (not significant) for overall effects on size; χ2 (df=1) = 4.71; P= 0.0299 (LDMCTX + LDMCPB vs vehicle). ",
    "molecules": "CPB, CTX"
  },
  {
    "caption": "B: HIF-1α levels in HT29 liver metastatic nodules. Left: vehicle- treated controls. Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB). Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Right: Automatic quantification of the effect of monotherapies or doublet regimens on HIF-1α+ areas in individual metastatic nodules. F(2,13) = 4.796 and P = 0.028 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.038 LDMCTX + MTDCPB vs vehicle or P= 0.0123 LDMCTX + LDMCPB vs vehicle; #, P= 0.0098 LDMCTX + LDMCPB vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test. ",
    "molecules": "capecitabine, CPB, CTX, cyclophosphamide"
  },
  {
    "caption": "B: Hypoxic fraction in lung metastatic nodules. Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule (rightmost panel). F(5,110) = 7.544 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicle; #, P= 0.0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini, Krieger and Yekuteli post hoc test.",
    "molecules": "capecitabine, cyclophosphamide, Pimonidazole"
  },
  {
    "caption": "D: Microvessel density in lung metastatic nodules. Automatic quantification of CD31 fractional areas per nodule (rightmost panel). F(5,124) = 7.531 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 (LDM capecitabine, cyclophosphamide, or doublet capecitabine + cyclophosphamide vs vehicle), #, P= 0.0417 (doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine) by Benjamini, Krieger and Yekuteli post hoc test.",
    "molecules": "capecitabine, cyclophosphamide"
  },
  {
    "caption": "D. Left, images showing apparent normal division of  retinal progenitors in  Rb∆K7 mice. Mice were labeled with BrdU for 2 hrs; retinal sections from indicated genotypes were stained  with  DAPI (blue), Ki67 (green), PH3  (red) or  BrdU (red) to mark nuclei, all dividing cells, mitotic  or S-phase cells, respectively. Right, quantification of  staining showing no significant difference among the genotypes. Bars denote mean ±SD; n=3 biological replicates). Scale bar: 20 μm.",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": "E. Left, top,  western blot analysis of Rb∆K7 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. Right, phospho-specific  immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in  Rb∆K7 thymocytes. Right, immuno-precipitation (IP)-Western blot  analysis demonstrating  phosphorylation of  pRb∆K7 on Ser243/Thr246 (top) and Ser605 (bottom). n.s., non-specific band.",
    "molecules": "Ala, Ser, Thr"
  },
  {
    "caption": "C. Left, representative flow cytometric analysis with  7-AAD  and AnnexinV, showing a significant decrease in apoptosis in  Rb∆K7 MEFs relative to control. Right, significant differences in average percentage of live (7-AAD-:AnnexinV-) vs. apoptotic (7-AAD+:AnnexinV+) cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test; n=3 independent MEFs.",
    "molecules": "7-AAD"
  },
  {
    "caption": "J. Hyperglycemia  in Rb∆K7 but not Rb∆K4 mice. Blood glucose in 10-12-month old fasting Rb∆K7 (left, n=11) or 18-month old fasting Rb∆K4 (right, n=15) mice versus control littermates (n=6 and n=8, respectively). Mean ±SD; P values by two-tailed unpaired student's t-test.",
    "molecules": "glucose"
  },
  {
    "caption": "B.  Glucose tolerance Test  (GTT) demonstrating progressive defect in clearance of blood glucose in Rb∆K7 mice. 6-7 week (left) or 6 month (right) old fasting  Rb∆K7 mice and control  littermates (n=5-6 each per group) were injected with  glucose, and blood glucose was determined at indicated intervals. Error bars represent SD.",
    "molecules": "glucose"
  },
  {
    "caption": "F. Loss of  pancreatic islet area in Rb∆K7 mice correlates with reduced proliferation and H3K36me3 histone marks, but not with increased cell death. TUNEL analysis showing apoptosis in 10 day-old Rb∆K7 islets  is even lower than in control littermates. Arrow points to a rare  apoptotic  cell in control islet. Serine800/804 phosphorylation of pRb in β-cells from 10 day-old control but not Rb∆K7 islets with a Ser-to-Ala substitution at this site. BrdU incorporation and  PH3 staining showing reduced S-phase and mitotic cells, respectively, in 2- and 3-week old Rb∆K7 vs control islets. Reduced H3K36me3 staining in  pancreatic islets from 3-week old Rb∆K7 mice  compared to control littermates (n>3 for each experiment). Scale bar, 100 μm.",
    "molecules": "BrdU, Ala, Ser, Serine"
  },
  {
    "caption": "H. Reduced ability of  Rb∆K7 β-cell  to re-enter the cell cycle in response to pregnancy (top) or mitogenic (bottom) signals. Top, cell proliferation in P14.5 pregnant  Rb∆K7 islet β-cell  vs. control was determined by double  immunostaining with BrdU  and insulin  (left); quantification (right). Results were normalized for the number of BrdU/Insulin positive cells in control islets. An independent experiment using ki67 to quantify cell cycle re-entry top. Bottom, cell proliferation in Rb∆K7 β-cell vs. control  islets following  Exendin4  treatment was determined by  immunostaining for  Ki67; results were normalized for the number of positive cells in control islets. Mean ±SD, P values by two-tailed unpaired student's t-test. Scale bar, 100 μm.",
    "molecules": "BrdU, Exendin4, insulin, Insulin"
  },
  {
    "caption": "D. Representative image of a  pancreatic islet  from a 9-month old  Rb∆K7 mouse immunestained for the SASP marker IL-6 (red) and  insulin (green). DAPI was used to stain nuclei (blue). Arrows point to IL-6+,  insulin+  pancreatic β-cells. Scale bar, 20 μm.",
    "molecules": "DAPI, insulin"
  },
  {
    "caption": "H. Vitamin C diet increases 5hmC  expression and improves  β-cell  morphology and islet size in  Rb∆K7 mice. Scale bar, 100 μm. I. Vitamin C diet reduces the difference in γ−H2A+ β-cells  in  Rb∆K7 vs control pancreatic islets relative to regular diet by 3.74 folds. Bars indicate mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test. J. Vitamin C diet increases  pancreatic islet cell cycle  re-entry during pregnancy of Rb∆K7 mice relative to control pregnant mice (no statistical difference, n.s.) or relative to pregnant  Rb∆K7 vs control mice fed on regular diet (P<0.0001) by 3.8 folds. Bars represent mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test.",
    "molecules": "5hmC, Vitamin C"
  },
  {
    "caption": "F. Vitamin C  reduces cellular senescence induced by  retroviral-mediated over-expression of Rb∆K11. Top - images of SA-βGAL staining in human diploid cells, IMR90, transduced with control or rRb∆K11 virus with or without vitamin C added at the time of infection. Arrows point to  SA-βGAL-positive cells. Bottom left, quantification of SA-βGAL staining (bars represent mean ±SEM; n=4 biological replicates). P values by one-way ANOVA, Tukey's multiple comparisons test. Bottom right, dot-blot  analysis with antibody for 5hmC on DNA extracted from  IMR90 cells treated or not with vitamin C. Original magnification: 200x.",
    "molecules": "5hmC, Vitamin C, vitamin C"
  },
  {
    "caption": "A Alkaline gel Southern blot  analysis  of AAV DNA. AAV DNA  was hybridized to a probe specific for the HLP  promoter. Neg, AAV DNA  treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6.",
    "molecules": "DNA"
  },
  {
    "caption": "E Haemoglobin   content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.",
    "molecules": "Haemoglobin"
  },
  {
    "caption": "A. Spike-binding  antibodies in serum. Binding  by antibodies of classes M, G, and A is detected with secondary antibodies conjugated to different fluorochromes, and the results are shown on the left, middle, and right plots, respectively. Binding  (geometric mean fluorescence intensity - GMFI- of corresponding secondary antibody) to TE  cells expressing  SARS-CoV-2 spike protein is plotted on the vertical axis, and binding to untransfected cells on the horizontal axis. Each point represents a value from one donor. Samples from donors with no known exposure to  SARS-CoV-2 antigens (n = 86) are plotted with blue circles, recently infected donors (n = 34) with red circles, and vaccinated  donors (n = 14) with green diamonds. Red circles with black triangles correspond to donors from whose  B cells monoclonal antibodies were isolated (n = 5, P values are derived from a two-way analysis of variance followed by Tukey's test to compare the specific binding, i.e., (binding  to spike-expressing cells)/(binding  to untransfected cells) between conditions (convalescent  or vaccinated  against unexposed) within each antibody class. The experiment was independently repeated three times, and results shown come from the third replicate.",
    "molecules": "fluorochromes"
  },
  {
    "caption": "E. Gating strategy in MACAC sorting. Single cells are selected based on scatter, antigen-expressing TE spike-mCherry  cells excluded on Cell Trace Violet label, and the spike-capturing (mCherry-high), activated (CD69-high) B cells (population labeled \"MACAC\" in red on the right-most plot) are sorted. The middle plot shows B cells that did not adhere to the TE-spike-cherry  antigen-expressing cells (putatively antigen-irrelevant), and the right plot those that did (putatively antigen-recognizing). Plots show data from one of five convalescent donors Cells in the MACAC  gate were singly distributed into wells of 384-well plates and cultured for 9 days with  IL-21 and CD40L, and then the single well culture supernatants were screened for anti-spike  antibody binding  and virus neutralization as described below. F. Results of single well supernatant screening for antibody binding  to SARS-CoV-2 spike  protein. Results from 3266 wells from 5 donors are shown for IgM  (left), IgG (middle), and IgA  (right).",
    "molecules": "Cell Trace Violet"
  },
  {
    "caption": "(A) RFP-ATG8g-labeled autophagosomes  were quantified from plants   syringe infected with mock or Xcv ΔxopQ (oD600=0.2) at 2 dpi in the presence or absence of ConA  (bars = 20 μm). Puncta were calculated from z-stacks (15) of n=6 individuals using ImageJ. Central band of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically different groups (P < 0.05) as determined by one-way ANOVA. The experiment was repeated twice with similar results.",
    "molecules": "ConA"
  },
  {
    "caption": "(C) Immunoblot  analysis of ATG8  protein levels in  N. benthamiana plants transiently expressing XopL  or GUS  control at 2dpi after ConA  or DMSO treatment. Expression  of GFP-XopL  was verified with an anti-GFP antibody, while expression  of GUS-HA  was confirmed with an anti-HA  antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.",
    "molecules": "ConA, DMSO"
  },
  {
    "caption": "(D) GFP-ATG8g-labeled autophagosomes  were quantified from plants infected with Xcv  or Xcv ΔxopL at 2 dpi in the presence or absence of ConA. Puncta were calculated from z-stacks (15) of n=12 individuals using ImageJ. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (** P < 0.01, *** P < 0.001) was determined by one way ANOVA. The experiment was repeated twice with similar results.",
    "molecules": "ConA"
  },
  {
    "caption": "(A) Interaction  of XopL  with SH3P2  in yeast two-hybrid assays. XopL  fused to the GAL4  DNA-binding   domain was expressed in combination with SH3P2  fused to the GAL4  activation domain (AD) in  yeast strain Y190. Cells were grown on  selective media before a LacZ  filter assay was performed. pSV40/p53  served as positive control, while the empty AD or BD vector served as negative control.  NtSH3P2 = Nicotiana tabacum SH3P2. -LT = yeast   growth on medium without Leu  and Trp, -HLT = yeast   growth on medium lacking His, Leu, and Trp, indicating expression of the HIS3  reporter gene. LacZ, activity of the lacZ  reporter gene.",
    "molecules": "His, Leu, Trp"
  },
  {
    "caption": "(A) SH3P2-GFP   was transiently coexpressed together with GUS-HA  and GFP-XopL  in N. benthamiana using agroinfiltration. At 42 hpi, 200 μM MG132 was infiltrated into  A. tumefaciens-inoculated leaves, and leaf  material was collected 48 hpi. Expression   of SH3P2-GFP  (lower band) and GFP-XopL  (upper band) was detected using an anti-GFP  antibody. GUS-HA expression   was confirmed with an anti-HA  antibody.  Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.",
    "molecules": "MG132"
  },
  {
    "caption": "(H) GFP-XopL, GFP-XopL ΔE3 were transiently expressed in N. benthamiana. RFP-AIMp  was co-infiltrated to stabilize both XopL  variants. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation  (IP) with GFP-Trap beads, followed by immunoblot  analysis of the precipitates using either anti-GFP  or anti-ubiquitin  antibodies. GFP  served as a negative control. RFP-AIMp expression was verified by an anti-RFP  antibody. GFP blot  was split for visualization purpose. Asterisk indicates the GFP-XopL  full-length protein. The experiment was repeated three times with similar results.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(I) GFP-XopL  was transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with the ubiquitin  pan selector, followed by immunoblot analysis of the precipitates using either anti-GFP  or anti-ubiquitin  antibodies. GFP  served as a control. Asterisk indicates the GFP-XopL  full-length protein. GFP blot  was split for visualization purpose. The experiment was repeated two times with similar results.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(K) HA-XopL  and GFP-Joka2  and its variants were transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation  (IP) with GFP-Trap beads, followed by immunoblot  analysis of the precipitates using either anti-GFP, anti-ubiquitin  and anti-NBR1  antibodies. GFP  served as a control.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "D. A549 cells were incubated with WSN/33 or HK/14 + AM-EVs for 40 min, washed, fixed, and permeabilized. Cells were subsequently stained with Anti-NP (green), Anti-Rab-7 (purple) Abs, and DAPI (blue). Images shown are representative of 8 independent experiments. Scale bars = 5 μm",
    "molecules": "DAPI"
  },
  {
    "caption": "E, F. Co-localization analysis of (E) NP:DAPI and (F) NP:Rab7 for each condition was performed on Z-stack confocal images images of WSN/33 (black) or HK/14 (red) infected A549s. Data represent the mean NP:DAPI co-localization ratios and the mean NP:Rab7 Mander's co-localization coefficient for individual cells from these 8 experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-D. Replication assessed by luminescence following 10 nM Baf-A1 addition at the indicated times following synchronized uptake of bound Luc-expressing virions. (A, B) Shown are examples from 1 experiment representative of a total of 3-11 independent experiments per strain. Data represent mean luminescence AUC normalized to the mean of the corresponding strain incubated in 0.1% DMSO (dashed line). (C, D) The kinetics of endosomal escape by Luc-WSN/33 and HK/14 strains are depicted by the loss of inhibition by high dose Baf-A1 between (C) 0-20 min and (D) 60-80 min. Data represent mean slope (change in % inhibition/time interval) from 5 wells per plate from 3-11 independent experiments.",
    "molecules": "Baf-A1, DMSO"
  },
  {
    "caption": "Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Baf-A1 on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.",
    "molecules": "Baf-A1"
  },
  {
    "caption": "F. Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Chl on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.",
    "molecules": "Chl"
  },
  {
    "caption": "B. Representative images of PBS- and DR-EV-treated MDCK-SIAT1 cells. Scale bars for full size images = 50 μm. Scale bars for cropped images = 10 μm. FDx particles are shown green and DR-EV particles are shown purple. White arrows are FDx endosomes containing DR-EVs.",
    "molecules": "PBS"
  },
  {
    "caption": "C. Measured pH of FDx endosomes in PBS- (left bar) and DR-EV-treated (right bar) MDCK-SIAT1 cells. The pH of FDx endosomes containing DR-EVs in DR-EV-treated cells (middle bar) (corresponding to white arrows in Fig 7B). Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments.",
    "molecules": "PBS"
  },
  {
    "caption": "Representative PET, MRI and fused PET/MRI images of the thoracic region of a mouse that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT and skeletal muscle throughout the dynamic scan was calculated and compared for each animal (n = 5). * P = 0.0488 (20 mins), * P = 0.0236 (25 mins), * P = 0.0167 (30 mins), ** P = 0.00301 (35 mins), ** P = 0.00269 (40 mins), ** P = 0.00215 (45 mins), ** P = 0.00129 (50mins) and ** P = 0.00141 (45mins) using unpaired 2-tailed Student's t tests.",
    "molecules": "AQ28A"
  },
  {
    "caption": "A. Representative PET images of a sagittal view of the thoracic region of fasted lean mice treated with either MP-10 (30mg/kg) (n = 3) or vehicle (n = 4) prior to receiving [18F]-FDG. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-FDG in BAT after both treatments was calculated for each group. ** P = 0.0013 using unpaired 2-tailed Student's t test.",
    "molecules": "18F, FDG, MP-10"
  },
  {
    "caption": "B. The percentage (%) change in core body temperature and associated area under the curve (AUC) of mice treated with MP-10 (30mg/kg) (n = 4) or vehicle (n=4) prior to a cold challenge study for 4 hours at 8oC. * P = 0.0108 (1st hour), * P = 0.0218 (2nd hour), * P = 0.0121 (3rd hour), * P = 0.0168 (4th hour) and * P = 0.0264 (AUC) using unpaired 2-tailed Student's t tests.",
    "molecules": "MP-10"
  },
  {
    "caption": "D Cyclic GMP (cGMP) and cyclic AMP (cGMP) concentrations normalized to protein concentration in SAT, VAT and BAT 30 minutes after treatment of lean mice with MP-10 (30 mg/kg) (n=6) or vehicle control (n = 6). ** P = 0.0085 (cGMP in VAT), ** P = 0.0011 (cGMP in BAT) and * P = 0.04 (cAMP in VAT) using unpaired 2-tailed Student's t tests.",
    "molecules": "cAMP, cGMP, cyclic AMP, Cyclic GMP, MP-10"
  },
  {
    "caption": "E RT-qPCR analysis of relative mRNA expression of thermogenic genes in SAT, VAT and BAT after treatment of lean mice treated with MP-10 (30 mg/kg) (n=7) or vehicle control (n = 6). **** P < 0.0001 (Pgc1alpha in VAT), ** P = 0.0012 (Ucp1 in VAT), ** P = 0.0071 (Pgc1alpha in BAT) and * P = 0.0486 (Ucp1 in BAT) using unpaired 2-tailed Student's t tests.",
    "molecules": "MP-10"
  },
  {
    "caption": "Representative fused PET/MRI images of the transverse view of the thoracic region of lean (n = 5), diet induced obese (DIO) (n = 4) and leptin deficient (ob/ob) (n = 5) mice that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT throughout the dynamic scan was calculated with associated area under the curve (AUC). * P = 0.0128 (lean vs DIO) and ** P = 0.0031 (lean vs ob/ob) using unpaired 2-tailed Student's t test.",
    "molecules": "AQ28A"
  },
  {
    "caption": "A, B The percentage (%) change in body weight and cumulative food intake of diet induced obese (DIO) mice in response to daily treatment with MP-10 (10mg/kg) or vehicle for a week (n= 4 per group). * P = 0.0264 using (day 4), *** P = 0.0002 (day 5), *** P < 0.0001 (day 6) and *** P < 0.0001 (day 7) using Two-Way analysis of variance (ANOVA) with Sidak's post-hoc test.",
    "molecules": "MP-10"
  },
  {
    "caption": "C. Upon completion of the chronic feeding study, an insulin tolerance test was performed on mice with single treatment of 0.75 U/kg insulin (n = 4 per group). * P = 0.0246 and * P = 0.0305 using unpaired 2-tailed Student's t tests.",
    "molecules": "insulin"
  },
  {
    "caption": "D, E, F Upon completion of the chronic feeding study, resting energy expenditure (REE), respiratory quotient (RQ) and locomotor activity of DIO mice treated with MP-10 (30mg/kg) compared to vehicle control were measured in metabolic cage experiments (n = 4 per group). * P = 0.0239 (02:00, RQ), * P = 0.0424 (02:30, RQ), * P = 0.0420 (03:00, RQ), * P = 0.0230 (03:30, RQ), * P = 0.0213 (04:00, RQ), ** P = 0.0116 (00:00, REE), ** P = 0.00823 (00:30, REE), ** P = 0.00969 (01:00, REE), ** P = 0.00274 (01:30, REE), ** P = 0.00323 (02:00, REE), ** P = 0.000441 (02:30, RE), ** P = 0.00390 (03:00, REE), ** P = 0.000211 (03:30, REE), ** P = 0.00216 (04:00, REE) and ** P = 0.0185 (04:30, REE) using unpaired 2-tailed Student's t tests.",
    "molecules": "MP-10"
  },
  {
    "caption": "H. Representative sections of inguinal SAT, peri-ovarian VAT and interscapular BAT immunostained for UCP1 of mice chronically treated with MP-10 (10 mg/kg) or vehicle control. Scale bars, 100µm.",
    "molecules": "MP-10"
  },
  {
    "caption": "Representative PET image of the upper body of a human subject who received the PDE10A radioligand [18F]-MNI-1869. Supraclavicular brown adipose tissue (BAT) is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-MNI-1869 in supraclavicular BAT and skeletal muscle was calculated in human subjects (n = 5). ** P = 0.00210 (5 mins), **** P < 0.0001 (15 mins), **** P < 0.0001 (30 mins), **** P < 0.0001 (50 mins) and ** P = 0.00462 (75 mins) using unpaired 2-tailed Student's t tests.",
    "molecules": "MNI-1869"
  },
  {
    "caption": "A. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human brown adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0278 vs (100nM MP-10 vs control; Pgc1alpha), **P = 0.0097 (10µM cA + cG vs control; Pgc1alpha), ** P = 0.0063 (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0017 (1nM MP-10 vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cAMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cGMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cA + cG vs 100nM MP-10; Ucp1), * P = 0.0112 vs (100nM MP-10 vs control; Cidea), *** P = 0.0007 (1nM MP-10 vs control; Dio2), **** P < 0.0001 (100nM MP-10 vs control; Dio2), # P = 0.0267 (1nM MP-10 vs 10µM cAMP; Dio2), §§§ P = 0.0001 (10µM cAMP vs 100nM MP-10; Dio2), §§§ P = 0.0007 (10µM cGMP vs 100nM MP-10; Dio2), * P = 0.0232 (10µM cA + cG vs control; Dio2) §§ P = 0.0058 (10µM cA + cG vs 100nM MP-10; Dio2), * P = 0.0416 (10µM cGMP vs control; Prdm16) and * P = 0.0105 (10µM cA + cG vs control; Prdm16) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.B. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human white adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0186 vs (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0016 (1nM MP-10 vs 100nM MP-10; Ucp1), *** P = 0.0003 (10µM cAMP vs control; Ucp1), §§§ P = 0.0002 (10µM cGMP vs 100nM MP-10; Ucp1) and *** P = 0.0003 (cA + cG vs control; Ucp1) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.",
    "molecules": "cAMP, cGMP, cyclic AMP, cyclic GMP, DMSO, MP-10"
  },
  {
    "caption": "B. Beta-hydroxybutyrate (ketone body) concentrations, plasma.",
    "molecules": "Beta-hydroxybutyrate"
  },
  {
    "caption": "D-G. Spiroergometry results from PEO patients and control subjects on normal diet (ND), as well as one month and six months after mAD initiation. PEO patients had higher serumlactate levels, and attenuated respiratory response to exercise. These effects were further enhanced after mAD and remained after 6 months of the study. mAD increased patients' serumammonium (NH4+) levels. Data presented as mean and SD.",
    "molecules": "ammonium, lactate"
  },
  {
    "caption": "C38 and IB3-1 cells transfected with GFP-LC3 plasmid in nutrient-rich or starvation medium. (a) Top: confocal microscopy of GFP. N, nucleus. Scale bar, 10 μm. Bottom: percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. (n = 5). Asterisk, P 0.001 versus C38 cells; analysis of variance (ANOVA).",
    "molecules": "nutrient"
  },
  {
    "caption": "(d, e) Immunoblot of LC3 in starved C38 and IB3-1 cells (d) and IB3-1 cells (e) after incubation with E64d and pepstatin A.",
    "molecules": "E64d, pepstatin A"
  },
  {
    "caption": "(a) IB3-1 cells cultured with 250 μM cystamine or transfected with either 50 nM human TG2 siRNA or scrambled oligonucleotides. IP, immunoprecipitation; IB, immunoblot.",
    "molecules": "cystamine"
  },
  {
    "caption": "(b) IB3-1 cells cultured with the proteasome inhibitor MG132 (50 μM).",
    "molecules": "MG132"
  },
  {
    "caption": "(c, d) IB3-1 cells transfected with either human TG2 siRNA or scrambled oligonucleotides followed by incubation with MG132. (c) Cells immunostained with anti-beclin 1 (green) and anti-HDAC-6 (red) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.",
    "molecules": "MG132"
  },
  {
    "caption": "(c, d) IB3-1 cells transfected with either humanTG2 siRNA or scrambled oligonucleotides followed by incubation with MG132. (d) FRET analysis of beclin 1-Alexa 546 (Al546) fluorescence after HDAC6-Cy5 photobleaching. F/F0 indicates the post-bleaching/pre-bleaching fluorescence ratio of Al546/Cy5.",
    "molecules": "MG132"
  },
  {
    "caption": "(g, h) IB3-1 cells were treated with cystamine or 10 mM EUK-134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides. (g) Percentage of cells containing more than five GFP-LC3 punctate dots. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA.",
    "molecules": "cystamine, EUK-134"
  },
  {
    "caption": "(g, h) IB3-1 cells were treated with cystamine or 10 mM EUK-134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides. (h) Left: electron micrographs of IB3-1 cells showing areas enriched in autophagosomes (arrows) on PIASy gene silencing, EUK-134 or cystamine. Scale bar, 100 nm. Right: number of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA.",
    "molecules": "cystamine, EUK-134"
  },
  {
    "caption": "(i) IB3-1 cells treated with cystamine. Immunoblot of p62.",
    "molecules": "cystamine"
  },
  {
    "caption": "(j) IB3-1 cells treated with cystamine or EUK-134 or transfected with human PIASy siRNA or scrambled oligonucleotides. Immunoblot of beclin 1 (left) and PIASy (right). Uncropped images of blots are shown in Supplementary Information, Fig. S10. The experiments were repeated three times. β-actin was used as loading control for all blots. Densitometric analysis of blots and quantitative measurement of co-localizations are shown in Supplementary Information, Fig. S9.",
    "molecules": "cystamine, EUK-134"
  },
  {
    "caption": "(a, b) C38 cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides (a, b) or cultured with CFTRinh-172 (b) in the presence or absence of cystamine (250 μM) or EUK-134 (10 mM). (a) Immunoblot of beclin 1, LC3 and p62 in C38 cells on starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (b) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus scrambled oligonucleotides; circle, P 0.01 versus CFTR siRNA or CFTRinh-172; ANOVA.",
    "molecules": "CFTRinh-172, cystamine, EUK-134"
  },
  {
    "caption": "(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine. (c) Immunoblot of TG2 and PIASy.",
    "molecules": "cystamine, EUK-134"
  },
  {
    "caption": "(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(d) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching.",
    "molecules": "cystamine, EUK-134"
  },
  {
    "caption": "(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(e) Immunoblot of beclin 1 and LC3 in A549 cells after starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10.",
    "molecules": "cystamine, EUK-134"
  },
  {
    "caption": "(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(f) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001; two asterisks, P 0.01; ANOVA. (g) Immunostaining with anti-p62 (red) and anti-GFP (green) antibodies in starved A549 cells. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. Each experiment was repeated three times.",
    "molecules": "cystamine, EUK-134"
  },
  {
    "caption": "(a, b) IB3-1 cells were transfected with either HA-tagged human beclin 1, empty vector, p62 siRNA or scrambled oligonucleotides followed by rosiglitazone (10 μM). (a) Cells were immunostained with anti-HDAC6 (red) and anti-PPAR-γ (green) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.",
    "molecules": "rosiglitazone"
  },
  {
    "caption": "(a, b) IB3-1 cells were transfected with either HA-tagged human beclin 1, empty vector, p62 siRNA or scrambled oligonucleotides followed by rosiglitazone (10 μM).(b) FRET analysis of increase in PPAR-γ-Alexa 546 fluorescence after N-CoR Cy5 photobleaching.",
    "molecules": "rosiglitazone"
  },
  {
    "caption": "(c) IB3-1 cells were transfected with either HA-tagged human beclin 1 or the empty vector under nutrient starvation. Cells were immunostained with anti-LC-3 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(d) IB3-1 cells were cultured under nutrient starvation with cystamine (250 μM) in the presence or absence of the PI(3)K inhibitor 3-MA. Immunostaining was with anti-LAMP-1 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.",
    "molecules": "3-MA, cystamine, nutrient"
  },
  {
    "caption": "(e) IB3-1 cells were transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of 250 μM cystamine under nutrient starvation. Cells were immunostained with anti-p62 (red) and anti-CFTR (green) antibodies. Yellow indicates co-localization. N, cell nucleus. Scale bar, 10 μm.",
    "molecules": "cystamine, nutrient"
  },
  {
    "caption": "(f, g) IB3-1 cells were transfected with 1 μg of pGFP-CFTRF508del or the empty vector, in the presence or absence of 250 μM cystamine. (f) Cells were immunostained with anti-GM-130 (red) and anti-GFP (green) antibodies. Yellow indicates co-localization (arrows). Scale bar, 10 μm. (g) FRET analysis of GFP fluorescence after HDAC6 Cy3 photobleaching. Each experiment was repeated three times. Quantitative measurement of co-localizations is shown in Supplementary Information, Fig. S9.",
    "molecules": "cystamine"
  },
  {
    "caption": "(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides. (a) Left: CFTR revealed with anti-GFP primary antibody in IB3-1 cell lysate. Top arrow, complex-glycosylated form (apparent Mr of band C about 170K, revealed as 210K with GFP tag); bottom arrow, core-glycosylated form (apparent Mr of band B about 150K, revealed as 190K with GFP tag). Uncropped images of blots are shown in Supplementary Information, Fig. S10. Right: quantification of bands B (black) and C (white) normalized to β-actin levels. Values are means ± s.d. for three independent experiments.",
    "molecules": "cystamine"
  },
  {
    "caption": "(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(b) Cells immunostained with an anti-GFP antibody under non-permeabilizing conditions. Serial confocal sections were collected from top to bottom of cell. (c) Side view (X-Z) of a z-stack of confocal images taken in b. (c) Side view (X-Z) of a z-stack of confocal images taken in b.",
    "molecules": "cystamine"
  },
  {
    "caption": "(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(d) Surface biotinylation assays was performed with membrane-impermeable sulpho-NHS-LC-biotin. The cells were fractioned to obtain the plasma membrane fraction, and biotinylated proteins were precipitated with streptavidin beads. Immunoblot of GFP to reveal the C form. E-cadherin and β-actin were used as positive and negative controls, respectively.",
    "molecules": "cystamine"
  },
  {
    "caption": "(g) IB3-1 cells transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. Immunoblot of phospho-p42/44.",
    "molecules": "cystamine"
  },
  {
    "caption": "(h) IB3-1 cells transfected with either 50 nM humanbeclin 1 siRNA or 50 nM humanATG5 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. TNF-α secretion. Each bar represents the mean ± s.d. for n = 3 experiments. Asterisk, P 0.01; ANOVA.",
    "molecules": "cystamine"
  },
  {
    "caption": "CF human nasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) (a) Immunoblot of beclin 1.",
    "molecules": "cystamine, NAC"
  },
  {
    "caption": "CF humannasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) in the presence or absence of 3-MA (patients 6-10 in Supplementary Information, Table S1) (b, c). (b) Fluorescence microscopy quantification of LC3-positive dots. Results represent means ± s.d. Asterisk, P 0.05 between groups of treatment; ANOVA.",
    "molecules": "3-MA, cystamine, NAC"
  },
  {
    "caption": "CF humannasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) in the presence or absence of 3-MA (patients 6-10 in Supplementary Information, Table S1) (b, c). (c) Confocal microscopy micrographs of p62. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative of five patients per group.",
    "molecules": "3-MA, cystamine, NAC"
  },
  {
    "caption": "(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (d) Immunoblot of beclin 1.",
    "molecules": "PBS, 3-MA, cystamine"
  },
  {
    "caption": "(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (e) Quantification of LC3 dots (per mm2 of lung tissue). Means ± s.d. Asterisk, P 0.01; ANOVA.",
    "molecules": "PBS, 3-MA, cystamine"
  },
  {
    "caption": "(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (f) Immunoblot of TG2. Uncropped images of blots are shown in Supplementary Information, Fig. S10.",
    "molecules": "PBS, 3-MA, cystamine"
  },
  {
    "caption": "(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (g) Confocal microscopy of SUMO-1 in lung tissues. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm.",
    "molecules": "PBS, 3-MA, cystamine"
  },
  {
    "caption": "(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (h) Number of CD68+ macrophages counted in 15-20 different randomly taken sections. Means ± s.d. per mm2 of lung tissue from seven mice in each group. Asterisk, P 0.01; ANOVA.",
    "molecules": "PBS, 3-MA, cystamine"
  },
  {
    "caption": "(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (i) MPO activity in lung homogenates (expressed as 10−3 × optical density (OD) s−1). Means ± s.d. for lung tissues from seven mice in each group. Asterisk, P 0.05; ANOVA.",
    "molecules": "PBS, 3-MA, cystamine"
  },
  {
    "caption": "CftrF508del mice (a-d)(a) Confocal microscopy of LC3 (top) and p62 (bottom) in lung tissues from CftrF508del mice. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative images of ten PBS-treated and ten NAC-treated mice. (b) Quantification of LC3 dots (per mm2 of tissue) in lung tissues from CftrF508del mice. Means ± s.d. Asterisk, P 0.001 versus PBS-treated mice; ANOVA.",
    "molecules": "PBS, NAC"
  },
  {
    "caption": "CftrF508del mice (a-d)(c) Number of macrophages per mm2 of lung tissue from CftrF508del mice (CD68-positive cells in 15-20 different randomly taken sections for each mouse lung for each condition); means ± s.d. for three separate experiments. Asterisk, P 0.05 versus PBS-treated mice; ANOVA.",
    "molecules": "PBS"
  },
  {
    "caption": "CftrF508del mice (a-d)(d) MPO activity in lung homogenates from CftrF508del mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA.",
    "molecules": "PBS"
  },
  {
    "caption": "Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (e) Confocal microscopy of LC3-positive dots in lung tissues from PBS-treated or NAC-treated mice-Tg LC3. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. (f) Number of LC3-positive dots in Scnn1b tissues from mice-Tg PBS. Means ± s.d. Asterisk, P 0.01 versus mice-treated mice; ANOVA.",
    "molecules": "NAC"
  },
  {
    "caption": "Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (g) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice.",
    "molecules": "PBS, NAC"
  },
  {
    "caption": "Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (h) Immunoblot of TG2 and phospho-p42/44 in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice. αβ-Tubulin was used as loading control.",
    "molecules": "PBS, NAC"
  },
  {
    "caption": "Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (i) Lung histology of PBS-treated and NAC-treated Scnn1b-Tg mice. Haematoxylin staining. Scale bar, 150 μm. Black arrows indicate decrease in lung infiltration in NAC-treated in comparison with PBS-treated mice (arrowheads).",
    "molecules": "PBS, NAC"
  },
  {
    "caption": "Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (j) Left: confocal microscopy of COX-2 staining (left) in PBS-treated and NAC-treated Scnn1b-Tg mice. Nuclei counterstained with DAPI (blue). Scale bar, 150 μm. Right; number of COX-2-positive cells per mm2 of lung tissue in NAC-treated and Scnn1b-treated mice-Tg PBS. Means ± s.d. Asterisk, P 0.05 versus mice-treated mice; ANOVA.1 versus PBS-treated mice; ANOVA.",
    "molecules": "PBS, NAC"
  },
  {
    "caption": "Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (k) MPO activity in lung homogenates from PBS-treated and NAC-treated Scnn1b-Tg mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. Densitometric analysis of the blot is shown in Supplementary Information, Fig. S9.",
    "molecules": "PBS, NAC"
  },
  {
    "caption": "(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm.",
    "molecules": "dextrose, lactate"
  },
  {
    "caption": "(d) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins.",
    "molecules": "proteins"
  },
  {
    "caption": "A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.",
    "molecules": "proteins"
  },
  {
    "caption": "WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.",
    "molecules": "lactate"
  },
  {
    "caption": "B. Average profile for H3K27me3, H2A.Z, H3K4me3 and H3K23ac at HVGs (blue), LVGs (green) and random genes (grey). C. Heatmap of the enrichment for H3K27me3, H2A.Z, H3K4me3 and H3K23ac for HVGs (top), random genes (middle) and LVG (bottom). Red means a high level and blue means a low level for the chromatin marks. ",
    "molecules": "H3K27me3, H3K4me3"
  },
  {
    "caption": "(A). A FRET assay was used to measure the difference in melting temperature (ΔTm) of (G4C2)4 RNA or DNA G-Qs in the presence of 2 μM of 138 different small molecules. An increase in ΔTm indicates stabilisation of the G-Q. Small molecules are ranked on the x axis according to their increasing thermal stabilisation of the DNA (G4C2)4 G-Q. Small molecules that preferentially stabilise RNA over DNA (G4C2)4 G-Qs reside in the upper part of the scatter plot, above the blue curve. An arbitrary ΔTm threshold of 13 °C greater than vehicle (grey line) and a differential binding to RNA over DNA (ΔTmRNA-ΔTmDNA ≥ 5 °C) were used to select candidate small molecules.",
    "molecules": "small molecules, Small molecules, (G4C2)4 RNA"
  },
  {
    "caption": "(C). FRET dose-response of DB1246, DB1247 and DB1273 on stabilisation of RNA or DNA (G4C2)4 G-Qs.",
    "molecules": "DB1246, DB1247, DB1273"
  },
  {
    "caption": "(D). Temperature unfold CD spectra for (G4C2)4 RNA alone (which shows a characteristic G-Q structure with minima at 237 nm, maxima at 264 nm and no additional signal), or in the presence of 2 μM DB1246, DB1247 or DB1273. A characteristic induced CD spectrum, in the 350 nm - 550 nm region, is observed only in the presence of each small molecule, confirming that each of these three compounds are binding to (G4C2)4 RNA G-Qs. Data in A represent mean ± s.d., n=1 with 3 technical replica. Data in C represent mean ± s.d., n=3 independent experiments. Data in D represent mean ± s.d., n=3 independent experiments.",
    "molecules": "DB1246, DB1247, DB1273, (G4C2)4 RNA"
  },
  {
    "caption": "(A) Representative images of C9orf72 iPSC-cortical neurons treated with DMSO (vehicle control) or 1 µM of DB1246, DB1247 or DB1273, for 4 days. RNA foci are shown in red and nuclei (DAPI) in blue. Scale bar represents 10 µm.",
    "molecules": "DB1246, DB1247, DB1273, DAPI"
  },
  {
    "caption": "(B) Quantification shows RNA foci are significantly reduced by all three compounds in iPSC-cortical neurons and by DB1246 and DB1273 in iPSC-motor neurons. Data are shown as the average percentage of neurons containing RNA foci relative to vehicle (DMSO). N=3 independent C9orf72 patient lines with 2-3 inductions per line and at least 70 neurons counted per induction. *p <0.05, **p <0.01, ***p <0.001, one sample two-tailed t-test versus normalised control. For cortical neurons, p=0.0124 (DB1246), p=0.0065 (DB1247), p=0.0096 (DB1273). For motor neurons, p=0.0004 (DB1246), p=0.0030 (DB1273).",
    "molecules": "DB1246, DB1247, DB1273"
  },
  {
    "caption": "(A) Poly(GP) levels were measured by MSD immunoassay in C9orf72 patient iPSC-motor neurons treated for 7 days with 8-16 µM of the G4C2 repeat G-Q binding small molecules DB1247, DB1247 and DB1273. Treatment with DB1246 or DB1273 leads to a significant reduction in poly(GP) levels relative to vehicle-treated controls. Data are shown as the mean and SEM of 3 independent C9orf72 iPSC lines, with 1-6 differentiations per line. *p<0.05, **p<0.01, *** p<0.001, one sample two-tailed t-test versus normalised control. p=0.0068 (DB1246, 12 µm), p=0.0417 (DB1246, 16 µm), p=0.0196 (DB1247, 8 µm), p=0.0002 (DB1273, 8 µm), p=0.0194 (DB1273, 12 µm), p=0.0062 (DB1273, 16 µm)",
    "molecules": "DB1246, DB1247, DB1273"
  },
  {
    "caption": "(C) XTT cell death assay for C9orf72 iPSC-motor neurons treated for 7 days with 0 - 40 µM of DB1246, DB1247, DB1273 or cisplatin as a positive control. Data are shown as the mean and SEM of two independent iPSC-motor neuron lines. Toxicity is only observed at the highest dose of 40 µM and not at the penultimate dose of 20 µM.",
    "molecules": "DB1246, DB1247, DB1273, cisplatin"
  },
  {
    "caption": "(D) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273, and the number reaching the pupal stage of development counted after 7 days. DB1273 treatment (n=1200 flies) significantly improves survival compared to vehicle (n=1150 flies), p=0.0320, chi-squared test. Data are shown as proportion reaching the pupal stage, with numbers within each group indicated on the bars. Genotype was w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).",
    "molecules": "DB1273"
  },
  {
    "caption": "(E) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats (36R) were treated with vehicle or 1 mM DB1273 for 5 days, until the third instar stage (L3), and poly(GR) measured by MSD immunoassay. Poly(GR) was also measured in control (w1118) larvae that do not express G4C2 repeats. DB1273 treatment significantly reduced poly(GR) expression. n=8 for 36R groups, n=7 for control group. **p<0.01, *** p<0.001, one way ANOVA with Dunnett's post hoc test. p=0.0001 (w1118 versus 36R 0 mM larvae), p=0.0019 (0 mM versus 1 mM 36R larvae). Genotypes were w1118; ; and w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).",
    "molecules": "DB1273"
  },
  {
    "caption": "A. HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (left) as well as IL6-OPT, IL6-WT and IL6-DE transcripts (right), post-doxycycline addition. Data information: data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***). The half-lives of the respective transcripts are indicated in brackets.",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).",
    "molecules": "CHX, cycloheximide, DMSO, doxycycline, Tet"
  },
  {
    "caption": "HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (B), under vehicle (DMSO)- and cycloheximide (CHX)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).",
    "molecules": "CHX, cycloheximide, DMSO, doxycycline, Tet"
  },
  {
    "caption": "HEK293 Tet-off experiments showing the degradation of REL-OPT and REL-WT transcripts (C) under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).",
    "molecules": "ANI, anisomycin, doxycycline, Tet"
  },
  {
    "caption": "HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT and IL6-DE transcripts (D), under vehicle (PBS)- and anisomycin (ANI)-treatment, post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).",
    "molecules": "ANI, anisomycin, doxycycline, Tet"
  },
  {
    "caption": "HEK293 Tet-off experiments showing the degradation of REL-OPT, REL-OPT (+1 Frameshift) and REL-WT transcripts (E) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "HEK293 Tet-off experiments showing the degradation of IL6-OPT, IL6-WT, IL6-DE and IL6-OPT (+1 Frameshift) transcripts (F) post-doxycycline addition. data is representative of 3 independent experiments each with 3 replicates. The data represents the mean ± SD for 3 replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**) and p<0.001 (***).",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "B, C. HEK293 Tet-off experiments showing the degradation of REL-OPT (B) and REL-WT (C) transcripts with ILF2 and ILF3 siRNA and Control (CTR) siRNA treatment, post-doxycycline addition. Data information: data is representative of 3 independent experiments in which the data represents the mean ± SD for 3 biological replicates. A two-way ANOVA with Holm-Sidak multiple comparisons was performed. P-values are denoted as follows: p < 0.05 (*), p<0.01 (**).",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "A) SEZ6KO and WT neurons were biotinylated with Sulfo-NHS-Biotin and surface proteins were enriched by streptavidin bead pull-down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma & Marques, 2013), therefore the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2 and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3 / SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± S.E.M., at least 10 replicates in 4 independent biological experiments, Mann-Whitney ****p-value<0.005).",
    "molecules": "biotinylated, streptavidin, Sulfo-NHS-Biotin"
  },
  {
    "caption": "(A) Left: Current trace resulting from whole-cell voltage-clamp recordings at a holding potential of -70 mV in a CA1 pyramidal neuron in a WT mouse hippocampal slice (P15). The current was recorded in the presence of 10 µM bicuculline, 20 µM GYKI53655 and 30 µM APV in the ACSF. Perfusion of the recording chamber with 10 µM kainate and 100 µM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) are indicated with green and blue bars, respectively. Right: Analogous experiment in a SEZ6KO mouse (P15). (B) Summary of the experiments in A. Bar graph illustrates mean charge with standard error of the mean carried by the inward currents (measured as area under the curve, AUC) for the two genotypes at P15 (WT 46.6 ± 0.5 nC (n=10 cells), SEZ6KO 30.8 ± 0.5 nC (n=9 cells), *p=0.0375 Mann-Whitney-U-test). ",
    "molecules": "2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, NBQX, APV, bicuculline, GYKI53655, kainate"
  },
  {
    "caption": "A) The glycome fingerprint of GluK2/3 was analyzed by lectin chip microarray (LecChip). Lectin PHAL and PHAE (indicated with asterisks) detected reduced amounts of the oligosaccharide Galβ1-4GlcNAcβ1 on GluK2/3 in SEZ6KO neurons (plot shows mean ± S.E.M., 3 WT replicates and 4 SEZ6KO replicates were used, discoveries were determined using the FDR method of Benjamini and Hochberg, with Q = 2%).",
    "molecules": "Galβ1-4GlcNAcβ1, oligosaccharide"
  },
  {
    "caption": "B) Immunoblot analysis revealed reduction of HNK-1 epitope on GluK2/3 in SEZ6KO brains. Left panels: Comparable amounts of GluK2/3 were immunoprecipitated (IP) in WT and SEZ6KO brains using excess of brain homogenates compared to beads. Anti-HNK-1 antibody was used for detection. HNK-1 band on GluK2/3 was detected only in WT brains but not in SEZ6KO brains (n=6). As a control, HNK-1 modification of NCAM-1 was unaltered, as revealed after IP of NCAM-1 from WT and SEZ6KO brains (n = 3), followed by detection with anti-HNK-1 antibody. Different isoforms of NCAM-1 are annotated with arrowheads (NCAM 180, NCAM 140 and NCAM 120). Mid panel: To prove the specificity of the HNK-1 antibody, GluK2/3 was immunoprecipitated and digested with Peptide-N-Glycosidase F (PNGase F), which removes N-linked oligosaccharides. Upon PNGase F digestion, HNK-1 was not detectable in WT brains and the molecular weight of GluK2/3 was reduced (n=2). Right panels: In total brain homogenates (input), no difference of general HNK-1 epitope or NCAM-1 levels was detected.",
    "molecules": "HNK-1, oligosaccharides"
  },
  {
    "caption": "B,C) The constitutively ER-retained SEZ6 mutant SEZ6∆cytoER-HA (B) and full length wild-type SEZ6FL-HA (C) were co-transfected with SBP-GFP-GluK2. Biotin was added to elicit release of SBP-GFP-GluK2 from the ER (0 min) and cells were fixed at different time points (0, 20 and 40 min). HA-tagged SEZ6FL and SEZ6∆cytoER transgenes were labeled with anti-HA (mouse) and anti-mouse-Alexa594 antibodies. Size bars represent 5 µm.",
    "molecules": "Alexa594, Biotin"
  },
  {
    "caption": "A) Representative pictures of WT hippocampal neurons transfected with the SBP-mCherry-GluK2 RUSH construct, followed by treating neurons with biotin and anti-SBP antibodies (mouse, green) for 20 min or 40 min to live-label the GluK2 transported to the neuronal cell surface. As a control (no biotin), neurons were incubated only with SBP antibody for 40 min. The surface/total ratio of SBP-mCherry-GluK2 constructs was estimated by calculating the ratio of SBP (green)/ mCherry (red) fluorescence signals in the same area chosen for quantification. B) Quantification results for A, indicating that incubation with biotin increases the surface/total ratio of SBP-mCherry-GluK2 over time. No Biotin n=16, 20 min n=13, 40 min n=15, all biological replicates. Graph shows mean±SEM, 1-way ANOVA p<0.0001 and Dunnett's post hoc test, No Biotin vs 20 min *p=0.0030; No Biotin vs 40 min ***p<0.0001.",
    "molecules": "biotin, Biotin"
  },
  {
    "caption": "C-D) SEZ6 flox/flox neurons were lentivirally infected with inactivated Cre (Y324F, Ctrl) or Cre recombinase (SEZ6KO), followed by transfection with the SBP-mCherry-GluK2 construct and treated with biotin and anti-SBP antibodies (mouse, green) for 20 min. Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti-Cre antibody (rabbit, blue). SEZ6KO neurons showed a significantly reduced surface/total ratio of SBP-mCherry-GluK2 (ctl n=25, SEZ6KO n=23 biological replicates. Graph shows mean ± SEM, unpaired t-test, **p=0.0068).",
    "molecules": "biotin"
  },
  {
    "caption": "DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Representative confocal microscopy images of CMAC-stained conjugates formed between B lymphocytes (isolated from DICER-KO mouse splenocytes and pre-activated with a mixture of LPS plus IL-4) and CD4+ T cells (from OVA-specific OT-II transgenic mice); conjugates were formed either in the presence or in the absence of OVA. A representative experiment of at least 3 independent experiments is shown.",
    "molecules": "CMAC, LPS"
  },
  {
    "caption": "DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively)",
    "molecules": "cell violet tracer"
  },
  {
    "caption": "DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. B cell proliferation after 24h, 48h and 72h co-culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively),",
    "molecules": "cell violet tracer"
  },
  {
    "caption": "DICER-KO B cells were co-cultured for 24 hours with OTII-derived T cells in the presence or absence of OVA. Flow cytometry analysis of B lymphocytes co-cultured with OT-II isolated T cells in the presence or absence of OVA survival (staining with the live marker DAPI)",
    "molecules": "DAPI"
  },
  {
    "caption": "Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated with LPS+IL-4 Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G.",
    "molecules": "LPS"
  },
  {
    "caption": "Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with LPS+IL-4 Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH.",
    "molecules": "LPS"
  },
  {
    "caption": "Fluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of nucleofected microRNA mimics in FACS-sorted FAM+ B cells with normalization to RNU1A1 and RNU5G",
    "molecules": "FAM"
  },
  {
    "caption": "Cells were fixed and the viral bait fused with the V5-tag was visualized by immunofluorescence staining using Alexa Fluor-488 labeled anti-V5 immunostaining (yellow), actin with Alexa Fluor594-labeled phalloidin (magenta), and nuclei were stained with DAPI (cyan) (Scale bar: 10 μm).",
    "molecules": "V5, Alexa Fluor-488, Alexa Fluor594, DAPI, phalloidin"
  },
  {
    "caption": "D Inhibition of interactions formed with DDX39B in the presence of MTX using co-IP assay. DDX39B is fused to the C terminus of a V5 tag, and the interaction partner is fused with a Strep-HA tag. The diagram (upper) shows the interaction pairs formed with DDX39B used for co-IP assay. Dot-blot results (lower) show the interaction pairs (arrangement as upper panel) in presence of different concentration of MTX as indicated on the top of the image. The image displayed is representative of 3 replicates (n = 3).",
    "molecules": "V5, MTX, Strep"
  },
  {
    "caption": "E Quantification of DDX39B binding to different interaction partners. Quantification of V5-tagged DDX39B protein amount was normalized over HA-tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells. Data that represent the mean of 3 replicates are highlighted in red. A fold change > 0.5 is considered significant.",
    "molecules": "V5"
  },
  {
    "caption": "T cell interface actin (phalloidin) re-organization during synapse symmetry breaking, imaged using SIM. Quantification of the shape elongation (AR; B, n= 44 for 5', 39 for 20'), or relative fluorescence signal of the indicated proteins in the SIM images (C, D) normalized to the mean of values at 5'; points represent values for individual cells. Arrowheads in (C) indicate foci. p values are: ***P< 0.0001 for AR, **P =0.003 for foci, **P= 0.008 for talin, and p =0.36 for total F-actin using Mann-Whitney two-tailed test between populations of cells within the same experiment. Relationship between AR and foci at individual cell level, each point in the scatter plot represents value obtained from a single cell.",
    "molecules": "phalloidin"
  },
  {
    "caption": "Integrin augmentation does not rescue synapse symmetry breaking. Cells were allowed to adhere to the APS for 5' or 10', and were then treated with vehicle control, 0.5mM MnCl2 or 100nM A286982 for subsequent 10'. Cells were fixed, stained with phalloidin and anti-Talin antibody, and imaged using SIM (F) and analyzed for AR (G) as well as talin, total F-actin and actin foci at the synapse (H). In (E-H), n for 5'= 40, for 20'= 62, for MnCl2= 59, for A286982= 53. The values in the plots represent the intensity values normalized to the mean of 5' in each set. p values in the graph, n.s. > 0.05; ***P <0.001; *P= 0.01; **P<0.009 using Mann-Whitney two-tailed test between populations of cells within the same experiment.",
    "molecules": "A286982, MnCl2, phalloidin"
  },
  {
    "caption": "Time-lapse imaging of LifeAct-GFP expressing T cell using Lattice lightsheet microscopy (LLSM) during synapse polarization. Shown are snapshots at selected timepoints (left) and a kymograph of actin foci (denoted by arrows in the first image panel) over time (right) for a representative T cell. Quantification of changes in actin foci and cellular aspect ratio in LLSM images during symmetry breaking. The values in the plot are the raw values normalized to their mean in each case.",
    "molecules": "LifeAct"
  },
  {
    "caption": "T cells incubated with APS for the indicated time periods were processed for phalloidin- or immune-staining (C, D) Graph in (D) shows relative levels of total and active WASP (WASP-phosphorylated at Y293, pWASP), normalized to the mean levels at 5'; for pWASP n in 5'= 49, in 20'= 44; for WASP, n in 5'= 56, 20'= 56. P values, P**= 0.0027 for pWASP and P**= 0.0035 for WASP using Mann-Whitney two-tailed test. The scatterplot in (D) shows the relationship between active WASP and foci on a per cell basis.",
    "molecules": "phalloidin"
  },
  {
    "caption": "Actin dynamics in mature synapse of LifeAct-GFP expressing T cell using TIRFM. The images show the foci (pseudocolored red) extracted from the total synaptic F-actin (F-G), and kymograph shows the time course of foci and lamellar activity over a period of 210 sec. Histograms of foci and lamellar protrusion and retraction (fluctuations) dynamics in WT T cell (H); Lifetime of individual foci and lamellar events (graph on the bottom right, each dot represents a single foci/lamellar protrusion-retraction event n= 126 for foci and n= 77 for lamellar protrusions). ***P<0.0001 using Mann-Whitney two-tailed test between populations of cells within the same experiment.",
    "molecules": "LifeAct"
  },
  {
    "caption": "Foci -deficient cells display poor traction forces in their synapse. WT or WASP-/- T cells were incubated on polyacrylamide substrates covalently functionalized with anti-CD3 and ICAM1, and traction force measurements were carried out as described in 'Methods'. The images in the right show traction force maps without (left panels) or with (right panels) force vectors. p value, **P<0.005 as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment, n = 9 and 11.",
    "molecules": "polyacrylamide"
  },
  {
    "caption": "SIM imaging of WT or WASP-/- T cells activated by APS for 5'. The graphs show quantification of actin foci (derived from phalloidin staining), pCasL and talin levels in the synapse normalized to the 'WT' mean value in each case (Middle graph; in the case of foci, n= 63 for WT and 86 for WASP-/-; in the case of pCasL n= 63 for WT and 78 for WASP-/-; in the case of Talin, n= 59 for WT and 73 for WASP-/-; in the case of AR, n= 46 for WT and 42 for WASP-/-); right graph shows AR measurement. ***P <0.0001; p value for talin *P=0.07, as determined by using Mann-Whitney two-tailed test between populations of cells within the same experiment.",
    "molecules": "phalloidin"
  },
  {
    "caption": "Schematic of the cytoskeletal inhibitors and their targets used in the experiments. SIM imaging of mouse T cells activated for 5' on APS, then treated with the indicated inhibitors or vehicle control. Shown are the representative SIM images (B), and quantification of actin foci (from the phalloidin images), talin, or pCasL in the contact interface (normalized to the mean value in the control case, C), and measured cell morphology aspect ratios (D, E). Since CK666 treatment reduces F-actin intensity Kumari et al., 2015(), the 'actin' image of the CK666 treated cell in (B) was contrasted differently than the 'control' and 'Blebb.' cases for a clearer visualization of F-actin. p values, *P =0.02, **P =0.001, n.s. >0.05. For the rest of the comparisons between the control and treatment cases, p <0.0001. For the bar graph in (B), n for foci groups are 214, 83, 92, 98; for pCasL groups are 100, 68, 91, 92; and for Talin groups are 102, 70, 89, 93 respectively. p value for the n.s. cases is > 0.05, *P =0.02. In the foci, pCasL and talin groups, when not indicated, the p values are <0.0001 using Mann-Whitney two-tailed test. Central values in the graphs represent Mean and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5µm.",
    "molecules": "Blebb, CK666, phalloidin"
  },
  {
    "caption": "Experimental confirmation of localized actomyosin crosslinking and symmetry breaking. LifeAct-GFP expressing T cells were incubated with activating substrates, treated with vehicle control or 1µM Azido-blebb and their contact interface imaged using low excitation laser power (0.5 mW), using a spinning disc confocal microscope (H). The montage shows the shape change and movement that synapse undergoes after it is exposed to a brief flash of blue light crosslinking Azido-Blebb and actomyosin network within the illuminated area (shown with a box in the image). The deflection in center of mass (COM) within a minute was calculated and plotted for at least 10 cells in each case (graphs in I). The left graphs in (I) show individual COM trajectory, and the graph on the right shows average COM movement of cells within a minute post photo-inactivation of myosinII. This experiment was repeated twice with similar results. Scale bar, 5µm.",
    "molecules": "LifeAct, Azido-blebb, Azido-Blebb"
  },
  {
    "caption": "T cells were activated using Alexa568-2C11 and ICAM1 reconstituted lipid bilayers for 5 min, fixed, stained with phalloidin and imaged using SIM. In the graph, n= 50 for WT and 61 for WASP-/-, P value, P***<0.0001, obtained using Mann-Whitney two-tailed test. The graphs in (B) show synaptic levels of indicated proteins, normalized to the mean levels of '+WASP' in each case. For the left graph, n= 48, 46, 52, 46 respectively, and p value, P *= 0.03. For the right graph, n= 50 for WT and n= 61 for WASP-/- in 'Actin', 'Foci', as well as 'pCasL' cases. P values P*** <0.0001, obtained using Mann-Whitney two-tailed test.",
    "molecules": "Alexa568, phalloidin"
  },
  {
    "caption": "Synapse breaking behavior of T cells on hydrogels of indicated stiffness, covalently conjugated with 2C11 and ICAM1. These experiments were repeated at least thrice with similar results. In the left graph, n for WT= 14, for WASP-/-, n= 19; P**= 0.0015; in the graph on the right, n for WT= 40, n for WASP-/-= 36, P***= 0.0006, as assessed using Mann-Whitney two-tailed test.",
    "molecules": "hydrogels"
  },
  {
    "caption": "(B) The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). Key residues involved in stabilizing the templating base (Y505 and R517) are shown in black.",
    "molecules": "8-oxo-dG"
  },
  {
    "caption": "(C) (Left panel) 8-oxo-dG (green) preferentially adopts the pro-mutagenic syn-conformation. A simulated annealing Fo-Fc omit density map is shown for 8-oxo-dG (contoured at 3σ). (Right panel) 8-oxo-dG(anti) (black, modeled using 2PFO)was overlayed with the Fo-Fc omit density map. In the anti-conformation, the C8-carbonyl would directly clash with the 5′-phosphate (red-dotted line).",
    "molecules": "8-oxo-dG"
  },
  {
    "caption": "(D) 8-oxo-dG(syn) (green) is stabilized by multiple interactions, including Tyr505 and Arg517 (grey).",
    "molecules": "8-oxo-dG"
  },
  {
    "caption": "(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming ddCTP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.",
    "molecules": "ddCTP, 8-oxo-dG, dATP, ddC"
  },
  {
    "caption": "(C) 8-oxo-dG (green) establishes a Watson-Crick pair with ddCTP (magenta). Binding of the 8-oxo-dG in the anti-conformation is only possible because the C8-carbonyl is far enough away not to clash with the 5′-phosphate. Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).",
    "molecules": "ddCTP, 8-oxo-dG"
  },
  {
    "caption": "(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and metal B sites, respectively.",
    "molecules": "dATP, Mg2+, water"
  },
  {
    "caption": "(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming dATP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.",
    "molecules": "8-oxo-dG, dATP, ddC"
  },
  {
    "caption": "(C) 8-oxo-dG (green) establishes a Hoogsteen base pair with dATP (magenta). Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).",
    "molecules": "8-oxo-dG, dATP"
  },
  {
    "caption": "(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and B sites, respectively.",
    "molecules": "dATP, Mg2+, water"
  },
  {
    "caption": "(A and B) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure (2PFO, black). Binding of 8-oxo-dG (green) in both the anti- (A) or syn-(B) conformation involves a repositioning of the 5′-phosphate (red arrow). Furthermore, the Arg514 side chain (purple) that normally stacks with the templating base and interacts with the 5′-phosphate (blue-dotted line), also repositions (black arrow). In both structures, Arg514 interacts with the C6-carbonyl of the templating 8-oxo-dG(anti) (A) or the C6-amino group of the incoming dATP (B). The incoming nucleotide is shown in magenta.",
    "molecules": "8-oxo-dG, dATP"
  },
  {
    "caption": "(B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dC and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513, R517 and Glu529) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.",
    "molecules": "dUMPNPP, dC, 8-oxo-dG"
  },
  {
    "caption": "(C) 8-oxo-dG (orange) establishes a Watson-Crick base pair with dC (cyan) at the primer terminus. Tyr505, Arg517, and Glu529 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).",
    "molecules": "dC, 8-oxo-dG"
  },
  {
    "caption": "(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dC in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.",
    "molecules": "dUMPNPP, dC, Mg2+"
  },
  {
    "caption": "(B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dA and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.",
    "molecules": "dUMPNPP, dA, dC, 8-oxo-dG"
  },
  {
    "caption": "(C) 8-oxo-dG (orange) establishes a Hoogsteen base pair with dA (cyan) at the primer terminus. Tyr505 and Arg517 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).",
    "molecules": "dA, 8-oxo-dG"
  },
  {
    "caption": "(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dA in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.",
    "molecules": "dUMPNPP, dA, Mg2+"
  },
  {
    "caption": "(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.",
    "molecules": "dG, dA, dC, 8-oxo-dG"
  },
  {
    "caption": "(A) Undamaged structure containing a templating dG at the primer terminus (2PFO) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus. (B) Undamaged structure containing a templating dA at the primer terminus (1XSN) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus.",
    "molecules": "dG, dA, dC, 8-oxo-dG"
  },
  {
    "caption": "(G) Preimplantation development of SCNT embryos. The percentage of embryos reaching each indicated stage is shown. The D-SCNT embryos produced by transfer of dox-Dux cumulus cell (B6D2F1 background) into WT enucleated oocytes (B6D2F1 background). Error bars, mean ± S.D.; n = 3 biological replicates per group. Scale bar, 100 μm.",
    "molecules": "dox"
  },
  {
    "caption": "(B) Durations of dox addition at different time points during chemical induction (upper) and assessment of AP+ colonies (bottom). Error bars, mean ± S.D.; n = 3 biological replicates per condition; **P < 0.01, ***P < 0.001 as compared with condition-1, by two-tailed Student's\u2028 t-test. n.s., not significant.",
    "molecules": "dox"
  },
  {
    "caption": "(E) RT-qPCR analysis of select ZGA-related genes in dox-untreated D-CiPSCs and canonical CiPSCs at passage 1. The value in CiPSCs was set as 1, and data shown are mean expression values relative to GAPDH. Error bars, mean ± S.E.M.; n = 3 biological replicates per group; ***P < 0.001 by two-tailed Student's t-test.",
    "molecules": "dox"
  },
  {
    "caption": "(A) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators (DFP, DFX or DFO). β-actin was used as loading control. Bafilomycin (-): without Bafilomycin, Bafilomycin (+): with 100 mM of Bafilomycin.",
    "molecules": "Bafilomycin, DFX, DFP, DFO, iron"
  },
  {
    "caption": "(B) Dual excitation (550/488 nm) of mt-mKeima transfected-Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment. Scale bar: 10 μm.",
    "molecules": "DFP"
  },
  {
    "caption": "(C) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt-mKeima at 633/488 nm for untreated (left panel) and DFP-treated (24 h, right panel) Huh7 cells.",
    "molecules": "DFP"
  },
  {
    "caption": "(D) Untreated Huh7 cells or those treated with DFP (10 μM), DFX (10 μM) or DFO (10 μM) were quantified for mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates). A high signal (633/488) area/ cell area indicates the proportion of cells undergoing mitophagy. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.",
    "molecules": "DFX, DFP, DFO"
  },
  {
    "caption": "(E) Electron microscopic images of untreated (left panel) and DFP-treated (right panel) Huh7 cells. Arrows indicate mitophagosome-like structures. Scale bar: 500 nm. The number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) between cells treated with DFP (1.0 mM) and those without DFP. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.",
    "molecules": "DFP"
  },
  {
    "caption": "(F) Quantification of mitophagy using mt-mKeima transfection and FACS analysis for Huh7 cells treated with DFP (0, 0.01. 0.5 or 1.0 mM as indicated) in the absence or presence of bafilomycin (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.",
    "molecules": "bafilomycin, DFP"
  },
  {
    "caption": "(A) Cytoplasmic and mitochondrial iron content was measured by a ferrozine-based assay for Huh7 cells with DFP, DFX or DFO treatment (0.01 mM) for 24 h (n=5, biological replicates). The cytoplasmic iron content was measured by subtracting the iron content of the mitochondrial fraction from the iron content of the whole lysate. The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.",
    "molecules": "DFX, DFP, DFO, iron"
  },
  {
    "caption": "(B) Cytoplasmic and mitochondrial iron content was measured for Huh7 cells with DFP treatment (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated) for 24 h. (n=5, biological replicates) The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.",
    "molecules": "DFP, iron"
  },
  {
    "caption": "(C) Immunoblots (left panel) for mitochondrial ferritin (FTMT), frataxin, mitoferrin, heat shock protein 60 (HSP60), ferritin heavy chain (FTH1) and transferrin receptor (TfR) and β-actin using the mitochondrial fraction or whole cell lysate of Huh7 cells following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM as indicated). HSP60 and β-actin were used as loading controls for mitochondrial and whole cell lysates, respectively. The expression level of FTMT was normalized to HSP60 (n=4, biological replicates, right panel). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05.",
    "molecules": "DFP"
  },
  {
    "caption": "(D) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before and after DFP (1.0 mM) treatment (n=4, biological replicates). The expression of FTMT mRNA level was normalized to GAPDH mRNA. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.",
    "molecules": "DFP"
  },
  {
    "caption": "(E) The mRNA expression of FTMT in Huh7 cells was quantified by real-time RT-PCR before DFP (1.0 mM) treatment and 3, 6, 12, and 24 h after DFP treatment (n=5, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01, ***: P<0.001.",
    "molecules": "DFP"
  },
  {
    "caption": "(F) DNA-binding activity of six transcription factors (specific protein 1 [SP1], cAMP response element binding protein [CREB], Ying yang 1 [YY1], GATA2, forkhead box protein A1 [FoxA1] and CCAAT-enhancer-binding protein β [ C/EBPβ]) to the FTMT promoter region was compared by chromatin immunoprecipitation before and after DFP (1.0 mM) treatment in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. *: P<0.05.",
    "molecules": "DFP"
  },
  {
    "caption": "(G) The DNA-binding activity of SP1 to the FTMT promoter region was assayed by chromatin immunoprecipitation following treatment with DFP (0, 0.01, 0.1, 0.5 or 1.0 mM) as indicated in Huh7 cells (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.",
    "molecules": "DFP"
  },
  {
    "caption": "(H) FTMT mRNA expression was quantified for Huh7 cells by real-time RT-PCR before and after DFP (1.0 mM) treatment with or without SP1 knockdown by siRNA (n=5, biological replicates). mRNA expression of FTMT level was normalized to GAPDH. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.",
    "molecules": "DFP"
  },
  {
    "caption": "(I) Immunoblots for SP1 using the cytoplasmic fraction and nuclear fraction before and after DFP (1.0 mM) treatment. β-actin and Lam B were used as loading control for the cytoplasmic and nuclear fractions, respectively.",
    "molecules": "DFP"
  },
  {
    "caption": "(J) Immunoblots for HIF1α using Huh7 cells and HepG2 cells before and after DFP (0.1 mM) treatment. β-actin was used as the loading control.",
    "molecules": "DFP"
  },
  {
    "caption": "(K) Immunoblots for FTMT, HIF1α and SP1 using the mitochondrial fraction lysate and whole cell lysate of Huh7 cells before and after DFP (1.0 mM) treatment with or without HIF1α or SP1 knockdown by siRNA. HSP60 and β-actin were used as loading controls for the mitochondrial fraction and whole cell lysates, respectively.",
    "molecules": "DFP"
  },
  {
    "caption": "(A) Immunoblots for mitochondrial ferritin (FTMT) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells (upper panels) and the quantification of mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates) (lower panels) before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.",
    "molecules": "DFP"
  },
  {
    "caption": "(B) Immunofluorescence staining for LC3 (red), the mitochondrial marker Tom20 (green) and FTMT (blue) in Huh7 cells before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. FTMT colocalize with LC3 at the mitochondria (white puncta indicated by arrows). Boxed areas are enlarged below. Scale bar: 5 μm",
    "molecules": "DFP"
  },
  {
    "caption": "(C) Immunoblots for mitochondrial ferritin (FTMT) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA-resistant FTMT (left panel). HSP60 as the loading control. Those cells were subjected to the quantification of mitophagy using mt-mKeima transfection and FACS analysis before and after DFP (1.0 mM) treatment (n=5, biological replicates) (right panel). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. ***: P<0.001.",
    "molecules": "DFP"
  },
  {
    "caption": "(D) Super resolution images of immunofluorescence staining for LC3 (red), FTMT (green) and Tom20 (blue) in FTMT knockdown-Huh7 cells transfected with FTMT-GFP or FTMT siRNA-resistant FTMT-GFP before and after DFP (1.0 mM) treatment. White arrows indicate colocalization of LC3, FTMT, and Tom20.",
    "molecules": "DFP"
  },
  {
    "caption": "(A) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti-FTMT-coupled magnetic beads and immunoblotted with the indicated antibodies (anti-NCOA4 or anti-LC3).",
    "molecules": "DFP"
  },
  {
    "caption": "(C) Immunofluorescence staining for Tom20 (red), NCOA4 (green) and FTMT (blue) in Huh7 cells before and after DFP (1 mM) treatment. Boxed areas are enlarged on the right. The white puncta indicated by white arrows show the colocalization of FTMT, NCOA4 and Tom20. Scale bar: 10 μm and 5 μm in enlarged images.",
    "molecules": "DFP"
  },
  {
    "caption": "(D) Super-resolution images of immunofluorescence staining for Tom20 (blue), NCOA4 (red) and FTMT (green) in Huh7 cells after DFP (1 mM) treatment, using the single-molecule fluorescence microscope. The white arrows indicate colocalization of these proteins.",
    "molecules": "DFP"
  },
  {
    "caption": "(F) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies. Co-immunoprecipitation revealed a specific interaction of NCOA4-mCherry with FTMT-GFP or FTMTD57A-GFP, which has a substitution of alanine (A) for aspartic acid (D) at position 57 in the FTMT mitochondrial leader sequence (lanes 1 and 3).",
    "molecules": "alanine, aspartic acid"
  },
  {
    "caption": "(G) Following DFP (1.0 mM) or ammonium iron sulfate (FAS) treatment as indicated, extracted proteins from Huh7 cells expressing FTMT-GFP and NCOA4-mCherry were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies.",
    "molecules": "DFP, ammonium iron sulfate, FAS"
  },
  {
    "caption": "(A) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria. The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC-1 for 1 h at 30°C and subjected to flow cytometric analysis (n=4, biological replicates) by excitation with a blue laser (488 nm) with simultaneous emission assessment using a 585/42 detector for JC-1 red and a 530/30 for JC-1 green. The trapezoid areas show the population of depolarized mitochondria. Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.",
    "molecules": "JC-1, CCCP, DFP"
  },
  {
    "caption": "(B) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer (n=4, technical replicates). Huh7 cells were incubated with 1 mM of DFP for different lengths of time (0, 3, 6, 18, and 24 h). Oxygen consumption was measured, and the respiration rate was analyzed by injecting of 1μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 10 μM rotenone/antimycin A. OCR: oxygen consumption rate. The central point and the error bards indicate mean ± SD.",
    "molecules": "antimycin A, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, FCCP, DFP, oligomycin, rotenone"
  },
  {
    "caption": "(E) Immunoblots for the precursor form and processed form of FTMT-GFP, endogenous FTMT, NCOA4, LC3, and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT-GFP with or without digestion with proteinase K in the presence or absence of DFP. mtHSP70 was used as the loading control.",
    "molecules": "DFP"
  },
  {
    "caption": "(F) Immunoblots for FTMT, VDAC, and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment. The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment. mtHSP70 was used as the non-membrane-integral protein control and VDAC as the membrane-integral protein control. mtHSP70 at the right-hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment.",
    "molecules": "DFP, sodium carbonate"
  },
  {
    "caption": "(A) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), and LC3 (green) in Huh7 cells untreated or treated with DFP (1 mM) for 24 h, or with CCCP (30 μM) for 6 h. Boxed areas are enlarged on the right. Scale bar: 10 μm, and 5 μm in enlarged images.",
    "molecules": "CCCP, DFP"
  },
  {
    "caption": "(C) STAM mice were fed a high-fat diet and orally administered 0.0375 mg/g body weight of DFP, 0.075 mg/g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age (n=6). Arrows indicate liver tumors. Scale bar: 1.0 cm. The number and maximum size of liver tumors were compared among the three groups. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.",
    "molecules": "DFP"
  },
  {
    "caption": "(D) Histology of the nontumorous liver (H&E and Sirius red stain, original magnification x40) and superoxide anion production in the livers of STAM mice fed a high-fat diet with or without DFP (0.075 mg/g) administration. Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion. Scale bar: 100 μm.",
    "molecules": "DFP, dihydroethidium, Sirius red, superoxide"
  },
  {
    "caption": "(F) Electron microscopic images of livers in control (C57BL/6) and DMBA + HFD mice that were orally administered 0.075 mg/g of DFP or distilled water. Scale bars: 2.0 μm. The boxed areas (x 1,500) are enlarged below (x6,000 on left and x10,000 on right). The white arrow indicates mitophagosome-like structures. The mean number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) among the four groups (control, DMBA + HFD mice treated with 0, 0.0375 or 0.075 mg/g of DFP) (n=6). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.",
    "molecules": "DMBA, DFP"
  },
  {
    "caption": "(A) Immunoblots for FTMT, frataxin, mitoferrin, HSP60, FTH1 and TfR using the hepatic mitochondrial fraction lysate and whole liver lysate in control (C57BL/6J) mice and STAM mice treated with DFP (0, 0.0375, or 0.075 mg/g body weight) (n=6). The expression of FTMT, frataxin, mitoferrin, FTH1, and TfR were normalized to HSP60 or β-actin, respectively. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. (B) The mean number of mitophagosome-like structures/100 μm2 from four randomly selected areas in the livers of DFP (0.075 mg/g body weight)-administered mice were compared between control and FTMT siRNA-treated groups for STAM mice (n=6) and DMBA + HFD mice (n=6). The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. NC: negative control siRNA, *: P<0.05. ",
    "molecules": "DMBA, DFP"
  },
  {
    "caption": "(C) Frozen liver sections of DFP (0.075 mg/g body weight)-administered mice (STAM mice and DMBA + HFD) with or without FTMT knockdown were stained with dihydroethidium. Red fluorescence indicates the ROS production. Scale bar: 100 μm. siNC: negative control siRNA.",
    "molecules": "DMBA, DFP, dihydroethidium, ROS"
  },
  {
    "caption": "(D, E) STAM mice (D) were fed a high-fat diet without DFP treatment or orally administered with 0.075 mg/g body weight of DFP from the age of 4 weeks, injected with FTMT siRNA (10 μl/g body weight) or negative control siRNA through the tail vein every 2 weeks for 8 weeks prior to sacrifice, and followed until 16 weeks of age (n=6). DMBA + HFD mice (E) were fed a high-fat diet, treated with DFP and siRNA in the same manner with STAM mice, and followed until 30 weeks of age (n=6). The number and maximum size of liver tumors were compared among the three groups (control without DFP, negative control siRNA, and FTMT siRNA with DFP). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01. Representative liver images are shown in the upper panel. Scale bar: 1 cm.",
    "molecules": "DMBA, DFP"
  },
  {
    "caption": "B Salt-induced vacuole fission. The indicated cells were logarithmically grown in YPD and stained with FM4-64. Vacuole morphology  was imaged as in A, before and after a mild salt shock with 0.5 M of NaCl  for 15 min. The look-up table has been inverted to allow better representation of the clusters of extremely small vacuolar  fragments in the SNX mutants. Scale bar: 5 µm. C The number of vacuoles  per cell was quantified for the samples from B. n=3 experiments with at least 100 cells per condition were evaluated; Error bars represent the SEM.",
    "molecules": "FM4-64, NaCl"
  },
  {
    "caption": "B Epistasis of ATG18  and retromer  genes concerning vacuolar morphology. The indicated cells were logarithmically grown in YPD medium, stained  with FM4-64 and  calcofluor white C Quantification of vacuole morphology. The number of vacuoles  per cell was measured in the cells from B. The graph shows the fractions of cells displaying the indicated numbers of  vacuolar vesicles (n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. ** p < 0.01",
    "molecules": "calcofluor white, FM4-64"
  },
  {
    "caption": "B Purified Atg18 and CSC-GFP were mixed in a 1:1 ratio, incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets.",
    "molecules": "PBS"
  },
  {
    "caption": "C  Pull-down. Cells (SEY6210 atg18∆, atg21∆) expressing genomically tagged Vps26yomCherry  and the indicated Atg18HA3-yEGFP variants were logarithmically grown in SC-URA media.  Vps26yomCherry was pulled down from whole-cell extracts with RFP-trap magnetic beads and analyzed by  SDS-PAGE and Western blotting against the indicated proteins. Bands were quantified on a LICOR fluorescence imager. Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry. Vps1 served as a loading control. n = 3 independent replicates. Data were subjected to an unpaired t-test. Bars represent the mean and errors bars the SEM, *** P < 0.001).",
    "molecules": "URA"
  },
  {
    "caption": "A, Effect of Atg18T56E on salt-induced vacuole fission. Cells expressing Atg18WTHA3-yEGFP or Atg18T56E-HA3-yEGFP from centromeric  plasmids in a SEY6210 atg18∆, atg21∆ strain were logarithmically grown in SD-URA medium. They were stained  and imaged before and after the induction of vacuole fission   by a short salt shock Calcofluor white-stained  cell walls are only represented in the merge (blue). Scale bar: 5 µm. B, Quantification of the number of vacuoles per cell from A, n = 3 biological experiments with at least 100 cells per condition were scored; bars represent the mean and error bars the SEM. C, Epistasis of atg18T56E over a vps17∆ mutation. The indicated variants of Atg18-HA3-yEGFP  were expressed from plasmids in a SEY6210 atg18∆, atg21∆, vps17∆ strain. Cells were logarithmically grown in SD-URA  and imaged Scale bar: 5 µm. D, Quantification of the experiments from C, n = 3 independent experiments with at least 100 cells per condition scored; bars represent the mean and error bars the SEM .",
    "molecules": "Calcofluor white, URA"
  },
  {
    "caption": "A. Atg18-dependent recruitment of CSC-GFP  to small liposomes. SUVs were incubated (10 min, 25°C) with purified CSC  (1.5 µM) alone or in combination with the indicated recombinant Atg18  variants (1.5 µM). The vesicles  were sedimented by centrifugation and supernatants   (Sup.) and pellets  were analyzed by SDS-PAGE  and Coomassie staining. B. Quantification of the Coomassie  signals for Vps35  by densitometry  on a LICOR Odyssey scanner. n = 3 independent experiments. Bars represent the mean and error bars the SEM, ** p < 0.01; * p < 0.05. C. Quantification as in B, but for the Atg18  variants.",
    "molecules": "Coomassie"
  },
  {
    "caption": "A, Tf  recycling. WIPI1-KO cells were transfected with WIPI1WT-eGFP, WIPI1S69E-eGFP  or  WIPI1S69A-eGFP for 18h. Then, they were serum-starved  for 1 h, loaded with  Alexa Fluor 568-conjugated Tf, chased at 37°C for 1 h without labeled Tf, and analyzed by  confocal microscopy. Scale bar: 10 μm. White dashed lines delineate the circumference of the cells. B, Quantification of Tf-fluorescence in the cells from a that expressed WIPI1  variants. Total cell fluorescence was integrated and corrected for background fluorescence. Mean values ± SD are shown. n = 3 independent experiments with a total of 150 cells analyzed per condition. P values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.",
    "molecules": "Alexa Fluor 568"
  },
  {
    "caption": "ER Proliferation under UPR-Inducing Conditions(A) Determination of ER abundance in control and UPR-induced cells. Representative cells are shown. The UPR was induced in wild-type cells by addition of DTT. Ultrastructure of control cells and UPR-induced cells was analyzed using ImageJ. The lower images show traces of cortical ER (represented in magenta) and the nuclear envelope (NE, in blue). Vacuoles, nuclei, and mitochondria are indicated as V, N, and M, respectively.(B) Quantification of the ER proliferation during the UPR. UPR was induced and cells were collected for EM at the indicated time points. Length of the ER (as traced in [A]) was measured and divided by the area of the section. Data are plotted relative to time 0. Measurements for each time point correspond to the mean of 25 independent cell images.(C) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h. ER was quantified as described above in (B).",
    "molecules": "DTT, DOC"
  },
  {
    "caption": "Characterization of ER-Containing Autophagosomes (ERAs) during the UPR(A) Images of representative DTT-treated wild-type cells that contain ERAs. Nuclei and cytoplasm are indicated as N and C, respectively.(B) Enlargement of representative images of ERAs from different cells. The bottom right image is likely to show a section through a cup-shaped ERA. Note that there are no connections between the stacked cisternae and the envelope.(C) High magnification of the ERA double membrane envelope.(D) Some ERAs are found attached to or are in close proximity to ER tubules/sheets (indicated by the arrow). Note that the section in (A) includes two such junctions.(E) High-pressure freezing/freeze substitution image of an ERA linked to an ER tubule/sheet. The osmium/lead staining used in this technique visualizes ribosomes and demonstrates that the outer ERA envelope membrane, but not the stacked internal cisternae, are tightly studded with ribosomes, indicating that they originate from ER membranes.(F) High-pressure freezing/freeze substitution image of an ER-ERA junction using an improved protocol to visualize membranes.(G) Using the same technique as in (F), we visualized the internal membrane content of an ERA. Note that both portions of the internal membranes and of the sequestering double membrane envelope contain bound ribosomes, and hence are likely derived from the ER.",
    "molecules": "DTT"
  },
  {
    "caption": "Fluorescence Visualization of an ER Marker after UPR Induction(A) Cells treated with the UPR-inducing drug DTT (+DTT) or with no drug were visualized using a fusion protein between the translocon component Sec61 and the red-fluorescent protein \"cherry.\" Top panels show untreated cells, and bottom panels show representative UPR-induced cells.(B) Representative images showing UPR-induced cells that contain ERAs (indicated by arrows).",
    "molecules": "DTT"
  },
  {
    "caption": "Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker(A) Representative section of a cell immunolabeled against a myc-tagged Sec63, an integral ER membrane protein. As a primary antibody, we used a rabbit polyclonal anti-myc and, as a secondary, we used 15-nm gold particles-conjugated anti-rabbit antibody. Nucleus, nuclear envelope, ER, and ERA are indicated as N, NE, ER, and ERA, respectively.(B) High magnification of an electron micrograph of a section of ER. Quantification showed that there are 5 ± 2 gold particles per linear micrometer of ER.(C) High magnification of ERAs. To predict how many gold particles one should expect in a particular ERA, we first calculated and averaged the amount of ER (expressed as length in linear micrometers) present in an ERA (similar to the ones shown in Figure 3B), and normalized the value for its area. These calculations determined that there are 20.8 ± 3.3 μm of ER per μm2 inside the ERAs. These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes. Two representative ERAs are shown. The ERA shown in the middle picture should hold 2.4 μm of ER inside and, therefore, should have 12 gold particles. We counted 12 gold particles. The ERA on the right could contain 2.7 μm of ER and should contain14 gold particles; we counted 16 gold particles. The image on the right shows a representative view of a nucleoplasmic region.(D) Quantification of gold-labeling density per area. To assess the signal-to-noise ratio of our immunogold-labeling procedure, we assessed background labeling by counting the number of gold particles over an areas of nucleoplasm (N) and over ERAs, and normalized the counts to the respective areas.",
    "molecules": "gold, gold particles"
  },
  {
    "caption": "(A) Wild-type cells transformed with a plasmid containing GFP-Atg8 were grown for 4 h in synthetic media with no drug, with UPR-inducing conditions (+DTT and +TM), or under nitrogen starvation conditions (N starv), and then harvested for protein preparation. Protein extracts were analyzed by Western blotting using antibodies against GFP (top panel) or Hac1 (bottom panel). Total protein concentration was measured by BCA protein assay. Same concentration of protein was loaded in each lane, and transfer efficiency was checked by Ponceau staining. The identities of the different bands are indicated.",
    "molecules": "DTT, nitrogen, TM"
  },
  {
    "caption": "(C) GFP-Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i (+DOC) by Western blotting using antibodies against GFP.",
    "molecules": "DOC"
  },
  {
    "caption": "(D) Western blot using antibodies against GFP of extracts from hac1Δ, ire1Δ, or vps4Δ pep4Δ cells expressing GFP-Atg8. Mutant cells were grown under regular conditions, UPR-inducing conditions (+DTT), or nitrogen starvation conditions (N starv).",
    "molecules": "DTT, nitrogen"
  },
  {
    "caption": "Localization of GFP-Atg8 during UPR InductionSome of the DTT-treated cells shown in Figure 4B expressing GFP-Atg8 and Sec61-cherry (as an ER marker) were visualized using fluorescence microscopy. GFP-Atg8 localizes in close proximity to the ERAs detected by the ER marker.",
    "molecules": "DTT"
  },
  {
    "caption": "Atg8 and Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild-type, hac1Δ, atg1Δ, atg8Δ, atg9Δ, atg16Δ, and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich-media plates with no drug (YPD) or with different concentrations of tunicamycin (TM; 0.2 or 1.0 μg/ml). atg19Δ gave an identical result to the other autophagy genes shown here (unpublished data).",
    "molecules": "TM, tunicamycin"
  },
  {
    "caption": "(B-D) Immunofluorescent  images of  mouse liver (B),  testis (C) and  heart (D). H3K27ac, PolII-S5P, and laminin were stained with specific primary antibodies and visualized using fluorescent labeled anti-mouse  ChIL-probe (red: H3K27ac and  PolIIS5P) and anti-rabbit IgG (green: laminin). DNA was counterstained with Hoechst 33342. Scale Bar: 20 µm (left images), 10 µm (right images).",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(A) Quantification of cell death-associated morphologies in 293 cells. Graphs show the percentage of cells harboring all spectrum of morphological changes occurring as a result of luciferase, p55/TNFR1, DRP-1 Δ73, or DAPk ΔCaM transfections in the presence or absence of the caspase inhibitors BD-fmk or z-VAD-fmk (100 μM; mean ± SD calculated from triplicates of 100 cells each). This experiment was repeated three times with reproducible results.",
    "molecules": "BD-fmk, z-VAD-fmk"
  },
  {
    "caption": "(B) Quantification of cell death-associated morphologies in MCF-7 cells. Experimental conditions were performed as in A in the presence or absence of BD-fmk (100 μM).",
    "molecules": "BD-fmk"
  },
  {
    "caption": "(D) Transmission electron micrographs of 293 cells transfected with p55/TNFR1 (1) or DRP-1 Δ73 (2a and 2b; parts of these figures are magnified at the right) and treated with the caspase inhibitor BD-fmk (100 μM). Immature autophagic vesicles (double arrow), autophagic vesicles (black arrow), and autolysosomes (white arrow) are shown. m, mitochondria; g, Golgi apparatus.",
    "molecules": "BD-fmk"
  },
  {
    "caption": "(A and B). Quantification of autophagy in MCF-7 cells. MCF-7 cells were transfected with luciferase or FLAG-DRP-1 K42A followed by steroid withdrawal (serum starvation plus 10−6 M tamoxifen treatment) (A) or by serum and amino acid starvation (B) (mean ± SD calculated from triplicates of 100 cells each). These experiments were repeated three times with reproducible results. Proteins extracted from the transfected cells were subjected to Western blot analysis with anti-FLAG and anti-β-tubulin antibodies.",
    "molecules": "amino acid, tamoxifen"
  },
  {
    "caption": "(A) Photographs of MDC-stained MCF-7 cells. (Top) Steroid withdrawal. Cells were either grown in complete medium (1), steroid depleted (2), or steroid depleted in the presence of the broad caspase inhibitor BD-fmk (100 μM) (3) for 3 d. (Bottom) amino acid starvation. Cells were either grown in complete medium (1-3) or under amino acid starvation condition (4-6) without (1 and 4) or with (2 and 5) the presence of the broad caspase inhibitor BD-fmk (100 μM) or the general inhibitor of autophagy 3-methyladenine (10 mM) (3 and 6) for 3 d.",
    "molecules": "3-methyladenine, amino acid, BD-fmk"
  },
  {
    "caption": "(C) Graph showing the cumulative phase microscopy scores of dead (shrunk and detached) MCF-7 cells in tamoxifen-treated (filled) or nontreated (unfilled) cultures in the presence (▴) or absence (•) of the indicated caspase inhibitors (25 μM).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(A) Quantitation of autophagy in HeLa cells. Cells were either grown in complete medium (1), treated with IFN-γ (1,000 U/ml) (2), or treated with IFN-γ in the presence of the broad caspase inhibitor BD-fmk (100 μM) (3) for 3 d. (B) Photographs of the MDC-stained HeLa cells corresponding to the experiment in A. Treatments: complete medium (1), IFN-γ (1,000 U/ml) (2), and IFN-γ (1,000 U/ml) + BD-fmk (3).",
    "molecules": "BD-fmk"
  },
  {
    "caption": "(E) DAPk antisense-transfected and control DHFR-transfected polyclonal populations of HeLa cells were incubated with hygromycin B (200 μg/ml) in the presence or absence of IFN-γ (1,000 U/ml) and BD-fmk (50 μM). Detection of autophagic vesicles was performed 2 d later using the MDC dye. Graphs represent mean ± SD of MDC-positive cells calculated from triplicates of 100 cells each.",
    "molecules": "BD-fmk, hygromycin B"
  },
  {
    "caption": "DRP-1 is localized to the lumen of autophagic vesicles. Immunogold staining of 293 cells expressing HA-DRP-1 reveals a specific staining of DRP-1 in the lumen of double membrane autophagosomes containing cytoplasmic material (A and B) in autophagosomes containing organelles and vacuoles (C) and inside the body of autolysosomes containing digested matter (D). The gold particles do not overlap with the phagocytosed organelles in C or the lysosomes in D.",
    "molecules": "gold"
  },
  {
    "caption": "B. The direct correlation between RNA (TPM) and protein abundances (copy number) for all quantified genes in the same tissues and cell lines. Spearman (ρ) and Pearson (r) correlation between the two values across the quantified genes are shown. The other seven tissues and five cell lines are shown in Figure EV4.",
    "molecules": "RNA"
  },
  {
    "caption": "F-G) HeLa cells were treated as in B) and then fixed and stained with ULK1 (green) (F) or ATG13 (red) antibodies (G) respectively. Hoechst was used to stain nuclei. Graphs (right) show the average of both ULK1 (F) (n=3 independent experiments, n=11 fields analysed) or ATG13 (n=3 independent experiments, n≥16 fields analysed) (G) puncta number per cell and both ULK1 (n=3 independent experiments) (F) or ATG13 (n=3 independent experiments) (G) fold of increase of the average size respectively. White arrows indicate most representative puncta of analysed conditions. All data are reported as the mean value ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. *, P<0.05; ****, P<0.0001. Scale bar 5 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed or starved conditions for 1h. Cells were fixed and co-labelled with ULK1 (red) antibody and A) anti-CALNEXIN (CANX) (green) antibody to highlight ER structures; (n=3 independent experiments, n≥16 fields analysed) B) GOLGIN97 (green) antibody to label GOLGI complex; (n=3 independent experiments, n=12 fields analysed) C) SEC31A (green) to point out COPII vesicles. (n=3 independent experiments, n≥16 fields analysed)",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "F) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Cells grown for 1h in starvation medium then fixed, permeabilized, and labelled with anti-ULK1 (green) and anti-ERGIC53 (red) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed as in D). Values of Mander's coefficient for ULK1, expressed as percentage, are mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Dunnetts's multiple comparison test. *, P<0,05. (n=3) (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed (FED) or starvation conditions (STV) in the presence or absence of CLQ for 1h. Cells were then fixed and stained using LC3B-II (red) antibody. Analysis of LC3B puncta number is reported (bottom). All data are expressed as the mean value ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. ****, P<0.0001. (n=4 independent experiments, n≥25 fields analysed). Scale bar 5 μm.",
    "molecules": "CLQ, oligonucleotides"
  },
  {
    "caption": "B) HeLa cells were transfected as in A) and cultured in starvation conditions (STV) in the presence or absence of Bafilomycin A1 (Baf A1) for 1h. Cells were then fixed and stained using LC3B-II (green) and LAMP1 (red) antibodies. Hoechst was used to mark nuclei. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for LC3B-II are expressed as mean ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; (n=3 independent experiments, n≥13 fields analysed). Scale bar 5 μm.",
    "molecules": "Hoechst, Baf A1, Bafilomycin A1"
  },
  {
    "caption": "A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR); after culturing cells in fed (FED) or starvation conditions (STV) for 1h, cells were fixed and labelled with the indicated antibodies. Co-localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander's coefficients of ULK1, expressed as percentage, is reported (bottom). White arrowheads point at co-localization events between ULK1 and LC3B; white arrows track puncta not co-localizing; N, nucleus. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; **, P<0.01; ****, P<0.0001. (n=3 independent experiments, n≥16 fields analysed). Scale bar 5 μm.",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "HEK293 cells stably expressing either GFP-ATG13 (B) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of ATG13 (B) are reported. Images of ATG13 (B) forming association with LC3B-II are also shown. The appearance of LC3B on ATG13 (B) (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "HEK293 cells stably expressing either GFP-DFCP1 (C) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of both ATG13 (C) are reported. Images of DFCP1 (C) forming association with LC3B-II are also shown. The appearance of LC3B on on DFCP1 (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "G) Control (TFG-WT) and patient's (TFG-R106C) fibroblasts were grown in fed or starvation conditions for 1h. Cells were fixed, permeabilized, and labelled with anti-ULK (red) antibody. Hoechst was used to stain nuclei. The average of ULK1 puncta number per cell is shown (bottom). All data are expressed as the mean value ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0.05; ***, P<0,001; ****; P<0,0001. (n=3 independent experiments, n≥14 fields analysed). Scale bar 5 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) Hoechst was used to stain nuclei. ULK1 puncta number (G) was analysed and reported as mean ± SEM in the graph (right) (n=3 independent experiments, n=24 fields analysed). Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) alone or ULK1 (red) and ERGIC53 (green) (H). Hoechst was used to stain nuclei. analysed). Co-localization analyses (H) were performed by Jacop plugin. Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A. Characterization of C. perfringens β-toxin (CPB) constructs with different N-termini by gel electrophoretic analysis under denaturing (A) conditions. SDS-PAGE gel analyis of CPB samples in cholate (1μg per lane). CPB samples containing cholate were boiled for 5 min at 95°C (+) or not (-).",
    "molecules": "cholate"
  },
  {
    "caption": "B-E) Expression of Hep-ID (n=13 genes), Hep-IDCONNECT (n=26 genes) and remaining TF-encoding genes from cluster G (Others; n=82 genes) was monitored in indicated transcriptomic data Box plots show log2 fold changes between adult versus newborn mouse livers (B), MPH of Hnf4ahep-/- (Hnf4a KO) versus wild-type mice (C), a meta-analysis of severe mouse liver injuries versus control livers and microdissected hepatocytes from alcohol-related human liver cirrhosis (alcoholic steatohepatitis) versus control livers (E). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Statistical significance was assessed using one-sided Wilcoxon rank sum test with Benjamini-Hochberg correction for multiple testing to determine if the mean log2 FC was statistically lower (B, D, E) or higher (C) than 0. *q<0.05.",
    "molecules": "alcohol"
  },
  {
    "caption": "E) Transcriptional modulation of Hep-ID TFs (n=13 genes) and a control group of non-Hep-ID TF-encoding genes matched for their promoter activity of similar size (n=13 genes) in Huh7 or HepG2 cells treated with GW3965, T3 or GC-1, respectively. The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of log2 fold changes is shown using box plots. Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction was used to define whether transcriptional regulation of Hep-ID TF genes was different from that of the control genes for each individual transcriptomic dataset. *q<0.05.",
    "molecules": "T3, GW3965, GC-1"
  },
  {
    "caption": "mRNA expression of the indicated genes was monitored using RT-qPCR in HepG2 cells treated with T3 or GC-1 for 24h or 96h. Bar graphs show mean ±SD (n=3 biological replicates) of log2 fold changes in treated versus untreated HepG2 cells. Gray dots show the results obtained from the 3 independent biological replicates (each performed in technical triplicates). One-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0. *q<0.05.",
    "molecules": "T3, GC-1"
  },
  {
    "caption": "mRNA expression of the indicated genes was monitored using RT-qPCR in HepG2 cells treated with T3 or GC-1 for 24h or 96h. For Hnf4a, the log2 fold change in the ratio of P1 over P2 promoter-derived isoforms is also shown. Gray dots show the results obtained from the 3 independent biological replicates (each performed in technical triplicates). One-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0. *q<0.05.",
    "molecules": "T3, GC-1"
  },
  {
    "caption": "I) Western blots assays performed using antibodies against the indicated proteins on extracts from HepG2 cells treated or not with T3 for 24h. Rep#1-3 indicates the 3 independent biological replicates analyzed.",
    "molecules": "T3"
  },
  {
    "caption": "L) Identity effector genes significantly downregulated in Nodules/Control (q<0.05) were split in 3 groups according to their log2 fold changes (i.e. low, intermediate and high repression; pink boxes) and then monitored for induction in Nodules+T3/Nodules (green boxes). Statistical differences between the High repression and the other groups regarding the Nodules/Control comparison, on the one hand, or the Nodules+T3/Nodules comparison, on the other hand, were defined using Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction for multiple testing correction. *q<0.05.",
    "molecules": "T3"
  },
  {
    "caption": "B-D) mRNA expression of the indicated genes was monitored in mouse livers using RT-qPCR. Mice treated with IL1B+T3 were subdivided into tertiles based on the mean expression of the Dio1 and Hectd3 genes and defined as low, intermediate or high T3 responsiveness groups (Low resp., Int. resp. and High resp., respectively). Fold change relative to the mean of the control group is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line (n=13 mice for the PBS group, 17 for the IL1B group and 10 for the other groups). Whiskers extend to the maximum and minimum values. Statistical differences between the High resp. and the other IL1B-treated groups were defined using Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction for multiple testing correction. *q<0.05.",
    "molecules": "T3, PBS"
  },
  {
    "caption": "(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom).",
    "molecules": "EtBr, ethidium bromide, dsRNA"
  },
  {
    "caption": "(C) mESCs stably expressing GFP (mESCs-GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA and the level of GFP in Cy5+cells was analysed 3 days later and is represented as histograms (left). Right panel, bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control (D) mESCs stably expressing d2GFP (mESCs-d2GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA. The level of d2GFP was analysed at the indicated timepoints post-transfection and displayed as in (C).",
    "molecules": "dsRNA"
  },
  {
    "caption": "(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs (top panels) or siRNAs (bottom panels). GFP level was measured in Cy5+ cells by flow cytometry 72 hours (hrs) post-transfection.",
    "molecules": "siRNAs, dsRNAs"
  },
  {
    "caption": "(B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated.",
    "molecules": "dsRNAs"
  },
  {
    "caption": "(C) WT, Ifnar1+/- or Ifnar1-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry at 48 hrs post-transfection.",
    "molecules": "dsRNAs"
  },
  {
    "caption": "(A) Mavs+/- and Mavs-/-MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+cells 72 hrs post-transfection.",
    "molecules": "dsRNA"
  },
  {
    "caption": "(B) Mavs-/-MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+cells.",
    "molecules": "dsRNA"
  },
  {
    "caption": "(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left).",
    "molecules": "siRNAs, dsRNA"
  },
  {
    "caption": "(C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP",
    "molecules": "dsRNA, dsRNAs"
  },
  {
    "caption": "(E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.",
    "molecules": "dsRNAs"
  },
  {
    "caption": "(B) Parental non-transduced (NT) Mavs-/- MEFs and complemented Mavs-/- Ago2-/- MEFs as described in (A) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.",
    "molecules": "dsRNAs"
  },
  {
    "caption": "(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment.",
    "molecules": "dsRNA"
  },
  {
    "caption": "C Representative confocal micrographs from lines co-expressing RPS5apro:HF-mScarlet-DCP1 and PIN2pro:PIN2-GFP (epidermal cells, region 3, Scale bars: 5 μm). Bottom: polarity index of DCP1 in root meristematic cells (compared to propidium iodide (PI) and tubulin staining of root cells; N, biological replicates = 3 roots, n = 13 cells). Polarity index calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells. The arrowhead in PIN2 indicates the cell plate or PM in DCP1. Right: representative confocal micrographs showing that PM localization is independent of the promoter used (DCP1pro, 35Spro; region 2, epidermal cells, or RPS5apro on the lower right). The details from the inset show increased localization at the cell edge (discussed later). mSc, mScarlet.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "B Representative confocal micrographs showing actin localization in WT, dcp1-1, dcp1-3, scar1234, dcp2-1, dcp5 and pat1 upon phalloidin staining and graph (right) indicating the percentage of cells in region 3 with an accumulation of actin at edges in various genotypes (N, biological replicates = 3, n = 7-9 roots, bars show means + s.d.).",
    "molecules": "phalloidin"
  },
  {
    "caption": "B DCP1 regulates cell expansion anisotropy. Representative confocal micrographs showing FM4-64 staining of the WT, dcp1-1 and dcp2-1 mutants (2 μM, 10 min). Scale bars, 20 μm. Right: percentage of isotropic cells per root meristem (%, epidermal cells) in each genotype (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-5 roots, bars show means ± s.d). Examples of isotropic or anisotropic cells are shown, along with the developmental axis offset at the x- and y-axes.",
    "molecules": "FM4-64"
  },
  {
    "caption": "G Assessment of the  blood-CSF  barrier permeability  to 4 kDa FITC-dextran  (n=5-9, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. AB, antibiotics-treated; ABR, recolonized AB; CSF,  cerebrospinal fluid; GF, germ-free; GFR, recolonized GF; SPF, specific pathogen-free",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "C, D Relative abundance of propionic acid  (C) and butyric acid (D) in mice fecal  samples (n=4-9, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. **p < 0.01. AB,  antibiotics-treated; SPF, specific pathogen-free.",
    "molecules": "butyric acid, propionic acid"
  },
  {
    "caption": "B,C TEER  (B) and assessment of the 70 kDa FITC-dextran  paracellular permeability  (C) of primary CPE  cells treated as showed in (A) (n=3, technical duplicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CPE, choroid plexus epithelial; LPS, lipopolysaccharides; TEER, trans-epithelial electrical resistance",
    "molecules": "dextran, FITC, lipopolysaccharides, LPS"
  },
  {
    "caption": "D Representative images of immunostainings  for ZO-1 and OCLN  in  primary CPE  cells following treatment for 6 h with 1 μM  sodium propionate  or 1 μM sodium butyrate, with 100 ng/ml LPS  stimulation. Scale bar: 50 μm. E The percentage of cells with intact the TJ immunostainings  in displayed (D) (n=3, technical duplicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CPE,  choroid plexus epithelial LPS, lipopolysaccharides TJ, tight junction.",
    "molecules": "lipopolysaccharides, LPS, sodium butyrate, sodium propionate"
  },
  {
    "caption": "G Representative images of immunostainings  for ZO-1  and OCLN  in  choroid plexus  of  sodium propionate or sodium butyrate-treated mice. Scale bar: 50 μm. H-J Quantification of continuous length (H), maximal length (I), and expressed area (J) of TJ immunostainings  in (G) (n=5, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. TJ, tight junction.",
    "molecules": "sodium butyrate, sodium propionate"
  },
  {
    "caption": "L Representative western blot  showing the IgG  levels in CSF  of  sodium propionate  or  sodium butyrate-treated mice  with disrupted gut  microbiota via antibiotics treatment. Data information: CSF, cerebrospinal fluid",
    "molecules": "sodium butyrate, sodium propionate"
  },
  {
    "caption": "H Representative images of immunostainings  for OCLN, CDH1  and ZO-1 in choroid plexus of sodium propionate or sodium butyrate-treated vagotomized mice. Scale bar: 50 μm. I-K Quantification of continuous length (I), maximal length (J), and expressed area (K) of the TJ immunostainings  displayed in (H) (n=4, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons *p < 0.05, **p < 0.01, ***p < 0.001. TJ, tight junction.",
    "molecules": "sodium butyrate, sodium propionate"
  },
  {
    "caption": "B Representative images of immunostainings  for OCLN, CDH1, ZO-1 and CLDN1  in choroid plexus of sodium propionate or sodium butyrate-treated AppNL-G-F mice. Scale bar: 50 μm. C-E Quantification of continuous length (C), maximal length (D), and expressed area (E) of theTJ immunostainings  displayed in (B) (n=5-6, biological replicates). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "sodium butyrate, sodium propionate"
  },
  {
    "caption": "I Representative images of 6E10 staining  in brain  of sodium propionate or  sodium butyrate-treated AppNL-G-F mice. Scale bar: 1 mm. J Quantification of Aβ  plaque area and number in whole sagittal section of the brain  (n=3 mice; one section per mouse). Data information: Bars represent mean ± SEM. Statistics were performed with one-way ANOVA Bonferroni's post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "sodium butyrate, sodium propionate"
  },
  {
    "caption": "A Fluorescence microscopy showing UBQLN2  (Alexa Fluor 647) in solutions contained 50 µM FL UBQLN2  with increasing amounts of K48- and K63-linked chains of different lengths in 20 mM NaPhosphate  and 200 mM NaCl  (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub   monomers and UBQLN2  molecules. The same image is used for the 0:1 condition. Scale bar, 5 µm. Yellow cartoons illustrate one major solution conformation of each chain.",
    "molecules": "Alexa Fluor 647, NaCl, NaPhosphate"
  },
  {
    "caption": "C 15N-1H SOFAST-HMQC NMR spectra of proximal Ub  of K48-Ub4   or K63-Ub4  at 50 µM in the absence and presence of UBQLN2  450-624 at a 2:1 UBQLN2:Ub4  ratio. Unbound monoUb spectrum (black) is overlaid for comparison. NMR spectra collected and processed under identical conditions.",
    "molecules": "1H, 15N"
  },
  {
    "caption": "F Normalized titration  curves of UBA  resonances showing more pronounced sigmoidal behaviors upon titration  of K48-Ub4  into UBQLN2  450-624 than upon titrations  of K11-Ub4, K63-Ub4, M1-Ub4  and HOTag6-Ub.",
    "molecules": "HOTag6"
  },
  {
    "caption": "A Fluorescence microscopy showing enhancement or inhibition of UBQLN2 droplet  formation at increasing amounts of chains at 50 µM UBQLN2  in 20 mM NaPhosphate  and 200 mM NaCl  (pH 6.8) at 30 ºC. [Ub]:[UBQLN2] reflects the ratio between Ub  monomers and UBQLN2  molecules. Solution is spiked with  Alexa Fluor 647-labeled UBQLN2. The same image is used for the 0:1 condition. For visual comparison, images for K48-Ub4  and  K63-Ub4  are identical to those shown in Figure 1A. Scale bar, 5 µm.",
    "molecules": "Alexa Fluor 647, NaCl, NaPhosphate"
  },
  {
    "caption": "E Fluorescence microscopy showing the distribution of Ub4  (spiked with Dylight 488-labeled chains) inside and outside droplets  containing 50 µM UBQLN2  (spiked with Alexa Fluor 647-labeled UBQLN2). Scale bar, 5 µm.",
    "molecules": "Dylight 488, Alexa Fluor 647"
  },
  {
    "caption": "A SDS-PAGE gels monitoring the formation of K63-linked polyUb chains and possibly ubiquitinated GST-Ubc13  over time, as indicated by asterisks and curly brackets. K63-Ub3  migrates on the gel similarly as His-Mms2, right below 20 kDa. Experimental conditions: ± 50 µM Ub  and UBQLN2, 30 nM  Dylight 650-labeled UBQLN2, 1 µM mE1, 2 µM His-Mms2, 4 µM GST-Ubc13, 10 mM ATP  and MgCl2, 3 mM TCEP, 50 mM Tris  pH8, 37 ºC. The K63-linked polyUb  chain reactions were done in the absence (top) and presence (bottom) of UBQLN2. Since UBQLN2  competes with E1  and E2  to bind  to Ub, the presence of UBQLN2  slowed down chain formation (indicated by comparing disappearance rates of Ub). The band intensity for GST-Ubc13  decreased with time. The time-dependent appearance of species between 50-85 kDa were obstructed in the presence of UBQLN2. These species are most likely ubiquitinated GST-Ubc13, and not free polyUb chains, since these bands were observed with BioRad Stain-free gels (right) that detect proteins with tryptophan  residues unlike Ub  or polyUb  chains.",
    "molecules": "Dylight 650, ATP, MgCl2, TCEP, Tris, tryptophan"
  },
  {
    "caption": "(A) Solubilized lysosomes cross‐linked with DSP were subjected to affinity chromatography with the antibody against the cytosolic tail of lamp2a (lanes 1 and 2). Eluted proteins were subjected to SDS-PAGE and silver stained (lane 1), or immunoblotted for lamp2a (lane 2). The sequence of the N‐terminus of the 32 kDa band is shown. Part of the solubilized lysosomes were directly subjected to non‐reducing SDS-PAGE (5%) and immunoblotted for lamp2a (lane 3) or PPCA (lane 4). Arrowheads indicate lamp2a multimeric complexes. The 600 kDa (*) and 200 kDa (**) molecular weight complexes of lamp2a were excised and subjected to reducing SDS-PAGE (12%) and silver staining (lanes 5 and 6). Molecular weight (kDa) is indicated on the left.",
    "molecules": "DSP"
  },
  {
    "caption": "(B) Immunoblot analysis of PPCA in rat liver lysosomes, their membranes (MB) and matrices (MTX) (100 μg of protein) incubated alone (None) or with 5 mM MgCl2. Arrowheads indicate the precursor (54 kDa) and heavy chain (32 kDa) of the mature PPCA.",
    "molecules": "MgCl2"
  },
  {
    "caption": "(C) Immunoblot analysis of hsc73 (upper) and PPCA (lower) in membranes from rat liver lysosomes incubated alone (None) or with 5 mM MgCl2, CaCl2, MnCl2 or 2 mM EDTA.",
    "molecules": "CaCl2, EDTA, MgCl2, MnCl2"
  },
  {
    "caption": "Levels of PPCA associated with the lysosomal membrane correlate with rates of CMA. Membranes of lysosomes incubated with increasing concentrations of GAPDH (A) or MgCl2 (B, top panel) as labeled, were analyzed by immunoblot for all forms of lamp2 (upper), lamp2a (middle) or PPCA (lower). The arrowhead indicates a previously identified truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). (B, bottom panel) Chart showing the effect of increasing concentrations of MgCl2, CaCl2 or EDTA on the degradation of [14C]GAPDH by intact ratliver lysosomes. Values are means ± SE of six different experiments. Differences from the control value were significant at P 0.001 (**) or P 0.01 (*).",
    "molecules": "[14C], CaCl2, EDTA, MgCl2"
  },
  {
    "caption": "(E) Correlation between CMA activity, measured as in (B, bottom panel) and levels of PPCA at the lysosomal membrane determined by denstometric quantification of immunoblots similar to those shown in Figures 1 and 2 in lysosomes from fed or 20 h starved rats (Starv 20h), highly active (H+) or less active (H−) lysosomes, or lysosomes incubated with 1 mM CaCl2, MgCl2, EDTA, or increasing amounts (2, 5, 10 μg) of GAPDH. Values are expressed as times the value in fed rats (control) and are means ± SE of 3-6 different experiments.",
    "molecules": "CaCl2, EDTA, MgCl2"
  },
  {
    "caption": "(B) Degradation of [14C]GAPDH after 30 min incubation with lysosomes isolated from wild‐type and PPCA(−/−) mice skin fibroblasts supplemented or deprived of serum was measured as described in Material and methods. Where indicated, 25 μg of RNase A were added. Values are means ± SE of four different experiments. Differences from the control value were significant to P 0.001 (**).",
    "molecules": "[14C]"
  },
  {
    "caption": "(C) Top: immunoblot analysis with an anti‐HA antibody of purified wild‐type (FL) and mutated (GL) mouse HA‐lamp2a incubated in an acidic pH buffer alone, or with PPCA. Bottom: wild‐type HA‐lamp2a was incubated as in the top panel without additions (None) or in the presence of 2 mM EDTA, 10 μM L‐trans‐epoxysuccinyl‐leucylamide‐(4‐guanido)‐butane (E‐64), 2 μM pepstatin A (Pepst) or 10 μM ABSF. The open arrowhead indicates the truncated form of lamp2a.",
    "molecules": "ABSF, E‐64, EDTA, pepstatin A"
  },
  {
    "caption": "(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise.",
    "molecules": "ub, ubiquitin"
  },
  {
    "caption": "(B) Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb-windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates.",
    "molecules": "ub, ubiquitin, Ubiquitin"
  },
  {
    "caption": "E) Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days.",
    "molecules": "3-amino-triazole, 3AT, histidine"
  },
  {
    "caption": "F) Euc1 is required for formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4).",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Euc1 interacts with SUMO pathway proteins in a yeast two-hybrid (Y2H) assay. A Y2H reporter strain (PJ69-7a) was transformed with Gal4 DNA-binding domain (BD) and Gal4 activation domain (AD) fusion constructs in the indicated combinations. AD-SUMO-GG can be conjugated to SUMOylation substrates, while AD-SUMO-AA is conjugation-deficient. Cells were spotted on control media or selective media (- His) and grown for 3 and 6 days, respectively.",
    "molecules": "His"
  },
  {
    "caption": "(C) Euc1 is predominantly monoSUMOylated on lysine 231. Denaturing NiNTA-pulldowns (NiNTA-PD) with strains expressing His-tagged SUMO (HisSUMO) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter. Covalently SUMO-modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1. Eluates were probed for SUMO to control for equal pull-down, Pgk1 was probed as input control. Euc1-KR denotes the K231R mutation here and hereafter.",
    "molecules": "lysine"
  },
  {
    "caption": "(B) The Slx5-SIMs and Slx5-Md are required for Euc1 ubiquitylation. NiNTA-PDs for His-ubiquitin as in (A). All strains expressed wild-type 3FLAGEuc1 from plasmids under the ADH promoter and His-ubiquitin. Slx5 constructs were expressed from plasmids under control of the endogenous promoter. WBs were probed as described in (A), Slx5-levels were probed using an HA antibody. Asterisk denotes a non-specific band. Slx5-SIM*: SIMs 1-4 were mutated as described (Xie et al, 2010), for SIM5, aa 477-479 (IIV) were mutated to alanines. To avoid possible mislocalization by deletion of the Slx5-Md, which overlaps with a putative NLS (Westerbeck et al, 2014), an N-terminal NLS was fused to all constructs. See Appendix Fig. S7C for HU complementation.",
    "molecules": "alanines"
  },
  {
    "caption": "(B) RNAseq transcriptome analysis of EUC1 overexpression as in (A). ∆euc1 cells with pGAL-EUC1 or pEUC1-EUC1 (control) integrated at the URA3-locus (YIplac211) were grown to mid-log phase and 2% galactose was added for 3h. See also Appendix Fig. S8E.",
    "molecules": "galactose"
  },
  {
    "caption": "(C) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol. For (C, D, F), serial dilutions of the indicated strains were spotted and grown on YPD control (or selective media) plates or under conditions as indicated. See also Fig. EV4A-B.",
    "molecules": "benzylalcohol"
  },
  {
    "caption": "(D) EUC1 overexpression toxicity depends on DNA binding and transactivation. The indicated EUC1 alleles with endogenous or galactose-inducible promoters were integrated at the URA3 locus (YIplac211, euc1∆ background) and spotted on glucose control or galactose plates to induce EUC1 overexpression. See also Appendix Fig. S9A-C.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "(E) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding. ChIP-qPCR of Euc1 after 3 hours galactose induction. Note that IP/input ratios of Euc1 signals are shown, also for the control locus (contr., TOS1 promoter) to highlight Euc1 binding at non-ub-HS sites. STI1-CIN5: intergenic region. TEC1-us: upstream (promoter) region. Data represent means ± SD (n = 3). See also Fig. EV4C-D.",
    "molecules": "galactose"
  },
  {
    "caption": "A. CCNA and CCNB2 expression transcripts were analyzed by RT-qPCR in cDNA derived from tumours of control (saline) or zoledronic acid (ZA)-treated mice (ref. 31). N=3 mice per group. P-values were calculated with two-tailed t-test.",
    "molecules": "ZA, zoledronic acid"
  },
  {
    "caption": "B. ChIP analysis of mutant p53 -bound chromatin from MDA-MB-231 cells treated or not with 1 µM cerivastatin (Cer) for 24h. The CCNB2 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The experiment was performed in biological triplicate. P-values were calculated with two-tailed t-test.",
    "molecules": "Cer, cerivastatin"
  },
  {
    "caption": "C. MDA-MB-231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures;p-values were calculated with two-tailed t-test.",
    "molecules": "Cer"
  },
  {
    "caption": "D. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231 (left panel) and SKBr3 (right panel) cell lines treated or not with 1 µM Cer for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.",
    "molecules": "Cer"
  },
  {
    "caption": "E. Western blot analysis for CCNA, CCNB and Actin (loading control) expression in lysates from MDA-MB-231 cells treated or not with 1 µM Cer.",
    "molecules": "Cer"
  },
  {
    "caption": "F. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231cell line treated or not with 30µM ZA for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.",
    "molecules": "ZA"
  },
  {
    "caption": "G. Viability assay of siGFP and sip53 -MDA-MB-231 cells after treatment with increasing amounts of cerivastatin (0, 0.01, 0.1, 1 and 10 µM) for 48 h. Data are normalized to untreated. Error bars represent mean + s.d., from three biological replicates.",
    "molecules": "cerivastatin"
  },
  {
    "caption": "(D and E) Growth comparisons of bir1∆-ad2 strains that contain (18 strains) or do not contain (28 strains) confirmed suppressor mutations as measured by area of colony growth after serial dilution. The mean (red line) is shown. 10-fold serial dilutions were made on YPAD plates containing either 0.1% DMSO (D) or 10µg/mL of benomyl (E).",
    "molecules": "benomyl, DMSO"
  },
  {
    "caption": "(B) The amount of mNeonGreen-Sli15 (CPC member) located at kinetochores (Nuf2-mCherry) was measured in prometaphase. Sgo1 was depleted by placing it under a galactose-inducible promoter and switching to glucose-containing media (YPAD). Representative images are on the left. Scale bar is 2µm. Averages from 3 independent experiments are shown.",
    "molecules": "galactose, glucose"
  },
  {
    "caption": "(a) Simultaneous loss of Lysotracker and AlPcS2a fluorescence immediately following light activation. A HeLa cell labelled with Lysotracker Red and AlPcS2a was 635-nm illuminated within the white square region (left panels). Lysotracker fluorescence within the illuminated region was immediately lost after light illumination (middle panels), indicating the successful triggering of LMP. Right panels: magnified view of Lysotracker fluorescence within the 635-nm illuminated region pre- and post light activation. (b) 635 nm illumination did not lead to complete lysosome rupture. A HeLa cell labelled with FITC-dextran (molecular weight=10,000) and AlPcS2a was 635-nm illuminated within the white square region (left panels). FITC-dextran remained within illuminated lysosomes (an increase in FITC fluorescence was observed, in accordance with an increase in pH in damaged lysosomes; middle panels). Right panels: magnified view of FITC-dextran fluorescence within the 635-nm illuminated region pre- and post light activation. (c) Light activation did not affect other cellular structures, such as mitochondria. A HeLa cell labelled with TMRE and AlPcS2a was 635-nm illuminated within the white square region (left panels). Illumination did not affect TMRE fluorescence, indicating that mitochondria within the same region were not affected/impaired. Right panels: magnified view of TMRE fluorescence within the 635-nm illuminated region pre- and post light activation. Scale bars, 10 μm.",
    "molecules": "dextran"
  },
  {
    "caption": "A) Representative P2YR12 immunostaining  (green) of microglia  of wild type male mice showing morphological  differences between control microglia  (a) and ischemic microglia  (b-d). Ischemic microglial cells show typical phagocytic pouches (arrows). Nuclei  are labeled with TO-PRO-3 (blue). Image (d) is a magnification of the area marked with a square in (c). Scale bar a, b: 20 μm; c: 10 μm; d: 4 μm.",
    "molecules": "TO-PRO-3"
  },
  {
    "caption": "D) Immunofluorescence  with antibodies against PLIN2  (green) and Iba-1 (red) in brain  tissue 1 day after induction of ischemia  in female mice. Nuclei  are stained  with DAPI  (blue). Control is the contralateral hemisphere. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "F) Flow cytometry  gates to identify Bodipy+ microglia  for control and ischemic brain  tissue. Gates were set based on fluorescence minus one (FMO) intensity values.",
    "molecules": "Bodipy"
  },
  {
    "caption": "G) Quantification of the percentage of CD11b+CD45low microglia  containing  lipid droplets as Bodipy+ microglia  using flow cytometry in male mice  deficient in Stat1  (Stat1-/-) (n=4) and corresponding Stat1+/+ mice  (n=7) shows ischemia-induced increases in Stat1+/+ mice  (** p=0.0086) but not in Stat1-/- mice  (p=0.927) (Two-way ANOVA and Šídák's multiple comparisons test). Graph shows values of the non-ischemic  (-) and ischemic  (+) brain  hemispheres of individual mice  and the mean±SD.",
    "molecules": "Bodipy"
  },
  {
    "caption": "D) Quantification of  flow cytometry  shows that microglia renewal  reduced the percentage of Bodipy+ microglia  in old mice  in sham (***p=0.0001) and ischemic  (*p=0.0057) conditions (one-way Anova and Holm-Šídák's multiple comparisons test). For illustrative purposes the values of young mice  are also shown (original  old  vs. young: ***p<0.001 in the sham group and **p=0.0026 in the ischemic  group). Values show individual mice  and bars show the mean±SD.",
    "molecules": "Bodipy"
  },
  {
    "caption": "E) Primary microglia  cultures were obtained using CD11b+ magnetic beads from old   male mice  with or without microglia renewal (n=2 per group). Cells were exposed to red fluorescent pHrodo E. coli bioparticles   and were stained  with green fluorescent Bodipy  and studied by  confocal microscopy. Black and white images illustrate raw intensity data of Bodipy staining. The lower images are corresponding merged channels showing Bodipy  in green, phagocytosed bioparticles   in red, and the DAPI+ nuclei  in blue. Repopulated microglia  show less  lipid droplets and the cytoplasm  is replenished with phagocytosed bioparticles. The square regions of the images indicate areas magnified at the side of each image. Scale bar = 20 µm.",
    "molecules": "Bodipy, DAPI"
  },
  {
    "caption": "SDS-PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated.",
    "molecules": "Lipid"
  },
  {
    "caption": "Western-blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross-linking product of ~80 kDa in presence of nanodisc-reconstituted SecYC282EG and SecYC283EG, presumably assigned to FtsQxSecY adduct. The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa-histidine tag by 3C protease.",
    "molecules": "hexa-histidine"
  },
  {
    "caption": "In-gel fluorescence imaging of SecYCF488A reveals a cross-linking product of ~80 kDa assigned to the FtsQC40xSecYC283 adduct (\"SecYxFtsQ\"). Bands for covalently cross-linked SecY dimer (\"SecYxSecY\"), and adducts of SecY and ribosomal proteins (\"SecYxRiboP\") are indicated. The nascent chain-specific adduct \"SecYxFtsQ\" does not depend on the presence of PE lipids.",
    "molecules": "lipids, PE"
  },
  {
    "caption": "G) Co-immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells. GFP-tagged Miro1 truncation constructs were pulled down with GFP-Trap agarose beads and Pex19 was probed with myc antibody.",
    "molecules": "agarose beads"
  },
  {
    "caption": "E) Representative blots of Miro1, Miro2 and actin from whole cell lysates of wild-type and Miro1-floxed ERT Cre-recombinase MEFs treated with and without 4-OH tamoxifen for 48 hours. Lysates were taken one day after the end of treatment.",
    "molecules": "4-OH tamoxifen"
  },
  {
    "caption": "A) Representative images of proximity-ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. B) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n=3 experiments). Data information: For B) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.",
    "molecules": "DAPI"
  },
  {
    "caption": "C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments). Data information: In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.",
    "molecules": "DAPI"
  },
  {
    "caption": "G) Zooms of endogenous Drp1, mitochondria (Mitotracker) and peroxisomes (PMP70) in WT and DKO MEFs. Scale bar is 5 μm. H) Integrated density of Drp1 signal on Mitotracker-positive and PMP70-negative structures in WT and DKO MEFs. I) Integrated density of Drp1 signal on PMP70-positive and Mitotracker-negative structures in WT and DKO MEFs. Data information G-I n=42 cells per condition over three independent experiments. For , H) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.",
    "molecules": "Mitotracker"
  },
  {
    "caption": "A) Western blot analysis of Mpl in healthy donors' (HD) platelets shows two distinct bands representing the mature glycoprotein (top band) and the ER‐core glycosylated form (bottom band). The ratio is ~ 1.",
    "molecules": "glycoprotein"
  },
  {
    "caption": "A) K562 cells labeled live for 30 min with an anti‐CD235a‐Atto655 (glycophorin A) antibody before treatment with the Duolink reagents.",
    "molecules": "Atto655"
  },
  {
    "caption": "C and D) HEL cells primed live for 30 min with an anti‐CD235a‐Atto655 antibody before treatment with the Duolink reagents.",
    "molecules": "Atto655"
  },
  {
    "caption": "A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl‐mKO2 cells after treatments for 60 min with Tpo, ionomycin (I), PMA, Mon or A23187.",
    "molecules": "A23187, ionomycin, Mon, PMA"
  },
  {
    "caption": "C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl‐mKO2 cells treated with Mon or A23187 for 15 h.",
    "molecules": "A23187, Mon"
  },
  {
    "caption": "D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng/mL for 30 min. The quantification of the blot is shown.",
    "molecules": "Bafilomycin A1"
  },
  {
    "caption": "E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3−MA (3−Methyladenine) or Rp.",
    "molecules": "3−MA, 3−Methyladenine, Rp"
  },
  {
    "caption": "A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C. trachomatis L2 (multiplicity of infection MOI=1) in the presence or not of BP. In the indicated samples CP4d was added 2 h before infection. Cell lysates were run on SDS-PAGE, proteins were transferred to a membrane and BP incorporation was revealed with HRP-conjugated streptavidin. BP incorporation is enhanced in infected samples, and is inhibited by CP4d. The two main bands present in all samples correspond to naturally biotinylated host proteins (Haneji & Koide, 1989). After blotting the membrane was stained with Coomassie blue to control for equal loading. B - Same as in (A), except that where indicated 250 μM doxycycline (doxy, left) or 7 μM cycloheximide (CHX, right) were added 24 h or 2 hpi, respectively.",
    "molecules": "Coomassie blue, CP4d, BP, CHX, cycloheximide, doxy, doxycycline, SDS"
  },
  {
    "caption": "C - Whole cell lysates were prepared with TG2+/+ and TG2-/- MEFs infected or not for 48 h with C. trachomatis L2 in the presence or not of BP, and analyzed as in (A).",
    "molecules": "BP"
  },
  {
    "caption": "E - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. Where indicated, 40 μM CP4d was added 2 hpi. tgm2 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least four times. P-values of Student's ratio-paired t-test <0.05 are shown.",
    "molecules": "CP4d"
  },
  {
    "caption": "A - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were disrupted and bacterial titers (IFU=inclusion forming unit) were determined by re-infecting fresh HeLa cells as described in the methods. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.",
    "molecules": "CP4d, DMSO"
  },
  {
    "caption": "D - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were fixed and analyzed by flow cytometry. The percentage of infected cells (left) and the mean fluorescence of the infected population (right) ± SD are shown for three independent experiments. P-values of Student's paired t-test are indicated when <0.05. A representative field for each condition is shown, scale bar = 10 μm.",
    "molecules": "CP4d, DMSO"
  },
  {
    "caption": "E - Primary epithelial cells isolated from fallopian tubes were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with C. trachomatis serovar L2 (left) or D (right). Twenty-four hours later the cells were fixed, bacteria were stained using FITC-labeled anti-Chlamydia-LPS antibodies, and the mean size of the inclusions manually determined using ImageJ, on twenty inclusions per experiment. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.",
    "molecules": "CP4d, DMSO, FITC, LPS"
  },
  {
    "caption": "A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria (MOI = 0.2). Cells were disrupted 30 h later and the bacterial titer determined by re-infecting fresh wild type cells. The mean ± SD of three independent experiments are shown.",
    "molecules": "glucose"
  },
  {
    "caption": "B - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 48 h. Where indicated 40 μM CP4d was added 2 hpi. GLUT-1 and GLUT-3 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated four times. P-values of Student's ratio-paired t-test are indicated when <0.05.",
    "molecules": "CP4d"
  },
  {
    "caption": "A - HeLa cells were infected with C. trachomatis (MOI = 1) and 40 µM CP4d was added or not 2 hpi. After 24 h 0.5 mM BP was added and cells were lysed at 48 hpi. Lysates were precipitated with streptavidin-coated beads. After separation with SDS-PAGE, proteins were transferred to a membrane and blotted with anti-GFPT antibody followed with HRP-conjugated secondary antibody.",
    "molecules": "CP4d, BP, SDS"
  },
  {
    "caption": "B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP. Samples were incubated for 3 h at 37°C before separation by SDS-PAGE. Proteins were transferred to a membrane and BP was revealed using HRP-conjugated streptavidin. rhGFPT1 is 77.5 kDa.",
    "molecules": "BP, SDS"
  },
  {
    "caption": "E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 °C for 45 min with fructose-6-P and glutamine. The production of glucosamine-6-P was measured using HPAEC-PAD. Results of three independent experiments are shown, with mean ± SD, and p-value of the Student's paired t-test is indicated (*P < 0.05).",
    "molecules": "fructose-6-P, glucosamine-6-P, glutamine, ionomycin"
  },
  {
    "caption": "B - Endocervical epithelial cells were pre-treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. Last the membrane was probed with anti-actin as a loading control.",
    "molecules": "CP4d, BP, DMSO, ionomycin"
  },
  {
    "caption": "C - The same experimental procedure as described in (B) was applied to HeLa cells treated for 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2.",
    "molecules": "ionomycin"
  },
  {
    "caption": "A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies.",
    "molecules": "SDS"
  },
  {
    "caption": "B - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A).",
    "molecules": "BP, DMSO, ionomycin"
  },
  {
    "caption": "C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies.",
    "molecules": "SDS"
  },
  {
    "caption": "(A) A3A co-precipitates  with CCT complex subunits. U2OS  and HepaRG cells with  dox-inducible A3A-HA  transgenes were treated with  dox prior to immunoprecipitation of HA. HA immunoprecipitates were analyzed by immunoblot with antibodies for  HA  and subunits of the CCT complex.",
    "molecules": "dox"
  },
  {
    "caption": "(B) Decreased cell viability  upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS  cells with dox-inducible  A3A transgenes. Immunoblot  analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in  A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A  cells were treated with  dox. Viability  was determined by  colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability  was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM.",
    "molecules": "dox, tetrazolium"
  },
  {
    "caption": "(D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4  by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS  results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control.",
    "molecules": "dox"
  },
  {
    "caption": "(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A  cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular  staining with anti-γH2AX  antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot  analysis of cell lysates showed decreased expression of CCT7  after siRNA targeting and A3A expression upon dox  treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.",
    "molecules": "dox"
  },
  {
    "caption": "(A) LC-MS/MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA. Signal intensities of the modifications are scaled to that for m1A, the ribonucleoside with the highest MS signal",
    "molecules": "tRNA, ribonucleoside, ribonucleosides"
  },
  {
    "caption": "(A) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC-MS/MS in total tRNA extracted from parasites across the IDC time course. The average fold-change values (relative to an arbitrary average control) were subjected to hierarchical clustering analysis (log2-transformed data). The scale of the heat map has saturated the fold-change values of some modifications. Refer to Appendix Table S6. (B) Three groups of modifications show differential regulation during the course of IDC. The majority (22) increase synchronously across the IDC (blue), while 4 show asynchronous or irregular behavior (purple) and 2 (orange) are uniquely up-regulated in the ring stage. Log2Fold-change values plotted in the three graphs represent mean ± SEM (N = 3). Differences between the highest value (denoted as peak) and the lowest value (denoted as trough) were subjected to a two-tailed Student's t-test: NS, not significant",
    "molecules": "tRNA, ribonucleosides"
  },
  {
    "caption": "LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of the unmodified (3055.425 Da) and modified (3143.43 Da) oligos for Glu-tRNAGAA (B",
    "molecules": "tRNA, Glu-tRNA"
  },
  {
    "caption": "Collision-induced dissociation of the parent molecular ions for m/z 1046.477-3 from Glu-tRNAGA produced expected -c, -w and -y ions for the sequences CU[mcm5s2U]UCACCC",
    "molecules": "Glu-tRNA"
  },
  {
    "caption": "LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of th unmodified (1835.20 Da) and modified (1923.262 Da) oligos for Gln-tRNACAA (E)",
    "molecules": "tRNA, Gln-tRNA"
  },
  {
    "caption": "Collision-induced dissociation of the parent molecular ions fo m/z 1923.262 from Gln-tRNACAA produced expected -c, -w and -y ions for the sequence CC[mcm5s2U]CCAA",
    "molecules": "Gln-tRNA"
  },
  {
    "caption": "Ratios of modified oligonucleotides from the anticodon stem-loop o (H)GluGA isoacceptors expressed as percentages of their sum total",
    "molecules": "GluGA"
  },
  {
    "caption": "Ratios of modified oligonucleotides from the anticodon stem-loop o (G)GlnCA",
    "molecules": "GlnCA"
  },
  {
    "caption": "Ratios of modified oligonucleotides from the anticodon stem-loop o (I)GlyGGA isoacceptors expressed as percentages of their sum total",
    "molecules": "GlyGGA"
  },
  {
    "caption": "A, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites. It is typically not described whether the metabolites that were not reported (white \"empty\" space) were not measured, or measured but not reported. Labelings: X-axis (top): ribbon colour code, denoting the cancer type (right; indicated by red arrow); X-axis (bottom): cohorts, arranged from 1 to 71; Y-axis (right): all 1,206 metabolites reported in at least one of the studies; Y-axis (left): ribbon colour code, denoting the metabolite class (amino acids, carbohydrates, etc; \"other\" refers to all other metabolites than the listed classes).",
    "molecules": "amino acids, carbohydrates, metabolites"
  },
  {
    "caption": "B, Vote counting of cancer blood metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrowheads denote metabolites mentioned in the main text.",
    "molecules": "metabolites"
  },
  {
    "caption": "C,D, Volcano plot of bloodmetabolites (C) and tissuemetabolites (D), reported in at least six cancer studies, with the vote counting score on the X-axis and the -log10 adjusted p-value on the Y-axis. Cyan indicates deregulated metabolites that show a trend (p < 0.1; for blood, corresponding to metabolites marked with * in panel 2B; for tissue, corresponding to metabolites marked with * in Figure 3) or statistical significance (p < 0.05; above black dashed horizontal line); red indicates metabolites with a p-value > 0.1. A subset of metabolites is annotated (see Table EV4 (blood) and EV5 (tissue) for full annotation).",
    "molecules": "metabolites"
  },
  {
    "caption": "Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).",
    "molecules": "metabolites"
  },
  {
    "caption": "A. Vps10yEGFP localization. Yeast cells carrying Vps10yEGFP and expressing the indicated vps35 alleles as the sole source of Vps35 were logarithmically grown in SC medium. Their vacuoles were labelled with FM4-64. Cells were harvested by brief centrifugation and immediately imaged by confocal microscopy. Single confocal planes are shown. A brightfield image was used to outline the cell boundaries (shown in the merged images). B. Co-localization of Vps10yEGFP and FM4-64 in cells from a was measured using Pearson's coefficient. 15 Confocal planes with 20 to 30 cells each from 3 independent experiments were analyzed. Means",
    "molecules": "FM4-64"
  },
  {
    "caption": "(E) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test. ",
    "molecules": "TTX"
  },
  {
    "caption": "(D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.",
    "molecules": "pSIVA"
  },
  {
    "caption": "(F) PSIVA staining in the dLGN. Exposed PS was detected by pSIVA on RGC inputs in Cdc50a-RNAi mice. Scale bar, 5 µm. Arrows indicate pSIVA+/mCherry+ RGC inputs. (G) Quantification of the percentage of pSIVA+/mCherry+ RGC inputs in total mCherry+ RGC inputs. N = 4 for biological replicates, mean ± SEM, **p < 0.01 by Student's t-test. ",
    "molecules": "PSIVA, pSIVA, PS"
  },
  {
    "caption": "E and F, Immunohistochemical staining  of ARG1  and ASS1  in fetal lungs  of WT and Src-1/-2 dKO mice  at 18.5 dpc. PBS  was used instead of the primary antibody as a negative control (NC). Scale bars: 50 μm in E and F.",
    "molecules": "PBS"
  },
  {
    "caption": "E and F, Immunohistochemical staining  of ARG1+ (E) or ASS1+ (F) cells in the fetal lungs  of WT mice  at 15.5 dpc, 17.5 dpc and labor. PBS  was used instead of the primary antibody in the NC group. Scale bars: 100 μm in E and F.",
    "molecules": "PBS"
  },
  {
    "caption": "D and E, Histograms showing the content of arginine, ornithine, ADMA  and the ratio of arginine  to ADMA  in the fetal lungs  of WT mice  and Src-1/-2 dKO mice  at 18.5 dpc. Mann Whitney test was used to analyze the data in panel D, and unpaired t test was used to analyze the data in panel E. n=5 biological replicates for each group, and the data are shown as the mean ± s.e.m. The central band indicates the median, the boxes indicate the upper quartile and lower quartile, and the whiskers indicate maximum value and minimum value of the data. *P<0.05, **P<0.01, compared with the WT fetal lungs.",
    "molecules": "arginine, ADMA, ornithine"
  },
  {
    "caption": "E, Multiple immunofluorescence staining  and densitometric  quantitation of TUNEL  with PDPN  antibody (type I alveolar epithelial cells  marker) on sections of fetal lungs  from AAV-shArg1-injected mice  vs. AAV-2/9-injected mice. Blue: DAPI  (nuclei); Green: PDPN  (type I cells); Red: TUNEL  positive. Unpaired t test was used to analyze the quantitation data and n=3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group. The data are shown as the mean ± s.e.m. ***P<0.001 compared with the AAV-2/9-injected group. F, Multiple immunofluorescence staining  and densitometric  quantitation of TUNEL  with LPCAT1  antibody (type II alveolar epithelial cells  marker) on sections of fetal lungs  from AAV-shArg1-injected mice  vs. AAV-2/9-injected mice. Blue: DAPI  (nuclei); Green: LPCAT1  (type II cells); Red: TUNEL  positive. Unpaired t test was used to analyze the quantitation data and n=3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group. The data are shown as the mean ± s.e.m. ***P<0.001 compared with the AAV-2/9-injected group. Data information: Scale bars in E and F: 50 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, Quantitative analysis of the gel area containing hTERT-HM cells treated with L-arginine  or vehicle control. Mann Whitney test was used to analyze the data, and n=9 biological replicates in control group, n=12 biological replicates in  L-Arginine-treated group. The data are shown as the mean ± s.e.m. **P < 0.01. Representative images of spontaneous contractions of the gel disks are shown.",
    "molecules": "L-arginine, L-Arginine"
  },
  {
    "caption": "p53 ChIPseq in MEFs of the indicated genotype treated with or without 10 µM nutlin-3a (Nutlin) for 16 hours. Shown are 2 kb regions surrounding the summit of the 468 p53 binding peaks called in Nutlin-treated p53+/+, but not p53-/- or p53EE/EE MEFs. For p53EE/EE MEFs three independent replicates are shown.",
    "molecules": "Nutlin, nutlin-3a"
  },
  {
    "caption": "RNAseq was performed with MEFs of the indicated genotype untreated or treated with 10 µM Nutlin for 16 hours. Shown are all Nutlin-regulated genes from the MSigDB gene set Hallmarks_P53_Pathway with a mean log2FC≥1 in p53+/+ cells. Shown are the z-transformed RNA expression values (FPKM).",
    "molecules": "Nutlin"
  },
  {
    "caption": "RNAseq data were subjected to gene set enrichment analysis (GSEA). Shown are enrichment plots for the indicated set of p53 downstream genes in pairwise comparisons of Nutlin-treated MEFs with the indicated p53 genotypes.",
    "molecules": "Nutlin"
  },
  {
    "caption": "Reverse transcription quantitative PCR (RTqPCR) analysis of p53 target genes in MEFs treated for 24 hours with 1 µg/ml doxorubicin (Doxo). Shown are expression values normalized to β-actin as mean ± SD (n=6).",
    "molecules": "Doxo, doxorubicin"
  },
  {
    "caption": "Western blots of protein lysates prepared from MEFs treated for 24 hours with (G) 10 µM Nutlin",
    "molecules": "Nutlin"
  },
  {
    "caption": "Western blots of protein lysates prepared from MEFs treated for 24 hours with 0.4 µg/ml Doxo.",
    "molecules": "Doxo"
  },
  {
    "caption": "Proliferation of primary MEFs. Cells were treated o/n with 0.2 µg/ml doxorubicin (Doxo) and pulse-labeled with 32 µM 5-Bromo-2-deoxyuridine (BrdU), fixed and processed for flow cytometry analysis. n=4.",
    "molecules": "5-Bromo-2-deoxyuridine, BrdU, Doxo, doxorubicin"
  },
  {
    "caption": "MEFs were immortalized with the adenoviral oncogene E1A.12S (E1A MEF) and treated with 0.4 µg/ml Doxo for 17 hours. Apoptosis (Annexin V) was quantified by flow cytometry. +/+ and -/-: n=3; EE/EE: n=6. Western blots show expression of E1A and β-actin as loading control.",
    "molecules": "Doxo"
  },
  {
    "caption": "Primary thymocytes were irradiated ex vivo with 6 Gy or treated with 1 µM dexamethasone as a control for p53-independent apoptosis. Cell survival relative to untreated samples was analyzed using CellTiter Glow assay (Promega). n=11 for each time point and genotype.",
    "molecules": "dexamethasone"
  },
  {
    "caption": "Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL)",
    "molecules": "BrdU"
  },
  {
    "caption": "Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for proliferation (BrdU); scale bars 50 µm.",
    "molecules": "BrdU"
  },
  {
    "caption": "Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for (F) apoptosis (TUNEL) Quantification for n=3 mice/genotype (150 crypts/mouse).",
    "molecules": "BrdU"
  },
  {
    "caption": "Mice of indicated genotype were subjected to 6 Gy whole-body irradiation and pulse-labeled with 120 mg/kg BrdU two hours before sacrifice at different time points. Small intestines were stained for proliferation (BrdU); scale bars 50 µm. Quantification for n=3 mice/genotype (150 crypts/mouse).",
    "molecules": "BrdU"
  },
  {
    "caption": "Mdm2-/-Trp53-/- (double knock-out, DKO) MEFs were transduced with pInducer20-p53EE lentivirus to enable tetracyclin (Tet)-inducible expression of mouse p53EE. Induction of p53EE sensitized cells to apoptosis induced by 24 hour treatment with 1 µg/ml doxorubicin (Doxo) as detected by flow cytometry for Annexin V.",
    "molecules": "Doxo, doxorubicin, Tet, tetracyclin"
  },
  {
    "caption": "mRNA expression of p53 target genes was measured in cells from (A) relative to β-actin by RTqPCR following treatment with Tet ± 1 µg/ml Doxo. Expression of the pro-apoptotic p53 target genes Bbc3 (Puma) and Bax is not significantly induced. n=6.",
    "molecules": "Doxo, Tet"
  },
  {
    "caption": "Proliferation of MEFs with indicated genotypes was analyzed by live-cell imaging in the presence of 10 µM nutlin-3a (Nutlin) and/or 0.05 µg/ml doxorubicin (Doxo). Shown is the median confluence ±SEM (n=12).",
    "molecules": "Doxo, doxorubicin, Nutlin, nutlin-3a"
  },
  {
    "caption": "Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0.4 µg/ml Doxo. n=3.",
    "molecules": "Doxo"
  },
  {
    "caption": "Cell viability assays for MEFs with indicated genotypes treated with Mdm2 inhibitors ± 0.4 µg/ml Doxo. n=3.",
    "molecules": "Doxo"
  },
  {
    "caption": "Western blot of E1A-MEFs with indicated genotypes treated with 10 µM Nutlin ± 0.4 µg/ml Doxo for 18 hours reveals induction of apoptosis (cCasp-3, cleaved caspase-3; cParp, cleaved Parp) in double-treated p53EE/EE MEFs in the absence of p53 target gene (p21, Mdm2) activation. *, 10 hours 0.4 µg/ml Doxo; **, 10 hours 0.2 µg/ml Doxo.",
    "molecules": "Doxo, Nutlin"
  },
  {
    "caption": "mRNA expression of p53 target genes was measured in E1A MEFs of indicated genotype relative to β-actin by RTqPCR following combined treatment with 10 µM Nutlin and 1 µg/ml Doxo. n=12.",
    "molecules": "Doxo, Nutlin"
  },
  {
    "caption": "Western blot of p53EE/EE E1A-MEFs with indicated genotypes treated for 18 hours with 10 µM Nutlin, 0.4 µg/ml Doxo and 30-100-500 nM Ganetespib as indicated. Comb, combined treatment with 3 drugs for 18 hours. Pre, pre-treatment with ganetespib for 24 hours followed by combined treatment with Nutlin and Doxorubicin for 18 hours.",
    "molecules": "Doxo, Doxorubicin, ganetespib, Ganetespib, Nutlin"
  },
  {
    "caption": "Western blot of cellular fractions from unstressed and Doxo-treated MEFs of indicated genotype identifies p53EE protein in the mitochondrial fraction. PCNA, Tom20/Bak are shown as nuclear and mitochondrial marker proteins.",
    "molecules": "Doxo"
  },
  {
    "caption": "Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo.",
    "molecules": "Doxo, propidium iodide, tet"
  },
  {
    "caption": "Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo. This effect is enhanced by addition of Nutlin and blocked by the pan-caspase inhibitor QVDOph, but not the ferroptosis inhibitor Ferrostatin-1 (Ferr) as control. (A) n=3; (B) n=2.",
    "molecules": "Doxo, Ferrostatin-1, Nutlin, propidium iodide, QVDOph, Ferr, tet"
  },
  {
    "caption": "mRNA expression analysis (RTqPCR) of p53 target genes following treatment with 0.5 µg/ml Doxo ± 10 µM Nutlin. KO, tet-induced H1299-pInd cells; EE, tet-induced H1299-pInd-p53EE cells; WT, H460 cells. mRNA expression was normalized to β-actin; n=3.",
    "molecules": "Doxo, Nutlin, tet"
  },
  {
    "caption": "Proximity ligation assay for p53 with Tom20 in indicated untreated (Mock) or Doxo-treated (0.1 µg/ml, 24 hours) H1299 cells following tet-induction. Shown are representative cells with PLA signals (red) counterstained with DAPI (blue). Quantification of proximity ligation assays for p53EE with Tom20, Bcl-2, Bcl-xL and Bak in H1299 cells. Doxo treatment (0.1 µg/ml, 24 hours) as indicated. Plotted is the average number of PLA signals per cell for 10 fields of view.",
    "molecules": "DAPI, Doxo, tet"
  },
  {
    "caption": "Mitochondrial membrane potential of control and p53EE expressing H1299 cells in the absence and presence of increasing concentrations of BID BH3 peptide. Treatment with the mitochondrial depolarizer CCCP is shown as a positive control; n=3.",
    "molecules": "CCCP"
  },
  {
    "caption": "Apoptosis measured by flow cytometry using Annexin V in H1299 cells with tet-inducible expression of the indicated human p53 mutants; n=3.",
    "molecules": "tet"
  },
  {
    "caption": "Apoptosis in Eµ-Myc lymphoma cells of indicated genotype was measured by flow cytometry (FITC-VAD-FMK staining) at indicated time points after treatment with 3 µg/ml MAF. Significance was calculated versus untreated. n≥5.",
    "molecules": "FITC-VAD-FMK, MAF"
  },
  {
    "caption": "Enrichment of p53 shRNA-expressing Eµ-Myc;p53EE lymphoma cells under MAF treatment. Non-silencing control shRNA served as control. n=3.",
    "molecules": "MAF"
  },
  {
    "caption": "mRNA expression of p53 target genes was measured relative to β-actin by RTqPCR following treatment of Eµ-Myc lymphoma cells of indicated genotype with 3 µg/ml MAF. n≥3.",
    "molecules": "MAF"
  },
  {
    "caption": "Kaplan-Meier survival plots for cyclophosphamide-treated versus untreated control mice transplanted with Eµ-Myc lymphoma cells of indicated genotype at day 0. Treatment started ~10 days later when peripheral lymph nodes became palpable. The time course of the experiment is illustrated in a scheme.",
    "molecules": "cyclophosphamide"
  },
  {
    "caption": "Representative bioluminescence imaging (BLI) pictures of mice transplanted with AE9+Nras AML cells. Day 0 indicates the start of combination therapy with cytarabine (AraC) + doxorubicin (Doxo).",
    "molecules": "AraC, cytarabine, Doxo, doxorubicin"
  },
  {
    "caption": "Kaplan-Meier survival plots for animals from (G). Statistical significance of differences calculated by Log-rank (Mantel-Cox) test between treated Eµ-Myc;p53EE/EE AML and all other groups is indicated in the table. C, untreated control; T, therapy (AraC+Doxo).",
    "molecules": "AraC, Doxo"
  },
  {
    "caption": "Antibacterial competition assay. E. coli K12 recipient cells (W3110 gfp+, kanR) were mixed with the indicated EAEC attacker cells (1:4 ratio): WT, ∆T6SS-1 and ∆tle1-tli1 carrying pBAD18 and pBAD33 vectors, or producing the indicated proteins, and spotted on SIM 0.02 % arabinose agar plates for 4-h at 37°C. The image of a representative bacterial spot is shown. The number of recovered prey cells is indicated in the upper graph (in log of colony-forming units (cfu)). The black, dark grey and light grey circles indicate values obtained from three different spots, and the average is indicated by the bar. Western blot analysis of the production of Tle1V and Tle1∆1-26 V is shown in the inset. The experiment was performed in triplicate and a representative result is shown.",
    "molecules": "kan"
  },
  {
    "caption": "(C) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A (ant.A).",
    "molecules": "ant.A, antimycin A, ATP"
  },
  {
    "caption": "(A) Timelapse of ATP sensor FRET efficiency after combined addition of 10 μM antimycin A (ant.A), 100 μM 3-Bromopyruvate (3BP), and 50 mM 2-deoxy-D-glucose (2DG). (B) Mean FRET efficiency of organizer (ORG) regions and non-organizer (NO) regions analyzed over time. Shaded regions indicate SD; dots indicate mean per timepoint, and solid lines indicate the fit to the mean. (C) Fit parameters of individual time traces for organizer and non-organizer regions. Each dot represents data from one disc, and lines connect the corresponding regions of the same disc. Box plots summarize the data: boxes encompass the 2nd-3rd quartiles, with whiskers indicating the 1st and 4th quartiles and the red line indicating the median. * = p-value < 0.05, ns = not significant p-value using a Kruskal-Wallis test (n=26 discs).",
    "molecules": "2-deoxy-D-glucose, 2DG, 3-Bromopyruvate, 3BP, ant.A, antimycin A, ATP"
  },
  {
    "caption": "(D) Time lapse montage of ATP sensor FRET efficiency in the wing pouch after 2DG addition. (E) Mean FRET efficiency time trace of the entire wing pouch; shaded region indicates SD; black arrows indicate the addition of the drugs. Brackets indicate the mean FRET values before and 2h after 2DG addition (1-10 min and 121-130 min, respectively). The distribution of mean values within these brackets are shown as violin plots in (F) (solid line indicates median, dotted indicate quartiles). These were compared using an unpaired t-test (ns = not significant p-value, n = 17 discs) including (G) Welch's correction and estimation plot (n=17 discs for each group). In (G), horizontal dotted lines run through the mean values for the two samples (before 2DG and 2hr after 2DG); on the right, the difference between the two means is plotted on a separate axis (right), where the bars indicate the upper and lower 95% confidence intervals.",
    "molecules": "2DG, ATP"
  },
  {
    "caption": "(C) Timelapse of ATP sensor FRET efficiency in the apGalts>PtcRNAi wing pouch after 10 μM antimycin A (ant.A) addition. Red dashed line indicates the position of the AP boundary.",
    "molecules": "ant.A, antimycin A, ATP"
  },
  {
    "caption": "(I) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition.",
    "molecules": "antimycin A, ATP"
  },
  {
    "caption": "(A) Timelapse montage of ATP sensor FRET efficiency upon induction of hypoxia in wing disc explants. To highlight the variability in this experiment, we show example discs from two separate experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "(B) CRMP2 expression in adult cortex (layers VI - IV), corpus callosum (CC) and hippocampal area CA1. CRMP2 (green) is expressed throughout the cortex and hippocampus, CRMP2A (red) is particularly strongly expressed in a subset of callosal axons. Nuclei are counterstained with Hoechst 33342. Scale bars: 50 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(G) DiI-labeled callosal axons from P9 oblique brain sections and their reconstruction in Neurolucida 360 (WT n=6, crmp2-/- n=9). Scale bars: 200 μm.",
    "molecules": "DiI"
  },
  {
    "caption": "(A, B) (A) Coronal sections of P14 brains stained for calbindin and VgluT1. Infrapyramidal bundle (IPB) progresses into hippocampal CA3 region in both WT and crmp2-/- mice. Nuclei are counterstained with Hoechst 33342. (B) Details from depicted regions in (A), arrowheads show the course of IPB. Scale bars: 100 μm.",
    "molecules": "Hoechst 33342"
  },
  {
    "caption": "(A) Upper row: DiI tracing of the visual cortex axons at P9 (before pruning), sagittal sections. Branching point of the tract is shown. Lower row: visual cortex axons in adult mice after pruning period. Note significantly reduced number of axons continuing into pyramidal tract in WT (dashed arrow). (arrows - 2 branches of corticospinal visual axons). Maximum projections are shown. Scale bars: 200 μm.",
    "molecules": "DiI"
  },
  {
    "caption": "A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice transfected with poly(I:C) ( 1 μg/ml) (C).",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "B. Immunoblot assay of RKIP+/+, RKIP+/- and RKIP-/- peritoneal macrophage cells treated with intracellular poly(I:C) (1 μg/ml).",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "E. Immunoblot analysis in lysates of RAW264.7 cells stably overexpressing RKIP treated with intracellular poly(I:C) (1 μg/ml).",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.",
    "molecules": "ATP"
  },
  {
    "caption": "D. Recombinant flag-tagged TBK1 was immunoprecipitated with anti-flag M2 beads in flag-TBK1 transfected 293T cells. Followed by in vitro kinase assay, which was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP together with 5 μM flag-TBK1 at 30 °C for 30 min. Assay mixtures were immunoblotted with the indicated antibody for phospho-RKIP, flag, GST or his.",
    "molecules": "ATP"
  },
  {
    "caption": "J. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST-TBK1 together with 100 μM GST-his-RKIP, 100 μM GST-his-S109D, 100 μM GST-his-S109A or 100 μM GST, respectively. React at 30 °C for 30 min. Then followed by immunoblotting with anti-p-TBK1or anti-GST antibody.",
    "molecules": "ATP"
  },
  {
    "caption": "C. Coomassie staining of SDS-PAGE-separated pulldown samples with/without cell extracts (C.E) using GST-tagged FET-NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E -). Several high-molecular weight proteins are retained by the FET-NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry",
    "molecules": "Sepharose"
  },
  {
    "caption": "E. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3 and C-terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS-DDIT3/BRG1 and FUS-DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645-94 and 402-91. C-terminal parts of FUS are not present in the FUS-DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": " B. Immunoblot analysis (IB) of GST-pulldown samples with deletion mutants of FUS‑NTD‑GST as baits with/without cell extract (C.E). ARID1A and BRG1 were used as tracers of SWI/SNF binding. Coomassie staining of recombinant proteins are shown in the lower panel. Amino acid numbers are indicated ",
    "molecules": "Amino acid"
  },
  {
    "caption": " D. Immunoblot analysis (IB) of SWI/SNF components (ARID1A, BRG1, BAF170, BAF155, BAF60A, SS18 and BAF47) and full-length FET proteins (EWSR1 and FUS) remaining on sepharose-bound FUS-NTD‑GST pulldown baits after increasing stringency washes with 100, 150, 250, 500 or 1000 mM NaCl. Note loss of BRG1 at NaCl concentration > 250mM ",
    "molecules": "sepharose, NaCl"
  },
  {
    "caption": "A. Immunoblot analysis (IB) of histone modifications in stably transfected HT1080 cell lines expressing EGFP, FUS‑DDIT3‑EGFP or EWSR1‑FLI1‑EGFP. Antibodies against H3K27Ac, H3K27me3, H3K4me3 and histone loading control H4 for detection of histone modifications and antibodies against EZH2 and loading control GAPDH to evaluate catalytic PRC2 amount. Immunoblot analysis with GFP antibody is shown to verify expression of FET fusion oncoproteins and EGFP. One representative immunoblot is shown. Graphs showing amount of protein (H3K27me3, H3K4me3, ratio H3K27me3/H3K4me3, H3K27Ac, and EZH2) quantified from immunoblots relative each corresponding loading control H4 or GAPDH, normalized to parental HT1080. Mean ± SEM is shown with individual replicates indicated by circles from two experiments with stable transfection and by squares from two experiments with transient transfection (24h and 48h , n=4. Student's t-test, ns= not significant, *p<0.05, **p<0.01, ***p<0.001. Original data for all quantifications, including p-values, are shown as source data.",
    "molecules": "H3K27Ac, H3K27me3, H3K4me3"
  },
  {
    "caption": "Quantification of Nanog-GFP positive cells after 10 days of AA+2i treatment of WT or Kdm3b-deleted pre-iPSCs.",
    "molecules": "2i, AA"
  },
  {
    "caption": "Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b-KO pre-iPSCs.",
    "molecules": "UNC0638"
  },
  {
    "caption": "Quantification of percent Nanog-GFP positive cells after 10 days of AA+2i or AA+2i with 5µM UNC0638 in WT or Kdm3b deleted pre-iPSCs.",
    "molecules": "2i, AA, UNC0638"
  },
  {
    "caption": "Pearson correlation of transcripts per million (TPM) of WT and Kdm3b-KO pre-iPSC upon DMSO or AA+2i treatment for 2 or 10 days and mESC, reprogramming populations from Chronis et. al (2017), (labelled as CC).",
    "molecules": "2i, AA, DMSO"
  },
  {
    "caption": "k-means clustering of the union of DEG (FDR > 0.99) between day 2 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 2 over DMSO. Key pluripotency genes are shown on the right. k-means clustering of the union of DEG (FDR > 0.99) between day 10 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 10 over DMSO. Key pluripotency genes or epigenetic regulators are shown on the right.",
    "molecules": "DMSO"
  },
  {
    "caption": "Expression of Tet enzymes and Tdg during AA+2i treatment in WT or Kdm3b-KO pre-iPSCs. Error bars represent the standard deviation of two biological replicates from the RNA-Seq sample.",
    "molecules": "2i, AA"
  },
  {
    "caption": "Representative 5hmC profiles during AA+2i conversion of WT pre-iPSCs. Illustration of the 5hmC pattern is shown in the bottom panel.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "Heatmap of k-means clustered 5hmC patterns with histone modifications in pre-iPSCs and mESCs from Chronis et al (2017).",
    "molecules": "5hmC"
  },
  {
    "caption": "Representative profiles of regions that undergo demethylation through a 5hmC intermediate. Top - 5hmC profile during AA+2i treatment. Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs, data from Rais et al 2013. Bottom - Percent 5hmC via Tet-assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days, data from Hu et al 2014.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "Left - Quantification of percent Nanog-GFP positive colonies. Right - Total Nanog-GFP positive colonies obtained after ten days of AA+2i treatment in Tdg-depleted pre-iPSC (Tdg) or control (Empty).",
    "molecules": "2i, AA"
  },
  {
    "caption": "Quantification of percent Nanog-GFP positive cells of WT or Kdm3b-KO pre-IPSC clones expressing Nanog, Tet1CD, or Nanog+Tet1CD after 15 days of AA+2i or AA+2i with 4 or 5µM of UNC0638.",
    "molecules": "2i, AA, UNC0638"
  },
  {
    "caption": "Left - Heatmap of K-mean clustered 5hmC patterns in WT pre-iPSC with 5hmC profile of Kdm3b-KO pre-iPSC treated with AA+2i for 0, 2 or 10 days. Middle - Metaplot of 5hmC signal on day 10 between WT (orange) and Kdm3b-KO (black) for each cluster. Y-axis scale is in normalized coverage data (1x106/Total Count). Right - Illustration of 5hmC pattern in WT and Kdm3b-KO during AA+2i treatment.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "Scheme for identifying genes with both misregulated 5hmC and expression. Heatmap of two-fold misregulated 5hmC and two-fold differential expression between WT and Kdm3b-KO after 10 days of AA+2i treatment (607 genes). Peaks are sorted by 5hmC signal in WT. RNA seq values represent a log2-fold change of day 10 over DMSO for either WT or Kdm3b KO pre-iPSCs.",
    "molecules": "2i, 5hmC, AA, DMSO"
  },
  {
    "caption": "Box plot of expression for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10 (607 genes).",
    "molecules": "5hmC"
  },
  {
    "caption": "Heatmap of POU5F1 bound peaks in both mESC and pre-IPSC (Cluster 1) and mESC alone (Cluster 2) with 5hmC levels during Day 0, 2 and 10 of AA+2i treatment.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "Metaplots of 5hmC Enrichment for Cluster 1 (Left) and Cluster 2 (Right) regions for Day 0, 2 and 10 of AA+2i treatment.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding. Right - Heatmap of 5hmC profile during AA+2i treatment of WT pre-iPSC and POU5F1 binding in pre-iPSCs or mESCs at the loci identified in the left panel.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC with POU5F1 binding in pre-iPSCs and mESCs. Loci were assigned to the nearest gene.",
    "molecules": "2i, 5hmC, AA"
  },
  {
    "caption": "ChIP-PCR for POU5F1 during AA+2i reprogramming of WT and Kdm3b-KO pre-iPSCs. Values are fold change of enrichment between WT and Kdm3b KO (WT/Kdm3bKO).",
    "molecules": "2i, AA"
  },
  {
    "caption": "Quantification of percent Nanog positive cells by intracellular flow cytometry in each genotype as indicated of Tet1WT/WT Tet2fl/fl, Tet1WT/WT Tet2KO/KO, Tet1KO/KO Tet2fl/fl, and Tet1KO/KO Tet2KO/KO after 10 days of AA+2i treatment.",
    "molecules": "2i, AA"
  },
  {
    "caption": "Immunofluorescence staining of Oct4 and hnRNPLL in WT and hnRNPLL-KO ES cells from two clones (H8 and G6). hnRNPLL was not expressed in either of the hnRNPLL-KO clones. DAPI stained the nuclei. Scale bar, 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Hematoxylin and eosin labeling and Oct4 immunohistochemical staining of teratomas derived from WT and hnRNPLL-KO ES cells (left). Representative Oct4-positive regions are shown. The percentage of Oct4 positive region (%) is plotted (right). The data represent the means ± SD, (n=3, from biological repeats). *P＜0.05; ***P＜0.001, T-test was used to determine the significance. Scale bar, 100 μm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "Silver nitrate-stained NuPAGE gel of hnRNPLL interacting proteins. Red star indicate hnRNPLL specific interacting proteins compared to IgG.",
    "molecules": "Silver nitrate"
  },
  {
    "caption": "Hematoxylin and eosin labeling of teratomas derived from WT and H8-Bptf or H8-Tbx3 exon-KO ES cells. The blue triangle represent ectoderm, the dark one represent mesoderm and the green one represent endoderm. Scale bar, 100 μm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "(A) Schematic drawing of the life cycle stages of Paramecium tetraurelia. MIC and parental MAC are represented in red, representing the DAPI signal, and developing MAC (dm) is represented in green until fully developed, representing the GFP signal. (B) ISWI1-tagged C-terminally with GFP localizes in the developing MAC as soon as developing new MACs (panel Early Development) become visible and remain there throughout late MAC development (panel Late Development). Red: DAPI, Green: ISWI1-GFP. Blue arrows identify developing MAC;",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Western blot analysis using anti-HA antibody after coimmunoprecipitation of ISWI1-3XFlagHA fusion protein. Non-transformed cells (WT) of the same strain were used as the negative control. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 12% SDS gel.",
    "molecules": "SDS"
  },
  {
    "caption": "(E) Western blot analysis using anti-HA and anti-GFP antibodies after coimmunoprecipitation of Ptiwi01-3XFlagHA fusion protein co-transformed with ISWI1-GFP. Non-transformed cells (WT) of the same strain and ISWI1-GFP fusion protein transformation were used as negative controls. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 10%SDS gel.",
    "molecules": "SDS"
  },
  {
    "caption": "(B) HeLa cells were transfected with siRNA duplexes targeting PELP1, TEX10, WDR18, MDN1 or a control siRNA as indicated. Downregulation of the respective proteins was analysed by western blotting as indicated (WB, three lower panels). At 72 h after transfection, cells were pulse labelled with 32P‐orthophosphate for 1 h and chased for 2.5 h. RNA was separated on a denaturing agarose gel and detected by ethidium bromide (EtBr) staining and autoradiography after drying of the gel.",
    "molecules": "32P"
  },
  {
    "caption": "(F) Nucleolar export of pre‐60S particles is strongly compromised in cells depleted of PELP1, TEX10, WDR18, MDN1 or SENP3. HeLa cells expressing YFP‐RpL27 from a tetracycline‐inducible promoter were transfected with the indicated siRNAs. After 24 h, expression of YFP‐RpL27 was induced and 72 h later the localization of RpL27 was monitored. Pictures were taken using identical exposure times.",
    "molecules": "tetracycline"
  },
  {
    "caption": "(C) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO conjugates were recovered on Ni‐NTA beads and subjected to western blotting using anti‐PELP1 antibodies.",
    "molecules": "Ni, NTA"
  },
  {
    "caption": "(A) LAS1L was generated by in vitro transcription/translation and incubated with recombinant E1 and E2 enzymes and SUMO1 or SUMO2, respectively, in the presence of ATP. In the control reaction (lane 1) SUMO was not added.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO2 conjugates were captured on magnetic Ni‐NTA beads and subjected to western blotting using anti‐LAS1L antibodies. Vertical lines indicate removal of irrelevant neighbouring lanes from the initial gel (see Supplementary Figure 14).",
    "molecules": "Ni, NTA"
  },
  {
    "caption": "Representative images of HeLa (B) or HCT-8 (C) cells infected with Shigella WT pre-treated or not with arsenite, analyzed at the indicated times post-infection Cfu quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with Shigella",
    "molecules": "arsenite"
  },
  {
    "caption": "Representative images (E) and cfu quantification (F) of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia and corresponding controls, analyzed at 0.5 hpi",
    "molecules": "anisomycin, H2O2"
  },
  {
    "caption": "Representative images (G) and cfu quantification (H) of intracellular Shigella in HeLa cells pre-treated with arsenite, anisomycin, stressors plus NAC, and corresponding controls",
    "molecules": "anisomycin, arsenite, NAC"
  },
  {
    "caption": "Representative images (A) and cfu quantification (B) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated or not with arsenite. Ruffle formation induced by Shigella WT in panel (A) is indicated by white arrowheads",
    "molecules": "arsenite"
  },
  {
    "caption": "Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated with anisomycin or DMSO (control)",
    "molecules": "anisomycin, DMSO"
  },
  {
    "caption": "Representative images (D) and cfu quantification (E) of Shigella WT or ΔipaB mutant bound to HCT-8 cells pre-treated or not with arsenite",
    "molecules": "arsenite"
  },
  {
    "caption": "Representative image ), cf and qRT-PCR quantification of intracellular Shigella in HeLa cells pre-treated with arsenite in the presence or not of amitriptyline, and corresponding controls",
    "molecules": "amitriptyline, arsenite"
  },
  {
    "caption": "Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells pre-treated wit anisomycin in the presence or not of amitriptyline, and corresponding controls",
    "molecules": "anisomycin, amitriptyline"
  },
  {
    "caption": "Representative image cf and  qRT-PC quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre-treated or not with arsenite prior to infection",
    "molecules": "arsenite"
  },
  {
    "caption": "3D reconstruction of representative images of ASM (J, K) or ceramide (L, M) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls. ASM, ceramide and Hoechst staining were surface converted by voxel distance",
    "molecules": "anisomycin, arsenite, ceramide, Hoechst"
  },
  {
    "caption": "Western blot analysis of p38 MAPK phosphorylation in HeLa cells treated with arsenite, TNF-α, H2O2 or anisomycin; β-actin was used as loading control",
    "molecules": "anisomycin, arsenite, H2O2"
  },
  {
    "caption": "Representative image of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls",
    "molecules": "arsenite, SB203580"
  },
  {
    "caption": "cfu quantificatio of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls",
    "molecules": "arsenite, SB203580"
  },
  {
    "caption": "Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not with arsenite prior to infection",
    "molecules": "arsenite"
  },
  {
    "caption": "cfu quantificatio of intracellular Shigella in HeLa cells pre-treated wit anysomycin in the absence or presence of the p38 inhibitor SB203580, and corresponding controls",
    "molecules": "anysomycin, SB203580"
  },
  {
    "caption": "Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not wit anisomycin prior to infection",
    "molecules": "anisomycin"
  },
  {
    "caption": "3D reconstruction of representative images of ceramide staining in HeLa cells treated with arsenite ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance",
    "molecules": "arsenite, ceramide, Ceramide, Hoechst, SB203580"
  },
  {
    "caption": "3D reconstruction of representative images of ceramide staining in HeLa cells treated wit anisomyci ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance",
    "molecules": "anisomyci, ceramide, Ceramide, Hoechst, SB203580"
  },
  {
    "caption": "Representative images (A), cfu (B and qRT-PCR quantification of intracellular Salmonella in HeLa cells pre-treated with arsenite or anisomycin and corresponding controls Infection was performed with Salmonella WT and analyzed at 0.5 hpi",
    "molecules": "anisomycin, arsenite"
  },
  {
    "caption": "Representative images of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10 min",
    "molecules": "arsenite"
  },
  {
    "caption": "Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT (E) or Δ4 mutant strain (F) for 10 min",
    "molecules": "arsenite"
  },
  {
    "caption": "Representative images (G) and cfu quantification (H) of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC, ΔfliC/pFliC or ΔflhC for 10 min",
    "molecules": "arsenite"
  },
  {
    "caption": "Representative images (I) and cfu quantification (J) of Listeria bound to HeLa cells pre-treated or not with arsenite followed by incubation with the bacteria for 20 min",
    "molecules": "arsenite"
  },
  {
    "caption": "Representative images (K) and cfu quantification (L) of Yersinia bound/internalized by HeLa cells pre-treated or not with arsenite followed by 70 min incubation with the bacteria",
    "molecules": "arsenite"
  },
  {
    "caption": "Cfu quantification of the secondary infection with Shigella WT in HeLa cells following amitriptyline",
    "molecules": "amitriptyline"
  },
  {
    "caption": "Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin SB203580 treatmen",
    "molecules": "SB203580"
  },
  {
    "caption": "3D reconstruction of representative images of ASM (A) or ceramide (B) staining in HeLa cells infected with Shigella WTor mock-treated, analyzed at 3 hpi. ASM, ceramide, Hoechst and Shigella staining were surface converted by voxel distance",
    "molecules": "ceramide, Hoechst"
  },
  {
    "caption": "ceramide measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi Ceramide is shown normalized to mock-treated cells",
    "molecules": "ceramide, Ceramide"
  },
  {
    "caption": "Mean fluorescence intensity of ceramide signal in surface epithelial cells (outermost tissue layer) of non-infected or Shigella-infected guinea pig colon tissue sections",
    "molecules": "ceramide"
  },
  {
    "caption": "A 1H, 15N‐BEST‐TROSY spectrum of phosphoUb (shades of blue) overlaid with wild‐type (wt) Ub (orange). The phosphoUb spectrum contains 130 non‐sidechain resonances that have been colored in dark blue for the major species and light blue for the minor species based on the assignment of each species in Supplementary Fig S2B-D.B-D Weighted chemical shift perturbation (CSP) graphs for (B) wt Ub versus major phosphoUb species, (C) wt Ub versus minor phosphoUb species, (D) major versus minor phosphoUb species.",
    "molecules": "1H, 15N"
  },
  {
    "caption": "A Structure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit (Table ). Ub molecules are colored differently, and atoms for pSer65 are shown as spheres with red oxygen and purple phosphorous atoms.",
    "molecules": "oxygen, phosphorous"
  },
  {
    "caption": "C Representative 2|Fo|‐|Fc| electron density, contoured at 1 σ, covering the phosphorylation site. pSer65 is shown in ball‐and‐stick representation, and neighboring side chains are shown in stick representation with red oxygen and blue nitrogen atoms.",
    "molecules": "oxygen, nitrogen"
  },
  {
    "caption": "D In wt Ub (left), Ser65 forms a hydrogen bond with the backbone amide of Gln62. Upon phosphorylation (right), an oxygen atom from the phosphate forms the same backbone hydrogen bond.",
    "molecules": "oxygen, hydrogen"
  },
  {
    "caption": "A Long‐range HNCO NMR experiments showing differences of hydrogen bonding patterns for major and minor phosphoUb species. Through H bond correlations (blue) recorded for residue Ile44 in the major and minor species of phosphoUb with reference to a control HNCO spectrum (red). In the major form of phosphoUb (left), the HN of Ile44 is a H bond donor to His68, as seen in wt Ub. In the minor form of phosphoUb (right), the HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70.",
    "molecules": "hydrogen"
  },
  {
    "caption": "C Structure of the major form of phosphoUb, where phosphoUb residues 1-61 are shown under a surface, and the β5 strand is shown in stick representation with red oxygen, blue nitrogen and purple phosphorus atoms. The four core Leu residues are shown in green.D Model of the phosphoUb minor species as in (C) in which the β5 strand is retracted into the protein core by two residues. This extends the β4-β5 loop and significantly shortens the C‐terminal tail of Ub.",
    "molecules": "oxygen, nitrogen, phosphorus"
  },
  {
    "caption": "(D-E) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in extracellular acidification rate (ECAR) after adding glucose was considered as glycolysis ability of the cells (D), while the decrease in oxygen consumption rate (OCR) was considered as the ATP production ability of the cells (E).",
    "molecules": "ATP, glucose"
  },
  {
    "caption": "(H-I) Expression of Pdk1/2 was modulated with a retrovirus system, and oligomycin (1 µM) and 2-DG (5 mM) were used during reprogramming (H). The numbers of Oct4GFP+ colonies were determined on day 15 (I).",
    "molecules": "2-DG, oligomycin"
  },
  {
    "caption": "Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers (A)",
    "molecules": "2-DG, oligomycin"
  },
  {
    "caption": "Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers cell migration (B) was determined with qPCR and live-cell imaging, respectively, on day 6.",
    "molecules": "2-DG, oligomycin"
  },
  {
    "caption": "Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. Except for OK+M+S group, the four Yamanaka factors were delivered simultaneously. Energy metabolism was determined on day 6 with Seahorse instrument (E-F).",
    "molecules": "Repsox"
  },
  {
    "caption": "Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. The expression of glycolysis markers was determined on day 6 with qPCR (G).",
    "molecules": "Repsox"
  },
  {
    "caption": "(C) Benign breast epithelial MCF10A and breast carcinoma BT-474 cells were embedded in 3D collagen as single cells or as spheroids, respectively, and the growth was followed for 5 days. Light micrographs show filamentous actin (phalloidin) and nuclei (Hoechst) in representative cell colonies. Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout. At 72 hours, MCF10A mock versus ECHDC1_sgRNA_1 and ECHDC1_sgRNA_2 P < 0.05; at 96 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.001; at 120 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.0001. Nuclei count relative to mock 0 hours. Error bars indicate mean ± SEM; n ≥ 10 colonies. Statistical significance was assessed with one-way ANOVA with Tukey's multiple comparison test. Scale bar 50 µm.",
    "molecules": "Hoechst, collagen, phalloidin"
  },
  {
    "caption": "(E) Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status (n=7 in both groups). Boxes represent the interquartile range, whiskers represent the range of the values and solid line within the box correspond to the median value. Outlier points indicates values not included between the whiskers. Statistical significance was assessed with Wilcoxon test.",
    "molecules": "propanoate"
  },
  {
    "caption": "Quantification of IL-1β concentration (pg/ml) in the supernatant of primary macrophage cultures from COVID-19 patients (n = 44; red bars) or SC-naïve individuals (n = 24; blue bars) stimulated with LPS or S-protein (0.1 µg/ml). To activate IL-1β secretion nigericin was added for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used. TNF-α (pg/ml) secreted from macrophages derived from SC-naïve and COVID-19 patients as described above. Cells were stimulated with LPS (SC-naïve/COVID-19 n = 4), LPS with nigericin (SC-naïve n = 4; COVID-19 n = 6), S-protein (SC-naïve n = 5; COVID-19 n = 4), S-protein with nigericin (SC-naïve n = 3; COVID-19 n = 4) or left unstimulated (SC-naïve n = 3; COVID-19 n = 4). Significances shown in the figure are always in comparison to the unstimulated control.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Representative confocal microscope pictures of macrophages from SC-naïve (top) and COVID-19 patients (bottom). Nuclei are stained with DAPI (blue) and ASC speck are labeled by immunofluorescence staining (green). Macrophages were stimulated with LPS (left) and S-Protein (right) for 4h followed by stimulation with nigericin in both cases. Pictures were taken with a 60x objective using the same microscope settings.",
    "molecules": "DAPI, LPS, nigericin"
  },
  {
    "caption": "Subgroup analysis of IL-1β secretion of macrophages after stimulation with S-protein and nigericin from COVID-19 patients with severe (red/black; n=23) or mild (white/red; n=21) disease. Student's t-test with Welsh corrections was used to calculate statistical differences. Graph shows mean ± SEM.",
    "molecules": "nigericin"
  },
  {
    "caption": "CD14+ cells of two convalescent individuals were isolated sequentially at d18, d26 and d56 after confirmation of SARS CoV2 infection by PCR. Following M-CSF differentiation, macrophages were stimulated with LPS (red triangle) or S-Protein (red circles) and IL-1β concentration (pg/ml) was quantified upon incubation with nigericin. Blue dashed line indicates mean IL-1β level of SC-naïve individuals stimulated with S-protein and nigericin.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "IL-1β concentration (pg/ml) in supernatants of COVID-19 patient derived macrophages pretreated with DMSO (n = 44), KINK-1 (n = 11) and MMG-11 (n = 19) and subsequent stimulation with LPS and nigericin. For statistical analysis, one-way ANOVA was used.",
    "molecules": "DMSO, KINK-1, LPS, MMG-11, nigericin"
  },
  {
    "caption": "IL-1β concentration (pg/ml) in supernatants from macrophages of SC naïve (blue; n = 7) and COVID-19 patients (red; n = 11). Macrophages were pre-treated with DMSO (SC-naïve n = 7; COVID-19 n = 11), KINK-1 (SC-naïve n = 6; COVID-19 n = 7) and MMG-11 (SC-naïve n = 4; COVID-19 n = 4) for 2h and then stimulated with zymosan (4h) and nigericin (2h) as indicated. For statistical analysis, two-way ANOVA with tukey post hoc test was used.",
    "molecules": "zymosan, DMSO, KINK-1, MMG-11, nigericin"
  },
  {
    "caption": "IL-1β concentration (pg/ml) in supernatants from COVID-19 patients which were stimulated for 2h with a blocking anti-TLR2 monoclonal antibody in different concentrations as indicated. Subsequently, macrophages were stimulated with LPS (1 ng/ml: n = 6; 10 and 100 ng/ml: n = 7) and S-Protein (1 ng/ml: n = 12; 10 and 100 ng/ml: n = 13) for 4h and both groups for 2h with nigericin. For statistical analysis, one-way ANOVA was used",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "IL-1β concentration (pg/ml) in supernatants of SC-naïve individuals (n = 7) which were pre-treated for 24h with FCCP (1µg/ml) and H2O2 (10µg/ml). Macrophages were then incubated with LPS or S-Protein (4h) and nigericin (2h) as indicated in the figure. Control cells were left unstimulated or stimulated with LPS or S-protein without pre-treatment with FCSP or H2O2 (n = 6). For statistical analysis, Brown-Forsythe and Welsh ANOVA test was used.",
    "molecules": "FCCP, FCSP, H2O2, LPS, nigericin"
  },
  {
    "caption": "Quantification of IL-1β concentrations (pg/ml) in the supernatant of primary macrophage cultures from patients with active/untreated tuberculosis (n = 6; red/black bars) or healthy/SARS-CoV-2 naïve individuals (n = 3; blue bars) or the same patients after 6 months of anti-tuberculous treatment; n = 6; white/red bars) stimulated with LPS or S-protein (0.1 µg/ml) for 4h and subsequent incubation with nigericin for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "(B)  YTS KIR2DL1 cells were transfected with either N.S siRNA (blue curve) or with SHP-1  and Cbls siRNA (red curve). After 48 hours, cells were loaded with  calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.",
    "molecules": "calcium, Fluo-3-AM"
  },
  {
    "caption": "(E) Killing assay. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with  [35S] Met-loaded 721.221 Cw4  or  Cw7 target cells. After 5 hours of co-culture, the supernatant was collected, and the radioactive  signal was measured. Data are means ± SEM of three independent experiments. P values were calculated by One-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05, **P < 0.01 .",
    "molecules": "Met, 35S"
  },
  {
    "caption": "(B) Characterization of the diameter and zeta potential (Z-potential) with the addition of each layer. Graph below presenting NP size distribution.",
    "molecules": "NP"
  },
  {
    "caption": "(A) NK92-NKp46low, NK92-NKp46high, YTS KIR2DL1,  K562 and  721.221 HLA-Cw4 cells were  stained with anti-NKp46  monoclonal antibody followed by staining  with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Cells were then analyzed using flow cytometry. (B) NK92-NKp46low, NK92-NKp46+, YTS KIR2DL1, K562, and 721.221 HLA-Cw4 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using  flow cytometry to confirm the internalization of the coated NPs in a NKp46-specific manner. ",
    "molecules": "Alexa568-Fluor, NPs, rhodamine"
  },
  {
    "caption": "(C-F) (C) NK92-NKp46low, (D) NK92-NKp46high, (E) YTS KIR2DL1 and (F) K562  cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using confocal microscopy. Upper panel shows wide field images. Lower panel shows single-cell images. The data shown are representative of three independent experiments.",
    "molecules": "NPs, rhodamine"
  },
  {
    "caption": "(A) YTS KIR2DL1 and  primary NK cells were incubated with NPs loaded with  Cbl-b, c-Cbl  and SHP-1 siRNA, or NPs loaded with 1500 pmol N.S siRNA. Following 48 hours of incubation, cells were lysed, and the membrane was  blotted with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. GAPDH served as loading control. Densitometric analysis of the bands was performed using ImageJ and normalized to the GAPDH densitometry values. Analysis by ImageJ densitometry  is summarized in the graph below. Data are means ± SEM of four independent experiments. (n=4). P values were calculated by One-sample t-tests and are indicated by asterisks. *P < 0.05, **P < 0.01.",
    "molecules": "NPs"
  },
  {
    "caption": "(B) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNAs-loaded NPs (red curve), with NS siRNA loaded NPs (green curve) or with empty NPs  (blue curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK  cells were then mixed with 721.221 Cw4  target cells at 37°C, and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.",
    "molecules": "calcium, Fluo-3-AM, NPs"
  },
  {
    "caption": "(D)  YTS KIR2DL1 cells were treated with SHP-1 and Cbls  siRNAs-loaded NPs  or N.S siRNA-loaded  NPs. After 48 hours, cells were incubated with either  721.221 Cw4  or Cw7 target cells, the supernatant was then collected, and granzyme B levels were evaluated using ELISA sandwich  assay. The levels of granzyme B were quantified based on a standard curve. Data are means ± SEM of five independent experiments. (n=5). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. **P < 0.01.",
    "molecules": "NPs"
  },
  {
    "caption": "(F) pNK cells were pretreated with either SHP-1 and Cbls siRNA-loaded NPs or N.S siRNA-loaded  NPs. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was quantitated by mean fluorescence intensity (MFI). The data represent five independent experiments (n=5) Data are shown as mean ± SEM.) (n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.05.",
    "molecules": "NPs"
  },
  {
    "caption": "(A)  YTS KIR2DL1 cells were incubated with SHP-1  and Cbls  siRNA-loaded NPs (red curve) or empty NPs (purple curve). After 48 hours, cells were loaded with  Fluo-3-AM and analyzed for intracellular calcium  levels for 20 min at 37°C.  YTS KIR2DL1 cells that were treated with PBS  served as a negative control (green curve). NK cells that were mixed with 721.221 Cw7  target cells served as a positive control (blue curve). Calcium levels were analyzed by spectrofluorometry. (n=3)",
    "molecules": "calcium, Calcium, Fluo-3-AM, NPs, PBS"
  },
  {
    "caption": "(B) Freshly isolated PBMCs were treated with SHP-1  and Cbls siRNA-loaded NPs, empty NPs, PBS as negative control or PHA as a positive control. Following incubation, supernatant from each sample was collected and the levels of  human  cytokines  TNF-α, IL-6, IL-10 and IFN-γ were determined using ELISA. The data represent four independent experiments (n=4). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.008. Data are shown as mean ± SEM.",
    "molecules": "NPs, PBS, PHA"
  },
  {
    "caption": "(E) Following 24 hours after the last treatment, extracted cells from tumors of either SHP-1 and Cbls or N.S siRNA loaded NKp46-NPs treated mice  were analyzed by flow cytometry.  Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the sample treated with N.S siRNA loaded  NPs. The data represent three independent experiments. Data are shown as mean ± SEMn=3; P value was calculated by one sample t-test and is indicated within the graph P =0.009.",
    "molecules": "NPs"
  },
  {
    "caption": "(F)  Survival  analysis in mice treated with NK cells' gene silenced for SHP-1  and Cbls using siRNA-loaded  NPs  (n=18) or N.S siRNA-loaded NPs (n=18) as demonstrated by Kaplan-Meier survival curve. P values were calculated using the log-rank test and is indicated within the graph P =0.00009.",
    "molecules": "NPs"
  },
  {
    "caption": "g, Degradation of long-lived proteins in STX17 knockdown cells. St, starved; wor, wortmannin. All error bars denote s.d. Scale bars, 1 µm (a, b), 200 nm (c) and 5 µm (d).",
    "molecules": "wortmannin"
  },
  {
    "caption": "Confocal images of the GFP fluorescent patterns in seedlings expressing ATG5-GFP or GFP-ATG8. (a,b) Control conditions: cytosolic signal associated with rare fluorescent puncta. (c,d) High increase of fluorescent puncta after 2 h of nutrient starvation. Note ring-like ATG5 structures in c. (e,f) Wortmannin treatment blocks the occurrence of fluorescent ATG5 or ATG8 structures. (g,h) Maximum intensity projections of vacuolar focal planes, allowing visualization of autophagic bodies after 4 h of nutrient starvation in the presence of concanamycin A. Vacuolar ATG5-GFP fluorescence is not detected (g), while GFP-ATG8 signals are abundant (h). An inset in g, corresponding to a cortical focal plane, shows that formation of cytosolic ATG5 structures in the cytosol is not inhibited by concanamycin A treatment. Maximum intensity projections of 10-μm-deep deconvolved stacks, of cortical (a-f, and inset in g) or vacuolar focal planes (g,h). Scale bar, 10 μm.",
    "molecules": "concanamycin A, nutrient, Wortmannin"
  },
  {
    "caption": "(a) Differential interference contrast micrographs of root epidermal cells incubated for 8 h with concanamycin A, in nutrient starvation conditions. Autophagic bodies accumulation in the vacuole is clearly visible in wild-type seedlings, absent in the atg5-1 mutant and restored in atg5-1 seedlings expressing ATG5-GFP. (b) Confocal microscopy. The ATG5-GFP fluorescence pattern in the atg5-1 background is identical to that observed in wild-type seedling, including ring-like structures. Scale bar, 15 μm.",
    "molecules": "concanamycin A, nutrient"
  },
  {
    "caption": "(a) Dual observation of ATG5-GFP and mCh-ATG8 under nutrient starvation reveals a partial colocalization of the two reporters. Merge of the GFP (green) and ATG8 (magenta) channels of maximum intensity projections of 20-μm-deep deconvolved stacks. Scale bar, 3 μm.",
    "molecules": "nutrient"
  },
  {
    "caption": "b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.",
    "molecules": "DAPI"
  },
  {
    "caption": "b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours.",
    "molecules": "Tanespimycin"
  },
  {
    "caption": "(A) Phenotypes of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on low-K+ (LK, 50 μM) or high-K+ (HK, 5 mM) medium for 7 d. Scale bar, 1 cm.",
    "molecules": "high-K+, low-K+"
  },
  {
    "caption": "(E) and (F) K+ content of the various plant materials tested in (A). Data are means ± SE (n = 3, biological replicates; each replicate contains 120-150 individual plants).",
    "molecules": "K+"
  },
  {
    "caption": "(G) and (H) Expression of KUP9 protein as determined in the kup9-1/ProKUP9:KUP9-GFP transgenic plants. Green fluorescence shows the subcellular localization of KUP9-GFP. PI (propidium iodide) and ER tracker were used to indicate the positions of the plasma membrane (PM) and endoplasmic reticulum (ER) and are shown as red and cyan fluorescence, respectively. Scale bars for root tip, 10 μm in (G) and 5 μm in (H). Scale bars for EZ and MAZ, 20 μm.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "(A) Auxin levels in root tips, as indicated by the ProDR5:GFP reporter line. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bar, 10 μm. (B) Quantification of GFP fluorescence in the QC cells shown in (A). Data are means ± SE (n = 20, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance. ",
    "molecules": "Auxin"
  },
  {
    "caption": "(A) Phenotypes of various plants. Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d. Scale bar, 1 cm.",
    "molecules": "NAA"
  },
  {
    "caption": "(C) QC cell observation using the QC marker line ProWOX5:GFP. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium with or without NAA (100 nM) for 96 h. Scale bar, 10 μm.",
    "molecules": "NAA"
  },
  {
    "caption": "Phenotypes of the kup9-1/ProWOX5:AMI1 (A) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.",
    "molecules": "IAM"
  },
  {
    "caption": "Phenotypes of the kup9-1/ProKUP9:AMI1 (C) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.",
    "molecules": "IAM"
  },
  {
    "caption": "(B) 3H-IAA accumulation in the BY-2 cells expressing the indicated proteins. PIN4-GFP and KUP10-GFP were used as positive control and negative control, respectively. Data are means ± SE (n = 3, biological replicates; each replicate contains 3-4 mL suspension cells). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.",
    "molecules": "IAA, 3H"
  },
  {
    "caption": "(D) 3H-IAA accumulation in the oocytes expressing KUP9-GFP and PIN4 at the indicated times. PIN4 was used as a positive control. The oocytes were incubated in the bath solution containing 3H-IAA for the indicated times. Data are means ± SE (n = 6, biological replicates; each replicate contains 6 oocytes).",
    "molecules": "IAA, 3H"
  },
  {
    "caption": "(E) IAA content in the oocytes expressing the indicated proteins. The oocytes were injected with the same amount of IAA and incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).",
    "molecules": "IAA"
  },
  {
    "caption": "(F) K+ efflux activity of the oocytes expressing KUP9 and NRT1.5. The oocytes were not injected with IAA. The oocytes were incubated in K+-free bath solution for 6 h. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).",
    "molecules": "IAA, K+"
  },
  {
    "caption": "(G) K+ efflux activity of the oocytes expressing the indicated proteins. The oocytes were injected with H2O, IAA, or BA. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).",
    "molecules": "BA, H2O, IAA, K+"
  },
  {
    "caption": "(H) K+ efflux activity of the oocytes expressing the indicated proteins. IAA was injected into the oocytes. Then the oocytes were incubated in K+-free bath solutions at different pH for 6 h. Data are means ± SE (n = 3-6, biological replicates; each replicate contains 6 oocytes).",
    "molecules": "IAA, K+"
  },
  {
    "caption": "(I) IAA efflux activity of oocytes expressing the indicated proteins under different external K+ conditions (-K, 0 mM; +K, 50 mM). The oocytes were injected with the same amount of IAA, and then incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.",
    "molecules": "IAA, K+"
  },
  {
    "caption": "(A) K+ levels of various materials using the fluorescent dye Asante Potassium Green-2 (AM). Scale bar, 50 μm. (B) Quantification of K+ concentration under LK and HK conditions. Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control. ",
    "molecules": "Asante Potassium Green-2 (AM), K+"
  },
  {
    "caption": "(C) to (E) The contents of free IAA (C), IA-Asp (D), and IA-Glu (E) in the root tips of wild type and the kup9-1 mutant. Data are means ± SE (n = 3, biological replicates; each replicate contains 1000-1200 individual plants). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represents control.",
    "molecules": "IAA, IA-Asp, IA-Glu"
  },
  {
    "caption": "(C) ARL11/14 co-localize with PLD1. Flag-tagged PLD1 was co-expressed with HA-tagged GTP-bound mutants of small GTPases in HEK293A cells as indicated. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. The Flag and HA-antibodies were detected by anti-mouse and anti-rabbit secondary antibodies, respectively. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) PLD1 preferentially binds to the GTP-form, not the GDP-form, of ARL11. Flag-tagged PLD1 was co-expressed with HA-tagged ARL11Q67L (GTP-form) or ARL11T26N (GDP-form) mutant in HEK293A cells. The result is a representative of two biologically independent experiments.",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "(F) The subcellular co-localization of ARL11 with PLD1 is GTP-dependent. HA-tagged GTP-bound ARL11Q67L, wild-type ARL11 or GDP-bound ARL11T26N was co-expressed with Flag-tagged PLD1. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments.",
    "molecules": "DAPI, GDP, GTP"
  },
  {
    "caption": "(C) Deletion of the loop region disrupts the PLD1 and ARL11 co-localization. Flag-tagged full-length or ∆Loop truncation of PLD1 was co-expressed with HA-tagged ARL11Q67L. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments. (D) Mander's overlap coefficient of (J). Mander's overlap coefficient calculated using JACoP plugin in ImageJ.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) ARL11Q67L, but not ARL11T26N, promotes phagocytosis of macrophages. Macrophages were differentiated from indicated THP1 cell pool, followed by incubated with green zymosan particles for 1 hour. Scale bars: 50 μm. The result is a representative of two biologically independent experiments.",
    "molecules": "zymosan"
  },
  {
    "caption": "(H) PLD1 knockout blocks the ARL11Q67L-stimulated phagocytosis. Macrophages were differentiated from indicated THP1 cell groups and then incubated with green zymosan particles for 1.5 hours. Scale bars: 50 μm. The result is a representative of two biologically independent experiments. (I) PLD1 is required for ARL11 to stimulate phagocytosis. Data are quantification of panel (H).",
    "molecules": "zymosan"
  },
  {
    "caption": "(B) Interaction of PI4KB with ARL5A/5B is stronger than ARF1, RAB11A/11B, and RAB14. Flag-tagged PI4KB was co-expressed with HA-tagged GTP-bound mutant of GTPases in HEK293A cells as indicated. PI4KB was immunoprecipitated with Flag beads, and the co-precipitated GTPases were detected by HA Western.",
    "molecules": "GTP"
  },
  {
    "caption": "(C) PI4KB preferentially binds to the GTP-form of ARL5B. Flag-tagged PI4KB was co-expressed with HA-tagged ARL5BQ70L (GTP-form) or ARL5BT30N (GDP-form) mutant in HEK293A cells. Interaction was measured similar to panel B. The result is a representative of two biologically independent experiments.",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "(D) The ARL5A colocalization with PI4KB is GTP-dependent. HA-tagged GTP-bound ARL5AQ70L, wild-type ARL5A or GDP-bound ARL5AT30N was co-expressed with Flag-tagged PI4KB. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. Shown is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient. Mean± SD; n= 6 technically independent samples.",
    "molecules": "DAPI, GDP, GTP"
  },
  {
    "caption": "(E) ARL5A/5B double knockout diminishes the colocalization of PI4KB and TGN46. Flag-tagged PI4KB was expressed in HEK293A wild-type or two ARL5A/5B double knockout clones as indicated. After 24 hours, cells were fixed, followed by incubation with anti-Flag and anti-TGN46 primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 5 μm. The result is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) The N1 region of PI4KB is required for the co-localization of ARL5A/B and PI4P. P4Mx2-mEGFP was co-expressed with HA-tagged ARL5AQ70L in HEK293A wild-type or PI4KB knockout cells (clone#2-5). HEK293A cells were co-transfected with indicated constructs. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-HA primary antibodies. DAPI (blue) was used for nuclei staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments. (I) Mander's overlap coefficient of (H).",
    "molecules": "DAPI, PI4P"
  },
  {
    "caption": "(B) ARL5A/5B recruitment to mitochondria induces PI4P accumulation at the mitochondria. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A cells. Cell samples were treated with or without 100nM rapamycin for 1 hour. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments. (C) PI4KB knockout blocks the PI4P accumulation induced by mitochondria anchored ARL5A/5B. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A PI4KB knockout cells (clone#2-5). Cell samples were treated with or without 100nM rapamycin for 1 hour before imaging. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments. (",
    "molecules": "PI4P, rapamycin"
  },
  {
    "caption": "(D) Mander's overlap coefficient of (B and C, result of rapamycin treatment). Mean± SD; n= 5 technically independent samples.",
    "molecules": "rapamycin"
  },
  {
    "caption": "F. HeLa cells expressing either control RNA or lncRNA-MIF were treated with or without MG132 (20 μM) for 6 h. Cell lysates were analyzed by Western blot with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "G. HeLa cells expressing either control shRNA or lncRNA-MIF shRNA were treated with MG132 for 6 h. Cell lysates were analyzed by Western blotting with the indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "H. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the c-Myc protein half-life. Protein band intensity were analyzed by ImageJ.I. Band intensity of c-Myc in Fig 1H was analyzed by ImageJ.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "J. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3 as indicated. Forty-eight hours after infection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the half-life of c-Myc protein.K. Band intensity of c-Myc in Fig 1J was analyzed by ImageJ.",
    "molecules": "CHX"
  },
  {
    "caption": "A. P493-6 cells (human B cell lymphoma cell line) carrying a c-Myc tet-off system were treated with Doxycycline (1 μg/ml) for the indicated periods of time. Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting with the indicated antibodies.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "G. P493-6 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after trasfection, cells were treated with doxycycline for 0 hour and 12 hours respectively. Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).",
    "molecules": "doxycycline"
  },
  {
    "caption": "A. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF or biotin-labeled antisense RNA for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies.",
    "molecules": "biotin"
  },
  {
    "caption": "E. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).",
    "molecules": "biotin"
  },
  {
    "caption": "F. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF and antisense RNA for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 and miR-34a levels. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).",
    "molecules": "biotin"
  },
  {
    "caption": "K. HeLa cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes of lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).",
    "molecules": "biotin"
  },
  {
    "caption": "A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By Forty-eight hours infection, cells were treated with MG132 or DMSO (vehicle) for 6 h. Cell lysates were then analyzed by Western blotting with the indicated antibodies.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. By 48 h infection, cells were treated with MG132 or DMSO for 6 h. Cell lysates were then analyzed by Western blot analysis with the indicated antibodies.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate.",
    "molecules": "lactate"
  },
  {
    "caption": "C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).",
    "molecules": "lactate"
  },
  {
    "caption": "D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).",
    "molecules": "lactate"
  },
  {
    "caption": "F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).",
    "molecules": "glucose"
  },
  {
    "caption": "B, C GOT2 can be acetylated. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag‐bead‐purified GOT2 were determined by Western blot analysis using a pan‐anti‐acetyllysine antibody (α‐Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag.",
    "molecules": "acetyllysine, NAM, TSA"
  },
  {
    "caption": "D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag‐GOT2 and Myc‐MDH2 were co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐bead‐purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis.",
    "molecules": "NAM"
  },
  {
    "caption": "E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag‐GOT2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐GOT2 and its association with endogenous MDH2 were determined by Western blot analysis.",
    "molecules": "NAM"
  },
  {
    "caption": "F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag‐MDH2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag‐MDH2 and endogenous GOT2 was determined by Western blot analysis.",
    "molecules": "NAM"
  },
  {
    "caption": "H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K‐to‐R mutants were examined for their protein association with ectopically expressed Myc‐MDH2 by Western blot analysis.",
    "molecules": "arginine, lysine"
  },
  {
    "caption": "I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag‐GOT2 was overexpressed in HEK293T cells without or with co‐overexpression of Myc‐MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag‐GOT2 from the Myc‐MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag‐GOT2 by Western blot analysis.",
    "molecules": "NAM"
  },
  {
    "caption": "A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2-MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis.",
    "molecules": "Glucose, glucose, glutamine"
  },
  {
    "caption": "C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels.",
    "molecules": "Glucose, glucose, glutamine"
  },
  {
    "caption": "E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.",
    "molecules": "glucose, glutamine"
  },
  {
    "caption": "G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.",
    "molecules": "glucose, glutamine"
  },
  {
    "caption": "F, G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.",
    "molecules": "glucose, glutamine"
  },
  {
    "caption": "A The malate-aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in .",
    "molecules": "EGCG, AOA, aspartate, malate, NADH, oxamate"
  },
  {
    "caption": "B, C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in .",
    "molecules": "NADH"
  },
  {
    "caption": "D GOT2 3K acetylation regulates mitochondrial NADH/NAD+ redox in the cell. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADH/NAD+ in the mitochondria of cells was measured as described in .",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "E GOT2 3K acetylation promotes ATP production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ATP production was determined in cell extracts as described in .",
    "molecules": "ATP"
  },
  {
    "caption": "F GOT2 3K acetylation promotes NADPH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADPH/NADP+ was determined in cell extracts as described in .",
    "molecules": "NADP+, NADPH"
  },
  {
    "caption": "G GOT2 3K acetylation promotes GSH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in .",
    "molecules": "GSH, GSSG"
  },
  {
    "caption": "H GOT2 3K acetylation suppresses cellular ROS levels. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ROS was determined as described in in cells under non‐stressed condition or exposed to menadione (50 μM for 30 min).",
    "molecules": "menadione, ROS"
  },
  {
    "caption": "I GOT2 3K acetylation protects cells from oxidative damage. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells.",
    "molecules": "menadione"
  },
  {
    "caption": "J, K Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were seeded in a 6‐well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1-2 days over a period of 7 days.",
    "molecules": "glucose"
  },
  {
    "caption": "L, M Panc‐1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in (M).",
    "molecules": "glucose"
  },
  {
    "caption": "C) Exemplar whole cell currents (Top) at the zero, three and seven minute time points. Current records (Bottom) from cells expressing the F514C:P538C and treated with extracellular 100 μM 1,2-methanediyl bismethanethiosulfonate (M2M).",
    "molecules": "1,2-methanediyl bismethanethiosulfonate, M2M"
  },
  {
    "caption": "(A) Chromosome 7 of S. bayanus is incompatible with the S. cerevisiae genome. The chromosome 7 replacement stain (Sc+Sb-chr7) exhibited serious growth defects when grown on the non-fermentable carbon source (glycerol). The respiration defects could be partially rescued when a whole set of S. bayanus chromosomes were provided (Sc+Sb-chr7 x Sb-ρ⁰), or fully rescued with the addition of S. bayanus mtDNA (Sc+Sb-chr7-ρ⁰ x Sb). Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. Sc, S. cerevisiae. Sb, S. bayanus. ρ⁰, mtDNA-less strains.",
    "molecules": "glucose, glycerol"
  },
  {
    "caption": "(E) CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates.",
    "molecules": "glycerol"
  },
  {
    "caption": "(D) Overexpressing Sb-CCM1 rescues the respiration defect of Sc+Sb-CCM1 cells. Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed.",
    "molecules": "glucose, glycerol"
  },
  {
    "caption": "(B) Seven of 12 tested PPR genes exhibit incompatibility between S. cerevisiae and at least one other Saccharomyces sensu stricto species. The doubling time of S. cerevisiae cells carrying the endogenous PPR genes (Sc) or orthologs of other yeast species (Sp, Sm, Sk and Sb) were measured in YPD (glucose) or YPG (glycerol) media. All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time. Graphs were plotted using data from at least three independent repeats for each strain. Sc, S. cerevisiae. Sp, S. paradoxus. Sm, S. mikatae. Sk, S. kudriavzevii. Sb, S. bayanus. n.d., growth not detected. p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001.",
    "molecules": "glucose, glycerol"
  },
  {
    "caption": "(B) Seven of 12 tested PPR genes exhibit incompatibility between S. cerevisiae and at least one other Saccharomyces sensu stricto species. The doubling time of S. cerevisiae cells carrying the endogenous PPR genes (Sc) or orthologs of other yeast species (Sp, Sm, Sk and Sb) were measured in YPD (glucose) or YPG (glycerol) media. All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time. Graphs were plotted using data from at least three independent repeats for each strain. Sc, S. cerevisiae. Sp, S. paradoxus. Sm, S. mikatae. Sk, S. kudriavzevii. Sb, S. bayanus. n.d., growth not detected. p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001.",
    "molecules": "glucose, glycerol"
  },
  {
    "caption": "H. Mass chromatograms of the nucleosides,  Um (Q1/Q3 = 259.1/113.1), Cm  (Q1/Q3 = 258.1/112.1), and G (Q1/Q3 = 284.1/152.2) of HctRNAGly(GCC), HctRNAGly(CCC), HctRNAGly(UCC), and HctRNAPros isolated from WT and hTrmt13  KO cells. Um, 2′-O-methyluridine,  Cm, 2′-O-methylcytidine. Q1/Q3: the mass of the precursor  ion and the mass of the product  ion. These endogenous  tRNAs were purified separately from total cellular RNAs using sequence-specific probes.",
    "molecules": "2′-O-methylcytidine, Cm, 2′-O-methyluridine, Um, tRNAGly, tRNAPros"
  },
  {
    "caption": "I. Quantification of the Um/G and Cm/G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells. Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.",
    "molecules": "Cm, Um"
  },
  {
    "caption": "A. De novo protein synthesis measured by puromycin incorporation in shSCR,  hTrmt13 knockdown cells (sh-1 and sh-2), and sh-1 with stable expression of wild type  hTrmt13 (sh-1+hTrmt13) or enzymatically-inactive hTrmt13 (sh-1+E463A). Puromycin-labeled proteins were detected by immunoblotting. Representative images (left) and statistical analysis (right) are shown. Knockdown and overexpression efficiency of hTrmt13 in MDA-MB-231 verified by Western blots. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.",
    "molecules": "puromycin, Puromycin"
  },
  {
    "caption": "G. Quantification of de novo protein synthesis in cells treated with different concentration of tRFs H. Quantification of de novo protein synthesis in cells treated with different concentration of  tRNAGly(CCC) Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.",
    "molecules": "tRNAGly"
  },
  {
    "caption": "A, B. Representative images showing endogenous hTrmt13  (A) and HA-tagged hTrmt13 (HA-hTrmt13, B) subcellular  localization by confocal microscopy analysis. DAPI staining was included to visualize the cell nucleus (Blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Representative  confocal microscopy images showing the subcellular  localization of hTrmt13 in MDA-MB-231  cells cultured with 10% FBS (left) or no FBS (right). DAPI staining  was included to visualize the cell nucleus  (Blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Confocal microscopy images of MDA-MB-231 cells were co-immunolabeled with antibodies against hTrmt13 and histone H3 (left). The images were captured by GE DeltaVision OMX SR. Co-localization of H3 and hTrmt13 was analyzed by ImageJ (right, n = 20 cells, center line, the median of the data). DAPI staining was included to visualize the cell nucleus  (Blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. The binding shift assays of hTrmt13, USF1, and their mixture for synthesized DNA from the TGFB1  promoter. In the reaction system, 10 ng/μL DNA was used, and 1 μM hTrmt13 or 1 μM USF1  or a mixture (0.5 μM hTrmt13 + 0.5 μM USF1) was applied. D. The binding shift assays of hTrmt13 for HctRNAGly(GCC) with the presence of various concentrations of USF1. In the reaction system, 0.3 μM HctRNAGly(GCC) and 0.5 μM hTrmt13 were used, and a varied amount of USF1 (0, 0.5, 1, 2, 3, 4 μM) were used. E. The binding shift assays  of hTrmt13 (0.5 μM), or USF1 (0.5 μM) or the hTrmt13-USF1 complex (0.5 μM) for both tRNA (0.1 μM) and  DNA (15 ng/μL) in the same reaction.",
    "molecules": "tRNAGly"
  },
  {
    "caption": "A Speed of propidium iodide (PI) penetration into fibroblasts during 60 min of fixation with either 4 % PFA or 3 % glyoxal. N = 3 independent experiments. Glyoxal fixation enables PI to penetrate far more rapidly into the cells.",
    "molecules": "glyoxal, Glyoxal, PFA, propidium iodide (PI)"
  },
  {
    "caption": "B Speed of FM 1-43 penetration in similar experiments. The arrowhead points to one example of ongoing endocytosis during PFA fixation. N = 3-4 independent experiments. The general pattern of FM 1-43 entry was similar to that of propidium iodide. Only the first 10 minutes are shown, to enable an optimal observation of the kinetics of the first stages of FM 1-43 entry. The results parallel those obtained with PI: faster penetration during glyoxal fixation. Scale bar = 40 µm, * p < 0.05, ** p < 0.01",
    "molecules": "FM 1-43, glyoxal, PFA"
  },
  {
    "caption": " Figure 2 A comparison of morphological changes taking place during fixation with PFA or glyoxal. The changes were visualized by DIC images taken at 5 minute intervals during fixation. The graph shows the correlation of each image to the first frame. N = 50 (PFA) and 54 (glyoxal) cellular regions analyzed, from 3 independent experiments. The higher correlation value indicates that glyoxal preserves the initial cell morphology with higher accuracy than PFA. Scale bar = 20 µm, * p < 0.05, ** p < 0.01 ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": "A SDS-PAGE gel showing rat brain cytoplasm incubated for 60 min with different fixatives. The graph shows the summed intensity of the bands in each lane. Fixed proteins either no longer run into the gel or form only smears. To compare the efficiency of fixation, the bands that survive fixation were summed and were expressed as % of an unfixed control. The intensity of PFA-fixed samples was significantly higher than that of glyoxal-fixed samples (N = 5 independent experiments). Glut = 0.2% glutaraldehyde.",
    "molecules": "glutaraldehyde, glyoxal, PFA"
  },
  {
    "caption": "B Staining of nucleic acids after fixation. The propidium iodide signal in fibroblasts was significantly higher for samples fixed with glyoxal pH 4 (N = 6-8). To test whether the fixed nucleic acids were still available for specific detection, we performed FISH for GAPDH in cultured neurons, using a standard protocol provided by the company Affymetrix. The fluorescence signal of the samples fixed with glyoxal (pH 4) was significantly higher than for PFA-fixed samples (N = 5-6). Scale bar = 10 µm, * p < 0.05, ** p < 0.01",
    "molecules": "glyoxal, nucleic acids, PFA, propidium iodide"
  },
  {
    "caption": " Figure 4 STED imaging of primary hippocampal neurons fixed with either PFA or glyoxal. Strong differences in labeling patterns can be observed. The images are brighter and less "spotty" for the glyoxal-fixed samples. Structures such as filaments or organelles are more easily detected. Quantification of the fluorescence signal (fold over background) shows that 16 out of 20 stainings are significantly brighter in glyoxal fixed samples compared to the PFA fixed samples. N = 35 - 132 objects. Scale bar = 6 µm for β-actin and α-tubulin, and 3 µm for the other proteins. ** p < 0.01, *** p < 0.001 ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": " Figure 5 Comparison of immunostaining NUP160 after fixation with either PFA or glyoxal. HeLa cells were stained for the nucleoporin complex protein NUP160 after fixation with either PFA, glyoxal pH 4 or glyoxal pH 5. Fluorescence intensities (fold over background) were compared and are shown in the graph. The quantification of fluorescence signals shows that glyoxal pH 4 fixation allows for significantly brighter stainings. N = 73 - 156 cells per condition analyzed. Scale bar = 10 µm. *** p < 0.001 ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": " Figure 6 Comparison of immunostained AtT20 cells after fixation with either PFA or glyoxal. AtT20 cells stained for the SNARE proteins syntaxin1 and SNAP25 and the autophagy marker LC3B, were compared with regard to the fluorescence intensity (fold over background) of the stainings. Quantification of the intensity shows that glyoxal fixation allows for significantly brighter stainings of the membrane SNARE proteins. LC3B staining is brighter in PFA fixed cells. N = 9 - 20 cells per condition. Scale bar = 5 µm. * p < 0.05, *** p < 0.001 ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": "A Primary hippocampal neurons were stained for Actin and Ankyrin G. A comparison between PFA- and glyoxal-fixed samples shows that actin staining with phalloidin works as least as well in both, showing the prominent actin rings. Ankyrin G staining is brighter in PFA-fixed cells.",
    "molecules": "glyoxal, PFA, phalloidin"
  },
  {
    "caption": "B Primary hippocampal neurons were stained for pan-Nav and Kv7.2. Both stainings seem to work at least as well for glyoxal fixed neurons as for PFA fixed neurons. Staining of K-channels shows a slightly more regular pattern in glyoxal fixed neurons.",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": "C Primary hippocampal neurons were stained for Neurofilament L and betaII spectrin. While the spectrin staining seems to be equally well in both fixation conditions, neurofilament staining is brighter in glyoxal fixed cells.",
    "molecules": "glyoxal"
  },
  {
    "caption": "D Growth cones of hippocampal neurons were stained for actin and βIII-tubulin after either glyoxal or PFA + glutaraldehyde fixation. The latter is a standard fixation used for the co-labeling of tubulin and actin, and is a stronger fixation than normal PFA fixation, which is incompatible with many organelle immunostainings (unlike glyoxal fixation). The filopodia and lamellipodia of the growth cones seem to be well stained for the samples fixed with glyoxal, whereas the samples fixed with PFA and glutaraldehyde seem to have lost some of the finer actin structures. Tubulin seems to be a bit better stained in samples fixed with PFA and glutaraldehyde. Scale bars = 1 µm.",
    "molecules": "glutaraldehyde, glyoxal, PFA"
  },
  {
    "caption": " Figure 8 Comparison of immunostained sepia fin after fixation with either PFA or glyoxal. Sepia fin was fixed with the respective fixative and stained for the neuropeptide FMRFamide. A clear change in morphology can be observed between samples fixed with PFA and samples fixed with glyoxal. The former appear broken and swollen, while the glyoxal-fixed ones appear complete. The effect is presumably due to the different speed of penetration into tissue and/or fixation. Scale bar = 5 µm. ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": " Figure 9 Comparison of immunostained ventricular myocytes fixed 10 min with either PFA or glyoxal. Freshly isolated murine ventricular myocytes were either fixed with 4% PFA or 3% glyoxal and immunostained for caveolin-3 or ryanodine receptor type 2. Quantification of the fluorescence intensity of the stainings show significantly brighter stainings for glyoxal fixed myocytes. The graph shows mean values and error bars represent standard deviations. N = 10 (RyR2) and 12 (Cav3) myocytes per condition. Scale bar = 2 µm. *** p < 0.001 ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": " Figure 10 Comparison of immunostained mouse inner hair cells after fixation with either PFA or glyoxal. Acutely dissected organs of corti were fixed in the respective fixative and immunostained for inner hair cell proteins and synaptic proteins. The quantification of fluorescence intensity for each staining shows a significant increase in signal to noise ratio for three target proteins (CtBP2, Calretinin and Homer1) fixed with glyoxal. None of the stained proteins shows a significant decrease in fluorescence after glyoxal fixation. Representative images show maximum intensity projections from z-stacks of inner hair cell ribbon synapses. N = 5 independent stainings from two animals per condition (PSD95, CtBP2, Otoferlin, Calretinin) and 10 - 15 images per condition (CaV1.3 and Homer1). Scale bar = 2 µm. ** p < 0.01, *** p < 0.001 ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": " Figure 11 Comparison of stained vimentin and α-synuclein in human neuroglioma cells after fixation with either PFA or glyoxal. Cells were fixed with PFA or glyoxal for 10 minutes, and were stained for endogenous vimentin, or for expressed alpha-synuclein. A quantification of the staining intensities indicates that glyoxal fixation allows for significantly brighter stainings for alpha-synuclein, but that PFA was superior for endogenous vimentin (leftmost graph). The fluorescence intensity of vimentin expressed with an mOrange2 tag was also analyzed after fixation with PFA or with glyoxal; the latter allowed more mOrange2 fluorescence to be detected (rightmost graph). N = 29 - 81 cell regions per condition. * p < 0.05, *** p < 0.001 Scale bar: 10 µm. ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": " Figure 12 Comparison of immunostainings for mitochondrial proteins after fixation with either PFA or glyoxal. Cells were stained with MitoTracker Orange prior to fixation with the respective fixative and immunostained for the mitochondrial proteins ATP5B, COA6, NDUFA9 and TIM23. Quantification of the staining intensity shows a significant increase of fluorescence (signal over background) for 2 markers (ATP5B and Mitotracker) after fixation with glyoxal, whereas for the remaining 3 proteins immunostainings seem to be more efficient after fixation with PFA, albeit the differences are small. N = 18 - 128 cells per condition. Scale bar = 5 µm. *** p < 0.001 ",
    "molecules": "glyoxal, Mitotracker, MitoTracker Orange, PFA"
  },
  {
    "caption": " Figure 13 Comparison of immunostained HeLa cells after fixation with either PFA or glyoxal. The fluorescence intensity of a variety of proteins was compared between cells fixed with PFA or glyoxal. Quantification is shown as percentage of signal derived from PFA fixed cells. 8 out of the 18 target proteins which were stained show significantly brighter signal when fixed with glyoxal. Only 4 proteins show significantly reduced staining intensity. For LC3B the intensity of less than 5 cells was quantified, therefore single data points were plotted in addition to the bars. N = 3 - 44 cells per condition. Scale bar = 10 µm. ** p < 0.01, *** p < 0.001 ",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": " Figure 14 Comparison of immunostained U2OS cells and primary hippocampal neurons after either PFA or glyoxal fixation. Immunostaining of the Na/K ATPase in primary hippocampal neurons shows a different distribution of the protein between PFA fixed and nucleus fixed samples. While in PFA-fixed neurons the antibody falsely stains the glyoxal as well as the cytoplasm (100% of the 82 cells we analyzed), in nucleus-fixed neurons the U2OS is devoid of signal, and the membrane appears to be correctly labeled (arrowheads; 100% of the 60 cells we analyzed). mitochondria cells were immunostained for KDEL (Tom20) and ER (EGFP-KDEL). The fixation and/or staining of mitochondria seems to be comparable in glyoxal and in PFA fixed cells. The staining of the ER shows an improved signal to noise ratio. The signal appears de-localized from the ER for multiple PFA-stained cells (25% of 36 analyzed cells, see arrows), while this is rare for the glyoxal-stained cells (3.4% of 58 analyzed cells). Scale bar = 10 µm.",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": "A - B Confocal images showing staining of Olfactory Marker Protein (OMP) and ß3-tubulin along the dorsal aspect of the mouse olfactory epithelium. While sections from both types of fixative show OMP signal in the olfactory sensory neuron somata, their dendrites, and axons, the axon bundles (green arrow) located above the olfactory epithelium exemplify the clear signal-to-noise ratio benefits of glyoxal fixation versus that of the PFA-fixative. Immunostaining with the ß3-tubulin antibody stains the dendrites and axons in both PFA and glyoxal-fixed tissue, but strong staining of the cilia can only be observed in the glyoxal-fixed sections (B).",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": "C - D Confocal images depicting bundles of axons belonging to olfactory sensory neurons on the path toward the olfactory bulb. Identities of the axons are in part defined by the neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2) expression levels, visualized here with antibodies raised against the two proteins. While complementary expression of the two molecules can be seen in the PFA-fixed sections (C), the glyoxal-fixed sections (D) exhibit profoundly improved signal-to-noise ratios for Nrp-1 (red arrows), and in the case of Nrp-2, also the segmentation of the axon bundle into varying levels of Nrp-2 (green arrows).",
    "molecules": "glyoxal, PFA"
  },
  {
    "caption": "E - F Confocal images of olfactory sensory neuron axons coalescing into glomeruli where they synapse with dendrites of olfactory bulb neurons. The axons of olfactory sensory neurons can be readily visualized with PFA staining (green) in the superficial olfactory nerve layer and terminating in glomeruli located below (green arrows). While sections fixed with either PFA or glyoxal display adequate staining levels, the signal distribution of the Immunostaining-fixed tissue appears more irregular (E), seemingly lacking the neurofilimentary morphology that appears preserved in the glyoxal-fixed sections (F). The glomeruli themselves are neuropil structures comprised primarily of olfactory sensory neurons forming synapses with dendrites of mitral/tufted cells as well as PFA of periglomerular neurons. Immunostaining with Vesicular Glutamate Transporter 2 (VGLUT2) allows visualization of these structures and is easily seen in the glyoxal-fixed section (F), while it appears the antigen was masked by PFA fixation as no signal above background can be seen in the PFA-fixed panel (red arrows in E). Note that a different polyclonal antibody for VGLUT2 from the same provider does provide signal with glyoxal, albeit weaker versus Glyoxal (inset). Staining with ß3-tubulin touts the benefits of glyoxal both due to the signal improvements in the case of the mild staining in the axons of the olfactory nerve layer that is only visible in the glyoxal-fixed tissue, but also in preserving tissue morphology as demonstrated by the dendritic processes inside glomeruli (blue arrows) and in the external plexiform layer located below the glomeruli.",
    "molecules": "glyoxal, Glyoxal, PFA"
  },
  {
    "caption": "NIH-3T3 cells were treated with TGFβ or mock for 48h and incubated with O-propargyl-puromycin (OPP) for the last 60 min. The translation rate was determined by flow cytometry for OPP incorporation into proteins. A representative plot is shown on the left, and the quantification is shown on the right. Values are relative to mock-treated cells.",
    "molecules": "O-propargyl-puromycin, OPP"
  },
  {
    "caption": "ECM was produced from NIH-3T3 cells according to (d), and collagen abundance was measured by picrosirius red staining, normalized to the packed cell volume before ECM extraction, and expressed relative to mock-treated cells.",
    "molecules": "picrosirius red, collagen"
  },
  {
    "caption": "ATP production rate from glycolysis (JATPgly) and mitochondrial oxidative phosphorylation (JATPox) from NIH-3T3 cells treated with TGFβ or mock for 48h, calculated using Seahorse data shown in (k) and EV Figure 1f.",
    "molecules": "ATP"
  },
  {
    "caption": "Pearson correlation of mitochondrial ATP production rate (JATPox) and OPP incorporation of NIH-3T3 cells treated with TGFβ or mock for 2h, 4h, 8h, 24h and 48h, relative to mock-treated cells. Also see EV Figure 1k and l. Note that the datapoints of the 2h and 4h measurements overlap.",
    "molecules": "ATP, OPP"
  },
  {
    "caption": "NIH-3T3 cells were treated with TGFβ or mock for 48h, and the oxygen consumption rate (OCR) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer. Oligo, Oligomycin; Rot/Anti-A, Rotenone/Antimycin-A; PCV, packed cell volume.",
    "molecules": "Anti-A, Antimycin-A, Oligo, Oligomycin, Rot, Rotenone"
  },
  {
    "caption": "NIH-3T3 cells were treated with TGFβ or mock for 48h and reduced (GSH) and oxidized (GSSG) glutathione were measured by LC-MS/MS. Shown is the GSH/GSSG ratio.",
    "molecules": "glutathione, GSH, GSSG"
  },
  {
    "caption": "NIH-3T3 cells were treated with TGFβ for 48h or mock and incubated with CM-H2DCFDA for the last 30 min. ROS were measured by flow cytometry for CM-H2DCFDA. Values are relative to mock-treated cells.",
    "molecules": "CM-H2DCFDA, ROS"
  },
  {
    "caption": "Comparison of the amino acid representation in all human proteins and all collagens. The x-axis denotes individual amino acids shown by single letter code.",
    "molecules": "amino acid, amino acids, collagens, proteins"
  },
  {
    "caption": "Glycine and proline representation in protein acid hydrolysates of untreated NIH-3T3 cells and their secreted ECM, produced as in Figure 1d. Amino acids were measured by GC-MS.",
    "molecules": "Amino acids, Glycine, proline"
  },
  {
    "caption": "Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells were treated with TGFβ (2 ng/mL for all experiments) or mock for 48h, and the medium was replaced (including all treatments) with DMEM lacking L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolic steady state was reached at this time point (see kinetic labeling curves in Figure EV3b). Metabolites were measured by LC-MS. The left graph shows the total pool size normalized to the packed cell volume; the right side shows the percent labeling. αKG, alpha-ketoglutarate; AU, arbitrary units.",
    "molecules": "alpha-ketoglutarate, αKG, 13C, Gln, L-glutamine"
  },
  {
    "caption": "Glutamine flux into glutamate, αKG and proline in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells.",
    "molecules": "αKG, glutamate, proline"
  },
  {
    "caption": "Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ([12C] Gln, left) and fully labeled glutamine ([U-13C] Gln, right). Peaks representing fully labeled (m+5, m+10) proline are highlighted in red. Peptides were measured by LC-MS.",
    "molecules": "12C, 13C, Gln, glutamine, proline"
  },
  {
    "caption": "Relative abundance of the analytical peptide containing unlabeled (m+0) or one/two fully labelled prolines (m+5/m+10) from the CO1A1 peptide in ECM derived from NIH-3T3 cells treated with TGFβ or mock in the presence of fully labelled L-glutamine ([U-13C] Gln). Peptides were measured by LC-MS. The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions.",
    "molecules": "13C, Gln, L-glutamine, prolines"
  },
  {
    "caption": "Left: NIH-3T3 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2 were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2.",
    "molecules": "proline"
  },
  {
    "caption": "Western blot of NIH-3T3 cells expressing sgCtrl or sgP5CS-2 and treated with TGFβ or mock for 48h in the presence or absence of 0.15 mM proline.",
    "molecules": "proline"
  },
  {
    "caption": "Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl or sgP5CS-2 grown in the presence or absence of TGFβ and 0.15 mM proline, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.",
    "molecules": "picrosirius red, Collagen, proline"
  },
  {
    "caption": "Proline abundance in NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.",
    "molecules": "Proline"
  },
  {
    "caption": "Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated",
    "molecules": "picrosirius red, Collagen"
  },
  {
    "caption": "Analysis of the indicated gene expression datasets for mRNA levels of P5CS: (i) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);",
    "molecules": "Bleo, bleomycin"
  },
  {
    "caption": "Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 48h and the medium was replaced (including all treatments) with DMEM without L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolites were measured by GC-MS.",
    "molecules": "13C, Gln, L-glutamine"
  },
  {
    "caption": "Mitochondrial basal respiration (a) and FCCP-induced maximal respiration (b) of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 2h, 4h, 8h, 24h or 48h, based on Seahorse OCR measurements. The relative values were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.",
    "molecules": "FCCP"
  },
  {
    "caption": "Left: western blot of NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX. MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG-tag. Right: NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are normalized to mock-treated empty vector expressing cells.",
    "molecules": "proline"
  },
  {
    "caption": "NIH-3T3 cells were treated with rotenone, antimycin, myxothiazol or vehicle control for 1h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.",
    "molecules": "antimycin, myxothiazol, proline, rotenone"
  },
  {
    "caption": "NIH-3T3 cells were treated with cobalt chloride (CoCl2) or vehicle control for 6h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.",
    "molecules": "cobalt chloride, CoCl2, proline"
  },
  {
    "caption": "NIH-3T3 cells were treated with TGFβ or mock for 48h, and FCCP or vehicle control was added for the last 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.",
    "molecules": "FCCP, Proline"
  },
  {
    "caption": "NIH-3T3 cells were treated with TGFβ or mock for 48h, and oligomycin or vehicle control was added for the last 1h or 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.",
    "molecules": "oligomycin, Proline"
  },
  {
    "caption": "NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment (Red line). Proline abundance was measured by GC-MS (Blue line). The relative levels were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.",
    "molecules": "CM-H2DCFDA, Proline, ROS"
  },
  {
    "caption": "NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are normalized to mock-treated sgCtrl expressing cells.",
    "molecules": "CM-H2DCFDA, ROS"
  },
  {
    "caption": "NIH-3T3 cells expressing mito-Grx1-roGFP2 or cyto-Grx1-roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control. Emission was measured with a 520/10nm filter after excitation with 405nm and 488nm lasers using flow cytometry. Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm. Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest.",
    "molecules": "H2O2"
  },
  {
    "caption": "NIH-3T3 cells expressing sgCtrl or sgP5CS-2 were treated with TGFβ or mock for 48h in the presence of 0.15 mM proline. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment.",
    "molecules": "CM-H2DCFDA, proline, ROS"
  },
  {
    "caption": "NIH-3T3 cells were treated with vehicle control or CTPi at increasing concentrations (0.1 mM, 0.2 mM, 0.5 mM) or TGFβ (2 ng/mL for all experiments) for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to control-treated cells. P-values represent comparison of individual samples to the control.",
    "molecules": "CM-H2DCFDA, CTPi, ROS"
  },
  {
    "caption": "Left: NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2.",
    "molecules": "CM-H2DCFDA, ROS"
  },
  {
    "caption": "NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells.",
    "molecules": "proline"
  },
  {
    "caption": "Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.",
    "molecules": "picrosirius red, Collagen"
  },
  {
    "caption": "NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated empty vector expressing cells.",
    "molecules": "CM-H2DCFDA, ROS"
  },
  {
    "caption": "NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.",
    "molecules": "proline"
  },
  {
    "caption": "Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated empty vector expressing cells.",
    "molecules": "picrosirius red, Collagen"
  },
  {
    "caption": "Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: (j) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);",
    "molecules": "Bleo, bleomycin"
  },
  {
    "caption": "A. Equal aliquots (2 mg) of a Triton X-100 solubilized lysate from rat brain were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "NP-40 soluble lysates were prepared from COS-7 cells transfected with siRNA as indicated and equal protein aliquots (1.5 mg) were incubated with GST-PAK-CRIB domain. Specifically bound proteins were detected by Western blot with antibody recognizing Rac1. An aliquot of the starting material (SM) equal to 5% of that added to GST-PAK-CRIB was analyzed in parallel.",
    "molecules": "NP-40"
  },
  {
    "caption": "(A, B) U2OS Flp-In/T-REx cells with integrated transgenes encoding GFP-BRCA1BRCT or -BRCA1BRCT S1655A were transfected with non-targeting siRNA (CTRL) or siRNA targeting RAP80 or BRCA1. Following doxycycline treatment to induce transgene expression (5 μg/mL, 24 h), cells were irradiated (10 Gy) and processed for immunofluorescence 1 h post-IR for GFP and antibodies against RAP80 and γH2AX. Shown in (A) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=4). Representative micrographs are shown in (B).",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C, D) U2OS Flp-In/T-REx cells stably integrated with the indicated transgenes were treated with doxycycline (5 μg/mL, 36 h) to induce protein expression and transfected with an siRNA targeting BRCA1 and also either non-targeting siRNA (CTRL) or siRNAs targeting RAP80. 1 h post-irradiation (10 Gy) cells were processed for immunofluorescence using GFP (to detect the BRCA1 fusions) and antibodies against γH2AX. Shown in (C) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A, B) MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies. Shown in (A) is the quantitation of a minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). Representative micrographs are shown in (B).",
    "molecules": "EdU"
  },
  {
    "caption": "MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies. Representative micrographs are shown in (D).",
    "molecules": "EdU"
  },
  {
    "caption": "(E) Clonogenic survival assays using the indicated dose of olaparib and MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions. Data is shown as the mean ± S.D (n=3 biological replicates). Representative clonogenic images are shown in Figure EV5D.",
    "molecules": "olaparib"
  },
  {
    "caption": " qPCR analysis of ERdj3 mRNA in HEK293T cells treated with or without thapsigargin (Tg; 6 h, 100 nM). Error bars represent the mean ± 95% confidence interval as calculated in DataAssist 2.0 (n = 3). ",
    "molecules": "Tg, thapsigargin"
  },
  {
    "caption": "  Representative immunoblot of lysates and media collected from HEK293T-Rex cells stably expressing non-silencing (NS) or ERdj3 shRNA. Cells were treated with the indicated concentration of Tg in fresh media for 16 h. Quantification of the fold increase of ERdj3 in lysates and media collected from HEK293T-Rex cells treated with the indicated concentration of Tg in fresh media for 16 h as in Figure 1C. *indicates a pvalue < 0.05; (n = 3).  ",
    "molecules": "Tg"
  },
  {
    "caption": "  Representative autoradiogram of [35S]-labeled ERdj3 in lysates and media collected from Huh7 cells incubated in the presence or absence of Tg (1 µM). The experimental paradigm for labeling is shown above. Quantification of fraction intracellular (dark grey) and extracellular (light grey) [35S]-labeled ERdj3 following a 4 h chase relative to total [35S]-labeled ERdj3 at 4 h (as shown in Figure 1E). Error bars represent standard error from biological replicates (n = 3). Quantification of total [35S]-labeled ERdj3 in media collected from cells pretreated with vehicle or Tg, as shown in Figure 1E. The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 4 h from vehicle-treated cells. Error bars represent standard error from biological replicates. *indicates a pvalue < 0.05; (n = 3).  ",
    "molecules": "35S, Tg"
  },
  {
    "caption": "Immunoblot in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP], as indicated (Shoulders et al, 2013). Immunoblots of the ATF6 target protein BiP (reactive with the KDEL antibody) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors.",
    "molecules": "TMP, dox"
  },
  {
    "caption": "  Representative autoradiogram of [35S]-labeled ERdj3 immunopurified from lysates and media collected from HEK293DAX cells following preactivation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP] (Shoulders et al, 2013). The experimental protocol is shown above. Quantification of relative amounts of [35S]-labeled ERdj3 in media at 8 h collected from cells treated as shown in Figure 2B (n=3). The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 8 h from vehicle-treated cells. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01; *** indicates p-value < 0.001 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 secreted from autoradiograms as shown in Figure 2B. The fraction of ERdj3 secreted was calculated by normalizing the recovered [35S]-labeled ERdj3 signal in the media at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition. Quantification of the fraction ERdj3 remaining from autoradiograms as shown in Figure 2B. The fraction of ERdj3 remaining was calculated by normalizing the sum of the recovered [35S]-labeled ERdj3 signal in the media and in the lysates at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition.  ",
    "molecules": "35S, TMP, dox"
  },
  {
    "caption": " Plot showing the time-dependent increase in ThioT fluorescence (ex:440 nm, em:485 nm) of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence (black) of recombinant ERdj3 (RERdj3). The RERdj3 concentrations and molar ratios of RERdj3 to A1-40 are as follows: Violet: 1.5 nM, 1:6561; light blue: 4.6 nM, 1:2187; dark blue: 14 nM, 1:729; dark green: 41 nM, 1:243; light green: 120 nM, 1:81; yellow: 370 nM, 1:27; orange: 1.1 M, 1:9; red: 3.3 M, 1:3, or maroon: 10 M, 1:1. All traces represent the average of three replicates. ",
    "molecules": "ThioT"
  },
  {
    "caption": " Plot showing the time-dependent increase in ThioT fluorescence of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence of BSA (14 µg/mL) or RERdj3 (14 µg/mL; 370 nM, RERdj3: A=1:27). All traces represent the average of three replicates. ",
    "molecules": "ThioT"
  },
  {
    "caption": "Immunoblot of M2 anti-FLAG immunopurifications from conditioned media of HEK293T cells overexpressing FTTTRA25T and ERdj3WT in the presence of the indicated concentrations of Tafamidis, a kinetic stabilizer of TTR tetramers. Media inputs (1:400) are shown as a control.",
    "molecules": "Tafamidis"
  },
  {
    "caption": "  Representative immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293DAX cells co-overexpressing FTTTRA25T and ERdj3WT or ERdj3H53Q. Media contained vehicle or trimethoprim (TMP, 10 M, 16 h; activates ATF6), as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of the ATF6 target proteins BiP and GRP94 in cells following TMP-dependent activation of ATF6. Bar graph depicting quantification of the change in the amount of ERdj3 co-immunoprecipitating with TTRA25T following activation of ATF6, as in Figure 6A. The ERdj3/TTR ratio is normalized to the ratio in the absence of ATF6 activation. *indicates a p-value < 0.05; n = 3.  ",
    "molecules": "TMP, trimethoprim"
  },
  {
    "caption": "A. N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159-175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs.",
    "molecules": "glycans"
  },
  {
    "caption": "B. The additional band of Δ159-167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159-167(169) the third glycosylation site is not present anymore.",
    "molecules": "glycan"
  },
  {
    "caption": "C. ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.",
    "molecules": "TX100"
  },
  {
    "caption": "A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159-175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non-infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30.",
    "molecules": "sarcosyl"
  },
  {
    "caption": "A. ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159-175. Det denotes detergent phase, Aq aqueous phase.",
    "molecules": "GdnHCl, TX114"
  },
  {
    "caption": "B. ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159-175 almost exclusively in detergent phase (lanes 1-4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions.",
    "molecules": "GdnHCl, TX114"
  },
  {
    "caption": "C. Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).",
    "molecules": "GdnHCl"
  },
  {
    "caption": "A. Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159-175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159-175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159-175 were statistically analyzed for mean and standard deviation (error bars).",
    "molecules": "3-methyladenine, bafilomycin A1, MG132"
  },
  {
    "caption": "B. ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.",
    "molecules": "GdnHCl, GndHCl"
  },
  {
    "caption": ". B. Δ31-160 variants are expressed at comparable levels and have EndoH-sensitive and -resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31-160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31-160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrPc was not detected under used conditions.",
    "molecules": "dithiothreitol, glycans"
  },
  {
    "caption": "METTL14  is  arginine methylated  at its C-terminal IDR in cells.  HEK293  cells expressing Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14 were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of  immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA. the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins.",
    "molecules": "arginine, Arginine, ADMA"
  },
  {
    "caption": "Knockdown of PRMT1 expression reduces METTL14 arginine methylation. HEK293 cells were transfected with control  siRNA  (siCtrl) and the siRNA targeting PRMT1 (siPRMT1). METTL14 was immunoprecipitated from these cells, and its methylation level was detected by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG  heavy chain.",
    "molecules": "arginine"
  },
  {
    "caption": "Arginine methylation  of the C-terminal IDR enhances the RNA methylation activity of the METTL14/METTL3  complex in vitro. In vitro RNA methylation assays were performed by incubating biotin-labeled RNA substrates with METTL3/METTL14 methyltransferase complexes containing WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 in various concentrations (10-100 nM). The methylation status of the METTL3/METTL14  complex was confirmed by Western blot analysis using an anti-ADMA antibody. Coomassie staining shows the purification of the enzyme complex. Enzymatic activity was measured in counts per minute (c.p.m.) using a scintillation counter. Data from three replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01; ns, not significant.",
    "molecules": "Arginine, arginine, biotin, MS023, ADMA"
  },
  {
    "caption": "Co-IP  assays were performed to compare the  interactions between METTL14 and RNAPII in control and MS023-treated HEK293 cells. Cells were treated with either DMSO or MS023 (1 μM) for 48 h before they were lysed. IP was performed using control  IgG and METTL14 antibodies, respectively. Western blot analysis was performed using anti-RNAPII, anti-ADMA, anti-METTL14, and anti-METTL3  antibodies.   Co-IP assays were performed to examine the involvement of RNA in the METTL14-RNAPII interaction. Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before  IP. Western blot analysis was performed using anti-RNAPII, anti-METTL3, and anti-METTL14 antibodies.   ",
    "molecules": "DMSO, RNase A, MS023, ADMA"
  },
  {
    "caption": "m6A  levels are reduced in mESCs expressing arginine methylation-deficient (RK) mutant METTL14. The mRNA purified from WT, Mettl14 KO, KO+WT, and KO+RK mESCs was subjected to  LC-MS/MS analysis to quantify m6A  levels (presented as the m6A/A ratio). data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.",
    "molecules": "arginine, m6A"
  },
  {
    "caption": "MeRIP (m6A-IP)-qPCR  assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to validate the MeRIP-seq results. m6A-negative regions of the transcripts (blue) were included as negative controls. : In (B) , data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.",
    "molecules": "m6A"
  },
  {
    "caption": "The expression  of ICL repair genes is reduced in mESCs expressing arginine methylation-deficient mutant (RK) METTL14. Total cell lysates from WT, Mettl14  KO, KO+WT, and KO+RK mESCs  were subjected to Western blot  analysis  using the indicated antibodies.",
    "molecules": "arginine"
  },
  {
    "caption": "mESCs  expressing arginine methylation-deficient mutant (RK) METTL14 are sensitive to ICL damage induced by MMC. WT, Mettl14 KO, KO+WT, and KO+RK  mESCs were treated with various concentrations of MMC  for 4 days before  cell viability was measured.   Similar to (A), except that mESCs were treated with cisplatin, another ICL-inducing chemical, at various concentrations.   In both (A) and (B), each point represents the average of three independent replicates and error bars represent standard deviation (SD). Statistical analysis was performed using Student's t-test. *, p < 0.05.",
    "molecules": "arginine, cisplatin, MMC"
  },
  {
    "caption": "Top: Schematic representation of the TEL7L::URA3 reporter used to assay silencing at telomeres. Bottom: wt cells (GA-503) transformed with either an empty plasmid (row 1), plasmid overexpressing wt Sir4 H‑BRCT (row 2), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 3). Cells lacking endogenous sir4 (GA-5822) (row 4). Cells were grown in the absence of uracil and plated as 1:5 serial dilutions onto control YPAD or YPAD lacking uracil plates and imaged after 2 days. Top: Schematic representation of the HMR-EΔB::TRP1 reporter gene locus used for assaying silencing at the HMR locus. Bottom: wt cells (GA-485) transformed with plasmids as in panel D. Cells lacking endogenous sir4 (GA-6888) (row 4). Cells were grown in the absence of tryptophan and plated as 1:5 serial dilutions onto YPAD or YPAD lacking tryptophan plates and imaged after 2 days.",
    "molecules": "tryptophan, uracil"
  },
  {
    "caption": "C-E, boxplots showing the serum  level of phenylacetylglutamine, mandelic acid  and homovanillic acid  in different CHF  subgroups in the REM-HF  cohort. NGT: n = 23; NGT+CHF: n = 48; Prediabetes+CHF: n = 83; T2D+CHF: n = 56; Prediabetes+CHF+CKD: n = 34; T2D+CHF+CKD: n = 16 (biological replicates). Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.",
    "molecules": "homovanillic acid, mandelic acid, phenylacetylglutamine"
  },
  {
    "caption": "F, the boxplot showing the serum  level of ImP  in the Swedish prediabetes  versus the Chinese REM-HF  cohorts. For Swedish prediabetes  cohort, NGT: n = 125; prediabetes: n = 262; T2D: n = 17 (biological replicates). For Chinese REM-HF  cohort: NGT: n = 23; NGT+CHF: n = 48; Prediabetes+CHF: n = 83; T2D+CHF: n = 56; Prediabetes+CHF+CKD: n = 34; T2D+CHF+CKD: n = 16. Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.",
    "molecules": "ImP"
  },
  {
    "caption": "G, differences in ImP  when stratified by left ventricular ejection fraction (LVEF; two-tailed Wilcoxon rank-sum tests). NGT: n = 23; HFpEF: LVEF  ≥50, n = 66; HFmEF: 40<LVEF<50, n = 68; HFrEF: LVEF ≤40, n = 103 participants. Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.",
    "molecules": "ImP"
  },
  {
    "caption": "B, representative   JC-1 staining  images showing red fluorescence of  JC-1  aggregates and green signal of monomers. Scale bars: 20 μm.",
    "molecules": "JC-1"
  },
  {
    "caption": "B, the  Kaplan-Meier survival curve  based on each microbially associated metabolite (grey colors except for imidazole propionic acid) and a combined metabolite risk score. 244 participants who completed the follow-up study were included in survival  analysis (biological replicates). P values were calculated based on the log-rank test.",
    "molecules": "imidazole propionic acid"
  },
  {
    "caption": "Confocal laser scanning fluorescence microscopy (CLSM) images showing DNA staining (blue in the merged images), rDNA FISH (green) and NCL immunofluorescence (red) in untreated HeLa cells or in cells treated with α‐amanitin (50 μg/ml), AMD (50 ng/ml) or the Pol III inhibitor ML‐60218 (200 μM) for 5 h.",
    "molecules": "AMD, α‐amanitin, DNA, ML‐60218"
  },
  {
    "caption": "CLSM images of propidium iodide‐stained RNA after microinjection of buffer or nucleolar RNA into α‐amanitin‐treated HeLa cells. Nucleoli were visualized by immunofluorescence of nucleophosmin (NPM). The graph represents the average number of nucleolar domains (± 95% CI) based on the analysis of 90, 87, 80 and 86 cells, respectively. **P‐value 0.01, t‐test.",
    "molecules": "α‐amanitin, RNA"
  },
  {
    "caption": "CLSM images showing nucleolar co‐localization of NCL (immunofluorescence) with aluRNA (RNA FISH) but not with L1‐repeat RNA. Cells were pre‐treated in situ with RNase A or an RNase inhibitor. Nuclei were counterstained with DAPI. The signal intensity of nucleolar aluRNA was two‐fold higher compared to nucleoplasmic signal (n = 92, P‐value 0.05, t‐test).",
    "molecules": "RNase inhibitor, RNA"
  },
  {
    "caption": "Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre‐treated with α‐amanitin (50 μg/ml) for 5 h or left untreated (control) (± 95% CI. **P‐value 0.01, n = 90, 87, 86, 83 or 86 cells, respectively). Representative CLSM images of propidium iodide‐stained RNA are shown on the right side.",
    "molecules": "α‐amanitin, RNA"
  },
  {
    "caption": "CLSMimages showing NPM (immunofluorescence, red) in HeLacells transfected with control ASO, aluRNAASO, or treated with transfection reagent only (mock). Nuclei were counterstained with DAPI (blue). A fraction of 80 ± 8% of the cells treated with aluRNAASO (n = 143) showed irregular nucleoli or dispersed nucleoli (53% of the cells). Only 26 ± 9% of the cells treated with control ASO presented abnormal nucleoli, with none of them showing a dispersion of nucleolar domains (n = 123), P‐value 0.01, t‐test.",
    "molecules": "ASO, RNA"
  },
  {
    "caption": "CLSM images showing NCL localization (immunofluorescence), aluRNA (RNA FISH) and DNA (DAPI). Transfection of HeLa cells was done as in (A).",
    "molecules": "DNA, RNA"
  },
  {
    "caption": "HeLa cells were transfected as in (A), and nascent RNAs were visualized by pulse labeling with ethynyl uridine (EU) for 30 min. Nuclei were counterstained with DAPI (DNA).",
    "molecules": "DNA, RNAs"
  },
  {
    "caption": "Levels of 47Spre‐rRNA in mock‐, control ASO‐ and aluRNAASO‐treated HeLacells. Top: Northern blot. Center: Agarose gel electrophoretic analysis of RNA. Bottom panel: Quantification from RT-qPCR levels of 47Spre‐rRNA normalized to 18SrRNA. Error bars represent the standard deviation (n = 6). **P‐value 0.01, t‐test.",
    "molecules": "ASO, RNA, 18S, 47S, rRNA"
  },
  {
    "caption": "HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1‐repeat RNA and GFP from a separate promoter. Transfected cells were identified by GFP fluorescence, and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM. The graphs show that the volume of nucleoli is increased by 30% in the presence of ectopic aluRNA (± 95% CI, n = 20). **P‐value 0.01, t‐test. Scale bar, 10 μm.",
    "molecules": "RNA"
  },
  {
    "caption": "CLSM live‐cell imaging of RFP‐NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h. The arrows indicate nucleolar domains displaying time‐dependent increase in the nucleolar size or fusion of two domains into one. Scale bars, 10 μm.",
    "molecules": "RNA"
  },
  {
    "caption": "HeLa cells were transfected as in (A) and incubated for 24 h. Nascent RNA was pulse‐labeled for 30 min, and nucleoli were visualized by immunofluorescence of NPM. Scale bars, 10 μm.",
    "molecules": "RNA"
  },
  {
    "caption": "Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27% increase in the presence of ectopic aluRNA (± 95% CI, n = 30). **P‐value 0.01, *P‐value = 0.01, t‐test.",
    "molecules": "RNA"
  },
  {
    "caption": "CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α‐amanitin to inhibit Pol II transcription show the dispersion of nucleoli, as previously also imaged via RNAstaining (Caudron‐Herger et al, ).",
    "molecules": "α‐amanitin, RNA"
  },
  {
    "caption": "CLSMimages showing the localization of NPM (immunofluorescence, red) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI (blue).",
    "molecules": "ASO"
  },
  {
    "caption": "CLSM images of MS2‐tagged aluRNA or MS2‐tagged control RNA (a fragment from the transcript of the CDV3 gene). MS2‐loop‐containing RNAs were visualized by RNA FISH using MS2‐loop‐specific FISH probes (green); nucleoli were visualized by immunofluorescence of NCL (red).",
    "molecules": "RNA, RNAs"
  },
  {
    "caption": "The top panel depicts the experimental approach used to tether MS2‐loop‐containing RNAs to lacO arrays via a GFP‐tagged LacI‐MS2 coat fusion protein. The lacO arrays were stably integrated in the genome of U2OS cells. In the bottom panel, CLSM images of U2OS cells are shown that were transfected with MS2‐aluRNAR. They reveal the localization of LacI‐GFP‐MS2 and NCL (immunofluorescence, red). The arrow indicates the lacO array, which is associated with a nucleolar domain.",
    "molecules": "RNA, RNAs"
  },
  {
    "caption": "MS2‐loop‐containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line. The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli (± 95% CI) and is plotted in the bar chart. Calculations are based on analysis of more than 100 cells. **P‐value 0.01, t‐test, from the analysis of two independent biological replicates. As controls, transcripts of the MS2 loops only, the MS2‐CDV3, MS2‐RepA, MS2‐STARD7 and MS2‐CORO1C RNAs were used. The inset shows a CLSM image with MS2‐GFP‐LacI (green) and NPM (red, immunofluorescence) after recruitment of MS2‐aluRNA.",
    "molecules": "RNA"
  },
  {
    "caption": "Top: Scheme illustrating the experimental approach used to tether GFP‐tagged LacI fusion proteins via a GFP‐binding protein (GBP) to lacO arrays. The lacO arrays are stably integrated in the genome of U2OS cells. Bottom: CLSM images showing the localization of GBP‐LacI‐RFP, GFP‐NCL, GFP‐NPM or GFP‐TIP5‐RBD fusion proteins relative to aluRNA that was visualized by RNA FISH. The insets contain an enlarged image of one of the lacO loci.",
    "molecules": "RNA"
  },
  {
    "caption": "CLSM images showing GBP‐LacI‐RFP and GFP‐NCL (green) recruited to lacO arrays as indicated by arrows. Immunofluorescence of endogenous NPM (red) marks nucleoli. The inset shows decondensation of lacO arrays in the nucleolus. The graph at the bottom depicts the average number of lacO loci tethered to nucleoli (± 95% CI) by the indicated proteins (n = 72). NCL‐∆RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding. **P‐value 0.01, t‐test.",
    "molecules": "RNA"
  },
  {
    "caption": "CLSM images of UBF (immunofluorescence, green) or rDNA (DNA FISH, green) and DNA (DAPI, blue) after knockdown of NCL and NPM by siRNA in HeLa cells.",
    "molecules": "DNA"
  },
  {
    "caption": "(E, F) Representative FACS plots (E) and quantification (F) of the proliferation  of EGFP+ CD4+ T cells expressing the indicated proteins using the Cell Trace Violet  method. In E, the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4+ T cells  before stimulation and retroviral transduction. In F, each point represents the values obtained with a single experimental mouse. n = 6 per each experimental condition, except in the case of  Vav1∆N (n = 3). Data information: values are shown as means ± SEM from three independent experiments.",
    "molecules": "Cell Trace Violet"
  },
  {
    "caption": "A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.",
    "molecules": "erastin"
  },
  {
    "caption": "B, H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.",
    "molecules": "erastin"
  },
  {
    "caption": "C, Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.",
    "molecules": "C11-BODIPY, Lipid, ROS"
  },
  {
    "caption": "E, Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.",
    "molecules": "erastin, ferrostatin-1"
  },
  {
    "caption": "B, Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.",
    "molecules": "erastin"
  },
  {
    "caption": "C, Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.",
    "molecules": "GSH"
  },
  {
    "caption": "D, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). E, Surviving cells from the assay shown in (D) were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.",
    "molecules": "erastin"
  },
  {
    "caption": "H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells.",
    "molecules": "iron"
  },
  {
    "caption": "I, Labile iron levels examined were quantified. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.",
    "molecules": "iron"
  },
  {
    "caption": "F, SMMC cells were treated with 100 μM etoposide (+) or untreated (-) for 24 h, and Western blot analysis was performed.",
    "molecules": "etoposide"
  },
  {
    "caption": "D, Co-IP analysis for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) treated with a USP7-specific inhibitor P5091.",
    "molecules": "P5091"
  },
  {
    "caption": "A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.",
    "molecules": "erastin"
  },
  {
    "caption": "B, H1299 cells were transfected with Flag-tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h. Co-IP analysis was performed with antibodies against Flag.",
    "molecules": "erastin"
  },
  {
    "caption": "C, H1299 cells transfected with Flag-tagged p53 were treated with erastin or untreated for 24 h. Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis.",
    "molecules": "erastin"
  },
  {
    "caption": "D, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA, or a control siRNA (siCont.) were treated with 20 μM erastin or untreated for 24 h as indicated. Western blot analysis was then performed.",
    "molecules": "erastin"
  },
  {
    "caption": "G, H1299 cells transfected with Flag-tagged p53 were treated with 12 μM erastin or untreated for 24 h. ChIP analysis with antibodies against Flag or USP7 was performed. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.",
    "molecules": "erastin"
  },
  {
    "caption": "H, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h. ChIP analysis was performed with anti-H2Bub1 antibodies. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.",
    "molecules": "erastin"
  },
  {
    "caption": "A, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as were treated with 12 μM erastin (+) or untreated (-) for 24 h as indicated. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.",
    "molecules": "erastin"
  },
  {
    "caption": "C, Lipid ROS levels were examined in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.",
    "molecules": "Lipid, ROS"
  },
  {
    "caption": "a, Flow chart of an in vivo screen to identify active DUBs by using biotin-Ub-VME pull-down assay (left panel), in which the USP8 peptide was identified by mass-spectrometry analysis (right panel). Silver staining and immunoblot (middle panel) of precipitants pulled down by biotin-Ub-VME probe of MDA-MB-231 cells.",
    "molecules": "biotin, Silver"
  },
  {
    "caption": "e, MCF10A-Ras cells stably expressing control vector (Co.vec), USP8 wt or USP8 cs were injected into the blood circulation of 48-hpf zebrafish embryos. SB431542 (5 μM) was added to the zebrafish environment. Representative images of zebrafish at 5 dpi (left panel); the experimental metastatic areas (middle panel) and percentage of embryos showing metastasis (right panel) are shown, n = 20.",
    "molecules": "SB431542"
  },
  {
    "caption": "j, Immunofluorescence and 4, 6-diamidino-2-phenylindole (DAPI) staining of HaCaT cells infected with control or USP8 wt/cs and treated with TGF-β (2.5 ng/ml) and SB431542 (10 μM) for 72 hrs. Scale bar, 20 μm.",
    "molecules": "4, 6-diamidino-2-phenylindole, DAPI, SB431542"
  },
  {
    "caption": "k, Immunoblot analysis of cell lysate of control and USP8 stably depleted-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) for 48 hrs (left); Immunoblot analysis of cell lysate of control and USP8 wt or USP8 cs stably expressed-MCF10AR-RAS (MII) cells treated with TGF-β (5 ng/ml) or SB431542 (10 μM) for 48 hrs (right).",
    "molecules": "SB431542"
  },
  {
    "caption": "a, Immunofluorescence and DAPI staining of HeLa cells transfected with TβRII-HA and Flag-USP8. Scale bar, 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "d, IB of Nickle-pull down precipitants derived from His-Ub stably expressed HEK293T cells incubated with purified USP8 protein as indicated time points.",
    "molecules": "Nickle"
  },
  {
    "caption": "e, IB of TCL and immunoprecipitants derived from HEK293T cells transfected with indicated plasmids and treat with or without MG132 (10 μM) for 4 h.",
    "molecules": "MG132"
  },
  {
    "caption": "g, IB of HeLa cells stably expressing TβRII-HA and transfected with control empty vector (Co.vector) and Flag-USP8 wt/cs plasmids and treated with CHX (20 μg/ml) for indicated time points.",
    "molecules": "CHX"
  },
  {
    "caption": "a, Comparison of the pre-treatment levels of USP8 mRNA in patient-derived tissue samples by qRT-PCR (left panel), enzyme activity of USP8 in patient-derived tissue samples by Ub-VME (middle panel) and TβRII+ crEVs level in the plasma from breast cancer patients by ELISA (right panel) between breast cancer patients with or without clinical response to PTX. R, responders; n = 16; NR, non-responders; n = 14.",
    "molecules": "PTX"
  },
  {
    "caption": "b, Pearson correlation of the USP8 mRNA or USP8 enzyme activity in patient-derived tissue samples with the EV-TβRII levels in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).",
    "molecules": "PTX"
  },
  {
    "caption": "d, Pearson correlation of the percentage of IFN-γ+ of CD8+ cells to the TβRII+ crEV level in the plasma from breast cancer patients without clinical response to PTX as in a (NR, n = 14).",
    "molecules": "PTX"
  },
  {
    "caption": "e, TβRII+ crEVs (left panel), the USP8 enzyme activity (midde panel) and USP8 mRNA (right panel) from breast cancer patients with or without clinical response to combined therapy using anti-PD-L1 (Atezolizumab) and paclitaxel. R, responders; n = 10 (left panel), n = 5 (middle and right panels, only 5 patient-derived tissue samples obtained). NR, non-responders; n = 10 (left panel), n = 6 (middle and right panels, only 5 patient-derived tissue samples obtained).",
    "molecules": "Atezolizumab, paclitaxel"
  },
  {
    "caption": "nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Normalized BLI signals (left panel) and representative mice (right panel) from each group at the indicated times (j).",
    "molecules": "doxycycline"
  },
  {
    "caption": "nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) Immunoblot analysis of the circulating EVs from plasma in mice (k).",
    "molecules": "doxycycline"
  },
  {
    "caption": "nude mice were tail vein-injected with 4T07-Luc cells (5 × 105 cells per mouse) expressing control vector or doxycycline-inducible USP8-wt/cs and tumors were grown for 2 weeks, followed by the administration of doxycycline (n = 6 for each group) ELISA analysis of circulating exosomal TβRII level in plasma samples of mice at the indicated times (l).",
    "molecules": "doxycycline"
  },
  {
    "caption": "h, Experimental procedure in vivo: BALB/c mice were nipple injected with 4T1-Luc cells (5 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting USP8 (shUSP8) and tumors were grown for 3 weeks, followed by the administration of doxycycline for 3 weeks (n = 6 for each group). i, Quantification of the percentage of TβRII+ crEVs in plasma samples from mice at day 24. j, Immunoblot analysis of tumor and the TEVs (left panel) and quantification of the percentage of TβRII+ EVs (right panel) in plasma samples from mice at day 42. k, Normalized photon flux at the indicated times. l,",
    "molecules": "doxycycline"
  },
  {
    "caption": "e, Heatmap shows differentially expressed genes in control DMSO-treated or USP8 inhibitor (5 μM)-treated MDA-MB-231 cells for 24 h (left panel). Volcano plot of transcriptome profiles between control and USP8 inhibitor treated MDA-MB-231 cells (right panel). Red and green dots represent genes significantly up-regulated and down-regulated in control MDA-MB-231 cells.",
    "molecules": "DMSO"
  },
  {
    "caption": "f, qRT-PCR analysis of control (DMSO-treated) or USP8 inhibitor (5 μM for 24 h) treated MDA-MB-231 cells. Relative mRNA levels are shown as a heatmap.",
    "molecules": "DMSO"
  },
  {
    "caption": "Interaction analysis for MIA40 and AK2. Stable inducible cell lines that express either different variants of MIA40-Strep (wildtype and a redox inactive mutant (MIA40C53S,C55S), (Fischer et al., 2013) or AK2-HA were treated with N-ethyl maleimide (NEM) to stop thiol-disulfide exchange processes and lysed under denaturing conditions. Lysates were analysed by subsequent affinity purification against Strep (MIA40) and immunoblotting. Like classical MIA40 substrates, AK2 interacted with the active site of MIA40 (lane 6). 0.5% input was loaded as control.",
    "molecules": "disulfide, N-ethyl maleimide, NEM, thiol"
  },
  {
    "caption": "Redox state analysis of endogenous AK2 by inverse redox shift assay. HEK293 cells were lysed by TCA precipitation after NEM treatment, lysates were reduced using TCEP and then thiols modified using the alkylating agent mmPEG12. With this "inverse" method, previously oxidized cysteine thiols are modified with mmPEG12, which results in a slower migration on SDS-PAGE. Treatment of intact cells with diamide and consequently modification of all cysteines served as control. Likewise, absence of NEM treatment served as 'max' control. Treatment lysates with NEM instead of mmPEG12 served as minimal shift (min) control. A, B, isoforms of AK2. Both isoforms of AK2 are semi-oxidized in intact cells.",
    "molecules": "diamide, cysteine, cysteines, NEM, mmPEG12, SDS, TCEP, thiols, TCA"
  },
  {
    "caption": "Oxidation kinetics experiment to investigate maturation of endogenous AK2. HEK293 cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by TCA precipitation, and then the lysate was treated with mmPEG12 to determine the AK2 redox states, followed by IP against AK2. Eluates were analyzed by Tris-Tricine-PAGE and autoradiography. With this \"direct\" redox state determination method reduced AK2 was modified with either three (isoform B) or four (isoform A) mmPEG12. Semi-oxidized AK2 was modified with only one (\"B\") or two (\"A\") mmPEG12, respectively. Unmodified and fully reduced cell lysates served as controls. Endogenous AK2 becomes oxidized with a half time of less than 20 min.",
    "molecules": "35S, methionine, mmPEG12, TCA, Tricine, Tris"
  },
  {
    "caption": "Influence of AK2 cysteines on the AK2 redox state. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were lysed after NEM treatment, and analyzed by inverse redox shift assay using mmPEG12, SDS-PAGE and immunoblot against the HA tag. AK2C42S and AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines.",
    "molecules": "cysteine, cysteines, disulfide, NEM, mmPEG12, serine, SDS"
  },
  {
    "caption": "Influence of AK2 cysteines on cellular localization. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were fixed and analyzed by immunofluorescence. The colocalization of AK2-HA variants with Mitotracker was quantified using Cellprofiler (Pearson's correlation coefficient). C42 and C92 are critical for mitochondrial localization of AK2. The C40S mutant exhibits significantly decreased colocalization with mitochondria. A Kruskall-Wallis rank sum test/ Dunn-test was performed. Numbers of analysed cells are indicated in the plot. *** represents p-value <0.001 compared to wild-type. The black horizontal line represents the median. Data information: Scale bars: 10 μm.",
    "molecules": "Mitotracker, cysteine, cysteines, serine"
  },
  {
    "caption": "Stability of endogenous AK2 in the presence and absence of MIA40. 72 h before experiments HeLa, cells were transfected with control siRNA or siRNAs directed against MIA40. Cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by cell lysis, and AK2 was isolated by denaturing immunoprecipitation. Eluates were analyzed by SDS-PAGE and autoradiography. Stability of endogenous AK2 is significantly decreased upon depletion of MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed; * represents p-value <0.05 compared to wild-type.",
    "molecules": "35S, methionine, SDS"
  },
  {
    "caption": "Stability of cytosolic AK2-HA cysteine variant. Experiment was performed as in (B) except that cells expressing AK2C40S,C42S,C92S-HA were analysed by IP against HA. AK2C40S,C42S,C92S-HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.",
    "molecules": "cysteine, MG132"
  },
  {
    "caption": "Steady state levels of AK2-HA cysteine variants. AK2C40S,C42S,C92S-HA and AK2WT-HA were expressed in the presence or absence of proteasomal inhibitor MG132. Protein levels at steady state were analysed by immunoblot. All variants are stabilized by proteasomal inhibition. Reported values are the mean of 3 (AK2WT-HA) or 5 (AK2C40S,C42S,C92S-HA) independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. *** represents p<0.001.",
    "molecules": "cysteine, MG132"
  },
  {
    "caption": "Steady state levels of AK2-HA cysteine variant. 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5. AK2C40S,C42S,C92S-HA was stabilized by decreased proteasome levels. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.",
    "molecules": "cysteine"
  },
  {
    "caption": "Steady state levels of AK2C40S,C42S,C92S-HA upon incubation with the DPP9 inhibitor 1G244 (Wu et al., 2009). As (B) except that cells were treated for 16 h with inhibitor or DMSO as control. AK2C40S,C42S,C92S-HA levels were strongly increased upon DPP8/9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. *** represents p<0.001.",
    "molecules": "1G244, DMSO"
  },
  {
    "caption": "Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244. As (D) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control. Endogenous AK2 levels were increased upon DPP8/9 inhibition. Reported values are the mean of 5 (3 d) and 6 (5 d) independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, and *** represents p<0.001.",
    "molecules": "1G244, DMSO"
  },
  {
    "caption": "Steady state levels of AK2-HA variants with mutated N-termini upon incubation with either 1G244 or vehicle control. HEK293 cells stably expressing AK2-HA variants were lysed and subjected to immunoblot against the indicated proteins. Levels of DPP8/9-insensitive AK2 variants are strongly increased. Reported values are the mean of 4 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01 and ###, *** represents p<0.001. Asterisk denominate statistical comparison with respective control, and hash tag represents comparison to N-WT.",
    "molecules": "1G244"
  },
  {
    "caption": "Cell fractionation upon inhibition of DPP9 activity. HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO. Cells were subjected to fractionation into 'cytosol' (supernatant) and 'mitochondria' (pellet) by differential digitonin lysis and centrifugation. The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration (lanes 2,5,8,11) irrespective of 1G244 treatment. Likewise, the cytosolic marker protein LDH localized under all conditions to the same fractions. Conversely, endogenous AK2 appeared at this digitonin concentration in the 'cytosol' fraction but only upon 1G244 treatment (compare lanes 2 and 5). Thus, inhibition of DPP9 results in appearance of AK2 in the cytosol. Quantification shows loading-control adjusted AK2-signals of 1G244-treated 'cytosol' fraction (lane 5) over DMSO-treated 'cytosol' fraction (lane 2). White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed.",
    "molecules": "1G244, digitonin, DMSO"
  },
  {
    "caption": "Localization of DPP9-insensitive AK2-HA variants. Experiment was performed as described in Figure 2C. Compared to the wild type, DPP9-insensitive and import-competent AK2-HA variants co-localized with cytosolic markers indicating partial cytosolic localization. The colocalization of AK2-HA variants with Mitotracker was quantified using Fiji (Pearson's correlation coefficient). A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. ** represents p<0.01. Data shown in Figures 2C, 5B, S2 and S5 are derived from the same experiments. The black horizontal line represents the median. Data information: Scale bars: 10 μm.",
    "molecules": "Mitotracker"
  },
  {
    "caption": "Localization of AK2C40S-HA upon proteasomal inhibition. Experiment was performed as described in Figure 2C. Upon MG132 treatment, colocalization of AK2C40S-HA with the cytosolic marker increased. A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. *** represents p<0.001.The black horizontal line represents the median. Data information: Scale bars: 10 μm.",
    "molecules": "MG132"
  },
  {
    "caption": "AK2 activity in dependence of its substrate AMP. Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0.012 mM AMP. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are superimposable. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   AK2 activity in dependence of its substrate ADP. Maximal activity was observed at around 1-2 mM ADP with an apparent KM of 0.12 mM and 0.1 mM ADP for AK2C40S,C232S and AK2C40S,C42S,C92S,C232S, respectively. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are thus highly similar. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   ",
    "molecules": "AMP, ADP"
  },
  {
    "caption": "Activity assay for AK2-HA variants isolated from HEK293 cells. Indicated AK2-HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants. Then, the AK2 activity assay described in (B) was performed. While import-competent AK2 variants exhibited full activity, a cytosolic AK2 variant with the native N-terminus showed very low activity. This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9-insensitive cytosolic AK2 variants. The enzymatic inactive K28Q variants served as negative control indicating that only AK2-HA-dependent activity ways measured by our assay. Reported values are the mean of 2 independent experiments; error bars represent ±SD.",
    "molecules": "1G244"
  },
  {
    "caption": "Steady state levels of NDUFB10, NDUFS5, NDUFA8, COA6, CHCHD2, and TIMM9 upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. Proteins except TIMM9 are stabilized upon DPP9 inhibition. Reported values are the mean of 3-5 independent experiments; error bars represent ±SD. Student's t-test was performed. ** represents p<0.01.",
    "molecules": "1G244, DMSO"
  },
  {
    "caption": "Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. NDUFB10C107S is stabilized upon DPP9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed for the respective control and treatment samples. ** represents p<0.01.",
    "molecules": "1G244, DMSO"
  },
  {
    "caption": "Localization of NDUFB10 variants upon 1G244 treatment. Experiment was performed as in (C) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins. Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition. Reported values are the mean of 2 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.",
    "molecules": "1G244"
  },
  {
    "caption": "D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen",
    "molecules": "T4"
  },
  {
    "caption": "A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student\"s test, ** p<",
    "molecules": "T4"
  },
  {
    "caption": "C) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat",
    "molecules": "paraformaldehyde"
  },
  {
    "caption": "C) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because",
    "molecules": "ADP"
  },
  {
    "caption": "Depletion of RBM14 induces ectopic formation of centriolar protein complexesA-E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase.",
    "molecules": "DNA"
  },
  {
    "caption": "C, D Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold).",
    "molecules": "HU"
  },
  {
    "caption": "E In vitro competitive binding assay. GST pull-down experiment was performed as in (B), with purified STIL[N] and GST-RBM14[C] in the presence of the indicated amount of purified His-CPAP[SBD], His-tagged STIL-Binding Domain of CPAP. The fraction of STIL[N] bound to GST-RBM14[C] in such conditions was monitored by Western blotting using STIL antibodies which recognize the N-terminal region of STIL. Input materials were analyzed by Western blotting using the STIL or CPAP antibodies. The precipitated GST-RBM14[C] was analyzed by SDS-PAGE, stained with SimplyBlue™ Safe (Invitrogen).",
    "molecules": "His"
  },
  {
    "caption": "A U2OS cells treated with control siRNA, RBM14 siRNA alone or RBM14 + HsSAS-6 siRNAs were stained with antibodies against centrin-2 (green) and HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). n.s., not significant (one-tailed t-test).",
    "molecules": "DNA"
  },
  {
    "caption": "D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment. Insets show threefold magnified images of fluorescent foci. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).",
    "molecules": "HU, lovastatin"
  },
  {
    "caption": "E Amorphous electron-dense structures containing microtubules are formed in RBM14-depleted cells (see also Supplementary Fig S5I). Electron micrographs from U2OS cells depleted of RBM14. Squares indicate the ectopic electron-dense structures, whereas circles indicate the pre-existing centrioles. Scale bar, 500 nm and 200 nm (magnified views). Average diameter of pre-existing centrioles and the amorphous electron-dense structures are 225 ± 8 nm (n = 10) and 130 + 11 nm (n = 16), respectively. Insets show magnified views of the squares. Note that microtubules (black arrowheads) are observed within the electron-dense structures (white arrowheads).",
    "molecules": "electron"
  },
  {
    "caption": "G U2OS cells expressing GFP-CPAP (green) and treated with RBM14 siRNA were analyzed using CLEM. Separate z-plane images of the cell with GFP-CPAP foci and the threefold magnified images are aligned from bottom to top along z-axis (left LM panels). White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. Corresponding regions of EM images are shown on the right. White squares (a-d) represent the region around GFP-CPAP foci. Note that other than pre-existing centrioles (a, 1-4), ectopic small CPAP foci are recognizable as amorphous electron-dense structures (b-d). One of pre-existing centrioles (1) is elongated because of exogenous GFP-CPAP expression. Scale bar, 10 μm (LM), 2 μm (EM, low magnified image), 500 nm [magnified images of (a) and (d)), 200 nm (magnified images of (b) and (c)].",
    "molecules": "electron"
  },
  {
    "caption": "B-E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30-60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P < 0.05, **P < 0.01 (one-tailed t-test).",
    "molecules": "DNA"
  },
  {
    "caption": "A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin-2 (green) as well as HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the frequency of mitotic cells with the indicated phenotype. Values are mean percentages ± SEM from three independent experiments (n = 60 for each condition).",
    "molecules": "DNA"
  },
  {
    "caption": "H HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA were analyzed using Live CLEM. Pre-existing GFP-centrin foci (white arrowheads) and ectopic GFP-centrin foci (red arrowheads) were traced throughout the time-lapse recording. Z-stacked confocal images spanning the entire height of the cells (< 30 μm) are shown (upper panels). Time is denoted in hh:min. Time zero corresponds to the onset of ectopic formation of GFP-centrin foci. The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images (lower panels). Red square (ectopically formed GFP-centrin foci) represents the region corresponding to that of the EM images acquired from serial sections. Note that ectopic GFP-centrin foci that form a spindle pole include not only electron-dense structures (1, 3) but also a morphologically recognizable centriole-like structure (2). Scale bars, 10 μm (live imaging) and 500 nm (EM).",
    "molecules": "electron"
  },
  {
    "caption": "(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox"
  },
  {
    "caption": "(D) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes. A dot displays measurements from each experiment. Experiments were performed in PLK4Dox polyclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox"
  },
  {
    "caption": "(F) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(G) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox"
  },
  {
    "caption": "(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox"
  },
  {
    "caption": "(B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm.",
    "molecules": "Dox"
  },
  {
    "caption": "(F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox"
  },
  {
    "caption": "(H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. (I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m. ",
    "molecules": "Dox, dox"
  },
  {
    "caption": "(C) Western blot showing the expression of ANKRD26. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes.",
    "molecules": "Dox"
  },
  {
    "caption": "(D) Quantification of the fraction of cells with ANKRD26 (left) and PIDD1 (right) localized to the mature mother centriole in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox"
  },
  {
    "caption": "(E) Graph showing the relative growth of doxycycline-treated, monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(F) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for 4 days. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes.",
    "molecules": "Dox, dox"
  },
  {
    "caption": "B The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45-/-mice correspond to 4 independent experiments (E1 to E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments (E1 and E2) involving a total of 5 individual mice. The Arhgap45-/- mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, **** P ≤ 0.0001; multiple unpaired T tests.",
    "molecules": "CMTPX, CTV"
  },
  {
    "caption": "E. Double  immunofluorescent staining of Fn12230, fadA-deletion mutant US1  (∆fadA) and spontaneous mutant  lam in log and stationary phases using mAb 7H7 at 1:800 dilution and anti-human β-amyloid polyclonal antibody A11 at 1:500 dilution, followed by incubation with Alexa Fluor 680-conjugated donkey anti-rabbit  and Alexa Fluor 555-conjugated goat anti-mouse, both at 1:1,000 dilution. Co-staining of FadA and A11 was observed in Fn in  stationary phase, not in log phase, or in US1  or lam. The imagines were taken under 60X objective. Scale bar equals 5 μm.",
    "molecules": "Alexa Fluor 680, Alexa Fluor 555"
  },
  {
    "caption": "E. Thioflavin-T binding assay in the presence of 0.1%  SDS. FadA proteins (0.8 mg/ml) were incubated with 10 μM Thioflavin-T  at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01 (compared to  BSA, t-test).",
    "molecules": "BSA, SDS, Thioflavin-T"
  },
  {
    "caption": "G. Analysis of amyloid-like  FadA  produced by  Fn 23726 and its ∆fap2  mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less  amyloid-like polymers than the wild type.",
    "molecules": "amyloid"
  },
  {
    "caption": "B. Double  immunofluorescence staining of paired normal and  carcinoma tissues from two  CRC patients. The frozen tissue sections were incubated with mAb 7H7 at 1:50 dilution and anti-amyloid  antibody A11  at 1:25 dilution, or mouse and rabbit IgG control, followed by incubation with Alexa Fluor 555-conjugated  goat anti-mouse and Alexa Fluor 680-conjugated donkey anti-rabbit, both at 1:1000 dilution. Co-staining of FadA (red) and  amyloid oligomers  (green) was observed in the carcinoma but not normal tissues. The  images were taken under 60X objective. Scale bar equals 10 μm.",
    "molecules": "Alexa Fluor 680, Alexa Fluor 555, oligomers"
  },
  {
    "caption": "B. Inhibition of biofilm formation mediated by detergent-resistant Fn  pellets by Congo Red. An aliquot of 10 μg detergent-resistant pellets prepared from Fn 12230 (pellet) was added in 96-well plates in the presence of 0, 10 or 50 µg/ml Congo Red (CR), followed by addition of Fn 12230 and incubated as described above. The results shown are the average of four experiments each performed in duplicate. The error bars indicate SD. A representative image following 72 hours of incubation is shown in the top panel. ** p<0.01, ***p<0.001 (t-test).",
    "molecules": "Congo Red, CR"
  },
  {
    "caption": "D. Effects of Congo Red on Fn survival in acidic environment. Wild type Fn 12230 (Fn) or its fadA deletion mutant US1 (∆fadA) grown to stationary phase were washed and incubated in PBS at pH4 in the presence of absence of 50 μg/ml Congo Red (CR) for 1 or 2 hours. The live bacterial counts   at time 0 were designated as 100%, and those after 1 or 2 hrs of incubation were expressed relative to  time 0. The results shown are the average of 5 independent experiments, each performed in duplicate. The error bars indicate SD. *p<0.05, ** p<0.01, ***p<0.001 (One-way ANOVA).",
    "molecules": "Congo Red, CR, PBS"
  },
  {
    "caption": "F. Inhibition of  Fn attachment by FadAc in the presence or absence of anti-amyloid antibody  OC. An aliquot of 50 μg purified recombinant FadAc was pre-incubated with  DLD1 for 45 min either alone, or mixed with 20 μl  OC or rabbit IgG control, before Fn was added and the attachment assay performed as above. The attachment value by Fn alone was designated as 100%, with all other values expressed relative to it. FadA mutant L14A was included as a negative control. Data shown are mean values ± SEM. The experiment was performed in duplicate and repeated three times. **p< 0.01 (One-way ANOVA).",
    "molecules": "OC"
  },
  {
    "caption": "D. Effect of detergent-resistant pellets prepared from Fn 12230 (Fn) and US1 (∆fadA) on tumor growth. HCT116 cells were inoculated into the nude mice as described above. In one group of mice (n=3), sarkosyl-resistant pellets prepared from  Fn 12230 and US1 (∆fadA) were injected into the tumors on opposite sides. In another group (n=5), sarkosyl-resistant pellets prepared from Fn 12230 were mixed with OC  or rabbit IgG control (rIgG) before injecting into the tumors on opposite sides. The tumor volumes were measured described above. The vertical lines represent the standard deviations. The individual tumor pairs are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 [compared to US1 (∆fadA), t-test].",
    "molecules": "OC, sarkosyl"
  },
  {
    "caption": "A. Approximately 1x109 CFU of  Fn 12230, US1 (∆fadA), and lam suspended in carboxymethylcellulose (CMC) were orally administered to C57BL/6 mice three times a week for 10 weeks. CMC alone was administered as a control. Maxillae from mice inoculated with CMC  alone (n=5),  Fn 12230 (n=5), US1 (∆fadA; n=5), and lam (n=4) were harvested and fixed in 4% paraformaldehyde  and stored in 70% ethanol, followed by microCT (μCT) scanning using a Scanco vivaCT 80 system at 55 kVp, 145 µA, and 250 ms integration time. Shown in the figure are reconstructed grayscale  images. ImageJ was used to measure difference in bone height from the cementoenamel junction (CEJ) to the alveolar crest  between palatal roots of first  and second molars (see arrows). B. The average bone loss of each group shown in (A), with the lines above each bar representing standard deviations. *p< 0.05. **p< 0.01 (t-test). ",
    "molecules": "carboxymethylcellulose, CMC, ethanol, paraformaldehyde"
  },
  {
    "caption": "F Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. G Sirius red staining in WT and cKI mice after UUO. Scale bar: 50μm.",
    "molecules": "Masson's trichrome, Sirius red"
  },
  {
    "caption": "A Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. B Sirius red staining in WT and cKO mice after UUO. Scale bar: 50μ",
    "molecules": "Masson's trichrome, Sirius red"
  },
  {
    "caption": "C Masson's trichrome staining of WT and cKI mice after 5/6Nx. Scale bar: 100μm.",
    "molecules": "Masson's trichrome"
  },
  {
    "caption": "G Masson's trichrome staining of WT and cKO mice after 5/6Nx. Scale bar: 100μm.",
    "molecules": "Masson's trichrome"
  },
  {
    "caption": "A Quantification of mitochondrial ROS production (n=4, biological replicates).",
    "molecules": "ROS"
  },
  {
    "caption": "J-N Representative Western blot and densitometric analysis of LONP1, FN1, α-SMA and Vimentin of each group was shown. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group). mPTCs were transfected with siNC or siLONP1, and then pre-treated with 84-B10 (10 μM) 2h before TGF-β1 (10ng/mL) treatment for another 24h.",
    "molecules": "84-B10"
  },
  {
    "caption": "A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model. Scale bar: 50μm. B Quantification of fibrotic area in UUO model (n=10 in each group, biological replicates).",
    "molecules": "Sirius red"
  },
  {
    "caption": "G, H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model (n=10 in each group, biological replicates). Scale bar: 50μm.",
    "molecules": "Sirius red"
  },
  {
    "caption": "Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).",
    "molecules": "PLGA, thapsigargin"
  },
  {
    "caption": "Thapsigargin or αGalCer treated J774.2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d-specific antibody (black circles) or isotype (red squares) for 16 hours. The addition of an anti-CD1d antibody blocked the activation of 2C12 demonstrating that the NKT-cell activation was CD1d-specific. Graphs show mean ± SEM from n=2 biological replicates.",
    "molecules": "αGalCer, Thapsigargin"
  },
  {
    "caption": "Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured murine macrophage cell line (J774.2) treated either with thapsigargin- or tunicamycin-treated CD1d knockdown (white bars) or untransfected (black bars). Knockdown of CD1d blocked IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.",
    "molecules": "thapsigargin, tunicamycin"
  },
  {
    "caption": "Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured either with thapsigargin- or tunicamycin-treated primary BMDM isolated from CD1d-/- (white bars) or wild type (black bars) mice. 2C12 cocultured with CD1d-/- BMDMs resulted in reduced IL-2 secretion. Graphs show mean ± SEM from n=3 biological replicates. ***P < 0.001 (Unpaired t test).",
    "molecules": "thapsigargin, tunicamycin"
  },
  {
    "caption": "qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate.",
    "molecules": "Ceapin-a7, 4μ8c, GSK, thapsigargin, tunicamycin"
  },
  {
    "caption": "Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.",
    "molecules": "DMSO, thapsigargin, tunicamycin"
  },
  {
    "caption": "Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).",
    "molecules": "thapsigargin"
  },
  {
    "caption": "Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.",
    "molecules": "DMSO, thapsigargin"
  },
  {
    "caption": "Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.",
    "molecules": "DMSO, thapsigargin"
  },
  {
    "caption": "qPCR analyses showing the expression (fold induction) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin or DMSO for 8 hours. In total n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.",
    "molecules": "DMSO, glycosphingolipid, thapsigargin"
  },
  {
    "caption": "Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells. IL-2 secretion in UPR-induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin. Graphs show mean ± SEM from n = 3 biological replicates. n.s (non-significant) (One-way ANOVA) biological replicates.",
    "molecules": "castanospermine, Glycolipid, deoxygalactonojirimycin"
  },
  {
    "caption": "L363 antibody does not impact UPR-induced NKT cell activation. 2C12 NKT cells were co-cultured with ER-stressed macrophages J774.2 cells and treated with L363 antibody or isotype control. α-GalCer induced responses served as positive control. In total, n=2 biological replicates were performed (Four technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.",
    "molecules": "α-GalCer"
  },
  {
    "caption": "Quantification of Gadd45a-ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc-/- MEF cells (n=3 for each genotype).",
    "molecules": "DNA"
  },
  {
    "caption": "Representative images of small intestine crypt sections stained with antibody against pATM (Ser-1981)(E). Quantification of frequencies of pATM (Ser-1981) positive crypt cells (n=4-6 of 7-month old mice per genotype) (F).",
    "molecules": "Ser"
  },
  {
    "caption": "Telomeric quantification and dot-bot assay of tri-methyl H3K9 (H3K9me3)-ChIP in indicated genotypes",
    "molecules": "H3K9me3"
  },
  {
    "caption": "Telomeric quantification and dot-bot assay of acetylated H3K9 (H3K9ac)-ChIP in indicated genotypes",
    "molecules": "H3K9ac"
  },
  {
    "caption": "Representative images of small intestine organoids from WT and G3Terc-/- mice incubated with or without APE1 inhibitor (CRT0044876, 10μM) treatment. Arrows indicate the crypt-like structure in organoid cultures (A). Quantification of organoid number (per well) and size (in 104 μm2) (n=3-4 mice per genotype/treatment) (B-C).",
    "molecules": "CRT0044876"
  },
  {
    "caption": "Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc-/- organoid cultures with or without APE1 inhibitor (CRT0044876, 10μM) (DNAs are stained with DAPI) (D). Quantification of γH2AX positive cells (n=3-4 mice per group) (E).",
    "molecules": "CRT0044876, DAPI, DNAs"
  },
  {
    "caption": "Telomeric DNA quantification and dot-blot assay of H3K9me3-ChIP",
    "molecules": "DNA"
  },
  {
    "caption": "Telomeric DNA quantification and dot-blot assay of H3K9ac-ChIP",
    "molecules": "DNA"
  },
  {
    "caption": "Telomeric DNA quantification and dot-blot assay of HP1a-ChIP",
    "molecules": "DNA"
  },
  {
    "caption": "Representative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor (CRT0044876, CRT). Arrows indicate Olfm4 positive intestine stem cells (I). Quantification of Olfm4 positive intestine stem cells (n=4 mice per group) (J).",
    "molecules": "CRT, CRT0044876"
  },
  {
    "caption": "A Immunofluorescence images of MMS22L foci formation at etoposide (ETP)-induced DSBs during S, M and G1 phases of the cell cycle in HeLa cells. Scale bar: 5 μm.B Quantification of a representative experiment from (A) shows number of MMS22L foci per nucleus during indicated cell cycle phases (n = 2; nnuclei ≥ 60).",
    "molecules": "etoposide, ETP"
  },
  {
    "caption": "C Immunofluorescence images of CPT-induced MMS22L, RPA2, BRCA2 and RAD51 foci in U2OS cells. Scale bar: 5 μm.D Quantification of a representative experiment (n = 3) from (C) shows percentage of γH2AX, RPA2, BRCA2 and RAD51 foci co-localizing with MMS22L foci (nnuclei = 30; nuclei with ≥ 25 MMS22L foci were analyzed).",
    "molecules": "CPT"
  },
  {
    "caption": "A Electrophoretic mobility shift assay monitoring MMS22L-TONSL binding to ssDNA without RPA and with RPA-coated ssDNA.B Quantification of data as in (A) shows percent MMS22L-TONSL binding to ssDNA with or without RPA, n = 2; error bars, SEM.",
    "molecules": "ssDNA"
  },
  {
    "caption": "C Schematic (left) and western blots (right) of streptavidin and NiNTA pulldowns of his-tagged MMS22L-TONSL, RPA and biotinylated ssDNA.",
    "molecules": "ssDNA"
  },
  {
    "caption": "D Yeast two-hybrid analysis shows the effect of deletion of the amino acids 384-522 of MMS22L on the interaction with RPA. The interaction between indicated MMS22L variants fused to LexA DNA-binding domain and RPA1 fused to GAL4 activating domain (GAD) is assessed by survival on media containing 3-aminotriazole (3-AT) in the absence of histidine and by β-galactosidase activity (X-Gal) assays.",
    "molecules": "3-aminotriazole, 3-AT, histidine"
  },
  {
    "caption": "E Images of MMS22L foci formed in U2OS cells stably expressing wild-type (WT) or Δ384-522 variants of siRNA-resistant HSS-MMS22L, depleted of endogenous MMS22L by RNAi and treated with 50 nM CPT for 18 hours. Scale bar: 5 μm.",
    "molecules": "CPT"
  },
  {
    "caption": "(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.A Abundance of MMS22L-TONSL at active and HU-stalled replication forks, and fully replicated DNA.",
    "molecules": "HU"
  },
  {
    "caption": "A Representative immunofluorescence images of foci induced by short CPT treatment (50 nM, 1 h) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci. Scale bar: 5 μm.B Boxplot quantification of representative experiment in (A) (n = 3; nnuclei ≥ 78). Boxes indicate the 25-75 percentile and whiskers the 10-90 percentile; horizontal lines mark \u2028the medians. Nuclei with ≥ 25 γH2AX foci (marker of replication stress sites) were analyzed. Statistical analysis: Mann-Whitney U test; *** P ≤ 0.0001.",
    "molecules": "CPT"
  },
  {
    "caption": "C, D Electron micrographs of a representative reversed fork (C) and a fork with extended ssDNA region at the junction (D) (arrow). P, parental duplex; R, regressed arm.E Frequency of reversed replication forks detected by EM. Similar results were obtained in one independent experiment; n = 2.F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h. In brackets, the total number of analyzed molecules is given. Statistical analysis according to Mann-Whitney U test; ** P ≤ 0.01, n = 2.",
    "molecules": "CPT, ssDNA"
  },
  {
    "caption": "C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.",
    "molecules": "3-AT, histidine"
  },
  {
    "caption": "C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.",
    "molecules": "3-AT, histidine"
  },
  {
    "caption": "E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.",
    "molecules": "ssDNA"
  },
  {
    "caption": "E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.",
    "molecules": "ssDNA"
  },
  {
    "caption": "H Representative immunofluorescence images of CPT-induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA-resistant variants of HSS-MMS22L treated with 50 nM CPT for 1 hour. Scale bar: 5 μm.",
    "molecules": "CPT"
  },
  {
    "caption": "I-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.",
    "molecules": "CPT, ETP"
  },
  {
    "caption": "B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).D Quantification shows averages, n = 2; error bars, SEM.",
    "molecules": "ssDNA"
  },
  {
    "caption": "E Averages of quantified strand exchange assays with ssDNA, RAD51 (270 nM) and varying concentrations of MMS22L-TONSL or MMS22L. n = 2; error bars, SEM.",
    "molecules": "ssDNA"
  },
  {
    "caption": "F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM.",
    "molecules": "dsDNA, ssDNA"
  },
  {
    "caption": "H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM).",
    "molecules": "dsDNA, ssDNA"
  },
  {
    "caption": "I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.",
    "molecules": "dsDNA, ssDNA"
  },
  {
    "caption": "(a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography.",
    "molecules": "Ganglioside, lipids, [1-3H]sphingosine"
  },
  {
    "caption": "(b) Activities of glycohydrolases (GCase, GBA2, β-galactosidase, β-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test.",
    "molecules": "glycohydrolases"
  },
  {
    "caption": "(a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean+s.d.; n=3, *P0.01, One-way ANOVA.",
    "molecules": "Glc-Cer"
  },
  {
    "caption": "(b) Control iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 μM) for 48 h. Representative western blot showing increased levels of α-syn in CBE-treated iPSC-derived neurons compared to untreated neurons.",
    "molecules": "CBE"
  },
  {
    "caption": "(c) Western blot analysis for LC3 in iPSC-derived neuronal cultures at DIV65, untreated (−) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h (+).",
    "molecules": "NH4Cl, leupeptin"
  },
  {
    "caption": "(a) Cytosolic calcium levels were evaluated in iPSC-derived neurons at basal conditions by measuring the 340/380 nm ratio of Fura-2 acetoxymethyl ester. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62). (b) Caffeine (10 mM) was applied to iPSC-derived neurons, and the peak cytosolic calcium concentration was measured as a change in Fura-2 fluorescence. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62).",
    "molecules": "Caffeine, calcium"
  },
  {
    "caption": "(c) iPSC-derived neurons were treated with A23187 (0-5 μM) for 4 h and cytotoxicity was evaluated by released LDH activity. LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X-100. Data are represented as mean+s.e.m.; experiments were independently repeated four times in triplicate. *P0.05, one-way ANOVA, control lines versus mutated lines; δP0.05, one-way ANOVA, mutated lines versus isogenic gene corrected controls.",
    "molecules": "A23187, Triton X-100"
  },
  {
    "caption": "(d) Quantification of TH+ cells treated with A23187 (1 μM) for 4 h expressed as a percentage of DMSO-treated controls. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01, one-way ANOVA.",
    "molecules": "A23187, DMSO"
  },
  {
    "caption": "(g) Quantification of TH+ cells in GBA-PD and GD iPSC-derived neuronal cultures infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Values were calculated as a percentage of scrambled shRNA-treated cells. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, Student's t-test.",
    "molecules": "A23187"
  },
  {
    "caption": "(h) Representative images showing GBA-PD iPSC-derived DA neurons infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Cells were stained for β-TubIII (green) and TH (red). Nuclei were counterstained with Hoechst (blue). (Bar, 20 μm.)",
    "molecules": "A23187"
  },
  {
    "caption": "(F) ChIP-seq tracks Brd4 on select genes (Foxp3, Nr4a2, Med1, Taf10, Ar, and Fbxw7) in Th2 cells, treated with or without JQ1.",
    "molecules": "JQ1"
  },
  {
    "caption": "(F) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 (500nM), and then treated with cycloheximide (CHX; 25μg/ml) for 0h, 0.5h, 1h and 2h before harvest to stop protein synthesis.",
    "molecules": "CHX, cycloheximide, JQ1"
  },
  {
    "caption": "(C) ChIP-qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 (250nM).",
    "molecules": "JQ1"
  },
  {
    "caption": "(F) ChIP-qPCR analysis of Brd4, Ezh2, Suz12 and EED binding on the gene loci of Il4, Il5, Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 (250nM).",
    "molecules": "JQ1"
  },
  {
    "caption": "(B) HEK293T cells are transfected with Flag-Brd4 and HA-EED, HA-Ezh2, and Suz12 plasmids. Lysates are immunoprecipitated with Flag-tagged Brd4, treated in-vitro with JQ1 (5μM), MS402 (5μM), and ABBV-744 (5μM), followed by Western blotting of HA.",
    "molecules": "ABBV-744, JQ1, MS402"
  },
  {
    "caption": "(B) qPCR analysis of Il4, Il5, Gata3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM.",
    "molecules": "ABBV-744"
  },
  {
    "caption": "(D) Western blotting of Gata3 of mouse Th2 cells treated with ABBV-744 (500nM) and MG132 (20μM).",
    "molecules": "ABBV-744, MG132"
  },
  {
    "caption": "(E) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (F) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (",
    "molecules": "ABBV-744"
  },
  {
    "caption": "C-E. MCU silencing reduces Ca2+mit uptake in TNBC cells. Cells were transfected with siMCU or siControl. After 48h, [Ca2+]mit uptake upon ATP (C, E) or Histamine (D) stimulation was measured (n=10). P-values: ***p= 0.0008, ***p<0.0001, ***p= 0.0001, respectively.",
    "molecules": "ATP, Ca2+, Histamine"
  },
  {
    "caption": "A-C. FLIM analysis of cellular NADH/NADPH levels. Fluorescence lifetimes of NAD(P)H autofluorescence in stable shControl and shMCU expressing cells were imaged. Representative images of the distribution of τbound on an intensity weighted pseudocolored scale (2.2-2.5 ns) are shown. Scale bars = 20 μm (A). Mean +/- SE of τbound (B) and relative NADH and NADPH intensities (C) calculated from equation in (Blacker et al., 2014) are shown (n=3). P-values: **p=0.01, **p=0.002, respectively.",
    "molecules": "NADH, NADPH"
  },
  {
    "caption": "D-E. Measurement of the redox state of the NADH/NAD+ couple. Representative measurements of NADH intensity at steady state and at minimal and maximal reduced state (D). Percentage of the steady state redox state (E) (n=3).",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "F. MCU depletion impairs the mitochondrial rate of ATP production. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 5.5 mM 2-deoxy-D-glucose for 1 hour and cellular ATP levels were quantified (n=6). P-value: ***p=0.0009.",
    "molecules": "2-deoxy-D-glucose, ATP"
  },
  {
    "caption": "A. Antioxidant treatments decrease cell migration. A linear scratch was obtained on cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with N-Acetylcysteine (NAC) or Dithioerythritol (DTE). Cell migration into the wounded area was monitored at 48-hour time-point and the covered area was measured (n=12). P-values: **p=0.008, ***p<0.0001, **p=0.005.",
    "molecules": "Dithioerythritol, DTE, N-Acetylcysteine, NAC"
  },
  {
    "caption": "B. Scavenging of mitochondrial ROS decreases cell migration. A linear scratch was obtained on a cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with 50 µM MitoTEMPO. Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001.",
    "molecules": "MitoTEMPO, ROS"
  },
  {
    "caption": "D-E. Mitochondrial H2O2 levels are critically blunted after MCU depletion. Cells were transfected with shMCU or shControl, together with the ratiometric YFP-based biosensor pHyper-dMito (D) or the mitochondrial H2O2-sensitive HyPerRed probe (E). 48 hours later, H2O2 production was measured (n=35). P-values: *p=0.02, *p=0.05, respectively.",
    "molecules": "H2O2"
  },
  {
    "caption": "F. Mitochondrial superoxide levels are critically blunted after MCU silencing. Cells were transfected with siMCU or siControl. 48 hours later, cells were loaded with the red dye MitoSOXTM and superoxide anion levels were measured (n=25). P-value: *p=0.04.",
    "molecules": "superoxide"
  },
  {
    "caption": "G. Mitochondrial GSSG/GSH ratio is critically reduced after MCU silencing. Cells were transfected with shMCU or shControl, together with the mitochondrial targeted mitGrx1-roGFP2 probe. 96 hours later, the glutathione redox potential (EGSH) was measured (n=46). P-value: ***p<0.0001.",
    "molecules": "GSSG, GSH"
  },
  {
    "caption": "B. MCU silencing reduces MG132-mediated HIF-1α and hydroxylated HIF-1α protein accumulation. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 10 μM of the proteasome inhibitor MG132. Left: Protein levels were revealed by western blot. Right: quantification by densitometry (n=5).",
    "molecules": "MG132"
  },
  {
    "caption": "C. ROS increase HIF-1α transcription. Cells were treated O/N with 100 µM paraquat to induce ROS production. HIF-1α mRNA levels were measured by RealTime-PCR (n=3). P-value: **p=0.002.",
    "molecules": "paraquat, ROS"
  },
  {
    "caption": "B HeLa cells expressing the indicated GFP-tagged Nups were semi-permeabilized and incubated with mitotic HeLa cell extract at 30ºC. NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC-dextran. Scale bar, 30 μm.",
    "molecules": "dextran, TRITC"
  },
  {
    "caption": "C Quantification of dextran-positive nuclei over time of the experiments in (B). N = 3 biological replicates, n > 100 cells. Error bars, SEM.",
    "molecules": "dextran"
  },
  {
    "caption": "B Confocal images of metaphase-arrested HeLa cells expressing 3GFP-Nup133, GFP-Nup107, GFP-Nup53 or 2GFP-Nup58 that were either directly fixed (untreated) or first semi-permeabilized and then fixed. The ER was visualized by immunostaining with an antibody targeting calnexin; DNA was visualized with Hoechst (visible only in the merged channels). Representative images of 3 biological replicates. Scale bar, 10 μm and inset scale bar, 1 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP-tagged Nup96. Cells were semi-permeabilized with digitonin and Nup96 was labelled with the BG-AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring-shaped structures built by Y-complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of 4 biological replicates. Inset scale bars, 200 nm.",
    "molecules": "AF647, digitonin, BG"
  },
  {
    "caption": "A 2D dSTORM images of a metaphase-arrested U2OS cell expressing Nup96 with a SNAP-tag, labelled with BG-Alexa Fluor 647 (orange), in combination with confocal imaging of the ER immunostained for Climp63 (red) and phospho-histone H3 (cyan) to label DNA. Inset 1 (mitotic clusters) and inset 2 (kinetochores) are indicated with white dashed boxes. SMLM data was reconstructed using Thunderstorm. Representative images of N = 4 biological replicates. Scale bars, 10 μm. Inset scale bars, 200 nm.",
    "molecules": "Alexa Fluor 647, BG"
  },
  {
    "caption": "Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm.",
    "molecules": "cellulose"
  },
  {
    "caption": "(C) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2. C-terminally 6xHA tagged Rx and C-terminally 4xMyc-tagged NRC2EEE were co-expressed with either free GFP or C-terminally GFP-tagged CP. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.",
    "molecules": "Ponceau, PS, Tris-HCl"
  },
  {
    "caption": "(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different heavy and light sensor-helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.",
    "molecules": "Ponceau, PS, Tris-HCl"
  },
  {
    "caption": "(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different \"heavy\" and \"light\" helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. A non-specific band was observed at ~110 kDa for the anti-FLAG antibody. Red asterisks indicate bands corresponding to the activated \"heavy\" NRC2 complexes. Blue asterisks indicate bands corresponding to \"light\" NRC2 complexes. Purple bands indicate intermediate molecular weight complexes combining \"heavy\" and \"light\" NRC2. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.",
    "molecules": "Ponceau, PS, Tris-HCl"
  },
  {
    "caption": "(B) BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.",
    "molecules": "Ponceau, PS, Tris-HCl"
  },
  {
    "caption": "BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC4AAA. Total protein was extracted with a HEPES-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC4 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.",
    "molecules": "HEPES, Ponceau, PS"
  },
  {
    "caption": "(C) Membrane enrichment assays are consistent with microscopy, showing that inactive NRC2EEE-GFP is mostly present in the soluble (cytoplasmic) fraction, whereas activated NRC2EEE-GFP exhibits equal distribution across soluble and membrane fractions. Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP. T = total, S = soluble, M = membrane. ATPase was used as a membrane marker. Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining (PS). Red asterisks indicate bands matching the expected MW for each protein. The experiment was repeated two times.",
    "molecules": "ATPase, Ponceau, PS"
  },
  {
    "caption": "(B) BN-PAGE and SDS-PAGE assays with infected and uninfected leaves expressing Rx and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.",
    "molecules": "Ponceau, PS, Tris-HCl"
  },
  {
    "caption": "STAT3 phosphorylation and gp130 expression of two representative cases (G) assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression; the p-value was calculated by chi-square test. Scale bar, 100 μm.",
    "molecules": "hematoxylin"
  },
  {
    "caption": "STAT3 phosphorylation and gp130 expression of two representative cases (G) and summarized for the entire cohort of 114 patients (H), as assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression",
    "molecules": "hematoxylin"
  },
  {
    "caption": "Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, on (E and F) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells (expanded by one serial passage through MISTRG6 mice),",
    "molecules": "tocilizumab"
  },
  {
    "caption": "Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, Representative microscopy images in F show STAT3 phosphorylation in spleen-colonizing primary cells exposed to either tocilizumab or isotype control antibody (in brown, counterstained in blue with hematoxylin), and an H&E-stained section from the same mouse. TCZ, tocilizumab. Scale bars, 100 μm.",
    "molecules": "hematoxylin, TCZ, tocilizumab"
  },
  {
    "caption": "Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI-Ly3-transplanted MISTRG mice (G) assessed at around four weeks post tumor cell injection.",
    "molecules": "tocilizumab"
  },
  {
    "caption": "Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the splenic lymphoma burden of RC-K8-transplanted MISTRG6 mice (H), all assessed at around four weeks post tumor cell injection.",
    "molecules": "tocilizumab"
  },
  {
    "caption": "(J)  Immunoblots  and quantifications of protein expression of mTOR targets two days after TAC surgery and after mTOR  inhibition with Torin 1.* p≤ 0.05 vs sham surgery, # p≤ 0.05 vs TAC 2d surgery. t-test, n=5 biological replicates per group. Data information: Error bars indicate means ± SEM",
    "molecules": "Torin 1"
  },
  {
    "caption": "(C)  Protein expression of  Cand2 in  NRCMs treated with PE for 3 and 24 h, and 24h of  Torin 1 treatment.  mTORC1 induction was monitored by downstream targets, p4EBP1 and pS6K proteins.  rpS5 - example of  mTOR-dependent mRNA used as a positive control. Csq  - calsequestrin used as a houskeeping protein.",
    "molecules": "PE, Torin 1"
  },
  {
    "caption": "(J) Schematic representation of reporter consisting of human Cand2 5'UTR and downstream  Renilla luciferase coding sequence. A potential TOP-like motif is highlighted in a grey box. Black arrows indicate transcription start sites in the adult heart  according to the Database of Transcriptional Start Sites (Release 10.1). The effect of Cand2  5'UTR on translation measured by luciferase activity in normal conditions and after  mTORC1  block with Torin 1. eEF2 - positive control of reporter with defined 5'TOP motif and non-TOP 5'UTR of  β-actin used as a negative control. Total protein content in lysates used for  luciferase assay. t-test, n=4-5 biological replicates per group, ** - P ≤ 0.01, *** - P ≤ 0.001.",
    "molecules": "Torin 1"
  },
  {
    "caption": "(C)  Immunofluorescent staining of paraffin  sections from hearts  from WT and Cand2 KO mice. CAND2 (stained in red), ACTIN (stained in green) and nuclei (stained with  DAPI, blue) in. Scale bar 20μm",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) NRCMs size measurement after Cand2 OE and 24h PE stimulation. Analyzed by One-way ANOVA. n>150 cells from n=3 independent experiments. Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001",
    "molecules": "PE"
  },
  {
    "caption": "(F) Nppa mRNA levels in vitro after  Cand2  OE and PE treatment measured by RT-qPCR. Analysed by One-way ANOVA. n=5-9 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001",
    "molecules": "PE"
  },
  {
    "caption": "(B) mRNA levels of MEF2-dependent genes Nr4a1  and Xirp2  in NRCMs with Cand2 KD for indicated groups. PE treatment for 24h (50uM). Analyzed by One-way ANOVA, n=6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001",
    "molecules": "PE"
  },
  {
    "caption": "(E)  Grk5  protein and mRNA levels in NRCMs as well as  MEF2-luciferase activity after 24h of Torin 1  treatment. Analyzed by t-test, n=4-6 biological replicates per conditions. Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein (S235/236rpS6). Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001",
    "molecules": "Torin 1, Torin1"
  },
  {
    "caption": "(B)  Cul1 neddylation  analyzed by  Western blot in NRCMs  depleted of  Cand2. NRCMs were treated with MLN4924 (100 nM) to block Cul1 neddylation. Quantification of neddylated  and unnedylated  Cul1  protein levels after MLN4924  treatment for 24h. t-test, n=4 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001",
    "molecules": "MLN4924"
  },
  {
    "caption": "(D) Grk5  expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition (MLN) analysed by  immunoblot  and RT-qPCR. Analyzed by One-way ANOVA, n=4-5 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001",
    "molecules": "MLN"
  },
  {
    "caption": "(D) Representative  immunoblot  and its quantification of protein levels of neddylated  and unnedylated  Cul1 in Cand2 KO  mice  compared to WT mice. Analyzed by t-test. n=5-8  mice per group. Cul1  neddylation was inhibited in C2C12 by MLN4924 (-/+ MLN) for neddylated Cul1 band detection. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.",
    "molecules": "MLN, MLN4924"
  },
  {
    "caption": "(a) Relative CK2α protein concentration over time following cytoplasmic microinjection of REF52 cells with the HA-tagged semisynthetic proteins bearing different modifications using immunocytochemistry with HA-specific antibody. Data represent mean values ± s.d. (b) Relative CK2α protein concentration following injection of β subunit together with CK2α proteins. Data represent mean values ± s.d. (c) Relative CK2α protein concentration following injection of proteasome inhibitor (1 μM MG132) together with unmodified CK2α protein. Data represent mean values ± s.d.",
    "molecules": "MG132"
  },
  {
    "caption": "(e) Western blot for total CK2α protein in HeLa cells following treatment with nocodazole to arrest cells in mitosis. Full gels corresponding to those in panels d and e are in Supplementary Figure 8.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(b) Coimmunoprecipitation of endogenous Pin1 with CK2α. Pfa-containing and unmodified semisynthetic HA-CK2α proteins were spiked into REF52 cell lysates and immobilized using HA-specific antibodies. Input samples are shown for loading control.",
    "molecules": "Pfa"
  },
  {
    "caption": "(d) Relative CK2α protein concentration over time following microinjection of Pfa344 CK2α with Pin1-specific IgY or nonspecific IgY (control). Data represent mean values ± s.d. Full gels corresponding to those in panels a-c are in Supplementary Figures 10 and 11.",
    "molecules": "Pfa344"
  },
  {
    "caption": "(a) Phosphorylation of C-terminal tail CK2α peptides (residues 337-352) by Cdk1-cyclin B. In vitro Cdk1-cyclin B kinase assays were performed using the following peptide substrate: SSMPGGSTPVXSANMMK(εBiotin), where X = serine or S-GlcNAc serine. After reactions were quenched, peptides were separated from the reaction mixture on Tris-tricine gels. Data represent mean values ± s.d.",
    "molecules": "serine"
  },
  {
    "caption": "(b) Immunoprecipitation and western blot for O-GlcNAc CK2α in HeLa cells after treatment with the O-GlcNAcase inhibitor TMG for 12 h. Numbers indicate relative quantification normalized for total CK2α concentrations.",
    "molecules": "TMG"
  },
  {
    "caption": "(c) Western blot for total CK2α protein in HeLa cells following treatment with TMG for 12 h or 16 h. Numbers indicate relative quantification normalized for loading control, mean values (n = 4) shown ± s.d. Full gels corresponding to those in panels b and c are in Supplementary Figures 14 and 15.",
    "molecules": "TMG"
  },
  {
    "caption": "Phosphorimage analysis of in vitro CK2 kinase assays with protein substrates. All reactions were performed at 30 °C. Numbers indicate the concentration (nM) of product for each kinase reaction. (a) AHCYL2 substrate (25 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (b) NAP1L3 substrate (15 ng μl−1) with 100 μM ATP and 10 nM CK2α ± 11 nM CK2β for 6 min. (c) NIPBL substrate (50 ng μl−1) with 100 μM ATP and 5 nM CK2α ± 11 nM Pin1 for 5 min. Full gels corresponding to those in panels a-c are in Supplementary Figures 19 and 25.",
    "molecules": "ATP"
  },
  {
    "caption": "(f) IL-17A increases resistance to nitrogen starvation. Cells were first grown in YPD medium overnight and then switched to nitrogen-free SLAD medium in the presence of 10 ng ml−1 IL-17A. Viability, expressed as a percentage of colony-forming units at zero time, was measured over a 15-day period. Cells were exposed to 0.1 (black circle), 1 (black triangle) or 10 ng ml−1 (black square) IL-17A or drug vehicle (open circle) at the beginning of the cultures or, in the case of 1 ng ml−1 (open triangle), also added every two other days. Data are mean±s.d. P-values ranged from 0.05 to 0.001 for all samples treated with 10 ng IL-17A; P0.05, on days 3 and 5 for samples treated 1 ng IL-17A every two other days (one-way analysis of variance Bonferroni post-test) (n=3).",
    "molecules": "nitrogen"
  },
  {
    "caption": "(g,l) C. albicans cells are metabolically active. Untreated (g-i) or IL-17A-treated (10 ng ml−1 in RPMI-1640 liquid media for 24 h) (j-l) cells were red after staining with FUN-1 (g,j), green with Con A (h,k) and yellow in the merged images (i,l). Metabolically active cells are shown in red, and cell wall polysaccharides are shown in green. Scale bars, 5 μm. Images from one representative experiment out of two are shown.",
    "molecules": "polysaccharides"
  },
  {
    "caption": "(g,h) TOR inhibition in cells treated with 10 ng ml−1 IL-17A, 15 nM rapamycin or vehicle (none) by western blotting after 4-h (g) or 24-h (h) exposure.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(s,t) Quantitative real-time PCR of orfs. 19.4325, 19.130, RIC1 on cDNA from Candida SC5314 (s) or the TOR1-1/TOR1 mutant (t) strains exposed to vehicle (open column), 10 ng ml−1 IL-17A (black column) or 15 nM rapamycin (grey column) for 4 h. The actual P-values are indicated (two-tailed t-test) (n=3).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a-g) GFP C. albicans cells were untreated (a-c) or treated (d-g) for 4 h with 10 ng ml−1 IL-17A followed by anti-IL17A-PE and examined by fluorescence microscopy (scale bars, 5 μm). Cells were green in (a,d), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Note the binding of IL-17A to hyphal tips (arrows and magnified in g) (n=3). Images are from one representative experiment out of three.",
    "molecules": "PE"
  },
  {
    "caption": "(j-o) Direct visualization of IL-17A on Candida freshly harvested from human vagina. Same experiment as in (a-g) on cells previously stained with Con A (j,m, green) and incubated with PE-anti-human IL-17A (k,n, red) and double stained in the merged images (l,o). Scale bars, 10 μm. Shown is one representative experiment out of three.",
    "molecules": "PE"
  },
  {
    "caption": "(a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle). The lack of binding of IL-17E to Crh11p (orange circle) serves as a negative control. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. Data are mean±s.d., n=3. P0.001, Crh11p and Gas1p without versus with IL-17A (one-way analysis of variance Bonferroni post-test).",
    "molecules": "zymosan, β-1,3-D-glucan, mannan, polysaccharides"
  },
  {
    "caption": "(d,e) Cellular localization of bound IL-17A. Electron micrographs of ultrafine sections of C. albicans MKY 378 wild-type (d) or the crh11Δ mutant (e) incubated with biotinylated IL-17A and streptavidin gold-conjugated particles. Arrows indicate immunogold particles. Scale bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "Fungi were harvested from the stomachs of C57BL/6 (a-c), IL-17F-deficient (d-f) or IL-17A-deficient (g-i) mice infected with GFP-Candida for 2 days, and stained using PE-labeled antibody to IL-17A. Cells were green in (a,d and g), red upon staining with anti-IL17A-PE (b,e and h) and double stained in the merged images (c,f and i). Scale bars, 10 μm. Images are from one representative experiment out of two. (j-l) SEM images showing fungal germination in the stomachs of C57BL/6 mice (j), filamentation in IL-17F-deficient mice (k) and neither one in IL-17A-deficient (l) mice intragastrically infected 2 days before. Scale bars, 10 μm. (a-l) Images are from one representative experiment out of two.",
    "molecules": "PE"
  },
  {
    "caption": "(a-f) GFPAspergillus conidia, incubated overnight at room temperature, were untreated (a-c) or exposed (d-f) to 10 ng ml−1 IL-17A for 4 h followed by anti-IL-17A-PE. Cells were green in (a,b), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Scale bars, 10 μm.",
    "molecules": "PE"
  },
  {
    "caption": "(i-k) IL-17A induces autophagy in Aspergillus. Representative electron micrographs of vacuoles (v) in A. fumigatus AF293 unexposed (k) or exposed to 10 ng ml−1 IL-17A (j) or 15 nM rapamycin (i) for 4 h before TEM. Scale bars, 500 nm.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(l) IL-17A binds the Aspergillus Crf1p protein. Dose-dependent binding of IL-17A to Crf1p (red circle), Gel1p (light blue circle) or the Aspergillus-derived polysaccharides β-1,3-D-glucan (blue circle), α-1,3-D-glucan (open circle) and galactomannan (grey circle). The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay (ELISA) on microtitre-coated plates. Data are mean±s.d. (n=5). P-values ranged from 0.01 to 0.001 for IL-17A-treated versus untreated (0) Crf1p (one-way analysis of variance, Bonferroni post-test).",
    "molecules": "α-1,3-D-glucan, β-1,3-D-glucan, galactomannan, polysaccharides"
  },
  {
    "caption": "(D) HeLa cells were transfected with 10 nM control (n=84) or  Nup50 siRNA (n=171), 20 nM control (n=101) or Nup50 siRNA (n=157) and 40 nM control (n=86) or  Nup50 siRNA (n=204). 72 h post transfection, cells were fixed with 4% PFA  and stained  with antibodies against Nup50 and mAB414, chromatin was labeled with  DAPI. Scale bars: 50 µm. Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations. The means are indicated as diamonds, error bars show the standard deviations. P-values have been calculated from a student t-test comparing the mean between the experimental conditions.",
    "molecules": "DAPI, PFA"
  },
  {
    "caption": "(A) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B  siRNA. After 72h cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is  stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative  nucleus for each picture. Scale bar: 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A, B) Demembranated sperm chromatin was preincubated in  Xenopus egg  extract, depleted for  MEL28/ELYS or Nup50 (B) or control treated (Mock, A). After 10 min,  membranes were added to the reaction. Reactions were stopped at the indicated time points  by fixation and analyzed by confocal microscopy after  immunostaining with α-MEL28/ELYS  (A), α-Nup50 (B), and mAb414 (A, B). Chromatin  was stained  with  DAPI (blue in the overlay). Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "3 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or  EGFP-tagged  Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg   extracts (right panel) or unchromatinized DNA-beads (left panel). After 3h the beads were re-isolated, washed, co-stained with DAPI  and analyzed by  confocal microscopy. Scale bar: 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Confocal microscopy images of  nuclei  assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50  or different mutants. Nuclei  were fixed in 4% PFA and 0.5 % glutaraldehyde, stained  for NPCs  (mAB414) and the chromatin  (DAPI). Scale bar: 10 µm. (B) Average percentage of mAB414 positive  nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated. ",
    "molecules": "DAPI, glutaraldehyde, PFA"
  },
  {
    "caption": "(B)  Confocal microscopy images of fixed  nuclei  assembled for 120 min in mock depleted (mock) and Nup50 depleted (∆Nup50)  Xenopus egg  extracts supplemented with recombinant  mouse Nup50  orthologues. Nuclei  were stained for Nup50 (Nup50  antibody for Xenopus protein,  His6  for  mouse protein) and NPCs (mAB414, red) on the chromatin  (DAPI). Scale bar: 10 µm. (C) The average percentage of mAB414 positive nuclei for 100 randomly chosen  chromatin substrates in each of three independent experiments (performed as in B) is shown. Data points from individual experiments are indicated. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(E) 2 µM recombinant Ran, loaded with MANT-GDP  was incubated with 2 mM GppNHp  in buffer control, supplemented with 2 nM recombinant RCC1, 20 nM recombinant Xenopus Nup50  proteins, or  RCC1 and  Nup50 together. GDP to GppNHp exchanged was monitored by the decrease in  MANT fluorescence of the liberated GDP-MANT. The lower panel shows the rate constant of each experimental condition with n=4 independent experiment per condition, bars represent the mean, individual data points are indicated.",
    "molecules": "MANT, GDP, GDP-MANT, MANT-GDP, GppNHp"
  },
  {
    "caption": "(G) Confocal microscopy images of nuclei  assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and  Nup50 depleted Xenopus egg extracts supplemented with recombinant  Xenopus  wild-type  Nup50 or RCC1 binding mutants. Nuclei  were fixed in 4 % PFA and 0.5 % glutaraldehyde, stained for NPCs  (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of  mAB414 positive  nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments. Individual data points are indicated (H) Confocal microscopy images of nuclei  assembled for 120 min in mock depleted, Nup50  depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with  RCC1  excess as indicated. Nuclei  were fixed in 4 %  PFA and 0.5 % glutaraldehyde,  stained for NPCs (mAB414) and the  chromatin (DAPI). Scale bar: 10 µm. Quantitation shows the average percentage of  mAB414 positive nuclei for 100 randomly chosen  chromatin substrates in each of three independent experiments. Individual data points are indicated. ",
    "molecules": "DAPI, glutaraldehyde, PFA"
  },
  {
    "caption": "DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (A) The scatter plot shows the apparent Young's moduli obtained by AFM analysis. Bars represent the median values of the distributions. The immunoblot shows UBTD1 siRNA knock-down efficiency.",
    "molecules": "fibronectin"
  },
  {
    "caption": "DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (B) Immunoblots (left) and quantification (right) show levels of GST-ROCK associated GTP-bound RhoA (RhoA-GTP) and total RhoA in lysates (RhoA) in confluent cells. Immunoblot of UBTD1 shows the level of siRNA depletion.",
    "molecules": "fibronectin, GTP"
  },
  {
    "caption": "DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (C) Representative confocal images of focal adhesion (paxillin) and actin cytoskeleton (phalloidin) and outlines of focal adhesions using threshold-based image. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. (D) Cell area, number and area of focal adhesion were quantified (ImageJ) using the analysis as shown (C).",
    "molecules": "fibronectin, phalloidin"
  },
  {
    "caption": "DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent DU145 (E) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.",
    "molecules": "fibronectin, hydrogel"
  },
  {
    "caption": "A549 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent A549 (F) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.",
    "molecules": "fibronectin, hydrogel"
  },
  {
    "caption": "DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (G) Confluent DU145 cells were seeded on 12 kPa matrix. Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel.",
    "molecules": "fibronectin, hydrogel"
  },
  {
    "caption": "DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (H) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness. DU145 cells were grown on collagen/acrylamide-coated glass coverslips (soft) or directly on glass coverslips (stiff). Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "acrylamide, collagen, DAPI, fibronectin"
  },
  {
    "caption": "Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 association with E-cadherin or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Representative confocal images of β-catenin and UBTD1 in DU145 under resting (Ctrl), after calcium chelator treatment (EGTA) and recovery (Rec) conditions. Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI, EGTA"
  },
  {
    "caption": "(H) Representative confocal images of β-catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment (24 h). Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (D) DU145 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) of YAP levels in confluent (E) A549 UBTD1 depleted cells treated or not with cycloheximide (CHX) for 2h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "DU145 cells or A549 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). Immunoblots (up) and quantification (down) showing YAP levels in UBTD1 depleted confluent DU145 A549 cells treated or not with MG132 for 6h. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading control.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 associated with YAP of DU145 and A549 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "Proximal ligation assay monitoring (F) of β-TrCP associated with Ubch5, YAP or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) Immunoblots show YAP ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors of histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of YAP were purified (IP: His) and the immunoblot of YAP showed YAP ubiquitylation. The immunoblot of YAP (lower panel) was performed in parallel to verify the amounts of YAP protein engaged in the His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion.",
    "molecules": "His, histidine"
  },
  {
    "caption": "DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (A) Representative wide-field immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells after UBTD1 depletion. Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) Representative confocal immunofluorescence images (left) and quantification (right) showing endogenous YAP nuclear translocation in DU145 cells depleted (siUBTD1single2) or not (siCTRL) for UBTD1 and transfected or not with GFP-UBTD1. Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative confocal immunofluorescence images (F) showing endogenous YAP nuclear translocation in DU145 control cells (siCTRL) or depleted for LATS1 and LATS2 (siLATS1+siLATS2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative confocal immunofluorescence images (I) showing endogenous YAP localization in DU145 control cells (siCTRL) or depleted for ROCK1 (siROCK1) or ROCK2 (siROCK2) and/or UBTD1 (siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.",
    "molecules": "DAPI"
  },
  {
    "caption": "Seedling phenotype under GA treatment. After germination, Five-day-old seedlings with the same growth stage were transferred to 0.5 MS liquid media without or with different concentrations of GA3. PA from soybean was added to the media at a final concentration of 20 μM. Pictures were taken 7 days after transfer. The horizontal red line separates different plants and the vertical red scale bar represents 2 cm.",
    "molecules": "GA, GA3, PA"
  },
  {
    "caption": "Total lipid levels in WT, pldα6 and COM without and with 10 μM GA3. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and lipids were extracted and profiled using ESI-tandem mass spectrometry. Values are means ± SD (n = 3 biological replicates)). MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; PA, phosphatidic acid.",
    "molecules": "DGDG, digalactosyldiacylglycerol, GA3, MGDG, monogalactosyldiacylglycerol, PA, phosphatidic acid, phosphatidylserine, PS, PC, phosphatidylcholine, PE, phosphatidylethanolamine, PG, phosphatidylglycerol"
  },
  {
    "caption": "Phospholipid species in WT, pldα6 and COM. Leaf samples (15 seedling each) from 4-leaf stage rice (Dongjin background) were collected and the different phospholipid species including carbon number and double bond number were determined. Values are means ± SD (n = 3 biological replicates). GA3 was dissolved in ethanol and seedlings treated with the same ethanol concentration were used as control.",
    "molecules": "carbon, ethanol, GA3"
  },
  {
    "caption": "Immunoblotting of His-GID1 proteins using constructs shown in (D). Proteins were separated by SDS-PAGE, followed by immunoblotting with anti-His-tag antibodies. The PA binding activity of different truncation mutations were analyzed by fat-immunoblotting. The red arrowheads indicate truncated proteins with different molecular weights.",
    "molecules": "fat, PA"
  },
  {
    "caption": "Immunoblotting of His-GID1 mutants and lipid-immunoblotting of PA binding by GID1 mutant proteins on a filter.",
    "molecules": "lipid, PA"
  },
  {
    "caption": "PLDα6-GFP distribution in rice protoplasts with different concentrations of GA3 or IAA. Protoplasts from 12-day old rice (ZH11 background) leaf sheath tissue were collected. Rice protoplasts were transfected with pM999-PLDα6 for 12 hours. After that, GA3 or IAA was added to protoplasts and 2 hours later, confocal images of protoplasts are shown. pM999-GFP refers to the empty vector with GFP only that was transformed as control and Ghd7-RFP was a nucleus marker. Scale bar = 10 μm.",
    "molecules": "GA3, IAA"
  },
  {
    "caption": "Immunoblotting of PLDα6 in subcellular fractions. Total (T), soluble (S) and nuclear (N) proteins were isolated from PLDα6:GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA. Equal amount of each sample was loaded for SDS-PAGE and blotting.",
    "molecules": "GA3, IAA"
  },
  {
    "caption": "Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments. Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours. Bars = 10 μm.",
    "molecules": "GA3"
  },
  {
    "caption": "Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts. After 2-hour treatment with 10 μM GA3, total (T), soluble (S) and nuclear (N) protein fractions were isolated from 10 samples of protoplasts. Equal amount of each sample was loaded for SDS-PAGE and immunoblotting.",
    "molecules": "GA3"
  },
  {
    "caption": "Protoplasts of WT and pldα6 from 12-day old stage leaf sheath were collected and transfected with a pM999-SLR1 construct, incubated for 12 hours and then treated with 10 μM GA3 for 0, 3, and 9 hours. The first column, fluorescence from GFP; the second, red fluorescence from the nucleus marker Ghd7; the third, bright field, and the fourth, overlay of the three channels. All confocal images were scanned using similar laser gain and offset settings. Bars = 10 μm.",
    "molecules": "GA3"
  },
  {
    "caption": "Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.",
    "molecules": "NM1267"
  },
  {
    "caption": "E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).  ",
    "molecules": "NM1267"
  },
  {
    "caption": "B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.",
    "molecules": "NM1267"
  },
  {
    "caption": "A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F ",
    "molecules": "NM1267"
  },
  {
    "caption": " (f) GFP in vivo fluorescence post intradermal injection of chitin oligomers into LysMEGFP/+ C57BL/6 mice (n=3/group) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic",
    "molecules": "chitin"
  },
  {
    "caption": "A. thalian leaf piece (K) ROS production (0-45 min, n=6) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate plant leave",
    "molecules": "ROS"
  },
  {
    "caption": " (B) IL-8 release and ROS production from primary PMNs (n=7) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su",
    "molecules": "ROS"
  },
  {
    "caption": " (F) Microarray intensities (excerpt) of chitin-specific signature genes (n=5) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) for details on data acquisition and analysis for panels (D-F) see Expanded View Information",
    "molecules": "chitin"
  },
  {
    "caption": "Flow cytometric quantificatio of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show p<0.05 according to Mann-Whitney U (A",
    "molecules": "Alexa647, C10-15"
  },
  {
    "caption": "microscale thermophoresis (MST) analysis (B) of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) I one representative of 'n' independent experiments is show",
    "molecules": "Alexa647, C10-15"
  },
  {
    "caption": "(C) Flow cytometr of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show one-sample t-test (C",
    "molecules": "Alexa488, Alexa594, CFW"
  },
  {
    "caption": "(D) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm I one representative of 'n' independent experiments is show",
    "molecules": "ConA, Alexa488, Alexa594, CFW"
  },
  {
    "caption": " (g) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc protein with SSL3 titration (n=2) Data information I one representative of 'n' independent experiments is show",
    "molecules": "Alexa647, C10-15"
  },
  {
    "caption": " (J) BALF TNF in C57BL/6 mice (n=5/group) upon C10-15 administration without or with SSL3 Data information: Quantificatio -panel represent data (mean+SE combined from 'n' biological replicate mic one-way ANOVA with Dunnett's multiple comparison (J)",
    "molecules": "C10-15"
  },
  {
    "caption": " (K) ROS production between 0 and 45 min post chitin application in A. thaliana leaf pieces (n=6) Data information: Quantificatio panels ( represent dat mean±SD in K combined from 'n' biological replicate plant leave",
    "molecules": "ROS"
  },
  {
    "caption": " (L) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc Protein with C5 titration (n=2) Data informatio I one representative of 'n' independent experiments is show",
    "molecules": "Alexa647, C5, C10-15"
  },
  {
    "caption": "HSATIII lncRNA-dependent localization of m6A-related factors. Control and HSATIII knockdown HeLa cells were exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C) and stained by HSATIII-FISH and immunofluorescence using an anti-WTAP antibody (B) or anti-YTHDC1 antibody (C). The nuclei were stained with DAPI. Scale bar: 10 µm.",
    "molecules": "DAPI, m6A"
  },
  {
    "caption": "Changes of nSB localization of m6A-related factors at various time points during and after thermal stress exposure. HeLa cells were exposed to thermal stress (42°C for 2 h) and cultured at 37°C for the indicated periods. m6A-related factors were visualized by HSATIII-FISH and IF using anti-WTAP antibody (D) or anti-YTHDC1 antibody (F). Nuclei were stained with DAPI. Scale bar: 10 µm. (E, G) Box plot of relative intensities of WTAP (D) and YTHDC1 (F) to HSATIII within nSB areas in individual nuclei. nSB areas were defined by binarized images of HSATIII-FISH. The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above (n=30).",
    "molecules": "DAPI, m6A"
  },
  {
    "caption": "RT-qPCR validation of splicing of nascent RNAs. Splicing efficiency of nascent RNAs was quantified as the ratio of the spliced form to the intron retaining isoform (IR) form and normalized to the control. Control siRNA and HSATIII control oligonucleotide are indicated with (-). Data are shown as the mean ± SD (n=3); p-values (Tukey's multiple comparison test) are shown above.",
    "molecules": "oligonucleotide"
  },
  {
    "caption": "Read maps represent RNA-seq data of nuclear poly (A)+ RNAs (upper panel, biological triplicate) and m6A-RIP RNAs (lower panel, biological duplicate) in control and HSATIII KD cells. The exon numbers of the FAM214A gene are indicated below. The retaining intron and the m6A peak are marked by a red line and arrow, respectively. For the entire FAM214A region, see also Figure EV4B.",
    "molecules": "m6A"
  },
  {
    "caption": "(A) Wild type (left and middle panels) and p53-/- (right panel) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 36 h, treated with 5 nM actinomycin D, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. Coomassie Brilliant Blue (CBB) staining serves as a loading control.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "(C) HCT116 cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1-targeting (mixture of siGRWD1-3 and 4) siRNAs for 24 h, treated with 50 µg/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(D) HFF2/T cells stably overexpressing HA-GRWD1 were established by retroviral infection. Cells were cultured in the presence or absence of actinomycin D (5 nM) for 12h and then treated with or without MG132 (20uM) for 6 h as indicated. Whole cell extracts were analyzed as above.",
    "molecules": "actinomycin D, MG132"
  },
  {
    "caption": "(E) Control and HA-GRWD1-overexpressing HFF2/T cells were first treated with actinomycin D (5nM) for 12 h, then further treated with 50 ug/ml cycloheximide, and harvested at the indicated times. Whole cell extracts were analyzed by immunoblotting with the indicated antibodies. The means and SDs from two independent experiments are shown.",
    "molecules": "actinomycin D, cycloheximide"
  },
  {
    "caption": "(C) HCT116 cells treated with actinomycinD (5nM) or vehicle (DMSO) for 12h were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 (green) and anti-fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. Scale bar, 20 um.",
    "molecules": "actinomycinD, DMSO, Triton X-100"
  },
  {
    "caption": "(C) HCT116 cells were treated with 5nM actinomycin D for 12 h or left untreated and subjected to immunoprecipitation with anti-GRWD1 antibody or control IgG. Immunoprecipitates (IPs) and 3.5% of inputs were immunoblotted with the indicated antibodies.",
    "molecules": "actinomycin D"
  },
  {
    "caption": "(G) HCT116 cells were transfected with RPL11-FLAG expression vector (0.56 ug) or empty vector (0.56 ug) for 24 h and then treated with 5 nM actinomycinD or vehicle (PBS) for 12 h. The cells were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 antibody (green) and anti-FLAG M2 antibody (red), and counterstained with DAPI. Scale bar, 20 um.",
    "molecules": "actinomycinD, Triton X-100"
  },
  {
    "caption": "(D) In vitro ubiquitination of p53 by immunopurified MDM2. His-Xpress-MDM2 was immunopurified from transfected 293T cells with anti-Omni probe antibody. Recombinant p53 was incubated with E1, E2 (UbcH5a), His-ubiquitin, ATP, GST-RPL11, GRWD1-His, and immunopurified His-Xpress-MDM2 or control immunoprecipitates at 30℃ for 120 min as indicated. The samples were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies.",
    "molecules": "ATP"
  },
  {
    "caption": ",B,C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd (gray, open squares, N=10). Control embryos were injected with DMSO (black, filled circles, N=9). All embryos are y/w and were injected while in cycle 14 and imaged 60 min later.",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "A, Wildtype or kit mutant stained with AFOG at 14 days and 30 dpi. Sham-operated zebrafish hearts serve as control. Scale bar = 100 μm.",
    "molecules": "AF, OG"
  },
  {
    "caption": "Q, Representative image of hearts from 7 dpi tcf21:nucGFP transgenic fish treated by PBS liposome, Clodronate liposome and Dex, respectively. The red boxes mark the peripheral area, and the white boxes mark the injury area and are shown in their respective zoom-in images to the right.. White dashed lines indicate approximate resection plane.",
    "molecules": "Clodronate, Dex, PBS"
  },
  {
    "caption": "A T98G cells were challenged or not with dsDNAs or with 2'3'cGAMP for 6 h prior to whole cell extraction and Western Blot (WB) analysis using indicated antibodies.",
    "molecules": "2'3'cGAMP, dsDNAs"
  },
  {
    "caption": "F T98G cells were challenged or not with dsDNA for 6 h, in the presence or not of the NU7441 DNA-PKcs inhibitor, prior WB analysis using indicated antibodies.",
    "molecules": "dsDNA, NU7441"
  },
  {
    "caption": "I T98G cells were treated or not with 0.16 µM camptothecin (CPT) for 48 h prior to immunofluorescence analysis using dsDNA- and 53BP1-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).",
    "molecules": "camptothecin, CPT, DAPI, dsDNA"
  },
  {
    "caption": "J Whole cell extracts from T98G cells treated or not for 72 h with 0.16 µM CPT were analyzed by WB using indicated antibodies.",
    "molecules": "CPT"
  },
  {
    "caption": "A T98G cells were treated or not with dsDNA for 6 h prior to IF analysis using DNA-PKcs- and pDNA-PKcs-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).",
    "molecules": "DAPI, dsDNA"
  },
  {
    "caption": "B Whole cell extracts from T98G cells were incubated with 80nt-long biotinylated ssDNA or dsDNA prior to pull-down using streptavidin-affinity beads. Input and eluates were analyzed by WB using indicated antibodies.",
    "molecules": "biotinylated, dsDNA, ssDNA, streptavidin"
  },
  {
    "caption": "A T98G cells stably expressing cGAS (T98GcGAS) or not (T98GEmpty) were transfected or not with dsDNA for 6 h prior to whole cell extraction and WB analysis using indicated antibodies.",
    "molecules": "dsDNA"
  },
  {
    "caption": "C T98GEmpty and T98GcGAS were transfected or not with biotinylated dsDNA prior to whole cell extraction and pull-down using streptavidin-affinity beads. Inputs and eluates were analyzed by WB using indicated antibodies.",
    "molecules": "biotinylated, dsDNA, streptavidin"
  },
  {
    "caption": "J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.",
    "molecules": "dsDNA, NU7441"
  },
  {
    "caption": "K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.",
    "molecules": "dsDNA, NU7441"
  },
  {
    "caption": "representative histological  images (B) of wild-type control mice  or BTNL2-KO mice  treated with 2% DSS  for 8 days were shown (n=15).",
    "molecules": "DSS"
  },
  {
    "caption": "representative H&E images (H) of wild-type control mice  or BTNL2-KO mice  treated with Fc or mIL-22-Fc (ip. 5 μg/mouse) at day 0, 2, 4, 6 during 2% DSS  treatment were shown (n=12). Data information Data are representative of three biological replicates H).",
    "molecules": "DSS"
  },
  {
    "caption": "F.  IL-22 production was analyzed by  Flow cytometry in LPLs  of wild-type mice  treated with Fc or mBTNL2-Fc (ip. 50 μg/mouse) on day 0, 2, 4, 6 during 2%DSS  treatment. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=12 for the Fc group and n=14 for the mBTL2-Fc group (F). Pooled data from three biological replicates were shown F.",
    "molecules": "DSS"
  },
  {
    "caption": "D-E. Expression of BTNL2   in normal colon  tissue or DSS+AOM  induced tumour tissue were measured by immunoblot  (D) and real-time PCR (E) (n=5). Data information: All data are mean ± s.e.m. NS, not significant., **P < 0.01 based on two-tailed Student's t test for (E). Each dot represents one mouse, n=5.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "(A Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 µM, 2h), scale bar: 7.5 µm. and the samples were processed for IF analysis with Ub and p62 antibody. (B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. (C The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.",
    "molecules": "MG132, Ub"
  },
  {
    "caption": "D) Representative confocal images of HeLa and TRIM16KO cells treated with (D) H2O2 (200 µM, 2h) and the samples were processed for IF analysis with Ub and p62 antibody. E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (E) H2O2, (200 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. F) The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.",
    "molecules": "H2O2, Ub"
  },
  {
    "caption": "(G) Western blot (WB) analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 µM, 1h) and probed with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.",
    "molecules": "MG132, Ub"
  },
  {
    "caption": "(K, L) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) Bafilomycin A1 (300 nM, 3h) alone or (L) with MG132 (10 µM, 4h) as indicated. L.E, Low exposure; H.E, high exposure.",
    "molecules": "Bafilomycin A1, MG132"
  },
  {
    "caption": "(M) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye, Ub, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells",
    "molecules": "Proteostat dye, H2O2, Ub"
  },
  {
    "caption": "(B) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(H) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 µM) for the indicated period and probed with different antibodies as indicated. The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X-ray film. (I) Quantification of NRF2, p62, and KEAP1 band intensities relative to actin. n=3, **p < 0.005, *p < 0.05 (Student's unpaired t-test).",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(J) Immunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 µM, 4h).",
    "molecules": "MG132"
  },
  {
    "caption": "IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence of MG132 (10 µM, 4h).",
    "molecules": "MG132"
  },
  {
    "caption": "IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (M) presence of MG132 (10 µM, 4h).",
    "molecules": "MG132"
  },
  {
    "caption": "(A-C) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co-IP assays using transiently transfected plasmid constructs as indicated. Two different variants of ubiquitin protein are used, one that can only be ubiquitinated at Lysine 48 residue (HA-K48-UB) and other that can be only ubiquitinated at Lysine 63 residue (HA-K63-UB). All other lysine residues are mutated. L.E, Low exposure; H.E, High Exposure.",
    "molecules": "Lysine, lysine, UB, ubiquitin"
  },
  {
    "caption": "Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.",
    "molecules": "lysines, ubiquitin"
  },
  {
    "caption": "Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.",
    "molecules": "lysines, ubiquitin"
  },
  {
    "caption": "(G) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(H) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(A) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated.",
    "molecules": "H2O2"
  },
  {
    "caption": "(B) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown. The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2. L.E, Low exposure; H.E, High Exposure.",
    "molecules": "H2O2"
  },
  {
    "caption": "(D) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2.5 µM (2 h) of As2O3.",
    "molecules": "As2O3"
  },
  {
    "caption": "(E-G) RNA isolated from HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h) were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05, **p < 0.005 (Student's unpaired t-test).",
    "molecules": "MG132, RNA"
  },
  {
    "caption": "RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).",
    "molecules": "RNA"
  },
  {
    "caption": "RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).",
    "molecules": "RNA"
  },
  {
    "caption": "(K) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated.",
    "molecules": "H2O2"
  },
  {
    "caption": "(M) Representative confocal images of control, Ubb and Ube2n siRNA transfected cells treated with H2O2 (200 µM, 2h) and IF was performed with Ub and p62 antibodies. Scale bar: 10 µm.",
    "molecules": "H2O2, Ub"
  },
  {
    "caption": "(B, C) RNA isolated from untreated or MG132-treated (20 µM, 2h) control or NRF2 siRNA transfected cells were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.",
    "molecules": "MG132, RNA"
  },
  {
    "caption": "WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with MG132 (20 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.",
    "molecules": "MG132"
  },
  {
    "caption": "WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with H2O2 (200 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.",
    "molecules": "H2O2"
  },
  {
    "caption": "Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (F) MG132 (20 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.",
    "molecules": "MG132, Ub"
  },
  {
    "caption": "Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (G) H2O2 (200 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.",
    "molecules": "H2O2, Ub"
  },
  {
    "caption": "(H) Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 or GFP-NRF2 were treated with MG132 (20 µM, 2h) and subjected to WB analysis with Myc, GFP and indicated antibodies.",
    "molecules": "MG132"
  },
  {
    "caption": "(I) Representative confocal images of Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 which were treated with MG132 (20 µM, 2h) and subjected to IF with Ub and p62 antibodies.",
    "molecules": "MG132, Ub"
  },
  {
    "caption": "(J) Graph represent % of cells with Ub-p62 dots, data from ≥ 10 microscopic fields (40X), n=3, mean ±SD, *p < 0.005 (one-way ANOVA test). Data information: Unless otherwise stated, scale bar: 10 µm.",
    "molecules": "Ub"
  },
  {
    "caption": "WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (C) GFP-polyQ74 probed with or GFP or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.",
    "molecules": "polyQ74"
  },
  {
    "caption": "(E) Confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm. (F) The graph shows the percentage of cells with TRIM16-Ub or TRIM16-Ub-p62 co-localized dots/aggregates. Data from ≥ 10 microscopic fields, n=2, mean ±SD. ",
    "molecules": "Bafilomycin A1, MG132, Ub"
  },
  {
    "caption": "(G) Representative confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm.",
    "molecules": "Bafilomycin A1, MG132"
  },
  {
    "caption": "(I) WB analysis of detergent-soluble and -insoluble DSS-cross-linked fractions of HeLa and TRIM16KO cells, untreated or treated with MG132 (10 µM, 4h) and probed with indicated antibodies.",
    "molecules": "DSS, MG132"
  },
  {
    "caption": "(A) MTT assays performed at different time points with HeLa and TRIM16KO cells, untreated or treated with H2O2 (400 µM, 2h). Data, mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).",
    "molecules": "H2O2"
  },
  {
    "caption": "(B) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm.",
    "molecules": "As2O3"
  },
  {
    "caption": "Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (C) As2O3 (2.5 µM, 4h)",
    "molecules": "As2O3, Propidium Iodide"
  },
  {
    "caption": "Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (D) H2O2 (400 µM, 2h)",
    "molecules": "H2O2, Propidium Iodide"
  },
  {
    "caption": "Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (E) puromycin (5 µg/ml, 6h)",
    "molecules": "Propidium Iodide, puromycin"
  },
  {
    "caption": "Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (F) MG132 (20 µM, 8h).",
    "molecules": "MG132, Propidium Iodide"
  },
  {
    "caption": "(G) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure. Arrowheads indicate the cleaved form of PARP-1 and Casapse-3.",
    "molecules": "As2O3"
  },
  {
    "caption": "(A, B) Clonogenic assays were performed with HeLa and TRIM16KO cells, untreated or treated with As2O3 or MG132.",
    "molecules": "As2O3, MG132"
  },
  {
    "caption": "(C) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points. As2O3 is injected when tumor volume of all groups was >100mm3 (day 0 in the graph). Tumor volumes were measured on an interval as indicated. Mean ±SE, n=6 (each group), *p < 0.05, **p < 0.005, ***p < 0.0005 (ANOVA). (D) Graph shows the % tumor regression (Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100). Mean ±SE, n=6 (each group), **p < 0.005 (Student's unpaired t-test). ",
    "molecules": "As2O3"
  },
  {
    "caption": "(E) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3. (F) Representative pictures of dissected tumors. ",
    "molecules": "As2O3"
  },
  {
    "caption": "(G) Immunohistochemistry analysis performed with KI-67 cell proliferation marker and hematoxylin nuclear stain. The graph represents the % of cells with KI-67 staining. >400 cells were counted for this analysis from three different sections from different animals, mean ±SD, *p < 0.05 (Student's unpaired t-test). Scale bar: 50 µm.",
    "molecules": "hematoxylin"
  },
  {
    "caption": "(B)  flg22-induced ROS production in Col-0 and fer-4 mutant roots  under  Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM  flg22, RALF23 or both  flg22 and RALF23 for 15 mins. Roots were harvested and stained with H2DCFDA. One representative root was shown. (C) Average H2DCFDA signal intensity in roots shown in (B). The fluorescence intensity was quantified with ImageJ. The data shown indicate the means ± SDs (n = 30, n refers to the number of  roots per group); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results. ",
    "molecules": "H2DCFDA, Pi, ROS"
  },
  {
    "caption": "(F)  RALF23 inhibits flg22-induced MAMP-responsive  FRK1 gene expression in a Pi  content-dependent manner. Seedlings  grown in either  HP or LP  media were treated with water or  flg22 for 15 mins and roots were harvested. The relative  expression of FRK1  was quantified via  RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results. ",
    "molecules": "Pi"
  },
  {
    "caption": "(B) PHR1 negatively regulates FLS2-BAK1 complex formation under  Pi-starvation conditions. Col-0,  phr1 mutant seedlings  were grown in either  HP or LP  media for 5 days, then lysing roots into  protoplasts, transformed with water or  PHR1-Myc plasmid and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2  and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as negative control. Data information: All experiments were repeated three times with similar results.",
    "molecules": "Pi"
  },
  {
    "caption": "(F) FER negatively regulates FLS2-BAK1  complex formation under Pi-starvation conditions. Col-0, fer-4  mutant and FER/fer-4  complementation lines seedlings  were grown in either HP or LP  media for 5 days and treated with  flg22 for 6 hours. The amount of  immunoprecipitated FLS2  and coimmunoprecipitated BAK1 was determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as a negative control. Data information: All experiments were repeated three times with similar results.",
    "molecules": "Pi"
  },
  {
    "caption": "(A-B) Relative  Pseudomonas  abundance in   rhizosphere  samples after (A), Pi-stress (LP) or (B) treatment with B. subtilis expressing  RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test). (C-D) Relative Bacillus abundance in  rhizosphere samples after (C),  Pi-stress (LP) or (D) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (E-F) Relative Geodermatophilus  abundance in rhizosphere samples after (E), Pi-stress (LP) or (F) treatment with B. subtilis expressing RALF23  (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (G-H) Relative  Methylobacillus  abundance in rhizosphere samples after (G), Pi-stress (LP) or (H) treatment with  B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test).",
    "molecules": "Pi"
  },
  {
    "caption": "Bacterial inoculation promotes  plant growth and alleviates LP stress.  Arabidopsis seeds  were germinated on  1/2-strength  MS media for 3 days and then transplanted to either  HP or LP  media for another 5 days. The seedlings were then transferred to  vermiculite and inoculated with  Pto DC3000 or B. subtilis. (A), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.",
    "molecules": "vermiculite"
  },
  {
    "caption": "(C) Pto DC3000  and B. subtilis upregulate  phosphate  absorption gene expression. Col-0, phr1  and  fer-4  mutant roots  were harvested 48 hours after inoculation with Pto DC3000 or B. subtilis  in Pi-sufficient or Pi-deficient condition, and the relative expression  of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.",
    "molecules": "phosphate, Pi"
  },
  {
    "caption": "The fer-4 rhizosphere microbiome promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Col-0 or fer-4 rhizosphere microbiomes. (B), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.",
    "molecules": "vermiculite"
  },
  {
    "caption": "(D) The  fer-4 rhizosphere microbiome  upregulated phosphate absorption  gene expression. The roots  of Col-0 were harvested after 4 weeks of inoculation with the Col-0 or  fer-4 rhizosphere microbiome  under LP  condition, and the relative  expression  of  Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.",
    "molecules": "phosphate, Pi"
  },
  {
    "caption": "E Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N-glycosidase pre-treatment to reveal untreated and deglycosylated placensin.",
    "molecules": "N-glycosidase"
  },
  {
    "caption": "G Immunoblotting of recombinant placensin generated in E. coli. and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]-CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N-glycosidase to remove N-linked carbohydrate side chains, prokaryotic- and eukaryotic cell-derived placensin proteins showed similar sizes.",
    "molecules": "N-glycosidase"
  },
  {
    "caption": "A Prokaryotic cell-derived placensin stimulated cAMP production (at 10 min.), and PKA activity (at 30 min.) by primary mouse hepatocytes and hepatocyte-derived AML12 cells (biological replicates, n=5).",
    "molecules": "cAMP"
  },
  {
    "caption": "B Placensin stimulation of glucose secretion (at 5h) and key gluconeogenesis gene transcripts (at 1h) in both cell types (biological replicates, n=4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT-PCR. Treatment with glucagon (10 nM) served as positive controls.",
    "molecules": "glucagon, glucose"
  },
  {
    "caption": "C Liver from adult female mice was perfused in situ with placensin (1.0 ug/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n=3).",
    "molecules": "glucose"
  },
  {
    "caption": "D Adult female mice were injected intraperitoneally with placensin (30 ug/injection) with blood sampling at different time points for glucose levels (biological replicates, n=5).",
    "molecules": "glucose"
  },
  {
    "caption": "C Placensin stimulation of cAMP production and PKA activities by HTR-8/SVneo cells. Cells were treated with placensin for 10 min. before cAMP measurement (biological replicates, n=5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 uM) served as positive controls.",
    "molecules": "cAMP, forskolin"
  },
  {
    "caption": "F Placensin stimulation of HTR-8/SVneo cell invasion. After placensin treatment for 24h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n=4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100μm. C: control.",
    "molecules": "mitomycin C"
  },
  {
    "caption": "F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.",
    "molecules": "AK-298, SK-153"
  },
  {
    "caption": "H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4-6).",
    "molecules": "RK-33"
  },
  {
    "caption": "I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.",
    "molecules": "RK-33"
  },
  {
    "caption": "J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.",
    "molecules": "RK-33"
  },
  {
    "caption": "Following injection of 20 mg/kg of RK-33, twice a week for 7 weeks, extensive histopathological examination was carried out following necropsy. Identical patterns were observed both in the control and in the treated mice (n = 2). Samples were stained with H&E. Scale bar is 50 μm.",
    "molecules": "RK-33"
  },
  {
    "caption": "Pharmacokinetics of RK-33 in SCID mice at various time intervals. Results are mean ± SD from 5 mice. LC-MS/MS method was used to determine concentration of RK-33 in mouse plasma and tissue.",
    "molecules": "RK-33"
  },
  {
    "caption": "HAPI cells were treated with RK-33 (10 μM), HA14-1 (25 μM), or DMSO vehicle, followed by oligomycin (oligo, 0.5 μg/ml), FCCP (3 μM), and antimycin A (AA, 1 μM) while oxygen consumption rate (OCR) was measured. Pyruvate (10 mM) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal OCR. Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages. OCR is baseline-normalized to the point prior to drug or vehicle addition.",
    "molecules": "HA14-1, antimycin A, FCCP, DMSO, oligomycin, Pyruvate, RK-33"
  },
  {
    "caption": "HAPI microglial cells were incubated for 1 h in glucose-free XF24 assay medium that was supplemented with 2-deoxyglucose (50 mM) and pyruvate (10 mM). RK-33 (10 μM), oligomycin (0.5 μg/ml), or vehicle control was additionally present as indicated. Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages. Significance was assessed by two-sided, unpaired t-test.",
    "molecules": "2-deoxyglucose, glucose, oligomycin, pyruvate, RK-33"
  },
  {
    "caption": "An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in (C). Figure displays H&E staining of lung sections from radiation-treated (upper panel) and RK-33- and radiation-treated mice (lower panel). Scale bar is 2 mm.Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic A549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.",
    "molecules": "RK-33"
  },
  {
    "caption": "B, C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK-33 (0, 2, 4, and 6 μM). RK-33 induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.",
    "molecules": "RK-33"
  },
  {
    "caption": "D, E Immunoblot of cell cycle-related proteins (Cyclin D1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in A549 and H1299 cells after treatment with RK-33 (10 μM). Initially, a strong decrease of Cyclin D1 was observed. After 8 and 24 h, cleaved caspases 9 and 7 were apparent.",
    "molecules": "RK-33"
  },
  {
    "caption": "F Immunoblot of MAPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in A549, H1299, and H3255 (RK-33 resistant) cells 24 h after treatment with RK-33 (7.5 μM or 10 μM). ERK2 and especially ERK1 become dephosphorylated after treatment with RK-33 in A549 and H1299 cells but not in H3255 cells. Outlined boxes indicate spliced lanes.",
    "molecules": "RK-33"
  },
  {
    "caption": "F-I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.",
    "molecules": "RK-33"
  },
  {
    "caption": "J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT-PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.",
    "molecules": "RK-33"
  },
  {
    "caption": "A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.",
    "molecules": "RK-33"
  },
  {
    "caption": "B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.",
    "molecules": "RK-33"
  },
  {
    "caption": "C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.",
    "molecules": "RK-33"
  },
  {
    "caption": "D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.",
    "molecules": "RK-33"
  },
  {
    "caption": "E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT-PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.",
    "molecules": "RK-33"
  },
  {
    "caption": "F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.",
    "molecules": "RK-33"
  },
  {
    "caption": "(A, B) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, middle row) and 4 hours after washing out of MYK-461 (Wash out, bottom row). These cells were stained with a phosphorylated AMPK antibody, an AMPKβ2 antibody and a N-cadherin antibody.",
    "molecules": "DMSO, MYK-461"
  },
  {
    "caption": "(C, D) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row) and stained with a connexin43 antibody, a plakoglobin antibody, an LKB-1 antibody and an N-cadherin antibody. The representative images from 4 independent experiments were shown.",
    "molecules": "DMSO, MYK-461"
  },
  {
    "caption": "(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0 min and 15 min after treatment with 20 μM Compound C (left side panel) and expressing EGFP-CLIP-170 S311A (right side panel). Higher magnification of white square (upper row) showing CLIP-170 migrated longitudinally toward the cell-cell junctions.",
    "molecules": "Compound C"
  },
  {
    "caption": "(B) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells in different fields was analyzed. Number of comets analyzed, Pre: n=32, Cpd. C: n=36, CLIP S311A: n=68. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Pre.",
    "molecules": "Cpd. C"
  },
  {
    "caption": "(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0, 2 and 3 hours after treatment with 2 μM MYK-461.",
    "molecules": "MYK-461"
  },
  {
    "caption": "(B) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row). These cells were stained with an α-tubulin (green) and a plakoglobin (red) antibody.",
    "molecules": "DMSO, MYK-461"
  },
  {
    "caption": "(C) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK-461, showing the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Adenovirus expressing EGFP-CLIP-170 (adCLIP) mutant was used (WT, S311A, S311D). Numbers in the graph indicate n number of the cells from 2 independent experiments. Control (-/-): n=784, MYK-461: n=939, CLIP WT: n=656, CLIP WT + MYK-461: n=619, CLIP S311A: n=405, CLIP S311A + MYK-461: n=635, CLIP S311D: n=613, CLIP S311D + MYK-461: n=735. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Control, ††, P<0.01 versus CLIP WT, n.s., not significant.",
    "molecules": "MYK-461"
  },
  {
    "caption": "(A) Beeswarm plots of the echocardiographic parameter (ejection fraction) of individual Cre control (CLIP-170 S311A +/+; α-MHC-MerCreMer+/−), Control mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer−/−), and CLIP-170 S311A overexpressing mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer+/−), before (Pre), 2 weeks (Tx2w), 8 weeks (Tx8w), 26 weeks (Tx26w) and over 1 year (Tx1y) after tamoxifen induction. Number of mice, (Cre control, Control, S311A), Pre (6, 2, 3), Tx 2w (9, 5, 5), Tx 8w (9, 10, 14), Tx1 2w (7, 10, 14), Tx 26w (3, 7, 8), Tx 52w (3, 7, 9).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(C) Representative long axis four-chamber cardiac magnetic resonance images of CLIP-170 S311A TG mice and control mice over 1 year after tamoxifen induction. Left column showed systole images and right column represented diastole images. Three mice from each group were analyzed for cardiac MRI. The representative images were shown.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(A) Masson's trichrome staining of the heart of CLIP-170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(C) Representative immunostained images with an α-tubulin antibody of CLIP-170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment. (D) Quantitative analysis of intensity of α-tubulin in (C). Number of mice, (Control: n=7, S311A: n=8). Average of 5 sections per animal were analyzed. **, P<0.01 versus Control. ",
    "molecules": "tamoxifen"
  },
  {
    "caption": "(G) Representative immunostained images of CLIP-170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment. These were stained with WGA (red) and a plakoglobin antibody (green).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and liver fat content (C). Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D).",
    "molecules": "fat"
  },
  {
    "caption": "Hepatocytes were treated with BSA, Palm or Palm+Rap for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates). Scale bar represents 100 µm.",
    "molecules": "Bodipy 493/503, Lipid, Hoechst, Rap, Palm"
  },
  {
    "caption": "Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm, Palm+Rap and stained as mentioned above (n=4 biological replicates).",
    "molecules": "Lipid, Rap, Palm"
  },
  {
    "caption": "Shown is one representative Western blot from two independent experiments and quantification of LC3-II/I and p62 protein levels in siCtrl (n=4 biological replicates) or siASK1 (n=4 biological replicates) cells treated with Palm.",
    "molecules": "Palm"
  },
  {
    "caption": "Colocalization of LC3-II punctate (red; arrow) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm or Palm+Rap. Scale bar represents 100 µm.",
    "molecules": "BODIPY 493/503, lipid, Hoechst, Rap, Palm"
  },
  {
    "caption": "Colocalization of LC3-II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; grey bars) or non-targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA+Baf for 24 hours (n=4 biological replicates).",
    "molecules": "Baf, BODIPY 493/503"
  },
  {
    "caption": "Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm or Palm+Baf and stained as mentioned above (n=4 biological replicates). Values are expressed as mean ± SEM. *",
    "molecules": "Baf, Lipid, Palm"
  },
  {
    "caption": "Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice).",
    "molecules": "TG, triglyceride"
  },
  {
    "caption": "Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm)",
    "molecules": "Sirius Red"
  },
  {
    "caption": "quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice).",
    "molecules": "Sirius Red"
  },
  {
    "caption": "Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #",
    "molecules": "Sirius Red, TG, triglyceride"
  },
  {
    "caption": "Representative immunofluorescence images of liver sections stained for LC3-II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Primary hepatocytes isolated from chow-fed ASK1F/F () and ASK1Δhep () mice were treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates).",
    "molecules": "Baf, Bodipy 493/503, Lipid, Hoechst, Rap, Palm"
  },
  {
    "caption": "Colocalization of LC3-II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm+Rap or Palm+Rap+Baf. Scale bar represents 100 µm. Values are expressed as mean ± SEM.",
    "molecules": "Baf, BODIPY 493/503, lipid, Hoechst, Rap, Palm"
  },
  {
    "caption": "liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment).",
    "molecules": "TG, triglyceride"
  },
  {
    "caption": "Liver triglyceride (TG) content (ASK1F/F n=11 mice; ASK1+hep n=10 mice) in livers of mice fed a HFD for 20 weeks.",
    "molecules": "TG, triglyceride"
  },
  {
    "caption": "Quantification of Sirius Red positive area (ASK1F/F n=11 mice; ASK1+hep n=9 mice) in livers of CCl4-injected mice.",
    "molecules": "CCl4, Sirius Red"
  },
  {
    "caption": "Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM. #",
    "molecules": "CCl4"
  },
  {
    "caption": "Relative mRNA expression (ASK1F/F n=13 mice; ASK1+hep n=14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4-injected mice. Values are expressed as mean ± SEM.",
    "molecules": "CCl4"
  },
  {
    "caption": "C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP  or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A). Data information: data are representative of two independent experiments.",
    "molecules": "1NM-PP1"
  },
  {
    "caption": "A. Imaging of  SPBs  (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus     hrr25-as. Cells were shifted from 24 to 36°C at 4 h in  SPM and treated with 1NM-PP1 to inhibit  Hrr25-as. Top, time-lapse series. Bottom,  Mpc70 loading to SPBs was quantified in cells synchronized  in silico to SPB  separation at entry into  metaphase I  (t = 0). Data information: data are representative of three independent experiments.",
    "molecules": "1NM-PP1"
  },
  {
    "caption": "C. Imaging of  SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆  spo13∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM. Mpc70  loading (bottom) was analyzed as in (A). PEST-SPC72 expression delays the  time of Mpc70 loading by 98 min (95%  CI, 67-129; P < 0.0001; Welch's t-test). Data information: data are representative of three independent experiments.",
    "molecules": "EST"
  },
  {
    "caption": "(B) Analysis of cells containing wild-type   Spo13, Spo13-mD, or Spo13-mD-m2.  Meiotic events  were quantified in cells synchronized  in silico to SPB reduplication at entry into metaphase II  (t = 0). Graphs show overlays of SPC72-GFP and  MPC70-GFP strains.  spo13-mD delays Spc72 removal and Mpc70  loading by 30 min (95%  CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of  Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and  Mpc70  loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95%  CI, 99-129; P < 0.0001; Welch's t-test), respectively.",
    "molecules": "EST"
  },
  {
    "caption": "E.  Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing  spo13∆ or  cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows  immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of  Ime2. Data information: data are representative of two independent experiments.",
    "molecules": "CMK"
  },
  {
    "caption": "A, B. Mitotic cdc20-3 cells were shifted to 36°C for 80 min. At t = 0, cells were treated with  estradiol  to induce expression from the PEST promoter. Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Spc72-GFP. Frame width, 19 μm. Bottom, quantification of cells with  Spc72-GFP at SPBs. (A) Expression  of  Ime2-∆C induces removal of Spc72 from SPBs, which is inhibited by co-expression of Spo13. (B) Expression of Cdc5  plus Ime2-∆C causes rapid removal of Spc72, which is inhibited by co-expression of Spo13. Data information: data are representative of three independent experiments.",
    "molecules": "EST, estradiol"
  },
  {
    "caption": "C. Mitotic cdc20-3 cells containing PEST-CDC5 plus PEST-IME2-∆C and/or PEST-NDT80 were shifted to 36°C for 30 min and subsequently treated with  estradiol (t = 0). Top, time-lapse  series from the imaging of SPBs  (Cnm67-RFP) and Mpc70-GFP. Frame width, 19 μm. Bottom, quantification of cells with Mpc70-GFP at SPBs. Data information: data are representative of two independent experiments.",
    "molecules": "EST, estradiol"
  },
  {
    "caption": "C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing  spo13∆ or  cdc5-as were treated with 1Na-PP1 at t = 3 h in  SPM to inhibit Cdc28-as2. At t = 9 h in  SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus  CMK to activate Cdc28-as2 and inhibit  Cdc5-as. Top,  time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of  spindle  formation and Mpc70  loading to  SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in  SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse  series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to  SPBs. Inhibition of  Cdc5-as in spc72-7 cells advances the  time of Mpc70  loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.",
    "molecules": "1Na-PP1, CMK"
  },
  {
    "caption": "A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM  (t = 0) and treated with 1NM-PP1  at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei  (TetR-RFP). Bottom, quantification of meiotic events.",
    "molecules": "1NM-PP1, CuSO4"
  },
  {
    "caption": "Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP) and western blotting. Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control. M.W. indicates protein molecular weight marker.",
    "molecules": "IgG"
  },
  {
    "caption": "Western blotting of endogenous Egg immunopurified from OSCs. Anti-Egg antibodies were used for immunoprecipitation (IP). Anti-Egg and anti-Ub antibodies were used for western blotting (WB). Input: OSC total lysates used for IP. n.i.: nonimmune IgG used as an IP negative control.",
    "molecules": "IgG, Ub"
  },
  {
    "caption": "Western blotting of Myc-Egg WT and its K1085R and S215/T217 (STAA) mutants expressed in OSCs. Egg modifications identified in this study are summarized on the right. P: phosphorylation. Ub: monoubiquitination.",
    "molecules": "Ub"
  },
  {
    "caption": "In vitro histone methyltransferase assays. Top and second from the top: silver staining of purified Myc-Egg and Myc-EGFP. Second from the bottom: CBB staining of histone H3.1. Bottom: 14C autoradiogram. EGFP: Myc-EGFP was used as a negative control. l.c.: light chain of antibody. Time: reaction time (min) of individual samples.",
    "molecules": "CBB, 14C"
  },
  {
    "caption": "Immunofluorescence analysis of OSCs using anti-Egg antibodies. Endogenous Egg is shown in red. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blotting of Myc-Egg expressed in OSCs before (siEGFP) and after (siUbc2) Ubc2 depletion. Anti-Myc-antibody was used for western blotting. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.43 (siEGFP) and 0.78 (siUbc2).",
    "molecules": "Ub"
  },
  {
    "caption": "Immunofluorescence analysis using anti-Flag antibody to identify Flag-Ubc2 (green) expressed in OSCs. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence analysis of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs using anti-Myc antibody. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blotting of Myc-Egg WT, Myc-SV40-NLS-Egg, and Myc-PKI-NES-Egg expressed in OSCs. Anti-Myc antibody was used. The ratios of mUb-Egg over non-mUb-Egg band signals were 1.50 (WT), 2.29 (SV40-NLS), and 0.95 (PKI-NES).",
    "molecules": "Ub"
  },
  {
    "caption": "In vitro HMT assays. Top: silver staining of purified Myc-Egg WT, Myc-Egg K1085R, Myc-SV40-NLS-Egg WT, and Myc-SV40-NLS-Egg K1085R. Middle: CBB staining of histone H3.1. Bottom: 14C autoradiogram. Myc-EGFP was used as a negative control (EGFP: not shown).",
    "molecules": "CBB, 14C"
  },
  {
    "caption": "Immunofluorescence analysis of endogenous Egg (red) in OSCs using anti-Egg antibody before (siEGFP) and after (siWde) Wde depletion. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence analysis of endogenous Egg (red) in OSCs upon Flag-Wde (green) co-expression. Anti-Egg and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Wde co-expression (green). Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence analysis of Myc-Egg WT (red) in OSCs upon Flag-Wde WT and ∆CC mutant (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence analysis of Myc-Egg WT, Myc-Egg K987A, and Myc-Egg R988A mutants (red) in OSCs before and after Flag-Wde (green) co-expression. Anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence analysis of Myc-Egg K987A and Myc-Egg R988A mutants (red) in OSCs. Both mutants were fused to SV40-NLS. Anti-Myc antibody was used for detection. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence analysis of H3K9me3 (green), Myc-Egg WT (red), and Myc-SV40-NLS-Egg WT (red) in OSCs before and after Flag-Wde (yellow) co-expression. Anti-H3K9me3, anti-Myc and anti-Flag antibodies were used. DAPI (blue) shows the nuclei. Scale bar: 10 μm.",
    "molecules": "H3K9me3, DAPI"
  },
  {
    "caption": "(C) Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red), and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0-3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient.",
    "molecules": "liposome, ACMA, NH4Cl, ATP, liposomes, H+, proton, NaCl"
  },
  {
    "caption": "(D) Michaelis-Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments errors bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions.",
    "molecules": "ACMA"
  },
  {
    "caption": "A) Native mass spectrum of NHE9* purified in the presence (top panel) or the absence of additional brain lipids (bottom panel). In the presence of brain lipids, NHE9* is detected as exclusively dimeric protein with poorly resolved peaks, indicating the presence of multiple lipid adducts. Without brain lipid addition, NHE9* appears as sharp peaks, revealing several adducts of approximately 1 kDa each, as well as a minor monomer population.",
    "molecules": "lipid adducts, lipid, lipids"
  },
  {
    "caption": "C) Thermal shift stabilization of purified dimeric NHE9*-GFP in the presence of PIP2 (red) compared to PIP2-free (black). Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.",
    "molecules": "PIP2"
  },
  {
    "caption": " E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. (F) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained. ",
    "molecules": "lipid adducts, PIP2"
  },
  {
    "caption": "(B) Comparison of cell proliferation rate between T- and B- lines. Cell proliferation assay was performed by MTS reagents. Data are the means ± SD (n=3 biological replicates).",
    "molecules": "MTS"
  },
  {
    "caption": "(D) Top: H2S catalyzing activity of cell lysates from PC3 T2, B2, T3, and B3 lines. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).",
    "molecules": "H2S, Lead acetate, PbS"
  },
  {
    "caption": "(B) Top: The H2S production capacity of cell lysates from PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).",
    "molecules": "H2S, Lead acetate, PbS"
  },
  {
    "caption": "(D) Cell proliferation rates of PC3-B2 cells with CTH knockdown. Cell proliferation assay was performed by MTS reagent. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).",
    "molecules": "MTS"
  },
  {
    "caption": "(G) PC3 cells were pre-treated with 10μM PAG for 16 hrs, and incubated for migration (16h) and invasion assay (24h) in the presence of 10μM PAG. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).",
    "molecules": "PAG"
  },
  {
    "caption": "(D) PC3 cells transfected with control or CTH siRNA were exposed to IL-1β (20ng/ml) for 1hr. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 25μm.",
    "molecules": "DAPI"
  },
  {
    "caption": " (A) PC3 cells were incubated for migration (16h) and invasion (24h) assay. DMEM/10% FBS, together with various concentration of NaHS (10nM-1mM) acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)",
    "molecules": "NaHS"
  },
  {
    "caption": " (B) PC3 cells transfected with control or CTH siRNA were incubated for invasion (24h) assay. DMEM/10% FBS, with or without NaHS (1μM), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)",
    "molecules": "NaHS"
  },
  {
    "caption": " (C) Top: PC3 cells treated with 100 μM NaHS for 30 min at 37°C. Cells were lyzed and treated with or without DTT for 10min at 4°C before subjecting to the modified biotin switch assay with the antibody against p65 to detect S-sulfhydration. Down: quantitative analysis of SSH-p65 protein level, and normalized with total p65 level Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)",
    "molecules": "DTT, NaHS"
  },
  {
    "caption": " (D) PC3 cells were incubate for invasion assay for 24 hrs. DMEM/10% FBS, together with 1μM NaHS and an NF-κB inhibitor (50μg/ml SN50, or 100nM QNZ), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)",
    "molecules": "QNZ, SN50, NaHS"
  },
  {
    "caption": " (E) Real-Time RT-PCR analysis for IL-1β, VEGF, and MMP-13 mRNA levels in PC3 cells treated with 1μM NaHS for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) Student's t-tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)",
    "molecules": "NaHS"
  },
  {
    "caption": " (F) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with various concentrations of NaHS (10nM-10μM) added for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)",
    "molecules": "NaHS"
  },
  {
    "caption": "(A-B) Hemoglobin  (A) and hematocrit  (B) levels of healthy subjects and cachectic cancer patients  presenting a body weight loss superior to 10% of initial body weight (19 healthy subjects, 17  cachectic patients). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "Hemoglobin"
  },
  {
    "caption": "(F) TFR1  mRNA levels in the gastrocnemius of  mice subjected to  iron deprivation by feeding with an  iron deficient diet (IDD) combined to a phlebotomy  (PHL) (n=5-6) Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "iron"
  },
  {
    "caption": "(L) Reprensentative pictures and diameter measurements of human myoblast-derived myotubes after 48h treatment with DFO 100µM (n=3). Scale bar=50µm. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "DFO"
  },
  {
    "caption": "(I-J)  ICP-MS  quantification of total (I) and  protein-bound (J) iron  in mouse quadriceps  (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "iron"
  },
  {
    "caption": "(A) ICP-MS quantification of mitochondrial iron  in mouse quadriceps (n=6-8). (B)  Gastrocnemius heme content quantified by fluorescent heme assay (n=4-6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "heme, iron"
  },
  {
    "caption": "(C-D) mRNA levels of mitochondrial iron  importer  MFRN2 (C) and the rate limiting enzyme of heme synthesis ALAS2 (D) normalized to  18s in mouse gastrocnemius (n=4-5). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "heme, iron"
  },
  {
    "caption": "(B) Western blot  of mitochondrial OXPHOS respiratory complexes in C2C12 myotubes treated for 48H with   C26CM and ferric citrate. (n=3)",
    "molecules": "ferric citrate"
  },
  {
    "caption": "(G-H) Representative microscopic  pictures and diameter of C2C12 myotubes stained  for myosin heavy chain (G) or human myoblast-derived myotubes  (H)after 48h treatment with  C26  CM and ferric citrate (n=3 per condition). Scale bars =50 and 100 µm, respectively. Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by one-way Anova with Bonferroni's correction Data are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and ##p < 0.01, ###p < 0.001 compared to C26 CM-treated group.",
    "molecules": "ferric citrate"
  },
  {
    "caption": "(E) Representative Western blot  and  densitometric  quantification of phospho-AMPK  and total AMPK (stripped and re-blotted) in the gastrocnemius  of  C26-tumor bearing mice after iron  carboxymaltose supplementation (n=6). Data information: For all data, n represents the number of biological replicates. Statistical significance was calculated by unpaired, two-tailed Student's T-test. Data are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to control and #p < 0.05, ##p < 0.01, ###p < 0.001 compared to C26 untreated group.",
    "molecules": "iron  carboxymaltose"
  },
  {
    "caption": "(A-B) Grip force of dominant  or non-dominant  arm in iron-deficient cancer patients, expressed in percentage of baseline force (A) and absolute values normalized to height (B) before/after single dose of iron carboxymaltose (15mg/kg, 7 subjects). Data information: For all data, n represents In graph (A), the boxes represent the range of values with the median value being the central band and whiskers the SEM.",
    "molecules": "iron carboxymaltose, iron"
  },
  {
    "caption": "(B) Drug response curves upon treatment of organoid models with nutlin‑3a, 5‑FU, oxaliplatin, ibrutinib, panobinostat and entinostat (biological replicates n=3, data are shown as mean ±SD).",
    "molecules": "5‑FU, entinostat, ibrutinib, nutlin‑3a, oxaliplatin, panobinostat"
  },
  {
    "caption": "Drug response curves upon treatment with inhibitors for (B) EGFR (afatinib, gefitinib), (biological replicates n=3, data are shown as mean ±SD).",
    "molecules": "afatinib, gefitinib"
  },
  {
    "caption": "Drug response curves upon treatment with inhibitors for (C) B‑RAF (LY3009120, TAK‑632, vemurafenib, dabrafenib), (biological replicates n=3, data are shown as mean ±SD).",
    "molecules": "dabrafenib, LY3009120, TAK‑632, vemurafenib"
  },
  {
    "caption": "Drug response curves upon treatment with inhibitors for (D) MEK1/2 (selumetinib, binimetinib) (biological replicates n=3, data are shown as mean ±SD).",
    "molecules": "binimetinib, selumetinib"
  },
  {
    "caption": "(B) Drug response curves of PDOs upon treatment with the MEK1/2 inhibitor trametinib (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (C) Drug response curves upon HDAC inhibition with panobinostat (PAN) and entinostat (ENT) (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2). (D) Comparison of the relative area under the curve (AUCrel) upon trametinib treatment in RTK/MAPK altered (mean AUCrel: 0.5308; biological replicate n=7) vs. non‑altered (mean AUCrel: 0.7101; biological replicates n=6) human PDOs (two‑tailed Student's t‑Test; *p= 0.0356).",
    "molecules": "ENT, entinostat, PAN, panobinostat, trametinib"
  },
  {
    "caption": "B. Analysis of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 and Trp53bp1. Left, metaphase spreads were prepared from B cells treated with olaparib. Arrows show examples of chromosome aberrations. Right, quantification of chromosome breaks (CSB), chromatid-type breaks (CTD), radial chromosomes, and other abnormalities in mouseB cells. Error bars indicate SD, N=3",
    "molecules": "olaparib"
  },
  {
    "caption": "D. Percentage of cells showing >5 RAD51 foci after 4hrs treatment with 10 μM olaparib (OLA) or 10 μM camptothecin (CPT). N=2.",
    "molecules": "camptothecin, CPT, OLA, olaparib"
  },
  {
    "caption": "F. Sister chromatid exchanges in cells that were not treated (NT) or exposed to 2 μM olaparib (OLA) for 16 hrs. Black arrows in images show SCEs in olaparib-treated cells. Scale bar: 5 μm. N=2.",
    "molecules": "OLA, olaparib"
  },
  {
    "caption": "A. Experimental scheme and representative images of DNA fibers. Scale bar: 5 μm.B. Analysis of fibers showing proportions of replication forks that showed restart after HU treatment or remained stalled. N=2, >500 fibers scored per experiment.C. Proportion of total forks showing de novo initiation after HU treatment. N=2, >500 fibers scored per experiment.D. Length of initial replication fork tracts (CldU tracts). N=2, >200 fibers measured per experiment.",
    "molecules": "CldU, DNA fibers, HU"
  },
  {
    "caption": "D. G2M checkpoint analysis in mouse B cells after IR treatment. Left, mitotic cells were identified by flow cytometry as having 4c DNA content (based on propidium iodide staining) and pSer10-H3+. Right, quantification of flow cytometry data, showing mitotic cells after IR as percentage of that seen in untreated cells. N≥2.",
    "molecules": "DNA"
  },
  {
    "caption": "(A)  Confocal  observation of  mitochondrial potential after TMRM staining of LMP1-negative or -positive   NPC   cells. Red fluorescence, which indicates normal mitochondrial potential, was converted into green fluorescence after a reduction in mitochondrial  potential. Images were analyzed using Image J software (scale bar, 10 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).",
    "molecules": "TMRM"
  },
  {
    "caption": "(C) Extent of  Ca2+-mediated  mitochondrial swelling in NPC cells. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).",
    "molecules": "Ca2+"
  },
  {
    "caption": "(B) The  interaction of LMP1 and endogenous ANT1 was analyzed by an  immunofluorescence confocal assay. HK1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-ANT1 (red) antibodies and then subjected to confocal microscopy，and use imageJ to calculate the  co-location  level of both. The nuclei were stained with  DAPI  (scale bar, 5 μm). The quantified graph on the top right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in HK1-LMP1 cells. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).",
    "molecules": "DAPI"
  },
  {
    "caption": "(A Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in  293T cells.",
    "molecules": "BKA, CATR"
  },
  {
    "caption": "B) Effect of inhibitors  BKA and CATR on ANT1-VDAC1 binding in 293T cells. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, ** p < 0.01).",
    "molecules": "BKA, CATR"
  },
  {
    "caption": "(G) 19F-NMR spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min. (H) 19F-NMR  spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1-ΔT. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min. ",
    "molecules": "F2Y"
  },
  {
    "caption": "(A-B) The ADP/ATP exchange rate in EBV-LMP1-positive nasopharyngeal carcinoma cells is decreased. The fluorescence value of Magnesium Green ™ (1 μM) was measured at 505 nm (Ex)/535 nm (Em) by enzymography (A), and the  ADP/ATP  exchange rate was calculated after conversion to ATP (B). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, ** p < 0.01).",
    "molecules": "ADP, ATP, Magnesium Green"
  },
  {
    "caption": "(C) LMP1-negative or -positive  NPC cells were treated with BKA (1 μM) or CATR (5 μM) for 24 h, Mitochondrial OCR assessed with the XF24 extracellular flux analyzer (Seahorse) under sequential treatment with oligomycin, FCCP, and antimycin A. Data are presented as means ± S.E.M. (n = 6, biological replicates per group). (D) Oxidative phosphorylation-related metrics based on OCR datas. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05,** p < 0.01). ",
    "molecules": "antimycin A, BKA, FCCP, CATR, oligomycin"
  },
  {
    "caption": "(A) HK1-LMP1  were treated with increasing concentrations of cisplatin for 24 h. Cell viability was determined by  MTS assay. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).",
    "molecules": "cisplatin"
  },
  {
    "caption": "(D) The IC50 value of the combination of CATR and cisplatin in CNE1-LMP1 cells was changed. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).",
    "molecules": "CATR, cisplatin"
  },
  {
    "caption": "(G) Flow cytometry  analysis of death of  NPC cells treated for 24 h with  CATR (1 μM) alone, cisplatin  (20 μM) alone, or cisplatin  (20 μM) combined with CATR  (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, * p < 0.05, ** p < 0.01).",
    "molecules": "CATR, cisplatin"
  },
  {
    "caption": "(H)  Trypan blue staining  was used to detect death of  NPC cells treated for 24 h with vehicle, cisplatin, CATR, or a combination of cisplatin and CATR (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).",
    "molecules": "CATR, cisplatin, Trypan blue"
  },
  {
    "caption": "(C) NAD+/NADH ratio was measured in mBA cell lines and 17.5 days embryo liver. Data represented as mean ±SEMs, and obtained from three different cell line and embryo liver. Significance determined by Student's t test, two-tailed, *P < 0.05, **P < 0.01.",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "(D) Immunostaining of 17.5 days embryo tissues (mid-brain, heart, liver, and skin) with anti-PAR antibody. Insets show higher magnification. Scale bar indicates 100 μm (insert).",
    "molecules": "PAR"
  },
  {
    "caption": "(A) Western blot analysis of Parp1 and DNA damage markers in Wild-type (WT) and Ews-/- mBA cells. Cells were treated with MMS (0.02%) in a time dependent manner. Proteins were fractionated into two groups, chromatin-bound (Chro) proteins and soluble (Solu) proteins. (B) After treatment with MMS (0.02%, 1hour), the media was replaced to release the DNA damage. The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot.",
    "molecules": "MMS"
  },
  {
    "caption": "(E) AsiSI endonuclease-integrated U2OS cells were transfected with siControl and siEWS. Cells were incubated with doxycycline for 4 hours to induce DSBs. With or without changing the Dox-added media to fresh media for 2 hours (for release samples), the amount of chromatin-associated PARP1 was measured using ChIP assay. (N.T: Non-treat, T: AsiSI treat, Release: Damage released samples). Data represented as mean ±SEMs, and technical repeats (n=3), significance determined by two-way ANOVA, ***P< 0.001.",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "(A) Total levels of PAR in whole cell lysate were measured using western blot analysis. Wild-type (WT) and Ews-/- mBA cells were treated with H2O2, (1 mM, 20 minutes) with or without Olaparib (5 μM, 7 hours).",
    "molecules": "H2O2, Olaparib, PAR"
  },
  {
    "caption": "(B) The kinetics of PAR accumulation was analyzed by western blotting. WT and Ews-/- cells were treated with H2O2 (1 mM, 20 minutes), followed by incubation in fresh media for the indicated times to allow release of DNA damage",
    "molecules": "H2O2, PAR"
  },
  {
    "caption": "(C) Immunohistochemistry detection of PAR by ADP-ribose antibody in WT and Ews-/- cells after H2O2 treatment (1 mM, 20 minutes) and recovery from DNA damage. Right graph displays mean of intensities measured from 500 cells. Data represented as mean ±SEMs, significance determined by One-way ANOVA, ***P < 0.001. Scale bar indicate 20 μm.",
    "molecules": "ADP-ribose, H2O2, PAR"
  },
  {
    "caption": "(D, E) Whole cell level of PAR was measured in WT and Ews-/- mBA cells upon treatment with (D) MMS (0.02%, 60 minutes) (E) with or without recovery from DNA damage.",
    "molecules": "MMS, PAR"
  },
  {
    "caption": "(F) NAD+/NADH ratio was measured in mBA cell lines following treatment of H2O2 (1 mM, 10 minutes). Data represented as mean ±SEMs, and technical repeats (n=3). Significance determined by Student's t test, two-tailed, **P < 0.01, ***P< 0.001.",
    "molecules": "H2O2, NAD+, NADH"
  },
  {
    "caption": "(A) PARP1 and EWS interaction was visualized in cells using the CUPID system. After transfection of mRCD-EWS and GC3-PARP1 (EGFP-PARP1) plasmid, the cells were treated with either H2O2 (1 mM, 10 minutes) or PMA. Scale bar indicates 5 μm.",
    "molecules": "H2O2, PMA"
  },
  {
    "caption": "(B) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis. Cells were treated with H2O2 (1 mM, 10 minutes) with or without Olaparib (5 μM, 7 hours).",
    "molecules": "H2O2, Olaparib"
  },
  {
    "caption": "(C) MMS-treated cells, with or without Olaparib, were immunoprecipitated using an anti-EWS antibody and immunoblotted by anti-PAR antibody (upper). Pre-Olaparib group was treated with Olaparib for 3 hours before MMS treatment (0.02% 30 minutes) and after-Olaparib group treated Olaparib after 10 minutes following treatment of MMS. Lower blot indicates input of anti-EWS antibody.",
    "molecules": "MMS, Olaparib, PAR"
  },
  {
    "caption": "(D) In vitro PAR binding assay. THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control.",
    "molecules": "PAR"
  },
  {
    "caption": "(E) Schematic map of nine EWS mutants (left) and IP-western blot analysis (right). GFP-PARP1 and siPARG were transfected into HEK-293 cells with each EWS mutants plasmid. After H2O2 treatment, the cells were conducted to IP with anti-Flag antibody.",
    "molecules": "H2O2"
  },
  {
    "caption": "(F) Western blot analysis of PARP1 accumulation on chromatin. EWS-WT and M4 mutants were transfected into EWS depleted HEK-293 cells. After treatment of MMS (0.02%, 1 hour), the proteins were fractionated to either chromatin-bound or soluble fraction. (G) Quantification of relative amount of PARP1 and γH2AX on chromatin, divided by chromatin H3, from three independent western experiments are presented. Data represented as mean ±SEMs, were measured from 3 independent experiments. Significance determined by t-test. * P < 0.05, **P < 0.01. n.s indicate non-significance",
    "molecules": "MMS"
  },
  {
    "caption": "(H) WT and Ews-/- cells were transfected with EWS-WT and -M4 mutant and cell viability was measured 24 hours after MMS treatment (indicated concentration). Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, **P < 0.01. n.s indicate non-significance,",
    "molecules": "MMS"
  },
  {
    "caption": "(A) Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP-1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD.",
    "molecules": "MMS"
  },
  {
    "caption": "(B) Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS-KO.",
    "molecules": "MMS"
  },
  {
    "caption": " C) After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co-depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot. ",
    "molecules": "MMS"
  },
  {
    "caption": "D) After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using western blot.",
    "molecules": "MMS"
  },
  {
    "caption": "E) Whole cell expression of PAR in mBA (WT: Ews-WT, KO: Ews-KO, DKO: double KO).",
    "molecules": "PAR"
  },
  {
    "caption": "F) Relative NAD+/NADH ratios were measured in WT, Ews-/- and DKO cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "H) Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews-/- cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.",
    "molecules": "MMS, NMN"
  },
  {
    "caption": "I) Chromatin-bound PARP1 was quantified by western blot in WT and Ews-/- cells treated with Olaparib (5 μM, 24 hours) treatment.",
    "molecules": "Olaparib"
  },
  {
    "caption": "J) Relative viability was measured in WT and Ews-/- cells upon treatment of Olaparib for 24 hours. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.",
    "molecules": "Olaparib"
  },
  {
    "caption": "(A) Western blot analysis of PARP1 in chromatin. Two Ewing Sarcoma cells (CHP100 and A4573) were treated with MMS (0.02%, 1 hour) with or without release (1 hour) from MMS (N.T: non treat, T: treat, R: release sample).",
    "molecules": "MMS"
  },
  {
    "caption": "(B) U2OS and two Ewing Sarcoma cells were treated MMS (0.02%, 30 minutes) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction. Scale bar indicates 20 μm (left). Red line indicates mean and more than 300 cells were analyzed. Significance determined by Two-way ANOVA, ***P < 0.001.",
    "molecules": "MMS"
  },
  {
    "caption": "(C) Human Ewing Sarcoma histology samples were analyzed by immuno-histochemistry using PAR antibody. Scale bar represents 100 mm.",
    "molecules": "PAR"
  },
  {
    "caption": "(a-d) Formation of protein inclusions in SY5Y cells expressing FL (a) or the indicated truncated forms of Sph1 (b-d) was induced by 12 h lactacystin treatment. After extensive washes, cells were maintained in serum-supplemented (serum+) or serum-free (serum−) media, and in the absence (Control) or presence of 10 mM 3-MA, an inhibitor of macroautophagy. Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (left) or Agg (right) observed under different recovery conditions. Values are mean+s.e. (n=4). *Significant versus serum+ condition. See also Supplementary Figs S1-S5.",
    "molecules": "3-MA, lactacystin"
  },
  {
    "caption": "(a-c) Top: fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL (a), or the indicated truncated Sph1 proteins (b-c) treated with lactacystin (12 h) and recovered in basal conditions (serum+). Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (middle) or Agg (bottom). Values are mean+s.e. (n=3). *Significant versus control.",
    "molecules": "lactacystin"
  },
  {
    "caption": "(a-d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b-d). Cells were treated with lactacystin (12 h) and recovered in the presence or absence of serum. Graphs: percentage of p38 existing alone in Agm or co-aggregating with Sph1 in the forms of Agm or Agg (left), and susceptibility of p38 existing on its own in Agm or co-aggregating with Sph1 proteins in Agm toward serum-stimulated autophagic clearance (right). Values are expressed as folds Agm in the presence of serum and are mean+s.e. (n=3). *Significant versus serum+ condition calculated using Student's t-test.",
    "molecules": "lactacystin"
  },
  {
    "caption": "(c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA-ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated.",
    "molecules": "lactacystin"
  },
  {
    "caption": "(a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b).",
    "molecules": "3-MA"
  },
  {
    "caption": "Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Zuc KD) Zuc knockdown in OSCs. Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi signal (green) is strongly overlapped with the mitochondrial signal (red) in Zuc-depleted OSCs (Zuc KD). Armi is localized to Yb bodies in control OSCs (control). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Gasz KD) Gasz depletion in Zuc-depleted OSCs (Zuc KD). Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi mitochondrial signal (green) found in Zuc-depleted cells (Zuc KD/control) mostly disappeared after additional Gasz depletion in the cells (Zuc KD/Gasz KD), but was found at Yb bodies. Mitochondrial signal is shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "Depletion of endogenous Piwi (Piwi KD) in Zuc-lacking OSCs (Zuc KD) caused Armi (green) to relocate to Yb bodies. Localization of Armi at Yb bodies is shown in B. Mitochondrial (upper panel) and Yb (lower panel) signals are shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "In vitro pull-down assays show that Armi-Flag (Armi-F) directly binds with GST-Gasz∆C113 but hardly with GST. Armi-F was immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F was detected by western blotting using anti-Flag antibodies.",
    "molecules": "CBB"
  },
  {
    "caption": "Armi localization to Yb bodies observed in Zuc- and Piwi-depleted OSCs (Zuc KD + Piwi KD) (Figs 3A and B) was restored by ectopic expression of Flag-Piwi WT and PAZmt but not of MIDmt (see also Fig EV4B). Armi, mitochondria, and F-Piwi are shown in green, red, and yellow, respectively. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F N756A mutant (green) localized to Yb bodies (see also Fig EV5D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and N756A mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and N756A mutant were detected by western blotting using anti-Flag antibodies.",
    "molecules": "CBB"
  },
  {
    "caption": "Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F ∆N34 mutant (green) localized to Yb bodies (see also Fig EV6D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.",
    "molecules": "DAPI"
  },
  {
    "caption": "In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and ∆N34 mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and mutant were detected by western blotting using anti-Flag antibodies.",
    "molecules": "CBB"
  },
  {
    "caption": "Immunoblot analysis of FLAG:AGO1 IPed in each root-tip layer. Scale bars: 50μM, NT: Non-transformed. CB: Coomassie blue.",
    "molecules": "CB, Coomassie blue"
  },
  {
    "caption": "qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17. Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2.",
    "molecules": "RNA"
  },
  {
    "caption": "Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions. Northern analysis of AGO1-loaded miR395 under the conditions in (C).",
    "molecules": "SO4"
  },
  {
    "caption": "Binding of miR395 by SHR::HA:P19 under SO4--sufficient (+) and -deficient (-) growth conditions.",
    "molecules": "SO4"
  },
  {
    "caption": "G PPAR-α and RIP140coimmunoprecipitate in WT but not FADD knockout MEFs upon WY-14,643 stimulation. Ligand-dependent interactions were examined in the presence of 100 μM WY-14,643 where indicated. Data shown are representative of three independent experiments having similar results.",
    "molecules": "WY-14,643"
  },
  {
    "caption": "A Left, Body weights of male WT and FADD-D mice on either a SD (n = 8 for each genotype) or a HFD (n = 6 for each genotype). Right, Food intake per mouse fed a HFD measured over 20 days normalized by body weight (n = 10 for each genotype). Results are means ± SEM. *P = 0.0007 (Student's t-test).",
    "molecules": "Food"
  },
  {
    "caption": "C Left, fat pad weights as a percentage of body weight (BW) and in absolute amounts (inset) from male FADD-D and WT mice. Mice were fed a SD or a HFD until 15 weeks of age (n = 6 for each genotype). Results are means ± SEM. *P = 0.0012, **P = 0.0002, ***P = 0.0027, ****P = 0.0014, ^P = 0.0038, ^^P = 0.0019, #P = 0.0045, ##P = 0.0007, *^P = 0.0252, *^^P = 0.0082, ###P = 0.0275, ####P = 0.0071 (one-way ANOVA). Middle, fat pad weights of male FADD-D and WT mice on a HFD at 30 weeks of age (n = 6 for each genotype). Inset, triacylglycerol (TAG) content in epididymal WAT. Data are expressed as means ± SEM. *P = 0.0003, **P = 0.0005, ***P = 0.0019, ****P = 0.0011 (Student's t-test). Right, weight of liver normalized by body weight for male FADD-D and WT littermates at the age of 15 weeks (n = 6 for each genotype). Data are expressed as means ± SEM. NS, not statistically significant (Student's t-test). Epi, epididymal fat; Ren, renal fat; Ing, inguinal fat.",
    "molecules": "TAG, triacylglycerol"
  },
  {
    "caption": "G Left, relative amounts of liver and muscle triacylglycerol contents in 10-month-old WT and FADD-D mice (n = 6 for each genotype). Right, relative amounts of cholesterol in the liver and muscle of the same group of WT and FADD-D mice (n = 6 for each genotype). Data are expressed as means ± SEM. *P = 0.0015, **P = 0.0027, ***P = 0.0318 (Student's t-test). NS, not statistically significant.",
    "molecules": "cholesterol, triacylglycerol"
  },
  {
    "caption": "H Left, plasma triacylglycerol levels in 10-month-old WT and FADD-D mice following fasting for 20 h (n = 5 for each genotype). Right, plasma cholesterol levels in the same group of WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0124 (Student's t-test). NS, not statistically significant.",
    "molecules": "cholesterol, triacylglycerol"
  },
  {
    "caption": "A Serum free fatty acid (FFA) levels and triacylglycerol levels in WT and FADD-D mice fed a HFD for 20 days (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0059, **P = 0.0012 (Student's t-test).",
    "molecules": "fatty acid, FFA, triacylglycerol"
  },
  {
    "caption": "C Morphology of inguinal WAT, BAT, and liver from WT and FADD-D mice fed a HFD for 20 days. WAT and BAT were stained with hematoxylin and eosin. Liver tissues were stained with Oil red O to demonstrate lipid accumulation and counterstained with hematoxylin. Scale bars = 50 μm. Shown are typical results from four different fields and three different experiments.",
    "molecules": "lipid"
  },
  {
    "caption": "F Whole-body oxygen consumption rate (VO2) during 24 hr in WT and FADD-D mice fed a SD (n = 4 for each genotype). Data are expressed as means ± SEM. *P = 0.0055, **P = 0.0081 (Student's t-test).",
    "molecules": "O2, oxygen"
  },
  {
    "caption": "G Average respiratory exchange ratio (VCO2/VO2) (RER) for the dark and light period in WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0042, **P = 0.0079 (Student's t-test).",
    "molecules": "CO2, O2"
  },
  {
    "caption": "F Oxygen consumption in isolated BAT from 10-week-old male WT and FADD-D mice on a SD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0025, **P = 0.0006 (Student's t-test).",
    "molecules": "Oxygen"
  },
  {
    "caption": "H β-oxidation analysis of white adipocytes isolated from WT (n = 5) and FADD-D mice (n = 4). 1-14C labeled oleic acid was added to the medium containing isolated adipocytes (5×105), and 14CO2 trapped by the filter paper soaked with hyamine hydroxide was measured after a 3 h-incubation by a scintillation counter. Data are expressed as mean ± SEM. *P = 0.0022 (Student's t-test).",
    "molecules": "hyamine hydroxide, CO2, oleic acid"
  },
  {
    "caption": "I Basal and stimulated (+ 100 nM isoproterenol) lipolysis, as measured by glycerol (left) and fatty acids (right) released from explants of epididymal WAT from overnight fasted 10-week-old male WT and FADD-D mice fed a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0078, **P = 0.0065, ***P = 0.0008, ^P = 0.0133, ^^P = 0.0053, ^^^P = 0.0006, ^^^^P = 0.0001, #P = 0.0082, ##P = 0.0051, ###P = 0.0009, *^P = 0.0151, *^^P = 0.0019, **^^P = 0.0005, ***^P = 0.0002 (Student's t-test).",
    "molecules": "fatty acids, glycerol, isoproterenol"
  },
  {
    "caption": "J Molar ratio of FFA to glycerol release from WAT explants (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0412, **P = 0.0033 (Student's t-test).",
    "molecules": "FFA, glycerol"
  },
  {
    "caption": "B cAMP abundance in epididymalWAT from 10-week-old male WT and FADD-D mice on a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0033 (Student's t-test).",
    "molecules": "cAMP"
  },
  {
    "caption": "C Basal and forskolin-stimulated adenylyl cyclase activity in epididymalWAT from 10-week-old male WT and FADD-D mice on a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0177, *P = 0.0091 (Student's t-test).",
    "molecules": "forskolin"
  },
  {
    "caption": "E Basal and stimulated (+200 nM isoproterenol) lipolysis in epididymalWAT of 10-week-old male WT and FADD-D mice (n = 4 for each genotype) treated with or without 10 μM BAY. Data are expressed as mean ± SEM. *P = 0.0019, **P = 00038, ^P = 0.0035, ^^P = 0.0053 (one-way ANOVA). NS, not statistically significant.",
    "molecules": "BAY, isoproterenol"
  },
  {
    "caption": "F Basal and stimulated (+200 nM isoproterenol) lipolysis in isolated adipocytes from 10-week-old WT and FADD-D mice (n = 4 for each genotype) treated with or without 10 μM BAY. Data are expressed as mean ± SEM. *P = 0.0033, **P = 0.0051, ^P = 0.004, ^^P = 0.0057 (one-way ANOVA). NS, not statistically significant.",
    "molecules": "BAY, isoproterenol"
  },
  {
    "caption": "C Food intake in 15-week-old male control and ad-FADDmice fed a HFD (n = 8 for each genotype).",
    "molecules": "Food"
  },
  {
    "caption": "A-C HFD-fed control and ad-FADDmice were housed in a computer-controlled open-circuit indirect calorimeter to determine (A) oxygen consumption, (B) carbon dioxide production, and (C) respiratory exchange ratio (RER) during the light (8 am - 8 pm) and dark (8 pm - 8 am) periods (n = 5 for each genotype). Data are expressed as mean ± SEM. *P = 0.0379, **P = 0.0453, ***P = 0.0445 (Student's t-test). NS, not statistically significant.",
    "molecules": "carbon dioxide, oxygen"
  },
  {
    "caption": "A and B Blood glucose levels and serum insulin levels in 2-h GTT 12 weeks after HFD. Inset graphs in (A) and (B) depict the respective analysis of the area under the curve (AUC). Data shown are from one experiment (n = 6 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0287, **P = 0.0173, ***P = 0.0332, ****P = 0.0453, #P = 0.0077, ##P = 0.0344, ###P = 0.0412, ####P = 0.0495, ^P = 0.0435, ^^P = 0.0304 (Student's t-test).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "D Insulin-stimulated Akt phosphorylation (Ser473) in WAT, liver, and skeletal muscle of control and ad-FADD mice. The experiments were performed in triplicate.",
    "molecules": "Insulin"
  },
  {
    "caption": "I Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes. Data shown are from one experiment (n = 4 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0073 (Student's t-test).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "J Basal and insulin-stimulated glucose uptake in 3T3L1 cells differentiated to adipocytes and infected with lentivirus containing FADD shRNA or scrambled shRNA control. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0092 (Student's t-test).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "K Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes treated with DMSO or 5 μM MK886. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0135 (one-way ANOVA). NS, not statistically significant.",
    "molecules": "MK886, DMSO, glucose, insulin"
  },
  {
    "caption": "C Basal and stimulated lipolysis measured by fatty acid release from explants of epididymalWAT in 10-week-old male ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0003, **P = 0.001 (one-way ANOVA).",
    "molecules": "fatty acid"
  },
  {
    "caption": "D cAMP level in WAT of 10-week-old male WT, FADD-D, ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0017, **P = 0.001 (one-way ANOVA).",
    "molecules": "cAMP"
  },
  {
    "caption": "E Whole-body oxygen consumption rate (VO2) during 24 hr in WT, FADD-D, ob/ob, and FADD-D/ob/ob mice fed a SD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0053, **P = 0.0025, ***P = 0.0062, ****P = 0.0011 (one-way ANOVA).",
    "molecules": "O2, oxygen"
  },
  {
    "caption": "I Oxidation of [1-14C] oleic acid to 14CO2by adipocytes isolated from WT, FADD-D, ob/ob, and FADD-D/ob/ob mice. Data shown are from one experiment (n = 3 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0031, **P = 0.0075 (one-way ANOVA). NS, not statistically significant.",
    "molecules": "CO2, oleic acid"
  },
  {
    "caption": "(B) Western blot showing levels of LC3-I and LC3-II in control and Epg5−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1).",
    "molecules": "Bafilo, bafilomycin A1, Rapa, rapamycin"
  },
  {
    "caption": "(F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles",
    "molecules": "nutrient"
  },
  {
    "caption": "HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.",
    "molecules": "carboplatin"
  },
  {
    "caption": "Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm.",
    "molecules": "DRAQ5"
  },
  {
    "caption": "COVID-19 severity is associated with significant changes in lipoprotein subclasses including high-density lipoprotein subclass-1 (HDL1), HDL4, low-density lipoprotein subclasses (LDL1, LDL4, LDL5), very low-density lipoprotein subclass-5 (VLDL5) and their compositional components (ApoA1, triglycerides, cholesterol). TG: triglycerides; FC: free cholesterol; CE: cholesteryl esters; CH: total cholesterol (i.e., FC + CE); PL: total phospholipids; A1: ApoA1; A2: ApoA2; L1TG: TG in LDL1; L1TG%: percentages of L1TG in total lipids of LDL1; L1%: percentage of LDL1 in all LDL; L-TG%: percentages of L-TG(TG in LDL) in total lipids of LDL; V5FC%, V5CE%: percentages of V5FC(FC in VLDL5) and V5CE(CE in VLDL5) in total lipids of VLDL5; L5CE%, L5CH%: percentages of L5CE(CE in LDL5) and L5CH(CH in LDL5) in total lipids of LDL5; H1FC%: percentages of H1FC (FC in HDL1) in total lipids of HDL1; H-A2: ApoA2 in both HDL and nascent HDL; H4A1, H4A2, H4CE, H4CH, H4FC, H4PL: ApoA1, ApoA2, CE, CH, FC and PL in HDL4;",
    "molecules": "H1FC, H4CE, H4CH, H4FC, H4PL, L1TG, L5CE, L5CH, V5CE, V5FC, CH, cholesterol, FC, free cholesterol, CE, cholesteryl esters, HDL, lipids, lipoprotein, LDL, low-density lipoprotein, phospholipids, PL, L-TG, TG, triglycerides"
  },
  {
    "caption": "Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID-19 severity. Data were represented as means ± SD and differences between groups were estimated using a Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Control, n=12; mild, n=29; severe, n=17; discharge, n=16; sLDLR: soluble low-density lipoprotein receptor; LCAT: lecithin-cholesterol acyltransferase; CEPT: cholesteryl-ester transfer protein.",
    "molecules": "lipoprotein"
  },
  {
    "caption": "HCT116 and U87 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.",
    "molecules": "LXR623"
  },
  {
    "caption": "HCT116 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.",
    "molecules": "LXR623"
  },
  {
    "caption": "U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin (Sim) for 48h. Thereafter, lysates were collected and analyzed for total cholesterol levels. Shown are means and SD (n = 3).",
    "molecules": "cholesterol, LXR623, Sim, Simvastatin"
  },
  {
    "caption": "GBM12 GBM cells (short term patient-derived xenograft), U87 GBM cells, HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263, LXR623 or the combination for 72h. Thereafter, cellular viability was analyzed and statistical analysis was performed.",
    "molecules": "LXR623, ABT263"
  },
  {
    "caption": "U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.",
    "molecules": "LXR623, ABT263, propidium iodide"
  },
  {
    "caption": ", Stem-like GBM cells, NCH644, NCH421k and NCH690 were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 48h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.",
    "molecules": "LXR623, ABT263, propidium iodide"
  },
  {
    "caption": "U87 (72h treatment) and HCT116 (48h treatment) were treated with 1 µM ABT263, 20 µM (U87) or 10 µM (HCT116) LXR623 or the combination of both, stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential.",
    "molecules": "LXR623, ABT263, TMRE"
  },
  {
    "caption": "HCT116 colonic carcinoma cells were treated with ABT263, LXR623 or the combination of both. Whole protein lysates were collected and subjected to capillary electrophoresis for the expression/cleavage of PARP, total caspase-9 (CP9) and Vinculin. CF: cleaved fragment, FL: full length.",
    "molecules": "LXR623, ABT263"
  },
  {
    "caption": "HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3-mimetics, WEHI-539 (Bcl-xL inhibitor), ABT199 (Bcl-2 inhibitor) or A1210477 (Mcl-1 inhibitor) in the presence or absence of LXR623 for 48h. Thereafter, cells were labeled with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.",
    "molecules": "A1210477, LXR623, propidium iodide, ABT199, WEHI-539"
  },
  {
    "caption": ", LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h. Thereafter, protein lysates were collected and subjected to standard western blot (LN229, T98G and U87) or capillary electrophoresis (U87-EGFR-vIII and HCT116) for the expression of Usp9X, Mcl-1, Bcl-2, Bcl-xL, Noxa, BIM and/or Actin and Vinculin. In U87-EGFRvIII and HCT116, Vinculin was used in lieu of Actin.",
    "molecules": "LXR623"
  },
  {
    "caption": "LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with LXR623 as described in A. Thereafter, protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin.",
    "molecules": "LXR623"
  },
  {
    "caption": "LN229 were transfected with siNT, siNoxa 1, siNoxa 2 or BAK-siRNA for 72h. Thereafter, cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h. After conclusion of the treatment, cells were harvested, fixed, stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation.",
    "molecules": "DNA, LXR623, ABT263, propidium iodide"
  },
  {
    "caption": "LN229 GBM cells were treated with 10 µM LXR623 (LXR), 1 µM ABT263 or the combination for 48h. Thereafter, protein lysates were prepared and immunoprecipitated with an antibody against BAK. Standard western blotting was performed (immunoprecipitation and the corresponding inputs) with antibody against BAK and Mcl-1. The arrows highlight the specific protein bands, while stars indicate the immunoglobulin light-chains.",
    "molecules": "LXR, LXR623, ABT263"
  },
  {
    "caption": "U87 cells were treated with 20 µM LXR623 for 7h and 24h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. The lower left quadrant indicates the percentage of viable cells.",
    "molecules": "LXR623, propidium iodide"
  },
  {
    "caption": "U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay. To account for dead cells, assays were normalized to DNA-content (cell number).",
    "molecules": "ATP, DNA, LXR623"
  },
  {
    "caption": "U87 cells were treated with LXR623 as indicated for 24h. Cell lysates were prepared and subjected to LC/MS for the determination of ATP levels.",
    "molecules": "ATP, LXR623"
  },
  {
    "caption": "U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.",
    "molecules": "LXR623, threonine"
  },
  {
    "caption": "U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4.",
    "molecules": "ATP, LXR623"
  },
  {
    "caption": "U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.",
    "molecules": "cholesterol, LXR623, threonine"
  },
  {
    "caption": "U87 cells were treated with increasing concentrations of LXR623. Thereafter, whole cell protein lysates were prepared and analyzed for classical ER-stress markers, GRP78, ATF4 and Noxa by capillary electrophoresis.",
    "molecules": "LXR623"
  },
  {
    "caption": "U87 cells were transfected with non-targeting siRNA (n.t.-siRNA), ATF3-siRNA, ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs. After 48h, cells were treated with LXR623, whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3, ATF4, Noxa and Vinculin.",
    "molecules": "LXR623"
  },
  {
    "caption": "U87 cells were treated as in A. Shown are the levels of TCA-cycle metabolites.",
    "molecules": "TCA"
  },
  {
    "caption": "U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non-labeled isotopologues (m+0).",
    "molecules": "U-13C-glucose, glucose, glutamine, LXR623, phenol red, pyruvate, TCA"
  },
  {
    "caption": "U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non-labeled isotopologues (m+0).",
    "molecules": "U-13C-glucose, glucose, glutamine, LXR623, phenol red, pyruvate, TCA"
  },
  {
    "caption": "U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid and aspartic acid).",
    "molecules": "U-13C-glucose, aspartic acid, glucose, glutamatic acid, glutamine, LXR623, phenol red, pyruvate"
  },
  {
    "caption": "U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid",
    "molecules": "U-13C-glucose, glucose, glutamatic acid, glutamine, LXR623, phenol red, pyruvate"
  },
  {
    "caption": "U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of glutathione and nucleotides.",
    "molecules": "U-13C-glucose, glucose, glutamine, glutathione, LXR623, phenol red, pyruvate"
  },
  {
    "caption": "U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool).",
    "molecules": "U-13C-glucose, glucose, glutamine, LXR623, phenol red, pyruvate"
  },
  {
    "caption": "U87 cells were treated with DMSO or LXR623 20 µM for 24h. Thereafter, cells were processed for polar metabolite analysis by LC/MS. Shown are the levels of NAD and NADH2.",
    "molecules": "DMSO, LXR623, NAD, NADH2"
  },
  {
    "caption": "U87 cells were treated with LXR623 for 1h, 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (A-C). From this assay, coupled respiration was calculated (D-F).",
    "molecules": "LXR623"
  },
  {
    "caption": "U87 cells were treated with LXR623, cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (G). From this assay, mitochondrial OCR and coupled respiration was calculated (H-I).",
    "molecules": "cholesterol, LXR623"
  },
  {
    "caption": "U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point. Thereafter, whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes (OXPHOS), Noxa, and ATF4. A star indicates an unspecific band.",
    "molecules": "DMSO, LXR623"
  },
  {
    "caption": "U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h. Thereafter, whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot.",
    "molecules": "cycloheximide, DMSO, LXR623"
  },
  {
    "caption": "U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes (OXPHOS) by standard western blot.",
    "molecules": "cholesterol, LXR623"
  },
  {
    "caption": "U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. Thereafter, extracellular flux analysis was performed in the context of a mitochondrial stress assay. From this assay, basal respiration and coupled respiration was calculated (M-N).",
    "molecules": "LXR623"
  },
  {
    "caption": "U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. RNA and protein were collected and analyzed for the expression of LXRβ mRNA (O) or LXRβ protein by capillary electrophoresis (P). Shown are means and SD (n = 5). Statistical significance was determined by two-sided student's t.test.",
    "molecules": "LXR623, RNA"
  },
  {
    "caption": "1x106 HCT116 colon carcinoma cells were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100-200 mg/kg), ABT263 (100 mg/kg) or both agents (3 days per week for 1.5 weeks. Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0063, ****p<0.0001. Statistical significance was determined by one-way ANOVA. B, E, GBM43 PDXs were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0031. Statistical significance was determined by one-way ANOVA. C, F, A375 BRAF V600E mutated melanomas were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.0109 (GW3965 vs Combination), *p=0.0166 (ABT263 vs Combination), **p=0.0047. Statistical significance was determined by one-way ANOVA. ",
    "molecules": "GW3965, LXR623, ABT263"
  },
  {
    "caption": "Related to the HCT116 colonic xenograft model, representative histopathological images (hematoxylin and eosin stain) are shown from each treatment group. Scale bar: 50 µm. Sections from the same treatment groups as in G were stained with TUNEL and SDHB, repectively. Displayed are representative images of the staining. Scale bar 50 µM.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(A) Immunofluorescence labeling of FTSJ1-Flag (red) in HEK293T cells. The nucleus was stained by DAPI (blue). Scale bar, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E) IP assays of FTSJ1-Asn221DIZPK. The empty vector was transfected as a negative control. (F) A list of top FTSJ1-Asn221DIZPK putative interacting proteins identified by IP-MS in HEK293T cells. ",
    "molecules": "Asn, DIZPK"
  },
  {
    "caption": "Mass chromatograms of the nucleosides, Cm (Q1/Q3=258.1/112.1), Gm (Q1/Q3=298.1/152.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. Cm, 2'-O-methylcytidine; Gm, 2'-O-methylguanosine; Q1/Q3: the mass of the precursor ion and the mass of the product ion.",
    "molecules": "2'-O-methylcytidine, Cm, 2'-O-methylguanosine, Gm, tRNAPhe"
  },
  {
    "caption": "Mass chromatograms of the nucleosides m1G (Q1/Q3=298.1/166.1), m7G (Q1/Q3=298.1/166.1) and m2G (Q1/Q3=298.1/166.1), o2yW (Q1/Q3=541.2/409.0), m5C (Q1/Q3=258.1/126.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. m2G, 2-methylguanosine; D, dihydrouridine; m22G, N2,N2-dimethylguanosine; m5C, 5-methylcytidine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m1A: 1-methyladenosine; ψ: pseudouridine; o2yW, peroxywybutosine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. Combined the retention time of standard product, we marked the o2yW in Figure 3G. Considering the change of this peak area was consistent with that of o2yW, we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled.",
    "molecules": "o2yW, peroxywybutosine, 1-methyladenosine, m1A, m1G, 5-methylcytidine, m5C, 7-methylguanosine, m7G, dihydrouridine, m22G, N2,N2-dimethylguanosine, 2-methylguanosine, m2G, pseudouridine, 5-methyluridine, m5U, tRNAPhe"
  },
  {
    "caption": "(B, C) Quantification of the Cm/A and Gm/A of tRNAPhe(GAA), △ftsj1_tRNAPhe(GAA), and m1G37_tRNAPhe(GAA) after incubation with FTSJ1, WDR6, and FTSJ1-WDR6 by UPLC-MS/MS analysis, respectively. Error bars represent the standard deviation of three independent experiments. n.d., not detected.",
    "molecules": "m1G, Cm, Gm, tRNAPhe"
  },
  {
    "caption": "(D) UPLC-MS/MS analysis of 2'-O-methylation of m1G37_tRNAPhe(GAA), -G34A, -G34C and G34U after incubation with FTSJ1-WDR6. 2 μL of Gm (1 ng/mL), Am (1 ng/mL), Cm (1 ng/mL) and Um (5 ng/mL) standards were injected to UPLC-MS/MS as control. cps, counts per second.",
    "molecules": "m1G, Cm, Gm, Um, Am, tRNAPhe"
  },
  {
    "caption": "A) The SAM binding affinity of FTSJ1 as measured by ITC.",
    "molecules": "SAM"
  },
  {
    "caption": "(B The binding affinity of FTSJ1 alone or FTSJ1-WDR6 for tRNA analyzed by the gel mobility shift assay.",
    "molecules": "tRNA"
  },
  {
    "caption": "C) For the reaction in figure C, 0.2 μΜ GST, 0.2 μΜ GST-WDR6, 1 μM FTSJ1, the mixture of 1 μM FTSJ1 with 0.2 μΜ GST-WDR6 or 0.2 μΜ GST, 0.5 mM SAM and 250 nM tRNAPhe(GAA) transcript were incubated.",
    "molecules": "SAM, tRNAPhe"
  },
  {
    "caption": "The representative graphs for quantification of the expected Nm32/A% and Nm34/A% ratios of G37-tRNAPhe(GAA), tRNALeu(UAA), tRNATrp(CCA), tRNAArg(ACG), tRNAArg(UCG), and A37-tRNAMet(CAU), which were isolated from WT, ftsj1 KO, and wdr6 KO cells by UPLC-MS/MS analysis. Error bars represent the standard deviation of three independent experiments. p values were determined using two-tailed Student's t-test for paired samples. ***p < 0.001. n.s., no significance. n.d., not detected.",
    "molecules": "tRNAArg, tRNALeu, tRNAMet, tRNAPhe, tRNATrp"
  },
  {
    "caption": "(A, B) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0.2 mg/mL paromomycin assayed by cell counting kit-8 proliferation analysis.",
    "molecules": "paromomycin"
  },
  {
    "caption": "(C, D) The growth curve of WT with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.",
    "molecules": "paromomycin, tRNAPhe"
  },
  {
    "caption": "(E, F) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.",
    "molecules": "paromomycin, tRNAPhe"
  },
  {
    "caption": "(A and B) FUS disrupts Ca2+ homeostasis. HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K as indicated. Release of ERCa2+ was induced by treatment of cells with OxoM. (A) shows cytosolicCa2+ levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right. Fluo4 fluorescence shows a transient increase in cytosolicCa2+ levels upon OxoM treatment but compared to control, wild-type and mutant FUS all increase peak cytosolicCa2+ levels. (B) shows mitochondrialCa2+ levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right. Data were analysed by one-way ANOVA and Tukey's post hoc test. (A) N=49-52 cells from 3 experiments; (B) N=50-52 cells from 5 experiments, error bars are s.e.m.; *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "Fluo4, Rhod2, Ca2+, OxoM"
  },
  {
    "caption": "(C) FUS reduces mitochondrial ATP production. ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1.03 and either control vector (CTRL), HA-FUS, HA-FUSR521C or HA-FUSR518K. Cells were imaged in time-lapse prior to and after KCN treatment to inhibit oxidative phosphorylation. Representative traces of YFP/CFP ratios are shown for the different samples; initial YFP/CFP ratios prior to KCN treatment and those after KCN treatment are indicated. The fall in YFP/CFP ratios correlates with ATP produced by oxidative phosphorylation. Bar chart shows relative ATP levels produced by oxidative phosphorylation (OXPHOS) in the different samples.",
    "molecules": "ATP, KCN"
  },
  {
    "caption": "(A) representative electron microscopy of ER-mitochondria associations in NSC34 cells treated with either vehicle or GSK-3β inhibitors AR-A014418 (1 M) or CT99021 (100 nM) for 16 h. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test. N=32 cells and 181-210 mitochondria. Error bars are s.e.m.; *p<0.05.",
    "molecules": "AR-A014418, CT99021"
  },
  {
    "caption": "(B) VAPB-PTPIP51 proximity ligation assays of NSC34 cells treated with either vehicle 1 M AR-A014418 or 100 nM CT99021 for 16 h. Cells were also stained for nuclei with DAPI. Bar chart shows relative number of proximity ligation assay signals/cell.",
    "molecules": "AR-A014418, CT99021"
  },
  {
    "caption": "Representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and treated with vehicle or 1 mM AR-A014418 for 16 hours. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples.",
    "molecules": "AR-A014418"
  },
  {
    "caption": "HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K and then treated with vehicle (DMSO) or 1 M AR-A014418 for 16 hours as indicated. Release of ER Ca2+ was induced by treatment of cells with OxoM. Representative Rhod2 fluorescence traces showing mitochondrial Ca2+ are shown along with bar chart displaying normalized peak values.",
    "molecules": "Rhod2, Ca2+, AR-A014418, DMSO, OxoM"
  },
  {
    "caption": "B) Expression of the 21U sensor (left) and DAPI staining (right) of gonad arms in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. Number of animals analysed and with indicated phenotype are given in the panel. Animals not showing the activation of the 21U sensor(+) were still silenced, and animals that did not show the silenced 21U sensor(RNAe) state were expressing weakly, comparable to the 21U sensor(+). Scale bar: 25 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "E) Crossing scheme to address if maternal 21U RNAs are sufficient to re-initiate the silencing of the 21U sensor. A pid-1 mutant male expressing the 21U sensor is crossed with a hermaphrodite, heterozygous for the same mutation. All their F1 offspring inherit a pool of 21U RNAs from the hermaphrodite, but in 50% of the F1, which is pid-1 homozygous mutant, no zygotic PID-1 is present, hence no zygotic 21U RNAs can be made. The silencing or expression of the 21U sensor in the pid-1 homozygous mutant F1 has been scored by microscopy, and depicted in a pie chart. At the bottom, a representative image of an animal carrying a silenced 21U sensor (lower: mCherry signal; upper: DAPI staining) in pid-1 mutant offspring. Gonads are outlined by a dashed line. Scale bar: 25 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Mean ΔF images of Rhod-FF and iGluSnFR signals in response to a voltage ramp and a 50-ms-long step depolarization, respectively. The synapse marked with * on the overlaid image is further analyzed in the following panels. (P: pillar side, M: modiolar side; Scale bar: 2 μm)",
    "molecules": "iGluSnFR, Rhod-FF"
  },
  {
    "caption": "Voltage command (top), corresponding whole-cell Ca2+-influx (middle) and the functional fluorescence responses (bottom) (bandstop filtered at 33.3 Hz) from Rhod-FF and iGluSnFR, respectively. A modified Boltzmann function (see Materials and Methods, R2 = 0.81) was fitted to a Rhod-FF fluorescence trace in response to a voltage ramp (C-C'). iGluSnFR-AUC was calculated per depolarization voltage and used for a Boltzmann fit (D', R2 = 0.92). The voltage of half-maximal activation (V1/2) and the dynamic range (10-90%) of synaptic Ca2+-influx and glutamate release were calculated from the fits and depicted as circle and bar in C' and D', respectively. The obtained fits from a Ca2+ \"hotspot\" (C') and from glutamate release (D') were plotted against each other in a voltage range from -57 mV to -17 mV in 1 mV increments. A power function was fitted to the 25% of the maximum iGluSnFR-AUC (R2 = 0.99) to obtain the m-estimate. (ruptured patch-clamp, 10 mM intracellular EGTA, 5 mM [Ca2+]e) (See also Fig EV4)",
    "molecules": "iGluSnFR, Rhod-FF, Ca2+, glutamate"
  },
  {
    "caption": "A voltage ramp was applied to obtain ΔF of Rhod-FF as a proxy synaptic Ca2+ influx. The V1/2 of synaptic Ca2+-influx was calculated from a modified Boltzman function (see Materials and Methods) fitted to ΔF/F0. (mean ± SD, n = 55 synapses; individual synapses are color coded) (see Appendix Fig S7 for individual fits).",
    "molecules": "Rhod-FF, Ca2+"
  },
  {
    "caption": "Normalized iGluSnFR-AUC, in response to 50-ms-long step depolarizations, same as A (see Appendix Fig S8 for individual fits).",
    "molecules": "iGluSnFR"
  },
  {
    "caption": "Left panel shows the linear regression analysis (solid lines) of V1/2 of synaptic Ca2+-influx (B), glutamate release (C), threshold (D) and dynamic range of release (E) as a function of position along the pillar-modiolar axis. Shaded areas depict the associated 95% confidence intervals. Significance of correlation coefficients is reported by a two-tailed p-value. Right panel shows box and whisker plots of these properties of synapses grouped into pillar and modiolar halves of the IHCs. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. For comparison of pillar and modiolar synapses, either student's t-test (for normally distributed data) or Mann-Whitney-U test (for non-normally distributed data) was applied. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001",
    "molecules": "Ca2+, glutamate"
  },
  {
    "caption": " Single  IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF  image of Rhod-FF  (red) and  iGluSnFR (green) shows  synaptic Ca2+-influx and glutamate release at three neighboring  modiolar synapses (individual synapses are color coded based on their clusters). E' The relation between Ca2+-influx and glutamate release of given synapses  in the voltage range of -57 to -17 mV plotted with 1 mV increments. A power function was fitted until the 25% of normalized  iGluSnFR AUC(ΔF/F0). Synapses  showed different  Ca2+-dependencies (m = 2.0, 1.3, 7.4; green, purple, orange). ",
    "molecules": "iGluSnFR, Rhod-FF, Ca2+, glutamate"
  },
  {
    "caption": " Mean glutamate release (iGluSnFR-AUC) as a function of depolarization voltage in the three identified clusters (mean ± SEM, Cluster 1: n = 27 synapses, Cluster 2: n = 22 synapses, Cluster 3: n = 6 synapses). ",
    "molecules": "iGluSnFR, glutamate"
  },
  {
    "caption": "(A and B) ARPE-19 cells were transfected with siRNA duplexes to raptor, RagA+B, p18, or nontarget. 60 h after transfection, cells were infected with adenovirus expressing either TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG (used to detect TFEB) and mTOR. Bars, 10 µm. (C) Quantification of A. (D) Quantification of B. Values are means ± SD of three independent experiments. ***, P < 0.001.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin.",
    "molecules": "DMSO, Torin-1"
  },
  {
    "caption": "(C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A.",
    "molecules": "Torin-1"
  },
  {
    "caption": "(D) HeLa cells were starved in serum- and amino acid-free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB.",
    "molecules": "amino acid"
  },
  {
    "caption": "(E) HeLa cells were incubated in normal medium (control) and serum- and amino acid-free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "(A) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-WT. 16 h later, cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 3 h. Cells were then fixed, permeabilized with 0.2% saponin, and double stained with antibodies against TFEB (used to detect recombinant TFEB) and Lamp1.",
    "molecules": "amino acid, saponin, Torin-1"
  },
  {
    "caption": "(B) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-S211A. 12 h later, cells were starved in serum- and amino acid-free medium for 4 h (starvation) or kept in complete medium (control). Cells were then fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB-S211A).",
    "molecules": "amino acid, Triton X-100"
  },
  {
    "caption": "(C) HeLa cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 4 h with the addition of Torin-1 during the last hour of starvation. Cells were then washed, fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against TFEB (used to detect endogenous TFEB) and Lamp1. Regions within the dotted boxes are magnified in the insets.",
    "molecules": "amino acid, Torin-1, Triton X-100"
  },
  {
    "caption": "(E and F) ARPE-19 cells were cotransfected with plasmids expressing TFEB-GFP together with inactive (E) or active Rag heterodimers (F). Cells were then incubated with 250 nM Torin-1 for 1 h (E) or starved in serum- and amino acid-free medium for 3 h (F). Anti-GST antibodies were used to detect Rag proteins. Arrows point to cells that do not express Rag heterodimers.",
    "molecules": "amino acid, Torin-1"
  },
  {
    "caption": "(A) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE-19 cells treated with either DMSO or Torin-1.",
    "molecules": "DMSO, Torin-1"
  },
  {
    "caption": "(D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB).",
    "molecules": "DMSO, Torin-1, Triton X-100"
  },
  {
    "caption": "(A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm.",
    "molecules": "saponin"
  },
  {
    "caption": "(B-D) ARPE-19 cells were infected with adenovirus expressing MITF1-FLAG. 16 h later, cells were incubated with DMSO or 250 nM Torin-1 for 1 h and analyzed by immunoblotting (C) or immunofluorescence (B and D) with anti-FLAG antibodies. The region within the dotted box is magnified in the insets. Bars: (B and D, main images) 10 µm; (D, insets) 5 µm.",
    "molecules": "DMSO, Torin-1"
  },
  {
    "caption": "(E) ARPE-19 cells were incubated with 250 nM Torin-1 for 1 h. Cells were then lysed and analyzed by immunoblotting with antibodies against MITF.",
    "molecules": "Torin-1"
  },
  {
    "caption": "(F) HEK-293T cells were incubated in medium containing DMSO or Torin-1 for 1 h. Cells were lysed, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF, Lamp1, and Histone H3.",
    "molecules": "DMSO, Torin-1"
  },
  {
    "caption": "(G) ARPE-19 cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were immunoprecipitated with the anti-FLAG antibody and immunoblotted with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively).",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "(H) HEK-293T cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were pulled down using glutathione-Sepharose beads and immunoblotted with antibodies against GST and MITF (used to detect Rag proteins and endogenous MITF, respectively). IP, immunoprecipitation.",
    "molecules": "GDP, GTP"
  },
  {
    "caption": "(A and B) ARPE-19 cells were nucleofected with the indicated Rag- and MITF-expressing plasmids. 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). Bars, 10 µm.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Male mice, housed at 30°C or 6°C for 10 days, were intraperitoneally (IP) administered with [U-13C]glucose (2 g/kg). 15 minutes after injection, BAT was harvested for metabolic enrichment assay. (A) m+6 glucose enrichment in serum and BAT. (B) relative glucose abundance in serum and BAT.",
    "molecules": "13C, glucose"
  },
  {
    "caption": "(D,E) Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates (D), m+2 TCA cycle intermediates (E).",
    "molecules": "13C"
  },
  {
    "caption": "(A) Representative immunofluorescence staining shows UCP1 (green), perilipin (red) and DAPI (blue) in sWAT of mice housed at 6°C.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B-G) Male mice, housed at 30°C or 6°C for 10 days, were administered with [U-13C]glucose (2 g/kg, IP). 15 minutes after injection, sWAT and gWAT were harvested for metabolic enrichment assay. Metabolic 13C enrichments in sWAT of male mice are shown as m+6 glucose (B), m+3 glycolytic intermediates (C), and m+2 TCA cycle intermediates (D). Metabolic 13C enrichments in gWAT of male mice are shown as m+6 glucose (E), m+3 glycolysis intermediates (F), and m+2 TCA cycle intermediates (G).",
    "molecules": "13C, glucose"
  },
  {
    "caption": "(H) Plasma 13CO2 enrichment, after normalizing to the [U-13C]glucose tracer enrichment.",
    "molecules": "13C, CO2, glucose"
  },
  {
    "caption": "Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (B) Glycolytic intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.",
    "molecules": "13C, glucose"
  },
  {
    "caption": "Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (C) TCA cycle intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.",
    "molecules": "13C, glucose"
  },
  {
    "caption": "(A) Fully differentiated brown adipocytes were treated with β3-AR agonist CL316,243 (10 µM). Relative mRNA levels of thermogenic markers were measured by qPCR.",
    "molecules": "CL316,243"
  },
  {
    "caption": "(B) Pyruvate and lactate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.",
    "molecules": "13C, CL316,243, glucose, lactate, Pyruvate"
  },
  {
    "caption": "(C) Citrate and malate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.",
    "molecules": "13C, Citrate, CL316,243, glucose, malate"
  },
  {
    "caption": "(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats.",
    "molecules": "CHC, UK5099"
  },
  {
    "caption": "(B) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates.",
    "molecules": "13C, CHC, CL316,243, glucose, UK5099"
  },
  {
    "caption": "(C) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7.",
    "molecules": "13C, CHC, glucose, PBS"
  },
  {
    "caption": "(D) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.",
    "molecules": "CHC"
  },
  {
    "caption": "(A) Oxidative  folding of Myc-tagged J-Chain (JcM). PDI+/+ and PDI-/- hMSCs  were transfected with JcM  for 48 hr, then were pulsed with DTT  and chased at indicated time  after DTT  removal. The lysates were analyzed using non-reducing SDS-15% PAGE and α-Myc western blotting. The mobility of reduced JcM  monomers (Red), oxidized  monomers (Oxi), homodimers (Dim), and high-molecular-weight (HMW) species are indicated.",
    "molecules": "DTT"
  },
  {
    "caption": "(D) Time-dependent changes of H2O2  levels in the ER  of PDI+/+ and PDI-/- hMSCs  at P8. Left, the fluorescence intensity ratio of HyPer-ER  at 535 nm with excitation at 488 and 405 nm were firstly monitored at resting state for 5 min, followed by addition of 0.5 mM DTT  (yellow bar) for another 15 min. Right, statistical analysis of the fluorescence intensity ratio at 488/405 nm excitation of HyPer-ER  probe at 15 min post DTT  addition. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.",
    "molecules": "DTT, H2O2"
  },
  {
    "caption": "(E) Analysis of the H2O2  levels in PDI+/+ and PDI-/- hMSCs at P8 using Amplex Red assays. n = 3 biological repeats. (F) Analysis of the cellular total ROS  levels in PDI+/+ and PDI-/- hMSCs  at P8 using H2DCFDA  probe. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.",
    "molecules": "H2DCFDA, H2O2, ROS"
  },
  {
    "caption": "(M) Confocal  imaging of PDI+/+ and PDI-/- hMSCs  expressing Hyper7-nucleus  at steady state and upon addition of 2 mM DTT. The fluorescence intensity ratio at 488/405 nm obtained from individual cells was calculated at indicated times  with a representative false color image. Scale bar, 10 μm.",
    "molecules": "DTT"
  },
  {
    "caption": "(K) Conjoint analysis showing the secretion levels of PDI  and those in (I) and various SASPs from multiple senescence inducers and in plasma  proteome from 3,087 healthy individuals RS, replicative senescence (PDI+/+, LP   vs. PDI+/+, EP). ATV, atazanavir  treatment; RAS, inducible RAS  overexpression; IR, X-irradiation. The color keys that change from dark blue to dark purple represent the fold change of gene and protein, change from white to red represent the change in protein levels per unit increase in the covariate in plasma  proteomic.",
    "molecules": "atazanavir, ATV"
  },
  {
    "caption": "(B) Western blotting analysis of SERPINE1  protein in PDI+/+ and PDI-/- hMSCs  at EP   and LP. GAPDH  was used as the loading control of cell lysates. Ponceau  was used as the loading control of conditional medium.",
    "molecules": "Ponceau"
  },
  {
    "caption": "(H) qPCR  analysis of the SERPINE1  mRNA expression in PDI+/+ and PDI-/- hMSCs  treated with the indicated concentration H2O2  for 4 days. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.",
    "molecules": "H2O2"
  },
  {
    "caption": "(J) H2O2  levels in PDI-/- hMSCs  transduced with lentiviruses  expressing Luc  or PDI   were determined by the fluorescence ratio of HyPer-ER  at 488/405 nm excitation after the addition of 0.5 mM DTT  for 15 min. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.",
    "molecules": "DTT, H2O2"
  },
  {
    "caption": "(K) H2O2  levels in PDI-/- hMSCs  transduced with lentiviruses  expressing Luc  or PDI   were determined by the fluorescence ratio of HyPer7-nucleus  at 500/400 nm excitation after the addition of 2 mM DTT  for 20 min. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.",
    "molecules": "DTT, H2O2"
  },
  {
    "caption": "B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Triglyceride levels were quantified by an enzymatic assay (C).",
    "molecules": "Triglyceride"
  },
  {
    "caption": "B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by triglyceride quantitation (C)",
    "molecules": "triglyceride"
  },
  {
    "caption": "F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).",
    "molecules": "polyG"
  },
  {
    "caption": "Western blots of FMRpolyG-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.",
    "molecules": "polyG"
  },
  {
    "caption": "Western blots of FMRpolyA-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.",
    "molecules": "polyA"
  },
  {
    "caption": "F Representative immunofluorescence staining of the p53 (green) and METTL14 (red) proteins in p53-WT HCT116, Lovo and RKO cells and p53-MT HT29 (p53R273H), SW480 (p53R273H/P309S), SW620 (p53R273H) and SW1116 (p53A159D) cells. Nuclei were stained with DAPI (blue). Scale bars = 10 μm. The relative mean fluorescence density was analyzed by ImageJ.",
    "molecules": "DAPI"
  },
  {
    "caption": "G Comparison of colorectum length between METTL14WT (n = 14, n = 24) and METTL14ΔIEC (n = 13, n = 21) mice from AOM/DSS-induced and AOM-induced CRC models, respectively. Data are expressed as mean ± SD.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "I Representative HE staining images of colorectum in METTL14WT (n = 14, n = 24) and METTL14ΔIEC (n = 13, n = 21) mice from AOM/DSS-induced and AOM-induced CRC models. Lower panels show enlarged images of indicated normal or CRC tissues. Scale bars = 2 mm (upper) and 40 μm (lower). Black dashed line refers to the border of tumor (T) and normal (N) tissues. Tumors are classified as adenomas with low to focal high-grade dysplasia. The percentages of mice with dysplasia are shown (right).",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "C Lactate production, ATP level, glucose uptake and pyruvate level in stably transfected Lv-vector and Lv-METTL14 or shNC and shMETTL14 HCT116 (p53-WT) cells. Data are presented as mean ± SD (biological replicates, n = 3; **P < 0.01).",
    "molecules": "ATP, glucose, Lactate, pyruvate"
  },
  {
    "caption": "H Western blot analysis of METTL14, SLC2A3 and PGAM1 protein levels of intestinal epithelial cells from AOM/DSS-induced Mettl14WT (n = 4) and Mettl14ΔIEC (n = 4) and mice.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "I Representative IHC staining images and quantitative analysis of SLC2A3 and PGAM1 in tumor tissues and non-tumor tissues from AOM/DSS-induced Mettl14ΔIEC and Mettl14WT mice CRC models. The insets show enlarged images of tumor tissues and non-tumor tissues, respectively. Scale bars = 40 μm and 4 μm (inset). Data are presented as mean ± SD (biological replicates, n = 6; **P < 0.01, ***P < 0.001).",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "F Representative IF staining and quantitative analysis of the SLC2A3 (green) and PGAM1 (green) proteins in HCT116 (p53-WT) cells transfected with control or miRNA mimics. Nuclei were stained with DAPI (blue). Scale bars = 20 μm. Data are presented as mean ± SD (biological replicates, n = 4; **P < 0.01). G Representative IF staining and quantitative analysis of the SLC2A3 (green) and PGAM1 (green) proteins in HT29 (p53-MT) cells transfected with control or miRNA mimics. Nuclei were stained with DAPI (blue). Scalebars = 20 μm. Data are presented as mean ± SD (biological replicates, n = 4; ns = no significance).",
    "molecules": "DAPI"
  },
  {
    "caption": "A Glucose uptake, Lactate, ATP, pyruvate levels were determined in HCT116 (p53-WT) cells treated with control, miR-6769b-3p mimics or inhibitor for 48h. B Glucose uptake, Lactate, ATP, pyruvate levels were determined in HCT116 (p53-WT) cells treated with control, miR-499a-3p mimics or inhibitor for 48h.",
    "molecules": "ATP, Glucose, Lactate, pyruvate"
  },
  {
    "caption": "E Glucose uptake, Lactate, ATP and pyruvate levels were determined in stably transfected Lv-vector and Lv-METTL14 HCT116 (p53-WT) cells treated with control or mixture inhibitors for 48h. Western blot analysis of the corresponding METTL14, SLC2A3 and PGAM1 protein levels in indicated treatment. F Glucose uptake, Lactate, ATP and pyruvate levels were determined in stably transfected Lv-vector and Lv-METTL14 HCT116 (p53-WT) cells treated with control or mixture mimics for 48h. Western blot analysis of the corresponding METTL14, SLC2A3 and PGAM1 protein levels in indicated treatment.",
    "molecules": "ATP, Glucose, Lactate, pyruvate"
  },
  {
    "caption": "(C-G)   ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into  S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression  was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.",
    "molecules": "MMS, sodium thiosulfate"
  },
  {
    "caption": "(C-E)  ssDNA repair efficiency  of the indicated TLS mutants as determined from the maximal and final percentages of cells with  Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into  S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the  MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel). Data information: The mean and SEM of and 3-12 12 (wild type), 6 (rev1∆,  rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆  rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.",
    "molecules": "MMS, sodium thiosulfate"
  },
  {
    "caption": "(C-D)  ssDNA repair efficiency  of the rad5∆ and  rev1∆ mutants as determined from the maximal and final percentages of cells with Rad54  foci during the time course (right panels). Cells were synchronized in G1  and released into S phase  in the presence of 0.033% MMS  for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Data information: The mean and SEM of independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.",
    "molecules": "MMS, sodium thiosulfate"
  },
  {
    "caption": "(F)   ssDNA repair efficiency  of the indicated mutants as determined by ChIP  against  Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of  Rfa1 at ssDNA was determined at 1 and 7 hours after MMS  release. RPA  enrichment was calculated as the ratio between  immunoprecipitate  and input values. All values were normalized to the wild type at 7 hours, taken as 1. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).",
    "molecules": "MMS, ssDNA, sodium thiosulfate"
  },
  {
    "caption": "Effect of the lack of HR factors in spontaneous and  MMS-induced mutagenesis. The frequency of spontaneous and MMS-induced forward  mutagenesis at the CAN1 locus was determined in mid-log phase cell cultures before (spontaneous mutagenesis) and after treatment with 0.01% MMS for 4 hours (DNA damage-induced). Thus, the frequency of DNA damage-induced mutagenesis includes the mutants that arose spontaneously before MMS addition. The frequency of mutagenesis  was determined at 0.01% MMS because at higher MMS  concentrations the loss of  viability of the rad mutants did not allow us to select for canavanine-resistant cells. Survival  (%) after 4 hours in  MMS  relative to the wild type (taken as 100) is shown below the genotypes. The mean and SEM of 6 (wild type), 9 (rad54∆) and 3 (rest) independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test (one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively). Only the statistical analysis of the effect of  rad52∆, rad57∆ and rad51∆ on rad54∆-mediated  mutagenesis  is included for clarity. All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS.",
    "molecules": "canavanine, MMS"
  },
  {
    "caption": "(A) Effect of the  rad54∆ and rad52∆ mutations in spontaneous and UV-induced uSCE. The frequency of spontaneous and UV-induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM without histidine  to calculate total and recombinant cells, respectively). The mean and SEM of 3-5 independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. Two and three asterisks represent p-values <0.01 and <0.001, respectively.",
    "molecules": "histidine"
  },
  {
    "caption": "(B)  Rad6 binds to DNA  in the absence and presence of MMS.  ChEC analysis of G1-synchronyzed cells released in the absence or the presence of 0.033% MMS for the indicated times. Data information: DNA gel  electrophoresis,  DNA content  (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.",
    "molecules": "DNA, MMS"
  },
  {
    "caption": "(C-F) Rad6 binding  to DNA depends on Rad18, Rad52  (C), Rad51 (D), Rad57  (E) but not  Rad54 (F) as determined by ChEC analyses of asynchronous cell cultures without or with 0.05% MMS for 2 hours. Data information: DNA gel  electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles  are shown. The experiments were repeated twice with similar results.",
    "molecules": "DNA, MMS"
  },
  {
    "caption": "(A-D) Effect of  rad52∆,  rad51∆ (A), rad57∆ (B) and rad54∆ (C) on PCNA ubiquitylation in cells synchronized in G1  and released into fresh medium in the presence of 0.025% MMS.  Cell cycle progression was followed by cell sorting  analysis and  western blot  against  Clb2. Pgk1 was used as loading control. The amounts of mono- and polyubiquitylated PCNA were normalized to the total amount of PCNA. For each time point, rad mutant values were relativized to those of the wild type, taken as 1. The mean and SEM of 3 independent experiments are shown (D). An asterisk indicates a mean significantly different than 1, according to a One-sample t-test.",
    "molecules": "MMS"
  },
  {
    "caption": "B CDF plot of relative abundance of transcripts with 3'UTRs > 1000 nt in K562 cells under conditions of UPF1 or non-targeting shRNA, for transcripts with hnRNP L eCLIP peaks in the indicated intervals Each trace represents a category of CLIP peak locations. For mRNAs with peaks > 400 nt from the TC or with peaks in the 5'UTR or CDS, transcripts with peaks at 3'UTR positions 1-400 were excluded. Statistical significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons and was determined based on comparison to the set with no 3'UTR CLIP peaks.",
    "molecules": "CDS, UTR"
  },
  {
    "caption": "C Representative northern blots assaying the stability of β-globin reporter transcripts containing the SMG5 3'UTR alone or supplemented with cassettes containing the indicated RSE variants in HeLa Tet-off cells. Half-lives were determined from at least 3 independent experiments and decay curves are shown with points representing mean and standard deviation at each timepoint. Significance was measured compared to the SMG5 3'UTR reporter using the sum-of-squares F test.",
    "molecules": "UTR, Tet"
  },
  {
    "caption": "D RNA immunoprecipitation experiments evaluating hnRNP L association with deletion and replacement reporter transcripts from (C). \"AS\" indicates a reporter in which the antisense sequence of the RSE was inserted in the SMG5 3'UTR in the same position as the RSE. The northern blot is representative of 3 independent experiments and quantification is presented in the accompanying bar graph with mean and standard deviation. * indicates P = 0.0146 in two-tailed Student's t-test. The reporter containing the GAP 3'UTR used here as a transfection control contains a predicted hnRNP L binding site, allowing its consistent recovery in the IPs.",
    "molecules": "UTR, RNA"
  },
  {
    "caption": "A RNA-seq analysis of cells treated with hnRNP L siRNA or a combination of siRNAs against hnRNP L and UPF1 identifies populations of transcripts that decreased in abundance upon hnRNP L knockdown in a UPF1-dependent manner. B CDF plot of transcript 3'UTR lengths for subsets identified in (A). Statistical significance was determined by two-sided K-S test. P value for decreased set is from comparison to the set of total transcripts. P value for rescued set is from comparison to decreased set. ",
    "molecules": "UTR"
  },
  {
    "caption": "B Half-lives of selected transcripts under conditions of non-targeting or hnRNP L knockdown. Measurements were calculated based on proportional recovery of transcript labeled with 5-EU in 1 hour versus total transcript. Significance was determined using two-tailed Student's t-test: *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Values represent mean and standard deviation of 3 independent replicates.",
    "molecules": "5-EU"
  },
  {
    "caption": "C The stability of β globin reporters with the CSRP1 3'UTR, along with several variants in which putative hnRNP L motifs in the indicated intervals were mutated, were evaluated in HeLa Tet-off cells. The schematic indicates areas of 3' UTR CA-enrichment in light blue and includes the sequence of the first 200 nucleotides of the CSRP1 3'UTR, where putative hnRNP L binding elements are in red typeface. The northern blot is a representative example from 3 independent replicates. Mean values plotted with SD at each timepoint were used to quantify decay (right panel). Significance is measured compared to the WT reporter using the sum-of-squares F test.",
    "molecules": "UTR, Tet"
  },
  {
    "caption": "D The stability of the WT and L mut 1-200 CSRP1 reporters in HeLa Tet-off cells treated with non-targeting RNAi or siRNA against UPF1 was evaluated. The northern blot is a representative example from at least 3 biological replicates. Decay quantification and statistics were performed as in (C), with significance measured compared to the non-targeting condition.",
    "molecules": "Tet"
  },
  {
    "caption": "C qPCR of selected transcripts in SU-DHL-4 cells under conditions of non-targeting RNAi or hnRNP L knockdown with or without 50 μg/mL cycloheximide treatment (for 4 hours; n ≥ 3). Significance of hnRNP L KD + DMSO and NT + CHX were compared to NT + DMSO, and hnRNP L KD + CHX was compared to hnRNP L KD + DMSO. Bars represent the mean ± SD. For clarity, only a single star was used to indicate significance; in all cases P ≤ 0.05 in two-tailed Student's t-tests.",
    "molecules": "CHX, cycloheximide, DMSO"
  },
  {
    "caption": "E Half-life determination from RT-qPCR detection of reporter constructs in HeLa Tet-off cells transfected with non-targeting or UPF1 siRNAs. For plots comparing non-targeting with UPF1 knockdown, significance is compared to the non-targeting condition using the the sum-of-squares F test. The fourth plot compares the non-targeting decay curves of the three reporters, and significance is compared to the decay of the fusion transcript. Points represent the mean ± SD from qRT-PCR at each time point of at least 3 independent experiments.",
    "molecules": "Tet"
  },
  {
    "caption": "B SU-DHL-4 and SU-DHL-6 cells were treated with varying doses of BCL2 inhibitor. Cell viability was measured after 24 hours of drug treatment using an assay for ATP concentration as a proxy for live cells. Traces were generated from at least 5 independent biological replicates. Points represent mean ± SD at each dose. P value from comparison of dose response between SU-DHL-4 and SU-DHL-6 cells calculated using sum-of-squares F test is given in the graph key. Significance of individual points calculated using two-tailed Student's t-tests is marked using asterisks on the graph. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.",
    "molecules": "ATP"
  },
  {
    "caption": "D Representative amplification of the region of chromosomal deletion in edited cells. PCR products were run on a 1% agarose gel to resolve the amplicons from edited and unedited chromosomes. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.",
    "molecules": "agarose gel"
  },
  {
    "caption": "H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.",
    "molecules": "SiR, Dextran, Dextran-568"
  },
  {
    "caption": "B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.",
    "molecules": "K+"
  },
  {
    "caption": "C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm. ",
    "molecules": "DA, dopaminergic"
  },
  {
    "caption": "(L), Diap1 is monitored by the GFP.4.3. (green) reporter (Djiane et al., 2013; Zhang et al., 2008). Diap1-GFP is detected in a subset of adult midgut esg+ cells that are negative for the mitotic marker PH3 (red, inset) and for EE marker Prospero (Pros, grey in inset). Diap1-GFP is not detected in mature EC (large nuclei cells, DAPI in blue). Data information: Scale bars, 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Lineage tracing using the initiator caspase sensor act-DBS-S-QF (14 days of tracing) combined with QUAS-FLP and act>FRT>lacZ are visualized by ßGalactosidase (ßGal, green). ISCs are labelled by anti-Dl (red, insets). Red arrowhead points to the ISC. Gut is counterstained with DAPI (blue) and DAPI (grey, in the inset). Data information: Scale bars, 100µm",
    "molecules": "DAPI"
  },
  {
    "caption": "(E-E'') Adult midgut klu>mCD8::GFP, GBE-Su(H)-lacZ stained for ß-Gal (red) and GFP (green). DAPI counterstaining in blue (E' and E''). The arrows point to examples of klu-negative Su(H)-lacZ-positive cells. The green label (asterisk, E) is in the overlying muscle. Data information: Scale bars 50µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Tryptophan-fluorescence time course associated to Na+ (blue) and K+ (black) binding, respectively, to detergent-purified apo EAAT1CRYST at pH 7.4. Horizontal and vertical scale bars represent time and ∆F/F0, respectively. B. Tryptophan-fluorescence time course associated to L-asp binding in presence of 1 mM (blue) and absence (black) of Na+, respectively, at pH 9. C",
    "molecules": "L-asp, K+, Na+, Tryptophan"
  },
  {
    "caption": "C. Na+ binding isotherms of EAAT1CRYST at pH 7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Na+ binding (green). Solid lines represent fits of Hill equation.",
    "molecules": "UPCH101, Na+"
  },
  {
    "caption": "A. Na+-induced L-glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K+. Choline (Ch+) condition was subtracted to the Na+ condition.",
    "molecules": "Ch+, Choline, L-glutamate, liposomes, K+, Na+"
  },
  {
    "caption": "B. Log-log plots of Na+ (red, n=19) and L-asp (blue, n=27) KD, as a function of pH at [Na+]=0.5 mM. Na+ (n=10) and L-asp (n=7) titration data at pH 7.4 and 9.0, respectively, are also reported in Fig. 1. Solid lines are fits of straight lines.",
    "molecules": "L-asp, Na+"
  },
  {
    "caption": "C. Log-log plots of L-asp KD for EAAT1CRYST-E406386Q (light blue, n=24) and EAAT1CRYST-Y405385F (dark blue, n=18), as a function of pH at [Na+]=0.5 mM..",
    "molecules": "L-asp, Na+"
  },
  {
    "caption": "A. Purified EAAT1CRYST takes up L-glutamate in liposomes loaded with K+ or Rb+, but not with choline (Ch+), or with Ch+ and Ca2+. Plots depict data as in Fig. 3A.",
    "molecules": "Ca2+, Ch+, choline, L-glutamate, liposomes, K+, Rb+"
  },
  {
    "caption": "B. Ca2+ binding isotherms of EAAT1CRYST at pH=7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Ca2+ binding (green). Symbols represent average and s.e.m. values of at least three independent experiments. Solid lines represent fits of Hill equation and dashed line is the fit in Fig. 1C (blue line) corresponding to Na+ binding.",
    "molecules": "UPCH101, Ca2+, Na+"
  },
  {
    "caption": "C. Log-log plot of L-asp KD as a function of Ca2+ (n=13).",
    "molecules": "Ca2+, L-asp"
  },
  {
    "caption": "(A-B) Coronal brain section, bregma: - 2.88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus (A). 8 h post-injection, majority of the injected Fib-Tau-1N3R-ATTO550 (red) is taken up by the corpus callosum of the injected side (B). Images are shown at low (left, 10X) and high (right, 63X) magnification. Data information: Scale bar, 100µm in the left panel in (B), 10µm everywhere else.",
    "molecules": "ATTO550"
  },
  {
    "caption": "(D-F) Single Particle Tracking using Quantum Dots (SPT-QD) of biotin tagged Fib-Tau. Representative single molecule trajectories of Fib-Tau following 10 or 60 min exposure are shown (D). Note after 60min exposure (0.36nM), single molecules are more confined suggesting they are trapped and clustered. Quantification of diffusion coefficient (E) and explored area (F, extracted from mean squared displacement (MSD), see methods) shows that both these parameters decrease after 60min exposure to Fib-Tau. Unpaired t-test, n is averaged value per cells imaged in 3 experiments (10 min: 22, 60 min: 23). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 2µm",
    "molecules": "biotin, Quantum Dots"
  },
  {
    "caption": "(G) Neurons were exposed for 60 min to Fib-Tau (0.36nM) labeled both with biotin and ATTO488 (red). Cell surface exposed biotin was labelled using streptavidin-550 (green) followed by live imaging. Note that most of the clusters of ATTO488 (red) are co-labelled with Streptavidin 550 (green) indicating that the clusters are at the cell surface. Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 5µm",
    "molecules": "550, ATTO488, streptavidin, biotin, Streptavidin"
  },
  {
    "caption": "(H-J) Clearance of Tau clusters from neurons. Neurons were exposed (0.36nM) to ATTO 550 labeled Fib-Tau for 10 min and the unbound fibrils were washed. Cells were fixed immediately (10min) or allowed to recover in culture medium for 60 min. Two representative images (H) and quantifications (I, J) show that following 60 min recovery most of the Tau clusters disappear/dissociate as indicated by a decrease in their density. Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test, n is number of images analyzed from 3 cultures (49 images). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar 2µm",
    "molecules": "ATTO 550"
  },
  {
    "caption": "(A) Super-resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib-Tau (0.36nM). Primary neurons were fixed 10, 60, 120 and 240 min after exposure to ATTO647N labeled Fib-Tau. Images rendered with a pixel size of 5nm show both clustered and diffused (non-clustered) distribution of exogenous Fib-Tau molecules. Scale bar, 1µm. (B-F) Quantification of various parameters from the super-resolution images. Averaged values per image is plotted. Increased "total detections per µm2" (B) and "cluster-density" (C) of Fib-Tau bound to neurons with increasing exposure time is observed. No change in the "size of cluster" (D) or "number of molecules within a cluster" (E) is seen with increased exposure time. The proportion of single molecule events detected within clusters remained nearly constant (~ 50%) in all conditions (F) indicating an equilibrium between clustered and non-clustered Fib-Tau single molecules. Unpaired t-test to compare difference with 10 min, n is number of images (8 images). *p<0.05; **p<0.01; ***p<0.001; ns= not significant. ",
    "molecules": "ATTO647N"
  },
  {
    "caption": "(D) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull-downs from neurons exposed to Fib-Tau. For each identified protein, the name of the protein, the gene name, the p-value (t-test with Benjamini-Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. In an independent analysis, after 10 min exposure of neurons to biotinylated Fib-Tau, a cross-linking step was performed during 20 min using 1mM of DTSSP added in the culture medium, in order to cross-link the protein complexes formed at the cell surface using a membrane impermeable cross-linker. After cross-linking, proteins were analyzed and identified exactly as non-cross-linked samples. Proteins identified with at least 2 peptides are labeled "+", the other are labeled "-".",
    "molecules": "biotinylated, DTSSP"
  },
  {
    "caption": "(E). Co-immunoprecipitation of exogenous biotin-labelled Fib-Tau with α3-NKA, GluA2 and GluN1. α3-NKA, GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section. The presence of Fib-Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin-HRP. A 2.4-, 2.3-, and 1.8- fold enrichment in Tau band-intensity is observed in α3-NKA, GluA2 and GluN1 immunoprecipitates, respectively, compared to controls performed with pre-immune goat or rabbit IgGs. Co-immunoprecipitates of exogenous biotin-labelled Fib-Tau with anti α3-NKA, GluA2 and GluN1 specific antibodies were also subjected to SDS-PAGE and Western blot analysis. The presence of Fib-Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin-HRP. Fib-Tau co-immunoprecipitates with α3-NKA and GluA2-AMPA receptor but not with GluN1-NMDA receptor.",
    "molecules": "AMPA, biotin, NMDA, nitrocellulose"
  },
  {
    "caption": "(A, B) Primary neurons were exposed to Fib-Tau-ATTO550 (0.36nM, red) for 10 (top row) or 60 min (bottom row) and excitatory (Homer, green) and inhibitory synapses (Gephyrin, blue) were immuno-labelled. Fib-Tau clusters co-localized and/or apposed to Homer (arrowheads) but not Gephyrin (A). Quantitative analysis shows that 10 to 20% of Fib-Tau clusters are localized at Homer containing synapses (B). Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test to compare difference between 10 and 60 min, n is number of images analyzed from 3-experiments (10 min: 49; 60 min: 50). Scale bar, 5µm.",
    "molecules": "ATTO550"
  },
  {
    "caption": "Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Arrows indicate excitatory synapses where Fib-Tau and α3-NKA/AMPA/NMDA are co-localized (C). Mann-Whitney-test, n is number of images analyzed from 3-4 experiments (α3-NKA: 75; GluA1: 95; GluA2: 50; GluN1/N2B: 45).",
    "molecules": "AMPA, NMDA"
  },
  {
    "caption": "Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Quantification of the fluorescence intensity (indicating size of clusters of synaptic α3-NKA/AMPA/NMDA spots (obtained after thresholding) following exposure to Fib-Tau showed a reduction in the size of α3-NKA and increase in the size of GluA2-subunit containing AMPA receptors (D). Box-plot represents: median, interquartile range and 10-90% distribution, **p<0.01; ns= not significant. Scale bar, 5µm.",
    "molecules": "ATTO550, AMPA, NMDA"
  },
  {
    "caption": "(A-C) SPT-QD of pHluorin tagged AMPA (GluA1 and GluA2) and NMDA (GluN1 and GluN2B) receptor subunits using GFP-antibody pre-coupled to QD655. Primary neurons were exposed or not to Fib-Tau (0.36nM, 60 min). Cumulative frequency of diffusion coefficient of all trajectories (extracted from MSD plot, inset, A) show a slow-down in diffusion of GluA2-subunit but not GluA1, GluN1 and GluN2B subunits (A). To compute statistical differences, the diffusion coefficient of all trajectories per movie (from 3 independent experiments) were averaged (B). To measure statistical differences in MSD, the explored area (area under curve during 600-900ms of MSD plot) per movie (from 3 independent experiments) were averaged (C). Decreased diffusion coefficient and area explored for GluA2-AMPA was observed indicating increased confinement concomitant with increased clustering (Fig 5C) following exposure to Fib-Tau. Mann-Whitney-test, n is number of movies (left to right): 14, 15, 14, 13, 15, 15, 15, and 15. **p<0.01; ns= not significant.",
    "molecules": "pHluorin, AMPA, NMDA, QD655, QD"
  },
  {
    "caption": "(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min). Scale bars 2µm",
    "molecules": "ATTO647, pHluorin, Paraformaldehyde"
  },
  {
    "caption": "(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta. Scale bars: 2µm",
    "molecules": "ATTO647, pHluorin"
  },
  {
    "caption": "DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn (shown here in green) or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of α-Syn fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn",
    "molecules": "ATTO550, ATTO647"
  },
  {
    "caption": "DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing Aβ (given in monomer-equivalent) of Aβ (B) fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ (shown here in green) or unlabeled Aβ fibrils]. concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils",
    "molecules": "ATTO550, ATTO647, Aβ, Aβ fibrils"
  },
  {
    "caption": "DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of both α-Syn fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 27, 25, 25, 26 and 28 in (C) , from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.",
    "molecules": "ATTO550, ATTO647"
  },
  {
    "caption": "DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of Aβ fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 18, 50, 50, 50, 25 in (D), from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.",
    "molecules": "ATTO550, ATTO647, Aβ, Aβ fibrils"
  },
  {
    "caption": "Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils The quantification in (E) includes all 5-experiments (n = number of images analyzed, left to right: 58, 54, 50, 62, 63, 65, 43, 35, 60, 60, 60, 35; One-Way ANOVA with Dunnette's post-hoc test was performed to compare the size of Fib-Tau clusters). Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.",
    "molecules": "Aβ fibrils"
  },
  {
    "caption": "A perfect co-localization between Fib-Tau and α-Syn clusters is also observed. The proportion of Fib-Tau clusters co-localizing with α-Syn fibrils increased with an increasing concentration of α-Syn fibrils reaching 60-70% overlap A weaker, concentration independent, overlap (10-20%) was observed with Aβ fibrils The quantification in (F) is derived from 3 to 4 independent experiments. The number of images analyzed is 27, 25, 25, 26, 28 for α-Syn and 18, 50, 50, 50, 25 for Aβ from left to right. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the co-localization differences from 100nM concentration. Box-plot represents: median, interquartile range and 10-90% distribution. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.",
    "molecules": "Aβ, Aβ fibrils"
  },
  {
    "caption": "(G-I) Primary neurons were co-exposed to Fib-Tau (0.36nM, ATTO550 labelled) and Fib-α-Syn (50nM, monomer-equivalent, unlabeled) for 60min and immunolabelled for homer (G) or α3-NKA (H). Quantification of Fib-Tau clusters co-localized with homer or α3-NKA (I). Note an increased co-localization of Fib-Tau with homer in presence of Fib-α-Syn. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.",
    "molecules": "ATTO550"
  },
  {
    "caption": "E Immunofluorescent  analyses of  USP33 (red) and the hypoxic cell population labeled by   pimonidazole  (green) in intracranial xenografts  derived from T3359 GSCs. Mice  bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of    pimonidazole  and were sacrificed 2 hours post injection. Hypoxic cells were labeled with the  FITC-conjugated  mouse monoclonal anti-pimonidazole antibody. Scale bar, 80 µm.",
    "molecules": "FITC, pimonidazole"
  },
  {
    "caption": "G  Immunoblot  analysis of USP33, HIF2α and HIF1α  protein levels in  GSCs, NSTCs  and NPCs in normoxic  and hypoxic  conditions. Hypoxia (5% O2) relative to normoxia (20% O2) clearly induced  USP33 and HIF2α expression in GSCs but not NSTCs or NPCs. The mild hypoxia of 5% O2 showed negligible induction of  HIF1α  in all cells.",
    "molecules": "O2"
  },
  {
    "caption": "A, Representative images of immunofluorescent analyses of USP33 (red) and HIF2α (green) in  human primary GBMs. Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α, and counterstained with DAPI to show nuclei (blue). Scale bar, 40 μm. (n = 5 sections; mean ± s.e.m.).",
    "molecules": "DAPI"
  },
  {
    "caption": "A,  Co-immunoprecipitation  (Co-IP) to detect the interaction  between endogenous HIF2α  and USP33 in GSCs in response to low oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours before harvest for the  Co-IP. Cell lysate was subjected to immunoprecipitation with anti-HIF2α antibodies.  Co-immunoprecipitated products were  immunoblotted  with the indicated antibodies. An increased  interaction between HIF2α  and USP33  was detected in  GSCs  cultured under 5% oxygen relative to 20%  oxygen.",
    "molecules": "oxygen"
  },
  {
    "caption": "D Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and  USP33 in GSCs in response to CoCl2 treatment. GSCs were treated with CoCl2 (300 µM) for 12 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-USP33 antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased  interaction  between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia.",
    "molecules": "CoCl2"
  },
  {
    "caption": "A  Immunoblot analysis of the  serine/threonine phosphorylation (pS/T) status of  HIF2α in GSCs in response to 5% oxygen. GSCs were cultured under 20% or 5%  oxygen for 24 hours, and then harvested for immunoprecipitation with anti-HIF2α antibodies or IgG control. Immunoprecipitated endogenous  HIF2α  were  immunoblotted with anti-pan-pS/T antibodies. An increased pS/T of  HIF2α was detected in GSCs  cultured under 5%  oxygen relative to 20% oxygen.",
    "molecules": "oxygen, serine, threonine"
  },
  {
    "caption": "D Co-immunoprecipitation to detect the interaction between endogenous HIF2α and USP33 in GSCs after inhibition of ERK1/2 activity. GSCs were cultured under 20% or 5% oxygen for 24 hours, then treated with the MEK  inhibitor U0126  (10µM) for 30 minutes to inhibit  ERK1/2 activity, and harvested for immunoprecipitation  with anti-USP33 antibodies. Co-immunoprecipitated  products were  immunoblotted with the indicated antibodies. Relative to 20%  oxygen treatment, 5% oxygen treatment promoted the  interaction between  HIF2α and USP33 in GSCs. However, inhibition of ERK1/2 activity by  U0126  treatment suppressed the  interaction between HIF2α and USP33.",
    "molecules": "oxygen, U0126"
  },
  {
    "caption": "G Co-immunoprecipitation to determine the  serine phosphorylation sites in human HIF2α responsible for its binding to USP33. Ectopic  GFP-tagged  USP33 along with HA-tagged wild type  HIF2α or HIF2α mutants with indicated  serine to  alanine point mutations were expressed in 293T cells. Cell lysates were subjected to  immunoprecipitation  with anti-HA agaroses Co-immunoprecipitated products were immunoblotted with the indicated antibodies. The S484A mutation largely attenuated the  interaction of HIF2α with  USP33, indicating that the  serine  484 site in HIF2α was the most important  phosphorylation site for the interaction  between  HIF2α and  USP33.",
    "molecules": "agaroses, alanine, serine"
  },
  {
    "caption": "B Representative images of   tumorsphere formation of T387  GSCs expressing USP33-targeting shRNA (shUSP33) or non-targeting shRNA (shNT). Twenty-four hours after  lentiviral infection, T387 GSCs expressing shUSP33 or shNT were planted in 96 well plates at the density of 2,000 cells per well and cultured for 48 hours under normoxia (20%  O2) or  hypoxia (5%  O2). Disruption of USP33 inhibited GSC tumorsphere  formation in normoxia  and dramatically suppressed  GSC tumorsphere  formation in hypoxia. Scale bar, 200 μm.",
    "molecules": "O2"
  },
  {
    "caption": "(A and B) Overexpressed LRRK2 levels in HEK 293T cells upon AICAr treatment. HEK293 cells were transfected with MYC-LRRK2 and treated with or without 1mM AICAr for 48 hours. LRRK2 levels were analyzed by Western blot.",
    "molecules": "AICAr"
  },
  {
    "caption": "(G-N) LRRK2 expression levels in primary neurons (G and H), microglia (I and J), astrocytes (K and L) and human fibroblasts (M and N) upon AICAr treatment. Mouse primary cortical neurons, microglia, astrocytes or human fibroblasts from a health control were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.",
    "molecules": "AICAr"
  },
  {
    "caption": "(Q and R) LRRK2 expression levels in mouse striatum upon AICAr treatment. AICAr (72μg/day/animal) or saline (used as a control) was injected into mouse striatum via osmotic pumps. One day after injection, striatal tissues near the injection sites were collected and LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM from total six mice (three male mice and three female mice). ****p < 0.0001 by Student's t tests.",
    "molecules": "AICAr, saline"
  },
  {
    "caption": "(S) Representative fluorescent images (green) showing that mouse primary cortical neurons were co-transfected with MYC-LRRK2 WT or G2019S (GS), and eGFP at a plasmid ratio of 10:1 at DIV 5, and treated with or without 1mM AICAr. Neuronal viability was analyzed at 48-hour post-transfection with non-viable neurons exhibiting no obvious neurite process (arrow). Scale bars, 100 μm. (T) Quantification of neuronal viability. Bars indicate the viability (n > 100) for each transfection condition expressed as a percent of control neurons (eGFP with pcDNA3.1 empty vector). Data represent the mean ± SEM from three independent experiments with n > 100 cells quantified in each experiment. *p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.",
    "molecules": "AICAr"
  },
  {
    "caption": "(A and B) LRRK2 protein levels upon treatment of AICAr together with a proteasome inhibitor MG132 or an autophagy inhibitor chloroquine (CQ). SH-SY5Y cells were treated with or without 1mM AICAr, 0.35μM MG132, and 5μM CQ as showed in (A) for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.",
    "molecules": "AICAr, chloroquine, CQ, MG132"
  },
  {
    "caption": "(C) LRRK2 and GBA mRNA levels upon AICAr treatment. SH-SY5Y cells were treated with or without 2mM AICAr for 24 hours. Total RNA was extracted. LRRK2 and GBA mRNA levels were analyzed by real-time PCR. Data are mean ± SEM, n=4 independent experiments. **p < 0.01 by Student's t tests. ns: not statistically significant. (D) LRRK2 mRNA level upon AICAr treatment in the presence of actinomycin D. SH-SY5Y cells were treated with or without 2mM AICAr for 24 hours and in the presence of 10μg/ml actinomycin D. Total RNA was extracted. LRRK2 mRNA levels were analyzed by real-time PCR. Data are mean ± SEM, n=4 independent experiments. **p < 0.01, ****p < 0.0001 by two-way ANOVA followed by Sidak's multiple comparison test.",
    "molecules": "actinomycin D, AICAr"
  },
  {
    "caption": "(E and F) LRRK2 expression levels from native LRRK2 cDNA and synthesized LRRK2 cDNA with synonymous codons upon AICAr treatment. HEK 293T cells were transfected with Flag-LRRK2 with native cDNA or with synthesized cDNA, and treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ***p < 0.001 by Student's t tests.",
    "molecules": "AICAr"
  },
  {
    "caption": "(I and J) The specific region (2221-3927bp) of the synthesized Flag-LRRK2 cDNA was replaced with the native sequence and the response of this chimeric LRRK2 cDNA expression to AICAr treatment was analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. ****p < 0.0001 by Student's t tests.",
    "molecules": "AICAr"
  },
  {
    "caption": "(B LRRK2 protein levels in AUF1 knockout (KO) cells upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.",
    "molecules": "AICAr"
  },
  {
    "caption": "(E and F) LRRK2 protein levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment. SH-SY5Y WT and KO cells were transfected AUF1-GFP or GFP and selected with 4μg/ml blasticidin. The resulting stable cell lines were treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot.",
    "molecules": "AICAr, blasticidin"
  },
  {
    "caption": "(G and H) Overexpressed LRRK2 levels in AUF1 KO cells with AUF1 expressing back upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were transfected AUF1-GFP or GFP and selected with 4μg/ml blasticidin. The resulting cell lines were transfected with a construct with native LRRK2 cDNA or synthesized LRRK2 cDNA with synonymous codons and treated with or without 1mM AICAr for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software.",
    "molecules": "AICAr, blasticidin"
  },
  {
    "caption": "(A and B) AUF1 binds to LRRK2 mRNA upon AICAr treatment. SH-SY5Y WT and AUF1 KO cells were treated with or without 1mM AICAr for 24 hours. RNA and protein complexes were immunoprecipitated using AUF1 antibody. The LRRK2 mRNA levels were analyzed by semiquantitative reverse transcription (RT)-PCR. 18S ribosomal RNA (rRNA) was as a loading control. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. *p < 0.05 by Student's t tests.",
    "molecules": "18S ribosomal RNA, AICAr"
  },
  {
    "caption": "(D) Direct interaction of LRRK2 mRNA and four AUF1 isoforms. Biotin-labelled 62-nt LRRK2 RNA probes from native LRRK2 cDNA or synthesized LRRK2 cDNA were incubated with purified AUF1 isoforms in an RNA electrophoretic mobility shift assay (EMSA). Free probes and AUF1 bound probes were detected using a chemiluminescent RNA EMSA kit.",
    "molecules": "chemiluminescent, Biotin"
  },
  {
    "caption": "(A and B) DCP1A and DCP2 expression levels upon AICAr treatment. SH-SY5Y cells were treated with or without 1mM AICAr for 48 hours. DCP1A and DCP2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software. Data are mean ± SEM, n=3 independent experiments. **p < 0.01 by Student's t tests.",
    "molecules": "AICAr"
  },
  {
    "caption": "(E) LRRK2 mRNA decapping by DCP1A and DCP2. Recombinant DCP1A and DCP2 proteins were purified from HEK 293T cells and incubated with [α-32P]cap-labeled LRRK2 RNA. LRRK2 mRNA decapping was analyzed by one-dimensional thin layer chromatography. The decapping activity is demonstrated by the increased m7GDP levels.",
    "molecules": "m7GDP, 32P"
  },
  {
    "caption": "(A and B) LRRK2 protein levels upon the treatment of AICAr, AICAr plus an adenosine transporter (AT) inhibitor ABT702 or an adenosine kinase (ADK) inhibitors NBT-1. SH-SY5Y cells were treated with or without 1mM AICAr, AICAr plus 1μM ABT-702 or 50μM NBT-1 for 48 hours.",
    "molecules": "NBT-1, ABT-702, ABT702, AICAr"
  },
  {
    "caption": "(C and D) LRRK2 protein levels upon the treatment of AICAr or plus AMPK direct activator A769662. SH-SY5Y cells were treated with or without 1mM AICAr, 10, 20, or 50 μM A769662 for 48 hours.",
    "molecules": "A769662, AICAr"
  },
  {
    "caption": "(E and F) LRRK2 protein levels upon the treatment of 1mM AICAr in WT and AMPK KO SH-SY5Ycells for 48 hours. LRRK2 expression levels were analyzed by Western blot. Images were quantified by ImageJ software.",
    "molecules": "AICAr"
  },
  {
    "caption": "(B) Representative confocal images (GFP) of dopamine neurons in each cluster from 9-week-old WT and LRRK2-G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion. Scale bars, 25 μm.",
    "molecules": "AICAr"
  },
  {
    "caption": "(C-E) Quantification of dopamine neurons per DA cluster in 9-week-old WT and LRRK2-G2019S transgenic flies with or without 1mM AICAr treatment after posteclosion. (F) Total numbers of dopamine neurons in four major DA clusters of 9-week-old WT and LRRK2-G2019S transgenic flies with or without AICAr treatment after posteclosion. Data are mean ± SEM, n=12 flies per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by a Tukey's post hoc test.",
    "molecules": "AICAr"
  },
  {
    "caption": "(A) Representative images of DAneuron staining. WT and LRRK-KO mice were injected with 1ul LPS (5ug/animal) or phosphate buffered saline (PBS) into the substantia nigra pars compacta (SNpc) and administrated with AICAr (2.4μg/day/animal) or saline to same stereotaxic coordinates using osmotic pumps. After 21 days of injection, DA neurons were stained by anti-tyrosine hydroxylase (TH) antibody. Scale bars, 500 μm.",
    "molecules": "AICAr, LPS, PBS, phosphate buffered saline, saline"
  },
  {
    "caption": "(B) Quantification of TH positive neurons in substantia nigra pars compacta (SNpc) using an unbiased stereological method with stereo investigator software from 7-8 animals per group. (C) Quantification of Nissl-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from 7-8 animals per group.",
    "molecules": "Nissl"
  },
  {
    "caption": "(E) CD68 levels in WT and LRRK-KO mice injected with 1μl LPS (5μg/animal) or PBS into striatum followed by administration of AICAr (2.4μg/day/animal) or saline to same stereotaxic coordinates using osmotic pumps. After 21 days of injection, LRRK2 and CD68 expression in striatal tissues near the needle sites was analyzed by Western blot. (F) Quantification of images from (E) using ImageJ software, n=3 animals per group.",
    "molecules": "AICAr, LPS, PBS, saline"
  },
  {
    "caption": "b, No significant inward current was detected in NMDG+ (Na+-free, Ca2+-free, pH 4.6) solution.",
    "molecules": "Ca2+, NMDG, Na+"
  },
  {
    "caption": "c, Inwardly rectifying step currents were evoked by 30 mM Fe2+ solution (pH 4.6) in the same cell as shown in a and b.",
    "molecules": "Fe2+"
  },
  {
    "caption": "e, Large IFe/TRPML2Va was seen in the presence of 30 and 105 mM Fe2+ (pH 4.6).",
    "molecules": "Fe2+"
  },
  {
    "caption": "a, Current densities (mean ± s.e.m., n = 4-10) of IFe (30 mM Fe2+, pH 4.6) for TRPML1Va and ML4 mutant TRPML1Va channels. Asterisk indicates statistical difference (P < 0.01) compared to TRPML1Va.",
    "molecules": "Fe2+"
  },
  {
    "caption": "b, 55Fe2+uptake (normalized) in HEK293T cells transfected with vector control, with TRPML1Va, with F408D-TRPML1Va and with T232P-TRPML1Va constructs. Error bars indicate the standard deviation on the basis of two independent triplicate experiments.",
    "molecules": "Fe2+"
  },
  {
    "caption": "c, [Fe2+]o-dependent quenching of Fura-2 fluorescence in TRPML1Va-transfected cells (arrows), but not in non-transfected control cells (arrowheads) or T232P-TRPML1Va-transfected cells (bottom row). The fluorescence intensity was measured at an excitation wavelength of 360 nm (F360). The original magnification used for all micrographs was ×200.",
    "molecules": "Fe2+"
  },
  {
    "caption": "d, Average normalized responses of EGFP-positive TRPML1Va-transfected cells (typically n = 20-40 cells) to 1 or 10 mM Fe2+ (pH 4.6).",
    "molecules": "Fe2+"
  },
  {
    "caption": "g, A small but significant quenching reaction was detected (with 10 mM Fe2+) in R403C-TRPML1Va-expressing cells.",
    "molecules": "Fe2+"
  },
  {
    "caption": "c, Lysosomal ITRPML1Va. Switching from lysosome-attached to (lysosome) luminal-side-out configuration significantly reduced the amplitude of the current. The luminal-side-out patch was exposed to the Tyrode's solution. A Cs+-based solution (147 mM Cs-methanesulphonate (Cs-MSA)) was used as a pipette solution for both configurations.",
    "molecules": "Cs+, methanesulphonate"
  },
  {
    "caption": "d, NMDG+-impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations (nominal divalent-free). e, Lowering pH potentiated lysosomal ITRPML1Va. f, IFe/TRPML1Va induced by 30 mM and 105 mM Fe2+.",
    "molecules": "Fe2+, NMDG"
  },
  {
    "caption": "g, Whole-lysosome current in an enlarged lysosome expressing wild-type TRPML1. The pipette (lumen) solution contained nominal divalent-free Tyrode solution. A Cs+-based bath solution (147 mM Cs-MSA) was used. h, Whole-lysosome ITRPML1Va. i, Whole-lysosome IFe/TRPML1. The pipette (lumen) solution contained 105 mM Fe2+ (pH 4.6).",
    "molecules": "Cs+, Fe2+"
  },
  {
    "caption": "a, Cultured TRPML1-/- (ML1-/-) skin fibroblasts showed less de-quenching of the iron-sensitive fluorescence than ML1+/- cells did. De-quenching was achieved by preloading the fibroblasts with an iron-sensitive dye, Phen Green SK (PG SK), and then adding the membrane-permeable transition metal chelator, 2,2′-bipyridyl (BPD). 2,2′-BPD is predicted to chelate free cellular iron (also referred to as chelatable or labile iron), which subsequently increases PG SK fluorescence. The original magnification used was ×200",
    "molecules": "2,2′-bipyridyl, 2,2′-BPD, iron"
  },
  {
    "caption": "b, The average 2,2′-BPD induced normalized change of fluorescence (Dgr;F/F0) for ML4 cells (ML1-/-; n = 6 experiments) is significantly (asterisk, P < 0.01) lower than for the parental ML1+/- cells. Fibroblast cells (n = 10-20) were analysed for each individual experiment. 4,4′-BPD, a 2,2′-BPD analogue that cannot bind Fe2+, did not induce any significant restoration of PG SK fluorescence. Error bars, s.e.m.",
    "molecules": "2,2′-BPD, Fe2+"
  },
  {
    "caption": "DNA-FISH detection of the number of X-linked Atrx gene foci. Representative images of the DNA-FISH are shown. Nuclei are surrounded by white dotted lines. Magenta: Atrx (white arrowheads), cyan: DAPI. N>100.",
    "molecules": "DAPI"
  },
  {
    "caption": "Clonogenic assay for ESCs in response to etoposide (A) or camptothecin (B). Non-treated cells were set to 1.",
    "molecules": "camptothecin, etoposide"
  },
  {
    "caption": "Detection of the number of X-linked Atrx gene foci by DNA-FISH with or without dox addition for 96 h (N >100), performed as described in Figure 1C.",
    "molecules": "dox"
  },
  {
    "caption": "Detection of the Xist cloud in #B3 by RNA-FISH, with or without dox addition for 96 h ( N >200 each). A representative image is shown [magenta: Xist (white arrowhead), cyan: DAPI. Scale bar: 10 μm].",
    "molecules": "DAPI, dox"
  },
  {
    "caption": "Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C. #B2 or #B3 were cultured with or without dox addition for 96 h. The combined number of GFP-positive cells and total colonies from all experiments are listed and summarized at right.",
    "molecules": "dox"
  },
  {
    "caption": "Detection of the Xist foci by RNA-FISH, with or without dox addition for 96 h (N >200 each). A representative image with percentage of Xist-positive cells is shown [magenta: Xist, cyan: DAPI.].",
    "molecules": "DAPI, dox"
  },
  {
    "caption": "Detection of the number of X-linked Atrx gene foci by DNA-FISH, with or without dox addition for 96 h (N >100 each), performed as described in Figure 1C.",
    "molecules": "dox"
  },
  {
    "caption": "Estimated repair events. The flow cytometry plots are shown in Figure EV4. The percentage of GFP-positive cells in TX-DRGFP#11 and TX-EJ5GFP#23 was normalized to the transfection efficiency detected by the VENUS expression vector. Dox (-) was set to 1 and the ratio of relative GFP-positive cells is shown (n=3).",
    "molecules": "Dox"
  },
  {
    "caption": "Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. All treatment groups were then analyzed by qPCR for gene expression (A) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7-54 and FA-TLR7-54 (n=3, technical replicates).",
    "molecules": "FA"
  },
  {
    "caption": "Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h or 2h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. cell supernatants were analyzed for secreted cytokines by ELISA. (B-C) Changes in CCL18 and IL-1β in the culture media induced upon treatment with TLR7-54 and FA-TLR7-54 for the treatment regimens (n=3, technical replicates).",
    "molecules": "FA"
  },
  {
    "caption": "M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. mRNA levels of profibrotic markers, Arg1 (A), CD206 (B) and CD163 (C) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).",
    "molecules": "FA, folate, glucosamine"
  },
  {
    "caption": "M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. protein levels of secreted profibrotic CCL18 (D) and antifibrotic cytokines, CXCL10 (E) and IL-6 (F) (n=3, technical replicates) were then determined. Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).",
    "molecules": "FA, folate, glucosamine"
  },
  {
    "caption": "(A) Healthy mice or BLM-induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200-fold excess FA-glucosamine to block all folate receptors. After 2h, mice were euthanized, resected and imaged for fluorescence intensity (n=5).",
    "molecules": "BLM, FA, folate, glucosamine, OTL38"
  },
  {
    "caption": "(B) Alternatively, lungs from mice injected with 100 nmol OTL38 in the present or absence of 200-fold excess FA-glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4/80 prior to FACS analysis (n=3). Representative plots showing the gating strategy yielding live macrophages (7AAD- CD11b+ F4/80+) and OTL38 positive macrophages are shown.",
    "molecules": "7AAD, FA, glucosamine, OTL38"
  },
  {
    "caption": "(C) Percentages of live macrophages in BLM-induced mice (n=3).",
    "molecules": "BLM"
  },
  {
    "caption": "(D) Percentages of lung macrophages that accumulated OTL38 in vivo (n=3).",
    "molecules": "OTL38"
  },
  {
    "caption": "Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (A) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR (n=3).",
    "molecules": "BLM, DMSO, FA"
  },
  {
    "caption": "Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (B) BALF cells were pelleted and similarly analyzed for the indicated mRNAs (n=3).",
    "molecules": "BLM, DMSO, FA"
  },
  {
    "caption": "Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (C) BALF supernatant was also collected and analyzed by ELISA for IL-6, IFNα and TNFα (n=3).",
    "molecules": "BLM, DMSO, FA"
  },
  {
    "caption": "(I) Lungs were resected and subjected to hematoxylin-eosin (H&E), trichome (collagen) and α-SMA IHC staining (scale bars, 200 μm),",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Lungs were (J) hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content (Healthy, n=5; others, n=10),",
    "molecules": "hydroxyproline"
  },
  {
    "caption": "Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (A) Analysis of body weight change versus time (n=10).",
    "molecules": "BLM, FA"
  },
  {
    "caption": "Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (B) Analysis of the number of cells per milliliter of BALF (n=5).",
    "molecules": "BLM, FA"
  },
  {
    "caption": "Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (C) Quantitation of total hydroxyproline content per right lung (Healthy control, n=5; others, n=7-9).",
    "molecules": "BLM, FA, hydroxyproline"
  },
  {
    "caption": "Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (D) H&E staining and trichrome staining of lung tissue (scale bars, 200 μm).",
    "molecules": "BLM, FA"
  },
  {
    "caption": "Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (A-C) Measurement of plasma IL-6 (A), IFNα (B) and TNFα (C) (n=3).",
    "molecules": "FA"
  },
  {
    "caption": "Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (D-F) Effect of drug concentration on plasma levels of IL-6 (D), IFNα (E), and TNFα (F) at 1.5h, 1h, or 1h after treatment, respectively (n=2).",
    "molecules": "FA"
  },
  {
    "caption": "Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (G) Change in body weight as a measure of systemic toxicity during alternate day dosing (n=2).",
    "molecules": "FA"
  },
  {
    "caption": "(I-K) Serum  levels of glucose (I), triglyceride (J) and free fatty acid (FFA) (K) in Ftoflox/flox and FtoAKO mice  fed a  HFD followed by fasting. Data information: The data were presented as the mean ± SD (n = 6). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "FFA, free fatty acid, glucose, triglyceride"
  },
  {
    "caption": "(A)  O2  consumption of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate  day  and night, respectively. (B)  CO2  generation of 16-week-old  Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and  night, respectively. ( Data information: The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "CO2, O2"
  },
  {
    "caption": "(C) Distribution of m6A peaks in  Hif1a mRNA transcript in siCtrl and siFto 3T3-L1 cells. Blue boxes represent exons, and blue lines represent introns. The m6A  peaks were in the dashed red box.",
    "molecules": "m6A"
  },
  {
    "caption": "(D) Methylated RNA immunoprecipitation  (MeRIP)-qPCR analysis of  m6A levels of Hif1a mRNA in siCtrl and siFto 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.",
    "molecules": "m6A"
  },
  {
    "caption": "I Live imaging  of GFP-RAB6A  in control, dynarrestin-treated and CC1-p150-expressing  aRG cells at E15.5. Scale bars = 5µm. Distance = 5µm, time  = 30 seconds. J. Number of RAB6A+ vesicles  in the apical  process of DMSO  and dynarrestin-treated mouse aRG cells. K. Relative time  spent by RAB6A+ vesicles  in apical  movement phase, in DMSO  and dynarrestin-treated mouse aRG cells. L. Number of RAB6A+ vesicles  in the apical  process of mCherry  and CC1-p150-expressing  aRG cells. M. Relative time  spent by RAB6A+ vesicles  in apical  movement phase, in mCherry  and CC1-p150-expressing aRG cells. DMSO  treatment slightly affected RAB6A  dynamics, as compared to mCherry  control. Data information: (J, K, L, M) 216 vesicles  from N=11 cells for DMSO, 145 vesicles  from N=25 cells for dynarrestin, 173 vesicles  from N=17 cells for mCherry  control, 71 vesicles  from N=15 cells for CC1-p150. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.",
    "molecules": "DMSO, dynarrestin"
  },
  {
    "caption": "(C-J) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4 labelled by the absence of  ubiGFP (green). The red channel shows ELAV, which labels the nuclei  of  photoreceptor cells. All UAS constructs were expressed under the control of GMR-GAL4. (C) Expression  of UAS-Rh1 (4C5, in blue) and (D)  Na+K+ATPase (A5-C, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (E) Expression of UAS-Xport-A-HA (anti-HA, in blue) and (F) UAS-fan-HA  (anti-HA, in blue) is strongly reduced in  EMC3Δ4 homozygous mutant cells. (G) Expression of UAS-rtv-HA (anti-HA, in blue) and (H) hid (anti-hid, in blue) is increased in  EMC3Δ4 homozygous mutant cells. (I-J) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells. (I) UAS-CG8814-HA (anti-HA, in blue) is a TA protein containing a TMD of low hydrophobicity (J) UAS-Syx13-HA is a TA protein with high hydrophobicity TMD. Scale bars represent 10 μm.",
    "molecules": "ubi"
  },
  {
    "caption": "(B-D) - Immunostaining  of  3rd instar   larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of  ubiGFP (green) shows reduced  expression of (B) UAS-HA-Xport-A (anti-HA, in blue) but normal expression of (C) UAS-HA-Xport-A4L (anti-HA, in blue) or (D) UAS-Xport-A4L-HA (anti-HA, in blue) in EMC3  mutant homozygous cells. ELAV (red in B and C) marks photoreceptor cells and Na+K+ATPase (red in D) acts as a positive control for the presence of EMC3Δ4 homozygous mutant clones. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.",
    "molecules": "ubi"
  },
  {
    "caption": "(E-F) Immunostaining of mosaic adult  retina with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (E) UAS-HA-Xport-A (anti-HA, in blue) in EMC3 mutant homozygous cells. (F)  Expression of UAS-HA-Xport-A4L (anti-HA, in blue) is normal in EMC3e02662 homozygous mutant clones. The rhabdomeres are stained for actin (Phalloidin, in green). Scale bars represent 10 μm.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "(A-B)  Immunostaining of  3rd instar larval eye imaginal discs with  eyeless-Flippase-induced clones of cells homozygous for  EMC3Δ4, labelled by the absence of ubiGFP (green) show loss of (A) UAS-Xport-A-HA (anti-HA, in blue) and UAS-Rh1 (4C5, in red). (B) Expression of UAS-Xport-A4L (anti-HA, in blue) and UAS-Rh1 (4C5, in red) is observed in  EMC3Δ4 homozygous cells. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.",
    "molecules": "ubi"
  },
  {
    "caption": "(C-D)  Immunostaining of mosaic adult retinas with  eyeless-Flippase-induced clones of cells homozygous for  EMC3e02662, labelled by the absence of RFP (red), show loss of (C) TRP (Mab83F6, in blue) when UAS-HA-Xport-A is expressed. (D)  Expression of TRP  (Mab83F6, in blue) is observed in EMC3e02662 homozygous cells, when HA-Xport-A4L is expressed. The rhabdomeres are stained  for actin  (Phalloidin, in green). The UAS constructs were expressed under the control of Rh1-GAL4. Scale bars represent 10 μm.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "(B) Autophagy‐negative phenotype of the apg16Δ strain. The autophagic ability of wild‐type (TN125), apg12Δ (YNM107) and apg16Δ cells (YNM114) was measured by the alkaline phosphatase assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate the standard deviation of three independent experiments.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(C) Loss of viability during starvation. Wild‐type (KA311B, ○), apg12Δ (YNM101, ●) and apg16Δ cells (YNM124, ▴) were cultured in nitrogen starvation medium, and their viability was determined by phloxine B staining.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A) PJ69‐4A cells were co‐transformed with each pGBD and pGAD plasmid as indicated. Transformants were selected on Trp− Leu− plates, and then two‐hybrid interaction (+ or −) was assessed for growth on Ade− Trp− Leu− plates.",
    "molecules": "Ade−, Leu, Trp"
  },
  {
    "caption": "(A) IDH1 acetylation levels upon treatment with NAM or TSA. Flag-tagged IDH1 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period.",
    "molecules": "NAM, TSA"
  },
  {
    "caption": "(B) Five putative lysine residues were mutated. Acetylation levels of Flag-bead-purified IDH1 were determined by Western blot analysis using a pan-anti-acetyllysine antibody. Relative IDH1 acetylation ratios were calculated after normalizing against Flag.",
    "molecules": "acetyllysine, lysine"
  },
  {
    "caption": "(A) Colony formation ability after transfection of SIRT2 plasmids, AGK2 and SIRT2 siRNA. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.",
    "molecules": "AGK2"
  },
  {
    "caption": "(B) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after overexpression or downregulation of SIRT2 or treated with AGK2. Stained cells in the lower chambers were quantified. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.",
    "molecules": "AGK2"
  },
  {
    "caption": "(D) Cell cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids, SIRT2 siRNA or AGK2. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.",
    "molecules": "AGK2"
  },
  {
    "caption": "(A-B) IDH1 K224 acetylation level was enhanced with increasing glucose concentration. Flag-IDH1 was overexpressed in cells treated with increased concentrations of glucose for 6 h. IDH1 proteins were purified by Flag beads, and then IDH1 K224 acetylation level was determined by Western blot and IDH1 catalytic activity was assessed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.",
    "molecules": "glucose"
  },
  {
    "caption": "(C-D) IDH1 K224 acetylation level was upregulated and IDH1 activity was weakened with increasing glutamine concentration. Flag-IDH1 was overexpressed in cells treated with increased glutamine for 6 h. IDH1 proteins were purified by Flag beads, and the K224 acetylation level of IDH1 was determined by Western blot analysis and then IDH1 catalytic activity was evaluated. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , D statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. P<0.05 and **P<0.01. n.s. = not significant.",
    "molecules": "glutamine"
  },
  {
    "caption": "Downregulation of SIRT2 diminished the effect of glucose on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glucose (E)",
    "molecules": "glucose"
  },
  {
    "caption": "Downregulation of SIRT2 diminished the effect of glutamine on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glutamine (F).",
    "molecules": "glutamine"
  },
  {
    "caption": "(G) IDH1 K224 deacetylation regulated cellular NADPH/NADP+ redox in cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of NADPH/NADP+ in cells was measured as followed by manufacture's instruction. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , G statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.",
    "molecules": "NADP+, NADPH"
  },
  {
    "caption": "(H) IDH1 K224 deacetylation promoted GSH production in HCT116 cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of GSH/GSSG was assessed as followed by manufacture's instruction. 1 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.",
    "molecules": "GSH, GSSG"
  },
  {
    "caption": "(I) IDH1 K224 deacetylation suppressed cellular ROS levels in HCT116 cells. ROS was determined in cells under non-stressed condition or exposed to menadione. *P<0.05 and **P<0.01 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure I, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.",
    "molecules": "menadione, ROS"
  },
  {
    "caption": "(F) HCT116 cells of IDH1 K224Q were cotreated with Octyl-α-KG and NAC, the expression of HIF1α and SRC was further evaluated.",
    "molecules": "Octyl-α-KG, NAC"
  },
  {
    "caption": "HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For G statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.",
    "molecules": "Octyl-α-KG, glucose, NAC"
  },
  {
    "caption": "HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for lactate levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.",
    "molecules": "Octyl-α-KG, lactate, NAC"
  },
  {
    "caption": "(D) Pictures of livers in nude mice and their respective representative images of the tissues by hematoxylin-eosin (HE) staining. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). Statistical significance was assessed with a two-tailed unpaired Student's t-test. **P<0.01. ",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(B) Changes in lncRNA expression  from HUVECs  exposed to hypoxia   (0.2 %  O2 for 12 h or 24 h), determined by ribo-minus RNA deep sequencing  (data acquired from GSE107033 ; NTRAS  is highlighted (n = 2 independent biological replicates).",
    "molecules": "O2"
  },
  {
    "caption": "(E) FITC-dextran-based in vitro permeability  comparing control and NTRAS-silenced HUVECs  (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test,",
    "molecules": "dextran, FITC"
  },
  {
    "caption": "(H) TMR-dextran-based assessment of vascular  permeability in vivo, comparing heart  homogenates from control and Ntras-silenced mice. Data normalized to organ and body weight (n = 11-12 mice  per group). Experimental outline on the left. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. H) two-tailed unpaired t-test",
    "molecules": "dextran, TMR"
  },
  {
    "caption": "(A) Separation of mock and proteinase K-treated HeLa nuclear  extracts by  sucrose  density gradient ultracentrifugation followed by NTRAS  detection using  RT-qPCR. Fractions 1 and 14 represent top and bottom of the gradient, respectively (n = 1).",
    "molecules": "sucrose"
  },
  {
    "caption": "(I) RT-PCR-based analysis of  TJP1 exon 20 inclusion  upon silencing of NTRAS  in HUVECs  (n = 7 independent biological replicates). Representative agarose  gels on the right. Data information: , I, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. I, two-tailed unpaired t-test.",
    "molecules": "agarose"
  },
  {
    "caption": "(J) RT-PCR-based analysis of TJP1 exon 20 inclusion upon silencing of hnRNPL  in HUVECs  (n = 4 independent biological replicates). Representative agarose  gels on the right. Data information: , J) data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. J) two-tailed unpaired t-test.",
    "molecules": "agarose"
  },
  {
    "caption": "(I) Analysis of TJP1 exon 20 inclusion by RT-PCR  upon transfection of HUVECs  with a control SSO or an SSO masking the exon 20 - intron 20 boundary (E20  SSO) (n = 9 independent biological replicates). Representative agarose  gel on the right. Schematic outline at the top right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,",
    "molecules": "agarose"
  },
  {
    "caption": "(G) FTSC  and TMR-dextran in vivo permeability  assays, comparing homogenates of hearts from NtrasCA/CA and Ntras∆CA/∆CA mice. Data normalized to organ and body weight (n = 11-18 mice  per group). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,",
    "molecules": "dextran, FTSC, TMR"
  },
  {
    "caption": "(H) Quantification of CD45+ cell (red) infiltration into cardiac  tissue from NtrasCA/CA and Ntras∆CA/∆CA mice  normalized to DAPI.  Isolectin B4 (green) was used to label endothelial cells  (n = 4-5 mice  per group). Representative micrographs  are shown. Scale bars are 50 µm. Insets and arrows indicate sites of CD45+ cell infiltration. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,",
    "molecules": "DAPI"
  },
  {
    "caption": "Plasma corticosterone levels at baseline, 30 minutes after induction of stress by placing mice in a clean cage, and after 90 minutes upon return in the home cage. DEX mice received an injection of dexamethasone at 0.1 mg/kg body weight 150 minutes before collecting the first blood sample (n = 5-6 animals per group). Flox and GRBATKO mice were injected with 0.02 mg/kg dexamethasone and the procedure described in (A) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in (A) (n = 6-7 animals per group).",
    "molecules": "corticosterone, DEX, dexamethasone"
  },
  {
    "caption": "GR mRNA levels in BAT of Flox and GRBATKO mice. Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage. Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure, controls received an injection with saline (n = 5-6 animals per group).",
    "molecules": "dexamethasone"
  },
  {
    "caption": "Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice (n = 5-6 animals per group). Blood glucose levels (n = 5-6 animals per group).",
    "molecules": "corticosterone, glucose"
  },
  {
    "caption": "Representative Hematoxilin and eosin staining image of BAT of cold-exposed Flox and GRBATKO mice. Scale bar 50 µm.",
    "molecules": "eosin, Hematoxilin"
  },
  {
    "caption": "Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group).",
    "molecules": "Triglyceride"
  },
  {
    "caption": "Serum corticosterone levels (n = 6 animals per group).",
    "molecules": "corticosterone"
  },
  {
    "caption": "Blood glucose (n = 6 per group).",
    "molecules": "glucose"
  },
  {
    "caption": "Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group).",
    "molecules": "corticosterone"
  },
  {
    "caption": "Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).",
    "molecules": "NEFA, triglycerides"
  },
  {
    "caption": "Oxygen consumption over a 90-hour period. Grays columns indicate dark phase (n = 6-8 mice per group). Average oxygen consumption specified for light phase, dark phase, and total (n = 6-8 animals per genotype).",
    "molecules": "Oxygen, oxygen"
  },
  {
    "caption": "Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight (n = 6-8 animals per group).",
    "molecules": "oxygen"
  },
  {
    "caption": "Intraperitoneal GTT after 19 weeks of HFD-feeding, glucose injections at 1.5 mg per g of body weight (n = 11-13 animals per group). Area under the curve calculated from (A) (n = 11-13 animals per group).",
    "molecules": "glucose"
  },
  {
    "caption": "Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).",
    "molecules": "Triglyceride"
  },
  {
    "caption": "Blood glucose in Flox and GRBATKO animals after 24 weeks HFD (n = 10-13 animals per group).",
    "molecules": "glucose"
  },
  {
    "caption": "(A) Representative MELC images showing the distribution of macrophage polarization markers at the indicated time points after injection of FITC-labeled zymosan (3 mg/ml, 10 µl) in one hind paw. White dotted lines show the position of zymosan.",
    "molecules": "zymosan, FITC"
  },
  {
    "caption": "Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of the number of macrophages and their subtypes 8, 24, 48 and 72 hours after zymosan injection based on the MELC images. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, ***p<0.001. #p<0.05, ###p<0.001 as compared to naïve mice.",
    "molecules": "zymosan"
  },
  {
    "caption": "FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "FACS analysis of macrophages and their subtypes 8, 24 and 48 hours after zymosan injection. Data are shown as mean (n=8 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "(A) Frequency of different immune cell types in the MELC images 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM (n.d., not determinable).",
    "molecules": "zymosan"
  },
  {
    "caption": "Representative MELC images showing the IL-4 and Siglec F expression 4 hours (panel C) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.",
    "molecules": "zymosan"
  },
  {
    "caption": "Representative MELC images showing the IL-4 and Siglec F expression 24 hours (panel D) after zymosan injection. The white dotted lines depict the area where the transition between the neighboring regions occurs.",
    "molecules": "zymosan"
  },
  {
    "caption": "(E) Number of IL-4-expressing eosinophils in the core region and the surrounding area 4 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, two-tailed Students t-test ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "(F) Number of IL-4-expressing eosinophils in the three regions 24 hours after zymosan injection. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni **p<0.01, ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "(G) FACS analysis of the number of eosinophils at the indicated time points after zymosan injection in the inflamed paw. Data are shown as mean (n=5 mice) ± SEM, One Way ANOVA/Bonferroni *p<0.05, **p<0.01, ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "FACS analysis of eosinophils in the blood B; n= 6 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.",
    "molecules": "zymosan"
  },
  {
    "caption": "FACS analysis of eosinophils in the paws (panel C n= 8 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.",
    "molecules": "zymosan"
  },
  {
    "caption": "Zymosan-induced mechanical (panel E) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.",
    "molecules": "Zymosan"
  },
  {
    "caption": "Zymosan-induced thermal (panel F) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.",
    "molecules": "Zymosan"
  },
  {
    "caption": "Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of eosinophils, the sum of all macrophages, M2-like, M1-like and M0-macrophages in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of eosinophils, the sum of all macrophages, as well as efferocytosis (panel H) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "Quantification of eosinophils, the sum of all macrophages, neutrophils (panel I) in the MELC images of paws from control or eosinophil-depleted mice 24 hours after zymosan injection. Data are presented as mean ± SEM (n=5). Student's t-test, **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "(A) IL-4 levels in paws from naive and zymosan-injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil-depleted mice. Data are shown as mean ± SEM (n=6 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: **p<0.01, ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "(B) FACS analysis of the eosinophil number in paws from control and eosinophil-depleted mice with or without IL-4c treatment 24h prior zymosan injection. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni ***p< 0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "(A) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil-depleted mice. Data are mean ± SEM (n=6 mice). Student's One-way ANOVA, significance between control and anti-Siglec F antibody treatment *p<0.05; **p<0.01; ***p<0.001.",
    "molecules": "zymosan"
  },
  {
    "caption": "(C) Zymosan-induced thermal hypersensitivity in control or eosinophil-depleted mice with or without IL-4c treatment 24h hours prior zymosan injection. The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points. Data are shown as the mean ± SEM (n=6 mice). Two-way ANOVA/Bonferroni, *p< 0.05, **p< 0.01; ns, not significant.",
    "molecules": "Zymosan, zymosan"
  },
  {
    "caption": "A. Spinning disk time-lapse microscopy of WT and UBAP2L KO HeLa cells synchronized with double thymidine block and release (DTBR) in mitosis. The selected frames of the movies are depicted and the corresponding time is indicated in minutes. SiR-DNA was used for DNA staining.",
    "molecules": "SiR-DNA, DNA"
  },
  {
    "caption": "I, J. The time of mitotic entry (I) and from prophase to anaphase (J) was quantified. At least 50 cells per condition were analyzed for each experiment (n=3). Red bars represent the mean. K-M. The percentages of cells with abnormal mitosis (chromosome misalignments and/or DNA bridges) (K), polylobed or multinucleated daughter cells (L) and mitotic cell death (M) were quantified. At least 50 cells per condition were analyzed. N. The nuclear size of at least 1000 control and UBAP2L-downregulated interphasic cells was measured. Red bars represent the mean.",
    "molecules": "DNA"
  },
  {
    "caption": "B. WB analysis of control or UBAP2L-silenced HeLa lysates from mitotic cells synchronized with 1µM paclitaxel and treated with 100µg/mL CHX for the indicated times. Proteins MW is indicated in kDa. WB is representative of three independent replicates.",
    "molecules": "CHX, paclitaxel"
  },
  {
    "caption": "C. WB analysis of WT or UBAP2L KO HeLa lysates from G1/S synchronized (DTB) cells treated with 25µM of the proteasomal inhibitor MG132 for the indicated times. Proteins MW is indicated in kDa. WB is representative of three independent replicates.",
    "molecules": "MG132"
  },
  {
    "caption": "D. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB, in S using 2mM hydroxyurea (HU) for 20h or in G2 using 10µM of CDK1 inhibitor RO 3306 for 20h.",
    "molecules": "HU, hydroxyurea, RO 3306"
  },
  {
    "caption": "IF representative pictures of WT or UBAP2L KO HeLa cells transfected with the indicated flag-tagged UBAP2L protein fragments for 48h and synchronized in mitosis using monastrol release (MR) (C) Scale bar, 5µm. At least 50 cells per condition were quantified for each experiment.",
    "molecules": "monastrol"
  },
  {
    "caption": "Representative IF images of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB and treated with vehicle (DMSO) or 25µM of MG132 for 4h (C)",
    "molecules": "DMSO, MG132"
  },
  {
    "caption": "Representative IF images of control or UBAP2L-downregulated cells synchronized in prometaphase using 1mM monastrol and released at the indicated time points.",
    "molecules": "monastrol"
  },
  {
    "caption": "E. Spinning disk time-lapse microscopy of PLK1-eGFP Knock-In (KI) HeLa cells synchronized with DTBR in mitosis. The selected frames of the movies are depicted and the corresponding time is indicated in minutes. SiR-DNA was used for DNA staining.",
    "molecules": "SiR-DNA, DNA"
  },
  {
    "caption": "D. WB analysis of control (siCTL) or siUBAP2L 48h-treated HeLa cells were synchronized with 1mM monastrol for 16h, treated with DMSO or with 10nM of the PLK1 inhibitor BI2536 for 45min, subsequently washed out from monastrol for the indicated time and collected for protein extraction. This moderate treatment is sufficient to restore the aberrant PLK1 catalytic activity observed in UBAP2L KO cells to the levels of the WT, enabling correct mitotic progression but prevents control cells to progress through mitosis. Proteins MW is indicated in kDa. WB is representative of three independent replicates.",
    "molecules": "BI2536, DMSO, monastrol"
  },
  {
    "caption": "DAPI staining of the experiment described in (D) showing different mitotic stages (E).",
    "molecules": "DAPI"
  },
  {
    "caption": "A) Average  survival curve of control (w1118) and chmRNAi male flies  upon ubiquitous knockdown of chm  with da-Gal4  (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); B) Average survival curve of control (w1118) and chmRNAi male flies  upon fatbody  knockdown of chm  with r4-Gal4  (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); C) Average survival curve  of control (w1118) and chmRNAi male flies  upon neuronal  knockdown of chm  with elav-Gal4  (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); D) Average survival curve of control (w1118) and chmRNAi male flies  upon muscle  knockdown of chm  with mef-Gal4  (N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); E, F Average survival curve  between E) ethanol  (N = 3, paired Log-rank test was performed for each biological replicate. p-values ranged between 0.2 to 0.05 across replicates) and F) RU486  (N = 4, paired, Log-rank test was performed for each biological replicate with all of them having a p-value < 0.05)) and administered control (w1118) and chmRNAi male flies  using Act-GS-Gal4  for adult-specific chm  knockdown; G) Average survival curve  of control (w1118) and chmMYST/+ male flies  (N = 5, paired Log-rank test was performed for each biological replicate with all of them having a p-value < 0.0001). Data information: All replicates are independent biological replicates and errors bars indicate standard error of the mean (SEM). For survival curves, log-rank test was performed for each biological replicate. The displayed p-value is based on all biological replicates.",
    "molecules": "ethanol, RU486"
  },
  {
    "caption": "(C) Confocal microscopy images of epidermal sheets from back skin of K5-R1/R2 and CTRL mice stained for IRF7 and K14, counterstained with DAPI. Arrows denote nuclear IRF7 (red) in basal and suprabasal keratinocytes of K5-R1/R2 mice. The strong red staining of the stratum corneum (sc) is unspecific background and is more pronounced in K5-R1/R2 mice due to the increased thickness of this layer.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) qRT-PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 (3.5 μM) or vehicle in the presence of serum.",
    "molecules": "BGJ398"
  },
  {
    "caption": "HaCaT keratinocytes were pre-treated for 2 h with the proteasome inhibitors MG132 (10 μm (G) followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.",
    "molecules": "MG132"
  },
  {
    "caption": "HaCaT keratinocytes were pre-treated for 2 h with epoxomicin (100 nM) (H), followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.",
    "molecules": "epoxomicin"
  },
  {
    "caption": "(A) Serum-starved HaCaT cells were treated for 24 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for IFNL1, IFNB, RSAD2, IRF1, IRF7, CGAS, TLR3 or DDX58, relative to RPLP0.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "(B) Serum-starved HaCaT cells were treated for 18 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for total and pSTAT1 (Y701 and S727), total and pSTAT2 (Y690), IRF1, IRF3, pIRF3 (S396), IRF9, RIG-1, RSAD2 and GAPDH.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 h post infection (hpi) by HSV-1 Glyc-D immunofluorescence (red) and DAPI counterstaining (blue) (B).",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Epidermal sheets from tail skin, infected ex vivo with HSV-1 (MOI = 2) +/- FGF7 (10 ng/ml), were stained 48 hpi for Glyc-D (red) using DAPI as counterstain (blue). The infected area (red staining) was measured using Image J software. Data are expressed as A.U. = arbitrary unit. Background staining of hairs was subtracted manually.",
    "molecules": "DAPI"
  },
  {
    "caption": "HaCaT cells were infected with ZIKV strains 976 (Uganda isolate) (MOI = 0.1) or PF13/251013-18 (French Polynesia isolate) (MOI = 20) and treated 2 hpi with FGF7 (20 ng/ml) or vehicle (CTRL). qRT-PCR analysis of RNA samples for ZIKV RNA (H) 48 hpi is shown.",
    "molecules": "RNA"
  },
  {
    "caption": "(A) Serum-starved HaCaT cells were infected with HSV-1 (MOI = 0.5) in the presence or absence of FGF7 (10 ng/ml) and the FGFR kinase inhibitors AZD4547 (1 μM) or BGJ398 (3.5 μM). DNA and protein lysates were analyzed by qPCR for Glyc-B relative to ACTB or by Western blot for Glyc-D and GAPDH, respectively, 16 hpi.",
    "molecules": "AZD4547, BGJ398"
  },
  {
    "caption": "HaCaT cells were cultured in DMEM/10% FCS (B) infected with HSV-1 (MOI=0.5) in the presence or absence of AZD45347 (1 μM) and/or BGJ398 (3.5 μM). Viral load was determined by qPCR for Glyc-D relative to ACTB (B)",
    "molecules": "BGJ398"
  },
  {
    "caption": "HaCaT cells were cultured in DMEM/5% FCS (C) and infected with HSV-1 (MOI=0.5) in the presence or absence of AZD45347 (1 μM) and/or BGJ398 (3.5 μM). Viral load was determined by measurement of viral titers (plaque forming units (PFU)) 14 hpi (C).",
    "molecules": "BGJ398"
  },
  {
    "caption": "(B) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, and then subjected to a pulse-labelling with IdU.(C) Analysis of replication fork velocity (fork speed) in the cells under unperturbed conditions. The length of the green tracks were measured. Mean values are represented as horizontal black lines (ns, not significant; Student's t-test).",
    "molecules": "CldU, IdU, DNA"
  },
  {
    "caption": "(E) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, treated with 4mM HU and then subjected to a pulse-labelling with IdU.(F) Graphs show the percentage of red (CldU) tracts (stalled forks) or red-green (CldU-IdU) contiguous tracts (restarting forks) in the cells. Mean shown, n = 3. Error bars represent standard error. (*, p < 0.05; **, p < 0.01; Student's t test). Representative DNA fiber images are shown. Scale bars, 10 µm.",
    "molecules": "CldU, IdU, DNA, HU"
  },
  {
    "caption": "(G) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were sequentially pulse-labelled with CldU and IdU as indicated, then treated or not with 4 mM HU.(H) Representative IdU tract length distributions in all cell lines under unperturbed conditions (left graph) or after HU treatment (right graph). Median tract lengths are given in parentheses. See also Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.",
    "molecules": "CldU, IdU, DNA, HU"
  },
  {
    "caption": "(A) Experimental scheme of dual labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were sequentially pulse-labelled with CldU and IdU as indicated, then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin.(B) Representative IdU tract length distributions in shWRNIP1WT (left graph) or shWRNIP1 cells (right graph) after treatment. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.",
    "molecules": "CldU, IdU, DNA, HU, Mirin"
  },
  {
    "caption": "(A) Evaluation of ssDNA accumulation at parental-strand by immunofluorescence microscopy analysis in wild-type (shWRNIP1WT) or WRNIP1-deficient (shWRNIP1) cells. Experimental design of ssDNA assay is shown. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU. In parallel samples, the MRE11 activity is chemically inhibited with 50 µM Mirin, alone or in combination with HU-induced replication stress. After treatment, cells were fixed and stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental ssDNA. Horizontal black lines represent the mean SE; n = 3. Error bars represent standard error (ns, not significant; **, p<0.01; ****, p<0.0001; two-tailed Student's t test). Representative images are shown. DNA was counterstained with DAPI (blue).",
    "molecules": "IdU, DNA, HU, Mirin, ssDNA"
  },
  {
    "caption": "(B) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells. Chromatin fractions of cells, treated or not with 4 mM HU, were analysed by immunoblotting. The membrane was probed with the anti-WRNIP1, anti-MRE11 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 and MRE11 (Input) in the cells was determined with the relevant antibodies. LAMIN B1 was used as a loading control. In the graph, the fold increase respect to the wild-type untreated of the normalized ratio of the chromatin-bound RAD51 (or MRE11)/ total RAD51 (or MRE11) is reported for each cell line.",
    "molecules": "HU"
  },
  {
    "caption": "(C) Analysis of DNA-protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay. Experimental designed used for the assay is given. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU for 4 h. Next, cells were fixed, stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental-strand ssDNA, and subjected to PLA assay as described in the \"Experimental procedures\" section. Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively. Each red spot represents a single interaction between ssDNA and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are given. Graph shows the number of PLA spots per cell. Horizontal black lines represent the mean value (ns, not significant; **, p < 0.01; two-tailed Student's t test); n = 3.",
    "molecules": "IdU, DNA, HU, ssDNA"
  },
  {
    "caption": "(D) Localization of WRNIP1, MRE11 and RAD51 to stalled replication forks. Forks were isolated by CldU-co-immunoprecipitation (CldU-IP). shWRNIP1WT or shWRNIP1 cells were pulse-labelled with CldU, then fixed or treated with HU. Cells were cross-linked, and the nuclear extracts were isolated (Input) and subjected to CldU-IP using an anti-CldU antibody (CldU-IP). The membranes were probed with the anti-WRNIP1 or anti-RAD51 antibodies. After stripping, the membranes were probed with an anti-MRE11 antibody. LAMIN B1 and GAPDH were used as loading controls (Input). Ponceau S was used as a loading control of CldU-IP. Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample. 10% of each IP was loaded on a nitrocellulose membrane. The membrane was probed with an anti-CldU antibody. The graph shows the normalized ratio of the proteins co-immunoprecipitated with CldU (CldUCo-IP proteins)/ the total of labelled DNAimmunoprecipitated with CldU (CldUIP) for each cell line after replication stress from two independent experiments. The dots in the graph represent the individual data points from each single experiment. Horizontal black line represents the mean value from two replicates; n = 2.",
    "molecules": "DNA, HU, CldU"
  },
  {
    "caption": "(A) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor, then treated or not with 4 mM HU.(B) Representative IdU tract length distributions in shWRNIP1WT cells (left graph) or shWRNIP1 cells (right graph). Median tract lengths are reported in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm.",
    "molecules": "IdU, DNA, HU"
  },
  {
    "caption": "(C) Scheme of DNA fiber tract analysis in shWRNIP1 cells. Cells were transfected with an empty vector or a plasmid expressing a wild-type human RAD51, and 48 h thereafter labelled with IdU and treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild-type RAD51 after HU exposure. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are given. Scale bars, 10 µm. Western blot shows the expression of the RAD51 protein in shWRNIP1 cells. The membrane was probed with an anti-RAD51. LAMIN B1 was used as a loading control.",
    "molecules": "IdU, DNA, HU"
  },
  {
    "caption": "(A) Co-immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG-WRNIP1 plasmid. Cells were treated or not with HU. After treatment, cell lysates were immunoprecipitated (FLAG-IP) using anti-FLAG antibody. The presence of WRNIP1, BRCA2 and RAD51 was assessed by immunoblotting using the anti-FLAG, anti-RAD51 and anti-BRCA2 antibodies, respectively. Whole cell extracts were analysed (Input). The membrane was probed with the same antibodies used for IP. GAPDH was used as a loading control.",
    "molecules": "HU"
  },
  {
    "caption": "(B) Analysis of protein-protein interactions between WRNIP1 and endogenous RAD51 in wild-type (shWRNIP1WT) or WRNIP1-mutant (shWRNIP1T294A) cells by in situ PLA assay. Cells were labelled with IdU for 24 h, washed and left to recover for 2 h, then treated or not with 4 mM HU. Antibodies raised against FLAG-Tag and RAD51 were used to reveal FLAG-WRNIP1 or endogenous RAD51 respectively. Each red spot represents a single interaction between WRNIP1 and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are shown. Graph shows the mean number of PLA spots per cell SE. Error bars represent standard error (ns, not significant; two-tailed Student's t test); n = 3.",
    "molecules": "IdU, DNA, HU"
  },
  {
    "caption": "(C) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were transfected with BRCA2 siRNA (siBRCA2), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1WT/siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells. The membrane was probed with an anti-BRCA2 or anti-WRNIP1. GAPDH was used as a loading control.",
    "molecules": "IdU, DNA, HU"
  },
  {
    "caption": "(E) Experimental scheme of pulse-labelling of DNA fibers in shWRNIP1 cells. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(F) Representative IdU tract length distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 cells with or without HU treatment. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test.",
    "molecules": "IdU, DNA, HU"
  },
  {
    "caption": "(G) Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h treated or not with HU for 4h. Chromatin fractions of cells were analysed by immunoblotting. The membrane was probed with the anti-FBH1 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 (Input) in the cells was determined with the relevant antibodies. GAPDH was used as a loading control. The ratio of the RAD51/LAMIN B1 signal (chromatin) is reported below each lane.",
    "molecules": "HU"
  },
  {
    "caption": "(A) Analysis of DNA damage accumulation. Wild-type (shWRNIP1WT), WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells were treated or not with 4 mM HU for 4 h, then subjected to -H2AXimmunofluorescence microscopy. Graph shows data presented as mean of -H2AX-positive cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of nuclei showing the different number of foci per nucleus are reported.",
    "molecules": "HU"
  },
  {
    "caption": "(B) Analysis of DNA breakage accumulation. shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells were treated as in (A), then subjected to alkaline Comet assay. Graph shows data presented as mean tail moment SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images are shown.",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Evaluation of cell death. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU for 16 h. Cell viability was evaluated by LIVE/DEAD fluorescent assay. Data are expressed as mean of dead cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of double-staining of viable (green) and dead (red) cells are shown.",
    "molecules": "HU"
  },
  {
    "caption": "(D) Experimental scheme for evaluation of the chromosomal aberrations is shown. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU, then left to recover for 16h in drug-free medium and metaphases collected with colcemid. Next, cells were fixed and processed as reported in \"Supplemental information\" section. Dot plot shows the number of chromosomal aberrations per cell. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of cells treated or not with 4 mM HU. Arrows indicate chromosomal aberrations.",
    "molecules": "colcemid, HU"
  },
  {
    "caption": "(E) Experimental scheme of the Mirin aberration analysis is given. The experiment was carried out as in (D) but cells were pre-treated or not with 50 µM Mirin. Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU. Arrows indicate chromosomal aberrations.",
    "molecules": "HU, Mirin"
  },
  {
    "caption": "(F) Experimental design of the chromosomal aberration assay is reported. shWRNIP1 cells were transfected with control siRNAs (siCtrl) or FBH1 siRNA (siFBH1). Fourth-eight hours thereafter, cells were treated or not with 4 mM HU and then left to recover for 16 h. Metaphases were collected with colcemid and prepared as reported in \"Supplemental information\" section. Dot plot shows the number of chromosomal aberrations per cell. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Horizontal black lines represent the mean SE. Error bars represent standard error. (**, p < 0.01; two-tailed Student's t test).",
    "molecules": "colcemid, HU"
  },
  {
    "caption": "A) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at B) pH 7.4 or C) pH 5.5, DIC micrographs of 50 μM MBP-N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.",
    "molecules": "MES, NaCl, Tris"
  },
  {
    "caption": "Phase separation over time as monitored by turbidity of 50 μM MBP-N in 50 mM Tris pH 7.4 after addition of TEV protease A) with varying torula yeast RNA (at 100 mM NaCl) or B) varying NaCl concentrations (at constant RNA concentration). Error bars represent standard deviation of three replicates.",
    "molecules": "NaCl, Tris"
  },
  {
    "caption": "A) DIC micrographs of 50 μM N and domain deletion variants in 50 mM Tris 70 mM NaCl pH 7.4 without and with TEV protease (to cleave MBP from N) and without or with 0.5 mg/mL desalted total torula yeast RNA. Scale bars represent 80 μm. Phase separation over time as monitored by turbidity of 50 μM full-length N or deletion variants after addition of TEV protease in the absence (B) or presence (C) of 0.5 mg/ml desalted total torula yeast RNA. Error bars represent standard deviation of three replicates.",
    "molecules": "NaCl, Tris"
  },
  {
    "caption": "A) Turbidity of N is increased in the presence of torula yeast RNA and homopolymeric RNAs. Error bars represent standard deviation of three replicates. B) DIC micrographs of MBP-N in the absence or presence of TEV (to cleave MBP from N to initiate with indicated RNA. In the absence of TEV, MBP-N in the presence of torula yeast RNA, polyC, or polyU RNA does not undergo LLPS. However, after cleavage of MBP, N phase separates. polyA RNA increases LLPS of N. Both in the absence and presence of TEV, polyG RNA induces aggregation of MBP-N. Sample conditions:50 µM MBP-N, 0.5 mg/mL RNA/polyX, 70 mM NaCl, 25˚C, 50 mM Tris pH 7.4. Scale bar represents 100 µm.",
    "molecules": "polyG RNA, polyA RNA, polyC, polyU RNA, NaCl, Tris"
  },
  {
    "caption": "Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules, in membranes of gallbladder epithelium, epididymis epithelium, testicular Sertoli cells and Leydig cells, a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes, with HPA000288 also staining the cardiac muscle fibers, while MAB933 only showed staining in a few cells. Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye, interlobular pancreatic ducts, as well as in placental villi, both in cytotrophoblasts, syncytiotrophoblasts, and also in extravillous trophoblasts, while placenta decidua was negative. ACE2 staining could be observed at the base of ciliated fallopian tube epithelium, however only for one of the antibodies. Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer. ACE2 also stained positive in endothelial cells and pericytes in several tissues, see Fallopian, thyroid, parathyroid, adrenal gland, pancreas and heart. Scale bar = 50µm. Scale bar in dashed squares = 10 µm. (Brunner = Brunner's gland, EVT = extravillous trophoblasts, endo=endothelial cells).",
    "molecules": "hematoxylin"
  },
  {
    "caption": "Representative images of human respiratory tissues, all stained on consecutive sections, with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Respiratory tissues were composed of different structures in nasal mucosa, bronchus, smaller bronchioles and lung tissue. ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium (arrowheads). Rare ACE2 staining was present in a few alveolar cells (arrows). No staining was observed in nasal squamous epithelium, bronchioles, or submucosal glands in either tissue. Gender and age are shown for all individuals. Red and green colored squares mark the positions in the TMA cores shown as magnifications. Scale bar = 50µm. Scale bar for images in dashed squares = 10 µm. (re=respiratory; sq=squamous).",
    "molecules": "hematoxylin"
  },
  {
    "caption": "Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic monogenic (db/db; BKS.Cg-m+/+ Lepr DB/J; N = 4/group, A-E), New Zealand Black (NZB) and polygenic obese/prediabetic New Zealand Obese (NZO; n=4/group; F-J), as well as young (i.e. 3 mo) and aged (i.e.22 mo; n = 5/group; K-O), mice were fed ad libitum (fed) or fasted for 24h (fasted). Serum non-esterified fatty acids (A, F, K), triglycerides (B, G, L) and ketone bodies (C, H, M) were measured. In addition, serumacylcarinitine profiling was conducted and medium-chain (D, I, N) and long-chain (E, J, O) acylcarnitine concentrations are shown.",
    "molecules": "acylcarinitine, acylcarnitine, non-esterified fatty acids, ketone bodies, triglycerides"
  },
  {
    "caption": "In another cohort of mice, ex vivo long-chain fatty acid (LCFA) metabolism, including uptake (P), oxidation (Q) and non-oxidative LCFA disposal (NOFAD; R), in precision cut liver slices from fed and fasted WT and db/db mice (n = 3/ group; 4 slices per mouse), were determined. In addition, in slices from fasted mice, glucose output was determined in the presence of incubation with NEFA (BSA-NEFA) or vehicle (BSA) (S).",
    "molecules": "NEFA, glucose, LCFA, long-chain fatty acid"
  },
  {
    "caption": "Male, GADD45β+/+ (WT; n=6) or GADD45β-/- mice (KO; n=4) were fed ad libitum (fed) or fasted for 24h (fasted), and subsequently refed for 24h. O2 consumption rate (A), CO2 production rate (B) and respiratory exchange ratio (C) were measured by indirect calorimetry.",
    "molecules": "O2, CO2"
  },
  {
    "caption": "In a distinct cohort, male GADD45β+/+ (WT; n=5) or GADD45β-/- mice (KO; n= 8) were fed ad libitum (fed) or fasted for 24h (fasted). Serumnon-esterified fatty acids (D) and ketone bodies (E) as well as serum (F) and liver (G) triglycerides (TG) were measured.",
    "molecules": "non-esterified fatty acids, ketone bodies, TG, triglycerides"
  },
  {
    "caption": "Serum TG (H), NEFA (I), and blood glucose (BG, J) concentrations during an oral lipid tolerance test in overnight fasted GADD45β+/+ (WT; n = 5) or GADD45β-/- (KO; n=5) mice.",
    "molecules": "NEFA, glucose, TG"
  },
  {
    "caption": "Bloodglucose excursion during and intraperitoneal insulin tolerance test (K) as well as fasting bloodglucose (L), seruminsulin (M), and HOMA-IR (N) in GADD45β+/+ (WT; n = 6) or GADD45β-/- mice (KO, n = 9) chronically fed a normal- (NFD) or high (HFD) fat diet. Data are mean ± SEM.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "Male, GADD45β+/+ (WT) or GADD45β-/- mice (KO) were fed ad libitum (fed) or fasted for 24h (fasted) and ex vivo liver slice long-chain fatty acid (LCFA) metabolism was measured including LCFA uptake (A), oxidation (B) and non-oxidative LCFA disposal (NOFAD) was calculated (C).",
    "molecules": "LCFA, long-chain fatty acid"
  },
  {
    "caption": "In addition, glucose production was measured (D) in the presence (BSA-NEFA) or absence (BSA) of extracellular fatty acids (n=4/group with 4 liver slices per mouse liver).",
    "molecules": "fatty acids, NEFA, glucose"
  },
  {
    "caption": "Male, C57Bl/6J mice with (AAV-G45b miR) or without (AAV-NC miR) liver-hepatocyte-restricted GADD45β silencing were fed or fasted for 24h (n = 6/group) and serum levels of non-esterified fatty acids (NEFA; F) as well as liver triglyceride (TG) concentration (G) were measured.",
    "molecules": "NEFA, non-esterified fatty acids, TG, triglyceride"
  },
  {
    "caption": "Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). LiverTG concentration (H) was measured.",
    "molecules": "TG"
  },
  {
    "caption": "Male, C57Bl/6J mice with (AAV-G45b miR; n= 15) or without (AAV-NC miR, n = 13) liver-hepatocyte-restricted GADD45β silencing were chronically fed a normal (NFD) or high (HFD) fat diet (n = 6-8/group). Fasting blood glucose (I) and serum insulin (J) were measured from which HOMA-IR was calculated (K).",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) prior liver-restricted GADD45β over-expression were fasted and blood glucose ( A) and serum insulin (B) were measured from which HOMA-IR was calculated (C) (n = 4-6/group). Data are mean ± SEM. Effect of genotype/viral manipulation, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "Scatter plots fasting plasma triglycerides (E) and glucose (F) in correlation with liver GADD45B mRNA expression (n = 37). Inserts show r2 values and p values from Spearman's correlation test. The statistical test used and respective p-value outputs can be found in Appendix Table S1.",
    "molecules": "glucose, triglycerides"
  },
  {
    "caption": "H: Liver long chain acyl-CoA (LC-acyl-CoA) concentrations were determined in GADD45β+/+ (WT) or GADD45β-/- (KO) mice (H; n=6/ group) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (I; n =5/group).",
    "molecules": "acyl-CoA"
  },
  {
    "caption": "Liver LC-acyl-CoA concentrations were determined in wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (J; n=4/group).",
    "molecules": "acyl-CoA"
  },
  {
    "caption": "Growth analysis of wild type Xcc8004, EPS deficient mutants (B5 and B12), ndvB and ndvB (pHMJ) grown in PS medium (A); PS medium containing 50 µm extracellularion chelator, diethylenetriaminepentaacetic acid (DETAPAC) (B); or supplemented with either with (C) secreted glucan (0.5 mg/ml); (D) xanthan (0.5 mg/ml); (E) FeSO4 (40µM); and (F) MnCl2 (40µM). Data shown are mean ± SD (n=3). From here onward, until not mentioned, the 'n' indicates three biological replicates.",
    "molecules": "xanthan, glucan, FeSO4, MnCl2, DETAPAC, diethylenetriaminepentaacetic acid"
  },
  {
    "caption": "(A) MALDI-MS analysis on the 2.1 RT eluted fraction from the wild type Xcc8004 EPS. Shown one quasimolecular ion at m/z (mass-to-charge ratio) 2632.8 which matched with the calculated mass for an [M+Na] + ion based on an unsubstituted 16-member cyclic glucan (inset) of Xanthomonas campestris. Peaks with m/z of below 1100 were matrix derived peaks after comparing the MALDI-MS of matrix only control (See also Fig. EV3A-B; Fig. EV4D).",
    "molecules": "EPS"
  },
  {
    "caption": "(B) Top: GC-MS analysis of acid hydrolyzed and BSTFA-derivatized, 2.1 RT eluted fraction from the EPS of wild type Xcc strain. Bottom: matching spectrum from NIST database, corresponding to glucopyranose pentakis-O-trimethyl silyl (See also Fig. EV3C).",
    "molecules": "glucopyranose pentakis-O-trimethyl silyl, EPS"
  },
  {
    "caption": "(C) Quantification of total EPS, secreted (S) and periplasmic (P) glucan by phenol-sulfuric acid colorimetric assay from 2-day-old cultures. Secreted xanthan and glucan was isolated from the cell-free culture supernatant by addition of KCl [1% (w/v)] and 2 volumes of ethanol, to precipitate xanthan. Supernatant containing cyclic-β-(1, 2) was purified by BioGel-P4 size-exclusion chromatography. To isolate periplasmic glucan, cell pellet was treated with 1% trichloroacetic acid (TCA) to release periplasmic glucans, purified by BioGel-P4 size-exclusion chromatography. Data shown are mean ± SD (n=3).",
    "molecules": "xanthan, glucan, EPS"
  },
  {
    "caption": "(A) Iron (Fe2+)-binding potential of different polysaccharide [used at 5.56 micromole (glucose equivalent)] using the Fe2+-ferrozine colorimetric assay. Lipopolysacchrides (LPS; E. coli 0111:B4), mannan (Saccharomyces cerevisiae), isolated and commercial (purchased from Sigma Aldrich) xanthan, extracellular glucan from Xcc. Data shown are mean ± SD (n=3).",
    "molecules": "Fe2+, Iron, Lipopolysacchrides, LPS, xanthan, ferrozine, glucan, mannan"
  },
  {
    "caption": "(B) Colorimetric iron-binding assay. Glucan from diverse source binds Fe2+ iron. Extracellular (ECG) or periplasmic (PPG) glucan isolated from the wild type Xcc8004 or the ndvB (pHM1J); commercial glucan from yeast, barley and Euglena purchased from Sigma Aldrich. Glucans from various sources were incubated with FeSO4 at room temperature for 15 min, amount of free iron was determined using the standard concentration of ferrozine-Fe2+ complex.",
    "molecules": "Fe2+, iron, ferrozine, Glucan, glucan, Glucans, FeSO4"
  },
  {
    "caption": "(C) Iron-binding measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Xanthan and various glucans were mixed with 2 mM FeSO4, dialysed against deionized water before determination of Fe2+ content by ICP-OES. Data shown are mean ± SD (n≥3). ** indicates p-value < 0.01 and *** indicates p-value < 0.001 by paired student t-test.",
    "molecules": "Fe2+, Xanthan, glucans, FeSO4"
  },
  {
    "caption": "(D) Specificity of glucan-Fe2+ interaction. Purified Xcc β-(1, 2)-glucan was incubated with 100 µM FeSO4 and increasing concentrations of Ca2+, Co2+, Ni2+, Mn2+, Mg2+ or Zn2+, and free or unbound iron was measured by Fe2+-ferrozine colorimetric assay. Data shown are mean ± SD (n=3).",
    "molecules": "Ca2+, Fe2+, iron, Mg2+, Ni2+, Zn2+, Co2+, ferrozine, glucan, Mn2+"
  },
  {
    "caption": "(A) Analysis of total iron levels measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Cells were grown under either rich-media (PS), iron-restricted (PS + 150 μM DP) or glucan supplemented iron-restricted media (PS + 150 μM DP + 0.5 mg/ml glucan) till the late exponential growth phase. Bacterial cells were harvested, washed and then iron content was determined using ICP-OES. Data shown are mean ± SD (n=3). ** indicates p value < 0.01 determined by paired student t-test (two tailed with equal variance).",
    "molecules": "iron, glucan"
  },
  {
    "caption": "(B) Co-elution of periplasmic glucan and radiolabellediron-glucan complex. Periplasmic glucan was isolated from the 55Fe2+-loaded cells, and analyzed by BioGel-P4 size-exclusion chromatography. Fractions (1.5 ml) were collected at a flow rate of 20 ml/h and assayed for glucan using the phenol-sulphuric acid colorimetric assay, and radioactivity was measured to detect the presence of 55Fe.",
    "molecules": "iron, glucan, 55Fe, 55Fe2+"
  },
  {
    "caption": "(C) Iron localization assay using atomic absorption spectroscopy. Two sets of bacterial cells were grown in iron-restricted media (PS + 150 μM DP) till late exponential phase. Cold iron (50 μM FeSO4) was added to the one set of bacterial cultures and incubated for one hour at 28°C. Bacterial cells were harvested, washed from the both sets of bacterial cultures. Periplasmic and cytoplasmic fractions were collected, freeze dried, and then iron content in each fraction was determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Iron content is represented on Y-axis as microgram of iron present per milligram of proteins in respective fractions. Data shown are mean ± S.E. (n = 3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 by paired student t-test.",
    "molecules": "Iron, iron, FeSO4"
  },
  {
    "caption": "Xanthomonas campestris: (A) wild type Xcc 8004; (B) B5 (EPS deficient) (C) B12EPS deficient); (D) ndvB (glucan deficient); and (E) ndvB(pHM1J)+ (complementing strain); cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+Dp (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.",
    "molecules": "DCF, dipyridyl, Dp, glucan, H2O2, FeSO4, EPS, ROS"
  },
  {
    "caption": "(A) Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± S.E. (n = 3).",
    "molecules": "DCF, H2O2, FeSO4, ROS"
  },
  {
    "caption": "(B) The percent of ROS positive Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.",
    "molecules": "Dp, H2O2, FeSO4, ROS"
  },
  {
    "caption": "(C) Iron sensitivity assay. Cells were grown overnight in PS medium and cultures were diluted in fresh media at an density of 1 x 106 cells/ml, treated in the absence or presence of 500 µM FeSO4. Viability of cultures was measured at the indicated time point by dilution plating. Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to FeSO4. Data shown are mean ± SE (n=3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 compared with data obtained from iron treated wild type and mutant by paired student t-test.",
    "molecules": "iron, FeSO4"
  },
  {
    "caption": "(A) Wild type E. coli (MG1655); ΔopgG (ΔmdoG; JW1035) and ΔopgH (ΔmdoH; JW1037), mutants blocked in periplasmic glucan synthesis were grown in LOS medium, and cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+DP (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.",
    "molecules": "DCF, dipyridyl, DP, glucan, H2O2, FeSO4, ROS"
  },
  {
    "caption": "(B) The percent of ROS positive E. coli cells treated with FeSO4, H2O2 and FeSO4+DP determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.",
    "molecules": "iron, DP, H2O2, FeSO4, ROS"
  },
  {
    "caption": "(C) E. coli cells treated with FeSO4, H2O2 and FeSO4+ Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.",
    "molecules": "iron, DCF, H2O2, FeSO4, ROS"
  },
  {
    "caption": "(C)  Min6  cells were treated with glucose  and palmitate  at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies. (D) Human islets  were treated with glucose and  palmitate  at the indicated dosages, followed by  immunoblotting  analysis. ( Data information: Data from 3 experiments are presented as the mean ± SD.",
    "molecules": "glucose, palmitate"
  },
  {
    "caption": "(B) Pancreata  of 18-week-old WT and Pdia4-/- (KO) mice  on BKS or Leprdb/db background were stained  with anti-insulin   (αIns) antibody and dihydroethidium (DHE) (left). Islet  area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate   islet  regions. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "DHE, dihydroethidium"
  },
  {
    "caption": "(D) The same batch mice  as in (B) were given water containing BrdU. The BrdU+ cells of the  islets  were visualized and quantified (BrdU+ labeling). TUNEL-positive cells in the  islets  of the mice (B) were visualized and quantified (TUNEL assay). BrdU+ cells per  islet area (0.01 mm2) and TUNEL+ cells per islet  area (0.05 mm2) are expressed in arbitrary units (AU). The dash circles indicate  islet regions and the black arrowheads indicate TUNEL+ cells. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "BrdU"
  },
  {
    "caption": "F) insulin  in supernatants of the mouse islets (F) in GSIS assays. High glucose (HG, 16.7 mM) or low  glucose (LG, 3.3) were used in GSIS assays. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "glucose"
  },
  {
    "caption": "(B) Pancreata of the same batch of mice as in (A), at the age of 14 weeks, were stained with anti-insulin (αIns) antibody and DHE (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar = 100 μm. The dash circles indicate  islet regions. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "DHE"
  },
  {
    "caption": "(D) The  islets  of the  mice  from (C) were tested for GSIS. High  glucose (HG, 16.7 mM) or low glucose (LG, 3.3 mM) were used in  GSIS assays. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "glucose"
  },
  {
    "caption": "(A) The islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice were isolated and grown in complete DMEM medium containing 3.3 mM (LG) and 30 mM glucose (HG) for 12 h. The  islets stained  with propidium iodide  (PI), photographed, and quantified. Scale bar = 50 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "glucose, PI, propidium iodide"
  },
  {
    "caption": "The islets from the mice (A) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). Mitochondrial ROS  in the  islets  were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "MitoGreen, MitoSOX, glucose, ROS"
  },
  {
    "caption": "The islets from the mice (A) were incubated with Hoechst 33342 (Ho) plus CM-H2DCFDA in the presence of glucose  at 3.3 mM (LG) and 16.7 mM (HG).  cytosolic ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "CM-H2DCFDA, Ho, Hoechst 33342, glucose, ROS"
  },
  {
    "caption": "(A)  Min6 cells infected with a lentivirus expressing a scramble RNAi (GK), a Pdia4 RNAi (KD) and a  Pdia4 cDNA (OVE) were sorted and tested for the Pdia4 protein level  (left). The cells were incubated with NAC (1 mM) and then  stained  with MitoSOX  or CellROX in response to 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. Signal from MitoSOX (middle) and CellROX  (right) was re-plotted into histograms. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "CellROX, MitoSOX, glucose, NAC"
  },
  {
    "caption": "(B) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK) and an RNAi of Ndufs3  (Ndufs3 KD) or p22phox (p22phox KD) were selected and tested for levels of Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX),  p22phox  (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG)  glucose. (C) The same experiments as (B) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 (tNdufs3) and p22phox (tp22phox). ( Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "CellROX, MitoSOX, glucose, ROS"
  },
  {
    "caption": "(D) 293T cells, which were transfected with the construct encoding Flag-Pdia4  and that expressing  Myc/Flag-tagged p22phox or Ndufs3, were treated with GHTT  (28 μM) for 30 min. The cells were lysed and incubated with anti-Pdia4 antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting  analysis with anti-Flag antibody.",
    "molecules": "GHTT"
  },
  {
    "caption": "(F) The sections of pancreata of the  mice (E) were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet  area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.",
    "molecules": "DHE, dihydroethidium"
  },
  {
    "caption": "A Localisation of transiently expressed GFP-Rab35 in IMCD3 cells after 48h serum starvation and staining for acetylated tubulin (acetyl. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "B Localisation of transiently expressed GFP-RAB35 in hTERT-RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin (polyglu. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "C, D hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 were treated and stained for GFP, acetylated tubulin (acetyl. tub.) and DNA. Representative images in (C) of cells treated with Neg or RAB35 siRNA. Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. Cilia length quantifications in (D) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0249, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0056)",
    "molecules": "DNA"
  },
  {
    "caption": "G-H Immunofluorescence of IMCD3 representative images treated cells are stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (H) is shown as box-and-whisker plots and is the result of 3 independent experiments (n ≥ 50 cilia per experimental condition). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to unpaired t-test with Mann-Whitney test (*** P = 0.0002)",
    "molecules": "DNA"
  },
  {
    "caption": "NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Quantification of ciliation in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 100 cilia per experimental condition).",
    "molecules": "DNA"
  },
  {
    "caption": "NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images in (K). Regions within white boxes shown at higher magnifications at the bottom. Scale bars, 10 µm. Cilia length quantifications in (L) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 100 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (**** P < 0.0001).",
    "molecules": "DNA"
  },
  {
    "caption": "A Representative images of hTERT-RPE1 cells transiently expressing wild type (WT), GDP-bound (S22N) or GTP-bound (Q67L) GFP-tagged RAB35. Cells were serum-starved for 24 h and stained for polyglutamylated tubulin (polyglu. tub.) and DNA. Higher magnification images of the cilia region shown in smaller panels. Scale bars; 10 µm.",
    "molecules": "DNA, GDP, GTP"
  },
  {
    "caption": "Analysis of cilia length in hTERT-RPE1 cells depleted of GEF DENND1B) and GAP (TBC1D10A regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (E). Regions within white boxes shown at higher magnifications to the right. Scale bars; 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "Analysis of cilia length in hTERT-RPE1 cells depleted of GEF (DENND1A, DENND1B) and GAP (TBC1D10A, TBC1D10B) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (F) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001; P-values: Neg vs. DENND1B P = 0.0033, Neg vs. TBC1D10A P = 0.0008)",
    "molecules": "DNA"
  },
  {
    "caption": "A, B hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 transfected with non-targeting siRNA control (Neg) or Rab35 siRNA, were serum-starved for 48 h and stained for ARL13B, GFP, polyglutamylated tubulin (polyglu. tub.) and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars; 10 µm. (B) Box-and-whisker plots show quantification of ciliary ARL13B intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P = 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0014, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0081).",
    "molecules": "DNA"
  },
  {
    "caption": "D,E NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines were serum-starved for 24h and stained for Arl13b, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (D). Higher magnifications of regions within white boxes shown at the bottom. Scale bars; 10 µm. (E) Box-and-whisker plots show quantification of ciliary Arl13b intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05; P-values: NIH3T3 WT vs. Rab35 KO#1 P = 0.0118, NIH3T3 WT vs. Rab35 KO#2 P = 0.0111).",
    "molecules": "DNA"
  },
  {
    "caption": "Quantification of ciliary ARL13B intensity in hTERT-RPE1 cells depleted of GEF (G) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for ARL13B, acetylated tubulin and DNA. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 30 cilia per experimental condition). Statistical significance according ANOVA followed by Bonferroni post-hoc test (*** P < 0.001; P-values: (G) P = 0.0005",
    "molecules": "DNA"
  },
  {
    "caption": "Quantification of ciliary ARL13B intensity in hTERT-RPE1 cells depleted of GAP (H) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for ARL13B, acetylated tubulin and DNA. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 30 cilia per experimental condition). Statistical significance according ANOVA followed by Bonferroni post-hoc test (*** P < 0.001; P-values: (H) P < 0.0001).",
    "molecules": "DNA"
  },
  {
    "caption": "I, J Ciliary ARL13B intensity quantification of hTERT-RPE1 cells transiently overexpressing GFP or GFP-RAB35. Cells were serum-starved for 24 h and stained for ARL13B, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (I). Higher magnification images of the cilia region are shown at the bottom. Scale bars; 5 µm. Cells with (RAB35+ cilia) and without (RAB35- cilia) GFP-RAB35 ciliary localisation are compared in (J). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (*** P =0.0004).",
    "molecules": "DNA"
  },
  {
    "caption": "D HEK293T cells were transiently transfected with ARL13B-FLAG and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with GFP-RAB35 bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0333).",
    "molecules": "GDP, GTPγS"
  },
  {
    "caption": "E HEK293T cells were transiently transfected with mCherry-RAB35 and serum-starved for the final 8 h before harvesting. HEK293T cell lysates expressing ARL13B-FLAG were subjected to pull-down with ARL13B-GFP bound to anti-GFP beads and preloaded with either no nucleotide, GTPγS or GDP. Bound proteins and cell lysates (1% of input) were analysed by immunoblotting. The graph shows the ratio of precipitated ARL13B and RAB35, normalized to the no nucleotide control. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according ANOVA followed by Tukey post-hoc test (* P =0.0466)",
    "molecules": "GDP, GTPγS"
  },
  {
    "caption": "A, B hTERT-RPE1 cells transfected with non-targeting siRNA control (Neg) or RAB35 siRNA, were serum-starved for 48 h and stained for INPP5E, ARL13B, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. (B) Box-and-whisker plots show quantification of ciliary INPP5E intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (*** P < 0.001; P-values: Neg vs. RAB35-1 P < 0.0001, Neg vs. RAB35-2 P = 0.0002).",
    "molecules": "DNA"
  },
  {
    "caption": "D, E NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines were serum-starved for 24h and stained for Innpp5e, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (D). Higher magnifications of regions within white boxes shown at the bottom. Scale bars; 10 µm. (E) Box-and-whisker plots show quantification of ciliary Inpp5e intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, *** P < 0.001; NIH3T3 WT vs. Rab35 KO#1 P < 0.0001, NIH3T3 WT vs. Rab35 KO#2 P = 0.0345).",
    "molecules": "DNA"
  },
  {
    "caption": "F, G Localisation of the PI(4,5)P2 sensor PH-PLCδ1-GFP to cilia in live NIH3T3 WT and Rab35 KO cells. Cilia are marked with HTR6-RFP. Representative images are shown in (F). Higher magnification images of the cilia region shown to the right. Scale bars; 10 µm. Quantification of localisation in (G). Data are mean ± S.E.M. of 3 independent experiments (n > 100 cilia per experimental condition). Statistical significance according to two-way ANOVA (* P = 0.0242).",
    "molecules": "PI(4,5)P2"
  },
  {
    "caption": "hTERT-RPE1 cells transfected with indicated siRNAs were serum-starved for 48 h and treated with SMO agonist (SAG) or vehicle control (DMSO) for the last 24 h. Cells were stained for SMO, acetylated tubulin and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. Graph in (B) shows the percentages of SMO-positive (SMO+) cilia. Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (*** P < 0.001; P-values: Neg+SAG vs. RAB35-1+SAG P < 0.0001, Neg+SAG vs. RAB35-2+SAG P = 0.0033)",
    "molecules": "SAG, DNA, DMSO"
  },
  {
    "caption": "hTERT-RPE1 cells transfected with indicated siRNAs were serum-starved for 48 h and treated with SMO agonist (SAG) or vehicle control (DMSO) for the last 24 h. (C) Box-and-whisker plots show quantification of the average SMO intensity in the ciliary area marked by acetylated tubulin staining. All cilia: cilia identified with acetylated tubulin staining. SMO+ cilia: cilia with discernible SMO localisation. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 30 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg+SAG vs. RAB35-1+SAG (all cilia) P < 0.0001, Neg+SAG vs. RAB35-2+SAG (all cilia) P = 0.0024, Neg+SAG vs. RAB35-1+SAG (SMO+ cilia) P < 0.0007, Neg+SAG vs. RAB35-2+SAG (SMO+ cilia) P = 0.0205)",
    "molecules": "SAG, DMSO"
  },
  {
    "caption": "Ciliary SMO intensity quantification in hTERT-RPE1 cells transiently expressing GFP, wild type ARL13B-GFP (WT) or the ciliary-targeting defective mutant ARL13B-V358A. Cells were serum-starved and treated with SAG for 24 h and stained for SMO, acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (G).",
    "molecules": "SAG, DNA"
  },
  {
    "caption": "Ciliary SMO intensity quantification in hTERT-RPE1 cells transiently expressing GFP, wild type ARL13B-GFP (WT) or the ciliary-targeting defective mutant ARL13B-V358A. Cells were serum-starved and treated with SAG for 24 h and stained for SMO Box-and-whisker plots in (H) show quantification of ciliary SMO intensity. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P = 0.0046).",
    "molecules": "SAG"
  },
  {
    "caption": "D. Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout (KO) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced. Cells were transfected to overexpress a control GFP plasmid, a plasmid encoding human wild type Cwh43 with GFP fused to the N-terminus, or a plasmid encoding human CWH43 harboring the iNPH-associated mutation (Lys696AsnfsTer23) with GFP fused to the N-terminus. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.",
    "molecules": "Triton X-114, GPI, Leu"
  },
  {
    "caption": "Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type, CWH43M533/M533 and CWH43WT/M533mouse brain or kidney. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.",
    "molecules": "Triton X-114, GPI"
  },
  {
    "caption": "Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. Arrowheads point to apical surfaces of ependymal and choroid plexus cells. Nuclei are counterstained using DAPI (blue). Scale bar is approximately 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.",
    "molecules": "N"
  },
  {
    "caption": "C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fraction",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.",
    "molecules": "sucrose"
  },
  {
    "caption": "Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.",
    "molecules": "formaldehyde, N, PMSF"
  },
  {
    "caption": "Apg9p does not comigrate with prAPI in a strain that accumulates Cvt/autophagic vesicles. Strain TK415 (ypt7Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, centrifuged at 100,000 g for 1 h, and the pellet fraction resuspended and centrifuged a second time. The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 16 h at 174,000 g as described in Materials and Methods. 16 fractions were collected starting from the top and subjected to SDS-PAGE and immunoblot with antisera against Apg9p and API. The graph represents an average of two experiments.",
    "molecules": "sucrose"
  },
  {
    "caption": "Ultrastructural localization of Apg9p for immunoelectron microscopy. Strain STY1 (pep4Δ) expressing 3×HA Apg9p was grown in YPD to log phase and transferred to SD(−N) medium for 2 h. The cells were fixed and stained with anti-HA antibody and 5-nm colloidal gold-conjugated goat anti-mouse IgG as described in Materials and Methods. A, Section showing autophagic bodies in the vacuole lumen. B, Section showing a cytosolic autophagosome. Apg9p is seen in patches near the vacuole, but not on autophagic bodies or autophagosomes. AB, autophagic body; AP, autophagosome; N, nucleus; V, vacuole; VM, vacuolar membrane. Arrows point to areas of concentrated Apg9p.",
    "molecules": "gold, N"
  },
  {
    "caption": "Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.",
    "molecules": "N, PMSF"
  },
  {
    "caption": "Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step. A, The nature of prAPI binding and pelleting in the apg9ts strain. Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38°C. The spheroplasts were osmotically lysed in a buffer containing no salt (20 mM Pipes, pH 6.8) or a physiological concentration of salt (100 mM KOAc, 50 mM KCl, 5 mM MgCl2, 20 mM Pipes, pH 6.8) and pelleted at 5,000 g. All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK. apg9ts retains the salt dependent binding and pelleting of prAPI. B, prAPI in the apg9ts strain accumulates in both a membrane-associated state and a large pelletable complex. Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 5,000 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 in gradient buffer (20 mM Pipes, 5 mM MgCl2, complete EDTA-free protease inhibitor cocktail) in the presence or absence of Triton X-100 and overlaid with 13 and 2% Ficoll-400 in gradient buffer. The step gradients were centrifuged at 13,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods. The position of prAPI is indicated. C, prAPI in the apg9ts strain is protease accessible. Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 5,000 g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. The resulting samples were immunoprecipitated with antiserum to API and PGK. The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager. The percent protease-protected prAPI was determined by dividing the value for prAPI in the protease-treated sample by the value for total prAPI before protease treatment. The percent PGK was determined by dividing the supernatant (S) or pellet (P) value over the sum of the S and P values.",
    "molecules": "protease, salt, Triton X-100"
  },
  {
    "caption": "(D) Live cell imaging of U2OS cells stably transfected with a GFP targeted to the mitochondrial outer membrane (OM-GFP), showing delayed FCCP-induced mitochondrial fission in Sept2 depleted cells compared to mock treated cells. White arrowheads point at fission examples, yellow arrowheads to looping. Scale bar: 10 µm, insets are enlarged twofold. (E) Quantification of mitochondrial elongation (long /short axis) in mock treated and FCCP treated U2OS cells showing increased elongation in Sept2 depleted cells compared to mock treated cells, mean of four independent experiments SEM.",
    "molecules": "FCCP"
  },
  {
    "caption": "(D) Sept2 immunoprecipitation from mock or CCCP-treated HeLa cells probed for Drp1 and Sept2. (E) Quantification of two independent experiments showing stimulated Drp1 co-immunoprecipitation with Sept2 in FCCP-treated cells.",
    "molecules": "FCCP, CCCP"
  },
  {
    "caption": "(F) Sept2 immunoprecipitation from mock or Mdivi-1-treated HeLa cells probed for Drp1 and Sept2. (G) Quantification of two independent experiments showing decreased Drp1 co-immunoprecipitation with Sept2 upon Mdivi-1 treatment.",
    "molecules": "Mdivi-1"
  },
  {
    "caption": "(C) Mock treated or Sept2 depleted Drp1 -/- MEF cells were labeled with mitotracker orange (red), treated for the indicated amount of time with 2µM FCCP and stained for actin with phalloidin (green). Insets are enlarged twofold.",
    "molecules": "FCCP"
  },
  {
    "caption": "(F) HeLa cells were mock treated or treated with cytochalasin D and subjected to Sept2 immunoprecipitation. Immunoprecipitates were analyzed by western blot for Drp1 and Sept2, showing that Drp1 co-immunoprecipitation with Sept2 is not affected by cytochalasin D treatment.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(D) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated; All scale bars are 20 μm. n=3 biological replicates.",
    "molecules": "DCFH-DA, ROS"
  },
  {
    "caption": "(B) Gpx4 protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647; n = 4 biological replicates.",
    "molecules": "GW7647"
  },
  {
    "caption": "(E) Chromatin immunoprecipitation assays were performed on soluble formaldehyde-crosslinked chromatin isolated from untreated and GW7647-treated WT or PPARα-/- livers with polyclonal anti-PPARα antibodies (anti-PPARα) or control IgG. The final DNA extraction was polymerase chain reaction-amplified with a primer pair that covered the sequence in intron 3 of Gpx4.",
    "molecules": "formaldehyde, GW7647"
  },
  {
    "caption": "(D) TRF protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647. n = 4 biological replicates.",
    "molecules": "GW7647"
  },
  {
    "caption": "(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. All scale bars are 20 μm. n = 3 biological replicates.",
    "molecules": "DCFH-DA, ROS"
  },
  {
    "caption": "(E) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.",
    "molecules": "DCFH-DA, ROS"
  },
  {
    "caption": "(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.",
    "molecules": "DCFH-DA, ROS"
  },
  {
    "caption": "H GFP fluorescence image of the Mx roots under different treatment. Nuclei were labeled using DAPI. Scale Bar: 100 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "H Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed in leaves Md/Mx. Scale bars: 100 μm.",
    "molecules": "BCECF"
  },
  {
    "caption": "I Rhizosphere acidification of MdCAX3-suppressed in leaves Md/Mx. Bromocresol purple was used as a pH indicator for visualization. Scale bars: 1 cm.",
    "molecules": "Bromocresol purple"
  },
  {
    "caption": "G Interaction between MdCXIP1 and MdCAX3 tested by yeast two-hybrid (Y2H) assays. A dilution series of NMY51 yeast cells co-expressing MdCXIP1 and MdCAX3 cultured on SD/II and SDIV screening media. pPBT3-N (empty vector) co-expressed with MdCXIP1 was used as a negative control. X-gal was used in SD/-Trp/-Leu/-His/-Ade screening medium to further test for possible interactions.",
    "molecules": "X-gal, Ade, His, Leu, Trp"
  },
  {
    "caption": "C Fluorescence ratio (488/440 nm excitations) of BCECF fluorescence in the roots of MdCAX3 and MdCXIP1 co-overexpressing Mb. The central band represents the median and the box ranges showing the interquartile range and covers the central 50% of the data. The whiskers showing the minimum and maximum of the data. Three biological replicates; 3 roots were quantified for each replicate. Asterisks indicate statistically significant differences (***, P < 0.001; ANOVA, Tukey correction).",
    "molecules": "BCECF"
  },
  {
    "caption": "D Ratio (488/440) image of BCECF fluorescence in MdCAX3 and MdCXIP1 co-overexpressing Mb. Scale Bar: 100 μm. E Rhizosphere acidification in MdCAX3 and MdCXIP1 co-overexpressing Mb. Bromocresol purple was used as a pH indicator for visualization. Scale Bar: 1 cm.",
    "molecules": "BCECF, Bromocresol purple"
  },
  {
    "caption": "D Cellular localization of Zn2+ (blue) in Mx and MdCAX3-suppressed Mx roots under Fe deficient conditions. Arrowhead indicates the cytoplasm. Scale Bars: 20 μm. E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D.",
    "molecules": "Fe, Zn2+"
  },
  {
    "caption": "G. Western blots against NPC1 and actin-β (ACTB) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX). Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt non cLTP induced) values (n=3 mice, 3 month-old, 2-way ANOVA, ***p < 0.001).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "H. To the left Western blots against NPC1 and actin-β (ACTB) in synaptosomal extracts from wt and NPC1nmf164 mice used as input for the immunoprecipitation assays shown in the right. Immunoprecipitates were pulled down with an anti-ubiquitin antibody and without it for negative control and analysed by western blot against NPC1. MG132 was used as proteasome inhibitor. Graphs show mean ± SEM levels of NPC1 normalized to ACTB in the inputs (left) (n=3, one-way ANOVA, *pWT+MG-132=0.0496, pNPC1nmf1+MG-132=0.0358) and of NPC1 associated to ubiquitin (right) (n=3 mice, 3 month-old, one-way ANOVA, *pNPC1nmf164= 0.0237, pNPC1nmf164+MG-132= 0.0386.",
    "molecules": "MG-132, MG132, ubiquitin"
  },
  {
    "caption": "G. Mean ± SEM cholesterol levels in synaptosomes (n=4 mice, 3 month-old, unpaired Student t test, *p = 0.0476) from wt and NPC1nmf164 mice expressed as percentage of wt mice.",
    "molecules": "cholesterol"
  },
  {
    "caption": "A. Mean ± SEM cholesterol level in plasma membrane fractions of hippocampal slice cultures from wt and NPC1nmf164 mice with or without cLTP induction (n=6, unpaired Student t test, *pwt = 0.0402",
    "molecules": "cholesterol"
  },
  {
    "caption": "B. Western blot of NPC1 and ACTB in extracts of cultured hippocampal neurons from wt mice transfected or not with sh-scr or sh-NPC1 RNAs. Black bars indicate that the sh-NPC1 lane was not consecutive to the others but belongs to the same Western blot. Graph shows the 2 biological replicates mean ± SEM of NPC1 level normalized to ACTB as a percentage of the wt, non-transfected controls.",
    "molecules": "RNAs"
  },
  {
    "caption": "C. Representative images of the cholesterol binding probe mCherry-D4 before and after cLTP in cultured hippocampal neurons from wt mice transfected with sh-scr or sh-NPC1 RNAs. Z-stack images acquired using a Nikon A1R+ confocal microscope were used to generate 3D images of the neurons.",
    "molecules": "cholesterol, RNAs"
  },
  {
    "caption": "Western blots against NPC1 actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface NPC1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA, *pNPC1 = 0.0251",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "Western blots against CYP46A1 and actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface CYP46A1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA , *pCYP46A1 = 0.0371).",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "F. Representative images of GluA1 immunocytochemical staining before and after cLTP in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs. Graph shows mean ± SEM GluA1 surface staining with respect to total in cLTP conditions, as a percentage of the sh-scr baseline (n=20 images per condition from 2 different experiments, 2-way ANOVA, ***psh-scr < 0.001).",
    "molecules": "RNAs"
  },
  {
    "caption": "C. Mean ± SEM cholesterol levels in EFV-treated or non-treated synaptosomes from wt (n=5 mice, 14-week-old, unpaired Student t test, *p = 0.0233) and NPC1nmf164 (n=4 mice, 14 week-old, unpaired Student t test, *p = 0.0188) mice. EFV values are expressed as percentage of their corresponding non-treated controls in wt and NPC1nmf164 conditions.",
    "molecules": "cholesterol, EFV"
  },
  {
    "caption": "D. Basal synaptic transmission in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM EPSP slope (n=9 slices from 5 WT mice, n=11 slices EFV-treated from 5 WT mice, n=8 slices from 5 NPC1nmf164 mice and n=13 slices EFV-treated from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, *p = 0.0117).",
    "molecules": "EFV"
  },
  {
    "caption": "E. LTP in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, ***p < 0.0001).",
    "molecules": "EFV"
  },
  {
    "caption": "F. Mean ± SEM paired pulse facilitation in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice (n=11 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, **pNPC1nmf164=0.0027, *pNPC1nmf164+EFV=0.0229).",
    "molecules": "EFV"
  },
  {
    "caption": "Representative images of GluA1 immunocytochemical staining before and after cLTP induction in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs and treated or not with EFV. Graph shows the mean ± SEM GluA1 surface staining with respect to total after cLTP, as a percentage of the sh-scr baseline (n=20 images per condition from 2 separate experiments, 2-way ANOVA, ***psh-scr < 0.001, **psh-scr+EFV < 0.01, ***psh-NPC1+EFV < 0.001).",
    "molecules": "EFV, RNAs"
  },
  {
    "caption": "The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV A. Mean ± SEM 24(S)-hydroxycholesterol plasma level after 6 and 8 weeks of oral EFV treatment in wt (n=6 mice, 14 week-old and 16 week-old, 2-way ANOVA, **p6 weeks = 0.0023) and NPC1nmf164 (n=5 mice, 14 week-old and 16 week-old, 2-way ANOVA, ***p6 weeks < 0.0001; ***p8 weeks < 0.0001) mice.",
    "molecules": "24(S)-hydroxycholesterol, EFV"
  },
  {
    "caption": "The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV B. Mean ± SEM body weight in grams in wt and NPC1nmf164 mice treated or not with EFV (n=4 mice, unpaired Student t-test, ***pNPC1nmf164 < 0.0001, **pNPC1nmf164+EFV = 0.0048)",
    "molecules": "EFV"
  },
  {
    "caption": "The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV C. Mean ± SEM discrimination index as a normalized ratio of time spent with novel and familiar objects in the Object Placement Recognition test (n=6 mice, 10 week-old, 2-way ANOVA, **pWT/NPC1nmf164 = 0.0065, ***pNPC1nmf164/NPC1nmf164+EFV = 0.0008).",
    "molecules": "EFV"
  },
  {
    "caption": "The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV D. Mean ± SEM percentage of entries in the novel arm of the Y maze test (n=10 mice, 10 week-old, 2-way ANOVA, ***p < 0.0001). E. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, **pNPC1 < 0.0025, *pNPC1+EFV < 0.0181). F. Mean ± SEM percentage of freezing time in the Cued Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, *pNPC1 < 0.0453, **pNPC1+EFV < 0.0054). ",
    "molecules": "EFV"
  },
  {
    "caption": "The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV G. Mean ± SEM cholesterol level in synaptosomes of wt and NPC1nmf164 mice treated or not with EFV expressed as percentage of the wt non-treated samples (n=4 mice, 14 week-old, 2-way ANOVA, *pNPC1 < 0.0476, *pNPC1+EFV < 0.0118).",
    "molecules": "cholesterol, EFV"
  },
  {
    "caption": "The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV H. Representative fluorescence images of the CA1 hippocampal region from wt and NPC1nmf164 mice treated or not with EFV and stained with filipin and an antibody against LAMP1. White arrows indicate lysosome enlargement in the hippocampus of non-treated NPC1nmf164 mice. Graph to the right shows mean ± SEM fluorescence intensity associated to filipin per area in arbitrary units (n=4 mice, 14-week old, unpaired Student t-test, **pNPC1nmf164 = 0.0011, *pNPC1nmf164+EFV = 0.0273)). I. Survival graph for wt and NPC1nmf164 mice treated or not with EFV (n=5 mice, 2-way ANOVA, ***p < 0.0001). ",
    "molecules": "EFV, filipin"
  },
  {
    "caption": "(a) Purified mitochondria were left untreated, or were treated with Proteinase K (Pr.K) following osmotic shock (OS), or solubilization of the total protein using Triton X-100 (TX-100), as indicated. Samples were analysed by immunoblotting with antibodies against the indicated proteins.",
    "molecules": "Proteinase K, Triton X-100"
  },
  {
    "caption": "(b) Mitochondrial preparations were subjected to membrane disruption by freeze-thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting.",
    "molecules": "Proteinase K"
  },
  {
    "caption": "(c) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density-gradient centrifugation. Numbers represent fractions from the gradient (1, top; 12, bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S8.",
    "molecules": "sucrose"
  },
  {
    "caption": "(e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST-IRGM. Binding was detected by immunoblotting.",
    "molecules": "lipids"
  },
  {
    "caption": "(f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST-IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads.",
    "molecules": "cardiolipin"
  },
  {
    "caption": "(g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e-g were probed with anti-GST antibody.",
    "molecules": "lipids"
  },
  {
    "caption": "(k) HeLa cells were transfected with vectors encoding GFP-IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.",
    "molecules": "z-VAD"
  },
  {
    "caption": "(e) Images of HeLa cells transfected with vectors encoding GFP-IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top).",
    "molecules": "Staurosporine"
  },
  {
    "caption": "(A-C) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS (Earle's Balanced Salt Solution) for 30 min. Total membranes (mem) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells. Reactions contained the indicated concentrations of PI3K inhibitor (PI3KI) 3-methyladenine (3-MA) (B) or FYVE protein (C). A diagram of the experimental scheme is shown in (A). RPN1, Ribophorin 1",
    "molecules": "3-MA, 3-methyladenine, PI3K inhibitor, PI3KI"
  },
  {
    "caption": "(D, E) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS in the absence or presence of 20 nM wortmannin (Wtm) or 10 mM 3-methyladenine (3-MA) for 30 min. Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K ×g, 25K ×g and 100K ×g pellet fractions followed by a lipidation assay as above (E). A diagram is shown in (D).",
    "molecules": "3-MA, 3-methyladenine, wortmannin, Wtm"
  },
  {
    "caption": "(F, G) Atg5 KO MEFs were starved for 30 min. Membranes in the 25K ×g and 100K ×g pellets from a differential centrifugation were collected as described above. A similar lipidation assay was performed in the presence of indicated concentrations (Conc in G) of 3-MA, wortmannin (F) and FYVE protein as well as a PI3P binding-deficient FYVE mutant protein (C/S) (G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).",
    "molecules": "3-MA, wortmannin"
  },
  {
    "caption": "(B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP).",
    "molecules": "3-MA, ATP, GTP, Wortmannin"
  },
  {
    "caption": "(E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml).",
    "molecules": "BFA, brefeldin A, wortmannin"
  },
  {
    "caption": "(F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction.",
    "molecules": "3-MA"
  },
  {
    "caption": "(A) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min. Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 (F2 in Figure 2C,D) of the OptiPrep gradient in the three-step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes.",
    "molecules": "wortmannin"
  },
  {
    "caption": "C The binding of His-SOS2 to different PA species. The amount of each PA spot is 5 nmol. PA species: natural PA from soy, dipalmitoyl PA (di16:0 PA), distearoyl PA (di18:0 PA), dioleoyl PA (di18:1 PA), dilinoleoyl PA (di18:2 PA), palmitoyl-oleoyl PA (16:0-18:1 PA), palmitoyl-linoleoyl PA (16:0-18:2 PA), stearoyl-oleoyl PA (18:0-18:1 PA), stearoyl-linoleoyl PA (18:0-18:2 PA). For Mock control, no PA was incubated with the proteins.",
    "molecules": "dioleoyl PA, 16:0-18:2 PA, 16:0-18:1 PA, 18:0-18:2 PA, stearoyl-linoleoyl PA, 18:0-18:1 PA, stearoyl-oleoyl PA, distearoyl PA, palmitoyl-oleoyl PA, palmitoyl-linoleoyl PA, dilinoleoyl PA, di18:1 PA, di18:0 PA, dipalmitoyl PA, di18:2 PA, di16:0 PA"
  },
  {
    "caption": "D The His-SOS2 binding to the liposome containing PA. Liposomes were made up of 16:0-18:2 PC only or the mixtures of 16:0-18:2 PC+16:0-18:2 PA. After incubation with the recombinant proteins, liposomes were pelleted by centrifugation. The liposome-conjugated proteins in pellet and proteins remaining in supernatant were detected by immunoblot using anti-His antibody. For the Mock control, no liposomes were incubated with the proteins.",
    "molecules": "16:0-18:2 PC, 16:0-18:2 PA, liposome, liposomes, Liposomes, PA"
  },
  {
    "caption": "E Microscale thermophoresis (MST) assay between His-SOS2 and PA. His-SOS2 was labeled and kept at a constant concentration (1.0 μM). His-RbohD1-100, as the negative control, was labeled and kept at a constant concentration (4.5 μM). PA was hydrated in TBST buffer (0.005% tween 20), and diluted 16 times in a 1:1 ratio.",
    "molecules": "PA"
  },
  {
    "caption": "A The PA synthesis defective mutants pldα1 pldδ-1 and pldα1 pldδ-2 are salt-sensitive. Six-d-old seedlings were grown in half-strength Murashige and Skoog (MS), and then transferred to half-strength MS medium with or without 100 mM NaCl for 7 d.",
    "molecules": "PA, NaCl, salt"
  },
  {
    "caption": "G, H Analysis of the average values of the net Na+ (G) and K+ (H) fluxes of Col-0, sos2, C-1 and C-2. Six-d-old seedlings were treated with or without 50 mM NaCl for 24 h. Error bars represent SD (n ≥ 3).",
    "molecules": "K+, Na+, NaCl"
  },
  {
    "caption": "I-N The contents of shoot Na+ (I), shoot K+ (J), root Na+ (K) and root K+ (L) as well as the relative ratios of shoot Na+/K+ (M) and root Na+/K+ (N) of Col-0 and pldα1 pldδ mutants. Six-d-old seedlings were transferred to half-strength MS medium with or without 100 mM NaCl treatment for 7 d. Error bars represent SEM of three independent biological repeats. O-T The contents of shoot Na+ (O), shoot K+ (P), root Na+ (Q) and root K+ (R) as well as the relative ratios of shoot Na+/K+ (S) and root Na+/K+ (T) of Col-0, sos2, C-1 and C-2 mutants. Six-d-old seedlings were transferred to half-strength MS medium with or without 50 mM NaCl treatment for 7 d. Error bars represent SEM of three independent biological repeats. D",
    "molecules": "K+, Na+, NaCl"
  },
  {
    "caption": "A PA promotes the phosphorylation of SCaBP8 by SOS2. The phosphorylation activity of His-SOS2 on His-SCaBP8 was detected with or without 1 or 10 μM PA. The solvent of methanol was used as mock control. Coomassie brilliant blue-stained (CBB) gel is shown in the upper panel, and autoradiograph (autorad) results are shown in the middle and lower panels. The numbers indicate the relative ratios of the signal intensity of autoradiograph. Statistical analysis of the relative ratios is shown B PA promotes the phosphorylation of SOS1 C300 by SOS2. CBB gel is shown in the upper panel, and autorad result is shown in the lower panel. The numbers indicate the relative ratios of the signal intensity of autoradiograph. Statistical analysis of the relative ratios is shown",
    "molecules": "CBB, methanol, PA"
  },
  {
    "caption": "C PA promotes the activity of SOS2 under salt stress. Ten-d-old transgenic plants expressing 35S:6×Myc-SOS2 in Col-0 or pldα1 pldδ-1 background were treated with or without 100 mM NaCl or 50 μM PA for 12 h. The total protein was extracted and immunoprecipitated with anti-Myc antibody-conjugated agarose, and then the purified Myc-SOS2 proteins at similar protein levels were incubated with His-SCaBP8 in the kinase assays. His-SCaBP8 was used as a specific substrate of SOS2. The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc-SOS2 bands (Autorad/Myc). Statistical analysis of the relative ratios is shown D PA promotes the PM localization of SOS2 under salt stress. Ten-d-old seedlings were treated as described in Fig 3C. GHR1 was used as a plasma membrane marker. PEPC was used as a cytoplasm marker. The numbers represent the relative ratios of the signal intensity between Myc-SOS2 and GHR1 bands (Myc/GHR1). Statistical analysis of the relative ratios is shown",
    "molecules": "agarose, PA, NaCl, salt"
  },
  {
    "caption": "B MST assays show that the site K57 is critical for SOS2 binding to PA. His-SOS2 with indicated point mutations were labeled and kept at a constant concentration (0.5 μM). PA was hydrated in TBST buffer and diluted 15 times in a 1:1 ratio.",
    "molecules": "PA"
  },
  {
    "caption": "D SOS2K57G has weak activity and cannot be activated by PA under salt stress. Ten-d-old seedlings expressing 35S:6×Myc-SOS2 or 35S:6×Myc-SOS2K57G in Col-0 or pldα1 pldδ-1 background were treated with or without 100 mM NaCl for 12 h. The brightness and contrast (B&C) of the autoradiograph was adjusted to clearly show the activity of SOS2K57G. The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc-SOS2 bands (Autorad/Myc). Statistical analysis of the relative ratios is shown",
    "molecules": "PA, NaCl, salt"
  },
  {
    "caption": "E The site K57 is important for the PM localization of SOS2 under salt stress. Ten-d-old seedlings were treated with or without 100 mM NaCl for 12 h. The numbers represent the relative ratios of the signal intensity between Myc-SOS2 and GHR1 bands (Myc/GHR1). The asterisk points to nonspecific band. Statistical analysis of the relative ratios is shown",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "A The loss-of-function mutants akt1-1 and akt1-2 are salt-sensitive. Six-d-old seedlings of Col-0 (WT) and akt1 mutants were treated with or without 100 mM NaCl for 7 d. B Analysis for primary root length, related to Fig 5A. Data was shown in a box and whiskers (min to max) form (n ≥ 10). Statistical analysis was done by two-way ANOVA with Holm-Sidak's multiple comparisons test (P ≤ 0.05). C",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "C, D Analysis of the average values of the net Na+ (C) and K+ (D) fluxes of Col-0 and akt1 mutants with or without 100 mM NaCl treatment for 24 h. Error bars represent SD (n ≥ 4).",
    "molecules": "K+, Na+, NaCl"
  },
  {
    "caption": "K Time- and voltage-dependent AKT1-mediated K+ current recordings from oocytes. Whole-cell K+ currents were recorded in oocytes that were co-injected with indicated mixture of cRNAs for AKT1, SCaBP8 and SOS2 (SOS2WT or SOS2K57G), in the presence of CIPK23 and CBL1. 30% SOS2 means that the cRNA injection amount of SOS2 was 30% of SCaBP8. The oocytes were treated with or without 50 μM PA for 1.5 h.",
    "molecules": "PA, K+"
  },
  {
    "caption": "(B) Dot-plot showing the fold change (log2) in number of reads between vehicle and AZD8186-treated conditions vs the p-value of the difference between the two treatment conditions for each shRNA. 9 out of 18 shRNAs targeting EGFR showed a p-value <0.2 and are highlighted in the plot. The plot was generated considering the results from biological triplicate of the experiment.",
    "molecules": "AZD8186"
  },
  {
    "caption": "(C MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. EGFR mRNA was then measured by RT-qPCR (C) Average ± SD of triplicates and representative of three independent experiments.",
    "molecules": "puromycin"
  },
  {
    "caption": "D) MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. cell viability was measured after 4 days of treatment with serial dilutions of AZD8186 (D). Average ± SD of triplicates and representative of three independent experiments.",
    "molecules": "AZD8186, puromycin"
  },
  {
    "caption": "(E MDA-MB-468 were treated with serial dilutions of gefitinib (E) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of gefitinib Average ± SD of triplicates and representative of two independent experiments.",
    "molecules": "MK2206, AZD8186, gefitinib, GDC0941"
  },
  {
    "caption": "F) MDA-MB-468 were treated with serial dilutions of lapatinib (F) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of lapatinib. Average ± SD of triplicates and representative of two independent experiments.",
    "molecules": "MK2206, AZD8186, lapatinib, GDC0941"
  },
  {
    "caption": "(G) Viability of six PTEN-null VS five PTEN-WT TNBC cell lines - not carrying other known mutations in PIK3CA, PIK3CB or PIK3R1 genes - treated with PI3Kβi (AZD8186 90 nM), EGFRi (gefitinib 3 μM) alone or in combination for 6 days. Mean of 3 independent experiments ± SD. Statistical significance of two-tailed unpaired student t-test in PTEN-null PI3Kbi versus PI3Kbi+EGFRi **P=0.0059, PTEN-null EGFRi versus PI3Kbi+EGFRi **P=0.0047, PTEN-null PI3Kbi+EGFRi versus PTEN-WT PI3Kbi+EGFRi *P=0.0459, PTEN-WT PI3Kbi versus PI3Kbi+EGFRi *P=0.0108, PTEN-WT EGFRi versus PI3Kbi+EGFRi n.s. P=0.1926. PTEN-null cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395 and BT-549; PTEN-WT cell lines were: MDA-MB-157, MDA-MB-231, HCC1187, HCC1428 and HCC1806.",
    "molecules": "AZD8186, gefitinib"
  },
  {
    "caption": "(H) Synergy score for combinations of serial dilutions of PI3Kβi (AZD8186) plus EGFRi (gefitinib) tested on six PTEN-null and five PTEN-WT TNBC cell lines for 6 days in three independent experiments. The score was obtained analysing the viability data through the software Chalice Analyser. Mean of the synergy scores ± SD. Statistical significance of Mann Whitney two-tailed test *P=0.0303.",
    "molecules": "AZD8186, gefitinib"
  },
  {
    "caption": "(A) Tumor volume of MDA-MB-468 mammary fat-pad xenografts treated with vehicle, AZD8186 (50 mg/kg, og twice/day), erlotinib (50 mg/kg IP once/day) alone or in combination (5-6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test **P=0.0028 and ***P<0.0001. (B) Tumor volume of HCC-70 mammary fat-pad xenografts treated with vehicle, AZD8186 (150 mg/kg, og once/day), erlotinib (50 mg/kg IP once/day) alone or in combination (6-7 mice per group, mean±SEM). Growth curves were compared using two-way ANOVA statistical test. Statistical significance of two-way ANOVA statistical test ***P=0.0006 and ****P<0.0001.",
    "molecules": "AZD8186, erlotinib"
  },
  {
    "caption": "(E) A cell line derived from a mammary tumor spontaneously developed in a Wap-cre:Ptenfl/fl:Tp53fl/fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6/J recipient mice. Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure. When tumours reached an average volume of 100mm3, they were treated with vehicle or a combination of AZD8186 (150mg/kg, og once/day) and erlotinib (50mg/kg IP once/day). Tumours were then measured during the treatment (6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test ****P<0.0001.",
    "molecules": "AZD8186, erlotinib"
  },
  {
    "caption": "(A) MDA-MB-468 cells were treated for 24h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM) or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies.",
    "molecules": "AZD8186, cetuximab, gefitinib"
  },
  {
    "caption": "(B) MDA-MB-468 cells were treated for 24h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM) or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.",
    "molecules": "MK2206, cetuximab, gefitinib"
  },
  {
    "caption": "(C, D) HCC70 (C) and ZR-75-1 (D) parental cells or PI3Kβi-Res (derivative cells with acquired resistance to AZD8186), or AKTi-Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM) or AKTi (MK2206 1 μM) for 24 hours. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P-S6 and S6 tot in (D) have been cut and reassembled for figure purposes.",
    "molecules": "MK2206, AZD8186"
  },
  {
    "caption": "(E) HCC70 parental, PI3Kβi-Res or AKTi-Res cells were treated with vehicle, AKTi (MK2206 250 nM) or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.",
    "molecules": "MK2206, gefitinib"
  },
  {
    "caption": "(F) HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments.",
    "molecules": "MK, MK2206, gefitinib"
  },
  {
    "caption": "(G) HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments.",
    "molecules": "AZD, AZD8186, gefitinib"
  },
  {
    "caption": "(H,I) p110β co-immunoprecipitates with EGFR in MDA-MB-468 (H) and in HCC70 (I). Cells were pre-treated with different inhibitors, including vehicle, pan-PI3Ki GDC0941 (1 μM for MDA-MB-468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM) or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti-EGFR antibody and the immuno-complexes or the total lysates were immune-blotted with the indicated antibodies.",
    "molecules": "AZD8186, gefitinib, GDC0941"
  },
  {
    "caption": "(J) EGF-induced increase in phospho-AKT is dependent on p110β kinase activity. MDA-MB-468 were starved in 0% FBS and pre-treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1h. Cell lysates were probed with the indicated antibodies. Phospho-AKT and pan-AKT bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT normalised to the control (left hand lane) is shown.",
    "molecules": "AZD8186"
  },
  {
    "caption": "(A Results of CRISPR-Cas9 screening in combination with AZD8186 100nM (A) The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions (AZD8186 and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). <0.05>",
    "molecules": "AZD8186, Gefitinib"
  },
  {
    "caption": "B) Results of CRISPR-Cas9 screening in combination with GDC0941 400nM (B). The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions GDC0941, and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. <0.05>",
    "molecules": "GDC0941"
  },
  {
    "caption": "(C) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941-treated conditions for MDA-MB-468 cells transduced with non-target Control, GNB2 or GNG5 sgRNAs (N=2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1.5 times the inter-quartile range. Statistical significance of unpaired t-test ****P<0.0001.",
    "molecules": "GDC0941"
  },
  {
    "caption": "(A) MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were treated with vehicle, GDC0941 1 μM, gefitinib 3 μM or lapatinib 1 μM for 24 hours. The cell lysates were probed with the indicated antibodies.",
    "molecules": "gefitinib, lapatinib, GDC0941"
  },
  {
    "caption": "(A) Schematic of the results from the drug screening with compounds targeting GPCR signaling. MDA-MB-468 cells were treated with the compounds of the GPCR-targeted library at three different concentrations (0.1, 1 or 10 μM) in combination with vehicle, GDC0941 450 nM or lapatinib 1 μM. The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI. Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle, GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot, respectively. Readings acquired following treatment with the three different concentrations of the GPCR-targeted drugs are reported in 3 different colours. Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot. The values reported are mean of a biological triplicate of the experiment.",
    "molecules": "DAPI, lapatinib, GDC0941, vorapaxar"
  },
  {
    "caption": "(B) MDA-MB-468 were treated for 24 h with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.",
    "molecules": "lapatinib, GDC0941, vorapaxar"
  },
  {
    "caption": "(C) MDA-MB-468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes, alone or in combination with vorapaxar. The cell lysates were probed with the indicated antibodies. Phospho-AKT, pan-AKT, phospho-ERK1/2 and pan-ERK1/2 bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT and phospho-ERK1/2 to pan-ERK1/2 normalised to the control peptide-treated conditions for WT and GNB2 KO cells (first and forth lanes, respectively) is shown.",
    "molecules": "vorapaxar"
  },
  {
    "caption": "(D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant.",
    "molecules": "MK2206, AZD8186, lapatinib, GDC0941, vorapaxar"
  },
  {
    "caption": "(E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown.",
    "molecules": "crystal violet, lapatinib, GDC0941, vorapaxar"
  },
  {
    "caption": "(A-D) The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies were measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal).",
    "molecules": "PBS"
  },
  {
    "caption": "(A) Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110-136) peptide or F 1-34 peptide (control) in ELISA.",
    "molecules": "F 1-34, F 110-136, F-p27, peptide"
  },
  {
    "caption": "(B) A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit anti-sera against F (center panels), or against F-p27 (110-136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice and variation between the two independent expeiremnts was <6%.",
    "molecules": "F-p27, Alexa 594, DAPI, peptide"
  },
  {
    "caption": "Mice lungs were collected from PBS-(mock) vaccinated BALB/c mice at day 5 post-RSV infection, fixed, paraffin embedded, and slides were processed for imaging. Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera (left panels) or rabbit anti-sera against RSV F protein (center panels), or against RSV F-p27 (110-136) peptide (right panels). Scale bar = 100 µm. The percentage of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) covering 500 x 500 µm of lung tissue for each mice for each antibody staining are shown. The experiments were performed twice and variation between the two independent expeiremnts was <9%.",
    "molecules": "F-p27, peptide, PBS"
  },
  {
    "caption": "(A) Lung tissues from individual mice (n=5) at day 5 post-RSV challenge (or uninfected control) were used to evaluate CD4/CD8 T cells separately for airway and distal lungs. Lung slides were fixed and stained with DAPI to visualize nuclei (blue) and either anti-CD4 (green) or anti-CD8 (red) T cells. The number of CD4/CD8 T cells were determined in each lung airway and distal region. Individual CD4+/CD8+ T cells data are presented as number of positive cells per 174 x 174 µm of lung tissue for each mice. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.",
    "molecules": "DAPI"
  },
  {
    "caption": "(c) Rates of arm-level and focal SCNVs (Online Methods) across different types of malignancies. Data regarding SCNVs of the following types were summarized from Zack et al.: RCC, renal cell carcinoma; GBM, glioblastoma multiforme; UCEC, uterine cervix; COAD, colorectal adenocarcinoma; BRCA, breast; HNSC, head and neck squamous cell; OV, ovary; LUAD, lung adenocarcinoma; BLCA, bladder; LUSC, lung squamous cell.",
    "molecules": "SCNVs"
  },
  {
    "caption": "Data matrix showing frequent SCNVs, significantly mutated genes (bold; Benjamini-Hochberg FDR 0.2 calculated with CHASM) and their associated pathway or family mutations discovered in cases with NPC analyzed by WES, targeted deep sequencing (TS) or SNP array (SNP-a). The frequencies of the alterations are plotted on the right. The colors and shapes denoting different types of somatic events are also applied in Figure 5a. Columns, examined cases; rows, genes; hot spot, identical mutations have been registered in COSMIC (see URLs); LOH, loss of heterozygosity. The asterisk indicates that this tumor was also subjected to SNP array profiling. PIK3CA amplification in C666 cells has been previously described.",
    "molecules": "SCNVs"
  },
  {
    "caption": "(G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1.",
    "molecules": "cisplatin"
  },
  {
    "caption": "(J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.",
    "molecules": "cisplatin"
  },
  {
    "caption": "D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel).",
    "molecules": "cisplatin"
  },
  {
    "caption": "(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.",
    "molecules": "cisplatin"
  },
  {
    "caption": "MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm.",
    "molecules": "cisplatin"
  },
  {
    "caption": "(H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA.",
    "molecules": "cisplatin"
  },
  {
    "caption": "(L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect.",
    "molecules": "cisplatin"
  },
  {
    "caption": "(C) MTT assays also confirmed that shFNTA can reverse oecircFNTA-increased chemo-resistance to cisplatin (2 μg/ml) in J82 and UMUC3 cells.",
    "molecules": "cisplatin"
  },
  {
    "caption": "Transwell assays (D, with quantitations at the right) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.",
    "molecules": "Tipifarnib"
  },
  {
    "caption": "MTT assays (E) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.",
    "molecules": "Tipifarnib"
  },
  {
    "caption": "(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6). (B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice. (C) Tumor weights also decreased in the shcircFNTA group. ",
    "molecules": "cisplatin"
  },
  {
    "caption": "F. Interactions  between ASF1A  and PIF7/HIRA  were detected in tobacco leaf  cells. The PIF7-Flash, HIRA-HA, and GFP-ASF1A  (GFP  as control) proteins were co-expressed in tobacco leaf  cells. Anti-GFP Sepharose  beads were used for Co-IP assay.",
    "molecules": "Sepharose"
  },
  {
    "caption": "(E) Top, Representative images of live HeLa  cells in cytokinesis transiently transfected with GFP-CENP-E2605-2701 and mutants (green), incubated with SiR-tubulin  (red). Scale bar, 10μm. Bottom, linescans showing the fluorescence intensity average and standard error of the mean (SEM) for the GFP-CENP-E2605-2701 and mutants and tubulin  across the cell midbody. For GFP-CENP-E2605-2701, n is the number of cells. n=11 and for the GFP-CENP-E2605-2701 mutants FDN2661LTT, YF2660AA, FF2644AA, n= 15, 16 and 25 respectively. Biological independent replicates were respectively 2, 3, 2 and 1.",
    "molecules": "SiR"
  },
  {
    "caption": "(B) Representative images of live HeLa  cells in mitosis transiently transfected with either GFP-Kif4A1133-1165 or GFP-Kif4A1133-1232 incubated with SiR-Tubulin. Scale bar, 10μm. Quantification of cells with GFP-Kif4A1133-1165 (n=12) or GFP-Kif4A1133-1232 (n=13) localization  to the  central spindle. Data represented from 2 independent experiments.",
    "molecules": "SiR"
  },
  {
    "caption": "(A) Representative images of GFP-PRC1  (magenta) mixed with rhodamine-microtubules. Fire blue-green intensity LUT used to show tubulin   intensity and microtubule  overlaps. (B) Representative images of 647GST-CENP-E2639-2671 (yellow) mixed with rhodamine-microtubules.",
    "molecules": "rhodamine"
  },
  {
    "caption": "(D) Representative immunofluorescence  images of HeLa  cells stained  for DNA, microtubules, PRC1  and with Alexa647-labelled GST-CENP-E2639-2671 showing it also recognizes PRC1  in cells. Experiments were repeated >3 times.",
    "molecules": "Alexa647"
  },
  {
    "caption": "(D) Live-cell imaging of  metaphase spindles in HeLa  cells transiently transfected wild-type, phosphomimetic and non-phosphorylatable mutants of GFP-CENP-E2605-2701 (monomeric) and GFP-GST-CENP-E2605-2701 (dimeric) and stained  for tubulin  using SiR-Tubulin. The fraction of cells localizing  to the overlapping microtubules  is represented as a percentage. Scalebar, 10 μm.",
    "molecules": "SiR"
  },
  {
    "caption": "(A) Representative immunofluorescence  images of HeLa  cells in  late anaphase and telophase, expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1  using a PRC1  siRNA and a PRC1  sgRNA after doxycycline-induced Cas 9 expression. Microtubules, GFP  and CENP-E are in white, green and red respectively. DNA  is in blue. (B) Graph showing the percentage of cells with a PRC1-localized  at the site of abscission for cells treated in (A). Mean and standard deviation are presented, n= 156 and 141 for cells expressing PRC1-WT and -MEE respectively. Data from 4 biological replicates and 1 technical replicate. Asterisks indicate a T-test significance value. ****P<0.0001. (C) Graph showing the percentage of cells with CENP-E localized  at the abscission site for cells treated in (A), mean and standard deviation are presented, n= 58 and 66 for cells expressing PRC1-WT and -MEE respectively. Data from 2 biological replicates. p-value (p=0.11) calculated for an unpaired T-test.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(G) Representative live-cell images of HeLa  cells expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1  using a PRC1  siRNA and a PRC1  sgRNA after doxycycline-induced Cas 9  expression. Microtubules  (SiR-Tubulin) and DNA  (SPY650) are shown in red. The cell membrane (CellMask) is highlighted in white and GFP-PRC1  is in green. Scalebar, 10 μm.",
    "molecules": "CellMask, SPY650, doxycycline, SiR"
  },
  {
    "caption": "(J) Representative immunofluorescence  images of cells expressing GFP-PRC1-WT or -MEE after 72 hours siRNA depletion and doxycycline-induced knockout of endogenous PRC1. DNA, microtubules  and GFP-PRC1/GFP-PRC1-MEE are in blue, red and green respectively. Scalebar, 10μm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "miR-181a/b-1-/- mice show increased mtDNA content vs. miR-181a/b-1+/+ mice as measured by q-PCR. N=4 animals/genotype.",
    "molecules": "mtDNA"
  },
  {
    "caption": "Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N≥7 independent experiments.",
    "molecules": "FCCP"
  },
  {
    "caption": "TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7b-MO-injected embryos. Scale bars are 20μm.",
    "molecules": "MO"
  },
  {
    "caption": "Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7b-MO-injected embryos. n≥5 embryos for each model.",
    "molecules": "MO"
  },
  {
    "caption": "Representative images of st30 medaka embryos injected with hccs-MO (B) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO embryos. hccs-MO/miR-181a/b-MO- b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in hccs-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model Scale bars are 100μm.",
    "molecules": "PD98059, Baf-A1, HA14-1, MO, MOs"
  },
  {
    "caption": "Representative images of st30 medaka embryos injected with cox7b-MO (H) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in cox7b-MO embryos. cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in cox7b-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation Scale bars are 100μm.",
    "molecules": "PD98059, Baf-A1, HA14-1, MO, MOs"
  },
  {
    "caption": "Percentage of embryos with or without MLS phenotype in the different conditions in hccs-MO embryos. N≥300 embryos/conditions.",
    "molecules": "MO"
  },
  {
    "caption": "Percentage of embryos with or without MLS phenotype in the different conditions in cox7b-MO (N) embryos. N≥300 embryos/conditions.",
    "molecules": "MO"
  },
  {
    "caption": "Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm.",
    "molecules": "DMSO, Rotenone, rotenone"
  },
  {
    "caption": "NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm.",
    "molecules": "DMSO, Rotenone"
  },
  {
    "caption": "Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm.",
    "molecules": "DMSO, Rotenone, rotenone"
  },
  {
    "caption": "Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.",
    "molecules": "rotenone"
  },
  {
    "caption": "Ndufs4-/-/miR-181a/b-1-/- mice show increased mtDNA content vs. Ndufs4-/- mice as measured by q-PCR. N≥4 animals/genotype.",
    "molecules": "mtDNA"
  },
  {
    "caption": "(E) Formalin-fixed, paraffin-embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis (n=16 and n=49, respectively) were stained with an anti-ESM1 antibody. Tissue-bound ESM1 is shown in brown. Scale bar: 50 μm. Arrowheads indicate positively stained nucleus of cancer cells. Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis. The scores are calculated by intensity (score 1-3) x percentage (score 1-4) of stained cells. The line in the middle of the box is plotted at the median. The box extends from the 25th to 75th percentiles. Whiskers above and below the box indicate the 5th and 95th percentiles.",
    "molecules": "Formalin"
  },
  {
    "caption": "(B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm.",
    "molecules": "PBS"
  },
  {
    "caption": "(A) Tumor spheroid formation. Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody.",
    "molecules": "PBS"
  },
  {
    "caption": "(C) 22Rv1-P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h. **",
    "molecules": "docetaxel"
  },
  {
    "caption": "(D) Suppression of β-catenin or NFκB in 22Rv1-P cells stably expressing ESM1-NLS were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector-shScramble cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments.",
    "molecules": "docetaxel"
  },
  {
    "caption": "(C) Human recombinant His-ESM1 and GST-β-catenin protein were pull down with either Ni sepharose or Glutathione sepharose.",
    "molecules": "sepharose, Glutathione, Ni"
  },
  {
    "caption": "(E) HEK293T cells were co-transfected with the indicated β-catenin-Flag and ESM1-HA constructs. Cell extracts were immunoprecipitated with Flag-M2 agarose beads. Light chain was labeled as l.c.",
    "molecules": "agarose"
  },
  {
    "caption": "Left: Representative time-lapse images showing the migration of single hRPE cells on the surface of polyacrylamide gels with uniform stiffness (top) and stiffness gradient (bottom), referring as random migration and durotaxis migration respectively. Yellow line is the cell outline and blue line is the cell migration trajectory. Scale bar: 50 μm. Middle: Angular displacement of hRPE cells on the uniform stiffness gels (top) and stiffness gradient gels (bottom). Right: y/x FMI (forward migration index), velocity and persistence of hRPE cells with random migration and durotaxis.",
    "molecules": "polyacrylamide"
  },
  {
    "caption": "Left: Representative immunofluorescent staining of F-actin in control and CK-666 (100 μM, 3 h) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and CK-666 (100 μM, pre-treated for 1h and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.",
    "molecules": "CK-666"
  },
  {
    "caption": "Left: Representative immunofluorescent staining of α-tubulin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and nocodazole (8 μM, pre-treated for 10 min and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Representative immunofluorescent staining of γ-tubulin (yellow) and DAPI (blue) in control and centrinone-B (500 nM, 7 days) treated hRPEs.",
    "molecules": "centrinone-B, DAPI"
  },
  {
    "caption": "Angular displacement and y FMI of control and centrinone-B (500 nM, 7 days) treated hRPEs moving on stiffness gradient gels.",
    "molecules": "centrinone-B"
  },
  {
    "caption": "Left: Representative immunofluorescent staining of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of microtubule fluorescence intensity.",
    "molecules": "centrinone-B"
  },
  {
    "caption": "Western blot showing the protein level of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.",
    "molecules": "centrinone-B"
  },
  {
    "caption": "Left: Representative live-cell fluorescence imaging of EGFP-EB1 in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of EB1 growth speed obtained by plusTipTracker. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.",
    "molecules": "centrinone-B"
  },
  {
    "caption": "Left: Representative immunofluorescent staining of pMLC (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of pMLC fluorescence intensity. Below: Western blot showing the protein level of pMLC in negative control and CAMSAP2 siRNA transfected hRPEs. α-tubulin is used as loading control",
    "molecules": "DAPI"
  },
  {
    "caption": "Left: Images showing collagen gel deformation at 0 h, 8 h and 24 h. HRPEs transfected with negative control, CAMSAP2 and AKAP450 siRNA were seeded in 1% collagen gel (yellow dashed lines). Scale bar: 5 mm. Right: Quantification of gel area.",
    "molecules": "collagen"
  },
  {
    "caption": "Left: Representative live-cell fluorescence imaging of RFP-paxillin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Quantification of FA area obtained by FAAS.",
    "molecules": "nocodazole"
  },
  {
    "caption": "Top: Schematic diagram of FA-MT contact analysis. Position of EB1 and paxillin were extracted from live-cell images, followed by automated co-localization identification. Scale bar: 20 μm. Bottom: Representative time-lapse fluorescence imaging of EGFP-EB1 and RFP-paxillin zoomed from the yellow box above showed the targeting of MT to FA. FA-MT contact ratio was calculated by the ratio of co-localization events to the total EB1 number.   FA-MT contact ratio of negative control, CAMSAP2, AKAP450 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.   ",
    "molecules": "centrinone-B"
  },
  {
    "caption": "Left: Representative immunofluorescent staining of GM130 (yellow), F-actin (gray) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h. Scale bar: 50 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the front (45°-135°) or the rear (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the front or rear of the cell.",
    "molecules": "DAPI"
  },
  {
    "caption": "Left: Representative immunofluorescent staining of GM130 (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs on the stiffness gradient gel. Scale bar: 20 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the stiff side (45°-135°) or the soft side (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the stiff or soft side.",
    "molecules": "DAPI"
  },
  {
    "caption": "Left: Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm laser irradiation. Middle: Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope. Right: Representative live-cell fluorescence imaging of cells marked with the Golgi (green) and nucleus (blue) on the soft region and the stiff island.",
    "molecules": "polyacrylamide"
  },
  {
    "caption": "Naïve CD4+ T cells were isolated from the spleen and LNs of WT, Stim2fl/fl Cd4Cre (S2), Orai1fl/fl Cd4Cre (O1) and Stim1fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17 and iTreg cells. (A) Fura-2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca2+ traces (left) and peak Ca2+ levels (right). Data are from the mean ± SEM of 4 mice per group.",
    "molecules": "Ca2+, Fura-2, ionomycin"
  },
  {
    "caption": "(A,B) Ca2+ influx in human lamina propria CD4+ and CD8+ T cells from 3 IBD patient samples analyzed by flow cytometry. (A) Experimental design. (B) Ca2+ influx rates in T cells pre-incubated for 4 h in the presence of 15-1000 nM BTP2, which was present until data acquisition. SOCE was induced by stimulation with the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG) in Ca2+ free buffer followed by the addition of 2 mM Ca2+. Bar graphs show the mean ± SEM of Ca2+ influx rates after Ca2+ readdition from 1 experiment (n=3 IBD patients).",
    "molecules": "Ca2+, TG, thapsigargin, BTP2"
  },
  {
    "caption": "(D) viSNE plots of CD45+CD3+ LPMCs stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 hours in the presence of 15-1000 nM BTP2. viSNE plots are colored according to the expression level of IL-2 (blue: low, red: high) and are representative of one CD patient.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(E) Dose-response curves of the frequencies of cytokine producing CD4+ and CD8+ T cells after PMA/ionomycin stimulation for 4h in the presence of increasing doses of BTP2. The frequencies of cytokine producing cells were normalized to control samples treated with PMA/ionomycin alone. Bar graphs show the mean ± SEM of 5 CD patient samples.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(F) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45+CD3+ LPMCs stimulated for 4h with PMA/ionomycin in the presence of BTP2; data are normalized to PMA/ionomycin treatment alone.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(G) Cytokine production by CD19+HLA-DR+ B cells and CD11c+ dendritic cells in the presence of increasing doses (15-1,000 nM) of BTP2. Left: Dose-response curves showing frequencies of cytokine-producing cells after 4h stimulation with PMA/ionomycin ex vivo. Data are normalized to control samples stimulated with PMA/ionomycin in the absence of BTP2. Error bars represent SEM obtained from 1 experiment (n=5 CD patients). Right: heatmaps showing the median fold change of cytokines and surface markers in CD19+HLA-DR+ B cells and CD11c+ dendritic cells treated as described for the left panel.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(A) Experimental setup for mass cytometric assays. Non-inflamed: n = 4, CD: n =6, UC: n =6. (B) FlowSOM plot of merged FCS files from LPMCs of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4h ex vivo. Colors of the t-SNE plot represent 20 clusters of distinct CD45+ LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(C) FlowSOM map of merged FCS files from CD45+ cells of non-inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. (D) viSNE plots of one exemplary non-inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high).",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(F) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45+ LPMCs that were isolated from non-inflamed, UC and CD samples and activated for 4h with PMA/ionomycin ex vivo. Statistical significance was calculated using an unpaired t test with FDR adjustment to 1% using the Benjamini, Krieger and Yekutieli procedure; *",
    "molecules": "ionomycin, PMA"
  },
  {
    "caption": "(A) FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3+ LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-inflamed controls, UC and CD patients after 4h stimulation with PMA/ionomycin in vitro. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(B) Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non-inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4h in vitro. Statistical significances were calculated using a one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cytokines after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(D) Heatmaps indicating the median fold change expression of cytokines and surface molecules in CD45+CD3+ LPMCs stimulated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(A) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3- LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-Inflamed, UC and CD samples after stimulation with PMA/ionomycin for 4h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(B) Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4h ex vivo. Statistical significances were calculated by one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cell markers after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(D) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45+CD3- LPMCs activated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.",
    "molecules": "ionomycin, PMA, BTP2"
  },
  {
    "caption": "(A-B) Adult stem cell-derived intestinal epithelial spheroids of wild-type C57BL/6 mice were cultured for 4 days in Wnt-free medium to induce differentiation. Initial viability was determined on day 0. DMSO control (vehicle) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy. Scale bar depicts 100 µm. (C) Transepithelial electrical resistance (TEER) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2.",
    "molecules": "DMSO, resazurin, BTP2"
  },
  {
    "caption": "(D) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt-free medium to induce differentiation into colonic organoids. Initial viability was determined on day 0. DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2, 4 and 6.",
    "molecules": "DMSO, BTP2"
  },
  {
    "caption": "(E) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2.",
    "molecules": "BTP2"
  },
  {
    "caption": "(F) Relative mRNA expression of markers for enterocytes (ALPI), goblet cells (MUC2), and entero-endocrine cells (CHGA) was quantified by RT-PCR in differentiated human colon monolayers after addition of 1 µM BTP2.",
    "molecules": "BTP2"
  },
  {
    "caption": "(A-C) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents (Icrac). (A) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1. (B) Current-voltage (I-V) relationship of ICRAC before and after CM4620 treatment. I-Vs were obtained at the time points indicated by the arrowheads in (A). (C) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration (arrowheads) using the formula 1- (ICM4620 / ICtrl). Data are from 8 cells per conditions and shown as the mean ± SEM.",
    "molecules": "CM4620"
  },
  {
    "caption": "(E) Relative weight loss of mice treated with vehicle or CM4620; the start of treatment is indicated by the white arrow. Data are the mean ± SEM of 9 mice per cohort, statistical analysis by unpaired student's t-test , *p<0.05.",
    "molecules": "CM4620"
  },
  {
    "caption": "(F) Concentrations of CM4620 in the serum of mice at day 49, data are the mean ± SEM. Statistical analysis by unpaired student's t-test, ***p<0.001.",
    "molecules": "CM4620"
  },
  {
    "caption": "(G) Representative H&E staining of the distal colon of Rag1-/- mice treated with vehicle or CM4620. Colitis scores of 9 mice per cohort. Each symbol represents one mouse.",
    "molecules": "CM4620"
  },
  {
    "caption": "(I) Frequencies of RORγt+ Th17 cells and Foxp3+ Treg cells in the CLP of mice treated with CM4620 or vehicle. Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.",
    "molecules": "CM4620"
  },
  {
    "caption": "(J) Frequencies of IFN-γ, TNF-α and IL-2 producing CD4+ T cells isolated from the CLP, restimulated ex vivo with PMA and ionomycin for 4 h (without addition of CM4620 during stimulation in vitro) and analyzed by flow cytometry. Bar graphs represent the percentages of IFN-γ+, TNF-α+, IL-2+ cells in CD4+ cells (top row) and mean fluorescent intensity (MFI) of IFN-γ, TNF-α, IL-2 on CD4+ cells (bottom row). Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.",
    "molecules": "CM4620, ionomycin, PMA"
  },
  {
    "caption": "A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown.",
    "molecules": "OxPAPC, DPPC, R837"
  },
  {
    "caption": "A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.",
    "molecules": "OxPAPC, DPPC, R837"
  },
  {
    "caption": "C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.",
    "molecules": "OxPAPC, PAPC, CuSO4, R837, FeSO4"
  },
  {
    "caption": "D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.",
    "molecules": "PAPC, CuSO4, R837"
  },
  {
    "caption": "E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.",
    "molecules": "OxPAPC, PAPC, CpG, CuSO4, R837, LPS, LTA, Poly I:C"
  },
  {
    "caption": "G-I Splenicdendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenicdendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenicdendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.",
    "molecules": "OxPAPC, DPPC"
  },
  {
    "caption": "Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown.",
    "molecules": "PAPC, CuSO4, OxPL"
  },
  {
    "caption": "Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.",
    "molecules": "OxPAPC, OxPL, thioglycollate"
  },
  {
    "caption": "Selected candidate lipids were tested for their inhibitory activity on R837-induced (5 μg/ml; 18 h) cytokine secretion in BMDCs. Concentrations of indicated lipids: PECPC (10 μM), PEIPC (10 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), and KOdiAPC (40 μM). Representative data (mean ± SD of triplicate determinations) from one of three independent experiments are shown. ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Sidak's multiple comparisons test.",
    "molecules": "OxPAPC, DPPC, POVPC, PEIPC, PGPC, KOdiAPC, R837, PECPC"
  },
  {
    "caption": "Dose-response curves of IL-12 secretion in BMDCs pulsed for 60 min with the indicated lipids, followed by stimulation with R837 (5 μg/ml) for 18 h. Toxic concentrations of lipids were excluded from analysis. Mean ± SEM of triplicate determinations are shown.",
    "molecules": "R837"
  },
  {
    "caption": "EC50 values of anti-inflammatory lipid products normalized to the capacity of 15d-PGJ2 to suppress IL-12 production in BMDCs (left panel) and their respective bioactivities (right panel) depicted as the fold increase relative to 15d-PGJ2. Mean ± SEM of triplicate determinations are shown.",
    "molecules": "15d-PGJ2"
  },
  {
    "caption": "IL-12 production of BMDC stimulated via TLR 4 (LPS; 100 ng/ml), TLR 9 (CpG; 100 nM), and TLR 7 (R837; 5 μg/ml) after pretreatment with the indicated free and esterified versions of EC and 15d-PGJ2. Lipids were used at 1 μM (EC), 10 μM (PECPC), or 20 μM (15d-PGJ2 and 15d-PGJ2PC). Data (mean ± SEM) are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.",
    "molecules": "15d-PGJ2, 15d-PGJ2PC, CpG, EC, PECPC, R837, LPS"
  },
  {
    "caption": "Analysis of the capacity of various OxPL-derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset. Concentrations of indicated lipids: EC (1 μM), PECPC (10 μM), 15d-PGJ2 (20 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), KOdiAPC (40 μM).",
    "molecules": "OxPAPC, DPPC, POVPC, 15d-PGJ2, PGPC, KOdiAPC, EC, PECPC, OxPL"
  },
  {
    "caption": "A IL-12 production of BMDC from wild-type, Nrf2−/−, Pparg−/−, and Pparg litter mate control mice normalized to medium control (open bars). Cells were treated with the indicated lipids (filled bars) for 60 min prior TLR 7 ligation with R837 (5 μg/ml) for 18 h. Lipids were used at starting concentrations of 40 μM (POVPC, PGPC, and KOdiAPC), 40 μg/ml (OxPAPC), 20 μM (15d-PGJ2), and 1.25 μM (EC), depicted as black bars, and 2-fold serial dilutions thereof (gray bars). Data represent mean ± SEM of triplicates from one of three independent experiments.",
    "molecules": "OxPAPC, POVPC, 15d-PGJ2, PGPC, KOdiAPC, EC, R837"
  },
  {
    "caption": "B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild-type and Nrf2−/−BMDM stimulated with EC (2 μM) for 60 min. Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx. Data (mean ± SEM) are representative of two independent experiments.",
    "molecules": "EC"
  },
  {
    "caption": "C, D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 (C) and of the pro-inflammatory cytokines IL-6 and IL-12 (D) in wild-type, PPAR-γ-deficient, and Nrf2-deficient BMDM after treatment with EC or 15d-PGJ2 for 60 min followed by LPS treatment for 3 h. Expression levels are normalized to G6pdx. Data represent mean ± SEM of triplicate cultures from one of two independent experiments.",
    "molecules": "15d-PGJ2, EC, LPS"
  },
  {
    "caption": "E mRNA expression of the indicated chemokines as determined by qPCR. Wild-type BMDCs were treated with EC (1 μM) or 15d-PGJ2 (20 μM) for 60 min followed by R837 stimulation (5 μg/ml) for 3 h. Expression levels are shown normalized to G6pdx. Data (mean ± SEM, n = 2) are representative of three independent experiments.",
    "molecules": "15d-PGJ2, EC, R837"
  },
  {
    "caption": "A C57BL/6mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 µm.B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test.",
    "molecules": "DPPC, EC, D-galactosamine, LPS"
  },
  {
    "caption": "D, E C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.",
    "molecules": "DPPC, EC, D-galactosamine, LPS"
  },
  {
    "caption": "C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.",
    "molecules": "DPPC, EC, D-galactosamine, LPS"
  },
  {
    "caption": "Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.",
    "molecules": "EC, R837, OxPL"
  },
  {
    "caption": "Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.",
    "molecules": "cEC, EC, R837"
  },
  {
    "caption": "mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression. BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.",
    "molecules": "DPPC, cEC, R837"
  },
  {
    "caption": "ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in (E).Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h.",
    "molecules": "EC, R837, OxPL"
  },
  {
    "caption": "mRNA quantification of the indicated chemokines relative to G6pdx expression. BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.",
    "molecules": "BisRed, cEC, R837"
  },
  {
    "caption": "Quantification and characterization of cellular infiltrates in BAL. Groups of six C57BL/6mice were pretreated i.t. with 50 μg cEC, EC, or BisRed 24 and 2 h before challenge with 150 ng/g LPS in the presence of 800 μg/g D-galactosamine. BAL was harvested 4 h after LPS injection, and inflammatory cells were characterized by FACS analysis. **P < 0.01; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test.",
    "molecules": "BisRed, cEC, EC, D-galactosamine, LPS"
  },
  {
    "caption": "Comparison of the capacity of cEC, EC, and PECPC (1 μM) to license splenicdendritic cells to polarize naïve CD4T cells into IFN-γ-producing (Th1) and IL-4-producing (Th2) effector cells. Data (mean ± SD) are representative of two independent experiments. One-way ANOVA adjusted by Dunnett's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.",
    "molecules": "cEC, EC, PECPC"
  },
  {
    "caption": "Representative immunofluorescence of primary cilium in WT or Mecp2 null MEF cells. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Merge of all channels and DAPI staining (blue) is depicted for both genotypes. Scale bar 20 µm, and 10 µm in the enlarged image. Arrows indicate not ciliated cells.",
    "molecules": "DAPI"
  },
  {
    "caption": "MECP2-silenced or control hTERT-RPE-1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies. The percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *** p<0.001, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA demonstrated a significant silencing effect (F (1, 12) = 160; p<0.0001), a significant SAG treatment (F (2, 12) = 259.4; p<0.0001) and a significant interaction (F (2, 12) = 36.85; p<0.0001).",
    "molecules": "SAG"
  },
  {
    "caption": "WT or Mecp2 null MEF cells were treated with SAG (200 and 500 nM) for 24 h and the percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant genotype effect (F (1, 12) = 11.22; p<0.01) and a significant SAG treatment (F (2, 12) = 11.7; p<0.0001).",
    "molecules": "SAG"
  },
  {
    "caption": "Gli1 protein levels were measured by Western blot in untreated (UT) and 100 nM SAG-treated WT and Mecp2 null MEF cells (n=5/6). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (**p<0.01, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant interaction (F (1, 18) = 6.652; p<0.05), a significant genotype effect (F (1,18) = 15.95; p<0.001) and a significant treatment (F (1, 18) = 20.15; p<0.001).",
    "molecules": "SAG"
  },
  {
    "caption": "Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons, following SAG treatment (200 and 400 nM). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (*p<0.05, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant interaction (F (2, 20) = 3.788; p<0.05), a significant genotype effect (F (1, 20) = 4.362; p<0.05) and a significant treatment (F (2, 20) = 13.5; p<0.001).",
    "molecules": "SAG"
  },
  {
    "caption": "Gli1 mRNA expression levels upon SAG exposure in MEF cells (n=7 WT and n=9 null samples) (F; SAG 100 nM) and cortical neurons (n= 15 WT and n=12 null samples) (G; SAG 400 nM). The graphs show the mean ± S.E. of the fold change derived from at least three independent experiments (mean ± S.E., **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test).",
    "molecules": "SAG"
  },
  {
    "caption": "Representative immunostaining of Arl13b (red) and γ-tubulin (green) in the P14 mouse cortical layers. Nuclei were stained with DAPI (blue). Scale bar 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative immunostaining of Arl13b (green) in the internal (IGL) and external (EGL) granular layers of Mecp2 null (n=3) and heterozygous (het) cerebella (n=3) at P7, and the corresponding WT (n=3 males and n=3 females). Nuclei were stained with DAPI (blue). Scale bar 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "WT and Mecp2 null MEF cells were starved for 48 h and then treated with the HDAC6 inhibitor tubacin (TUB) (1 μM) for 48 h or with DMSO (VEH) or left untreated (UT). Cells were fixed, stained for anti-acetylated α-tubulin and cilia were counted. The graph shows the percentage of ciliated cells out of three independent experiments (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F(1, 10) = 17.94; p<0.01), a significant tubacin treatment (F(2, 10) = 4.425; p<0.05) and a significant interaction (F(2, 10) = 7.266; p<0.05).",
    "molecules": "DMSO, TUB, tubacin"
  },
  {
    "caption": "Primary cortical neurons were treated at DIV5 with DMSO (VEH) or tubacin (TUB, 1 μM) for 48 h, or left untreated (UT), fixed at DIV7 and stained with anti-AC3 antibody. The graph shows the percentage of ciliated cells obtained from three independent experiments counting n=300 UT cells, n=216 VEH treated cells, n=267 TUB treated cells for each genotype (mean ± S.E., *p<0.05, **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant genotype effect (F (1, 15) = 23.42; p<0.001), a significant treatment effect (F (2, 15) = 11.25; p<0.01) and a significant interaction (F (2, 15) = 4.853; p<0.05).",
    "molecules": "DMSO, TUB, tubacin"
  },
  {
    "caption": "MECP2 silenced or control hTERT-RPE-1 cells were starved for 48 h, treated with tubacin (TUB, 1 µM) for 24 h and then exposed to 200 nM SAG for 24 h in the presence of tubacin. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies and the percentages of Smo-positive cilia were calculated. The graph shows the averages of three independent experiments (mean ± S.E., **p<0.01, by three-way ANOVA followed by Tukey's post hoc test). Statistical analysis reported a significant difference between genotype (F (1, 2) = 21.47; p<0.001), a significant SAG effect (F (2, 2) = 154.5; p<0.0001) and a significant tubacin effect (F (1, 2) = 25.18; p<0.001). In details, a significant interaction between genotype and SAG treatment (F (2, 2) = 5.413; p<0.05) and between SAG and tubacin treatment (F (2, 2) = 6.939; p<0.01) were reported, demonstrating respectively a difference between CTRL and MeCP2 depleted cells in response to SAG and a different SAG effect when cells were treated with tubacin.",
    "molecules": "SAG, TUB, tubacin"
  },
  {
    "caption": "Representative immunostaining of Smo (green) and acetylated α-tubulin (red) in control (CTRL) and silenced (siMeCP2) hTERT-RPE-1 cells, after stimulation with 200 nM SAG in the presence or absence of the HDAC6 inhibitor tubacin (TUB). Scale bar 50 µm, and 20 µm in the enlarged image.",
    "molecules": "SAG, TUB, tubacin"
  },
  {
    "caption": "Representative images of MAP2 positive WT and Mecp2 null neurons (DIV7). At DIV5 neurons were treated with tubacin (TUB, 1µM for 48 h), Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h) or left untreated and morphologically analyzed by Sholl analysis plug-in. Scale bar 20 µm. The graph reports the mean ± S.E. of the number of intersections measured 85 to 145 nm from the soma calculated by Sholl analysis for WT (n=39 neurons) and Mecp2 null neurons, when left untreated (n=57 neurons) or treated with tubacin (n=53 neurons) or TC-S 7010 (n=38 neurons). Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, **p<0.01).",
    "molecules": "TC-S, TC-S 7010, TUB, tubacin"
  },
  {
    "caption": "Representative images of WT and Mecp2 null neurons (DIV14) immunostained for MAP2 (white), Synapsin1/2 (green) and Shank2 (red). Mecp2 null cells were treated at DIV12 with tubacin (TUB, 1µM for 48 h) or Aurora A inhibitor TC-S 7010 (TC-S, 7 nM for 48 h). Scale bar 15 µm, and 5 µm in the enlarged image.",
    "molecules": "TC-S, TC-S 7010, TUB, tubacin"
  },
  {
    "caption": "The graphs represent the mean ± S.E. of the Synapsin1/2 and the Shank2 puncta density in WT (n=67 neurons) and Mecp2 null neurons, when untreated (n=51 neurons) or treated with tubacin (n=59 neurons) or TC-S 7010 (n=55 neurons). Neurons derived from at least 6 different biological replicates. Data from WT and null neurons were compared by Mann-Whitney test (**p<0.01, ***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05, ***p<0.00­­1). The graph depicts the mean ± S.E. of the number of co-localized pre- and post-synaptic puncta (n=20 neurons deriving from 3 different biological samples). Data from WT and null neurons were compared by Mann-Whitney test (***p<0.001); drug effects on null cells were analyzed by one-way ANOVA followed by Dunn's post hoc test (*p<0.05).",
    "molecules": "TC-S 7010, tubacin"
  },
  {
    "caption": "Representative immunostaining of primary cilia and MeCP2 in cultured human fibroblasts from healthy controls (CTRL, n=3) and RTT patients. Fibroblasts were starved for 24 h and then stained for acetylated α-tubulin (green), MeCP2 (red) and DAPI (blue). Scale bar 50 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "The effect of tubacin (TUB) treatment (250 nM, 24 h) on primary cilium length of controls (CTRL) and RTT fibroblasts is reported. The graph indicates the mean ± S.E of the cilium length analyzed in untreated (n=130 CTRL; n=73 705delG; n=57 Q244X; n=68 R255X) and treated cells (n=25 CTRL; n=26 705delG; n=26 Q244X; n=49 R255X) (***p<0.001 vs untreated CTRL, §p<0.05 vs corresponding mutated untreated fibroblasts by two-way ANOVA, followed by Bonferroni post hoc test). Statistical analysis reported a significant treatment effect (F (1,429) = 26.56; p<0.0001) and a significant genotype (F(3,429) = 42.85; p<0.001).",
    "molecules": "TUB, tubacin"
  },
  {
    "caption": "B  Western blots of whole-cell extracts showing efficient degradation of PDS5A   or PDS5B  (left, right) with dTAG  treatment (at different  time points).",
    "molecules": "dTAG"
  },
  {
    "caption": "D  ChIP-seq  signal for FLAG-PDS5A  and FLAG-PDSB  (ChIP  assays were performed with antibodies against Flag  and PDS5A/B) centered at TSS of promoters (± 3 kb) across all peaks called for each of the proteins in DMSO  treated PDS5A-dTAG  or PDS5B-dTAG  cells, showing the drastic reduction in PDS5A/B enrichment on chromatin  after degradation. Top: normalized signal intensities for each protein.",
    "molecules": "DMSO, dTAG"
  },
  {
    "caption": "A Validation of PDS5A;PDS5B-dTAG  cells for simultaneous depletion of PDS5A  and PDS5B.",
    "molecules": "dTAG"
  },
  {
    "caption": "D Changes of RAD21  signals at all loop anchors defined in PDS5A-dTAG  cells or PDS5B-dTAG  cells with DMSO  treatment after PDS5A  and PDS5B  co-depletion, or either depletion. For PDS5A;PDS5B-dTAG  cells, we used the genomic positions of loop anchors identified in DMSO  treated PDS5A-dTAG  cells. E Differential RAD21 ChIP-seq signals of representative regions showing significantly down-regulated peaks after co-depletion of PDS5A  and PDS5B. F Differential RAD21 ChIP-seq signals of representative regions showing significantly up-regulated peaks after co-depletion of PDS5A  and PDS5B.",
    "molecules": "DMSO, dTAG"
  },
  {
    "caption": "(c) P- and total MEK and ERK localize on to the cytoplasmic/extra-luminal face of APh in vivo. Left; model depicting localization of MEK and ERK on the extra-luminal face of autophagic vesicles and the ability of trypsin to degrade extra-luminal MEK and ERK, and right; immunoblots for indicated proteins in APh fractions from mice livers untreated (−) or treated with increasing amounts of trypsin for 15 min. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with corresponding trypsin-untreated value; ANOVA-Bonferroni post hoc test, n=3. The p44, p42 forms of ERK, LC3-I and membrane-associated LC3-II, and 37 kDa and 25 kDa forms of cathepsin (Cath) B are indicated.",
    "molecules": "trypsin"
  },
  {
    "caption": "(c) ATG4B depletion does not increase autophagic flux in serum-fed cells. Immunoblots for indicated proteins in lysates from scr or siATG4B NIH/3T3 cells in presence/absence of lysosomal inhibitors, ammonium chloride and leupeptin (Inh) for 2 h. Scale bars, 1 μm. Bars represent mean±s.e.m., n=3.",
    "molecules": "ammonium chloride, leupeptin"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/mL 555-EGF for the indicated durations. Scale bar: 10 μm. ",
    "molecules": "555, Alexa 555"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.",
    "molecules": "555"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Quantification of percentage of total EGFR vesicles that colocalise with 555-EGF Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.",
    "molecules": "555"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. A: Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/mL EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3)P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm. B: Quantification of PI(3)P+ label intensity per cell (in A). C: Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "3'MA, Alexa 488, PI(3)P"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. F: Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/mL EGF for 15 min then lysed in CHAPS-containing detergent buffer and binding partners detected by western blotting. G: Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "detergent buffer, CHAPS"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells transiently expressing YFP-Gal8 were stimulated with 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min. Scale bar: 10 μm. D: Quantification of the percentage of YFP-Gal8-labelled vesicles that colocalise with 555-EGF in control or ATG7-deficient cells (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.",
    "molecules": "555, Alexa 555"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Cells transiently expressing YFP-Gal8 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. F: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Gal8 upon monensin treatment (in E). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001. ",
    "molecules": "monensin"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells stably expressing GFP-LC3 were treated with 100 μM monensin in the presence or absence of bafilomycin A1 (20 nM) for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. White arrows indicate colocalisation. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with GFP-LC3 (in A). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "bafilomycin A1, monensin"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells were treated for 1 hr with 100 μM monensin then stimulated with 2 ng/mL EGF for 15 min. LAMP2 and EEA1 were then detected by immunofluorescence staining. White arrows indicate colocalisation. Scale bar: 10 μm. D: Quantification of the percentage of total EEA1 vesicles that colocalise with LAMP2 (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "monensin"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). G: Cells stably expressing Flag-S-ATG16L1 were treated for 1 hr with either 100 μM monensin or 30 μM Dynasore, then stimulated with 2 ng/mL EGF for 15 min. Cells were then stained by immunofluorescence against EEA1 and Flag tag. Scale bar: 10 μm. H: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 (in G). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "Dynasore, monensin"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). I: Untreated control or sgAtg7 cells, or control cells pre-treated with 100 μM monensin 1 hr, stably expressing Flag-S-ATG16L1 were stimulated 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min before fixation and immunofluorescence staining against Flag tag and EEA1. Cells were then imaged by structured illumination microscopy (SIM) and images were reconstructed in Nikon Elements software. Scale bar: 10 μm. J: Quantification of the percentage of EGF-EEA1 colocalised vesicles that stained triple-positive with ATG16L1 by SIM (in I). Due to the low throughput nature of this assay, the following cell numbers were counted: control untreated (9 cells), sgAtg7#1 (10 cells), sgAtg7#2 (9 cells), and control + monensin: (11 cells). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "555, Alexa 555, monensin"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells stably expressing Flag-S-ATG16L1 were pre-treated for 1 hr with TBK1 inhibitors (100 μM MRT68601 or 5 μM momelotinib). Control cells were also treated 100 μM monensin for 1 hr as indicated. All cells were stimulated for 15 min with 2 ng/mL EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm.",
    "molecules": "momelotinib, monensin, MRT68601"
  },
  {
    "caption": "The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Control and sgGal8 cells stably expressing Flag-S-ATG16L1 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 and Flag tag. White arrows indicate colocalisation. Scale bar: 10 μm. G: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 in sgGal8 cells (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.",
    "molecules": "monensin"
  },
  {
    "caption": "A: Glial shNf-1/shTp53 control and sgAtg7 cells were serum starved 4 hr then stimulated with 20 ng/mL TexasRed-EGF (TxRed-EGF) for 30 min. Scale bar: 10 μm. B: Quantification of TxRed-EGF puncta per cell, relative to the number taken up by control cells (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.",
    "molecules": "TexasRed, TxRed"
  },
  {
    "caption": "C: Endocytosis rate of wild type (WT) EGFR was assayed in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells by cell surface biotinylation and one application of 2 ng/mL EGF for 15 min. Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.",
    "molecules": "Monensin"
  },
  {
    "caption": "D: Plasma membrane recycling rate of WT EGFR in control, sgAtg7, or sgAtg16l1 glial shNf-1/shTp53 or EGFRvIII-expressing cells was assayed by cell surface biotinylation and successive applications of 2 ng/mL EGF treatments for 15 min Monensin treatment was added as indicated. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, *** p<0.001.",
    "molecules": "Monensin"
  },
  {
    "caption": "H: Control or sgAtg7 glial shNf-1/shTp53 cells were serum starved for 24 hr in the presence or absence of 20 ng/mL EGF. Quantification of the percentage of propidium iodide (PI)-positive cells is shown. Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.",
    "molecules": "PI, propidium iodide"
  },
  {
    "caption": "HT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1",
    "molecules": "coumermycin A1, BV-6, z-VAD-fmk"
  },
  {
    "caption": "Thioflavin T fluorescence as a function of time, following dilution of WT or AAAA His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 from 8 M to 200 mM urea-containing buffer. Buffer sample contains ThT but no protein. Samples were tested in triplicate and solid line is the average of the triplicates and the error bars (in grey) represent +/- s.d",
    "molecules": "buffer, Buffer, His, Thioflavin T, ThT, Ub, urea"
  },
  {
    "caption": "Absorbance spectra from solutions containing Congo red with WT (black lines) or AAAA mutant (dark grey lines) His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 that have been dialysed against Assembly Buffer. Congo red sample alone with no protein (light grey line) served as a control for the experiment. Experiment conducted once",
    "molecules": "Buffer, Congo red, His, Ub"
  },
  {
    "caption": "Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown here",
    "molecules": "FITC"
  },
  {
    "caption": "Representative electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparations",
    "molecules": "His, Ub"
  },
  {
    "caption": "SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane.",
    "molecules": "SDS, urea"
  },
  {
    "caption": "SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45.",
    "molecules": "SDS"
  },
  {
    "caption": "SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.",
    "molecules": "SDS"
  },
  {
    "caption": "A-B. Wild-type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al., 2018). Shown are volcano plots of two replicates depicting changes in: protein abundance (A) and thermostability (B). A local FDR (false discovery rate) <0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were colored grey and not labelled to increase the plot clarity.",
    "molecules": "TMT"
  },
  {
    "caption": "C-D. Affinity chromatography with immobilised NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 p-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were colored grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins.",
    "molecules": "NaCl"
  },
  {
    "caption": "A. HPLC-based PG digestion assay representing EPase activity. The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods. MepM and PBP4 were incubated with sacculi, whilst MepS and PBP7 were incubated with soluble muropeptides, both from E. coli MC1061, respectively. Values are mean ± standard deviation of three independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)",
    "molecules": "muropeptide, muropeptides"
  },
  {
    "caption": "G. Relative fitness of ΔnlpI, ΔmepS and ΔnlpIΔmepS mutants. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10% sucrose, or LB agar plates containing 0 mM or 500 mM NaCl. Plates were incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants.",
    "molecules": "NaCl, sucrose"
  },
  {
    "caption": "H. Cells of wildtype (WT), ΔnlpI, ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity. CPRG (yellow) cannot penetrate intact Gram-negative envelopes. Its conversion by intracellular β-galactosidase to CPR (red) indicates loss of envelope integrity.",
    "molecules": "agar, CPR, CPRG"
  },
  {
    "caption": "B GFP‐KSHV‐TK‐induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y‐27632 and blebbistatin, respectively. Error bars represent SEM from three independent experiments, n = 213 for DMSO, n = 189 for Y‐27632 and n = 245. ***P 0.001.",
    "molecules": "blebbistatin, DMSO, Y‐27632"
  },
  {
    "caption": "C Immunoblot analysis of rhotekinpull‐down (P.D.) assays reveals that GFP‐KSHV‐TK increases the level of GTP‐bound RhoA (GTP‐RhoA). Actin is included as a loading control, and the input lysates are indicated.",
    "molecules": "GTP"
  },
  {
    "caption": "D Immunofluorescence microscopy analysis reveals that GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) does not alter focal adhesion integrity (paxillin) in the presence of the ROCK inhibitor Y‐27632.",
    "molecules": "Y‐27632"
  },
  {
    "caption": "E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV.219/Vero cell line in the presence or absence of Y‐27632.",
    "molecules": "Y‐27632"
  },
  {
    "caption": "F Immunoblot analysis of level of GTP‐bound and total RhoA in cells expressing the indicated GFP‐tagged KSHV‐TK proteins.",
    "molecules": "GTP"
  },
  {
    "caption": "C. N2a cells transfected with Atoh1-GST plasmids were treated with DMSO(Control), Palbociclib (Palb) or Palb and 10 uM MG132 (MG132) for 6hrs before harvest. Lysates of N2a were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between Control, Palb, MG132. Bars and error bars are representing as Mean ± SEM from three trials of each condition. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Three slots of each experimental condition represent the three biological replicants of each condition.",
    "molecules": "DMSO, MG132, Palb, Palbociclib"
  },
  {
    "caption": "G. Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids (4D). N2a cells were treated with 10 nM, 100 nM, 1000 nM Palb for 6 hrs before harvest. Lysates from N2a cells were subjected to immunoblotting with anti-Atoh1, p-S309-Ab and β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between 0, 10, 100, 1000nM. Bars and error bars are representing as Mean ± SEM from three trials of each condition. **p < 0.01, *p < 0.05, N.S.: Not Significant.",
    "molecules": "Palb"
  },
  {
    "caption": "L. ChIP assay in P7 mouse cerebellum with anti-Prox1 or goat IgG as a negative control. Primer set used in this experiment was designed to amplify Ccnd1 promoter region. M-R. Administration of Valproic acid (VPA) to electroporated mice. (M) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation. Immunostaining for CCND1 (magenta) was performed the day following the last VPA treatment. Nuclei of electroporated cells were detected with GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with CCND1. (N-Q) Images of H2B-GFP with Saline (N), PROX1 with Saline (O), H2B-GFP with VPA (P), and PROX1 with VPA (Q). (R) The relative proportions of CCND1+ cells among the electroporated cells (normalized to the Control).Tukey-Kramer test among Control+saline (N=4), PROX1+saline (N=3), Control +VPA (N=3) and PROX1 +VPA (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, *p <0.05, N.S.: Not Significant. Number of cells: 120, 488, 934 and 211 from N=4 mice (Control+saline), 630, 356 and 545 from N=3 mice (Control+VPA), 363, 127 and 219 from N=3 mice (Prox1+saline), 110, 116 and 202 from N=3 mice (Prox1+VPA).",
    "molecules": "Saline, saline, Valproic acid, VPA"
  },
  {
    "caption": "RT-PCR analysis (top) and representative direct sequencing (bottom) of Ttn exon 326 transcripts from HL-1 cardiomyocytes transiently transfected with different 2OMePS AONs. Only 2OMePS-AON1 and 2OMePS-AON1 + 3 (*) lead to a correct excision of exon 326 as confirmed by direct sequencing.",
    "molecules": "2OMePS"
  },
  {
    "caption": "Immunofluorescence images of sarcomeric α-actinin in untransfected and 2OMePS-AON1 + 3-transfected HL-1 cardiomyocytes. Scale bars, 10 μm.",
    "molecules": "2OMePS"
  },
  {
    "caption": "RT-PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.",
    "molecules": "2OMePS"
  },
  {
    "caption": "Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.",
    "molecules": "Iso"
  },
  {
    "caption": "B, C, Analysis of GFP transfer after transplantation of Nrl::GFP donor photoreceptors into wildtype C57BL/6J (B) and Nrl-/- (C) recipient retinas after 14 (wt, n=9; Nrl-/-, n=7) and 21 (wt, n=6; Nrl-/-, n=4) and 90 (wt, n=5; Nrl-/-, n=4) days. Tissue is stained for GFP and nuclei (Hoechst). In this and all following figures, donor cells (D) are located above, and acceptor cells with GFP (A) are located below the outer limiting membrane (white dashed line), which delimits the apical border of the outer nuclear layer (ONL). In wildtype recipients, donor cells are typically located above the outer segment region (between the grey dashed lines). In Nrl-/- recipients, donor cells are located in the inner segment (IS) region. Plotted is the ratio of the numbers of GFP+ photoreceptors in the ONL (acceptor photoreceptors) and GFP+ donor photoreceptors in the subretinal space (SRS) over time.",
    "molecules": "Hoechst"
  },
  {
    "caption": "F, G, C57BL/6J cells with mitochondria labelled with MitoTrackerTM-Green (MTG) or MitoTrackerTM-Red (MTR) exchange mitochondria after 3 DIV (0 DIV, n=6; 3 DIV, n=6). White dashed line delimitates both cell somas and the protrusion connecting them (F). No exchange is observed when the two photoreceptor populations are separated by a transwell (n=3), nor in co-cultures of live MTG-labelled and lysed MTR-labelled cells (n=3) (G).",
    "molecules": "MitoTrackerTM-Green, MTG, MitoTrackerTM-Red, MTR"
  },
  {
    "caption": "A, B, Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin+ (SirActin) and GFP+ (A) and tubulin+ (ViaFluor) and MitoTracker Green+ (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy.",
    "molecules": "ViaFluor, MitoTracker Green, MTG"
  },
  {
    "caption": "E, Time lapse imaging showing movement of MitoTracker Green+ puncta in photoreceptors connected by a protrusion. White arrows point MTG+ puncta inside of the protrusion during the time lapse imaging.",
    "molecules": "MitoTracker Green, MTG"
  },
  {
    "caption": "(A) Expression in E. coli and purification of human GS (hGS). Protein was separated by SDS-PAGE and stained with Coomassie Blue dye. M: marker Precision Plus Protein Dual Color Standard (Biorad). From right Lanes 1-4: E. coli Bl21(DE3) cells containing the expression vector without (lanes 1 and 2) and with (lanes 3 and 4) the coding sequence of hGS. Samples were taken immediately before (lanes 1 and 3) and 3,5 h later (lanes 2 and 4) the induction of expression with isopropil-β-D-1-tiogalattopiranoside (IPTG) 0.7mM. The same number of bacteria was analyzed in each sample. Lane 5: isolated and purified inclusion bodies (4 μg). Adiacent boxed lane: Western Blotting analysis of GS.",
    "molecules": "Coomassie Blue, IPTG, isopropil-β-D-1-tiogalattopiranoside"
  },
  {
    "caption": "Kinetic study of the reaction catalyzed by recombinant hGS. The reaction, started by adding ATP, was linear for at least 10 min at 25°C (B).",
    "molecules": "ATP"
  },
  {
    "caption": "Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of glufosinate (C) Symbols- (): 0.025 glufosinate; or 0.050 mM of glufosinate; or 0.065 mM of glufosinate; 0.075 mM of glufosinate.",
    "molecules": "glufosinate, glutamate"
  },
  {
    "caption": "Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of MSO Symbols- (): 2 mM MSO ; () 3 mM of MSO ; () 4 mM of MSO ; () 5 mM of MSO",
    "molecules": "glutamate, MSO"
  },
  {
    "caption": "Kinetic study of the reaction catalyzed by recombinant hGS. The insets represent the secondary plot of the slopes of Lineweaver-Burk plot obtained at the indicated concentrations of glufosinate (C) or MSO (D) used for determining the inhibitor constant Ki. GS inhibition (%) in presence of increasing concentrations of glufosinate () or MSO () results from the average of at least three independent experiments (E). The control value for uninhibited hGS activity is 0.51 ±0.08 μmol/(min x mg protein).",
    "molecules": "glufosinate, MSO"
  },
  {
    "caption": "Evaluation of M1 markers in macrophages by real-time PCR. Fold change of TNFA, CD80, CXCL9, and CXCL10 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3). Evaluation of M2 markers in macrophages by real-time PCR. Fold change of MRC1, MSR1, mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).",
    "molecules": "glufosinate, MSO"
  },
  {
    "caption": "Evaluation of M2 markers in macrophages by real-time PCR. Fold change of CCL17 and CCL18 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).",
    "molecules": "glufosinate, MSO"
  },
  {
    "caption": "Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of TNFA, CXCL10, CD86 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).",
    "molecules": "acriflavine, glufosinate"
  },
  {
    "caption": "Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of CD80 and CXCL9 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3). Evaluation of M2 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of MRC1, MSR1, and CCL18 mRNA in IL10, glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).",
    "molecules": "acriflavine, glufosinate"
  },
  {
    "caption": "(Q) Quantification of cancer cell motility through a matrigel coated membrane in presence of IL10, MSO/IL10 and glufosinate (10 and 20µM)- IL10 treated macrophages after 24 h of incubation (n=6).",
    "molecules": "glufosinate, MSO"
  },
  {
    "caption": "(R) Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 after 4 h of incubation with HUVEC cells (n=6).",
    "molecules": "glufosinate, MSO"
  },
  {
    "caption": "(S) CD8+ T cell suppression by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.",
    "molecules": "glufosinate, MSO, thymidine"
  },
  {
    "caption": "(T) CD8+ T cell recruitment in a transwell system by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h versus macrophages treated with LPS/IFNγ; the migration of T cells cultured without macrophages (Mφ-) in presence of CXCL10 was used as positive control (n=4).",
    "molecules": "glufosinate, LPS, MSO"
  },
  {
    "caption": "(U) Representative image of Western blotting analysis of HIF1α, REDD1, 4E-BP1, S6 and P70S6K (in their phosphorylated and unphosphorilated form) to test mTOR activation in normoxic (NRX) and hypoxic (HYP) IL10 macrophages treated with glufosinate (20µM), rapamycin (20nM) and a combination of both (n=3).",
    "molecules": "glufosinate, rapamycin"
  },
  {
    "caption": "(V) CD8+ T cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate (20µM), rapamycin and a combination of both for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.",
    "molecules": "glufosinate, rapamycin, thymidine"
  },
  {
    "caption": "Quantification of glufosinate (B) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Quantification of glutamine (GLN) (C) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).",
    "molecules": "glufosinate, GLN, glutamine"
  },
  {
    "caption": "number of lung metastatic nodules in vehicle and glufosinate (10 and 20mg/kg) treated mice (E) (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Quantification and representative images of F4/80+ CD11c+ cells in the lung metastasis (F), F4/80+ CD206+ (G) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Quantification and representative images of F4/80 CD11c+ (H) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "RT-PCR quantification of M2 (Ccl22, Arg1 and Ccl17) (I) markers in vehicle and glufosinate treated mice (n=4).",
    "molecules": "glufosinate"
  },
  {
    "caption": "RT-PCR quantification of M1 (Tnfa, Cxcl9, Nos2, Cd86 and Cd80) (J) markers in vehicle and glufosinate treated mice (n=4).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Quantification of total length of vessels (A) in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Quantification of CD31+ tumor vessel area (B) and of αSMA+ pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(D) Quantification and representative images of Lectin+ tumor vessel area over the total number of CD31+ vessels in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate, Lectin"
  },
  {
    "caption": "(E) Quantification and representative images of PIMO+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate, PIMO"
  },
  {
    "caption": "FACS quantification of TCRbeta+ CD8+ cytotoxic T cells in tumors of vehicle and glufosinate-treated mice (F).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(I) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate-treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(B) Evaluation of body weight in vehicle and glufosinate (20mg/kg) treated mice (n=8). (C) Representative images of metastasis at the beginning and end of therapy. Evaluation of the metastatic burden following glufosinate treatment by H&E staining (n=8). ",
    "molecules": "glufosinate"
  },
  {
    "caption": "(D) Evaluation of lung total metastatic area and of lung metastatic lesions per size by H&E staining in vehicle and glufosinate (20mg/kg) treated mice (n=8).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Quantification (F) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).",
    "molecules": "glufosinate"
  },
  {
    "caption": "representative images (G) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Intradermal YUMM 1.7 (CD90.1+) tumor growth over the time (B) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "number of metastatic lesions per lung (C) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(D) Representative images of metastatic lesions checked by DAB-H stain for CD90.1 positivity in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per lung were analysed. Scale bar: 2mm. Zoomed area scale bar: 50µm.",
    "molecules": "DAB-H, glufosinate"
  },
  {
    "caption": "(E, F) FACS quantification of M1-like MHC class IIhigh (E) and MHC class IIlow (F) CD11c+ CD206- TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(G) Quantification and representative images of CD11c+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.",
    "molecules": "glufosinate"
  },
  {
    "caption": "FACS quantification of M2-like MHC class IIhigh CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "FACS quantification of M2-like MHC class IIlow (I) CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(J) Quantification and representative images of CD206+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.",
    "molecules": "glufosinate"
  },
  {
    "caption": "FACS quantification of M1-like MHC class IIhigh (K) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "FACS quantification of M1-like M2-like MHC class IIlow (L) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(N) RT-PCR quantification of M2 (Arg1 and Cxcr4) and M1 (Cxcl9 and Nos2) markers in vehicle and glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "CD31+ tumor vessel area (B) and of αSMA+pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.",
    "molecules": "glufosinate"
  },
  {
    "caption": "(D) Quantification and representative images of CA9+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.",
    "molecules": "glufosinate"
  },
  {
    "caption": "(E, F) FACS quantification of CD8+ cytotoxic T cells (E) and CD69+ CD8+ T cells (F) over the total number of CD45+ in vehicle or glufosinate (20mg/kg) treated mice (n=6).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Evaluation of 4T1 tumor growth over the time (B) , in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "mice body weight (C) in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "number of metastatic nodules (D), in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Total metastatic area (E) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.",
    "molecules": "glufosinate"
  },
  {
    "caption": "number of lung metastatic lesions per size by H&E staining (F) with representative images (G) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.",
    "molecules": "glufosinate"
  },
  {
    "caption": "(H, I) Quantification of F4/80+ macrophage density (H), of F4/80+ CD206+ cells infiltration in the lung metastasis and representative images (I), in vehicle and glufosinate-treated mice (n=6). Six images per lung were analysed. Scale bar: 20µm.",
    "molecules": "glufosinate"
  },
  {
    "caption": "(O) GFP+ cancer cells amount in lungs, expressed as GFP expression levels, was determined by qPCR in vehicle and glufosinate (10mg/kg) treated mice (n=8). The results were normalized to the vehicle group.",
    "molecules": "glufosinate"
  },
  {
    "caption": "(A) Time course evaluation of murine body weight during treatment in vehicle and glufosinate (10 and 20 mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "(B) Blood counts of WBC and RBC in vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Evaluation of liver toxicity following glufosinate treatment. Quantification of liver glufosinate (C) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Evaluation of liver toxicity following glufosinate treatment. hepatotoxicity markers ALT (Alanine Aminotransferase) and AST (Aspartate aminotransferase) (D) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Evaluation of neuronal toxicity following glufosinate treatment. Quantification of brain glufosinate (E)",
    "molecules": "glufosinate"
  },
  {
    "caption": "Evaluation of neuronal toxicity following glufosinate treatment. grip strength for front paws (triangular bar) (F), as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "Evaluation of neuronal toxicity following glufosinate treatment. grip strength for all paws (rectangle grid) (G), of the latency to fall off an accelerating rotarod (H), and of the mean paw withdrawal force that caused animals' response in the von Frey test (left hindpaw, I; right hindpaw, J) as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).",
    "molecules": "glufosinate"
  },
  {
    "caption": "B SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment for one hour with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-).",
    "molecules": "SDS"
  },
  {
    "caption": "C Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg2+. Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1).",
    "molecules": "EDTA, Mg2+"
  },
  {
    "caption": "C Effect of an RNase A inhibitor (RNasin) on RNase A mediated protein aggregation in neuronal cell lysate.",
    "molecules": "RNasin"
  },
  {
    "caption": "B Effect of RNA/protein ratio on protein aggregation after renaturing.",
    "molecules": "RNA"
  },
  {
    "caption": "C Coomassie-stained gel of soluble (Sup 1/2) and aggregated (Pel 1/2) proteins after removal of GuHCl in the presence (+) or absence (-) of total RNA. Asterix (*) denotes added RNase A.",
    "molecules": "GuHCl, RNA"
  },
  {
    "caption": "soluble (Sup 1/2) and aggregated (Pel 1/2) proteins after removal of GuHCl in the presence (+) or absence (-) of total RNA. D As in (c) but analysed by Western blot.",
    "molecules": "GuHCl, RNA"
  },
  {
    "caption": "E ATP-hydrolysing activity of proteins renatured with total RNA or buffer (Ve-) after removal of GuHCl.",
    "molecules": "GuHCl, RNA"
  },
  {
    "caption": "A Genomic attributes of RNA associated with renatured NF-H, Tau, or Aβ. Inset shows which fractions of the peaks that derive from exons or introns have their origin in coding or non-coding transcripts, respectively.",
    "molecules": "RNA"
  },
  {
    "caption": "B Characterization of DNA associated with soluble, renatured Aβ or all proteins (Total), captured either by immunoprecipitation (Aβ ) or absorption to membranes (Total).",
    "molecules": "DNA"
  },
  {
    "caption": "A Proportion of aggregated proteins after renaturation with either single-stranded (Forward or Reverse) or complementary strands (For/Rev) of the selected M1x4 or the control oligonucleotide, M2x4.",
    "molecules": "oligonucleotide"
  },
  {
    "caption": "B Western blot analysis of proteins renatured with either single-stranded or double-stranded M1x4 or M2x4 DNA oligonucleotides.",
    "molecules": "DNA oligonucleotides"
  },
  {
    "caption": "C The effect on protein re-aggregation from varying the number of DNA motif repeats when complementary oligonucleotides are given together.",
    "molecules": "complementary oligonucleotides"
  },
  {
    "caption": "D Protein aggregation after renaturation with the M1x4 DNA oligonucleotides pre-annealed with 100 mM NaCl or vehicle (None).",
    "molecules": "DNA oligonucleotides, NaCl"
  },
  {
    "caption": "B Proportion of protein aggregation following renaturing with the ds/ss/ds oligonucleotides shown in (A).",
    "molecules": "oligonucleotides"
  },
  {
    "caption": "C Renaturing capacity of structurally different oligonucleotides. The diagrams on the right show a theoretical structure of each oligonucleotide. All oligonucleotides, except the 3x-loop and 3x-bulges, contain a stretch of 30 Ts in the single-stranded region and the same sequences (15 nucleotides each) in the double stranded regions. The 3x-loops and bulges oligonucleotides have 3 stretches of 9 Ts and the same sequence in the ds-regions.",
    "molecules": "oligonucleotide, oligonucleotides, Ts"
  },
  {
    "caption": "D Proportion of protein aggregation in Jurkat cell lysate supplemented with various amounts and configurations of the M1x4 or M2x4 DNA oligonucleotides.",
    "molecules": "DNA oligonucleotides"
  },
  {
    "caption": "e Insoluble proteins from two ALS brain tissues were chemically denatured in guanidine hydrochloride and treated with either buffer (Vehicle), total RNA from Jurkat cells or the complementary strands of the M1x4 DNA oligonucleotides (M1 F/R) (Pellet 1, top panel). After removal of GuHCl, the soluble fraction from these samples was treated with RNase and any aggregated proteins (Pellet 2), analysed by western blot (middle panel). Remaining supernatants were then treated with Benzonase to degrade any remaining nucleic acids and aggregated proteins collected by centrifugation and analysed by western blot (bottom panel). Proteins aggregated by enzymatic RNA degradation in Jurkat cell lysates, which do not contain NF-H, were used as a control.",
    "molecules": "guanidine hydrochloride, GuHCl, DNA oligonucleotides, RNA"
  },
  {
    "caption": "A. EGFR (pY1068, left), Akt (pS473, middle) and Erk (pT202 and pY204, right) phosphorylation response in WT (red) compared to p22phox-KO (green) MCF7 cells as function of EGF concentration (ng/ml; nM) upon 5' stimulation with different doses of EGF-Alexa647 quantified from Western blot analysis. N=4 biological replicates with mean±SD, P:unpaired two-tailed t-test. B. Same as (A) comparing WT (red) to RPTPγ-KO (blue) MCF7 cells. N=3 biological replicates with mean±SD, P: unpaired two-tailed t-test. C. Quantitative Western blot analysis as in (A) comparing WT (red) and RPTPγ-KO (blue) MCF7 cells after EGF stimulus (20, 80, 160 ng/ml from (B), left column: w/o gef.) to the cells from the corresponding cell line treated with 10 μM of the EGFR-inhibitor Gefitinib for 1 h and the indicated EGF concentration for the last 5' (ng/ml). N=3 biological replicates with mean±SD, P: unpaired two-tailed t-test. D.",
    "molecules": "Alexa647, Gefitinib"
  },
  {
    "caption": "E. Comparison of normalized EGFR- (pY1068-) phosphorylation as a function of EGF concentration (N=10, from Figs 1A, 1B, EV1E, red) to EGF-Alexa647 bound to WT MCF7 cells at the corresponding, indicated concentrations normalized to the 160 ng/mL, measured by fluorescence microscopy. N=5 biological replicates, n=16-19 fields of view, mean±SD, P: One-way ANOVA with Tukey's multiple comparison test. ",
    "molecules": "Alexa647"
  },
  {
    "caption": "A. Left panel: comparison of normalized EGF-Alexa647 (160 ng/ml; 5') fluorescence intensity bound to individual endogenous EGFR expressing MCF10A (yellow), to exogenous EGFR-mCitrine expressing EmCit_MCF7 cells (black) and WT MCF7 cells (red); Right panel: normalized EGF-Alexa647 fluorescence plotted against normalized EGFR-mCitrine fluorescence intensity in WT (red) and EmCit_MCF7 (black, with 2nd order polynomal fit: grey line) cells. N=3 biological replicates, n>75 cells, mean±SD.",
    "molecules": "Alexa647"
  },
  {
    "caption": "D. Gray: αL upon each administered dose for individual EmCit_MCF7 cells to cumulative doses of EGF-Alexa647 (2.5-640 ng/ml). N=3 biological replicates, n=13 cells. Black: EGF-Alexa647 bound to WT MCF7 cells at the indicated concentrations normalized to the mean fluorescence intensity at 160 ng/ml EGF-Alexa647 (Fig 1E; mean±SD, N=5 biological replicates, n=16-19 fields of view).",
    "molecules": "Alexa647"
  },
  {
    "caption": "F. Left: EGFR- (pY1068-) phosphorylation response in WT MCF7 cells obtained from western blots normalized to maximal phosphorylation obtained by inhibiting all phosphatases by 0.33 mM pervanadate (N=6; red symbols with mean±SD and fit to the hill equation (solid line)) at 0 (plotted as 0.001 to fit the logarithmic x-axis), 0.5, 1, 2, 5, 10, 20, 40 and 80 ng/ml plotted against corresponding αL (obtained from in cell dose response experiments in EmCit_MCF7 cells (D)). Correspondig molecular RPTPγ/EGFR-ratio (see G, H, Fig EV2H) is depicted above the graphs; Inserted into each graph are the values of Hill coefficient (HC) and EC50 of the fitted hill equation (95% confidence interval). 2nd graph: Same as left graph with αp plotted vs αL both obtained from in cell dose response experiments in EmCit_MCF7 cells. 3rd-5th graph: Same as 2nd graph for EmCit_MCF7 RPTPγ-KO with RPTPγ-mTFP ectopic expression clustered by RPTPγ/EGFR-expression ratio and HC (Fig EV2G).",
    "molecules": "pervanadate"
  },
  {
    "caption": "C-E (C) in cell EGF-dose response imaging for RPTPγ-mCitrine oxidation. Left panel: Representative confocal micrographs of RPTPγ-mCitrine in EmTFP_MCF7 cells (top row) together with its oxidized fraction estimated using DyTo-FLIM (αox, bottom row), upon 10' stimulation with EGF-Alexa647 (0-160 ng/ml) including 5' together with 0.5 mM DyTo. Scale bar: 10 μm. Right panel: Quantification depicting the PM-proximal (orange) and PM-distal (blue) oxidized fractions as functions of receptor occupancy (αL) and corresponding EGF-Alexa647, or H2O2 concentration in EmTFP MCF7 cells expressing RPTPγ-mCitrine (WT) or RPTPγC1060S-mCitrine (C1060S) as well as WT cells treated with 0.5 mM atto590 instead of DyTo (atto590). Mean of individual cells (symbols) with mean±SD (black lines), N=3 biological replicates, n=13-15 cells per EGF dose. P: unpaired two-tailed t-test, between PM (serpentine peripheral structures) and endosomal (vesicular structures) fractions. (D) Same as in (C), for RPTPγ-mCitrine oxidation in p22phox-KO cells. N=3 biological replicates, n=14-26 cells per EGF dose. (E) Same as in (C), for TCPTP-mCitrine or TCPTPC216S-mCitrine (C216S) oxidation in EmTFP_MCF7 cells. N=3 biological replicates, n=18-21 cells per EGF dose. ",
    "molecules": "Alexa647, atto590, H2O2, serpentine"
  },
  {
    "caption": "A. Representative confocal micrographs of MCF7 WT cells showing the co-localization of RPTPγ-mCitrine (1st column: green; 3rd column: blue) and EGFR-mCherry (2nd column: green; 3rd column: yellow) with recycling-endosome defined by immunostaining against Rab11a (magenta), without (top row) or after 30' EGF-DyLight405 stimulus (160 ng/ml; bottom row). Scale bar: 10 μm.",
    "molecules": "DyLight405"
  },
  {
    "caption": "E. Upper panel: Dual-color widefield images (1st column), SRRF reconstructions (2nd column) with magnifications of boxed areas (3rd column) of Alexa647-SNAP-EGFR (green) and RPTPγ-mCitrine (magenta) of cryo-arrested MCF7 cells, unstimulated (top row) or stimulated with 100 ng/ml EGF (bottom row) for 15'. Scale bar: 10 μm. Lower panel: corresponding Manders colocalization coefficients for Alexa647-Snap-EGFR/RPTPγ-mCitrine from SRRF reconstructions on intracellular compartments or PM area for unstimulated (n=12-18) and 15' EGF-stimulated (n=13-14) cells. mean±SD, P: unpaired two-tailed t-test.",
    "molecules": "Alexa647"
  },
  {
    "caption": "F. Left: Representative IP-western blot showing co-IP of EGFR (2nd row) upon RPTPγ-mCitrine (1st row: lanes 1-6) or RPTPγC1060S-mCitrine (lane 7) pull-down by anti-GFP antibody from lysates of MCF7 cells co-transfected with EGFR and RPTPγ-mCitrine or RPTPγC1060S-mCitrine: without stimulus (0 ng/ml), upon 10' stimulus with EGF-Alexa647 (5-320 ng/ml, also displayed as corresponding receptor-occupancy αL, Fig 2D) or 8 mM of H2O2. 3rd and 4th row: total protein concentrations of RPTPγ-mCitrine and EGFR in the lysate measured by western blot as input control for the Co-IP. Right: corresponding ratiometric quantification of co-immunoprecipitated EGFR over pulled down RPTPg-mCitrine or RPTPγC1060S-mCitrine protein bands (mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test)",
    "molecules": "Alexa647, H2O2"
  },
  {
    "caption": "C. Top: Representative Western blot against Erk and phosphorylated Erk (pT202 and pY204) in WT (red) compared to p22phox-KO (green) MCF7 cells after the indicated times of sustained stimulation with 20 ng/ml EGF-Alexa647. Bottom: Corresponding quantification of the fraction of phosphorylated ERK as a function of stimulation time. Mean±SD, N=4 biological replicates, P: unpaired two-tailed t-test.",
    "molecules": "Alexa647"
  },
  {
    "caption": "D. Quantification of cell proliferation using retinoblastoma (Rb) protein phosphorylation detected by immunofluorescence, for WT (red), RPTPγ-KO (blue) and p22phox-KO (green) MCF7 cells without or post 24 h of EGF-Alexa647 treatment (1, 20, 160 ng/ml). Mean±SEM, N=3 biological replicates, n>2000 cells per EGF stimulus per cell line, P: two-way ANOVA with Tukey multiple comparisons.",
    "molecules": "Alexa647"
  },
  {
    "caption": "Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1.",
    "molecules": "Poly(I:C)"
  },
  {
    "caption": "Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (C) Quantification of the presence of SARS-CoV-2.",
    "molecules": "Poly(I:C)"
  },
  {
    "caption": "Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (B) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (C) ISG response measured by ISG15, RSAD2, and IFIT1 (Donors marked in green and red were not quantifiable in all test conditions).",
    "molecules": "Poly(I:C)"
  },
  {
    "caption": "Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2.",
    "molecules": "Poly(I:C)"
  },
  {
    "caption": "Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.",
    "molecules": "Poly(I:C)"
  },
  {
    "caption": "A-F, Staining for Isolectin B4 (A-C) and oxidized thiols (D-F) in WT cortices at E10.5 (A,D), E11.5 (B,E) and E12.5 (C,F). Panels D-F are pseudocolor conversions of staining intensity (from low [purple] to high [red]). Arrowheads denote the position of the vascular front.",
    "molecules": "thiols"
  },
  {
    "caption": "B-E, Staining for pimonodazole (PIMO, green) and Isolectin B4 (IB4,red) in Gpr124KO (B,C) and control (D,E) cortices after exposure of the pregnant dams to 21% O2 (B,D) or 80% O2 (C,E). Compared to the pimonidazole staining in panels B,C, the intensity of this staining is much weaker in panels D,E. For reasons of clarity, the pimonidazole staining in panels D,E was therefore enhanced, equally in both panels.",
    "molecules": "O2"
  },
  {
    "caption": "F,G, Immunostaining for HIF-1 (red) and DAPI (blue) in Gpr124KOcortices after exposure of the pregnant dams to 21% O2 (F) or 80% O2 (G). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm",
    "molecules": "O2"
  },
  {
    "caption": "H,I, Immunostaining for Tbr2 (red) and Ngn1 (green) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 (H) or 80% O2 (I). J-L Quantification of neurogenic (Ngn1+) RGs and BPs (J), newborn BPs (K) or expanding (Tbr2- Ngn1-) RGs (L) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=4 for 21% O2 and N=5 for 80% O2; * p<0.05, ** p<0.01). M,N, Immunostaining for Tbr2 (red) and Ngn1 (green) in control cortices after exposure of the pregnant dams to 21% O2 (M) or 80% O2 (N). O-Q Quantification of neurogenic (Ngn1+) RGs and BPs (O), newborn BPs (P) or expanding (Tbr2-Ngn1-) RGs (Q) in control cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=5; ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm",
    "molecules": "O2"
  },
  {
    "caption": "B, Measurement of lactate secretion from proliferating NSCs, and of NSCs differentiating for 3 days (switch to neurogenesis) or for 7 days (fully differentiated), normalized to cellular protein content (mean±SEM; N=6; *** p<0.001).",
    "molecules": "lactate"
  },
  {
    "caption": "D, Measurement of lactate secretion from proliferating NSCs transduced with scr or Pfkfb3shRNA#1, normalized to cellular protein content (mean±SEM; N=3; * p<0.05).",
    "molecules": "lactate"
  },
  {
    "caption": "Tissue microarrays (TMAs) from primary PCa (n=132) and CRPC (n=148) were stained via immunohistochemistry for poly(ADP-ribose) (PAR), and scored by a clinical pathologist (T. Parsons) for intensity (0-3) and percentage (0-3) PAR score was generated via the equation: (intensity x 1) + (percentage x 2). PAR scores were compared between primary and CRPC. ****=p value<0.0001 by Chi-square test",
    "molecules": "PAR, poly(ADP-ribose)"
  },
  {
    "caption": "Manual PAR scores were divided in to quartiles and then were compared to progression-free survival in the CRPC TMAs. *=p<0.05, ns=not statistically significant by Log-rank (Mantel-Cox)",
    "molecules": "PAR"
  },
  {
    "caption": "Representative image of one TMA core after multiplex fluorescent IHC for γH2AX (green), PAR (red), PARP-1 (purple), with DNA (blue). Top Right: Insets of parent image at left. Numbers above inset columns coincide with numbers on image at left that were chosen for further magnification and representation (boxed areas). Bottom Left: Percent positive staining for PAR for the entirety of each TMA cohort. Bottom Middle: Percent positive staining for PARP-1. Bottom right: γH2AX for the entirety of each TMA cohort. Data was considered after a median intensity cutoff, and analyzed for statistical significance using two-tailed Student"s t-test for PAR, PARP-1, and γH2AX, respectively. Exact p values are indi",
    "molecules": "DNA, PAR"
  },
  {
    "caption": "Two-tailed Spearman correlation test between PAR and γH2AX (% positive with a median intensity cut-off). Exact p values are indicated when available",
    "molecules": "PAR"
  },
  {
    "caption": "Top, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of HT-sensitive LNCaP cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for 1 hour, then subsequently treated with either 1nM DHT or vehicle control (EtOH) for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated by DHT v. EtOH (left) or DHT v. PARPi followed by DHT (right). Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changed",
    "molecules": "DHT, DMSO, EtOH, veliparib"
  },
  {
    "caption": "Top, left: Schematic representing the conditions utilized for transcriptomic analyses (n=2) of CRPC C4-2 cells. Cells were deprived of hormones for 72 hours, followed by either treatment with 2.5uM veliparib (PARPi) or vehicle control (DMSO) for for 16 hours. Top, right: Immunoblot with the indicated antisera. Bottom: Volcano plots of transcripts found to be differentially regulated PARPi v. vehicle control. Red dots indicate transcripts that were both statistically significantly altered (p<0.05) and more than 1.5-fold changed",
    "molecules": "DMSO, veliparib"
  },
  {
    "caption": "Athymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumors",
    "molecules": "veliparib"
  },
  {
    "caption": "Prostatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples Each individual tissue is depicted by a separate bar color",
    "molecules": "veliparib"
  },
  {
    "caption": "Indicated cell lines were treated as depicted in Figure 2, and labeled with bromodeoxyuridine (BrdU), harvested at indicated time points and utilized for FACS analyses",
    "molecules": "BrdU, bromodeoxyuridine"
  },
  {
    "caption": "Athymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumors",
    "molecules": "veliparib"
  },
  {
    "caption": "Prostatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples. Each individual tissue is depicted by a separate bar color. Statistical analyses were performed by Wilcoxon signed rank test",
    "molecules": "veliparib"
  },
  {
    "caption": "C4-2 cells treated with 2.5uM veliparib (Vel.) or vehicle control (Veh.) for 24 hours. Cells were then harvested, lysed, and differentially centrifuged as described in the material and methods section, resulting in a soluble fraction (Sol.) (GAPDH serves as control) or a chromatin-tethered fraction (Teth.) (histone H4 serves as control). Immunoblots were performed for the indicated proteins",
    "molecules": "veliparib"
  },
  {
    "caption": "Indicated cell lines were transfected with indicated constructs, and treated with veliparib. Cell growt was assessed LNCaP cell growth: Control transfection, p=0.0220; BRCA1 transfection, p=0.67787; BRCA2 transfection, p=0.4676. C4-2 cell growth: Control transfection, p=0.0354; BRCA1 transfection, p=0.1638; BRCA2 transfection, p=0.2519. 22Rv1 cell growth: Control transfection, p=0.0039; BRCA1 transfection, p=0.1085; BRCA2 transfection, p=0.2781",
    "molecules": "veliparib"
  },
  {
    "caption": "Indicated cell lines were transfected with indicated constructs, and treated with veliparib DDR via γH2AX was assessed LNCaP γH2AX: Control transfection, p=0.0008; BRCA1 transfection, p=0.9035; BRCA2 transfection, p=0.4685. C4-2 γH2AX: Control transfection, p=0.0009; BRCA1 transfection, p=0.6362; BRCA2 transfection, p=0.4217. 22Rv1 γH2AX: Control transfection, p<0.0001; BRCA1 transfection, p=0.4698; BRCA2 transfection, p=0.4937",
    "molecules": "veliparib"
  },
  {
    "caption": "F. Confocal fluorescence images of Proteostat (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. G. Quantitation of aggregates/cell in IGRs cell lines by immunofluorescence analysis, left panel; fluorescence gain of soluble proteins treated with Proteostat reagent, right panel. (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). ",
    "molecules": "Proteostat, DAPI"
  },
  {
    "caption": "A. Absolute quantitation of 24, 25 and 27-hydroxycholesterol in primitive and metastatic melanoma cells as indicated. (T-test analysis, ***p-value<0.001, N=3 biological replicates, data are mean ±SD).",
    "molecules": "27-hydroxycholesterol"
  },
  {
    "caption": "B. Histogram representing R square of correlation analysis between absolute quantitation of 24, 25 and 27-hydroxycholesterol and label free quantitation of APOE in a cohort of primitive (A375, IPC298) and metastatic (SKMEL5, SKMEL28) melanoma cell lines.",
    "molecules": "27-hydroxycholesterol"
  },
  {
    "caption": "C. Confocal fluorescence images of anti-HMB45 PMEL antibody signal (green) and DAPI staining (blue) in IGR37 and IGR39. Scale bar is 10µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-D. Immunofluorescence images with Proteostat (red) and DAPI (blue) staining on (A) human normal skin, (B) samples of primitive melanomas, (C) melanoma metastases in brain and (D) melanoma  metastases in lung.",
    "molecules": "DAPI, Proteostat"
  },
  {
    "caption": "E. Details of brain metastases. Scale Bar is 30µm. F. Quantitation of Proteostat positive dots in primitive vs metastatic melanoma tissues: 6 tissues from metastatic lesions and 6 from primitive melanoma lesions were analysed. For each tissue two sections were quantified. T-test analysis was applied. T-test analysis, ** = p-value<0.01 (N=6, data are mean ±SEM).",
    "molecules": "Proteostat"
  },
  {
    "caption": "A. Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB-360.",
    "molecules": "DMSO, NB-360"
  },
  {
    "caption": "B. Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB-360.",
    "molecules": "DMSO, NB-360"
  },
  {
    "caption": "D. Confocal fluorescence images of Proteostat signal (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. E. Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software. (T-test analysis, ** = p-value<0.01, N=3 biological replicates, data are mean ±SD). ",
    "molecules": "Proteostat, DAPI"
  },
  {
    "caption": "G. mRNA levels of CTGF measured by real-time PCR in IGR37 treated with DMSO or NB-360 (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).",
    "molecules": "DMSO, NB-360"
  },
  {
    "caption": "J. CTGF mRNA level measured by real-time PCR in IGR37 treated with DMSO, NB-360 or NB-360 plus recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis* = p-value<0.05, ** = p-value<0.01. Data are mean ±SD.",
    "molecules": "DMSO, NB-360"
  },
  {
    "caption": "A. MTT assay for IGRs treated with DMSO or NB-360 (25µM). Biological replicates N=4 biological replicates. T-test analysis ** = p-value<0.01, *** p-value <0.001. Data are mean ±SD. B. MTT assay for IGRs cells treated with DMSO or inhibitor 3I (3µM). Biological replicates N=3 biological replicates. T-test analysis ** = p-value<0.01. Data are mean ±SD. ",
    "molecules": "3I, DMSO, NB-360"
  },
  {
    "caption": "C-D. MTT assay of IGRs treated with NB-360 (25µM) and different concentration of doxorubicin as indicated. Biological replicates N=4 biological replicates. T-test analysis: * = 0.01<p-value<0.05, ** = p-value<0.01. Data are mean ±SD.",
    "molecules": "doxorubicin, NB-360"
  },
  {
    "caption": "E. T-test analysis of IC50 values for doxorubicin used alone or in combination with NB-360 (combo). Data are mean ±SD. T-test analysis: * = 0.01<p-value<0.05; ** = 0.001<p-value<0.01.",
    "molecules": "doxorubicin, NB-360"
  },
  {
    "caption": "(b) Adult weight in the offspring of tesaglitazar-injected males (Tesa-inj, n = 16) compared to the offspring of vehicle-injected males (Vehicle-inj, n = 23). Two-tailed Student's t-test, P = 0.019, t = 2.44, df = 37.",
    "molecules": "Tesa, tesaglitazar"
  },
  {
    "caption": "(c) Glucose level in the offspring of Tesa-inj (n = 14) and Vehicle-inj (n = 21) males during a glucose tolerance test. Repeated measures ANOVA, treatment effect P = 0.0083, F (1, 33) = 7.877, time effect P < 0.0001, F (4, 132) = 347.7, interaction P = 0.0776, F (4, 132) = 2.155 Conc.; concentration.",
    "molecules": "Glucose, Tesa"
  },
  {
    "caption": "(a Differentially expressed transposable elements in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.",
    "molecules": "Tesa"
  },
  {
    "caption": "b) mRNAs/lincRNAs in sperm from Tesa-inj and MSUS males. Data represent genes with P < 0.05 and similar fold change. Fold change in heat map represents log2(fold change) respective to the corresponding control group. Total overlap is presented in Extended Data Fig. 11.",
    "molecules": "Tesa"
  },
  {
    "caption": "(c,d) (c) Volcano plot and (d) heat map of differentially expressed (FDR < 0.05) mRNA/lincRNA in sperm from Tesa-inj males. Dashed black lines in (c) represent y = 1.3, equivalent to FDR = 0.05, and x = ±0.03, equivalent to FC = 1.23 and 0.81. Black dots represent top candidates, which all have FC > 2 or FC < 0.5, and FDR < 0.05. Teal dots represent non-significant genes.",
    "molecules": "Tesa"
  },
  {
    "caption": "(c,d) Blood glucose levels in MSUS offspring and the offspring of MSUS serum-injected males following a 30-min restraint challenge. MSUS offspring n = 13, Control offspring n = 12, repeat measures ANOVA, treatment effect P = 0.016, F (1, 23) = 6.704, time effect P < 0.0001, F (3, 69) = 53.13, interaction P = 0.027, F (3, 69) = 3.25, at 15-min adjusted P = 0.0015, t = 3.693, df = 92. MSUS serum-injected offspring n = 17, and Control serum-injected offspring n = 14, repeat measures ANOVA, treatment effect P = 0.023, F (1,14) = 6.493, time effect P < 0.0001, F (3, 42) = 48.4, interaction P = 0.29, F (3, 42) = 1.29. Conc., concentration.",
    "molecules": "glucose"
  },
  {
    "caption": "F ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM",
    "molecules": "AHT, thiamine"
  },
  {
    "caption": "A. Representative pictures of immunostaining for F4/80 (green) in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury. B. Quantification of F4/80 staining/damaged area (μm2) at day 2 (top graph) and 5 (bottom graph) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Representative pictures of immunostaining for CD206 in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury, respectively. D-E. Quantification of CD206+ MPs per area (mm2) at day 2 (D) and 5 (E) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).",
    "molecules": "DAPI"
  },
  {
    "caption": "C. Representative pictures of immunostaining for GFP (green), CD206 (red), pSMAD3 (white) in GFP-Control and GFP-CriptoMy-LOF TA sections at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. D. Quantification of GFP+/CD206±/pSMAD3± cell distribution in TA sections from GFP-Control and GFP-CriptoMy-LOF at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean±SEM (n=5 biological replicates; **P<0.01, Student's t-test). ",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Representative pictures of double immunostaining with Laminin (green) and embryonic myosin heavy chain (eMHC; red) at day 5 (left panel) and 30 (right panel) after cardiotoxin (CTX) injection. Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Representative pictures of double immunostaining with Laminin (green) and eMHC (red) at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "B. Representative pictures of immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections with GFP (green) and CD206 (white; left panel), pSMAD3 (red; middle panel) and GFP and pSMAD3 (right panel). C-E. Quantification of GFP (C), GFP/CD206 (D) and GFP/pSMAD3 (E) positive cells per area (mm2) in mdx-Control and mdx-CriptoMy-LOF diaphragms. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.",
    "molecules": "DAPI"
  },
  {
    "caption": "F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test).",
    "molecules": "Picrosirius red, picrosirius red"
  },
  {
    "caption": "H. Hydroxyproline (HOP) concentration in mdx-Control and mdx-CriptoMy-LOF diaphragm muscle sections. Data are expressed as HOP levels (μg) per muscle section volume (mm3) and as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01, Student's t-test).",
    "molecules": "HOP, Hydroxyproline"
  },
  {
    "caption": "Representative images of Laminin (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections of centrally nucleated myofibers. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.",
    "molecules": "DAPI"
  },
  {
    "caption": "L-M. Representative images of eMHC (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (L) and quantification of eMHC+ myofibers per area (mm2, M). Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05; Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I). B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.",
    "molecules": "DAPI"
  },
  {
    "caption": "F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.",
    "molecules": "DAPI"
  },
  {
    "caption": "A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury. B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury. Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury. F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I). Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF Data information: Nuclei were counterstained with DAPI (blue).",
    "molecules": "DAPI"
  },
  {
    "caption": "(D) miR-574-5p and miR-574-3p were induced in the hearts of mice with isoproterenol (ISO) infusion (4 weeks; n=10-12).",
    "molecules": "ISO, isoproterenol"
  },
  {
    "caption": "(A) H&E of hearts from WT and miR-574-/- mice with or without isoproterenol (ISO) treatment for 4 weeks. Mice were between 8-10 weeks old females.",
    "molecules": "ISO, isoproterenol"
  },
  {
    "caption": "(B) Ratio of HW/TL (heart weight/tibia length) in WT and miR-574-/- mice (n=8/7/8/6 for 4 groups of WT, Veh.; WT, ISO; KO, Veh.; KO, ISO).",
    "molecules": "ISO"
  },
  {
    "caption": "(D) Picrosirius red staining in WT and miR-574-/- mice (n=6 per group). Scale bar: 1 mm.",
    "molecules": "Picrosirius red"
  },
  {
    "caption": "(E) RT-qPCR of fetal cardiac genes in WT and miR-574-/- mice with ISO treatment (n=3 per group). ANF, atrial natriuretic factor; BNP, B-type natriuretic peptide; Myh6/Myh7, myosin heavy polypeptide 6/7; Col1a2/Col3a1, procollagen, type I, α2; type III, α1.",
    "molecules": "ISO"
  },
  {
    "caption": "(F) TUNEL assay for heart tissue sections from WT and miR-574-/- mice under ISO versus vehicle treatment (n=6 per group). Scale bar: 10 μm.",
    "molecules": "ISO"
  },
  {
    "caption": "(D) Picrosirius red staining of hearts from WT and miR-574-/- mice under TAC surgery (n=8 per group). Scale bar: 1 mm.",
    "molecules": "Picrosirius red"
  },
  {
    "caption": "(C) Picrosirius red staining of murine hearts in the therapeutic models. Scale bar: 1 mm. The fibrotic area was quantified in the right panel (n=4-10 per group).",
    "molecules": "Picrosirius red"
  },
  {
    "caption": "(E,F) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment (10 μM for 24 hr) with overexpression of miR-574-5p or miR-574-3p. >120 cells/group were quantified in (E) and n=4 biological replicates in (F). The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.",
    "molecules": "ATP, ISO"
  },
  {
    "caption": "(G,H) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR-574-/- mice under ISO treatment. Scale bar: 10 μm. >110 cells/group were quantified in bottom panel of (G) and n=5 in (H).",
    "molecules": "ATP, ISO"
  },
  {
    "caption": "(H)  Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 (UCP1 (red); DNA staining  by DAPI (green); phase contrast (PC) (gray)).",
    "molecules": "DAPI"
  },
  {
    "caption": "Impact of Ctcflos KD on  mitochondrial  biogenesis. (R) Microscopic images of Mito Tracker stained brite adipocytes (Mito tracker (blue); DNA staining by DAPI (green);  phase contrast  (PC) (gray))",
    "molecules": "DAPI, Mito Tracker, Mito tracker"
  },
  {
    "caption": "(B-D) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis, (B) expressed as  Ucp1  relative to Fabp4 mRNA levels, (C) Ucp1 relative to  Gtf2b and (D) Fabp4  relative to Gtf2b. Mean values ± SD, n=3 (biological replicates), one-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.",
    "molecules": "isoginkgetin"
  },
  {
    "caption": "Effect of Ctcflos KD on  Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (E) Immunocytochemistry of SC35. SC35 signal (blue) alone (middle images) and SC35 (blue),  DNA staining by DAPI (green) and phase contrast (PC) (gray) combined (outer  images).",
    "molecules": "Arginine, DAPI, Serine"
  },
  {
    "caption": "(C) FD20 pre-incubation with SARS-CoV-2 pp in the absence of cells strongly impairs entry. To test direct action on particles, SARS-CoV-2 pp and FD20 were co-incubated for 1 h, then the premixes were used to infect VeroE6-hACE2 cells for 6 h at 37 °C. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate the gained neutralization (%) by pre-incubation. As control, cells were incubated at each step with an equal volume of PBS or Y112R. Percentages of primary infection were calculated according to viral titers of PBS control conditions. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test). ns, not significant.",
    "molecules": "PBS"
  },
  {
    "caption": "(D) Inhibition of cell surface binding of SARS-CoV-2 VLP by FD20. VLP-GFP were preincubated, or not, with FD20 and RBM-targeting mAbs (CB6, REGN10933, REGN10987) for 30 min at 37 °C. Then the VLP premixes were incubated with VeroE6-hACE2 for 1 h at 37 °C and the binding detected by flow cytometry. Control binding assays were performed using soluble ACE2 as a competitor. Data are representatives from three experiments.",
    "molecules": "VLP"
  },
  {
    "caption": "(E) Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band.",
    "molecules": "Coomassie"
  },
  {
    "caption": "(A) Comparison of RNA isoforms and sRNA patterns generated by 35S:GFP-GUS and 35S:GFP-EVDint/ter-GUS. High and low molecular-weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background. mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig.2A. EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs, respectively.",
    "molecules": "EtBr"
  },
  {
    "caption": "(B). Western blot analysis of the translation products from GFP and GFP-GUS transcripts. Coomassie (coom.) staining as a loading control. Black arrow: GFP-GUS fusion protein; white arrow: GFP protein.",
    "molecules": "Coomassie"
  },
  {
    "caption": "(C) Nucleo-cytosolic distribution of 35S:EVD and 35S:GFP-EVDint/ter-GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR. RNA extracted from Total, nuclear (Nucl) and cytoplasmic (Cyto) fractions was reverse transcribed with random hexamers and oligo(dT). snoRNA U5 is shown as a nuclear-only RNA control. (D) Same as in (C) but using exclusively oligo(dT) to reverse transcribe poly(A)+ RNAs.",
    "molecules": "poly(A), snoRNA"
  },
  {
    "caption": "B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths. Yeast cells expressing clogger proteins (cytochrome b2-DHFR and cytochrome b2∆19-DHFR) or cytosolic DHFR for comparison under control of the galactose-inducible promoter were grown to mid-log phase on lactate medium. Ten-fold serial dilutions were dropped on lactate (no induction) or lactate with 0.5% galactose medium (induction). MTS, matrix-targeting signal.",
    "molecules": "galactose, lactate"
  },
  {
    "caption": "E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with 0.5% galactose before samples were harvested and lysed. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.",
    "molecules": "galactose, lactate"
  },
  {
    "caption": "A The clogger was expressed in wild type (WT) and ∆rpn4 (∆) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a non-inducing lactate medium. Precursor (pre) and mature (m) forms of the b2∆-DHFR clogger were visualized by Western blotting using a DHFR-specific antibody.",
    "molecules": "galactose, lactate"
  },
  {
    "caption": "G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆rpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.",
    "molecules": "galactose, lactate"
  },
  {
    "caption": "B Hsp104-GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures. SN, supernatant representing the non-bound fraction. The signals for Ssa1 and Sod1 are shown for control.",
    "molecules": "sepharose"
  },
  {
    "caption": "A The mitochondrial proteins Pdb1, Mam33 and Aim17 were co-expressed for 4.5 h with clogger as fusions with the red fluorescent protein RFP (mCherry). Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4.5 h after induction of the clogger or cytosolic DHFR for control. For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17-expressing plasmids and grown to mid-log phase in lactate medium. Clogger expression was induced for 4.5 h with 0.5 % galactose before microscopy was perfomed. Please note that in the absence of Hsp42 and Hsp104, no Aim17-RFP-containing granules are formed. C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N-terminally truncated version of Mam33-RFP (both under control of GAL promoter) together with constitutively expressed Hsp104-GFP (TPI promoter) . The cells were grown on lactate plus 0.5% galactose-containing medium for 4.5 h. The distribution of the fluorescent proteins was visualized by microscopy.",
    "molecules": "galactose, lactate"
  },
  {
    "caption": "D Wild type cells expressing Hsp104-GFP were grown on glucose or lactate medium to log phase. Cells were diluted and visualized by life cell imaging. Please note, that in glucose media, Hsp104-GFP is initially dispersed in cells and only aggregates when cells become stationary. In contrast, Hsp104-GFP-containing granules are frequent on lactate medium even at log phase. See movie EV1 for life cell imaging of glucose-grown cells.",
    "molecules": "glucose, lactate"
  },
  {
    "caption": "F The mitochondria used for E were treated with proteinase K (PK) to remove surface-exposed proteins. Mitochondrial membranes were lysed with NP-40 and soluble (S) and aggregated (P) proteins were separated by centrifugation. T, total.",
    "molecules": "NP-40"
  },
  {
    "caption": "G To measure the toxicity of clogger expression, clogger proteins were induced for 24 h with lactate medium containing 0.5 % galactose in the mutants indicated. Aliquots were removed and the number of living cells were assessed by a plating assay on glucose-containing plates. The resistance of ∆rpn4 to clogger expression depends on Hsp104 and Hsp42. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.",
    "molecules": "galactose, glucose, lactate"
  },
  {
    "caption": "A BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 μM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least 3 independent experiments.",
    "molecules": "carboxyfluorescein, Ni, NTA"
  },
  {
    "caption": "B BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of 3 independent experiments.",
    "molecules": "dextran, digitonin"
  },
  {
    "caption": "B BAK, or BAK with cysteine introduced at the indicated positions (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and assess for BAK expression by immunoblotting (inset panel; * indicates non-specific band.) and apoptotic activity in response to etoposide treatment.",
    "molecules": "Cys, cysteine, etoposide"
  },
  {
    "caption": "C E24 and R169 are proximal in inactive BAK on mitochondria. Mitochondria-enriched membrane fractions from cells were incubated with oxidant (CuPhe) and induced intramolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.",
    "molecules": "CuPhe"
  },
  {
    "caption": "D E24 and R169 dissociate during BAK activation. Mitochondria-enriched membrane fractions from cells were incubated with cBID (100 nM) prior to oxidant (CuPhe) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.",
    "molecules": "CuPhe"
  },
  {
    "caption": "C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake.",
    "molecules": "Dox, doxycycline, PI"
  },
  {
    "caption": "D Mutation of BH4 promotes BAK activating conformation change. Cells were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172).",
    "molecules": "doxycycline"
  },
  {
    "caption": "D BAK wild-type, or BAK with an engineered α1-α2 loop thrombin cleavage site were stably expressed in Bax-/-Bak-/- MEFs. Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline (CuPhe) to induce disulphide-linkage. Samples were run on SDS-PAGE under non-reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N-terminus upstream of the thrombin cleavage site (aa23-38) or an antibody recognising the BH3 domain (4B5) downstream of the cleavage site. Schematic indicates the nature of the BAK protein detected by each antibody. Disulphide-linkage indicated by a hashed line. Note that the N-terminal portion of thrombin cleaved BAK could not be detected with the N-terminal antibody unless it was intramolecularly disulphide-linked. *cBID (100 nM) was added to activate BAK during thrombin cleavage. Data representative of two independent experiments.",
    "molecules": "copper phenanthroline, CuPhe, thrombin"
  },
  {
    "caption": "E Cleavage of the BAK α1-α2 loop potentiates MOMP. Membrane fractions from Bax-/-Bak-/- MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID (10 nM) for the indicated times. Membrane (P) and supernatant (S) fractions were separated and immunoblotted for cytochrome c or BAK 4B5.",
    "molecules": "thrombin"
  },
  {
    "caption": "B) Bar chart indicating  protein quantification, protein localization  (according to UniProt) in the secretome of primary neurons, comparing the previous SPECS   (blue) to the new hiSPECS   method using DDA  (light green) or DIA  (dark green). Proteins were counted if quantified in at least 9 of the 11 biological replicates of hiSPECS   or 4 of 5 biological replicates The category glycoprotein* includes UniProt annotations and proteins annotated as glycoproteins   Proteins can have multiple UniProt annotations, e.g. for APP membrane, TM, cytoplasm  and nucleus, because distinct proteolytic fragments are found in different organelles, so that some proteins in categories cytoplasm  and nucleus  may overlap with the categories secreted and TM+GPI. Data information: TM: single-pass transmembrane protein; GPI: Glycosylphosphatidylinositol-anchored membrane protein.",
    "molecules": "glycoprotein, glycoproteins"
  },
  {
    "caption": "B) Volcano plot showing changes in protein levels in the secretome of primary neurons upon BACE1  inhibitor C3  treatment using the hiSPECS DIA  method. The negative log10 p-values (two sample t-test) of all proteins are plotted against their log2 fold changes (C3  vs control) (N=11). The grey hyperbolic curves depict a permutation based false discovery rate estimation (p = 0.05; s0 = 0.1). Significantly regulated proteins (p<0.05) are indicated with a dark blue dot and known BACE1  substrates are indicated with blue letters. The two newly validated BACE1  substrates CD200  and ADAM22  are indicated in red.",
    "molecules": "C3"
  },
  {
    "caption": "C) Independent validation of the novel BACE1  substrate candidates CD200  and ADAM22  by Western blotting in supernatants  and lysates  of primary neurons  incubated with or without the BACE1  (B1) inhibitor C3  for 48h. Full-length (fl) ADAM22  (mainly mature ADAM22, lower band) and CD200  levels in the neuronal lysate  and were mildly increased upon BACE1  inhibition, as expected due to reduced cleavage by BACE1. Calnexin  served as a loading control. The soluble ectodomain of ADAM22   (sADAM22) was strongly reduced in the conditioned medium upon BACE1  inhibition. Ectodomain levels of the known BACE1  substrate SEZ6   (sSEZ6) were strongly reduced upon BACE1  inhibition and served as positive control. Arrows indicate the mature (m) and immature (im) (before prodomain cleavage) form of ADAM22.",
    "molecules": "C3"
  },
  {
    "caption": "D) Quantification of the Western blots in (C) (N=6). Signals were normalized to calnexin  levels and quantified relative to the control (ctr) condition. Statistical testing was performed with N=6 biological replicates, using the one sample t-test with the significance criteria of p < 0.05. According to this criterion, ADAM22  and CD200  were significantly increased in total lysates upon C3  treatment (flADAM22: *p-value 0.0251, flCD200: **p-value 0.011). Soluble ADAM22   was significantly reduced in the supernatant upon C3  treatment (****p-value <0.0001). The black central horizontal line indicates the mean and error bars, mean ± SD.",
    "molecules": "C3"
  },
  {
    "caption": "A) Protein dynamic range plot of the log10 transformed LFQ intensities of the murine CSF  proteins quantified in at least 3 of 4 biological replicates measured with DIA. The proteins are split into quartiles according to their intensities, with the 1st quartile representing the 25% most abundant proteins. The percentage of proteins annotated in UniProt with the following subcellular locations/keywords are visualized for: membrane, secreted, cytoplasm, and glycoprotein. Examples of cell type specifically  secreted  proteins  are indicated with circles colored according to the UniProt annotation (purple: secreted, blue: membrane). B) Protein dynamic range plot of the log10 transformed LFQ intensities specifically of glycoproteins  in the murine CSF quantified in at least 3 of 4 biological replicates measured with DIA   (N=4). The proteins are split into quartiles according to their intensities. The percentage of proteins identified in the secretome  of astrocytes, microglia, neurons  or oligodendrocytes  in at least 5 of 6 biological replicates is illustrated below. Selected proteins specifically secreted from one cell type are indicated with the color code of the corresponding cell type.",
    "molecules": "glycoprotein, glycoproteins"
  },
  {
    "caption": "D) Volcano plot showing changes in protein levels in the secretome of primary cultured brain slices upon 6 h LPS  treatment in a 24 h collection window using the hiSPECS DIA  method. The negative log10 p-values (two sample t-test) of all proteins are plotted against their log2 fold changes (LPS  vs control) (N=6). The grey hyperbolic curves depict a permutation based false discovery rate estimation (p = 0.05; s0 = 0.1). Significantly regulated proteins (p<0.05) are indicated with a dark blue dot. Proteins highlighted in red indicate proteins known to increase upon LPS  treatment, whereas proteins labeled in blue are not upregulated in this study",
    "molecules": "LPS"
  },
  {
    "caption": "(E) Weight  and survival curves  of neutrophil  specific  Nlrp3A350VneoR Gsdmd-/- mice. n≥18",
    "molecules": "neo"
  },
  {
    "caption": "(A) Collagen  deposition assessed by Sirius red staining  Data information: Representative images shown, bar indicates 250 μM, n≥6 per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats",
    "molecules": "Collagen, Sirius red"
  },
  {
    "caption": "Northern blot analysis of tlh1/2+, Tf2 retrotransposons, and 18S rRNA (loading control) in wt, cay1Δ, and trichostatin A (TSA)‐treated wt cells. Black arrowhead indicates tlh1/2+ mRNA; red arrowhead unprocessed Tf2 transcripts; and blue arrowhead processed Tf2 transcripts.",
    "molecules": "trichostatin A, TSA"
  },
  {
    "caption": "Western blot analysis of Rap1-YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition. Membranes were probed with antibodies against GFP and Act1 (loading control).Quantification of Rap1-YFP protein levels in experiments as in (C). Rap1-YFP levels are expressed as fold increase over time 0 after normalization through Act1. Data points and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t-test. *P 0.05, **P 0.01 for cay1+ versus cay1∆ samples.",
    "molecules": "thiamine"
  },
  {
    "caption": "PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1‐arrested by nitrogen starvation (G1). Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused).",
    "molecules": "nitrogen"
  },
  {
    "caption": "To measure calcium influx cells were loaded with Indo-1. Shown is the mean calcium concentration as ratio of bound to unbound Indo-1 versus time in s. First the basal level was monitored for 50 s in Krebs-Ringer solution. Subsequently, the BCR was stimulated with 13 µg/ml anti-IgM F(ab)2 (shown by the black arrow). Shown is one representative result of four independent experiments. Mice were between 36 and 48 weeks of age. To measure intracellular signaling splenic cells were pre-gated on single, living cells and lymphocytes, then B220+ cells, including B220low CD5+ lymphocytes were selected. ",
    "molecules": "calcium, Indo-1"
  },
  {
    "caption": "(C) Overexpression of MIR376A resulted in a decrease in the autophagic activity of MCF-7 cells. Starvation-induced conversion of LC3-I to LC3-II in MCF-7 cells was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.",
    "molecules": "E64d, Pepstatin A"
  },
  {
    "caption": "(C) Overexpression of MIR376A resulted in decreased autophagic flux in Huh-7 cells. Starvation-induced conversion of LC3-I to LC3-II was analyzed. Tests were performed in the presence or absence of E64d (10 µg/ml) and Pepstatin A (10 µg/ml) (E+P). LC3-II/LC3-I densitometric ratios were marked. ACTB was used as a loading control.",
    "molecules": "E64d, Pepstatin A"
  },
  {
    "caption": "(a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.), clathrin heavy chain (cla.), AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 (+/- BafA1), after which they were lysed for western blot analysis with anti-LC3 and anti-tubulin antibodies. Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we use. However, this is not a problem because it is advisable to relate LC3-II to tubulin/actin.",
    "molecules": "BafA1, bafilomycin A1"
  },
  {
    "caption": "(b) Ratio of LC3-II to tubulin on knockdown (KD) of control, epsin, clathrin heavy chain, AP2 or AP1, with or without bafilomycin A1, quantified from three independent experiments (two for epsin) and represented in the graph. Asterisk, P 0.01; two asterisks, P 0.001; three asterisks, P 0.0001.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(c) HeLa cells stably expressing GFP-mRFP-LC3 (ref. 17) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA, during which they were either left untreated (UT) or treated with bafilomycin A1 (BafA1) for the last 15 h. Cells were then fixed and analysed on a Cellomics ArrayScan system. Quantification of autophagic vacuoles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph. Asterisk, P 0.01; n.s., not significant. n = 2,000 cells.",
    "molecules": "BafA1, bafilomycin A1"
  },
  {
    "caption": "(c) HeLa cells transfected for 24 h with GFP-Atg16L1 were either left untreated or treated with 50 μM dynasore (Sigma) for 4 h and processed for immunogold electron microscopy with anti-GFP antibodies (15-nm gold particles) and anti-clathrin antibodies (10-nm gold particles). Co-localization can be seen in the boxed areas. Quantification of clathrin-coated structures (CCS) that were associated with Atg16L1 per 1,000 μm2 is shown in the graph. Three asterisks, P 0.0001. Scale bar, 100 nm. All error bars represent s.e.m.",
    "molecules": "dynasore, gold particles"
  },
  {
    "caption": "(b) HeLa cells transfected for 24 h with GFP-Atg16L1 were incubated for 15 min with horseradish peroxidase (HRP)-conjugated cholera toxin subunit B at 4 °C. The cells were then incubated for 10 min at 37 °C, after which they were processed for double immunogold labelling with anti-HRP antibodies (15-nm gold particles) and anti-GFP antibodies (10-nm gold particles) for electron microscopy. PM, plasma membrane. Scale bar, 100 nm. Co-localization can be seen in boxed areas. n = 49 fields. All error bars represent s.e.m.",
    "molecules": "gold particles"
  },
  {
    "caption": "(c) HeLa cells treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot analyses for total lysate (TL) and IP were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the strong Atg16L1-clathrin interaction with dynasore treatment. The graph shows quantification from two independent experiments. All error bars represent s.e.m.",
    "molecules": "dynasore"
  },
  {
    "caption": "  (D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)  ",
    "molecules": "AS, AS605240"
  },
  {
    "caption": " (C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples. ",
    "molecules": "T-Lipo, AS, cyclosporin A"
  },
  {
    "caption": " (F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately. ",
    "molecules": "T-Lipo, AS, fMLP"
  },
  {
    "caption": " (G) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments. ",
    "molecules": "T-Lipo, AS, DY-635"
  },
  {
    "caption": " (B Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure). ",
    "molecules": "EDTA"
  },
  {
    "caption": " C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure). ",
    "molecules": "EDTA"
  },
  {
    "caption": " (A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS). ",
    "molecules": "T-Lipo, AS, CDFDA"
  },
  {
    "caption": " (B, C) Survival analysis of PCI and sham animals. Subgroups were treated with (B) free AS605240 or vehicle (Veh; i.p.) or (C) with liposomal formulated AS605240 (T-LipoAS) or T-Lipo (i.v.) as the specific vehicle control once per day. *p<0.05, Log-Rank Test. Animal numbers: PCI Veh: 25 (%male: 44, %female: 56), PCI AS: 43 (%male: 44, %female: 56), PCI: T-Lipo 24 (%male: 50, %female: 50), PCI-T-LipoAS: 35 (%male: 49, %female: 51), sham Veh: 7 (%male: 100), sham AS: 7 (%male 57, %femla: 43), sham T-Lipo (%male 25, %femla: 75): 4, and sham T-LipoAS 7 animals (%male 29, %femla: 71).",
    "molecules": "liposomal, T-Lipo, AS, AS605240"
  },
  {
    "caption": "(f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).",
    "molecules": "saline"
  },
  {
    "caption": "(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA).",
    "molecules": "WX-340"
  },
  {
    "caption": "(d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA).",
    "molecules": "WX-340"
  },
  {
    "caption": "(f) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX-340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).",
    "molecules": "WX-340"
  },
  {
    "caption": "a, Analysis of BCL2 phosphorylation (detected by anti-BCL2 immunoprecipitation and autoradiography of 32P-labelled cells) and beclin 1 co-immunoprecipitation with BCL2 in wild-type (WT) or BCL2 AAA MEFs grown in normal media or subjected to 4 h Earle's balanced salt solution (EBSS) starvation. p-BCL2, phospho-BCL2.",
    "molecules": "32P"
  },
  {
    "caption": "c, d, Plasma glucose (c) and insulin (d) levels in mice of indicated genotype at rest, after 80 min exercise (∼900 m), or maximal exercise. Data represent combined mean ± s.e.m. for 9-11 mice per group from three independent cohorts; similar results were observed in each cohort.",
    "molecules": "glucose, insulin"
  },
  {
    "caption": "h, Soleus muscle14C-deoxyglucose uptake during treadmill exercise in mice of indicated genotype. Data represent mean ± s.e.m. of 3 mice per group. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA for comparison between groups; †P<0.05, ††P<0.01, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes. NS, not significant.",
    "molecules": "14C, deoxyglucose"
  },
  {
    "caption": "a, b, Oral glucose tolerance test (OGTT) before (a, week 0) and after (b, week 4) 4 weeks of HFD.",
    "molecules": "glucose"
  },
  {
    "caption": "c, d, OGTT (c) and serumleptin and adiponectin levels (d) after 8 weeks of daily exercise. For a-d, results represent the mean ± s.e.m. for 4-5 mice per group. E, exercise; NE, no exercise; RD, regular diet . *P  0.05, **P  0.01, (c, one-way ANOVA; d, Wilcoxon rank test). NS, not significant.",
    "molecules": "adiponectin, leptin"
  },
  {
    "caption": "B HeLa cells expressing GFP-parkin (green), OCT-DsRed2 (red) and CFP-Drp1K38E were left untreated (untreated) or treated for 2 h with 10 µM CCCP (CCCP), then fixed and stained for TOM20 (blue). Scale bars, 30 µm.",
    "molecules": "CCCP"
  },
  {
    "caption": "C Cells prepared as in (B) were treated with 50 µM antimycin A for 2 h. Arrows indicate OCT-DsRed2-positive/TOM20-negative MDVs that colocalize with GFP-parkin, while circles indicate MDVs that are parkin-negative. Arrowheads indicate “nascent” vesicles, adjacent to mitochondria, exhibiting cargo selectivity and parkin recruitment. Open arrowheads indicate parkin-negative MDVs containing the reciprocal cargo (TOM20-positive/OCT-DsRed2-negative). Cell boundaries are delineated in the GFP-parkin single-channel image. Scale bars, 30 µm.",
    "molecules": "antimycin A"
  },
  {
    "caption": "A Quantification of the number of OCTμDsRed2μpositive/TOM20μnegative vesicles in cells expressing GFP, GFPμparkinWT, GFPμparkinR42P, GFPμparkinK211N, or GFPμparkinC431F, treated with or without antimycin A (anti A) as in (C); both the total number (white bars) and the number colocalizing with GFPμparkin (gray bars) are indicated. Bars represent the mean ± s.e.m. Pμvalues are given first for GFPμ/GFPμparkinμpositive vesicles, then for total vesicle number (n = 49-68 cells in 2-3 experiments); ns, not significant; **P < 0.01; ***P < 0.001).",
    "molecules": "antimycin A"
  },
  {
    "caption": "C HeLa cells expressing various GFP-parkin mutant contructs (green), pOCT-DsRed2 (red) and CFP-Drp1K38E were treated with 50 µM antimycin A for 2 h, then fixed and immunostained against TOM20. Arrows indicate matrix-positive/TOM20-negative structures colocalizing with GFP-parkin. Scale bars, 30 µm.",
    "molecules": "antimycin A"
  },
  {
    "caption": "D Immunoblot of whole-cell lysates of HeLa cells expressing various GFP-parkin constructs, treated with 50 µM antimycin A (anti A, +) or DMSO (-) for 2 h.",
    "molecules": "antimycin A, DMSO"
  },
  {
    "caption": "B U2OS:GFP−parkin cells transfected with siRNA targeting Drp1 were treated with DMSO (upper panels) or 25 μM antimycin A (anti A, lower panels) for 90 min, then fixed and immunostained against PDH (red) and TOM20 (blue). PDHE2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm (first panels on left) and 2 μm.",
    "molecules": "antimycin A, DMSO"
  },
  {
    "caption": "C Quantification of PDH−positive/TOM20−negative vesicles in U2OS:GFP and U2OS:GFP−parkin cells transfected with the indicated siRNA, treated with DMSO or antimycin A (anti A) as in (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 48-85 cells in 2-3 experiments); ns, not significant; ***P < 0.001).",
    "molecules": "antimycin A, DMSO"
  },
  {
    "caption": "D 5 μm‐long profile of the parkin‐positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A‐treated cell from (B) (left). Increasing position values on the x‐axis of the fluorescence intensity plot (right) correspond to moving from the top to the bottom of the profile. *MDV indicated by an arrow in (B).",
    "molecules": "antimycin A"
  },
  {
    "caption": "E Quantification of the distance between the centres of GFP−parkin−negative (black bar) or −positive (gray bar) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A−treated U2OS:GFP−parkin cells transfected with siDrp1 in (C). Error bars represent the mean ± s.e.m.; *P < 0.05 (obtained by Student's t−test).",
    "molecules": "antimycin A"
  },
  {
    "caption": "F U2OS:GFP‐parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 μM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20‐negative MDVs containing the specified cargo and colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP‐parkin and the indicated mitochondrial marker. Scale bars, 20 μm (first two columns of panels) and 2 μm (remaining columns of panels).",
    "molecules": "antimycin A"
  },
  {
    "caption": "G Quantification of TOM20−negative structures that stained positively for the indicated cargo in U2OS:GFP (GFP) and U2OS:GFP−parkin (GFP−parkin) cells, transfected with siRNA targeting Drp1, treated with antimycin A (anti A); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 24-59 cells in two experiments; ns, not significant; ***P < 0.001).",
    "molecules": "antimycin A"
  },
  {
    "caption": "AHeLa cells, transfected with siRNA targeting Drp1 and GFP‐parkin (green), were treated with 25 μM antimycin A for 90 min following a 30‐min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bar, 30 μm.",
    "molecules": "antimycin A, bafilomycin A1"
  },
  {
    "caption": "B Quantification of PDH E2/E3bp‐positive/TOM20‐negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E‐64d; both total number (white bars) and the number colocalizing with GFP‐parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P‐values are given for total vesicle number (n = 19 to 120 cells in 2-3 experiments; **P < 0.01; ***P < 0.001).",
    "molecules": "antimycin A, bafilomycin A1, E‐64d, pepstatin A"
  },
  {
    "caption": "CHeLa cells, transfected with GFP‐parkin (green) and siRNA targeting Drp1 and Atg5, were treated with 25 μM antimycin A for 90 min, then fixed, immunostained for PDH (red) and TOM20 (blue), and counterstained for Hoescht (gray). PDH E2/E3bp bp‐positive/TOM20‐negative MDVs colocalizing with GFP‐parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm.",
    "molecules": "antimycin A"
  },
  {
    "caption": "A Representative immunoblot of whole‐cell lysates from HeLa cells transfected with non‐targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 μM CCCP for 6 h in order to stabilize the PINK1 full‐length band.",
    "molecules": "CCCP"
  },
  {
    "caption": "B HeLa cells transfected with GFP−parkin (green) and siRNA targeting PINK1 (siPINK1) or non−targeting control (ctrl siRNA), were treated with 25 μM antimycin A for 90 min, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp−positive/TOM20−negative MDVs colocalizing with GFP−parkin (arrows) or not (circles) are indicated. Arrowheads indicate parkin−positive MDVs adjacent to mitochondria, possibly budding. Scale bar, 20 μm.",
    "molecules": "antimycin A"
  },
  {
    "caption": "A Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP‐parkin cells treated with 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), and 20 μM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO‐treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments).",
    "molecules": "antimycin A, CCCP, DMSO, oligomycin"
  },
  {
    "caption": "B U2OS:GFP‐parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 μm.",
    "molecules": "antimycin A, CCCP, DMSO, oligomycin"
  },
  {
    "caption": "D U2OS:GFP−parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp−positive/TOM20−negative structures colocalizing with GFP−parkin (green). Scale bars, 20 μm.",
    "molecules": "antimycin A, CCCP, DMSO, oligomycin"
  },
  {
    "caption": "A Representative immunoblot of whole‐cell lysates from U2OS:GFP and GFP‐parkin cells treated with DMSO, 25 μM antimycin A (anti A), 25 μM antimycin A with 10 μM oligomycin (anti A + oligo), or 20 μM CCCP for the indicated time period.",
    "molecules": "antimycin A, CCCP, DMSO, oligomycin"
  },
  {
    "caption": "A Representative images of Bem1-GFP and the PS marker LactC2-GFP in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks. Black and white pixels were inverted to improve contrast for display. B Frequency of cells displaying polarized Bem1-GFP signal in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells in each of 3 independent experiments. Paired Student's t-tests were performed. ",
    "molecules": "PS"
  },
  {
    "caption": "C Imaging of the PI4P probe GFP-2xPHOsh2 in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks in which black and white pixels were inverted.",
    "molecules": "PI4P"
  },
  {
    "caption": "D Scatter dot plot showing PI4P levels at the plasma membrane (see experimental procedure for details of the quantification) in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells, observed over 3 experiments. Mann-Whitney tests were performed.",
    "molecules": "PI4P"
  },
  {
    "caption": "E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1∆ mutant. Values are means ± SD, n > 100 cells, observed over 3 experiments. A value > 1 indicates enrichment of the fluorescence in the bud.",
    "molecules": "PI4P"
  },
  {
    "caption": "F Images of Bem1-GFP in cho1∆ 9xMyc-AID-stt4 cells after 30 min treatment with or without 0.5 mM auxin. Images are average intensity projections of deconvolved z-stacks.",
    "molecules": "auxin"
  },
  {
    "caption": "G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed.",
    "molecules": "auxin"
  },
  {
    "caption": "H Images of Bem1-GFP (cyan) and Cdc24-mCherry (red) signals in cho1∆ 9xMyc-AID-stt4 cells with or without auxin. Images display maximum intensity projections of z-stacks.",
    "molecules": "auxin"
  },
  {
    "caption": "B Upper panel. SDS-PAGE stained with Coomassie blue in which Bem1 is indicated. Lower panel. The liposomes were composed of 100% phosphatidylcholine (PC), 80% PC and 20% phosphatidylethanolamine (PE), 95% PC and 5% phosphatidic acid (PA), 95% PC and 5% phosphoatidylinositol (PI), 95% PC and 5% PI(4,5)P2 (PI(4,5)P2), 95% PC and 5% PI4P (PI4P), 80% PC and 20% phosphatidylserine (PS) or 75% PC 20% PS 5% PI4P (PS+PI4P). (S) supernatant, (P) pellet.",
    "molecules": "PI(4,5)P2, PA, phosphatidic acid, phosphatidylserine, PS, PC, phosphatidylcholine, PE, phosphatidylethanolamine, phosphoatidylinositol, PI, PI4P"
  },
  {
    "caption": "C Liposomes composed of either 100% PC ± 40% ergosterol or 75% PC, 20% PS, 5% Bem1 ± 40% ergosterol replacing PC were incubated with Bem1 and treated as in panel (B).",
    "molecules": "ergosterol, PS, PC"
  },
  {
    "caption": "E Percentage of the indicated bem1 constructs associated with liposomes containing 75% PC, 20% PS and 5% PI4P.",
    "molecules": "PS, PC, PI4P"
  },
  {
    "caption": "F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately.",
    "molecules": "POPC-d31, Lipid, POPS"
  },
  {
    "caption": "Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/PI4P (19:1 molar ratio).",
    "molecules": "POPC-d31, Lipid, PI4P"
  },
  {
    "caption": "Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPC/PI4P (15:4:1 molar ratio).",
    "molecules": "POPC-d31, Lipid, PI4P, POPC"
  },
  {
    "caption": "Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).",
    "molecules": "POPC-d31, ergosterol, Lipid, POPS"
  },
  {
    "caption": "B Percentage of the different full-length bem1 bc mutants associated with liposomes containing 75% PC 20% PS and 5% PI4P.",
    "molecules": "PS, PC, PI4P"
  },
  {
    "caption": "C Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 to reactions containing Mant-GTP (100 nM), GMP-PNP (100 µM) and the proteins indicated.",
    "molecules": "GDP, GMP-PNP, Mant-GTP, mant-GTP"
  },
  {
    "caption": "D Ten-fold serial dilutions of cells and subsequent colony formation on the indicated plates, where expression of wild type GALp-CDC42 is either induced in the presence of Gal or repressed in Dex. Note how mutation of the wild type Cdc42 (KKSKK) to MMSMM is lethal (see blue box), whereas appending the Bem1 BC-1 motif to this cdc42 mutant restores viability (red box).",
    "molecules": "Dex, Gal"
  },
  {
    "caption": "E Representative images of the mEOS-cdc42 mutants signal (cyan) after inducing the expression of GAL1p-CDC42 in the presence of Gal or repressing it in the presence of Dex. The cells in the blue and red boxes correspond to the cells in the blue and red box in panel (D). Images are average fluorescence intensity projections in which black and white pixels have been inverted.",
    "molecules": "Dex, Gal"
  },
  {
    "caption": "B Percentage of full-length Cdc24 and cdc24 ph domain mutant (cdc24 ph) associated with liposomes containing 75% PC 20% PS and 5% PI4P.",
    "molecules": "PS, PC, PI4P"
  },
  {
    "caption": "C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes.",
    "molecules": "lipid"
  },
  {
    "caption": "F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants.",
    "molecules": "lipid"
  },
  {
    "caption": "F Active Cdc42-GTP levels were quantified in the strains indicated using a gic2(1-208)-yeGFP probe. Values are mean ± SD for n = 30 cells observed over 2 experiments. Data were compared using a Mann-Whitney test.",
    "molecules": "GTP"
  },
  {
    "caption": "A Super-resolution fluorescence microscopy  images showing relative distribution between MATR3  and DAPI. Scale bars, 5μm (Upper) or 0.5μm (Lower). Arrows point to the representative MATR3/DAPI co-staining regions. B Coefficient of correlation between MATR3  and histone  modification H3K9me3 (n=105), H3K9me2 (n=98), H3K27me3 (n=107), H3K27ac (n=97) and H3K4me3 (n=99) in AML12  cells. Quantifications were performed on randomly selected ROIs in cell nuclei. Each point represents one cell.",
    "molecules": "DAPI"
  },
  {
    "caption": "C (Upper) Schematic diagram of dox-inducible shRNA system for MATR3  knockdown and MATR3  rescue in AML12  cells. (Lower)  Western blotting  detected the expression level of MATR3  after 3 days of Dox  treatment (+Dox) and followed by 3 days of Dox  removal (±Dox) in AML12  cells. Rep, replicate.",
    "molecules": "dox, Dox"
  },
  {
    "caption": "D (Upper) Representative cross-section images showing distribution of histone  modifications  upon Ctrl and MATR3  knock down (+Dox). (Lower) Quantify the distribution pattern of histone  modifications by Standard Deviation of Pixel Intensity in cell nuclei. For H3K9me3, n=102 (Ctrl) or 84 (+Dox); for H3K9me2, n=100 (Ctrl) or 101 (+Dox); for H3K27me3, n=98 (Ctrl) or 98 (+Dox); for H3K27ac, n=117 (Ctrl) or 124 (+Dox); for H3K27me3, n=98 (Ctrl) or 98 (+Dox); for H3K4me3, n=107 (Ctrl) or 97 (+Dox). Each point represents one cell. The P values were calculated using unpaired two-tailed Student's t test; ns, not significant, *p<0.05, ****p<0.0001. Error bars indicate mean ± s.e.m.",
    "molecules": "Dox"
  },
  {
    "caption": "K Representative cross-section images showing nuclear localization of H3K27me3 in ES  cells after 6h addiction of 500μM IAA  (+IAA  6h) or equal-volume of alcohol  (-IAA). Middle: The zoom-in view of H3K27me3 in one ES  cell. The outer dotted line colocalizes  with nuclear membrane; the inner dotted line has the 80% diameter of the outer dotted line. The average pixel intensity of regions within outer dotted line (ROI.o) and regions within inner dotted line (ROI.i) were measured separately for each cell. Scale bars, 5μm.",
    "molecules": "alcohol, IAA"
  },
  {
    "caption": "L Statistics for relative intensity (ROI.o/ ROI.i) after 6h addiction of 500μM IAA  (+IAA  6h) (n=46) or equal-volume of alcohol (-IAA) (n=38). Each point represents one cell. The P values were calculated using unpaired two-tailed Student's t test; ****p<0.0001. Error bars indicate mean ± s.e.m.",
    "molecules": "IAA"
  },
  {
    "caption": "A Western blotting showing the distribution of MATR3  proteins in  chromatin-non-associated and chromatin-associated extracts before and after DRB  (75μM for 12h) or RNase A (pre-treat with 0.05% Triton x-100 for 30s, followed by 10μg/ml RNase A for 1h) treatment in AML12  cells. Representative of two independent replicates with similar results.",
    "molecules": "DRB, Triton x-100"
  },
  {
    "caption": "B (Left) The representative cross-section image showing nuclear  distribution of DAPI, MATR3  and H3K27me3 before and after 24h treating of 75μM DRB  in AML12  cells. (Right) Line charts showing pixel intensity of each channel on the ROIs. r, coefficient of correlation. Scale bars, 5μm.",
    "molecules": "DRB, DAPI"
  },
  {
    "caption": "C (Left) The representative cross-section image showing nuclear  distribution of H3K27me3 and AS  L1  RNA before and after MATR3  knockdown (Dox  treatment for 3d) in AML12  cells. (Right) The normal distribution curve for the AS  L1  pixel intensity. Scale bars, 5μm.",
    "molecules": "Dox"
  },
  {
    "caption": "C Representative images of  droplet  formation assays with different concentrations of GFP-MATR3  proteins. NaCl  concentration, 50mM. Scale bars, 5μm. D Representative images of droplet  formation assays  with different NaCl  concentrations. GFP-MATR3  protein concentration, 3 μM. Scale bars, 5μm. E Areas of MATR3  protein droplets  formed in different protein concentration (3 μM: n = 315; 2 μM: n = 196; 1 μM: n = 165; 0.5 μM: n = 50). The P values were calculated using unpaired two-tailed Student's t test; ****p<0.0001. F Areas of MATR3  protein droplets  formed in different NaCl  concentration (50 mM: n = 248; 100 mM: n = 196; 400 mM: n = 127; 800 mM: none). The P values were calculated using unpaired two-tailed Student's t test; ****p<0.0001.",
    "molecules": "NaCl"
  },
  {
    "caption": "H Representative images of  droplet formation assays by GFP-MATR3  with different concentration (0nM, 5nM, 50nM, 200nM, 500nM) of AS L1  RNAs. GFP-MATR3  protein concentration, 3 μM. NaCl  concentration, 50mM. Scale bars, 5μm.",
    "molecules": "NaCl"
  },
  {
    "caption": "(E) dose response of MyD88 flies after ∆gliP (gliotoxin) mutant or wild-type [∆akuB] A. fumigatus infection; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each; wild-type flies are used as a control for the dose of 250 conidia. (F-H) dose response of MyD88 and wild-type flies after gliotoxin (F), fumagillin (G), and helvolic acid (H) injection at the indicated concentrations (20 flies per condition).",
    "molecules": "fumagillin, gliotoxin, helvolic acid"
  },
  {
    "caption": "(A) survival of MyD88 or wild-type flies to 250 injected ∆aspf1 (restrictocin mutant) or wild-type [∆akuB] A. fumigatus conidia (20 flies per condition); MyD88: ∆aspf1 vs. ∆akuB (***p=0.0007). (B) survival of MyD88 flies after the injection of different concentrations of restrictocin (R) (20 flies per condition ). (C-D) survival of antibiotics-treated (C) and axenic (D) MyD88 mutant flies after restrictocin injection (20 flies per condition).",
    "molecules": "restrictocin"
  },
  {
    "caption": "(E-F) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild-type (E) and MyD88 (F) flies; the arrowheads show the position of the 28S RNA-derived α-sarcin fragments whereas arrows on the right show its electrophoretic band position.",
    "molecules": "PBS, restrictocin"
  },
  {
    "caption": "(H) SDS-PAGE analysis of 35S-labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G-capped reporter RNA containing the 5'UTR of β-globin followed by the Renilla luciferase coding sequence (arrow), in the presence of increasing concentrations (0.125 to 25 nM) of restrictocin.",
    "molecules": "m7G, restrictocin, 35S"
  },
  {
    "caption": "(C-D) rescue of the sensitivity of Bom∆55C flies to verruculogen (C) or to restrictocin (D) by the transgenic expression of individual 55C locus genes (caption in D also applies to (C)). 55C flies vs. BomS1, *p=0.0495, vs. BomS6 *p=0.011 for verruculogen assay (C); 55C flies vs. BomBc1 or BomS3, ****p<0.0001, 55C flies vs. BomS6, **p=0.0028 for restrictocin assay (D) (20 flies per condition).",
    "molecules": "restrictocin, verruculogen"
  },
  {
    "caption": "(E-F) expression levels of BomBc1, and BomS3 measured by RT-digital PCR 48 hours after challenge; BomBc1 PBST vs. Af, *p=0.015, PBST vs. restrictocin (R), *p=0.015; BomS3: PBST vs. Af, *p=0.02, PBST vs. R, *p=0.03 (pooled data of n=3 experiments, biological replicates).",
    "molecules": "restrictocin"
  },
  {
    "caption": "(A-B) tremor rate (A) and survival (B) of flies (20 flies per condition) overexpressing Tl[10B] in neurons compared to wild-type after injection of verruculogen. (A) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. elav> UAS-Toll10B, **p=0.002. (C-D) tremor rate (C) and survival (D) of flies (20 flies per condition) overexpressing Tl[10B] in glia compared to wild-type after injection of verruculogen. (C) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. repo> UAS-Toll10B, **p=0.002. (D) Survival wt V vs. repo> UAS-Toll10B V, **p=0.005.",
    "molecules": "verruculogen"
  },
  {
    "caption": "(E-G) recovery time from tremor (E,F) and survival (G) of single flies overexpressing BomS6 ubiquitously (E,G) or in neurons (F-G) (biological replicates) compared to wild-type after injection of verruculogen; in (G) the inset represents the survival of vehicle control groups.",
    "molecules": "verruculogen"
  },
  {
    "caption": "(H-I) recovery time from tremor (H) and survival (I) of single flies overexpressing BomS6 in glia (pooled data from n=3 experiments, biological replicates) compared to wild-type after injection of verruculogen; in (I) the inset represents the survival of vehicle control groups. Recovery time (H) repo>mCherry vs. repo>BomS6, p=0.058, survival (I) repo>mCherry V vs. repo>BomS6 V, *p=0.016.",
    "molecules": "verruculogen"
  },
  {
    "caption": "(J-M) expression of BomS4 (J) and BomS6 (K) in head after verruculogen powder challenge, and BomS4 (L) and BomS6 (M) in head after A. fumigatus (Af), restrictocin or M. luteus injection (pooled data from n=3 experiments, biological replicates).",
    "molecules": "restrictocin, verruculogen"
  },
  {
    "caption": " B In vitro substrate ubiquitylation assay for Hrd1 with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; \"no E3\" reactions do not contain S-Hrd1, \"no ATP\" reactions do not contain ATP, but S-Hrd1. The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the \"no E3\" control of a given E2 are shown. ",
    "molecules": "ATP, Ub"
  },
  {
    "caption": " C In vitro Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (top - yellow) and Ubc6 (bottom - green) with ethanolamine as nucleophile. Representative Coomassie gels are shown. ",
    "molecules": "ethanolamine, Ub"
  },
  {
    "caption": " D Quantification of Ub nucleophile discharge assays for Hrd1 with U7BR/Ubc7 (left) and Ubc6 (right). Plots of E2~Ub discharge (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as means ± standard deviation (n = 3). Insets show reaction rates derived from these fits. Significances were determined by student T-test; * p < 0.05. (E) Stimulation of U7BR/Ubc7 and Ubc6 discharge activities by Hrd1. E2 stimulation is reported as the ratio of rates derived from RING-catalyzed reactions and "no E3" controls in D. Values are reported as means ± standard deviation (n = 3). Significance was determined by student T-test; * p < 0.05. ",
    "molecules": "Ub"
  },
  {
    "caption": " A In vitro Ub nucleophile discharge assays for U7BR/Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2C-D). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, reactions for Hrd1(400R) and Doa10(94R) are significantly faster than all other reactions and the reaction for Doa10(94H) is significantly faster than that for Doa10(94E). The wild-type E3s are identified by black frames for each set. ",
    "molecules": "Ub"
  },
  {
    "caption": " B In vitro Ub chain formation assay by Ubc7 with indicated Hrd1 (left) and Doa10 (right) variants in the presence of indicated Cue1 variants. Rates for reactions of mono-Ub to di-Ub and di-Ub to tri-Ub with fluorescently-labeled Ub are shown on a logarithmic scale. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control of given reaction set are shown. ",
    "molecules": "di-Ub, tri-Ub, mono-Ub, Ub"
  },
  {
    "caption": " D Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2). ",
    "molecules": "CHX"
  },
  {
    "caption": " A Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrate PrA*-3xHA (left) and by CHX decay assays for the Doa10 model substrate Deg1-eGFP2 (right). Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA and n = 3 for Deg1-eGFP2). Data for control strains (Hrd1(400R) wt, Δhrd1, Doa10(94H) wt and Δdoa10) is the same as in Fig. 3D. ",
    "molecules": "CHX"
  },
  {
    "caption": " B In vitro substrate ubiquitylation assay for Hrd1 variants with Ubc7. Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; \"no E3\" reaction does not contain any S-Hrd1, \"no ATP\" reaction does not contain ATP, but wild-type S-Hrd1(400R). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the \"no E3\" control are shown. In addition, the reaction containing S-Hrd1(400R) shows significantly more ubiquitylation than those with other E3 variants. ",
    "molecules": "ATP, Ub"
  },
  {
    "caption": " C In vitro Ub nucleophile discharge assays for Ubc6 with indicated Doa10 variants. E2 stimulation is shown as the ratio of rates derived from RING-catalyzed reactions and the "no E3" control (see Fig. EV2E). Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity only significances related to the "no E3" control are shown. In addition, the reaction for Doa10(94R) is significantly faster than that for Doa10(94H) and (94E). The wild-type E3 is identified by a black frame. The scale of the y-axis was set identical to that of plots from other discharge assays ",
    "molecules": "Ub"
  },
  {
    "caption": " D In vitro substrate ubiquitylation assay for Doa10 variants with Ubc7 (left) and Ubc6 (right). Ubc7 reactions contained equimolar amounts of Cue1 and were performed with Ub(K48R). Top: representative immunoblot using a poly-clonal α-RNase A antibody; \"no E3\" reaction does not contain any S-Doa10, \"no ATP\" reaction does no contain ATP, but wild-type S-Doa10(94H). The RNase-Ub2 band co-migrates with a nonspecific band (#) common to all samples. Bottom: Quantification of RNase-Ub signals is shown. Values are reported as means ± standard deviation (n = 3). Significances for pairwise comparisons were determined by One-way ANOVA Test; * p < 0.05. For clarity, only significances related to the \"no E3\" control of a given E2 are shown. In addition, the reaction containing Ubc7 and S-Hrd1(400R) shows significantly more ubiquitylation than those with Ubc7 and other E3 variants. ",
    "molecules": "ATP, Ub"
  },
  {
    "caption": " A E2~Ub hydrolysis assays for U7BR/Ubc7 and Ubc6. E3-independent Ub discharge assays for both E2s in PBS (absence of nucleophile = hydrolysis) were performed at 32°C. Left: representative Coomassie gels. Right: Plots of E2~Ub hydrolysis (dots) as a function of time with first-order reaction models fitted to the discharge data (lines) are shown. Values for each time point are reported as mean ± standard deviation (n = 3). ",
    "molecules": "Ub"
  },
  {
    "caption": " B HSQC-TROSY experiments comparing free and E2-conjugated (15N)Ub. Top: CSPs of (15N)Ub(G76C) conjugated to indicated E2s via disulfide bond (775 µM each) compared to free (15N)Ub(G76C) are shown; Ub resides known to be involved in the closed conformation interface are underlined in grey; * = number of resonances with perturbations too large to be assigned confidently (shown as off-scale in diagram). Bottom: Spectra of resonances for selected residues in Ub's hydrophobic patch for free Ub(G76C) (gray) and Ub conjugated to Ubc6 (green) and U7BR/Ubc7 (yellow). ",
    "molecules": "15N, Ub, Ub's"
  },
  {
    "caption": " Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates Deg1-eGFP2 Values for each time point are reported as means ± standard deviation n = 3 for Deg1-eGFP2 ",
    "molecules": "CHX"
  },
  {
    "caption": " Protein degradation in indicated yeast strains monitored by CHX decay assays for the Doa10 model substrates FLAG-Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG-Sbh2 ",
    "molecules": "CHX"
  },
  {
    "caption": " Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the lysine-less CPY*-K0-HA Values for each time point are reported as means ± standard deviation ",
    "molecules": "lysine"
  },
  {
    "caption": "b, Autophagicbody formation in yeast deprived of nitrogen in the presence (+) and absence (-) of 1 mM PMSF. Arrows denote cells that would be scored as positive in the experiment shown in c.",
    "molecules": "nitrogen, PMSF"
  },
  {
    "caption": "c, Quantitative effects of apg6/vps30 and beclin 1 transformation on autophagicbody formation in apg6/vps30-disrupted yeast in the presence of PMSF. Cells with one or more autophagic bodies within the vacuole were scored as positive (see arrows in b). A minimum of 100 cells was counted for each sample. Results represent the mean (±s.e.m.) percentage of cells with autophagic bodies within the vacuole for triplicate samples. Similar results were obtained in five independent experiments.",
    "molecules": "PMSF"
  },
  {
    "caption": "a-f, Electron micrographs of MCF7.control (clone 38) (a, b) and MCF7.beclin1 (clone 17) cells (c- f) grown in nutrient-rich media (a, c) or subjected to 4 h of serum and amino-acid deprivation (b, d-f). Asterisks in e and f denote autophagic vacuoles that would be counted in the experiment shown in g. Autophagic vacuoles were defined as double-membrane vacuolar structures containing recognizable cytoplasmic contents. Scale bars, 1 µm.",
    "molecules": "amino-acid, nutrient"
  },
  {
    "caption": "g, Quantitative effects of beclin 1 on basal and nutrient-deprivation-induced autophagy of MCF7 cells. Bars indicate mean (±s.e.m.) number of autophagic vacuoles per cell for cells growing in normal media (black), for cells subjected to 4 h of serum and amino-acid deprivation (grey), and for cells pre-treated for 30 min with 10 mM 3-MA and subjected to 4 h of serum and amino-acid deprivation in the presence of 3-MA (open). Mean was determined by counting the total number of autophagic vacuoles in each cell for 100 cells per clone per treatment.",
    "molecules": "3-MA, amino-acid, nutrient"
  },
  {
    "caption": "h, Comparison of the rates of degradation of long-lived proteins in MCF7.control cells (squares, clone 38), MCF7.beclin1 cells (circles, clone 17) and MCF7.beclin1stop cells (triangles, clone 70), in EBSS + 10% serum and complete amino acids (open symbols, solid lines) in EBSS alone (closed symbols, solid lines), and in EBSS + 10 mM 3-MA (closed symbols, dotted lines). Results are mean (±s.e.m.) of triplicate wells. Similar results were obtained in three independent experiments. Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g.",
    "molecules": "3-MA, amino acids"
  },
  {
    "caption": "(A) The growth rate of NME6 KO and NME6 KO + NME6-MycFlag HeLa relative to WT HeLa cells incubated in DMEM containing 25 mM glucose (log2; n=13 independent cultures).",
    "molecules": "glucose"
  },
  {
    "caption": "(D) Representative live-cell images of the indicated cell lines grown in Human Plasma Like Medium (HPLM) (left) and calculated cell death after 96 h (right). Cell confluency is depicted with the yellow mask and dead cells are identified by SYTOX green staining in purple (n=3 independent cultures).",
    "molecules": "SYTOX green"
  },
  {
    "caption": "(A) Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of the indicated HeLa cell lines during a mitochondrial stress test with inhibitor treatments at the indicated timepoints. P-values for OCR: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001; P-values for ECAR:two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.001, P-value (interaction) = 0.0517; P-values for genotype are shown (Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot+AA, rotenone and antimycin a; n=3 independent experiments).",
    "molecules": "AA, antimycin a, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, FCCP, Oligo, oligomycin, Rot, rotenone"
  },
  {
    "caption": "(H) The relative growth of WT and NME6 KO liver cancer cell lines monitored on each day (d) using an ATP luminescence assay. P-values for HLE cells: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = <0.0001; P-values for Huh6 cells: two-way ANOVA P-value (time) = <0.0001, P-value (genotype) = 0.0005, P-value (interaction) = <0.0001;p-values for genotype are shown (n=4 independent cultures).",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Representative mitochondrial translation assay monitored by the incorporation of 35S methionine and cysteine into the indicated mtDNA-encoded proteins followed by autoradiography (top panel). MtDNA encoded proteins are labelled according to expected size and NME6 and SDHA immunoblots are shown below. (B) Quantification of 35S methionine and cysteine incorporation into all mtDNA-encoded proteins labelled in (B) relative to WT HeLa cells at 15 min (log2; mitochondrial preparations from n=3 independent cultures).",
    "molecules": "cysteine, methionine, 35S"
  },
  {
    "caption": "(C) Immunofluorescence of NME6 with mtDNA (top) or bromouridine (BrU) labelled nascent mtRNA (bottom) in HeLa cells imaged by confocal microscopy. Relative fluorescence intensities were calculated from linescans generated within the mitochondrial regions indicated by dotted lines.",
    "molecules": "bromouridine, BrU"
  },
  {
    "caption": "(A) Heat map of log2 transformed mean mitochondrial transcript levels of WT and NME6 KO HeLa cells incubated with 100 µM rNTPs or dNTPs for 48 h relative to untreated WT cells analysed by qRT-PCR (n=3 independent cultures).",
    "molecules": "dNTPs, rNTPs"
  },
  {
    "caption": "(B) Volcano plot representation of log2 fold change in proteins between NME6 KO cells treated with 100 µM rNTPs for 120 h and untreated NME6 KO HeLa cells determined by quantitative mass spectrometry. OXPHOS subunits are highlighted in teal (n=3 independent cultures)",
    "molecules": "rNTPs"
  },
  {
    "caption": "(G) CYTB (top) and ND5 (bottom) transcript levels analysed by qRT-PCR in WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 48 h. P-values were calculated using a one-way ANOVA (A, adenosine; G, guanosine; C, cytidine; U, uridine; log2; 100 µM; n=4 independent cultures).",
    "molecules": "adenosine, cytidine, guanosine, uridine"
  },
  {
    "caption": "(H) CTP (top) and dCTP (bottom) levels in the mitochondria enriched fraction of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for 120 h as determined by quantitative mass spectrometry. P-values for CTP: two-way ANOVA P-value (supplementation) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = 0.3179; P-values for dCTP: two-way ANOVA P-value (supplementation) = <0.0001, P-value (genotype) = <0.0001, P-value (interaction) = 0.0569; P-values for supplementation are shown (C, cytidine; U, uridine; log2; 100 µM; n=4 independent cultures).",
    "molecules": "CTP, cytidine, dCTP, uridine"
  },
  {
    "caption": "(I) Basal (top) and maximal (bottom) oxygen consumption rates of WT and NME6 KO HeLa cells incubated with the indicated nucleoside species for a minimum of 120 h relative to untreated WT cells (A, adenosine; G, guanosine; C, cytidine; U, uridine; log2; 100 µM; n=4 independent experiments). P-values were calculated using a one-way ANOVA.",
    "molecules": "adenosine, cytidine, guanosine, uridine"
  },
  {
    "caption": "(B) Histopathological scoring of edema, inflammation in different regions of the joint footpad and muscle pathology of WT-CHIKV challenged animals (n=5) at 6 dpc, performed blind. Three sections from each joint footpad were scored. The data are expressed as the means ± SD. Data were analyzed by one-way ANOVA with Tukey post-test; Edema (***P =0.000002 WT-CHIKV versus PBS, ***P =0.000004 RH-CHIKV versus PBS), Muscle necrosis (**P =0.005521 WT-CHIKV versus PBS, *P =0.02628 RH-CHIKV versus PBS), Muscle regeneration (***P <0.000001 WT-CHIKV versus PBS, ***P <0.000001 WT-CHIKV versus RH-CHIKV), Muscle inflammation (*P =0.040258 WT-CHIKV versus PBS, *P =0.040258 RH-CHIKV versus PBS), Synovial membrane inflammation (*P =0.014457 WT-CHIKV versus PBS, *P =0.014457 RH-CHIKV versus PBS), Subcutaneous area inflammation (***P =0.00004 WT-CHIKV versus PBS, ***P =0.000074 RH-CHIKV versus PBS).",
    "molecules": "PBS"
  },
  {
    "caption": "S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. A) Puromycin was added 20 min. before cell harvesting. Total puromycin incorporation and Actin level were revealed by western blot.",
    "molecules": "O2, Puromycin"
  },
  {
    "caption": "S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. (C) LDH and RpL32 protein levels were detected by western blot in total cell extracts from S2 cells exposed to normoxic conditions or to 1% O2 environment for 24 and 48 hours.",
    "molecules": "O2"
  },
  {
    "caption": "S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. (E) One representative experiment performed as in (D) is shown.",
    "molecules": "O2"
  },
  {
    "caption": "S2 cells were transiently transfected with the indicated constructs and exposed to 1% O2 for 24h. (A, B) (A) Activities of Firefly (Fluc) and Renilla (Rluc) luciferase, produce from reporter genes under control of the Ldh gene promoter, were measured in total cell extracts by dual luciferase assay and the ratio of both measures was calculated for each transfection. (B) Relative FLuc and RLuc mRNA levels were measured by RT-qPCR.",
    "molecules": "O2"
  },
  {
    "caption": "S2 cells were transiently transfected with the indicated constructs and exposed to 1% O2 for 24h. (D) Indicated stably transfected cell lines were exposed to 21% or 1% O2 for 24h before fractionation of cell extracts by ultracentrifugation on linear sucrose gradients (15%-50% The distribution of FLuc mRNA, produced from reporter genes under control of the pMT promoter was measured by northern blot in each cell types. Fractions numbers from top to bottom of the gradient are indicated.",
    "molecules": "O2"
  },
  {
    "caption": "(A) Control (TM3>UAS-eIF4EHP_RNAi) or eIF4EHP KD (Tub-GAL4>UAS-eIF4EHP_RNAi) flies were maintained in either normoxia or hypoxia (1%O2, 24h). Protein extracts were prepared from whole flies and eIF4EHP, LDH and Actin were detected by western blot. (B) Quantification of LDH/Actin ratio in 3 independent experiments performed as in A. Values represent mean +/- SD, n=3 biological replicates, P-value of two-ways ANOVA.",
    "molecules": "O2"
  },
  {
    "caption": "(E) Cell extracts from Yellow, eIF4EHP or eIF4E6 KO cells incubated at 21 or 1% O2 were separated by centrifugation on linear sucrose gradients and Ldh mRNA was detected by northern blot in the different fractions. Fractions numbers from top to bottom of the gradient are indicated.",
    "molecules": "O2"
  },
  {
    "caption": "(G) Indicated S2 cell lines were cultivated at 1% O2 for 24h and labelled with puromycin (5µg/ml) for 20 min. before harvesting. Puromycin signal in total cell extracts was quantified by western blot and expressed relative to the level detected in wild-type cells (n=6 biological replicates, t-test, two-sided).",
    "molecules": "O2, puromycin, Puromycin"
  },
  {
    "caption": "(E, F) 24h after transfection, cells were cultivated in hypoxia (1%O2) for an additional 24h before measurement of luciferase activity by dual luciferase reporter assay (D) and luciferase mRNA levels by qPCR (E). Values represent mean ± SD, n=3 biological replicates, P-value of one-way ANOVA, *-P ≤ 0.05, ns- P>0.05.",
    "molecules": "O2"
  },
  {
    "caption": "(C) Combined detection of eIF4EHP by immunofluorescence (green) and polyA mRNA by oligo-dT FISH (red) in normoxic and hypoxic WT S2 cells. nuclei were counterstained with DAPI (blue). Red and green signal intensities were quantified with the Zen software (Zeiss).",
    "molecules": "oligo-dT, DAPI, polyA"
  },
  {
    "caption": "(D) Combined detection of GFP-Rox8 or GFP-Dcp1 (green) and endogenous eIF4EHP (red) in transfected S2 cells cultivated in normoxia (21% O2), in hypoxia (1% O2 for 24h) or after oxidative stress (250μM arsenite for 1h). Representative images of confocal imaging (Z-stack maximum intensity projection) and zooms are shown. , nuclei were counterstained with DAPI (blue). Red and green signal intensities were quantified with the Zen software (Zeiss).",
    "molecules": "arsenite, DAPI, O2"
  },
  {
    "caption": "(B) Adult Canton S flies were kept at 21% or 1% O2 for 24h. Total protein extracts were prepared as described in the method section and fractionated by ultracentrifugation on sucrose gradients (15-50%). OD at 260 nm was measured in line during fractionation (upper panels). EIF4EHP was detected by western blot in representative fractions of the gradients.",
    "molecules": "O2"
  },
  {
    "caption": "(C) Cross-Linking and Immunoprecipitation experiment (CLIP) was performed on cell extracts from normoxic and hypoxic (1% O2, 24h) UV irradiated WT or eIF4EHP KO S2 cells. eIF4EHP was immunoprecipitated with eIF4EHP antibody-coated protein G sepharose beads before Ldh and Rpl32 mRNA detection by RT-qPCR in total extract (input) or immunoprecipitation eluate. mRNA enrichment levels in eluate as compared to input from 3 independent experiments were calculated as described Data represent mean +/- SD. Statistical significance was tested by two-way ANOVA followed by Sidak's multiple comparisons test, * P<0.05.",
    "molecules": "sepharose, O2"
  },
  {
    "caption": "(A) Female or male flies with chromosomal insertions of an UAS_eIF4EHP (BDSC 36876) RNAi construct were crossed with male or female Tub>Gal4/Tm3 (BDSC 5138) driver flies. Two hours after mating, embryos were transferred to 6% O2 (lowest tolerated O2 concentration) or kept in normoxia until eclosion. Tub-Gal4/UAS-eIF4EHP_RNAi and Tm3/UAS-eIF4EHP_RNAi F1 individuals were counted based on the presence (control) or absence (eIF4EHP KD) of the Tm3 SB marker and results were expressed as % of total eclosions for each condition. Data represent mean +/-SD fro 2 independent biological replicates. Statistical significance was tested by two-way ANOVA followed by Sidak's multiple comparisons test. **** P<0.0001; *** P<0.001; ** P<0.01; * P<0.05.",
    "molecules": "O2"
  },
  {
    "caption": "(B) Mobility of age-matched Tub-Gal4/UAS-eIF4EHP_RNAi (eIF4EHPRNAi), TM3/UAS-eIF4EHP_RNAi (control) (upper panels) or Tub-Gal4/UAS-mCherry_RNAi (m-cherryRNAi) (lower panel) females was assessed by negative geotaxis assay in normoxic conditions (21% O2) or 40 min. and 48h after an initial exposure to 1% O2 for 24h. Flies climbing past threshold within 120s were counted and expressed as % of total flies. Each assay was performed in triplicate with 20 flies. Data are mean +/- SD.",
    "molecules": "O2"
  },
  {
    "caption": "Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (A) proliferation by flow cytometry for Ki-67 staining after four days of culture (n=6, biological replicates),",
    "molecules": "PGE2"
  },
  {
    "caption": "Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for , (B) ability to kill K562 cells after two days of culture in a 4-hour chromium-assay at an E:T ratio of 5:1 (n=4, biological replicates).",
    "molecules": "PGE2"
  },
  {
    "caption": "Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (C) Viability by flow cytometry using fixable live/dead staining after two days of culture (n=7, biological replicates).",
    "molecules": "PGE2"
  },
  {
    "caption": "D) Intracellular cAMP detected in two-day IL-2 or IL-15 activated NK cells before and 5 minutes after the addition of PGE2 (1 µM) (n=3, biological replicates)",
    "molecules": "cAMP, PGE2"
  },
  {
    "caption": "E) mRNA levels of selected PGE2 response elements as measured by qPCR in NK cells (n=3-4, biological replicates).",
    "molecules": "PGE2"
  },
  {
    "caption": "A-B) NK cells were activated with IL-2 or IL-15 for two days in the presence or absence of PGE2 (1µM) and analyzed for A) the levels of phosphorylated S6 (pS235/pS236), STAT3 (S727), STAT5 (Y694), Akt (S473) (n=4, biological replicates) and B)16 categories of phosphorylation array showed. Red box indicates category 1 (pS6+, pSTAT3+, pSTAT5+, pAKT+).",
    "molecules": "PGE2"
  },
  {
    "caption": "C) Relative changes in frequency of NK cells expressing pS6 in the presence and absence of PGE2 (n=4, biological replicates).",
    "molecules": "PGE2"
  },
  {
    "caption": "D) Flow cytometry analysis of PDE4A in the presence or absence of Torin-1 (1µM) (left, n=7, biological replicates) with representative histograms (right).",
    "molecules": "Torin-1"
  },
  {
    "caption": "E) NK cell-mediated cytotoxicity of K562 cells by 4-hour chromium-release assay of two-day cytokine activated NK cells in the presence or absence of Torin-1 (1µM) and PGE2 (1µM) (n=6, biological replicates). E:T ratio=5:1.",
    "molecules": "PGE2, Torin-1"
  },
  {
    "caption": "F, G) (F) Flow cytometry analysis of pS6 and (G) 4-hour chromium-release assay of PGE2-treated NK cells against K562 measured after 48 hours treatment with Roflumilast (PDE4i, 1µM) (n=3, biological replicates) E:T ratio=5:1.",
    "molecules": "PGE2, Roflumilast"
  },
  {
    "caption": "D) Representative flow cytometry graphs for CD25 and CD54 staining in IL-2 and IL-15 NK cells in the presence or absence of PGE2.",
    "molecules": "PGE2"
  },
  {
    "caption": "F) Representative phase contrast image of IL-2 and IL-15 activated NK cells in the presence or absence of PGE2. Scale bar 50 µm.",
    "molecules": "PGE2"
  },
  {
    "caption": "G) Chromium-release cytotoxicity assay by two-day cytokine activated NK cells in the presence or absence of PGE2 (1µM) and anti-CD54 (20 µg/ml) (n=4, biological replicates), E:T ratio=5:1.",
    "molecules": "PGE2"
  },
  {
    "caption": "A) NK cells were stimulated with either IL-2 or IL-15 in the presence or absence of PGE2 for two days and thereafter purified as CD25+/CD54+ and CD25-/CD54- and tested in a 51Cr-release assay against K562. (E: T=5:1, n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54- NK cells for each condition.",
    "molecules": "PGE2"
  },
  {
    "caption": "J) NK cells were first stimulated with IL-15 for two days in the absence of PGE2, then purified as CD25+/CD54+ or CD25-/CD54- NK cells and tested in a 51Cr-release cytotoxicity assay against K562 cells at an E:T ratio of 5:1 in the presence (filled bars) or absence (open bars) of PGE2 (1 µM) (n=3, biological replicates).",
    "molecules": "PGE2"
  },
  {
    "caption": "L) Representative image of Mitotracker Red FM in CD25+/CD54+ NK cells and CD25-/CD54- NK cells. Scale bar: 20 μm.",
    "molecules": "Mitotracker Red FM"
  },
  {
    "caption": "B) NK cells were stimulated with IL-2 or IL-15 in the presence or absence of PGE2 and then purified as CD25+/CD54+ or CD25-/CD54- cells and tested for cytotoxicity against A549 tumor cell in a 51Cr-release assay. E:T ratio = 5:1 (n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54-.",
    "molecules": "PGE2"
  },
  {
    "caption": "Death rates of wild-type and GlpK22 mutants plotted versus growth rates. Wild type is grown in glycerol minimal medium in batch cultures (black circle) and in 'chemostat' continuous cultures with growth rates coded in colors, see legend on the center right. GlpK22 mutants (white circle) are grown in glycerol minimal medium in batch culture. Data shown as mean ± (standard deviation) SD. Two replicates per condition.",
    "molecules": "glycerol"
  },
  {
    "caption": "Example experiment from a starved culture previously grown at a rate of 0.3 h − 1 with a viability after 5 days of starvation. After (1) extraction and (2) addition of 40 µM of glycerol the decay of viability is delayed (black circles) compared to a control without glycerol (green symbols). Per viable cell, the glycerol addition in this experiment is 0.33 fmol CFU − 1. After an initial period of survival, the culture with added glycerol (black) dies at the same rate as the control (green). The 'lag time' fitted to this data is 1 day.",
    "molecules": "glycerol"
  },
  {
    "caption": "Lag time of the experiments of panel B for different glycerol concentrations. Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits.",
    "molecules": "glycerol"
  },
  {
    "caption": "Adaptation at the end of growth. When the nutrient concentration decreases, the nutrient uptake rate slows down with a Michaelis-Menten type kinetics (top). Depending on the uptake affinity, bacteria will experience either a sharp or smooth decrease of growth rate (bottom left, bright orange: 10 µM, dark orange: 1 mM). During this slow down, the proteome adapts (bottom center), with lower affinities (dark orange) showing a slight improvement of proteome adaptation, which results in a slightly lower death rate (bottom right).",
    "molecules": "nutrient"
  },
  {
    "caption": "Adaptation in complex media. A medium supplemented with multiple nutrients (one - red, two - green, ten - blue) leads to a step-wise decrease of growth rate during nutrient depletion (bottom left). Due to the extended periods of slow growth after exhaustion of primary nutrients, bacteria can adapt their proteome substantially (bottom center), and decrease their death rate (bottom right).",
    "molecules": "nutrient"
  },
  {
    "caption": "(G) Representative confocal  images of RIPosomes  in mouse BMDMs  infected with S. flexneri  ( MOI 1:25, 8 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained  with DAPI (pseudo-colored red for better contrast).",
    "molecules": "DAPI"
  },
  {
    "caption": "(I) Representative confocal  images of RIPosomes  in  THP-1 cells infected with RFP expressing Salmonella typhimurium (MOI 1:5, 4 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 8 µm. Zoom panels are digital magnifications. DNA is stained  with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(J) Left panel, representative  high-content microscopy images (yellow masks represent software algorithms-defined cell boundaries) of RIPosomes in THP-1  cells infected with S. typhimurium (MOI 1:5, 4 h). Right panel, the graph depicts an average number of RIPosomes/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test. DNA is stained  with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative confocal  images of RIPosomes  in THP-1 cells infected with (K) E. coli LF82 strain (MOI 1: 10, 8h) (LPS antibody is used to stain LF82) Line profile: co-localization  analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained  with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative confocal  images of (M) GFP  expressing M. tuberculosis (MOI 1:10, 8 h). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained  with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(L-M) Representative immunofluorescence  images of doxycycline-inducible GFP-RIPK2  expressing HeLa  cells. (L) Upper panel, uninfected. Lower panel, S. flexneri -infected MOI 1:25, 4h). Immunostaining  was performed with the p65  antibody (red) and DNA stained  with DAPI  (Blue). (M) The graph indicates % of cells that are RIPosomes  positive or negative with nuclear/cytoplasmic p65  (5 fields (each group), Mean ± SD, n=3). ****p < 0.00005, Student's unpaired t-test.",
    "molecules": "DAPI, doxycycline"
  },
  {
    "caption": "(C) Left panel, representative  high-content microscopy images of RIPosomes  in HEK293T  cells that are control cells or cells treated with rapamycin   (500 nM, 4h) or cells treated with rapamycin (500 nM, 4h) and bafilomycin A1  (BafA1, 300 nM). Right panel, the graph depicts the average number of RIPosomes/cell. About 15000 cells were plated per well and RIPosomes  were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), **p < 0.005 and, ***p < 0.0005, ordinary one-way ANOVA (Tukey's multiple comparisons test).",
    "molecules": "BafA1, bafilomycin A1, rapamycin"
  },
  {
    "caption": "(J) GST pull-down assay using purified GST  or GST-RIPK2  proteins with in-vitro translated 35S  radiolabeled myc-ULK1  or myc-ATG16L  or myc-BECLIN1.",
    "molecules": "35S"
  },
  {
    "caption": "(A-B) The  THP-1  cell lysates were subjected to immunoprecipitation  analysis (A) untreated and treated with iE-DAP (40 μg/ml, 6 h), (B) untreated and treated with MDP  (40 μg/ml, 6 h). IP  was performed with isotype control IgG or IRGM  antibody and Western blotting  was performed with indicated antibodies. IgG (H); IgG heavy chain.",
    "molecules": "iE-DAP, MDP"
  },
  {
    "caption": "(E) GST pull-down assay using purified GST  and GST-IRGM  and in-vitro translated 35S  radiolabeled  myc-NOD2, myc-NOD1, and myc-RIPK2.",
    "molecules": "35S"
  },
  {
    "caption": "(C) Doxycycline-inducible stable GFP-RIPK2 HeLa  cells were transfected with control siRNA or IRGM  siRNA followed by infection with S. flexneri  (MOI 1:25, 4h). The cells were fixed and subjected to  high-content microscopy  to quantitate the number of RIPosomes  formed. The graph depicts an average number of RIPosome/cell. About 10000 cells were plated per well and RIPosomes  were screened in 35 fields per well. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test.",
    "molecules": "Doxycycline"
  },
  {
    "caption": "(C) Left panel, representative pictures of colons  of Irgm1+/+ and Irgm-/- mice  untreated or treated with GSK583  infected with  S. flexneri. Right panel, the graph depicts the average colon  lengths of the mice  groups. Mean ± SD, n=6, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.",
    "molecules": "GSK583"
  },
  {
    "caption": "(D) Representative microscopic images of H&E staining  of colon  tissues of Irgm1+/+ and Irgm-/- mice  untreated or treated with GSK583  administrated with DSS. The graph depicts the combined  histological scores. Mean ± SD, n=3 (DSS), **p < 0.005, Student's unpaired t-test. Scale bar, 200 µm",
    "molecules": "DSS, GSK583"
  },
  {
    "caption": "(E) The soluble  and  insoluble fractionations of lysates from colon  tissues of Shigella-infected or DSS-treated Irgm1+/+ and Irgm-/- mice  treated with GSK583  as indicated, were subjected to immunoblot  analysis with indicated antibodies.",
    "molecules": "DSS, GSK583"
  },
  {
    "caption": "(H-I) Western blot  analysis with the colon  tissue lysates of DSS-treated or S. flexneri  infected mice  groups as indicated.",
    "molecules": "DSS"
  },
  {
    "caption": "(a) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo (left panel); Draq5 live nuclear stain with nuclear segmentation overlay (right panel); scale bar 30 μm.",
    "molecules": "Draq5"
  },
  {
    "caption": "(a) Start:Finish Venus::HES5 intensity ratio in E10.5 Venus::HES5 heterozygous spinal cord slices treated with control (DMSO) and Notch inhibitor DBZ (2 μM) observed over 16h; bars indicate mean and standard deviation of DMS0 (n=3 experiments) and DBZ (n=4 experiments); 2-tailed t-test ****p=0.0001.",
    "molecules": "DBZ, DMS0, DMSO"
  },
  {
    "caption": "a, Plasma and tissue amino acid concentrations. Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions. 'Total' indicates the sum of the Asp, Thr, Ser, Asn, Glu, Gln, Pro, Gly, Ala, Val, Cys, Met, Ile, Leu, Tyr, Phe, Lys, His and Arg concentrations; 'Essential' indicates the sum of Thr, Val, Met, Ile, Leu, Phe, Lys, His and Arg concentrations; 'BCAA' indicates the sum of the Val, Ile and Leu concentrations. Tissue amino acid concentrations are expressed as mmol kg-1 of wet weight. Bars represent mean ± s.d. of three mice. Single and double asterisks indicate a significant difference between mutant and control (wild-type plus heterozygous) mice at P 0.05 and P 0.01, respectively.",
    "molecules": "amino acid, Amino acid"
  },
  {
    "caption": "b, Activation of AMPK in Atg5-/- mice. Hearts were isolated from unfed neonates at 0 h (lanes 1, 3, 5) and 10 h (lanes 2, 4, 6), and force-fed mice at 10 h (lane 7) after the caesarean delivery. Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti-AMPK and anti-phospho-AMPK antibodies.",
    "molecules": "phosphatase inhibitors"
  },
  {
    "caption": "a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX.",
    "molecules": "CHX, staurosporine"
  },
  {
    "caption": "c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &mgr;g ml−1 CHX.",
    "molecules": "CHX"
  },
  {
    "caption": "d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.",
    "molecules": "CHX, staurosporine"
  },
  {
    "caption": "a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose.",
    "molecules": "bafilomycin A1, glucose"
  },
  {
    "caption": "e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units.",
    "molecules": "glucose"
  },
  {
    "caption": "a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments.",
    "molecules": "glucose"
  },
  {
    "caption": "F, G 6-week MECP2-KO neurons (F) showed a pharmacologically rescuable reduction of co-localized synaptic puncta (G; one-way ANOVA, F16,118 = 9.148, *P < 0.05; Dunnet's multiple comparisons test vs untreated KO (WT83/Q83X cell lines were used; N = 7-8 neurons/condition). *P < 0.05, **P < 0.01, ***P < 0.001. Z scores relative to KO untreated: Control = 3.903; Nefiracetam = 3.560; Carbamoylcholine = 2.705; Pirenzepine = 0.0448; PHA543613 = 2.121; Acamprosate = 3.339; Baclofen = 0.5672; GR73632 = 2.237; Hyperforin = 3.016, and IGF-1 = 3.791. Scale bar = 5 µm.",
    "molecules": "Acamprosate, Baclofen, Carbamoylcholine, GR73632, Hyperforin, Nefiracetam, PHA543613, Pirenzepine"
  },
  {
    "caption": "H Drug treatment increased calcium transient frequency in 6-week neurons. Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest (one-way ANOVA, F4,52 = 20.28, Dunnett's multiple comparisons test vs untreated KO: Nefiracetam, **P < 0.01 and Z = 2.364; PHA 543613, ***P < 0.001 and Z = 3.391; Acamprosate, ***P < 0.001 and Z = 3.153; WT83/Q83X cell lines were used; N = 10-14 neurons/condition) and the percentage of active neurons (one-way ANOVA, F4,52 = 23.11; Dunnett's multiple comparison test vs untreated KO: Nefiracetam, ***P < 0.001 and Z = 3.144; PHA 543613, **P < 0.01 and Z = 2.567; **Acamprosate, P < 0.01 and Z = 2.285; N = 10-14 neurons/condition).",
    "molecules": "Acamprosate, calcium, Nefiracetam, PHA 543613"
  },
  {
    "caption": "I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2-KO neurons on MEA (two-sided unpaired Student's t-test compared to KO untreated: Nefiracetam, *P = 0.016 and Z = 2.418; PHA 543613, *P = 0.027 and Z = 2.147; Acamprosate, P = 0.39 and Z = 0.477; WT83/Q83X and WT82/K82fs cell lines were used; N = 4-11 MEA wells/condition).",
    "molecules": "Acamprosate, Nefiracetam, PHA 543613"
  },
  {
    "caption": " E Calcium imaging analysis showing calcium transients normalized by control (one-way ANOVA, F2,72 = 15.62, P < 0.0001; Dunnett's multiple comparisons test vs control: MECP2-mosaic, ***P < 0.001; MECP2-KO, ***P < 0.001). WT83/Q83X cell lines were used; N = 44 Q83X neurospheres and 65 WT83 neurospheres. ",
    "molecules": "Calcium, calcium"
  },
  {
    "caption": " G Cell viability was improved or unharmed in treated 8-week neurospheres (one-way ANOVA, F9,104 = 6.81; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi + PHA 1 µM, **P < 0.01 and Z = 2.974; Nefi + PHA 10 µM, ***P < 0.001 and Z = 3.733; N = 8-24 neurospheres per condition). ",
    "molecules": "Nefi, PHA"
  },
  {
    "caption": "H, I Calcium transient frequency (number of peaks in 10 mins recording) normalized by CTR/KO untreated (H; one-way ANOVA, F9,239 = 6.18; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi 1 µM, *P < 0.05 and Z = 0.780; Nefi + PHA 10 µM, ***P < 0.001 and Z = 1.972; N = 15-61 neurospheres per condition) and calcium transient amplitude normalized by CTR/KO untreated (I; one-way ANOVA, F9,241 = 1.27, P = 0.255; N = 16-62 neurospheres per condition).",
    "molecules": "Calcium, calcium, Nefi, PHA"
  },
  {
    "caption": "J, K MEA spike frequency heatmap (J) and quantification (K) normalized by CTR/KO untreated (one-way ANOVA, F5,38 = 1.21, P = 0.321; Dunnett's multiple comparisons test vs CTR/KO untreated: PHA, P = 0.65 and Z = 1.134; Nefi, P = 0.98 and Z = 0.06; PHA + Nefi, P = 0.86 and Z = 0.94; N = 7-8 wells per condition). 'X' on activity heatmap signifies absence of a neurosphere in that position.",
    "molecules": "Nefi, PHA"
  },
  {
    "caption": "B 2-month old cortical organoid diameter (one-way ANOVA, F2,173 = 91.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, ***P < 0.001 and Z = 5.414; PHA 543613, ***P < 0.001 and Z = 5.351; WT83/Q83X and WT82/K82fs cell lines were used; N = 28-90 organoids per condition). Left: representative brightfield images of cortical organoids; Right: cortical organoid size quantification. Scale bar = 200 µm.",
    "molecules": "Nefiracetam, PHA 543613"
  },
  {
    "caption": "RNA sequencing (D, left: heatmap) and GO analysis (D, right) of untreated MECP2-KO organoids vs Nefiracetam (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated genetic expression in synapse-relevant pathways.",
    "molecules": "Nefiracetam"
  },
  {
    "caption": "RNA sequencing (F, left: heatmap) and GO analysis (F, right) of untreated MECP2-KO organoids vs PHA 543613 (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated expression of genes in synapse-relevant pathways.",
    "molecules": "PHA 543613"
  },
  {
    "caption": "H Synapsin1+ puncta quantitation (one-way ANOVA, F3,34 = 10.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, **P < 0.01 and Z = 2.998; PHA 543613, P > 0.05 and Z = 1.266; WT83/Q83X cell lines were used; N = 8-10 each). Left: representative image of Synapsin1+ immunofluorescent staining; Right: Synapsin1+ puncta quantification. Scale bar = 20 µm.",
    "molecules": "Nefiracetam, PHA 543613"
  },
  {
    "caption": "Drug treatment increased spiking network activity of MECP2-KO cortical organoids to a level not significantly different from that of control organoids (M; one-way ANOVA, F3,31 = 3.775, Dunnett's multiple comparisons test vs control: untreated KO, **P < 0.01 and Z = 3.192; Nefiracetam, P = 0.98 and Z = 2.117; PHA 543613, P = 0.12 and Z = 1.101, WT83/Q83X cell lines were used; N = 7-10 MEA wells per condition).",
    "molecules": "Nefiracetam, PHA 543613"
  },
  {
    "caption": "A) Immunoelectron microscopy of HeLa cells infected with S. Typhimurium strains for 12 h (ubiquitin - 10 nm gold particles; scale bars, 0.5 µm). Arrows indicate ubiquitin accumulations near bacteria within vacuoles.",
    "molecules": "gold"
  },
  {
    "caption": "(B) Quantification of infected macrophages containing ubiquitinated aggregates 10 h after bacterial uptake. Cells were processed as in (A) and analysed by fluorescence microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of at least 3 independent experiments. * p<0.05; *** p<0.001. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. (C) Immunoelectron microscopy of RAW264.7macrophages infected with the ΔsseL mutant bacteria for 12 h. Arrows and arrowheads indicate ubiquitin (15 nm gold particles) and p62 (10 nm gold particles), respectively (scale bar, 0.5 µm).",
    "molecules": "gold"
  },
  {
    "caption": "(B) Quantification of the number of puromycin-induced ubiquitin inclusions in cells. HeLa cells were transfected with a vector expressing myc-SseL or myc-SseLC/A for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc, anti-p62 and anti-ubiquitin. 50 individual cells were counted per condition in each experiment. Values are the means ± SEM of at least 3 independent experiments. ** p<0.01.",
    "molecules": "puromycin"
  },
  {
    "caption": "(C) Single confocal sections of HeLa cells transfected with a vector expressing myc-SseL for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc (blue), anti-p62 (green) and anti-ubiquitin (Ub, red) (scale bars, 5 µm). The lower panels show a merged image of p62, ubiquitin and myc-SseL. Arrows indicate ubiquitin and p62 aggregates present in untransfected cells and arrowheads indicate cells expressing myc-SseL",
    "molecules": "puromycin"
  },
  {
    "caption": "SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.",
    "molecules": "3-MA"
  },
  {
    "caption": "(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting.",
    "molecules": "NH4Cl"
  },
  {
    "caption": "C. Comparison of kinase inhibition profile of Ponatinib and Baricitinib.",
    "molecules": "Baricitinib, Ponatinib"
  },
  {
    "caption": "B. Dose-response curves of ponatinib treatment on SARS-Cov2  NTD (Wuhan)-mediated cytokine release in PBMCs. Right, a table showing EC50 (nM) values of ponatinib-mediated inhibition of cytokine release in response to indicated SARS-Cov2 variant NTD treatments in PBMCs.",
    "molecules": "ponatinib"
  },
  {
    "caption": "A. Comparison of  LPS and full length S1 spike protein-mediated changes in cytokines in THP1 macrophages. Data information, , data are shown as the mean of three technical replicates. Error bars denote SEM.",
    "molecules": "LPS"
  },
  {
    "caption": "D. Representative H&E  images showing Ponatinib alleviates  LPS-induced inflammatory cell infiltration, septal thickening, alveolar edema in  mouse lungs. Scale bars 100 µm (upper) and 50 µm (lower).",
    "molecules": "LPS, Ponatinib"
  },
  {
    "caption": "E. Plots showing pre-treatment (60 min) with Ponatinib (35 mg/Kg) inhibits  LPS-induced cytokine release in BALF. Cytokine levels were measured using Luminex. Data information, data are mean of five biological replicates from two independent studies, error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Welch's t-test.",
    "molecules": "LPS, Ponatinib"
  },
  {
    "caption": "C Percentage of BrdU positive cb9Δmyc cells, in the presence or absence (24h after removal) of doxycycline. Data are presented as mean ± SD; n = 3. Two-tailed Student t-test was used to compare between two groups and expressed as p-values.",
    "molecules": "BrdU, doxycycline"
  },
  {
    "caption": "A  3T9 fibroblasts expressing the indicated MycER proteins and control cells (EV) were treated with OHT (4h) and subjected to biochemical fractionation in three independent experiments. The quantity of MycER protein in the different fractions was quantified by western blotting (shown in Fig. EV4A) and the relative proportion in the various fractions plotted as average and standard deviation.",
    "molecules": "OHT"
  },
  {
    "caption": "Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of Dieldrin for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.",
    "molecules": "Dieldrin, DMSO"
  },
  {
    "caption": "Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of PMA for the indicated times. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.",
    "molecules": "DMSO, PMA"
  },
  {
    "caption": "Jurkat T cells were treated with either DMSO (vehicle) or with 100μM of either Dieldrin or PMA for the indicated times. Abundance of mRNA for the indicated genes by RT-qPCR.",
    "molecules": "Dieldrin, DMSO, PMA"
  },
  {
    "caption": "Jurkat cells were exposed to either DMSO, PMA in DMSO, or dieldrin in DMSO for 30 min in triplicates. RNA-seq was then performed on cDNA libraries prepared with poly(A) selection: principal component analysis on the 485 genes the most affected by DMSO, PMA, or Dieldrin.",
    "molecules": "dieldrin, Dieldrin, DMSO, PMA"
  },
  {
    "caption": "Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing an example of a HERV becoming a site of divergent transcription upon exposure of the Jurkat cells to dieldrin. Top tracks show coverage from sequencing of poly(A)-enriched libraries for comparison. All coverage tracks are at the same scale and all the sequencing result files are of comparable size. Blue and red reads (rds) are in opposite orientation. CAGE peaks use the same color code. Reads mapping inside a series of 4686 HERVs located away (30Kb) from protein-coding genes were quantified both in the poly(A)-enriched and the ribo-depleted data. Read counts from each replicate were then summed and plotted.",
    "molecules": "dieldrin, DMSO, ribosomal RNAs"
  },
  {
    "caption": "Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Distribution of regions annotated \"LTR\" in RepeatMasker relative to sites of divergent transcription in Jurkat cells treated with either DMSO or dieldrin in DMSO.",
    "molecules": "dieldrin, DMSO, ribosomal RNAs"
  },
  {
    "caption": "Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Jurkat T cells were treated with either DMSO (vehicle) or 100μM dieldrin for the indicated times. Abundance of IL2 mRNA was assessed by RT-qPCR. Data shown are means +/- SEM from four independent experiments. Significance (p-value) was estimated using the two-sided student t test appropriate for small sample numbers. ***: p-value<0.001.",
    "molecules": "dieldrin, DMSO, ribosomal RNAs"
  },
  {
    "caption": "Jurkat cells were exposed to either DMSO, or dieldrin in DMSO for 30 min. RNA-seq was then performed on cDNA libraries prepared after depletion of ribosomal RNAs. Screen captures from IGV showing increased divergent transcription inside a HERV sequence upon exposure of the Jurkat cells to dieldrin in the intergenic region between MUC21 and MUC22. Color code as in (A).",
    "molecules": "dieldrin, DMSO, ribosomal RNAs"
  },
  {
    "caption": "A human phospho-kinase antibody array was probed with extracts from Jurkat cells treated with either DMSO, dieldrin, or PMA for 30 min as indicated. The array processed as a western and revealed by ECL. Graphic shows selected antibodies. Values shown are means +/- SEM from two measures.",
    "molecules": "dieldrin, DMSO, PMA"
  },
  {
    "caption": "Jurkat cells were treated with either DMSO (vehicle), dieldrin, or PMA for the indicated times. Cell extracts were resolved by PAGE and western blotting was carried out with the indicated antibodies. Nearest size marker is indicated to the right (kDa).",
    "molecules": "dieldrin, DMSO, PMA"
  },
  {
    "caption": "Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.",
    "molecules": "dieldrin"
  },
  {
    "caption": "Jurkat cells were cultured in the absence or in the presence of the indicated small molecule inhibitors for 1h then exposed to 100μM dieldrin for 30 min. Abundance of mRNA for the indicated genes or HERVs was assessed by RT-qPCR.",
    "molecules": "dieldrin"
  },
  {
    "caption": "GSC neurospheres were dissociated to single cell suspension and transfected with either miR-10b inhibitor (labeled \"miR-10b-i\") or non-targeting control oligonucleotide, or treated with Lipofectamine 2000 only (Mock). Statistical significance of the difference was determined by Student's t test, with p-values < 0.001 indicated by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4.A. Cell viability was monitored at day 5 after transfection as described in Materials and Methods.",
    "molecules": "Lipofectamine 2000, oligonucleotide"
  },
  {
    "caption": "Three types of GSC (GBM4, GBM6, and GBM8) were transfected with miR-10bASO, and gene expression was analyzed 24 hours later by the Affymetrix microarrays. The heatmaps colors intensity demonstrate altered expression of the genes (up- or down-regulated relative to the mock treated samples) with the fold change > 1.2 and p < 0.05 in at least two out of the three GSC cultures.A. The genes associated with \"cell cycle\" bioterm have been selected using Ingenuity Pathway Analysis. The treatment with the miR-10b inhibitor is indicated as \"miR-10b-i\".",
    "molecules": "ASO"
  },
  {
    "caption": "C. miR-10b binding motifs are enriched in 5'UTRs of the genes up-regulated by miR-10b ASO. The graph shows the probability that enrichment of the miR-10b motifs in mRNAs up-regulated vs. unchanged (p<0.05) by anti-miR-10b does not occur by chance.",
    "molecules": "ASO"
  },
  {
    "caption": "B. qRT-PCR analysis validates that mRNA of MBNL1-3, SART3, RSRC1 and other splicing factors are de-repressed by miR-10b ASO in different GSC and GBM cell lines. mRNA expression levels were normalized to GAPDH expression.",
    "molecules": "ASO"
  },
  {
    "caption": "B. 2'-O-MOE-POmiR-10b inhibitor (miR-10b-i) or non-targeting control (1 μg of each) formulated with InVivo-jet PEI were injected intratumorally at days 20 and 25 after cells implantation. The efficacy of miR-10b inhibition was assessed by qRT-PCR analysis of the resected tumors, with miR-10b expression levels normalized to miR-125b.",
    "molecules": "2’-O-MOE-PO"
  },
  {
    "caption": "A. Intravenously injected Cy5-labeled 2'-O-MOE-PSoligonucleotide is distributed to intracranialGBM8tumor. In the normal brain, the signal is observed in blood vessels but not within brain parenchyma. \"T\" - tumor, \"B\" - brain tissue. Each image is representative of three mice analyzed.",
    "molecules": "Cy5, 2’-O-MOE-PS, oligonucleotide"
  },
  {
    "caption": "B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6",
    "molecules": "2’-O-MOE-PS"
  },
  {
    "caption": "B-D. Systemic inhibition of miR-10b markedly reduces tumor burden. Uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control of the same chemistry were injected at 80 mg/kg through the tail vein at the days indicated by arrows.B. The left panels illustrate tumorimages of representative animals at day 34, and average signals (photons/sec) are indicated below the images. The bars represent average signal ratios for each group at day 34, relative to day 6.C. Each mouse was sacrificed when the tumor-generated signal reached 5X107 photons/sec, and Kaplan-Meier survival plots were built retrospectively.D. Growth curves of individual tumors based on the ratios of bioluminescence signals to the baseline signals at day 6.",
    "molecules": "2’-O-MOE-PS"
  },
  {
    "caption": "A. Uptake of the uncomplexed Cy5-labeled 2'-O-MOE-PSoligonucleotide (80 mg/kg injected via the tail vein) by normal extracranial tissues was examined by fluorescence microscopy24 h after injections.",
    "molecules": "Cy5, 2’-O-MOE-PS, oligonucleotide"
  },
  {
    "caption": "B-D. Systemic treatment of intracranialGBM8tumors with uncomplexed 2'-O-MOE-PSmiR-10b inhibitor (miR-10b-i) or non-targeting control oligonucleotide (at 80 mg/kg) was not associated with toxic effects.B. No significant difference in average mice weight was observed between the anti-miR-10b and control treatment groups.C. No significant difference in average organs' weight was observed between the anti-miR-10b and control treatment groups.D. No significant difference in tissue histology using Hematoxylin and Eosin staining was observed between the anti-miR-10b and control treatment groups.",
    "molecules": "2’-O-MOE-PS, oligonucleotide"
  },
  {
    "caption": "A. Continuous osmotic delivery of miR-10b inhibitor markedly reduces tumor burden. The osmotic pumps, loaded with lipid nanoparticles formulated with 2'-O-MOE-POmiR-10b inhibitor or non-targeting control infused 2 μg of the oligonucleotides per day introtumorally, over 13 days. Tumors growth was monitored by the WBI, and the left panels illustrate tumor imaging of representative animals at the end of treatment. The bars represent average signal ratios for each group at day 13 (end of treatment), relative to day 2 (treatment initiation).",
    "molecules": "lipid nanoparticles, 2’-O-MOE-PO"
  },
  {
    "caption": "A, B. miR-10b inhibition decreases GL261 cellviability. Cellviability was measured at days 3-7 after transfection with miR-10b inhibitor, non-targeting control, or Lipofectamine 2000 alone (mock).A. Phase contrast photographs of GL261 cultures at day 6 post-transfection.B. Growth curves of cultured GL261 cells, based on the viability assay.",
    "molecules": "Lipofectamine 2000"
  },
  {
    "caption": "C. InVivo-jet PEI - formulated 2'-O-MOE-PS/PO miR-10b inhibitor or non-targeting control were infused to orthotopic GL261 tumors by osmotic pumps, starting at day 6 after cell implantation. The uptake of ASOs was confirmed by IHC for PS-containing oligonucleotides (green). GL261 tumor cells expressing M-Cherry are red.",
    "molecules": "ASO, 2’-O-MOE-PS, PO, PS"
  },
  {
    "caption": "D AID−/− or AID+/− mice were treated with 3% DSS for 10 cycles, and colonic sections were analyzed by histology inspection after H/E staining. Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments. n = 28 (AID−/− males); 35 (females); 23 (AID+/− males); 25 (females). P-value: male: 0.8; female: 0.246.",
    "molecules": "DSS"
  },
  {
    "caption": "qRT-PCR analysis of AID expression in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. n = 5 (R26AID+/+ VillinCRE+/TG); 4 (R26AID+/KIVillinCRE+/TG); 2 (R26AID+/+p48CRE+/KI); 2 (R26AID+/KIp48CRE+/KI). LPS+IL4-stimulated B cells are shown as a positive control (n = 2). Bars show mean values ± SEM normalized to LPS+IL4-treated B cells.",
    "molecules": "LPS"
  },
  {
    "caption": "(A-D) Activation of NPF neurons  with dTrpA1 induced Ca2+ rise in pC1 dsx neurons  in 4-6d males but not in 3-6h males. (A and B) Ca2+  imaging of pC1 neurons  from 22˚C to 27˚C and 30˚C in 3-6h (A) and 4-6d (B) males. (C) Time-course of fluorescent changes in pC1 neurons  upon activation of NPF neurons  in 3-6h and 4-6d males (UAS-GCaMP6m/LexAop-dTrpA1; NPF-lexA/dsxGAL4), and in 4-6d control males (UAS-GCaMP6m/+; dsxGAL4/+) without NPF neuronal  activation, which are quantified in (D). n = 8 for 4-6d control males, 15 for 3-6h and 8 for 4-6d males with dTrpA1. n.s., not significant, *p < 0.05, **p < 0.01, ***p<0.001, unpaired t test. Data information: Scale bars, 10 μm. Error bars indicate SEM.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(E-H) Activation of TH neurons  with dTrpA1 triggered  Ca2+ increase in pC1 dsx neurons  in both 3-6h and 4-6d males. (E and F) Ca2+ imaging of pC1 neurons  from 22˚C to 27˚C and 30˚C in 3-6h (E) and 4-6d (F) males. (G) Time-course of fluorescent changes in pC1 neurons  upon activation of TH neurons  in 3-6h and 4-6d males (UAS-dTrpA1/LexAop-GCaMP6s; dsxLexA/TH-GAL4), and in 4-6d control males (LexAop-GCaMP6s/+; dsxLexA/+) without activating TH neurons, which are quantified in (H). n = 8 for each. n.s., not significant, *p < 0.05, Mann-Whitney U test. Data information: Scale bars, 10 μm. Error bars indicate SEM.",
    "molecules": "Ca2+"
  },
  {
    "caption": "B. Wild type (WT) and NCOA4 KO MEFs were pretreated with 25 μg/ml ferric ammonium citrate (FAC) for 16 h and then chased with 20 μg/ml cycloheximide (CHX) for the indicated times. Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies.",
    "molecules": "CHX, cycloheximide, FAC, ferric ammonium citrate"
  },
  {
    "caption": "C. WT MEFs untreated or treated with 10 μg/ml FAC for 12 h were lysed with SDS-containing sample buffer or Triton buffer, and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "FAC, SDS, Triton"
  },
  {
    "caption": "E. Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12 h, or 100 μM Deferoxamine (Dfo) for 6 h after treatment with 10 μg/ml FAC for 12 h were prepared by differential centrifugation as described in (D). Each fraction was subjected to SDS-PAGE  and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "Deferoxamine, Dfo, FAC"
  },
  {
    "caption": "F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test).",
    "molecules": "E64d, FAC, pep A, pepstatin A"
  },
  {
    "caption": "G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm. H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates.",
    "molecules": "FAC"
  },
  {
    "caption": "B. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with anti-myc antibody. Representative images are shown. Scale bar, 10 μm.",
    "molecules": "FAC"
  },
  {
    "caption": "D. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "FAC"
  },
  {
    "caption": "F. WT MEFs stably expressing NCOA4 IDR (aa 167-334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6h. The cells were fixed for imaging  and immunostained with anti-myc to analyze puncta of the fusion protein. Scale bar, 10μm. G. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P<0.0001 (Dfo+ligand vs FAC+ligand) and P=0.048 (FAC vs FAC+ligand) (Kruskal-Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.",
    "molecules": "AP20187, Dfo, FAC"
  },
  {
    "caption": "H. Coomassie-stained SDS-PAGE gel of purified proteins. I. The amount of co-purifying  iron in purified proteins in (H) was measured by  inductively coupled plasma mass spectrometry (ICP-MS). Data are shown as means ± SD of three biological replicates. P=0.032 (Welch Two Sample t-test).",
    "molecules": "iron"
  },
  {
    "caption": "A. Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated. Samples were subjected to SDS-PAGE  and stained with Coomassie Brilliant Blue. B. Quantitative results in (A) are shown as means ± SEM of three biological replicates.",
    "molecules": "Coomassie Brilliant Blue, FeSO4"
  },
  {
    "caption": "A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by  SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1",
    "molecules": "FAC"
  },
  {
    "caption": "B. MEFs  were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin  band, and the white arrowhead indicates a partial degradation band of ferritin.",
    "molecules": "E64d, DMSO, FAC, pepA, pepstatin A"
  },
  {
    "caption": "C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm.",
    "molecules": "FAC"
  },
  {
    "caption": "E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies.",
    "molecules": "biotin, DMSO, FAC, streptavidin, Triton"
  },
  {
    "caption": "F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "CHX, FAC"
  },
  {
    "caption": "G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging  and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.",
    "molecules": "BafA, bafilomycin A1, CHX, FAC"
  },
  {
    "caption": "A. WT, ATG7 KO, TAX1BP1 KO, and FIP200KO MEFs were treated or not treated with 10 μg/ml FAC for 12 h. Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "FAC"
  },
  {
    "caption": "WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg/ml FAC for 12 h. (D, F) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "FAC"
  },
  {
    "caption": "WT MEFs  and  TAX1BP1  KO MEFs  reconstituted with the indicated TAX1BP1 variants were treated with 10 μg/ml  FAC for 12 h. (E) Cells were fixed for imaging  and immunostained  to analyze ferritin localization. Scale bar, 10μm.",
    "molecules": "FAC"
  },
  {
    "caption": "A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates  were analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "Dfo, FAC, SDS, Triton"
  },
  {
    "caption": "B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis.",
    "molecules": "Dfo, FAC, Triton"
  },
  {
    "caption": "C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates  were fractionated and then analyzed by immunoblotting with the indicated antibodies. D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates.",
    "molecules": "Dfo, FAC"
  },
  {
    "caption": "E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1  were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM  5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degron",
    "molecules": "5-Ph-IAA, Auxin, auxin, Dfo, DMSO, FAC"
  },
  {
    "caption": "F. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "Dfo, FAC"
  },
  {
    "caption": "G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "Dfo, FAC"
  },
  {
    "caption": "H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.",
    "molecules": "CHX, Dfo, FAC"
  },
  {
    "caption": "J. MEFs expressing hNCOA4-TurboID  were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous  NCOA4, respectively.",
    "molecules": "biotin, DFO, DMSO, FAC, streptavidin, Triton"
  },
  {
    "caption": "(G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). *p<0.05, **p<0.005 by Student t test.",
    "molecules": "CCCP"
  },
  {
    "caption": "A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1/S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment.",
    "molecules": "nocodazole, thymidine"
  },
  {
    "caption": "C Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG-tagged XIAP or empty vector (EV) and synchronized in mitosis using nocodazole or left untreated.",
    "molecules": "nocodazole"
  },
  {
    "caption": "E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG-tagged XIAP and transfected with siRNA oligonucleotides as specified. Cells were synchronized in mitosis using sequential thymidine/nocodazole treatment, as indicated. Subsequent to treatment with MG132, whole cell extracts (WCE) were prepared and ubiquitylated XIAP was isolated by anti-FLAGimmunoprecipitation (IP) under denaturing conditions.",
    "molecules": "MG132, nocodazole, thymidine"
  },
  {
    "caption": "G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine/nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated.",
    "molecules": "DMSO, nocodazole, thymidine, WP1130"
  },
  {
    "caption": "A Immunoblot analysis of WT, Xiap-/-, Mcl1-/- or cIap2-/- mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non-relevant mRNA (Ctrl) or against Usp9X mRNA and treated with taxol as specified.",
    "molecules": "taxol"
  },
  {
    "caption": "C Immunoblot analysis of HeLa cells transfected with a FLAG-tagged USP9X expression construct or empty vector (EV) and treated with taxol for the indicated times.",
    "molecules": "taxol"
  },
  {
    "caption": "F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non-relevant mRNA (Ctrl) or against Xiap mRNA and treated with taxol as specified.",
    "molecules": "taxol"
  },
  {
    "caption": "G Representative PET images of animals, injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection (pre treatment) and three days following intraperitoneal vincristine administration (post treatment). Bone marrow infiltration, representative for systemic lymphoma manifestation, is indicated by a blue asterisk.",
    "molecules": "vincristine"
  },
  {
    "caption": "H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden (sh_Ctrl, black, n=6; sh_Usp9X, white, n=8; sh_Xiap, grey, n=8). Within the sh_Usp9X and sh_Xiap groups all animals lived up to control imaging, while two of eight animals of the sh_ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden. These animals had to be excluded from final analysis, suggesting an underestimation of the actual effect. Data was obtained from two independent experiments. ****, P < 0.0001; *, P = 0.0167, student's t-test",
    "molecules": "18FDG"
  },
  {
    "caption": "B FACS analysis (propidium iodide (PI)) of USP9X high and low expressing DLBCL cells shown in (A) to determine cell survival. Cells were treated with taxol and collected at the specified time points. PI negative cells are indicated relative to time point 0 (n=3 ± SD).",
    "molecules": "taxol"
  },
  {
    "caption": "C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES-GFP shRNA constructs against USP9X or a non-relevant mRNA, FACS sorted for GFP+ PI- cells and exposed to taxol for the indicated periods of time. The quantification of cleaved caspase 3 (CC3) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA. **, P = 0.0099, student's t-test",
    "molecules": "taxol"
  },
  {
    "caption": "D Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time. Three hours before collecting, WP1130 at a concentration of 5μM or DMSO was added as specified.",
    "molecules": "taxol, DMSO, WP1130"
  },
  {
    "caption": "E FACS analysis (propidium iodide (PI) uptake) of DLBCL cell lines treated with taxol and/or the SMAC mimetic BV6 as indicated. Results displayed are from three independent experiments (n=3 ± SD). ***, P = 0.00085; ***, P = 0.00529; Student's t test.",
    "molecules": "taxol, BV6"
  },
  {
    "caption": "G Expression data derived from (E) was correlated with clinical follow-up. Within a cohort of 58 patients receiving vincristine-containing chemotherapy (CHOP regimen: cyclophosphamide, doxorubicin, vincristine, prednisone) without the addition of the anti-CD20 antibody rituximab (left panel) overexpression of USP9X and XIAP (n = 14, straight line) was associated with a significantly shortened event-free survival (EFS) as compared to all remaining patients (n = 44, dotted line) (*, P = 0.050; log rank test). Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti-CD20 antibody rituximab overexpression of USP9X and XIAP (n = 14, straight line) was not associated with a shortened event-free survival (EFS) as compared to all remaining patients (n = 49, dotted line) (P = 0.454).",
    "molecules": "cyclophosphamide, doxorubicin, prednisone, rituximab, vincristine"
  },
  {
    "caption": "B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) ib DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc) and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons.",
    "molecules": "Ca2+"
  },
  {
    "caption": "F-H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRGneurons as in (B-E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments.",
    "molecules": "Ca2+"
  },
  {
    "caption": "A, B. FM 1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm.",
    "molecules": "FM 1-43, K+"
  },
  {
    "caption": "C. Quantification of KCl-evoked FM 1-43 uptake with different stimulation times.",
    "molecules": "FM 1-43, KCl"
  },
  {
    "caption": "F. Basal [Ca2+]i in DRG neurons measured with Fura-2.",
    "molecules": "Ca2+"
  },
  {
    "caption": "G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm.",
    "molecules": "FM1-43, K+"
  },
  {
    "caption": "H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm.",
    "molecules": "FM4-64"
  },
  {
    "caption": "I. Time-course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (6 coverslips from at least 3 biological repeats each).",
    "molecules": "FM4-64"
  },
  {
    "caption": "A. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynasore. Endocytic inhibition by dynasore (Dyna) was estimated by comparison of Cm traces recorded during 1-3 min (KD) and 5-10 min (KD + Dyna and Ctrl + Dyna) after whole-cell dialysis.",
    "molecules": "dynasore"
  },
  {
    "caption": "D. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynole-34-2TM (Dynole). Cm traces and endocytic rate were measured as in (A) except that 10 μM dynole-34-2TM with 0.1% DMSO was dialyzed into patched cells.",
    "molecules": "DMSO, Dynole, dynole-34-2TM"
  },
  {
    "caption": "G, H. Averaged ∆Cm traces and Endo-5s recorded from KD neurons under control conditions (0.1 % DMSO).",
    "molecules": "DMSO"
  },
  {
    "caption": "I. KCl-evoked FM 1-43 uptake in DRG neurons after 10-min pre-incubation with 0.1% DMSO (control) or with 100 μM dynasore.",
    "molecules": "DMSO, dynasore, KCl"
  },
  {
    "caption": "C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves).",
    "molecules": "MDC"
  },
  {
    "caption": "E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.",
    "molecules": "DMSO, FM 1-43, MDC, KCl"
  },
  {
    "caption": "G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.",
    "molecules": "dextran, KCl, tetramethylrhodamine"
  },
  {
    "caption": "D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).",
    "molecules": "K+"
  },
  {
    "caption": "A. Representative electron micrographs of HRP uptake from standard (SE) or 100 mM K+ (100K) bath solution. Scale bar, 2 μm.",
    "molecules": "K+"
  },
  {
    "caption": "Figure 9. Functional domains of Syt11 in endocytosisA. Diagram of the mutant forms of Syt11 used for rescue experiments.B-D. Statistics of FM4-64 (5 min), Tf, and large dextran (40-KD) uptake into KD neurons with or without rescue by indicated forms of Syt11.All data are presented as mean ± s.e.m. of 3 independent experiments. One-way ANOVA, values labeled with different letters (a, b, and c) are significantly different from each other, ***P < 0.001. Student's t-test for the comparison between KD and control cells in (B-D), **P < 0.01, ***P < 0.001.",
    "molecules": "dextran, FM4-64"
  },
  {
    "caption": "Western blotting in the WT and the Trim71(R783H) mESCs with dox-inducible expression of Lsd1-GFP.",
    "molecules": "dox"
  },
  {
    "caption": "Western blotting in the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.",
    "molecules": "dox"
  },
  {
    "caption": "Exit pluripotency assay for the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.",
    "molecules": "dox"
  },
  {
    "caption": "Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.",
    "molecules": "dox"
  },
  {
    "caption": "Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.",
    "molecules": "dox"
  },
  {
    "caption": "Degranulation of primary human NK  cells after incubation with plate-coated antibodies (A) Comparison of CD107a expression  after incubation in either uncoated wells (PBS) or wells coated with αTRAIL, αNKG2D, αNKp46, or isotype using flow cytometry  (n = 12 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression  as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml antibody concentration. Right panel: Box plots showing relative frequency of CD107a+ NK  cells (y-axis) after incubation with plate-coated antibodies of different concentrations (x-axis). (B) Comparison of CD107a expression  after incubation with plate-coated DR4  protein, DR5  protein or human  IgG using  flow cytometry (n = 11 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression  as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml protein concentration. Right panel: Box plots showing relative frequency of CD107a+ NK  cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. (A, B, Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.",
    "molecules": "PBS"
  },
  {
    "caption": "(E) Expression levels of phosphorylated  signaling proteins. Upper panel: Concatenated contour plots of one donor depicting the expression  of phosphorylated  signaling proteins Syk, p38 MAPK, Akt  and PLC-γ2 for the following culture conditions and controls (x-axis: left to right): FMO, PBS, isotype, αTRAIL, αNKp46, αNKG2D, αCD16. Lower panel: Bar graphs showing the percentage of p-Syk+, p-p38 MAPK+, p-Akt+ and p-PLC-γ2+ NK  cells after 30 min of stimulation (n = 3 different donors). Bar graphs represent the median, whiskers display minimum and maximum data points.",
    "molecules": "PBS"
  },
  {
    "caption": "(D-F) Upper panels: Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs treated with SB431542 (10 m), TGFR2 or Smad2 shRNA lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to -tubulin, HSP90, or Smad2 and expressed as a fraction of a control value.",
    "molecules": "SB431542"
  },
  {
    "caption": "(A) Dissected mouse aorta demonstrating lipid-rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe-/- mice. (b) & (d) Cross-section of brachiocephalic artery from (a) & (c) stained with Oil Red O. L: lumen. Scale bar: 4 mm. 3 mice per group.",
    "molecules": "fat, lipid"
  },
  {
    "caption": "(C-F) Analysis of brachiocephalic artery of Apoe-/- mice maintained for 4 months on either normal or high fat diet using anti-CD31 (green), anti-p-FGFR1 (red), anti-FGFR1 (red), anti-p-Smad2 (red), and anti-p-Smad3 (red) antibodies. Nuclei counterstained with DAPI (blue). Scale bar: 62 m. L: lumen. M: Media. 6 mice per group.",
    "molecules": "fat"
  },
  {
    "caption": "(G-J) Quantification of the number of medial smooth muscle cells expressing p-FGFR1, FGFR1, p-Smad2, and p-Smad3 (***p<0.001 compared to ND, NS: not significant compared to ND; unpaired two-tailed Student's t test.). ND: Normal diet. HFD: High fat diet. A full table of p-values for this figure is shown in Appendix Table S1.",
    "molecules": "fat"
  },
  {
    "caption": "(A) Representative photomicrographs of Oil Red O-stained atherosclerotic lesions in the aortic arch, of Apoe-/- or Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet. 3 mice per group. Scale bar: 5 mm.",
    "molecules": "fat"
  },
  {
    "caption": "(B) (Left) Microphotographs of aortas (en face) from Apoe-/- and Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet after staining with Oil Red O. (Right) Lesion area quantification. All data shown as mean ± SD. (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test). 12 mice per group.",
    "molecules": "fat"
  },
  {
    "caption": "(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype upon inhibition of protein synthesis with cycloheximide (chx). A plasmid-borne 3HA-Pgc1 expressed from the endogenous promoter was used. 3HA-Pgc1 was detected with anti-HA antibodies. Kar2 was used as a loading control and detected with anti-Kar2 antibodies. The graph on the right shows the average of four independent experiments; error bars represent the standard deviation.",
    "molecules": "chx, cycloheximide"
  },
  {
    "caption": "(C) The degradation of 3HA-Pgc1 was analyzed as in (A) in cells bearing the CDC48 temperature-sensitive allele cdc48-6. Cells were grown 2,5 hours at 37C prior addition of cycloheximide. Inactivation of Cdc48 mutant protein was confirmed by stabilization of the ERAD substrate Erg1 in the same cells.",
    "molecules": "cycloheximide"
  },
  {
    "caption": "(B) GFP-Pgc1 was expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δ. Fluorescence microscopy was used to follow GFP-Pgc1 localization over time. LDs were visualized upon staining with the neutral lipid dye MDH. Scale bar: 5 µm.",
    "molecules": "galactose"
  },
  {
    "caption": "(C) Photo-conversion of tdEOS-Pgc1 expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells. The red square marks the photo-converted region. The time (in minutes) after photoconversion is indicated. Arrowheads point LDs containing photoconverted tdEOS-Pgc1. Scale bar 5µm.",
    "molecules": "galactose"
  },
  {
    "caption": "(B) The degradation of the Doa10 substrates 3HA-Pgc1 and 3HA-Vma12-Ndc10C' was analyzed in cells of the indicated genotypes treated with oleic acid. 3HA-Pgc1 and 3HA-Vma12-Ndc10C' were detected with anti-HA antibodies. Dpm1 was used as a loading control and detected with anti-Dpm1 antibodies. The graph shows the average of three independent experiments; error bars represent the standard deviation.",
    "molecules": "oleic acid"
  },
  {
    "caption": " A Bar graph showing the ROS levels in the NFIB models. The fluorescence signal was normalized to the blank. n = 2 biological replicates and n = 2 technical replicates, means ± s.d., two-tailed Student's t-test. ",
    "molecules": "ROS"
  },
  {
    "caption": " B Left panel: representative images of HIF1α immunofluorescence. HIF1α was stained with Alexa-633 (red) and nucleus with DAPI (blue). Scale bar 100 μm. Right panel: bar graph showing quantification of HIF1α nuclear/cytoplasmatic ratio. n = 5 biological replicates, means ± s.d., two-tailed Mann-Whitney U-test. ",
    "molecules": "DAPI, Alexa-633"
  },
  {
    "caption": "(B) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CGA inhibitory pair (red) and the CGC optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).",
    "molecules": "Met, Puro, puromycin"
  },
  {
    "caption": "(C) Representative eTLCs (left) and corresponding elongation kinetics (right) for the CGA-CCG inhibitory pair (red) and the CGC-CCA optimal pair (green). Product formation is normalized to the fraction of Met ICs that form Met-Puro when reacted with puromycin (Fig EV1A).",
    "molecules": "Met, Puro, puromycin"
  },
  {
    "caption": "(A) Comparison of observed rates of elongation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).",
    "molecules": "tRNA"
  },
  {
    "caption": "(B) Comparison of total peptide formation for inhibitory pairs (red) and their optimal controls (green) at limiting tRNA concentrations (hatched bars, 15 to 25 nM aa-tRNA) and saturating tRNA concentrations (solid bars, 150 to 250 nM aa-tRNA).",
    "molecules": "tRNA"
  },
  {
    "caption": "(C) Elongation kinetics for the CGA-CGA inhibitory codon pair with the native arginine ICGtRNA (red) or the non-native arginine UCGtRNA (pink) and for the CGC-CGC optimal control pair with the native arginine ICGtRNA (green).",
    "molecules": "arginine, ICGtRNA, UCGtRNA"
  },
  {
    "caption": "(C) Comparison of 21 nt RPFs aligned at all 17 inhibitory codons from libraries made with CHX/ANS (blue) and CHX/TIG (black) (left) to their corresponding optimal pairs with the same antibiotic combination (right). No significant difference found (n.s.: p=0.9961, n=2) between CHX-TIG and CHX-ANS.",
    "molecules": "ANS, CHX, TIG"
  },
  {
    "caption": "Whole-mount fluorescence images of E14.5 Npr3-CreER;R26-Confetti and Npr3-sCreER;R26-Confetti mouse hearts. No Tam is used as control for leakiness of Npr3-sCreER.",
    "molecules": "Tam"
  },
  {
    "caption": "Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial cell labeling in Npr3-sCreER;R26-Confetti heart compared with Npr3-CreER;R26-Confetti heart (Tam). *P < 0.05; Data are mean ± s.e.m.; n = 5. Scale bars, 500 µm in c; 100 µm in d. Each image is representative of 5 individual biological samples.",
    "molecules": "Tam"
  },
  {
    "caption": "Whole mount fluorescent images of E14.5 or E15.5 Npr3-sCreER;R26-LZLT and Npr3-CreER;R26-LZLT mouse hearts. No tam is as control for detecting leakiness of sCreER or CreER. Representative Immunostaining images for tdTomato, ESR and CDH5 on heart sections.",
    "molecules": "tam"
  },
  {
    "caption": "Immunostaining for VEGFR2 on Npr3-sCreER;Kdrflox/flox heart section from mice without tamoxifen treatment (No Tam).",
    "molecules": "Tam, tamoxifen"
  },
  {
    "caption": "Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium.",
    "molecules": "Tam, tamoxifen"
  },
  {
    "caption": "Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2-sCreER;R26-Confetti heart compared with Col1a2-CreER;R26-Confetti heart (Tam). Quantification of the percentage of fibroblasts expressing fluorescent reporters in cardiac valves. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm in c-e; 100 µm in h. Each figure is a representative of 5-6 individual biological samples.",
    "molecules": "Tam"
  },
  {
    "caption": "B IFNAR1 mRNA stability after DRB treatment. 293T cells were transfected with  RBM47-Flag or empty vectors and treated with DRB (50 μg/mL). Total RNA was harvested at 0, 15, 30, 45, and 60 min, and IFNAR1 mRNA levels were measured using qRT-PCR and normalized to  β-actin. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).",
    "molecules": "DRB"
  },
  {
    "caption": "A mRNA of  IFNAR1, but not IFNAR2  or STAT1, was stabilized by  RBM47 ectopic expression in 293T cells with or without IFN-α  stimulation. Cells were treated with IFN-α or  PBS for 6 h, and the relative gene expression levels were determined using qRT-PCR.The PCR  results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.",
    "molecules": "PBS"
  },
  {
    "caption": "F, G  Protein levels of IFNAR1 and phosphorylation levels of TYK2, STAT1, and STAT2 in RBM47-overexpressing (F) or knockdown (G) 293T  cells. Cells were treated with PBS or IFN-α for 6 h, and the relative band densities were analyzed using ImageJ software and normalized to GAPDH. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.",
    "molecules": "PBS"
  },
  {
    "caption": "Plot showing the average Z-scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one-sample t-test of whether average Z-score ≠ 0. Green circles represent significantly upregulated genes (Z > 0 and P < 0.05). Magenta circles represent significantly downregulated genes (Z < 0 and P < 0.05). Grey circles represent genes whose expression is not significantly changed (P > 0.05). All comparisons were between cancers and normal cells from the same tissue. Dashed line shows position on Y-axis that corresponds to P = 0.05. Positions of MCOLN1 (red circle), VAC14 (blue circle), and MTM1 (yellow circle) are indicated. Inset, schematic showing that Mtm1 and Vac14 regulate the levels of PI(3,5)P2, and thereby, influence TRPML1 activity.",
    "molecules": "PI(3,5)P2"
  },
  {
    "caption": "Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 and GPN. Arrows serve as event markers that denote the points of time at which the drugs were added. Grey lines on top represent the time at which the bath Ca2+ was removed. All values represent mean±SEM from 3 biological replicates.",
    "molecules": "Ca2+, GPN, ML-SA1, fura-2"
  },
  {
    "caption": "Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to ML-SA1 except that the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release in response to the application of ML-SA1. All values represent mean±SEM from 3 biological replicates.",
    "molecules": "Ca2+, ML-SA1, fura-2"
  },
  {
    "caption": "Graphs showing the changes in the fura-2 ratio in cells of the indicated genotypes in response to GPN. the baseline ratios for both genotypes were adjusted to 0 at the start of the experiment. Lines on the right indicate peak amplitudes of Ca2+ release Ca2+ release was evoked using GPN.",
    "molecules": "Ca2+, GPN, fura-2"
  },
  {
    "caption": "Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 (E) All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.",
    "molecules": "Ca2+, ML-SA1"
  },
  {
    "caption": "Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to GPN (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.",
    "molecules": "Ca2+, GPN"
  },
  {
    "caption": "Ratio of the amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.",
    "molecules": "Ca2+, GPN, ML-SA1"
  },
  {
    "caption": "Total Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.",
    "molecules": "Ca2+, GPN, ML-SA1"
  },
  {
    "caption": "Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML-SI1 as indicated. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (A). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.",
    "molecules": "ML-SI1"
  },
  {
    "caption": "Weighted mean univariate K-function curves of immunogold labeled GFP-HRASG12V under indicated conditions, n ≥ 15 for each condition. The gold point patterns were analyzed with univariate K-functions expressed as L(r)-r. The bar graph on the right shows the peak value Lmax of the L(r)-r curve, which represents the extent of GFP-HRASG12V nanoclustering. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.",
    "molecules": "DMSO"
  },
  {
    "caption": "Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. Values are normalized to DMSO average and represent mean±SEM, n ≥ 15 for each condition. Statistical test employed was Student's t-test with Bonferroni post hoc correction to account for multiple pairwise comparisons.",
    "molecules": "DMSO, gold"
  },
  {
    "caption": "The extent of GFP-HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.",
    "molecules": "DMSO"
  },
  {
    "caption": "Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. but in cells exposed to the indicated perturbations. Values shown are normalized to DMSO average.",
    "molecules": "DMSO, gold"
  },
  {
    "caption": "Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Primary antibodies used are indicated on the right.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Bar graph showing quantification of the Western blots in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was ANOVA.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Bar graph showing the relative levels of free/total cholesterol in HN31 cell extracts exposed to the indicated perturbagens Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Bar graph showing the relative levels of free/total cholesterol in larval brain extracts from animals of the indicated genotypes Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Confocal images of MDCK cells expressing mCherry-D4H. Colocalization with the cortical actin (phalloidin) indicates that mCherry-D4H is predominantly localized to the plasma membrane in DMSO treated cells. Within 24 hours of ML-SI1 treatment, the mCherry-D4H redistributes to intracellular punctae. Although minimal mCherry-D4H signal remained at the plasma membrane in ML-SI1-treated cells, the phalloidin signal (cortical actin) remained unchanged. Scale bar (10μm) shown in the panel on the top right applies to all panels. Bar graph showing quantification of mCherry-D4H punctae in DMSO or ML-SI1-treated cells. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.",
    "molecules": "ML-SI1, DMSO, phalloidin"
  },
  {
    "caption": "Confocal images of MDCK cells expressing mCherry-D4H and LAMP1-GFP treated with ML-SI1. Scale bar (10μm) shown in the panel on the top right applies to all panels.",
    "molecules": "ML-SI1"
  },
  {
    "caption": "Confocal images of control and MCOLN1 shRNA-expressing HN31 cells treated with the cholesterol sensor, filipin. Scale bar (10μm) shown in the panel on the top applies to both panels. Bar graph showing the quantification of the internalized filipin staining in HN31 cells after MCOLN1 knockdown. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.",
    "molecules": "cholesterol, filipin"
  },
  {
    "caption": "f-j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j).",
    "molecules": "DHT"
  },
  {
    "caption": "k-p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1-GFP (l, negative control). CL1-GFP accumulates in DTS7flies with temperature-dependent proteasome impairment (compare m to n) and in AR52flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)",
    "molecules": "DHT"
  },
  {
    "caption": "a-e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59-82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies.",
    "molecules": "DHT"
  },
  {
    "caption": "g-l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)",
    "molecules": "DHT"
  },
  {
    "caption": "a, b, d, Western blots from flies expressing the indicated transgenes. a, Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6, but are elevated in flies in which atg6 or atg12 has been knocked down. b, Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression. AR52 protein became detectable by 2.5 h after treatment with RU486, reached a peak at 10 h, and then slowly decayed. c, A logarithmic plot of AR52/actin ratios was used to determine the line of best fit by regression analysis (y = Ae-Kx). R2 = 0.9117 (AR52 - DHT), R2 = 0.7808 (AR52 + DHT), R2 = 0.9719 (HDAC6 + (AR52 - DHT)), R2 = 0.9644 (HDAC6 + (AR52 + DHT)). Half-life was determined by the slope of the best fit line with the equation t1/2 = 0.693/K. Half-life of AR52 in vivo was reduced ∼2-fold in flies co-expressing Drosophila HDAC6 and did not differ significantly in the presence (broken lines) or absence (solid lines) of DHT. Plots of the mean AR52/actin ratios are shown in Supplementary Fig. S12. d, Flies co-expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b, but AR protein decayed at an accelerated rate. Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope.",
    "molecules": "DHT, RU486"
  },
  {
    "caption": "a-l, SEM images of fly eyes expressing the indicated transgenes. The rough eye phenotypes caused by proteasome mutation (a) or by expression of polyQ-expanded AR (b) were both suppressed by rearing flies on the TOR inhibitor rapamycin (c, d). e, f, Rapamycin failed to suppress degeneration in an autophagy-deficient background created by knockdown of atg12, confirming that rescue by rapamycin is autophagy-dependent. g, h, Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down, demonstrating that suppression via the TOR pathway is HDAC6-dependent. k, j, HDAC6 failed to suppress degeneration in an autophagy-deficient background, confirming that rescue by HDAC6 is dependent on autophagy (compare k, l to i, j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2.",
    "molecules": "rapamycin, Rapamycin"
  },
  {
    "caption": "Ltbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild‐type mice were treated with the LTβR‐Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA. ANA were tested with LIA at the age of 3 months.",
    "molecules": "antibiotics"
  },
  {
    "caption": "A. Cytosolicnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. Two-tailed unpaired T-test was performed. N=4 mice for each group.B. Mitochondrialnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. * P=0.024 with two-tailed unpaired T-test. N=4 mice for each group.",
    "molecules": "non-heme iron"
  },
  {
    "caption": "C. Mitochondrial labile iron in H9c2 cells with or without H2O2 treatment measured using RPA fluorescence. * P<0.0001 with two-tailed unpaired T-test. N=8 independent samples for PBS group and N=10 independent samples for H2O2 group.D. Cytosoliclabile iron in H9c2 cells with or without H2O2 treatment measured using calcein fluorescence. * P=0.0042 with two-tailed unpaired T-test. N=11 independent samples in each group.",
    "molecules": "calcein, RPA, H2O2, labile iron"
  },
  {
    "caption": "E. Mitochondrialiron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). * P=0.041 with two-tailed unpaired T-test. N=4 independent samples in each group.F. Cytosoliciron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). Two-tailed unpaired T-test was performed. N=4 independent samples in each group. All data are expressed as mean ± SEM.",
    "molecules": "iron"
  },
  {
    "caption": "A. Cytosolic iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators. ANOVA followed by post-hoc Tukey test was performed. * P=7.9E-8 PBS vs. DFO. * P=9.4E-6 BPD vs. DFO. * P=0.003 PBS vs. BPD. N=4 independent samples for PBS group and N=6 independent samples for other groups.B. Nuclear iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators. ANOVA followed by post-hoc Tukey test was performed. * P=0.0003 PBS vs. BPD. * P=5.58E-5 PBS vs. DFO. N=4 independent samples for PBS group and N=6 independent samples for other groups.",
    "molecules": "Fe, BPD, DFO, iron"
  },
  {
    "caption": "C. Representative RPA fluorescence staining for labile mitochondrial iron in H9c2 cells with indicated iron chelator treatment. Scale bar = 100μm.D. Labile mitochondrial iron measured by RPA fluorescence in H9c2 cells with indicated iron chelator treatment. ANOVA followed by post-hoc Tukey test was performed.* P=0.016 PBS vs. BPD. * P=0.04 DFO vs. BPD. N=8 independent samples for each group.",
    "molecules": "RPA, BPD, DFO, iron"
  },
  {
    "caption": "E. H2O2-induced cell death in H9c2 cells with indicated treatments. ANOVA followed by post-hoc Tukey test was performed.* P=5E-8 PBS-PBS vs. PBS-H2O2. * P=4E-6 PBS-H2O2 vs. DFO-H2O2. * P=5E-8 PBS-H2O2 vs. BPD-H2O2. * P=1E-7 BPD-H2O2 vs. DFO-H2O2. N=6 independent samples for each group.",
    "molecules": "BPD, DFO, H2O2"
  },
  {
    "caption": "F. Labile mitochondrial iron in H9c2 cells with indicated treatment. PBS with and without H2O2 data were copied from Fig 1C. ANOVA followed by post-hoc Tukey test was performed. * P=1E-8 PBS-PBS vs. PBS-H2O2. * P=3E-9 PBS-H2O2 vs. BPD-H2O2. N=8 independent samples for PBS-PBS, and N=12 for the other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.",
    "molecules": "BPD, H2O2, iron"
  },
  {
    "caption": "A. Mitochondrial non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. * P=0.021 with two-tailed unpaired T-test.B. Cytosolic non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. Two-tailed unpaired T-test was performed.",
    "molecules": "non-heme iron"
  },
  {
    "caption": "E. Lipid peroxidation products in hearts of TG and NTG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.",
    "molecules": "Lipid"
  },
  {
    "caption": "A. Mitochondrial non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=0.03. N=4 mice for PBS and DFO, and N=5 mice for BPD.B. Cytosolic non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=5E-4 PBS vs. DFO. * P=0.01 DFO vs. BPD. P=0.06 PBS vs. DFO. N=4 mice for PBS and DFO, and N=5 mice for BPD.C. Nuclear non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. N=4 mice for PBS and DFO and N=5 mice for BPD.",
    "molecules": "BPD, DFO, non-heme iron"
  },
  {
    "caption": "E. Cardiac function of chelator-treated mice after I/R. ANOVA followed by post-hoc Tukey test was performed for each time point. * P<0.0001 compared to PBS-I/R group at the same time point. # P<0.0001 compared to PBS-sham group at the same time point. Exact P-values are included in Appendix Table S3. N=5 micePBS-sham and BPD-I/R, N=6 mice for all other groups.",
    "molecules": "BPD"
  },
  {
    "caption": "F. Representative Hematoxylin & Eosin (H&E) and Masson Trichrome (MT) staining of heart sections in mice with indicated chelator treatment undergone sham or I/R. Scale bar = 1100μm. Bar graph represent quantification of tissue fibrosis. ANOVA followed by post-hoc Tukey test was performed. * P=0.048. 2-3 sections from each mice were quantified, N=4 mice for PBS, N=5 mice for DFO and N=6 mice for BPD.",
    "molecules": "BPD, DFO"
  },
  {
    "caption": "G. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 PBS-sham vs. PBS-I/R. * P=0.02 DFO-sham vs. DFO I/R. * P=0.007 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.H. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P= 0.0007 PBS-sham vs. PBS-I/R. * P= 0.014 DFO-sham vs. DFO I/R. * P= 0.003 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.I. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-sham vs. PBS-I/R. * P=0.038 DFO-sham vs. DFO I/R. * P=0.006 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.",
    "molecules": "BPD, DFO"
  },
  {
    "caption": "A. Cardiac mitochondrial iron in ABCB8 KO and WT mice with indicated treatment harvested 4 weeks after the tamoxifen treatment. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 WT-vehicle vs. WT-BPD. * P= 0.001 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.004 ABCB8 KO-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.",
    "molecules": "BPD, iron, tamoxifen"
  },
  {
    "caption": "B. Ejection fraction in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. ABCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.",
    "molecules": "BPD"
  },
  {
    "caption": "C. Fractional shortening in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. ABCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.",
    "molecules": "BPD"
  },
  {
    "caption": "D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.017 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.01 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.0003 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.011 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.045 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.",
    "molecules": "BPD"
  },
  {
    "caption": "B. Complex I ROS production in mitochondria with ABCB8 downregulation with or without rotenone. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.",
    "molecules": "ROS, rotenone"
  },
  {
    "caption": "C. Complex III ROS production in mitochondria with ABCB8 downregulation with or without antimycin A. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.",
    "molecules": "antimycin A, ROS"
  },
  {
    "caption": "D. Complex I ROS production in mitochondria with ABCB8 overexpression with or without rotenone. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.",
    "molecules": "ROS, rotenone"
  },
  {
    "caption": "E. Complex IIIROS production in mitochondria with ABCB8 overexpression with or without antimycin A. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.",
    "molecules": "antimycin A, ROS"
  },
  {
    "caption": "F. Mitochondrial ROS in H9c2 cells with various treatments with or without oxidative stress. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=2E-8 PBS-PBS vs. PBS-H2O2. * P=2E-8 DFO- H2O2 vs. BPD-H2O2.",
    "molecules": "BPD, DFO, H2O2, ROS"
  },
  {
    "caption": "G. Mitochondrial membrane potential as measured by TMRE intensity in H9c2 cells treated with indicated iron chelator with or without oxidative stress. N= 6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=1E-8 PBS-PBS vs. PBS-H2O2. * P=1.8E-8 DFO-PBS vs. DFO-H2O2.",
    "molecules": "DFO, H2O2"
  },
  {
    "caption": "H. The levels of lipid peroxidation products in ABCB8 KO mice with or without chelator treatment. N=4 mice for WT-vehicle, N=6 mice for ABCB8 KO-vehicle, and N=5 mice for all other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.004 WT-vehicle vs. ABCB8 KO-vehicle. * P=1E-6 ABCB8 KO-vehicle vs. ABCB8 KO-BPD.",
    "molecules": "BPD, lipid"
  },
  {
    "caption": "I. Lipid peroxidation products in ABCB8 TG and NTG mice two days after I/R. N=6 mice for NTG-sham, and N=4 mice for the other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.024 NTG-sham vs. NTG-I/R. * P=0.016 NTG-I/R vs. TG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.",
    "molecules": "Lipid"
  },
  {
    "caption": "A. Mitochondrial aconitase activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=4 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 EV-PBS vs. EV-H2O2. * P=0.0001 EV-H2O2 vs. ABCB8-H2O2.",
    "molecules": "H2O2"
  },
  {
    "caption": "B. Complex I activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0005 EV-PBS vs. EV-H2O2. * P=0.00001 EV-H2O2 vs. ABCB8-H2O2.C. Complex II activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 EV-PBS vs. EV-H2O2. * P=0.001 EV-H2O2 vs. ABCB8-H2O2.",
    "molecules": "H2O2"
  },
  {
    "caption": "D. Complex IV activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0008 EV-PBS vs. EV-H2O2. * P=0.0006 EV-H2O2 vs. ABCB8-H2O2.",
    "molecules": "H2O2"
  },
  {
    "caption": "E. Mitochondrial aconitase activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=4 independent samples in each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.036 PBS-PBS vs. PBS-H2O2. * P=0.0047 DFO-PBS vs. DFO-H2O2. * P=0.035 PBS- H2O2 vs. BPD-H2O2.",
    "molecules": "BPD, DFO, H2O2"
  },
  {
    "caption": "F. Complex I activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 PBS-PBS vs. PBS-H2O2. * P=0.0003 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.G. Complex II activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-PBS vs. PBS-H2O2. * P<0.0001 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.H. Complex IV activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 PBS-PBS vs. PBS-H2O2. * P=0.0009 DFO-PBS vs. DFO-H2O2. * P=0.006 PBS- H2O2 vs. BPD-H2O2. All data are expressed as mean ± SEM.",
    "molecules": "BPD, DFO, H2O2"
  },
  {
    "caption": "Changes in relative body weight and ear thickness upon CARD14E138A induction with tamoxifen. Malt1EKO = Malt1fl/fl. The combined results of five independent experiments are shown (Malt1+/+ WT n=17, Malt1fl/fl WT n=8, Malt1+/+ ieCARD14E138A n=18, Malt1fl/fl ieCARD14E138A n=12).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative H&E-stained histological sections of ear tissue of tamoxifen-treated mice (Scale bar represents 200 μm). Arrowheads indicate infiltrates of immune cells.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Analysis of infiltrating immune cells in the ears of tamoxifen-treated mice using flow cytometry. Cell count of neutrophils (CD45+ CD3/CD19- CD64- CD11b+ Ly6G+), eosinophils (CD45+ CD3/CD19- CD64- Ly6G- SiglecF+), T cells (CD45+ CD3/CD19+, MHCII-), DCs (CD45+ CD3/CD19- CD64- MHCII+CD11c+) in single cell suspensions of the ear (n≥4).",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Cleavage of MALT1 substrates CYLD and BCL10 as analyzed by western blotting of lysates of ear tissue of mice four days after treatment with tamoxifen and MALT1 inhibitor or vehicle. Actin is shown as a loading control. Each lane represents one mouse.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative histological sections of ear tissue stained with hematoxylin and eosin",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "measurements of epidermal thickness of tamoxifen-treated mice. Combined results of two independent experiments are shown. Each symbol represents the mean of at least ten epidermal thickness measurements for each ear; the line represents the mean value.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "B Density plots of changes in relative mRNA abundance as determined by RNA-seq following treatment of  HEK-293  cells with 1 µM thapsigargin for 6hr (left) or 9hr (right). Genes were categorized as up-regulated by siUPF1total only or siUPF1LL only under basal conditions. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.",
    "molecules": "thapsigargin"
  },
  {
    "caption": "D RT-qPCR  analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 1 µM thapsigargin for 6hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons",
    "molecules": "thapsigargin"
  },
  {
    "caption": "C RT-qPCR  analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons",
    "molecules": "puromycin"
  },
  {
    "caption": "A mRNA decay  measurement using Roadblock-qPCR RNA was isolated from HEK-293 cells at indicated timepoints following transfection with indicated siRNAs and treatment with 400 µM 4sU and 50 µg/mL puromycin.  mRNA half-lives were estimated by fitting the data to a single-phase exponential decay model. Puromycin treatment was compared to the vehicle control and siUPF1LL was compared to the siNT in the absence and presence of puromycin treatment using the extra-sum-of-squares F test. Error bars indicate mean±SD (n = 4 biological replicates).",
    "molecules": "4sU, puromycin, Puromycin"
  },
  {
    "caption": "B RT-qPCR  analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons",
    "molecules": "puromycin"
  },
  {
    "caption": "C Density plot of changes in relative mRNA abundance as determined from RNA-seq  following treatment of HEK-293 cells with 50 µg/mL of puromycin for 4hr. mRNAs were subdivided by PTBP1 and/or  hnRNP L motif density within the first 400 nt of 3'UTR. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons. D Density plot as in (C), following  UPF1LL-specific knockdown.",
    "molecules": "puromycin"
  },
  {
    "caption": "A, Cyclin E overexpression results in increased RMD when the DSB is close to one repeat. The U2OS (EGFP-SSA-3427-3) cell line carrying Tet-on-cyclin E plasmid was used, and RMD frequency was determined with or without induction of cyclin E expression by doxycycline (DOX, 0.5 µg/mL), which was added 24 hours before transfection of the indicated sgRNAs/Cas9. Cyclin E expression levels were validated by Western blot, using KU70 as the loading control.",
    "molecules": "DOX, doxycycline, Tet"
  },
  {
    "caption": "B, ATR is required for BIR/RMD upon cyclin E overexpression. RMD frequency was determined in U2OS (EGFP-SSA-3427-3) cells infected with lentivirus-expressed ATR shRNA or the control vector (Ctrl) with and without induction of cyclin E expression by DOX (0.5 µg/ml) after R29 sgRNA/Cas9 cleavage. ATR knockdown was shown by Western blot with KU70 as the loading control.",
    "molecules": "DOX"
  },
  {
    "caption": "F, p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP-SSA-3427-3) cells carrying the Tet-on-cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 µg/mL) added 24 hours before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.",
    "molecules": "DOX, Tet"
  },
  {
    "caption": "SSA/RMD and BIR/SSA are both dependent on ATM. SSA/RMD and BIR/SSA frequencies in U2OS (EGFP-SSA-3427-17) cells were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9",
    "molecules": "DMSO, ku55933"
  },
  {
    "caption": "BIR/SSA are both dependent on ATM. BIR/RMD in mES cells carrying the RMD-GFP reporter were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 or in ATM KO mES cells carrying the RMD-GFP reporter (right).",
    "molecules": "DMSO, ku55933"
  },
  {
    "caption": "B and C, The effects of RAD52, POLD3, ATM and H2AX inactivation on BIR/RMD with cleavage at L17/D10k. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter was determined using the sgRNAs/Cas9 at L17/D10k in RAD52 KO cells or after POLD3 depletion by siRNA (B), or after treatment with ATM inhibitor (ku55933, 15mM) or H2AX depletion by siRNA (C).",
    "molecules": "ku55933"
  },
  {
    "caption": "A Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres.",
    "molecules": "DNA, Zn"
  },
  {
    "caption": "B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.",
    "molecules": "DNA"
  },
  {
    "caption": "E Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′-TTGCCTACCTGGGTGGTCTCTCCACTT-3′) or mutated GliBS sequence used as control (5′-TTGCCTACCTCCCACTTCTCTCCACTT-3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242-424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.",
    "molecules": "DNA"
  },
  {
    "caption": "A Inhibition of Gli1‐induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only.",
    "molecules": "DMSO, GANT61"
  },
  {
    "caption": "C Inhibition of Gli1‐induced transcription in transfected Smo−/−MEF cells. Smo−/−MEF cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control.",
    "molecules": "DMSO"
  },
  {
    "caption": "D The graphs show the Hh target gene expression levels in Ptch1−/−MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real‐time PCR (qRT-PCR) normalized to endogenous control (β2‐microglobulin and HPRT). Pfkfb3 gene was used as a negative control.",
    "molecules": "DMSO"
  },
  {
    "caption": "F SuFu−/−MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1mRNA levels were determined by qRT-PCR normalized to β2‐microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control.",
    "molecules": "DMSO"
  },
  {
    "caption": "H Ptch1 mRNA expression levels (left panel) were determined by qRT-PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non‐specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT-PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression.",
    "molecules": "DMSO"
  },
  {
    "caption": "A The predicted binding mode of GlaB (green sticks) to Gli1ZF (blue cartoons). Residues highlighted by the single‐point mutation study to impact on Gli1 binding to DNA are shown as blue sticks; K340 and K350 of the GlaB binding site are colored magenta. GlaB protons highlighted by the NMR study are shown as orange spheres.",
    "molecules": "DNA"
  },
  {
    "caption": "C Inhibition of Gli1/DNA binding by GlaB. EMSA using recombinant GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A in the presence of different concentrations of GlaB or with DMSO only. The shifted complex is competed with a 50× excess of cold probe. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding in absence of GlaB. *P 0.05 versus DMSO; **P 0.05 K340A + GlaB versus Gli1 WT + GlaB.",
    "molecules": "DNA, DMSO"
  },
  {
    "caption": "D HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty vector) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB. Treatment time was 24 h, and control cells were treated with DMSO only. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO; **P 0.05 K340A versus Gli1 WT.",
    "molecules": "DMSO"
  },
  {
    "caption": "D BrdU assay in GCPs. Cerebellargranule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouseSonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh.",
    "molecules": "DMSO"
  },
  {
    "caption": "A, B Ex vivo cell cultures from Ptch1+/−mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells.",
    "molecules": "DMSO, GANT61"
  },
  {
    "caption": "A, B Ex vivo cell cultures from Ptch1+/−miceMBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, Gli1 mRNA expression levels were determined by qRT-PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT).",
    "molecules": "DMSO, GANT61"
  },
  {
    "caption": "C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right).",
    "molecules": "DMSO"
  },
  {
    "caption": "C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT).",
    "molecules": "DMSO"
  },
  {
    "caption": "C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels.",
    "molecules": "DMSO"
  },
  {
    "caption": "C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.",
    "molecules": "DMSO"
  },
  {
    "caption": "G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.",
    "molecules": "DMSO"
  },
  {
    "caption": "A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "C Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.",
    "molecules": "DMSO"
  },
  {
    "caption": "(A) WT and CITK -/- embryonic (E14.5) mousecerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars = 5 μm.(B) Quantification of vertical divisions of apical progenitors in WT and CITK -/- mice. n= 3 per each genotype.",
    "molecules": "DNA"
  },
  {
    "caption": "(D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments).",
    "molecules": "DNA"
  },
  {
    "caption": "(A) HeLa cells pre-extracted for 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), γ-tubulin (red) and DNA (blue). Scale bars, 10μm",
    "molecules": "DNA, Triton X-100"
  },
  {
    "caption": "(B) HeLa cells expressing CITK-GFP (green) from a BAC transgene treated as in (A) and immunostained for γ-tubulin (red) and DNA (blue). Scale bars, 5μm",
    "molecules": "DNA"
  },
  {
    "caption": "(D) HeLa cells pre-extracted 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), ASPM (red) and DNA (blue).",
    "molecules": "DNA, Triton X-100"
  },
  {
    "caption": "(E) HeLa cells expressing CITK-GFP treated as in (A) and immunostained for GFP (green), ASPM (red) and DNA (blue).",
    "molecules": "DNA"
  },
  {
    "caption": "(G) Control or CITK-depleted cells immunostained for ASPM (green), γ-tubulin (red) and DNA (blue).",
    "molecules": "DNA"
  },
  {
    "caption": "(H) Control or ASPM-depleted cells treated as in (A) and immunostained for CITK (green), γ-tubulin (red) and DNA (blue).(I) Quantification of CITK positive centrosomes and of the ratio of CITKspindle pole intensity versus total cell mean intensity, in control and ASPM-depleted cells. (n ≥ 75 cells, 4 independent experiments).",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Control, CITK or ASPM-depleted HeLa cells immunostained for α-tubulin (red) and DNA (blue). Maximum intensity projections of confocal z-stacks are shown.(B) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL, CITK or ASPM siRNA (n > 130 cells in 3 independent experiments).",
    "molecules": "DNA"
  },
  {
    "caption": "(E) Synchronized HeLa transfected with control, ASPM- or CITK-specific siRNAs treated with DMSO or with 250 pM Paclitaxel (taxol) 30 min before immunostaining for α-Tubulin. Spindle angles were measured as above (n > 100 cells in 6 independent experiments).",
    "molecules": "DMSO, Paclitaxel"
  },
  {
    "caption": "(D) Immunofluorescence microscopy images showing MT growth at mitotic spindle poles 0, 1, and 2 min after nocodazole washout. Control and CITK-depleted cells were immunostained for α-tubulin (red) and DNA (blue).(E) Percentage of cells showing detectable MT nucleation (aster size > 1 µm) 0, 1, and 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).(F) Quantification of aster size 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).",
    "molecules": "DNA, nocodazole"
  },
  {
    "caption": "(B) Representative leak-subtracted ClC-3, ClC-4 and ClC-5 whole-cell currents measured with (upper row) or without (lower row) 5 mM ATP added to the pipette solution. Currents were elicited every 10 s by voltage steps from 0 mV to +140 mV, followed by a step to -100 mV to maximize the off-gating currents visible as sharp peaks at the end of the pulses. The recording started shortly (10s - 30s) after obtaining a whole-cell configuration. The first and last recorded currents in a series are depicted in green and red, respectively. NMDG-based solutions were used to characterize ClC-4.",
    "molecules": "ATP, NMDG"
  },
  {
    "caption": "(C) Effects of various adenine nucleotides or their washout on the ionic transport of ClC-3, ClC-4 and ClC-5. The current amplitudes were measured at the end of the test pulses from experiments as shown in (B) and normalized to the initial current amplitudes obtained after establishing the whole-cell configuration. Some of the experimental data points have been omitted for clarity. Monoexponential fits to the data are depicted as red lines. Asterisks indicate statistical significances evaluated by two-sample t-test analysis of the residual currents determined at the end of each 10-minute measurement (*, p < 0.05; ***, p < 0.001, n.s., not significant; n= 5-11).",
    "molecules": "adenine"
  },
  {
    "caption": "(B) Voltage dependence of the WT ClC-5 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=7-12). Error bars represent SEM. Red lines depict Boltzmann fits to the data; fit parameters are provided in Appendix Table S1.",
    "molecules": "adenine"
  },
  {
    "caption": "(C) Voltage dependence of the WT ClC-3 off-gating charge normalized to the ionic current at +165 mV in the absence or presence of adenine nucleotides (n=4-5, Error bars represent SEM.). Red lines represent Boltzmann fits to the data",
    "molecules": "adenine"
  },
  {
    "caption": "(A) Representative whole-cell recordings of cells expressing E211G ClC-5 upon voltage pulses to +140 mV from a -60-mV holding potential applied in 10-s intervals starting shortly after obtaining the whole-cell configuration with (left) or without (right) 5 mM ATP in the pipette solution. Leak subtraction was not applied. Asymmetric [Cl-] was used (110 mM internal NaCl substituted by TrisSO4) to achieve a stable negative reversal potential of <-50 mV allowing to recognize recordings with high unspecific leak current contamination.",
    "molecules": "TrisSO4, ATP, Cl-, NaCl"
  },
  {
    "caption": "(B) Normalized time course of the steady-state E211G ClC-5 current amplitudes measured with (n=5) or without (n=4) internal ATP. Error bars show SEM. Red line represents a monoexponential fit to the data with a time constant of 108±8 s (SEM); currents measured with ATP-free pipette solution declined to 57.9±2.6% (SEM) of the initial amplitude.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) Effects of different adenine nucleotides or their washout on the ClC-5 current amplitudes (n=4-7). The data were normalized to the initial current amplitude determined shortly after establishing whole-cell configuration. Some of the data points are omitted for clarity. For comparison, the ADP data set from Figure 1 is also depicted. Red lines indicate monoexponential fits to the averaged data.",
    "molecules": "adenine"
  },
  {
    "caption": "(f) A549-ACE2 or control A549-Venus cells were pretreated with Tubercidin or vehicle (DMSO) 3h prior to infection with SARS-CoV-2 at MOI 0.1. After 24 hours, abundances of SARS-CoV-2 nucleoprotein (N), ACE2, Venus and β-actin (ACTB, loading control) were visualized using Western blotting. Presented data is representative of 3 independent repeats.",
    "molecules": "DMSO, Tubercidin"
  },
  {
    "caption": "(i) NHBEs derived from three independent donors were pretreated for 6 h with indicated concentrations of DZNep or vehicle and infected with SARS-CoV-2 for 24 h. Cells were fixed and the abundance of SARS-CoV-2 N was quantified by immunofluorescent staining. Shown are vehicle-normalized integrated anti-N fluorescent intensity and cell confluence, error bars represent mean +/- sd of n=3 donors.",
    "molecules": "DZNep"
  },
  {
    "caption": "Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (b) Number of significantly up- or downregulated proteins in indicated comparisons according to (a).",
    "molecules": "DZNep"
  },
  {
    "caption": "Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (c) Donor-normalized LFQ abundances of viral nucleoprotein (N) and spike (S) in the indicated conditions. Error bars represent mean +/- sd of n=4 donors (NHBE) or n=4 independently infected A549-ACE2 cultures. Statistics were calculated using Student's two-sided t-test as indicated.",
    "molecules": "DZNep"
  },
  {
    "caption": "(e) NHBEs (6 independent donors) were pretreated for 6 h with 0.75 μM DZNep or vehicle and infected with SARS-CoV or SARS-CoV-2 at MOI 3. 24 h later, accumulation of IL-6 and IP-10 was measured in the supernatant by ELISA. Donor-wise IL-6 and IP-10 secretion, normalized to vehicle treated uninfected controls (as further described in materials and methods) is shown.",
    "molecules": "DZNep"
  },
  {
    "caption": "(F) Representative Western blot (n = 3) for LC3. Chloroquine was added 12 h before cell harvest.",
    "molecules": "Chloroquine"
  },
  {
    "caption": "(A, left) MitoTracker staining. (right) MitoTracker staining after bezafibrate treatment. (B) Quantification of myotubes with perinuclear MitoTracker-positive aggregates in untreated (vehicle) or bezafibrate-treated myotubes at DM-2. Data represent mean ± SD (n = 3). *, P < 0.001 by Student's t test.",
    "molecules": "bezafibrate"
  },
  {
    "caption": "(C) MitoTracker (red) and LC3immunostaining (green) of myotubes at DM-2. Chloroquine and 3-MA (where indicated) were added 12 h before staining. Note the extensive overlap of MitoTracker and LC3 in Rb−/−myotubes (yellow), which is inhibited by 3-MA. (D) Mean percentage of MitoTracker (red) to LC3 (green) overlap in 25 myotubes from two independent cultures in control versus Rb−/−myotubes (total) or Rb−/−myotubes with perinuclear mitochondrial aggregation (high). *, P = 7 × 10−5 by Student's t test.",
    "molecules": "3-MA, Chloroquine"
  },
  {
    "caption": "(E) Ultrastructural analysis of myotubes treated or not with 3-MA at DM-2. Arrowheads point to mitochondria. Asterisks indicate electron-dense autolysosomes. N, nucleus. (F) Ratio of mitochondrialDNA/nuclearDNA in the indicated cultures (n = 3). (G) Ratio of mitochondrialDNA/nuclearDNA in E16.5 skeletal muscle (n = 3; *, P = 0.009). (H) Quantification of ATP in E16.5 skeletal muscle (n = 3; *, P = 0.011). (F-H) Error bars represent SD. *, P < 0.001.",
    "molecules": "3-MA, DNA"
  },
  {
    "caption": "(D) Mean number of Rb−/− myotubes after 3-MA treatment, bezafibrate, or DMSO/vehicle as indicated. Counts are mean ± SD of six fields (n = 4).",
    "molecules": "3-MA, bezafibrate, DMSO"
  },
  {
    "caption": "(E, left and middle) Brightfield images of the indicated myotubes treated or not with 3-MA at DM-8. (right) Immunostaining for MHC (red) at DM-14 in the presence of 3-MA. Arrowheads label 3-MA-rescued Rb−/− myotubes at DM-8. 3-MA-rescued, twitching Rb−/− myotubes are shown in Video 4.",
    "molecules": "3-MA"
  },
  {
    "caption": "(F) Immunostaining for BrdU (green) and MHC (red) of 3-MA-treated cultures at DM-2 or DM-10. Rb−/− myotubes incorporated BrdU at DM-2 (arrowheads) but not DM-10.",
    "molecules": "3-MA"
  },
  {
    "caption": "(G) Brightfield images (left and middle) and MHC staining (right) of the indicated cultures treated or not with bezafibrate. Arrowheads point to bezafibrate-rescued Rb−/− myotubes.",
    "molecules": "bezafibrate"
  },
  {
    "caption": "(A) Immunostaining for MHC (green) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine. Note the shortened myotubes in the presence of lonidamine.",
    "molecules": "lonidamine"
  },
  {
    "caption": "Effector T (Teff) cells isolated from the spleen of a WT mouse were stimulated with soluble anti-CD3 (5 μg/ml) in the presence of mitomycin C-treated splenocytes and were cultured with regulatory T (Treg) cells isolated from WT and Cd4cre A1fl/fl mice at the indicated ratios. After 72 h, the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, N.S.; not significant). The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0%.",
    "molecules": "mitomycin C"
  },
  {
    "caption": "(B) Maximum Ca2+ flux (peak height) at each anti-IgM concentration for FOB and MZB cells. Paired t test, ns = not significant, *P < 0.05, and ***P < 0.001.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(E) Wdr37+/+ and Wdr37−/− splenocytes were labeled with Indo-1, stained for cell surface markers to identify FOB cells, and stimulated with the indicated amounts of anti-IgM (black arrowhead). Normalized traces from three (2.5 mcg/ml anti-IgM) or four independent experiments (10 mcg/ml and 5 mcg/ml anti-IgM) are shown (Wdr37+/+ gray, Wdr37−/− pink). Mean Ca2+ flux for each genotype is overlaid in bold (Wdr37+/+ black, Wdr37−/− red).",
    "molecules": "Ca2+, Indo-1"
  },
  {
    "caption": "(D) Peak cytosolic Ca2+ concentration based on aequorin measurements as performed in (C). Symbols represent individual wells containing ~5 x 105 transfected 3T3 or Pacs1-/- cells from one experiment. Horizontal bars indicate mean. Two-tailed unpaired t test, **P < 0.01.",
    "molecules": "Ca2+"
  },
  {
    "caption": "(E) Pacs1+/+ and Pacs1-/- NIH-3T3 cells (C1 and C2 from (A)) were transfected with ER-GCamP6-210. ER Ca2+ was measured before and after treatment with 10 µM ATP. Kinetic traces show the mean 488/405 nm excitation ratio of each cell line with error bars indicating SEM. N=30 cells (3T3), 16 cells (C1), 25 cells (C2). Data are representative of 2 independent experiments.",
    "molecules": "GCamP6-210, ATP, Ca2+"
  },
  {
    "caption": "(B, C) Pacs1+/+ and Pacs1−/− mice were immunized with alum-ova and one week later with NP-Ficoll. Anti-ova IgG and anti-NP IgM titers were measured at 14 days and 7 days after immunization, respectively. Each symbol represents an individual mouse.",
    "molecules": "NP, alum, Ficoll, ova"
  },
  {
    "caption": "(H, I) Pacs1+/+ and Pacs1-/- mice were injected with EdU and the fraction of EdU+ FOB and MZB cells were measured in the spleen at 1, 4, and 7 days post-injection. Data are from one experiment.",
    "molecules": "EdU"
  },
  {
    "caption": "Binding assay with different dilutions of Apg2 incubated with DNAJB1/Hsc70 and (A) with αSyn fibres or (B) without αSyn fibres (control). 5 mM ATP was present in the buffer throughout.",
    "molecules": "ATP"
  },
  {
    "caption": "Representative 18F-FDG images with microPET in the coronal and axial planes in Wt and LXRα-Tg mice 5 weeks post-TAC.",
    "molecules": "18F, FDG"
  },
  {
    "caption": "Myocardial FDG uptake measured as standard uptake value (SUV); n = 4-6/group. *P = 0.04 versus Wt sham, **P = 0.02 versus LXRα-Tg sham, #P = 0.03, ##P = 0.01.",
    "molecules": "FDG"
  },
  {
    "caption": "Quantification of myocardial glycogen content shows no significant differences; n = 5 per sham group, n = 7 per TAC group.",
    "molecules": "glycogen"
  },
  {
    "caption": "I Representative 18F-FDG-microPET scans.",
    "molecules": "FDG"
  },
  {
    "caption": "J Myocardial FDG uptake quantified as standard uptake value (SUV); n = 6/group. *P = 0.0001 versus WT sham, #P = 0.047.",
    "molecules": "FDG"
  },
  {
    "caption": "A Assessment of 2-deoxyglucose (2-DG) uptake from 4 independent experiments. *P = 0.03 versus Ad-cont.",
    "molecules": "2-deoxyglucose, 2-DG"
  },
  {
    "caption": "D Western blot indicating Ad-LXRα- and PE-induced increases in global protein O-GlcNAcylation, which was abrogated following inhibition of HBP with DON. LXRα protein expression is shown, and GAPDH served as a loading control.",
    "molecules": "DON, GlcNAc, PE"
  },
  {
    "caption": "E, F Modulation of Anp and Bnp mRNA levels by Ad-LXRα-induced O-GlcNAc signaling. Gene expression as determined by RT-PCR normalized to 36b4, n = 5 per condition in the absence of PE, n = 4 per condition in the presence of PE. *P = 0.02 versus Ad-cont, **P = 0.008 versus Ad-cont, #P = 0.03, ##P = 0.02.",
    "molecules": "GlcNAc, PE"
  },
  {
    "caption": "H Representative images for the determination of cell size. Cells were stained with an antibody specific for LXRα (green, indicated by arrow), DAPI for nuclei (blue), and rhodamine-phalloidin for F-actin (red); scale bar = 50 μm.",
    "molecules": "rhodamine"
  },
  {
    "caption": "A Protein synthesis determined via leucine incorporation, n = 2-4 independent experiments. *P = 0.03 versus Ad-cont, §P = 0.06 versus Ad-cont.",
    "molecules": "leucine"
  },
  {
    "caption": "B Global O-GlcNAc protein expression with Western blot, GAPDH served as a control.",
    "molecules": "GlcNAc, protein"
  },
  {
    "caption": "A, B Western blot analyses of global protein O-GlcNAc levels in left ventricles of mice with either (A) cardiac-specific LXRα overexpression or (B) LXRα deficiency and subjected to 5 weeks TAC.",
    "molecules": "GlcNAc"
  },
  {
    "caption": "A-H Effect of CCCP on the localisation of Noc and NocNΔ10. Cellular localisation of Noc‐mYFP (DWA206) and NocNΔ10‐mYFP (DWA382) either with no additions (NA) or after CCCP treatment (5 min; 100 μM), as indicated. Scale bar, 2.5 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "I-L Effect of Noc overproduction on cell division. Exponentially growing cultures of DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were examined after growth for 1 h with 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Insets show corresponding phase contrast light microscopy images. Scale bar, 5 μm.",
    "molecules": "DNA, IPTG"
  },
  {
    "caption": "M Complementation of noc in a Δnoc ΔminCD background. Strains DWA564 (Δnoc, ΔminCD, Pxyl‐noc‐myfp) and 566 (Δnoc, ΔminCD, Pxyl‐nocNΔ10‐myfp) were streaked on nutrient agar (NA) plates in the presence of 0.5% w/v xylose and incubated at 30 and 39°C, as indicated.",
    "molecules": "xylose"
  },
  {
    "caption": "N Effect of Noc and NocNΔ10 overproduction on sporulation. Strains DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were streaked on NA plates in the absence and presence of 1 mM IPTG, as indicated, and photographed after 48 h at 37°C.",
    "molecules": "IPTG"
  },
  {
    "caption": "B Growth of strains DWA350 (Δnoc ΔminCD) and DWA307 (Δnoc ΔminCD, Pxyl‐HCVAH‐NocNΔ10) on nutrient agar plates at 30 and 48°C in the absence and presence of 0.5% w/v xylose, as indicated.",
    "molecules": "xylose"
  },
  {
    "caption": "C-F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30°C (C and D) and at 42°C in LB + 0.5% w/v xylose (E and F). Arrowheads indicate minicells. Cell membranes were stained with FM5-95. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.",
    "molecules": "xylose"
  },
  {
    "caption": "G, H Cellular localisation of HCVAH‐NocNΔ10‐YFP (G) in strain DWA193 (Δnoc, Pxyl‐HCVAH‐nocNΔ10‐yfp) and overlay showing DAPI‐stained DNA (H). The strain was grown at 30°C in CH medium. Inset shows the corresponding phase contrast light microscopy image. Scale bar, 5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "G Ability of ParB‐box mutants to rescue the growth defect of Δnoc ΔminCD. Strains DWA564 (Pxyl‐noc‐myfp), 590 (Pxyl‐nocQ68R‐myfp), 568 (Pxyl‐nocG86S‐myfp), 598 (Pxyl‐nocR88A‐myfp), 600 (Pxyl‐nocR89A‐myfp) and 602 (Pxyl‐nocR91A‐myfp) were streaked on plates containing 0.5% w/v xylose and incubated for 18 h at either 30 or 39°C, as indicated, before being photographed.",
    "molecules": "xylose"
  },
  {
    "caption": "H-O ParB‐box mutants are dominant‐negative. Cells of strains DWA362 (ΔminCD, Pspac(hy)‐noc), 363 (ΔminCD, Pspac(hy)‐nocNΔ10), 364 (ΔminCD, Pspac(hy)‐nocQ68R) and 365 (ΔminCD, Pspac(hy)‐nocG86S) were examined after growth for 2 h at 42°C with either no additions (NA) (H-K) or in the presence of 1 mM IPTG (L-O), as indicated. Cell membranes were stained with FM5‐95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.",
    "molecules": "IPTG"
  },
  {
    "caption": "A-C Representative images of TetR‐mCherry in cells lacking (A) or containing (B and C) the NBS plasmid pDWA117. Strains DWA427 (A) and 429 (B and C) (both strains contain Pspac‐noc) were examined after growth for 2 h in the absence (A and B) and presence (C) of 1 mM IPTG. DNA was stained with DAPI.",
    "molecules": "DNA, IPTG"
  },
  {
    "caption": "A, B Effects of Noc overproduction on cell division and nucleoid morphology in E. coli. Cells of strain DWA261 carrying pDWA37 (PA1/04/03‐noc) were examined after growth in LB with either no additions (A) or after induction for 1 h with 1 mM IPTG (B).",
    "molecules": "IPTG"
  },
  {
    "caption": "C-E Effects of overproduction of Noc variants on nucleoid morphology. Cells of strains DWA266 (PA1/04/03‐nocNΔ10) (C), 270 (PA1/04/03‐HCVAH‐nocNΔ10) (D) and 267 (PA1/04/03‐nocCΔ50) (E) carrying plasmids for the overproduction of the indicated mutants (see cartoons underneath panels) were grown in LB and examined after growth for 1 h in the presence of 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Scale bar, 5 μm.",
    "molecules": "DNA, IPTG"
  },
  {
    "caption": "A, B Effects of Spo0J and Noc30‐Spo0J overproduction on cell division and nucleoid morphology in E. coli. Cells of strains DWA271 (PA1/04/03‐spo0J) (A) and 272 (PA1/04/03‐noc‐spo0J) (B) were grown in LB in the presence of 1 mM IPTG, to induce the expression of either Spo0J (A) or the Noc‐Spo0J hybrid (B), and were examined 1 h post‐induction. Cell membranes and DNA were stained with FM5-95 and DAPI, respectively. Scale bar, 5 μm.",
    "molecules": "DNA, IPTG"
  },
  {
    "caption": "C, D Genomic views of relevant TF binding to Cebpa and Gata3 loci (supporting direct regulatory interactions identified in Figs 3A,B,C). The red arrow indicates a putative enhancer element, which is bound by Ebf1 and flanked by AcK27-rich regions.",
    "molecules": "AcK27"
  },
  {
    "caption": "B . A competition curve for the Dvl-CXXC5 interaction by KY-02061.",
    "molecules": "KY-02061"
  },
  {
    "caption": "C-E. NMR titration analyses for Dvl PZD domain with KY-02061. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02061. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02061) are displayed as red (1:0), orange (1:10), purple (1:20), cyan (1:40), green (1:60) and blue (1:80) (C, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:80 (D, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (E).",
    "molecules": "15N, KY-02061"
  },
  {
    "caption": "F. Molecular docking of Dvl binding motif (DBM) or KY-02061 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of DBM (green) and KY-02061 (yellow) on the surface of Dvl PDZ domain (gray) was visualized.",
    "molecules": "KY-02061"
  },
  {
    "caption": "A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with DMSO, KY-02061 in DMSO, or 5 µM PolyR-DBM for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]",
    "molecules": "KY-02061, DMSO"
  },
  {
    "caption": "B . Primary calvaria cells were isolated from the calvariae of 4-day-old mice (Manton et al, 2007), and treated with DMSO or KY-02061 in DMSO for 4 days. ALP activity levels were visualized by ALP staining.",
    "molecules": "KY-02061, DMSO"
  },
  {
    "caption": "C. Primary calvaria cells were treated with DMSO or 5 µM KY-02061 in DMSO for 14 days. The cells were subjected to immunofluorescence analyses to visualize Runx2 (green) and β-catenin (red). The cell nuclei were counterstained with DAPI (blue).",
    "molecules": "KY-02061, DMSO"
  },
  {
    "caption": "D, E. Calvariae from 4-day-old mice were cultured ex vivo for 7 days with KY-02061 in DMSO (D). The calvaria thicknesses were measured from the stained sections using Image Pro software (E). [n=3]",
    "molecules": "KY-02061, DMSO"
  },
  {
    "caption": "B. A competition curve for the Dvl-CXXC5 interaction by KY-02327.",
    "molecules": "KY-02327"
  },
  {
    "caption": "C. Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY-02327. From top to bottom final moral ratio between Dvl PDZ and KY-02327 was, 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20 and 1:25, respectively.",
    "molecules": "KY-02327"
  },
  {
    "caption": "D-F. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02327. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02327) are displayed as red (1:0), yellow (1:5), green (1:10), and magenta (1:20) (D, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:20 (E, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (F).",
    "molecules": "15N, KY-02327"
  },
  {
    "caption": "G. Molecular docking of KY-02061 or KY-02327 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of KY-02061 (yellow) and KY-02327 (cyan) on the surface of Dvl PDZ domain (gray) was visualized.",
    "molecules": "KY-02061, KY-02327"
  },
  {
    "caption": "A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with indicated dose of KY-02327 for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]",
    "molecules": "KY-02327"
  },
  {
    "caption": "B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot.",
    "molecules": "KY-02327"
  },
  {
    "caption": "B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot (B) or immunofluorescence (C) analyses. Nuclei were counterstained with DAPI (C, blue).",
    "molecules": "KY-02327"
  },
  {
    "caption": "D. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 14 days. The mRNA level of collagen 1a (Col1a) was measured by quantitative real-time qRT-PCR. [n=3]",
    "molecules": "KY-02327"
  },
  {
    "caption": "E. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 21 days. The mRNA level of osteocalcin (OCN) was measured by qRT-PCR. [n=3]",
    "molecules": "KY-02327"
  },
  {
    "caption": "A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. After calcein injection, the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein (A, green, arrows). Nuclei were counterstained with DAPI (A, blue). Osteoblast numbers (B) and calcein double labelled surfaces (C) were measured in the femoral sections.",
    "molecules": "KY-02327, calcein"
  },
  {
    "caption": "A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The mouse femurs were analyzed using micro-CT and BMD (D) and BV/TV (E) were calculated from the micro-CT data.",
    "molecules": "KY-02327"
  },
  {
    "caption": "A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The three-dimensional images of femoral trabecular bone were reconstructed (F), and trabecular number (G) and trabecular separation (H) were calculated.",
    "molecules": "KY-02327"
  },
  {
    "caption": "Two-fold serial dilutions of the total protein extracts from the wild-type, ash122/ash19011 (ash1-), ash122,NSDds46/ash19011,NSDds46 (ash1-, NSD-) and Set21 (Set2-) larval brains, imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 (A), H3K36me2 (B) and H3K36me3 (C). Note the strong (>10-fold) reduction of H3K36me3 signal in the Set2- extract and the slight (~2-fold) reduction of H3K36me1 signal in the ash1- and ash1-, NSD- extracts. The protein extracts from the wild-type, double ash1-, NSD- and single NSD- and Set2- mutants (right panels) were analyzed together on the same membrane, however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1-,NSD- and the Set2- extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls.",
    "molecules": "H3K36me1, H3K36me3"
  },
  {
    "caption": "(D) Reverse transcription and quantitative PCR (RT-qPCR) measurement of Abd-B expression in His3.2-,His3.3-,H3K36R embryos. Expression of Abd-B in stage 16 embryos, which are homozygous for His3.3A, His3.3B, and ∆HisC deletions and carry 12xH3K36R transgene (mutant), is not reduced compared to their wild-type counterparts (control 1) or embryos heterozygous for His3.3A, and ∆HisC deletions (control 2). Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results.",
    "molecules": "H3K36R"
  },
  {
    "caption": "Immunocytochemistry using the same antibody along with Mitotracker red was performed in COS7 cells, 24 hours after transfection. Nuclei were counterstained with DAPI (blue) 11.5RVD and 8.5RVD mitoTev-TALEs both co-localized with mitochondria, as seen in the merge image. Scale bar: 10 µm",
    "molecules": "DAPI, Mitotracker red"
  },
  {
    "caption": "MtDNA heteroplasmy analyzed by PCR/RFLP, 24 hours post mitoTev-TALE and mitoTALEN transfection. The RFLP analysis shows increased %WT mtDNA in sorted cells when compared to the the untransfected. mitoTALEN monomers positive for both eGFP and mCherry were isolated as "Yellow". The "Black" cells represent the mitoTALEN sorted population of cells negative for eGFP and mCherry and the "GFP-+" population represents mitoTev-TALE sorted cells with low levels of eGFP fluorescence. GFP++ were cells positive for GFP after mitoTev-TALEs transfections",
    "molecules": "MtDNA, mtDNA"
  },
  {
    "caption": "Determination of the levels of mtDNA (ND1/ACTB) by qPCR of mutant cybrids transfected with MERRF mitoTev-TALE",
    "molecules": "mtDNA"
  },
  {
    "caption": "mtDNA levels in wild-type sorted cells transfected with the MERRF mitoTev-TALE. Data is expressed as percentage of the Untransfected cells (%UNT)",
    "molecules": "mtDNA"
  },
  {
    "caption": "Total mtDNA levels determined by qPCR in 6-7 separate sortings/experiments",
    "molecules": "mtDNA"
  },
  {
    "caption": "Quantification of mtDNA levels by PrimeTime qPCR probes. The mtDNA levels were analyzed in the different populations of sorted cells. Data are expressed as mean±SEM of 3-4 separate experiments mtDNA levels were also determined in the WT cybrids as described above. Data are expressed as mean±SEM of 4-7 separate experiments",
    "molecules": "mtDNA"
  },
  {
    "caption": "Oxygen consumption rate (OCR) upon sequential injection of oligomycin (Oligo), FCCP and rotenone (Rot) + antimycin (AA), in untransfected, sorted "GFP-+" and "GFP++". The error bars represent the mean±SEM of 5-7 separate experiments",
    "molecules": "AA, antimycin, FCCP, Oligo, oligomycin, Rot, rotenone"
  },
  {
    "caption": "E, Immunofluorescence of MFG-E8, a marker of programmed cell death, showed that MFG-E8 is not expressed at the implantation sites of Rb1f/f mice at 0900h on day 5 and Rb1d/d mice at 0900h and 2000h on day 5, but is expressed at the implantation sites of Rb1f/f mice at 2000h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; arrowhead, embryo.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, Serum estradiol-17β (E2) levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice (mean ± SEM, Student's t test; n=4 mice for each group).",
    "molecules": "E2, estradiol-17β"
  },
  {
    "caption": "B, Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice. In Rb1d/d mice, pre-implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 (mean ± SEM, Student's t test; n=4 mice for each group).",
    "molecules": "P4"
  },
  {
    "caption": "D, Persistent Ki67 expression in the epithelium of Rb1d/d mice on day 4 was recovered by pre-implantation P4 treatment on days 2 and 3. Scale bar = 100μm; le, luminal epithelium; s, stroma. E, Ratio of Ki67-positive epithelial cells in Rb1d/d mice was completely suppressed by pre-implantation P4 supplementation on days 2 and 3. The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2D were used (mean ± SEM, Student's t test; n=5 different mice for each group). Three different high-powered fields per mouse were analyzed and each of the Ki67-posivie ratios was demonstrated. ",
    "molecules": "P4"
  },
  {
    "caption": "G, Resorption rate was reduced on day 8 in Groups 1 (n=41 different implantation sites) and 2 (n=30 different implantation sites), but not in Group 3 (n=40 different implantation sites). The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2I were used. H&E staining was performed using all sections that shows the implantation sites, and resorption rate was evaluated. *P<0.05 vs Rb1f/f mice; **P<0.05 vs Rb1d/d mice; Fisher's exact test.",
    "molecules": "P4"
  },
  {
    "caption": "H, Number of pups delivered by Rb1d/d dams was normalized by pre-implantation P4 treatment on days 2 and 3 (Group 2)(mean ± SEM, Student's t test). The same data of Rb1f/f and Rb1d/d dams without P4 treatment in Fig 1E were used. The numbers of pups in 20 Rb1f/f and 37 Rb1d/d dams without P4 treatment and 7 Rb1d/d dams with P4 treatment were evaluated.",
    "molecules": "P4"
  },
  {
    "caption": "A, pRIP3, a central mediator of necroptosis, was not expressed at the implantation sites of Rb1d/d mice but was expressed at those of Rb1f/f mice at 1500h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo; green dotted line, luminal epithelium; red dotted line, stroma.",
    "molecules": "DAPI"
  },
  {
    "caption": "B, TNFα stimulated the expression of pMLKL, a critical mediator of necroptosis, in the primary mouse uterine epithelial cells. Scale bar = 50μm; blue signal, nuclei stained by DAPI; red signal, pMLKL.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-B, In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V, a marker of PS presentation at the outer membrane of Rb1f/f epithelial cells but does not in Rb1d/d ones, and P4 restores TNFα-primed annexin V expression in Rb1d/d uterine epithelial cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.",
    "molecules": "PS, P4"
  },
  {
    "caption": "C-D, Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth-arrested mouse epithelial cells with thymidine treatment, but does not in the control cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.",
    "molecules": "thymidine"
  },
  {
    "caption": "E, The expression of TNF receptor type 2 (TNFR2) was upregulated in growth-arrested epithelial cells with thymidine and P4 supplementation (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d groups, qPCR was performed in duplicate.",
    "molecules": "P4, thymidine"
  },
  {
    "caption": "A, In Rb1d/d mice with pre-implantation P4 treatment, the fragmented uterine epithelial cells with the cytoplasmic lipid droplets were engulfed by the trophoblast cells. Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; dotted line circle, lipid droplets in the cytoplasm; tr, trophoblast; le, luminal epithelium; s, stroma; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.",
    "molecules": "P4"
  },
  {
    "caption": "B, Pre-implantation P4 treatment rescued the expression of MFG-E8 on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm. Blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; le, luminal epithelium; s, stroma; arrowhead, embryo. C, Pre-implantation P4 treatment recovered the expression of pRIP3, a mediator of necroptosis, on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo. ",
    "molecules": "DAPI, P4"
  },
  {
    "caption": "Raji   cells expressing HIV-1YU-2 envelope  (Raji-Env) or Raji  control cells (Raji-Ctr) were incubated with 10-1074  or scFv10-1074  or with controls (only secondary antibody or streptavidin  added). Binding  of  10-1074 and scFv10-1074  was assessed by flow cytometry  using a BV421-conjugated anti-IgG Fc antibody or streptavidin, respectively. The numbers indicate the % of cells with bound antibody. One experiment is shown.",
    "molecules": "streptavidin"
  },
  {
    "caption": "Histological analysis of colon tumors was shown by hematoxylin and eosin (HE) staining. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Scale bars, 25 µm.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "GF mice were orally gavaged with C.albicans (twice a week, 1×107) during administration with AOM-DSS (n=8, each group). Mice were euthanized on Day 100, tumor load in each mouse were measured.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "GFP-C. albicans (5×106) were added onto 1×106 BMDMs, and incubated at 37°C for 1h. Wells were washed and fresh media containing fluconazole (300 μg/ml) was added. At 6 hours and 18 hours, C. albicans CFU inside BMDMs were photographed and calculated by plating on YPD agar. Scale bars, 25 µm.",
    "molecules": "fluconazole"
  },
  {
    "caption": "Mice were treated as described in Fig.2A. LP cells were isolated from each mouse and were culture for 48h. Cytokine and chemokine production of LP cells were detected using multiplex cytokine assay. Color from blue to red indicates enrichment of gene expression.",
    "molecules": "chemokine, Cytokine"
  },
  {
    "caption": "WT and Dectin-3-/- mice were intraperitoneally treated with anti-IL22 antibody or anti-IgG antibody as control during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, tumor number, tumor size, and tumor load in colons were measured.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "WT and Dectin-3-/- mice were intraperitoneally treated with anti-CD90 antibody or anti-IgG antibody during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, feces were collected in colon from each mouse. Total fungal burden in feces were quantified using 18S rDNA qPCR.",
    "molecules": "AOM, DSS"
  },
  {
    "caption": "BMDM cells were stimulated with C.albicans (5×106), Curdlan, and α-mannan for 24h. Glucose uptake, pyruvate level, lactate production, and ATP level were determined using assay kit.",
    "molecules": "Curdlan, α-mannan, ATP, Glucose, lactate, pyruvate"
  },
  {
    "caption": "BMDMs acquired from WT and Dectin-3-/- mice were stimulated with C.albicans for 24h. Cytokine and chemokine production of BMDMs were detected using multiplex cytokine assay.",
    "molecules": "chemokine, Cytokine"
  },
  {
    "caption": "WT-derived BMDMs were stimulated with C.albicans, curldan, or α-mannan in combination with or without 2-DG (2.5mM) for 24h. mRNA expression of Il-7 were detected by using qPCR. BMDMs were acquired from WT and HIF-1-/- mice and were stimulated with C.albicans, curldan, or α-mannan for 24h. mRNA expression of Il-7 were detected by using qPCR.",
    "molecules": "curldan, α-mannan, 2-DG"
  },
  {
    "caption": "Primary ILC3 cells were sorted from colonic LP cells and were stimulated with indicated cytokines. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 were detected by qPCR.",
    "molecules": "cytokines"
  },
  {
    "caption": "Primary ILC3 cells were sorted from LPs and were stimulated with IL-7 in combined with FIZC and AhR Inh. Cells without stimulation were used as control (None). Production of IL-22 was detected by ELISA.",
    "molecules": "FIZC"
  },
  {
    "caption": "Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins. Amino acids for the identified LIRs from positive signals are marked on each SNARE. Amino acids are denoted as amino acid single letter codes in the blot.",
    "molecules": "amino acid, Amino acids"
  },
  {
    "caption": "WT or STX17-knockout (STX17KO) HeLa cells were starved with or without the presence of bafilomycin A1 (Baf A1, 100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B and p62.   Quantifications of LC3B-II levels normalized to β-actin from cells treated as in (A); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; *, p < 0.05 (one-way ANOVA).   ",
    "molecules": "Baf A1, bafilomycin A1"
  },
  {
    "caption": "WT, STX16KO, STX17KO or STX16/STX17 double KO (STX16/STX17DKO) HeLa cells were starved with or without the presence of Baf A1 (100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B.   Quantifications of LC3B-II levels normalized to β-actin from (C); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; †, not significant; **, p < 0.01 (one-way ANOVA).   ",
    "molecules": "Baf A1"
  },
  {
    "caption": "WT or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.   Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).   ",
    "molecules": "antimycin A, CCCP, DNA, mtDNA, oligomycin A"
  },
  {
    "caption": "WT or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.   Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).   ",
    "molecules": "H2O2"
  },
  {
    "caption": "WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LysoTracker Red DND-99 (LTR) (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.   Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in (A). Data shown as means ± SEM of LTR puncta per cell or object total area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant (two-way ANOVA).   ",
    "molecules": "LTR, LysoTracker Red DND-99"
  },
  {
    "caption": "WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of the colocalization between LTR and TGN46. Masks: white, cells identified based on nuclei; red, LTR puncta; green, TGN46 puncta; yellow, overlap between LTR and TGN46. Scale bar: 20 µm.   Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in (C). Data shown as means ± SEM of LTR and TGN46 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).   ",
    "molecules": "LTR, LysoTracker Red"
  },
  {
    "caption": "WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.",
    "molecules": "LTR"
  },
  {
    "caption": "Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA).",
    "molecules": "LTR"
  },
  {
    "caption": "b. Multiplexed Error Robust Fluorescent in situ hybridization (MERFISH) and DAPI stained nuclei in the mouse hippocampus. Each gene is represented by a different color. For the entire hippocampus (left), only the mRNA spots are shown with a scale bar of 500 microns. On the zoomed-in section (right), each gene is represented by a different color dot, and the DAPI intensity is displayed in white. The scale bar is 20 microns.",
    "molecules": "DAPI, mRNA"
  },
  {
    "caption": "D-E. Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13). F. Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4). ",
    "molecules": "DMSO, LPS, Pinometostat"
  },
  {
    "caption": "D. Statistical analysis of serum titers of NP-specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge. Adjusted p-value were calculated using two-way ANOVA. Error bars indicate mean ± standard error of mean (SEM). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).*** denotes p- value < 0.0001 and ** denotes p- value < 0.001.",
    "molecules": "NP"
  },
  {
    "caption": "A. Representative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).",
    "molecules": "DMSO, LPS, Pinometostat"
  },
  {
    "caption": "B. Representative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).",
    "molecules": "DMSO, LPS, Pinometostat"
  },
  {
    "caption": "C. Representative flow cytometry plots showing the relative surface density for B220 (CD45R) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analysis of MFI of B220 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).",
    "molecules": "DMSO, LPS, Pinometostat"
  },
  {
    "caption": "(a) Ba/F3 cells expressing myrPI(3)K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3)K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(b) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ± s.e.m. normalized for B2M (n=4). *P0.05, **P0.01.",
    "molecules": "cytokine, 4-OHT"
  },
  {
    "caption": "(c) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).",
    "molecules": "cytokine, 4-OHT"
  },
  {
    "caption": "(d) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ±  s.e.m. values normalized for B2M (n=3). **P0.01.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(e) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(f) Wild-type Ba/F3 cells were cytokine starved overnight and stimulated with mIL-3. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).",
    "molecules": "cytokine"
  },
  {
    "caption": "(g) Wild-type Ba/F3 cells were incubated with LY294002 in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).",
    "molecules": "LY294002"
  },
  {
    "caption": "(h) FOXO1,3,4−/− MEFs and wild-type MEFs were incubated with LY294002 (10 μM) for the indicated times. Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR. Data are represented as mean values normalized for B2M (n=2). Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "LY294002"
  },
  {
    "caption": "(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and actinomycin D (1 μg ml−1) or dimethylsulphoxide (DMSO) as a control in the presence of mIL-3 for 16 h. Cell lysates were analysed for protein levels of glutamine synthetase (GS), p27 and actin. Shown are representative blots (n=3).",
    "molecules": "actinomycin D, 4-OHT, dimethylsulphoxide, DMSO"
  },
  {
    "caption": "(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.",
    "molecules": "4-OHT, glutamine, MSO"
  },
  {
    "caption": "(d) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=4).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(e) The osteosarcoma cell line U2OS expressing FOXO(A3)-ER was stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(f) MSCs expressing FOXO(A3)-ER were stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin. Shown are representative blots (n=3).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(g) Wild-type, daf-2 and daf-16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity.",
    "molecules": "hypochlorite, imidazole"
  },
  {
    "caption": "(h) Wild-type N2 worms or daf-2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF-16 double-stranded RNA. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity. (g,h) Shown are the means of five independent plates for each condition. Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "hypochlorite, imidazole"
  },
  {
    "caption": "(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for glutamine synthetase (GS) activity in an enzyme assay (left). In addition, the expression of glutamine synthetase, p27 and actin was determined using western blot (right). Shown are the mean ± s.e.m. (n=3) and representative blots of these experiments. **P0.01.",
    "molecules": "4-OHT"
  },
  {
    "caption": "(b) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT for 16 h in the presence of mIL-3 together with MSO, as indicated. Cell lysates were analysed for glutamine synthetase activity in an enzyme assay (left). Furthermore the expression levels of glutamine synthetase, p27 and actin were determined using western blot (right). Shown are the mean ±  s.e.m. (n=3) and representative blots of these experiments. *P0.05, **P0.01.",
    "molecules": "4-OHT, MSO"
  },
  {
    "caption": "(c) Ba/F3 cells expressing myrPI(3)K-ER were cytokine-starved overnight. The next day, cells were washed in PBS and put in a medium without serum, with or without 4-OHT. At the times indicated, medium samples were taken and analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho-PKB (S473), phospho-FOXO3 (T32) and actin (inset). Shown are the mean of relative amino acid levels, compared with t=0 (n=2), and representative blots of these experiments.",
    "molecules": "cytokine, 4-OHT, amino acid"
  },
  {
    "caption": "(d) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in serum-free medium containing mIL-3. The medium was analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and analysed for protein levels of glutamine synthetase, p27 and actin (inset). Shown are the mean ±  s.e.m. of relative amino acid levels, compared with t=0 (n=4), and representative blots of these experiments. **P0.01. Uncropped images of blots are shown in Supplementary Fig. S7.",
    "molecules": "4-OHT, amino acid"
  },
  {
    "caption": "(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO. After 24 h, the cells were starved of serum, amino acids and glucose in D-PBS containing the indicated inhibitors and stimulated with amino acids for 10 min. Cell lysates were analysed for protein levels of phospho-S6K (Thr 389) and actin. Shown are representative blots of three independent experiments.",
    "molecules": "4-OHT, amino acids, glucose, MSO"
  },
  {
    "caption": "(b,c) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO (0.25 M) for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy. (b) Shown are representative pictures (n=4). Scale bars, 100 μm. (c) Quantification of co-localization between mTOR and LAMP2 as seen in b. Depicted are the mean ±  s.e.m. of the percentage of co-localized pixels from four experiments. *P0.05, **P0.01.",
    "molecules": "4-OHT, glutamine, MSO"
  },
  {
    "caption": "(d) DLD1 cells expressing FOXO3(A3)-ER were treated with or without 4-OHT, MSO and BafA1 (200 nM). After 24 h, cell lysates were analysed for protein levels of LC3 and tubulin. Shown are representative blots (n=4).",
    "molecules": "4-OHT, BafA1, MSO"
  },
  {
    "caption": "(e) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase (GS) siRNA or a non-targeting siRNA and stimulated with 4-OHT in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of glutamine synthetase, LC3 and actin. Shown are representative blots (n=2)",
    "molecules": "4-OHT, glutamine"
  },
  {
    "caption": "(f) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetasesiRNA or non-template (NT) control siRNA and stimulated with 4-OHT for 16 h. Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase. Shown are representative blots (n=2).",
    "molecules": "4-OHT"
  },
  {
    "caption": "(g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.",
    "molecules": "PKB inhibitor VIII, LY294002"
  },
  {
    "caption": "(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and ER and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) Quantification of LC3-positive spots as seen in a using ImageJ software. Depicted are mean ±  s.e.m. of the number of LC3-positive spots divided by the number of DAPI-positive cells (n=4). *P0.05.",
    "molecules": "4-OHT, glutamine, MSO"
  },
  {
    "caption": "(c) MSCs expressing FOXO3(A3)-ER were treated with or without 4-OHT and BafA1 (100 nM). At the indicated time points, cell lysates were analysed for protein levels of glutamine synthetase (GS), LC3, pS6 (Ser 235/236) and actin. Shown are representative blots (n=2).",
    "molecules": "4-OHT, BafA1"
  },
  {
    "caption": "(d) IL-3 was withdrawn from Ba/F3 cells in the presence or absence of BafA1 (100 nM) as indicated. Cell lysates were analysed for protein levels of glutamine synthetase, LC3 and hsp90. Shown are representative blots (n=2).",
    "molecules": "BafA1"
  },
  {
    "caption": "(e) DLD1 cells were stimulated with l-glutamine (l-Gln) in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of LC3, p62, p-S6K (Thr 389) and actin. Shown are representative blots (n=3).",
    "molecules": "glutamine, l-glutamine"
  },
  {
    "caption": "(f) DLD1 cells were starved and stimulated with glutamine (5 and 10 mM). Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy, and the co-localization between mTOR and LAMP2 was quantified by ImageJ. Depicted are the means of the percentage of co-localized pixels (n=2).",
    "molecules": "glutamine"
  },
  {
    "caption": "(g) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently stimulated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS or medium without amino acids for 24 h. GFP-WIPI-1 puncta formation analysis was performed by confocal microscopy. Depicted are the percentages of cells positive for GFP-WIPI-1 puncta. Shown are the mean ±  s.e.m. (n=4). *P0.05 and **P0.01. Uncropped images of blots are shown in Supplementary Fig. S8.",
    "molecules": "4-OHT, amino acids, glutamine, MSO"
  },
  {
    "caption": "(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.",
    "molecules": "4-OHT, glutamine, MSO"
  },
  {
    "caption": "(b) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently treated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h, cells were analysed for GFP and p62 expression by confocal microscopy. Shown are representative pictures (n=3). Scale bar, 20 μm.",
    "molecules": "4-OHT, glutamine, MSO"
  },
  {
    "caption": "(c) DLD1 cells expressing FOXO3(A3)-ER were treated with 4-OHT and MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h cells were analysed for endogenous Atg12 puncta formation by confocal microscopy. Shown are the mean ± s.d. (n=3). *P0.05.",
    "molecules": "4-OHT, glutamine, MSO"
  },
  {
    "caption": "(d) DLD1 cells expressing FOXO(A3)-ER were transfected with glutamine synthetase (GS) siRNA or non-template (NT) control siRNA, and stimulated with 4-OHT for 24 h with or without 3-methyladenine (3-MA). Apoptosis was determined by FACS analysis after labelling cells with annexin V (AxV)-phycoerythrin and DAPI. The graph shows relative fold versus control samples, mean ±  s.e.m. (n=3). *P0.05.",
    "molecules": "3-methyladenine, 4-OHT"
  },
  {
    "caption": "C Conversion of GDP-D-mannose into GDP-L-fucose. The conversion GDP-D-[14C]mannose to GDP-L-[14C]fucose was analyzed in an in-vitro assay. After 1 h nearly half of GDP-D-mannose was transformed to GDP-fucose  in the control cell line, whereas the patient´s fibroblasts  were nearly unable to catalyze the reaction. Data were obtained from fibroblasts; n = 1.",
    "molecules": "GDP-D-[14C]mannose, GDP-D-mannose, GDP-fucose, GDP-L-[14C]fucose, GDP-L-fucose"
  },
  {
    "caption": "D Lectin binding studies and quantification in fibroblasts. Aleuria aurantia lectin (AAL) was used to address the fucosylation  level in  fibroblasts. AAL staining was performed before and after treatment of the patient  cell line with 100 µM L-fucose. Before sugar supplementation a significant loss of AAL signal strength was measured, whereas after treatment a significant re-fucosylation was detected. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD.",
    "molecules": "L-fucose"
  },
  {
    "caption": "E Complementation study with patient-derived fibroblasts. Viral infection was used to introduce an empty cloning vector and the wild-type GFUS cDNA in  patient cells, respectively. For analysis lysates of a control cell-line, a control cell-line supplemented with 100 µM fucose, patient  cells, patient cells supplemented with 100 µM fucose, patient cells transfected with an empty vector and patient  cells transfected with wild type GFUS  were analyzed by AAL staining clearly indicating the disease-causing influence of the defective GFUS in the patient. Western blot analysis against  GFUS further showed expression of wildtype-GFUS  protein in the infected patient  derived fibroblasts. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 2 times, for statistics an one-way ANOVA was performed. Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD. ",
    "molecules": "fucose"
  },
  {
    "caption": "A Aleuria aurantia lectin (AAL) was used to address the fucosylation level in sera of controls and the patient  (left) which revealed significant loss of fucose residues in case of the patient. Data were obtained from  serum; n = 6; experiment was independently repeated 3 times, for statistics an unpaired t test was performed. Data information: **p < 0.01. Bars and error bars represent mean ± SD. Exact p‑values are reported in the results part.",
    "molecules": "fucose"
  },
  {
    "caption": "B FACS analysis of  PBMCs. Results of flow cytometry  analysis of patient  (red) and control (green) PBMCs stained  with 1 µg/ml FAA lectin  without (dark) or with 200 mM L-fucose  (light) before the start of fucose therapy. C  FACS  analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained  with 1 µg/ml FAA lectin without (dark) or with 200 mM  L-fucose (light) 8 weeks after therapy start ",
    "molecules": "L-fucose"
  },
  {
    "caption": "D, E MAGE-A3/6 protein levels are actively degraded upon nutrient deprivation. HeLa cells were treated for the indicated times with complete media (DMEM + 10% FBS) or EBSS containing 100 µg/mL translation inhibitor cycloheximide (CHX). Protein degradation rates are shown (E). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01. ns indicates p>0.05 using 2-way ANOVA.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown.",
    "molecules": "NEAA, non-essential amino acids, glucose"
  },
  {
    "caption": "FBS (10%), but not non-essential amino acids (NEAA; 10 mM) or glucose (4.5 g/L), rescues MAGE-A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.",
    "molecules": "NEAA, non-essential amino acids, glucose"
  },
  {
    "caption": "D, E MAGE-A3/6 protein stability is controlled by a <3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal-dextran stripped FBS, 10% boiled FBS, 10% dialyzed >3 kDa FBS, or 10% dialyzed <3 kDa FBS for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.",
    "molecules": "charcoal, dextran"
  },
  {
    "caption": "F MAGE-A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/mL, testosterone, or bovine pituitary extract 25 µg/mL.",
    "molecules": "testosterone"
  },
  {
    "caption": "Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown.",
    "molecules": "NEAA, Insulin, insulin"
  },
  {
    "caption": ", H Insulin partially rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing insulin (10 µg/mL). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate ** p<0.01; *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.",
    "molecules": "Insulin, insulin"
  },
  {
    "caption": "A MAGE-A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT-QPCR analysis was performed to detect both MAGE-A3/6 with a common primer set. Data was normalized to 18S rRNA levels. Quantitation represents average of n=3 with standard deviation shown.",
    "molecules": "18S rRNA"
  },
  {
    "caption": "B, Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM).",
    "molecules": "BafA1, bafilomycin A1, MG132"
  },
  {
    "caption": "C Proteasome inhibition rescues MAGE-A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 µM). Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.",
    "molecules": "BafA1, bafilomycin A1, MG132"
  },
  {
    "caption": "D Nutrient deprivation increases ubiquitination of endogenous MAGE-A3/6. HeLa cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.",
    "molecules": "agarose, MG132"
  },
  {
    "caption": "F, MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times.",
    "molecules": "MLN4924"
  },
  {
    "caption": "G MAGE-A3/6 degradation upon nutrient starvation is dependent on a Cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 µM) that blocks the activity of Cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n=3 with standard deviation shown. Data information: Asterisks indicate *** p<0.001. ns indicates p>0.05 using 2-way ANOVA.",
    "molecules": "MLN4924"
  },
  {
    "caption": "B MAGE-A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc-MAGE-A3 and HA-DCAF5 or HA-DCAF12). Two days later cells were treated with complete media or EBSS for 4 hrs. Cells were treated in the presence of 1 µM MLN4924 to stabilize MAGE-A3. Cell extracts were subjected to anti-Myc IP.",
    "molecules": "MLN4924"
  },
  {
    "caption": "C Nutrient deprivation increases endogenous DCAF12 binding to MAGE-A3. HeLa cells were transfect with Myc-vector or Myc-MAGE-A3 for 48 hrs. Cells were then treated with 10 µM MG132 in complete media, EBSS, or EBSS containing 20 µg/mL insulin for 6 hrs. Cell extracts were subjected to anti-Myc IP.",
    "molecules": "insulin, MG132"
  },
  {
    "caption": "Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).",
    "molecules": "MLN4924"
  },
  {
    "caption": "E Knockdown of DCAF12, but not DCAF5, altered MAGE-A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control or DCAF12 siRNAs for 72 hrs before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 µM).",
    "molecules": "MLN4924"
  },
  {
    "caption": "G Ubiquitination of MAGE-A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 µM) in complete media or EBSS for 6 hrs. Cell extracts were incubated with control agarose or agarose-TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.",
    "molecules": "agarose, MG132"
  },
  {
    "caption": "b) Activation of the hybridoma as described in a, with the addition of dimethyl sulfoxide (DMSO; negative control), bafilomycin A (Baf), brefeldin A (BFA) or MG-132 at 2 h after macrophage infection.",
    "molecules": "bafilomycin, brefeldin A, DMSO, MG-132"
  },
  {
    "caption": "(c) Activation of the hybridoma as described in a, with the addition of bafilomycin A at 2 h after macrophage infection.",
    "molecules": "bafilomycin A"
  },
  {
    "caption": "(b) Activation of the gB-specific CD8+ T cell hybridoma (described in Fig. 1a) by macrophages infected for various times (horizontal axis) with HSV-1, with (DMEM + 3-MA) or without (DMEM) the addition of 3-methyladenine 2 h after infection, then incubated for 12 h at 37 °C with the hybridoma.",
    "molecules": "3-MA, 3-methyladenine"
  },
  {
    "caption": " (e) Activation of the hybridoma (as described in b) by macrophages infected with HSV-1 and incubated at 37 °C (Basal), incubated for 12 h at 39 °C before being infected with HSV-1 (heat shock (HS)), or treated with rapamycin during HSV-1 infection (Rapa), with (+ Baf) or without the addition of bafilmycin A at 2 h after infection. ",
    "molecules": "bafilmycin A, rapamycin"
  },
  {
    "caption": " (f) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control siRNA or Atg5-specific siRNA, then infected for 8 h with HSV-1, with (+) or without (−) the addition of rapamycin at 2 h after infection. Results in b,c,e,f are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 8 h at 37 °C without further treatment (b,e) or infected macrophages treated with control siRNA (c,f) and are presented in arbitrary units. Data are representative of three independent experiments (a,d) or are from three independent experiments (mean and s.e.m. of triplicate samples; b,c,e,f). ",
    "molecules": "rapamycin"
  },
  {
    "caption": " (a-c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3. ",
    "molecules": "rapamycin"
  },
  {
    "caption": " (i) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (below graph) with wild-type or Δ34.5 HSV-1, with (+) or without (-) the addition of bafilomycin A at 2 h after infection. Results in f,i are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 6 h with wild-type virus (f) or with infected macrophages incubated without bafilomycin (i) and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples). ",
    "molecules": "bafilomycin"
  },
  {
    "caption": " Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a-c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin-gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a-c) or two (d) independent experiments. ",
    "molecules": "gold"
  },
  {
    "caption": " (a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages exposed to DMSO (negative control), mild heat shock, IL-1β or IFN-γ. ",
    "molecules": "DMSO"
  },
  {
    "caption": " (c-f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages incubated at 37 °C (c) or exposed to mild heat shock (d), IL-1β (e) or IFN-γ (f) and infected for 8 h with wild-type HSV-1 with the addition of DMSO (negative control), 3-methyladenine, bafilomycin, brefeldin A or MG-132 at 2 h after infection. Results in a,c-f are normalized to results obtained for CD8+ T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples). ",
    "molecules": "3-methyladenine, bafilomycin, brefeldin A, DMSO, MG-132"
  },
  {
    "caption": "(a-b) eIF4A knockdown in either S2 (a) or Kc167 (b) cells blunts the inactivation of TORC1 upon amino acid removal. Cells were treated with dsRNA targeting GFP as a negative control, or three independent, non-overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes. Representative of 3 biological replicates.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "(d) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids, caused by removal of amino acid subsets. After 5 days of knockdown, S2 cells were treated for 30 min with either complete Schneider's medium (+aa), Schneider's medium lacking all amino acids (-aa) or various subsets of amino acids, as indicated (where EAA \"essential amino acids\" = H,I,L,K,M,T,W,V). Error bars: std dev. n=3 biological replicates.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "(e) Time course of amino-acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying (see drop in S6K and tubulin levels). Representative of two biological replicates.",
    "molecules": "amino-acid"
  },
  {
    "caption": "(f) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids. Control (w1118) or eIF4A1006/1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS/1% agarose+2%sucrose for 1h prior to lysis and immunoblot analysis. Two biological replicates are shown. Representative of 3 biological replicates.",
    "molecules": "agarose, amino acids, food, sucrose"
  },
  {
    "caption": "(a-a') Although knockdown of eIF4A or eIF3-S2 equally blunt translation of EGFP from an inducible plasmid (a), only knockdown of eIF4A but not eIF3-S2 impairs TORC1 inactivation upon amino acid removal (a'). (a) S2 cells, treated with indicated dsRNAs for 5 days, on day 3 transfected with an inducible EGFP plasmid (pMT-EGFP) and on day 4 induced for 18h. Non-targeting LacZ dsRNA used as a negative control. (a') S2 cells treated with indicated dsRNAs for 5 days, then incubated with medium lacking amino acids for indicated time prior to lysis. Representative of two biological replicates.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "(b) Knockdown of eIF4A, but not other translation initiation factors, blunts TORC1 inactivation upon amino acid withdrawal. Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider's medium or Schneider's medium lacking the indicated amino acids for 30 minutes prior to lysis.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "(c) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells. Kc167 cells treated with either eIF4A dsRNA for 4 days, or cycloheximide (50 µg/mL, \"CHX\") for 5 min prior to, as well as during removal of amino acids (-LIVASTQP). CHX treated cells still inactivate TORC1 (compare lanes 8 to 7) whereas eIF4A knockdown cells do not (lanes 14 vs 13). Quantifications of two biological replicates are shown. CHX and eIF4A samples were normalized to their respective control conditions.",
    "molecules": "amino acid, amino acids, CHX, cycloheximide"
  },
  {
    "caption": "(d) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min. Quantification of total intracellular amino acids, analyzed in a blinded fashion. Levels of individual amino acids shown in Figure S2D. For CHX samples, cycloheximide (50µg/mL) was added 5 minutes prior to, and during treatment with medium containing or lacking amino acids. Statistical significance tested by ANOVA2 using Scheffé's multiple comparisons method (p<0.05). Error bars: std. dev. n=5.",
    "molecules": "amino acids, CHX, cycloheximide"
  },
  {
    "caption": "(a) Knockdown of eIF4F components (eIF-4E and eIF-4G) partially phenocopy the eIF4A knockdown, leading to elevated TORC1 activity upon amino acid removal (30 min). Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal (main Figures 1c, 2a' and 2b). Error bars: std. dev. n=3 biological replicates.",
    "molecules": "amino acid"
  },
  {
    "caption": "(b) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown, explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity (panel a) are a bit milder than the eIF4A knockdown. Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click-it OPP reagent 30min before fixation and fluorescent labeling. Quantification of OPP fluorescence per cell (nuclear count) for two independent experiments is displayed (3 independent images per condition), normalized to the no dsRNA condition. Scale bars: 25µm.",
    "molecules": "OPP, protein"
  },
  {
    "caption": "(a) Binding of eIF4G to the Rag GTPases detected by co-immunoprecipitation from Kc167 cells in absence or presence of the chemical cross-linker DSP. Representative of >3 biological replicates.",
    "molecules": "DSP"
  },
  {
    "caption": "(c) Interaction between eIF4A and Raptor detected via co-immunoprecipitation of epitope-tagged proteins. Proteins were cross-linked with DSP prior to lysis and immunoprecipitation. Representative of 3 biological replicates.",
    "molecules": "DSP"
  },
  {
    "caption": "(e-f) The eIF4A-Raptor interaction does not take place on lysosomes (e) or the endoplasmic reticulum (ER) (f). Kc167 cells were either incubated with 100 µg/mL Dextran for 1 hour, washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes (e), or transfected to express an ER-resident GFP (f). Amino acid removal was for 45 min. Pearson's correlation coefficients for eIF4A-Raptor PLA and Dextran in panel (e) are 0.09 and 0.08 for +aa and -LIVASTQP respectively (calculated for all cells in the acquired fields, which were chosen randomly, n=40). Scale bars: 5µm.",
    "molecules": "Dextran, Amino acid"
  },
  {
    "caption": "(b) Activation of TSC2 with the p90RSK inhibitor BI-D1870 rescues the elevated TORC1 activity caused by eIF4A knockdown in Kc167 cells, consistent with TSC2 acting downstream of eIF4A. BI-D1870 leads to inhibition of TORC1 (lanes 1-4) in a TSC2-dependent manner (see also Appendix Figure S5A). Representative of two biological replicates.",
    "molecules": "BI-D1870"
  },
  {
    "caption": "(c) FLAG-eIF4A and TSC2-V5 co-immunoprecipitate in Kc167 cells in the absence of chemical cross-linker. Cells were transfected with FLAG-eIF4A and TSC2-V5 expression vectors and treated with media containing or lacking amino acids for indicated timepoints prior to lysis and anti-FLAG immunoprecipitation. The experiment was performed in absence of the chemical cross-linker DSP. Representative of 3 biological replicates.",
    "molecules": "DSP, amino acids"
  },
  {
    "caption": "(d) Expression of TSC2-insensitive (S15H or Q63L), but not wild-type (WT) Rheb causes TORC1 activity to remain high upon amino acid removal. Kc167 cells were transfected to express either wild-type or mutant Rheb and then incubated with Schneider's medium either containing or lacking amino acids for 30 minutes. Elevated TORC1 activity can be observed either by looking at phosphorylation of endogenous S6K, or phosphorylation of an HA-tagged S6K that was co-transfected with the Rheb constructs to assay specifically the transfected cells. Representative of two biological replicates.",
    "molecules": "amino acid, amino acids"
  },
  {
    "caption": "(a) Mass spectrometry analysis of proteins co-immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins. eIF4A immunoprecipitations were performed in triplicate, from cells treated with medium either containing (+aa) or lacking amino acids (-aa), and average values are shown. Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts. Raw peptide counts shown in parentheses.(a') Schematic diagram of eIF4G and NAT1 primary protein structures. Binding sites for other initiation factors are shown.",
    "molecules": "amino acids"
  },
  {
    "caption": "(b) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1-independent fashion. Co-immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times. Representative of 3 biological replicates.",
    "molecules": "amino acid, amino acids, rapamycin"
  },
  {
    "caption": "(c) Knockdown of NAT1 using 4 independent, non-overlapping dsRNAs leads to reduced TORC1 activity. Kc167 cells, treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.",
    "molecules": "amino acids"
  },
  {
    "caption": "(d) eIF4A is epistatic to NAT1 for TORC1 regulation. In the absence of amino acids, eIF4A knockdown cells have elevated TORC1 activity, NAT1 knockdown cells have reduced TORC1 activity, and eIF4A & NAT1 double-knockdown cells have elevated TORC1 activity compared to control cells. Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.",
    "molecules": "amino acids"
  },
  {
    "caption": "(A-C) Distribution of spindle pole factors in centrosomal and acentrosomal spindles. DMSO-treated control mitotic spindles (2-centrosomes) and centrinone-treated acentrosomal spindles (0-centrosome) of HeLa cells. (A) Red, gray, green, and blue represent NuMA, α-tubulin, Cep152, and DNA, respectively. Z-projections of eight sections, every 0.3 μm. Scale bar, 5 μm. (B) Red, gray, and blue represent NuMA, Katanin p60, and DNA, respectively. Z-projections of five sections, every 0.3 μm. Scale bar, 5 μm. (C) Red, gray, and blue represent p150glued, ASPM, and DNA, respectively. Z-projections of 10 (2-centrosomes) or five (0-centrosome) sections, every 0.3 μm. Scale bar, 5 μm.",
    "molecules": "centrinone, DNA, DMSO"
  },
  {
    "caption": "(D) The structure of NuMA and microtubules in centrinone-treated acentrosomal spindles of HeLa cells. Two-way arrows indicate the bipolarity of elongated NuMA structures. Gray, red, green and blue represent α-tubulin, NuMA, Cep152 and DNA, respectively. Scale bar, 5 μm.",
    "molecules": "centrinone, DNA"
  },
  {
    "caption": "(E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm.",
    "molecules": "centrinone, DNA"
  },
  {
    "caption": "(G) Representative images of NuMA (arrowheads) in centrosomal and acentrosomal cells before and after photobleaching. DMSO- or centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed. Time after photobleaching (sec) is indicated. Scale bar, 5 μm.",
    "molecules": "centrinone, DMSO"
  },
  {
    "caption": "(B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.",
    "molecules": "Centrinone, DNA"
  },
  {
    "caption": "(D) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells. Two-way arrow indicates the bipolarity of elongated NuMA structures. Red, gray, green, and blue represent NuMA, CENP-C, GT335, and DNA, respectively. Scale bar, 5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(E, F) Time-lapse observation of the structure of NuMA and microtubules upon centrosome removal. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed with a 60× objective. Red and green represent NuMA and SiR-tubulin, respectively. Z-projections of 17 sections, every 1 μm. Scale bar, 5 μm. Time zero corresponds to NEBD. (F) An example of NuMA formed several asters (arrowheads) at the time of NEBD.  ",
    "molecules": "Centrinone"
  },
  {
    "caption": "(A) NuMA structure upon microtubule depolymerization. Nocodazole-treated HCT116 CMV-OsTIR1 CEP152-mClover-mAID cells were treated with 100 ng/ml nocodazole. Gray, red, green and blue represent α-tubulin, NuMA, CEP152 (mClover) and DNA, respectively. Scale bar, 5 μm.",
    "molecules": "DNA, Nocodazole, nocodazole"
  },
  {
    "caption": "(B) Immunostaining of DHC1 and NuMA in centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells. Gray, green, red and blue represent DHC1 (mClover), GT335, NuMA, and DNA, respectively. Scale bar, 5 μm.",
    "molecules": "centrinone, DNA"
  },
  {
    "caption": "(D) NuMA structure upon DHC1 depletion. Centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM indole-3-acetic acid (IAA). Arrowhead indicates the spindle pole. Green, red, gray and blue represent DHC1 (mClover), NuMA, GT335 and DNA, respectively. Scale bar, 5 μm. (E) Frequency of acentrosomal pole patterns upon DHC1 depletion in (D). Values are mean percentages ± SD. from three independent experiments (N≧24 spindles in each experiment). ( ",
    "molecules": "Centrinone, DNA, Dox, doxycycline, IAA, indole-3-acetic acid"
  },
  {
    "caption": "(F) The distribution of NuMA upon DHC1 depletion. Arrowheads indicate the presence of NuMA on microtubules. Green, red, and blue represent α-tubulin, NuMA, and DNA, respectively. Scale bars, 5 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(G) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA-mAID-mClover-FLAG with either mCherry-NuMA WT or 5A-3. Arrowheads indicate the assembled NuMA structure. Green, red, gray and blue represent endogenous NuMA (mClover), expressed NuMA (mCherry), GT335 and DNA, respectively. Z-projections of 21 sections, every 1 μm. Scale bar, 5 μm. (H) The number of NuMA structure in (G). Values are mean percentages ± SD. from four independent experiments (N≧15 spindles in each experiment). One-way ANOVA with Tukey's multiple comparisons test was used to obtain a P value.  ",
    "molecules": "DNA"
  },
  {
    "caption": "(A, B) Structure of the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM IAA. Two-way arrow indicates the bipolarity. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (C) Frequency of spindle structure patterns upon NuMA depletion in (A, B). Values are mean percentages ± SD. from three independent experiments (N=30 spindles in each experiment). ( ",
    "molecules": "Centrinone, DNA, Dox, doxycycline, IAA"
  },
  {
    "caption": "(D) The localization of Eg5 in centrinone-treated acentrosomal HeLa cells. Red, gray, green and blue represent NuMA, Eg5, GT335 and DNA, respectively. Scale bar, 5 μm.",
    "molecules": "centrinone, DNA"
  },
  {
    "caption": "(E) The localization of Eg5 in the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with Dox and 500 μM IAA. Gray, green, red, and blue represent Eg5, NuMA (mClover), CP110, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.",
    "molecules": "Centrinone, DNA, Dox, IAA"
  },
  {
    "caption": "(F) Structure of the spindle upon Eg5 inhibition. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 30 μM monastrol. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm. (G) Frequency of spindle structure patterns upon Eg5 inhibition in (F). Values are mean percentages ± SD. from three independent experiments (N≧23 spindles in each experiment).  ",
    "molecules": "Centrinone, DNA, monastrol"
  },
  {
    "caption": "(A) Time-lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed in the presence of 50 μM of monastrol with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads and two-way arrows indicate the monopolar and bipolar states, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (B) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (A). Each plot shows the cumulative percentage of each event at each time point (N=total 75 cells from two independent experiments). ( ",
    "molecules": "monastrol"
  },
  {
    "caption": "(C) Time-lapse observation of the dynamics of mClover-NuMA and chromosomes. HeLa cells expressing mClover-NuMA were observed in the presence of SiR-DNA with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads indicate initial bipolarity in the acentrosomal spindle pole (8 min), dispersed chromosomes (328 min), and the monopolar-like state of the acentrosomal spindle pole (332 min), respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (D) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (C). Each plot shows the cumulative percentage of each event at each time point (N=total 35 cells from three independent experiments).  ",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Structure of the spindle upon NuMA and Eg5 inhibition. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline (Dox), 500 μM IAA and monastrol. Green, red, and blue represent NuMA (mClover), α-tubulin, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (B) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in (A). Values are mean percentages ± SD. from three independent experiments (N≧22 spindles in each experiment). ( ",
    "molecules": "DNA, Dox, doxycycline, IAA, monastrol"
  },
  {
    "caption": "(C) The localization of Eg5 in the spindle upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline Dox and 500 μM IAA. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), Eg5 and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.",
    "molecules": "DNA, Dox, doxycycline, IAA"
  },
  {
    "caption": "(D) Time-lapse observation of the establishment of spindle bipolarity upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml Dox and 500 μM IAA and observed with a 40× objective. Red and green represent SiR-tubulin and NuMA, respectively. Z-projections of 14 sections, every 2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (E) The time required for the initial establishment of spindle bipolarity in (D). Line and error bars represent the mean and SD. (N≧60 cells from two independent experiments). The Mann-Whitney U test (two-tailed) was used to obtain a P value.  ",
    "molecules": "Dox, IAA"
  },
  {
    "caption": "D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from (B). Tub1 served as a loading control.",
    "molecules": "DNA"
  },
  {
    "caption": "A MET3pr‐CDC20cdc15‐as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM‐PP1 to inhibit Cdc15‐as1. Cells were released from the metaphase arrest by Cdc20 re‐induction (left graph), or Cdc14 and/or Sic1 expression were induced from the GAL1 promoter while cells remained arrested (right graph). The fraction of large‐budded cells after spheroplastation (n = 100) was quantified. Representative photographs 120 min after galactose addition are shown. Scale bar, 10 μm.",
    "molecules": "1NM‐PP1, galactose"
  },
  {
    "caption": "B Western blot analysis of cells expressing conditional Clb2m (+) or Clb2mΔCdk (Δ) fusions to the indicated proteins, before (−) and 5 h after (+) β‐estradiol treatment. Tub1 served as a loading control. c, control cells.",
    "molecules": "β‐estradiol"
  },
  {
    "caption": "C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins. Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates, with red dots representing individual and bars representing average values. Bnr1 fusions were included as controls for cytokinetic defects. Student's t‐test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains. ‐P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.",
    "molecules": "DNA"
  },
  {
    "caption": "D Serial dilutions of cells from (C) were plated 6 h following β‐estradiol treatment and grown for 1 day at 36°C.",
    "molecules": "β‐estradiol"
  },
  {
    "caption": "A Genes corresponding to the indicated proteins were replaced by wild‐type (wt) or Cdk phospho‐site mutant (xA) sequences and then conditionally fused to Clb2m or Clb2mΔCdk. Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C. The individual values from two independent clones (red dots) as well as their average (bars) are shown. Student's t‐test was applied to compare matched wt and xA strains. c, control cells.",
    "molecules": "DNA"
  },
  {
    "caption": "C The indicated strains were spotted in serial dilutions onto auxin‐containing plates and grown for 2 days.",
    "molecules": "auxin"
  },
  {
    "caption": "D Auxin was added to cultures of the indicated strains, and after 6 h, cells were sonicated and the number of attached cell bodies was counted (n > 200). Significance was calculated using a Wilcoxon rank‐sum test.",
    "molecules": "Auxin"
  },
  {
    "caption": "E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis (n = 3). Error bars represent SD.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) HOIP, HOIL-1L, and SHARPIN co-localize with Htt-Q97 aggregates. SH-SY5Y cells co-expressing Htt-Q25-GFP or Htt-Q97-GFP (green) and HOIP, HOIL-1L, SHARPIN, or HHARI (red); DAPI (blue). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "HOIP co-localizes with Htt inclusion bodies in human HD frontal cortex. Perinuclear Htt aggregates visualized by DAB immunohistochemistry (C) in human HD frontal cortex. Scale bar, 15 µm (C).",
    "molecules": "DAB"
  },
  {
    "caption": "M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured SH-SY5Y cells (A) Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 20 µm (A)",
    "molecules": "polyQ, lipofuscin, M1-linked ubiquitin"
  },
  {
    "caption": "M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured R6/2 mouse cortex and striatum (B) Scale bar 10 µm",
    "molecules": "polyQ, M1-linked ubiquitin"
  },
  {
    "caption": "M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured human HD frontal cortex (C). Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 10 µm",
    "molecules": "polyQ, lipofuscin, M1-linked ubiquitin"
  },
  {
    "caption": "(D) Htt-Q25- or Htt-Q97-expressing HEK293T were analyzed by filter retardation assays. As indicated, the M1 ubiquitin antibody was preincubated with recombinant tetra-ubiquitin linked via M1 (4 x M1 ubiquitin) or K63 (4 x K63 ubiquitin).",
    "molecules": "K63, K63 ubiquitin, M1, M1 ubiquitin, tetra-ubiquitin"
  },
  {
    "caption": "(C) Htt-Q97-GFP (green) was co-expressed with HA-tagged WT or HOIP mutants (red) in SH-SY5Y cells and analyzed by immunocytochemistry using an antibody against HA. DAPI (blue). Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) The interaction of soluble Htt-Q60 with p97/VCP is dependent on HOIP. WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt-Q60-HA. 48 h after transfection cells were lysed with 1% Triton X-100, followed by an immunoprecipitation of Htt-Q60 via the HA tag. Immunopurified proteins were detected by Western blotting using an anti-VCP and anti-HOIP antibody.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "(F) SDS-soluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with Htt-Q60-GFP (as a control for specific immunoprecipitation of HA-tagged Htt-Q60), Htt-Q60-HA or Htt-Q60-3R (3 lysine residues replaced by arginines) and the plasmids indicated. Cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of Htt via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3 and anti-HA.",
    "molecules": "arginines, polyQ, lysine, SDS, Triton X-100, linear ubiquitin, M1 ubiquitin"
  },
  {
    "caption": "(H) SDS-insoluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells expressing Htt-Q97-HA or Htt-Q97-GFP were lysed under denaturing conditions in 1.5% SDS. After centrifugation, the pellets containing the SDS-insoluble aggregates (SDS-insoluble fraction) were dissolved in formic acid. Formic acid-dissolved aggregates were analyzed by immunoblotting using the M1 ubiquitin-specific 1F11/3F5/Y102L antibody.",
    "molecules": "formic acid, Formic acid, polyQ, SDS, linear ubiquitin, M1 ubiquitin"
  },
  {
    "caption": "(I) Endogenous NEMO co-localizes with Htt-Q97 aggregates. NEMO (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm. (J) Endogenous Optineurin co-localizes with Htt-Q97 aggregates. Optineurin (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) HOIP is recruited to disease-associated protein aggregates formed by Ataxin-3-Q84, SOD1 G85R, TDP-43 Q331K or Optineurin R96L. SH-SY5Y cells co-expressing disease-associated proteins (green) and HOIP (red) were analyzed by immunocytochemistry; DAPI (blue). Scale bar, 20 µ",
    "molecules": "DAPI"
  },
  {
    "caption": "Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in SH-SY5Y cells using the 1E3 antibody (Millipore) (B) DAPI (blue). Scale bar, 20 µm.",
    "molecules": "DAPI, Linear ubiquitin, M1-linked ubiquitin"
  },
  {
    "caption": "Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in primary striatal neurons using the 1F11/3F5/Y102L antibody (Genentech) (C). DAPI (blue). Scale bar, 20 µm.",
    "molecules": "DAPI, Linear ubiquitin, M1-linked ubiquitin"
  },
  {
    "caption": "(D) SDS-soluble SOD G85R and TDP-43 Q331K species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with disease-associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA. 48 h after transfection cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of SOD G85R and TDP-43 Q331K via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3.",
    "molecules": "SDS, Triton X-100, linear ubiquitin, M1 ubiquitin"
  },
  {
    "caption": "(D) Catalytically active HOIP decreases the number of cells with Htt-Q97 aggregates independently of autophagy. WT and ATG5 KO MEFs were transiently transfected with Htt-Q97-GFP and either control vector (CO), HOIP or HOIP C885A. Cells were treated 24 h after transfection with the proteasomal inhibitor MG132 (1 µM, 16 h). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 7. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.",
    "molecules": "MG132"
  },
  {
    "caption": "(F) Catalytically active HOIP decreases the number of cells with TDP-43 aggregates dependent on proteasomal degradation. SH-SY5Y cells were transfected with TDP-43 Q331K and either control vector (CO), HOIP or HOIP C885A. Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 (1 µM) or the p97/VCP inhibitor NSM-873 (1 µM) for 3 h. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.",
    "molecules": "MG132, NSM-873"
  },
  {
    "caption": "(G) Inhibition of the proetasome and p97/VCP do not have additive effects on the degradation of misfolded TDP-43 species. SH-SY5Y cells expressing TDP-43 Q331K were treated 48 h after transfection with MG132 (1 µM) and/or NSM-873 (1 µM) for 3 h. All data are mean ± SD, data were analyzed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.",
    "molecules": "MG132, NSM-873"
  },
  {
    "caption": "(C) Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1-478, 554-900) and hCAP-H (residues 394-515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6) or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506-514 residues were deleted from 394-515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS-PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9).",
    "molecules": "resin, agarose, Ni, NTA"
  },
  {
    "caption": "(C) Add-back assay using the WT and mutant condensin I complexes. Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes (from top to bottom; WT, IV-5Q, ∆G, ∆G[IV-5Q]). The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes. The samples were fixed and labeled with an antibody against mSMC4 (red). DNA was counterstained with DAPI (blue). The data from a single representative experiment out of two repeats are shown. In the experiment shown here, multiple images were collected for condensin-depleted extracts supplemented with the WT (n = 17), IV-5Q (n = 22), ∆G (n = 20) and ∆G(IV-5Q) (n = 25) complexes. The scale bar represents 10 μm.",
    "molecules": "DAPI, DNA"
  },
  {
    "caption": "(B) Double-stranded DNA (dsDNA) and single-stranded DNA binding assay of the hCAP-G. 30-bp dsDNA was incubated with no protein (lanes 1) or increasing amounts of WT hCAP-G (WT; lanes 2-4). 30-mer ssDNA was incubated with no protein (lanes 5) or increasing amounts of WT hCAP-G (WT; lanes 6-8).",
    "molecules": "Double-stranded DNA, dsDNA, single-stranded DNA, ssDNA"
  },
  {
    "caption": "(C) The dsDNA binding assay for the hCAP-G-H subcomplexes. 30-bp dsDNA was incubated with no protein (lanes 1, 5 and 9), increasing amounts of WT hCAP-G-H subcomplex (WT; lanes 2-4), CAP-G K60D/R848E double mutant (K60D/R848E; lanes 6-8) or CAP-G R168E mutant (R168E; lanes 10-12).",
    "molecules": "dsDNA"
  },
  {
    "caption": "(D) The ssDNA binding assay for the hCAP-G-H subcomplexes",
    "molecules": "ssDNA"
  },
  {
    "caption": "Hydroxyproline assay (Cell biolabs Inc.) for total hydroxyproline activity of cells. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "hydroxyproline"
  },
  {
    "caption": "Western blot indicating levels of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination.",
    "molecules": "DPCA"
  },
  {
    "caption": "Quantification of immunofluorescence staining of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. H1299RASSF1A cell line was used as negative control for comparison. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "DPCA"
  },
  {
    "caption": "Invasion of H1299control and H1299RASSF1A cells through three-dimensional collagen matrix coated inserts, over 24 hours (n=3). Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "collagen"
  },
  {
    "caption": "Representative images and quantification of H1299 cells treated with siNT, siRNA against P4HA2 or in the presence of the prolylhydroxylase inhibitor DPCA (4μM), and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "DPCA, Matrigel"
  },
  {
    "caption": "Representative images and quantification of HOP92 cells stably transfected with shcontrol or shRASSF1A and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "Matrigel"
  },
  {
    "caption": "3D collagen contraction assay: 5x105/ml cells were embedded into collagen rat tail I matrix (2mg/ml) and analysed after 4 days. White circles indicates diameter of gel plugs at time 0. Representative bright-field images (bottom) with quantification, showing the effect of H1299RASSF1A or H1299control cells on collagen gel remodelling and after cells pre-treatment with siRNA against P4HA2, 4μM DPCA or combination as indicated. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "DPCA"
  },
  {
    "caption": "Upper image: Cartoon of hanging drops method for spheroids formation and embedding collagen rat tail I matrix (2mg/ml). Bottom: Representative immunofluorescence images of YAP distribution and its nuclear quantification (right bars) by Pearson's coefficient for correlation of YAP and DAPI co-staining in H1299control and H1299RASSF1A 3D spheroids grown in 3D collagen matrix (2mg/ml). Scale bars 10µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative immunofluorescence images of YAP distribution (right) and its nuclear quantification by Pearson's coefficient for correlation (left) of YAP and DAPI co-staining in H1299control and H1299RASSF1A plated on extracted ECM from H1299control or H1299RASSF1A cells with or without treatment with siP4HA2 or 4μM DPCA. (Correlation, Pearson's coefficient). (n=3, 300 cells per experiment) Scale bars 10µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.",
    "molecules": "DPCA, DAPI"
  },
  {
    "caption": "Representative atomic force microscopy (AFM) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices (2mg/ml).",
    "molecules": "collagen"
  },
  {
    "caption": "Second harmonic generation (SHG) representative Images and Quantification (bars) showing deposition and organization of collagen fibres produced by H1299control and H1299RASSF1A spheroids embedded in collagen I matrix (2mg/ml), treated with siNT, siRNA against P4HA2 or 4μM DPCA to restrict P4HA2 activity. Red arrowheads show highly organized, long collagen fibres. Scale bars 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.",
    "molecules": "DPCA"
  },
  {
    "caption": "Representative H&E and quantification (bars) of picrosirius red staining of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm, zoom 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.",
    "molecules": "picrosirius red"
  },
  {
    "caption": "Left: Representative images of H1299 cells on 3D collagen wells with defined stiffness with NANOG (green) or DAPI (blue). Scale bars 10µm. Right: Quantification of NANOG:DAPI nuclear co-localization (upper graph) and total NANOG fluorescence intensity (bottom graph). Quantification of nuclear co-localization (n=300cells/experiment) is represented by Pearson's coefficient. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.",
    "molecules": "collagen, DAPI"
  },
  {
    "caption": "Representative immunofluorescence images show merge of NANOG (green), DAPI (blue) distribution in H1299control and H1299RASSF1A cells cultured on 2D glass. Scale bars 10µm. Right: Quantification of nuclear co-localization (n=200cells/experiment) is represented by Pearson's coefficient.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative immunofluorescence images of CD133 in H1299control and H1299RASSF1A cells grown on 3D collagen wells with defined stiffness. Scale bars 10µm. Bottom: Quantification of n=200 cells/experiment. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.",
    "molecules": "collagen"
  },
  {
    "caption": "Representative images of immunofluorescence staining for the pluripotency marker NANOG and β-catenin in three-dimensional spheroids embedded in collagen matrix (2mg/ml). Scale bars 50µm.",
    "molecules": "collagen"
  },
  {
    "caption": "Representative immunofluorescence images of NANOG and YAP in the presence of siNT or siYAP in H1299control and H1299RASSF1A cells embedded and grown in three-dimensional collagen matrix (2mg/ml). Scale bars 10µm.",
    "molecules": "collagen"
  },
  {
    "caption": "Representative immunofluorescence images of H1299control and H1299RASSF1A spheroids embedded and grown in collagen matrix (2mg/ml) and stained for NANOG (Green) and YAP (Red) with or without 4μM DPCA. Scale bars 10µm.",
    "molecules": "DPCA, collagen"
  },
  {
    "caption": "Western blots of H1299control lysates for NANOG, SOX2 and OCT4 in the presence of siNT, siP4HA2 or 4μM DPCA.",
    "molecules": "DPCA"
  },
  {
    "caption": "Representative images of immunofluorescence staining of spheroids grown in collagen matrix (2mg/ml) for differentiation markers TTF-1 (Green) and Mucin 5B (Red). Scale bars 50µm, Zoom 10µm.",
    "molecules": "collagen"
  },
  {
    "caption": "Bright field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three-dimensional substrate. Scale bars 200µm.",
    "molecules": "Matrigel"
  },
  {
    "caption": "HeLa  cells co-transfected with EMTB-CFP-FRB  and YFP-FKBP  were treated with 100 nM rapamycin  (Rapa). Addition of rapamycin  rapidly translocated YFP-FKBP  onto EMTB-CFP-FRB-labeled microtubules  and increased the FRET  signal. The scale for the FRET/CFP  intensity ratio is shown. Scale bar, 10 μm. The normalized intensity of FRET/CFP  in cells before and after rapamycin  (blue) and 0.1% DMSO  (control; red) treatment. n = 6 and 10 cells in the rapamycin  and DMSO  groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.",
    "molecules": "DMSO, Rapa, rapamycin"
  },
  {
    "caption": "HeLa  cells co-transfected with the indicated constructs were treated with nocodazole  (Noc, 3.3 µM; left) or rapamycin  (Rapa, 100 nM; right) for 0, 30, and 60 min. Transfected cells were then fixed and labeled with anti-acetylated tubulin  (red). Dotted lines indicate the boundary of transfected cells. Scale bars, 10 μm. The intensity of acetylated tubulin  in cells transfected with EMTB-CFP-FRB  (orange) or cotransfected with EMTB-CFP-FRB and dNSpastin3Q-YFP-FKBP  (blue) after addition of nocodazole  (Noc, 3.3 µM) or rapamycin  (Rapa, 100 nM) for the indicated times. n = 123 and 81 cells in the nocodazole  and dNSpastin3Q groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M. Student's t-tests were performed, with p-values indicated.",
    "molecules": "Noc, nocodazole, Rapa, rapamycin"
  },
  {
    "caption": "NIH3T3 fibroblasts  co-transfected with 5HT6-mCherry  (5HT6-mCh; a ciliary membrane  marker; red), CFP-FRB-MAP4m  (blue), and dNSpastin3Q-YFP-FKBP  (green) were serum-starved for 24 hr to induce ciliogenesis. Ciliated  cells were then treated with rapamycin  (Rapa, 100 nM) to induce dNSpastin3Q-YFP-FKBP  recruitment to  axonemal microtubules. Dotted lines indicate the cell boundaries. Scale bar, 5 μm. The normalized length of axonemes  (blue), primary cilia (red), and dNSpastin3Q-YFP-FKBP  in cilia  (green) upon rapamycin  treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": "HeLa  cells co-transfected with H2B-mCherry  (H2B-mCh; a chromosome  marker; red), CFP-FRB-MAP4m  (a marker of mitotic spindles; blue), and dNSpastin3Q-YFP-FKBP  (green) were synchronized in metaphase  and treated with rapamycin  (100 nM) to induce dNSpastin3Q-YFP-FKBP  recruitment to  mitotic spindles. Dotted lines indicate the cell boundaries. Scale bar, 10 μm. The normalized area of the mitotic spindle  (blue) and intensity of dNSpastin3Q-YFP-FKBP  in the mitotic spindle  (green) upon rapamycin  treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.",
    "molecules": "rapamycin"
  },
  {
    "caption": "HeLa  cells co-transfected with H2B-mCherry  (red), CFP-FRB-MAP4m  (intercellular bridges; blue), and dNSpastin3Q-YFP-FKBP  (green) were treated with rapamycin  (100 nM) to induce dNSpastin3Q-YFP-FKBP  recruitment to intercellular bridges. Arrows indicate the  intercellular bridges. Dotted lines indicate the cell boundaries. Scale bar, 10 μm.   The normalized area of intercellular bridges (blue) and intensity of dNSpastin3Q-YFP-FKBP  at intercellular bridges  (green) upon rapamycin  treatment. n = 5 cells from three independent experiments. Data are shown as the mean ± S.E.M.  ",
    "molecules": "rapamycin"
  },
  {
    "caption": "COS7  cells co-transfected with TagRFP-FRB-A1AY1   (red) and TagCFP-FKBP   (green) were treated with rapamycin  (100 nM). The addition of rapamycin  rapidly recruits TagCFP-FKBP  from cytosol  onto A1AY1-labeled microtubules  (arrows). Scale bar, 10 µm. The normalized intensity of TagCFP-FKBP  at A1AY1-labeled microtubules  upon rapamycin  treatments. n=7 cells from three independent experiments. Data are shown as the mean ± S.E.M.",
    "molecules": "rapamycin"
  },
  {
    "caption": "COS7  cells co-transfected with TagRFP-FRB-A1AY1   (red) and dNSpastin3Q-TagCFP-FKBP  (cyan) were treated with 0.1% DMSO  or rapamycin  (100 nM) for 1 hr and followed by immunostaining  with anti-tyrosinated tubulin  antibody (green). Dotted lines highlight the transfected cells. Scale bar, 20 μm. The normalized intensity of tyrosinated microtubules  in TagRFP-FRB-A1AY1  and dNSpastin3Q-TagCFP-FKBP  co-transfected cells upon 0.1% DMSO  or rapamycin  treatment for 1 hr. n=31 and 26 cells in DMSO  and rapamycin  treated groups, respectively, from three independent experiments. Data (blue) represent as mean ± S.E.M. Student's t-tests were performed with p values indicated.",
    "molecules": "DMSO, rapamycin"
  },
  {
    "caption": "COS7  cells co-transfected with EMTB-YFP-CIBN  and mCh-Cry2  (red) were incubated with SPY650-tubulin    (SPY650-tub) to visualize microtubules. The cells were illuminated by  blue light within a specified region (indicated by the dotted circle) for the indicated time period. Scale bar, 10 μm. The normalized intensity of mCh-Cry2  at microtubules  in illuminated regions (red) and non-illuminated regions (green). n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.",
    "molecules": "SPY650"
  },
  {
    "caption": "COS7  cells co-transfected with EMTB-YFP-CIBN  and dNSpastin3Q-mCh-Cry2  were incubated with SPY650-tubulin   to visualize microtubules. The cells were illuminated by  blue light as in (A). Scale bar, 10 μm. The normalized intensity of dNSpastin3Q-mCh-Cry2  and mCh-Cry2  at microtubules  and the normalized area of SPY650-tubulin   in illuminated regions and non-illuminated regions. n = 6 and 6 cells in dNSpastin3Q-mCh-Cry2  and mCh-Cry2  groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.",
    "molecules": "SPY650"
  },
  {
    "caption": "E-F. COS7  cells co-transfected with EMTB-CFP-FRB, dNSpastin3Q-mCh-FKBP, and Tom20-Neon   were treated with rapamycin  (100 nM) for acute microtubule disruption. The morphology  of mitochondria  at different levels of microtubule  disruption is shown. Scale bar, 10 µm. The ratio of the long axis to the short axis of each mitochondrion  and microtubule filament area was determined. n= 4 different cells, three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.",
    "molecules": "rapamycin"
  },
  {
    "caption": "G-H. The normalized intensity of MitoTracker Red  in non-transfected cells (Mock) or cells co-transfected with EMTB-CFRP-FRB  and dNSpastin3Q-YFP-FKBP  or dNSpastin3QED-YFP-FKBP  after rapamycin  (Rapa, 100 nM) treatment for the indicated time. Scale bar, 10 µm. n = 41, 6, and 18 cells from three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.",
    "molecules": "MitoTracker Red, Rapa, rapamycin"
  },
  {
    "caption": "COS7  cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP  (red), and Lifeact-Neon  (heatmap and green) were treated with rapamycin  (Rapa, 100 nM) to induce microtubule  disassembly. Arrows and arrowheads indicate lamellipodia  and  stress fibers, respectively. Insets show images of lamellipodia morphology  from regions corresponding to the dotted squares with overexposed CFP and mCherry  signals. Scale bar, 10 µm. The normalized level of microtubule  area, lamellipodia  length, and filopodia  density of COS-7 cells treated as in (A). n = 4 cells from three independent experiments. Data are shown as the mean ± S.E.M.",
    "molecules": "Rapa, rapamycin"
  },
  {
    "caption": "E. Analysis of GFP-IDH1 acetylation  by immunoprecipitation  with the GFP  antibody followed by immunoblotting  with the Ace-lysine antibody. HEK293T  cells were treated with tubacin, tubastatin A  (TubA), trichostatin A (TSA), or sodium butyrate (NaB) (E).",
    "molecules": "tubacin, NaB, sodium butyrate, trichostatin A, TSA, TubA, tubastatin A"
  },
  {
    "caption": "Immunoprecipitation  and immunoblotting  showing the level of IDH1 acetylation  in HEK293T  cells transfected with wild-type GFP-IDH1  or various mutants, These cells were treated with the HDAC6  inhibitor, tubacin",
    "molecules": "tubacin"
  },
  {
    "caption": "C. Analysis of IDH1  activity in HEK293T  cells treated with the indicated concentration of tubacin  (n = 6-9 tests from three independent experiments). D. Analysis of the α-KG  level in HEK293T  cells treated with the indicated concentration of tubacin  (n = 4 independent experiments). Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA test",
    "molecules": "α-KG, tubacin"
  },
  {
    "caption": "K. Representative images of the colony formation assay. Scale bar, 200 µm. L. Number of colonies formed by wild-type and Hdac6  knockout mice bone marrow cells treated or untreated with 10 µM GSK321  (n = 6 mice  per group). Data information: All values are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 ANOVA test",
    "molecules": "GSK321"
  },
  {
    "caption": "qPCR  showing that knockdown of HDAC6  significantly upregulates the expression of TET2  target genes with hyper-hydroxymethylation. qPCR  showing that treatment with 10 µM GSK321  inhibits the upregulation of the indicated genes in HDAC6-knockdown HEK293T  cells. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).",
    "molecules": "GSK321"
  },
  {
    "caption": "qPCR  showing that treatment with 10 µM GSK321  inhibits the upregulation of the indicated genes in HDAC6-knockdown HEK293T  cells. Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (ANOVA test).",
    "molecules": "GSK321"
  },
  {
    "caption": "isolates.BALB/c mice and Wistar rats were immunized with various doses of PiCoVacc or control (adjuvant only as the sham group) (n=10). Serums from recovered COVID19 patients (RECOV) and non-infected people (NI) act as positive and negative controls, respectively. The antibody responses were analyzed in mice (A), humans (B) and rats (C). Top: SARS-CoV-2-specific IgG response measured by ELISA",
    "molecules": "adjuvant"
  },
  {
    "caption": "primates.Macaques were immunized three times through the intramuscular route with various doses of PiCoVacc or adjuvant only (sham) or placebo (n=4). SARS-CoV-2-specific IgG response (A) and neutralizing antibody titer (B) were measured. Points represent individual macaques; dotted lines indicate the limit of detection; horizontal lines indicate the geometric mean titer (GMT) of EC50 for each group. Protective efficacy of PiCoVacc against SARS-CoV-2 challenge at week 3 after immunization was evaluated in macaques",
    "molecules": "adjuvant"
  },
  {
    "caption": "Histopathological examinations (F) in lungs from all the inoculated macaques at day 7 post infection. Lung tissue was collected and stained with hematoxylin and eosin.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "primates.Macaques were immunized three times at day 0, 7 and 14 through the intramuscular route with low dose (1.5 μg per dose) or high dose (6 μg per dose) of PiCoVacc or adjuvant only (sham) or placebo. (A and B) Hematological analysis in all four groups of macaques (n=4). Lymphocyte subset percents (A), including CD3+, CD4+ and CD8+ were monitored at day −1 (1 day before vaccination), 18 (3 day after the second vaccination) and 29 (7 day after the third vaccination). Key cytokines (B), containing TNF-α, IFN-γ and IL-2 were examined at day −1, 1 (the day for the first vaccination), 4, 18 and 29 after vaccination. Values are mean ± s.d.",
    "molecules": "adjuvant"
  },
  {
    "caption": "(C) Histopathological evaluations in lungs from four groups of macaques at day 29. Lung tissue was collected and stained with hematoxylin and eosin.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(A, B and C)  Polyphenols TA , PGG and EGCG activate  rhpR-lux  in wild type but not rhpS  deletion Psph  strains. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).",
    "molecules": "EGCG, PGG, Polyphenols, TA"
  },
  {
    "caption": "(D, E and F) Polyphenols repress  T3SS induction in P. syringae in MM. Transcripts of the indicated genes in the presence of 100-μM TA (D), 100-µM PGG (E) or 2-mM EGCG (F) were determined by RT-qPCR  6 hours after culturing in  MM. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).",
    "molecules": "EGCG, PGG, Polyphenols, TA"
  },
  {
    "caption": "(D, E and F)  Polyphenols inhibit the phosphatase activity  of RhpS. The GST-RhpR was phosphorylated by adding 5-μM   PSPPH_3550c protein as well as 10-μCi [γ-32P]-ATP for 10 minutes. The  RhpS-FL protein was treated in the presence of different concentrations of TA (D), PGG (E), or EGCG (F) for 10 min. Then, the full-length  RhpS protein was added into the mixture phosphorylated GST-RhpR for 1 hour before stopping the reaction. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE  gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results. Data information: the reaction was stopped by adding 5 × loading buffer before SDS-PAGE. Abbreviation is as follows: CBB, coomassie bright blue stain; CFU, colony-forming unit.",
    "molecules": "EGCG, PGG, ATP, CBB, coomassie bright blue stain, γ-32P, Polyphenols, TA"
  },
  {
    "caption": "(I, J and K) Polyphenols inhibits the  virulence levels and in planta growth of  Psph in  bean plants. Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants. Disease symptoms were recorded 5 days after inoculation. Leaf  disks (1 cm2) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).",
    "molecules": "Polyphenols, polyphenols"
  },
  {
    "caption": "(B, C and D) Polyphenols bind  to the  RhpS-FL protein. The concentration of  RhpS-FL protein was consistently measured as 0.6-μM. The  binding  curves of TA (B), PGG (C) and EGCG (D) are shown. The individual data points were shown (n=2 biological replicates).",
    "molecules": "EGCG, PGG, Polyphenols, TA"
  },
  {
    "caption": "(E) Alanine-scanning mutagenesis of the RhpS sensor. RhpS containing a corresponding mutation was provided in the  rhpS deletion strain on a pHM1 vector. LUX activity was estimated in the absence (black frame) and presence  TA  (50-μM), PGG (100-μM) or EGCG (2-mM). Error bars represent standard deviations (n=3 biological replicates). Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001).",
    "molecules": "EGCG, PGG, Alanine, TA"
  },
  {
    "caption": "B Following treatment with 10 mM metformin overnight, NIH3T3 reporter cells were heat-shocked at 43°C for 30 min and recovered at 37°C for 5 h. GFP induction was quantitated by flow cytometry. HS: heat shock.",
    "molecules": "metformin"
  },
  {
    "caption": "C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting.",
    "molecules": "metformin"
  },
  {
    "caption": "D, E Following heat shock and recovery for 4 h, mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). β-actin was used as the internal control.",
    "molecules": "metformin"
  },
  {
    "caption": "F Reporter cells were treated with 10 mM metformin overnight, and viability was measured using Guava ViaCount® reagents (mean ± SD, n = 5, n.s., not significant, Student's t-test).",
    "molecules": "metformin"
  },
  {
    "caption": "G Left panel, graphic depiction of the ELISA-based DNA binding assay. HSE: heat shock element. Right panel: Immediately following heat shock at 43°C for 30 min, nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). Experimental details are described in Materials and Methods.",
    "molecules": "metformin"
  },
  {
    "caption": "I HEK293T cells stably expressing a scramble or HSF1-targeting shRNA were treated with 10 mM metformin overnight and heat-shocked at 43°C for 30 min. Cells were fixed for PLA to detect HSF1-DNA interactions. Experimental details are described in Materials and Methods. Scale bars: 50 μm for low magnification and 10 μm for high magnification.",
    "molecules": "DNA, metformin"
  },
  {
    "caption": "J Immediately following heat shock, cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting. LDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. C: cytoplasm; N: nucleus.",
    "molecules": "metformin"
  },
  {
    "caption": "K Reporter mice (ventral side shown) were pre-treated with metformin for 3 days, 2 mg/mouse/day, via i.p. injection. Following a single i.p. injection of velcade (5 mg/kg) and recovery for 6 h, whole-body luciferase activities were quantitated by bioluminescence imaging. The photon flux of each mouse was expressed as photon/s/cm2/steradian (right panel, mean ± SD, n = 3, one-way ANOVA, *P < 0.05; **P < 0.01).",
    "molecules": "velcade, metformin, photon"
  },
  {
    "caption": "A-C Reporter cells were treated with and without 1 μM A-769662 for 3 h followed by heat shock at 43°C for 30 min and recovery at 37°C for 5 h. Hspa1a and Hspb1 mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001). ACC phosphorylation was detected by immunoblotting (C).",
    "molecules": "A-769662"
  },
  {
    "caption": "Following viral transductions, Hsp mRNA levels were quantitated by qRT-PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43°C heat shock for 30 min and recovery for 4 h (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).",
    "molecules": "metformin"
  },
  {
    "caption": "G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids: the HSF1-dependent reporter pHSE-SEAP, in which ideal HSEs drive the expression of secreted alkaline phosphatase (SEAP), and the control reporter pCMV-Gaussia Luc, in which a CMV promoter drives the expression of secreted Gaussia luciferase. In HEK293T cells, either LacZ or GST-AMPKα1CA plasmid was co-transfected with the two reporter plasmids. After 24 h, the transfected cells were heat-shocked at 44°C for 45 min. The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock. SEAP signals were normalized to Gaussia luciferase signals (mean ± SD, n = 5, Student's t-test, **P < 0.01, ***P < 0.001).",
    "molecules": "Gaussia, Gaussia luciferase, GST"
  },
  {
    "caption": "H, I Following treatment with 10 mM metformin overnight, endogenous AMPKα and HSF1 proteins were co-precipitated using anti-AMPKα agarose conjugates from lysates of immortalized Hsf1+/+MEFs (H).",
    "molecules": "metformin"
  },
  {
    "caption": "A In HEK293T cells that stably express an shRNA targeting the 3′ UTR of human HSF1, HSF1WT or HSF1S121A was expressed. Following treatment with 10 mM metformin overnight, the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho-HSF1 Ser121 antibody (A8041, Assay Biotechnology).",
    "molecules": "metformin"
  },
  {
    "caption": "B Primary Ampkα1fl/fl; Ampkα2fl/fl MEFs were transduced with adenoviral GFP or Cre. Following treatment with 10 mM metformin overnight or treatment with 10 μM A-769662 for 3 h, HSF1 Ser121 phosphorylation was examined by immunoblotting.",
    "molecules": "A-769662, metformin"
  },
  {
    "caption": "C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.",
    "molecules": "GST"
  },
  {
    "caption": "D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight. Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His-tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C (CC). HSF1 Ser121 phosphorylation was detected by immunoblotting.",
    "molecules": "His, AMP, compound C, metformin"
  },
  {
    "caption": "E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ or AMPKα1CA (E). Following expression of HSF1WT or HSF1S121A, cells were treated with and without 10 mM metformin overnight (F) (mean ± SD, n = 5, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).",
    "molecules": "metformin"
  },
  {
    "caption": "G, H FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with LacZ or AMPKα1CA plasmids into HSF1-deficient HEK293T cells for 3 days (G). Following transfection with FLAG-HSF1WT or FLAG-HSF1S121A plasmids for 3 days, HSF1-deficient HEK293T cells were treated with and without 10 mM metformin overnight (H). Nuclear proteins were extracted to measure HSF1-DNA binding by the ELISA-based DNA binding assay using anti-FLAG antibodies (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).",
    "molecules": "DNA, metformin"
  },
  {
    "caption": "I, J Following transfections and metformin treatment as described in (G) and (H), cytoplasmic and nuclear proteins were extracted for immunoblotting using anti-FLAG antibodies.",
    "molecules": "metformin"
  },
  {
    "caption": "A, B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine (A) or 4.5 g/l glucose (B). Following heat shock at 43°C for 30 min and recovery for 5 h, GFP induction was quantitated by flow cytometry.",
    "molecules": "glucose, leucine"
  },
  {
    "caption": "C, D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA-based DNA binding assay (mean ± SD, n = 3, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001).",
    "molecules": "DNA, nutrient"
  },
  {
    "caption": "E, F Following nutrient deprivations overnight, the reporter cells were heat-shocked at 43°C for 30 min and recovered overnight. Individual proteins were detected by immunoblotting.",
    "molecules": "nutrient"
  },
  {
    "caption": "G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.",
    "molecules": "nutrient"
  },
  {
    "caption": "G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.",
    "molecules": "nutrient"
  },
  {
    "caption": "D, E Immediately following heat shock at 43°C for 40 min, HEK293 cells were subjected to nutrient deprivations for 4 h. AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting.",
    "molecules": "nutrient"
  },
  {
    "caption": "F-H HSF1WT or HSF1S121A plasmids were transfected into HSF1-deficient HEK293T cells. Transfected cells were heat-shocked at 43°C for 40 min. HSP mRNAs were quantitated by qRT-PCR following overnight recovery. HSF1-DNA binding was measured immediately after heat shock (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001).",
    "molecules": "DNA"
  },
  {
    "caption": "A, B Following treatment with 10 μM metformin for 3 days, the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP (mean ± SD, n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001). Normal rabbit IgG served as the negative control (A). Following the same treatment, HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001) (B).",
    "molecules": "metformin"
  },
  {
    "caption": "C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).",
    "molecules": "metformin"
  },
  {
    "caption": "C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).",
    "molecules": "metformin"
  },
  {
    "caption": "E Following treatment with 10 μM metformin for 7 days, protein levels in WM115 and S462 cells were measured by immunoblotting.",
    "molecules": "metformin"
  },
  {
    "caption": "F, G Following treatment with 10 μM metformin for 7 days, levels of polyubiquitinated proteins were detected in both detergent-soluble and detergent-insoluble fractions in WM115 and A2058 cells using a Lys48-specific polyubiquitin antibody.",
    "molecules": "metformin"
  },
  {
    "caption": "H A2058 cells were treated with 10 μM metformin alone or co-treated with 2 μM compound C for 7 days. Individual proteins were detected by immunoblotting.",
    "molecules": "compound C, metformin"
  },
  {
    "caption": "I HEK293T cells stably expressing either scramble or HSF1-targeting shRNAs were treated with 10 μM metformin for 7 days. Individual proteins were detected by immunoblotting.",
    "molecules": "metformin"
  },
  {
    "caption": "J In HEK293T cells, a plasmid encoding HA-polyQ79-GFP was co-transfected with the indicated plasmids. Following treatment with and without 10 μM metformin for 5 days, the sizes of aggregates in detergent-insoluble fractions were quantitated by a Multisizer™ 3 Coulter Counter. Experimental details are described in Materials and Methods.",
    "molecules": "metformin"
  },
  {
    "caption": "K, L Immortalized and RAS-transformed MEFs were treated with either 10 μM metformin (K) or different concentrations of glucose (L) for 7 days. Individual proteins were detected by immunoblotting.",
    "molecules": "glucose, metformin"
  },
  {
    "caption": "M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.",
    "molecules": "glucose, polyQ"
  },
  {
    "caption": "A, B A2058 cells were transduced with lentiviral scramble or HSF1-targeting (hA6 and hA9) shRNAs. (A) Cell numbers were quantitated by Hoechst 33342 DNA staining (mean ± SD, n = 4, two-way ANOVA, ***P < 0.001). (B) Protein levels were measured by immunoblotting.",
    "molecules": "DNA"
  },
  {
    "caption": "C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4.5, 1.0, or 0.45 g/l glucose. Following treatment with 10 μM metformin, cell proliferation was measured by Hoechst 33342 DNA staining (mean ± SD, n = 5, two-way ANOVA, ***P < 0.001).",
    "molecules": "DNA, glucose, metformin"
  },
  {
    "caption": "D, E 1 × 106 LacZ- or HSF1-expressing A2058 cells were transplanted into female NOD/SCID mice. One day after transplantation, metformin was administered via drinking water at 1 mg/ml. Tumor incidence (log-rank test; D) and volumes (E) were measured (mean ± SEM, two-way ANOVA n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time (DT).",
    "molecules": "metformin"
  },
  {
    "caption": "H A2058 cells stably expressing FLAG-HSF1WT or FLAG-HSF1S121A were treated with 10 μM metformin for 7 days. Protein levels were measured by immunoblotting.",
    "molecules": "metformin"
  },
  {
    "caption": "I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning®ultra-low attachment 96-well culture plates with 10 μM metformin and different concentrations of glucose for 7 days, 10,000 cells per well. Viable cells were counted by Guava flow cytometer using ViaCount® reagent (mean ± SEM, n = 8, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).",
    "molecules": "glucose, metformin"
  },
  {
    "caption": "E Distribution of endogenous WIPI2 and STING in MEFs. F Statistical analysis of the number of STING- and WIPI2-positive puncta in cells treated as in (E). G Distribution of endogenous ATG16L1 in HEK293T cells stably expressing STING-HA. H Statistical analysis of the number of STING- and ATG16L1-positive puncta in cells treated as in (G). All the cells were treated with or without cGAMP for 2 h. All statistical data are presented as mean ± SEM of three independent experiments.",
    "molecules": "cGAMP"
  },
  {
    "caption": "A Co-immunoprecipitation of endogenous STING with WIPI1/2-Myc in MEFs transfected with Myc-tagged WIPI1 or WIPI2. The cells were treated with or without cGAMP for 2 h and the WIPI1/2-Myc precipitates were analyzed by western blot using anti-STING. B Co-immunoprecipitation of endogenous WIPI2 with HA-tagged STING, STING-R238A or STING-S366A from HEK293T cells stably expressing STING-HA STING-R238A-HA or STING-S366A-HA. The cells were treated with or without cGAMP for 2 h.",
    "molecules": "cGAMP"
  },
  {
    "caption": "J Purified GST-tagged STING-153-379 or STING-153-379-3A-2 was incubated with recombinant WIPI2 or the indicated WIPI2 mutants, and then precipitated with glutathione sepharose beads. The bound WT or mutant WIPI2 was detected by western blot using anti-WIPI2. K Statistical analysis of (J).",
    "molecules": "sepharose, glutathione"
  },
  {
    "caption": "A WIPI2-GFP puncta in MEFs stably expressing GFP-tagged WT or mutant WIPI2. The cells were treated with cGAMP for 2 h or Torin1 for 1 h. B Statistical analysis of the number of WIPI2-GFP puncta in cells treated as in (A).",
    "molecules": "cGAMP, Torin1"
  },
  {
    "caption": "F LC3 and p62 level in HEK293T cells stably expressing HA-tagged WT or mutant STING. The cells were treated with or without cGAMP for 4 h.",
    "molecules": "cGAMP"
  },
  {
    "caption": "C Co-immunoprecipitation of endogenous WIPI2 with STING-HA from HEK293 cells stably expressing STING-HA with or without BECN1 deletion. The cells were treated with or without Torin1 for 3 h.",
    "molecules": "Torin1"
  },
  {
    "caption": "E Co-immunoprecipitation of endogenous STING with Myc-tagged WT or mutant WIPI2 from MEFs treated with or without VPS34-IN1 for 2 h.",
    "molecules": "VPS34-IN1"
  },
  {
    "caption": "G GFP-LC3 punctum formation in MEFs stably expressing GFP-LC3 and STING-HA. The cells were treated with or without cGAMP or VPS34-IN1 for 2 h. Scale bars, 10 µm.",
    "molecules": "cGAMP, VPS34-IN1"
  },
  {
    "caption": "I Western blot assay of p62 and phospho-TBK1 (Ser172) in MEFs. The cells were pretreated with or without VPS34-IN1 for 1 h and then co-treated with cGAMP or Torin1 for another 4 h along with or without CQ. CQ, chloroquine. J Statistical analysis of p62 protein level in cells treated as in (I).",
    "molecules": "cGAMP, chloroquine, CQ, Torin1, VPS34-IN1"
  },
  {
    "caption": "A, B Localization of endogenous WIPI2, WIPI1, ATG16L1 (A) or Myc-tagged WT WIPI2 or WIPI2 mutants (B) in MEFs transfected with Cy3 labeled-ISD (1 μg/ml). ISD, interferon stimulatory DNA.",
    "molecules": "interferon stimulatory DNA, ISD, Cy3"
  },
  {
    "caption": "D Statistical analysis of cytoplasmic DNA puncta in cells treated as in (C).",
    "molecules": "DNA"
  },
  {
    "caption": "F Statistical analysis of cytoplasmic DNA puncta in cells treated as in (E).",
    "molecules": "DNA"
  },
  {
    "caption": "A Wipi2- or Sting-deleted MEFs were treated with Ara-C for 12 h, and then cultured in Ara-C-free medium. After 24 h, the cells were lysed and analyzed by western blot using the indicated antibodies. B Statistical analysis of the protein level of phospho-TBK1 (Ser172) and STING in cells treated as in (A). The relative phospho-TBK1 level was normalized to TBK1, the relative STING level was normalized to Actin.",
    "molecules": "Ara-C"
  },
  {
    "caption": "C-E MEFs with Wipi2 or Sting deletion (C), Wipi2-KO MEFs with transfection of Myc-tagged WT or mutant WIPI2 (D), or Sting-KO MEFs with transfection of HA-tagged WT or mutant STING (E), were treated with Ara-C for 12 h and then cultured in Ara-C-free medium for another 24 h. Total mRNA was extracted and the Ifnb1 mRNA level was measured by real-time PCR and normalized to Actb mRNA. 2RE, WIPI2-R108E/R125E.",
    "molecules": "Ara-C"
  },
  {
    "caption": "F Sting-KO MEFs with transfection of HA-tagged WT or mutant STING were treated with cGAMP for the indicated time. The cells were lysed and analyzed by western blot using the indicated antibodies.",
    "molecules": "cGAMP"
  },
  {
    "caption": "A-D PBMCs from vaccine breakthrough cases (n=15) and close contacts (n=26) were examined for T cell responses. PBMCs were left unstimulated (Unstim), were stimulated with pooled SARS-CoV-2 PepTivator® S, S1, M and N peptides for 6 hours (SARS-CoV-2 Peptide Stim), or non-specifically stimulated with phorbol 12-myristate 13-acetate (PMA), then assessed by high-dimensional flow cytometry. CD4+ T cells positive for intracellular staining of IFNγ (a), IL-2 (b), TNF (c), or CD8+ T cells positive for intracellular staining of granzyme B (d) are shown. Error bars indicate median and interquartile range.",
    "molecules": "phorbol 12-myristate 13-acetate, PMA"
  },
  {
    "caption": "Western blot showing co-immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent (Triton X-100). Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial (VDAC and Tom20), endoplasmic reticulum (calnexin), and lysosomal markers (LAMP1). ctr: control. n = 2.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Measurement of mitochondrial ROS production (using Mitosox) in different HD models. Results are presented as percent of control [Q23, normal fibroblast (Nor), Q7]. n = 3. *P = 0.001; **P = 0.0001; ***P = 0.0001; #P = 0.0045.",
    "molecules": "ROS"
  },
  {
    "caption": "ATP levels in HD patient-derived fibroblast cells. n = 2. *P = 0.003; **P = 0.004.",
    "molecules": "ATP"
  },
  {
    "caption": "Representative immunofluorescence of normal and HD patient-derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox. Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson's correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS. #P = 0.03; ##P = 0.02; *P = 0.006; **P = 0.005. Images were acquired at 63× magnification, and brightness and contrast of images were adjusted by 50%.",
    "molecules": "ROS"
  },
  {
    "caption": "Sub-mitochondrial localization of GAPDH and expanded polyglutamine repeats. Isolated mitochondria were left untreated or subjected to the proteinase K treatment in the presence or absence of detergent (Triton X-100). n = 3.",
    "molecules": "proteinase K, Triton X-100"
  },
  {
    "caption": "Determination of mitochondrial ROS production in different HD models. Results are shown as percent of control. n = 3. *P = 0.008; #P = 0.054; **P = 0.03.",
    "molecules": "ROS"
  },
  {
    "caption": "ATP levels in normal and HD patient-derived fibroblasts. The difference in ATP levels between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in HD patient-derived fibroblasts was not statistically significant. n = 2.",
    "molecules": "ATP"
  },
  {
    "caption": "Representative Western blot showing levels of mitochondrial proteins (aconitase and Tom20) in the total lysates of PC12 cells. The cells were treated with 1 mM of 3-methyladenine (3MA) for 5 h prior to the micro-mitophagy reconstitution assay. n = 2. *P = 0.001; **P = 0.01; *#P = 0.003; **#P = 0.01; #P = 0.02; §P = 0.049; #§P = 0.004; ##§P = 0.007.",
    "molecules": "3-methyladenine, 3MA"
  },
  {
    "caption": "Example time trace when no binding event occurs (green laser excitation). EmCCD image shows signal from green and red channels before the addition of Cy3-labelled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.   Example time trace showing a binding event (green laser excitation). EmCCD image shows signal from green and red channels after the addition of Cy3-labeled DNA when illuminated with green laser. Size of EmCCD image is 25µm x 50 µm.   ",
    "molecules": "Cy3, DNA"
  },
  {
    "caption": "Example time traces when DNA containing 3 PAM sequences is added (top) and when DNA containing a single PAM is added showing both short and long binding events (bottom).",
    "molecules": "DNA"
  },
  {
    "caption": "Bar plot of all dwelltime values for all constructs showing short binding events for all DNA and long events only for the constructs containing PAM sequences. The times shown are mean values of dwelltimes obtained during four different experiments on four different days. Error bars represent standard error of the mean.",
    "molecules": "DNA"
  },
  {
    "caption": "Example dwelltime histograms from negative control and a construct containing a single PAM. Negative control dwelltime distribution is characterized by a single exponential decay (top) and dwelltime distribution for PAM-containing DNA is fitted by a double-exponential decay (blue line) (bottom). Equation for the double exponential decay fit: y=A1e-t/τ1 + A2e-t/τ2; ΔA1= 796.4 ΔA2= 28.3. Red line shows what a single-exponential decay fit for such distribution would be. Errors represent standard error of the mean.",
    "molecules": "DNA"
  },
  {
    "caption": "Scatter plot showing the average dwelltime (Δτav) values for the cases of 2 (d2) and 4 (d4) nucleotide separation between the PAM sites. The values are averages of four measurements over four different days for each case. Error bars represent the standard error of the mean. The average dwelltime was obtained using the equation τav=(A1Δτ12+A2τ22)/(A1Δτ1+A2Δτ2)",
    "molecules": "nucleotide"
  },
  {
    "caption": "Example trace showing direct FRET transitions for a 5-PAM containing DNA construct with separation between PAM sites being 4 nucleotides.",
    "molecules": "DNA, nucleotides"
  },
  {
    "caption": "Histogram showing the percentage of events that are longer than τ1 showing FRET fluctuations for multiple PAM constructs with PAM separation of 4 nucleotides. The values are averages of four measurements over four different days. The error bars represent standard error of the mean.",
    "molecules": "nucleotides"
  },
  {
    "caption": "An example trace showing the addition of Cy3-labeled DNA target and the first binding event. Δτon is indicated.",
    "molecules": "Cy3, DNA"
  },
  {
    "caption": "Scatter plot showing the on-target binding rate (kon-target) for each construct. Binding rate decreases moderately with increasing number of adjacent PAMs. The values are averages of four measurements over three days. Error bars represent standard error of the mean. kon-target was calculated using the equation kon-target=1/ (CxΔτon) where C is the molarity of DNA target in the channel.",
    "molecules": "DNA"
  },
  {
    "caption": "(H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.",
    "molecules": "HA, DAPI, Phalloidin"
  },
  {
    "caption": "All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.B) Western blot analysis with protein extracts obtained from ex vivo-purified intestinal crypts and probed with the indicated antibodies.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "A) PAS and (B) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "C) Quantification (mean ± SD) of Goblet cells in crypts and villi from the same mice sown in A and B. More than 100 crypts and villi were scored for each condition after 15 days from the first β-naphthoflavone administration.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "D) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice injected with β-naphthoflavone and sacrificed after 15 days using a lysozyme-specific antibody (upper panels: LYZ, a Paneth cell-specific marker). Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels (ALPI, an enterocyte-specific marker). Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels (Alcian/KI67).",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "E) GFP expression from an Lgr5-eGFPtransgene identifying LGR5+ ISCs in near-native agarose-embedded sections from Eed+/+ and Eed-/- mice at 30 days from the first β-naphthoflavone administration. Cell nuclei were counterstained with DAPI.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "A) Radiation-induced intestinal regeneration: AhCre Eed+/+ and AhCre Eedfl/fl mice were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 4 and 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (n=6).",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "D) In vitro organoids formation using crypts isolated from AhCre Eed+/+ and AhCre Eed-/- mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in AhCre Eed-/- crypts relative to AhCre Eed+/+ crypts 15 days after β-naphthoflavone administration.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "E) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "F) Expression analysis by qRT-PCR (mean ± SD) of both Cdkn2a isoforms (p16Ink4A and p19Arf) in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5).",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemistry analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.",
    "molecules": "β-naphthoflavone"
  },
  {
    "caption": "C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (Atoh1, Gfi1, Spdef), early secretory precursor cells (Dll1), NOTCH targets (Hes1, Olfm4) and intestinal stem cells (Lgr5, Rnf43) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4).",
    "molecules": "DBZ, DMSO"
  },
  {
    "caption": "(B) Representative immunoblot analysis of CALHM6 expression in BMDM and BMDC stimulated for 6 or 24 h with Poly(I:C) (50 μg/ml) or LPS (100 ng/ml) (one representative experiment of two is shown, n = 2 mice).",
    "molecules": "LPS, Poly(I:C)"
  },
  {
    "caption": "(G and H) Calhm6 mRNA expression measured by qPCR in naïve or IFN-γ (10 ng/ml) primed (6 h) BMDM stimulated with (G) Poly(I:C) (50 μg/ml) (one representative experiment of two is shown, n = 2 mice, biological replicates), or (H) Zymosan (100 μg/ml) for 2 to 24h (one representative experiment is shown, n = 3 mice, biological replicates).",
    "molecules": "Zymosan, Poly(I:C)"
  },
  {
    "caption": "(F-J) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS for 3 h (F-H) or 12 h (I-J), spleens were collected and processed as in (A-E). Representative contour plots (F, I), pooled frequency of IFN-γ+ in CD3-NK1.1+ cells of WT vs Calhm6-/- mice (G,J) and ratio of IFN-γ+ cells that are NK1.1+ or CD3+ normalised as in C (H) (For Poly(I:C) 3 h: pooled results from two independent experiments, Poly(I:C) WT mice = 10, Calhm6-/- mice = 10, control WT mice = 2, Calhm6-/- mice = 2, One way ANOVA with multiple comparisons. For Poly(I:C) 12 h: results from one experiment, Poly(I:C) WT mice = 6, Calhm6-/- mice = 5, control WT mice = 1, Calhm6-/- mice = 1 mice, One-way ANOVA with multiple comparisons).",
    "molecules": "PBS, Poly(I:C)"
  },
  {
    "caption": "(A-B) WT and Calhm6-/- mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS. On day 3 spleens were collected, stained with antibodies against NK cell-maturation markers and analysed by flow cytometry. Representative contour plots (A) and pooled flow cytometry results (B) are shown (results from one experiment, Poly(I:C) WT mice = 5, Calhm6-/- mice = 5, control WT mice = 2, Calhm6-/- mice = 2 mice, One-way ANOVA with multiple comparisons).",
    "molecules": "PBS, Poly(I:C)"
  },
  {
    "caption": "(F) NO production in WT and Calhm6-/- BMDM treated overnight with LPS or Poly(I:C) quantified with a Griess test on cell supernatant (results from one experiment, WT mice = 3, Calhm6-/- mice = 3, Kruskal Wallis test with multiple comparisons).",
    "molecules": "LPS, NO, Poly(I:C)"
  },
  {
    "caption": "(G) BMDM with or without IFN−γ priming were grown overnight in antibiotic free medium. Cells were infected with L. monocytogenes (MOI 1) for 30 min. Gentamicin (5 ng/ml) was then added to the medium to kill extracellular bacteria. At 0, 4 and 8 h cells were harvested, lysed with 0.1% Triton X-100 and the lysate was plated on antibiotic free BHI plates for 24 h, and CFU were quantified (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-way ANOVA test).",
    "molecules": "Gentamicin, Triton X-100"
  },
  {
    "caption": "(D-G) BMDM were transduced with a retroviral vector expressing CALHM6-eGFP. BMDM (3x105) were plated on coverslips and pre-treated with IFN-γ and LPS for 6 h. (D) Freshly isolated primary CD3-NK1.1+ cells isolated from mouse spleens were added for 0, 10, 20, or 40 min on top of plated BMDMs that were generated from bone marrow cells transduced with a retroviral vector expressing CALHM6-eGFP. The interacting cells were then fixed and stained with phalloidin to identify actin and DAPI to identify nucleus. Synaptic interaction between BMDM and NK cell is indicated by white arrowheads; entire collapsed imaging Z-stack is shown. (E) Images were blinded and CALHM6-eGFP polarisation towards CD3-NK1.1+ cells manually scored only in Z-slices where BMDM-NK interaction was identified (p-value calculated by two-way ANOVA of polarisation and time points). Numbers on top of each bar indicate number of synapses scored for that time point. (F) IgG2a-opsonised sRBC labelled with PHK26 were added on top of plated CALHM-eGFP-expressing BMDM. One single Z-slice containing BMDM-sRBC interaction plane is shown. Synaptic interaction between BMDM and sRBC cell is indicated by white arrowheads (G) Quantified results of experiment in F. Numbers on top of each bar indicate number of synapses scored for that time point. Longer time points were not possible to score for opsonised sRBC because of the speed of the phagocytosis as sRBC were quickly internalised. Images taken with 60x objective (p-value calculated by two-way ANOVA of polarisation and time points).",
    "molecules": "PHK26, DAPI, LPS, phalloidin"
  },
  {
    "caption": "(B) WT or Calhm6-/- BMDM stimulated with IFN-γ (10 ng/ml) for 24 h were lysed with 1% Triton X100-containing buffer with cOmplete protease inhibitor cocktail and treated with increasing concentrations of DTT. The samples, without boiling, were resolved by SDS-PAGE gel (one representative experiment of two is shown, n = 4 mice). Lack of DTT influences solubilisation of GADPH monomer, and its detection for immunoblotting.",
    "molecules": "DTT, Triton X100"
  },
  {
    "caption": "(G) Current-voltage (I-V) relations for WT-mCALHM6, E119R-mCALHM6 and control (ASO) in 2-mM Ba2+ bath solution (pooled individual whole-cell patches, WT-mCALHM6 n = 13, E119R-mCALHM6 n = 4, control = 4, SEM error bars).",
    "molecules": "Ba"
  },
  {
    "caption": "(A) Fura-2 fluorescence ratio (F340/380) recorded in N2a cells expressing human CALHM1 (hCALHM1), mouse CALHM6 (mCALHM6) or control (vector) in response to removal and subsequent add-back of extracellular Ca2+. Traces representative of four independent experiments for each, normalized to baseline ratio. Right: Resting F340/380 ratio in N2a cells expressing hCALHM1, mCALHM6 or vector in 2 mM Ca2+ (the error bars represent SEM).",
    "molecules": "Ca2+, Fura-2"
  },
  {
    "caption": "(E) Steady-state ATP release from activated macrophages: BMDMs were plated at 150,000 cells/well in a 48-well plate, and incubated for 3 h with Poly(I:C) (50 μg/ml), LPS (100 ng/ml) and ATPase inhibitor ARL67156 (1 mM) in culture medium. Control cells were incubated with 1 mM ARL67156 only. ATP release (nM) measured using Sigma ATP Bioluminescent Assay: WT without stimulation: 9.12 ± 0.76; WT with stimulation: 22.16 ± 1.83; Calhm6-/- cells without stimulation: 8.76 ± 0.55; KO cells with stimulation: 12.19 ± .75. (pooled results from three independent experiments, WT mice = 3, Calhm6-/- mice = 3, Two-tailed unpaired Student's T test, error bars represent SEM).",
    "molecules": "ARL67156, ATP, ATPase, LPS, Poly(I:C)"
  },
  {
    "caption": "(G) Representative images of tissue stained  with H&E and AB/PAS, and for ROR1  in the PDO#1 xenograft  and of tumor derived from ROR1high or ROR1low cells in the PDO#1 xenograft. Data information: Scale bars, 100 µm (G).",
    "molecules": "PAS, AB"
  },
  {
    "caption": "(J) Treatment of PDO#1 and S2-VP10 organoids   with DMSO  (vehicle) or  Aurora kinase  inhibitors (n = 3, biological replicates). Tozasertib, pan-Aurora kinase inhibitor; Barasertib, selective  Aurora B kinase inhibitor. Representative images of organoids  are shown. An area of 2,000 μm2 and more was identified as an organoid. The area of organoids  is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars 1 mm (J) Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.",
    "molecules": "Barasertib, DMSO, Tozasertib"
  },
  {
    "caption": "(C)  Tumor growth curve of the four groups (control, Dox  only, gemcitabine  only, and Dox  + gemcitabine) (n = 8). The endpoint for each group is shown by a slash. Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05.",
    "molecules": "Dox, gemcitabine"
  },
  {
    "caption": "(I) Relative expression  of ROR1  and YAP   target genes (CTGF  and CYR61) in S2-VP10  cells treated with DMSO  or verteporfin  (VP) (n = 3, biological replicates). mRNA levels are normalized to that of RPS18.",
    "molecules": "DMSO, verteporfin, VP"
  },
  {
    "caption": "(M) Relative luciferase activity using the ROR1   reporter in S2-VP10 cells treated with DMSO  or BET  inhibitor (JQ1) (n = 3, biological replicates). Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (M)",
    "molecules": "DMSO, JQ1"
  },
  {
    "caption": "(O), Treatment of PDO#1 and S2-VP10 organoids  with DMSO  or JQ1  (n = 3, biological replicates). Relative expression  of ROR1  (O), mRNA levels are normalized to that of RPS18. Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (O)",
    "molecules": "DMSO, JQ1"
  },
  {
    "caption": "(P) Treatment of PDO#1 and  S2-VP10 organoids  with DMSO  or JQ1  (n = 3, biological replicates). representative images of organoids, and organoid  area (P) are shown. The area of organoids  is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars, 1 mm (P). Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (P).",
    "molecules": "DMSO, JQ1"
  },
  {
    "caption": "(C) Blood parasitemia (mean +/- SEM) monitored by flow cytometry after inoculation of 5x104 PbA.GFP spz in B6 mice, treated (open squares) or not (black squares) with CQ at day 6 and 7 pi, in order to avoid ECM without blunting infection. Day 6, P = 0.29 ; Day 7, P = 0.0013 by multiple unpaired t-tests.",
    "molecules": "CQ"
  },
  {
    "caption": "A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h).",
    "molecules": "KU0063794"
  },
  {
    "caption": "D Co‐immunoprecipitation of HA‐Flag‐TBC1D5 with myc‐VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated.",
    "molecules": "DMSO, KU0063794"
  },
  {
    "caption": "E, F Co‐immunoprecipitation of endogenous TBC1D5 and ATG9A with myc‐VPS29(E) transiently overexpressed in 293T cells or with HA‐Flag‐VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h).",
    "molecules": "DMSO, KU0063794"
  },
  {
    "caption": "G Co‐immunoprecipitation of endogenous VPS29 and ATG9A with HA‐Flag‐TBC1D5stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h).",
    "molecules": "DMSO, KU0063794"
  },
  {
    "caption": "H Co‐immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA‐Flag‐TBC1D5, stably expressed in T‐REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.",
    "molecules": "DMSO, doxycycline, KU0063794"
  },
  {
    "caption": "I Magnified still images extracted from Supplementary Movie S2. mCherry‐TBC1D5 co‐localizes with GFP‐ATG9 upon autophagy induction by mTOR inhibitor, KU0063794.",
    "molecules": "mCherry, KU0063794"
  },
  {
    "caption": "H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.",
    "molecules": "DMSO, doxycycline, KU0063794"
  },
  {
    "caption": "A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS-PAGE.",
    "molecules": "DMSO, KU0063794"
  },
  {
    "caption": "B U2OS cells stably expressing mCherry‐ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti‐LAMP1 antibody.",
    "molecules": "mCherry, DMSO, KU0063794"
  },
  {
    "caption": "E Quantification of ATG9 and LAMP1 co−localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc−TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post−transfection, fixed, and immunostained with anti−LAMP1 and anti−myc antibodies.",
    "molecules": "KU0063794"
  },
  {
    "caption": "G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA‐resistant myc‐TBC1D5, treated with KU0063794 (6 h). 20 h post‐transfection cells were treated, subsequently fixed, and immunostained with anti‐myc and anti‐LAMP1 antibodies.",
    "molecules": "KU0063794"
  },
  {
    "caption": "C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h).",
    "molecules": "DMSO, KU0063974"
  },
  {
    "caption": "E U2OS cells stably expressing HA‐Flag‐ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti‐AP2A1 antibody.",
    "molecules": "KU0063794"
  },
  {
    "caption": "F U2OS cells stably expressing mCherry‐ATG9A were treated with KU0063794 (6 h), lysed in co‐immunoprecipitation buffer. Lysates from DMSO‐ or KU0063794‐treated cells were split and equal volumes were incubated with RFP‐Trap beads or GFP‐Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS-PAGE.",
    "molecules": "DMSO, KU0063794"
  },
  {
    "caption": "A U2OS cells stably expressing HA‐Flag‐ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post‐transfection, fixed, and stained with anti‐HA antibody.",
    "molecules": "DMSO, KU0063794"
  },
  {
    "caption": "A U2OS cells stably expressing HA−Flag−ATG9A treated with DMSO or Dynasore (100 μM) 15 min, fixed and immunostained with anti−HA and anti−TBC1D5 antibodies.",
    "molecules": "DMSO, Dynasore"
  },
  {
    "caption": "D Quantification of ATG9A and AP2 co‐localization. Cells were starved in EBSS media (45 min) or pretreated with Dynasore (15 min) and subsequently starved for 45 min in combination with Dynasore. Control cells were treated with DMSO.",
    "molecules": "DMSO, Dynasore"
  },
  {
    "caption": "F T‐REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo#1 targeting (40 nM). 72 h post−transfection, expression of HA‐Flag−TBC1D5 was induced with doxycycline (50 ng/ml), and 96 h post−transfection cells were treated with Dynasore for 15 min, or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min. Cells were lysed in co‐immunoprecipitation buffer, and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4°C. Beads were washed 3 times with incubation buffer and subjected to SDS‐PAGE.",
    "molecules": "doxycycline, Dynasore"
  },
  {
    "caption": "Immunofluorescence and reporter expression in spheroids derived from single WT and Hdac3-/- naïve mESCs. DNA counterstain is Hoechst. Dashed white lines indicate the embryonic Gata6::mCherry negative part. Scale bar: 10 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Nanog>GFP and Gata6::mCherry fluorescence intensities of WT and Hdac3-/- Fraction of Gata6::mCherry positive cells in indicated genotypes and conditions after 4d Jak(i) blocks LIF, LDN19 BMP4 and PD03 FGF signaling. Average and SD of at least two independent clones.",
    "molecules": "LDN19, PD03"
  },
  {
    "caption": "E. Co-IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM. N. benthamiana leaves were agroinfiltrated and treated with 2 mM 3-MA at 48 hpi, and then for IP with anti-Flag beads 12 h later.",
    "molecules": "3-MA"
  },
  {
    "caption": "Confocal microscopy of YFP-ATG8e-labeled autophagic bodies in root cells of Col-0/YFP-ATG8e and VISP1OE/YFP-ATG8e plants. 7-day-old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark. Scale bar = 20 μm.   Puncta numbers of YFP-ATG8e-labeled autophagic bodies per 10 cells in panel A. Data points represent means of three biological repeats. Error bars indicate SD. ***p < 0.001 (Student's t-test).   ",
    "molecules": "ConA"
  },
  {
    "caption": "Western blotting analysis of accumulation of ATG8a and ATG8a-PE in leaves of 4-week-old Col-0, VISP1OE, VISP1mUIM/OE and VISP1mARM/OE plants.",
    "molecules": "PE"
  },
  {
    "caption": "Representative TEM images of autophagic structures in inoculated leaves of Col-0 and VISP1OE plants treated with mock buffer or CMV-2blm infection at 4 dpi. The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling. Magenta arrowheads indicate autophagic bodies inside the vacuoles. Scale bar = 2 μm. Cp, chloroplast; CW, cell wall; V, vacuole.   Autophagic structure numbers per 10 cells in panel F. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).   ",
    "molecules": "ConA"
  },
  {
    "caption": "Northern blotting analyzing accumulation of genomic RNAs (gRNAs) in systemically leaves of Col-0, OE1 (VISP1OE1), OE2 (VISP1OE2), rdr6-15, and sgs3-1 plants infected by CMV-2blm at 14 dpi. Methylene blue-stained rRNA were used as loading controls.   Northern blotting analyzing accumulation of RNA3-derived   vsiRNAs in the same samples as shown in panel D.",
    "molecules": "Methylene blue, rRNA"
  },
  {
    "caption": "Co-immunoprecipitation analyses of the interactions between SGS3, SGS3ΔCC, or SGS3CC and VISP1 or VISP1mARM. N. benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3-MA at 48 hpi, and collected for IP with anti-Flag beads 12 h later.",
    "molecules": "3-MA"
  },
  {
    "caption": "Confocal analysis of co-localization of CFP-NbATG8f-labelled autophagic bodies with the VISP1-YN and SGS3-YC bodies in N. benthamiana leaves. The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi. Scale bar = 20 μm.",
    "molecules": "E64d, DMSO"
  },
  {
    "caption": "Northern blotting detecting accumulation of TAS1-TasiR255, TAS2-TasiR1511, TAS3-TasiR5′D8, miR173, and miR390 in leaves of Arabidopsis plants shown in panel A. Values represent relative accumulation (RA) of siRNAs and values in Col-0 plants were set as 1. rRNA served as loading controls.",
    "molecules": "rRNA"
  },
  {
    "caption": "(A) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by fluorescent microscopy with MitoTracker Orange straining. Scale bar for all micrographs is 10 µm.",
    "molecules": "MitoTracker Orange"
  },
  {
    "caption": "(B) Characterization of Mrp17 translocation signal using an in vitro import assay: shown are autoradiographs of full-length Mrp17 or its truncations fused to DHFRmut, translated in vitro with radiolabeled amino-acids, incubated with isolated yeast mitochondria for 2, 5, or 10 min, treated with proteinase K (PK) to remove nonimported proteins and visualized by 16% SDS-PAGE/autoradiography. As a negative control, mitochondria were treated with valinomycin, antimycin and oligomycin (VAO) that eliminate membrane potential. For comparison, 20% of the protein used per import reaction was loaded on the first lane.",
    "molecules": "antimycin, PK, proteinase K, oligomycin, valinomycin"
  },
  {
    "caption": "(B) In vitro mitochondria translocation capacity of WT Mrp17, Mrp17K-R with all lysines (K) substituted with arginines (R), and Mrp17K-A with all lysines (K) substituted with alanines (A) fused to DHFRmut. Import was performed as described in Fig. 2 legend.",
    "molecules": "alanines, arginines"
  },
  {
    "caption": "(E) Mrp17 variants rescue ∆mrp17 strain growth defect on respiratory media: the indicated variants or empty vector (EV) were introduced in WT yeast and then the genomic MRP17 was disrupted by knock out, the resulting mutants were serially diluted 10× and spotted on media containing glucose or glycerol as a sole carbon source.",
    "molecules": "glucose, glycerol"
  },
  {
    "caption": "(A) The in vitro import of Mrp17-DHFRmut is sensitive to the elimination of outer mitochondrial membrane proteins by trypsinization (left), more so than the model import substrate Atp1 (right). Mitochondria were incubated with the indicated concentrations of trypsin and the import assay was performed as described in the legend for Fig. 2. Proteinase K (PK) was not added to the sample without mitochondria. (B) In vitro translocation of Mrp17-DHFRmut into mitochondria isolated from WT and ∆tom20 yeast showing reduced translocation in ∆tom20 background.",
    "molecules": "PK, Proteinase K, trypsin"
  },
  {
    "caption": "(G),(H) Import of Mrp17-DHFRmut and control proteins Atp1 and Aac1 into WT, tim17-ts (G) and tim22-ts (H) mitochondria. Import was performed at indicated temperatures, proteinase K was added to all samples except the loading control in the first lane of each autoradiograph (20% of protein amount used for each import reaction). The full autoradiograph for Aac1 with molecular weight markers is shown in Appendix Figure S6D.",
    "molecules": "proteinase K"
  },
  {
    "caption": "(B) On the right: MRP N-termini structural environment (solvent exposure): average number of atoms belonging to the same protein, other protein chains or RNA in the 13Å radius from the first structured amino acid Cα atom, plotted separately for proteins with their most N-terminus appearing in the structure vs proteins with precisely cleaved MTS, and other proteins which have a longer unstructured extension (see the classification legend on the bottom) calculated based on PDB:5MRC; on the right: same as on the left but only coordination number (number of protein Cα atoms in the 13Å radius (Hamelryck, 2005)) is calculated, ** p-value<0.01 (Mann-Whitney test), absolute values were measured without replicates; on the bottom: MRPs were divided in three groups depending on relative position of the MTS and structured residues: "No MTS (str)" - structure starts at the very N-terminus without a cleavable MTS, "Precise MTS" - MTS is cleaved off 1-2 amino acids before the structure starts, "Other" - unstructured uncleaved amino acids present before the structure start.",
    "molecules": "Cα atom, Cα atoms"
  },
  {
    "caption": "(a) Fbxo7 relocates from the cytosolic to the mitochondrial fractions of HEK293T cells treated with CCCP (10 μM). IB, immunoblot.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) Fbxo7 levels are increased in Flag-Parkin complexes immunoprecipitated (IP) from the mitochondrial fraction of HEK293T cells transfected with Flag-Parkin and untagged Fbxo7 following 1 or 3 h treatment with CCCP (10 µM).",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) Parkin localization at the mitochondria was assessed by immunocytochemistry in SH-SY5Y cells transfected with Flag-Parkin plus scrambled (scr) or Fbxo7 siRNA, following 1 or 3 h treatment with CCCP (10 μM). Cells were scored visually for the colocalization of Flag-Parkin with HtrA2, a mitochondrial marker. Images are displayed for cells transfected as indicated, following 0 or 3 h CCCP treatment. For corresponding images at 1 h treatment, see Supplementary Figure 2c. Nuclei (blue) were stained with DAPI. Scale bars, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) PINK1 localization at the mitochondria was assessed by immunocytochemistry in SH-SY5Y cells transfected with PINK1-HA plus scrambled (scr) or Fbxo7 siRNA following 1 or 3 h treatment with CCCP (10 μM). Cells were scored visually for the colocalization of PINK1-HA with complex Vβ subunit (CxVβ), a mitochondrial marker. Nuclei (blue) are stained with DAPI. Histograms indicate the percentage of cells in which PINK1-HA accumulated at the mitochondria. Data are presented as mean ± s.e.m.; *P 0.05. Representative images are displayed for cells transfected as indicated, following 0 or 3 h CCCP treatment. For corresponding images at 0 h and 1 h treatment, see Supplementary Figure 5a. Scale bars, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) Fbxo7 accumulation in the mitochondrial fraction following treatment with CCCP (10 μM) is impaired in SH-SY5Y cells transfected with PINK1 siRNA compared to scrambled siRNA (scr). IB, immunoblot. Full-length blots are presented in Supplementary Figure 9.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a,b) Ubiquitination of Mfn1 following treatment with CCCP (10 μM) is reduced in the mitochondrial fraction of both SH-SY5Y cells stably expressing Fbxo7 short hairpin RNA (Fbxo7 KD) compared to an empty vector control line (a) and in patient fibroblasts with homozygous R378G mutation compared to fibroblasts from healthy controls (b). Arrowheads indicate ubiquitinated Mfn1. IB, immunoblot.",
    "molecules": "CCCP"
  },
  {
    "caption": "(a) Treatment with CCCP (10 μM) results in an increase in LC3-II in the mitochondrial but not the cytosolic fraction of cells stably expressing the empty shRNA vector (control), and this is delayed in stable Fbxo7 knockdown (KD) SH-SY5Y cells.",
    "molecules": "CCCP"
  },
  {
    "caption": "(b) As in a, an accumulation of LC3-II was observed in the mitochondrial fraction of healthy control fibroblasts following 1 and 5 h CCCP treatment, but this was reduced in fibroblasts from a patient carrying the R378G mutation. Western blots were performed a minimum of three times.",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) Mitochondrial accumulation of p62 following CCCP treatment is inhibited by Fbxo7 siRNA. Flag-Parkin overexpressing SH-SY5Y cells were transfected with scrambled (scr) or Fbxo7 siRNA as indicated and treated with either DMSO or CCCP (10 μM) for 6 h. Colocalization of p62 with HtrA2, a mitochondrial marker, was assessed by Pearson's correlation coefficient (Rr) on a cell-by-cell basis. Histogram shows the percentage of cells in which Rr was greater than 0.5. Data are represented as mean ± s.e.m., *P 0.05. Scale bars, 10 μm.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "(d) Mitophagy was analyzed in untransfected (UT) SH-SY5Y cells or in stable Flag-Parkin overexpressing SH-SY5Y cells transfected with either scrambled (scr) or Fbxo7 siRNA. Histogram indicates the percentage of cells with no remaining mitochondria following 24 h treatment with CCCP (10 μM) for each condition. Complex Vβ subunit (CxVβ) was used as a mitochondrial marker. Data are presented as mean ± s.e.m., **P 0.01. Scale bars, 10 μm.",
    "molecules": "CCCP"
  },
  {
    "caption": "(e) Mitochondrial mass was measured in Flag-Parkin overexpressing SH-SY5Y cells transfected with scrambled (scr) or Fbxo7 siRNA and treated for 24 h with either dimethylsulfoxide (DMSO) vehicle or CCCP (10 μM). For representative images, see Supplementary Figure 8d. Full-length blots are presented in Supplementary Figure 9.",
    "molecules": "CCCP, dimethylsulfoxide, DMSO"
  },
  {
    "caption": "(C) Immunofluorescence assays of fixed RBCs infected with trophozoite or schizont stage SMC3-3HA-glmS parasites. DNA was stained with DAPI (blue) and SMC3-3HA was detected with anti-HA (green in B and magenta in C) antibody. HP1 was detected with anti-HP1 antibody (green in C). DIC, differential interference contrast. Scale bars equal 10 µm (B) and 5 µm (C).",
    "molecules": "DAPI"
  },
  {
    "caption": "Western blot analysis of nuclear extracts at 12, 24, and 36 hpi from a clonal population of SMC3-3HA-glmS parasites in the absence (-) or presence (+) of glucosamine (GlcN). SMC3-3HA is detected with an anti-HA antibody. An antibody against histone H3 is used as a control. Molecular weights are shown to the right.",
    "molecules": "GlcN, glucosamine"
  },
  {
    "caption": "Growth curve showing parasite growth rate (parasitemia at Day X/parasitemia at Day 1) over five days for WT and two clones of SMC3-3HA-glmS parasites in the absence or presence of glucosamine (GlcN). Glucosamine treatment was started 96 h (two IDC cycles) before Day 1 to ensure SMC3 knockdown during the days sampled Uninfected red blood cells (Blood) served as reference of background. Error bars indicate standard deviation of three technical replicates (n = 3). A two-way ANOVA with Tukey post hoc test was used for statistical analysis. No significant differences were found.",
    "molecules": "GlcN, glucosamine, Glucosamine"
  },
  {
    "caption": "RNA-seq of an SMC3-3HA-glmS clone shows smc3 transcript levels (FPKM) at 12 (blue, q = 8.5 x 10-3), 24 (coral, q = 1.3 x 10-39), and 36 (green, q = 4.1 x 10-54) hpi in the absence (filled circles) or presence (empty circles) of glucosamine (GlcN). Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites, for which there were two replicates. P-values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 q = Bonferroni corrected P-value. Asterisks indicate q values < 0.5.",
    "molecules": "GlcN, glucosamine"
  },
  {
    "caption": "RNA-seq of an SMC3-3HA-glmS clone shows transcript levels (FPKM) for rap2 (PF3D7_0501600) at 12 (q = 3 x 10-2) and 24 (q = 3.3 x 10-2) hpi and gap45 (PF3D7_1222700) at 12 (q = 8 x 10-1) and 24 (q = 1.6 x 10-2) hpi in the absence (black) or presence (grey) of glucosamine (GlcN). Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites, for which there were two replicates. P-values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected P-value.",
    "molecules": "GlcN, glucosamine"
  },
  {
    "caption": "(B) Different constructs fused to the Gal4VP16 domain were used in a γ-secretase reporter assay. Control transfections (pIPspAdApt empty vector and pIPspAdApt-APP without Gal4VP16) show no luciferase activity. APP-Gal4VP16 and NotchΔE-Gal4VP16 activate the luciferase gene upon cleavage and are inhibited by DAPT, L658,458, or compound 32 (125 nM). No luciferase activity was detected using TLNΔE or APP in which the transmembrane domain was replaced by that of TLN (APP/TLNTMR chimaera), indicating that this region is not cleaved by γ-secretase.",
    "molecules": "luciferase, DAPT, L658,458"
  },
  {
    "caption": "Wild-type and PS1−/− cortical neurons were transduced with SFV-TLN (A) or -APP (B), pulse labeled for 15 min with [35S]methionine, and chased for different time periods. ImmunoprecipitatedTLN and APP were treated with EndoH and analyzed by SDS-PAGE and phosphorimaging. The accumulation of an EndoH-resistant band indicates progressive maturation during Golgi passage; however, the ratio of EndoH-resistant to -sensitive TLN (A, top) and APP (B, top) revealed no difference (mean ± SEM, n = 3). Instead, the half-life of overexpressed TLN (A, bottom), but not full-length APP (B, bottom), is significantly prolonged in neurons−/−neurons (mean ± SEM, n = 3).",
    "molecules": "methionine"
  },
  {
    "caption": "TLN accumulation occurs in a compartment distinct from classic biosynthetic and endosomal pathways. (A-F) Early compartments. No colocalization was observed between TLN (green in all panels) and nuclei (A, TOPRO-3), ER-markers BIP (B) and calnexin (C), ERGIC-53 (D), β-COP (E) or GM130 (F), and labeling cis-Golgi. (G-L) Late compartments. TLN accumulations are not accessible for exogenous biotin (G). Early and recycling endosomes (H, EEA1) and transferrin receptor (I, TFR), late endosomes (J, LBPA), and lysosomes (K, Lamp-2; L, catD) also stained negative for TLN. Vertical sections (x-z, arrowheads in I and J) clearly distinguish TLN accumulations from recycling (I, TFR) and late endosomes (J, LBPA). Bars, 10 μm.",
    "molecules": "biotin, LBPA"
  },
  {
    "caption": "Correlative light immuno-EM analysis of TLN accumulations. (A) Insert: overview of the selected neuron as seen by immunocytochemistry. The bright signal for TLN reflects the numerous membranous accumulations seen at the ultrastructural immuno-EM level (star). (B) Higher magnification of the boxed area in A. (C) High magnification of the boxed structure seen in B. 10-nm gold particles label TLN. Note the complex composition of the internal membranes of the TLN-positive compartments. (D) Example of a TLN (15-nm gold)-containing structure obtained by the conventional cryosectioning technique. Note some heterogeneity in the internal membranes between C and D. Lamp-1 (10-nm gold) is absent from the TLN-containing compartment. Arrows point to invaginations of the cytoplasm. N, nucleus; P, plasma membrane. Bars: A, 5 μm; B, 2 μm; C, 500 nm; D, 200 nm.",
    "molecules": "gold particles"
  },
  {
    "caption": "TLN accumulations are distinct from lysosomes. A TLN (15-nm gold)-containing structure as obtained by the conventional cryosectioning technique double labeled with Lamp-1 (10-nm gold). The TLN-positive compartment is clearly distinct in size and from lysosomes (L) and lacks Lamp-1. P, plasma membrane. Bar, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "(B-D) TLN localizes to autophagic vacuoles of catD−/−hippocampal neurons. Lysotracker staining revealed sparsely distributed small-sized organelles in wild-type and PS1−/− neurons in contrast to catD−/−neurons where high numbers of large Lysotracker-positive organelles were found (B, arrowheads). Although TLN accumulations in PS1−/− were fully negative for Lysotracker, some acidic organelles tended to closely associate (C, arrowheads, middle). In catD−/− neurons, TLN was clearly detected in the large acidic organelles (C, bottom). Bars, 20 μm. (D) At the immuno-EM level, the large acidic organelles seen in catD−/−neurons represent dense autophagic vacuole-like structures (star) that label positive for TLN (10-nm gold). M, mitochondrion. Bars, 200 nm.",
    "molecules": "gold"
  },
  {
    "caption": "TLN mediates phagocytic uptake of microbeads in hippocampal neurons. (A) Microbeads align along neurites of hippocampal neurons as demonstrated by phase contrast (DIC). Already after 4 h, many beads stain positive for TLN (arrowheads, top). At 48 h (bottom), most TLN immunoreactivity was associated with microbeads at the expense of its typical plasma membrane staining. *, cell body. Note that microbeads were not found associated with preexisting TLN accumulations (asterisk in top panels, 4 h). (B) Microbeads (24 h) accumulate TLN and actin, as shown by phalloidin-Alexa 568. An overview of the neuron is given in each top left inset, next to two detailed areas (white boxes). The differential interference contrast (DIC) clearly demonstrates a complete colocalization of microbeads with TLN and phalloidin (arrowheads). TOPRO-3 (blue) marks the nucleus (see also Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200406060/DC1). (C) Similar as in B, but immunostained for TLN and endogenous PIP2. The overview demonstrates the overall recruitment of PIP2 to microbeads (arrows). The parallel insets show a detailed area (white box) with PIP2 being colocalized with TLN on individual microbeads (arrowheads). Note that PIP2 occasionally colocalizes with TLN at the plasma membrane (asterisk). Bars (A-C), 10 μm.",
    "molecules": "PIP2"
  },
  {
    "caption": " E. Binding of σ28-RNAP with fliCp as detected by EMSA (left). The heparin-resistant RNAP-promoter open complex is indicated as \"RPo\", and the heparin-sensitive RNAP-promoter closed complex is shown as \"RPc\". Percentages of RNAP-promoter open complex (RPo) and closed complex (RPc) in EMSA performed using σ28-RNAP and fliCp (right). ",
    "molecules": "heparin"
  },
  {
    "caption": " G. Activities of fliCp in Ec-K74A-K87A-C and Ec-K74A-K87A-V strains. L-arabinose was used at final concentration of 0.002%. Data are mean ± SD from three colonies. ",
    "molecules": "L-arabinose"
  },
  {
    "caption": "A-CConfirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected either with HA‐Rab8A (A), HA‐Rab8B (B) or HA‐Rab13 (C) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (10 mg) were immunoprecipitated with anti‐HA agarose, resolved by SDS-PAGE and stained with colloidal Coomassie blue (second panel). Coomassie‐stained bands migrating with expected molecular mass of HA‐Rabs were excised, in‐gel digested with trypsin and subjected to high‐performance liquid chromatography with LC‐MS/MS on an LTQ‐Orbitrap mass spectrometer. Upper panel shows the extracted ion chromatogram (XIC) analysis of Ser111‐containing phosphopeptides (8A, NIEEHApSADVEK; 8B, NIEEHApSSDVER; 13, SIKENApSAGVER) with the combined signal intensity of the 2+ and 3+ forms of the peptide indicated on the y‐axis. Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1‐FLAG‐expressing cells following CCCP stimulation.",
    "molecules": "agarose, CCCP, doxycycline"
  },
  {
    "caption": "DCharacterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.",
    "molecules": "agarose, CCCP, doxycycline"
  },
  {
    "caption": "PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls.",
    "molecules": "agarose, CCCP, DMSO"
  },
  {
    "caption": "Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.",
    "molecules": "agarose, CCCP, DMSO"
  },
  {
    "caption": "Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.",
    "molecules": "agarose, CCCP, DMSO"
  },
  {
    "caption": "Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.",
    "molecules": "agarose, CCCP, DMSO"
  },
  {
    "caption": "Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS-PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.",
    "molecules": "32P, Mg2, ATP"
  },
  {
    "caption": "Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.",
    "molecules": "agarose, CCCP, doxycycline"
  },
  {
    "caption": "Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.",
    "molecules": "agarose, CCCP"
  },
  {
    "caption": "Rabin8‐catalysed mant‐GDP release from mant‐GDP loaded WT, S111A and S111E mutants of Rab8A. The Rab proteins (1 μM) were incubated with 100 μM GDP in buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE), and the reaction was started by the addition of 0.5 μM Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.",
    "molecules": "DTE, GDP, MgCl2, NaCl"
  },
  {
    "caption": "Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells. Rab8A KO HeLa cells were transfected with wild‐type (WT), S111E or S111A HA‐Rab8A. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rabin8 or anti‐HA antibody. Lysates were immunoblotted with Rabin8 or anti‐HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA‐Rab8A in extracts.",
    "molecules": "agarose"
  },
  {
    "caption": "(C) Tomato leaves were either pre-infiltrated with water (control) or autophagy inhibitors Wortmannin, LY294002, Chloroquine (CQ), and 3-methyladenine (3-MA). Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection. (B) 48 hours post inoculation; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol (1: 3, v/v). Images were taken 48 hours post inoculation.",
    "molecules": "3-methyladenine, Chloroquine, LY294002, water, Wortmannin"
  },
  {
    "caption": "(A) NBT treated Arabidopsis (Col-0 and two independent atg8a mutant lines) following agar plug inoculation with the A2 mutant. Images were collected 48 hours post inoculation. Dotted lines represent the edge of the observable legion.",
    "molecules": "NBT"
  },
  {
    "caption": "D. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 0.36μM IBL-202 or 0.05 μM IBL-301. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified. IBL-301 treated cells were stained for Tuj1. DAPI was used to visualize nuclei. E. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) cells treated with IBL-301. For LU-NB-3 PDX cells, representative areas (n=2) were used and n=344 and n=240 cells/condition for CTRL and IBL-301, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=2 and n=3 for CTRL and IBL-301 respectively) were used and n=141 and n=130 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-tes. P=0.003 for LU-NB-3 and p=0.08 for SK-N-BE(2)c.",
    "molecules": "IBL-202, IBL-301, DAPI"
  },
  {
    "caption": "A. Viability IBL-202 or IBL-301 treated cells determined by CellTiterGlo.",
    "molecules": "IBL-202, IBL-301"
  },
  {
    "caption": "E. Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL-202 or 50 nM IBL-301. F. Quantification of live and dead cells from the AnnexinV/PI stainings. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.",
    "molecules": "IBL-202, IBL-301"
  },
  {
    "caption": "Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours A. Expression of pAkt(Ser473) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. B. Expression of pAkt(Thr308) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls.",
    "molecules": "IBL-302"
  },
  {
    "caption": "Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours C. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 50 nM IBL-302. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). D. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2)c cells treated with IBL-302. For LU-NB-3 PDX cells, representative areas (n=4) were used and n=460 and n=216 cells/condition for CTRL and IBL-302, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=5 and n=7 for CTRL and IBL-302 respectively) were used and n=124 and n=260 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-test. p=0.009 for LU-NB-3 and p=0.0003 for SK-N-BE(2)c.",
    "molecules": "IBL-302"
  },
  {
    "caption": "Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours E. N-Myc protein expression determined by western blotting. SDHA was used as loading control.",
    "molecules": "IBL-302"
  },
  {
    "caption": "Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 72 hours F. Cell viability determined by CellTiterGlo.",
    "molecules": "IBL-302"
  },
  {
    "caption": "Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours G. Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL-302 H. Quantification of live and dead cells. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.",
    "molecules": "IBL-302"
  },
  {
    "caption": "I. Neuroblastoma SK-N-BE(2)c carrying mice (n=5 in each group) were treated with vehicle (CTRL) or 40 mg/kg IBL-302 for up to 35 days and tumor growth was followed over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.",
    "molecules": "IBL-302"
  },
  {
    "caption": "J. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL) or IBL-302. Log-rank test was used to determine statistical significance. p=0.0577.",
    "molecules": "IBL-302"
  },
  {
    "caption": "A-B. LU-NB-3 PDX, SK-N-BE(2)c and SK-N-SH cells were treated with indicated concentrations of cisplatin and/or 0.36 μM IBL-202, 0.05 μM IBL-301, or 0.05 μM IBL-302 for 48 hours. Cell viability determined by CellTiterGlo. Graphs show mean values and SEM from three independent experiments. Theoretical additive curves were evaluated by calculating the combination index (CI) based on the Bliss Independence model.",
    "molecules": "IBL-202, IBL-301, IBL-302, cisplatin"
  },
  {
    "caption": "C-D. Cell cycle distribution in LU-NB-3 PDX cells after treatment with 320 nM cisplatin, 50 nM IBL-302 or the combination of these, determined by flow cytometry. Bars show mean values from two independent experiments.",
    "molecules": "IBL-302, cisplatin"
  },
  {
    "caption": "E. Tumor size in individual mice from Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.",
    "molecules": "IBL-302, cisplatin"
  },
  {
    "caption": "F. Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.",
    "molecules": "IBL-302, cisplatin"
  },
  {
    "caption": "G. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL), IBL-302, cisplatin, or the combination. Log-rank test was used to determine statistical significance. p=0.0014.",
    "molecules": "IBL-302, cisplatin"
  },
  {
    "caption": "LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. A-C. Heat map of significantly differentially expressed genes across treatment groups from RNAseq data (FDR<0.1) (A). Genes defined as differentially expressed that were also present in MS and phospho-MS are visualized in (B) and (C), respectively.",
    "molecules": "IBL-302"
  },
  {
    "caption": "(H) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelialNMuMG cells (d0) and cells treated with TGF-β for seven days in the presence of JNK inhibitor (d7 JNKi) or control (d7 DMSO). Staining was performed with antibodies against Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. It is important to note that the untreated epithelial cells are highly proliferative and appear small in size, round in shape and adhere to surrounding cells whereas day 7 TGF- β-treated (mesenchymal) cells are much bigger in size, elongated in shape and detached from the nearby cells. Scale bar, 100 μm, 63X magnification.",
    "molecules": "DMSO, JNKi"
  },
  {
    "caption": ". (E) ChIP assay using H3K27me3-specific antibody in epithelial cells (day 0)) or NMuMG cells treated for seven days with TGF-β (mesenchymal) and DMSO or JNKi. qRT-PCRs were performed for indicated gene promoters and enrichments are plotted on the right y-axis as ratio of precipitated DNA (bound) to total input DNA. On the left y-axis RNA levels of analyzed genes derived from RNA-seq data (fold change to untreated normalized tag counts ) are plotted. Mean and SEM is plotted from two independent biological replicates.",
    "molecules": "DMSO, JNKi"
  },
  {
    "caption": "PRKRIR, PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate. (A) Scratch assay in MDA-MB-231 cells treated for four days with DMSO or JNKi. Scale bar, 200 μm; 20 X magnification. Mean and SEM is plotted from three independent biological replicates.",
    "molecules": "DMSO, JNKi"
  },
  {
    "caption": "(B) mRNA levels for key EMT marker genes in MDA-MB-231 treated with DMSO or JNKi for 4 days were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates",
    "molecules": "DMSO, JNKi"
  },
  {
    "caption": "(F) mRNA levels of key EMT marker genes in MDA-MB-231 and MDA-MB-231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor (SP600125) for 24 hours were measured by qRT-PCRs relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.",
    "molecules": "SP600125"
  },
  {
    "caption": "(D) RNA levels of Pcbp4, Prkrir, Tsc22d1, represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 (JNKi) for 6 hours.",
    "molecules": "SP600125, DMSO"
  },
  {
    "caption": "(X) Muscle fractional incorporation of M+6 of glutamate, M+5 of αKG, M+4 of fumarate, M+4 of malate, M+3 of alanine and M+3 of lactate, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-glutamate. In panels X data are presented as Mean ± SEM. In panel X, COX10 KO (n=4), CTL (n=4). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.",
    "molecules": "13C5, αKG, alanine, fumarate, glutamate, lactate, malate, 15N"
  },
  {
    "caption": "(Z) Muscle fractional incorporation of M+6 of valine, M+5 of α-KIV, M+4 of β-ΗΙΒ, M+3 of propionyl carnitine, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-valine. In panels Z, data are presented as Mean ± SEM. In panel Z, COX10 KO (n=3), CTL (n=3). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.",
    "molecules": "13C5, β-ΗΙΒ, α-KIV, 15N, propionyl carnitine, valine"
  },
  {
    "caption": "(N) Levels of metabolites of trans-sulfuration pathway by LC-MS analysis in MERRF muscle and plasma expressed relative to CTL muscle and plasma. α-HB, α-hydroxybutyrate; GSH, glutathione. In panel N, data are presented as Mean ± SEM. Muscle: MERRF (n=10), CTL (n=15). Plasma: MERRF (n=9), CTL (n=25). * p<0.05 MERRF vs. CTL. Statistically significant differences between the two groups for all panels were estimated by unpaired two-tailed Student's test.",
    "molecules": "α-HB, α-hydroxybutyrate, glutathione, GSH"
  },
  {
    "caption": "(K, L) Levels of acylcarnitine, 3-OH acylcarnitine, fatty acid and 3-OH fatty acid in MERRF muscle (K) and plasma (L) by LC-MS analysis, expressed relative to CTL. In panels data by LC-MS analysis are presented as Mean ± SEM. In panels A and C, 50, 100, 200d: COX10 KO (n=6), CTL (n=6). *, p<0.05 COX10 KO vs. same age CTL. In panels K and L, muscle: MERRF (n=10), CTL (n=15); plasma: MERRF (n=9), CTL (n=25). *, p<0.05 MERRF vs. CTL.",
    "molecules": "3-OH fatty acid, fatty acid, 3-OH acylcarnitine, acylcarnitine"
  },
  {
    "caption": "(M) MERRF plasma levels of 4-hydroxyl-L-proline by LC-MS analysis, expressed relative to CTL value set at 1.",
    "molecules": "4-hydroxyl-L-proline"
  },
  {
    "caption": "(G) Levels of glucocorticoids in MERRF plasma by LC-MS analysis, expressed relative to CTL value set at 1. In panels G data are presented as Mean ± SEM. MERRF (n=9), CTL (n=25).",
    "molecules": "glucocorticoids"
  },
  {
    "caption": "(N) Age-dependent grip strength of COX10 KO mice treated with RU-486 and vehicle. The black arrow indicates the beginning of the treatment at 43 days. In panel data are presented as Mean ± SEM. Panel N: KO+RU486 (n=6), KO (n=4).",
    "molecules": "RU-486, RU486"
  },
  {
    "caption": "(P) Visceral fat deposits of COX10 KO mice treated with RU-486 and vehicle at 100 days of age. In panel P, data are presented as Mean ± SD. KO+RU486 (n=6), KO (n=6). *, p<0.05 KO+RU486 vs. KO. Statistically significant differences between the two groups for all panels were estimated by unpaired two-tailed Student's test.",
    "molecules": "RU-486, RU486"
  },
  {
    "caption": "A. Jurkat cells were pre-labeled with light or medium L-Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N-terminal COFRADIC fractionation and LC-MS/MS analysis.",
    "molecules": "Arg, L-Arg"
  },
  {
    "caption": "A. Jurkat cells were treated with 100 µg/ml CHX for 0, 0.5, 1.5, 4, 8, 12 or 24 hours. Protein degradation was monitored by Western blotting and stabilities of several short-lived endogenous proteins were confirmed using antibodies (including lamin B, securin, β-catenin, GCIP interacting protein p29) and compared to stable proteins (such as GAPDH and actin). Proteoform-specific bands were used to calculate protein half-lives which were in a good agreement with turnover times obtained from pSILAC",
    "molecules": "CHX"
  },
  {
    "caption": "B-C. Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E. Selective C-terminal V5-tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse-chase experiments were performed as described for Jurkat cells. Degradation of overexpressed proteoforms was monitored by an anti-V5 antibody and compared to the turnover of stable proteins such as actin and tubulin.",
    "molecules": "CHX"
  },
  {
    "caption": "C Growth phenotype of H subunit mutants. Deletion of V-ATPase subunits from yeast results in a conditional lethal phenotype (Vma-) characterized by an inability to grow at pH 7.5 in the presence of 60 mM CaCl2 (upper panel), but growth is observed at pH 5 (lower panel). Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed. Both humanH subunit isoforms (HsH1, HsH2), a chimeric H subunit (HChim) containing ScHNT and HsHCT, mutations in yeast intended to mimic the length of the human H loop sequence and accessibility (Hloop), and a point mutation of the conserved aspartic acid residue in the loop sequence (HD410A) are shown compared to the H subunit deletion (ΔH) and wild type H on a plasmid (H WTpl). All grow on pH 5 medium, but the human isoforms failed to complement the deletion phenotype.",
    "molecules": "CaCl2"
  },
  {
    "caption": "(B) Structures of S. cerevisiae Hrr251-394 K38R bound to CK1-7 (Chijiwa et al, 1989; Xu et al, 1996) and C. glabrata Hrr251-403 K38R bound to ADP, with domains colored as in (A). Bound PO4-/SO4- ions are shown as spheres. See Figure EV2A for additional C. glabrataHrr25 structures, and Figure EV3A for SO4- ion electron density.",
    "molecules": "ADP, CK1-7, PO4, SO4"
  },
  {
    "caption": "(C) Overlay of the kinase domain of C. glabrataHrr25 (Apo form, crystallized with 1.2 M PO4-) with ratCK1δ crystallized in the presence of tungstate ions (WO4; PDB ID 1CKJ, (Longenecker et al, 1996)) See Figure EV2B for detailed views of ion binding.",
    "molecules": "PO4, WO4"
  },
  {
    "caption": "(D) Stereo view of CK1-7 binding to S. cerevisiae Hrr25. Bound drug is positioned identically to a previous structure of S. pombe Cki1 bound to CK1-7 (Xu et al, 1996).",
    "molecules": "CK1-7"
  },
  {
    "caption": "(E) Stereo view of ADP binding to C. glabrata Hrr25 (formate structure; SO4- structure is equivalent, but the GxGxxG motif is disordered in that structure). All active-site residues shown in panels (D) and (E) are conserved between S. cerevisiae and C. glabrata Hrr25.",
    "molecules": "ADP"
  },
  {
    "caption": "(A) Two views of C. glabrata Hrr25 (Apo form) with domains colored as in Figure 1, showing bound PO4- ions and the previously-identified Mam1-binding residues His25 and Glu34 (Petronczki et al, 2006).",
    "molecules": "PO4"
  },
  {
    "caption": "(E) Geometry of zinc binding in Mam1. Zn-S bonds and the Zn-N bond were restrained to ~2.3 Å and ~2.0 Å, respectively, during refinement.",
    "molecules": "Zn-N, Zn-S"
  },
  {
    "caption": "(A-C) Ribbon views of the S. cerevisiae Hrr251-394:Mam187-191 structure (form 1), CK1-7 bound S. cerevisiae Hrr251-394, and ADP-bound C. glabrata Hrr251-403 (formate condition), with Hrr25 colored according to main-chain B-factor from low (purple) to cyan (high). For each, coloring is normalized to correspond to the average of the 20 lowest (purple) or highest (cyan) main-chain B-factors in Hrr25. Mam1 (panel A) is colored in light gray.",
    "molecules": "ADP, CK1-7"
  },
  {
    "caption": "(B) Stimulation of Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A by ATP, measured using an enzyme-coupled ATPase assay.",
    "molecules": "ATP"
  },
  {
    "caption": "(C) ADP-Glo assay showing the effect of added CK1-7 on Hrr251-394, Hrr251-394:Mam187-191, and Hrr251-394:Mam187-191 R131A.",
    "molecules": "CK1-7"
  },
  {
    "caption": "(D) Km, Vmax, and IC50 values for tested enzymes. Vmax was calculated from curves in (A) at 100 M ATP.",
    "molecules": "ATP"
  },
  {
    "caption": "B A549 cells were transfected with a plasmid encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 (25 μM) or vehicle for 6 h before harvesting. PB1-F2 transcripts were analyzed by semi-quantitative RT-PCR (top panel), and The PB1-F2 protein was analyzed by Western blotting with monoclonal anti-Flag antibody.",
    "molecules": "MG132"
  },
  {
    "caption": "C Representative immunofluorescence images showing expression of PB1-F2 (red). A549 cells were transfected with plasmids encoding indicated PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis. Magnification, ×100; scale bar, 100 μm.",
    "molecules": "MG132"
  },
  {
    "caption": "D Each HA-tagged PB1-F2 was transfected into 293T cells with or without Flag-ubiquitin. At 18 h after transfection, cells were treated with MG132 for 6 h. The lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting with anti-Flag antibody. PR8, A/Puerto Rico/8/34 (H1N1); 1918, A/Brevig Mission/1/1918 (H1N1).",
    "molecules": "MG132, ubiquitin"
  },
  {
    "caption": "B-D A549 cells were transfected with the indicated plasmids encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and chimeric PB1-F2 mutants. At 18 h after transfection, cells were treated with or without MG132 for 6 h. The expression of PB1-F2 and control genes was detected by semi-quantitative RT-PCR (upper panel) or Western blotting (lower panel). Results shown are representative of three independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "C U937 cells were co-transfected with the IFNβ luciferase reporter plasmid and the PB1-F2 or NS1 expressing plasmid and treated with polyI:C for 12 h before harvesting. IFNβ promoter activity was determined by luciferase reporter assay. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).",
    "molecules": "polyI:C"
  },
  {
    "caption": "E Cells were transfected with each plasmid encoding PB1-F2. 18 h after transfection, cells were treated with MG132 for 6 h and the level of IFNβ mRNA was determined by semi-quantitative RT-PCR (left) and qPCR (right). PB1-F2 protein expression was assessed by Western blotting. All data are shown as mean (±SEM) from at least three independent experiments (*p < 0.05, **p <0.01, ***p <0.001).",
    "molecules": "MG132"
  },
  {
    "caption": "A SDS-PAGE analysis of proteins immunoprecipitated from A549 cell lysates using HA-antibody. Bands were stained by Coomassie Blue. Results were confirmed in three independent experiments, and representative data are shown.",
    "molecules": "Coomassie Blue"
  },
  {
    "caption": "E Analysis of interaction between DDX3 and 1918 PB1-F2. Cells were transfected with indicated plasmids; 18 h later, cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-HA antibody. Proteins were detected by immunoblotting with the indicated antibodies (left panel). IAV-infected cell lysates were immunoprecipitated with anti-PB1-F2 antibody and immunoblotted with the indicated antibodies (middle panel). Purified recombinant 1918 PB1-F2 protein was incubated with recombinant LysRS or LysRS-DDX3, and the interaction was analyzed by immunoblotting (right panel). Results were confirmed in three independent experiments, and representative data are shown.",
    "molecules": "MG132"
  },
  {
    "caption": "F Analysis of interaction between DDX3 and PB1-F2 mutants. A549 cells were transfected with the indicated clones; 18 h after transfection, cells were treated with MG132 for 6 h and analyzed by immunoprecipitation. Results were confirmed in three independent experiments, and representative data are shown.",
    "molecules": "MG132"
  },
  {
    "caption": "H Representative fluorescence images showing expression level of GFP-DDX3 (green). A549 cells were co-transfected with plasmids encoding the GEP-DDX3 and Flag-tagged PR8 or 1918 PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to fluorescence analysis. Results were confirmed in three independent experiments, and representative data are shown.",
    "molecules": "MG132"
  },
  {
    "caption": "J A549 cells were transfected with the indicated plasmids or treated with poly I:C. Cytoplasmic and nuclear fractions were separated and subjected to Western blotting. Results were confirmed in three independent experiments, and representative data are shown.",
    "molecules": "poly I:C"
  },
  {
    "caption": "A Representative picture of plants carrying pASDOG1::LUC sprayed with mock solution, ABA or GA and imaged 24 h later. The full pictures are shown in Fig EV2 and Appendix Fig S1. Scale bar, 1 cm.",
    "molecules": "ABA, GA"
  },
  {
    "caption": "B Quantification of emitted light intensity per plant after treatment with ABA and GA, compared with mock treatment.",
    "molecules": "ABA, GA"
  },
  {
    "caption": "F The asDOG1 expression level (black line) is reduced while DOG1 sense expression (grey line) is increased after application of ABA. Leaves of mature Col-0 (WT) 40-day-old plants were sprayed with ABA and collected 0, 6 and 10 h later for RNA extraction and RT-qPCR. Signals were normalized against the level of the UBC transcript. The data points are the averages for at least three biological replicates and are normalized against the mock-treated value. Error bars represent standard deviation.* and *** represent t-test P-values of < 0.05 and < 0.001, respectively.",
    "molecules": "ABA"
  },
  {
    "caption": "A Plants expressing LUC fused with full-length DOG1 (pDOG1LUC::DOG1) were sprayed with mock solution or ABA 10, 20 and 40 days after germination and analysed 24 h after treatment. The graphs show mean emitted light intensity per plant.",
    "molecules": "ABA"
  },
  {
    "caption": "B Representative picture of mock- and ABA-treated pDOG1LUC::DOG1 plants. The full picture is shown in Fig EV2C and D. Scale bar, 1 cm.",
    "molecules": "ABA"
  },
  {
    "caption": "C A truncated construct that lacks the DOG1 antisense promoter region (pDOG1shDOG1::LUC) is not induced by ABA and is highly expressed throughout development. The graphs show mean emitted light intensity per plant.",
    "molecules": "ABA"
  },
  {
    "caption": "D Representative picture of mock- and ABA-treated pDOG1shDOG1::LUC plants. Scale bar: 1cm.",
    "molecules": "ABA"
  },
  {
    "caption": "F Strand-specific RT-qPCR analysis of antisense levels after 10 h of ABA treatment in 10-, 20- and 40-day-old Col-0 plants.",
    "molecules": "ABA"
  },
  {
    "caption": "G The pASDOG1::LUC reporter is silenced by the application of ABA, while mutation of TATA elements in the asDOG1 promoter (pASDOG1∆TATA::LUC) leads to attenuation of expression in the presence and absence of ABA.",
    "molecules": "ABA"
  },
  {
    "caption": "H Mutation of TATA elements in the asDOG1 promoter leads to high-level DOG1 sense expression and non-responsiveness to ABA. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.",
    "molecules": "ABA"
  },
  {
    "caption": "B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.",
    "molecules": "ABA"
  },
  {
    "caption": "(a) Chemical structure of PIK-III. (b) The indicated kinases were incubated with different doses of PIK-III and IC50 values were determined.",
    "molecules": "PIK-III"
  },
  {
    "caption": "(a) General domain structure of human VPS34 kinase comprising the helical domain (yellow) and the kinase domain (green). The ATP-binding pocket and active site lie between the amino-terminal lobe and the carboxy-terminal lobe of the kinase domain. (b) Close-up view of the VPS34 active site with PIK-III bound in the ATP-binding pocket. PIK-III forms two hydrogen-bond interactions between the compound's donor/acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685. A prominent hydrophobic pocket, bounded in part by the side chains of Phe 612, Pro 618 and Phe 684 accommodates the cyclopropyl functional group. An extended solvent-mediated hydrogen-bonding network bridges interactions between the PIK-III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644. (c) The binding properties surface of the VPS34 active site is superimposed on the solvent-accessible surface. Green surfaces are regions with hydrophobic binding properties; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits. Blue and red surfaces represent hydrogen bond donor and acceptor regions, respectively. PIK-III is shown bound in the active site. (d) Superposition of the PIK-III-bound VPS34 pocket surface with a mesh (white) mapping the human PI(3)Kα active site. The displacement of the PI(3)Kα hydrophobic pocket away from the hinge (relative to that of VPS34) is readily apparent.",
    "molecules": "PIK-III, ATP"
  },
  {
    "caption": "b) HeLa cells expressing GFP-Parkin were treated with PIK-III for 12 h followed by the addition of CCCP for 12 h, fixed, stained for endogenous Tom20 and imaged. Representative images of an overlay of Tom20 (red) and Hoechst (blue) are shown with a scale bar equivalent to 50 μm. Bars represent the mean cellular Tom20 staining intensity per cell and data points represent values from four wells from one experiment.",
    "molecules": "PIK-III, CCCP"
  },
  {
    "caption": "(d) Steady-state levels of autophagy markers visualized by western blotting in H4 GFP-p62 cells treated overnight with 0.5 μM AZD8055 or 5 μM PIK-III.",
    "molecules": "PIK-III, AZD8055"
  },
  {
    "caption": "(e) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in PSN-1 cells treated for 48 h with the indicated doses of PIK-III. (f) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in Panc10.05 cells treated for 48 h with the indicated doses of PIK-III.",
    "molecules": "PIK-III"
  },
  {
    "caption": "(g) RKO cells expressing inducible HA-ATG4B-C74A were treated with doxycycline (DOX induction) for five days and then incubated in DOX-free medium for 24 h (DOX washout). Bafilomycin A1 was added with or without PIK-III at the beginning of the washout period (lanes 5 and 6). Steady-state levels of ATG4B, LC3 and GAPDH were visualized by western blotting. Uncropped images of blots are shown in Supplementary Fig. 7.",
    "molecules": "PIK-III, Bafilomycin A1, DOX, doxycycline"
  },
  {
    "caption": "(c) Steady-state levels of NCOA4 in DLD1 cells treated with bafilomycin A1 or PIK-III for 20 h.",
    "molecules": "PIK-III, bafilomycin A1"
  },
  {
    "caption": "e) DLD1 cells cultured in rich medium (Fed) were deprived of serum and amino acids (Starve) for the indicated times (hours, h) and levels of NCOA4, p62, LC3 and GAPDH were determined by western blotting.",
    "molecules": "amino acids"
  },
  {
    "caption": "(f) Confocal microscopy was used to evaluate the cellular distribution of endogenous NCOA4, LC3, LAMP2 and EEA1 in cells treated overnight with bafilomycin A1 or PIK-III. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full panels on left, and zoomed panels on right correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.",
    "molecules": "PIK-III, bafilomycin A1"
  },
  {
    "caption": "(c) DLD1 cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous FTH1 or FTL with LC3, LAMP2 or EEA1. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(e) DLD1 parental or NCOA4−/− cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous ferritin (FTH1-FTL) and LAMP2. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(a) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot. Proteins elute in high- to low-molecular-weight fractions from 18 to 33, respectively. Dashed lines are for visual comparison only.",
    "molecules": "bafilomycin A1, DMSO"
  },
  {
    "caption": "(a) DLD1 cells were incubated in the presence or absence of DFX (30 μM) and PIK-III (5 μM) and the levels of FTH1 and FTL were analysed using western blotting.",
    "molecules": "PIK-III, DFX"
  },
  {
    "caption": ". (b) DLD parental or NCOA4−/− cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined.",
    "molecules": "DFX"
  },
  {
    "caption": "(e) The presence of iron in spleens from control or Ncoa4−/− mice was determined using Perls' Prussian blue staining. Representative micrographs (left panels) or magnifications (right panels) are shown. Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively. (f) Quantitative image analysis of the data shown in e was performed as described in the Methods. Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group (n = 6 mice per group; ∗P 0.0006 for unpaired t-test). Uncropped images of blots are shown in Supplementary Fig. 7.",
    "molecules": "iron"
  },
  {
    "caption": "(B) Representative immunofluorescence images of HeLa cells in various mitotic stages. Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue). Scale bar, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Representative immunofluorescence images of HeLa cells that were fixed and co-stained for Cyclin B2 (green), mAb414 (red), and DNA (blue). Scale bar, 10 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "Representative immunofluorescence images of HeLa cells treated with an indicated siRNA Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).",
    "molecules": "DNA"
  },
  {
    "caption": "Representative immunofluorescence images of HeLa cells treated with an indicated various drugs (E). Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).",
    "molecules": "DNA"
  },
  {
    "caption": "(F) HeLa cells were synchronized by double thymidine, then released and collected at selected time points. Cell lysate samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B1 antibody, anti-Cyclin B2 antibody, or anti-β-Actin antibody.",
    "molecules": "thymidine"
  },
  {
    "caption": "(B) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies. Nocodazole-arrested mitotic cells were collected and lysed. Immunoprecipitation was accomplished using control IgG, anti-Cyclin B2-1 (Abcam, ab185622), and anti-Cyclin B2-2 (Proteintech, 21644-1-AP). Immunoprecipitation samples were resolved by Western blotting using a mouse anti-Cyclin B2 antibody ( Santa Cruz, sc-28303) and a mouse anti-Mad2 antibody (Santa Cruz, sc-47747)",
    "molecules": "Nocodazole"
  },
  {
    "caption": "(E) GST pulldown assay. Purified GST, GST-Cyclin B1-165-433, and GST-Cyclin B2-130-398-bound agarose beads were used as affinity matrices to absorb purified Trx-6×His-tagged C-Mad2 recombinant protein. Absorbed proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining",
    "molecules": "Coomassie Brilliant Blue"
  },
  {
    "caption": "(F-I) Representative immunofluorescence images of HeLa cells stably expressing GFP-Cyclin B2 (F) or GFP-Cyclin B1 (H). The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h. Then cells were fixed and co-stained for Mad2 (red) and DNA (blue). Scale bar, 10 µm. Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2 (G) or GFP-Cyclin B1 (I) in cells treated as in F and H, respectively. Bars represent the mean kinetochore intensity (±SD) normalized to values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments)",
    "molecules": "DNA, MG132, nocodazole"
  },
  {
    "caption": "(D) Representative immunofluorescence images of HeLa cells transfected with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for ACA (red) and DNA (blue). (E) Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2-WT/4A treated as in D. Bars represent the mean kinetochore intensity (±SD) normalized to the values of GFP-Cyclin B2-WT. Each dot represents one cell (≥30 cells from three independent experiments).",
    "molecules": "DNA, nocodazole"
  },
  {
    "caption": "Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (green) in D, ACA (red), and DNA (blue) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (red) in DNA (blue)",
    "molecules": "DNA, nocodazole"
  },
  {
    "caption": "Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (green) in D, ACA (red), and DNA (blue) E) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (red) in E, and DNA (blue)",
    "molecules": "DNA, nocodazole"
  },
  {
    "caption": "(G) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12 h",
    "molecules": "nocodazole"
  },
  {
    "caption": "A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆rapZ mutant Z28 under normal growth and GlcN6P starvation conditions. Both strains, which also carried a chromosomal glmS'-lacZ fusion, were treated with 60 μg/ml Nva-FMDP or H2O (\"mock\"). Samples were harvested hourly for Northern analysis and determination of β-galactosidase activity. Growth curves are shown in Appendix Fig S1. Blots were re-probed using a 5S rRNA specific probe to provide loading controls.",
    "molecules": "GlcN6P, Nva-FMDP, H2O"
  },
  {
    "caption": "B The purification profile of Strep-RapZ from the ∆glmS strain Z904 under GlcN6P replete and depletion conditions is shown (top). The cleared lysate (CL), flow through (FT), washing steps (W) and the elution fractions (E1-3) from StrepTactin affinity chromatography were separated on 12.5 % SDS-PAA gels and stained with Coomassie blue. Metabolites were extracted from E2 and analyzed by HILIC-MS/MS. The extracted ion chromatograms of the LC-MS analysis targeting GlcN6P (retention time 16.6 minutes) are shown below. The samples derived from purification of Strep-RapZ (panels i and ii) or Strep-KdpE (panels iii and iv) were analyzed with the SRM transition m/z 258.1 to m/z 97 in the negative ion mode. The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg/μl (panel v).",
    "molecules": "GlcN6P, PAA, SDS"
  },
  {
    "caption": "C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites. The Strep-tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite (i.e. 100, 500, 1000, 2500 and 5000 nM) were injected using a single cycle kinetics approach.",
    "molecules": "GlcN6P"
  },
  {
    "caption": "Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z197 and Z360 were used, which harbor glmY'-lacZ and glmZ'-lacZ fusions, respectively.",
    "molecules": "GlcN6P, Nva-FMDP"
  },
  {
    "caption": "Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Expression of glmY'-lacZ in strain Z197 and the ∆rapZ mutant Z225 is compared.",
    "molecules": "GlcN6P, Nva-FMDP"
  },
  {
    "caption": "Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and Z201 were addressed, which transcribe glmY'-lacZ either from the σ54-promoter or the σ70 promoter, respectively.",
    "molecules": "GlcN6P, Nva-FMDP"
  },
  {
    "caption": "Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and the ∆qseF mutant Z196 are compared, both of which transcribe the glmY'-lacZ fusion solely from the σ54 promoter.",
    "molecules": "GlcN6P, Nva-FMDP"
  },
  {
    "caption": "Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures. Strains Z197 and the ∆rapZ mutant Z225 were used. Tested plasmids were pFDX4291 (vector control for pFDX4324 and pYG82 = VC1), pFDX4324 (rapZ), pYG82 (rapZquad), pBGG237 (vector control for pBGG164 = VC2) and pBGG164 (strep-rapZ).",
    "molecules": "GlcN6P, Nva-FMDP"
  },
  {
    "caption": "D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10-tagged QseE' (aa 196-475) in presence of various concentrations of RapZ. Samples were removed following [γ-32P]-ATP addition at indicated times and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.",
    "molecules": "ATP, 32P, PAA, SDS"
  },
  {
    "caption": "F To analyse the role of GlcN6P, 5 μM RapZ or the equivalent volume of buffer was pre-incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE'-His10 was added. Following an additional incubation for 5 min, [γ-32P]-ATP was added and the reactions were stopped after 1 min.",
    "molecules": "ATP, GlcN6P, 32P"
  },
  {
    "caption": "B Strains Z197 (wild type) and Z225 (∆rapZ) were transformed with the following plasmids expressing the mentioned sRNAs: pBR-plac (vector control = VC), pYG83 (glmY), pYG84 (glmZ) and pSD69 (gcvB). sRNA expression was induced with 1 mM IPTG and β-galactosidase activities were determined in the exponential growth phase.",
    "molecules": "IPTG"
  },
  {
    "caption": "C To assess the impact of GlmY* and GlmZ on stimulation of QseE' autophosphorylation by RapZ, 1 μM QseE'-His10 was incubated with [γ-32P]-ATP in absence or presence of 5 μM Strep-RapZ and/or the sRNAs GlmY*, GlmZ and GcvB. In lanes 3-11 QseE' was co-incubated with RapZ as well as the indicated sRNAs provided at 0.5 μM, 1.25 μM and 2.5 μM. In lanes 14-16, QseE' was incubated with 2.5 μM of each sRNA without RapZ. Samples were removed 1 min after addition of [γ-32P]-ATP and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.",
    "molecules": "ATP, 32P, PAA, SDS"
  },
  {
    "caption": "Northern blot experiments assessing the half-lives of GlmY* and GlmZ under normal growth and GlcN6P starvation conditions. Bacterial cultures were either treated with 100 μg/ml Nva-FMDP or H2O (mock). Transcription was stopped by rifampicin addition when cultures attained OD600 = 1.0 and samples were removed at indicated times for Northern analysis. A Analysis of GlmY* and GlmZ decay in the wild type strain Z8. B Analysis of GlmY* and GlmZ decay in the ∆rapZ mutant Z28. C Semi-logarithmic plots of GlmY* and full-length GlmZ decay for half-life determination. ",
    "molecules": "GlcN6P, Nva-FMDP, rifampicin, H2O"
  },
  {
    "caption": "Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis. A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow). B The ∆glmS ∆rapZ double mutant Z1127 was tested. C Semi-logarithmic plots of GlmY* decay for half-life determination. ",
    "molecules": "GlcN, GlcN6P, rifampicin"
  },
  {
    "caption": "D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow.",
    "molecules": "GlcN6P, PAA"
  },
  {
    "caption": "E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow.",
    "molecules": "GlcN6P"
  },
  {
    "caption": "MCF7 cells were infected with control shRNA (sh-CTL) or two independent shRNAs specifically targeting circPVT1 (sh-circPVT1#1 and sh-circPVT1#2) were subjected to colony formation assay (I) (n = 3 biological replicates, ± s.e.m., ***P < 0. 001 by two-tailed Student's t-test). (J) Quantification of the crystal violet dye as shown in (I) (n = 3 biological replicates, ± s.e.m., ***P < 0.001 by two-tailed Student's t-test).",
    "molecules": "crystal violet"
  },
  {
    "caption": "(O) Tamoxifen-resistant MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were treated with or without tamoxifen (Tam, 5 μM, 72 h) followed by cell proliferation assay",
    "molecules": "Tam, Tamoxifen, tamoxifen"
  },
  {
    "caption": "(B) Total RNAs extracted from MCF7 cells were incubated with or without RNase R (10 units/μg RNA) at 37℃ for duration as indicated, followed by RT-qPCR analysis to examine the expression of PVT1 or circPVT1 (C) MCF7 cells were treated with Actinomycin D (10 g/ml) for duration as indicated, followed by RT-qPCR analysis to examine the expression of PVT1 or circPVT1",
    "molecules": "Actinomycin D"
  },
  {
    "caption": "(G) MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were subjected to RNA-FISH analysis using probe specifically targeting circPVT1. Red: circPVT1; Blue: DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F) MCF7 cells transfected with si-CTL, si-circPVT1#1, or si-circPVT1#2 were treated with or without estrogen (E2, 10-7 M, 6 h) followed by RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated (G) MCF7 cells infected with sh-CTL, sh-circPVT1#1, or sh-circPVT1#2 were treated with or without estrogen (E2, 10-7 M, 6 h) followed by RNA extraction and RT-qPCR analysis to examine the expression of genes as indicated",
    "molecules": "E2, estrogen"
  },
  {
    "caption": "MCF7 cells transfected with control siRNA (si-CTL) or siRNAs specifically targeting circPVT1 (si-circPVT1#1 and si-circPVT1#2) were treated with or without estrogen (E2, 10-7 M, 6 h) followed by immunoblotting analysis (D) to examine the expression of ERα",
    "molecules": "E2, estrogen"
  },
  {
    "caption": "(P) MCF7 cells were infected with control shRNA (sh-CTL) or shRNAs specifically targeting circPVT1 (sh-circPVT1) in the presence or absence of miR-181a-2-3p inhibitor were subjected to colony formation assay. (Q) Quantification of the crystal violet dye as shown in (P) (n = 3 biological replicates, ± s.e.m., **P < 0.01 by two-tailed Student's t-test).",
    "molecules": "crystal violet"
  },
  {
    "caption": "(L) HEK293T cells transfected with HA-tagged MAVS and Flag-tagged RIGI in the presence or absence of circPVT1 were subjected to immunoprecipitation (IP) with anti-Flag M2 agarose followed by immunoblotting analysis using antibodies as indicated.",
    "molecules": "agarose"
  },
  {
    "caption": "(I, J) MCF7 cells were treated with ASO-CTL, ASO-circPVT1 (50 nM), or fulvestrant (ICI, 1 μM) for duration as indicated followed by cell proliferation assay (I) and colony formation (J) (n = 3 biological replicates, ± s.e.m., **p < 0.01, ***p < 0.001, day 4 by two-tailed Student's t-test).",
    "molecules": "fulvestrant, ICI"
  },
  {
    "caption": "E. HeLa cells expressing Arf1-HA were serum starved overnight (E, top) and subsequently stimulated with EGF for 5 min (E, bottom) prior to fixation with PFA. Fixed cells were stained for Arf1 (HA; green) and GIV (red) and nuclei (DAPI; blue). Panels on the left show overlay of all 3 stains and representative RGB plots of sections through the Arf1-stained pixels. Panels on the right display the magnified 3D surface plots of the boxed regions in the left panels. Scale bar = 10 µm.",
    "molecules": "DAPI, PFA"
  },
  {
    "caption": "F. HeLa cells starved with 0.2% FBS overnight or stimulated subsequently with 50 nM EGF were fixed and stained for active Gαi (green; anti-Gαi:GTP mAb) and Man II (red) and analyzed by confocal microscopy. Activation of Gαi was detected exclusively after EGF stimulation. When detected, active Gαi colocalizes with Man II (yellow pixels in merge panel). See also Figure EV2C-D for additional time points and stimulus. Scale bar = 7.5 µm.",
    "molecules": "GTP"
  },
  {
    "caption": "G-H. Control (parental) and GIV-depleted (GIV KO) HeLa cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as indicated. Line graphs in 3D (G) depict the formazan absorbance expressed as a measure of cell viability from the HeLa cells in various conditions tested. Bar graphs (H) depict the cell number in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA.",
    "molecules": "BFA, formazan"
  },
  {
    "caption": "I-K. Control (parental) and GIV-depleted (GIV KO) MDA MB-231 cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as in G-H. Line graphs in 3D (I) depict the formazan absorbance expressed as a measure of cell viability from the MDA MB-231 cells in various conditions tested. Bar graphs (J) depict the viability of the MDA MB-231 cells in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA. Immunoblots (K) of equal aliquots of whole cell lysates confirm the depletion of GIV compared to tubulin (loading control). See also Appendix Figure S5D-H for dot plots and early and late apoptotic fractions. Results are expressed as mean ± S.E.M; n = 3 biological replicates.",
    "molecules": "BFA, formazan"
  },
  {
    "caption": "Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diplotene Arrows in (I-L) indicate the position of some chromocenters. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.",
    "molecules": "DAPI"
  },
  {
    "caption": "O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.",
    "molecules": "DAPI"
  },
  {
    "caption": "Q, R Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.",
    "molecules": "DAPI"
  },
  {
    "caption": "S, T Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.",
    "molecules": "DAPI"
  },
  {
    "caption": "G-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.",
    "molecules": "DAPI"
  },
  {
    "caption": "K-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red).",
    "molecules": "histone"
  },
  {
    "caption": "D 300% magnification of selected zygotene-like and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals. E Quantification of telomeres with a regular or altered disposition of telomeric DNA in Pds5AB cKO spermatocytes at the indicated stages (n=1198 telomeres in zygotene-like spermatocytes and 574 in pachytene-like ones).",
    "molecules": "DNA"
  },
  {
    "caption": "(B-E) Co-localization of EGFP and the two CA isoforms with filamentous actin studied in fibroblasts expressing (B) EGFP, (C) EGFP-CA2, or (D, E) EGFP-CA7 and stained with phalloidin-594 to visualize F-actin (n = 4, 10 and 8 independent replicates, respectively). A magnification of the area marked with the yellow rectangle in (C) and (D) shows the localization of EGFP-CA2 and EGFP-CA7 compared to phalloidin-594. (E) EGFP-CA7 caused a prominent overexpression phenotype with thick and curvy cytosolic actin bundles (arrow) and plasmalemmal protrusions (arrowhead).",
    "molecules": "phalloidin-594"
  },
  {
    "caption": "(B) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the absence (PBS control, upper panel) or presence of mCA7 (lower panel). Numbers in images indicate the time after the onset of the experiment (0, 5 and 23 min). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. Scale bar 10 µm. (C) Quantification of the mean increase in filament length (n = 10 filaments at each time point) and the mean relative fluorescence intensity values of cross-sections for individual filaments /bundles (n = 30 - 31) in the absence and presence of mCA7 (1.12 µM). The data were analyzed using a general mixed model with time as a within-unit factor and the presence of CA7 as a between-unit factor. n = 3 independent repetitions, experiment repeats were included as a covariate and were non-significant. ",
    "molecules": "Rhodamine"
  },
  {
    "caption": "(E) Fluorescence time‐lapse images of F-actin bundling in an in vitro bundling assay. A mixture of unlabeled and Rhodamine labelled non-muscle actin was polymerized in the presence of mCA7 (0.11 µM). Numbers above images indicate the time after the onset of the experiment (22 min -23 min 20 sec). Intensity based Fire-coloring (Fiji) was used to visualize intensity changes. White arrows highlight the bundling filaments. Scale bar 5 μm. This is a representative video from 8 similar experiments.",
    "molecules": "Rhodamine"
  },
  {
    "caption": "(A,B) NIH3T3 cells transfected with DsRed (A) or DsRed-CA7 (B) were incubated in growth medium with 5 µM Latrunculin B for 0, 2, 5, 10, or 30 minutes, or in an equal amount of DMSO for 60 minutes. Analyses of experiments show that in cells transfected with DsRed-CA7 F-actin structures collapse more slowly (0 min: 83% \"normal\"; 2 min: 80%, p = 0.44; 5 min: 74%, p = 0.39; 10 min: 27%, p = 0.08; 30 min: 16%, p = 0.02; DMSO: 82%, p = 0.99; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) than in the DsRed-transfected ones (0 min: 89% \"normal\"; 2 min: 48%, p = 0.14; 5 min: 31%, p = 0.04; 10 min: 2%, p = 0.001; 30 min: 0.7%, p = 0.001; DMSO: 88%, p = 0.8; tested against 0 min with two-way ANOVA, Dunnett's multiple comparison test) . For the analysis, cells were categorized to three groups as \"normal\", \"some shape/F-actin left\" and \"round\". The upper panel shows example images of the cells in all three categories for (A) DsRed- or (B) DsRed-CA7 transfected cells (actin visualized with Phalloidin-488).",
    "molecules": "Phalloidin-488, DMSO, Latrunculin B"
  },
  {
    "caption": "(A-C) NIH3T3 fibroblasts expressing EGFP-CA7-mutant1 (A), EGFP-CA7-mutant2 (B), and EGFP-CA7-mutant3 (C). F-actin is visualized with Phalloidin-594. In the right-most panel of (A-C) are the normalized fluorescence intensity profiles of the mutated CA7 EGFP signal (black) and actin (red) and the yellow line in left-most panels indicates the cross-section from which the pixel intensities were measured. Scale bars 20 µm.",
    "molecules": "Phalloidin-594"
  },
  {
    "caption": "(D) Analysis of the mutated CA7 and F-actin co-localization in cultured fibroblasts. Scatterplots of fluorescent intensities per pixel (EGFP vs. Phalloidin-594) along a cross section through a representative cell. Pearson's correlation coefficient (r) for the analyzed cell is given in each panel.",
    "molecules": "Phalloidin-594"
  },
  {
    "caption": "(B) Representative confocal images of apical dendrites from Lucifer Yellow injected cortical layer 2/3 pyramidal neurons from WT and CA7 KO mice. The dendritic spine density and spine head size were examined in fixed slice preparations from P34 - P37 mice. Scale bar 2 µm.",
    "molecules": "Lucifer Yellow"
  },
  {
    "caption": "(C) Summary of the spine density analysis done from the Lucifer Yellow injected neurons. Spine density was a higher in CA7 KO neurons both in apical and basal dendrites (n = 28 neurons for both) compared to WT (n= 29 neurons for apical and n=30 for basal dendrite analysis) (P = 0.000002 for apical dendrites, analyzed with Mann-Whitney test, and P = 6,8 x 10-8 for basal dendrites, Student's t-test with Welch-correction.) A total of 8279 spines were analyzed from four CA7 KO mice and 8730 spines from two WT control mice.",
    "molecules": "Lucifer Yellow"
  },
  {
    "caption": "B 18F-FDG PET-MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX.",
    "molecules": "18F-FDG, FOLFIRINOX"
  },
  {
    "caption": "D Phase-contrast images of organoids (upper panel) and respective 2D cultures (isolated from the biopsy before FOLFIRINOX (ID188) or after the neoadjuvant therapy from the resected cancer (ID211). Scale bar represents 200µm.",
    "molecules": "FOLFIRINOX"
  },
  {
    "caption": "A Dose response curves of ID188 (p27-29) and ID211 (p26-28) organoids treated with FOLFIRINOX over three days. ATP was measured with CellTiter-Glo assays. Shown is the mean ± SD of three independent experiments.",
    "molecules": "ATP, FOLFIRINOX"
  },
  {
    "caption": "E - J Dose response curves of ID188 and ID211 organoids (ID188 p27-29, ID211 p26-28) or 2D lines (ID188 p5+29, ID211 p9+13) treated with (E and F): Cobimetinib or (H and I): Biminetinib. (G) Cobimetinib and (J) Biminetinib: GI50 values. Shown is the mean ± SD of three independent experiments.",
    "molecules": "Cobimetinib"
  },
  {
    "caption": "K Illustration of a cohort of patients (n=15) with available primary 2D PDAC cell lines. This cohort includes the ID188 (pre-FFX (FOLFIRINOX), red) and ID211 (post-FFX, blue) lines. These lines were screened for sensitivity towards three MEKi. L 15 primary human PDAC 2D cell lines were screened for MEKi (as indicated) sensitivity. The determined AUC was variance scaled and the z-scores are depicted. The ID188 and ID211 identity is color coded.",
    "molecules": "FFX, FOLFIRINOX"
  },
  {
    "caption": "B) SPPL2c impairs secretion and maturation of glycan-modifying enzymes. Protein levels of endogenous EXTL3, B4GALT1, OGFOD3 and GnT-V were monitored in lysates of cells treated with control siRNA (siCtr) or with siRNA targeting SPPL3 (siSPPL3) and in cells ectopically expressing either SPPL2c or SPPL3 by Western Blot. In addition, the secreted forms (sEXTL3, sB4GALT1, sOGFOD3, sGnTV,) were analyzed in the corresponding conditioned media using Western Blot. Note that SPPL2c reduces secretion of the glycosyltransferases similar to SPPL3 knock down, but additionally induces a decrease of the mature glycosyltransferases in the cell lysate similar to ectopic expression of SPPL3. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control",
    "molecules": "glycan"
  },
  {
    "caption": "After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A) Recombinant proteins were mixed, as indicated, in the presence (lane 5) or absence (lane 4) of ATP and incubated for 1 h at 30°C before being separated by SDS-PAGE and analyzed by western blotting using the α-His antibody. Components present (+) or absent (−) from the assay are indicated.",
    "molecules": "ATP"
  },
  {
    "caption": "(A) The occurrence of mC-ATG5 puncta in WT L. major promastigotes expressing mC-ATG5 incubated in nutrient-rich medium at log phase at 26°C.",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) Co-labelling of puncta with mC-ATG5 and GFP-ATG12 co-expressed in promastigotes incubated in PBS for 2 h at 26°C. (D) In nutrient-rich medium, GFP-ATG8 punctum without mC-ATG5 staining is arrowed. (E) Incubated in PBS for 30 min at 26°C. (F) In nutrient-rich medium at 26°C. (G) Incubated in PBS for 30 min at 26°C, the small panels are enlargements of the merged panels. (H-I) Promastigotes in nutrient-rich medium. (J-K) Promastigotes at late log phase in nutrient-rich conditions. (L) Promastigotes at stationary phase in nutrient-rich conditions. Scale bar throughout, 10 µm.",
    "molecules": "nutrient"
  },
  {
    "caption": "(B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm",
    "molecules": "nutrient"
  },
  {
    "caption": "(C) Occurrence of GFP-ATG8 puncta in promastigotes when incubated in the conditions detailed in (B). Means ± SD from four independent experiments. * and **, occurrence of GFP-ATG8 puncta in Δatg5 were significantly different from in WT in nutrient-deprived and nutrient-rich conditions (p<0.05).",
    "molecules": "nutrient"
  },
  {
    "caption": "Phospholipid accumulation in Δatg5 promastigotes.Negative ion survey scans (650-900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).",
    "molecules": "lipids"
  },
  {
    "caption": "A) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient-rich medium. Shown are ovoid and amastigote-like form (left); spindled-shaped form without an external flagellum (centre left panel) and with an external flagellum of varying lengths (centre right and right panels). Scale bar, 10 µm.",
    "molecules": "nutrient"
  },
  {
    "caption": "G. β-catenin-mediated reporter activity in HEK293T cells expressing Wnt3a and WT RNF43, oncogenic RNF43 (R519X) or a LOF RNF43 variant (I48T) after o/n treatment with DMSO or the PORCN inhibitor C59 (1 μM).",
    "molecules": "DMSO, C59"
  },
  {
    "caption": "A. Volcano plot showing proteins enriched after streptavidin pull-down of biotin-treated HEK293 cells stably expressing dox-inducible RNF43-BirA*. Striped line demarcates the empirical 0.01 False discovery rate (FDR) cut off. Significantly enriched Wnt/β-catenin pathway components are highlighted in red and RNF43 in orange.",
    "molecules": "biotin, dox"
  },
  {
    "caption": "B. Relative outgrowth of WT, TP53KO and onco-RNF43/TP53KO organoid lines treated with the PORCN inhibitor C59 (1 μM) for 7 days. Graph shows the number of organoids at 5 days after splitting (day 14) relative to the number of organoids at day 2. Error bars represent ±s.d. of the mean of n = 3 experiments.",
    "molecules": "C59"
  },
  {
    "caption": "Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.",
    "molecules": "DMSO, PP2"
  },
  {
    "caption": "In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.",
    "molecules": "DMSO, PP2"
  },
  {
    "caption": "A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L-MICA-129Met and an L-MICA-129Val clone. L-con cells served as a negative and K562 cells as a positive control. The means of specific lysis of triplicates plus SD at different E:T ratios (200:1 to 3:1) were measured in an 51chromium-release assay. The MICA expression intensity and the binding of a recombinant NKG2D-Fc fusion protein to the target cells were determined in parallel by flow cytometry, and the MFIs are indicated.",
    "molecules": "51chromium"
  },
  {
    "caption": "MACS-separated CD8+ T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed (n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (*P < 0.05, t-test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484).",
    "molecules": "thymidine"
  },
  {
    "caption": "B,C HR and R-R interval of WT (black bars) and Adcy9-/- mice (red bars) before and after ISO (1 μg/g) injection",
    "molecules": "ISO"
  },
  {
    "caption": "D Representative ECG recordings of 7 mo WT and Adcy9-/- mice, ~55 min post ISO-injection.",
    "molecules": "ISO"
  },
  {
    "caption": "E,F HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences (RMSSD; panel (E)) and percentage of sequential R-R intervals differing by >6 ms (pNN6; panel (F)) methods.",
    "molecules": "ISO"
  },
  {
    "caption": "A,B PLA assay performed in neonatal cardiomyocytes expressing GFP-Flag or AC9-Flag using antibodies against Flag and endogenous POPDC1, POPDC2 or Gβγ. Images (A); scale bar is 20 um; nuclear staining by DAPI shown in blue) and quantitation (B) of PLA signal are shown.",
    "molecules": "DAPI"
  },
  {
    "caption": "A HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or -2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=6 experiments, P=0.003 between all groups) with multiple comparisons to AC9 control by Dunn's method and Mann-Whitney Rank Sum Test (**P=0.002).",
    "molecules": "GTPγS"
  },
  {
    "caption": "A IP-AC assay with IgG versus anti-TREK-1 in heart homogenates from WT and Adcy9-/- mice; AC activity stimulated with 300 nM Gαs-GTPγS. IP-AC with anti-AKAP150 is shown as a positive control. Two-way ANOVA (overall P<0.001, n=3 mice per genotype) with multiple comparisons by Holm-Sidak test; #P<0.001 for indicated comparison or to respective IgG controls. IgG versus anti-TREK-1 in Adcy9-/- was analyzed by Student's t-test (**P=0.009, n=3). A portion of the co-IP was subjected to western blotting with anti-TREK-1 and anti-AKAP150.",
    "molecules": "GTPγS"
  },
  {
    "caption": "C Total AC activity in heart homogenates from WT or Popdc1-/- (P1-/-) mice stimulated with 300 nM Gαs-GTPγS or 100 μM calcium and 300 nM calmodulin.",
    "molecules": "calcium, calmodulin, GTPγS"
  },
  {
    "caption": "D IP-AC assay with anti-MYC from cell lysates expressing AC1 or AC8 in the presence or absence of Myc-tagged POPDC1. AC activity stimulated with 100 μM calcium and 300 nM calmodulin. Student's t-test (n=3 experiments, **P=0.0011 and *P=0.012).",
    "molecules": "calcium, calmodulin"
  },
  {
    "caption": "A Treatment of HEK293 cells for 10 min in the presence of 10 µM ISO reduced the amount of Flag-AC9 and POPDC1-Myc pulled down by TREK-1 co-IP.",
    "molecules": "ISO"
  },
  {
    "caption": "B, C Quantification (IP/input) of Flag-AC9 (B) and POPDC1-Myc (C) between ISO treatment groups by two-tailed paired Student's t-test (*P=0.028, **P=0.007; n=3 biological replicates).",
    "molecules": "ISO"
  },
  {
    "caption": ", B BiFC signal between the indicated VN- and VC-tagged proteins (POPDC1, P1). B Percent decrease of BiFC signal in (A) with ISO treatment (10 μM, 10 min at 37°C). Paired t-test for each condition (vehicle versus ISO) using raw data prior to calculation of percent of vehicle control (n=6; *P<0.05; **P<0.01",
    "molecules": "ISO"
  },
  {
    "caption": "C ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9D).",
    "molecules": "ISO"
  },
  {
    "caption": "E Overexpression of POPDC1 truncation of Popeye domain (POPDC1-Δ172), but not WT POPDC1, abolishes ISO reduction of TREK-1:AC9 BiFC signal. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4 experiments; ***P<0.001 compared to control).",
    "molecules": "ISO"
  },
  {
    "caption": "C Relative currents normalized to TREK-1 plus POPDC1 in the absence and presence of theophylline. Data are plotted as mean ± SEM.",
    "molecules": "theophylline"
  },
  {
    "caption": "D, E Oocytes were treated with 0.5 mM theophylline prior to voltage-clamp measurements as in (A, B).",
    "molecules": "theophylline"
  },
  {
    "caption": "A Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti-puromycin antibody. Scale bar, 10 μm. B Quantification of fluorescence intensity per cell of experiment shown in (A). AU, arbitrary units. (n= 90-110 cells per treatment isolated from 4 mice). Data information data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values.",
    "molecules": "puromycin"
  },
  {
    "caption": "C Quantification of the number of puromycin granules under indicated conditions (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test).",
    "molecules": "puromycin"
  },
  {
    "caption": "D Confocal images of primary hepatocytes stained with anti-puromycin and anti-CLUH antibodies. The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S1B for each individual channel. Scale bar, 4 μm. Cells analyzed were isolated from 4 different mice with similar results. Arrows point to colocalizing particles.",
    "molecules": "puromycin"
  },
  {
    "caption": "E Manders' colocalization coefficient between puromycin and CLUH from experiment shown in (D) (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (E) ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).",
    "molecules": "puromycin"
  },
  {
    "caption": "F Quantification of puromycin granules containing CLUH signal (n ≥ 80 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test).",
    "molecules": "puromycin"
  },
  {
    "caption": "D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and incubated in HBSS medium with or without CHX. Cells were recorded for a maximum of 2 h. Insets show 2.5x enlarged areas. Scale bar, 10 µm. E Total number of cells analyzed by life imaging for the indicated experiments shown in (D). \"Positive\" relates to a cell which formed G3BP1 granules at the end of the recording. ",
    "molecules": "CHX"
  },
  {
    "caption": "H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin. More than 100 cells per replicate were analyzed in a blind fashion (n=3 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values.",
    "molecules": "rapamycin"
  },
  {
    "caption": "H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies. Two different exposure times are shown for LC3. I refers to total LC3 and II indicates lipidated form of LC3. Asterisk indicates signal of previous incubation. I Quantification of LC3 levels in western blots as shown in (H) (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).",
    "molecules": "bafilomycin A"
  },
  {
    "caption": "J Quantification of autophagic flux of western blot as shown in (H). The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition. The flux in KO cells was normalized to flux in WT cells. (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).",
    "molecules": "bafilomycin A"
  },
  {
    "caption": "C Western blot analysis of isolated mitochondria from Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. PonceauS staining was used as a loading control.",
    "molecules": "PonceauS"
  },
  {
    "caption": "G Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. When indicated cells were treated with rapamycin (200 nM for 4h). Bottom panels show 2.3x magnified areas for each channel of indicated boxes. Arrows point to colocalizing spots. Scale bar, 10 μm. H Manders´ colocalization coefficient from experiment shown in (G) (n ≥ 50 cells isolated from 3 mice per genotype). Data information: In (H), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).",
    "molecules": "rapamycin"
  },
  {
    "caption": "I Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10μm. J Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).",
    "molecules": "antimycin A, galactose, oligomycin"
  },
  {
    "caption": "K Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152, incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10 μm. L Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).",
    "molecules": "antimycin A, galactose, oligomycin, rapamycin"
  },
  {
    "caption": "A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-TOM20 antibody. Scale bar, 25 µm. B Quantification of morphological parameters of experiments shown in (A). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype).Data information: data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).",
    "molecules": "rapamycin"
  },
  {
    "caption": "C Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-LAMP1 antibody. Scale bar, 25 µm. D Quantification of morphological parameters of experiments shown in (C). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(A) Proof-of-principle experiment. Time-lapse images show overexpression of Myc-tagged CPAP-WT (i) or CPAPΔT (ii) has no effect on two centrosome-containing MCF10A cells (-Dox, two centrosomes). The cells were imaged from interphase to Cytokinesis. Bar graph at right quantifies mitotic duration of cells overexpressing CPAP-WT and CPAPΔT. We define mitotic duration as time consumed from the onset of cell rounding until cytokinesis. Number of cells (n) analyzed in each condition is indicated at the top of each bar. Number of independent experiments (N), (N)=3. Error bars, mean ± SEM. Unpaired t-test. CPAP-WT (iii) or CPAPΔT (iv) expressing extra centrosome-containing MCF10A cells (+Dox, extra centrosomes). Note compared to CPAP-WT expressing centrosomes (iii) CPAPΔT expressing centrosomes nucleate enhanced levels of microtubule asters already at prophase-like stage (iv). White arrows indicate centrosomal dots. Red arrows indicate centrosomes nucleating microtubule asters causing multipolar spindles. Scale bar, 2µm. Bar graph at right quantifies mitotic duration and percentage of cells exhibiting prolonged mitosis in cells expressing CPAP-WT and CPAPΔT. (N)=3. Error bars, mean ± SEM. Unpaired t-test ***P < 0.0001",
    "molecules": "Dox"
  },
  {
    "caption": "(A) 1H NMR spectra of free CCB02 (black) and bound to tubulin in 50:1 ratio (orange). Broadening of CCB02 proton peaks indicate its binding ability to tubulin",
    "molecules": "CCB02"
  },
  {
    "caption": "(B) NOESY spectrum recorded for a solution comprising tubulin (6.5µM), CPAP peptide (400µM) and CCB02.1 (200µM) with 70ms mixing time. The signals colored in green and (highlighted by boxes) correspond to inter-ligand trNOE signals arising due to protein-mediated spin-diffusion (INPHARMA NOEs), while the black peaks represent intra-ligand trNOE signals.",
    "molecules": "CCB02.1"
  },
  {
    "caption": "(A) 14 day CCB02 treatment of MDA-MB-231, HCC827-GR, Calu-6 and MCF10A (+Dox, extra centrosomes) mostly eliminated cells with extra centrosomes. At the end of the treatment, cells were fixed and stained for centrosomes. Bar graph shows the percentage of cells displaying two- and more than two centrosomes. 'Vehicle' at the bar graph indicates percentages of cells with extra centrosome Note that percentages of cells containing more than two centrosomes are significantly reduced upon CCB02-treatment (open bars). Data represent mean ± SEM of three independent experiments. (N)=3, (n=70). p values were obtained using unpaired t test **P < 0.001, ***P < 0.0001",
    "molecules": "CCB02, Dox"
  },
  {
    "caption": "(B) CCB02 treatment prevents centrosome clustering in HCC827-GR cells. Activated centrosomes (arrows) fail to cluster leading to multipolar mitosis. Cells were stained with Cep152 (green), CPAP (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm, Insets, 0.5µm.",
    "molecules": "CCB02"
  },
  {
    "caption": "Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression of MCF10A (+Dox, extra centrosomes) (C cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment. Cartoons of a cell with extra centrosomes below to the panel (C) represent various events occurring upon vehicle or CCB02 treatment. Scale bar, 2µm. Bar graphs at right to panel (C quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv) At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001",
    "molecules": "CCB02, Dox"
  },
  {
    "caption": "Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression o HCC827-GR (D) cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment Bar graphs at right to pane (D) quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv). At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001",
    "molecules": "CCB02"
  },
  {
    "caption": "(i) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes (+Dox, extra centrosomes) (A Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP",
    "molecules": "CCB02, Dox"
  },
  {
    "caption": "Experimental strategy to test if the effect of CCB02 is CPAP dependent i MDA-MB-23 cell (B). Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP",
    "molecules": "CCB02"
  },
  {
    "caption": "(C) Fractions of cells containing centrosomes after siRNA treatment. 48 hours of siRNA treatment still contained a fraction of cells with extra centrosomes that were analyzed in the following experiments. Bar graph shows the percentage of cells with extra centrosomes in MCF10A (+Dox, extra centrosomes) in the presence of control siRNA or CPAP siRNA. Data represent mean ± SEM of three independent experiments. (N)=3, (n=300 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01, **P < 0.001",
    "molecules": "Dox"
  },
  {
    "caption": "(D) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A (i) MDA-MB-231 cells (ii) CPAP depletion does not prevent centrosomes from clustering (second rows of i and ii). Note, in CPAP depleted cultures, centrosomes are marked with Cep152 (green) where CPAP (magenta) is detected faintly. In CPAP depleted cells, CCB02 does not prevent extra centrosomes from clustering (fourth rows of i and ii). Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA (third rows of i and ii). Cells are stained for Cep152 (green), CPAP (magenta), microtubules (α-tubulin, red) and DNA (DAPI, blue). Scale bar, 2µm",
    "molecules": "CCB02, DNA"
  },
  {
    "caption": "(F) Bar graph shows the percentage of living (TUNEL-negative) and dyeing (TUNEL-positive) cells under various conditions tested. Higher percentage of TUNEL-positive cells is observed in CCB02-treated cultures. Data represent mean ± SEM of three independent experiments. (N)=3, (n=500 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01.",
    "molecules": "CCB02"
  },
  {
    "caption": "(A) Interphase centrosomes of CCB02-treated two-centrosome containing MCF10A cells display an enhanced level of PCM proteins. CPAP (green) intensity remains same. Cells are stained for CPAP (green), PCM proteins (magenta) Cep152 (i), PCNT (ii) and CDK5RAP2 (iii) and microtubules (α-tubulin, red). Scale bars, 2µm for left panels and 0.2µm for right panels. Heat map images show intensity saturation (blue) for CPAP, Cep152, PCNT and CDK5RAP2 upon CCB02 treatment. Line graphs at right to each panels indicate the heat map intensity of single centrosomes of cells (given in boxes at heat map panels) treated with vehicle (blue line) or CCB02 (red line). Images were recorded with low laser excitation and high zoom factor",
    "molecules": "CCB02"
  },
  {
    "caption": "(B) Bar graph quantifies relative intensity of CPAP-interacting proteins in interphase centrosomes compared to vehicle treatment. At least 150 centrosomes (for vehicle); 130 centrosomes (for CCB02) were analyzed in each condition. Error bars, mean ± SEM. (N)=3. Ordinary Two-way ANOVA test ***P < 0.0001",
    "molecules": "CCB02"
  },
  {
    "caption": "(C) Immunopurification of CPAP complexes from CCB02-treated cell extracts. (i) CCB02 specifically prevents CPAP-Tubulin interaction. This allows CPAP to bind an enhanced amount of Cep152 and γ-tubulin. Control IP experiment is shown below. (ii) Plot showing PCM protein intensities co-immunoprecipitated with CPAP at various concentrations of CCB02 treatment. CPAP amount does not change with CCB02 treatment. Data represent mean ± SEM. (N)=3.",
    "molecules": "CCB02"
  },
  {
    "caption": "(B-D) MT-regrowth assays at 0, 1.5 and 3.0 min using MCF10A (-Dox, two centrosomes), MCF10A (+Dox, extra centrosomes) and MDA-MB-231 cells. MT nucleation panel is shown in grey scale (inset images are inverted) and γ-tubulin is shown in red. Note that in contrast to vehicle treatment, CCB02 treatment caused centrosomes to nucleate an enhanced level of microtubules already at 1.5 min after induction of regrowth with simultaneous increase in γ-tubulin recruitment. All these cells were stained with γ-tubulin (red), microtubules (α-tubulin, grey) and DNA (DAPI blue). Scale bar, 2µm and insets, 0.5 µm. Bar graphs show MT and γ-tubulin intensities at 3 min after induction of MT regrowth. (N)=3. At least 80 centrosomes were considered to calculate intensities from each cell line. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001.",
    "molecules": "CCB02, DNA, Dox"
  },
  {
    "caption": "(A) Bar graph quantifies relative intensity of PCM proteins at interphase centrosomes of cells treated with vehicle, CCB02, and known tubulin binders. Two centrosomes containing MCF10A (-Dox, two centrosomes) cells were used. Elevated PCM proteins intensities were detected only with CCB02 compared to vehicle treated cells. However, a slight increase in PCNT was observed with Docetaxel treatment. Number of centrosomes tested, n=100 (vehicle); n=100 (for CCB02, Taxol, Bacatalin III, Docetaxel and Vinblastine). (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001, ***P < 0.0001",
    "molecules": "Bacatalin III, CCB02, Docetaxel, Dox, Taxol, Vinblastine"
  },
  {
    "caption": "(B) CCB02 but not conventional tubulin binders (Taxol, Bacatalin III, Docetaxel and Vinblastine; concentrations used 30nM) causes activation of extra centrosomes and prevents them from clustering. MCF10A cells with extra centrosomes (+Dox, extra centrosomes) were used. In contrast to CCB02-treatment, interphase centrosomes of tubulin binders-treated cells consistently display clustered centrosomes. Note, disrupted mitotic spindles were observed in tubulin binders-treated cells due to their general toxicity to microtubules. Cells were stained with CPAP (green), Cep152 (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm. Three independent experiments (N)=3 with each at least 100 cells were examined per treatment",
    "molecules": "Bacatalin III, CCB02, Docetaxel, Dox, Taxol, Vinblastine"
  },
  {
    "caption": "(C) Bar graph quantifies percentages of cells exhibiting multipolar spindles in interphase and mitosis of vehicle, CCB02 and conventional tubulin binders-treated cells. At least 150 cells were analyzed for each independent experiment (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001",
    "molecules": "CCB02"
  },
  {
    "caption": "(D) Kymographs (i) and averaged microtubule polymerization velocities mediated by vehicle and CCB02. Graph below represents the microtubule growth velocities mediated by vehicle (red) and CCB02 (blue) during microtubule end-tracking assay (ii). CCB02 does not alter the microtubule growth although there is a slight increase in growth at 5µM. At least 75 filaments were analyzed for each condition. The error bars represent mean ± SEM of three independent experiments (N)=3",
    "molecules": "CCB02"
  },
  {
    "caption": "(E) Snap-shot images show the effect of CCB02 on microtubule dynamics in two-centrosome containing MCF10A cells. The microtubule was stained with SiR-tubulin and +end tip of microtubule was marked with EB3-EGFP. A 5µM CCB02 did not alter localization and dynamics of EB3 at the microtubules indicating that the CCB02 does not affect microtubule dynamic At least, 50 cells were analyzed for each condition. Scale bar, 2µm",
    "molecules": "CCB02, SiR"
  },
  {
    "caption": "(F) Box plots represent the speed (µm/sec) (i), lifetime (sec) (ii), and length (µm) (iii) of growing microtubule tracks in MCF10A cells treated with 1, 2 and 5µM of CCB02. At least, 60 filaments were analyzed from each cell from a total of 30-40 cells. (N)=3. Error bars in boxplots mean ± SD. Ordinary One-way ANOVA test was performed. Boxes in all plots show the growing microtubule parameters such as speed (µm/sec), lifetime (sec) and length (µm/sec). Horizontal lines in boxes show the median value. Whiskers at both extremes of boxes indicate minimum and maximum values.",
    "molecules": "CCB02"
  },
  {
    "caption": "(A) Real time live imaging of NSCLC (H1975T790M) 3D spheroids with vehicle, 5µM erlotinib or 5µM CCB02. At the end of live imaging, spheroids were fixed and stained for F-actin (green). Arrow indicates invasive (vehicle-treated) and non-invasive (CCB02-treated) structures in spheroids. Scale bar, 100µm",
    "molecules": "CCB02, erlotinib"
  },
  {
    "caption": "(B) Time series plot shows the spheroid growth rate measurements. CCB02 treatment prevents the spheroid growth rate by inhibiting the formation of invasive protrusions At least 10 spheroids were measured for each condition. Error bars, mean ± SEM. Unpaired t-test ***P < 0.0001",
    "molecules": "CCB02"
  },
  {
    "caption": "(D) Antitumor activity of CCB02 in vivo. Left: Subcutaneous xenograft tumor volume measurements of H1975T790M (30mg/kg of weight, daily) in nude mice treated with vehicle or CCB02. Right: Bar graph shows total tumor volume at day 1 and day 24 of vehicle or CCB02 treated xenograft. Error bars, mean ± SD with n = 8 for control vehicle and n = 8 for CCB02 treatment. Unpaired t-test *P < 0.01.",
    "molecules": "CCB02"
  },
  {
    "caption": "b. SARS-CoV-2 S protein-mediated cell-cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 μm).",
    "molecules": "EK1, EK1C, EK1P"
  },
  {
    "caption": "c. Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell-cell fusion.",
    "molecules": "EK1, EK1C, EK1P"
  },
  {
    "caption": "e. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell-cell fusion.",
    "molecules": "EK1"
  },
  {
    "caption": "f. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar).",
    "molecules": "EK1"
  },
  {
    "caption": "HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SARS-CoV (a)",
    "molecules": "EK1C4"
  },
  {
    "caption": "HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of MERS-CoV (b)",
    "molecules": "EK1C4"
  },
  {
    "caption": "HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-OC43 (c)",
    "molecules": "EK1C4"
  },
  {
    "caption": "HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-229E (d)",
    "molecules": "EK1C4"
  },
  {
    "caption": "to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-NL63 (e)",
    "molecules": "EK1C4"
  },
  {
    "caption": "to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of WIV1 (f)",
    "molecules": "EK1C4"
  },
  {
    "caption": "to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of Rs3367 (g)",
    "molecules": "EK1C4"
  },
  {
    "caption": "to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SHC014 (h).",
    "molecules": "EK1C4"
  },
  {
    "caption": "i to o. Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i)",
    "molecules": "EK1C4"
  },
  {
    "caption": "to o. Inhibitory activity of EK1C4 in PsV infection assays against MERS-CoV (j)",
    "molecules": "EK1C4"
  },
  {
    "caption": "Inhibitory activity of EK1C4 in PsV infection assays against HCoV-OC43(k)",
    "molecules": "EK1C4"
  },
  {
    "caption": "Inhibitory activity of EK1C4 in PsV infection assays against HCoV-229E (l)",
    "molecules": "EK1C4"
  },
  {
    "caption": "Inhibitory activity of EK1C4 in PsV infection assays against , NL63 (m)",
    "molecules": "EK1C4"
  },
  {
    "caption": "Inhibitory activity of EK1C4 in PsV infection assays against WIV1 (n)",
    "molecules": "EK1C4"
  },
  {
    "caption": "Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 (o).",
    "molecules": "EK1C4"
  },
  {
    "caption": "Inhibitory activity of EK1 on live HCoV replication for SARS-CoV-2 (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), and HCoV-NL63 (e).",
    "molecules": "EK1"
  },
  {
    "caption": "In vivo prophylactic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (f) and survival curves (g) of mice challenged with HCoV-OC43. h-i. In vivo therapeutic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (h) and survival curves (i) of mice challenged with HCoV-OC43. Experiments were repeated twice, and the data are expressed as means ± SD.",
    "molecules": "EK1C4"
  },
  {
    "caption": "Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions. Vegetative cells grown in SD medium were observed (A-E). For starvation, logarithmically growing cells in SD were transferred to SD(-N) for 25 min (F-H) or 1 h (I-K) in the presence of 1 mM PMSF. Samples were prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image of Cvt vesicle (marked with an arrow). (B) Immunostaining of a Cvt vesicle (marked with an arrow). (C and D) Higher magnification image of Cvt vesicle. Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle, respectively. (E) Freeze-substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex (marked with an arrow). (F) Freeze-substitution image of the wrapping of a Cvt complex by the isolation membrane (marked with an arrow). (G) Freeze-substitution image of a Cvt vesicle (marked with an arrow) fusing to a vacuole. (H) Freeze-substitution image of autophagic body containing a Cvt complex. (I) Immunostaining image depicting the wrapping of a Cvt complex. The arrow marks the enwrapping membrane. (J) Immunostaining of an autophagosome containing a Cvt complex. (K) Immunostaining of an autophagic body containing a Cvt complex in the vacuole. V, vacuole.",
    "molecules": "nitrogen, PMSF"
  },
  {
    "caption": "Cvt complexes in TVY1 (pep4) cells under nitrogen starvation conditions. Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium (SD[-N]) at 30°C for 30 min (A and B) or 60 min (C and D) and prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image showing a Cvt complex in an autophagosome (marked by an arrow) in the cytosol. (B) Freeze-substitution fixation image showing a Cvt complex in an autophagic body in the vacuole. (C) Immunostaining of a Cvt complex in an autophagic body in the vacuole. (D) Immunostaining of Cvt vesicles (marked with arrows) in the vacuole. AB, Autophagic body; AP, autophagosome; V, vacuole.",
    "molecules": "nitrogen"
  },
  {
    "caption": "Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes. (A) SEY6210 (wild-type), cvt3, and cvt7 yeast were grown in SD to 1 OD600 U/ml and transferred to SD (-N). Aliquots were collected after 0 h (+) and 4 h (−) incubation and subjected to immunoblotting. The positions of precursor (pro) and mature (m) API are indicated. (B) cvt3 and cvt7 cells were pulse labeled for 10 min. After addition of cold cysteine and methionine, the cells were harvested, washed, and resuspended in either SD or SD(-N) and chased for the indicated times. API was recovered by immunoprecipitation, resolved on SDS polyacrylamide gels, and quantified using a phosphorimager (STORM; Molecular Dynamics, Sunnyvale, CA).",
    "molecules": "cysteine, methionine"
  },
  {
    "caption": "(A) Representative single immunofluorescent staining for dsDNA at 6 h, 24 h, and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain. Scale bars, 5 μm.",
    "molecules": "dsDNA"
  },
  {
    "caption": "(B) Representative images of double-immunofluorescent stained for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel). Scale bars, 5 μm.",
    "molecules": "dsDNA"
  },
  {
    "caption": "Mice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for 3 consecutive days after MCAO induction. (A) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT-PCR. n = 6 mice per group. **",
    "molecules": "A151"
  },
  {
    "caption": "(D) Representative images of double-immunofluorescent stained for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. (E) Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. DAPI staining: nuclei.",
    "molecules": "A151, DAPI"
  },
  {
    "caption": "(C) The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by western blot. (D) Quantitative analysis for Western blot analysis of caspapse-1 and IL-1β. *P < 0.05, **P<0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group.",
    "molecules": "LPS, poly(dA:dT)"
  },
  {
    "caption": "(B) Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving A151, or vehicle at days 1, 3, 7, and 14 after MCAO. n = 10 mice per group.",
    "molecules": "A151"
  },
  {
    "caption": "(C) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO, the infarct area is shown in white.",
    "molecules": "TTC"
  },
  {
    "caption": "(E) Flow cytometry plots and (F) summarized results show percentages of Annexin V+ PI+ cells in the brains of MCAO mice receiving A151 or vehicle. n = 6 mice per group.",
    "molecules": "A151, PI"
  },
  {
    "caption": "(F) Representative images of TTC staining of brain sections from vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice on day 3 after tMCAO. Three representative rostrocaudal levels of TTC staining are shown. (G) Bar graph shows percentages of infarct volume of indicated group. n = 6 mice per group. (H) Bar graph shows modified Neurological Severity Score (mNSS) in vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice n = 10 mice per group.",
    "molecules": "A151, TTC"
  },
  {
    "caption": "B The formation of ceramide was determined by a diacylglycerol (DAG) kinase assay (upper panel) and mass spectrometry (lower panel). In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.",
    "molecules": "ceramide"
  },
  {
    "caption": "C, D For determination of the secretion of Asm by platelets into the supernatants (C), tumor cells and wild-type (WT) or Asm-deficient (Asm−/−) platelets were co-incubated for 20 s, the samples were pelleted, the supernatants were removed and acidified, and the Asm activity was measured in the presence or absence of Zn2+. For measurement of surface Asm and ceramide, samples were co-incubated as indicated (C, D) and pelleted; the supernatants were discarded, incubated with anti-Asm antibodies, washed, and lysed; and Asm immunocomplexes were immobilized and subjected to immunocomplex enzyme assays. Surface ceramide was measured by incubation of intact cells with DAG kinase in the presence of [32P]γATP followed by extraction and measurement of [32P]-ceramide. Omission of one cell type indicated by \"−\" here and thereafter.",
    "molecules": "Zn2+, γATP, ceramide"
  },
  {
    "caption": "E Treatment of B16F10tumor cells for 2 min with 1 U/ml ASM or 10 μM C16ceramide restores in vivo metastasis in Asm-deficient mice. After treatment, the cells were injected intravenously into Asm-deficient (Asm−/−) mice. Controls were left untreated prior to injection. The number of metastases was determined 14 days after tumor cell injection.",
    "molecules": "C16 ceramide"
  },
  {
    "caption": "Incubation of human melanoma (HM) cells with human platelets results in the release of Zn2+-dependent ASM into the supernatant, Zn2+-dependent activity of ASM on cell surfaces, and the formation of ceramide. The assay buffer contained 100 μM Zn2+.",
    "molecules": "Zn2+, ceramide"
  },
  {
    "caption": "Addition of human or mouse recombinant ASM to human melanoma or B16F10 cells, respectively, results in binding of ASM to the tumorcell surfaces as determined by flow cytometry. Cytochalasin B was added to control for internalization of added ASM.",
    "molecules": "Cytochalasin B"
  },
  {
    "caption": "Aggregation properties of platelets after co-incubation with collagen or ADP are independent of Asm as determined by aggregometry measurements.",
    "molecules": "ADP, collagen"
  },
  {
    "caption": "4 × 104B16F10tumor cells were incubated with 2 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts) or a 45:55 of WT:Asm−/−platelet mixture in the presence or absence of 50 ng/ml PGE1. Controls were stimulated with Mn2+. Tumor cells were then incubated for 60 s on fibronectin-coated cover slips, washed, and fixed, and adhesion of the tumor cells was determined. The graph displays the mean ± SD of tumor cells adhering to fibronectin-coated cover slips, n = 7 for WT-platelets, n = 9 for Asm-deficient-platelets, n = 4 for 45:55 WT:Asm−/−platelets, and all others n = 3. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. P-values are indicated.",
    "molecules": "fibronectin, Mn2+, PGE1"
  },
  {
    "caption": "A-C 1 × 105 B16F10 cells were incubated with (A) 1 U/ml purified acid sphingomyelinase (ASM) or (B) 5 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts), or a mixture of 45:55 wild-type:Asm-deficient platelets. Cells were fixed with 2% paraformaldehyde for 15 min and stained with fluorescein isothiocyanate (FITC)-coupled anti-α5 integrin, Cy3-labeled anti-β1 integrin, and Cy5-labeled anti-ceramide antibodies. Samples were analyzed by confocal microscopy. Shown are representative examples from four independent experiments (A, B) or the quantitative analysis of cells positive for ceramide/β1 integrin clusters from at least 100 cells/sample (C). Given is the mean ± SD, n = 4, ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.",
    "molecules": "ceramide"
  },
  {
    "caption": "D ASM-induced B16F10 tumorcell adhesion to fibronectin-coated cover slips is abrogated by the inhibition of integrins with RGD peptides. Shown is the mean ± SD of the number of cells adherent to the cover slip. Statistical significance was determined by ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.",
    "molecules": "RGD, fibronectin"
  },
  {
    "caption": "E Intravenous injection of CFSE-labeled B16F10 cells into wild-type mice results in formation of ceramide-enriched domains that contain β1 integrin clusters on tumor cells in vivo, while injection of tumor cells into Asm-deficient mice does not result in ceramide and integrin clustering. Please note that the cell in the lower panel still binds a platelet. Shown are representative results from four independent experiments each.",
    "molecules": "ceramide"
  },
  {
    "caption": "Human melanoma cells were stimulated for 10 min with 1 U/ml purified ASM or 10 μM C16 ceramide (C16-Cer) in the presence or absence of 500 ng ceramidase (CDase) or 1 μg/ml neutralizing anti-ceramide antibodies (anti-Cer), and lysed and active β1 integrin was immunoprecipitated using the HUTS-4 antibody. Octylglucopyranoside (OGP, final concentration 0.01%) served to resuspend C16 ceramide. Internalization was excluded by addition of cytochalasin B (CTB) as indicated. Samples were separated by SDS-PAGE and blotted with an activation-independent anti-β1 Integrin antibody to detect the amount of immunoprecipitated, that is active β1 integrin. Control immunoprecipitates (control Ipt) were performed with an irrelevant isotype control antibody. Activation of β1 integrin in human melanoma cells by incubation with Asm or C16 ceramide was confirmed by FACS analysis upon staining with FITC-labeled HUTS-4 antibodies.",
    "molecules": "Octylglucopyranoside, OGP, ceramide, CTB, cytochalasin B, C16 ceramide, C16ceramide, C16-Cer"
  },
  {
    "caption": "Incubation of human melanoma with human platelets or ASM for 5 min triggers co-clustering of ceramide and activated β1 integrin on the surface of the tumor cells. Cells were stained with FITC-coupled anti-active β1 integrin (HUTS-4) antibodies and Cy3-coupled anti-ceramide antibodies. Shown are representative stainings from each four independent experiments.",
    "molecules": "ceramide"
  },
  {
    "caption": "B16F10tumor cells or B16F10 transfected with siRNA targeting Asm expression or with scrambled control siRNA were labeled with [3H]thymidine, washed, and treated (+) for 10 min with 1 U/ml ASM, 10 μM C16ceramide, 10 μM S1P, 10 μM PDMP, 20 μM sphingosine kinase inhibitor SKI II, and 10 μM myriocin, or left untreated (−). The cells were then injected intravenously into wild-type or Asm-deficient mice as indicated. If indicated, 10 μg of arginine-glycine-aspartic acid (RGD) peptide or 10 μg/ml neutralizing anti-β1 integrin antibodies were added to B16F10tumor cells together with ASM for 15 min prior injection. Mice were sacrificed 30 min after tumorcell injection, the lungs carefully flushed via the right heart, and the radioactivity in the lung as a measurement for adherent tumor cells was determined. Shown is the mean ± SD of the counts per minute (cpm) in the lung lysates from three independent experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's test for multiple comparison to determine P-values.",
    "molecules": "thymidine, SKI II, RGD, myriocin, C16ceramide, S1P, PDMP"
  },
  {
    "caption": "A Amitriptyline (2 mg/kg, Ami) was intraperitoneally injected into C57BL/6mice for five times every 12 h. Sixty minutes after the last injection, 1 × 105B16F10tumor cells were intravenously injected. Control experiments confirmed that amitriptyline inhibited Asm activity in the blood by approximately 85%. Asm heterozygous mice were injected with 1 × 105B16F10tumor cells. The number of lungmetastases was determined after 14 days. Shown is the mean ± SD from six mice each, ANOVA followed by Tukey's test for multiple comparison. P-values are indicated.",
    "molecules": "Amitriptyline, amitriptyline"
  },
  {
    "caption": "E To measure secretion of Asm by platelets, tumor cells and platelets were co-incubated for 30 s, the samples were pelleted, the supernatants were acidified, and the Asm activity was measured. All Asm activity measurements were performed in the presence of 100 μM Zn2+.",
    "molecules": "Zn2+"
  },
  {
    "caption": "B. BrdU-sensitized  U-2-OS cells stably expressing GFP-NLS-CDKL5  were pre-incubated with DMSO  (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273  (0.3 μM) for 1 h prior to  micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μm",
    "molecules": "BrdU, DMSO, olaparib, PDD00017273, talazoparib"
  },
  {
    "caption": "E.  BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ,  PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP  NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μm",
    "molecules": "BrdU"
  },
  {
    "caption": "B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the  GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL)  GFP-NLS-CDKL5 were subjected to line  micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μm",
    "molecules": "BrdU"
  },
  {
    "caption": "F. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on  nitrocellulose membrane and then incubated with synthetic  PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown",
    "molecules": "PAR, nitrocellulose"
  },
  {
    "caption": "(G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2  for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting  using the indicated antibodies. One of two independent experiments is shown",
    "molecules": "PAR, H2O2"
  },
  {
    "caption": "A. (Top)  BrdU-sensitized  U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot  micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated   cells per point. Scale bar is 10 μm",
    "molecules": "BrdU"
  },
  {
    "caption": "BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5,  mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation  (355 nm) and time lapse  imaging. One of three independent experiments is shown. Scale bar is 10 μm",
    "molecules": "BrdU"
  },
  {
    "caption": "E. Same as B-D. except that BrdU-sensitized cells stably expressing  GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect  immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μm",
    "molecules": "PAR, BrdU, olaparib"
  },
  {
    "caption": "F. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm",
    "molecules": "PBS"
  },
  {
    "caption": "B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates.",
    "molecules": "Ser"
  },
  {
    "caption": "C. Boxplots of the VSN-adjusted TMT  reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400,  FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT  channel represents a single biological replicate.",
    "molecules": "TMT"
  },
  {
    "caption": "(C-E). Wild type (WT), CDKL5­-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of  ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm",
    "molecules": "Ser"
  },
  {
    "caption": "(A) Representative images of anterior, dorsal, ventral and lateral lobe sections from 30-week-old ELAC2 WT (n=7), KO (n=7) and A537T (n=5) mice. 5 µm sections of each lobe were cut and stained with haematoxylin and eosin.",
    "molecules": "eosin, haematoxylin"
  },
  {
    "caption": "(A) Representative images of anterior, dorsal, ventral and lateral lobe sections from 30-week-old WT-TRAMP (n=5), KO-TRAMP (n=5) and A537T-TRAMP mice (n=5). 5 µm sections of each lobe were cut and stained with haematoxylin and eosin. (B) Histological changes scored according to Suttie et al. with additional categories (n=6-7 biological replicates), values are means ± SD of biological replicates. Anterior lobe * p =0.013, Dorsal lobe ** p = 0.003, * p = 0.025, Ventral lobe * p =0.031 and Lateral lobe ** p = 0.0038, * p = 0.032, compared with WT-TRAMP by two-tailed Student's t-testing.",
    "molecules": "eosin, haematoxylin"
  },
  {
    "caption": "Kainic acid mouse model of TLE: Daily EEG recordings obtained from the epileptogenic focus starting from one month after KA injection and spanning the period from 2 days before to 7 days after AAV-pDyn delivery (2x109 gp).",
    "molecules": "KA, Kainic acid"
  },
  {
    "caption": "Injection of norBNI (20 mg/kg; i.p.) results in a transient reappearance of HPDs immediately and 24 hrs after application. One week after norBNI application (washout) suppression of HPDs was re-established. Data obtained from 4 epileptic animals before (black) and after (red) AAV-pDyn delivery (2x109 gp) are depicted. *",
    "molecules": "norBNI"
  },
  {
    "caption": "Spatial learning and memory was tested on the Barnes maze. Quadrant 1 (Q1) contains the target hole (red; A). Unilateral injection of AAV-pDyn (B) or AAV-eGFP (C) into naïve young adult mice (12 weeks age) did not influence the performance as compared to naïve controls (D) when tested 4 weeks after AAV injection. Mice treated two weeks after KA with AAV-pDyn (E,H) performed equally to age-matched naïve controls (D,G) 2 weeks (E) and 5.5 months (H) after treatment. By contrast, animals treated two weeks after KA with AAV-ΔGFP (F,I) gradually lost this ability. Two-way ANOVA revealed significance between AAV-ΔGFP and AAV-pDyn treated groups for interaction 2 weeks (p = 0.0349) and 5.5 months (p = 0.0311) after AAV, respectively, at each time interval and quadrant (p < 0.0001) 2 weeks after AAV. Comparison of AAV-pDyn injected with naïve animals revealed no differences.",
    "molecules": "KA"
  },
  {
    "caption": "epileptic mice, which were not able to learn the Barnes maze task one month after KA (J, M), AAV-pDyn application restored spatial memory one (K) and 2 months (L) after treatment.",
    "molecules": "KA"
  },
  {
    "caption": "Double-immunofluorescence labeling is depicted for pDyn and NeuN (A-C) or GFAP (D-F) in the ipsilateral dentate gyrus of KA-treated and AAV-pDyn-injected mice. Enlarged view in (F) represents 15 x 30 µm.",
    "molecules": "KA"
  },
  {
    "caption": ": mature Dyn B content (measured by a Dyn B specific EIA) in the dorsal hippocampus of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n = 6) and 6 (filled symbols; n = 3) months after vector treatment. Naïve animals were age matched to the 1.5 months after AAV group. iH stands for ipsilateral hippocampus and cH for contralateral hippocampus.* = p<0.05; ** = p<0.01; paired t-test was used for comparison of ipsi- and contralateral hippocampi. Two-way ANOVA was used to compare Dyn levels between the early and late time interval.",
    "molecules": "KA"
  },
  {
    "caption": "Mature Dyn B content in the CSF of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n= 6) and 7 (filled symbols; n = 4) months after vector treatment. ** = p<0.01; one-way ANOVA with Dunnett post hoc test",
    "molecules": "KA"
  },
  {
    "caption": ": depicts representative traces under conditions of increased KCl. B: Same as A together with Dyn A/B and C: same as A under co-application of Dyn A/B with 5'-GNTI. D: Burst events and E: interictal spikes were recorded from area CA1.",
    "molecules": "5'-GNTI, KCl"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Serum SUA level",
    "molecules": "SUC, SUA"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. body weight gain",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fat",
    "molecules": "fat, SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lean mass",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. gastrocnemius index.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Gastrointestinal muscle fiber immunofluorescent laminin staining",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. frequency histogram of fiber cross-sectional area.",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. The muscle grip strength",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in low speed",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. four-limb handing time",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. running time in high speed.",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. serum concentration of RBC",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. HGB in whole blood",
    "molecules": "HGB, SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle force",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. ex vivo gastrocnemius muscle force",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. fatigability",
    "molecules": "SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. glucose consumption",
    "molecules": "glucose, SUC"
  },
  {
    "caption": "male C57BL/6J mice fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. lactate production were tested.",
    "molecules": "lactate, SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. mRNA expression of MyHC I, MyHC IIa, PGC-1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx and TnnT3 in the gastrocnemius muscle (n=5-6).",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Immunoblots and quantification of MyHC-I, IIa and IIb protein expression in gastrocnemius (n=3-4).",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, 1% SUC for 8 weeks. Representative images and quantification of Laminin (green), MyHC-I and IIb immunofluorescent staining (red) in gastrocnemius (n=3). Scale bar in (C) represents 100 µm.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The O2 consumption (VO2) and respiratory exchange ratio (RER)",
    "molecules": "O2, SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Serum concentration of (E) NEFA in whole blood.",
    "molecules": "NEFA, SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. The enzymes activity of (F) SDH, (G) HK, and (H) LDH in gastrocnemius.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Immunoblots and quantification of of p-AMPK，PGC-1α and myoglobin in gastrocnemius.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. Quantification of mitochondrial and electron transport chain (ETC) related genes expression in gastrocnemius.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were fed with chow diet supplemented with 0 and1% SUC for 6 weeks. OCRs were measured under basal condition in gastrocnemius.",
    "molecules": "SUC"
  },
  {
    "caption": "C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Representative images of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).",
    "molecules": "SUC"
  },
  {
    "caption": "C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. quantification of MyHC-I and IIb immunofluorescent staining (green) in C2C12 cells (n=16).",
    "molecules": "SUC"
  },
  {
    "caption": "C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. The enzymes activity of (C) SDH, (D) LDH",
    "molecules": "SUC"
  },
  {
    "caption": "C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. lactate production in C2C12 cells.",
    "molecules": "lactate, SUC"
  },
  {
    "caption": "C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Quantification of mitochondrial DNA contents in C2C12 cells.",
    "molecules": "DNA, SUC"
  },
  {
    "caption": "C2C12 cells were treated with 0, 0.5 and 2 mM SUC for 48 h. Mitochondrial electron microscopy showed the (H) mitochondrial density, (I) mitochondrial coverage, and (J) average mitochondrial area in C2C12 cell. Scale bar in (A) represents 50 μm, scale bar in (G) represents 0.5 μm.",
    "molecules": "SUC"
  },
  {
    "caption": "[Ca2+]i in C2C12 cells treated with 0 or 2 mM SUC (n=18-20).",
    "molecules": "Ca2+, SUC"
  },
  {
    "caption": "NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0.5 h or 3 h after i. p. injection of 15 mg/kg succinate in C57BL/6J mice (n=4).",
    "molecules": "succinate"
  },
  {
    "caption": "[Ca2+]i, in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).",
    "molecules": "Ca2+, SUC"
  },
  {
    "caption": "enzymes activity (n=9-10)of (J) HK, (K) LDH, and (L) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n=5-6).",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The O2 consumption (VO2), RER",
    "molecules": "O2, SUC"
  },
  {
    "caption": "Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing time",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. low speed running time.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (H) HK, (I) LDH, and (J) SDH in gastrocnemius.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Representative images and quantification of Laminin (green), or MyHC-I and IIb (red) immunofluorescent staining in gastrocnemius muscle (n=3). Scale bar in (M) represents 100 µm.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV-shScrambled (n=3).",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The running time in low speed",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. four-limb handing time",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. muscle grip strength of both control and gastrocnemius-specific SUNCR1 knockdown mice.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. The enzymes activity of (F) HK, (G) LDH, (H) SDH in gastrocnemius.",
    "molecules": "SUC"
  },
  {
    "caption": "Male C57BL/6J mice were injected with LV-shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After two weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. Immunoblots and quantification of MyHC-I, IIb, NFAT, and PGC-1α protein in gastrocnemius (n=3).",
    "molecules": "SUC"
  },
  {
    "caption": "B,C Effects of H2A mutations (B) and H2A.B mutations (C) on nucleosome resistance to MNase digestion. MNase digested nucleosomal DNA are analyzed by 10% Native-PAGE. All MNase digestion experiments are repeated twice.",
    "molecules": "DNA"
  },
  {
    "caption": "D,E Graph presentation of effects of H2A mutations (D) and H2A.B mutations (E) on nucleosome resistance to MNase digestion. MNase digestion efficiency for each sample is calculated from quantitation of the digested DNA against total DNA. All experiments are repeated twice. Data are mean ± SD, n = 2.",
    "molecules": "DNA"
  },
  {
    "caption": "B Effect of the canonical and noncanonical H2A ROF on histone octamer assembly. Left: Gel filtration chromatography profiles of octamers assembled by core histones including various H2A family members or mutants. Highlighted in blue shadow are chromatography fractions subject to SDS-PAGE analysis. Right: SDS-PAGE analysis of histone octamer integrity by visualizing histone stoichiometry. The stars indicate lnkH2B-H2A containing different H2A family members or mutants present in chromatography fractions. The triangles indicate the chromatography fractions containing the majority of the lnkH2B-H2A or their mutants.",
    "molecules": "histone, histones"
  },
  {
    "caption": "D,E Effects of the canonical and noncanonical H2A ROF on nucleosome resistance to MNase digestion. Displayed are Native-PAGE results (D) and graph of MNase digestion efficiency calculated from quantitation of the digested DNA against total DNA (E). All experiments are repeated twice. Data are mean ± SD, n = 2.",
    "molecules": "DNA"
  },
  {
    "caption": "E Effect of H2A.Z.2.2 ROF on facilitating histone replacement. Incorporations of yeast H2A.Z-H2B dimers containing either the wild type H2A.Z or Z.2.2-like H2A.Z were analyzed by SWR1-catalyzed histone replacement assay. H2A.Z and Z.2.2-like H2A.Z incorporated into nucleosome were resolved by 6% Native-PAGE and detected by SYBR Green staining and western blotting.",
    "molecules": "histone, SYBR Green"
  },
  {
    "caption": "a, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*P 0.02, **P 0.002, n = 3).",
    "molecules": "3-methyladenine, 3MA, oleate, TG"
  },
  {
    "caption": "b, TG levels in vector-infected (VEC) and siAtg5 cells in RM, OL or in MCDM (*P  0.001, n = 5). OL values are in mM.",
    "molecules": "TG"
  },
  {
    "caption": "c, TG levels in wild-type (WT) or Atg5 knockout mice embryonic fibroblasts (Atg5-/-) (*P 0.01 or **P 0.0001, n = 5).",
    "molecules": "TG"
  },
  {
    "caption": "a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 3–4).",
    "molecules": "oleate, TG"
  },
  {
    "caption": "c, d, Rates of TG decay in OL (c) and MCDM (d) (*P  0.05, **P  0.01, ***P  0.001 as compared to VEC cells, n = 3–7).",
    "molecules": "TG"
  },
  {
    "caption": "e, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*P  0.00001 with DMSO-treated cells, #P  0.003 with 3MA-treated cells, n = 6). Error bars, s.e.m.",
    "molecules": "3-methyladenine, 3MA, DEUP, diethylumbelliferyl phosphate, dimethyl sulphoxide, DMSO, TG"
  },
  {
    "caption": "g, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.",
    "molecules": "vinblastine"
  },
  {
    "caption": "b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P  0.01, **P  0.001 with cells in RM, n = 4–6). Left: representative examples. Error bars, s.e.m.",
    "molecules": "gold particles, lipid"
  },
  {
    "caption": "b, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P  0.001, **P  0.0001, n = 4–6). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid)",
    "molecules": "lipid"
  },
  {
    "caption": "f, Total hepatic TG and cholesterol content (*P 0.01, **P 0.00001, n = 817).",
    "molecules": "cholesterol, TG"
  },
  {
    "caption": "g, Hepatic TG concentration (*P  0.05, n = 3).",
    "molecules": "TG"
  },
  {
    "caption": "h, Percentage of cellular cholesterol in lysosomes (*P  0.02, n = 4).",
    "molecules": "cholesterol"
  },
  {
    "caption": "C Percentage of CXCR2highneutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0005 (ZT13).",
    "molecules": "SB225002, DMSO"
  },
  {
    "caption": "D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0079 (ZT13).",
    "molecules": "SB225002"
  },
  {
    "caption": "E Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0002 (ZT13).",
    "molecules": "SB225002"
  },
  {
    "caption": "B Percentage of CXCR2hineutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0046 (ZT13); ZT5 vs. ZT13: *P = 0.0153 (DMSO).",
    "molecules": "SB225002, DMSO"
  },
  {
    "caption": "C Flow cytometry quantification of cardiacneutrophils 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0006 (ZT13); ZT5 vs. ZT13: *P = 0.0001 (DMSO).",
    "molecules": "SB225002, DMSO"
  },
  {
    "caption": "D Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0,0486 (ZT13).",
    "molecules": "SB225002"
  },
  {
    "caption": "a, Representative mitochondrial morphology in Smurf1+/+ (wild-type) and Smurf1−/− (KO) MEFs transfected with indicated construct and treated with DMSO or 10 µM CCCP for 24 h. b, Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance. Results shown represent combined data from 3-5 experiments per condition with triplicate wells (of at least 100 cells per well) analysed for each condition per experiment. Shown are mean ± s.e.m. for average values from each experiment. Similar results were observed in each independent experiment. *P  0.001, Students t-test. c, Measurement of mitochondrial fractional area (percentage of total cellular area) in MEFs treated as in a. Results shown represent mean ± s.e.m. for 50 cells per condition.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "d, Representative confocal micrographs of KO MEFs transfected with YFP-SMURF1 wild-type or YFP-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with DMSO or CCCP.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "e, Representative confocal micrographs of KO MEFs transfected with GFP-LC3 and wild-type mCherry-SMURF1 (WT) or mCherry-SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with CCCP. Inset, upper right, formation of completed autophagosome around a damaged mitochondrion associated with wild-type SMURF1; insets, lower right, incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr;C2. See also Supplementary Figs 10 and 11 for enlarged images.",
    "molecules": "CCCP"
  },
  {
    "caption": "A Germination of dog1-3 (knock-out), WT, and dog1-5 (DOG1 upregulation) seeds in media supplemented with NaCl for the indicated number of days after stratification (DAS). Scale bar represents 5 mm. B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes. Lines represent the fitted curves with a 95% confidence interval (grey area). ∗ p-value < 0.05 from two-tailed Student's t-test. Data for WT is the same as plotted in Fig 3C. C",
    "molecules": "NaCl"
  },
  {
    "caption": "C shDOG1 expression levels normalized to UBC21 (AT5G25760) in dog1-3 and dog1-5 relative to WT. D shDOG1 and lgDOG1 expression levels normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock. E",
    "molecules": "NaCl"
  },
  {
    "caption": "Germination time-course in 100 mM NaCl after stratification for puppies-1 (C) relative to WT. Lines represent fitted curves with a 95% confidence interval (grey area), dots represent data points, ∗ p-value < 0.05 from the two-tailed Student's t-test. (C) Data for WT is the same as plotted",
    "molecules": "NaCl"
  },
  {
    "caption": "F RT-qPCR fold-change induction of PUPPIES-uns, PUPPIES-prom, PUPPIES-fusion, shDOG1, and lgDOG1 in puppies-ox relative to WT (blue dashed line), in dry (light grey) and imbibed seeds in the presence of 100 mM NaCl (red). Significance from the two-tailed Student's t-test for comparing dry seeds of WT and puppies-ox is represented with light grey asterisks, and salt-imbibed seeds of WT and puppies-ox are represented with red asterisks. Black asterisks represent significance from two-way ANOVA with Tukey's multiple comparisons test for dry seeds vs imbibed seeds. ns p-value > 0.05, ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001. Error bars represent the mean ± SD. n = 4 biological replicates.",
    "molecules": "NaCl, salt"
  },
  {
    "caption": "G RT-qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies-ox relative to WT. Nascent RNA levels were normalized to UBC21. Bars and error bars represent the mean ± SD. Points represent biological replicates. Statistical significance from two-tailed Student's t-test. ∗ p-value < 0.05.",
    "molecules": "nascent RNA, Nascent RNA, NaCl"
  },
  {
    "caption": "A z-stack max-projection image of smFISH for DOG1 RNA. The \"Inferno\" colour scale is used for the intensity of fluorescence from Quasar670 fluorophore (DOG1). The blue colour shows fluorescence from DAPI (nuclei staining). Arrowheads point to foci corresponding to transcription sites (TS). The scale bar is 20 μm. B Distribution of cytoplasmic DOG1 foci per cell in WT. The blue vertical dashed line indicates the average. C",
    "molecules": "Quasar670, DAPI"
  },
  {
    "caption": "G z-stack max-projection images from seeds imbibed in 100 mM NaCl of WT (left) versus puppies-ox (right). Arrowheads point to foci corresponding to TS.",
    "molecules": "NaCl"
  },
  {
    "caption": "F, G RT-qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies-ox, and puppies-1 relative to WT, in seeds treated with 100 mM NaCl.",
    "molecules": "NaCl"
  },
  {
    "caption": "C.)  Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and the P. falciparum DPCK-mCherry protein (red) with DAPI marking the nuclei (blue). The borders of the red blood cell and parasite are marked with dotted blue and white lines, respectively. Microscopy images represent fields that are 10µm long by 10µm wide.",
    "molecules": "DAPI"
  },
  {
    "caption": "B.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG  (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.",
    "molecules": "DAPI"
  },
  {
    "caption": "D.)  Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck parasite line that is expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). E.) Live epifluorescence microscopy of the  PfMev api-EcDPCK-mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate. This parasite line expresses  api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.",
    "molecules": "azithromycin, DAPI, mevalonate"
  },
  {
    "caption": "F.) Attempted  PCR detection of the ldh,  sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We failed to amplify sufB from the azithromycin-treated (+ Azith) PfMev api-EcDPCK-mCherry Δdpck parasites, indicating the loss of the  apicoplast  organellar genome.",
    "molecules": "Azith, azithromycin"
  },
  {
    "caption": "B.) Live epifluorescence microscopy of the  PfMev EcDPCK-mCherry line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei  (blue). D.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry Δdpck line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei  (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.",
    "molecules": "DAPI"
  },
  {
    "caption": "E.) Attempted  PCR detection of the  ldh, sufB, and cox1 genes from the nuclear (N), apicoplast  (A), and mitochondrial (M) genomes, respectively. We were successful in amplifying  sufB from the PfMev EcDPCK-mCherry Δdpck parasite line grown under continuous supplementation with 50μM mevalonate, indicating retention of the  apicoplast  organellar genome.",
    "molecules": "mevalonate"
  },
  {
    "caption": "C.)  Live epifluorescence microscopy of the PfMev CLD-EcDPCK-mCherry-apt Δdpck line. This parasite line expresses api-SFG (green) and CLD-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). This parasite line was grown in the presence of 0.5μM aTc. D.) The  PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. E.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the presence of 0.5μM aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. F.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. Data information: All microscopy images represent fields that are 10µm long by 10µm wide.",
    "molecules": "aTc, DAPI, Shield1, Shld"
  },
  {
    "caption": "G.) Growth curve of the  PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown either in the presence of 0.5μM  aTc  and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM  aTc and the presence of 0.5μM Shield1  (non-permissive condition, red). Pantothenate was supplied at 50nM in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 1 (two-way ANOVA, followed by Bonferroni's correction; *, P < 0.05). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.",
    "molecules": "aTc, Pantothenate, Shield1"
  },
  {
    "caption": "A.) Attempted  PCR detection of the  ldh, sufB, and cox1 genes from the  nuclear  (N), apicoplast (A), and  mitochondrial  (M) genomes, respectively. We were unsuccessful in amplifying sufB  from the  PfMev CLD-EcDPCK-mCherry-apt Δdpck  parasite line after treatment with 100nM azithromycin  (Azith) in the presence of 50μM mevalonate, indicating the disruption of the  apicoplast organelle.",
    "molecules": "Azith, azithromycin, mevalonate"
  },
  {
    "caption": "B.) Live epifluorescence microscopy of the apicoplast-negative PfMev CLD-EcDPCK-mCherry-apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate. This parasite line expresses  api-SFG (green) and the CLD-EcDPCK-mCherry construct (red) with DAPI  marking the nuclei  (blue).  Images represent fields that are 10µm long by 10µm wide.",
    "molecules": "azithromycin, DAPI, mevalonate"
  },
  {
    "caption": "C.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate (50nM) and mevalonate  (50μM) were supplied in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 2 (two-way ANOVA, followed by Bonferroni's correction; **, P < 0.01). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.",
    "molecules": "aTc, mevalonate, Pantothenate, Shield1"
  },
  {
    "caption": "Relative gene expression of AXIN2 (*P=0.0129), P21(*P=0.0103), and HSPB1(*P=0.0236) (b) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.",
    "molecules": "doxycycline"
  },
  {
    "caption": "protein expression of HSP27 (c) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.",
    "molecules": "doxycycline"
  },
  {
    "caption": "Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR-99021 (**P=0.0016, one way ANOVA, n=4) (d)",
    "molecules": "CHIR-99021"
  },
  {
    "caption": "i, j, Representative images of Apc-/- organoids cultured in the absence or presence of 60 μM BVDU (i), and quantification of their clonogenic potential (**P=0.0039) (j).",
    "molecules": "BVDU"
  },
  {
    "caption": "k, l, Relative gene expression of Wnt target genes Axin2 (**P=0.0080) and Lgr5 (*P=0.0142) in Apc-/- organoids cultured in the absence or presence of 60 μM BVDU.",
    "molecules": "BVDU"
  },
  {
    "caption": "m, RNA-ISH for Lgr5 in control or BVDU-treated Apc-/- organoids. Scale bar, 50 μm, zoom panel 20 μm.",
    "molecules": "BVDU"
  },
  {
    "caption": "b, c, Visualization of proliferating cells in tissues incubated with EdU for 2-hours (b), and quantification of the hyperproliferative zone (**P=0.0057, n=3 mice) (c).",
    "molecules": "EdU"
  },
  {
    "caption": "e, f, RNA-ISH for Notum reveals Apc-mutant clones in crypt bottoms of control or BVDU-treated mice (e), and quantification of the abundance of mutant crypts (n=2 mice per condition, n=5 technical replicates per mouse) (f).",
    "molecules": "BVDU"
  },
  {
    "caption": "g, h, Clone size distributions of Notum+ crypts in control (n=265 crypts) or BVDU-treated (n=260 crypts) mice, the average clone size and number of fixed clones are included in the figure.",
    "molecules": "BVDU"
  },
  {
    "caption": "Survival curves for control and BVDU-treated mice (P=0.0351, Mantel-Cox test) (j)",
    "molecules": "BVDU"
  },
  {
    "caption": "(H) Western-blotting of Axin2 and p-GSK3β(S9) in the total proteins, and β-catenin(S33) in the nuclear protein of ACC of WT or VPA-treated mice. 3 in H) mice Mean ratio ± SEM. Two-Tailed unpaired T-test. *P<0.05. **P<0.01. ***P<0.001. Con, control.",
    "molecules": "VPA"
  },
  {
    "caption": "(F) ECAR assay of primary WT and Shank3-/- neurons. Notice the higher ECAR values of Shank3-/- neurons upon glucose and oligomycin stimulation. 5 batches of cells *P<0.05. **P<0.01. ***P<0.001. WT, wild type. KO, Shank3-/",
    "molecules": "glucose, oligomycin"
  },
  {
    "caption": "(G) LC-MS measurement of lactic acid and pyruvic acid in the ACC of control and VPA-treated mice. Lactic acid is increased in VPA-treated mice. Two-Tailed unpaired T-test. Welch's t-test Pyruvic acid analysis in G *P<0.05. **P<0.01. ***P<0.001. Con, control.",
    "molecules": "pyruvic acid, Pyruvic acid, lactic acid, Lactic acid, VPA"
  },
  {
    "caption": "(A) Typical images of Golgi staining and reconstructed neurons in the ACC of Shank3-/- mice treated with 2-DG (KO+2-DG) or vehicle (KO+Veh). Sholl analysis of pyramidal neurons in the ACC of Shank3-/- mice treated with 2-DG or vehicle. Bar = 50 μm in (A) N = 24-28 neurons from 4 mice in KO, Shank3-/-. Veh, vehicle.",
    "molecules": "Golgi, 2-DG"
  },
  {
    "caption": "(G) Western-blotting of GluR1, PSD95 and Homer1 in the total proteins of Shank3-/- ACC with or without 2-DG treatment. 3 mice in (G) KO, Shank3-/-. Veh, vehicle.",
    "molecules": "2-DG"
  },
  {
    "caption": "resident-intruder assay of Shank3-/- mice treated with 2-DG or vehicle. Notice the improvement of social preference and social interaction by 2-DG treatment. 10-12 mice in KO, Shank3-/-. Veh, vehicle.",
    "molecules": "2-DG"
  },
  {
    "caption": "(A) ECAR assay of primary Shank3-/- neurons treated with or without XAV939. Notice the lower levels of glycolysis in XAV939 treated cells. (B) ECAR assay of primary Shank3-/- neurons treated with or without ICG-001. N = 4-5 batches of cells in Two-Tailed unpaired t-test *P<0.05. **P<0.01. KO, Shank3-/-.",
    "molecules": "ICG-001, XAV939"
  },
  {
    "caption": "(E) Double-immunostaining of ENO1/Axin2 in WT neurons, Shank3-/- neurons, and XAV939 treated Shank3-/- neurons. Notice the colocalization of Axin2/ENO1-immunoreactivity in Shank3-/- neurons and the membrane localization of Axin2-immunoreactivity in XAV939 treated Shank3-/- neurons. Arrows point to membrane Axin2-immunoreactivity. Bar = 5 μm in E. WT, wild type. KO, Shank3-/-.",
    "molecules": "XAV939"
  },
  {
    "caption": "(G) Protein CO-IP of Axin2/ENO1 in WT and Shank3-/- neurons treated with or without XAV939. Notice the strong interaction of Axin2/ENO1 in Shank3-/- neurons and diminished interaction upon XAV939 treatment. per group and 3 samples from 9 mice per group in One-way ANOVA with Tukey's multiple-comparison test WT, wild type. KO, Shank3-/-.",
    "molecules": "XAV939"
  },
  {
    "caption": "Spine length, spine density and spine subtypes of Shank3-/- ACC neurons treated with or without XAV939. Notice the increase of total spine density, stubby and mushroom-like spines and the decrease of average spine length in XAV939 treated Shank3-/- mice. 32 spines from 3 mice in Filopodia analysis in F. Spines analysis excepting filopodia analysis in F. KO, Shank3-/-.",
    "molecules": "XAV939"
  },
  {
    "caption": "(H) Western-blotting of p-PSD95 and Homer1 in WT and Shank3-/- ACC treated with or without XAV939. 4-6 mice in One-way ANOVA with Tukey's multiple-comparison test WT, wild type. KO, Shank3-/-.",
    "molecules": "XAV939"
  },
  {
    "caption": "(I, J) Western-blotting of GluR1 in the total and membrane protein of WT and Shank3-/- neurons treated with or without XAV939. Notice the rescuing effects of XAV939 on the expression of GluR1 in Shank3-/- neurons. 4-6 mice in and 3 samples from 12 mice in (J) per group. One-way ANOVA with Tukey's multiple-comparison test WT, wild type. KO, Shank3-/-.",
    "molecules": "XAV939"
  },
  {
    "caption": "Calcium response of WT and Shank3-/- neurons treated with or without XAV939. Notice that XAV939 treatment significantly restored the amplitude, rising and decay time of calcium response in Shank3-/- neurons. Bar = 15 μm in (A) and 30 μm in magnified images of (A). N = 39 neurons from 3 batches of cells in Kruskal-Wallis H test with Dunn's multiple-comparison test KO, Shank3-/-. Veh, vehicle.",
    "molecules": "Calcium, calcium, XAV939"
  },
  {
    "caption": "Calcium response of WT and Shank3-/- neurons treated with or without XAV939. Notice that XAV939 treatment significantly restored the amplitude, rising and decay time of calcium response in Shank3-/- neurons. N = 39 neurons from 3 batches of cells in Dunn's multiple-comparison test KO, Shank3-/-. Veh, vehicle.",
    "molecules": "Calcium, calcium, XAV939"
  },
  {
    "caption": "(D) Patch-clamp recording of mEPSCs in Shank3-/- ACC treated with or without XAV939. Both the frequency and amplitude of Shank3-/- neurons were increased by XAV939. , 7-8 neurons from 3 mice in (D) KO, Shank3-/-. Veh, vehicle.",
    "molecules": "XAV939"
  },
  {
    "caption": "resident-intruder assays of Shank3-/- mice treated with or without XAV939. Notice the improvement of social preference, social novelty and social interaction of Shank3-/- mice by XAV939 treatment. 8-10 mice KO, Shank3-/-. Veh, vehicle.",
    "molecules": "XAV939"
  },
  {
    "caption": "(H) ECAR assay of naive human neurons, VPA-pretreated human neurons with or without XAV939 treatment. Notice the higher levels of glycolysis in VPA-pretreated human neurons and the inhibitory effects of XAV939. , 4-5 batches of cells in per group.",
    "molecules": "VPA, XAV939"
  },
  {
    "caption": "H&E stained sections of back skin from indicated mice treated with IMQ for 2, 3 or 5 days. Bright-field images, Magnification: 20x, Scale bar: 100 µm.",
    "molecules": "IMQ"
  },
  {
    "caption": "Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were dermal γδ T cells (γδ TCRint+) (F) Graphs show immune cells as % of live, single cells. (n = 10-28 (F), ; ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.",
    "molecules": "IMQ"
  },
  {
    "caption": "Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were Monocytes (CD11b+Ly6Chi) (G), and Neutrophils (CD11b+Ly6G+) (H). Graphs show immune cells as % of live, single cells. n = 10-21 (G), n = 4-25 (H); ≥ 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Bonferroni multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.",
    "molecules": "IMQ"
  },
  {
    "caption": "H-I. Western blot analysis of Phospho- or total JNK and c-Jun in BMDCs of indicated genotype pre-treated with DMSO (1: 1000) or SP600125 (JNKi, 25 µM) for 1h and stimulated with IMQ (5 µg/mL) for 15 min.",
    "molecules": "DMSO, IMQ, SP600125"
  },
  {
    "caption": "Flow cytometry of back skin 12h after intradermal (i.d.) injection of 1 µg rCCL2 and/or IMQ treatment. Analyzed were pDCs (BST-2+B220+CD11cintCD11b-) shown as % of live, single cells. (n = 6; 2 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by Tukey multiple comparison test. Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.",
    "molecules": "IMQ"
  },
  {
    "caption": "Luciferase activity was quantified in lysates of RAW 264.7 cells transfected with pGL3 basic vector harboring wild-type (wt) or mutated (mut1 or 2) IL-23p19 promoter. Transfected cells were pre-treated with JNKi (SP600125, 25 µM, 1h) and stimulated with IMQ (6h). Results are shown as fold change to pGL-3-wt transfected, LAL (Limulus amebocyte lysate) treated RAW264.7 cells. (n = 4; 2 independent experiments).",
    "molecules": "IMQ, SP600125"
  },
  {
    "caption": "Epidermal thickness of back skin was quantified in H&E stained sections of indicated mice treated with rIL-23 (i.d., 1µg) and/or IMQ for two consecutive days. (n = 4-7; 3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by one-way ANOVA with Tukey multiple comparison test Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.",
    "molecules": "IMQ"
  },
  {
    "caption": "DC subsets (cDC1, cDC2), Macrophages (MP) and Langerhans cells (LC) were sorted from IMQ treated back skin (5h). c-Jun, Ccl2 and Il23p19 mRNA was analyzed by qRT-PCR. Sort strategy is shown in (B). (10 mice were pooled per condition, 1 experiment).",
    "molecules": "IMQ"
  },
  {
    "caption": "Immunofluorescence of c-Jun (green), CD1a or CD1c or CD14 (red) and DAPI in psoriatic lesions. Shown is a representative image (Magnification 25x, Scale Bar: 100µm) with an Inset (Magnification 63x, Scale Bar: 20 µm, Deconvoluted). Arrows indicate c-Jun+ DCs. Asterisk highlights a DC with prominent c-Jun expression as shown enlarged in 2 white-framed windows (DC Marker (red) + c-Jun (green, left) or DAPI (blue, right)).",
    "molecules": "DAPI"
  },
  {
    "caption": "D-E. Representative immunofluorescence of c-Jun (green), CD1a (red), CCL2 (D) or Il23p19 (E) (white) and DAPI in psoriatic lesions. Arrows indicate triple-positive cells and an asterisk highlights a representative one that is shown enlarged in a white-framed inlet. Magnification: 40x. Scale bar: 50 µm. (n = 2 patient samples).",
    "molecules": "DAPI"
  },
  {
    "caption": "B-C Human mo-DCs pre-treated with JNKi (SP600125, 25µM) (B) or AP-1 Inhibitor (T-5224, 20 µM) (C) for 1h were stimulated with R848 (10 µg/ml). CCL2 and IL-23 were analyzed by ELISA. (n = 5-11; 2-3 independent experiments). Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test (B, C, Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.",
    "molecules": "R848, SP600125, T-5224"
  },
  {
    "caption": "(F) Immunofluorescence analysis of the old APPPS1 slice in co-culture (14 DIV) treated with cytochalasin D (CytoD) and vehicle control (Ctr) and immunostained with M3.2 (green) and CD68 (red). Inhibition of phagocytosis by CytoD blocks amyloid plaque clearance. Scale bar: 50 µm.(G) Quantitative analysis of core-only plaques in the old APPPS1 slice in co-culture (14 DIV) treated with CytoD and Ctr reveals a decreased number of core-only plaques upon CytoD treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques. The values represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).",
    "molecules": "cytochalasin D, CytoD"
  },
  {
    "caption": "Either young WT (A, B) or old APPPS1 (C-E) tissue was treated with clodronate (Clo) to remove CD68 positive cells or vehicle Ctr from 1 until 7 DIV. Treatment was stopped and subsequently old (A, B) or young (C-E) tissue was added to the culture as schematically indicated in A and C. and analyzed 14 days after. Removal of CD68 positive cells in either young WT or old APPPS1 tissue prevents amyloid plaque clearance in the co-culture model.(A) Immunofluorescence analysis of the old APPPS1 slice co-cultured with the young WT slice pre-treated with Clo and Ctr and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(B) Quantitative analysis of core-only plaques in the old APPPS1 tissue co-cultured with the young WT tissue pre-treated with Clo and Ctr as indicated in A reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(C) Immunofluorescence analysis of the old APPPS1 slice treated with Clo and Ctr and subsequently co-cultured with the young WT slice and immunostained with CD68 (red) and M3.2 (green). Scale bar: 50 µm.(D) Area of CD68 positive cells (CD68 coverage) in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C. CD68 coverage is reduced upon Clo treatment. The values are normalized to CD68 coverage of the Ctr and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (***P < 0.001).(E) Quantitative analysis of core-only plaques in the old APPPS1 tissue treated with Clo and Ctr and subsequently co-cultured with the young WT tissue as indicated in C reveals a decreased number of core-only plaques upon Clo treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, including total of 6 independent slice culture dishes (**P < 0.01).",
    "molecules": "Clo, clodronate"
  },
  {
    "caption": "(B) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices previously treated with clodronate (old alone + young CM + Clo, grey bars ) and vehicle control (old alone + young CM + Ctr, white bars). Quantitative analysis at 7, 11 and 14 DIV reveals decreased numbers of core-only plaques upon incubation of the old APPPS1 tissue with conditioned media collected from young WT slices pre-treated with Clo. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each experiment including at least 2 independent slice culture dishes (n.s.= not significant, ***P < 0.001).",
    "molecules": "Clo, clodronate"
  },
  {
    "caption": "(E) Old APPPS1 tissue was incubated with conditioned media collected from young WT slices and in addition treated with proliferation inhibitor AraC (young CM + AraC) or vehicle Ctr (young CM + Ctr). Immunofluorescence analysis was performed at 14 DIV using CD68 (red) and M3.2 (green). Scale bar: 50 µm.(F) Quantitative analysis of core-only plaques in the old APPPS1 tissue (14 DIV) incubated with conditioned media from young WT slices and in addition treated with AraC or Ctr reveals a decreased number of core-only plaques upon AraC treatment. The values are expressed as percentages of core-only plaques from the total number of amyloid plaques and represent mean ± SEM from 3 independent experiments, each including 2 independent slice culture dishes (***P < 0.001).",
    "molecules": "AraC"
  },
  {
    "caption": "(C) Cell morphology and SA-β-Gal activity of mouse or NMR fibroblasts 12 or 21 days after DXR treatment. Arrowheads indicate dying cells. The number in the upper left corner indicates Hoechst-positive nuclei. Scale bar, 100 μm.",
    "molecules": "Hoechst, DXR"
  },
  {
    "caption": "Time-course analysis of mouse or NMR fibroblasts after DXR treatment quantification of BrdU-positive cells (%) (E)",
    "molecules": "BrdU, DXR"
  },
  {
    "caption": "Time-course analysis of mouse or NMR fibroblasts after DXR treatment qRT-PCR analysis of the expression of INK4a (F) normalized to ACTB mRNA levels",
    "molecules": "DXR"
  },
  {
    "caption": "qRT-PCR analysis of the expression of INK4a (J) in shINK4a-transduced NMR fibroblasts at 21 days after DXR treatment.",
    "molecules": "DXR"
  },
  {
    "caption": "quantification of Annexin V-positive cells (%) (K) in shINK4a-transduced NMR fibroblasts at 21 days after DXR treatment.",
    "molecules": "DXR"
  },
  {
    "caption": "(C) Cell morphology and SA-β-Gal activity of mouse or NMR fibroblasts 12 days after INK4a transduction. Arrowheads indicate dying cells. ​​The number in the upper left corner indicates Hoechst-positive nuclei. Scale bar, 100 μm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C and D) Quantification of SA-β-Gal-positive cells (%) (C) and Annexin V-positive cells (%) (Annexin V+/PI- as early apoptotic and Annexin V+/PI+ double-positive as late apoptotic) (D) in NMR-fibroblasts transduced with different forms of SV40 Large T antigen (LT, LT∆, and LTK1) and INK4a. OE; overexpression.",
    "molecules": "PI"
  },
  {
    "caption": "(F) Quantification of SA-β-Gal-positive cells (%) at 21 days after DXR treatment in NMR-fibroblasts transduced with different forms of SV40 Large T antigen (LT, LT∆, and LTK1). (G) Quantification of Annexin V-positive cells after the same treatment as in F. Annexin V+/PI- cells were counted as early apoptotic cells and Annexin V+/PI- cells as late apoptotic cells.",
    "molecules": "DXR, PI"
  },
  {
    "caption": "(C) Quantification of PI-positive cells in NMR and mouse fibroblasts 18 h after 6 h of treatment with the indicated doses of hydrogen peroxide (H2O2). Data are expressed as the mean ± SD from n = 3 biological replicates.",
    "molecules": "H2O2, hydrogen peroxide, PI"
  },
  {
    "caption": "(C) Quantification of PI-positive cells in NMR fibroblasts treated for 24 h with N-acetyl L-cysteine (NAC), Trolox, or Tempol at 20 days after INK4a transduction (%). Data are expressed as the mean ± SD from n = 6 biological replicates except for Trolox and Tempol (n = 4).",
    "molecules": "Tempol, N-acetyl L-cysteine, NAC, PI, Trolox"
  },
  {
    "caption": "(G) Western blot of MAO-A and MAO-B in mouse or NMR fibroblasts at 21 days after doxorubicin (DXR) treatment. ACTIN was used as a loading control. Numbers below the gel images indicate quantification of MAO-A or -B/ACTIN intensity (n = 3 average).",
    "molecules": "doxorubicin, DXR"
  },
  {
    "caption": "(D) SA-β-Gal activity (SA-β-Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 2, 7, 14, and 21 days after Bleo administration.",
    "molecules": "Bleo"
  },
  {
    "caption": "Time-course analysis of mice or NMR lungs after Bleo administration: qRT-PCR analysis of the expression of INK4a normalized to ACTB mRNA levels (F)",
    "molecules": "Bleo"
  },
  {
    "caption": "Time-course analysis of mice or NMR lungs after Bleo administration ; quantification of TUNEL-positive cells (%) (G).",
    "molecules": "Bleo"
  },
  {
    "caption": "(B) SA-β-Gal activity (SA-β-Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 21 days after Bleo administration, with or without Phe are shown.",
    "molecules": "Bleo, Phe"
  },
  {
    "caption": "qRT-PCR analysis of INK4a expression normalized to ACTB mRNA levels (D) in mouse or NMR lungs at 21 days after Bleo administration, with or without Phe.",
    "molecules": "Bleo, Phe"
  },
  {
    "caption": "quantification of TUNEL-positive cells (%) (E) in mouse or NMR lungs at 21 days after Bleo administration, with or without Phe.",
    "molecules": "Bleo, Phe"
  },
  {
    "caption": "(B) qPCR analysis of the enrichment±s.d. of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts. Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced, with respect to the amount immunoprecipitated from uninduced cell extracts according to the (Atro-No Ab)induced/(Atro−No Ab)uninduced formula. The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al, 2007) and has been used as a negative control.",
    "molecules": "DNA"
  },
  {
    "caption": "C) Time course analysis of triubiquitin (2 µM) hydrolysis using 250 nM SARS2 PLpro, resolved on a Coomassie stained SDS-PAGE gel. Linkage specific cleavage of Lys48-linked triubiquitin to di- and monoubiquitin resembles SARS PLpro activity (Békés et al, 2015; 2016). See Source Data for uncropped gels, and Appendix Fig S1B-D for gel-based cleavage quantification.",
    "molecules": "triubiquitin, Lys, monoubiquitin"
  },
  {
    "caption": "B) Fluorescence polarisation assays against ubiquitin-TAMRA and ISG15-TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n=2 for each mutant, and compared to wild-type PLpro as shown in Appendix Fig S1A. Catalytic efficiencies were calculated as described in Methods.",
    "molecules": "TAMRA, ubiquitin"
  },
  {
    "caption": "C) Gel based analysis of PLpro variant activity against Lys48-triubiquitin and proISG15. Experiments were performed in duplicate, see Fig EV3 and Source Data for uncropped gels.",
    "molecules": "triubiquitin, Lys"
  },
  {
    "caption": "C) Calculated enzymatic efficiencies for K48-diUb-TAMRA cleavage, followed by fluorescence polarisation with PLpro wild-type (reproduced from Appendix Fig S1A), and PLpro F69S. (Left) Using the same concentration range of SARS2 PLpro, the F69S mutant activity could not be fitted; (middle) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin-TAMRA (45 vs. 86 M-1s-1, compare Fig 1D, Appendix Fig S1A). (Right) A ~3-fold lower efficiency for PLpro F69S cleaving ISG15-TAMRA yields values similar to cleavage of ISG15CTD-TAMRA (Appendix Fig S1A), suggesting that the S2 site contributes the difference in binding for the N-terminal Ubl-fold. Experiments for F69S were performed in technical triplicate and biological duplicate; wild-type data is reproduced from experiments shown in Fig 1D, Appendix Fig S1A.",
    "molecules": "diUb, TAMRA, ubiquitin"
  },
  {
    "caption": "D-F) Gel based analysis showing hydrolysis time course of triubiquitin (D), proISG15CTD (E) and proISG15 (F) using wild-type PLpro (left, wild-type gels reproduced from Appendix Fig S1B-D to enable direct comparison) or PLpro F69S (right). PLpro F69S has a strong effect on triubiquitin hydrolysis (D), no marked effect on hydrolysis of proISG15CTD (E) and reduces proISG15 cleavage to the same levels as proISG15CTD (compare E, F), Experiments were performed in duplicate, see Source Data for uncropped gels.",
    "molecules": "triubiquitin"
  },
  {
    "caption": "B) Hit compounds and compound rac5c (see Fig 5), were further assessed in 10-point IC50 titrations using the Ub-Rhodamine assay, using a starting concentration of 100 µM serially diluted in 1:3 steps. Degree of inhibition is shown as a titration heat-map from dark (full inhibition) to light blue (low/no inhibition). The catalytic domain of human USP21 (Ye et al, 2011) was used as a counterscreen. Each PLpro hit compound showed either no activity in the titration analysis, or an identical inhibition profile against PLpro and USP21, suggesting assay interference. Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM. IC50 assays were performed in technical triplicate in two independent experiments.",
    "molecules": "rac5c, Rac5c, Rhodamine, Ub"
  },
  {
    "caption": "C) In vitro inhibition (IC50) for rac5c inhibiting SARS2 PLpro. Experiments were performed using the HTS assay (Fig 4), in technical triplicate in three independent experiments. A geometric mean was used to determine IC50.",
    "molecules": "rac5c"
  },
  {
    "caption": "D) Full-length nsp3 was expressed from a C-terminally GFP-tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h. GFP is released from the C-terminus, presumably by nsp3 protease activity. Nsp3 can be detected by a SARS/SARS2 PLpro antibody (see Fig EV5E for antibody validation). Lysates were blotted for Lys48-linked polyubiquitin with a linkage specific antibody (K48). Experiments were performed in duplicate with similar results. Also see Fig EV5F, G and Source Data for uncropped blots.",
    "molecules": "polyubiquitin, Lys, rac5c"
  },
  {
    "caption": "B) Reduction in SARS-CoV-2 induced cytopathic effect with rac5c, Remdesivir (RDV) and hydroxychloroquine (HCQ) treatment. DMSO 0.3% (v/v) was required to keep 33 µM rac5c in solution (see Fig EV6A, B). Mean (black line) is provided for 18 samples in each group, representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c. HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats. P values were calculated using a one-way ANOVA, with regular Dunnet's post-hoc test for multiple comparisons between treatment arms and infected/vehicle treated control using a single pooled variance.",
    "molecules": "DMSO, HCQ, hydroxychloroquine, rac5c, RDV, Remdesivir"
  },
  {
    "caption": "B. Two-hybrid interaction between GBD-AC40 and WT or mutant GAD-IN1578-635. Alanine substitutions in IN1578-635 are indicated. Cells were plated in two-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1A-B). +, interaction; -, no interaction.",
    "molecules": "Alanine, His, Leu, Trp"
  },
  {
    "caption": "C. Two-hybrid interaction between GBD-AC40 and different IN1 regions fused to GAD, as indicated. Cells were plated in ten-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1C-D). +, interaction; -, no interaction.",
    "molecules": "His, Leu, Trp"
  },
  {
    "caption": "A. Co-immunoprecipitation of ectopic IN1 using C160-HA as bait, from yeast protein extracts expressing WT or mutated IN1-Strep (K617A, S621A, or L622A) from the GAL1 promoter in the presence of galactose. Expected sizes are 160 kDa for C160-HA and 100 kDa for IN1-Strep (WT and mutants).",
    "molecules": "galactose"
  },
  {
    "caption": "B. Quantitative ChIP analysis of HA-IN1 enrichment at Pol III-transcribed genes. Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input. Pol III transcribed-genes: tDNA-Ile and tDNA-Leu families (16 and 22 genes, respectively) and the unique SCR1 gene. GAL1 ORF serves as a control. Data represent means ± SD (n ≥ 3).",
    "molecules": "Ile, Leu"
  },
  {
    "caption": "A. Co-immunoprecipitation of ectopic IN1 using TAP-tagged-A190 as bait from yeast protein extracts expressing WT or the indicated IN1-HBH mutants from a pTet-Off promoter in the absence of doxycycline. Expected sizes are 204 kDa for A190-HA and 82 kDa for IN1-Strep (WT and mutants).",
    "molecules": "doxycycline, Tet"
  },
  {
    "caption": "A. Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 (red triangle) and a primer in the locus of interest (blue triangle). Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant (IN1 K617A, S621A and L622A) Ty1his3AI from the GAL1 promoter. Total genomic DNA was extracted from His+ cells obtained from independent cultures.",
    "molecules": "His"
  },
  {
    "caption": "A. Two-hybrid interaction between GBD-AC40 (left) or GBD-Sir4 (right) and different GAD-IN5 or GAD-IN1 constructions. Cells were plated in five-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected",
    "molecules": "His, Leu, Trp"
  },
  {
    "caption": "D. Detection of Ty5, Ty5ΔTD5+bNLS or Ty1 de novo integration events at HMR and HML loci, SCR1 and upstream of all glycine tDNAs, by PCR using a primer in HIS3 and a primer in the locus of interest. Retrotransposition was induced in cells transformed with WT or mutated pGAL1-Ty5his3AI (Ty5 or Ty5ΔTD5+bNLS) and pGAL1-Ty1his3AI (Ty1). Total genomic DNA was extracted from His+ cells obtained from independent cultures. Nucleosome position (green circles) is indicated on the right of the panels (Brogaard et al, 2012).",
    "molecules": "glycine, His"
  },
  {
    "caption": "C. Infected midgut  showing oocysts stained  with mercurochrome; scale bar, 100 μm. The graph shows oocyst  numbers per infected mosquito. Numbers of mosquitoes  that were analyzed are indicated above the graph. Data derived from at least two independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (C) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.",
    "molecules": "mercurochrome"
  },
  {
    "caption": "B. Scheme showing a sporozoite  moving through a   polymeric network in 3D. The graph shows sporozoite  speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.",
    "molecules": "hydrogel"
  },
  {
    "caption": "B. Representative Puro-PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes. Puro: puromycin; Aniso: anisomycin; 1ary Abs: primary antibodies. white arrowheads indicate Puro-PLA punctae; yellow dashed lines outline protrusion borders (B);",
    "molecules": "Aniso, anisomycin, Puro, puromycin"
  },
  {
    "caption": "C. Quantification of RAB13 Puro-PLA punctae normalised to protrusion area (n≥40 protrusions; *P<0.05, ****P<0.0001; Kruskal-Wallis test with Dunn's correction).",
    "molecules": "Puro"
  },
  {
    "caption": "qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX (n=3 biological replicates for each sample).",
    "molecules": "DOX"
  },
  {
    "caption": "Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX.",
    "molecules": "DOX"
  },
  {
    "caption": "qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells (n=3 biological replicates for each group).",
    "molecules": "Tet"
  },
  {
    "caption": "qPCR quantification of retinal marker expression in PAX6D TetOn cells w/o DOX treatment (n=3 biological replicates for each group).",
    "molecules": "DOX, Tet"
  },
  {
    "caption": "Verification of PAX6D target genes using PAX6D TetOn cells. Two sets of genes (retinal and non-retinal neural genes) are directly regulated by PAX6D in opposite directions (n=3 biological replicates for each group).",
    "molecules": "Tet"
  },
  {
    "caption": "qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation (n=3 biological replicates for each group).",
    "molecules": "CHIR99021, IWR1"
  },
  {
    "caption": "qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation (n=3 biological replicates for each group).",
    "molecules": "IWR1"
  },
  {
    "caption": "qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 (n=3 biological replicates for each group).",
    "molecules": "CHIR99021, DOX, Tet"
  },
  {
    "caption": "B. In vitro cross-linking experiments of purified His6-TssA1. About 30 μg of purified His6-TssA1 was cross-linked (30 min, 1h and 2h) at room temperature using increasing amount of ethylene glycol-bis(succinimidylsuccinate) EGS (2 mM and 5 mM) where indicated. Western blot analysis of cross-linked products using an anti-His6 monoclonal antibody is shown. The cross-linked species are highlighted with stars with the corresponding oligomeric state indicated on the right (1x = monomer; 2x = dimer; 6x = hexamer). Molecular weight markers (kDa) are indicated on the left.",
    "molecules": "EGS, ethylene glycol-bis(succinimidylsuccinate)"
  },
  {
    "caption": "C. Representative micrographs of the immunogold-labelled complex His6-TssA1/TssB1C1. The presence of gold particles is indicated with white arrows. Two close-up views of TssB1C1 sheath displaying gold particles at one extremity are shown (inset panels). Scale bars are 2,000 Å and 500 Å for images 1 and 2-6, respectively.",
    "molecules": "gold"
  },
  {
    "caption": "B. Cycloheximide chase analysis on N2a cells expressing Sig1R-FLAG. Quantitative data of immunoblotting for the levels of Sig1R-FLAGprotein and its variants during the cyclohexamide chase were plotted as mean ± SEM of three independent experiments (upper panel). Representative immunoblots for the levels of Sig1R-FLAGproteins were shown (lower panel). **: p < 0.01, *: p = 0.0216 in E102Q vs. wild-type (WT); ##: p < 0.01, #: p < 0.05 in L95fs vs. WT. A one-way ANOVA with subsequent post hoc Tukey's test.",
    "molecules": "Cycloheximide"
  },
  {
    "caption": "C. N2a cells transfected with Sig1R-FLAG variants were incubated with MG-132 (10 µM) or the combination of E64d and pepstatin A (5 µg/mL each) (E64d/PepA) for 8 h. The relative mean levels of Sig1R-FLAG variants determined by immunoblotting from three independent experiments were plotted as mean ± SEM (upper panel). The representative immunoblots were shown (lower panel). *: p < 0.05 vs. no inhibitor control. A one-way ANOVA with subsequent post hoc Tukey's test.",
    "molecules": "PepA, E64d, MG-132, pepstatin A"
  },
  {
    "caption": "I and J. Cytoplasmic calcium (Ca2+) flux in N2a or human IP3R3 stably expressing N2a (N2a-IP3R3) cells. siCtrl or siSig1R was transfected to N2a or N2a-IP3R3 cells, then cytoplasmicCa2+ flux were determined with fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized to the intensity in resting state at 0 s, and plotted as mean ± SD.",
    "molecules": "Ca2+"
  },
  {
    "caption": "I and J. mitochondrial (J) calcium (Ca2+) flux in N2a or human IP3R3 stably expressing N2a (N2a-IP3R3) cells. siCtrl or siSig1R was transfected to N2a or N2a-IP3R3 cells, then mitochondrial Ca2+ flux were determined with fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized to the intensity in resting state at 0 s, and plotted as mean ± SD.",
    "molecules": "Ca2+"
  },
  {
    "caption": "K and L. Cytoplasmic (K) or mitochondrial (L) Ca2+ flux in N2a-IP3R3 cells. siCtrl or siSig1R was transfected with or without the Sig1R-FLAG variants. The data are obtained and plotted as described above. n = 10 each. **: p < 0.01, *: p < 0.05 (I-L). Two-way ANOVA with subsequent post hoc Tukey's test (I-J).",
    "molecules": "Ca2+"
  },
  {
    "caption": "B-G. intracellular ATP levels (E-G) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.",
    "molecules": "ATP"
  },
  {
    "caption": "A and B. Cytoplasmic (A) or mitochondrial (B) Ca2+ flux was measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 1 h prior to fluorescent imaging. Cytoplasmic and mitochondrialCa2+ flux were detected by fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized by the resting state at 0 s, and plotted as mean ± SD. n = 10 each. **: p < 0.01, *: p < 0.05. Two-way ANOVA with subsequent post hoc Tukey's test.",
    "molecules": "Ca2+, PRE-084, NE-100"
  },
  {
    "caption": "C and D. Calpain activity were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.",
    "molecules": "PRE-084, NE-100"
  },
  {
    "caption": "C and D. intracellular ATP levels were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.",
    "molecules": "PRE-084, ATP, NE-100"
  },
  {
    "caption": "E and F. SOD1G93Amice were intraperitoneally administered with saline or PRE-084 (0.25 mg/kg, 3 times per week) from postnatal day 35 to 95. Lumbar spinal cord sections were stained by using anti-Sig1R (white), IP3R3 (green), and βIII-tubulin (red) at 95 days old. Arrowheads indicate the neurons with normal distribution of Sig1R and IP3R3. Scale bars: 50 µm.",
    "molecules": "PRE-084"
  },
  {
    "caption": "C. Interaction strength (Gibbs free energy) of all RNA-RNA interactions recovered by RNase E-CLASH (solid line) and for randomly paired hybrid read halves (dashed line).",
    "molecules": "RNA"
  },
  {
    "caption": "D. Interaction strength of sRNA-mRNA hybrid read halves (solid line) and randomly paired sRNA and mRNA read halves (dashed line).",
    "molecules": "sRNA, mRNA"
  },
  {
    "caption": "E. Position of sRNA-mRNA interactions relative to the mRNA start codon.",
    "molecules": "sRNA, mRNA"
  },
  {
    "caption": "C-E Mapping of the binding  region between myoVa and caldendrin using co-immunoprecipitation of cald-tagRFP  and  GFP-myoVa fragments expressed in HEK293T  cells. Arrows in D indicate the size of the respective myosin fragment. F Summary of the results from C - E.  Caldendrin binds  specifically to a fragment containing  IQ-motif 1 (amino acids  742-791), but not to any other region of myoVa, in a Ca2+-dependent manner.",
    "molecules": "Ca2+"
  },
  {
    "caption": "B Western blot  analysis of competition co-immunoprecipitation experiment in HEK293T cells. In the presence of Ca2+, caldendrin binds  strongly to the IQ1 fragment (upper arrow), while tagRFP  by itself does not  bind (lower arrow). Detection using a calmodulin antibody indicated that Ca2+-dependent binding of  caldendrin reduced the amount of CaM bound to the IQ1 motif relative to the control (tagRFP only). MW = molecular weight marker. C Quantification of the normalized relative intensity of cald-tagRFP (left) and  CaM  bands (right) in western blot  analysis of three independent experiments as shown in B. Shown is mean ± SEM. Upper: Ca2+ greatly increases cald-tagRFP binding to GFP-myoVa-IQ1. Lower: When Ca2+ is available, the simultaneous presence of  cald-tagRFP leads to a reduced binding of CaM  compared to the control (tagRFP). D",
    "molecules": "Ca2+"
  },
  {
    "caption": "F Native  MS analysis of ion saturation for  CaM.  Ca2+ carried along from purification is detected on CaM  in  native  MS. Without further supplementation with ions, intact  CaM  shows primarily its high affinity calcium binding sites occupied (left), while in the same analysis  myoVa peptide selectively and preferentially  binds only to the fully Ca2+-saturated CaM  species (right). Additional unassigned peaks represent unspecific adducts of Na+ and Mg2+ ions.",
    "molecules": "Ca2+, calcium, Mg2+, Na+"
  },
  {
    "caption": "G Native  MS analysis of ion saturation for caldendrin. Supplemented by an excess of 200 µM Ca2+  and 100 µM Mg2+, caldendrin displays myoVa peptide  binding to both fully  Ca2+-saturated and 2 Ca2+-bound species (right), which are also both detectable for the intact  caldendrin  protein in the same analysis (left).",
    "molecules": "Ca2+, Mg2+"
  },
  {
    "caption": "B Co-sedimentation assay of GFP-tagged myosin fragment lacking the  globular tail  domain (GFP-myoVa-motor+CC) with  F-actin shows that the presence of Ca2+-caldendrin does not prevent myoV from binding  to  actin filaments. GFP-myoVa-motor+CC, as visualized by an anti-GFP antibody via western blot, co-sediments with  F-actin  (comassie blue stained SDS-PAGE), both in presence and absence of Ca2+, regardless of the presence of caldendrin.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D Left: Representative image of COS7  cell expressing the inducible dimerization  system described in C. Red: Peroxisomes (PEX-RFP-FKBP), Green: KIF17-GFP-PEX13 and myoV-GFP-FRB. Scale bar = 10 µm. Middle and right: Individual tracks of  kinesin-driven peroxisome  movements reconstructed over 20 seconds (yellow) before (middle) and after (right) addition of  rapalog indicate that the recruitment of myoV  slows down the  motility of peroxisomes. Blue outlines represent manually drawn cellular border and nucleus  (excluded from analysis).",
    "molecules": "rapalog"
  },
  {
    "caption": "E Analysis of the mean squared displacement (MSD) of peroxisomes over time  (left) suggests a linear increase for all conditions. Slope analysis for MSD curves for individual cells (right) indicates that addition of  rapalog induces efficient stalling (2-way ANOVA, *** p<0.0001 as compared before and after rapalog). However, no difference between control and  caldendrin (cald) transfected cells could be observed (2-way ANOVA, Mean ± SEM; n control=14 cells, n cald=23 cells; 3 experiments).",
    "molecules": "rapalog"
  },
  {
    "caption": "C Representative  confocal  images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons expressing an  ER-marker (ER-tDimer). To pre-load overexpressed  caldendrin with Ca2+, neurons  were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and  stained with a homer1-antibody to visualize synapse-containing spines. Arrows indicate spines  that contain ER. Scale bar = 5 µm. D - F Quantification of protrusion- and spine  density and the number of SER-positive spines as shown in C. (mean ± SEM, n GFP ctrl = 24 cells, n Cald-GFP = 25 cells; 3 experiments). (D) The density of  dendritic protrusions (spines and filopodia) is increased in  cald-GFP overexpressing neurons compared to the control. (* p=0.0119, unpaired t-test). (E) The density of homer1-positive dendritic spines is increased in cald-GFP overexpressing  neurons  compared to the control. (** p=0.0049, unpaired t-test). (F) The percentage of ER-containing  dendritic spines is increased in  cald-GFP overexpressing neurons compared to the control. (* p=0.0179, unpaired t-test) G",
    "molecules": "bicuculline, Ca2+"
  },
  {
    "caption": "G Representative confocal images of control (GFP) or  caldendrin  (cald-GFP) transfected rat primary hippocampal neurons pre-treated with bicuculline as in C and stained  against homer1 and the spine  apparatus-marker  synaptopodin. Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft, blue arrows indicate spine-localized synaptopodin. Scale bar = 5 µm. H - J Quantification of spine density, synaptopodin  clusters and synaptopodin-positive spines as shown in G. (mean ± SEM, n GFP ctrl = 21 cells, n cald-GFP = 30 cells; 2 experiments). (H) Spine density is increased in  cald-GFP overexpressing neurons  compared to the control (in line with 5e). (** p = 0.0024, unpaired t-test). (I) The total number of synaptopodin clusters is not affected by caldendrin overexpression. (n.s., unpaired t-test). (J) The percentage of synapotpodin-positive  spines is increased in  cald-GFP overexpressing  neurons compared to the control. (**** p < 0.0001, unpaired t-test).",
    "molecules": "bicuculline"
  },
  {
    "caption": "A 10 min  time-lapse  imaging of DIV12 wild-type (cald WT), caldendrin  knock-out (cald KO), caldendrin knock-out expressing  caldendrin-GFP (cald  KO + cald-GFP) or caldendrin knock-out treated with  jasplakinolide  (cald  KO + JPK) mouse primary hippocampal neurons, additionally expressing a cell fill (maxGFP) and an ER-marker (ER-tDimer). Yellow arrows indicate protrusions with a stably anchored  ER (>10 min). Blue arrows indicate protrusions that show transient presence of  ER. Scale bar = 5 µm.",
    "molecules": "jasplakinolide, JPK"
  },
  {
    "caption": "A Representative confocal  images of  mouse primary hippocampal neurons stained  against homer1 and synaptopodin. The groups compared are wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald  KO cald), and caldendrin knock-out treated with  jasplakinolide (cald  KO  JPK). Scale bar = 5 µm.",
    "molecules": "jasplakinolide, JPK"
  },
  {
    "caption": "B - C Quantification of spine density, synaptopodin clusters and synaptopodin-positive  spines  as shown in A (mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparisons post-test. *** p < 0.0001. cald wt n = 34 cells, 2 experiments; cald  KO n = 33 cells, 3 experiments; cald  KO cald n = 10 cells, 1 experiment; cald  KO  JPK n = 14 cells, 1 experiment). (B) The total number of synaptopodin  clusters is unaffected by the loss of caldendrin. (C) The number of synaptopodin-positive  spines is reduced in cald KO compared to the WT neurons. This phenotype could be rescued by over-expression of caldendrin (cald KO + cald), but not by treatment with JPK (cald KO + JPK).",
    "molecules": "JPK"
  },
  {
    "caption": "Two examples of retinas  at 48 hpf in (left) control and (right) Prdm1a  morphant (MO). Atoh7+ cells (magenta), inhibitory neurons  (yellow) and photoreceptors  (cyan) are labelled. Scale bar 50 µm. A') Close up of Crx  (cyan) signal (upper panel) and DAPI  (grey, lower panel) from Fig A, for controls (left) and Prdm1a  morphant (right). Scale bar 20 µm. Staining  for the photoreceptor cell marker  zpr-1 at 72 hpf in (left) control and (right) Prdm1a  knockdown. Atoh7+ cells (magenta),  inhibitory neurons (yellow), photoreceptors  (cyan) and zpr-1 (grey). Scale bar 50 µm. Arrowheads indicate zpr-1 staining. B') Close up of Atoh7  (magenta) and Crx  (cyan) signal (upper panel), together with Zpr-1 (grey, lower panel) from Fig B, for controls (left) and Prdm1a  morphant (right). Scale bar 20 µm. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(H)  Ang-2 dose and time-dependently activates p-EphrinB, p-TIE2 and p-AKT in HUVEC;  immunoblotting results;  NA3VO4: sodium orthovanadate.",
    "molecules": "NA3VO4, sodium orthovanadate"
  },
  {
    "caption": "(I) Tumor vessels in  B16F10 tumors are mostly p-TIE2 (Tyr992)+ and pSHP2(Tyr542)+;  confocal images  (scale bars: 200μm).",
    "molecules": "Tyr"
  },
  {
    "caption": "(C,D) The JAK inhibitor Tofacitinib (Tofa, 50nM) enhances the  proliferation of HUVEC and BMEC compared to control (C) and reduces endogenous p-STAT3 levels in HUVEC and BMEC compared to control (D) after 72-hour culture. SHP099 (5μM HUVEC; 20μM  BMEC) reduces cell proliferation and increases p-STAT3 in HUVEC and BMEC compared to control; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.",
    "molecules": "SHP099, Tofa, Tofacitinib"
  },
  {
    "caption": "(E,F) The  MAP kinases inhibitor  PD098059 (PD0980, 10μM) and SHP099 (5μM HUVEC; 20μM BMEC) inhibit HUVEC and  BMEC proliferation compared to control (E) and reduce p-ERK1/2 levels by immunoblotting after 72-hour culture (F). Results of  proliferation in panels C and E (% of control) from triplicate cultures are representative of 3-4 experiments; *P<0.05, **P<0.01 ***P<0.001; two-tailed Student's t-test; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.",
    "molecules": "PD0980, PD098059, SHP099"
  },
  {
    "caption": "(G,H) The ERK1/2 inhibitors  AZD6244 and Tamnitinib dose-dependently  reduce B16F10 cell proliferation (G); representative experiment of 3 independent experiments; the results reflect the means of 5 replicate/condition; error bars: ± S.D.; and levels of endogenous p-STAT3  and p-ERK in B16F10 cells (representative of 3 blots) (H).",
    "molecules": "AZD6244, Tamnitinib"
  },
  {
    "caption": "(A-C) SHP099 (A, 75mg/kg; B, C: 100mg/kg) reduces B16F10 tumor growth in syngeneic mice. Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; no. mice/group: 6-11 control; 7-9 SHP099.",
    "molecules": "SHP099"
  },
  {
    "caption": "(E) \"Vascular sleeves\" (Collagen IV+ CD31-) identified in a representative  SHP099-treated tumor. Representative  confocal images from 6 tumors (scale bar: 50μm). (F) Reduced CD31+ vascular  area in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ. (G) The ratio of Collagen IV+/CD31+ area is increased in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ; Coll: collagen. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.",
    "molecules": "SHP099"
  },
  {
    "caption": "(H) Cleaved caspase-3 detection in a CD31+ vessel (representative of 6 SHP099-treated tumors); arrowheads point to cleaved caspase-3+CD31+ cells. Cleaved caspase-3+ tumor cell is marked by *. Confocal images (scale bar: 10μm). (I) Increased cleaved caspase-3 fluorescence intensity in CD31+ cells from  SHP099-treated tumors compared to controls (n=3/group); at least 100 cells counted/sample. (J) Ki67+ proliferating cells in representative of 3 SHP099-treated and 3 control tumors; CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm). (K) Quantification of proliferating cells within  SHP099 and control (n=4/group) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.",
    "molecules": "DAPI, SHP099"
  },
  {
    "caption": "(L) Hypoxic tumor identified Hypoxyprobe (green) representative of 3 SHP099-treated tumors; confocal images (scale bar: 100μm), left; quantification of hypoxic areas in control (n=3) and  SHP099-treated (n=3) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.",
    "molecules": "Hypoxyprobe, SHP099"
  },
  {
    "caption": "(M) Representative control (of 5) and SHP099-treated (of 5)  B16F10 tumor sections through the maximum diameter; H&E staining (scale bar: 10mm). (N) Cleaved caspase-3+ fluorescence intensity in SHP099 and control tumors (n=3/group). Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001. ",
    "molecules": "SHP099"
  },
  {
    "caption": "(B) Extravasation of  FITC-dextran (green) in tumors established in NSG  mice treated with SHP099; scale bar (50μm). Representative  images from 3 mice/group. (C) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=5/group); ImageJ quantification. P values from two-tailed Student's t-test; *P<0.05. Error bars: ± S.D. ",
    "molecules": "dextran, FITC, SHP099"
  },
  {
    "caption": "E) SHP099 reduces  weight of control (p-LKO vector) and SHP2-depleted (shSHP2)  B16F10 tumors compared to untreated controls: p-LKO untreated (n=5), p-LKO treated with SHP099 (n=9), shSHP2 untreated (n=5), and shSHP2 treated with SHP099  (n=13). P values by analysis of variance with Dunnett's multiple comparison test; ***P<0.001. (F) Persistent depletion of SHP2 in tumors induced by shSHP2-B16F10 cells (n=5) compared to control tumors induced by p-LKO-B16F10 (n=5); tumors removed from mice 18 days after cell inoculation. ",
    "molecules": "SHP099"
  },
  {
    "caption": "(N) Representative  popliteal lymph nodes from control and SHP099-treated  mice with microscopic evidence of metastases; H&E stain; scale bars 500 μm (low magnification) 20 μm (high magnification).",
    "molecules": "SHP099"
  },
  {
    "caption": "(C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative  confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001. (E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001. ",
    "molecules": "AMG386"
  },
  {
    "caption": "(G) Vessel  perfusion visualized by FITC-dextran  (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3  mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes.",
    "molecules": "DAPI, dextran, FITC"
  },
  {
    "caption": "(H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells  counted: control (n=311); SHP099  (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.",
    "molecules": "SHP099, AMG386"
  },
  {
    "caption": "(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm.",
    "molecules": "SHP099, AMG386"
  },
  {
    "caption": "FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images",
    "molecules": "SHP099, AMG386"
  },
  {
    "caption": "(C) Vessel  perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the  nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation.",
    "molecules": "DAPI, dextran, FITC"
  },
  {
    "caption": "Levels of the cellular housekeeping genes GAPDH and 18S rRNA, negative-strand E (negE), and sgE relative to total levels of E RNA were measured in infected cells, clarified supernatants and virus concentrated through a sucrose  cushion by ultracentrifugation. Data points represent four technical replicates from two independent experiments (Cell and Supernatant) or three replicates (Concentrated Virus) and are normalized to the total levels of E for each sample, plotted as mean with error bars plotted as +/- SEM.",
    "molecules": "sucrose"
  },
  {
    "caption": "Maximum 18F-FDG-glucose uptake by Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−tumors 22 weeks after infection by positron emission tomography (PET).Representative PET-CT image of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs.",
    "molecules": "FDG-glucose"
  },
  {
    "caption": "Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1-positive cells in skin and intestine sections of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Survival curve of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Quantification of the histological alterations observed in tamoxifen-treated Trf1lox/lox hUBC-CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild-type counterparts.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Quantification of blood cell populations in wild-type and Trf1lox/lox hUBC-CreERT2mice subjected to a tamoxifen-containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Trf1 expression levels in blood, intestine, skin, and bone marrow of Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild-type mice.",
    "molecules": "tamoxifen"
  },
  {
    "caption": "Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right.",
    "molecules": "ETP-47037, ETP-47228, DMSO"
  },
  {
    "caption": "Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right.",
    "molecules": "ETP-47037, ETP-47228, DMSO"
  },
  {
    "caption": "Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.",
    "molecules": "ETP-47037, ETP-47228, DMSO"
  },
  {
    "caption": "Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.",
    "molecules": "ETP-47037, ETP-47228"
  },
  {
    "caption": "Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).",
    "molecules": "ETP-47037"
  },
  {
    "caption": "Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).",
    "molecules": "ETP-47037"
  },
  {
    "caption": "A-C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lungcarcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.D Representative Ki67 and p-H3 images.",
    "molecules": "ETP-470037"
  },
  {
    "caption": "E Representative H&E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures.",
    "molecules": "ETP-47037"
  },
  {
    "caption": "F Representative H&E images of bone marrow and skin samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown indicating the presence of necrosis, hemosiderosis, multinucleated cells, and giant nuclei. Bone marrow showed moderated aplasia.",
    "molecules": "ETP-47037"
  },
  {
    "caption": "G Telomere length in untreated and ETP-47037-treated lung tumor samples. Representative images are shown to the right.",
    "molecules": "ETP-47037"
  },
  {
    "caption": "IL-1β concentrations in supernatants of macrophages, unstimulated (control) (B) or stimulated with SP/N (C) or LPS/N (D).",
    "molecules": "LPS"
  },
  {
    "caption": "F. Immunofluorescence microscopy images of ASC specks (green) in T2 macrophages. Nuclei were stained with DAPI (blue). Scale bar indicates 20 µm. Arrow indicates ASC speck formation with magnified illustration in the right corner of the image.",
    "molecules": "DAPI"
  },
  {
    "caption": "I. Cleaved gasdermin D (lysate) and cleaved IL-1β (supernatant) detected by Western Blot in macrophages stimulated with LPS/N or SP/N at T0 and T2.",
    "molecules": "LPS"
  },
  {
    "caption": "J, K. Cell death quantification of macrophages by flow cytometry in unstimulated or SP/N stimulated cells at T0 (n = 5 individuals) (J) and T2 (n = 5 individuals) (K) using the viability dye Zombie UV. LPS/N was used as positive control.",
    "molecules": "Zombie UV, LPS"
  },
  {
    "caption": "L. LDH release of macrophages (n = 6 individuals) stimulated with SP/N at T2 compared to unstimulated cells. M. IL-1β concentrations of MCC950 treated T2 macrophage (n = 44 individuals) stimulation with SP/N.",
    "molecules": "MCC950"
  },
  {
    "caption": "E, F. SP or LPS as a control PAMP were incubated with affinity purified CLRs (ratio: 10 vs. 1 for SP) for 1h and then added to primary T2 macrophages (n = 5 individuals). Subsequently, IL-1β was quantified in the supernatants. (E) shows absolute values for SP; (F) shows the fold change calculated with the corresponding unstimulated control for SP and LPS.",
    "molecules": "LPS"
  },
  {
    "caption": "G. Monocytes were isolated prior (n = 5 individuals) or after vaccination (n = 5 individuals) and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin. H. Monocytes were isolated prior (T0; n = 35 individuals) or after vaccination [2 weeks after 1st (T1; n = 28 individuals), 2 weeks (T2; n = 38 individuals) and 10 weeks (T3; n =33 individuals) after 2nd vaccination] and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages, stimulated with the SP S2 subunit SP are shown.",
    "molecules": "nigericin"
  },
  {
    "caption": "B. Phospho-SYK Immunofluorescence-microscopy in macrophages before and after vaccination. Nuclei stained with DAPI. Scale bars indicate 20 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "D. Western blot analysis of phospho-NF-κB and NLRP3 in cell lysates of macrophages (T0; T2) stimulated with LPS/N or SP/N.",
    "molecules": "LPS"
  },
  {
    "caption": "E. Macrophages were incubated with IKK-β-inhibitor KINK-1 (n = 10 individuals) and SYK-inhibitors entospletinib (n = 20 individuals), R406 (n = 13 individuals) prior to SP/nigericin stimulation. IL-1β concentrations measured with ELISA. DMSO-treated, SP/N-stimulated and untreated unstimulated macrophages were used as control (both n = 44 individuals).",
    "molecules": "DMSO, entospletinib, KINK-1, nigericin, R406"
  },
  {
    "caption": "F. IL-1β quantification in THP-1 wild-type cells incubated with SYK inhibitors entospletinib (n = 4 independent experiments) and R406 (n = 2 independent experiments) or in THP-1 SYKKO cells stimulated with SP/N (n = 5 independent experiments).",
    "molecules": "entospletinib, R406"
  },
  {
    "caption": "H. Quantification of cell death in T2 macrophages (n = 4 individuals) treated with R406 prior to SP/N-stimulation.",
    "molecules": "R406"
  },
  {
    "caption": "I-K. mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages without inhibitor (I [exemplary results], J [T0: n = 5 individuals; T2: n = 5 individuals])",
    "molecules": "MitoSOX red, mROS"
  },
  {
    "caption": "mROS quantified MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages with R406 treatment (n = 4 individuals) (K).",
    "molecules": "MitoSOX red, R406, mROS"
  },
  {
    "caption": "L. Western blot analysis of SOD2 in macrophages at T0 and T2 treated with SP/N or LPS/N.",
    "molecules": "LPS"
  },
  {
    "caption": "M. Mitochondrial membrane potential determined with flow cytometry of tetramethylrhodamine methyl ester (TMRM) stained macrophages at T0 (n = 5 individuals) and T2 (n = 5 individuals) stimulated with SP/N. N. T2-macrophages (n = 4 individuals) were treated with the SYK-inhibitor R406. Data information: For statistical analysis, one- or two-way ANOVA with tukey post hoc test was used. Box plots indicate the median and the upper and lower quartile. Outliers are plotted as individual dots (outside the 10-90 percentile). Scatter dot plots show mean ± SD. *p<0.05; **p<0.01, ***p<0.001. (S-protein: SP, lipopolysaccharide: LPS, nigericin: N)",
    "molecules": "tetramethylrhodamine methyl ester, TMRM, lipopolysaccharide, LPS, nigericin, R406"
  },
  {
    "caption": "A-H. (A) T cells at T0 (n = 6 individuals) and T2 (n = 10 individuals) were incubated with SP/N-stimulated macrophages. Co-cultures of unstimulated macrophages were used as control (con). CD4+ subpopulation analyses performed with flow cytometry (Experimental setup: Fig EV3). (B) Quantification of fold change of the CD4+ T cell subpopulation upon SP/N stimulation (T0; T2). Dotted line indicates the baseline (fold change = 1.0). (C) Fold change of CD4+ naïve T cell and CD4+ effector memory T cell populations (D) upon co-culture of macrophages stimulated with SP/N in presence of MCC950 or R406. Identical analyses performed with CD8+ T cells (E, F, G, H).",
    "molecules": "MCC950, R406"
  },
  {
    "caption": "K, L. Interferon-γ quantified in supernatants of corresponding co-cultures, untreated (T0 [n = 6 individuals] and T2 [n = 11 individuals]) (K) and treated with MCC950 or R406 (L) at T2 (n = 11 individuals).",
    "molecules": "MCC950, R406"
  },
  {
    "caption": "A-D Measurement of phosphorylating respiration (represented as State 3o, in the presence of ADP and substrates) in kidney (A) and skeletal muscle (C) from male and female Coq9+/+, Coq9Q95X and Coq9R239X mice at 3 months of age. Representative O2 consumption graphic in kidney (B) and skeletal muscle (D) from female Coq9+/+, Coq9Q95X and Coq9R239X mice.Data information: All values are presented as mean ± SD. (A, C) *P < 0.05; **P < 0.01; ***P < 0.001; Coq9Q95X and Coq9R239Xmice versus Coq9+/+mice. #P < 0.05; Coq9Q95X versus Coq9R239Xmice. One-way ANOVA with a Tukey's post hoc test. Numbers above columns indicate P-values of the one-way ANOVA test (n = 3 for each group).",
    "molecules": "ADP, O2"
  },
  {
    "caption": "A, B KidneyCoQ9 levels in Coq9+/+, Coq9Q95X and Coq9R239Xmice treated with 2,4-diHB (+2,4-diHB) compared with the non-treated littermate (vehicle). Statistical analysis was performed on +2,4-diHBCoq9+/+, Coq9Q95X and Coq9R239Xmice versus vehicle Coq9+/+, Coq9Q95X and Coq9R239Xmice, respectively (n = 3 for each group).",
    "molecules": "2,4-diHB"
  },
  {
    "caption": "C, D CoQ10 levels in COQ9R244X skinfibroblasts treated with 2,4-diHB (+2,4-diHB, blue bars) compared with the non-treated controls (vehicle, yellow bars). Statistical analysis was performed on +2,4-diHBCOQ9R244X versus vehicle COQ9R244X (n = 4 for each group).",
    "molecules": "2,4-diHB"
  },
  {
    "caption": "(E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation.",
    "molecules": "AnTc"
  },
  {
    "caption": "(C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared).",
    "molecules": "AnTc"
  },
  {
    "caption": "HeLa and cyclin F Knock-Out (CCNF K/O) cells were treated with Kinase ChemoGenomic Set (KSGS) in 384 well format. After 72 Hours (h) viability was measured using rezasurin. Flowchart representation (left). Robust Z-score difference is plotted for data acquired at 1 μM concentration (right).",
    "molecules": "rezasurin"
  },
  {
    "caption": "Cell survival measured using rezasurin and compared to controls treated with DMSO (expressed as relative proliferation %). Cells were treated with the indicated inhibitors at 0.1 and 1 μM concentration.",
    "molecules": "DMSO, rezasurin"
  },
  {
    "caption": "Cell survival measured using rezasurin and compared to controls (0) treated with DMSO (expressed as relative proliferation %). Cells were treated with UCN-01 at the indicated concentrations.",
    "molecules": "UCN-01, DMSO, rezasurin"
  },
  {
    "caption": "Differences in viability of HeLa and CCNF K/O cells after treatment with specific Chk1i (LY2603618) at the indicated concentrations plotted on a log10 scale to measure differences in IC50.",
    "molecules": "LY2603618"
  },
  {
    "caption": "Number of cells was measured in HeLa and CCNF K/O cells left untreated or treated with Chk1i (LY2603618) for 24h, 48h, or 72h as indicated. (n.s. = non-significant).",
    "molecules": "LY2603618"
  },
  {
    "caption": "Cell viability measurements using Propidium Iodide staining for HeLa and CCNF K/O cells after treatment with Chk1i (LY2603618).",
    "molecules": "LY2603618"
  },
  {
    "caption": "U-2-OS cells were seeded and transfected with non-targeting siRNA siNC (Negative Control) or siCyc F. 24 hours after transfection, cells were treated with UCN-01, or Chk1i (LY2603618) at indicated concentrations for 3 days before viability was measured.",
    "molecules": "UCN-01, LY2603618"
  },
  {
    "caption": "Cell viability measurements using Propidium Iodide staining in U-2-OS cells transfected with non-targeting siNC and siCyc F and treated with Chk1i (LY2603618) as indicated.",
    "molecules": "LY2603618"
  },
  {
    "caption": "Cells treated with 1μM Chk1i (LY2603618) for the indicated hours (h) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by Western blot (WB). TFIIH was used as a loading control.",
    "molecules": "LY2603618, SDS"
  },
  {
    "caption": "Percentage (%) of γ-H2AX positive cells in G1 cells after Chk1i (LY2603618) treatment for the indicated hours (h) measured using Fluorescence Activated Cell Sorting (FACS). G1 cells were considered having DAPI 2n staining and EdU negative. Percentage (%) of γ-H2AX positive cells in replicating (EdU+) cells after Chk1i (LY2603618) treatment at the indicated time points. Percentage (%) of γ-H2AX positive cells in G2/M cells after Chk1i (LY2603618) treatment. G2/M cells were considered having DAPI 4n staining and EdU negativity.",
    "molecules": "DAPI, EdU, LY2603618"
  },
  {
    "caption": "Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in early S phase cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+ cells with 2N DAPI content. Intensity of γ-H2AX measured as Relative Fluorescence Units (RFU) in replicating cells treated with Chk1i. Mean intensity of γ-H2AX fluorescence was evaluated from EdU+. Overall, cell cycle profile of HeLa and CCNF K/O cells after Chk1i treatment. Cells with 2n DAPI staining but EdU- were gated as G1 cells; cells with >2n but <4n DAPI staining but EdU- were gated as EdU- S cells; cells with >2n but <4n DAPI staining but EdU+ were gated as EdU+ S phase; cells with 4n DAPI and pH3 S10 negative staining were gated as G2 phase and cells with 4n DAPI and pH3 S10 positive as M cells. Data from at least 3 independent replicates and plotted as Mean ± SD.",
    "molecules": "DAPI, EdU"
  },
  {
    "caption": "Representative plots of γ-H2AX vs RPA2 signal of HeLa and CCNF K/O cells after treatment with 1μM Chk1i (LY2603618) at the indicated hours (h). Blue labeled dots represent γ-H2AX positive cells. Relates to materials section for details on representation.",
    "molecules": "LY2603618"
  },
  {
    "caption": "Representative plots of γ-H2AX intensity vs DAPI signal of CCNF K/O cells transfected with empty GFP and GFP-CCNF untreated (top panels) and after treatment with 1μM Chk1i (LY2603618) for 20h (bottom panels). Normalised γ-H2AX intensity in CCNF K/O cells transfected with either GFP empty vector or GFP-Cyc F and treated with 1μM Chk1i for 20 hours. GFP positive cells were gated. Data from 3 independent experiments were plotted with Mean % ± SD.",
    "molecules": "DAPI, LY2603618"
  },
  {
    "caption": "Representative images of neutral and alkaline comet assay from HeLa and CCNF K/O cells treated with Chk1i. Cells were treated with 1μM Chk1i for 24h before being harvested for neutral and alkaline comet assays as indicated. Quantification of comets in alkaline gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100. Quantification of comets in neutral gels. At least 100 cells, across two slides, were analysed in each condition in two biological replicates. Data are shown as medians, with 25/75% percentile range (box) and 10-90% percentile range (whiskers). Fold changes of median values are shown in bold. p-values were calculated using the Mann-Whitney test (two-tailed). Olive tail moment = (Tail.mean - Head.mean)*Tail%DNA/100.",
    "molecules": "DNA"
  },
  {
    "caption": "Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618)",
    "molecules": "DAPI, LY2603618"
  },
  {
    "caption": "HeLa and CCNF K/O cells transfected with the indicated siRNA and treated with 1μM Chk1i (LY2603618) for 20 hours were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. TFIIH served as a loading control.",
    "molecules": "LY2603618, SDS"
  },
  {
    "caption": "HeLa and CCNF K/O cells were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. Ponceau S staining was used as a loading control.",
    "molecules": "SDS"
  },
  {
    "caption": "U-2-OS cells transfected with non-targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS. Whole cell lysates were analyzed by immunoblotting 48h after transfection. H3 was used as a loading control.",
    "molecules": "SDS"
  },
  {
    "caption": "HEK293T cells transfected with empty vector (EV), FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated.",
    "molecules": "MLN4924"
  },
  {
    "caption": "HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h=hours) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. H2B served as a loading control.",
    "molecules": "LY2603618, SDS"
  },
  {
    "caption": "HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min) Quantification of E2F1 half-life",
    "molecules": "CHX, Cycloheximide"
  },
  {
    "caption": "Relative survival (%) of HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations.",
    "molecules": "rezasurin"
  },
  {
    "caption": "(A) Internalization of apoptotic or necrotic GFP-expressing RAW264.7 cells. (B) Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres (B) in primary M2 (IL-4) and M0 macrophages. Cytochalasin D (Cyto) (6 µM) was used as an inhibitor of phagocytosis, 1 h before phagocytosis. Data are shown as means of relative fluorescence units (RFU) ± standard error of the mean (SEM).",
    "molecules": "amino microspheres, Cyto, Cytochalasin D"
  },
  {
    "caption": "(C-E) Real-time fluorescence assays for intraphagosomal proteolysis (C), acidification (D) and lipolysis (E) show substantially increased proteolysis, acidification and lipolysis in the phagosomes of M2(IL-4) macrophages. The kinetics of proteolysis, acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence. Beads were added to macrophages at 0 min. Shaded area in (C) & (D) represents SEM. (E) is a representative of three independent experiments. Leupeptin (100nM) and Bafilomycin (100nM) treatment serve as negative controls in (C) and (D), respectively.",
    "molecules": "Bafilomycin, Leupeptin"
  },
  {
    "caption": "Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1, TAB2, TAK1 and MKK7 to the phagosome of M2(IL-4) macrophages, indicating a K63-polyubiquitylation dependent translocation for these proteins.",
    "molecules": "NSC697923"
  },
  {
    "caption": "(A) Immunoblot against MSR1 of TUBE pulldowns of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL.",
    "molecules": "fucoidan, oxLDL"
  },
  {
    "caption": "(B) Msr1+/+ (WT) and Msr1-/- (KO) M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL (50 μg/ml, 30 min) were analysed for the phosphorylated and the total forms of JNK1/2 and MSR1. ITGAM (CD11b) serves as a loading control. Both fucoidan and oxLDL activate JNK in a MSR1-dependent manner.",
    "molecules": "fucoidan, oxLDL"
  },
  {
    "caption": "(C) IL4-activated MSR1 knock-out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg/ml Fucoidan for 1 hr. Mutation of the ubiquitylation site K27 abolishes MSR1 signalling.",
    "molecules": "Fucoidan"
  },
  {
    "caption": "(D) qPCR data of Tnfa, Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2(IL-4) BMDMs shows an MSR1-dependent increase of pro-inflammatory cytokines in response to MSR1 ligation by fucoidan.",
    "molecules": "fucoidan"
  },
  {
    "caption": "(E) Inhibition of JNK by JNK-IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation, showing that it is JNK-dependent.",
    "molecules": "JNK-IN8"
  },
  {
    "caption": "(F) Flow cytometry analysis of cell surface markers in WT and MSR1-/- M0 macrophages and M2(IL-4) macrophages untreated or stimulated with the MSR1 ligands fucoidan (50 μg/ml, 24 h). Data shows MSR1-dependent increase of the early activation markers CD54, CD69 and CD86 and a decrease of the M2 marker CD301b/Mgl2. CD11b serves as a control.",
    "molecules": "fucoidan"
  },
  {
    "caption": "(B) Pull-downs of K63 polyubiquitin chains and IB analysis of five human primary cancers shows a correlation between the amount of polyubiquitylated MSR1 and JNK activation in an ovarian tumour.",
    "molecules": "polyubiquitin"
  },
  {
    "caption": "A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and tubulin were used as loading controls. *non‐specific band.",
    "molecules": "puromycin"
  },
  {
    "caption": "A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO‐FISH analysis. Metaphase chromosome spreads were stained with Cy3‐conjugated leading strand telomeric PNA probe (red) and FITC‐conjugated lagging strand telomeric PNA probe (green). DNA was counter‐stained with DAPI (blue).B The frequency of chromosome‐type telomeric fusions in cells treated as in (A) was quantified as a percentage of total number of chromosomes (illustrated in Supplementary Fig S2A). A minimum of 2,000 chromosomes were scored for each treatment. Error bars represent SD of at least two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; **P ≤ 0.01; NS, P > 0.05.",
    "molecules": "DNA, puromycin"
  },
  {
    "caption": "A Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.",
    "molecules": "puromycin"
  },
  {
    "caption": "B Immortalized Brca1F/C61G MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. The frequency of end‐to‐end chromosome‐type fusions was analysed as in (A).",
    "molecules": "puromycin"
  },
  {
    "caption": "C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.",
    "molecules": "puromycin"
  },
  {
    "caption": "C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.",
    "molecules": "puromycin"
  },
  {
    "caption": "C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.",
    "molecules": "puromycin"
  },
  {
    "caption": "C-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.",
    "molecules": "puromycin"
  },
  {
    "caption": "A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.B Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (A) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.",
    "molecules": "puromycin"
  },
  {
    "caption": "D Immortalized Brca1F/−MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.E Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.",
    "molecules": "puromycin"
  },
  {
    "caption": "A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and GAPDH were used as loading controls. *non‐specific band.",
    "molecules": "puromycin"
  },
  {
    "caption": "B MboI‐ and AluI‐digested DNA from cells treated as in (A) was resolved by pulsed‐field gel electrophoresis and probed with end‐labelled (AACCCT)4 probe. Representative pulsed‐field gel samples run under native and denatured conditions are shown.",
    "molecules": "DNA"
  },
  {
    "caption": "C Quantification of the 3′ overhang in cells treated as in (B). For each sample, the ss/total DNA ratios were expressed relative to the GFP shRNA‐treated control. Error bars represent SD of two independent experiments.",
    "molecules": "DNA"
  },
  {
    "caption": "A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.",
    "molecules": "puromycin"
  },
  {
    "caption": "A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.",
    "molecules": "puromycin"
  },
  {
    "caption": "A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.",
    "molecules": "puromycin"
  },
  {
    "caption": "Western blot analysis of cytoplasmic (Cty) and nuclear extracts from a bulk population of ISWI-3HA-ribo parasites in the absence (-) or presence (+) of glucosamine (GlcN). ISWI-3HA is detected with an anti-HA antibody. Antibodies against aldolase (Ald.) and histone H3 are controls for the cytoplasmic and nuclear extracts, respectively. Molecular weights are shown to the right.",
    "molecules": "GlcN, glucosamine"
  },
  {
    "caption": "MA plot of log2[glucosamine-treated/untreated, M] plotted over the mean abundance of each gene (A) at 12 hpi. Transcripts with a significantly higher (above x-axis) or lower (below x-axis) abundance in the presence of glucosamine are highlighted in red (q ≤ 0.05). iswi is highlighted in blue (q = 6.71x10-10) and the active var gene is highlighted in green (q = 2.53x10-5). Two and three replicates were used for untreated and glucosamine-treated parasites, respectively. p-values were calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected p-value.",
    "molecules": "glucosamine"
  },
  {
    "caption": "(A) Normalized and rescaled ParBF binding profiles at different ParBF/parSF ratio. ChIP-seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced (16, 28) or not (0.4) with IPTG (100 and 500 µM), or carrying HCN plasmids pZC302 (0.04) or pJYB57 (0.016), normalized as in Fig. 1C, E with the amplitudes of the curves rescaled by the indicated factors (1.2, 10 or 50) to overlap with the curves of highest amplitude. The ParB/parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses (Appendix Fig. S2B). Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig. 1E. Note (i) that the dips at ~9-kbp are not visible for the low levels of available ParB since the signal is close to the basal level and (ii) that the ChIP-seq data at 100 µM IPTG induction (16) are the same as in Fig. 1E. Inset; Same as in the main to display the density without rescaling. (B) ParBF are dispersed in the cell upon titration by HCN plasmids. ParBF-mVenus expressed from pJYB294 were imaged as in Fig. 1D in DLT3577 (left) and DLT3576 (right) carrying pZC302 and pJYB57, respectively. The number of extra parSF per cell, indicated on top of each raw, are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites. White bars: 1 µm. (C) The size of ParBF clusters is independent of the intracellular ParBF concentration. We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of "Nucleation & caging" with corresponding schematics drawn on the right. For direct comparison with (A), all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level. Top: constant ParB concentration; supposing that clusters are compact, the cluster radius σ would depend on the number m of ParB like$\ \sigma = m^{\frac{1}{3}}$. Predictions profiles, plotted at different ratio of ParB/parS, vary within the range of the experimental levels tested. Bottom: constant cluster size; ParB concentrations vary but the range of exploration remains the same resulting in overlappin",
    "molecules": "IPTG"
  },
  {
    "caption": "(A) The formation of secondary ParBF-DNA complexes requires the box II motif. EMSA were performed with a 144-bp 32P-labelled DNA fragments (C144) carrying a single 16-bp parS binding motif. Reaction mixtures containing 100 µg.ml-1 sonicated salmon sperm DNA were incubated in the absence (-) or the presence of increasing concentrations (grey triangle; 10, 30, 100, 300, and 1000 nM) of ParBF or ParBF-3R*. Positions of free and bound probes are indicated on the left. B1 represents complexes involving the specific interaction on the 16-bp binding site, while B\"2 and B\"3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA-binding interactions, respectively (Sanchez et al,",
    "molecules": "salmon sperm DNA"
  },
  {
    "caption": "ChIP-sequencing assays were performed on DLT2075 (xylE::parSF) expressing ParBF grown in exponential (expo) or stationary (stat) phases with addition of rifampicin when indicated (+Rif). The assays have been performed in duplicate for the +Rif and once for the stationary phase experiments. (A) ParBF DNA binding around parSF is independent of active transcription. The color-coded ParBF profiles are represented over 50-Kbp as the relative ParB density normalized to 1 at the first bp after the last parSF binding site. Loci A, C, E and F are defined in Appendix Fig. S1A. (B) The dips and peaks are highly similar in the three indicated conditions. Same as in (A) with zoom in on the right side of parSF up to 9-Kpb and normalization to 1 at genomic coordinate 230. The dotted line corresponds to the analytics description of "Nucleation and caging" (see details in Fig. 1C-E). (C) ParBF binding profile upstream of the locus A. Same as in (A) with zoom in from -6.5 to -16.5-Kbp by normalization to 1 at genomic coordinate -6.5-Kbp (upstream of the dip at the locus A). The ParBF DNA binding profile remains compatible to a power-law, represented by the analytics description (dotted line), upstream of the locus A in stationary phase (black) and in exponential phase (blue). Also, the dips and peaks are highly similar in both conditions. These data are not in favor of the "1D-spreading" or the "Spreading and bridging" models that predicts a basal uniform distribution or a linear decrease after a barrier, respectively (Broedersz et al, 2014). (D) The promoter region at locus A prevents ParBF DNA binding. Chip-seq assays were performed in isogenic xylE::parSF strains (DLT2075; black curve) in which the locus A is replaced by a kanamycin gene (DLT3651; red curve). The assay in the ∆(locus A) genomic context has been performed once. The relative ParB density as a function of the distance from parSF is",
    "molecules": "kanamycin, Rif, rifampicin"
  },
  {
    "caption": "(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.",
    "molecules": "DMSO, doxycycline"
  },
  {
    "caption": "(b) Densitometric analysis relative to actin of soluble EGFP-HDQ74 clearance in stable inducible PC12 cell line expressing EGFP-HDQ74. Transgene expression was induced with doxycycline for 8 h, and then switched off (by removing doxycycline) with drug (all 1 μM) or DMSO (control) treatment for 96 h. Control condition is set to 100%. Error bars show s.e.m.",
    "molecules": "DMSO, doxycycline"
  },
  {
    "caption": "(c) SK-N-SH cells transfected with EGFP-HDQ74 construct for 4 h were treated with drugs (all 1 μM) or DMSO (control) for 48 h post-transfection. The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1. Error bars show 95% confidence interval.",
    "molecules": "DMSO"
  },
  {
    "caption": "(d) PC12 cells were treated with clonidine, minoxidil and verapamil (all 1 μM) or DMSO (control) for 24 h. Endogenous LC3-II levels were detected with anti-LC3 antibody and quantified relative to actin. Rapamycin (0.2 μM) was positive control. Error bars show s.e.m.",
    "molecules": "clonidine, DMSO, minoxidil, Rapamycin, verapamil"
  },
  {
    "caption": "(e) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs, transfected with EGFP-HDQ74 for 4 h and then treated for 48 h with drugs (all 1 μM) or DMSO (control). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.",
    "molecules": "DMSO"
  },
  {
    "caption": "(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.",
    "molecules": "DMSO, doxycycline"
  },
  {
    "caption": "(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 1 μM rilmenidine, 1 mM dibutyl-cAMP (db-cAMP), 24 μM forskolin or 500 μM 2′5′-dideoxyadenosine (2′5′ddA) for 24 h. Note that control clearance levels for a given substrate may vary from figure to figure, due to different exposures of blots, which aid illustration of agents that either increase or retard clearance.",
    "molecules": "db-cAMP, dibutyl-cAMP, 2′5′-dideoxyadenosine, 2′5′ddA, forskolin, rilmenidine"
  },
  {
    "caption": "(b) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH (for rilmenidine) and COS-7 (for db-cAMP, forskolin and 2′5′ddA) cells as in Figure 1c, treated with concentrations used in a for 48 h post-transfection. Error bars show 95% confidence interval.",
    "molecules": "db-cAMP, 2′5′ddA, forskolin, rilmenidine"
  },
  {
    "caption": "(c) Endogenous LC3-II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2′5′ddA for 24 h.",
    "molecules": "2′5′ddA, rilmenidine"
  },
  {
    "caption": "(d) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 8-CPT-2Me-cAMP or 6-Bnz-cAMP (1 μM or 10 μM).",
    "molecules": "6-Bnz-cAMP, 8-CPT-2Me-cAMP"
  },
  {
    "caption": "(e) The proportions of EGFP-positive COS-7 cells with aggregates as in Figure 1c, treated with or without 10 μM 8-CPT-2Me-cAMP or 6-Bnz-cAMP for 48 h post-transfection. Error bars show 95% confidence interval.",
    "molecules": "6-Bnz-cAMP, 8-CPT-2Me-cAMP"
  },
  {
    "caption": "(h) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or dominant-negative Rap2B (1:3 ratio) for 4 h, were treated with or without 10 μM 8-CPT-2Me-cAMP for 48 h. Error bars show 95% confidence interval.",
    "molecules": "8-CPT-2Me-cAMP"
  },
  {
    "caption": "(a) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Calpain activity was detected by immunoblotting with anti-calpain small subunit antibody. Densitometry analysis is relative to actin. Error bars show s.e.m.",
    "molecules": "Bay K8644"
  },
  {
    "caption": "(b) SK-N-SH cells transfected with EGFP-HDQ74 for 4 h, then pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 48 h post-transfection, were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval.",
    "molecules": "Bay K8644"
  },
  {
    "caption": "(c) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Endogenous LC3-II levels were detected by immunoblotting with anti-LC3 antibody.",
    "molecules": "Bay K8644"
  },
  {
    "caption": "(d) Comparison between calpain activity, EGFP-HDQ74 aggregation and autophagosomes as seen in a-c. The control condition for all the assessments was set at 100%. Calpastatin lowered the increased calpain activity and EGFP-HDQ74 aggregation caused by (±)-Bay K8644 in a dose-dependent manner with a simultaneous increase in LC3-II levels (autophagosomes).",
    "molecules": "Bay K8644"
  },
  {
    "caption": "(e) COS-7 cells transfected with empty vector (pcDNA3.1) or wild-type PLC-ε for 4 h and treated with or without 50 μM calpeptin for 48 h post-transfection were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.",
    "molecules": "calpeptin"
  },
  {
    "caption": "(a) The proportions of EGFP-positive SK-N-SH cells with EGFP-HDQ74 aggregates or cell death as in Figure 1c, treated with or without 10 μM calpastatin, 50 μM N-acetyl-Leu-Leu-methional (ALLM) or 50 μM calpeptin for 48 h post-transfection. DMSO was control for ALLM and calpeptin. Error bars show 95% confidence interval.",
    "molecules": "N-acetyl-Leu-Leu-methional, ALLM, calpeptin, DMSO"
  },
  {
    "caption": "(g) SK-N-SH cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active m-calpain (1:3 ratio) for 4 h, were subsequently treated with DMSO (control), 1 μM verapamil or 1 μM clonidine for 48 h. The proportions of EGFP-positive cells with aggregates were assessed as in Figure 1c. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05.",
    "molecules": "clonidine, DMSO, verapamil"
  },
  {
    "caption": "(d) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin, 50 μM ALLM or 50 μM calpeptin. Calpain inhibitors were treated for 24 h before bafilomycin A1 addition. EGFP-LC3-II was detected with anti-EGFP antibody. Data from different calpain inhibitors are from different blots.",
    "molecules": "bafilomycin A1, ALLM, calpeptin"
  },
  {
    "caption": "(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 100 nM PACAP, with or without 10 μM calpastatin or 500 μM 2′5′ddA for 24 h.",
    "molecules": "2′5′ddA"
  },
  {
    "caption": "(b) The proportion of EGFP-positive cells with aggregates in SK-N-SH cells as in Figure 1c, treated for 48 h with 1 μM PACAP or 200 μM NF449. Error bars show 95% confidence interval.",
    "molecules": "NF449"
  },
  {
    "caption": "(c) Aggregation in COS-7 cells transfected with either pcDNA3:1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for 4 h and then treated with or without 500 μM 2′5′ddA for 48 h. Error bars show 95% confidence interval.",
    "molecules": "2′5′ddA"
  },
  {
    "caption": "(f) Endogenous LC3-II levels in PC12 cells treated with or without 200 μM NF449 for 24 h.",
    "molecules": "NF449"
  },
  {
    "caption": "(g) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h. Densitometric analysis is relative to actin. Error bars show s.e.m.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(a) The proportions of EGFP-positive COS-7 cells with aggregates or cell death, after transfection with EGFP-HDQ74 and rheb or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post-transfection. The control condition for assessing the effect of calpain inhibitors in rheb-transfected cells was taken as 1. Error bars show 95% confidence interval.",
    "molecules": "calpeptin"
  },
  {
    "caption": "(b) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or rheb (1:3 ratio) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post-transfection. Error bars show 95% confidence interval.",
    "molecules": "calpeptin"
  },
  {
    "caption": "(c) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 and constitutively active (CA) m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 48 h post-transfection. Error bars show 95% confidence interval.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(d) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and CA m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 24 h post-transfection. Error bars show 95% confidence interval.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(e,f) The proportions of EGFP-positive COS-7 cells with aggregates (e) and cell death (f) as in Figure 1c, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for 48 h. Error bars show 95% confidence interval.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(g) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 48 h switch-off period. Error bars show s.e.m.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(h) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1a, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 8 h switch-off period. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(a) Western blot showing EGFP-HDQ71-induced degeneration of rod photoreceptors at 9 d.p.f. as indicated by the decrease in the abundance of of monomeric (∼36 kDa) and dimeric (∼72 kDa) rhodopsin. Rhodopsin expression is further reduced by 3 μM (±)-Bay K8644, and recovered upon treatment with 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin. No general toxicity to the zebrafish at these concentrations was observed. (b) Expression of EGFP-HDQ71 significantly reduces the levels of rhodopsin expression when compared to wild-type (WT) nontransgenic controls. This effect is further abrogated by 3 μM (±)-Bay K8644, whereas 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin all protected against rod photoreceptor degeneration. Error bars show s.e.m.",
    "molecules": "2′5′ddA, Bay K8644, clonidine, verapamil"
  },
  {
    "caption": "(c) Magnified bright field and EGFP images of zebrafish eye sections, taken at the ventral marginal zone for each of the indicated treatments (with concentrations as in b), are shown. Scale bar, 10 μm. ***P 0.001; **P 0.01; *P 0.05. (d) The total number of aggregates across 8 × 12 μm sections was counted and compared to the EGFP-HDQ71 DMSO-treated control. 3 μM (±)-Bay K8644 was found to significantly increase the aggregate burden, whereas 3 μM verapamil, 1 μM calpastatin, 3 μM clonidine and 100 μM 2′5′ddA decreased the aggregate load. As in our cell models, these aggregates were insoluble in 4% triton/SDS. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05.",
    "molecules": "2′5′ddA, Bay K8644, clonidine, DMSO, verapamil"
  },
  {
    "caption": "E. Polarographic profiles of isolated mitochondria from wt (lower trace) and ATPIF1H49K|T/H (upper trace) animals. Histograms show a reduction in state 3 respiration consistent with the inhibition of ATP synthase in ATPIF1H49K|T/H mice. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A.",
    "molecules": "Ant A, antimycin A, OL, oligomycin"
  },
  {
    "caption": "A. Representative images of mice and body weight graph following the expression of mitochondrial ATPIF1H49K (days of doxycycline) (wt, n= 12; LowOXPHOS, n= 12).",
    "molecules": "doxycycline"
  },
  {
    "caption": "B. Representative images of hindlimb muscles. Arrows point to the soleus. (C, D) Transversal slices of red fibers from soleus stained with hematoxylin/eosin (C). Higher im-WAT infiltrations in LowOXPHOS mice are shown. Quantifications in D (wt, n= 6; LowOXPHOS, n= 6; 10 fields/mouse). ",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "H. Skm acetyl-CoA amounts (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "acetyl-CoA"
  },
  {
    "caption": "J. Myocyte rates of aerobic glycolysis to lactate production. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "lactate"
  },
  {
    "caption": "(K, L). 14C(u)-glucose uptake (K) and oxidation to CO2 (L) in myocytes expressing or not the ATP synthase inhibitor ATPIF1H49K. Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "CO2, 14C, glucose"
  },
  {
    "caption": "N. Skm levels of lactate (wt, n= 6; LowOXPHOS, n= 6). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "lactate"
  },
  {
    "caption": "O. FFA β-oxidation in primary cultures of myocytes derived from wt or LowOXPHOS mouse hindlimbs. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "FFA"
  },
  {
    "caption": "P. FFA amounts in Skm extracts (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "FFA"
  },
  {
    "caption": "Transversal slices of soleus stained with hematoxylin/eosin (Q). Arrows indicate LDs in LowOXPHOS mice. Images are representative of n= 4 mice/genotype, 10 images/mouse.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "Oil Red O staining and enzymatic activity of respiratory CI in transverse contiguous slices (R). Images are representative of n= 4 mice/genotype, 10 images/mouse. Note the colocalization of oxidative fibers with LDs (R).",
    "molecules": "Oil Red O"
  },
  {
    "caption": "T. LD formation upon ATP synthase inhibition (ATPIF1H49K) in starved myocytes after 24 hours of palmitate supplementation in the presence or absence of BCAA. Blue: DAPI, nuclei; green: BODIPY-positive LDs. Histograms show the quantification expressed as the number of LDs/nuclei. Bars are the mean ± s.e.m. of n= 3 experiments, 12 fields/condition. U. LD formation upon ATP synthase inhibition (5µM oligomycin) in myocytes after 24 hours of palmitate supplementation. Histograms show the quantification expressed as fold of control of the BODIPY/DAPI fluorescence intensity. 7 fields/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "BODIPY, BCAA, DAPI, palmitate, oligomycin"
  },
  {
    "caption": "Plasma (A) BCAA levels in wt and LowOXPHOS mice (wt, n= 4; LowOXPHOS, n= 4). Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.",
    "molecules": "BCAA"
  },
  {
    "caption": "Skm (C) BCAA levels in wt and LowOXPHOS mice (wt, n= 4; LowOXPHOS, n= 4). Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.",
    "molecules": "BCAA"
  },
  {
    "caption": "D, E). oxidation (D) and incorporation into lipids (E) in myocytes expressing or not ATPIF1H49K. Bars are the mean ± s.e.m. of n= 3 experiments, 6 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.",
    "molecules": "lipids"
  },
  {
    "caption": "F. Longitudinal (left panels) and transversal (right panels) slices of soleus stained with hematoxylin/eosin. im-WAT infiltrations and fiber shrinkage (arrows) in HFD-fed LowOXPHOS mice (wt + HFD, n= 4; LowOXPHOS + HFD, n= 4; 10 fields/mouse).",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(J, K) Quantitative Skm lipidomics at day 80 of HFD (J). The color scale (brown to blue) in the heat-map represents LowOXPHOS/wt amounts of detailed lipid species. A p-value ≤ 0.05 was considered statistically significant (red). In the lower heat-map (K), the color scale (yellow to blue) highlights an increase in DAG in HFD-fed LowOXPHOS mice. Each sample is a pool from 4 mouse extracts. Histograms show the total amounts of Skm DAG (wt, n= 8; LowOXPHOS, n= 8). L. Blood triglycerides on the 80th day of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype *p <0.05 when compared to wt by ANOVA or Student's t-test.",
    "molecules": "DAG, lipid, triglycerides"
  },
  {
    "caption": "O. Blood glucose following the administration of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). T2D onset occurred on day 80 in wt and day 60 in LowOXPHOS mice.",
    "molecules": "glucose"
  },
  {
    "caption": "P. Insulin (ITT) and glucose (GTT) tolerance tests on day 60 of HFD (wt + HFD, n= 10; LowOXPHOS + HFD, n= 10). LowOXPHOS but not wt mice appeared diabetic.",
    "molecules": "glucose"
  },
  {
    "caption": "A. v-WAT iTRAQ ratio of proteins from BCAA catabolism (wt, n= 4; LowOXPHOS, n= 4). Higher intensities of blue colors represent lower LowOXPHOS/wt expression ratios.",
    "molecules": "BCAA"
  },
  {
    "caption": "B. Odd chain FAs in Skm and WAT from wt (n= 8) or LowOXPHOS (n= 8) mice. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "Odd chain FAs"
  },
  {
    "caption": "Representative WB expression (C) of v-WAT proteins from lipid metabolism. Each sample contains extracts from 3 mice. The histogram shows the quantifications (wt, n= 6; LowOXPHOS, n= 6). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "lipid"
  },
  {
    "caption": "iTRAQ analysis (D) of v-WAT proteins from lipid metabolism. Each sample contains extracts from 3 mice. The histogram shows the quantifications (wt, n= 6; LowOXPHOS, n= 6).",
    "molecules": "lipid"
  },
  {
    "caption": "E. v-WAT weight expressed as a percentage of the wt (wt, n= 12; LowOXPHOS, n= 12). F. Quantitative lipidomics in v-WAT. The color scale (brown to blue) in the heat map represents LowOXPHOS/wt amounts of specific lipid species. A p-value ≤ 0.05 was considered statistically significant (red). Each sample is a pool from 4 mouse extracts, n= 8/genotype. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "lipid"
  },
  {
    "caption": "G. v-WAT amounts of triacylglycerides (TAGs) and diacylglycerides (DAGs) (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "DAGs, diacylglycerides, TAGs, triacylglycerides"
  },
  {
    "caption": "H. Higher saturated lipid species in LowOXPHOS mice compared to wt (wt, n= 8; LowOXPHOS, n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "Higher saturated lipid"
  },
  {
    "caption": "I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "MitoSox, ROS"
  },
  {
    "caption": "J. Representative WB of Skm lipid peroxidation and redox system proteins. Two samples per condition are shown. Each sample contains extracts from 3 mice. Histograms represent quantification (wt, n= 6; LowOXPHOS, n= 6). 4-Hydroxynonenal (4HN), peroxiredoxin 2, 3 and 6 (PRX), superoxide dismutase 1 and 2 (SOD), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. βF1 is presented as a loading control. Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "4-Hydroxynonenal, 4HN"
  },
  {
    "caption": "L. Skm iTRAQ ratio of FADH2-binding proteins (wt, n= 12; LowOXPHOS, n= 12). A higher intensity of red color represents a higher LowOXPHOS/wt expression ratio.",
    "molecules": "FADH2"
  },
  {
    "caption": "M. Oxidized FAD levels and the reduced/oxidized FAD ratio in Skm extracts (wt, n= 8; LowOXPHOS n= 8). Data information: Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by Student's t-test.",
    "molecules": "FAD"
  },
  {
    "caption": "B. Polarographic profiles of isolated mitochondria from wt (lower trace) and LowOXPHOS (upper trace) animals using succinate (left graph) or palmitoyl-carnitine (right graph) as a substrate. Quantification of maximal respiration in the right histogram. Bars are the mean ± s.e.m. of n= 3 mice/genotype, 3 traces/mouse; OL, oligomycin; Ant A, antimycin A. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.",
    "molecules": "Ant A, antimycin A, carnitine, palmitoyl, OL, oligomycin, succinate"
  },
  {
    "caption": "I. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each ETC inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.",
    "molecules": "MitoSox, ROS"
  },
  {
    "caption": "J. MitoSox staining in myocytes expressing or not ATPIF1H49K. The left scheme illustrates where each CII inhibitor works. The right histogram shows the quantification of mitochondrial ROS. Bars are the mean ± s.e.m. of n= 3 experiments, 12 replicas/condition. Data information: *, # p <0.05 when compared to wt or LowOXPHOS, respectively, by ANOVA and Student's t-test.",
    "molecules": "MitoSox, ROS"
  },
  {
    "caption": "(A, B) The schematic illustrates the screening process of 702 drugs related to the mitochondrial respiratory capacity assessed by Seahorse XFe96 in C2C12 myocytes. Palmitate is used as a substrate. A total of 41 hits were identified. Maximum respiration (Max R, % of untreated cells) of compounds categorized by their therapeutic use (B).",
    "molecules": "Palmitate"
  },
  {
    "caption": "(C, D) The schematic illustrates the screening process of the 41 hits on FFA β-oxidation using 9,103H(N) palmitic acid as a substrate in C2C12 myocytes. Ten enhancers were identified. D. Correlation between the maximum respiration (x) and palmitate β-oxidation (y). Colors identify classes of pharmaceuticals as in B. Edaravone was selected as a hit.",
    "molecules": "FFA, palmitate, 9,103H(N) palmitic acid"
  },
  {
    "caption": "E. Representative respiratory profile of C2C12 myocytes treated (green trace) or not treated (black trace) with 2 µM edaravone. The CPT1 inhibitor etomoxir was used as a negative control. OCR, oxygen consumption rate; OL, oligomycin; DNP, 2,4-dinitrophenol; Rot, rotenone; Ant A, antimycin A. Quantification in right histogram. Bars are the mean ± s.e.m. of 12 replicas/condition. Data information ;0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "2,4-dinitrophenol, DNP, Ant A, antimycin A, edaravone, etomoxir, OL, oligomycin, Rot, rotenone"
  },
  {
    "caption": "(F, G) FFA β-oxidation in C2C12 myocytes transfected with CRL or ATPIF1H49K plasmids (F) and in primary myotubes from wt and LowOXPHOS mice (G), treated (green bars) or not with 2 µM edaravone. Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition. Data information: *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "edaravone, FFA"
  },
  {
    "caption": "A. Mitochondrial ROS after 3 hours treatment with 2 μM edaravone (green bars), 10 nM MitoQ (purple bars) or 1 mM NAC (blue bars). Bars are the mean ± s.e.m. of n= 3 experiments, 9 replicas/condition.",
    "molecules": "edaravone, MitoQ, NAC, ROS"
  },
  {
    "caption": "B. Representative WB expression of Skm ROS system proteins in mice administered (30 days) edaravone. Two samples per condition are shown. Each sample contains extracts from 3 mice (wt + edaravone, n= 6; LowOXPHOS + edaravone n= 6). Peroxiredoxin 2 and 3 (PRX), catalase (CATA) and glutathione reductase (GSR) immunoblots are shown. GAPDH is presented as a loading control.",
    "molecules": "edaravone, ROS"
  },
  {
    "caption": "C. qPCR relative expression of TNFα and PGC1α in myocytes treated for 24 h with 2μM edaravone (green bars), 10nM MitoQ (purple bars) or 1 mM NAC (blue bars). 6 replicas/condition.",
    "molecules": "edaravone, MitoQ, NAC"
  },
  {
    "caption": "Representative WB expression of Skm de novo lipid synthesis (D) One month of edaravone treatment downregulated FASN and ACLY in both wt and LowOXPHOS mice (D) Two samples per condition are shown. Each sample contains extracts from 3 mice. βF1 is presented as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6; wt + edaravone, n= 6; LowOXPHOS + edaravone, n= 6).",
    "molecules": "edaravone, lipid"
  },
  {
    "caption": "Representative OXPHOS (E) proteins. One month of edaravone treatment in both wt and LowOXPHOS mice and rewired the upregulation of ETF-A, ETF-B and SDHA proteins to the levels of wt (E). Two samples per condition are shown. Each sample contains extracts from 3 mice. Tubulin is presented as a loading control. Quantifications in lateral histograms (wt, n= 6; LowOXPHOS, n= 6; wt + edaravone, n= 6; LowOXPHOS + edaravone, n= 6).",
    "molecules": "edaravone"
  },
  {
    "caption": "F. 2D-PAGE of Skm extracts from wt (n= 3) and LoxOXPHOS (n= 3) mice treated with edaravone. The pI of SDHA, calculated by protein migration in pH 3-10 NL strips, was the same for both genotypes.",
    "molecules": "edaravone"
  },
  {
    "caption": "G. Representative blue native immunoblots (BN) of Skm mitochondrial membrane proteins from 2-month-old mice treated with edaravone for 30 days (wt + edaravone, n= 3; LowOXPHOS + edaravone, n= 3). The migration of the respiratory complexes/supercomplexes CI-CIV and hATPIF1 is indicated. VDAC is shown as a loading control.",
    "molecules": "edaravone"
  },
  {
    "caption": "H. Mouse body weight following the administration of HFD and edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "edaravone"
  },
  {
    "caption": "I. v-WAT amounts at day 60th of HFD in mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "edaravone"
  },
  {
    "caption": "J. Insulin (ITT) and glucose (GTT) tolerance tests after 60 days of HFD in mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "edaravone, glucose"
  },
  {
    "caption": "K. GTT after 80 days of HFD (left) and ITT after 90 days of HFD (right) in wt and LowOXPHOS mice treated with edaravone, showing that the compound has an improving effect itself. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "edaravone"
  },
  {
    "caption": "L. Blood glucose following the administration of HFD. T2D onset occurred on day 90 in wt and LowOXPHOS mice treated with edaravone. Data information: H-L: wt + HFD, n= 5; LowOXPHOS + HFD n= 4; wt + HFD + edaravone, n= 10; LowOXPHOS + HFD + edaravone n= 9. Bars are the mean ± s.e.m. of the indicated (n) mice/genotype. *p <0.05 when compared to wt by ANOVA and Student's t-test.",
    "molecules": "edaravone, glucose"
  },
  {
    "caption": "E. Survival curves for WT (n=12) and Spns2-/- (n=17) CLP  models receiving meropenem  treatment within 7 days. Data information: Data are presented as percentage E) N represents biological replicates. P values were determined by log-rank test E) *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.",
    "molecules": "meropenem"
  },
  {
    "caption": "G. Concentrations of serum  cytokines in meropenem-treated CLP  models (n=6 per time point  for each group). Data information: Data are presented as mean ± s.e.m. G). N represents biological replicates. P values were determined by unpaired t-test G). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.",
    "molecules": "meropenem"
  },
  {
    "caption": "A. Gene expression of inflammatory cytokines in isolated PMs  challenged with 10 ng/ml LPS  (n=3 per  time point  for each group). Data information: Data are presented as mean ± s.e.m. (A, N represents biological replicates. P values were determined by unpaired t-test (A, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.",
    "molecules": "LPS"
  },
  {
    "caption": "D. Survival curves for bone marrow transplanted CLP  models receiving meropenem  treatment within 7 days (n=8 for each group). Data information: Data are presented as percentage D, N represents biological replicates. P values were determined by log-rank test D, *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.",
    "molecules": "meropenem"
  },
  {
    "caption": "B. Oxygen consumption rates  (OCR) and quantifications of  basal respiration, maximal respiration, ATP  production, and proton leakage in resting WT, Spns2-/-, and 1 μM S1P  pre-treated Spns2-/- PMs  (n=4 for each group). Data information: Data are presented as mean ± s.e.m. (B N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.",
    "molecules": "ATP, S1P"
  },
  {
    "caption": "C. Total glucose  consumption in 72 hours (n=4 to 5 for each group), the ratio of NAD+ to NADH  (n=4 for each group), and the levels of intracellular lactate  (n=4 to 5 for each group) in resting PMs. Data information: Data are presented as mean ± s.e.m. C). N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.",
    "molecules": "glucose, lactate, NAD+, NADH"
  },
  {
    "caption": "H. Protein levels of Slc25a12  and Slc25a13  in WT, Spns2-/-, and 1 μM S1P  pre-treated Spns2-/- PMs  (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.",
    "molecules": "S1P"
  },
  {
    "caption": "A. Elevated levels of intracellular lactate  in resting WT PMs  treated with 5 and 10 nM rotenone  for 16 hours (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "lactate, rotenone"
  },
  {
    "caption": "C. Enhanced activities  of total superoxide dismutase (SOD) and catalase  in rotenone-treated WT PMs  (n=3 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "rotenone"
  },
  {
    "caption": "F. Attenuated activities  of total SOD  and catalase  in oxamate- and S1P-treated Spns2-/- PMs  (n=3 to 4 for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "oxamate, S1P"
  },
  {
    "caption": "A, B. Reduced gene expression  of inflammatory cytokines in Spns2-/- PMs  treated with either oxamate  (A) or mitoquinone  (B) (n=3 per time point  for each group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. **P < 0.01; ***P < 0.001.",
    "molecules": "oxamate, mitoquinone"
  },
  {
    "caption": "C. Activation of Spns2/S1P  signaling and suppression of the lactate-ROS  axis promoted the survival of Spns2-/- sepsis  models induced by heat-killed E. coli through the alleviation of hyerinflammation  (n=5 for Spns2-/- control group; n=4 for each treatment group). Data information: Data are presented as mean ± s.e.m. N represents biological replicates. P values were determined by unpaired t-test. **P < 0.01; ***P < 0.001.",
    "molecules": "lactate, ROS, S1P"
  },
  {
    "caption": "A. S1P  treatment restored inflammatory response in Spns2-/- PMs  after 6 hours post- LPS  challenge (n=3 per time point for each group). Data information: Data are presented as mean ± s.e.m. (A N represents biological replicates. P values were determined by unpaired t-test (A *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "LPS, S1P"
  },
  {
    "caption": "B. Enhancement of Spns2/S1P  signaling significantly improved the survival  of CLP  models without antibiotic therapy (n=8 for Spns2-/- saline  group; n=14 for Spns2+/- group; n=12 for Spns2-/- S1P  treatment group). Data information: Data are presented as percentage (B). N represents biological replicates. P values were determined by log-rank test (B). *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "saline, S1P"
  },
  {
    "caption": "C. Enhancement of Spns2/S1P  signaling restored inflammatory response in Spns2-/- CLP  models at 12 hours post-infection (n=6 for each group). Data information: Data are presented as mean ± s.e.m. C) N represents biological replicates. P values were determined by unpaired t-test C) *P < 0.05; **P < 0.01; ***P < 0.001.",
    "molecules": "S1P"
  },
  {
    "caption": "(A) Transfer of K18-ATTO 594 fibrils from donor cells to H2B-GFP-expressing acceptor cells. Representative confocal images of each population are in the upper panels, below are pictures after 24 hours of co-culture of the two populations (left), and of acceptor cells treated with conditioned medium from donor cells for 24 hours (SN, right). In white is the cell membrane labelling with WGA (wheat germ agglutinin) coupled to Alexa 647. The arrows point to acceptor cells containing fibrils, scale bars are 10 μm.",
    "molecules": "ATTO 594, Alexa 647"
  },
  {
    "caption": "(B) Quantification by flow cytometry of the percentage of K18-ATTO 594-positive acceptor cells after co-culturing donor and acceptor cells (total), or culturing acceptor cells with donor-conditioned medium for 24 hours (secretion). The total transfer is arbitrarily set at 100%, and cell-cell contact transfer is calculated by subtracting secretion transfer from total transfer. Data represent the means (+SD) of 4 independent experiments, with statistical analysis by two-tailed unpaired t-test (****P = 4.64E-08).",
    "molecules": "ATTO 594"
  },
  {
    "caption": "(C) Quantification by flow cytometry of the relative percentage of K18-ATTO 594-positive acceptor cells upon treatment with CK666 during the co-culture. Left, data represent the means (132%) +SD, normalized to non-treated co-culture arbitrarily set at 100%, of 3 independent experiments, with statistical analysis by two-tailed unpaired t-test (*P = 0.015). The hatched area of each bar represents the part due to secretion (respectively 4.8 and 4.3% in the absence and presence of CK666). Right is the same analysis when the cells were cultured in sparse conditions, not allowing cell-to-cell contacts (mean= 47.7%, 3 independent experiments, *P = 0.020).",
    "molecules": "ATTO 594, CK666"
  },
  {
    "caption": "(D) Representative confocal images (40x) of CAD cells treated with 1 µM K18-ATTO 594 fibrils, 24h after fibrils addition, fixed and stained with WGA-Alexa 488 (green) and DAPI (blue in the merge panels). Bottom panels are a bottom slice corresponding to the substrate-attached surface of cells (z3), upper panels correspond to slice 10 of the same picture, (z10), not attached to the substrate). On the right and below z10 pictures, are the orthogonal views (xz and yz) of the same region covering 27 slices over 11 μm in total. The arrows point to red fibrils into a WGA-positive TNT. Scale bars are 10 μm.",
    "molecules": "ATTO 594, Alexa 488, DAPI"
  },
  {
    "caption": "(A) Representative confocal images of DS1 cells (expressing soluble Tau RD-YFP), non-treated and treated with increasing concentrations of non-labelled K18 fibrils, fixed after two days and stained with WGA-Alexa 594 (red). The arrows point to examples of aggregate-containing cells, scale bars are 40 μm. On the right is the quantification of the mean percentage +SEM of cells where green aggregates were detected (the total number of cells analyzed over 3 independent experiments were 1188 for non-treated, 1552 for 0.3 μM, and 1506 for 1 μM), with statistical analysis by one-way ANOVA and Bonferroni post hoc test (****P = 6.20E-06 and 6.74E-05 for 0 to 0.3 and 0 to 1 comparisons respectively).",
    "molecules": "Alexa 594"
  },
  {
    "caption": "(B) Representative confocal images (40x) of RD-YFP SH cells, non-treated and treated with 1 μM K18-ATTO 594 fibrils, 48h after fibrils addition. Right insets are threefold enlargements of the boxed region of treated cells, merge and single channel pictures; white arrows point to partial colocalization. Scale bars are 10 μm, 2 μm in the insets. On the right is the quantification of the mean percentage (24%) +SD of cells where green aggregates were detected (2874 treated cells analyzed over 6 independent experiments), with statistical analysis by two-tailed unpaired t-test (****P = 3.75E-07).",
    "molecules": "ATTO 594"
  },
  {
    "caption": "(D) Monitoring the conversion of DS1 cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. The scatter plot represents means of 5 independent experiments (± SD for green and black curves).",
    "molecules": "ATTO 594"
  },
  {
    "caption": "Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. (E) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. the scatter plot shows means of 3 independent experiments ±SD for the green and black curves.",
    "molecules": "ATTO 594"
  },
  {
    "caption": "Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. (F) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with a cortex crude extracts from a AD-deceased patient. , the scatter plot shows means of 2 independent experiments ±SD for the green curve. IncuCyte (20x objective) was set to acquire images every 30 minutes, phase-contrast and green channel (Excitation 440-480 nm, 400 ms) were acquired (9 images/well). Analysis was performed with IncuCyte software to give the relative proportion of cells being converted to inclusion-containing cells (the end point of treated cells was arbitrarily set at 100%, black curve: non-treated cells, green curve: treated cells).",
    "molecules": "ATTO 594"
  },
  {
    "caption": "(A) RD-YFP SH cells were challenged with non-labelled K18 fibrils, incubated 2 days later with Bafilomycin A1 (second line of panels), Bortezomide (bottom panels) or left non-treated (NT, upper panels) for 4 hours before fixation, saponin-permeabilization and staining with antibody recognizing p62 (red) and WGA (white in left panels). Images are confocal pictures after deconvolution (63x, zoom 1.8, px size = 60 nm, slice of 0.43 μm) representative of three independent experiments. Insets are threefold enlargements of the boxed regions of second column; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in insets.",
    "molecules": "Bafilomycin A1, Bortezomide, saponin"
  },
  {
    "caption": "(A) RD-YFP SH cells were challenged with non-labelled K18 fibrils, incubated 2 days later with Bafilomycin A1 (second line of panels), Bortezomide (bottom panels) or left non-treated (NT, upper panels) for 4 hours before fixation, saponin-permeabilization and staining with antibody and WGA (white in left panels). Images are confocal pictures after deconvolution (63x, zoom 1.8, px size = 60 nm, slice of 0.43 μm) representative of three independent experiments. Insets are threefold enlargements of the boxed regions of second column; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in insets. anti-LAMP1 antibody was used",
    "molecules": "Bafilomycin A1, Bortezomide, saponin"
  },
  {
    "caption": "(D) RD-YFP SH cells were challenged with AD-derived brain extracts, trypsinized and replated 3 days later for an additional 24h before fixation, saponin-permeabilization and staining with antibody recognizing p62 or LAMP1 (in red). Images are confocal pictures (63x, zoom 1.6, px size = 60 nm, slice of 0.43 μm) representative of two independent experiments. Insets are threefold enlargements of the boxed regions; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in the insets.",
    "molecules": "saponin"
  },
  {
    "caption": "(F) Confocal pictures of RD-YFP cells, treated with Bortezomide phalloidin-Texas red was incubated together with the secondary Alexa 647 antibody. Upper panels are the bottom slice corresponding to the substrate-attached surface of cells (z1), below are projections of slices 6 to 9 of the same pictures (z6-z9, not attached to the substrate). Below is a two-fold magnification of the area framed in the merged projection, showing a TNT. White arrows point to overlapping between RD-YFP aggregates and p62, and arrowheads to aggregates not colocalizing with p62 inside the TNT; scale bars are 10 μm, 5 μm in the insets. Bottom is a 3D reconstruction (using Imaris software) of the same region.",
    "molecules": "Alexa 647, Bortezomide, phalloidin, Texas red"
  },
  {
    "caption": "(A) Below the schematic representation of the experiment are representative confocal images of two-day co-cultures of RD-YFP SH cells (acceptor cells) with K18-ATTO 594 fibrils-treated SH-SY5Y cells (donor cells, upper panel). Bottom panels are acceptor cells cultured with the supernatant of K18-ATTO 594 fibrils-treated SH-SY5Y cells (donor SN). WGA (white) labels cell membrane and DAPI the nuclei in the merge panels. Arrows point to cells with green and red aggregates, scale bars are 20 μm. (B) Quantification of seeding after transfer in RD-YFP SH cells in the experiments described in (A), depending on the condition (donor cells or donor SN). Analysis was performed using ICY software, data represent the number of aggregate-containing cells over the total number of green cells ±SD (the total number of RD-YFP cells analyzed over 3 independent experiments were 1011 for co-culture and 1265 for SN) with statistical analysis by two-tailed unpaired t-test (****P =4.18E-07). ",
    "molecules": "ATTO 594, DAPI"
  },
  {
    "caption": "(C) Below the schematic representation of the experiment are representative confocal images showing maximal intensity projections of 6 z-slices (covering 2 μm of thickness) of 24-hour co-culture of RD-YFP SH cells (acceptor cells) with RD-YFP SH cells expressing nls-Red and challenged with K18-ATTO 594 fibrils (donor cells). WGA (white) labels cell membrane and DAPI the nuclei in the merge panel. Stars label donor cells containing green and red aggregates, arrows indicate an acceptor cell with green and red aggregates, and the arrowheads show an acceptor cell with green aggregates but devoid of red fibrils. Below are two-fold enlargement of the respective framed cells. The apparent discontinuity in the picture corresponds to the boundary between two adjacent tiles. Scale bars are 10 μm, 5 μm in the enlargements.",
    "molecules": "ATTO 594, DAPI"
  },
  {
    "caption": "(A) Below the schematics of the experiment are representative confocal images of donor neuron-like cells (CAD cells labelled with Cell Tracker Green (CTG)) and acceptor primary neurons after 24 hours in co-culture. The upper panels show control conditions (non-challenged CAD cells, CTL), and the bottom panels show donor CTG-CADs that were loaded with K18-ATTO 594 fibrils prior to co-culture with primary cortical neurons. The images are representative Z-stack projections covering the whole cell body of donor and acceptor cells. In the merged images, white show neurons labelled with MAP-2, green are CTG-labelled donor CAD cells, red corresponds to K18-ATTO 594 puncta and nuclei are stained in blue. The single channel pictures are gray scale images. Yellow arrowheads point to K18 puncta inside acceptor cells. MAP-2 and K18-ATTO 594 panels are threefold enlargements of the boxed regions in the merged pictures. On the right are the orthogonal views of the same regions covering 14 slices. Scale bars are 20 μm in the merge panels, and 5 μm in the insets and orthogonal views.",
    "molecules": "ATTO 594, Cell Tracker Green, CTG"
  },
  {
    "caption": "(C) Below the schematics of the co-culture experiment are representative confocal images of neuron-like cells (CTG-CADs growing on top of organotypic slices (n=6), in which the astrocytes were labelled with GFAP. The upper panels show control CTG-CADs and the middle panels show donor CTG-CADs loaded with K18-ATTO 594 fibrils, co-cultured with an acceptor hippocampal slice. The images are representative Z-stack projections covering the whole cell body of donor cells. In the merged images, white are acceptor astrocytes (GFAP positive), green are donor CTG-CADs, red are K18-ATTO 594 puncta and nuclei are stained in blue. Insets are threefold enlargements of the boxed region in the merged picture. The orthogonal views of the bottom panels show 25 slices of K18-ATTO 594 fibrils in donor cells (1), or transfer of K18-ATTO 594 fibrils from donor CTG-CADs to hippocampal astrocytes of the organotypic slice (2 to 5). Yellow arrowheads point to Tau puncta inside acceptor astrocytes. Orthogonal views show each of the five K18-ATTO 594 positive-puncta contained inside acceptor cells as indicated in the inset of the middle panel. Scale bars are 100 μm in the merged panel and 10 μm in the insets.",
    "molecules": "ATTO 594, CTG"
  },
  {
    "caption": "(C) Below the schematics of the co-culture experiment are representative confocal images of neuron-like cells (CTG-CADs as in A) growing on top of organotypic slices (n=6), in which the astrocytes were labelled with The upper panels show control CTG-CADs and the middle panels show donor CTG-CADs loaded with K18-ATTO 594 fibrils, co-cultured with an acceptor hippocampal slice. The images are representative Z-stack projections covering the whole cell body of donor cells. In the merged images, white are acceptor astrocytes , green are donor CTG-CADs, red are K18-ATTO 594 puncta and nuclei are stained in blue. Insets are threefold enlargements of the boxed region in the merged picture. of K18-ATTO 594 fibrils in donor cells (1), or transfer of K18-ATTO 594 fibrils from donor CTG-CADs to hippocampal astrocytes of the organotypic slice (2 to 5). Yellow arrowheads point to Tau puncta inside acceptor astrocytes. Orthogonal views show each of the five K18-ATTO 594 positive-puncta contained inside acceptor cells as indicated in the inset of the middle panel. Scale bars are 100 μm in the merged panel and 10 μm in the insets. the neurons in the slices were labelled with the dendrite marker MAP-2 and the axonal marker β-III-tubulin (white), and the orthogonal views show 26 slices.",
    "molecules": "ATTO 594, CTG"
  },
  {
    "caption": "(C) End fusion analysis for domain deletion mutants of Rap1. NotI-digested genomic DNA  from nitrogen-starved cells was analyzed by pulsed-field gel electrophoresis  (PFGE) and Southern blot with probes specific for the terminal C, I, L, and M fragments from  chromosomes  I  and II.",
    "molecules": "DNA, nitrogen"
  },
  {
    "caption": "(D) PFGE  analysis as in (C) for nitrogen-starved cells harboring N-terminal truncation mutants of Rap1. All mutants were V5-tagged at their C-terminus.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A)  Telomere   length analysis of cells from different sequential restreaks following introduction of Poz1-V5-Rap1_491-693 into rap1∆ cells. Telomere  length was assessed by Southern blotting  of EcoRI-digested genomic DNA  probed with a telomere  specific probe. (B) PFGE  analysis of nitrogen-starved Poz1-V5-Rap1_491-693 cells after 5 and 14 restreaks.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(C) Telomere   length analysis of cells with  Rap1∆RCT fused to Taz1  or Poz1  with intervening V5  tag. The fusion protein was integrated at the endogenous rap1  locus in the context of taz1∆ or poz1∆, respectively. Different sequential restreaks following introduction of the fusion construct were analyzed. (D) PFGE  analysis for nitrogen-starved Rap1∆RCT-V5-Taz1  and Rap1∆RCT-V5-Poz1  from 2nd restreak.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(B) PFGE  analysis of cells containing plasmids of  Rap1_440-693 with mutations of the amino acids identified in (A) in the context of rap1∆. Cells were subjected to nitrogen-starvation after recovery from transformation (Restreak 0). (C) PFGE  analysis of nitrogen-starved cells harboring the same mutations after 5 restreaks on plates (approximately 110 generations). (D) Quantification of (B) and (C) using Fiji. The y-axis shows the percentage of intensity represented by the three fusion bands (I+L, I+M and L+M) relative to the total intensity of the fused and unfused signals. WT: wildtype  Rap1_440-693.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(D) PFGE  analysis of the same strains in (C) that were nitrogen-starved. (E) PFGE  analysis of nitrogen-starved internal deletion mutants in poz1∆ background.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A) PFGE  analysis of nitrogen-starved N-terminally V5-tagged full length Rap1  mutants. All mutants were integrated at the endogenous rap1  locus. M5A is the mutation of all 5 non-alanine amino acids under selection in the p-patch to alanine. (B) PFGE  analysis of nitrogen-starved N-terminally V5-tagged rap1_440-693 mutants integrated at the endogenous rap1  locus.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(C) PFGE  analysis of nitrogen-starved rap1  mutants in poz1∆ background.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(D) PFGE  analysis of nitrogen-starved rap1  mutants in  ppm1∆ background. Untagged Rap1   are in lanes 1-2 and N-terminally V5-tagged in lanes 3-7.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(A) ChIP-qPCR  analysis of N-terminally V5-tagged WT, F545L and M6 mutants of Rap1. Untagged Rap1   was used as a control. Bars represent mean of percentage of DNA   precipitated with anti-V5  antibody conjugated beads relative to input DNA. Error bars represent SEM. N= 4 biological replicates. Two-tailed unpaired Student's t-test was performed and significance is indicated. ***: p < 0.001, **: 0.001 ≤ p < 0.01, n.s.: p>0.05. (B) ChIP-qPCR  analysis of N-terminally V5-tagged WT, F545L and M6 mutants of Rap1  in poz1∆ background performed as in (A). N= 4 biological replicates. Statistical analysis is the same as (A).",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Yeast two hybrid assay  probing interaction  between Rap1  and Bqt4. WT and mutants of Rap1  were fused to the DNA-binding domain (BD) in pGBKT7. Bqt4   and Taz1   were fused to the Gal4  activation domain (AD) in pGADT7. Cells (5μl) carrying both plasmids at 5 x106 cells/ml were spotted onto -Leu-Trp dropout plates and -Leu-Trp-Ade-His  dropout plates to select for ADE2  and HIS3  markers. \"-\": Empty BD or AD plasmid.",
    "molecules": "Ade, His"
  },
  {
    "caption": "(D) PFGE  analysis of nitrogen-starved bqt4∆ and bqt3∆ cells in the presence or absence of poz1.",
    "molecules": "nitrogen"
  },
  {
    "caption": "(E) End fusions in bqt4∆poz1∆ and bqt3∆poz1∆ are dependent on the presence of ligase IV. PFGE  analysis of nitrogen-starved cells in the presence or absence of lig4.",
    "molecules": "nitrogen"
  },
  {
    "caption": "A A representative photograph showing the co-cultured DRG and DH neurons. The DRG neurons were expressed with Spy-pHluorin for imaging the synaptic transmission. Scale bar, 10 μm. B Images of a presynaptic bouton marked in (A) showing the Spy-pHluorin fluorescence at 20 s before (-20 s, pre-stimulus), 20 s, 40 s, and 120 s after electrical stimulation (post-stimulus) in 0Ca2+ (upper panel) or 2Ca2+ (lower panel) solution. C Averaged fluorescence changes (ΔF/F0) of Spy-pHluorin in 0Ca2+ (left) or 2Ca2+ solution (right) in response to the same electrical stimulation (20 Hz, 20 s) (n = 45 puncta from 6 cells for 0Ca2+ and 57 puncta from 6 cells for 2Ca2+). The shadow in the traces represents the error bars (s.e.m) of each point.",
    "molecules": "Ca2+"
  },
  {
    "caption": "D-F The same as in (A-C), but the experiments were performed in cultured hippocampal neurons (n = 70 puncta from 3 cells for 0Ca2+ and 72 puncta from 3 cells for 2Ca2+).",
    "molecules": "Ca2+"
  },
  {
    "caption": "C Evoked EPSCs and statistics from DH neurons co-cultured with control (Ctrl) or Syb2-cKO DRG cells (cKO) in 0Ca2+ solution (n = 13 cells for Ctrl and 11 for cKO).",
    "molecules": "Ca2+"
  },
  {
    "caption": "D Evoked EPSCs and statistics from DH neurons co-cultured with DRG neurons before (black) and after (red) applying 1 μM ω-conotoxin-GVIA (GVIA) in 0Ca2+ bath (n = 9 cells).",
    "molecules": "Ca2+, GVIA, ω-conotoxin-GVIA"
  },
  {
    "caption": "F Left panels, evoked EPSCs from DH neurons co-cultured with scrambled shRNA control (Ctrl) or Cav2.2-KD (sh-1/sh-2) DRG neurons in 0Ca2+ solution. Right panel, quantification of evoked EPSCs (n = 17 cells for Ctrl, 12 for sh-1, and 19 for sh-2). EPSCs were evoked by local electrical stimulation (Estim, at arrows).",
    "molecules": "Ca2+"
  },
  {
    "caption": "(J, K) Intracellular levels of Aβ1-40 are significantly increased in AP-2µ KO axons compared to the WT (WTAβ1-40: 1.82±0.12, KOAβ1-40: 2.45±0.12, p=0.000, WT=24 and KO=24 neurons, N=3 biological replicates). Scale bars, 5µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.",
    "molecules": "Aβ1-40"
  },
  {
    "caption": "Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(N) Significantly increased ratio of Αβ1-42/ Αβ1-40 measured by ELISA in the media of cultured AP-2μ KO neurons compared to the WT set to 100% (KO:124.57±10.97%, p=0.037, N=6 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.",
    "molecules": "Αβ1-40, Αβ1-42"
  },
  {
    "caption": "Accumulation of intracellular Aβ1−42 in neurons expressing the AP2-α−WT (WT: 1.14±0.18 , WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts).",
    "molecules": "Aβ1−42"
  },
  {
    "caption": "Accumulation of intracellular Aβ1−42 in neurons overexpressing the AP2-α-Mut comparing to neurons expressing the AP2-α−WT (WT: 1.14±0.18, Mut: 2.06±0.29, p=0.011, WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.",
    "molecules": "Aβ1−42"
  },
  {
    "caption": "Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down (WTScr: 2.76±0.20 WTshBACE1: 2.52±0.12 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down in AP-2µ KO neurons (WTScr: 2.76±0.20, KOScr: 3.94±0.21; WTshBACE1: 2.52±0.12; KOshBACE1: 2.74±0.21; pWTScr vs pKOScr=0.000, pKOscr vs pKOshBACE1=000; pWTscr vs pWTshBACE1=0.826; pWTshBACE1 vs pKOshBACE1=0.863; 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (T) n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.",
    "molecules": "Aβ1-42"
  },
  {
    "caption": "(U, V) Levels of intracellular Aβ1−42 are significantly upregulated in neurons overexpressing the HA-BACE1-LL/AA-GFP comparing to neurons expressing the HA-BACE1-GFP (BACE1: 0.78±0.90, BACE1-LL-AA: 1.99±0.33, p=000, 28-29 neurons per condition, N=3 biological replicates). Scale bar, 50µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001.",
    "molecules": "Aβ1−42"
  },
  {
    "caption": "AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (B) murine hair follicles, P9; DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. Arrowheads mark the CH, cortex of the hair; Scale bars, 20 µm.",
    "molecules": "Hoechst dye 33258, DNA"
  },
  {
    "caption": "AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (C) developing murine epidermis, E16.5; (D) human epidermis, adult; (E) murine thymus, P0, outer region. DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. In panel C, the fluorescent signal in the periderm (P) is nonspecific, as it is present when primary antibodies are omitted from the staining. Inner regions of the murine thymus (not shown) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E. Arrowheads mark the dermal/epidermal border (C, D). CT, cortex of the thymus. Scale bars, 20 µm.",
    "molecules": "Hoechst dye 33258, DNA"
  },
  {
    "caption": "Representative skin phenotypes of Foxn1nu/nu (Nude), or KRT14-cre; Aff4flox/flox (Aff4 cko) mice are shown macroscopically or histologically after hematoxylin and eosin staining (H-N). The phenotypes compared are as follows: hair coats, 4-month-old adults; (E-G) vibrissae, P7; (H) hair follicles with growing hairs, juveniles; (I-K) regressing hair follicles, juveniles; (L-N) epidermis, P9. In panel H, micrographs from left to right are WT, Nude, and Aff4 cko. In panels I-K, HF marks examples of hair follicles. In panels L-M, arrowheads indicate the dermal/epidermal border; arrows mark examples of bent hair shafts. Scale bars: H, L-N, 20 µm; I-K, 40 µm.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(C-E) KRT86 (red) is shown by immunofluorescence in skin sections from wild-type (WT), Foxn1 null, or Aff4 cko mice at P9. DNA is stained blue. HS marks examples of hair shafts. Scale bar, 40 µm.",
    "molecules": "DNA"
  },
  {
    "caption": "D. Quantification of CD206+ area as a percentage of total dermal area in wt (N=7) and Act (N=6) mice in back skin treated 8x with TPA (Antsiferova et al, 2011).",
    "molecules": "TPA"
  },
  {
    "caption": "F, G. Inflammatory thioglycollate-elicited peritoneal macrophages (F) or mouse peripheral blood monocytes (G) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay. Activin A 0.1ng/ml (A0.1), 1ng/ml (A1), or 10ng/ml (A10) was added to the lower well. Alternatively, 10ng/ml was added to the lower well and the insert (A10/A10). Random migration to medium (ctrl) was set to 1 for each mouse; fold over control is shown. Log-transformed data were used for statistical analysis. Results in (F) are representative of 4 independent experiments performed with 8 mice in total (results obtained in one experiment performed with 3 mice are shown). Results in (G) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice.",
    "molecules": "thioglycollate"
  },
  {
    "caption": "E. Differential GSEA was performed on combined genotype comparison [all]/Act vs [all]/wt and two datasets of human AK vs normal skin (GSE2503; Nindl et al, 2006) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets (top); and gene sets derived from top upregulated genes in datasets of differential human macrophage activation (IFN-γ, TNF-α, LPS, IL-4 vs non-treated) and selected human myeloid/lymphoid cell type-specific gene expression profiles (macrophage vs monocyte, dendritic cell, and T-cell) (Xue et al, 2014; bottom). Activin target genes enriched in the AK transcriptomes are shown (right). Data are presented as pseudo-heatmaps with magnitudes of Normalized Enrichment Scores (NES) and Log2(Fold Change) color-coded as indicated in the respective legends; all non-grey comparisons are FDR<0.05 for NES and p<0.10 for Fold Change.",
    "molecules": "LPS"
  },
  {
    "caption": "G. In situ gelatin zymography (top panel) and methylene blue staining (bottom panel) of the ear skin from 10 week-old mice. Representative images of 2-3 mice per genotype are shown. Scale bar = 500μm.",
    "molecules": "gelatin"
  },
  {
    "caption": "H,I. Gelatin zymography of ear skin lysates from 10 week-old mice. Inverted image of a representative gel with 2 (wt/wt) or 3 (HPV8/wt, wt/Act, HPV8/Act) mice per genotype is shown in (H), quantification of the results obtained in 2 independent experiments is shown in (I). N=5 wt/wt, N=6 HPV8/wt, wt/Act and HPV8/Act mice.",
    "molecules": "Gelatin"
  },
  {
    "caption": "heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. A Injection of 100 nM FGF2.",
    "molecules": "biotinylated, heparin"
  },
  {
    "caption": "heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. B 64 nM S1 RBD protein. C 64 nM S1 RBD protein over a streptavidin surface (no heparin functionalization).",
    "molecules": "biotinylated, heparin"
  },
  {
    "caption": "The structural change of the SARS-CoV-2 S1 RBD observed in the presence of heparin by circular dichroism (CD) spectroscopy.(A) CD spectra of 200 μg/mL spike 1 alone (purple) or with 30 μg heparin (orange) in phosphate buffered saline pH 7.4. Theoretical sum of spike 1 alone and 30 μg of heparin (control) if no interaction was observed (black). (B) Δ secondary structure (%) of (A). A 2.4% increase in helix and 2.2% decrease in antiparallel secondary structural features were calculated (BestSel) for the observed spectrum compared to that of the theoretical, summative spectrum of the SARS-CoV-2 S1 RBD in the presence of heparin.",
    "molecules": "heparin"
  },
  {
    "caption": "(A) Representative micrographs  of human skin  sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients  (n = 11) and tumors of cSCC patients  (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean  IL-38 expression   per high-powered field in tissues. (B) Relative expression   of  IL-38 in human  normal tissues (n = 9) and cSCC  (n = 18) were analyzed using Geo Datasets (GSE98767). Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(C and D) The  dorsal hair of normal C57/BL6 mice  were shaved and treated with DMBA/TPA  twice a week for 32 weeks to induce skin  tumors. (C) Representative micrographs   of mouse normal skin  (n = 6) and tumor (n = 6) sections stained  with anti-IL-38  antibody. The graph shows the quantification of mean  IL-38 expression   per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression   levels of Il-38 in normal skin  (n = 5) and tumors (n = 5) of mice  were quantified using qPCR. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.",
    "molecules": "DMBA, TPA"
  },
  {
    "caption": "(F) Representative western blot bands indicating  IL-38 in mouse normal skin  (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.",
    "molecules": "DMBA, TPA"
  },
  {
    "caption": "(B) Representative photographs  of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice  (n = 8) treated with DMBA/TPA for 32 weeks. White triangles indicate the tumors  on the back of mice. (C Average tumor number (C) per mouse  from Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice  (n = 8) treated with DMBA/TPA.. Error bars represent the mean ± SD (C) All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test (C",
    "molecules": "DMBA, TPA"
  },
  {
    "caption": "(A) Representative immunohistochemical staining micrographs  of γH2AX  in the skin  of Il-38f/f (n = 5) and   K14Cre/+-Il-38f/f (n = 5) mice  after treated with DMBA  for 24h. Red triangles indicate the γH2AX+ positive cells. Scale bars represent 100 μm. The graph shows the number of  γH2AX+ cells per high-powered field. Data information: Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.",
    "molecules": "DMBA"
  },
  {
    "caption": "(A The  dorsal hair  of normal C57/BL6 mice  were shaved and treated with DMBA/TPA  twice a week for 3 weeks to induce the skin inflammation. (A) Representative micrograph  sections stained  with hematoxylin-eosin  (H&E) from the skin of Il-38f/f (n = 5) and  K14Cre/+-Il-38f/f mice (n = 5) Scale bars represent 100 μm. The graph shows average epidermal  thickness.",
    "molecules": "DMBA, eosin, hematoxylin, TPA"
  },
  {
    "caption": "B) The dorsal hair  of normal C57/BL6 mice  were shaved and treated with DMBA/TPA  twice a week for 3 weeks to induce the skin inflammation. (B) Representative micrograph  sections stained  with anti-Ki67  antibody from the skin  of Il-38f/f (n = 6) and K14Cre/+-Il-38f/f mice  (n = 6). Scale bars represent 100 μm. The graph shows average numbers of Ki67+ cells per high-powered field.",
    "molecules": "DMBA, TPA"
  },
  {
    "caption": "Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (A) DSBs quantified by qPCR analysis of Igk (Jκ1) genomic DNA. Schematic shows germline (GL) Igk locus, unrepaired Jκ1 coding end (Cut) and primer location. Results are normalized to Rag1-/-:Bcl2 abl pre-B cells, which do not generate RAG DSBs and have only germline Igk DNA. Loss of Igk germline product is representative of DSB generation.",
    "molecules": "imatinib"
  },
  {
    "caption": "Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (B) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.",
    "molecules": "imatinib"
  },
  {
    "caption": "Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (C) Western blot analysis of KAP1 phosphorylation (p-KAP1), NF-κB2 (p100 and p52) and PIM2. GAPDH is shown as loading control.",
    "molecules": "imatinib"
  },
  {
    "caption": "Rag1-/-:Bcl2 and Art-/-:Bcl2 abl pre-B cells were treated with imatinib for 48 h to induce G1 arrest. (D) Cd40 and Pim2 mRNA expression.",
    "molecules": "imatinib"
  },
  {
    "caption": "Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (E) DSBs quantified by qPCR analysis of Igh (JH1) genomic DNA and analyzed as in A with results normalized to empty vector control, which has only germline Igh DNA.",
    "molecules": "imatinib"
  },
  {
    "caption": "Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (F) Representative images of 53BP1 foci. Scale bar denotes 8 μm. Bar graph shows percentage of cells with indicated number of foci from three biological replicates. In each replicate, 100 cells per condition were quantified.",
    "molecules": "imatinib"
  },
  {
    "caption": "E-H. Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (G) Western blot analysis of FLAG-RAG1, p-KAP1, NF-κB2, and PIM2. GAPDH is shown as loading control.",
    "molecules": "imatinib"
  },
  {
    "caption": "Rag1-/-:Lig4-/-:Bcl2 abl pre-B cells were transduced with an empty vector (control) or vector expressing RAG1 then treated with imatinib for 48 h. (H) Cd40 and Pim2 mRNA expression.",
    "molecules": "imatinib"
  },
  {
    "caption": "A. Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with (+) or without (-) 2 μM doxycycline (Dox) to induce Cas9 as indicated. Western blot shows FLAG-Cas9 and GAPDH (loading control).",
    "molecules": "Dox, doxycycline"
  },
  {
    "caption": "Rag1-/-:Lig4-/-:Bcl2:iCas9 abl pre-B cells were treated with doxycycline as in A and imatinib to trigger cell cycle arrest for 24 h. Cells were then transfected with indicated gRNA and maintained in doxycycline and imatinib. All analyses were completed 48 h after gRNA transfection. DSBs quantified by qPCR analysis of Eb (B) and Gapdh (C) genomic DNA. Results are normalized to cells without gRNA (-). Schematic shows germline (GL) locus, unrepaired cut end (Cut) and location of gRNA and primers. Loss of germline product is representative of DSB generation.",
    "molecules": "doxycycline, imatinib"
  },
  {
    "caption": "B Plot of the whole-cell current (Vhold -65 mV) in control conditions (ctrl) and after application of NASPM (NSP, p<0.0001, n=18).",
    "molecules": "NASPM, NSP"
  },
  {
    "caption": "B Current-voltage relationship of the tonic Ba2+-sensitive (100 µM) outward GIRK current demonstrating reversal near expected EK and inward rectification (n=6 biological replicates).",
    "molecules": "Ba2+"
  },
  {
    "caption": "A With GDPβS-containing internal solution, noradrenaline-induced GluD1R current (INA) was diminished by >20 minutes post-dialysis, shown in a representative trace.",
    "molecules": "GDPβS, noradrenaline"
  },
  {
    "caption": "C Plot of the magnitude of tonic GluD1R current measured after dialysis with GTP + LiCl-, GDPβS-Li3-, and GTP-containing internal solution, displaying no significant difference between the groups (p=0.46, n=9, 13, 8 biological replicates respectively).",
    "molecules": "GDPβS, GTP, Li3, LiCl"
  },
  {
    "caption": "B-C Plot of the amplitude of the α1-AR-EPSC in control conditions (ctrl) and in glycine (1 mM, p=0.43, n=10 biological replicates, B), or D-serine (1 mM, p=0.13, n=9 biological replicates, C).",
    "molecules": "D-serine, glycine"
  },
  {
    "caption": "E Glycine had no significant effect on the amplitude of the α1-AR-EPSC (arrow) or whole-cell current, shown in a representative trace.",
    "molecules": "Glycine"
  },
  {
    "caption": "A Increasing extracellular calcium to 4.8 mM reduced the amplitude of the α1-AR-EPSC, shown in a time-course plot (n=18 and 26 biological replicates for 2.4 and 4.8 mM respectively).",
    "molecules": "calcium"
  },
  {
    "caption": "F Plot of the magnitude of tonic GluD1R current measured by application of NASPM in control conditions (1.2 mM), 2.4 mM and 4.8 mM calcium. The magnitude of tonic GluD1R current in 2.4 and 4.8 mM calcium was reduced relative to control conditions (1.2 mM: n=18; 2.4 mM: p=0.03, n=17; 4.8 mM: p=0.005, n=22 biological replicates).",
    "molecules": "NASPM, calcium"
  },
  {
    "caption": "J Elevating extracellular calcium to 4.8 mM reduces the change in whole-cell current to NASPM and accelerates recovery of the α1-AR-EPSC from NASPM-block in control conditions (1.2 mM extracellular calcium), shown in a representative whole-cell voltage-clamp recording.",
    "molecules": "NASPM, calcium"
  },
  {
    "caption": "A Representative traces of whole-cell current clamp recordings of membrane potential and AP firing evoked by current injection (1.5 s) demonstrating hyperpolarization by NASPM. Dashed line is at -80 mV.",
    "molecules": "NASPM"
  },
  {
    "caption": "C Plot of the membrane potential (Vm) versus injected current relative to rheobase (rb) in control and NASPM, demonstrating that NASPM produced a hyperpolarization at subthreshold potentials (n=10 biological replicates).",
    "molecules": "NASPM"
  },
  {
    "caption": "A-C Volcano plots depicting significantly up (blue) and down (pink) regulated phosphorylation events for DMXAA vs DMSO groups for 5 min (A), 10 min (B) and 30 min (C) timepoints. -Log10(p-value) is plotted on the y-axis vs Log2(Fold change [FC]) on the x-axis. Significantly regulated events display p-value>0.01 and Log2(FC) >1 or<-1.",
    "molecules": "DMSO, DMXAA"
  },
  {
    "caption": "A Sting-/- iBMDMs expressing mRuby3-STING (magenta) and eGFP-Golgin84 (green) were imaged live on the 3i marianas spinning disk microscope. Z stack images were acquired either before (i.e., untreated) or after 50 μg/mL DMXAA treatment (i.e., image captured at 12 min post DMXAA treatment). Data is shown as a maximum intensity projection (MIP) of Z stack images.",
    "molecules": "DMXAA"
  },
  {
    "caption": "D Sting-/- iBMDMs expressing mRuby3-STING were imaged live on the spinning disk microscope. Representative images of timelapse showing STING vesicles exiting the Golgi for a recording starting 27 min after 50 μg/mL DMXAA treatment. Data is shown as a MIP of Z stack images. Square region of interest (ROI) indicates zoomed insert. Scale bar = 5 μm.",
    "molecules": "DMXAA"
  },
  {
    "caption": "F Sting-/- iBMDMs expressing eGFP-STING were imaged live on the spinning disk microscope. A representative image of timelapse showing transport of STING vesicles for a recording starting 1 h 18 min after 50 μg/mL DMXAA treatment. Data is shown as a MIP of Z stack images. Square ROI indicates zoomed insert. Scale bar = 5 μm.",
    "molecules": "DMXAA"
  },
  {
    "caption": "A Primary BMDMs were left untreated or treated with 25 μg/mL DMXAA for 2, 4 or 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments. B Primary BMDMs were left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 2, 4 or 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.",
    "molecules": "2'3'-cGAM(PS)2, DMXAA"
  },
  {
    "caption": "D Sting-/-, WT or TBK1/IKKε dKO iBMDMs were left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 1, 2, or 3 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown representative of 3 independent experiments.",
    "molecules": "2'3'-cGAM(PS)2"
  },
  {
    "caption": "B Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 25 μg/mL DMXAA for 1, 2 or 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments. C Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.",
    "molecules": "2'3'-cGAM(PS)2, DMSO, TAK243, DMXAA"
  },
  {
    "caption": "D Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/mL DMXAA for 1, 2 or 4 h. Cell were fixed and stained for p-STING (white) and the nucleus (DAPI; blue), before Z stack images were acquired on the LSM980 confocal microscope. Images are displayed as a maximum intensity projection (MIP) of Z stack images. Scale bar = 5 μm. Data shown is representative of 3 independent experiments.",
    "molecules": "DAPI, DMSO, TAK243, DMXAA"
  },
  {
    "caption": "G-I Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/mL DMXAA or 10 μg/mL 2'3'-cGAM(PS)2 for 4 h. Cells were lysed for RNA purification and the expression of Ifnb1 (G) , Isg15 (H) and Il6 (I) was analysed by qPCR. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Bonferroni's multiple comparisons test, where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.",
    "molecules": "2'3'-cGAM(PS)2, DMSO, TAK243, DMXAA"
  },
  {
    "caption": "B Sting-/- iBMDMs expressing HA-STING were left UT or treated with 50 μg/mL DMXAA for 1 or 3 h. Cells were lysed and a portion of lysate underwent immunoblot with the indicated antibodies (i.e., lysate). The remaining lysate underwent immunoprecipitation with an anti-HA antibody (i.e., HA pull down). Samples then underwent immunoblot with the indicated antibodies. Data representative of 3 independent experiments.",
    "molecules": "DMXAA"
  },
  {
    "caption": "A dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/mL doxycycline (Dox) to induce sgRNA expression. Cells were then left untreated (UT), or treated with 20 μg/mL DMXAA or 10 μg/mL 2'3'-cGAM(PS)2 for 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.",
    "molecules": "2'3'-cGAM(PS)2, Dox, doxycycline, DMXAA"
  },
  {
    "caption": "B-C dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/mL Dox to induce sgRNA expression. Cells were then left UT, or treated with 20 μg/mL DMXAA or 5 μg/mL 2'3'-cGAM(PS)2 for 6 h. Cell supernatant was collected and assayed for secreted IFNβ (B) and IL6 (C) by ELISA. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Bonferroni's test, where * p<0.05, *** p<0.001, **** p<0.0001. n.d. = not detected",
    "molecules": "2'3'-cGAM(PS)2, Dox, DMXAA"
  },
  {
    "caption": "D-F dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 24 h with 1 μg/mL Dox to induce sgRNA expression. iBMDMs were further treated with 1 μg/mL (i.e. 1000 ng/mL) or 100 ng/mL H-151 as indicated, and incubated for a further 24 h. Cells were lysed for RNA purification and the expression of Ifnb1 (D), Isg15 (E) and Irf7 (F) was analysed by qPCR. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Tukey's multiple comparisons test, where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Bars display significant differences between dCas9 and sgRNA knockdowns in the absence of H-151. Additional asterisks represent significant differences between the absence or presence of H-151 across the same cell line.",
    "molecules": "H-151, Dox"
  },
  {
    "caption": "A-B VPS4aWT or VPS4aE228Q dominant negative BMDMs were left untreated (UT) or treated with 1 μg/mL doxycycline (Dox) for 4 h. BMDMs were then further left UT or treated with 25 μg/mL DMXAA (A) or 10 μg/mL 2'3'-cGAM(PS)2 (B) for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 2 independent experiments for A and 3 independent experiments for B.",
    "molecules": "2'3'-cGAM(PS)2, Dox, doxycycline, DMXAA"
  },
  {
    "caption": "C-D VPS4aWT or VPS4aE228Q dominant negative BMDMs were left UT or treated with 1 μg/mL Dox for 4 h. BMDMs were then further left UT or treated with 25 μg/mL DMXAA for 4 h. Cell supernatant was collected and secreted IFNβ (C) or IL-6 (D) was measured by ELISA. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using one-way ANOVA. using Bonferroni's test, where * p<0.01. n.d. = not detected",
    "molecules": "Dox, DMXAA"
  },
  {
    "caption": "RAP1 expression in HeLa cells treated with the telomerase inhibitor BIBR1532 for 25 days (20 µM final concentration). Fifteen days before cell harvesting, doxycycline (DOX; 1 µg/µl final concentration) was added to deplete the expression of RAP1.",
    "molecules": "BIBR1532, DOX, doxycycline"
  },
  {
    "caption": "Number of fusions in HeLa cells after 25 days in culture. Cells were maintained with BIBR1532 during the whole period of the experiment, while doxycycline (1 µg/µl final concentration) and the infection with shControl, shLIG3 or shLIG4 was carried out for the last 15 days of the experiment. Data represent mean +/- SD of three biological replicates are shown. Statistical analyses were performed using Mann-Whitney U test (*p < 0.05; **p < 0.001; ***p < 0.0001).",
    "molecules": "BIBR1532, doxycycline"
  },
  {
    "caption": "Examples of metaphase spreads hybridized with a telomeric PNA probe (green) when RAP1 was depleted (+DOX). Telomerase was inhibited with BIBR1532 for 25 days. Scale bar = 10 µm. Quantification of chromosome aberrations observed in RAP1 depleted cells (+DOX) or control (-DOX). Data represent mean +/- SD of three biological replicates. Approximately 15 metaphase spreads were analyzed per replicate with a total of 2300 chromosomes examined per condition. *p < 0.05; two-tailed Student's t test.",
    "molecules": "BIBR1532, DOX"
  },
  {
    "caption": "Dividing cells (magenta) were identified by EdU staining (1 µM EdU for 24 h). The percentage of dividing cells after 15 dpi is shown (n = 3). Scale bar = 10 µm.",
    "molecules": "EdU"
  },
  {
    "caption": "Senescent cells, transduced with shp21CIP1 or shp21CIP1+shRAP1 for 15 days, were stained with CytoCalcein violet (live cells), Apopxin Green (apoptotic cells) and 7-aminoactinomycin D (7-AAD) (late apoptostic/necrotic cells) and visualized by flow cytometry. Quantification of apoptotic cells from the conditions described in E. Data represent mean +/- SD of three biological replicates (**P < 0.001; two-tailed Student's t test).",
    "molecules": "Apopxin Green, CytoCalcein violet, 7-AAD, 7-aminoactinomycin D"
  },
  {
    "caption": "Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm.",
    "molecules": "SiR, Hoechst"
  },
  {
    "caption": "SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm.",
    "molecules": "SiR, Hoechst"
  },
  {
    "caption": "Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm.",
    "molecules": "SiR, Hoechst"
  },
  {
    "caption": "Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.",
    "molecules": "SiR, Hoechst, DNA"
  },
  {
    "caption": "3D reconstructions from spinning disc confocal microscopy z-stacks across an early oocyst labelled with the DNA dye Hoechst (left). 50 and 19 images with a z-distance of 0.5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Right: 3D reconstructions from late WT and α1-tubulin(-) oocysts labelled with Hoechst (blue) and SiR-tubulin (green). Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites. 10 and 28 images with a z-distance of 1,5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Sb: sporoblast; scale bars: 5 µm.",
    "molecules": "SiR, Hoechst, DNA"
  },
  {
    "caption": "Fluorescence images showing the SiR-tubulin stained microtubules, nuclei (Hoechst) and cytoplasmic shape (mCherry) of the parasite lines. Note the small amount of DNA (black arrowhead) and the reduced level of SiR-tubulin fluorescence in the α1cm&∆introns-expressing sporozoites (lower graph). See also Fig S6. Quantitative data was derived from 96-120 sporozoites per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. Scale bar: 5 μm. **** indicates p<0.0001, Kruskal-Wallis-test. Note that α1cm&∆introns-expressing sporozoites do not express mCherry; their autofluorescence (auto-fluo) indicates an aberrant shape.",
    "molecules": "SiR, Hoechst, DNA"
  },
  {
    "caption": "SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.",
    "molecules": "SiR"
  },
  {
    "caption": "Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.",
    "molecules": "SiR"
  },
  {
    "caption": "MDM were incubated with 1,25D3 for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h, washed then incubated with or without 1,25D3 for 7 days. (A) Top, extracellular release of HIV p24 antigen into the cell supernatant at days 0, 4 and 7 was detected by ELISA. Bottom, MDM were harvested and stained for HIV p17. Histograms are shown for a representative donor.",
    "molecules": "1,25D3"
  },
  {
    "caption": "HIV and/or M. tuberculosis (TB) infected MDM were treated for 7 days with 100 pmol/L 1,25D3. (A) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β-actin performed.",
    "molecules": "1,25D3"
  },
  {
    "caption": "B) Cells were incubated with 10 µg/mL pepstatin A for 4 h on day 7 prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.",
    "molecules": "pepstatin A"
  },
  {
    "caption": "C) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.",
    "molecules": "1,25D3, saponin"
  },
  {
    "caption": "(D) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d followed by 10 µg/mL pepstatin A for a further 4 h. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.",
    "molecules": "1,25D3, pepstatin A, saponin"
  },
  {
    "caption": "(E) Immunoblots of p62 using antibody to p62 or β-actin 7 d after macrophages treated with 1,25D3.",
    "molecules": "1,25D3"
  },
  {
    "caption": "1,25D3 induces autophagy in human macrophages co-infected with HIV and M. tuberculosis.HIV (A), M. tuberculosis (TB) (B), and HIV/TB dual infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Representative density plots from three donors are shown. (D) HIV/M. tuberculosis infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Left, representative density plots from three donors from HIV/M. tuberculosis infected MDM are shown for HIV/M. tuberculosis co-infection. Right, histograms of saponin-resistant LC3B-II in macrophages that were HIV+M. tuberculosis− or HIV+day 7+ at day 7 post-infection.",
    "molecules": "1,25D3, saponin"
  },
  {
    "caption": "MDM were transduced with non-specific scrambled shRNA (shNS) or Beclin-1 shRNA (shBCLN1) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to Beclin-1 or β-actin after initial pathogen exposure (Day 0) or after 7 days.",
    "molecules": "1,25D3, puromycin"
  },
  {
    "caption": "MDM were transduced with non-specific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to ATG5 or β-actin after initial pathogen exposure (Day 0) or after 7 days",
    "molecules": "1,25D3, puromycin"
  },
  {
    "caption": "MDM were pretreated with bafilomycin A1 (Baf A1) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7.",
    "molecules": "bafilomycin A1, 1,25D3"
  },
  {
    "caption": "SID 26681509 inhibits the 1,25D3 mediated inhibition of HIV and M. tuberculosis replication.MDM were pretreated with SID 26681509 (SID) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7.",
    "molecules": "1,25D3, SID 26681509"
  },
  {
    "caption": "Inhibition of HIV and M. tuberculosis by 1,25D3 is CAMP and autophagy dependent.MDM were transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to CAMP or β-actin after initial pathogen exposure (Day 0) or after 7 days.",
    "molecules": "1,25D3, puromycin"
  },
  {
    "caption": "(B) Day 7 HIV-1Ba-L and TB co-infected cells were harvested and stained for saponin-resistant LC3B-II at day 7. A representative histogram from three donors are shown.",
    "molecules": "saponin"
  },
  {
    "caption": "Macroscopic appearance of livers in 12-month-old DEN-treated lncHand2+/+ and lncHand2 knockout (lncHand2-/-) mice (left panel). Black arrows indicate liver tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers (right panel). Results are shown as means ± SD (n=12).",
    "molecules": "DEN"
  },
  {
    "caption": "Representative H&E and immunohistological staining with Ki67 antibody of liver sections of 12-month-old DEN-treated lncHand2+/+ and lncHand2-/- mice. Scale bar, 100 μm.",
    "molecules": "DEN"
  },
  {
    "caption": "Quantitation of Ki67-positive cells in non-treated and DEN-treated mice. Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice. Data are shown as means ± SD.",
    "molecules": "DEN"
  },
  {
    "caption": "Representative immunofluorescence staining of livers in 5.5-month-old DEN-treated and untreated lncHand2RFP reporter mice for indicated molecules (lower panel). Scale bar, 100 μm. Upper panel: scheme of targeting strategy for IRES-RFP knockin allele.",
    "molecules": "DEN"
  },
  {
    "caption": "Biotin-RNA pulldowns were performed with nuclear extracts of oncosphere cells using full-length HAND2-AS1 transcript (sense), antisense, and one HAND2-AS1 intron control, followed by mass spectrometry. Band 1: INO80, band 2: RUVBL2.",
    "molecules": "Biotin"
  },
  {
    "caption": "Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates.",
    "molecules": "biotin"
  },
  {
    "caption": "EMSA of biotin-labeled HAND2-AS1 (nt 83-242) probes incubated with INO80 protein.",
    "molecules": "biotin"
  },
  {
    "caption": "Macroscopic appearance of livers in 10.5-month-old DEN-treated Ino80flox/flox (Ino80+/+) and Ino80 knockout (Ino80-/-) (left panel). Black arrows indicate tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers. Results are shown as means ± SD (n=10).",
    "molecules": "DEN"
  },
  {
    "caption": "Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors.",
    "molecules": "DEN"
  },
  {
    "caption": "Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5.",
    "molecules": "DEN"
  },
  {
    "caption": "BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).",
    "molecules": "noggin"
  },
  {
    "caption": "A-C Confocal images of Stage 12 embryos  from control, P{GT1}CG9005BG02278 P-element mutant (CG9005PBG), and CG9005PBG with CG9005 expression restored in macrophages. Macrophage: red. Phalloidin to visualize embryo: green. Germband edge: dotted white line. Data information: Scale bars: 50µm (A-C)",
    "molecules": "Phalloidin"
  },
  {
    "caption": "C-D Confocal images or (D) quantification of the macrophages in germband in Stage 12 embryos from the control,  atosPBG, and  atosPBG expressing Atos itself or variants lacking particular domains. Transgene expression directly from macrophage specific promoter (mac-). For control (n=32 embryos) vs atosPBG mutant (n=56) p<0.0001; vs rescue with mac-atos (n=18) p>0.99; vs rescue with mac-atosDUF- (n=17) p=0.0003; vs rescue with mac-atosChrSeg-(n=21) p=0.0003; vs rescue with mac-atosDUF-/ChrSeg-(n=19) p=0,00014; vs rescue with mac-atosTAD1-/ TAD2- (n=25) p=0.0009, atosPBG mutant vs. rescue with mac-atos p=0.0031. Data information: Germband edge: dotted white line. Mac- indicates direct expression from the  srpHemo promoter. (C, macrophage nuclei (red), actin by Phalloidin staining (green). (D One-way ANOVA with Tukey. Mean±SEM, ns=p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars: 50µm (C",
    "molecules": "Phalloidin"
  },
  {
    "caption": "E Confocal images of atosPBG rescued by expressing Atossa's murine orthologs, mFAM214A or B (mFAMA-B) in macrophages, F Quantification of macrophages in the germband in Stage 12 embryos from the control, atosPBG, and atosPBG embryos expressing mFAM214A or B specifically in macrophages (mac-). For control (n=24 embryos) vs. atosPBG mutant (n=56) p<0.0001; vs mac-atos rescue (n=18) p=0.7; vs mac-mFAMA rescue (n=22) p=0.6; vs mac-mFAMB rescue (n=25) p=0.086. For atosPBG mutant vs mac-atos rescue p=0.0006; vs mac-mFAMA  rescue p=0.0002; vs mac-mFAMB rescue p=0.0043. Data information: Germband edge: dotted white line. Mac- indicates direct expression from the  srpHemo  promoter. E) macrophage nuclei (red), actin by Phalloidin staining (green). F) One-way ANOVA with Tukey. Mean±SEM, ns=p>0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars: 50µm E).",
    "molecules": "Phalloidin"
  },
  {
    "caption": "I Confocal images of early Stage 12 embryos  from control, atosPBG, and atosPBG expressing atos::FLAG::HA (mac>atos) or  pths::FLAG::HA (mac>pths) in macrophages (red). Embryo detected by phalloidin staining (green). White dotted line: germband  edge.. J Quantification of macrophages  in the  germband  of an atosPBG mutant rescued by expressing pths::FLAG::HA in macrophages. For control (n=15 embryos) vs atosPBG mutant (n=22) and  mac>atos  rescue (n=27) both p<0.0001, vs mac>pths rescue (n=30) p=0.0007. Data information: Movies in each analysis set are from independent embryos. Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. one-way ANOVA with Tukey Scale bars: 50µm",
    "molecules": "phalloidin"
  },
  {
    "caption": "B The Oxygen Consumption Rate (OCR, pmols  O2/min) assessed as a representative parameter of OxPhos in control and pths-KD S2R+ cells by a Seahorse efflux assay. n≥3 independent biological experiments each with n>6 technical replicate. Data information: Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests",
    "molecules": "O2"
  },
  {
    "caption": "I Single plane  confocal microscope  image during germband entry from control or atosPBG embryos, or those expressing  pths-RNAi or CV-DN in macrophages. Antibodies against S293-phosphorylated inactivated  Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in  macrophages (red). Higher pPDH levels are usually found when  ATP/ADP  levels are high and input into the TCA cycle is being downregulated Data information: show Stage 12 embryos. Scale bars: 10µm",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "J Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (I) during initial germband  invasion. The pPDH/PDH ratio is significantly reduced, arguing that decreased function of CV, Atos  or Pths in macrophages results in lower cellular ATP/ADP ratios compared to the control. N=3 independent experiments. Ctrl 1 (n=10 embryos) vs CV-DN  (n=9) and vs.  atos  mutant (n=13) both p=0.0002; Ctrl 2 (n=7 embryos) vs pths RNAi (n=8) p=0.0001.*** shown above columns in J are for comparison to relevant control. Data information: Mean±SEM, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-tests",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "A Single plane confocal microscope  image during germband entry from control or atosPBG embryos, or those expressing pths, GR/HPR, and LKR/SDH in macrophages (mac>). Embryos were stained  for antibodies against S293-phosphorylated inactivated Pyruvate Dehydrogenase (pPDH, green) or total PDH (magenta) in macrophages (red). Higher pPDH/PDH ratios are consistent with higher  ATP/ADP  levels. Data information: Scale bars: 10µm",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "B Quantification of normalized pPDH/PDH levels calculated from fluorescence intensities in macrophages from the genotypes in (A) during initial germband invasion. The pPDH/PDH ratio is significantly increased in  atosPBG embryos expressing either  pths, GR/HPR, or LKR/SDH in macrophages compared to the atosPBG embryos. This argues that the decreased Atos  function in atosPBG macrophages, resulting in lower cellular ATP/ADP ratios, was restored by expressing either its targets or murine orthologs. N=3 independent experiments. Control (n=9 embryos) vs.  atos mutant (n=10) p<0.0001. Control vs. atos mutant rescued with mac>pths (n=10) p=0.77; rescued with  mac>GRHPR (n=9) p=0.48; rescued with mac>LKRSDH (n=10) p=0.012. For  atos mutant vs. atos rescued with  mac>pths or  mac>GRHPR p<0.0001. vs. atos rescued with  mac>LKRSDH p=0.0014. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "Cellular metabolites were measured by  LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. (B) Normalized ATP/ADP ratio values are decreased in atosPBG compared to control embryos  (p=0.028). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test",
    "molecules": "ADP, ATP"
  },
  {
    "caption": "Cellular metabolites were measured by LC-MS-based untargeted metabolomics from extracts of Stage 11 embryos; biological replicates for control n=5, for atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos shown with average log2fold change (FC) in the non-targeted  LC-MS/MS analysis reveals (C) an increase in atosPBG in substrates of the dGR/HPR  enzyme, including 4-hydroxy α-ketoglutarate and hydroxyproline (HLP) and a smaller decrease in its products, glycolate and glycerate, (D) a significant increase in some dipeptides including those containing  hydroxyproline. Data information: Values in heat maps are obtained from untargeted  metabolomic analysis, unpaired t-test",
    "molecules": "4-hydroxy α-ketoglutarate, HLP, hydroxyproline, dipeptides, glycerate, glycolate"
  },
  {
    "caption": "Cellular metabolites were measured by LC-MS-based untargeted metabolomics  from extracts of Stage 11 embryos; biological replicates for control n=5, for  atosPBG n=7. (E) Quantification shows an increase in atosPBG in the pyruvate/glucose ratio (p=0.035), but none for the  lactate/glucose ratio (p=0.65). Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ****p<0.0001. unpaired t-test",
    "molecules": "glucose, lactate, pyruvate"
  },
  {
    "caption": "Cellular metabolites were measured by LC-MS-based untargeted metabolomics  from extracts of Stage 11 embryos; biological replicates for control n=5, for  atosPBG n=7. Heatmap of non-targeted metabolites in atosPBG compared to wild-type embryos  shown with average log2fold change (FC) in the non-targeted  LC-MS/MS analysis (F) We observe increases in atosPBG in intermediates of mitochondrial fatty acid β-oxidation (FAO), including different carnitine-conjugated lipids, and (F-G) a significant increase in ketone body substituents compared to the control. Data information: Values in heat maps are obtained from untargeted metabolomic analysis, unpaired t-test",
    "molecules": "fatty acid β, carnitine, ketone body, lipids"
  },
  {
    "caption": "(D-F) Confluent layers of WM983 cells treated as indicated were 'scratched' followed by a 20 hour incubation at 37°C with 5% CO2. (D) Representative images of cells after being scratched after being incubated for 0, 10 or 20 hours in the absence (-) or presence (+) of 2uM IA65. (E-F) The cell-free area was quantified by ImageJ (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed by 2-way ANOVA. In the absence of IA65, SOCE suppression effect was p<0.05; effect of time was p < 0.0001; interaction was p<0.05. In the presence of IA65, SOCE suppression effect was p>0.05; effect of time was p < 0.0001; interaction was p>0.05. Within all panels, differences established through multiple comparisons are labeled as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.",
    "molecules": "IA65, CO2"
  },
  {
    "caption": "WM983 and UACC257 human melanoma cells were transfected with Perfringolysin O (PFO)-mCherry D4H cholesterol sensor and plated on glass coverslips. Cells were exposed to UV (0 or 175 J/m2) and incubated for 0, 2 or 5 weeks as indicated. (H) Representative cell images. (I) Fluorescence intensity from cells analyzed in (H) was measured and quantified using LASX analysis software (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed using 1-way ANOVA with multiple comparisons (WM983, p < 0.001; UACC257 p > 0.05). **p<0.01, ***p<0.001, ****p<0.0001.",
    "molecules": "cholesterol"
  },
  {
    "caption": "O1C78 WM983 cells expressing the PFO-mCherry D4H cholesterol sensor were examined as in panels H and I (N≥8 biological replicates); data displayed are mean±SEM.",
    "molecules": "cholesterol"
  },
  {
    "caption": "Patient surgical explants were received from the biosample repository of FCCC. Cells were isolated from tumors using 50 μM Medicons and the Medimachine system and grown for 3 days before experimentation to eliminate non-melanoma cells. (A-B) Plated control or UV irradiated (175J/m2) cells were then incubated overnight on glass coverslips prior to Fura2 loading and administration of Tg in Ca2+-free media for 15 minutes. (A) Representative examples of SOCE responses in patient samples following the addition of Ca2+ (1 mM). Each trace represents 5 to 10 individual cells; solid line represents the mean and the dotted line represents SEM. (B) Each dot represents SOCE in cells obtained from a different patient sample (n=20; data displayed as mean±SEM). The effect of UV on SOCE was determined by one way ANOVA (p > 0.05). (C) Comparison of SOCE in cells collected from both primary tumors and metastatic lymph nodes from the same patient (from patients analyzed in panels A, B).",
    "molecules": "Ca2+, Fura2, Tg"
  },
  {
    "caption": "(E) Single cells from patient samples were treated with or without the indicated drug in the presence of Tg in nominally Ca2+-free media for 15 minutes prior to the addition of Ca2+ (1 mM). The number on each bar represents the number of patient specimens tested. Data were compared against their matched control analyzed by the unpaired T test (Tak475, p = 0.015; compactin, p = 0.0049; IA65, p = 0.0390).",
    "molecules": "IA65, Ca2+, compactin, Tak475, Tg"
  },
  {
    "caption": "(F) Patient cells were placed in Matrigel transwell inserts in OptiMEM reduced serum media with or without TAK475 (10uM), compactin (2μM), IA65 (2μM) or OSMI1 (40μM); DMEM with 10% FBS was used as a chemoattractant. Crystal violet-stained invaded cells were measured 20 hrs after initiation of assay. Each dot represents 1 patient sample. Data analyzed by 2-way ANOVA (cholesterol inhibition was p < 0.0001; patient samples were p < 0.001; interaction was p < 0.01). For IA65, (inhibition was p < 0.0001; patient samples were p < 0.01; interaction was p < 0.0001). For OSMI1, (inhibition was p < 0.05; patient samples were p > 0.05; interaction was p < 0.05). *p<0.05, **p<0.01, ****p<0.0001.",
    "molecules": "IA65, cholesterol, Crystal violet, compactin, OSMI1, TAK475"
  },
  {
    "caption": "Immunoblotting of BM67 cell lines extract and its quantitation following incubation with 100 µg/mL cycloheximide as indicated. Graphs represent mean ± standard deviation (n = 5).",
    "molecules": "cycloheximide"
  },
  {
    "caption": "For each codon, the average frequency (per thousand) is compared between transcripts induced but offset vs non-offset (i.e. abundance mode of regulation) upon ERα depletion in BM67 cells. Codons for the same amino acid are connected by a gray line. Heatmap of standardized residuals from a chi-squared contingency table test. Shown in red and blue are cells with counts significantly higher and lower, respectively, than expected counts under the null hypothesis (i.e. independence between mode for regulation of gene expression and codon composition). A correspondence analysis for average codon frequency among transcripts annotated to each GO term. Each gray dot corresponds to one GO term. The gene expression modes observed upon ERα depletion (Fig 1E) were then projected into the same dimensions based on the average codon frequencies of included transcripts. A correspondence analysis for codon frequency in each regulated mRNA (from Fig 1E). Each dot represents one mRNA and is colored according to its mode of regulation. Codons identified as over-represented among translationally offset mRNAs are also indicated. Unsupervised clustering of gene level codon usage normalized by amino acid counts. All regulated mRNAs (from Fig 1E) are shown in rows and all codons in columns. Codons identified as over-represented among mRNAs whose levels were induced but offset or suppressed but offset are indicated in light and dark blue, respectively.",
    "molecules": "amino acid"
  },
  {
    "caption": "Boxplots (plotted as in Fig 3B) of gene expression of ELP3, ALKBH8 and CTU2 upon treatment with 17β-E2 and/or ICI-182780 in MCF7 cells (extracted from (Wardell et al, 2012; Data Ref: McDonnell et al, 2012)). Wilcoxon-Mann-Whitney tests (two-sided) were used to assess differences between conditions (n = 10) and a global Bonferroni adjustment was applied on p-values.",
    "molecules": "17β-E2, ICI-182780"
  },
  {
    "caption": "Immunoblotting of MCF7 cell extracts. MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours and then treated with 1 nM E2 and/or 100 nM ICI-182780 for 24 hours. Graphs represent mean ± SD of n=5 experiments. Data were analyzed using two-sided paired Student's t tests.",
    "molecules": "E2, charcoal, ICI-182780, phenol red"
  },
  {
    "caption": "Quantification of mcm5S2-U, mcm5U and cm5U levels in ELP3-null MCF-7 cells and in MCF-7 cells treated with 100 nM ICI-182780 for 72 hours versus their respective controls by liquid chromatography-mass spectrometry (LC-MS) analysis. The MCF-7 cells were grown in phenol red-free RPMI media supplemented with 5% charcoal-stripped serum for 24 hours prior to ICI-182780 treatment. Graphs represent mean ± SD of n=4. Data were analyzed using two-sided paired Student's t tests. Vh, Vehicle; ICI, ICI-182780.",
    "molecules": "cm5U, mcm5S2-U, mcm5U, charcoal, ICI, ICI-182780, phenol red"
  },
  {
    "caption": "A Representative kymographs of  LysoTracker-labeled  lysosomes in  proximal dendrites  of  hippocampal neurons  infected with scrambled shRNA (shSc), LAMTOR2  shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement  from kymographs (N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Sidak's post-test ***P < 0.001 as compared to shSc; n.s., not significant.",
    "molecules": "LysoTracker"
  },
  {
    "caption": "E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594-conjugated transferrin (red, Tf  594) in dendrites. Shown are dendritic segments (upper) and kymographs (lower). Scale bar, 5 µm. F Quantitative analysis of  vesicular movement from  kymographs (N = 10 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.",
    "molecules": "Alexa 594"
  },
  {
    "caption": "A Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV  expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker  to visualize lysosomal  trafficking in dendrites. N = 17-31  neurons  from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.",
    "molecules": "LysoTracker"
  },
  {
    "caption": "C-F Quantification of the percent of lysosomes moving in the anterograde  or  retrograde direction. C, E, F Cultured  hippocampal neurons  were infected with AAV  expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML-SI1 (20 µM, C, n = 16-49 neurons from 3-10 independent experiments), or  ML-SA1 (20 µM, E, n = 13-49  neurons from 3-10 independent experiments), or  CilioD (20 µM, F, n = 12-49  neurons from 3-10 independent experiments), and imaged with  LysoTracker to visualize lysosomal  trafficking in dendrites. D Neurons  were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.",
    "molecules": "ML-SI1, ML-SA1, CilioD, LysoTracker"
  },
  {
    "caption": "A  Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding  of  LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT-2031 peptide. Wes protein analysis with anti-LAMTOR1  and -TRPML1  antibodies of  immunoprecipitation performed with anti-LAMTOR1  antibodies or negative control anti-HA  antibodies using whole  hippocampal  homogenates from naïve, TAT or TAT-2031-treated mice. B Quantification of the relative abundance of  TPRML1 pulled down by  LAMTOR1 in naïve, TAT or TAT-2031-treated  mice. N = 3 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.",
    "molecules": "TAT, TAT-2031"
  },
  {
    "caption": "C Representative images from  proximity ligation assay  (PLA) performed on  brain  slices from naïve, TAT  or  TAT-2031-treated  mice. Evidence of proximity between  LAMTOR1  and  TRPML1 is indicated by the appearance of red puncta. Nuclei  are counterstained  with DAPI (blue). Scale bar, 10 μm. D Quantification of the number of PLA signals in CA1 from naïve, TAT  or TAT-2031-treated mice. N = 6 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.",
    "molecules": "TAT, TAT-2031, DAPI"
  },
  {
    "caption": "D  TAT-2031 treatment (10 µM) increased ML-SA1-induced   TRPML1 Ca2+  release in both control and  LAMTOR1 KD cells. Data information: traces represent the mean values of each group.",
    "molecules": "TAT-2031, ML-SA1, Ca2+"
  },
  {
    "caption": "G Treatment with GPN (200 µM), BAPTA-AM  (20 µM), or  ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in  neurons. Data information: traces represent the mean values of each group.",
    "molecules": "GCaMP6m, ML-SI1, BAPTA-AM, ML-SA1, GPN"
  },
  {
    "caption": "I Both LAMTOR1 KD and TAT-2031  treatment (10 µM) increased ML-SA1-induced TRPML1-GCaMP6m responses in neurons. J Quantification of peak responses as shown in I. N = 6-13 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.",
    "molecules": "GCaMP6m, TAT-2031, ML-SA1"
  },
  {
    "caption": "K LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)-induced  TRPML1-GCaMP6m responses and the blocking effect of ML-SI1  treatment (20 µM). L Quantification of peak  TRPML1-GCaMP6m responses as shown in K. N = 6-16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.",
    "molecules": "GCaMP6m, ML-SI1, PI(3,5)P2"
  },
  {
    "caption": "A-D Effects of  LAMTOR1 KD and ML-SI1 treatment on TBS-induced LTP (A) or  LFS-induced  LTD (C) in CA1. B, D Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4-5 slices from 4-5 mice) or LFS (D, n = 4-8 slices from 4-8 mice) in different groups. Data information: Slopes of fEPSPs  were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms.",
    "molecules": "ML-SI1"
  },
  {
    "caption": "G-J Effects of TAT-2031 treatment on  TBS-induced LTP  (G) or  LFS-induced LTD (I) in  CA1. H, J Means ± SEM of  fEPSPs  measured 40 min after TBS (H, n = 6 slices from 6  mice) or LFS (J, n = 3-4 slices from 3-4 mice) in different groups. Data information: Slopes of fEPSPs  were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT Insets show representative traces of evoked fEPSPs before and 40 min after  TBS/LFS. Scale bar 0.5 mV/10 ms.",
    "molecules": "TAT, TAT-2031"
  },
  {
    "caption": "K Effects of LAMTOR1 KD and treatment with a calcineurin  inhibitor, FK506, on LFS-induced  LTD in  CA1. L Means ± SEM of fEPSPs measured 45 min after LFS in different groups (n = 3-4 slices from 3-4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after  LFS. Scale bar 0.5 mV/10 ms.",
    "molecules": "TAT, FK506"
  },
  {
    "caption": "LAMTOR1  KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. A Representative images of CA1 pyramidal neurons stained with anti-p-GluA1  S845 (magenta), anti-GluA1 (red), and anti-GFP (green) antibodies. Scale bar = 20 µm.",
    "molecules": "ML-SI1"
  },
  {
    "caption": "E Representative images of proximal dendrites of hippocampal neurons stained with anti-GluA1 (green), anti-LAMP2  (red), and anti-GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false-colored green and  GFP  false-colored gray to better show colocalization  of GluA1 with  LAMP2. Arrowheads indicate clearly co-localized puncta. Scale bar, 5 µm.",
    "molecules": "Alexa Fluor 633"
  },
  {
    "caption": "A  Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA-injected mice  exhibited a significant deficit 24 h after training, which was reversed by ML-SI1 treatment (N = 7-18 mice). B % freezing for different experimental groups in context memory (N = 7-19 mice). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post-test. *P < 0.05, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1.",
    "molecules": "ML-SI1"
  },
  {
    "caption": "X-gal staining in Axin2Lac/+-Lgr5-GFP-CreERT2 WT or KO ileums.",
    "molecules": "X-gal"
  },
  {
    "caption": "Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP duodenums. Cell membranes are shown with b-catenin and nuclei were counterstained with DAPI. Quantification of Olfm4+ve cells per intervilli regions in duodenum and ileum. Each symbol indicates the value for a given embryo.",
    "molecules": "DAPI"
  },
  {
    "caption": "Gene expression analysis by qRT-PCR of stem cell and differentiation markers in Lgr5-DTReGFP ileums (vehicle-treated: 7 WT and 7 KO; LGK974-treated: 3 WT and 7 KO).",
    "molecules": "LGK974"
  },
  {
    "caption": "Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP WT and KO organoids cultured in Rspo2 conditions. Epithelium is delineated with b-catenin and nuclei counterstained with DAPI (Merge). Insets: white arrowheads evidence Olfm4+ve cells concentrated in organoid protrusions. Quantification of Olfm4+ve area with respect to the total epithelial area. Each symbol indicates the value for an organoid culture originating from a given embryo.",
    "molecules": "DAPI"
  },
  {
    "caption": "A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A)",
    "molecules": "5-AzadC, 5-AzaC"
  },
  {
    "caption": "A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, metabolic activity was assessed by a WST-1 assay (B)",
    "molecules": "5-AzadC, 5-AzaC"
  },
  {
    "caption": "A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment viral protein expression was analyzed by FACS (C)",
    "molecules": "5-AzadC, 5-AzaC"
  },
  {
    "caption": "A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment; and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR (D).",
    "molecules": "5-AzadC, 5-AzaC"
  },
  {
    "caption": "E J-Lat 8.4 cells were mock-treated or treated with 5-AzadC (400nM) or TNFα (10ng/ml) as positive control. At 24 h, 48 h or 72 h post-treatment, initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. Viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)",
    "molecules": "5-AzadC, SAHA"
  },
  {
    "caption": "A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. metabolic activity was assessed by a WST-1 assay (C, D). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 100% (C, D).",
    "molecules": "5-AzadC, SAHA"
  },
  {
    "caption": "E From data of ex vivo cultures of CD8+-depleted PBMCs isolated from 24 HIV+ patients presented in Appendix Table S2, the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented. One night after cell purification, cells were mock-treated or simultaneously treated with 5-AzadC (1µM) and/or SAHA (1µM). Six days after treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.",
    "molecules": "5-AzadC, SAHA"
  },
  {
    "caption": "A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: viral p24 production in cell supernatant was measured (A and B)",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows:FACS analyses were performed and the percentages of GFP+ cells are presented as histograms (C and D)",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR and results were normalized using the β-actin gene primers and are presented as histograms representing fold-inductions compared to the mock-treated condition (E and F). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (E, F).",
    "molecules": "5-AzadC"
  },
  {
    "caption": "One night after cell purification, CD8+-depleted PBMCs from 12 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added to the cultures. Six days after 5-AzadC treatment, WST-1 assay was performed. Mock value was arbitrary fixed at 100% for each individual.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "Figure 6: Representation of reactivation status of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients.A-C From data of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients (Table 2), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "Figure 8: Representation of reactivation status of ex vivo cultures of resting CD4+ T cells.A-C From data of ex vivo cultures of resting CD4+ T cells isolated from HIV+ patients (Table 3), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "One night after cell purification, HLA DR-CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The median fluorescence intensity of CD4 receptor of viable CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.",
    "molecules": "5-AzadC"
  },
  {
    "caption": "A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 for the indicated times, then fixed and immunostained for TOMM20 (Alexa Fluor 488 - Green) and B. abortus LPS (Alexa Fluor 568 - Magenta). DNA was stained with Hoechst 33258 (Blue). Scale bars: 20 µm.",
    "molecules": "Alexa Fluor 488, Alexa Fluor 568, LPS, Hoechst 33258"
  },
  {
    "caption": "A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP for 48 h, then fixed and immunostained for the β-subunit of the ATP synthase (Alexa Fluor 633 - Magenta) and LC3 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LC3-ATP synthase β-positive punctae. Scale bars: 20 µm. B. Quantification of the number of LC3-β-subunit of the ATP synthase-positive punctae per HeLa cell infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A) Data are presented as means ± SD from n=3 (biological replicates independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; ***: p <0.001; ****: p <0,0001; hashtags indicate significant differences between infected conditions throughout time; #: p <0.05; ##: p <0.01.",
    "molecules": "Alexa Fluor 568, Alexa Fluor 633, Hoechst 33258"
  },
  {
    "caption": "C. Representative confocal micrographs of HeLa cells transfected with a FIS1-GFP(Green)-mCherry(Magenta) expression construct, infected or not with B. abortus 544 for 48 h, then fixed and immunostained for B. abortus LPS (Alexa Fluor 633 - Red). DNA was stained with Hoechst 33258 (Blue). Arrows indicate FIS1-mCherry-positive-GFP-negative punctae. Scale bars: 20 µm. D. Quantification of the number of FIS1-mCherry-positive-GFP-negative punctae per HeLa cell infected or not (NI) with B. abortus 544 for 48 h from micrographs shown in (C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0008).",
    "molecules": "Alexa Fluor 633, LPS, Hoechst 33258"
  },
  {
    "caption": "A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Red) for 24 h, 48 h, and 72 h then fixed and immunostained for HIF-1α (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). B. Quantification of the percentages of cells positive for a nuclear localisation of HIF-1α from HeLa cells infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A.B.C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ****: p <0.0001.",
    "molecules": "Alexa 568, Hoechst 33258"
  },
  {
    "caption": "C. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Red) for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green) and TOMM20 (Alexa 633 - Magenta). DNA was stained with Hoechst 33258 (Blue). D. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=4 (biological replicates) independent experiments (8,296 cells analysed in total per condition). Statistical analyses were performed using a one sample t-test; *: p <0.05 (p = 0.0351).",
    "molecules": "Alexa 568, Alexa 633, Hoechst 33258"
  },
  {
    "caption": "D. Representative confocal micrographs of HeLa cells infected or not (NI) with B. abortus 544 GFP (Red) treated or not (ctrl) with 500 µM FeCl2 for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). E. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 µM FeCl2 for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=3 (biological replicates) independent experiments (10,199 cells analysed in total per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; *: p <0,05; hashtags indicate significant differences compared to the infected condition without FeCl2; #: p <0.05.",
    "molecules": "Alexa 568, FeCl2, Hoechst 33258"
  },
  {
    "caption": "F. Representative confocal micrographs of iBMDM infected or not (NI) with B. abortus 544 GFP (Red) treated or not (ctrl) with 500 µM FeCl2 for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). G. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from iBMDM infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 µM FeCl2 for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=3 (biological replicates) independent experiments (14,152 cells analysed in total per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; *: p <0,05; hashtags indicate significant differences compared to the infected condition without FeCl2; ###: p <0.001.",
    "molecules": "Alexa 568, FeCl2, Hoechst 33258"
  },
  {
    "caption": "A. Representative confocal micrographs of HeLa cells transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM), infected or not (NI) with B. abortus 544 GFP (Red) for 48 h, then fixed and immunostained for BNIP3L (Alexa Fluor 568 - Magenta) and TOMM20 (Alexa Fluor 633 - Green). DNA was stained with Hoechst 33258 (Blue). B.,C. Quantification of the mitochondrial population morphology by assessing the AR (B) and EBR (C) of the mitochondria of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a multiple Mann-Whitney test followed by a Holm-Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); *: p <0.05; **: p <0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; #: p <0.05.",
    "molecules": "Alexa Fluor 568, Alexa Fluor 633, Hoechst 33258"
  },
  {
    "caption": "D. Representative confocal micrographs of HeLa cells transfected with a FIS1-GFP(Green)-mCherry(Magenta) expression construct, infected or not (NI) with B. abortus 544 for 48 h while being transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM), then fixed and immunostained for B. abortus LPS (Alexa Fluor 633 - Red). DNA was stained with Hoechst 33258 (Blue). Arrows indicate FIS1-mCherry-positive-GFP-negative punctae. Scale bars: 20 µm. E. Quantification of the number of FIS1-mCherry-positive-GFP-negative punctae per cell of HeLa cells from micrographs t shown in (D). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; **: p <0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; #: p <0.05.",
    "molecules": "Alexa Fluor 633, LPS, Hoechst 33258"
  },
  {
    "caption": "A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Green) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 48 h, then incubated under reinfection-permissive conditions for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. B. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; **: p <0.01 (p = 0.0032). C. CFU assay expressing Log (CFU/ml) from the supernatant of HeLa cells infected with B. abortus 544 GFP (Green) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 48 h, then incubated under reinfection-permissive conditions for 24 h before collecting the supernatant at 72 h pi for analysis. Data are presented as means ± SD from n=5 (biological replicates) independent experiments; Statistical analyses were performed using an unpaired two-tailed Student's t-test; (p = 0.0042).",
    "molecules": "Hoechst 33258"
  },
  {
    "caption": "D. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Red) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 72 h pi, then fixed and immunostained for LAMP-1 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LAMP-1-positive BCVs (aBCVs). Scale bars: 20 µm. E. Quantification of the number of LAMP-1-positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p <0.05 (p = 0.0117).",
    "molecules": "Alexa Fluor 568, Hoechst 33258"
  },
  {
    "caption": "A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl2 (500 µM) for 48 h, then incubated under reinfection-permissive conditions (with or without FeCl2) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. B. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0008).",
    "molecules": "FeCl2, Hoechst 33258"
  },
  {
    "caption": "C. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Magenta) treated or not (ctrl) with FeCl2 (500 µM) for 72 h, then fixed and immunostained for LAMP-1 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LAMP-1-positive BCVs (aBCVs). D. Quantification of the number of LAMP-1-positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p <0.05 (p = 0.0368).",
    "molecules": "Alexa Fluor 568, FeCl2, Hoechst 33258"
  },
  {
    "caption": "E. Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl2 (500 µM) for 48 h, then incubated under reinfection-permissive conditions (with or without FeCl2) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. F. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of iBMDM from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; **: p <0.01 (p = 0.0043).",
    "molecules": "FeCl2, Hoechst 33258"
  },
  {
    "caption": "A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 µ B. Quantification of the percentage of infected HeLa cells displaying TOMM20-positive BCVs (mBCVs) at the indicated times, from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0001). C. Quantification of the number of TOMM20-positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p = 0.05.",
    "molecules": "Alexa Fluor 647, Hoechst 33258"
  },
  {
    "caption": "D. Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (Magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 µm. E. Quantification of the percentage of infected iBMDM displaying TOMM20-positive BCVs (mBCVs) at the indicated times, from micrographs shown in (D). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0009). F. Quantification of the number of TOMM20-positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ns: not significant (p = 0.3739).",
    "molecules": "Alexa Fluor 647, Hoechst 33258"
  },
  {
    "caption": "(B) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine (Rv) for 12, 24, 48, and 72 hours. Thapsigargin  (Tg) is shown as positive control.",
    "molecules": "Reversine, Rv, Tg, Thapsigargin"
  },
  {
    "caption": "(E) Detection of mRNA  expression (by  RT-PCR) of UPR-associated genes (Ern1, Af4, Ddit3 and Atf6) in tumor cells treated with Reversine  (Rv) at 4µM (DLD1  cells) or 0.5µM (SKOV3  cells) for 6 hours. Data points refer to triplicate samples collected at the same time point, run in duplicate, and expressed as means ± SD.",
    "molecules": "Reversine, Rv"
  },
  {
    "caption": "(F)  Western Blot analysis of the activation of IRE1, PERK and ATF branches of the UPR in DLD1  and SKOV3 cells treated or not with Rv at 4µM for 24H (DLD1 cells) or 0.5µM for 3 days (SKOV3 cells). Thapsigargin (Tg) was used as positive control.",
    "molecules": "Rv, Tg, Thapsigargin"
  },
  {
    "caption": "(G) Western Blot  analysis of phosphorylation  of GCN2 and PKR defining the integrated stress response (ISR) in DLD1 and SKOV3 cells treated or not with  Rv as in F). Cells starved for 24 hours then treated for 30 min with Calyculin-A (Cal-A) were used as positive control.",
    "molecules": "Cal-A, Calyculin-A, Rv"
  },
  {
    "caption": "(A)  Xbp1 splicing analysis (left panel) and quantification (right panel) of bone marrow-derived macrophages (BMDM) cultured in conditioned medium (CM) of Rv-treated  DLD1 and SKOV3  cells or respective control medium (culture medium of cancer cells not treated with Rv), and CM of fused B16 melanoma  cells or their nonfused parental cells.",
    "molecules": "Rv"
  },
  {
    "caption": "(B, C) mRNA expression (RT-PCR) of Il6 (B) and Arg1 (C) in BMDM  cultured in the CM of  Rv-treated  SKOV3  cells (0.5µM for 3 days) or control cells, and CM of fused B16 cells or their nonfused parental cells. Data points refer to triplicate experiments expressed as means ± SD.",
    "molecules": "Rv"
  },
  {
    "caption": "(E)  Flow cytometry analysis of  IFNγ  and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in the presence of CM from Rv-treated  SKOV3 cells or control SKOV3 cells (cultured without Rv). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B  (lower panel). Percentages in parenthesis refer to change from control. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.",
    "molecules": "Rv"
  },
  {
    "caption": "(F) Flow cytometry  analysis of IFNγ and Granzyme B production by  human T cells  activated by anti-CD3/anti-CD28 Dynabeads in complete medium spiked with Rv (0.5µM). Column graphs represent the percentage expression of IFNγ  (upper panel) and Granzyme B (lower panel). Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.",
    "molecules": "Rv"
  },
  {
    "caption": "(d) Immunofluorescence with antibodies that recognize PS-CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction.",
    "molecules": "methanol"
  },
  {
    "caption": "(b, c) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 16 h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean+s.e. of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars, 5 μm.",
    "molecules": "3-MA, 3-methyladenine"
  },
  {
    "caption": "(e-g) Effect of inhibition of macroautophagy by treatment with 3-methyladenine (3-MA) on the lysosomal content of KFERQ-PS-CFP2 in cells maintained in serum-free media for 16 h. (e) Immunoblot for the indicated proteins of the same fractions as described in (b). The amount of KFERQ-PS-CFP2 associated to lysosomes (f) and the changes in the intracellular content of KFERQ-PS-CFP2 were calculated as in (c, d). Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.032 (t-test).",
    "molecules": "3-MA, 3-methyladenine"
  },
  {
    "caption": "(d) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.",
    "molecules": "3-MA, 3-methyladenine, H2O2, MG-132"
  },
  {
    "caption": "(B) Confocal microscopy analysis of SB and 10C cells grown to 20% (top panels) or 100% (bottom panels) confluency followed by immuno-fluorescence staining of Annexin A1 (green). The nuclei were stained with DAPI (blue). A series of 20-50 consecutive images in the z-axis were stacked together to generate the 3-D figure at 400x magnification. Annexin A1 is most abundantly expressed on the outer layer of 20% confluent 10C (pointed by arrows compared to 100% confluent 10C, or SB of either confluency. Scale bars, x = 1 μm, y = 1 μm, z = 1.6 μm. The experiment was repeated at least twice.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Flow cytometry analysis of Annexin A1 expression in 10C cells either untreated, or incubated with BSA (1000 µg/ml), or mFadA (1000 µg/ml), or FadAc (100, 300, or 1000 µg/ml) for 1 hr. Data are mean values ± SD. The experiment was performed in triplicates and repeated more than three times. **p<0.001 (one-way ANOVA).",
    "molecules": "BSA"
  },
  {
    "caption": "(C) Flow cytometry analysis of Annexin A1 in 10C cells transfected with control siRNA (dotted black line) or CDH1-specific siRNA (dotted red line) followed by no treatment, or incubation with BSA (1000 µg/ml) or FadAc (1000 µg/ml) for 1 hr. C, untreated control. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. ***p<0.001 (two-way ANOVA).",
    "molecules": "BSA"
  },
  {
    "caption": "(D) Confocal microscopy analysis of SB and 10C cells either untreated or following incubation with CFSE-labeled F. nucleatum 12230 (Fn) for 1 hr at MOI of 5:1. Annexin A1 was stained green and E-cadherin blue. Imagines are 800x magnification. Note the enhanced expression of Annexin A1 in 10C compared to SB and its location on the outer layer of the cell mass. The experiment was repeated three times. Scale bar, 250 nm.",
    "molecules": "CFSE"
  },
  {
    "caption": "(B) Co-immunoprecipitation with Annexin A1. DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with rabbit anti-Annexin A1 polyclonal antibody (α-Annexin A1) or control rabbit IgG. FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in the eluates were detected by Western blot. C, untreated control. The experiment was repeated three times.",
    "molecules": "agarose"
  },
  {
    "caption": "(C) Co-immunoprecipiation with FadA. DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with mouse anti-FadA monoclonal antibody (α-FadA) or control mouse IgG. FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in the eluates were detected by Western blot. The experiment was repeated three times.",
    "molecules": "agarose"
  },
  {
    "caption": "(D) Confocal microscopy analysis of 10C cells either untreated (top panel) or following incubation with CFSE-labeled F. nucleatum 12230 (red, bottom panel) for 3 hrs and then immunofluorescent-stained for Annexin A1 (green) and E-cadherin (blue). Images are 1200x magnification. A side view of the enlarged image is shown on the far right. Note the enhanced expression of Annexin A1 in the F. nucleatum-bound cells and the co-localization of Annexin A1, E-cadherin and F. nucleatum on the cell membranes (arrows). The experiment was repeated more than three times. Scale bar, 500 nm.",
    "molecules": "CFSE"
  },
  {
    "caption": "(E) Confocal microscopy analysis of 10C cells following incubation with Alexa Fluor™ 488-conjugated BSA, mFadA, or FadAc (red) for 1 hr and immuno-staining of Annexin A1 (green) and E-cadherin (blue). Images are 1200x magnification. Note the enhanced expression of Annexin A1 and its co-localization with E-cadherin in the presence of FadAc (arrows), but not with BSA and mFadA. The experiment was repeated twice. The side views are shown to the right and bottom of each image. Scale bar, 500 nm.",
    "molecules": "BSA, Alexa Fluor™ 488"
  },
  {
    "caption": "Flow cytometry analysis of SB (A), 10C (B), DLD1 (C) and HCT116 (D) cells incubated with CFSE-labeled F. nucleatum 12230 (Fn) or its fadA-deletion mutant US1 (US1) at MOI of 10-20:1 for the indicated time and immuno-stained with anti-Annexin A1 antibodies. Shown on the top panels are the density plots. x-axis, CFSE-labeled F. nucleatum or US1 (CFSE-Fn); y-axis, Annexin A1 (ANXA1). Shown on the bottom panels are the percentages of Annexin A1-positive (solid bars) or negative (clear bars) cells bound by F. nucleatum or US1 out of the total number of cells analyzed. Data are mean values ± SD. The experiments were performed in triplicates and repeated 2-3 times. *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA).",
    "molecules": "CFSE"
  },
  {
    "caption": "(A) Colorectal tumors generated in Apcmin/+ mice following treatment with PBS, E. coli DH5α (E. coli), fadA-deletion mutant US1 (US1) or F. nucleatum 12230 (Fn). Each symbol represents one mouse. Horizontal lines represent mean values. Representative tumors formed in the mouse colon are shown on the right, pointed by blue arrows on the right. *p<0.05, **p<0.01 (one-way ANOVA).",
    "molecules": "PBS"
  },
  {
    "caption": "(G) Confocal microscopy analysis of paired normal and carcinoma tissues from two colon cancer patients. The frozen sections were incubated with rabbit anti-AnnexinA1 polyclonal antibodies and 5G11 mouse anti-FadA monoclonal antibodies. The slides were then stained with Alexa Fluor®680-conjugated donkey anti-rabbit and Alexa Fluor®555-conjugated goat anti-mouse, washed, and covered in mounting medium containing DAPI. The scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for the normal tissues and 400x for the carcinomas. Scale bar, 50 μm. Co-localization of FadA (red) and Annexin A1 (green) was observed in carcinomas but not in the paired normal tissues.",
    "molecules": "Alexa Fluor®680, Alexa Fluor®555, DAPI"
  },
  {
    "caption": "(B) Immuno-staining of β-catenin in 10C cells following transfection with control siRNA or ANXA1-specific siRNA and incubation with F. nucleatum 12230 (Fn) at MOI of ~100:1 for 2 hrs. β-catenin was stained with Alexa Fluor®680 (red) and the nuclei with DAPI (blue). The images were captured with confocal microscope at 800x magnification. -, no bacteria added. Note the increased expression of β-catenin and its nucleus translocation in response to F. nucleatum in control siRNA-treated cells, but not in ANXA1 siRNA-treated cells. The experiment was repeated twice. Scale bar, 200 nm.",
    "molecules": "Alexa Fluor®680, DAPI"
  },
  {
    "caption": "(A) Representative kymographs of tetramethylrhodamine-labelled microtubule movement driven by the WT and the E239K mutant. The arrowheads represent the end positions of the WT and E239K.",
    "molecules": "tetramethylrhodamine"
  },
  {
    "caption": "(F) Rate constants (kobs) determined for the reaction of mant-ATP with nucleotide-free KIF1A(WT) and KIF1A(E239K) were plotted against concentration of mant-ATP. Data are represented as the mean ± SD. The data from three independent measurements were analysed.",
    "molecules": "mant-ATP, nucleotide"
  },
  {
    "caption": "(A) The positions of the β6-L10-β7 and neck-coil peptides are shown in brown and green. The positions of the neck-linker peripheral peptides (α1-β3-P loop, α4-L12 and L12-α5) are shown in light blue. (B-D) Quantitation of site-specific intramolecular interactions. Representative MS/MS spectra of the indicated peptides, including the mutated amino acid (β6-L10-β7) (B and C) and neck-coil (D). In the inserted graphs, the intensities in the ADP-BeFx and ADP-AlFx states were measured with respect to the nucleotide-free state as 1.0. Grey bar graphs, WT; black bar graphs, E239K; (-), nucleotide-free.",
    "molecules": "ADP, AlFx, BeFx"
  },
  {
    "caption": "(B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(A) Inter‐kinetochore distance is reduced in unaligned chromosomes of Beclin‐1‐depleted cells. The distance between each kinetochore pair was quantified in siRNA‐treated and control cells treated with nocodazole for 3 h before fixation. Cells were stained with anti‐CREST antibody and DAPI. Quantification of the distance between kinetochore pairs (indicated by coloured bars) is reported at the bottom and done on sister kinetochores present in the same focal plane. One asterisk indicates significant result (P=0.0365). Graphs represent three independent experiments (error bars=s.d.).",
    "molecules": "nocodazole"
  },
  {
    "caption": "(C) Zwint‐1 levels do not change in Beclin‐1‐depleted cells. Cells were treated with nocodazole before fixation. On the right, magnified individual kinetochore pairs stained for Zwint‐1 (red) and CREST (green), and quantification of Zwint‐1 levels normalized to CREST signal from three independent experiments (error bars=s.d.).",
    "molecules": "nocodazole"
  },
  {
    "caption": "(D) Beclin‐1 is associated with both the centrosome and the mitotic spindle during mitosis. HeLa cells were permeabilized before fixation and immunostained for Beclin‐1 (green), α‐tubulin (red) and DNA (DAPI, blue).",
    "molecules": "DNA"
  },
  {
    "caption": "(F) Beclin‐1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule‐dependent manner. Untreated cells (left) and nocodazole‐treated cells (right) were pre‐extracted and fixed before labelling with Beclin‐1 (green) and Zwint‐1 (red) antibodies.",
    "molecules": "nocodazole"
  },
  {
    "caption": "(H) Beclin‐1 localization close to the kinetochore does not depend on the ZW10 protein. Cells treated with Control, Beclin‐1 and ZW10 siRNA were pre‐extracted and fixed before labelling with Beclin‐1 (green), CREST (blue) and ZW10 (red) antibodies. DNA was visualized with DAPI. (C-H) Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; DNA, deoxyribonucleic acid; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin.",
    "molecules": "DNA"
  },
  {
    "caption": "B. Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells. C. Similar experiment using TPO-independent  TF-1 TpoR CALRdel52 cells at 8 hours. D. Similar experiment using PBMNCs from CALRdel52 PMF  primary cells at 8 hours. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "G. Western blot of  TpoR immunoprecipitation under non-reducing conditions showing associated CALR  50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-hour treatment with 20 µg/mL 4D7 but not  PBS  or 20 µg/mL IgG. CALR  monomers & dimers are detectable by polyclonal anti-wild type CALR or anti-mutant CALR  monoclonal antibodies. Red arrowheads, detected mutant CALR  protein; brown arrowheads, detected wildtype CALR protein, asterisk, non-specific bands.",
    "molecules": "PBS"
  },
  {
    "caption": "C. Number of CD41+ megakaryocytes  derived from isolated  CD34+ progenitors from myelofibrosis patients. Number of  CD41/CD61+ cells counted on day 12 using  trypan blue  exclusion (n=1 technical replicate). D. Summary of fold change reduction of  CD41/CD61+ megakaryocytes by 4D7 in all tested CALR  mutated patient  samples compared to CALR wild type, normalised to IgG (n=11 samples from different patients, with 4 technical replicates per sample). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.",
    "molecules": "trypan blue"
  },
  {
    "caption": "E Number of  CD41+ megakaryocyte  colonies from patient  samples after  4D7  treatment. CD34+ from  patients with myelofibrosis  with  CALRdel52 or CALRins5 were plated on  collagen-based matrix in presence of 20 µg/mL  4D7  or IgG control (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.",
    "molecules": "collagen"
  },
  {
    "caption": "F. Western blot  showing signalling in ruxolitinib-resistant TF-1 TpoR CALRdel61 compared to ruxolitinib sensitive TF-1 TpoR CALRdel61 cells after treatment with 100nM  ruxolitinib for 16 hours or 30 minutes and blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and  actin  as indicated. Ruxolitinib-sensitive  cells were non-viable after 16 hours of treatment.",
    "molecules": "ruxolitinib, Ruxolitinib"
  },
  {
    "caption": "G. Comparison of  cell growth after DMSO, 20 µg/mL   4D7, IgG or 100 nM ruxolitinib  treatment over 4 days of ruxolitinib-resistant  TF-1 TpoR CALRdel61. Cells counted using Trypan Blue  exclusion (n=3 biological replicates with 3 technical replicates). H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL  4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates). Data information: Error bars on represent standard deviation. All P values are unpaired Student's unpaired t-test.",
    "molecules": "DMSO, ruxolitinib, Trypan Blue"
  },
  {
    "caption": "F. Kaplan-Meier survival curve of TF-1 TpoR CALRdel61 chloroma mice  treated with  4D7 or IgG until humane killing due to tumour diameter > 30 mm or ulceration. (n=3  mice  per treatment) G. Kaplan-Meier survival curve of NSG mice engrafted  with  ruxolitinib-resistant TF-1 TpoR CALRdel61 treated with 12.5 mg/kg  4D7  or IgG twice weekly (n=5 and 6 mice  for IgG and   4D7 respectively). Data information For all  survival curves  the log-rank Mantel-Cox test P value is shown.",
    "molecules": "ruxolitinib"
  },
  {
    "caption": "DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with OP-puro (25 µM) for 2 h. Where indicated cells were allowed to recover in drug-free medium (control) or in presence of MG132 (10 µM) for 5 h. Scale bars: 5 µm. GFP-NCL HeLa Kyoto cells were treated as described in A, with the exception that a lower concentration of OP-puro was used (5 µM). The distribution of DRiPs and GFP-NCL was analyzed by STED super-resolution microscopy. Scale bars: 5 µm.",
    "molecules": "MG132, OP-puro"
  },
  {
    "caption": "Isolation of nucleoli from HeLa Kyoto cells untreated or treated with puromycin (25 µM) for 2 h. Puromycylated proteins were detected by immunoblotting (right panel). The left panel shows the purity of isolated nucleoli by western blotting, where fibrillarin was used as nucleolar marker, FUS as nucleoplasmic marker and TUBA4A as cytoplasmic marker.",
    "molecules": "puromycin"
  },
  {
    "caption": "HeLa cells were left untreated or treated with heat shock (HS) at 42˚C for 3 h, alone or with cycloheximide (CHX; 50 μg/ml), or Actinomycin D (Act. D; 3 µM) followed by staining with the dye Amylo-glo.",
    "molecules": "Amylo-glo, Act. D, Actinomycin D, CHX, cycloheximide"
  },
  {
    "caption": "DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42˚C and OP-puro (25 µM) for 2 h. Where indicated, cells were cotreated with CHX (50 μg/ml).",
    "molecules": "CHX, OP-puro"
  },
  {
    "caption": "HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42˚C for 2 h, alone or combined with CHX, in GFP-NCL HeLa Kyoto cells.",
    "molecules": "CHX"
  },
  {
    "caption": "DRiP labelling in HeLa cells treated with MG132 (10 µM) alone or with CHX (50 μg/ml) for 8 h; where indicated, OP-puro (25 µM) was added during the last 45 min, prior to cell fixation.",
    "molecules": "CHX, MG132, OP-puro"
  },
  {
    "caption": "Amylo-glo staining of HeLa cells left untreated or exposed to MG132 (10 µM) alone or combined with CHX (50 μg/ml) for 8 or 13 h.",
    "molecules": "Amylo-glo, CHX, MG132"
  },
  {
    "caption": "Amylo-glo staining of HeLa cells left untreated or subjected to transcriptional stress (MG132 10 µM and Act. D 4 µM) for 6 hrs. Where indicated, translation was concomitantly inhibited with CHX (50 μg/ml). Amylo-Glo staining is also shown in HeLa cells treated with Act.D alone or with CHX for 6 hrs, as control.",
    "molecules": "Amylo-glo, Amylo-Glo, Act. D, Act.D, CHX, MG132"
  },
  {
    "caption": "Amylo-glo staining of HeLa cells exposed to HS at 42˚C for 3 h and let to recover for 3 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 3 h).",
    "molecules": "Amylo-glo, VER"
  },
  {
    "caption": "Amylo-glo staining of HeLa cells treated with MG132 (10 µM) for 13 h and let to recover for 5 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 5 h).",
    "molecules": "Amylo-glo, MG132, VER"
  },
  {
    "caption": "HeLa cells were transfected with vectors encoding for GFP, GFP-GR50 or RPL23a-GFP. 24 hrs post-transfection cells were fixed and stained with Amylo-glo. The percentage of transfected cells with Amylo-Glo positive nucleoli is shown. Number of cells counted/condition: GFP, 412; RPL23a-GFP, 482; GFP-GR50, 359, in three independent experiments; statistical significance via One-way ANOVA; p < 10-7, ± s.e.m.",
    "molecules": "Amylo-glo, Amylo-Glo"
  },
  {
    "caption": "DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h; where indicated cycloheximide was also added (CHX; 50 μg/ml). Scale bars: 5 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and heat shock (HS) at 42˚C for 2 h. PML-NBs were visualized using a PML specific antibody, followed by incubation with alexa fluor 647. For convenience the alexa fluor 647 signal is shown in green. Scale bars: 5 µm.",
    "molecules": "alexa fluor 647, CHX, cycloheximide, OP-puro, polyUb"
  },
  {
    "caption": "PolyUb proteins (FK1) labelling in HeLa cells left untreated or treated with HS at 42˚C for 2 h, alone or combined with CHX (50 μg/ml). Scale bars: 5 µm.",
    "molecules": "CHX, PolyUb"
  },
  {
    "caption": "STED super-resolution microscopy showing colocalization of endogenous polyUb proteins (FK1) and PML-NBs in PML-GFP HeLa cells treated with HS at 42˚C for 2 h. Scale bars: 5 µm.",
    "molecules": "polyUb"
  },
  {
    "caption": "DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h; where indicated, cycloheximide was also added (CHX; 50 μg/ml). Asterisks (*) indicate nucleoli. Scale bars: 10 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and MG132 (10 µM) for 4 hrs. Scale bars: 5 µm. Quantitation of cells shown in E. Number of cells counted/condition: 298 - 1713 in three independent experiments; statistical significance via One-way ANOVA; p ≤ 0.001, ± s.e.m.",
    "molecules": "CHX, cycloheximide, MG132, OP-puro, polyUb"
  },
  {
    "caption": "Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Asterisks (*) indicate nucleoli. Scale bars: 10 µm.",
    "molecules": "MG132, OP-puro"
  },
  {
    "caption": "HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, cells were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Left panel: automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 334 - 364; p < 10-10. Right panel: automated quantitation of the number of DRiP foci/nucleus. Cell number analyzed: 334 - 364; p < 10-10. Automated DRiP segmentation is based on azide signal.",
    "molecules": "azide, MG132, OP-puro"
  },
  {
    "caption": "DRiPs and polyUb (FK1) protein distribution in cells treated as described in (A). Automated DRiP segmentation is based on azide signal. Quantitation of the number of DRiP foci enriched for polyUb (>2) is shown; n = 590 - 2114; statistical significance via 2‐tailed Student's t‐test; p < 10-10. Scale bars: 10 µm.",
    "molecules": "azide, polyUb"
  },
  {
    "caption": "Cells lipofected for 72 h with non-targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 151 - 225; p < 10-10 (see panel G).",
    "molecules": "MG132, OP-puro"
  },
  {
    "caption": "Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal. Representative pictures showing that Ubc9-depleted cells cannot efficiently induce the self-assembly of PML-NBs upon treatment with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Scale bars: 10 µm.",
    "molecules": "MG132, OP-puro"
  },
  {
    "caption": "PolyUb protein (FK1) labelling in PML-GFP HeLa Kyoto cells that were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Cells were either immediately fixed or let to recover for 5 h in drug-free medium (Control), with VER (40 µM) or Eeyarestatin I (5 µM). Scale bars: 10 µm. Quantitation of the % of cells with polyUb (FK1) nuclear foci shown in (A). Number of cells counted/condition: 310 - 1258 in three independent experiments; statistical significance via One-way ANOVA; p = 0.0003 or 10-6, ± s.e.m.",
    "molecules": "Eeyarestatin I, MG132, OP-puro, PolyUb, polyUb, VER"
  },
  {
    "caption": "Quantitation of the number of PML-NBs enriched for polyUb (>2); n = 611 - 2612; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.",
    "molecules": "polyUb"
  },
  {
    "caption": "Quantitation of the % of cells with polyUb proteins (FK1) sequestered in nuclear foci. HeLa cells were treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h. Then, cells were either immediately fixed or let to recover for 3 h in drug-free medium (control), with VER (40 µM) or Eeyarestatin I (5 µM). Number of cells counted/condition: 593 - 1016 in three independent experiments; statistical significance via One-way ANOVA; p = 10-3, ± s.e.m.",
    "molecules": "Eeyarestatin I, OP-puro, polyUb, VER"
  },
  {
    "caption": "Quantitation of the number of PML-NBs enriched for polyUb (>2) in HeLa cells treated as described in (D) but with a recovery time of 2 h; n = 1155 - 3283; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.",
    "molecules": "polyUb"
  },
  {
    "caption": "Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to the following treatments: untreated (Control); MG132 (10 µM) and OP-puro (25 µM) for 4 h, alone or combined with CHX (50 μg/ml). Where indicated cells were allowed to recover for 5 hrs in drug-free medium (Control) or with VER (40 µM). Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.",
    "molecules": "Amylo-Glo, CHX, MG132, OP-puro, VER"
  },
  {
    "caption": "Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to heat shock (HS) at 42˚C for 2 hrs. Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.",
    "molecules": "Amylo-Glo"
  },
  {
    "caption": "PML-GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry-VHL. 24 h post-transfection, cells were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Where indicated (OP-puro + MG132 + CHX), translation was inhibited during stress with cycloheximide (CHX; 50 μg/ml). In the lower panel, cells were allowed to recover for 5 h in drug-free medium (Control), or with VER (40 µM). The subcellular distribution of PML-GFP and mCherry-VHL was studied by live-cell confocal imaging. Representative pictures are shown. Scale bars: 5 µm. Quantitation of the fluorescence intensity recovery after bleach of PML-GFP. PML-GFP HeLa Kyoto overexpressing mCherry-VHL for 24 h and treated as described in (A) were analyzed. The mean of 19-23 FRAP curves and the fitting curves are shown in black and red respectively. In gray, the SD is shown.",
    "molecules": "CHX, cycloheximide, MG132, OP-puro, VER"
  },
  {
    "caption": "Intranuclear distribution of 20S proteasomes, HSP70, VCP and DRiPs in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h and allowed to recover for 5 h in drug-free medium (control), with VER (40 µM), Eeyarestatin I (5 µM) or MG132 (10 µM). Scale bars: 5 µm.",
    "molecules": "Eeyarestatin I, MG132, OP-puro, VER"
  },
  {
    "caption": "Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h, followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI). Where indicated, cells were treated with MG132, VER or EeyI alone for 5 h. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (A) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 1112 - 1537 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.",
    "molecules": "Eeyarestatin I, EeyI, MG132, OP-puro, VER"
  },
  {
    "caption": "HeLa cells were lipofected with cDNAs encoding for HA-Ubiquitin, Flag-Ubiquitin or Flag-VCP 24 h post-transfection, cells were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM). Cells were processed for staining of HA, Flag and endogenous 53BP1. * indicates cells that do not express HA- or Flag-Ubiquitin and show no rescue of 53BP1 foci formation after stress. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (C) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 710 - 938 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.",
    "molecules": "MG132, OP-puro, Ubiquitin"
  },
  {
    "caption": "Colony formation assay. The number of colonies surviving 10 days after plating is shown. Control cells or cells treated with MG132 (10 µM) and puromycin (5 µg/ml), followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI) are shown. Where indicated, cells were treated with VER or EeyI alone for 5 h. Number of colonies counted/condition: up to 526 in three independent experiments; statistical significance via One-way ANOVA; p < 0.03, ± s.e.m.",
    "molecules": "Eeyarestatin I, EeyI, MG132, puromycin, VER"
  },
  {
    "caption": "(F) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (G) Normalized density of REC8 in (F). n=3 mice.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(E) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (F) Normalized density of SECURIN and CYCLIN B1 in (E). n=3 mice.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Representative immunofluorescence images of Sec31A marked ERES in HeLa cells cultured on crossbow shaped micropatterned surface of small (upper panel) or large (lower panel) size for 4 hours. Cells were treated with DMSO (Ctrl) or 50 µM NSC23766 (Rac1 inhibitor) for 4 hours prior to fixation and immunostaining. The number of ERES per cell for each pattern size in HeLa (B) The number of ERES was quantified from at least 74 cells per condition for HeLa (B) Each dot represents an individual cell.",
    "molecules": "DMSO, NSC23766"
  },
  {
    "caption": "(A) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were transfected with 10 nM non-targeting control (siCtrl) or co-transfected with siRNAs targeting Sar1A and Sar1B. After 48 h, cells were trypsinized and 80,000 cells were seeded on micropatterned chips. Cells were allowed to attach for 3 h, stained with CellMask for 5 min and processed for microscopy. Scale bars: 5 µm (B) Quantification graph showing the percentage of cells failing to entirely cover the different geometries (described in A). Data derived from at least 100 cells from a set of three independent experiments. Error bars represent standard deviation. Asterisks (*) denote statistical significance (p-value < 0.05; chi-squared test).",
    "molecules": "CellMask"
  },
  {
    "caption": "(D) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were treated with 50 µM of the Rac1 inhibitor NSC23766 for 4 hours prior to staining with CellMask and processing for immunofluorescence microscopy. (E) Quantification of (D). Percentage of cells failing to cover entire micropatterned surface is shown for control (ctrl) and the Rac1 inhibitor NSC23766. Between 113 - 117 cells were counted in total from a set of three independent experiments.",
    "molecules": "CellMask, NSC23766"
  },
  {
    "caption": "(A) HeLa cells were transfected with 10 nM non-targeting control siRNA (siCtrl) or siRNAs targeting Rac1 (siRac1). After 72 h, cells were fixed and processed for immunostaining against Sec31A to label ERES. Representative confocal microscopy images are shown. (B) Quantification graph shows the number of ERES/cell in cells transfected with control siRNA (siCtrl) or siRNA against Rac1 (two siRNAs #1 and #2). Values are expressed as % of siCtrl. From three independent experiments, with >30 cells per condition were counted. (C) Representative immunofluorescence images showing ERES in HeLa cells treated with DMSO (Ctrl), Rac1 inhibitor NSC23766 (50 µM, 4 h), or NSC23766 washout (50 µM for 4h, then washout for 2h). (D) Graph shows the number of ERES per cell (displayed as % of Ctrl) derived from at least 30 cells per condition from three experiments.",
    "molecules": "DMSO, NSC23766"
  },
  {
    "caption": "(E) Fluorescence recovery after photobleaching (FRAP) of GFP-Sec16A marked ERES in HeLa cells. Images show an individual ERES before (-1), immediately after (0) and 28 sec after photobleaching. The graph illustrates FRAP analysis of individual ERES from 23 (for control) and 24 (for NSC23766) regions in 3 experiments. Line connects individual time points. Data are ± SD. (F) Quantification of the mobile fraction of GFP-Sec16A in control and Rac1 inhibited cells. Mobile fraction is derived from a total of 23 (Ctrl) and 24 (NSC23766) individual ERES from three independent experiments.",
    "molecules": "NSC23766"
  },
  {
    "caption": "(G) The rate of ER-export was monitored using GFP-ManII-RUSH in HeLa cells after perturbation of Rac1 with siRNA (siRac1), or Rac1 inhibitor NSC23766, or a combination of both. Representative images show GFP-ManII-RUSH distribution in HeLa cells at indicated time points. (H) Quantification shows ratio of ManII fluorescence intensity within Golgi to outside Golgi region after addition of biotin at indicated time points. Between 76 - 104 cells were used for measurement of ManII intensity in 3 experiments.",
    "molecules": "biotin, NSC23766"
  },
  {
    "caption": "(A and B) Representative immunofluorescence images showing Sec31A labelled ERES in HeLa cells treated with DMSO (Ctrl), 0.5 µM cytochalasin D (CytoD) and 0.5 µM latrunculin A (LatA) for 15 and 30 min (A). Scale bar: 5 µm. The number of ERES per cell in HeLa cells treated with different doses of CytoD and LatA for 30 min are displayed as % of control ± standard deviation (90 -100 cells from 3 experiments) (B).",
    "molecules": "cytochalasin D, CytoD, DMSO, LatA, latrunculin A"
  },
  {
    "caption": "(C) Distribution of ManII-RUSH in HeLa cells. Cells were pre-treated with DMSO (Ctrl) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII-trafficking. Golgi arrival for ManII-RUSH was monitored for 30 min.",
    "molecules": "biotin, DMSO, LatA"
  },
  {
    "caption": "(D-E) Graph showing ratio of ManII within Golgi to outside Golgi. Values were derived from HeLa cells treated with LatA or CytoD as described in C. At least 30 cells per condition were used for quantification in each experiment (n = 3).",
    "molecules": "CytoD, LatA"
  },
  {
    "caption": "(F) HeLa cells cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D (CytoD) and ERES were labelled with anti-Sec31A antibody before (no strain) or after 15% equibiaxial strain (15% strain). Anti-phalloidin staining was performed to visualize actin in cells. Representative images are shown. (G) Quantification of experiment described in (F). The graph shows average ERES/Cell. Data were derived from a total of 90 - 115 cells from 3 experiments. Each dot represents several cells and error bars show standard deviation.",
    "molecules": "cytochalasin D, CytoD, PDMS, DMSO, phalloidin"
  },
  {
    "caption": "(B) Microsomes (M) from HeLa cells stably expressing mCherry-ManII-RUSH were incubated with ATP and an ATP-regenerating system, GTP, biotin and cytosol in the presence or absence of recombinant Rac1 for 30 min at 25°C. COPII vesicles (V) were pelleted by ultracentrifugation (100,000 xg) and immunoblotted for anti-mCherry to detect ManII-RUSH and anti-calnexin (ER marker). (C) Quantification data of two independent vesicle budding assays. All values were normalized to 4 ℃ Ctrl condition.",
    "molecules": "ATP, biotin, GTP"
  },
  {
    "caption": "Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. Fasting and fed blood glucose.",
    "molecules": "glucose"
  },
  {
    "caption": "Eight-week-old male WT and DOCK5-/- mice were fed a standard diet (SD) or high-fat diet ( HFD) for 3 months. 24-h oxygen consumption. Respiratory exchange ratio (RER: VCO2/VO2). Energy expenditure.",
    "molecules": "CO2, O2"
  },
  {
    "caption": "Eight-week-old male WT and DOCK5-/- mice were fed a SD or HFD for 3 months. Blood glucose levels and area under curve (AUC) during the glucose tolerance test. Blood glucose levels and AUC during the insulin tolerance test.",
    "molecules": "glucose"
  },
  {
    "caption": "Eight-week-old male WT and DOCK5-/- mice Glucose infusion rates (GIR) during lipid infusion.",
    "molecules": "lipid"
  },
  {
    "caption": "Eight-week-old male WT and DOCK5-/-mice were fed a SD or a HFD for 3 months. Mice were treated with insulin (1 U/kg) or control (PBS) by intraperitoneal injection for 10 minutes; animals were killed and livers were collected. Total and phosphorylated insulin receptor (InsR), IRS-1ser1101 and Akt.",
    "molecules": "ser"
  },
  {
    "caption": "Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in Hep1-6 cells.",
    "molecules": "GlcN"
  },
  {
    "caption": "Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. - mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in Hep1-6 cells.",
    "molecules": "GlcN, Ser"
  },
  {
    "caption": "Hep1-6 cells were infected with pCDNA3.1-DOCK5 (DOCK5 +) or pCDNA3.1 (DOCK5-) for 48 h. mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in Hep1-6 cells.",
    "molecules": "GlcN"
  },
  {
    "caption": "MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. The mRNA and protein expression of PEPCK and G6Pase in MPHs.",
    "molecules": "GlcN"
  },
  {
    "caption": "MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in MPHs.",
    "molecules": "GlcN, Ser"
  },
  {
    "caption": "MPHs isolated from WT and DOCK5-/- mice were incubated in control (GlcN-) or GlcN (GlcN+) medium for indicated times, followed with (Ins+) or without (Ins-) 100 nmol/L insulin stimulation for 20 min. Total and phosphorylated mTOR and S6K1, and the levels of Raptor and Rictor protein in MPHs.",
    "molecules": "GlcN"
  },
  {
    "caption": "Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Fasting and fed blood glucose 14 days post-infection.",
    "molecules": "glucose"
  },
  {
    "caption": "Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Blood glucose levels and AUC during glucose tolerance tests. Blood glucose levels and AUC during insulin tolerance tests. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP). Percentage of suppression of hepatic glucose production (HGP).",
    "molecules": "glucose"
  },
  {
    "caption": "Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in the liver.",
    "molecules": "Ser"
  },
  {
    "caption": "MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. Total and phosphorylated InsR, IRS-1Ser1101 and Akt.",
    "molecules": "Ser"
  },
  {
    "caption": "SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes (MPHs) of DOCK5-/- mice.",
    "molecules": "SEW2871"
  },
  {
    "caption": "(A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real‐time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h).",
    "molecules": "MG132, vinblastine"
  },
  {
    "caption": "(D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online). Diagram shows the percentage of aggresome‐positive cells.",
    "molecules": "MG132"
  },
  {
    "caption": "(E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome‐positive cells at 4, 24 and 48 h after MG132 wash out.",
    "molecules": "MG132"
  },
  {
    "caption": "(F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome‐positive cells and the mean size of aggresomes ( supplementary Fig S1H online). Images are shown in supplementary Fig S1G online.",
    "molecules": "BafA1, MG132"
  },
  {
    "caption": "(G) Dot‐blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40‐soluble and ‐insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online.",
    "molecules": "NP40"
  },
  {
    "caption": "(H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40‐soluble and ‐insoluble proteins were analysed by immunoblotting.",
    "molecules": "DMSO, MG132, NP40"
  },
  {
    "caption": "(I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST‐DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole‐cell lysates and in precipitates obtained by GST pull‐down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; Hsp70, heat‐shock protein 70; nons, nonsense; siRNA, short‐interfering RNA; vinblast, vinblastine.",
    "molecules": "MG132"
  },
  {
    "caption": "(E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or GST as vehicle control was added for GST and GST pull‐down analysis was performed as in (D).",
    "molecules": "lactacystin"
  },
  {
    "caption": "(F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14.",
    "molecules": "sucrose"
  },
  {
    "caption": "(H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.",
    "molecules": "PYR41"
  },
  {
    "caption": "(C) HEK cells transfected for 8 h with SODG85R‐GFP along with FLAG‐BAG3 and HA‐Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R‐GFP levels in BafA1‐treated cells to those in untreated cells.",
    "molecules": "BafA1"
  },
  {
    "caption": "(F) HEK cells were transfected with SODG85R‐GFP and indicated FLAG‐tagged BAG3 constructs together with HA‐Hsp70, and lysosomal degradation of SODG85R‐GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat‐shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.",
    "molecules": "bafilomycin A1"
  },
  {
    "caption": "(B) Nissl-stained paraffin sections show intact retinal layers in 20 months old Clcn3unc/un mice. Neurodegeneration in Clcn3-/- mice however, results in a loss of retinal structure already at 11 weeks of age (scale bar: 100 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.",
    "molecules": "paraffin"
  },
  {
    "caption": "(B) Nissl-stained paraffin sections show progressive neuronal cell loss (arrows) that begins in hippocampal CA1 region of P14 Clcn3unc/unc/Clcn4-/- mice and results in a complete loss of the hippocampus at P28. Neurodegeneration progresses slower in Clcn3-/- mice Stobrawa et al, 2001() (scale bar: 200 µm).",
    "molecules": "paraffin"
  },
  {
    "caption": "(D) GFP antibody immunolabels VenusClC-3 (green) in neurites of cultured neurons. EEA1 and VGLUT1 are co-stained (both in red; left and right panel, respectively). The dendritic marker MAP2 is stained in blue (right panel) (scale bars: 10 µm). DNA stained with DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Acidification of synaptic vesicle LP2 fractions from Clcn3unc/unc and Clcn3+/+ mice at one year of age (upper panel, n= 2 animals each and ≥3 measurements per animal), 10-weeks-old Clcn4-/- and WT mice (middle panel, n= 3 animals each and ≥3 measurements per animal), and 4-6-weeks-old Clcn3unc/unc/Clcn4-/- and control mice (lower panel, 6 animals each with ≥2 measurements per animal). A decrease in fluorescence reflects acidification. The protonophore FCCP dissipated the pH gradient. Mean values ± s.e.m.",
    "molecules": "FCCP"
  },
  {
    "caption": "(C) Quantification of ATP-induced acidification of LP2 fractions derived from 2, 6, and 10-12 weeks-old Clcn3-/- compared to wild-type mice. Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl. At least 2 animals in at least 2 independent experiments were pooled. Each measurement was performed at least 3 times. Mean ± s.e.m. is shown.* p < 0.05, *** p < 0.0005 (two-tailed unpaired t test).",
    "molecules": "acridine orange, ATP, KCl"
  },
  {
    "caption": "A. Representative images of immunofluorescence of α-smooth muscle actin (α-SMA), pan Cytokeratin, Pan-hMENA, hMENA11a and E-cadherin expression in CAFs and autologous cancer cells (Ep-PDAC) obtained from enzymatically digested primary PDAC tissue of patient #36. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm.",
    "molecules": "4′,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "A. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA‑positive CAFs are also positive for Pan-hMENA (arrow).",
    "molecules": "4′,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "B. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow).",
    "molecules": "4′,6-diamidino-2-phenylindole, DAPI"
  },
  {
    "caption": "D. Immunoblot analysis with the indicated antibodies of PANC-1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6-mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (-) or rGAS6 (200 ng/mL), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported.",
    "molecules": "DMSO"
  },
  {
    "caption": "Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of Ca2+ monitored at 280 nm absorption wavelength. Size exclusion chromatography (SEC) results for MICU2_WT and a set of mutants in the presence of EGTA monitored at 280 nm absorption wavelength.",
    "molecules": "Ca2+, EGTA"
  },
  {
    "caption": "MICU2_WT (A), MICU2_K172A (B) were pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+. MICU2_EF1mut was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+ (C), while MICU2_EF2mut was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (D). MICU2_R352A was not pulled-down by GST-MICU1 in the absence of 2 mM Ca2+ (E), while MICU2_E329A was pulled-down by GST-MICU1 both in the absence and presence of 2 mM Ca2+ (F). MICU2_D185A was partially pulled-down by GST-MICU1 in the presence of Ca2+, while they showed a strong interaction in the absence of 2 mM Ca2+. MICU2_E196K was not pulled-down by GST-MICU1 in the presence of 2 mM Ca2+, while a strong interaction was observed in the absence of 2 mM Ca2+.",
    "molecules": "Ca2+"
  },
  {
    "caption": "GST-MICU1_EF1mut failed to pull down MICU2_WT in the absence of 2 mM Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A & H385A failed to pull-down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. C GST-MICU1_E242K failed to pull-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_D231A partly pulled-down MICU2_WT in the absence of Ca2+, whereas they displayed a strong interaction in the presence of 2 mM Ca2+. GST-MICU1_F383A failed to pull down MICU2_WT in the presence of Ca2+, whereas they displayed a strong interaction in the absence of 2 mM Ca2+. GST-MICU1_H385A exhibited a strong interaction with MICU2 both in the absence and presence of 2 mM Ca2+.",
    "molecules": "Ca2+"
  },
  {
    "caption": "ITC analyses of MICU1 and MICU2 binding properties in the presence of 2 mM EGTA (G) and CaCl2 (H).",
    "molecules": "CaCl2, EGTA"
  },
  {
    "caption": "K-L) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone (Dex) injections in chow-fed controls (K) and after 4 weeks on HFD (L). M) Comparison of average responses in the two conditions, n=4 each, data presented as mean +/- SEM, **P=0.0012, paired Student's t tests.",
    "molecules": "Dex, Dexamethasone"
  },
  {
    "caption": "(I-J) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (I), and comparison of responsiveness to water spray between NachBac and control mice (J).Gray traces showed Ca2+ independent signal for system stability and red ones showed Ca2+ dependent signals for neuron activity, N=5-7 each, males, data presented as mean +/-SEM, **p<0.0001 in both (H and J), unpaired Student's t tests.",
    "molecules": "Ca2+"
  },
  {
    "caption": "Quantitative expression of Lif mRNA in the colon of DSS-challenged and control mice (n=6 per group).",
    "molecules": "DSS"
  },
  {
    "caption": "LIF measured by ELISA in mouse colon explant supernatants from DSS-challenged and control mice (n=4 per group).",
    "molecules": "DSS"
  },
  {
    "caption": "Quantitative expression of Lif mRNA in LPLs and IECs from DSS-challenged and control mice (n=4 per group).",
    "molecules": "DSS"
  },
  {
    "caption": "mRNA expression of the indicated genes in IECs stimulated by LPS (2.5μg/ml), PGN (3μg/ml) or LTA (2μg/ml) for 2 hours.",
    "molecules": "LPS, LTA, PGN"
  },
  {
    "caption": "Serum concentrations of LIF in IEC supernatants untreated (G) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.",
    "molecules": "LPS, LTA"
  },
  {
    "caption": "Serum concentrations of LIF in IEC supernatants pretreated with monensin (H) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.",
    "molecules": "LPS, LTA, monensin"
  },
  {
    "caption": "Weight loss of wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS.",
    "molecules": "PBS, DSS"
  },
  {
    "caption": "wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. Macroscopic changes in the colon of the mice on day 10 of colitis induction Comparison of the colon length in the mice on day 10 of colitis induction",
    "molecules": "PBS, DSS"
  },
  {
    "caption": "wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. DAI (stool consistency and bleeding score) of colitis mice treated",
    "molecules": "PBS, DSS"
  },
  {
    "caption": "wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. H&E histology of representative colons from colitis mice. Scale bar, 20μm.",
    "molecules": "PBS, DSS"
  },
  {
    "caption": "Weight loss of Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks.",
    "molecules": "PBS"
  },
  {
    "caption": "Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. Macroscopic changes in the colon of Rag1-/- mice treated Comparison of colon lengths in the Rag1-/- mice at week 8 (n=5).",
    "molecules": "PBS"
  },
  {
    "caption": "Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. H&E histology of representative mouse colons on day 54 of colitis induction",
    "molecules": "PBS"
  },
  {
    "caption": "FACS staining of LPLs isolated on day10 from the colon of WT or Stat4-KO colitis mice receiving PBS or LIF (n=4 per group). The percentage of IL-17A+ and/or IFNγ+ CD4+ T cells in vivo was analyzed.",
    "molecules": "PBS"
  },
  {
    "caption": "Immunoblot analysis of STAT4 phosphorylation in CD4+ T cells pretreated with PBS or LY2784544 (1μM) for 12 hours followed by LIF for the indicated number of minutes.",
    "molecules": "PBS, LY2784544"
  },
  {
    "caption": "The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus.",
    "molecules": "serine"
  },
  {
    "caption": "ChIP analysis of naïve CD4+ T cells obtained from IL-17A-eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. GFP+ cells isolated by flow cytometry were restimulated with IL-6 in the presence or absence of LIF, crosslinked with formaldehyde and immunoprecipitated with anti-STAT3 (top) or anti-STAT4 (bottom) antibodies, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.",
    "molecules": "formaldehyde"
  },
  {
    "caption": "ChIP analysis of naïve WT or Stat4-KO CD4+ T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. The cells were restimulated with IL-6 or LIF for 30 minutes, crosslinked with formaldehyde and immunoprecipitated with the anti-STAT3 antibody, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.",
    "molecules": "formaldehyde"
  },
  {
    "caption": "C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group.",
    "molecules": "Formic acid"
  },
  {
    "caption": "D. ELISA of insoluble (Formic acid-soluble) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM.",
    "molecules": "Formic acid"
  },
  {
    "caption": "GAG content as assessed by Alcian blue staining with densitometry (A) A HAC micromasses treated with a GCP-2 neutralizing antibody (n=4) or IgG control (n=4); p values were determined by the Mann-Whitney U test.",
    "molecules": "Alcian blue, GAG"
  },
  {
    "caption": "GAG content as assessed by Alcian blue staining with spectrophotometric B C28/I2 micromasses treated with GCP-2 siRNA (n=12) or scrambled control siRNA (Scr; n=14); p values were determined by unpaired two-tailed Student's t test.",
    "molecules": "Alcian blue, GAG"
  },
  {
    "caption": "GAG content as assessed by Alcian blue staining spectrophotometric C C3H10T½ micromasses treated for 3 days with recombinant GCP-2 100 ng/ml (n=4) or vehicle (n=4) or BMP-2 (n=4); p values were determined by ANOVA followed by Tukey HSD post hoc test.",
    "molecules": "Alcian blue, GAG"
  },
  {
    "caption": "E spectrophotometric quantification of GAG release (dimethylmethylene blue assay) in supernatant of human cartilage explants treated with recombinant GCP-2 (n=5) or vehicle (n=5) and incubated for 24h",
    "molecules": "GAG"
  },
  {
    "caption": "F Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co-implantation of HACs in a 10:1 ratio with growth-arrested COS7 expressing either GFP (n=3) or GCP-2 (n=3) and quantification - on the right-hand side - of the metachromatically stained area as % of total area; scale bar =200µm;",
    "molecules": "Toluidine blue"
  },
  {
    "caption": "C Alkaline phosphatase staining (red) in HAC monolayer after 3 week treatment with T3 hormone (100 ng/ml), T3 hormone + GCP-2 (100ng/ml), or control medium; scale bar 100µm.",
    "molecules": "T3 hormone"
  },
  {
    "caption": "E qPCR for Col10A1 (n=3) of HAC 3 weeks after treatment with vehicle, T3 (100 ng/ml) or T3+GCP-2 (100 ng/ml); n=3;",
    "molecules": "T3"
  },
  {
    "caption": "F, H C3H10T½ monolayers were treated with osteogenic medium (OM) or OM + GCP-2 (100 ng/ml): (F) number of cells per well positive for alkaline phosphatase staining (n=3 in OM and n=4 in OM+GCP-2), (G) number of alkaline phosphatase positive nodules per well (n=7), and (H) spectrophotometric quantification of alizarin red staining at 570nm (n=6)",
    "molecules": "alizarin red"
  },
  {
    "caption": "GAG binding capacity of wildtype GCP-2, and mutants (K101E single mutant, K105E single mutant, GCP-2-D = K101E_K105E double mutant and GCP-2-T = triple mutant K100E_ K101E_K105E) was assessed by (B) a heparin microtiter plate binding assay measuring the interaction of biotinylated-heparin (b-heparin) with immobilized proteins",
    "molecules": "biotinylated, GAG, heparin"
  },
  {
    "caption": "(C) heparin affinity chromatography equilibrated and run in PBS, pH 7.4. In (C) the proteins are eluted with a salt gradient from 0-2 M NaCl monitored by the conductivity (Cond), where 20 and 90 mS/cm correspond to 193 and 1360 mM NaCl, respectively; GCP-2-T, GCP-2-D, K101E, K105E and WT proteins elute at 530 mM, 617 mM, 815 mM, 850 mM and 1024 mM NaCl, respectively.",
    "molecules": "heparin, PBS, NaCl"
  },
  {
    "caption": "C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed G spectrophotometric quantification of proteoglycan content by Alcian blue staining. p values were determined by Kruskal-Wallis test with Dunn's post-hoc test; (n=8).",
    "molecules": "Alcian blue, proteoglycan"
  },
  {
    "caption": "E, F Osteoarthritis severity assessed using (E) OARSI scoring system 10 weeks after MLI; GFP (n= 7), GCP-2 (n=8) and GCP-2-T (n=7); p values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means. F representative image (Safranin O staining) for each treatment; arrows indicate the tidemark;",
    "molecules": "Safranin O"
  },
  {
    "caption": "FMR1 mRNA levels were analyzed via RT-qPCR in EBV-transformed lymphocyte cells derived from the patient's blood samples. Patient cells showed a ˜60% decrease in FMR1 mRNA compared to control cells (*P = 0.010, 0.383 ± 0.110 SD, n = 3). Treatment with translational blocker puromycin restored FMR1 mRNA levels in patient cells (*P = 0.048, 0.383 ± 0.110 SD versus 1.25 ± 0.166 SD, n = 3), suggesting that the reduction of FMR1 mRNA in these cells is primarily due to nonsense-mediated decay. RT-qPCR reactions were run in triplicate in three independent experiments. Fold changes in FMR1 expression, normalized to HRPT expression, were calculated using the ΔΔCT method, and analyzed statistically with a two-tailed t-test (GraphPad). Error bars represent mean values with SD.",
    "molecules": "puromycin"
  },
  {
    "caption": "Transfected HEK293 cells expressing GFP-tagged wild-type FMRP fused to the patient NLS motif (GFP-FMR1wt+NLS). Unlike the patient FMRP, GFP-FMR1wt+NLS protein is predominantly cytoplasmic and does not aggregate in the nucleus. However, treatment of HEK293 cells expressing GFP-FMR1wt+NLS with leptomycin B-an inhibitor of nuclear export-resulted in the appearance of nucleolar inclusions. This suggests that the presence of a full-length C-terminus facilitates the nuclear export of FMRP in this context.Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP, in which the truncation of the C-terminus is reverted by restoring the open reading frame (GFP-FMR1G-ins.[Ct restored]). The patient protein is not detected in the nucleus when the C-terminus is intact; however, nucleolar retention of the protein is observed upon leptomycin B treatment of the transfected cells. In line with results from (A), this suggests that an intact C-terminus enables nuclear export of the FMR1 protein.Data information: Scale bar represents 10 μm.",
    "molecules": "leptomycin B"
  },
  {
    "caption": " B. Representative traces of the forward DNA unzipping in the presence of dSaCas9/sgRNA showing the force versus the number of unzipped base pairs. The naked DNA unzipping signatures are also presented for comparison (gray). Insets: zoomed-in view of the regions with increases in force. The dashed lines illustrate the interaction sites between dSaCas9 and the DNA. Black arrows indicate the force peaks of the pre-PAM interaction. C. Representative traces of the reverse DNA unzipping in the presence of dSaCas9/sgRNA showing the force versus the number of unzipped base pairs. Insets: zoom-in view of the regions with increases in force. The dashed lines illustrate the interaction sites between dSaCas9 and the DNA. Black and blue arrows indicate the force peaks of the pre-PAM and post-PAM interactions, respectively. ",
    "molecules": "DNA"
  },
  {
    "caption": " D. Positions and strengths of the two interactions between dSaCas9 and DNA. The black and blue triangles indicate the pre- and post-PAM interactions, respectively. In total, 27 and 34 traces were collected in the forward and reverse unzipping assays, respectively. pre-PAM interactions (black) were detected in both the forward and reverse DNA unzipping assays. The error bars represent the SD. ",
    "molecules": "DNA"
  },
  {
    "caption": " E. Exonuclease III footprinting of dSaCas9 on the DNA target with Cy5 labeled on the 5' end of the nontarget strand. F. Exonuclease III footprinting of dSaCas9 on the DNA target with Cy5 labeled on the 5' end of the target strand. ",
    "molecules": "Cy5, DNA"
  },
  {
    "caption": "  A. DNA unwinding by DnaB was initiated from the upstream side of the PAM in the presence of dSaCas9 (n = 10). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. For clarity, the traces have been shifted along the time axis. The dashed lines indicate the expected dSaCas9 binding positions. B. DNA unwinding by DnaB was initiated from the downstream side of the PAM in the presence of dSaCas9 (n = 11). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. C. DNA unwinding by BLM was initiated from the upstream side of the PAM in the presence of dSaCas9 (n = 12). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. D. DNA unwinding by BLM was initiated from the downstream side of the PAM in the presence of dSaCas9 (n = 14). Representative traces show the number of unwound base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. E. Phi29 DNAP strand-displacement synthesis was initiated from the upstream side of the PAM (n = 15). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9. F. Phi29 DNAP strand-displacement synthesis was initiated from the downstream side of the PAM (n = 19). Representative traces show the number of unwound/synthesized base pairs versus the time under an assisting force of 12 pN in the absence (black) or presence (red) of the prebound dSaCas9.  ",
    "molecules": "DNA"
  },
  {
    "caption": " Schematic representation of the sequences of the DNA target and sgRNAs. The PAM is shown in yellow. The nontarget DNA strand was labeled with Cy5. The matched and mismatched sequences in sgRNAs are shown in purple and blue, respectively. Representative gel shows DNA cleavage by SaCas9 guided by partially matched sgRNAs. ",
    "molecules": "Cy5, DNA"
  },
  {
    "caption": " A. DNA binding fractions by dSaCas9 at the fully matched (target) and partially matched DNA target positions, as revealed by the reverse DNA unzipping assays (n = 15, 16, 22, 26 and 46 from left to right). The subscript "MM" represents the mismatches of the DNA sequence with the sgRNAs. ",
    "molecules": "DNA"
  },
  {
    "caption": " B. Positions and strengths of the pre-PAM interactions (n7-22MM = 21, n11-22MM = 41, n15-22MM = 39) and post-PAM interactions (n7-22MM = 21, n11-22MM = 25, n15-22MM = 45) between dSaCas9/sgRNA and mismatched DNA targets. The error bars represent the SD. ",
    "molecules": "DNA"
  },
  {
    "caption": " C. Schematic depiction of the stopped-flow experiment. 2-AP was substituted at position +18 of the nontarget strand. The 2-AP fluorescence signals as a function of time are shown for four sgRNAs. The solid lines are double-exponential fits of the data ",
    "molecules": "2-AP"
  },
  {
    "caption": " E. Cleavage activities of SaCas9 on DNA targets with sgRNA containing bases mismatched to DNA. The reactions were quenched at five time points (0, 15, 30, 45, and 60 min). The experiment was performed in triplicate, and a representative gel is shown. ",
    "molecules": "DNA"
  },
  {
    "caption": " A. Representative traces of the reverse DNA unzipping in the absence (gray) and presence (blue) of wild-type SaCas9 showing the force versus number of base pairs unzipped. In total, 23 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA. The blue arrow indicates the force peak of the post-PAM interaction. B. Representative traces of the forward DNA unzipping in the absence (gray) and presence (black) of wild-type SaCas9 showing the force versus number of base pairs unzipped. In total, 49 traces were collected in this assay. The dashed line shows the expected interaction sites between dSaCas9 and DNA.",
    "molecules": "DNA"
  },
  {
    "caption": " C. Confocal images of fluorescently labeled λ DNA with dSaCas9 (n = 11) and wild-type SaCas9 (n = 16) bound to its DNA target. DNA-bound SaCas9 was visualized by labeling the 5' end of the crRNA with Cy3. The λ DNA molecule was either suspended between two microspheres held by two optical traps under 5 pN or stretched by laminar flow. The fluorescence intensity of Cy3 alongside the image is shown to indicate the on-target binding of SaCas9. ",
    "molecules": "Cy3, DNA, λ DNA"
  },
  {
    "caption": "
 D. DNA unwinding by DnaB was initiated from the downstream side of the PAM after the association of the SaCas9 protein (n = 11). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN. For clarity, the traces have been shifted along the time axis. The dotted lines indicate the expected SaCas9 binding positions. E. Phi29 DNAP-mediated strand-displacement synthesis was initiated from the downstream side of the PAM after the association of the SaCas9 protein (n = 13). Representative traces show the number of unwound/synthesized base pairs versus time under an assisting force of 12 pN. F. DNA unwinding by BLM was initiated from the downstream side of the PAM after the association of the SaCas9 protein (n = 23). Representative traces show the number of unwound base pairs versus time under an assisting force of 12 pN. 
",
    "molecules": "DNA"
  },
  {
    "caption": "(B-E)  B16F0 knockout created with Homology-Directed Repair approach with controls for  puromycin selection (B16F0-Ctr) created with pBabe-puro retrovirus. YUMM1.7  knockout created with double nickase approach with KO1 and KO2 indicating two different clones. Wildtype (WT: red)  B16F0 (B,C) and  YUMM1.7 (D,E) cells and CCN4  knockout variants (KO1: blue, KO2: black) were subcutaneously injected into (B,D) C57BL/6 immunocompetent and (C,E) NSG immunocompromised mice. Tumor volumes measured by caliper as a function of  time after tumor challenge, with n expressed as the number of mice with tumors over the number of mice injected.",
    "molecules": "puro, puromycin"
  },
  {
    "caption": "(K) Kaplan-Meier summary of the fraction tumor free in a 2x2 factor experimental design (n = 5 / group), where Tet-on vector control (red) versus inducible mCCN4 vector (black) and in the presence (dotted lines) or absence (solid lines) of doxycycline were the two factors.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(M) Representative flow cytometry data of the frequency of tumor-infiltrating  MDSC (GR1+ CD11c−/lo : dotted box) from the live  CD45+ CD11b+ compartment isolated from  CCN4-induced rescue (ID mCCN4 +  Dox) and CCN4 knockout (YM1.7-KO1) YUMM1.7 tumors.",
    "molecules": "Dox"
  },
  {
    "caption": "(A) ELISpot  for  IFNγ  release by CD8+ T cells using parental YUMM1.7 and CCN4-KO YUMM1.7  (KO1) cells as targets and different amount of in vivo activated CD8+ T cells. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001. Results of three biological replicates summarized as mean ± SEM. (B) ELISpot for IFNγ release by in vivo activated CD8+ T cells  with CCN4-inducible cells as targets in the presence or absence of 0.5 mg/ml doxycycline. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001. Results of biological replicates summarized as mean ± SEM.",
    "molecules": "doxycycline"
  },
  {
    "caption": "(C) CD8+ T cells isolated from the  spleens of C57BL/6 mice that rejected  YUMM1.7  tumors were assayed by in vitro ELISpot using variants of the YUMM1.7 cell line as targets (WT YUMM1.7 (Ym1.7) - yellow, CCN4-KO YUMM1.7 (Ym1.7-KO1)- light green,  CCN4-KO YUMM1.7 with a blank inducible expression vector (Ym1.7-KO1-IDvector) - dark green and blue, CCN4-KO YUMM1.7 with a CCN4 inducible expression vector (Ym1.7-KO1-IDmCCN4) - purple and red). Representative images shown under indicated condition with scale bar indicating 1 mm. Variants containing the inducible expression vector were also cultured in the absence (dark green and purple) or presence of 0.5 μg/ml  doxycycline (blue and red).  CD8+ T cells expressing IFNγ  and TNFα were quantified following 24 hour co-culture (bar graph). Results shown as mean ± S.D. for three biological replicates. Statistical significance was assessed using a Student's t-test with results annotated with ***: p<0.001 and n.s.: p>0.05.",
    "molecules": "doxycycline"
  },
  {
    "caption": "A, Representative images of DIV 3+2 cortical neurons transfected with FlucSM-EGFP (green) and subjected to the indicated treatments: 5 µM MG-132 for 4 hours; 10 nM Bafilomycin A1 (BafA1) for 24 hours; 0.5 µM 17-AAG for 4 hours; heat shock at 43°C for 30 min. Nuclei were labeled with DAPI (blue). Insets show higher magnification of the areas outlined by the boxes.",
    "molecules": "BafA1, Bafilomycin A1, DAPI, MG-132, 17-AAG"
  },
  {
    "caption": "D, DIV 3+2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q97-mCherry (magenta) and treated with 1 mM 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cells were stained for MAP2 (grey) as a neuronal marker, nuclei were labeled with DAPI (blue). Arrowhead points to Fluc-EGFP foci. Corresponding cultures transfected with control HTT are shown in Fig. EV1D. E, Quantification of the fraction of double-transfected cells showing Fluc-EGFP foci. N=4 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, **p=0.0046; 4-PBA, n.s.; HTT x 4-PBA, n.s. Significant pairwise comparisons are indicated on the graph. D",
    "molecules": "4-PBA, DAPI"
  },
  {
    "caption": "C, Left, Western blot of acute brain slice lysates from Fluc-EGFP mice and non-transgenic littermates. The slices were treated with 1 mM 4-PBA or vehicle control, and subjected to heat shock at 43°C for 15 min, as indicated above the blot. Middle, quantification of specific luciferase activity of Fluc-EGFP upon indicated treatments, normalized to vehicle-treated slices kept at 37°C. Right, quantification of Fluc-EGFP protein levels in the indicated conditions. N=3 mice. Colored asterisks indicate comparisons to the corresponding vehicle-treated 37°C control group (one-sample t-test), black asterisk indicates comparison between 4-PBA and vehicle-treated heat shock groups (two-tailed t-test).",
    "molecules": "4-PBA"
  },
  {
    "caption": "A, Cortical sections from rTg4510:Fluc-EGFP mice (right) and control littermates (left) stained for total human tau (HT7, magenta) and p-tau (AT8, yellow). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were labeled with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes, with neuronal cell bodies outlined. Note the presence of EGFP foci (arrowheads) in the majority of neurons in rTg4510:Fluc-EGFP mice. Occasional foci of lower relative fluorescence intensity can be observed in control mice (arrow).",
    "molecules": "DAPI"
  },
  {
    "caption": "A, Cortical (upper row) and striatal (lower row) sections from 12-week-old R6/2:Fluc-EGFP mice (right) and control WT:Fluc-EGFP littermates (left) stained for aggregated mHTT (EM48, magenta). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were counterstained with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes. Continuous and stippled lines mark cell bodies and nuclei, respectively.",
    "molecules": "DAPI"
  },
  {
    "caption": "A, B, Primary cortical neurons transfected with cyt-Fluc-EGFP (A) or nuc-Fluc-EGFP (B) (green), in combination with nuc-mHTT (upper rows) or cyt-mHTT (lower rows) (magenta). Cells were fixed at DIV 3+2 and stained for the neuronal marker MAP2 (grey). Nuclei were labeled with DAPI (blue). Arrowheads point to Fluc-EGFP foci. Dashed lines mark the nuclei. Schemes on the right summarize the cellular distribution of the respective Fluc-EGFP (green) and mHTT (magenta) proteins. Corresponding cultures transfected with control HTT are shown in Appendix Fig. S3A.",
    "molecules": "DAPI"
  },
  {
    "caption": "B Identification of RabGGTβ as a binding protein of PTAR1. Anti-FLAG immunoprecipitates from control HeLa S3 cells or FLAG-PTAR1-expressing HeLa S3 cells were analyzed by SDS-PAGE and silver staining. The 35 kDa protein was identified as RabGGTβ by mass spectrometry. The asterisk denotes a degradation product of FLAG-PTAR1.",
    "molecules": "silver"
  },
  {
    "caption": "C Identification of Ykt6 as a protein biotin-geranylated by GGTase-III. Cytosolic proteins extracted from statin-treated HeLa S3 cells were applied to GST or GST-GGTase-III affinity columns. Bound proteins were eluted and incubated with recombinant GGTase-III and BGPP. Reaction products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase (HRP)-labeled avidin to detect biotinylated proteins. Mass spectrometry identified the 25 kDa protein as Ykt6.",
    "molecules": "biotin, statin"
  },
  {
    "caption": "D Biotin-geranylation of recombinant Ykt6 by GGTase-III. Bacterially produced recombinant untagged Ykt6 (left) was incubated with buffer, GGTase-III, or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC. Biotin-geranylated Ykt6 was detected",
    "molecules": "Biotin"
  },
  {
    "caption": "C Farnesylation of recombinant Ykt6 by FTase. Recombinant Ykt6 (5 μM) was incubated with FTase (0 or 50 nM) and 3H-FPP (1 μM; ~3,500 dpm/pmol) at 37ºC. Reactions were stopped at the indicated time points, and the amount of 3H-farnesyl transferred to Ykt6 was quantified by scintillation counting (mean ± SEM, n = 3).",
    "molecules": "FPP, farnesyl, 3H"
  },
  {
    "caption": "D Geranylgeranylation of recombinant Ykt6 by GGTase-III. Recombinant Ykt6 (5 μM) was incubated with increasing concentrations of GGTase-III (0-100 nM) and 3H-GGPP (1 μM; ~3,000 dpm/pmol) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).",
    "molecules": "GGPP, geranylgeranyl, 3H"
  },
  {
    "caption": "E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).",
    "molecules": "GGPP, Cys, geranylgeranyl, Ser, 3H"
  },
  {
    "caption": "Effects of C-terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase-III. Purified recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, and Cys195-farnesyl Ykt6ΔAIM (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified. (mean ± SEM, n = 3).",
    "molecules": "GGPP, farnesyl, geranylgeranyl, 3H"
  },
  {
    "caption": "I Gel filtration chromatography of purified unprenyl Ykt6, Cys195-farnesyl Ykt6ΔAIM, and Cys194/195-diprenyl Ykt6ΔAIM (135 μg each) on a Superdex 200 column. Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison. The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6, suggesting more compact and stable folds.",
    "molecules": "farnesyl"
  },
  {
    "caption": "E Geranylgeranylation activity of WT GGTase-III and the indicated mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).",
    "molecules": "GGPP, farnesyl, 3H"
  },
  {
    "caption": "F Kinetic analysis of WT GGTase-III and the site 1 or site 2 mutant (100 nM each) using increasing concentrations of Cys195-farnesyl Ykt6ΔAIM (mean ± SEM, n = 3 for WT and Y306A, n = 1 for V48A/V50A).",
    "molecules": "farnesyl"
  },
  {
    "caption": "Geranylgeranylation of unprenyl WT Ykt6 and the site 1 or site 2 mutants by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM), and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).",
    "molecules": "GGPP, geranylgeranyl, 3H"
  },
  {
    "caption": "H Geranylgeranylation activity of WT GGTase-III and tunnel mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).",
    "molecules": "GGPP, farnesyl, 3H"
  },
  {
    "caption": "I GGPP binding to GGTase-III. WT GGTase-III and tunnel mutants (1 μM each) were incubated with 3H-GGPP (2 μM) at 4ºC for 10 min. After desalting, enzyme-bound 3H-GGPP was quantified by scintillation counting (mean ± SEM, n = 3).",
    "molecules": "GGPP, 3H"
  },
  {
    "caption": "B Prenylation status of rat brain Ykt6. Recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, Cys194/195-diprenyl Ykt6 samples (1 ng each), and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate (DOC)-containing buffer and analyzed by immunoblotting with anti-Ykt6 antibody (DOC-PAGE; upper panel). The same samples were analyzed by conventional SDS-PAGE and immunoblotting with anti-Ykt6 antibody (SDS-PAGE; lower panel).",
    "molecules": "deoxycholate, DOC, farnesyl"
  },
  {
    "caption": "D In vitro reconstitution of Ykt6 double prenylation. Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37ºC for the indicated times with recombinant GGTase-III (100 nM) or RabGGTase (100 nM) in the absence or presence of GGPP (10 μM). After incubation, reaction products were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.",
    "molecules": "GGPP"
  },
  {
    "caption": "Defect in intra-Golgi trafficking in PTAR1 KO cells. (D) VSVG-GFP expressing WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were cultured at 40ºC and then shifted to 32ºC. At the indicated time points, cell surface proteins were biotinylated using sulfo-NHS-LC-biotin. Biotinylated VSVG-GFP was purified from cell lysates using avidin agarose and analyzed by immunoblotting with anti-GFP antibody. The right panel shows quantification of the cell surface biotinylated VSVG-GFP (means ± SEM, n = 3). Data were analyzed by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, ***p < 0.001.",
    "molecules": "agarose, sulfo-NHS-LC-biotin"
  },
  {
    "caption": "E Analysis of LAMP1 glycosylation. Cell lysates of WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were analyzed by immunoblotting with anti-LAMP1 antibody (upper). Sialylated LAMP1 was precipitated from the cell lysates using Maackia amurensis leucoagglutinin (MAL) agarose and analyzed by immunoblotting (lower).",
    "molecules": "agarose"
  },
  {
    "caption": "F Defect in the Golgi SNARE assembly in PTAR1 KO cells. The Golgi SNARE complex was immunoprecipitated from NEM-treated WT HAP1 cells, PTAR1 KO HAP1 cells, and PTAR1 KO HAP1 cells stably expressing PTAR1 (KO + PTAR1) using control mouse IgG, anti-GS28 IgG, or anti-syntaxin 5 IgG. Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5, GS28, GS15, and Ykt6. Syntaxin 5 has two isoforms with different translation initiation sites. Inputs were 20% (syntaxin 5, GS28, and GS15) and 2% (Ykt6). The data shown are representative of three independent experiments with similar results.",
    "molecules": "NEM"
  },
  {
    "caption": "Micrographs of S2 cells stained with PicoGreen, following 5 days of treatment with the dsRNA indicated. GFP and tamas (Polg, CG8987) were used as negative and positive controls, respectively. In rescreening, CG5924 was also used as a positive control. Both were detected in the blinded screen as positives. Other images show a typical negative (CG31380), a typical positive (CG6413) and a typical case of a target classed as abnormal, in this case CG2028 (CkIα), which showed a decreased number of nucleoid signals per cell. Images are optimized for brightness and contrast but with no other manipulations. Scale bar indicates 50 μm.",
    "molecules": "dsRNA, PicoGreen"
  },
  {
    "caption": "A-C Relative mtDNA level (A), TMRM (B) and MitoSox (C) following treatment for 5 days with dsRNA against the genes indicated.",
    "molecules": "MitoSox, TMRM, dsRNA"
  },
  {
    "caption": "E Whole cell respiration following treatment for 5 days with dsRNA against the genes indicated.",
    "molecules": "dsRNA"
  },
  {
    "caption": "F, G Relative mtDNA level (F) and MitoSox fluorescence (G) after 5 days of treatment with oligomycin.",
    "molecules": "MitoSox, oligomycin"
  },
  {
    "caption": "A‐D Relative mtDNA level (A, B) and MitoSox fluorescence (C, D) following 5 days of treatment with dsRNA against the genes indicated.",
    "molecules": "MitoSox, dsRNA"
  },
  {
    "caption": "E‐G Relative mtDNA level (E), MitoSox fluorescence (F) and TMRM fluorescence (G) of cells stably expressing AOX or empty vector, as indicated, after 5 days of treatment with dsRNA against CG2968, as shown. Note that these cells were cultured in the presence of hygromycin to maintain the AOX‐expressing or control plasmid.",
    "molecules": "MitoSox, TMRM, dsRNA"
  },
  {
    "caption": "H Indicated parameters, following 5 days of treatment with dsRNA against CG2968, in cells grown in galactose‐containing (Gal) medium.",
    "molecules": "dsRNA, galactose"
  },
  {
    "caption": "A. LysoTracker Red and NAO fluorescence of cells treated as indicated for 5 days with dsRNA against the indicated genes, normalized against the values for untreated cells. Mean ± SD for four independent experiments; asterisks indicate significant differences from untreated, P < 0.05.",
    "molecules": "NAO, dsRNA"
  },
  {
    "caption": "B. Representative confocal microscopy images of living cells treated for 5 days with dsRNA against the indicated genes and stained with both MitoTracker Green and LysoTracker Red. The scale bar is 10 μm.",
    "molecules": "dsRNA"
  },
  {
    "caption": "C. Mean areas of LysoTracker red staining in cells treated for 5 days with dsRNA against the indicated genes, or with chloroquine. Mean ± SD for three independent experiments, in each of which ≥ 50 cells were analyzed by confocal microscopy in a single plane. Analysis of z−stacks in ≥ 30 cells gave similar results. Note that the increase in lysosome content was intermediate between that of untreated cells and cells treated with chloroquine to block lysosomal turnover of autophagosomes. Asterisks indicate significant differences from untreated, P < 0.01.",
    "molecules": "chloroquine, dsRNA"
  },
  {
    "caption": "(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP, panels d, e, and f) for 30 min and then were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) in the continuous presence of dbcAMP. Cells without any treatment were used as control (−dbcAMP, panels a, b, and c). Cells were immediately analyzed by confocal microscopy. A quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown in the right panel (n = 100 cells/condition, * p<0.05 paired Student's t-test). These data are representative of three independent experiments.",
    "molecules": "dbcAMP, dybutiryl cAMP"
  },
  {
    "caption": "(B) GFP-LC3 CHO cells were incubated with complete medium in the absence (−dbcAMP) or presence of dbcAMP (+dbcAMP) and treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) with (lower panel) or without (upper panel) bafilomycin A1 to block lysosomal degradation. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of three independent experiments. (C) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).",
    "molecules": "bafilomycin A1, dbcAMP"
  },
  {
    "caption": "(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM H89, a PKA inhibitor, in the presence (panels a, b, and c) or absence (panels d, e, and f) of 1 mM dbcAMP. Subsequently, they were incubated for 2 h in starvation medium (Stv, panels b and e) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels c and f) in full nutrient media. Cells without any treatment were used as control (Ctr, panels a and d). Cells were finally analyzed by confocal microscopy. Images are representative of three independent experiments.",
    "molecules": "dbcAMP, H89, nutrient"
  },
  {
    "caption": "(C) GFP-LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89, and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-phosphoCREB and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantified with the Adobe Photoshop program (lower panel). These data are representative of two independent experiments.",
    "molecules": "dbcAMP, H89"
  },
  {
    "caption": "(A) GFP-LC3 CHO cells were preincubated with 10 µM 8-pCPT-2′-O-Me-cAMP (8-pCPT) for 30 min and then they were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv). Cells without any treatment were used as control. Cells were immediately analyzed by confocal microscopy. Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel (n = 50 cells/condition). These data are representative of two independent experiments.",
    "molecules": "8-pCPT, 8-pCPT-2′-O-Me-cAMP"
  },
  {
    "caption": "(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (left panel) or GFP-Rap2b ΔAAX (right panel). Twenty-four hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) in full nutrient media, or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of three independent experiments.",
    "molecules": "nutrient, rapamycin"
  },
  {
    "caption": "(C) CHO cells were transfected with GFP-Rap2b wt or GFP-Rap2b ΔAAX and incubated with complete medium without (Ctr) or with 10 µg/ml of α-hemolysin (Hla) for 4 h, with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h in the absence or presence of bafilomycin A1. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.",
    "molecules": "HRP, bafilomycin A1, rapamycin"
  },
  {
    "caption": "(A) HeLa cells were cotransfected with Rap2b siRNA and GFP-LC3 (right panel) or irrelevant siRNA and GFP-LC3 (left panel). Forty-eight hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "(D) HeLa cells were cotransfected with GFP-LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence (Ctr) or presence of 10 µg/ml of α-hemolysin (Hla), with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.",
    "molecules": "rapamycin"
  },
  {
    "caption": "cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.",
    "molecules": "dbcAMP"
  },
  {
    "caption": "(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin (a calpain inhibitor, panels b, f, and j), with 1 mM dbcAMP (panels d, h and l), or with Calpeptin+dbcAMP (panels c, g, and k). Afterwards, they were incubated for 2 h in starvation medium (Stv, panels e, f, g, and h) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels i, j, k, and l). Cells without any treatment were used as control (Ctr, panels a, b, c and d). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. * p<0.05 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.",
    "molecules": "dbcAMP, calpeptin"
  },
  {
    "caption": "(C) Upper panel: CHO cells were preincubated for 30 min with 10 µM of calpeptin, with 1 mM dbcAMP, or with calpeptin+dbcAMP, and then they were incubated for 4 h in complete medium in the presence (Hla) or absence (Ctr) of α-hemolysin. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. Lower panel: Quantification of the band intensities with the Adobe Photoshop program. * p<0.05 (paired Student's t-test). These data are representative of three independent experiments.",
    "molecules": "dbcAMP, calpeptin"
  },
  {
    "caption": "(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−) +pHla). Cells without any treatment were used as control (−dbcAMP). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods, and immediately visualized by confocal microscopy. Images are representative of two independent experiments.",
    "molecules": "dbcAMP, dybutiryl cAMP"
  },
  {
    "caption": "Pre-enriched thymocytes from WT BALB/c thymi as in (a) were labeled by CFSE (CFSE+), mixed with pre-enriched thymocytes from SKG thymi (CFSE-), loaded with Indo-1, and stimulated as in (a). Ratio of Indo (Violet) and Indo (Blue) was analyzed by flow cytometry. Comparison of cytoplasmic Ca2+ profiles between WT and SKG NKT1 (c) and NKT2 (d) cells in the same test tube are shown. Data are representative from one of at least three independent experiments. Graphs represent the average Ca2+ levels.",
    "molecules": "CFSE, Ca2+, Indo, Indo-1"
  },
  {
    "caption": "Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet, and activated by anti-CD3ε/CD28 Dynabeads for 2 days, in the presence of different [EGTA] in the medium. CellTrace Violet/CTV signal in living cells at the end of the culture is shown. Cell proliferation was quantified as division index based on CTV signals in (a).",
    "molecules": "CellTrace Violet, EGTA"
  },
  {
    "caption": "Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. EGTA was added 48h after the start of culture (day 2), and the expression of CXCR3 (g), PLZF (h) and PD1 (i) in total iNKT cells are shown.",
    "molecules": "EGTA"
  },
  {
    "caption": "Confocal images of Tim 23 (Alexa Fluor 488, green) and MitoTracker deep red (Alexa Fluor 647, red) in freshly isolated thymic NKT1 (c1, 2) and NKT2 (c5, 6) cells using Airyscan technology. 3D images reconstructed by Imaris software in NKT1 (c3, 4) and NKT2 cells(c7, 8). Dotted line in panel (5-8) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells.",
    "molecules": "Alexa Fluor 488, Alexa Fluor 647, MitoTracker deep red"
  },
  {
    "caption": "Maximal cytoplasmic [Ca2+] in single, freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 (1uM);",
    "molecules": "BTP2, Ca2+"
  },
  {
    "caption": "BTP2 (1uM) was added 48h after the start of culture, and the expression of Tbet in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).",
    "molecules": "BTP2"
  },
  {
    "caption": "BTP2 (1uM) was added 48h after the start of culture, and the expression of CXCR3 (d), PLZF (e), and PD1 (f) in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).",
    "molecules": "BTP2"
  },
  {
    "caption": "Profiles showing average Ca2+ influx induced by Thapsigargin (TG) in freshly sorted NKT1 (Red) and NKT2 (Black) cells. The timing of TG (1uM), ionomycin (Iono) (2uM) and CCCP (1uM) addition, and extracellular [Ca2+] in mM are shown.",
    "molecules": "Ca2+, CCCP, ionomycin, TG, Thapsigargin"
  },
  {
    "caption": "B Measured free PF-670 exposure in the brain tissue of mice and NHPs. Drug exposure in NHPs after administration of 10 mpk PF-670 (AUC=3.6μMh) is ~7-fold higher than that in mice given 32 mpk PF-670 (AUC=0.5 μMh) (mean±SEM).",
    "molecules": "PF-670"
  },
  {
    "caption": "C, D Double-plotted actograms of NHPs' activity for 16 days. NHPs were treated with 10 mpk PF-670 for 3 consecutive days at the same solar time of day: 4 pm for the DD experiment (C) and 4:30 pm (ZT11) for the LD experiment (D), respectively, which are highlighted as red lines. White and black rectangles indicate the times of light going on and off (LD 12:12).",
    "molecules": "PF-670"
  },
  {
    "caption": "E, F Phase delay induced by the 3-day dosing under DD (E) or LD (F). The phase of activity onset in NHPs (10 mpk) is more delayed than that in mice (32 mpk) under DD (E), but not under LD (F) (P=0.03, one-way analysis of variance (ANOVA); n.s., no significant difference). Veh denotes vehicle. The error bars represent mean±SEM. The mouse data are adopted from (Kim et al, 2013). G The quantification of (E) and (F) indicates that light has a stronger attenuating effect on the PF-670-induced phase delay in NHPs than in mice (P=0.036, two-way ANOVA). ",
    "molecules": "PF-670"
  },
  {
    "caption": "E NHPs were treated with 10 mpk PF-670 for 3 consecutive days at ZT4 in LD 12:12, which are highlighted as the red line.",
    "molecules": "PF-670"
  },
  {
    "caption": "E Consistently, higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF-670-induced phase delay which was also captured by the NHP model when 20 mpk dosing was used.",
    "molecules": "PF-670"
  },
  {
    "caption": "Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times.",
    "molecules": "Baf A1, bafilomycin A1, MG132"
  },
  {
    "caption": "Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In E, endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.",
    "molecules": "Baf A1, bafilomycin A1, MG132"
  },
  {
    "caption": "G Same cells as in E were left untreated or treated with Baf A1 for 6 hours, and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.",
    "molecules": "Baf A1"
  },
  {
    "caption": "J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.",
    "molecules": "Baf A1"
  },
  {
    "caption": "A GST pulldown binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by coomassie brilliant blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR .",
    "molecules": "35S"
  },
  {
    "caption": "GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.",
    "molecules": "35S"
  },
  {
    "caption": "GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.",
    "molecules": "35S"
  },
  {
    "caption": "GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.",
    "molecules": "35S"
  },
  {
    "caption": "GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.",
    "molecules": "35S"
  },
  {
    "caption": "GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.",
    "molecules": "35S"
  },
  {
    "caption": "GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right).",
    "molecules": "35S"
  },
  {
    "caption": "B GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins",
    "molecules": "35S"
  },
  {
    "caption": "C GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 and 35S-Myc-p62 with recombinant GST-GABARAPL2 (WT and indicated mutants).",
    "molecules": "35S"
  },
  {
    "caption": "D GST pulldown assays of in vitro transcribed/translated 35S-Myc-TAX1BP1 (WT and indicated mutants) with recombinant GST-GABARAP (WT and indicated mutants).",
    "molecules": "35S"
  },
  {
    "caption": "Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691. Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In F, the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, *p ˂ 0.01; ns is not significant.",
    "molecules": "Baf A1, MG132, tetracycline"
  },
  {
    "caption": "G HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm.",
    "molecules": "Baf A1"
  },
  {
    "caption": "C, D GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-VAPA or GST-VAPB constructs.The scheme is a representation of CALCOCO1 domains and motifs.",
    "molecules": "35S"
  },
  {
    "caption": "A, B Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.",
    "molecules": "Baf A1, MG132"
  },
  {
    "caption": "D, E Immunoblot analysis of HeLa cells transfected with the indicated siRNAs and treated with Baf A1 as indicated. In D, the panels are from more than one western blot experiment but only a single GAPDH loading control is shown. In E, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005; ns is not significant.",
    "molecules": "Baf A1"
  },
  {
    "caption": "A, B Immunoblot analysis of HeLa WT and HeLa CALCOCO1 KO cells. The cells were starved for 6 hours (HBSS) as indicated and treated with Baf A1 as indicated. Numbers below the blots in A represent relative intensity of the bands in the shown blots normalized against GAPDH loading control. In A, the panels are collected from more than one western blot experiment but for clarity, only a single GAPDH loading control is shown. In B, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.",
    "molecules": "Baf A1"
  },
  {
    "caption": "C, D Immunoblot analysis of HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1. Expression of EGFP-CALCOCO1 was induced or not with tetracycline and the cells were treated with MG132 or Baf A1 as indicated. Numbers below the blots in C represent relative intensity of the bands in the shown blots normalized against actin loading control. In C, the panels are collected from more than one western blot experiment but only a single actin loading control is shown. In D, the bars represent the mean±sd of band intensities of three independent experiments as quantified using ImageJ. Statistical comparison was analyzed as in B and significance displayed as **p ˂ 0.005, *p ˂ 0.01; ns is not significant.",
    "molecules": "Baf A1, MG132, tetracycline"
  },
  {
    "caption": "E HeLa CALCOCO1 KO cell lines reconstituted with EGFP-CALCOCO1 were treated with tetracycline or not to induce expression of EGFP-CALCOCO1. Abundance of the ER was quantified from widefield fluorescence images of endogenous RTN3 staining (see Materials and methods). Data are presented as mean ± sd of three independent experiments. Statistical comparison was analyzed as in B and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.",
    "molecules": "tetracycline"
  },
  {
    "caption": "A Immunoblot analysis of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 in fed or starved conditions and treated as indicated with Baf A1 or PI3KC3 inhibitor SAR405.",
    "molecules": "Baf A1, SAR405"
  },
  {
    "caption": "D Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf discs treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~200 µmol m-2 s-1) for 3 days after agroinfiltration and then leaf circles were treated with W+FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2-way ANOVA: * p-value <0.05) between ChHFR1 and AtHFR1 at the same time point.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "F Relative chlorophylls levels of (left) AtWT, pifq, ChWT, (middle) ΔNtHFR1, hfr1-5 and (right) chfr1-1 and chfr1-2 lines after DIS was promoted for the indicated time. For each genotype, values are relative to pigment levels at time 0 (7 days in W). Data are the means ± SE of four independent biological replicates.",
    "molecules": "chlorophylls"
  },
  {
    "caption": "(A) NIH-3T3 cells were transduced with retroviral vectors expressing M45 or GFP. Two days after transduction cells were stimulated with the TLR4 agonist LPS (10 µg/ml), the TLR2 agonist LTA-SA (10 µg/ml), or IL-1β (20 ng/ml). IκBα levels were determined by immunoblotting.",
    "molecules": "LPS, LTA-SA"
  },
  {
    "caption": "(C) NIH-3T3 cells expressing an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 (Pam.,1 µg/ml), the TLR4 agonist LPS (0.1 µg/ml), or IL-1β (5 ng/ml). SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non-stimulated cells (mean ± SD)",
    "molecules": "Pam3CSK4, LPS"
  },
  {
    "caption": "(A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting.",
    "molecules": "Pam3CSK4, LPS"
  },
  {
    "caption": "(B) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD).",
    "molecules": "R848"
  },
  {
    "caption": "(C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).",
    "molecules": "Pam3CSK4, CpG, brefeldin A, R837, LPS, Malp-2"
  },
  {
    "caption": "(D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).",
    "molecules": "CpG, brefeldin A, R838"
  },
  {
    "caption": "(D) NIH-3T3 cells were mock infected or infected with RM45 (MOI of 5) and treated 2 hpi with 10 mM ammonium chloride (NH4Cl) or a mix of lysosomal protease inhibitors (PI-mix). 9 hpi, levels of IKK subunits, M45, IE1, and actin were determined by immunoblotting. (",
    "molecules": "ammonium chloride, NH4Cl"
  },
  {
    "caption": "(E) NIH-3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP, and treated for the indicated times with PI-mix, 10 mM NH4Cl, or 5 µM lactacystin, respectively. Levels of the indicated proteins were analyzed by immunoblotting.",
    "molecules": "NH4Cl, lactacystin"
  },
  {
    "caption": "(B) 10.1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5. Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3-II. Cells were harvested at the indicated time points, and LC3-II levels in cell lysates were analyzed by immunoblotting using an LC3-II-specific antibody. LC3-II levels were quantified by densitometric analysis and normalized using the corresponding actin levels. Fold increases are shown relative to mock-infected cells.",
    "molecules": "NH4Cl"
  },
  {
    "caption": "Quantification of mitotic defects as in (B) of H2B-mNeon-expressing HeLaEGFP-AID-CENATAC cells treated as indicated (three or five biological replicates, >85 cells in total per condition). For 2xZF the four zinc-finger cysteines were mutated to alanines; for Δ1-4 the corresponding motif was removed.",
    "molecules": "alanines, cysteines, mNeon"
  },
  {
    "caption": "Representative stills of HeLaEGFP-AID-CENATAC cells expressing H2B-mNeon and depleted of CENATAC. Microtubules were visualized with SiR-Tubulin. Arrowheads and arrows indicate non-congressed chromosomes and supernumerary spindle poles, respectively. Scale bar, 5 μm. Time in hours. See Figure EV2 for the control condition. See also Movies EV1 and EV2. IAA, 3-indoleacetic acid.",
    "molecules": "3-indoleacetic acid, IAA, mNeon, SiR"
  },
  {
    "caption": "C. RBM3 E3a regulation is conserved in humans. HEK293 cells were incubated at the indicated temperatures for 12h (DMSO/CHX last 4h) and investigated for E3a inclusion as in B (mean ± s.d., n= 3, all individual data points are shown).",
    "molecules": "CHX, DMSO"
  },
  {
    "caption": "D. ASOs targeting M2-9, M4-7 or the 5'ss (see Figure EV4A and Table EV2) prevent endogenous RBM3 E3a inclusion in human HeLa cells. ASO-transfected cells were kept for 24 hours at 40°C. Control samples at 37°C and 40°C are shown; CHX was added for the last 4 hours. Exon 3a inclusion was investigated by splicing sensitive RT-PCR, a representative gel and phosphorimager quantification are shown (mean ± s.d., n= 3). The hashtag marks the use of internal 5' and 3'ss that is promoted by all ASOs targeting the M4 region. ASOs targeting the 5'ss induced the usage of an internal 5'ss (marked by two asterisks).",
    "molecules": "ASO, ASOs, CHX"
  },
  {
    "caption": "M2D induces RBM3 mRNA (E) expression in human HeLa cells. ASOs were transfected for 24 hours at 37°C (grey) or at 40°C (red). RBM3 induction was measured relative to GAPDH expression (mean ± s.d., n= 3, all individual data points are shown).",
    "molecules": "ASOs"
  },
  {
    "caption": "M2D induces RBM3 protein (F) expression in human HeLa cells. ASOs were transfected for 24 hours at 37°C (grey) or at 40°C (red). RBM3 induction was measured relative to GAPDH expression (mean ± s.d., n= 3, all individual data points are shown).",
    "molecules": "ASOs"
  },
  {
    "caption": "G. M2D  induces RBM3 protein expression in vivo. Hippocampus samples from two mice per condition were analyzed by Western blotting (left) and RBM3 protein was quantified relative to actin and PBS (right, mean ± s.d., n= 2, all individual data points are shown).",
    "molecules": "PBS"
  },
  {
    "caption": "B. Western blot of hippocampal lysates from prion-infected mice treated with non-targeting control ASO (n= 5 mice) or M2D  (n= 8 mice). M2D  increases RBM3 expression by 2-fold compared to control ASO-treated mice, 9 weeks after ASO injection at 12 w.p.i. ** p= 0.0058, calculated using student's t-test.",
    "molecules": "ASO"
  },
  {
    "caption": "C. Representative images of haematoxylin and eosin stained brain slices from NBH (control), and prion-infected control ASO- and M2D-treated mice at 12 w.p.i. when control ASO-treated mice were culled for prion signs. M2D  confers marked neuroprotection in the hippocampus, with conservation of CA1-3 pyramidal layer, protection from shrinkage of the whole hippocampus, as well as reduced spongiform change.",
    "molecules": "ASO, eosin, haematoxylin"
  },
  {
    "caption": "D. NeuN counts of pyramidal neurons in NBH (n= 5 mice) versus control ASO-treated (n= 5 mice) and M2D-treated prion mice (n= 8 mice). M2D  confers neuroprotection close to levels seen in NBH mice. control ASO-treated versus M2D-treated prion mice ** p= 0.0014. One-way ANOVA.",
    "molecules": "ASO"
  },
  {
    "caption": "F. Total PrP and proteinase K-resistant PrPSc  levels in NBH-injected mice and in prion-diseased mice injected with control - or M2D ASOs. PrPSc levels are unaffected by M2D-mediated RBM3 induction.",
    "molecules": "ASOs"
  },
  {
    "caption": "(D-F) Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α-tubulin (Ace-α-tubulin) and γ-tubulin and DAPI. Scale bar, 2 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(K-M) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (L), >200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B-D) Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), >100 cells were analyzed for each experiment. Scale bar, 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), >150 cells were analyzed for each experiment. Scale bar, 5 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(I, J) Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), >80 cells were analyzed for each experiment. Scale bar, 2 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(K, L) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP-ENKD1, GFP-ENKD1-N, GFP-ENKD1-M, or GFP-ENKD1-C, cultured in serum-free medium, and stained with the antibody against acetylated α-tubulin and DAPI. The siRNA-resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), >120 cells were analyzed for each experiment. Scale bar, 3 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(G) Immunofluorescence images of RPE1 cells cultured in normal serum or serum-free medium, and stained with antibodies against ENKD1 and γ-tubulin and DAPI. Scale bar, 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(H) Immunofluorescence images of RPE1 cells cultured in serum-free medium, and stained with antibodies against ENKD1 and acetylated α-tubulin and DAPI. Scale bar, 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(I) Immunofluorescence images of RPE1 cells transfected with GFP-ENKD1 or GFP vector, cultured in a serum-starved condition, and stained with the antibody against acetylated α-tubulin and DAPI. Scale bar, 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Immunofluorescence images (B) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI.",
    "molecules": "DAPI"
  },
  {
    "caption": "(D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Decreased Drp1S616 phosphorylation in PINK1-null HEK293 cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) HEK293 cells (a) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO HEK293 cell lines (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "Ser"
  },
  {
    "caption": "Decreased Drp1S616 phosphorylation in PINK1-null MEF cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) MEF cells (c) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "Ser"
  },
  {
    "caption": "Decreased Drp1S616 phosphorylation in PINK1-null mouse substantial nigra tissues. Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) mouse substantial nigra tissues (e), and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO substantial nigra from two separate PINK1KO and parkinKO mice (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "Ser"
  },
  {
    "caption": "(g, h). Overexpression of PINK1, not PINK1 kinase mutants, reverses Drp1S616 phosphorylation in PINK1KO HEK293 cells. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells expressing PINK1 variants were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1) and an anti-PINK1 antibody (PINK1). Cells with mock transfection (Blank) and transfected with an empty plasmid (3.1) were included as controls (g). Quantitation of pS616/Drp1 is shown (h). PINK1: wildtype PINK1; D384N: PINK1 kinase-dead mutant; G309D, pathogenic PINK1 mutant; ∆110: PINK1 mito-target deficient mutant. Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "Ser"
  },
  {
    "caption": "(i, j). PINK1 deficiency does not affect expression and kinase activity of CDK1 and ERK1/2. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1), an anti-PINK1 antibody (PINK1), an anti-CDK1 antibody(CDK1), an anti-p44/p42 MAPK antibody (ERK1/2), an anti-phospho(Thr161)-CDK1 antibody (pThr161), and an anti-phospho(Thr202/204)-p44/p42 MAPK antibody (pThr202/204). β-actin was detected as a loading control (i). Quantitation of CDK1Thr161 phosphorylation (pThr161) and ERK1/2Thr202/204 phosphorylation (pThr202/204) was shown (j). Student's test. ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "Ser, Thr"
  },
  {
    "caption": "(l, m). PINK1 phosphorylates Drp1S616 in vitro. Recombinant TcPINK1 was incubated with either GST-Drp1518-736 (WT) or GST-Drp1518-736, S616A (S616A) in the presence (+)/absence (-) of ATP. After heat inactivation, reactions were treated with (+)/without (-) shrimp alkaline phosphatase (SAP). Phosphorylation of Drp1S616 (pS616) was immuno-detected (l, upper panel). Total Drp1 (l, middle panel) and protein loading stained with Coomassie blue staining (l, lower panel) were shown. Quantitation of pS616/Drp1 ratio was shown (m). One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "(n, o). Label-free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation. Representative spectra for the Drp1 phospho-peptide (S616) SKPIPMPA(p)SPQK and non-phospho-peptide SKPIPMPA(p)SPQK. Spectra obtained for peptides from PINK1WT HEK293 cells are shown (n). The relative ratios of the phospho-peptides containing phosphor-Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO HEK293 samples were calculated and plotted (o).",
    "molecules": "Ser"
  },
  {
    "caption": "(p, q). Km and Kcat for PINK1-mediated phosphorylation of ubiquitin and Drp1. In vitro kinase assays were performed with different concentrations of ubiquitin (p) or Drp1 (q) with TcPINK1 at fixed ATP concentration (500 μM). Results were analyzed on phos‐tag gels and modeled to the Michaelis-Menten equation. The given graphs represent global fits to data collected from 3 sets of reactions for both Ub and Drp1 performed independently. Km: michaelis constant. Kcat: catalytic constant. Bars represent the mean ± SEM.",
    "molecules": "ATP, Ub, ubiquitin"
  },
  {
    "caption": "(a, b). Decreased Drp1S616 phosphorylation in PINK1-null primary neurons. Lysates of DIV6 neurons generated from PINK1KO and PINK1WT control mice were immune-detected with antibodies against either phospho (Ser616)-Drp1 (pS616) or Drp1. Tuj1 was detected as a loading control (a). Quantitation of pS616/Drp1 for each experiment is shown (b). Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "Ser"
  },
  {
    "caption": "(a-c). Drp1 is required for mitochondrial fission induced by PINK1. Drp1-null HeLa cells (Drp1KO) and their wildtype controls (Drp1WT) were co-transfected Δ110-PINK1-GFP-FKBP with FRB-MTS. Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (Δ110-PINK1, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown (a, upper panel, scale bar=25 μm). Higher magnification images are also included (a, lower panel, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified. (b, c). Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.",
    "molecules": "rapalog, DAPI, DMSO"
  },
  {
    "caption": "(d-f). Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1. Drp1KO HeLa cells were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A. Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, blue), exogenous Drp1 (myc, red), Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm (d). Higher magnification images are also included (d, Zoom, scale bar=5 μm). Mitochondrial morphology in different transfections was quantified (e, f). Student's test. **p<0.01, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.",
    "molecules": "rapalog, DMSO"
  },
  {
    "caption": "(g, h). Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616. HEK293 cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP were treated with 250 nM rapalog (Rapalog). After 2hours treatment, cytosol protion (cyto) and mitochondria (Mito) were fractionated and immunobloted with antibodies against either phospho (Ser616)-Drp1 (pS616), Drp1 (Drp1), or GFP (PINK1). Tom20 and β-actin were detected as mitochondrial loading control and cytosol loading control, respectively (g). Quantitation of pS616/Drp1 in various conditions was shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "rapalog, Rapalog, Ser"
  },
  {
    "caption": "(i, j). Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1. HeLa cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP (FKBP-PINK1) were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cells were immunostained with antibodies against either Tom20, Drp1 and GFP (PINK1-FKBP). Representative images were shown, scale bar=2 μm (i). Higher magnification images from gated box are shown (bottom panels). Arrows indicate Drp1 puncta. Mitochondria-associated (yellow) and non-associated Drp1(Red) are shown, scale bar=1 μm. Mitochondria-associated Drp1 puncta were analyzed by using an ImageJ plugin-Coloc 2 and expressed as the Manders' Colocalization Coefficients (MCC) (j). Three different regions from each cell and >15 cells for each experimental group were analyzed. Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "rapalog, Rapalog, DMSO"
  },
  {
    "caption": "(k, l). PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms. DRP1KO HEK293 cells co-transfected FRB-MTS/Δ110-PINK1-GFP-FKBP (PINK1WT) with either myc-Drp1WT or myc-Drp1S616A were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cell lysates were immunoblotting with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho-(Ser637)-Drp1 (pS637), Drp1 and GFP (to detect PINK1-FKBP) (k). β-actin was detected as a loading control. Quantitation of pS637/Drp1 in various conditions was shown (l). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "rapalog, Rapalog, DMSO, Ser"
  },
  {
    "caption": "(a-e). ATG5 is dispensable for PINK1-mediated mitochondrial fission. MEF cells derived from ATG5-null (ATG5KO) and their wildtype control mice (ATG5WT) were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either PINK1WT or a kinase-dead mutant PINK1D384N. Cells were induced with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images are shown, scale bar=25 μm (a). Higher magnification images are also included (panels beneath the large cell images, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified (b-e). Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.",
    "molecules": "rapalog, Rapalog, DMSO"
  },
  {
    "caption": "(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 20 μM CCCP for 2 hours (f) followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.",
    "molecules": "CCCP, DAPI, DMSO"
  },
  {
    "caption": "(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 150 μM actinonin for 6 hours (h), followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.",
    "molecules": "actinonin, DAPI, DMSO"
  },
  {
    "caption": "(c) Drp1WT and Drp1S616D, but not Drp1S616A, restore abnormal ATP production in PINK1-null flies. ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "(h, i). Drp1WT and Drp1S616D, but not Drp1S616A, suppress cell death in PINK1KO flies. Representative TUNEL staining (Red) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Nuclei were counter-stained with DAPI (blue), scale bar=10 μm. Apoptotic cells (%) in different genotype flies were quantified (i). >5 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat.One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "DAPI"
  },
  {
    "caption": "(j). Expression and phosphorylation of hDrp1 in Drosophila. Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc-gal4 driven hDrp1WT (WT) and hDrp1S616A (S616A) were immunoblotted with antibodies against either phospho(Ser616)-Drp1(pS616) (to detect phosphorylated hDrp1S616), Drp1 (to detect both endogenous and exogenous Drp1), myc-tag (to detect exogenous Drp1). α-tubulin was detected as a loading control. Flies expressing mhc-gal4 (-) were included as an expression control.",
    "molecules": "Ser"
  },
  {
    "caption": "(e). Drp1 rescues PINK1KO induced ATP reduction in Drosophila. ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.",
    "molecules": "ATP"
  },
  {
    "caption": "(b) shows succinate  quantification by NMR  from tracheal  aspirates collected in mechanically-ventilated patients with (pink bar, n=9) or without (orange bar, n=7) diagnostic of IAV pneumonia. *P < 0.05. (c)  IL-6 and IL-8 measurements by ELISA  from these same tracheal  aspirates. Data information: Data are mean ± SEM and statistical analysis was performed using the Mann-Whitney U-test.",
    "molecules": "succinate"
  },
  {
    "caption": "8-week-old female C57Bl/6 mice  were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate  (Suc; by the intranasal route). Some mice  were euthanized at 4 days post-infection and lungs  were collected to determine: ; (b) the viral protein expression by Western blotting followed by a relative quantification (c), Data information: Statistical analysis was performed using the Mann-Whitney test c, (*P < 0.05).",
    "molecules": "Suc, succinate"
  },
  {
    "caption": "8-week-old female C57Bl/6 mice  were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice  were euthanized at 4 days post-infection and lungs  were collected to determine: (h) tissue lesions  in lung  sections stained  with hematoxylin-eosin  and further assessed by microscopy. Scale bar: x6/20 µm. All data are represented as the mean or the mean ± SEM and are cumulative of 2 independent experiments with 5 animals each. Data information: Statistical analysis was performed using the Mann-Whitney test , h, , (*P < 0.05).",
    "molecules": "eosin, hematoxylin, Suc, succinate"
  },
  {
    "caption": "Human bronchial epithelial BEAS-2B  cells were infected with  A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1. After 4 h, cells were treated or not with succinate  (Suc; 4 mg/ml) up to 20 h. protein expression (c) were assessed by Western blotting to detect viral proteins (β-actin was used as a loading control), respectively.",
    "molecules": "Suc, succinate"
  },
  {
    "caption": "Human bronchial epithelial BEAS-2B  cells were infected with  influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate  (Suc; 4 mg/mL) for 20 h. (a Expression  of IAV  proteins (i.e. NP, NS1) was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained  in green, DNA in blue and actin  in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM. (b) Relative nuclear  intensity of NS1  or NP  was determined by using the Intensity Ratio Nuclei Cytoplasm  Tool. Seven (NS1) or nine (NP) random images were collected per treatment with at least 5 cells per field. Data are represented as the mean ± SEM. Statistical analysis was performed using the Mann-Whitney test, (*P < 0.05).",
    "molecules": "Suc, succinate"
  },
  {
    "caption": "Human bronchial epithelial BEAS-2B  cells were infected with the  influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate  (Suc) for 20 h. (a, b) Cells were stained  using a monoclonal antibody that specifically recognizes \"NP-vRNA\" complexes, but not RNA-free NP, and confocal microscopy (a) was performed (green staining). The value raw integrated density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured (b). Data information: Data are represented as the mean ± SEM of 3 biological replicates or without (a 2 technical replicates each. 8 (b) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (b, (*P < 0.05).",
    "molecules": "Suc, succinate"
  },
  {
    "caption": "Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate  (Suc) for 20 h. (e) Human bronchial epithelial BEAS-2B  cells were infected with a A/Scotland/20/74 (H3N2) virus  (IAV) carrying a wild-type NP  (NP  WT) or with the corresponding mutated virus bearing a NP  with a K87R substitution (NP  K87R). Cells were infected by either virus at MOI=1 for 4 h, then washed and treated or not with 4 mg/mL of succinate  for 20 h. Localization  of NP  proteins was analyzed by confocal immunofluorescence microscopy. Scale bar: 10 µM. Pictures are representative of three independent experiments. (f) Relative nuclear  intensity of NP  was determined by using the Intensity Ratio Nuclei Cytoplasm  Tool. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. at least 10 (f) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05).",
    "molecules": "Suc, succinate"
  },
  {
    "caption": "B. On the top are the results of DNA affinity pull-down followed by western blot with ROW antibody. None is a pull-down with no probe, AT-rich is a pull-down with a biotin-labeled AT-rich dsDNA probe, and control is a pull-down with a probe composed of 49% A/T bases. On the bottom is the stain-free gel as a loading control.",
    "molecules": "biotin"
  },
  {
    "caption": "(G) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (H) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA. (I) Quantitative analysis of the spine density. Representative data of five samples, Data are shown as mean ± SEM. ****P < 0.0001, one-way ANOVA. ",
    "molecules": "DSS"
  },
  {
    "caption": "(E) Gastrointestinal permeability barrier defect as determined by FITC-dextran translocation in pre-vagotomy DSS-treated, DSS-treated and vehicle-treated 3xTg mice. Data represent the mean ± SEM; representative data of three samples; *P=0.0126 (*P < 0.001), ***P=0.0002 (***P < 0.001) compared with control, one-way ANOVA.",
    "molecules": "dextran, DSS"
  },
  {
    "caption": "(F) Pro-inflammatory cytokines IL-6, IL1-β, and TNFα concentrations in colon lysates of vehicle-treated 3xTg mice, DSS-treated single side vagotomy 3xTg and DSS-treated 3xTg, respectively. Representative data of three samples; data are shown as mean ± SEM. *P=0.0372 (*P < 0.05), **P=0.0050 (**P < 0.01), ***P=0.0008 (***P <0.001), ****P<0.0001 compared with control, two-way ANOVA.",
    "molecules": "DSS"
  },
  {
    "caption": "(H) Representative electron microscopy of the synaptic structures in hippocampus. Red stars indicate the synapses. (Scale bar: 1 μm). (I) The dendritic spines from the apical dendritic layer of the cerebral cortex region were analyzed by Golgi staining. (Scale bar: 5 μm). (J) Quantitative analysis of the synaptic densities in DSS-treated mice. Representative data of five samples, data are shown as mean ± SEM. ****P < 0.0001, unpaired t tests. (K) Quantitative analysis of the spine density. Representative data of five samples, data are shown as mean ± SEM. ***P =0.0001 (***P < 0.001), unpair t tests. ",
    "molecules": "DSS"
  },
  {
    "caption": "(A&B) Cued and Contextual Fear-conditioning tests. Data represent the mean ± SEM of n = 6-8 mice per group; **P=0.0067 (3xTg+DSS vs 3xTg ctrl), **P=0.0059 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0009 (***P < 0.001), two-way ANOVA (A); **P=0.0086 (3xTg+DSS vs 3xTg ctrl), **P=0.0085 (3xTg+DSS vs 3xTg R-vagotomy+DSS) (**P< 0.01), ***P=0.0005 (***P < 0.001), one-way ANOVA (B).",
    "molecules": "DSS"
  },
  {
    "caption": "(B) Immunofluorescent staining showed ATTO 550-labeled Aβ PFFs and Tau N368 PFFs spread into different brain regions in WT mice 3 months after colonic injection. Scale bar: 20 μm.",
    "molecules": "ATTO 550"
  },
  {
    "caption": "(C) Immunohistochemistry staining of human Aβ propagating along the vagus nerve from WT mice colonic-injected with PBS, AD brain extracts or 5xFAD brain extracts, using human specific Aβ antibody (BAN50). Black arrows indicate the human Aβ in mouse vagus nerve. (D) Immunohistochemistry staining of human Tau propagating along the vagus nerve from WT mice colonic-injected with PBS or AD brain extracts, using human Tau specific antibody (HT7). Black arrows indicate the human Tau in mouse vagus nerve.",
    "molecules": "PBS"
  },
  {
    "caption": "(A) Immunofluorescent staining of AEP and p-C/EBP β in cerebral cortex of brains of WT mice 6 months after colonic inoculation with either Aβ PFFs or Tau N368 PFFs. Scale bar: 20 μm. White arrows indicate that AEP and p-C/EBP β signals (green) were colocalized with ATTO 550 signals (red), respectively.",
    "molecules": "ATTO 550"
  },
  {
    "caption": "(A) Immunofluorescent staining of AEP (red) and C/EBP β (green) in CA1 region from hippocampus of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. Scale bar: 20μm. (B) Quantitative analysis of AEP positive cells and C/EBP β positive cells, respectively. The density of both AEP and C/EBP β positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. **P =0.016 (**P < 0.01), ****P<0.0001, one-way ANOVA. ",
    "molecules": "PBS"
  },
  {
    "caption": "(C) Immunofluorescent staining of cleaved APPC586 (red) and Aβ (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (D) Quantitative analysis of cleaved APP C586 positive cells and Aβ positive cells, respectively. The density of both cleaved APP C586 positive cells and Aβ positive cells were significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ****P<0.0001, one-way ANOVA. ",
    "molecules": "PBS"
  },
  {
    "caption": "(E) Immunofluorescent staining of T22 (red) and HT7 (green) in cerebral cortex of brains from colonic-injected PBS, colonic-injected AD brain extracts, and pre-Vagotomy colonic injected AD brain extracts 3xTg mice. (F) Quantitative analysis of T22 positive cells. The density of T22 positive cells was significantly increased by colonic-injection of AD brain extracts and decreased through vagotomy before colonic-injection. Representative data of three samples, data are shown as mean ± SEM. ***P=0.0007 (***P < 0.001), ****P<0.0001, one-way ANOVA. ",
    "molecules": "PBS"
  },
  {
    "caption": "(B) Levels of PIN2 phosphopeptide in our experiments. Bars represent the mean plus standard error of replicates (2 biological replicates for nitrate treatments at 20 min (Col-0 roots) and 3 biological replicates for all other experimental conditions). Each independent biological replicate consisted of a pool of 4.500 roots collected from Arabidopsis plants grown independently under the same experimental conditions. The asterisk indicates statistically significant differences in phosphoproteomic analysis (multiple t-test comparison without testing corrections, assuming same variance, p < 0.05).",
    "molecules": "nitrate"
  },
  {
    "caption": "(C) Detection of phosphorylation PIN2 by Phos-tag Western blotting. Arabidopsis plants (Col-0) were growth in ammonium as only nitrogen source, and treated with 5 mM KNO3 or 5mM KCl as control. Total protein from roots were analyzed in SDS PAGE using Phos-tag to detect changes in phosphorylation status. Immunoblotting was performed with PIN2 antibody. Total proteins isolated from eir1.1 roots were used as a negative control. White and red asterisks indicate a slow- or fast-mobility band corresponding to a more or less phosphorylated PIN2, respectively.",
    "molecules": "ammonium, nitrogen, KCl, KNO3"
  },
  {
    "caption": "(D) Western blot against PIN2 protein comparing nitrate treated (KNO3) and control (KCl) condition in Arabidopsis roots for all genotypes (eir1-1 mutant background was complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation).",
    "molecules": "nitrate, KCl, KNO3"
  },
  {
    "caption": "(A) Primary root length of Col-0 wild type plants or eir1-1 mutant plants was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).",
    "molecules": "KCl, KNO3"
  },
  {
    "caption": "(B) Number of initiating and emerging lateral roots of Col-0 or eir1-1 mutant plants were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).",
    "molecules": "KCl, KNO3"
  },
  {
    "caption": "(A) Primary root length of the different genotypes was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).",
    "molecules": "KCl, KNO3"
  },
  {
    "caption": "(B) Number of initiating and emerging lateral roots for all genotypes were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 8-10 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicate statistically significant difference between means analyzed by unpaired, two-tailed and assuming equal variance t-test (*p < 0.05, **p < 0.01).",
    "molecules": "KCl, KNO3"
  },
  {
    "caption": "(A) Homer1c-GFP  signal (magenta) and immunostaining  for surface AMPARs  (cyan) and SP  (green) in neurons  treated for 48h with TTX, or untreated (UT). Scale bars: 10 µm (low magnification), 5 µm (insets). Dotted lines indicate the outline of dendrites. Arrowheads indicate SP+ synapses. (B) Percentage of SP+ synapses  for untreated (UT) and TTX-treated neurons  (dot plots represent different cells; UT: n = 26; TTX: n = 33, n indicates the number of cells, from 2 cultures). % SP+ spines: **P = 0.019 (Mann Whitney test). Data information: Data are represented as mean ± SEM.",
    "molecules": "TTX"
  },
  {
    "caption": "(C) AMPAR synaptic  fluorescence intensity for SP+ versus SP- synapses  in UT and TTX-treated neurons. AMPAR synaptic  fluorescence intensity was normalized to SP- or small synapses, respectively, from UT condition. (UT: SP-, n = 1455, SP  +, n = 516; TTX: SP-, n = 1529, SP+, n = 724, n indicates the number of synapses, from 2 cultures). **P < 0.01, ****P < 0.0001, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.",
    "molecules": "TTX"
  },
  {
    "caption": "(F) Homer1c-DsRed (red) and immunostained  surface AMPARs  (green) in dendrites  from neurons  transfected with either SP-shRNA-GFP  or empty vector (EV) with GFP   reporter (blue) and treated with TTX  or left untreated (UT). Scale bar: 5 µm. (G) AMPAR synaptic  fluorescence intensity normalized to untreated empty vector (EV) condition (EV: UT, n = 42, TTX, n = 33; SP-shRNA: UT, n = 30, TTX, n = 31, n indicates the number of cells, from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.",
    "molecules": "TTX"
  },
  {
    "caption": "(A) Micrographs showing Homer1c-GFP  (green) and immunostaining  for surface AMPARs  (blue) and endogenous SP  (red) in neurons  transfected with miR-124  or control miR-67 (miR-Ctrl), and treated with TTX, or left untreated (UT). Scale bar: 5 µ (B) Percentage of SP+ synapses  for same conditions as in (A) (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ****P < 0.0001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (C) Homer1c-GFP  intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (D) AMPAR synaptic  fluorescence intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26, miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ***P < 0.001, *P < 0.01, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.",
    "molecules": "TTX"
  },
  {
    "caption": "(A) Micrographs showing dendrites  from neurons  transfected with Homer1c-DsRed  (magenta) and SEP-GluA2  constructs containing wild-type (WT) or mutated (MUT) 3'UTR (green) and treated with TTX  for 48 h, or left untreated (UT). Scale bar: 10 µm. (B) SEP-GluA2 synaptic  fluorescence intensity for each condition, normalized to GluA2-3'UTR-WT untreated neurons  (3'UTR-WT: UT, n = 29, TTX, n = 37; 3'UTR-MUT: UT, n = 18, TTX, n = 14; n indicates the number of cells, from 3 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.",
    "molecules": "TTX"
  },
  {
    "caption": "(C) Micrographs showing dendrites  from neurons  transfected with Homer1c-BFP  (magenta), GFP-SP-shRNA (gray) and a rescue RFP-SP  construct containing wild-type (WT) or mutated (MUT) 3'UTR (geen) and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (D) Percentage of SP+ synapses  for each condition (3'UTR-WT: UT, n = 26, TTX, n = 28; 3'UTR-MUT: UT, n = 26, TTX, n = 24; n indicates the number of cells, from 3 cultures). **P < 0.01, ***P < 0.001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.",
    "molecules": "TTX"
  },
  {
    "caption": "(E) Micrographs showing Homer1c-GFP  (magenta) and immunostaining  for surface AMPARs  (cyan) and endogenous SP  (green) in neurons  transfected with or without 50 nM SP TSB-LNA and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (F) Percentage of SP+ synapses  for each condition (Control: UT, n = 36, TTX, n = 52; SP TSB-LNA: UT, n = 62, TTX, n = 29; n indicates the number of cells from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (G) AMPAR synaptic  fluorescence intensity for each condition, normalized to control untreated neurons  (Control: UT, n = 30, TTX, n = 36; SP TSB-LNA: UT, n = 37, TTX, n = 20; n indicates the number of cells from 2 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.",
    "molecules": "LNA, TTX"
  },
  {
    "caption": "(A) Micrographs showing puro-PLA staining  of newly synthesized SP  (green) in neurons immunostained  for MAP-2 (magenta) and DAPI  (blue) and treated with TTX  for 24 h or left untreated. The images on bottom panels show no staining  in the absence of puromycin  treatment or when omitting SP  primary antibody. Scale bars: 30 µm (large view), 5 µm (insets).",
    "molecules": "DAPI, puro, puromycin, TTX"
  },
  {
    "caption": "(C) Density of SP puro-PLA  puncta, normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n represents the number of cells, from 3 cultures). *P < 0.05 (Mann-Whitney test). (D) SP puro-PLA  cluster fluorescence intensity normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n indicates the number of cells, from 3 cultures). ****P < 0.0001 (Mann-Whitney test). Data information: Data are represented as mean ± SEM.",
    "molecules": "puro, TTX"
  },
  {
    "caption": "(E) Micrographs showing puro-PLA staining  of newly synthesized SP  (green) in neurons  transfected with Homer1c-GFP  (magenta) and treated with TTX  for 24 h or left untreated. Scale bar: 5 µm. Arrowheads indicate puro-PLA+ spines. (F) Percentage of SP puro-PLA+ synapses  (UT, n = 26, TTX, n = 33; n indicates the number of cells, from 2 cultures). **P < 0.01, ns, not significant (Mann-Whitney test). (G) Homer1c-GFP  integrated fluorescence intensity at SP puro-PLA- vs SP puro-PLA+ synapses  (n = 32 cells, from 2 cultures). ****P < 0.0001, ns, not significant (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.",
    "molecules": "puro, TTX"
  },
  {
    "caption": "A. Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "C. Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker) and SGs (blue, eIF3η as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358 positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean SD (n = 3).",
    "molecules": "sodium arsenite"
  },
  {
    "caption": "A. HeLa cells were transfected with control, Dicer or Nup358 specific siRNAs, as indicated. Upper panel, total RNA was isolated and analyzed by northern blotting for let-7a using radio-labeled probe. Ethidium bromide (EtBr) stained gel indicates equal loading of RNA samples.",
    "molecules": "EtBr, Ethidium bromide"
  },
  {
    "caption": "B OsPRR73 confers salinity stress tolerance in  rice. The seedlings of Dongjin (DJ, the wild type control), osprr73, and the complementation line of  osprr73 (abbreviated as Com-L1) grown under 12 h light/12 h dark conditions for 28 days (left panel), transferred to 180 mM NaCl  for 21 days (middle panel) and recovered for 12 and 24 days (the two right panels) respectively. Scale bar, 5 cm. C Statistical analysis of the survival rate of DJ, osprr73 and Com-L1  plants in (B) after recovery 12 days. Data are presented as mean ± SD. (n from 4 biological replicates, and 24 plants were tested in each of biological replicates). (***) P ≤ 0.001 were generated by student's t-test.",
    "molecules": "NaCl"
  },
  {
    "caption": "E Null mutant of osprr73 in Nipponbare (NIP) background by genome editing displayed hypersensitivity to NaCl treatment. Scale bar, 5 cm. F Survival rate of NIP (wild type of CRISPR lines) and osprr73-C (C stands for CRISPR) plants in (E) after recovery 9 days. Data are presented as mean ± SD. n = 4 biological replicates, 24 plants for each biological replicate. (*) P ≤0.05 and (***) P ≤ 0.001 were generated by student's t-test.",
    "molecules": "NaCl"
  },
  {
    "caption": "Quantification of  chlorophyll content of WT and  osprr73 mutants Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.",
    "molecules": "chlorophyll"
  },
  {
    "caption": "Quantification of electrolyte leakage   of WT and  osprr73 mutants with or without 180 mM NaCl  treatment for 7 days. Data are presented as mean ± SD. n = 3, biological replicates, and asterisks represent significant difference among means by student's t-test with (*) P ≤0.05, (**) P ≤ 0.01, (***) P ≤ 0.001.",
    "molecules": "electrolyte, NaCl"
  },
  {
    "caption": "A, B The Na+ and  K+  contents in the  shoots and roots  of DJ and osprr73 mutant plants with or without 180 mM NaCl treatment for 7 days. Na+ (A) and  K+ (B) contents were measured with ICP method. Data represent means ± SD, (n = 3, biological replicates). (**) P ≤ 0.001 and (*) P ≤ 0.001 indicate significant difference by student t-test. The abbreviation of n.s. stands for no significant. C Ratio of Na+ to  K+  was calculated with their respective content in (A) and (B). Data represent mean ± SD (n = 3, biological replicates). For each of biological replicates, 6 individual  plants were measured. The asterisk represents significant difference among means by student's t-test with (*) P ≤0.05, and n.s. indicates no significant.",
    "molecules": "K+, NaCl, Na+"
  },
  {
    "caption": "D Four-weeks old seedlings  of DJ and  osprr73  were treated with 90 mM Na2SO4 for 14 d (middle panel) and recovered for additional 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rates with  Na2SO4 treatment in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants for each biological replicate. (***) P ≤ 0.001 indicates significant difference by student t-test. F The  seedlings  of DJ and osprr73 mutant were treated with 90 mM  MgCl2 for 14 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. G Statistical analysis of survival rates  with MgCl2  treatment in (F). The survival rate  of DJ and osprr73 plants in  MgCl2 stress were calculated after recovery 7 days. Data represent means ± SD. n = 2 biological replicates, 24 plants of each replicate. H The  seedlings of DJ and osprr73 mutant were treated with 180 mM  mannitol for 36 days (middle panel) and recovered for 7 days (right panel). Scale bar, 5 cm. I The survival rate  of DJ and osprr73 plants in mannitol stress. Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. The abbreviation of n.s. stands for no significant generated by student t-test.",
    "molecules": "MgCl2, mannitol, Na2SO4"
  },
  {
    "caption": "E ChIP-qPCR assay showing the enriched DNA fragments including the S3 and S4 regions of OsHKT2;1 promoter by OsPRR73, compared to wild type DJ controls. The amplicon of  Ubiquitin was taken as a negative control. Two-week-old seedlings were harvested at ZT12. Data represent means ± SD (n =3, technical repeats). The experiments were performed at least two biological replicates with similar result. Top scheme indicates the locations of amplicons for ChIP  analysis. The asterisks represent significant difference among means by student's t-test with (**) P ≤ 0.01.",
    "molecules": "Ubiquitin"
  },
  {
    "caption": "D The  oshkt2;1 seedling is more tolerant to the treatment of NaCl. The oshkt2;1 and NIP, its wild type, four-week-old seedlings  grown under NaCl conditions for 0 days (left panel), then were transferred to 200 mM NaCl  for 28 days (middle panel), and recovered for 7 days (right panel). Scale bar, 5 cm. E Statistical analysis of survival rate in (D). Data represent means ± SD. n = 3 biological replicates, 24 plants of each replicate. (*) P ≤ 0.05 was generated by student's t-test.",
    "molecules": "NaCl"
  },
  {
    "caption": "A Malondialdehyde  (MDA) content in leaves  of 14-d-old DJ and osprr73 plants  treated by 180 mM NaCl for 3 d and normal condition. Data represent means ± SD. n = 6, biological replicates. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.",
    "molecules": "Malondialdehyde, MDA, NaCl"
  },
  {
    "caption": "B DAB staining showing the greater ROS accumulation in the leaves of osprr73 mutant treated by 180 mM  NaCl treatment for 3 days. Scale bar, 1 cm.",
    "molecules": "DAB, ROS, NaCl"
  },
  {
    "caption": "C  H2O2  concentration was detected by using  H2DCFDA in the root tip of DJ and  osprr73 mutant  plants. Scale bar, 5 mm. -NaCl  and +NaCl  represented the untreated or treated plants  by NaCl respectively.",
    "molecules": "H2DCFDA, H2O2, NaCl"
  },
  {
    "caption": "D Quantitative analysis of H2O2 concentration in the root  of DJ and osprr73 mutant. Data represent means ± SD. n ≥ 6. The asterisks indicate the significant difference (***) P ≤ 0.001 by student's t-test.",
    "molecules": "H2O2"
  },
  {
    "caption": "E, F Measurement of SOD  (Superoxide dismutase) (E) and CAT (Catalase) (F) enzymes activity in  shoots  of DJ and osprr73 seedlings  with or without  NaCl stress. Data represent means ± SD. n = 3 technical replicates. Data are representative from two independent biological replicates with similar result. The asterisks indicate the significant difference (*) P ≤ 0.05 by student's t-test.",
    "molecules": "NaCl"
  },
  {
    "caption": "A: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a screen of 23 pediatric cancer cell lines. Rhabdoid tumor cell lines (red) cluster towards the left of the graph indicating these cell lines are more sensitive to mithramycin. These results confirm a previously published screen (Osgood et al., 2016).",
    "molecules": "mithramycin"
  },
  {
    "caption": "B: Graph of the ratio of the median IC50 of the entire panel to that of each cell line generated from a published screen of 445 agents in 62 sarcoma cell lines (Teicher et al., 2015). The rhabdoid tumor cell line, G401 (red), appears on the left side of the graph indicating this cell line is more sensitive to mithramycin.",
    "molecules": "mithramycin"
  },
  {
    "caption": "C: Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines. RT cell lines (black) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells (grey). RT cell lines are not sensitive to three broadly-active chemotherapeutic agents: etoposide, doxorubicin or SN38. Data represents mean with standard deviation derived from three independent experiments.",
    "molecules": "doxorubicin, etoposide, mithramycin, SN38"
  },
  {
    "caption": "A, B: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to etoposide in BT12 and G401 RT cells (A). Red bar indicates the measured IC50 (Figure 1C). Despite induction of DNA damage, 15 μM etoposide does not lead to apoptosis as indicated by live cell imaging in the presence of cleaved caspase 3,7 (CC3,7) reagent that fluoresces with caspase activation (B). Scale bar (lower left): 150μm.",
    "molecules": "etoposide"
  },
  {
    "caption": "  C, D: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to mithramycin (MMA) in BT12 and G401 RT cells (C). Red bar indicates the measured IC50 (Figure 1C). 100 nM Mithramycin induces apoptosis at 8-hours as measured by CC3,7 fluorescence without the presence of DNA damage (D). Scale bar (lower left): 150μm. ",
    "molecules": "Mithramycin, mithramycin, MMA"
  },
  {
    "caption": " E: BT12 cells treated with 20nM MMA show a different cellular phenotype than with 100nM. Beyond 24h of exposure, there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes. Scale bar (lower left): 150μm. ",
    "molecules": "MMA"
  },
  {
    "caption": " F, G: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to EC8042 in BT12 and G401 RT cells. Red bar indicates the measured IC50 (Figure 1C). In contrast to apoptotic induction, 75nM EC8042 exhibits evidence of mesenchymal differentiation, similar to 20nM MMA (E,G). Please note, solvent control for (E) and (G) is the same although different fields are shown. Scale bar (lower left): 150μm. ",
    "molecules": "EC8042, MMA"
  },
  {
    "caption": " H: Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 and 48-hours. Please note, solvent control for 20nM MMA and 75nM EC8042 is the same. Scale bar (lower left): 150μm. ",
    "molecules": "EC8042, lipid, MMA, oil red O"
  },
  {
    "caption": " I: PPARγ mRNA expression is induced following 20nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). **, P = 0.002, ****, P = 0.0001. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. ",
    "molecules": "mithramycin"
  },
  {
    "caption": " A, B: Mithramycin displaces SMARCC1 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in BT12 rhabdoid tumor (A) but not U20S osteosarcoma (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCC1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control). ",
    "molecules": "mithramycin, Mithramycin"
  },
  {
    "caption": "C: SMARCC1 and SMARCE1 mRNA expression does not change following 100nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). Data represents mean with standard deviation derived from three independent experiments.",
    "molecules": "mithramycin"
  },
  {
    "caption": " D: Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8 or 18-hour mithramycin treatment. BT12 cells were treated for 8 or 18-hours with solvent (S), mithramycin (M, 100nM), bortezomib (PI, 2.5μM), or combination of 100nM mithramycin and 2.5μM bortezomib (C). ",
    "molecules": "bortezomib, PI, mithramycin"
  },
  {
    "caption": " E: Loss of SWI/SNF occupancy at defined loci in the genome as measure by chromatin immunoprecipitation qPCR (ChIP-qPCR) at previously described SWI/SNF target genes, MYT1 (8-hour, P=0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.0001; 18-hour P=0.0001). ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. ",
    "molecules": "DNA"
  },
  {
    "caption": " F: Chromatin immunoprecipitation qPCR (ChIP-qPCR) of H3K27me3 at MYT1 (8-hour, P= 0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.02; 18-hour P=0.0001). H3K27me3 occupancy is increased in a time-dependent manner. ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. ",
    "molecules": "DNA"
  },
  {
    "caption": "Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in BT12 cells relative to loading control (H3).",
    "molecules": "H3K27me3, mithramycin"
  },
  {
    "caption": " Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in G401 cells relative to loading control (H3). Re-expression of SMARCB1 following doxycycline treatment inhibits the mithramycin-dependent effects on H3K27me3 amplification ",
    "molecules": "doxycycline, H3K27me3, mithramycin"
  },
  {
    "caption": " A, B: Mithramycin displaces BRD9 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in G401 (A) but only BRD9 with SMARCB1 re-expression in G401 (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCB1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control). ",
    "molecules": "mithramycin, Mithramycin"
  },
  {
    "caption": " C: BT12 cells show a threshold of exposure that leads to irreversible growth inhibition. The cells were exposed to 100nM MMA for the indicated times followed by replacement with drug-free medium. Beyond 8h (red) of mithramycin exposure, the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death. Data represents mean with standard deviation derived from three independent experiments. ",
    "molecules": "mithramycin, MMA"
  },
  {
    "caption": " D: Mithramycin leads to H3K27me3 amplification in a time-dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP. Western blot lysates collected at 2h, 4h, 6h, 8h, 12h, 18h, 24h of continuous 100nM mithramycin treatment. ",
    "molecules": "H3K27me3, mithramycin, Mithramycin"
  },
  {
    "caption": " E: Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ-6438 relative to mithramycin alone (media) or a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments. ",
    "molecules": "mithramycin, EPZ-6438"
  },
  {
    "caption": " F: Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells (wild type SWI/SNF) to mithramycin. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI/SNF subunit SMARCB1 relative to a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments. ",
    "molecules": "mithramycin"
  },
  {
    "caption": " G. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in SMARCB1- silenced U2OS cells relative to loading control (H3). Knockdown of SMARCB1 following siRNA suppression triggers mithramycin-dependent H3K27me3 amplification, while siNegative (control) does not have an effect on H3K27me3 following mithramycin exposure. ",
    "molecules": "H3K27me3, mithramycin"
  },
  {
    "caption": "A-C: Volcano plot showing gene expression trends at 8h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (A) or 1.5 log2FC (drug over control) (B) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (C). Labels indicate names of genes that meet the logFC and q-value threshold.",
    "molecules": "MMA"
  },
  {
    "caption": "D-F: Volcano plot showing gene expression trends at 18h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (D) or 1.5 log2FC (drug over control) (E) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (F). Labels indicate names of genes that meet the logFC and q-value threshold.",
    "molecules": "MMA"
  },
  {
    "caption": " G: SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100nM mithramycin exposure for 1h, 4h, 8h, 12h, 18h. ",
    "molecules": "mithramycin"
  },
  {
    "caption": " The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 (8-hour, P= 0.0006; 18-hour P=0.0001) . ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. ",
    "molecules": "DNA, mithramycin"
  },
  {
    "caption": " I: The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 (8-hour, P= 0.009; 18-hour P=0.0001) ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. ",
    "molecules": "DNA, mithramycin"
  },
  {
    "caption": "A: Heatmaps depicting ATAC-seq global chromatin structure following 8-hours (middle) and 18-hours (right) 100nM MMA treatment. A 2kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right.",
    "molecules": "MMA"
  },
  {
    "caption": " E: IGV tracks of rhabdoid tumor genes previously identified to be occupied by non-canonical SWI/SNF (Michel et al., 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8-hours or 18-hours. ",
    "molecules": "H3K27ac, mithramycin"
  },
  {
    "caption": " F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment. ",
    "molecules": "H3K27ac, H3K27me3, mithramycin"
  },
  {
    "caption": " G: IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively. ",
    "molecules": "H3K27ac, mithramycin"
  },
  {
    "caption": " A: Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis) or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50μm. ",
    "molecules": "H3K27me3, mithramycin"
  },
  {
    "caption": " B: Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg/kg EC8042 administered continuously over 72-hours. Treatment duration indicated by gray shaded box. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text). ",
    "molecules": "EC8042"
  },
  {
    "caption": " C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text). ",
    "molecules": "EC8042"
  },
  {
    "caption": " D: 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72-hours (3-day pump). H3K27me3 increases and correlates with apoptosis (CC3), however induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50μm. ",
    "molecules": "DNA, EC8042, H3K27me3"
  },
  {
    "caption": " Immunohistochemistry analysis of H&E stains from G401 xenograft tumors on 1, 3, and 7-days after treatment with vehicle or 3-day EC8042 infusion. EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle. microCT analysis of xenograft tumors on 7-days after treatment exhibit enhanced calcification compared to vehicle. IHC scale bar (lower left): 100μm. ",
    "molecules": "EC8042"
  },
  {
    "caption": "E and F. TLC  analysis of cGAMP, which is indicative of cGAS  activation, upon incubation with E) tRNA  or F) total RNA  (n=2, biological replicates, data from one representative independent biological replicate are shown). The FL-hcGAS concentration was 0.265 mg/mL. The tRNA  concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.85 mg/mL. The extracted total RNA  concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.5 mg/mL. NC indicates the negative control that has only the FL-hcGAS. dsDNA  ISD at a concentration of 0.05 mg/mL was used as a positive control. All the samples were prepared in 20 mM HEPES  at pH 7.5 and 150 mM NaCl.",
    "molecules": "cGAMP, dsDNA, HEPES, RNA, NaCl, tRNA"
  },
  {
    "caption": "G. The three images on the left showing fluorescence signals of endogenous cGAS  and RNA   in HeLa  cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS  versus RNA   at each pixel in the indicated cytoplasmic  area (yellow circle). H. The three images on the left showing the fluorescence signals of mCherry  and RNA   in HEK293T  cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry  versus RNA   at each pixel in the indicated cytoplasmic  area (yellow circle). I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS  vs. RNA  (n=13, biological replicates), and mCherry  vs. RNA  (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).",
    "molecules": "RNA"
  },
  {
    "caption": "D. Fluorescence microscopy images showing the replacement of FTSC-tRNA  by Cy5-ISD  at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA  (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. E. Fluorescence microscopy  images showing the replacement of FTSC-tRNA  by TAMRA-380 bp-dsDNA  at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA  (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. F. Quantification of the replacement in \"D\". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of Cy5-ISD  over FTSC-tRNA  in the four parts were calculated, averaged and plotted. G. Quantification of the replacement in \"E\". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of TAMRA-380 bp-dsDNA  over FTSC-tRNA  in the four parts were calculated, averaged and plotted.",
    "molecules": "FTSC, ISD, Cy5, dsDNA, TAMRA, tRNA"
  },
  {
    "caption": "The siRNA-transfected HUVECs were plated inside a transwell chamber and incubated with serum for 18 h. The cells that invaded were observed after staining with crystal violet (CV) solution. Scale bars, 600 μm. The numbers of cells that invaded in each field of view was counted with the ImageJ software in (D).",
    "molecules": "crystal violet, CV"
  },
  {
    "caption": "Blood vessels were observed in MOs (control, cep41 AUG (2.5 ng), or cep41 SB (2 ng))-injected or cep41-mutated Tg(kdrl:eGFP) zebrafish at 40 hours post-fertilization (hpf) by fluorescent microscopy. Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. The representative images for analysis of ISV lumen diameter are indicated by dotted rectangles. A, anterior; P, posterior; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel. Scale bars, 100 μm. Quantification of ISV lumen diameter in (B), the numbers of defective ISVs in (C), the numbers of embryos with aberrant DLAVs in (D), and the numbers of ruptured DLAVs in (E) from data observed in equivalent fields of view (within eight somites). The severity of blood vessel defects in cep41-deficient zebrafish: B (narrowed ISVs) < C (shorten, fused, and missing ISVs) < D and E (ruptured DLAVs). Data are shown as mean ± SD of three independent experiments with ≥ 20 embryos per condition.",
    "molecules": "MOs"
  },
  {
    "caption": "The blood vessels in the trunks of cep41-mutant/morphant zebrafish were compared to those of zebrafish co-injected with ccp5 MOs (2 ng) at 40 hpf in (F). Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. A, anterior; P, posterior. Scale bars, 100 μm. Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view (within eight somites). Data are median of three independent experiments with ≥ 20 embryos per condition.",
    "molecules": "MOs"
  },
  {
    "caption": "The mRNA levels of zebrafish vegfa (E) and vegfr2 (F) were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish at 18 hpf (low shear stress) and 26 hpf (high shear stress). The expression of vegfa and vegfr2 was quantified in neuronal cells from control MO-injected zebrafish for comparisons with that of ECs. The expression of zebrafish β-actin was quantified for the normalization of those qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).",
    "molecules": "MO"
  },
  {
    "caption": "Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 26 hpf. The insets indicate the representative area from each immunostaining experiment and the red dots indicate EC aurka activation. Scale bars, 40 μm. Quantification of the phospho-aurka-positive ECs (H) in equivalent fields of view for each MO-injected zebrafish in images in (G) are the result of three independent experiments with ≥ 10 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. **P < 0.01 (Unpaired Student's t-test with Welch's correction).",
    "molecules": "DAPI, MO, MOs"
  },
  {
    "caption": "Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and then incubated under hypoxia at 28 hpf (a stage of high shear stress) for 2 h. They were subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 30 hpf. The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs. Scale bars, 40 μm. Quantification of phospho-aurka-positive ECs (E) in equivalent fields of view for each MO-injected zebrafish in (D) are the result of three independent experiments with ≥ 20 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. *P < 0.05 (Unpaired Student's t-test).",
    "molecules": "DAPI, MO, MOs"
  },
  {
    "caption": "Zebrafish vegfa mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.",
    "molecules": "MO"
  },
  {
    "caption": "vegfr2 (G) mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results.",
    "molecules": "MO"
  },
  {
    "caption": "Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.",
    "molecules": "MG132"
  },
  {
    "caption": "(C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm. (D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test. ",
    "molecules": "DAPI"
  },
  {
    "caption": "(A) Overexpression of Wip1 down-regulates Smad4 phosphorylation at Thr277. MOCK- or Flag-Wip1-transfected HEK293T cells were treated with 1 h pulse of FGF2 (10 ng/ml), followed by addition of U0126 (40 μM) and harvesting for western blotting at the indicated times. - FGF2, no treatment with FGF2.",
    "molecules": "U0126"
  },
  {
    "caption": "(C) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay. Myc-tagged Smad4 was expressed in HEK293T cells, which were then treated or not with FGF2 (40 ng/ml, 2 h), and the Myc-Smad4 was immunoprecipitated using anti-Myc antibody and subsequently incubated with or without recombinant human Wip1 protein (0.5 μg) in the absence or presence of Mg2+ as indicated.",
    "molecules": "Mg2+"
  },
  {
    "caption": "(E) Polyubiquitination of wt Smad4 is promoted in Wip1-silenced cells. HEK293T cells were transfected with the indicated combinations of Flag-ubiquitin, Co siRNA, Wip1 siRNA, Myc-Smad4 and Myc-Smad4(T277A) and then treated with FGF2 (10 ng/ml, 4 h) in the presence of the proteasome inhibitor MG132, and Myc-Smad4 and its phosphorylation-resistant mutant were immunoprecipitated with anti-Myc antibody. Ubiquitin-conjugated Smad4 (Smad4-Ubn) was detected by western blotting with anti-Flag antibody.",
    "molecules": "MG132, Ubn, Ubiquitin"
  },
  {
    "caption": "(F) Wip1 prolongs the half-life of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide (CHX, 100 ng/ml) for the indicated times and harvested for western blotting. An arrow indicates non-specific bands.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(B) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs without heparin.",
    "molecules": "sdAbs, heparin, thrombin"
  },
  {
    "caption": "(C) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs and heparin.",
    "molecules": "sdAbs, heparin, thrombin"
  },
  {
    "caption": "(D) Bar graph representing residual activated factor X (FXa) activity (ΔOD/min) in presence of the sdAbs and heparin.",
    "molecules": "sdAbs, heparin"
  },
  {
    "caption": "(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.",
    "molecules": "KB-AT-23, FXa, heparin, thrombin"
  },
  {
    "caption": "(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.",
    "molecules": "KB-AT-23, FXa, heparin, thrombin"
  },
  {
    "caption": "(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.",
    "molecules": "KB-AT-23, Thrombin"
  },
  {
    "caption": "(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.",
    "molecules": "KB-AT-23, Thrombin"
  },
  {
    "caption": "(B) Purified KB-AT-23-fus and KB-hFX-11-fus were given intravenously to wild-type C57B6 mice (10 mg/kg). At indicated time-points, blood was collected and residual protein was measured. Plotted is residual protein versus time after injection. Green circles: KB-AT-23-fus; Red circles: KB-hFX-11-fus.",
    "molecules": "KB-AT-23, KB-hFX-11"
  },
  {
    "caption": "(D) Graph reporting the volume of blood loss (µL) in treated mice (n=4-9 per group) observed during 30 minutes post TVT. Dosing was 10 mg/kg for KB-AT-23 and 1 mg/kg for FVIIa. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016).",
    "molecules": "KB-AT-23"
  },
  {
    "caption": "(B) Western blot analysis of KB-AT-23 protein secretion in conditioned media 72 hours post-transfection of HuH7 cells. In the lower panel the western blot quantification via densitometry analysis is depicted. Data are presented as mean ±SD, and were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. ****p<0.001.",
    "molecules": "KB-AT-23"
  },
  {
    "caption": "(C) HuH7-celles were collected 72 hours post-transfection. Presented is the Western blot analysis of lysed cells for the presence of KB-AT-23. Cntr- refers to non-transfected cells, while Cntr+ refers to purified recombinant KB-AT-23.",
    "molecules": "KB-AT-23"
  },
  {
    "caption": "(B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker.",
    "molecules": "KB-AT-23"
  },
  {
    "caption": "(D) Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=6-9/group) 8 weeks post-AAV administration. Data represent mean±SD.",
    "molecules": "KB-AT-23"
  },
  {
    "caption": "G Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=5/group) 8 weeks post-AAV administration. Data represent mean±SD.",
    "molecules": "KB-AT-23"
  },
  {
    "caption": "(C) Representative western blot analyses on plasma samples (n=4) collected from animals 4 weeks post-AAV injection. Cntrl +: positive controls represented by the loading of the purified KB-AT-23 or KB-AT-113 proteins.",
    "molecules": "KB-AT-113, KB-AT-23"
  },
  {
    "caption": "(D-H) Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 (D)",
    "molecules": "KB-AT-01, sdAbs"
  },
  {
    "caption": "Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 , -02 (E), -03 (F), -23 (G) or -113 (H) sdAbs.",
    "molecules": "KB-AT-01, sdAbs"
  },
  {
    "caption": " (B) Western blotting of WT and Drp1-KO MEFs with or without FCCP treatment (30 min) using the indicated antibodies. ",
    "molecules": "FCCP"
  },
  {
    "caption": " (B) Western blotting of WT MEFs, Drp1-KO MEFs, and Drp1-KO MEFs carrying Drp1, all of which express L2-HA, using the indicated antibodies. The expression of L2-HA was induced for 16 h (0.1 µg/ml doxycycline). The asterisk indicates non-specific bands of anti-Oma1 antibodies. ",
    "molecules": "doxycycline"
  },
  {
    "caption": "(A) The mitochondrial membrane potential was measured in WT and Drp1-KO MEFs using a membrane potential-dependent dye (MitoLite NIR) and flow cytometry.",
    "molecules": "MitoLite NIR"
  },
  {
    "caption": " (B) Drp1-KO MEFs carrying Su9-GFP were treated with DMSO (control) or 10 nM antimycin A or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1-EV4). Scale bar, 10 µm. ",
    "molecules": "antimycin A, oligomycin, TMRE"
  },
  {
    "caption": " (F) Drp1-KO MEFs were treated with DMSO or 10 nM antimycin A or 10 ng/ml oligomycin or 10 µM FCCP for 1 h. The mitochondrial membrane potential was analyzed using MitoLite NIR and flow cytometry. ",
    "molecules": "MitoLite NIR, antimycin A, FCCP, oligomycin"
  },
  {
    "caption": " (G) Drp1-KO MEFs expressing L2-HA (induced for 16 h) were treated with 10 nM antimycin A along with 10 µM FCCP. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (H) Quantification of band intensity. Values are average ± SD (n = 3). ",
    "molecules": "antimycin A, FCCP"
  },
  {
    "caption": " (I) Western blotting of Drp1-KO MEFs with or without 10 ng/ml oligomycin treatment. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (J) Quantification of band intensity. Values are average ± SD (n = 3). ",
    "molecules": "oligomycin"
  },
  {
    "caption": " (K) WT MEFs carrying Su9-GFP were treated with DMSO or 10 ng/ml oligomycin for 1 h and viewed by laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The arrows indicate mitochondria that showed flickering. Three frames from the time-lapse analysis are shown (please also see Movie EV1 and EV5). Scale bar, 10 µm. ",
    "molecules": "oligomycin, TMRE"
  },
  {
    "caption": " (L) WT MEFs expressing L2-HA (induced for 16 h) were treated with 10 ng/ml oligomycin. Western blotting was performed using the indicated antibodies. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (M) Quantification of band intensity. Values are average ± SD (n = 3). ",
    "molecules": "oligomycin"
  },
  {
    "caption": " (C) Drp1flox/floxOpa1flox/flox MEFs and Drp1Opa1-KO MEFs were transduced with the indicated Opa1 constructs. The cells were observed using laser scanning confocal microscopy for 30 min with 10-s intervals in the presence of TMRE. The percentage of cells that exhibited flickering is shown. Values are average ± SD (n = 3 experiments). In each experiment, 48-62 cells were analyzed. ",
    "molecules": "TMRE"
  },
  {
    "caption": " (C) The fluorescence intensity of Su9-GFP and TMRE in mitochondria was quantified. ",
    "molecules": "TMRE"
  },
  {
    "caption": " (D) Western blotting of WT MEFs after the indicated FCCP treatments. The asterisk indicates non-specific bands of anti-Oma1 antibodies. (E) Quantification of band intensity. Values are average ± SD (n = 3). Significance was calculated using ANOVA with post-hoc Tukey: **p<0.01, ***p<0.001. ",
    "molecules": "FCCP"
  },
  {
    "caption": " WT which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment. ",
    "molecules": "TMRE"
  },
  {
    "caption": " Drp1-KO MEFs which carry shRNAs (Oma1 or scramble) and ectopic Mfn1, were stained with TMRE. The arrows indicate cells that lost the mitochondrial membrane potential in (C). Scale bar, 10 µm. The percentage of cells that maintained the membrane potential is shown. Values are average ± SD (n = 3). 100-150 cells were analyzed in each experiment. ",
    "molecules": "TMRE"
  },
  {
    "caption": "(c). Dynamic exchange assay of wild-type Beclin 1. His-tagged and untagged Beclin 1 CC domain were co-incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2+- nitrilotriacetic acid (NTA) agarose beads. The presence of heterodimeric Beclin 1 CC domain, consisting of one monomer with his-tag and the other monomer without tag, was checked by SDS gel and indicated by the arrow.",
    "molecules": "nitrilotriacetic acid, NTA"
  },
  {
    "caption": "c) Pull-down assay to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes. Increasing amount of His6-tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region. The Beclin 1-Atg14L/UVRAG complexes were pulled down by Ni2+- nitrilotriacetic acid (NTA) agarose beads and checked by SDS gel.",
    "molecules": "His6, Ni2+, nitrilotriacetic acid, NTA"
  },
  {
    "caption": "Analysis of the interaction between CtUba4 and GST-CtUrm1 (ratio 2:1 or 0.5:1 when marked with an asterisk) in the presence of nucleotide derivates by GST pull-down resolved by SDS-PAGE and visualized with Coomassie stain.",
    "molecules": "Coomassie, SDS"
  },
  {
    "caption": "Functional analyses of ScUba4 mutant yeast using APM-gel retardation (top) and viability in response to rapamycin (bottom). PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti-tRNA<sub>UU<em>C</em></sub><sup>Gl<em>u</em></sup>; probe. APM: ([N-Acryloyl-amino]phenyl)mercuric chloride; YPD: yeast extract peptone dextrose. All residue numbering follows the CtUba4 sequence, but the respective ScUba4 numbering is added in subscript.",
    "molecules": "tRNA, N-Acryloyl-amino]phenyl)mercuric chloride, APM, dextrose, peptone, rapamycin"
  },
  {
    "caption": "Interaction analysis of WT and mutated CtUba4 with GST-CtUrm1 by GST pull-down in the presence of 1mM ATP. CtUba4C202K covalently linked to GST-CtUrm1 is marked by an asterisk.",
    "molecules": "ATP"
  },
  {
    "caption": "Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated (-OH) or thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH, DTT, TCEP and HA, respectively. ATP: Adenosine triphosphate TBH: tert-Butyl hydroperoxide HA: Hydroxylamine; DTT: 1,4-Dithiothreitol; TCEP: Tris(2-carboxyethyl)phosphine.",
    "molecules": "1,4-Dithiothreitol, DTT, Adenosine triphosphate, ATP, HA, Hydroxylamine, TCEP, Tris(2-carboxyethyl)phosphine, tert-Butyl hydroperoxide, TBH"
  },
  {
    "caption": "Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide.",
    "molecules": "Diamide, cysteine, HP, Hydrogen peroxide"
  },
  {
    "caption": "Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin.",
    "molecules": "rapamycin"
  },
  {
    "caption": "Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.",
    "molecules": "Cys, HA, TBH"
  },
  {
    "caption": " Figure 1. DENND2B binds ITSN  Purified GST-SH3-containing proteins were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.  ",
    "molecules": "Ponceau S staining"
  },
  {
    "caption": "Purified GST-SH3 domains of ITSN were incubated with lysates of HEK-293T cells expressing Flag-DENND2B. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.",
    "molecules": "Ponceau S staining"
  },
  {
    "caption": "D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.",
    "molecules": "AlexaFluor561"
  },
  {
    "caption": "D,E HeLa cells expressing the indicated constructs. Flag-ITSN-s was visualized with anti-Flag and AlexaFluor561 or AlexaFluor488 in D and E respectively. Scale bar: 5 µm. Boxed regions are magnified on the bottom. White arrows and red arrows point to colocalizing and non-colocalizing structures, respectively.",
    "molecules": "AlexaFluor488"
  },
  {
    "caption": " Figure 2. DENND2B phosphorylation by PKD disrupts ITSN interaction A HEK-293T cells were treated with DMSO or okadaic acid (OA), lysed and analyzed by Western blot. ",
    "molecules": "okadaic acid"
  },
  {
    "caption": "B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA, and cell lysates were incubated with GST-proteins. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.",
    "molecules": "OA, Ponceau S staining"
  },
  {
    "caption": "C Percentage of phosphorylated peptides following DMSO or OA treatment as determined by mass spectrometry. Low abundance peptides not included. Refer to Table EV1 for a complete peptide list.",
    "molecules": "DMSO, OA"
  },
  {
    "caption": "E HEK-293T cells expressing Flag-DENND2B were treated with DMSO, 250 nM OA or with increasing concentrations of PKD inhibitor CID755673. Cell lysates were analyzed by Western blot.",
    "molecules": "CID755673, OA"
  },
  {
    "caption": "F HEK-293T cells expressing Flag-DENND2B or control cells were treated with 250 nM OA or DMSO, incubated with protein G beads coupled to anti-Flag and analyzed by Western blot.",
    "molecules": "OA"
  },
  {
    "caption": " Figure 3. 14-3-3 outcompetes ITSN for binding to phosphorylated DENND2B A Purified GST-14-3-3 WT or K50E were incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively. ",
    "molecules": "Ponceau S staining"
  },
  {
    "caption": "B HEK-293T cells expressing Flag-DENND2B were treated with DMSO or 250 nM OA and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.",
    "molecules": "OA, Ponceau S staining"
  },
  {
    "caption": "D HEK-293T cells expressing Flag-DENND2B were treated with 250 nM OA alone or with 50 µm PKD inhibitor CID755673 and cell lysates were incubated with GST-14-3-3. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.",
    "molecules": "CID755673, OA, Ponceau S staining"
  },
  {
    "caption": "F GST-SH3A was incubated with lysates of HEK-293T cells expressing GFP-DENND2B WT or S30A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.",
    "molecules": "Ponceau S staining"
  },
  {
    "caption": "G Lysates of HEK-293T cells expressing Flag-DENND2B were pre-incubated with increasing concentrations of purified 14-3-3 WT and subsequently incubated with GST-SH3A (top panel) or GST-Grb2 (bottom panel). Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.",
    "molecules": "Ponceau S staining"
  },
  {
    "caption": "E Transduced MCF10A cells, were biotinylated and cell lysates were incubated with streptavidin beads. Bound proteins were detected by Western blot.",
    "molecules": "biotinylated"
  },
  {
    "caption": "E HEK-293T cells were serum starved, treated with 100 ng/mL EGF for 5 min and cell lysates were incubated with GST-SH3A. Total proteins and bound proteins were detected by Ponceau S staining and Western blot, respectively.",
    "molecules": "Ponceau S staining"
  },
  {
    "caption": "Immunostaining for CCN1 (green) in the adult brain coronal section. Scale bar: 50 μm. The insert refers to the same image with DAPI staining to show cell nuclei (blue). LV, lateral ventricle.",
    "molecules": "DAPI"
  },
  {
    "caption": "V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm.",
    "molecules": "TAM"
  },
  {
    "caption": "Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 2 days after TAM treatment. n=4, *p=0.0141. V-SVZ whole-mounts stained for GFAP (red) and Ki67 (cyan). Arrowheads point to dividing GFAP+ cells. Scale bar: 10 μm.",
    "molecules": "TAM"
  },
  {
    "caption": "Numbers of ASCL1+ cells in V-SVZ coronal sections 2 days after TAM treatment. n=3, **p= 0.0087. V-SVZ coronal sections stained for ASCL1 (red) and nuclei (blue). Scale bar: 50 μm.",
    "molecules": "TAM"
  },
  {
    "caption": "Densities (cells/mm2) of BrdU+ cells in the OB granule cell layer (GCL) 2 days after TAM treatment. n=4 (Ctrl) and 3 (Ccn1cKO), **p=0.0086. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.",
    "molecules": "BrdU, TAM"
  },
  {
    "caption": "Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 4 weeks after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.",
    "molecules": "TAM"
  },
  {
    "caption": "Numbers of ASCL1+ cells in V-SVZ coronal sections 4 weeks after TAM treatment. n=3, n.s. not significant. Scale bar: 20 μm.",
    "molecules": "TAM"
  },
  {
    "caption": "Densities (cells/mm2) of BrdU+ cells in the OB GCL 4 weeks after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.",
    "molecules": "BrdU, TAM"
  },
  {
    "caption": "Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 16 months after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.",
    "molecules": "TAM"
  },
  {
    "caption": "Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.",
    "molecules": "BrdU, TAM"
  },
  {
    "caption": "Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm.",
    "molecules": "BrdU"
  },
  {
    "caption": "Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm.",
    "molecules": "TMZ"
  },
  {
    "caption": "Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm.",
    "molecules": "TMZ"
  },
  {
    "caption": "Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.",
    "molecules": "BrdU, TMZ"
  },
  {
    "caption": "Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm.",
    "molecules": "TMZ"
  },
  {
    "caption": "Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm.",
    "molecules": "TMZ"
  },
  {
    "caption": "Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.",
    "molecules": "BrdU, TMZ"
  },
  {
    "caption": "Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001.",
    "molecules": "erlotinib"
  },
  {
    "caption": "Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002.",
    "molecules": "erlotinib"
  },
  {
    "caption": "(A) Dually labeled D. discoideum amoebae producing MCS components fused to GFP, and either calnexin  (CnxA)-mCherry  (pAW012), P4C-mCherry  (pWS032), or AmtA-mCherry  were infected (MOI 5, 2 h) with mCerulean-producing  L. pneumophila JR32   (pNP99), fixed with 4 % PFA, and imaged by confocal fluorescence microscopy. Merged images are shown. Scale bars: 3 μm (insets: 1 μm).",
    "molecules": "PFA"
  },
  {
    "caption": "(A) Dually labeled  D. discoideum Ax3  producing mCherry-Vap  (pSV048) and OSBP8-GFP  (pMIB89) or GFP-OSBP11  (pMIB39) were infected (MOI 5, 1-8 h) with mCerulean-producing  L. pneumophila JR32  (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm (insets: 1 μm).",
    "molecules": "PFA"
  },
  {
    "caption": "(C) RAW 264.7 macrophages  were treated with increasing concentrations of OSW-1 (0-100 nM, 1-60 h), infected (MOI 1) with GFP-producing L. pneumophila JR32 (pNT28), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to infected cells treated with 0 nM OSW-1; *P<0.05; ***P<0.001, Student`s t-test).",
    "molecules": "OSW-1"
  },
  {
    "caption": "(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1  mutants producing P4C-mCherry  (pWS032) and CnxA-GFP  (pAW016), or Ax3  producing P4C-mCherry  and either GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed  time points  are shown. Scale bars: 3 μm. (B) LCV  area was measured using ImageJ (n=100-200 per condition from 3 independent biological replicates). Means and SEM of single cells are shown (**P<0.01; ***P<0.001, Student`s t-test).",
    "molecules": "PFA"
  },
  {
    "caption": "(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1  mutants producing P4C-mCherry  (pWS032) and GFP-Sac1  (pLS037) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed  time points are shown. Scale bars: 3 μm. (B, C) Imaging flow cytometry  (IFC) analysis of dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry  (pWS032) and either GFP-Sac1  (pLS037) or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Data information (B, C, : Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).",
    "molecules": "PFA"
  },
  {
    "caption": "(D) Dually labeled  D. discoideum Ax3 producing GFP  fusions of MCS components and either mCherry-Sac1  (pSV044) or mCherry-Sac1_ΔTMD (pSV045) were infected (MOI 5, 2 h) with mCerulean-producing  L. pneumophila JR32  (pNP99) and fixed with 4 % PFA. Merged images for the analyzed  time points are shown. Scale bars: 3 μm. (E) IFC  analysis of dually labeled D. discoideum Ax3  producing GFP  fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32  (pAW014). Quantification of (B) P4C-mCherry  (C) Sac1-GFP  or (E) GFP  fusions of MCS components localizing  to LCVs  at 2 h p.i. Due to the lower resolution of IFC, \"LCVs\" designates the LCV  limiting membrane and tightly attached ER. Number of events per sample, n = 5000. Data information E): Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).",
    "molecules": "PFA"
  },
  {
    "caption": "(A) Dually labeled  D. discoideum Ax3 producing GFP-D4H* (pSV046) or P4C-GFP  (pWS034) and P4C-mCherry  (pWS032), CnxA-mCherry  (pAW012), or AmtA-mCherry  were infected (MOI 5, 15 - 120 min) with mCerulean-producing L. pneumophila JR32  (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B-C) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for (B) AmtA-mCherry  with respect to P4C-GFP  or GFP-D4H* (n=100-150 per   time point), and (C) GFP-D4H* with respect to P4C-mCherry  or CnxA-mCherry  (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (*P<0.05, Student`s t-test).",
    "molecules": "PFA"
  },
  {
    "caption": "(D) Single labeled D. discoideum Ax3 producing either CnxA-GFP  (pAW016) or P4C-GFP  (pWS034) were infected (MOI 5, 15-120 min) with mCherry-producing L. pneumophila JR32 (pNP102), fixed with 4 % PFA  and stained  with filipin. Merged images for the analyzed time points  are shown. Scale bars: 3 μm. (E) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for filipin  with respect to CnxA-GFP  or P4C-GFP  (n=80-90 each per  time point). Data represent mean and SEM of the means of three independent biological replicates (***P<0.001, Student`s t-test).",
    "molecules": "filipin, PFA"
  },
  {
    "caption": "(A) Dually labeled  D. discoideum Ax3  producing P4C-mCherry  (pWS032) and either CnxA-GFP  (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32, ΔlepB or ΔsidC (pNP99) and fixed with 4 % PFA. Merged images for the analyzed  time points are shown. Scale bars: 3 μm.",
    "molecules": "PFA"
  },
  {
    "caption": "(B) GALpr-Dfm1-BirA-Flag and GALpr-BirA-Flag levels were measured by western blotting with α -FLAG at 0 (uninduced) vs. 5 hours post-galactose induction (3 biological replicates; n=3).",
    "molecules": "galactose"
  },
  {
    "caption": "(C) Dfm1-BirA is still functional and able to degrade Hmg2-GFP. dfm1Δ+Hmg2-GFP strains containing DFM1-BIRA, empty vector, or BIRA only addbacks were grown to log phase and degradation was measured by CHX. After CHX addition, cells were lysed at the indicated times and analyzed by SDS-PAGE and immunoblotted for Hmg2-GFP with α-GFP.",
    "molecules": "CHX"
  },
  {
    "caption": "(D) Yeast strains expressing Dfm1-Bira and BirA only negative control were incubated with different amounts of biotin: 0, 0.1, and 1 mM. dfm1Δ+Hmg2-GFP. Microsomes were isolated from each strain and subjected to streptavidin pulldown (3 biological replicates; n=3). Flow-through and pull-down fractions were detected by western blotting for Dfm1-BirA with α-Flag and Cdc48 with α-Cdc48 antibodies.",
    "molecules": "biotin, streptavidin"
  },
  {
    "caption": "(A) Indicated strains were spotted 5-fold dilutions on synthetic complete (SC) plates , and plates were incubated at room temperature, 30oC, and 37oC (3 biological replicates, 2 technical replicates; n=5). WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ were compared for growth in the dilution assay. Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells. (B) dfm1∆tsc3∆ confers resistance to myrocin. WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ strains were grown to log-phase in YPD medium, and 5-fold serial dilutions of cultures were spotted on (SC) plates containing either drug vehicle alone, 1 µM of myriocin and 10 μM of PHS (3 biological replicates, 2 technical replicates; n=5). Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells.",
    "molecules": "myriocin, myrocin, PHS"
  },
  {
    "caption": "(A) Indicated strains were spotted 5-fold dilutions on SC plates in 3 biological replicates and 2 technical replicates (n=5), and plates were incubated at room temperature, 30oC, and 37oC. Upper panel: WT, dfm1∆, orm1∆, and dfm1∆orm1∆ were compared for growth by dilution assay. Middle panel: WT, dfm1∆, orm2∆, and orm2∆tsc3∆ were compared for growth by dilution assay. Bottom panel: WT, dfm1∆, orm1∆, orm2∆, and orm2∆, and orm1∆orm2∆ were compared for growth by dilution assay. Upper, middle, and lower panel: gray arrowhead depicts growth phenotype of dfm1∆orm1∆, dfm1∆orm2∆, and orm1∆orm2∆ respectively. (B) dfm1∆orm1∆ confers resistance to myriocin and sensitivity to PHS. WT, dfm1∆, orm1∆, and dfm1∆orm1∆ strains were grown to log-phase in SC medium, and 5-fold serial dilutions of cultures were spotted on YPD plates containing either drug vehicle alone, 1 mM of myriocin and 10 μM of PHS with each condition performed in 3 biological replicates and 2 technical replicates (n=5). Plates were incubated at room temperature and photographed after 3 days. Open circle indicates growth phenotype of dfm1∆orm1∆ cells.",
    "molecules": "myriocin, PHS"
  },
  {
    "caption": "(A) Degradation of Orm2 depends on Dfm1 and not Der1. The indicated strains expressing Orm2-RFP were grown into log phase and degradation was measured by cycloheximide chase (CHX). After CHX addition, cells were lysed at the indicated times, and analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates (n=3). (B) Same as (A) except degradation of Orm2-RFP was measured in WT and der1∆ cells (3 biological replicates; n=3).",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(D) Dfm1's SHP box is not required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).",
    "molecules": "CHX"
  },
  {
    "caption": "(G) Dfm1's WR motif, GxxxG motif, substrate binding and lipid thinning function are required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).",
    "molecules": "CHX"
  },
  {
    "caption": "(A) E3 ligase Tul1 is required for Orm2 degradation. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.",
    "molecules": "CHX"
  },
  {
    "caption": "(C) Dfm1 does not function in the post-ubiquitination step of Orm2 degradation pathway. Indicated strains expressing Orm2-RFP were grown into log phase. Cells were lysed, and microsomes were collected and immunoprecipitated with α-RFP conjugated to agarose beads. Samples were then subjected to SDS-PAGE and immunoblot by α-Ubiquitin and α-RFP (3 biological replicates; n=3).",
    "molecules": "Ubiquitin"
  },
  {
    "caption": "(A) Phosphorylated Orm2 accumulates in the absence of Dfm1. Phos-tag western blot analysis shows that there is an accumulation of phosphorylated Orm2 in dfm1∆ cells. The indicated strains were grown to log phase, treated with vehicle or 1.5 μM myriocin for 1 hour and subjected to SDS-PAGE or Phos-tag western blot analysis via blotting for Orm2 with α-RFP and PGK1 with α-PGK1 antibodies (2 biological replicates; n=2).",
    "molecules": "myriocin"
  },
  {
    "caption": "(C) dfm1∆ cells block the degradation of phosphorylated mimic of Orm2 (Orm2-3D). In the indicated strains, degradation of Orm2-3A-GFP and Orm2-3D-GFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted α-GFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.",
    "molecules": "CHX"
  },
  {
    "caption": "(f) Left panel: Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead-based sprouting assays over 24h (representative images; scale bar, 100 µm). Right panel: Box-whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h. n = 3 healthy, n = 3 NAFLD, with 4-5 beads analyzed per donor and 5-11 sprouts per bead.",
    "molecules": "fibrin"
  },
  {
    "caption": "(a) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in NAFLD (n = 10) BOECs, normalized to healthy (n = 12) BOECs, with 2 technical replicates per donor. (c) Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20 and CX3CL1 in LPS+FFA-treated or non-treated NAFLD (n = 5) and healthy (n = 5) BOECs, normalized to non-treated healthy BOECs, with 3 technical replicates per donor.",
    "molecules": "FFA, LPS"
  },
  {
    "caption": "(b) Left: Representative images showing non-treated and LPS+FFA-treated NAFLD and healthy BOECs, stained with Nile Red (scale bars, 100 µm). Right: Fluorescence intensity of intracellular Nile Red stain was quantified at 515/585nm, and readings were normalized with blanks (n = 3 donors/ group).",
    "molecules": "FFA, LPS, Nile Red"
  },
  {
    "caption": "(e) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of liver sections from chow diet-fed and HFHC diet-fed humanized mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Right: Box-whisker plot of Mander's Coefficient (M2) that was a measure of the degree of CXCL12 overlap with CDH5-positive vasculatures in the livers. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 4) and HFHC diet-fed (n = 4) humanized mice were analysed, with 10-20 independent regions of interest per mouse were analysed for image quantification.",
    "molecules": "DAPI"
  },
  {
    "caption": "(f) Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of aortic sections from chow diet-fed and LIDPAD mice (scale bar, 40 μm). Boxed regions were further magnified (scale bar, 30 µm). Arrows point at aortic endothelial cells. AW: Aortic wall; L: lumen. Right: Box-whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia. CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). Chow diet-fed (n = 3) and LIDPAD diet-fed (n = 3) mice were analysed, with 10-20 delimitated endothelial regions per mouse analysed for image quantification.",
    "molecules": "DAPI"
  },
  {
    "caption": "(d) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with AMD3100 (CXCR4 small molecule inhibitor). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 6 NAFLD and n = 4 healthy, with 3-4 technical replicates per donor.",
    "molecules": "AMD3100"
  },
  {
    "caption": "(f) Left: Schematic of transendothelial migration assay with patient-derived BOECs and immune cells. Right: Transendothelial electric resistance (TEER) measurements of BOEC barrier tightness in the presence and absence of PBMCs and/or AMD3100. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample size is n = 5 patient samples, with 3 technical TEER readings recorded per sample.",
    "molecules": "AMD3100"
  },
  {
    "caption": "A, B Functional freeMitos association with PMN. Overlay dot plot population, representative of 4 biological replicates of the flow cytometry  gating strategy used to quantify the interaction  between PMN  (anti-CD11b-PE) and freeMitos. Freshly isolated platelets  were labeled with MitoTrackerTM Deep Red  and freeMitos isolation was subsequently performed. CD11b-labeled PMN  were incubated in absence (A) or presence (B) of freeMitos at a 1:5 ratio (PMN:mitochondria).",
    "molecules": "MitoTrackerTM Deep Red, PE"
  },
  {
    "caption": "C Confocal microscopy  imaging of PMN cell membrane  (red) labeled with CellMask Orange in absence (upper panel) or presence (lower panel) of MitoTrackerTM Deep Red-labeled freeMitos (magenta). PMN nuclei  were stained  with DAPI  (cyan). The white scale bars shown in the lower right of the images represent 10 μm in size.",
    "molecules": "CellMask Orange, DAPI, MitoTrackerTM Deep Red"
  },
  {
    "caption": "FreeMitos effect on the respiratory state of PMN. O2 consumption rates  of resting  or activated  (incubated with TNF-a and GM-CSF) PMN  were measured using high-resolution O2  respirometry (Oroboros Oxygraph) and revealed no significant differences between the basal state (left panel), leak state (middle panel) and the maximum electron transport system capacity (ETS, right panel) of cellular respiration (F). Data information: For panels F data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different (P < 0.05).",
    "molecules": "O2"
  },
  {
    "caption": "A, B FreeMitos induce PMN  intracellular calcium  release in PMN. Representative visualization of intracellular calcium  monitoring by flow cytometry of 1 x 106 PMN  co-incubated with 5 x 106 mitochondria  (A). Calcium mobilization  was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo-3 AM. The gray line represents PMN  with the vehicle (HBSS) while the dark line represents PMN  incubated with mitochondria. PMN  intracellular calcium mobilization  induced by either the vehicle (HBSS), freeMitos or mitochondrial DAMPs  and quantified by flow cytometry (B). Data information: Data are shown as means ± SEM of 3 biological replicates (panel B, One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels B, Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.",
    "molecules": "calcium, Calcium, Fluo-3 AM"
  },
  {
    "caption": "Functional freeMitos induce the release of PMN-derived microvesicles. A dose-response analysis of various mitochondria  to PMN  ratios was performed to measure the release of PMN microvesicles. PMN microvesicles  labeling was performed with either anti-CD66b-FITC  labelling (C, left panel) or anti-CD11b-PE  (C, right panel). Data information: Data are shown as means ± SEM of 4 biological replicates (panel C two-way (panel C) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.",
    "molecules": "FITC, PE"
  },
  {
    "caption": "PMN-derived microparticle gating strategy is shown in panel D. Flow cytometry  panel established to distinguish the PMNL-MVs  from the background using the leucocyte  specific  integrin alpha-M beta-2 (CD11b), the granulocyte  specific carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b), and size reference beads. PMN  were either incubated with the vehicle (Tyrode's Buffer, upper panels) or freeMitos (lower panels). Following co-incubation, PMN  were washed twice and resuspended in HBSS  before processing on  flow cytometer. Only single events, identified by the singlets gate (left upper and lower panels) using side scatter area (SSC-A) vs side scatter heights (SSC-H) were analyzed. Light scatter (side scatter (SSC) and forward scatter (FSC)) approach for determining the background noise in HBSS. Histogram displaying the fluorescence intensity of samples labelled with either CD11b-FITC  or CD66b-PE. Overlay dot plot of PMNL-MVs  population and size reference beads, representative of 4 biological replicates, each performed in 2 technical replicates.",
    "molecules": "FITC, PE"
  },
  {
    "caption": "E Visual confirmation of PMN  and PMN-derived microvesicles. Transmission electron microscopy  (TEM) imaging of PMN-derived microvesicles  (black arrowhead), representative of 3 biological replicates, from resting  or activated PMN  (106 cells) incubated with either mitochondria, mitochondrial DAMPs  or fMLP  as positive control. The black scale bars are shown in the lower right of the images for the vehicles (400 nm), mitochondria  (600 nm), DAMPs  (600 nm) and fMLP  (500 nm) conditions.",
    "molecules": "fMLP"
  },
  {
    "caption": "G Quantification of calpain  activity, by fluorescence assay, in cell lysate of either resting   (left panel) or activated   (right panel) PMN  (2 x 105 cells) incubated in presence of freeMitos for 2 hr. Data information: Data are shown as means ± SEM of 4 biological replicates (panel G). One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels G. Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.",
    "molecules": "Mitos"
  },
  {
    "caption": "H ELISA  quantification of  IL-8 concentrations present in the supernatant following the incubation of PMN  (106 cells) with freeMitos (107), DAMPs  (25 µg) or fMLP  (1 µM) for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel H) One-way (panel H) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.",
    "molecules": "fMLP"
  },
  {
    "caption": "F-M) Total cellularity of F) HSPCs, G) LT-HSCs, H) ST-HSCs, I) Tom+ HSCs, J) GFP+ drHSCs, K) MPP2, L) MPP3, M) MPP4  in E16.5 fetuses  following saline  or maternal infection with Pru  or RH  as shown in Fig. 1A. n = 9-15 fetuses  from at least 3 litters/condition.",
    "molecules": "saline"
  },
  {
    "caption": "N-O) Frequency of N) HSCs  (CD150hi) O) MPPs  (CD150- HSPCs) expressing Ki67  (G1+G2-M-S) at E16.5 following saline  or maternal infection with Pru  or RH  n = 9-15 fetuses  from at least 3 litters/condition. For all analysis above bars represent mean + SEM. One-way ANOVA with Tukey's test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.",
    "molecules": "saline"
  },
  {
    "caption": "C-H) Total cellularity of C) HSPCs, D) LT-HSCs, E) ST-HSCs, F) MPP2, G) MPP3, and H) MPP4  in saline  or IFNγ  exposed littermates of each genotype (IFNγ  +/- or -/-), as derived from the cross (IFNγR +/- dam). n = 19-23 fetuses  from 3 litters/condition.",
    "molecules": "saline"
  },
  {
    "caption": "J-O) Total cellularity of J) HSPCs, K) LT-HSCs, L) ST-HSCs, M) MPP2, N) MPP3, and O) MPP4  in saline  or IFNγ  exposed littermates of each genotype (IFNγ  +/- or -/-), (IFNγR  -/- dam). n = 19-21 fetuses  from 3 litters/condition. For all analysis bars represent mean. Statistical significance was determined by unpaired student's t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.",
    "molecules": "saline"
  },
  {
    "caption": "(B) Quantitative PCR analysis of post-natal lungs for mitochondrial DNA (mtDNA). Actin was used as a normalization control. (mean ± SD, : p<0.05;: p<0.01; N=3).",
    "molecules": "DNA"
  },
  {
    "caption": "(C) Quantitative analysis of oxidative stress in post-natal pups of indicated age (in days) using the peroxide linked oxidative stress marker DHR-123, as described in methods (mean ± SD, : p<0.01; N=8).",
    "molecules": "DHR-123, peroxide"
  },
  {
    "caption": "(E) Immunofluorescence analysis of oxidative DNA damage (left panel) and the DNA double-strand breaks sensor kinase marker pATM (right panel) in post-natal murine lungs. Here anti-8-oxo-7, 8-dihydroguanine was used for the detection of oxidative DNA modification (8-oxoG, left panel), and anti-pATM Serine 1981 was used for the analysis of the activation of the DDR (pATM, right panel). Green cytoplasmic staining represents α-Tubulin; blue DAPI staining represents nucleus (scale 10μm). No primary antibody control served as a negative control (P14) of the staining.",
    "molecules": "8-oxo-7, 8-dihydroguanine, 8-oxoG, DAPI, Serine"
  },
  {
    "caption": "(F) Mean percentage of nuclei positive for oxidative DNA damage marker 8-oxoG in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; : p<0.001; N=8). (G) Mean percentage of nuclei positive for DNA damage marker pATM in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; N=8). ",
    "molecules": "8-oxoG"
  },
  {
    "caption": "(H) Quantitative analysis of the NAD+/NADH ratio in post-natal lungs after homogenization in extraction buffer (mean ± SD, : p<0.01; : p<0.001).",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "(A) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma (A549) cells, cultured either in low glucose (5.5mM, left panel) or high glucose (30mM, right panel) for 5 days, then treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment, CML (marked in green) served as induction control (scale 10µm). (B) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre-treatment of different reducing carbohydrates at 0, 3 or 24 hours. More than 400 cells were analyzed for each bar (mean ± SD, : p<0.01;: p<0.001). ",
    "molecules": "etoposide, glucose"
  },
  {
    "caption": "(D) Quantitative analysis of the efficiency of NHEJ-repair in a reporter cell line, as described in Methods. Cells were cultured in low glucose (5.5mM) or high glucose (30mM) for 5 days, fructose (30mM), ribose (20mM), for 3 days (graph bar 4). I-SceI, untransfected cells served as a negative control. Shown is the average from three independent experiments (mean ± SEM, : p<0.01 : p<0.001).",
    "molecules": "fructose, glucose, ribose"
  },
  {
    "caption": "(E) Quantitative analysis of the NAD+/NADH ratio in A549 cells, cultured in either low (5.5mM, LG), or high glucose (30mM, HG) for 5 days, fructose (30mM, FR), ribose (20mM, RI), for 3 days (graph bar 4). Quantification was performed after homogenization in extraction buffer (Data represent mean ± SD, : p<0.01;: p<0.001; N=3).",
    "molecules": "fructose, glucose, NAD+, NADH, ribose"
  },
  {
    "caption": "(F) A549 cells were cultured in either low (5.5mM; lane 1) or high glucose (30mM; lane 2) for 5 days, fructose (30mM; lane 3), ribose (20mM; lane 4), for 3 days. The cell extracts were then for immunoprecipitation by using anti-PARP, or a non-specific species control antibody. The PARP, or its interacting partner DBC1 was then detected using PARP, or a DBC1 specific antibody.",
    "molecules": "fructose, glucose, ribose"
  },
  {
    "caption": "(A) Representative images of lungs for nuclei positive for the DNA-DSBs marker γH2AX, versus DAPI, in age-matched 3-, or 6- months control, versus STZ induced diabetic mice, as determined by immunofluorescence analysis (scale 10μm). (B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in lungs of age-matched control versus 6-month STZ induced diabetic mice, as determined by immunofluorescence analysis of lungs (mean ± SEM, :p<0.05;: p<0.01; : p<0.001; N=6). ",
    "molecules": "DAPI, STZ"
  },
  {
    "caption": "(C) Representative images of lungs from age-matched control versus 3 or 6-month STZ mice, stained for the peroxide linked oxidative stress marker DHR-123(in red), as determined by mean fluorescence intensity. Blue nuclear staining represents DAPI (Scale 10µm). (D) Quantitative analysis of respectively age-matched control versus 3 or 6-month STZ induced diabetic lungs for persistent DNA damage signaling associated inflammatory marker IL-6, as determined by mean fluorescence intensity of respective group lungs (mean ± SD; : p<0.01: p<0.001; N=6). ",
    "molecules": "DAPI, DHR-123, peroxide, STZ"
  },
  {
    "caption": "(E) Representative images of lungs from age-matched controls, versus 3 or 6-month STZ induced diabetic mice. Sections stained for cellular senescence-associated β-galactosidase [β-Gal] as described in Methods and visualized by bright field and polarized light, the senescent areas are recognized by its bluish-green staining (Scale 40µm).",
    "molecules": "STZ"
  },
  {
    "caption": "(A) Pressure-volume curves were determined using the FlexiVent system in age-matched control versus 3-month STZ induced diabetic mice. The curves represent group averages (mean ± SD; : p<0.05; N=5).",
    "molecules": "STZ"
  },
  {
    "caption": "(B) Quantitative analysis of static pulmonary compliance in age-matched control versus 3 months STZ induced diabetic mice, as described in Figure 3a. The curves represent group averages (mean ± SD; : P<0.05, : p<0.01; N=5).",
    "molecules": "STZ"
  },
  {
    "caption": "(C) Representative images of lungs from age (3- or 6- month) matched control versus STZ induced diabetic mice, stained for Masson's Trichrome stain, as described in Methods and visualized by bright field and polarized light, the accumulated ECM is recognized by its blue staining (Scale 40µm).",
    "molecules": "Masson's Trichrome stain, STZ"
  },
  {
    "caption": "(F) Quantitative analysis of the NAD+/NADH ratio in lungs harvested from age-matched 3, or 6 months control, or STZ induced diabetic mice, after homogenization in extraction buffer (mean ± SD; : p<0.01, : p<0.001; N=5).",
    "molecules": "NAD+, NADH, STZ"
  },
  {
    "caption": "(G) Quantitative analysis of the NAD+/NADH ratio determined in serum from patients with diabetes, as described in methods (N=3; mean ± SD, : p<0.01, : p<0.001).",
    "molecules": "NAD+, NADH"
  },
  {
    "caption": "(A) Representative images of γH2AX positive nuclei in lungs of 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. The lungs were harvested 6-weeks after viral transduction. The empty vector served as control. Red represents γH2AX foci green cytoplasmic staining represents α-tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (from control lung) of the staining.",
    "molecules": "DAPI, STZ"
  },
  {
    "caption": "(B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in transduced lungs of 6-month STZ diabetic mice as described in Figure 5a (mean ± SEM; : p<0.01; N=8).",
    "molecules": "STZ"
  },
  {
    "caption": "(C) Representative images of lungs from 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. Lungs were stained for cellular senescence-associated β-galactosidase as described in Figure 5a and visualized by bright field and polarized light, where the accumulated senescent areas are recognized by its blue staining (Scale 40µm).",
    "molecules": "STZ"
  },
  {
    "caption": "(D) Quantitative analysis of persistent DNA damage associated inflammatory marker IL-6, in transduced lungs from 6-month STZ-diabetic mice, as determined by mean fluorescence intensity of respective group lungs (mean ± SD,: p<0.05, : p<0.001; N=8).",
    "molecules": "STZ"
  },
  {
    "caption": "(E) Quantitative analysis of Masson's Trichrome stain for lungs of 6-month STZ induced diabetic mice with respective RAGE virions as described in Figure 5F (mean ± SD, : p<0.001; N=8).",
    "molecules": "Masson's Trichrome stain, STZ"
  },
  {
    "caption": "(F) Representative images of Masson's Trichrome stain for lungs transduced with the respective RAGE expressing virions as described in Figure 5a and visualized by bright field and polarized light, where the accumulated ECM areas are recognized by its blue staining (Scale 40µm). The dotted lines represent the respective zoomed window.",
    "molecules": "Masson's Trichrome stain"
  },
  {
    "caption": "d) representative images of Ki67 and p16Ink4a immunofluorescence and SA-β-Gal staining; e) % of Ki67 positive cells (28 days after co-culture); f) % of p16INK4A positive cells (28days after co-culture); g) % of SA-β-Gal positive cells (28 days after co-culture); h) Western blotting for p21 and quantification; i) MitoSOX fluorescence; ",
    "molecules": "MitoSOX"
  },
  {
    "caption": "h) Representative Immuno-FISH micrographs (using telomere‐specific (CCCTAA) peptide nucleic acid probe and anti-53BP1 antibody) 20 days following co-culture; i) quantification of mean TAF and % TAF in MRC5 fibroblasts 8 and 20 days following neutrophil co-culture. Data are mean±S.E.M. of 3 independent experiments; ",
    "molecules": "nucleic acid"
  },
  {
    "caption": "m) Representative immunoFISH (Telomere-FISH and 8-oxodG); n) mean number of 8-oxodG co-localizing with telomeres in hepatocytes; o) Mean telomere FISH intensity in hepatocytes. ",
    "molecules": "8-oxodG"
  },
  {
    "caption": "i) Comparison between p16Ink4a mRNA levels of young (3 months old) and old INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-8 mice per age group; j) % of TAF positive hepatocytes in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-6 mice per group; k) Liver neutrophils (Ly6G) in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-7 mice per group; ",
    "molecules": "AP20187"
  },
  {
    "caption": "A Lysates from RK13 cells transfected with WT, S1, S3 and S3.F88W PrP were probed with antibody Sha31, 9A2 and 12B2 after digestion with PNGaseF.",
    "molecules": "PNGaseF"
  },
  {
    "caption": "B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).",
    "molecules": "PNGaseF"
  },
  {
    "caption": "B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).",
    "molecules": "PNGaseF"
  },
  {
    "caption": "D Lysates of N2a, HEK, SMB-PS and SH-SY5Y cells not transfected (−) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31.",
    "molecules": "PNGaseF"
  },
  {
    "caption": "E Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP, S1 PrP and S3 PrP. Lysates were PNGaseF-digested before Western blot using the Sha31 antibody. F1: A114F, F2: G118F, F3: G122F, F4: G126F",
    "molecules": "PNGaseF"
  },
  {
    "caption": "A Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls.",
    "molecules": "PNGaseF"
  },
  {
    "caption": "C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.",
    "molecules": "PNGaseF"
  },
  {
    "caption": "B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.",
    "molecules": "PNGaseF"
  },
  {
    "caption": "A, B Infected brain homogenates were PK-digested and analysed by Western blot using (A) Sha31 antibody and (B) after PK and PNGaseF digestion using Sha31.",
    "molecules": "PNGaseF, PK"
  },
  {
    "caption": "C Conformational stability assay on RML-infected brain homogenates incubated with increasing concentrations of GdnHCl. The best-fit alignments of the data are presented. Genotypes are noted on the individual panels.Source data are available online for this figure.",
    "molecules": "GdnHCl"
  },
  {
    "caption": "C: Representative images of FISH analysis with an RNA probe of ELDR (red) and DAPI staining (blue) in formalin fixed paraffin embedded OSCC tissue. An unrelated probe was used as negative control. Magnification: 40X; inset magnification 100X and scale bar 10 µm.",
    "molecules": "DAPI, formalin"
  },
  {
    "caption": "A: Control or ELDR plasmid DNA stably transfected NOKs were analyzed for cell proliferation using Trypan blue exclusion at the indicated time points. Live cell numbers are presented. n = 4 biological replicates. Data are represented as the mean ± SD,",
    "molecules": "Trypan blue"
  },
  {
    "caption": "B: Control or ELDR plasmid DNA overexpressed NOK (3×102) were seeded and allowed to form colonies. After two weeks, colonies were stained with crystal violet and counted. Representative images of colonies in control and ELDR overexpressed cells are presented. The right panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,",
    "molecules": "crystal violet"
  },
  {
    "caption": "F: Control, ELDR overexpressed and ELDR-depleted Cal27 cells were probed with an antibody against ILF3 (red) and DAPI (blue). Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3. Arrows indicate cytoplasmic expression. Magnifications 60X, scale bar 20 μ.",
    "molecules": "DAPI"
  },
  {
    "caption": "H: Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested, fixed and stained with propidium iodide. DNA content was analyzed by flow cytometry. Results are represented cell population in G1, S, and G2/M phases of the cell cycle. Right panels show percentage of cell population in different phases of cell cycle. n = 4 biological replicates. Data are represented as the mean ± SD,",
    "molecules": "propidium iodide"
  },
  {
    "caption": "D-F. Volcano plots showing RNA-seq data in mouse islets    (20% vs 5% O2  for 24 hours; n = 3 mice/group; D), human islets    (20% [n = 2 biological replicates from donor 1 sample] vs 2% [n = 3 biological replicates from donor 1 sample] O2  for 24 hours; E), and MIN6  cells (20% vs 5% O2  for 6 hours; n = 3 biological replicates; F). Atf3, Bhlhe40, and other reported hypoxia-inducible transcriptional repressor genes (Cavadas et al., 2017) are shown (red, significantly upregulated genes).",
    "molecules": "O2"
  },
  {
    "caption": "Western blots of BHLHE40 expression  across tissues (A), BHLHE40 expression  in MIN6  cells cultured under 20% or 5% O2  for the indicated time  (B) and the quantification of B (n = 3 biological replicates) Data information: Glyceraldehyde-3-phosphate dehydrogenase  (GAPDH) or  β-actin was used as a loading control. HPT, hypothalamus",
    "molecules": "O2"
  },
  {
    "caption": "D-F. The effect of oxidative stress, endoplasmic reticulum stress, and energy stress on BHLHE40  expression. qRT-PCR analysis of Bhlhe40  in MIN6  cells incubated with 10μM H2O2  (n = 3 biological replicates; D), 2μM thapsigargin  (Thap) or 5 μg/ml tunicamycin (Tun) (n = 7 biological replicates; E), or 2mM metformin  (Met) for 24 hours (n = 3 biological replicates; F). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.",
    "molecules": "H2O2, Met, metformin, Thap, thapsigargin, Tun, tunicamycin"
  },
  {
    "caption": "A. Glucose-stimulated insulin  secretion in MIN6  cells expressing short hairpin RNA against a non-targeting Ctrl or Bhlhe40   knockdown (B40  KD) were cultured under 20% or 5% O2  for 24 hours (n = 8 biological replicates). B. Glucose-stimulated insulin  secretion in MIN6  cells infected with retroviruses  generated with pMx-Ctrl (Ctrl) or pMx-Bhlhe40  (B40  OE; n = 4 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.",
    "molecules": "Glucose, O2"
  },
  {
    "caption": "D. KCl-stimulated insulin  secretion in Ctrl and B40   OE MIN6  cells (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.",
    "molecules": "KCl"
  },
  {
    "caption": "G. Cellular ATP  content in Ctrl and B40   KD MIN6  cells cultured under 20% or 5% O2  for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.",
    "molecules": "ATP, O2"
  },
  {
    "caption": "I-K. Mitochondrial DNA (mtDNA) content (n = 4 biological replicates; I), mitochondrial  mass (n = 4 biological replicates; J), and mitochondrial membrane potential (n = 4 biological replicates; K) in Ctrl and B40   KD MIN6  cells cultured under 20% or 5% O2  for 24 hours. Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.",
    "molecules": "O2"
  },
  {
    "caption": "D. qRT-PCR of MAFA  target genes in Ctrl and B40  KD MIN6  cells cultured under 20% or 5% O2  for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test.",
    "molecules": "O2"
  },
  {
    "caption": "G. Western blot of oxidative phosphorylation complex proteins and TOM20  protein in Ctrl and B40  KD MIN6  cells cultured under 20% or 5% O2  for 24 hours, and quantification of the band intensities (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test. β-actin  was used as a loading control. Ctrl, control.",
    "molecules": "O2"
  },
  {
    "caption": "C. Luciferase reporter assay  was performed with MIN6  cells transfected with BHLHE40   expression plasmids, pRL-SV40 plasmid, and pGL3-Mafa  plasmids (wildtype [black] and E-box mutated [red] sites; n = 4 biological replicates). D. Luciferase reporter activity  in MIN6  cells cultured under 20% or 5% O2  for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.",
    "molecules": "O2"
  },
  {
    "caption": "H-J. Proteins sampled from Ctrl and B40  OE MIN6  cells with FLAG-Pdx1   expression (H) or from MIN6  cells with FLAG-Pdx1   expression pre-cultured under 20% or 5% O2  for 24 hours (I) were  immuno-precipitated  by IgG or anti-FLAG  antibody, after which qRT-PCR  was performed for the indicated regions (n = 9 biological replicates for H and n = 6 biological replicates for I). FLAG  (PDX1) expression in I was confirmed by Western blotting (J). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.",
    "molecules": "O2"
  },
  {
    "caption": "E. Glucose-stimulated insulin  secretion in Ctrl:ob/ob  and βB40KO:ob/ob mice  (n = 9 mice  and n = 6 mice, respectively; 8 weeks old). F. Glucose-stimulated insulin  secretion in isolated islets   from Ctrl and βB40KO mice  after culture under 5% O2  for 24 hours (n = 8 biological replicates). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.",
    "molecules": "Glucose, O2"
  },
  {
    "caption": "B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells.",
    "molecules": "Gold particles"
  },
  {
    "caption": "A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey's test for multiple comparisons.",
    "molecules": "Rapamycin"
  },
  {
    "caption": "B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections).",
    "molecules": "Rapamycin"
  },
  {
    "caption": "The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).",
    "molecules": "rapamycin"
  },
  {
    "caption": "C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.",
    "molecules": "Bafilomycin"
  },
  {
    "caption": "A) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (E. coli LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour.",
    "molecules": "Pam3csk4, LPS"
  },
  {
    "caption": "B) Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&2 ligands (E. coli LPS and Pam3csk4)",
    "molecules": "LPS, Pam3csk4"
  },
  {
    "caption": "C Si-TRAF3IP3 (oligo #1) were transfected into HEK 293 cells, which were then treated with mock, VSV, SeV, poly(I:C) or HSV-1. After various treatments, IFNB inductions were measured by qPCR.",
    "molecules": "poly(I:C)"
  },
  {
    "caption": "E Wild type and Traf3ip3-/- HEK293T cells were treated with or without VSV, SeV or poly (I:C) as indicated for twelve hours. IFNB induction was measured by qPCR.",
    "molecules": "poly (I:C)"
  },
  {
    "caption": "C shRNA targeting TRAF3IP3 (sh-TRAF3IP3) was transduced into Mavs-/- HEK293T cells. Twenty-four hours after transduction, cells were treated with puromycin (2 μg/mL) for forty-eight hours and then transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours after transfection, cells were infected with or without VSV for twelve hours. Cells were then collected and subjected to immunoprecipitation assay and immunoblotting.",
    "molecules": "puromycin"
  },
  {
    "caption": "C-E Immunofluorescent microscopic imaging for TRAF3IP3. HeLa cells were transfected with pcDNA3-FLAG-TRAF3IP3. Twenty-four hours after transfection, cells were infected with or without SeV for twelve hours. Anti-FLAG M2 (FITC) was used for immunofluorescence staining of FLAG-TRAF3IP3. Nuclei were stained with DAPI. Mitochondria was stained with Mitotracker Red (C). Calnexin (an ER protein) was stained for ER (D). GM130 (a Golgi protein) was stained for Golgi (E). Scale bar represents 5 micrometers. F Quantitative analysis of yellow color as shown in Fig 5C, D and E, indicating TRAF3IP3 localization on various organelles with or without virus infection. The quantification was performed with ImageJ. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. ***P<0.001. NS indicates no statistically significant difference.",
    "molecules": "DAPI, FITC, Mitotracker Red"
  },
  {
    "caption": "BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Ifnb (A), Ifna4 (B), Il6 induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.",
    "molecules": "poly (I:C)"
  },
  {
    "caption": "BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Il6 (C), Isg54 (D), and Cxcl10 (E) induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.",
    "molecules": "poly (I:C)"
  },
  {
    "caption": "(C) Treatment by sodium periodate followed by qPCR does not support 2'-OH modification of either ssc-miR-205  or  bta-miR-10b miRNA. Shown are relative levels of the  sodium periodate-treated samples to non-treated samples, which were set to one. The experiment was performed three times. Error bars = SD. Efficiency of  periodate treatment was confirmed using non-methylated and methylated miR-221  RNA  oligonucleotides",
    "molecules": "periodate, sodium periodate"
  },
  {
    "caption": "(D) 3′ tailing of small RNAs in  murine, bovine, and porcine oocytes Shown are percentages of miRNAs tailed at 3′ end with mono- and oligonucleotides.",
    "molecules": "mono, oligonucleotides"
  },
  {
    "caption": "E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites.",
    "molecules": "LY294002"
  },
  {
    "caption": "F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1.",
    "molecules": "LY294002"
  },
  {
    "caption": "A, HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.",
    "molecules": "Akt inhibitor IV"
  },
  {
    "caption": "C, RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.",
    "molecules": "Akt inhibitor IV"
  },
  {
    "caption": "D, mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite.",
    "molecules": "Akt inhibitor IV"
  },
  {
    "caption": "E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole.",
    "molecules": "Akt inhibitor IV"
  },
  {
    "caption": "F, Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite",
    "molecules": "Akt inhibitor IV"
  },
  {
    "caption": "G, H, mHEVc cells were transfected with Beclin 1 siRNA (G), Atg7 siRNA (H) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 24 h.",
    "molecules": "Akt inhibitor IV"
  },
  {
    "caption": "I, mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii. 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge.",
    "molecules": "Akt inhibitor IV, leupeptin, pepstatin"
  },
  {
    "caption": "J, Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii. Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.",
    "molecules": "3-methyl adenine, Akt inhibitor IV"
  },
  {
    "caption": "A, HBMEC were treated with or without pertussis toxin (PTx; 100 ng/mL) for 4 h prior to challenge with T. gondii. Cell lysates were obtained at 5, 15 or 30 min post-incubation with T. gondii and used to examine total Akt and phospho-Akt serine 473 by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and PTx-treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. HBMEC were also treated with or without LPA (10 µM) in the presence or absence of PTx. Cell lysates were obtained at 5 min and subjected to immunoblotting",
    "molecules": "LPA"
  },
  {
    "caption": "B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.",
    "molecules": "AG1478"
  },
  {
    "caption": "A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.",
    "molecules": "AG1478"
  },
  {
    "caption": "C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.",
    "molecules": "AG1478"
  },
  {
    "caption": "F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined.",
    "molecules": "AG1478"
  },
  {
    "caption": "G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.",
    "molecules": "AG1478"
  },
  {
    "caption": "HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.",
    "molecules": "GM6001"
  },
  {
    "caption": "A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-T. gondii. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii.",
    "molecules": "rapamycin, CD154, AG1478, EGF"
  },
  {
    "caption": ". C, hmCD40 mHEVc-LC3-EGFP cells were treated with or without CD154 followed by challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko, MIC1-3 ko+MIC1-3 tachyzoites. LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post-challenge. D, hmCD40 mHEVc-LC3-EGFP treated with or without CD154 were challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko or MIC1-3 ko+MIC1-3 tachyzoites. Parasite load was examined 24 h post-challenge.",
    "molecules": "CD154"
  },
  {
    "caption": "E, HBMEC cells were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites followed by treatment with vehicle or rapamycin. Parasite load was examined 24 h post-challenge. F, HBMEC treated with or without CD154 or IFN-γ/TNF-α were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites. Monolayers were examined microscopically at 24 h post-challenge.",
    "molecules": "rapamycin, CD154"
  },
  {
    "caption": "G, hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1-3 ko tachyzoites in the presence of complete medium (CM) alone or medium plus MIC3, MIC4, M2AP or MIC6 (all 10 nM). Parasite load was determined microscopically 24 h post challenge. Results shown are representative of 3-4 independent experiments.",
    "molecules": "CD154"
  },
  {
    "caption": "Viability curve of cells following 24h of recombinant Pol II expression induction by removal of tetracycline (Tet-Off system) and treatment with α-amanitin (black arrow-head). Untransfected Raji (WT: black) is shown for reference. For all experiments presented in this article, cells are collected after 24 hours of α-amanitin treatment for CTD-∆5 and YFFF mutants expressing cells since CTD-∆5 is unstable after 48h (see panel C). Error bars representing top and bottom values are based on the average of 3 independent experiments.",
    "molecules": "α-amanitin, Tet, tetracycline"
  },
  {
    "caption": "Western blot analysis of the recombinant RPB1 expression (rWT, YFFF, and ∆5) assessed by (HA)-tag expression after 24 or 48 hours of α-amanitin treatment following 24h of induction. Untransfected Raji extracts are shown for comparison (WT). Total RPB1 levels are represented by the F12 western blot. Tubulin was used as a loading control.",
    "molecules": "α-amanitin"
  },
  {
    "caption": "Volcano plot of differentially expressed genes in chromatin associated RNA-seq (ChrRNA) data sets: protein coding genes UP (purple) and DOWN (pink) regulated in B relative to A after 24h hours α-amanitin treatment (n=22810). Experiments were done in biological duplicates. Threshold: log2 fold change ≥ 3, p-value < 0.05.",
    "molecules": "α-amanitin"
  },
  {
    "caption": "Example showing ChrRNA-seq (top) and its associated PolyA RNA-seq signals (bottom) in rWT and CTD-∆5.",
    "molecules": "PolyA"
  },
  {
    "caption": "Boxplots of PolyA read-through (RT) indexes based on signal over 20kb upstream (anti-sense) and 20kb downstream (sense) regions of coding genes in rWT (dotted grey line) and CTD-∆5 mutant (blue). Units are asinh transformed. Experiments were done in biological duplicates. Boxplots represent minimal and maximal values, first and third quartiles with median value as central band. P-values were calculated using two-sided Wilcoxon tests.",
    "molecules": "PolyA"
  },
  {
    "caption": "Volcano plot comparing the Pol II interactome in rWT and CTD-∆5 mutant. Left: Proteins lost in CTD-∆5, Right: Proteins enriched in CTD-∆5. Highlighted are the subunits of the Mediator (purple) and Integrator complexes (orange) CTD phosphatases scaffolds (pink), Ubiquitin ligases (light blue) and SOSS complex subunits (red). In black are highlighted some chosen interactors gained by the CTD-∆5 Threshold: log2 fold change ≥ 4; p-value < 0.05. Data based on three independent biological replicates. The table in the figure sums up the proteins that do not change significantly; dotted lines connected to the table represent the region where these proteins are found in the volcano",
    "molecules": "phosphatases"
  },
  {
    "caption": "(A) Total circRNAs detected in the SN, MTG and AMG of both PD and control donors, p*=0.02 p*=0.026 and p*=0.022 for comparison between PD and CT in SN, MTG and AMG. T-test for total SN vs MTG p*=2.27E-8 and SN vs AMG p*=5.86E-10. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values. P value was calculated by T-test.",
    "molecules": "circRNAs"
  },
  {
    "caption": "(C) Global RNA editing levels based on the Alu editing index, studied only in Alu elements within circRNA exons and their flanking introns, in the 3 brain regions, for circRNAs corrected p*= 0.025 and 0.040 for the AMG and MTG and p=0.3 for SN. For flanking introns p*= 0.02 and 0.040 for AMG and MTG and p=0.34 for SN, Wilcoxon test. n=8 for Amygdala control, 15 for Amygdala PD, 8 for MTG control and 13 for MTG PD, 10 for SN control and 15 for SN PD. The box is drawn from Q1 to Q3 with a horizontal line drawn in the middle to denote the median and x marks the average. Whiskers mark minimum or maximum values.",
    "molecules": "circRNA, circRNAs"
  },
  {
    "caption": "(F,G) Correlations between circRNAs detected in each sample and the age of the control (green, correlation = 0.68, p-value = 0.032) and PD donors (blue, correlation = -0.14 p value = 0.62).",
    "molecules": "circRNAs"
  },
  {
    "caption": "(H) Volcano plot of DE circRNAs in PD vs control tissues, red dots indicate statistically significant DE circRNAs according to FDR correction of Wald test (DEseq2 analysis). CircSLC8A1 is marked is red.",
    "molecules": "circRNAs"
  },
  {
    "caption": "(H) Increased CircSLC8A1 RNA levels in PQ-exposed SH-SY cells (normalized to Tubb3 and RPL19) while other circRNAs remain unchanged",
    "molecules": "PQ, circRNAs"
  },
  {
    "caption": "(I,J) Immunoblot decline (western blot and quantification) of the SLC8A1 protein in PQ-exposed SH-SY cells, with Tubulin as a loading control, T-test p*=0.015. n=3 biological replicas for each condition. Data presented as mean ± SD.",
    "molecules": "PQ"
  },
  {
    "caption": "(A) qPCR measurements of circSLC8A1 and SLC8A1 mRNA after SH-SY treatment of statins or the PF LRRK2 inhibitor, T-test p**=0.007 and p**=0.01 for PF-06447475 and p*=0.047 and p=*0.0136 for Statins for cirSLC8A1 and SLC8A1 mRNA respectively",
    "molecules": "PF, PF-06447475, statins, Statins"
  },
  {
    "caption": "(B,C) Protein gel and quantification of SLC8A1 and Tubulin as a loading control in Statin and PF treated neuronal cell cultures, T-test p*=0.027 for Statins and p*=0.011 for PF-06447475, n=3 biological replicas for each condition. Data presented as mean ± SD.",
    "molecules": "PF, PF-06447475, Statin, Statins"
  },
  {
    "caption": "(D) SLC8A1 immunostaining of statin-treated SH-SY cells.",
    "molecules": "statin"
  },
  {
    "caption": "(H) relative expression of known miR-128 targets in SH-SY cells treated with Paraquat, T-test p*=0.03, p*=0.02 and p*=0.002 for AXIN1, BM1 and SIRT1 respectively, Data presented as mean ± SD with 4-6 biological replicas for each condition.",
    "molecules": "Paraquat"
  },
  {
    "caption": "Luciferase-based imaging of drug response in Vegfr3Luc;Tyr:CreERT2;BrafV600E; Ptenflox/flox mice. Panels labeled as \"basal\" and \"induced\" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) Treatment with anti-PD-L1 antibody (αPD-L1; clone 10F.9G2, 3 weeks) or the corresponding control IgG (200 ug/dose, twice per week, 3 weeks); vemurafenib (Vem, 50 mg/Kg, oral once per day, 3 weeks) or BO-110 (BO, 0.8 mg/kg, twice per week, 3 weeks). Scale: p/s/cm2/sr (x106).",
    "molecules": "4OH-tamoxifen, BO, BO-110, Tyr, Vem, vemurafenib"
  },
  {
    "caption": "Treatment with BO-110 of human patient-derived xenografts (PDX) implanted in Vegfr3Luc nu/nu. 42 days after implantation (when systemic luciferase was detected), animals were randomized for treatment with vehicle (V) or with 0.8 mg/kg BO-110 (BO, twice per week), and luciferase emission was acquired at the indicated  times. Scale, p/s/cm2/sr (x106).",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0.5 ug/ml  BO-110 (BO), 10 µM vemurafenib  (Vem), or vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by t-test.   qRT-PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0.5 or 1 ug/ml BO-110 (VO), 10 µM vemurafenib (Vem), or the corresponding vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by ANOVA.   ",
    "molecules": "BO, BO-110, Vem, vemurafenib"
  },
  {
    "caption": "Luciferase signal driven by FLT4 (VEGFR3)-promoter transduced into HLEC treated with vehicle (v) or BO-110 (BO) at the indicated doses (ug/ml) as indicated in methods. Results were normalized to vehicle control. N=4 biological replicates. Error bars correspond to mean ± SD. Statistical significance was determined by ANOVA.",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "Relative mRNA levels of VEGFC  and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle (V) or 0.5 µg/ml BO-110 (BO), as determined by qRT-PCR. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "Inhibitory effect of the indicated doses of BO-110 (in µg/ml) or vehicle (V) on MDK mRNA  expression  determined by qRT-PCR in SK-Mel-147 (16 h after treatment). Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD. Statistical significance was determined by ANOVA.   qRT-PCR  analysis of relative mRNA levels of MDA5 16 h after treatment of SK-Mel-147 with the indicated doses of BO-110 (in µg/ml) (BO). Data correspond to the mean ± SD of three experiments with three technical replicates. Statistical significance was determined by ANOVA.   ",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "Analysis of apoptotic cells at the indicated time points. SK-Mel-147 cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "Immunohistochemical analysis of MDK repression (pink staining) in SK-Mel-147 xenografts  and PDX lesions after treatment with BO-110 (BO, 0.8 mg/kg, 2 doses/week). Histological staining in tumors extracted from animals treated with vehicle control (V) are included as a reference. Nuclei were counterstained  in with  hematoxylin.",
    "molecules": "hematoxylin, BO, BO-110"
  },
  {
    "caption": "Type I IFN mRNA induction (IFNA2 and IFNB1) in SK-Mel-147 melanoma cells treated for 16 h with the indicated amounts of BO-110 (in mg/ml). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Statistical significance was determined by ANOVA.",
    "molecules": "BO-110"
  },
  {
    "caption": "Heatmap depicting expression changes in interferon-related genes (GO:0034340) in SK-Mel-147 melanoma cells (left panel) and HLEC (right panel) treated with vehicle or 0.5 g/ml of BO-110 for 10 hours.  CD274, LAG3 and PDCD1 genes were also included as a reference.",
    "molecules": "BO-110"
  },
  {
    "caption": "IFNB1  mRNA induction analyzed by qPCR at the indicated times  after BO-110 treatment (0.5 µg/ml) of SK-Mel-147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control.",
    "molecules": "BO-110"
  },
  {
    "caption": "Quantification of the impact of  BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on  MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3  mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate.",
    "molecules": "BO-110"
  },
  {
    "caption": "Growth  of B16 melanoma xenografts in siblings of  Ifnar1+/+, Ifnar1+/- or Ifnar1-/- mice. Treatment started 10 days after tumor cell implantation. BO-110 was administered at 0.8 mg/kg, every third day for 2 weeks. N= 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by Two-Way ANOVA.",
    "molecules": "BO-110"
  },
  {
    "caption": "Impact of BO-110 on tumor-bearing mice implanted with syngeneic B16R2L (5 x 105 cells). When tumors have an average size on 100 mm3, mice were randomized into two groups and treated with BO-110  (BO) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points. , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "Efficacy of BO-110  as an adjuvant (preventing relapse after surgical removal of the primary lesion). Shown are representative examples of  Vegfr3Luc mice implanted with mCherry-SK-Mel-147 and imaged for luciferase  emission (prior to and after tumor removal. Animals were left to recover from surgery (4 days) and then treated for 2 weeks (4 dosis) with 0.8 mg/Kg  BO-110 or vehicle control (n= 8 for control and n=10 for treatment arm). Scale, p/s/cm2/sr x106.   mCherry emission from tumor cells of the animals in (A). Scale, p/s/cm2/sr x109.   ",
    "molecules": "BO-110"
  },
  {
    "caption": "Kaplan-Meier survival curves of animals treated as in (A). 8/8 animals treated with vehicle (V) control had to be sacrificed for humane reasons 110 days after surgery. 9/10 animals in the BO-110 arm (BO) remained tumor-free 8 months after stopping treatment. The grey box marks the period of treatment with BO-110. Statistical significance was determined by logrank test.",
    "molecules": "BO, BO-110"
  },
  {
    "caption": "(F) Confocal  images of DRAQ5 staining, showing double stranded DNA (top), immunostaining  of H3K27ac (middle), and Nanog-EGFP  (bottom), along with the progression of differentiation (+2i, +LIF, −LIF, and −LIF 2d).",
    "molecules": "double stranded DNA, DRAQ5"
  },
  {
    "caption": "(G) Swarmplots and violinplots of coefficient of variation of DRAQ5  fluorescence in a nucleus  of an mESC  expressing Nanog-EGFP   at the condition of +2i  (red, n = 20 cells), +LIF  (blue, n = 20), −LIF (green, n = 23), −LIF+TSA  (light green, n = 22), −LIF2d (purple, n = 21), and −LIF2d+TSA  (pink, n = 19). Double asterisks and single asterisks indicate p-value < 0.05 and 0.05 ≤ p-value < 0.1, respectively, in Student's t-test.",
    "molecules": "DRAQ5, TSA"
  },
  {
    "caption": "(D) Effect of 0.5 µM TSA addition on log10g0 of Nanog-EGFP  (left) and Oct4-EGFP  (right) in +LIF  (filled) and -LIF  (opened). Error bars represent standard deviations. For Nanog-EGFP, N = 65 for +LIF  -TSA, 91 for +LIF  +TSA, 66 for -LIF  -TSA, and 88 cells for -LIF  +TSA. For Oct4-EGFP, N = 53 for +LIF  -TSA, 42 for +LIF  +TSA, 58 for -LIF  -TSA, and 39 cells for -LIF  +TSA. Exclamation marks and double exclamation marks indicate p-values of less than 0.05 and 0.01, respectively, in Mann Whitney's U-test.",
    "molecules": "TSA"
  },
  {
    "caption": "(C) Immunofluorescence  analysis of TFAM  and Phalloidin staining  in human HCC  cells from paired clinical samples (n=100). The 3D reconstruction of z-stacks from nuclear  region is showed in the right side. Red: LifeAct, Blue: DAPI, Green: TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.",
    "molecules": "LifeAct, DAPI, Phalloidin"
  },
  {
    "caption": "(F) Western blot  analysis for the distribution of  mDia2  in SNU-368 cells incubated with malonyl-CoA  (5 μM) or  ND-630 (10 μM) (n = 3 independent experiments).",
    "molecules": "ND-630, malonyl-CoA"
  },
  {
    "caption": "(A) Flow cytometry analysis of l(2)mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2)mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006).",
    "molecules": "3MA, Baf, nutrient"
  },
  {
    "caption": "(B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium.",
    "molecules": "nutrient"
  },
  {
    "caption": "(A) Germaria of the nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 B. DAPI staining of nuclei is shown in blue. (B) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 C.",
    "molecules": "nutrient"
  },
  {
    "caption": "(A) Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient-deprived w1118 files. In Dcp-1Prev flies, fewer TUNEL-positive cysts in region 2 were observed. Under well-fed conditions, numerous TUNEL-positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. (B) Numerous degenerating stage 8 egg chambers (arrows) with Bruce-positive staining (green) were observed in well-fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right.",
    "molecules": "DNA, nutrient"
  },
  {
    "caption": "(A) TUNEL-positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14). DAPI staining of nuclei (white) is shown on the right. (B) In nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. (C) Dying stage 8 egg chambers (arrows) from nutrient-deprived control siblings (Atg1Δ3D/TM3) generated from the same cross in D had abundant TUNEL-positive staining. (D) In nutrient-deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls.",
    "molecules": "DNA, nutrient"
  },
  {
    "caption": "Primary wild type MEF were treated with DMSO or Aurora kinase inhibitor ZM447439 (ZM, 2μM) for 72h, followed by RNA isolation and mRNA-sequencing. (A) Volcano plot representation of differentially expressed genes (DEGs) between ZM and DMSO treatment. Adjusted p value ≤ 0.01 and log2 FC ≥ 1 or log2 FC ≤ -1.",
    "molecules": "DMSO, ZM, ZM447439"
  },
  {
    "caption": "(A) Time-lapse analysis of NF-κB-driven EGFP reporter activity in A549kB cells where cytokinesis failure was induced by treatment with ZM (2μM ) or DHCB (4μM). Single cells were imaged every 20 min using IncuCyte life cell imaging technology. Left, representative images of A549kB cells 48h after starting the treatment. Scale bars: 10 μm. Right, EGFP integrated fluorescence intensity of individually tracked cells was measured over time. Time zero was determined as the moment of cytokinesis failure (CF) of each individual cell analyzed. Dashed lines indicate the integrated EGFP intensity of the cells at time zero that was used to calculate the fold-change over time. Solid lines indicate the mean EGFP integrated fluorescence intensity of the cells 24 h after failing cytokinesis. An n ≥ 10 cells per condition were followed individually in a single experiment, data points represent means ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "DHCB, ZM"
  },
  {
    "caption": "(B) Top, experimental design. Left, densitometric analysis of IKBα protein levels in synchronized A549 cells detected over time after release. Bars represent ZM values normalized with respect to DMSO. n=3 independent experiments, data represent mean values ± SEM. Right, time course immunoblot analysis of synchronized A549 cells, released in DMSO or ZM (2μM). Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "DMSO, ZM"
  },
  {
    "caption": "(D, E) (D) Western blot analysis confirming lack of PIDD1 expression in A549 cells and (E) MDM2 processing after induction of cytokinesis failure using ZM (2µM) for 48h. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "ZM"
  },
  {
    "caption": "(F) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal A549κB cells. NF-κB activity of ZM-treated cells is represented as fold-change with respect their untreated counterparts (DMSO-treated cells, grey lines). n=5 independent experiments. Error bars represent ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "DMSO, ZM"
  },
  {
    "caption": "(A) Representative immunofluorescence images of γ-tubulin staining to mark and enumerate centrosomes in A549κB reporter cells. Cells were pre-treated for 24 h with centrinone (125nM) or solvent control (DMSO) and cultured further in medium either containing DMSO or ZM to promote cytokinesis failure. Size bar: 10µM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "centrinone, DMSO, ZM"
  },
  {
    "caption": "(B) Cells treated as indicated in A were analyzed for NF-κB-driven EGFP-reporter activation by IncuCyte live-cell imaging. Left, representative pictures of live-cell imaging NF-κB activation after treatment with DMSO, ZM (2μM, 48 h) or TNF (20 ng/mL, 24h). Size bar: 20µm. Right, EGFP MFI quantification of ZM-treated cells was plotted as fold-change expression relative to DMSO-treated controls. Data points represent means of n=4 biological replicates ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "DMSO, ZM"
  },
  {
    "caption": "(C) Left, representative immunofluorescence images of supernumerary centrosomes originated from Dox-inducible PLK4 overexpression in CRISPR-edited polyclonal U2OSPLK4 cells. Size scale bar: 10µm. Right, representative cell cycle profiles of U2OSPLK4 control and PIDD1-deficient cells. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "Dox"
  },
  {
    "caption": "(E) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal PIDD1-competent or -deficient U2OSκB-PLK4 and A549κB-PLK4 cells. NF-κB activity of ZM, Dox or TNF-treated cells is represented as fold-change with respect to their untreated counterparts. Bars represent means of n=5 independent experiments ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "Dox, ZM"
  },
  {
    "caption": "(B) Left, immunoblot of CRISPR-edited polyclonal RIPK1- or NEMO-deficient A549κB derivatives. Right, luciferase assay of A549 cells indicated genotypes, treated for 48h with either ZM (2 μM) or DHCB (4 μM). n=4 independent experiments. Data are represented as the mean values ± SEM. The statistical significance was determined using two-way ANOVA; p< 0.01 (**);p< 0.001 (***); p< 0.0001 (****).",
    "molecules": "DHCB, ZM"
  },
  {
    "caption": "(C) Representative confocal fluorescence images documenting centrosome accumulation, as identified by CEP63 staining in 293T-Trex-Flip-in cells. Time course analysis was performed in cells synchronized in G1/S after a 24h single thymidine block and release in either DMSO or ZM (2 μM). Cells were fixed at the indicated times for IF analyses. Scale bars: 10 μm.",
    "molecules": "DMSO, thymidine, ZM"
  },
  {
    "caption": "(D) Different time point immunoprecipitations (IP) after release in ZM (2 μM) of endogenous RAIDD, RIPK1 and NEMO proteins in PIDD1-deficient 293T-Trex-Flip-in cells re-expressing a FLAG-tagged PIDD1-FL protein. One representative experiment out of three is shown.",
    "molecules": "ZM"
  },
  {
    "caption": "(E) Analysis of NEMO-PIDDosome formation in PIDD1-deficient 293Trex-Flip-In cells re-expressing different PIDD1 mutants depicted in G. Cells were treated as in C and processed for FLAG-IP 11h after release into DMSO or ZM (2 μM). One out of two independent experiments is shown.",
    "molecules": "DMSO, ZM"
  },
  {
    "caption": "Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (B) qRT-PCR analysis confirm expression of different pro-inflammatory genes in Pidd1+/+ but not Pidd1-/- MEF. Hprt1, β-actin and Gapdh were used as housekeeping gene expression controls. n=3 independent samples; data represent mean values ± SEM. The statistical significance was determined using two-way ANOVA test; p ≤ 0.01 (**), p ≤ 0.001 (***).",
    "molecules": "DMSO, ZM"
  },
  {
    "caption": "Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (D) qRT-qPCR analysis of macrophage activation markers indicative of a pro-inflammatory state. Relative expression values are plotted as the fold-change expression relative to BMDM stimulated with conditioned medium from untreated MEFPLK4. β-actin was used as housekeeping gene for normalization. Bars represent mean ± SEM (n=9) biological replicates. The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).",
    "molecules": "DMSO, ZM"
  },
  {
    "caption": "A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (A) Representative images of target A549EGFP cells pre-treated for 72 h with ZM (2µM) and co-cultured subsequently with NK-92 cells in a 1:10 target to effector ratio. Interactions were recorded by live-cell imaging for 18 h at a 1 min interval. White arrow indicate polyploid cell undergoing cell death. EGFP release coincides with loss of cell membrane integrity. All images were acquired at the same exposure time and light intensity. Scale bar: 10 µm. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).",
    "molecules": "DMSO, ZM"
  },
  {
    "caption": "A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (D) Confluence of ZM pre-treated A549EGFP cells expressing or lacking PIDD1 was measured over time in presence of NK cells and normalized with respect to their DMSO treated counterparts. Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥4 independent experiments performed in duplicates, mean ± SEM. (E) Confluence measurement over time of A549EGFP derivatives impaired in either NEMO-PIDDosome (RIPK1 KO) or Caspase-2-PIDDosome formation (RAIDD KO). Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥3 independent experiments; mean ± SEM; unpaired t-test. (F) Confluence analysis of Dox pre-treated A549EGFP cells overexpressing PLK4 was performed over time in the presence of NK cells and normalized with respect to their DMSO treated controls. n=5 independent experiments; mean ± SEM. (G) Confluence analysis of centriole-depleted and ZM pre-treated A549EGFP cells cultured in presence of NK cells. Values are normalized with respect to their DMSO ± Centrinone (125nM) treated controls. n=4 independent experiments; mean ± SEM. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).",
    "molecules": "Centrinone, DMSO, Dox, ZM"
  },
  {
    "caption": "C, D Cr-release assay showing % specific lysis of MCF7 cells by survivin-specific T cells at different ratios upon CCR9 knockdown (C) or overexpression (D). MCF7 cells were transfected with either CCR9 siRNA s1 (Δ), pooled siRNA sequences (○), positive control PD-L1 (□), and non-specific control siRNA (▪) (C) or with pCMV6-AC-His control vector (▪) and pCMV6-AC-His-CCR9 expression construct (○) (D) 72 h prior to the assay.",
    "molecules": "His"
  },
  {
    "caption": "c and d. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG old Col-0 roots 12 HAT to ammonium (C) or nitrate (D) supplemented media. The first 20 epidermal (e) and cortex (c) cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (C), and in blue and green on nitrate (D), respectively. Scale bar=30 µm. Column bars denote the geometric mean of the cell lengths at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 3.32639+0.17172 (ammonium, epidermis), 1.22033+0.08754 (ammonium, cortex) and 1.70502+0.09532 (nitrate, epidermis), 0.82342+0.06973 (nitrate, cortex). Data are derived from 3 biological replicates, n=18 roots in each case.",
    "molecules": "ammonium, nitrate"
  },
  {
    "caption": "a and b. Maximum intensity Z-stack projection images of 5 DAG old roots expressing the R2D2 auxin signaling reporter 12 HAT to ammonium (a) or nitrate (b) supplemented media. White arrows mark the position of the 10th and 20th cells from QC; "e" and "c" mark epidermis and cortex, respectively. Scale bar=50 µm. Graphs denote normalized relative auxin levels at the respective positions. Lines represent polynomial regression fit with 95% confidence band. Data are derived from 5 roots per condition from 3 biological replicates.",
    "molecules": "R2D2, ammonium, auxin, nitrate"
  },
  {
    "caption": "b and c. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG eir1-4 roots 12 HAT to ammonium (B) or nitrate (C) supplemented media. The first 20-20 epidermal and cortex cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (B) and in blue and green on nitrate (C), respectively. Scale bar=30 µm. Column bars denote the geometric mean of cell length at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 0.75884+0.02624 (ammonium, epidermis), 1.13088+0.08446 (ammonium, cortex) and 2.06912+0.10341 (nitrate, epidermis), 0.99878+0.07278 (nitrate, cortex). Data are derived from 3 biological replicates, n=9 (ammonium) and 8 (nitrate) roots.",
    "molecules": "ammonium, nitrate"
  },
  {
    "caption": "b. Higher magnification of pseudo-colored confocal images of 5 DAG old roots expressing PIN2-GFP 12 HAT to ammonium or nitrate supplemented media. \"e\" denotes epidermis and \"c\" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=12 µm. White stars mark PIN2-GFP protein localization on the lateral membranes. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes divided by the signal value at inner/outer membranes) of roots on ammonium (n=31 cells from 6 roots) or nitrate (n=24 cells from 6 roots) supplemented medium. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.",
    "molecules": "ammonium, nitrate"
  },
  {
    "caption": "d. Microscopic images showing PIN2-Dendra fluorescent signal five hours after photoconversion of PIN2-Dendra into its red form. Depletion of the red signal and recovery of the green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media. Note the increase in the intensity of the green signal in roots transferred to nitrate. Graph represents the mean signal + SD (n=6 roots per condition, 20 cells per root analyzed). The experiment was repeated 3 times. Scale bar=20 µm.",
    "molecules": "ammonium, nitrate"
  },
  {
    "caption": "d. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) in roots transferred to ammonium ((grey, epidermis (ep) and red, cortex (co) and to nitrate (blue, epidermis (ep) and green, cortex (co)). 5 cells per roots were analyzed in at least 9 roots per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.",
    "molecules": "ammonium, nitrate"
  },
  {
    "caption": "g. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes vs inner/outer membranes) of P2wt, P2D and P2A roots transferred to ammonium (grey) or nitrate (red) supplemented medium. At least 24 cells from 5 roots were analyzed per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.",
    "molecules": "ammonium, nitrate"
  },
  {
    "caption": "Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7 or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment.",
    "molecules": "SYBR Gold, agarose, DNA"
  },
  {
    "caption": "U2OS cells were transfected with the indicated siRNA. 48h post-transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0, 1, 2, 4 and 24 hr post NCS treatment. Flow Cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal.",
    "molecules": "NCS"
  },
  {
    "caption": "U2OS cells stably expressing HA-REV7 (Top) or HA-SHLD2 (Bottom) were pre-sensitized with 10ug/mL Hoescht 33342 before exposed to UV micro-irradiation. Immunofluorescence against endogenous HA and γ-H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.",
    "molecules": "Hoescht 33342"
  },
  {
    "caption": "U2OS LacR-Fok1 cells were transfected with GFP or GFP-SHLD2 and 24 h later DNA damage was induced using Shield-1 and 4-OHT. The cells were then processed for GFP and mCherry immunofluorescence. Shown are representative micrographs. Scale bar = 5µm. Quantification of the experiments shown in (C). Data are represented as the mean ± SD (n=3). At least 100 cells per condition were counted.",
    "molecules": "4-OHT, Shield-1"
  },
  {
    "caption": "293T cell lines expressing ER-AsiSI with Flag-SHLD2 and treated with 1 µM of 4-OHT. 6h later the cells were processed and immunoprecipitated with Anti-FLAG Magnetic Beads and anti-γ-H2AX.x/Protein A/G magnetic beads. DNA was purified and subjected to qPCR detection. Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input (Bottom).",
    "molecules": "Protein A/G, 4-OHT, DNA"
  },
  {
    "caption": "U2OS mCherry-LacR-Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP-SHLD2 construct. 24 h post-transfection DNA damage was induced using Shield-1 and 4-OHT. The cells were then fixed and analyzed for the intensity of the GFP-SHLD2 signal at mCherry-LacR-Fok1 focus. Shown is the quantification of the GFP-SHLD2 signal at the Fok1 focus. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. At least 75 cells were counted per condition.",
    "molecules": "4-OHT, Shield-1"
  },
  {
    "caption": "293T cells were transfected with Flag-REV7 and GFP-SHLD2 expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP, REV7 and p-Chk1 antibodies.",
    "molecules": "DMSO, KU-60019"
  },
  {
    "caption": "CH12F3-2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.",
    "molecules": "IgA"
  },
  {
    "caption": "U2OS cells were transfected with the indicated siRNAs and then treated with CldU, IdU and NCS 48h post-transfection as indicated. The slides were stained, dehydrated, mounted and visualized and shown is the quantification of CldU/IdU tract length in order to visualize DNA end-resection (Right). At least 500 DNA tracks were measured per condition.",
    "molecules": "IdU, CldU, NCS"
  },
  {
    "caption": "Genes significantly enriched or dropped out after a 14-day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated. Ranking was determined based on the Log2 fold score (Left). The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change (Fc) in Log2.",
    "molecules": "Doxorubicin"
  },
  {
    "caption": "293T cells were transfected with Flag-REV7 and GFP-SHLD1 (Left) or Flag-SHLD2 and GFP-SHLD1 (Right) expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies.",
    "molecules": "DMSO, KU-60019"
  },
  {
    "caption": "CH12F3-2 cells stably expressing either shCTRL, shSHLD1#1 or shSHLD1#2 were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.",
    "molecules": "IgA"
  },
  {
    "caption": "OTULIN DNA sequence chromatograms showing the homozygous single base substitution (c.841G>A, p.Gly281Arg, arrowhead). Data are representative to two independent experiments.",
    "molecules": "DNA"
  },
  {
    "caption": "Affinity measurements by fluorescence polarisation (FP) with recombinant catalytically inactive OTULINC129A or OTULINC129A/G281R and FlAsH-labelled M1-linked diUb. Data represent mean ± SD of one experiment performed in technical triplicate. Data are representative of three independent experiments. a.u., arbitrary units. K­D­, dissociation constant.",
    "molecules": "FlAsH, M1, Ub"
  },
  {
    "caption": "M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.",
    "molecules": "M1, silver, Ub"
  },
  {
    "caption": "First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.­",
    "molecules": "tryptophan"
  },
  {
    "caption": "Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.",
    "molecules": "MG132"
  },
  {
    "caption": "Immunoblot (left) and densitometry (right) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), as indicated was analysed using MTT reduction assays.",
    "molecules": "CHX"
  },
  {
    "caption": "Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.",
    "molecules": "CHX, Nec-1, Q-VD-OPh"
  },
  {
    "caption": "Viability of shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL),",
    "molecules": "CHX"
  },
  {
    "caption": "shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.",
    "molecules": "CHX, Nec-1, Q-VD-OPh"
  },
  {
    "caption": "Increased apoptotic cell death in the skin of ORAS patient III.2. Serial sections of normal skin (top panels) and a skin biopsy from patient III.2 taken at an inflammatory flare (bottom panels) were immunostained for cleaved caspase-3 (centre panels) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (right panels). Arrowheads indicate cleaved caspase-3-positive mesenchymal cells. None \u2028of these markers were present in the healthy control skin. H&E, haematoxylin and eosin. Boxes in H&E panels indicate the areas magnified in the cleaved caspase-3 and TUNEL panels. Scale bars, 100 μm. \u2028Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III.2.",
    "molecules": "dUTP, eosin, haematoxylin"
  },
  {
    "caption": "Photograph of patient III.2 at age ~10 years at an episode of inflammation caused by delayed etanercept administration. Arrows indicate the erythematous subcutaneous nodules (panniculitis).",
    "molecules": "etanercept"
  },
  {
    "caption": "D qRT-PCR analysis of G-rich TERRA, polyA+ TERRA and ARRET/αARRET levels in cells as in (B). Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains (ter1+ and est1+) grown in parallel. Bars and error bars are averages and SD from 4 (ter1Δ clones) or 3 (est1Δ clones) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to wt; two-tailed Student's t test).",
    "molecules": "G-rich, polyA"
  },
  {
    "caption": "H qRT-PCR analysis of total TERRA and polyA+ TERRA in cellular fractions as in (G). Values are expressed after normalization to total cellular extracts. Bars and error bars are averages and SD from 3 independent experiments.",
    "molecules": "polyA"
  },
  {
    "caption": "A RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).",
    "molecules": "G-rich, polyA"
  },
  {
    "caption": "B RIP experiments as in (A) where, prior to RNA extraction, washed beads were resuspended in DNaseI buffer (DNbuff) and incubated for 1 hour at 30° C in presence or absence of DNaseI. Values represent fractions of input polyA+ TERRA detected in immunoprecipitated material expressed as fold increase over an untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).",
    "molecules": "polyA"
  },
  {
    "caption": "C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student's t test).",
    "molecules": "polyA"
  },
  {
    "caption": "D qRT-PCR analysis of tiTERRA in cells carrying no tiTEL (CAF13), one tiTEL (CAF545) or two tiTELs (CAF110) cultured in EMM medium in presence or absence of Thiamine (THI; repressed and induced condition, respectively) for 24 h. RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA (graph on the left) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA (on the right). Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions (THI+). Bars and error bars are averages and SD from 3 independent experiments. * P < 0.05, ** P < 0.01 (relative to CAF545 THI+; two-tailed Student's t test).",
    "molecules": "THI, Thiamine"
  },
  {
    "caption": "E Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs (CAF110) treated with combinations of THI and Trichostatin A (TSA) for 24 h. Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C-rich (oC) or G-rich (oG) telomeric repeats. To confirm similar hybridization efficiencies of the two oligonucleotides, northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA (upper right insets). After signal detection, RNAmembranes were stripped and re-hybridized with U6 probes to assure equal loading. Marker molecular weights are on the left in kilobases.",
    "molecules": "DNA, G-rich, THI, Trichostatin A, TSA"
  },
  {
    "caption": "A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs (CAF110) grown for 24 h in EMM medium containing THI and TSA as indicated. DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe. Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.",
    "molecules": "THI, TSA"
  },
  {
    "caption": "C ter1+ cells carrying two tiTELs (CAF110, left panel) or trt1Δ cells carrying one tiTEL (MKSP2104, central panel) were cultured in EMM medium in presence of THI for 24 h (THI+) or in absence of THI for 24, 48 or 72 h. In the right panel, cells carrying two tiTELs (CAF110) were treated with TSA for 24 h in the presence or absence of THI. TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform. The statistical analysis for each set of conditions is displayed above each panel. P values for each comparison were derived from the Student's t test between each condition and the THI+ sample using Welch's correction when appropriate. The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red (center panel).",
    "molecules": "THI, TSA"
  },
  {
    "caption": "A qRT-PCR analysis of G-rich and polyA+ tiTERRA in cells carrying one tiTEL and expressing Trt1-myc (CAF610) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h. Values are normalized to ACT1 mRNA and expressed as fold increase over THI+ TSA- samples. Bars and error bars are averages and SD from 4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, (relative to THI+ TSA-; two-tailed Student's t test).",
    "molecules": "G-rich, polyA, THI, TSA"
  },
  {
    "caption": "C RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test).",
    "molecules": "G-rich, polyA"
  },
  {
    "caption": "D ChIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qPCR using tiTEL specific oligonucleotides. Values correspond to fraction of input DNA expressed as fold increase over THI+ TSA-. Unt: untagged strain (CAF545). Bars and error bars are averages and SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (relative to THI+ TSA-; two-tailed Student's t test).",
    "molecules": "THI, TSA"
  },
  {
    "caption": "except that cells were preextracted in 0.2% Triton X-100, fixed and immunostained with SUMO2/3 antibody. Representative images are shown. Scale bar, 5 μm.",
    "molecules": "Triton X-100"
  },
  {
    "caption": "Relative DPC levels in cells treated were quantified using a KCl/SDS precipitation assay (mean±SD; n=3 independent experiments).",
    "molecules": "KCl, SDS"
  },
  {
    "caption": "HeLa cells were treated with formaldehyde for 1 h, and where indicated propagated for an additional h in the absence of formaldehyde (recovery). Cells were then fractionated into soluble and chromatin-enriched fractions and immunoblotted with SUMO2/3 and SUMO1 antibodies.",
    "molecules": "formaldehyde"
  },
  {
    "caption": "Mass spectrometry-based analysis of formaldehyde-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database, at the default interaction confidence setting of 0.4. Proteins not connected to the network were omitted.",
    "molecules": "Ni-NTA, formaldehyde"
  },
  {
    "caption": "SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies.",
    "molecules": "formaldehyde"
  },
  {
    "caption": "HeLa cells were transfected with non-targeting control (CTRL) or DNMT1 siRNAs, exposed or not to 5-azadC and collected 2 h later. Chromatin-enriched fractions were immunoblotted with antibodies to SUMO2/3, DNMT1 and MCM6 (loading control).",
    "molecules": "5-azadC"
  },
  {
    "caption": "Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with indicated siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).",
    "molecules": "5-azadC"
  },
  {
    "caption": "Extracts of HeLa/GFP-DNMT1 cells treated with 5-azadC for the indicated times were subjected to GFP immunoprecipitation (IP) under denaturing conditions followed by immunoblotting with SUMO2/3 and GFP antibodies.",
    "molecules": "5-azadC"
  },
  {
    "caption": "HeLa cells were subjected to consecutive rounds of transfection with DNMT1 siRNA targeting the UTR and expression plasmid encoding WT or catalytically inactive (CI) GFP-DNMT1. Cells were then left untreated or incubated with 5-azadC for 2 h, and SUMOylation of GFP-DNMT1 was analyzed Asterisk denotes unmodified GFP-DNMT1 recognized by the SUMO2/3 antibody due to weak cross-reactivity.",
    "molecules": "5-azadC"
  },
  {
    "caption": "Mass spectrometry analysis of 5-azadC-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to 5-azadC were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. Proteins displaying significantly altered SUMOylation in response to 5-azadC treatment relative to the control condition were visualized through two-sample t-testing using permutation-based FDR to achieve q-values of <0.05",
    "molecules": "Ni-NTA, 5-azadC"
  },
  {
    "caption": "SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown. Both control (CTRL) and DNMT1 siRNA-transfected cells were treated with 5-azadC.",
    "molecules": "5-azadC"
  },
  {
    "caption": "Chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5-azadC for 2 h were analyzed by immunoblotting with SUMO2/3 and Actin antibodies.",
    "molecules": "5-azadC"
  },
  {
    "caption": "HeLa cells stably expressing GFP-DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5-azadC for 2 h. Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal. Mean detergent-resistant GFP signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.",
    "molecules": "5-azadC"
  },
  {
    "caption": "Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).",
    "molecules": "5-azadC"
  },
  {
    "caption": "untransfected HeLa cells were exposed or not to 5-azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor (SUMO-E1i).",
    "molecules": "5-azadC, SUMO-E1i"
  },
  {
    "caption": "HeLa cells treated with 5-azadC for 2 h in the presence or absence of SUMO-E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal, and immunostained with DNMT1 antibody. Mean detergent-resistant DNMT1 signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.",
    "molecules": "5-azadC, SUMO-E1i"
  },
  {
    "caption": "Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5-azadC doses for 2 h was assayed",
    "molecules": "5-azadC"
  },
  {
    "caption": "iPOND analysis of 5-azadC-induced DNMT1 trapping and SUMOylation. HeLa cells were mock-treated or pre-incubated with 5-azadC for 5 min before addition of EdU for 10 min. Cells were then washed and incubated with thymidine for 0 (pulse) or 60 min (chase) before performing iPOND.",
    "molecules": "5-azadC, EdU, thymidine"
  },
  {
    "caption": "HeLa cells stably expressing GFP-DNMT1 were pre-incubated or not with SUMO-E1i for 30 min, and where indicated 5-azadC was added to the medium for an additional 60 min. Cells were then processed for GFP immunoprecipitation (IP) followed by immunoblotting for the indicated proteins.",
    "molecules": "5-azadC, SUMO-E1i"
  },
  {
    "caption": "Immunoblot analysis of chromatin-enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block, pulse-labeled with 5-azadC for 30 min and grown in the presence or absence of proteasome inhibitor (MG132) for the indicated times.",
    "molecules": "5-azadC, MG132, thymidine"
  },
  {
    "caption": "cells were treated or not with aphidicolin following pulse-labeling with 5-azadC.",
    "molecules": "5-azadC, aphidicolin"
  },
  {
    "caption": "Representative images of U2OS cells stably expressing GFP-SPRTN or GFP-ACRC that were transfected with control (CTRL) or UBC9 siRNAs, exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme (UBA1) inhibitor TAK-243 (Ub-E1i) as indicated and fixed one h later. Scale bar, 10 μm.",
    "molecules": "formaldehyde, TAK-243, Ub-E1i"
  },
  {
    "caption": "U2OS cells expressing GFP-ACRC were exposed to the indicated genotoxic agents (formaldehyde: 600 μM, 1 h; UV: 20 J/m2, 6 h recovery; hydroxyurea (HU): 2mM, 24 h; mitomycin C (MMC): 40 ng/mL, 24 h), preextracted and fixed, and analyzed by microscopy. Representative images are shown. Scale bar, 10 μm.",
    "molecules": "formaldehyde, HU, hydroxyurea, mitomycin C, MMC"
  },
  {
    "caption": "Representative images of U2OS cells expressing indicated GFP-ACRC alleles that were treated with 5-azadC in the presence or absence of SUMO inhibitor (SUMO-E1i), fixed 2 h later and immunostained with DNMT1 antibody. Scale bar, 10 μm.   Quantification of data in (G), showing proportion of cells displaying GFP-ACRC co-localization with DNMT1 foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).   ",
    "molecules": "5-azadC, SUMO-E1i"
  },
  {
    "caption": "Formaldehyde survival of wild type (wt), dvc-1, gcna-1 loss-of-function (lof) and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=4 independent experiments).",
    "molecules": "Formaldehyde"
  },
  {
    "caption": "Cisplatin survival of wild type, dvc-1, gcna-1 and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=3 independent experiments).",
    "molecules": "Cisplatin"
  },
  {
    "caption": "Formaldehyde survival of gcna-1 deletion (del) and E364Q mutant C. elegans (mean±SEM; n=2 independent experiments).",
    "molecules": "Formaldehyde"
  },
  {
    "caption": "Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).",
    "molecules": "Formaldehyde"
  },
  {
    "caption": "Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or gei-17 RNAi bacteria (mean±SEM; n=2 independent experiments).",
    "molecules": "Formaldehyde"
  },
  {
    "caption": "Formaldehyde survival of wild type and gcna-1 deletion (del) mutant C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).",
    "molecules": "Formaldehyde"
  },
  {
    "caption": "LC-MS/MS measurements of m6A levels in total RNA (B) or in poly(A)+ RNA (C) upon KD of predicted methyltransferases in Drosophila S2R+ cells. m6A abundance in total RNA is significantly reduced when Mettl5 is depleted, while its depletion has no effect on m6A level in mRNA. As expected, the KD of Mettl3 and Mettl14 reduce m6A levels in mRNA. Bar chart represents the mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).",
    "molecules": "m6A, poly(A)"
  },
  {
    "caption": "LC-MS/MS measurements of m6A levels in total RNA of WT and Mettl5 mutant flies. Bars represent mean ± standard deviation of measurements of three biological replicates. ***P<0.001 (two-tailed t-test).",
    "molecules": "m6A"
  },
  {
    "caption": "Purified 18S rRNA analyzed for its m6A content by quantitative HPLC. The 18S rRNA was extracted from 40S subunits isolated on sucrose gradients. The calibration control is a commercial source of m6A (in grey). m6A elutes at 48 min.",
    "molecules": "m6A"
  },
  {
    "caption": "Mature rRNA analysis on ethidium-stained denaturing agarose gels. The same amounts of total RNA extracted from the indicated flies, and from S2R+ cells depleted or not of Mettl5, were loaded. The 28S/18S ratio was established by densitometry.",
    "molecules": "ethidium"
  },
  {
    "caption": "Trmt112 depletion reduces m6A levels in total RNA from Drosophila S2R+ cells. Mean ± standard deviation of three technical measurements from three biological replicates. *P < 0.05 (two-tailed t-test).",
    "molecules": "m6A"
  },
  {
    "caption": "C57Bl/6 were infected with 107 CFUs of WT or CV8  strains and (B) Inflammatory profile of mouse lungs  inoculated with WT (dark blue), CV8  (light blue), or PBS  (grey). Gene expression  was analysed by RT-qPCR  using gapdh  as an endogenous control (see Materials and Methods). Data are shown as average ± SD of fold-change (FC) in mRNA expression (One-way ANOVA + Tukey post-hoc test; *P < 0.05).",
    "molecules": "PBS"
  },
  {
    "caption": "C57Bl/6 were infected with 107 CFUs of WT or CV8  strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (C) Histological  findings of mouse lung  samples at the analysed time points. Left, plots representing the quantitative evaluation (score: 0-5) of alveolar  infiltrate (top panel) and peribronquial/olar  infiltrate (FC) (bottom panel) (see Materials and Methods). Parameters were normalised using the average of the PBS  group (FC = sample value/average PBS; FC control group, ~1). Data are shown as average ± SD of FC (one-way ANOVA + Tukey post-hoc test) (*P < 0.05). Right, representative images of lungs stained  with hematoxylin-eosin  (the line represents 250 μm).",
    "molecules": "eosin, hematoxylin, PBS"
  },
  {
    "caption": "U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu-3 cells transfected with mCherry and SARS-CoV-2 Spike (S) or vector control. Cells were co-cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye. A) Representative example fluorescent imaging of the indicated co-cultures showing the presence of EGFP/mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs. Scale bar indicates 50 μm. B) Quantification of nuclei per EGFP/mCherry double positive cell syncytia in co-cultures involving the indicated IFITM3 constructs as compared to vector control. Bars depict averages of three independent experiments with circles representing the individual data points. For each condition, nuclei within 50-75 individual syncytia were counted. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test. ",
    "molecules": "Hoechst dye"
  },
  {
    "caption": " (D) Reno1 smFISH signal (red) and DAPI staining (blue) in WT and Reno1m/m cells at day eight of neuronal differentiation, imaged using 100x objective. ",
    "molecules": "DAPI"
  },
  {
    "caption": " (E) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation. Y axis: forward scatter; X axis: PI fluorescence intensity. ",
    "molecules": "PI"
  },
  {
    "caption": " (D) Expression levels of Reno1 following Dox addition in WT and Reno1m/m cells. Mean ± SEM is shown for three independent experiments, * P<0.05 (unpaired two sample t-test). ",
    "molecules": "Dox"
  },
  {
    "caption": " (E) Cell count of WT and Reno1m/m cells following four days of differentiation, with or without Dox addition. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. * P<0.05 (unpaired two sample t-test). ",
    "molecules": "Dox"
  },
  {
    "caption": " (F-G) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of WT (F) or Reno1m/m (G) cell treated with Dox for the first two days of differentiation compared to untreated cells, in genes which were significantly (P<0.05) downregulated (n=252) or upregulated (n=223) by at least two out of three Reno1 perturbations at day 2 in fig. 5A, compared to all genes (n=16,511). ** P<0.01 (two-sided Wilcoxon rank sum test). ",
    "molecules": "Dox"
  },
  {
    "caption": " (D) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells (H3K4me3 is from ENCODE project and Fam60a from (Streubel et al., 2017)). Coefficient and P-value computed using Spearman's correlation. n=49,722 peaks. ",
    "molecules": "H3K4me3"
  },
  {
    "caption": "(a) [3H]thymidine incorporation at 72 h by wild-type (WT) and Irgm1-/- CD4+ T cells activated with soluble mAb to CD3 and irradiated syngenic wild-type splenocytes.",
    "molecules": "[3H]thymidine"
  },
  {
    "caption": "(b) [3H]thymidine incorporation at 72 h by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA protein (OVA) or OVA peptide of amino acids 323-339 (OVA(323-339)) in the presence of syngenic splenic DCs.",
    "molecules": "[3H]thymidine"
  },
  {
    "caption": "(c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide.",
    "molecules": "[3H]thymidine"
  },
  {
    "caption": "(g) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b-d,g).",
    "molecules": "[3H]thymidine"
  },
  {
    "caption": "(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture.",
    "molecules": "[3H]thymidine"
  },
  {
    "caption": "(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells in the presence or absence of wortmannin or Ly294002 (g), assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).",
    "molecules": "Ly294002, wortmannin"
  },
  {
    "caption": "(I) Kymographs  of the SPB   (Sfi1-mCherry, yellow) and  histone H2B (Htb1-CFP, blue) in dhc1∆ mutant. At 20 min (white solid lines), dimethyl sulfoxide  (DMSO, as a negative control) or MBC  (final concentration of 20 µg/mL) were added. Dashed lines indicate cell shape. Data information: Scale bars, 5 µm.",
    "molecules": "MBC, dimethyl sulfoxide, DMSO"
  },
  {
    "caption": "A-D) Confocal image analysis of cryostat sections from adult CX3CR1+/GFP;Sfrp1+/βgal (A,B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1-/- (D) and mouse brains three days after intra-cortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A;B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar 25μm. E) ELISA determination of Sfrp1 levels in brain extracts from 3 months-old wt and Sfrp1-/- mice. WT mice were injected either with saline or LPS. Three days after injection the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1-/- mice used as negative control (n=5 mice for each group). Error bars represent Standard Error. Statistical significance: ns P>0.5 ****P<0.0001; One-way ANOVA followed by Bonferroni multiple comparisons test. ",
    "molecules": "LPS, saline"
  },
  {
    "caption": "C, D) Coronal sections from wt and Sfrp1-/- mouse brains three days after infusion of saline or LPS, immunostained for GFAP (cyan, C) or CD45 (magenta, D). The images are high power views of the somatosensory cortex (lower power view in Fig EV1D). Scale bar 60μm. E, F) The graphs show the normalised levels of immunoreactivity (IR) for GFAP (E) and CD45 (F, P=0,006) present in cortical sections (n=24 acquisitions white dots; from N=3 animals, black dots, per group) from wt and Sfrp1-/- animals infused with saline or LPS. Bars represent Standard Error. Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black. ** or ## P<0.01; *** or ### P<0.001; **** or #### P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. * and # indicate significance between genotypes and treatments, respectively. ",
    "molecules": "LPS, saline"
  },
  {
    "caption": "D) ELISA determination of SFRP1 levels present in the media of microglia (n=4 cultures per genotype), astrocytes (n=4 cultures per genotype) or mixed astrocytes and microglial (2:1, n=7 cultures per genotype) cultures derived from wt or Sfrp1-/- pups exposed for 24h to saline or LPS (1μg/ml). Error bars represent Standard Error. Statistical significance: *P<0.05; ***P<0.001; ****P<0.0001; One-way ANOVA followed by Bonferroni Test.",
    "molecules": "LPS, saline"
  },
  {
    "caption": "B, C) Volcano plots of differential gene expression from CX3CR1GFP/+ (B) or CX3CR1GFP/+;Sfrp1-/- (C) microglial cell in response to LPS. Data are represented as Log2 Fold Change vs -Log10 adjusted p-value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. Blue vertical lines represent an increase of 75 and 400% in the expression levels respectively. Green horizontal lines represent a 0.05 or 0.01 adjusted p-value of statistical significance respectively. Genes with an expression change higher than 75% and an adjusted p-value lower than 0.05 are highlighted in dark red (+FC) and dark blue (-FC). Genes with an expression change higher than 400% and an adjusted p-value lower than 0.01 are coloured in light red (+FC) and light blue (-FC).",
    "molecules": "LPS"
  },
  {
    "caption": "B) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains. Scale bar: 2 Kbp.",
    "molecules": "LPS, saline"
  },
  {
    "caption": "(C) The 3.1 Å 2Fo-Fc map (1.2 σ) around the DNA of the final refined ATPγS-MjMR-DNA complex structure.",
    "molecules": "ATPγS, DNA"
  },
  {
    "caption": "(D) A ribbon representation of the 3.1 Å ATPγS-MjMR-DNA structure showing the Mre11 nuclease, capping, and C-terminal three helix-bundle domain (light pink), Rad50 molecules (green and light blue), and the DNA (yellow and orange). ATPγS is shown in spheres. See Movie EV 1 for the movement of the complex.",
    "molecules": "ATPγS, DNA"
  },
  {
    "caption": "(A) A ribbon diagram showing the overall DNA recognition by the ATPγS-Rad50 dimer.",
    "molecules": "ATPγS, DNA"
  },
  {
    "caption": "(B) Close-up view of the interaction between the internal segment (major and minor grooves) of the DNA strand and the edge of lobe I of Rad50A (green). Loop α1-α2 wedge faces the minor groove between the 10th and 13' th phosphates of the template (orange) and non-template (yellow) strands, respectively. (C) Close-up view of the interaction between the DNA strand and the α1-α2 loop in an orientation different from Fig 2B.",
    "molecules": "DNA"
  },
  {
    "caption": "(B, C) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA (φx174RFII) in the absence (B) or presence (C) of AMP-PNP/Mg2+. Each protein sample was incubated with φx174RFII dsDNA (3.5 nM) for 30 min on 4°C. The following molar ratio of protein: DNA is used in Fig 3B-3E; lane 2, 50:1; lane 3, 100:1; lane 4, 200:1; lane 5, 500:1; lane 6, 1000:1; lane 7, 2000:1. We note that some MRcd variants may form a second form of the complex with DNA.",
    "molecules": "AMP-PNP, DNA, Mg2+"
  },
  {
    "caption": "(D, E) Interactions of the WT or five full-length TmMR mutants with φx174RFII were examined in the absence (D) or presence of AMP-PNP/Mg2+ (E). Reaction conditions are same as those in Fig 3B.",
    "molecules": "AMP-PNP, Mg2+"
  },
  {
    "caption": "(F) Nuclease activities of free MjMre11 (lane 3, 4), nucleotide unbound- (5, 6) and -bound MjMRcd complex (7, 8) towards a hairpin DNA. Each protein sample was incubated with a hairpin DNA (10 nM) in a 1:10 or 1:20 molar ratio (protein: DNA) for 30 min on 55°C. The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment.",
    "molecules": "DNA"
  },
  {
    "caption": "(G) Nuclease activities of free TmMre11 alone (lane 3, 4), nucleotide unbound- (lane 5, 6) and -bound (lane 7, 8) full-length TmMR complex towards a hairpin DNA.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Superposition of the ATPγS-MjMR-DNA complex (green) and apo-MjMR (3AV0, magenta) structures. Alignment was done using their NBDs (1.3 Å rmsd in the positions of 311 Cα atoms). The coiled-coils of the DNA-bound Rad50 molecules are shifted (arrow) to more parallel orientation. The entire structures can be superimposed with a 1.9 Å rmsd in 692 Cα positions.",
    "molecules": "ATPγS, DNA"
  },
  {
    "caption": "(B, C) Comparison of the local structures between the apo (B) and the DNA-bound MjMR complex (C). Upon DNA binding, helix α6 translates to DNA backbone, resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II. Arrows indicate the direction of the helix movement by DNA binding. See Fig EV5 for a close-up view.",
    "molecules": "DNA"
  },
  {
    "caption": "(A) Stereo view of the superposition of the ADP-MjRad50 (3AUX) complex (black) onto the ATPγS-MjMR-DNA complex (green and blue) by aligning their lobe II domains. The α1-α2 loop that could collide with parts of the DNA molecule is indicated. (B) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11, which could allow the access of the unwound DNA. Color schemes are same as those in Fig 5A.",
    "molecules": "ATPγS, ADP, DNA"
  },
  {
    "caption": "(C) DNA-unwinding activities of TmMR. Lane 2, no protein; lanes 3 and 4, no ATP; lanes 5 and 6, with ATPγS; lanes 7 and 8, with ATP. The DNA: protein ratio was 1:2 for lanes 3, 5, and 7, and 1:5 for lanes 4, 6, and 8. Plotted data represent the mean of three experiments, with error bars indicating one standard deviation. A graph of the quantified data is shown in the lower panel.",
    "molecules": "ATPγS, ATP, DNA"
  },
  {
    "caption": "(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.",
    "molecules": "ATP, DNA"
  },
  {
    "caption": "(A) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence (left panel) or presence of ATP (right panel). Major products are marked with dots. The 5'-32P-label is indicated by a star. See Appendix Fig S4A and S4B for the quantified values.",
    "molecules": "ATP, DNA"
  },
  {
    "caption": "(B) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP-PNP. See Appendix Fig S3E and S3F for the original data.",
    "molecules": "AMP-PNP, DNA"
  },
  {
    "caption": "(C) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence (left panel) or presence of ATP (right panel). See Appendix Fig S4C and S4D for the quantified values.",
    "molecules": "ATP, DNA"
  },
  {
    "caption": "(D) The effects of single or multiple point mutations in the DNA-contacting or neighboring residues of S. cerevisiae rad50 on the sensitivity to chemical drugs. Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT, PHL, and MMS were examined. (E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.",
    "molecules": "CPT, MMS, PHL"
  },
  {
    "caption": "(D) Histological  sections of patient  biopsies stained  with hematoxylin  and eosin (left panel) or with an  actin  antibody (right panel). Scale bars: skin: left 1000µm, right 100µm;  lung: left 500µm, right 250µm; spleen: left 1000µm, right 75µm.",
    "molecules": "eosin, hematoxylin"
  },
  {
    "caption": "(B) FLAG-tagged  tandem ubiquitin binding entity (TUBE) assay was performed to  pull down linear  ubiquitin linkages in patient-derived and control fibroblasts and B cells. One representative out of three independent experiments is shown.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "Biochemical  characterization of recombinant OTULIN  variants by means of surface plasmon resonance (SPR) measurements (B) SPR  measurements and steady-state binding  curves with calculated dissociation constants (Kd) after injection of a concentration series of  OTULINC129A, OTULINC129A/M86I or OTULINC129A/W167S to CM5-immobilized di-ubiquitin chains.",
    "molecules": "CM5, ubiquitin"
  },
  {
    "caption": "(C) Linear  ubiquitin linkages isolated from  A549 OTULIN KO cells by M1 TUBE assay were incubated with increasing concentrations of recombinant OTULINWT, OTULINM86I and OTULINW167S or catalytically inactive OTULINC129A as control for 1 hour. Afterwards, samples were subjected to analysis by western blot  for the indicated proteins. Images are representative of three independent experiments.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(A - C)  A549 OTULIN  KO cells were transfected with empty vector or different OTULIN  constructs as indicated and analyzed by  western blot. (D) FLAG-tagged tandem ubiquitin binding entity  (TUBE) assay was performed in B cells  from control, patient, mother and father to pull down linear ubiquitin  linkages. One representative (A - D) of three independent experiments is shown.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "Patient-derived and control fibroblasts  were used. (A) FLAG-tagged  tandem ubiquitin binding entity (TUBE) assay was performed to pull down linear ubiquitin linkages upon stimulation with 500ng/ml  TNF. (B) The TNFR1-SC was isolated using 500ng/ml TAP-TNF. (C) Cells were stimulated with 100ng/ml TNF for the indicated  times and analyzed by western blot. One representative (A - C) of three independent experiments is shown.",
    "molecules": "ubiquitin"
  },
  {
    "caption": "(E) B cells  were incubated with 50µg/ml cycloheximide  (CHX) for the indicated times  and subjected to analysis by  western blot. One representative (left panel) out of 4 individual experiments analyzed by densitometry (right panel) is shown. Data are presented as mean ± SD. *, P = 0.02, unpaired t-test.",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": "(G) Fibroblasts were incubated with 50µg/ml cycloheximide (CHX) or DMSO and stimulated with  TNF as indicated. Viability  was measured after 24 hours. Data are presented as mean ± SD of 5 individual experiments performed in three technical replicates; ***, P = 0.0008, mixed-effects analysis with Tukey's multiple comparisons test.",
    "molecules": "CHX, cycloheximide, DMSO"
  },
  {
    "caption": "A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005.",
    "molecules": "LY294002, rapamycin"
  },
  {
    "caption": "B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005.",
    "molecules": "FCS"
  },
  {
    "caption": "C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.",
    "molecules": "CCCP"
  },
  {
    "caption": "2A) SH-SY5Y cells were starved for 2 h in HBSS with Bafilomycin (+Baf) or left untreated in RPMI+5% FCS (- Baf). The LC3-II and actin content were determined by western blotting. A representative blot is shown on the right, showing only LC3-II bands in the Bafilomycin-treated samples. The LC3-II bands of Bafilomycin treated samples were normalized to actin and the relative LC3-II content of nt cells was set as 1. Cells with stable PINK1 knockdown exhibited a reduced LC3-II/actin ratio compared to the control (nt) cells; n = 4, p<0.005.",
    "molecules": "Bafilomycin"
  },
  {
    "caption": "3B) Transduced nt and PINK1 knockdown (kd) SH-SY5Y cells were cultivated either in RPMI+5% FCS or starved for 24 h and 40 h in HBSS. The LAMP-2 and actin content were determined by western blotting. A representative blot after 40 h starvation in HBSS is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP-2/actin ratio compared to the nt cells; n = 4, p<0.05.",
    "molecules": "FCS"
  },
  {
    "caption": "4A) Oxygen consumption rate (OCR) was measured in nt and PINK1 knockdown (kd) SH-SY5Y cells without (nt) or with PINK1 knockdown (kd) cultivated in RPMI medium with 5% FCS. Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd, no significant changes were observed; n = 1 (quadruplicates).",
    "molecules": "oxygen"
  },
  {
    "caption": "(C) pS65-Ub (left) and total Ub (right) immunoblots of mitochondria-enriched fractions from wild-type flies treated 3 days with either paraquat or vehicle (sucrose).",
    "molecules": "paraquat, sucrose"
  },
  {
    "caption": "(D) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days. T, total lysate; N, nuclear-enriched fraction; M, mitochondria-enriched fraction; S, post-mitochondrial supernatant.",
    "molecules": "paraquat"
  },
  {
    "caption": "(C) pS65-Ub immunoblot of mitochondria-enriched fractions following the paraquat pulse-chase assay in wild-type and park-/- flies. UT, untreated; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food. Asterisk (*) denotes non-specific band. (D) pS65-Ub lane densitometry analysis of n = 3 independent replicates from (C), expressed relative to the most intense lane signal in each blot.",
    "molecules": "paraquat, PQ"
  },
  {
    "caption": "(A) pS65-Ub immunoblot of whole-animal lysates following paraquat pulse-chase assay of Atg5- flies with parkin overexpression (OE) (Atg55cc5 daG4>UAS-park) compared with Atg5- flies overexpressing mitochondrially targeted GFP (Atg55cc5 daG4>UAS-mito-HA-GFP). UT, untreated flies; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food.",
    "molecules": "paraquat, PQ"
  },
  {
    "caption": "(B) pS65-Ub lane densitometry, normalised to total protein (RevertTM stain), of n = 3 independent replicates of (A), expressed as pS65-Ub intensity relative to the most intense band in each blot.",
    "molecules": "Revert"
  },
  {
    "caption": "(D) Western blotting analysis of proteins involved in the amino-acid sensing and integrated stress response: GCN2, SLC7A11 and ATF4 in control derived muscle (n=3), patient derived muscle during the symptomatic phase (n=3) and patient derived muscle after recovery (n=1). All samples were normalized to VCL which was used as a loading control. (E) Densitometry analysis of proteins from (D) showing a significant increase of major transducers of ISR such as GCN2 and ATF4. Consistent with ISR activation is also the increase in SLC7A1.",
    "molecules": "amino-acid"
  },
  {
    "caption": "(A) Patient n= 4 (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts (n=2) were grown for 12 days in media containing 0.02 mM cysteine and 5% dialyzed FBS. Proteins belonging to mitochondrial complexes (NDUFA9-Complex I, SDHA-Complex II, MTCO1-Complex IV and MTCO2-Complex IV) were analysed via Western Blotting. VCL was used as a loading control and the densitometry analysis was done based on the mitochondrial proteinTOM20. (B) Densitometry analysis of proteins from (A) showing no significant changes in OXPHOS proteins upon glutamine/ glutamic acid depletion. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). ",
    "molecules": "cysteine, glutamic acid, glutamine"
  },
  {
    "caption": "(C) Patient (F7/7F-healthy carrier, F7/2M-RIRCD, F7/5F-healthy carrier, F7/1M-RIRCD without a clear second mutation) and control fibroblasts were grown for 12 days in media with no added glutamine or glutamic acid and 5% dialyzed FBS. Cells were lysed and the above mentioned mitochondrial proteins were checked via Immunoblotting. VCL was used as a loading control while the densitometry analysis (D) was done based on the mitochondrial proteinTOM20. Control bar represents an average of the two control fibroblast lines employed (F008 and F154). Although we could observe significant changes upon cysteine depletion in all conditions, the phenotype was exacerbated in the digenic fibroblasts compared to the controls.",
    "molecules": "cysteine, glutamic acid, glutamine"
  },
  {
    "caption": "(B) Analysis of altered mitochondrial gene expression resulted from our RNAseq showing that affected genes and their coding proteins carry a high numbers of glutamic acid and glutamine residues, in accordance to the mitochondrial translational defect.",
    "molecules": "glutamic acid, glutamine"
  },
  {
    "caption": "(a) Z-projection of an E7.25 embryo mosaically labelled through OH-tamoxifen injection, imaged by two-photon microscopy. Scale bar: 50 μm. Data information: n embryos=54. (a') Mouse line strategy used to produce embryos ubiquitous for membrane tdTomato (grey) and mosaic for membrane GFP (mGFP, green) in the epiblast. (b) Z-projections of stills from live imaging recording of a bottle-shaped cell (indicated by a white arrow) that delaminates through the basal membrane at time 0min, 25 min, 50 min, 1h15min and 1h40min. (b') Top: Bottle shape events: GFP+ bottle shape cells over total GFP+ cells, expressed in percentage, in anterior versus posterior region of the epiblast. The posterior region includes the PS region, as it could not be precisely discriminated. Percentage of bottle shape cells exiting the epiblast among total number of GFP+ bottle shape cells, in anterior versus posterior epiblast. white dotted lines outline basal (top) and apical (bottom) borders of the epiblast.",
    "molecules": "OH-tamoxifen"
  },
  {
    "caption": "(c) Z-projections of transverse sections from E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. Samples were stained for F-actin (Phalloidin, grey) and mitosis (Phh3, magenta) on the left and nuclei (DAPI, cyan) and basement membrane (collagen IV, yellow) on the right. Yellow arrowheads show non-apical mitosis. White arrows delimitate borders of the basement membrane degradation region. A: anterior, P: posterior.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "(d) Mitotic Index (Phh3/DAPI) in percentage at the anterior, posterior, and PS regions of E6.5 (top), E6.75 (mid) and E7 (bottom) embryos. The posterior region quantification excludes counts from the PS region.",
    "molecules": "DAPI"
  },
  {
    "caption": "(a) 3D reconstruction from wholemount lightsheet imaging of an E5.75 Hex-GFP (green, anterior marker) embryo, stained for nuclei (DAPI, grey) on the left, mitosis (Phh3, magenta) and F-actin (Phalloidin, grey) on the right. (b) Z-slice (left) and 3D reconstruction (right) from wholemount lightsheet imaging of an E6.25 embryo stained for a posterior marker (Eomesodermin, cyan), mitosis (Phh3, magenta) and F-actin (Phalloidin, grey). (a', b') Mitotic Index (Phh3/DAPI) in percentage at the anterior and posterior regions of E5.75 (a') and (b') E6.25 embryos. Posterior region includes the PS region, as it could not be precisely discriminated. (a\", b\") Ratio of non-apical mitosis (non-apical Phh3 over total Phh3, normalized by number of nuclei) in percentage in the anterior and posterior region of E5.75 (a\") and E6.25 (b\") embryos.",
    "molecules": "DAPI, Phalloidin"
  },
  {
    "caption": "(b, c) 3D reconstruction (b) and confocal Z-slice (c) of E6.25 WT (left) and RhoAVE-deleted (right) embryos stained for an AVE marker (Cerberus 1, yellow), F-actin (Phalloidin, grey) and mitosis (Phh3, magenta). Scale bar: 25 μm. Blue dotted line delimitates the pro-amniotic cavity (apical side of the epiblast). Red arrowheads point to non-apical mitosis. White lines delimitates the boundary between extraembryonic (up) and embryonic (down) regions of the embryo.",
    "molecules": "Phalloidin"
  },
  {
    "caption": "D. The relative abundance of valine  (Val) and leucine-isoleucine  (Leu-Ile). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data E. The relative abundance of 3-hydroxybutyrate (3-HOB) and acetylcarnitine  (AC). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "3-HOB, 3-hydroxybutyrate, Ile, isoleucine, Leu, leucine, AC, acetylcarnitine, Val, valine"
  },
  {
    "caption": "A and B. The serum  concentration of FSH  and the changes of follicles  in mice  on low BCAA   diet for 1.5 months. A. Control, N=10; low BCAA, N=8; B. Control, N=4; low BCAA, N=4; Data information: S1, Primordial; S2, Primary; S3, Secondary; S4, Antral; S5, Atretic. Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "BCAA"
  },
  {
    "caption": "C. The serum  concentration of FSH  in mice  on a different batch of the low BCAA  diet. N=5; D. The serum  concentration of FSH  in mice  on the low BCAA  diet from Research Diet. N=10; Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "BCAA"
  },
  {
    "caption": "E and F. The serum  concentration of AMH  and E2  in mice  on the low BCAA  diet for 1.5 months. Control, N=5; low BCAA, N=3 Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "E2, BCAA"
  },
  {
    "caption": "A-B. Elevation of serum ceramide  in POI patients  from the Fudan Cohort or the Shandong Cohort. Left, the relative abundance of total ceramide; right, the relative abundance of ceramide  with specific acyl chain. A. N=18; B. N=10; Truncated violin plot, central band stands for median, dotted lines stand for the upper quartile or the lower quartile of the data; Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "ceramide"
  },
  {
    "caption": "D. Elevation of ceramide in the ovaries  of mice  on a low BCAA  diet. N=10; Truncated violin plot, central band stands for median, dotted lines stand for the upper quartile or the lower quartile of the data Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "BCAA"
  },
  {
    "caption": "F. The serum  concentration of FSH  in mice  with ceramide  treatment. N=7 Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "ceramide"
  },
  {
    "caption": "H. The changes in follicle  count from mice  with ceramide  treatment. N=7. Data information: S1, Primordial; S2, Primary; S3, Secondary; S4, Antral; S5, Atretic. Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "ceramide"
  },
  {
    "caption": "C, Relative expression  of genes related to ROS  in granulosa cells Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "ROS"
  },
  {
    "caption": "E-F. Relative abundance of GSH  and GSSG. N=3; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "GSH, GSSG"
  },
  {
    "caption": "G. The concentration of E2  secreted by KGN cells with ceramide  treatment w/o NAC. N=4; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "E2, ceramide, NAC"
  },
  {
    "caption": "H- J. Serum  concentration of FSH, AMH, and E2. H. Control, N=10; BCAA, N=5; TG=9; TG+BCAA=8; I. Control, N=5; BCAA, N=5; TG=6; TG+BCAA=6; J. N=5; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "E2, TG, BCAA"
  },
  {
    "caption": "K. The changes in follicle  count. Control, N=5; BCAA, N=4; TG=5; TG+BCAA=5; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.",
    "molecules": "TG, BCAA"
  },
  {
    "caption": "(b) RT-PCR analysis using the blue primer set (left) and green primer set (right) from a. Whereas control cDNA samples showed a single product corresponding to the wild-type allele (WT), an apparently longer product was observed in subjects 1, 2 and 5, indicating that only the transcripts from the mutant allele were expressed. In subject 3, both wild-type and mutant alleles were expressed. Template without reverse transcriptase was used as a negative control, RT(-).",
    "molecules": "cDNA"
  },
  {
    "caption": "(a-e) T1-weighted imaging shows hyperintensity of the substantia nigra with a central band of T1-weighted hypointensity (arrowheads). Images are shown for subject 1 at 33 years (a), subject 2 at 25 years (b), subject 3 at 39 years (c), subject 4 at 46 years (d) and subject 5 at 33 years (e). (f-h) T2-weighted imaging shows marked hypointensity of the globus pallidus (arrows), suggesting iron deposition. Cerebral atrophy and mild cerebellar atrophy are also seen. Images are shown for subject 1 (f), subject 2 (g) and subject 3 (h). (i,j) The fluid attenuated inversion recovery (FLAIR) image of subject 1 (i) and the T1-weighted FLAIR coronal image of subject 2 (j) also show cerebral atrophy.",
    "molecules": "iron"
  },
  {
    "caption": "(b) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies to LC3, WIPI4 and HSP90. The positions of LC3-I (cytosolic) and LC3-II (membrane bound) are indicated.",
    "molecules": "chloroquine, Torin1"
  },
  {
    "caption": "(c-e) Cells were cultured in the presence of Torin1 for 2 h. (c) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A. Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects. Scale bars, 10 μm and 1 μm in the inset. (d,e) The numbers of LC3+ (d) and LC3+ATG9A+ (e) foci were quantified from more than 20 images from 3 independent samples (Online Methods). Data are presented as mean ± s.e.m. *P 0.05, ANOVA followed by Bonferroni-Dunn post-hoc test.",
    "molecules": "Torin1"
  },
  {
    "caption": "C, Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (C) Tumor volumes were measured starting from day 0 and during the course of treatment as shown. Data points represent the mean ± SD. n = 7-8 tumors/group. p-values indicate significant difference compared to the vehicle group.",
    "molecules": "ETC-159, Olaparib"
  },
  {
    "caption": "H, I. Wnt inhibition reduces the expression of HR and FA pathway genes in a RNF43-mutant pancreatic cancer patient-derived xenograft and AsPC-1 cells. (H) Pancreatic canc­er PDX with G371fs RNF43 mutation treated with vehicle or ETC-159 (30 mg/kg) for 21 days were analysed for changes in the expression of the indicated genes measured by qRT-PCR. Each data point represents an individual tumor. The horizontal lines represent mean of 3 tumors/group. (I) AsPC-1 cells were seeded in low adherence plates, treated with DMSO or ETC-159 (100 nM) for 72 hours and the expression of indicated genes was measured by qRT-PCR.",
    "molecules": "DMSO, ETC-159"
  },
  {
    "caption": "Olaparib and ETC-159 combination is more effective in preventing the growth of PaTu8988T xenografts in mice. NSG mice with established PaTu8988T orthotopic xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (30 mg/kg), olaparib (50 mg/kg) or a combination of ETC-159 and olaparib. Treatment was initiated after the tumors were established. Tumor weights in the respective groups at the end of the 18 days treatment are shown. Each dot represents an individual tumor and the horizontal lines represent the mean. p-values were calculated with Mann-Whitney U test.",
    "molecules": "ETC-159, Olaparib, olaparib"
  },
  {
    "caption": "Wnt inhibition through ETC-159 does not change the expression of HR and FA pathway genes in Panc 08.13 cells. Panc 08.13 cells were cultured in low adherence plates and treated with ETC-159 for 72 hours. Total RNA was then isolated and expression of indicated genes was measured by qRT-PCR. The horizontal lines represent mean of replicates.",
    "molecules": "ETC-159"
  },
  {
    "caption": " Stabilized β-catenin prevents the downregulation of HR and FA pathway genes upon Wnt inhibition. Mice bearing HPAF-II xenografts without or with stabilized β-catenin were treated with ETC-159 (37.5mg/kg bid) for 56 hours before tumors were harvested and the expression of indicated genes was measured by qRT-PCR. ETC-159 induced reduction in the expression of HR and FA pathway genes was blocked in xenografts with stabilized β-catenin. The horizontal lines represent mean of replicates",
    "molecules": "ETC-159"
  },
  {
    "caption": " E, F. G007-LK and olaparib synergistically inhibit the growth of COLO320HSR colorectal cancer cells with APC mutation. COLO320HSR cells were plated at a low density and treated with G007-LK, olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A. (E) Representative image from two independent experiments is shown. (F) The Combination Index (CI) values of olaparib and G007-LK calculated from two independent experiments using the Chou-Talalay CompuSyn software ",
    "molecules": "G007-LK, olaparib"
  },
  {
    "caption": "β-catenin regulates the expression of MYBL2 in HPAF-II cells. HPAF-II cells were either treated with ETC-159 (100 nM) or transfected with siRNA against β-catenin alone or in the presence of ETC-159 for 48 hours. The relative expression of MYBL2 as measured by qRT-PCR is shown. The horizontal lines represent mean of replicates.",
    "molecules": "ETC-159"
  },
  {
    "caption": "Expression of DNA repair genes in Wnt-addicted cells is regulated by MYBL2. HPAF-II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC-159 (100 nM) for 48 hours. Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT-PCR. Data are representative of three independent experiments. The horizontal lines represent mean of replicates.",
    "molecules": "ETC-159"
  },
  {
    "caption": "H-K. Expression of MYBL2 is downregulated upon ETC-159 or G007-LK treatment in multiple Wnt-high tumors. Expression of MYBL2 in AsPC-1 xenograft tumors and colorectal cancer PDX (as measured by RNA-seq) or in pancreatic cancer PDX with RNF43 mutation (measured by qRT-PCR) is reduced with ETC-159 treatment. G007-LK reduces MYBL2 expression in COL320HSR cells (measured by qRT-PCR). p-values were calculated with Mann-Whitney U test. Each data point represents an individual tumor or replicate. n = 4-6 samples/group. The horizontal lines represent median of 4-6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1.5x interquartile range.",
    "molecules": "ETC-159, G007-LK"
  },
  {
    "caption": "F, Wnt inhibition induces senescence, which is further enhanced by co-treatment with olaparib. (F) Representative images of tumor sections from the four treatment groups (treated for 21 days) stained for SA-β-galactosidase, a senescence marker, and counterstained with nuclear fast red. Blue color indicates positive staining for senescent cells. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.",
    "molecules": "olaparib"
  },
  {
    "caption": "Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (H) HPAF-II cells were treated with DMSO, ETC-159 (50 nM), olaparib (20 µM) or both for 7 days. 53BP1 foci per cell were then assessed by immunofluorescence. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.",
    "molecules": "DMSO, ETC-159, olaparib"
  },
  {
    "caption": "I. Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (I) Representative immunofluorescence images of 53BP1 foci (red) and nuclei counterstained with DAPI (blue) are shown for each group.",
    "molecules": "DAPI, olaparib"
  },
  {
    "caption": "PORCN inhibition reduces the expression of HR and FA pathway proteins in the normal mouse intestine. C57BL/6J mice were treated with vehicle or ETC-159 (60 mg/kg) for 3 days. Small intestines were sectioned and analyzed by immunostaining for BRCA1, FANCD2, RAD51 and phosphorylated-BRCA1.",
    "molecules": "ETC-159"
  },
  {
    "caption": "E Drug-response curves showing increased resistance of shTINCR-WM902B cells after treatment with BRAF (Vemurafenib; from 10 to 10,000 nM) and MEK (Trametinib; from 4 to 2,000 nM) inhibitors. The shSCR- (control) and shTINCR-WM902B cells were treated with increasing concentrations of drugs and their viability assayed after 72 hours using CellTiter-Glo luminescent kit (Promega). Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).",
    "molecules": "Trametinib, Vemurafenib"
  },
  {
    "caption": "D Drug-response curves showing sensitization to MEK inhibitor Trametinib of MM13 (NRAS-mutant PDX) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells (BRAF-mutant PDX). The Empty (control) and TINCR expressing MM13 cells were treated with increasing concentrations of drug (ranging from 5 to 5000 nM) and cell viability was assayed after 72 hours by CellTiter-Glo viability assay. Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).",
    "molecules": "Trametinib, Vemurafenib"
  },
  {
    "caption": "G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown. Hematoxylin-eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells. Metastasis formation was evaluated 8 weeks after transplantation into NSG mice. Pictures of representative lungs and livers infiltrated with metastases are shown in the inset. Scale bars: 200μm.",
    "molecules": "eosin, Hematoxylin"
  },
  {
    "caption": "(B-D) GFP-Atg8, overexpressed using the Dmef2-Gal4 driver, labels autophagosomes. (B), starved on low-nutrientfood for 6 h (C), or starved on low-nutrientfood +2.5 mg/ml CQ for 6 h (D). GFP-Atg8-labeled vesicles appeared only in the starved animals (C-D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP-Atg8-labeled vesicles.",
    "molecules": "CQ, food, nutrient"
  },
  {
    "caption": "(E-G) Dmef2-Gal4, UAS-GFP-Atg8/UAS-HRP-Lamp1 animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP-Atg8 and HRP-Lamp1-labeled vesicles. (F) Colocalization of GFP-Atg8 and HRP-Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP-Atg8 and HRP-Lamp-labeled vesicles, but they failed to colocalize.",
    "molecules": "CQ, food, nutrient"
  },
  {
    "caption": "(H) Quantification of the number of GFP-Atg8, HRP-Lamp1, or GFP-Atg8+HRP-Lamp1 vesicles in starved or starved +CQ muscles.",
    "molecules": "CQ"
  },
  {
    "caption": "(I-M) The core Atg genes are required for starvation-induced autophagy in both wild-type and CQ-treated skeletal muscles. Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1 larvae were starved on low-nutrient food for 6 h (I) or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (J). Note that Atg1 knockdown completely abolished the formation of GFP-Atg8-labeled autophagosomes (compare I-J to C-D). (K-L) Dmef2-Gal4, UAS-GFP-Atg8, Atg1Δ3d larvae failed to form GFP-Atg8 vesicles when starved or starved and treated with CQ. (M) Quantification of autophagy changes due to Atg gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. Each of the 10 UAS-Atg RNAi transgenes tested caused a highly significant decrease (p<.01) in the total area of GFP-Atg8vesicles. SEM is indicated, with n = 5 ventral longitudinal muscles from individual animals.",
    "molecules": "CQ, food, nutrient"
  },
  {
    "caption": ". (N-O) EM of muscles from Dmef2-Gal4, UAS-whitei larvae. Animals starved on low-nutrient food +2.5 mg/ml CQ (O) accumulated vesicles in the intermyofibril spaces (red asterisk), disrupting the integrity of the sarcomere compared to non-CQ-treated control muscles (N).",
    "molecules": "food, nutrient, CQ"
  },
  {
    "caption": "(P) CQ treatment increased the larval crawling time of Dmef2-Gal4, UAS-whitei larvae in starved animals, and weakly in fed animals. (Q) CQ treatment increased the larval righting time of Dmef2-Gal4, UAS-whitei larvae in starved but not fed animals. For both locomotor assays, SEM is indicated for n = 10 larvae (*p<.05, **p<.01).",
    "molecules": "CQ"
  },
  {
    "caption": "(A) Sectioned third instar OreR larva stained with Periodic acid-Schiff (PAS). The muscles, but not the fat body, are stained purple, indicating high levels of glycogen (m, muscle; bw, body wall; fb, fat body).",
    "molecules": "glycogen"
  },
  {
    "caption": "(B) Glycogen was also detected in muscle from Dmef2-Gal4, UAS-GFP-Atg8 larvae, immunostained with an antiglycogen monoclonal antibody.",
    "molecules": "Glycogen"
  },
  {
    "caption": "(C) GFP-Atg8 vesicles colocalized with glycogen in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h (GFP, green; antiglycogen, red).",
    "molecules": "CQ, food, glycogen, nutrient"
  },
  {
    "caption": "D) HRP-Lamp1 vesicles show less colocalization with glycogen in UAS-HRP-Lamp1;Dmef2-Gal4 larvae starved and treated with CQ.",
    "molecules": "CQ, glycogen"
  },
  {
    "caption": "(E) Quantification of GFP-Atg8 or HRP-Lamp1 vesicles with glycogen.",
    "molecules": "glycogen"
  },
  {
    "caption": "(F-G) EM from Dmef2-Gal4, UAS-whitei larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Double- and single-membrane vesicles containing glycogen granules accumulated between myofibers (s, sarcomere; m, mitochondrion; AVs, autophagic vesicles). (G) Higher magnification view of region outlined in (E). (H) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2-Gal4, UAS-whitei larva starved on low-nutrient food for 6 h. Arrow points to double membrane.",
    "molecules": "CQ, food, glycogen, nutrient"
  },
  {
    "caption": "(A-D) Time course of autophagy induction in Dmef2-Gal4, UAS-GFP-Atg8 muscles, accompanied by quantification of GFP-Atg8 and glycogen colocalization. Animals were fed for 18 h in high-nutrient food +2.5 mg/ml CQ, then starved on low-nutrient food +2.5 mg/ml CQ for 0-8 h (antiglycogen, red; GFP, green; DAPI, blue). (A) At time point 0, following 18 h in high-nutrient food +CQ, the muscles contained large amounts of glycogen with no apparent autophagy. (B) At 3 h of starvation, glycogen stores were still high, and GFP-Atg8-labeled vesicles began to appear. (C-D) At 6 and 8 h of starvation, the majority of GFP-Atg8-labeled vesicles colocalized with glycogen.",
    "molecules": "CQ, food, glycogen, nutrient"
  },
  {
    "caption": "(E) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall). Animals were fed for 24 h in high-nutrient food, then starved on low-nutrient food +/− 2.5 mg/ml CQ for 0-24 h. Starvation caused reduction of glycogen levels in both untreated and CQ-treated larvae over time. However, after 6 h of starvation, CQ treatment significantly increased glycogen levels compared to controls. SEM is indicated for n = 5-8 samples (*p<.05, **p<.01).",
    "molecules": "CQ, food, glycogen, nutrient"
  },
  {
    "caption": "(F-G) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Autophagy levels were high in control Dmef2-Gal4/UAS-whitei larvae. Muscles from (G) UAS-Rheb/+; Dmef2-Gal4/+, (H) Dmef2-Gal4/UAS-Tsc1i, and (I) Dmef2-Gal4/UAS-gigi all failed to induce autophagy.",
    "molecules": "CQ, food, nutrient"
  },
  {
    "caption": "(A-B) Glycogen phosphorylase is not required for glycogen autophagy (antiglycogen, red; GFP, green; DAPI, blue). (A) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h exhibited high levels of colocalization between GFP-Atg8 and glycogen.(B) Higher magnification of region outlined in (A)",
    "molecules": "food, glycogen, nutrient"
  },
  {
    "caption": "C-F) Dmef2-Gal4, UAS-GFP-Atg8 larvae with GlyP and/or Atg1 knockdown were fed on high-nutrient food for 18 h before being starved on low-nutrient food (antiglycogen, red; GFP, green; DAPI, blue). (C) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larval muscle contained high levels of glycogen prior to starvation, indicating no defect in glycogen synthesis. (D) Following 24 h starvation UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ muscles contained no glycogen detectable by antibody staining. (E) Following 24 h of starvation Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i muscles contained no glycogen. (F) Double-mutant larvae UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i larval muscles contained high levels of glycogen after 24 h of starvation, indicating an inability to break down glycogen.",
    "molecules": "food, glycogen, nutrient"
  },
  {
    "caption": "(G) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall) with expression of UAS-RNAi transgenes targeting white, GlyP, Atg1, or GlyP+Atg1. Simultaneous knockdown of GlyP and Atg1, but not either gene alone, significantly reduced glycogen degradation compared to the white control after 24 h of starvation, consistent with immunostaining (C-F). Between 6 and 12 h of starvation, individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels, indicating a reduced rate of glycogen degradation. SEM is indicated for n = 5-8 samples. The p values were calculated relative to white RNAi control at each time point (*p<.05, **p<.01).",
    "molecules": "glycogen"
  },
  {
    "caption": "(A-D) Glycogen synthase (GlyS) is required for glycogen synthesis in D. melanogaster muscles. PAS staining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (B) compared to control Dmef2-Gal4/UAS-whitei muscles (A). Antiglycogen immunostaining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (D) compared to Dmef2-Gal4/UAS-whitei control muscles (C).",
    "molecules": "glycogen"
  },
  {
    "caption": "(C). (E-K) GlyS is required for the formation of large CQ-induced autophagosomes. Vesicles are much smaller in Dmef2-Gal4, UAS-GFP-Atg8/UAS-GlySi larval muscle starved 6 h in low-nutrient food +2.5 mg/ml CQ (G) than in control Dmef2-Gal4, UAS-GFP-Atg8/UAS-whitei larval muscle (E). (F, H) The difference in autophagosome size is clearly evident at high magnification.",
    "molecules": "CQ, food, nutrient"
  },
  {
    "caption": "(I-K) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. SEM is indicated, with n = 5 (I) or n = 10 (J-K) ventral longitudinal muscles from individual animals (*p<.05, **p<.01). (I) Each of the four UAS-GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP-Atg8vesicles in the muscle compared to the UAS-whitei control. (J) Vesicle number was unchanged by GlyS knockdown. (K) UAS-GlyS RNAi caused a highly significant decrease in the mean vesicle size (area) compared to the control.",
    "molecules": "CQ, food, nutrient"
  },
  {
    "caption": "N-O) Larvae were starved on low-nutrient food for 6 h prior to dissection of the fat bodies. Autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-GlySi (O) was not substantially different from autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-whitei control fat bodies (GFP, green; DAPI, blue).",
    "molecules": "food, nutrient"
  },
  {
    "caption": ". (P) EM of muscle from Dmef2-Gal4/UAS-GlySi animal starved on low-nutrient food +CQ. Note that the intermyofibril spaces (red asterisk) and sarcomere structure are not distorted.",
    "molecules": "CQ, food, nutrient"
  },
  {
    "caption": "Q) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h. SEM is indicated for n = 10 larvae. The p values were calculated relative to white RNAi control larvae (*p<.05, **p<.01).",
    "molecules": "CQ"
  },
  {
    "caption": "(B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag-Atg8 binds to a Venus-GlyS or Venus-GlyS (S651A) protein complex in response to starvation. Flag-Atg8 is unable to co-IP with either Venus-GlyS (R593A) or Venus-GlyS (W609A). Venus-GlyS and Venus-GlyS mutants were co-IP'd from muscle lysate from Dmef2-Gal4/UAS-Flag-Atg8 or UAS-Venus-GlyS(WT or mutant)/+;Dmef2-Gal4/UAS-Flag-Atg8third instar larvae. These were fed on high-nutrientfood for 18 h, and then transferred to fresh high-nutrientfood or low-nutrientfood for 6 h.",
    "molecules": "food, nutrient"
  },
  {
    "caption": "M Left: RT-PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs. Right: Quantification of relative spliced AXIN1a/AXIN1b expression ratio normalized to actin from RT-PCR analysis (left). (n = 3 biological replicates).",
    "molecules": "estrogen"
  },
  {
    "caption": "(A). Human neutrophils stained for CD66b after incubation with RBCs infected with GFP+ P. falciparum parasites (white arrows). Nuclei were stained with DAPI (blue), neutrophils were stained with CD66b (red), GFP expressing parasites (green). Scale bar, 5 µm. The upper and lower panel show two different cells.",
    "molecules": "DAPI"
  },
  {
    "caption": "(B). Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P. falciparum parasites (black arrows).",
    "molecules": "Giemsa"
  },
  {
    "caption": "(F). Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression. Neutrophils were stained with an anti-CD11b antibody, while iRBCs were labeled with SYBR green. Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population, excluding the neutrophils that were SYBR green negative, but CD11b positive.",
    "molecules": "SYBR green"
  },
  {
    "caption": "(G). Short term neutrophil killing of late-stage NF54-luciferase positive RBCs in the presence (red) or absence (blue) of catalase.",
    "molecules": "catalase"
  },
  {
    "caption": "(H). Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase.",
    "molecules": "catalase"
  },
  {
    "caption": "(I). Short-term luciferase-based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D.",
    "molecules": "cytochalasin D"
  },
  {
    "caption": "(B). Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP+ iRBC.",
    "molecules": "trypsin"
  },
  {
    "caption": "(C). Flow cytometric quantification of neutrophil interaction with MitoTracker (APC+) stained NF54 iRBCs and antibody-selected PFD1235w expressing iRBC.",
    "molecules": "APC, MitoTracker"
  },
  {
    "caption": "(A) EM analysis of KMnO4-fixed pex3 atg1 and WT cells grown for 16 h on MM-M/G. The inset shows a cluster of vesicles (enlarged from the boxed region).",
    "molecules": "KMnO4"
  },
  {
    "caption": "(A) FM images of pex3 cells with Pex14-mCherry upon Pex3-eGFP reintroduction after shifting cells from MM-Glu with ammonium sulfate to MM-M/G with methylamine.",
    "molecules": "ammonium sulfate, methylamine"
  },
  {
    "caption": "(G) EM analysis of KMnO4-fixed pex3 atg1 pex25 cells grown for 16 h on MM-M/G-showing membrane vesicles (arrows).",
    "molecules": "KMnO4"
  },
  {
    "caption": "The expression levels of viral copy number (d) Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n= 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). 'd.p.i.' stands for 'day post-infection'.",
    "molecules": "saline"
  },
  {
    "caption": "cytokines and chemokines in lung were measured by qPCR (e). Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n= 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). 'd.p.i.' stands for 'day post-infection'.",
    "molecules": "saline"
  },
  {
    "caption": "(a) AAV-hACE2 mice were treated with saline and challenged with SARS-CoV-2 (8 × 104 PFU) for 5 days. Lung tissue sections were stained with DAPI (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (Cit-H3) antibody (red), anti-CD42b antibody (yellow) and anti-CD31 antibody (gray). yellow arrow: NETs (DNA+Gr-1+MPO+Cit-H3+); red arrow: immunothrombosis (NETs + thrombus (CD42b+)), Scale bar is 100 μm.",
    "molecules": "DAPI, DNA, saline"
  },
  {
    "caption": "b & c) For prophylactic treatment, CLEC2.Fc (or vehicle) was given at 1 h before virus challenge; for therapeutic treatment, CLEC2.Fc was given at 8 hours post-infection. The area of NET (colocalization area of MPO and Cit-H3) (b) and immunothrombosis (colocalization area of Cit-H3 and CD42b) (c) were measured using MetaMorph software. Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n=5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01 (Two-way ANOVA).",
    "molecules": "saline"
  },
  {
    "caption": "a, b) AAV-hACE2 mice were treated with saline and challenged with SARS-CoV-2 (8 × 104 PFU) for 5 days. Samples were collected at day 5 post-infection, and the collagen were stained with Picro Sirius red (a), while collagen deposition was quantified using MetaMorph software and presented as area (μm2) (b). The images were captured by light microscopy (upper panel) and polarized light microscopy (lower panel). Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n=5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). ***p < 0.001, ****p < 0.0001 (Two-way ANOVA).",
    "molecules": "Picro Sirius red, saline"
  },
  {
    "caption": "A) Deletion of the N-terminal of Sen1 is lethal. A ∆sen1 strain (YDL2767) covered by an URA3-containing plasmid (pFL38) expressing wt Sen1 was transformed with a TRP1-plasmid (pFL39) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left. After over-night growth in non-selective medium, cells were plated on minimal medium (CSM) containing 5-fluorootic acid (5-FOA) to select those that have lost the URA3 plasmid. The absence of growth in 5-FOA implies that the SEN1 version expressed in the TRP1-plasmid does not support viability.",
    "molecules": "5-fluorootic acid, 5-FOA"
  },
  {
    "caption": "B) Deletion of the N-terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo. Northern blot analyses of three well-characterized NNS-targets, the CUT NEL025c and the snoRNAs SNR13 and SNR33, in a Sen1-AID (auxin-induced degron) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1. Sen1-AID was depleted for 2h by the addition of 500 μM indole-3-acetic acid (IAA) to monitor the capacity of the plasmid-borne versions of SEN1 to induce transcription termination. The expected RNA species resulting from inefficient termination (RT for readthrough species) are indicated. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.",
    "molecules": "auxin, IAA, indole-3-acetic acid, snoRNAs"
  },
  {
    "caption": "C) Overexpression of sen1∆Nter restores viability. Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter (pGAL) in the presence of galactose.",
    "molecules": "galactose"
  },
  {
    "caption": "D) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N-terminal domain. Northern blot analysis of a typical NNS-target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.",
    "molecules": "galactose"
  },
  {
    "caption": "RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS, divided by the average signal over the mature snoRNA.",
    "molecules": "snoRNA, snoRNAs"
  },
  {
    "caption": "RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs.",
    "molecules": "snoRNAs"
  },
  {
    "caption": "A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.",
    "molecules": "IAA"
  },
  {
    "caption": "B) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.",
    "molecules": "galactose"
  },
  {
    "caption": "C) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1-plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1∆Nter chimera in 5-FOA implies that this gene can support viability.",
    "molecules": "5-FOA"
  },
  {
    "caption": "E) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro. Pull-down experiments using either a wt or a ∆NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments.",
    "molecules": "sepharose, glutathione"
  },
  {
    "caption": "M-O) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs.",
    "molecules": "snoRNAs"
  },
  {
    "caption": "P) Comparison of the total levels of the different Sen1 variants at mRNAs, CUTs and the termination region of snoRNAs (i.e. a window of 200 nt downstream of the 3' end of the mature snoRNA, snoRNA-ter). Boxes in box-plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR. The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. Datasets for the different Sen1 versions were compared using a t-test (bilateral distributions, unpaired data). p-values≥ 0.05 are indicated as ns (not significant), one asterisk is used when 0.05>p-value≥0.01, two asterisks when 0.01>p-value≥0.001 and three asterisks for p-value <0.001.",
    "molecules": "snoRNA, snoRNAs"
  },
  {
    "caption": "A) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter. Northern blot assays performed in a Sen1-AID, ∆rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments. Where indicated, cells were treated with 50 mg/L of 6-azauracile (6AU) for 2h. Representative gel of one out of two independent biological replicates.The U4 RNA is used as a loading control.",
    "molecules": "6-azauracile, 6AU"
  },
  {
    "caption": "(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).",
    "molecules": "rapamycin"
  },
  {
    "caption": "(b-e) Induction of conventional macroautophagy in WT and Ulk1gt/gt, but not in Atg5−/−, embryonic erythroid cells. Ter119+ erythroid cells from WT, Atg5−/−, and Ulk1gt/gt embryonic mice (E18.5) were treated with 1 μM rapamycin (b,c) and 1 μM STS (d,e), and then harvested at the indicated times. (b,d) Representative protein expression of LC3 and p62 measured by western blot. Actin was a loading control. Uncropped images are shown in Supplementary Fig. 10. (c,e) Semi-quantitative analysis of LC3-II/LC3-I and p62 protein expression (n=3, mean±s.d.). Asterisks indicate a significant difference at P0.05 (analysis of variance (ANOVA)). #P0.05 versus value of WT 24 h (ANOVA). 'NS' indicates not significant (ANOVA).",
    "molecules": "STS"
  },
  {
    "caption": "(a) Induction of alternative macroautophagy in WT and Atg5−/−, but not Ulk1gt/gt and DKO, erythroid cells. Erythroblasts and reticulocytes from the liver of embryonic mice (E18.5) were incubated with or without STS (1 μM) for 24 h, followed by staining with anti-Lamp2 (red), anti-Ter119 (green) and DAPI (blue). DAPI-positive erythroblasts and DAPI-negative reticulocytes are shown. Lamp2 image and merged image (DAPI, Ter119 and Lamp2) are shown. Scale bar, 1 μm. Large dots for Lamp2 are observed in STS-treated WT and Atg5−/− cells, but not STS-treated Ulk1gt/gt or DKO cells.",
    "molecules": "STS"
  },
  {
    "caption": "(b) Representative electron micrographs of EC incubated with or without STS. Erythroid cells were harvested from the liver of embryonic mice (E18.5), incubated with or without STS (1 μM) for 24 h, and analysed by electron microscopy (EM). Scale bar, 1 μm. Insets of WT and Atg5−/− cells show mitophagy (Scale bar, 0.5 μm). Inset of a Ulk1gt/gt cell showing mitochondria that have not been engulfed (Scale bar, 0.5 μm). Arrows point to non-engulfed mitochondria and the arrowheads indicate engulfed mitochondria. (c-e) Quantitative analysis of mitophagy after STS treatment, calculated from EM photos. Population of reticulocytes with mitophagy (c), number of mitochondria per reticulocytes (d) and population of reticulocytes showing macroautophagy (e) were calculated (n>35 cells per mouse). The data are shown as mean±s.d. (n=3). *P0.05 versus value of WT (analysis of variance (ANOVA)); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).",
    "molecules": "STS"
  },
  {
    "caption": "(e) Percentage of mitochondrialow cells in Syto16low cells. Liver erythroid cells (E18.5) were stained with Syto16 (DNA) and Mitotracker Deep Red. Representative dot plots of the mitochondrial content of Syto16low cells are demonstrated in Supplementary Fig. 5, the percentage of Syto16low cells without mitochondria was determined by gating the mitochondrialow fraction (mean±s.d., n=6). *P0.05 versus value of WT (ANOVA); #P0.05 versus value of Atg5−/− (ANOVA); 'NS' indicates not significant versus value of WT (ANOVA).",
    "molecules": "DNA"
  },
  {
    "caption": "(c) Modification of Ulk1 and Atg13 during erythrocyte maturation. Cell lysates were treated with λ-phosphatase (400 U) for 45 min at 30 °C, and band shift was examined by anti-Ulk1 and anti-Atg13 antibodies. Asterisk indicates non-specific band. Uncropped images are shown in Supplementary Fig. 12.",
    "molecules": "λ-phosphatase"
  },
  {
    "caption": "(e-h) Impact of various drugs on mitochondrial clearance during in vitro differentiation. Differentiated cells from WT embryos were incubated with or without wortmannin (e), 3-methyladenine (3-MA) (f), rapamycin (g) or compound C (h) at the indicated concentrations and time periods. Percentage of mitochondrialow cells/Syto16low cells was measured by flow cytometry (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).",
    "molecules": "3-MA, 3-methyladenine, compound C, rapamycin, wortmannin"
  },
  {
    "caption": "(i)Impact of various drugs on mitochondrial clearance during in vitro differentiation.with cells from Atg5−/− embryos exposed to wortmannin or 3-MA (mean±s.d., n=3). *P0.05 versus value of no drug (ANOVA).",
    "molecules": "3-MA, wortmannin"
  },
  {
    "caption": "(a) Number of EC and haemoglobin content in the blood of the indicated mice (10-20 weeks old; mean±s.d., n=6). *P0.05 versus value of WT (Student's t-test).",
    "molecules": "haemoglobin"
  },
  {
    "caption": "(h,i) The impact of Ulk1 on mitochondrial clearance during stress erythropoiesis. (h) Representative images of erythrocyte in PHZ-treated WT and Ulk1gt/gt mice (14 weeks old). Blood cells were stained with anti-Ter119 (erythroid cell marker) and anti-Tom20 (mitochondrial marker) antibodies and observed by fluorescence microscopy. Green and red indicate Ter119 and Tom20, respectively. Scale bar, 10 μm. (i) Percentage of mitochondrialow cells among Syto16low cells. Erythroid cells were stained with Syto16 (DNA) and Mitotracker Deep Red (mean±s.d., n=3). *P0.05 versus value of WT (Student's t-test). Mitochondrial retention was observed in Ulk1gt/gt mice.",
    "molecules": "DNA, PHZ"
  },
  {
    "caption": "(C, HEK293T  cells stably expressing a  type I IFN  sensitive response element (ISRE) driven Fluc  gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60  wt, or RIG-I  in combination with DDX60  E890A while supplementing with GFP  plasmid to equalize the total amount of DNA transfected. Transfected cells were then treated with either PBS  (negative control) or transfected with LMW poly(I:C). In parallel, untransfected cells were treated with either PBS  (mock), transfected with LMW poly(I:C), or treated with IFN-ß (positive control). Cells were subsequently used for a luciferase assay  to assess ISRE activity (C) Data information: (C) Mean ± SD from at least n=3 biological replicates; *p < 0.05 using unpaired t-test with Welch's correction, ns, not significant using repeated measures one-way ANOVA with Geisser-Greenhouse correction comparing RIG-I transfected vs RIG-I + DDX60  wt or RIG-I + DDX60  E890A transfected cells.",
    "molecules": "PBS, poly(I:C)"
  },
  {
    "caption": "(B) Cells depicted in (A) were subjected to  polysome  profiling by lysing in the presence of cycloheximide  and protease  and phosphatase   inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. Centrifuged gradients were run through a fractionator and total RNA  in each fraction was measured by UV absorbance (254 nm).",
    "molecules": "phosphatase, cycloheximide, protease, RNA"
  },
  {
    "caption": "(C) Representative results of RNA electrophoresis  analysis of individual fractions. Shown are results from one replicate of DDX60-transfected, EMCV-IRES-PV-infected cells. RNA  was analyzed by Bioanalyzer to visualize 28S, and 18S ribosome   subunit distribution. RIN, RNA  integrity number.",
    "molecules": "RNA"
  },
  {
    "caption": "Left image: SUD generally stimulates protein translation level detected by the luciferase-pcDNA3 reporter (n=6). Middle image: SUD does not increase the amounts of total protein synthesis (host and viral proteins) in the replicon-transfected cells. Right image: SUD augments viral protein synthesis (n=8). HEK-293 cells growing in 12-well plates were transfected with the indicated plasmids and replicon DNA. Twenty-four hours post-transfection, cells were harvested for Renilla luciferase activity measurement (left and right image). For the ribopuromycylation assay, 24 hours post-transfection cells were pulsed with 3 µM puromycin for 1 hour at 37°C before harvesting for western blot analysis (middle image).",
    "molecules": "puromycin"
  },
  {
    "caption": "(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of  AG for 3 d. Lung  sections stained  with Haematoxylin and eosin(H&E) (A) and quantification of  lung lesion burden from H&E-stained sections (B). Data information: Data are means + SD of indicated numbers of  mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.",
    "molecules": "AG, eosin, Haematoxylin"
  },
  {
    "caption": "(D-G) H&E staining of lung sections from mice 4 weeks after intranasal infection with  M. bovis BCG (D and E) or  Mtb  H37Rv (F and G) in the absence or presence of  intranasally administrated  AG aptamers (1 μg) once at a 1 week-interval. Quantification of lung inflamed regions shown in (E and G). Data information: Data in are means + SD of indicated numbers of mice  from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test and Student's t test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.",
    "molecules": "AG"
  },
  {
    "caption": "(B) Quantitative polymerase chain reaction  (qPCR) analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times. (C) Immunoblots of cell supernatants to analyze secreted MMP2, MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated  times; GADPH of cell lysates served as a loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.",
    "molecules": "AG"
  },
  {
    "caption": "(H) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h (MOI=5) in absence or presence of AG  aptamers (1 μg/ml). (I) Immunoblots  of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in the absence or presence of AG  aptamers (0.5 μg/ml); GADPH of cell lysates served as the loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.",
    "molecules": "AG"
  },
  {
    "caption": "(G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv  for indicated times (MOI=5) in absence or presence of inhibitor targeting  ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.",
    "molecules": "PD98059"
  },
  {
    "caption": "(A, B) C57BL/6 mice were left untreated (NT) or were  intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat  (10 mg/kg) given intraperitoneally prior to  AG stimulation. Lung sections stained  with H&E (A) and quantification of lung lesion burden from H&E stained  sections (B). Data information: Data in (A) are representative of n=3 independent experiments. Data in (B) are means + SD of indicated numbers of mice from one of n=3 independent experiments and each symbol represents data from 1 mouse. One-way ANOVA followed by Dunnett's post hoc test (B) was used for statistical analysis. ns, not significant; ****, p<0.0001. Scale bar, 200 μm. ",
    "molecules": "AG, marimastat"
  },
  {
    "caption": "(A) Surface plasmon resonance (SPR) assay of the direct interaction of AG with galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.",
    "molecules": "AG"
  },
  {
    "caption": "(C) SPR assay of the interaction of AG with CRD2 of galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.",
    "molecules": "AG"
  },
  {
    "caption": "(I) Immunoblot of lysates of shCtrl and shGalectin-9 THP-1  cells stimulated with AG (1 μg/ml) for indicated  times. Data are representative of n=3 independent experiments.",
    "molecules": "AG"
  },
  {
    "caption": "(K) qRT-PCR detection of MMP transcripts including Mmp9, Mmp10, and Mmp12  in shCtrl and shGalectin-9 THP-1  cells stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001.",
    "molecules": "PD98059, AG"
  },
  {
    "caption": "(D) Confocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 μg/mL) for 2 h (middle row) or infected with H37Rv for 3 h (MOI=5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 μm. Data in the right graph show mean + SD of n=12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field.",
    "molecules": "AG, DAPI"
  },
  {
    "caption": "(F) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) in absence or presence of  TAK1 inhibitor 5Z-7-OZ (1 μM) for indicated  times. Data are representative of n=3 independent experiments.",
    "molecules": "5Z-7-OZ, AG"
  },
  {
    "caption": "(B, WT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given  intraperitoneally prior to AG  stimulation.  Lung sections  stained with H&E Data information: Data are representative of n=3 independent experiments.",
    "molecules": "AG, marimastat"
  },
  {
    "caption": "(D, WT or Galectin-9 KO mice were intranasally infected with  H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.",
    "molecules": "AG"
  },
  {
    "caption": "Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row).",
    "molecules": "Ficoll 70, dextran, dextran 500"
  },
  {
    "caption": "Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.",
    "molecules": "Ficoll 70, PEG 8, buffer, dextran 500"
  },
  {
    "caption": "Formation of condensates as a function of FtsZ and SlmA·SBS concentration, as measured by turbidity, in working buffer (300 mM KCl). SlmA concentration was fivefold that of SBS (except at 0.5 μM SBS, where SlmA concentration was 3 μM). Data are the average of 2 independent measurements. Errors (S.D.), symmetrical, are depicted as white discs.",
    "molecules": "buffer, KCl"
  },
  {
    "caption": "Representative confocal images of the FtsZ·SlmA·SBS condensates at the specified salt concentrations. The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 µM, respectively. Scale bars: 5 μm. Data information: All measurements in 150 g/L dextran 500.",
    "molecules": "dextran 500"
  },
  {
    "caption": "Representative confocal images showing final state after addition of FtsZ-Alexa 488 to FtsZ·SlmA·SBS complexes (FtsZ labeled with Alexa 647) in 150 g/L dextran. Below, images showing the stepwise diffusion of FtsZ-Alexa 488 into the condensates containing FtsZ-Alexa 647 at the indicated times in seconds (time zero, beginning of visualization for that particular condensate) and corresponding intensity profiles at selected times in the green channel. The profile in the red channel is shown as a reference and varies slightly among images. Scale bars: 5 μm (top row) and 4 μm (bottom row).",
    "molecules": "Alexa 488, Alexa 647, dextran"
  },
  {
    "caption": "Stepwise diffusion of FtsZ-Alexa 488 into FtsZ·SlmA·SBS condensates (FtsZ labeled with Alexa 647) at the indicated times in seconds (time zero, beginning of visualization for those particular condensates) in 100 g/L PEG. Scale bars: 4 μm.",
    "molecules": "Alexa 488, Alexa 647, PEG"
  },
  {
    "caption": "Assembly of FtsZ fibers upon GTP addition (0.5 mM) to FtsZ·SlmA·SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes (time zero, GTP addition) in 150 g/L dextran. Scale bars: 5 μm. Scheme of the dynamic process on the right. The number of condensates decreases upon fibers formation, and they rearrange upon GTP depletion and fibers disassembly.",
    "molecules": "dextran, GTP"
  },
  {
    "caption": "B-D. Representative confocal images of the microdroplets without (B) and with GTP (C and D). The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 μM, respectively (B and C) or 6, 3 and 0.5 μM respectively (D). 1 mM (C) or 2 mM GTP (D). Scale bars: 40 μm except in images on the far right (20 μm), which are either a magnification of the indicated region in the merged image (B) or independent images at higher magnification (C, D).",
    "molecules": "GTP"
  },
  {
    "caption": "B,C. Representative confocal images of the microdroplets stabilized by the E. coli lipid mixture containing the biphasic PEG/DNA mixture and the FtsZ·SlmA·SBS complex without and with 2 mM GTP, respectively. Last image in (B) focuses in the lipid interface to show the high density of condensates. Scale bars: 20 μm except in images on the far right (10 μm), which are an independent image at higher magnification (B) or a magnification of the indicated region in the merged image (C).",
    "molecules": "GTP, lipid, PEG"
  },
  {
    "caption": "B. Still images of GFP (syncytia) and Propidium Iodide (PI) (cell death) at different time-points. Scale bar: 100 µm.",
    "molecules": "PI, Propidium Iodide"
  },
  {
    "caption": "D. S staining of infected U2OS-ACE2 cells analyzed by immunofluorescence. The Hoechst dye stains the nuclei. Scale bar: 40 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. A. TMPRSS2 increases fusion and cell mortality. Right panel: Areas of GFP+ cells and PI+ cells",
    "molecules": "PI"
  },
  {
    "caption": "G-J) ChIP-qPCR for indicated antibodies after treatment with 5nM rapamycin for the indicated time points. Samples were either normalized to input or to the core  histone. n=3, biological replicates, error bars are standard error of the mean. *, p<0.05, relative to t=0, based on unpaired, 2-tailed t-test",
    "molecules": "rapamycin"
  },
  {
    "caption": "C) Peptide pulldown using recombinantly purified Nhp2. Eluted peptides were  slot blotted  and detected using streptavidine-HRP.",
    "molecules": "HRP, streptavidine"
  },
  {
    "caption": "G. IncuCyte live-imaging analysis of WT and Casp11-/- BMDM viability  after infection with  M. catarrhalis as in D, or transfection with LPS. Data information: Data are pooled from two (G) independent experiments mean and s.e.m. in G).",
    "molecules": "LPS"
  },
  {
    "caption": "A. Immunoblot  analysis of caspase-1 (Casp-1),  caspase-11 (Casp-11) and  gasdermin D (GSDMD) in WT, Nlrp3-/-, Casp11-/- and Aim2-/- BMDMs  left untreated (Med.) or 5 h after transfection with 5 µg of LOS  from M. catarrhalis (O35E) or 5 µg of LPS  from  E. coli. Data information: Data are from three independent experiments (A,",
    "molecules": "LOS, LPS"
  },
  {
    "caption": "A. Immunoblot  analysis of caspase-1  (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Gbp1-/-, Gbp2-/-, Gbp3-/-, Gbp5-/-, Gbp7-/-, Gbp4/8/9-/-, Gbp11­­-/- and Casp11-/- BMDMs  left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 µg LPS  from E. coli.",
    "molecules": "LPS"
  },
  {
    "caption": "B. Quantification of OD600  of M. catarrhalis in BHI media over 6 h in the presence of solvent control (Sol. Ctrl.), 80 µg/mL recombinant GBP2  or 50 µg/mL kanamycin. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B Data are from one experiment representative of two (A) or three (B, Each independent experiment consists of three (B, technical replicates.",
    "molecules": "kanamycin"
  },
  {
    "caption": "C. Quantification of ATP  from M. catarrhalis in BHI media over 6 h in the presence of Sol. Ctrl., 30 µg/mL recombinant GBP2  or 50 µg/mL kanamycin. Data information: Data are pooled from two (C) independent experiments Each independent experiment consists of two (C) technical replicates.",
    "molecules": "ATP, kanamycin"
  },
  {
    "caption": "(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for U2OS-ACE2 20h post infection at MOI 0.001. Right Panel: Representative images of U2OS-ACE2 20h post infection, GFP-Split (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(C) Left Panel: Quantified fusion of Vero cells infected at MOI 0.01. Right Panel: Representative images of Vero cells 48h post infection, GFP-Split (Green) and Hoechst (Blue).",
    "molecules": "Hoechst"
  },
  {
    "caption": "(B) Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(B) Left Panel: Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S. Right Panel: Representative images of fusion at 20h. Scale bar: 200 µm. Top and bottom are the same images with and without Hoechst channel.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(A) Vero GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.",
    "molecules": "Hoechst"
  },
  {
    "caption": "(D) Caco2 GFP-split cells were transfected with variant S proteins and imaged 18h post-transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Caco2 GFP-split cells 18h post-transfection, GFP (Green) and Hoechst (Blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm",
    "molecules": "Hoechst"
  },
  {
    "caption": "Representative H&E (top) and immunofluorescent (IF) images (bottom) of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (for secretory cell, white), Acetylated tubulin (Act-Tub, for ciliated cell, green), and DAPI (blue). Scale bar, 100μm. Statistical quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (B).",
    "molecules": "DAPI, tamoxifen"
  },
  {
    "caption": "Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (for AT1 cell, white), and DAPI (blue). Scale bar, 100μm. Quantification of Scgb1a1 lineage-labeled tdTomato+AQP5+ AT1 cells in (D). Data are presented as mean ± SEM (n=5 mice for each group).",
    "molecules": "DAPI, tamoxifen"
  },
  {
    "caption": "Representative H&E (top) and IF images (bottom) of control, dKO, and qKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (white), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (G). Data are presented as mean ± SEM (n=3 mice for each genotype).",
    "molecules": "DAPI, tamoxifen"
  },
  {
    "caption": "Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CLDN4 (for DATPs, white), CC10 (G, green), AGER (H, for AT1 cell, green), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CLDN4+ DATPs in (G and H). Data are presented as mean ± SEM (n=5 mice for each genotype).",
    "molecules": "DAPI, tamoxifen"
  },
  {
    "caption": "Representative brightfield and H&E images of 3D organoids. Arrow indicates transitioned 3D organoids retaining DATP and AT1 cells. Scale bars, 1mm for brightfield images, 200μm for H&E images. Representative IF images of secretory, DATP, or AT1 cells in secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), CC10 (E, green), CLDN4 (E, white), AGER (F, green), and DAPI (blue). Quantification of transitioned 3D organoids in 3D normal media (3D), amino acid-limited Basal MEM media (BME), or affluent repletion of EAA in BME media (BME+EAA) with 4-OHT or EtOH treatment. Representative IF images of YAP and TAZ expression of secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), YAP (green), TAZ (white), and DAPI (blue). Scale bars, 100μm.",
    "molecules": "4-OHT, amino acid, EAA, DAPI, EtOH"
  },
  {
    "caption": "Representative H&E and IF images of control, Lats1/2 dKO, and Raptor;Lats1/2 tKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green, upper), AGER (green, lower), and DAPI (blue). Scale bar, 100μm.",
    "molecules": "DAPI, tamoxifen"
  },
  {
    "caption": "Measurement of L-amino acid or essential amino acids (leucine, isoleucine, valine) contents in Scgb1a1 lineage-labeled tdTomato+ organoids derived from control, Lats1/2 dKO or Raptor;Lats1/2 tKO mice. Data are presented as mean ± SEM (n=3 technical replicates for each genotype). *p<0.05, **p<0.001 (Student's t-test).",
    "molecules": "isoleucine, L-amino acid, leucine, valine"
  },
  {
    "caption": "Concentration of L-amino acids (L-AA, left) or EAA (right; leucine, isoleucine, valine) in cell lines in (A). Data are presented as mean ± SEM (n=3 biological replicates for each group). *p<0.05, NS=not significant (Student's t-test).",
    "molecules": "isoleucine, L-AA, L-amino acids, leucine, valine"
  },
  {
    "caption": "Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP cell marker CLDN4, and AT1 cell marker CAV1 in organoids derived from Lats1/2 dKO secretory cells with shscramble, shAtf4, or shSlc7a5. CC10 (green), CLDN4 (white), CAV1 (white) and DAPI (blue). Quantification of Scgb1a1+ lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (green), and tdTomato+CAV1+ AT1 cells (blue) in (E). Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP cell marker CLDN4, or AT1 cell marker CAV1 in organoids derived from Scgb1a1 lineage-labeled secretory cells with or without hydroxytamoxifen-induced ATF4 overexpression. CC10 (green), CLDN4 (white), CAV1 (white), and DAPI (blue). Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (green), and tdTomato+CAV1+ AT1 cells (blue) in (G).",
    "molecules": "DAPI, hydroxytamoxifen"
  },
  {
    "caption": "Representative IF images showing the expressions of nuclear YAP, DATP cell marker CLDN4, and AT1 cell marker AQP5 in the airways of normal background (top) and BO (bottom) human lungs. CLDN4 (white), AQP5 (red), YAP (green), and DAPI (blue). Of note, a flattened airway layer in BO lungs. Scale bars, 100μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF images showing the expressions of secretory cell marker CC10 and mTORC1 activation marker p-S6 in the airways of normal background (top) and BO (bottom) human lungs. CC10 (green), p-S6 (white), and DAPI (blue). Scale bars, 100μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "Representative IF images showing the expression of nuclear YAP, ATF4, and AQP5 in the airways of normal background (top) and BO (bottom) human lungs. YAP (green), ATF4 (white), AQP5 (red), and DAPI (blue). Of note, co-expressions of nuclear YAP, nuclear ATF4, and AQP5 in the airways of BO lungs. Scale bars, 100μm.",
    "molecules": "DAPI"
  },
  {
    "caption": "(C) Top panel: Representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged images of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows point to the enlarged PS+ RGC inputs in the insets. (D) Quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and P13.",
    "molecules": "CTB488, PS"
  },
  {
    "caption": "(E) Co-labeling of vGlut2 and PSVue in the dLGN at P6. Circles indicate colocalized vGlut2 and PSVue.",
    "molecules": "PSVue"
  },
  {
    "caption": "(F) Co-labeling of Homer1 and PSVue in the dLGN at P6. Circles indicate colocalized Homer1 and PSVue.",
    "molecules": "PSVue"
  },
  {
    "caption": "(G) Orthogonal sections showed a triple positive PS+/CTB488+/CD68+ RGC inputs inside of microglia.",
    "molecules": "CTB488, PS"
  },
  {
    "caption": "(H) Orthogonal sections showed a triple positive PS+/vGlut2+/CD68+ synapse inside of microglia.",
    "molecules": "PS"
  },
  {
    "caption": "(I) A representative image of microglia (upper-left) is surface rendered (bottom-left). The white arrow points to cells which might be apoptotic cells labeled by PSVue. RGC inputs and PSVue outside or inside of microglia are shown in the two right panels. Scare bar, 20 µm.(J) Quantification of the percentage of PS+ and PS- RGC inputs outside of microglia in total inputs. N = 4. (K) Quantification of the percentage of engulfed PS+ and PS- RGC inputs in total engulfed inputs. N=4. ",
    "molecules": "PSVue, PS"
  },
  {
    "caption": "(D) The binding experiment using flow cytometry show that only the GAIN domain binds to PS, similar as Annexin V binding.",
    "molecules": "PS"
  },
  {
    "caption": "(E) In the competition experiment, AF647 channel shows that GAIN domain binds 60.1% of the PS+ Ba/F3 cells (left). Correspondingly, FITC channel reveals 39.7% Annexin V binding to PS+ Ba/F3 cells (right).",
    "molecules": "AF647, FITC, PS"
  },
  {
    "caption": "(G) Direct binding of hFc-tagged GPR56 GAIN proteins to PS and other phospholipids. HFc was used as a negative control, and GAS6 as a positive control. ",
    "molecules": "PS, phospholipids"
  },
  {
    "caption": "(N) Representative images and surface rendered microglia (green) in which CTB+(red) RGC inputs were engulfed. (O) Quantification of the percentage of engulfed RGC inputs in controls, Gpr56 null and Gpr56 S4 microglia.",
    "molecules": "CTB"
  },
  {
    "caption": "(B) CTB-labeled dLGN shows reduced eye-segregation at P30 in CKO mice. The left column shows contralateral dLGN labeled with CTB488 (green), and the middle one is ipsilateral dLGN with CTB647 (magenta). The right column represents the dLGN pseudocolored according to the R-value for each pixel (R = log(Fipsi/Fcontra)). (C) A histogram distribution chart of R-value for all pixels within dLGN represents the degree of eye-specific segregation. When R is 0, it means equal ipsilateral and contralateral fluorescence intensity at a pixel. A greater R-value means a bigger difference of ipsi-to-contraleteral fluorescence intensity. The narrower distribution of CKO in the inset indicates reduced segregation. (D) The variance of R distributions in P30 control and CKO mice. N = 4 (Ctrl), N = 6 (CKO), p = 0.004, mean ± SD. ",
    "molecules": "CTB, CTB488, CTB647"
  },
  {
    "caption": "(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls.",
    "molecules": "PSVue, PS"
  },
  {
    "caption": "(C) Representative images of engulfed PS+ and PS- RGC inputs by microglia in control and CKO. (D) Quantification of engulfed PS+ RGC inputs by microglia, which is calculated as: Volume of engulfed PS+ RGC inputs / Volume of microglia cell. (E) Quantification of engulfed PS- RGC inputs in control and CKO. ",
    "molecules": "PS"
  },
  {
    "caption": "D: Analytical ultracentrifugation absorbance data analysis of N-NTD. The clear peak at ~15.4 kDa molecular weight (MW) corresponds to the N-NTD monomer. E: Analytical ultracentrifugation absorbance data analysis of N-CTD. The clear peak at ~28.7 kDa MW corresponds to the N-CTD dimer. ",
    "molecules": "dimer, monomer"
  },
  {
    "caption": "(G) Baf A1 sensitivity assay for NDST3  KO cells after the NDST3 restoration. The NDST3  KO cells with stable expression of Flag-NDST3 or Flag-EGFP (control) were treated with 25 nM  Baf A1 for 72 h, and cell survival was measured using  calcein AM staining. The cell survival  was calculated as the fluorescence intensity of the treated cells relative to the untreated cells (n = 4 independent cultures, ****P < 0.0001). Data information: Error bars represent ± standard deviation. Scale bar, 200 μm. ",
    "molecules": "Baf A1, calcein AM"
  },
  {
    "caption": "(C) Lysosomal pH  values in WT and NDST3  KO RPE1  cells calculated from the fluorescence ratio of LysoSensor Yellow/Blue dextran staining against the pH calibration curve in (B) (n = 3 independent cultures, *P = 0.0242). (D) Lysosomal  pH values measured for WT and NDST3 KO  RPE1 cells after the treatment with Baf A1  (100 nM, 1h) (n = 3 independent cultures, *P = 0.0305). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.",
    "molecules": "Baf A1, Blue dextran, LysoSensor Yellow"
  },
  {
    "caption": "(F) Magic Red assay for Cat B proteolytic activity. WT and NDST3 KO RPE1 cells were treated with vehicle (VEH) or Baf A1 (100 nM, 1 h) and then stained with MR-(RR)2 to measure the Cathepsin B activity (n = 48 cells in the VEH-treated WT group, n = 37 cells in the VEH-treated NDST3 KO group, n = 64 cells in the Baf A1-treated WT group, n = 42 cells in the Baf A1-treated NDST3  KO group, ****P < 0.0001).",
    "molecules": "Magic Red, MR, Baf A1"
  },
  {
    "caption": "(G) Alexa Fluor (AF) 488-dextran degradation assay. WT and NDST3 KO  RPE1 cells were loaded with AF488-dextran in culture medium and observed for fluorescence. The AF488  fluorescence signals without a chase period, with a 4-h chase period, and with the 4-h chase period in the presence of Baf A1 (100 nM, 1h) were recorded as fluorescence of uptake, chase, and chase with Baf A1, respectively. The AF488 intensity per cell was quantified and calculated into  degradation percentage according to the equation in Materials and Methods (n = 62 cells in the VEH-treated WT group, n = 36 cells in the VEH-treated  NDST3  KO group, n = 65 cells in the Baf A1-treated WT group, n = 78 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).",
    "molecules": "AF488, Alexa Fluor (AF) 488, Baf A1, dextran"
  },
  {
    "caption": "(J) The LC3  turnover assay after a 48-h Baf A1 treatment. WT and NDST3 KO  RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B-II  fold change as in (I) (n = 6 independent experiments, **P = 0.0014). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm. ",
    "molecules": "Baf A1"
  },
  {
    "caption": "(A, B)  Perinuclear  enrichment and colocalization of acetylated microtubules,  lysosomes, and V-ATPase V1 subunits. RPE1 cells stably expressing LAMP1-GFP were treated with vehicle (VEH) or 10 μg/mL nocodazole (Noco) for 6 h before being fixed and subjected to  immunofluorescence staining with antibodies against ATP6V1C1 and acetylated-α-tubulin (Ac-α-tubulin). The nucleus was  stained with DAPI. Representative images of  LAMP1-GFP,  ATP6V1C1, and Ac-α-tubulin immunostaining are shown in (A). Representative distribution profiles of  Ac-α-tubulin, LAMP1-GFP and ATP6V1C1 were plotted by Fiji software in (B).",
    "molecules": "DAPI, Noco, nocodazole"
  },
  {
    "caption": "(C) In vitro enzymatic assay quantitating the effect of NDST3 on α-tubulin acetylation. Flag-NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti-Flag beads. Flag-EGFP was used as a control. The immunoprecipitated  protein was incubated with purified tubulin heterodimers in vitro, with or without 1 mM NAD+, 400 nM TSA, or 5 mM nicotinamide (NAM) for 2 h at room temperature. The reaction products were examined by  immunoblotting against  Ac-α-tubulin or total α-tubulin (n = 3 independent experiments, *P = 0.0185, **P < 0.01, ****P < 0.0001). (D) The same in vitro enzymatic assay as in (C), except that the purified  tubulin  was polymerized in the presence of 20 μM Taxol  and 1 mM GTP (n = 3 independent experiments, **P < 0.01, ***P = 0.0001). Data information: Error bars represent ± standard deviation. ns means non-significant. ",
    "molecules": "GTP, NAD+, NAM, nicotinamide, Taxol, TSA"
  },
  {
    "caption": "(M, N) Baf A1 sensitivity assay for WT,  NDST3 KO, and Noco-treated NDST3 KO  RPE1 cells expressing proline-arginine poly-dipeptide (PR82). 24 h after the transfection of  PR82, WT and NDST3  KO cells were treated with vehicle or 10 μg/mL Noco overnight. The cell survival measured using calcein AM staining at 48 h post-transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells (n = 3 independent cultures, *P = 0.0369, ***P = 0.0007). Scale bar, 200 μm. Data information: Error bars represent ± standard deviation.",
    "molecules": "arginine, Baf A1, calcein AM, Noco, proline"
  },
  {
    "caption": "(A) RCC1.24‐NeoR and ‐NucNeoR cells were incubated for 48 h with IFN‐γ and in addition with 7.5 mM 3‐MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with 20‐4/A4 cells in a GM‐CSF release assay. Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing.",
    "molecules": "3‐MA, chloroquine, leupeptin"
  },
  {
    "caption": "(C) To exclude toxic side effects of the inhibitors, LCL1.11 cells were incubated for 20 h with 200 ng/mL recombinant NeoR protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the NeoR‐specific CD4+ T cell clone 20‐4/A4. The presentation of exogenous NeoR on MHC class II was not affected by 3‐MA, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin.",
    "molecules": "3‐MA"
  },
  {
    "caption": "(A) RCC1.24 cells were transfected with the NES‐GFP‐NeoR‐GFP‐NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence. As compared to untreated cells (a), cells treated with 8 nM LMB for 20 h (b) showed a strong accumulation of the protein in the nucleus. (a') and (b') are the corresponding daylight photographs of the transfected cells.",
    "molecules": "LMB"
  },
  {
    "caption": "(B) NeoR‐ and NucNeoR‐transfected LCL1.11 cells were either left untreated or incubated for 20 h with 8 nM LMB. Subsequently, the cells were fixed with 0.5% paraformaldehyde and probed with the NeoR‐specific T cells. LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II, indicating that CRM1‐dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II.",
    "molecules": "LMB"
  },
  {
    "caption": "(A) The indicated cell lines stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to confocal immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial or complete translocation to mitochondria in each cell was scored as separate phenotypes. Partial and complete denote that Parkin translocates to some of or all mitochondria, respectively. The error bars represent ±SD from three independent experiments. Over 100 cells were counted in each of three separate wells. (C) YFP-LC3 morphologies in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate. For the criteria of LC3 morphology, see Figure 2-figure supplement 1.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(D) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the absence or presence of HA-tagged TBC1D15 WT or Δ221-250 mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (E) The YFP-LC3 morphology of cells in (D) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(F and G) YFP-LC3 and mCherry-Parkin stably expressing WT (F) and TBC1D15−/− (G) cells were treated with valinomycin for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. The square in panel a shows enlarged areas in panel b Scale bars, 500 nm.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(A) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti-Cytochrome c antibody. Confocal images were acquired as z-stacks comprising 6 sequential sections with 0.8 μm z-intervals. Scale bars, 10 μm.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(D) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr. YFP-LC3 and mCherry-Parkin images are merged in the right panels. Scale bars, 10 μm. (E) YFP-LC3 morphologies of cells in (D) were quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.",
    "molecules": "valinomycin, nocodazole"
  },
  {
    "caption": "(A and B) YFP-Parkin stably expressing cells were treated with valinomycin for indicated times. Total cell lysates were subjected to immunoblotting. I and II denote cytosolic and lipidated LC3B, respectively. An asterisk indicates ubiquitinated TOMM20. (C) Indicated protein amounts as in (B) were quantified. The amount of protein without valinomycin treatment was set to 100%. The error bars represent ±SD from three independent experiments.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(D) YFP-Parkin stably expressing WT HCT116 cells were treated with or without valinomycin for 40 hr. Total cell lysates were analyzed by immunoblotting.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(A) WT, MFF−/− or DRP1−/− cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial and complete denote that Parkin translocates to some of and all mitochondria, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate. (C) YFP-LC3 morphologies of cells in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(A) The indicated cells transiently expressing YFP-GABARAPL1 (green) and mCherry-Parkin (red) were treated with valinomycin for 3 hr. Scale bars, 10 μm.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(J) LC3-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(B) The indicated cells stably expressing YFP-LC3 (green) and mCherry-Parkin were treated with control (NTC) or Rab7_#5 siRNA. After 3 hr valinomycin treatment, cells were analyzed by immunofluorescence microscopy with anti-TOMM20 antibody (red). Z-stacks of confocal images are shown. Magnified images are also shown. Scale bars, 10 μm. (C) YFP-LC3 morphologies of cells in (B) were quantified. Percentages of cells harboring diffuse, punctuate or accumulated/tubulated YFP-LC3 are shown. Data and error bars were obtained from at least 50 cells in each of three independent replicates.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(D) The indicated cells stably expressing YFP-LC3 (green), mCherry-Parkin, and 2HA-Rab7 (Red) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti-HA antibody. Magnified images are also shown. Scale bars, 10 μm.",
    "molecules": "valinomycin"
  },
  {
    "caption": "(E) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (F) The YFP-LC3 morphology of cells in (E) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate.",
    "molecules": "valinomycin"
  },
  {
    "caption": "E. Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog. Oct4 expression was confirmed by the Oct4-GFP transgene. DNA stained by DAPI. Scale bars: 100 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "A. In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry: endoderm (α-fetoprotein, AFP), mesoderm (α-smooth muscle actin, SMA), and ectoderm (α-tubulin, TUJ1). Nuclei (DNA) were stained by Hoechst (blue). Scale bars: 200 μm.",
    "molecules": "DNA"
  },
  {
    "caption": "(F) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.",
    "molecules": "RDEA119"
  },
  {
    "caption": "B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student's t test.",
    "molecules": "2APB, dantrolene"
  },
  {
    "caption": "C. Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co-cultures with either no treatment, 30 min DHPG, or 2APB and dantrolene pretreatment followed by DHPG. D. Quantified expression of GluA1 under each condition, n = 9 replicates of 10 cells, p < 0.001, ANOVA. E. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. F. Quantified expression of GluA2 under each condition, n = 12 replicates of 10 cells each, p < 0.001, ANOVA. G. Representative images of surface myc-GluA2, internal myc-GluA2, and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. H. Quantified expression of surface myc:total myc under each condition, n = 4 replicates of 10 cells each, p < 0.05, ANOVA. All error bars represent s.e.m. Abbr. A1 = GluA1, A2 = GluA2, D = DARPP32, s-myc = surface myc, i-myc = internal myc, m = merge.",
    "molecules": "2APB, DHPG, dantrolene"
  },
  {
    "caption": "A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student's t test.",
    "molecules": "DHPG, S107, XeC, Xestospungin C"
  },
  {
    "caption": "B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA.",
    "molecules": "DHPG, S107, XeC, Xestospungin C"
  },
  {
    "caption": "C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student's t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student's t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.",
    "molecules": "DHPG, BAPTA, BAPTA-AM, TG, Thapsigargin"
  },
  {
    "caption": "A. Top. Representative image of a radiograph of mature and immature bands of GluA2 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA2 band to the total GluA2 for each condition. For DHPG, n = 16; for DHPG + 2APB/dan, n = 11, p < 0.05, ANOVA.",
    "molecules": "2APB, DHPG, dan"
  },
  {
    "caption": "B. Top. Representative image of a radiograph of mature and immature bands of GluA1 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA1 band to the total GluA1 for each condition, For DHPG, n = 4; for DHPG + 2APB/dan, n = 4, ANOVA.",
    "molecules": "2APB, DHPG, dan"
  },
  {
    "caption": "C. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment. D. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.",
    "molecules": "DHPG, brefeldin A"
  },
  {
    "caption": "A Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and 2APB/dantrolene followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 10 min DHPG, n = 8; for 10 min DHPG + 2APB/dan, n = 6, p < 0.05, ANOVA.",
    "molecules": "2APB, DHPG, dan, dantrolene"
  },
  {
    "caption": "B. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and and cycloheximide followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions, For 10 min DHPG, n = 8; for 10 min DHPG + CHX, n = 5, p < 0.05, ANOVA.",
    "molecules": "DHPG, CHX, cycloheximide"
  },
  {
    "caption": "C. Top. Representative immunoblots from GluA2 co-IPs probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and 2APB/dantrolene followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 17; For 30 min DHPG + 2APB/dan, n = 14, p < 0.05, ANOVA.",
    "molecules": "2APB, DHPG, dan, dantrolene"
  },
  {
    "caption": "D. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and cycloheximide followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 12; For 30 min DHPG + CHX, n = 11 p < 0.01, ANOVA.",
    "molecules": "DHPG, CHX, cycloheximide"
  },
  {
    "caption": "E. Top. Representative immunoblot demonstrating IgG and GluA2 coIP after no treatment and 30 min DHPG. Bottom. Quantification of Sec23 after subtracting the non-specific signal, n = 3, p < 0.01, Student's t test.",
    "molecules": "DHPG"
  },
  {
    "caption": "A. Representative immunoblots from GluA2 coIP probed for Sec23, Sec13, and GluA2 after no DHPG, 10, 30, and 60 minutes of treatment with DHPG. B. Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A. For Sec23, 10 min DHPG, n = 16; 30 min DHPG, n = 15; 60 min DHPG, n = 7, p < 0.0001, ANOVA. For Sec13, 10 min DHPG, n = 7; 30 min DHPG, n = 14; 60 min DHPG, n = 6, p < 0.01, ANOVA.",
    "molecules": "DHPG"
  },
  {
    "caption": "C. Representative images of total expression levels of Sec23 (top) and Sec13 (bottom). Expression levels do not change in the presence of DHPG. All error bars represent s.e.m.",
    "molecules": "DHPG"
  },
  {
    "caption": "A. Individual traces of mEPSCs of both control and naspm treated neurons after TTX, TTX + DHPG, and TTX + DHPG + 2APB/dan treatment.",
    "molecules": "naspm, 2APB, DHPG, dan, TTX"
  },
  {
    "caption": "B. Averaged mEPSCs and an enlarged time scale from cells under the same conditions as A. Traces from cells that were not treated with naspm are in black and naspm treated traces are in grey.",
    "molecules": "naspm"
  },
  {
    "caption": "C. Average amplitude of mEPSCs (pA) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 17; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.005, ANOVA. D. Average frequency of mEPSC (Hz) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 12; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.0001, ANOVA.",
    "molecules": "naspm, 2APB, DHPG, dan, dantrolene, TTX"
  },
  {
    "caption": "E. Average amplitude of mEPSCs (pA) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. F. Average frequency of mEPSC (Hz) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene.",
    "molecules": "naspm, 2APB, DHPG, dantrolene, TTX"
  },
  {
    "caption": "G. Average rise time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. H. Average decay time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. All error bars represent s.e.m.",
    "molecules": "naspm, 2APB, DHPG, dantrolene, TTX"
  },
  {
    "caption": "B) Percentage specific binding of [3H]dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence (control) or presence of 10 and 50 µM of LUF compounds. The specific binding of [3H]dofetilide in the absence of test compounds was set as Bcontrol, while the specific binding in their presence was set as B. * = p < 0.05 vs control; N: 3-4.",
    "molecules": "dofetilide"
  },
  {
    "caption": "C) Representative traces of hERG activation (left), inactivation (middle), and deactivation (right) measured in HEK293 hERG cells under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). Insets: voltage clamp protocols.",
    "molecules": "LUF7346"
  },
  {
    "caption": "D) Steady-state activation (empty symbols) and inactivation (filled symbols) curves for IKr under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). The corresponding Boltzmann's fittings are superimposed to data points.* = p < 0.05 vs baseline. N: 11-14.",
    "molecules": "LUF7346"
  },
  {
    "caption": "E) Plot of the time constants (τ) of deactivation derived from biexponential fittings under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p < 0.05 vs respective baseline. N: 8.",
    "molecules": "LUF7346"
  },
  {
    "caption": "C) Representative traces of IKr steady state activation in WT hiPSC-CMs in Tyrode (left), in the presence of 5 µM LUF7346 (middle) and after the application of 5 µM E4031 to selectively block IKr (right). Inset: voltage clamp protocol.",
    "molecules": "LUF7346, E4031"
  },
  {
    "caption": "D) Average I/V relationships (left) and steady-state activation curves with superimposed Boltzmann's fittings (right) under baseline conditions (black) and in the presence of 5 µM LUF7346 (red). * = p<0.05. N: 19.",
    "molecules": "LUF7346"
  },
  {
    "caption": "E) Plot of the time constants (τ) of deactivation derived from biexponential fits (left) and representative examples (right) under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p<0.05. N: 17-21.",
    "molecules": "LUF7346"
  },
  {
    "caption": "A) Representative MEA trace (left) showing the effect of increasing concentrations of LUF7346 on FP contour, measured in LQT2N996I-CMs. Average data (right) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs baseline. Colour in the heatmap defines the magnitude of QT shortening (blue) after treatment with increasing concentrations of LUF7346. N: 7-14.",
    "molecules": "LUF7346"
  },
  {
    "caption": "B) Representative AP from LQT2corr-, LQT2N996I-, LQT1corr-, and LQT1R190Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of increasing concentrations of LUF7346 (colour-code is shown).",
    "molecules": "LUF7346"
  },
  {
    "caption": "C) Effect of LUF7346 on APD90, APD70, and APD50, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), LQT1R190Q- (dark blue) paced at 1 Hz; * = p<0.05 vs baseline. The colour of the asterisk indicates comparisons and respective statistical significance. ° = p<0.05 vs the genetically-matched corrected control. # = p<0.05 LQT1corr vs LQT2corr. N: 12-15.",
    "molecules": "LUF7346"
  },
  {
    "caption": "D) Effect of LUF7346 on APA and Ediast, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), and LQT1R190Q-CMs (dark blue) paced at 1 Hz. N: 12-15.",
    "molecules": "LUF7346"
  },
  {
    "caption": "A) Representative MEA trace (left) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour, measured in LQT1R594Q-CMs. Average data (right) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs respective baseline. ° = p<0.05 vs AST. Colour in the heatmap defines the magnitude of QT prolongation (red) and QT shortening (blue), respectively, after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST. N: 5-10.",
    "molecules": "LUF7346, AST"
  },
  {
    "caption": "B) Representative AP from WT-, LQT1R594Q- and JLNSR594Q-CMs, paced at 1 Hz, under baseline conditions (black), after application of 100 nM AST (red), and after addition of increasing concentrations of LUF7346 in the presence of AST (colour-code is shown).",
    "molecules": "LUF7346, AST"
  },
  {
    "caption": "C) Effect of AST and LUF7346 in the presence of AST on APD90, APD70, and APD50 in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. * = p<0.05 vs respective baseline; ° = p<0.05 vs AST. N: 11-16.",
    "molecules": "LUF7346, AST"
  },
  {
    "caption": "D) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. N: 11-16.",
    "molecules": "LUF7346, AST"
  },
  {
    "caption": "A: Representative AP traces measured in JLNSR594Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of either AST alone (red), AST+LUF7346 3 µM (green) and AST+LUF7346 5 µM (blue).",
    "molecules": "LUF7346, AST"
  },
  {
    "caption": "C: STV changes (left) and representative examples of QT interval oscillations plotted over 30 consecutive beats (right) in the presence of AST and AST+LUF7346 20 µM. * = p<0.05 vs Baseline. ° = p<0.05 vs AST. N = 7-24.",
    "molecules": "LUF7346, AST"
  },
  {
    "caption": "(A) Silver-stained PAGE gel of proteins from a nuclear extract that interact with either GST-VgpA or GST glutathione agarose. The protein bound by GST-VgpA (red arrow), but not GST, was identified as EBP2 by mass spectrometry; GST-VgpA is labeled with a blue arrow.",
    "molecules": "glutathione"
  },
  {
    "caption": " (D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes primarily to the nucleoplasm (white arrow, lower panel).",
    "molecules": "DAPI"
  },
  {
    "caption": " (A) Fluorescence micrographs of uninfected Caco-2 cells or Caco-2 cells infected with the indicated V. parahaemolyticus strains. Cells were stained with DAPI (blue) and anti-BrdU antibody (green). ",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": " (B) Fluorescence micrographs of Caco-2 cells transfected with siRNA targeting EBP2 (siEBP2) or control siRNA (siNC) and subsequently infected with vopZ'ΔvscN1. Cells were stained with anti-BrdU antibody (green) and DAPI (blue). ",
    "molecules": "BrdU, DAPI"
  },
  {
    "caption": " (C) The percentage of BrdU-positive cells (green) among the total cells (green + blue) was analyzed for 100 cells in three experiments. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). One-way analysis of variance (ANOVA) was used for statistical analysis. *P<0.05 (when compared to the uninfected Caco-2 cells). ",
    "molecules": "BrdU"
  },
  {
    "caption": " (D) The percentage of BrdU+ cells was analyzed as above for cells treated with siNC or siEBP2. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). Two-way ANOVA was used for statistical analysis. *P<0.05 (when compared to siNC-transfected cells that are infected with vopZ'ΔvscN1). ",
    "molecules": "BrdU"
  },
  {
    "caption": " (A) Localization of c-Myc in uninfected Caco-2 cells (first row), Caco-2 cells infected with V. parahaemolyticus vopZ'ΔvscN1 (wild type VgpA, second row), Caco-2 cells infected with vopZ'ΔvscN1vgpA' (third row), Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpA (fourth row) and Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpAL10A (fifth row). Fixed cells were stained with DAPI (blue), and anti-EBP2 (red), and anti c-Myc (green) antibodies. White arrows are examples indicating the co-localization of EBP2 and c-Myc. ",
    "molecules": "DAPI"
  },
  {
    "caption": " (E) Caco-2 cells were infected with the indicated strains and treated with cycloheximide (CHX) for the indicated time. Whole cells lysates were analyzed by western blot using anti-c-Myc and anti-Actin antibody. ",
    "molecules": "CHX, cycloheximide"
  },
  {
    "caption": " (A) Amounts of rRNA were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells). ",
    "molecules": "rRNA"
  },
  {
    "caption": " (B) Amounts of rRNA in uninfected or infected Caco-2 cells transfected with control (siNC) or EBP2-targeting (siEBP2) siRNAs. *P<0.05 (when compared to the siNC-transfected cells that are infected with vopZ'ΔvscN1). ",
    "molecules": "rRNA"
  },
  {
    "caption": " (D). Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 and 38 h after infection. *P<0.05 (when compared to the uninfected rabbits at 18 hours). ΔP<0.05 (when compared to the uninfected rabbits at 38 hours post infection). ",
    "molecules": "rRNA"
  },
  {
    "caption": " (C) Frozen sections from uninfected rabbits or rabbits infected with the indicated strains for 18 hours were stained with anti-Ki67 antibodies to detect actively dividing cells (green), phalloidin to visualize F-actin (red) and DAPI to detect nuclei (blue). ",
    "molecules": "DAPI, phalloidin"
  },
  {
    "caption": " (D) Eighteen hours after infant rabbits were infected with WT (middle panel) or vgpAL10A (lower panel) V. parahaemolyticus, fixed sections from the small intestine were stained with antibodies to c-Myc (green), EBP2 (red), and DAPI (blue). Uninfected rabbits were processed similarly and were included as a control (upper panel). White arrows indicate co-localization of c-Myc and EBP2 (middle panel). ",
    "molecules": "DAPI"
  },
  {
    "caption": "(B) Representative confocal microscopy sections of m6A-Tracer signal in HAP-1 cells following pA-DamID with indicated antibodies. pA-Dam only: primary antibody was omitted, the dotted yellow line indicates DAPI segmentation. Free Dam: permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min. Scale bar corresponds to 2 µm. (C) Quantification of peripheral enrichment of antibody staining and m6A-Tracer signals after pA-DamID with indicated antibodies. The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2-ratio. The nuclear rim mask extends slightly beyond the NL, resulting in underestimation of the real enrichment. ",
    "molecules": "DAPI, m6A, SAM"
  },
  {
    "caption": "(D) HT1080 cells expressing inducible Dam-Lamin B1 were treated with Shield1 to induce m6A methylation. Cells were either fixed immediately or processed according to the pA-DamID protocol (as negative controls) and then fixed on poly-L-lysine coated cover slips. Cells were imaged by confocal microscopy for the NL (Lamin B2 antibody) and methylation (m6A-Tracer) for both conditions. Laser settings were changed between images to optimize image quality. The mask for defining the nuclear rim was obtained by segmentation of the DAPI image.The scale bar corresponds to 2 µm.",
    "molecules": "DAPI, m6A, poly-L-lysine, Shield1"
  },
  {
    "caption": "(E) For every cell, the 50% decay distance from the nuclear periphery was determined for Lamin B2 and m6A-Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50% decay distance between Lamin B2 and m6A-Tracer was used as a measure of the thickness of the m6A-Tracer layer.",
    "molecules": "DAPI, m6A"
  },
  {
    "caption": "(F) Distribution of the m6A-Tracer layer thickness for HT1080 cells expressing Dam-Lamin B1, visualized before and after the pA-DamID protocol. For comparison, a similar analysis was performed with HT1080 cells not expressing Dam-LaminB1, subjected to Lamin B2 pA-DamID.",
    "molecules": "m6A"
  },
  {
    "caption": "(A) Example of raw pA-DamID data tracks from 2 million HAP-1 cells for Lamin B2, H3K27me3 and the Dam control from a single experiment. Sequenced reads are counted in 20kb bins and normalized for library size. (B) Same pA-DamID tracks of Lamin B2 and H3K27me3 as in (A) but after normalization to the Dam-only control (to correct for accessibility and amplification biases) and log2-transformation. ",
    "molecules": "H3K27me3"
  },
  {
    "caption": "(C) Correlation between the median peripheral enrichment of m6A-Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP-1 cells. The LAD definition is based on conventional Lamin B1 DamID data. Every point represents a single pA-DamID experiment for which both microscopy and sequencing data were generated, colored by the antibody used. The blue line represents a linear model with a standard error confidence interval in grey. The red dashed line represents the diagonal. The Pearson correlation coefficient was converted to a t-statistic and a one-sided t-test with n-2 degrees of freedom was used to determine statistical significance.",
    "molecules": "m6A"
  },
  {
    "caption": "(D) RT-qPCR were performed on RNA extracted from non-Group 3 (blue) and Group 3 (yellow) MB cell lines to compare expression levels of INHBB (left) and TGFB3 (right). The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "RNA"
  },
  {
    "caption": "(D) RT-qPCR were performed on total RNA extracted from 1603MED cells 48 hours after transfection with siRNA targeting INHBB. Relative INHBB expression was assessed. siCTRL condition was set at 1. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "RNA"
  },
  {
    "caption": "D458 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "7AAD"
  },
  {
    "caption": "D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "7AAD"
  },
  {
    "caption": "1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon inhibition of TGFβ/Activin signaling using LY364947 and SB431542 inhibitors for 24 hours. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "7AAD, DMSO, LY364947, SB431542"
  },
  {
    "caption": "1603MED cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48 hours after TGFβ/Activin signaling inhibition. Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon inhibition of TGFβ/Activin signaling using LY364947 and SB431542 inhibitors for 24 hours. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition. The p-values were determined by unpaired t-test and two-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "7AAD, DMSO, LY364947, SB431542"
  },
  {
    "caption": "D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48 hours after TGFβ/Activin signaling inhibition. Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "(F) Immunoblots of phosphorylated Smad2 (P-Smad2), total Smad2, MYC, PMEPA1, OTX2 and β-actin were performed on extracts from 1603MED or D283 or D458 cells treated with either DMSO (vehicle), LY364947, SB431542, blocking antibody against ActivinB (Ab α-ActB), Follistatin, PBS or ActivinB (ActB) for 24 hours.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "(E) Immunoblots of phosphorylated Smad2 (P-Smad2), total Smad2, MYC, PMEPA1, OTX2 and β-actin were performed on extracts from cell cultures of PDX4, PDX3 and PDX7 treated by either DMSO (vehicle), LY364947, SB431542, blocking antibody against ActivinB (Ab α-ActB), Follistatin, PBS or ActivinB for 24 hours.",
    "molecules": "DMSO, LY364947, SB431542"
  },
  {
    "caption": "(A) Kaplan-Meier representing survival of mice treated with either vehicle (black) or Galunisertib (LY2157299, red) or Cisplatin (blue) or a combination of Galunisertib and cisplatin (purple) after orthotopic grafting of PDX4 cells into the cerebellum. The pink rectangle represents Galunisertib treatment duration, while the blue dotted lines represent the 3 cisplatin administrations. The p-values were determined by Log-Rank (Mantel-Cox) test on panel A *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.",
    "molecules": "Cisplatin, cisplatin, Galunisertib, LY2157299"
  },
  {
    "caption": "TEM image and (B) size distribution of pcMNPs; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles.",
    "molecules": "pcMNPs, nanoparticles"
  },
  {
    "caption": "Zeta potentials of bare MNPs, NH2-MNPs and pcMNPs in water. (D) The magnetic response property of bare MNPs, NH2-MNPs and MBs in 30 s.",
    "molecules": "NH2-MNP, pcMNP, MNP"
  },
  {
    "caption": "RNA binding affinity of pcMNPs analysed by native PAGE;",
    "molecules": "pcMNP"
  },
  {
    "caption": "Dispersion stability of pcMNPs in a high-salt solution (50 mM NaCl).",
    "molecules": "NaCl, pcMNPs"
  },
  {
    "caption": "RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.",
    "molecules": "pcMNPs"
  },
  {
    "caption": "(D)  Two-color dSTORM imaging  of RIM1/2 and  gephyrin  in cryosections. From left to right: rendered images of RIM1/2 and  gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labelled with Alexa 647, gephyrin (mAb7a) with Cy3B  in these experiments.",
    "molecules": "Alexa 647, Cy3B"
  },
  {
    "caption": "(A-C) Alignment of SSDs of pre-synaptic RIM1/2 and SSDs of post-synaptic gephyrin, GlyRs, and  GABAARs, respectively. (A1-C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual-color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labelled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labelled with Cy3B, while GlyRs or GABAARs were labelled with Alexa 647  in (B1) and (C1). (A2-C2) Trans-synaptic alignment of SSDs of gephyrin, GlyRs, GABAARs and RIM1/2. SSDs were segmented by H-watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX  treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All  images are adjusted to the same scale. Scale bar: 200 nm.",
    "molecules": "Alexa 647, Cy3B, TTX"
  },
  {
    "caption": "(A-B) Representative dSTORM images of  GlyRs and GABAARs at synapses in side view (A) or en face view (B). The top row of images shows the pointillist representation of the detections, the second row the rendered images and the bottom row the corresponding segmented SSDs. GlyRs were labelled with Alexa 647 and GABAARs with Cy3B  in these experiments. Scale bar: 200 nm.",
    "molecules": "Alexa 647, Cy3B"
  },
  {
    "caption": "Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses. (A) Synaptic receptor cluster rendered from detections of both  GABAARs (labelled with Alexa 647) and GlyRs (labelled with Cy3B). (B) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with binary  synaptic mask (white).",
    "molecules": "Alexa 647, Cy3B"
  },
  {
    "caption": "(A) The number of receptor SSDs, segmented from the combined receptor clusters of GABAARs and GlyRs, was decreased after 4-AP  treatment (MW test, p < 0.0001). (B) This was mostly due to the loss of GABAAR SSDs  that were segmented from the rendered  images of GABAAR detections only (MW, ****p < 0.0001). (C) The number of GlyR SSDs (based on GlyR detections) were only marginally reduced (MW, **p < 0.01). Data information: Number  of synapses: n = 452 in TTX, n = 583 in  4-AP  condition, from three independent experiments. Mean ± SEM. ",
    "molecules": "4-AP, TTX"
  },
  {
    "caption": "(A-C) Reduced immuno-reactivity of S270 phosphorylated gephyrin but not total gephyrin levels after 4-AP treatment, revealed by conventional fluorescence microscopy (KS test, p < 0.001 in B1 and p < 0.0001 in B2, p < 0.0001 in C). Total gephyrin was probed with polyclonal rabbit primary antibody (rbGPHN), and pS270  phosphorylated gephyrin with monoclonal mouse primary antibody (m7a).  Number of synapses: n = 4040 in TTX and n = 3818 in  4-AP conditions from two independent experiments. Scale bar: 2 µm.",
    "molecules": "4-AP, TTX"
  },
  {
    "caption": "(D-F) Reduced  numbers of pS270 gephyrin (m7a) detections were recorded by dSTORM for the entire synaptic area (D, KS test, p < 0.01). The number of detections of pS270  gephyrin per  SSD (E, KS, p = 0.18) and the number of SSDs (F, MW, p = 0.11) were not changed by 4-AP treatment. Gephyrin was probed with mAb7a antibody and Alexa 647 dye. Number of synapses: n = 810 in TTX and n = 727 in  4-AP conditions from three independent experiments; (F) mean ± SEM. Scale bar: 100 nm.",
    "molecules": "4-AP, Alexa 647 dye, TTX"
  },
  {
    "caption": "G) LIF  and VEGF  synergistically stimulated BCE cell growth. Cell proliferation was analyzed after 6 days using alamar blue as described in Materials and Methods, n=3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ns, not statistically significant. ",
    "molecules": "alamar blue"
  },
  {
    "caption": "A) The JAK  inhibitor baricitinib (Ba) blocked LIF-induced STAT3 phosphorylation. BCE cells were pre-incubated with  DMSO, baricitinib (2 μM), cobimetinib  (Co) (150 nM) or BEZ235 (BE) (5 nM) for 1 hour and were then treated with vehicle or  LIF (10 ng/ml, Sigma) for 15 minutes. Ctrl, no pre-incubation with inhibitors.",
    "molecules": "Ba, baricitinib, Co, cobimetinib, BE, BEZ235, DMSO"
  },
  {
    "caption": "B).  JAK  inhibitor  baricitinib blocked activation of STAT3 by  LIF. BAE  cells pre-incubated with DMSO and inhibitors for 1 hour were treated with vehicle and LIF  (10 ng/ml) for 15 minutes. Whole-cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no pre-incubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib  (150 nM); BE, BEZ235 (5 nM).",
    "molecules": "Ba, baricitinib, Co, cobimetinib, BE, BEZ235, DMSO"
  },
  {
    "caption": "C, D)  OCT-A imaging of LIF-treated mouse retina. Adult mice were intravitreally injected with 1 μl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT-A  images were obtained 7 days after the injection and representative images are shown. Blood vessel  density was determined as percentage of vessel-covered area/total area surface using ImageJ software and shown in D), n=7-8. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test.",
    "molecules": "PBS"
  },
  {
    "caption": "E).  STAT3 was phosphorylated in retinal ECs after LIF  treatment. 50 ng of our in-house  LIF with low-endotoxin levels was injected. Phospho-Stat3 was examined on cryosection of retina after 2 hour by  IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm.",
    "molecules": "endotoxin"
  },
  {
    "caption": "F). Five-day old neonatal  mice  were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight-488-labeled lectin. Representative images for similar  ocular  loci and quantification of lectin-labeled area using ImageJ software were shown, n=4. Scale bar = 100 µm. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ",
    "molecules": "Dyight-488, lectin, PBS"
  },
  {
    "caption": "A).  LIF enhanced laser-induced CNV. Intravitreal administration of LIF (10 ng and 100 ng, Sigma) and OSM (10 ng) after laser-induced CNV. PBS was used as vehicle control. OCT-A imaging and  immunostaining of choroid  flatmounts was carried out 10 days after laser induction. Quantification of CNV area is shown. Dashed line circles indicate the CNV  in OCT-A images. n = 5. Dot plot shows all data points from three independent experiments. Scale bar = 100 µm.",
    "molecules": "PBS"
  },
  {
    "caption": "D).  Avascular area in choroid in sodium iodate model was determined and quantified using Image J. E). Thickness of retina in  H&E  staining was quantified and bar graph was shown using ImageJ. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two-way ANOVA was used as statistical test. ",
    "molecules": "sodium iodate"
  },
  {
    "caption": "(G) Human choroidal EC  (HCEC) and mouse retinal EC (MREC) were treated with  LIF 10 ng, LIF  100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation  was detected by  western blot.",
    "molecules": "PBS"
  },
  {
    "caption": "Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (A) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation. Ribosomal RNAs (rRNAs) stained with ethidium bromide were used as loading controls. Black lines indicate probes used to detect nam1 and byr2 RNAs.",
    "molecules": "rRNAs"
  },
  {
    "caption": "Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (B) Northern blot showing the level of nam5/6/7 lncRNA population in mmi1-ts3, dcr1∆, clr4∆ single mutant cells and in mmi1-ts3 dcr1∆ and mmi1-ts3 clr4∆ double mutant cells, at the permissive (25ºC) and restrictive (36ºC) temperatures.",
    "molecules": "lncRNA"
  },
  {
    "caption": "(a) WT and Pgam5 KO MEFs evaluated by TEM. The black arrows indicate mitochondria; (b) TEM analysis after 12 h CCCP treatment; black arrowheads indicate DMS. Inset shows a DMS with an encapsulated mitochondrion. The black arrows indicate mitochondria. Scale bar=0.5 micron;",
    "molecules": "CCCP"
  },
  {
    "caption": "(c) WT and KO MEFs were treated with CCCP for 0, 3 or 6 h and cytosolic fractions were immunoblotted with anti-LC-3, anti-p62 or anti-actin as indicated.",
    "molecules": "CCCP"
  },
  {
    "caption": "(d) Similar to c. WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti-COXIV, anti-Tomm20 or anti-actin as indicated. For c and d, the numbers below each lane indicate the fractional band density compared with the 0 h time point, which has been set at 1.0;",
    "molecules": "CCCP"
  },
  {
    "caption": "(e) Confocal photomicrographs of mt-Keima-transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h (six individual cells from each group were scanned and representative cells are shown); Intracellular mt-Keima excited at 450 nm was shown in green colour, while red colour indicated the excitation by 550 nm in the same cell. Scale bar=20 μm.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "(f) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (−) or CCCP (+) for 3 h and analysed by immunoblot as above. (Note that mouse PINK1 antibody can only detect the full-length PINK1). Arrow indicates the 63 kDa form of PINK1.",
    "molecules": "CCCP, DMSO"
  },
  {
    "caption": "(g) Hela cells transduced with either nonspecific (NS) or PGAM5 shRNA viruses were treated with CCCP for 3 h, and mitochondrial fractions were immunoblotted for the proteins indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are indicated. For f and g, the numbers below indicate the full-length PINK1 band density compared with the first lane that has been set at 1.0",
    "molecules": "CCCP"
  },
  {
    "caption": "(h) Confocal micrographs of Hela cells as in g were transfected with PARKIN-YFP (green) plasmid before CCCP treatment, then (i) treated with CCCP for 12 h. Mitochondria were stained with anti-Tomm20 (red) 4',6-diamidino-2-phenylindole stained nucleus was shown in blue. Co-localization was indicated by white colour. Scale bar=10 μm. (j) Quantification of the co-localization percentage of Tomm20 (red) with parkin (green) per cell by Imaris software (n=7 for NS and PGAM5 shRNA group separately. Results are presented as mean and s.d. values and were analysed by the student t-test). All the blots and images are representative of three independent experiments. DMSO, dimethylsulphoxide.",
    "molecules": "CCCP, dimethylsulphoxide, DMSO"
  },
  {
    "caption": "(a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.K) for 30 min on ice and then immunoblotted for the indicated proteins. Full-length and cleaved PGAM5 bands are indicated with arrows.",
    "molecules": "Prot.K, proteinase K"
  },
  {
    "caption": "(b) Pgam5 WT and KO MEFs were transduced with either nonspecific (NS) or Parl shRNA and either treated (+) or not (−) with CCCP for 3 h. Mitochondria were then purified and analysed by immunoblot for the indicated proteins.",
    "molecules": "CCCP"
  },
  {
    "caption": "(d) Anti-myc immunoprecipitates (IP) of lysates from 293T cells transfected with combinations of myc-tagged PGAM5 and PINK1-V5 with or without CCCP treatment and immunoblotting with anti-PINK1 or anti-myc.",
    "molecules": "CCCP"
  },
  {
    "caption": "WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.",
    "molecules": "CCCP, digitonin, DMSO, Triton X-100"
  },
  {
    "caption": "High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice.",
    "molecules": "dihydroxyphenylacetic acid, DOPAC, DA, Homovanillic acid, 3MT"
  },
  {
    "caption": "The 18-month-old Pgam5 WT and KO mice received either water as control (n=4), or 5 mg kg−1 L-DOPA and 12.5 mg kg−1 Benserazide (n=5) by intraperitoneal injection. Twenty minutes later, mice were subjected to the open-field test and the centre area time in seconds (s) as well as the centre area distance in centimeters (cm) were measured as described in the Materials and Methods. Graphs show the mean and s.d. values and were analysed by the student t-test, NS, no significance; *P0.05; **P0.01.",
    "molecules": "Benserazide, L-DOPA, water"
  },
  {
    "caption": "(j) NRK or stable clathrin-GFP-expressing NRK cell lines were analysed by pre-embedding-immuno-electron microscopy using antibodies against GFP. Scale bar, 100 nm. (k) Quantification of the average number of gold particles per image from control or clathrin-GFP cells. n = 25 images from three independent experiments. The error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.",
    "molecules": "gold"
  },
  {
    "caption": "(f) NRK cells were starved for 4 h, autolysosomes were purified by density fractionation and the quality of purified autolysosomes was monitored by TEM (left panel). Right panel, purified autolysosomes stained with antibodies against PI(4)P and PtdIns(4,5)P2. Scale bar, 500 nm.",
    "molecules": "PtdIns(4,5)P2, PI(4)P"
  },
  {
    "caption": "(g) NRK cells were starved for 0 or 4 h and stained with antibody against PI(4)P. Scale bar, 5 μm.",
    "molecules": "PI(4)P"
  },
  {
    "caption": "(h) Purified reformation tubules stained with antibodies against PI(4)P, PtdIns(4,5)P2 and PIP5K1A. Scale bar, 250 nm.",
    "molecules": "PtdIns(4,5)P2, PI(4)P"
  },
  {
    "caption": "(h) Stripped autolysosomes were pretreated with GFP or PtdIns(4,5)P2 antibody and then incubated with rat brain cytosol. Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and LAMP2.",
    "molecules": "PtdIns(4,5)P2"
  },
  {
    "caption": "(c) Trypsin-treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP, clathrin, AP2, AP3, AP4 and PtdIns(4,5)P2, and then analysed by western blotting.",
    "molecules": "PtdIns(4,5)P2"
  },
  {
    "caption": "Volumetric measurements based on the PET and CT scans could be generated from M032, M032R1, and M032R5. Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment, but regressed upon vemurafenib treatment. M032R5 showed progressive disease after 4 months, whereas M032R1 still displayed stable disease. M032 was excised and no recurrence was observed.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "Hematoxylin-eosin (H&E) and p-ERK stainings on FFPE material of all metastases showed that all vemurafenib-resistant tumors had reactivation of the MAPK pathway. Scale bar represents 100 μm",
    "molecules": "vemurafenib"
  },
  {
    "caption": "qPCR was performed on gDNA retrieved from each of the metastases, using primers for BRAF and CRAF and normalized on LINE levels. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in M032R1, M032R2, and M032R5.",
    "molecules": "gDNA"
  },
  {
    "caption": "Dose-response curves for A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.",
    "molecules": "SCH772984, trametinib, vemurafenib"
  },
  {
    "caption": "Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).",
    "molecules": "dabrafenib, DMSO, trametinib"
  },
  {
    "caption": "A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean.",
    "molecules": "dabrafenib"
  },
  {
    "caption": "Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX.",
    "molecules": "vemurafenib"
  },
  {
    "caption": "D. Enrichment of phosphor-tyrosine (pY) at CAR microclusters. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-pan phosphor-tyrosine antibody. No CD19 was included in the control experiment (only ICAM-1). Scale bar: 2 μm.",
    "molecules": "phosphor-tyrosine, pY"
  },
  {
    "caption": "A. WT or LAT-deficient Jurkat cells (Jcam2.5) expressing CAR-GFP were stimulated on SLB-coated with OKT3 (anti-TCR antibody) or CD19 (CAR antigen); in both conditions, the lipid bilayers were also coated with ICAM-1 to facilitate cell attachment. TIRF microscopy revealed clustering of Ax647-labeled streptavidin-Biotin-OKT3 or CD19, which serves as a probe for TCR or CAR respectively. Scale bar, 2 µm. B. Quantification of clustering as normalized variance. n = 100 cells. Shown are the mean ± SD. Statistical test: unpaired two-tailed t-test. **** p < 0.0001. ** 0.005 < p < 0.05. ",
    "molecules": "Ax647, Biotin"
  },
  {
    "caption": "B. WT or LAT-deficient (Jcam2.5) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM-1. TIRF microscopy revealed clustering of CAR and Ax647-labeled streptavidin-Biotin-CD19. Scale bar, 2 µm. C. Quantification of clustering level of Ax647-labeled streptavidin-Biotin-CD19. Shown are means ± SD. Clustering is quantified as normalized variance. N = 50 cells for CAR 3rd WT and 1st WT, 63 cells for CAR 3rd lat-, and 51 cells for CAR 1st lat-. **: 0.0005 < p < 0.005. Statistical test: unpaired two-tailed t-test. ",
    "molecules": "Ax647, Biotin"
  },
  {
    "caption": "B. CAR T cells were stimulated on SLBs coated with biotin-OKT3 (left) or biotin-CD19 (right) supplemented with ICAM-1. TIRF microscopy revealed clustering of Alexa647-labeled streptavidin-Biotin-OKT3 or CD19. Scale bar, 5 µm. C. Quantification of clustering of OKT3 or CD19 as normalized variance. n > 200 cells from two independent donors were scored for each condition. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.48. ",
    "molecules": "Alexa647, biotin, Biotin"
  },
  {
    "caption": "LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. CBA analysis of IL-1β (A) in the culture supernatants.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. ELISA of IL-18 (B) in the culture supernatants.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "CBA analysis of IL-1β secretion from Abro1+/+ and Abro1−/− BMDMs transduced with GFP- or ABRO1-GFP-expressing lentiviruses prior to stimulation with LPS and ATP. Cell lysates were immunoblotted with anti-ABRO1 antibody.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "ELISA analysis of IL-1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium (Con), LPS or LPS plus ATP. Cell lysates were immunoblotted with anti-ABRO1 and anti-GAPDH antibodies.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "Abro1+/+ and Abro1−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with nigericin, silica, MSU, MDP, Alum, poly (dA: dT), flagellin or LTx (Anthrax Lethal Factor). CBA analysis of IL-1β secretion in the culture supernatants.",
    "molecules": "LTx, poly (dA: dT), Alum, flagellin, LPS, MSU, MDP, nigericin, silica"
  },
  {
    "caption": "Representative immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Abro1+/+ and Abro1−/− BMDMs treated with ATP, nigericin, MSU, flagellin or poly (dA: dT).",
    "molecules": "poly (dA: dT), ATP, flagellin, LPS, MSU, nigericin"
  },
  {
    "caption": "Flow cytometric analysis of caspase-1 activity in LPS-primed Abro1+/+ and Abro1−/− BMDMs stimulated with nigericin for indicated times.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Immunostaining of endogenous ASC specks in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin or poly (dA:dT). Scale bars, 10 μm. Quantification of ASC specks from (H) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei. At least 100 cells from each treatment condition were quantified. UD, undetectable.",
    "molecules": "poly (dA:dT), LPS, nigericin"
  },
  {
    "caption": "LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti-ASC antibody.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (A). n = 6 for PBS groups and n = 12 for MSU groups.",
    "molecules": "PBS, MSU"
  },
  {
    "caption": "WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups.",
    "molecules": "PBS, MSU"
  },
  {
    "caption": "Abro1+/+ and Abro1−/− mice were intraperitoneally injected with MSU (1 mg per mouse). Flow cytometric analysis of caspase-1 activity in peritoneal exudate monocytes and neutrophils. n=12 per group.",
    "molecules": "MSU"
  },
  {
    "caption": "Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β in the peritoneal lavage fluid (D) n = 6 for PBS groups and n = 12 for Alum groups.",
    "molecules": "Alum, PBS"
  },
  {
    "caption": "Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (E). n = 6 for PBS groups and n = 12 for Alum groups.",
    "molecules": "Alum, PBS"
  },
  {
    "caption": "Survival of Abro1+/+ and Abro1−/− mice subjected to LPS (15 mg/kg). n=20 per group.",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.",
    "molecules": "PBS, LPS"
  },
  {
    "caption": "HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.",
    "molecules": "agarose"
  },
  {
    "caption": "HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.",
    "molecules": "agarose"
  },
  {
    "caption": "WT BMDMs were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.",
    "molecules": "Anisomycin"
  },
  {
    "caption": "WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.",
    "molecules": "SP600125, LPS"
  },
  {
    "caption": "HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194A) were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.",
    "molecules": "Anisomycin"
  },
  {
    "caption": "HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194D) were treated with or without 100 nM SP600125 for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.",
    "molecules": "SP600125"
  },
  {
    "caption": "WT and Nlrp3S194A/S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.",
    "molecules": "Anisomycin, LPS"
  },
  {
    "caption": "HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.",
    "molecules": "agarose, MG132"
  },
  {
    "caption": "HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.",
    "molecules": "agarose, MG132"
  },
  {
    "caption": "LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.",
    "molecules": "ATP, LPS, nigericin"
  },
  {
    "caption": "Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.",
    "molecules": "LPS"
  },
  {
    "caption": "HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.",
    "molecules": "Anisomycin, SP600125"
  },
  {
    "caption": "LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.",
    "molecules": "ATP, LPS"
  },
  {
    "caption": "WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody.",
    "molecules": "LPS"
  },
  {
    "caption": "Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.",
    "molecules": "LPS, MG132"
  },
  {
    "caption": "Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody.",
    "molecules": "LPS, MG132"
  },
  {
    "caption": "Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.",
    "molecules": "poly (dA:dT), Alum, ATP, flagellin, LPS, MSU, nigericin"
  },
  {
    "caption": "Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT).",
    "molecules": "poly (dA: dT), ATP, flagellin, LPS, MSU, nigericin"
  },
  {
    "caption": "Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable.",
    "molecules": "poly (dA:dT), LPS, nigericin"
  },
  {
    "caption": "LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.",
    "molecules": "LPS, nigericin"
  },
  {
    "caption": "Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group.",
    "molecules": "LPS"
  },
  {
    "caption": "ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.",
    "molecules": "PBS, LPS"
  },
  {
    "caption": "Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G)",
    "molecules": "PBS, MSU"
  },
  {
    "caption": "Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.",
    "molecules": "PBS, MSU"
  },
  {
    "caption": "(B) Signal developed with oligo(dT)25-TexasRed probes on GFP-Trap_M beads binding EmGFP-Tm1-I or FlagMycGFP after denaturing washes. 594 nm reflection marks bead boundaries.(B') Quantification of mean oligo(dT) signal measured on beads.",
    "molecules": "oligo(dT)"
  },
  {
    "caption": "(D) Images of beads binding to EmGFP-Tm1-I (green) and DIG labelled in vitro transcribed RNA fragments (magenta).(E, F) Mean DIG-Cy5 fluorescence measured on beads capturing the RNA fragment, with or without UV cross-linking, indicated below the charts. P values of pairwise Mann-Whitney U tests are indicated. In panel F, none of the non-cross-linked samples differ significantly from the no RNA control (p>0.05). Numbers below the plots indicate the number of beads analysed.",
    "molecules": "RNA"
  }
]